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Title:
RENIN-INHIBITORY PEPTIDES HAVING AN EPOXIDE OR GLYCOL MOIETY
Document Type and Number:
WIPO Patent Application WO/1988/004664
Kind Code:
A2
Abstract:
Novel renin-inhibitory peptides of the formula X-A6-B7-C8-D9-E10-F11, containing C-terminus truncated epoxy or azido or cyano groups or containing a position 10-11 diol and a position 11-12 retro bond. The present invention further provides novel transition-state analogue inserts which are useful as intermediates for the preparation of renin-inhibitory peptides.

Inventors:
THAISRIVONGS SUVIT (US)
Application Number:
PCT/US1987/003007
Publication Date:
June 30, 1988
Filing Date:
November 23, 1987
Export Citation:
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Assignee:
UPJOHN CO (US)
International Classes:
C07D295/18; A61K38/55; A61P5/38; A61P9/00; A61P9/12; C07C59/245; C07C235/06; C07C235/08; C07C237/12; C07C237/22; C07C259/06; C07D233/54; C07D263/04; C07D263/06; C07D295/185; C07D413/04; C07K5/02; C07K5/06; C07K5/065; C07K14/81; A61K38/00; (IPC1-7): C07K5/02; C07K5/06; C07D233/64; C07D203/06; C07D295/18
Foreign References:
EP0189203A21986-07-30
EP0173481A21986-03-05
Download PDF:
Description:
RENIN-INHIBITORY PEPTIDES HAVING AN EPOXIDE OR GLYCOL MOIETY DESCRIPTION BACKGROUND OF THE INVENTION The present invention provides novel compounds . More par- ticular ly, the present invention provides novel renin inhibitory peptides and intermediates thereto . Most particularly , the present invention provides renin- inhibitory peptides containing C- terminus truncated epoxy or azido or cyano groups or containing a position lu¬ ll diol and a position 11-12 retro bond as compared to the renin substrate . These renin- inhibitory peptides are useful for the diagnosis and control of renin- dependent hypertension. The present invention further provides novel transition- state analogue inserts which are useful as intermediates to renin- inhibitory peptides .

Renin is an endopeptidase which specifically cleaves a par- ticular peptide bond of its substrate (angiotensinogen) , of which the N- terminal sequence in equine substrate is for example :

Renin 4. Asp-Arg-Val-Tyr- Ile-His-Pro-Phe-His -Leu-Leu-Val-Tyr- Ser- IA 1 2 3 4 5 6 7 8 9 10 11 12 13 14

as found by L. T. Skeggs et al . , J . Exper. Med. 106 , 439 (1957) . Human renin substrate has a different sequence as recently discovered by D .A. Tewkesbury et al . , Bioche . Biophys . Res . Comm. 99 , 1311 (1981) . It may be represented as follows : Renin

-Val-Ile-His-

11 12 13 IB

and having the sequence to the left of the arrow (4) being as designated in formula IA above.

Renin cleaves angiotensinogen to produce angiotensin I, which is converted to the potent pressor angiotensin II. A number of an- giotensin I converting enzyme inhibitors are known to be useful in the treatment of hypertension. Inhibitors of renin are also useful in the treatment of hypertension.

A number of renin-inhibitory peptides have been disclosed. Thus, U.S. patent 4,424,207; European published applications 45,665;

104,041; and 156,322; and U.S. patent application, Serial No. 825,250, filed 3 February 1986 (Case 4119.2.1); disclose certain peptides with the dipeptide at the 10,11-position containing an isostere bond. A number of statine derivatives stated to be renin inhibitors have been disclosed, see, e.g., European published applications 77,028; 81,783; 114,993; 156,319; and 156,321; and U.S. patents 4,478,826; 4,470,971; 4,479,941; and 4,485,099. Terminal disulfide cycles have also been disclosed in renin inhib¬ iting peptides; see, e.g., U.S. patents 4,477,440 and 4,477,441. Aromatic and aliphatic amino acid residues at the 10,11 position of the renin substrate are disclosed in U.S. patents 4,478,827 and 4,455,303. C-teπninal amide cycles are disclosed in U.S. patent 4,485,099 and European published applications 156,320 and 156,318. Certain tetrapeptides are disclosed in European publications 111,266 and 77,027. Further, European published application No. 118,223 discloses certain renin inhibiting peptide analogs where the 10-11 peptide link is replaced by a one to four atom carbon or carbon- nitrogen link. Additionally, Holladay et al. , in "Synthesis of

Hydroxyethylene and Ketomethylene Dipeptide Isosteres", Tetrahedron Letters, Vol. 24, No. 41, pp. 4401-4404, 1983 disclose various inter¬ mediates In a process to prepare stereo-directed "ketomethylene" and "hydroxyethylene" dipeptide isosteric functional groups disclosed in the above noted U.S. Patent No. 4,424,207.

Additionally, published European Applications 45,161 and 53,017 disclose amide derivatives useful as inhibitors of angiotensin converting enzymes.

Certain dipeptide and tripeptldes are disclosed in U.S. patents 4,514,332; 4,510,085; and 4,548,926 as well as in European published applications 128,762; 152,255; and 181,110. Pepstatin derived renin inhibitors have been disclosed In U.S. patent 4,481,192. Retro- inverso bond modifications at positions 10-11 have been disclosed In U.S. patent 4,560,505 and in European published applications 127,234 and 127,235. Derivatives of isosteric bond replacements at positions 10-11 have been disclosed in European published applications 143,746 and 144,209; and U.S. patent application, Serial No. 833,993, filed 27 February 1986 (Case 4212) . Isosteric bond modifications at positions 11-12 and 12-13 have been disclosed in European published application 179,352. Certain peptides containing 2-substituted

statine analogues have been disclosed in European published applica¬ tion 157,409. Certain peptides containing 3-aminodeoxystatine have been disclosed in European published application 161,588. Certain peptides containing l-amino-2-hydroxybutane derivatives at positions 10-11 have been disclosed in European published application 172,346. Certain peptides containing l-amino-2-hydroxypropane derivatives at positions 10-11 have been disclosed in European published application 172,347. Certain peptides containing N-terminal amide cycles have been disclosed in U.S. patent application, Serial No. 844,716, filed 27 March 1986 (Case 4293). Certain peptides containing dihalostatine have been disclosed in PCT application, Serial No. 000,713, filed 7 April 1986 (Case 4156.P).

European published applications 156,322;.114,993; and 118,223; and U.S. patent application, Serial No. 798,459, filed 15 November 1985 (Case 4137); U.S. patent application, Serial No. 825,250, filed 3 February 1986 (Case 4119.2.1); U.S. patent application, Serial No. 833,993, filed 27 February 1986 (Case 4212); and U.S. patent applica¬ tion, Serial No. 844,716, filed 27 March 1986 (Case 4293); disclose hydroxamic acids or esters at the C-terminus. INFORMATION DISCLOSURE

E.P. 189,203 discloses new N-dihydrox alkyl peptide derivatives which are useful as inhibitors of renin for treating hypertension.

E.P. 184,855 discloses new hydroxy substituted-statine peptide derivatives which are useful as inhibitors of renin for treating hypertension.

Derivatives of isosteric bond replacements at positions 10-11 as dihydroxy ethylene isosteres have been disclosed in U.S. patent application, Serial No. 833,993, filed 27 February 1986 (Case 4212).

