INDOLINONE COMPOUNDS AS KINASE INHIBITORS

COMPOSES D'INDOLINONE TELS QUE DES INHIBITEURS DE KINASE

English Abstract

The invention relates to certain indolinone compounds, their method of
synthesis, and a combinatorial library consisting of the indolinone compounds
of the invention. The invention also relates to methods of modulating the
function of protein kinases using indolinone compounds of the invention and
methods of treating diseases by modulating the function of protein kinases and
related signal transduction pathways.

French Abstract

L'invention concerne certains composés d'indolinone, leur méthode de synthèse, et une bibliothèque combinatoire contenant les composés d'indolinone de cette invention. Cette dernière concerne également des méthodes de modulation de la fonction des protéines kinases au moyen des composés d'indolinone de cette invention, et des méthodes de traitement de maladies par modulation de la fonction des protéines kinases et des voies relatives de transduction de signaux.

Claims

190

What is claimed is:

CLAIMS

1. A compound having the chemical structure set forth in formula I:
Image
or a pharmaceutically acceptable salt thereof wherein,
n and m are independently 0 or 1;

when n is 1, then A, B, D, E and F are independently selected from the group
consisting of carbon and nitrogen; however, no more than three of A, B, D, E
and
F are nitrogen at the same time and, when A, B, D, E, or F is nitrogen, then
R4,
R5, R6, R7 or R8, respectively, does not exist;

when m is 1, then G, H, J, K and L are independently selected from the group
consisting of carbon and nitrogen; however, at least one and no more than
three of
G, H, J, K and L are nitrogen at the same time and, when G, H, J, K or L is
nitrogen, then R9, R10, R11, R12 or R13, respectively, does not exist;


when n is 0, then A is selected from the group consisting of carbon and
nitrogen,
B and F are selected from the group consisting of carbon, nitrogen, NH, oxygen
and sulfur, provided that when B or F is NH, the other cannot be NH, and E is


191


selected from the group consisting of carbon, nitrogen, oxygen and sulfur,
further
provided that no more than one of B, E or F is oxygen or sulfur and provided
also
that at least one of A, B, E or F is a heteroatom (i.e., not carbon);
when m is 0, then G is selected from the group consisting of carbon and
nitrogen,
H, K and L are selected from the group consisting of carbon, nitrogen, NH,
oxygen and sulfur, provided that when H or L is NH, the other cannot be NH,
and
K is selected from the group consisting of carbon, nitrogen, oxygen and
sulfur,
further provided that no more than one of H, K or L is oxygen or sulfur and
provided also that at least one of G, H, K or L is a heteroatom (i.e., not
carbon);
R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently
selected
from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl,
alkenyl,
alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, mercapto,
alkylthio,
aryloxy, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl,
C-
carboxy, O-carboxy, carboxyalkyl, cyano, nitro, halo, O-carbamyl, N-carbamyl,
C-amido, N-amido and -NR14R15;
R4 and R5 or R5 and R6 or R6 and R7 or R8 and R8 may combine to form a five-
member or a six-member aryl or heteroaryl ring; and,
R14 and R15 are independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, carbonyl, sulfonyl,
and,
combined, a five-member or a six-member heteroalicyclic ring.
2. The compound or salt of claim 1 wherein,
R1, R2 and R3 are independently selected from the group consisting of,
hydrogen;
lower alkyl, optionally substituted with one or more halo groups;
lower alkoxy, optionally substituted with one or more halo groups;
halo;


192


(lower alkyl)-S-sulfonamido;
(aryl)-S-sulfonamido; and,
-NR14R15;
R4, R5, R6, R7, R8, R9, R10, R11, R12 and R13 are independently selected from
the
group consisting of:
hydrogen,
lower alkyl, optionally substituted with one or more groups selected from the
group consisting of aryl, heteroaryl, heteroalicyclic, halo, hydroxy, lower
alkoxy,
mercapto, lower alkylthio, C-carboxy and -NR14R15;
cycloalkyl,
hydroxy,
lower alkoxy, optionally substituted with one or more groups selected from the
group consisting of one or more halo groups, aryl, heteroaryl and
heteroalicyclic,
halo,
cyano,
nitro,
carboxyalkyl,
aryl, optionally substituted with one or more groups selected from the group
consisting of,
lower alkyl optionally substituted one or more halo groups;
halo;
hydroxy;
lower alkoxy optionally substituted with one or more halo groups,
aryloxy; and,
-NR10R11;
aryloxy, optionally substituted with one or more groups selected from the
group
consisting of,
lower alkyl optionally substituted with one or more halo groups;
halo;
hydroxy;
lower alkoxy optionally substituted with one or more halo groups; and,


193


-NR14R15;
heteroaryl, optionally substituted with one or more groups
selected from the group consisting of,
lower alkyl optionally substituted with one or more halo groups;
halo;
hydroxy;
lower alkoxy optionally substituted with one or more halo groups; and,
-NR14R15,
heteroalicyclic, optionally substituted with one or more groups selected from
the
group consisting of,
lower alkyl optionally substituted with one or more halo groups;
halo;
hydroxy;
lower alkoxy optionally substituted with one or more halo groups; and,
-NR14R15;
(lower alkyl) C-carboxy,
(lower alkyl)carbonyl,
aryl carbonyl,
(lower alkyl)-S-sulfonamido,
aryl-S-sulfonamido,
(lower alkyl)-N-sulfonamido,
aryl-N-sulfonamido,
(lower alkyl)-N-carbamoyl,
(lower alkyl)-C-amido,
(lower alkyl)-N-amido,
(cycloalkyl)-N-amido, and,
-NR14R15;
R14 and R15 being independently selected from the group consisting of hydrogen
and
lower alkyl.
3. The compound or salt of claim 1 wherein,


194


n is 1;
A, B, D, E and F are carbon;
m is 1; and,
one of G, H, J, K and L is nitrogen.
4. The compound or salt of claim 1 wherein,
n is 1;
A, B, D, E and F are carbon;
m is 0;
G, H and K are carbon; and,
L is NH.
5. The compound or salt of claim 1 wherein,
n is 0;
A, B and E are carbon;
F is NH;
R4 and R7 are independently selected from the group consisting of hydrogen and
lower alkyl;
R5 is selected from the group consisting of,
lower alkyl optionally substituted with one or more groups selected from the
group consisting of,
hydroxy;
heteroalicyclic containing at least one NH group;
C-carboxy; and,
-NR14R15;
carboxyalkyl;
(lower alkyl)-C-carboxy; and,
-NR14R15;
m is 1;
one of G, H, J, K and L is nitrogen; and,
R14 and R15 are independently selected from the group consisting of hydrogen,


195


lower alkyl and carbonyl.
6. The compound or salt of claim 1 wherein,
n is 0;
A, B and E are carbon;
F is NH;
R4 and R7 are independently selected from the group consisting of hydrogen and
lower alkyl;
R5 is selected from the group consisting of,
lower alkyl optionally substituted with one or more groups selected from the
group consisting of,
hydroxy;
heteroalicyclic containing at least one NH group;
C-carboxy; and,
-NR14R15;
carboxyalkyl;
(lower alkyl)-C-carboxy; and,
-NR14R15;
m is 0;
G, H and K are carbon;
L is NH, oxygen or sulfur; and,
R14 and R15 are independently selected from the group consisting of hydrogen,
lower alkyl and carbonyl.
7. A 3-aralkyl-2-indolinone having the chemical structure set forth in
formula II:


196


Image
or a physiologically acceptable salt thereof wherein,
n is 0 or 1;
when p is 1, then M, Q, T, Y, and V are independently selected from the group
consisting of carbon and nitrogen, it being understood that, when M, Q, T, Y,
or
V is nitrogen, R20, R21, R22, R23, or R24, respectively, do not exist;
when p is 0, then M, Q, Y, and V are independently selected from the group
consisting of carbon, nitrogen, oxygen and sulfur, it being understood that,
when
M, Q, Y, or V is oxygen or sulfur or nitrogen (wherein said nitrogen is
participating in a double bond), R20, R21, R22, R23, or R24, respectively, do
not
exist;
R16, R17, R18, R19, R20, R21, R22, R23, or R24 are independently selected from
the
group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,
alkynyl,
aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, mercapto, alkylthio,
aryloxy,
sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl, C-carboxy, O-
carboxy, carboxyalkyl, cyano, nitro, halo, O-carbamyl, N-carbamyl, C-amido, N-
amido and -NR25R26;
R20 and R21 or R21 and R22 or R23 and R23 or R23 and R24 may combine to form a


197


five-member or a six-member aryl or heteroaryl ring; and,
R25 and R26 are independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, carbonyl, sulfonyl,
and,
combined, a five-member or a six-member heteroalicyclic ring.
8. The compound of claim 7 wherein, R16, R17, R18, R19, R20, R21, R22,
R23, or R24 are independently selected from the group consisting of:
hydrogen,
lower alkyl, optionally substituted with one or more groups selected from the
group consisting of cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,
hydroxy, C-
carboxy and
-NR25R26,
cycloalkyl,
hydroxy,
lower alkoxy,optionally substituted with one or more groups selected from the
group consisting of one or more halo groups, aryl, heteroaryl and
heteroalicyclic,
trihalomethyl,
trihalomethoxy,
halo,
carboxyalkyl,
aryl,optionally substituted with one or more groups selected from the group
consisting of lower alkyl, lower alkyl substituted with one or more halo
groups,
trihalomethyl, trihalomethoxy, halo, hydroxy, lower alkoxy, aryloxy and -
NR25R26,
aryloxy,
heteroaryl, optionally substituted with one or more groups
selected from the group consisting of lower alkyl, trihalomethyl, halo,
hydroxy,
(lower alkyl)alkoxy, amino and
-NR25R26,
heteroalicyclic,
(lower alkyl)carboxy,


198


(lower alkyl)carbonyl,
aryl carbonyl,
(lower alkyl)-S-sulfonamido,
aryl-S-sulfonamido,
(lower alkyl)-N-sulfonamido,
aryl-N-sulfonamido,
(lower alkyl)-N-carbamoyl,
(lower alkyl)-C-amido,
(lower alkyl)-N-amido,
(cycloalkyl)-N-amido, and
-NR25R26.
9. The compound of claim 7 wherein,
p is 1; and,
M, Q, T, U, and V are carbon.
10. The compound of claim 9 wherein,
R16, R17, R18, and R19 are independently selected from the group consisting
of:
hydrogen,
halo,
hydroxy,
-NR25R26,
S-sulfonamido optionally substituted with one or more groups selected from the
group consisting of hydrogen, lower alkyl and aryl,
lower alkyl optionally substituted with a group selected from the group
consisting
of:
hydroxy,
one or more halo groups,


199


C-carboxy,
C-amido,
heteroalicyclic and
-NR25R26,
lower alkoxy optionally substituted with one or more halo groups,
aryl optionally substituted with one or more groups selected from lower alkyl,
lower alkoxy, halo, hydroxy and -NR25R26 and,
N-amido optionally substituted with one or more groups selected from the group
consisting of hydrogen, lower alkyl, cycloalkyl and aryl; and,
R20, R21, R22, R23, and R24 are independently selected from the group
consisting of:
hydrogen,
halo,
hydroxy,
-NR25R26,
lower alkyl optionally substituted with one or more groups selected from the
group consisting:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and
-NR26R26,
cycloalkyl,
lower alkoxy optionally substituted with one or more groups selected from the
group consisting of:
one or more halo groups,
aryl,
-NR25R26, and
heteroaryl,
aryl optionally substituted with one or more groups selected from the group


200


consisting of:
halo,
hydroxy,
lower alkoxy,
-NR25R26 and
C-carboxy.
11. The compound of claim 7 wherein,
p is 1; and,
one or two of M, Q, T, U, or V is nitrogen.
12. The compound of claim 11 wherein,
R16, R17, R18, and R19, are independently selected from the group consisting
of:
hydrogen,
halo,
hydroxy,
-NR25R26,
S-sulfonamido optionally substituted with one or more groups selected from the
group consisting of hydrogen, lower alkyl and aryl,
lower alkyl optionally substituted with a group selected from the group
consisting
of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,


201


lower alkoxy optionally substituted with one or more halo groups,
aryl optionally substituted with one or more groups selected from the group
consisting of lower alkyl, lower alkoxy, halo, hydroxy and -NR25R26,
N-amido optionally substituted with one or more groups selected from the group
consisting of hydrogen, lower alkyl, cycloalkyl and aryl; and,
R20, R21, R22, R23, and R24, whichever of these are not nitrogen, are
independently
selected from the group consisting of:
hydrogen,
halo,
hydroxy,
-NR25R26,
lower alkyl optionally substituted with one or more groups selected from the
group consisting of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
cycloalkyl,
lower alkoxy optionally substituted with one or more groups selected from the
group consisting of:
one or more halo groups,
aryl,
-NR25R26 and,
heteroaryl,
aryl optionally substituted with one or more groups selected from the group
consisting of:
halo,
hydroxy,


202


lower alkoxy,
-NR25R26 and,
C-carboxy.

13. The compound of claim 7 wherein,
p is 1; and,
R21 and R22 combine to form a fused pyrrolo group.

14. The compound of claim 13 wherein,
R16, R17, R18, and R19 are independently selected from the group consisting
of:
hydrogen,
halo,
hydroxy,
-NR25R26,
S-sulfonamido optionally substituted with one or more groups selected from the
group consisting of hydrogen, lower alkyl and aryl,
lower alkyl optionally substituted with a group selected from the group
consisting
of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
lower alkoxy optionally substituted with one or more halo groups,
aryl optionally substituted with one or more groups selected from the group
consisting of lower alkyl, lower alkoxy, halo, hydroxy and -NR25R26,
N-amido optionally substituted with one or more groups selected from the group
consisting of hydrogen, lower alkyl, cycloalkyl and aryl; and,


203


R20, R23, and R24are independently selected from the group consisting of:
hydrogen,
halo,
hydroxy,
-NR25R26,
lower alkyl optionally substituted with one or more groups selected from the
group consisting of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
cycloalkyl,
lower alkoxy optionally substituted with one or more groups selected from the
group consisting of:
one or more halo groups,
aryl,
-NR25R26 and,
heteroaryl,
aryl optionally substituted with one or more groups selected from the group
consisting of:
halo,
hydroxy,
lower alkoxy,
-NR25R26 and,
C-carboxy.
15. The compound of claim 7 wherein,


204


p is 0;
M or Q is -NH, oxygen or sulfur; and,
M or Q, whichever is not -NH, oxygen or sulfur, and U and V are carbon.
16. The compound of claim 15 wherein,
R16, R17, R18, and R19 are independently selected from the group consisting
of:
hydrogen,
halo,
hydroxy,
-NR25R26,
S-sulfonamido optionally substituted with one or more groups selected from the
group consisting of hydrogen, lower alkyl and aryl,
lower alkyl optionally substituted with a group selected from the group
consisting
of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
lower alkoxy optionally substituted with one or more halo groups,
aryl optionally substituted with one or more groups selected from the group
consisting of lower alkyl, lower alkoxy, halo, hydroxy and -NR25R26,
N-amido optionally substituted with one or more groups selected from hydrogen,
lower alkyl, cycloalkyl and aryl; and,
R20, R21, R23, and R24 are independently selected from the group consisting
of:
hydrogen,


205


halo,
hydroxy,
-NR25R26,
lower alkyl optionally substituted with one or more groups selected from the
group consisting of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
cycloalkyl,
lower alkoxy optionally substituted with one or more groups selected from the
group consisting of:
one or more halo groups,
aryl,
-NR25R26 and,
heteroaryl.
17. The compound of claim 7 wherein,
R16, R17, R18, and R19 are independently selected from the group consisting
of:
hydrogen,
halo,
hydroxy,
-NR25R26,
S-sulfonamido optionally substituted with one or more groups selected from the
group consisting of hydrogen, lower alkyl and aryl,
lower alkyl optionally substituted with a group selected from the group
consisting
of:
hydroxy,


206


one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
lower alkoxy optionally substituted with one or more halo groups,
aryl optionally substituted with one or more groups selected from the group
consisting of lower alkyl, lower alkoxy, halo, hydroxy and -NR25R26,
N-amido optionally substituted with one or more groups selected from hydrogen,
lower alkyl, cycloalkyl and aryl; and,
R20, R23, and R24 are independently selected from the group consisting of:
hydrogen,
halo,
hydroxy,
-NR25R26,
lower alkyl optionally substituted with one or more groups selected from the
group consisting of:
hydroxy,
one or more halo groups,
C-carboxy,
C-amido,
heteroalicyclic and,
-NR25R26,
cycloalkyl,
lower alkoxy optionally substituted with one or more groups selected from the
group consisting of:
one or more halo groups,
aryl,
-NR25R26 and,
heteroaryl,


207


aryl optionally substituted with one or more groups selected from the group
consisting of:
halo,
hydroxy,
lower alkoxy,
-NR25R26 and,
C-carboxy.

18. An indolinone compound having a structure set forth in formula III:
Image
wherein
(a) R27 is selected from the group consisting of
(i) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl, alkoxy,
carboxylate,
amino, nitro, ester, and a five-membered or six-membered aromatic,
heteroaromatic,
aliphatic, or heteroaliphatic ring moiety,
wherein said ring moiety is optionally substituted with one, two, or
three substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties; and
(ii) an aromatic or heteroaromatic ring optionally substituted with one, two,
or
three substituents independently selected from the group consisting of alkyl,
alkoxy,


208


halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;

(b) R32, R33, and R34 are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with one or more
substituents selected from the group consisting of halogen, trihalomethyl,
carboxylate, amino, nitro, ester, and a five-membered or six-membered
aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moiety,
wherein said ring moiety is optionally substituted with one or more
substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents


209
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(v) an amine of formula -(X1)n1-NX2X3, wherein
X, is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
or aliphatic ring moieties,
n 1 is 0 or 1,and
X2 and X3 are independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula -NO2;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula -(X4)n4-CO-X5, wherein
X4 and X5 are independently selected from the group consisting of
alkyl and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one, two, or three substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester moieties, and
n 4 is 0 or 1;


210
(ix) a carboxylic acid of formula -(X6)n6-COOH or ester of formula -(X7)n7,-
COO-
X8, wherein
X6, X7, and X8 and are independently selected from the group
consisting of alkyl and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties, and
n6 and n7 are independently 0 or 1;
(x) an alcohol of formula -(X9)n9-OH or an alkoxyalkyl moiety of formula
-(X10)n10-O-X11, wherein
X9, X10, and X11 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n9 and n10 are independently 0 or 1;
(xi) an amide of formula -(X12)n12-NHCOX13, or of formula -(X14)n14-CONX15X16,
wherein
X12 and X14 are each independently selected from the group consisting
of alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,


211
said ring is optionally substituted with one or more
substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
and ester, and
n 12 and n 14 are independently 0 or 1,
X13, X15, and X16 are each independently selected from the group
consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said ring is optionally substituted with one or more
substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
and ester;
(xii) a sulfonamide of formula -(X17)n17-SO,NX18X19, wherein
X17 is selected from the group consisting of alkyl, and five-membered
or six-membered aromatic, heteroaromatic, aliphatic, or
heteroaliphatic ring moieties
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester,
n17 is 0, 1, or 2,


212

X18, and X19 are independently selected from the group consisting of
hydrogen, alkyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties
said alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
or
wherein X18 and X19 taken together form a five-membered or six-
membered aliphatic or heteroaliphatic ring optionally substituted with
one or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
and ester;
(xiii) an aldehyde of formula -(X20)n20-CO-H wherein
X20 is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n20 is 0 or 1;
(xiv) a sulfone of formula -(X21)n21-SO2-X22, wherein
X21 and X22 are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered




213

aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n21 is 0 or 1; and
(xv) a thiol of formula -(X23)n23-SH or a thioether of formula -(X24)n24-S-
X25,
wherein
X23, X24, and X25 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n23 and n24 are independently 0 or 1;
or
R3, and R34 taken together form a six-membered aliphatic or aromatic ring,
optionally substituted with one or more substituents independently selected
from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
and
ester; and


214
(c) R28, R29, R30, and R31 are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with one or more
substituents selected from the group consisting of hydroxy, halogen,
trihalomethyl,
carboxylate, amino, nitro, ester, and a five-membered or six-membered
aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moiety,
said ring moiety is optionally substituted with one or more
substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(v) an amine of formula -(X1)n1-NX2X3, wherein
X1 is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
or aliphatic ring moieties,


215
n1 is 0 or 1, and
X2 and X3 are independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula -NO2;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula -(X4)n4-CO-X5, wherein
X4 and X5 are independently selected from the group consisting of
alkyl and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one, two, or three substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester moieties, and
n4 is 0 or 1;
(ix) a carboxylic acid of formula -(X6)n6-COOH or ester of formula -(X7)n7-COO-

X8, wherein
X6, X7, and X8 and are independently selected from the group
consisting of alkyl and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties, and


216
n6 and n7 are independently 0 or 1;
(x) an alcohol of formula -(X9)n9-OH or an alkoxyalkyl moiety of formula
-(X10)n10-O-X11, wherein
X9, X10, and X11 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n9 and n10 are independently 0 or 1;
(xi) an amide of formula -(X12)n12-NHCOX13, or of formula -(X14)n14-CONX15X16,
wherein
X12 and X14 are each independently selected from the group consisting
of alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester,
n12 and n14 are independently 0 or 1, and


217
X13, X15, and X16 are each independently selected from the group
consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties, and
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester;
(xii) a sulfonamide of formula -(X17)n17-SO2NX18X19, wherein
X17 is selected from the group consisting of alkyl, five-membered or
six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
ring moieties
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n17 is 0, 1, or 2, and
X18, and X19 are independently selected from the group consisting of
hydrogen, alkyl, five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, or


218
X18 and X19 taken together form a five-membered or six-membered
aliphatic or heteroaliphatic ring optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester;
(xiii) an aldehyde of formula -(X20)n20-CO-H wherein
X2O is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n20 is 0 or 1;
(xiv) a sulfone of formula -(X21)n21-SO2-X22, wherein
X21 and X22 are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n21 is 0 or 1; and


219
(xv) a thiol of formula -(X23)n23-SH or a thioether of formula -(X24)n24-S-
X25,
wherein
X23, X24, and X25 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n23 and n24 are independently 0 or 1.
19. The compound of claim 18, wherein
(a) R6 is hydrogen;
(b) R7, R8, and R9 are each independently selected from the group consisting
of
(i) hydrogen;
(ii) saturated alkyl optionally substituted with a six-membered
heteroaliphatic
ring moiety;
(iii) an amine of formula -(X1)n1-NX2X3, wherein X1 is saturated alkyl, n1 is
0 or
1, and X2 and X3 are independently selected from the group consisting of
hydrogen
and saturated alkyl;




220

(iv) a carboxylic acid of formula -(X6)n6-COOH or ester of formula -(X7)n7-COO-

X8, wherein X6, X7, and X8 and are alkyl, and n6 and n7 are independently 0 or
1;
and

(v) an amide of formula -(X14)n14-CONX15X16, wherein X14 is alkyl, n12 and n14
are independently 0 or 1, and X15 and X16 are each independently selected from
the
group consisting of hydrogen and alkyl;

or

R8 and R9, taken together form an optionally substituted six-membered
aliphatic or
aromatic ring; and

(c) R3, R4, and R5 are each independently selected from the group consisting
of
(i) hydrogen;
(ii) saturated alkyl;
(iii) a halogen or trihalomethyl;
(iv) a carboxylic acid of formula -(X6)n6-COOH or ester of formula -(X7)n7-COO-

X8, wherein X6, X7, and X8 and are alkyl, and n6 and n7 are independently 0 or
1;
(v) an alcohol of formula -(X9)n9-OH or an alkoxyalkyl moiety of formula
-(X10)n10-O-X11, wherein X9, X10, and X11 are alkyl, and n9 and n10 are
independently
0 or 1;

(vi) an amide of formula -(X12)n12-NHCOX13, or of formula -(X14)n14-CONX15X16,
wherein X12 and X14 are alkyl, n12 and n14 are independently 0 or 1, and X13,
X15,
and X16, are each independently selected from the group consisting of hydrogen
and


221
alkyl; and
(vii) a sulfonamide of formula -(X17)n17-SO2NX18X19, wherein X17 is alkyl, and
n17
is 0, 1, or 2, and X18, and X19 are independently selected from the group
consisting of
hydrogen and alkyl.
20. The compound of claim 18, wherein
(a) R3 is selected from the group consisting of hydrogen, methyl, and 2-
hydroxyethyl;
(b) R4 is selected from the group consisting of hydrogen, methyl, chloro,
bromo,
carboxy, methoxy, -NHC(O)-CH3, and -SO2N(CH3)2;
(c) R5 is selected from the group consisting of hydrogen, chloro, methoxy, and
-NHC(O)-CH3;
(d) R6 is hydrogen;
(e) R7 is selected from the group consisting of hydrogen, methyl, -
CH2CH2C(O)OH, -CH2CH2C(O)NH2, -CH2CH2CH2N(CH3)2, and 3-
morpholinopropyl; and
(f) R8 and R9, are each independently selected from the group consisting of
hydrogen, methyl, -CH2CH2C(O)OH, -CH2CH2CH2N(CH3)2, and 3-
morpholinopropyl, or R8 and R9, taken together form a six-membered aromatic or
aliphatic ring.
21. An indolinone compound, wherein said compound is formed by the
reaction of a ketone with and oxindole,
wherein said ketone is selected from the group consisting of


222

Image


223
Image
wherein R1 is selected from the group consisting of
(i) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl, alkoxy,
carboxylate,
amino, nitro, ester, and a five-membered or six-membered aromatic,
heteroaromatic,
aliphatic, or heteroaliphatic ring moiety,
wherein said ring moiety is optionally substituted with one, two, or
three substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties; and
(ii) an aromatic or heteroaromatic ring optionally substituted with one, two,
or
three substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties; and
wherein said oxindole is selected from the group consisting of 1,3-dihydro-


224
indol-2-one, 4-methyl-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-1,3-dihydro-

indol-2-one, 5-chloro-1,3-dihydro-indol-2-one, 5-bromo-1,3-dihydro-indol-2-
one, 5-
methoxy-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-carboxylic
acid,
2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide, N-(2-oxo-2,3-
dihydro-
1H-indole-5-yl)-acetamide, 6-chloro-1,3-dihydro-indol-2-one, 6-methoxy-1,3-
dihydro-indol-2-one, and N-(5-methyl-2-oxo-2,3-dihydro-1H-indole-6-yl)-
acetamide.
22. A compound having a structure set forth in formula IV or formula V:
Image
wherein
(a) R1, R2, and R7 are each independently selected from the group consisting
of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl, alkoxy,
carboxylate,
amino, nitro, ester, and a five-membered or six-membered aromatic,
heteroaromatic,
aliphatic, or heteroaliphatic ring moiety,
wherein said ring moiety is optionally substituted with one, two, or
three substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties; and
(iii) an aromatic or heteroaromatic ring optionally substituted with one, two,
or


225
three substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(b) R3 is an ethyl-2-oxy group of formula -CH2CH2-O-R, wherein R is selected
from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl,
carboxylate, amino, nitro, ester, and a five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moiety, and
said ring moiety is optionally substituted with one, two, or
three substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of
alkyl, aryl, alkoxy, halogen, trihalomethyl, carboxylate, amino, nitro,
and ester moieties;
(iv) a substituent of formula -C(E)NX15X16, wherein
X15, and X16 are each independently selected from the group
consisting of hydrogen, alkyl, hydroxyl, sulfone of formula -
SO2-X22, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said ring is optionally substituted with one or more
substituents independently selected from the group
consisting of alkyl, aryl, halogen, trihalomethyl,
carboxylate, amino, nitro, and ester,
X22 is selected from the group consisting of saturated
or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or


226
heteroaliphatic ring moieties,
said ring is optionally substituted with one or
more substituents independently selected from
the group consisting of alkyl, halogen,
trihalomethyl, carboxylate, amino, nitro, and
ester,
E is selected from the group consisting of oxygen and sulfur,
and;
(c) R4, R5, and R6 are each hydrogen;
(d) Z is a 5, 6, 7, 8, 9, or 10 membered, monocyclic or bicyclic, aromatic or
heteroaromatic, ring moiety, optionally substituted with one or more
substituents
selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with one or more
substituents selected from the group consisting of hydroxy, halogen,
trihalomethyl,
carboxylate, amino, nitro, ester, and a five-membered or six-membered
aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moiety,
said ring moiety is optionally substituted with one or more
substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(v) an amine of formula -(X1)n1-NX2X3, wherein




227

X1 is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
or aliphatic ring moieties,
n1 is 0 or 1, and
X2, and X3 are independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula -NO2;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula -(X4)n4-CO-X5, wherein
X4 and X5 are independently selected from the group consisting of
alkyl and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one, two, or three substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester moieties, and
n4 is 0 or 1;
(ix) a carboxylic acid of formula -(X6)n6-COOH or ester of formula -
(X7)n7-COO-X8, wherein
X6, X7, and X8 and are independently selected from the group
consisting of alkyl and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties, and
n6 and n7 are independently 0 or 1;
(x) an alcohol of formula -(X9)n9-OH or an alkoxyalkyl moiety of formula
-(X10)n10-O-X11, wherein
X9, X10, and X11 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said alkyl and ring moieties are optionally substituted with one or




228

more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n9 and n10 are independently 0 or 1;
(xi) an amide of formula -(X12)n12-NHCOX13, or of formula -(X14)n14-CONX15X16,
wherein
X12 and X14 are each independently selected from the group consisting
of alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester,
n12 and n14 are independently 0 or 1, and
X13, X15, and X16 are each independently selected from the group
consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties, and
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester;
(xii) a sulfonamide of formula -(X17)n17-SO2NX18X19, wherein
X17 is selected from the group consisting of alkyl, five-membered or
six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
ring moieties
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, or ester, and
n17 is 0, 1, or 2, and




229

X18, and X19 are independently selected from the group consisting of
hydrogen, alkyl, five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, or ester, or
X18 and X19 taken together form a five-membered or six-membered
aliphatic or heteroaliphatic ring optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester;
(xiii) an aldehyde of formula -(X20)n20-CO-H wherein
X20 is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n20 is 0 or 1;
(xiv) a sulfone of formula -(X21)n21-SO2-X22, wherein
X21 and X22 are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n21 is 0 or 1; and
(xv) a thiol of formula -(X23)n23-SH and a thioether of formula -(X24)n24-S-
X25,
wherein



230

X23, X24, and X25 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
said alkyl and ring moieties are optionally substituted with
one or more substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n23 and n24 are independently 0 or 1.

23. The compound of claim 22, wherein
(a) R1, R2, and R7 are hydrogen;
(b) R3 is an ethyl-2-oxy group of formula -CH2CH2-O-R, wherein R is selected
from the group consisting of hydrogen, saturated alkyl, an aromatic ring
optionally
substituted with one or more substituents independently selected from the
group
consisting of alkyl, aryl, and alkoxy moieties, and a substituent of formula
-C(E)NHX15, wherein
X15 is selected from the group consisting of alkyl, sulfone of formula -SO2-
X22, and six-membered aromatic or aliphatic ring moieties,
said ring is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl and aryl,
X22 is selected from the group consisting of saturated alkyl, and
optionally substituted six-membered aromatic ring moieties, and
E is selected from the group consisting of oxygen and sulfur, and;
(c) Z is a 5, 6, or 9 membered, monocyclic or bicyclic, aromatic or
heteroaromatic, ring moiety, optionally substituted with one or more
substituents
selected from the group consisting of
(i) hydrogen;
(ii) saturated alkyl,
(iii) an optionally substituted aromatic or heteroaromatic ring;
(iv) an amine of formula -(X1)n1-NX2X3, wherein




231

X1 is alkyl,
n1 is 0 or 1, and
X2 and X3 are independently selected from the group consisting of
hydrogen and saturated alkyl;
(v) a halogen or trihalomethyl;
(vi) a carboxylic acid of formula -(X6)n6-COOH, wherein
X6 is alkyl, and
n6 is 0 or 1; and
(vii) an alcohol of formula -(X9)n9-OH or an alkoxyalkyl moiety of formula
-(X10)n10-O-X11, wherein
X9, X10, and X11 are alkyl, and
n9 and n10 are independently 0 or 1.

24. The compound of claim 22, wherein
(a) R3 is an ethyl-2-oxy group of formula -CH2CH2-O-R, wherein R is selected
from the group consisting of hydrogen, methyl, ethyl, 2-isopropylphenyl, 3-
isopropylphenyl, 4-isopropylphenyl, 4-methoxyphenyl, biphen-3-yl, 5-
chloropyridin-3-yl, ethylcarbamyl, tert-butylcarbamyl, cyclohexylcarbamyl,
phenylcarbamyl, benzene sulfonylcarbamyl, biphen-2-yl-carbamyl, and
phenylthiocarbamyl;
(b) Z is selected from the group consisting of 4-bromophenyl, 2-pyridyl, 6-
methyl-2-pyridyl, 1H-indol-5-yl, 4-methoxy-3-thiophenphenyl, 4-(3-
dimethylamino)-3,5-dimethyl-1H-pyrrol-2-yl, 3-hydroxy-6-methyl-pyridin-2-yl, 4-

(3-dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-yl, and 3-carboxy-2,4-
dimethyl-
1H-pyrrol-2-yl.

25. The compound of claim 22, wherein said compound is selected from
the group consisting of 4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-
one,
4-[2-(2-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(biphenyl-3-
yloxy)-ethyl]-1,3-dihydro-indol-2-one, 3-(4-bromo-benzylidene)-4-(2-hydroxy-
ethyl)-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-pyridin-2-ylmethylene-
1,3-



232

dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-(6-methyl-pyridin-2-ylmethylene)-
1,3-
dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-(1H-indol-5-ylmethylene)-1,3-
dihydro-
indol-2-one, 4-(2-hydroxy-ethyl)-3-(4-methoxy-3-thiophen-2-yl-benzylidene)-1,3-

dihydro-indol-2-one, 3-[4-(3-dimethylamino-propyl)-3,5-dimethyl-1H-pyrrol-2-
yhnethylene]-4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one, phenyl-thiocarbamic
acid O-[2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl] ester, phenyl-carbamic acid
2-
(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, tert-butyl-carbamic acid 2-(2-
oxo-
2,3-dihydro-1H-indol-4-yl)-ethyl ester, cyclohexyl-carbamic acid 2-(2-oxo-2,3-
dihydro-1H-indol-4-yl)-ethyl ester, benzene sulfonyl-carbamic acid 2-(2-oxo-
2,3-
dihydro-1H-indol-4-yl)-ethyl ester, biphenyl-2-yl-carbamic acid 2-(2-oxo-2,3-
dihydro-1H-indol-4-yl)-ethyl ester, ethyl-carbamic acid 2-(2-oxo-2,3-dihydro-
1H-
indol-4-yl)-ethyl ester, 4-[2-(4-methoxy-phenoxy)-ethyl]-1,3-dihydro-indol-2-
one,
4-(2-methoxy-ethyl)-1,3-dihydro-indol-2-one, 4-(2-ethoxy-ethyl)-1,3-dihydro-
indol-
2-one, 4-[2-(4-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(5-
chloro-
pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-(3-
hydroxy-
6-methyl-pyridin-2-ylmethylene)-1,3-dihydro-indol-2-one, 3-[4-(3-dimethylamino-

propyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-4-[2-(3-isopropyl-phenoxy)-
ethyl]-
1,3-dihydro-indol-2-one, and 3-(5-{4-[2-(4-isopropyl-phenoxy)-ethyl]-2-oxo-1,2-

dihydro-indol-3-ylidenemethyl}-2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid.

26. A compound selected from the group consisting of 4-[2-(3-isopropyl-
phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(2-isopropyl-phenoxy)-ethyl]-1,3-

dihydro-indol-2-one, 4-[2-(biphenyl-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one, 3-
(4-
bromo-benzylidene)-4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one, 4-(2-hydroxy-
ethyl)-3-pyridin-2-ylmethylene-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-
(6-
methyl-pyridin-2-ylmethylene)-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-
(1H-
indol-5-ylmethylene)-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-3-(4-methoxy-
3-
thiophen-2-yl-benzylidene)-1,3-dihydro-indol-2-one, 3-[4-(3-dimethylamino-
propyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-4-(2-hydroxy-ethyl)-1,3-dihydro-
indol-2-one, phenyl-thiocarbamic acid O-[2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-
ethyl] ester, phenyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl
ester,




233

tert-butyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester,
cyclohexyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester,
benzene
sulfonyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester,
biphenyl-2-
yl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, ethyl-
carbamic
acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, 4-[2-(4-methoxy-phenoxy)-

ethyl]-1,3-dihydro-indol-2-one, 4-(2-methoxy-ethyl)-1,3-dihydro-indol-2-one, 4-
(2-
ethoxy-ethyl)-1,3-dihydro-indol-2-one, 4-[2-(4-isopropyl-phenoxy)-ethyl]-1,3-
dihydro-indol-2-one, 4-[2-(5-chloro-pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-
2-
one, 4-(2-hydroxy-ethyl)-3-(3-hydroxy-6-methyl-pyridin-2-ylmethylene)-1,3-
dihydro-indol-2-one, 3-[4-(3-dimethylamino-propyl)-3,5-dimethyl-1H-pyrrol-2-
ylmethylene]-4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, and 3-
(5-
{4-[2-(4-isopropyl-phenoxy)-ethyl]-2-oxo-1,2-dihydro-indol-3-ylidenemethyl}-
2,4-
dimethyl-1H-pyrrol-3-yl)-propionic acid.

27. A compound of any one of claims 1, 7, 18, 21, 22, or 26, wherein
said compound is capable of inhibiting the catalytic activity of a protein
kinase.

28. The compound of claim 27 wherein said protein kinase is selected
from the group consisting of a receptor protein tyrosine kinase, a cellular
tyrosine
kinase and a serine-threonine kinase.

29. A method for the modulation of the catalytic activity of a protein
kinase comprising contacting said protein kinase with said compound of any one
of
claims 1, 7, 18, 21, 22, or 26.

30. A method of modulating signal transduction pathways in cells with a
compound according to any one of claims 1, 7, 18, 21, 22, or 26, comprising
the step
of contacting said cells with said compound.

31. The method of claim 30, wherein said cells express a protein kinase
and wherein said compound modulates the function of said protein kinase.





234

32. A method of identifying indolinone compounds that modulate the
function of protein kinase, comprising the following steps:
(a) contacting cells expressing said protein kinase with a compound of
any one of claims 1, 7, 18, 21, 22, or 26; and
(b) monitoring an effect upon said cells.

33. The method of claim 32, wherein said effect is selected from the
group consisting of a change in cell phenotype, a change in cell
proliferation, a
change in the catalytic activity of said protein kinase, and a change in the
interaction
between said protein kinase and a binding partner.

34. A method of regulating an unregulated protein kinase signal
transduction comprising administering to a subject a therapeutically effective
amount of a compound according to any one of claims 1, 7, 18, 21, 22, or 26.

35. The method of claim 34, wherein unregulated protein kinase signal
transduction leads to a disease or an abnormal condition in an organism and
said
method leads to the treatment or prevention of said disease or abnormal
condition;
wherein said disease or abnormal condition is associated with an aberration
in a signal transduction pathway characterized by an interaction between a
protein
kinase and a binding partner, and wherein said method further comprises the
steps of
promoting or disrupting said abnormal interaction.

36. The method of claim 35, wherein said disease or abnormal
condition is a cancer.

37. The method of claim 35 wherein said disease or abnormal
condition is selected from the group consisting of an immunological disorder,
a
hyperproliferation disorder, a cardiovascular disorder, an inflammatory
disorder,
restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis,
aetherosclerosis,




235

diabetes, and angiogenesis.

38. The method of any one of claims 29, 31, 32, or 34, wherein said
protein kinase is selected from the group consisting of receptor protein
tyrosine
kinase, cellular tyrosine kinase and serine-threonine kinase.

39. A pharmaceutical composition comprising
(i) a physiologically acceptable carrier, diluent, or excipient or a
combination thereof; and
(ii) a compound according to any one of claims 1, 7, 18, 21, 22, or 26.

Description

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DESCRIPTION
INDOLINONE COMPOUNDS AS KINASE INHIBITORS
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided to
aid in understanding the invention, but is not admitted to describe or
constitute prior
art to the invention.
Protein kinases ("PKs") are enzymes that catalyze the phosphorylation of
hydroxy groups on tyrosine, serine and threonine residues of proteins. The
consequences of this seemingly simple activity are staggering. Cell growth,
differentiation and proliferation, i.e., virtually all aspects of cell life,
in one way or
another depend on PK activity. Furthermore, abnormal PK activity has been
related
to a host of disoruers, ranging from relatively non-life-threatening diseases
such as
psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).
The PKs can conveniently be broken down into two classes, the protein
tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).
One of the prime aspects of PK activity is involvement with growth factor
receptors. Growth factor receptors are cell-surface proteins. When bound by a
growth factor ligand, growth factor receptors are converted to an active form
that can
interact with proteins on the inner surface of a cell membrane. This
interaction leads
to phosphorylation on tyrosine residues of the receptor as well as other amino
acids
and to the formation inside the cell of complexes with a variety of
cytoplasmic
signaling molecules. In turn, these complexes affect numerous cellular
responses
such as cell division (proliferation), cell differentiation, cell growth,
expression of
metabolic effects on the extracellular microenvironment, etc. For a more
complete
discussion, see Schlessinger and Ullrich, Neuron, 1992, 9:303-391 which is
incorporated by reference, including any drawings, as if fully set forth
herein.
Receptor tyrosine kinases (RTKs) are growth factor receptors with PK
activity. They comprise a large family of transmembrane receptors with diverse
biological activity. At present, at least nineteen (19) distinct subfamilies
of RTKs
have been identified. An example of these is the subfamily designated the
"HER"



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2
RTKs, which includes EGFR (epithelial growth factor receptor), HER2, HERS and
HER4. These RTKs consist of an extracellular glycosylated ligand binding
domain,
a transmembrane domain and an intracellular cytoplasmic catalytic domain that
can
phosphorylate tyrosine residues on proteins.
Another RTK subfamily consists of insulin receptor (IR), insulin-like growth
factor I receptor (IGF-1R) and insulin receptor related receptor (IRR). IR and
IGF-
1R interact with insulin, IGF-I and IGF-II to form a heterotetramer composed
of two
entirely extracellular glycosylated a subunits and two (3 subunits which
contain the
tyrosine kinase domain.
A third RTK subfamily is referred to as the platelet derived growth factor
receptor ("PDGFR") group, which includes PDGFRa,, PDGFR(3, CSFIR, c-kit and
c-fms. These receptors consist of a glycosylated extracellular domain composed
of
variable numbers of immunoglobin-like loops, a transmembrane domain and an
intracellular domain having a tyrosine kinase domain interrupted by unrelated
amino
acid sequences.
Another group which, because of its similarity to the PDGFR subfamily, is
sometimes subsumed in the latter group, is the fetus liver kinase ("flk")
receptor
subfamily. This group is believed to be composed of kinase insert domain-
receptor
fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and fins-like tyrosine kinase
1 (flt-
1 ).
One further member of the tyrosine kinase growth factor receptor family is
the fibroblast growth factor ("FGF")receptor group. This group consists of
four
receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well
characterized, it
appears that the receptors also consist of a glycosylated extracellular domain
containing a variable number of immunoglobin-like loops, a transmembrane
domain
and an intracellular domain in which the tyrosine kinase domain is interrupted
by
regions of unrelated amino acid sequences.
A more complete listing of the known RTK subfamilies is described in
Plowman et al., DN&P, 1994, 7(6):334-339 which is incorporated by reference,
including any drawings, as if fully set forth herein.
In addition to the RTKs, there also exists a family of entirely intracellular



CA 02368041 2001-09-24
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PTKs called "non-receptor tyrosine kinases" or "cellular tyrosine kinases"
("CTK").
CTKs do not contain extracellular and transmembrane domains. At present, over
24
CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and
Ack)
have been identified. The Src subfamily appears so far to be the largest group
of
CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more
detailed discussion of CTKs, see Bolen, Oncogene, 1993, 8:2025-2031, which is
incorporated by reference, including any drawings, as if fully set forth
herein.
The serine-threonine kinases or STKs, like the CTKs, are predominantly
intracellular although there are a few STK receptor kinases. STKs are the most
common of the cytosolic kinases; i.e., kinases that perform their function in
that part
of the cytoplasm other than the cytoplasmic organelles and cytoskelton. The
cytosol
is the region within the cell where much of the cell's intermediary metabolic
and
biosynthetic activity occurs; e.g., it is in the cytosol that proteins are
synthesized on
ribosomes.
RTKs, CTKs and STKs have all been implicated in a host of pathogenic
conditions including, significantly, cancer. Other pathogenic conditions which
have
been associated with PTKs include, without limitation, psoriasis, hepatic
cirrhosis,
diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases,
rheumatoid
arthritis and other inflammatory disorders, immunological disorders such as
autoimmune disease, cardiovascular diseases such as atherosclerosis and a
variety of
renal disorders.
With regard to cancer, two of the major hypotheses advanced to explain the
excessive cellular proliferation that drives tumor development relate to
functions
known to be PK regulated. That is, it has been suggested that malignant cell
growth
is the result of a breakdown in the mechanisms that control cell division
and/or
differentiation. It has been shown that the protein products of a number of
proto-
oncogenes are involved in the signal transduction pathways that regulate cell
growth
and differentiation. These protein products of proto-oncogenes include the
extracellular growth factors, transmembrane growth factor PTK receptors
(RTKs),
cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
In view of the apparent link between PK-related cellular activities and a



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4
number of human disorders, it is no surprise that a great deal of effort is
being spent
to identify ways to modulate PK activity. Some of these efforts have been
directed
at biomimetic approaches using large molecules patterned on those involved in
the
actual cellular processes (e.g., mutant ligands (U.S. App. No. 4,966,849);
soluble
receptors and antibodies (App. No. WO 94/10202, Kendall and Thomas, Proc.
Nat'l
Acad. Sci., 1994, 90:10705-09, Kim, et al., Nature, 1993, 362:841-844); RNA
ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al., Mol.
Bio. Cell,
1993, 4:358A; Kinsella, et al., Exp. Cell Res., 1992, 199:56-62; Wright, et
al., J.
Cellular Phys., 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO
92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al.,
Proc. Am. Assoc. Cancer Res., 1994, 35:2268).
More recently, attempts have been made to identify small molecules that act
as PK inhibitors. For example, bis- monocylic, bicyclic and heterocyclic aryl
compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO
94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have
been described as PTK inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999),
styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), quinazoline
derivatives (EP App. No. 0 566 266 A1), selenaindoles and selenides (PCT WO
94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and
benzylphosphonic acid compounds (PCT WO 91/15495) have: alI been described as
PTK inhibitors useful in the treatment of cancer.
SUMMARY OF THE INVENTION
The present invention is directed in part towards indolinone compounds and
methods of modulating the function of protein kinases with these compounds.
The
methods incorporate cells that express a protein kinase. In addition, the
invention
describes methods of preventing and treating protein kinases-related abnormal
conditions in organisms with a compound identified by the methods described
herein. Furthermore, the invention pertains to pharmaceutical compositions
comprising compounds identified by methods of the invention.
The present invention features indolinone compounds that potently inhibit



CA 02368041 2001-09-24
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protein kinases and related products and methods. Inhibitors of protein
kinases can
be obtained by adding chemical substituents to an indolinone compound. The
compounds of the invention represent a new generation of therapeutics for
diseases
associated with one or more functional or non-functional protein kinases.
Neuro-
degenerative diseases and certain types of cancer fall into this class of
diseases.
Other diseases or disorders include dermatologic, ophthalmic, nurologic,
cardiovascular, and immune disorders as well as disorders associated with
abnormal
angiogenesis and/or vasculogenesis. The compounds can be modified such that
they
are specific to their target or targets and will subsequently cause few side
effects and
thus represent a new generation of potential cancer therapeutics. These
properties
are significant improvements over the currently utilized cancer therapeutics
that
cause multiple side effects and deleteriously weaken patients.
It is beliwed the compounds of the invention will minimize or obliterate
solid tumors by inhibiting the activity of the protein kinases, or will at
least
modulate or inhibit tumor growth and/or metastases. Protein kinases regulate
proliferation of blood vessels during angiogenesis, among other functions.
Increased
rates of angiogenesis accompany cancer tumor growth in cells as cancer tumors
must
be nourished by oxygenated blood during growth. Therefore, inhibition of the
protein kinase and the corresponding decreases in angiogenesis will starve
tumors of
nutrients and most likely obliterate them.
While a precise understanding of the mechanism by which compounds
inhibit PTKs (e.g., the fibroblast growth factor receptor 1 [FGFR1]) is not
required
in order to practice the present invention, the compounds are believed to
interact
with the amino acids of the PTKs' catalytic region. PTKs typically possess a
bi-
lobate structure, and ATP appears to bind in the cleft between the two lobes
in a
region where the amino acids are conserved among PTKs; inhibitors of PTKs are
believed to bind to the PTKs through non-covalent interactions such as
hydrogen
bonding, Van der Waals interactions, and ionic bonding, in the same general
region
that ATP binds to the PTKs. More specifically, it is thought that the oxindole
component of the compounds of the present invention binds in the same general
space occupied by the adenine ring of ATP. Specificity of a PTK inhibitor for
a



CA 02368041 2001-09-24
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particular PTK may be conferred by interactions between the constituents
around the
oxindole core with amino acid domains specific to individual PKs. Thus,
different
substitutents may contribute to preferential binding to particular PKs. The
ability to
select those compounds active at different ATP binding sites makes them useful
in
targeting any protein with such a site, including not only protein tyrosine
kinases,
but also serine/threonine kinases. Thus, such compounds have utility for in
vitro
assays on such proteins and for in vivo therapeutic effect through such
proteins.
Usually, indolinone compounds that are synthesized by the condensation of
an oxindole compound and a ketone compound display a mixture of the possible E
and Z isomers, making the isolation of the isomer of choice difficult. The
compounds of the present invention feature an intermolecular hydrogen bond
between the carbonyl of the oxindole compound and the hydrogen of the 1
position
of the pyrrole moiety of the ketone compounds. Said hydrogen bond eliminates
the
problem of having a mixture of isomers by locking the intermediates in the
synthesis
1 S of the indolinone compounds in the preferred conformation.
I. DEFENITIONS
The term "compound" refers to the compound or a pharmaceutically
acceptable salt, ester, amide, prodrug, isomer, or metabolite, thereof.
The term "pharmaceutically acceptable salt" refers to a formulation of a
compound that does not abrogate the biological activity and properties of the
compound. Pharmaceutical salts can be obtained by reacting a compound of the
invention with inorganic or organic acids such as hydrochloric acid,
hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic
acid, p-toluenesulfonic acid, salicylic acid and the like.
A "prodrug" refers to an agent that is converted into the parent drug in vivo.
Prodrugs are often useful because, in some situations, they may be easier to
administer than the parent drug. They may, for instance, be bioavailable by
oral
administration whereas the parent is not. The prodrug may also have improved
solubility in pharmaceutical compositions over the parent drug. An example,
without limitation, of a prodrug would be a compound of the present invention



CA 02368041 2001-09-24
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which is administered as an ester (the "prodrug") to facilitate transmittal
across a cell
membrane where water solubility is detrimental to mobility but which then is
metabolically hydrolyzed to the carboxylic acid, the active entity, once
inside the
cell where water solubility is beneficial. A fruther example of a prodrug
might be a
short peptide (polyaminoacid) bonded to an acid group wherein the peptide is
metabolized to reveal the active moiety.
The term "indolinone" is used as that term is commonly understood in the art
and includes a large subclass of substituted or unsubstituted compounds that
are
capable of being synthesized from an aldehyde moiety and an oxindole moiety.
The term "oxindole" refers to an oxindole compound substituted with
chemical substituents. Oxindole compounds are of the general structure:
O
N
H
As used herein a "fused pyrrolo" group refers to the structure in brackets:
N
H
The term "substituted", in reference to the invention, refers to an oxindole
compound that is derivatized with any number of chemical substituents.
As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group.
The alkyl moiety may be a "saturated alkyl" group, which means that it does
not
contain any alkene or alkyne moieties. The alkyl moiety may also an be
"unsaturated alkyl" moiety, which means that it contains at least one alkene
or
alkyne moiety. An "alkene" moiety refers to a group consisting of at least two
carbon atoms and at least one carbon-carbon double bond, and an "alkyne"
moiety
refers to a groupconsisting of at least two carbon atoms and at least one
carbon-
carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be
branched, non-branched, or cyclic.
Preferably, the alkyl group has 1 to 20 carbon atoms (whenever it appears
herein, a numerical range such as "1 to 20" refers to each integer in the
given range;



CA 02368041 2001-09-24
WO 00/56709 PCT/US00/07704
e.g., "1 to 20 carbon atoms" means that the alkyl group may consist of 1
carbon
atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon
atoms,
although the present definition also covers the occurrence of the term "alkyl"
where
no numerical range is designated). More preferably, it is a medium size alkyl
having
1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4
carbon
atoms. The alkyl group may be substituted or unsubstituted. When substituted,
the
substituent groups) is(are) preferably one or more groups) individually and
independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic,
hydroxy,
alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl,
thiocarbonyl,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-
sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato,
isothiocyanato, nitro, silyl and amino, including mono- and di-substituted
amino
groups.
The term "aromatic" refers to an aromatic group which has at least one ring
having a conjugated pi electron system and includes both carbocyclic aryl
(e.g.,
phenyl) and heterocyclic aryl groups (e.g., pyridine). The term includes
monocyclic
or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon
atoms)
groups. The term "carbocyclic" refers to a compound which contains one or more
covalently closed ring structures, and that the atoms forming the backbone of
the
ring are all carbon atoms. The term thus distinguishes carboc:yclic from
heterocyclic
rings in which the ring backbone contains at least one atom which is different
from
carbon. The term "heteroaromatic" refers to an aromatic group which contains
at
least one heterocyclic ring.
The term "aliphatic ring" refers to a compound which contains one or more
covalently closed ring structures, and that at least one of the atoms forming
the
backbone is a saturated carbon atom (e.g., cyclohexane). The term
"heteroaliphatic
ring" refers to a ring system in which at least one of the atoms forming the
backbone
is a heteroatom (e.g., tetrahydropyran).
The term "amine" refers to a chemical moiety of formula -NR,Rz where R,
and RZ are independently selected from the group consisting of hydrogen,
saturated
or unsaturated alkyl, and five-membered or six-membered aryl or heteroaryl
ring



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9
moieties, where the ring is optionally substituted with one or more
substituents
independently selected from the group consisting of alkyl, halogen,
trihalomethyl,
carboxylate, nitro, and ester moieties.
The term "imine" refers to a chemical moiety of formula -N=R, where R, is
selected from the group consisting of hydrogen, saturated or unsaturated
alkyl, and
aryl or heteroaryl ring moieties (monocyclic or bicyclic), where the ring is
optionally
substituted with one or more substituents independently selected from the
group
consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester
moieties.
The term "halogen" or "halo" refers to an atom selected from the group
consisting of fluorine, chlorine, bromine, and iodine. The term
"trihalomethyl"
refers to the -CX3 group, where X is a halogen.
The term "carboxylic acid" refers to a chemical moiety with formula
-(R)n COOH, ~.,vlz~re R is selected from the group consisting of of alkyl,
cycloalkyl,
aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded
through
a ring carbon), as those terms are defined herein, and where n is 0 or 1.
The term "ester" refers to a chemical moiety with formula -(R)~ COOR',
where R and R' are independently selected from the group consisting of alkyl,
cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and
heteroalicyclic
(bonded through a ring carbon), as those terms are defined herein, and where n
is 0
or 1. As used herein, the term "carboxyalkyl" also falls within this
definition.
The term "aldehyde" refers to a chemical moiety with formula -(R)"-CHO,
where R is as defined herein and where n is 0 or 1.
The term "sulfone" refers to a chemical moiety with formula -SOZ-R, where
R is as defined herein.
The term "acyl" refers to chemical moieties of the general formula -C(O)R.
When R is hydrogen the molecule containing the acyl group is an aldehyde. When
R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl
(bonded
through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as
those
terms are defined herein, then the molecule containing the acyl group is a
ketone.
A "thiocarbonyl" group refers to a -C(=S)-R group, where R is as defined
herein.



