U.S. patent application number 11/255827 was filed with the patent office on 2006-03-16 for dipeptide derivatives.
Invention is credited to Cynthia Anne Fink.
Application Number | 20060058242 11/255827 |
Document ID | / |
Family ID | 26966569 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060058242 |
Kind Code |
A1 |
Fink; Cynthia Anne |
March 16, 2006 |
Dipeptide derivatives
Abstract
Compounds of the formula ##STR1## wherein R, R.sub.1,
COOR.sub.2, R.sub.3-R.sub.7, alk, and X have meaning as defined,
such being useful as dual inhibitors of angiotensin converting
enzyme and neutral endopeptidase, as well as inhibitors of
endothelin converting enzyme.
Inventors: |
Fink; Cynthia Anne;
(Lebanon, NJ) |
Correspondence
Address: |
NOVARTIS;CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 104/3
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
26966569 |
Appl. No.: |
11/255827 |
Filed: |
October 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10884150 |
Jul 2, 2004 |
6992105 |
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11255827 |
Oct 21, 2005 |
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10142693 |
May 9, 2002 |
6777443 |
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10884150 |
Jul 2, 2004 |
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60339575 |
Dec 11, 2001 |
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60291088 |
May 15, 2001 |
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Current U.S.
Class: |
514/10.9 ;
514/12.4; 514/15.7; 514/16.2; 514/16.4; 514/17.4; 514/21.91;
514/542; 562/450 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
9/12 20180101; A61P 9/04 20180101; A61K 45/06 20130101; C07K
5/06026 20130101; A61K 38/05 20130101; A61K 31/235 20130101; A61P
9/00 20180101; C07K 5/06034 20130101; A61P 43/00 20180101; A61K
2300/00 20130101; C07K 5/06191 20130101; A61K 2300/00 20130101;
A61K 31/235 20130101; A61K 38/05 20130101; A61P 3/12 20180101 |
Class at
Publication: |
514/019 ;
514/542; 562/450 |
International
Class: |
A61K 38/04 20060101
A61K038/04; A61K 31/235 20060101 A61K031/235 |
Claims
1. A pharmaceutical composition comprising (a) a compound of the
formula (I) ##STR55## wherein R represents hydrogen, lower alkyl,
carbocyclic or heterocyclic aryl-lower alkyl or cycloalkyl-lower
alkyl; R.sub.1 represents lower alkyl, cycloalkyl, carbocyclic or
heterocyclic aryl, or biaryl; or R.sub.1 represents (cycloalkyl,
carbocyclic aryl, heterocyclic aryl or biaryl)-lower alkyl; alk
represents lower alkylene; R.sub.3 represents hydrogen or acyl;
R.sub.4 represents hydrogen, optionally substituted lower alkyl,
carbocyclic or heterocyclic aryl, (carbocyclic or heterocyclic
aryl)-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, biaryl,
biaryl-lower alkyl; oxacycloalkyl, thiacycloalkyl, azacycloalkyl,
or (oxacycloalkyl, thiacycloalkyl or azacycloalkyl)-lower alkyl;
R.sub.5 represents hydrogen or lower alkyl; or R.sub.4 and R.sub.5
together with the carbon atom to which they are attached, represent
cycloalkylidene, benzo-fused cycloalkylidene; or 5- or 6-membered
(oxacycloalkylidene, thiacycloalkylidene or azacycloalkylidene),
each optionally substituted by lower alkyl or aryl-lower alkyl;
R.sub.6 represents lower alkyl, carbocyclic or heterocyclic aryl,
(carbocyclic or heterocyclic aryl)-lower alkyl, cycloalkyl,
cycloalkyl-lower alkyl, biaryl or biaryl-lower alkyl; R.sub.7
represents lower alkyl, (carbocyclic or heterocyclic aryl)-lower
alkyl, cycloalkyl-lower alkyl or biaryl-lower alkyl; or R.sub.6 and
R.sub.7, together with the carbon atom to which they are attached,
represent 3- to 10-membered cycloalkylidene which may be
substituted by lower alkyl or aryl-lower alkyl or may be fused to a
saturated or unsaturated carbocyclic 5- to 7-membered ring; or 5-
or 6-membered (oxacycloalkylidene, thiacycloalkylidene or
azacycloalkylidene), each optionally substituted by lower alkyl or
aryl-lower alkyl; or 2,2-norbonylidene; X represents --O--,
--S(O).sub.r--, --NHSO.sub.2--, or --NHCO--; n is zero, one or two;
and COOR.sub.2 represents carboxyl or carboxyl derivatized in form
of a pharmaceutically acceptable ester; or a disulfide derivative
derived from a said compound wherein R.sub.3 is hydrogen; or a
pharmaceutically acceptable salt thereof; and (b) at least one
second therapeutic agent selected from angiotensin II receptor
antagonists, renin inhibitors, calcium channel blockers,
aldosterone synthase inhibitors, diuretics, vasopressin receptor
antagonists, cardiotonic drugs, endothelin antagonists and ECE
inhibitors, anti-atherosclerotic agents, cholesterol absorption
inhibitors, statin HMG CoA reductase inhibitors, nicotinic acid
derivatives, thyromimetic agents, and antidiabetic agents.
2. The composition of claim 1, wherein the compound has the formula
(Ia): ##STR56##
3. The composition of claim 1, wherein the compound has the
structure: ##STR57##
4. The composition of claim 1, wherein the angiotensin II receptor
antagonists are selected from valsartan, losartan, candesartan,
eprosartan, irbesartan and telmisartan.
5. The composition of claim 1, wherein the blockers are selected
from bisoprolol, propanolol, atenolol, sotalol and metoprolol.
6. The composition of claim 1, wherein the calcium channel blockers
are selected from amlodipine, verapamil, diltiazem, bepridil,
felodipine, isradipine, nicardipine, nifedipine, nimodipine and
nisoldipine.
7. The composition of claim 1, wherein the aldosterone synthase
inhibitors are selected from eplerenone, (+)-fadrozole (WO
01/76574), spironolactone and canrenone.
8. The composition of claim 1, wherein the diuretics are selected
from furosemide, hydrochlorothiazide, indapamide, metazolone,
amiloride and triamterene
9. The composition of claim 1, wherein the vasopressin receptor
antagonists are selected from OPC 21268, SR 49059, SR121463A,
SR49059, VPA985, OPC31260 and YM087.
10. The composition of claim 1, wherein the statin HMG CoA
reductase inhibitors are selected from atorvastatin, fluvastatin,
lovastatin, pravastatin, simvastatin and pitavastatin.
11. The composition of claim 1, wherein the antidiabetic agents are
selected from repaglinide, nateglinide, metformin, rosiglitazone,
pioglitazone, glyburide, glipizide, glimepiride, DPP728, LAF237,
NH622 and DRF4158.
12. A method of treating or inhibiting cardiovascular disorders in
mammals comprising administering to a mammal in need thereof an
effective amount of the pharmaceutical composition of claim 1.
13. The method according to claim 12 for the treatment of
hypertension, edema, salt retention or congestive heart failure.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
application No. 60/291,088 filed May 15, 2001 and of provisional
application No. 60/339,575 filed Dec. 11, 2001, the contents of
which are incorporated herein by reference.
SUMMARY OF THE INVENTION
[0002] The present invention directed to novel vasopeptidase
inhibitors described below which are useful as dual inhibitors of
both angiotensin converting enzyme (ACE) and neutral endopeptidase
(NEP, EC 3.4.24.11). The compounds of the invention are
particularly useful for the treatment and/or the prevention of
conditions which are responsive to ACE and NEP inhibition,
particularly cardiovascular disorders, such as hypertension,
isolated systolic hypertension, renal failure (including edema and
salt retention), pulmonary edema, left ventricular hypertrophy,
heart failure (including congestive heart failure) and
atherosclerosis.
[0003] The compounds of the invention are also useful for reducing
elevated cholesterol plasma levels in mammals. Furthermore, they
also inhibit endothelin converting enzyme (ECE) and are useful for
the treatment and/or prevention of conditions which are responsive
to ECE inhibition.
[0004] By virtue of their inhibition of neutral endopeptidase, the
compounds of the invention may also be useful for the treatment of
pain, depression, certain psychotic conditions, and cognitive
disorders. Other potential indications include the treatment of
angina, premenstrual syndrome, Meniere's disease,
hyperaldosteronism, hypercalciuria, ascites, glaucoma, asthma and
gastrointestinal disorders such as diarrhea, irritable bowel
syndrome and gastric hyperacidity.
[0005] By virtue of their inhibition of ECE, the compounds of the
invention may also be useful for the treatment and/or prevention of
endothelin dependent conditions and diseases, including cerebral
ischemia (stroke), subarachnoid hemorrhage, traumatic brain injury,
cerebral vasospasm, arterial hypertrophy, restenosis, Raynaud's
disease, myocardial infarction, obesity; also prostate hyperplasia,
migraine, diabetes mellitus (diabetic nephropathy), preeclampsia,
glaucoma, and transplantaUon rejection such as in aorta or solid
organ transplantation; as well as erectile dysfunction.
DETAILED DESCRIPTION OF THE INVENTION
[0006] The present invention relates to compounds of formula I
##STR2## [0007] wherein [0008] R represents hydrogen, lower alkyl,
carbocyclic or heterocyclic aryl-lower alkyl or cycloalkyl-lower
alkyl; [0009] R.sub.1 represents lower alkyl, cycloalkyl,
carbocyclic or heterocyclic aryl, or biaryl; or R.sub.1 represents
(cycloalkyl, carbocyclic aryl, heterocyclic aryl or biaryl)-lower
alkyl; [0010] alk represents lower alkylene; [0011] R.sub.3
represents hydrogen or acyl; [0012] R.sub.4 represents hydrogen,
optionally substituted lower alkyl, carbocyclic or heterocyclic
aryl, (carbocyclic or heterocyclic aryl)-lower alkyl, cycloalkyl,
cycloalkyl-lower alkyl, biaryl, biaryl-lower alkyl, oxacycloalkyl,
thiacycloalkyl, azacycloalkyl, or (oxacycloalkyl, thiacyctoalkyl or
azacycloalkyl)-lower alkyl; [0013] R.sub.5 represents hydrogen or
lower alkyl; or [0014] R.sub.4 and R.sub.5 together with the carbon
atom to which they are attached, represent cycloalkylidene,
benzo-fused cycloalkylidene; or 5- or 6-membered
(oxacycloalkylidene, thiacycloalkylidene or azacycloalkylidene),
each optionally substituted by lower alkyl or aryl-lower alkyl;
[0015] R.sub.6 represents lower alkyl, carbocyclic or heterocyclic
aryl, (carbocyclic or heterocyclic aryl)-lower alkyl, cycloalkyl,
cycloalkyl-lower alkyl, biaryl or biaryl-lower alkyl; [0016]
R.sub.7 represents lower alkyl, (carbocyclic or heterocyclic
aryl)-lower alkyl, cycloalkyl-lower alkyl or biaryl-lower alkyl; or
[0017] R.sub.6 and R.sub.7, together with the carbon atom to which
they are attached, represent 3- to 10-membered cycloalkylidene
which may be substituted by lower alkyl or aryl-lower alkyl or may
be fused to a saturated or unsaturated carbocyclic 5- to 7-membered
ring; or 5- or 6-membered (oxacycloalkylidene, thiacycloalkylidene
or azacycloalkylidene), each optionally substituted by lower alkyl
or aryl-lower alkyl; or 2,2-norbonylidene; [0018] X represents
--O--, --S(O).sub.n--, --NHSO.sub.2--, or --NHCO--; [0019] n is
zero, one or two; and [0020] COOR.sub.2 represents carboxyl or
carboxyl derivatized in form of a pharmaceutically acceptable
ester; [0021] disulfide derivatives derived from said compounds
wherein R.sub.3 is hydrogen; and pharmaceutically acceptable salts
thereof.
