U.S. patent application number 13/556670 was filed with the patent office on 2012-11-08 for bradykinin receptor agonists and uses thereof to treat ocular hypertension and glaucoma.
This patent application is currently assigned to ASTELLAS PHARMA INC.. Invention is credited to Hwang-Hsing Chen, Keith D. Combrink, Mark R. Hellberg, Suchismita Mohapatra, Abdelmoula Namil, Iok-Hou Pang, Ganesh Prasanna, Bryon Severns, Najam A. Sharif.
Application Number | 20120283260 13/556670 |
Document ID | / |
Family ID | 44720101 |
Filed Date | 2012-11-08 |
United States Patent
Application |
20120283260 |
Kind Code |
A1 |
Combrink; Keith D. ; et
al. |
November 8, 2012 |
BRADYKININ RECEPTOR AGONISTS AND USES THEREOF TO TREAT OCULAR
HYPERTENSION AND GLAUCOMA
Abstract
The invention provides compositions and methods for treating
and/or preventing ocular disorders associated with increased
intraocular pressure. In particular, the compounds are bradykinin
agonists.
Inventors: |
Combrink; Keith D.;
(Arlington, TX) ; Mohapatra; Suchismita;
(Arlington, TX) ; Hellberg; Mark R.; (Arlington,
TX) ; Sharif; Najam A.; (Arlington, TX) ;
Prasanna; Ganesh; (Fort Worth, TX) ; Pang;
Iok-Hou; (Grand Prairie, TX) ; Severns; Bryon;
(Arlington, TX) ; Chen; Hwang-Hsing; (Fort Worth,
TX) ; Namil; Abdelmoula; (Arlington, TX) |
Assignee: |
ASTELLAS PHARMA INC.
Chuo-ku Tokyo
TX
ALCON RESEARCH, LTD.
Fort Worth
|
Family ID: |
44720101 |
Appl. No.: |
13/556670 |
Filed: |
July 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13211827 |
Aug 17, 2011 |
8252793 |
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13556670 |
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61374740 |
Aug 18, 2010 |
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Current U.S.
Class: |
514/234.5 ;
514/253.04; 514/253.09; 514/256; 514/275; 514/300; 514/302;
514/312; 514/313; 514/318; 514/333; 514/338; 514/395; 544/131;
544/331; 544/333; 544/362; 544/364; 546/116; 546/121; 546/153;
546/162; 546/194; 546/256; 546/273.7; 548/306.1 |
Current CPC
Class: |
C07D 413/14 20130101;
A61P 27/02 20180101; A61P 27/06 20180101; C07D 401/12 20130101;
A61P 27/00 20180101; C07D 498/04 20130101; C07D 401/06 20130101;
A61P 9/12 20180101; C07D 403/06 20130101; C07D 401/14 20130101 |
Class at
Publication: |
514/234.5 ;
546/273.7; 514/338; 546/256; 514/333; 546/116; 514/302; 544/364;
514/253.09; 546/194; 514/318; 544/331; 514/275; 544/131; 546/121;
514/300; 544/362; 514/253.04; 546/153; 514/312; 544/333; 514/256;
546/162; 514/313; 548/306.1; 514/395 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439; C07D 401/14 20060101 C07D401/14; A61K 31/444 20060101
A61K031/444; C07D 498/04 20060101 C07D498/04; A61K 31/496 20060101
A61K031/496; A61K 31/4545 20060101 A61K031/4545; A61K 31/506
20060101 A61K031/506; C07D 413/14 20060101 C07D413/14; A61K 31/5377
20060101 A61K031/5377; C07D 471/04 20060101 C07D471/04; C07D 401/12
20060101 C07D401/12; A61K 31/4709 20060101 A61K031/4709; C07D
403/04 20060101 C07D403/04; A61K 31/4184 20060101 A61K031/4184;
A61P 27/02 20060101 A61P027/02; A61P 27/06 20060101 A61P027/06;
A61P 9/12 20060101 A61P009/12; C07D 401/06 20060101 C07D401/06 |
Claims
1. A compound of Formula 1: ##STR00143## wherein, R.sup.1, R.sup.2
independently=--CH.sub.3 or --Cl; R.sup.3.dbd.C.sub.1-C.sub.3alkyl;
A is: ##STR00144## R.sup.4=--OR.sup.5, --NR.sup.7R.sup.10 or
--R.sup.5; R.sup.5.dbd.C.sub.1-C.sub.3 alkyl;
X=--(CH.sub.2).sub.n--, --CF.sub.2CH.sub.2--; n=1-3; Y is:
##STR00145## D.sub.1=N, CH, CR.sup.5, or COR.sup.5;
R.sup.6=--C(O)OR.sup.10, --N(R.sup.10)C(O)R.sup.11,
--N(R.sup.10)S(O.sub.2)R.sup.11, --C(O)N(R.sup.10)(R.sup.11),
--N(R.sup.10)C(O)OR.sup.11, --N(R.sup.10)C(O)NR.sup.7R.sup.11,
NR.sup.10R.sup.12, or --(CH.sub.2).sub.mNR.sup.10R.sup.12;
R.sup.7.dbd.H or C.sub.1-C.sub.3 alkyl; R.sup.8.dbd.O,
NC(O)R.sup.11, NS(O.sub.2)R.sup.11, NC(O)OR.sup.11,
NC(O)NR.sup.7R.sup.11, or NR.sup.11; R.sup.9.dbd.NC(O)R.sup.11,
NS(O.sub.2)R.sup.11, NC(O)OR.sup.11, NC(O)NR.sup.7R.sup.11, or
NR.sup.11; R.sup.10.dbd.H or C.sub.1-C.sub.3 alkyl; R.sup.11.dbd.H,
C.sub.1-C.sub.4 alkyl, or --(CH.sub.2).sub.p--Z; R.sup.12.dbd.H,
C.sub.1-C.sub.3 alkyl, or --C(O)R.sup.7; m=1-3; p=2-4; Z=--OH or
--OR.sup.12; R.sup.13=--N(R.sup.10)C(O)R.sup.11,
--N(R.sup.10)S(O.sub.2)R.sup.11, --C(O)N(R.sup.10)(R.sup.11),
--N(R.sup.10)C(O)OR.sup.11, or --N(R.sup.10)C(O)NR.sup.7R.sup.11;
R.sup.14=--H, --CH.sub.3, or -cyclopropyl R.sup.15=--H,
C.sub.1-C.sub.4 alkyl, C(O)OR.sup.11, --C(O)N(R.sup.10)(R.sup.11)
or --(CH.sub.2).sub.mNR.sup.10R.sup.12; W=--O-- or --NH--; B.sup.1
is: ##STR00146## D.sup.2=N, CH or CF; ; and B.sup.2 is:
##STR00147## D.sup.2=N, CH or CF; or a prodrug thereof.
2. A compound of claim 1, wherein: R.sup.1, R.sup.2
independently=--CH.sub.3 or --Cl; R.sup.3.dbd.C.sub.1-C.sub.3
alkyl; A is: ##STR00148## R.sup.4=--OR.sup.5 or --NR.sup.7R.sup.10;
R.sup.5.dbd.C.sub.1-C.sub.3 alkyl; X=--(CH.sub.2).sub.n--; n=1-3; Y
is: ##STR00149## D.sub.1.dbd.N, CH, CR.sup.5, or COR.sup.5;
R.sup.6.dbd.--N(R.sup.10)C(O)R.sup.11, --C(O)N(R.sup.10)(R.sup.11),
or --N(R.sup.10)C(O)OR.sup.11; R.sup.7=--H or C.sub.1-C.sub.3
alkyl; R.sup.8=--O--, --NC(O)R.sup.11, --NC(O)OR.sup.11, or
--NC(O)NR.sup.7R.sup.11; R.sup.9.dbd.NC(O)R.sup.11 or
NC(O)OR.sup.11; R.sup.10=--H or C.sub.1-C.sub.3 alkyl;
R.sup.11=--H, --C.sub.1-C.sub.4 alkyl or --(CH.sub.2).sub.p--Z;
R.sup.14=--H, --CH.sub.3, or -cyclopropyl; p=2-4; Z=--OH or
--OR.sup.12; W=--O-- or --NH--; B.sup.1 is: ##STR00150## and
B.sup.2.dbd.B.sup.1; or a prodrug thereof.
3. A compound of claim 2, wherein the compound is selected from the
group consisting of: Compound 7,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpiperazine-1-carboxamide Compound 8,
3-(4-acetamidopiperidin-1-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyrid-
in-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-
-oxoethyl)propanamide; Compound 28,
3-(4-acetamidophenyl)-N-(2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylme-
thoxy)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)propanamid-
e; Compound 32,
3-(6-acetamidopyridin-3-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-
-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-o-
xoethyl)propanamide; Compound 33,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylbenzamide; Compound 34, (S)-tert-butyl
3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imi-
dazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)carbamoyl)pyrrolidi-
ne-1-carboxylate; Compound 44,
4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoethy-
l)-N-methylmorpholine-2-carboxamide; Compound 56,
4-(3-((2-((3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl-
)oxy)methyl)-2,4-dimethylphenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylbenzamide; Compound 64,
5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpicolinamide; Compound 74,
3-(6-(2-aminoacetamido)pyridin-3-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-
-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)a-
mino)-2-oxoethyl)propanamide; and Compound 78,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-((1-methyl-1H-imidazol-4-yl)meth-
yl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)a-
mino)-3-oxopropyl)-N-methylbenzamide.
4. A pharmaceutical composition comprising a compound of claim
1.
5. A topical ophthalmic formulation comprising a therapeutically
effective amount of a compound of claim 1, and one or more
ingredients selected from the group consisting of surfactants,
tonicity agents, buffers, preservatives, co-solvents, and viscosity
building agents.
6. The formulation of claim 5, wherein the therapeutically effect
amount of the compound is between 0.001-1.0%.
7. The formulation of claim 6, wherein the therapeutically effect
amount of the compound is 0.005%.
8. A method for controlling intraocular pressure in a patient
comprising administering to the patient a composition comprising a
pharmaceutically acceptable ophthalmic carrier and a
therapeutically effective amount of a compound of claim 1.
9. The method of claim 8, wherein: R.sup.1, R.sup.2
independently=--CH.sub.3 or --Cl; R.sup.3.dbd.C.sub.1-C.sub.3
alkyl; A is: ##STR00151## R.sup.4=--OR.sup.5 or --NR.sup.7R.sup.10;
R.sup.5.dbd.C.sub.1-C.sub.3 alkyl; X=--(CH.sub.2).sub.n--; n=1-3; Y
is: ##STR00152## D.sub.1=N, CH, CR.sup.5, or COR.sup.5;
R.sup.6=--N(R.sup.10)C(O)R.sup.11, --C(O)N(R.sup.10)(R.sup.11), or
--N(R.sup.10)C(O)OR.sup.11; R.sup.7=--H or C.sub.1-C.sub.3 alkyl;
R.sup.8=--O--, --NC(O)R.sup.11, --NC(O)OR.sup.11, or
--NC(O)NR.sup.7R.sup.11; R.sup.9.dbd.NC(O)R.sup.11 or
NC(O)OR.sup.11; R.sup.10=--H or C.sub.1-C.sub.3 alkyl;
R.sup.11=--H, --C.sub.1-C.sub.4 alkyl or --(CH.sub.2).sub.p--Z;
R.sup.14=--H, --CH.sub.3, or -cyclopropyl; p=2-4; Z=--OH or
--OR.sup.12; W=--O-- or --NH--; B.sup.1 is: ##STR00153## and
B.sup.2.dbd.B.sup.1; or a prodrug thereof.
10. The method of claim 9, wherein the compound is selected from
the group consisting of: Compound 7,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpiperazine-1-carboxamide; Compound 8,
3-(4-acetamidopiperidin-1-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyrid-
in-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-
-oxoethyl)propanamide; Compound 28,
3-(4-acetamidophenyl)-N-(2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylme-
thoxy)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)propanamid-
e; Compound 32,
3-(6-acetamidopyridin-3-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-
-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-o-
xoethyl)propanamide; Compound 33,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylbenzamide; Compound 34, (S)-tert-butyl
3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imi-
dazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)carbamoyl)pyrrolidi-
ne-1-carboxylate; Compound 44,
4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoethy-
l)-N-methylmorpholine-2-carboxamide; Compound 56,
4-(3-((2-((3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl-
)oxy)methyl)-2,4-dimethylphenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylbenzamide; Compound 64,
5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpicolinamide; and Compound 78,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-((1-methyl-1H-imidazol-4-yl)meth-
yl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)a-
mino)-3-oxopropyl)-N-methylbenzamide.
11. The method of claim 8, wherein the patient has glaucoma.
12. The method of claim 8, wherein the patient has ocular
hypertension.
13. The method of claim 8, further comprising administering to the
patient a therapeutically effective amount of an intraocular
(IOP)-lowering agent in combination with a non-peptidic BK
agonist.
14. The method of claim 13, wherein the IOP-lowering agent is an
aqueous humor production (inflow) inhibitor.
15. The method of claim 8, wherein the composition comprises one or
more ingredients selected from the group consisting of surfactants,
tonicity agents, buffers, preservatives, co-solvents, and viscosity
building agents.
16. The method of claim 8, wherein the therapeutically effect
amount of a non-peptidic BK agonist is between 0.001-1.0%.
17. The method of claim 16, wherein the therapeutically effect
amount of the non-peptidic BK agonist is 0.005%.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application, which claims
priority to U.S. patent application Ser. No. 13/211,827 filed Aug.
17, 2011, which claims priority under 35 U.S.C. .sctn.119 to U.S.
Provisional Patent Application No. 61/374,740, filed Aug. 18, 2010,
the entire contents of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods and compositions to treat
ocular disorders associated with elevated intraocular pressure
(IOP). More specifically the invention relates to disorders
including, but not limited to, ocular hypertension and
glaucoma.
BACKGROUND OF THE INVENTION
[0003] Bradykinin (BK) is an endogenous nonapeptide
(H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH; SEQ ID NO: 1) that is
generated by cleavage of the precursor polypeptide (kininogen) by
specific proteases (kallikriens) within numerous tissues of the
body (Regoli, D. and Barabe, J. Pharmacol. Rev., 32, 1-46, 1980;
Hall, J. M., Pharmacol. Ther., 56, 131-190, 1992; Leeb-Lundberg et
al., Pharmacol. Rev. 57: 27-77, 2005). Certain enzymes of the
kininase family degrade BK and related peptides and thus inactivate
these peptides. All components of the kallkrien/kinin system,
including specific receptors activated by BK and related peptides,
are present in the human eye cells and tissues (Ma et al., Exp. Eye
Res. 63: 19-26, 1996; Sharif and Xu, Exp. Eye Res. 63: 631-637,
1996). BK and another endogenous peptide (Lys-BK;
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; SEQ ID NO: 2) interact
with two major BK receptor-subtypes, namely B.sub.1 and B.sub.2 to
produce their biological effects (Regoli and Barabe, Pharmacol.
Rev., 32, 1-46, 1980; Hall, Pharmacol. Ther., 56, 131-190, 1992;
Leeb-Lundberg et al., Pharmacol. Rev. 57: 27-77, 2005).
[0004] The B.sub.2-subtype is found under normal physiological
conditions, while the B.sub.1-subtype is typically induced during
injury or trauma (Hall, J. M., Pharmacol. Ther., 56, 131-190, 1992;
Leeb-Lundberg et al., Pharmacol. Rev. 57: 27-77, 2005). While the
B.sub.1-subtype has a low affinity for BK and a high affinity for
Des-Arg.sup.9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe; SEQ ID NO: 3)
and Lys-BK, the B.sub.2-subtype has a high affinity for BK and
Lys-BK but a low affinity for Des-Arg.sup.9-BK. Both receptor
subtypes have been cloned and shown to be coupled to G-proteins and
phospholipase C and their activation results in the generation of
the second messengers inositol trisphosphate (IP.sub.3) and
diacylglycerol (DAG) (Bhoola et al., Pharmacol. Rev. 44: 1080,
1992; Hall, J. M., Pharmacol. Ther., 56, 131-190, 1992;
Leeb-Lundberg et al., Pharmacol. Rev. 57: 27-77, 2005). While
IP.sub.3 mobilizes intracellular Ca.sup.2+ ([Ca.sup.2+].sub.i) DAG
phosphorylates protein kinase C and together these events lead to
the final biological response such as cell shape change, tissue
contraction or fluid secretion.
[0005] Additional events ensuing from elevation of
[Ca.sup.2+].sub.i include activation of nitric oxide synthase (NOS)
to produce nitric oxide (NO) that in turn activates guanylyl
cyclase to produce cyclic guanosine monophosphate (cGMP), and
activation of cycloxygenases and/or phospholipase A.sub.2 that
produce endogenous prostaglandins that in turn elevate
intracellular cyclic adenosine monophosphate (cAMP) (Leeb-Lundberg
et al., Pharmacol. Rev. 57: 27-77, 2005). Activation of the
B.sub.2-receptor can also lead to inhibition of cAMP production in
host cells transfected with the human recombinant B.sub.2 receptors
(Meini et al., Brit. J. Pharmacol. 143: 938-941, 2004). The
majority of the physiological and pathological effects of BK are
mediated by the B.sub.2-receptor-subtype. However, pharmacological
evidence has pointed to two additional BK-receptor subtypes, namely
B.sub.3 and B.sub.4 (Hall, Pharmacol. Ther., 56, 131-190, 1992;
Sharma, Gen. Pharmacol., 24, 267-274, 1993). B.sub.3 and B.sub.4
receptor subtypes are actually stimulated by certain peptide BK
antagonists whereas the B.sub.1 and B.sub.2 subtypes are blocked by
the latter antagonists (Sharma, J. N., Gen. Pharmacol., 24,
267-274, 1993). While the presence of B.sub.3 or B.sub.4 receptor
subtypes in the eye has not been investigated to-date, there is
precedence for their existence in this organ since there is a
robust BK-precursor and BK-generating enzyme pool in human ocular
tissues and the presence of B.sub.1 and B.sub.2 receptors (Ma et
al., Exp. Eye Res., 63: 19-26, 1996).
[0006] Two new families of peptides related to BK, namely ovikinins
(Schroder et al. J. Biol. Chem. 272: 12475-12481, 1997) and
bombinakinins (Lai et al., Biochem. Biophys. Res. Comm. 286: 259,
2001; Lai et al. Peptides, 24: 199, 2003; O'Rouke et al., Regul.
Peptides 121: 65, 2004; Lee et al., Regul. Peptides, 127: 207,
2005) have been discovered recently that may react with BK
receptors or similar receptors. Additionally, a new receptor termed
GPR100 has been recently discovered with which BK also interacts
(Boels and Schaller, Br. J. Pharmacol. 140: 932-938, 2003).
[0007] Additional useful properties imparted by BK or BK mimetics
include the lowering of mRNA of connective tissue growth factor
(CTGF) (Huang et al. Am. J. Physiol. Lung Cell Mol. Physiol. 290:
L1291-L1299, 2006), a fibrotic cytokine that has been implicated in
the possible etiology of ocular hypertension by promoting
deposition of collagen and fibronectin in the TM area
(International Patent Application No. PCT/US2003/012521 to Fleenor
et al. published Nov. 13, 2003 as WO 03/092584); BK-induced
inactivation of RhoA (Am. J. Physiol. Lung Cell Mol. Physiol. 290:
L129-L1299, 2006); BK-induced blunting of systemic hypertension
(Majima et al., Hypertension 35: 437-442, 2000) and BK-induced
increase in blood flow (Ito et al. Br. J. Pharmacol. 138: 225-233,
2003), which can be beneficial for retinoprotection (Tamaki et al.,
J. Ocular Pharmacol. Ther. 15: 313-321, 1999). In addition, BK has
been shown to attenuate the release of pro-inflammatory cytokines
from activated microglial cells (Noda et al., J. Neurochem. 101:
397-410, 2007).
[0008] There are a number of ocular conditions that are caused by,
or aggravated by, damage to the optic nerve head, degeneration of
ocular tissues, and/or elevated intraocular pressure (IOP). For
example, "glaucomas" are a group of debilitating eye diseases that
are a leading cause of irreversible blindness in the United States
and other developed nations. Primary Open Angle Glaucoma ("POAG")
is the most common form of glaucoma (Quigley, Br. J. Ophthalmol.,
80: 389-393, 1996). The disease is characterized by the
degeneration of the trabecular meshwork, leading to obstruction of
the normal ability of aqueous humor to leave the eye without
closure of the space (e.g., the "angle") between the iris and
cornea (Rohen, Ophthalmol. 90: 758-765, 1983; Quigley, Br. J.
Ophthalmol., 80: 389-393, 1996). A characteristic of such
obstruction in this disease is an increased IOP, resulting in
progressive visual loss and blindness if not treated appropriately
and in a timely fashion. The disease is estimated to affect between
0.4% and 3.3% of all adults over 40 years old. Moreover, the
prevalence of the disease rises with age to over 6% of those 75
years or older. Thus, close to 70 million are afflicted by glaucoma
(Quigley, Br. J. Ophthalmol., 80: 389-393, 1996).
