INHIBITORS OF FARNESYL-PROTEIN TRANSFERASE

INHIBITEURS DE LA TRANSFERASE DE LA FARNESYL-PROTEINE

English Abstract

The present invention is directed to compounds which inhibit farnesyl-protein
transferase (FTase) and the farnesylation of the oncogene protein Ras. The
invention is further directed to chemotherapeutic compositions containing the
compounds of this invention and methods for inhibiting farnesyl-protein
transferase and the farnesylation of the oncogene protein Ras.

French Abstract

La présente invention concerne des composés qui inhibent la transférase de la farnésyl-protéine (FTase) et la farnésylation de la protéine Ras oncogène. L'invention concerne par ailleurs des compositions chimiothérapeutiques renfermant ces composés et des procédés permettant d'inhiber la transférase de la farnésyl-protéine ainsi que la farnésylation de la protéine Ras oncogène.

Claims

WHAT IS CLAIMED IS:
1. A compound which inhibits farnesyl-protein
transferase of the formula A:
<IMG>
wherein:
R1a, R1b and R1c are independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl,
C2-C6 alkynyl, R8O-, R9S(O)m-, R8C(O)NR8-, CN, NO2,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or
R9OC(O)NR8-,
c) C1-C6 alkyl unsubstituted or substituted by unsubstituted or
substituted aryl, heterocyclic, C3-C10 cycloalkyl, C2-C6
alkenyl, C2-C6 alkynyl, R8O-, R9S(O)m-, R8C(O)NR8-, CN,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or
R9OC(O)-NR8-;
R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
unsubstituted or substituted C2-8 alkenyl, unsubstituted or substituted aryl,
unsubstituted or substituted heterocycle, <IMG>, <IMG> and
-S(O)2R6,
wherein the substituted group is substituted with one or more of:
-80-

1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)p OR6,
c) (CH2)p NR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) C3-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
5) -NR6R7
6) <IMG>
7) <IMG>
8) <IMG>
9) <IMG>
10) <IMG>
11) -SO2-NR6R7,
12) <IMG>,
-81-

<IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a and R3b are independently absent or selected from: H, unsubstituted or
substituted alkyl, unsubstituted or substituted aryl, unsubstituted or
substituted heteroaryl, unsubstituted or substituted aralkyl and
unsubstituted or substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl,
C2-C6 alkynyl, perfluoroalkyl, F, Cl, Br, R8O-, R9S(O)m-,
R8C(O)NR8-, CN, NO2, R8 2N-C(NR8)-, R8C(O)-, R8OC(O)-,
N3, -N(R8)2, or R9OC(O)NR8-, and
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6
alkynyl, perfluoroalkyl, F, Cl, Br, R8O-, R9S(O)m-,
R8C(O)NH-, CN, H2N-C(NH)-, R8C(O)-, R8OC(O)-, N3,
-N(R8)2, or R8OC(O)NH-;
R5 is independently selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl,
perfluoroalkyl, F, Cl, Br, R8O-, R9S(O)m-, R8C(O)NR8-, CN,
NO2, (R8)2N-C-(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or
R9OC(O)NR8-, and
-82-

c) C1-C6 alkyl, unsubstituted or substituted by perfluoroalkyl,
F, Cl, Br, R8O-, R9S(O)m-, R8C(O)NR8-, CN, (R8)2N-
C(NR8)-, R8C(O)-, R8OC(O)-, N3, -N(R8)2, or R9OC(O)NR8-;
R6, R7 and R7a are independently selected from: H; C1-4 alkyl, C3-6
cycloalkyl, heterocycle, aryl, C1-4 perfluoroalkyl, unsubstituted or
substituted with one or two substituents selected from:
a) C1-4 alkoxy,
b) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle,
c) halogen,
d) HO,
<IMG>
g) -S(O)mR9 , or
h) N(R8)2; or
R6 and R7 may be joined in a ring;
R7 and R7a may be joined in a ring;
R8 is independently selected from hydrogen, C1-C6 alkyl, benzyl,
2,2,2-trifluoroethyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, unsubstituted or substituted C3-6 cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
-83-

aroyl, unsubstituted or substituted heteroaryl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) -S(O)m Rs
h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C~C-,
-C(O)-, -C(O)NR8-, -NR8C(O)-, O, -N(R8)-, -S(O)2N(R8)-, -
N(R8)S(O)2-, or S(O)m;
J and K are independently selected from N, NH or CHy;
V is selected from:
a) hydrogen,
b) heterocycle,
-84-

C) aryl,
d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a a heteroatom selected from O, S, and N, and
e) C2-C20 alkenyl,
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
W is a heterocycle;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m-, -NR10-, O or -C(=O)-;
m is 0,1 or 2;
n is 0,1, 2, 3 or 4;
p is 0,1, 2, 3 or 4;
q is 0,1, 2, 3 or 4;
r is 0 to 5, provided that r is 0 when V is hydrogen;
s is 1 or 2;
t is 0 or 1; and
y is 1 or 2;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
2. The compound according to Claim 1, which inhibits
farnesyl-protein transferase, of the formula B:
<IMG>
-85-

wherein:
R1a and R1c are independently selected from: hydrogen, C3-C10
cycloalkyl, R8O-, -N(R8)2, F or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C6 cycloalkyl, R8O-, -N(R8)2 or
C2-C6 alkenyl,
c) C1-C6 alkyl unsubstituted or substituted by unsubstituted or
substituted aryl, heterocycle, C3-C6 cycloalkyl, C2-C6
alkenyl, R8O-, or -N(R8)2;
R2 is selected from:
a) C1-8 alkyl, unsubstituted or substituted with one or
more of:
1) aryl or heterocycle, unsubstituted or substituted
with:
i) C1-4 alkyl,
ii) (CH2)pOR6,
iii) (CH2)pNR6R7,
iv) halogen,
v) C1-4 perfluoroalkyl,
2) OR6,
3) SR6, SO2R6, or
<IMG>
-86-

c) aryl, unsubstituted or substituted with one or
more of:
1) C1-8 alkyl,
2) C1-8 perfluoroalkyl,
3) OR6,
4) SR6, SO2R6, or
<IMG>
d) -SO2R6;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2, (R8)2N-
C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or R9OC(O)NR8-, and
c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, R8O-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2,
or R9OC(O)NR8-;
R5 is selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6
perfluoroalkyl, F, Cl, R8O-, R9S(O)m-, R8C(O)NR8-, CN,
NO2, (R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or
R9OC(O)NR8-, and
c) C1-C6 alkyl unsubstituted or substituted by C1-C6
perfluoroalkyl, F, Cl, R8O-, R9S(O)m-, R8C(O)NR8-, CN,
-87-

(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or
R9OC(O)NR8-;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl, benzyl,
2,2,2-trifluoroethyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, unsubstituted or substituted C3-6 cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) - S(O)m R9 ,
-88-

h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C~C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
J and K are independently selected from N or CH y;
V is selected from:
a) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl, and
b) aryl;
W is a heterocycle selected from pyrrolidinyl, triazolyl, imidazolyl,
pyridinyl, thiazolyl, indolyl, quinolinyl, or isoquinolinyl;
X is a bond, -C(=O)NR10-,-NR10C(=O)-,S(O)m- or -NR10-;
m is 0,1 or 2;
n is 0,1,2,3or4;
p is 1,2 or 3;
q is 0 or 1;
r is 0 to 5, provided that r is 0 when V is hydrogen;
s is 1 or 2;
t is 1; and
y is 1 or 2;
-89-

the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
3. The compound according to Claim 1, which inhibits
farnesyl-protein transferase, of the formula C:
<IMG>
wherein:
R1a and R1c are independently selected from: hydrogen, C3-C10
cycloalkyl, R8O-, -N(R8)2, F or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C6 cycloalkyl, R8O-, -N(R8)2 or
C2-C6 alkenyl,
c) C1-C6 alkyl unsubstituted or substituted by unsubstituted or
substituted aryl, heterocycle, C3-C6 cycloalkyl, C2-C6
alkenyl, R8O-, or -N(R8)2;
R2 is selected from:
a) C1-8 alkyl, unsubstituted or substituted with one or
more of:
-90-

1) aryl or heterocycle, unsubstituted or substituted
with:
i) C1-4 alkyl,
ii) (CH2)p OR6,
iii) (CH2)p NR6R7,
iv) halogen,
v) C1-4 perfluoroalkyl,
2) OR6,
3) SR6, SO2R6, or
<IMG>
c) aryl, unsubstituted or substituted with one or
more of:
1) C1-8 alkyl,
2) C1-8 perfluoroalkyl,
3) OR6,
4) SR6, SO2R6, or
<IMG>
d) -SO2R6;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
-91-

b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
perfluoroalkyl, F, C1, R8O-, R8C(O)NR8-, CN, NO2,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or R9OC(O)NR8-, and
c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, R8O-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2,
or R9OC(O)NR8-;
R5 is selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6
perfluoroalkyl, F, C1, R8O-, R9S(O)m-, R8C(O)NR8-, CN,
NO2, (R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or
R9OC(O)NR8-, and
c) C1-C6 alkyl unsubstituted or substituted by C1-C6
perfluoroalkyl, F, C1, R8O-, R9S(O)m-, R8C(O)NR8-, CN,
(R8)2N-C(NR8)-, R8C(O)-, R8OC(O)-, -N(R8)2, or
R9OC(O)NR8-;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl, benzyl,
2,2,2-trifluoroethyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, unsubstituted or substituted C3-6 cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
-92-

aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) -S(O)m R9 ,
h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C~C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
V is selected from:
a) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl; and
b) aryl;
-93-

W is a heterocycle selected from pyrrolidinyl, triazolyl, imidazolyl,
pyridinyl, thiazolyl, indolyl, quinolinyl, or isoquinolinyl;
X is a bond, -C(=O)NR10-,-NR10C(=O)-, -S(O)m- or -NR10-;
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 1, 2 or 3;
q is 0 or 1;
r is 0 to 5, provided that r is 0 when V is hydrogen;
s is 1 or 2; and
t is 1;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
4. The compound according to Claim 1, which inhibits
farnesyl-protein transferase, of the formula D:
<IMG>
wherein:
R1a and R1c are independently selected from: hydrogen, C3-C10
cycloalkyl, R8O-, -N(R8)2, F or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
-94-

b) aryl, heterocycle, C3-C10 cycloalkyl, R8O-, -N(R8)2, F or
C2-C6 alkenyl,
c) unsubstituted or substituted C1-C6 alkyl wherein the
substituent on the substituted C1-C6 alkyl is selected from
unsubstituted or substituted aryl, heterocycle, C3-C10
cycloalkyl, C2-C6 alkenyl, R8O- and -N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
unsubstituted or substituted C2-8 alkenyl, unsubstituted or substituted aryl,
unsubstituted or substituted heterocycle, <IMG>
and-S(O)2R6,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)p OR6,
c) (CH2)p NR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) C3-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
5) -NR6R7 ,
<IMG>
-95-

<IMG>
11) -SO2-NR6R7 ,
<IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
-96-

perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2,
(R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or R9OC(O)NR8-, and
c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, R8O-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R9OC(O)NR8-;
R5a and R5b are independently hydrogen, C1-C6 alkyl, cyclopropyl,
trifluoromethyl and halogen;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl,
2,2,2-trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, unsubstituted or substituted C3-6 cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
-97-

<IMG>
g) -S(O)m R9 ,
h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C~C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
J and K are independently selected from N or CH y;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl,
c) aryl,
d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a a heteroatom selected from O, S, and N, and
e) C2-C20 alkenyl, and
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
-98-

X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10-;
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
q is 0 or 1;
r is 0 to 5, provided that r is 0 when V is hydrogen; and
y is 1 or 2;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
5. The compound according to Claim 1, which inhibits
farnesyl-protein transferase, of the formula E:
<IMG>
wherein:
R1a and R1c are independently selected from: hydrogen, C3-C10
cycloalkyl, R8O-, -N(R8)2, F or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, R8O-, -N(R8)2, F or
C2-C6 alkenyl,
c) unsubstituted or substituted C1-C6 alkyl wherein the
substituent on the substituted C1-C6 alkyl is selected from
-99-

unsubstituted or substituted aryl, heterocycle, C3-C10
cycloalkyl, C2-C6 alkenyl, R8O- and -N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
unsubstituted or substituted C2-8 alkenyl, unsubstituted or substituted aryl,
<IMG>
unsubstituted or substituted heterocycle,
S(O)2R6,
wherein the substituted group is substituted with one or more of:
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)pOR6,
c) (CH2)pNR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) C3-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
5) -NR6R7
<IMG>
-100-

<IMG>
11) -SO2-NR6R7 ,
<IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
-101-

perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2, (R8)2N-
C(NR8)-, R8C(O)-, -N(R8)2, or R9OC(O)NR8-, and
c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, R8O-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R9OC(O)NR8-;
R5a and R5b are independently hydrogen, C1-C6 alkyl, cyclopropyl,
trifluoromethyl and halogen;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl,
2,2,2-trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, unsubstituted or substituted C3-6 cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
-102-

<IMG>
g) -S(O)mR9 ,
h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl,
c) aryl,
d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a a heteroatom selected from O, S, and N, and
e) C2-C20 alkenyl, and
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m,- or -NR,10-;
-103-

m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
q is 0 or 1; and
r is 0 to 5, provided that r is 0 when V is hydrogen;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
6. The compound according to Claim 5, which inhibits
farnesyl-protein transferase, of the formula F:
<IMG>
wherein:
R1a and R1c are independently selected from: hydrogen, C3-C10
cycloalkyl or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, R8O-, -N(R8)2, F or
C6-alkenyl,
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, R8O-, or
- N(R8)2;
-104-

R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
<IMG>
unsubstituted or substituted aryl, <IMG> and -S(O)2R6,
wherein the substituted group is substituted with one or more of:
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)pOR6,
c) (CH2)pNR6R7,
d) halogen,
e) C14 perfluoroalkyl,
2) C3-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
<IMG>
-105-