SUMMARY OF THE INVENTION The present invention particularly provides: A renin inhibitory peptide of the formula I χ - A 6 _B 7 _c 8 _D 9- E 10 _F ll ~ wherein X is

(a) hydrogen, (b) Ci-Cs lk l

(c) R 5 -0-(CH 2 ) q -C(0)-,

(d) R 5 -(CH 2 ) q -0-C(0)-,

(e) R 5 -0-C(0)-,

(f) R 5 -(CH 2 ) n -C(0)- f

(g) R 4 N(R 4 )-(CH 2 ) n -C(0)-, (h) R 5 -S0 2 -(CH 2 ) q -C(0)-, (i) R 5 -SO 2 -(CH 2 ) q -0-C(0)-, (j) R 5 -S-(CH 2 ) q -C(0)- r

(k) R 5 -(CH 2 ) q -S-(CH 2 ) q -CCO)- f

CD R 5 -(CH 2 ) q -0-(CH 2 ) q -C(0)-, or

( ) (R 3 ) p+1 C(H) 2 . p -NH-C(0)- ; wherein Y is (a) -0-,

(b) -NH-, or

(c) -0NH- ; wherein Ag is absent or a divalent moiety of the formula L^, XL 2 , or XL 2a ; wherein B is absent or a divalent moiety of the formula XL b ; wherein Cg is absent or a divalent moiety of the formula XL_, XL 2 or XL 7 ; or wherein Cg is a monovalent moiety of the formula XL a , when X, Ag and By are all absent; wherein D9 is a divalent moiety of the formula XL3 or XL 2a ; or wherein Cg-Dg is a divalent moiety of the formula XL4 or XL 4a ; or wherein Cg-Dg is a monovalent moiety of the formula 4b when X, Ag, and B7 are all absent; wherein EiO'^ll i- s a divalent moiety of the formula XLg, XLg a , XL 6b , or XL 6c ; wh&rein * indicates an asymmetric center which is either In the R or S configuration; wherein G^ is absent or a divalent moiety of the formula XL5, XL 5a or X 5b ; wherein H^ β is absent or a divalent moiety of the formula XL5, XL 5a or XL 5b ; wherein R^ is

(a) hydrogen,

(c) aryl,

(d) C3-C 7 cycloalkyl,

(e) -Het,

(f) C^-C3alko y, or

(g) C 1 -C3alkylthlo; wherein R 2 is

(a) hydrogen,

(b) C ] _-C5 alkyl, or

(c) aryl-C 1 -C 5 alkyl; wherein R3 is

(a) -(CH 2 ) q -0H,

(b) -(CH 2 ) q -NH 2 , or

(c) -(CH 2 ) q -C0 2 H; wherein R 4 at each occurrence is the same or different and is

(a) hydrogen, or

(b) C 1 -C 5 alkyl; wherein R5 is

(a) C 1 -C alkyl,

(b) C3-C 7 cycloalkyl, .

(c) aryl,

(d) -Het, or

(e) 5-oxo-2-pyrrolidinyl; wherein Rg is

(a) hydrogen,

(b) C2.-C5alkyl,

(c) -(CH 2 ) p -aryl,

(d) -(CH 2 ) p -Het,

(e) C3-C 7 cycloalkyl,

(f) 1- or 2-adamantyl, or

(g). -C(CH 2 0H) 3 ; wherein R 7 is

(a) hydrogen,

(c) hydroxy,

(d) amino C^-C 4 alkyl- ,

(e) guanidinyl C^^al yl-,

(f) aryl,

(g) -Het,

(h) methylthio,

(i) C3-C 7 cycloalkyl,

(j) amino, or

(k) -(CH 2 ) n C0 2 Hr wherein Rg-Y taken together can be

(a) -NR 2 R ,

(b) N-morpholino, (c) N-piperidyl,

(d) N-prolinol,

(e) N-proline-Y-Rg; wherein m is one or two; wherein for each occurrence n Is independently an integer of zero to five, inclusive; wherein p is zero to 2, inclusive; wherein q is 1 to 5, inclusive; wherein Q Is

(a) -CH 2 -, (b) -CH(OH)-,

(c) -0-, or

(d) -S- ; wherein M is

(a) -CO-, or (b) -CH 2 -; wherein aryl is phenyl or naphthyl substituted by zero to 3 of the following;

(a) C 1 -C 3 alkyl,

(b) hydroxy, (c) C 1 -C 3 alkoxy,

(d) halo,

(e) amino,

(f) mono- or di-C ] _-C3alkylamino,

(g) -CHO, (h) -C00H,

(i) C00R 2 f

(j ) C0NHR 2 j

(k) nitro ,

(1) mercapto , (m) C^ -C3 alkyl thio ,

(π) C1-C2 alley lsulfinyl ,

(o) C 1 -C3alkylsulfonyl r

(p) C 1 -C 3 alkylsulfonyl-N(R 4 ) - ,

(q) S0 3 H, (r) S0 2 NH 2 , (s) -CN, or (t) -CH 2 NH 2 ; wherein -Het is a 5- or 6-membered saturated or unsaturated ri containing from one to three heteroatoms selected from the gro consisting of nitrogen, oxygen, and sulfur; and including a bicyclic group in which any of the above heterocyclic rings is fus to a benzene ring, which heterocyclic moiety is substituted with ze to 3 of the following:

(i) Ci-Cgalkyl, (ii) hydroxy, (ill) trifluoromethyl, (iv) C_-C 4 alkoxy, (v) halo,

(vi) aryl, (vii) aryl-C 1 -C 4 alkyl-, (viii) amino, or

(Ix) mono- or di-C^-C alkylamino; or a carboxy-, amino-, or other reactive group-protected for thereof; or a pharmaceutically acceptable acid addition salt thereof; with the overall provisos that

(1) when ~±~-~~.l i X ~ *6a. and ^8 ^ s - " ^2 ' then B 7 must b present, and M must be -CH -;

(2) when E]_o" F H is X^ * c 8 is ~ ~ -l and bo h Ag and B 7 ar absent, then X in formula I is not hydrogen;

(3) when E]_o- F ll is X g b , Cg is XL , both G 12 and H 13 are absent, and X in XLg b is hydrogen, C]_-C 5 alkyl, or R 5 -(CH 2 ) n -C(0)-, then B 7 must be present and M must be -CH 2 -;

(4) when E-LO" F 11 is XLg b , Cg is XL^, both G 12 and H 13 are absent, X in XLg b is hydrogen, C]_-C 5 alkyl, or R5-(CH ) n -C(0)- , and both Ag and B are absent, then X in formula I is not hydrogen, C^-Cς alkyl, or 5 -(CH 2 ) n -C(0)- ; and (5) when Eio" F ll is XLg and Cg is XL 2 , then Rg in XL 2 of Cg is neither hydrogen nor C1-C5 alkyl.

These compounds are shown in relation to the human renin substrate as follows:

6 7 8 9 10 11 12 13

-His Pro Phe His Leu Val lie His-

A 6 »7 c 8 Dg E 10 F U

The present invention provides peptide inhibitors of renin which contain right -hand- terminating epoxy or azido or cyano groups or containing a position 10-11 diol and a position 11-12 retro bond as compared to the renin substrate.