[0009] Glaucoma affects three separate tissues in the eye. The
elevated IOP associated with POAG is due to morphological and
biochemical changes in the trabecular meshwork (TM), a tissue
located at the angle between the cornea and iris, and ciliary
muscle (CM) bundles. Most of the nutritive aqueous humor exits the
anterior segment of the eye through the TM. The progressive loss of
TM cells and the build-up of extracellular debris in the TM of
glaucomatous eyes leads to increased resistance to aqueous outflow,
thereby raising IOP. Elevated IOP, as well as other factors such as
ischemia, cause degenerative changes in the optic nerve head (ONH)
leading to progressive "cupping" of the ONH and loss of retinal
ganglion cells and axons. The detailed molecular mechanisms
responsible for glaucomatous damage to the TM, ONH, and the retinal
ganglion cells are unknown.
[0010] Twenty years ago, the interplay of ocular hypertension,
ischemia and mechanical distortion of the optic nerve head was
heavily debated as the major factors causing progression of visual
field loss in glaucoma. Since then, other factors including
excitotoxicity, nitric oxide, absence of vital neurotrophic
factors, abnormal glial/neuronal interplay and genetics have been
implicated in the degenerative disease process. The consideration
of molecular genetics deserves some discussion insofar as it may
ultimately define the mechanism of cell death, and provide for
discrimination of the various forms of glaucoma. Within the past 10
years, over 15 different glaucoma genes have been mapped and 7
glaucoma genes identified. However, despite such progress, the
glaucomas still remain poorly understood.
[0011] Glaucoma is a progressive disease which leads to optic nerve
damage and, ultimately, total loss of vision. Since there is a good
correlation between IOP control and prevention/reduction of
glaucomatous damage in POAG patients (Mao et al., Am. J.
Ophthalmol. 111: 51-55, 1991), several therapeutic agents have been
developed to treat ocular hypertension (Clark and Yorio, Nature
Rev. Drug Discovery, 2: 448-459, 2003; Sharif and Klimko,
Ophthalmic Agents, in Comprehensive Medicinal Chemistry II., Vol.
6, Chapter 6.12, p. 297-320; Eds: D. J. Triggle and J. B. Taylor,
Elsevier Oxford, 2007). Thus, it is known that elevated IOP can be
at least partially controlled by administering drugs which either
reduce the production of aqueous humor within the eye, such as
beta-blockers and carbonic anhydrase inhibitors, or increase the
outflow of aqueous humor from the eye, such as miotics and
sympathomimetics. Unfortunately, many of the drugs conventionally
used to treat ocular hypertension have a variety of problems. For
instance, miotics such as pilocarpine can cause blurring of vision
and other visual side effects, which may lead either to decreased
patient compliance or to termination of therapy. Systemically
administered carbonic anhydrase inhibitors can also cause serious
side-effects such as nausea, dyspepsia, fatigue, and metabolic
acidosis, which can affect patient compliance and/or necessitate
the termination of treatment. Another type of drug, beta-blockers,
has increasingly become associated with serious pulmonary side
effects attributable to their effects on beta-2 receptors in
pulmonary tissue. Sympathomimetics may cause tachycardia,
arrhythmia and hypertension. Recently, certain prostaglandins and
prostaglandin derivatives have been described in the art as being
useful in reducing IOP. Typically, however, prostaglandin therapy
for the treatment of elevated IOP is attended by undesirable
side-effects, such as irritation and hyperemia of varying severity
and duration. There is, therefore, a continuing need for therapies
that control elevated IOP associated with glaucoma without the
degree of undesirable side-effects attendant to most conventional
therapies.
SUMMARY OF THE INVENTION
[0012] The invention provides compounds that are bradykinin
agonists. The invention further provides compositions and methods
for treating and/or preventing an ocular condition associated with
elevated intraocular pressure (IOP), such as ocular hypertension
and glaucoma. In certain aspects, a method of the invention
comprises administering to a patient a composition comprising a
therapeutically effective amount of a compound of Formula I in a
pharmaceutically acceptable ophthalmic vehicle.
[0013] Specific preferred embodiments of the invention will become
evident from the following more detailed description of certain
preferred embodiments and the claims.
DETAILED DESCRIPTION OF THE INVENTION
[0014] There are conflicting reports in the literature as to the
functions and involvement of BK in the modulation of IOP in various
animal models. For example, while intravenous infusion of BK
apparently lowered IOP (Chiang et al., Arch. Int. Pharmacodyn.
Ther. 207: 131-138, 1974; Funk et al., Graefes Arch. Clin. Exp.
Ophthalmol. 223: 145-149, 1985), injection of BK directly into the
anterior chamber of the eye raised IOP and caused intense miosis
(Cole and Ungar, Ophthalmic Res. 6: 308-314, 1974; Yokahama et al.,
Jpn. J. Ophthalomol. 34: 245-255, 1990) and increased both aqueous
humor inflow and outflow (Green and Elijah, Exp. Eye Res. 33:
239-245, 1981). Furthermore, BK either had no effect on aqueous
humor outflow (no ciliary muscle retrodisplacement) or decreased
outflow (with ciliary muscle retrodisplacement) in cynomolgus
monkey eyes upon injection of BK into the eye anterior chamber
(Kaufman et al. Exp. Eye Res. 35: 191-199, 1982). Additionally, in
perfused human and bovine anterior eye segments BK decreased
outflow facility (Llobet et al., Invest. Ophthalmol. Vis. Res., 40:
113-125, 1999), while another group has recently demonstrated an
apparent increase in outflow in bovine eyes (Webb et al., J. Ocular
Pharmacol. Ther. 22: 310-316, 2006). Such conflicting data coupled
with an existing patent (U.S. Pat. No. 6,500,831) that contemplated
the need for BK antagonists to elicit ocular hypotension has
resulted in confusion about the potential role of endogenous BK and
related peptides and their receptors in the modulation of IOP and
ocular hypertension.
[0015] Some obvious drawbacks and reasons for the lack of
consistent observations noted above are probably related to species
differences in the effects of BK on IOP changes and on the fact
that BK is a labile peptide that can easily and rapidly be
inactivated by kininases and other proteases when it comes into
contact with body fluids (Hall, Pharmacol. Ther., 56, 131-190,
1992).
[0016] In certain embodiments, the invention provides compounds
that can be represented by the general Formula I:
##STR00001## [0017] wherein, [0018] R.sup.1, R.sup.2
independently=--CH.sub.3 or --Cl; [0019]
R.sup.3.dbd.C.sub.1-C.sub.3alkyl; [0020] A is:
[0020] ##STR00002## [0021] R.sup.4=--OR.sup.5, --NR.sup.7R.sup.10
or --R.sup.5; [0022] R.sup.5.dbd.C.sub.1-C.sub.3alkyl; [0023]
X=--(CH.sub.2).sub.n-- or --CF.sub.2CH.sub.2--; [0024] n=1-3;
[0025] Y is:
[0025] ##STR00003## [0026] D.sub.1=N, CH, CR.sup.5, or COR.sup.5;
[0027] R.sup.6=--C(O)OR.sup.10, --N(R.sup.10)C(O)R.sup.11,
--N(R.sup.10)S(O.sub.2)R.sup.11, --C(O)N(R.sup.10)(R.sup.11),
--N(R.sup.10)C(O)OR.sup.11, --NR.sup.10)C(O)NR.sup.7R.sup.11,
NR.sup.10R.sup.12, or --(CH.sub.2).sub.mNR.sup.10R.sup.12; [0028]
R.sup.7.dbd.H or C.sub.1-C.sub.3 alkyl; [0029] R.sup.8=0,
NC(O)R.sup.11, NS(O.sub.2)R.sup.11, NC(O)OR.sup.11,
NC(O)NR.sup.7R.sup.11, or NR.sup.11; [0030]
R.sup.9.dbd.NC(O)R.sup.11, NS(O.sub.2)R.sup.11, NC(O)OR.sup.11,
NC(O)NR.sup.7R.sup.11, or NR.sup.11; [0031] R.sup.10.dbd.H or
C.sub.1-C.sub.3 alkyl; [0032] R.sup.11.dbd.H, C.sub.1-C.sub.4
alkyl, or --(CH.sub.2).sub.p--Z; [0033] R.sup.12.dbd.H,
C.sub.1-C.sub.3 alkyl, or --C(O)R.sup.7; [0034] m=1-3; p=2-4;
[0035] Z=--OH or --OR.sup.12; [0036]
R.sup.13=--N(R.sup.10)C(O)R.sup.11,
--N(R.sup.10)S(O.sub.2)R.sup.11, --C(O)N(R.sup.10)(R.sup.11),
--N(R.sup.10)C(O)OR.sup.11, or --N(R.sup.10)C(O)NR.sup.7R.sup.11;
[0037] R.sup.14=--H, --CH.sub.3, or -cyclopropyl; [0038]
R.sup.15=--H, C.sub.1-C.sub.3 alkyl, C(O)OR.sup.11,
--C(O)N(R.sup.10)(R.sup.11), or
--(CH.sub.2).sub.mNR.sup.10R.sup.12; [0039] W=--O-- or --NH--;
[0040] B.sub.1 is:
[0040] ##STR00004## [0041] D.sup.2=N, CH or CF; [0042] ; and [0043]
B.sub.2 is:
[0043] ##STR00005## [0044] D.sup.2=N, CH or CF;
[0045] Preferred compounds of Formula I are those in which: [0046]
R.sup.1, R.sup.2 independently=--CH.sub.3 or --Cl; [0047]
R.sup.3.dbd.C.sub.1-C.sub.3 alkyl; [0048] A is:
[0048] ##STR00006## [0049] R.sup.4=--OR.sup.5 or
--NR.sup.7R.sup.10; [0050] R.sup.5.dbd.C.sub.1-C.sub.3 alkyl [0051]
X=--(CH.sub.2).sub.n--; [0052] n=1-3; [0053] Y is:
[0053] ##STR00007## [0054] D.sub.1.dbd.N, CH, CR.sup.5, or
COR.sup.5; [0055] R.sup.6=--N(R.sup.10)C(O)R.sup.11,
--C(O)N(R.sup.10 (R.sup.11), or)-N(R.sup.10)C(O)OR.sup.11; [0056]
R.sup.7=--H or C.sub.1-C.sub.3 alkyl; [0057] R.sup.8=--O--,
--NC(O)R.sup.11, --NC(O)OR.sup.11, or --NC(O)NR.sup.7R.sup.11;
[0058] R.sup.9.dbd.NC(O)R.sup.11 or NC(O)OR.sup.11; [0059]
R.sup.10=--H or C.sub.1-C.sub.3 alkyl; [0060] R.sup.11=--H,
C.sub.1-C.sub.4 alkyl or --(CH.sub.2).sub.p--Z; [0061]
R.sup.14=--H, --CH.sub.3, or -cyclopropyl; [0062] p=2-4 [0063]
Z=--OH or --OR.sup.12 [0064] W=--O-- or --NH--; [0065] B.sub.1
is:
[0065] ##STR00008## [0066] and [0067] B.sup.2.dbd.B.sup.1.
[0068] Other preferred compounds of Formula I are:
[0069] Compound 7,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpiperazine-1-carboxamide;
[0070] Compound 8,
3-(4-acetamidopiperidin-1-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyrid-
in-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-
-oxoethyl)propanamide;
[0071] Compound 28,
3-(4-acetamidophenyl)-N-(2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylme-
thoxy)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)propanamid-
e;
[0072] Compound 32,
3-(6-acetamidopyridin-3-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-
-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-o-
xoethyl)propanamide;
[0073] Compound 33,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylbenzamide;
[0074] Compound 34, (S)-tert-butyl
3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imi-
dazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)carbamoyl)pyrrolidi-
ne-1-carboxylate;
[0075] Compound 44,
4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoethy-
l)-N-methylmorpholine-2-carboxamide;
[0076] Compound 56,
4-(3-((2-((3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl-
)oxy)methyl)-2,4-dimethylphenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylbenzamide;
[0077] Compound 64,
5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpicolinamide;
[0078] Compound 74,
3-(6-(2-aminoacetamido)pyridin-3-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-
-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)a-
mino)-2-oxoethyl)propanamide; or
[0079] Compound 78,
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-((1-methyl-1H-imidazol-4-yl)meth-
yl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)a-
mino)-3-oxopropyl)-N-methylbenzamide.
[0080] In certain embodiments, a BK agonist of the invention is a
compound shown in Table 1.
TABLE-US-00001 TABLE 1 Structure Compound # ##STR00009## 1
##STR00010## 2 ##STR00011## 3 ##STR00012## 4 ##STR00013## 5
##STR00014## 6 ##STR00015## 7 ##STR00016## 8 ##STR00017## 9
##STR00018## 10 ##STR00019## 11 ##STR00020## 12 ##STR00021## 13
##STR00022## 14 ##STR00023## 15 ##STR00024## 16 ##STR00025## 17
##STR00026## 18 ##STR00027## 19 ##STR00028## 20 ##STR00029## 21
##STR00030## 22 ##STR00031## 23 ##STR00032## 24 ##STR00033## 25
##STR00034## 26 ##STR00035## 27 ##STR00036## 28 ##STR00037## 29
##STR00038## 30 ##STR00039## 31 ##STR00040## 32 ##STR00041## 33
##STR00042## 34 ##STR00043## 35 ##STR00044## 36 ##STR00045## 37
##STR00046## 38 ##STR00047## 39 ##STR00048## 40 ##STR00049## 41
##STR00050## 42 ##STR00051## 43 ##STR00052## 44 ##STR00053## 45
##STR00054## 46 ##STR00055## 47 ##STR00056## 48 ##STR00057## 49
##STR00058## 50 ##STR00059## 51 ##STR00060## 52 ##STR00061## 53
##STR00062## 54 ##STR00063## 55 ##STR00064## 56 ##STR00065## 57
##STR00066## 58 ##STR00067## 59 ##STR00068## 60 ##STR00069## 61
##STR00070## 62 ##STR00071## 63 ##STR00072## 64 ##STR00073## 65
##STR00074## 66 ##STR00075## 67 ##STR00076## 68 ##STR00077## 69
##STR00078## 70 ##STR00079## 71 ##STR00080## 72 ##STR00081## 73
##STR00082## 74 ##STR00083## 75 ##STR00084## 76 ##STR00085## 77
##STR00086## 78 ##STR00087## 79 ##STR00088## 80 ##STR00089## 81
##STR00090## 82 ##STR00091## 83 ##STR00092## 84 ##STR00093## 85
##STR00094## 86 ##STR00095## 87
[0081] As used herein, a "bradykinin agonist" or "BK agonist"
refers to a compound of Formula 1.
[0082] A BK agonist of the invention can be generated, for example,
as described in the following Schemes.
##STR00096##
##STR00097##
##STR00098## ##STR00099##
##STR00100##
##STR00101##
##STR00102##
##STR00103##
[0083] The invention provides methods for treating or preventing
ocular hypertension and ocular diseases associated with elevated
intraocular pressure (IOP), such as glaucoma. In certain
embodiments, the methods of the invention comprise the step of
administering a pharmaceutical composition to the eye of a patient,
wherein the composition comprises a therapeutically effective
amount of a non-peptide bradykinin (BK) agonist and a
pharmaceutically acceptable ophthalmologic carrier.
[0084] In particular embodiments, the invention provides
pharmaceutical compositions comprising at least one non-peptide BK
agonist. The pharmaceutical compositions of the invention can be
used to control IOP in a patient, thereby treating or preventing
ocular hypertension and diseases associated with elevated IOP, such
as glaucoma. As used herein, the phrases "control IOP" and
"controlling IOP" refer to the ability of a pharmaceutical
composition of the invention to prevent an increase of a patient's
IOP and/or to lower a patient's existing IOP. Thus, a
pharmaceutical composition of the invention can be used, for
example, to prevent ocular hypertension from progressing into an
ocular disease associated with elevated IOP, such as glaucoma.
Alternatively, a pharmaceutical composition of the invention can be
used, for example, to treat a patient who has been diagnosed with
glaucoma, thereby reducing the patient's elevated IOP and improving
and/or restoring vision to the patient.
[0085] As used herein, the term "patient" includes human and animal
subjects.
[0086] The term "therapeutically effective amount" refers to the
amount of a pharmaceutical composition of the invention determined
to produce a therapeutic response in a mammal. Such therapeutically
effective amounts are readily ascertained by one of ordinary skill
in the art and using methods as described herein.
[0087] As used herein, the term "pharmaceutically acceptable
ophthalmic carrier" refers to those carriers that cause at most,
little to no ocular irritation, provide suitable preservation if
needed, and deliver one or more non-peptide BK agonists of the
present invention in a homogenous dosage.
[0088] The terms "pharmaceutical composition" and "composition" as
used herein refer to a composition comprising a pharmaceutically
acceptable ophthalmologic carrier, excipient, or diluent and a BK
agonist as described herein that is capable of inducing a desired
therapeutic effect (e.g. lowering IOP or preventing an increase in
IOP) when properly administered to a patient.
[0089] Compounds of the invention may exist as one or more
stereoisomers and mixtures thereof, wherein asymmetric or chiral
centers are present. Stereoisomers include enantiomers and
diastereomers, and mixtures of enantiomers or diastereomers.
[0090] Various geometric isomers and mixtures thereof may exist in
the compounds of the present invention, resulting from the
disposition of substituents around a carbon-carbon double bond, a
carbon-nitrogen double bond, a cycloalkyl group, or a heterocycle
group.
[0091] All such isomers and mixtures thereof are specifically
included within the scope of this invention. Individual
stereoisomers of compounds of the present application may be
prepared synthetically using techniques well known to those of
skill in the art.
[0092] It is to be understood that compounds disclosed herein may
exhibit the phenomenon of tautomerism.
[0093] Thus, the formulae drawings depicting chemical structures of
certain compounds of the invention can represent only one of the
possible tautomeric or stereoisomeric forms. It is to be understood
that the invention encompasses any tautomeric or stereoisomeric
form, and mixtures thereof, and is not to be limited merely to any
one tautomeric or stereoisomeric form utilized within the naming of
the compounds or formulae drawings.
[0094] Furthermore, certain embodiments of the present invention
comprise pharmaceutically acceptable salts of compounds according
to Formula I. Pharmaceutically acceptable salts comprise, but are
not limited to, soluble or dispersible forms of compounds according
to Formula I that are suitable for treatment of disease without
undue undesirable effects such as allergic reactions or toxicity.
Representative pharmaceutically acceptable salts include, but are
not limited to, acid addition salts such as acetate, citrate,
benzoate, lactate, or phosphate and basic addition salts such as
lithium, sodium, potassium, or aluminum.
[0095] In order to reduce possible ocular side-effects such as
redness (hyperemia) or irritation, the compositions of this
invention can be converted to suitable prodrugs by incorporation of
acceptable functional groups to the non-peptide BK agonists, such
that the prodrug would readily and rapidly penetrate the cornea and
be then hydrolyzed to release the active species of the conjugate
molecule into the aqueous humor. In this way, the residence time
for the drug on the ocular surface is reduced along with the
reduced extent and duration of ocular surface side-effects. A
prodrug can be identified and generated using techniques well known
to those skilled in the art (e.g. for ocular hypotensive
prostaglandins isopropyl esters or amides are known; Stjernschantz
et al. Adv. Prostaglandin. Thrombox. Leukotr. Res., 23: 513-518,
1995; Woodward et al. J. Pharmacol. Exp. Ther. 305:772-785,
2003).
[0096] The compositions of the invention can be administered to an
eye of a patient as solutions, suspensions, or emulsions
(dispersions) in a suitable ophthalmic carrier. For example, the
compositions can be delivered topically to the eye in the form of
drops, sprays, or gels. Alternatively, the compositions can be
administered by injection (e.g., intravitreal, intracameral,
intraocular, intraorbital, and/or subconjunctival and/or sub-tenon
injection). The compositions can also be administered by means of
an implantable device, which can be attached, for example, to a
subconjunctival, intracameral, or intravitreal region of the
eye.
[0097] In preparing compositions for topical administration, the BK
agonists are generally formulated from about 0.00005 to about 1.0
percent by weight (wt %). The BK agonists are preferably formulated
between about 0.0003 to about 0.3 wt % and, more preferably,
between about 0.0005 and about 0.03 wt %. In a particular
embodiment, the formulation is about 0.003% or 0.005%. In general,
the compositions will be solutions, having a pH between about 4.5
and about 7.4. While the precise regimen is left to the discretion
of the clinician, the resulting formulation(s) are preferably
administered by placing one drop of each solution(s) in each eye
one to four times a day, or as directed by the clinician.
