Note: Descriptions are shown in the official language in which they were submitted.
CA 02338741 2001-O1-23
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IL-8 RECEPTOR ANTAGONISTS
s
FIELD OF THE INVENTION
This invention relates to novel benzoisothiazole substituted compounds,
pharmaceutical compositions, processes for their preparation, and use thereof
in
treating IL-8. GROG, GROG. GROy and NAP-2 mediated diseases.
BACKGROUND OF THE INVENTION
Many different names have been applied to Interleukin-8 (IL-8), such as
neutrophil attractant/activation protein-1 (NAP-1), monocyte derived
neutrophil
chemotactic factor (MDNCF), neutrophil activating factor (NAF), and T-cell
lymphocyte chemotactic factor. Interleukin-8 is a chemoattractant for
neutrophils,
basophils, and a subset of T-cells. It is produced by a majority of nucleated
cells
including macrophages, fibroblasts, endothelial and epithelial cells exposed
to TNF,
IL-la, IL-lp or LPS, and by neutrophils themselves when exposed to LPS or
chemotactic factors such as FMLP. M. Baggiolini et al, J. Clin. Invest. 84,
L045
( 1989); J. Schroder et al., J. Immunol. 139, 3474 (1987) and J. Immunol. 144,
2223
( 1990): Strieter et al., Science 243, 1467 ( 1989) and J. Biol. Chem. 264,
10621
( 1989); Cassatella et al., J. Imm~ol. 148, 3216 ( 1992).
GROa, GR0~3, GROy and NAP-2 also belong to the chemokine a family.
Like IL-8, these chemokines have also been referred to by different names. For
instance GROa, (3, y have been referred to as MGSAa, (3 and y respectively
(Melanoma Growth Stimulating Activity), see Richmond et al., J. Cell
Physiology
129. 375 ( 1986) and Chang et al:, J. Immunol 148, 451 ( 1992). All of the
chemokines of the a-family which possess the ELR motif directly preceding the
CXC motif bind to the IL-8 B receptor.
3o IL-8, GROa, GROG, GROy, NAP-2 and ENA-78 stimulate a number of
functions in vitro. They have all been shown to have chemoattractant
properties for
neutrophils, while IL-8 and GROG have demonstrated T-lymphocytes, and
basophilic chemotactic activity. In addition IL-8 can induce histamine release
from
basophils -from both normal and atopic individuals GRO-a and IL-8 w~ i:.
addition,
induce lysozomal enzyme release and respiratory burst from neutrophils. IL-8
has
also been shown to increase the surface expression of Mac-1 (CDIIb/CD18) on
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WO 00/05216 PC'fltJS98/15533
neutrophils without de novo protein synthesis. This may contribute to
increased
adhesion of the neutrophils to vascular endothelial cells. Many known diseases
are
characterized by massive neutrophil infiltration. As IL.-8, GROG, GR0~3, GRO~y
and
NAP-2 promote the accumulation and activation of neutrophils, these chemokines
5 have been implicated in a wide range of acute and chronic inflammatory
disorders
including psoriasis and rheumatoid arthritis, Baggiolini et al., FEBS Lett.
307, 97
( 1992); Miller et al., Crit. Rev. Immunol. 12, 17 ( 1992); Oppenheim et al.,
A~,uj
Rev. Immunol. 9, 617 ( 1991 ); Seitz et al., ). CJ"in. Invest. 87, 463 ( 1991
); Miller et
al., Am. Rev. Red ir.~Di~. 146, 427 ( 1992); Donnely et al., lancet 341, 643 (
1993).
to In addition the ELR chemokines (those containing the amino acids ELR motif
just
prior to the CXC motif) have also been implicated in angiostasis, see Strieter
et al.,
Science 258, 1798 ( 1992).
In vitro, IL-8, GROG, GROG, GROy and NAP-2 induce neutrophil shape
change, chemotaxis, granule release, and respiratory burst, by binding to and
t5 activating receptors of the seven-transmembrane, G-protein-linked family,
in
particular by binding to IL-8 receptors, most notably the B-receptor. Thomas
et al.,
J. Biol. Chem. 266, 14839 ( 1991 ); and Holmes et al., Science 253, 1278 (
1991 ). The
development of non-peptide small molecule antagonists for members of this
receptor
family has precedent. For a review see R. Freidinger in: ~r~rgss in Drug
Research,
2o Vol. 40, pp. 33-98, Birkhauser Verlag, Basel 1993. Hence, the IL-8 receptor
represents a promising target for the development of novel anti-inflammatory
agents.
Two high affinity human IL-8 receptors (77% homology) have been
characterized: IL-8Ra, which binds only IL-8 with high affinity, and IL-8R(i,
which
has high affinity for IL-8 as well as for GRO-a, GROG, GROy and NAP-2. See
25 Holmes et al., supra; Murphy et al., Science 253, 1280 (1991); Lee et al.,
J. Biol.
Chem. 267, 16283 ( 1992); LaRosa et al., J. Biol. Chem. 267, 25402 ( 1992);
and
Gayle et al., J. Biol. Chem. 2~8, 7283 ( 1993).
There remains a need for treatment, in this field, for compounds which are
capable of binding to the IL-8 a or (3 receptar. Therefore, conditions
associated with
3o an increase in IL,-8 production (which is responsible for chemotaxis of
neutrophil
and T-cells subsets into the inflammatory site) would benefit by compounds
which
are inhibitors of IL-8 receptor binding.
SUMMARY OF THE INV NTION
35 This invention provides for a method of treating a chemokine mediated
disease, wherein the chemokine is one which binds to an IL-8 a or ~i receptor
and
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which method comprises administering an effective amount of a compound of
Formula (I) or (II) or a pharmaceutically acceptable salt thereof. In
particular the
chemokine is IL-8.
This invention also relates to a method of inhibiting the binding of IL-8 to
its
receptors in a mammal in need thereof which comprises administering to said
mammal an effective amount of a compound of Formula (I) or (II).
The present invention also provides for the novel compounds of Formula (I),
and (II) and pharmaceutical compositions comprising a compound of Formula (I),
and (II) and a pharmaceutical carrier or diluent.
1o Compounds of Formula (I) useful in the present invention ace represented by
the structure:
(R~)m
.X
~NH
R (I)
wherein
R is -NH -C(X2)-NH- (CR 13R 14)v - Z:
~ (CR~sR~s)P
Z is W, HET, (~" , optionally substituted C 1 _ 10 alkyl, optionally
substituted C~_ 10 alkenyl, or optionally substituted C2_ 10 alkynyl;
X is C(X 1 )2, O, N-R 1 g, C=O, or S(O)m ;
XI is independently hydrogen, halogen, CI-10 ~kyl, NR4R5, C(O}NR4R5,
optionally substituted C 1 _ I p alkyl, C 1 _ 10 alkoxy, halosubstituted C 1 _
10 alkoxy,
hydroxy, aryl, aryl C 1 _4 alkyl, aryloxy, aryl C 1 ~. alkyloxy, heteroaryl,
heteroarylalkyl, heterocyclic, heterocyclic C 1..4alkyl, or heteroaryl C 1~
alkyloxy; provided that both of X1 are not hydrogen;
X2 is =O, or =S;
A is CH2, C(O) or C(S);
R 1 is independently selected from hydrogen, halogen, nitro, cyano,
halosubstituted
CI-10 ~kYl, CI-10 alkyl, C2_10 alkenyl, CI-10 alkoxy, halosubstituted CI-10
alkoxy, azide, (CRBRg)q S(O)tR4, hydroxy, hydroxyC 1_ l0alkyl, aryl, arylC l~.
