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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/08—Vasodilators for multiple indications
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to novel polypeptides. These compounds are useful for treatment of hypertension, congestive heart failure, cardiac hypertrophy including left ventricular hypertrophy, peripheral vascular complications, diabetic complications including renal disease, and other degenerative disorders mediated by angiotensin II.
• chymases a subfamily of serine proteases, including human heart chymase, human mast cell chymase, and human skin chymase (which if not all the same, are very closely related enzymes), have been found to cleave the naturally occurring decapeptide known as angiotensin I to the octapeptide known as angiotensin II, without substantially degrading angiotensin II. These enzymes are termed herein angiotensin I chymases.
• Angiotensin II is known to be a potent pressor substance, i.e. a substance that is capable of inducing a significant increase in blood pressure and is believed to act by causing the constriction of blood vessels and the release of the sodium-retaining hormone aldosterone from the adrenal gland.
• Angiotensin II is also produced from the decapeptide angiotensin I by the action of angiotensin converting enzyme (ACE).
• ACE angiotensin converting enzyme
• ACE pathway is the target of a number of therapeutically useful antihypertensive agents.
• angiotensin I chymases including human heart chymase, provide an ACE-independent pathway for formation of angiotensin II, and are thus implicated as a causative factor in certain forms of hypertension and congestive heart failure.
• Angiotensin II is also implicated as a causative agent in other diseases or risk factors associated with hypertension, such as cardiac hypertrophy (enlargement of the heart), myocardial infarction, vascular hypertrophy (proliferation of vascular smooth muscle cells), diabetic and nondiabetic renal disease (caused in part by hypertension in the kidney), and restenosis or vascular damage in blood vessels of patients suffering from aetheroma treated with angioplasty techniques or thrombolytics.
• Angiotensin I chymases including human heart chymase
• Angiotensin II is the administration of a substance capable of Inhibiting the angiotensin I cleaving action of chymase, including human heart chymase.
• n is one or two
• R 1 is phenyl, naphthyl, (C 3 -C 7 )cycloalkyl, unsaturated heterocycle, or benzo- fused unsaturated heterocycle; wherein said unsaturated heterocycle is selected from pyrrolyl, pyrrolinyl, furyl, dihydrofuryl, thienyl, dihydrothienyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolinyl, imidazolyl, imidazolinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, 1 ,3,5-oxadiazolyI, 1 ,2,4- oxadiazolyl, 1 ,3,5-thiadiazolyl, 1 ,2,4-thiadiazolyl, pyridyl
• R 3 is (C 1 -C 5 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C C 5 )alkyl, (C,- C 5 )alkoxy(C 1 -C 2 )alkyl, (C.-C 5 )alkylthio(C 1 -C 2 )alkyl, phenyl, unsaturated heterocycle, phenyl(C.-C 2 )alkyl, or unsaturated heterocycle(C.-C 2 )aIkyl; wherein said unsaturated heterocycle is as defined for R 1 ; wherein said unsaturated heterocycle(C.-C 2 )alkyl is an unsaturated heterocycle moiety as defined in R ⁇ wherein any one of the carbon atoms of said unsaturated heterocycle moiety is substituted with (C C 2 )alkyl; wherein said (C-- C 5 )alkyl, (C 8 -C
• R 7 C-N-(C 2 -C 4 )alkyl, unsaturated heterocycle(C 2 -C 4 )alkyl, amino(C 2 -C 4 )alkyl,
• R 7 is azetidino, pyrrolidino, piperidino, piperazino, 4-(N)-(C 1 -C 5 )alkylpiperazino, thiomorpholino, oxothiomorpholino, dioxothiomorpholino or morpholino;
• A is carbonyl or sulfonyl
• D is NH, N(C C 5 )alkyl, CH 2 , oxygen, CH(OH), or CH-0-(C,-C 5 )alkyl;
• X is proline, 2-piperidinecarboxylic acid or 2-azetidinecarboxylic acid, wherein said proline, 2-piperidinecarboxylic acid and 2-azetidinecarboxylic acid may optionally be substituted with one or two substituents, said substituents being independently selected from bromine, chlorine, fluorine, (C,-C 5 )alkyl, (C,-C 3 )alkoxy, oxo, and hydroxy;
• Y is BF 2 , B(OM) 2 , -C-Z or -C(OH) 2 Z, wherein M is hydrogen, or (C.-C 5 )alkyl, wherein the two M substituents may optionally form, together with the boron atom and the two oxygen atoms to which they are attached, a saturated heterocyclic ring containing the boron atom, 2 oxygen atoms and 2 or 3 carbon atoms, and wherein any of the carbon atoms of said heterocyclic ring may optionally be substituted with one or two (C,-C 5 )alkyl groups; O O O O
• Z is selected from CF 2 R , CF 2 C-N-R 12 , -C-N-R 12 , -C-O-R 12 or a
• R 13 R 13 heterocycle selected from 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-thienyl, 2-furyl, 2- pyrrolyl, 5-tetrazolyl, 2-benzothiazolyl, 2-benzoxazolyl, 2-benzimidazolyl, 2-benzofuryl, 2-benzothienyl and 2-indolyl; wherein said heterocycle may optionally be substituted with one to three substituents, said substituents being independently selected from (C,- C 3 )alkoxy, bromo,
• R 11 is hydrogen, fluorine, (C T -CgJalkyl, (C--C ⁇ )perfluoroalkyl, amino (C--C ⁇ )alkyl, (C.-C 5 )a!kylamino(C 1 -C 5 )alkyl, di(C 1 -C 5 )alkylamino(C 1 -C 5 )alkyl, (C 1 -C 5 )alkoxy(C 1 -C 5 )- alkyl or hydroxy(C 1 -C 5 )alkyl;
• R 12 and R 13 are independently selected from hydrogen, (C ⁇ C g Jalkyl, (C 3 - C 5 )alkenyl, and R 7 (C 2 -C 4 )alkyl, wherein R 7 is defined as above; with the proviso that (a) no carbon alpha to a ring nitrogen in the substituent R 4 may be directly bonded to a halogen, oxygen or nitrogen substituent, (b) when X is substituted proline, 2-piperidinecarboxylic acid or 2-azetidinecarboxylic acid, then no fluorine, oxo, (C--C 3 )alkoxy or hydroxy substituent is present on either of the ring carbon atoms adjacent to the nitrogen atom of said proline, 2-piperidinecarboxylic acid or 2-azetidinecarboxylic acid, and (c) the compound of formula I can not be defined as a compound wherein n is one, R 1 is phenyl, R 3 is phenyl(C,-C 2
• Preferred compounds of formula I are those wherein: n is 1 ;
• R 1 is phenyl or (C 3 -C 7 )cycloalkyl, wherein said phenyl or (C 3 -C 7 )cycloalkyl may optionally be substituted as defined in R 1 of formula I above;
• R 3 is (C-Cgjalkyl, hydroxy(C C 5 )aIkyl, (C.-C 5 )alkoxy(C C 2 )alkyl, (C,- C g JalkylthiofC.-C ⁇ alkyl, phenylmethyl, 4-imidazolylmethyl or4-thiazolylmethyl; wherein any of the carbon atoms of said (C--C 5 )alkyl may optionally be substituted with one or more fluorine atoms; and wherein from one to three carbon atoms of the phenyl moiety of said phenylmethyl may optionally be substituted with any of the functionalities set forth in the definition of R 1 of formula I for the substituents on said phenyl;