[0022] The present invention is also directed to pharmaceutical
compositions comprising said compounds; methods for preparation of
said compounds; intermediates; and methods of treating disorders in
mammals which are responsive to ACE and NEP inhibition by
administration of said compounds to mammals in need of such
treatment.
[0023] Encompassed by the instant invention are also any prodrug
derivatives of compounds of the invention having a free carboxyl,
sulfhydryl or hydroxy group, said prodrug derivatives being
convertible by solvolysis or under physiological conditions to be
the free carboxyl, sulfhydryl and/or hydroxy compounds. Prodrug
derivatives are, e.g., the esters of free carboxylic acids and
S-acyl and O-acyl derivatives of thiols, alcohols or phenols,
wherein acyl has meaning as defined herein.
[0024] Pharmaceutically acceptable esters are preferably prodrug
ester derivatives, such being convertible by solvolysis or under
physiological conditions to the free carboxylic acids of formula
I.
[0025] Pharmaceutically acceptable prodrug esters are preferably,
e.g., lower alkyl esters, aryl-lower alkyl esters, .alpha.-(lower
alkanoyloxy)-lower alkyl esters such as the pivaloyloxymethyl
ester, and .alpha.-(lower alkoxycarbonyl, morpholinocarbonyl,
piperidinocarbonyl, pyrrolidinocarbonyl or di-lower
alkylaminocarbonyl)-lower alkyl esters.
[0026] Pharmaceutically acceptable salts are salts derived from
pharmaceutically acceptable bases for any acidic compounds of the
invention, e.g., those wherein COOR.sub.2 represents carboxyl. Such
are, e.g., alkali metal salts (e.g., sodium, potassium salts),
alkaline earth metal salts (e.g., magnesium, calcium salts) amine
salts (e.g., tromeihamine salts).
[0027] Compounds of formula I, depending on the nature of
substituents, possess two or more asymmetric carbon atoms. The
resulting diastereomers and optical antipodes are encompassed by
the instant invention. The preferred configuration is indicated in
formula Ia. ##STR3## wherein asymmetric carbons carrying the
substituents--alk-X--R, and R.sub.4 typically have the
S-configuration.
[0028] Preferred are the compounds of formula I and Ia wherein R
and RS represent hydrogen; R.sub.1 represents lower alkyl, C.sub.5-
or C.sub.6-cycloalkyl, carbocyclic or heterocyclic aryl, or
(carbocyclic or heterocyclic aryl)-lower alkyl; alk represents
lower alkylene; X represents --O-- or --S(O).sub.n wherein n
represents zero or two; R.sub.3 represents hydrogen or acyl;
R.sub.4 represents optionally substituted lower alkyl,
oxacycloalkyl, oxacycloalkyl-lower alkyl, or (carbocyclic or
heterocyclic aryl)-lwer alkyl; R.sub.5 represents hydrogen, or
R.sub.4 and R.sub.5 combined with the carbon atom to which they are
attached represent C.sub.5 or C.sub.6-cycloalkylidene; R.sub.6 and
R.sub.7 represent lower alkyl; or R.sub.6 and R.sub.7, together
with the carbon atom to which they are attached, represent 5- or
6-membered cycloalkylidene; COOR.sub.2 represents carboxyl or
carboxyl derivatized in form of a pharmaceutically acceptable
ester; disulfide derivatives derived from said compounds wherein
R.sub.3 is hydrogen; and pharmaceutically acceptable salts
thereof.
[0029] Further preferred are said compounds of formula I and Ia
wherein R and R.sub.5 represent hydrogen; R.sub.1 represents
carbocyclic or heterocyclic aryl or (carbocyclic or heterocyclic
aryl)-lower alkyl; R.sub.3 represents hydrogen or optionally
substituted lower alkanoyl; R.sub.4 represents lower alkyl,
cycloalkyl, tetrahydropyranyl or C.sub.1-C.sub.4-lower alkoxy-lower
alkyl; R.sub.6 and R.sub.7 both represent C.sub.1-C.sub.4-alkyl and
are identical; X represents --O-- or --S--; alk represents
methylene; COOR.sub.2 represents carboxyl, lower alkoxycarbonyl,
(di-lower alkylaminocarbonyl)-lower alkoxycarbonyl or
(morpholinbcarbonyl, piperidinocarbonyl or
pyrrolidinocarbonyl)-lower alkoxycarbonyl; and pharmaceutically
acceptable salts thereof.
[0030] Particularly preferred are said compounds of formula I or Ia
wherein R and R.sub.5 represent hydrogen; R.sub.1 represents
carbocyclic aryl or carbocyclic aryl-lower alkyl in which
carbocyclic aryl represents phenyl or phenyl substituted by one or
two of hydroxy, lower alkanoyloxy, lower alkyl, lower alkoxy,
trifluoromethyl, trifluoromethoxy or halo; R.sub.3 represents
hydrogen, lower alkanoyl or lower alkanoyl substituted by lower
alkoxy; R.sub.4 represents lower alkyl, 4-tetrahydropyranyl or
C.sub.1-C.sub.4-lower alkoxy-C.sub.1-C.sub.4-lower alkyl; R.sub.6
and R.sub.7 represent methyl; X represents --O--; alk represents
methylene or ethylene; and COOR.sub.2 represents carboxyl or lower
alkoxycarbonyl; and pharmaceutically acceptable salts thereof. An
embodiment thereof relates to compounds wherein R.sub.3 represents
hydrogen or lower alkanoyl.
[0031] Further preferred are the above compounds of formula I or Ia
wherein R and R.sub.5 represent hydrogen; R.sub.1 represents phenyl
fluorophenyl, benzyl or fluorobenzyl; R.sub.3 represents hydrogen,
lower alkanoyl or lower alkanoyl substituted by lower alkoxy;
R.sub.4 represents isopropyl, tert-butyl, 1-methoxyethyl or
4-tetrahydropyranyl; R.sub.6 and R.sub.7 represent methyl; X
represents --O--; alk represents methylene; and COOR.sub.2
represents carboxyl or lower alkoxycarbonyl; and pharmaceutically
acceptable salts thereof. An embodiment thereof relates to
compounds wherein R.sub.3 represents hydrogen or lower
alkanoyl.
[0032] Preferred particular embodiments relate to compounds of
formula I or Ia wherein R and R.sub.5 represent hydrogen; R.sub.1
represents benzyl; R.sub.3 represents hydrogen, acetyl or
methoxyacetyl; R.sub.4 represents isopropyl or tert-butyl; R.sub.6
and R.sub.7 represent methyl; X represents --O--; alk represents
methylene; and COOR.sub.2 represents carboxyl or ethoxycarbonyl; or
a pharmaceutically acceptable salt thereof.
[0033] The definitions as such or in combination as used herein,
unless denoted otherwise, have the following meanings within the
scope of the present invention.
[0034] Aryl represents carbocyclic or heterocyclic aryl, either
monocyclic or bicyclic.
[0035] Monocyclic carbocyclic aryl represents optionally
substituted phenyl, being preferably phenyl or phenyl substituted
by one to three substituents, such being advantageously lower
alkyl, hydroxy, lower alkoxy, acyloxy, halogen, cyano,
trifluoromethyl, trifluoromethoxy, amino, lower alkanoylamino,
lower alkyl-(thio, sulfinyl or sulfonyl), lower alkoxycarbonyl,
mono- or di-lower alkylcarbamoyl, or mono- or di-lower alkylamino;
or phenyl substituted by lower alkylenedioxy.
[0036] Bicyclic carbocyclic-aryl represents 1- or 2-naphthyl or 1-
or 2-naphthyl preferably substituted by lower alkyl, lower alkoxy
or halogen.
[0037] Monocyclic heterocyclic aryl represents preferably
optionally substituted thiazolyl, pyrimidyl, triazolyl, thienyl,
furanyl or pyridyl.
[0038] Optionally substituted furanyl represents 2- or 3-furanyl or
2- or 3-furanyl preferably substituted by lower alkyl.
[0039] Optionally substituted pyridyl represents 2-, 3- or
4-pyridyl or 2-, 3- or 4-pyridyl preferably substituted by lower
alkyl, halogen or cyano.
[0040] Optionally substituted thienyl represents 2- or 3-thienyl or
2- or 3-thienyl preferably substituted by lower alkyl.
[0041] Optionally substituted pyrimidyl represents, e.g.,
0.2-pyrimidyl or 2-pyrimidyl substituted by lower alkyl.
[0042] Optionally substituted thiazolyl represents, e.g.,
-2-thiazolyl or 2-thiazolyl substituted by lower alkyl.
[0043] Optionally substituted triazolyl represents, e.g.,
1,2,4-triazolyl or 1,2,4-triazolyl preferably substituted by lower
alkyl.
[0044] Bicyclic heterocyclic aryl represents preferably indolyl,
benzothiazolyl, quinolinyl or isoquinolinyl optionally substituted
by hydroxy, lower alkyl, lower alkoxy or halogen, advantageously
3-indolyl, 2-benzothiazolyl or 2- or 4-quinolinyl.
[0045] Aryl as in aryl-lower alkyl is preferably phenyl or phenyl
substituted by one or two of lower alkyl, lower alkoxy, hydroxy,
lower alkanoyloxy, halogen, trifluoromethyl, cyano, lower
alkanoylamino or lower alkoxycarbonyl; also, optionally substituted
naphthyl.
[0046] Aryl-lower alkyl is advantageously benzyl or 1- or
2-phenethyl optionally substituted on phenyl by one or two of lower
alkyl, lower alkoxy, hydroxy, lower alkanoyloxy, halogen or
trifluoromethyl.
[0047] The term "lower" referred to herein in connection with
organic radicals or compounds respectively defines such with up to
and including 7, preferably up and including 4 and advantageously
one or two carbon atoms. Such may be straight chain or
branched.
[0048] Optionally substituted lower alkyl refers to lower alkyl or
lower alkyl substituted by, e.g., halo, hydroxy, lower alkoxy,
amino, (mono- or di-lower alkyl) amino, acylamino, 1-lower
alkyl-piperazino, morpholino, piperidino, pyrrolidino and the
like.
[0049] Lower alkylene refers to a straight or branched carbon chain
having preferably 1 to 4 carbon atoms, which may be substituted,
e.g., by lower alkoxy, for example, --CH.sub.2--, --CH
(CH.sub.3)--, --CH.sub.2CH.sub.2-- and the like.
[0050] A lower alkyl group preferably contains 1-4 carbon atoms
which may be straight chain or branched and represents, for
example, ethyl, propyl, butyl or advantageously methyl.
[0051] A lower alkoxy group preferably contains 1-4 carbon atoms
which may be straight chain or branched and represents, for
example, methoxy, propoxy, isopropoxy or advantageously ethoxy.
[0052] Cycloalkyl represents a saturated cyclic hydrocarbon radical
which preferably contains 5- to 7-ring carbons, preferably
cyclopentyl or cyclohexyl.
[0053] Oxacycloalkyl represents preferably 5- to 7-membered
oxacycloalkyl, e.g., tetrahydropyranyl, such as
4-tetrahydropyranyl.
[0054] Thiacycloalkyl represents preferably 5- to 7-membered
thiacycloalkyl, e.g., tetrahydrothiopyranyl, such as
4-tetrahydrothiopyranyl.
[0055] Azacycloalkyl represents preferably 5- to 7-membered
azacycloalkyl, e.g., pyrrolidinyl or piperidinyl in which the
nitrogen may be substituted by lower alkyl or aryl-lower alkyl.