alkyl, aryloxy, aryl Cl_4 alkyloxy, heteroaryl, heteroarylalkyl, heterocyclic,
heterocyclic C 1 alkyl, heteroaryl C I ~ alkyloxy, aryl C2_ 10 alkenyl,
heteroaryl
3o C2_ 10 alkenyl, heterocyclic C2_ 10 alkenyl, (CRBRg)qNR4R5, C~_ 10 alkenyl
C(O)NR4R5, (CRBRg)q C(O)NR4R5, (CRBRg)q C(O)NR4R10, S(O)3H,
-3-
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S(O)3Rg, (CR8R8)q C(O)R11, C?-10 alkenyl C(O)R11, C?_10 alkenyl
C(O)OR11, C(O)R11, (CRBRg)q C(O)OR12, (CRBRg}q OC(O) R11, (CRBRg)q
NR4C(O)R11, (CRgRg)qC(NR4.)NR4Rg, (CRgRg)q NR4C(NRS)R11~ (CR8R8}q
NHS(O)~R1~, or (CRBRg)q S(O)~NR4R5, or two R1 moieties together may form
O-(CH~)s0- or a 5 to 6 membered saturated or unsaturated ring;
n is an integer having a value of 1 to 3;
m is an integer having a value of 1 to 3;
m' is an integer having a value of 1 or 2;
p is an integer having a value of 1 to 3;
t o q is 0, or an integer having a value of 1 to 10;
s is an integer having a value of 1 to 3;
t is 0, or an integer having a value of 1 or 2;
v is 0, or an integer having a value of 1 to 4;
HET is an optionally substituted heteroaryl;
t5 R4 and RS are independently hydrogen, optionally substituted C1~ alkyl,
optionally
substituted aryl, optionally substituted aryl C1_4alkyl, optionally
substituted
heteroaryl, optionally substituted heteroaryl C 1 alkyl, heterocyclic, or
heterocyclic C 1_4 alkyl, or R.~ and RS together with the nitrogen to which
they
are attached form a 5 to 7 member ring which may optionally comprise an
20 additional heteroatom selected from O/N/S;
Y is independently selected from hydrogen, halogen, nitro, cyano,
halosubstituted
C 1 _ 10 alkyl, C 1 _ 10 alkyl, C2_ 10 alkenyl, C 1 _ 10 alkoxy,
halosubstituted C 1-10
alkoxy, azide, (CRBRg)q S (O)tR4, hydroxy, hydroxyC 1 _ l palkyl, aryl, aryl C
1 ~
alkyl, aryloxy, arylC 1 _q. alkyloxy, heteroaryl, heteroarylalkyl, heteroaryl
C 1~
25 alkyloxy, heterocyclic, heterocyclic C 1 alkyl, aryl C2_ 10 alkenyl,
heteroaryl
C~_10 alkenyl, heterocyclic C?_10 alkenyl, (CRBRg)q NR4R5, Cz_10 alkenyl
C(O)NR4R5, (CRBRg)q C(O)NR4R5, (CRBRg)q C(O)NRq.R 10, S(O)3Rg,
(CRBRg)q C(O)R11, C2-10 ~kenyl C(O)R11, C2-10 ~kenyl C(O)OR11,
(CRBRg)q C(O)OR12, (CRBRg)q OC(O) R11, (CRBRg)q NR4C(O)R11,
30 (CRBRg)qC(NR4)NR4R5, (CRBRg)q NR4C(NRS)R1 l, (CRBRg)q NHS(O)~Ra,
or (CRBRg)q S(O)2NR4R5, or two Y moieties together may form O-(CH~)s -O
or a S to 6 membered saturated or unsaturated ring;
R( and R~ are independently hydrogen or a C 1 ~ alkyl group, or R6 and R~
together
with the nitrogen to which they are attached form a 5 to 7 member ring which
ring
35 may optionally contain an additional heteroatom which heteroatom is
selected from
oxygen, nitrogen or sulfur;
-4-
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Rg is independently hydrogen or C 1 ~ alkyl;
R 10 is C 1_ 10 alkyl C(O}2Rg;
R 11 is hydrogen, C 1 _~. alkyl, optionally substituted aryl, optionally
substituted aryl
C 1 alkyl, optionally substituted heteroaryl, optionally substituted
5 heteroarylCl_~alkyl, optionally substituted heterocyclic, or optionally
substituted heterocyclicC I _~.alkyl;
R 12 is hydrogen, C 1 _ 10 alkyl, optionally substituted aryl or optionally
substituted
arylalkyl;
R 13 and R 14 are independently hydrogen, optionally substituted C 1 _:~
alkyl, or one
!o of R13 and R14 may be an optionally substituted aryl;
R 1 g and R 16 are independently hydrogen, or an optionally substituted C 1 ~
alkyl;
R l~ is C 1 _4alkyl, aryl, arylalkyl, heteroaryl, heteroarylC 1 alkyl,
heterocyclic, or
heterocyclicCl_4alkyl, wherein the aryl, heteroaryl and heterocyclic
containing
rings may all be optionally substituted;
t s R 1 g is hydrogen, C 1 _4 alkyl, aryl, arylC 1 ~ alkyl, heteroaryl,
heteroarylC 1 ~ alkyl,
heterocyclic, or heterocyclicC 1 ~, alkyl, all of which may be optionally
substituted;
Ra is NR6R~, alkyl, arylC 1 _~ alkyl, arylC2~. alkenyl, heteroaryl, heteroaryl-
C 1 alkyl, heteroarylC2_4 alkenyl, heterocyclic, or heterocyclicC 1 _4 alkyl,
2o wherein the aryl, heteroaryl and heterocyclic containing rings may all be
optionally substituted;
n
i
W is ~ , or
2s the E containing ring is optionally selected from
(Y)n
* *
* ! I
,/
o ; (Y)n o ; n(Y) ; or n(Y) /
the asterix * denoting point of attachment of the ring;
-5.
CA 02338741 2001-O1-23
WO 00/05216 PCT/US98/15533
or a pharmaceutically acceptable salt thereof.
Compounds of Formula (II) useful in the present invention are represented
by the structure:
rr
\ ( N~X
H
(II)
wherein
R is -NH -C(X2)-NH- (CR13R14)v - Z;
OR,sR,s)P
Z is W, HET, «" , optionally substituted C I _ IO alkyl, optionally
substituted C~_ IO alkenyl, or optionally substituted C2_ I O alkynyl;
X is C(X I ) or N;
XI is hydrogen, halogen, CI-10 ~kYl, NR4R5, C(O)NR4R5, optionally substituted
C 1-10 ~kYl, C I -10 alkoxy, halosubstituted C I _ 10 alkoxy, hydroxy, aryl,
aryl
C 1 _4 alkyl, aryloxy, aryl C 1 _4 alkyloxy, heteroaryl, heteroaryl C 1 ~
alkyl,
heterocyclic, heterocyclic C I _4alkyl, or heteroaryl C I ~ alkyloxy;
X~ is =O, or =S;
A is CRIB;
R 1 is independently selected from hydrogen, halogen, vitro, cyano,
halosubstituted
C 1-10 ~kYl, C 1-10 ~kYl, C2_ I p alkenyl, C I _ I O alkoxy, halosubstituted C
1 _ 10
alkoxy, azide, (CRgRg)q S(O)tR4, hydroxy, hydroxy C 1 _ Ipalkyl, aryl, aryl C
1 _4
?o alkyl, aryloxy, aryl C I _4 alkyloxy, heteroaryl, heteroaryl C I ~ alkyl,
heterocyclic, heterocyclic C I _4alkyl, heteroaryl C 1 ~ alkyloxy, aryl C~_ 10
alkenyl, heteroaryl C2_ IO alkenyl, heterocyclic C2_ IO alkenyl,
(CRgRg)qNR4R5,
CZ_ IO alkenyl C(O)NR4R5, (CRgRg)q C(O)NR4R5, (CRgRg)q C(O)NR4R 10,
S(O)3H, S(O)3Rg, (CRgRg)q C(O)RI I, C2-10 ~kenyl C(O)RI l, C?_IO alkenyl
z5 C(O)ORI I, C(O)Rl I, (CRgRg)q C(O)OR12, (CRgRg)q OC(O) RI1, (CRgRg)q
NRq.C(O)RI l, (CRgRg)qC(NR4)NR4R5, (CRgRg)q NR4C(NRg)RI 1,
(CRgRg)q NHS(O)2R1~, or (CRgRg)q S(O)~NR4R5, or two RI moieties
together may form O-(CH~)s0 or a 5 to 6 membered saturated or unsaturated
rmg;
30 m is an integer having a value of 1 to 3;
n is an integer having a value of I to 3;
-6-
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p is an integer having a value of 1 to 3:
q is 0, or an integer having a value of I to 10;
s is an integer having a value of I to 3;
t is 0, or an integer having a value of 1 or 2;
v is 0, or an integer having a value of I to 4;
HET is an optionally substituted heteroaryl:
R4 and RS are independently hydrogen, optionally substituted CI_4 alkyl,
optionally
substituted aryl, optionally substituted aryl C I alkyl, optionally
substituted
heteroaryl, optionally substituted heteroaryl CI_q.alkyl, heterocyclic, or
t0 heterocyclic CIA. alkyl, or R.~ and R5 together with the nitrogen to which
they
are attached form a 5 to 7 member ring which may optionally comprise an
additional heteroatom selected from O/N/S;
Y is independently selected from hydrogen, halogen, nitro, cyano,
halosubstituted
C 1-10 alkyl, C I -10 alkyl, C?_ I 0 alkenyl, C I -10 alkoxy, halosubstituted
C I-10
t5 alkoxy, azide, (CRgRg)q S(O)tR4, hydroxy, hydroxyCl_l0alkyl, aryl, aryl CIA
alkyl, aryloxy, arylC I _4 alkyloxy, heteroaryl, heteroaryl C I _q. alkyl,
heteroaryl
C I _:~ alkyloxy, heterocyclic, heterocyclic C I _4alkyl, aryl C2_ Ip alkenyl,
heteroaryl C~_ 10 alkenyl, heterocyclic C2_ IO alkenyl, (CRgRg)q NR4Rg, C2_ 10
alkenyl C(O)NR4R~, (CRgRg)q C(O)NR4RS, (CRgRg)q C(O)NR4R10,
2o S(O)3Rg, (CRgRg)q C(O)RI I, C2-10 ~kenyl C(O)RI I, C2-10 alkenyl
C(O)ORI I, (CRgRg)q C(O)ORI2, (CRgRg)q OC(O) RI I, (CRgRg)q
NR4C(O)R I I , (CRgRg)qC(NR4)NR4R5, (CRgRg)q NR4C(NRS)R I 1.
(CRgRg)q NHS(O)~Ra, or (CRgRg)q S(O)2NR4R5, or two Y moieties together
may form O-(CH?)s0 or a 5 to 6 membered saturated or unsaturated ring;
25 R( and R7 are independently hydrogen or a C I _4 alkyl group, or R6 and R7
together
with the nitrogen to which they are attached form a 5 to 7 member ring which
ring may optionally contain an additional heteroatom which heteroatom is
selected from oxygen, nitrogen or sulfur;
Rg is independently hydrogen or C I _~ alkyl;
3o R I0 is C I _ I O alkyl C(O)2Rg;
R I I is hydrogen, C I _4 alkyl, optionally substituted aryl, optionally
substituted aryl
CI_4alkyl, optionally substituted heteroaryl, optionally substituted
heteroarylC I alkyl, optionally substituted heterocyclic, or optionally
substituted heterocyclicC I alkyl;
35 R 12 is hydrogen, C I _ I p alkyl, optionally substituted aryl or
optionally substituted
arylalkyl;
_7_
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R 13 and R l~ are independently hydrogen, optionally substituted C 1 ~ alkyl,
or one
of R 13 and R l4 may be an optionally substituted aryl;
R 15 and R 16 are independently hydrogen, or an optionally substituted C 1 ~
alkyl;
R 17 is C 1 alkyl, aryl, arylalkyl, heteroaryl, heteroarylC 1 _4alkyl,
heterocyclic, or
heterocyclicC 1 _q,alkyl, wherein the aryl, heteroaryl and heterocyclic
containing
rings may all be optionally substituted;
R 1 g is hydrogen, C 1 _4 alkyl, aryl, arylC 1 ~, alkyl, heteroaryl,
heteroarylC l _4 alkyl,
heterocyclic, or heterocyclicC 1 _4 alkyl, all of which may be optionally
substituted;
1o Ra is NR6R~, alkyl, arylC l ~ alkyl, arylC2_4 alkenyl, heteroaryl,
heteroaryl-
C 1..4a1ky1, heteroarylC~_4 alkenyl, heterocyclic,or heterocyclicC 1 ~ alkyl,
wherein the aryl, heteroaryl and heterocyclic rings may all be optionally
substituted;
Y)nw
15 Wis ~ ,or
the E containing ring is optionally selected from
O
('fin *
*
f
Y n.
o ( ) o . n(Y) n(Y) ,/
;or ;
the asterix * denoting point of attachment of the ring;
2o or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION OF THE INVENTIOI'~V
The compounds of Formula (I) and (II) may also be used in association with
the veterinary treatment of mammals, other than humans, in need of inhibition
of
IL-8 or other chemokines which bind to the IL-8 a and (3 receptors. Chemokine
25 mediated diseases for treatment, therapeutically or prophylacticaIly, in
animals
include disease states such as those noted herein in the Methods of Treatment
section.
