Renin inhibitors containing a C-terminal amide cycle

Renin inhibitory peptides of the formula ##STR1## and analogs thereof inhibit renin and are useful for treating various forms of renin-associated hypertension and hyperaldosteronism.

BACKGROUND OF THE INVENTION 
1. Field of The Invention 
The present invention is concerned with novel peptides which inhibit renin. 
The pesent invention is also concerned with pharmaceutical compositions 
containing the novel peptides of the present invention as active 
ingredients, with methods of treating renin-associated hypertension and 
hyperaldosteronism, with diagnostic methods which utilize the novel 
peptides of the present invention, and with methods of preparing the novel 
peptides of the present invention. 
Renin is a proteolytic enzyme of molecular weight about 40,000, produced 
and secreted by the kidney. It is secreted by the juxtaglomerular cells 
and acts on the plasma substrate, angiotensinogen, to split off the 
decapeptide angiotensin I, which is converted to the potent pressor agent 
angiotensin II. Thus, the renin-angiotensin system plays an important role 
in normal cardiovascular homeostasis and in some forms of hypertension. 
In the past, attempts to modulate or manipulate the renin-angiotensin 
system have met with success in the use of inhibitors of angiotensin I 
converting enzyme. In view of this success, it seems reasonable to 
conclude that a specific inhibitor of the limiting enzymatic step that 
ultimately regulates angiotensin II production, the action of renin on its 
substrate, would be at least equally successful. Thus, an effective 
inhibitor of renin has been long sought as both a therapeutic agent and as 
an investigative tool. 
2. Brief Description of the Prior Art 
There has been substantial interest in the synthesis of useful renin 
inhibitors for many decades; and the following table lists the major 
classes of renin inhibitors that have been studied, as well as their 
inhibition constants (K.sub.i): 
______________________________________ 
Class K.sub.i (M) 
______________________________________ 
Renin antibody probably 10.sup.-6 
Pepstatin 10.sup.-6 -10.sup.-7 
Phospholipids 10.sup.-3 
Substrate analogs 
Tetrapeptides 10.sup.-3 
Octa- to tridecapeptides 
10.sup.-5 -10.sup.-6 
______________________________________ 
Umezawa et al., in J. Antibiot. (Tokyo) 23: 259-262, 1970, reported the 
isolation of a peptide from actinomyces that was an inhibitor of aspartyl 
proteases such as pepsin, cathepsin D, and renin. This peptide, known as 
pepstatin, was found by Gross et al., Science 175:656, 1971, to reduce 
blood pressure in vivo after the injection of hog renin into 
nephrectomized rats. However, pepstatin has not found wide application as 
an experimental agent because of its limited solubility and its inhibition 
of a variety of other acid proteases in addition to renin. The structure 
of pepstatin is shown below: 
##STR2## 
To date, many efforts have been made to prepare a specific renin inhibitor 
based on substrate analogy. Since the human renin substrate has only 
recently been elucidated (Tewksbury et al., Circulation 59, 60, Supp. II: 
132, October 1979), heretofore substrate analogy has been based on the 
known pig renin substrate. While the human and pig renin substrates are 
not the same, the substrate analogy based on pig renin has always been 
considered acceptable in the art as predictive of human renin inhibitory 
activity because of the closely related activity of the two renins. Thus, 
while pig renin does not cleave the human renin substrate, human renin, on 
the other hand, does cleave the pig renin substrate. See Poulsen et al., 
Biochim. Biophys. Acta 452:533-537, 1976; and Skeggs, Jr. et al., J. Exp. 
Med. 106:439-453, 1957. Moreover, the human renin inhibitory activity of 
the peptides of the present invention most active in inhibiting pig renin 
has been confirmed, thus providing further evidence of this accepted 
correlation between human and pig renin activity. 
It has been found, for example, using pig renin substrate analogy, that the 
octapeptide sequence extending from histidine-6 through tyrosine-13 has 
kinetic parameters essentially the same as those of the full 
tetradecapeptide renin substrate. The amino acid sequence of the 
octapeptide in pig renin substrate as is follows: 
##STR3## 
Renin cleaves this substrate between Leu.sup.10 and Leu.sup.11. 
Kokubu et al., Biochem. Pharmacol. 22: 3217-3223, 1973, synthesized a 
number of analogs of the tetrapeptide found between residues 10 to 13, but 
while inhibition could be shown, inhibitory constants were only of the 
order of 10.sup.-3 M. 