• R 4 is selected from the functionalities listed in groups (a)-(d) below: a) piperidino, pyrrolidino, 3-azabicyclo[3.1.0]hex-3-yl and azetidino; wherein the nitrogen in the 4-position of said piperazino may optionally be substituted with any of the functionalities set forth in the definition of R 4 (a) of formula I for the substituents on the nitrogen in the 4-position of piperazino; wherein any of the ring carbon atoms of said piperazino, piperidino, pyrrolidino, 3-azabicyclo[3.1.0]hex-3-yl and azetidino may optionally be substituted with one or two substituents, said substituents being independently selected from (C--C 5 )alkyl, amino (C 1 -C 3 )alkyl, (C 1 -C 5 )alkyiamino(C 1 - C 3 )alkyI,(C,-C 5 )
• R 7 C is azetidino, pyrrolidino, piperidino, piperazino, 4-N-(C.-C 5 )alkylpiperazino, thiomorpholino, oxothiomorpholino, dioxothiomorpholino or morpholino;
• A is carbonyl or sulfonyl;
• D is NH, CH 2 or oxygen;
• X is proline; O
• Y is -C-Z or -C(OH) 2 Z;
• Z is CF 3' CO 2 R 12 , -C-N-R 12 -CF 2 CN-R 12 ,
• R 13 R 13 or a heterocycle selected from 2-oxazolyl, 2-benzoxazolyl, 2-thiazolyl, 2-benzothiazoly!, 2-furyl, 2-benzofuryl, 2-thienyl and 2-benzothienyl; wherein said 2-oxazolyl, 2- benzoxazolyl, 2-thiazolyl, 2-benzothiazolyl, 2-furyl, 2-benzofuryl, 2-thienyl and 2- benzothienyl may optionally be substituted with from one to three substituents independently selected from (C,-C 3 )alkoxy, bromo,
• R 1 ? and R 13 are independently selected from hydrogen, (CvCgJalkyl, (C 3 -
• R 1 is cyclohexyl or phenyl
• R 3 is (C--C 5 )alkyl, phenylmethyl, 4-imidazolylmethyl, or 4-thiazolylmethyl
• R 4 is piperazino, 4-N-(C 1 -C 5 )alkylpiperazino, morpholino, piperidino, 3- azabicyclo[3.1.0]hex-3-yl, 2-(C 1 -C 5 )dialkylamino(C l -C 3 )alkylmo ⁇ holino, or (C 3 - C 7 )cycloalkyl
• said piperidino and 3-azabicyclo[3.1.0]hex-3-yl may optionally be substituted with one or two substituents, said substituents being independently selected from (C--C 5 )alkyl, amino(C 1 -C 3 )alkyl, (C 1 -C 5 )al
• Y is -C-Z or -C(OH) 2 Z;
• Z is CF 3 , CO 2 R 12 , -C-N-R 12 , -CF 2 CN-R 12 ,
• R 12 and R 13 are independently selected from hydrogen, (C,-C 5 )alkyl, (C 3 -
• Y is , n is 1 , R 3 is isopropyl and R 1 is phenyl; or
• R 4 is 4-(methylamino)piperidin-1-yl, A is carbonyl, D is NH, R 3 is isopropyl, X is proline, Y is -BF 2 , n is 1 and R 1 is phenyl;
• the present invention also includes a method of treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising administering to a mammal, preferably a human, in need of such treatment a chymase inhibiting effective amount of a chymase inhibiting compound , or pharmaceutically acceptable salt thereof.
• a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma)
• a chymase inhibiting effective amount of a chymase inhibiting compound or pharmaceutically acceptable salt thereof.
• the present invention also includes a pharmaceutical composition for treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising a chymase inhibiting effective amount of a chymase inhibiting compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising a chymase inhibiting effective amount of a chymase inhibiting compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the present invention also includes a method for treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease which comprises administering to a mammal, preferably a human, in need of such treatment an amount of a compound of the formula I or a pharmaceutically acceptable salt thereof, that is effective in treating or preventing such disease.
• a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease
• a mammal preferably a human
• the present invention also includes a pharmaceutical composition for treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising an amount of a compound of the formula I that is effective in treating or preventing such disease, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising an amount of a compound of the formula I that is effective in treating or preventing such disease, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the present invention also includes a method for treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease which comprises administering to a mammal, preferably a human, in need of such treatment a chymase inhibiting amount of a compound of the formula I or a pharmaceutically acceptable salt thereof.
• a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma)
• a chymase inhibiting amount of a compound of the formula I or a pharmaceutically acceptable salt thereof comprising to a mammal, preferably a human, in need of such treatment a chymase inhibiting amount
• the present invention also includes a pharmaceutical composition for treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising a chymase inhibiting amount of a compound of the formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• a pharmaceutical composition for treating or preventing a disease selected from hypertension, cardiac and left ventricular hypertrophy, coronary artery disease (including myocardial infarction, vascular hypertrophy, and vascular damage such as restenosis following angioplasty or aetheroma), diabetic renal disease, and non-diabetic renal disease comprising a chymase inhibiting amount of a compound of the formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the pharmaceutically acceptable salts of the present invention are those which are non-toxic at the dosages administered. Since compounds of the invention may contain acidic or basic groups, acid or base addition salts are possible.
• Pharmaceutically acceptable acid addition salts include, for example, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, maleate, mesylate, fumarate, citrate, acid citrate, tartrate, bitartrate, succinate, gluconate and saccharate salts.
• Pharmaceutically acceptable base addition salts are for example sodium, potassium, calcium and magnesium salts.
• alkyl may be straight or branched.
• di(C 1 -C 5 )alkylamino ordi(C--C 7 )alkylamino refer to two alkyl groups independently selected from ( ⁇ -C ⁇ alky! or (C ⁇ C ⁇ alkyl.
• Scheme I refers to the synthesis of compounds of formula I wherein Y is -C-Z. These compounds are prepared from compounds of formula VIII.
• the basic sub-unit of the preferred chemical synthesis is the coupling or acylation of the unprotected amino group of the amine residue of a compound of formula VIII with an amino acid (e.g., proline) having an activated (for acylation purposes) carboxylic function and a suitable protecting group bonded to its own alpha-nitrogen to form a peptide bond between the two amino acid residues, followed by the removal of said protecting group.
• an amino acid e.g., proline
• Such a coupling reaction is generally conducted at a temperature of about -30 to about 80°C, preferably about 0 to about 25°C.
