U.S. patent application number 10/432092 was filed with the patent office on 2004-03-18 for novel compounds.
Invention is credited to Adams, Jerry L., Bryan, Deborah L..
Application Number | 20040053943 10/432092 |
Document ID | / |
Family ID | 22945749 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040053943 |
Kind Code |
A1 |
Adams, Jerry L. ; et
al. |
March 18, 2004 |
Novel compounds
Abstract
The present invention is directed to novel compounds of Formula
(I) for use in the treatment of diseases in a mammal, in which
inappropriate, excessive or undesirable angiogenesis has occurred
and/or where excessive Tie2 receptor activity has occurred.
Inventors: |
Adams, Jerry L.; (King of
Prussia, PA) ; Bryan, Deborah L.; (King of Prussia,
PA) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION
CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Family ID: |
22945749 |
Appl. No.: |
10/432092 |
Filed: |
May 20, 2003 |
PCT Filed: |
November 20, 2001 |
PCT NO: |
PCT/US01/43814 |
Current U.S.
Class: |
514/256 ;
514/341; 544/294; 544/333; 546/272.7 |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 27/02 20180101; A61P 35/00 20180101; A61P 3/10 20180101; C07D
401/04 20130101; A61P 19/02 20180101; A61P 9/10 20180101; C07D
405/14 20130101; C07D 401/14 20130101; C07D 409/14 20130101 |
Class at
Publication: |
514/256 ;
514/341; 544/333; 546/272.7; 544/294 |
International
Class: |
A61K 031/505; C07D
43/04; C07D 43/14; A61K 031/4745; A61K 031/4439 |
Claims
What is claimed is:
1. A compound of formula (I): 26wherein Ar is an optionally
substituted napth-2-yl, or napth-1-yl, an optionally substituted
bicyclic or tricyclic carbocyclic ring, an optionally substituted
bicyclic or tricyclic heteroaromatic ring, or an optionally
substituted bicyclic or tricyclic heterocyclic ring; V is CH or N;
X is O, CH.sub.2, S or NH; Z is oxygen or sulfur; n is 0, or an
integer having a value of 1 to 4; t is 0 or an integer having a
value of 1 to 10; Q is hydrogen, (CR.sup.13R.sup.14).sub.t
OR.sup.9, (CR.sup.13R.sup.14).sub.t OR.sup.11, C.sub.1-10 alkyl,
halo-substituted C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10
alkynyl, C.sub.3-7 cycloalkyl, C.sub.3-7cycloalkylC.sub.1-10 lkyl,
C.sub.5-7 cycloalkenyl, C.sub.5-7 cycloalkenyl C.sub.1-10 alkyl,
aryl, arylC.sub.1-10 alkyl, heteroaryl, heteroarylC.sub.1-10 alkyl,
heterocyclyl, heterocyclylC.sub.1-10 alkyl,
(CR.sup.13R.sup.14).sub.tS(O)- .sub.mR.sup.8,
(CR.sup.13R.sup.14).sub.t NHS(O).sub.2R.sup.8,
(CR.sup.13R.sup.14).sub.tNR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.t NO.sub.2, (CR.sup.13R.sup.14).sub.tCN,
(CR.sup.13R.sup.14).sub.tS(O).sub.- 2NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tC(Z)R.sup.11,
(CR.sup.13R.sup.14).sub.tOC(Z)R.sup.11,
(CR.sup.13R.sup.14).sub.tC(Z)OR.s- up.11,
(CR.sup.13R.sup.14).sub.tC(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tC(Z)NR.sup.11R.sup.9,
(CR.sup.13R.sup.14).sub.tN- R.sup.10C(Z)R.sup.11,
(CR.sup.13R.sup.14).sub.t NR.sup.10C(Z)NR.sup.10R.su- p.11,
(CR.sup.13R.sup.14).sub.tN(OR.sup.10)C(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tN(OR.sup.10)C(Z)R.sup.11,
(CR.sup.13R.sup.14).sub.tC(.dbd.NOR.sup.10)R.sup.11,
(CR.sup.13R.sup.14).sub.tNR.sup.10C(.dbd.NR.sup.15)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tOC(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.- tNR.sup.10C(Z)NR.sup.10R.sup.11, or
(CR.sup.13R.sup.14).sub.tNR.sup.10C(Z)- OR.sup.10; wherein the
cycloalkyl, cycloalkyl alkyl, aryl, arylalkyl, heteroaryl,
heteroaryl alkyl, heterocyclic and heterocyclic alkyl may be
optionally substituted; R.sup.1 is hydrogen, X--R.sup.4, halogen,
hydroxy, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.1-6alkylsulfinyl, CH.sub.2OR.sup.5, amino, mono
or di-C.sub.1-6alkylamino, N(R.sup.6)C(O)R.sup.7,
N(R.sup.6)S(O).sub.2R.sup.- 8, or a 5 to 7-membered N-heterocyclyl
ring which optionally contains an additional heteroatom selected
from O, S and NR.sup.9; R.sup.2 and R.sup.3 independently represent
an optionally substituted C.sub.1-6alkyl, or R.sup.2 and R.sup.3
together with the carbon atom to which they are attached form an
optionally substituted C.sub.3-7cycloalkyl or C.sub.5-7
cycloalkenyl ring, or R.sup.2 and R.sup.3 together with the carbon
atom to which they are attached form an optionally substituted 5 to
7-membered heterocyclyl ring containing up to 3 heteroatoms
selected from N, O and S; R.sup.4 is independently C.sub.1-6allyl,
aryl, arylC.sub.1-6alkyl, heterocyclyl, heterocyclylC.sub.1-6alkyl,
heteroaryl, or a heteroarylC.sub.1-6alkyl moiety, and wherein any
of these moieties may be optionally substituted; R.sup.5 is
hydrogen, C(Z)R.sup.10 or optionally substituted C.sub.1-6alkyl,
optionally substituted aryl, optionally substituted
arylC.sub.1-6alkyl, or S(O).sub.2R.sup.8; R.sup.6 is hydrogen or
C.sub.1-6alkyl; R.sup.7 is hydrogen, C.sub.1-6alkyl,
C.sub.3-7cycloalkyl, aryl, arylC.sub.1-6alkyl, heteroaryl,
heteroarylC.sub.1-6alkyl, heterocyclyl, or
heterocyclylC.sub.1-6alkyl; R.sup.8 is C.sub.1-6alkyl,
C.sub.3-7cycloalkyl, aryl, arylC.sub.1-6alkyl, heteroaryl,
heteroarylC.sub.1-6alkyl, heterocyclyl, or
heterocyclylC.sub.1-6alkyl; R.sup.9 is hydrogen, C.sub.1-4alkyl,
C.sub.3-7cycloalkyl or aryl; R.sup.10 is selected from hydrogen,
C.sub.1-6alkyl, C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl
C.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl, heterocyclyl,
heterocyclylC.sub.1-6alkyl, heteroaryl and
heteroarylC.sub.1-6alkyl, any of which may be optionally
substituted; and R.sup.13 and R.sup.14 are independently hydrogen,
C.sub.1-6alkyl, C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl
C.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl, heterocyclyl,
heterocyclylC.sub.1-6alkyl, heteroaryl, or
heteroarylC.sub.1-6alkyl, any of which may be optionally
substituted; or R.sup.11 and R.sup.12 together with the nitrogen to
which they are attached form a 5 to 7 membered heterocyclic ring
optionally containing an additional heteroatom selected from O, S,
or NR.sup.9; R.sup.15 is hydrogen, cyano, C.sub.1-4 alkyl,
C.sub.3-7 cycloalkyl or aryl; or a pharmaceutically acceptable salt
thereof.
2. The compound according to claim 1 wherein V is CH.
3. The compound according to claim 1 wherein V is N.
4. The compound according to any one of claims 1 to 3 wherein
R.sup.1 is hydrogen, or the moiety X--R.sup.4.
5. The compound according to claim 4 wherein X is oxygen or
nitrogen.
6. The compound according to claim 5 wherein R.sup.4 is an
optionally substituted alkyl, aryl, arylC.sub.1-6alkyl, or
heterocyclicC.sub.1-6alky- l.
7. The compound according to claims 6 wherein R.sup.4 is an
optionally substituted heterocyclicC.sub.1-6alkyl which is a
piperidine, morpholine, pyrrolidine, piperazine, or a
pyrrolidinone.
8. The compound according to any one of the preceding claims
wherein Ar is an optionally substituted naphthyl, benzothiophene or
benzofuran ring.
9. The compound according to claim 8 wherein the Ar ring is
substituted by up to 3 substituents independently selected from
halo, hydroxy, hydroxy C.sub.1-6allyl, or C.sub.1-6alkoxy.
10. The compound according to claim 9 wherein Ar is a napth-2-yl
optionally substituted by a C.sub.1-6alkoxy group.
11. The compound according to claim 1 wherein R.sup.2 and R.sup.3
independently an optionally substituted C.sub.1-6alkyl.
12. The compound according to claim 1 wherein R.sup.2 and R.sup.3
together with the carbon atom to which they are attached form an
optionally substituted C.sub.3-7cycloalkyl or C.sub.5-7cycloalkenyl
ring.
13. The compound according to claim 1 wherein R.sup.2 and R.sup.3
together with the carbon atom to which they are attached form an
optionally substituted 5 to 7 membered heterocyclyl ring containing
up to 3 heteroatoms selected from N, O and S.
14. The compound according to claim 1 which is:
2-tert-Butyl-4-naphthalen-- 2-yl-5-pyridin-4-yl-imidazole;
2-tert-Butyl-4-(6-Ethoxynaphthalen-2-yl)-5--
pyridin-4-yl-imidazole;
4-(2-tert-butyl-5-(6-methoxy-napthalen-2-yl)-3H-im-
adazol-4-yl)-pyridine;
2-tert-Butyl-4-Inden-2-yl)-5-pyridin-4-yl-1H-imidaz- ole
2-tert-Butyl-4-Benzofuran-2-yl-5-pyridin-4-yl-1H-imidazole;
4-(2-tert-Butyl-5-dibenzothiophen-4-yl-H-imidazol-4-yl)-pyridine;
4-(2-tert-Butyl-5-dibenzofuran-4-yl-1H-imidazol-4-yl)-pyridine;
4-(5-Benzo[b]thiophen-2-yl-2-tert-butyl-1H-imidazol-4-yl)-pyridine;
4-(5-Benzo[b]thiophen-3-yl-2-tert-butyl-1H-imidazol-4-yl)-pyridine;
4-(2-tert-Butyl-5-thianthren-1-yl-1H-imidazol-4-yl)-pyridine;
4-(2-tert-Butyl-5-phenoxathin-4-yl-1H-imidazol-4-yl)-pyridine;
4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-1-methyl-1H-imidazol-4-yl]--
pyridine; or a pharmaceutically acceptable salt thereof.
15. The compound according to claim 1 which is:
4-[2-tert-Butyl-5-(6-metho-
xy-naphthalen-2-yl)-3-H-imidazol-4-yl]-pyridin-2-yl}-(3-morpholin-4-yl-pro-
pyl)-amine;
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4--
yl]-pyridin-2-yl}-(3-morpholin-4-yl-ethyl)-amine;
{4-[2-tert-Butyl-5-(6-me-
thoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-pyridin-2-ylamino}-propyl)-pyrro-
lidin-2-one;
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-
-yl]-pyridin-2-yl}-[3-(2-methyl-piperidin-1-yl)-propyl]-amine;
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-pyridin-
-2-yl}-N,N-diethyl-butane-1,4-diamine;
{4-[2-tert-Butyl-5-(6-methoxy-napht-
halen-2-yl)-3-H-imidazol-4-yl]-pyridin-2-yl}-(3-pyrrolidin-1-yl-propyl)-am-
ine;
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-pyr-
idin-2-yl}-[3-(4-methyl-piperazin-1-yl)-propyl]-amine;
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-pyridin-
-2-y}-N,N-diethyl-ethane-1,2-diamine; or a pharmaceutically
acceptable salt thereof.
16. A pharmaceutical composition comprising a compound according to
any one of claims 1 to 15 or a pharmaceutically acceptable salt
thereof and a pharmaceutically acceptable carrier.
17. A method of treating, including prophylaxis, of a TIE2 receptor
mediated disease in a mammal in need thereof, which comprises
administering to said mammal an effective amount of a compound
according to any one of claims 1 to 15.
18. The method according to claim 17 wherein the disease is
characterized by excessive, undesired, or inappropriate
angiogenesis.
19. The method according to claim 18 wherein the disease is
diabetic retinopathy, macular degeneration, or other ocular
neovascularizations.
20. The method according to claim 17 wherein the disease is
characterized by excessive or increased proliferation of
vasculature.