Analogs of a larger segment of renin substrate were also synthesized: 
Burton et al., Biochemistry 14: 3892-3898, 1975, and Poulsen et al., 
Biochemistry 12: 3877-3882, 1973. Two of the major obstacles which had to 
be overcome to obtain an effective renin inhibitor useful in vivo were 
lack of solubility and weak binding (large inhibitory constant). 
Modifications to increase solubility soon established that the inhibitory 
properties of the peptides are markedly dependent on the hydrophobicity of 
various amino acid residues, and that increasing solubility by replacing 
lipophilic amino acids with hydrophilic isosteric residues becomes 
counterproductive. Other approaches to increasing solubility have had 
limited success. Various modifications designed to increase binding to 
renin have also been made, but here too, with only limited success. For a 
more detailed description of past efforts to prepare an effective 
inhibitor of renin, see Haber and Burton, Fed. Proc. Fed. Am. Soc. Exp. 
Biol. 38: 2768-2773, 1979. 
More recently, Hallett, Szelke, and Jones, in work described in European 
Patent Publication No. 45,665 Nature, 299, 555 (1982), and Hypertension, 
4, Supp. 2, 59 (1981), have replaced the Leu-Leu site of renin cleavage by 
isosteric substitution, and obtained compounds with excellent potency. 
Powers et al., in Acid Proteases, Structure, Function and Biology, Plenum 
Press, 1977, 141-157 have suggested that in pepstatin, statine occupies 
the space of the two amino acids on either side of the cleavage site of a 
pepsin substrate, and Tang et al., in Trends in Biochem. Sci., 1: 205-208 
(1976) and J. Biol. Chem., 251: 7088-94, 1976, have proposed that the 
statine residue of pepstatin resembles the transition state for pepsin 
hydrolysis of peptide bonds. However, the applicability of these concepts 
to renin inhibitors is not taught in any of these references, and would be 
speculative due to the known high degree of specificity of the renin 
enzyme. 
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, Sept. 1980; Suketa et al., 
Biochemistry 14: 3188, 1975; Swales, Pharmac. Ther. 7: 173-201, 1979; 
Kokubu et al., Nature 217: 456-457, Feb. 3, 1968; Matsushita et al., J. 
Antibiotics 28: 1016-1018, December 1975; Lazar et al., Biochem. Pharma. 
23: 2776-2778, 1974; Miller et al., Biochem. Pharma. 21: 2941-2944, 1972; 
Haber, Clinical Science 59: 7s-19s, 1980; and Rich et al., J. Org. Chem. 
43: 3624, 1978, and J. Med. Chem. 23: 27, 1980.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS 
In accordance with the present invention there are provided renin 
inhibitory peptides of the formula: 
##STR4## 
wherein: A is hydrogen; or 
##STR5## 
where X is 
##STR6## 
and R.sub.a.sup.1 and R.sub.b.sup.1 may be the same or different and are 
hydrogen; Y--(CH.sub.2).sub.p -- or Y--(CH.sub.2).sub.p' 
--CH.dbd.CH--(CH.sub.2).sub.p", where Y is C.sub.1-4 alkyl; hydrogen; 
aryl; C.sub.3-7 cycloalkyl; or 
C.sub.3-7 cycloalkyl or aryl substituted with up to five members 
independently selected from the group consisting of C.sub.1-8 alkyl, 
trifluoromethyl, hydroxy, C.sub.1-4 alkoxy, and halo; 
p is 0 to 5; and p' and p" are independently 0 to 2; except that where X is 
--O--, only one of R.sub.a.sup.1 or R.sub.b.sup.1 is present; 
B is absent; glycyl; sarcosyl; or 
##STR7## 
where R.sup.2 is hydrogen; C.sub.1-4 alkyl; hydroxy C.sub.1-4 alkyl; 
aryl; aryl substituted with up to three members selected from the group 
consisting of C.sub.1-4 alkyl, trifluoromethyl, hydroxy, C.sub.1-4 alkoxy, 
and; halo; indolyl; 4-imidazolyl; amino C.sub.2-4 alkyl; guanidyl 
C.sub.2-3 alkyl; or methylthiomethyl; 
D is absent; or 
##STR8## 
where Z is --(CH.sub.2).sub.1 -- and l is 1 or 2; or --S--; E is absent; 
or 
##STR9## 
where R.sup.6 is hydrogen; C.sub.1-4 alkyl; aryl; aryl C.sub.1-4 alkyl; 
or aryl C.sub.1-4 alkyl or aryl where the aryl portion is substituted with 
up to three members selected from the group consisting of C.sub.1-4 alkyl, 
trifluoromethyl, hydroxy, C.sub.1-4 alkoxy, and halo; or indolyl; 
F is absent; or glycyl; 
R.sup.3 is C.sub.3-6 alkyl; C.sub.3-7 cycloalkyl; aryl; or C.sub.3-7 
