FIELD OF THE INVENTION

Renin-inhibiting compounds are known for control of hypertension. Of particular interest herein are non-peptidyl compounds useful as renin inhibiting agents.

BACKGROUND OF THE INVENTION

Renin is a proteolytic enzyme produced and secreted into the bloodstream by the juxtaglomerular cells of the kidney. In the bloodstream, renin cleaves a peptide bond in the serum protein angioten- sinogen to produce a decapeptide known as angiotensin I. A second enzyme known as angiotensin converting enzyme, cleaves angiotensin I to produce the octapeptide known as angiotensin II. Angiotensin II is a potent pressor agent responsible for vasoconstriction and elevation of cardiovascular pressure. Attempts have been made to control hypertension by blocking the action of renin or by blocking the formation of angiotensin II in the body with inhibitors of angio¬ tensin I converting enzyme.

Classes of compounds published as inhibitors of the action of renin on angiotensinogen include renin antibodies, pepstatin and its analogs, phospho-

lipids, angio ensinogen analogs, pro-renin related analogs and peptide aldehydes.

A peptide isolated from actinomyces has been reported as an inhibitor of aspartyl proteases such as pepsin, cathepsin D and renin [Umezawa et al, in J. Antibiot. (Tokyo), 23, 259-262 (1970)]. This peptide, known as pepstatin, was found to reduce blood pressure in vivo after the the injection of hog renin into nephrectomized rats [Gross et al, Science, 175, 656 (1971)]. Pepstatin has the disadvantages of low solubility and of inhibiting acid proteases in addition to renin. Modified pepstatins have been synthesized in an attempt to increase the specificity for human renin over other physiologically important enzymes. While some degree of specificity has been achieved, this approach has led to rather high molecular weight hepta- and octapeptides [Boger et al, Nature, 303, 81 (1983)]; high molecular weight peptides are generally considered undesirable as drugs because gastrointestinal absorption is impaired and plasma stability is compromised.

Short peptide aldehydes have been reported as renin inhibitors [Kokubu et al, Biochim. Biophys. Res. Commun. , 118, 929 (1984); Castro et al, FEBS Lett. , 167, 273 (1984)]. Such compounds have a reactive

C-terminal aldehyde group and would likely be unstable in vivo.

Other peptidyl compounds have been described as renin inhibitors. EP Appl. #128,762, published 18 December 1984, describes dipeptide and tripeptide glycol-containing compounds as renin inhibitors [also see Hanson et al, Biochm. Biophys. Res. Comm. , 132, 155-161 (1985), 146, 959-963 (1987)]. EP Appl. #181,110, published 14 May 1986, describes dipeptide

histidine derivatives as renin inhibitors. EP Appl. #189,203, published 30 July 1986, describes peptidyl- aminodiols as renin inhibitors. EP Appl. #200,406, published 10 December 1986, describes alkylnaphthyl- methylpropionyl-histidyl aminohydroxy alkanoates as renin inhibitors. EP Appl. #216,539, published 1 April 1987, describes alkylnaphthylmethylpropionyl aminoacyl aminoalkanoate compounds as renin inhibitors orally administered for treatment of renin-associated hypertension. EP Appl. #229,667, published 22 July 1987, describes acyl α-aminoacyl aminodiol compounds having a piperazinylcarbonyl or an alkylaminoalkylcarbonyl terminal group at the N-amino acid terminus, such as 2(S)-{ [(l-piperazinyl)carbonyl]- oxy]-3-phenylpropionyl}-Phe-His amide of 2(S)-amino-l- cyclohexyl-3(R), (S)-dihydroxy-6-methylheptane. PCT Application No. WO 87/04349, published 30 July 1987, describes aminocarbonyl aminoacyl hydroxyether derivatives having an alkylamino-containing terminal substitutent and which are described as having renin- inhibiting activity for use in treating hypertension.

EP Appl. #300,189 published 25 January 1989 describes

^• amino acid monohydric derivatives having an alkylamino- alkylamino N-terminus mentioned as useful in treating hypertension.

For other articles describing previous efforts to devise renin inhibitors, see Marshall, Federation Proc. , 35, 2494-2501 (1976); Burton et al, Proc. Natl. Acad. Sci. USA, 77, 5476-5479 (1980); Suketa et al, Biochemistry, 14, 3188 (1975); Swales, Pharmac. Ther. , 7, 173-201 (1979); Kokubu et al, Nature, 217, 456-457 (1986); Matsushita et al, J. Antibiotics, 28, 1016-1018 (1975); Lazar et al, Biochem. Pharma., 23, 2776-2778 (1974); Miller et al., Biochem. Pharm . , 21, 2941-2944 (1972); Haber,

Clinical Science, 59, 7s-19s (1980); Rich et al,

J. Org. Chem., 43, 3624 (1978); J. Med. Chem., 23 _, , 27 (1980); especially Haber, Clin. and Exper. Hyper., A5(7&8), 1193 (1983); and European Patent Applications #172,346 and #172,347 published 26 February 1986.