21. The method according to claim 20 wherein the disease is tumor
growth and metastasis.
22. The method according to claim 17 wherein the disease is
atherosclerosis.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the treatment of diseases,
in a mammal, in which inappropriate, excessive or undesirable
angiogenesis has occurred and/or where excessive Tie2 receptor
activity has occurred.
BACKGROUND OF THE INVENTION
[0002] Chronic proliferative diseases are often accompanied by
profound angiogenesis, which can contribute to or maintain an
inflammatory and/or proliferative state, or which leads to tissue
destruction through the invasive proliferation of blood vessels.
(Folkman, EXS 79:1-8, 1997; Folkman, Nature Medicine 1:27-31, 1995;
Folkman and Shing, J. Biol. Chem. 267:10931, 1992).
[0003] Angiogenesis is generally used to describe the development
of new or replacement blood vessels, or neovascularisation. It is a
necessary and physiological normal process by which the vasculature
is established in the embryo. Angiogenesis does not occur, in
general, in most normal adult tissues, exceptions being sites of
ovulation, menses and wound healing. Many diseases, however, are
characterized by persistent and unregulated angiogenesis. For
instance, in arthritis, new capillary blood vessels invade the
joint and destroy cartilage (Colville-Nash and Scott, Ann. Rheum.
Dis., 51, 919,1992). In diabetes (and in many different eye
diseases), new vessels invade the macula or retina or other ocular
structures, and may cause blindness (Brooks et al., Cell, 79, 1157,
1994). The process of atherosclerosis has been linked to
angiogenesis (Kahlon et al., Can. J. Cardiol. 8, 60, 1992). Tumor
growth and metastasis have been found to be angiogenesis-dependent
(Folkman, Cancer Biol, 3, 65, 1992; Denekamp, Br. J. Rad. 66, 181,
1993; Fidler and Ellis, Cell, 79, 185, 1994).
[0004] The recognition of the involvement of angiogenesis in major
diseases has been accompanied by research to identify and develop
inhibitors of angiogenesis. These inhibitors are generally
classified in response to discrete targets in the angiogenesis
cascade, such as activation of endothelial cells by an angiogenic
signal; synthesis and release of degradative enzymes; endothelial
cell migration; proliferation of endothelial cells; and formation
of capillary tubules. Therefore, angiogenesis occurs in many stages
and attempts are underway to discover and develop compounds that
work to block angiogenesis at these various stages.
[0005] There are publications that teach that inhibitors of
angiogenesis, working by diverse mechanisms, are beneficial in
diseases such as cancer and metastasis (O'Reilly et al., Cell, 79,
315, 1994; Ingber et al., Nature, 348, 555, 1990), ocular diseases
(Friedlander et al., Science, 270, 1500, 1995), arthritis (Peacock
et al., J. Exp. Med. 175, 1135, 1992; Peacock et al., Cell. Immun.
160, 178, 1995) and hemangioma (Taraboletti et al., J. Natl. Cancer
Inst. 87, 293, 1995).
[0006] Angiogenesis signals result from the interaction of specific
ligands with their receptors. The Tie1 and Tie2 receptors are
single-transmembrane, tyrosine kinase receptors (Tie stands for
Tyrosine kinase receptors with immunoglobulin and EGF homology
domains). Both were recently cloned and reported by several groups
(Dumont et al., Oncogene 8:1293-1301, 1993; Partanen et al., Mol.
Cell Biol. 12:1698-1707, 1992; Sato et al., Proc. Natl. Acad. Sci.
USA 90:9355-9358, 1993).
[0007] Based upon the importance of Tie2 receptors in angiogenesis,
inhibition of Tie2 kinase activity is predicted to interrupt
angiogenesis, providing disease-specific therapeutic effects.
Recently, Lin et al. (J. Clin. Invest. 100:2072-2078, 1997) has
shown that exogenously administered soluble Tie2 receptor inhibited
angiogenesis and cancer growth in animal models. Thus inhibition of
Tie2 receptors by other means, such as inhibition of Tie2 receptor
kinase activity, is expected to have therapeutic benefit in
proliferative diseases involving angiogenesis.
[0008] The current application teaches the novel finding that
compounds of specific structure can inhibit the kinase activity of
the Tie2 receptor, block its signal transduction and thus may be
beneficial for proliferative diseases via inhibition of signals for
angiogenesis.
SUMMARY OF THE INVENTION
[0009] The present invention is the finding that novel compounds
can inhibit Tie2 kinase, and that these compounds can be used for
inhibition of angiogenesis for the treatment of chronic
inflammatory or proliferative or angiogenic diseases which are
caused by excessive or inappropriate angiogenesis. The preferred
compounds for use as Tie2 receptor kinase inhibitors are those
compounds of Formula (I) as noted herein.
[0010] This invention relates to the novel compounds of Formula (I)
and pharmaceutical compositions comprising a compound of Formula
(I) and a pharmaceutically acceptable diluent or carrier.
[0011] Accordingly, the present invention provides for the novel
compounds of Formula (I) represented by the structure: 1
[0012] wherein
[0013] V is CH or N;
[0014] X is O, CH.sub.2, S or NH;
[0015] n is 0, or an integer having a value of 1 to 4;
[0016] t is 0 or an integer having a value of 1 to 10;
[0017] Ar is a napth-2-yl, napth-1-yl, a bicyclic or a tricyclic
carbocyclic ring, a bicyclic or a tricyclic heteroaromatic ring, or
a bicyclic or a tricyclic heterocyclic which ring may be optionally
substituted;
[0018] Q is hydrogen, (CR.sup.13R.sup.14).sub.t OR.sub.9,
(CR.sup.13R.sup.14).sub.t OR.sub.11, C.sub.1-10 alkyl,
halo-substituted C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10
alkynyl, C.sub.3-7 cycloalkyl, C.sub.3-7cycloalkylC.sub.1-10 alkyl,
C.sub.5-7 cycloalkenyl, C.sub.5-7 cycloalkenyl C.sub.1-10 alkyl,
aryl, arylC.sub.1-10 alkyl, heteroaryl, heteroarylC.sub.1-10 alkyl,
heterocyclyl, heterocyclylC.sub.1-10alkyl,
(CR.sup.13R.sup.14).sub.tS(O).sub.mR.sup.8,
(CR.sup.13R.sup.14).sub.tNHS(O).sub.2R.sup.8,
(CR.sup.13R.sup.14).sub.tNR- .sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.t NO.sub.2, (CR.sup.13R.sup.14).sub.tCN,
(CR.sup.13R.sup.14).sub.tS(O).sub.2NR.sup.10- R.sup.11,
(CR.sup.13R.sup.14).sub.tC(Z)R.sup.11, (CR.sup.13R.sup.14).sub.t-
OC(Z)R.sup.11, (CR.sup.13R.sup.14).sub.tC(Z)OR.sup.11,
(CR.sup.13R.sup.14).sub.tC(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.t- C(Z)NR.sup.11OR.sup.9,
(CR.sup.13R.sup.14).sub.tNR.sup.10C(Z)R.sup.11,
(CR.sup.13R.sup.14).sub.t NR.sup.10C(Z) NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tN(OR.sup.10)C(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tN(OR.sup.10)C(Z)R.sup.11,
(CR.sup.13R.sup.14).sub.tC(.dbd.NOR.sup.10)R.sup.11,
(CR.sup.13R.sup.14).sub.tNR.sup.10C(.dbd.NR.sup.15)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tOC(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.- tNR.sup.10C(Z)NR.sup.10R.sup.11, or
(CR.sup.13R.sup.14).sub.tNR.sup.10C(Z)- OR.sup.10; wherein the
cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocyclic and heterocyclicalkyl groups may be
optionally substituted;
[0019] R.sup.1 is independently hydrogen, X--R.sup.4, halogen,
hydroxy, optionally substituted C.sub.1-6 alkyl, optionally
substituted C.sub.1-6 alkylsulfinyl, CH.sub.2OR.sup.5, amino, mono
or di-C.sub.1-6alkylamino, N(R.sup.6)C(O)R.sup.7,
N(R.sup.6)S(O).sub.2R.sup.8, or a 5 to 7-membered N-heterocyclyl
ring which optionally contains an additional heteroatom selected
from O, S and NR.sup.9;
[0020] R.sup.2 and R.sup.3 independently represent an optionally
substituted C.sub.1-6alkyl, or R.sup.2 and R.sup.3 together with
the carbon atom to which they are attached form an optionally
substituted C.sub.3-7cycloalkyl or C.sub.5-7 cycloalkenyl ring, or
R.sup.2 and R.sup.3 together with the carbon atom to which they are
attached form an optionally substituted 5 to 7-membered
heterocyclyl ring containing up to 3 heteroatoms selected from N, O
and S;
[0021] R.sup.4 is independently C.sub.1-6alkyl, aryl,
arylC.sub.1-6alkyl, heterocyclyl, heterocyclylC.sub.1-6alkyl,
heteroaryl, or a heteroarylC.sub.1-6alkyl moiety, and wherein any
of these moieties may be optionally substituted;
[0022] R.sup.5 is hydrogen, C(Z)R.sup.1 or optionally substituted
C.sub.1-6alkyl, optionally substituted aryl, optionally substituted
arylC.sub.1-6alkyl, or S(O).sub.2R.sup.8;
[0023] R.sup.6 is hydrogen or C.sub.1-6alkyl;
[0024] R.sup.7 is hydrogen, C.sub.1 alkyl, C.sub.3-7cycloalkyl,
aryl, arylC.sub.1-6alkyl, heteroaryl, heteroarylC.sub.1-6alkyl,
heterocyclyl, or heterocyclylC.sub.1-6alkyl;
[0025] R.sup.8 is C.sub.1-6alkyl, C.sub.3-7cycloalkyl, aryl,
arylC.sub.1-6alkyl, heteroaryl, heteroarylC.sub.1-6alkyl,
heterocyclyl, or heterocyclylC.sub.1-6alkyl;
[0026] R.sup.9 is hydrogen, C.sub.1-4alkyl, C.sub.3-7cycloalkyl or
aryl;
[0027] R.sup.10 is selected from hydrogen, C.sub.1-6alkyl,
C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl C.sub.1-6alkyl, aryl,
arylC.sub.1-6alkyl, heterocyclyl, heterocyclylC.sub.1-6alkyl,
heteroaryl and heteroarylC.sub.1-6alkyl, any of which may be
optionally substituted; and
[0028] R.sup.13 and R.sup.14 are independently hydrogen,
C.sub.1-6alkyl, C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl
C.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl, heterocyclyl,
heterocyclylC.sub.1-6alkyl, heteroaryl, or
heteroarylC.sub.1-6alkyl, any of which may be optionally
substituted; or R.sup.11 and R.sup.12 together with the nitrogen to
which they are attached form a 5 to 7 membered heterocyclic ring
optionally containing an additional heteroatom selected from O, S,
or NR.sup.9;
[0029] R.sup.15 is hydrogen, cyano, C.sub.1-4 alkyl, C.sub.3-7
cycloalkyl or aryl;
[0030] Z is oxygen or sulfur;
[0031] or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention is the directed to novel compounds
which can inhibit Tie2 kinase, and use of these compounds for
inhibition of angiogenesis in the treatment of chronic inflammatory
or proliferative or angiogenic diseases which are caused by
excessive or inappropriate angiogenesis in a mammal in need
thereof.
[0033] In the compounds of formula (I), V is suitably CH or N,
preferably carbon.
[0034] Suitably, the pyridyl or pyrimidine ring is optionally
substituted, independently, one to two times independently by the
R.sup.1 moiety.
[0035] Suitably, R.sup.1 is independently selected from hydrogen,
X--R.sup.4, halogen, hydroxy, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.1-6alkylsulfinyl,
CH.sub.2OR.sup.5, amino, mono or di-C.sub.1-6alkylamino,
N(R.sup.6)C(O)R.sup.7, N(R.sup.6)S(O).sub.2R.sup.5, or a 5 to
7-membered N-heterocyclyl ring which optionally contains an
additional heteroatom selected from O, S and NR.sup.9. Preferably,
the pyridyl or pyrimidine is substituted in the 2-position.
Preferably, R.sup.1 is hydrogen or X--R.sup.4.
[0036] X is suitably, O, CH.sub.2, S or NH. Preferably X is oxygen
or nitrogen.
[0037] R.sup.4 is independently C.sub.1-6alkyl, aryl,
arylC.sub.1-6alkyl, heterocyclyl, heterocyclylC.sub.1-6alkyl,
heteroaryl, or a heteroarylC.sub.1-6alkyl moiety, and wherein any
of these moieties may be optionally substituted. Preferably R.sup.4
is an optionally substituted alkyl, aryl, arylalkyl, or
heterocyclic alkyl group.