_g_
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As readily seen, the difference between compounds of Formula (I) and (II)
lies in the unsaturation of the A containing ring, and hence the substitutions
on the X
and A moieties. The remaining terms, defined below, are the same for both
compounds of Formula (I) and (II) unless otherwise indicated.
5 Suitably, R 1 is independently selected from hydrogen; halogen; nitro;
cyano;
halosubstituted CI_10 alkyl, such as CF3; Cl-10 alkyl, such as methyl, ethyl,
isopropyl, or n-propyl; C2-10 alkenyl; C 1-10 alkoxy, such as methoxy, or
ethoxy;
halosubstituted CI-10 alkoxy, such as trifluoromethoxy; azide; (CRgRg)q
S(O)tRq.,
wherein t is 0, 1 or 2: hydroxy; hydroxy CI-l0alkyl, such as methanol or
ethanol;
to aryl, such as phenyl or naphthyl; aryl CI_4 alkyl, such as benzyl; aryloxy,
such as
phenoxy; aryl CI_4 alkyloxy, such as benzyloxy; heteroaryl; heteroaryl CI_4
alkyl;
heteroaryl C 1..~ alkyloxy; aryl C2_ 10 alkenyl; heteroaryl C2_ 10 alkenyl;
heterocyclic
C?_ 10 alkenyl; (CRgRg)qNR4R~; C?_ 10 alkenyl C(O)NR4R5; (CRgRg)q
C(O)NR4R5; (CRgRg)q C(O)NR4R10; S(O)3H; S(O)3Rg; (CRgRg)q C(O)R11;
15 C~_Ip alkenyl C(O)R11; C2-10 ~kenyl C(O)OR11; C(O)RI1; (CRgRg)q C(O)OR12;
(CRgRg)q OC(O) R11; (CkgRg)q NR4C(O)R11; (CRgRg)qC(NR4)NR4R5;
(CRgRg)q NR4C(NRS)RI 1; (CRgRg)q NHS(O)2R1~; or (CRgRg)q S(O)~NR4Rg;
or two R 1 moieties together may form O-(CH2)s-O or a 5 to 6 membered
saturated or
unsaturated ring. All of the aryl, heteroaryl, and heterocyclic containing
moieties
20 above may be optionally substituted as defined herein below. Preferably, R
1 is other
than azido for methods of use herein.
It is recognized that the R 1 moiety may be substituted on the benzene ring or
the A containing ring, if possible. Suitably, s is an integer having a value
of 1 to 3,
and m is an integer having a value of 1 to 3.
25 When RI forms a dioxybridge, s is preferably 1. When RI forms an
additional saturated or unsaturated ring, it is preferably 6 membered ring,
preferably
resulting in a naphthylene ring system. These additional rings may be
substituted
independently, 1 to 3 times, by the other R 1 moieties as defined above.
Preferably, RI is hydrogen, halogen, cyano, nitro, CF3, (CRgRg)q
3o C(O)NR4R5, C2_10 alkenyl C(O)NR4R5, (CRgRg)q C(O)Rq.Rlp, C?_10 alkenyl
C(O)OR12, heteroaryl, heteroaryl CI_4 alkyl, heteroaryl C2_10 alkenyl, or
S(O)?NR4R5.
Suitably, R4 and RS are independently hydrogen, optionally substituted CI-4
alkyl, optionally substituted aryl, optionally substituted aryl CI~aIkyl,
optionally
35 substituted heteroaryl, optionally substituted heteroaryl Cl~alkyl,
heterocyciic, or
heterocyclicC 1 _4 alkyl, or R4 and RS together with the nitrogen to which
they are
-9-
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attached form a 5 to 7 member ring which may optionally comprise an additional
heteroatom selected from O/N/S.
Suitably, R6 and R~ are independently hydrogen or a C1_~ alkyl group, or R6
and R7 together with the nitrogen to which they are attached form a ~ to 7
member ring
which ring may optionally contain an additional heteroatom which heteroatom is
selected from oxygen, nitrogen or sulfur.
Suitably, Rg is independently hydrogen or C1_4 alkyl.
Suitably, q is 0 or an integer having a value of 1 to 10.
Suitably, R 10 is C 1-10 alkyl C(O)2Rg, such as CH2C(O)2H or
to CH2C(O)2CH3.
Suitably, R 11 is hydrogen, C 1 _4 alkyl, aryl, aryl C 1 ~ alkyl, heteroaryl,
heteroaryl C 1 _4alkyl, heterocyclic, or heterocyclic C 1 alkyl.
Suitably, R12 is hydrogen, Cl_lp alkyl, optionally substituted aryl or
optionally substituted aryl C 1 ~. alkyl.
t5 Suitably, R13 and R14 are independently hydrogen, an optionally substituted
C 1 ~ alkyl which may be straight or branched as defined herein, or one of R
13 and
R14 are an optionally substituted aryl; v is 0, or an integer having a value
of 1 to 4.
When R 13 or R 14 are an optionally substituted alkyl, the alkyl moiety may
be substituted one to three times independently by halogen; halosubstituted C1-
4
2o alkyl such as trifluoromethyl; hydroxy; hydroxy C 1 _.~alkyl, C 1 ~ alkoxy;
such as
methoxy, or ethoxy, halosubstituted C 1_10 alkoxy, S(O)tR4; aryl; NIt4Rg;
NHC(O)R4; C(O)NR4Rg; or C(O)ORg.
Suitably, R 17 is C 1 alkyl, aryl, arylalkyl, heteroaryl, heteroarylC 1 alkyl,
heterocyclic, or heterocyclicCl_4alkyl, wherein the aryl, heteroaryl and
heterocyclic
25 containing rings may all be optionally substituted.
Suitably, Y is independently selected from hydrogen, halogen, vitro, cyano,
halosubstituted C 1 _ l p alkyl, C 1 _ l0 alkyl, C2_ 10 alkenyl, C 1 _ 10
alkoxy,
halosubstituted C I _ 10 alkoxy, azide, (CRgRg)q S(O)tR4, hydroxy,
hydroxyC 1 _ l0alkyl, aryl, aryl C 1 ~, alkyl, aryloxy, arylC 1 ~ alkyloxy,
heteroaryl,
3o heteroaryl C1~ alkyl, heteroaryl C1_4 alkyloxy, heterocyclic, heterocyclic
C 1 _4alkyl, aryl C?_ 10 alkenyl, heteroaryl C2_ 10 alkenyl, heterocyclic C?_
10
alkenyl, (CRgRg)q NR4R5, C2_ 10 alkenyl C(O)NR4R5, (CRgRg)q C(O)NR4R5,
(CRgRg)q C(O)NRq.RIO~ S(O)3Rg, (CRgRg)q C(O)R1 l, C2-10 alkenyl C(O)R11,
C2_10 alkenyl C(O)OR11, (CRgRg)q C(O)OR12, (CRgRg)q OC(O) R11, (CRgRg)q
35 NR4C(O)Rl 1, (CRgRg)qC(NRq.)NR4Rg, (CRgRg)q NR4C(NRS)R1 I, (CRgRg)q
NHS(O)~Ra, or (CRgRg)q S(O)2NR4R5, or two Y moieties together may form
10-
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O-(CH~)s -O or a 5 to 6 membered saturated or unsaturated ring. Preferably, Y
is
other than azido for methods of use herein.
Suitably, n is an integer having a value of 1 to 3.
When Y forms a dioxybridge, s is preferably 1. The aryl, heteroaryl and
5 heterocyclic containing moieties noted above for Y may all be optionally
substituted
as defined herein. When Y forms an additional saturated or unsaturated ring,
it is
preferably 6 membered ring, more preferably resulting in a naphthylene ring
system.
These additional rings may be optionally substituted I to 3 times by other Y
moieties as defined above.
t o Suitably, Ra is NR6R~, alkyl, aryl C I _4 alkyl, arylC 2-4 alkenyl,
heteroaryl,
heteroary!-C I alkyl, heteroarylC~~ alkenyl, heterocyclic, or heterocyclicC I-
4
alkyl, wherein the aryl, heteroaryl and heterocyclic containing rings may all
be
optionally substituted.
Y is preferably a halogen. C I _~. alkoxy, optionally substituted aryl.
optionally
15 substituted aryloxy or aryl C I ~ alkoxy, methylenedioxy, NR4R5, thin C I-
4alkyl,
thioaryl, halosubstituted CI-10 alkoxy, CI-10 alkyl, or hydroxy CI-10 alkyl. Y
is
more preferably mono-substituted halogen, disubstituted halogen, mono-
substituted
alkoxy, disubstituted alkoxy, methylenedioxy, aryl, or alkyl. More preferably
these
groups are mono or di-substituted in the 2'- position or 2'-, 3'-position when
Z is W
2o and W is a phenyl ring (such as when no E group is present).
While Y may be substituted in any of the 5 ring positions when W is a
phenyl moiety, Y is preferably mono-substituted in the 2'-position or 3'-
position,
with the 4'- preferably being unsubstituted. If the phenyl ring is
disubstituted, the
substituents are preferably in the 2' or 3' position of a monocyclic ring.
While both
~5 R I and Y can both be hydrogen, it is preferred that at least one of the
rings be
substituted, preferably both rings are substituted.
In compounds of Formula (I), A is suitably CH2, C(S) or C(S). It is noted
that in Formula (I) the A containing ring is saturated. In compounds of
Formula (II),
A is suitably CRIB. It is also noted that in Formula (II) the A containing
ring
30 contains unsaturation.
Suitably, R I g is hydrogen. C I _4 alkyl, aryl, arylC I -4 alkyl, heteroaryl,
heteroarylC I ~ alkyl, heterocyclic, or heterocyclicC I-4 alkyl, all of which
may be
optionally substituted.