8) <IMG>
9)<IMG>
10)<IMG>
11) -SO2-NR6R7,
12) <IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a is selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2, (R8)2N-
C(NR8)-, R8C(O)-, -N(R8)2, or R9OC(O)NR8-, and
-106-

c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl,
R8O-,R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R9OC(O)NR8-;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl,
2,2,2-trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) -S(O)mR9
-107-

h) N(R8)2, or
i) C3-5 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 is selected from: a bond, -C(O)-, O, -N(R8)-, or S(O)m;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10-;
n is 0 or 1; provided that n is not 0 if A1 is a bond, O, -N(R8)-, or
S(O)m;
m is 0, 1 or 2;
p is 0, 1, 2, 3 or 4;
r is 1 or 2; and
q is 0 or 1;
or an optical isomer or pharmaceutically acceptable salt thereof.
7. The compound according to Claim 1, which inhibits
farnesyl-protein transferase, of the formula G:
<IMG>
-108-

wherein:
R1a and R1c are independently selected from: hydrogen, R8O-, -N(R8)2,
F, C3-C10 cycloalkyl or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, R8O-, -N(R8)2, F or
C2-C6 alkenyl,
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, R8O-, or
-N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
unsubstituted or substituted aryl, <IMG> and -S(O)2R6,
wherein the substituted group is substituted with one or more of:
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)p OR6,
c) (CH2)p NR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) C3-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
-109-

5) -NR6R7 ,
<IMG>
11) -SO2-NR6R7,
<IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a is selected from:
-110-

H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
perfluoroalkyl, F, Cl, R8O-, R8C(O)NR8-, CN, NO2,
(R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or R9OC(O)NR8-, and
c) C1-C6 alkyl substituted by C1-C6 perfluoroalkyl, R8O-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R9OC(O)NR8-;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl,
2,2,2-trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1-4 alkoxy,
-111-

b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) -S(O)m R9 ,
h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10-;
n is 0 or 1;
m is 0, 1 or 2;
p is 0, 1, 2, 3 or 4, provided that p is not 0 if X is a bond, -NR8- or
O;
q is 0 or 1; and
r is 1 or 2;
or an optical isomer or pharmaceutically acceptable salt thereof.
8. The compound according to Claim 6, which inhibits
farnesyl-protein transferase, of the formula H:
-112-

<IMG>
wherein:
R1a and R1c are independently selected from: hydrogen, C3-C10
cycloalkyl or C1-C6 alkyl;
R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, R8O-, -N(R8)2 or F,
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, R8O-, or -N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
unsubstituted or substituted aryl, <IMG> and -S(O)2R6,
wherein the substituted group is substituted with one or more of:
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)p OR6,
c) (CH2)p NR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) C3-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
-113-

<IMG>
11) -SO2-NR6R7
<IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a is selected from:
-114-

H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl,
2,2,2-trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) -S(O)m R9 ,
-115-

h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10-;
m is 0, 1 or 2;
p is 0, 1, 2, 3 or 4;
q is 0 or 1; and
or an optical isomer or pharmaceutically acceptable salt thereof.
9. The compound according to Claim 7, which inhibits
farnesyl-protein transferase, of the formula I:
<IMG>
wherein:
R1a and R1c are independently selected from: hydrogen, R8O-, -N(R8)2,
F, C3-C10 cycloalkyl or C1-C6 alkyl;
-116-

R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle or C3-C10 cycloalkyl,
c) C1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, R8O-, or
-N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1-8 alkyl,
<IMG>
unsubstituted or substituted aryl, end -S(O)2R6,
wherein the substituted group is substituted with one or more of:
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1-4 alkyl,
b) (CH2)pOR6,
c) (CH2)pNR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) C-6 cycloalkyl,
3) OR6,
4) SR6, S(O)R6, SO2R6,
5) -NR6R7 ,
<IMG>
-117-

<IMG>
11) -SO2-NR6R7 ,
<IMG>
15) C1-8 alkyl, or
16) C1-8 perfluoroalkyl;
R3a is selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C1-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
-118-

a) C1-4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl,
2,2,2-trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1-4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
<IMG>
g) ~S(O)m R9 ,
h) N(R8)2, or
i) C3-6 cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
-119-

A1 is selected from: a bond, -C(O)-, O, -N(R8)-, or S(O)m;
X is a bond, -C(=O)NR10-,-NR10C(=O)-, -S(O)m- or -NR10-;
m is 0,1 or 2;
n is 0 or 1;
p is 1, 2 or 3; and
q is 0 or 1;
or an optical isomer or pharmaceutically acceptable salt thereof.
10. A compound which inhibits farnesyl-protein
transferase which is:
<IMG>
4-phenyl-4,5,6,7-tetrahydro-1H-imidazo[4,5] pyridine-6(S)-carboxylic acid
{2-[3-(4-cyano-benzyl)-3H -imidazol-4-yl]-ethyl}-amide;
or an optical isomer or pharmaceutically acceptable salt thereof.
11. A pharmaceutical composition comprising a
pharmaceutical carrier, and dispersed therein, a therapeutically
effective amount of a compound of Claim 1.
12. A pharmaceutical composition comprising a
pharmaceutical carrier, and dispersed therein, a therapeutically
effective amount of a compound of Claim 3.
-120-

13. A pharmaceutical composition comprising a
pharmaceutical carrier, and dispersed therein, a therapeutically
effective amount of a compound of Claim 4.
14. A pharmaceutical composition comprising a
pharmaceutical carrier, and dispersed therein, a therapeutically
effective amount of a compound of Claim 5.
15. A pharmaceutical composition comprising a
pharmaceutical carrier, and dispersed therein, a therapeutically
effective amount of a compound of Claim 10.
16. A method for inhibiting farnesyl-protein
transferase which comprises administering to a mammal in need
thereof a therapeutically effective amount of a composition of Claim
11.
17. A method for inhibiting farnesyl-protein
transferase which comprises administering to a mammal in need
thereof a therapeutically effective amount of a composition of Claim
12.
18. A method for inhibiting farnesyl-protein
transferase which comprises administering to a mammal in need
thereof a therapeutically effective amount of a composition of Claim
13.
19. A method for inhibiting farnesyl-protein
transferase which comprises administering to a mammal in need
thereof a therapeutically effective amount of a composition of Claim
14.
20. A method for inhibiting farnesyl-protein
transferase which comprises administering to a mammal in need
-121-

thereof a therapeutically effective amount of a composition of Claim
15.
21. A method for treating cancer which comprises
administering to a mammal in need thereof a therapeutically
effective amount of a composition of Claim 11.
22. A method for treating cancer which comprises
administering to a mammal in need thereof a therapeutically
effective amount of a composition of Claim 12.
23. A method for treating cancer which comprises
administering to a mammal in need thereof a therapeutically
effective amount of a composition of Claim 13.
24. A method for treating cancer which comprises
administering to a mammal in need thereof a therapeutically
effective amount of a composition of Claim 14.
25. A method for treating cancer which comprises
administering to a mammal in need thereof a therapeutically
effective amount of a composition of Claim 15.
26. A method for treating neurofibromin benign
proliferative disorder which comprises administering to a mammal in
need thereof a therapeutically effective amount of a composition of Claim
11.
27. A method for treating blindness related to retinal
vascularization which comprises administering to a mammal in need
thereof a therapeutically effective amount of a composition of Claim 11.
28. A method for treating infections from hepatitis delta
and related viruses which comprises administering to a mammal in
-122-

need thereof a therapeutically effective amount of a composition of Claim
11.
29. A method for preventing restenosis which comprises
administering to a mammal in need thereof a therapeutically effective
amount of a composition of Claim 11.
30. A method for treating polycystic kidney disease which
comprises administering to a mammal in need thereof a therapeutically
effective amount of a composition of Claim 11.
31. A pharmaceutical composition made by combining
the compound of Claim 1 and a pharmaceutically acceptable carrier.
32. A process for making a pharmaceutical composition
comprising combining a compound of Claim 1 and a pharmaceutically
acceptable carrier.
-123-

Description

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
~TTT F OF THE INVENTION
INHIBITORS OF FARNESYL-PROTEIN TRANSFERASE
_B_ACKGROUND OF ~IEiE INVENTION
The Ras protein is part of a signalling pathway that
links cell surface growth factor receptors to nuclear signals initiating
cellular proliferation. Biological and biochemical studies of Ras
action indicate that Ras functions like a G-regulatory protein. In the
inactive state, Ras is bound to GDP. Upon growth factor receptor
activation Ras is induced to exchange GDP for GTP and undergoes a
conformational change. The GTP-bound form of Ras propagates the
growth stimulatory signal until the signal is terminated by the
intrinsic GTPase activity of Ras, which returns the protein to its
inactive GDP bound form (D.R. Lowy and D.M. Willumsen, Ann.
Rev. Biochem. 62:851-891 (1993)). Mutated rays genes are found in
many human cancers, including colorectal carcinoma, exocrine
pancreatic carcinoma, and myeloid leukemias. The protein products
of these genes are defective in their GTPase activity and constitutively
transmit a growth stimulatory signal.
Ras must be localized to the plasma membrane for both
normal and oncogenic functions. At least 3 post-translational
modifications are involved with Ras membrane localization, and all 3
modifications occur at the C-terminus of Ras. The Ras C-terminus
contains a sequence motif termed a "CAAX" or "Cys-Aaal-Aaa2-
Xaa" box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa is
any amino acid) (Willumsen et al., Nature 310:583-586 ( 1984)).
Depending on the specific sequence, this motif serves as a signal
sequence for the enzymes farnesyl-protein transferase or
geranylgeranyl-protein transferase, which catalyze the alkylation of
the cysteine residue of the CAAX motif with a C15 or C20 isoprenoid;
respectively. (S. Clarke., Ann. Rev. Biochem. 61:355-386 (1992); W.R.
Schafer and J. Rine, Ann. Rev. Genetics 30:209-237 (1992)). The Ras
protein is one of several proteins that are known to undergo post-
translational farnesylation. Other farnesylated proteins include the
-1-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
Ras-related GTP-binding proteins such as Rho, fungal mating
factors, the nuclear lamins, and the gamma subunit of transducin.
James, et al., J. Biol. Chem. 269, 14182 (1994) have identified a
peroxisome associated protein Pxf which is also farnesylated. James,
et al., have also suggested that there are farnesylated proteins of
unknown structure and function in addition to those listed above.
Inhibition of farnesyl-protein transferase has been
shown to block the growth of Ras-transformed cells in soft agar and to
modify other aspects of their transformed phenotype. It has also been
demonstrated that certain inhibitors of farnesyl-protein transferase
selectively block the processing of the Ras oncoprotein intracellularly
(N.E. Kohl et al., Science, 260:1934-1937 (1993) and G.L. James et al.,
science, 260:1937-1942 (1993). Recently, it has been shown that an
inhibitor of farnesyl-protein transferase blocks the growth of ras-
dependent tumors in nude mice (N.E. Kohl et al., Proc. Na~tl. Aca~d.
Sci U.S.A. , 91:9141-9145 ( 1994) and induces regression of mammary
and salivary carcinomas in ras transgenic mice (N.E. Kohl et al.,
Nature Medicine, 1:792-797 ( 1995).
Indirect inhibition of farnesyl-protein transferase in vivo
has been demonstrated with lovastatin (Merck & Co., Rahway, NJ)
and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et acl.,
Science 245:379 (1989)). These drugs inhibit HMG-CoA reductase, the
rate limiting enzyme for the production of polyisoprenoids including
farnesyl pyrophosphate. Farnesyl-protein transferase utilizes
farnesyl pyrophosphate to covalently modify the Cys thiol group of the
Ras CAAX box with a farnesyl group (Reins et cal., Cell, 62:81-88
(1990); Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et
acl., Science, 249:1133-1139 (1990); Manne et ail., Proc. Natl. Acad. Sci
USA, 87:7541-7545 (1990)). Inhibition of farnesyl pyrophosphate
biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane
localization in cultured cells. However, direct inhibition of farnesyl-
protein transferase would be more specific and attended by fewer side
effects than would occur with the required dose of a general inhibitor
of isoprene biosynthesis.
-2-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
Inhibitors of farnesyl-protein transferase (FPTase) have
been described in two general classes. The first are analogs of
farnesyl diphosphate (FPP), while the second class of inhibitors is
related to the protein substrates (e.g., Ras) for the enzyme. The
5 peptide derived inhibitors that have been described are generally
cysteine containing molecules that are related to the CAAX motif that
is the signal for protein prenylation. (Schaber et al., ibid; Reiss et.
al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Such inhibitors may
inhibit protein prenylation while serving as alternate substrates for
10 the farnesyl-protein transferase enzyme, or may be purely
competitive inhibitors (U.S. Patent 5,141,851, University of Texas;
N.E. Kohl et al., Science, 260:1934-1937 (1993); Graham, et al., J. Med.
Chem., 37, 725 (1994)). In general, deletion of the thiol from a CAAX
derivative has been shown to dramatically reduce the inhibitory
15 potency of the compound. However, the thiol group potentially places
limitations on the therapeutic application of FPTase inhibitors with
respect to pharmacokinetics, pharmacodynamics and toxicity.
Therefore, a functional replacement for the thiol is desirable.
It has recently been shown that farnesyl-protein
20 transferase inhibitors are inhibitors of proliferation of vascular
smooth muscle cells and are therefore useful in the prevention and
therapy of arteriosclerosis and diabetic disturbance of blood vessels
(JP H7-112930). It has also recently been disclosed that certain
1,2,3,4-tetrahydroisoquinoline peptidomimetic compounds, some of
25 which incorporate an imidazole moiety, are inhibitors of FPTase
(U.S. Pat. No. 5,439,918, EP 0 618 221 A2 and EP 0 675112 A1 ).
It is, therefore, an object of this invention to develop novel
peptidomimetic compounds that do not have a thiol moiety, and that
will inhibit farnesyl-protein transferase and thus, the post-
30 translational farnesylation of proteins. It is a further object of this
invention to develop chemotherapeutic compositions containing the
compounds of this invention and methods for producing the
compounds of this invention.
-3-

CA 02311921 2000-OS-26
WO 99/Z7928 PCT/US98/25325
SUMMARY OF THE INVENTION
The present invention comprises non-thiol compounds
which inhibit farnesyl-protein transferase. Further contained in this
invention are chemotherapeutic compositions containing these
farnesyl transferase inhibitors and methods for their production.
The compounds of this invention are illustrated by the
formula A:
R3~- J . ~ R3b
{R4)~ {R5) 11
V - A1{CR182)nA2{CRla2)n W - (CRlb2)PW 1~ N'R2
X-(CR
A
DETAILED DESCRIPTION OF THE INVENTION
The compounds of this invention are useful in the
inhibition of farnesyl-protein transferase and the farnesylation of the
oncogene protein Ras. In a first embodiment of this invention, the
inhibitors of farnesyl-protein transferase are illustrated by the
formula A:
R3~ K Rab
(R4)~ {R5) 11
V - A1{CR182)nA2{CRla2)n W t (CRlb2)Pw X-{CR1°2) N~R2
4
A
wherein:

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
Rla, Rlb and Rlc are independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C10 cycloalkyl, C2-Cg alkenyl,
C2-Cg alkynyl, R80-, R9S(O)m-, R8C(O)NR8-, CN, N02,
(R8)2N-C(NR8)-, R8C(O)-, R80C(O)-, Ng, -N(R$)2, or
R90C(O)NR8-,
c) C1-Cg alkyl unsubstituted or substituted by unsubstituted or
substituted aryl, heterocyclic, Cg-C10 cycloalkyl, C2-Cg
10 alkenyl, C2-Cg alkynyl, R80-, R9S(O)m-, R8C(O)NR8-, CN,
(R8)2N-C(NR~)-, R8C(O)-, R80C(O)-, N3, -N(R$)2, or
R90C(O)-NR8-;
R2 is selected from: H; unsubstituted or substituted C1_g alkyl,
15 unsubstituted or substituted C2_g alkenyl, unsubstituted or substituted
aryl,
~s ~RsR~
unsubstituted or substituted heterocycle, ~.0 ' ~ and
-S(O)2R6~
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
20 one or two groups selected from:
a) C1~ alkyl,
b) (CH2)pORS,
c) (CH2)pNR6R7,
d) halogen,
25 e) C1-4 perfluoroalkyl,
2) Cg_6 cycloalkyl,
3) OR6,
4) SR6, S(O)RS, S02R6,
-5-

CA 02311921 2000-OS-26
- WO 99/27928 PCT/US98/25325
5) -NRsR~
,
Rs
-N R7
6) ~ ,
0
s
7) ~ 7 7a '
-N~ NR R
'IO
-O~ NRsR~
'IO
-O~ORs ,
I IO
10) ~ N RsR~ ,
O
11) -g02-NRsR~ ,
Rs
12) -N-g02 R7 ,
13) ~ Rs ,
O
14) ~ ORs ,
O
15) C1_g alkyl, or
16) C 1-g perfluoroalkyl;
R3a and R3b are
independently
absent or selected
from: H, unsubstituted
or
substituted alkyl,unsubstituted or substituted aryl, unsubstituted
or
-6-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
substituted heteroaryl, unsubstituted or substituted aralkyl and
unsubstituted or substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, Cg-C10 cycloaikyl, C2-Cg alkenyl,
C2-Cg alkynyl, perfluoroalkyl, F, Cl, Br, R80-, R9S(O)m-,
R8C(O)NR8-, CN, N02, R82N-C(NR8)-, R8C(O)-, R80C(O)-,
N3, -N(R8)2, or R90C(O)NRg-, and
c ) C 1-Cg alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, C2-Cg alkenyl, C2-Cg
alkynyl, perfluoroalkyl, F, Cl, Br, R80-, R9S(O)m-,
R8C(O)NH-, CN, H2N-C(NH)-, R8C(O)-, R80C(O)-, Ng,
-N(R8)2, or R80C(O)NH-;
R5 is independently selected from:
a) hydrogen,
b) C2-Cg alkenyl, C2-Cg alkynyl, C3-C6 cycloalkyl,
20 perfluoroalkyl, F, Cl, Br, R80-, R9S(O)m-, R8C(O)NRg-, CN,
N02, (R8)2N-C-(NR8)-, R8C(O)-, R80C(O)-, N3, -N(R8)2, or
R90C(O)NR8-, and
c) C1-C6 alkyl, unsubstituted or substituted by perfluoroalkyl,
F, Cl, Br, R80-, R9S(O)m-, R8C(O)NR8-, CN, (R8)2N
C(NR8)-, R8C(O)-, R80C(O)-, N3, -N(R8)2, or R90C(O)NR8-;
R6, R7 and R7a are independently selected from: H; C1_4 alkyl, C3-6
cycloalkyl, heterocycle, aryl, C 1-4 perfluoroalkyl, unsubstituted or
substituted with one or two substituents selected from:
a) C 1_4 alkoxy,
b) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle,
c) halogen,
d) HO,

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
Rs
e)
O
f) \/ORs
O
9) -S~O)mRs ~ or
h) N(R8)2; or
Rs and R7 may be joined in a ring;
R7 and R7a may be joined in a ring;
R8 is independently selected from hydrogen, CZ-Cg alkyl, benzyl, 2,2,2-
trifluoroethyl and aryl;
R9 is independently selected from C~-Cg alkyl and aryl;
R10 is selected from: H; RaC(O)-; R9S(O)m-; unsubstituted or substituted
C1_4 alkyl, unsubstituted or substituted C3_g cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C 1_4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
_g_

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
Rs
e)
0
~oR8
0
9) -S~~)mRs '
h) N(R$)2, or
i) C3-g cycloalkyl;
S
Rll is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR8-, -NR8C(O)-, O, -N(R8)-, -S(O)2N(R8)-, -
N(R8)S(O)2-, or S(O)m;
J and K are independently selected from N, NH or CHy;
V is selected from:
a) hydrogen,
b) heterocycle,
c) aryl,
d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a a heteroatom selected from O, S, and N, and
e) C 2-C 20 alkenyl,
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
W is a heterocycle;
-9-

CA 02311921 2000-OS-26
- WO 99/27928 PCTNS98/25325
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m-, -NR10-, O or -C(=O)-;
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
q is 0, 1, 2, 3 or 4;
r is 0 to 5, provided that r is 0 when V is
hydrogen;
s is 1 or 2;
t is 0 or l; and
y is 1 or 2;
the dashed
lines
represent
optional
double
bonds;
R3~--~ K - R3b
R4 ~
)r ~R5) ~11
~N
V - A1~CR1a2)nA2(CRla2)n W t (CRlb2)pwX-(CR1~2)q ~R2
B
or an optical isomer or pharmaceutically acceptable salt thereof.
In a further embodiment of this invention, the inhibitors of
farnesyl-protein transferase are illustrated by the formula B:
wherein:
Rla and Rlc are independently selected from: hydrogen, Cg-C10
cycloalkyl, R80-, -N(R8)2, F or C1-C6 alkyl;
Rlb is independently selected from:
a) hydrogen,
-10-

CA 02311921 2000-OS-26
WO 99127928 PCTNS98/25325
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C6 cycloalkyl, R80-, -N(R8)2 or
C2-Cg alkenyl,
c) C1-Cg alkyl unsubstituted or substituted by unsubstituted or
substituted aryl, heterocycle, Cg-CS cycloalkyl, C2-Cg
alkenyl, R80-, or -N(R8)2;
R2 is selected from:
a) C1_g alkyl, unsubstituted or substituted with one or
more of:
1) aryl or heterocycle, unsubstituted or substituted
with:
i) C1_4 alkyl,
ii) (CH2)pORS,
iii) (CH2)pNR6R7,
iv) halogen,
v) C1_4 perfluoroalkyl,
2) ORS,
3) SRS, S02R6, or
0
b)
~6
c) aryl, unsubstituted or substituted with one or
more of:
1) C 1_g alkyl,
2) . C 1_g perfluoroalkyl,
3) OR6,
4) SR6, S02R6, or
-lI-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
d) -so2RS;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) C1-C6 alkyl, C2-Cg alkenyl, C2-C6 alkynyl, C1-Cg
perfluoroalkyl, F, Cl, R80-, R$C(O)NRg-, CN, N02, (R8)2N-
C(NR8)-, R8C(O)-, R80C(O)-, -N(R8)2, or R90C(O)NR8-, and
c ) C 1-Cg alkyl substituted by C 1-Cg perfluoroalkyl, R80-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, RgOC(O)-, -N(R8)2,
or R90C(O)NR8-;
R5 is selected from:
a) hydrogen,
b) C2-C6 alkenyl, C2-CS alkynyl, Cg-C6 cycloalkyl, C1-C6
perfluoroalkyl, F, Cl, R80-, R9S(O)m-, R8C(O)NR8-, CN,
N02, (R8)2N-C(NR8)-, RgC(O)-, R80C(O)-, -N(R8)2, or
R90C(O)NRg-, and
c) C1-Cg alkyl unsubstituted or substituted by C1-Cg
perfluoroalkyl, F, Cl, R80-, R9S(O)m-, R8C(O)NR8-, CN,
(R8)2N-C(NR8)-, R8C(O)-, R80C(O)-, -N(R8)2, or
R90C(O)NR8-;
R6, R7 and R7a are independently selected from:
H; C1_4 alkyl, Cg_6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1_4 alkoxy,
b) halogen, or
-12-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-Cg alkyl, benzyl, 2,2,2-
trifluoroethyl and aryl;
R9 is independently selected from C1-C6 alkyl and aryl;
R1~ is selected from: H; R8C(O)-; R9S{O)m-; unsubstituted or substituted
C1-4 alkyl, unsubstituted or substituted Cg-g cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C 1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
R9
e)
O
f) I I ORs
O
9) -S~O)mR9 '
h) N(R$)2, or
i) Cg_g cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
-13-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
J and K are independently selected from N or CHy;
V is selected from:
a) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl, and
b) aryl;
W is a heterocycle selected from pyrrolidinyl, triazolyl, imidazolyl,
pyridinyl, thiazolyl, indolyl, quinolinyl, or isoquinolinyl;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10_;
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 1, 2 or 3;
q is 0 or 1;
r is 0 to 5, provided that r is 0 when V is
hydrogen;
s is 1 or 2;
t is 1; and
y is 1 or 2;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
In another embodiment of this invention, the inhibitors of
farnesyl-protein transferase are illustrated by the formula C:
-14-

CA 02311921 2000-OS-26
' WO 99/27928 PCTNS98/25325
Rs3-NON-Rsb
R4
( )r (R5) R11
V - A1(CRla2)nA2(CRla2)n W - (CRlb2)p~ 1~ N~ 2
X-(CR 2)q R
C
wherein:
Rla and R1c are independently selected from: hydrogen, Cg-C10
cycloalkyl, R80-, -N(R8)2, F or C1-Cg alkyl;
Rlb is independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or
substituted heterocycle, C3-C6 cycloalkyl, R80-, -N(R8)2 or
C2-Cg alkenyl,
c ) C 1-Cg alkyl unsubstituted or substituted by unsubstituted or
substituted aryl, heterocycle, Cg-Cg cycioalkyl, C2-C6
alkenyl, R80-, or -N(R8)2;
R2 is selected from:
a) C1_g alkyl, unsubstituted or substituted with one or
more of:
1) aryl or heterocycle, unsubstituted or substituted
with:
i) C 1-4 alkyl,
ii) (CH2)pORS,
iii) (CH2)pNRSR7,
iv) halogen,
v) C1-4 perfluoroalkyl,
2) ORS,
3) SRS, S02R6, or
-15-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
~NRsR~
O
b)
~s
O
c) aryl, unsubstituted or substituted with one or
more of:
1) C 1-g alkyl,
2) C ~,-g perfluoroalkyl,
3) ORS,
4) SR6, S02R6, or
O
d) -S02R6;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) C1-Cg alkyl, C2-CS alkenyl, C2-Cg alkynyl, C1-Cg
perfluoroalkyl, F, Cl, R80-, R8C(O)NR8-, CN, N02, (R8)2N-
C(NR8)-, R8C(O)-, R80C(O)-, -N(R8)2, or R90C(O)NR8-, and
c ) C 1-Cg alkyl substituted by C 1-Cg perfluoroalkyl, R80-,
R8C(O)NR$-, (R8)2N-C(NR8)-, R8C(O)-, RaOC(O)-, -N(R8)2,
or R90C(O)NR8-;
R5 is selected from:
a) hydrogen,
-16-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
b) C2-Cg alkenyl, C2-Cg alkynyl, Cg-Cg cycloalkyl, C1-Cg
perfluoroalkyl, F, Cl, R80-, R9S(O)m-, RgC(O)NR$-, CN,
N02, (R8)2N-C(NR8)-, R8C(O)-, R80C(O)-, -N(R8)2, or
R90C(O)NR8-, and
c) C1-Cg alkyl unsubstituted or substituted by C1-Cg
perfluoroalkyl, F, Cl, R80-, R9S(O)m-, R8C(O)NR8-, CN,
(R8)2N-C(NR8)-, R8C(O)-, R80C(O)-, -N(R8)2, or
R90C(O)NR8-;
R6, R7 and R7a are independently selected from:
H; C1_4 alkyl, Cg-6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1_4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-Cg alkyl, benzyl, 2,2,2-
trifluoroethyl and aryl;
R9 is independently selected from C1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1_4 alkyl, unsubstituted or substituted Cg_s cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C 1_4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
-17-

CA 02311921 2000-OS-26
' WO 99/27928 PCTNS98/25325
Rs
e)
O
f) I I OR8
O
J) -S~O)mRs '
h) N(R8)2, or
i) C3_s cycloalkyl;
Rll is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
V is selected from:
a) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl; and
b) aryl;
25
W is a heterocycle selected from pyrrolidinyl, triazolyl, imidazolyl,
pyridinyl, thiazolyl, indolyl, quinolinyl, or isoquinolinyl;
X is a bond, -C(=O)NR10_~ _Ng,lOC(=O)-, -S(O)m- or -NR10_;
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 1, 2 or 3;
-18-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
q is 0 or 1;
r is 0 to 5, provided that r is 0 when V is hydrogen;
s is 1 or 2; and
t is 1;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
In a preferred embodiment of this invention, the inhibitors
of farnesyl-protein transferase are illustrated by the formula D:
(R4)r R5a R3a-~~ ~(_ R3b
1 1a 2 is ~N 11
V - A (CR 2)nA (CR 2)n N/v'J ~'-R
R5b (,CR3b2~X- (CR1~2) N~R2
4
D
wherein:
R1a and Rlc are independently selected from: hydrogen, C3-C10
cycloalkyl, R80-, -N(R8)2, F or C1-C6 alkyl;
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, R80-, -N(R8)2, F or C2-
Cg alkenyl,
c) unsubstituted or substituted C1-C6 alkyl wherein the
substituent on the substituted C1-C6 alkyl is selected from
unsubstituted or substituted aryl, heterocycle, C3-C 10
cycloalkyl, C2-C6 alkenyl, R$O- and -N(R8)2;
R2 is selected from: H; unaubstituted or substituted C 1-g alkyl,
unsubstituted or substituted C2-g alkenyl, unsubstituted or substituted aryl,
-19-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
Il s II RsR7
unsubstituted or substituted heterocycle, O ' O and -
S(O)2R6,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1_4 alkyl,
b) (CH2)pOR6,
c) (CH2)pNRSR7,
d) halogen,
e) C1_4 perfluoroalkyl,
2) Cg_g cycloalkyl,
3) ORE,
4) SRS, S(O)RE, S02RE,
5) -NRsR~ ,
Rs
g) -N~ R~
I IO
Rs
) -N NR7R7a
O
-20-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
) -O~ NR6R~
O
9) -O~ORs
I IO
10) ~ NRsR~ ,
O
11) -S02-NR6R~ '
Rs
12) -N-S02 R'
13) II Rs ,
O
14) ,.,~ ORs ,
O
15) C 1-g alkyl, or
16) C 1-g perfluoroalkyl;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6
-21-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98125325
perfluoroalkyl, F, Cl, R80-, R8C(O)NR8-, CN, N02, (R8)2N-
C(NR8)-, R8C(O)-, -N(R8)2, or R90C(O)NR8-, and
c) C1-Cg alkyl substituted by C1-Cg perfluoroalkyl, R80-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R90C(O)NR8_;
R5a and R5b are independently hydrogen, C1-Cg alkyl, cyclopropyl,
trifluoromethyl and halogen;
Rs, R7 and R7a are independently selected from:
H; C1_4 alkyl, C3_6 cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1_4 alkoxy,
b) halogen, or
I S c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl, 2,2,2-
trifluoroethyl, benzyl and aryl;
R9 is independently selected from C 1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1_4 alkyl, unsubstituted or substituted Cg-g cycloalkyl, unsubstituted or
substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1_4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
-22-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
Rs
e)
O
f) I I OR8
O
9) -S~O)mRs
h) N(R8)2, or
i) Cg_g cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
J and K are independently selected from N or CHy;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl,
c) aryl,
d) C 1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a a heteroatom selected from O, S, and N, and
e) C2-C2p alkenyl, and
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
-23-