DESCRIPTION OF THE INVENTION

Non-peptidyl ethereal N-terminal aminodiol amino acid derivatives having utility as renin inhibitors for treatment of hypertension in mammals constitute a family of compounds of general Formula I:

wherein X is selected from oxygen atom, methylene and J^NR ₁₀ with R ₁₀ selected from hydrido, alkyl ^' and benzyl; wherein R _x is a group selected from alkyl, alkoxycarbonyl, loweralkanoyl, cycloalkyl, alkoxyacyl, haloalkylacyl, phenyl, heterocyclicalkyl, dialkyl- - aminoalkyl, benzyl, benzyloxycarbonyl, naphthyl and naphthylmethyl, any one of which groups having a substitutable position may be optionally substituted with one or more radicals selected from alkyl, alkoxy, alkenyl, alkynyl, halo, haloalkyl, cyano and phenyl; wherein R ₂ is selected from hydrido, alkyl, alkylamino- alkyl, alkylacylaminoalkyl, benzyl and cycloalkylalkyl; wherein R ₃ is selected from alkyl, cycloalkyl, cycloalkylalkyl, acylaminoalkyl, phenylalkyl, naphthyl- methyl, aryl and heterocyclicalkyl, wherein the aromatic portion of any of said phenylalkyl, naphthyl¬ methyl, aryl and heterocyclicalkyl may be substituted by one or more halo or alkyl or by both; wherein each of R ₄ and R ₆ is independently selected from hydrido, alkyl, benzyl and cycloalkyl; wherein R ₇ is selected

from substituted or unsubstituted cycloalkyl, phenyl, cycloalkylalkyl and phenylalkyl, any one of which may be substituted with one or more groups selected from alkyl, alkoxy, halo, haloalkyl, alkenyl, alkynyl and cyano; wherein R ₈ is selected from hydrido, alkyl, haloalkyl, alkylcycloalkyl, alkylcycloalkenyl and alkoxycarbonyl; wherein each of R ₉ and R _1X is independ¬ ently selected from hydrido, alkyl, dialkylaminoalkyl and phenyl; and wherein m is zero or one and n is a number selected from zero ψrough five;

with the proviso that where m is zero, then R ₅ is selected from hydrido, alkyl, benzyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio- alkyl, heterocyclicalkyl, sulfonylheterocyclicalkyl and acylheterocyclicalkyl; and ^•

with the further proviso that when m is one, then R ₅ is selected from hydrido, alkyl, benzyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, ^' alkylthioalkyl and imidazolernethy1.

Two distinct families of renin-inhibiting compounds are specified within Formula I, namely, those families being defined by the values of m. A first family of compounds consists of those c.-amino acid derivatives defined by the condition where m is zero. A second family of compounds consists of those β-amino acid derivatives defined by the condition where m is one.

A preferred family of compounds consists of those compounds of Formula I wherein X is selected from oxygen atom, methylene and __^NR ₁₀ with R ₁₀ selected from hydrido, alkyl and benzyl; wherein Rj is selected from lower alkyl, alkoxycarbonyl, loweralkanoyl, cycloalkyl, dialkylaminoalkyl,

heterocyclicalkyl, phenyl and benzyl; wherein R ₂ is selected from hydrido, methyl, ethyl and isopropyl; wherein R ₃ is selected from phenylalkyl, cyclohexyl- alkyl, naphthylmethyl, pyridylmethyl, pyridylethyl and pyridylpropyl; wherein each of R ₄ and R ₆ is independ¬ ently selected from hydrido and methyl; wherein R ₇ is selected from substituted or unsubstituted cyclohexyl- methyl and benzyl, either one of which may be substitu¬ ted with one or more groups selected from alkyl, alkoxy, halo and haloalkyl; wherein R ₈ is selected from hydrido, ethyl, n-propyl, n-butyl, isobutyl and fluoroalkyl; wherein each of R ₉ and R _X1 is independently selected from hydrido and lower alkyl; wherein m is zero or one and n is a number selected, from zero through five; or a pharmaceutically-acceptable salt thereof;

with the proviso that where m is zero, then R ₅ is selected from hydrido, alkyl, benzyl, cycloalkyl, cycloalkylalkyl, imidazolemethyl, imidazoleethyl, thiazolemethyl, pyridylmethyl, sulfonylimidazolemethyl and acylimidazolemethyl; and

with the further proviso that when m is one, then

R ₅ is selected from hydrido, alkyl and imidazolemethyl.

A further preferred family of compounds consists of those compounds of Formula I wherein X is selected from oxygen atom, methylene and _ NR ₁₀ with Rio selected from hydrido, alkyl and benzyl; wherein Rj. is selected from lower alkyl, alkoxycarbonyl, loweralkanoyl, benzyl, imidazolealkyl and pyrimidyl- alkyl; wherein R ₂ is selected from hydrido, methyl, ethyl and isopropyl; wherein R ₃ is selected from benzyl, phenethyl, phenpropyl, cyclohexylmethyl, pyridylmethyl and 2-pyridylethyl; wherein each of R and R _β is independently selected from hydrido and

methyl; wherein R ₇ is cyclohexylmethyl; wherein R ₈ is selected from ethyl, n-propyl, isobutyl and perfluoro- propyl; wherein each of R ₉ and R _u is independently selected from hydrido and methyl; wherein m is zero or one and n is a number selected from zero through five; or a pharmaceutically-acceptable salt thereof;

with the proviso that where m is zero, then R ₅ is selected from imidazolemethyl, thiazolemethyl and isobutyl; and

with the further proviso that when m is one, then R ₅ is methyl or ethyl.