35 R is suitably -NH -C(X~)-NH- (CR 13R 14)v - Z
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WO 00/05216 PCT/US98/15533
(CR, sR, s)P
Suitably, Z is W, HET, (fin , an optionally substituted C I _ 10
alkyl. an optionally substituted C~_ I0 alkenyl, or an optionally substituted
C~_ 10
alkynyl. Suitably p is an integer having a value of I to 3.
Y)n
*
Suitably, W is ~ , or
n
Suitably, the E containing ring is optionally selected from
o
(Y)n
* *
* f i
O ; (Y) : n~Y) / ~ or n~Y)
the asterix * denoting point of attachment of the ring.
n(Y)
~* i
t0 Preferably, Z is
The E ring, denoted by its point of attachment through the asterix (*) may
optionally be present. If it is not present, the ring is a phenyl moiety which
is
substituted by the Y terms as shown above. The E ring may be substituted by
the Y
moiety in any ring, saturated or unsaturated, and is shown for purposes herein
~5 substituted only in the unsaturated ring(s).
Suitably X2 is =O, or =S.
In compounds of Formula (I), X is suitably C(X I )2, N-R 1 g, O, C=O, or
S(O)m~, and m' is an integer having a value of I or 2. Preferably X is O, N-
Rlg,
2o C=O, or S(O)m~. More preferably, X is S(O)m . Most preferably X is S(O)m,
and
m' is 2, when A is CH2. When X is C(X 1 )2, both of X 1 can not be hydrogen.
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Preferably in compound of Formula (I} when X is C(X 1)2 then one of X 1 is
an electron withdrawing group such as a halosubstituted alkyl, such as CF3, or
C(O)NR~RS.
5 In compounds of Formula (II), X is suitably C(X 1 ) or N, preferably C(X 1
).
Suitably, X 1 is independently hydrogen, halogen, NRq,RS, C(O)NR4R5,
optionally substituted C 1 _ 10 alkyl, C 1-10 alkoxy, halosubstituted C 1 _ 10
alkoxy, aryl,
aryl C 1 _4 alkyl, aryloxy, aryl C 1 _4 alkyloxy, heteroaryl, heteroaryl C 1 ~
alkyl,
heterocyclic, heterocyclic C 1 _4alkyl; or heteroaryl C 1 ~ alkyloxy. The
alkyl group
10 may be optionally substituted one or more times by hydroxy, NR4R5, or
halogen.
Preferably, for compounds of Formula (I), when X is C(X 1 )z, at least one of
X 1 is
hydrogen.
For compounds of Formula (II}, X 1 is preferably hydrogen or an electron
withdrawing group, such as a halosubstituted alkyl, such as CF3, or C(O)NR4R5,
l5 HET is an optionally substituted heteroaryl moiety, as defined below both
for
optional substituents and for particular heteroaryl moieties.
Suitably R 15 and R 16 are independently hydrogen, or an optionally
substituted C 1 ~. alkyl as defined above for R 13 and R 14.
As used herein, "optionally substituted" unless specifically defined shall
20 mean such groups as halogen, such as fluorine, chlorine, bromine or iodine;
hydroxy; hydroxy substituted C1-lO~kYl; Cl-10 alkoxy, such as methoxy or
ethoxy;
S(O)m~~ C1-10 alkyl, wherein m" is 0, 1 or 2, such as methyl thio, methyl
sulfinyl or
methyl sulfonyl; amino, mono & di-substituted amino, such as in the NR4R5
group;
NHC(O)R4; C(O)NR4R5; C(O)OH; S(O)2NRq.RS; NHS(O)2R19; C1-10 ~kYl,
25 such as methyl, ethyl, propyl, isopropyl, or t-butyl; halosubstituted C1-10
~kYl, such
CF3; an optionally substituted aryl, such as phenyl, or an optionally
substituted
arylalkyl, such as benzyl or phenethyl; optionally substituted heterocylic;
optionally
substituted heterocyclicalkyl; optionally substituted heteroaryl; optionally
substituted heteroaryl alkyl; and wherein these aryl , heteroaryl, or
heterocyclic
30 containing moieties may be substituted one to two times by halogen,
hydroxy,
hydroxy substituted alkyl, C 1 _ 10 aikoxy, S(O)m~~C 1 _ 10 alkyl, amino, mono
& di-
substituted amino, such as in the NR4R5 group, C 1 _ 10 alkyl, or
halosubstituted
C 1-10 alkyl, such as CF3.
R 19 is suitably C 1 ~ alkyl, aryl, aryl C 1 _4alkyl, heteroaryl,
35 heteroarylC 1 _4alkyl, heterocyclic, or heterocyclicC 1 alkyl.
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Suitable pharmaceutically acceptable salts are well known to those skilled in
the art and include basic salts of inorganic and organic acids, such as
hydrochloric
acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic
acid,
ethane sulphonic acid, acetic acid. malic acid, tartaric acid, citric acid,
tactic acid,
5 oxalic acid, succinic acid, fumaric acid, malefic acid, benzoic acid,
salicylic acid,
phenylacetic acid and mandelic acid. In addition, pharmaceutically acceptable
salts
of compounds of Formula (I) may also be formed with a pharmaceutically
acceptable cation, for instance, if a substituent group comprises a carboxy
moiety.
Suitable pharmaceutically acceptable cations are well known to those skilled
in the
to art and include alkaline, alkaline earth, ammonium and quaternary ammonium
cations.
The following terms, as used herein, refer to:
"halo" - all halogens, that is chloro, fluoro, bromo and iodo.
"CI-IOalkYI" or "alkyl" - both straight and branched chain radicals of 1 to
i5 10 carbon atoms, unless the chain length is otherwise limited, including,
but not
limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-
butyl, tert-
butyl, n-pentyl and the like.
~ "cycloalkyl" is used herein to mean cyclic radicals, preferably of 3 to 8
carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl,
and the
20 like.
~ "alkenyl" is used herein at all occurrences to mean straight or branched
chain radical of 2-10 carbon atoms, unless the chain length is limited
thereto,
including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-
propenyl,
1-butenyl, 2-butenyl and the like.
?5 ~ "aryl" - phenyl and naphthyl;
"heteroaryl" (on its own or in any combination, such as "heteroaryloxy", or
"heteroaryl alkyl") - a 5-10 membered aromatic ring system in which one or
more
rings contain one or more heteroatoms selected from the group consisting of N,
O or
S, such as, but not limited, to pyrroIe, pyrazole, furan, thiophene,
quinoline,
30 isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole,
thiadiazole,
triazole, imidazole, or benzimidazole.
~ "heterocyclic" (on its own or in any combination, such as
"heterocyclicalkyl") - a saturated or partially unsaturated 4-10 membered ring
system in which one or more rings contain one or more heteroatoms selected
from
35 the group consisting of N, O, or S; such as, but not limited to,
pyrrolidine,
piperidine, piperazine, morpholine, tetrahydropyran, or imidazolidine.
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~ "arylalkyl" or "heteroarylalkyl" or "heterocyclicalkyl" is used herein to
mean C1-IO alkyl, as defined above, attached to an aryl, heteroaryl or
heterocyclic
moiety, as also defined herein, unless otherwise indicated.
~ "sulfinyl" - the oxide S (O) of the corresponding sulfide, the term "thio"
refers to the sulfide, and the term "sulfonyl" refers to the fully oxidized
S(O)2
moiety.
~ "wherein two Rl moieties (or two Y moieties) may together form a 5 or 6
membered saturated or unsaturated ring" is used herein to mean the formation
of a
bicyclo ring system or a phenyl moiety having attached a 5 or 6 membered
saturated
l0 or unsaturated ring such as a C6 cycloalkenyl, i.e. hexene, or a CS
cycloalkenyl
moiety, cyclopentene, or a fully unsaturated ring 5 or 6 membered ring such as
benzene, i.e. resulting in a naphtylene ring.
Exemplified compounds of Formula (I) include:
t5 N-(2-Bromophenyl) -N' -(1, 3-Dihydro-2, 2-dioxo-2,1-benzisothiazol-7-yl-
)urea
N-[( 1,3-dihydro-2 2-dioxo-4-floro-2,1-benzisothiazo)-7-ylJ-N'-(2-
bromophenyl)urea
N-[( 1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-ylJ-N'-(2,3-
dichlorophenyl)urea
20 N-[( 1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl]-N'-(2-
chlorophenyl)urea
N-[( 1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl]-N'-(2-
methoxyphenyl)urea
N-[( 1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl)-N'-isopropylurea
25 N-[( 1,3-Dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo}-7-yIJ-N'-
(2-bromophenyl)urea
N-[( 1,3-Dihydro-2,2-dioxo-4-cyano-2,1-benzisothiazo)-7-yl)-N'-
(2-bromophenyl)urea
N-[( 1,3-Dihydro-2,2-dioxo-4-bromo-2,1-benzisothiazo)-7-ylJ-N'-
30 (2-bromophenyl)urea
Additional exemplified compounds of Formula (I) include:
N-(4-Bromophenyl) -N' -( 1, 3-Dihydro-2, 2-dioxo-2,1-benzisothiazol-7-yl-)urea
35 Exemplified compounds of Formula (II) include:
N-(2-indazole)-N'-(2-bromophenyl)urea
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Methods of Preparation
The compounds of Formula (I) and (II) may be obtained by applying
synthetic procedures, some of which are illustrated in the Schemes below. The
synthesis provided for in these Schemes is applicable for the producing of
Formula
(I) and (II) having a variety of different Z, R1, and E groups which are
reacted,
employing optional substituents which are suitably protected to achieve
compatibility with the reactions outlined herein. Subsequent deprotection, in
those
cases, then affords compounds of the nature generally disclosed. Once the urea
to nucleus has been established, further compounds of these formulas may be
prepared
by applying standard techniques for functional group interconversion, well
known in
the art. While the schemes are shown with various compounds of Formula (I) and
(II) this is merely for illustration purposes only and not a limitation on the
extent of
synthesis available using these methods.