[0098] Other ingredients which may be desirable to use in the
ophthalmic preparations of the present invention include
preservatives, surfactants (such as tyloxapol), co-solvents (such
as polyethylene glycol, including, but not limited to, PEG 400),
buffers, tonicity building agents, viscosity building agents and
penetration enhancers. Viscosity building agents, such as xanthan
gum, carbomer, hydroxymethyl cellulose, hydroxyethyl cellulose,
methylcellulose, polyvinylpyrrolidine, a polymer matrix such as
CAPA4101 or the like, may be added to the compositions of the
present invention to improve the retention of the compound in the
conjunctival sac or surrounding area. In order to prepare sterile
ophthalmic ointment formulations, the BK agonist may be combined
with a preservative in an appropriate vehicle, such as white
petroleum, mineral oil or liquid lanolin. Sterile ophthalmic gel
formulations may be prepared by suspending the BK agonist in a
hydrophilic base prepared from the combination of, for example,
carbopol-940, or the like, according to the methods known in the
art for other ophthalmic formulations. Other compositions of the
present invention may contain penetration enhancing agents such as
cremephor and tween 80, in the event the BK agonists are less
penetrating in the eye.
[0099] In other embodiments, a pharmaceutical composition of the
invention comprising a non-peptide BK agonist can be administered
to a patient alone or in combination with other IOP-lowering agents
to increase the potency, efficacy and/or duration of the IOP
reduction. Numerous agents known to lower IOP have been previously
described (Sugrue, J. Med. Chem. 40: 2793-2809, 1997; Clark and
Pang, Expert Opin. Emerg. Drugs, 7: 141-163, 2002; Sharif and
Klimko, Ophthalmic Agents, in Comprehensive Medicinal Chemistry
II., Vol. 6, Chapter 6.12, p. 297-320; Eds: D. J. Triggle and J. B.
Taylor, Elsevier Oxford, 2007) including, but not limited to,
carbonic anhydrase inhibitors, beta-blockers, prostaglandins,
alpha-2 agonists, serotonin-2 agonists, alpha-1 antagonists,
dopamine agonists, Rho kinase inhibitors, myosin-II
Ca.sup.2+-ATPase inhibitors, matrix metalloproteinase activators,
Activator protein-1 (AP-1) activators (U.S. Pat. No. 7,005,446),
natriuretic peptide receptor-B agonists (Potter and Hunter, J.
Biol. Chem. 276: 6057-6060, 2001; Scotland and Ahluwalia,
Pharmacol. Ther. 105: 85-93, 2005), K.sup.+-channel blockers
(European patent EP1772514) and maxi-K-channel activators (Park et
al. J. Pharmacol. Sci., 92: 218-227, 2003; Stumpff et al., Exp. Eye
Res. 80: 697-708, 2005), phosphodiesterase inhibitors (Menniti et
al. Nat. Rev. Drug Discov., 5: 660-670, 2006),
stimulators/activators of membrane-bound and cytosolic soluble
adenylyl and/or guanylyl cyclases (Evgenov et al. Nature Rev. Drug
Discovery 5: 755-768, 2006). Other compounds and compound classes
described for lowering IOP are also useful for the current
invention (Clark and Yorio, Nature Rev. Drug Discovery, 2: 448-459,
2003; and in Sharif and Klimko, Ophthalmic Agents, in Comprehensive
Medicinal Chemistry II., Vol. 6, Chapter 6.12, p. 297-320; Eds: D.
J. Triggle and J. B. Taylor, Elsevier Oxford, 2007; International
Publication No. WO 2006/041875; U.S. Pat. No. 7,005,446). Dual and
multipharmacophoric agents can be also contemplated and synthesized
by those skilled in the art of conjugating BK agonists with one or
more of the agents mentioned above or cited in the publications
above.
[0100] Just as nitric oxide (NO) liberated de novo from NO-donors
or other biological processes have demonstrated physiological and
pathological roles, two other compounds, carbon monoxide (CO;
Snyder et al., Brain Res. Rev. 26: 167-175, 1998) and hydrogen
sulfide (H.sub.2SS; Boehning and Snyder, Ann. Rev. Neurosci. 26:
1050131, 2003; Kimura et al., Antioxid. Redox Signal. 7: 795-803,
2005), produced endogenously or delivered exogenously also mediate
important biological functions. While CO appears to be able to
activate soluble guanylyl cyclase (sGC), and NO can increase CO
production (Leffler et al., Am. J. Physiol. Heart Circ. Physiol.
289: H1442-H1447, 2005), CO and NO can also act synergistically
(Stone and Marletta, Chem. Biol. 5: 255-261, 1998; Sharma and
Magde, Methods: 19: 494-505, 1999) Thus, sGC may be activated by NO
and CO. Although H.sub.25 has not been shown yet to activate sGC,
because it relaxes smooth muscle (Kimura et al., Antioxid. Redox
Signal. 7: 795-803, 2005), it is likely that sGC is involved in
this process. Also, H.sub.25 has recently been shown to lower IOP
in rabbits (PCT Application WO 2006/119258). Therefore, in some
embodiments, the bradykinin receptor agonists may be advantageously
combined with or conjugated with NO-donors and/or CO-donors
(tricarbonylchloro [glycinato] ruthenium, tricarbonylchloro
[glutamic acidato] ruthenium, tricarbonylchloro [lysinato]
ruthenium; tricarbonylchloro [alanato] ruthenium, (Rodella et al.,
Free Radical Biol. Med. 40: 2198-2205, 2006);
tricarbonyldichlororuthenium (II) dimer, (Srisook et al., Biochem.
Pharmacol. 71: 307-318, 2006)), and/or H.sub.2S-donors (NaHS; NaS)
to achieve synergistic or additive reduction in IOP.
[0101] A pharmaceutical composition of the invention may also
include an agent that is a source of NO. NO-donors usually cause
hyperemia and typically do not exhibit high efficacy in vivo for
IOP-lowering. However, a derivative of the anti-epileptic drug
gabapentin, NCX8001 ([1-(aminomethyl-cyclohexane acetic acid
3-(nitroxymethyl)phenyl ester]), has been synthesized and shown to
be bioavailable and to be a slow releaser of NO and that activates
soluble guanylyl cyclase (Wu et al Br. J. Pharmacol. 141: 65-74,
2003). The slow NO-releasing compound NCX8001 may readily penetrate
the cornea and be hydrolyzed in the aqueous humor to release NO to
lower IOP, thereby limiting its hyperemic potential. Therefore, in
some embodiments, NCX8001 may be included in a pharmaceutical
composition for ocular administration to lower IOP. Other agents
that may prove suitable NO donors for lowering IOP include
nitroparacetamol and nitroflurbiprofen (Eur. J. Pharmacol. 483:
317-322, 2004) and nitroglycerin, isosorbide, sodium nitropruside,
minoxidil and molsidomine also lower IOP (Nathanson Eur. J.
Pharmacol. 147: 155-156, 1988; Nathanson, J. Pharmacol. Exp. Ther.
260: 956-965, 1992; U.S. Pat. No. 5,500,230).
[0102] A phosphodiesterase inhibitor is a drug that blocks one or
more of the subtypes of the enzyme phosphodiesterase (PDE),
therefore preventing the inactivation of the intracellular second
messengers, cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP), by the respective PDE subtype(s).
Of these, there are at least two types, non-selective and
selective. Since cAMP and cGMP are known to relax ciliary muscle
(Stumpff et al. Exp. Eye Res. 80: 697-708, 2005; Wiederholt et al.
Prog. Retinal Eye Res. 19: 271-295, 2000), a process that leads to
change of the TM topography and thus resulting in an increase in
the aqueous humor outflow (Stumpff et al. Exp. Eye Res. 80:
697-708, 2005; Wiederholt et al. Prog. Retinal Eye Res. 19:
271-295, 2000). Thus, PDE inhibitors could be advantageously
combined with BK agonists to enhance the efficacy and/or duration
of IOP-lowering induced the BK agonists. Various examples of
non-selective phosphodiesterase inhibitors include (1) caffeine;
(2) bronchodilator theophylline; and, (3) IBMX
(3-isobutyl-1-methylxanthine), the latter of which, at least, is
used as investigative tool in pharmacological research. Of the
selective inhibitors, there are various ones typically related to
the inhibitors subtype.
[0103] Examples of PDE1-selective inhibitors are Vinpocetine and
IC224. (Menniti et al., Nature Rev. Drug Discov., 5: 660-670,
2006). Examples of PDE2-selective inhibitors are
erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and BAY 60-7550.
(Menniti et al., Nature Rev. Drug Discov., 5: 660-670, 2006).
[0104] Examples of PDE3-selective inhibitors are enoximone,
milrinone, and cilostamide. All are used clinically for short-term
treatment of cardiac failure. Clinically these drugs mimic
sympathetic stimulation and increase cardiac output. (Menniti et
al., Nature Rev. Drug Discov., 5: 660-670, 2006) Further suitable
examples are disclosed in U.S. Pat. No. 6,156,753, the contents of
which are hereby incorporated by reference as if it were presented
herein in its entirety.
[0105] An example of a PDE4-selective inhibitor is rolipram. It is
used as investigative tool in pharmacological research. PDE4 is the
major cAMP-metabolizing enzyme found in inflammatory and immune
cells. PDE4 inhibitors have potential as anti-inflammatory drugs
especially in airway diseases. They suppress the release of
inflammatory signals, such as, but not limited to, cytokines, and
inhibit the production of reactive oxygen species. PDE4 inhibitors
have a high therapeutic and commercial potential as non-steroidal
disease controllers in inflammatory airway diseases such as asthma,
COPD and rhinitis (Menniti et al., Nature Rev. Drug Discov., 5:
660-670, 2006). Further suitable examples are disclosed in U.S.
Pat. No. 6,127,363, the contents of which are hereby incorporated
by reference as if it were presented herein in its entirety.
[0106] Examples of PDE5-selective inhibitors are sildenafil,
tadalafil, vardenafil, udenafil, and avanafil. A main use for these
PDE5 inhibitors are remedies for erectile dysfunction (Menniti et
al., Nature Rev. Drug Discov., 5: 660-670, 2006). Further suitable
compounds are those disclosed in WO 94/28902, WO 96/16644, and WO
01/19802, the contents of all which are hereby incorporated by
reference as if they were presented herein their entirety,
including, but not limited to the griseolic acid derivatives,
2-phenylpurinone derivatives, phenylpyridone derivatives, fused and
condensed pyrimidines, pyrimidopyrimidine derivatives, purine
compounds, quinazoline compounds, phenylpyrimidinone derivative,
imidazoquinoxalinone derivatives, pyrazolopyrimidinones, such as,
but not limited to,
5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H--
pyrazolo[4,3d]pyrimidin-7-one,
5-(5-morpholinoacetyl-2-n-propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro--
7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)-phenyl]-1-methyl-3-n-propy-
l-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[2-allyloxy-5-(4-methyl-1-piperazinylsulfonyl)-phenyl]-1-methyl-3-n-pro-
pyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[2-ethoxy-5-[4-(2-propyl)-1-piperazinylsulfonyl)-phenyl]-1-methyl-3-n-p-
ropyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[2-ethoxy-5-[4-(2-hydroxyethyl)-1-piperazinylsulfonyl)phenyl]-1-methyl--
3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[5-[4-(2-hydroxyethyl)-1-piperazinylsulfonyl]-2-n-propoxyphenyl]-1-meth-
yl 1,3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[2-ethoxy-5-(4-methyl-1-piperazinylcarbonyl)phenyl]-1-methyl-3-n-propyl-
-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
5-[2-ethoxy-5-(1-methyl-2-imidazolyl)phenyl]-1-methyl-3-n-propyl-1,6-dihy-
dro-7H-pyrazolo[4,3-d]pyrimidin-7-one,
1,3-dimethyl-5-benzylpyrazolo[4,3-d]pyrimidine-7-one,
2-(2-propoxyphenyl)-6-purinone,
6-(2-propoxyphenyl)-1,2-dihydro-2-oxypyridine-3-carboxamide,
2-(2-propoxyphenyl)-pyrido[2,3-d]pyrimid-4(3H)-one,
7-methylthio-4-oxo-2-(2-propoxyphenyl)-3,4-dihydro-pyrimido[4,5-d]pyrimid-
ine, 6-hydroxy-2-(2-propoxyphenyl)pyrimidine-4-carboxamide,
1-ethyl-3-methylimidazo[1,5a] quinoxalin-4(5H)-one,
4-phenylmethylamino-6-chloro-2-(1-imidazoloyl)quinazoline,
5-ethyl-8-[3-(N-cyclohexyl-N-methylcarbamoyl)-propyloxy]-4,5-dihydro-4-ox-
o-pyrido[3,2-e]-pyrrolo[1,2-a]pyrazine,
5'-methyl-3'-(phenylmethyl)-spiro[cyclopentane-1,7'(8'H)-(3'H)-imidazo[2,-
1-b]purin]4'(5'H)-one,
1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-yl)piperidine--
4-carboxylic acid,
(6R,9S)-2-(4-trifluoromethyl-phenyl)methyl-5-methyl-3,4,5,6a,7,8,9,9a-oct-
ahydrocyclopent[4,5]-imidazo[2,1-b]-purin-4-one,
1-t-butyl-3-phenylmethyl-6-(4-pyridyl)pyrazolo[3,4-d]-pyrimid-4-one,
1-cyclopentyl-3-methyl-6-(4-pyridyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimi-
d-4-one, 2-butyl-1-(2-chlorobenzyl)-6-ethoxy-carbonylbenzimidaole,
2-(4-carboxy-piperidino)-4-(3,4-methylenedioxy-benzyl)amino-6-nitroquinaz-
oline and 2-phenyl-8-ethoxycycloheptimidazole.
[0107] The PDE6's are distributed in the retina and have been
implicated with retinal degeneration (Menniti et al., Nature Rev.
Drug Discov., 5: 660-670, 2006). Selective inhibitors comprise
sildenafil, zaprinast, and dipyridamole.
[0108] An example of a PDE7-selective inhibitor is Dipyridamole.
The PDE8's are distributed throughout the cortex, striatum,
hippocampus, and cerebellum and have been implicated with
Parkinson's disease and psychosis (Menniti et al., Nature Rev. Drug
Discov. 5: 660-670, 2006).
[0109] An example of a PDE8-selective inhibitor is Dipyridamole.
The PDE8's are distributed throughout the cortex, striatum, and
hippocampus and have been implicated with Alzheimer's disease
(Menniti et al., Nature Rev. Drug Discov., 5: 660-670, 2006).
[0110] An example of a PDE9-selective inhibitor is BAY 73-6691. The
PDE9's are distributed throughout the brain and have been
implicated with neurodegeneration and cognitive issue (Menniti et
al., Nature Rev. Drug Discov., 5: 660-670, 2006).
[0111] Examples of PDE10-selective inhibitors include papaverine
and PQ-10. The PDE10's have been implicated with psychosis (Menniti
et al., Nature Rev. Drug Discov., 5: 660-670, 2006).
[0112] An example of a PDEll-selective inhibitor is tadalafil
(Menniti et al., Nature Rev. Drug Discov., 5: 660-670, 2006).
[0113] Pharmaceutical compositions of the invention can also be
advantageously combined with suitable neuroprotective agents such
as memantine, eliprodil, Ca.sup.2+-channel blockers, betaxolol,
etc. (Clark and Yorio, Nature Rev. Drug Discovery, 2: 448-459,
2003; and in Sharif and Klimko, Ophthalmic Agents, in Comprehensive
Medicinal Chemistry II., Vol. 6, Chapter 6.12, p. 297-320; Eds: D.
J. Triggle and J. B. Taylor, Elsevier Oxford, 2007) to obtain
IOP-lowering and protection of retinal ganglion cells (RGC). Since
angiotensin converting enzyme (ACE) inhibitors appear to potentiate
the effects of endogenous and exogenous BK independent of blocking
BK inactivation, and ACE and B.sub.2-receptors form a complex (Chen
et al., FASEB J. 13: 2261-2270, 2006), a combination of BK agonists
and ACE inhibitors (e.g. captopril; omapatrilat; enalapril, etc)
may also be useful for lowering IOP and RGC protection.
[0114] As demonstrated in the Examples below, BK agonists can
increase outflow of aqueous humor in the eye, thereby lowering
intraocular pressure (IOP). In certain embodiments, the invention
provides methods for lowering IOP comprising administering to a
patient in need thereof a therapeutically effective amount of a
non-peptide BK agonist in combination with an aqueous humor
production (inflow) inhibitor. In other embodiments, the invention
provides pharmaceutical compositions comprising at least one
non-peptide BK agonist and/or at least one aqueous humor inflow
inhibitor. The combination therapy of the invention provides the
benefit of lowering IOP by two mechanisms, including inducing
uveoscleral outflow of aqueous humor and inhibiting aqueous humor
inflow, which can allow for reduced dosages of the compounds
thereby lowering the risk of side effects. In certain embodiments,
the BK agonist and aqueous humor inflow inhibitor are administered
concurrently in separate pharmaceutical compositions. In other
embodiments, the BK agonist and aqueous humor inflow inhibitor are
administered formulated together in a pharmaceutical composition.
In yet other embodiments, the BK agonist and aqueous humor inflow
inhibitor are administered sequentially in separate pharmaceutical
compositions.
[0115] Non-limiting examples of "aqueous humor inflow inhibitors"
include .beta.-blockers (e.g. betaxolol; timolol; levobunolol; U.S.
Pat. Nos. 4,883,814, 6,399,605; Clark and Yorio, Nature Rev. Drug
Discovery, 2: 448-459, 2003; and in Sharif and Klimko, Ophthalmic
Agents, in Comprehensive Medicinal Chemistry II., Vol. 6, Chapter
6.12, p. 297-320; Eds: D. J. Triggle and J. B. Taylor, Elsevier
Oxford, 2007), .alpha.-2 agonists (e.g. brimonidine; apraclonidine;
U.S. Pat. Nos. 5,212,196; 5,612,364; U.S. Pat. Nos. 4,883,814,
6,399,605; Clark and Yorio, Nature Rev. Drug Discovery, 2: 448-459,
2003; and in Sharif and Klimko, Ophthalmic Agents, in Comprehensive
Medicinal Chemistry II., Vol. 6, Chapter 6.12, p. 297-320; Eds: D.
J. Triggle and J. B. Taylor, Elsevier Oxford, 2007), carbonic
anhydrase inhibitors (e.g. brinzolamide; dorzolamide; U.S. Pat.
Nos. 5,153,192; 5,240,923; 5,464,831; 5,538,966; 5,620,970;
6,242,441; 6,242,442; 6,316,441); serotonin-2 agonists (e.g. R-DOI;
.alpha.-methyl-serotonin; U.S. Pat. No. 6,664,286), and other
classes of compounds that exert their IOP-lowering effects in whole
or in part by inhibiting the production of aqueous humor (inflow
pathway) (Clark and Yorio, Nature Rev. Drug Discovery, 2: 448-459,
2003; and in Sharif and Klimko, Ophthalmic Agents, in Comprehensive
Medicinal Chemistry II., Vol. 6, Chapter 6.12, p. 297-320; Eds: D.
J. Triggle and J. B. Taylor, Elsevier Oxford, 2007).
Preparing Compounds
[0116] The procedures below are representative of the methods used
to prepare these compounds. One skilled in the art of organic
synthesis could prepare these compounds in a number of ways using
known methods. The methods are provided for the illustration
purposes only.
Preparation of
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-(2-methoxyethyl)benzamide (1)
##STR00104##
[0118] A solution of (E)-benzyl
3-(4-((2-methoxyethyl)carbamoyl)phenyl)acrylate (1.0 g, 3.38 mmol)
in ethanol (50 ml) is hydrogenated over 10% Pd/C (100 mg) at 50 psi
for 24 h. The catalyst is removed by filtration and the solvent
removed under vacuum to give
3-(4-((2-methoxyethyl)carbamoyl)phenyl)propanoic acid as a clear
oil that solidifies on standing. MS (ESI) 251 (M+), .sup.1H NMR
(CDCl.sub.3); 1.24 (t, 3H, J=6.8 Hz), 2.686 (t, 2H, J=7.2 Hz), 3.00
(t, 2H, J=7.2 Hz), 3.39 (s, 3H), 3.57 (t, 2H, J=5.2 Hz), 3.63 (t,
2H, J=5.2 Hz), 3.69-3.74 (m, 1H), 7.27 (d, 2H, J=8.4 Hz), 7.73 (d,
2H, J=8.4 Hz). A mixture of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (400 mg, 0.8
mmol), 3-(4-((2-methoxyethyl)carbamoyl)phenyl)propanoic acid (251
mg, 1.0 mmol) and TBTU
(O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate) (321 mg, 1.0 mmol) and diisopropyl amine (0.42
ml) are dissolved in DMF (20 mL) and stirred for 12 h at 23.degree.