A more preferred family of compounds consists of those compounds of Formula I wherein X is selected from oxygen atom, methylene and^NR ₁₀ with R ₁₀ selected from hydrido and methyl; wherein R _x is selected from methyl, ethyl, benzyl, t-butyloxycarbonyl and 2-(N-piperidinyl)ethyl; wherein R _x and R ₉ may be taken together to form an alkylene chain to form a cyclic structure having 6 ring members; wherein R ₂ is - selected from hydrido, methyl, ethyl and isopropyl; wherein R ₃ is selected from benzyl, phenethyl, pyridyl¬ methyl, cyclohexylmethyl and 2-pyridylethyl; wherein each of R ₄ and R ₆ is independently selected from hydrido and methyl; wherein R ₇ is cyclohexylmethyl; wherein R ₈ is independently selected from ethyl, n-propyl and isobutyl; wherein each of R ₉ and R _1X is hydrido; wherein m is zero or one and n is a number selected from zero through five; or a pharmaceutically- acceptable salt thereof; with the proviso that where m is zero, then R ₅ is selected from imidazolemethyl, thiazolemethyl and isobutyl; and with the further proviso that when m is one, then R ₅ is methyl or ethyl.

A particularly preferred family of compounds consists of those compounds of Formula I wherein X is selected from oxygen atom and methylene; wherein R _x is selected from methyl, ethyl, t-butyloxycarbonyl and 2-(N-piperidinyl)ethyl; wherein R ₂ is selected from hydrido, methyl, ethyl and isopropyl; wherein R ₃ is selected from benzyl, cyclo¬ hexylmethyl, phenethyl, pyridylmethyl and 2-pyridyl¬ ethyl; wherein each of R ₄ and R ₆ is independently selected from hydrido and methyl; wherein R ₇ is cyclohexylmethyl; wherein R ₈ is isobutyl; wherein each of R ₉ and R _π is hydrido; wherein m is zero or one and n is a number selected from zero through five; or a pharmaceutically-acceptable salt thereof; with the ^' proviso that where m is zero, then R ₅ is selected from imidazolemethyl, thiazolemethyl and isobutyl; and with the further proviso that when m is one, then R ₅ is methyl or ethyl.

A more particularly preferred family of compounds consists of those compounds of Formula I wherein X is selected from oxygen atom and methylene; wherein R _x is methyl or t-butyloxycarbonyl; wherein R ₂ is selected from hydrido, methyl, ethyl and isopropyl; wherein R ₃ is selected from benzyl, phenethyl, pyridyl- methyl and 2-pyridylethyl; wherein each of R ₄ and R ₆ is independently selected from hydrido and methyl; wherein R ₇ is cyclohexylmethyl; wherein R ₈ is isobutyl; wherein each of R ₉ and R _u is hydrido; wherein m is zero or one and n is a number selected from zero through three; or a pharmaceutically-acceptable salt thereof; with the proviso that where is zero, then R ₅ is selected from imidazolemethyl and isobutyl; and with the further proviso that when m is one, then R ₅ is methyl or ethyl.

A most preferred family of compounds of Formu-1-a I consists of the following compounds:

0-[N-methyl-N-(2-me hoxyethyl)ammocarbonyl]-3-L-phenyl- lactyl-L-histidineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane;

0-[N-methyl-N-(2-ethoxyethyl)ammocarbonyl]-3-L-phenyl- lactyl-L-histidineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane;

O-[N-methyl-N-(2-methoxyethyl)ammocarbonyl]-3-L-phenyl- lactyl-L-leucineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane;

O-[N-methyl-N-(2-ethoxyethyl)aminocarbonyl]-3-L-phenyl- lactyl-L-leucineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane;

O-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-3-L-phenyl- lactyl-α-(R)-methyl-β-alanineamide of (2S,3R,4S)-2- amino-l-cyclohexyl-3,4-dihydroxy-6-methylheptane;

0-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-3-L-homo- phenyllactyl-α-(R)-methyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane;

0-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-3-L-homo- phenyllactyl-α-(R)-ethyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane; and

3-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-2-(R)- (2-pheny1ethy1)-propionyl- -(R)-ethyl-β-alanineamide of (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6- methylheptane.

Unless otherwise described, the chemical groups recited herein shall have meanings as follows: "Alkyl" includes linear and branched radicals; "lower alkyl" means alkyl radicals containing one to about 10 carbon atoms in a linear or branched config¬ uration, examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, 1-methylhexyl, n-heptyl, ^" 2-ethylheptyl, n-octyl, 3-propyloctyl, n-nonyl, 4-butylnonyl, n-decyl and the like.

"Haloalkyl" means alkyl radicals substituted at one or more substitutable positions with one or more halo groups. Preferred haloalkyl group are those provided by lower alkyl- radicals substituted at least at one position with one, two or three halo groups such as fluoro or chloro, a specific example of which is trifluoromethyl. "Alkylcycloalkyl" means a cyclized alkyl having from four to about nine ring carbon atoms, any one or more of the substitutable ring carbon atoms being substituted with an alkyl group, preferably a lower alkyl group. "Alkoxycarbonyl" means an oxycarbonyl radical having an alkyl, preferably lower alkyl, group attached to the oxygen atom.

"Aryl" means an aromatic hydrocarbon radical provided by a homocyclic or heterocyclic ring system, such as phenyl, naphthyl, and pyridyl. "Acyl" means a carbonyl moiety attached to a hydrocarbon moiety, typically an alkyl or lower alkyl group, of which "loweralkanoyl" is a preferred subclass including, for example, acetyl group. "Heterocyclicalkyl" means a cyclized group having three to about ten ring members, of which one to about three of such ring members is a hetero atom selected from oxygen, nitrogen and sulfur, with the remaining ring members being carbon atoms and such cyclized group being fully unsaturated, or partially saturated, or fully saturated, and having an alkyl group attached to any

ring member of the heterocyclic ring, through which alkyl group the heterocyclic ring is attached to the Formula I backbone. Examples of heterocyclicalkyl are imidazolemethyl, ^' 2-imidazoleethyl, pyrimidmemethyl, 2-pyrimidineethyl, pyridylmethyl and 2-pyridylethyl.