CA 02311921 2000-OS-26
' WO 99/27928 PCTNS98/25325
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10_
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
q is 0 or 1;
r is 0 to 5, provided that r is 0 when V is hydrogen; and
y is 1 or 2;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
In a preferred embodiment of this invention, the inhibitors
of farnesyl-protein transferase are illustrated by the formula E:
(R4)r R5a R3a_ N~ N-R3b
/' N
V - AIURla2)nA2(CR~a2)n Nw~J
5b N
(CR~b2jP X- (CR1~2)q R2
E
wherein:
Rla and Rlc are independently selected from: hydrogen, Cg-C10
cycloalkyl, R80-, -N(R8)2, F or C1-Cg alkyl;
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, Cg-C10 cycloalkyl, R80-, -N(R8)2, F or C2-
Cg alkenyl,
c) unsubstituted or substituted C1-Cg alkyl wherein the
substituent on the substituted C~-Cg alkyl is selected from
-24-

CA 02311921 2000-OS-26
' WO 99/27928 PCT/US98/25325
unsubstituted or substituted aryl, heterocycle, Cg-C10
cycloalkyl, C2-Cg alkenyl, R80- and -N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1_g alkyl,
unsubstituted or substituted C2_g alkenyl, unsubstituted or substituted aryl,
~RsR~
unsubstituted or substituted heterocycle, O ' O and -
S(O)2R6,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C 1_4 alkyl,
b) (CH2)pORS,
c) (CH2)pNRSR7,
d) halogen,
e) C1_4 perfluoroalkyl,
2) C3_6 cycloalkyl,
3) ORS,
4) SRS, S(O)RS, S02R6,
-NRsR~
Rs
-N~ R7
I IO
Rs
-N NR~R7a '
O
-25-

CA 02311921 2000-OS-26
' WO 99/27928 PCTNS98/25325
-O~ NRsR~
O
-O~ORs
,
O
10) ~ NRsR~ ,
O
11) -Sp2-NRsR~ ,
Rs
12) -N-S02 R' ,
13) ~ Rs ,
O
14) ~ ORs ,
O
15) C1_g alkyl, or
16) C1_g perfluoroalkyl;
R3a and R3b are independently absent or selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-C6 alkyl, C2-Cg alkenyl, C2-Cg alkynyl, C1-Cg
-26-

CA 02311921 2000-OS-26
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perfluoroalkyl, F, Cl, R80-, R8C(O)NR8-, CN, N02, (R8)2N-
C(NR8)-, R8C(O)-, -N(R8)2, or R90C(O)NR8-, and
c) C1-CS alkyl substituted by C1-C6 perfluoroalkyl, R80-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R90C(O)NR8-;
R5a and R5b are independently hydrogen, C1-Cg alkyl, cyclopropyl,
trifluoromethyl and halogen;
R6, R7 and R7a are independently selected from:
H; C1_4 alkyl, C3-g cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C 1_4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl, 2,2,2-
trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O)m-; unsubstituted or substituted
C1_4 alkyl, unsubstituted or substituted Cg-g cycloalkyl, unsubstituted or
25 substituted heterocycle, unsubstituted or substituted aryl, substituted
aroyl, unsubstituted or substituted heteroaroyl, substituted arylsulfonyl,
unsubstituted or substituted heteroarylsulfonyl, wherein the substituted
group is substituted with one or two substituents selected from:
a) C1-4 alkoxy,
30 b) aryl or heterocycle,
c) halogen,
d) HO,
-27-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
Rs
e)
O
f) II OR8
O
-S~O)mRs
h) N(R8)2, or
i) Cg_g cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 and A2 are independently selected from: a bond, -CH=CH-, -C=C-,
-C(O)-, -C(O)NR8-, O, -N(R8)-, or S(O)m;
V is selected from:
a) hydrogen,
b) heterocycle selected from pyrrolidinyl, imidazolinyl,
pyridinyl, thiazolyl, oxazolyl, indolyl, quinolinyl,
isoquinolinyl, triazolyl and thienyl,
c) aryl,
d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced
with a a heteroatom selected from O, S, and N, and
e) C2-C20 alkenyl, and
provided that V is not hydrogen if A1 is S(O)m and V is not hydrogen if
A1 is a bond, n is 0 and A2 is S(O)m;
X is a bond, -C(=O)NR10_~ _Ng,lOC(=O)-, -S(O)m- or -NR10_;
-28-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
m is 0, 1 or 2;
n is 0, 1, 2, 3 or 4;
p is 0, 1, 2, 3 or 4;
q is 0 or 1; and
r is 0 to 5, provided that r is 0 when V is hydrogen;
the dashed lines represent optional double bonds;
or an optical isomer or pharmaceutically acceptable salt thereof.
In a preferred embodiment of this invention, the inhibitors
of farnesyl-protein transferase are illustrated by the formula F:
R5a R3a-
V
A~OR~a2)ri N~
Nr
R5b (CR~b2)p x- (CRS°2)q ~R2
CR4/ r F
wherein:
Rla and R1c are independently selected from: hydrogen, Cg-C10
cycloalkyl or C1-C6 alkyl;
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-C10 cycloalkyl, R80-, -N(R8)2, F or C2-
Cg alkenyl,
c) C1-Cg alkyl unsubstituted or substituted by aryl,
heterocycle, Cg-C10 cycloalkyl, C2-Cg alkenyl, R80-, or -
N(R8)2
-29-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
R2 is selected from: H; unsubstituted or substituted C 1_g alkyl,
s RsR~
unsubstituted or substituted aryl, O ' O and -S(O)2RE,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1_4 alkyl,
b) (CH2)pORE,
c) (CH2)pNR6R7,
d) halogen,
e) C1_4 perfluoroalkyl,
2) C3_6 cycloalkyl,
3) ORE,
4) SRE, S(O)RE, S02RE,
'E) - N RsR~ ,
Rs
E) -NU R~
O
s
7) ~ 7 7a '
-N~ NR R
'IO
-30-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
-O~ NR6R~
O
-O~ORs
O
10) ~ NRsR~
O
11) -S02_NRsR7 ,
Rs
l
12) -N-S02 R7
13) ~ Rs ,
O
14) ~ ORs ,
O
15) C 1-g alkyl, or
16) C 1-g perfluoroalkyl;
R3a is selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-Cg alkyl, C2-Cg alkenyl, C2-Cg alkynyl, C1-Cg
perfluoroalkyl, F, Cl, R80-, R8C(O)NR8-, CN, N02, (R8)2N-
C(NR8)-, R8C(O)-, -N(R8)2, or R90C(O)NR8-, and
-31-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
c) C1-Cg alkyl substituted by C1-CS perfluoroalkyl, R$O-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(R8)2, or
R90C(O)NR8-;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C1_4 alkyl, Cg-s cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1_4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
IS R8 is independently selected from hydrogen, C1-Cg alkyl, 2,2,2-
trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)=; R9S(O)m-; unsubstituted or substituted
C1_4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C 1-4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
Rs
e)
O
f) I I OR8
O
9) -S~O)mR9
-32-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
h) N(R8)2, or
i) C3_6 cycloalkyl;
Rll is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
A1 is selected from: a bond, -C(O)-, O, -N(R8)-, or S{O)m;
X is a bond, -C(=O)NR10_, _~,lOC(=O)-, -S(O)m- or -NR10_;
n is 0 or 1; provided that n is not 0 if A1 is a bond, O, -N(R8)-, or
S{O)m
m is 0, 1 or 2;
p is 0, 1, 2, 3 or 4;
r is 1 or 2; and
q is 0 or 1;
or an optical isomer or pharmaceutically acceptable salt thereof.
In a preferred embodiment of this invention, the inhibitors
of farnesyl-protein transferase are illustrated by the formula G:
R$a~N ~N
R5a
1 1a ~~ ~R11
~CR 2)n ~~.~N~ N,
R5b (CR~b2)P X- (CR1~2)q R2
~R~r
-33-

CA 02311921 2000-OS-26
' WO 99/27928 PCTNS98/25325
wherein:
Rla and R1c are independently selected from: hydrogen, R80-, -N(R8)2,
F, C3-C10 cycloalkyl or C1-Cg alkyl;
R1b is independently selected from:
a) hydrogen,
b) aryl, heterocycle, C3-Clp cycloalkyl, R80-, -N(R8)2, F or C2-
Cg alkenyl,
c ) C 1-C6 alkyl unsubstituted or substituted by aryl,
heterocycle, Cg-C10 cycloalkyl, C2-C6 alkenyl, R80-, or -
N(R8)2
R2 is selected from: H; unsubstituted or substituted C1_g alkyl,
~s ~ RsR~
unsubstituted or substituted aryl, ~ ' 0 and -S(O)2R6,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1_4 alkyl,
b) (CH2)pOR6,
c) (CH2)pNR6R7,
d) halogen,
e) C1-4 perfluoroalkyl,
2) Cg-g cycloalkyl,
3) OR6,
4) SR6, S(O)RE, S02R6,
-34-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
5) -NR6R~
,
Rs
g) -N~ R~
O
s
I
) ~ ~a
-N
NR R
U
I I
O
-O~ NR6R~
O
9) -O II ORs
O
10) ~ NRsR~ ,
O
11) -S02-NRsR~ '
Rs
12) -N-S02 R~
13) ~ Rs ,
O
14) ~ ORs
O
15) C1_g alkyl, or
16) C 1_g perfluoroalkyl;
R3a is selected
from:
-35-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98125325
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R4 is independently selected from:
a) hydrogen,
b) aryl, substituted aryl, heterocycle, substituted heterocycle,
C1-Cg alkyl, C2-C6 alkenyl, C2-Cg alkynyl, C1-Cg
perfluoroalkyl, F, Cl, R80-, R8C(O)NR8-, CN, N02, (R8)2N-
C(NR8)-, R8C(O)-, -N(R8)2, or R90C(O)NR8-, and
c) C1-Cg alkyl substituted by C1-C6 perfluoroalkyl, R80-,
R8C(O)NR8-, (R8)2N-C(NR8)-, R8C(O)-, -N(Rg)2, or
R90C(O)NR8-;
Rya and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C1_4 alkyl, C3_g cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a) C1_4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-CS alkyl, 2,2,2-
trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S{O~-; unsubstituted or substituted
C1_4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1_4 alkoxy,
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CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
b) aryl or heterocycle,
c) halogen,
d) HO,
Rs
0
f) II OR8
O
9) -S~O)mR9
h) N(R8)2, or
i) Cg_g cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10_
n is 0 or 1;
m is 0, 1 or 2;
p is 0, 1, 2, 3 or 4, provided that p is not 0 if X is a bond, -NR8- or
O;
q is 0 or 1; and
r is 1 or 2;
or an optical isomer or pharmaceutically acceptable salt thereof.
In a preferred embodiment of this invention, the inhibitors
of farnesyl-protein transferase are illustrated by the formula H:
-37-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
Rsa . R3a-N/~ N
~-= N
Rla2 N ~ R5b ~ R11
_ N
2
/ (CRlb2)P X- (CR1~2)q 'R
NC
wherein:
Rla and Rlc are independently selected from: hydrogen, Cg-C10
cycloalkyl or C1-Cg alkyl;
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle, Cg-C 10 cycloalkyl, R80-, -N(R8)2 or F,
c) C1-Cg alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, R80-, or -N(R8)2;
R2 is selected from: H; unsubstituted or substituted C1_g alkyl,
~s ~RsR~
unsubstituted or substituted aryl, ~ ~ O and -S(O)2R6,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1~ alkyl,
b) (CH2)pORS,
c) (CH2)pNRSR7,
d) halogen,
e) C 1-4 perfluoroalkyl,
2) C3-S cycloalkyl,
3) ORS,
4) SRS, S(O)RS, S02R6,
-38-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
5) -NR6R~
Rs
i
-N~ R~
O
s
~
7) 7 7a
-N
NR R
~
O
-O~ NRsR~
O
9) -O II ORs
O
10) N R6R> >
O
11) -S02-NRsR> >
Rs
12) -N-S02 R~
13) ~ Rs ,
O
14) ~ORs
I I
O
15) C 1-g alkyl, or
16) C 1_g perfluoroalkyl;
R3a is selected
from:
-39-

CA 02311921 2000-OS-26
- WO 99/27928 PCT/US98/25325
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
Rs, R7 and R7a are independently selected from:
H; C 1_4 alkyl, C3_g cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
a ) C 1 _4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-CS alkyl, 2,2,2-
trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)-; R95(O~-; unsubstituted or substituted
C1_4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1_4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
R9
e)
O
~oR$
0
9) -S~O)mR9
-40-