[0038] When R.sup.4 is aryl, it is preferably an optionally
substituted phenyl. When R.sup.4 is aryl alkyl, it is preferably an
optionally substituted benzyl or phenethyl.
[0039] When R.sup.4 is a heteroaryl or heteroarylC.sub.1-6alkyl
moiety, it is as defined below. Preferably, it is an optionally
substituted pyrrole, quinoline, isoquinoline, pyridine, pyrimidine,
oxazole, thiazole, thiadiazole, triazole, imidazole, benzimidazole,
isoxazole, thiophene, benzothiophene, furan, benzofuran, pyrazole,
pyran, quinazolinyl, pyridazine, pyrazine, uracil, oxadiazole,
oxathiadiazole, isothiazole, tetrazole, and indazole.
[0040] When R.sup.4 is a heterocyclyl, or
heterocyclylC.sub.1-6alkyl it is as defined below. Preferably, it
is an optionally substituted tetrahydropyrrole, tetrahydropyran,
tetrahydrofuran, tetrahydrothiophene (including oxidized versions
of the sulfur moiety), pyrrolidine, piperidine, piperazine,
morpholine, thiomorpholine (including oxidized versions of the
sulfur moiety), imidazolidine and pyrazolidine ring.
[0041] These R.sup.4 moieties may be optionally substituted one or
more times, preferably 1 to 3 times, independently with halogen;
C.sub.1-4 alkyl, such as methyl, ethyl, propyl, isopropyl, or
t-butyl; halosubstituted alkyl, such as CF.sub.3; hydroxy; hydroxy
substituted C.sub.1-4 alkyl; C.sub.1-4 alkoxy, such as methoxy or
ethoxy; S(O).sub.malkyl and S(O).sub.m aryl (wherein m is 0, 1, or
2); C(O)OR.sup.11, such as C(O)C.sub.1-6alkyl or C(O)OH moieties;
C(O); C(O)R.sup.11; OC(O)R.sup.8; O--(CH.sub.2)s-O--, such as in a
ketal or dioxyalkylene bridge (s is a number having a value of 1 to
5); amino; mono- and di-C.sub.1-6 alkylsubstituted amino;
N(R.sup.10)C(O)R.sup.7; C(O)NR.sup.1OR.sup.11; cyano; nitro; or an
N-heterocyclyl ring which ring has from 5 to 7 members and
optionally contains an additional heteroatom selected from oxygen,
sulfur or NR.sup.9; optionally substituted aryl, such as phenyl; an
optionally substituted aryl C.sub.1-6alkyl, such as benzyl or
phenethyl; aryloxy, such as phenoxy; or arylC.sub.1-6 alkyloxy such
as benzyloxy; and wherein these aryl and arylalkyl moieties may
themselves by optionally substituted with halogen, C.sub.1-6 alkyl,
alkoxy, S(O).sub.m alkyl, amino, or mono- and di-C.sub.1-6
alkylsubstituted amino.
[0042] Preferably the R.sup.4 moieties are substituted with an
amino, mono- or di-C.sub.1-6alkylsubstituted amino, a heterocyclic
alkyl ring, more preferably an N-heterocyclyl (alkyl) ring which
ring has from 5 to 7 members and optionally contains an additional
heteroatom selected from oxygen, sulfur or NR.sup.9, such as
morpholino, pyrollidine, piperazine, piperidine, or
pyrrolidinone.
[0043] Suitably, R.sup.2 and R.sup.3 independently represent an
optionally substituted C.sub.1-6alkyl, or R.sup.2 and R.sup.3
together with the carbon atom to which they are attached form an
optionally substituted C.sub.3-7cycloalkyl or
C.sub.5-7.cycloalkenyl ring, or R.sup.2 and R.sup.3 together with
the carbon atom to which they are attached form an optionally
substituted 5 to 7-membered heterocyclyl ring containing up to 3
heteroatoms selected from N, O and S. Preferably R.sup.2 and
R.sup.3 independently represent optionally substituted
C.sub.1-6alkyl.
[0044] Suitably, n is 0, 1, 2, 3 or 4. Preferably, n is 1.
[0045] Suitably, R.sup.11 is hydrogen, C.sub.1 alkyl,
C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl C.sub.1-6alkyl, aryl,
arylC.sub.1-6alkyl, heterocyclyl, heterocyclylC.sub.1-6alkyl,
heteroaryl, or heteroarylC.sub.1-6alkyl, any of which may be
optionally substituted; or R.sup.10 and R.sup.11 together with the
nitrogen to which they are attached form a 5 to 7 membered
heterocyclic ring optionally containing an additional heteroatom
selected from O, S, or NR.sup.9.
[0046] Suitably, R.sup.13 and R.sup.14 are independently hydrogen,
C.sub.1-6alkyl, C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl
C.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl, heterocyclyl,
heterocyclylC.sub.1-6alkyl, heteroaryl, or
heteroarylC.sub.1-6alkyl, any of which may be optionally
substituted; or R.sup.11 and R.sup.12 together with the nitrogen to
which they are attached form a 5 to 7 membered heterocyclic ring
optionally containing an additional heteroatom selected from O, S,
or NR.sup.9.
[0047] Suitably, R.sup.12 is selected from hydrogen, C.sub.1 alkyl,
C.sub.3-7cycloalkyl, C.sub.3-7cycloalkyl C.sub.1-6alkyl,
heterocyclyl, heterocyclylC.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl,
heteroaryl or heteroarylC.sub.1-6alkyl, any of which may be
optionally substituted. The R.sup.10 group and R.sup.12 moieties
may be optionally substituted as defined for the alkyl term.
[0048] Suitably, R.sup.5 is hydrogen, C(Z)R.sup.12 or optionally
substituted C alkyl, optionally substituted aryl, optionally
substituted arylC.sub.1-6alkyl, or S(O).sub.2R.sup.8.
[0049] Suitably, R.sup.6 is hydrogen or C.sub.1-6alkyl.
[0050] Suitably, R.sup.7 is hydrogen, C.sub.1-6alkyl,
C.sub.3-7cycloalkyl, aryl, arylC.sub.1-6alkyl, heteroaryl,
heteroarylC.sub.1-6alkyl, heterocyclyl, or
heterocyclylC.sub.1-6alkyl.
[0051] Suitably, R.sup.8 is C.sub.1-6alkyl, C.sub.3-7cycloalkyl,
aryl, arylC.sub.1-6alkyl, heteroaryl, heteroarylC.sub.1-6alkyl,
heterocyclyl, or heterocyclylC.sub.1-6alkyl.
[0052] Suitably, R.sup.9 is hydrogen, cyano, C.sub.1-4alkyl,
C.sub.3-7cycloalkyl or aryl.
[0053] Suitably, R.sup.10 and R.sup.12 are independently selected
from hydrogen, C.sub.1-6alkyl, C.sub.3-7cycloalkyl, heterocyclyl,
heterocyclylC.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl, heteroaryl or
heteroarylC.sub.1-6alkyl, any of which may be optionally
substituted.
[0054] Suitably, Z is oxygen or sulfur.
[0055] Ar is suitably a napth-2-yl, napth-1-yl, a bicyclic or a
tricyclic carbocyclic ring, a bicyclic or a tricyclic
heteroaromatic ring, or a bicyclic or a tricyclic heterocyclic
ring, and wherein any ring in the system may be optionally
substituted one or more times as defined herein below. A bicyclic
or tricyclic heteroaromatic or heterocyclic ring system may be a
fused ring system which includes a carbocyclic ring. For the
heteroaromatic ring system, it will be an aromatic ring, for the
heterocyclic ring system the carbocyclic ring may be saturated,
contain some unsaturation, or be aromatic.
[0056] More specifically, Ar as a bicyclic or a tricyclic
heterocyclic, or a bicyclic or tricyclic heteroaromatic ring, which
includes, but is not limited to, quinoline, isoquinoline,
benzoxazole, benzthiazole, benzofuran, dibenzofuran, benzthiaphene,
dibenzothiaphene, benzthiadiazole, benztriazole, benzimidazole,
thioanthracene, phenoxathine, benzimidazole, indolyl, or
quinazolinyl. Ar is preferably a bicyclic or a tricyclic
heteroaromatic ring.
[0057] Ar as a heterocyclic ring system includes a bicyclo or a
tricyclo fused ring system, containing at least one or more
aromatic or heteroaromatic rings, and one or more saturated or
unsaturated carbocyclic 4-7 membered rings, or one or more
saturated or unsaturated rings containing 4-7 members with one or
more heteroatoms selected from oxygen, nitrogen or sulfur
(including oxidized versions thereof). The ring system must contain
at least one heteroatom selected from oxygen, nitrogen or sulfur.
The ring system may include from 7 to 14 ring members.
[0058] Ar as a heteroaromatic ring system may include at least one
aromatic ring and/or one or more heteroaromatic rings, but the ring
system must contain one or more heteroatoms selected from oxygen,
nitrogen or sulfur. The ring system may include from 8 to 12
members.
[0059] Ar as a bicyclic or a tricyclic carbocyclic ring, includes
but is not limited to indene, tetraline, or indane derivatives, in
addition to the specific napthyl rings as indicated separately.
This carbocyclic ring system includes at least one aromatic ring
and one or more saturated or unsaturated carbocyclic 4-7 membered
rings attached thereto.
[0060] The Ar ring may be optionally substituted one or more times,
preferably 1 to 3 times, independently, in any ring. Suitable
substituents include halogen, C.sub.1-6alkyl, aryl,
arylC.sub.1-6alkyl, cycloalkyl, cycloalkyl C.sub.1-6alkyl,
C.sub.1-6alkoxyC.sub.1-6alkyl, halosubstituted C.sub.1-6alkyl,
arylC.sub.1-6alkoxy, (CR.sup.13R.sup.14).sub.t OR.sup.12, nitro,
cyano, (CR.sup.13R.sup.14).sub.t NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.t NR.sup.10C(Z)R.sup.12,
(CR.sup.13R.sup.14).sub.tC(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tCOR.sup.12,
(CR.sup.13R.sup.14).sub.tZC(Z)R.sup.- 12,
(CR.sup.13R.sup.14).sub.tC(Z)OR.sup.12,
(CR.sup.13R.sup.14).sub.tC(O)N- R.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tNR.sup.10C(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.t NR.sup.10C(.dbd.NH)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tC(.dbd.NH)--NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tNR.sup.10S(O).sub.2R.sup.8,
(CR.sup.13R.sup.14).sub.tS(O).sub.2NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tS(O).sub.mR.sup.12, heterocyclyl,
heteroaryl, heterocyclylC.sub.1-6alkyl or
heteroarylC.sub.1-6alkyl.
[0061] Suitably t is 0, or an integer having a value of 1 to 10.
Preferably t is 0, or 1, more preferably 0.
[0062] Suitably, R.sup.13 and R.sup.14 are independently hydrogen,
or a C.sub.1-6 alkyl.
[0063] Preferably, the Ar group is substituted, in any ring, one or
more times by halo, cyano,
(CR.sup.13R.sup.14).sub.tC(Z)NR.sup.10R.sup.11,
(CR.sup.13R.sup.14).sub.tC(Z)OR.sup.12,
(CR.sup.13R.sup.14).sub.tCOR.sup.- 12,
(CR.sup.13R.sup.14).sub.tS(O).sub.mR.sup.12,
(CR.sup.13R.sup.14).sub.t OR.sup.12, halo-substituted-C.sub.1-6
alkyl, C.sub.1-6 allyl,
(CR.sup.13R.sup.14).sub.tNR.sup.10C(Z)R.sup.12,
(CR.sup.13R.sup.14).sub.t NR.sup.10S(O).sub.2R.sup.8,
(CR.sup.13R.sup.14).sub.tS(O).sub.2NR.sup.1OR- .sup.11,
(CR.sup.13R.sup.14)tZC(Z)R.sup.12, or (CR.sup.13R.sup.14).sub.tN
R.sup.10R.sup.11.
[0064] More preferably, the Ar ring is substituted one or more
times by halogen, S(O).sub.mR.sup.12, OR.sup.12, C.sub.1-6alkyl,
halosubstituted C.sub.1-6alkyl, hydroxy C.sub.1-6alkyl, and
C.sub.1-6alkoxy, NR.sup.10S(O) 2R.sup.8, NR.sup.10C(Z)R.sup.12 or N
R.sup.10R.sup.11. More preferred substitution is hydroxy; a
C.sub.1-6alkoxy group, such as methoxy; a C.sub.1-6 alkyl, such as
methyl; or halogen, such as fluorine or chlorine.