C. The mixture was poured into water and extracted with ethyl
acetate (3.times., 50 mL). The ethyl acetate layer is washed with
saturated sodium bicarbonate, 1 N HCl then dried over sodium
sulfate and concentrated to give a tan oil. The oil was purified by
silica gel chromatography using 10% methanol in dichloromethane
with 0.1% NH.sub.4OH as eluant to give
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-(2-methoxyethyl)benzamide (1) as a tan solid, 250 mg (43%).
MS (ESI) 733 (M.sup.+); .sup.1H NMR (CDCl.sub.3) 2.53 (d, 2H, J=6.8
Hz), 2.89 (d, 2H, J=6.8 Hz), 3.23 (s, 3H), 3.37 (s, 3H), 3.54-3.55
(m, 2H), 3.56-3.62 (m, 2H), 4.21 (s, 3H), 5.29-5.28 (m, 2H), 5.67
(s, 2H), 6.31-6.37 (m, 1H), 6.42-6.55 (m, 1H), 6.75-6.85 (m, 2H),
6.89-6.92 (m, 1H), 7.02-7.08 (m, 1H), 7.15-7.28 (m, 6H), 7.45-7.46
(m, 1H), 7.51-7.59 (m, 1H), 7.67-7.69 (m, 1H), 8.55-8.58 (m,
1H).
Preparation of methyl
(5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d-
]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopro-
pyl)pyridin-2-yl)carbamate (2)
##STR00105##
[0120]
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-be-
nzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
3-(6-((methoxycarbonyl)amino)pyridin-3-yl)propanoic acid were
combined as previously described for compound 1 to give 2. LCMS
(+APCI) 706 (M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.58
(m, 1H), 8.06 (d, J=2.0 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.72 (bs,
1H), 7.58 (dt, J=1.6, 7.6 Hz, 1H), 7.51 (2.0, 8.4 Hz, 1H), 7.47 (d,
J=8.4 Hz, 1H), 7.21 (d, J=8.4 Hz, 1H), 7.19 (m, 1H), 7.04 (t, J=8.0
Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.83 (d, J=7.6 Hz, 1H), 6.81 (d,
J=8.0 Hz, 1H), 6.40 (bt, 1H), 5.67 (d, J=1.6 Hz, 2H), 5.28 (s, 2H),
4.21 (s, 3H), 3.78 (s, 3H), 3.75 (m, 1H), 3.50 (dd, J=4.0, 17.6 Hz,
1H), 3.23 (s, 3H), 2.88 (dd, J=7.6 Hz, 2H), 2.49 (t, J=7.6 Hz, 2H).
Calcd. for C.sub.34H.sub.33Cl.sub.2N.sub.7O.sub.6+1H.sub.2O: C,
56.36; H, 4.87; N, 13.53. Found: C, 56.34; H, 4.68; N, 13.24.
N-(2-((3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)-
methyl)-2,4-dimethylphenyl)(methyl)amino)-2-oxoethyl)-3-(2-oxo-2,3-dihydro-
oxazolo[4,5-b]pyridin-6-yl)propanamide (3)
##STR00106##
[0122]
2-amino-N-(2,4-dimethyl-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-be-
nzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
3-(2-oxo-2,3-dihydrooxazolo[4,5-b]pyridin-6-yl)propanoic acid were
used to prepare 3 as described above for compound 1. LCMS (+APCI)
650 (M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.59 (m, 1H),
7.85 (d, J=2.0 Hz, 1H), 7.60 (dt, J=2.0, 7.6 Hz, 1H), 7.22 (m, 2H),
7.05 (t, J=8.0 Hz, 1H), 6.95 (d, J=7.6 Hz, 1H), 6.90 (d, J=8.0 Hz,
1H), 6.82 (m, 3H), 6.73 (bt, 1H), 5.30 (m, 4H), 4.21 (s, 3H), 3.72
(dd, 4.0, 18.0 Hz, 1H), 3.44 (dd, J=4.4, 18.0 Hz, 1H), 3.25 (s,
3H), 2.95 (m, 2H), 2.56 (m, 2H), 2.33 (s, 3H), 2.27 (s, 3H). Calcd.
for C.sub.35H.sub.35N.sub.7O.sub.6+0.3H.sub.2O: C, 64.17; H, 5.48;
N, 14.97. Found: C, 64.06; H, 5.46; N, 14.96.
methyl
(5-(3-((2-((3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidaz-
ol-4-yl)oxy)methyl)-2,4-dimethylphenyl)(methyl)amino)-2-oxoethyl)amino)-3--
oxopropyl)pyridin-2-yl)carbamate (4)
##STR00107##
[0124]
2-amino-N-(3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazo-
l-4-yl)oxy)methyl)-2,4-dimethylphenyl)-N-methylacetamide and
3-(6-((methoxycarbonyl)amino)pyridin-3-yl)propanoic acid were used
to prepare 4 using the method described for 1. LCMS (+APCI) 666
(M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): .sup.1H-NMR
(CDCl.sub.3, .delta. ppm): 8.58 (m, 1H), 8.06 (d, J=1.6 Hz, 1H),
7.85 (d, J=8.4 Hz, 1H), 7.55 (m, 3H), 7.20 (m, 1H), 7.13 (d, J=8.0
Hz, 1H), 7.03 (m, 2H), 6.93 (d, J=8.0 Hz, 1H), 6.81 (m, 2H), 6.43
(bt, J=4.0 Hz, 1H), 5.41 (s, 2H), 5.28 (s, 2H), 4.20 (s, 3H), 3.79
(s, 3H), 3.69 (dd, J=4.4, 18.0 Hz, 1H), 3.47 (dd, J=4.0, 18.0 Hz,
1H), 3.21 (s, 3H), 2.88 (t, J=7.6 Hz, 2H), 2.49 (s, 3H), 2.47 (m,
2H), 2.31 (s, 3H). Calcd. for
C.sub.36H.sub.39N.sub.7O.sub.6+0.8H.sub.2O: C, 63.57; H, 6.02; N,
14.42. Found: C, 63.64; H, 5.93; N, 14.53.
Preparation of
N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imid-
azol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)-3-(6-(methylsulfona-
mido)pyridin-3-yl)propanamide (5)
##STR00108##
[0126] Compound 5 was prepared from
3-(6-(methylsulfonamido)pyridin-3-yl)propanoic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide described for
compound 1. MS (APCI) 737 (M+), .sup.1H NMR (CDCl.sub.3) 2.41-2.51
(m, 2H), 2.65 (s, 3H), 2.81-2.92 (m, 2H), 3.05 (s, 3H), 3.22 (s,
3H), 3.33-3.52 (m, 1H), 3.37-3.90 (m, 1H), 5.40 (s, 2H), 5.61 (s,
2H), 6.50-6.61 (m, 1H), 6.71 (s, 1H), 7.17-7.3 (m, 4H), 7.34-7.42
(m, 1H), 7.42-7.52 (m, 1H), 7.55-7.63 (m, 2H), 7.70-7.80 (m, 1H),
7.82-7.95 (m, 1H), 8.04 (s, 1H), 8.61-8.68 (m, 1H).
Preparation of
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpiperazine-1-carboxamide (7)
##STR00109##
[0128] To a cold solution (ice bath) of piperazine-1-carboxylic
acid tert-butyl ester (9.0 g, 48.32 mmol) and triethylamine (9.8 g,
96.64 mmol) in dichloromethane (100 mL) was slowly added a solution
of methylaminoformyl chloride (5.0 g, 53.5 mmol) in dichloromethane
at 0.degree. C. The reaction mixture was allowed to warm to room
temperature and stirred overnight, and then partitioned in
dichloromethane (200 mL) and water (100 mL). Organic layer was
separated, dried (MgSO.sub.4), and concentrated to yield tert-butyl
4-(methylcarbamoyl)piperazine-1-carboxylate as a solid which was
dissolved in dichloromethane (100 mL) and TFA (20 mL). The solution
was stirred for 12 h and the volatiles were evaporated under
pressure, the oily residue was dissolved in methanol and 1N
hydrogen chloride solution was added. The solid formed was filtered
and washed with ether, dried to give
N-methylpiperazine-1-carboxamide hydrochloride, 8.12 g (93%)
[0129] To a suspension of N-methylpiperazine-1-carboxamide
hydrochloride (6 g, 33.52 mmol) and K.sub.2CO.sub.3 (9.26 g, 67.0
mmol) in acetonitrile (100 mL) was added bromopropionic acid
tert-butyl ester (8.4 g, 40.2 mmol). The reaction mixture was
stirred at reflux for 12 h, cooled to room temperature and
partitioned in ethyl acetate (200 mL) and water (100 mL). Organic
layer was separated, dried (MgSO.sub.4), and concentrated to give
tert-butyl 3-(4-(methylcarbamoyl)piperazin-1-yl)propanoate as an
oil which was dissolved in a mixture of dichloromethane (100 mL)
and TFA (trifluoroacetic acid) (20 mL) and then stirred overnight.
The volatiles were removed under vacuum. The residue was dissolved
in methanol, 1N hydrogen chloride in ether was added, and then the
solution was concentrated to give
3-(4-(methylcarbamoyl)piperazin-1-yl)propanoic acid as a solid.
LCMS 216 (M+1)
[0130] A mixture of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
3-(4-(methylcarbamoyl)piperazin-1-yl)propanoic acid was coupled as
described for the preparation of 2 to give
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylpiperazine-1-carboxamide (7).
[0131] .sup.1HNMR (400 MHz, CDCl.sub.3) .delta. 2.39 (t, 2H), 2.62
(m, 4H), 2.66 (t, 2H), 2.82 (s, 3H), 3.48 (s, 3H), 3.49 (m, 4H),
3.50-3.56 (dd, 1H), 4.20 (s, 3H), 5.29 (s, 2H), 5.65 (m, 2H),
6.91-8.59 (9Har), 8.80 (1H, NH), LC/MS 697 (M+1);
C.sub.33H.sub.38Cl.sub.2N.sub.8O.sub.5+0.54H.sub.2O; Calculated: C,
56.04; H, 5.57; N, 15.84. Found: C, 56.05; H, 5.57; N, 15.66
3-(4-acetamidopiperidin-1-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridi-
n-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2--
oxoethyl)propanamide (8)
##STR00110##
[0133] A stirred mixture of 4-acetamidopiperidine (1.82 g, 12.8
mmol) and benzyl acrylate (2.57 g, 15.4 mmol) in acetonitrile (50
mL) was stirred overnight under nitrogen and at 40.degree. C. for 1
h. The mixture was evaporated to dryness and the residue was
purified by silica gel chromatograph eluting with a gradient of 0%
to 30% dichloromethane/methanol/NH.sub.4OH (10:1:0.1) and
dichloromethane to give benzyl
3-(4-acetamidopiperidin-1-yl)propanoate as an oil (3.81 g,
98%).
[0134] A mixture of benzyl 3-(4-acetamidopiperidin-1-yl)propanoate
(3.81 g, 12.5 mmol) and Pd/C (10%, 0.20 g) in methanol (100 mL) was
stirred under hydrogen atmosphere overnight. The solid was filtered
and the filtrate was evaporated to give
3-(4-acetamidopiperidin-1-yl)propanoic acid (2.50 g). LCMS (+ESI)
215 (M+1).
[0135] To a stirred mixture of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (0.30 g, 0.60
mmol) in anhydrous DMF (15 mL) was added triethyl amine (0.067 g,
0.66 mmol) under nitrogen at 0.degree. C. The mixture was stirred
for 30 min and was added 3-(4-acetamidopiperidin-1-yl)propanoic
acid (0.257 g, 1.20 mmol) and HBTU
(O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate)
(0.296 g, 0.78 mmol). The mixture was stirred at room temperature
overnight, evaporated, mixed with a saturated brine solution and
extracted with ethyl acetate. Evaporation gave a crude oil that was
purified using silica chromatography eluting with a gradient of 2%
to 30% DCM(dichloromethane)/MeOH/NH.sub.4OH (10:1:0.1). After
drying at 78.degree. C. under high vacuum,
3-(4-acetamidopiperidin-1-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyrid-
in-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-
-oxoethyl)propanamide, 8, was isolated as a solid (0.23 g, 55%).
LCMS (+ESI) 696 (M+1). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.96
(bs, 1H), 8.59 (m, 1H), 7.58 (t, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.27
(m, 1H), 7.19 (t, J=8.0 Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.82 (m,
1H), 5.66 (m, 2H), 5.48 (bd, 1H), 5.28 (s, 2H), 4.20 (s, 3H), 3.85
(m, 2H), 3.54 (dd, J=18, 3.6 Hz, 1H), 3.26 (s, 3H), 2.96 (bd, 2H),
2.62 (m, 2H), 2.37 (m, 2H), 2.14 (m, 2H), 1.97 (s, 3H), 1.96 (m,
3H). Calcd. for C.sub.34H.sub.39Cl.sub.2N.sub.7O.sub.5+1.4H.sub.2O:
C, 56.57; H, 5.84; N, 13.58. Found: C, 56.44; H, 5.59; N,
13.48.
Ethyl
(5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-be-
nzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-o-
xopropyl)-1,4,5,6-tetrahydropyrimidin-2-yl)carbamate (9)
##STR00111##
[0137]
3-(2-((ethoxycarbonyl)amino)-1,4,5,6-tetrahydropyrimidin-5-yl)propa-
noic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide were used to
prepare compound 9 as described for compound 1. LCMS (+ESI) 725
(M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta.): 8.58 (d, J=4.0 Hz,
1H), 8.37 (bs, 1H), 7.58 (t, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.29 (d,
J=8.4 Hz, 1H), 7.18 (m, 1H), 7.03 (t, 1H), 6.92 (d, 1H), 6.80 (m,
2H), 6.62 (bt, 1H), 5.66 (d, J=2.8 Hz, 2H), 5.27 (s, 2H), 4.21 (s,
3H), 4.03 (t, J=7.2 Hz, 2H), 3.81 (dd, J=5.2, 17.6 Hz, 1H), 3.51
(dd, 1H), 3.40 (dd, J=4.8, 12.4 Hz, 2H), 3.24 (s, 3H), 2.98 (dd,
J=9.2, 12.4 Hz, 2H), 2.27 (t, J=7.6 Hz, 2H), 1.96 (m, 1H), 1.80 (m,
2H), 1.67 (q, J=8.4 Hz, 2H), 1.23 (t, J=7.2 Hz, 3H).
2-(2-((4-acetamidocyclohexyl)amino)acetamido)-N-(2,4-dichloro-3-(((2-metho-
xy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-me-
thylacetamide (10)
##STR00112##
[0139] 2-((4-acetamidocyclohexyl)amino)acetic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide were used to
prepared compound 10 as described for compound 1. LCMS (+ESI) 696
(M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta.): .sup.1H-NMR
(CDCl.sub.3, .delta. ppm): 8.58 (dd, J=0.8, 4.8 Hz, 1H), 7.98 (t,
4.4 Hz, 1H), 7.58 (dt, J=1.6, 7.6 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H),
7.29 (d, J=8.4 Hz, 1H), 7.19 (m, 1H), 7.04 (t, J=8.0 Hz, 1H), 6.92
(d, J=8.0 Hz, 1H), 6.81 (d, J=7.2 Hz, 1H), 6.79 (d, J=7.6 Hz, 1H),
5.65 (dd, J=10.4, 13.6 Hz, 2H), 5.28 (s, 2H), 5.26 (m, 1H), 4.21
(s, 3H), 3.84 (dd, J=5.2, 17.6 Hz, 1H), 3.73 (m, 1h), 3.54 (dd,
J=4.0, 17.6 Hz, 1H), 3.31 (m, 2H), 3.25 (s, 3H), 2.36 (m, 1H), 1.99
(m, 4H), 1.94 (s, 3H), 1.16 (m, 4H). Calcd. for
C.sub.34H.sub.39Cl.sub.2N.sub.7O.sub.5+1.0H.sub.2O: C, 57.14; H,
5.78; N, 13.72. Found: C, 56.87; H, 5.75; N, 13.54.
1-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoethyl-
)-N-methylpiperidine-4-carboxamide (13)
##STR00113##
[0141] 2-(4-(methylcarbamoyl)piperidin-1-yl)acetic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide were combined as
described for compound 1 to give 13. LCMS (+ESI) 682 (M.sup.+).
.sup.1H-NMR (CDCl.sub.3, .delta.): 8.58 (m, 1H), 7.88 (bt, 1H),
7.58 (t, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.31 (d, J=8.4 Hz, 1H), 7.19
(m, 1H), 7.03 (t, J=8.0 Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.83 (d,
J=8.0 Hz, 1H), 6.81 (d, J=8.0 Hz, 1H), 5.67 (s, 2H), 5.28 (s, 2H),
4.20 (d, J=2.8 Hz, 3H), 3.82 (dd, 1H), 3.56 (dd, 1H), 3.24 (s, 3H),
2.94 (m, 4H), 2.81 (d, J=4.8 Hz, 3H), 2.18 (m, 3H), 1.85 (m, 5H).
Calcd. for C.sub.33H.sub.37Cl.sub.2N.sub.7O.sub.5+3.1H.sub.2O: C,
53.67; H, 5.90; N, 13.28. Found: C, 53.29; H, 5.52; N, 12.92.
N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol--
4-yl)oxy)methyl)phenyl)-N-methyl-2-(2-morpholinoacetamido)acetamide
(14)
##STR00114##
[0143]
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-be-
nzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
2-morpholinoacetic acid were combined to give 14 as previously
described. LCMS (+ESI) 627 (M.sup.+). .sup.1H-NMR (CDCl.sub.3,
.delta.): 8.58 (dd, J=0.8, 4.8 Hz, 1H), 7.88 (bt, 1H), 7.58 (td,
J=1.6, 8.0 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.19 (m, 1H), 7.03 (t,
J=8.0 Hz, 1H), 6.92 (d, J=7.6 Hz, 1H), 6.83 (d, J=7.6 Hz, 1H), 6.81
(d, J=7.6 Hz, 1H), 5.67 (d, J=3.2 Hz, 2H), 5.28 (s, 2H), 4.21 (s,
3H), 3.87 (dd, J=5.2, 17.6 Hz, 1H), 3.77 (m, 4H), 3.54 (dd, J=4.4,
17.6 Hz, 1H), 3.25 (s, 3H), 2.55 (m, 4H). Calcd. for
C.sub.30H.sub.32Cl.sub.2N.sub.6O.sub.5: C, 57.42; H, 5.14; N,
13.39. Found: C, 57.23; H, 5.22; N, 13.23.
2-(2-(4-acetamidopiperidin-1-yl)acetamido)-N-(2,4-dichloro-3-(((2-methoxy--
1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methy-
lacetamide (15)
##STR00115##
[0145] 2-(4-acetamidopiperidin-1-yl)acetic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide are combined as
previously described to give 15. LCMS (+ESI) 682 (M.sup.+).
.sup.1H-NMR (CDCl.sub.3, .delta.): 8.58 (m, 1H), 7.97 (bt, 1H),
7.58 (t, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.29 (d, J=8.4 Hz, 1H), 7.19
(m, 1H), 7.03 (t, J=8.0 Hz, 1H), 6.92 (d, J=7.6 Hz, 1H), 6.83 (d,
J=8.0 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H), 5.67 (s, 2H), 5.33 (bd, 1H),
5.28 (s, 2H), 4.21 (s, 3H), 3.82 (dd, 1H), 3.81 (m, 1H), 3.56 (dd,
1H), 3.25 (s, 3H), 2.99 (d, 2H), 2.80 (m, 2H), 2.32 (m, 2H), 1.96
(s, 3H), 1.92 (m, 2H), 1.52 (m, 2H).
3-(4-acetylpiperazin-1-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-
-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxo-
ethyl)propanamide (16)
##STR00116##
[0147]
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-be-
nzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
3-(4-acetylpiperazin-1-yl)propanoic acid were used to prepare 16 as
described for compound 1. LCMS (+ESI) 668 (M.sup.+). .sup.1H-NMR
(CDCl.sub.3, .delta.): 8.58 (m, 1H), 7.84 (bt, 1H), 7.58 (t, J=2.0
Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.30 (d, J=8.8 Hz, 1H), 7.18 (m,
1H), 7.03 (t, J=8.0 Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.82 (m, 2 H),
5.68 (d, J=4.0 Hz, 2H), 5.28 (s, 2H), 4.21 (s, 3H), 3.89 (dd, 1H),
3.69 (m, 2H), 3.25 (s, 3H), 2.53 (m, 4H), 2.04 (s, 3H). Calcd. for
C.sub.33H.sub.37Cl.sub.2N.sub.7O.sub.5+1H.sub.2O: C, 56.57; H,
5.61; N, 13.99. Found: C, 56.33; H, 5.48; N, 14.21.