Based upon the foregoing, the meanings of the following terms should be readily discernible, namely, "acylaminoalkyl", "cycloalkyl", "alkoxyacyl", "cycloalkylalkyl", "phenylalkyl" and "alkoxy".

Compounds of Formula I have at least five asymmetric carbons. Such compounds whether in their pure form or as diastereomeric mixtures are embraced in the Formula I compounds of the invention. Many of the more active renin inhibitors are provided by compounds having a specific arrangement of stereogenic carbons. Within Formula I, reading from the N terminus to the C terminus (terminating with the diol moiety), the preferred configurations for the asymmetric carbons are as follows:

Compounds of Formula I have been found to inhibit renin and thus limit the production of angiotensin I which, in turn, limits the production of angiotensin II in mammals. Angiotensin II is a potent vasoconstrictor and participates in the formation of aldosterone which regulates sodium and water balance in mammals. Thus, compounds of Formula I are

therapeutically useful in methods for treating hyper¬ tension by administering to a hypertensive patient a therapeutically-effective amount of a compound of Formula I. The phrase "hypertensive patient" means, in this context, a mammalian subject suffering from the effects of hypertension or susceptible to a hypertensive condition if not treated to prevent or control such hypertension.

These compounds can be formulated into pharmaceutically-acceptable dosage forms by any of a number of well-known carriers or diluents. The compounds can be formulated using pharmacologically- acceptable acid addition salts and can be used in a suitable hydrated form. The formulated compounds can be administered in oral dosage forms such as tablets, capsules, pills, powders, or granules. The compounds can also be administered intramuscularly, using forms known to the pharmaceutical art. In general, the preferred form of administration is oral. A thera- peutically effective but non-toxic quantity of the compound is employed in treatment of high blood pressure - in mammals. The dosage regimen for preventing or treating hypertension with the compounds of Formula I is selected upon consideration of a variety of factors, including the type, age, weight, sex, and medical condition of the patient, the severity of the hypertension, the route of administration, and the particular compound employed. Dosages of the compounds are ordinarily in the range from about 0.5 to about 100 mg/kg (active compound-to-body weight), and preferably from about 1.0 to about 20 mg/kg given orally or by injection.

Compounds of Formula I are also useful as diagnostic agents for identification of hypertension due to renin excess.

Compounds of Formula I can be administered as prodrugs. Preferably, esterification of one or more of the hydroxyl groups of the compounds of Formula I is accomplished with amino acids to make aminoesters, succinates to make succinic acid esters, alkanoic acids to make carboxylic acid esters such as valerates, or phosphates to make phosphoric acid esters. Aminoesters and valerates of the Formula I compounds are more preferred.

Procedures for preparation of compounds of

Formula I are set forth in the schemes and descriptions under General Synthetic Schemes I & II taken with the specific procedures described in Examples 1-16 which follow thereafter. The substituents X and R _x through R _u are as described above for the Formula I substituents.

GENERAL SYNTHETIC SCHEMES I & II

A suitably protected amino aldehyde 1 • (Scheme I) is treated with a Grignard reagent, prefer- ably vinylmagnesium bromide to obtain vinyl carbinol 2. This material, suitably protected, is oxidized, preferably with ozone, followed by dimethyl sulfide treatment to ^' give 3. This aldehyde is reacted with an organometallic reagent such as isobutylmagnesium chloride to give compound 4. This intermediate is deprotected, then coupled, using standard amide/peptide coupling methodology, to either alpha or beta amino acid derivatives, suitably protected, to give compound 5. This intermediate is deprotected then coupled using standard amide/peptide coupling methodology to intermediate 9 (shown in Scheme II) to give renin inhibitor 6. Synthetic Scheme II shows synthetic routes to intermediate 9, using the reaction of

intermediates 7 and 8 followed by deprotection. The synthesis of various types of intermediates 8 (shown more explicitly as intermediates 11,- 13 and 15) is depicted, depending on whether X is 0, CH ₂ or NHR ₁₀ . If X = 0, a suitably protected lactic acid derivative is treated with phosgene or carbonyl diimidazole to give intermediate 11. If X = CH ₂ / a suitably protected succinic acid derivative is activated by treatment with base/isobutylchloroformate or other standard activating agent to give intermediate 13. If X = NHR ₁₀ , a suitably protected amino acid derivative is activated by treatment with phosgene or other activating agent to give intermediate 15.

Synthetic Scheme I

P, is an N-protecting group P ₂ is H or N-protecting group P3 is H or oxygen protecting group

-15/1-

Synthetlc Scheme II

X- NR ₁₀ H:

Q is an activating group such as Cl, imi azole P ₄ is afkyl. benzyl, oxygen protecting group

-16-

The following examples are provided to illustrate the present invention and are not intended to limit the scope thereof. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures expressed are in degrees Centigrade. Within the foregoing synthetic description and examples which follow, abbreviations have meanings as indicated below:

Example 1

(3S,4S)-N-[(tert-Butyloxy)carbonyl]-4-amino-3-acetoxy- 5-phenylpentene

The preparation of the above intermediate was carried out using the procedure described in Hanson, et al., (1985) J. Org. Chem. 50, 5399.