• suitable coupling reagents which activate the carboxylic functionality of the amino acid are dicyclohexylcarbodiimide/hydroxybenzotriazole (HBT), N-3-dimethylaminopropyl-N'- ethylcarbodiimide/HBT, 2-ethoxy-1-ethoxycarbonyl-1 ,2-dihydroquinoline (EEDQ), carbonyl diimidazole (CDI)/HBT, and diethylphosphorylcyanide.
• HBT dicyclohexylcarbodiimide/hydroxybenzotriazole
• EEDQ 2-ethoxy-1-ethoxycarbonyl-1 ,2-dihydroquinoline
• CDI carbonyl diimidazole
• diethylphosphorylcyanide diethylphosphorylcyanide
• the coupling is conducted in an inert solvent, preferably an aprotic solvent.
• suitable solvents include acetonitrile, dichloromethane, chloroform, and dimethylformamide.
• the preferred solvent is dichloromethane.
• the protecting group, of the compound of the formula VII is carbobenzyloxy
• the latter may be removed by hydrogenation with a noble metal catalyst such as palladium or palladium hydroxide on carbon in the presence of hydrogen.
• the hydrogenation is generally conducted at a temperature of about 0 to about 100°C, preferably about 20 to about 50° C.
• the protecting group, R" is t-butoxycarbonyl group, such group may be removed by acidolysis.
• Acidoiysis may be conducted with HCi in dioxane or with neat trifiuoracetic acid at a temperature of about -30 to about 70°C, preferably about - 5 to about 35 °C.
• R a is 9-fluorenylmethylenoxycarbonyl
• such group may be removed by treatment with an amine base, preferably piperidine. This reaction may be run in piperidine as solvent at 10°C to about 100°C, preferably at 25°C.
• the compounds of formula VI are oxidized to compounds of formula I by methods commonly known to those skilled in the art.
• oxidation reactions are the Swern oxidation and variants thereof, chromium based oxidations (preferably pyridinium dichromate), Pfitzner-Moffatt and modified variants thereof, and the Dess- Martin periodinane oxidation.
• a preferred method, to form the compounds of formula I, wherein Y is not COCF 3 uses a Swern oxidation involving treatment of the alcohol of formula VI with oxalyl chloride, dimethyl sulfoxide and triethylamine in methylene chloride. In some cases, up to 10-20 equivalents of oxidizing agent is preferred.
• the more preferred oxidant is the Dess- Martin periodane.
• a dry solution of the peptide in a non polar solvent such as dichloromethane is added to a dry solution of the peptide in a non polar solvent such as dichloromethane and the mixture is stirred for a period of one to twenty-four hours. This procedure is described below as General Procedure C.
• Suitable protecting groups for this purpose are those suitable protecting groups commonly used for amino group protection in peptide synthesis (such as N-tert-butoxycarbonyl, N-carbo-benzyloxy, and 9-fluorenylmethylenoxy- carbonyl) which are also not chemically reactive under the coupling, protection and deprotection, or oxidation conditions described or referred to for the synthesis of compounds herein (for example, the oxidative conditions employed to convert the thus- protected intermediate of formula VI to the thus protected intermediate of formula I).
• the thus-protected intermediate of formula I is deprotected to the compound of formula I wherein the protecting group has been replaced by hydrogen. Methods are commonly known to those skilled in the art and are described as above.
• Examples 19 and 20 contained below illustrate the synthesis of such a secondary amine-containing compound of formula I wherein the intermediates of formula V and VI are employed having a methylamino functionality protected with the N-tert-butoxycarbonyl protecting group which is removed by treatment with HCI-dioxane to give a compound of formula I.
• All compounds of formula I containing a tertiary amino functionality, in R 4 , R 3 , or R- including such compounds wherein Y is COF 3 can also be prepared by reductive amination of a compound of formula I having secondary amine functionality at the corresponding position of R 4 , R 3 , or R, with an appropriate carbonyl compound.
• a suitable procedure for this reductive alkylation is the addition of the appropriate carbonyl compound (1 to 100 equivalents) to a mixture of the compound of formula I having a secondary amine functionality, sodium cyanoborohydride (1.4-2 equivalents), and powdered 3 angstrom (A) molecular seives in absolute methanol at 0-50° C, preferably at 20-25°C.
• amino, alkylamino, and dialkylamino substituted compounds of the formulae VI, V and C-terminally protected compounds of formulae V, XVI and XI including the properly protected variants thereof as described herein may be synthesized from the corresponding oxo- substituted compounds, by reductive amination of the latter with a salt of ammonia (e.g., NH 4 CI), an alkylamine, or a dialkylamine according to methods familiar to those skilled in the art, such as is illustrated for the conversion of an oxo-substituted compound of formula V to the corresponding methylamino-substituted compound of formula V by reductive amination with methylamine in Example 19a, and conversion of the latter to a suitably protected R 4 amine-containing compound of formula V in Example 19b.
• a salt of ammonia e.g., NH 4 CI
• an alkylamine e.g., NH 4 CI
• this reductive amination/protection method can be used not only for the synthesis of C-terminally protected compounds of formula V, but also for the synthesis of amine-containing compounds of formula VI, C-terminally unprotected compounds of formulae V, XI, and XVI and the properly N-protected variants thereof wherein the primary or secondary amine functionality is protected with one of the groups commonly employed for amine protection.
• Conditions for the introduction and removal of such groups are summarized by Greene in "Protecting Groups in Organic Synthesis", Wiley, NY, 1981.
• compounds of formula VI may be synthesized as shown in Scheme 2, wherein a compound of formula XI is first coupled by standard peptide coupling methods referred to above to a C-terminally protected ⁇ -amino acid X such as, for example, proline benzyl ester to give a C-terminally protected compound of formula V.
• Suitable protecting groups are those commonly used for carboxyl group protection in peptide synthesis. ⁇ Examples of such groups are benzyl ester and t-butyl ester groups.
• the C-terminally protected compound of the formula V is deprotected using conventional methods to provide the C-terminally unprotected compound of formula V. For example:
• the carboxyl group of the compound of the formula V is protected as a benzyl ester, the latter may be removed by hydrogenation with a noble metal catalyst such as palladium on carbon in the presence of hydrogen.
• the hydrogenation is generally conducted at a temperature of about 0 to about 100°C, preferably about 20 to about 50°C.
• Acidolysis may be conducted with HCl in dioxane or with neat trifluoracetic acid at a temperature of about -30 to about 70 °C, preferably about - 5 to about 35 °C.
• the carboxyl protecting group is an alkyl ester
• the group may be removed by basic hydrolysis.
• Basic hydrolysis may be conducted with a suitable base (e.g., sodium hydroxide) at a temperature of about -30 to about 120°C, preferably about 0 to 80 °C.
• the solvents used for removal of the protecting group should be inert solvents.
• Suitable and preferred solvents are as described for the deprotection in Scheme 1.
• the C-terminally unprotected compounds of the formula V so formed are then coupled with a compound of the formula VIII, from Scheme I by conventional peptide coupling reactions as described above (e.g., Procedure A described below) to yield compounds of the formula VI.