Examples of pharmaceutically acceptable acid addition salts include: acetate, adipate, alginate, aspartate, benzoate, ben- zenesulf onat , bisulfate, butyrate, citrate, camphorate, camphorsul- f onate, cyclopentanepropionate, dlgluconate, dodecylsulf te , ethane - sulf onate, fumarate, glucoheptanoate , glycerophosphate , hemisulf te, heptanoate, hexanoate, hydrochloride , hydrobromide , hydro iodide, 2- hydroxyethanesulf onate , lac ate, maleate, me thanesulf onate, 2- naphthalenesulf onate , nicotinate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyaπate, tosylate, and undecanoate.

The carbon atom content of various hydrocarbon-containing moieties is Indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix indicates a moiety of the Integer "i" to the integer "j n carbon atoms, inclusive. Thus (Ci"C 4 ) lkyl refers to alkyl of one to 4 carbon atoms, inclusive, or methyl, ethyl, propyl, butyl, and isomeric forms thereof. C 4 -C 7 cyclic amino indicates a monocyclic group containing one nitrogen and 4 to 7 carbon atoms .

Examples of CC3-C^ 0 )cyclo lkyl, which include .alkyl- substituted cycloalkyl containing a total of up to 10 total carbon atoms, are cyclopropyl, 2-methylcyclopropyl, 2,2-dimethylcyclopropyl, 2,3- diethylcyclopropyl , 2-butylcyclopropyl, cyclobutyl, 2-methyl- cyclobutyl, 3 -pr opylcyclobutyl , cyclopentyl, 2, 2 -dimethyl cyclopentyl, cyclohexyl, cycloheptyl , cyclooctyl, cyclononyl, cyclodecyl and isomeric forms thereof.

Examples of aryl include phenyl, naphthyl, (o- , - , or p-)tolyl, (o-, m-, or p-)ethylphenyl, 2-ethyl-tolyl, 4-ethyl-o-tolyI, 5- ethyl -m- to lyl, (o-, - , or p-)propylphenyl, 2-propyl-(o- , m-, or p-)tolyl, 4-isopropyI-2,6-xylyl, 3-propyl-4-ethylphenyl, (2,3,4- 2,3,6-, or 2 , 4 , 5 - ) trimethylphenyl , (o-, m- , or p-) fluorophenyl, (o-, m-, or p-trifluoromethyl) phenyl, 4-fluoro-2,5-xylyl, (2,4-, 2,5-,

2,6-, 3,4-, or 3,5-)difluorophenyl, (o-, m- , or p-)chlorophenyl, 2 chloro-p-tolyl, (3-, 4-, 5- or 6-)chloro-o-tolyl, 4-chloro-2-propyl phenyl, 2-isopropyl-4-chlorophenyl, 4-chloro-3-fluorophenyl, (3- o 4-)chloro-2-fluorophenyl, (o-, m- , or p-)trifluoro-meth lphenyl, (o-, m- , or p-)ethoxyphenyl, (4- or 5-)chloro-2-methoxy-phenyl, and 2,4- dichloro(5- or 6-)meth lphenyl, and the like.

Examples of -Het include: 2-, 3-, or 4-pyridyl, imidazolyl, indolyl, N in -formyl-indolyl, N in -C 1 -C 5 alkyl-C(0)-indolyl, 1,2,4- triazolyl, 2-, 4-, or 5-pyrimidinyl, 2- or 3-thienyl, piperidinyl, pyrryl, pyrrollnyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidi- nyl, imidazolinyl, imidazolidinyl, pyrazinyl, piperazinyl, pyridazin- yl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, quinolinyl"; isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, and benzothienyl. Each of these moieties may be substituted as noted above.

As would be generally recognized by those skilled in the art of organic chemistry, a heterocycle as defined herein for -Het would not be bonded through oxygen or sulfur or through nitrogen which is within a ring and part of a double bond.

Halo is halogen (fluoro, chloro, bromo, or iodo) or tri- fluoromethyl.

Examples of pharmaceutically acceptable cations include: pharmacologically acceptable metal cations, ammonium, amine cations, or quaternary ammonium cations. Especially preferred metal cations are those derived from the alkali metals, e.g., lithium, sodium, and potassium, and from the alkaline earth metals, e.g., magnesium and calcium, although cationic forms of other metals, e.g., aluminum, zinc, and iron are also within the scope of this invention. Phar- macologically acceptable amine cations are those derived from primary, secondary, or tertiary amines.

The novel peptides herein contain both natural and synthetic amino acid residues. These residues are depicted using standard amino acid abbreviations (see, e.g., IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) , "Nomenclature and Symbolism for Amino Acids and Peptides," Eur. J. Biochem. 138:9-37 (1984) unless otherwise indicated.

The renin inhibitors of this invention are useful for treating

any medical condition-for which it is beneficial to reduce the levels of active circulating renin. Examples of such conditions include renin-dependent hypertension, hypertension, hypertension under treatment with another antihypertensive and/or a diuretic agent, congestive heart failure, renin-dependent hyperaldosterism, angina, post-myocardial infarction and other renin-dependent cardiovascular disorders. The renin-angiotension system may play a role in main¬ tenance of Intracellular homeostasls: see Clinical and Experimental Hypertension, 86, 1739-1742 (1984) at page 1740 under Discussion. The compounds of the present invention are preferably orally administered to humans to effect renin inhibition for the purpose of favorably affecting blood pressure. For this purpose, the compounds are administered from 0.1 mg to 1000 mg per kg per dose, administered from 1 to 4 times daily. Equivalent dosages for other routes of administration are also employed.

The exact dose depends on the age, weight, and condition of the patient and on the frequency and route of administration. Such variations are within the skill of the practitioner or can readily be determined. The compounds of the present invention may be in the form of pharmaceutically acceptable salts both those which can be produced from the free bases by methods well known in the art and those with which acids have pharmacologically acceptable conjugate bases.

Conventional forms and means for administering renin-inhibiting compounds may be employed and are described, e.g., in U.S. Patent No. 4,424,207 which is incorporated by reference herein. Likewise, the amounts disclosed in the U.S. Patent No. 4,424,207 are examples applicable to the compounds of the present invention.

The compounds of the present invention are preferably orally administered in the form of pharmacologically acceptable acid addition salts. Preferred pharmacologically acceptable salts for oral administration include the citrate and aspartate salts, although any pharmacologically acceptable salt Is useful in this invention, Including those listed above. These salts may be in hydrated or solvated form.

For these purposes the compounds of the present invention may be administered parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically

acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramus¬ cular, intrasternal injection or infusion techniques. In addition to the treatment of warm-blooded animals such as mice, rats, horses, dogs, cats, etc., the compounds of the invention are effective in the treatment of humans.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example as a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated accord- ing to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenteral- ly-acceptable diluent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or digly- cerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The peptides of this invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non- irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

The renin-inhibiting compounds of this invention may be adminis¬ tered in combination with other agents used in antihypertensive therapy such as diuretics, o and/or / S-adrenergic blocking agents, CNS-acting agents, adrenergic neuron blocking agents, vasodilators, angiotensin I converting enzyme inhibitors, and the like as described for example in published European patent application 156,318.

The present invention is also directed to combinations of the novel renin-inhibitory peptides of Formula I with one or more antihypertensive agents selected from the group consisting of diuretics, α and/or ^-adrenergic blocking agents, CNS-acting agents, adrenergic neuron blocking agents , vasodilators, angiotensin I

conver ing enzyme inhibitors, and other antihypertensive agents.