-17-

Example 2

(2R,3S)-N-[(tert-Butyloxy)carbonyl]-3-amino-2-acetoxy- 4-phenylbutanal

The preparation of the above intermediate was carried out as described in Hanson, et al. above. Ozone/oxygen was bubbled at -70° into a solution of 2.55g (8.0 mmol) of the allylic acetate of Example 1 in lOOmL of methylene chloride until a deep blue color persisted. Oxygen was introduced until the blue color completely faded, then 3.0 mL of Me ₂ S was added and the solution was allowed to warm to 0-5° and stand overnight. The solvent was removed at 0° under vacuum yielding the title compound as a thick yellow oil which was used in the following step without purifi- cation.

Example.3

(2S,3R,4S)-N-[(tert-Butyloxy)carbonyl]-2-amino-1-phenyl- 3, -dihydroxy-6-methylheptane

The oil prepared in Example 2 was dissolved under nitrogen in lOOmL of dry THF and cooled to -70°. To this solution was added 13mL (26mmol) of a 2.0M solution of isobutylmagnesiu chloride in ether and the stirred mixture was allowed to warm to room temperature and stir for 2 hrs. After decomposition with MeOH/H ₂ 0 the mixture was diluted with ether, washed with saturated NH ₄ C1 solution twice, then dried and the solvents stripped off under vacuum. The residue was allowed to stand overnight in 80% MeOH- H ₂ 0 containing excess ammonium hydroxide. The MeOH was stripped off and the mixture was extracted with ether. These extracts were combined, washed with water,

-18-

dilute KHS0 ₄ , then dried and evaporated to give 2.36g of a yellow glass which crystallized from 50mL of pentane on standing overnight. The yellow-white powder obtained was recrystallized from ether-hexane and furnished the title compound (0.41g) as white, hairy needles, mp 134-136°, Rf (ether): single spot, 0.6. By chromatography of the mother liquors and crystalli¬ zation of the appropriate fractions, an additional 0.22g of product, mp 138-139°, was obtained. Anal: Calc'd. for C ₁₉ H ₃₁ N0 ₄ (337.45): C, 67.62; H, 9.26; N, 4.15. Found: C, 67.51; H, 9.43; N, 4.24.

Example 4

(2S,3R,4S)-N-[(tert-Butyloxy)carbonyl]-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane

The diol of Example 3, 0.27g, was reduced in

MeOH with 60psi H2 at 60° in 3 hrs using 5% Rh/C catalyst. After filtering, the solvent was stripped off and the white crystals were recrystallized from CH ₂ Cl ₂ -hexane to furnish tiny needles of the title compound, 0.19g, mp 126-128°; further recrystalliza¬ tion gave mp 128.5-129.5°. Rf (ether): single spot, 0.8. Anal: Calc'd. for C ₁₉ H ₃₇ N0 ₄ (343.50): C, 66.43; H, 10.86; N, 4.08. Found: C, 66.43; H, 11.01; N, 4.03.

-19-

Example 5

L-leucineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4- dihydroxy-6-methylheptane

The title compound of Example 4 was treated with trifluoroacetic acid (TFA) for 30 minutes at room temperature and the solvent evaporated. The residue was neutralized with aqueous potassium carbonate and the free amine was extracted with ethyl acetate. This amine was then coupled to Boc- L-leucine-OH following the general procedure given in Example 6. The resulting amide was treated with TFA for 30 minutes at room temperature and the solvent evaporated. The residue was neutralized with aqueous potassium carbonate and the mixture extracted with ethyl acetate. After evaporation, the title free base was obtained:

Rf = 0.45 (single spot, 9:1 methylene chloride-MeOH, silica); 400 MHz NMR (DMSO) spectrum: consistent with proposed structure. Anal: Calc'd. for C ₂₀ H ₄₀ N ₂ 0 ₃ + 0.5 H ₂ 0: C, 65.70; H, 11.31; N, 7.67. Found: C, 65.62; H, 11.01; N, 7.49.

Example 6

Boc-(im-Tosyl)-L-histidineamide of (2S,3R,4S)-2-amino- l-cyclohexyl-3,4-dihydroxy-6-methylheptane

To a stirred solution of N-Boc-(im-tosyl)-L- histidine (809mg, l.δeq) in methylene chloride (5ml) cooled with an ice/salt bath was added N-methylpiperi- dine (0.240ml, 1.6eq) followed by isobutylchloroformate (0.224ml, 1.4eq). After 5 minutes, the free base (300mg, 1.23mmol), which had been previously formed by treating the title compound of Example 4 with trifluoro-

-20-

acetic acid followed by potassium carbonate as described in Example 5, dissolved in methylene chloride (5ml) was added and the reaction mixture was stirred at 0° overnight ca. 15h. The methylene chloride was evapor- ated in vacuo to afford an oily residue which was partitioned between ethyl acetate and saturated sodium bicarbonate. The organic layer was separated and further washed with KHS0 solution (1M) followed by NaHC0 ₃ (1M). The ethyl acetate, layer was dried (MgS0 ₄ ) and evaporated in vacuo to afford a white solid, which was recrystallized from methanol/diethyl ether. This gave the title compound; (560mg, 72% yield), 300MHz ^l E NMR was fully consistent with the proposed structure. Anal: Calc'd. for C ₃₂ H ₅₀ 0 ₇ N ₄ S + 0.75 H ₂ 0: C, 59.28; H, 8.01; N, 8.64. Found: C, 59.31; H, 7.98; N, 8.63.