• Scheme 3 refers to the synthesis of compounds of formula VIII wherein Z is CF 3 .
• Compounds of formula VIII can be prepared from compounds of formula II.
• compounds of formula VIII wherein Z is CF 3 can also be prepared by the method of Kolb et. al. (Liebings Ann. Chem. 1990, 1-6) as adapted by Peet e a (J. Med. Chem. 1990, 33, 394-407) which involves a) dehydration of an N-aroyl (e.g. N-benzoyl) amino acid derivative (Aryl)-CONH-CHC0 2 H with acetic anhydride, to
• Aldehydes of formula XII are readily prepared from protected ⁇ -amino esters of formula Xa by reduction with diisobutylaluminum hydride (DIBAH) or from analogous N-methoxymethylamides by reduction with lithium aluminum hydride (LAH).
• Aldehydes of formula XII may be converted to cyanohydrins of formula XIII by treating the aldehyde with a salt of cyanide, preferably potassium or sodium cyanide, in an aqueous solution with a cosolvent such as, tetrahydrofuran, ethyl acetate, or dioxane.
• the cyanohydrin of formula XIII so formed can be converted by alcoholysis to
• amides of formula VIII wherein Z is -CON-R 12 may be
• the compound of formula XIV is then amidated by mixing with an amine of the formula R 12 R 13 NH in a suitable polar, preferably protic, solvent such as ethanol or methanol to give a compound of formula XIV wherein Z is CF 2 CONR 12 R 13 .
• a suitable polar, preferably protic, solvent such as ethanol or methanol.
• the 2-metallo heterocycle is preferably a 2-lithio heterocycle and is obtained by treatment of the parent heterocycle with a suitable organolithium reagent, such as n-butyllithium, methyllithium, sec-butyllithium or tert- butyllithium.
• 2-Metallo heterocycles may be alternatively prepared by transmetallation of a 2-bromo or 2-iodo heterocycle with an organolithium reagent. Conditions for the formation and use of 2-metallo-heterocyclic reagents are dependent on the particular heterocycle. Methods for the formation of 2-metallo heterocycles are familiar to those skilled in the art.
• the metallo heterocycle is formed and allowed to react with the aldehyde within 30 minutes at -78 °C.
• Conditions for the formation and reaction of various 2-substituted heterocycles are described in Organic Reactions (Volume 26). Specific applications of this reaction to the synthesis of compounds of formula XIV wherein Z is 2-thiazolyl or 2-benzothiazoyl are Examples 3a and 13c herein.
• Compounds of formula XIV so formed are then converted to compounds of formula VIII by removing the protecting group by the methods described in Scheme 1 , above.
• Parent heterocycles having a free NH such as imidazole, tetrazole, or indole are N-protected with an appropriate protecting group, such as 1 -ethoxyethyl or trimethylsilylethyl prior to metallation of the heterocycle.
• an appropriate protecting group such as 1 -ethoxyethyl or trimethylsilylethyl prior to metallation of the heterocycle.
• compounds of formula VIII wherein Z is substituted or unsubstituted 2-benzoxazolyl or substituted or unsubstituted 2-oxazolyl may be prepared from a cyanohydrin of formula XIII and an appropriate ortho-aminophenol or aminoethanol derivative, respectively, by the method of Edwards et. al. (J. Am. Chem. Soc. 1992, 114, 1854-1863).
• the synthesis of compounds of formula I wherein Y is B(OM) 2 is accomplished by coupling a compound of formula V with an aminoboronic acid ester of the formula NH 2 CHB(OM 2 ).
• Compounds of the formula I wherein Y is B(OH) 2 are synthesized
• a compound of formula XV may be directly activated with phosgene or trichloromethyl chloroformate to yield an isocyanate (Lombardino and Gerber, J. Med. Chem. 1964, 7, 97-101), N-carbamoylchloride, or chloroformate derivative wherein D is NH, N-alkyl, or O, respectively, and condensed with amine or protected amine of the formula R 4 H to yield a compound of the formula XVI.
• Many compounds of formula XI wherein R 4 , A, D, and R 3 are as described instantly above are known in the literature and have been prepared by one of these methods.
• Compounds of formula XI so formed can be converted to compounds of formula I by the methods of Schemes 1 and 2. .
• Acylations of this type are accomplished via the acid chloride R COCI or by one of the peptide coupling methods described above. Sulfonylation is accomplished in an inert solvent, preferably dichloromethane, in the presence of a tertiary amine base, preferably triethylamine or diisopropylethylamine.
• a tertiary amine base preferably triethylamine or diisopropylethylamine.
• Compounds of formula XI and XVI wherein R 4 A is substituted or unsubstituted alkoxycarbonyl may be prepared by acylation of a compound of the formula XVI with the appropriate chloroformate or carbonate derivatives.
• European Patent 416,393 describes the synthesis of compound of formula XVI wherein R 4 is 4- trifluoroethylpiperazino, 4-methylpiperazino, and 2-(2-pyridyl)ethyl(methyl)amino by this method.
• Compounds of formula XI wherein A is CO and D is CH 2 are also prepared by other methods, such as a) Stobbe condensation of a carbonyl compound with a succinic acid diester to give a 2-dihydrosuccinate 1-monoester, b) coupling of the free carboxyl to an amine R -H, and c) reduction (e.g., catalytic hydrogenation) of the olefin to a compound of formula XI.
• Steps a and b of this method are described by Plattner et al. (J. Med. Chem. 1988, 31 , 2277-2288) for the synthesis of an olefin which upon hydrogenation would give a compound XI wherein R 4 is morpholino, A is CO, D is CH 2 , and R 3 is phenylmethyl.
• Plattner et al. J. Med. Chem. 1988, 31 , 2277-2288
• European Patent 416,393 describes methods for the synthesis of compounds of formula XI and XVI wherein R 4 is unsaturated heterocyclicethyl(methyl)amino, A is carbonyl, D is CH 2 , and R 3 is phenylmethyl, by N-alkylation of a five membered unsaturated heterocycle such as imidazole and pyrazole.
• R 4 is unsaturated heterocyclicethyl(methyl)amino
• A is carbonyl
• D is CH 2
• R 3 is phenylmethyl
• N-alkylation of a five membered unsaturated heterocycle such as imidazole and pyrazole.
• One skilled in the art can use this method to prepare other such compounds of formulae XVI and XI by varying the R 3 -substituted succinate monoester, and by choosing the appropriate ⁇ -hydroxy-(C 2 - C 4 )alkyl(C,-C 5 )alkylamine, and by choosing
• Bromoketones of formula R 4 COCH 2 Br may be prepared, as will be known to one skilled in the art, by many methods such as a) reaction of an activated acid R 4 COOH (such as the acid chloride or mixed anhydride) with diazomethane to give the diazomethyl ketone which is b) treated with anhydrous hydrogen bromide.