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An "O-carboxy" group refers to a RC(=O)O- group, where R is as defined
herein.
A "C-carboxy" group refers to a -C(=O)OR groups where R is as defined
herein.
5 An "acetyl" group refers to a -C(=O)CH3 group.
A "trihalomethanesulfonyl" group refers to a X3CS(=O),- group where X is a
halogen.
A "cyano" group refers to a -C---N group.
An "isocyanato" group refers to a -NCO group.
10 A "thiocyanato" group refers to a -CNS group.
An "isothiocyanato" group refers to a -NCS group.
A "sulfinyl" group refers to a -S(=O)-R group, with R as defined herein.
A "S-sulfonamido" group refers to a -S(=O)~NRZ group, with R as defined
herein.
A "N-sulfonamido" group refers to a RS(=O)zNH- group with R as defined
herein.
A "trihalomethanesulfonamido" group refers to a X3CS(=O)zNR- group with
X and R as defined herein.
An "O-carbamyl" group refers to a -OC(=O)-NRZ group with R as defined
herein.
An "N-carbamyl" group refers to a ROC(=O)NH- group, with R as defined
herein.
An "O-thiocarbamyl" group refers to a -OC(=S)-NR, group with R as
defined herein.
An "N-thiocarbamyl" group refers to an ROC(=S)NH- group, with R as
defined herein.
A "C-amido" group refers to a -C(=O)-NRz group with R as defined herein.
An "N-amido" group refers to a RC(=O)NH- group, with R as defined
herein.
By "combined," when referring to two adjacent "R" groups herein is meant that
the two "R" groups are covalently bonded to each other so as to form a ring
system. The



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11
ring system may be cycloalkyl, aryl, heteroaryl or heteroalicyclic.
A "combinatorial library" refers to all the compounds formed by the reaction
of each compound of one dimension with a compound in each of the other
dimensions in a mufti-dimensional array of compounds. In the context of the
present
invention, the array is two dimensional and one dimension represents all the
oxindoles of the invention and the second dimension represents all the
aldehydes of
the invention. Each oxindole may be reacted with each and every aldehyde in
order
to form an indolinone compound. All indolinone compounds formed in this way
are
within the scope of the present invention. Also within the scope of the
present
invention are smaller combinatorial libraries formed by the reaction of some
of the
oxindoles with all of the aldehydes, all of the oxindoles with some of the
aldehydes,
or some of the oxindoles with some of the aldehydes.
The term "pharmaceutical composition" refers to a mixture of a compound of
the invention with other chemical components, such as diluents, excipients, or
1 S carriers. The pharmaceutical composition facilitates administration of the
compound
to an organism. Multiple techniques of administering a compound exist in the
art
including, but not limited to, oral, aerosol, parenteral, and topical
administration.
Pharmaceutical compositions can also be obtained by reacting compounds with
inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric
acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic
acid, salicylic acid and the like.
The term "physiologically acceptable" defines a Garner or diluent that does
not abrogate the biological activity and properties of the compound.
The term "Garner" defines a chemical compound that facilitates the
incorporation of a compound into cells or tissues. For example dimethyl
sulfoxide
(DMSO) is a commonly utilized Garner as it facilitates the uptake of many
organic
compounds into the cells or tissues of an organism.
The term "diluent" defines chemical compounds diluted in water that will
dissolve the compound of interest as well as stabilize the biologically active
form of
the compound. Salts dissolved in buffered solutions are utilized as diluents
in the
art. One commonly used buffered solution is phosphate buffered saline because
it



CA 02368041 2001-09-24
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12
mimics the salt conditions of human blood. Since buffer salts can control the
pH of
a solution at low concentrations, a buffered diluent rarely modifies the
biological
activity of a compound.
The term "pharmaceutical composition" refers to a mixture of a compound of
the invention with other chemical components, such as diluents, excipients, or
Garners. The pharmaceutical composition facilitates administration of the
compound
to an organism. Multiple techniques of administering a compound exist in the
art
including, but not limited to, oral, aerosol, parenteral, and topical
administration.
Pharmaceutical compositions can also be obtained by reacting compounds with
inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric
acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic
acid, salicylic acid and the like.
The term "physiologically acceptable" defines a carrier or diluent that does
not abrogate the biological activity and properties of the compound.
The term "function" refers to the cellular role of a protein kinase. The
protein kinase family includes members that regulate many steps in signaling
cascades, including cascades controlling cell growth, migration,
differentiation, gene
expression, muscle contraction, glucose metabolism, cellular protein
synthesis, and
regulation of the cell cycle.
The term "catalytic activity", in the context of the im,.°~:,srtion,
defines the rate
at which a protein kinase phosphorylates a substrate. Catalytic activity can
be
measured, for example, by determining the amount of a substrate converted to a
product as a function of time. Phosphorylation of a substrate occurs at the
active-
site of a protein kinase. The active-site is normally a cavity in which the
substrate
binds to the protein kinase and is phosphorylated.
The term "substrate" as used herein refers to a molecule phosphorylated by a
protein kinase. The substrate is preferably a peptide and more preferably a
protein.
The term "activates" refers to increasing the cellular function of a protein
kinase. The protein kinase function is preferably the interaction with a
natural
binding partner and most preferably catalytic activity.
The term "inhibit" refers to decreasing the cellular function of a protein



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13
kinase. The protein kinase function is preferably the interaction with a
natural
binding partner and most preferably catalytic activity.
The term "modulates" refers to altering the function of a protein kinase by
increasing or decreasing the probability that a complex forms between a
protein
kinase and a natural binding partner. A modulator preferably increases the
probability that such a complex forms between the protein kinase and the
natural
binding partner, more preferably increases or decreases the probability that a
complex forms between the protein kinase and the natural binding partner
depending
on the concentration of the compound exposed to the protein kinase, and most
preferably decreases the probability that a complex forms between the protein
kinase
and the natural binding partner. A modulator preferably activates the
catalytic
activity of a protein kinase, more preferably activates or inhibits the
catalytic activity
of a protein kinase depending on the concentration of the compound exposed to
the
protein kinase, or most preferably inhibits the catalytic activity of a
protein kinase.
The term "complex" refers to an assembly of at least two molecules bound to
one another. Signal transduction complexes often contain at least two protein
molecules bound to one another.
The term "natural binding partner" refers to polypeptides that bind to a
protein kinase in cells. Natural binding partners can play a role in
propagating a
signal in a protein kinase signal transduction process. A change in the
interaction
between a protein kinase and a natural binding partner can manifest itself as
an
increased or decreased probability that the interaction forms, or an increased
or
decreased concentration of the protein kinase/natural binding partner complex.
The term "contacting" as used herein refers to mixing a solution comprising
a compound of the invention with a liquid medium bathing the cells of the
methods.
The solution comprising the compound may also comprise another component, such
as dimethylsulfoxide (DMSO), which facilitates the uptake of the compound or
compounds into the cells of the methods. The solution comprising the compound
of
the invention may be added to the medium bathing the cells by utilizing a
delivery
apparatus, such as a pipet-based device or syringe-based device.
The term "monitoring" refers to observing the effect of adding the compound



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14
to the cells of the method. The effect can be manifested in a change in cell
phenotype, cell proliferation, protein kinase catalytic activity, or in the
interaction
between a protein kinase and a natural binding partner.
The term "effect" describes a change or an absence of a change in cell
phenotype or cell proliferation. "Effect" can also describe a change or an
absence of
a change in the catalytic activity of the protein kinase. "Effect" can also
describe a
change or an absence of a change in an interaction between the protein kinase
and a
natural binding partner.
The term "cell phenotype" refers to the outward appearance of a cell or tissue
or the function of the cell or tissue. Examples of cell phenotype are cell
size
(reduction or enlargement), cell proliferation (increased or decreased numbers
of
cells), cell differentiation (a change or absence of a change in cell shape),
cell
survival, apoptosis (cell death), or the utilization of a metabolic nutrient
(e.g.,
glucose uptake). Changes or the absence of changes in cell phenotype are
readily
measured by techniques known in the art.
The term "antibody" refers to an antibody (e.g., a monoclonal or polyclonal
antibody), or antibody fragment, having specific binding affinity to protein
kinase or
its fragment.
By "specific binding affinity" is meant that the antibody binds to target
(protein kinase) polypeptides with greater affinity than it binds to other
polypeptides
under specified conditions. Antibodies having specific binding affinity to a
protein
kinase may be used in methods for detecting the presence and/or amount of a
protein
kinase in a sample by contacting the sample with the antibody under conditions
such
that an immunocomplex forms and detecting the presence and/or amount of the
antibody conjugated to the protein kinase. Diagnostic kits for performing such
methods may be constructed to include a first container containing the
antibody and
a second container having a conjugate of a binding partner of the antibody and
a
label, such as, for example, a radioisotope. The diagnostic kit may also
include
notification of an FDA approved use and instructions therefor.
The term "polyclonal" refers to antibodies that are heterogenous populations
of antibody molecules derived from the sera of animals immunized with an
antigen



CA 02368041 2001-09-24
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or an antigenic functional derivative thereof. For the production of
polyclonal
antibodies, various host animals may be immunized by injection with the
antigen.
Various adjuvants may be used to increase the immunological response,
depending
on the host species.
"Monoclonal antibodies" are substantially homogenous populations of
antibodies to a particular antigen. They may be obtained by any technique
which
provides for the production of antibody molecules by continuous cell lines in
culture. Monoclonal antibodies may be obtained by methods known to those
skilled
in the art. See, for example, Kohler, et al., Nature 256:495-497 (1975), and
U.S.
10 Patent No. 4,376,110.
The term "antibody fragment" refers to a portion of an antibody, often the
hypervariable region and portions of the surrounding heavy and light chains,
that
displays' specific binding affinity for a particular molecule. A hypervariable
region
is a portion of an antibody that physically binds to the polypeptide target.
15 The term "aberration", in conjunction with a signal transduction process,
refers to a protein kinase that is over- or under-expressed in an organism,
mutated
such that its catalytic activity is lower or higher than wild-type protein
kinase
activity, mutated such that it can no longer interact with a natural binding
partner, is
no longer modified by another protein kinase or protein phosphatase, or no
longer
interacts with a natural binding partner.
The term "promoting or disrupting the abnormal interaction" refers to a
method that can be accomplished by administering a compound of the invention
to
cells or tissues in an organism. A compound can promote an interaction between
a
protein kinase and natural binding partners by forming favorable interactions
with
multiple atoms at the complex interface. Alternatively, a compound can inhibit
an
interaction between a protein kinase and natural binding partners by
compromising
favorable interactions formed between atoms at the complex interface.
"In vitro" refers to procedures performed in an artificial environment, such
as, without limitation, in a test tube, in a cell, or culture medium. As used
herein,
"in vivo" refers to procedures performed within a living organism such as,
without
limitation, a mouse, rat, or rabbit.



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16
II. COMPOUNDS OF THE INVENTION
A. 3- liden~-6-Heterocvclyl-2-Indolinone Derivatives
In one aspect, the present invention relates to 3-arylidenyl-6-heterocyclyl-
2-indolinone derivatives having the chemical structure set forth in formula I:
Rs
R
R~~ iB D 6 n
~~E~R
F
l
R8
~I~ R~owH~
J ~ L~ R3
Rat ~ R~3
m
R~2
or a salt or prodrug thereof where n and m are independently 0 or 1.
When n is 1, then A, B, D, E and F are independently selected from the
group consisting of carbon and nitrogen; however, no more th~?yi three of A,
B, D,
E and F are nitrogen at the same time and, when A, B, D, E, or F is nitrogen,
then
R4, RS, R6, R~ or R8, respectively, does not exist.
When m is 1, then G, H, J, K and L are independently selected from the
group consisting of carbon and nitrogen; however, at least one and no more
than
three of G, H, J, K and L are nitrogen at the same time and, when G, H, J, K
or L
is nitrogen, then R" R,o, R", R,2 or R,~, respectively, does not exist.
When n is 0, then A is selected from the group consisting of carbon and
nitrogen, B and F are selected from the group consisting of carbon, nitrogen,
NH,
oxygen and sulfur, provided that when B or F is NH, the other cannot be NH,
and
E is selected from the group consisting of carbon, nitrogen, oxygen and
sulfur,
further provided that no more than one of B, E or F is oxygen or sulfur and



CA 02368041 2001-09-24
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17
provided also that at least one of A, B, E or F is a heteroatom (i.e., not
carbon).
When m is 0, then G is selected from the group consisting of carbon and
nitrogen, H, K and L are selected from the group consisting of carbon,
nitrogen,
NH, oxygen and sulfur, provided that when H or L is NH, the other cannot be
NH, and K is selected from the group consisting of carbon, nitrogen, oxygen
and
sulfur, further provided that no more than one of H, K or L is oxygen or
sulfur
and provided also that at least one of G, H, K or L is a heteroatom (i. e.,
not
carbon).
R" R2, R3, R4, R5, R6, R,, R8, R<" R,o, R", R,, and R,3 are independently
selected from the group consisting of hydrogen, alkyl, trihaloalkyl,
cycloalkyl,
alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,
mercapto,
alkylthio, aryloxy, arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-
sulfonamido,
carbonyl, C-ca~~boxy, O-carboxy, carboxyalkyl, cyano, nitro, halo, O-carbamyl,
N-carbamyl, C-amido, N-amido and -NR,4R,5.
1 S R4 and RS or RS and R6 or R~ and R, or R8 and R8 may combine to form a
five-member or a six-member aryl or heteroaryl ring.
R,4 and R,5 are independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, carbonyl,
sulfonyl,
and, combined, a five-member or a six-member heteroalicyclic ring.
A presently preferred embodiment of this invention is a compound of
formula I wherein,
R,, Rz and R3 are independently selected from the group consisting of (i)
hydrogen, (ii) lower alkyl optionally substituted with one or more halo
groups,
(iii) lower alkoxy optionally substituted with one or more halo groups, (iv)
(lower alkyl)-S-sulfonamido, (v) aryl-S-sulfonamido, (vi) halo, and (vii) -
NR,4R,5; and,
R4, R5, R~, R" R8, R~" R,o, R", R", and R,3 are independently selected
from the group consisting of (i) hydrogen; (ii) lower alkyl, optionally
substituted
with one or more groups selected from the group consisting of aryl,
heteroaryl,
heteroalicyclic, halo, hydroxy, lower alkoxy, mercapto, lower alkylthio, C-
carboxy and -NR,4R,5; (iii) cycloalkyl; (iv) hydroxy; (v) lower alkoxy,
optionally



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18
substituted with one or more groups selected from the group consisting of one
or
more halo groups, aryl, heteroaryl and heteroalicyclic; (vi) halo; (vii)
carboxyalkyl; (viii) aryl, optionally substituted with one or more groups
selected
from the group consisting of lower alkyl optionally substituted one or more
halo
groups, halo, hydroxy, lower alkoxy optionally substituted with one or more
halo
groups, aryloxy and -NR,QR"; (ix) aryloxy, optionally substituted with one or
more groups selected from the group consisitng of lower alkyl optionally
substituted with one or more halo groups, halo, hydroxy, lower alkoxy
optionally
substituted with one or more halo groups, aryloxy and -NR,4R"; (x) heteroaryl,
optionally substituted with one or more groups selected from the group
consisting
of lower alkyl optionally substituted with one or more halo groups, halo,
hydroxy,
lower alkoxy optionally substituted with one or more halo groups and -NR,4R";
(xi) heteroalicyclic; (xii) (lower alkyl)-C-carboxy; (xiii) (lower
alkyl)carbonyl;
(xiv) aryl carbonyl; (xv) (lower alkyl)-S-sulfonamido; (xvi) aryl-S-
sulfonamido;
1 S (xvii) (lower alkyl)-N-sulfonamido; (xviii) aryl-N-sulfonamido; (xix)
(lower
alkyl)-N-carbamoyl; (xx) (lower alkyl)-C-amido; (xxi) (lower alkyl)-N-amido;
(xxii) (cycloalkyl)-N-amido; and, (xxiii) -NR,4R,5; and
R,4 and R,; are independently selected from the group consisting of hydrogen
and lower alkyl.
Another presently preferred embodiment of this invention is a compound
of formula I in which n is l; A, B, D, E and F are carbon, m is 1 and one of
G, H,
J, K and L is nitrogen.
It is also a presently preferred embodiment of this invention that, in a
compound of formula I, n is 1; A, B, D, E and F are carbon, m is 0, G, H and K
are carbon, and L is NH, oxygen or sulfur.
A still further preferred embodiment of this invention is a compound of
formula I in which n is 0; A, B and E are carbon; F is NH; R4 and R~ are
independently selected from the group consisting of hydrogen and lower alkyl;
R;
is selected from the group consisting of (i) lower alkyl optionally
substituted with
one or more groups selected from the group consisting of, hydroxy,
heteroaromatic containing an NH group in the ring, heteroalicyclic containing
at



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19
least one NH group in the ring, C-carboxy, and, -NR,4R,5; (ii) carboxyalkyl;
(iii)
C-carboxy; and, (iv) -NR,4R,5, wherein R,~ and R,5 are independently selected
from the group consisting of hydrogen, lower alkyl and carbonyl; m is l; and,
one
of G, H, I, J, K or L is nitrogen.
It is likewise a presently preferred embodiment of this invention that, in a
compound of formula I, n is 0; A, B and E are carbon; F is NH; R4 and R, are
independently selected from the group consisting of hydrogen and lower alkyl;
RS
is selected from the group consisting of (i) lower alkyl optionally
substituted with
one or more groups selected from the group consisting of, hydroxy,
heteroaromatic containing an NH in the ring, heteroalicyclic containing at
least
one NH group in the ring, C-carboxy, and, -NR,4R,5; (ii) carboxyalkyl; (iii) C-

carboxy; and, (iv) -NR,4R,5, wherein R,4 and R,5 are independently selected
from
the group consisting of hydrogen, lower alkyl and carbonyl; m is 0; G, H and K
are carbon; and, L is NH, oxygen or sulfur.
B. 3-Aralkyl-2-Indolinone Derivatives
In another aspect, the present invention relates to 3-aralkyl-2-indolinone
derivatives having the chemical structure set forth in formula II:
i z~
R2ow i.Q T~R22
II
~ U~
V R23
R
(II) Rza
R
R~~
or a physiologically acceptable salt or prodrug thereof where n is 0 or 1.
When p is 1, then M, Q, T, U and V are independently selected from the
group consisting of carbon and nitrogen, it being understood that, when M, Q,
T,
U, or V is nitrogen, Rio, R," R~~, R,3, or R,4, respectively, do not exist.
When p is 0, then M, Q, U, and V are independently selected from the



CA 02368041 2001-09-24
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group consisting of carbon, nitrogen, oxygen and sulfur, it being understood
that,
when M, Q, U, or V is oxygen or sulfur or nitrogen (wherein said nitrogen is
participating in a double bond), R,o, R~,, R", R," or R,4, respectively, do
not
exist.
5 R,6, R", R,B, R,~, R,o, Rz" R", R,3, or R~4 are independently selected from
the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,
alkynyl,
aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, mercapto, alkylthio,
aryloxy,
sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl, C-carboxy, O-
carboxy, carboxyalkyl, cyano, nitro, halo, O-carbamyl, N-carbamyl, C-amido, N-
10 amido and -NR~SR,~.
R,o and R,, or R~, and Rz~ or R,3 and R~3 or R,3 and Rz4 may combine to
form a five-member or a six-member aryl or heteroaryl ring.
R,5 and R~~ are independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, carbonyl,
sulfonyl,
15 and, combined, a five-member or a six-member heteroalicyclic ring.
A presently preferred embodiment of this invention is a compound
wherein R,6, R", R,g, R,9, R,o, RZ" R", R23, and R,4 are independently
selected
from the group consisting of (i) hydrogen; (ii) lower alkyl, optionally
substituted
with one or more groups selected from the group consisting of cycloalkyl,
aryl,
20 heteroaryl, heteroalicyclic, halo, hydroxy, C-carboxy and -NRZSR,6; (iii)
cycloalkyl; (iv) hydroxy; (v) lower alkoxy,optionally substituted with one or
more groups selected from the group consisting of one or more halo groups,
aryl,
heteroaryl and heteroalicyclic; (vi) trihalomethyl; (vii) trihalomethoxy;
(viii) halo;
(ix) carboxyalkyl; (x) aryl,optionally substituted with one or more groups
selected
from the group consisting of lower alkyl, lower alkyl substituted with one or
more
halo groups, trihalomethyl, trihalomethoxy, halo, hydroxy, lower alkoxy,
aryloxy
and -NR,SRzb; (xi) aryloxy; heteroaryl, optionally substituted with one or
more
groups
selected from the group consisting of lower alkyl, trihalomethyl, halo,
hydroxy,
(lower alkyl)alkoxy, amino and -NR,SR~~; (xii) heteroalicyclic; (xiii) (lower
alkyl)carboxy; (xiv) (lower alkyl)carbonyl; (xv) aryl carbonyl; (xvi) (lower
alkyl)-S-



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21
sulfonamido; (xvii) aryl-S-sulfonamido; (xviii) (lower alkyl)-N-sulfonamido;
(xix)
aryl-N-sulfonamido; (xx) (lower alkyl)-N-carbamoyl; (xxi) (lower alkyl)-C-
amido;
(xxii) (lower alkyl)-N-amido; (xxiii) (cycloalkyl)-N-amido; and, (xxiv) -
NRz5Rz6.
Another presently preferred embodiment of this invention is a compound
wherein p is 1 and M, Q, T, U, and V are carbon.
It is a further presently preferred embodiment of this invention that, when
p is 1 and M, Q, T, U, and V are carbon; R,6, R", R,B, and R,~ are
independently
selected from the group consisting of (i) hydrogen; (ii) halo; (iii) hydroxy;
(iv)
-NRz5Rz6; (v) S-sulfonamido optionally substituted with one or more groups
selected from the group consisting of hydrogen, lower alkyl and aryl; (vi)
lower
alkyl optionally substituted with a group selected from the group consisting
of
hydroxy, one or more halo groups, C-carboxy, C-amido, heteroalicyclic, and
-~25R26~ (vii) to wer alkoxy optionally substituted with one or more halo
groups;
(viii) aryl optionally substituted with one or more groups selected from the
group
1 S consisting of lower alkyl, lower alkoxy, halo, hydroxy and -NRz5Rz6, (ix)
N-
amido optionally substituted with one or more groups selected from the group
consisting of hydrogen, lower alkyl, cycloalkyl and aryl; and Rzo, Rz" Rzz,
R23
and Rz4 are independently selected from the group consisting of (i) hydrogen;
(ii)
halo; (iii) hydroxy; (iv) -NRz5Rz6; (v) lower alkyl optionally substituted
with one
or more groups selected from the group consisting of hydroxy, one or more halo
groups, C-carboxy, C-amido, heteroalicyclic, and -NRz5Rz6; (vi) cycloalkyl;
(vii)
lower alkoxy optionally substituted with one or more groups selected from the
group consisting of one or more halo groups, aryl, -NRz5Rz6, and heteroaryl,
(viii)
aryl optionally substituted with one or more groups selected from the group
consisting of halo, hydroxy, lower alkoxy, -NRz5Rz6, and C-carboxy.
It is yet another presently preferred embodiment of this invention that p is
1 and one or two of M, Q, T, U, or V are nitrogen.
It is likewise presently preferred that, when p is 1 and one or two of , Q, T,
U, and V are nitrogen, R,6, R", R,B, and R,~ are independently selected from
the
group consisting of (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) -NRz5R26, (v)
S-
sulfonamido optionally substituted with one or more groups selected from the



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22
group consisting of hydrogen, lower alkyl and aryl; (vi) lower alkyl
optionally
substituted with a group selected from the group consisting of hydroxy, one or
more halo groups, C-carboxy, C-amido, heteroalicyclic, and -NRzsRzb; (vii)
lower
alkoxy optionally substituted with one or more halo groups; (viii) aryl
optionally
substituted with one or more groups selected from the group consisting of
lower
alkyl, lower alkoxy, halo, hydroxy and -NRz5Rz6; (ix) N-amido optionally
substituted with one or more groups selected from the group consisting of
hydrogen, lower alkyl, cycloalkyl and aryl; and, Rzo, Rz" Rzz, Rz3, and Rz4,
whichever of these are not nitrogen, are independently selected from the group
consisting of (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) -NRz5Rz6; (v) lower
alkyl
optionally substituted with one or more groups selected from the group
consisting
of hydroxy, one or more halo groups, C-carboxy, C-amido, heteroalicyclic, and
-~zsRz6~ (vi) cycloalkyl; (vii) lower alkoxy optionally substituted with one
or
more groups selected from the group consisting of one or more halo groups,
aryl,
-NRz5Rz6, and heteroaryl; and (viii) aryl optionally substituted with one or
more
groups selected from the group consisting of halo, hydroxy, lower alkoxy,
-~25R26~ ~d C-carboxy.
It is a presently preferred embodiment of this invention that p is 1 and Rz,
and Rzz combine to form a fused pyrrolo group.
In further preferred embodiments of the invention, R,6, R,~, R,B, and R,9
are independently selected from the group consisting of (i) hydrogen; (ii)
halo;
(iii) hydroxy; (iv) -NRz5Rz6; (v) S-sulfonamido optionally substituted with
one or
more groups selected from the group consisting of hydrogen, lower alkyl and
aryl; (vi) lower alkyl optionally substituted with a group selected from the
group
consisting of hydroxy, one or more halo groups, C-carboxy, C-amido,
heteroalicyclic and, -NRz5Rz6; (vii) lower alkoxy optionally substituted with
one
or more halo groups; (viii) aryl optionally substituted with one or more
groups
selected from the group consisting of lower alkyl, lower alkoxy, halo, hydroxy
and -NRz5Rz6; (ix) N-amido optionally substituted with one or more groups
selected from the group consisting of hydrogen, lower alkyl, cycloalkyl and
aryl.
Similarly it is preferred that Rzo, Rz3, and Rz4 are independently selected



CA 02368041 2001-09-24
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23
from the group consisting of (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) -
NRz5Rz6;
(v) lower alkyl optionally substituted with one or more groups selected from
the
group consisting of hydroxy, one or more halo groups, C-carboxy, C-amido,
heteroalicyclic and, -NR,SRz~; (vi) cycloalkyl; (vii) lower alkoxy optionally
substituted with one or more groups selected from the group consisting of one
or
more halo groups, aryl, -NR,SRz~ and, heteroaryl; (viii) aryl optionally
substituted
with one or more groups selected from the group consisting of halo, hydroxy,
lower alkoxy, -NRz5Rz6 and, C-carboxy.
In another preferred embodiment, p is 0, M or Q is -NH, oxygen or sulfur;
and, M or Q, whichever is not -NH, oxygen or sulfur, and U and V are carbon.
Other preferred embodiments include compounds in which R,6, R", R,B,
and R,9 are independently selected from the group consisting of (i) hydrogen;
(ii)
halo; (iii) hydroxy; (iv) -NRz5Rz6; (v) S-sulfonamido optionally substituted
with
one or more groups selected from the group consisting of hydrogen, lower alkyl
and aryl; (vi) lower alkyl optionally substituted with a group selected from
the
group consisting of hydroxy, one or more halo groups, C-carboxy, C-amido,
heteroalicyclic and, -NRz5Rz6; (vii) lower alkoxy optionally substituted with
one
or more halo groups; (viii) aryl optionally substituted with one or more
groups
selected from the group consisting of lower alkyl, lower alkoxy, halo, hydroxy
and -NRz5Rz6; (ix) N-amido optionally substituted with one or more groups
selected from hydrogen, lower alkyl, cycloalkyl and aryl.
In these preferred compounds Rzo, Rz" Rz3, and Rz4 are independently
selected from the group consisting of (i) hydrogen; (ii) halo; (iii) hydroxy;
(iv)
-NRz5Rz6; (v) lower alkyl optionally substituted with one or more groups
selected
from the group consisting of hydroxy, one or more halo groups, C-carboxy, C-
amido, heteroalicyclic and, -NRz5Rz6; (vi) cycloalkyl; (vii) lower alkoxy
optionally substituted with one or more groups selected from the group
consisting
of one or more halo groups, aryl, -NRzSRzb, and, heteroaryl.
In still further preferred embodiments, R,6, R", R,B, and R,9 are
independently selected from the group consisting of (i) hydrogen; (ii) halo;
(iii)
hydroxy; (iv) -NRz5Rz6; (v) S-sulfonamido optionally substituted with one or



CA 02368041 2001-09-24
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24
more groups selected from the group consisting of hydrogen, lower alkyl and
aryl; (vi) lower alkyl optionally substituted with a group selected from the
group
consisting of hydroxy, one or more halo groups, C-carboxy, C-amido,
heteroalicyclic and, -NRzSRzb; (vii) lower alkoxy optionally substituted with
one
or more halo groups; (viii) aryl optionally substituted with one or more
groups
selected from the group consisting of lower alkyl, lower alkoxy, halo, hydroxy
and -NRz5Rz6; (ix) N-amido optionally substituted with one or more groups
selected from hydrogen, lower alkyl, cycloalkyl and aryl.
Preferably, Rzo, Rz3, and Rz4 are independently selected from the group
consisting of (i) hydrogen; (ii) halo; (iii) hydroxy; (iv) -NRz5Rz6, (v) lower
alkyl
optionally substituted with one or more groups selected from the group
consisting
of hydroxy, one or more halo groups, C-carboxy, C-amido, heteroalicyclic and,
-~zsRz6~ (vi) cycloalkyl; (vii) lower alkoxy optionally substituted with one
or
more groups selected from the group consisting of one or more halo groups,
aryl,
-NRz5Rz6 and, heteroaryl; (viii) aryl optionally substituted with one or more
groups selected from the group consisting of halo, hydroxy, lower alkoxy,
-~zsRz6 and, C-carboxy.
C. Diaryl Indolinone Compounds
In another aspect, the invention provides a compound ~ awing a structure set
forth in formula III
R34
(III)
R2~
R3a
where
(a) is selected from the group consisting of
(i) saturated or unsaturated alkyl optionally substituted with substituents
v R'~ 3



CA 02368041 2001-09-24
WO 00/56709 PCT/US00/07704
selected from the group consisting of halogen, trihalomethyl, alkoxy,
carboxylate,
amino, nitro, ester, and a five-membered or six-membered aromatic,
heteroaromatic,
aliphatic, or heteroaliphatic ring moiety,
where the ring moiety is optionally substituted with one, two, or three
5 substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties; and
(ii) an aromatic or heteroaromatic ring optionally substituted with one, two,
or
three substituents independently selected from the group consisting of alkyl,
alkoxy,
10 halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
15 independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(b) R3z, R33, and R34 are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with one or more
20 substituents selected from the group consisting of halogen, trihalomethyl,
carboxylate, amino, nitro, ester, and a five-membered or six-membered
aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moiety,
where the ring moiety is optionally substituted with one or more
substituents independently selected from the group consisting of
25 alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,



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2G
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(v) an amine of formula -(X,)",-NX,X3, where
X, is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
or aliphatic ring moieties,
nlis0orl,and
X, and X3 are independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and five-membered or six-
rr~embered aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula -NO~;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula -(X4)na-CO-X5, where
X4 and XS are independently selected from the group consisting of
alkyl and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one,
two, or three substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester moieties and where, and
n4is0orl;
(ix) a carboxylic acid of formula -(X6)"~ COOH or ester of formula -(X,)",-COO-

XR, where
X6, X,, and X$ and are independently selected from the group
consisting of alkyl and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties, and
n6 and n7 are independently 0 or 1;
(x) an alcohol of formula -(X~)"~-OH or an alkoxyalkyl moiety of formula
-(X,o)",o-O-X", where



CA 02368041 2001-09-24
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27
X~, X,o, and X" are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n9 and n10 are independently 0 or l;
(xi) an amide of formula -(X,2)",Z-NHCOX,3, or of formula -(X,4)n14-CONX,SX,6,
where
X,~ and X,4 are each independently selected from the group consisting
of alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
1 S the ring is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n12 and n14 are independently 0 or l,
X,3, X,S, and X,6 are each independently selected from the group
consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the ring is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester;
(xii) a sulfonamide of formula -(X")",~-SO,NX,~X,~, where
X" is selected from the group consisting of alkyl, and five-membered
or six-membered aromatic, heteroaromatic, aliphatic, or
heteroaliphatic ring moieties
the alkyl and ring moieties are optionally substituted with one



CA 02368041 2001-09-24
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28
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester,
n17is0, l,or2,
X,B, and X,~ are independently selected from the group consisting of
hydrogen, alkyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
or
where X,g and X,~ taken together form a five-membered or six-
membered aliphatic or heteroaliphatic ring optionally substituted with
one or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
and ester;
(xiii) an aldehyde of formula -(XZo)"ZO CO-H where
X,o is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n20 is 0 or 1;
(xiv) a sulfone of formula -(X2,)"2~-SOZ-X22, where
X2, and Xz2 are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group



CA 02368041 2001-09-24
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29
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n21 is 0 or 1; and
(xv) a thiol of formula -(X23)",3-SH or a thioether of formula -(X,4)n24-S-
X?SW'here
X~,, X,4, and X,5 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n23 and n24 are independently 0 or l;
or
R33 and R34 taken together form a six-membered aliphatic or aromatic ring,
optionally substituted with one or more substituents independently selected
from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro,
and
ester; and
(c) R,B, R,~, R3o, and R3, are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with one or more
substituents selected from the group consisting of hydroxy, halogen,
trihalomethyl,
carboxylate, amino, nitro, ester, and a five-membered or six-membered
aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moiety,
the ring moiety is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;



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(iv) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
S independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(v) an amine of formula -(X,)",-NX,X3, where
X, is selected from the group consisting of saturated or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
10 or aliphatic ring moieties,
nl is 0 or 1, and
X2, and X3 are independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, or aliphatic ring moieties;
15 (vi) a nitro of formula -NOZ;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula °(X4)n4 CO-X5, where
X4 and XS are independently selected from the group consisting of
alkyl and five-membered or six-membered aromatic, heteroaromatic,
20 aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one,
two, or three substituents independently selected from the
group consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester moieties, and
25 n4is0orl;
(ix) a carboxylic acid of formula -(X6)"6-COOH or ester of formula -(X~)"; COO-

X8, where
X6, X,, and X$ and are independently selected from the group
consisting of alkyl and five-membered or six-membered aromatic,
30 heteroaromatic, aliphatic, or heteroaliphatic ring moieties, and
n6 and n7 are independently 0 or l;



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31
(x) an alcohol of formula -(X9)"~-OH or an alkoxyalkyl moiety of formula
OX~o)n,o-O-X", where
X~, X,o, and X" are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n9 and n 10 are independently 0 or 1;
(xi) an amide of formula -(X,2)",2-NHCOX,3, or of formula -(X,4)",4-CONX"X,6,
where
X,, and X,4 are each independently selected from the group consisting
of alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester,
nl2 and n14 are independently 0 or l, and
X,3, X,;, and X,6 are each independently selected from the group
consisting of hydrogen, alkyl, hydroxyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties, and
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester;
(xii) a sulfonamide of formula -(X")""-SOZNX,BX,~, where
X" is selected from the group consisting of alkyl, five-membered or



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32
six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic
ring moieties
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n17is0, l,or2,and
X,A, and X,9 are independently selected from the group consisting of
hydrogen, alkyl, five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, or
X,g and X,9 taken together form a five-membered or six-membered
aliphatic or heteroaliphatic ring optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester;
(xiii) an aldehyde of formula -(XZO)",o CO-H where
XZO is selected from the group consisting of sat~a-uEed or unsaturated
alkyl, and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n20 is 0 or 1;
(xiv) a sulfone of formula -(X2,)",,-SO~-X22, where
X2, and X2z are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,



CA 02368041 2001-09-24
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33
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
n21 is 0 or 1; and
(xv) a thiol of formula -(X,3)nz3-SH or a thioether of formula -(X24)n24-s-
X25~ where
X,3, X,4, and X~5 are independently selected from the group consisting
of saturated or unsaturated alkyl, and five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the alkyl and ring moieties are optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
amino, nitro, and ester, and
1 S n23 and n24 are independently 0 or 1.
In preferred embodiments, the invention relates to a compound of formula III
where
(a) R3, is hydrogen;
(b) R32, R33, and R34 are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated alkyl optionally substituted with a six-membered
heteroaliphatic
ring moiety;
(iii) an amine of formula -(X,)",-NX~X3, where X, is saturated alkyl, nl is 0
or l,
and XZ and X3 are independently selected from the group consisting of hydrogen
and
saturated alkyl;
(iv) a carboxylic acid of formula -(X6)"6-COOH or ester of formula -(X,)",-COO-

X~, where X6, X,, and Xg are alkyl, and n6 and n7 are independently 0 or 1;
and
(v) an amide of formula -(X,4)"14-CONX,SX,6, where X,4 is alkyl, n12 and n14
are independently 0 or 1, and X,5 and X,6 are each independently selected from
the
group consisting of hydrogen and alkyl;
or R, is as described herein and R8 and R<, taken together form an optionally



CA 02368041 2001-09-24
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34
substituted six-membered aliphatic or aromatic ring; and
(c) R,B, R2~, and R,o are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated alkyl;
(iii) a halogen or trihalomethyl;
(iv) a carboxylic acid of formula -(X6)"6-COOH or ester of formula -(X,)~,-COO-

Xg, where X~, X,, and X8 and are alkyl, and n6 and n7 are independently 0 or
1;
(v) an alcohol of formula -(X9)9-OH or an alkoxyalkyl moiety of formula
-(X,o)",0 0-X", where X~, X,o, and X" are alkyl, and n9 and n10 are
independently 0
or l;
(vi) an amide of formula -(X,z)",z-NHCOX,3, or of formula -(X,4)m4 CONX,SX,6,
where X,~ and X,4 are alkyl, n12 and n14 are independently 0 or l, and X,3,
X,S, and
X,6 are each independently selected from the group consisting of hydrogen and
alkyl; and
(vii) a sulfonamide of formula -(X")""-SOZNX,8X,9, where X" is alkyl, and n17
is
0, 1, or 2, and X,g, and X,9 are independently selected from the group
consisting of
hydrogen and alkyl.
More preferably, in the compounds of formula III
(a) R,8 is selected from the group consisting of hydrogen, methyl, and 2-
hydroxyethyl;
(b) Rz9 is selected from the group consisting of hydrogen, methyl, chloro,
bromo, carboxy, methoxy, -NHC(O)-CH3, and -SO~N(CH3)2;
(c) R3o is selected from the group consisting of hydrogen, chloro,
methoxy, and -NHC(O)-CHj;
(d) R" is hydrogen;
(e) R32 is selected from the group consisting of hydrogen, methyl, -
CH,CH,C(O)OH, -CHZCHzC(O)NH" -CH,CH~CH~N(CH3)2, and 3-
morpholinopropyl; and
(f) R33 and R34 are each independently selected from the group consisting
of hydrogen, methyl, -CH,CHZC(O)OH, -CH~CH~CH,N(CH3)Z, and 3-
morpholinopropyl, or R8 and R~, taken together form a six-membered aromatic or



CA 02368041 2001-09-24
WO 00/56709 PCT/US00/07704
aliphatic ring.
The preferred diaryl indolinone compounds of the invention are those which
are preferrably formed by the reaction of a ketone compound with and oxindole
compound. The ketone compound is preferrably selected from the group
consisting
of
OH
O
R27 / ~ R27 / ~ R2~ / ~ R27 /
N N N N
O H O H O H O H
> >
\N' NH2 OH \N ~
O O
/~ /~ /~ /
Rz~ N R2~ N R2~ N R27 N
O H O H O H O H
NH2 N
O O
OH
/~ /~ /~
Rz~ N R27 N Rz~ N
O H O H O H
O
N/ N ' OH
_. 1 _. ~O _,
Rz~ /N~ R2~ /N~ R2z /Nl
O H O H O H
> >
OH
O
N/ N
~O
/~ /
R2~ N Rz~ N R27 N
10 0 H ~ O H ~ O H



CA 02368041 2001-09-24
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36
,_
NHZ N N
O
/Nl Ri /Nl R2~ /N/
O H , o H , and O H
In the above structures R,, is selected from the group consisting of
(i) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl, alkoxy,
carboxylate,
S amino, nitro, ester, and a five-membered or six-membered aromatic,
heteroaromatic,
aliphatic, or heteroaliphatic ring moiety,
wherein said ring moiety is optionally substituted with one, two, or
three substituents independently selected from the group consisting of
alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester
moieties; and
(ii) an aromatic or heteroaromatic ring optionally substituted with one, two,
or
three substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting waf alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties.
The oxindole compound is preferrably selected from the group consisting of
1,3-dihydro-indol-2-one, 4-methyl-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-

1,3-dihydro-indol-2-one, 5-chloro-1,3-dihydro-indol-2-one, 5-bromo-1,3-dihydro-

indol-2-one, 5-methoxy-1,3-dihydro-indol-2-one, 2-oxo-2,3-dihydro-1H-indole-5-
carboxylic acid, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide, N-
(2-
oxo-2,3-dihydro-1H-indole-5-yl)-acetamide, 6-chloro-1,3-dihydro-indol-2-one, 6-




CA 02368041 2001-09-24
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37
methoxy-1,3-dihydro-indol-2-one, and N-(5-methyl-2-oxo-2,3-dihydro-1H-indole-6-

yl)-acetamide.
D. 4-Substituted Indolinone Compounds
S In a further aspect, the invention provides a compound having a structure
set
forth in formula IV or formula V:
R3s Z
R37 R3G R37
1 R35
R38\/\X R3aw
/~ ~N /~~N
R3~ ~ R39
(IV) R4o Rai (V) R4o Ray
where
O
(a) R35, R36, and R4, are each independently selected from the group
consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl, alkoxy,
carboxylate,
amino, nitro, ester, and a five-membered or six-membered aromatic,
heteroaromatic,
aliphatic, or heteroaliphatic ring moiety,
where the ring moiety is optionally substituted with one, two, or three
substituents independently selected from the group consisting of alkyl,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties; and
(iii) an aromatic or heteroaromatic ring optionally substituted with one, two,
or
three substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(b) R3, is an ethyl-2-oxy group of formula -CH=CHz-O-R, where R is selected
from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with substituents
selected from the group consisting of halogen, trihalomethyl, carboxylate,
amino,
nitro, ester, and a five-membered or six-membered aromatic, heteroaromatic,