[0056] The term cycloalkyl-lower alkyl represents preferably
(cyclopentyl or cyclohexyl)-methyl, 1- or 2-(cyclopentyl or
cyclohexyl)ethyl, 1-, 2- or 3-(cyclopentyl or cyclohexyl)propyl, or
1-, 2-, 3- or 4-(cyclopentyl or cyclohexyl)-butyl. Similarly
(oxacyclyl, thiacycloalkyl or azacycloalkyl)-lower alkyl.
[0057] A lower alkoxycarbonyl group preferably contains 1 to 4
carbon atoms in the alkoxy portion and represents, for example,
methoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl or
advantageously ethoxycarbonyl.
[0058] Cycloalkylidene is 3- to 10-membered, preferably 5- or
6-membered, and represents a cycloalkane linking group in which the
two attached groups are attached to the same carbon of the
cycloalkane ring.
[0059] 5- or 6-membered oxacycloalkylidene represents a
tetrahydrofuran or tetrahydropyran linking group, i.e.,
tetrahydrofuranylidene or tetrahydropyranylidehe, in which the two
attached groups are attached to the same carbon atom of the
respective rings, e.g., at the 3- or 4-position thereof.
[0060] 5- or 6-membered thiacycloalkylidene represents a
tetrahydrothiophene or tetrahydrothiopyran linking group in which
the two attached groups are attached to the same carbon atom of the
respective rings, e.g., at the 3- or 4-position thereof.
[0061] 5- or 6-membered azacycloalkylidene represents a pyrrolidine
or piperidine linking group in which the two attached groups are
attached to the same carbon atom of the respective rings, e.g., at
the 3- or 4-position thereof, and the nitrogen may be substituted
by lower alkyl, e.g., methyl, or by aryl-lower alkyl, e.g.,
benzyl.
[0062] Benzo-fused cycloalkylidene represents, e.g., 1,1- or
2,2-tetralinylidene or 1,1- or 2,2-indanylidene.
[0063] Halogen (halo) preferably represents fluoro or chloro, but
may also be bromo or iodo.
[0064] Acyl is derived from a carboxylic acid and represents
preferably optionally substituted lower alkanoyl, carbocyclic
aryl-lower alkanoyl, aroyl, lower alkoxycarbonyl or aryl-lower
alkoxycarbonyl, advantageously optionally substituted lower
alkanoyl, or aroyl.
[0065] Lower alkanoyl is preferably acetyl, propionyl, butyryl, or
pivaloyl.
[0066] Optionally substituted lower alkanoyl, for example,
represents lower alkanoyl or lower alkanoyl substituted by, e.g.,
lower alkoxycarbonyl, lower alkanoyloxy, lower alkanoylthio, lower
alkoxy, lower alkylthio, hydroxy, di-lower alkylamino, lower
alkanoylamino, morpholino, piperidino, pyrrolidiho, 1-lower
alkylpiperazino, aryl or heteroaryl.
[0067] Aroyl is carbocyclic or heterocyclic aroyl, preferably
monocyclic carbocyclic or monocyclic heterocyclic aroyl.
[0068] Monocyclic carbocyclic aroyl is preferably benzoyl or
benzoyl substituted by lower alkyl, lower alkoxy, halogen or
trifluoromethyl.
[0069] Monocyclic heterocyclic aroyl is preferably pyridylcarbonyl
or thienylcarbonyl.
[0070] Acyloxy is preferably optionally substituted lower
alkanoyloxy, lower alkoxycarbonyloxy, monocyclic carbocyclic
aroyloxy or monocyclic heterocyclic aroyloxy.
[0071] Aryl-lower alkoxycarbonyl is preferably monocyclic
carbocyclic-lower alkoxycarbonyl, advantageously
benzyloxycarbonyl.
[0072] Biaryl represents monocarbocyclic aryl substituted by
monocyclic carbocyclic or monocyclic heterocyclic aryl, and
preferably represents biphenylyl, advantageous 4-biphenylyl
optionally substituted on one or both benzene rings by lower alkyl,
lower alkoxy, halogen or trifluoromethyl.
[0073] Biaryl-lower alkyl is preferably 4-biphenylyl-lower alkyl,
advantageously 4-biphenylyl-methyl.
[0074] The novel compounds of the invention are ACE inhibitors
inhibiting the conversion of angiotensin I to the pressor substance
angiotensin II and thus decrease blood pressure in mammals.
Furthermore, compounds of the invention demonstrate inhibition of
NEP and thus potentiate the cardiovascular (e.g., diuretic and
natriuretic) effects of atrial natriuretic factors (ANF). The
combined effect is beneficial for the treatment of cardiovascular
disorders in mammals, in particular, hypertension, cardiac
conditions such as congestive heart failure, and renal failure. A
further beneficial effect of the compounds of the invention in the
treatment of said cardiovascular disorders is the inhibition of
ECE.
[0075] The above-cited properties are demonstrable in vitro and in
vivo tests, using advantageously mammals, e.g., mice, rats, dogs,
monkeys, or isolated organs, tissues and preparations thereof. Said
compounds can be applied in vitro in the form of solutions, e.g.,
preferably aqueous solutions, and in vivo either enterally,
parenterally, advantageously, orally (p.o.) or intravenously
(i.v.), e.g., as a suspension or in aqueous solution. The dosage in
vitro may range between about 10.sup.-6 molar and 10.sup.-9 molar
concentrations. The dosage in vivo may range, depending on the
route of administration, between about 0.01 and 50 mg/kg,
advantageously between about 0.1 and 25 mg/kg.
[0076] In vitro testing is most appropriate for the free carboxylic
acids of the invention. The test compound is dissolved in dimethyl
sulfoxide, ethanol or 0.25 M sodium bicarbonate solution, and the
solution is diluted with buffer to the desired concentration.
[0077] The in vitro inhibition of the AGE by the compounds of this
invention can be demonstrated by a method analogous to that given
in Biochem. Pharmacol., Vol. 20, p. 1637 (1971). The buffer for the
ACE assay is 300 mM NaCl, 100 mM KH.sub.2PO.sub.4 (pH 8.3). The
reaction is initiated by the addition of 100 .mu.L of
hippuryl-histidyl-leucine (2 mg/mL) to tubes containing enzyme and
drug in a volume of 150 .mu.L and tubes are incubated for 30
minutes at 37.degree. C. The reaction is terminated by the addition
of 0.75 mL 0.6 N NaOH. 100 .mu.L of freshly prepared O-pthaldehyde
solution (2 mg/mL in methanol) is added to the tubes, the contents
are mixed and allowed to stand at room temperature. After 10
minutes, 100 .mu.L of 6 N HCl is added. The tubes are centrifuged
and the supernatant optical density is read at 360 nM. The results
are plotted against drug concentration to determine the IC.sub.50,
i.e., the drug concentration which gives half the activity of the
control sample containing no drug.
[0078] Typically, the compounds of invention demonstrate an
IC.sub.50 in the range of about 0.1-50 nM for ACE inhibition.
[0079] Illustrative of the invention, the compound of Example 6(a)
demonstrates an IC.sub.50 of about 20 nM in the ACE in vitro
assay.
[0080] Inhibition of ACE can be demonstrated in vivo on p.o. or
i.v. administration by measuring inhibition of the angiotensin I
induced pressor response in normotensive rats.
[0081] The in vivo test for i.v. administered compounds is
performed with Male, normotensive rats, which are conscious. A
femoral artery and femoral vein are cannulated respectively for
direct blood pressure measurement on i.v. administration of
angiotensin I and i.v. or p.o. admiriistration of a compound of
this invention. After the basal blood pressure is stabilized,
pressor responses to 3 or 4 challenges of 300 ng/kg angiotensin I
i.v., at 15-minute intervals, are obtained. Such pressure responses
are usually again obtained at 15, 30, 60 and 90 minutes, and then
every hour up to 6 hours after i.v. or p.o. administration of the
compound to be tested, and compared with the initial responses. Any
observed decrease of said pressor response is an indication of ACE
inhibition.
[0082] Illustrative of the invention, the compound of Example 6(a)
inhibits the angiotensin I induced pressor response for 3 hours at
a dose of 10 mg/kg i.v. Similarly, the compound of Example 1 (a)
inhibits the angiotensin I induced pressor response for 6 hours at
a dose of 11.8 mg/kg p.o.
[0083] The in vitro inhibition of NEP (EC 3.4.24.11) can be
determined as follows:
[0084] NEP 3.4.24.11 activity is determined by the hydrolysis of
the substrate glutaryl-Ala-Ala-Phe-2-naphthylamide (GAAP) using a
modified procedure of Orlowski and Wilk (1981). The incubation
mixture (total volume 125 .mu.L) contains 4.2 .mu.L of protein (rat
kidney cortex membranes prepared by method of Maeda et al., 1983),
50 mM tris buffer, pH 7.4 at 25.degree. C., 500 .mu.M substrate
(final concentration), and leucine aminopeptidase M (2.5 .mu.g).
The mixture is incubated for 10 minutes at 25.degree. C., and 100
.mu.L of fast garnet (250 .mu.g fast garnet/mL of 10% Tween 20 in 1
M sodium acetate pH 4.2) is added. Enzyme activity is measure
spectrophotometrically at 540 nM. One unit of NEP 24.11 activity is
defined as 1 nmol of 2-naphthylamine released per minute at
25.degree. C. at pH 7.4. IC.sub.50 values are determined, i.e., the
concentration of test compound required for 50% inhibition of the
release of 2-naphthylamine.
[0085] NEP activity can also be determined using ANF as a
substrate. ANF degrading activity is determined by measuring the
disappearance of rat-ANF (r-ANF) using a 3-minute reverse
phase-HPLC separation. An aliquot of the enzyme in 50 mM tris HCl
buffer, pH 7.4, is pre-incubated at 37.degree. C. for 2 minutes and
the reaction is initiated by the addition of 4 nmol of r-ANF in a
total volume of 50 .mu.L. The reaction is terminated after 4
minutes with the addition of 30 .mu.L of 0.27% trifluoroacetic acid
(TFA). One unit of activity is defined as the hydrolysis of 1 nmol
of r-ANF per minute at 37.degree. C. at pH 7.4. IC.sub.50 values
are determined, i.e., the concentration of test compound required
for 50% inhibition of the hydrolysis of ANF.
[0086] Typically, the compounds of the invention demonstrate an
IC.sub.50 in the range of about 0.1-50 nM for NEP inhibition.
[0087] Illustrative of the invention, the compound of Example 6(a)
demonstrates an IC.sub.50 of about 5 nM in the GMP in vitro
assay.
[0088] The effect of the compounds of the invention on rat plasma
ANF concentration can be determined as follows:
[0089] Male Sprague-Dawley rats (275-390 g) are anesthetized with
ketamine (150 mg/kg)/acepromazine (10%) and instrumented with
catheters in the femoral artery and vein to obtain blood samples
and infuse ANF, respectively. The rats are tethered with a swivel
system and are allowed to recover for 24 hours before being studied
in the conscious, unrestrained state.
[0090] In the assay, plasma ANF levels are determined in the
presence and absence of NEP inhibition. On the day of study, all
rats are infused continuously with ANF at 450 ng/kg/min. i.v. for
the entire 5 hours of the experiment. Sixty minutes after beginning
the infusion, blood samples for baseline ANF measurements are
obtained (time 0) and the rats are then randomly divided into
groups treated with the test compound or vehicle. Additional blood
samples are taken 30, 60,120,180 and 240 minutes after
administration of the test compound.