• R 4 COOH such as the acid chloride or mixed anhydride
• European Patent 476,515 describes a method for the preparation of compounds of the formulae XVI and XI wherein R 4 is 2-(R 7 CON(CH 3 ))ethyl(methyl)amino, A is carbonyl, D is NH or O, and R 3 is phenylmethyl, wherein R 7 is thiomorpholino, piperidino , or dialkylamino , by acylation of an appropriate methylaminoethyl(methyl)aminocarbonyl-phenylalanine or phenyllactate derivative with R 7 COCI.
• This method may be used by one skilled in the art for the synthesis of other compounds of the formula XVI and XI wherein the precursor containing R 3 , the monoprotected (C 1 -C 5 )alkylamino(C 2 -C 4 )alkyl(C 1 -C 5 )alkylamine, and the R 7 COCI reagent are appropriately chosen.
• the pressures of the foregoing reactions are not critical. Generally, the reaction pressures will be about 0.5 to about 2 atmospheres, preferably ambient pressure ( , generally at about one atmosphere).
• the activity of the active compounds of the present invention as inhibitors of the angiotensin i-cleaving activity of angiotensin I chymase(s) may be determined by studying their ability to inhibit the angiotensin l-cleaving activity of an angiotensin I chymase isolated and semipurified from the heart of the marmoset. Thus, left ventricles were removed from necropsied marmoset monkeys. Tissues were frozen in liquid nitrogen and stored at -70°C.
• the tissue was thawed and homogenized in 10 volumes (wN) of 20 mM Tris-HCl, pH 7.4 with a polytron set at 8. The homogenate was centrifuged at 40,000 Xg for 30 min. The pellet was washed twice by homogenization and centrifugation. The final pellet was suspended in 10 volumes of 20 mM Tris-HCl, pH 7.4 with 1% Triton X-100 and 10 mM KCI using a polytron. The homogenate was incubated at 4°C for 1 hr and centrifuged at 40,000 Xg for 30 min.
• the pellet was homogenized in 20 mM Tris-HCl, pH 8.0 with 1% Triton X-100 and 0.5 M KCI, incubated and centrifuged. The resulting pellet was suspended in 20 mM Tris, HCl, pH 8.0 with 1% Triton X-100 and 2 M KCI, incubated and centrifuged. The supernatant was the source of chymase and was frozen in liquid nitrogen and stored at -70° C. Protein concentration was determined (Bradford, Anal. Biochem. 1976, 72, 248-254). Inhibition of angiotensin I chymases can be determined by an angiotensin-radioreceptor assay.
• angiotensin I is incubated with the chymase in 20 mM Tris-HCl, pH 8.0 with 0.25% Triton X-100 and 0.5 M KCI in a final volume at 100 /I. Samples were incubated at 37° C and a 4°C control was included. The reaction was terminated by the addition of 100 //I of 2 mM phenylmethylsulfonyl chloride (PMSF) in 50 mM Tris-HCl, pH 7.2 with 5 mM MgCI 2 and 0.25% bovine serum albumin and placing on ice.
• PMSF phenylmethylsulfonyl chloride
• the concentration of angiotensin II formed was measured by displacement of radiolabeled angiotensin II from preformed rat liver microsomes saturated with radiolabeled angiotensin II.
• Rat liver microsomes are a known source of angiotensin II receptors. These microsomes were isolated and purified from the livers of sacrificed rats which were removed and homogenized in 10 volumes of 10 mM Tris-HCl, pH 7.4 with 200 mM sucrose and mM ethylenediaminetetraacetic acid (EDTA) using 10 strokes of a teflon pestle in a glass tube. The homogenate was centrifuged at 3000 Xg for 10 min.
• EDTA ethylenediaminetetraacetic acid
• the resulting supernatant was centrifuged at 12,000 Xg for 13 minutes. This supernatant was separated and centrifuged at 104,000 Xg for 1 hour. The resulting pellet was suspended in 50 mM Tris-HCl, pH 7.2 with 5 mM MgCI 2 . The microsomes were assayed for protein (Bradford, ibid) and frozen at -20°C until use.
• Radiolabeled 125 l S late angiotensin II (0.125 nM) was incubated with the rat microsomes (30 /g, 100 ⁇ l) in 50 mM Tris-HCl, pH 7.2 with 5 mM MgCI 2 , 1 mM PMSF and 0.25% BSA for 40 min at ambient temperature at a final volume of 200 ⁇ .
• the reaction was terminated by filtration of the suspension through GF/B filters pretreated with 0.2% PEI and dried.
• the angiotensin II levels were determined from an angiotensin II standard curve.
• the IC 50 of chymase inhibition was defined as the concentration of the inhibitor that inhibited
• the colorimetric assay is a less time intense alternative method for measuring the inhibitory activity of the compounds of this invention against the angiotensin I cleaving action of chymases.
• the experimental sample is prepared by mixing inhibitor (90 ⁇ , in 10% methanol) with enzyme, (90 yl, in 20 mM Tris, pH 8.0, 2 M KCI, 1% Triton X-100 (47 //g/well)) and is pre-incubated at 37 °C for 20 minutes.
• a control sample of the enzyme, (90 ⁇ , in 20 mM Tris, pH 8.0, 2 M KCI, 1% Triton X-100 (47 /g/well)) is separately prepared.
• a solution of a peptidyl para nitroanilide substrate (N-succinoyl-Phe-Val-Pro-Phe-p-Nitroanilide) (180 ⁇ volume of 400 ⁇ M) in 30 mM Tris, pH 8.0 (200 ⁇ M final concentration).
• the final buffer concentration is 20 mM Tris, pH 8.0 with 0.5 M KCI and 0.25% Triton X-100.
• Cleavage of the para nitroanilide moiety by the chymase produces a color change.
• the reaction of experimental and control samples are incubated at 37 °C for 3 hours, the color change is continuously recorded by the increase in absorbance at 410 nanometers (NM).
• the rate reaction is expressed as mOD/minutes.
• the IC50 of the chymase inhibitors was defined as the concentration of the inhibitor that inhibited 50% of the enzyme activity and was determined by increasing concentration of inhibitor.
• the following examples illustrate the invention but are not to be construed as limiting the same. All melting points are uncorrected.
• "boc” refers to t-butoxycarbonyl and "diboc" to di-t-butoxy-carbonyl.
• the active compounds of the present invention can be administered as antihypertensive agents, agents for the treatment of congestive heart failure, cardiac and vascular hypertrophy including left ventricular hypertrophy and diabetic and non ⁇ diabetic renal disease by either the oral or parental routes of administration, with the former being preferred for reasons of patient convenience and comfort.
• these compounds are normally administered orally in dosages ranging from about 0.1 to about 50 mg per kg of body weight per day, preferably about 0.1 to about 20 mg per kg of body weight per day, and about 0.05 mg to about 10 mg per kg of body weight per day, preferably about 0.05 to about 2 mg per kg of body weight per day, when given parenterally; variations will necessarily occur depending upon the condition of the subject being treated and the particular compound being administered.
• treatment is commenced at a low daily dosage and increased by the physician only if necessary.
