CA1142868A - Aminopeptidase inhibitor - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A reversible inhibitor of an aminopeptidase specific for N-terminal aspartylpeptides having the chemical formula:

where, in aqueous solution at physiological pH, Z is an anionic group Y is a cationic group, n is 1 or 2, and X
is either hydrogen or halogen is described.
Preferably, the inhibitor is:

Description

2&'~3 The present invention relates to aminopeptidase inhibitor.
Hypertension is a major factor leading to heart disease and stroke. Extensive efforts have been made to understand the etiology of this condition to develop drugs capable of treating it. The physiological regulation of blood pressure is in part medicated by the renin-angioten-sin system. See Freeman, R.H., Davis, J.O., Lohusen, T.E.
and Spielman, W.S., Fed. Proc. 36, 1766 (1977).
.
Among the compounds believed to affect blood pres-sure in the renin-angiotensin system are three related pep-tides designated angiotensin I, II and III. Abbreviations used in describing the amino acid sequences of peptides are shown in Table I.
TABLE I

Arg = L-arginine Asp = L-aspartic acid Gly = glycine His = L-histidine Ile = L-isoleucine Leu = L=leucine Phe = L-phenylalanine Pro = L-proline Tyr = L-tyrosine Val = L-valine EDTA = Ethylene diamine tetraacetic acid Tris = 2-Amino-2-hydroxyethyl-1,3-propanediol Angiotensin I, hereinafter "I", is a decapeptide having the sequence AspArgValTyrIleHisProPheHisLeu. By convention, the reading order of the sequence is from the N-terminal amino acid on the left to the C-terminal amino acid on the right. Angiotensin II, hereinafter "II", is the same structure as above, .. . ~

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except lacking the C-terminal HisLeu. Angiotensin III, hereinafter "III", is des-Aspl angiotensin II, that is, angiotensin II lacking the N-terminal Asp.
I is converted to II by the action of angiotensin converting enzyme~ which removes the C-terminal dipeptide by catalyzing the hydrolysis of the peptide bond between Phe8 and His of I. III may be formed from I or II by the selective removal of the N-terminal Asp to form des-Asp angiotensin I or III. The des-Asp1-angiotensin I may be converted to III by angiotensin converting enzymeO
Angiotensin I appears to have no effect on blood pressure. However, angiotensin II is known to have a powerful blood-pressure elevating effect. Compounds found to be inhibitors of angiotensin converting enzyme are effective in lowering blood pressure and thus alleviating hypertension. See Ondetti, et al., 4,0~6,889, issued September 6, 1977, Cushman et al., 4,~52,511, issued October 4, 1977, and Ondetti, et al., 4,053,651, issued October 11, 1977.
The physiological role of angiotensin III is less well characterized. However, it is a normal constituent of blood, and it has demonstrable effects of significance in elevating blood pressure. Very little is known of how III might be formed _ vivo. In either of the two routes of synthesis from angiotensin I, described supra, a specific cleavage of the N-terminal Asp residue is required, either from I or II.
Therefore, an aminopeptidase enzyme specific for an N-terminal aspartyl peptide (any peptide having an N-terminal aspartic acid residue) is likely involved in the production of angiotensin III. Suitable inhibitors of such an en~yme are expected to have 30 anti-hypertensive properties. The inhibitors are also expected to be useful in the treatment of non-hypertensive edematous states in which the renin-angiotensin system plays a role.