Preparation of
4-(3-((2-((2,4-dichloro-3-(((2-ethyl-1-(pyridin-2-ylmethyl)-1H-benzo[d]im-
idazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl-
)-N-methylpiperazine-1-carboxamide (19)
##STR00117##
[0149] 2,3-diaminophenol (5 g, 40.3 mmol) was dissolved in
propionic acid (10 mL) and stirred at 150.degree. C. for 3 h. The
reaction mixture was cooled to room temperature and the volume
reduced under high vacuum to give a brown solid which was
partitioned between chloroform and a saturated solution of
bicarbonate, the chloroform layer was separated, dried and
concentrated to give a brown solid. The brown solid (1.3 g, 8.0
mmol) was dissolved in DMF (40 mL) and K.sub.2CO.sub.3 (2.8 g, 0.02
mmol) was added followed by benzylbromide (1.5 g, 8.8 mmol). The
reaction mixture was stirred at room temperature overnight,
partitioned with water and ethyl acetate, ethyl acetate layer was
separated, dried (MgSO.sub.4), concentrated, and purified by flash
chromatography to give 7-benzyloxy-2-ethyl-1H-benzimidazole (0.9 g,
44%). LCMS 253 (M.sup.+)
[0150] To a solution cold solution (ice bath) of
7-benzyloxy-2-ethyl-1H-benzimidazole prepared above (0.8 g, 3.17
mmol) in DMF (10 mL) was added sodium hydride (0.41 g, 10.47 mmol),
followed by 2-(bromomethyl)pyridine hydrobromide (0.88 g, 3.49
mmol). After addition of the bromide the reaction mixture was
allowed to warm to room temperature and stirred for 16 h. The
reaction mixture was partitioned between water and ethyl acetate.
Ethyl acetate layer was separated, dried (MgSO.sub.4), and
concentrated to give
4-benzyloxy-2-ethyl-1-pyridin2-ylmethyl-1H-benzoimidazole as a
solid which was crystallized in ethyl acetate to give
4-benzyloxy-2-ethyl-1-pyridin2-ylmethyl-1H-benzoimidazole, 0.35 g.
LCMS 344 (M+1). .sup.1HNMR (400 MHz, CDCl.sub.3) .delta.1.38 (t,
3H), 2.90 (q, 2H), 5.40-5.42 (m, 4H), 6.65-8.60 (12H, Ar)
4-benzyloxy-2-ethyl-1-pyridin2-ylmethyl-1H-benzoimidazole (0.35 g,
1.2 mmol) was dissolved in ethanol and in presence of Pd/C 10% and
stirred under a hydrogen balloon overnight. The solution was
filtered through celite and concentrated to give
2-ethyl-1-pyridin-2-ylmethyl-1H-benzoimidazole-4-ol. .sup.1HNMR
(400 MHz, CDCl.sub.3) .delta. 1.28 (t, 3H), 2.87 (q, 2H), 5.40 (s,
2H), 6.49 (d, 1Har), 6.51 (m, 2H, Ar), 6.92 (m. 1H Ar), 7.09 (m,
1H, Ar), 7.73 (m, 1H, Ar), 8.49 (m, 1H, Ar), 9.57 (1H, OH).
[0151] To a solution of
N-(2,4-Dichloro-3-hydroxymethyl-phenyl)-2-(1,3-dioxo-1,3-dihydro-isoindol-
-2-yl)-N-methyl-acetamide (1.0 g, 2.54 mmol) in anhydrous
dichloromethane (40 mL) and two drops of DMF was added thionyl
chloride (0.6 g, 5.08 mmol), and the reaction mixture was stirred 3
h at room temperature. The volatiles were evaporated and, toluene
(50 mL) was added and evaporated, this was repeated twice to get
rid of traces of thionyl chloride. To the solid residue dissolved
in acetonitrile (100 mL) was added K.sub.2CO.sub.3 (0.7 g, 5.1
mmol) and 2-ethyl-1-pyridin-2-ylmethyl-1H-benzoimidazole-4-ol (0.70
g, 2.76 mmol). The reaction mixture was stirred at 70.degree. C.
for 4 h then cooled. The reaction mixture was partitioned between
water and ethyl acetate. The ethyl acetate layer was separated,
dried (MgSO.sub.4) and concentrated to give a solid, which was
dissolved in 10 ml of 2 N methyl amine as a solution in ethanol,
and stirred at 40.degree. C. for 2 h. After, the solution was
cooled and concentrated to give a solid which was purified by
column chromatography using a mixture of dichloromethane and 5%
ammonium in methanol (9:1) to yield
2-Amino-N-[2,4-dichloro-3-(2-ethyl-1-pyridin-2-ylmethyl-1H-benzoimidazol--
4-yloxymethyl)-phenyl]-N-methyl-acetamide. LCMS 498 (M.sup.+)
[0152]
2-Amino-N-[2,4-dichloro-3-(2-ethyl-1-pyridin-2-ylmethyl-1H-benzoimi-
dazol-4-yloxymethyl)-phenyl]-N-methyl-acetamide and
3-(4-(methylcarbamoyl)piperazin-1-yl)propanoic acid were coupled as
described for compound 1 above to give 19. MS (APCI) 694 (M.sup.+),
.sup.1HNMR (400 MHz, MeOD) .delta. 2.12 (t, 3H), 2.49 (t, 2H), 2.71
(m, 4H), 2.89 (s, 3H), 2.89 (m, 2H), 2.93 (q, 2H), 3.2 (s, 3H),
3.31 (m, 4H), 3.46-3.84 (dd, 3H), 5.55 (m, 4H), 6.93-8.51 (m,
9Har), LCMS 695 (M+1);
C.sub.34H.sub.38Cl.sub.2N.sub.8O.sub.4+1.73H.sub.2O; Calculated: C,
56.69; H, 5.98; N, 15.56. Found: C, 56.69; H, 6.18; N, 15.81.
4-((2-((2-((3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl-
)oxy)methyl)-2,4-dimethylphenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoethy-
l)amino)-N-methylpiperidine-1-carboxamide (21)
##STR00118##
[0154] 2-((1-(methylcarbamoyl)piperidin-4-yl)amino)acetic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide were used to
prepare compound 21 as described for compound 1. LCMS (+ESI) 657
(M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta.): .sup.1H-NMR
(CDCl.sub.3, .delta. ppm): 8.51 (dd, J=0.8, 4.0 Hz, 1H), 7.85 (t,
1H), 7.52 (t, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.11 (m, 1H), 7.07 (d,
J=8.0 Hz, 1H), 6.96 (m, 2H), 6.86 (d, J=7.6 Hz, 1H), 6.74 (m, 2H),
5.33 (s, 2H), 5.21 (s, 2H), 4.37 (bq, 1H), 4.13 (s, 3H), 3.79 (m,
2H), 3.67 (dd, 1H), 3.46 (dd, 1H), 3.23 (s, 2H), 3.15 (s, 3H), 2.75
(m, 2H), 2.72 (d, J=4.8 Hz, 3H), 2.53 (m, 1H), 2.43 (s, 3H), 2.27
(s, 3H), 1.82 (m, 2H), 1.24 (m, 3h).
Preparation of
N-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imida-
zol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)-1,3'-bipyrrolidine-3--
carboxamide (22)
##STR00119##
[0156] A solution of
l'-(tert-butoxycarbonyl)[1,3'-bipyrrolidine]-3-carboxylic acid (0.5
g, 1.0 mmol) in anhydrous DMF (20 ml) was treated with HOBT
(Hydroxybenzotriazole) (0.21 g, 1.52 mmol), EDCI
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), hydrochloride
(0.24 g, 1.27 mmol) then
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (0.50 g, 1.0
mmol) and the mixture was stirred at room temperature overnight.
The reaction was treated with water (100 mL) and the resulting
precipitate was filtered. The aqueous mixture was treated with
aqueous saturated sodium bicarbonate (100 mL), extracted with ethyl
acetate (200 mL.times.2). The combined organic layers were dried
over magnesium sulfate, filtered and evaporated to give a white
solid, 0.62 g that was purified by chromatography (silica gel,
dichloromethane and methanol gradient) to afford tert-butyl
3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imi-
dazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)carbamoyl)-[1,3'-bi-
pyrrolidine]-1'-carboxylate as a white solid, 0.41 g (53%), that
was pure by LC/MS (+ESI) m/z 766 (M.sup.+).
[0157] A solution of tert-butyl
3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imi-
dazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)carbamoyl)-[1,3'-bi-
pyrrolidine]-1'-carboxylate (0.27 g, 0.35 mmol) in dichloromethane
(10 ml) was cooled in an ice bath and treated with dropwise
addition of trimethylsilyl triflate (0.07 mL, 0.39 mmol) and
stirred at 0.degree. C. for ten minutes. The reaction was treated
with another dropwise addition of trimethylsilyl triflate (0.06 mL,
0.35 mmol) stirring at 0.degree. C. for one hour. The reaction was
quenched with triethyl amine (1 mL), evaporated, treated with
aqueous saturated sodium bicarbonate (10 mL) and brine (100 mL) and
extracted with dichloromethane (100 mL.times.3). The combined
organic layers were dried over magnesium sulfate, filtered and
evaporated to afford
N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imid-
azol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)-[1,3'-bipyrrolidine-
]-3-carboxamide (22) as a tan solid, 0.22 g (94%), that was about
90% pure by LC/MS (+ESI) m/z 666 (M'+1) with about 10% impurity of
MW 786 (M.sup.+). 786 (M'+1). .sup.1HNMR (400 MHz, CDCl.sub.3)
.delta. 8.59 (s, 1H), 7.93 (m, 1H), 7.59 (m, 1H), 7.45 (m, 1H),
7.32 (m, 1H), 7.19 (m, 1H), 7.04 (m, 1H), 6.95 to 2.00 (many m,
17H). .sup.13CNMR (100 MHz, CDCl.sub.3) .delta.175.6, 169.5, 169.2,
156.9, 156.1, 149.6, 149.1, 138.4, 137.1, 136.4, 135.8, 135.4,
130.1, 129.8, 122.6, 121.7, 120.8, 107.4, 103.1, 67.1, 62.6, 57.5,
55.0, 54.9, 53.4, 51.4, 50.4, 47.7, 46.1, 44.0, 43.9, 41.9, 30.9,
9.0.
Preparation of
N3-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imid-
azol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)-N1'-methyl-1,3'-bipy-
rrolidine-1',3-dicarboxamide (24)
##STR00120##
[0159] A solution of
N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imid-
azol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)-[1,3'-bipyrrolidine-
]-3-carboxamide (0.20 g, 0.3 mmol) in dichloromethane (15 ml) was
treated with methyl isocyanate (0.023 mL, 0.39 mmol) with stirring
at room temperature for ten minutes. The reaction was evaporated,
treated with methanol and evaporated to a tan solid, 0.20 g. This
residue was purified by chromatography (silica gel, dichloromethane
and methanol gradient) to afford 24 as a white solid, 0.06 g (28%),
that was pure by LC/MS (+ESI) m/z 723 (M.sup.+), and was pure by
.sup.1HNMR (400 MHz, CDCl.sub.3) .delta. 8.58 (s, 1H), 7.59 (m,
1H), 7.49 (m, 2H), 7.29 (m, 1H), 7.19 (m, 1H), 7.05 (m, 1H), 6.80
(m, 2H), 5.69 (s, 2H), 5.29 (s, 2H), 4.20 (m, 4H), 3.80 (m, 1H),
3.50 (m, 3H), 3.25 (m, 5H), 2.85 (m, 7H), 2.55 (m, 2H), 2.10 (m,
5H). .sup.13CNMR (100 MHz, CDCl.sub.3) 8150, 148, 131, 130, 121,
122, 123, 108, 103, 65, 63, 58, 57, 51, 50, 48, 43, 42, 41, 35, 31,
30, 29, 28. Anal. Calcd for
C.sub.15H.sub.21N.sub.3O.sub.3+1.0H.sub.2O: C, 56.68; H, 5.71; N,
15.11. Found: C, 56.73; H, 5.51; N, 14.82.
3-(2-acetamido-1,4,5,6-tetrahydropyrimidin-5-yl)-N-(2-((2,4-dichloro-3-(((-
2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)pheny-
l)(methyl)amino)-2-oxoethyl)propanamide (25)
##STR00121##
[0161]
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-be-
nzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
3-(2-acetamido-1,4,5,6-tetrahydropyrimidin-5-yl)propanoic acid are
used to prepare compound 25 as described for compound 1. LCMS
(+ESI) 695 (M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.58
(dd, J=0.4, 4.8 Hz, 1H), 7.58 (dt, J=1.6, 7.6 Hz, 1H), 7.47 (d,
J=8.8 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.19 (m, 1H), 7.03 (t, J=8.0
Hz, 1H), 6.92 (d, J=8.0 Hz, 1H), 6.82 (m, 2H), 6.68 (bs, 1H), 5.66
(dd, J=2.4, 14.4 Hz, 2H), 5.27 (s, 2H), 4.21 (s, 3H), 3.80 (dd,
1H), 3.51 (dd, 1H), 3.439 (dd, J=4.4, 12.8 Hz, 2H), 3.24 (s, 3H),
2.99 (dd, J=9.2, 12.0 Hz, 2H), 2.29 (t, J=7.6 Hz, 2H), 2.00 (s,
3H), 1.98 (m, 1H), 1.68 (m, 2H). Calcd. for
C.sub.35H.sub.36Cl.sub.2N.sub.8O.sub.5+1.2H.sub.2O: C, 55.26; H,
5.40; N, 15.62. Found: C, 55.32; H, 5.25; N, 15.38.
5-(3-((2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-8-yl-
)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)-N-methylp-
icolinamide (27)
##STR00122##
[0163] Compound 27 was prepared from
3-(6-(methylcarbamoyl)pyridin-3-yl)propanoic acid and
2-amino-N-(2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-8-y-
l)oxy)methyl)phenyl)-N-methylacetamide in a as described for
compound 1. MS (APCI) 700 (M.sup.+), .sup.1H NMR (CDCl.sub.3) 2.17
(s, 3H), 2.48 (t, 2H, J=5.8 Hz), 2.65 (s, 3H), 2.89 (t, 2H, J=5.8
Hz), 3.22 (s, 3H), 3.4-3.5 (m, 1H), 3.7-3.81 (m, 1H), 5.41 (s, 2H),
5.61 (s, 2H), 7.2-7.3 (m, 1H), 7.31-7.40 (m, 1H), 7.44-7.62 (m,
5H), 7.75-7.76 (m, 1H), 7.9-8.06 (m, 4H), 8.63-8.65 (m, 1H).
3-(4-acetamidophenyl)-N-(2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmet-
hoxy)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)propanamide
(28)
##STR00123##
[0165] Compound 28 was prepared from 3-(4-acetamidophenyl)propanoic
acid and
2-amino-N-(2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-
-8-yl)oxy)methyl)phenyl)-N-methylacetamide in a as described for
compound 1. MS (APCI) 700 (M+), .sup.1H NMR (CDCl.sub.3) 1.91 (s,
3H), 2.4-2.6 (m, 5H), 2.80-2.93 (m, 2H), 3.10 (s, 3H), 3.32-3.45
(m, 1H), 3.61-3.71 (m, 1H), 5.41 (s, 2H), 5.45-5.56 (m, 2H),
6.41-6.45 (m, 1H), 6.71 (s, 1H), 6.91-7.05 (m, 3H), 7.18-7.21 (m,
1H), 7.22-7.28 (m, 1H), 7.31-7.50 (m, 2H), 7.55-7.60 (m, 1H),
7.65-7.75 (m, 1H), 7.91-7.99 (m, 1H), 8.61-8.69 (m, 1H), 9.14 (s,
1H).
N-(2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-8-yl)oxy-
)methyl)phenyl)(methyl)amino)-2-oxoethyl)-3-(4-(methylsulfonamido)phenyl)p-
ropanamide (29)
##STR00124##
[0167] Compound 29 was prepared from
3-(4-(methylsulfonamido)phenyl)propanoic acid and
2-amino-N-(2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-8-y-
l)oxy)methyl)phenyl)-N-methylacetamide in a as described for
compound 1. MS (APCI) 736 (M+), .sup.1H NMR (CDCl.sub.3) 2.45-2.56
(m, 2H), 2.65 (s, 3H), 2.83-2.92 (m, 5H), 3.15 (s, 3H), 3.35-3.48
(m, 1H), 3.75-3.85 (m, 1H), 5.41 (s, 2H), 5.61 (s, 2H), 6.61-6.65
(m, 1H), 6.71 (s, 1H), 7.15 (s, 4H), 7.24-7.34 (m, 2H), 7.3-7.45
(m, 5H), 7.56-7.61 (d, 1H, J=7.8 Hz), 7.66-7.78 (m, 1H), 7.91-7.94
(dd, 1H, J=1, 7.74 Hz), 8.64 (d, 1H, J=4.14 Hz).
4-(3-((2-((2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-8-yl-
)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)-N-methylb-
enzamide (31)
##STR00125##
[0169] Compound 31 was prepared from
3-(4-(methylcarbamoyl)phenyl)propanoic acid and
2-amino-N-(2,4-dichloro-3-(((2-methyl-4-(pyridin-2-ylmethoxy)quinolin-8-y-
l)oxy)methyl)phenyl)-N-methylacetamide in a as described for
compound 1. MS (APCI) 700 (M+), .sup.1H NMR (CDCl.sub.3) 2.42-2.52
(m, 2H), 2.61 (s, 3H), 2.85-2.95 (m, 5H), 3.15 (s, 3H), 3.32-3.45
(m, 1H), 3.62-3.68 (m, 1H), 5.41 (s, 2H), 5.61 (s, 2H), 6.41-6.51
(m, 1H), 6.55-6.62 (m, 1H), 6.71 (s, 1H), 7.15-7.31 (m, 4H),
7.31-7.45 (m, 2H), 7.55-7.62 (m, 1H), 7.62-7.71 (m, 2H), 7.71-7.81
(m, 1H), 7.91-7.95 (m, 1H), 8.65-8.72 (m, 1H).
3-(6-acetamidopyridin-3-yl)-N-(2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin--
2-ylmethyl)-1H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-ox-
oethyl)propanamide (32)
##STR00126##
[0171] Compound 32 is prepared
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and
3-(6-acetamidopyridin-3-yl)propanoic acid as described for compound
1. MS (APCI) 690 (M+), 1H NMR 2.18 (s, 3H), 2.49 (t, 2H, J=7.4 Hz),
2.89 (t, 2H, J=7.4 Hz), 3.32 (s, 3H), 3.4-3.55 (m, 1H), 3.7-3.82
(m, 1H), 4.21 (s, 3H), 5.28 (s, 2H), 5.66 (s, 2H), 6.45-6.52 (m,
1H), 6.81 (t, 2H, J=8.2 Hz), 6.92 (d, 1H, J=7.86 Hz), 7.01-7.06 (m,
1H), 7.17-7.19 (m 1H), 7.4-7.6 (m, 4H), 7.9-7.95 (m, 1H), 8.06-8.09
(m, 2H), 8.55-8.59 (m, 1H).
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropy-
l)-N-methylbenzamide (33)
##STR00127##
[0173] Compound 33 is prepared from
3-(4-(methylcarbamoyl)phenyl)propanoic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-
-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide as
described for compound 1. MS (ESI) 689 (M+) 1HNMR (CDCl3),
2.42-2.58 (m, 2H), 2.80-2.90 (m, 5H), 3.18 (s, 3H), 3.3-3.5 (m,
1H), 3.65-3.72 (m, 1H), 4.14 (s, 3H), 5.25 (s, 2H), 5.5-5.7 (m,
2H), 6.4-6.6 (m, 2H), 6.75-6.82 (m, 2H), 6.85-6.91 (m, 1H),
6.95-7.10 (m, 3H), 7.38-7.45 (m, 1H), 7.52-7.62 (m, 1H), 6.8-6.72
(m, 2H), 8.52-8.60 (m, 1H).
Preparation of (S)-tert-butyl
3-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imida-
zol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylcarbamoyl)pyrrolidine-1-
-carboxylate (34)
##STR00128##
[0175] A solution of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (2.00 g, 4 mmol)
in anhydrous DMF (12 ml) was treated with HBTU (2.12 g, 5.6 mmol),
(S)-1-N-boc-.beta.-proline (0.95 g, 4.4 mmol) and diisopropylethyl
amine (2.09 mL, 12 mmol) with stirring at room temperature
overnight. To the reaction was added ethyl acetate (200 mL) and the
mixture was washed with aqueous saturated sodium bicarbonate (100
mL.times.3). The organic layer was dried over magnesium sulfate,
filtered and evaporated to a tan solid, 2.43 g that was purified by
chromatography (silica gel, ethyl acetate and methanol gradient) to
afford (S)-tert-butyl
3-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imida-
zol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylcarbamoyl)pyrrolidine-1-
-carboxylate (34) as a tan solid, 1.68 g (60%), that had one peak
by LC/MS (+ESI) m/z 697 (M.sup.++1) and 711 (M.sup.++1) (both ions
in the same LC peak), and was pure by .sup.1HNMR (400 MHz,
CDCl.sub.3) .delta. 8.60 (m, 1H), 7.60 (m, 1H), 7.48 (m, 1H), 7.28
(m, 1H), 7.19 (m, 1H), 7.04 (m, 1H), 6.93 (m, 1H), 6.81 (m, 2H),
6.50 (m, 1H), 5.67 (s, 2H), 5.28 (s, 2H), 4.22 (s, 3H), 3.81 (m,
1H), 3.50 (m, 4H), 3.25 (m, 4H), 2.91 (m, 1H), 2.08 (m, 3H), 1.44
(s, 9H). .sup.13CNMR (100 MHz, CDCl.sub.3) .delta.168.2, 156.8,
156.1, 154.3, 149.6, 149.0, 138.4, 138.1, 137.0, 136.5, 135.8,
135.4, 130.1, 129.8, 122.6, 121.7, 120.8, 107.4, 103.1, 79.3, 67.1,
64.3, 60.4, 57.5, 48.6, 47.7, 41.9, 35.9, 30.7, 28.5, 21.0, 19.1,
14.2, 13.7. LN-15495-57.