[0091] Plasma ANF concentrations are determined by a specific
radioimmunoassay. The plasma is diluted (.times.12.5, .times.25 and
.times.50) in buffer containing: 50 mM tris (pH 6.8), 154 mM NaCl,
0.3% bovine serum albumin, 0.01% EDTA. One hundred microliters of
standards [rANF (99-126)] or samples are added to 100 .mu.L of
rabbit anti-rANF serum and incubated at 4.degree. C. for 16 hours.
Ten thousand cpm of [.sup.125I]rANF are then added to the reaction
mixture which is incubated at 4.degree. C. for 16 hours. Ten
thousand cpm of [.sup.125I]rANF are then added to the reaction
mixture which is incubated at 4.degree. C. for an additional 24
hours. Goat anti-rabbit IgG serum coupled to paramagnetic particles
is added to the reaction mixture and bound [.sup.125I]rANF is
pelleted by exposing the mixture to an attracting magnetic rack.
The supernatant is decanted and the pellets counted in a gamma
counter. All determinations are performed in duplicate. Plasma ANF
levels are expressed as a percent of those measured in
vehicle-treated animals which received ANF alone (450 ng/kg/min.
i.v.)
[0092] Illustrative of the invention, the compound of Example 1 (a)
increases plasma ANF levels by about 70% at a dose of 11.8 mg/kg
p.o.
[0093] The anti-hypertensive activity can be determined, e.g., in
the spontaneously hypertensive rat (SHR) and the DOCA-salt
hypertensive rat, e.g., according to Bazil et al., J. Cardiovasc.
Pharmacol., Vol. 22, pp. 897-905 (1993) and Trapani et al., J.
Cardiovasc. Pharmacol., Vol. 14, pp. 419-424 (1989),
respectively.
[0094] Illustrative of the invention, the compound of example 1(a)
reduces mean arterial pressure in conscious SHR at once daily
administration of 11.8 mg/kg p.o.
[0095] The anti-hypertensive effect can be determined in
desoxy-corticosterone acetate (DOCA)-salt hypertensive rats as
follows:
[0096] DOCA-salt hypertensive rats (280-380 g) are prepared by the
standard method. Rats undergo a unilateral nephrectomy and one week
later are implanted with silastic pellets containing 100 mg/kg of
DOCA. The rats are maintained on 1% of NaCl/0.2% KCl drinking water
for three to five weeks until sustained hypertension is
established. The anti-hypertensive activity is evaluated at this
time.
[0097] Two days before an experiment, the rats are anesthetized
with methoxyflurane and instrumented with catheters in the femoral
artery to measure arterial blood pressure. Forty-eight hours later,
baseline arterial pressure and heart rate are recorded during a one
hour period. The test compound or vehicle is then administered and
the same cardiovascular parameters are monitored for an additional
5 hours.
[0098] The diuretic (saluretic) activity can be determined in
standard diuretic screens, e.g., as described in "New
Anti-hypertensive Drugs", Spectrum Publications, pp. 307-321
(1976), or by measuring the potentiation of ANF-induced natriuresis
and diuresis in the rat.
[0099] The potentiation of the natriuretic effect of ANF can
determined as follows:
[0100] Male Sprague-Dawley rats (280-360 g) are anesthetized with
inactin (100 mg/kg i.p.) and instrumented with catheters in the
femoral artery, femoral vein and urinary bladder to measure
arterial pressure, administer ANF and collect urine, respectively.
A continuous infusion of normal saline (33 .mu.L/min.) is
maintained throughout the experiment to promote diuresis and sodium
excretion. The experimental protocol consists of an initial
15-minute collection period (designated as pre-control) followed by
three additional collection periods. Immediately after completion
of the pre-control period, test compound or vehicle is
administered; nothing is done for the next 45 minutes. Then, blood
pressure and renal measurements are obtained during a second
collection period (designated control, 15 minutes). At the
conclusion of this period, ANF is administered (1 .mu.g/kg i.v.
bolus) to all animals and arterial pressure and renal parameters
are determined during two consecutive 15-minute collection periods.
Mean arterial pressure, urine flow and urinary sodium excretion are
determined for all collection periods. Blood pressure is measured
with a Gould p50 pressure transducer, urine flow is determined
gravimetrically, sodium concentration is measured by flame
photometry, and urinary sodium excretion is calculated as the
product of urine flow and urine sodium concentration.
[0101] The in vitro inhibition of ECE can be determined as
follows:
[0102] ECE is partially purified from porcine primary aortic
endothelial cells by DE52 anion exchange column chromatography and
its activity is quantified by radioimmunoassay (RIA) as described
in Anal. Biochem., Vol., 212, pp. 434-436(1993). Alternatively, the
native enzyme can be substituted by a recombinant form of ECE, as
described, for example, in Cell, Vol. 78, pp. 473-485 (1994). Human
ECE-1 has been described by several groups (Schmidt et al., FEBS
Letters, Vol. 356, pp. 238-243 (1994); Kaw et al., 4th Int. Conf.
on Endothelin; April 23-25, London (UK) (1995) C6; Valdenaire et
al., J. Biol. Chem., Vol. 270, pp. 29794-29798 (1995); Shimada et
al., Biochem. Biophys. Res. Commun., Vol. 207, pp. 807-812 (1995)).
The ECE inhibition can be determined as described in Biochem. Mol.
Biol. Int., Vol. 31, No. 5, pp. 861-867 (1993), by RIA to measure
ET-1 formed from big ET-1.
[0103] Alternatively, recombinant human ECE-1 (rhECE-1) can be
used, as follows:
[0104] Chinese hamster ovary cells expressing rhECE-1 (Kaw et al.,
4th Int. Conf. on Endothelin; April 23-25, London (UK), (1995) C6)
are cultured in DMEM/F12 medium containing 10% fetal bovine serum
and 1.times. antibiotic-antimycotic. Cells are harvested by
scraping, pelleted by centrifugation, and homogenized at 4.degree.
C. in a buffer containing 5 mM MgCl.sub.2, 1 .mu.M pepstatin A, 100
.mu.M leupeptin, 1 mM PMSF, and 20 mM Tris, pH 7.0, with a ratio of
2 mL of buffer/mL of cells. The cell debris is removed by brief
centrifugation, and the supernatant is centrifuged again at
100,000.times.g for 30 minutes. The resulting pellet is
re-suspended in a buffer containing 200 mM NaCl and 50 mM Tes, pH
7.0, at a protein concentration about 15 mg/mL and stored in
aliquots at -80.degree. C.
[0105] To assess the effect of an inhibitor on ECE-1 activity, 10
.mu.g of protein is pre-incubated with the compound at a desired
concentration for 20 minutes at room temperature in 50 mM TES, pH
7.0, and 0.005% Triton X-100 in a volume of 10 .mu.L. Human big
ET-1 (5 .mu.L) is then added to a final concentration of 0.2 .mu.M,
and the reaction mixture is further incubated for 2 hours at
37.degree. C. The reaction is stopped by adding 500 .mu.L of RIA
buffer containing 0.1% Triton X-100, 0.2% bovine serum albumin, and
0.02% NaN.sub.3 in phosphate-buffered saline.
[0106] Diluted samples (200 .mu.L) obtained from the above enzyme
assay are incubated at 4.degree. C. overnight with 25 .mu.L each of
[.sup.125I]ET-1 (10,000 cpm/tube) and 1:20,000-fold diluted rabbit
antibodies that recognize specifically the carboxyl terminal
tryptophan of ET-1. Goat anti-rabbit antibodies coupled to magnetic
beads (70 .mu.g) are then added to each tube, and the reaction
mixture is further incubated for 30 minutes at room temperature.
The beads are pelleted using a magnetic rack. The supernatant is
decanted, and the radioactivity in the pellet is counted in a gamma
counter. Total and nonspecific binding are measured in the absence
of non-radioactive ET-1 and anti-ET antibodies, respectively. Under
these conditions, ET-1 and big ET-1 displace [.sup.125I]ET-1
binding to the antibodies with IC.sub.50 values of 21.+-.2 and
260,000.+-.66,000 fmol (mean.+-.SEM, n=3-5), respectively.
[0107] In order to determine the IC.sub.50 value of an inhibitor, a
concentration-response curve of each inhibitor is determined. An
IBM-compatible version of ALLFIT program is used to fit data to a
one-site model.
[0108] ECE inhibition can also be determined in vivo by measuring
the inhibition of big ET-1-induced pressor response in the
anesthesized or conscious rat, as described below. The effect of
the inhibitors on the pressor response resulting from big ET-1
challenge is measured in Sprague-Dawley rats as described in
Biochem. Mol. Biol. Int., Vol. 31, No. 5, pp. 861-867 (1993).
Results are expressed as percent inhibition of the big ET-1-induced
pressor response as compared to vehicle.
[0109] Male Sprague-Dawley rats are anesthetized with Inactin (100
mg/kg i.p.) and instrumented with catheters in the femoral artery
and vein to record mean arterial pressure (MAP) and administer
compounds, respectively. A tracheostomy is performed and a cannula
inserted into the trachea to ensure airway patency. The body
temperature of the animals is maintained at 37.+-.1.degree. C. by
means of a heating blanket. Following surgery, MAP is allowed to
stabilize before interrupting autonomic neurotransmission with
chlorisondamine (3 mg/kg i.v.). Rats are then treated with the test
compound at 10 mg/kg i.v. or vehicle and challenged with big ET-1
(1 nmol/kg i.v.) 15 and 90 minutes later. Generally, the data are
reported as the maximum increase in MAP produced by big ET-1 in
animals treated with the test compound or vehicle.
[0110] Male Sprague-Dawley rats are anesthetized with methohexital
sodium (75 mg/kg i.p.) and instrumented with catheters in the
femoral artery and vein to measure MAP and administer drugs,
respectively. The catheters are threaded through a swivel system
that enables the rats to move freely after regaining consciousness.
The rats are allowed to recover from this procedure for 24 hours
before initiating the study. On the following day, MAP is recorded
via the femoral artery catheter and a test compound or vehicle is
administered via the femoral vein. Animals are challenged with big
ET-1 at 1 nmolkg i.v. at various times after dosing. After an
adequate washout period, depending upon the dose and regimen,
animals can be re-tested at another dose of test compound or
vehicle. Generally, the data are reported as the change in MAP
produced by big ET-1 at 2-minute intervals in animals treated with
the test compound as compared to vehicle.
[0111] ECE inhibition can also be determined in vivo by measuring
the inhibition of the big ET-1 induced pressor response in
conscious SHR, e.g. as described in Biochem. Biophys. Res. Commun.,
Vol. 204, pp. 407-412 (1994).
[0112] Male SHR (16-18 weeks of age) are administered either test
compound or vehicle (1 M NaHCO.sub.3) via an osmotic minipump
implanted subcutaneously. On day 5, femoral arterial and venous
catheters are placed in anesthetized rats for the measurement of
MAP and for test compound administration, respectively. After a
48-hour recovery period, MAP is recorded (day 7) through the
arterial catheter connected to a pressure transducer. Blood
pressure and heart rate are allowed to stabilize for 30 minutes
before ganglion blockade is performed using chlorisondamine (10
mg/kg i.v.). Approximately 15 minutes later, a bolus dose of big
ET-1 (0.25 nmol/kg i.v.) is administered to both vehicle- and test
compound-treated rats. The change in blood pressure in response to
big ET-1 is then compared between the two groups of rats.
[0113] The inhibition of cerebral vasospasm is demonstrated by
measuring the inhibition of experimentally induced constriction of
basilar cerebral arteries in the rabbit (Caner et al., J.
Neurosurg., Vol. 85, pp.-917-922 (1996).