CA 02311921 2000-OS-26
- WO 99/27928 PCT/US98/25325
h) N(R8)2, or
i) Cg-g cycloalkyl;
Rll is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;
X is a bond, -C(=O)NR10-, -NR10C(=O)-, -S(O)m- or -NR10_;
m is 0, 1 or 2;
p is 0, 1, 2, 3 ox 4;
q is 0 or 1; and
or an optical isomer or pharmaceutically acceptable salt thereof.
In a preferred embodiment of this invention, the inhibitors
of farnesyl-protein transferase are illustrated by the formula I:
Rsa_ N~ N
R5a
N,
NyCRlb2)P X- (CRS°2)q
NC ~ ~ A1(CR~a2)n
wherein:
Rla and R1c are independently selected from: hydrogen, R80-, -N(R8)2,
F, Cg-C10 cycloalkyl or C1-Cg alkyl;
-4.1-

CA 02311921 2000-OS-26
WO 99/27928 PCTNS98/25325
Rlb is independently selected from:
a) hydrogen,
b) aryl, heterocycle or Cg-C10 cycloalkyl,
S c) C1-Cg alkyl unsubstituted or substituted by aryl,
heterocycle, C3-C10 cycloalkyl, C2-Cg alkenyl, R80-, or -
N(R$)2
RZ is selected from: H; unsubstituted or substituted C 1_g alkyl,
~s ~RsR~
unsubstituted or substituted aryl, IO ' O and -S(O)ZRE,
wherein the substituted group is substituted with one or more of
1) aryl or heterocycle, unsubstituted or substituted with
one or two groups selected from:
a) C1_4 alkyl,
1 S b) (CH2)pORE,
c) (CH2)pNRSR7,
d) halogen,
e) C 1-4 perfluoroalkyl,
2) C3-E cycloalkyl,
3) ORS,
4) SRS, S(O)RE, S02RE,
5) -NRsR~
Rs
g) -N~ R~
I IO
s
~ ~a
-N~ NR R
IIO
-42-

CA 02311921 2000-OS-26
- WO 99/27928 PCTNS98/25325
) -O~ NR6R~
,
O
9) -OUORs
,
O
10) ~ NRsR~ ,
O
11) -S02-NRsR~ ,
Rs
12) -N-S02 R~ ,
13) ~ Rs ,
O
14) ~ ORs ,
O
15) C1_g alkyl, or
16) C1_g perfluoroalkyl;
R3a is selected from:
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted heteroaryl,
unsubstituted or substituted aralkyl and unsubstituted or
substituted heteroaralkyl;
R5a and R5b are independently hydrogen, ethyl, cyclopropyl or methyl;
R6, R7 and R7a are independently selected from:
H; C 1_4 alkyl, Cg_s cycloalkyl, aryl, heterocycle,
unsubstituted or substituted with:
-43-

CA 02311921 2000-OS-26
- WO 99/27928 PCT/US98125325
a) C1_4 alkoxy,
b) halogen, or
c) substituted or unsubstituted aryl or substituted or
unsubstituted heterocycle;
R8 is independently selected from hydrogen, C1-C6 alkyl, 2,2,2-
trifluoroethyl, benzyl and aryl;
R9 is independently selected from C1-Cg alkyl and aryl;
R10 is selected from: H; R8C(O)-; R9S(O~-; unsubstituted or substituted
C 1_4 alkyl, wherein the substituted alkyl group is substituted with one or
two substituents selected from:
a) C1_4 alkoxy,
b) aryl or heterocycle,
c) halogen,
d) HO,
R9
e)
O
f) II ORa
O
9) -S~O)mR9
h) N(R8)2, or
i) C3_g cycloalkyl;
R11 is selected from
H, unsubstituted or substituted alkyl, unsubstituted or
substituted aryl, unsubstituted or substituted aralkyl,
unsubstituted or substituted heteroaryl and unsubstituted or
substituted heteroaralkyl;

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98l25325
A1 is selected from: a bond, -C(O)-, O, -N(R8)-, or S(O)m;
X is a bond, -C(=O)NR10-, _NR,lOC(-O)-, -S(O)m- or -NR10_;
m is 0, 1 or 2;
n is 0 or 1;
p is 1, 2 or 3; and
q is 0 or 1;
or an optical isomer or pharmaceutically acceptable salt thereof.
Specific examples of the compounds of the invention are:
NC
HN~N
N N ,,,,, H
O
N
4-phenyl-4,5,6,7-tetrahydro-1H-imidazo[4,5] pyridine-6(S)-carboxylic acid
(2-[3-(4-cyano-benzyl)-3H -imidazol-4-yl]-ethyl}-amide;
or an optical isomer or a pharmaceutically acceptable salt thereof.
The compounds of the present invention may have
asymmetric centers and occur as racemates, racemic mixtures, and
as individual diastereomers, with all possible isomers, including
optical isomers, being included in the present invention. When any
substituent, term, or variable (e.g. aryl, heterocycle, Rla, R4 etc.)
occurs more than one time in any formula or generic structure its
definition on each occurence is independent from the definition at
every other occurence. Also, combinations of substituents/or
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CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
variables are permissible only if such combinations result in stable
compounds.
As used herein, "alkyl" is intended to include both
branched and straight-chain saturated aliphatic hydrocarbon groups
having the specified number of carbon atoms; "alkoxy" represents an
alkyl group of indicated number of carbon atoms attached through an
oxygen bridge. "Halogen" or "halo" as used herein means fluoro,
chloro, bromo and iodo.
As used herein, "aryl" is intended to mean any stable
monocyclic, bicyclic or tricyclic carbon ring of up to 7 members in each
ring, wherein at least one ring is aromatic. Examples of monocyclic and
bicyclic aryl elements include phenyl, naphthyl, tetrahydronaphthyl,
indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. Examples of
tricyclic aryl elements include 10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-yl (which is also known as dibenzylsuberyl),
9-fluorenyl and 9,10-dihydroanthracen-9-yl. Preferably, "aryl" is a
monocyclic or bicyclic carbon ring.
The term heterocycle or heterocyclic, as used herein,
represents a stable 5- to 7-membered monocyclic or stable 8- to
11-membered bicyclic heterocyclic ring or stable 13- to 15-membered
tricyclic heterocyclic ring, which is either saturated or unsaturated, and
which consists of carbon atoms and from one to four heteroatoms
selected from the group consisting of N, O, and S, and including any
bicyclic group in which any of the above-defined heterocyclic rings is
fused to a benzene ring. The heterocyclic ring may be attached at any
heteroatom or carbon atom which results in the creation of a stable
structure. Examples of monocyclic and bicyclic heterocyclic elements
include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl,
benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl,
benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl,
dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl,
dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl,
-46-