CA 02368041 2001-09-24
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38
aliphatic, or heteroaliphatic ring moiety, and
the ring moiety is optionally substituted with one, two, or three substituents
independently selected from the group consisting of alkyl, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl, aryl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties
(iv) a substituent of formula -C(E)NX,SX,6, where
X,S, and X,6 are each independently selected from the group consisting of
hydrogen, alkyl, hydroxyl, sulfone of formula -SOZ-X22, and five-membered
or six-membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the ring is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, aryl,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
X22 is selected from the group consisting of saturated or
unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
moieties,
the ring is optionally substituted with one or more
substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl,
carboxylate, amino, nitro, and ester,
E is selected from the group consisting of oxygen and sulfur; and
(c) R38, R3~, and R4o are each hydrogen;
(d) Z is a 5, 6, 7, 8, 9, or 10 membered, monocyclic or bicyclic, aromatic or
heteroaromatic, ring moiety, optionally substituted with one or more
substituents
selected from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl optionally substituted with one or more
substituents selected from the group consisting of hydroxy, halogen,
trihalomethyl,



CA 02368041 2001-09-24
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39
carboxylate, amino, nitro, ester, and a five-membered or six-membered
aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moiety,
the ring moiety is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iii) an aromatic or heteroaromatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester moieties;
(iv) an aliphatic or heteroaliphatic ring optionally substituted with one or
more
substituents independently selected from the group consisting of alkyl,
alkoxy,
halogen, trihalomethyl, carboxylate, amino, nitro, ester, and an aromatic or
heteroaromatic ring optionally substituted with one or more substituents
independently selected from the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, amino, nitro, and ester moieties;
1 S (v) an amine of formula -(X,)",-NXZX3, where
X, is selected from the group consisting of saturated or unsaturated alkyl,
and
five-membered or six-membered aromatic, heteroaromatic, or aliphatic ring
moieties,
nl is 0 or 1, and
X" and X, are independently selected from the group consisting of hydrogen,
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, or aliphatic ring moieties;
(vi) a nitro of formula -NOZ;
(vii) a halogen or trihalomethyl;
(viii) a ketone of formula -(X~)"4 CO-X5, where
X4 and X5 are independently selected from the group consisting of alkyl and
five-membered or six-membered aromatic, heteroaromatic, aliphatic, or
heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one, two,
or three substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester



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moieties, and
n4is0orl;
(ix) a carboxylic acid of formula -(X~)"6-COOH or ester of formula -(X~)"~-COO-

Xg, where
5 X6, X" and X$ and are independently selected from the group consisting of
alkyl and five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties, and
n6 and n7 are independently 0 or 1;
(x) an alcohol of formula -(X9)~g-OH or an alkoxyalkyl moiety of formula
10 -(X,o)n,o O-X", where
X~, X,o, and X" are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or more
15 substituents independently selected from the group consisting of alkyl,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester, and
n9 and n10 are independently 0 or 1;
(xi) an amide of formula -(X,z)~,2-NHCOX,3, or of formula -(X,4)m4-CONX,SX,6,
where
20 X,, and X,4 are each independently selected from the g~~uup consisting of
alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
25 of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
nl2 and n14 are independently 0 or l, and
X,3, X,S, and X,6 are each independently selected from the group consisting
of hydrogen, alkyl, hydroxyl, and five-membered or six-membered aromatic,
heteroaromatic, aliphatic, or heteroaliphatic ring moieties, and
30 the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting



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41
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester;
(xii) a sulfonamide of formula -(X")""-SO,NX,gX,9, where
X" is selected from the group consisting of alkyl, five-membered or six-
membered aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring
S moieties
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, or ester,
and
n17 is 0, 1, or 2, and
X,B, and X,~ are independently selected from the group consisting of
hydrogen, alkyl, five-membered or six-membered aromatic, heteroaromatic,
aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or
1 S more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, or ester, or
X,8 and X,9 taken together form a five-membered or six-membered
aliphatic or heteroaliphatic ring optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester;
(xiii) an aldehyde of formula -(Xzo)"zo-CO-H where
Xzo is selected from the group consisting of saturated or unsaturated alkyl,
and five-membered or six-membered aromatic, heteroaromatic, aliphatic, or
heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or more
substituents independently selected from the group consisting of alkyl,
halogen, trihalomethyl, carboxylate, amino, nitro, and ester, and
n20 is 0 or l;
(xiv) a sulfone of formula -(Xz,)",,-SOz-Xzz, where
Xz, and Xzz are independently selected from the group consisting of saturated
or unsaturated alkyl, and five-membered or six-membered aromatic,



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42
heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n21 is 0 or 1; and
(xv) a thiol of fornmla -(X,3)"23-SH and a thioether of formula -(Xz4)n2a-S-
X?5~
where
X23, X24, and X25 are independently selected from the group consisting of
saturated or unsaturated alkyl, and five-membered or six-membered
aromatic, heteroaromatic, aliphatic, or heteroaliphatic ring moieties,
the alkyl and ring moieties are optionally substituted with one or
more substituents independently selected from the group consisting
of alkyl, halogen, trihalomethyl, carboxylate, amino, nitro, and ester,
and
n23 and n24 are independently 0 or 1.
In preferred embodiments, the invention relates to a compound of formula IV
or formula V, where
(a) R,S, R36, and R4, are hydrogen;
(b) R" is an ethyl-2-oxy group of formula -CH~CHz-O-R, where R is selected
from the group consisting of hydrogen, saturated alkyl, an aromatic ring
optionally
substituted with one or more substituents independently selected from the
group
consisting of alkyl, aryl, and alkoxy moieties, and a substituent of formula
-C(E)NHX,S, where
X,5 is selected from the group consisting of alkyl, sulfone of formula -SO,-
XZ" and six-membered aromatic or aliphatic ring moieties,
the ring is optionally substituted with one or more substituents
independently selected from the group consisting of alkyl and aryl,
X2~ is selected from the group consisting of saturated alkyl, and
optionally substituted six-membered aromatic ring moieties, and
E is selected from the group consisting of oxygen and sulfur, and;



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43
(c) Z is a 5, 6, or 9 membered, monocyclic or bicyclic, aromatic or
heteroaromatic, ring moiety, optionally substituted with one or more
substituents
selected from the group consisting of
(i) hydrogen;
(ii) saturated alkyl,
(iii) an optionally substituted aromatic or heteroaromatic ring;
(iv) an amine of formula -(X,)",-NX,X3, where
X, is alkyl,
nl is 0 or 1, and
XZ and X3 are independently selected from the group consisting of hydrogen
and saturated alkyl;
(v) a halogen or trihalomethyl;
(vi) a carboxylic acid of formula -(X6)"6-COOH, where
X6 is alkyl, and
n6is0orl;and
(vii) an alcohol of formula -(X9)n9-OH or an alkoxyalkyl moiety of formula -
(X,o)",o O-X", where
X~, X, o, and X" are alkyl, and
n9 and n10 are independently 0 or 1.
More preferably, in the compounds of formula IV or formula V, R3, is an
ethyl-2-oxy group of formula -CH,CH,-O-R, wherein R is selected from the group
consisting of hydrogen, methyl, ethyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-

isopropylphenyl, 4-methoxyphenyl, biphen-3-yl, 5-chloropyridin-3-yl,
ethylcarbamyl, tert-butylcarbamyl, cyclohexylcarbamyl, phenylcarbamyl, benzene
sulfonylcarbamyl, biphen-2-yl-carbamyl, and phenylthiocarbamyl, and Z is
selected
from the group consisting of 4-bromophenyl, 2-pyridyl, 6-methyl-2-pyridyl, 1H-
indol-5-yl, 4-methoxy-3-thiophenphenyl, 4-(3-dimethylamino)-3,5-dimethyl-1H-
pyrrol-2-yl, 3-hydroxy-6-methyl-pyridin-2-yl, 4-(3-dimethylaminopropyl)-3,5-
dimethyl-1H-pyrrol-2-yl, 3-carboxy-2,4-dimethyl-1H-pyrrol-2-yl, and
isopropylcarbamyl.
The preferred compounds of the invention are listed in Table 1.



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Table 1
Compound
Number Compound Name


IN-001 4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one


IN-002 4-[2-(2-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one


IN-003 4-[2-(biphenyl-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one


IN-004 3-(4-bromo-benzylidene)-4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one


IN-005 4-(2-hydroxy-ethyl)-3-pyridin-2-ylmethylene-1,3-dihydro-indol-2-one


IN-006 4-(2-hydroxy-ethyl)-3-(6-methyl-pyridin-2-ylmethylene)-1,3-dihydro-
indol-2-one


IN-007 4-(2-hydroxy-ethyl)-3-(IH-indol-5-ylmethylene)-1,3-dihydro-indol-2-one


4-(2-hydroxy-ethyl)-3-(4-methoxy-3-thiophen-2-yl-benzylidene)-1,3-dihydro-
indol-2-
IN-008 one


3-[4-(3-dimethylamino-propyl)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-4-(2-
hydroxy-
IN-009 ethyl)-1,3-dihydro-indol-2-one


IN-010 phenyl-thiocarbamic acid O-[2-(2-oxo-2,3-dihydro-IH-indol-4-yl)-ethyl]
ester


IN-011 phenyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl
ester


IN-012 tert-butyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-s~la-ethyl
ester


IN-013 cyclohexyl-carbamic acid 2-(2-oxo-2,3-dihydro-IH-indol-4-yl)-ethyl
ester


IN-014 benzene sulfonyl-carbamic acid 2-(2-oxo-2,3-dihydro-IH-indol-4-yl)-
ethyl
ester


IN-015 biphenyl-2-yl-carbamic acid 2-(2-oxo-2,3-dihydro-IH-indol-4-yl)-ethyl
ester


IN-016 ethyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl
ester


IN-017 4-[2-(4-methoxy-phenoxy)-ethyl]-1,3-dihydro-indol-2-one


IN-018 4-(2-methoxy-ethyl)-1,3-dihydro-indol-2-one


IN-019 4-(2-ethoxy-ethyl)-1,3-dihydro-indol-2-one


IN-020 4-[2-(4-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one






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Compound


Number Compound Name


IN-021 4-[2-(5-chloro-pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one


4-(2-hydroxy-ethyl)-3-(3-hydroxy-6-methyl-pyridin-2-ylmethylene)-1,
3-dihydro-


IN-022 indol-2-one


3-[4-(3-dimethylamino-propyl)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-4-[2-(3-


IN-023 isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one


3-( 5- { 4-[2-(4-isopropyl-phenoxy)-ethyl]-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl
}


IN-024 2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid



IN-025 Isopropyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl
ester


Some of the above compounds have the structure of formula VI, with the R
substituent as defined in Table 2.
R
H
(VI)
H
H
Table 2
Compound R


Number


IN-001 3-isopropyl-phenoxy


IN-002 2-isopropyl-phenoxy


IN-003 biphenyl-3-yloxy


IN-O 10 N
0-


~
~


S


IN-O11 N
O


~
~


O


IN-012 . Nup-


CH3) 3C
II


O


IN-O 13 N ~ 0


0





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46
Compound R
Number
IN-014 'I
S H~0
0
IN-015
/
I / 0
IN-O1G CH3CH2 Nu0
~I0
IN-017 4-methoxy-phenoxy
IN-018 methoxy
IN-019 ethoxy
IN-()20 4-isopropyl-phenoxy
IN-021 5-chloro-pyridin-3-yloxy
IN-025 H
CH3) ZCH~ N~0
O
Other compounds of the invention have the structure set forth in formula VII,
with
the substituents as defined in Table 3.
(VII)
H ~.
Table 3
Compound R Z
Number


IN-004 hydroxy 4-bromophenyl


IN-005 hydroxy pyridin-2-yl


IN-006 hydroxy 6-methyl-pyridin-2-yl






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47
Compound R Z
Number


IN-007 hydroxy 1H-indol-5-yl


IN-008 hydroxy 4-methoxy-3-thiophen-2-yl-phenyl


IN-009 hydroxy 4-(3-dimethylamino-propyl)-3,5-dimethyl-
1 H-pyrrol-2-yI


IN-022 hydroxy 3-hydroxy-6-methyl-pyridin-2-yl


IN-023 3-isopropyl-phenoxy4-(3-dimethylamino-propyl)-3,5-dimethyl-
1 H-pyrrol-2-yl


IN-024 4-isopropyl-phenoxy4-carboxy-3,5-dimethyl-1H-pyrrol-2-yl


III. COMBINATORIAL LIBRARIES AND SYNTHETIC PROCEDURES
A. General Notes on Synthetic Procedures
To synthesize the compounds of the invention a base may be used. The base
is preferably a nitrogen base or an inorganic base. "Nitrogen bases" are
commonly
used in the art and are selected from acyclic and cyclic amines. Examples of
nitrogen bases include, but are not limited to, ammonia, methylamine,
trimethylamine, triethylamine, aniline, 1,8-diazabicyclo[5.4.0]undec-7-ene,
diisopropylethylamine, pyrrolidine, piperidine, and pyridine or substituted
pyridine
(e.g., 2,6-di-tertbutylpyridine). "Inorganic bases" are bases that do not
contain any
carbon atoms. Examples of inorganic bases include, but are not limited to,
hydroxide, phosphate, bisulfate, hydrosulfide, and amide anions. Those skilled
in
the art know which nitrogen base or inorganic base would match the
requirements of
the reaction conditions. In certain embodiments of the invention, the base
used may
be pyrrolidine or piperidine. In other embodiments the base may be the
hydroxide
anion, preferably used as its sodium or potassium salt.
The synthesis of the compounds of the invention may take place in a solvent.
The solvent of the reaction is preferably a protic solvent or an aprotic
solevent.
"Protic solvents" are those that are capable of donating a proton to a solute.
Examples of protic solvents include, but are not limited to, alcohols and
water.
"Aprotic solvents" are those solvents that, under normal reaction conditions,
do not
donate a proton to a solute. Typical organic solvents, such as hexane,
toluene,



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48
benzene, methylene chloride, dimethylformamide, chloroform, tetrahydrofuran,
are
some of the examples of aprotic solvents. Other aprotic solvents are also
within the
scope used by the present invention. In some preferred embodiments, the
solvent of
the reaction is an alcohol, which may preferably be isopropanol or most
preferably
ethanol. Water is another preferred erotic solvent. Dimethylformamide, known
in
the chemistry art as DMF, is a preferred aprotic solvent.
The synthetic method of the invention calls for the reaction to take place at
elevated temperatures which are temperatures that are greater than room
temperature. More preferably, the elevated temperature is preferably about 30-
150
°C, more preferably about 80-100 °C, and most preferably about
80-90 °C, which is
about the temperature at which ethanol boils (i.e., the boiling point of
ethanol). By
"about" a certain temperature it is meant that the temperature range is
preferably
within 10 °C of the listed temperature, more preferably within 5
°C of the listed
temperature, and most preferably within 2 °C of the listed temperature.
Therefore,
by way of example, by "about 80 °C" it is meant that the temperature
range is
preferably 80110 °C, more preferably 805 °C, and most preferably
8012 °C.
The synthetic method of the invention may be accompanied by the step of
screening a library for a compound of the desired activity and structure -
thus,
providing a method of synthesis of a compound by first screening for a
compound
having the desired properties and then chemically synthesizing that compound.
B. 3-Arylidenyl-6-Heterocyclyl-2-Indolinone Derivatives
An additional aspect of this invention is a combinatorial library of at least
ten
3-arylidenyl-6-heterocyclyl-2-indolinone compounds that can be formed by
condensing oxindoles of structure 2 with aldehydes of structure 3.
As used herein, "condensing" or "condensation" refers to a reaction by which
two molecules are combined to give one molecule. In particular with regard the
present invention a condensation refers to the reaction shown in Scheme I.



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49
R~
RS ~ D , R~
B E base
R,o' H/ O + / AI % F\ soQ ent'
Ra ~ Rs
J / L~ R3 CHO
R~ ~ ~ Rt3
m
3
R~Z
2
SCHEME I
The oxindole in the above combinatorial library is preferrably selected from
the group consisting of 6-(pyridin-2-yl)-2-oxindole, 6-(pyridin-3-yl)-2-
oxindole, 6-
(pyridine-4-yl)-2-oxindole, 6-(pyrimidin-2-yl)-2-oxindole, 6-(pyrimidin-4-yl)-
2-
oxindole, 6-(pyrimidin-5-yl)-2-oxindole, 6-(triazinyl)-2-oxindole, 6-(pyrrol-2-
yl)-2-
oxindole, 6-(pyrrol-3-yl)-2-oxindole, 6-(thiophen-2-yl)-2-oxindole, 6-
(thiophen-3-
yl)-2-oxindole, 6-(furan-2-yl)-2-oxindole, 6-(furan-3-yl)-2-oxindole, 6-
(imidazol-2-
yl)-2-oxindole, 6-(imidazol-4-yl)-2-oxindole, 6-(thiazol-2-yl)-2-oxindole, 6-
(thiazol-
4-yl)-2-oxindole, 6-(oxazol-2-yl)-2-oxindole, 6-(oxazol-4-yl)-2-oxindole, 6-
(thiadiazol-2-yl)-2-oxindole, 6-(oxadiazolyl)-2-oxindole and 6-(triazol-2-yl)-
2-
oxindole, 6-(3methylisoxazole-5-yl)-2-oxindole,6-(3-methylisothiazole-5-yl)-2-
oxindole, 6-(3,5-dimethyl-isoxazole-4-yl)-2-oxindole, 6-(thiazole-2-yl)-2-
oxindole,
6-(thiazole-4-yl)-2-oxindole, 6-(thiazole-S-yl)-2-oxindole, 6-(3-
methylthiophene-2-
yl)-2-oxindole, 6-(4-methylthiophene-2-yl)-2-oxindole, 6-(5-methylthiophene-2-
yl)-
2-oxindole, 6-(5-chlorothiophene-2-yl)-2-oxindole, 6-(4-methylfuran-2-yl)-2-
oxindole
The aldehyde in the above combinatorial library is preferably selected from
the group consisting of, without limitation, benzaldehyde, 3-isopropyl-p-
anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,5-diisopropyl-p-
anisaldehyde,
3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-anisaldehyde, 3-phenyl-p-
anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-dichlorobenzaldehyde,
2-
hydroxy-5-chlorobenzaldehyde, 2-hydroxy-4-methoxy-5-(4-
methoxyphenyl)benzaldehyde, 3-cyclopentyl-4-methoxybenzaldehyde, 3-



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cyclopentyl-4-hydroxybenzaldehyde, 3-(2-thienyl)-4-methoxybenzaldehyde, 2-
hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-S-
bromobenzaldehyde, 2-methylpyridine-6-carboxaldehyde, 3-tent-butyl-4-hydroxy-5-

bromobenzaldehyde, 3-bromobenzaldehyde, 3,5-diisopropyl-4-[2-(N-
5 morpholino)ethyl]benzaldehyde,indole-S-carboxaldehyde, 2-hydroxy-3-
fluorobenzaldehyde, 3-cyclohexyl-4-[2-(N-morpholino)ethyl]benzaldehyde, 3-(3-
thienyl)-4-methoxybenzaldehyde, 2-methyl-5-isopropyl-4-methoxy- benzaldehyde,
2,3-dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde,
3-(thiophen-3-yl)-4-methoxybenzaldehyde, 3-(3-acetylaminophenyl)-4-methoxy-
10 benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2-(3-methoxyphenyl)-4,5-

dimethoxybenzaldehyde, 3-(pyridin-3-yl)-p-anisaldehyde, 2-(3-ethoxyphenyl)-4,5-

dimethoxybenzaldehyde, 3-isopropyl-4-[2-(N--morpholino)ethoxy]benzaldehyde,
3,5-diisopropyl-~-'.-[2-(N-morpholino)ethoxy)benzaldehyde, 2-(2-methoxyphenyl)-

4,5-dimethoxybenzaldehyde, 3-(3-ethoxyphenyl)-p-anisaldehyde, 3-(thiophen-2-
yl)-
15 4-[2-(N-morpholino)ethoxy]- benzaldehyde, 2-(thiophen-2-yl)-4,5-
dimethoxybenzaldehyde, 2,4-dimethyl-3-[3-dimethylaminopropyl]-pyrrole-5-
carboxaldehyde, 2,4-dimethyl-3-[3-carboxypropyl]-pyrrole-S-carboxaldehyde, 2,4-

dimethyl-3-[3-(N-morpholino)prop-1-yl]-pyrrole-5-carboxaldehyde, 2,4-dimethyl-
3-
[2-dimethylaminoethyl]-pyrrole-5-carboxaldehyde, 2,4-dimethyl-3-[2-
20 carboxyethyl]-pyrrole-5-carboxaldehyde and 2,4-dimethyl-3-[2-(N-
morpholino)propyl]-pyrrole-5-carboxaldehyde, 3-isopropyl-4-[2-(N-
morpholino)propoxy]benzaldehyde, 3,5-diisopropyl-4-[2-(N-
morpholino)propoxy]benzaldehyde, 3-(thiophen-2-yl)-4-[2-N-
morpholino)propoxy]benzaldehyde,.
25 Another aspect of this invention provides a method for the synthesis of 3-
arylidenyl-6-heterocyclyl-2-indolinone of formula I comprising condensing an
oxindole of formula 2 with an aldehyde of formula 3 in a solvent, preferably
in the
presence of a base.
Examples of the oxindoles of formula 2 that may be condensed with an
30 aldehyde of formula 3 to give a 3-arylidenyl-6-heterocyclyl-2-indolinones
of
formula I are 6-(pyridin-2-yl)-2-oxindole, 6-(pyridin-3-yl)-2-oxindole, 6-
(pyridine-



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51
4-yl)-2-oxindole, 6-(pyrimidin-2-yl)-2-oxindole, 6-(pyrimidin-4-yl)-2-
oxindole, 6-
(pyrimidin-5-yl)-2-oxindole, 6-(triazinyl)-2-oxindole, 6-(pyrrol-2-yl)-2-
oxindole, 6-
(pyrrol-3-yl)-2-oxindole, 6-(thiophen-2-yl)-2-oxindole, 6-(thiophen-3-yl)-2-
oxindole, 6-(furan-2-yl)-2-oxindole, 6-(furan-3-yl)-2-oxindole, 6-(imidazol-2-
yl)-2-
oxindole, 6-(imidazol-4-yl)-2-oxindole, 6-(thiazol-2-yl)-2-oxindole, 6-
(thiazol-4-yl)-
2-oxindole, 6-(oxazol-2-yl)-2-oxindole, 6-(oxazol-4-yl)-2-oxindole, 6-
(thiadiazol-2-
yl)-2-oxindole, 6-(oxadiazol-2-yl)-2-oxindole and 6-(triazol-2-yl)-2-oxindole,
6-
(3methylisoxazole-5-yl)-2-oxindole, G-(3-methylisothiazole-5-yl)-2-oxindole, 6-

(3,5-dimethyl-isoxazole-4-yl)-2-oxindole, 6-(thiazole-2-yl)-2-oxindole, 6-
(thiazole-
4-yl)-2-oxindole, 6-(thiazole-5-yl)-2-oxindole, 6-(3-methylthiophene-2-yl)-2-
oxindole, 6-(4-methylthiophene-2-yl)-2-oxindole, 6-(5-methylthiophene-2-yl)-2-
oxindole, 6-(5-chlorothiophene-2-yl)-2-oxindole, 6-(4-methylfuran-2-yl)-2-
oxindole.
Examples of aldehydes of structure 3 which may be condensed with oxindoles
of structure 2 to give a compound of this invention are, without limitation,
benzaldehyde, 3-isopropyl-p-anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde,
3,5-
diisopropyl-p-anisaldehyde, 3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-
anisaldehyde, 3-phenyl-p-anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-
3,5-
dichlorobenzaldehyde, 2-hydroxy-5-chlorobenzaldehyde, 2-hydroxy-4-methoxy-S-(4-

methoxyphenyl)benzaldehyde, 3-cyclopentyl-4-methoxybenzaldehyde, 3-cyclopentyl-

4-hydroxybenzaldehyde, 3-(2-thienyl)-4-methoxybenzaldehyde, 2-
hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-S-
bromobenzaldehyde, 2-methylpyridine-6-carboxaldehyde, 3-tent-butyl-4-hydroxy-5-

bromobenzaldehyde, 3-bromobenzaldehyde, 3,5-diisopropyl-4-[2-(N-
morpholino)ethyl]benzaldehyde,indole-5-carboxaldehyde, 2-hydroxy-3-
fluorobenzaldehyde, 3-cyclohexyl-4-[2-(N-morpholino)ethyl]benzaldehyde, 3-(3-
thienyl)-4-methoxybenzaldehyde, 2-methyl-5-isopropyl-4-methoxy- benzaldehyde,
2,3-dihydrobenzofuran-~-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde,
3-
(thiophen-3-yl)-4-methoxybenzaldehyde, 3-(3-acetylaminophenyl)-4-methoxy-
benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2-(3-methoxyphenyl)-4,5-
dimethoxybenzaldehyde, 3-(pyridin-3-yl)-p-anisaldehyde, 2-(3-ethoxyphenyl)-4,5-

dimethoxybenzaldehyde, 3-isopropyl-4-[2-(N-morpholino)ethoxy]benzaldehyde, 3,5-




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diisopropyl-4-[2-(N-morpholino)ethoxy]benzaldehyde, 2-(2-methoxyphenyl)-4,5-
dimethoxybenzaldehyde, 3-(3-ethoxyphenyl)-p-anisaldehyde, 3-(thiophen-2-yl)-4-
[2-
(N-morpholino)ethoxy]- benzaldehyde, 2-(thiophen-2-yl)-4,5-
dimethoxybenzaldehyde, 2,4-dimethyl-3-[3-dimethylaminopropyl]-pyrrole-5-
carboxaldehyde, 2,4-dimethyl-3-[3-carboxypropyl]-pyrrole-5-carboxaldehyde, 2,4-

dimethyl-3-[3-(N-morpholino)propyl]-pyrrole-5-carboxaldehyde, 2,4-dimethyl-3-
[2-
dimethylaminoethyl]-pyrrole-5-carboxaldehyde, 2,4-dimethyl-3-[2-carboxyethyl]-
pyrrole-5-carboxaldehyde and 2,4-dimethyl-3-[2-(N-morpholino)ethyl]prop-1-yl]-
pyrrole-5-carboxaldehyde, 3-isopropyl-4-[2-(N-morpholino)propoxy]benzaldehyde,
3,5-diisopropyl-4-[2-(N-morpholino)propoxy]benzaldehyde, 3-(thiophen-2-yl)-4-
[2-
N-morpholino)propoxy]benzaldehyde.
C. 3-Aralkyl-2-Indolinone Derivatives
Another aspect of this invention is a combinatorial library of at least ten 3-
aralkyl-2-indolinone compounds that can be formed by condensing oxindoles of
structure 4 with aldehydes of structure 5 and then reducing the 3-position
double
bond of the resultant 3-arylidene-2-oxindole. In particular, the condensation
refers
to reaction "A" in Scheme II. Compound 4 is the "3-arylidene-2-oxindole"
referred
to above.
"Reducing," as used herein, refers to the addition of hydaogen across the
double bond at the 3-position of compound 6



CA 02368041 2001-09-24
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53
Rzn_ i B~ i Rz2J
P
Rm i 20
Rig ~ Rz» ~M CHO
\ N - C /T~ ~Vw
R i s ~ H Rzz p i Rza
R» Rz3
i z~
Rzo~A~B~ D
R' 6
mZ3
Ri;
Rza
~O
R~s \ H
II
R~~
SCHEME II
(Reaction "B") to give compound II in Scheme II.
The oxindole in the above combinatorial library is preferrably selected from
the group consisting of oxindole itself and substituted oxindoles such as,
without
limitation, 5-fluorooxindole, 6-fluorooxindole, 7-fluorooxindole, 6-
trifluoromethyloxindole, 5-chlorooxindole, 6-chlorooxindole, indole-4-
carboxylic
acid, 5-bromooxindole, 6-(acetamido)- oxindole, 4-methyloxindole, 5-
methyloxindole, 4-methyl-5-chlorooxindole, 5-ethyloxindole, 6-hydroxyoxindole,
G-
(cyclopentylcarboxamido)oxindole, 5-acetyloxindole, oxindole-S-carboxylic
acid, S-
methoxyoxindole, 6-methoxyoxindole, 5-aminooxindole, 6-aminooxindole, 4-[2-(N-
morpholino)ethyl]- oxindole, 7-azaoxindole, oxindole-4-carabamic acid t-butyl
ester,
oxindole-6-carbamic acid t-butyl ester, 4-(2-carboxyethyl)oxindole, 4-n-
butyloxindole, 4,5-dimethoxyoxindole, 6-(methanesulfonamido)oxindole, 6-
1 S (benzamido)oxindole, 5-ethoxyoxindole, 6-phenyloxindole, 4-(2-hydroxyeth-1-




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54
yl)oxindole, 6-(2-methoxyphen-1-yl)oxindole, 6-(3-methoxyphen-1-yl)oxindole
and
6-(4-methoxyphen-1-yl)oxindole.
The aldehyde in the above combinatorial library is preferably selected from
the group consisting of, without limitation, benzaldehyde itself as well as 3-
isopropyl-p-anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,5-diisopropyl-
p-
anisaldehyde, 3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-anisaldehyde, 3-
phenyl-p-anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-
dichlorobenzaldehyde, 2-hydroxy-S-chlorobenzaldehyde, 2-hydroxy-4-methoxy-5-
(4-methoxyphenyl)benzaldehyde, 3-cyclopentyl-4-methoxybenzaldehyde, 3-
cyclopentyl-4-hydroxybenzaldehyde, 3-(2-thienyl)-4-methoxybenzaldehyde, 2-
hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-S-
bromobenzaldehyde, 2-methylpyridine-6-carboxaldehyde, 3-tert-butyl-4-hydroxy-5-

bromobenzaldehy de, 3-bromobenzaldehyde, 3,5-diisopropyl-4-[2-(N-
morpholino)ethyl]benzaldehyde,indole-5-carboxaldehyde, 2-hydroxy-3-
fluorobenzaldehyde, 3-cyclohexyl-4-[2-(N-morpholino)ethyl]benzaldehyde, 3-(3-
thienyl)-4-methoxybenzaldehyde, 2-methyl-5-isopropyl-4-methoxy- benzaldehyde,
2,3-dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde,
3-(thiophen-3-yl)-4-methoxybenzaldehyde, 3-(3-acetylaminophenyl)-4-methoxy-
benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2-(3-methoxyphenyl)-4,5-
dimethoxybenzaldehyde, 3-(pyrid-3-yl)-p-anisaldehyde, 2-(3-ezi~.oxyphenyl)-4,5-

dimethoxybenzaldehyde, 3-isopropyl-4-[2-(N-morpholino)ethoxy]benzaldehyde,
3,S-diisopropyl-4-[2-(N-morpholino)ethoxy]benzaldehyde, 2-(2-methoxyphenyl)-
4,5-dimethoxybenzaldehyde, 3-(3-ethoxyphenyl)-p-anisaldehyde, 3-(thiophen-2-
yl)-
4-[2-(N-morpholino)ethoxy]- benzaldehyde, 2-(thiophen-2-yl)-4,5-
dimethoxybenzaldehyde, 2,4-dimethyl-3-[3-dimethylaminoprop-1-yl]-pyrrole-5-
carboxaldehyde, 2,4-dimethyl-3-[3-carboxyprop-1-yl]-pyrrole-5-carboxaldehyde,
2,4-dimethyl-3-[3-(N-morpholino)prop-1-yl]-pyrrole-5-carboxaldehyde, 2,4-
dimethyl-3-[2-dimethylaminoethyl]-pyrrole-5-carboxaldehyde, 2,4-dimethyl-3-[2-
carboxyethyl]-pyrrole-5-carboxaldehyde and 2,4-dimethyl-3-[2-(N-
morpholino)ethyl]prop-1-yl]-pyrrole-5-carboxaldehyde.



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Another aspect of this invention provides a method for the synthesis of 3-
aralkyl-2-indolinone of formula II comprising condensing an oxindole of
formula 4
with an aldehyde of formula 5 in a solvent, preferably in the presence of a
base,
optionally isolating the resultant 3-arylidene-2-oxindole and then reducing
the 3-
5 arylidene-2-oxindole.
Examples of the oxindoles of formula 4 that may be condensed with an
aldehyde of formula 5 and the product reduced to give the 3-aralkyl-2-
indolinones of
formula II are oxindole itself and substituted oxindoles such as, without
limitation,
5-fluorooxindole, 6-fluorooxindole, 7-fluorooxindole, 6-
trifluoromethyloxindole, S-
10 chlorooxindole, 6-chlorooxindole, indole-4-carboxylic acid, 5-
bromooxindole, 6-
(acetamido)- oxindole, 4-methyloxindole, 5-methyloxindole, 4-methyl-5-
chlorooxindole, 5-ethyloxindole, 6-hydroxyoxindole, G-
(cyclopentylcarboxamido)oxindole, 5-acetyloxindole, oxindole-S-carboxylic
acid, 5-
methoxyoxindole, G-methoxyoxindole, 5-aminooxindole, 6-aminooxindole, 4-[2-(N-
15 morpholino)ethyl]- oxindole, 7-azaoxindole, oxindole-4-carabamic acid t-
butyl ester,
oxindole-G-carbamic acid t-butyl ester, 4-(2-carboxyethyl)oxindole, 4-n-
butyloxindole, 4,5-dimethoxyoxindole, G-(methanesulfonamido)oxindole, 6-
(benzamido)oxindole, S-ethoxyoxindole, 6-phenyloxindole, 4-(2-hydroxyeth-1-
yl)oxindole, 6-(2-methoxyphen-1-yl)oxindole, 6-(3-methoxyphen-1-yl)oxindole
and
20 6-(4-methoxyphen-1-yl)oxindole.
Examples of aldehydes of structure 3 which may be condensed with
oxindoles of structure 4 and the product reduced to give a compound of this
invention are, without limitation, benzaldehyde itself as well as 3-isopropyl-
p-
anisaldehyde, 2-methyl-5-isopropyl-p-anisaldehyde, 3,5-diisopropyl-p-
anisaldehyde,
25 3-cyclopentyl-p-anisaldehyde, 3-cyclohexyl-p-anisaldehyde, 3-phenyl-p-
anisaldehyde, 3,5-dimethyl-p-anisaldehyde, 2-hydroxy-3,5-dichlorobenzaldehyde,
2-
hydroxy-5-chlorobenzaldehyde, 2-hydroxy-4-methoxy-5-(4-
methoxyphenyl)benzaldehyde, 3-cyclopentyl-4-methoxybenzaldehyde, 3-
cyclopentyl-4-hydroxybenzaldehyde, 3-(2-thienyl)-4-methoxybenzaldehyde, 2-
30 hydroxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-5-
bromobenzaldehyde, 2-methylpyridine-G-carboxaldehyde, 3-tert-butyl-4-hydroxy-5-




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56
bromobenzaldehyde, 3-bromobenzaldehyde, 3,5-diisopropyl-4-[2-(N-
morpholino)ethyl]benzaldehyde,indole-5-carboxaldehyde, 2-hydroxy-3-
fluorobenzaldehyde, 3-cyclohexyl-4-[2-(N-morpholino)ethyl]benzaldehyde, 3-(3-
thienyl)-4-methoxybenzaldehyde, 2-methyl-5-isopropyl-4-methoxy- benzaldehyde,
2,3-dihydrobenzofuran-5-carboxaldehyde, 2,2-dimethylchroman-6-carboxaldehyde,
3-(thiophen-3-yl)-4-methoxybenzaldehyde, 3-(3-acetylaminophenyl)-4-methoxy-
benzaldehyde, 2-naphthyl-4,5-dimethoxybenzaldehyde, 2-(3-methoxyphenyl)-4,5-
dimethoxybenzaldehyde, 3-(pyrid-3-yl)-p-anisaldehyde, 2-(3-ethoxyphenyl)-4,5-
dimethoxybenzaldehyde, 3-isopropyl-4-[2-(N-morpholino)ethoxy]benzaldehyde,
3,5-diisopropyl-4-[2-(N-morpholino)ethoxy]benzaldehyde, 2-(2-methoxyphenyl)-
4,5-dimethoxybenzaldehyde, 3-(3-ethoxyphenyl)-p-anisaldehyde, 3-(thiophen-2-
yl)-
4-[2-(N-morpholino)ethoxy]- benzaldehyde, 2-(thiophen-2-yl)-4,5-
dimethoxybenzaldehyde, 2,4-dimethyl-3-[3-dimethylaminoprop-1-yl]-pyrrole-5-
carboxaldehyde, 2,4-dimethyl-3-[3-carboxyprop-1-yl]-pyrrole-S-carboxaldehyde,
2,4-dimethyl-3-[3-(N-morpholino)prop-1-yl]-pyrrole-5-carboxaldehyde, 2,4-
dimethyl-3-[2-dimethylaminoethyl]-pyrrole-5-carboxaldehyde, 2,4-dimethyl-3-[2-
carboxyethyl]-pyrrole-5-carboxaldehyde and 2,4-dimethyl-3-[2-(N-
morpholino)ethyl]prop-1-yl]-pyrrole-5-carboxaldehyde.
D. Diaryl Indolinone Compounds
In another aspect, the invention provides a combinatorial library of at least
10 indolinone compounds that can be formed by reacting an oxindole with a
ketone,
where the oxindole has the following structure
R2s
R2~ ~ CHZ
~O
R3o ~ _ H
R3 i
and the ketone has the following structure



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57
R32 R33
Rz~ I
N R3a
O H
and R2,, R,B, RZ~, R,o, R3" R3" R33, and R34 are as described herin.
The oxindole in the above combinatorial library is preferrably selected from
the group consisting of 1,3-dihydro-indol-2-one, 4-methyl-1,3-dihydro-indol-2-
one,
4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one, 5-chloro-1,3-dihydro-indol-2-one,
5-
bromo-1,3-dihydro-indol-2-one, 5-methoxy-1,3-dihydro-indol-2-one, 2-oxo-2,3-
dihydro-1H-indole-S-carboxylic acid, 2-oxo-2,3-dihydro-1H-indole-5-sulfonic
acid
dimethylamide, N-(2-oxo-2,3-dihydro-1H-indole-5-yl)-acetamide, 6-chloro-1,3-
dihydro-indol-2-one, 6-methoxy-1,3-dihydro-indol-2-one, and N-(S-methyl-2-oxo-
2,3-dihydro-1H-indole-6-yl)-acetamide.
The ketone is preferably selected from the group consisting of
OH
O
R2z ~N~ Rzz ~N~ R2z ~N~ R2~ ~N~
O H O H O H O H
> > >
\N' NHz OH \N ~
O O
R2~ ~N~ Rzz I N~ R2z I N~ R2z ~N~
O H O H O H O H
NHz N
O O
OH
Rzz I N~ Rzz ~N~ R2z ~N~
O H O H O H



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58
O
N~ N o OH
Rz~ ~N~ R2~ ~N~ Rz~ ~N~
O H O H O H
> >
OH
O
N/ N
~O
R2~ ~N~ R2~ ~N~ R2~ ~N~
O H O H O H
> >
,_
NHz N N
O
Rz~ /Nl R1 /Nl Rz~ /N/
O H , ~ H , and O H
Another aspect of the invention provides for a method for synthesizing a
compound of formula III, as described herein, comprising the step of reacting
a first
reactant with a second reactant in a solvent and in the presence of a base at
elevated
temperatures, where the first reactant is an oxindole having the following
structure
Rzs
Rz~ ~ CHz
/ ~O
R3o ~ _ H
R3~
and the second reactant is a ketone having the following structure
R32 R33
R2~
N R3a
O H
and Rz~, R28, Rz~, R3~, R3" R3z, R3j, and R34 are as described herin.
The first reactant is preferrably an oxindole selected from the group
consisting of 1,3-dihydro-indol-2-one, 4-methyl-1,3-dihydro-indol-2-one, 4-(2-



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59
hydroxy-ethyl)-1,3-dihydro-indol-2-one, 5-chloro-1,3-dihydro-indol-2-one, 5-
bromo-1,3-dihydro-indol-2-one, 5-methoxy-1,3-dihydro-indol-2-one, 5-carboxy-
1,3-
dihydro-indol-2-one, 5-dimethylsulfonamido-1,3-dihydro-indol-2-one, 5-
formamido-1,3-dihydro-indol-2-one, 6-chloro-1,3-dihydro-indol-2-one, 6-methoxy-

1,3-dihydro-indol-2-one, and 5-methyl-6-formamido-1,3-dihydro-indol-2-one.
The second reactant is preferrably a ketone selected from the group
Rz~ I ~ Rz7 I ~ Rzz I
N N N
consisting of O H O H O H
> > >
OH N' NHz OH
O O O
Rz~ I N~ Rz~ I N~ Rz~ I Nh Rz~ I N
O H O H O H O H
\N ~ NHz N
O O
'OH
R I ~ R I ~ R,z I ~ Rzz
z~ N z~ N ' N N
O H O H O H O H
O
N/ N ' OH
~o
R2~ I ~ Rz~ l, ~ Rzz
N N N
O H ~ O H ~ O H
OH
O
N/ N
~O
R2~ I Nl Rz~ I Nl Rz~ I Nl
O H O H O H
> >



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NHz N N
O
R2~ /Nl Ri /Nl Rz~ /N/
p H , ~ ~ , arid p H
E. 4-Substituted Indolinone Compounds
5 In another aspect, the invention provides a combinatorial library of at
least
10 indolinone compounds that can be formed by reacting an oxindole with an
aldehyde, where the oxindole has a structure set forth in formula I, as
defined herein,
and where the aldehyde has the formula
Z-C(O)-H
10 where Z is as defined herein.
The oxindole in the above combinatorial library is preferrably selected from
the group consisting of 4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-
one,
4-[2-(2-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(biphenyl-3-
yloxy)-ethyl]-1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-
one,
15 phenyl-thiocarbamic acid O-[2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl]
ester,
phenyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, tert-
butyl-
carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, cyclohexyl-
carbamic
acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, benzene sulfonyl-
carbamic
acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, biphenyl-2-yl-carbamic
acid 2-
20 (2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, ethyl-carbamic acid 2-(2-oxo-
2,3-
dihydro-1H-indol-4-yl)-ethyl ester, 4-[2-(4-methoxy-phenoxy)-ethyl]-1,3-
dihydro-
indol-2-one, 4-(2-methoxy-ethyl)-1,3-dihydro-indol-2-one, 4-(2-ethoxy-ethyl)-
1,3-
dihydro-indol-2-one, 4-[2-(4-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-
one, 4-
[2-(5-chloro-pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one, and isopropyl-
25 carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester.



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61
The aldehyde is preferably selected from the group consisting of
~N~ O.CH, F /-O O.CH,
O
\ \ \ I ~ ~ I \ \ \ \ \
\ O I/i ~ H,C,O I'i F Ho I/~ CI I
° O O o O O CH' O
7 7 7 7 7 7 7 7
Br F
\ CI \ H~C~N~CFh
A O\ CHI O - O ~ O - F I i I ~ O
HO~ \ I NCH \ / I \ \ / N F \ / -N
O a ~OH O ~ ~ O o
7 7 7 7 7 7 7 7
H,C~N~CH, / \ OH CI (,O O O ,O
\ ~ I ~ O- . I \ ~ N- F
HO I i \ I N ~ N / \ N\% ~ \ ~ N ~ I I .0
/ \ ~ N~ O F F
o c H, a
7 7 7 7 7 7 7
CI O F O
/ \ / \ - F
O Ct O' ~s O Ha~
CI ~ I I ~ I N I I ~ N \ / F F O / \ O ~C.N / OH C ~ /N\ CHI
CH, O CH, O CI I Cue, CH3
7 7 7 7 7 7
O
CH,
O O / I H,C \ CHo HaC \ \ CH,
~N \ CH, O~ / ~ \ N \
HaC.OH,C CH, ° 0 O \ / O \ /
7 7 7 7 7
O O~ / I \ p~ i \ O' / I \ O' / \
H~C~N \ CH ~ N _ \ I N \ N \ I N Br
3
O ~ / F ~ / F ~ /
7 7 7 7 9
I
/ _ O
c~ / I \ o~ ~ I \ I \ / \
\ N \ N ~ N
CH, N
o v , o \ , cH, o ~ / H H3cJ
7 7 7 7 7
0
o I
o I o O. / I \ / \
I O / \ I \ N \ ( N
o / I \ I / \ I N ~ I \
/
/ I \ N \ I N / N / I \ I
N \I
i
CH \ I O~ ~ H,C, I i CH O'CH
a7 7 7 F 7 O 7 3 7 a7
° O O
-\,N ° p \ ~ cH, o ° ° o
H'C ~ J H C I \ O,CH, I \ O,CH '\ OH I i
°~ ~N~ O~ ~ \ CH, S ~ / ~ / I I ~ I i O,~H
CH, N \ +
IO CHn CH, O,CH7 g O,CH' g O,CH o,N,,
7 7 7 7 7 7
O Nw F F F
I \ O,CH, OH ( ~ CH,
I I
\O
\ I i O,CH Br CI i N N ~ ~ \ \
O.CH ~ ~ I / ~ / i
O o o ~o N
7 7 7 7 7 7 7



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62
.cH, o O
O O HsC H C OH H C OH
~ ~O ~ ~ \O ~\ ~ 0~3 / ~ 0~3 /
O N N CH3 N oH~ CH,
N N CI CH, H H H
> > > > > ,
HaC O O 0' i I \ O / O.CH3
N OH 9 I ~ N O
\ i ~ \ ~ Ha0 ~/
I ~ i ~ O~ /
0 H,CJ CH, H,C.S i O.CH ' N CHa
, ~ ' ~ O a H ,
O
CH, I p HO
HC C
HOC N I ~ C~ ~ ~ ~ Br ~ N ~ OH ~ OH
OH3 ~ ~ , ~~ ~~ ~O ~ , H,C N
0~ I ~ HO HO HO' Y HO' Y ~OH H
N OHa ii
H O O O O 0 CI
> > > > > > >
O
O \ / NCH, HO I w w
HO , and ' ' .
Another :~ 5pect of the invention provides for a method for synthesizing a
compound of formula IV or formula V, as described herein. The method of
synthesizing a compound of formula IV, comprises the step of reacting a first
reactant with a second reactant in a solvent and in the presence of a mixture
of other
reagents. The reaction may take place at ambient temperatures or at elevated
temperatures.
The first reactant is preferably an oxindole, more preferably 4-(2-
hydroxyethyl)-1,3-dihydro-indol-2-one. The second reactant i:. preferably
selected
from the group consisting of an alcohol, an iodoalkyl, an alkyl or aryl
isocyanate,
and an alkyl or aryl isothiocyanate, and more preferably is selected from the
group
1 S consisting of 3-isopropylphenol, 2-isopropylphenol, 3-phenylphenol, 4-
methoxyphenol, 5-chloro-3-pyridinol, methyl iodide, ethyl iodide, phenyl
isocyanate, tent-butyl isocyanate, cyclohexyl isocyanate, benzenesulfonyl
isocyanate,
2-biphenylyl isocyanate, ethyl isocyanate, isopropyl isocyanate, and phenyl
isothiocyanate.
The "mixture of other reagents" generally refers to a mixture of synthetic
reagents or solvents that would facilitate the synthesis of the compounds of
the
invention, and may include a mixture of diethyl azodicarboxylate with
triphenylphosphine in a solvent, or silver trifluoromethanesulfonate, by
itself or with



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63
a base, in a solvent, or a mixture of solvents.
The method of synthesizing a compound of formula V, comprises the step of
reacting a first reactant with a second reactant in a solvent and in the
presence of a
base at elevated temperatures, where the first reactant is an oxindole having
a
structure set
forth in formula I, as defined herein, and the second reactant is an aldehyde,
having
the formula
Z-C(O)-H
where Z is as defined herein.
The first reactant is preferrably an oxindole selected from the group
consisting of 4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-
(2-
isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-(biphenyl-3-yloxy)-
ethyl]-
1,3-dihydro-indol-2-one, 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one, phenyl-
thiocarbamic acid O-[2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl] ester, phenyl-
carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, tent-butyl-
carbamic
acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, cyclohexyl-carbamic acid
2-
(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, benzene sulfonyl-carbamic acid
2-(2-
oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester, biphenyl-2-yl-carbamic acid 2-(2-
oxo-
2,3-dihydro-1H-indol-4-yl)-ethyl ester, ethyl-carbamic acid 2-(2-oxo-2,3-
dihydro-
1H-indol-4-yl)-ethyl ester, 4-[2-(4-methoxy-phenoxy)-ethyl]-1,3-dihydro-indol-
2-
one, 4-(2-methoxy-ethyl)-1,3-dihydro-indol-2-one, 4-(2-ethoxy-ethyl)-1,3-
dihydro-
indol-2-one, 4-[2-(4-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one, 4-[2-
(5-
chloro-pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one, and isopropyl-carbamic
acid 2-(2-oxo-2,3-dihydro-IH-indol-4-yl)-ethyl ester.
The second reactant is preferrably an aldehyde selected from the group
consisting of 4-bromobenzaldehyde, 2-pyridinecarboxaldehyde, 6-methyl-2-
pyridine-carbaldehyde, 1H-indole-5-carbaldehyde, 4-methoxy-3-thiophen-2-yl-
benzaldehyde, 4-(3-dimethylamino-propyl)-3,5-dimethyl-IH pyrrole-2-
carbaldehyde, 3-hydroxy-6-methyl-pyridine-2-carbaldehyde, 4-(3-dimethylamino-
propyl)-3,5-dimethyl-IH-pyrrole-2-carbaldehyde, and 4-carboxyethyl-3,5-
dimethyl-
2-formylpyrrole.