cycloalkyl or aryl substituted with up to three members selected from the 
group consisting of C.sub.1-4 alkyl, trifluoromethyl, hydroxy, C.sub.1-4 
alkoxy, and halo; 
R.sup.4 is hydrogen; or 
##STR10## 
where R.sup.7 is hydrogen; C.sub.1-4 alkyl; hydroxy; or C.sub.3-7 
cycloalkyl; and R.sup.6 is as defined above; 
R.sup.5 is hydrogen; 
##STR11## 
where R.sup.6 and R.sup.7 are as defined above; or --(CH.sub.2).sub.q 
--R.sup.8, where q is 0 or 1-4; and R.sup.8 is heterocyclic; heterocyclic 
substituted with up to five members independently selected from the group 
consisting of C.sub.1-6 alkyl, hydroxy, trifluoromethyl, C.sub.1-4 alkoxy, 
halo, aryl, aryl C.sub.1-4 alkyl, amino, and mono- or di-C.sub.1-4 
alkylamino; guanidyl C.sub.2-3 alkyl; or amino C.sub.1-4 alkyl; 
m is 1 to 4; 
n is 0 to 4; and 
wherein all of the asymmetric carbon atoms have an S configuration, except 
for those in the A, B and D substituents, which may have an S or R 
configuration; and a pharmaceutically acceptable salt thereof. 
While both the S and R chiralities for asymmetric carbon atoms in the B and 
D substituents are included in the peptides of the present invention, 
preferred chiralities are indicated in the description which follows. 
In the above definitions, the term "alkyl" is intended to include both 
branched and straight chain hydrocarbon groups having the indicated number 
of carbon atoms. 
The term "halo" means fluoro, chloro, bromo and iodo. 
The aryl substituent represents phenyl, and naphthyl. 
The heterocyclic substituent recited above represents any 5- or 6-membered 
aromatic ring containing from one to three heteroatoms selected from the 
group consisting of nitrogen, oxygen, and sulfur; having various degrees 
of saturation; and including any bicyclic group in which any of the above 
heterocyclic rings is fused to a benzene ring. Heterocyclic substituents 
in which nitrogen is the heteroatom are preferred, and of these, those 
containing a single nitrogen atom are preferred. Fully saturated 
heterocyclic substituents are also preferred. Thus, piperidine is a 
preferred heterocyclic substituent. Other preferred heterocyclic 
substituents are: pyrryl, pyrrolinyl, pyrrolidinyl, pyrazolyl, 
pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, 
pyridyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, 
oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, 
thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, 
quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, 
furyl, thienyl and benzothienyl. 
Where the heterocyclic substituent itself is substituted, it is preferred 
that the substituent be arylC.sub.1-4 alkyl. 
The novel renin inhibitory peptides of the present invention may also be 
described in terms of common amino acid components and closely related 
analogs thereof, in accordance with the following formula: 
##STR12## 
The A, B, D, E and F components correspond to the same portions of Formula 
I. 
In Formula II, Sta represents the unusual amino acid statine and its 
closely related analogs, and its presence constitutes a unique feature of 
the renin inhibitory peptides of the present invention. Statine may be 
named as 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid, and may be 
represented by the following formula: 
##STR13## 
As shown in Formula III above, the delta-substituent is 
naturally-occurring statine is isopropyl, or a leucine sidechain, 
essentially. As shown in Formula I by the R.sup.3 substituents, the 
isopropyl group may be replaced by higher alkyl groups up to six carbon 
atoms, cycloalkyl groups containing from three to seven carbon atoms, 
aryl, and C.sub.3-7 cycloalkyl or aryl substituted with up to three 
members selected from the group consisting of C.sub.1-4 alkyl, 
trifluoromethyl, hydroxy, C.sub.1-4 alkoxy, fluoro, chloro, bromo, and 
iodo. A phenyl substituent and a cyclohexyl substituent are especially 
preferred. These modifications of the naturally-occurring statine 
structure are in accordance with the hydrophobicity considered necessary 
to maintain the inhibitory activity of the total peptide. 
