CA1275350C - Lisinopril - Google Patents

Abstract

A B S T R A C T

The invention relates to new carboxyalkyl dipeptide derivatives of the formula:

Description

~ 16271Y

BACKGROUND_OF INVENTION
The invention in its broad aspects relates to carboxyalkyl dipeptides and derivatives thereof which are useful as converting enzyme inhibitors and as anti-hypertensives. The compounds of this invention can beshown by the following formula:
o 71 R3 R IR5 O
R- C- C- NH- C~I- C- N-C- C- R

I
wherein:
R and R6 are the same or different and are hydroxy or loweralkoxy;
Rl is a substituted lower alkyl wherein the sub-stituent is phenyl or halophenyl;
R2 and R7 are hydrogen;
R3 is lower alkyl amino;
R is lower alkyl;
R5 is lower alkyl;
R4 and R5 may be connected together to form an alkylene bridge of from 2 to 4 carbon atoms;
and the pharmaceutically acceptable salts thereof.
The lower alkyl groups except where noted otherwise represented by any of the variables include straight and branched chain hydrocarbon radicals from one to six carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl or vinyl allyl, butenyl and the like.
The R substituted lower alkyl moieties are exemplified by groups such as ~CH2--:' ~ .

.

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R4 and R when joined through the carbon and nitrogen atoms to which they are attached form a 4 to 6 membered ring. Preferred ring has the formula:
r~
-N

COOH
S Preferred are those compounds of Formula I
wherein:
R is hydroxy;
R6 is hydroxy;
R2 and R7 are hydrogen;
R3 is amino lower alkyl;
R4 and R5 are joined to form the preferred ring as defined above;
Rl is as defined previously.
Still more preferred compounds are those pre-ferred compounds of Formula I wherein further Rl is a substituted lower alkyl wherein the alkyl group has 1-4 carbon ato~s and the substituent i5 phenyl.
Most preferred are compounds of Formula I
wherein:
R is hydroxy;
R6 is hydroxy;
R2 and R7 are hydrogen;
R is ~mino lower alkyl;
R4 and R5 are joined through the carbon and nitrogen atom to form proline;
Rl is a substituted lower alkyl wherein the alkyl group has 1-4 carbon atoms and the substituent is phenyl~

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- .: . .. , : . ..
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~ 3 - 16271 The preferred, more preferred and most pre-ferred compounds also include the pharmaceutically accepta~le salts thereof.
The products of Formula I and the pre:~erred 5 subgroups can be produced by one or more of the me~hods and subroutes depicted in the following equations:
O Rl R3 0 R4 R5 0

2 l7 ~ . . . . .
_ ~

-:, , : ... : :

.

. . . . , . :

~! 2~;i3~iÇD

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As will be evident to those skilled in the art and as demonstrated in the Exa~ples, reactive groups not involved in the condensa~ions, such as amino, carboxy, mercapto, etc., may be protected by methods standard in peptide chemistry prior to the coupling reactions and sub-sequently deprotected to obtain the dPsired products.

~ethod I, Route 1 tR2 = H) _ _ ", R30 R4 RS 6 NaBH3CN
R-C-C = 0 + H2NCHC - N - C - C - R
II III

Keto acid (or ester, amide or hydroxamic acid) II is condensed with dipeptide III in aqueous solution, optimally near neutrality, or in suitable organic solvent (CH3C~ for example) in the presence of sodium cyano borohydride to give I (R = H). Alter-natively the intermediate Schiff base, enamine,or aminol may be catalytically reduced to yield produc_ I, for example, by hydrogen in the presence of 10~ palladium on 1~ carbon or of Raney nickel. The ratio of diasterivm~ric products formed may be altered by choice of catalyst.
If R and R6 are carboxy protecting groups such as alkoxy or benzyloxy or the like, they can be converted by well-known methods such as hydrolysis or hydrogenation to (I), where R and R6 are hydroxy.
This is ~rue in all the following methods where the above situation exists.