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64
IV. BIOLOGICAL ACTIVITY AND PHARMACEUTICAL COMPOSITIONS
In another aspect, the invention features a pharmaceutical composition
comprising (i) a physiologically acceptable Garner, diluent, or excipient; and
(ii) a
compound as described herein.
The invention also features a method of modulating the function of a protein
kinase with a compound of the invention, comprising the step of contacting
cells
expressing the protein kinase with the compound.
A still further aspect of this invention is that the protein kinase whose
catalytic activity is being modulated by a compound of this invention is
selected
from the group consisting of receptor protein tyrosine kinases, cellular
tyrosine
kinases and serine-threonine kinases.
It is an aspect of this invention that the receptor protein kinase whose
catalytic activity is modulated by a compound of this invention is selected
from the
group consisting of EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRa,,
PDGFR~3, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-l, FGFR-1R, FGFR-
2R, FGFR-3R and FGFR-4R. In addition, it is an aspect of this invention that
the
cellular tyrosine kinase whose catalytic activity is modulated by a compound
of this
invention is selected from the group consisting of Src, Frk, Btk, Csk, Abl,
ZAP70,
Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. Another
aspect
of this invention is that the serine-threonine protein kinase whose catalytic
activity is
modulated by a compound of this invention is selected from the group
consisting of
CDK2 and Raf.
A protein kinase natural binding partner can bind to a protein kinase's
intracellular region with high affinity. High affinity represents an
equilibrium
binding constant on the order of 10-6 M or less. In addition, a natural
binding partner
can also transiently interact with a protein kinase intracellular region and
chemically
modify it. Protein kinase natural binding partners are chosen from a group
that
includes, but is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains,
other
phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange
factors,



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GS
protein phosphatases, and other protein kinases. Methods of determining
changes in
interactions between protein kinases and their natural binding partners are
readily
available in the art.
The compounds of the invention preferably modulate the activity of the
protein tyrosine kinase in vitro. These compounds preferably show positive
results
in one or more in vitro assays for an activity corresponding to treatment of
the
disease or disorder in question (such as the assays described in the Examples
below).
The invention also features a method of identifying compounds that
modulate the function of protein kinase, comprising the following steps: (a)
contacting cells expressing the protein tyrosine kinase with the compound; and
(b)
monitoring an effect upon the cells. The effect upon the cells is preferably a
change
or an absence of a change in cell phenotype, more preferably it is a change or
an
absence of a change in cell proliferation, even more preferably it is a change
or
1 S absence of a change in the catalytic activity of the protein kinase, and
most
preferably it is a change or absence of a change in the interaction between
the
protein kinase with a natural binding partner, as described herein.
In a preferred embodiment, the invention features a method for identifying
the compounds of the invention, comprising the following steps: (a) lysing the
cells
to render a lysate comprising protein tyrosine kinase; (b) adsorbing the
protein
tyrosine kinase to an antibody; (c)incubating the adsorbed protein tyrosine
kinase
with a substrate or substrates; and (d) adsorbing the substrate or substrates
to a solid
support or antibody; where the step of monitoring the effect on the cells
comprises
measuring the phosphate concentration of the substrate or substrates.
In yet another aspect, the invention features a method for treating a disease
related to unregulated kinase signal transduction, where the method includes
the step
of administering to a subject in need thereof a therapeutically effective
amount of a
compound of the invention as described herein.
The invention also features a method of regulating kinase signal transduction
comprising administering to a subject a therapeutically effective amount of a
compound of the invention as described herein.



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Furthermore, the invention features a method of preventing or treating an
abnormal condition in an organism, where the abnormal condition is associated
with
an aberration in a signal transduction pathway characterized by an interaction
between a protein kinase and a natural binding partner, where the method
comprises
the following steps: (a) administering a compound of the invention as
described
herein; and (b) promoting or disrupting the abnormal interaction. The organism
is
preferably a mammal and the abnormal condition is preferably cancer. The
abnormal condition may also preferably be selected from the group consisting
of
hypertension, depression, generalized anxiety disorder, phobias, post-
traumatic
stress syndrome, avoidant personality disorder, sexual dysfunction, eating
disorders,
obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer's
disease, obsessive-compulsive disorder, panic disorder, memory disorders,
Parkinson's disease, endocrine disorders, vasospasm, cerebellar ataxia, and
gastrointestinal tract disorders.
As used herein, "PK related disorder," "PK driven disorder," and "abnormal
PK activity" all refer to a condition characterized by inappropriate; i.e.,
under or,
more commonly, over, PK catalytic activity, where the particular PK can be an
RTK,
a CTK or an STK. Inappropriate catalytic activity can arise as the result of
either:
( 1 ) PK expression in cells which normally do not express PKs; (2) increased
PK
expression leading to unwanted cell proliferation, differentiatic:-~:~ and/or
growth; or,
(3) decreased PK expression leading to unwanted reductions in cell
proliferation,
differentiation and/or growth. Over-activity of a PK refers to either
amplification of
the gene encoding a particular PK or production of a level of PK activity
which can
correlate with a cell proliferation, differentiation and/or growth disorder
(that is, as
the level of the PK increases, the severity of one or more of the symptoms of
the
cellular disorder increases). Under-activity is, of course, the converse,
wherein the
severity of one or more symptoms of a cellular disorder increase as the level
of the
PK activity decreases.
The term "therapeutically effective amount" as used herein refers to that
amount of the compound being administered which will relieve to some extent
one
or more of the symptoms of the disorder being treated. In reference to the
treatment



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67
of cancer, a therapeutically effective amount refers to that amount which has
the
effect of (1) reducing the size of the tumor; (2) inhibiting (that is, slowing
to some
extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent
(that is,
slowing to some extent, preferably stopping) tumor growth; and/or, (4)
relieving to
some extent (or, preferably, eliminating) one or more symptoms associated with
the
cancer.
It is an aspect of this invention that the above-referenced protein kinase
related disorder is selected from the group consisting of a receptor protein
tyrosine
kinase related disorder, a cellular tyrosine kinase disorder and a serine-
threonine
kinase related disorder.
In yet another aspect of this invention, the above referenced protein kinase
related disorder is selected from the group consisting of an EGFR related
disorder, a
PDGFR related disorder, an IGFR related disorder and a flk related disorder.
The above referenced protein kinase related disorder is a cancer selected
from the group consisting of squamous cell carcinoma, astrocytoma,
glioblastoma,
lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer,
prostate cancer, breast cancer, small-cell lung cancer and glioma in a further
aspect
of this invention.
The above referenced protein kinase related disorder is selected from the
group consisting of diabetes, immunlogical disorders such as autoimmune
disorder,
a hyperproliferation disorder, restinosis, fibrosis, psoriasis,
osteoarthritis, rheumatoid
arthritis, an inflammatory disorder, angiogenesis, cardiovascular disease such
as
aetherosclerosis, and renal disease, in yet another aspect of this invention.
In addition to modulating PK activity, the compounds of this invention may
inhibit the activity of protein phosphatases which are enzymes which remove
phosphate groups from phosphorylated proteins. Thus the compounds disclosed
herein may also represent a new generation of therapeutic compounds for
diseases
and disorders associated with abnormal phosphatase activity (such as, without
limitation, diabetes, cell proliferation disorders and inflammatory
disorders). The
terms defined herein with respect to PKs would be understood by one skilled in
the
art to have the same or similar meaning with regard to phosphastases.



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The summary of the invention described above is non-limiting and other
features and advantages of the invention will be apparent from the following
description of the preferred embodiments, and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compounds capable of regulating and/or
modulating cellular signal transduction and, in preferred embodiments,
receptor and
non-receptor tyrosine kinase signal transduction.
Receptor kinase mediated signal transduction is initiated by extracellular
interaction with a specific growth factor (ligand), followed by receptor
dimerization,
transient stimulation of the intrinsic protein kinase activity and
phosphorylation.
Binding sites are thereby created for intracellular signal transduction
molecules and
lead to the formation of complexes with a spectrum of cytoplasmic signaling
molecules that facilitate the appropriate cellular response (e.g., cell
division,
metabolic effects to the extracellular microenvironment). See, Schlessinger
and
Ullrich, 1992, Neuron 9:303-391.
It has been shown that tyrosine phosphorylation sites in growth factor
receptors function as high-affinity binding sites for SH2 (SYC homology)
domains of
signaling molecules. Fantl et al., 1992, Cell 69:413-423; Songyang et al.,
1994,
Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and
Koch
et al., 1991, Science 252:668-678. Several intracellular substrate proteins
that
associate with receptor kinases have been identified. They may be divided into
two
principal groups: (1) substrates which have a catalytic domain; and (2)
substrates
which lack such domain but serve as adapters and associate with catalytically
active
molecules. Songyang et al., 1993, Cell 72:767-778. The specificity of the
interactions between receptors and SH2 domains of their substrates is
determined by
the amino acid residues immediately surrounding the phosphorylated tyrosine
residue. Differences in the binding affinities between SH2 domains and the
amino
acid sequences surrounding the phosphotyrosine residues on particular
receptors are
consistent with the observed differences in their substrate phosphorylation
profiles.
Songyang et al., 1993, Cell 72:767-778. These observations suggest that the



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69
function of each receptor kinase is determined not only by its pattern of
expression
and ligand availability but also by the array of downstream signal
transduction
pathways that are activated by a particular receptor. Thus, phosphorylation
provides
an important regulatory step which determines the selectivity of signaling
pathways
recruited by specific growth factor receptors, as well as differentiation
factor
receptors.
Kinase signal transduction results in, among other responses, cell
proliferation, differentiation and metabolism. Abnormal cell proliferation may
result
in a wide array of disorders and diseases, including the development of
neoplasia
such as carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis,
arteriosclerosis, arthritis and diabetic retinopathy (or other disorders
related to
uncontrolled angiogenesis and/or vasculogenesis).
This invention is therefore directed to compounds which regulate, modulate
and/or inhibit kinase signal transduction by affecting the enzymatic activity
of the
receptor kinases (RKs) and/or the non-receptor kinases and interfering with
the
signal transduced by such proteins. More particularly, the present invention
is
directed to compounds which regulate, modulate and/or inhibit the RK and/or
non-
receptor kinase mediated signal transduction pathways as a therapeutic
approach to
cure many kinds of solid tumors, including but not limited to carcinoma,
sarcoma,
erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and
myoblastoma. Indications may include, but are not limited to brain cancers,
bladder
cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers,
blood
cancers, lung cancers and bone cancers.
I. Target Diseases to be Treated by the Compounds of the Invention.
The compounds described herein are useful for treating disorders related to
unregulated kinase signal transduction, including cell proliferative
disorders, fibrotic
disorders and metabolic disorders.
Cell proliferative disorders which can be treated or further studied by the
present invention include cancers, blood vessel proliferative disorders and
mesangial
cell proliferative disorders.



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Blood vessel proliferative disorders refer to angiogenic and vasculogenic
disorders generally resulting in abnormal proliferation of blood vessels. The
formation and spreading of blood vessels, or vasculogenesis and angiogenesis,
respectively, play important roles in a variety of physiological processes
such as
5 embryonic development, corpus luteum formation, wound healing and organ
regeneration. They also play a pivotal role in cancer development. Other
examples
of blood vessel proliferation disorders include arthritis, where new capillary
blood
vessels invade the joint and destroy cartilage, and ocular diseases, like
diabetic
retinopathy, where new capillaries in the retina invade the vitreous, bleed
and cause
10 blindness. Conversely, disorders related to the shrinkage, contraction or
closing of
blood vessels, such as restenosis, are also implicated.
Fibrotic disorders refer to the abnormal formation of extracellular matrix.
Examples of fi.brotic disorders include hepatic cirrhosis and mesangial cell
proliferative disorders. Hepatic cirrhosis is characterized by the increase in
15 extracellular matrix constituents resulting in the formation of a hepatic
scar. Hepatic
cirrhosis can cause diseases such as cirrhosis of the liver. An increased
extracellular
matrix resulting in a hepatic scar can also be caused by viral infection such
as
hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Other
fibrotic
disorders implicated include atherosclerosis (see, below).
20 Mesangial cell proliferative disorders refer to disorders :~rought about by
abnormal proliferation of mesangial cells. Mesangial proliferative disorders
include
various human renal diseases, such as glomerulonephritis, diabetic
nephropathy,
malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant
rejection, and glomerulopathies. The PDGF-R has been implicated in the
25 maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney
International 43:47S-54S.
PKs have been associated with such cell proliferative disorders. For
example, some members of the receptor tyrosine kinase family have been
associated
with the development of cancer. Some of these receptors, like the EGFR (Tuzi
et
30 al., 1991, Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719)
HER2/neu (Slamon et al., 1989, Science 244:707-712) and the PDGF-R (Kumabe et



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71
al., 1992, Oncogene 7:627-633) are overexpressed in many tumors and/or
persistently activated by autocrine loops. In fact, in the most common and
severe
cancers these receptor overexpressions (Akbasak and Suner-Akbasak et al.,
1992, J.
Neurol. Sci. 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-
273;
Korc et al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine loops (Lee and
Donoghue, 1992, J. Cell. Biol. 118:1057-1070; Korc et al., supra; Akbasak and
Suner-Akbasak et al., supra) have been demonstrated. For example, the EGFR
receptor has been associated with squamous cell carcinoma, astrocytoma,
glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has
been associated with breast, ovarian, gastric, lung, pancreas and bladder
cancer. The
PDGF-R has been associated with glioblastoma, lung, ovarian, melanoma and
prostate cancer. The RK c-met has been generally associated with
hepatocarcinogenesis and thus hepatocellular carcinoma. Additionally, c-met
has
been linked to malignant tumor formation. More specifically, the RK c-met has
been associated with, among other cancers, colorectal, thyroid, pancreatic and
gastric
carcinoma, leukemia and lymphoma. Additionally, over-expression of the c-met
gene has been detected in patients with Hodgkin's disease, Burkitt's disease,
and the
lymphoma cell line. Flk has likewise been associated with a broad spectrum of
tumors including, without limitation, mammary, ovarian and lung tumors as well
as
gliomas such as glioblastoma.
The IGF-IR, in addition to being implicated in nutritional support and in
type-II diabetes, has also been associated with several types of cancers. For
example, IGF-I has been implicated as an autocrine growth stimulator for
several
tumor types, e.g., human breast cancer carcinoma cells (Arteaga et al., 1989,
J. Clin.
Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990,
Cancer
Res. 50:2511-2517). In addition, IGF-I, integrally involved in the normal
growth
and differentiation of the nervous system, appears to be an autocrine
stimulator of
human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The
importance of the IGF-IR and its ligands in cell proliferation is further
supported by
the fact that many cell types in culture (fibroblasts, epithelial cells,
smooth muscle
cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells
of the



CA 02368041 2001-09-24
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72
bone marrow) are stimulated to grow by IGF-I. Goldring and Goldring, 1991,
Eukaryotic Gene Expression 1:301-32G. In a series of recent publications,
Baserga
even suggests that IGF-I-R plays a central role in the mechanisms of
transformation
and, as such, could be a preferred target for therapeutic interventions for a
broad
spectrum of human malignancies. Baserga, 1995, Cancer Res. 55:249-252;
Baserga,
1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol. 14:4588-4595.
Some protein kinases (PKs) have been implicated in many types of cancer
including, notably, breast cancer (Cance, et al., Int. J. Cancer, 54:571-77
(1993)).
The association between abnormalities in RKs and disease are not restricted
to cancer, however. For example, RKs have been associated with metabolic
diseases
like psoriasis, diabetes mellitus, wound healing, inflammation, and
neurodegenerative diseases. These diseases include, but are not limited to
hypertension, depression, generalized anxiety disorder, phobias, post-
traumatic
stress syndrome, avoidant personality disorder, sexual dysfunction, eating
disorders,
obesity, chemical dependencies, cluster headache, migraine, pain, Alzheimer's
disease, obsessive-compulsive disorder, panic disorder, memory disorders,
Parkinson's disease, endocrine disorders, vasospasm, cerebellar ataxia, and
gastrointestinal tract disorders. For example, the EGF-R is indicated in
corneal and
dermal wound healing. Defects in the Insulin-R and the IGF-1R are indicated in
type-II diabetes mellitus. A more complete correlation between specific RKs
and
their therapeutic indications is set forth in Plowman et al., 1994, DN&P 7:334-
339.
Not only receptor type kinases, but also many cellular kinases (CKs)
including src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by
Bolen et al.,
1992, FASEB J. 6:3403-3409) are involved in the proliferative and metabolic
signal
transduction pathway and thus in indications of the present invention. For
example,
mutated src (v-src) has been demonstrated as an oncoprotein (pp60"-S'~) in
chicken.
Moreover, its cellular homolog, the proto-oncogene pp60'-S'~ transmits
oncogenic
signals of many receptors. For example, overexpression of EGF-R or HER2/neu in
tumors leads to the constitutive activation of pp60~-S", which is
characteristic for the
malignant cell but absent from the normal cell. On the other hand, mice
deficient for
the expression of c-src exhibit an osteopetrotic phenotype, indicating a key



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participation of c-src in osteoclast function and a possible involvement in
related
disorders. Similarly, Zap 70 is implicated in T-cell signaling.
Furthermore, the identification of CTK modulating compounds to augment
or even synergize with RK aimed Mockers is an aspect of the present invention.
Additionally, both RKs and non-receptor type kinases have been connected
to hyperimmune disorders.
Further, the compounds of the present invention are also effective in treating
diseases that are related to the PYK-2 protein. This protein, its cellular
function, and
diseases related to them are set forth in detail in U.S. Applications Serial
Number
08/357,642, filed December 15, 1994, by Lev et al., and entitled "PYK2 RELATED
PRODUCTS AND METHODS" (Lyon & Lyon Docket No. 209/070), and Serial
Number 08/460,626, filed June 2, 1995, by Lev et al., and entitled "PYKZ
RELATED PRODUCTS AND METHODS" (Lyon & Lyon Docket No. 211/121),
both of which are hereby incorporated by reference herein in their entirety,
including
any drawings.
Finally, the present invention is directed towards oxindole and indolinone
compounds and methods of modulating the functions of protein phosphatases, as
well as methods of preventing and treating protein phosphatase related
abnormal
conditions in organisms with a compound of the method identified above. The
compounds of the invention, as well as compounds obtained by adding chemical
substituents, may potently inhibit the action of phosphatases and may
represent a
new generation of therapeutics for diseases associated with defects in said
phosphatases. Terms defined above with respect to kinases have a similar
meaning
to one skilled in the art with respect to phosphatases. Both RKs and non-
receptor
type kinases have been connected to hyperimmune disorders.
The compounds of the present invention are also effective in treating diseases
that are related to the PYK-2 protein. This protein, its cellular function,
and diseases
related to them are set forth in detail in U.S. Patent Number 5,837,524,
issued
November 17, 1998, to Lev et al., and entitled "PYKZ RELATED PRODUCTS
AND METHODS," and U.S. Patent Number 5,837,815, issued November 17, 1998,
to Lev et al., and entitled "PYK2 RELATED PRODUCTS AND METHODS," both



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of which are hereby incorporated by reference herein in their entirety,
including any
drawings.
II. KDR/FLK-1
a. The KDR/FLK-1 Receptor and VEGF
Normal vasculogenesis and angiogenesis play important roles in a variety of
physiological processes such as embryonic development, wound healing, organ
regeneration and female reproductive processes such as follicle development in
the
corpus luteum during ovulation and placental growth after pregnancy. Folkman
and
Shing, 1992, J. Biological Chem. 267:10931-34. However, many diseases are
driven by persistent unregulated or inappropriate angiogenesis. For example,
in
arthritis, new capillary blood vessels invade the joint and destroy the
cartilage. In
diabetes, new capillaries in the retina invade the vitreous, bleed and cause
blindness.
Folkman, 1987, in: Congress of Thrombosis and Haemostasis (Verstraete, et. al,
eds.), Leuven University Press, Leuven, pp.583-596. Ocular neovascularization
is
the most common cause of blindness and dominates approximately twenty (20) eye
diseases.
Moreover, vasculogenesis and/or angiogenesis have been associated with the
growth of malignant solid tumors and metastasis. A tumor must continuously
stimulate the growth of new capillary blood vessels for the tumor itself to
grow.
Furthermore, the new blood vessels embedded in a tumor provide a gateway for
tumor cells to enter the circulation and to metastasize to distant sites in
the body.
Folkman, 1990, J. Natl. Cancer Inst. 82:4-6; Klagsbrunn and Soker, 1993,
Current
Biology 3:699-702; Folkman, 1991, J. Natl., Cancer Inst. 82:4-6; Weidner et
al.,
1991, New Engl. J. Med. 324:1-S.
Several polypeptides with in vitro endothelial cell growth promoting activity
have been identified. Examples include acidic and basic fibroblastic growth
factor
(aFGF, bFGF), vascular endothelial growth factor (VEGF) and placental growth
factor. Unlike aFGF and bFGF, VEGF has recently been reported to be an
endothelial cell specific mitogen. Ferrara and Henzel, 1989, Biochem. Biophys.
Res.
Comm. 161:851-858; Vaisman et al., 1990, J. Biol. Chem. 265:19461-19566.



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Thus, the identification of the specific receptors to which VEGF binds is an
important advancement in the understanding of the regulation of endothelial
cell
proliferation. Two structurally closely related RKs have been identified to
bind
VEGF with high affinity: the flt-1 receptor (Shibuya et al., 1990, Oncogene
5 5:519-524; De Vries et al., 1992, Science 255:989-991) and the KDR/FLK-1
receptor, discussed in the U.S. Patent Application No. 08/193,829.
Consequently, it
had been surmised that these RKs may have a role in the modulation and
regulation
of endothelial cell proliferation.
Evidence, such as the disclosure set forth in copending U.S. Application
10 Serial No. 08/193,829, strongly suggests that VEGF is not only responsible
for
endothelial cell proliferation, but also is a prime regulator of normal and
pathological angiogenesis. See generally, Klagsburn and Soker, 1993, Current
Biology 3:699-702; Houck et al., 1992, J. Biol. Chem. 267:26031-26037.
Moreover,
it has been shown that KDR/FLK-1 and flt-1 are abundantly expressed in the
15 proliferating endothelial cells of a growing tumor, but not in the
surrounding
quiescent endothelial cells. Plate et al., 1992, Nature 359:845-848; Shweiki
et al.,
1992, Nature 359:843-845.
b. Identification Of Agonists And Antagonists To The KDR/FLK-1
20 Receptor
In view of the deduced importance of RKs in the control, regulation and
modulation of endothelial cell proliferation and potentially vasculogenesis
and/or
angiogenesis, many attempts have been made to identify RK "inhibitors" using a
variety of approaches. These include the use of mutant ligands (U.S. Patent
No.
25 4,966,849); soluble receptors and antibodies (Application No. WO 94/10202;
Kendall and Thomas, 1994, Proc. Natl. Acad. Sci. USA 90:10705-10709; Kim et
al.,
1993, Nature 362:841-844); and RNA ligands (Jellinek et al., 1994,
Biochemistry
33:10450-10456).
Furthermore, kinase inhibitors (WO 94/03427; WO 92/21660; WO
30 91/15495; WO 94/14808; U.S. Patent No. 5,330,992; Mariani et al., 1994,
Proc. Am.
Assoc. Cancer Res. 35:2268), and inhibitors acting on receptor kinase signal



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transduction pathways, such as protein kinase C inhibitors have been
identified
(Schuchter et al., 1991, Cancer Res. 51:682-687); Takano et al., 1993, Mol.
Bio.
Cell 4:358A; Kinsella et al., 1992, Exp. Cell Res. 199:56-62; Wright et al.,
1992, J.
Cellular Phys. 152:448-57).
More recently, attempts have been made to identify small molecules which
act as kinase inhibitors for use in the treatment of cancer. Consequently,
there is an
unmet need for the identification and generation of effective small compounds
which
selectively inhibit the signal transduction of the KDR/FLK-1 receptor in order
to
effectively and specifically suppress vasculogenesis.
Consequently, there is an unmet need for the identification and generation of
effective small compounds which selectively inhibit the signal transduction of
the
KDR/FLK-1 receptor in order to effectively and specifically suppress
vasculogenesis.
Some of the compounds of the present invention demonstrate excellent
activity in biological assays and thus these compounds and related compounds
are
expected to be effective in treating Flk related disorders such as those
driven by
persistent unregulated or inappropriate angiogenesis.
III. Pharmaceutical Formulations And Routes Of Administration
The compounds described herein can be administered to a human patient per
se, or in pharmaceutical compositions where they are mixed with other active
ingredients, as in combination therapy, or suitable Garners or excipient(s).
Techniques for formulation and administration of the compounds of the instant
application may be found in "Remington's Pharmaceutical Sciences," Mack
Publishing Co., Easton, PA, latest edition.
a) Routes Of Administration
Suitable routes of administration may, for example, include oral, rectal,
transmucosal, or intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intravenous, intramedullary injections, as well
as
intrathecal, direct intraventricular, intraperitoneal, intranasal, or
intraocular



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inj ections.
Alternately, one may administer the compound in a local rather than systemic
manner, for example, via injection of the compound directly into a solid
tumor, often
in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system,
for example, in a liposome coated with tumor-specific antibody. The liposomes
will
be targeted to and taken up selectively by the tumor.
b) Composition/Formulation
The pharmaceutical compositions of the present invention may be
manufactured in a manner that is itself known, e.g., by means of conventional
mixing, dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one or more
physiologically acceptable Garners comprising excipients and auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Proper formulation is dependent upon the route of
administration
chosen.
For injection, the agents of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks's
solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation.
Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by
combining the active compounds with pharmaceutically acceptable Garners well
known in the art. Such Garners enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries,
suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained by mixing one or more
solid excipient with one or more compound of the invention, optionally
grinding the



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78
resulting mixture, and processing the mixture of granules, after adding
suitable
auxiliaries, if desired, to obtain tablets or dragee cores. Suitable
excipients are, in
particular, fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol;
cellulose preparations such as, for example, maize starch, wheat starch, rice
starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added,
such as
the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as
sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium
dioxide, lacqucr solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active compound
doses.
Pharmaceutical preparations which can be used orally include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of gelatin and
a
plasticizes, such as glycerol or sorbitol. The push-fit capsules can contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches, and/or
lubricants such as talc or magnesium stearate and, optionally, :.~abilizers.
In soft
capsules, the active compounds may be dissolved or suspended in suitable
liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition,
stabilizers may be added. All formulations for oral administration should be
in
dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present invention are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a



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79
pressurized aerosol the dosage unit may be determined by providing a valve to
deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in
an
inhaler or insufflator may be formulated containing a powder mix of the
compound
and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by
injection, e.g:, by bolus injection or continuous infusion. Formulations for
injection
may be presented in unit dosage form, e.g., in ampoules or in mufti-dose
containers,
with an added preservative. The compositions may take such forms as
suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions
of the active compounds may be prepared as appropriate oily injection
suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame
oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides, or
liposomes.
Aqueous injection suspensions may contain substances which increase the
viscosity
of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or
dextran.
Optionally, the suspension may also contain suitable stabilizers or agents
which
increase the solubility of the compounds to allow for the preparation of
highly
concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases
such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may
also be formulated as a depot preparation. Such long acting formulations may
be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with
suitable polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble derivatives,
for



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example, as a sparingly soluble salt.
A pharmaceutical Garner for the hydrophobic compounds of the invention is
a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-
miscible organic polymer, and an aqueous phase. The cosolvent system may be
the
5 VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of
the
nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene glycol 300,
made
up to volume in absolute ethanol. The VPD co-solvent system (VPD:DSW) consists
of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent
system
dissolves hydrophobic compounds well, and itself produces low toxicity upon
10 systemic administration. Naturally, the proportions of a co-solvent system
may be
varied considerably without destroying its solubility and toxicity
characteristics.
Furthermore, the identity of the co-solvent components may be varied: for
example,
other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80;
the
fraction size of polyethylene glycol may be varied; other biocompatible
polymers
15 may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or
polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds may be employed. Liposomes and emulsions are well known examples
of delivery vehicles or Garners for hydrophobic drugs. Certain organic
solvents such
20 as dimethylsulfoxide also may be employed, although usually at the cost of
greater
toxicity. Additionally, the compounds may be delivered using a sustained-
release
system, such as semipermeable matrices of solid hydrophobic polymers
containing
the therapeutic agent. Various sustained-release materials have been
established and
are well known by those skilled in the art. Sustained-release capsules may,
25 depending on their chemical nature, release the compounds for a few weeks
up to
over 100 days. Depending on the chemical nature and the biological stability
of the
therapeutic reagent, additional strategies for protein stabilization may be
employed.
Many of the PK modulating compounds of the invention may be provided as
salts with pharmaceutically compatible counterions. Pharmaceutically
compatible
30 salts may be formed with many acids, including but not limited to
hydrochloric,
sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be
more soluble in



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81
aqueous or other protonic solvents than are the corresponding free base forms.
c) Effective Dosaee.
Pharmaceutical compositions suitable for use in the present invention include
compositions where the active ingredients are contained in an amount effective
to
achieve its intended purpose. More specifically, a therapeutically effective
amount
means an amount of compound effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the
capability of
those skilled in the art, especially in light of the detailed disclosure
provided herein.
For any compound used in the methods of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays. For
example, a
dose can be formulated in animal models to achieve a circulating concentration
range that includes the ICS° as determined in cell culture (i.e., the
concentration of
the test compound which achieves a half maximal inhibition of the PK
activity).
Such information can be used to more accurately determine useful doses in
humans.
Toxicity and therapeutic efficacy of the compounds described herein can be
determined by standard pharmaceutical procedures in cell cultures or
experimental
animals, e.g., for determining the LDS° (the dose lethal to 50% of the
population) and
the EDSO (the dose therapeutically effective in 50% of the population). The
dose
ratio between toxic and therapeutic effects is the therapeutic index and it
can be
expressed as the ratio between LDS° and EDS°. Compounds which
exhibit high
therapeutic indices are preferred. The data obtained from these cell culture
assays
and animal studies can be used in formulating a range of dosage for use in
human.
The dosage of such compounds lies preferably within a range of circulating
concentrations that include the EDS° with little or no toxicity. The
dosage may vary
within this range depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration and
dosage
can be chosen by the individual physician in view of the patient's condition.
(See
e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.
1 p.l).



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Dosage amount and interval may be adjusted individually to provide plasma
levels of the active moiety which are sufficient to maintain the kinase
modulating
effects, or minimal effective concentration (MEC). The MEC will vary for each
compound but can be estimated from in vitro data; e.g., the concentration
necessary
to achieve 50-90% inhibition of the kinase using the assays described herein.
Dosages necessary to achieve the MEC will depend on individual characteristics
and
route of administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds
should be administered using a regimen which maintains plasma levels above the
MEC for 10-90% of the time, preferably between 30-90% and most preferably
between 50-90%.
In cases of local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the
subject being treated, on the subject's weight, the severity of the
affliction, the
manner of administration and the judgment of the prescribing physician.
d) Packaging
The compositions may, if desired, be presented in a pay:': or dispenser device
which may contain one or more unit dosage forms containing the active
ingredient.
The pack may for example comprise metal or plastic foil, such as a blister
pack. The
pack or dispenser device may be accompanied by instructions for
administration.
The pack or dispenser may also be accompanied with a notice associated with
the
container in form prescribed by a governmental agency regulating the
manufacture,
use, or sale of pharmaceuticals, which notice is reflective of approval by the
agency
of the form of the polynucleotide for human or veterinary administration. Such
notice, for example, may be the labeling approved by the U.S. Food and Drug
Administration for prescription drugs, or the approved product insert.
Compositions
comprising a compound of the invention formulated in a compatible
pharmaceutical
earner may also be prepared, placed in an appropriate container, and labeled
for



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treatment of an indicated condition. Suitable conditions indicated on the
label may
include treatment of a tumor, inhibition of angiogenesis, treatment of
fibrosis,
diabetes, and the like.
IV. Biological Activity of the Compounds of the Invention
The compounds of the present invention were tested for their ability to
inhibit most of protein kinase activity. The biological assays and results of
these
inhibition studies are reported herein. The methods used to measure modulation
of
protein kinase function are similar to those described in International
Publication
No. WO 98/07695, published March 26, 1998, by Tang et al., and entitled
"Indolinone Combinatorial Libraries and Related Products and Methods for the
Treatment of Disease," with respect to the high throughput aspect of the
method.
The WO 98/07695 publication is incorporated herein by reference in its
entirety,
including any drawings.
V. Pharmaceutical Compositions and Administration of Compounds of the
Invention
Methods of preparing pharmaceutical formulations of the compounds,
methods of determining the amounts of compounds to be administered to a
patient,
and modes of administering compounds to an organism are disclosed in the WO
98/07695 publication, and International patent publication number WO 96/22976,
by
Buzzetti et al., and entitled "Hydrosoluble 3-Arylidene-2-Oxindole Derivatives
as
Tyrosine Kinase Inhibitors," published August l, 1996, both of which are
incorporated herein by reference in their entirety, including any drawings.
Those
skilled in the art will appreciate that such descriptions are applicable to
the present
invention and can be easily adapted to it.
EXAMPLES
The examples below are non-limiting and are merely representative of
various aspects and features of the present invention. The examples describe



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84
methods for synthesizing compounds of the invention and methods for measuring
an
effect of a compound on the function of protein kinases.
The cells used in the methods are commercially available. The nucleic acid
vectors harbored by the cells are also commercially available and the
sequences of
genes for the various protein kinases are readily accessible in sequence data
banks.
Thus, a person of ordinary skill in the art can readily recreate the cell
lines in a
timely manner by combining the commercially available cells, the commercially
available nucleic acid vectors, and the protein kinase genes using techniques
readily
available to persons of ordinary skill in the art.
SYNTHETIC PROCEDURES
EXAMPLE 1: PROCEDURE FOR SYNTHESIZING THE COMPOUNDS
OF THE INVENTION
The compounds of this invention, as well as the precursor 2-oxindoles and
aldehydes, may be readily synthesized using techniques well known in the
chemical
arts. It will be appreciated by those skilled in the art that other synthetic
pathways
for forming the compounds of the invention are available and that the
following is
offered by way of example and not limitation. Furthermore, the compounds whose
syntheses are described below are likewise not to be construed as limiting the
scope
of this invention in any manner whatsoever.
A. 3- lidenyl-6-Heterocyclyl-2-Indolinone Derivatives
Compound AHI-1: 3-(4-Methoxy-3-thiophen-2-yl-benzylidene)-6-pyridin-3-yl-
1,3-dihydroindol-2-one
To a warm, stirred solution of 6-bromo-2-oxindole (4 g, 26.3 mmol) in 60
mL toluene and 60 mL ethanol was added
tetrakis(triphenylphosphine)palladium(0)
(2.3 g, 1.9 mmol) followed by 2M aqueous sodium carbonate (50 mL, 100 mmol)
and pyridine-3-boronic acid, propanediol ester (5 g, 30.7 mmol). The mixture
was
stirred at 100 °C in an oil bath for 12 hours. The reaction mixture was
cooled,



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diluted with ethyl acetate (500 mL) and washed with saturated sodium
bicarbonate
(200 mL), water (200 mL) and brine (200 mL). The organic layer was separated,
dried over anhydrous magnesium sulfate and concentrated to afford a brown
solid.
The solid was triturated with methylene chloride/diethyl ether to give 2.32 g
(42%)
S of 6-pyridin-3-yl-1,3-dihydro-indol-2-one as a brown solid.
'H NMR (360 MHz, DMSO-db) 8 10.51 (s, 1H, NH), 8.81 (d, J= 2.SHz, 1H,
Ar-H), 8.55 (dd, J= 1.8 and 5.7Hz, 1H, Ar-H), 8 (m, 1H, Ar-H), 7.45 (dd, J=
5.7
and 9.3Hz, 1H, Ar-H), 7.3 (m, 2H, Ar-H), 7.05 (s, 1H, Ar-H), 3.51 (s, 2H,
CHZCO).
MS: 210 (M]+.
10 A mixture of 6-pyridin-3-yl-1,3-dihydro-indol-2-one (100 mg, 0.5 mmol), 4-
methoxy-3-thiophen-2-yl-benzaldehyde (110 mg, 0.5 mmol) and piperidine (0.23
mL) in ethanol (4 mL) was heated to reflux and stirred overnight. The reaction
mixture was then cooled and diluted with ether to afford a precipitate which
removed by filtration. Thefiltrate was column chromatographed (eluant -
1 S isopropanol/dichloromethane) to give 30 mg (15%) of 3-(4-methoxy-3-
thiophen-2-
yl-benzylidene)-6-pyridin-3-yl-1,3-dihydroindol-2-one as a yellow solid.
'H NMR (360 MHz, DMSO-db) b 10.72 (s, 1H, NH), 8.86 (d, J = 2.2 Hz,
1H), 8.57 (m, 1H), 8.12 (d, J = 1.8 Hz, 1H), 8.04 (m, 1H), 7.76 (m, 2H), 7.68
(s,
1H), 7.65 (d, J = 2.9 Hz, 1H), 7.58 (d, J = 5 Hz, 1H), 7.46-7.49 (m, 1H), 7.32
(d, J =
20 8.6 Hz, 1H), 7.25 (m, 1H), 7.13-7.16 (m, 2H), 4.0 (s, 3H, OCH3).
MS EI: 410 [M]+.
Compound AHI-2: 3-(2-H~rox~benzylidene)-6-pyridin-3-yl-1,3-dihydroindol-2-
one
25 A mixture of 6-pyridin-3-yl-1,3-dihydroindol-2-one (100 mg, 0.5 mmol), 2-
hydroxybenzaldehyde (60 mg, 0.5 mmol) and piperidine (0.23 mL) in ethanol (4
mL) was heated to reflux and stirred overnight. The reaction mixture was
concentrated and column chromatographed (eluant - isopropanol/dichloromethane)
to give 100 mg (64%) of 3-(2-hydroxybenzylidene)-6-pyridin-3-yl-1,3-
dihydroindol-
30 2-one as a yellow solid.
'H NMR (360 MHz, DMSO-dG) 6 10.68 (s, 1H, NH), 10.19 (s, br, 1H, OH),



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8.84 (d, J = 2.2 Hz, 1H), 8.56 (m, 1H), 8.01-8.05 (m, 1H), 7.73 (s, 1H, H-
vinyl), 7.65
(d, J = 7.2 Hz, 1 H), 7.60 (d, J = 8.3 Hz, 1 H), 7.45-7.49 (m, 1 H), 7.32 (m,
1 H), 7.22
(dd, J = 1.8 & 7.9 Hz, 1H), 7.13 (d, J = 1.4 Hz, 1H), 6.98 (d, J = 7.9 Hz,
1H), 6.94 (t,
J = 7.9 Hz, 1 H).
S MS EI: 314 [M]+.
Compound AHI-3: 3-(4-Methoxy-3-thiophen-2-yl-benzylidene)-6-thiophen-2-yl-
1,3-dihydroindol-2-one
To a warm, stirred solution of 6-bromo-2-oxindole (4 g, 26.3 mmol)
dissolved in 60 mL toluene and 60 mL ethanol was added
tetrakis(triphenylphosphine)palladium(0) (2.3 g, 1.9 mmol) followed by 2M
aqueous
sodium carbonate (50 mL, 100 mmol) and thiophene-2-boronic acid (4.38 g, 34.2
mmol) in three portions over 1.5 hours. The mixture was stirred at 100
°C in an oil
bath for 12 hours. The mixture was then diluted with ethyl acetate (400 mL)
and
washed with saturated sodium bicarbonate (200 mL), water (200 mL) and brine
(200
mL). The organic layer was separated, dried over anhydrous magnesium sulfate
and
concentrated to give 2.53 g (42%) of 6-thiophen-2-yl-1,3-dihydro-indol-2-one a
tan
solid.
'H NMR (360 MHz, DMSO-db) 8 10.42 (s, 1H, NH), 7.50 (dd, J = 0.83 and
4.97Hz, 1H, Ar-H), 7.43 (dd, J = 0.89 and 3.52Hz, 1H, Ar-H), '.'..'.~," 1 (s,
2H, Ar-H),
7.10 (dd, J = 3.31 and 4.82Hz, 1H, Ar-H), 7.01 (s, 1H, Ar-H), 3.47 (s, 2H,
CHzCO).
MS EI: 21 S [M]+.
A mixture of 6-thiophen-2-yl-1,3-dihydroindol-2-one (100 mg, 0.5 mmol), 4-
methoxy-3-thiophen-2-benzaldehyde (110 mg, 0.5 mmol) and piperidine (0.23 mL)
in ethanol (4 mL) was stirred at reflux overnight. The reaction mixture was
concentrated and column chromatographed (eluant - isopropanol/dichloromethane)
to give 100 mg (48%) of 3-(4-methoxy-3-thiophen-2-ylbenzylidene)-6-thiophen-2-
yl-1,3-dihydroindol-2-one as a yellow-brown solid.
'H NMR (360 MHz, DMSO-d6) 8 10.63 (s, br, 1H, NH), 8.10 (d, J = 2.2 Hz,
1H), 7.51-7.74 (m, 7H), 7.31 (d, J = 8.3 Hz, 1H), 7.21 (dd, J = 1.8 & 7.9 Hz,
1H),
7.10-7.15 (m, 3H), 4.0 (s, 3H, OCH3).



CA 02368041 2001-09-24
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87
MS EI: 415 [M]+.
Compound AHI-4: 3-~2-Hydroxybenzylidene)-6-thiophen-2-yl-1,3-dihydroindol-
2-one
A mixture of 6-thiophen-2-yl-1,3-dihydroindol-2-one (100 mg, 0.5 mmol), 2-
hydroxybenzaldehyde (60 mg, 0.5 mmol) and piperidine (0.23 mL) in ethanol (4
mL) was stirred at reflux overnight. The reaction mixture was concentrated and
column chromatographed (eluant - isopropanol/dichloromethane) to give 80 mg
(50%) of 3-(2-hydroxybenzylidene)-6-thiophen-2-yl-1,3-dihydroindol-2-one as a
yellow-orange solid.
'H NMR (360 MHz, DMSO-d6) 8 10.60 (s, br, 1H, NH), 10.17 (s, br, 1H,
OH), 7.68 (s, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.48-7.56 (m, 3H), 7.31 (m, 1H),
7.19
(dd, J = 1.8 & 7.9 Hz, 1H), 7.13 (m, 1H), 7.08 (d, J = 0.7 Hz, 1H), 6.90-6.98
(m,
2H).
MS EI: 319 [M]+.
Compound AHI-5: 3-(4-Methoxy-3-thiophen-2-yl-benzylidene)-6-thiophen-3-yl-
1,3-dihydroindol-2-one
To a warm, stirred solution of 6-bromo-2-oxindole (4 g, 26.3 mmol)
dissolved in 60 mL toluene and 60 mL ethanol was added
tetrakis(triphenylphosphine)palladium(0) (2.3 g, 1.9 mmol) followed by 2M
aqueous
sodium carbonate (50 mL, 100 mmol) and thiophene-3-boronic acid (4.3 g, 33.6
mmol). The mixture was stirred at 100 °C in an oil bath for 12 hours.
The reaction
mixture was cooled, diluted with ethyl acetate (500 mL), washed with 1N
hydrochloric acid (200 mL), water (200 mL), saturated sodium bicarbonate (200
mL) and brine (200 mL). The organic layer was separated, dried over anhydrous
magnesium sulfate and concentrated to give a black solid. The solid was
triturated
with methylene chloride to give 2.02 g (36%) of 6-thiophen-3-yl-1,3-
dihydroindol-
2-one as a purple-gray solid.
'H NMR (360 MHz, DMSO-d~) 8 10.49 (s, 1H, NH), 7.77 (s, 1H, Ar-H),
7.59 (m, 1H, Ar-H), 7.45 (m, 1H, Ar-H), 7.24 (m, 2H, Ar-H), 7.07 (m, 1H, Ar-
H),



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88
3.46 (s, 2H, CHZCO).
MS m/z: 215 [M]+.
A mixture of 6-thiophen-3-yl-1,3-dihydroindol-2-one (100 mg, 0.5 mmol), 4-
methoxy-3-thiophen-2-benzaldehyde ( 110 mg, 0.5 mmol) and piperidine (0.23 mL)
in ethanol (4 mL) was stirred at reflux overnight. The precipitate was
collected by
vacuum filtration, washed with ethanol and dried to give 100 mg (48%) of 3-(4-
methoxy-3-thiophen-2-yl-benzylidene)-6-thiophen-3-yl-1,3-dihydroindol-2-one as
a
yellow-brown solid.
'H NMR (360 MHz, DMSO-d~) b 10.63 (s, br, 1H, NH), 9.06 (d, J = 2.2 Hz,
1H), 8.37 (d, 1H), 7.86 (m, 1H), 7.82 (s, 1H, H-vinyl), 7.72 (d, J = 7.9 Hz,
1H), 7.63
(m, 2H), 7.58 (m, 1H), 7.53 (m, 1H), 7.37 (m, 1H), 7.26 (d, J = 7.9 Hz, 1H),
7.16 (m,
1H), 7.11 (m, 1H), 4.0 (s, 3H, OCH3).
MS EI: 415 [M]+.
Compound AHI-6: 3-(2-Hydroxybenzylidene)-6-thiophen-3-yl-1,3-dihydroindol-
2-one
A mixture of 6-thiophen-3-yl-1,3-dihydroindol-2-one (100 mg, 0.5 mmol),2-
hyroxy-benzaldehyde (60 mg, 0.5 mmol) and piperidine (0.23 mL) in ethanol (4
mL)
was stirred at reflux overnight. The precipitate was collected by vacuum
filtration,
washed with ethanol and dried to give 110 mg (69%) of 3-(2-hydroxybenzylidene)-

6-thiophen-3-yl-1,3-dihydroindol-2-one as yellow-red crystals.
'H NMR (360 MHz, DMSO-d~) 8 10.60 (s, 1H, NH), 10.16 (s, br, 1H, OH),
7.85 (m, 1H), 77.62-7.67 (m, 3H), 7.49-7.54 (m, 2H), 7.29 (m, 1H), 7.22 (m,
1H),
7.13 (d, 1H), 6.90-6.98 (m, 2H).
MS EI: 319 [M]+.
Compound AHI-7: 3-f4-(3-Dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-
ylmethylene]-6-pyridin-3 yl-1,3-dihydroindol-2-one
A mixture of 6-pyridin-3-yl-1,3-dihydroindol-2-one (100 mg, 0.48 mmol), 4-
(3-dimethylaminopropyl)-3,5-dimethyl-1H-pyrrole-2-carboxaldehyde (100 mg, 0.48
mmol), and piperidine ( 1 drop) in ethanol (2 mL) was stirred at reflux
overnight. The



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89
precipitate was collected by vacuum filtration, washea wnn ethanol and dried
to give
3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-6-pyridin-
3-
yl-1,3-dihydroindol-2-one.
'H NMR (360 MHz, DMSO-d~) 8 13.40 (s, 1H, NH), 10.85 (s, 1H, NH), 8.85
(d, J = 1.8 Hz, 1H), 8.52 (dd, J = 1.8 & 5.2 Hz, 1H), 8.01-8.04 (m, 1H), 7.82
(d, J =
8.2 Hz, 1H), 7.61 (s, 1H, H-vinyl), 7.43-7.47 (m, 1H), 7.32 (dd, J = 1.6 & 8.2
Hz,
1H), 7.13 (d, J = 1.6 Hz, 1H), 2.4 (t, J = 7.3 Hz, 2H, CHZCH~CHZN(CH3)Z), 2.29
(s,
3H, CH3), 2.26 (s, 3H, CH3), 2.18 (t, J = 7.3 Hz, CHzCH2CHzN(CH3)Z), 2.12 (s,
6H,
CH,CH~CHzN(CH,)z), 1.49-1.57 (m, 2H, CHZCHzCHZN(CH3)2).
MS EI 400 [M]+.
Compound AHI-8: 3 j4-(3-Dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-
ylmethylenel-6-thiophen-2-yl-1,3-dihydroindol-2-one
A mixture of 6-thiophen-2-yl-1,3-dihydroindol-2-one (100 mg, 0.46 mmol),
4-(3-dimethylaminopropyl)-3,5-dimethyl-1H pyrrole-2-carboxaldehyde (100 mg,
0.48 mmol), and piperidine ( 1 drop) in ethanol (2 mL) was stirred at reflux
overnight. The precipitate was collected by vacuum filtration, washed with
ethanol
and dried to give 3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-
ylmethylene]-6-thiophen-2-yl-1,3-dihydro-indol-2-one.
'H NMR (360 MHz, DMSO-d~) 8 13.35 (s, 1H, NH), 10.76 (s, 1H, NH), 7.72
(d, J = 7.9 Hz, 1H), 7.55 (s, 1H, H-vinyl), 7.47 (dd, J = 1.1 & 5.0 Hz, 1H),
7.43 (dd,
J = 1.1 & 3.6 Hz, 1 H), 7.27 (dd, J = 1.5 & 7.9 Hz, 1 H), 7.11 (dd, J = 3.6 &
5.0 Hz,
1H), 7.08 (d, J = 1.5 Hz, 1H), 2.4 (t, J = 7.3 Hz, 2H, CHzCH2CH2N(CH3)z), 2.29
(s,
3H, CH3), 2.26 (s, 3H, CH3), 2.17 (t, J = 7.3 Hz, CH,CHzCH2N(CH3)2), 2.11 (s,
6H,
CH,CH~CH,N(CH3)2), 1.49-1.57 (m, 2H, CHzCHzCH2N(CH3)z).
MS EI 405 [M]+.
B. 3-Aralkyl-2-Indolinone Derivatives
General Synthesis