• these compounds may be administered in combination with pharmaceutically acceptable carriers by either of the routes previously indicated, and that such administration can be carried out in both single and multiple dosages.
• the active compounds of the present invention can be orally administered in a wide variety of different dosage forms, i.e., they may be formulated with various pharmaceutically acceptable inert carrier in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, aqueous suspensions, elixirs, syrups and the like.
• Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc.
• such oral pharmaceutical formulations can be suitably sweetened and/or flavored by means of various agents of the type commonly employed for such purposes.
• the active compounds of the present invention are present in such oral dosage forms at concentration levels ranging from about 0.5% to about 90% by weight of the total composition, in amounts which are sufficient to provide the desired unit dosages.
• tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch (preferably potato or tapioca starch), alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
• disintegrants such as starch (preferably potato or tapioca starch), alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
• lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc and compositions of a similar type may also be employed.
• Lactose or milk sugar as well as high molecular weight polyethylene glycols may be employed as fillers in soft and hard-filled gelatin capsules.
• the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying agents and/or solvents such as water, ethanol, propylene glycol, glycerin or combinations thereof.
• One or more other active compounds may be added to the formulations described above to provide formulations for combination therapy.
• Such compounds include antihypertensives such as diuretics, beta-adrenergic blocking agents, central nervous system-acting agents, adrenergic neuron blocking agents, vasodilators, renin inhibitors, angiotensin II antagonists, and angiotensin I converting enzyme inhibitors.
• a preferred antihypertensive agent for administration together with a compound of the present invention is a diuretic.
• HPLC HPLC was performed with 214 nm detection on a (system A) 150 mm Waters Novapak C18 column eluted at 0.8 ml/min, or (System B) 250 mm Rainin Microsorb C18 column eluted at 1.0 ml/min by a two-pump/mixer system supplying the indicated mixture (v:v) of acetonitrile and aqueous pH 2.1 (H 3 PO 4 ) 0.1 M KH 2 PO 4 respectively.
• concentration and “coevaporated” refer to removal of solvent at water aspirator pressure on a rotary evaporator with a bath temperature of less than 40° C. Organic solutions were dried over magnesium sulfate unless specified otherwise.
• a solution of the primary amine (0.2-0.5 M, 1.0 equivalent) in dichloromethane (or a primary amine hydrochloride and 1.0-1.3 equivalents of triethylamine) is treated sequentially with the carboxylic acid coupling partner (1.0-1.2 equivalents), hydroxybenzotriazole hydrate (HBT) (1.5-1 .8 equivalents), and 1 -(3- dimethylamincpropyl)-3-ethylcarbodiimide hydrochloride (DEC) (1.0-1.2 equivalents, stoichiometrically equivalent to the quantity of carboxylic acid) and the mixture is stirred overnight in an ice bath.
• the carboxylic acid coupling partner 1.0-1.2 equivalents
• HBT hydroxybenzotriazole hydrate
• DEC 1 -(3- dimethylamincpropyl)-3-ethylcarbodiimide hydrochloride
• the ice bath is allowed to warm, thus the reaction mixture is typically held at 0-20 °C for 4-6 hours and 20-25 °C for the remaining period.
• the mixture is diluted with ethyl acetate or other solvent as specified, and the resulting mixture was washed twice with 1 N NaOH, twice with 1 N HCl, once with brine, then dried over magnesium sulfate (MgSO , and concentrated to give the crude product which is purified as specified.
• the carboxylic acid component can be used as the dicyclohexylamine salt in coupling to the primary amine or hydrochloride of the latter; in this case no triethylamine is employed.
• reaction was not complete (TLC)
• additional periodinane was added as specified.
• the mixture was diluted with the specified extraction solvent and water (20-100 mL each/mmol substrate), Na 2 S 2 O 3 »5H 2 O (1.3-3 g/mmol substrate) and NaHCO 3 (2.5-3 g/mmol substrate) were added, and the resulting solution stirred 1 to 2 hours or until both layers clarified.
• the separated organic layer was washed with aqueous NaHCO 3 , brine, and combined with one extract (same solvent) of the separated aqueous layers.
• the combined organic layers were dried (MgSO 4 ), concentrated, and the residue chromatographed on silica eluted with the specified solvent mixture.
• Procedure B A 5°C solution of anhydrous hydrogen chloride in p-dioxane (30 mL of 4M) was added in one portion to the product of the preceding example (3.24 g, 7.97 mmol) and the resulting solution was warmed to 25°C. After 30 minutes, the mixture was concentrated, and the residue dried in vacuo and triturated with ether (3 x 6 mL). The resulting colorless solid was dried in vacuo at 56°C for 1.5 hours (2.82 g, 103%).
• L-proline phenylmethyl ester hydrochloride (8.0 g) was coupled to N 0 N r -bis-[(1 ,1-dimethylethoxy)carbonyl]-L-histidine (11.8 g) and the crude product (11.1 g, oil) was chromatographed on 200 g silica eluted with 2:1 ethyl acetate-hexanes followed by ethyl acetate.
• the compound was prepared in a manner according to the procedure described as General Procedure C, except that only 2 equiv of periodinane was used, and that chloroform, rather than ethyl acetate, was used as the extraction solvent.
• the product of the preceding example (642 mg) was converted to the title substance (530 mg, 83%) which was not further purified. (TLC R, 0.22, ethyl acetate).
• Example 13B (free acid, 304 mg) using General Procedure A, giving 265 mg of crude product which was purified on silica eluting with a gradient (1-16%) of ethanol in dichloromethane. Yield, 194 mg, 43%, TLC Rf 0.73 (9:1 dichloromethane-ethanol). LSIMS 592 (MH + , 40%), 309 (45%).
• Example 1 C The compound was prepared in a manner according to the procedure described as General Procedure A.
• the product of Example 1 C 250 mg was coupled to Boc-L- leucine to yield 346 mg of crude product which was purified on silica eluting with ethyl acetate-hexanes and ethyl acetate to yield the title substance (303 mg, 80%).
• the compound was prepared in a manner according to the procedure described as General Procedure C, with the following exception: 200 mg of the product of the preceding Example was treated with a total of 6 equiv of the periodinane which was added in 2 equiv portions over 7 days, until the reaction was complete by HPLC. Ethyl acetate was employed in the workup and the crude product (164 mg) was chromatographed on silica eluted with a gradient of 0.5-4% ethanol in dichloromethane to yield the title substance (148 mg). (TLC Pt, 0.52, ethyl acetate).
• LSIMS 672 (20%, MH + +matrix), 564 (35%, MH + +C 2 H 5 OH), 518 (50%, MH + ), 462 (35%), 418 (100%).
• Example 1 C The product of Example 1 C (266 mg) was coupled to Boc-3- (4-thiazolyl)-L-aianine) (200 mg) and the crude product (370 mg) purified by chromatography on silica eluted with ethyl acetate-hexanes, ethyl acetate, and ethanol- ethyl acetate to yield the title substance (195 mg, 46%), (TLC R, 0.25, ethyl acetate). LSIMS 561 (MH + , 100%).