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1 ~ 4 ~3$~3 Aminopeptidases are hydrolytic enzymes which remove an N-terminal amino acid from a peptide or protein. The substrate specificity is variable from enzyme to enzyme, for example, the well-known leucîne !
aminopeptidase is relatively non-specific, being capable of sequentially ¦
hydrolyzing an entire peptide. Two aminopeptidases specific for N-terminal aspartylpeptides have been described. Aminopeptidase A, first isolated by Glenner, G.G., McMillan, P.J. and Folk, J.E., Nature 194, 867 (1962), preferentially removes N-terminal dicarboxylic acids. The enzyme is activated by Ca~+ and inhibited by EDTA. A second enzyme, called aspartate aminopeptidase~ has been described by Cheung, H.S. and Cushman, D.W., Biochim. _iophys. Acta 242, 190 (1971). Aspartate aminopeptidase is not _ .
affected by Ca+~ or EDTA, but is activated by MnC12. Both enzymes hydrolyze cc-L-aspartyl-~ -naphthylam;de, which reaction may be measured fluorimetrically to provide a convenient assay. The two enzymes are further distinguished by their ability to hydrolyze c~-L-glutamyl-,67 -naphthylamide. Aminopeptidase A hydrolyzes the glutamyl substrate and the aspartyl substrate equally well. The aspartate aminopeptidase hydrolyzes the aspartyl substrate three times faster than the glutamyl substrate.
lt remains to be determined whether one of the known aminopeptidases or some other is responsible for the in vivo formation of angiotensin III.
In the meantime, a specific inhibitor of aminopeptidases acting only on N-terminal aspartylpeptides is useful for differential analysis of s~ch enzymes in biological materials, for in vitro and in vivo research on angiotensin III-mediated hypertension and for conversion of angiotensin III
from purified angiotensins I or II.
A reversible inhibitor is useful in research on the kinetics of enzyme activity and on relative abundance of the enzyme compared to less specific aminopeptidases. Additionallyg a . .

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reversible inhibitor will be useful as a probe to distinguish between different N-terminal aspartylpeptide amiropeptidases coexisting in the same biological sample.
An irreversible inhibitor is useful for labeling the active-S site on the enzyme, for tissue localization of the enzyme, both grossly and microscopically~ and for molecular ablation studies.
Ultimately, it is expected that such inhibitors will prove useful as antihypertensive drugs.
Specific or at leas~ highly selective enzyme inhibitors have been developed for certain enzymes, based upon detailed - background knowledge of the nature of the active site, including details of the ionic and hydrophobic binding sites and their steric relationship, whether a metal atom is involved in binding or catalysis, and the location o~ the metal atom relative to the bound substrate. Such detailed knowledge has been obtained from extensive enzymological studies, including X-ray diffraction, and kinetic data obtained with a variety of sub-strate analogs. Exploiting such inormation, Byersr L.D. and Wolfe~den, R., Biol.Chem. 247, 606 (1972~ and Byers, L.D. and Wolfenden, R., J.Biochemistry 12, 2070 C1973? have developed an unusually potent competitive inhibitor of carboxypep~idase A, D-2-benzylsuccinic acid. Ondetti, M.A., Rubin, B. and Cushman, D.W., Science 196, 441 (1977) and Cushman, D.W., Cheung, H.S., Sabo, E.F., and Ondetti, M.A., Biochemistry 16, 5484 (1977~
2~ have extended this approach to include the development of highly selective inhibitors o~ angiotensin converting enzyme. The most potent of these inhibitors is ~-3-mercapto-2-methylpropanoyl-L-proline. Certain chloromethyl ketones have been observed to inhibit certain proteolytic enzymes. Trypsin was s~own to be irreversibly inhibited by N-tosyl-L-lysine chloromethyl ketone ~142~

by Shaw, E. and Glover, G., Arch.Biochem.Biophys. 139, 298 (1970). The inhibitor acted as an alkylating agent of a histidine moiety at or near the active site of the enzyme.
However 7 chloromethylketone analogs of DL-leucine and DL-alanine were observed to he reversible inhibitors of leucine aminopeptidase. See Birch, P.L., El-Obied, H.A. and Akhtar~
M., Arch.Biochem.Biophys. 148, 447 (1972).
Relatively little is known about the substrate binding sites of the aminopeptidases specifically active on N-terminal aspartyl peptides. It remains to be seen whether the two enzymes that have been isolated, amino-peptidase A and aspartate aminopeptidase, are the major enzymes responsible for ln vivo removal of N-terminal aspar-tyl groups, or for the d-aspartyl-~-naphthylamide hydro-lytic activity of biological materials such as rat serum.
Although there is suggestive evidence implicating one or both of the two enzymes in the in vivo formation of angio-tensin III, neither has been clearly shown to be involved.
Additional unknown factors include the range of substrate specificity, whether a metal atom such as Zn++ is present at the active site, and what amino acid side chains con-tribute to catalysis.
In accordance with the present invention, there is provided an inhibitor of aminopeptidase capable of cataly-zing the removal of an N-terminal aspartyl residue from a peptide, the essential active ingredient of which has the chemical formulao Z-(CH ) ~-C*H-C-CH X
Y O
wherein Z is an anionic group in a~ueous solution at phys-iological pH; Y is a cationic group in aqueous solution -2~