- . . : . - :

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~ lternatively II can be condensed with an amino acid IV
__ R3 0 Rl ~3 H2NCH-COOH + II Na~H3CN~ R-C-CHNHCHCOOH
IV

under the same conditions to yield amino acid V. Sub-s~quent coupling by known methods with amino acid derivative VI gives I
The ~nown methods encompass reactive group pro-tection during the coupling reaction, for example, by N-formyl, N-t-butoxycarbonyl and N-carbobenzyloxy groups followed by their removal to yield I. Furthermore, the R
functi~n may include removable ester groups such as benzyl, ethyl, or t-butyl. Condensin~ agents in this synthetic route are typically those useful in peptide chemistry such as dicyclohexylcarbodiimide (DCC) or diphenylphosphoryl azide (DPPA) or V may be activated via the intermediacy of active esters such as that derived from l-hydroxybenzotri-azole.

: R4 ~5 V ~ HN - C - CO - R6 DCC ~
~7 (DCC = Dicyclohexylcarbodiimide) or (~I) DPPA
(DPPA = Diphenylph~sphoryl azide) ~ .
.

: ~ , : - . ~ .: .
-. - ~ . . .. . . .

- ' ', -, ii3S;~

O Rl R30 R4 R5 ~ 6 R-C - C - NH2 + O=C-C - N - C - CO - R
~2 R7 V VIII

Amino acid (or ester, amide or hydroxamic acid~ VII
is condensed with ketone VIII under conditions described for Route I to give I.
Alternatively the synthesis can be performed in a step-wise fashion by condensing VII with keto acid IX.

R3 Rl R3 .. . .
VII ~ O = C - COO~ 3 RC - C - N~CH COOH

lX X

t~ yield amino acid X. By known methods as indicated above under R~ute 1, X can be condensed with amino acid derivative VI to give I.
R4 R5 0 Rl R O R4R O
~; n ~ ~ n t ~ n L
X + HN - C CO - R ~ R - C~ C - NHCHC-N-C-C-RV

VI
_ In the special case of Rl bearing an Q-amino substituent, the carbonyl and amino groups can be convenient-ly protected as a ~-lactam function.

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Method 2 Route 1 _ R3 o R4 P~5 Rl H2N - CH - C - N - C - COR6 ~ X-C - COR

III XI

O Rl R3 R4 R5 R-C - C - NH - CH - C - N - C - CORV
~2 R7 The dipeptide III is alkylated with the appropriate Q-haloacid (ester or amide) or a-sulfonyloxy acid (ester or amide) XI under basic conditions in water or an organic solvent.
X is chlorine, bromine, iodine or alkyl sulfonyl-oxy or aryl sulfonyloxy.
Alternatively the synthesis can be performed in a stepwise fashion R3 Rl Rl ~3 H N-CH-COOH ~ X-C - COR --~RCO - C - NH-CH - COOH

IV XI X

R4 R5 o Rl R3 O R4 R5 f HN - C - COR6 ~ R-C-C -NH-CH-C-N -C -CGR6 R7 ~2 R7 VI
.
X = Cl , Br, I , alkylsulfonyloxy or arylsulfonyloxy.

.

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The aminoacid IV is alkylated by the Q-halo-acid (ester or amide) or ~-sulfonyloxy acid (ester or amide) XI under basic conditions to yield compounds X.
This is condensed by standard methods as indicated under Route 1 with the aminoacid ~ester or amide) Vl to afford I. _ Reductive cleavage of a benzyl ester I (where R6 is benzyloxy and R is alkoxy) will yiel~ compounds of Formula I wherein R is alkoxy and R6 is hydroxy, and where R6 is alkoxy and R is benzyloxy, will yield com-pounds of ~ormula I wherein R is hydroxy and R6 is alkoxy.

Route 2 RlR3 O R4RS
R-C - C -NH2 + X-CH-C-N-C -COR

VI I XI I

R R O R R O
R-C-C-NH-CH-C-N-C -CRV
.. . .
o R2 R7 X = Cl, Br, I, alkyl sulfonyloxy or aryl sulfonyloxy~
The aminoacid or derivative VII is alkylated with the appropriately substituted c-haloacetyl or a~
sulfonyloxy acetyl aminoacid XII under basic conditions in water or other solvent to obtain compounds of Formula I.
Alternatively, the synthesis can be performed in a step-wise fashion by condensing an aminoacid ester VII with a substituted -- .

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Rl R3 Rl R3 RCO -C-NH2 + X-CH-COOH ~ RCO-C-NH-CH-COOH
R2 ~2 VII XIII X
.