[0114] The degree or lack of undesirable immunostimulatory
potential of the compounds of the invention can be determined with
the murine popliieal lymph node assay described in Toxicology
Letters, Vols. 112/113, pp. 453-459 (2000).
[0115] The compounds of the invention, e.g., can be prepared [0116]
a) by condensing a compound of formula II ##STR4## wherein the
symbols alk, X, R, R.sub.1, R.sub.6 and R.sub.7 have the meaning as
defined above and COOR.sub.2 represents esterified carboxyl, with a
carboxylic acid of the formula III ##STR5## or a reactive
functional derivative thereof, wherein R.sub.4 and R.sub.5 have
meaning as defined above; R.sub.3' represents hydrogen or a labile
S-protecting group, e.g., acyl, t-butyl or optionally substituted
benzyl; or [0117] b) by condensing a compound of the formula IV
##STR6## or a reactive functional derivative thereof wherein the
symbols R.sub.3', R.sub.4-R.sub.5 and R.sub.6-R.sub.7 have meaning
as defined above, with an amino acid ester of the formula V
##STR7## wherein alk, X, R and R.sub.1 have meaning as defined
above and COOR.sub.2 represents esterified carboxyl; or [0118] c)
by condensing under basic conditions a compound of the formula VI
##STR8## wherein the symbols R, R.sub.1, COOR.sub.2,
R.sub.4-R.sub.7, alk and X have meaning as defined above and Y
represents a reactive esterified hydroxyl group (e.g., chloro or
bromo) as a leaving group, with a compound of the formula
R.sub.3'SH (VII) or a salt thereof, wherein R.sub.3 represents a
labile S-protecting group, e.g., acyl, t-butyl or optionally
substituted benzyl; and converting a resulting product to a
compound of formula I wherein R.sub.3 is hydrogen; and in above
said process, if temporarily protecting any interfering reactive
group(s), removing said protecting group(s), and then isolating the
resulting compound of the invention; and, if desired, converting
any resulting compound of the invention into another compound of
the invention; and/or, if desired, converting a free carboxylic
acid function into a pharmaceutically acceptable ester derivative,
or converting a resulting ester into the free acid or into another
ester derivative; and/or, if desired, converting a resulting free
compound into a salt or a resulting salt into the free compound or
into another salt, and/or, if desired, separating a mixture of
isomers or racemates, and/or, if desired, resolving a racemate
obtained into the optical antipodes.
[0119] In starting compounds and intermediates which are convened
to the compounds of the invention in manner described herein,
functional group present, such as thiol, carboxyl, amino and
hydroxy groups, are optionally protected by conventional protecting
groups that are common in preparative organic chemistry. Protected
thiol, carboxyl, amino and hydroxy groups are those that can be
converted under mild conditions into free thiol, carboxyl, amino
and hydroxy groups without other undesired side reactions taking
place.
[0120] The purpose of introducing protecting groups is to protect
the functional groups from undesired reactions with reaction
components and under the conditions used for carrying out a desired
chemical transformation. The need and choice of protecting groups
for a particular reaction is known to those skilled in the art and
depends on the nature of the functional group to be protected
(thiol, carboxyl, amino group, etc.), the structure and stability
of the molecule of which the substituent is a part, and the
reaction conditions.
[0121] Well-known protecting groups that meet these conditions and
their introduction and removal are described, for example, in J. F.
W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press,
London, N.Y. (1973), T. W. Greene and P. G. M. Wuts, "Protective
Groups in Organic Synthesis", Wiley, N.Y. 3.sup.rd Ed. (1999), and
also in "The Peptides", Vol. I, Schroeder and Luebke, Academic
Press, London, N.Y. (1965).
[0122] The preparation of compounds of the invention according to
process (a) involving the condensation of an amine of formula II
with the acid of formula III or a functional reactive derivative
thereof, is carried out by methodology well-known for peptide
synthesis.
[0123] The condensation according to process (a) of an amino ester
of formula II with a free carboxylic acid of formula III is carried
out advantageously in the presence of a condensing agent such as
dicyclohexylcarbodiimide,
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide,
hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole,
chlorodimethoxytriazine,
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP Reagent), or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU), either alone or in combination, and
triethylamine or N-methylmorpholine, in an inert polar solvent,
such as ethyl acetate, acetonitrile, dimethylformamide or methylene
chloride, preferably at room temperature.
[0124] The condensation of an amino ester of formula II with a
reactive functional derivative of an acid of formula III in the
form of an acid halide, advantageously an acid chloride, or mixed
anhydride, is carried out in an inert solvent such as toluene or
methylene chloride, advantageously in the presence of a base, e.g.,
an inorganic base such as potassium carbonate or an organic base
such as triethylamine, N-methylmorpholine or pyridine, preferably
at room temperature.
[0125] Reactive functional derivatives of carboxylic acids of
formula III are preferably acid halides (e.g., the acid chloride)
and mixed anhydrides, such as the pivaloyl or isobutyloxycarbonyl
anhydride, or activated esters such as benzotriazole,
7-azabenzotriazole or hexafluorophenyl ester.
[0126] The starting material of formula II can be prepared
according to methods described herein and illustrated in the
examples.
[0127] The preparation of a starting material of formula II
involves the acylatidn of an ester of formula VIII ##STR9## wherein
alk, X, R and R.sub.1 have meaning as defined hereinabove and
COOR.sub.2 represents esterified carboxyl (e.g., wherein R.sub.2 is
lower alkyl or benzyl) with an appropriately N-protected amino acid
(or a reactive functional derivative) of formula IX ##STR10##
wherein R.sub.6 and R.sub.7 have meaning as defined hereinabove and
R.sub.8 is a labile amino protecting group, e.g., t-butoxycarbonyl,
to obtain the corresponding N-protected compound of formula II.
[0128] The condensation of a compound of formula VIII with a
compound of formula IX is carried out by methodology well-known in
peptide synthesis, e.g., as described above for the condensation of
a compound of formula II with a compound of formula III. The
N-protecting group is removed according to methods well-known in
the art, e.g., the t-butoxycarbonyl is removed with anhydrous acid
such as trifluoroacetic acid or HCl.
[0129] The starting amino esters and acids of compounds of formula
VIII and IX, respectively, are either known in the art, or if new,
can be prepared according to methods well-known in the art, e.g.,
or illustrated herein. The amino acid esters of formula VIII are
preferably the S-enantiomers.
[0130] The starting materials of formula III are known, or if new,
may be prepared according to conventional methods. The starting
materials are prepared, e.g., from the corresponding racemic or
optically active .alpha.-amino acids, by conversion thereof to the
.alpha.-bromo derivative followed by displacement thereof with
inversion of configuration using the appropriate thiol derivative
of formula VII, under basic conditions, for example, as illustrated
in European Patent Application No. 524,553 published Jan. 27, 1993.
S-debenzylation of the resulting final products is carried out by
reductive cleavage, e.g., with Raney nickel in ethanol.
S-deacylation is carried out by, e.g., base catalyzed hydrolysis
with dilute aqueous sodium hydroxide. Cyclic starting materials of
formula III can be prepared by treatment of the cyclic carboxylic
acid (e.g., cyclopentanecarboxylic acid) with sulfur in the
presence of a strong base such as lithium diethylamide.
[0131] The preparation of the compounds of the invention according
to process (b) involving the condensation of an acid of formula IV
with an amino acid ester of formula V is carried out in a similar
fashion to process (a). Similarly, the starting materials of
formula IV are prepared by condensation of an acid of formula III
with an ester corresponding to gem-disubstituted amino acids of
formula IX (wherein R.sub.8 is now hydrogen) under conditions
similar to those described above, followed by removal of the
carboxylprotecting group.
[0132] The preparation of the compounds of the invention according
to process (c) involving the displacement of a leaving group Y in a
compound of formula VI with a thiol derivative R.sub.3'-SH as a
salt thereof is carried out according to methods well-known in the
art.
[0133] A reactive esterified hydroxyl group, represented by Y, is a
hydroxyl group esterified by a strong inorganic or organic acid.
Corresponding Y groups are in particular halo, for example, chloro,
bromo or iodo, also sulfonyloxy groups, such as lower alkyl- or
arylsulfonyloxy groups, for example, (methane-, ethane-, benzene-
or toluene-) sulfonyloxy groups, also the
trifluoromethylsulfonyloxy group.
[0134] The displacement is carried out in an inert solvent, such as
dimethylformamide or methylene chloride in the presence of a base
such as potassium carbonate, triethylamine, diisopropylethylamine,
N-methylmorpholine, and the like at room or elevated temperature.
Using a salt of R.sub.3'SH (e.g., potassium thioacetate), the
reaction is carried out in the absence of a base, in an inert
solvent such as tetrahydrofuran or dimethylformamide.
[0135] Similarly, the starting materials of formula VI can be
prepared by reacting the dipeptide derivative of formula II with an
acid of the formula ##STR11## wherein R.sub.14 and R.sub.5 and Y
have meaning as defined above, under conditions described for
process (a).
[0136] The compounds of formula X wherein Y is halo, such as the
.alpha.-bromocarboxylic acids are known and are prepared, e.g., as
described in International Application WO 99/55726 published Nov.
4, 1999.
[0137] The compounds of the invention and intermediates, e.g.,
those of formulas II, V and VI, having the side chain
alk-X--R.sub.1 are prepared from the corresponding compounds having
the alk-X' side chain wherein X' represents amino, hydroxy, thiol
or a suitable leaving group according to methodology known in the
art and illustrated herein. For example, the acids and esters of
formula V can be obtained starting with serine, homoserine,
threonine, cysteine and the like, preferably in optically active
form.
[0138] Certain compounds of the invention and intermediates can be
converted to each other according to general reactions well-known
in the art.
[0139] The free mercaptans may be converted to the S-acyl
derivatives by reaction with a reactive derivative of a carboxylic
acid (corresponding to R.sub.3 being acyl in formula I), such as an
acid anhydride or said chloride, preferably in the presence of a
base such as triethylamine in an inert solvent such as acetonitrile
or methylene chloride.
[0140] Free alcohols and phenols can be converted to the
corresponding acyl derivatives, e.g., by reaction with a
corresponding acid chloride in the presence of a base, such as
triethylamine.
[0141] The free mercaptans, wherein R.sub.3 represents hydrogen,
may be oxidized to the corresponding disulfides, e.g., by air
oxidation or with the use of mild oxidizing agents such as iodine
in alcoholic solution. Conversely, disulfides may be reduced to the
corresponding mercaptans, e.g., with reducing agents such as sodium
borohydride, zinc and acetic acid or tributylphosphine.
[0142] Carboxylic acid esters may be prepared from a carboxylic
acid by condensation with, e.g., the halide corresponding to
R.sub.2--OH, in the presence of a base, or with an excess of the
alcohol in the presence of an acid catalyst, according to methods
well-known in the art.
[0143] Carboxylic acid esters and S-acyl derivatives may be
hydrolyzed, e.g., with aqueous alkali such as alkali metal
carbonates or hydroxides. S-acyl and ester groups can be
selectively removed as illustrated herein.
[0144] Preferably, and wherever possible, the preferred isomers of
the invention of formula Ia are prepared from pure enantiomers.
[0145] In case mixtures of stereoisomers (e.g., diastereomers) are
obtained, these can be separated by known procedures such as
fractional crystallization and chromatography (e.g., thin layer,
column, flash chromatography). Racemic free acids can be resolved
into the optical antipodes by fractional crystallization of d- or
I-(.alpha.-methylbenzylamine, cinchonidine, cinchonine, quinine,
quinidine, dehydroabiethylamine, brucine or strychnine) salts and
the like. Racemic products, if not diastereoisomers, can first be
converted to diastereoisomers with optically active reagents (such
as optically active alcohols to form esters) which can then be
separated as described above, and, e.g., hydrolyzed to the
individual enantiomer. Racemic products can also be resolved by
chiral chromatography, e.g., high pressure liquid chromatography
using a chiral absorbent; also by enzymatic resolution, e.g., of
esters with alkalase.