S02C1 /
N. ,O _ a,b ~ ( ~ S02
i N
O- H
1
a)SnCl2, EtOAc b)Et3N, CHZC12
If the desired cyclic sulfonamide 2-scheme I is not commercially available
the corresponding heterocyclic sulfamide can be prepared from the commercially
20 available compound I-scheme 1 with reduction of the vitro group using
suitable
reducing conditions such as SnCh, hydrogen and Pd/C or zinc metal in a
suitable
organic solvent such as, ethyl acetate and cyclization using triethylamine in
methylene chloride. Alternative reaction conditions for the cyclic sulfonamide
2-
scheme 1 can be achieved by cyclization of 2-chlorobenzylsulfonamide with
25 potassium carbonate and copper-bronze powder in under pressure using a
polar
solvent or using a high boiling solvent such as 2, 3-dimethylaniline at 100-
180oC,
preferably about 180°C, or by reacting the 2-aminobenzylsulfonic acid
sodium satt
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with phosphorus oxychIoride at 30-I70oc, preferably about 170°C, for
between I-24
hours, preferably about 3 hours.
O
OH
NH~
NH
O
2
a) Ph3P oxide, Et3N, TFAA, 1,2 dichloroethane
If the desired heterocyclic compound 2-scheme 2 is not commercially
available then it can be prepared from the commercially available compound j_
scheme 2 with triphenyl phosphine oxide, triethylamine, trifluoroacetic
anhydride in
an aprotic solvent such as 1, 2 - dichloroethane.
~h_eme 3
O
O
~OH
N~ :O
I \ NHO
2
a) Zn, NH4C1, THF/H20
l0
If the desired heterocyclic compound 2-scheme 3 is not commercially
available then it can be prepared from the commercially available compound 1-
sc with zinc and ammonium chloride in THF/H20.
t5
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Sc eme 4
O
~H
a
Nt :O --t ~ NHO
I.
O
1
2
a) Aluminum amalgam, Et20
If the desired heterocyclic compound 2-scheme 4 is not commercially
available then it can be prepared from the commercially available compound ~
scheme 4 with aluminum amalgam in diethyl ether or THF.
5
OH ~ ~ ( . OH - c ~ / ~
NH2 \ NH ~ NH NH
I
NHZ
1 2
a) HONG b) Na2N03 c)H2S04
If the desired heterocyclic compound 3-scheme 5 is not commercially
available then it can be prepared from the commercially available compound 1-
10 ,s~heme,~ with nitrous acid to the diazonium salt, followed by treating
with sodium
sulfite to the hydrazine 2-scheme ~. Cyclization can be achieved with sulfuric
acid
to give 3-scheme 5. An alternative method to obtain 3-scheme 5 would be
reduction
of indazolinone with lithium aluminum hydride.
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_ a b
\ ~ . SOZ ~ \ ( SO ~ / ( SO
~N N~ z \ N.
H H H
O .N; O ~ NH2
a) NaN03, 3M HzS04, CH2CI2, 23°C b) Pd/C, MeOH
If the desired aniline 3-scheme 6 is not commercially available the
corresponding nitro compound can be prepared from 1-scheme 6, under standard
nitration conditions (using HN03 or NaN03) at 0-100oC, preferably about
23°C,
under acid conditions such as acetic acid, acetic anhydride, or under biphasic
condition such as aqueous sulphuric acid and a chlorinated solvent such as
methylene chloride. The nitro compound is then reduced to the corresponding
aniline using suitable redut;ing agents, such as H2/Pd in an organic solvent,
such as
1o MeOH, DMF or ethylacetate (alternately SnCl2 in EtOH, or LiAlH4 or zinc
metal in
acetic acid ) at 0-100oC. Other commercially available heterocylic compounds
such
as Indoline, Indole, Oxindole, Isatin, Indazole and Indazolinone can be
converted to
the desired anilines through this procedure.
l5
i ~ ~ S02
NHSOZ - a / I NHO
NH2 \ NH~NH
1
a) PhNCO, DMF, 80°C
Ortho substituted heterocyclic phenyl ureas in 2-scheme 7 may be prepared
by standard conditions involving the condensation of the commercially
available
20 optionally substituted aryl isocyanate(Aldrich Chemical Co., Milwaukee, Wi)
with
the corresponding aniline 1-scheme 7 in an aprotic solvent such as DMSO, DMF,
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toluene or methylene chloride at room temperature or elevated temperature from
I h
to 24 h.
Alternatively the desired isocyanates can be made by condensing the amine
with triphosgene in the presence of base (such as potassium carbonate) or by
reacting the carboxylic acid with diphenyl phosphoazide in the presence of a
base
(such as triethyl amine).
The aromatic rings of the compounds can be further functionalized by
conditions well known in the art such as bromination or other electrophilic
substitution reactions. These substituents can be further manipulated using
standard
to nucleophilic substitutions such as reaction with an anion (such as sodium
methoxide), or in palladium catalyzed coupling reaction chemistry.
Another aspect of the invention is the novel process for making compounds
of Formula (I), in particularly when A is CH2 and X is S(O)m which process
~ 5 comprises a process for producing a compound of Formula (I), as defined
above,
wherein A is CH2 and X i's S(O)m, which process comprises
a) reacting a compound of the formula
(R,)m ~ I
\ N . SOZ
H
NH2 (A)
20 with a compound of the formula:
C(X~)-N- (CR13R14)v - Z
to yield a compound of Formula (I).
25 Another aspect of the present invention are the novel compounds of Formula
(A) and the novel process to make compounds of Formula (A) which process
comprises reacting the corresponding nitro compound, Formula (B) under
conventional reducing conditions to yield the corresponding aniline
derivative.
30 Another aspect of the present invention is the novel process of producing
novel compounds of the formula:
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(R,)m /
\ ~ N~SOx
H
..N .
O . O (B)
which process comprises reacting a compound of the formula
(R,)m /
\ N . SOz
H (C)
under standard nitrating conditions to yield a compound of Formula (B).
Yet another aspect of the present invention is a process for producing a
compound of the Formula (C), which process comprises reacting a compound of
the
formula
/ ~ S02CI
\ ~ N+-.O
i_
O (D)
to under reducing conditions and cyclization of the corresponding reduced
amine to
yield a compound of Formula (D).
SYNTHETIC EXAMPLES
The invention will now be described by reference to the following examples
which
15 are merely illustrative and are not to be construed as a limitation of the
scope of the present
invention. All temperatures are given in degrees centigrade, all solvents are
highest
available purity and all reactions run under anhydrous conditions in an argon
atmosphere
unless otherwise indicated.
In the Examples, all temperatures are in degrees Centigrade (°C). Mass
spectra were
2o performed upon a VG Zab mass spectrometer using fast atom bombardment,
unless
otherwise indicated. 1 H-NMR (hereinafter "NMR") spectra were recorded at 250
MHz
using a Bruker AM 250 or Am 400 spectrometer. Multiplicities indicated are:
s=singles,
d=doublet, t=triplet, q=quartet, m=multiplet and br indicates a broad signal.
Sat. indicates a
saturated solution, eq indicates the proportion of a molar equivalent of
reagent relative to
25 the principal reactant.
General Method: Synthesis of N, N'- phenyl urea To a solution of phenyl
isocyanate( 1.0 equiv.) in dimethyl formamide ( 1 ml) the corresponding
aniline ( 1.0
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WO 00/05216 PCT/US98/15533
equiv.) was added. The reaction mixture was stirred at 80°C until
complete (3-16
hrs.), then removed solvent under vacuum. The purification, yields and
spectral
characteristics for each individual compound are listed below. Additional
synthetic
methods are provided for in PCT US96/02260 filed 16 February 1996 whose
5 disclosure is incorporated herein by reference.
el
Preparation of N-l2-Bromophenyll -N' -( 1. 3-Di~ydro-2. 2-dioxo-2.1-
(~enzisothiazol-
7- - urea '
to a)Preparation of 1, 3-Dihydro-1, ?-benzisothiazole-2, 2-dioxide
To a solution of 2-vitro-alpha-toluenesulfonyl chloride(S.Og, 2 I.3mmo1) in
250m1 of ethyl acetate was added tin II chloride( 19.2g, 85mmo1). The reaction
was
stirred at 70oC overnight then poured into ice and neutralized with sodium
bicarbonate. The solution was then extracted with ethyl acetate and the
solvents
~ 5 were evaporated. The crude reaction mixture was then diluted with
methylene
chloride and excess triethyl amine was added. The solution was stirred at 25oC
overnight and the solvent was evaporated and the product was obtained by
chromatography of the resulting solid on silica gel(EtOAc/
hexane(lequiv./lequiv.)). (500mg, 14%). 'H NMR (CDCl3): 87.25 (d, IH), 7.24
20 (t, 1 H), 7.07 (t, 1 H), 6.91 (d, 1 H), 6.62 (s, I H), 4.40 (s, 2H).
b)Preparation of 4-vitro-1, 3-Dihydro-1, 2-benzisothiazole-2, 2-dioxide
1, 3-Dihydro-1, 2-benzisothiazole-2, 2-dioxide (400mg, 2.40mmol) was
dissolved in methylene chloride(40m1) followed by the addition of sodium
nitrate
(0.22g, 2.60mmo1). The addition of sulfuric acid (S.OmI/ 3M) is then made,
25 followed by addition of a catalytic amount of sodium nitrite. The mixture
is allowed
to stir. After 24 hr., the reaction mixture is diluted with methylene chloride
and
extracted with water. The organic layer is dried over MgSO, and filtered. The
solvent was evaporated and chromatography of the resulting solid on silica gel
(4%MeOH/ CH,CI=) gave the desired product( 150 mg, 29 %). 'H NMR (CD30D): 8
30 8.1 I (d, l H), 7.57 d, I H), 7.09 (t, 1 H), 4.50 (s, 2H).
c)Preparation of 4-amino-l, 3-Dihydro-1, 2-benzisothiazole-2, 2-dioxide
To a solution of 4-vitro-1, 3-Dihydro-l, 2-benzisothiazole-2, 2-dioxide
( 100mg, S.Ommol} in methanol (50m1) and was added 10% Pd/C (50mg). The
mixture was flushed with argon, then hydrogen was bubbled through the solution
for
35 10 min. and a hydrogen atmosphere was maintained at balloon pressure
overnight.
The mixture was filtered through celite and the celite was washed with
methanol.