X ~HN-c~-coR6 _ Rl ' VI

Rl R30 R R O
R-c-c-NH-c-c-N-c --CR6 ,. . . .
O R R
I

a-haloacetic acid ox q-sulfonyloxy acetic acid (XIII) to yield the intermediate X. By known methods described under Route 1, X can be coupled with an aminoacid VI or derivative to give I.
As desired, protecting groups may be removed by ~nown methods.
The starting materials which are required for the above processes herein described are known in the literature or can be made by known methods from known ; 10 ~ starting materials.
: In products of general Formula I, the carbon atoms to which Rl, R3 and ~ are attached may be asymmetric. The compounds accordingly exist in disastereoisomeric forms or in mixtures thereof. The a~ove described syntheses can ;
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utilize racemates, enantiomers or diastereomers as starting materials. When diastereomeric products result from the synthetic procedures, the diastereomeric products can be separated by conventional chromatographic or frac-5 tional crystallization methods. In general, the aminoacidpart-structures, i.e., O Rl ~3 R
n ~ ~ 1 7 n R-C-C-NH- , ~NH-CHCO -- and -N-C -C-R ! R7 of Formula (I) are preferred in the S-configuration.
The compounds of this invention form salts with various inorganic and organic acids and bases which are 10 also within the scope of the invention. Such salts include ammonium salts, alkali metal salts like sodium and potassium salts (which are preferred), alkaline earth metal salts like the calcium and magnesium salts, salts with or~anic bases e.g., dicyclohexylamine salts, N-methyl-15 D-glucamine, sal~s with amino acids like arginine, lysine and the like. Also salts with organic and inorganic acids may be p~-epared, e.g., HCl, HBr, H2SO4, H3PO4, methane-sulfonic, toluensulfonic, maleic, fumaric, camphorsulfonic.
The non-toxic phvsiologically acceptable salts are pre-20 ferred, although other salts are also useful, e.g., inisolating or purifying the product.
The salts may be formed by conventional means, as by reac~ing the free acid or free base forms of the product with one or more equivalents of the appropriate 25 base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed _ vacuo or by freeze-drying or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.

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~2~

The compounds of this invention inhibit angio-tensin converting enzyme and thus block conversion of the decapeptide angiotensin I to angiotensin II. Angiotensin II is a ~otent pressor substance. Thus blood-pressure lowering can result from inhibition of its biosynthesis especially in animals and humans whose hypertension is angiotensin II related. Furthermore, converting enzyme degrades ~he vasodepressor substance, bradykinin. There-fore, inhibitors of angiotensin converting enzy~e may lower blood-pressure also by potentiation of bradykinin. Al-thou~h the relative importance of these and other possible mechanisms remains to be established, inhibitors of angio-tensin converting enzyme are effective antihypertensive agents in a variety of animal models and are useful clinically, for example, in many human patients with r no-vascular, malignant and essential hypertension. See, for example, D. W. Cushman et al., Biochemistry 16, 5484 (1977~.
The evaluation of converting enzyme inhibitoxs is guided by in vitro enzyme inhibition assays. For example, a useful method is that of Y. Piquilloud, A. Reinharz and M. ~oth, Biochem. Biophys. Acta, 206, 136 (1970) in which the hydrolysis of carbobenzyloxyphenyl-alanyl~istidinylleucine is measured. In vivo evaluations may be made, for example, in normotensive rats challenged with angiotensin I by the technique of J. R. Weeks and J. A. Jones, Proc. Soc. Exp. Biol. Med., 104, 646 ~1~60) or in a high renin rat model such as that of S. Koletsky et al., Proc. Soc. Exp. Biol. Med., 125, 96 (1967).
Thus, the compounds of this invention are useful as antihypertensives in treating hypertensive mammals, including humans and they can be utilized to achieve the reduction of blood pressure by formulating in compositions such as tablets, capsules or elixirs for oral administra-tion or in ~terile solutions or suspensions for parenteral administration. The compounds of this in~ention can be : , : . ................... . : . . , -:, :
. ~ ' '- , : . . :, ~27~35~) administered to patients tanimals and human) in need of such treatme-t in a dosage range of 5 to S00 mg per patient generally given several times, thus giving a total daily dose of from 5 to 2000 mg per day. The dose will S vary depending on severity of disease, weight of patient and other factors which a person skilled in the art will recognize.
Also the compounds of this invention may be given in combination with other diuretics or antihyper-tensives. Typically these are combinations whoseindi~idual per day dosages range from one-fifth of the minimally reco~mended clinical dosages to the maximum reco~mended levels for the entities when they are given singly. To illustrate these combinations, one of the anti-lS hypertensives of this invention effective clinically inthe range 15-200 milligrams per day can be effectively combined at levels ranging from 3-200 milligrams per day with the following antihypertensives and diuretics in dose ranges per day as indicated:
hydrochlorothiazide (15-200 mg), chlorothiazide (125-2000 mg), ethacrynic acid (15-200 mgJ, amiloride ~5-20 mg), furosemide (5-80 ma), propanolol (20-4~0 mg), timolol ~5-50 mg.) and metnyldopa (65-2000 mg). In addition, the triple drug combinations of hydrochlorothiazide (15-200 mg) plus amiloride (5-20 mg) plus converting enzyme inhibitor of this invention (3-200 mg) or hydrochlorothiazide (15-200 mg) plus timolol (5-50 mg) plus the converting enzyme inhibitor of this invention (3-200 mg) are effect-ive combinations to control blood pressure in hypertensive patients. The above dose ranges will be adjusted on a unit basis as necessary to permit divided daily dosage.
~lso, the dose will vary depending on the severity of the disease, weight of patient and other factors which a person skilled in the art will recognize.
Typically the combinations shown above are formulated into pharmaceutical compositions as discussed below.