[0146] The above-mentioned reactions are carried out according to
standard methods, in the presence or absence of diluents,
preferably such as are inert to the reagents and are solvents
thereof, of catalysts, alkaline or acidic condensing or said other
agents respectively and/or inert to the reagents and are solvents
thereof, of catalysts, alkaline or acidic condensing or said other
agents respectively and/or inert atmospheres, at low temperatures,
room temperature or elevated temperatures, preferably near the
boiling point of the solvents used, at atmospheric or
superatmospheric pressure.
[0147] The invention further includes any variant of said
processes, in which an intermediate product obtainable at any stage
of the process is used as a starting material and any remaining
steps are carried out, or the process is discontinued at any stage
thereof, or in which the starting materials are formed under the
reaction conditions, or in which the reaction components are used
in the form of their salts or optically pure antipodes. Mainly
those starting materials should be used in said reactions, that
lead to the formation of those compounds indicated above as being
preferred.
[0148] The present invention additionally relates to the use in
mammals of the compounds of the invention and their
pharmaceutically acceptable, non-toxic acid addition salts, or
pharmaceutical compositions thereof, as medicaments, for inhibiting
both ACE and NEP, and, e.g., for the prevention or treatment of
cardiovascular disorders such as hypertension, edema, salt
retention and congestive heart failure, either alone or in
combination with one or more other agents which are useful for the
treatment of such disorders. Such may be anti-hypertensive agents,
anti-atherosclerotic agents, cardiac agents, diuretic agents,
antidiabetic agents, cholesterol-lowering agents and the like. When
used in combination with other therapeutic agents such can be
administered separately or in a fixed combination.
[0149] Examples of therapeutic agents which can be used in
combination are angiotensin II receptor antagonists, such as
valsartan, losartan, candesartan, eprosartan, irbesartan and
telmisartan; .beta.-blockers, such as bisoprolol, propanolol,
atenolol, sotalol and metoprolol; renin inhibitors; calcium channel
blockers, such as amlodipine, verapamil, diltiazem, bepridil,
felodipine, isradipine, nicardipine, nifedipine, nimodipine and
nisoldipine; aldosterone synthase inhibitors/aldosterone
antagonists, such as eplerenone, (+)-fadrozole (WO 01/76574),
spironolactone and canrenone; diuretics, such as furosemide,
hydrochlorothiazide, indapamide, metazolone, amiloride and
triamterene; vasopressin receptor antagonists, such as OPC 21268,
SR 49059, SR121463A, SR49059, VPA985, OPC31260 and YM087;
cardiotonic drugs, such as enoximone and levosimendan; endothelin
antagonists and ECE inhibitors, such as bosentan, BMS193884,
TBC3711 and compounds disclosed in WO 99/55726;
anti-atherosclerotic agents, particularly cholesterol lowering
agents, such as bile acid sequestrants (e.g., cholestyramine and
colestipol); cholesterol absorption inhibitors, such as ezetimibe;
fibrates, such as fehofibrate and gemfibrozil; statin HMG CoA
reductase inhibitors, such as atorvastatin, fluvastatin,
lovastatin, pravastatin, simvastatin and pitavastatin; and
nicotinic acid derivatives; thyromimetic agents, such as those
disclosed in U.S. Pat. No. 5,569,674 and WO 00/58279; also
antidiabetic agents, such as repaglinide, nateglinide, metformin,
rosiglitazone, pioglitazone, glyburide, glipizide, glimepiride,
DPP728, LAF237, NH622 and DRF4158.
[0150] The present invention also relates to the use of the
compounds of the invention for the preparation of pharmaceutical
compositions, especially pharmaceutical compositions having ACE and
NEP inhibiting activity, and, e.g., anti-hypertensive activity.
[0151] The pharmaceutical compositions according the invention are
those suitable for enteral, such as oral or rectal, transdermal and
parenteral administration to mammals, including man, for the
treatment of cardiovascular disorders, such as hypertension,
comprising an effective amount of a pharmacologically active
compound of the invention or a pharmaceutically acceptable salt
thereof, alone or in combination with one or more pharmaceutically
acceptable carriers, as well as in combination with other
therapeutic agents also useful for the treatment of cardiovascular
disorders, as indicated above.
[0152] The pharmacologically active compounds of the invention are
useful in the manufacture of pharmaceutical compositions comprising
an effective amount thereof in conjunction or admixture with
excipients or carriers suitable for either enteral or parenteral
application. Preferred are tablets and gelatin capsules comprising
the active ingredient, together with a) diluents, e.g., lactose,
dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)
lubricants, e.g., silica, talcum, stearic acid, its magnesium or
calcium salts and/or polyethyleneglycol; for tablets also c)
binders, e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone; if desired, d) disintegrants, e.g., starches,
agar, alginic acid or its sodium salt, or effervescent mixtures;
and, if desired, absorbents, colorants, flavors and sweeteners.
Injectable compositions are preferably aqueous isotonic solutions
or suspensions, and suppositories are advantageously prepared from
fatty emulsions or suspensions. Said compositions may be sterilized
and/or contain adjuvants, such as preserving, stabilizing, wetting
or emulsifying agents, solution promoters, salts for regulating the
osmotic pressure and/or buffers. In addition, the compositions may
also contain other therapeutically valuable substances. Said
compositions are prepared according to conventional mixing,
granulating or coating methods, respectively, and contain about
0.1-75%, preferably about 1-50%, of the active ingredient.
[0153] Suitable formulations for transdermal application include an
effective amount of a compound of the invention with carrier.
Advantageous carriers include absorbable pharmacologically
acceptable solvents to assist passage through the skin of the host.
Characteristically, transdermal devices are in the form of a
bandage comprising a backing member, a reservoir containing the
compound, optionally with carriers, optionally a rate controlling
barrier to deliver the compound to the skin of the host at a
controlled and predetermined rate over a prolonged period of time,
and means to secure the device to the skin.
[0154] A unit dosage for a mammal of about 50-70 kg may contain
between about 10 and 200 mg of the active ingredient. The dosage of
active compound is dependent on the species of warm-blooded animal
(mammal), the body weight, age and individual condition, and on the
form of administration.
[0155] The following examples are intended to illustrate the
invention and are not to be construed as being limitations thereon.
Temperatures are given in degrees Centigrade. If not mentioned
otherwise, all evaporations are performed under reduced pressure,
preferably between about 15 and 100 mm Hg. Optical rotations
(expressed in degrees) are measured at room temperature at 589 nM
(D line of sodium) or other wave lengths as specified in the
examples. The structure of the compounds are confirmed by standard
analytical methods such as mass spectrum, elemental analysis, NMR,
IR spectroscopy and the like.
[0156] The prefixes R and S are used to indicate the absolute
configuration at each asymmetric center.
EXAMPLE 1
(a)
N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-ben-
zyl-L-serine ethyl ester
[0157] ##STR12##
[0158]
N-[2-[-[(R)-3-bromo-3-methylbutanoylamino]-2-methylproprionyl-O-be-
nzyl-L-serine ethyl ester (4.96 g, 10.5 mmol) is dissolved in
tetrahydrofuran (100 mL) and potassium thioacetate (6.00 g, 52.5
mmol) is added. The mixture is stirred at room temperature for 4
hours, then diluted with ethyl acetate (500 mL) and washed with
water (100 mL), sodium bicarbonate solution (2.times.100 mL), water
(2.times.100 mL) and then brine (50 mL). The solution is dried over
sodium sulfate and concentrated in vacuo. The crude material is
purified by flash chromatography (silica gel, 3:2 hexane/ethyl
acetate) to yield title compound; m.p. 55-570C;
[.alpha.].sup.20.sub.D-63.5.degree. (c=0.99, CH.sub.3OH); MS (M+H):
467.
[0159] Alternately, the above displacement can be carried out with
2 equivalents of potassium thioacetate in ethyl acetate at room
temperature and the resulting product can be crystallized from
t-butyl methyl ether/heptane (40/60) to give the title compound
having m.p. of 68.degree. C.
[0160] The starting material is prepared as follows:
[0161] A solution of O-benzyl-L-serine (9.75 g, 50 mmol) in ethanol
(200 mL) is saturated with HCl gas for 8 minutes. The mixture is
stirred overnight at room temperature, and then concentrated in
vacuo. The solid is washed with diethyl ether and collected by
filtration to yield O-benzyl-L-serine ethyl ester hydrochloride as
a white solid.
[0162] To a solution of BOC-.alpha.-methylalanine (3.05 g, 15
mmol), O-benzyl-L-serine ethyl ester hydrochloride (3.89 g, 15
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCl, 2.88 g,
15 mmol) and 1-hydroxy-7-azabenzotriazole (HOAT, 2.04,15 mmol) in
methylene chloride (150 mL) is added triethylamine (1.52 g, 15
mmol). The mixture is stirred overnight and then concentrated in
vacua. The residue is re-dissolved in ethyl acetate and washed with
water, 1 N HCl, water, and brine. The solution is dried over sodium
sulfate and concentrated to yield
N-[2-(BOC-amino)-2-methylpropionyl]-O-benzyl-L-serine ethyl ester
of the formula ##STR13##
[0163] Alternately, the above carbamate can be prepared by
condensing O-benzyl-L-serine ethyl ester hydrochloride with
BOC-.alpha.-methylalanine in the presence of 1 equivalent of
2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, see Synthesis, pp.
917-920 (1987)) and N-methylmorpholine (2.5 equivalents) in ethyl
acetate at room temperature.
[0164] The above carbamate (6.12 g, 15 mmol) is dissolved in
methylene chloride (200 mL) and chilled in an ice bath. The
solution is saturated with HCl gas for 10 minutes and then stirred
at room temperature overnight. The residue is concentrated.
Methylene chloride is added and the residue is concentrated again
to give N-(2-amino-2-methylpropionyl)-O-benzyl-L-serine ethyl ester
hydrochloride as a foam; MS (M+H): 309.
[0165] Alternately, N-(2-amino-2-methylpropionyl)-O-benzyl-L-serine
hydrochloride can be prepared by treating the carbamate with HCl
gas (3 equiv.) in ethyl acetate at a temperature of 25-50.degree.
C. for 3 hours.
[0166] To a solution of the above amine hydrochloride (4.90 g, 14
mmol) in methylene chloride (150 mL) is added
(R)-2-bromo-3-methylbutanoic acid diisopropyl amine salt (4.03 g,
14 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCl, 2.70 g, 14 mmol) and
1-hydroxy-7-azabenzotriazole (HOAT, 1.90 g, 14 mmol). The mixture
is stirred at room temperature overnight and then concentrated in
vacua. The residue is dissolved in ethyl acetate and washed with
water, dilute sodium bicarbonate, water, 1 N HCl, and then brine.
The solution is dried over sodium sulfate and concentrated to give
a solid. The solid is purified by flash chromatography (silica gel,
2:1 hexane/ethyl acetate) to give
N-[2-(R)-2-bromo-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-ser-
ine ethyl ester of the formula ##STR14##
[0167] Alternately, the above condensation of
(R)-2-bromo-3-methylbutanoic acid diisopropylamine salt with the
amine hydrochloride can be carried out in acetonitrile in the
presence of CDMT (1.05 equiv.) and N-methyl-morpholine (1.5 equiv.)
at a temperature of 5-25.degree. C.