For example, the compounds of this invention can be given in combination with such compounds or salt or other derivative forms thereof as: Diuretics: acetazolamide; amlloride; bendroflumethiazide; benzthia- zide; bumetanide; chlorothiazide; chlorthalidone ; cyclothiazide ; ethacrynic acid; furose ide; hydrochlorothiazide; hydrof lumethiazide ; iπdacrinone (racemic mixture, or as either the (+) or (-) enantlomer alone, or a manipulated ratio, e.g., 9:1 of said enantiomers, respec- tively) ; metolazone; methyclothiazide; muzolimine; polythiazide; quinethazone ; sodium ethacrynate; sodium nitroprusside; spironol- actone; ticrynaten; trimaterene; trichlormethiazide ; α-Adrenergic Blocking Agents: dib en amine; phentolamine; phenoxyben- zamine; prazosin; tolazoline; β -Adrenergic Blocking Agents: atenolol; metoprolol; nadolol; propranolol; timolol;

((±) -2- [3- ( tert-butylamino) -2-hydroxypropoxy] -2-furananIlide)

(ancarolol) ;

(2-acetyl-7- (2-hydroxy-3-isopropylaminopropoxy)benzofuran HC1) (befun- olol) ;

( (±) -1- ( isopropylamino) -3- (p- (2-cyclopropylmethoxyethyl) -phenoxy) -2- propranol HC1) (betaxolol) ;

Cl- [ (3 , -dimethoxyphenethyl) amino ] - 3- (m- tolyloxy) - 2 -propanol HC1) (be- vantolol) ; ( ( (±) -1- (4-C(2-isoρropoxyethoxy)methyl)phenoxy) -3 -isopropylamino- 2- propanol)fumarate) (bisoprolol) ;

(4- (2-hydrσxyτ3- [4- (phenoxymethyl -piperidino] -propoxy)-indole) ;

(carbazolyl-4-oxy-5 , 2- (2-me hoxyphenoxy) -ethylamino- 2 -propanol) ;

(l-((l,l-dimethylethyl)amino) -3- C(2-methyl 'H-indol-4-yl)oxy)-2-pro- panol benzoate) (bopindolol) ;

( l- ( 2-exobicyclo[2.2.1J-hept-2-ylphenoxy)-3-[(l-methylethy l)-amino]- 2-propanol HC1) (bornaprolol) ;

( o-[2-hydroxy-3-[(2-indol-3-yl-l,l-dimethylethyl)-amino ]propoxy]ben- zonitrile HC1) " (bucindolol) ; (o-[(tert.butylamino)methyl] -7-ethyl-2-benzofuranmethanol) (bufur- alol) ;

(3- [3-acetyl-4- [3- (tert .butylamino) - 2-hydroxypropyl] -phenyl] -1,1- diethylurea HC1) (celiprolol) ;

((±)-2-[2-[3-[ (1, 1 - dime thy le thy 1) amino] - 2 -hydroxypropoxy ] phenoxy ] -N methylaceta ide HC1) (cetamolol) ;

(2-benzimidazolyl-phenyl(2-isopropylaminoproρanol) ) ;

( (±) -3 ' -acetyl-4 ' - (2-hydroxy-3 -isopropyla inopropoxy) -acetanilid HC1) (diacetolol) ;

(methyl-4- [2-hydroxy-3- [ (l-methylethyl)aminopropoxyl] ] -benzene propanoate HC1) (esmolol) ;

(erythro-DL-1- (7-methylindan-4-yloxy) -3-isopropylaminobutan-2-ol) ; (1- (tert. butylamino) -3- [0-(2-propynyloxy)phenoxy] -2-propanol (pargo- lol) ;

(1- (tert .butylamino) -3- [o- (6 -hydrazino-3 -pyridazinyl)phenoxy] -2- propanol diHCl) (prizidilol) ;

((-)-2-hydroxy-5- [ (R)-l-hydroxy-2- [ (R) - (1 -methyl- 3 -phenylpropyl) - amino] ethyl ]benzamide) ; (4-hydroxy-9 - [ 2-hydroxy- 3- (isopropylamino) -propoxy] - 7 -methyl -5H- furo[3,2-g] [1] -benzopyran-5-one) (iprocrolol) ;

((-)-5-( tert. butylamino) -2 -hydroxypropoxy] -3 , 4-dihydro-l-(2H) - naphthalenone HC1) (levobunolol) ;

(4- (2 -hydroxy- 3 -isopropylamino -propoxy) -1,2-benzisothiazole HC1); (4- [ 3- (tert .butylamino) -2-hydroxypropoxy ] -N-methylisocarbostyrll HC1);

((±)-N-2-[4- (2 -hydroxy- 3 -isopropylaminopropoxy) phenyl ] ethyl -N'- isopropylurea) (pafenolol) ;

(3- [ [ (2 -trifluoroacetamido) ethyl] amino] -l-phenoxypropan-2-ol) ; (N-(3-(o-chlorophenoxy)-2-hydroxypropyl)-N' -(4' -chloro-2 , 3-dihydro-3- oxo-5-pyridazinyl)ethylenediamine) ;

((±)-N-[3-acetyl-4-[2-hydroxy-3- [ (1-methylethyl) amino Jpropoxyphenyl] - butanamide) (acebutolol) ;

( (±) -4 ' - [ 3 - ( tert -butylamino) - 2 -hydroxypropoxy ] spiro [ cyclohexane-1 , 2 ' - indan] -1' -one) (spirendolol) ;

(7 - [ 3- [ [ 2 -hydroxy- 3 - [ (2-methylindol -4-yl) oxylpropyl ] amino ] butyl ] thio- phylline) (teoprolol) ;

( (±) - 1 - tert . butylamino-3 - ( thiochroman- 8 -yloxy ) - 2 -propanol ) ( tertato - lol) ; ((±)-l-tert.butylamino-3-(2,3-xylyloxy)-2-propanol HC1) (xibenolol) ; (8- [3- (tert. butylamino)- 2 -hydroxypropoxy] -5-methylcoumarin) (bucumo- lol); (2-(3- (tert.butylamino)-2-hydroxy-propoxy)benzonitrile HC1) (bunitro-

lol) ;

((±)-2' -[3-(tert-butylamino)-2-hydroxypropoxy-5' -fluorobutyrophenone)

(butofilolol) ;

(l-(carbazol-4-yloxy)-3-(isopropylamino)-2-propanol) (carazolol) ; (5-(3-tert.butylamino-2-hydroxy)propoxy-3,4-dihydrocarbotyri l HC1)

(carteolol) ;

(1-(tert.butylamino)-3-(2,5-dichlorophenoxy)-2-propanol) (clorano- lol) ;

(l-(inden-4(or 7) -yloxy) -3- (Isopropylamino) -2-propanol HC1) (indeno- lol) ;

(l-isoρropylamino-3- [(2-methylindol-4-yl)oxy] -2-propanol) (mepindo- lol) ;

(1- (4- acetoxy- 2 , 3 , 5 - trimethylphenoxy) -3-isopropylaminopropan-2-ol)

(metipranolol) ; (1- (isopropylamino) -3- (o-methoxyphenoxy) -3- [ (l-methylethyl)amino] -2- propanol) (moprolol) ;

( (1- tert . butylamino) - 3 - [ (5 , 6 , 7, 8 - tetrahydro- cis - 6 , 7-dIhydroxy-l- naphthyl)oxy] -2-propanol) (nadolol) ;