[0065] Preferably the Ar ring is a napthyl ring, more preferably a
napth-2-yl ring. If Ar is a bicyclo heteroaryl ring it is
preferably a benzothiophene or a benzofuran ring. A preferred ring
placement for the napth-2-yl ring is in the 6-position.
[0066] For use herein, the term "alkyl", and "alkenyl" groups,
individually or as part of a larger group e.g. "alkoxy", may be
straight or branched chain radicals containing up to six carbon
atoms, unless otherwise limited, including, but not limited to,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,
tert-butyl, and the like. The alkyl and alkenyl groups may be
optionally substituted as herein defined.
[0067] For use herein, "cycloalkyl" includes cyclic radicals having
from three to eight ring carbon atoms, including but not limited to
cyclopropyl, cyclopentyl, cyclohexyl, and the like. The cycloalkyl
groups may be optionally substituted as herein defined.
[0068] For use herein, "cycloalkenyl" includes cyclic radicals,
preferably of 5 to 8 carbons, which have at least one bond
including but not limited to cyclopentenyl, cyclohexenyl, and the
like. The cycloalkenyl groups may be optionally substituted as
herein defined.
[0069] For use herein the term "aryl" (on its own or in any
combination, such as "arylalkyl" or "aryloxy") includes a single or
fused ring system, suitably containing from 4 to 7, preferably 5 or
6 ring atoms in each ring, which rings, may each be unsubstituted
or substituted by, independently for example, up to three
substituents. A fused ring system may include an aliphatic ring,
such as a saturated or partially saturated ring, and need include
only one aromatic ring. Suitable aryl groups include phenyl and
naphthyl such as 1-naphthyl or 2-naphthyl. The aryl rings may be
optionally substituted as herein defined unless otherwise
indicated.
[0070] For use herein the term "heterocyclyl" (on its own or in any
combination, such as "heterocyclyl alkyl" or "heterocyclyl oxy")
suitably includes, unless otherwise defined, non-aromatic,
saturated or partially unsaturated, single and fused rings,
suitably containing up to four heteroatoms in each ring, each of
which independently selected from O, N and S or S(O).sub.m, and m
is 0 or an integer having a value of 1 or 2, and which rings, may
be unsubstituted or substituted independently by, for example, up
to three substituents. Each heterocyclic ring suitably has from 4
to 7, preferably 5 or 6 ring atoms, or a fused ring system
containing from 7 to 14 ring members. A fused heterocyclic ring
system may include carbocyclic rings and need include only one
heteroatom, as a heterocyclic ring. Examples of heterocyclyl groups
include but are not limited to, the saturated or partially
saturated versions of the heteroaryl moieties as defined herein,
such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran,
tetrahydrothiophene (including oxidized versions of the sulfur
moiety), pyrrolidine, piperidine, piperazine, morpholine,
thiomorpholine (including oxidized versions of the sulfur moiety),
imidazolidine and pyrazolidine. The heterocyclic rings may be
optionally substituted as herein defined below, unless otherwise
indicated.
[0071] When used herein, the term "heteroaryl" (on its own or in
any combination, such as "heteroaryloxy" or "heteroarylalkyl")
suitably includes, unless otherwise defined, mono- and bicyclic
heteroaromatic ring systems comprising up to four, preferably 1 or
2, heteroatoms each selected from O, N and S. Each ring may have
from 4 to 7, preferably 5 or 6, ring atoms, or be a fused ring
system containing from 8 to 12 ring members. A bicyclic
heteroaromatic ring system may include a carbocyclic ring. Examples
of heteroaryl groups include but are not limited to pyrrole,
quinoline, isoquinoline, pyridine, pyrimidine, oxazole, thiazole,
thiadiazole, triazole, imidazole, benzimidazole, isoxazole,
thiophene, benzothiophene, furan, benzofuran, pyrazole, pyran,
quinazolinyl, pyridazine, pyrazine, uracil, oxadiazole,
oxathiadiazole, isothiazole, tetrazole, and indazole. The
heteroaryl rings may be optionally substituted as defined herein
below, unless otherwise indicated.
[0072] Suitably the when the term "optionally substituted" is used
herein, such as on the alkyl, alkenyl, cycloalkyl, cycloalkenyl,
aryl, arylalkyl, heterocyclyl, heterocyclicalkyl, heteroaryl, and
heteroarylalkyl groups, unless otherwise defined, shall mean that
the group may be optionally substituted one or more times,
preferably by one to three substituents, each independently
selected from halogen, C.sub.1-6alkyl, aryl, arylC.sub.1-6alkyl,
cycloalkyl, cycloalkyl C.sub.1-6alkyl,
C.sub.1-6alkoxyC.sub.1-6alkyl, halosubstituted C.sub.1-6alkyl,
arylC.sub.1-6alkoxy, OR.sup.12, nitro, cyano,
(CR.sub.1-R.sub.20).sub.n NR.sup.10R.sup.11,
(CR.sub.10R.sub.20).sub.nNR.sup.10C(Z)R.sup.12,
(CR.sub.10R.sub.20).sub.nC(Z)NR.sup.10R.sup.11,
(CR.sub.10R.sub.20).sub.n- COR.sup.12, (CR.sub.10R.sub.20).sub.n
ZC(Z)R.sup.12, (CR.sub.10R.sub.20).sub.nC(Z)OR.sup.12,
(CR.sub.10R.sub.20).sub.nC(O)NR.s- ub.10R.sup.11,
(CR.sub.10R.sub.20).sub.n NR.sup.10C(Z)NR.sup.10R.sup.11,
(CR.sub.10R.sub.20).sub.nNR.sup.10C(--NH)NR.sup.10R.sup.11,
(CR.sub.10OR.sub.20).sub.n C(.dbd.NH)--NR.sup.10R.sup.1,
(CR.sub.10R.sub.20).sub.nNR.sup.10S(O).sub.2R.sup.8,
(CR.sub.10R.sub.20).sub.nS(O).sub.2NR.sup.10R.sup.11,
S(O).sub.mR.sup.12, heterocyclyl, heteroaryl,
heterocyclylC.sub.1-6alkyl or heteroaryl C.sub.1-6alkyl. In
addition, two adjacent ring carbon atoms may be linked to form a
bicyclic system.
[0073] Particular compounds according to the invention include
those mentioned in the examples and their pharmaceutically
acceptable salts.
[0074] It will be appreciated that for use in medicine the salts of
the compounds of formula (I) should be pharmaceutically acceptable.
Suitable pharmaceutically acceptable salts will be apparent to
those skilled in the art and include those described in J. Pharm.
Sci., 1977, 66, 1-19, such as acid addition salts formed with
inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitric
or phosphoric acid; and organic acids e.g. succinic, maleic,
acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic,
methanesulfonic or naphthalenesulfonic acid. Other salts e.g.
oxalates, may be used, for example in the isolation of compounds of
formula (I) and are included within the scope of this
invention.
[0075] The compounds of this invention may be in crystalline or
non-crystalline form, and, if crystalline, may optionally be
hydrated or solvated. This invention includes within its scope
stoichiometric hydrates as well as compounds containing variable
amounts of water.
[0076] The invention extends to all isomeric forms including
stereoisomers and geometric isomers of the compounds of formula (I)
including enantiomers and mixtures thereof e.g. racemates. The
different isomeric forms may be separated or resolved one from the
other by conventional methods, or any given isomer may be obtained
by conventional synthetic methods or by stereospecific or
asymmetric syntheses.
[0077] Since the compounds of formula (I) are intended for use in
pharmaceutical compositions it will readily be understood that they
are each preferably provided in substantially pure form, for
example at least 60% pure, more suitably at least 75% pure and
preferably at least 85%, especially at least 98% pure (% are on a
weight for weight basis). Impure preparations of the compounds may
be used for preparing the more pure forms used in the
pharmaceutical compositions.
[0078] Compounds of Formula (I) are imidazole derivatives which may
be readily prepared using procedures well-known to those skilled in
the art, and described in, for instance, Comprehensive Heterocyclic
Chemistry, Editors Katritzky and Rees, Pergamon Press, 1984, 5,
457-497, from starting materials which are either commercially
available or can be prepared from such by analogy with well-known
processes. A key step in many such syntheses is the formation of
the central imidazole nucleus, to give compounds of formula (I).
Suitable procedures are described in inter alia U.S. Pat. Nos.
3,707,475 and 3,940,486 which are herein incorporated by reference
in their entirety. These patents describe the synthesis of
a-diketones and .alpha.-hydroxyketones (benzoins) and their
subsequent use in preparing imidazoles and N-hydroxyl imidazoles.
Thereafter, further compounds of formula (I) may be obtained by
manipulating substituents in any of the groups R.sup.1, R.sup.2,
and R.sup.3 using conventional functional group interconversion
procedures. 2
[0079] Compounds of the general formula I with Q=H can be prepared
as outlined in Scheme I. Condensation of the anion of
4-methyl-2-(methylthio)pyrimidine with the Weinreb amide of an aryl
acid will yield the ketone, which upon oxidation with sodium
nitrite affords the ketooxime. Heating this product with an alkyl
aldehyde and ammonium acetate in acetic acid allows access to the
imidazole nucleus. Reduction of the hydroxyimidazole may be
accomplished with heating with a trialkyl phosphite or stirring at
ambient temperature with titanium trichloride. Replacement of the
methylthio-group (R.sup.1=SMe) with nucleophiles (R.sup.1=OR.sup.4,
NHR.sup.4, SR.sup.4) can be effected by oxidation to the
methylsulfinyl derivative with 3-chloroperoxybenzoic acid or oxone,
followed by displacement with nucleophiles with or without the
addition of bases such as sodium hydride, organolithiums or
trialkylamines. In the case of amines (R.sup.1=NHR.sup.4), aluminum
amide derivatives can be used to effect the displacements. 3
[0080] Alternative methods for preparing compounds of this
invention are as outlined in the Scheme II. .alpha.-Diketones are
prepared by condensation of the anion of, for example, a
4-substituted pyridine derivative (V=CH, R.sup.1=H) with the
Weinreb amide of an aryl acid or an aryl-aldehyde, followed by
oxidation of the intermediate product. Heating the diketone with an
aldehyde and ammonium acetate in acetic acid allows access to the
imidazole nucleus.
[0081] Compounds of the general formula (R.sup.1=OR.sup.4,
NHR.sup.4, SR.sup.4) can be prepared as in Schemes I or II except
substituting 4-methyl-2-chloropyridine or 4-methyl-2-fluoropyridine
for 4-methylpyridine (Gallagher et al Bioorg. Med. Chem. 5, 49,
1997). Nucleophilic substitution of the resulting
2-halopyridinylimidazole can be effected by the procedure described
in U.S. Pat. No. 5,670,527.
[0082] Alternatively compounds may be prepared as in Scheme III
wherein the Aryl (Ar) group is added last. 4
[0083] Oxidation of a 4-acetyl substituted pyridine derivative
(V=CH, R.sup.1=H) with sodium nitrite affords the ketooxime.
Heating this product with an alkyl aldehyde and ammonium acetate in
acetic acid allows access to the imidazole nucleus. Reduction of
the hydroxyimidazole may be accomplished with heating with a
trialkyl phosphite or stirring at ambient temperature with titanium
trichloride. Treatment of the imidazole with N-iodosuccinimide
gives the iodoimidazole with then may be reacted with various
boronic acids under palladium catalysis to give the aryl or
heteroaryl imidazoles. Alternative biaryl coupling reactions may
also be used.
[0084] The synthetic schemes described above afford a compound of
Formula I with Q=H. Alkylation of this intermediate with an alkyl
halide (Cl, Br, I or F) or other reactive alkylating agent (for
example, an sulfonate ester, such as mesylate or tosylate) in the
presence of an appropriate base and solvent and affords compounds
of Formula I in which Q now fits the generally outlined structural
requirements outlined or compounds of this invention.
Alternatively, biaryl coupling chemistry may be employed to prepare
Q is a directly attached aryl, heteroaryl, alkenyl or alkynyl. Such
chemistry is well known to those skilled in the art and has been
reported for coupling to heterocyclic nitrogens, such as those in
imidazole. It should be appreciated that both the alkylation and
coupling reactions may also give rise to variable amounts of the
undesired regioisomers, from which the desired product may be
obtained using any number of standard separation techniques,
including chromatography and crystallisation. By variation of the
alkylation conditions and dependent on the particular structure of
both reactants, the ratio of desired formula I to undesired
products may be greatly influenced. Examples of generally effective
conditions for the alkylation reaction and conditions which favor
the production of the regioisomer of Formula I are presented in
Liverton, et. al., J. Med. Chem. Vol. 42, No. 12, p2180-2190,
1999.