The remaining common amino acid components of Formula II are as follows: 
A has the same meaning as above in Formula I; 
B is Ala, Leu, Ser, Thr, Phe, Tyr, Trp, His, Lys, Orn, Arg, or Met; 
D is Pro; 
E is Ala, Leu, Phe, Tyr, or Trp; 
G is one end of the cyclical structure: Lys, Orn, HLys 
(2S-amino-6-amino-heptanoic acid) or DAB (2S-amino-4-butyric acid); 
H is Gly, Ala, Val, Leu, Ile, Ser, Thr, Phe, Tyr, or Trp; 
I is the same as H and may also be Lys, Orn, Arg, or His; and 
F is Gly and the other end of the cyclical structure, or, when absent, I is 
that other end. 
It will be understood that closely related analogs of the above common 
amino acids, for example, aliphatic amino acids in addition to Ala, Val, 
Leu, and Ile, such as .alpha.-aminobutyric acid (Abu), and substituted 
phenyl derivatives of Phe, are included in the broad description of the 
novel inhibitory peptides of the present invention represented by Formula 
I and its definitions. Thus, the peptides of Formula II and its 
definitions, including the derivatives of naturally-occurring statine 
represented by the definitions of the R.sup.3 substituent in Formula I, 
represent preferred peptides of the present invention. 
Preferred inhibitory peptides of the present invention are the following: 
##STR14## 
The inhibitory peptides of the present invention may be better appreciated 
in terms of substrate analogy from the following illustration of Formula I 
alongside the octapeptide sequence of a portion of the pig renin 
substrate, which renin cleaves between Leu.sup.10 and Leu.sup.11 : 
##STR15## 
As can be seen, a unique aspect and essential feature of the present 
invention is the substitution of the single statine amino acid component 
for the double amino acid sequence: Leu.sup.10 -Leu.sup.11 in the 
endogenous pig renin substrate. It is believed that substitution of 
statine for both leucine amino acids rather than just one leucine results 
in an improved substrate analogy due to the greater linear extent of 
statine as compared to a single leucine component. Thus, statine more 
closely approximates Leu-Leu in linear extent, and thereby provides a 
better "fit" to the renin enzyme. 
The inhibitory peptides of the present invention may also be better 
appreciated in terms of substrate analogy from the following illustration 
of Formula I alongside the octapeptide sequence of a portion of the human 
renin substrate, which renin cleaves between Leu.sup.10 and Val.sup.11 : 
##STR16## 
As can be seen, a unique aspect and essential feature of the present 
invention is the substitution of the single statine amino acid component 
for the double amino acid sequence: Leu.sup.10 -Val.sup.11 in the 
endogenous human renin substrate. It is believed that substitution of 
statine for both the leucine and valine amino acids rather than just the 
leucine results in an improved substrate analogy due to the greater linear 
extent of statine as compared to a single leucine component. Thus, statine 
more closely approximates Leu-Val in linear extent, and thereby provides a 
better "fit" to the human renin enzyme. 
In the endogenous substrate it is also preferred to substitute Leu for 
Val.sup.12 and Phe for Tyr.sup.13 in order to enhance the inhibitory 
activity of the resulting peptide. 
The Formula I compounds can be used in the form of salts derived from 
inorganic or organic acids and bases. Included among such acid addition 
salts are the following: acetate, adipate, alginate, aspartate, benzoate, 
benzenesulfonate, bisulfate, butyrate, citrate, camphorate, 
camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, 
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, 
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 
2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, 
persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, 
tartrate, thiocyanate, tosylate, and undecanoate. Base salts include 
ammonium salts, alkali metal salts such as sodium and potassium salts, 
alkaline earth metal salts such as calcium and magnesium salts, salts with 
organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and 
salts with amino acids such as arginine, lysine, and so forth. Also, the 
basic nitrogen-containing groups can be quaternized with such agents as 
lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, 
bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; 
and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl 
and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl 
and phenethyl bromides and others. Water or oil-soluble or dispersible 
products are thereby obtained. 
The novel peptides of the present invention possess an excellent degree of 
activity in treating renin-associated hypertension and hyperaldosteronism. 