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Method A: Suzuki coupling
A mixture of tetrakis(triphenylphosphine)palladium(0) (3% equiv.), aryl
bromide (1 equiv.), 2 M aqueous sodium carbonate (2.5 equiv.) and boronic acid
(1.2 equiv.) in equal amount of toluene and ethanol is refluxed for 12 hours.
The
5 reaction is poured into water and extracted with ethyl acetate. The organic
layers are
washed with base and brine, dried and concentrated. The residue is purified by
column chromatography to give the product.
Method B: Condensation of oxindoles and aldehydes
10 One equivalent of oxindole, 1 equivalent of the aldehyde and 1 - 5
equivalents of piperidine (or pyrrolidine) in enough ethanol to make a
solution
which is 0.5 - 1.0 M in the oxindole are stirred at 90-100 °C for 1-18
hours. The
mixture is cooler to room temperature and, if a precipitate forms, it is
collected by
vacuum filtration, washed with ethanol and dried to give the product. When no
15 precipitate forms upon cooling of the reaction mixture, the mixture is
concentrated
and the residue purified by column chromatography.
Method C: Reduction using Palladium on Carbon
A solution of the indolinone in methanol containing a couple of drops of
20 acetic acid is hydrogenated over palladium on carbon overnight at room
temperature.
The catalyst is removed by filtration, rinsed with methanol and the filtrate
concentrated to give the reduced product.
Method D: Reduction using sodium borohydride
25 To a mixture of the indolinone (1 equiv.) in methanol and dimethylforamide
is added sodium borohydride (10 equiv.). The mixture is stirred at room
temperature
for '/Z - 3 hours. The reaction is then poured into water, extracted with
ethyl acetate,
washed with brine, dried and concentrated to give the reduced product.
30 Method E: Reduction using Pearlman's catalyst
A solution of the indolinone in methanol is hydrogenated over Pearlman's



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catalyst at room temperature for %2 - 10 hours. The catalyst is removed by
filtration,
rinsed with methanol and the filtrate concentrated to give the reduced
product.
b. Specific compound syntheses
The specific syntheses of compounds of this invention which follow are
presented by way of example only and are not to be construed as limiting the
scope
of this invention in any manner.
Compound AAI-1: 6-Methoxv-3-(4-methoxy-3-thiophen-3-ylbenzyl)-1,3-
dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (1.35 g, 1.17 mmol) was added to
a solution of 4-methoxy-3-bromobenzaldehyde (6.72 g, 31.26 mmol) dissolved in
toluene (45 mL) and ethanol (45 mL). To the solution was added 2M aqueous
sodium carbonate (80 mL, 78 mmol). Thiophene-3-boronic acid (S g, 39.07 mmol)
1 S was then added to the mixture. The mixture was refluxed for 12 hours after
which it
was poured into water (200 mL) and extracted with ethyl acetate (2 x 150 mL).
The
organic layer was washed with saturated aqueous sodium bicarbonate (150 mL)
and
brine (150 mL), dried over anhydrous magnesium sulfate and concentrated.
Chromatography (silica, 20% ethyl acetate/hexanes) afforded 6 g (88%) of 3-(3-
thiophene)-4-methoxybenaldehyde as a yellow oil.
'H NMR (360 MHz, DMSO-d~) 8 9.92 (s, 1H, CHO), 8.04 (d, 1H, J= 2.4
Hz, lxAr-H), 7.8 (m, 2H, Ar-H), 7.58 (dd, 1H, J= 3.1 and S.5 Hz, thiophene and
Ar-
H), 7.50 (dd, 1H, J= 1.6 and 5.1 Hz, Ar-H), 7.28 (d, 1H, J= 8.7 Hz,
thiophene),
3.94 (s, 3H, OCH3).
MS m/z: 219.2 [M+1 ]+.
A mixture of 3(3-thiophene)-4-methoxybenzaldehyde (0.53 g, 2.45 mmol),
6-methoxy-2-oxindole (0.4g, 2.45 mmol) and piperidine (1.2 mL, 12.25 mmol) in
S
mL of ethanol was held in a sealed tube at 100 °C for 12 hours. The
reaction was
cooled and added to diethyl ether (150 mL) and hexanes (150 mL). The solid
which
formed was removed by filtration, washed with diethyl ether and then hexanes
and
dried to give 0.57 g (64%) of 6-methoxy-3-(4-methoxy-3-thiophen-3-



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ylbenzylidene)-1,3-dihydroindol-2-one as a mustard yellow solid.
'H NMR (360 MHz, DMSO-d~) 8 10.45 (s, 1H, NH), 8.78 (d, J= 2.3 Hz, 1H,
Ar-H), 8.30 (dd, J= 2.1 and 8.7 Hz, 1H, Ar-H), 7.8 (dd, 1H, J= 1.5 and 2.9 Hz,
Ar-
H), 7.5 (m, 3H, 2xAr-H and Ar-CH=C), 7.18 (d, J= 8.9 Hz, 1H, Ar-H), 6.55 (dd,
1H, J= 2.1 and 8.5 Hz, Ar-H), 6.39 (d, 1H, J= 2.3 Hz, Ar-H), 3.91 (s, 3H,
OCH3),
3.76 (s, 3H, OCH3), 2.04 (s, 3H, NHCOCH3).
MS m/z: 364.1 [M+1 ]+.
To a mixture of 6-methoxy-3-(4-methoxy-3-thiophen-3-ylbenzylidene)-1,3-
dihydroindol-2-one (363 mg, 1 mmol) in methanol (10 mL) and dimethylforamide
(5 mL) was added sodium borohydride (380 mg, 10 mmol) in portions. The
reaction was stirred at room temperature for 3 hours. The reaction mixture was
quenched with water ( 100 mL) and extracted into ethyl acetate (200 mL). The
organic layer was washed with brine, dried over anhydrous sodium sulfate and
concentrated. The residue was chromatographed on silica gel (ethyl
acetate:hexane
2:3 then 1:1 ) to give 215 mg (59%) of the title compound as a faint orange
crystalline solid.
'H NMR (360 MHz, DMSO-db) 8 10.2 (s, br, 1H, NH), 7.61 (dd, J= 1.4 &
2.8 Hz, 1H), 7.51 (dd, J= 3.2 & 4.7 Hz, 1H), 7.31 (dd, J= 1.3 & 4.9 Hz, 1H),
7.25
(d, J= 2.5 Hz, 1H), 7.0 (dd, J= 2.2 & 8.6 Hz, 1H), 6.85, 6.88, 6.90, 6.92 (m,
2H),
6.42 (dd, J= 2.5 & 8.3 Hz, 1H), 6.28 (d, J= 2.2 Hz, 1H), 3.76 ;s, 3H, OCH3),
3.67-
3.73 (m, 1H, Ar-CHCO), 3.67 (s, 3H, OCH3), 3.24 (dd, J= 4.7 & 13.7 Hz, 1H,
1 xArCHz), 2.91 (dd, J = 7.2 & 13.7 Hz, 1 H, 1 xArCH,).
MS-EI m/z: 365 [M]+.
Compound AAI-2: Cyclopentanecarboxvlic acid [3-(3 5_dichloro-2-
hydroxybenzyl)-2-oxo-2,3-dihydro-1 H-indol-6-yl]-amide
Cyclopentanecarboxylic acid (2-oxo-2,3-dihydro-1H-indol-6-yl)-amide was
condensed with 3,5-dichlorosalicylaldehyde using method B to give a mixture of
isomers of cyclopentanecarboxylic acid [3-(3,5-dichloro-2-hydroxy-benzylidene)-
2-
oxo-2,3-dihydro-1H-indol-6-yl]-amide as an orange solid which was reduced
using
method D to give 10 mg (40%) of cyclopentanecarboxylic acid [3-(3,5-dichloro-2-




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hydroxybenzyl)-2-oxo-2,3-dihydro-1H-indol-6-yl]-amide as an off white
crystalline
solid.
'H NMR (360 MHz, DMSO-d~) 8 10.2 (s, br, 1H, NH), 10.39 (s, br, 1H,
NH), 9.72 (s, br, 1H), 9.54 (s, br, 1H), 7.35 (m, 2H), 7.09 (m, 1H), 6.91 (d,
J= 7.2
Hz, 1H), 6.55 (d, J= 7.2 Hz, 1H), 3.73 (m, 1H, lxAr-CHCO), 3.23 (m, 1H,
lxArCH~), 2.8 (m, 1H, lxArCHz), 2.72 (m, 1H, CH-cpentyl), 1.8 (m, 2H,
cpentyl),
1.65 (m, 4H, cpentyl), 1.53 (m, 2H, cpentyl).
MS-EI m/z: 419 [M]+.
Compound AAI-3: Cyclopentanecarboxylic acid [3-(5-chloro-2-hvdroxybenzyl)-
2-oxo-2,3-dihydro-1 H-indol-6~1]-amide
Cyclopentanecarboxylic acid (2-oxo-2,3-dihydro-1H-indol-6-yl)-amide was
condensed with 5-chlorosalicylaldehyde using method B to give
cyclopentanecarboxylic acid [3-(5-chloro-2-hydroxy-benzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-amide which was reducted using method D to give 34.4 mg
(68%) cyclopentanecarboxylic acid [3-(5-chloro-2-hydroxybenzyl)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-amide as an off white crystalline solid.
'H NMR (360 MHz, DMSO-db) 8 10.33 (s, br, 1H, NH), 9.72 (s, br, 1H,
NH), 9.71 (s, br, 1H, OH), 7.34 (m, 1H), 7.07 (dd, J= 2.5 & 8.3 Hz, 1H), 7.03
(d, J
= 2.5 Hz, 1 H), 6. 8 8 (m, 1 H), 6.79 (d, J = 8.3 Hz, 1 H), 6.43 (d, J = 6. 8
Hz, 1 H), 3 . 72
(m, 1H, lxAr-CHCO), 3.20 (dd, J= 5.4 & 13.7 Hz, 1H, lxArCH2), 2.72 (m, 1H,
CH-cpentyl), 2.62 (dd, J= 9.2 & 13.7 Hz, 1H, lxArCHz), 1.81 (m, 2H, cpentyl),
1.65 (m, 4H, cpentyl), 1.52 (m, 2H, cpentyl).
MS-EI m/z: 384 & 386 [M]+.
Compound AAI-4: 3-(4-Hydroxy-6,4'-dimethoxybiphenyl-3-ylmethyl)-1 3-
dihydroindol-2-one
2-Hydroxy-4-methoxybenzaldehyde (20 g, 131.4 mmol) was brominated to
give 5-bromo-2-hydroxy-4-methoxybenzaldehyde as a light yellow solid, followed
by coupling with 4-methoxyphenylboronic acid (750 mg, 4.94 mmol) using method
A to give 800 mg (75%) of 4-hydroxy-6,4'-dimethoxybiphenyl-3-carbaldehyde as a



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light yellow solid.
'H NMR (360 MHz, DMSO-d~) 8 11.06 (s, br, 1H, OH), 10.04 (s, 1H, CHO),
7.55 (s, 1H), 7.35 (d, J= 8.6 Hz, 2H), 6.94 (d, J= 8.6 Hz, 2H), 6.63 (s, 1H),
3.81 (s,
3H, OCHj), 3.77 (s, 3H, OCH3).
MS-EI m/z: 258 [M]+.
2-Oxindole was condensed with 4-hydroxy-6,4'-dimethoxybiphenyl-3-
carbaldehyde using method B to give a mixture of isomers of 3-(4-hydroxy-6,4'-
dimethoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-2-one, followed by reduction
using method E to give 4.5 mg (75%) of 3-(4-hydroxy-6,4'-dimethoxybiphenyl-3-
ylmethyl)-1,3-dihydroindol-2-one as a white solid.
MS-EI m/z: 375 [M]+.
Compound AAI-~: 3-(3-Cvclopentvl-4-methoxvbenzvll-5-fluoro-1.3
dihydroindol-2-one
5-Fluoro-2-oxindole was condensed with 3-cyclopentyl-4-
methoxybenzaldehyde using method B to give 3-(3-cyclopentyl-4-
methoxybenzylidene)-5-fluoro-1,3-dihydro-indol-2-one as a yellow-orange solid,
followed by reduction using method E to give 0.78 g (78%) of 3-(3-cyclopentyl-
4-
methoxybenzyl)-5-fluoro-1,3-dihydroindol-2-one as a white solid.
'H NMR (360 MHz, DMSO-d6) 8 10.19 (s, 1H, NH), 6.8 (m, 4H, 4xAr-H),
6.73 (d, J= 7.92 Hz, 1H, Ar-H), 6.64 (dd, J= 4.32 and 8.28 Hz, 1H, Ar-H), 3.73
(m,
1H, lxAr-CH-CO), 3.68 (s, 3H, OCH,), 3.25 (dd, J= 5.4 and 14.0 Hz, 1H, lxAr-
CH,), 3.15 (m, 1H, CH-cpentyl), 2.93 (dd, J= 7.2 and 14.0 Hz, 1H, lxAr-CHZ),
1.8
(br m, 2H, cpentyl), 1.6 (br m, 4H, cpentyl), 1.37 (br m, 1H, cpentyl), 1.25
(br m,
1H, cpentyl).
MS m/z: 339 [M]+.
Compound AAI-6: N-[3-(4-Methoxv-3-thiophen-2- lbenzyl)-2-oxo-2 3-dihydro-
1 H-indol-6-yl]-acetamide
4-Methoxy-3-bromobenzaldehyde (1.5 g) was coupled with thiophene-2-
boronic acid (0.98 g) using method A to give 1.3 g (87%) of 3-(2-thiophene)-4-



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methoxybenzaldehyde as a yellow oil.
'H NMR (360 MHz, DMSO-d~) 8 9.92 (s, 1H, CHO), 8.2 (d, 1H, J= 3Hz,
lxAr-H), 7.8 (dd, 1H, J= 10 and 10 Hz, SCHCHCH), 7.65 (m, 1H, Ar-H), 7.60 (dd,
1H, J= 2 and 6 Hz, Ar-H), 7.32 (d, 1H, J= IOHz, SCHCHCH), 7.13 (d, 1H, J= 10
5 and 6 Hz, SCHCHCH), 3.99 (s, 3H, OCH3).
6-Acetamido-2-oxindole was condensed with 3-(2-thiophene)-4-
methoxybenzaldehyde to give N [3-(4-methoxy-3-thiophen-2-ylbenzylidene)-2-oxo-
2,3-dihydro-1H indol-6-yl]-acetamide as a yellow solid which was reduced using
method E to give 0.04 g (80%) of N-[3-(4-methoxy-3-thiophen-2-ylbenzyl)-2-oxo-
10 2,3-dihydro-1H-indol-6-yl]-acetamide as a white solid.
'H NMR (360 MHz, DMSO-d6) 8 10.19 (s, 1H, NH), 9.80 (s, 1H, NHAc),
7.59 (dd, J= 1.08 and 2.88 Hz, 1H, Ar-H), 7.49 (dd, J= 5.04 and 2.88 Hz, 1H,
Ar-
H), 7.30 (dd, J= 1.08 and 5.04 Hz, 1H, Ar-H), 7.2 (m, 2H, 2xAr-H), 6.98 (dd,
J=
2.16 and 8.28 Hz, 1H, Ar-H), 6.9 (m, 3H, 3xAr-H), 3.75 (s, 3H, OCH3), 3.7 (m,
1H,
15 Ar-CH-CO), 2.93 (dd, J= 7.2 and 13.7 Hz, 1H, Ar-CH), 1.98 (s, 3H, NHCOCH3).
MS m/z: 392 [M]+.
Compound AAI-7: N-~3-[3-Cyclohexyl-4-(2-morpholin-4-ylethoxy)-benzYll-2-
oxo-2,3-dih~ro-1 H-indol-6-~) -acetamide
20 6-Acetamido-2-oxindole was condensed with 3-cyclohexyl-4-
morpholinoethoxybenzaldehyde using method B to give a mixture of isomers of N-
{3-[3-cyclohexyl-4-(2-morpholin-4-ylethoxy)-benzylidene]-2-oxo-2,3-dihydro-1H-
indol-6-yl}-acetamide as a mustard yellow solid which was reduced using method
E
to give 0.04 g (80%) of N-{3-[3-cyclohexyl-4-(2-morpholin-4-ylethoxy)-benzyl]-
2-
25 oxo-2,3-dihydro-1H-indol-6-yl}-acetamide as a white solid.
'H NMR (360 MHz, DMSO-db) b 10.14 (s, 1H, NH), 9.79 (s, 1H, NHAc),
7.22 (s, 1H, Ar-H), 6.8 (m, SH, SxAr-H), 4. 0 (br s, 2H), 3.6 (br m, SH), 3.1
(m, 2H),
2.7 (m, SH), 1.97 (s, 3H, NHCOCH3), 1.7 (br m, 6H), 1.2 (br m, 6H).
MS m/z: 491.9 [M]+.
Compound AAI-8: 3-(4-Methoxy-3-thiophen-3-ylbenzyl)-S-(2-morpholin-4-



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ylethyl)-1,3-dihydroindol-2-one
A solution of 5-(2-chloroethyl)-2-oxindole (2.3 g), morpholine (1.2 mL) and
diisopropylethylamine (1.2 mL) in dimethylsulfoxide (10 mL) was stirred
overnight
at 100°C. The mixture was cooled, poured into water and extracted with
ethyl
acetate. The organic layer was washed with brine, dried and evaporated. The
residue was chromatographed (silica gel, 5 % methanol in chloroform) to give
0.9 g
(31 %) of S-(2-morpholin-4-ylethyl)-2-oxindole as a white solid.
'HNMR (360 MHz, DMSO-db) 8 10.21 (s, br, 1H, NH), 7.06 (s, br, 1H, H-4),
7.0 (d, J= 8Hz, 1H, H-6), 6.71 (d, J= 8Hz, 1H, H-7), 3.55-3.58 (m, 4H), 3.41
(s,
2H, H-3), 2.63-2.68 (m, 2H), 2.39-2.47 (m, 6H).
5-(2-Morpholin-4-ylethyl)-2-oxindole was condensed with 3(3-thiophene)-4-
methoxybenzaldehyde using method B to give 3-(4-methoxy-3-thiophen-3-yl-
benzylidene)-5-(2-morpholin-4-ylethyl)-1,3-dihydroindol-2-one as an orange-
yellow
solid which was reduced using method E to give 0.03 g (60%) of 3-(4-methoxy-3-
thiophen-3-yl-benzyl)-5-(2-morpholin-4-ylethyl)-1,3-dihydroindol-2-one as a
white
solid.
'H NMR (360 MHz, DMSO-db) 8 10.16 (s, 1H, NH), 7.57 (br d, J= 1.8 Hz,
1 H, Ar-H), 7.51 (dd, J = 3.24 and 5.4 Hz, 1 H, Ar-H), 7.27 (br d, J = 5.4 Hz,
1 H, Ar-
H), 7.21 (br d, J= 1.8 Hz, 1H, Ar-H), 6.9 (m, 3H, 3xAr-H), 6.7 (m, 1H, Ar-H),
6.62
(d, J= 7.9 Hz, 1H, Ar-H), 3.75 (s on m, 4H, OCH3 and Ar-CII--~;O), 3.67 (br m,
6H),
2.98 (m, 3H), 2.7 (m, 4H).
MS m/z: 448.9 [M]+.
Compound AAI-9: 3-(5-Isopropyl-4-methoxy-2-methylbenz,~l,)-1,3-dihydroindol-
2-one
2-Oxindole was condensed with 5-isopropyl-4-methoxy-2-
methylbenzaldehyde using method B to give 0.25 g of 3-(5-Isopropyl-4-methoxy-2-

methylbenzylidene)-1,3-dihydroindol-2-one as a yellow-orange solid which was
reduced using method E to give 2 g (100%) of 3-(5-isopropyl-4-methoxy-2-
methylbenzyl)-1,3-dihydroindol-2-one as a white solid.
'H NMR (360 MHz, DMSO-d6) b 10.30 (s, 1H, NH), 7.10 (m, 1H, Ar-H),



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6.78 (m, 3H, Ar-H), 6.68 (m, 2H, Ar-H), 3.7 (s, 3H, OCH3), (m, 1H, Ar-CH-CO),
3.62 (dd, J= 4.6 and 9.3 Hz, 1H, Ar-CH-CO), 3.23 (dd, J= 4.6 and 14 Hz, 1H,
1 xAr-CHZ), 3.1 (m, 1 H, CH-isopropyl), 2.75 (dd, J = 9.36 and 14 Hz, 1 H, 1
xAr
CHz), 2.2 (s, 3H, Ar-CHj), 1.02 (d, J= 6.8 Hz, 3H, isopropyl), 0.97 (d, J= 7.5
Hz,
3H, isopropyl).
Compound AAI-10: 3-(3,5-Diisopropyl-4-methoxybenzyl)-1,3-dihydroindol-2-one
2-Oxindole was condensed with 3,5-diisopropyl-4-methoxybenzaldehyde
using method B to give 3-(3,5-diisopropyl-4-methoxy-benzylidene)-1,3-dihydro-
indol-2-one as a yellow-orange solid which was reduced using method E to give
2 g
(100%) of 3-(3,5-diisopropyl-4-methoxybenzyl)-1,3-dihydroindol-2-one as a
white
solid.
'H NMR (360 MHz, DMSO-d~) 8 10.21 (s, 1H, NH), 7.07 (m, 1H, Ar-H), 6.8
(m, 4H, Ar-H), 6.69 (d, J= 7.5 Hz, 1H, Ar-H), 3.7 (m, 1H, Ar-CH-CO), 3.57 (s,
3H,
OCH3), 3.2 (dd, J= 4.6 and 13 Hz, 1H, lxAr-CHZ), 3.14 (m, 2H, CH-isopropyl),
2.86 (dd, J= 7.5 and 13.6 Hz, 1H, lxAr-CHZ), 1.08 (d, J= 6.8 Hz, 6H,
isopropyl),
1.02 (d, J= 6.8 Hz, 6H, isopropyl).
Compound AAI-1 l: 3-(3-Cyclopent~h~droxybenzyl)-5-fluoro-1,3-
dihydroindol-2-one
3-(3-Cyclopentyl-4-hydroxybenzylidene)-5-fluoro-1,3-dihydroindol-2-one
(0.045 g) was reduced using method E to give 0.025 g (56%) of 3-(3-cyclopentyl-
4-
hydroxy-benzyl)-S-fluoro-1,3-dihydroindol-2-one as a tan foam.
'H NMR (360 MHz, DMSO-db) b 10.18 (s, 1H, NH), 8.9 (s, 1H, Ar-H), 6.89
(m, 1H, Ar-H), 6.7 (m, 3H, Ar-H), 6.56 (d, J= 8.28 Hz, 1H, Ar-H), 3.68 (m,
1H),
3.16 (m, 2H), 2.84 (m, 1H), 1.8 to 1.2 (m, 8H, cpentyl).
MS m/z: 325 [M]+.
Compound AAI-12: 3-Bend-4-(2-hydroxyethyl)-1 3-dihydroindol-2-one
4-(2-Hydroxyethyl)-1,3-dihydroindol-2-one was condensed with 4-
bromobenzaldehyde using method B to give 3-(4-bromobenzylidene)-4-(2-hydroxy-



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ethyl)-1,3-dihydroindol-2-one as a yellow solid which was reduced using method
E
to give 0.2 g (91%) of 3-benzyl-4-(2-hydroxyethyl)-1,3-dihydroindol-2-one as a
yellow/orange foam.
'H NMR (360 MHz, DMSO-d~,) 8 10.09 (s, 1H, NH), 7.0 (m, 3H, Ar-H), 6.99
(m, 1H, Ar-H), 6.91 (m, 2H, Ar-H), 6.45 (d, J= 7.9 Hz, 1H, Ar-H), 3.8 (m, 1H),
3.67 (m, 2H), 3.3 (m, 2H), 2.9 (m, 1H), 2.7 (m, 1H), 2.75 (dd, J= 9.36 and 14
Hz,
1H, lxAr-CHz), 2.2 (s, 3H, Ar-CH3), 1.02 (d, J= 6.8 Hz, 3H, isopropyl), 0.97
(d, J=
7.5 Hz, 3H, isopropyl).
MS m/z: 267 [M]+.
Compound AAI-13: 3-(2-H d~ybenzyl)-6-(3-methoxmhenyl)-1,3-dihydroindol-
2-one
6-(3-Methoxyphenyl)-1,3-dihydroindol-2-one was condensed with
salicylaldehyde using method B to give 3-(2-hydroxybenzylidene)-6-(3-
methoxyphenyl)-1,3-dihydro-indol-2-one as a yellow solid which was reducted
using method E to give 0.03 g (66%) of 3-(2-hydroxy-benzyl)-6-(3-methoxy-
phenyl)-1,3-dihydro-indol-2-one as a white solid.
Compound AAI-14: 3-(4-Bromobenzyl)-4-(2-hydroxyethyl)-1,3-dihydroindol-2-
one
3-(4-Bromobenzylidene)-4-(2-hydroxyethyl)-1,3-dihydroindol-2-one (0.18 g,
0.52 mmol) was reduced using method D to give 0.17 g (95%) of 3-(4-
bromobenzyl)-4-(2-hydroxyethyl)-1,3-dihydroindol-2-one as a white solid.
MS m/z: 345/347 [M]+.
Compound AAI-15: 5-Chloro-3-[3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzyl]-1,3-dihydroindol-2-one
5-Chloro-2-oxindole was condensed with 3,5-diisopropyl-4-(2- morpholin-4-
yl-ethoxy)-benzaldehyde using method B to give S-chloro-3-[3,5-diisopropyl-4-
(2-
morpholin-4-ylethoxy)-benzylidene]-1,3-dihydroindol-2-one as a brownish-orange
solid which was reduced using method E to give 1.3 g (100%) of 5-chloro-3-[3,5-




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diisopropyl-4-(2-morpholin-4-ylethoxy)-benzyl]-1,3-dihydroindol-2-one as a
yellow/orange foam.
MS m/z: 471 [M]+.
Compound AAI-16: 3-(1H-Indol-S-ylmethyl)-4-methyl-1,3-dihydroindol-2-one
4-Methyl-2-oxindole was condensed with 1H-indole-S-carbaldehyde using
method B to give 3-(1H-indol-S-ylmethylene)-4-methyl-1,3-dihydroindol-2-one as
a
white solid which was reduced using method C to give 20 mg (40%) of 3-(1H-
indol-
S-ylmethyl)-4-methyl-1,3-dihydroindol-2-one.
MS-EI mlz:276 [M]+.
Compound AAI-17: 3-(1H-Indol-S-ylmethyl~-1,3-dihydroindol-2-one
2-Oxindole (133 mg, lmmol) was condensed withlH-indole-S-carbaldehyde
(14S mg, 1 mmol) using method B to give 1SS mg (60%) of 3-(1H-indol-S-
1S ylmethylene)-1,3-dihydroindol-2-one as a white solid.
'H NMR (360 MHz, DMSO-db) b 11.38 (s, 1H, NH), 10.47 (s, 1H, NH), 7.96
(s, 1H), 7.76 (m, 2H), 7.47-7.54 (m, 2H), 7.43 (t, 1H), 7.19 (t, 1H), 6.82-
6.88 (m,
2H), 6.54 (m, 1H).
3-(1H-Indol-S-ylmethylene)-1,3-dihydroindol-2-one (80 mg, 0.31 mmol) was
reduced using method C to give 76 mg (9S%) of 3-(1H-indol-S-ylmethyl)-1,3-
dihydro-indol-2-one.
MS-EI m/z: 262 [M]+.
Compound AAI-18: 3-L4-(3-Dimethylaminopropyl)-3,S-dimethyl-1H-nyrrol-2-
2S l~hyll-1,3-dihydroindol-2-one
2-Oxindole (133 mg, 1.0 mmol) was condensed with 4-(3-
dimethylaminopropyl)-3,S-dimethyl-1H pyrrole-2-carbaldehyde (208 mg, 1.0 mmol)
using method B to give 168.3 mg (S2%) of 3-[4-(3-dimethylaminopropyl)-3,S-
dimethyl-1H-pyrrol-2-ylmethylene]-1,3-dihydro-indol-2-one as a yellow solid.
'HNMR (360 MHz, DMSO-d~,) 8 13.38 (s, 1H, NH), 10. 70 (s, 1H, NH), 7.68
(d, J = 7.54 Hz, 1 H, H-4), 7.53 (s, 1 H, H-vinyl), 7.06 (t, J = 7.54 Hz, 1 H,
H-6), 6.94



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(t, J= 7.54 Hz, 1H, H-5), 6.85 (d, J= 7.54 Hz, 1H, H-7), 2.38 (t, J= 7.25 Hz,
2H,
(CH,)ZNCH,CHZCH,), 2.27 (s, 3H, CH,), 2.23 (s, 3H, CH3), 2.17 (t, J= 7.25 Hz,
2H,
(CH,)zNCHzCH,CH2), 2.11 (s, 6H, (CH3)zNCH,CH,CHz), 1.52 (quint., J= 7.25 Hz,
2H, (CH,)ZNCH,CH,CHz).
S MS m/z: 323 [M]+.
3-[4-(3-Dimethylaminopropyl)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-1,3-
dihydro-indol-2-one (13 mg, 0.04 mmol) was reduced using method C to give 6 mg
(46%) of 3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-ylmethyl]-1,3-
dihydroindol-2-one.
MS-EI m/z: 325 [M]+.
Compound AAI-19: 5-Bromo-314-(3-dimethylaminopropyl)-3,5-dimethyl-1H-
pyrrol-2-ylmethyll-1,3-dihydroindol-2-one
5-Bromo-1,3-dihydroindol-2-one was condensed with 4-(3-dimethylamino-
propyl)-3,5-dimethyl-1H pyrrole-2-carbaldehyde (208 mg, 1.0 mmol) using method
B to give 284.9 mg (71 %) of S-bromo-3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-

1H pyrrol-2-ylmethylene]-1,3-dihydroindol-2-one as a red solid.
'HNMR (360 MHz, DMSO-db) 8 13.42 (s, 1H, NH), 10.81 (s, 1H, NH), 7.98
(d, J= 1.89 Hz, 1H), 7.66 (s, 1H, H-vinyl), 7.17 (dd, J= 1.89& 8.23 Hz, 1H),
6.79
(d, J= 8.23 Hz, 1H), 2.38 (t, J= 7.23 Hz, 2H, (CH3)~NCHZCH,~:H,), 2.27 (s, 3H,
CH3), 2.25 (s, 3H, CH,), 2.16 (t, J= 7.23 Hz, 2H, (CH,)ZNCHZCHZCHZ), 2.10 (s,
6H,
(CH3)ZNCHZCH,CHZ), 1.51 (quint., J= 7.23 Hz, 2H, (CH3)ZNCH,CHZCHZ).
MS m/z: 401/403 [M]+.
5-Bromo-3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1 H-pyrrol-2-
ylmethylene]-1,3-dihydroindol-2-one (50 mg, 0.12 mmol) was reduced using
method D to give 5-bromo-3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-
2-ylmethyl]-1,3-dihydro-indol-2-one.
MS m/z: 404 [M]+.
Compound AAI-20: 3-[4-(3-Dimethylamino-propyl)-3,5-dimethyl-1 H-pyrrol-2-
ylmethyll-6-(2-methox~phenyl)-1,3-dihydro-indol-2-one



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6-(2-Methoxyphenyl)-1,3-dihydroindol-2-one (239 mg, 1.0 mmol)was
condensed with 4-(3-dimethylaminopropyl)-3,5-dimethyl-1H pyrrole-2-
carbaldehyde (208 mg, 1.0 mmol) using method B to give 333 mg (83%) of 3-[4-(3-

dimethylaminopropyl)-3,5-dimethyl-1H pyrrol-2-ylmethylene]-6-(2-
methoxyphenyl)-1,3-dihydroindol-2-one as a yellow solid.
'HNMR (360 MHz, DMSO-d~) 8 13.38 (s, 1H, NH), 10.72 (s, 1H, NH), 7.70
(d, J= 8.06 Hz, 1H), 7.55 (s, 1H, H-vinyl), 7.28-7.36 (m, 2H), 7.14 (d, J=
8.32 Hz,
1 H), 7. 04 (dd, J = 1.21, 8.06 Hz, 1 H), 6.99 (d, J = 7.42 Hz, 1 H), 6. 99
(d, J = 1.21
Hz, 1H) 3.76 (s, 3H, OCH,), 2.39 (t, J= 7.24 Hz, 2H, (CH3)ZNCH,CH,CHZ), 2.28
(s,
3H, CHj), 2.25 (s, 3H, CHj), 2.18 (t, J= 7.24 Hz, 2H, (CH3)ZNCH~CH~CHZ), 2.11
(s,
6H, (CH3)ZNCH,CH~CHZ), 1.53 (quint., J = 7.24 Hz, 2H, (CH3)zNCHZCH~CH,).
MS m/z: 429 [M]+.
3-[4-(3-Dimethylaminopropyl)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-6-(2-
methoxyphenyl)-1,3-dihydroindol-2-one (54 mg, 0.126 mmol) was reduced using
method C to give 45 mg (86%) of 3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-
pyrrol-2-ylmethyl]-6-(2-methoxyphenyl)-1,3-dihydroindol-2-one.
MS-EI m/z: 431 [M]+.
Compound AAI-21: 3-j4-(3-Dimethylaminopro~,yl)-3,5-dimethyl-1H-pyrrol-2-
ylmethyl]-6-(3-methoxyphenyl)-1,3-dihydroindol-2-one
6-(3-Methoxyphenyl)-1,3-dihydroindol-2-one (239 mg, 1.0 mmol) was
condensed with 4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-pyrrole-2-
carbaldehyde (208 mg, 1.0 mmol) using method B to give 333 mg (83%) of 3-[4-(3-

dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-6-(3-
methoxyphenyl)-1,3-dihydroindol-2-one as a red solid.
'HNMR (360 MHz, DMSO-d~,) 8 13.38 (s, 1H, NH), 10.80 (s, 1H, NH), 7.60
(d, J= 8.06 Hz, 1H), 7.57 (s, 1H, H-vinyl), 7.35 (t, J= 8.15 Hz, 1H), 7.26
(dd, J=
1.39, 8.06 Hz, 1H), 7.19 (d, br, J= 8.15 Hz, 1H), 7.13 (m, 1H), 7.09 (d, J=
1.39 Hz,
1H), 6.90 (dd, J= 2.57, 8.15 Hz, 1H), 3.81 (s, 3H, OCH3), 2.39 (t, J= 7.17 Hz,
2H,
(CH3),NCH,CH~CH,), 2.29 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.17 (t, J= 7.17 Hz,
2H,
(CH3)ZNCHZCH,CH,), 2.11 (s, 6H, (CH3)zNCH~CH,CH2), 1.53 (quint., J= 7.17 Hz,



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2H, (CH3)2NCHzCH~CH2).
MS m/z: 429 [M]+.
3-[4-(3-Dimethylaminopropyl)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-6-(3-
methoxyphenyl)-1,3-dihydroindol-2-one (50 mg, 0.12 mmol) was reduced using
method C to give 40 mg (77%) of 3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-
pyrrol-2-ylmethyl]-6-(3-methoxyphenyl)-1,3-dihydroindol-2-one.
MS-EI m/z: 429 [M-2]+.
Compound AAI-22: 3-[4-(3-Dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-
ylmethyll-6-(4-metho~henyl)-1,3-dihydroindol-2-one
6-(4-Methoxyphenyl)-1,3-dihydroindol-2-one (239 mg, 1.0 mmol) was
condensed with 4-(3-dimethylaminopropyl)-3,5-dimethyl-1H pyrrole-2-
carbaldehyde (2~.'.3 mg, 1.0 mmol) using method B to give 333 mg (83%) of 3-[4-
(3-
dimethylaminopropyl)-3,5-dimethyl-1H pyrrol-2-ylmethylene]-6-(4-
methoxyphenyl)-1,3-dihydroindol-2-one as a brown solid.
'HNMR (360 MHz, DMSO-db) 8 13.35 (s, 1H, NH), 10.77 (s, 1H, NH), 7.73
(d, J= 7.82 Hz, 1H), 7.56 (d, J= 8.83 Hz, 2H), 7.54 (s, 1H, H-vinyl), 7.20
(dd, J=
1.64, 7.82 Hz, 1H), 7.04 (d, J= 1.64 Hz), 7.00 (d, J= 8.83 Hz, 2H), 3.78 (s,
3H,
OCH3), 2.39 (t, J= 7.24 Hz, 2H, (CH,),NCH,CH~CHZ), 2.28 (s, 3H, CH3), 2.25 (s,
3H, CH3), 2.17 (t, J= 7.24 Hz, 2H, (CH,)zNCH,CH,CH2), 2.11 (s, 6H,
(CH3)zNCH~CH~CH,), 1.52 (quint., J= 7.24 Hz, 2H, (CH3)~NCH,CHZCHZ).
MS m/z: 429 (M]+.
3-[4-(3-Dimethylaminopropyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-6-(4-
methoxyphenyl)-1,3-dihydroindol-2-one (69 mg, 0.16 mmol) was reduced using
method C to give 65 mg (94%) of 3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-
pyrrol-2-ylmethyl]-6-(4-methoxyphenyl)-1,3-dihydroindol-2-one.
MS-EI m/z: 431 [M]+.
Compound AAI-23: 5-Chloro-3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-
pyrrol-2-ylmethyll-1,3-dihydroindol-2-one
5-Chloro-1,3-dihydroindol-2-one (167 mg, 1.0 mmol) was condensed with 4-



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(3-dimethylaminopropyl)-3,5-dimethyl-1H pyrrole-2-carbaldehyde (208 mg, 1.0
mmol) using method B to give 188.7 mg (53%) of 5-chloro-3-[4-(3-
dimethylaminopropyl)-3,5-dimethyl-1H pyrrol-2-ylmethylene]-1,3-dihydroindol-2-
one as a brown solid.
'HNMR (360 MHz, DMSO-d6) 8 13.43 (s, 1H, NH), 10.84 (s, 1H, NH), 7.87
(d, J= 1.85 Hz, 1H), 7.66 (s, 1H, H-vinyl), 7.05 (dd, J= 1.85, 8.15 Hz, 1H),
6.83 (d,
J= 8.15 Hz, 1H), 2.36-2.45 (m, 4H, (CH,),NCHZCH,CHZ), 2.30 (s, 6H,
(CH,),NCHZCH~CH,), 2.28 (s, 3H, CH3), 2.26 (s, 3H, CH3), 1.58 (quint., J= 7.52
Hz, 2H, (CH3)2NCH,CH,CHz).
MS m/z: 357 [M-1]+.
5-Chloro-3-[4-(3-dimethylaminopropyl)-3,5-dimethyl-1 H-pyrrol-2-
ylmethylene]-1,3-dihydroindol-2-one (63 mg, 0.18 mmol) was reduced using
method C to give 15 mg (24%) of 5-chloro-3-[4-(3-dimethylaminopropyl)-3,5-
dimethyl-1 H-pyrrol-2-ylmethyl]-1,3-dihydroindol-2-one.
MS-EI m/z: 325 [M-Cl]+.
Compound AAI-24: 3-(3-Fluoro-2-hvdroxvbenzvl)-6-nhenvl-1,3-dihvdroindol-2-
one
6-phenyl-2-oxindole (50 mg, 0.3 mmol) was condensed with 3-fluoro-2-
hydroxybenzaldehyde (50 mg, 0.36 mmol) using method B to give 52 mg (53%) of
3-(3-fluoro-2-hydroxybenzylidene)-6-phenyl-1,3-dihydroindol-2-one as a
yellow/orange solid.
'HNMR (300 MHz, DMSO-d~) 8 10.71 (s, 1H, NH), 10.32 (s, 1H, OH), 7.60
7.64 (m, 3H), 7.43-7.51 (m, 4H), 7.37 (d, J= 7.2 Hz, 1H), 7.3 (m, 1H), 7.16
(m, 1H),
7.09 (d, J = 1.2 Hz, 1 H), 6.92-6.99 (m, 1 H).
MS-EI m/z: 331 [M]+.
3-(3-Fluoro-2-hydroxybenzylidene)-6-phenyl-1,3-dihydroindol-2-one (26
mg, 0.08 mmol) was reduced using method E to give 26 mg ( 100%) 3-(3-fluoro-2-
hydroxy-benzyl)-6-phenyl-1,3-dihydroindol-2-one.
MS m/z: 333 [M]+.



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Compound AAI-25: 3-(2-Hydroxy-4-methoxybenzyl~phenyl-1,3-dihydroindol-
2-one
6-Phenyl-2-oxindole (SO mg, 0.3 mmol) was condensed with 2-hydroxy-4-
methoxybenzaldehyde (50 mg, 0.33 mmol) using method B to give 70 mg (68%) of
3-(2-hydroxy-4-methoxybenzylidene)-6-phenyl-1,3-dihydroindol-2-one as a
yellow/orange solid.
'HNMR (300 MHz, DMSO-d~) 8 10.60 (s, 1H, NH), 9.80 (s, 1H, OH), 7.35-
7.38 (m, SH), 7.45 (t, J= 7.5 Hz, 2H), 7.37 (d, J= 7.5 Hz, 1H), 7.17 (d, J=
7.5 Hz,
1H), 7.08 (s, 1H), 6.53-6.58 (m, 2H), 3.78 (s, 3H, OCH3).
MS-EI m/z: 343 [M]+.
3-(2-Hydroxy-4-methoxybenzylidene)-6-phenyl-1,3-dihydroindol-2-one (80
mg, 0.23 mmol) was reduced using method E to give 80 mg ( 100%) 3-(2-hydroxy-4-

methoxybenzyl)-6-phenyl-1,3-dihydroindol-2-one.
MS m/z: 345 [M]+.
Compound AAI-26: 3-(5-Bromo-2-hvdroxybenzyl)-6-phenyl-1,3-dihydroindol-2-
one
6-Phenyl-2-oxindole (50 mg, 0.3 mmol) was condensed with S-
bromosalicylaldehyde (50 mg, 0.25 mmol) using method B to give 80 mg (82%) of
3-(S-bromo-2-hydroxybenzylidene)-6-phenyl-1,3-dihydroindoi -?-one as a
yellow/orange solid.
'HNMR (300 MHz, DMSO-db) 8 10.70 (s, 1H, NH), 10.52 (s, 1H, OH), 7.74
(d, J= 2 Hz, 1H), 7.62-7.65 (m, 2H), 7.56 (s, 1H, H-vinyl), 7.43-7.49 (m, 4H),
7.37
(m, 1H), 7.20 (dd, J= 2, 8 Hz, 1H), 7.09 (d, J= 1.2 Hz, 1H), 6.95 (d, J= 8 Hz,
1H).
MS-EI m/z: 391/393 [M]+.
3-(5-bromo-2-hydroxybenzylidene)-6-phenyl-1,3-dihydroindol-2-one (23
mg, 0.06 mmol) was reduced using method E to give 23 mg ( 100%) 3-(5-bromo-2-
hydroxy-benzyl)-6-phenyl-1,3-dihydroindol-2-one.
MS m/z: 393/395 [M]+.
Compound AAI-27: 4-(2-Hydroxyethyl)-3-(6-methylpyridin-2-ylmethyl)-1 3-



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dihydroindol-2-one
4-(2-Hydroxyethyl)-1,3-dihydroindol-2-one (89 mg, 0.5 mmol) was
condensed with 6-methyl-2-pyridinecarbaldehyde (61 mg, 0.5 mmol) using method
B to give 4-(2-hydroxyethyl)-3-(6-methylpyridin-2-ylmethylene)-1,3-
dihydroindol-
2-one as a yellow solid.
'HNMR (360 MHz, DMSO-db) 8 10.53 (s, 1H, NH), 8.18 (d, J= 7.5 Hz, 1H),
7.68 (t, J = 7.5 Hz, 1 H), 7.64 (s, 1 H), 7.19 (d, J = 7.5 Hz, 1 H), 7.14 (t,
J = 7.5 Hz,
1H), 6.82 (d, J= 7.5 Hz, 1H), 6.68 (m, 1H), 4.78 (t, J= 5 Hz, 1H, OH), 3.68-
3.74
(m, 2H, CHZCHZOH), 3.02 (t, J= 7 Hz, 2H, CH~CHzOH), 2.49 (s, 3H, CH3).
MS-EI m/z: 280 [M]~.
4-(2-Hydroxyethyl)-3-(6-methylpyridin-2-ylmethylene)-1,3-dihydroindol-2-
one (16 mg, 0.06 mmol) was reduced using method E to give 16 mg (100%) of 4-(2-

hydroxy-ethyl)-3-(6-methylpyridin-2-ylmethyl)-1,3-dihydroindol-2-one.
MS m/z: 282 [M]+.
Compound AAI-28: 3-(3-Bromo-5-tert-butyl-4-hydroxybenzyl)-5-chloro-1 3-
dihydroindol-2-one
5-Chloro-2-oxindole ( 167 mg, 1 mmol) was condensed with 3-bromo-5-tert-
butyl-4-hydroxybenzaldehyde (308.5 mg, 1.2 mmol) using method B to give 329 mg
(81%) of 3-(3-bromo-5-tert-butyl-4-hydroxybenzylidene)-5-chloro-1,3-
dihydroindol-2-one as a mixture of isomers.
MS m/z: 407 [M+1 ]+.
3-(3-bromo-5-tent-butyl-4-hydroxybenzylidene)-5-chloro-1,3-dihydroindol-
2-one (1 g, 2.46 mmol) was reduced using method C to give 1.005 g (100%) of 3-
(3-
bromo-5-tert-butyl-4-hydroxybenzyl)-5-chloro-1,3-dihydroindol-2-one as a
mixture
of isomers.
'H NMR (360 MHz, DMSO-d~) 8 10.29 &10.16 (2s, 1H, NH), 9-9.01 (s, br,
1H, OH), 7.05-7.13 (m, 1H), 6.8-6.9 (m, 1H), 6.66-6.77 (m, 3H), 6.55-6.6 (m,
1H),
3.65 (m, 1H), 3.19 (m, 1H), 2.8 (m, 1H), 1.2 & 1.21 (2s, 9H, C(CH3)3).
MS m/z: 408/410 [M]+.