[0085] Regioselective placement of Q in compounds of Formula (I) is
possible as illustrated in Scheme IV. Addition of a primary amine
to the aldehyde prior to addition of the ketooxime results in
selective N-Q formation adjacent to the Aryl ring of the
hydroxyimidazole. Reduction as in Scheme III with trialkyl
phosphite or titanium trichloride affords compounds of Formula (I).
5
[0086] Suitable protecting groups for use with hydroxyl groups and
the imidazole nitrogen are well known in the art and described in
many references, for instance, Protecting Groups in Organic
Synthesis, Greene T W, Wiley-Interscience, New York, 1981. Suitable
examples of hydroxyl protecting groups include silyl ethers, such
as t-butyldimethyl or t-butyldiphenyl, and alkyl ethers, such as
methyl connected by an alkyl chain of variable link,
(CR.sup.13R.sup.14).sub.n. Suitable examples of imidazole nitrogen
protecting groups include tetrahydropyranyl.
SYNTHETIC EXAMPLES
[0087] The invention will now be described by reference to the
following examples which are merely illustrative and are not to be
construed as a limitation of the scope of the present
invention.
[0088] All temperatures are given in degrees centigrade (.degree.
C.), all solvents are highest available purity and all reactions
run under anhydrous conditions in an argon atmosphere unless
otherwise indicated. Mass spectra were performed upon a VG Zab mass
spectrometer using fast atom bombardment or on a micromass platform
electrospray ionization mass spectrometer in the positive ion mode
using 95:5 CH.sub.3CN/CH.sub.3OH with 1% formic acid as the carrier
solvent, unless otherwise indicated. .sup.1H-NMR (hereinafter
"NMR") spectra were recorded at 250 MHz or 400 Mz using a Bruker A
M 250 or Am 400 spectrometer. Multiplicities indicated are:
s=singlet, 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 the principal reactant. Flash chromatography is run over Merck
Silica gel 60 (230-400 mesh).
Example 1
[0089] Preparation of
2-tert-Butyl-4-naphthalen-2-yl-5-pyridin-4-yl-imidaz- ole 6
[0090] a) 2-Oxo-2-pyridin-4-yl-acetaldehyde Oxime.
[0091] Isoamyl nitrite (11.7 grams (hereinafter "g"), 0.1
millimoles (hereinafter "mmol")) was added to alkaline ethanol
[NaOH (4 g) in EtOH (50 milliliters (hereinafter "ml"))] followed
by cooling to 0.degree. and addition dropwise addition of
4-acetylpyridine (12.1 g in 95% ethanol 10 ml) with vigorous
stirring. After complete addition the reaction was placed in the
refrigerator overnight. The mixture was diluted with ether and
filtered to give a light reddish-brown solid. This was dissolved in
water and at 0.degree. acidified to pH 5.0 with glacial HOAc to
give an orange solid 6.76 g. The solid was recrystallized from
30/70 EtOH/H.sub.2O to give 2-Oxo-2-pyridin-4-yl-acetaldehyde oxime
as needles (5.0 g 33%). .sup.1H NMR(400 MHz, CDCl.sub.3) .delta.
8.61 (dd, J=4.6 Hz, 1.42 2H), 7.85 (s, 1H), 7.80 (dd, J=4.6, 1.5
Hz, 2H).
[0092] b) 2-tert-Butyl-5-pyridin-4-yl-imidazol-1-ol
[0093] A solution of 2-Oxo-2-pyridin-4-yl-acetaldehyde oxime (9.87
g, 65.8 mmol), trimethylacetaldehyde (6.8 g, 79 mmol), and ammonium
acetate (50.7 g) in glacial acetic acid (600 ml) was kept at 870
for 4 h, then at 800 for 4 h after the addition of trimethyl
acetaldehyde (1 ml). The yellow solution was cooled to 0.degree.
and carefully neutralized with conc. ammonium hydroxide with
vigorous stirring. At pH 7.0-7.2 a copious precipitate formed and
the mixture was stirred for 30 min. The beige solid was filtered
off and dried in a vacuum oven at 40.degree. to yield the title
compound (11.0 g). The aqueous filtrate was further extracted with
CH.sub.2Cl.sub.2 to give an additional yellow oil (3.12 g) as a
mixture of title compound and corresponding imidazole.
[0094] c) 4-(2-tert-Butyl-1-H-imidazol-4-yl)-pyridine
[0095] The compound in Example 1(b) was treated with trimethyl
phosphite (25 ml) in DMF (200 ml) and kept at 90.degree. for 3 hr.
The DMF was distilled off under reduced pressure and the residue
partitioned between water (pH 8) and chloroform and extracted
several times into chloroform. The organic portions were dried over
MgSO.sub.4 and evaporated then triturated with ether to give the
title compound. (9.33 g) .sup.1H NMR(400 MHz, CDCl.sub.3) .delta.
9.35 (br s, 1H), 8.56 (d, J=5.6 Hz, 2H), 7.68 (d, J=4.9, 21) 7.38
(s, 2H), 1.44 (s, 9H).
[0096] d) 4-(2-tert-Butyl-5-iodo-3-H-imidazol-4-yl)-pyridine
[0097] The compound in Example 1(c) (3.)g, 14.92 mmol in EtOH (60
ml) was treated with N-iodosuccinimide at room temperature
(hereinafter "rt") for 4 hours (hereinafter "h"). Thin layer
chromatography (Tlc) showed complete reaction and the mixture was
diluted with water slowly. The resultant solid was filtered and
dried under high vacuum overnight at 60.degree. to afford the title
compound. (4.39 g, 90%) MS m/e: 328 [M+H].sup.+, 326
[M-H].sup.-;
[0098] e) 2-tert-Butyl-4-naphthalen-2-yl
5-pyridin-4-yl-imidazole
[0099] In a heavy-walled pressure vessel
4-(2-tert-Butyl-5-iodo-3-H-imidaz- ol-4-yl)-pyridine (100
milligrams (hereinafter "mg"), 0.3 mmol) and
tetrakis-triphenylphosphine palladium (0) catalyst was dissolved in
toluene (2-3 ml) and argon was bubbled through the solution while
the other reagents were added: 1-naphthyl boronic acid (86 mg, 0.5
mmol), solid NaHCO.sub.3 (84 mg), water (500 uL), EtOH (500 uL).
The vessel was sealed and the reaction heated to 100.degree.
overnight. The reaction mixture was partitioned between 5%
NaHCO.sub.3 and EtOAc and extracted several times with EtOAc. The
combined organic layers were washed with brine, dried over
MgSO.sub.4, filtered, and evaporated. The crude product was
chromatographed on silica gel (chromatotron, eluting with 1-4%
MeOH/CH.sub.2Cl.sub.2) to give the title compound as a yellow
solid. (58 mg, 59%). mp 218-220.degree.; MS m/e: 328 [M+H].sup.+,
326 [M-H].sup.-; .sup.1H NMR(400 MHz, CDCl.sub.3) .delta. 9.63 (br
s, 1H), 8.16 (app d, J=4.8 Hz, 2H), 7.92 (br d, J=8.3 Hz, 2H), 7.64
(br d, 1H), 7.52 (m, 3H), 7.41 (t, J=6 Hz, 1H), 7.29 (br s, 2H),
1.49 (s, 9H).
Example 2
[0100] Preparation of
2-tert-Butyl-4-(6-Ethoxynaphthalen-2-yl)-5-pyridin-4- -yl-imidazole
7
[0101] a) 2-Bromo-6-ethoxy-naphthalene
[0102] To a suspension of sodium hydride (800 mg, 60% oil
dispersion) in DMF (20 ml) was added a solution of
6-Bromo-2-naphthol dropwise with stirring at 0.degree.. After gas
evolution had ceased, neat ethyl iodide (1.6 ml) was added dropwise
to the stirred solution. The reaction was stirred overnight at rt.
The reaction mixture was poured into water and extracted several
times with tert-butyl methyl ether. The combined organic extracts
were washed with water and brine, then dried over MgSO.sub.4,
filtered and evaporated under reduced pressure to give tan solid.
This was recrystallized from MeOH and dried under high vacuum to a
constant wt. giving a white solid (3.11 g, 60%). .sup.1H NMR(400
MHz, CDCl.sub.3) .delta. 7.92 (d, J=1.7 Hz, 1H), 7.65 (d, J=8.9 Hz,
1H), 7.59 (d, J=8.7 Hz, 2H), 7.16 (dd, J=8.9 Hz, 2.48, 11), 7.09
(d, J=2.4 Hz, 1H), 4.15 (q, 2H), 1.49 (t, 3H).
[0103] b) 6-Ethoxy-naphth-2-yl boronic Acid
[0104] To a solution of Example 2(a) (2.51 g, 10 mmol) in dry THF
(15 ml, freshly distilled from Na) at -78.degree. C. was added
dropwise 2.5 M n-butyl lithium (4 ml, 10 mmol) over .about.5 min.
The reaction was stirred at -78.degree. C. for 10 min then
triisopropyl borate (3.46 ml) was added and the reaction was
allowed to come to rt. The reaction was partitioned between 10%
ammonium chloride and tert-butyl methyl ether and further extracted
with and tert-butyl methyl ether (.times.3), washed with brine,
dried with MgSO.sub.4 and evaporated to a white solid. The solid
was triturated with hot hexanes to give pure title compound. (1.9
g, 88%) MS(ES) m/e: 217 [M+H].sup.+.
[0105] c)
2-tert-Butyl-4-(6-Ethoxynaphthalen-2-yl)-5-pyridin-4-yl-imidazol-
e
[0106] Following the procedure of Example 1(e), except substituting
6-Ethoxy-naphth-2-yl boronic acid for 1-naphthyl boronic acid the
title compound was prepared. (34 mg, 34%) mp 258-259.degree.; MS
m/e: 372 [M+H].sup.+, 370 [M-H].sup.-;
Example 3
[0107] Preparation of 4-(2-tert-but
1-5-(6-methoxy-napthalen-2)-3H-imadazo- l-4-yl)-pyridine 8
[0108] a) 6-Methoxy-2-naphthoic-N-methoxy-N-methyl Amide
[0109] At 0.degree. C., SOCl.sub.2(1.15 mL, 15.8 mmol) was added
very slowly to a stirred solution of 6-methoxy-2-naphthoic acid
(2.9 g, 14.3 mmol) in CH.sub.2Cl.sub.2 (40 mL) and Et.sub.3N (7.9
mL, 57.2 mmol). The solution became dark and homogeneous. After
stirring at room temperature for 40 min., N,O-dimethylhydroxylamine
hydrochloride (1.86 g, 17.16 mmol) was added. After the reaction
was stirred for 2 h, it was quenched with water, then extracted
with CH.sub.2Cl.sub.2 (3.times.). The organic layer was washed with
saturated Na.sub.2CO.sub.3(3.times.), dried (Na.sub.2SO.sub.4),
concentrated to afford 6-methoxy-2-naphthoic-N-methox- y-N-methyl
amide.(3.65 g, 94%) MS(ES) m/e 246 [M+H].sup.+.
[0110] b) 6-Methoxynaphth-2-yl-4-pyridylmethyl Ketone
[0111] At 0.degree. C., n-BuLi (2.5 M in hexane, 8.12 mL, 20.3
mmol) was added to a solution of diisopropylamine (3.32 mL, 23.6
mmol) in THF (20 mL) to generate LDA. The solution was cooled to
-78.degree. C., 4-picoline (2.00 mL, 20.3 mmol) was added to the
solution, the solution was kept at -78.degree. C., and stirred for
15 min, then compound in Example 3(a) (3.65 g, 14.9 mmol) was
added. The reaction was warmed to rt over 0.5 h, and stirred for
another 1 h. The reaction was quenched by NH.sub.4Cl (5 mL),
extracted with CH.sub.2Cl.sub.2 (3.times.). The organic layer was
washed with brine, dried (MgSO.sub.4), and concentrated. The
obtained residue was subjected to flash column (from 1% MeOH in
CH.sub.2Cl.sub.2 to 4% MeOH in CH.sub.2Cl.sub.2) to afford
6-methoxynaphth-2-yl-4-pyridylmethyl ketone. (3.3 g, 70%) MS(ES)
m/e 278 [M+H].sup.+.
[0112] c)
2-Hydroxyimino-1-(6-methoxynaphth-2-yl)-2-(4-pyridyl)ethan-1-one
[0113] NaNO.sub.2 (0.9 g, 14.3 mmol) was added to a suspension of
compound in Example 3(b) (3.3 g, 11.9 mmol) in 3N HCl (70 mL), and
H.sub.2O (70 mL). The slurry was stirred for 3 h, filtered, washed
with water, air dried to give
2-hydroxyimino-1-(6-methoxynaphth-2-yl)-2-(4-pyridyl)ethan-- 1-one.
(3.4 g, 93%) MS(ES) m/e 307 [M+H].sup.+.