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- 13 - 16~7~Y

~ bout 10 to 500 mg. of a compound or mixture of compounds of ~ormula I or a physiologically acceptable salt is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice, The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.
Illustrative of the adjuvants which may be incorporated in tablets, capsules and the like are the following: a binder such as gum,tragacanth, acacia, corn starch or gelatin; an excipient such as microcrystal-line cellulose; a disintegrating agent such as corn starch, pregelatinized starch, alqinic acid and the like7 a lubri-cant such as magnesium stearate; a sweetening a~ent suchas sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry. When the dosage unit form is a capsule, it may contain in addition to mate-rials of the above type, a liquid carrier such as fatty oil.
~o Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
~or instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active com-pound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
Sterile compositions for injection can be formulated according ~o conventional pharmaceutical practice by dissolving or suspending the ac~ive sub-stance in a vehicle such as water for injection, anaturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc. or a - ' - ' , ' ' ' '' ~ ':
. . . - . ~ . . .
.. . . . .

ii35~
- 14 - lfi~71Y

synthetic fatty vehicle like ethyl oleate or the like.
Buffers, preservatives, antioxidants and the like can be incorporated as required.
Th~ following examples are illustrative of the invention and constitute especially preferrea embodi-ments~ The preferred diastereomers of these examplas axe isolated by column chromatography or fractional crystallization.

A. N-(l-Carbox~-3-phenylpro~yl)-L-lysyl-L-proline A solution of the sodium salt of 2-oxo-4-phenylbutyric acid and N-t-Boc-L-lysyl-L-proline is adjusted to pH 7 with caustic and treated with sodium cyanoborohydride at room temperature for several days.
Essentially all of the t-Boc protecting group is cleaved when the product is absorbed on strong acid ion exchange resin. The crude N-(l-carboxy-3-phenyl-propyl)-L-lysyl-L-proline is eluted from the resin with 10% ammonia, freeze dried, and purified by gel fil-tration chromatography (LH-20). A minute peak for t-Boc protons in the Nmr spectrum disappears when the product is treated with ethyl acetate that is 4 N in hydrogen chloride gas. ~he nmr spectrum of th~ resulting HCl salt of the product is consistent with structure. The mass spectrum shows a molecular ion at 693 m/e for the tetrasilylated species. Chromatography on XAD-2 resin using 3.5% acetcnitrile in 0.1 molar ammonium hydroxide affords N-~-(l(S)-carboxy-3-phenylpropyl)-L~lysyl-L-proline.
B. N-a-(l(S)-Car~oxy-3-phenylpropyl)-L-lysyl-L-proline A solution of the sodium salt of 2-oxo-4-phenylbutyric acid and N-t-Boc-L-lysyl-L-proline is adjusted to pH 7 with caustic and treated with sodium cyanoborohydride at room temperature for several days.