((S) -1- (2-cyclopentylphenoxy) -3- [ (1, 1- dime thy le thy 1) amino] -2-propanol sulfate (2:1) ) (penbutolol) ;

(4' - [l-hydroxy-2- (amino) ethyl] me thanesulfonanilide) (sotalol) ;

(2-methyl-3- [4- (2 -hydroxy- 3- tert. butylaminopropoxy) phenyl] -7-methoxy- isoquinolin-1- (2H) -one) ;

(l- (4- (2- (4- fluorophenyloxy) ethoxy) phenoxy) -3 - isopropylamino -2- propanol HCl) ;

( (-) -p- [3- [(3 , 4-dime thoxyphenethyl) amino ] - 2 -hydroxypropoxy ] -;S -methyl - cinnamonitrile) (pacrinolol) ;

( (±) -2- (3 ' -tert. butylamino- 2' -hydroxypropylthio) -4- (5 ' -carbamoyl-2 ' - thIenyl)thiazoIe HCl) (arotinolol) ; ( (±) - l- [p- [2-( cyclopropylme thoxy ) ethoxy] phenoxy ] - 3 - ( isopropylamino ) -

2-propanol) (clcloprolol) ;

((±) -1- [ (3-chloro- " 2-methyllndol-4-yl)oxy] -3- [(2-phenoxyethyl)amino] -

2-propanoI) (indopanolol) ;

( (±) -6 - [ [ 2- [ [3- (p-butoxyphenoxy) -2-hydroxypropyl] amino] ethyl] amino] - 1, 3-dimethyluracil) (pirepolol) ;

(4- (cyclohexylamino) -1- (1-naphtholenyloxy) -2-butanol) ;

(l-phenyl- 3- [2- [3- (2-cyanophenoxy) -2-hydroxypropyl]aminoethyl]hydan- toin HCl) ;

(3 ,4-dihydro-8 - (2 -hydroxy- 3- isopropylaminopropoxy) -3-nitroxy-2H-l- benzopyran) (nipradolol) ;

Angiotensin I Converting Enzyme Inhibitors: l-(3-mercapto-2-methyl-l-oxopropyl)-L-proline (captopril) ; (1- (4-ethoxycarbonyl-2 ,4(R,R) -dimethylbutanoyl)indoline-2(S)-car- boxylic acid) ;

(2-[2-[(l- (ethoxycarbonyl) - 3 -phenyl -propyl] amino ] -1-oxopropyl] - 1,2,3 ,4-tetrahydro-3-isoquinoline carboxylic acid);

((S)-l-[2-[(l- (ethoxycarbonyl) -3-phenylpropyl]amino] -l-oxopropyl]oc- tahydro-lH-indole-2-carboxylic acid HCl) ;

(N- cyclopentyl -N- (3- (2,2-dimethyl-l-oxopropyl)thiol-2-methyl-l-oxo- propyl)glycine) (pivalopril) ;

( (2R.4R) -2-(2-hydroxyphenyl) -3- (3-mercaptopropionyl) -4-thiazolidine- carboxylic acid) ; (1- (N- [l(S)-ethoxycarbonyl-3-phenylpropyl] -(S)-alanyl) -cis,syn-octa- hydroindol - 2 (S) -carboxylic acid HCl);

((-)-(S)-l-[(S) - 3 -mercapto- 2 -methyl -1-oxopropyl] indoline- 2 -carboxylic acid) ;

( [1(S) ,4S] -1- [3- (benzoylthio)-2-methyl-l-oxopropyl] -4-phenylthio-L- proline;

(3-([l-ethoxycarbonyl-3-phenyl- (IS) -propyl] amino) -2,3 ,4,5-tetrahydro- 2-oxo-l-(3S)-benzazepine-l-acetic acid HCl);

(N-(2-benzyl-3-mercaptopropanoyl) -S-ethyl-L-cysteine) and the S- methyl analogue; (N- (1(S) -ethoxycarbonyl-3-phenylpropyl) -L-alanyl-L-proline aleate) (enalapril) ;

N- [l-(S)-carboxy-3-phenylpropyl] -L-alanyl-1 -proline; N 2 - [l-(S)-carboxy-3-phenylpropyl] -L-lysyl-L-proline (lysinopril) ; Other Antihypertensive Agents: aminophylline ; cryptenamine acetates and tannates; deserpldine; meremethoxylline procaine; pargyline; tri- methaphan camsylate; and the like, as well as admixtures and combina¬ tions thereof.

Typically, the individual daily dosages for these combinations can range from, about one-fif h of the minimally recommended clinical dosages to the maximum recommended levels for the entities when they are given singly. Coadministration is most readily accomplished by combining the active ingredients into a suitable unit dosage form containing the proper dosages of each. Other methods of coad-

ministration are, of course, possible.

The novel peptides of the present invention possess an excellent degree of activity in treating renin-associated hypertension and hyperaldosteronlsm. The compounds of the present invention are prepared as depicted in the charts and as described more fully in the Preparations and Examples.

The process of the present invention is more completely under¬ stood by reference to the charts below. In these charts, any variables are as defined above.

Chart A Chart A describes the preparation of compounds which are useful as intermediates for the preparation of renin inhibitory peptides.

The known aldehyde of formula A-l is reacted with sulfonium ylide to afford a mixture of epimeric epoxides of formula A-2. Azide opening then proceeds to give an epimeric mixture of alcohols of formula A-3.

Chart B Chart B describes the incorporation of amino acids into the compound of formula A-3 of Chart A.

The compound of formula B-l (A-3) is deprotected with acidic methanol to the corresponding free amine which is coupled to Boc- His(T≤)-0H to give an epimeric mixture. The mixture is separated into the compounds of formulas B-2A and B-2B. The stereochemistry at the hydroxyl group at C-2 has not been assigned. The compound of formula B-2A Is deprotected and the resulting amine coupled to Boc- Phe-OH to give the compound of formula B-3A.

Chart C Chart C describes the preparation of the acid of formula C-4. Diastereoselective alkylation of the known dianion of the diester of formula C-l with 1-bromomethylnaphthalene gives the compound of formula C-2. Selective ester hydrolysis, followed by amide formation with morpholine, gives the ester of formula C-3. Hydrolysis of the ester then affords the acid of formula C-4. Chart D

Chart D describes the preparation of a representative renin- inhibitory peptide of formula D-4.

The compound of formula D-l (B-3A) is hydrogenolyzed to the

amine of formula D-2. Reaction with isopropylisocyanate gives the compound of formula D-3. Removal of the tosyl group affords the peptide of formula D-4.

Chart E Chart E describes the preparation of the compound of formula E-2 and its incorporation into a representative renin-inhibitory peptide. The epimeric epoxides of formula E-l (A-2) are opened with cyanide to give a mixture of nitriles of formula E-2. The mixture is then deprotected with acidic methanol to give the corresponding free amine which is coupled to Boc-His(Ts)-OH to give an epimeric mixture. The mixture is separated into the compounds of formulas E-3A and E- 3B. The assignment of stereochemistry of the C-3 hydroxy1 group is tentative. Compound E-3A is deprotected with trifluoroacetic acid in dichloromethane. The corresponding free amine is coupled to Boc-Phe- OH to give the compound of formula E-4A. Removal of the tosyl group affords the renin-inhibitory peptide of formula E-5A.

Chart F Chart F describes the incorporation of the acid of formula C-4 of Chart C into a representative renin-inhibitory peptide of formula F-3.