[0114] d)
4-(2-tert-Butyl-5-(6-methoxy-naphalen-2-1-5-pyridin-4-yl-imidazo-
l-1-ol
[0115] Acetic acid (3 mL) was added to a mixture of
trimethylacetaldehyde (21 .mu.L, 1.37 mmol), ammonium acetate (125
mg, 1.6 mmol), and compound in Example 3(c) (50 mg, 0.16 mmol).
After the resulting solution was heated at 110.degree. C.
overnight, it was cooled to 0.degree. C., then NH.sub.4OH was added
to the solution slowly with stirring. The precipitate formed was
filtered, and dried to afford
4-(2-tert-butyl-5-(6-methoxy-napthalen-2-yl)-5-pyridin-4-yl)-imidazol-1-o-
l. (35 mg, 57%) MS(ES) m/e 375 [M+H].sup.+.
[0116] e)
4-(2-tert-Butyl-5-(6-methoxy-napthalen-2-yl)-3H-imidazol-4-yl)-p-
yridine
[0117] Triethylphosphite (63 mg, 0.4 mmol) was added to a stirred
solution of the compound in Example 3(d) (50 mg, 0.13 mmol) and
N,N-dimethylformamide (2 mL) at room temperature. The reaction was
stirred for 8 hours. Then excess water was added and the mixture
stirred for 3 hours. The solid precipitate was filtered and dried
under vacuum to afford the title compound. (27 mg, 57%) MSES) m/e
358 [M+H].sup.+.
Example 4
[0118] Preparation of
2-tert-Butyl-4-Inden-2-yl)-5-pyridin-4-yl-1H-imidazo- le 9
[0119] 2-tert-Butyl-4-Inden-2-yl)-5-pyridin-4-yl-1H-imidazole
Trifluoracetate Salt
[0120] A mixture of indene (175 uL, 1.5 mmol), tetrabutylammonium
chloride (83 mg, 0.3 mmol), palladium acetate (3 mg),
tri-o-tolylphosphine (6 mg), and the compound in Example 1(d) (100
mg, 0.3 mmol) in DMF (1 ml) were microwaved for 10 min at 30 W and
for 24 min at 45W. Both tlc and lc/ms were indicative of complete
reaction. The mixture was partitioned between CH.sub.2Cl.sub.2 and
5% NaHCO.sub.3. The aqueous layer was extracted with
CH.sub.2Cl.sub.2 (.times.4), washed with H.sub.2O, dried
(MgSO.sub.4), and concentrated. The crude product was
chromatographed on silica gel (chromatotron, from 1% MeOH in
CH.sub.2Cl.sub.2 to 4% MeOH in CH.sub.2Cl.sub.2) to afford title
compound (27 mg) contaminated with tetrabutylammonium chloride.
Preparative hplc (YMS-combiprep ODS-A, 20-80% acetonitrile/water
0.1% TFA) afforded pure title compound as a bright yellow solid
(9.2 mg, 10%). MS(ES) m/e 358 [M+H].sup.+. 370 [M-H].sup.-, 350
[+Cl--H].sup.-, 442 [M+I--H].sup.-.
Example 5
[0121] 2-tert-Butyl4-Benzofuran-2-yl-5-pyridin-4-yl-1H-imidazole
10
[0122] Following the procedure of Example 1(e), except substituting
Benzofuran-2-boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
by microwave for 16 min at 45W. (34 mg, 66%) MS(ES) m/e 318
[M+H].sup.+, 316 [M-H].sup.-.
Example 6
[0123]
4-(2-tert-Butyl-5-dibenzothiophen-4-yl-1H-imidazol-4-yl)pridine
11
[0124] Following the procedure of Example 1(e), except substituting
Dibenzothiaphene-4-boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
in a 96 well-plate format with microwave for 2 hr at 100-200W with
temperature not exceeding 65.degree.. Purified by hplc
(YMS-combiprep ODS-A, 20-80% acetonitrile/water 0.1% TFA (6 mg,
10%) Lc-MS(ES) m/e 384 [M+H].sup.+, 97%.
Example 7
[0125]
4-(2-tert-Butyl-5-dibenzofuran-4-yl-1H-imidazol-4-yl-pyridine
12
[0126] Following the procedure of Example 1(e), except substituting
Dibenzofuran-4-boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
in a 96 well-plate format with microwave for 2 hr at 100-200W with
temperature not exceeding 65.degree.. Purified by hplc
(YMS-combiprep ODS-A, 20-80% acetonitrile/water 0.1% TFA (9 mg,
16%) Lc-MS(ES) m/e 368 [M+H].sup.+, 100%
Example 8
[0127]
4-(5-Benzo[b]thiophen-2-yl-2-tert-butyl-1H-imidazol-4-yl-pyridine
13
[0128] Following the procedure of Example 1(e), except substituting
thiaphene-2-boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
in a 96 well-plate format with microwave for 2 hr at 100-200W with
temperature not exceeding 65.degree.. Purified by hplc
(YMS-combiprep ODS-A, 20-80% acetonitrile/water 0.1% TFA (34 mg,
64%) Lc-MS(ES) m/e 334 [M+H].sup.+, 100%
Example 9
[0129]
4-(5-Benzo[b]thiophen-3-yl-2-tert-butyl-1H-imidazol-4-yl-pyridine
14
[0130] Following the procedure of Example 1(e), except substituting
thiaphene-3-boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
in a 96 well-plate format with microwave for 2 br at 100-200W with
temperature not exceeding 65.degree.. Purified by hplc
(YMS-combiprep ODS-A, 20-80% acetonitile/water 0.1% TFA (5 mg, 8%)
Lc-MS(ES) m/e 334 [M+H].sup.+, 100%.
Example 10
[0131] 4-(2-tert-Butyl-5-thianthren-1-yl-1H-imidazol-4-yl)-pyridine
15
[0132] Following the procedure of Example 1(e), except substituting
thianthrene-1-boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
in a 96 well-plate format with microwave for 2 hr at 100-200W with
temperature not exceeding 65.degree.. Purified by hplc
(YMS-combiprep ODS-A, 20-80% acetonitrile/water 0.1% TFA (3 mg, 5%)
Lc-MS(ES) m/e 416 [M+H].sup.+, 100%.
Example 11
[0133]
4-(2-tert-Butyl-5-phenoxathin-4-yl-1H-imidazol-4-yl)-pyridine
16
[0134] Following the procedure of Example 1(e), except substituting
4-phenoxathine boronic acid for 1-naphthyl boronic acid and
Na.sub.2CO.sub.3 for NaHCO.sub.3, the title compound was prepared
in a 96 well-plate format with microwave for 2 hr at 100-200W with
temperature not exceeding 65.degree.. Purified by hplc
(YMS-combiprep ODS-A, 20-80% acetonitrile/water 0.1% TFA (4 mg, 6%)
Lc-MS(ES) m/e 400 [M+H].sup.+, 93%.
Example 12
[0135]
4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-1-methyl-1H-imidazol--
4-yl]-pyridine 17
[0136] Methylamine (2 mL of a 1M solution in methanol) was added to
an acetic acid solution (2 ml) of trimethylacetaldehyde (353 .mu.L,
3.2 mmol). After stirring at room temperature for 1 hour, the
keto-oxime was added (200 mg, 0.65 mmol). The resulting solution
was heated at 80.degree. C. for 24 hours. The reaction was allowed
to cool to room temperature, then the solution was neutralized with
NH.sub.4OH. The reaction mixture was extracted with methylene
chloride and the combined organics were dried over anhydrous
magnesium sulfate.
[0137] The crude product was dissolved in 5 mL of methanol and 1 mL
of TiCl.sub.3 (>10% in HCl) was added. The reaction was stirred
at room temperature for 18 hrs. The methanol was removed under
reduced pressure and the slurry was diluted with water. The pH was
adjusted to 8 with the addition of NH.sub.4OH, and the solution was
extracted with EtOAc. The combined organic extracts were dried over
MgSO.sub.4, filtered and concentrated. The crude product was
purified via flash chromatography (silica gel, 5%
methanol/methylene chloride) to afford 75 mg (0.2 mmol, 31% yield);
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.25 (d, 2H), 7.80 (d,
1H), 7.70 (s, 1H), 7.65 (d, 1H), 7.25 (d, 1H), 7.10 (m, 4H), 3.90
(s, 3H), 3.10 (s, 3H), 0.20 (s, 3H); MS (ES) 372 [M+H].sup.+.
Example 13
[0138]
4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-py-
ridin-2-yl}-(3 morpholin-4-yl-propyl)-amine 18
[0139] a) 6-Methoxynaphth-2-yl-(2-fluoropyridin-4-yl)-methyl
Ketone
[0140] At 0.degree. C., n-BuLi (2.5 M in hexane, 19.6 mL, 48.92
mmol) was added to a solution of diisopropylamine (8.0 mL, 57.08
mmol) in THF (60 ML) to generate LDA. After 15 min stirring at
0.degree. C., the solution was cooled to -78.degree. C. and
2-fluoropicoline (5.44 mL, 48.92 mmol) in THF (30 ml) was added to
the solution. The solution was kept at -78.degree. C., and stirred
for 30 min, then compound in Example 3(a) (10.0 g, 40.77 mmol) was
added. The reaction was warmed to room temperature (hereinafter
"rt") over 0.5 h, and stirred for another 40 min. The reaction was
quenched by KHPO.sub.4 (0.5 M), extracted with EtOAc (3.times.).
The organic layer was washed with brine, dried (MgSO.sub.4), and
concentrated then pumped under high vacuum to afford
6-methoxynaphth-2-yl-4-pyridylmethyl ketone as pale yellow
solid.(11.82 g, 98%) MS(ES) m/e 296 [M+H].sup.+.
[0141] b)
2-Hydroxyimino-1-(6-methoxynaphth-2-yl)-2-(2-fluoropyridin-4-yl)-
ethan-1-one
[0142] At 0.degree. C., t-butylnitrite (3.46 mL, 28.5 mmol) was
added dropwise to a solution of Example x(1) (8.0 g, 27.1 mmol) in
THF (400 mL) under Argon. After 3 min stirring at 0.degree. C., an
HCl solution (2M in ether, 32.0 mL, approx. 64 mmol) was added
dropwise to the solution. The solution was kept at rt for 2 h, then
the solvent was removed under reduced pressure and the brown oily
residue was partitioned between water and CH.sub.2Cl.sub.2 after
adjusting the pH to 8, extracted further with CH.sub.2Cl, washed
with brine, dried (MgSO.sub.4), and concentrated then pumped under
high vacuum overnight to afford on oil which was recrystallized
from t-butyl-O-methyl ether/hexane to give as an orange solid
2-hydroxyimino-1-(6-methoxynaphth-2-yl)-2-(2-fluoropyridin-4-yl)eth-
an-1-one. (7.66 g, 87%) MS(FS) m/e 325 [M+H].sup.+.
[0143] c)
{4-[2-tert-Butyl-5-(6-methoxy-naphtalen-2-yl-3-H-imidazol-4-yl]--
pyridin-2-yl}-(3-morpholin-4-yl-propyl)-amine
[0144] A mixture of trimethylacetaldehyde (60 .mu.L, 0.55 mmol),
ammonium trifluoroacetate (500 mg), N-(3-aminopropyl)morpholine
(200 .mu.L, >1.2 mmol) and compound in Example 2(x) (130 mg,
0.40 mmol) was heated as a melt at 150.degree. C. for several
hours. After cooling to rt, the reaction mixture was dissolved in
water and extracted with EtOAc (3.times.). The combined extracts
were concentrated and redissolved in DMSO and purified by hplc to
afford after lyopholization pure
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-pyridin-
-2-yl}-(3-morpholin-4-yl-propyl)-amine. (68.4 mg, 57%) MS(ES) m/e
500 [M+H].sup.+100%.
Example 14
[0145]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-yl}-(3-morpholin-4-yl-ethyl)-amine 19
[0146] Following the procedure of Example 13(c), except
substituting N-(3-aminoethyl)morpholine for
N-(3-aminopropyl)morpholine, the title compound was prepared. (124
mg, 43%) Lc-MS(ES) m/e 486 [M+H].sup.+, 100% uv, 100% els.
Example 15
[0147]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-ylamino}-propyl)-pyrrolidin-2-one 20
[0148] Following the procedure of Example 13(c), except
substituting N-(3'-aminopropyl)-2-pyrrolidinone for
N-(3-aminopropyl)morpholine, the title compound was prepared. (99.1
mg, 41%) Lc-MS(ES) m/e 498 [M+H].sup.+, 89% uv, 100% els.