at physiological pH; n is 1 or 2, and X is hydrogen or halogen,and C* is an asymmetric center in the L-configura-tion.
Preferably, the essential active ingredient has the formula:
~ o Il ~ OOC- ~CH2 ) n-c*H-c-cH2x : NH 2 where X is Cl or Br and n is 1 or 2. The asterisk denotes 10 an asymmetric centre in the L-configuration.
Aminopeptidases specific for N-terminal aspartyl peptides are inhibited by compounds of the present inven-` tion. The inhibiting compounds have the general formula:
Y O

Z-(CH2) -C*H-C-CH -X
where Z is an anionic group in aqueous solution at phys-iological pH, Y is a cationic group in aqueous solution at physiological pH, n is 1 or 2, X is either hydrogen or halogen, and the asymmetric carbon denoted by an asterisk 20 is in the L-form. In preferred compounds, Z is COO , Y
is NH3+, n is 1 and Z is eithér Cl or Br. The most pre-ferred compound is L-aspartyl bromomethane, O.
HOOC-CH2-CH-C-CH2Br.

In aqueous solution at physiological pH (approximately pH
6.0 - pH 8.0) the ionic structure is OOC - CH2 - CH - C - CH2Br The inhibitor may be used _ vitro or in vivo to inhibit the aminopeptidase-catalyzed removal of an N-terminal aspartyl residue of a peptide, or to inhibit the hydrolysis of ~ -L-aspartyl- ~ -naphtylamide, or to inhibit the hydrolysis of ~ -L-aspartyl-3[H]benzylamideO The inhi-bitor is introduced by appropriate means, such as pipetting, perfusion, injection and the like, into reactive proximity with the enzyme. By reactive proximity it will be under-stood that - 7a -the mode of introduction is suitable to provide an adequate concentration of inhibitor capable of diffusing to, and interacting with, the active site of ~he enzyme. The interaction may be a binding or a formation of a coordinate or covalent chemical bond or the disruption of such a bondO For example, in an n vitro reaction, merely pipetting an amount of inhibitor sufficient to provide a predetermined final inhibitor concentration will ordinarily suffice. For ln vivo inhibition, such factors as the route of administration, the approximate dilution factor to be expected, rates of metabolism and excretion of the inhibitor and the like, must be taken into account, as is well understood in pharmacology, in order to introduce the inhibitor in an appropriate amount into reactive proximity with the enzyme.
Synthesis of the inhibitor is carried out starting with the appropriate dicarboxylic amino acid, having protecting groups attached to the amino group and to the ~- or ~ - carboxyl group, an expedient well-known in the art of peptide chemistry.
Suitable protecting groups include, for example tert-butyl, benzyl, or 2-methoxybenzyl groups for the carboxyl group, and tert-butoxycarbonyl (also known as tert-butyloxycarbonyl), carbobenzoxy (also known as benzyloxycarbonyl) or tert-amyloxycarbonyl groups for the amino group. Choice of protecting group will be based upon considerations of convenience, ease of deprotection, availability and the like.
The key intermediate for the synthesis of all inhibitors of the present invention is the diazomethane derivative. The structure of the key intermediate is given by the formula r NH-B
A-OOC-(CH2)n-CH-C-CHN2 o where A is a carboxyl protecting group, B is an amino protecting group, and n is 1 or 2.