[0176] To obtain an analytical sample, the above method was
repeated and the product (0.2 g) was treated with methanol (10 mL)
and potassium carbonate (0.2 g) with stirring at room temperature
overnight. The mixture was filtered and evaporated to give
(S)-tert-butyl
3-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]imida-
zol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylcarbamoyl)pyrrolidine-1-
-carboxylate as white solid, 0.12 g. Anal. Calcd for
C.sub.34H.sub.38Cl.sub.2N.sub.6O.sub.6: C, 58.54; H, 5.49; N,
12.05. Found: C, 58.14; H, 5.57; N, 11.80.
Preparation of
1-(2-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]im-
idazol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylamino)-2-oxoethyl)-N-
-methylpiperidine-3-carboxamide (35)
##STR00129##
[0178] A solution of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (0.45 g, 0.9
mmol) in anhydrous DMF (10 ml) was treated with HBTU (0.48 g, 1.2
mmol), 2-(3-(methylcarbamoyl)piperidin-1-yl)acetic acid (0.20 g,
0.99 mmol) and diisopropylethyl amine (0.47 mL, 2.7 mmol) with
stirring at room temperature overnight. To the reaction was added
ethyl acetate (100 mL) and the mixture was washed with aqueous
saturated sodium bicarbonate (100 mL.times.3). The organic layer
was dried over magnesium sulfate, filtered and evaporated to a
sticky yellow residue, 0.63 g that was purified by chromatography
(silica gel, dichloromethane and methanol gradient) to afford
1-(2-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-ben-
zo[d]imidazol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylamino)-2-oxoe-
thyl)-N-methylpiperidine-3-carboxamide (35) as a white solid, 0.17
g (28%), that was pure by LC/MS (+APCI) m/z 682 (M'+1), .sup.1HNMR
(400 MHz, CDCl.sub.3) .delta.8.60 (m, 1H), 7.60 (m, 2H), 7.50 (m,
1H), 7.30 (m, 1H), 7.35 (m, 1H), 7.19 (m, 1H), 7.04 (m, 1H), 6.92
(m, 1H), 6.81 (m, 2H), 6.70 (broad m, 1H), 5.67 (s, 2H), 5.29 (s,
2H), 4.21 (two s, 3H), 4.1 to 1.4 (many multiplets, 19H).
.sup.13CNMR (100 MHz, CDCl.sub.3) .delta.174.4, 170.5, 168.8,
156.8, 156.1, 149.6, 149.0, 138.4, 138.2, 137.0, 136.5, 135.8,
135.3, 130.3, 122.6, 121.7, 120.8, 107.3, 103.1, 67.0, 61.6, 57.5,
56.5, 56.2, 54.3, 53.4, 47.7, 45.4, 41.3, 36.0, 30.9, 26.3, 26.1,
24.5. Anal. Calcd for
C.sub.33H.sub.37Cl.sub.2N.sub.6O.sub.5+0.57CH.sub.2Cl.sub.2: C,
55.16; H, 5.26; N, 13.41. Found: C, 55.18; H, 5.14; N, 13.35.
Preparation of
(S)-1-(2-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[-
d]imidazol-4-yloxy)methyl)phenyl)methyl)amino)-2-oxoethylamino)-2-oxoethyl-
)-N-methylpiperidine-3-carboxamide (36)
##STR00130##
[0180] A solution of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (0.45 g, 0.9
mmol) in anhydrous NMP (10 ml) was treated with HBTU (0.48 g, 1.2
mmol), (S)-2-(3-(methylcarbamoyl)piperidin-1-yl)acetic acid (0.21
g, 1.08 mmol) and diisopropylethyl amine (0.47 mL, 2.7 mmol) with
stirring at room temperature overnight. To the reaction was added
ethyl acetate (100 mL) and the mixture was washed with aqueous
saturated sodium bicarbonate (100 mL.times.3). The organic layer
was dried over magnesium sulfate, filtered and evaporated to an off
white solid, 0.34 g that was purified by chromatography (silica
gel, ethyl acetate and methanol gradient) to afford
(S)-1-(2-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-
-benzo[d]imidazol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylamino)-2--
oxoethyl)-N-methylpiperidine-3-carboxamide (36) as a white solid,
0.12 g (20%), that was about 90 pure by LC/MS (+APCI) m/z 682
(M.sup.+), .sup.1HNMR (400 MHz, CDCl.sub.3) .delta. 8.60 (m, 1H),
7.60 (m, 2H), 7.50 (m, 1H), 7.30 (m, 1H), 7.35 (m, 1H), 7.19 (m,
1H), 7.04 (m, 1H), 6.92 (m, 1H), 6.81 (m, 2H), 6.70 (broad m, 1H),
5.67 (s, 2H), 5.29 (s, 2H), 4.21 (s, 3H), 4.1 to 1.4 (many
multiplets, 19H). .sup.13CNMR (100 MHz, CDCl.sub.3) .delta.174.4,
170.5, 168.8, 156.8, 156.1, 149.6, 149.0, 138.4, 138.2, 137.0,
136.5, 135.8, 135.3, 130.3, 122.6, 121.7, 120.8, 107.3, 103.1,
67.0, 61.6, 57.5, 56.5, 56.2, 54.3, 53.4, 47.7, 45.4, 41.3, 36.0,
30.9, 26.3, 26.1, 24.5.
Preparation of methyl
4-(3-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]im-
idazol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylamino)-3-oxopropyl)b-
enzoate (37)
##STR00131##
[0182] A solution of
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (0.45 g, 0.9
mmol) in anhydrous NMP (12 ml) was treated with HBTU (0.48 g, 1.2
mmol), 3-(4-(methoxycarbonyl)phenyl)propanoic acid (0.22 g, 1.08
mmol) and diisopropylethyl amine (0.47 mL, 2.7 mmol) with stirring
at room temperature overnight. To the reaction was added ethyl
acetate (100 mL) and the mixture was washed with aqueous saturated
sodium bicarbonate (100 mL.times.3). The organic layer was dried
over magnesium sulfate, filtered and evaporated to a yellow
residue, 0.59 g that was purified by chromatography (silica gel,
ethyl acetate and methanol gradient) to afford methyl
4-(3-(2-((2,4-dichloro-3-((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]im-
idazol-4-yloxy)methyl)phenyl)(methyl)amino)-2-oxoethylamino)-3-oxopropyl)b-
enzoate (37) as a white solid, 0.18 g (29%), LC/MS (+APCI) m/z 690
(M'+1) and 704 (M'+1), .sup.1HNMR (400 MHz, CDCl.sub.3) .delta.8.60
(m, 1H), 7.92 (m, 2H), 7.60 (m, 1H), 7.49 (m, 1H), 7.29 (m, 1H),
7.19 (m, 1H), 7.02 (m, 1H), 6.91 (m, 1H), 6.82 (m, 2H), 6.35 (m,
1H), 5.67 (s, 2H), 5.28 (s, 2H), 4.21 (s, 3H), 3.89 (s, 3H), 4.1 to
2.5 (many multiplets, 10H). .sup.13CNMR (100 MHz, CDCl.sub.3)
.delta. 171.4, 168.2, 156.1, 149.6, 149.0, 146.2, 138.4, 138.1,
137.0, 135.8, 135.3, 130.2, 129.9, 128.4, 128.2, 122.6, 121.7,
120.8, 107.3, 103.0, 67.0, 57.5, 51.9, 47.7, 41.9, 37.4, 35.9,
31.4
(2R,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-b-
enzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3--
oxopropyl)-N-methylpiperidine-2-carboxamide and
(2S,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide (38 and 39)
##STR00132##
[0184] A mixture of tert-butyl
3-(6-(methylcarbamoyl)pyridin-3-yl)propanoate (2.16 g, 8.18 mmol),
PtO.sub.2 (0.40 g) and 1,1,2-trichloroethane (1.0 mL, 12.4 mmol) in
methanol (50 mL) was placed in Parr reactor under 70 psi hydrogen
atmosphere for 3.5 days. To the mixture was added 1.25 N HCl/MeOH
(6 mL, 7.5 mmol) and the reaction was allowed to continue
overnight. The reaction was filtered and evaporated to dryness to
give tert-butyl 3-(6-(methylcarbamoyl)piperidin-3-yl)propanoate/HCl
salt as a foamy solid (2.80 g). LCMS (+APCI) 271 (M.sup.+). The
solid give tert-butyl
3-(6-(methylcarbamoyl)piperidin-3-yl)propanoate/HCl was mixed with
dichloromethane (100 mL) at 0.degree. C. and to this mixture was
added DIPEA (diisopropylethylamine) (6.37 mL) and trifluoroacetic
anhydride (3.44 g, 16.4 mmol) with stirring for 30 min. The mixture
was warmed to room temperature, stirred for 30 min and evaporated
to dryness. The crude was neutralized with a saturated aqueous
sodium bicarbonate solution (100 mL) and extracted with ethyl
acetate. Evaporation gave tert-butyl
3-(6-(methylcarbamoyl)-1-(2,2,2-trifluoroacetyl)piperidin-3-yl)propanoate
as an oil that was purified by silica chromatography eluting with a
gradient of 5% to 100% EtOAc/Hex then 0% to 10% MeOH/EtOAc to give
0.84 g of the ester as an oil. LCMS (+APCI) 367 (M+1).
[0185] tert-butyl
3-(6-(methylcarbamoyl)-1-(2,2,2-trifluoroacetyl)piperidin-3-yl)propanoate
was treated with trifluoroacetic acid (5 mL) for 10 min, evaporated
to dryness and placed in vacuum overnight. The crude
3-(6-(methylcarbamoyl)-1-(2,2,2-trifluoroacetyl)piperidin-3-yl)propanoic
acid was mixed with dichloromethane (30 min) and to this mixture
was added DIPEA (3 mL),
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (1.03 g, 2.07
mmol) and HBTU (1.13 g, 2.99 mmol) with stirring. The mixture was
stirred for 1 h and was added a saturated aqueous sodium
bicarbonate solution (100 mL) and extracted with dichloromethane.
The combined extracts was dried and evaporated to dryness. Silica
chromatography eluting with a gradient of 3% to 10% of MeOH/DCM
gave a mixture of
(2R,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide and
(2S,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide, 1.01 g as a foamy
solid (62%). LCMS (+ESI) 792 (M.sup.+).
[0186] The mixture of
(2R,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide and
(2S,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide (1.01 g, 1.27 mmol)
was diluted with ethanol (20 mL). To the solution was added
NaBH.sub.4 (10% on Al.sub.2O.sub.3, 0.48 g, 1.27 mmol) with
stirring. After 1.5 h a saturated aqueous sodium bicarbonate
solution (50 mL) was added and the mixture was extracted with
dichloromethane. Evaporation gave a crude mixture that was
separated by chromatography eluting with a gradient of 10% to 40%
MeOH/DCM to give two isomers (0.19 g and 0.12 g respectively). NMR
analysis showed the first isomer was the cis-isomer
(2S,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide and the second isomer
was the trans-isomer
(2R,5R)-5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-
-oxopropyl)-N-methylpiperidine-2-carboxamide. 2S,5R (cis) isomer:
LCMS (+APCI) 696 (M+1). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.58
(d, J=4.0 Hz, 1H), 7.58 (m, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.28 (d,
J=8.4 Hz, 1H), 7.19 (m, 1H), 7.04 (t, J=8.0 Hz, 1H), 6.92 (d, J=7.6
Hz, 1H), 6.80 (dd, 2H), 6.48 (bs, 1H), 6.38 (bt, 1H), 5.66 (dd,
2H), 5.28 (s, 2H), 4.21 (s, 3H), 3.82 (dt, 1H), 3.50 (dt, 1H), 3.35
(t, 1H), 3.25 (s, 3H), 2.81 (d, J=4.8 Hz, 3H), 2.80 (m, 1H), 2.48
(m, 1H), 2.22 (m, 2H), 2.07 (m, 1H), 1.69 (m, 1H), 1.60 (m, 1H),
1.50 (m, 1H), 1.39 (m, 1H), 1.29 (m, 1H). Calcd. for
C.sub.34H.sub.39Cl.sub.2N.sub.7O.sub.5+1.8H.sub.2O: C, 56.01; H,
5.89; N, 13.45. Found: C, 56.04; H, 5.59; N, 13.15. 2R,5R (trans
isomer) .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.59 (d, J=4.0 Hz,
1H), 7.59 (m, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.28 (d, J=8.8 Hz, 1H),
7.19 (m, 1H), 7.03 (t, J=8.0 Hz, 1H), 6.92 (d, 1H), 6.80 (dd,
J=13.8, 7.6 Hz, 2H), 6.63 (bs, 1H), 6.38 (bt, 1H), 5.66 (dd, 2H),
5.28 (s, 2H), 4.21 (s, 3H), 3.82 (dd, J=4.4, 17.6 Hz, 1H), 3.50
(dd, J=3.6, 18.0 Hz, 1H), 3.25 (s, 3H), 3.11 (dd, J=2.8, 11.2 Hz,
1H), 3.07 (m, 1H), 2.78 (d, J=4.8 Hz, 3H), 2.28 (t, J=11.2 Hz, 1H),
2.23 (m, 2H), 2.07 (m, 1H), 1.99 (bd, J=13.6 Hz, 1H), 1.50 (m, 2H),
1.37 (m, 1H), 1.28 (m, 1H), 1.05 (m, 1H). Calcd. for
C.sub.34H.sub.39Cl.sub.2N.sub.7O.sub.5+1.8H.sub.2O: C, 56.01; H,
5.89; N, 13.45. Found: C, 56.15; H, 5.63; N, 13.18.
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2,2-difluo-
ro-3-oxopropyl)-N-methylpiperazine-1-carboxamide (40)
##STR00133##
[0188] Benzotriazolmethanol (5.00 g, 33.5) and t-boc-piperazine
(6.70 g, 33.5) were stirred in methanol at room temperature for 2 h
to give 4-benzotriazol-ylmethylpiperazine-1-carboxylic acid
tert-butyl ester. This solid was filtered and crystallized from
methanol.
[0189] To a suspension of zinc dust (1.93 g, 29.5 mmol) in dry THF
(30 mL), under nitrogen, was added TMS-Cl (trimethylsilyl chloride)
(1.28 g, 11.8 mmol). The reaction mixture was stirred for 15 min,
and then ethylbromodifluoroacetate (4.49 g, 22.1 mmol) was slowly
added, followed 15 min later by a solution
4-benzotriazol-ylmethylpiperazine-1-carboxylic acid tert-butyl
ester (4.68 g, 14.8 mmol). After, 3 h stirring at room temperature,
the mixture was poured onto 5% aqueous NaHCO.sub.3 (40 mL) and
filtered on celite. The filtrate was diluted with ethyl acetate and
organic layer was separated, dried (MgSO.sub.4) and concentrated to
give an oil which was purified by flash chromatography
(Hexane-Ethyl acetate (8:2) to give 1.05 g of
4-(2-ethoxycarbonyl-2,2-difluoro-ethyl)-piperazine-1-carboxylic
acid tert-butyl ester as an oil.
[0190] .sup.1HNMR (400 MHz, CDCl.sub.3) .delta. 1.37 (t, 3H), 1.44
(s, 9H), 2.58 (m, 4H), 3.01 (dd, 2H), 3.38 (m, 4H), 4.35 (q,
2H)
[0191]
4-(2-ethoxycarbonyl-2,2-difluoro-ethyl)-piperazine-1-carboxylic
acid tert-butyl ester prepared was dissolved in dichloromethane (10
mL) and TFA (5 mL). The solution was stirred for 2 h at room
temperature, concentrated to dryness, and dissolved in
dichloromethane (20 mL) and triethylamine (0.69 g, 6.84 mmol),
after stirring for 5 min, a solution of methylaminoformyl chloride
(0.34 g, 3.64 mmol) was slowly added. The reaction mixture was
stirred for 2 h, diluted with dichloromethane (100 mL), washed with
aqueous solution of NaHCO.sub.3. Organic layer was dried
(MgSO.sub.4), and concentrated to ethyl
2,2-difluoro-3-(4-(methylcarbamoyl)piperazin-1-yl)propanoate as an
oil, which was purified by flash chromatograph using ethyl
acetate.
[0192] .sup.1HNMR (400 MHz, CDCl.sub.3) .delta. 1.37 (t, 3H), 2.59
(m, 4H), 2.79 (d, 3H), 2.99 (dd, 2H), 3.3 (m, 4H), 4.31 (q, 2H)
[0193] To ethyl
2,2-difluoro-3-(4-(methylcarbamoyl)piperazin-1-yl)propanoate (1.0
g, 3.7 mmol) in THF (15 mL) and water (18 mL) was added LiOH (0.6
g, 14.29 mmol), after 18 h stirring at room temperature, the
volatiles were removed by high vacuum and the solid residue
2,2-difluoro-3-(4-(methylcarbamoyl)piperazin-1-yl)propanoic acid
was used without further purification. LCMS 250 [M-H].sup.-
[0194] 2,2-difluoro-3-(4-(methylcarbamoyl)piperazin-1-yl)propanoic
acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benz-
o[d]imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide are coupling
using TBTU as described for compound 1 to give
4-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2,2-diflu-
oro-3-oxopropyl)-N-methylpiperazine-1-carboxamide. LCMS (APCI) 733
(M.sup.+).
[0195] .sup.1HNMR (400 MHz, MeOD) .delta. 2.48 (m, 4H), 2.59 (s,
3H), 2.89 (t, 3H), 3.11 (m, 3H), 3.21 (s, 3H), 3.5-3.76 (m, 2H),
4.03 (s, 3H), 5.21 (s, 2H), 5.48 (m, 2H), 6.74 (m, 2H), 6.93 (m,
2H), 6.97 (m, 1H), 7.50 (m, 2H), 7.64 (m, 1H), 8.40 (d, 1H).
5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropy-
l)-N-methyl-1,4,5,6-tetrahydropyrimidine-2-carboxamide (41)
##STR00134##
[0197] To a solution of 5-bromopyrimidine-2-carboxylic acid methyl
ester (5.0 g, 23.0 mmol) in THF (10 mL) was added 8 N solution of
methyl amine in ethanol (11.5 mL). The reaction mixture was heated
20 min in a microwave oven, cooled down to room temperature and
concentrated under pressure to give 5 g of
5-bromo-N-methylpyrimidine-2-carboxamide as a solid LCMS: 216, 218
(M+1).
[0198] To a solution of 5-bromo-N-methylpyrimidine-2-carboxamide
(0.14 g, 0.64 mmol) in DMF (5 mL) was added acrylic acid benzyl
ester (0.13 g, 0.78 mmol), Pd(OAc).sub.2 (14.3 mg), tri-o-tolyl
phosphine (49 mg, 0.16 mmol), tributyl amine (0.48 g, 2.59 mmol).
The reaction mixture was heated in microwave oven at 110.degree. C.
for 1 h. The reaction mixture was cooled to room temperature,
partitioned in water (50 mL) and ethyl acetate (50 mL). Ethyl
acetate layer was separated, dried (MgSO.sub.4), concentrated to
give 3-(2-methylcarbamoyl-pyrimidin 5-yl)acrylic acid benzyl ester
as a solid, this was washed with hexanes and dried to give the
0.122 g of the product. LCMS (APCI) 298 (M.sup.+)
[0199] 3-(2-methylcarbamoyl-pyrimidin 5-yl)acrylic acid benzyl
ester was dissolved in ethanol (20 mL) in presence of 15 mg of Pd/C
10% and HCl.sub.c (2 mL). The mixture was stirred under hydrogen
balloon overnight to give
3-(2-methylcarbamoyl-pyridine-5-yl)-propionic acid which was used
without further purification.
[0200] 3-(2-methylcarbamoyl-pyridine-5-yl)-propionic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide were coupled as
described for compound 1. MS (APCI) 695 (M.sup.+), .sup.1HNMR (400
MHz, MeOD) .delta. 1.57-1.69 (m, 3H), 2.31 (m, 2H), 2.78 (s, 3H),
2.98 (m, 2H), 3.19 (s, 3H), 3.34-3.824 (m, 4H), 4.30 (s, 3H), 5.30
(s, 2H), 5.55 (m, 2H), 6.83 (m, 2H), 7.05 (m, 2H), 7.30 (m, 1H),
7.57 (m, 2H), 7.63 (m, 1H), 8.49 (d, 1H).