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WO 00/05216 PCT/US98/15533
The solvent was evaporated and chromatography of the resulting solid on silica
gel
( 10%MeOH/ CH=CL) gave the desired product (64mg, 74 %). 'H NMR (CD,OD): 8
6.86 (t, IH), 6.62 (d, IH), 6.55 (d, IH), 4.35 (s, 2H).
d)Preparation of N-[1,3-Dihydro-I, 2-benzisothiazole-3, 3-dioxide]-N'-[2-
bromophenyl] urea
N-[ 1,3-Dihydro-1, 2-benzisothiazole-3, 3-dioxide]-N'-[2-bromophenyl] urea
was prepared from 4-amino-1, 3-Dihydro-1, 2-benzisothiazole-2, 2-dioxide
(64mg,
0.35 mmol) according to the procedure in General Method B. The product was
purified by chromatography of the resulting solid on silica gel(EtOAc/ hexane
'
to ( lequiv./lequiv.)). (45mg, 34%). 'H NMR (CD3S02CD3): b 9.70 (s, IH), 9.04
(s,
IH), 8.51 (s, 1H), 8.08 (d,lH), 7.67 (t, 1H), 7.60 (d, 1H), 7.34 (t, 1H), 7.04
(d, 2H),
6.96 (t, IH), 4.58 (s, 2H).
Using analogous methods to those described above or in the schematics, the
is following compounds may be synthesized:
Example 2: N-(l, 3-Dihydro-4-bromo-I, 2-benzisothiazole-3, 3-dioxide) N'-
phenylurea
Example 3: N-[(1,3-dihydro-2,2-dioxo-4-floro-2,1-benzisothiazo)-7-yl]-N'-(2-
bromophenyl)urea; (M-: 398.1, 400.1)
20 Example 4: N-[( 1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl]-N'-
(2,3-
dichlorophenyl)urea; (M- : 403.9, 406.2, 408.0)
Example 5: N-[( 1,3-dihydro-2,2-dioxo-4.-chloro-2,1-benzisothiazo)-7-ylJ-N'-(2-
chlorophenyl)urea; (M- : 370.1, 372.1)
Example 6: N-[(1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl]-N'-(2-
25 methoxyphenyl)urea; (M- : 366.2, 358.1)
Example 7: N-[(1,3-dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl]-N'-
isopropylurea; (M- : 304.1, 306.2)
Example 8: N-[( 1,3-Dihydro-2,2-dioxo-4-chloro-2,1-benzisothiazo)-7-yl]-N'-
(2-bromophenyl)urea: (M- : 414, 416)
3o Example 9: N-[(1,3-Dihydro-2,2-dioxo-4-cyano-2,1-benzisothiazo)-7-yl]-N'-
(2-bromophenyl)urea; (M- : 404.9, 407.1)
Example 10: N-[(1,3-Dihydro-2,2-dioxo-4-bromo-2,1-benzisothiazo)-7-yl]-N'-
{2-bromophenyl)urea: (M- : 457.9, 459.9, 461.9)
Example 1 I: N-(2-indazole)-N'-(2-bromophenyl)urea; MS (M +H = 331.1; M-H =
35 329.2)
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iVIETHOD OF TREATMENT
The compounds of Formula (I), and (II) or a pharmaceutically acceptable salt
thereof can be used in the manufacture of a medicament for the prophylactic or
therapeutic treatment of any disease state in a human, or other mammal, which
is
exacerbated or caused by excessive or unregulated IL,-8 cytokine production by
such
mammal's cell, such as but not limited to monocytes and/or macrophages, or
other
chemokines which bind to the IL-8 a or ~i receptor, also referred to as the
type I or
type II receptor.
For purposes herein, the term Formula (I) will also refer to compounds of
to Formula (II) unless otherwise indicated.
Accordingly, the present invention provides a method of treating a
chemokine mediated disease, wherein the chemokine is one which binds to an IL-
8
a or ~3 receptor and which method comprises administering an effective amount
of a
compound of Formula (I) or a pharmaceutically acceptable salt thereof. In
~5 particular, the chemokines are 1L-8. GROG, GROG, GROy, NAP-2 or ENA-78.
The compounds of Formula (I) are administered in an amount sufficient to
inhibit cytokine function, in particular IL-8, GROG, GRO~i, GROy, NAP-2 or
ENA-78, such that they are biologically regulated down to normal levels of
physiological function, or in some case to subnormal levels, so as to
ameliorate the
2o disease state. Abnormal levels of IL-8, GROG, GRO~i, GRO~y, NAP-2 or ENA-78
for instance in the context of the present invention, constitute: (i) levels
of free IL-8
greater than or equal to 1 picogram per mL; (ii) any cell associated IL,-8,
GROG,
GR0~3, GROy, NAP-2 or ENA-78 above normal physiological levels; or (iii) the
presence IL-8, GROG, GROG, GROy, NAP-2 or ENA-78 above basal levels in cells
25 or tissues in which IL-8, GROa, GROG, GROy, NAP-2 or ENA-78 respectively,
is
produced.
There are many disease states in which excessive or unregulated IL-8
production is implicated in exacerbating and/or causing the disease. Chemokine
mediated diseases include psoriasis, atopic dermatitis, arthritis, asthma,
chronic
30 obstructive pulmonary disease, adult respiratory distress syndrome,
inflammatory
bowel disease, Crohn's disease, ulcerative colitis, stroke, septic shock,
endotoxic
shock, gram negative sepsis, toxic shock syndrome, cardiac and renal
reperfusion
injury, glomerulonephritis, thrombosis, graft vs. host reaction, Alzheimer's
disease,
allograft rejections, malaria, restinosis, angiogenesis or undesired
hematopoietic
35 stem cells release, rhinovirus infections, and various bone resorptive
indications,
such as osteoporosis or osteoarthritis.
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The association of interleukin-8 and rhinovirus may be found in articles such
as: Tumer,et al., Clin. Infect. Dis. ( 1998), 26(4), 840-846; Sanders, et al.,
J. Virol.
( 1998), 72(2), 934-942; Sethi, et al., Clin. Exp. Immunol. ( 1997), 110(3),
362-369;
Zhu, et al., Am. J. Physiol. (1997), 273(4, Pt. 1), L814-L824; Terajima, et
al., Am. J.
Physiol. ( 1997), 273(4, Pt. 1), L749-L759; Grunberg, et al., Clin. Exp.
Allergy
( 1997), 27( 1 ), 36-45; and Johnston, et aL, J. Infect. Dis. ( 1997), 175(2),
323-329.
The association of interleukin-8 and osteoporosis may be found in articles
such as: Streckfus et al., J. Gerontol., Ser. A (1997), 52A(6), M343-M351;
Hermann, T. WO 95/31722; and Chaudhary, et al., Endocrinology (Baltimore)
t0 (1992), 130(5), 2528-34.
These diseases are primarily characterized by massive neutrophil infiltration,
T-cell infiltration, or neovascular growth, and are associated with IL-8,
GROG,
GROG, GROy, NAP-2 or ENA-78 production which is responsible for the
chemotaxis of neutrophils into the inflammatory site or the directional growth
of
t5 endothelial cells. In contrast to other inflammatory cytokines (IL-8, GROa,
GROG,
GROy, or NAP-2) has the unique property of promoting neutrophil chemotaxis,
enzyme release including but not limited to elastase release as well as
superoxide
production and activation. The a-chemokines but particularly, GROG. GROG,
GROy, or NAP-2, working through the IL-8 type I or II receptor can promote the
20 neovascularization of tumors by promoting the directional growth of
endothelial
cells. Therefore, the inhibition of IL-8 induced chemotaxis or activation
would lead
to a direct reduction in the neutrophil infiltration.
Recent evidence also implicates the role of chemokines in the treatment of
HIV infections, Littleman et al., Nature 381, pp. 661 ( 1996) and Koup et al.,
Nature
25 38 l, pp. 667 ( 1996).
The present invention also provides for a means of treating, in an acute
setting, as
well as preventing, in those individuals deemed susceptible to, CNS injuries
by the
chemokine receptor antagonist compounds of Formula (I).
CNS injuries as defined herein include both open or penetrating head trauma,
3o such as by surgery, or a closed head trauma injury, such as by an injury to
the head
region. Also included within this definition is ischemic stroke, particularly
to the
brain area.
Ischemic stroke may be defined as a focal neurologic disorder that results
from insufficient blood supply to a particular brain area, usually as a
consequence of
35 an embolus, thrombi, or local atheromatous closure of the blood vessel. The
role of
inflammatory cytokines in this are has been emerging and the present invention
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provides a mean for the potential treatment of these injuries. Relatively
little
treatment, for an acute injury such as these has been available.
TNF-a is a cytokine with proinflammatory actions, including endothelial
leukocyte adhesion molecule expression. Leukocytes infiltrate into ischemic
brain
lesions and hence compounds which inhibit or decrease levels of TNF would be
useful for treatment of ischemic brain injury. See Liu et al., Stoke, Vol.
25., No. 7,
pp. 1481-88 (1994) whose disclosure is incorporated herein by reference.
Models of closed head injuries and treatment with mixed 5-LOCO agents is
discussed in Shohami et al., J. of Vaisc & Clinical Physiology and
Pharmacology,
io Vol. 3, No. 2, pp. 99-I07 (1992) whose disclosure is incorporated herein by
reference. Treatment which reduced edema formation was found to improve
functional outcome in those animals treated.
Present evidence also indicates the use of IL-8 inhibitors in the treatment of
atherosclerosis. The first reference, Boisvert et al., J Clin Invest, 1998,
101:353-363
~5 shows, through bone marrow transplantation, that the absence of IL-8
receptors on
stem cells (and, therefore, on monocytes/macrophages) leads to a reduction in
the
development of atherosclerotic plaques in LDL receptor deficient mice.
Additinal
supporting references are: Apostolopoulos, et al., Arterioscler Thromb Vasc
Biol.
1996, 16:1007-1012: Liu, et al., Arterioscler Thromb Vasc Biol, 1997, 17:317-
323;
2o Rus, et al., Atherosclerosis. 1996, 127:263-271.; Wang et al., J Biol Chem.
1996,
271:8837-8842: Yue, et aL, Eur J Pharmacol. 1993, 240:81-84; Koch, et al.. Am
J
Pathol, 1993, 142:1423-1431.; Lee, et al., Immunol Lett, 1996, 53, 109-113.:
and
Terkeltaub et al., Arterioscler Thromb, 1994, 14:47-53.