~r`

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Example 1 ~-L-Aspartyl chloromet_ane.
A solution of ~-carbobenzoxy-L-aspartic acid-~-benzvl ester (ha~ing a free ~ -carboxyl group) in tetrahydrofuran was cooled to ~10C prior to addition of N-ethylmorpholine and reaction with isobutyl chloroformate to form a mixed anhydride. The reaction mixture was stirred at -10C for 12 minutes, then warmed to 0C and allowed to react for an additional 10 minutes. A pre-cooled excess of ethyl ether at -10C was added to the reaction mixture, which was then filtered.
The resulting mixed anhydride solution was added dropwise into a solution of diazomethane in ether at 4C and allowed to stand for 2 hours after which the mixture was slowly warmed to room temperature and washed sequentially with acetic acid, sodium bicarbonate solution, saturated sodium chloride and dried over anhydrous magnesium sulfate. The mixture was then filtered and excess solvent and fre~ diazomethane removed by rotary evaporation. The resulting c:Lear oil, ~ -benzyl ester of ~-carbobenzoxy- ~ -L aspartyl diazomethane, was analyzed by infrared spectrophotometry, in chloroform. The compound had a sharp absorption band at 2117 cm corresponding to CH=N =N , and lacked any bands in the region 2300-2800 cm , correspondin~
to a free carboxyl group.
The ~-benzyl ester of ~-carbobenzoxy- ~-L-aspartyl ~ diazomethane was decomposed by dropwise addition of 3.5 ~ HCl in ethyl ether at -10C. The mixture was stirred at -10C until there was no further evolution of nitrogen gas. A yellow, oily product was obtained in 82~ yield, having an infrared spectrum (in chloroform) almost identical with ~-carbobenzoxy~L-aspartic acid- ~-benzyl ester except that no absorption was observed in the 2300-2800 cm 1 region of free carboxyl absorption. The product, ~-benzyl ester of ~ -carbobenzoxy- ~ -L-aspartyl chloromethane, was pure as judged by migration as a single _g _ ~42E3~i~

discrete component on thin layer chromatography in five separate solvent systems.
The product was deprotected by txeatment with anhydrous HF in the presence of anisole to yield ~ -L-aspartyl chloromethane.
Example 2 ~-L-aspartyl bromomethane.
The ~-benzyl ester of ~ -carbobenzoxy -~ -L-aspartyl diazomethane, prepared as described in Example 1, was decomposed by dropwise addition of HBr in ether, under conditions essentially identical with those described in Example 1 for decomposition with HCl. The resulting product was the ~-benzyl ester o ~-carbobenzoxy - a-L-aspartyl bromomethane, which was deprotected to yield a-L-aspartyl bromomethane.
Following the procedure of Examples 8 and 9 using ~ -L-aspartyl-3[H]benzylamide and ~ -L-lysyl- [H]
benzylamide as the substrates, the I50 was determined to be 2 x 10 6M for rat lung or serum aminopeptidase-catalyzed a -L-aspartyl-3[H]benzylamide hydrolysis and 8 x 10 3~ for a -L-lysyl- ~H]benzylamide hydrolysis.
Example 3 -L-a_ ~ ethane.

3 -benzyl ester of a -carbobenzoxy- ~ -L-aspartyl chloromethane, prepared as described in Example 1, was dissolved in methanol and subjected to catalytic hydrogenolysis in the presence of 10% palladium on barium sulfate.
Example 4 -L-aspartyl aminomethane.
The ~-benzyl ester of ~-benzyloxycarbonyl-L-aspartyl bromomethane, as prepared in Example 2 r is mixed with potassium phthalimide in redistilled dimethylformamide and stirred at room temperature overnight. Ethylacetate was added to the reaction mixture. The organic phase was washed three times with water, -1~
- ~ ~