For these purposes the compounds of the present invention may be 
administered parenterally, by inhalation spray, or rectally in dosage unit 
formulations containing conventional non-toxic pharmaceutically acceptable 
carriers, adjuvants and vehicles. The term parenteral as used herein 
includes subcutaneous injections, intravenous, intramuscular, intrasternal 
injection or infusion techniques. In addition to the treatment of 
warm-blooded animals such as mice, rats, horses, dogs, cats, etc., the 
compounds of the invention are effective in the treatment of humans. 
The pharmaceutical compositions may be in the form of a sterile injectable 
preparation, for example as a sterile injectable aqueous or oleagenous 
suspension. This suspension may be formulated according to the known art 
using suitable dispersing or wetting agents and suspending agents. The 
sterile injectable preparation may also be a sterile injectable solution 
or suspension in a non-toxic parenterally-acceptable diluent or solvent, 
for example as a solution in 1,3-butanediol. Among the acceptable vehicles 
and solvents that may be employed are water, Ringer's solution and 
isotonic sodium chloride solution. In addition, sterile, fixed oils are 
conventionally employed as a solvent or suspending medium. For this 
purpose any bland fixed oil may be employed including synthetic mono- or 
diglycerides. In addition, fatty acids such as oleic acid find use in the 
preparation of injectibles. 
The peptides of this invention may also be administered in the form of 
suppositories for rectal administration of the drug. These compositions 
can be prepared by mixing the drug with a suitable non-irritating 
excipient which is solid at ordinary temperatures but liquid at the rectal 
temperature and will therefore melt in the rectum to release the drug. 
Such materials are cocoa butter and polyethylene glycols. 
Dosage levels of the order of 2 to 35 grams per day are useful in the 
treatment of the above indicated conditions. For example, renin-associated 
hypertension and hyperaldosteronism are effectively treated by the 
administration of from 30 milligrams to 0.5 grams of the compound per 
kilogram of body weight per day. 
The amount of active ingredient that may be combined with the carrier 
materials to produce a single dosage form will vary depending upon the 
host treated and the particular mode of administration. 
It will be understood, however, that the specific dose level for any 
particular patient will depend upon a variety of factors including the 
activity of the specific compound employed, the age, body weight, general 
health, sex, diet, time of administration, route of administration, rate 
of excretion, drug combination and the severity of the particular disease 
undergoing therapy. 
Thus, in accordance with the present invention there is further provided a 
pharmaceutical composition for treating renin-associated hypertension and 
hyperaldosteronism, comprising a pharmaceutical carrier and a 
therapeutically effective amount of a peptide of the formula: 
##STR17## 
wherein A, B, D, E, R.sup.3, R.sup.4, R.sup.5 and F have the same meaning 
as recited further above for Formula I; wherein all of the asymmetric 
carbon atoms have an S configuration, except for those in the A, B and D 
substituents, which may have an S or R configuration; and a 
pharmaceutically acceptable salt thereof. 
Also, in accordance with the present invention there is still further 
provided a method of treating renin-associated hypertension and 
hyperaldosteronism, comprising administering to a patient in need of such 
treatment, a therapeutically effective amount of a peptide of the formula: 
##STR18## 
wherein A, B, D, E, R.sup.3, R.sup.4, R.sup.5 and F have the same meaning 
as recited further above for Formula I; wherein all of the asymmetric 
carbon atoms have an S configuration, except for those in the A, B and D 
substituents, which may have an S or R configuration; and a 
pharmaceutically acceptable salt thereof. 
The renin inhibitory novel peptides of the present invention may also be 
utilized in diagnostic methods for the purpose of establishing the 
significance of renin as a causative or contributory factor in 
hypertension or hyperaldosteronism in a particular patient. For this 
purpose the novel peptides of the present invention may be administered in 
a single dose of from 0.1 to 10 mg per kg of body weight. 
Both in vivo and in vitro methods may be employed. In the in vivo method, a 
novel peptide of the present invention is administered to a patient, 
preferably by intravenous injection, although parenteral administration is 
also suitable, at a hypotensive dosage level and as a single dose, and 
there may result a transitory fall in blood pressure. This fall in blood 
pressure, if it occurs, indicates supranormal plasma renin levels. 