Example 7

(im-Tosyl)-L-histidineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane

To a stirred solution of the title compound of Example 6 (3.78g, 5.96mmol) in methylene chloride (20mL) and methanol (5mL) was added trifluoroacetic acid (25mL). The reaction mixture was stirred at room temperature for 30min and then poured onto saturated sodium bicarbonate solution. The solution was adjusted to pH>12 by addition of potassium carbonate and then extracted with ethyl acetate. The organic extracts were dried (MgS0 ₄ ), and evaporated to afford a solid white residue. Recrystallization from methanol/diethyl

-21-

ether gave the title compound; (2.8g, 88% yield); 300MHz ¹ H NMR spectrum: consistent with the proposed structure. Anal. Calc'd. for C ₂₇ H ₄₂ N ₄ 0 ₅ S + 0.7 H ₂ 0: C, 59.25; H, 7.99; N, 10.24. Found: C, 59.29; H, 7.75; N, 10.15.

Example 8

O-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-3-L-phenyl- lactyl-L-histidineamide of (2S,3R,4S)-2-a_τ_ino-l-cyclo- ^' hexyl-3,4-dihydroxy-6-methylheptane

To a stirred solution of 0-(N-methyl-2- methoxyethylaminocarbonyl)-3- L-phenyllactic acid (200mg, 0.375 mmol) in methylene chloride (5mL) cooled to -10° was added N-methylpiperidine (0.08mL, 1.6eq) followed by isobutylchloroformate (0.07ml, 1.4eq) . After 5 min, the title compound of Example 7 (300mg, 1.23mmol) in methylene chloride (5ml) was added and the reaction mixture was stirred at 0° for ca 15 hours. The methylene chloride was evaporated in vacuo to afford an oily residue which was partitioned between ethyl acetate and saturated sodium bicarbonate. The organic layer was separated and dried (MgS0 ₄ ) . After evaporation the crude residue was dissolved in

-22-

methanol (4mL) to which potassium hydroxide solution (lmL,lM) was added. The reaction mixture was stirred for 30 minutes, evaporated to dryness and the residue extracted into ethyl acetate . The organic extracts were washed with water followed by saturated aqueous sodium bicarbonate and dried over MgS0 ₄ . Evaporation of the solvent gave a yellow residue which was recrystallized from diethyl ether to afford the title compound (120mg, 50% yield). Anal: Calc'd. for C ₃₄ H ₅₃ N ₅ 0 ₇ + 1.8 H ₂ 0: C, 60.39; H, 8.44; N, 10.36.- Found: C, 60.23; H, 8.28; N:10.49. 300 MHz -E NMR was consistent with the proposed structure.

Example 9

0-(N-methyl-N-(2-methoxyethyl)aminocarbonyl)-3-L-phenyl- lactyl-L-leucineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane

0-(N-methyl-2-methoxyethylaminocarbonyl)-3- L-phenyllactic acid (Example 10) (94mg, 0.33mmol) and N-methylpiperidine (31mg, 0.31mmol) was dissolved in CH ₂ C1 ₂ (2mL) and the solution was cooled to -10°C. To

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this cooled solution was added isobutylchloroformate (42mg, 0.31mmol) and the mixture stirred for 5 minutes at which time a solution of the title compound of Example 5 (76mg, 0.21mmol) in CH ₂ C1 ₂ (1.5mL) was added 5 via pipette. The resulting solution was stirred at -10°C for 2 hours followed by 18 hours at +5°C. The solvent was then removed in vacuo and the residue dissolved in ethyl acetate/H ₂ 0. After partitioning, the organic layer was washed in succession with: 0.5M 0 citric acid, sat'd aq. NaHC0 ₃ and-brine. Drying over MgS0 ₄ , filtering and stripping off the solvent yielded 129mg of a sticky oil. Dissolving this oil in a minimum of warm hexane followed by cooling yielded the title compound (43mg) as small prisms: mp: -5 72-76°C. 200 MHz -E NMR was consistent with proposed structure. Anal: Calc'd. for C ₃₄ H ₅₇ N ₃ 0 ₇ + 1/2H ₂ 0 C, 64.94; H, 9.30; N, 6.68. Found: C, 64.91; H, 9.23; N, 6.34.

Example 10

0 ^" 0-(N-methyl-2-methoxyethylaminocarbonyl)-3-L-phenyl- lactic acid

Methyl L-phenyllactate (5.4g, 30mmol) was dissolved in 2.5M phosgene in toluene (140 mL). The solution was cooled to -15° and triethylamine (6.14g, 5 61mmol) was added over a 5 minute period. The solution was allowed to warm to room temperature and stirred overnight. The solvent was removed in vacuo and the residue was taken up in methylene chloride (100 mL) and filtered. The filtrate was cooled to -15° and

30 a solution of N-methyl-2-methoxyethylamine (3.77g) and triethylamine (6.14g) in methylene chloride (50 mL) was added dropwise over a 15 minute period. After 1

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hour at -15°, the solution was stirred overnight ^' at room temperature. Water was added, the organic phases separated, washed with dilute HC1, brine, dried over sodium sulf te and the solvent removed in vacuo. The residue was chromatographed on silica to give an ester (1.68g): Anal: Calc'd.: C, 61.00; H, 7.17; N, 4.74. Found: C, 60.82; H, 7.03; N, 4 .67. This ester was dissolved in methanol (5.7mL) and 1.5N NaOH (5.7mL) was added. The mixture was stirred at room temperature for 15 minutes.. Water was added followed by washing with methylene chloride. The aqueous phase was acidified with 5% HC1, extracted with methylene chloride, washed with brine, dried over sodium sulfate and the solvent removed in vacuo to the title compound. Anal: Calc'd. for C ₁₄ H ₁₉ N0 ₅ + 0.5 H ₂ 0: C, 57.92; H, 6.94; N, 4.82. Found: C, 57.72; H, 6.87; ^' N, 4.78. 300 MHz -E NMR: consistent with proposed structure.