• B The product of Example 1 C (266 mg) was coupled to Boc-3- (4-thiazolyl)-L-aianine) (200 mg) and the crude product (370 mg) purified by chromatography on silica eluted with ethyl acetate-hexanes, ethyl acetate, and ethanol- ethyl acetate to yield the title substance (195 mg, 46%), (TLC R, 0.25,
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (190 mg) was oxidized with the periodinane (ethyl acetate used for extraction) to yield 183 mg of crude product which was purified on silica eluted with ethyl acetate-hexanes followed by ethyl acetate, to yield 136 mg (72%) of the title substance (TLC R, 0.35 in ethyl acetate).
• LSIMS 713 (MH + +C 4 H, 0 O 2 S 2 , 40%), 559 (MH + , 90%), 127 (100%).
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (130 mg) was treated with periodinane (4.6 equiv) and the crude product, isolated by chloroform extraction, was purified on silica eluted with ethyl acetate-hexanes followed by ethyl acetate, to yield 86 mg (67%) of the title substance (TLC R, 0.34 in ethyl acetate).
• Example 1 C The compound was prepared in a manner according to the procedure described as General Procedure A.
• the product of Example 1 C (250 mg) was coupled to Boc-L- proline (173 mg) to yield 291 mg of crude product which was purified on silica eluted with ethyl acetate-hexanes followed by ethyl acetate to yield 245 mg (67%) of the title product.
• Example 1 C The compound was prepared in a manner according to the procedure described as General Procedure A.
• the product of Example 1 C 250 mg was coupled to Boc-L- valine (174 mg) to yield 276 mg of crude product which was purified on silica eluted with ethyl acetate-hexanes to yield 189 mg (51 %) of the title product.
• Example 1 C The product was prepared in a manner according to the procedure described as Procedure A.
• the product of Example 1 C 500 mg was coupled to N-(morpholino- 1-carbonyl)-L-phenylalanine (USP 4,814,342, 446 mg) and the crude product (828 mg) was purified by chromatography on silica and eluted with 1%-32% ethanol in dichloromethane to yield the title substance (186 mg, 22%), (TLC Ft, 0.41 in 5% ethanol- dichloromethane).
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (168 mg) was treated with periodinane and the crude product (146 mg) was isolated by extraction using ethyl acetate and was purified on silica eluted with 1-32% ethanol in dichloromethane to yield the title substance (107 mg, 63%).
• N-(4-oxopiperidine-1-carbonyl)-L-phenylalanine benzyl ester L-Phenylalanine benzyl ester p-toluenesulfonate (420 g, 0.982 mol) was added to a stirred mixture of 1 N NaOH (1.5 L) and dichloromethane (0.5 L) at 25° C. After the solid dissolved the organic layer was separated, dried over MgSO 4 , and added over 1 hour to a stirred 0-5°C slurry of imidazole (135 g, 1.96 mol, 2.0 equiv) and carbonyldiimidazole (175 g, 1.08 mol) in dichloromethane (1.6 L).
• the compound was prepared in a manner according to the procedure described as General Procedure A.
• the product of the preceding Example (281 mg) was coupled with the product of Example 1 C (300 mg) to yield crude product (427 mg) which was chromatographed on silica eluted with ethyl acetate and a 1-4% ethanol in ethyl acetate gradient to yield the title compound (254 mg), (TLC R, 0.5 in 18/2/1 CHCI 3 -ethanol- NH 4 OH).
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (246 mg) was treated with periodinane.
• the product (215 mg, 87%), was isolated by extraction using ethyl acetate.
• Example 1C The product of Example 1C (85 mg) and 4-[4-[N-[(1 ,1- dimethylethoxy)carbonyl]methylamino]piperidinyl]-2(R)-(phenylmethyl)-1 ,4-butanedioic acid (100 mg) were coupled to give 160 mg of crude product which was purified on silica eluted with ethyl acetate-hexanes followed by ethyl acetate, to yield 110 mg (64%) of product, (TLC R, 0.85 in 18/2/1 HCCI 3 -ethanol-NH 4 OH). LSIMS 693 (MH + , 100%), 387 (90%).
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (90 mg) was treated with the periodinane, and the crude product (78 mg) isolated by ethyl acetate extraction was purified on silica, eluted with ethyl acetate followed by 2% and 4% ethanol in ethyl acetate to yield the title product (45 mg, 50%), (TLC R, 0.22 in ethyl acetate).
• LSIMS 691 (MH + , 40%), 387 (100%).
• the compound was prepared in a manner according to the procedure described as General Procedure A.
• the product of the preceding example 60 mg was coupled to Boc-L-leucine (38 mg) and the crude product (84 mg) was purified on silica eluted with ethyl acetate-hexane to yield the title substance (49 mg, 58%), TLC (R f 0.32 ethyl acetate).
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example 46 mg was treated with the periodinane, and the crude product (54 mg), isolated by ethyl acetate extraction, was purified on silica eluted with ethyl acetate-hexanes, to yield the title product (28 mg, 62%).
• LSIMS 585 (MH + +H 2 O, 20%), 567 (MH + , 60%), 467 (100%), 354 (30%).
• N-(morpholino-1-carbonyl)-L-phenylalanine (12.6 g) and L-proline benzyl ester hydrochloride (10.0 g) were coupled according to General Procedure A and the crude product purified on silica gel eluted with ethyl acetate-hexanes followed by ethyl acetate to yield 10.0 g (53%) of the title product, (TLC R, 0.26 in ethyl acetate).
• LSIMS 466 (MH + , 28%), 309 (50%), 119 (100%).
• Example 13B DCHA salt
• Example 13B DCHA salt
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (191 mg) was treated with the periodinane, and the crude product (100 mg), isolated by ethyl acetate extraction, was purified on silica eluted with ethyl acetate to yield the title product (30 mg, 16%), (TLC Ft, 0.56 in 18/2/1 CH 2 CI 2 -ethanol-NH 4 OH).
• Example 13B The product of Example 13B (free acid, 404 mg) and the product of the preceding Example (250 mg) were coupled according to General Procedure A, to yield 500 mg of crude product which was purified on silica eluting with ethyl acetate-hexanes. Three fractions were thus obtained (distinguished by TLC): less polar material (135 mg, 24%, TLC Ft, 0.21 in ethyl acetate), more polar material (164 mg, 29%, TLC R, 0.16 in ethyl acetate), and a mixture (119 mg, 21%) of the above. Less polar band: LSIMS 577 (100%, MH + ), 317 (50%), 261 (50%), 233 (40%). More polar band 577 (MH + , 100%), 317 (80%), 261 (80%), 233 (65%). ⁇
• Example 10B The product of Example 10B (30.5 g) was coupled to proline benzyl ester using General Procedure A and the crude product (45.3 g) triturated three times with hexanes to yield 43.4 g of an orange solid. A portion (38.1 g) of this material was dissolved with heating in 260 mL ethyl acetate and 100 mL hexanes was added. The resulting solution on standing 15 hours at 25 °C deposited light beige crystals which were collected by filtration and washed with hexanes to yield 5.44 g of the title substance, (TLC R, 0.16 in ethyl acetate). Anal. Calcd for C 27 H 31 N 3 O 5 : C, 67.91 ; H, 6.54; N, 8.80. Found: C, 67.35; H, 6.37; N, 8.67.