[0168] Similarly prepared are:
(b)
N-[2-[(S)-2-acetylthio-3,3-dimethylbutanoylamino]-2-methylpropinyl]-O--
benzyl-L-serine ethyl ester
[0169] ##STR15##
(c)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-ben-
zyl-L-threonine ethyl ester; m.p. 121-122.degree. C.
[0170] ##STR16##
(d)
N-[2-[(S)-2-acetylthio-3-methoxybutanoylamino]-2-methylpropionyl]-O-be-
nzyl-L-serine ethyl ester; [.alpha.].sub.D.sup.20+14.9.degree.
(c=1.04, DMSO)
[0171] ##STR17##
(e)
N-[2-[(S)]-2-acetylthio-3-methylpentanoylamino]-2-methylpropionyl]-O-b-
enzyl-L-serine ethyl ester; [.alpha.].sub.D.sup.20-6.93.degree.
(c=1.09, CH.sub.3OH)
[0172] ##STR18##
(f)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(3--
trifluoromethylbenzyl)-L-serine ethyl-ester
[0173] ##STR19##
[0174] The starting O-(3-trifluoromethylbenzyl)-L-serine ethyl
ester hydrochloride is prepared as follows:
[0175] To a suspension of sodium hydride (60% in oil, 3.04 g, 76
mmol) in N,N-dimethylformamide (60 mL) at 0.degree. C. is added
BOC-L-serine (7.80 g, 38 mmol). The mixture is stirred for 1 hour
and then m-trifluoromethylbenzyl chloride (7.39 g, 38 mmol) is
added. The mixture is allowed to warm to room temperature and is
stirred overnight. The mixture is quenched with water. Ethyl
acetate is added and the mixture is washed with brine, dried over
MgSO.sub.4 and concentrated to give a yellow oil which is purified
by flash chromatography (SiO.sub.2; hexane/ethyl acetate) to give a
clear oil. The residue is dissolved in ethanol (120 mL), the
solution is cooled to 0.degree. C. and saturated with HCl gas for 5
minutes. The mixture is allowed to warm to room temperature and
stirred overnight. The mixture is concentrated to give
O-(4-trifluoromethylbenzyl)-L-serine ethyl ester hydrochloride.
(g)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(4--
fluorobenzyl)-L-serine ethyl ester as an oil
[0176] ##STR20##
(h)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(4--
fluorophenyl)-L-homoserine ethyl ester as an oil
[0177] ##STR21##
[0178] The starting O-(4-fluorophenyl)-L-homoserine ethyl ester
hydrochloride is prepared as follows:
[0179] To a solution of BOC-L-homoserine t-butyl ester (3.2 g, 11.6
mmol) in tetrahydrofuran is added triphenylphosphine (7.59 g, 29
mmol), p-fluorophenol (2.08 g, 18.6 mmol) and
1,1'-azobis(N,N-dimethylformamide) (3.2 g, 18.6 mmol). The mixture
is stirred overnight, washed with brine, dried over MgSO.sub.4, and
the solvent is removed to give an orange oil. The oil is purified
by flash chromatography (SiO.sub.2, 85% hexane/15% ethyl acetate)
to give a clear oil which is dissolved in ethanol (100 mL) and the
solution is saturated with HCl gas, then stirred overnight. The
mixture is concentrated to give O-(4-fluorophenyl)-L-homoserine
ethyl ester hydrochloride as a white solid.
(i)
N-[2-[(S)-2-acetylthio-3-methyl-butanoylamino]-2-methylpropionyl]-O-(3-
-fluorophenyl)-L-homoserine ethyl ester
[0180] ##STR22##
(j)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-ben-
zyl-L-serine morpholinocarbonylmethyl ester, purified by
chromatography on silica gel with hexane, ethyl acetate, methanol
(20:70:10) as a white solid
[0181] ##STR23##
[0182] The starting material is prepared as follows:
[0183] O-Benzyl-L-serine (10.0 g, 51.3 mmol),
di-tert-butyl-dicarbonate (11.2 g, 51.4 mmol) and 1 N sodium
hydroxide (103 mL, 103 mmol) are stirred together in 100 mL of
dioxane at room temperature for 16 hours. The mixture is
concentrated in vacuo, taken up in water, acidified to pH 1 with 6
N HCl and extracted with ethyl acetate. The organic layer is washed
with water, then brine, and dried over anhydrous magnesium sulfate.
The mixture is filtered and concentrated in vacuo to give
BOC-O-benzyl-L-serine as an oil. 4-(2-Chloroacetyl)morpholine (1.22
g, 7.48 mmol) is added to a solution of BOC-O-benzyl-L-serine (2.20
g, 7.46 mmol), triethylamine (0.75 g, 7.43 mmol) and sodium iodide
(0.11 g, 0.73 mmol) in 5 mL of N,N-dimethylformamide and the
mixture stirred at room temperature for 2 hours. The mixture is
diluted with ethyl acetate, washed with water, then with brine, and
dried over anhydrous magnesium sulfate. The mixture is filtered and
concentrated in vacuo to give a yellow oil. The oil is
chromatographed on silica gel with hexane:ethyl acetate:methanol
(35:60:5) to give BOC-O-benzyl-L-serine morpholinocarbonylmethyl
ester as a colorless oil. HCl gas is bubbled through a solution of
the carbamate ester (1.72 g, 4.08 mmol) in methylene chloride (50
mL) for 5 minutes and the mixture is stirred at room temperature
for 3 hours. The resulting mixture is concentrated in vacuo to
yield O-benzyl-L-serine morpholinocarbonylmethyl ester as a
foam.
(k)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-ben-
zyl-L-serine dimethylaminocarbonylmethyl ester, prepared and
purified as described for compound of Example 1(j)
[0184] ##STR24##
(l) N-[2-[(S)-2-acetylthio-3-methyl
butanoylamino]-2-methylpropionyl]-O-benzyl-L-serine
diethylaminocarbonylmethyl ester, prepared as described for
compound of Example 1(j) and purified by chromatography on silica
gel with hexane, ethyl acetate, methanol (35:60:5)
[0185] ##STR25##
(m)
N-[2-[(S)-2-[(methoxyacetyl)thio]-3-methylbutanoylamino]-2-methylpropi-
onyl]-O-benzyl-L-serine ethyl ester; [.alpha.].sub.D-55.27.degree.;
(c=1.084, CH.sub.3OH)
[0186] ##STR26##
(n)
N-[2-[(S)-2-[(morpholinoacetyl)thio]-3-methylbutanoylamino]-2-methylpr-
opionyl]-O-benzyl-L-serine ethyl ester; [a].sub.D-48.61.degree.
(c=1.098, CH.sub.3OH)
[0187] ##STR27##
(o)
N-[2[(S)-2-acetylthio-2-(4-tetrahydropropanyl)acetylamino]2-methylprop-
ionyl]-O-benzyl-L-serine ethyl ester;
[.alpha.].sub.D.sup.20-55.4.degree. (c=0.83, DMSO)
[0188] ##STR28##
[0189] The starting
(D)-.alpha.-bromo-.alpha.-(4-tetrahydropyranyl)-acetic acid can be
prepared as follows:
[0190] A solution of sodium nitrite (4.71 g, 68.3 mmol) in 35 mL of
water is added dropwise to a chilled (0.degree. C.) solution of
(D)-.alpha.-bromo-.alpha.-(4-tetrahydropyranyl)-glycine (J. Am.
Chem. Soc., Vol. 117, pp. 9375-9376 (1995) (7.05 g, 44.3 mmol) and
48% HBr (aq) (70 mL) in 35 mL of water. Upon completion of the
addition, the mixture is allowed to warm to room temperature and
stirred at room temperature for 3 hours. The mixture is extracted
with ethyl acetate; the organic layer is washed sequentially with
water, 5% aqueous sodium thiosulfate, and brine, then dried over
anhydrous magnesium sulfate. The mixture is filtered and
concentrated in vacuo to yield
(D)-.alpha.-bromo-.alpha.-(4-tetrahydropyranyl)-acetic acid as a
solid.
(p)
N-[2-[(S)-2-[(1-(1,2,4)-triazolyl)acetylthio]-3-methylbutanoylamino]-2-
-methylpropionyl]-O-benzyl-L-serine ethyl ester; m.p.
106-107.degree.; [.alpha.].sub.D-61.46.degree. (c=1.09,
CH.sub.3OH)
[0191] ##STR29##
(q)
N-[2-[(S)-2-[(4-methylpiperazino)acetylthio]-3-methylbutanoylamino]-2--
methylpropionyl]-O-benzyl-L-serine ethyl ester; m.p. 95-96.degree.;
[.alpha.].sub.D-48.5.degree. (c=0.935, CH.sub.3OH)
[0192] ##STR30##
(r)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-ethylbutanoyl]-O-benzy-
l-L-serine ethyl ester; [.alpha.].sub.D-83.6.degree. (c=1.07,
CH.sub.3OH).
[0193] ##STR31##
(s)
N-[2-[(S)-2-(morpholinoacetylthio)-3,3-dimethylbutanoylamino]-2-methyl-
propionyl]-O-benzyl-L-serine ethyl ester;
[.alpha.].sub.D-55.5.degree. (c 1.008, DMSO)
[0194] ##STR32##
(t)
N-[2-[(S)-2-[(methoxyacetyl)thio]-3,3-dimethylbutanoylamino]-2-methylp-
ropionyl]-O-benzyl-L-serine ethyl ester;
[.alpha.].sub.D-61.67.degree. (c=1.024, DMSO)
[0195] ##STR33##
(u)
N-[2-[(S)-2-(acetylthio)pentanoylamino]-2-methylpropionyl]-O-benzyl-L--
serine ethyl ester
[0196] ##STR34##
(v)
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(4--
biphenylylmethyl)-L-serine ethyl ester
[0197] ##STR35##
EXAMPLE 2
N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-S-benzyl--
L-cysteine ethyl ester
[0198] ##STR36## The title compound is prepared similarly to
Example 1 and re-crystallized from methyl t-butyl ether/hexane,
m.p. 69.71.degree. C.
[0199] The starting material is prepared as follows:
[0200] HCl (g) is bubbled into a solution of
BOC-S-benzyl-L-cysteine (9.33 g, 30 mmol) in ethanol (200 mL) for
15 minutes. The container is stoppered and stirred at room
temperature overnight. The solvent is evaporated in vacuo and the
residue stirred in diethyl ether (150 mL) for 1.5 hours to yield
S-benzyl-L-cysteine ethyl ester hydrochloride as a solid.
[0201] A mixture of S-benzyl-L-cysteine ethyl ester hydrochloride
(7.98 g, 29 mmol), BOC-.alpha.-methylalanine (5.89 g, 29 mmol),
triethylamine (2.93 g, 29 mmol), 1-hydroxybenzotriazole (HOBT, 3.92
g, 29 mmol) and EDCl (5.57 g, 29 mmol) in methylene chloride (200
mL) is stirred under an argon atmosphere at room temperature
overnight. The reaction mixture is evaporated to dryness and the
residue is dissolved in ethyl acetate (200 mL). The solution is
washed with water (50 mL), 1 N HCl (50 mL), water (50 mL), 5%
sodium bicarbonate (50 mL), water (50 mL) and finally brine (25
mL). The solution is then dried over sodium sulfate, filtered and
evaporated to dryness to give
N-[2-(BOC-amino)-2-methylpropionyl]-S-benzyl-L-cysteine ethyl
ester.
EXAMPLE 3
N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-ami-
no-3-(benzylsulfonyl)-propionic acid ethyl ester
[0202] ##STR37##
[0203] The above compound is prepared similarly to Example 1.