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C. Diaryl Indolinone Compounds
General Synthesis
Condensation of ketones and oxindoles:
A reaction mixture of the proper indolin-2-ones (1.0 equiv.), the appropriate
ketone (1.2 equiv.), and piperidine or pyrrolidine (0.1 equiv.) in ethanol (1-
2 mL/1.0
mmol oxindole) is stirred at 90 °C for 3-5 h. After cooling, the
precipitate is filtered,
washed with cold ethanol, and dried to yield the target compound.
4-(3,5-dimethyl-1H-pyrrol-2-vl)-4-oxo-butyric acid
0 0
1 . AIC~3
~~o-'w + / \ .~ o / \
N N
2. KOH o
To a mixture of 13.3 g (0.1 mol) of aluminum chloride in dichloromethane
(150 mL) was added 10 g (0.105 mol) of 2,4-dimethylpyrrole at room
temperature.
The resulting solution was stirred t room temperature for 15 minutes and added
with
16.4 g (0.1 mol) of ethyl succinyl chloride. The reaction mixture was stirred
at room
temperature for 2 days and quenched with saturated sodium carbonate. The
mixture
was then diluted with water (100 mL) and dichloromethane (300 mL). The organic
layer was separated and concentrated. The residue was taken into ethanol (30
mL)
and 1 N potassium hydroxide (30 mL) and heated at 90 °C for 2 hours.
The cooled
mixture was then diluted with water and extracted with ethyl acetate. The
basic
aqueous layer was acidified with 6 N hydrochloric acid until pH 3. The mixture
was
then extracted with ethyl acetate (3x). The combined organic extracts were
washed
with brine, dried over sodium sulfate and concentrated. The residue was
stirred with
ethyl acetate (20 mL) and the solid was collected by filtration to give 820 mg
of 4-
(3,5-dimethyl-1H-pyrrol-2-yl)-4-oxo-butyric acid as a dark solid.
'HNMR (300 MHz, DMSO-d~) b 12.01 (s, br, 1H, COOH), 11.18 (s, 1H,
NH), 5.75 (d, J = 2.1 Hz, 1H, H-pyrrole), 2.88 (t, J = 6.6 Hz, 2H, CH,CHz),
2.49 (t, J
= 6.6 Hz, 2H, under DMSO), 2.24 (s, 3H, CH3), 2.15 (s, 3H, CH3). MS EI 195



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[M]+.
This will then be condensed with oxindole.
3-f(3,5-Dimethyl-1 H-pyrrol-2-yl)-(4-methoxy-phenyl)-methylene]-1,3-dihydro-
indol-2-one (DAI-I)
p ~ \ ~ ~ AICI~ O ~ ~ I
CI N ~ ~N
O O
oxindole
~O
Aluminum chloride ( 1.3 equiv.) in dichloromethane was added to the
benzoyl chloride (1 equiv.) at room temperature, followed by a solution of 2,4-

dimethylpyrrole (1.6 equiv.) in dichloromethane. The mixture was then stirred
at
room temperature for overnight. The reaction was diluted with ethyl acetate,
washed
with brine, dried and concentrated. The residue was chromatographed eluting
with
ethyl acetate and hexane to give the ketone.
(3,5-Dimethyl-1H-pyrrol-2-yl)-(4-methoxy-phenyl)-methanone (50 mg, 0.22
mmol), oxindole (50 mg, 0.37 mmol), pyrrolidine (0.3 mL) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (0.1 mL) in dimethylforamide (1 mL) was heated
at
150-160 °C for 1 day. Sodium hydride (12 mg) was added to the reaction
mixture
and the heating was continued for 3 days. The reaction was poured into water
and
extracted with ethyl acetate. The organic layer was washed with water, dried
and
concentrated. The residue was chromatographed eluting with ethyl acetate and
dichloromethane to give 6 mg of 3-[(3,5-dimethyl-1H-pyrrol-2-yl)-(4-methoxy-
phenyl)-methylene]-1,3-dihydro-indol-2-one as a yellow solid.
'H NMR (300 MHz, DMSO-d~) 8 14.06 (s, 1H, NH), 10.79 (s, 1H, NH), 7.18



CA 02368041 2001-09-24
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(d, J = 9 Hz, 2H), 7.10 (d, J = 9 Hz, 2H), 6.89 (t, J = 8 Hz, 1H), 6.75 (d, J
= 8 Hz,
1H), 6.45 (m, 1H), 5.87 (d, J = 2.7 Hz, 1H, H-pyrrole), 5.40 (d, J = 8 Hz,
1H), 3.84
(s, 3H, OCH3), 2.27 (s, 3H, CH,), 1.30 (s, 3H, CH3). MS EI 344 [M]+.
3-[(3,4-dimethoxy-phenyl(3,5-dimethyl-1H-pyrrol-2-yl)-methylene]-1,3-dih
,o
~o
\O \ ~ + ~ AIC13 \
CI N CHzCIz rt o/n O
O N
O
oxindole
O
indol-2-one (DAI-II)
~o
~o
\O \ ~ + ~ AIC13 \
CI N CHZCIz rt o/n O..
1
O N
O
oxindole
O
Aluminum chloride (1.7 g, 12.75 mmol) in dichloromethane (100 mL) was
added to the 3,4-dimethoxybenzoyl chloride (2.0 g, 10 mmol) at room
temperature,
followed by a solution of 2,4-dimethylpyrrole (1.5 g, 15.8 mmol) in



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dichloromethane (5 mL). The mixture was then stirred at room temperature for
overnight. The reaction was diluted with ethyl acetate, washed with brine,
dried and
concentrated. The residue was chromatographed eluting with ethyl acetate and
hexane to give 1 g (39%) of (3,4-dimethoxy-phenyl)-(3,5-dimethyl-1H-pyrrol-2-
yl)-
methanone as a solid.
'H NMR (300 MHz, DMSO-db) b 11.15 (s, 1H, NH), 7.18 (m, 2H, Ar-H),
7.03 (d, 1H, Ar-H), 5.80 (d, 1H, H-pyrrole), 3.81 (s, 3H, OCH3), 3.77 (s, 3H,
OCH,),
2.17 (s, 3H, CH3), 1.97 (s, 3H, CH3). MS 260.2 [M+1]+.
(3,4-Dimethoxy-phenyl)-(3,S-dimethyl-1H-pyrrol-2-yl)-methanone (100 mg,
0.4 mmol), oxindole (100 mg, 0.8 mmol), pyrrolidine (0.2 mL) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (a few drops) in dimethylforamide (1.5 mL) was
heated at 160 °C for 2 hours. Sodium hydride ( 18 mg) was added to the
reaction
mixture and the heating was continued at 160 °C for 3 days. The
reaction was
poured into water and extracted with ethyl acetate. The organic layer was
washed
with water, dried and concentrated. The residue was chromatographed eluting
with
ethyl acetate and hexane to give 5 mg of 3-[(3,4-dimethoxy-phenyl)-(3,5-
dimethyl-
1H-pyrrol-2-yl)-methylene]-1,3-dihydro-indol-2-one. MS EI 374 [M]+.
D. 4-Substituted Indolinone Compounds
Compound IN-001:
4-[2-(3-Isopropyl-phenoxyl-ethyll-1,3-dihydro-indol-2-one
0
0
N
H
Diethyl azodicarboxylate (0.47 mL, 3 mmol) was added to a solution of
triphenylphosphine (0.786 g, 3 mmol) in tetrahydrofuran (10 mL) under nitrogen
atmosphere. The mixture was stirred for 15 minutes. To it was then added 4-(2-
hydroxy-ethyl)-1,3-dihydro-indol-2-one (0.53 g, 3 mmol) (Hayler, J. D.; Howie,
S.
L. B.; Giles, R. G.; Negus, A.; Oxley, P. W.; et al; J. Heteocycl. Chem.; 32;
3; 1995;



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110
875-882), followed by 3-isopropylphenol (0.41 mL, 3 mmol). The mixture was
stirred at room temperature for one day and the solvent was evaporated. The
residue
was chromatographed on silica gel eluting with ethyl acetate: hexane 1:9 to
give 120
mg (14%) of 4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one.
'HNMR (360 MHz, DMSO-d6) 8 10.31 (s, br, 1H, NH), 7.09-7.12 (m, 2H,
Ar-H), 6.88 (d, J= 7.6 Hz, 1H, Ar-H), 6.67-6.79 (m, 4H, Ar-H), 4.16 (t, J= 6.7
Hz,
2H, OCHZCHZ), 3.5 (s, 2H, H-3, CH~), 2.93 (t, J= 6.7 Hz, 2H, OCH,CHZ), 2.82
(m,
1H, CH(CH3)z), 1.16 (d, J= 6.8 Hz, 6H, CH(CH3)2). MS-EI 295 [M]+.
Compound IN-002
4-[2-(2-Isopr ~yl-phenoxy)-ethyl-1,3-dihydro-indol-2-one
0
0
N
H
Diethyl azodicarboxylate (1.58 mL, 10 mmol) was added to a solution of
triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under nitrogen
atmosphere. The mixture was stirred for 15 minutes. To it was then added 4-(2-
hydroxy-ethyl)-1,3-dihydro-indol-2-one (1.77 g, 10 mmol) followed by 2-
isopropylphenol (1.36 mL, 10 mmol). The mixture was stirred at room
temperature
for 18 hours and the solvent was evaporated. The residue was chromatographed
on
silica gel eluting with ethyl acetate: hexane 2:8 to give 0.26 g (9%) of 4-[2-
(2-
isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one as a light yellow solid.
'HNMR (360 MHz, DMSO-cl~) 8 10.29 (s, br, 1H, NH), 7.08-7.14 (m, 3H,
Ar-H), 6.83-6.93 (m, 3H, Ar-H), 6.67 (d, J= 7.2 Hz, 1H, Ar-H), 4.18 (t, J= 6.4
Hz,
2H, CHZCH20), 3.47 (s, 2H, H-3, CH,), 3.11 (m, 1H, CH(CH3)Z), 2.96 (t, J= 6.4
Hz,
2H, CHZCH,O), 1.05 (d, J= 6.8 Hz, 6H, CH(CH3)z). MS-EI 295 [M]+.
Compound IN-003
4-f 2-(Biphenyl-3 .yloxy)-eth,~ll-1,3-dihydro-indol-2-one



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H
Diethyl azodicarboxylate (1.58 mL, 10 mmol) was added to a solution of
triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under nitrogen
atmosphere. The mixture was stirred for 15 minutes. To it was then added 4-(2-
hydroxy-ethyl)-1,3-dihydro-indol-2-one (1.77 g, 10 mmol) followed by 3-
phenylphenol (1.7 g, 10 mmol). The mixture was stirred at room temperature for
one day and the solvent was evaporated. The residue was dissolved in ethyl
acetate
(150 mL) and the organic solvent was washed with 2 N hydrochloric acid (3x SO
mL), saturated sodium bicarbonate solution and brine. The residue was
chromatographed on silica gel eluting with ethyl acetate: hexane 2:8 to give
1.19 g
(36%) of 4-[2-(biphenyl-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one.
'HNMR (360 MHz, DMSO-d6) b 10.30 (s, br, 1H, NH), 7.63-7.65 (m, 2H,
Ar-H), 7.41-7.45 (m, 2H, Ar-H), 7.32-7.36 (m, 2H, Ar-H), 7.2 (m, 1H, Ar-H),
7.16
(m, 1H, Ar-H), 7.12 (t, J= 7.9 Hz, 1H, Ar-H), 6.89-6.93 (m, 2H, Ar-H), 6.68
(d, J=
7.2 Hz, 1H, Ar-H), 4.26 (t, J= 6.8 Hz, 2H, CH,CHzO), 3.52 (s, 2H, H-3, CHZ),
2.98
(t, J= 6.8 Hz, 2H, CHZCH~O).
Compound IN-004
3-(4-Bromo-benzylidene)-4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one
Br
HO ~
O
N
H
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (3.0 g, 17 mmol),
4-bromobenzaldehyde (3.1 g, 17 mmol) and piperidine (8.4 mL, 85 mmol) in
ethanol
( 113 mL) was heated at 90 °C for overnight. The reaction mixture was
concentrated
and the residue was chromatographed on a column of silica gel to give 2.4 g
(41%)



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of 3-(4-bromo-benzylidene)-4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one as a
yellow orange solid.
'HNMR (360 MHz, DMSO-d~,) 8 10.53 (s, 1H, NH), 7.96 (d, J= 7.4 Hz, 2H),
7.69 (s, 1H, H-vinyl), 7.61 (d, J= 7.4 Hz, 2H, Ar-H), 7.12 (m, 1H, Ar-H), 6.81
(d, J
= 7.4 Hz, 1H, Ar-H), 6.67 (d, J= 7.4 Hz, 1H, Ar-H), 4.80 (m, 1H, OH), 3.69 (m,
2H,
CH,CHZOH), 3.04 (m, ZH, CH2CH,OH). MS-EI 343/345 [M]+.
Compound IN-005
4~2-Hydroxy-ethyl)-3-pyridin-2-ylmethylene-1,3-dihydro-indol-2-one
OH
N I
I\
O
N
lO H
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (80 mg, 0.45
mmol) and 2-pyridinecarboxaldehyde (60 mg, 0.56 mmol) in 1 % of piperidine in
ethanol (S mL) was heated at 90 °C for 8 hours. The reaction mixture
was
concentrated and the residue was chromatographed on a column of silica gel. It
was
then triturated with ethyl acetate and hexane to give 55 mg (46%) of 4-(2-
hydroxy-
ethyl)-3-pyridin-2-ylmethylene-1,3-dihydro-indol-2-one.
'HNMR (360 MHz, DMSO-d~,) 8 10.58 (s, br, 1H, NH~3; 8.64 (d, J= 4.7 Hz,
1 H, Ar-H), 8.40 (d, J = 8.3 Hz, 1 H), 7. 81 (dt, J = 1.8 & 7.7 Hz, 1 H, Ar-
H), 7.7 (s,
1H, H-vinyl), 7.34 (m, 1H, Ar-H), 7.16 (t, J= 7.6 Hz, 1H), 6.83 (d, J= 7.6 Hz,
1H,
Ar-H), 6.69 (d, J= 7.6 Hz, 1H, Ar-H), 4.80 (t, J= 5.2 Hz, 1H, OH), 3.71 (m,
2H, -
CHZCHZOH), 3.03 (t, J= 7.2 Hz, 2H, -CHZCHZOH). MS-EI 267 [M+1]+.
Compound IN-006
4-(2-Hydroxy-ethyl)-3~6-methyl-pyridin-2-ylmethylene)-1,3-dihydro-indol-2-one
OH
N
I\
0
N
H



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113
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (89 mg, O.S
mmol), 6-methyl-2-pyridine-carbaldehyde (61 mg, O.S mmol) and piperidine (0.1
mL) in 1.0 mL of ethanol was heated at 100 °C for 1 hour and cooled to
room
temperature. The precipitate was filtered and recrystallized from ethyl
acetate and
S hexanes to give 4-(2-hydroxy-ethyl)-3-(6-methyl-pyridin-2-ylmethylene)-1,3-
dihydro-indol-2-one as a yellow solid.
'HNMR (360 MHz, DMSO-cl6) 8 10.53 (s, 1H, NH), 8.18 (d, J= 7.S Hz,
1H), 7.68 (t, J= 7.S Hz, 1H), 7.64 (s, 1H), 7.19 (d, J= 7.S Hz, 1H), 7.14 (t,
J= 7.S
Hz, 1H), 6.82 (d, J= 7.5 Hz, 1H), 6.68 (m, 1H), 4.78 (t, J= S Hz, 1H, OH),
3.68-
3.74 (m, 2H, CH,CHzOH), 3.02 (t, J= 7Hz, 2H, CHZCH,OH), 2.49 (s, 3H, CH3).
MS-EI 280 [M]+.
Compound IN-007
4-(2-Hydroxy-ethyl_)-3-( 1 H-indol-S- l~ylene)-1.3-dihydro-indol-2-one
OH H
N
O
N
IS H
Indole-S-carboxylic acid (5.02 g, 31.3 mmol) in tetrahydrofuran (40 mL) was
added slowly to the lithium aluminium hydride (1.88 g, 49.5 mmol) stirred in
tetrahydrofuran (12S mL) under nitrogen with external heating so as to
maintain a
gently reflux. The mixture was heated at reflux for 2 hours and cooled to room
temperature. 2 N sodium hydroxide ( 10 mL) was then added dropwise to the
reaction mixture. The precipitate was removed by filtration and the filtrate
was
concentrated. It was dissolved in dichloromethane (80 mL), washed with 2 N
sodium hydroxide ( 10 mL), water (20 mL) and brine (20 mL), dried over
anhydrous
sodium sulfate and concentrated to give about S g of (1H-indol-S-yl)-methanol.
2S A mixture of (1H-indol-S-yl)-methanol (3.8 g, 25.8 mmol) and manganese
oxide (S g, S7.S mmol) in dichloromethane (SO mL) was stirred at room
temperature
for 60 hours. After the usual work-up and chromatography, 1.S g (40%) of 1H-
indole-S-carbaldehyde was obtained.



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A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (39 mg, 0.22
mmol), 1H-indole-5-carbaldehyde (32 mg, 0.22 mmol) and 1 drop of piperidine in
ethanol was heated at 90 °C for overnight and cooled to room
temperature. The
reaction mixture was concentrated and the residue was purified on silica gel
column
to give 48 mg (72%) of 4-(2-hydroxy-ethyl)-3-(1H-indol-5-ylmethylene)-1,3-
dihydro-indol-2-one as a yellow solid.
'HNMR (360 MHz, DMSO-d6) 11.28 (s, br, 1H, NH), 10.48 (s, br, 1H,
NH), 8.59 (s, 1H), 8.06 (d, 1H), 7.89 (s, 1H, H-vinyl), 7.4 (m, 2H), 7.06 (t,
J= 7 Hz,
1H, H-6), 6.09 (d, J= 7 Hz, 1H, H-S), 6.68 (d, J= 7 Hz, 1H, H-7), 6.53 (s,
1H), 4.86
(t, 1H, OH), 3.75 (m, 2H, -CHZCHZOH), 3.11 (m, 2H, -CHZCHzOH). MS-EI 304
[M]+.
Compound II~i-0~~8
4-(2-Hydroxy-ethyl)-3-(4-methoxy-3-thiophen-2-yl-benzylidene)-1 3-dihydro-
indol-
2-one
o~
H
Tetrakis(triphenylphosphine)palladium(0) (0.24 g, 0.21 mmol) was added to
a solution of4-methoxy-3-bromobenzaldehyde (1.5 g, 6.98 mmol) in toluene (15
mL) and ethanol (15 mL), followed by a 2 M aqueous solution of sodium
carbonate
( 14 mL, 28 mmol). To this mixture was added thiophene-2-boronic acid (0.98 g,
7.68 mmol), and the mixture was heated to reflux. After 3 hours, the reaction
was
partitioned between water (100 mL) and ethyl acetate (250 mL). The organic
layer
was washed with saturated aqueous sodium bicarbonate (75 mL) and brine (75
mL),
dried with magnesium sulfate and concentrated. Chromatography (silica, 20%
ethyl
Y
acetate / hexanes) afforded 1.3 g (87%) of 4-methoxy-3-thiophen-2-yl-
benzaldehyde
as a yellow oil.
'H NMR (360 MHz, DMSO-d6) 8 9.92 (s, 1H, CHO), 8.2 (d, 1H, J= 3Hz,



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lxAr-H), 7.8 (dd, 1H, J= 10 and IOHz, SCHCHCH), 7.65 (m, 1H, Ar-H), 7.60 (dd,
1H, J= 2 and 6Hz, Ar-H), 7.32 (d, 1H, J= IOHz, SCHCHCH), 7.13 (d, 1H, J= 10
and 6Hz, SCHCHCH), 3:99 (s, 3H, OCH3).
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (1.6 g, 9.2 mmol),
4-methoxy-3-thiophen-2-yl-benzaldehyde (2 g, 9.2 mmol) and piperidine (4.5 mL,
85 mmol) in ethanol (46 mL) was heated to reflux for overnight. The reaction
mixture was concentrated and the residue was chromatographed on a column of
silica gel to give 1.63 g (47%) of 4-(2-hydroxy-ethyl)-3-(4-methoxy-3-thiophen-
2-
yl-benzylidene)-1,3-dihydro-indol-2-one as a yellow orange solid.
'HNMR (300 MHz, DMSO-d6) 8 10.53 (s, br, 1H, NH), 8.73 (d, J= 2.1 Hz,
1H), 8.10 (dd, J= 2.2 & 9.1 Hz, 1H), 7.74 (s, 1H, H-vinyl), 7.56-7.65 (m, 2H),
7.07-
7.22 (m, 3H), 6.81 (d, J= 7.5 Hz, 1H), 6.68 (dd, J= 0.6, 7.5 Hz, 1H), 4.87 (t,
J= 4.5
Hz, 1H, OH), 3.97 (s, 3H, OCH3), 3.68-3.75 (m, 2H, CHZCHZOH), 3.09 (t, J= 4.5
Hz, 2H, CHZCHZOH).
MS-EI 377 [M]+.
Compound IN-009
3-[4-(3-Dimethylamino-propel)-3,5-dimeth~pyrrol-2-ylmethylene]-4-~2-
h dery-ethyl)-1,3-dihydro-indol-2-one
_ i
OH N
N
H
O
N
H
To the suspension of 3-(2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid (10 g,
60.8 mmol) in 60 mL of dichloromethane was added 1,1'-carbonyldiimidazole
(11.6
g, 71.8 mmol) followed by the addition of 2 M dimethylamine solution in
tetrahydrofuran (30 mL) and N,N-diisopropylethylamine (Hunig's base, 10 mL,
60.8
mmol). The dark red reaction mixture was stirred at room temperature overnight
and poured into ice water. The organic layer was washed with brine until pH of
6,
dried over anhydrous sodium sulfate, and concentrated. The crude product was



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purified on a silica gel column eluting with dichloromethane-methanol (98:2)
to give
9.8 g (83%) of 3-(2,4-dimethyl-1H pyrrol-3-yl)-N,N dimethyl-propionamide as a
yellow oil.
To the suspension of lithium aluminum hydride (2.3 g, 60 mmol) in
tetrahydrofuran (50 mL) was added a solution of 3-(2,4-dimethyl-1H pyrrol-3-
yl)-
N,N dimethyl-propionamide (9.81 g, 50 mmol) in THF (50 mL) dropwise. The
reaction mixture was stirred at 80 °C for lhour and cooled with ice
bath. Ice cube
was added into the reaction mixture slowly until no more gas generated. A few
drops of 2 N sodium hydroxide was added and the reaction mixture was stirred
at
room temperature for 30 minutes, extracted with ethyl acetate, dried over
anhydrous
sodium sulfate and concentrated to give 6.8 g (75%) of [3-(2,4-dimethyl-1H
pyrrol-
3-yl)-propyl]-dimethyl-amine as an orange oil which was used without further
purification.
To the ice-cooled solution of N,N-dimethylformamide (6.0 mL, 76 mmol) in
dichloromethane (30 mL) was added phosphorus oxychloride (7.0 mL, 76 mmol)
dropwise. Upon addition of phosphorus oxychloride, the reaction mixture was
stirred
at room temperature for 15 minutes and a solution of [3-(2,4-dimethyl-1H
pyrrol-3-
yl)-propyl]-dimethyl-amine (6.8 g, 38 mmol) in dichloromethane (20 mL) was
added
dropwise at 0 °C. The final reaction mixture was refluxed at 60
°C for 4 hours and
cooled with ice bath. Ice cube was added to the reaction mixt~~~.> slowly
followed by
addition of 2 N sodium hydroxide to adjust pH to 12. The reaction mixture was
stirred at room temperature for 30 minutes and extracted with ethyl acetate.
The
organic layer was washed with brine, dried over anhydrous sodium sulfate and
concentrated to give crude product which was purified on a silica gel column
eluting
with dichloromethane-methanol-ammonium hydroxide (9:1:0.01) to give 5 g (63%)
of 4-(3-dimethylamino-propyl)-3,5-dimethyl-1H pyrrole-2-carbaldehyde as a dark
red oil.
'HNMR (360 MHz, DMSO-c16) 8 11.33 (s, br, 1H, NH-1), 9.40 (s, 1H, CHO
2), 2.30 (t, J= 7.42 Hz, 2H, (CH,)ZNCH~CH,CHZ-4), 2.18 (s, 3H, CH,-3), 2.15
(t, J=
7.42 Hz, 2H, (CH3),NCHzCH~CH,-4), 2.14 (s, 3H, CH3-5), 2.10 (s, 6H,
(CH3)ZNCH,CH,CH,-4), 1.47 (quint., J= 7.42 Hz, 2H, (CH3),NCH~CHZCHz-4); MS



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208 ([M+1]~.
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (177 mg, 1.0
mmol), 4-(3-dimethylamino-propyl)-3,5-dimethyl-1H pyrrole-2-carbaldehyde (208
mg, 1.0 mmol) and 3 drops of pyrrolidine in 2.0 mL of ethanol was refluxed at
90 °C
for 4 hours and cooled to room temperature. The precipitate was filtered,
washed
with cold ethanol and hexane, and dried in a vacuum oven overnight to give
360.6
mg (98%) of 3-[4-(3-dimethylamino-propyl)-3,5-dimethyl-1H pyrrol-2-
ylmethylene]-4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one.
'HNMR (300 MHz, DMSO-d6) 8 13.47 (s, br, 1H, NH), 10.79 (s, br, 1H,
NH), 7.56 (s, 1H, H-vinyl), 6.99 (m, 1H, Ar-H), 6.76 (m, 2H, Ar-H), 4.9 (s,
br, 1H,
OH), 3.7 (m, 2H, CHz), 3.07 (m, 2H, CHZ), 2.93 (m, 2H, CHz), 2.67 (s, 6H,
2xCHj),
2.42 (m, 2H, CHI), 2.29 (s, 3H, CH3), 2.20 (s, 3H, CH3), 1.73 (m, 2H, CHZ).
Compound IN-O10
Phenyl-thiocarbamic acid O-f2-(2-oxo-2.3-dihydro-1H-indol-4-yl)-ethyl] ester
0~ N
H
O
N
H
Phenyl isothiocyanate (0.45 mL, 3.75 mmol) was added dropwise to a stirred
mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (443 mg, 2.5 mmol) in
tetrahydrofuran (5 mL). The mixture was stirred at room temperature for 20
hours
and then 70 °C for 8 hours. One mL of dimethylforamide was added to the
mixture
to make it homogenous and the heating was continued for another 42 hours. The
reaction was cooled, poured into 1 N sodium hydroxide solution ( 100 mL) and
extracted with ethyl acetate (200 mL). The organic layer was washed with 1 N
hydrochloric acid (100 mL) and brine, dried over anhydrous sodium sulfate and
concentrated. The residue was recrystallized from methanol/ethyl
acetate/hexane to
give 452 mg (58%) of phenyl-thiocarbamic acid O-[2-(2-oxo-2,3-dihydro-1H-indol-




CA 02368041 2001-09-24
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118
4-yl)-ethyl] ester as a white solid.
'HNMR (360 MHz, DMSO-cl6) 8 11.00 (s, br, 1H, NH), 10.30 (s, br, 1H,
NH), 7.58 (s, br, 1H, Ar-H), 7.25 (s, br, 3H, Ar-H), 7.11 (t, J= 7.9 Hz, 2H,
Ar-H),
6.85 (m, 1H, Ar-H), 6.68 (d, J= 7.2 Hz, 1H, Ar-H), 4.67 (m, 2H, OCHZCHZ), 3.27
(s, 2H, H-3), 2.95 (m, 2H, OCHZCHZ). MS-EI 312 [M]+.
Compound IN-011
Phenvl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester
0~ N
H
O
N
H
Phenyl isocyanate (0.652 mL, 6 mmol) was added dropwise to a stirred
mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (709 mg, 4 mmol) in
tetrahydrofuran (8 mL), dimethylforamide (2 mL) and pyridine (3 drops). The
mixture was heated at 70 °C for 15 hours. The reaction was cooled,
poured into 1 N
sodium hydroxide solution (100 mL) and extracted with ethyl acetate (200 mL).
The
1 S organic layer was washed with 1 N hydrochloric acid ( 100 mL) and brine,
dried over
anhydrous sodium sulfate and concentrated. The residue was recrystallized from
methanol/ethyl acetate/hexane to give 953 mg (80%) of phenyl-carbamic acid 2-
(2-
oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester as an off white powder.
'HNMR (360 MHz, DMSO-d6) 8 10.30 (s, br, 1H, NH), 9.52 (s, br, 1H,
NH), 7.42 (d, J= 7.9 Hz, 2H, Ar-H), 7.24 (m, 2H, Ar-H), 7.11 (t, J= 7.5 Hz,
1H,
Ar-H), 6.96 (m, 1 H, Ar-H), 6.84 (d, J = 7.5 Hz, 1 H, Ar-H), 6.69 (d, J = 7.5
Hz, 1 H,
Ar-H), 4.28 (t, J= 6.8 Hz, 2H, OCHZCHZ), 3.48 (s, 2H, H-3), 2.85 (t, J= 6.8
Hz, 2H,
OCHZCHZ). MS-EI 296 [M]+.



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Compound IN-012
tent-Butyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester
0
O~N
H
O
N
H
Tert-butyl isocyanate (0.685 mL, 6 mmol) was added dropwise to a stirred
mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (709 mg, 4 mmol) in
tetrahydrofuran (8 mL), dimethylforamide (2 mL) and pyridine (3 drops). The
mixture was heated at 70 °C for 15 hours and 80 °C for 39 hours.
0.457 mL of tert-
butyl isocyanate was added to the reaction and continued to heat at 90
°C for 24
hours. More tert-butyl isocyanate (0.457 mL) was added and continued to heat
at 90
°C for 37 hours. The reaction was cooled, poured into 1 N sodium
hydroxide
solution (100 mL) and extracted with ethyl acetate (200 mL). The organic layer
was
washed with 1 N hydrochloric acid ( 100 mL) and brine, dried over anhydrous
sodium sulfate and concentrated. The residue was recrystallized from
methanol/ethyl acetate/hexane to give 130 mg (12%) of tert-butyl-carbamic acid
2-
(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester.
'HNMR (300 MHz, DMSO-ct6) 8 10.30 (s, br, 1H, NH), 7.09 (t, J= 7.6 Hz,
1H, Ar-H), 6.81 (s, 1H, NHC(CH3)3, 6.80 (d, J= 7.6 Hz, 1H, Ar-H), 6.65 (d, J=
7.6
Hz, 1H, Ar-H), 4.07 (m, 2H, OCHZCH,), 3.45 (s, 2H, H-3), 2.73-2.74 (m, 2H,
OCH2CH2), 1.18 (s, 3H, 3xCH3). MS-EI 276 [M]+.



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Compound IN-013
Cyclohex~l-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester
0
O~ N
H
O
N
H
Cyclohexyl isocyanate (0.766 mL, 6 mmol) was added dropwise to a stirred
mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (709 mg, 4 mmol) in
tetrahydrofuran (8 mL), dimethylforamide (2 mL) and pyridine (3 drops). The
mixture was heated at 70 °C for 16 hours. 0.511 mL of cyclohexyl
isocyanate was
added to the reaction and continued to heat at 80 °C for 24 hours. More
cyclohexyl
isocyanate (0.255 mL) was added and continued to heat at 85 °C for 24
hours. The
reaction was cooled, poured into 1 N sodium hydroxide solution (100 mL) and
extracted with ethyl acetate (200 mL). The organic layer was washed with 1 N
hydrochloric acid (100 mL) and brine, dried over anhydrous sodium sulfate and
concentrated. The residue was recrystallized from methanol/ethyl
acetate/hexane to
give 317 mg of the product. A second crop of product (32 mg) was obtained from
I S the mother liquor followed by column chromatography (1:1 ethyl acetate:
hexane).
A total of 349 mg (29%) of cyclohexyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-
indol-4-yl)-ethyl ester as a white powder was obtained.
'HNMR (360 MHz, DMSO-cl6) 8 10.28 (s, br, 1H, NH), 7.09 (t, J= 7.6 Hz,
1H, Ar-H), 6.97 (d, br, J= 6.6 Hz, 1H, CONHCH), 6.79 (d, J= 7.6 Hz, 1H, Ar-H),
6.65 (d, J = 7.6 Hz, 1 H, Ar-H), 4.11 (t, J = 6.6 Hz, 2H, OCHZCHZ), 3.44 (s,
2H, H
3), 3.25-3.35 (m, 1H, CONHCH), 2.75 (t, J= 6.6 Hz, 2H, OCHZCHZ), 1.5-1.8 (m, c-

hexyl). MS-EI 302 [M]+.



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Compound IN-014
Benzene sulfonyl-carbamic acid 2-(2-oxo-2 3-dihydro-1H-indol-4-yl)-ethyl ester
0
~o:s:o
I\
i
I~ o
N
H
Benzene sulfonyl isocyanate (1.07 mL, 8 mmol) was added dropwise to a
stirred mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (709 mg, 4
mmol) in
tetrahydrofuran (8 mL), dimethylforamide (2 mL) and pyridine (3 drops) under
nitrogen atmosphere. The mixture was heated at 80 °C for 15 hours. The
reaction
was cooled, poured into 1 N sodium hydroxide solution (100 mL) and extracted
with
ethyl acetate (200 mL). The organic layer was discarded and the basic aqueous
layer
was acidified with 6 N hydrochloric acid (18 mL) followed by extraction into
ethyl
acetate. The organic layer was washed with brine, dried over anhydrous sodium
sulfate and concentrated. The residue was recrystallized from methanol/ethyl
acetate/hexane to give 390 mg (27%) of benzene sulfonyl-carbamic acid 2-(2-oxo-

2,3-dihydro-1H-indol-4-yl)-ethyl ester as an off white powder.
'HNMR (360 MHz, DMSO-d~) 8 12.03 (s, br, 1H, NH), 10.30 (s, br, 1H,
NH), 7.83 (d, J= 7.2 Hz, 2H, Ar-H), 7.68-7.72 (m, 1H, Ar-H), 7.58-7.62 (m, 2H,
Ar-H), 7.05 (t, J= 7.7 Hz, 1H), 6.71 (d, J= 7.7 Hz, 1H), 6.64 (d, J= 7.7 Hz,
1H),
4.17 (t, J= 6.3 Hz, 2H, OCH~CHZ), 3.42 (s, 2H, H-3), 2.71 (t, J= 6.3 Hz, 2H,
OCHZCHZ). MS 360 [M]+.
Co found IN-Ol 5
Biphenyl-2-yl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-vl)-ethyl ester
0
O~N \ I
H
I
I~ o
N
H
2-Biphenylyl isocyanate (1.15 mL, 6.7 mmol) was added dropwise to a



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stirred mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (709 mg, 4
mmol) in
tetrahydrofuran (8 mL), dimethylforamide (2 mL) and pyridine (3 drops) under
nitrogen atmosphere. The mixture was heated at 80 °C for 15 hours. The
reaction
was cooled, quenched with 1 N sodium hydroxide solution (100 mL) and extracted
with ethyl acetate (200 mL). The organic layer was washed with brine, dried
over
anhydrous sodium sulfate and concentrated. The residue was chromatographed on
silica gel eluting with ethyl acetate: hexane 3:2 to give 1.188 g (80%) of
biphenyl-2-
yl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester as a white
fluffy
solid.
'HNMR (360 MHz, DMSO-d6) 8 10.28 (s, br, 1H, NH), 8.57 (s, 1H, NH),
7.25-7.41 (m, 9H, Ar-H), 7.08 (t, J= 7.6 Hz, 1H, Ar-H), 6.73 (d, J= 7.6 Hz,
1H, Ar-
H), 6.66 (d, J= 7.6 Hz, 1H, Ar-H), 4.09 (t, J= 6.4 Hz, 2H, OCHZCHZ), 3.35 (s,
2H,
CH,, H-3), 2.69 (~, J= 6.4 Hz, 2H, OCHZCHZ). MS-EI 372 [M]+.
Compound IN-016
Ethyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester
0
o~ N''
H
O
N
H
Ethyl isocyanate (0.633 mL, 8 mmol) was added dropwise to a stirred
mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (709 mg, 4 mmol) in
tetrahydrofuran (8 mL) and dimethylforamide (2 mL) under nitrogen atmosphere.
The mixture was heated at 80 °C for 48 hours. The reaction was cooled,
poured into
water (100 mL) and extracted with ethyl acetate (200 mL). The organic layer
was
washed with brine, dried over anhydrous sodium sulfate and concentrated. The
residue was chromatographed on silica gel eluting with ethyl acetate/ hexane
to give
158 mg (16%) of ethyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl
ester as a white powder.
'HNMR (360 MHz, DMSO-d6) 8 10.28 (s, br, 1H, NH), 7.08 (t, J= 7.7Hz,



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1 H, Ar-H), 7.03 (m, br, 1 H, NH), 6.78 (d, J = 7. 7Hz, 1 H, Ar-H), 6.64 (d, J
= 7.7Hz,
1H, Ar-H), 4.10 (t, J= 6.5 Hz, 2H, OCHzCH2), 3.43 (s, 2H, H-3), 2.91-2.99 (m,
2H,
CHZCH3), 2.74 (t, J= 6.5 Hz, 2H, OCH,CHZ), 0.96 (t, J= 7.3 Hz, 3H, CH,CH,).
MS-EI 248 [M]+.
Compound IN-017
4-[2-(4-Methoxy-phenoxy)-ethyl-1,3-dihydro-indol-2-one
~ I o~
0
0
I
N
H
Diethyl azodicarboxylate (0.47 mL, 3 mmol) was added to a solution of
triphenylphosphine (0.786 g, 3 mmol) in tetrahydrofuran (10 mL) under nitrogen
atmosphere. The mixture was stirred for 15 minutes. To it was then added 4-(2-
hydroxy-ethyl)-1,3-dihydro-indol-2-one (0.53 g, 3 mmol) followed by 4-
methoxyphenol (0.372 g, 3 mmol). The mixture was stirred at room temperature
for
18 hours and the solvent was evaporated. The residue was dissolved in ethyl
acetate
(150 mL) and the organic solvent was washed with 2 N hydrochloric acid (3x 30
mL), saturated sodium bicarbonate solution and brine. The residue was
chromatographed on silica gel eluting with ethyl acetate: hexane 2:8 to give
0.14 g
(16%) 4-[2-(4-Methoxy-phenoxy)-ethyl]-1,3-dihydro-indol-2-one.
'HNMR (300 MHz, DMSO-d6) b 10.33 (s, br, 1H, NH), 7.10 (t, J= 7.8 Hz,
1H, Ar-H), 6.83-6,87 (m, SH, Ar-H), 6.67 (d, J= 7.5 Hz, 1H, Ar-H), 4.10 (t, J=
6.9
Hz, 2H, OCHZCHZ), 3.67 (s, 3H, OCH3), 3.49 (s, 2H, CHz, H-3), 2.91 (t, J= 6.9
Hz,
2H, OCHZCHZ).
Compound IN-018
4-(2-Methoxy-ethyl)-1,3-dihydro-indol-2-one



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o~
0
N
H
Methyl iodide (1.41 g, 10 mmol) was added to a stirred mixture of silver
trifluoromethanesulfonate (2.56 g, 10 mmol) and 4-(2-hydroxy-ethyl)-1,3-
dihydro-
indol-2-one (0.88 g, S mmol) in dichloromethane (20 mL) at 0 °C. The
precipitate
formed after 5-10 minutes changed color from yellow to gray then dark purple.
The
mixture was stirred at room temperature for 2 hours. The reaction mixture was
diluted with dichloromethane and filtered through celite. The filtrate was
concentrated and chromatographed on silica gel to give 4-(2-methoxy-ethyl)-1,3-

dihydro-indol-2-one.
'HNMR (360 MHz, DMSO-d6) 8 10.27 (s, br, 1H, NH), 7.07 (t, J= 7.6 Hz,
1 H, Ar-H), 6.78 (d, J = 7.6 Hz, 1 H, Ar-H), 6.65 (d, J = 7.6 Hz, 1 H, Ar-H),
3.51 (t, J
= 6.7 Hz, 2H, OCHzCH2), 3.42 (s, 2H, H-3, CHZ), 3.22 (s, 3H, OCH3), 2.71 (t,
J=
6.7 Hz, 2H, OCHZCHZ). MS-EI 191 [M)+.
Compound IN-019
4-(2-Ethoxy-ethyl)-1,3-dihydro-indol-2-one
o~
0
N
H
Ethyl iodide (0.47 mL, 6 mmol) was added to a stirred mixture of silver
trifluoromethanesulfonate (1.35 g, 5.2 mmol) 2,6-di-tert-butylpyridine (1 g,
5.2
mmol) and 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (0.53 g, 3 mmol) in
dichloromethane ( 10 mL) at 0 °C. The mixture was warmed to room
temperature
and stirred for 2 hours. The precipitate formed after S-10 minutes changed
color
from yellow to brown. The reaction mixture was diluted with dichloromethane
(100
mL) and washed with 1 N hydrochloric acid, saturated sodium bicarbonate and
brine, dried and concentrated. The residue was chromatographed on silica gel
to



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give 180 mg (29%) of 4-(2-ethoxy-ethyl)-1,3-dihydro-indol-2-one as a purple
oil.
'HNMR (360 MHz, DMSO-d6) 8 10.27 (s, br, 1H, NH), 7.07 (t, J= 7.8 Hz,
1 H, Ar-H), 6.78 (d, J = 7.8 Hz, 1 H, Ar-H), 6.65 (d, J = 7.8 Hz, 1 H, Ar-H),
3.54 (t, J
= 6.9 Hz, 2H, OCHzCH,), 3.38-3.45 (m, 2H, OCHZCH3), 3.43 (s, 2H, H-3, CHZ),
2.71 (t, J= 6.9 Hz, 2H, OCH,CHZ), 1.07 (t, J= 7.0 Hz, 3H, OCHZCH3). MS 206.2
[M+1 ]+.
Compound IN-020
4-[2-(4-Isopropyl-phenoxy)-ethyl-1,3-dihydro-indol-2-one
0
0
N
lO
Diethyl azodicarboxylate (1.58 mL, 10 mmol) was added to a solution of
triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under nitrogen
atmosphere. The mixture was stirred for 15 minutes. To it was then added 4-(2-
hydroxy-ethyl)-1,3-dihydro-indol-2-one (1.77 g, 10 mmol) followed by 4-
isopropylphenol (1.36 g, 10 mmol). The mixture was stirred at room temperature
for
one day and the solvent was evaporated. The residue was chromatographed on
silica
gel eluting with ethyl acetate: hexane 2:8 to give 1.1 g (37%) of 4-[2-(4-
isopropyl-
phenoxy)-ethyl]-1,3-dihydro-indol-2-one as a light yellow solid.
'HNMR (360 MHz, DMSO-d6) 8 10.30 (s, br, 1H, NH), 7.11 (d, J= 7.2 Hz,
2H), 7.1 (t, J= 7.6 Hz, 1H, Ar-H), 6.87 (d, J= 7.6 Hz, 1H), 6.82 (d, J= 7.2
Hz, 2H,
Ar-H), 6.68 (d, J= 7.6 Hz, 1H, Ar-H), 4.14 (t, J= 6.8 Hz, 2H, CHZCH20), 3.49
(s,
2H, H-3, CHZ), 2.93 (t, J= 6.8 Hz, 2H, CHzCH,O), 2.81 (m, 1H, CH(CH3)Z), 1.15
(d,
J= 6.5 Hz, 6H, CH(CH3)z).
MS-EI 295 [M]+.
Compound IN-021



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4-(2-(5-Chloro-pyridin-3-yloxy)-ethyl]-1,3-dihydro-indol-2-one
c1
'1
\ N
0
0
N
H
Diethyl azodicarboxylate (1.74 g, 10 mmol) was added to a solution of
triphenylphosphine (2.62 g, 10 mmol) in tetrahydrofuran (20 mL) under nitrogen
S atmosphere. The mixture was stirred for 15 minutes. To it was then added 4-
(2-
hydroxy-ethyl)-1,3-dihydro-indol-2-one (1.77 g, 10 mmol) followed by 5-chloro-
3-
pyridinol (1.29 g, 10 mmol). The mixture was stirred at room temperature for 2
days. The precipitate was collected by vacuum filtration, washed with ethyl
acetate
and dried to give 1.05 g (36%) of 4-[2-(5-chloro-pyridin-3-yloxy)-ethyl]-1,3-
dihydro-indol-2-one as a light brown solid.
'HNMR (360 MHz, DMSO-d6) 8 10.31 (s, br, 1H, NH), 8.23 (d, J= 2.3 Hz,
1 H, Ar-H), 8.17 (d, J = 2.3 Hz, 1 H, Ar-H), 7.5 8 (t, J = 2.3 Hz, 1 H, Ar-H),
7.11 (t, J
= 7.9 Hz, 1 H, Ar-H), 6.87 (d, J = 7.9 Hz, 1 H, Ar-H), 6.68 (d, J = 7.9 Hz, 1
H, Ar-H),
4.30 (t, J= 6.9 Hz, 2H, CHZCH20), 3.51 (s, 2H, CHI, H-3), 2.97 (t, J= 6.9 Hz,
2H,
CHZCH,O). MS-EI 288 [M]+.
Compound IN-022
4-(2-Hydroxy-ethyl)-3-(3-l~droxy-6-methyl-~rridin-2-ylmethylene)-1,3-dihydro-
indol-2-one
OH
N'
\ ~ H
N
H
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (51 mg, 0.3
mmol), 3-hydroxy-6-methyl-pyridine-2-carbaldehyde (45 mg, 0.33 mmol) and 1
drop of pyrrolidine in 2.0 mL of ethanol was heated at 90 °C for 4
hours and cooled



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to room temperature. The precipitate was filtered, washed with cold ethanol
and
hexane, and dried in a vacuum oven overnight to give mixture of isomers of 4-
(2-
hydroxy-ethyl)-3-(3-hydroxy-6-methyl-pyridin-2-ylmethylene)-1,3-dihydro-indol-
2-
one.
MS-EI 296 [M]+.
Compound IN-023
3-[~3-Dimethylamino-propel)-3,5-dimethyl-1 H-pyrrol-2-ylmethylene]-4-(2-(3-
isopropyl-phenox~)-ethyll-1,3-dihydro-indol-2-one
i
0 N
_ 1
N
H
0
N
H
A mixture of 4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one (28
mg, 0.095 mmol), 4-(3-dimethylamino-propyl)-3,5-dimethyl-1H pyrrole-2-
carbaldehyde (20 mg, 0.095 mmol) and 1 drop of pyrrolidine in 1.0 mL of
ethanol
was heated at 90 °C for 4 hours and cooled to room temperature. The
precipitate
1 S was filtered, washed with cold ethanol and hexane, and dried in a vacuum
oven
overnight to give 40 mg (87%) of 3-[4-(3-dimethylamino-propyl)-3,5-dimethyl-1H-

pyrrol-2-ylmethylene]-4-[2-(3-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-
one.
'HNMR (360 MHz, DMSO-d6) b 13.44 (s, 1H, NH), 10.77 (s, 1H, CONH),
7.59 (s, 1H, H-vinyl), 7.16 (t, J= 7.2 Hz, 1H), 7.04 (t, J= 7.5 Hz, 1H), 6.89
(d, J=
7.5 Hz, 1H, H-5), 6.79 (d, J= 7.1 Hz, 1H), 6.77 (d, J= 7.2 Hz, 1H), 6.75 (s,
1H),
6.72 (d, J= 7.5 Hz, 1H), 4.23 (t, J= 6.8 Hz, 2H), 3.39 (t, J= 7.4 Hz, 2H),
2.80 (m,
1H, CH(CH3)z), 2.34 (t, J= 7.4 Hz, 2H), 2.27 (s, 3H, CH3-pyrrole), 2.13 (t, J=
7.4
Hz, 2H), 2.08 (s, 6H, (CH3)ZNCHzCH~CHz), 2.04 (s, 3H, CH3-pyrrole), 1.47
(quint.,
J= 7.4 Hz, 2H, (CH3)zNCHZCHZCH,), 1.13 (d, J = 7.2 Hz, 6H,CH(CH3)2). MS-EI
485 [M]+.
Compound IN-024



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3-(5-14-f 2-(4-Isopropyl-phenoxy)-ethyl]-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl ~-
2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid
0
O OH
N
H
O
N
H
2-Benzyloxycarbonyl-3,5-dimethyl-4-(2-methoxycarbonylethyl)pyrrole
(Aldrich, 2.0 g) was hydrogenated over 0.2 g of 10 % palladium on carbon in 40
mL
of methanol for 2 hours at room temperature. The catalyst was removed by
filtration and washed with 40 mL of methanol. The filtrate was evaporated to
dryness and dried overnight in a vacuum oven to give 1.3 g (92 % yield) of 2-
carboxy-3,5-dimethyl-4-(2-methoxycarbonylethyl)pyrrole.
2-Carboxy-3,5-dimethyl-4-(2-methoxycarbonylethyl)pyrrole (1.3 g) was
ground with 0.5 g of anhydrous sodium acetate and then heated at 100 °C
for 3 days.
The mixture was dissolved in water, extract with ethyl acetate and
chromatographed
on a column of silica gel in ethyl acetate: hexane 1:3 to give 0.4 g (38 %
yield) of
2,4-dimethyl-3-(2-methoxycarbonylethyl)pyrrole as a thick pale yellow oil.
2,4-Dimethyl-3-(2-methoxycarbonylethyl)pyrrole (0.35 g) and 0.5 g of the
Vilsmeier reagent (Aldrich) in 10 mL of dichloroethane were stirred at room
temperature under nitrogen for 2 hours. The reaction mixture was concentrated
and
treated with 10 mL of 6 M sodium hydroxide solution and then extracted three
times
with 10 mL of ethyl acetate. The ethyl acetate extracts were combined, dried
over
anhydrous sodium sulfate and evaporated to dryness to give 0.24 g (60 % yield)
of
2,4-dimethyl-5-formyl-3-(2-methoxycarbonylethyl)pyrrole as a brown oil.
2,4-Dimethyl-5-formyl-3-(2-methoxycarbonylethyl)pyrrole (0.23 g, 1.2
mmol) in 6N sodium hydroxide (10 mL) was heated at 100 °C for 2 hours.
The
reaction mixture was cooled to room temperature, acidified with 6 N
hydrochloric
acid and extracted three times with 10 mL of ethyl acetate. The combined
organic
layers were washed with 10 mL of water and 5 mL of brine, dried over anhydrous



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sodium sulfate and evaporated to dryness to give 230 mg (106 % yield) of crude
4-
carboxyethyl-3,5-dimethyl-2-formylpyrrole as a brown oil.
A mixture of 4-[2-(4-isopropyl-phenoxy)-ethyl]-1,3-dihydro-indol-2-one
(118 mg, 0.4 mmol), 4-carboxyethyl-3,5-dimethyl-2-formylpyrrole (78 mg, 0.4
mmol) and piperidine (0.3 mL) in 1.0 mL of ethanol was heated in a sealed tube
at
90 °C for 18 hours and cooled to room temperature. The reaction mixture
was
concentrated and the residue was dissolved in methylene chloride followed by
the
addition of diethyl ether. The precipitate was filtered and washed with ethyl
acetate
to give 110 mg (58%) of 3-(5- f 4-[2-(4-isopropyl-phenoxy)-ethyl]-2-oxo-1,2-
dihydro-indol-3-ylidenemethyl}-2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid
(piperidine salt).
'HNMR (300 MHz, DMSO-d6) 8 13.42 (s, 1H, NH), 10.82 (s, br, 1H, NH),
7.55 (s, 1H, H-vinyl), 7.09 (d, J= 8.7 Hz, 2H), 7.02 (t, J= 7.5 Hz, 1H), 6.87
(d, J=
7. S Hz, 1 H), 6.83 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 7.5 Hz, 1 H), 4.21 (t, J
= 7.4 Hz,
1 S 2H, CH,CHZO), 3.37 (t, J= 6.7 Hz, 2H, CHZ), 2.72-2.82 (m, 1H, CH(CH3)2),
2.56 (t,
J= 6.7 Hz, 2H, CH,), 2.26 (s, 3H, CH3), 2.14 (t, J= 7.4 Hz, 2H, CHZCH20), 2.04
(s,
3H, CH3), 1.13 (d, J= 7 Hz, 6H, CH(CH3)Z). MS-EI 472 [M]+.
Compound IN-025
Isopropyl-carbamic acid 2-(2-oxo-2,3-dihydro-1H-indol-4-~)-~,;~lwl ester
0'I
O~N
H
0
N
H
A mixture of 4-(2-hydroxy-ethyl)-1,3-dihydro-indol-2-one (3 g, 16.93 mmol)
and isopropyl isocyanate (2.49 mL, 25.40 mmol) in tetrahydrofuran (16 mL) and
dimethylformamide (6 mL) was heated at 80 °C for 64 hours. The reaction
mixture
was poured into water and extracted with ethyl acetate (400 mL), washed with
brine,
dried (sodium sulfate), and concentrated. The residue was recrystallized form



CA 02368041 2001-09-24
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130
methanol, ethyl acetate and hexanes to give 1.136 g (26%) of isopropyl-
carbamic
acid 2-(2-oxo-2,3-dihydro-1H-indol-4-yl)-ethyl ester as crystalline solid.
ASSAY PROCEDURES
The following in vitro assays may be used to determine the level of activity
and effect of the different compounds of the present invention on one or more
of the
PKs. Similar assays can be designed along the same lines for any PK using
techniques well known in the art.
The cellular/catalytic assays described herein are performed in an ELISA
format. The general procedure is a follows: a compound is introduced to cells
expressing the test kinase, either naturally or recombinantly, for some period
of time
after which, if the test kinase is a receptor, a ligand known to activate the
receptor is
added. The cells are lysed and the lysate is transferred to the wells of an
ELISA
plate previously coated with a specific antibody recognizing the substrate of
the
enzymatic phosphorylation reaction. Non-substrate components of the cell
lysate are
washed away and the amount of phosphorylation on the substrate is detected
with an
antibody specifically recognizing phosphotyrosine compared with control cells
that
were not contacted with a test compound.
The cellular/biologic assays described herein measure the amount of DNA
made in response to activation of a test kinase, which is a general measure of
a
proliferative response. The general procedure for this assay is as follows: a
compound is introduced to cells expressing the test kinase, either naturally
or
recombinantly, for some period of time after which, if the test kinase is a
receptor, a
ligand known to activate the receptor is added. After incubation at least
overnight, a
DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or 3H-thymidine is
added. The amount of labeled DNA is detected with either an anti-BrdU antibody
or
by measuring radioactivity and is compared to control cells not contacted with
a test
compound.