CA 02311921 2000-OS-26
WO 99/27928 PCT/US98/25325
naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, 2-oxopyrrolidinyl, pyridyl,
pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl,
pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl,
tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,
thienofuryl, thienothienyl, and thienyl. Examples of tricyclic
heterocyclic elements include, but are not limited to, 6,11-dihydro-5H-
benzo[5,6]cyclohepta[1,2-b]pyridine, 9,10-dihydro-4H-3-thia-
benzo [f1 azulen-4-yl and 9-xanthenyl. The 6,11-dihydro-5H-
benzo[5,6]cyclohepta[1,2-b]pyridine moiety has the following structure:
1
N ,~",,
Preferably, "heterocyclic" is a monocyclic or bicyclic moiety.
As used herein, "heteroaryl" is intended to mean any stable
monocyclic, bicyclic ar tricyclic carbon ring of up to 7 members in each
ring, wherein at least one ring is aromatic and wherein from one to four
carbon atoms are replaced by heteroatoms selected from the group
consisting of N, O, and S. Examples of monocyclic and bicyclic
heteroaryl elements include, but are not limited to, benzimidazolyl,
benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,
benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,
cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,
dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl,
imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,
isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl,
pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl,
quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiazolyl,
thienofuryl, thienothienyl, and thienyl. Examples of tricyclic heteroaryl
elements include, but are not limited to, 6,11-dihydro-5H-
benzo[5,6)cyclohepta[1,2-b)pyridine. Preferably, "heteroaryl" is a
monocyclic or bicyclic moiety.
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As used herein, "aralkyl" is intended to mean an aryl
moiety, as defined above, attached through a C1-C6 alkyl linker, where
alkyl is defined above. Examples of aralkyls inlcude, but are not limited
to, benzyl and naphthylmethyl.
As used herein, "heteroaralkyl" is intended to mean a
heteroalkyl moiety, as defined above, attached through a C1-Cg alkyl
linker, where alkyl is defined above. Examples of heteroaralkyls
include, but are not limited to, 2-pyridylmethyl, 2-imidazolylethyl and 2-
quinolinylmethyl.
As used herein, the term "substituted alkyl" is
intended to include the branch or straight-chain alkyl group of 1 to 6
carbon atoms unless otherwise indicated, wherein the carbon atoms
may be substituted with F, Cl, Br, CF3, N3, N02, NH2~ oxo, -OH,
-O(C1- Cg alkyl), S(O)p_2, (C1- Cg alkyl)S(O)p-Z-, (C1- Cg alkyl)S(O)0.
2(C1- Cg alkyl)-, C3-Clp cycloalkyl, C2-Cg alkenyl, C2-Cg alkynyl,
-C(O)NH, (C1- Cg alkyl)C(O)NH-, H2N-C(NH)-, (C1- C6 alkyl)C(O)-,
-O(C1-C6 alkyl)CFg, (C1- C6 alkyl)OC(O)-, (C1- C6 alkyl)O(C1- Cg
alkyl)-, (C1- Cg alkyl)C(O)2(C1- Cg alkyl)-, (C1- Cs alkyl)OC(O)NH-,
aryl, benzyl, heterocycle, aralkyl, heteroaralkyl, halo-aryl, halo-
benzyl, halo-heterocycle, cyano-aryl, cyano-benzyl and cyano-
heterocycle.
As used herein, the terms "substituted aryl",
"substituted heterocycle", "substituted aralkyl ", "substituted
heteroaralkyl " and "substituted cycloalkyl" are intended to include
the cyclic group containing from 1 to 3 substitutents in addition to the
point of attachment to the rest of the compound. Such substitutents
are preferably selected from the group which includes but is not
limited to F, Cl, Br, CFg, NH2, N(C1-Cg alkyl)2, N02, CN, (C1-Cg
alkyl)O-, -OH, (C1-C6 alkyl)S(O)m-, (C1-Cg alkyl)C(O)NH-, H2N-
C(NH)-, (C1-CS alkyl)C(O)-, (C1-C6 alkyi)OC(O)-, N3,(C1-Cs
alkyl)OC(O)NH- and C1-CZO alkyl.
When Rs and R7 or R7 and R7a are combined to form a
ring, cyclic amine moieties are formed. Examples of such cyclic
moieties include, but are not limited to:
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~~N '~~ N
U
In addition, such cyclic moieties may optionally include
another heteroatom(s). Examples of such heteroatom-containing
cyclic amine moieties include, but are not limited to:
~si'rN ~s.'rN ~~~ ~~~ N
» >c
O S o S
~sss'N ~s.s's ~s~~ N ~~~ N
1
S
S N
O
O COR9
Lines drawn into the ring systems from substituents (such
as from R2, R3, R4 etc.) indicate that the indicated bond may be attached
to any of the substitutable ring carbon or nitrogen atoms.
Preferably, R1a and Rlb are independently selected from:
hydrogen, -N(R8)2, R8C(O)NR$- or C1-Cg alkyl which is unsubstituted
or substituted by -N(R8)2, R80- or R8C(O)NR8-.
Preferably, R2 is selected from:
a) C 1-g alkyl, unsubstituted or substituted with one or
more o~
1) aryl or heterocycle, unsubstituted or substituted
with:
i) C1-4 ~Yl~
ii) (CH2)pOR6,
iii) (CHZ)pNR6R7,
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iv) halogen,
v) C1_4 perfluoroalkyl,
2) OR6,
3) SR6, SOZRS, or
) ~ NRsR~
O
b)
~s
O
c) aryl, unsubstituted or substituted with one or
more of:
1) C1_g alkyl,
2) C 1-g perfluoroalkyl,
3) ORS,
4) SR6, S02R6, or
5) II NR6R7.
O
d) -S02R6.
Preferably, R2 comprises at least one unsubstituted or
substituted phenyl.
Preferably, R4 is selected from: hydrogen, perfluoroalkyl, F,
Cl, Br, R$O-, R9S(O)m-, CN, N02, R82N-C(NR8)-, RgC(O)-, Ng, -N(R8)2,
R90C(O)NR8- and C1-Cg alkyl.
Preferably, R5 is hydrogen.
Preferably, R7b is C 1-Cg alkyl substituted with hydrogen or
an unsubstituted or substituted aryl group.
Preferably, R8 is selected from H, C1-CS alkyl and benzyl.
Preferably, A1 and A2 are independently selected from: a
bond, -C(O)NR8-, -NR8C(O)-, O, -N(R8)-, -S(O)ZN(R8)- and-N(R8)S(O)2-.
Preferably, V is selected from hydrogen, heterocycle and
aryl.
Preferably, W is imidazolyl.
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Preferably, X is a bond, -C(=O)NR10-, -NR10C(=O)- or -
Ng,lO-,
Preferably, n, p and r are independently 0, 1, or 2. More
preferably, r is 1.
5 Preferably t is 1.
Preferably, the moiety
~R4)~
V - A~(CRla2)nA2~CRla2)n ' W - (CR~b2)p-X- (CR1~2)q ~-
t
is selected from:
~R4~ N \ ~R4)/ / N~''L,
sb and ~ Rsa
Rsa N R Rsb N
The pharmaceutically acceptable salts of the compounds
of this invention include the conventional non-toxic salts of the
compounds of this invention as formed, e.g., from non-toxic
inorganic or organic acids. For example, such conventional non-
15 toxic salts include those derived from inorganic acids such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric
and the like: and the salts prepared from organic acids such as
acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic, malefic, hydroxymaleic, phenylacetic,
20 glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,
toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,
isethionic, trifluoroacetic and the like.
It is intended that the definition of any substituent or
variable (e.g., Rla, Z, n, etc.) at a particular location in a molecule be
25 independent of its definitions elsewhere in that molecule. Thus,
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- N(R8)2 represents -NH2, -NHCHg, -NHC2H5, etc. It is understood
that substituents and substitution patterns on the compounds of the
instant invention can be selected by one of ordinary skill in the art to
provide compounds that are chemically stable and that can be readily
synthesized by techniques known in the art, as well as those methods
set forth below, from readily available starting materials.
The pharmaceutically acceptable salts of the compounds
of this invention can be synthesized from the compounds of this
invention which contain a basic moiety by conventional chemical
methods. Generally, the salts are prepared either by ion exchange
chromatography or by reacting the free base with stoichiometric
amounts or with an excess of the desired salt-forming inorganic or
organic acid in a suitable solvent or various combinations of solvents.
Abbreviations used in the description of the chemistry
and in the Examples that follow are:
Ac20 Acetic anhydride;
Boc t-Butoxycarbonyl;
CBz Carbobenzyloxy;
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene;
DMAP 4-Dimethylaminopyridine;
DME 1,2-Dimethoxyethane;
DMF Dimethylformamide;
EDC 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide-
hydrochloride;
Et3N Triethylamine;
EtOAc Ethyl acetate;
FAB Fast atom bombardment;
HOBT 1-Hydroxybenzotriazole hydrate;
HOOBT 3-Hydroxy-1,2,2-benzotriazin-4(3I~-one;
HPLC High-performance liquid chromatography;
MCPBA m-Chloroperoxybenzoic acid;
MsCl Methanesulfonyl chloride;
NaHMDS Sodium bis(trimethylsilyl)amide;
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Py Pyridine;
TFA Trifluoroacetic acid;
THF Tetrahydrofuran.
5 The compounds of this invention are prepared by employing
reactions as shown in Schemes 1-3, in addition to other standard
manipulations such as ester hydrolysis, cleavage of protecting groups,
etc., as may be known in the literature or exemplified in the
experimental procedures. While stereochemistry is shown in the
10 Schemes, a person of ordinary skill in the art would understand that the
illustrated compounds represent racemic mixtures which may be
separated at a subsequent purification step or may be utilized as the
racemic mixture.
These reactions may be employed in a linear sequence to
15 provide the compounds of the invention or they may be used to synthesize
fragments which are subsequently joined by the reductive alkylation or
acylation reactions described in the Schemes.
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SCHEME 1
O
N OH H R KOH / EtOH
<N I NH2 + 70° C
2_
O O
Boc
OH 3 Boc20 N OH
<N I NH ~~ I N
N ~ ~ Boc
R R
N
'N' NH2 Boc,
NC N~ N
5 ' / CN
H ~--R
N , N
HOBt / EDC <N I ~ o~~ ~ Boc
N . 6
TFA
NC ~ HN~N
R
H N
N N ~I' H
0
* ~ Diastereomer A N
8 Diastereomer B
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SCHEME 2
NC HN~N
H ~R NaCNBH3
,. N
/N ( N ~~ H O
\N .~.$ O H
R
NC HN~N
R
H '
N N~~', NCR
O
N
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SCHEME 2A
NC HN~N ~ NEt
+ CI-S- R'
R p CH2CI2
H N
,
N N ~~ H
I o
N 7.8
NC ~ HN~N
R
H N
N I N ~~,,, O S-O
O R.
N
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NC HN ~ N HOBt / EDC
*R
N N ; H HO R.
O
N 7 .$
NC HN~N
R
H '
N
N ~ N ~~;, ~ R.
O O
N
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O O
/N OH MeOH /N OCH3
v I ~ v
\N I NH HCI \N NH
R R
O
H
Bra N ( OCH3 Br~ R'
NEt3 N N NaH
R
R~ O O
N OCH3
/N I ~~OCH3 and ~~ ~ N
N N
N R ~ i R,~ R
NaOH
i
R' O
N OH
N OH ~~ ~ I
and N
N N
R R
N
~N~ NH2
HOBt / EDC
5 ~ / CN
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SCHEMA 3 (CONT'D.)
O I N
ON ~ H N CN
N N
R'-'~ R
R~~ O
AND N I N N
H
~N N ~ ~ CN
R
Pd(PPh3)4
NDMBA
o IN
N ~~N N
i I H
<N NH ~ ~ CN
R~,~ R
R'
AND 1 O
~~ N N
H CN
N NH
R
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In the above Schemes, it is understood that
R is Rll or a protected precursor thereof; and
R' is R3a or R3b or protected precursor thereof.
The instant compounds are useful as pharmaceutical
agents for mammals, especially for humans. These compounds may be
administered to patients for use in the treatment of cancer. Examples of
the type of cancer which may be treated with the compounds of this
invention include, but are not limited to, colorectal carcinoma, exocrine
pancreatic carcinoma, myeloid leukemias and neurological tumors.
Such tumors may arise by mutations in the rocs genes themselves,
mutations in the proteins that can regulate Ras activity (i.e.,
neurofibromin (NF-1), neu, scr, abl, lck, fyn) or by other mechanisms.
The compounds of the instant invention inhibit farnesyl-
protein transferase and the farnesylation of the oncogene protein Ras.
The instant compounds may also inhibit tumor angiogenesis, thereby
affecting the growth of tumors (J. Rak et al. Cacncer Research, 55:4575-
4580 (1995)). Such anti-angiogenesis properties of the instant compounds
may also be useful in the treatment of certain forms of blindness related
to retinal vascularization.
The compounds of this invention are also useful for
inhibiting other proliferative diseases, both benign and malignant,
wherein Ras proteins are aberrantly activated as a result of oncogenic
mutation in other genes (i.e., the Ras gene itself is not activated by
mutation to an oncogenic form) with said inhibition being accomplished
by the administration of an effective amount of the compounds of the
invention to a mammal in need of such treatment. For example, the
compounds are useful in the treatment of neurofibromatosis, which is a
benign proliferative disorder.
The instant compounds may also be useful in the treatment
of certain viral infections, in particular in the treatment of hepatitis
delta and related viruses (J.S. Glenn et al. Science, 256:1331-1333 (1992).
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The compounds of the instant invention are also useful in
the prevention of restenosis after percutaneous transluminal coronary
angioplasty by inhibiting neointimal formation (C. Indolfi et al. Nature
medicine, 1:541-545(1995).
The instant compounds may also be useful in the treatment
and prevention of polycystic kidney disease (D.L. Schaffner et al.
American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley, Jr.
et al.FASEB Journal, 2:A3160 (1988)).
The instant compounds may also be useful for the
treatment of fungal infections.
In a preferred embodiment of the instant invention the
compounds of this instant invention are selective inhibitors of farnesyl-
protein transferase. A compound is considered a selective inhibitor of
farnesyl-protein transferase, for example, when its in vitro farnesyl-
protein transferase inhibitory activity, as assessed by the assay described
in Example 2, is at least 100 times greater than the in vitro activity of the
same compound against geranylgeranyl-protein transferase-type I in
the assay described in Example 3. Preferably, a selective compound
exhibits at least 1000 times greater activity against one of the enzymatic
activities when comparing geranylgeranyl-protein transferase-type I
inhibition and farnesyl-protein transferase inhibition.
In another preferred embodiment of the instant invention
the compounds of this instant invention are dual inhibitors of farnesyl
protein transferase and geranylgeranyl-protein transferase type I. Such
a dual inhibitor will exhibit certain characteristics when assessed in in
vitro assays, which are dependent on the type of assay employed.
In a SEAP assay, such as described in Example 6, it is
preferred that the dual inhibitor compound has an in vitro inhibitory
activity (IC50) that is less than about 12 ~t,M against K4B-Ras dependent
activation of MAP kinases in cells. More preferably, the dual inhibitor
compound has an in vitro inhibitory activity (IC50) against K4B-Ras
dependent activation of MAP kinases in cells which is more than about 5
times lower than the inhibitory activity (IC50) against Myr-Ras
dependent activation of MAP kinases in cells. Also more preferably, in a
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SEAP assay, the dual inhibitor compound has an inhibitory activity
(ICSp) that is less than about 10 nM against H-Ras dependent activation
of MAP kinases in cells.
In a GGTase plus anion assay, such as described in
Example 3, it is preferred that the dual inhibitor compound has an in
vitro inhibitory activity (ICSp) that is less than about 5 E,~M against
transfer of a geranylgeranyl residue to a protein or peptide substrate
comprising a CAAXG motif by geranylgeranyl-protein transferase type I
in the presence of a modulating anion. More preferably, the dual
inhibitor compound has an in vitro inhibitory activity {IC50) that is less
than about 1 ~.M against transfer of a geranylgeranyl residue to a protein
or peptide substrate comprising a CAAXG motif by geranylgeranyl-
protein transferase type I in the presence of a modulating anion.
Preferably, the dual inhibitor compound has an in vitro inhibitory
activity (IC50) in the in vitro assay as described in Example 2 that is less
than about 1 N.M against transfer of a farnesyl residue to a protein or
peptide substrate, comprising a CAAXF motif, by farnesyl-protein
transferase. more preferably, the dual inhibitor compound has an in
vitro inhibitory activity (IC50) that is less than about 100nM against
transfer of a farnesyl residue to a protein or peptide substrate,
comprising a CAAXF motif, by farnesyl-protein transferase. Also
preferably, the dual inhibitor compound has an in vitro inhibitory
activity (ICSp) in the in vitro assay as described in Example 5, that is less
than about 100 nM against the anchorage independent growth of H-ras-
transformed mammalian fibroblasts.
The protein or peptide substrate utilized in the instant assay
may incorporate any CAAX motif that is geranylgeranylated by GGTase-
I. The term "CAAXG" will refer to such motifs that may be
geranylgeranylated by GGTase-I. It is understood that some of the
"CAAXG" containing protein or peptide substrates may also be
farnesylated by farnesyl-protein transferase. In particular such
"CAAXG" motifs include (the corresponding human protein is in
parentheses): CVIM (K4B-Ras) SEQ.ID.NO. 1, CULL (mutated H-Ras)
SEQ.ID.NO. 2, CWM (N-Ras) SEQ.ID.NO. 3, CIIM (K4A-Ras)
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SEQ.ID.NO 4, CLLL (Rap-IA) SEQ.ID.NO. 5, CALL (Rap-IB)
SEQ.ID.NO. 6, CSIM SEQ.ID.NO. 7, CAIM SEQ.ID.NO. 8, CKVL
SEQ.ID.NO. 9 and CLIM SEQ.ID.NO. 10 (PFX). Preferably, the CAAX
motif is CVIM SEQ.ID.NO. 1.
5 As used herein, the term "CAAXF" is used to designate a
protein or peptide substrate that incorporates four amino acid C-
terminus motif that is farnesylated by farnesyl-protein transferase. It is
understood that certain of the "CAAXF" containing protein or peptide
substrates Fy also be geranylgeranylated by GGTase-I. In particular
10 such "CAAX " motifs include (the corresponding human protein is in
parentheses): CVLS (H-ras) SEQ.ID.NO. I1, CVIM (K4B-Ras)
SEQ.ID.NO. 1 and CVVM (N-Ras) SEQ.ID.N0.3.
The compounds of this invention may be administered to
mammals, preferably humans, either alone or, preferably, in
15 combination with pharmaceutically acceptable carriers or diluents,
optionally with known adjuvants, such as alum, in a pharmaceutical
composition, according to standard pharmaceutical practice. The
compounds can be administered orally or parenterally, including the
intravenous, intramuscular, intraperitoneal, subcutaneous, rectal
20 and topical routes of administration.
For oral use of a chemotherapeutic compound according
to this invention, the selected compound may be administered, for
example, in the form of tablets or capsules, or as an aqueous solution
or suspension. In the case of tablets for oral use, carriers which are
25 commonly used include lactose and corn starch, and lubricating
agents, such as magnesium stearate, are commonly added. For oral
administration in capsule form, useful diluents include lactose and
dried corn starch. When aqueous suspensions are required for oral
use, the active ingredient is combined with emulsifying and
30 suspending agents. If desired, certain sweetening and/or flavoring
agents may be added. For intramuscular, intraperitoneal,
subcutaneous and intravenous use, sterile solutions of the active
ingredient are usually prepared, and the pH of the solutions should
be suitably adjusted and buffered. For intravenous use, the total
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PCTNS98125325
concentration of solutes should be controlled in order to render the
preparation isotonic.
The present invention also encompasses a
pharmaceutical composition useful in the treatment of cancer,
S comprising the administration of a therapeutically effective amount
of the compounds of this invention, with or without pharmaceutically
acceptable carriers or diluents. Suitable compositions of this
invention include aqueous solutions comprising compounds of this
invention and pharmacolo-gically acceptable carriers, e.g., saline, at
a pH level, e.g., 7.4. The solutions may be introduced into a patient's
intramuscular blood-stream by local bolus injection.
As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specific amounts, as well as any product which results, directly or
indirectly, from combination of the specific ingredients in the
specified amounts.
When a compound according to this invention is
administered into a human subject, the daily dosage will normally be
determined by the prescribing physician with the dosage generally
varying according to the age, weight, and response of the individual
patient, as well as the severity of the patient's symptoms.
In one exemplary application, a suitable amount of
compound is administered to a mammal undergoing treatment for
cancer. Administration occurs in an amount between about 0.1
mg/kg of body weight to about 60 mg/kg of body weight per day,
preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of
body weight per day.
The compounds of the instant invention are also useful
as a component in an assay to rapidly determine the presence and
quantity of farnesyl-protein transferase (FPTase) in a composition.
Thus the composition to be tested may be divided and the two portions
contacted with mixtures which comprise a known substrate of
FPTase (for example a tetrapeptide having a cysteine at the amine
terminus) and farnesyl pyrophosphate and, in one of the mixtures, a
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compound of the instant invention. After the assay mixtures are
incubated for an sufficient period of time, well known in the art, to
allow the FPTase to farnesylate the substrate, the chemical content of
the assay mixtures may be determined by well known
S immunological, radiochemical or chromatographic techniques.
Because the compounds of the instant invention are selective
inhibitors of FPTase, absence or quantitative reduction of the amount
of substrate in the assay mixture without the compound of the instant
invention relative to the presence of the unchanged substrate in the
assay containing the instant compound is indicative of the presence
of FPTase in the composition to be tested.
It would be readily apparent to one of ordinary skill in
the art that such an assay as described above would be useful in
identifying tissue samples which contain farnesyl-protein
transferase and quantitating the enzyme. Thus, potent inhibitor
compounds of the instant invention may be used in an active site
titration assay to determine the quantity of enzyme in the sample. A
series of samples composed of aliquots of a tissue extract containing
an unknown amount of farnesyl-protein transferase, an excess
amount of a known substrate of FPTase (for example a tetrapeptide
having a cyateine at the amine terminus) and farneayl pyrophosphate
are incubated for an appropriate period of time in the presence of
varying concentrations of a compound of the instant invention. The
concentration of a sufficiently potent inhibitor (i.e., one that has a Ki
substantially smaller than the concentration of enzyme in the assay
vessel) required to inhibit the enzymatic activity of the sample by 50%
is approximately equal to half of the concentration of the enzyme in
that particular sample.
EXAM~'LES
Examples provided are intended to assist in a further
understanding of the invention. Particular materials employed,
species and conditions are intended to be further illustrative of the
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CA 02311921 2000-OS-26
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invention and not limitative of the reasonable scope thereof.
Purification by HPLC was utilized for Example 1 as set forth below.