The compound of formula F-l (B-2A) is deprotected and the resulting amine is coupled to the acid of formula C-4 to give the compound of formula F-2. Tosyl group removal then gives the peptide of formula F-3. Other renin-inhibitory peptides of this invention can be prepared according to the processes of these charts by using dif¬ ferent amino acids known in the art.

Generally, the renin inhibiting polypeptides may be prepared by solution phase peptide synthetic procedures analogous to those described hereinafter or to those methods known in the art. Appro¬ priate protecting groups, reagents, and solvents can be found in "The Peptides: Analysis, Synthesis, and Biology," Vols. 1-5, eds. E. Gross and T. Meienhofer, Academic Press, NY, 1979-1983; "The Practice of Peptide Synthesis", M. Bodansky and A. Bodansky, Springer-Verlag, New York, 1984; "The Principles of Peptide Syn¬ thesis", M. Bodansky, Springer-Verlag, New York, 1984. Thus, for example, the carboxylic moiety of t^-t-butyloxycarbonyl (Boc)-substi ¬ tuted amino acid derivatives having suitable side chain protecting

groups, If necessary, may be condensed with the amino functionality of a suitably protected amino acid or peptide using a conventional coupling protocol such as dicyclohexylcarbodiimide and 1-hydroxyben- zotriazole or diethylphosphoryl cyanide and trialkylamine in methyl- 5 ene chloride or dimethylformamide.

Following coupling reaction completion, the N'^-Boc moiety may be selectively removed with 50% trifluoroacetic acid with or without 2% anisole (v/v) in methylene chloride. Neutralization of the resultant trifluoroacetate salt may be accomplished with 10% diisopropyl-

10 ethylamine or sodium bicarbonate in methylene chloride.

The incorporation of N ιn -formyl-Trp into compounds of the present invention is easily accomplished because of the commercial availability of N α -Boc-N in -formyl-Trp-OH. However, the N in -formyl moiety may be introduced into indolyl-substituted amino acid deriva-

15 tives or related compounds by reaction with hydrochloric-formic acid as reported in the literature,- see A. Previero et al, Biochim. Bio- phys. Acta 147, 453 (1967); Y.C.S. Yang et al, Int. J. Peptide Protein Res. 15, 130 (1980) .

Generally, methods of alkylation useful in alk lating histidlne

20 for use in the present invention are found in Cheung, S.T. et al, Can. J. Che . , Vol 55, pp. 906-910 (1977). However it is now found that in the Cheung, S. T. et al, method, It is critical that the reaction conditions for the alkylation of histidine be anhydrous. Further, it is now found also that during work-up instead of adding

25 water directly to the reaction mixture, it is preferred that a buffered aqueous solution be added to the reaction mixture, for example, aqueous sodium or potassium hydrogen sulfate.

Variations in the above description for starting materials, reactants, reaction conditions and required protecting groups to

30 obtain other such N-alkylated compounds are known to an ordinarily skilled chemist or are readily available in the literature.

The compounds of the present invention may be in either free form or in protected form at one or more of the remaining (not previously protected) peptide, carboxyl, amino, hydroxy, or other

3.5 reactive groups. The protecting groups may be any of those known in the polypeptide art. Examples of nitrogen and oxygen protection groups are set forth in T.V. Greene, Protecting Groups In Organic Synthesis, Wiley, New York, (1981); J.F.W. McOmie, ed. Protective

Groups in Organic Chemistry, Plenum Press (1973); and J. Fuhrhop an G. Benzlin, Organic Synthesis, Verlag Chemie (1983). Included amon the nitrogen protective groups are t-butoxycarbonyl (Boc) , ben- zyloxycarbonyl, acetyl, allyl, phthalyl, benzyl, benzoyl, trityl an the like.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following Preparations and Examples illustrate the present invention.

In the Preparations and Examples below and throughout this document:

^ H-NMR is proton nuclear magnetic resonance (δ is relative to tetramethylsilane) ;

-" •■ ^C-NMR is carbon-13 nuclear magnetic resonance; BOC is t-butoxycarbonyl; C is centigrade;

CDCI3 is deuteriochloroform; Celite is a filter aid; g is grams; His is histidine; IR is infrared spectra; M or mol is mole; min is minute; ml is milliliter;

FAB HRMS is fast-atom bombardment high-resolution mass spec- troscopy;

Phe is phenylalanine; Ts is p-toluenesulfonyl.

The wedge-shape line indicates a bond which extends above the plane of the paper relative to the plane of the compound thereon. The dotted line indicates a bond which extends below the plane of the paper relative to the plane of the compound thereon. Preparation 1 IR and IS-(3-tert-Butyloxycarbonyl-2,2-dimethyl-

4S-cyclohexylmethyl-5R-oxazolidinyl) -ethylene oxide (Formula A-2) Refer to Chart A. To a stirred suspension of 3.3 g of trimethylsulfonium iodide in

14.6 ml of dry dimethylsulfoxide and 14.6 ml of anhydrous tetrahydro- furan, cooled to -5 β C, is added dropwise, via addition funnel, 13.5 ml of a 1.0 M solution of sodium bistrimethylsilylamide in tetrahy-

drofuran. After 1 min, 2.9 g of the aldehyde of formula A-l in 12 ml of anhydrous tetrahydrofuran is added rapidly via cannula. After 2 h, the ice bath Is removed and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture is quenched with 30 ml of pH 7 phosphate buffer and then diluted with dichloromethane. The aqueous phase is extracted with four portions of dichloromethane. The combined organics are dried (magnesium sulfate) , filtered and concentrated. The residue is purified by flash chromatography on silica gel using 5% ethyl acetate in hexane to afford 2.7 g of the title product as a mixture of isomers.

Physical characteristics are as follows:

^-NMR (5, CDC1 3 ): 0.96-2.0, 1.48, 1.52, 1.63, 2.66, 2.80, 2.86, 3.06, 4.0.

13 C-NMR (5, CDCI3): 29.74, 29.97, 30.13, 31.40, 32.10, 36.02, 38.04, 38.66, 38.94, 45.23, 49.37, 56.28, 61.52, 83.29, 85.39, 97.89, 155.07.

IR (cm -1 mull): 2950, 2926, 1703. FAB HEMS: [M+H] + at m/z - 340.2509. Anal. Found: C, 67.65; H, 9.65; N, 4.09. Preparation 2 2-Azido-lR and lS-(3-tert-butyloxycarbonyl-2,2- dimethyl-4S-cyclohexylmethyl-5R-oxazolidinyl)- ethanol (Formula A-3) Refer to Chart A.

To a stirred solution of 2.4 g of the epoxide of formula A-2 of

Preparation 1 in 13 ml of dry methanol is added 1.06 g of magnesium sulfate and 1.15 g of sodium azide. The reaction mixture is heated to reflux for 6 h, then cooled to room temperature. The reaction mixture is partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous phase is extracted with four portions of dichloromethane. The combined organics are dried (magnesium sulfate), filtered and concentrated. The residue Is purified by flash chromatography on silica gel using 10% ethyl acetate in hexane to afford 2.17 g of the title product as a mixture of isomers.