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Cellular/Catalytic Assts
Enzyme linked immunosorbent assays (ELISA) may be used to detect and
measure the presence of PK activity. The ELISA may be conducted according to
known protocols which are described in, for example, Voller, et al., 1980,
"Enzyme-
Linked Immunosorbent Assay," In: Manual of Clinical Immunology, 2d ed., edited
by Rose and Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C.
The disclosed protocol may be adapted for determining activity with respect
to a specific PK. For example, the preferred protocols for conducting the
ELISA
experiments for specific PKs is provided below. Adaptation of these protocols
for
determining a compound's activity for other members of the RTK family, as well
as
for CTKs and STKs, is well within the scope of knowledge of those skilled in
the
art.
EXAMPLE 2: FLK-1
An ELISA assay was conducted to measure the kinase activity of the FLK-1
receptor and more specifically, the inhibition or activation of TK activity on
the
FLK-1 receptor. Specifically, the following assay was conducted to measure
kinase
activity of the FLK-1 receptor in cells genetically engineered to express Flk-
1.
Materials and Methods.
Materials.
The following reagents and supplies were used:
a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);
b. Cappel goat anti-rabbit IgG (catalog no. 55641);
c. PBS (Gibco Catalog No. 450-1300EB);
d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCI and 0.1% Tween-20);
e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4 °C);
f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCI, 0.2% Triton
X-100, and 10% glycerol);
g. EDTA (0.5 M (pH 7.0) as a 100X stock);



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h. Sodium orthovanadate (0.5 M as a 100X stock);
i. Sodium pyrophosphate (0.2 M as a 100X stock);
j. NUNC 96 well V bottom polypropylene plates (Applied Scientific Catalog
No. AS-72092);
k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);
1. DMEM with 1X high glucose L-Glutamine (catalog No. 11965-050);
m. FBS, Gibco (catalog no. 16000-028);
n. L-glutamine, Gibco (catalog no. 25030-016);
o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 ~g/100 ~L stock in
Milli-Q dH~O and stored at -20 °C;
p. Affinity purified anti-FLK-1 antiserum;
q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley, et
al., 1990, Cancer Research 50:1550-1558);
r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);
s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution (100
mM citric acid (anhydrous), 250 mM Na,HP04 (pH 4.0), 0.5 mg/mL ABTS (Sigma
catalog no. A-1888)), solution should be stored in dark at 4 °C until
ready for use;
t. H20z (30% solution) (Fisher catalog no. H325);
u. ABTS/H20, (15 mL ABTS solution, 2 ~L H,Oz) prepared S minutes before
use and left at room temperature;
v. 0.2 M HCl stock in HBO;
w. dimethylsulfoxide (100%) (Sigma Catalog No. D-8418); and
y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).
Protocol
The following protocol was used for conducting the assay:
1. Coat Corning 96-well ELISA plates with 1.0 pg per well Cappel Anti-rabbit
IgG antibody in 0.1 M NazC03 pH 9.6. Bring final volume to 150 ~.L per well.
Coat plates overnight at 4 °C. Plates can be kept up to two weeks when
stored at 4
°C.
2. Grow cells in Growth media (DMEM, supplemented with 2.0 mM L-



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133
Glutamine, 10% FBS) in suitable culture dishes until confluent at 37
°C, 5% CO~.
3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene 96-
well round bottom cell plates, 25.000 cells/well in 200 ~L of growth media.
4. Grow cells at least one day at 37 °C, S% CO,.
5. Wash cells with D-PBS 1X.
6. Add 200 ~.L/well of starvation media (DMEM, 2.0 mM 1-Glutamine, 0.1
FBS). Incubate overnight at 37 °C, 5% COZ.
7. Dilute Compounds 1:20 in polypropylene 96 well plates using starvation
media. Dilute dimethylsulfoxide 1:20 for use in control wells.
8. Remove starvation media from 96 well cell culture plates and add 162 ~L of
fresh starvation media to each well.
9. Add 18 ~.L of 1:20 diluted Compound dilution (from step 7) to each well
plus the 1:20 dimethylsulfoxide dilution to the control wells (+/- VEGF), for
a final
dilution of 1:200 after cell stimulation. Final dimethylsulfoxide is 0.5%.
Incubate
1 S the plate at 37 °C, 5% COZ for two hours.
10. Remove unbound antibody from ELISA plates by inverting plate to remove
liquid. Wash 3 times with TBSW + 0.5% ethanolamine, pH 7Ø Pat the plate on a
paper towel to remove excess liquid and bubbles.
11. Block plates with TBSW + 0.5% Ethanolamine, pH 7.0, 150 ~L per well.
Incubate plate thirty minutes while shaking on a microtiter plai.4shaker.
12. Wash plate 3 times as described in step 10.
13. Add 0.5 ~g/well affinity purified anti-FLU-1 polyclonal rabbit antiserum.
Bring final volume to 150 ~,L/well with TBSW + 0.5% ethanolamine pH 7Ø
Incubate plate for thirty minutes while shaking.
14. Add 180 ~L starvation medium to the cells and stimulate cells with 20
~L/well 10.0 mM sodium ortho vanadate and 500 ng/mL VEGF (resulting in a final
concentration of 1.0 mM sodium ortho vanadate and SO ng/mL VEGF per well) for
eight minutes at 37 °C, S% COz. Negative control wells receive only
starvation
medium.
15. After eight minutes, media should be removed from the cells and washed one
time with 200 pL/well PBS.



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134
16. Lyse cells in 150 ~L/well HNTG while shaking at room temperature for five
minutes. HNTG formulation includes sodium ortho vanadate, sodium
pyrophosphate and EDTA.
17. Wash ELISA plate three times as described in step 10.
18. Transfer cell lysates from the cell plate to ELISA plate and incubate
while
shaking for two hours. To transfer cell lysate pipette up and down while
scrapping the
wells.
19. Wash plate three times as described in step 10.
20. Incubate ELISA plate with 0.02 ~g/well UB40 in TBSW + OS%
ethanolamine. Bring final volume to 150 pL/well. Incubate while shaking for 30
minutes.
21. Wash plate three times as described in step 10.
22. Incubate ELISA plate with 1:10,000 diluted EIA grade goat anti-mouse IgG
conjugated horseradish peroxidase in TBSW + 0.5% ethanolamine, pH 7Ø Bring
1 S final volume to 150 p.L/well. Incubate while shaking for thirty minutes.
23. Wash plate as described in step 10.
24. Add 100 ~L of ABTS/HZO~ solution to well. Incubate ten minutes while
shaking.
25. Add 100 pL of 0.2 M HCl for 0.1 M HCl final to stop the color development
reaction. Shake 1 minute at room temperature. Remove bubbles with slow stream
of air and read the ELISA plate in an ELISA plate reader at 410 nm.
EXAMPLE 3: HER-2 ELISA
Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells.
HERZ kinase activity in whole EGFR-NIH3T3 cells was measured as
described below:
Materials and Reagents
The following materials and reagents were used to conduct the assay:
a. EGF: stock concentration: 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.



CA 02368041 2001-09-24
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135
b. OS-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular
domain).
c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see, Fendley, et
al.,
supra).
d. Detection antibody: Goat anti-rabbit 1gG horse radish peroxidase conjugate,
TAGO, Inc., Burlingame, CA.
e. TBST buffer:
Tris-HCI, pH 7.2 50 mM
NaCI 150 mM
Triton X-100 0.1
f. HNTG SX stock:


HEPES 0.1 M


NaCI 0.75 M


Glycerol 50%


Triton X-100 1.0%


g. ABTS stock:


Citric Acid 100 mM


Na~HP04 250 mM


HCI, conc. 0.5 pM


ABTS' 0.5 mg/mL


* (2,2'-azinobis(3-ethylbenzthiazolinesulfonic
acid)). Keep solution
in dark at 4 C


until use.


h. Stock reagents o~
EDTA 100 mM pH 7.0
NajVOa 0.5 M
Naa(P20,) 0.2 M
Procedure
The following protocol was used:
A. Pre-coat ELISA Plate
1. Coat ELISA plates (Corning, 9G well, Cat. #25805-9G) with OS-101 antibody
at 0.5 g per well in PBS, 100 pL final volume/well, and store overnight at 4
°C.
Coated plates are good for up to 10 days when stored at 4 °C.
2. On day of use, remove coating buffer and replace with 100 p.L blocking
buffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubate the plate,
shaking,
at room temperature (about 23 °C to 25 °C) for 30 minutes. Just
prior to use, remove



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136
blocking buffer and wash plate 4 times with TBST buffer.
B. Seeding Cells
1. An NIH3T3 cell line overexpressing a chimeric receptor containing the
EGFR extracellular domain and intracellular HER2 kinase domain can be used for
this assay.
2. Choose dishes having 80-90% confluence for the experiment. Trypsinize
cells and stop reaction by adding 10% fetal bovine serum. Suspend cells in
DMEM
medium ( 10% CS DMEM medium) and centrifuge once at 1500 rpm, at room
temperature for 5 minutes.
3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count
the cells using trypan blue. Viability above 90% is acceptable. Seed cells in
DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 p.L
per well, in a 96 well microtiter plate. Incubate seeded cells in 5% CO~ at 37
°C for
about 40 hours.
C. Assay Procedures
1. Check seeded cells for contamination using an inverted microscope. Dilute
drug stock (10 mg/mL in DMSO) 1:10 in DMEM medium, then transfer 5 pL to a
TBST well for a final drug dilution of 1:200 and a final DMSO concentration of
1%.
Control wells receive DMSO alone. Incubate in 5% CO~ at 37 °C for two
hours.
2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10
pL dilute EGF (1:12 dilution), 100 nM final concentration is attained.
3. Prepare fresh HNTG' sufficient for 100 ~L per well; and place on ice.
HNTGk (10 ml):
HNTG stock 2.0 mL
milli-Q H20 7.3 mL
EDTA, 100 mM, pH 7.0 0.5 mL
Na3 V 04, 0. 5 M 0.1 mL
Na4(P,O,), 0.2 M 0.1 mL
4. After 120 minutes incubation with drug, add prepared SGF ligand to cells,
10
~L per well, to a final concentration of 100 nM. Control wells receive DMEM
alone.



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137
Incubate, shaking, at room temperature, for 5 minutes.
5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer
HNTG' to cells, 100 p.L per well. Place on ice for 5 minutes. Meanwhile,
remove
blocking buffer from other ELISA plate and wash with TBST as described above.
G. With a pipette tip securely fitted to a micropipettor, scrape cells from
plate
and homogenize cell material by repeatedly aspirating and dispensing the HNTGi
lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate.
Incubate
shaking at room temperature for one hour.
7. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-
Ptyr antibody to ELISA plate at 100 yL per well. Incubate shaking at room
temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000
dilution in TBST).
8. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the
freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at 100 pL per
well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG
antibody:
1:3000 dilution in TBST).
9. Remove TAGO detection antibody and wash 4 times with TBST. Transfer
freshly prepared ABTS/HZO~ solution to ELISA plate, 100 ~L per well. Incubate
shaking at room temperature for 20 minutes. (ABTS/HZOZ solution: 1.0 ~L 30%
H,O, in 10 mL ABTS stock).
10. Stop reaction by adding 50 ~L SN H=SO~ (optional), and determine O.D. at
410 nm.
11. The maximal phosphotyrosine signal is determined by subtracting the value
of the negative controls from the positive controls. The percent inhibition of
phosphotyrosine content for extract-containing wells is then calculated, after
subtraction of the negative controls.
EXAMPLE 4: PDGF-R ELISA
All cell culture media, glutamine, and fetal bovine serum were purchased
from Gibco Life Technologies (Grand Island, NY) unless otherwise specified.
All
cells were grown in a humid atmosphere of 90-95% air and 5-10% CO, at 37
°C.



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All cell lines were routinely subcultured twice a week and were negative for
mycoplasma as determined by the Mycotect method (Gibco).
For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU)
were grown to 80-90% confluency in growth medium (MEM with 10% FBS,
NEAR, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in
0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in
0.5%
serum-containing medium, cells were changed to serum-free medium and treated
with test compound for 2 hr in a 5% CO,, 37 °C incubator. Cells were
then
stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM
Hepes, 150 mM NaCI, 10% glycerol, 5 mM EDTA, 5 mM Na3V04, 0.2% Triton X-
100, and 2 mM NaPyr). Cell lysates (0.5 mg/well in PBS) were transferred to
ELISA plates previously coated with receptor-specific antibody and which had
been
blocked with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCI and 0.1%
Triton X-100) at room temperature for 30 min. Lysates were incubated with
shaking
for 1 hour at room temperature. The plates were washed with TBST four times
and
then incubated with polyclonal anti-phosphotyrosine antibody at room
temperature
for 30 minutes. Excess anti-phosphotyrosine antibody was removed by rinsing
the
plate with TBST four times. Goat anti-rabbit IgG antibody was added to the
ELISA
plate for 30 min at room temperature followed by rinsing with TBST four more
times. ABTS (100 mM citric acid, 250 mM Na,HP04 and 0.5 mg/mL 2,2'-azino-
bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H,Oz (1.2 mL 30% H,O~ to 10
mL
ABTS) was added to the ELISA plates to start color development. Absorbance at
410 nm with a reference wavelength of 630 nm was recorded about 1 S to 30 min
after ABTS addition.
EXAMPLE 5: IGF-I RECEPTOR ELISA
The following protocol may be used to measure phosphotyrosine level on
IGF-I receptor, which indicates IGF-I receptor tyrosine kinase activity.
Materials and Reagents
The following materials and reagents were used:



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139
a. The cell line used in this assay is 3T3/IGF-1R, a cell line genetically
engineered to overexpresses IGF-1 receptor.
b. NIH3T3/IGF-1R is grown in an incubator with 5% CO~ at 37 °C. The
growth
media is DMEM + 10% FBS (heat inactivated)+ 2 mM L-glutamine.
c. Affinity purified anti-IGF-1R antibody 17-69.
d. D-PBS:
KH,POa 0.20 g/L
K,HP04 2.16 g/L
KCl 0.20 g/L
NaCI 8.00 g/L(pH 7.2)
e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat Dry
Milk).
~ TBST buffer:
Tris-HCl 50 mM
NaCI 150 mM (pH 7.2/HCl 10 N)
Triton X-100 0.1%
Stock solution of TBS (10X) is prepared, and Triton X-100 is added to the
buffer
during dilution.
g. HNTG buffer:
HEPES 20 mM
NaCI 150 mM (pH 7.2/HCl 1N)
Glycerol 10%
Triton X-100 0.2%
Stock solution (SX) is prepared and kept at 4 °C.
h. EDTA/HC1: 0.5 M pH 7.0 (NaOH) as 100X stock.
i. Na3V04: 0.5 M as 100X stock and aliquots are kept in -80 °C.
j. Na4PZ0,: 0.2 M as 100X stock.
k. Insulin-like growth factor-1 from Promega (Cat# G5111).
1. Rabbit polyclonal anti-phosphotyrosine antiserum.
m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat. No.
4520, Lot No. 1802): Tago, Inc., Burlingame, CA.
n. ABTS (2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution:
Citric acid 100 mM
Na,HP04 250 mM (pH 4.0/1 N HCl)



CA 02368041 2001-09-24
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140
ABTS 0.5 mg/mL
ABTS solution should be kept in dark and 4°C. The solution should be
discarded
when it turns green.
0. Hydrogen Peroxide: 30% solution is kept in the dark and at 4 °C.
Procedure
All the following steps are conducted at room temperature unless it is
specifically
indicated. All ELISA plate washings are performed by rinsing the plate with
tap
water three times, followed by one TBST rinse. Pat plate dry with paper
towels.
A. Cell Seedin.~:
I. The cells, grown in tissue culture dish (Corning 25020-100) to 80-90%
confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 mL/D-100, GIBCO).
2. Res~as..;::nd the cells in fresh DMEM + 10% FBS + 2 mM L-Glutamine, and
transfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000
cells/well (100
~L/well). Incubate for 1 day then replace medium to serum-free medium (90/p,L)
and incubate in S% CO2 and 37 °C overnight.
B. ELISA Plate Coating and Blocking:
1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at 0.5
yg/well in 100 pL PBS at least 2 hours.
2. Remove the coating solution, and replace with 100 pL Blocking Buffer, and
shake for 30 minutes. Remove the blocking buffer and wash the plate just
before
adding lysate.
C. Assay Procedures:
1. The drugs are tested in serum-free condition.
2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-
propylene plate, and transfer 10 pL/well of this solution to the cells to
achieve final
drug dilution 1:100, and final DMSO concentration of 1.0%. Incubate the cells
in
5% CO, at 37 °C for 2 hours.
3. Prepare fresh cell lysis buffer (HNTG*)
HNTG 2 mL
EDTA 0.1 mL
Na3V04 0.1 mL



CA 02368041 2001-09-24
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141
Na4(P~O,) 0.1 mL
H,O 7.3 mL
4. After drug incubation for two hours, transfer 10 pL/well of 200 nM IGF-1
Ligand in PBS to the cells (Final Conc. = 20 nM), and incubate at 5% CO, at 37
°C
for 10 minutes.
S. Remove media and add 100 pL/well HNTG* and shake for 10 minutes.
Look at cells under microscope to see if they are adequately lysed.
6. Use a 12-channel pipette to scrape the cells from the plate, and homogenize
the lysate by repeated aspiration and dispensing. Transfer all the lysate to
the
antibody coated ELISA plate, and shake for 1 hour.
7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 with TBST)
100 ~L/well, and shake for 30 minutes.
8. Remove anti-pTyr, wash the plate, transfer TAGO (1:3,000 with TBST) 100
~L/well, and shake for 30 minutes.
9. Remove detection antibody, wash the plate, and transfer fresh ABTS/HZOZ
(1.2 p.L H,Oz to 10 mL ABTS) 100 pL/well to the plate to start color
development.
10. Measure OD at 410 nm with a reference wavelength of 630 nm in Dynatec
MR5000.
EXAMPLE 6: EGF Receptor ELISA
EGF Receptor kinase activity in cells genetically engineered to express
human EGF-R was measured as described below:
Materials and Reagents
The following materials and reagents were used:
a. EGF Ligand: stock concentration = 16.5 ~M; EGF 201, TOYOBO, Co., Ltd.
Japan.
b. OS-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular
domain).
c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).
d. Detection antibody: Goat anti-rabbit 1gG horse radish peroxidase conjugate,



CA 02368041 2001-09-24
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142
TAGO, Inc., Burlingame,
CA.


e. TBST buffer:


Tris-HCI, pH 7 50 mM


NaCI 150
mM


Triton X-100 0.1


f. HNTG SX stock:


HEPES 0.1
M


NaCI 0.75
M


Glycerol 50


Triton X-100 1.0%


g. ABTS stock:
Citric Acid 100 mM
Na,HP04 250 mM
HCI, conc. 4.0 pH
ABTS' 0.5 mg/mL
Keep solution in dark at 4 °C until used.
h. Stock reagents of:
EDTA 100 mM pH 7.0
Na3V04 0.5 M
Na4(PzO,) 0.2 M
Procedure
The following protocol was used:
A. Pre-coat ELISA Plate
1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with OS-101 antibody
at 0.5 ug per well in PBS, 150 ~,L final volume/well, and store overnight at 4
°C.
Coated plates are good for up to 10 days when stored at 4 °C.
2. On day of use, remove coating buffer and replace with blocking buffer (5%
Carnation Instant NonFat Dry Milk in PBS). Incubate the plate, shaking, at
room
temperature (about 23 °C to 25 °C) for 30 minutes. Just prior to
use, remove
blocking buffer and wash plate 4 times with TBST buffer.
B. Seeding Cells
1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can be use
for this assay.
2. Choose dishes having 80-90% confluence for the experiment. Trypsinize



CA 02368041 2001-09-24
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143
cells and stop reaction by adding 10% CS DMEM medium. Suspend cells in DMEM
medium (10% CS DMEM medium) and centrifuge once at 1000 rpm at room
temperature for S minutes.
3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count
the cells using trypan blue. Viability above 90% is acceptable. Seed cells in
DMEM
medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 ~.L per
well,
in a 96 well microtiter plate. Incubate seeded cells in 5% CO, at 37 °C
for about 40
hours.
C. Assay Procedures.
1. Check seeded cells for contamination using an inverted microscope. Dilute
drug stock ( 10 mg/mL in DMSO) 1:10 in DMEM medium, then transfer 5 ~L to a
test well for a final drug dilution of 1:200 and a final DMSO concentration of
1%.
Control wells receive DMSO alone. Incubate in 5% CO, at 37 °C for
one hour.
2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10
pL dilute EGF (1:12 dilution), 25 nM final concentration is attained.
3. Prepare fresh 10 mL HNTG~ sufficient for 100 ~L per well wherein HNTG*
comprises: HNTG stock (2.0 mL), milk-Q HZO (7.3 mL), EDTA, 100 mM, pH 7.0
(0.5 mL), Na3V0~ 0.5 M (0.1 mL) and Naa(PZO,), 0.2 M (0.1 mL).
4. Place on ice.
5. After two hours incubation with drug, add prepared EGF ligand to cells, 10
~L per well, to yield a final concentration of 25 nM. Control wells receive
DMEM
alone. Incubate, shaking, at room temperature, for 5 minutes.
6. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer
HNTG' to cells, 100 ~L per well. Place on ice for 5 minutes. Meanwhile, remove
blocking buffer from other ELISA plate and wash with TBST as described above.
7. With a pipette tip securely fitted to a micropipettor, scrape cells from
plate
and homogenize cell material by repeatedly aspirating and dispensing the HNTGk
lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate.
Incubate
shaking at room temperature for one hour.
8. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti
Ptyr antibody to ELISA plate at 100 ~L per well. Incubate shaking at room



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temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000
dilution in TBST).
9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the
freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plate at 100 ~L
per
well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG
antibody:
1:3000 dilution in TBST).
10. Remove detection antibody and wash 4 times with TBST. Transfer freshly
prepared ABTS/H,O, solution to ELISA plate, 100 pL per well. Incubate at room
temperature for 20 minutes. ABTS/H,O, solution: 1.2 p,L 30% H~O~ in 10 mL
ABTS stock.
11. Stop reaction by adding 50 p.L SN H,S04 (optional), and determine O.D. at
410 nm.
12. The maximal phosphotyrosine signal is determined by subtracting the value
of the negative controls from the positive controls. The percent inhibition of
phosphotyrosine content for extract-containing wells is then calculated, after
subtraction of the negative controls.
EXAMPLE 7: Met Autophosphorylation Assay - ELISA
This assay determines Met tyrosine kinase activity by analyzing Met protein
tyrosine kinase levels on the Met receptor.
Materials and Reagents
The following materials and reagents were used:
a. HNTG (SX stock solution): Dissolve 23.83 g HEPES and 43.83 g NaCI in
about 350 mL dH,O. Adjust pH to 7.2 with HC1 or NaOH, add 500 mL glycerol and
10 mL Triton X-100, mix, add dH,O to 1 L total volume. To make 1 L of 1X
working solution add 200 mL SX stock solution to 800 mL dH,O, check and adjust
pH as necessary, store at 4 °C.
b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco Cat. # 450-1300EB (1X
solution).
c. Blocking Buffer: in 500 mL dH,O place 100 g BSA, 12.1 g Tris-pH7.5,



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58.44 g NaCI and 10 mL Tween-20, dilute to 1 L total volume.
d. Kinase Buffer: To S00 mL dH,O add 12.1 g TRIS pH7.2, 58.4 g NaCI, 40.7
g MgCI, and 1.9 g EGTA; bring to 1 L total volume with dH~O.
e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. # P-7626, to 43S.S mg,
S add 100% ethanol, to 2S mL total volume, vortex.
f. ATP (Bacterial Source), Sigma Cat. # A-7699, store powder at -20 °C;
to
make up solution for use, dissolve 3.31 mg in 1 mL dH~O.
g. RC-20H HRPO Conjugated Anti-Phosphotyrosine, Transduction
Laboratories Cat. # E120H.
h. Pierce 1-Step (TM) Turbo TMB-ELISA (3,3',S,S'-tetramethylbenzidine,
Pierce Cat. # 34022.
i. H,S04, add 1 mL conc. (18 N) to 3S mL dH~O.
j. TRIS HCL, Fischer Cat. # BP1S2-S; to 121.14 g of material, add 600 mL
MilliQ HBO, adjust pH to 7.S (or 7.2) with HCl , bring volume to 1 L with
MilliQ
1S H,O.
k. NaCI, Fischer Cat. # 5271-10, make up S M solution.
1. Tween-20, Fischer Cat. # 5337-500.
m. Na3V04, Fischer Cat. # S4S4-S0, to 1.8 g material add 80 mL MilliQ H,O,
adjust pH to 10.0 with HCl or NaOH, boil in microwave, cool, check pH, repeat
procedure until pH stable at 10.0, add MilliQ HZO to 100 m .., total volume,
make 1
mL aliquots and store at -80 °C.
n. MgCl2, Fischer Cat. # M33-500, make up 1 M solution.
o. HEPES, Fischer Cat. # BP310-500, to 200 mL MilliQ H,O, add 59.6 g
material, adjust pH to 7.5, bring volume to 2S0 mL total, sterile filter.
2S p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30 grams material add
sterile distilled water to make total volume of 300 mL, store at 4 °C.
q. TBST Buffer: to approx. 900 mL dH~O in a 1 L graduated cylinder add 6.OS7
g TRIS and 8.766 g NaCI, when dissolved, adjust pH to 7.2 with HC1, add 1.0 mL
Triton X-100 and bring to 1 L total volume with dH,O.
r. Goat Affinity purified antibody Rabbit IgG (whole molecule), Cappel Cat. #
SS641.



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s. Anti h-Met (C-28) rabbit polyclonal IgG antibody, Santa Cruz Chemical Cat.
# SC-161.
t. Transiently Transfected EGFR/Met chimeric cells (EMR) (Komada, et al.,
Oncogene, 8:2381-2390 (1993).
u. Sodium Carbonate Buffer, (Na,C04, Fischer Cat. # S495): to 10.6 g material
add 800 mL MilliQ H,O, when dissolved adjust pH to 9.6 with NaOH, bring up to
1 L
total volume with MilliQ HBO, filter, store at 4 °C.
Procedure
All of the following steps are conducted at room temperature unless it is
specifically indicated otherwise. All ELISA plate washing is by rinsing 4X
with
TBST.
A. EMR Lysis
This procedure can be performed the night before or immediately prior to the
start of receptor capture.
1. Quick thaw lysates in a 37 °C waterbath with a swirling motion until
the last
crystals disappear.
2. Lyse cell pellet with 1X HNTG containing 1 mM PMSF. Use 3 mL of
HNTG per 15 cm dish of cells. Add '/z the calculated HNTG volume, vortex the
tube for 1 min., add the remaining amount of HNTG, vortex for another min.
3. Balance tubes, centrifuge at 10,000x g for 10 min at 4 °C.
4. Pool supernatants, remove an aliquot for protein determination.
5. Quick freeze pooled sample in dry ice/ethanol bath. This step is performed
regardless of whether lysate will be stored overnight or used immediately
following
protein determination.
6. Perform protein determination using standard bicinchoninic acid (BCA)
method (BCA Assay Reagent Kit from Pierce Chemical Cat. # 23225).
B. ELISA Procedure
1. Coat Corning 96 well ELISA plates with 5 pg per well Goat anti-Rabbit



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antibody in Carbonate Buffer for a total well volume of 50 pL. Store overnight
at 4
°C.
2. Remove unbound Goat anti-rabbit antibody by inverting plate to remove
liquid.
3. Add 150 pL of Blocking Buffer to each well. Incubate for 30 min. at room
temperature with shaking.
4. Wash 4X with TBST. Pat plate on a paper towel to remove excess liquid and
bubbles.
5. Add 1 pg per well of Rabbit anti-Met antibody diluted in TBST for a total
well volume of 100 ~.L.
6. Dilute lysate in HNTG (90 pg lysate/100 ~L)
7. Add 100 ~L of diluted lysate to each well. Shake at room temperature for 60
mm.
8. Wash 4X with TBST. Pat on paper towel to remove excess liquid and
bubbles.
9. Add SO p,L of 1X lysate buffer per well.
10. Dilute compounds/extracts 1:10 in 1X Kinase Buffer in a polypropylene 96
well plate.
11. Transfer 5.5 ~L of diluted drug to ELISA plate wells. Incubate at room
temperature with shaking for 20 min.
12. Add 5.5 ~L of 60 pM ATP solution per well. Negative controls do not
receive any ATP. Incubate at room temperature for 90 min., with shaking.
13. Wash 4X with TBST. Pat plate on paper towel to remove excess liquid and
bubbles.
14. Add 100 ~L per well of RC20 ( 1:3000 dilution in Blocking Buffer).
Incubate 30 min. at room temperature with shaking.
15. Wash 4X with TBST. Pat plate on paper towel to remove excess liquid and
bubbles.
16. Add 100 pL per well of Turbo-TMB. Incubate with shaking for 30-60 min.
17. Add 100 ~L per well of 1 M H,S04 to stop reaction.
18. Read assay on Dynatech MR7000 ELISA reader. Test Filter = 450 nm,



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reference filter = 410 nm.
EXAMPLE 8: Biochemical src assay - ELISA
This assay is used to determine src protein kinase activity measuring
phosphorylation of a biotinylated peptide as the readout.
Materials and Reagents
The following materials and reagents were used:
a. Yeast transformed with src.
b. Cell lysates: Yeast cells expressing src are pelleted, washed once with
water,
re-pelleted and stored at -80 °C until use.
c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by
standard procedures well known to those skilled in the art.
d. DMSO: Sigma, St. Louis, MO.
e. 96 Well ELISA Plate: Corning 96 Well Easy Wash, Modified flat Bottom
Plate, Corning Cat. #25805-96.
f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:
Applied Scientific Cat. # A-72092.
g. Vecastain ELITE ABC reagent: Vector, Burlingame, CA.
h. Anti-src (327) mab: Schizosaccharomyces Pombe was used to express
recombinant Src (Superti-Furga, et al., EMBO J., 12:2625-2634; Superti-Furga,
et
al., Nature Biochem., 14:600-605). S. Pombe strain SP200 (h-s leu1.32 ura4
ade210)
was grown as described and transformations were pRSP expression plasmids were
done by the lithium acetate method (Superb-Furga, supra). Cells were grown in
the
presence of 1 ~M thiamine to repress expression from the nmtl promoter or in
the
absence of thiamine to induce expression.
i. Monoclonal anti-phosphotyrosine, UBI OS-321 (UB40 may be used instead).
j. Turbo TMB-ELISA peroxidase substrate: Pierce Chemical.
Buffer Solutions:
a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO Cat. #



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450-1300EB.
b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS.
c. Carbonate Buffer: Na,C04 from Fischer, Cat. # S495, make up 100 mM
stock solution.
d. Kinase Buffer: 1.0 mL (from 1 M stock solution) MgCI,; 0.2 mL (from a 1
M stock solution) MnCI,; 0.2 mL (from a 1 M stock solution) DTT; S.0 mL (from
a
1 M stock solution) HEPES; 0.1 mL TX-100; bring to 10 mL total volume with
MilliQ H=O.
e. Lysis Buffer: 5.0 HEPES (from 1 M stock solution.); 2.74 mL NaCI (from 5
M stock solution); 10 mL glycerol; 1.0 mL TX-100; 0.4 mL EDTA (from a 100 mM
stock solution); 1.0 mL PMSF (from a 100 mM stock solution); 0.1 mL Na3V04
(from a 0.1 M stock solution); bring to 100 mL total volume with MilliQ H,O.
~ ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/mL).
g. TRIS-HCI: Fischer Cat. # BP 152-5, to 600 mL MilliQ H,O add 121.14 g
material, adjust pH to 7.5 with HCI, bring to 1 L total volume with MilliQ
H20.
h. NaCI: Fischer Cat. # S271-10, Make up 5 M stock solution with MilliQ
H,O.
i. Na3V04: Fischer Cat. # 5454-50; to 80 mL MilliQ H20, add 1.8 g material;
adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool; check pH,
repeat
pH adjustment until pH remains stable after heating/coolin~cycle; bring to 100
mL
total volume with MilliQ H,O; make 1 mL aliquots and store at -80 °C.
j. MgCI,: Fischer Cat. # M33-500, make up 1 M stock solution with MilliQ
H,O.
k. HEPES: Fischer Cat. # BP 310-500; to 200 mL MilliQ HZO, add 59.6 g
material, adjust pH to 7.5, bring to 250 mL total volume with MilliQ H,O,
sterile
filter ( 1 M stock solution).
1. TBST Buffer: TBST Buffer: To 900 mL dH,O add 6.057 g TRIS and 8.766
g NaCI; adjust pH to 7.2 with HCI, add 1.0 mL Triton-X100; bring to 1 L total
volume with dH20.
m. MnCIZ: Fischer Cat. # M87-100, make up 1 M stock solution with MilliQ
H,O.



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n. DTT; Fischer Cat. # BP 172-5.
o. TBS (TRIS Buffered Saline): to 900 mL MilliQ H,O add 6.057 g TRIS and
8.777 g NaCI; bring to 1 L total volume with MilliQ H20.
p. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 mL
Kinase Buffer, 200 yg GST-~, bring to final volume of 8.0 mL with MilliQ H,O.
q. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide stock solution
(1 mM, 2.98 mg/ml) in water fresh just before use.
r. Vectastain ELITE ABC reagent: To prepare 14 mL of working reagent, add
1 drop of reagent A to 15 mL TBST and invert tube several times to mix. Then
add
1 drop of reagent B. Put tube on orbital shaker at room temperature and mix
for 30
minutes.
Procedures
a. Preparation of src coated ELISA plate.
1. Coat ELISA plate with 0.5 ~g/well anti-src mab in 100 ~L of pH 9.6 sodium
carbonate buffer at 4 °C overnight.
2. Wash wells once with PBS.
3. Block plate with 0.15 mL 5% milk in PBS for 30 min. at room temperature.
4. Wash plate SX with PBS.
5. Add 10 ~g/well of src transformed yeast lysates diluted in Lysis Buffer
(0.1
mL total volume per well). (Amount of lysate may vary between batches.) Shake
plate for 20 minutes at room temperature.
b. Preparation of phosphotyrosine antibody-coated ELISA plate.
1. 4610 plate: coat 0.5 ~g/well 4610 in 100 ~L PBS overnight at 4 °C
and
block with 150 pL of 5% milk in PBS for 30 minutes at room temperature.
c. Kinase assay procedure.
1. Remove unbound proteins from step 1-7, above, and wash plates SX with
PBS.
2. Add 0.08 mL Kinase Reaction Mixture per well (containing 10 ~L of lOX
Kinase Buffer and 10 ~M (final concentration) biotin-EEEYEEYEEEYEEEYEEEY
per well diluted in water.



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3. Add 10 ~L of compound diluted in water containing 10% DMSO and pre-
incubate for 1S minutes at room temperature.
4. Start kinase reaction by adding 10 pL/well of O.OS mM ATP in water (S pM
ATP final).
S S. Shake ELISA plate for 1S min. at room temperature.
6. Stop kinase reaction by adding 10 ~L of O.S M EDTA per well.
7. Transfer 90 pL supernatant to a blocked 4610 coated ELISA plate from
section B, above.
8. Incubate for 30 min. while shaking at room temperature.
9. Wash plate SX with TBST.
10. Incubate with Vectastain ELITE ABC reagent (100 p,L/well) for 30 min. at
room temperature.
11. Wash the wells SX with TBST.
12. Develop with Turbo TMB.
1S
EXAMPLE 9: Biochemical lck Assay - ELISA
This assay is used to determine Ick protein kinase activities measuring
phosphorylation of GST-~ as the readout.
Materials and Reagents
The following materials and reagents were used:
a. Yeast transformed with lck. Schizosaccharomyces Pombe was used to
express recombinant Lck (Superb-Furga, et al., EMBO J, 12:2625-2634; Superti-
Furga, et al., Nature BiotecJz., 14:600-60S). S. Pombe strain SP200 (h-s
leul.32 ura4
2S ade210) was grown as described and transformations with pRSP expression
plasmids were done by the lithium acetate method (Superti-Furga, supra). Cells
were grown in the presence of 1 ~.M thiamine to induce expression.
b. Cell lysates: Yeast cells expressing lck are pelleted, washed once in
water,
re-pelleted and stored frozen at -80 °C until use.
c. GST-~: DNA encoding for GST-~ fusion protein for expression in bacteria
obtained from Arthur Weiss of the Howard Hughes Medical Institute at the



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University of California, San Francisco. Transformed bacteria were grown
overnight
while shaking at 25 °C. GST-~ was purified by glutathione affinity
chromatography,
Pharmacia, Alameda, CA.
d. DMSO: Sigma, St. Louis, MO.
e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified Flat Bottom
Plate, Corning Cat. #25805-96.
f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:
Applied Scientific Cat. # AS-72092.
g. Purified Rabbit anti-GST antiserum: Amrad Corporation (Australia) Cat.
#90001605.
h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301
i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. # 5215-005-003.
j. Anti-L.~k (3A5) mab: Santa Cruz Biotechnology Cat # sc-433.
k. Monoclonal anti-phosphotyrosine UBI OS-321 (UB40 may be used instead).
Buffer solutions:
a. PBS (Dulbecco's Phosphate-Buffered Saline) 1X solution: GIBCO PBS,
GIBCO Cat. # 450-1300EB.
b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS-pH7.5, 58.44 g NaCI, 10 mL
Tween-20, bring up to 1 L total volume with MilliQ H,O.
c. Carbonate Buffer: Na,C04 from Fischer, Cat. # S495; make up 100 mM
solution with MilliQ H~O.
d. Kinase Buffer: 1.0 mL (from 1 M stock solution) MgCIZ; 0.2 mL (from a 1
M stock solution) MnCl2; 0.2 mL (from a 1 M stock solution) DTT; 5.0 mL (from
a
1 M stock solution) HEPES; 0.1 mL TX-100; bring to 10 mL total volume with
MilliQ H,O.
e. Lysis Buffer: 5.0 HEPES (from 1 M stock solution.); 2.74 mL NaCI (from 5
M stock solution); 10 mL glycerol; 1.0 mL TX-100; 0.4 mL EDTA (from a 100 mM
stock solution); 1.0 mL PMSF (from a 100 mM stock solution); 0.1 mL Na3V04
(from a 0.1 M stock solution); bring to 100 mL total volume with MilliQ H20.
f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/mL).



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g TRIS-HCI: Fischer Cat. # BP 152-5, to 600 mL MilliQ Hz0 add 121.14 g
material, adjust pH to 7.5 with HCI, bring to 1 L total volume with MilliQ
H20.
h. NaCI: Fischer Cat. # 5271-10, Make up 5 M stock solution with MilliQ
H,O.
i. Na,VO4: Fischer Cat. # S454-50; to 80 mL MilliQ H,O, add 1.8 g material;
adjust pH to 10.0 with HCI or NaOH; boil in a microwave; cool; check pH,
repeat pH
adjustment until pH remains stable after heating/cooling cycle; bring to 100
mL total
volume with MilliQ HBO; make 1 mL aliquots and store at -80 °C.
j. MgClz: Fischer Cat. # M33-500, make up 1 M stock solution with MilliQ
H,O.
k. HEPES: Fischer Cat. # BP 310-500; to 200 mL MilliQ HzO, add 59.6 g
material, adjust pH to 7.5, bring to 250 mL total volume with MilliQ H,O,
sterile
filter (1 M stock solution).
1. Albumin, Bovine (BSA), Sigma Cat. # A4503; to 150 mL MiIIiQ H,O add
30 g material, bring 300 mL total volume with MilliQ H,O, filter through 0.22
p.m
filter, store at 4 °C.
m. TBST Buffer: To 900 mL dH,O add 6.057 g TRIS and 8.766 g NaCI; adjust
pH to 7.2 with HCI, add 1.0 mL Triton-X100; bring to 1 L total volume with
dHzO.
n. MnCh: Fischer Cat. # M87-100, make up 1 M stock solution with MiIIiQ
H,O.
o. DTT; Fischer Cat. # BP172-5.
p. TBS (TRIS Buffered Saline): to 900 mL MilliQ H,O add 6.057 g TRIS and
8.777 g NaCI; bring to 1 L total volume with MilliQ HzO.
q Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 mL
Kinase Buffer, 200 pg GST-~, bring to final volume of 8.0 mL with MiIIiQ HzO.
Procedures
a. Preparation of Lck coated ELISA plate.
1. Coat 2.0 p.g/well Sheep anti-mouse IgG in 100 pL of pH 9.6 sodium
carbonate buffer at 4 °C overnight.
2. Wash well once with PBS.



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154
3. Block plate with 0.15 mL of blocking Buffer for 30 min. at room temp.
4. Wash plate SX with PBS.
5. Add 0.5 ~g/well of anti-lck (mab 3A5) in 0.1 mL PBS at room temperature
for 1-2 hours.
6. Wash plate SX with PBS.
7. Add 20 ~g/well of lck transformed yeast lysates diluted in Lysis Buffer
(0.1
mL total volume per well). (Amount of lysate may vary between batches) Shake
plate at 4 °C overnight to prevent loss of activity.
b. Preparation of phosphotyrosine antibody-coated ELISA plate.
1. UB40 plate: 1.0 pg/well UB40 in 100 L of PBS overnight at 4 °C and
block
with 150 ~L of Blocking Buffer for at least 1 hour.
c. Kinase assay procedure.
1. Remove unbound proteins from step 1-7, above, and wash plates SX with
PBS.
2. Add 0.08 mL Kinase Reaction Mixture per well (containing 10 pL of l OX
Kinase Buffer and 2 p.g GST-~ per well diluted with water).
3. Add 10 ~L of compound diluted in water containing 10% DMSO and pre-
incubate for 15 minutes at room temperature.
4. Start kinase reaction by adding 10 ~L/well of 0.1 mM ATP in water (10 ~M
ATP final).
5. Shake ELISA plate for 60 min. at room temperature.
6. Stop kinase reaction by adding 10 ~,L of 0.5 M EDTA per well.
7. Transfer 90 pL supernatant to a blocked 4610 coated ELISA plate from
section B, above.
8. Incubate while shaking for 30 min. at room temperature.
9. Wash plate SX with TBST.
10. Incubate with Rabbit anti-GST antibody at 1:5000 dilution in 100 ~L TBST
for 30 min. at room temperature.
11. Wash the wells SX with TBST.
12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilution in 100 p,L of



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TBST for 30 min. at room temperature.
13. Wash the wells SX with TBST.
14. Develop with Turbo TMB.
EXAMPLE 10: ASSAY MEASURING PHOSPHORYLATING FUNCTION
OF RAF
The following assay reports the amount of RAF-catalyzed phosphorylation
of its target protein MEK as well as MEK's target MAPK. The RAF gene sequence
is described in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407, and is
readily
accessible in multiple gene sequence data banks. Construction of the nucleic
acid
vector and cell lines utilized for this portion of the invention are fully
described in
Morrison et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8855-8859.
Materials and Reagents
1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, MD.
2. RIPA buffer: 20 mM Tris/HC1 pH 7.4, 137 mM NaCI, 10% glycerol, 1 mM
PMSF, 5 mg/L Aprotenin, 0.5 % Triton X-100;
3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and
purification by affinity chromatography were performed according to the
manufacturer's procedures. Catalog# K 350-O1 and R 350-40, Invitrogen Corp.,
San
Diego, CA
4. His-MAPK (ERK 2); His-tagged MAPK was expressed in XLl Blue cells
transformed with pUCl8 vector encoding His-MAPK. His-MAPK was purified by
Ni-affinity chromatography. Cat# 27-4949-O1, Pharmacia, Alameda, CA, as
described herein.
5. Sheep anti mouse IgG: Jackson laboratories, West Grove, PA. Catalog, #
S 15-006-008, Lot# 28563
6. RAF-1 protein kinase specific antibody: URP2653 from UBI.
7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg,
MD
8. Wash buffer: TBST - 50 mM Tris/HCL pH 7.2, 150 mM NaCI, 0.1 % Triton



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X-100
9. Block buffer: TBST, 0.1 % ethanolamine pH 7.4
10. DMSO, Sigma, St. Louis, MO
11. Kinase buffer (KB): 20 mM HEPES/HC1 pH 7.2, 150 mM NaCI, 0.1
Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium ortho vanadate, 0.5
MM DTT and 10 mM MgCI,.
12. ATP mix: 100 mM MgCl2, 300 mM ATP, 10 mCi 3'P ATP (Dupont-
NEN)/mL.
13. Stop solution: 1 % phosphoric acid; Fisher, Pittsburgh, PA.
14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland.
15. Filter wash solution: 1 % phosphoric acid, Fisher, Pittsburgh, PA.
16. Tomtec plate harvester, Wallac, Turku, Finnland.
17. Wallav beta plate reader # 1205, Wallac, Turku, Finnland.
18. NLTNC 96-well V bottom polypropylene plates for compounds Applied
Scientific Catalog # AS-72092.
Procedure
All of the following steps were conducted at room temperature unless
specifically indicated.
1. ELISA plate coating: ELISA wells are coated with 100 mL of Sheep anti
mouse affinity purified antiserum (1 mg/100 mL coating buffer) over night at 4
°C.
ELISA plates can be used for two weeks when stored at 4 °C.
2. Invert the plate and remove liquid. Add 100 mL of blocking solution and
incubate for 30 min.
3. Remove blocking solution and wash four times with wash buffer. Pat the
plate on a paper towel to remove excess liquid.
4. Add 1 mg of antibody specific for RAF-1 to each well and incubate for 1
hour. Wash as described in step 3.
5. Thaw lysates from RAS/RAF infected Sf~3 cells and dilute with TBST to 10
mg/100 mL. Add 10 mg of diluted lysate to the wells and incubate for 1 hour.
Shake the plate during incubation. Negative controls receive no lysate.
Lysates



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from RAS/RAF infected Sf~ insect cells are prepared after cells are infected
with
recombinant baculoviruses at a MOI of S for each virus, and harvested 48 hours
later. The cells are washed once with PBS and lysed in RIPA buffer. Insoluble
material is removed by centrifugation (5 min at 10 000 x g). Aliquots of
lysates are
frozen in dry ice/ethanol and stored at -80 °C until use.
6. Remove non-bound material and wash as outlined above (step 3).
7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjust the
volume to 40 mL with kinase buffer. Methods for purifying T-MEK and MAPK
from cell extracts are provided herein by example.
8. Pre-dilute compounds (stock solution 10 mg/mL DMSO) or extracts 20 fold
in TBST plus 1% DMSO. Add 5 mL of the pre-diluted compounds/extracts to the
wells described in step 6. Incubate for 20 min. Controls receive no drug.
9. Start the kinase reaction by addition of 5 mL ATPmix; Shake the plates on
an ELISA plate shaker during incubation.
10. Stop the kinase reaction after 60 min by addition of 30 mL stop solution
to
each well.
11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate
harvester. Harvest and wash the filter with the filter wash solution according
to the
manufacturers recommendation. Dry the filter mats. Seal the filter mats and
place
them in the holder. Insert the holder into radioactive detecticui apparatus
and
quantify the radioactive phosphorous on the filter mats.
Alternatively, 40 mL aliquots from individual wells of the assay plate can be
transferred to the corresponding positions on the phosphocellulose filter mat.
After
air drying the filters, put the filters in a tray. Gently rock the tray,
changing the wash
solution at 15 min intervals for 1 hour. Air-dry the filter mats. Seal the
filter mats
and place them in a holder suitable for measuring the radioactive phosphorous
in the
samples. Insert the holder into a detection device and quantify the
radioactive
phosphorous on the filter mats.
EXAMPLE 11: CDK2/Cyclin A - Inhibition Assay
This assay analyzes the protein kinase activity of CDK2 in exogenous



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substrate.
Materials and Reagents
A. Buffer A (80 mM Tris ( pH 7.2), 40 mM MgCl2): 4.84 G. Tris (F.W. =121.1
g/mol), 4.07 g. MgCI, (F.W.=203.31 g/mol) dissolved in 500 mL HBO. Adjust pH
to
7.2 with HCI.
B. Histone H1 solution (0.45 mg/mL Histone H1 and 20 mM HEPES pH 7.2
(pH 7.4 is OK): 5 mg Histone H1 (Boehinger Mannheim) in 11.111 mL 20 mM
HEPES pH 7.2 (477 mg HEPES (F.W.= 238.3 g/mol) dissolved in 100 mL ddH,O,
stored in 1 mL aliquots at -80 °C.
C. ATP solution (60 pM ATP, 300 g/mL BSA, 3 mM DTT): 120 ~L 10 mM
ATP, 600 pL 10 mg/mL BSA to 20 mL, stored in 1 mL aliquots at -80
°C.
D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCI, 0.5
mM DTT, 10% glycerol, stored in 9 pL aliquots at -80 °C.
Description of Assay:
1. Prepare solutions of inhibitors at three times the desired final assay
concentration in ddH~O/15 % DMSO by volume.
2. Dispense 20 qL of inhibitors to wells of polypropylene 96-well plates (or
20
uL 15% DMSO for positive and negative controls).
3. Thaw Histone H1 solution (1 mL/plate), ATP solution (1 mL/plate plus 1
aliquot for negative control), and CDK2 solution (9 laL/plate). Keep CDK2 on
ice
until use. Aliquot CDK2 solution appropriately to avoid repeated freeze-thaw
cycles.
4. Dilute 9 pL CDK2 solution into 2.1 mL Buffer A (per plate). Mix. Dispense
20 ~L into each well.
S. Mix 1 mL Histone Hl solution with 1 mL ATP solution (per plate) into a 10
mL screw cap tube. Add y33P ATP to a concentration of 0.15 p,Ci/20 p.L (0.15
~Ci/well in assay). Mix carefully to avoid BSA frothing. Add 20 pL to
appropriate
wells. Mix plates on plate shaker. For negative control, mix ATP solution with
an
equal amount of 20 mM HEPES pH 7.2 and add y'3P ATP to a concentration of 0.15



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p.Ci/20 ~,L solution. Add 20 ~L to appropriate wells.
6. Let reactions proceed for 60 minutes.
7. Add 35 pL 10% TCA to each well. Mix plates on plate shaker.
8. Spot 40 ~L of each sample onto P30 filter mat squares. Allow mats to dry
(approx. 10-20 minutes).
9. Wash filter mats 4 X 10 minutes with 250 mL 1 % phosphoric acid ( 10 mL
phosphoric acid per liter ddH,O).
10. Count filter mats with beta plate reader.
Cellular/Biolo~ic Assays
EXAMPLE 12: PDGF-Induced BrdU Incorporation Assay
Materials and Rea eg nts:
(1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,
Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No.
1 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution : 1X PBS, pH 7.4.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
USA.
(8) 3T3 cell line genetically engineered to express human PDGF-R.
Protocol
(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96
well plate. Cells are incubated overnight at 37 °C in 5% CO~.