Example 11

N-Boc-α-(R)-methyl-β-alanineamide of (2S,3R,4S)-2-amino- 1-cyclohexyl-3,4-dihydroxy-6-methylheptane

To a solution of N-Boc- -(R,S)-methyl-β- alanine (137mg, 0.67mmol) in methylene chloride (4mL) at -10°C was added N-methylpiperidine (61mg, 0.61mmol) followed by isobutylchloroformate (75mg, 0.55mmol).

After stirring for 5 min., a solution of (2S,3R,4S)-2- amino-l-cyclohexyl-3,4-dihydroxy-6-methylheptane (lOlmg, 0.41mmol) [prepared from the title compound of Example 4 by treatment with trifluoroacetic acid, followed by aqueous potassium carbonate] in methylene chloride (2mL) was added. The resulting solution was stirred for 3 hours at -10°C, followed by 2 hours at

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room temperature at which time a white solid was isolated by filtration (60mg, 34% yield): Rf = 0.3 (5% MeOH/methylene chloride, silica gel); mp 197-200° ^X E NMR (CDC1 ₃ ): consistent with proposed structure. Anal: Calc'd. for C ₂₃ H ₄₄ N ₂ 0 ₅ + 0.25 H ₂ 0: C, 63.77; H, 10.35; N, 6.46. Found: C, 63.84; H, 10.50; N, 6.45.

Example 12

α-(R)-Methyl-β-alanineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane

The title compound of Example 11 (53mg, 0.12mmol) was stirred with a mixture of trifluoroacetic acid and methanol (9:1, 5mL). The resulting solution was allowed to stand at room temperature for 20 minutes, then the solvent was evaporated. The resulting oil was stirred for 2 hours with aqueous potassium carbonate (5%, lOmL). This mixture was then extracted with ethyl acetate which was dried, filtered and evaporated to give the title compound (40mg, 100%): Rf: 0.10 (5% MeOH/ methylene chloride, silica gel). This material was used without further purification.

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Example 13

0-(N-methyl-N-(2-methoxyethyl)aminocarbonyl)-3-L-phenyl- lactyl-α-(R)-methyl-β-alanineamide of (2S,3R,4S)-2- amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane

0-(N-methyl-2-methoxyethylaminocarbonyl)-3-

L-phenyllactic acid [the title compound of Example 10] (548mg, 1.95 mmol) and N-methylpiperidine (189mg, 1.91 mmol) were dissolved in CH ₂ C1 ₂ (8.0mL) and cooled to -10° with a salt/ice bath. To this solution was added isobutyl chloroformate (221mg, 1.62mmol) in

CH ₂ C1 ₂ (5.0mL) and the resulting solution was stirred at -10° for 5 minutes. Next, a solution of the title compound of Example 12 (402mg, 1.22mmol) in CΞ ₂ C1 ₂ (4.0 mL) was added via pipette, and this solution was stirred at -10° for 2 hours followed by 15 hours at 5°. The solvent was then removed in vacuo and the residue dissolved in ethyl acetate/water. After partitioning, the organic layer was washed twice with 0.5M citric acid, twice with saturated NaHC0 ₃ and once with brine. The organics were dried over MgS0 , filtered and the solvent removed in vacuo to yield 790mg of a yellow oil. Chromatography on silica gel (eluting with 5% CH ₃ 0H/CH ₂ C1 ₂ ) afforded the title compound (404mg) as a

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colorless oil. 200 MHz ^X H NMR is consistent with the proposed structure. Anal: Calc'd. for C ₃₁ H ₅₃ N ₃ 0 ₇ + 0.25 H ₂ 0: C, 63.72; H,-9.16; N, 7.19; Found C, 63.77; H, 9.16; N, 6.99.

Example 14

3-(N-methyl-2-methoxyethylaminocarbonyl)-2R- phenethylpropionic acid

2(R)-phenethyl-mono-methyl succinate (160 mg, 0.68 mmol) was coupled to N-2-methoxyethyl-N-methylamine hydrochloride (85 mg, 0.68 mmol) via the mixed carbonic anhydride method. The crude product was dissolved in a mixture of methanol (3 mL) and IN KOH (1.5 mL), and stirred at r.t. overnight. After evaporation of the solvent, the residue was dissolved in water (3 mL). The aqueous solution was washed with ethyl acetate, then acidified to pH 2 with 0.5 M citric acid. The resulting mixutre was extracted with methylene chloride (3 X 5 mL). The organic layers were combined, dried (MgS0 ₄ ), and evaporated to give the title acid (160 mg, 77% yield). --E NMR: 300 MHz spectrum consistent with proposed structure.