Physical characteristics are as follows: ^-H-NMR (δ , CDCI3): 0.96-1.0, 1.47, 1.52, 1.58, 3.61, 4.0. IR (cm -1 mull): 3393, 2926, 2853, 2099, 1661. FAB HRMS: [M+H] + at m/z - 383.2663. Preparation 3 N α -tert-Butyloxycarbonyl-N im -tosyl-L-histidyl-4S-

amino-1-azido-(2R and 2S) ,3R-dihydroxy-5-cyclo- hexylpentane (Formulas B-2A and B-2B) Refer to Chart B. To 7.0 ml of dry methanol, cooled to 0 β C, is added 0.50 ml of acetyl chloride. After stirring for 10 min at room temperature, this solution is added to 541.1 mg of the azide of formula B-l (A-3) of Preparation 2. After stirring at room temperature for 3 h, 5.0 g of Amberlite exchange resin (hydroxide form) is added. After 45 min, the reaction mixture is filtered and the resin is washed with several portions of methanol. The filtrate is concentrated. The residue is chromatographed on 33 g of silica gel using 5% methanol (saturated with ammonia) in dichloromethane to afford 252.4 mg of a mixture of the title product as a mixture of isomers.

Physical characteristics are as follows: FAB HRMS: [M+H] + at m/z - 243.1814.

To a stirred solution containing 250.1 mg of a mixture of the isomers and 507.6 mg of Boc-His(Ts)-0H in 4.1 ml of dichloromethane is added 0.25 ml of diisopropylethylamine followed by 0.19 ml of diethylcyanophosphonate. After 4 h at room temperature, the reaction mixture is partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous phase is extracted with five portions of dichloromethane. The combined organics are dried

(magnesium sulfate), filtered and concentrated. The residue is chromatographed on 67 g of silica gel using 40 to 50 to 65% ethyl acetate in hexane to afford 292.9 mg of the title product B-2A and

94.2 mg of the title product B-2B. Note: assignment of 2S to B-2A and 2R to B-2B. is tentative.

Physical characteristics are as follows : FAB HRMS : [M+H] + at m/z - 634.3015. Preparation 4 N α -tert-Butyloxycarbonyl - L-phenylalanyl -N im - tosy 1 - L-hlst idyl -4S - amino- l-azido- 2"S π , 3R-dihy- droxy-5-cyclohexylpentane (Formula B-3A) Refer to

Chart B .

The peptide of formula B-2A of Preparation 3, 193.9 mg, is dis- solved in 1.0 ml of dichloromethane and 1.0 ml of trifluoroacetic acid. After 45 min at room temperature, the reaction mixture is slowly pipetted into a stirred solution containing 1.5 g of solid sodium bicarbonate in 15 ml of water. After 10 min, the aqueous

phase is extracted with five portions of dichloromethane. The combined organic phases are dried (magnesium sulfate) , filtered and concentrated to afford 150.4 mg of the free amine. A solution of this material and 105.2 mg of N^-tert-butyloxycarbonyl-L-phenylal- anine, 110 μl of diisopropylethylamine and 70 μl of diethylphosphor- ylcyanide in 1.2 ml of dichloromethane affords 204.1 mg of the title product after chromatography on silica gel with 50 to 85% ethyl acetate in hexane.

Physical characteristics are as follows: FAB HRMS: [M+H] + at m/z - 781,3678.

Preparation s Diethyl 3S-hydroxy-2R-(1'-naphthylmethyl)sue- cinate (Formula C-2) Refer to Chart C. A solution of 2.85 g of diethyl S-malate of formula C-l in 5 ml of tetrahydrofuran is slowly added to a solution of 30.6 mmol of lithium diisopropylamlde in 20 ml of tetrahydrofuran at -78 β C under argon. The resulting mixture is stirred at -20°C for 30 min and then recooled to -78 β C. A solution of 5 g of 1-bromomethyl-naphthalene in 5 ml of tetrahydrofuran is slowly added to the reaction mixture. The resulting mixture Is allowed to warm slowly overnight to -5*C and then quenched with 3 ml of acetic acid. The reaction mixture is partitioned between ether and saturated aqueous sodium bicarbonate. The organic phase is dried (magnesium sulfate) , filtered and then concentrated. The residue is chromatographed on silica gel with 20- 25% ethyl acetate in hexane to give 3.13 g of the title product. Physical characteristics are as follows:

X H-NMR (£, CDC1 3 ): 1.20, 1.22, 1.24, 3.3-3.6, 3.68-3.74, 4.1- 4.3, 7.4-8.2.

Preparation 6 Ethyl 3S-hydroxy-4-morphollno-2R- (l' -naphthyl¬ methyl) -succinate (Formula C-3) Refer to Chart C.

A solution of 369 mg of compound of formula C-2 of Preparation 5 in 2.3 ml of tetrahydrofuran is added 1.12 ml of a 1M solution of potassium hydroxide. After stirring at room temperature for 1 h, the reaction mixture is partitioned between dichloromethane and aqueous potassium bisulfate. The organic phase is dried (magnesium sulfate) , filtered and then concentrated to give 340 mg of an acidic residue.

To a stirred solution of this residue in 5 ml of dichloromethane is added 0.13 ml of morpholine, 0.21 ml of diisopropylethylamine and

0.21 ml of diethylphosphoryl cyanide. After stirring at room temperature for one day, the reaction mixture is applied to a silica gel column and eluded with 40-50% ethyl acetate in hexane to give 310 mg of the title product. Physical characteristics are as follows: -H-NMR (δ , CDC1 3 ): 1.20, 1.22, 1.24, 2.8-3.1, 3.2-3.7, 4.0-4.3, 7.4-8.2.

Preparation 7 3S-Hydroxy-4-morpholino-2R-(l' -naphthylmethyl) - succinic acid (Formula C-4) Refer to Chart C. A solution of 310 mg of compound of formula C-3 of Preparation 6 in 2 ml of tetrahydrofuran is added 0.91 ml of a 1M solution of aqueous potassium hydroxide. After stirring at room temperature for 2 h, the reaction mixture is partitioned between dichloromethane and aqueous potassium bisulfate. The organic phase is dried (magnesium sulfate) , filtered and then concentrated to give 280 mg of the title product.

Physical characteristics are as follows:

^-H- MR (δ , CDCI3): 2.4-2.5, 2.8-2.9, 3.0-3.2, 3.4-3.7, 7.3-8.1. Preparation 8 N α -tert-Butyloxycarbonyl-L-phenylalanyl-N lm - tosyl-L-histidyl-4S-amino-2"S" ,3R-dihydroxy-5- cyclohexylpenta ine (Formula D-2) Refer to Chart D. To 100 mg of the peptide of formula B-3A (D-l) of Preparation 4 in 0.65 ml of dry methanol is added 30 mg of 10% palladium-on-carbon. The reaction mixture is flushed with nitrogen and evacuated several times, and then filled with hydrogen to an initial pressure of 55 psi. The reaction mixture is hydrogenated on a Parr shaker over¬ night. The reaction mixture is filtered through a pad of Celite. The catalyst is washed with several portions of methanol. The filtrate is concentrated and the residue is purified on 11 g of silica gel using 7% methanol saturated with ammonia in dichloromethane to afford 58.2 mg of the title product.

Physical characteristics are as follows: FAB HRMS: ' [M+H] + at m/z - 755.3784. Preparation 9 N α -tert-Butyloxycarbonyl-L-phenylalanyl-N im - tosy1-L-histidyl-4S-amino-2"S" ,3R-dihydroxy-5- cyclohexyl-1-isopropylaminocarbonylamino-pentane (Formula D-3) Refer to Chart D.