[0204] The starting material is prepared as follows:
[0205] To a solution of
N-[2-(BOC-amino)-2-methylpropionyl]-S-benzyl-L-cysteine ethyl ester
(7.21 g, 17 mmol) in methylene chloride (250 mL) under an argon
atmosphere is added m-chloro-perbenzoic acid (8.77 g, 51 mmol) and
the mixture is stirred overnight at room temperature. The mixture
is evaporated to dryness and the residue is dissolved in ethyl
acetate (300 mL). The solution is washed with 5% sodium bicarbonate
(3.times.50 mL), water (50 mL) and brine (25 mL). The solution is
dried over sodium sulfate, filtered and evaporated in vacuo to give
N-[2-(BOC-amino)-2-methylpropionyl]-(S-)-2-amino-3-(benzylsulfonyl)-propi-
onic acid ethyl ester.
EXAMPLE 4
(a)
N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-
-amino-3-(benzoylamino)-propionic acid methyl ester
[0206] ##STR38##
[0207] A mixture of benzoyl chloride (0.085 mL, 0.73 mmol),
N.sup.2-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S-
)-2,3-diaminopropionic acid methyl ester hydrochloride (0.29 g,
0.73 mmol) and triethylamine (0.15 mL, 1.49 mmol) in methylene
chloride (10 mL) is stirred at room temperature for 16 hours. The
reaction mixture is evaporated to dryness in vacuo, the residue is
dissolved in ethyl acetate, and the solution is washed with water,
then with saturated sodium bicarbonate solution and brine, dried
over anhydrous magnesium sulfate, and evaporated to dryness to give
an oil. The oil is chromatographed on silica gel with hexane, ethyl
acetate (50:50) to yield the title compound as a white foam; m.p.
48-54.degree. C.
(b) Similarly prepared is
N.sup.2-[2[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-
-2-amino-3-(benzenesulfonamido)propionic acid methyl ester; m.p.
47-51.degree. C.; [.alpha.].sub.D.sup.20-41.72 (c=1.03,
CH.sub.3OH).
[0208] The starting material is prepared as follows:
[0209] A mixture of (S)-2-amino-3-(BbC-amino)-propionic acid methyl
ester hydrochloride (4.6 g, 2.1 mmol), N-CBZ-a-methylalanine (5.0
g, 2.1 mmol), HOAT (2.87 g, 2.1 mmol), EDCl (4.02 g, 2.1 mmol) and
triethylamine (2.93 g, 2.1 mmol) in methylene chloride (50 mL) is
stirred at room temperature for 16 hours. The reaction mixture is
washed with brine, dried over anhydrous magnesium sulfate and
concentrated in vacuo. The resulting oil is chromatographed on
silica gel with hexane and ethylacetate (1:1) to yield
N.sup.2-[2-(CBZ-amino)-2-methylpropionyl]-(S)-2-amino-3-(BOC-amino)-
-propionic acid methyl ester as a white foam; m.p. 100-101.degree.
C.
[0210] A mixture of the above product (2.14 g, 4.90 mmol) and 10%
palladium on charcoal (0.27 g) in ethanol (50 mL) is hydrogenated
under 45 psi pressure in a Parr bottle for 4 hours. The mixture is
filtered through a pad of Celite and concentrated in vacuo to give
N.sup.2-[2-amino-2-methylpropionyl]-(S)-2-amino-3-(BOC-amino)-propionic
acid methyl ester hydrochloride as an oil.
[0211] A solution of the above product (2.28 g, 8.09 mmol),
(R)-2-bromo-3-methylbutanoic acid diisopropyl amine salt (2.16 g,
7.13 mmol), EDCl (1.43 g, 7.49 mmol) and HOAT (1.15 g, 8.52 mmol)
in methylene chloride (75 mL) is stirred at room temperature for 16
hours. The reaction mixture is evaporated to dryness in vacuo and
the residue taken up in ethyl acetate. The ethyl acetate solution
is washed with water, saturated sodium bicarbonate solution and
brine, and then dried over anhydrous magnesium sulfate, and
concentrated in vacuo. The resulting oil is chromatographed on
silica gel with hexane and ethyl acetate (40:60) to give
N.sup.2-[2-[(R)-2-bromo-3-methylbutanoylamino]-2-methylpropionyl]-(S-
)-2-amino-3-(BOC-amino)-propionic acid methyl ester as a white
foam.
[0212] A mixture of the above product (1.31 g, 2.82 mmol) and
potassium thioacetate (1.28 g, 11.2 mmol) in tetrahydrofuran (50
mL) is stirred at room temperature for 4 hours and diluted with
ethyl acetate. The mixture is washed with water, saturated sodium
bicarbonate solution, brine and then dried over magnesium sulfate.
The reaction mixture is concentrated to dryness in vacuo and the
resulting oil is chromatographed on silica gel with hexane and
ethyl acetate (40:60) to give
N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-am-
ino-3-(BOC-amino)-propionic acid methyl ester.
[0213] Hydrogen chloride gas is bubbled through a solution of the
above compound (1.01 g, 2.19 mmol) in 50 mL of methylene chloride
for about 5 minutes, the mixture is stirred at room temperature for
3 hours, and then concentrated in vacub to yield
N.sup.2-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S-
)-2,3-diaminopropionic acid methyl ester hydrochloride.
EXAMPLE 5
[0214] To a solution of
1-[(R)-2-bromo-3-methylbutanoylamino]cyclopentanecarboxylic acid (1
g, 3.42 mmol), O-benzyl-L-serine ethyl ester hydrochloride (0.89 g,
3.42 mmol), dicyclohexylcarbodiimide (0.7 g, 3.42 mmol) and
1-hydroxy-7-azabenzotriazole (0.47 g, 3.42 mmol) in methylene
chloride is added triethylamine (0.48 mL, 3.42 mmol). The mixture
is stirred for 24 hours and then washed with brine and concentrated
in vacuo to give a light yellow oil. The residue is purified by
flash chromatography (silica gel hexane/ethyl acetate) to give
N-[1-(R)-2-bromo-3-methylbutanoylamino]-cyclopentanecarbonyl]-O-benzyl-L--
serine ethyl ester of the formula ##STR39##
[0215] The bromo compound (0.7 g, 1.41 mmol) is dissolved in
tetrahydrofuran (50 mL) and potassium thioacetate (0.19 g, 1.69
mmol) is added. The mixture is stirred at room temperature for 18
hours and then diluted with ethyl acetate and washed with brine,
dried over magnesium sulfate and concentrated in vacuo to give
yellow oil. The crude material is purified by flash chromatography
(silica gel, hexane/ethyl acetate) to give a semi-solid which is
triturated with hexane to yield
N-[1-[(S)-2-acetylthio-3-methylbutanoylamino]-cyclopentanecarbonyl]-O-ben-
zyl-L-serine ethyl ester of the formula ##STR40##
[0216] The
1-[(R)-2-bromo-3-methylbutanoylamino]cyclopentanecarboxylic acid
starting material is prepared essentially by methodology described
in WO 99/55726 by condensation of (R)-2-bromo-3-methylbutanoic acid
diisopropylamine salt (prepared from L-valine) with cycloleucine
methyl ester hydrochloride.
EXAMPLE 6
(a)
N-[2-[(S)-2-Mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-benzy-
l-L-serine
[0217] ##STR41##
[0218] To a solution of the S-acetyl ethyl ester of Example 1 (0.47
g, 1 mmol) in methanol (10 mL) is added 1 N sodium hydroxide (5.0
mL, 5 mmol). The mixture is stirred at room temperature for 4
hours, acidified to pH 1 with 1 N HCl and then concentrated in
vacuo. To the residue is added ethyl acetate. The mixture is washed
with 1 N NaOH. The combined aqueous phase is then acidified and
extracted with ethyl acetate. The organic phase is washed with
brine, dried over sodium sulfate and then concentrated in vacuo.
Trituration with hexane yields a white foam; m.p. 57-70.degree. C.;
[.alpha.].sub.D.sup.20-16.8.degree. (c=1.032, DMSO); MS (M+H):
397.
[0219] Similarly prepared are the following:
(b)
N-[1-[(S)-2-mercapto-3-methylbutanoylamino]-cyclopentanecarbonyl]-O-be-
nzyl-L-serine; m.p. 132-136.degree. C. (crystallized from
hexane/t-butylmethyl ether)
[0220] ##STR42##
(c)
N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-S-benzy-
l-L-cysteine; m.p. 81-87.degree. C.; [.alpha.].sub.D.sup.20-37.87
(c=0.545, DMSO)
[0221] ##STR43##
(d)
N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-benzy-
l-L-threonine; m.p. 61-64.degree. C.
[0222] ##STR44##
(e)
N-[2-[(S)-2-mercapto-3,3-dimethylbutanoylamino]-2-methylpropionyl]-O-b-
enzyl-L-serine; m.p. 128-130.degree. C.; [.alpha.].sub.D-2.46
(c=1.06, DMSO)
[0223] ##STR45##
(f)
N-[2-[(S)-2-mercapto-3-methibutanoylamino]-2-methylpropionyl]-O-(4-flu-
orobenzyl)-L-serine; m.p. 50-54.degree. C.
[0224] ##STR46##
(g)
N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-(4-fl-
uorophenyl)-L-homoserine; m.p. 127-128.degree. C.
[0225] ##STR47##
(h)
N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-(3-fl-
uorophenyl)-L-homoserine; m.p. 5.degree.-56.degree. C.
[0226] ##STR48##
(i)
N-[2-[(S)-2-mercapto-3-methoxybutanoylamino]-2-methylpropionyl]-O-benz-
yl-L-serine; [.alpha.].sub.D.sup.20+18.85 (c=0.997, DMSO)
[0227] ##STR49##
(j)
N-[2-[(S)-2-mercapto-2-(4-tetrahydropyranyl)acetylamino]-2-methylpropi-
onyl]-O-benzyl-L-serine; m.p. 184-189.degree. C.;
[.alpha.].sub.D.sup.20-24.94 (c=1.013, DMSO)
[0228] ##STR50##
EXAMPLE 7
(a)
N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-S-benzy-
l-L-cysteine ethyl ester
[0229] ##STR51##
[0230] Under an argon atmosphere, the thioacetyl compound of
Example 2 (0.48 g, 1.0 mmol) is dissolved in absolute EtOH (5 mL)
and treated with 1 N NaOH of (1.0 mL, 1.0 mmol). The mixture is
stirred for 4 hours at room temperature before treatment with 1 N
HCl until pH 3. The mixture is evaporated to remove most of the
EtOH and the aqueous residue is extracted with EtOAc (2.times.10
mL). The combined extracts are washed with H.sub.2O (5 mL) and then
with brine solution (5 mL). The solution is dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The product
solidifies from tert-butyl methyl ether/hexane to give product;
m.p. 87-91.degree. C.
(b) Similarly prepared is
N-[2-[(S)-2-mercapto-2-(4-tetrahydropyranyl)acetylamino]-2-methylpropiony-
l]-O-benzyl-L-serine ethyl ester; m.p. 85-93.degree. C.;
[.alpha.].sub.D-37:21.degree. (c=1.012, DMSO)
[0231] ##STR52##
(c) Similarly Prepared is
N-[2-[(S)-2-mercapto-3,3-dimethylbutanoylamino]-2-methylpropionyl]-O-benz-
yl-1-serine ethyl ester; oil; [.alpha.].sub.D-20.90 (c=1.025,
DMSO)
[0232] ##STR53##
(d) Similarly Prepared is
N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-ethylbutanoyl]-O-benzyl-1-s-
erine ethyl ester; [.alpha.].sub.D-31.48.degree. (c=0.955,
CH.sub.3OH)
[0233] ##STR54##
* * * * *