four times with lN NaHCO3 and finally three times with saturated NaCl solution. The organic layer was dried over anhydrous MgSO4, filtered, and the solvent was removed with a rotary evaporator. The residue was crystallized from isopropanol to yield a semi-solid material. The product was identified by infrared spectroscopy and behaved as a pure substance on thin layer chromatography using silica gel and three solvent systems.
The phthaloyl group may be removed by hydrazinolysis or acid hydrolysis. The resulting compound may be deprotected by (a) anhydrous HF in the presence of anisole, (b) acid hydrolysis using HCl, (c) HBx in glacial acetic acid, or (d) hydrogenation (10~ Pd/C) in a solution of acetic acid-H2O-methanol to yield a -L-aspartyl aminomethane.
Example 5 L-3-amino-5-benzoylthio-4-oxo~ _anoic acid .
The ~-t-butyl ester of ~-t-butyloxycarbonyl-L-aspartyl chloromethane is prepared as in Example 1 using the ~ -t-butyl ester of ~ -t-butyloxycarbonyl-L-aspartic acid as the starting matexial.- A mixture of thiobenzoic acid, anhydrous potassium carbonate and the ~-t-butyl ester of a-t-butyloxycarbonyl-L-aspartyl chloromethane in redistilled dimethylformamide was stirred overnight at room temperature. Ethyl acetate was added to the reaction mixture and the organic solvent was washed thoroughly with water. The organic phase was dried over anhydrous MgSO4, filtered and the solvent removed with a rotary evaporator. The crude material was purified by LH-20 column chromatography, eluted with isopropanol. This product was identified by infrared spectroscopy and behaved as a pure substance on thin layer chromatography using silica gel in four solvent systems. The ~ t-butyloxycarbonyl and t-butyl ester protecting aroups were removed by arhydrous trifluoroacetic acid in the presence of anisole. The resulting compound was purified by column chromatography and identified as L-3-amino-5~

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~ ~2~613 benzoylthio-4-oxo-pentanoic acid. The I50 for this compound is approximately 1 x 10 5M when assayed follo~ing the procedure of Example 8 using ~ -L-aspartyl-3[H]benzylamide as the substrate.
Example 6 -L-aspartyl mercaptom thane.
Alkaline hydrolysis with NH3 in methanol of L-3-amino-5-benzoylthio-4-oxo-pentanoic acid gives a -L-aspartyl mercaptomethane. The I50 for this compound is approximately 1 x 10 6M when assayed following the procedure of Example 8 using 0 a -L-aspartyl-3[H]benzylamide as the substrate.
Example 7 Glutamic acid analogs.
Analogs of the compounds synthesized according to Examples 1-5 are readily prepared starting from ~-carbobenzoxy-I.-glutamic acid- y -benzyl ester or preferably y -t-butyl ester of a-t-butoxycarbonyl-L-glutamic acid thaving a free a-carboxyl group~. The reactions of Example 1 yield y-benzyl ester of ~ -carbobenzoxy-~ -L-glutamyl diazomethane or preferably y -t-butyl ester of 6-diazo-4-N-t-butoxycarbonyl-5-oxo-L-hexanoate~
i.e. y-t-butyl ester of a -t-butoxycarbonyl- a -L-glutamyl diazomethane. The corresponding chloromethane and bromomethane derivatives are prepared as described in Examples 1 and 2, respectively. The compound ~-L-glutamyl methane is prepared from the ~ -benzyl ester of a -carbobenzoxy- ~ -L-glutamyl bromomethane or preferably the y -t butyl ester of a-t-butoxycarbonyl- ~ ~L-glutamyl bromomethane as described in Example 3. The aminomethane and mercaptomethane derivatives are prepared as described in Examples 4 and 5 respectively.
Example 8 Ami o ~ e inhibition by a-L-aspartyl_chloromethane.
An aminopeptidase assay was employed, using ~-aspartyl- ~-naphthylamide as substrate for N-terminal aspartic-!

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specific aminopeptidase activity and ~-arginyl- ~ ~naphthyl-amlde as substrate for nonspecific aminopeptidase activity.
Hydrolysis of the substrate amide bond releases the fluorophor, -naphthyl-amine.
Rat serum, 25 ul, was added to an assay mixture containing 90 ~moles of Trls-HCl buffer, pH 7.0, 0.45 ~moles of substrate, 0.9 ~moles calcium chloride and sufficient water to give 0.9 ml total reaction volume. The concentration of inhibitor was varied from 10 1OM to 10 3M, and a control reaction was assayed in the absence of added inhibitor. The percent of inhibition was determined as a function of inhibitor concentration in order to establish I50, the concentration required to inhibit the reaction rate 50%, under the reaction conditions used. The I50 of a-L-aspartyl chloromethane was 3 x 10 M for rat serum aminopeptidase-catalyzed ~ -L-aspartyl--naphthylamide hydrolysis, and 6 x ]0 3M for ~ -L-arginyl--naphthylamide hydrolysis.
Example 9 Aminope~tidase inhibition by a-L-aspartylmethane.
The inhibitor, prepared as described in Example 3, was tested for activity in the assay system described in Example 8.
The I50 for a-L-aspartyl- ~ ~naphthylamide hydrolysis was 8 ~
6 x 10-5 M, indicating the methane derivative was only about 1~20th as effective a$ the chloromethane derivative.