To 72.4 mg of the compound of formula D-2 of Preparation 8 in 1 ml of dichloromethane at 0 β C is added 10 μl of isopropylisocyanate. After stirring at room temperature for one day, the reaction mixture is then applied to a silica gel column and eluded with 4-6% methanol in dichloromethane to give 42.8 mg of the title product. Physical characteristics are as follows: X H-NMR (5 , CDC1 3 ) : 1.10 , 1.17 , 1.38 , 2.40 , 7.0-8.0. Example 1 N^-tert-Butyloxycarbonyl-L-phenylalanyl-L-histidyl^S- amino - 2"S " , 3R- dihydroxy-5 - cyclohexyl - 1- isopropylamino - carbonylamino-pen ane (Formula D-4) Refer to Chart D.

54 mg of the compound of formula D-3 of Preparation 9 and 34 mg of 1-hydroxybenzotriazole are dissolved in 0.5 ml of methanol. After stirring at room temperature overnight, the reaction mixture is concentrated and the residue chromato graphed on silica gel with 3-5% methanol (saturated with ammonia) in dichloromethane to afford 31.9 mg of the title product.

Physical characteristics are as follows: -NMR (δ , CDCI3) : 1.10 , 1.17 , 1.38 , 6.83 , 7.27 , 7.57. FAB HRMS : [M+H] + at m/z - 686.4211. Preparation 10 3 - ( 3 - tert- Butyloxycarbonyl- 2 , 2 -dimethyl-4S - cyclohexylmethyl-5R-oxazolidinyl) -3R and 3S- hydroxypropionitrile (Formula E-2) Refer to

Chart E.

To a stirred solution of 4.18 g of the epoxide of formula E-l (A-2) of Preparation 1 in 25 ml of dry methanol is added 1.85 g of magnesium sulfate and 2.0 g of potassium cyanide . The reaction mixture is heated to reflux for 4 h, then cooled to room temperature .

The reaction mixture is partitioned between dichloromethane and saturated aqueous sodium bicarbonate. The aqueous phase is extracted with four portions of dichloromethane . The combined organics are dried (magnesium sulfate) , filtered and concentrated. The residue is purified by flash- chromatography on silica gel using 15% ethyl acetate in hexane to afford 3.65 g of the title product as a mixture of isomers. Physical characteristics are as follows:

- " .-UMR (5, CDCI3): 1.35, 1.38, 1.39, 1.40, 2.4, 3.5, 3.75. IR (cm -1 , mull): 3413, 2924, 2264, 1700. FAB HRMS: {M+H] + at m/z - 367.2571.

Anal. found: C, 65.13; H, 9.41; N, 7.34. Preparation 11 N α -tert-Butyloxycarbonyl-N im -tosyl-L-histidyl-5S- amino-6-cyclohexyl-3R and 3S,4R-dihydroxy- hexanenitrile (Formulas E-3A and E-3B) Refer to Chart E.

By the same procedure as in the preparation of the compounds of formulas B-2A and B-2B of Preparation 3, 88 mg of the compound of formula E-2 of Preparation 10 is treated with methanol and acetyl chloride to give 53.7 mg of the free amine. A solution of this material, 118.1 mg of N^-tert-butyloxycarbonyl-N 1111 - osyl-L-histidine, 0.08 ml of diisopropylethylamine and 0.05 ml of diethylcyanophos- phonate in 0.9 ml of dichloromethane affords 98.5 mg of the title product E-3A and 26.2 mg of the title product E-3B after chromatog¬ raphy on silica gel with 50 to 70% ethyl acetate in hexane. The assignment of stereochemistry to the title products is tentative. Physical characteristics are as follows: FAB HRMS: [M+H] + at m/z - 618.2953. Preparation 12 N α -tert Butyloxycarbonyl-L-phenylalanyl-N im - tosyl-L-histidyl-5S-amino-6-cyclohexyl-3 π S",4R- dihydroxy-hexanenitrile (Formula E-4A) Refer to

Chart E. By the same procedure as in the preparation of the compounds of formula B-3A of Preparation 4, 95.1 mg of the compound of formula E- 3A of Preparation 11 is treated with trifluoroacetic acid and dichloromethane to give the free amine. A solution of this material, 53.1 mg of N α -tert-butyloxycarbonyl-L-phenylalanine, 56 μl of diisopropylethylamine and 37 μl of diethylcyanophosphonate in 0.62 ml of dichloromethane affords 95.7 mg of the title product after chromatography on silica gel with 65 to 70% ethyl acetate in hexane. Physical characteristics are as follows: FAB HRMS: [M+H] + at m/z - 765.3617. Example 2 N α -tert-Butyloxycarbonyl-L-phenylalanyl-L-histidyl-5S- amino-6-cyclohexyl-3"S" ,4R-dihydroxy-hexanenitrile (Formula E-5A) Refer to Chart E. 91.7 mg of the peptide of formula E-4A of Preparation 12 and 64.8 mg of 1-hydroxybenzotriazole hydrate are dissolved in 0.5 ml of dry methanol. After stirring at room temperature overnight, the reaction mixture is concentrated and chromatographed on silica gel

wlth 5% methanol (saturated with ammonia) in dichloromethane to afford 51.4 mg of the title product.

Physical characteristics are as follows: FAB HRMS: [M+H] + at m/z - 611.3568. Preparation 13 3S-Hydroxy-4-morpholino-2R-(l'-naphthylmethyl)- succinoyl-N lm -tosyl-L-histid l-4S-amino-1-azido- 2"S",3R-dihydroxy-5-cyclohexylpentane (Formula F- 2) Refer to Chart F. To 89 mg of the amine derived from the compound of formula F-l (B-2A) of Preparation 3 and 52 mg of the compound of formula C-4 of Preparation 7 in 1 ml of dichloromethane is added 29 μl of diisopro¬ pylethylamine and 28 μl of diethylphosphoryl cyanide. After stirring at room temperature for one day, the reaction mixture Is applied to a silica gel column and eluded with 3-4% methanol in dichloromethane to give 54.5 mg of the title product.

Physical characteristics are as follows:

^ H-NMR (S, CDC1 3 ): 0.8-1.8, 2.36, 2.5-3.8, 4.2-4.4, 4.6-4.8, 7.2-8.2.

FAB MS: [M+H] + at m/z - 860. Example 3 3S-Hydroxy-4-morpho1ino-2R-(1' -naphth lmethyl)-sue- cinoyl-L-histidyl-4S-amino-l-azldo-2' , S",3R-dlhydroxy- 5-cyclohexylpentane (Formula F-3) Refer to Chart F. A solution of 53.8 mg of the compound of formula F-2 of Prepara¬ tion 13 and 34 mg of 1-hydroxybenzotriazole in 0.5 ml of methanol is stirred at room temperature for one day. The concentrated reaction mixture is chromatographed on silica gel with 3-4% methanol (satura¬ ted with ammonia) in dichloromethane to give 26.8 mg of the title product.

Physical characteristics are as follows: L H-NMR (δ, CDCI3): 0.6-1.8, 2.6-3.8, 4.0-4.5, 4.5-4.8, 6.9-8.5. FAB MS: [M+H] + at m/z - 706.

FORMUIA CHART

X-A 6 -B 7 -C 8 -D 9 -E 10 -F n

Rg

CHART A

15

35

CHART B

B-3A

CHART C

CHART D

Boc-Phe-His D-l (B-3A)

CHART E

CHART