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(2) After 24 hours, the cells are washed with PBS, and then are serum starved
in
serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) and
test compounds are added to the cells simultaneously. The negative control
wells
receive serum free DMEM with 0.1 % BSA only; the positive control cells
receive
the ligand (PDGF) but no test compound. Test compounds are prepared in serum
free DMEM with ligand in a 96 well plate, and serially diluted for 7 test
concentrations.
(4) After 20 hours of ligand activation, diluted BrdU labeling reagent ( 1:100
in
DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final
concentration=10 ~.M) for 1.5 hours.
(5) After incubation with labeling reagent, the medium is removed by decanting
and tapping rl~;: inverted plate on a paper towel. FixDenat solution is added
(50 ~L
/well) and the plates are incubated at room temperature for 45 minutes on a
plate
shaker.
(6) The FixDenat solution is thoroughly removed by decanting and tapping the
inverted plate on a paper towel. Milk is added (S% dehydrated milk in PBS, 200
~L
/well) as a blocking solution and the plate is incubated for 30 minutes at
room
temperature on a plate shaker.
(7) The blocking solution is removed by decanting and the wells are washed
once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
added (100 ~L /well) and the plate is incubated for 90 minutes at room
temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by decanting and rinsing the
wells 5 times with PBS, and the plate is dried by inverting and tapping on a
paper
towel.
(9) TMB substrate solution is added (100 ~.L /well) and incubated for 20
minutes
at room temperature on a plate shaker until color development is sufficient
for
photometric detection.



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(10) The absorbance of the samples are measured at 410 nm (in "dual
wavelength" mode with a filter reading at 490 nm, as a reference wavelength)
on a
Dynatech ELISA plate reader.
EXAMPLE 13: EGF-Induced BrdU Incorporation Assay
Materials and Reagents
( 1 ) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,
Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No.
1 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution : 1X PBS, pH 7.4.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
USA.
(8) 3T3 cell line genetically engineered to express huma~~ EGF-R.
Protocol
( 1 ) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a 96
well plate. Cells are incubated overnight at 37 °C in 5% COZ.
(2) After 24 hours, the cells are washed with PBS, and then are serum starved
in
serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA) and test
compounds are added to the cells simultaneously. The negative control wells
receive serum free DMEM with 0.1% BSA only; the positive control cells receive
the ligand (EGF) but no test compound. Test compounds are prepared in serum
free
DMEM with ligand in a 96 well plate, and serially diluted for 7 test
concentrations.



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(4) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100
in
DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final
concentration=10 ~M) for 1.5 hours.
(5) After incubation with labeling reagent, the medium is removed by decanting
and tapping the inverted plate on a paper towel. FixDenat solution is added
(50
pL/well) and the plates are incubated at room temperature for 45 minutes on a
plate
shaker.
(6) The FixDenat solution is thoroughly removed by decanting and tapping the
inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200
~L/well) as a blocking solution and the plate is incubated for 30 minutes at
room
temperature on a plate shaker.
(7) The blocking solution is removed by decanting and the wells are washed
once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
added (100 ~L/well) and the plate is incubated for 90 minutes at room
temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by decanting and rinsing the
wells 5 times with PBS, and the plate is dried by inverting and tapping on a
paper
towel.
(9) TMB substrate solution is added (100 p.L/well) and incubated for 20
minutes
at room temperature on a plate shaker until color development is sufficient
for
photometric detection.
(10) The absorbance of the samples are measured at 410 nm (in "dual
wavelength" mode with a filter reading at 490 nm, as a reference wavelength)
on a
Dynatech ELISA plate reader.
EXAMPLE 14: EGF-Induced Her2-Driven BrdU Incorporation
Materials and Reagents:
(1) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,
Boehringer Mannheim, Germany.



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(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No.
1 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution : 1X PBS, pH 7.4, made in house.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
USA.
(8) 3T3 cell line engineered to express a chimeric receptor having the extra-
cellular domain of EGF-R and the intra-cellular domain of Her2.
Protocol:
( 1 ) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96-
well plate. Cells are incubated overnight at 37 °C in 5% COZ.
(2) After 24 hours, the cells are washed with PBS, and then are serum starved
in
serum free medium (0%CS DMEM with 0.1 % BSA) for 24 hours.
(3) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and test
compounds are added to the cells simultaneously. The negative control wells
receive serum free DMEM with 0.1 % BSA only; the positive cc:,~~trol cells
receive
the ligand (EGF) but no test compound. Test compounds are prepared in serum
free
DMEM with ligand in a 96 well plate, and serially diluted for 7 test
concentrations.
(4) After 20 hours of ligand activation, diluted BrdU labeling reagent ( 1:100
in
DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final
concentration = 10 p.M) for 1.5 hours.
(5) After incubation with labeling reagent, the medium is removed by decanting
and tapping the inverted plate on a paper towel. FixDenat solution is added
(50
pL/well) and the plates are incubated at room temperature for 45 minutes on a
plate
shaker.
(6) The FixDenat solution is thoroughly removed by decanting and tapping the
inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200



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164
pL/well) as a blocking solution and the plate is incubated for 30 minutes at
room
temperature on a plate shaker.
(7) The blocking solution is removed by decanting and the wells are washed
once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
added (100 ~L/well) and the plate is incubated for 90 minutes at room
temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by decanting and rinsing the
wells 5 times with PBS, and the plate is dried by inverting and tapping on a
paper
towel.
(9) TMB substrate solution is added (100 pL/well) and incubated for 20 minutes
at room temperature on a plate shaker until color development is sufficient
for
photometric detection.
( 10) The a~:.sorbance of the samples are measured at 410 nm (in "dual
wavelength" mode with a filter reading at 490 nm, as a reference wavelength)
on a
Dynatech ELISA plate reader.
EXAMPLE 15: IGFI-Induced BrdU Incorporation Assay
Materials and Reagents:
(1) IGFI Ligand: human, recombinant; 6511, Promega Corp, USA.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,
Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No.
1 647 229, Boehringer Mannheim, Germany.
(6) PBS Washing Solution: 1X PBS, pH 7.4.
(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
USA.



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(8) 3T3 cell line genetically engineered to express human IGF-1 receptor.
n..~4.~.~~~.
(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96-
well plate. Cells are incubated overnight at 37 °C in 5% CO,.
(2) After 24 hours, the cells are washed with PBS, and then are serum starved
in
serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) and
test compounds are added to the cells simultaneously. The negative control
wells
receive serum free DMEM with 0.1% BSA only; the positive control cells receive
the ligand (IGF1) but no test compound. Test compounds are prepared in serum
free
DMEM with ligand in a 96 well plate, and serially diluted for 7 test
concentrations.
(4) After 16 hours of ligand activation, diluted BrdU labeling reagent ( 1:100
in
DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final
concentration=10 pM) for 1.5 hours.
(5) After incubation with labeling reagent, the medium is removed by decanting
and tapping the inverted plate on a paper towel. FixDenat solution is added
(50
p.L/well) and the plates are incubated at room temperature for 45 minutes on a
plate
shaker.
(6) The FixDenat solution is thoroughly removed by decanting and tapping the
inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200
p,L/well) as a blocking solution and the plate is incubated for 30 minutes at
room
temperature on a plate shaker.
(7) The blocking solution is removed by decanting and the wells are washed
once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
added (100 pL/well) and the plate is incubated for 90 minutes at room
temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by decanting and rinsing the
wells S times with PBS, and the plate is dried by inverting and tapping on a
paper
towel.
(9) TMB substrate solution is added (100 pL/well) and incubated for 20 minutes



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at room temperature on a plate shaker until color development is sufficient
for
photometric detection.
( 10) The absorbance of the samples are measured at 410 nm (in "dual
wavelength" mode with a filter reading at 490 nm, as a reference wavelength)
on a
Dynatech ELISA plate reader.
EXAMPLE 16: HUV-EC-C Assay
The following protocol may also be used to measure a compound's activity
against PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally
expressed by HUV-EC cells.
nav n
1. Wash and trypsinize HIJV-EC-C cells (human umbilical vein
endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL).
Wash with Dulbecco's phosphate-buffered saline (D-PBS; obtained from Gibco
BRL; catalogue no. 14190-029) 2 times at about 1 mL/10 cm2 of tissue culture
flask.
Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution
(Sigma Chemical Company; catalogue no. C-1544). The 0.05% trypsin was made
by diluting 0.25% trypsin/1 mM EDTA (Gibco; catalogue no. 25200-049) in the
cell
dissociation solution. Trypsinize with about 1 mL/25-30 cmz of tissue culture
flask
for about 5 minutes at 37 °C. After cells have detached from the flask,
add an equal
volume of assay medium and transfer to a 50 mL sterile centrifuge tube (Fisher
Scientific; catalogue no. OS-539-6).
2. Wash the cells with about 35 mL assay medium in the 50 mL sterile
centrifuge tube by adding the assay medium, centrifuge for 10 minutes at
approximately 200 g, aspirate the supernatant, and resuspend with 35 mL D-PBS.
Repeat the wash two more times with D-PBS, resuspend the cells in about 1 mL
assay medium/15 cm' of tissue culture flask. Assay medium consists of F12K
medium (Gibco BRL; catalogue no. 21127-014) + 0.5% heat-inactivated fetal
bovine
serum. Count the cells with a Coulter Counter Coulter Electronics, Inc.) and
add
assay medium to the cells to obtain a concentration of 0.8-1.0x105 cells/mL.



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3. Add cells to 96-well flat-bottom plates at 100 pL/well or 0.8-1.0x10''
cells/well; incubate ~24h at 37 °C, 5% CO,.
DAY 1
1. Make up two-fold drug titrations in separate 96-well plates, generally
50 ~M on down to 0 ~M. Use the same assay medium as mentioned in day 0, step 2
above. Titrations are made by adding 90 pL/well of drug at 200 pM (4X the
final
well concentration) to the top well of a particular plate column. Since the
stock drug
concentration is usually 20 mM in DMSO, the 200 pM drug concentration contains
2% DMSO.
Therefore, diluent made up to 2% DMSO in assay medium (F12K + 0.5%
fetal bovine serum) is used as diluent for the drug titrations in order to
dilute the
drug but keep the DMSO concentration constant. Add this diluent to the
remaining
wells in the column at 60 pL/well. Take 60 p,L from the 120 p.L of 200 p.M
drug
dilution in the top well of the column and mix with the 60 ~L in the second
well of
the column. Take 60 ~L from this well and mix with the 60 ~,L in the third
well of
the column, and so on until two-fold titrations are completed. When the next-
to-the-
last well is mixed, take 60 ~L of the 120 ~L in this well and discard it.
Leave the
last well with 60 pL of DMSO/media diluent as a non-drug-containing control.
Make 9 columns of titrated drug, enough for triplicate wells ea~;t~ for 1)
VEGF
(obtained from Pepro Tech Inc., catalogue no. 100-200, 2) endothelial cell
growth
factor (ECGF) (also known as acidic fibroblast growth factor, or aFGF)
(obtained
from Boehringer Mannheim Biochemica, catalogue no. 1439 600); or, 3) human
PDGF B/B (1276-956, Boehringer Mannheim, Germany) and assay media control.
ECGF comes as a preparation with sodium heparin. .
2. Transfer 50 p,L/well of the drug dilutions to the 96-well assay plates
containing the 0.8-l.OxlO~ cells/100 ~.L/well of the HUV-EC-C cells from day 0
and
incubate ~2 h at 37 °C, 5% CO,.
3. In triplicate, add 50 pL/well of 80 pg/mL VEGF, 20 ng/mL ECGF, or
media control to each drug condition. As with the drugs, the growth factor
concentrations are 4X the desired final concentration. Use the assay media
from day



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168
0 step 2 to make the concentrations of growth factors. Incubate approximately
24
hours at 37 °C, 5% CO,. Each well will have 50 ~L drug dilution, 50 ~L
growth
factor or media, and 100 pL cells, = 200 ~L/well total. Thus the 4X
concentrations
of drugs and growth factors become 1 X once everything has been added to the
wells.
DAY 2
1. Add'H-thymidine (Amersham; catalogue no. TRK-686) at 1 Ci/well
( 10 1/well of 100 Ci/mL solution made up in RPMI media + 10% heat-inactivated
fetal bovine serum) and incubate ~24 h at 37 °C, 5% CO,. Note: 'H-
thymidine is
made up in RPMI media because all of the other applications for which we use
the
'H-thymidine involve experiments done in RPMI. The media difference at this
step
is probably not significant. RPMI was obtained from Gibco BRL, catalogue no.
11875-051.
DAY 3
1. Freeze plates overnight at -20 °C.
DAY 4
1. Thaw plates and harvest with a 96-well plate harvester (Tomtec
Harvester 96~R~) onto filter mats (Wallac; catalogue no. 1205-401 ); read
counts on a
Wallac Betaplate~TM~ liquid scintillation counter.
EXAMPLE 17: FGF-Induced BrdU incorporation Assay
This assay measures FGF-induced DNA synthesis in 3Tc7/EGFr cells that
express endogenous FGF receptors.
Materials and Rea e~ nts:
1. FGF: human FGF2/bFGF (Gibco BRL, No. 13256-029).
2. BrdU Labeling reagent, (10 mM PBS (pH 7.4), Boehringer
Mannheim Cat No. 1 647 229).
3. FIXDENAT fixation solution (Boehringer Mannheim Cat No. 1 647
229).



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169
4. Anti-BrdU-POD (mouse monoclonal antibody conjugated with
peroxidase, Boehringer Mannheim Cat. No. 1 647 229).
5. TMB (tetramethylbenzidine, Boehringer Mannheim Cat. No. 1 647
229).
6. PBS washing solution, pH 7.4.
7. Albumin, bovine (BSA), fraction V powder (Sigma Chemical Co.,
Cat. No. A-8551)
Procedure
1. 3T3 engineered cell line: 3T3c7/EGFr.
2. Cells are seeded at 8,000 cells/well in DMEM, 10% CS and 2 mM
Gln in a 96-well plate. Incubate 24 hours at 37 °C in 5% COZ.
3. After 24 hours, wash cells with PBS then serum starve in serum free
medium (0% DMEM, 0.1% BSA) for 24 hours.
4. Add ligand (FGF2 (1.5 nM in DMEM with 0.1% BSA) and test
compound simultaneously. Negative control wells receive serum free DMEM with
0.1 % BSA only; positive control wells receive FGF2 ligand but no test
compound.
Test compounds are prepared in serum-free DMEM with ligand in a 96-well plate
and serially diluted to make seven (7) test concentrations.
5. After 20 hours, add diluted BrdU labeling reagent (1:100
BrdU:DMEM, 0.1 % BSA, final concentration is 10 pM) to the cells and incubate
for
1.5 hours.
6. Decant medium. Remove traces of material with paper towel. Add
FIXDENAT (50 ~l/well) and incubate at room temperature for 45 minutes on a
plate
shaker.
7. Remove FIXDENAT solution. Add blocking solution (5%
dehydrated milk in PBS (200 ~l/well)) and incubate for 30 minutes at room
temperature on a plate shaker.
8. Decant blocking solution; wash wells once with PBS. Add anti-
BrdU-POD solution ( 1:100 dilution in PBS, 0.1 % BSA); incubate for 90 minutes
at
room temperature on a plate shaker.



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9. Decant antibody conjugate; rinse wells 5 times with PBS. Dry plate
by inverting on paper towel and tapping.
10. Add TMB solution (100 ~L/well); incubate 20 minutes at room
temperature on a plate shaker until color development is sufficient for
photometric
detection.
11. Measure absorbance at 410 nM on a Dynatech ELISA plate reader
using "Dual wavelength" mode with a f lter at 490 nM.
EXAMPLE 18: Biochemical EGFR Assay
This assay measures the in vits~o kinase activity of EGFR using ELISA.
Materials And Reagents
1. Corning 96-well ELISA plates (Corning Catalog No. 25805-96).
2. SUMOI monoclonal anti-EGFR antibody.
1 S 3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No.
450-1300EB).
4. TBST Buffer
Reagent M.W. Working ConcentrationAmount per
~ L


Tris 121.14 50 mM 6.057 g


NaCI 58.44 150 mM 8.766 g


Triton X-100NA 0.1% 1.0 mL


5. Blocking Buffer:
Reagent M.W. Working Amount per
Concentration'
100 mL


Carnation InstantNA 5% 5.0 g
Non-Fat Milk


PBS NA NA 100 mL



6. A431 cell lysate
7. TBS Buffer:
Reagent M.W. Working ConcentrationAmount per
L


Tris 121.14 __ SO mM 6.057 g
~


NaCI 58.44 150 mM 8.766 g





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8. TBS + 10% DMSO
Reagent M. Working ConcentrationAmount per
W. L


Tris 121.1450 mM 1.514 g


NaC 1 5 8.44150 mM 2.192 g


DMSO NA 10% 25 mL


9. Adenosine-S'-triphosphate (ATP, from Equine muscle, Sigma Cat.
No. A-5394).
Prepare a 1.0 mM solution in dH,O. This reagent should be made up
immediately prior to use and kept on ice.
10. MnC 1,.
Prepare a 1.0 M stock solution in dH~O.
11. ATP/MnCI, phosphorylation mix
Reagent M. W. Amount per 10 Working
mL Concentration


ATP 1.0 300 ~L 30 ~M
mM


MnCI, 1.0 500 ~L 50 mM
M


dH,O 9.2 mL


This reagent should be prepared immediately before use and kept on
ice
12. NI1NC 96-well V bottom polypropylene plates (Applied Scientific
Cat. No. AS-72092).
13. Ethylenediaminetetraacetic acid (EDTA)
Prepare 200 mM working solution in dH~O. Adjust to pH 8.0 with 10
N NaOH.
14. Rabbit polyclonal anti-phosphotyrosine serum.
15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.
ALI0404)
16. ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), Sigma
Cat. No. A-1888).
Reagent M.W. Working Concentration Amount per L
Citric Acid 192.12 100 mM 19.21 g



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Na.,HPO~ 141.9_6 250 mM 35.49 g
ABTS NA 0.5 mg/mL 500 mg
Mix first two ingredients in about 900 mL dH,O, adjust pH to 4.0 with
phosphoric acid. Add ABTS, cover, let sit about 0.5 hr., filter. The solution
should
be kept in the dark at 4 °C until ready to use.
17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325)
18. ABTS/H,Oz
Mix 15 mL ABTS solution and 2.0 ~L H,O=. Prepare S minutes before use.
19. 0.2 M HCl
Procedure
1. Coat Corning 96 well ELISA plates with 0.5 ~g SUMO1 in 100 ~L
PBS per well, store overnight at 4 °C.
2. Remove unbound SUMO1 from wells by inverting plate to remove
liquid. Wash lx with dH20. Pat the plate on a paper towel to remove excess
liquid.
3. Add 150 pL of Blocking Buffer to each well. Incubate for 30 min. at
room temperature with shaking.
4. Wash plate 3x with deionized water, then once with TBST. Pat plate
on a paper towel to remove excess liquid and bubbles.
5. Dilute lysate in PBS (7 ~g lysate/100 ~L PBS).
6. Add 100 ~.L of diluted lysate to each well. Shake at room temperature
for 60 min.
7. Wash plates as described in 4, above.
8. Add 120 ~L TBS to ELISA plate containing captured EGFR.
9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates (ie. 10
yL compound + 90 ~L TBS).
10. Add 13.5 p.L diluted test compound to ELISA plate. To control wells
(wells which do not receive any test compound), add 13.5 ~L TBS + 10% DMSO.
11. Incubate for 30 minutes while shaking at room temperature.
12. Add 15 ~L phosphorylation rnix directly to all wells except negative



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control well which does not receive ATP/MnC 1 ~ (final well volume should be
approximately 150 ~L with 3 ~M ATP/5 mM MnCI, final concentration in each
well.) Incubate S minutes while shaking.
13. After 5 minutes, stop reaction by adding 16.5 ~.L of 200 mM EDTA
(pH 8.0) to each well, shaking continuously. After the EDTA has been added,
shake
for 1 min.
14. Wash 4x with deionized water, twice with TBST.
15. Add 100 ~,L anti-phosphotyrosine (1:3000 dilution in TBST) per
well. Incubate 30-45 min. at room temperature, with shaking.
16. Wash as described in 4, above.
17. Add 100 ~L Biosource Goat anti-rabbit IgG peroxidase conjugate
(1:2000 dilution in TBST) to each well. Incubate 30 min. at room temperature,
with
shaking.
18. Wash as described in 4, above.
19. Add 100 p.L of ABTS/H,O, solution to each well.
20. Incubate S to 10 minutes with shaking. Remove any bubbles.
21. If necessary stop reaction with the addition of 100 pL 0.2 M HCl per
well.
22. Read assay on Dynatech MR7000 ELISA reader. Test Filter: 410 nM
Reference Filter: 630 Nm.
EXAMPLE 19: Biochemical PDGFR Assay
This assay measures the in vita°o kinase activity of PDGFR using
ELISA.
Materials And Reagents
Unless otherwise noted, the preparation of working solution of the following
reagents is the same as that for the Biochemical EGFR assay, above.
1. Corning 96-well Elisa plates (Corning Catalog No. 25805-96).
2. 28D4C 10 monoclonal anti-PDGFR antibody.
3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No.
450-1300EB)



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4. TBST Buffer.


5. Blocking Buffer.


6. PDGFR-(3 expressing NIH 3T3 cell lysate.


7. TBS Buffer.


8. TBS + 10% DMSO.


9. Adenosine-5'-triphosphate (ATP, from Equine
muscle, Sigma Cat.


No. A-5394).


10. MnCl,.


11. Kinase buffer phosphorylation mix.


Reagent Stock Amount Working


solution per 10 mL Concentration


Tris 1 M 250 pL 25 mM


NaCI 5 M 200 q,L 100 mM


MnCI, 1 M 100 pL 10 mM


TX-100 100 mM 50 qL 0.5 mM


12. NUNC 96-well V bottom polypropylene plates (Applied Scientific
Cat. No. AS-72092).
13. Ethylenediaminetetraacetic acid (EDTA).
14. Rabbit polyclonal anti-phosphotyrosine serum.
15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.
ALI0404).
16. 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma
Cat. No. A-1888).
17. Hydrogen peroxide 30% solution (Fisher Cat. No. H325).
18. ABTS/H,Oz.
19. 0.2 M HCI.
Procedure
1. Coat Corning 96 well ELISA plates with 0.5 qg 28D4C10 in 100 qL
PBS per well, store overnight at 4° C.
2. Remove unbound 28D4C 10 from wells by inverting plate to remove
liquid. Wash lx with dH,O. Pat the plate on a paper towel to remove excess
liquid.



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3. Add 150 qL of Blocking Buffer to each well. Incubate for 30 min. at
room temperature with shaking.
4. Wash plate 3x with deionized water, then once with TBST. Pat plate
on a paper towel to remove excess liquid and bubbles.
5. Dilute lysate in HNTG (10 pg lysate/100 ~L HNTG)
6. Add 100 ~L of diluted lysate to each well. Shake at room temperature
for 60 min.
7. Wash plates as described in 4, above.
8. Add 80 p,L working kinase buffer mix to ELISA plate containing
captured PDGFR.
9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates
(i.e., 10 pL compound + 90 q,L TBS).
10. Add 10 ~L diluted test compound to ELISA plate. To control wells
(wells which do not receive any test compound), add 10 ~L TBS + 10% DMSO.
11. Incubate for 30 minutes while shaking at room temperature.
12. Add 10 ~L ATP directly to all wells except negative control well
(final well volume should be approximately 100 ~L with 20 p,M ATP in each
well.)
Incubate 30 minutes while shaking.
13. After 30 minutes, stop reaction by adding 10 ~L of 200 mM EDTA
(pH 8.0) to each well.
14. Wash 4x with deionized water, twice with TBST.
15. Add 100 ~L anti-phosphotyrosine (1:3000 dilution in TBST) per
well. Incubate 30-45 min. at room temperature, with shaking.
16. Wash as described in 4, above.
17. Add 100 ~L Biosource Goat anti-rabbit IgG peroxidase conjugate
(1:2000 dilution in TBST) to each well. Incubate 30 min. at room temperature,
with
shaking.
18. Wash as described in 4, above.
19. Add 100 ~L of ABTS/H,O~ solution to each well.
20. Incubate 10 to 30 minutes with shaking. Remove any bubbles.
21. If necessary stop reaction with the addition of 100 ~L 0.2 M HCl per



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well.
22. Read assay on Dynatech MR7000 ELISA reader: test filter: 410 nM,
reference filter: 630 nM.
EXAMPLE 20: Biochemical FGFR Assay
This assay measures in vitro kinase activity of the Myc-GyrB-FGFR fusion
protein using ELISA.
Materials And Reagents
1. HNTG
Reagent M.W. Sx Stock Amountlx Working


Concentration per Concentration
L


HEPES 238.3 100 mM 23.83 g 20
mM


NaCI 58.44 750 150 mM
mM
43.83
g


Glycerol NA 50% 500 mL 10%


Triton X-100 NA 5% 10 mL 1.0%


To make a liter of Sx stock solution, dissolve HEPES and NaCI in about 350
mLl dH20, adjust pH to 7.2 with HCl or NaOH (depending on the HEPES that is
used), add glycerol, Triton X-100 and then dH~O to volume.
2. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco Catalog #
450-1300EB).
3. Blocking Buffer.
4. Kinase Buffer.
Reagent M.W. lOx Stock lx Working
Concentration Concentration
HEPES (pH 7.2) 238.3 500 mM 50 mM
MnCI, 20 mM 2 mM
MgCI, - 203.32 200 mM 10 mM
Triton-X-100 1 % 0.1%
DTT 380.35 5 mM 0.5 mM
5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Cat. No. P-7626):
Working solution: 100 mM in ethanol.
6. ATP (Bacterial source, Sigma Cat. No. A-7699)



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Use 3.31 mg per mL MilliQ H,O for a stock concentration of 6 mM.
7. Biotin conjugated anti-phosphotyrosine mab (clone 4610, Upstate
Biotechnology Inc. Cat. No. 16-103, Ser. No. 14495).
8. VECTASTAIN ELITE ABC reagent (Avidin peroxidase conjugate,
Vector Laboratories Cat. No. PK-6 100).
9. ABTS Solution.
10. Hydrogen peroxide 30% solution ( Fisher Catalog # H325).
11. ABTS/H~O,.
12. 0.2 M HC1.
13. TRIS HC1 (Fischer Cat. No. BP 152-5).
Prepare 1.0 mM solution in MilliQ H,O, adjust pH to 7.2 with HC1.
14. NaCI (Fisher Cat. No. S271-10).
Prepare 5 M solution in MilliQ H,O.
15. MgClz (Fisher Cat. No. M33-S00).
Prepare 1 M solution in MilliQ H,O.
16. HEPES (Fisher Cat. No. BP310-S00).
Prepare 1 M solution in MilliQ HZO, adjust pH to 7.5, sterile filter.
17. TBST Buffer.
18. Sodium Carbonate Buffer (Fisher Cat. No. 5495).
Prepare 0.1 M solution in MilliQ H,O, adjust pH to 9.6 with NaOH,
filter.
19. Dithiothreitol (DTT, Fisher Cat. No. BP172-25).
Prepare 0.5 mM working solution in MilliQ H20 just prior to use.
Store at -20° C until used, discard any leftover.
20. MnCl,.
21. Triton X-100.
22. Goat a-Rabbit IgG (Cappel).
23. Affinity purified Rabbit a GST GyrB.
Procedure
All of the following steps are conducted at room temperature unless



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178
otherwise indicated.
1. Coat Corning 9G-well ELISA plates with 2 ~g Goat a-Rabbit
antibody per well in Carbonate Buffer such that total well volume is 100 ~1.
Store
overnight at 4° C.
2. Remove unbound Goat a-Rabbit antibody by inverting plate to
remove liquid. Pat plate on a paper towel to remove excess liquid and bubbles
3. Add 150 ~L Blocking Buffer (5% Low Fat Milk in PBS) to each
well. Incubate while shaking on a micro-titer plate shaker for 30 min.
4. Wash 4x with TBST. Pat plate on a paper towel to remove excess
liquid and bubbles.
S. Add 0.5 ~g Rabbit a-GyrB antibody per well. Dilute antibody in
DPBS to a final volume of 100 pL per well. Incubate with shaking on a micro-
titer
plate shaker at room temperature for 1 hour.
6. Wash 4x with TBST as described in step 4.
7. Add 2 ~g COS/FGFR cell lysate (Myc-GyrB-FGFR source) in
HNTG to each well to give a final volume of 100 ~L per well. Incubate with
shaking
on a micro-titer plate shaker for 1 hour.
8. Wash 4X with TBST as described in step 4.
9. Add 80 p,L of lx kinase buffer per well.
10. Dilute test compound 1:10 in 1 x kinase buffer + 1 % DMSO in a
polypropylene 96-well plate.
11. Transfer 10 pL of diluted test compound solution and control wells
from polypropylene plate wells to the corresponding ELISA plate wells,
incubate
with shaking on a micro-titer plate shaker for 20 minutes.
12. Add 10 p.L of 70 p,M ATP diluted in kinase buffer to positive control
and test wells (final ATP concentration is 7 pM/well). Add 10 ~L 1x kinase
buffer
to negative control wells. Incubate with shaking on a micro-titer plate shaker
for 15
mm.
13. Stop kinase reaction by adding 5 ~L 0.5 M EDTA to all wells.
14. Wash 4x with TBST as described in step 4.
15. Add 100 pL biotin conjugated a-phosphotyrosine mab (b4G10)



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179
diluted in TBST to each well. Incubate with shaking on a micro-titer plate
shaker
for 30 minutes.
16. Prepare VECTASTAIN ABC reagent. Add 1 drop reagent A to 15
mL TBST. Mix by inverting tube several times. Add 1 drop reagent B and mix
again.
17. Wash 4x with TBST as described in step 4.
18. Add 100 ~L ABC HRP reagent to each well. Incubate with shaking
on a micro-titer plate shaker for 30 minutes.
19. Wash 4x with TBST as described in step 4.
20. Add 100 ~tL of ABTS/H,O, solution to each well.
22. Incubate 5 to 15 minutes with shaking. Remove any bubbles.
23. If necessary stop reaction by adding 1 00 p,L of 0.2 M HCl/well.
24. Read assay on Dynatech MR7000 ELISA Plate Reader; test filter:
410 nM, reference filter: 630 nM.
EXAMPLE 21: Biochemical FLK-1 Assay
This assay evaluates flk-1 autophosphorylation activity in vitro using ELISA.
Materials And Reagents
1. 15 cm tissue culture dishes
2. Flk-11NIH cells: NIH fibroblast line over-expressing human flk-1
clone 3.
3. Growth medium: DMEM plus heat inactivated 10% FBS and 2 mM
Glutamine (Gibco-BRL).
4. Starvation medium: DMEM plus 0.5% heat-inactivated FBS, 2 mM
Glutamine (Gibco-BRL).
5. Corning 96-well ELISA plates (Corning Cat. No. 25805-96).
6. L4 or E38 monoclonal antibody specific for flk-l; Purified by
Protein-A agarose affinity chromatography.
7. PBS (Dulbecco's Phosphate-Buffered Saline) Gibco Cat. No.
450-1300EB).



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8. HNTG (see BIOCHEMICAL FGFR for preparation).
9. Pierce BCA protein determination kit.


10. Blocking buffer


11. TBST (pH 7.0)


12. Kinase Buffer


13. Kinase Stop Solution: 200 mM EDTA.


14. Biotinylated 4610, specific for phosphotyrosine
(UBI, Cat. No. No.


16-103).
15. AB kit (Vector Laboratories Cat. No. PK 4000).
16. DMSO
17. NUNC 96-well V bottom polypropylene plates (Applied Scientific
Cat. No. AS-72092).
18. TURBO-TMB (Pierce).
19. TURBO-TMB stop solution: 1 M H,S04.
20. ATP (Sigma Cat. No. A-7699).
21. 20% DMSO in TBS (pH 7.0).
Procedure
Cell Growth and Lysate Preparation.
1. Seed cell into growth medium and grow for 2-3 days to 90-100%
confluency at 37° C and 5% CO,. Do not exceed passage #20.
2. Remove the medium and wash the cells twice with PBS. Lyse with
HNTG lysis buffer. Collect all lysates and vortex mix them for 20-30 seconds.
3. Remove insoluble material by centrifugation (5-10 min at about
10,000 xg).
4. Determine the protein concentration using BCA kit.
5. Partition lysate into 1 mg aliquots, store at -80° C.
Assay Procedure
1. Coat Corning 96-well ELISA plates with 2 ug/well purified L4 (or E
38) in 100 ~.L of PBS. Store overnight at 4° C.



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2. Remove unbound proteins from wells by inverting the plate to
remove the liquid. Wash one time with dH,O, pat plate on paper towel to remove
excess liquid.
3. Block plates with 1SO qL blocking buffer per well. Incubate for
S 4S-60 minutes with shaking at 4° C.
4. Remove the blocking buffer and wash the ELISA plate three times
with dH=O and one time with TBST. Pat plate on paper towel to remove excess
liquid.
S. Dilute lysate in PBS to give final concentration of SO p.g/100 pl. Add
100 qL of diluted lysate to each well. Incubate with shaking at 4° C
overnight.
6. Remove unbound proteins from wells by inverting the plate. Wash as
in step 4.
7. Add 80 qL of kinase buffer to wells (90 ~L to negative control
wells).
1 S 8. Dilute test compounds (normally 10-fold) into wells of a
polypropylene plate containing 20% DMSO in TBS.
9. Add 10 q.L of the diluted compounds to the ELISA wells containing
immobilized flk-1 and shake. Control wells receive no compounds.
10. From stock 1 mM ATP, prepare 0.3 mM ATP solution in dH~O
(alternatively, kinase buffer may be used).
11. Add 10 ~L of 0.3 mM ATP to all wells except the negative controls.
Incubate for 60 min. at room temperature with shaking.
12. After 1 hr stop the kinase reaction by adding 11 pL 200 mM EDTA.
Shake for 1-2 min.
2S 13. Wash the ELISA plate 4 times with dH,O and twice with TBST.
14. Add 100 pL of 1:5000 biotinylated 4G10:TBST to all wells. Incubate
4S min with shaking at room temperature.
1S. While the above is incubating, add SO qL of solutions A & B from
the ABC kit to 10 mL of TBST. These solutions must be combined approximately
30 min prior to use.
16. Wash plates as in step 4



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17. Add 100 ~L of the preformed A & B complex to
all wells. Incubate


30 min with
shaking
at room
temperature.


18. Wash plates as in step 4.


19. Add 100 ~L TURBO-TMB. Shake at room temperature
for 10- 15


min.


20. When the color in the positive control wells
reaches an absorbance of


about 0.35 0.4, stop the reaction with 100 ~L of TURBO-TMB
- stop solution.


21. Read plates on Dynatech MR7000 ELISA reader;
test filter: 450 nM,


reference
filter:
410 nM.



In Vivo Animal Models
EXAMPLE 22: Xenograft Animal Models
The ability of human tumors to grow as xenografts in athymic mice (e.g.,
Balb/c, nu/nu) provides a useful in vivo model for studying the biological
response
to therapies for human tumors. Since the first successful xenotransplantation
of
human tumors into athymic mice, (Rygaard and Povlsen, 1969, Acta Pathol.
Microbial. Scand. 77:758-760), many different human tumor cell lines (e.g.,
mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma,
bone,
and malignant melanomas) have been transplanted and successfully grown in nude
mice. The following assays may be used to determine the level of activity,
specificity and effect of the different compounds of the present invention.
Three
general types of assays are useful for evaluating compounds:
cellular/catalytic,
cellular/biological and in vivo. The object of the cellular/catalytic assays
is to
determine the effect of a compound on the ability of a TK to phosphorylate
tyrosines
on a known substrate in a cell. The object of the cellular/biological assays
is to
determine the effect of a compound on the biological response stimulated by a
TK in
a cell. The object of the in vivo assays is to determine the effect of a
compound in
an animal model of a particular disorder such as cancer.
Suitable cell lines for subcutaneous xenograft experiments include C6 cells
(glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619), A431



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cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB
56), PC3 cells (prostate, ATCC # CRL 1435) and NIH 3T3 fibroblasts genetically
engineered to overexpress EGFR, PDGFR, IGF-1R or any other test kinase. The
following protocol can be used to perform xenograft experiments:
Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen
Laboratories (Gilroy, CA). All animals are maintained under clean-room
conditions
in Micro-isolator cages with Alpha-dri bedding. They receive sterile rodent
chow
and water ad libitum.
Cell lines are grown in appropriate medium (for example, MEM, DMEM,
Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine serum (FBS) and 2 mM
glutamine (GLN)). All cell culture media, glutamine, and fetal bovine serum
are
purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise
specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10%
CO, at 37 °C. All cell lines are routinely subcultured twice a week
and are
negative for mycoplasma as determined by the Mycotect method (Gibco).
Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and
pelleted at 450 x g for 10 min. Pellets are resuspended in sterile PBS or
media
(without FBS) to a particular concentration and the cells are implanted into
the
hindflank of the mice (8 - 10 mice per group, 2 - 10 x l OG cells/animal).
Tumor
growth is measured over 3 to 6 weeks using venier calipers. T~;.z~~or volumes
are
calculated as a product of length x width x height unless otherwise indicated.
P
values are calculated using the Students t-test. Test compounds in 50 - 100 ~L
excipient (DMSO, or VPD:DSW) was delivered by IP injection at different
concentrations generally starting at day one after implantation.
EXAMPLE 23: Tumor Invasion Model
The following tumor invasion model has been developed and maybe used
for the evaluation of therapeutic value and efficacy of the compounds
identified
to selectively inhibit KDR/FLK-1 receptor.
3O
Procedure



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8 week old nude mice (female) (Simonsen Inc.) were used as experimental
animals. Implantation of tumor cells was performed in a laminar flow hood. For
anesthesia, Xylazine/Ketamine Cocktail ( 100 mg/kg ketamine and 5 mg/kg
Xylazine) are administered intraperitoneally. A midline incision is done to
expose the abdominal cavity (approximately 1.5 cm in length) to inject 10'
tumor
cells in a volume of 100 ~L medium. The cells are injected either into the
duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum
and muscles are closed with a 6-0 silk continuous suture and the skin was
closed
by using wound clips. Animals were observed daily.
Analysis
After 2-6 weeks, depending on gross observations of the animals, the mice
are sacrificed, and the local tumor metastases, to various organs (lung,
liver,
brain, stomach, spleen, heart, muscle) are excised and analyzed (measurements
of
tumor size, grade of invasion, immunochemistry, and in situ hybridization).
EXAMPLE 24: Measurement of Cell Toxicity
Therapeutic compounds should be more potent in inhibiting protein kinase
activity than in exerting a cytotoxic effect. A measure of the effectiveness
and cell
toxicity of a compound can be obtained by determining the therapeutic index:
IC;°/LD;°. IC;°, the dose required to achieve 50%
inhibition, can be measured using
standard techniques such as those described herein. LD;°, the dosage
which results in
50% toxicity, can also be measured by standard techniques (Mossman, 1983, J.
Irnmunol. Methods, 65:55-63), by measuring the amount of LDH released
(Korzeniewski and Callewaert, 1983, J. Immunol. Methods, 64:313; Decker and
Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by measuring the
lethal
dose in animal models. Compounds with a large therapeutic index are preferred.
The therapeutic index should be greater than 2, preferably at least 10, more
preferably at least 50.



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EXAMPLE 25: The Activity of the Compounds of the Invention
The biological or biochemical activity of some of the compounds of the
invention were tested using the assays described above. The ICSO values were
measured for several of the compounds of the invention. The results are shown
in
the tables below.
TABLE 4
Compound Her-2 kinase bio PDGFr bio EGFr
Number ICso (~M) ICso (~M) ICso (~M)


AHI-1 > 100 > 100 > 100


AHI-2 31.51 49.88 >100


AHI-3 > 100 > 100 > 100


AHI-4 > 100 > 100 > 100


AHI-5 > 100 > 100 > 100


AHI-6 5 7.31 76.5 6 > 100


AHI-7 16.76 0.02 12.08


AHI-8 70.97 0.52 >100


TABLE 5
PDGFr EGFr SKOV3 BrdU BrdU BrdU
C HER2 kmase A431 growth ~ DNA DIVA DNA
d ! syn.
'


om oun bi ochembiochem IC rowth IC s n. s n. PDGFr
P IC IC so g so IC HER2 EGFr IC
(I~NI) so (w~4) ~ Y so
y


so (w~) so ICso ICso ' (uM)
(w~~) (wM) (N~) (wM)


AAI-1 >100 >100 >100


AAI-2 50.5 > > I 00
100


AAI-3 >100 >I00 >100


AAI-4 >100 >100 >100


AAI-5 1G.4 >100 >100 17.86 5.2G 32.67 29.95 32.7


AAI-G >100 >100 >100 27 3 -~~0.._._.._.._j50...__._...





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186
AAI-7 > I > I > I G.8 28. I 31. I
00 00 00 1 !


AAI-8 > 100 > 100 > I 1 I >50 >50
00 .5


AAI-9 >100 >100 >100 5.2 5.25


AAI-10 22.2 >100 >100 17.27 17.7 34.OG >50 >50


AAI-11 >100 >100 >100 2(.78 29.64 >50 >50 >50


AAI-12 >100 >100 >100 >50 >50


AAI-13 >100 >100 >100 >50 >50 >50 >50 >50


AAI-14 >100 >100 >100


AAI-15 >100 >100 >100


AAI-1G >100 >100 >100 >50 >50 >50
'


AAI-17 >100 >100 >100 >SO >50 >50


AAI-18 28.47 O.SG >100 42.97 >SO >50 >50 3.87


AAI-19 >100 0.25 >100


AAI-20 20.95 2.13 >100 8.81 11.OG 29.24 38.5 8.81


AAI-21 52.92 3.42 48.GG >50 27.77 1.2


AAI-22 21.45 80.03 >100 >50 32.94 >50 27.SG 1.15
' ' ~


_....-..-_..__....~__....._._...-~.............__.- __~- -.
AAI-23 54.09 0.81 > 100 >50 32.21 0.35


AAI-24 26.12 >100 >100 30.94 >50 >50


AAI-25 >100 >100 >100 >50 >50 >50


AAI-2G 67.62 >100 >100


AAI-27 >100 >100 >100 >50 >50 >50


AAI-28 48 >100 >100


TABLE 6
Compound No. Her2-Driven EGF-Driven PDGF-Driven
BrdU BrdU BrdU


Incorp. ICso Incorp. ICSO Incorp. ICso
(~M) (~tM) (~M)



DAI-I 46.9 > 50 0.77


DAI-II 30.2 38.3 0.26


TABLE 7



CA 02368041 2001-09-24
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Comp'dHER2 PDGFr EGFr SKOV3 A431 BrdU BrdU BrdU
No. Kinase biochembiochemgrowth growthDNA DNA DNA
ICS" ICso ICSO ICSO ICt~ syn. syn. syn.
(~M) (~M) (~M) (~M) (pM) HER2 EGFr PDGFr
ICso ICSa ICso
(pM) (~M) (~M)


IN-00116.6 > 100 > 100 27.8 12.3 28.9 32.5 35.2


IN-00218 > 100 > 100 24.5 8.2 30 41.5 37.1


IN-00315.5 20.2 > 100 17.8 8.7 12.1 29 22


IN-00410.6 > 100 > 100 2.9 1.1 3.6 4.7 3.3


IN-00512.9 > 100 > 100 N/A* N/A* 1.6 3 1.7


IN-00610.6 > 100 > 100 5 6 6.6 11.1 4.6


IN-00722.7 > 100 > 100 9.7 4.2 N/A* N/A* N/A*


IN-0088.5 > 100 > 100 1.5 2.5 2.3 31.3 5.5


IN-009> 100 0.43 > 100 25.2 13.6 4.2 25.3 0.56


IN-010> 100 N/A* > 100 > 25 > 25 N/A* N/A* N/A*


IN-O1 > 100 N/A* > 100 > 50 > 50 N/A* N/A* N/A*
I


IN-012> 100 N/A* > 100 > 50 > 50 N/A* N/A* N/A*


IN-013> 100 N/A* > 100 > 50 > 50 N/A* N/A* N/A*


IN-014> 100 N/A* > 100 > 50 > 50 N/A* N/A* N/A*


IN-015> 100 N/A* > 100 20.5 21 N/A* N/A* N/A*


IN-016> 100 N/A* > 100 > 50 > 50 N/A* N/A* N/A*


IN-017> 100 N/A* > 100 > 50 N/A* N/A* N/A* N/A*


IN-018> 100 N/A* > 100 N/A* N/A* N/A* N/A* N/A*


IN-019> 100 N/A* > 100 N/A* N/A* '~~3!!~,*N/A* N/A*


IN-020> 100 N/A* > 100 > 50 15.2 N/A* N/A* N/A*
i


IN-021> 100 N/A* > 100 N/A* N/A* N1A* N/A* N/A*


IN-022> 100 > 100 > 100 N/A* N/A* > 50 > 50 > 50


IN-023> 100 49.1 > 100 N/A* N/A* 33.8 28.7 23.6


IN-024> 100 97.5 > 100 N/A* N/A* > 50 > 50 49.6


IN-0253.9 70.8 > 100 N/A* N/A* N/A* N/A* N/A*


*Designation "N/A" indicates that no data is available for this compound.



CA 02368041 2001-09-24
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CONCLUSION
One skilled in the art would readily appreciate that the present invention is
well adapted to carry out the objects and obtain the ends and advantages
mentioned,
as well as those inherent therein. The molecular complexes and the methods,
procedures, treatments, molecules, specific compounds described herein are
presently representative of preferred embodiments are exemplary and are not
intended as limitations on the scope of the invention. Changes therein and
other
uses will occur to those skilled in the art which are encompassed within the
spirit of
the invention are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions
and modifications may be made to the invention disclosed herein without
departing
from the scope and spirit of the invention.
All patents and publications mentioned in the specification are indicative of
the levels of those skilled in the art to which the invention pertains. All
patents and
publications are herein incorporated by reference to the same extent as if
each
individual publication was specifically and individually indicated to be
incorporated
by reference.
The invention illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which is not
specifically disclosed herein. Thus, for example, in each instance herein any
of the
terms "comprising", "consisting essentially of and "consisting of may be
replaced
with either of the other two terms. The terms and expressions which have been
employed are used as terms of description and not of limitation, and there is
no
intention that in the use of such terms and expressions of excluding any
equivalents
of the features shown and described or portions thereof, but it is recognized
that
various modifications are possible within the scope of the invention claimed.
Thus,
it should be understood that although the present invention has been
specifically
disclosed by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by those skilled
in the
art, and that such modifications and variations are considered to be within
the scope
of this invention as defined by the appended claims.



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In addition, where features or aspects of the invention are described in terms
of Markush groups, those skilled in the art will recognize that the invention
is also
thereby described in terms of any individual member or subgroup of members of
the
Markush group. For example, if X is described as selected from the group
consisting of bromine, chlorine, and iodine, claims for X being bromine and
claims
for X being bromine and chlorine are fully described.
The invention has been described broadly and generically herein. Each of
the narrower species and subgeneric groupings falling within the generic
disclosure
also form part of the invention. This includes the generic description of the
I 0 invention with a proviso or negative limitation removing any subject
matter from the
genus, regardless of whether or not the excised material is specifically
recited herein.
Other embodiments are within the following claims.