C.sub.33H.sub.36Cl.sub.2N.sub.8O.sub.5+0.4H.sub.2O, Calculated: C,
56.4; H, 5.28; N, 15.94. Found: C, 56.4; H, 5.35; N, 15.90.
5-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropy-
l)-N-methylpyrimidine-2-carboxamide (42)
##STR00135##
[0202] 3-(2-methylcarbamoyl-pyrimidin 5-yl)acrylic acid benzyl
ester in ethanol (20 mL) in presence of 15 mg of Pd/C 10%, and
stirred under hydrogen balloon overnight to give
3-(2-methylcarbamoyl-pyridine-5-yl)-propionic acid which was used
without further purification.
[0203] 3-(2-methylcarbamoyl-pyridine-5-yl)-propionic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide and coupled
together as described for compound 1 above. LCMS (APCI) 691 (M+1);
.sup.1HNMR (400 MHz, MeOD) .delta. 2.90-3.0 (m, 5H), 3.21-3.25 (m,
5H), 3.66 (d, 1H), 3.96 (d, 1H), 4.15 (s, 3H), 5.33 (s, 2H), 5.58
(m, 2H), 6.86 (m, 2H), 7.08 (m, 2H), 7.32 (m, 1H), 7.55-7.62 (m,
2H), 7.76 (m, 1H), 8.51 (d, 1H), 9.08 (s, 2H)
6-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropy-
l)-N-methylnicotinamide (43)
##STR00136##
[0205] Methyl 6-formylnicotinate (1.0 g, 6.06 mmol) and tert-butyl
(triphenylphosphoranylidene)acetate (2.73 g, 7.27 mmol) were put in
suspension in water (50 mL) and stirred at 90.degree. C. for 2 h.
The reaction mixture was cooled to room temperature and
concentrated under pressure. The residue was triturated in ethyl
acetate and filtered. The filtrate was concentrated and purified by
flash chromatography using hexanes-ethyl acetate 10% to give
(E)-methyl 6-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)nicotinate. MS
(APCI) 264 (M.sup.+). (E)-methyl
6-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)nicotinate was dissolved in
ethanol in the presence of 50 mg Pd/C 10% and stirred under
hydrogen balloon overnight to give
6-2(tert-butoxycarbonyl-ethyl)nicotinic methyl ester. LCMS: 266
(M.sup.+)
[0206] To a solution of 6-2(tert-butoxycarbonyl-ethyl)nicotinic
methyl ester (1.7 g, 6.4 mmol) in methanol (10 mL) was added 3.2 mL
of 8 N solution of methyl amine in methanol. The reaction vial was
sealed and stirred at 70.degree. C. overnight, cooled to room
temperature and concentrated under reduced pressure. The residue
was purified by flash chromatography using ethyl acetate to give
tert-butyl-3(5-methylcarbamoyl pyridine-2-yl) propionate. LCMS 265
(M+1)
[0207] tert-Butyl-3(5-methylcarbamoyl-pyridin-2-yl)propionate (0.29
g, 1.10 mmol) was dissolved in 10 mL of a 4 N hydrogen chloride
solution in dioxane and stirred overnight. Evaporation of the
solvent gave 0.27 mg of 3(5-methylcarbamoyl-pyridin-2-yl)propionic
acid hydrochloride. LCMS 209 (M+1)
[0208] To a suspension of
3(5-methylcarbamoyl-pyridin-2-yl)propionic acid hydrochloride (0.26
g, 1.06 mmol) in dichloromethane (20 mL) was added
ethyldiisopropylamine (0.74 mL, 4.26 mmol), the solution was
stirred for 5 min, and then
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide (0.5 g, 1.0
mmol) was added followed by HOBT (0.135 g, 1.0 mmol) and EDCI (0.28
mL, 1.59 mmol). The reaction mixture was then stirred overnight at
room temperature and partitioned in dichloromethane (100 mL) and a
saturated solution of NaHCO.sub.3 (100 mL). Organic layer was
separated, dried (MgSO.sub.4) and concentrated to give oil, which
was purified by flash chromatography using a mixture of
dichloromethane- and a 5% ammonium solution in methanol (9:1) to
give
6-(3-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxoprop-
yl)-N-methylnicotinamide as foam. LCMS (APCI) 690 (M.sup.+);
.sup.1HNMR (400 MHz, MeOD) .delta. 2.60 (t, 2H), 3.07 (s, 3H), 3.19
(t, 2H), 3.20 (s, 3H), 3.32-3.69 (dd, 2H), 4.01 (s, 3H), 5.19 (s,
2H), 5.46 (dd, 2H), 6.71-8.74 (m, 12H)
4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]i-
midazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoethyl-
)-N-methylmorpholine-2-carboxamide (44)
##STR00137##
[0210] To a cold solution (ice bath) of
N-Boc-2-morpholinecarboxylic acid (0.5 g, 2.16 mmol) in DMF (10 mL)
was added HATU
((2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) (0.98 g, 2.58 mmol), N,N diisopropylethylamine
(2.2 g, 17.3 mmol), and 1 mL of 8 N solution of methylamine in
ethanol. After the addition was finished the ice bath was removed,
and the heterogeneous solution was stirred overnight at room
temperature. The solution was dissolved in ethyl acetate (50 mL)
and washed with a saturated solution of NaHCO.sub.3. Ethyl acetate
layer was separated, dried (MgSO.sub.4) and concentrated under
reduced pressure to give tert-butyl
2-(methylcarbamoyl)morpholine-4-carboxylate as an oil. MS (APCI)
245 (M+)
[0211] tert-butyl 2-(methylcarbamoyl)morpholine-4-carboxylate
prepared above (0.5 g, 0.20 mmol) was dissolved in DCM (10 mL) and
TFA (2 mL). The reaction mixture was stirred at room temperature
for 4 h and then concentrated; excess 1N HCl solution in ether was
added to the residue and concentrated under high vacuum. The
residue was dissolved in acetonitrile (20 mL) and K.sub.2CO.sub.3
(0.56 g, 40.8 mmol) was added followed by bromo-acetic acid benzyl
ester (0.7 g, 3.07 mmol). The reaction mixture was heated at
60.degree. C. overnight. The solution was cooled to room
temperature and diluted with Ethyl acetate (50 mL) and water (50
mL), organic layer was separated, dried (MgSO.sub.4) and
concentrated to give an oil which was purified by column
chromatography using ethyl acetate-hexanes (1:1) to give benzyl
2-(2-(methylcarbamoyl)morpholino)acetate. MS (APCI) 293
(M.sup.+).
[0212] benzyl 2-(2-(methylcarbamoyl)morpholino)acetate was
dissolved in ethanol (40 mL), Pd/C 10% (100 mg) was added to the
solution, and the mixture was stirred overnight under hydrogen
(balloon) then filtered through celite and concentrated to give
2-(2-(methylcarbamoyl)morpholino)acetic acid. MS (APCI) 203
(M+1)
[0213] 2-(2-(methylcarbamoyl)morpholino)acetic acid and
2-amino-N-(2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo[d]-
imidazol-4-yl)oxy)methyl)phenyl)-N-methylacetamide were coupled
using EDCI to give
4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H--
benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-
-oxoethyl)-N-methylmorpholine-2-carboxamide. MS (APCI) 684
(M.sup.+); 1H NMR (CDCl.sub.3) 2.26-2.40 (m, 1H), 2.61-2.82 (m,
4H), 2.95-3.15 (m, 2H), 3.15-3.28 (m, 4H), 3.40-3.49 (m, 1H),
3.61-3.91 (m, 2H), 3.90-4.01 (m, 1H), 4.08-4.18 (m, 1H), 4.22 (s,
3H), 5.28 (s, 2H), 5.62-5.70 (m, 2H), 6.51-6.58 (m, 1H), 6.75-6.87
(m, 2H), 6.91 (d, 1H, J=5.7 Hz), 7.02-7.06 (t, 1H, J=6.2 Hz),
7.19-7.21 (m, 1H), 7.31-7.33 (m, 1H), 7.46-7.49 (m, 1H), 7.55-7.62
(m, 1H), 7.78-7.90 (m, 1H), 8.57 (m, 1H).
C.sub.32H.sub.35Cl.sub.2N.sub.7O.sub.6+0.8H.sub.2O, Calculated: C,
54.97; H, 5.28; N, 14.03. Found: C, 54.99; H, 5.20; N, 13.85.
(S)-4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1H-benzo-
[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-2-oxoe-
thyl)-N-methylmorpholine-2-carboxamide (45)
##STR00138##
[0215]
(S)-4-(2-((2-((2,4-dichloro-3-(((2-methoxy-1-(pyridin-2-ylmethyl)-1-
H-benzo[d]imidazol-4-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-
-2-oxoethyl)-N-methylmorpholine-2-carboxamide was prepared from
N-Boc-2S-morpholinecarboxylic acid as described for the 44 above.
MS (APCI) 684 (M.sup.+)
[0216] .sup.1HNMR (400 MHz, MeOD) .delta. 2.18-2.24 (m, 2H), 2.64
(s, 3H), 2.66 (t, 1H), 3.11 (m, 3H), 3.23 (s, 3H), 3.5-4.10 (m,
5H), 4.16 (s, 3H), 5.21 (s, 2H), 5.46 (m, 2H), 6.76 (m, 2H), 6.99
(m, 2H), 7.22 (m, 1H), 7.48 (m, 2H), 7.66 (m, 1H), 8.39 (d,
1H).
Preparation of ethyl
2-((8-((2,6-dichloro-3-(N-methyl-2-(3-(6-(methylcarbamoyl)pyridin-3-yl)pr-
opanamido)acetamido)benzyl)oxy)-2-methylquinolin-4-yl)oxy)acetate
(46)
##STR00139##
[0218] To 4-chloro-2-methylquinolin-8-ol (10.20 g, 52.7 mmol),
potassium carbonate (10.9 g, 79.0 mmol) in 2-butanone (100 mL) was
added benzyl bromide (6.89 mL, 58.0 mmol) vial syringe with
stirring. The suspension was heated at 70.degree. C. for 4 h,
stirred at ambient temperature overnight and poured into ice water
(200 mL). The precipitate was filtered, washed with a little
EtOAc/water and dried to give
8-(benzyloxy)-4-chloro-2-methylquinoline (5.82 g). The filtrate was
extracted with EtOAc, dried, filtered and evaporated. Solid from
Hex/EtOAc (10/1) was filtered and dried to give additional
8-(benzyloxy)-4-chloro-2-methylquinoline, 13.72 g (92% total crude
yield).
[0219] To a mixture of 8-(benzyloxy)-4-chloro-2-methylquinoline
(3.09 g, 10.9 mmol), potassium carbonate (3.22 g, 32.8 mmol) in
DMSO (30 mL) was added water (1.00 mL) to dissolve the solid. The
mixture was heated at 140.degree. C. for 2 h and was added 2N NaOH
(12 mL, 24.0 mmol) and heated at 130.degree. C. overnight. The
mixture was poured into ice/NaHCO.sub.3. Solid precipitated and was
filtered, washed with water and dried to give
8-(benzyloxy)-2-methylquinolin-4-ol (2.34 g, 81%).
[0220] To a mixture of give 8-(benzyloxy)-2-methylquinolin-4-ol
(2.34 g, 8.83 mmol) and potassium carbonate (1.83 g, 13.2 mmol) in
anhydrous DMF (50 mL) was added ethyl bromoacetate (1.77 g, 10.6
mmol) with stirring under nitrogen. The mixture was heated at
80.degree. C. for 1 h, cooled, mixed with a saturated aqueous
sodium bicarbonate and extracted with EtOAc. Evaporation gave a
crude product that was dissolved in dichloromethane and
precipitated by the addition of a mixture of Hex/EtOAc (10/1). The
precipitate was filtered and dried to give ethyl
2-((8-(benzyloxy)-2-methylquinolin-4-yl)oxy)acetate (2.13 g, 69%).
A mixture of ethyl
2-((8-(benzyloxy)-2-methylquinolin-4-yl)oxy)acetate (0.70 g, 1.99
mmol), Pd/C (10%, 0.07 g) in EtOH/dioxane (20 mL/20 mL) was placed
under hydrogen atmosphere with stirring overnight. The mixture was
filtered and evaporated to give ethyl
2-((8-hydroxy-2-methylquinolin-4-yl)oxy)acetate (0.24 g, 46%).
[0221] To a mixture of
2-((8-hydroxy-2-methylquinolin-4-yl)oxy)acetate (0.24 g, 0.92 mmol)
and
5-(3-((2-((2,4-dichloro-3-(chloromethyl)phenyl)(methyl)amino)-2-oxoethyl)-
amino)-3-oxopropyl)-N-methylpicolinamide (0.28 g, 0.61 mmol) in
anhydrous DMF (30 mL) was added potassium carbonate (0.25 g, 1.8
mmol). The mixture was heated at 80.degree. C. for 3 h, cooled,
mixed with a saturated aqueous sodium bicarbonate and extracted
with EtOAc. The crude mixture was purified by silica chromatography
eluting twice with a gradient of 3% to 25% of MeOH/DCM and a
gradient of 5% to 15% of MeOH/DCM to give ethyl
2-((8-((2,6-dichloro-3-(N-methyl-2-(3-(6-(methylcarbamoyl)pyridin-3-yl)pr-
opanamido)acetamido)benzyl)oxy)-2-methylquinolin-4-yl)oxy)acetate
as a powder (0.16 g, 38%) after drying at high vacuum at 78.degree.
C. overnight. LCMS (+ESI) 696 (M+1). .sup.1H-NMR (CDCl.sub.3,
.delta.): 8.37 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.95 (bs, 1H), 7.91
(d, J=7.2 Hz, 1H), 7.63 (m, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.38 (t,
J=8.0 Hz, 1H), 7.25 (m, 3H), 6.50 (s, 1H), 6.40 (bt, 1H), 5.62 (s,
2H), 4.82 (s, 2H), 4.31 (q, J=7.2 Hz, 2H), 3.76 (dd, J=4.8, 18 Hz,
1H), 3.43 (dd, J=4.0, 18 Hz, 1H), 3.21 (s, 3H), 3.01 (m, 6H), 2.66
(s, 3H), 2.54 (t, J=7.6 Hz, 2H), 1.30 (t, J=7.2 Hz, 3H). Calcd. for
C.sub.34H.sub.35Cl.sub.2N.sub.5O.sub.7+0.7H.sub.2O: C, 57.58; H,
5.17; N, 9.88. Found: C, 57.30; H, 5.30; N, 10.11.
5-(3-((2-((2,4-dichloro-3-(((4-((2-fluorobenzyl)oxy)-2-methylquinolin-8-yl-
)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)-N-methylp-
icolinamide (47)
##STR00140##
[0223] To a stirred solution of 2-fluorobenzyl alcohol (1.95 g,
15.5 mmol) in anhydrous DMF (35 mL) was added sodium hydride (60%
in mineral oil, 0.62 g, 15.5 mmol). After 30 min the
4-chloro-2-methylquinolin-8-ol (1.00 g, 5.16 mmol) was added and
resulting mixture was heated at 130.degree. C. for 20 h under
nitrogen atmosphere. The mixture was cooled, poured into
ice/NaHCO.sub.3 and extracted with EtOAc. Evaporation gave a crude
oil that was purified by silica chromatography eluting with a
gradient of 1% to 10% of MeOH/DCM to give
4-((2-fluorobenzyl)oxy)-2-methylquinolin-8-ol as an oil that
solidified on standing (0.39 g, 27%). LCMS (+APCI) 284 (M+1), mp
124-125.degree. C. .sup.1H-NMR (CDCl.sub.3, .delta.): 7.75 (d,
J=8.4 Hz, 1H), 7.50 (d, J=7.6 Hz, 2H), 7.43 (s, 1H), 7.35 (m, 3H),
7.20 (d, J=7.6 Hz, 1H), 7.06 (d, J=8.0 Hz), 5.45 (s, 2H), 2.78 (s,
3H).
[0224] 4-((2-fluorobenzyl)oxy)-2-methylquinolin-8-ol from step 1
was reacted with
5-(3-((2-((2,4-dichloro-3-(chloromethyl)phenyl)(methyl)amino)-2-oxoethyl)-
amino)-3-oxopropyl)-N-methylpicolinamide as described previously to
give
5-(3-((2-((2,4-dichloro-3-(((4-(2-fluorobenzyl)oxy)-2-methylquinolin-8-yl-
)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)-N-methylp-
icolinamide in 53% yield. MS (+APCI) 718 (M.sup.+). .sup.1H-NMR
(CDCl.sub.3, .delta. ppm): 8.36 (s, 1H), 8.07 (d, J=8.0 Hz, 1H),
7.95 (bd, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.63 (m, 1H), 7.58 (m, 1H),
7.45 (d, J=8.4 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.25 (m, 4H), 7.15
(m, 1H), 7.05 (m, 1H), 6.77 (bs, 1H), 6.40 (bt, 1H), 5.62 (s, 2H),
5.38 (s, 2H), 3.76 (dd, J=5.2, 17.6 Hz, 1H), 3.46 (dd, J=3.6, 18
Hz, 1H), 3.22 (s, 3H), 3.00 (m, 6H), 2.69 (m, 2H), 2.56 (t, 2).
5-(3-((2-((2,4-dichloro-3-(((2-methyl-4-((1-methyl-1H-imidazol-4-yl)methox-
y)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopr-
opyl)-N-methylpicolinamide (48)
##STR00141##
[0226]
5-(3-((2-((2,4-dichloro-3-(((2-methyl-4-((1-methyl-1H-imidazol-4-yl-
)methoxy)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)--
3-oxopropyl)-N-methylpicolinamide was prepared from
2-methyl-4-((1-methyl-1H-imidazol-4-yl)methoxy)quinolin-8-ol and
5-(3-((2-((2,4-dichloro-3-(chloromethyl)phenyl)(methyl)amino)-2-oxoethyl)-
amino)-3-oxopropyl)-N-methylpicolinamide as previously described.
MS (+ESI) 704 (M+1). .sup.1H-NMR (CDCl.sub.3, .delta. ppm): 8.36
(s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.94 (bs, 1H), 7.98 (dd, J=1.2,
9.2 Hz, 1H), 7.64 (dd, J=2.4, 8.4 Hz, 1H), 7.44 (m, 2H), 7.25 (m,
3H), 7.02 (s, 1H), 6.82 (s, 1H), 6.49 (bt, 1H), 5.61 (s, 2H), 5.22
(s, 2H), 3.75 (dd, 1H), 3.71 (s, 3H), 3.38 (dd, J=4, 17.6 Hz, 1H),
3.20 (s, 3H), 3.01 (d, J=5.2 Hz, 3H), 2.99 (m, 2H), 2.67 (s, 3H),
2.54 (t, J=7.6 Hz, 2H).
4-(3-((2-((2,4-dichloro-3-(((2-methyl-4-((1-methyl-1H-imidazol-2-yl)metho-
xy)quinolin-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxop-
ropyl)-N-methylbenzamide was prepared from
2-methyl-4-((1-methyl-1H-imidazol-2-yl)methoxy)quinolin-8-ol and
5-(3-((2-((2,4-dichloro-3-(chloromethyl)phenyl)(methyl)amino)-2-oxoethyl)-
amino)-3-oxopropyl)-N-methylpicolinamide as previously described.
LCMS (+ESI) 704 (M.sup.+).
5-(3-((2-((2,4-dichloro-3-(((2-methyl-4-((pyridin-2-ylmethyl)amino)quinoli-
n-8-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)-N-m-
ethylpicolinamide (49)
##STR00142##
[0228] A mixture of the 4-chloro-2-methylquinolin-8-ol (2.00 g,
10.3 mmol) and 2-aminomethyl pyridine (3.35 g, 30.9 mmol) in DMSO
(5 mL) was heated at 160.degree. C. for 2 h under nitrogen
atmosphere. The mixture was cooled, poured into ice/NaHCO.sub.3 and
extracted with ethyl acetate and dichloromethane. Evaporation gave
a crude mixture that was mixed with ethyl acetate. The yellow solid
precipitated and was filtered and dried to give 1.20 g of
2-methyl-4-((pyridin-2-ylmethyl)amino)quinolin-8-ol (44%). MS
(+APCI) 266 (M.sup.+). .sup.1H-NMR (CDCl.sub.3, .delta.): 8.56 (m,
1H), 7.79 (t, J=6.0 Hz, 1H), 7.75 (dt, J=2.0, 7.6 Hz, 1H), 7.63
(dd, J=1.2, 8.4 Hz, 1H), 7.34 (d, J=7.6 Hz, 1H), 7.25 (m, 2H), 6.96
(d, J=7.6 Hz, 1H), 6.29 (s, 1H), 4.62 (d, J=6.0 Hz, 2H), 2.41 (s,
3H).