Preparation of 4-phenyl-4,5,6,7-tetrahydro-1H-imidazo[4,5] pyridine-6(S)-
~ ra_boxvlic acid f2-f3-(4-cvano-benzvl)-3H -imidazol-4- l~l-ethyll-amide
Step A: 4-phenyl-6,7-dihydro-4-H-imidazo[4,5]pyridine-1,5,6(S)-
tricarboxylic acid 1~5-di-tert-butyl ester
To a solution of L-histidine (3.1g, 0.02mo1) and KOH (1.12g,
0.02mo1) in water (50m1) and EtOH (50m1) was added benzaldehyde
(3.06m1, 0.03mo1). The resulting solution was heated at 70°C for 18h.
The solvents were removed in vacuo. The residue was dissolved in THF
IS (70m1) and water (30m1) and Boc anhydryde (4.37g, 0.04mo1) was added
and the mixture was stirred for 18h at 25°C. The solvents were removed
in vacuo and the residue was partitioned with EtOAc and water. The
water layer was adjusted to pH=5 with 1M HCl and extracted twice with
EtOAc. The EtOAc layers from the pH=5 extraction were combined and
dried with brine and magnesium sulfate. The EtOAc was removed in
vacuo to obtain the title compound as a solid which was used in the next
step as is.
FAB mas spectrum m/e 444 (m+1).
Ste~B: 6(S)-(2-[3-(4-cyano-benzyl)-3H-imidazol-4y1]ethylcarbamoyl}-
4-phenyl-6,7-dihydro-4H-imidazo[4,5-] pyridine-1,5-
dicarboxvlic acid di-tert-buts 1~ estg~
To a solution of the product as described in Step A above, [4-
phenyl-6,7-dihydro-4-H-imidazo[4,5]pyridine-1,5,6(S)-tricarboxylic acid
1,5-di-tert-butyl ester] (0.34g, 0.826mm), cyanobenzyl histamine
(5)(0.187g, 0.826mm), HOBt (0.126g, 0.826mm), EDC (0.158g, 0.826mm) in
DMF (5m1) was added NMM (0.27m1, 2.48mm). The solvents were
removed in vacuo and the residue was partitioned with EtOAc and
saturated sodium bicarbonate. The EtOAc layer was dried with brine
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CA 02311921 2000-OS-26
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and magnesium sulfate. The EtOAc was removed in vacuo to obtain the
title compound as a solid which was used in the next step as is.
Step C: 4-phenyl-4,5,6,7-tetrahydro-1H-imidazo[4,5] pyridine-6(S)-
carboxylic acid {2-[3-(4-cyano-benzyl)-3H -imidazol-4-yl]-
ethyl )-amide
To a solution of 6(S)-{2-[3-(4-cyano-benzyl)-3H-imidazol-
4y1]ethylcarbamoyl]-4-phenyl-6,?-dihydro-4H-imidazo[4,5-] pyridine-1,5-
dicarboxylic acid di-tert-butyl ester (0.58g) in CH2CI2 (lOml) was added
TFA (5m1) and the solution was stirred 45min. The solvents were
removed in vacuo and the crude product was purified by preparative
HPLC to obtain diastereomer A and diastereomer B as the title
compounds.
EXAMPLE 2
~In vitro inhibition of ras farnesyl transferase
Assays of farnesyl-protein transferase. Partially purified
bovine FPTase and Ras peptides (Ras-CVLS SEQ.ID.NO. 11, Ras-CVIM
SEQ.ID.NO. 1 and Ras-CAIL SEQ.ID.NO. 12) were prepared as
described by Schaber et ~1_., J. Biol. Chem. 265:14701-14704 (1990),
Pompliano, gt ~1_., Biochemistry 31:3800 (1992) and Gibbs et al., P_ NAS
U.S.A. 86:6630-6634 (1989), respectively. Bovine FPTase was assayed in a
volume of 100 ml containing 100 mM N-(2-hydroxy ethyl) piperazine-N'-
(2-ethane sulfonic acid) (HEPES), pH 7.4, 5 mM MgCl2, 5 mM
dithiothreitol (DTT), 100 mM [3H]-farnesyl diphosphate ([3H]-FPP; 740
CBq/mmol, New England Nuclear), 650 nM Ras-CVLS and 10 mg/ml
FPTase at 31°C for 60 min. Reactions were initiated with FPTase
and
stopped with 1 ml of 1.0 M HCL in ethanol. Precipitates were collected
onto filter-mats using a TomTec Mach II cell harvestor, washed with
100% ethanol, dried and counted in an LKB b-plate counter. The assay
was linear with respect to both substrates, FPTase levels and time; less
than 10% of the [3H]-FPP was utilized during the reaction period.
Purified compounds were dissolved in 100% dimethyl sulfoxide (DMSO)
and were diluted 20-fold into the assay. Percentage inhibition is
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measured by the amount of incorporation of radioactivity in the presence
of the test compound when compared to the amount of incorporation in
the absence of the test compound.
Human FPTase was prepared as described by Omer et al. ,
~3iochemistry 32:5167-5176 (1993). Human FPTase activity was assayed
as described above with the exception that 0.1% (w/v) polyethylene glycol
20,000, 10 mM ZnCl2 and 100 nM Ras-CVIM were added to the reaction
mixture. Reactions were performed for 30 min., stopped with 100 ml of
30% (v/v) trichloroacetic acid (TCA) in ethanol and processed as
described above for the bovine enzyme.
The compounds of the instant invention described in the
above Examples were tested for inhibitory activity against human
FPTase by the assay described above and were found to have IC50 of <50
~.M .
EXAMPLE 3
'M~~~~~pd In vitro GGTase inhibition asssav
The modified geranylgeranyl-protein transferase inhibition
assay is carried out at room temperature. A typical reaction contains (in
a final volume of 50 mL): [3H]geranylgeranyl diphosphate, biotinylated
Ras peptide, 50 mM HEPES, pH 7.5, a modulating anion (for example 10
mM glycerophosphate or 5mM ATP), 5 mM MgCl2, 10 mM ZnCl2, 0.1%
PEG (15-20,000), 2 mM dithiothreitol, and geranylgeranyl-protein
transferase type I(GGTase). The GGTase-type I enzyme employed in the
assay is prepared as described in U.S. Pat. No. 5,470,832, incorporated by
reference. The Ras peptide is derived from the K4B-Ras protein and has
the following sequence: biotinyl-G KTKCVIM (single amino
acid code) (SEQ.ID.NO.: 13). Reactions are initiated by the addition of
GGTase and stopped at timed intervals (typically 15 min) by the addition
of 200 mL of a 3 mg/mL suspension of streptavidin SPA beads
(Scintillation Proximity Assay beads, Amersham) in 0.2 M sodium
phosphate, pH 4, containing 50 mM EDTA, and 0.5% BSA. The
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quenched reactions are allowed to stand for 2 hours before analysis on a
Packard TopCount scintillation counter.
For inhibition studies, assays are run as described above,
except inhibitors are prepared as concentrated solutions in 100%
dimethyl sulfoxide and then diluted 25-fold into the enzyme assay
mixture. IC50 values are determined with Ras peptide near KM
concentrations. Enzyme and nonsaturating substrate conditions for
inhibitor IC50 determinations are as follows: 75 pM GGTase-I, 1.6
mM Ras peptide, 100 nM geranylgeranyl diphosphate.
~'l-based in vitro ras nreny,~lation assay
The cell lines used in this assay consist of either Ratl or
NIH3T3 cells transformed by either viral H-ras; an N-ras chimeric gene
in which the C-terminal hypervariable region of viral-H-ras was
substituted with the corresponding region from the N-ras gene; or
ras-CULL, a viral-H-ras mutant in which the C-terminal exon encodes
leucine instead of serine, making the encoded protein a substrate for
geranylgeranylation by GGTase-I. The assay can also be performed
using cell lines transformed with human H-ras, N-ras or I~4B-ras.
The assay is performed essentially as described in DeClue, J.E. et al.,
Cancer Research 51:712-717, (1991). Cells in 10 cm dishes at 50-75%
confluency are treated with the test compounds) (final concentration
of solvent, methanol or dimethyl sulfoxide, is 0.1%). After 4 hours
at 37°C, the cells are labelled in 3 ml methionine-free DMEM supple-
mented with 10% regular DMEM, 2% fetal bovine serum, 400
mCi[35S]methionine (1000 Ci/mmol) and test compound(s). Cells treated
with lovastatin, a compound that blocks Ras processing in
cells by inhibiting the rate-limiting step in the isoprenoid biosynthetic
pathway (Hancock, J.F. et al. Cell, 57:1167 (1989); DeClue, J.E.
et al. Cancer Res., 51:712 (1991); Sinensky, M. et al. J. Biol. Chem.,
265:19937 (1990)), serve as a positive control in this assay. After an
additional 20 hours, the cells are lysed in 1 ml lysis buffer (1% NP40/20
mM HEPES, pH 7.5/5 mM MgCl2/1mM DTT/10 mg/ml aprotinen/2
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mg/ml leupeptin/2 mg/ml antipain/0.5 mM PMSF) and the lysates
cleared by centrifugation at 100,000 x g for 45 min. Alternatively, four
hours after the additon of the labelling media, the media is removed, the
cells washed, and 3 ml of media containing the same or a different test
compound added. Following an additional lfi hour incubation, the lysis
is carried out as above. Aliquots of lysates containing equal numbers of
acid-precipitable counts are bought to 1 ml with IP buffer (lysis buffer
lacking DTT) and immunoprecipitated with the ras-specific monoclonal
antibody Y13-259 (Furth, M.E. et al., J. Virol. 43:294-304, (1982)).
Following a 2 hour antibody incubation at 4°C, 200 ml of a 25%
suspension of protein A-Sepharose coated with rabbit anti rat IgG is
added for 45 min. The immunoprecipitates are washed four times with
IP buffer (20 nM HEPES, pH 7.5/1 mM EDTA/1% Triton X-100Ø5%
deoxycholate/0.1%/SDS/0.1 M NaCl) boiled in SDS-PAGE sample buffer
and loaded on 13% acrylamide gels. When the dye front reached the
bottom, the gel is fixed, soaked in Enlightening, dried and
autoradiographed. The intensities of the bands corresponding to
prenylated and nonprenylated Ras proteins are compared to determine
the percent inhibition of prenyl transfer to protein.
EXAMPLE 5
Cell based in vitro anchorage independent ~owth assay (SALSA)
SALSA (loft agar-dike Surrogate Assay) measures the
inhibition of anchorage-independent growth by prenyl-transferase
inhibitors. Only transformed cells are able to grow anchorage-
independently in the SALSA format. Additionally, cells growing in
the SALSA format grow in clumps, resembling the colonies formed
in soft agar. SALSA may been used to measure the growth inhibition
by prenyl-transferase inhibitors in a variety of transformed cell lines,
including R,at1 fibroblasts transformed with viral-H-ras (H-ras/ratl),
as well as a panel of human tumor cell lines (HTL's).
SALSA is performed in 96-well plates that are coated
with a thin film of the polymer, PolyHEMA (Poly(2-hydroxyethyl
methacrylate)), which prevents cells from attaching to the plate.
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Ratl fibroblast cells transformed with v-Ha-ras (this cell line has
been deposited in the ATCC on August 19, 1997 under the terms of the
Budapest convention and has been given a designation of ATCC CRL
12387) are seeded at 5000 cells/well, grown for 4 hr, then vehicle or
half log dilutions of test compound (in either an 8 or 12 point titration)
are added. The cells are then grown for 6 days at 37 degrees, without
changing the growth media or adding fresh compound. At day 6, cell
growth is assessed via a colorimetric assay that measures the
cleavage of the tetrazolium dye, MTT, to an insoluble purple
formazan, a reaction dependent upon mitochondria)
dehydrogenases. At day 6, the cells are incubated for 4 hr with 0.5
mg/ml MTT, and then SDS is added to 9% w/v to lyre the cells and
solubilize the insoluble MTT-formazan. The amount of MTT
metabolism is quantitated via spectrophotometric detection at 570 nM.
Dose-inhibition curves and IC50's are determined.
(construction of SEAP reuorter nlasmid pDSE100
The SEAP reporter plasmid, pDSE100 was constructed by
ligating a restriction fragment containing the SEAP coding sequence
into the plasmid pCMV-RE-AKI. The SEAP gene is derived from the
plasmid pSEAP2-Basic (Clontech, Palo Alto, CA). The plasmid pCMV-
RE-AKI was constructed by Deborah Jones (Merck) and contains 5
2S sequential copies of the 'dyad symmetry response element' cloned
upstream of a 'CAT-TATA' sequence derived from the cytomegalovirus
immediate early promoter. The plasmid also contains a bovine growth
hormone poly-A sequence.
The plasmid, pDSE100 was constructed as follows. A
restriction fragment encoding the SEAP coding sequence was cut out of
the plasmid pSEAP2-Basic using the restriction enzymes EcoRl and
HpaI. The ends of the linear DNA fragments were filled in with the
HIenow fragment of E. coli DNA Polymerase I. The 'blunt ended' DNA
containing the SEAP gene was isolated by electrophoresing the digest in
an agarose gel and cutting out the 1694 base pair fragment. The vector
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plasmid pCMV-RE-AKI was linearized with the restriction enzyme Bgl-
II and the ends filled in with Klenow DNA Polymerase I. The SEAP
DNA fragment was blunt end ligated into the pCMV-RE-AKI vector and
the ligation products were transformed into DH5-alpha E. coli cells
(Gibco-BRL). Transformants were screened for the proper insert and
then mapped for restriction fragment orientation. Properly oriented
recombinant constructs were sequenced across the cloning junctions to
verify the correct sequence. The resulting plasmid contains the SEAP
coding sequence downstream of the DSE and CAT-TATA promoter
elements and upstream of the BGH poly-A sequence.
Cloning of vristylated viral-H-ras expression plasmid
A DNA fragment containing viral-H-ras can be PCRed from plasmid "H-
1" (Ellis R. et al. J. Virol. 36, 408, 1980) using the following oligos.
Sense strand:
5'TCTCCTCGAGGCCACCATGGGGAGTAGCAAGAGCAAGCCTAA
GGACCCCAGCCAGCGCCGGATGACAGAATACAAGCTTGTGGTG
G 3'. (SEQ.ID.NO.: 14)
Antisense: 5'CACATCTAGATCAGGACAGCACAGACTTGCAGC 3'.
(SEQ.ID.NO.: 15)
A sequence encoding the first 15 aminoacids of the v-src gene,
containing a myristylation site, is incorporated into the sense strand
oligo. The sense strand oligo also optimizes the 'Kozak' translation
initiation sequence immediately 5' to the ATG start site.To prevent
prenylation at the viral-ras C-terminus, cysteine 186 would be mutated
to a serine by substituting a G residue for a C residue in the C-terminal
antisense oligo. The PCR primer oligos introduce an XhoI site at the 5'
end and a Xbal site at the 3'end. The XhoI-XbaI fragment can be ligated
into the mammalian expression plasmid pCI (Promega) cut with XhoI
and XbaI. This results in a plasmid in which the recombinant myr-
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viral-H-ras gene is constitutively transcribed from the CMV promoter of
the pCI vector.
Cloning of a viral-H-rah-CVLL expression nlasmid
A viral-H-ras clone with a C-terminal sequence encoding the amino
acids CULL can be cloned from the plasmid "H-1" (Ellis R. et al. J. Virol.
36, 408, 1980) by PCR using the following oligos.
Sense strand:
5'TCTCCTCGAGGCCACCATGACAGAATACAAGCTTGTGGTGG-3'
(SEI~,I.ID.NO.: 16)
Antisense strand:
5'CACTCTAGACTGGTGTCAGAGCAGCACACACTTGCAGC-3'
(SEQ.ID.NO.: 17)
The sense strand oligo optimizes the 'Kozak' sequence and adds an XhoI
site. The antisense strand mutates serine 189 to leucine and adds an
XbaI site. The PCR fragment can be trimmed with XhoI and XbaI and
ligated into the XhoI-XbaI cut vector pCI (Promega). This results in a
plasmid in which the mutated viral-H-ras-CVLL gene is constitutively
transcribed from the CMV promoter of the pCI vector.
30
Cloning of c H ras-Leu61 expression plasmid
The human c-H-ras gene can be PCRed from a human cerebral cortex
cDNA library (Clontech) using the following oligonucleotide primers.
Sense strand:
5'-GAGAGAATTCGCCACCATGACGGAATATAAGCTGGTGG-3'
(SEQ.ID.NO.: 18)
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Antisense strand:
5'-GAGAGTCGACGCGTCAGGAGAGCACACACTTGC-3'
(SEQ.ID.NO.: 19)
The primers will amplify a c-H-ras encoding DNA fragment with the
primers contributing an optimized 'Kozak' translation start sequence,
an EcoRI site at the N-terminus and a Sal I stite at the C-terminal end.
After trimming the ends of the PCR product with EcoRI and Sal I, the c-
H-ras fragment can be ligated ligated into an EcoRI -Sal I cut
mutagenesis vector pAlter-1 (Promega). Mutation of glutamine-61 to a
leucine can be accomplished using the manufacturer's protocols and the
following oligonucleotide:
5'-CCGCCGGCCTGGAGGAGTACAG-3' (SEQ.ID.N0.:20)
After selection and sequencing for the correct nucleotide substitution,
the mutated c-H-ras-Leu61 can be excised from the pAlter-1 vector,
using EcoRI and Sal I, and be directly ligated into the vector pCI
(Promega) which has been digested with EcoRI and Sal I. The new
recombinant plasmid will constitutively transcribe c-H-ras-Leu61 from
the CMV promoter of the pCI vector.
Cloning of a c-N-ras-Val-12 expression plasmid
The human c-N-ras gene can be PCRed from a human cerebral cortex
cDNA library (Clontech) using the following oligonucleotide primers.
Sense strand:
5'-GAGAGAATTCGCCACCATGACTGAGTACAAACTGGTGG-3'
(SEQ.ID.N0.:21)
Antisense strand:
5'-GAGAGTCGACTTGTTACATCACCACACATGGC-3' (SECg,I.ID.NO.:
22)
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The primers will amplify a c-N-ras encoding DNA fragment with the
primers contributing an optimized 'Kozak' translation start sequence,
an EcoRI site at the N-terminus and a Sal I stite at the C-terminal end.
After trimming the ends of the PCR product with EcoRI and Sal I, the c-
N-ras fragment can be ligated into an EcoRI -Sal I cut mutagenesis
vector pAlter-1 (Promega). Mutation of glycine-12 to a valine can be
accomplished using the manufacturer's protocols and the following
oligonucleotide:
5'-GTTGGAGCAGTTGGTGTTGGG-3' (SEQ.ID.NO.: 23)
After selection and sequencing for the correct nucleotide substitution,
the mutated c-N-ras-Val-12 can be excised from the pAlter-1 vector,
using EcoRI and Sal I, and be directly ligated into the vector pCI
(Promega) which has been digested with EcoRI and Sal I. The new
recombinant plasmid will constitutively transcribe c-N-ras-Val-12 from
the CMV promoter of the pCI vector.
Cloning_of a c K ras Val-12 expression ~~lasmid
The human c-K-ras gene can be PCRed from a human cerebral cortex
cDNA library (Clontech) using the following oligonucleotide primers.
Sense strand:
5'-GAGAGGTACCGCCACCATGACTGAATATAAACTTGTGG-3'
(SEQ.ID.NO.: 24)
Antisense strand:
5'-CTCTGTCGACGTATTTACATAATTACACACTTTGTC-3'
(SEQ.ID.NO.: 25)
The primers will amplify a c-K-ras encoding DNA fragment with the
primers contributing an optimized 'Kozak' translation start sequence, a
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KpnI site at the N-terminus and a Sal I stite at the C-terminal end.
After trimming the ends of the PCR product with Kpn I and Sal I, the c-
K-ras fragment can be ligated into a KpnI -Sal I cut mutagenesis vector
pAlter-1 (Promega). Mutation of cysteine-12 to a valine can be
accomplished using the manufacturer's protocols and the following
oligonucleotide:
5'-GTAGTTGGAGCTGTTGGCGTAGGC-3' (SEQ.ID.NO.: 26)
After selection and sequencing for the correct nucleotide substitution,
the mutated c-K-rocs-Val-12 can be excised from the pAlter-1 vector,
using Kpnl and Sal I, and be directly ligated into the vector pCI
(Promega) which has been digested with KpnI and Sal I. The new
recombinant plasmid will constitutively transcribe c-K-ras-Val-12 from
the CMV promoter of the pCI vector.
SEAP assay
Human C33A cells (human epitheial carcenoma - ATTC
collection) are seeded in lOcm tissue culture plates in DMEM + 10% fetal
calf serum + 1X Pen/Strep + 1X glutamine + 1X NEAR. Cells are grown
at 37oC in a 5% C02 atmosphere until they reach 50 -80% of conflunecy.
The transient transfection is performed by the CaP04
method (Sambrook et al., 1989). Thus, expression plasmids for H-ras, N-
ras, K-ras, Myr-ras or H-ras-CVLL are co-precipitated with the DSE-
SEAP reporter construct. For lOcm plates 600m1 of CaCl2 -DNA solution
is added dropwise while vortexing to 600m1 of 2X HBS buffer to give 1.2m1
of precipitate solution (see recipes below). This is allowed to sit at room
temperature for 20 to 30 minutes. While the precipitate is forming, the
media on the C33A cells is replaced with DMEM (minus phenol red;
Gibco cat. # 31053-028)+ 0.5% charcoal stripped calf serum + 1X
(Pen/Strep, Glutamine and nonessential aminoacids). The CaP04-DNA
precipitate is added dropwise to the cells and the plate rocked gently to
distribute. DNA uptake is allowed to proceed for 5-6 hrs at 37oC under a
5% C02 atmosphere.
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Following the DNA incubation period, the cells are washed
with PBS and trypsinized with 1m1 of 0.05% trypsin. The 1 ml of
trypsinized cells is diluted into lOml of phenol red free DMEM + 0.2%
charcoal stripped calf serum + 1X (Pen/Strep, Glutamine and NEAR ).
Transfected cells are plated in a 96 well microtiter plate (100m1/well) to
which drug, diluted in media, has already been added in a volume of
100m1. The final volume per well is 200m1 with each drug concentration
repeated in triplicate over a range of half log steps.
Incubation of cells and drugs is for 36 hrs at 37o under C02.
At the end of the incubation period, cells are examined microscopically
for evidence of cell distress. Next, 100m1 of media containing the
secreted alkaline phosphatase is removed from each well and
transferred to a microtube array for heat treatment at 65oC for 1 hr to
inactivate endogenous alkaline phosphatases (but not the heat stable
secreted phosphatase).
The heat treated media is assayed for alkaline phosphatase
by a luminescence assay using the luminescence reagent CSPD~
(Tropix, Bedford, Mass.). A volume of 50 ml media is combinRased with
200 ml of CSPD cocktail and incubated for 60 minutes at room
temperature. Luminesence is monitored using an ML2200 microplate
luminometer (Dynatech). Luminescence reflects the level of activation of
the fos reporter construct stimulated by the transiently expressed
protein.
DNA-CaPO~prec:~tate for lOcm plate of cells
Ras expression plasmid ( lmg/ml) lOml
DSE-SEAP Plasmid (1mg/ml) 2m1
Sheared Calf Thymus DNA (1mg/ml) 8ml
2M CaCl2 74m1
dH20 506m1
~X HBS Buffer
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280mM NaCl
lOmM KCl
l.5mM Na2HP04 2H20
l2mM dextrose
50mM HEPES
Final pH = 7.05
T uminesence Buffer (26m1)
Assay Buffer 20m1
Emerald ReagentT"' (Tropix) 2.5m1
100mM homoarginine 2.5m1
CSPD Reagent~ (Tropix) l.Om1
Assay Buffer
Add 0.05M Na2C03 to 0.05M NaHCOg to obtain pH 9.5. Make 1mM in
MgCl2
1EXAMPLE 7
In vivo tumor~owth inhibition assay (nude mouse)
In vivo efficacy as an inhibitor of the growth of cancer cells
may be confirmed by several protocols well known in the art. Examples
of such in vivo efficacy studies are described by N. E. Kohl et al. (Nature
Medicine, 1:792-797 (1995)) and N. E. Kohl et al. (Proc. Nat. Acad. Sci.
U.S.A., 91:9141-9145 (1994)).
Rodent fibroblasts transformed with oncogenically mutated
human Ha-ras or Ki-ras (106 cellslanimal in 1 ml of DMEM salts) are
injected subcutaneously into the left flank of 8-12 week old female nude
mice (Harlan) on day 0. The mice in each oncogene group are randomly
assigned to a vehicle, compound or combination treatment group.
Animals are dosed subcutaneously starting on day 1 and daily for the
duration of the experiment. Alternatively, the farnesyl-protein
tranaferase inhibitor may be administered by a continuous infusion
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pump. Compound, compound combination or vehicle is delivered in a
total volume of 0.1 ml. Tumors are excised and weighed when all of the
vehicle-treated animals exhibited lesions of 0.5 - 1.0 cm in diameter,
typically 11-15 days after the cells were injected. The average weight of
the tumors in each treatment group for each cell line is calculated.
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SEQUENCE LISTING
<110> Ciccarone, Terrence M.
deSolms, Jane S. J.
Merck & Co., Inc.
<120> INHIBITORS OF FARNESYL-PROTEIN
TRANSFERASE
<130> 20121Y
<150> 60/067,552
<151> 1997-12-04
<160> 26
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 4
<212> PRT
<213> Homosapien
<400> 1
Cys Val Ile Met
1
<210> 2
<211> 4
<212> PRT
<213> Homosapien
<400> 2
Cys Val Leu Leu
1
<210> 3
<211> 4
<212> PRT
<213> Homosapien
<400> 3
Cys Val Val Met
1
<210> 4
<211> 4
<212> PRT
<213> Homosapien
<400> 4
Cys Ile Ile Met
1
- 1-