Example 16
[0149]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-yl}-[3-(2-methyl-piperidin-1-yl-propyl]-amine 21
[0150] Following the procedure of Example 13(c), except
substituting 1-(3-aminopropyl)-2-pipecoline for
N-(3-aminopropyl)morpholine, the title compound was prepared. (57.1
mg, 23%) Lc-MS(ES) m/e 512 [M+H].sup.+, 96% uv, 100% els.
Example 17
[0151]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-yl}-N,N-diethyl-butane-1,4-diamine 22
[0152] Following the procedure of Example 13(c), except
substituting 4-(diethylamino)butylamine for
N-(3-aminopropyl)morpholine, the title compound was prepared. (51.4
mg, 21%) Lc-MS(ES) m/e 500 [M+H].sup.+, 91% uv, 100% els.
Example 18
[0153]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-yl}-(3-pyrrolidin-1-yl-propyl)-amine 23
[0154] Following the procedure of Example 13(c), except
substituting n-(2-aminopropyl)pyrrolidine for
N-(3-aminopropyl)morpholine, the title compound was prepared. (10.2
mg, 4%) Lc-MS(ES) m/e 484 [M+H].sup.+, 100% uv, 100% els.
Example 19
[0155]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-yl}-[3-(4-methyl-piperazin-1-yl)-propyl]-amine 24
[0156] Following the procedure of Example 13(c), except
substituting 1-(3-aminopropyl)-4-methylpiperazine for
N-(3-aminopropyl)morpholine, the title compound was prepared. (54.1
mg, 22%) Lc-MS(ES) m/e 513 [M+H].sup.+, 100% uv, 100% els
Example 20
[0157]
{4-[2-tert-Butyl-5-(6-methoxy-naphthalen-2-yl)-3-H-imidazol-4-yl]-p-
yridin-2-yl}-N,N-diethyl-ethane-1,2-diamine 25
[0158] Following the procedure of Example 13(c), except
substituting N,N-diethylethylenediamine for
N-(3-aminopropyl)morpholine, the title compound was prepared. (23.2
mg, 100%) Lc-MS(ES) m/e 472 [M+H].sup.+, 89% uv, 100% els
[0159] Methods of Treatment
[0160] The Tie receptors are proteins of approximately 125 kDa,
with a single putative transmembrane region. The extracellular
domain of these receptors is divided into several regions: there
are 3 regions that have a pattern of cysteine expression found in
EGF-like domains; there are 2 regions that have some weak homology
to and structural characteristics of immunoglobulin-like domains;
and there are 3 regions with homology to the fibronectin III repeat
structure. This particular combination of extracellular domains is
unique to the Tie receptors. The intracellular portion of Tie2 is
most closely related (.about.40% identity) to the kinase domains of
FGF-R1, PDGF-R and c-kit. The intracellular portions of Tie2
contain all of the features of tyrosine kinases, including a GXGXXG
ATP binding site consensus sequence and typical tyrosine kinase
motifs (i.e., HRDLAARN and DFGL).
[0161] These receptors have sparked interest because they are the
only receptor tyrosine kinases, other than those receptors for
vascular endothelial cell growth factor (VEGF), that are largely
restricted to endothelial cells in their expression. There are
several lines of evidence showing that Tie2 is important in
angiogenesis, detailed in the following paragraphs.
[0162] a. Tie1 and Tie2 Receptor Location
[0163] i. Embryological Vascular Development
[0164] The location of the Tie receptors in the embryo has been
studied by a number of investigators using in situ hybridization.
Korhonen et al. (Blood 80:2548-2555, 1992) showed that the mRNA for
Tie receptors is located in endothelial cells of all forming blood
vessels and in the endocardium of mouse embryos. During embryonic
development, expression of the Tie receptors is seen in angioblasts
and all developing vasculature. Expression of the Tie receptors
follows expression of the major VEGF receptor, Flk-1, by 12-24
hours during mouse embryogenesis, perhaps suggesting sequential and
different actions of these receptor systems (Schnurch and Risau,
Development 119: 957-968, 1993). Cloning of a 1.2 Kb genomic 5'
flanking region of Tie2, coupled to a lacZ gene and expressed in
transgenic mice, demonstrated a selective pattern of expression in
endothelial cells during embryonic development (Schlaeger et al.,
Development 121:1089-1098, 1995). Thus, the Tie2 promoter acts to
assure endothelial cell-specific expression of Tie2.
[0165] ii. In Adult Tissues
[0166] The similarities between embryonic angiogenesis and
pathologic angiogenesis yields the hypothesis that blocking Tie2
function, in tumors or chronic inflammatory settings, for examples,
may block angiogenesis, thus blocking further cell proliferation
and provide therapeutic benefit. Tie mRNA cannot be observed in
adult skin, except at sites of active wound healing, where the
proliferating capillaries in the granulation tissue contain
abundant Tie mRNA (Korhonen et al., Blood 80:2548-2555, 1992). PCR
amplification of cDNA from normal skin fails to show a signal for
Tie receptor (Kaipainen et al., Cancer Res. 54:6571-6577, 1994). In
contrast, a strong signal is seen with cDNA from metastasizing
melanomas, where in situ studies localize this signal to the
vascular endothelium. While Tie receptor expression is
down-regulated in the established vasculature, it is upregulated in
the angiogenesis that occurs in the ovary during ovulation, in
wounds and in tumor (breast, melanoma and renal cell carcinoma)
vasculature, consistent with prevailing views that angiogenesis in
the adult borrows from embryonic angiogenic mechanisms.
[0167] b. Tie Knockout Animals
[0168] Homozygous mice with a Tie2 knockout, or carrying a
transgene encoding a "dominant-negative" Tie2 receptor, confirmed
that the Tie2 receptor is critical for embryonic development
(Dumont et al., Genes Dev. 8:1897-1909, 1994; Sato et al., Nature
376:70-74, 1995). Embryonic death in these mice occurred due to
vascular insufficiency and there were dramatically reduced numbers
of endothelial cells. Vasculogenesis--that is the differentiation
of endothelial cells and the in situ formation of vessels--appeared
relatively normal in mice lacking Tie2. The subsequent sprouting
and remodeling resulting in formation of vessel branches
(angiogenesis) was drastically reduced in the Tie2 mutant mice
embryos. This lack of sprouting and angiogenesis resulted in
substantial growth retardation, particularly of the brain, neural
tube and heart, resulting in lack of viability. This exemplifies
the critical importance of Tie2 in angiogenesis. This is
significant, as angiogenesis is regulated by a number of growth
factors. Interestingly, Flk1 (VEGF receptor) knockout mice exhibit
embryo lethal defects in vasculogenesis that occur earlier than
those of Tie2 disruption. Disruption of the Tie1 receptor yields a
much different, and later, defective phenotype; the mouse embryo
dies late in development due to hemorrhage resulting from defective
integrity of an otherwise well formed vasculature. Taken together,
these studies suggest that the VEGF/Flk1 and Tie systems operate in
sequential fashion, with Tie2 having a critical role in
angiogenesis.
[0169] c. Tie2 Ligands
[0170] Recently, two ligands for the Tie2 receptor have been
reported. Angiopoietin-1 binds and induces the tyrosine
phosphorylation of Tie2 and its expression in vivo is in close
proximity with developing blood vessels (Davis et al., Cell
87:1161-1169,1996). Mice engineered to lack Angiopoietin-1 display
angiogenic deficits reminiscent of those previously seen in mice
lacking Tie2 receptors, demonstrating that Angiopoietin-1 is a
primary physiologic ligand for Tie2 and that Tie2 has critical in
vivo angiogenic actions (Suri et al., Cell 87:1171-1180, 1996).
Angiopoietin-2 was identified by homology screening and shown to be
a naturally occurring antagonist for Tie2 receptors. Transgenic
overexpression of Angiopoietin-2 disrupts blood vessel formation in
the mouse embryo (Maisonpierre et al., Science 277:55-60, 1997).
Together, these results support a role for Tie2 receptors in
angiogenesis.
[0171] In order to use the compounds of formula (I) in therapy,
they will normally be formulated into a pharmaceutical composition
in accordance with standard pharmaceutical practice.
[0172] According to a further aspect of the invention there is
provided a pharmaceutical composition comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier.
[0173] No toxicological effects are indicated/expected when a
compound of formula (I) is administered in the above mentioned
dosage range.
[0174] 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
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
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
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not deleterious
to the recipient thereof.
[0175] The pharmaceutical carrier employed may be, for example,
either a solid or liquid. Exemplary of solid carriers is lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, stearic acid and the like. Exemplary of liquid carriers
is syrup, peanut oil, olive oil, water and the like. Similarly, the
carrier or diluent may include time delay material well known to
the art, such as glyceryl mono-stearate or glyceryl distearate
alone or with a wax.
[0176] 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 carrier will vary
widely but preferably will be from about 25 mg. to about 1 g. 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.
[0177] Compounds of Formula (I) may be administered topically, that
is by non-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.
[0178] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of inflammation 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.
[0179] 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 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.
[0180] 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 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 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 silicas, and other ingredients such
as lanolin, may also be included.
[0181] 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 include a surface active
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.degree. C. 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 phenylmercuric nitrate or acetate
(0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate
(0.01%). Suitable solvents for the preparation of an oily solution
include glycerol, diluted alcohol and propylene glycol.
[0182] 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 as an
aerosol formulation or a metered dose inhaler, may be prepared by
conventional techniques.
[0183] The compounds of Formula (I) may also be used topically in
the treatment or prophylaxis of disease states exacerbated by
excessive or inappropriate angiogenesis.
[0184] The compounds of Formula (I) are administered in an amount
sufficient to inhibit Tie2 receptor activity such that it is
regulated down to normal levels, or in some case to subnormal
levels, so as to ameliorate or prevent the disease state.
[0185] Chronic diseases which have an inappropriate angiogenic
component are various ocular neovasularizations, such as diabetic
retinopathy and macular degeneration. Other chronic diseases which
have an excessive or increased proliferation of vasculature are
tumor growth and metastasis, atherosclerosis, and certain arthritic
conditions. Therefore Tie2 tyrosine kinase receptor inhibitors will
be of utility in the blocking of the angiogenic component of these
disease states.
[0186] The term "excessive or increased proliferation of
vasculature inappropriate angiogenesis" as used herein includes,
but not limited to, diseases which are characterized by hemangiomas
and ocular diseases. The term "inappropriate angiogenesis" as used
herein includes, but not limited to, diseases which are
characterized by vessel proliferation with accompanying tissue
proliferation, such as occurs in cancer, metastasis, arthritis,
psoriasis and atherosclerosis.
[0187] For all methods of use disclosed herein for the compounds of
Formula (I), the daily oral dosage regimen will preferably be from
about 0.1 to about 80 mg/kg of total body weight, preferably from
about 0.2 to 30 mg/kg, more preferably from about 0.5 mg to 15 mg.
The daily parenteral dosage regimen will be from about 0.1 to about
80 mg/kg of total body weight, preferably from about 0.2 to about
30 mg/kg, and more preferably from about 0.5 mg to 15 mg/kg. 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 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 compound of Formula (I) or a pharmaceutically acceptable salt
thereof given per day for a as defined number of days, can be
ascertained by those skilled in the art using conventional course
of treatment determination tests.
[0188] Pharmacological Test Methods
[0189] A. Measurement of Tie2 Kinase Activity
[0190] A partial cDNA clone for the Tie2 receptor was used to make
protein for Tie kinase studies. In order to generate the primary
screening assay, a baculovirus expressed GST fusion for Tie2 kinase
domain was constructed and expressed using the commercial vector
pAcG1 (Pharmingen).
[0191] This final construct was transfected into Baculovirus and
soluble GST fusion products used in the screening assay. Prior work
had demonstrated the use of a baculovirus expressed GST fusion for
the murine Tie2 kinase domain to screen for candidate
target/signaling molecules (Huang et al., Oncogene 11:2097-2103,
1995). Tie2 kinase activity assay: The Tie2 kinase activity assay
was typically run in one of 2 ways described as follows. Minor
variations in the assay give similar results.
[0192] 1. Autophosphorylation Flashplate Assay
[0193] Materials:
[0194] Kinase buffer (final 20 mM Tris-HCl, pH 7.0, 100 mM NaCl, 12
mM MgCl2, 1 mM DTT).
[0195] Gamma .sup.33p-ATP (usually final amount of 0.5-1
uCi/well)
[0196] ATP (final 30 uM, or other desired concentration)
[0197] Flashplate (96-well, polystyrene microplate with plastic
scintillator coated wells)
[0198] TopCount (microplate scintillation counter)
[0199] Procedures:
[0200] Turn on incubator shaker and adjust temperature to
30.degree. C.
[0201] Add 20 ul of 3.times.kinase buffer per well to the
Flashplate
[0202] Add 20 ul of protein per well except for background.