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Example 15

3-(N-methyl-N-(2-methoxyethyl)aminocarbonyl)-2-(R)- (2-phenylethyl)propionyl-α-(R)-ethyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy- 6-methylheptane

Following the coupling procedure of Example 13, the title acid of Example 14 (31 mg, 0.1 mmol) was coupled to α-(R)-ethyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane (34 mg, 0.1 mmol). The crude product was purified by flash chromatography on silica gel, eluting with 20:1 CH ₂ Cl ₂ :MeOH to give pure title compound (20 mg, 32% yield). ^: H NMR: 300 MHz spectrum consistent with proposed structure.

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Example 16

3-(N-methyl-N-(2-methoxyethyl)aminocarbonyl)-2-(R)-2- phenethylpropionyl- -(R)-methyl-β-alanineamide of (2S,3R, S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane

Following the coupling procedure of Example 13, the title compound of Example 14 (73 mg, 0.25 mmol) was coupled to α-(R)-methyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane (55 mg, 0.17 mmol). The crude product was purified by flash chromatography on silica gel, eluting with 20:1 CΞ ₂ Cl ₂ :MeOH to give pure title compound (30 mg, 30% yield). ¹ H NMR: .300 MHz spectrum consistent with proposed structure.

Anal.: C ₃₄ H ₅₇ N ₃ 0 ₆ + 1 H ₂ 0 + 0.3 CH ₂ C1 ₂

Calc. : C 63.64 Found: C 63.94 H 9.28 H 8.99 N 6.49 N 6.20

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Other compounds of Formula I which can be prepared in accordance with the above-described general and specific procedures are as follows:

0-[N-methyl-N-(2-ethoxyethyl)aminocarbonyl]-3-L-phenyl- lactyl-L-histidineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane;

0-[N-methyl-N-(2-ethoxyethyl)aminocarbonyl]-3-L-phenyl- lactyl-L-leucineamide of (2S,3R,4S)-2-amino-l- cyclohexyl-3,4-dihydroxy-6-methylheptane;

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0-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-3-L-homo- phenyllactyl-α-(R)-methyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3, -dihydroxy-6- methylheptane;

O-[N-methyl-N-(2-methoxyethyl)aminocarbonyl]-3-L-homo- phenyllactyl-α-(R)-ethyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane; and

3-[N-methyl-N-(2-ethoxyethyl)aminocarbonyl]-2-(R)- (2-phenylethyl)-propionyl-α-(R)-ethyl-β-alanineamide of (2S,3R,4S)-2-amino-l-cyclohexyl-3,4-dihydroxy-6- methylheptane.

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BIOLOGICAL EVALUATION

Compounds of Formula I were evaluated as inhibitors of human renin in an in vitro assay, as follows: This human renin inhibition test has been previously described in detail [Papaioannou et al. ,

Clinical and Experimental Hypertension, A7(9), 1243-1257 (1985)]. Human renin was obtained from the National Institute for Biological Standards, London. An incubation mixture was prepared containing in total volume of 0.25mL 100 mM Tris-acetate buffer at

—6 pH 7.4, 25 x 10 Goldblatt units of renin, 0.05mL of plasma from human volunteers taking oral contraceptives,

6.0 mM sodium EDTA, 2.4 mM phenyl ethyl sulfonyl fluoride, 1.5 mM 8-hydroxyquinoline, 0.4 mg/mL BSA, and 0.024 mg/mL neomycin sulfate. This mixture was incubated for two hours at 37°C in the presence or absence of renin inhibitors. The produced angiotensin I was determined by radioimmunoassay (New England Nuclear kit) . Test compounds to be assayed were solubilized in DMSO and diluted with lOOmM Tris-acetate - buffer at pH 7.4 containing 0.5% bovine serum albumin (BSA) to the appropriate concentration. The final concentration of organic solvent in the reaction mixture was less than 1%. Control incubations at 37°C were used to correct for effects of organic solvent on renin activity.

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Biological Results:

TABLE I

In Vitro Effect of Compounds on Renin Activity

Inhibition of

Compound Human Renin IC 50

Example 8 1.2 nM Example 9 10 nM Example 13 62 nM

,-8. Example 15 17% inhibition @ 5 X l(T ^ϋ M Example 16 - 45 nM

The oral activity of compounds of Formula I was determined in vivo in Marmoset monkeys in accordance with the following procedure: Common marmosets (Callithrix jacchus, Charles River) were placed on a modified high protein low sodium diet (Purina, St.

Louis, MO) for 1 to 2 weeks. On the day of the test an animal was anesthetized with isoflurane and cannu- lated in the femoral artery and vein for blood pressure monitoring, intravenous saralasin infusion and blood sampling. After allowing the animal to recover from surgery for 2 hr, saralasin was infused at 1 microgram/ min for 15 minutes to confirm that the animal's blood pressure was dependent on angiotensin II levels. The marmoset was allowed to stabilize for 30 min after the saralasin infusion. The test compound was admin¬ istered orally and blood pressure was monitored for 2 hr. Blood samples were taken in K-EDTA for plasma renin activity before, 30 min, and 1 hr after compound administration. Results are shown in Table II.

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Table II Oral Effect of Compounds on Plasma Renin Activity in Sodium Depleted Marmoset

% Reduction of Plasma Renin Activity Compound Tested @ lh (3 mg/kg dose)

Example 8 %

Example 9 79%

Although this invention has been described with respect to specific embodiments , the details of these embodiments are not to be construed as limita¬ tions . Various equivalents , changes and modifications may be made without departing from the spirit and scope of this invention, and it is understood that such equivalent embodiments are part of this invention.