The compounds of Formula (I) are administered in an amount sufficient to
25 inhibit IL-8, binding to the IL-8 alpha or beta receptors, from binding to
these
receptors, such as evidenced by a reduction in neutrophil chemotaxis and
activation.
The discovery that the compounds of Formula (I) are inhibitors of IL-8 binding
is
based upon the effects of the compounds of Formulas (I) in the in vitro
receptor
binding assays which are described herein. The compounds of Formula (I) have
30 been shown to be inhibitors of type II IL-8 receptors.
As used herein, the term "IL-8 mediated disease or disease state" refers to
any and all disease states in which IL-8, GROG, GRO~i, GROy, NAP-2 or ENA-78
plays a role, either by production of IL-8, GROG, GROø, GROy, NAP-2 or ENA-78
themselves, or by IL-8, GROa, GR0~3, GRO~y, NAP-2 or ENA-78 causing another
35 monokine to be released, such as but not limited to IL-1, IL-6 or TNF. A
disease
state in which, for instance, IL-1 is a major component, and whose production
or
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WO 00/05216 PCT/US98I15533
action, is exacerbated or secreted in response to IL-8. would therefore be
considered
a disease stated mediated by IL-8.
As used herein, the term "chemokine mediated disease or disease state" refers
to any and all disease states in which a chemokine which binds to an IL-8 a or
[3
5 receptor plays a role, such as but not limited IL-8, GROa, GR0~3, GROy, NAP-
2 or
ENA-78. This would include a disease state in which, IL-8 plays a role, either
by
production of IL-8 itself, or by IL-8 causing another monokine to be released,
such
as but not limited to IL-l, IL-6 or TNF. A disease state in which, for
instance, IL.-1
is a major component, and whose production or action, is exacerbated or
secreted in
1o response to IL-8, would therefore be considered a disease stated mediated
by IL-8.
As used herein, the term "cytokine" refers to any secreted polypeptide that
affects the functions of cells and is a molecule which modulates interactions
between
cells in the immune, inflammatory or h.: -~atopoietic response. A cytokine
includes,
but is not limited to, monokines and lymphokines, regardless of which cells
produce
t5 them. For instance, a monokine is generally referred to as being produced
and
secreted by a mononuclear cell, such as a macrophage andlor monocyte. Many
other
cells however also produce monokines, such as natural killer cells,
fibroblasts,
basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow
stromal cells,
epideral keratinocytes and B-lymphocytes. Lymphokines are generally referred
to as
2o being produced by lymphocyte cells. Examples of cytokines include, but are
not
limited to, Interleukin-1 (IL-1 ), Interleukin-6 (IL.-6), Interleukin-8 (1I,-
8), Tumor
Necrosis Factor-alpha (TNF-a) and Tumor Necrosis Factor beta (TNF-Li).
As used herein, the term "chemokine" refers to any secreted polypeptide that
affects the functions of cells and is a molecule which modulates interactions
between
25 cells in the immune, inflammatory or hematopoietic response, similar to the
term
"cytokine" above. A chemokine is primarily secreted through cell
transmembranes
and causes chemotaxis and activation of specific white blood cells and
leukocytes,
neutrophils, monocytes, macrophages, T-cells, B-cells, endothelial cells and
smooth
muscle cells. Examples of chemokines include, but are not limited to, IL-8,
GRO-a,
30 GRO-~3, GRO-'y, NAP-2, ENA-78, IP-10, MIP-1 a, MIP-~3, PF4, and MCP 1, 2,
and 3.
In order to use a compound of Formula (I) or a pharmaceutically acceptable
salt thereof in therapy, it will normally be formulated into a pharmaceutical
composition in accordance with standard pharmaceutical practice. This
invention,
therefore, also relates to a pharmaceutical composition comprising an
effective, non-
35 toxic amount of a compound of Formula (I) and a pharmaceutically acceptable
carrier or diluent.
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Compounds of Formula (I), pharmaceutically acceptable salts thereof and
pharmaceutical compositions incorporating such may conveniently be
administered
by any of the routes conventionally used for drug administration, for
instance, orally,
topically, parenterally or by inhalation. The compounds of Formula (I) may be
5 administered in conventional dosage forms prepared by combining a compound
of
Formula (I) with standard pharmaceutical carriers according to conventional
procedures. The compounds of Formula (I) may also be administered in
conventional dosages in combination with a known, second therapeutically
active
compound. These procedures may involve mixing, granulating and compressing or
t0 dissolving the ingredients as appropriate to the desired preparation. It
will be
appreciated that the form and character of the pharmaceutically acceptable
character
or diluent is dictated by the amount of active ingredient with which it is to
be
combined, the route of administration and other well-known variables. The
carriers) must be "acceptable" in the sense of being compatible with the other
t5 ingredients of the formulation and not deleterious to the recipient
thereof.
The pharmaceutical carrier employed may be, for example, either a solid or
liquid. Exemplary of solid carriers are lactose, terra albs, sucrose, talc,
gelatin, agar,
pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of
liquid
carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the
carrier or
2o diluent may include time delay material well known to the art, such as
glyceryl
mono-stearate or glyceryl distearate alone or with a wax.
A wide variety of pharmaceutical forms can be employed. Thus, if a solid
carrier is used, the preparation can be tableted, placed in a hard gelatin
capsule in
powder or pellet form or in the form of a troche or lozenge. The amount of
solid
25 carrier will vary widely but preferably will be from about 25mg. to about
lg. When
a liquid carrier is used, the preparation will be in the form of a syrup,
emulsion, soft
gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous
liquid
suspension.
Compounds of Formula (I) may be administered topically, that is by non-
3o systemic administration. This includes the application of a compound of
Formula (I)
externally to the epidermis ~or the buccal cavity and the instillation of such
a
compound into the ear, eye and nose, such that the compound does not
significantly
enter the blood stream. In contrast, systemic administration refers to oral,
intravenous, intraperitoneal and intramuscular administration.
35 Formulations suitable for topical administration include liquid or semi-
liquid
preparations suitable for penetration through the skin to the site of
inflammation
_2g.
CA 02338741 2001-O1-23
WO 00/05216 PCT/US98/15533
such as liniments, lotions, creams, ointments or pastes, and drops suitable
for
administration to the eye, ear or nose. The active ingredient may comprise,
for
topical administration, from 0.001 % to 10% w/w, for instance from 1 % to 2%
by
weight of the formulation. It may however comprise as much as 10% w/w but
preferably will comprise less than 5% w/w, more preferably from 0.1 % to 1 %
w/w
of the formulation.
Lotions according to the present invention include those suitable for
application to the skin or eye. An eye lotion may comprise a sterile aqueous
solution
optionally containing a bactericide and may be prepared by methods similar to
those
I o for the preparation of drops. Lotions or liniments for application to the
skin may also
include an agent to hasten drying and to cool the skin, such as an alcohol or
acetone,
and/or a moisturizer such as glycerol or an oil such as castor oil or arachis
oil.
Creams. ointments or pastes according to the present invention are semi-solid
formulations of the active ingredient for external application. They may be
made by
15 mixing the active ingredient in finely-divided or powdered form, alone or
in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of suitable
machinery, with a greasy or non-greasy base. The base may comprise
hydrocarbons
such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a
mucilage;
an oil of natural origin such as almond, corn, arachis, castor or olive oil;
wool fat or
2o its derivatives or a fatty acid such as steric or oleic acid together with
an alcohol
such as propylene glycol or a macrogel. The formulation may incorporate any
suitable surface active agent such as an anionic, cationic or non-ionic
surfactant such
as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents
such
as natural gums, cellulose derivatives or inorganic materials such as
silicaceous
25 silicas, and other ingredients such as lanolin, may also be included.
Drops according to the present invention may comprise sterile aqueous or
oily solutions or suspensions and may be prepared by dissolving the active
ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal
agent
and/or any other suitable preservative, and preferably including a surface
active
30 agent. The resulting solution may then be clarified by filtration,
transferred to a
suitable container which is then sealed and sterilized by autoclaving or
maintaining
at 98-100 oC. for half an hour. Alternatively, the solution may be sterilized
by
filtration and transferred to the container by an aseptic technique. Examples
of
bactericidal and fungicidal agents suitable for inclusion in the drops are
35 phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01 %)
and
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WO 00/05216 PCT/US98/15533
chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an
oily
solution include glycerol, diluted alcohol and propylene glycol.
Compounds of formula (I) may be administered parenterally, that is by
intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal
or
intraperitoneal administration. The subcutaneous and intramuscular forms of
parenteral administration are generally preferred. Appropriate dosage forms
for such
administration may be prepared by conventional techniques. Compounds of
Formula (I) may also be administered by inhalation, that is by intranasal and
oral
inhalation administration. Appropriate dosage forms for such administration,
such
t0 as an aerosol formulation or a metered dose inhaler, may be prepared by
conventional techniques.
For all methods of use disclosed herein for the compounds of Formula (I),
the daily oral dosage regimen will preferably be from about 0.01 to about 80
mg/kg
of total body weight. The daily parenteral dosage regimen about 0.001 to about
80
15 mg/kg of total body weight. The daily topical dosage regimen will
preferably be
from 0.1 mg to 150 mg, administered one to four, preferably two or three times
daily. The daily inhalation dosage regimen will preferably be from about 0.01
mg/kg to about 1 mg/kg per day. It will also be recognized by one of skill in
the art
that the optimal quantity and spacing of individual dosages of a compound of
20 Formula (I) or a pharmaceutically acceptable salt thereof will be
determined by the
nature and extent of the condition being treated, the form, route and site of
administration, and the particular patient being treated, and that such
optimums can
be determined by conventional techniques. It will also be appreciated by one
of skill
in the art that the optimal course of treatment, i.e., the number of doses of
a
25 compound of Formula (I) or a pharmaceutically acceptable salt thereof given
per day
for a defined number of days, can be ascertained by those skilled in the art
using
conventional course of treatment determination tests.
The invention will now be described by reference to the following biological
30 examples which are merely illustrative and are not to be construed as a
limitation of
the scope of the present invention.