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: - . . .
- . .
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The product is absorbed on strong acid ion exchange resin, and eluted with 2% pyridine in water.
Product-rich cuts are stripped to a glass and treatPd with 4 N HCl in ethyl acetate to remove the t-Boc pro-tecting group. The resulting hydrochloride salt isconverted to the free base by absorbing on strong acid ion exchange resin and eluting with 2~ pyridine in water. Freeze drying of product-rich cuts affords N-a-(l-carboxy-3-phenylpropyl)-L-lysyl-L-proline as a white fluffy solid. The nmr spectrum is consistent with structure. The mass spectrum shows a molecular ion at 549 for the disilylated species. Chromatography affords the desired isomer.

N-~-(l(S)-Carboxy-3-~henylE~opyl)-L-lys~ -proline N-a- ( l-Carboxy-3-phenylpropyl)-L-lysyl-L-proline, a mixture of diastereomers prepared as de-scribed in Exæmple lB is purified by gel filtration chromatography in methanol (LH-20). The XAD-2 column prepared as described in Example 1 is equilibxated at 53C with 0.1 M NH40H containing 4% acetonitrile. The isomer mixture from above (250 mg) is dissolved in 10 ml of the same solvent and added to the column. When the col~unn is eluted with this solvent, the first isomer emerges in the volume range 320-360 ml of eluate. The second isomer emerges in the range 450-540 ml of eluate.
Intermediate fractions contain a mixture of isomers.
When fractions containing the first isomer are freeze-dried, 72 mg of fluffy white solid is obtained. This is the more active isomer and is the S,S,S confi~uration by analogy to the more active isomer of N-~ (1-carboxy-3-phenylpropyl)-~-alanyl-L-proline which was established by X-ray analysis to have the S,S,S configuration. By thin layer chromatography on silica gel in 1~
ethylacetate/n-butanol/water/acetic acid, this solid is - . . ................ .
. .
-, ' - : . . .

ii35~3 a singl~ spot having an Rf value of 0.43. The 300 M~z nmr spectrum shows a triplet for the methine proton y to tha phenyl substituent at 3.40 ppm. When the fractions containing the second isomer are freeze-dried, 72 mg of white fluffy solid is obtained. This solid by thin layer chromatography is a single spot of Rf value 0.39.
The 300 MHz nmr spectrum shows the triplet for the methine proton y to the phenyl substituent at 3.61 ppm.
EXAMPLE_3 Benzyl N-(l(S)-carboxy-3-phenylpropyl) L-alanyl-L-prolinate Thionyl chloride ~13.1 ml) was added to 150 ml of benzyl alrohol keeping the temperature below 0 (exothermic reaction). N-(l(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline (15 g) was added portionwise to the cold solution. The cooling bath was removed and the mixture was stirred at room temperature overnight.
After heating to 45 under vacuum to remove dissolved gases the reaction was diluted with 500 ml e~her and washed with 10 x 100 ml of water. A solid appeared in the organic layer which was filtered and dried to yield 4.9 g of crude monobenzyl ester.
The combined aqueous extracts neutralized with NaHCO3 gave a second crop weighing 1.4 g. The two crops were combined and a portion (1.0 g~ was recrystallized from ethanol-water to yield 0~95 g of pure monobenzyl ester, m.p. 120-125. Mass spectral analysis indicated the benzyl ester was attached to the proline ring.
This is a divisional of Canadian Patent Application S.N. 341,340 filed on December 6, 1979, ~ , , .
- - . .
.

, .

Claims (6)

16271Y (Div. A) The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound of the formula:

I

wherein:
R and R6 can be the same or different and are hydroxy or loweralkoxy;
R1 is a substituted lower alkyl wherein the substituent is phenyl or halophenyl;
R2 and R7 are hydrogen;
R3 is amino lower alkyl;
R4 and R5 are lower alkyl;
R4 and R5 may be connected together to form an alkylene bridge of from 2 to 4 carbon atoms;
and the pharmaceutically acceptable salts thereof.

2. N-.alpha.-(1(S)-carboxy-3-phenylpropyl)-L-lysyl-L-proline.

3. The pharmaceutically acceptable acid addition salt of N-.alpha.-(1(S)-carboxy-3-phenylpropyl)-L-lysyl-L-proline.

4. The N-?-(1-carboxy-3-phenylpropyl)-L-Lysyl-L-proline hydrochloride salt.

5. A pharmaceutical composition for reducing hypertension comprising an effective amount of a compound of Claim 1, 2 or 4 and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition for reducing hypertension comprising an effective amount of a compound of Claim 4 and a pharmaceutically acceptable carrier.