CA 02311921 2000-OS-26
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<210> 5
<211> 4
<212> PRT
<213> Homosapien
<400> 5
Cys Leu Leu Leu
1
<210> 6
<211> 4
<212> PRT
<213> Homosapien
<400> 6
Cys Gln Leu Leu
1
<210> 7
<211> 4
<212> PRT
<213> Homosapien
<400> 7
Cys Ser Ile Met
1
<210> 8
<211> 4
<212> PRT
<213> Homosapien
<400> 8
Cys Ala Ile Met
1
<210> 9
<211> 4
<212> PRT
<213> Homosapien
<400> 9
Cys Lys Val Leu
1
<210> 10
<211> 4
<212> PRT
<213> Homosapien
<400> 10
Cys Leu Ile Met
1
<210> 11
<211> 4
- 2-

CA 02311921 2000-OS-26
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<212> PRT
<213> Homosapien
<400> 11
Cys Val Leu Ser
1
<210> 12
<211> 4
<212> PRT
<213> Homosapien
<400> 12
Cys Ala Ile Leu
1
<210> 13
<211> 15
<212> PRT
<213> Homosapien
<400> 13
Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met
1 5 10 15
<210> 14
<211> 86
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 14
tctcctcgag gccaccatgg ggagtagcaa gagcaagcct aaggacccca gccagcgccg 60
gatgacagaa tacaagcttg tggtgg 86
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 15
cacatctaga tcaggacagc acagacttgc agc 33
<210> 16
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
- 3-

CA 02311921 2000-OS-26
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<400> 16
tctcctcgag gccaccatga cagaatacaa gcttgtggtg g 41
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 17
cactctagac tggtgtcaga gcagcacaca cttgcagc 38
<210> 18
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 18
gagagaattc gccaccatga cggaatataa gctggtgg 38
<210> 19
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 19
gagagtcgac gcgtcaggag agcacacact tgc 33
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 20
ccgccggcct ggaggagtac ag 22
<210> 21
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 21
- 4-

CA 02311921 2000-OS-26
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gagagaattc gccaccatga ctgagtacaa actggtgg 38
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 22
gagagtcgac ttgttacatc accacacatg gc 32
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 23
gttggagcag ttggtgttgg g 21
<210> 24
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 24
gagaggtacc gccaccatga ctgaatataa acttgtgg 38
<210> 25
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 25
ctctgtcgac gtatttacat aattacacac tttgtc 36
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> completely synthesized
<400> 26
gtagttggag ctgttggcgt aggc 24