BIOLOGICAL EXAMPLES
The IL-8, and GRO-a chemokine inhibitory effects of compounds of the
present invention are determined by the following in vitro assay:
35 Receptor Binding Assays:
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~ 135I~ u,_g (human recombinant) is obtained from Amersham Corp.,
Arlington Heights. IL, with specific activity 2000 Ci/mmol. Gro-a is obtained
from
NEN- New England Nuclear. All other chemicals are of analytical grade. High
levels of recombinant human IL-8 type a and (3 receptors were individually
expressed in Chinese hamster ovary cells as described previously (Holmes, et
al.,
Science, 1991, 253, 1278). The Chinese hamster ovary membranes were
homogenized according to a previously described protocol (Haour, et al., J
Biol
Chem., 249 pp 2195-2205 ( 1974)). Except that the homogenization buffer is
changed to IOmM Tris-HCL, ImVI MgS04, O.SmM EDTA (ethylene-diaminetetra-
to acetic acid), Im MPMSF (a-toluenesulphonyl fluoride), 0.5 mg/L Leupeptin,
pH
7.5. Membrane protein concentration is determined using Pierce Co. micro-assay
kit
using bovine serum albumin as a standard. All assays are performed in a 96-
well
micro plate format. Each reaction mixture contains 125I IL-8 (0.25 nM) or 125I
GRO-a and 0.5 trg/mL of IL-8Ra or 1.0 pg/mL of IL-8R(3 membranes in 20 mM
t5 Bis-Trispropane and 0.4 mM Tris HCI buffers, pH 8.0, containing 1.2 mM
MgS04,
0. I mM EDTA, 25 mM NaCI and 0.03010 CHAPS. In addition, drug or compound of
interest is added which has been pre-dissolved in DMSO so as to reach a final
concentration of between O.OlnN1 and 100 uM. The assay is initiated by
addition of
125I_IL-8. After 1 hour at room temperature the plate is harvested using a
Tomtec
20 96-well harvester onto a glass fiber filtermat blocked with 1 %
polyethylenimine/
0.5% BSA and washed 3 times with 25 mM NaCI, 10 mM TrisHCl> 1 mM MgS04,
0.5 mM EDTA, 0.03 % CHAPS, pH 7.4. The filter is then dried and counted on the
Betaplate liquid scintillation counter. The recombinant IL-8 Ra, or Type I.
receptor
is also referred to herein as the non-permissive receptor and the recombinant
IL-8
25 R(3, or Type II, receptor is referred to as the permissive receptor.
Representative compounds of Formula (I), Example 1, 3 to 10, and a
representative compound of Formula (II), Example 11, have been found to have
positive inhibitory activity of < 30 pmg in this assay.
3o Chemotaxi~ Assav
The in vitro inhibitory properties of these compounds are determined in the
neutrophil chemotaxis assay as described in Current Protocols in Immunology,
vol.
I, Suppl 1, Unit 6.12.3., whose disclosure is incorporated herein by reference
in its
entirety. Neutrophils where isolated from human blood as described in Current
35 Protocols in Immunology Vol. I, Suppl 1 Unit 7.23.1, whose disclosure is
incorporated herein by reference in its entirety. The chemoattractants IL-8,
GRO-a,
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WO 00/05216 PCT/US98/15533
GRO-~3, GRO-y and NAP-2 are placed in the bottom chamber of a 48 multiwell
chamber (Neuro Probe, Cabin John. MD) at a concentration between 0.1 and 100
nM. The two chambers are separated by a Sum polycarbonate filter. When
compounds of this invention are tested, they are mixed with the cells (0.001 -
1000
5 nM) just prior to the addition of the cells to the upper chamber. Incubation
is
allowed to proceed for between about 45 and 90 min. at about 37oC in a
humidified
incubator with 5% C02. At the end of the incubation period, the polycarbonate
membrane is removed and the top side washed, the membrane then stained using
the
Diff Quick staining protocol (Baxter Products, McGaw Park, IL, USA). Cells
which
to have chemotaxed to the chemokine are visually counted using a microscope.
Generally, four fields are counted for each sample, these numbers are averaged
to
give the average number of cells which had migrated. Each sample is tested in
triplicate and each compound repeated at least four times. To certain cells
(positive
control cells) no compound is added, these cells represent the maximum
chemotactic
15 response of the cells. In the case where a negative control (unstimulated)
is desired,
no chemokine is added to the bottom chamber. The difference between the
positive
control and the negative control represents the chemotactic activity of the
cells.
;~~stase Release Assav:
2o The compounds of this invention are tested for their ability to prevent
Elastase release from human neutrophils. Neutrophils are isolated from human
blood as described in Current Protocols in Immunology Vol. I, Suppl 1 Unit
7.23.1.
PMNs 0.88 x 106 cells suspended in Ringer's Solution (NaCI 118, KCl 4.56,
NaHC03 25, KH2P04 1.03, Glucose 11.1, HEPES 5 mM, pH 7.4) are placed in
25 each well of a 96 well plate in a volume of 50 ul. To this plate is added
the test
compound (0.001 - 1000 nM) in a volume of 50 ul, Cytochalasin B in a volume of
50 ul (20ug/ml) and Ringers buffer in a volume of SO uI. These cells are
allowed to
warm (37 oC, 5% C02, 95% RH) for 5 min. before IL-8, GROG, GROG, GROy or
NAP-2 at a final concentration of 0.01 - 1000 nM was added. The reaction is
3o allowed to proceed for 45 min. before the 96 well plate is centrifuged (800
xg 5
min.) and 100 ul of the supernatant removed. This supernatant is added to a
second
96 well plate followed by an artificial elastase substrate (MeOSuc-Ala-Ala-Pro-
Val-
AMC, Nova Biochem, La Jolla, CA) to a final concentration of 6 ug/ml dissolved
in
phosphate buffered saline. Immediately, the plate is placed in a fluorescent
96 well
35 plate reader (Cytofluor 2350, Millipore, Bedford, MA) and data collected at
3 min.
intervals according to the method of Nakajima et al J. Biol Chem 2~4 4027 (
1979).
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The amount of Elastase released from the PMNs is calculated by measuring the
rate
of MeOSuc-Ala-Ala-Pro-Val-A~~fC degradation.
TNF-a in Traumatic Brain Injury Assay
5 This assay provides for examination of the expression of tumor necrosis
factor
mRNA in specific brain regions which follow experimentally induced lateral
fluid-
percussion traumatic brain injury (TBI) in rats. Since TNF- a is able to
induce nerve
growth factor (NGF~ and stimulate the release of other cytokines from
activated
astrocytes, this post-traumatic alteration in gene expression of TNF- a plays
an
to important role in both the acute and regenerative response to CNS trauma. A
suitable
assay may be found in WO 97/35856 or WO 97/49286 whose disclosures are
incorporated herein by reference.
CNS Injury model for IL-~i mRNA
15 This assay characterizes the regional expression of interleukin-1B (IL-1B)
mRNA in specific brain regions following experimental lateral fluid-percussion
traumatic brain injury (TBI) in rats. Results from these assays indicate that
following
TBI, the temporal expression of IL-1B mRNA is regionally stimulated in
specific brain
regions. These regional changes in cytokines, such as IL-1B play a role in the
post-
2o traumatic pathologic or regenerative sequelae of brain injury. A suitable
assay may be
found in WO 97/35856 or WO 97/49286 whose disclosures are incorporated herein
by
reference.
In vivo - athereoschlerosis assay:
25 In vivo models for measuring atherosclerosis in mice is based on the assay
of Paigen
et al with small modifications as described below. See Paigen B, Morrow A,
Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic
lesions in mice. Atherosclerosis 68: 231-240 (1987); and Groot PHE, van
Vlijmen
BJM, Benson GM, Hofker MH, Schiffelers R, Vidgeon-Hart M, Havekes LM.
3o Quantitative assessment of aortic atherosclerosis in APOfi*3 Leiden
transgenic mice
and its relationship to serum cholesterol exposure. Arterioscler Thromb Vasc
Biol.
16: 926-933 (1996).
Sectioning and staining of the aortic sinus
Cross-sections of the aortic root are taken as has been described previously (
1,2).
35 Briefly, the hearts are bisected just below the level of the atria and the
base of the heart
plus aortic root are taken for analysis. After equilibrating the tissue in OCT
compound
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WO 00/05216 PCTIUS98/15533
overnight the hearts are immersed in OCT compound on a cryostat chuck (Bright
Instrument Company Ltd., UK) with the aorta facing the chuck. The tissue is
frozen by
surrounding the chuck with dry ice. The hearts are then sectioned
perpendicular to the
axis of the aorta, starting within the heart and working in the direction of
the aorta. Once
the aortic root has been identified by the appearance of the three valve
leaflets, alternate
10 mm sections are taken and mounted on gelatinised slides. Sections are air
dried for 1
hour and subsequently rinsed briefly in 60% isopropyl alcohol. The sections
are stained
with Oil Red O, counterstained with Mayer's haematoxylin, cover slipped using
glycerol
gelatine and sealed with nail varnish.
10
Quantification of atherosclerosiS in the aortic root
Ten alternate sections of the aortic root are imaged using an Olympus BH-?
microscope
equipped with an 4x objective and a video camera (Hitachi, HV-CIO). Twenty-
four bit
colour images are acquired and analysed using a PC (Datacell Pentium P5-133.
Datacell,
15 Berks, U.K.) fitted with a framegrabbing board (Snapper, Active Imaging
Ltd, Berks,
U.K.) and running Optimas software (version 5.1, Optimas Corp., WA, U.S.A.).
The
images are captured under identical lighting, microscope, camera and PC
conditions.
Quantification of the atherosclerotic lesion areas is performed by drawing
around the
lesions by hand using the Optimas software. Colour thresholds are set that
quantify the
2o areas that are stained red within the lesions. Absolute values for the
cross-sectional
areas of the lesions and the areas stained red are obtained by calibrating the
software
using an image of the grid on a haemocytometer slide.
All publications, including but not limited to patents and patent
applications,
25 cited in this specification are herein incorporated by reference as if each
individual
publication were specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
The above description fully discloses the invention including preferred
embodiments thereof. Modifications and improvements of the embodiments
3o specifically disclosed herein are within the scope of the following claims.
Without
further elaboration, it is believed that one skilled in the are can, using the
preceding
description, utilize the present invention to its fullest extent. Therefore
the
Examples herein are to be construed as merely illustrative and not a
limitation of the
scope of the present invention in any way. The embodiments of the invention in
35 which an exclusive property or privilege is claimed are defined as follows.
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