Example 10 Inhibition of Rat Lung Aminopeptidase.
A homogenate of rat lung tissue, pumped free of blood, was fractio~ated by low speed centrifugation at 1600 x 9 for 10 minutes at 4C. The supernatant was further fractionated by high speed centrifugation in 0.1 M Tris-HCl, pH 7.4, and MgC12, 10 mM, at 30,000 x 9 for 30 minutes at 4C. The majority of the aminopeptidase activity was calcium dependent, requiring 5 mM to 10 mM Ca++ for optimal activity, resembling~ in this regard, aminopeptidase A. The Iso f c~-L aspartyl chloromethane for the rat lung enzyme was substantially the same as that of the rat serum activity described in Example 8, using the same assay system.

~ Example 11 I.
i5 In vivo aminopeptidase inhib;tion by c{ -L-aspartyl bromomethane.
It is genèrally agreed that the effects of angiotensin II on systemic arterial blood pressure are two to Four times as great as those of angiotensin TII. Further, it is often assumed that angiotensin III
is among the first degradation products~of angiotensin II. Thus, after a dose of angiotensin II is given to an animal, the blood pressure should rise and then drop as the angiotensin II is degraded or eliminated.
I~ an inhibitor against the aminopeptidase which degrades angiotensin II to III is administered, angiotensin II will be degraded less rapidly resulting in a higher blood pressure response at a given time interval.
In this example the in vivo effect of GC-L-aspartyl bromomethane was examined by measuring the difference in the blood pressure response after administration of angiotensin II.

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Sprague-Dawley rats were anesthetized with pentobarbital. A common carotid artery and a femoral vein were exposed. The vein was cannulated and heparin was injected. The artery was cannulated and connected to a pressure transducer. Angiotensin II, 40 ng/kg, was injected intravenously at 5 minute intervals until a reproducible blood pressure response was obtained (usually 3-4 injections). An arterial blood sample was collected at this time. Four doses of angiotensin II - 40, 80, 160 and 320 ng/kg -were injected at 5 minute intervals in order to construct a log dose-response curve. The effect of these doses is shown in Table I. The blood pressure (BP~ response is shown by the systolic (Sys.), diastolic (Dias.) and mean arterial pressure (MAP).
A second blood sample was collected. ~ ~L-aspartyl bromomethane ~8 mg/kg) - ;ndicated infra as either inhibitor or drug - was injected and followed by ;njections of 160 ng/kg of anglotensin II at 2~ 7, 17 and 28 minutes following the inhibitor administration. Blood samples were also collected at timed intervals. The bloocl pressure was measured after each inject;on and the results are shown in Table II. Table III shows the percent change in blood pressure response when compared to a control.
Table IV shows the blood pressure response in terms of equivalents of angiotensin II as interpolated from the log dose-response curve.
The blood samples which were collected were centrifuged to obtain the plasma. The plasmas were then assayed for residual aminopeptidase A-like activity using cC L-aspartyl~[3H~benzylamide as substrate. To test for specificity of the oC-L-aspartyl bromomethane, the plasmas were also assayed for lysine aminopeptidase using cC-L-lysyl-C3H~benzylamide.
As shown in Table III, at 2 and 7 minutes after the injection of O~ -L-aspartyl bromomethane~ the mean arterial blood pressure showed an increase of 17% over the control in response to the injection of angiotensin II. The systolic blood pressure showed an even more marked _15_ ~ ~ b .~ ~``
~14 TABLE I
.
Before Drug Dose of BP response (m~ Hg) an~iotensin II (ng/kg) MAP _ Sys. Dias.
5 . 40 2~ 32 22 . 320 61 76 40 TABLE II
.
Af~er Drug Dose of BP response (m~ Hg) . Time (min)angiotensin II ~ng/kg) MAP Sys. Dias.