[0229] 2-methyl-4-((pyridin-2-ylmethyl)amino)quinolin-8-ol and
5-(3-((2-((2,4-dichloro-3-(chloromethyl)phenyl)(methyl)amino)-2-oxoethyl)-
amino)-3-oxopropyl)-N-methylpicolinamide were reacted as previously
described to give
5-(3-((2-((2,4-dichloro-3-(((4-((2-fluorobenzyl)amino)-2-methylquinolin-8-
-yl)oxy)methyl)phenyl)(methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)-N-meth-
ylpicolinamide in 39% yield. MS (+ESI) 700 (M.sup.+). .sup.1H-NMR
(CDCl.sub.3, .delta.): 8.64 (s, 1H), 8.35 (d, J=1.6 Hz, 1H), 8.07
(d, J=8.0 Hz, 1H), 7.94 (bs, 1H), 7.70 (t, J=6.0 Hz, 1H), 7.62 (dd,
J=2, 8 Hz, 2H), 7.43 (d, J=8.8 Hz, 1H), 7.35 (m, 2H), 7.25 (m, 3H),
7.19 (m, 1H), 6.71 (bs, 1H), 6.50 m (bt, 1H), 6.36 (s, 1H), 5.61
(s, 2H), 4.60 (d, J=4.0 Hz, 2H), 3.77 (dd, J=4.8, 17.6 Hz, 1H),
3.43 (dd, J=3.6, 17.6 Hz, 1H), 3.20 (s, 3H), 3.00 (d, J=5.2 Hz,
3H), 2.99 (m, 2H), 2.58 (s, 3H), 2.51-2.69 (m, 3H).
[0230] All references cited in this application are expressly
incorporated by reference herein for any purpose.
[0231] Unless otherwise required by context, singular terms used
herein shall include pluralities and plural terms shall include the
singular.
EXAMPLES
[0232] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting the invention.
Example 1
Compounds of Formula 1 Bind to Bradykinin B.sub.2-Receptor
[0233] Compounds of Formula 1 were tested for ability to
specifically bind to human recombinant BK B.sub.2-receptor, and
thus displace radiolabeled BK (for B.sub.2-receptors) from the
receptor, using competition radioligand binding techniques as
described by Sharif and Whiting (Neurochem. Int. 18: 89-96, 1991)
and Wiernas et al. (Brit. J. Pharmacol. 123: 1127-1137, 1998).
[0234] Chinese hamster ovary (CHO) cell membrane homogenates (2.5
.mu.g protein) expressing the recombinant human bradykinin B2
receptor were incubated for 60 min at 23.degree. C. with 0.2 nM
[.sup.3H]-bradykinin (95.5 Ci/mmole, PerkinElmer) in the absence or
presence of the test compound in a buffer containing 50 mM Tris/HCl
(pH 7.4), 0.2 g/L 1-10-phenanthroline and 0.1% BSA. All reagent and
test compound additions to the assay plates were made using Biomek
3000 robotic workstations (Beckman Instruments, Fullerton, Calif.).
Nonspecific binding was determined in the presence of 1 .mu.M
unlabeled bradykinin. Following the incubation, the assay was
terminated by rapid filtration under vacuum through glass fiber
filters (GF/C, PerkinElmer) presoaked with 0.3% polyethyleneimine
and rinsed several times with an ice-cold 50 mM Tris-HCl buffer
using a 96-sample cell harvester (Tomtec). The filters were
air-dried and the radioactivity counted in a beta-scintillation
counter (Topcount, PerkinElmer) using a scintillation cocktail
(Microscint 20, PerkinElmer) (Sharif and Whiting, Neurochem. Int.
18: 89-96, 1991). Competitive binding inhibition curves were
generated using a 7-point concentration--response of test compound
in duplicate. Bradykinin peptide was also tested as an internal
control in the assay. The data were analyzed using nonlinear,
iterative curve-fitting to obtain the potency and intrinsic
activities of the test agents as previously described (Sharif et
al. J. Pharmacol. Exp. Ther. 286: 1094-1102, 1998; Sharif et al.
Invest Ophthalmol. Vis. Sci. 39:2562-2571, 1998; Kelly et al. J.
Pharmacol. Exp. Ther. 304: 238-245, 2003). Determined IC.sub.50
values were converted to K.sub.i values using the standard
Cheng-Prusoff equation.
[0235] The results shown in Table 2 indicated that the tested
compounds were able to interact with the agonist binding pocket of
the B.sub.2-receptor with greater affinity that Des-Arg.sup.9-BK,
which has high affinity for BK B.sub.1-receptor. Furthermore, these
compounds exhibited a reasonable affinity for the B.sub.2-receptor
as indicated by a low inhibition constant value (K) for competing
for [.sup.3H]-BK binding to the latter receptor.
TABLE-US-00002 TABLE 2 BK B.sub.2-receptor binding affinity of
bradykinin and some selected non-peptide BK agonists BK and Its
[.sup.3H]-BK Binding [.sup.3H]-BK Binding Analogs & Non- to
Human Cloned to Human Cloned peptide BK B.sub.2 Receptor B.sub.2
Receptor Compounds # (K.sub.i, nM) (K.sub.i, nM) BK 0.5 .+-. 0.05
+++ Lys-BK 1.8 .+-. 0.7 +++ Hyp.sup.3-BK 1.9 .+-. 1.1 +++
Met-Lys-BK 73 .+-. 25 +++ Des-Arg.sup.9-BK >10,000.sup. + 1 54
+++ 2 101 ++ 3 273 ++ 4 158 ++ 5 71 .+-. 30 +++ 6 10 .+-. 2 +++ 7
118 .+-. 2 ++ 8 68 .+-. 16 +++ 9 667 .+-. 101 ++ 10 76 .+-. 16 +++
11 391 .+-. 35 ++ 12 261 .+-. 195 ++ 14 197 ++ 15 467 ++ 16 985
.+-. 473 ++ 17 274 .+-. 69 ++ 18 239 .+-. 150 ++ 19 262 .+-. 11 ++
20 1086 .+-. 138 + 21 881 .+-. 263 ++ 22 160 .+-. 47 ++ 23 1734
.+-. 428 + 24 171 .+-. 97 ++ 25 74 .+-. 6 ++ 26 2142 .+-. 223 + 27
11 +++ 28 .sup. 3 .+-. 0.4 +++ 29 8 .+-. 3 +++ 30 .sup. 3 .+-. 0.1
+++ 32 25 .+-. 8 +++ 33 7 .+-. 1 +++ 34 77 +++ 35 396 +++ 36 220
+++ 37 55.2 +++ 38 332 +++ 39 514 +++ 40 450 +++ 41 300 ++ 42 194
+++ 43 NT NT 44 102 +++ 45 40 +++ 46 NT NT 47 NT NT 48 NT NT 49 NT
NT 50 15 +++ 51 701 ++ 52 15.7 +++ 53 340 ++ 54 455 ++ 55 984 ++ 56
42 +++ 57 852 ++ 58 3150 + 59 2140 + 60 NT NT 61 451 ++ 62 105 ++
63 1190 + 64 40.3 +++ 65 98.4 +++ 66 NT NT 67 NT NT 68 59.5 +++ 69
178 ++ 70 599 ++ 71 17 +++ 72 NT NT 73 184 ++ 74 384 ++ 75 544 ++
76 237 ++ 77 229 ++ 78 NT NT 79 859 ++ 80 279 ++ 81 108 ++ 82 205
++ 83 329 ++ 84 78.3 +++ 85 NT NT 86 NT NT 87 NT NT +++ = <0.1
.mu.M; ++ = >0.1 .mu.M but <1 .mu.M; + = >1 .mu.M, NT =
not tested Values are mean .+-. SEM or singular where they are
averaged values from multiple experiments or from a single
experiment. K.sub.i is the equilibrium concentration required to
inhibit the binding of [.sup.3H]-BK to the B.sub.2-receptor and is
inversely related to the receptor affinity. All values are in
nM.
Example 2
Compounds of Formula 1 are BK Receptor Agonists--Functional Assay
Measuring [Ca.sup.2+].sub.i Mobilization in Cultured Cells
[0236] Agents that can specifically activate native or recombinant
BK receptors present in isolated animal or human tissues [strips or
rings] (Sharif and Whiting, Neurochem. Int. 18: 89-96, 1991; Rizzi
et al. Naunyn-Schmiedeberg Arch. Pharmacol. 360: 361-367, 1999), in
cultured cells of animal or human tissue source (Sharif et al.,
Neurosci. Lett. 86: 279-283, 1988; Sharif and Whiting, Neurochem.
Res. 12: 1313-1320, 1993), especially primary or immortalized
ocular cells involved in aqueous humor dynamics such as human
trabecular meshwork (h-TM) (Sharif and Xu, Exp. Eye Res. 63:
631-637, 1996), human ciliary muscle (h-CM; Sharif et al. J. Ocular
Pharmacol. Ther. 19: 437-455, 2003), and non-pigmented ciliary
epithelial (NPE; Crider and Sharif, J. Ocular Pharmacol. Ther. 18:
221-230, 2002) cells can be identified by measuring second
messengers such as intracellular Ca.sup.2+ ([Ca.sup.2+].sub.i)
(Kelly and Sharif, J. Pharmacol. Expt. Ther. 317:1254-1261, 2006)
produced after stimulation of the BK receptor(s) using well
documented procedures.
[0237] To determine whether the compounds of the invention could
activate BK receptors, a functional assay was conducted to measure
[Ca.sup.2+].sub.i mobilized by BK or compounds of the invention in
human ciliary muscle cells (h-CM) as previously described (Sharif
et al. J. Ocular Pharmacol. Ther. 19: 437-455, 2003; Sharif et al.,
J. Ocular Pharmacol. Ther. 18: 141-162, 2002; Sharif et al. Invest.
Ophthalmol. Vis. Res. 47: 4001-4019, 2006; Sharif et al., J. Ocular
Pharmacol. Ther. 22: 291-309, 2006). BK-induced [Ca.sup.2+].sub.i
mobilization was examined using the Fluorescence Imaging Plate
Reader (FLIPR) instrument (Kelly and Sharif, J. Pharmacol. Expt.
Ther. 317: 1254-1261, 2006).
[0238] h-CM cells expressing BK receptors were seeded at a density
of about 20,000 cells/well in a black-wall, 96-well tissue culture
plates and grown for 2 days. On the day of the experiment, media
was removed, cells were rinsed and 50 .mu.L of serum-free media
added. One vial of FLIPR Calcium Assay Kit dye was reconstituted in
50 mL of a FLIPR buffer consisting of Hank's Balanced Salt Solution
(HBSS), 20 mM HEPES, and 2.5 mM probenecid, pH 7.4. Cells were
loaded with the calcium-sensitive dye by addition of an equal
volume (50 .mu.L) to each well of the 96-well plate and incubated
with dye for 1 h at 23.degree. C. Test compounds were stored at 25
.mu.M in 100% DMSO solvent with intermediate dilution in 25%
DMSO/25% Ethanol and final dilutions in FLIPR buffer. For agonist
concentration response experiments, compounds were serially diluted
10-fold to give 5-point activity curves. After this time, the test
compound plate and cell plate were placed in a Fluorescence Imaging
Plate Reader (FLIPR-Tetra.RTM.) instrument (Molecular
Devices/MDSPharma, Sunnyvale, Calif.). At the beginning of an
experimental run, a signal test was performed to check the basal
fluorescence signal from the dye-loaded cells and the uniformity of
the signal across the plate. Instrument settings for a typical
assay were the following: LED .lamda..sub.ex=470-495 nm,
.lamda..sub.em=515-575 nm, camera F-stop F/2, and exposure time 0.4
sec. An aliquot (25 .mu.L) of the test compound was added to the
existing 100 .mu.L dye-loaded cells at a dispensing speed of 50
.mu.L/sec. Intracellular calcium mobilization was measured by
monitoring real-time changes in cellular fluorescence upon test
compound addition. Fluorescence data were collected in real-time at
1.0 sec intervals for the first 60 seconds and at 6.0 second
intervals for an additional 120 seconds. Baseline fluorescence
readings were normalized across the plate and background-corrected
from negative control wells. Responses were measured as peak
fluorescence intensity and where appropriate were expressed as a
percentage of a maximum BK-induced response [E.sub.max %].
EC.sub.50 values were determined by 4-parameter nonlinear curve
fitting of the concentration-response curve. Calibration of the
instrument was performed using manufacturer's standard
procedures.
[0239] The sources of materials and reagents for such assays can be
found in the references cited above. The results demonstrated that
the compounds of the invention are functional BK agonists (Table
3).
TABLE-US-00003 TABLE 3 Compound CaM # (EC.sub.50, nM) 1 +++ 2 +++ 3
+++ 4 ++ 5 ++ 6 +++ 7 +++ 8 +++ 9 +++ 10 +++ 11 +++ 12 +++ 13 ++ 14
++ 15 ++ 16 ++ 17 ++ 18 ++ 19 +++ 20 ++ 21 ++ 22 +++ 23 + 24 +++ 25
+++ 26 ++ 27 ++ 28 ++ 29 ++ 30 ++ 31 +++ 32 +++ 33 +++ 34 +++ 35
+++ 36 +++ 37 +++ 38 +++ 39 ++ 40 +++ 41 +++ 42 +++ 43 +++ 44 +++
45 +++ 46 ++ 47 ++ 48 +++ 49 ++ 50 ++ 51 ++ 52 ++ 53 ++ 54 ++ 55 ++
56 +++ 57 ++ 58 + 59 ++ 60 ++ 61 +++ 62 +++ 63 +++ 64 +++ 65 +++ 66
+++ 67 +++ 68 +++ 69 +++ 70 +++ 71 +++ 72 +++ 73 +++ 74 +++ 75 +++
76 +++ 77 +++ 78 +++ 79 ++ 80 +++ 81 +++ 82 +++ 83 +++ 84 +++ 85
+++ 86 +++ 87 ++ +++ = <0.1 .mu.M; ++ = >0.1 .mu.M but <1
.mu.M; + = >1 uM
Example 3
Compounds of Formula 1 Stimulate Production of Endogenous
Prostaglandin in h-CM Cells
[0240] BK agonist compounds of Formula 1 were evaluated for the
ability to induce prostaglandin (PG) release from primary human
ciliary muscle cells (h-CM). Briefly, h-CM cells were grown to
confluence in FNC-coated 24-well culture plates and serum-starved
(0.8% FBS) for 24 hours prior to the experiment. Cells were
stimulated with a 9-pt concentration-response (0.1 nM-10 .mu.M) of
bradykinin or BK agonist for 1 hour at 37.degree. C. with
subsequent sample recovery and processing for total PG measurement.
Total PG measurement was determined using a total Prostaglandin
Screening EIA kit (cat#514012) purchased from Cayman Chemical (Ann
Arbor, Mich.). The assay was conducted according to the package
insert from the manufacturer. Potency values (EC.sub.50) defined as
the concentration of compound required to obtain 50% of maximal
activity were determined by nonlinear curve fitting of the dose
response data. Efficacy values (E.sub.max, %) defined as the
maximal response of the test compound relative to a high
concentration of bradykinin set to 100%.
[0241] As shown in Table 4, a number of compounds of the present
invention caused the production of endogenous PGs in h-CM
cells.
TABLE-US-00004 TABLE 4 Compound PG Release # (EC.sub.50, nM) 1 +++
2 +++ 3 + 4 + 5 NT 6 +++ 7 +++ 8 +++ 9 ++ 10 + 11 + 12 + 13 + 14 NT
15 NT 16 NT 17 NT 18 NT 19 + 20 + 21 NT 22 + 23 NT 24 ++ 25 ++ 26
NT 27 ++ 28 +++ 29 + 30 ++ 31 ++ 32 +++ 33 +++ 34 +++ 35 ++ 36 ++
37 +++ 38 +++ 39 ++ 40 ++ 41 ++ 42 ++ 43 +++ 44 +++ 45 +++ 46 +++
47 +++ 48 ++ 49 ++ 50 NT 51 NT 52 NT 53 NT 54 NT 55 NT 56 +++ 57 +
58 NT 59 NT 60 NT 61 + 62 ++ 63 + 64 +++ 65 +++ 66 ++ 67 + 68 ++ 69
+++ 70 ++ 71 +++ 72 + 73 ++ 74 ++ 75 ++ 76 + 77 ++ 78 +++ 79 + 80
++ 81 + 82 + 83 + 84 +++ 85 +++ 86 +++ 87 +++ +++ = <0.1 .mu.M;
++ = >0.1 .mu.M but <1 .mu.M; + = >1 .mu.M
[0242] The results are relevant since PGs are highly potent
IOP-lowering agents in ocular hypertensive monkeys (Hellberg et al.
J Ocular Pharmacol Ther. 17: 421-432, 2001; Sharif et al. J Ocular
Pharmacol Ther. 20: 489-508, 2004) and in humans (Alm, Prog Ret Eye
Res. 17: 291-312, 1998). Thus, the compounds of the present
invention can be expected to lower IOP in experimental animals, as
shown below.
Example 4
Compounds of Formula 1 Reduce IOP Response in Lasered
(Hypertensive) Eyes of Cynomolgous Monkeys
[0243] Intraocular pressure (IOP) was determined with an Alcon
Pneumatonometer (Alcon Laboratories, Inc., Fort Worth, Tex.) after
light corneal anesthesia with 0.1% proparacaine. Right eyes were
hypertensive as a result of laser trabeculoplasty. After a baseline
IOP measurement, compounds of Formula 1 were administered topical
ocularly in various formulations (including hydroxypropyl
methylcellulose (0.5%), anhydrous dibasic sodium phosphate
(0.2-0.5%), sodium chloride (0.5-0.75%), disodium EDTA (Edetate
disodium) (0.01%), polysorbate 80 (0.05%), benzalkonium chloride
(0.01%), sodium hydroxide/hydrochloric acid (for adjusting pH to
7.3-7.4), purified water (q.s. to 100%), and xanthan gum
(0.5-6.0%), anhydrous dibasic sodium phosphate (0.2%), sodium
chloride (0.5%), disodium EDTA (Edetate disodium) (0.01%),
polysorbate 80 (0.05%), benzalkonium chloride (0.01%), sodium
hydroxide/hydrochloric acid (for adjusting pH to 7.3-7.4), purified
water (q.s. to 100%) with PEG400 (0-8.0%)) in conscious ocular
hypertensive Cynomolgus monkeys. Vehicles were formulated and used
in the current studies (including hydroxypropyl methylcellulose
(0.5%), anhydrous dibasic sodium phosphate (0.2-0.5%), sodium
chloride (0.5-0.75%), disodium EDTA (Edetate disodium) (0.01%),
polysorbate 80 (0.05%), benzalkonium chloride (0.01%), sodium
hydroxide/hydrochloric acid (for adjusting pH to 7.3-7.4), purified
water (q.s. to 100%), and xanthan gum (0.5-6.0%), anhydrous dibasic
sodium phosphate (0.2%), sodium chloride (0.5%), disodium EDTA
(Edetate disodium) (0.01%), polysorbate 80 (0.05%), benzalkonium
chloride (0.01%), sodium hydroxide/hydrochloric acid (for adjusting
pH to 7.3-7.4), purified water (q.s. to 100%) with PEG400
(0-8.0%)). Vehicle was instilled in the right eyes of 5-8
additional animals. Subsequent IOP measurements were taken at 24
hours. IOP measurements are also taken on left eyes (normotensive
and untreated) at each of these time points. The percent change in
IOP from baseline was determined for each animal for every IOP
measurement (Sharif et al. J. Ocular Pharmacol. Ther. 17: 305-317,
2001; May et al., J Pharmacol Exp Ther. 306: 301-309, 2003; Sharif
et al. Invest. Ophthalmol. Vis. Res. 47: 4001-4010, 2006). Group
mean and standard error of the mean (SEM) were calculated.
[0244] As shown in Table 5, several of the compounds reduced monkey
IOP (MIOP) by at least 15%.
TABLE-US-00005 TABLE 5 MIOP Compound % change # at 24 h 1 - 4 - 7 +
8 + 9 - 10 - 12 - 28 + 32 + 33 + 34 + 38 - 44 + 45 - 56 + 64 + 65 -
73 - 74 + 76 - 78 + 84 - + = 15% or better reduction - = <15%
reduction
[0245] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims, including the use of different formulations shown for
instance in the examples above.
Sequence CWU 1
1
319PRTHomo sapiens 1Arg Pro Pro Gly Phe Ser Pro Phe Arg1
5210PRTArtificialSynthetic peptide 2Lys Arg Pro Pro Gly Phe Ser Pro
Phe Arg1 5 1038PRTArtificialSynthetic peptide 3Arg Pro Pro Gly Phe
Ser Pro Phe1 5
* * * * *