• Example 16D The more polar product of Example 16D was (153 mg) was oxidized according to General Procedure C, and the crude product (90 mg), isolated by chloroform extraction, was purified on silica eluted with ethyl acetate-hexanes, ethyl acetate, and
• LSIMS 633 (MH + +C 2 H 5 OH, 40%), 605 (30%, MH + +H 2 O), 587 (MH + , 40%), 361 (60%), 333 (40%), 315 (60%), 273 (70%), 245 (70%).
• Example 13B The product of Example 13B (363 mg) was coupled to the product of the preceding Example (239 mg) by General Procedure A, and the crude product (514 mg) was purified on silica and sequentially eluted with ethyl acetate-hexanes, ethyl acetate, 1% and 2% ethanol in ethyl acetate to yield 446 mg (79%) of the title substance as a mixture of (presumably four) isomers, (TLC R, 0.61 and 0.59 in 18/2/1 CH 2 CI 2 -ethanol- NH 4 OH).
• LSIMS 583 (MH + , 60%), 323 (50%), 307 (100%).
• the second product to elute from the chromatography of the product of the preceding example was a mixture of the above substance and the more polar 1 (R)
• Example 16A Sodium cyanoborohydride (441 mg, 1.2 equiv) was added to a mixture of the product of Example 16A (2.79 g, 5.84 mmol), methylamine hydrochloride (1.972 g, 29.2 mmol), anhydrous sodium acetate (4.79 g, 58.4 mmol), and 3A molecular seives (600 20 mesh, 2.8 g) in methanol (30 mL) at 0°C and the mixture was stirred at 25 °C for 16 hours. The mixture was filtered through Supercel (trademark) and the filtrate concentrated.
• 3A molecular seives 600 20 mesh, 2.8 g
• Example 14B (300 mg) by General Procedure A and the crude product (765 mg) was purified on silica and sequentially eluted with 2.5%, 5%, 10% and 20% ethanol in 1 :1 dichloromethane-ethyl acetate to yield the less polar, first-titled product (78 mg, 9%), (TLC R, 0.19 in 0.1 :1 :1 ethanol-dichloromethane-ethyl acetate).
• the first-titled, less polar product of the preceding Example was oxidized by General Procedure C to yield crude product (67%), which was isolated by chloroform extraction.
• the crude product was purified on silica and eluted with ethyl ac ⁇ tate-dichloromethane to yield 52 mg of the title substance, (TLC Ft, 0.24 in 0.1 :1 :1 ethanol-dichloromethane-ethyl acetate).
• Example 19D mixture of first and second-titled products of Example 19D
• Example 19D mixture of first and second-titled products of Example 19D
• General Procedure C to yield crude product (191 mg) which was isolated by chloroform extraction and purified on silica, and sequentially eluted with 1%, 2% and 4% ethanol in 1 :1 dichloromethane-ethyl acetate.
• the less polar substance (91 mg) was identical by TLC and NMR to that obtained immediately above and different from the product of Example 20A. A mixture of this substance and a more polar product was also obtained (99 mg).
• Example 20B The product of the preceding Example (78 mg) was dissolved in trifluoroacetic acid (1 mL) at 25°C for 30 minutes. The mixture was concentrated, the residue dissolved in ethanol (1 mL) and the resulting solution treated with 0.20 mL 1 N HCl. The solution was concentrated and the crude product ground to a fine powder, triturated with ether and dried (61 mg, 87%). The product of Example 20B could not be detected in this material by RP-HPLC (30/70), System B.
• Example 19E The second-titled, more polar product of Example 19E (174 mg) was oxidized by General Procedure C to yield crude product (168 mg), isolated by chloroform extraction, which was purified on silica eluted with 1%, 2%, and 4% ethanol- dichloromethane to yield the title substance (124 mg, 71%), (TLC Ft, 0.11 in 0.1 :1 :1 ethanol-dichloromethane-ethyl acetate) .
• Example 21 h By the procedure of the preceding example (21 h), the product of Example 21 g (99 mg, 0.137 mmol) was deprotected with trifluoroacetic acid and converted to the title hydrochloride, which was triturated with ether, and thus obtained as a colorless solid (70 mg, 86%). HPLC (retention time 6.77 min, 30/70 system B) showed 10% of the isomeric product of Example 21 h (5.9 min retention time).
• 1 H NMR (D 2 0, 300 mHz) ⁇ 0.8-1.1 (overlapping d, 6H total), 1.5 (m, 2H), 1.61 (m, 1 H), 1.75 (m, 1 H), 1.93 (m, ca. 3H), 2.12 (m, ca.
• Example 21 d The product of Example 21 d (766 mg, 1.68 mmol) was coupled to isopropyl 3(S), 2(R)-3-amino-2-hydroxy-4- phenylbutanoate (USP 4,814,342, 400 mg, 1.68 mmol) to give 1.08 g (97%) of crude product which was purified on silica gel eluted with 50%, 67%, 75%, and 100% ethyl acetate in hexanes giving the title substance (669 mg, 60%).
• Example 16b The compound was prepared in a manner according to the procedure described as General Procedure A.
• the product of Example 16b 250 mg, 0.645 mmol was coupled with isopropyl 3(S),2(R)-3-amino-2-hydroxy-4- phenylbutanoate (EP 4 814 342, 168 mg, 0.710 mmol) to yield crude product (316 mg) which was chromatographed on silica packed in 2:1 ethyl acetate-hexanes and eluted with ethyl acetate followed by 3%, 10%, and 20% ethanol in ethyl acetate, giving the title substance ' (242 mg, 62%).
• the compound was prepared in a manner according to the procedure described as General Procedure C.
• the product of the preceding Example (172 mg, 0.283 mmol) was treated with the periodinane, and the crude product, isolated by chloroform extraction, was purified on silica eluted with 2%, 4%, and 10% ethanol in dichloromethane giving the title substance as a colorless foam (110 mg, 64%).
• LSIMS 605 24, M + + H), 333(50), 207(55), 147 (100).
• Example 16b The product of Example 16b (250 mg, 0.645 mmol) was coupled with 3(S),2(R)-3-amino-2-hydroxy-4-phenylbutyramide (USP 4 668 769, 164 mg, 0.710 mmol) to yield crude product in unexpectedly low yield (30 mg, 9%).
• the aqueous layers from the extractions were brought to pH 7 and saturated with NaCl, and the resulting solution was repeatedly extracted with chloroform. These extracts were combined, dried and concentrated giving 102 mg of a yellow foam which was chromatographed on silica eluted with 2%, 6%, and 10% ethanol in dichloromethane giving 26 mg of the title substance.

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