17 160 50 6~ 34 .

.
_ , TAB~E III
; `
. After Drug Dose of BP response, as % of control 20 Time ~min)angiotensin II, ng/kg MAP Sys. _Dias.

: 7 160 117 127 125 17 160 10~ 113 106 .
TABLE IV .

ter Drug BP response, in terms of angio-tensin II equivalents Time (min) a~ n ~ (estimated from ~MAP) - _ 2 160 248 ng/kg 2~ 160 160 _ , . . __. _._ .

increase. Ta~le IV indicates that the 17~ increase in the response of the MAP to the angiotensin ~I at a dose of 160 ng/kg is the pressure response expected of a dose of 24~ ng/kg of angiotensin II without administration of the inhibitor. This is a 60~ increase in apparent potency. In four similar experiments, the increase in apparent potency ranged from 48%~135%.
~ -L-aspartyl bromomethane had the expe~ted result on residual plasma aminopeptidase A-like activity. One minute after inhibitor injection, enzyme activity fell by 48%. At 5 minutes, enzyme activity was at 64% of control, and at 15 minutes it was at 68% of control. The activity of plasma lysine aminopeptidase was not changed when compared to the control.
In an additional experiment, norepinephrine or angiotensin III was examined in place of angiotensin II.
~ -L aspartyl bromomethane showed no effects on the blood pressure response to these two compounds.
While tlle invention has been described in connection with specific embodiments thereof, it will be understood that it is capable o~ further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as , come within known or customary practice within the art to which the-invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (11)

1. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
   l. A process of formation of a compound of the formula:
   
   wherein Z is an anionic functional group, in aqueous solution at physiological pH; Y is a cationic functional group, in aqueous solution at physiological pH; n is 1 or 2; X is hydro-gen or halogen; and C* is an asymmetric centre in the L-con-figuration, comprising:
   (a) blocking, if necessary, the anionic functional group and the cationic functional group of the compound:
   
   with the groups A and B, respectively;
   (b) forming the mixed anhydride of the compound formed in step (a);
   (c) reacting the product formed in step (b) with diazomethane;
   (d) decomposing the product formed in step (c) in halogen acid; and (e) removing the blocking groups A and B, if necessary.
2. 2. The process of claim 1 wherein said compound has the formula:
   
   in which X is a halogen and n is 1 or 2.
3. 3. The process of claim 2 wherein the halogen acid used in step (d) is hydrochloric acid in ethyl ether.
4. 4. The process of claim 2 wherein the halogen acid used in step (d) is hydrobromic acid in ethyl ether, whereby said halogen is bromine.
5. 5. The process of claim 1 wherein said compound has the formula:
   in which X is Cl or Br and n is 1 or 2.
6. 6. A compound of the formula:
   
   wherein Z is an anionic functional group, in aqueous solution at physiological pH; Y is a cationic functional group, in aqueous solution at physiological pH; n is 1 or 2; X is hydrogen or halogen; and C* is an asymmetric centre in the L-configura-tion, said compound being useful as the essential active ingredient of an inhibitor of an aminopeptidase capable of catalyzing the removal of an N-terminal aspartyl residue from a peptide, whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
7. 7. A compound of the formula:
   
   wherein n is 1 or 2 and X is a halogen, whenever prepared by the process of claim 2 or by an obvious chemical equivalent thereof.
8. 8. The compound of claim 7 wherein X is bromine, whenever prepared by the process of claim 4 or by an obvious chemical equivalent thereof.
9. 9. A compound of the formula:
   
   wherein n is 1 or 2 and X is Cl or Br, whenever prepared by the process of claim 5 or by an obvious chemical equivalent thereof.
10. 10. A method for inhibiting an enzyme capable of removing the N-terminal aspartyl residue from an N-terminal aspartylpeptide comprising, providing an inhibitor of the enzyme, said inhibi-tor having the chemical formula wherein X is hydrogen or halogen, and introducing the inhibitor into in vitro reactive proximity with the enzyme whereby the enzyme is inhibited.
11. 11. The method of claim 10 wherein X is Cl or Br.