Compounds added, typically in DMSO stocks, at 1-2 ul.
[0203] Add 20 ul mixture of gamma .sup.33p-ATP and cold ATP per
well.
[0204] Total volume is 60 ul.
[0205] Cover with transparent polyester film.
[0206] Incubate at 30.degree. C. for two hours in shaker, or
desired time.
[0207] Take Flashplate out of the shaker, wash five times (for
example, with 300 ul of 10 uM ATP in 1.times.PBS per well).
[0208] Read plate on TopCount or other counting instrument. Results
are calculated as % inhibition
[0209] and IC50, using normal calculation methods.
[0210] 2. Fluorescence Polarization for Tie 2 Kinase
[0211] Final Assay conditions:
[0212] 50 mM HEPES pH 7.5
[0213] 2% DMSO (when screening compounds)
[0214] 250 uM ATP
[0215] 2 mM MgCl.sub.2
[0216] 1 mM DTT
[0217] 50 uM NaVanidate
[0218] 10 uM peptide substrate
[0219] activated tie 2 kinase * see activation protocol below
[0220] Peptide Substrate:
[0221] RFWKYEFWR-OH
[0222] MW (TFA salt)=1873 Da
[0223] Make a 1 mM peptide stock and store at -20.degree. C.
[0224] Dilute to 100 uM just prior to use.
[0225] 9.times.Kinase buffer:
[0226] 450 mM HEPES pH 7.5
[0227] 900 mM NaCl
[0228] 450 uM NaVanidate
[0229] 18 mM MgCl.sub.2
[0230] 100 mM DTT
[0231] Can be made up ahead of time and stored in aliquots at
-20.degree. C.
[0232] ATP Stock:
[0233] Make a 25 mM ATP stock and store in aliquots at -20.degree.
C. until needed. Dilute to 2.5 mM prior to use.
[0234] Procedure:
[0235] For a 50 ul reaction add the following to each well of a
96-well black half-area plate (Costar, cat# 3694)
[0236] 1. 5 ul of compound in 20% DMSO
[0237] 2. 5 ul 9.times.kinase buffer.
[0238] 3. 5 ul 2.5 mM ATP.
[0239] 4. 5 ul 100 uM peptide substrate.
[0240] 5. 25 ul PTK detection mix (Panvera, P-2652, 50 ml--UK
distributor is Cambridge Bioscience)
[0241] 6. 5 ul activated tie 2 kinase (protocol below) diluted in
1.times. buffer to initiate the reaction.
[0242] 7. Read polarization on an FP instrument cycling for 30-50
minutes in accordance with enzyme activity.
[0243] Representative compounds of Formula (I), Examples 1 to 20
were found to be active in this fluorescence assay, having an IC50
of <1 uM.
[0244] Activation of Tie 2 Kinase Protocol:
[0245] Final Buffer Conditions:
[0246] 20 mM Tris-HCl pH 7.5
[0247] 12 mM MgCl.sub.2
[0248] 100 mM NaCl
[0249] 20 uM NaVanidate
[0250] 1 mM DTT
[0251] 300 uM ATP
[0252] Procedure
[0253] 1. Incubate 5 uM tie 2 kinase in the 300 uM ATP and the
buffering conditions described above.
[0254] 2. Allow to incubate at 27.degree. C. for 2 hours.
[0255] 3. Add 2.5 ml reaction to a NAP-25 desalting column
(Pharmacia Biotech cat. no. 17-0852-O.sub.2) pre-equilibrated in 20
mM Tris-HCl pH 7.5, 100 mM NaCl.sub.2 to separate the ATP from the
enzyme.
[0256] 4. Elute the enzyme with 5.0 ml 20 mM Tris-HCl pH 7.5, 100
mM NaCl.sub.2; the protein concentration should be 2.5 uM at this
point.
[0257] 5. Aliquot out the enzyme and store at -80.degree. C. as
soon as possible.
[0258] B. Measurement of Tie2 Receptor Signal Transduction--a
Cellular Assay
[0259] HEL cells (ATCC # TIB180) are cultured at between 1 and
5.times.10.sup.5 ml in RPMI-1640 medium supplemented with 2 mM
glutamine and 10% FBS as a suspension culture. Sixteen to
thirty-six hours prior to an experiment, the necessary number of
cells are passaged into 0.5% FBS/RPMI medium. On the day of an
experiment, cells are harvested and resuspended at a density of
0.5-1.0.times.10.sup.7 cells ml in 0.5% FBS RPMI and seeded at 2-3
ml/well in six well plates.
[0260] Alternatively, Human Umbilical Vein Endothelial Cells
(HUVECs) (Clonetics--Walkersville, Md.) may be used for the assay.
HUVECs between passages 2 and 12, are plated at 2.times.10.sup.5
and 1.times.10.sup.6 cells per well in a six well plate in
supplemented EGM (Clonetics). After 24 hours the media is changed
to EBM containing 3% BSA (Clonetics), and the cells are cultured
overnight and used for assay the following day.
[0261] Cells are treated with inhibitory compounds at appropriate
concentrations for 30-45 minutes. The contents of the wells are
mixed briefly on a rocker (approx. 30 seconds) and then incubated
at 37.degree. C. The cells are then treated with a source of native
ligand, such as serum or fibroblast conditioned medium for 10
minutes
[0262] At the end of 10 minute incubation period the plate is
placed on ice. The cells are harvested and the media is removed.
The cells are lysed in denaturing sample buffer
Invitrogen--Carlsbad, Calif.). The suspension is sonicated for 5
pulses at a medium setting and returned to ice. The phosphorylation
state of the Tie2 receptor is determined Western blotting-and
detection by an anti-phospho-Tie2 antibody, as detailed below
(Harlow, E., and Lane, D. P., Antibodies--A Laboratory Manual, Cold
Spring Harbor Laboratory Press: New York, 1988.). Thirty ul of the
lysate are run on a 7.5% SDS/polyacrylamide gel. The gel is then
transferred to a nitrocellulose or PVDF membrane as per the
manufacturer's instructions for Western blotting.
[0263] The blots are washed with PBS 0.05% between-20 and then
blocked with 3% BSA/PBS/Tween for 1 hour at room temperature. The
blots are then incubated with 1 ug/ml anti-phospho-Tie2 antibody
(SmithKline Beecham) in PBS/0.05% Tween for 1 hour. The blot is
then washed 4 times with PBS/Tween for 5 minutes each. The blot is
incubated with an anti-mouse-HRP conjugate secondary antibody at
the dilution recommended by the manufacturer, in PBS/Tween for 1
hour. The blot is washed in PBS/Tween, 4 times for 5 minutes each.
After the last wash, the blot is developed by the ECL method
(Amersham) or some equivalent.
[0264] Using a densitometer or graphics program (e.g.
ImageQuant--Molecular Dynamics), each blot is scanned. The Tie-2
band is isolated and "boxed out" for each lane. The pixel volume or
comparable measure for each sample is analyzed. Also, an
appropriate background region of the same dimensions is determined
for each sample. After adjusting for background, phosphorylation is
expressed as the ratio of phosphotyrosine staining, relative to the
untreated control.
[0265] Angiogenesis In Vivo Model
[0266] Measurement of Angiogenesis In Vivo--Murine Air Pouch
Granuloma Model:
[0267] Described below a model of inflammatory angiogenesis used to
show that inhibition of Tie2 will stop the tissue destruction of
excessive or inappropriate proliferation of blood vessels. The
murine airpouch granuloma model of chronic inflammation (Kimura et
al., 1985, J. Pharmacobio-Dyn., 8:393-400; Colville-Nash et al.,
1995, J. Pharm. and Exp. 772er., 274:1463-1472) whose disclosure is
incorporated herein by reference in its entirety, is characterized
by inflammatory cell influx, fibrous tissue proliferation and
intense angiogenesis. It is representative of inflammatory
angiogenesis and demonstrates that the angiogenic component can be
pharmacologically modulated independently of granuloma growth and
size. In addition, angiogenesis can be accurately quantitated by a
vascular casting method.
[0268] Day 1, mice are anesthetized using Aerrane (isoflurane) gas
(5%) or other approved methods, after which 3 mls of air is
injected into the dorsal subcutaneous tissue using a 27 g needle.
Mice are allowed to recover.
[0269] Day 0, mice are again anesthetized using Aerrane or other
approved methods, once anesthetized 0.5 ml of Freunds complete
adjuvant with 0.1% v/v croton oil is injected into the air pouch
formed on Day -1. The animals also begin their dosing regime
(number of day's dependent upon study) with the animals typically
receiving compound in 0.2 ml N,N, Dimethyl Acetoacetamide(DMA)
(Sigma, St. Louis, Mo.)/Cremephor E1 (Sigma, St. Louis, Mo.),
saline (10/10/80) or other appropriate vehicle. The animals are
allowed to recover and all subsequent dosing is performed on the
animals in the absence of anesthetics.
[0270] Days 1-5, animals are dosed according to schedule.
[0271] On Day 6 the animals are again anesthetized after which a
vascular cast is made (Kimura et al., 1986, J. Pharmacobio-Dyn.,
9:442-446); this involves a 1 ml tail vein i.v. injection of a
Carmine Red (100%) (Sigma, St. Louis, Mo.)/gelatin (5%) (Sigma, St.
Louis, Mo.) solution. The animals are then sacrificed by lethal
dose of anesthesia and chilled at 4.degree. C. for 2 hours prior to
the removal of the granuloma tissue.
[0272] When the granuloma is removed it is weighed and then dried
for 3 days at 45.degree. C. and reweighed. The dried tissue is then
digested in 0.9 ml of a 0.05M phosphate buffer pH 7.0 containing 12
U/ml-1 papain (Sigma, St. Louis, Mo.) and 0.33 g/L.sup.-1
N-acetyl-1-Cysteine (Sigma, St. Louis, Mo.) at 57.degree. C. for 3
days. After 3 days digestion the carmine red is solublized by the
addition of 0.11 ml 5 mM NaOH. Samples are centrifuged and then
filtered using 0.2 um acrodiscs. The carmine content is then
determined against a carmine red standard curve (0.5 to 2 mg/ml)
generated in extracted tissue from non carmine treated animals and
read at 490 nm. Sample and standard values are determined typically
using DeltaSoft Elisa analysis software (Biometallics Inc.,
Princeton, N.J.). The carmine content is then used to determine the
vascular indexes for the various treatments, vascular index being
the mg carmine dye/gm dry tissue.
[0273] The effect of compounds on vascular density was typically
measured for 6 days after induction of the granuloma. This time
point has been determined to be at or near the peak of
angiogenesis. As a positive control medroxyprogesterone, an
angiostatic steroid (Gross et al., 1981, Proc. Natl. Acad. Sci.
USA, 78:1176-1180), whose disclosure is hereby incorporated by
reference in its entirety, was utilized. This control demonstrated
a maximum reduction of 50% in this model. Medroxyprogesterone had
no effect on granuloma size as measured by dry weight.
[0274] Angiogenesis Model--In Vivo
[0275] Measurement of Angiogenesis In Vivo--Matrigel Model:
[0276] Angiogenesis is modeled in-vivo by placing an extra-cellular
matrix gel, beneath the skin of a mouse for approximately one week,
and then employing several measures to quantitate angiogenic
invasion of the gel (Biancone, L, et. al. Development of
Inflammatory Angiogenesis by Local Stimulation of Fas In Vivo. J.
Exp. Med. 186:147, 1997.). Briefly, reduced growth factor,
endotoxin free Matrigel.RTM. (Becton-Dickinson, Bedford, Mass.) is
a gel at low temperatures. Antibodies or known angiogenic agents
are mixed with the gel, such that they do not constitute more than
2% of the total volume. Eight week old or older, C57 female mice
are administered 0.5 ml of the Matrigel.RTM. by dorsal subcutaneous
injection through chilled syringes. At physiological temperature,
the liquid Matrigel.RTM. rapidly forms a solid and cohesive gel.
During the course of the experiment, mice receive test compounds or
controls administered as described above. After six days, the mice
are sacrificed and the Matrigel.RTM. plugs recovered. Angiogenesis
is quantitated by analyzing the hemoglobin content of the gel by
the method of Drabkin (Drablin, D L and Austin, J H:
Spectrophotometric Studies II. Preparations from washed blood
cells; nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem
112:51, 1935.) (Sigma, St. Louis, Mo.), or by staining and
quantitating blood vessel with CD31 staining as described
above.
[0277] All publications, including but not limited to patents and
patent applications, 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.
[0278] The above description fully discloses the invention
including preferred embodiments thereof. Modifications and
improvements of the embodiments 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
which an exclusive property or privilege is claimed are defined as
follows.
* * * * *