An in vitro method which may be employed involves incubating a body fluid, 
preferably plasma, with a novel peptide of the present invention and, 
after deproteinization, measuring the amount of angiotensin II produced in 
nephrectomized, pentolinium-treated rats. Another in vitro method involves 
mixing the plasma or other body fluid with a novel peptide of the present 
invention and injecting the mixture into a test animal. The difference in 
pressor response with and without added peptide is a measure of the renin 
content of the plasma. 
Pepstatin may be employed in the methods described above as an active 
control. See, e.g., U.S. Pat. Nos. 3,784,686 and 3,873,681 for a 
description of the use of pepstatin in diagnostic methods of this type. 
The novel peptides of the present invention may be prepared in accordance 
with well-known procedures for preparing peptides from their constituent 
amino acids, which will be described in more detail below. The unusual 
amino acid, statine, may be prepared in accordance with the procedure 
described by Rich et. al., J. Org. Chem. 43: 3624 (1978). 
A general method of preparation may be described in the following terms; 
wherein amino acids forming peptides of various lengths are sequentially 
assigned a Roman numeral for each peptide, rather than on the basis of a 
position in the overall peptide Formula I: 
A method of preparing a peptide of formula I, said peptide being comprised 
of from four to nine amino acids identified as I through IX, amino acid 
(AA) I being at the C-terminus of said peptide, and amino acid (AA) IV 
through IX, depending upon the number of amino acids present, being at the 
N-terminus of said peptide, to which substituent A is attached, said 
peptide of Formula I being cyclical by virtue of a peptide bond between AA 
I and AA IV or AA V, comprising the steps of: 
(A) treating an ester of the C-terminus amino acid (AA I) with the next 
adjacent amino acid (AA II) of said peptide, the amino group of said amino 
acid being protected by a protecting group, in the presence of a 
condensing agent, whereby a dipeptide of the two amino acids (AA I and II) 
is formed; 
(B) deprotecting the dipeptide formed in Step (A) by removing the 
protecting group from the amino group of AA II; 
(C) treating the dipeptide of AA I and AA II with AA III, the amino group 
of which is protected by a protecting group, in the presence of a 
condensing agent, whereby a tripeptide of AA I, AA II and AA III is 
formed; 
(D) deprotecting the tripeptide formed in Step (C) by removing the 
protecting group from the amino group of AA III; 
(E) treating the tripeptide of AA's I-II-III with AA IV, the amino group of 
which is protected by a protecting group, in the presence of a condensing 
agent, whereby a quadripeptide of AA's I-II-III-IV is formed; 
(F) deprotecting the quadripeptide formed in Step (E) by removing the 
protecting group from the amino group of AA IV; 
(G) forming the methyl ester of AA I if said ester is not employed 
initially; 
(H) cyclizing the quadripeptide by forming a peptide bond between AA I and 
AA IV in the presence of a condensing agent to give the peptide of Formula 
I wherein A is hydrogen; 
(I) treating the cyclical quadripeptide formed in Step (H) with 
##STR19## 
where X, R.sub.a.sup.2, and R.sub.b.sup.2, are as defined above and W is 
an acid halide, anhydride, or other carbonyl activating group, to give the 
peptide of Formula I wherein A is other than hydrogen; and optionally 
(J) forming a cyclical pentapeptide up to a nonapeptide of AA I, through AA 
IV or AA IX, by repeating the procedure of Step (E) using protected AA V 
through protected AA IX, followed by deprotecting of the pentapeptide 
through nonpeptide to give the peptide of Formula I wherein A is hydrogen, 
and optionally treating the pentapeptide through nonapeptide as in Step 
(I) above to give the peptide of Formula I wherein A is other than 
hydrogen; the step of cyclizing being carried out as recited in Steps (F), 
(G), and (H) above, preferably after formation of the complete linear 
pentapeptide up to nonapeptide, but optionally prior thereto, and also 
before or after formation of the A substituent; said method also 
comprising, where necessary, protection of sidechain substituents of the 
component amino acids AA I through AA IX, with deprotection being carried 
out as a final step; said method also comprising any combination of the 
steps set out above, whereby the amino acids I through IX and substituent 
A is assembled in any desired order to prepare the peptide of Formula I; 
said method also comprising employment of the steps set out above in a 
solid phase sequential synthesis, whereby in the initial step the carboxyl 
group of the selected amino acid is bound to a synthetic resin substrate 
while the amino group of said amino acid is protected, followed by removal 
of the protecting group, the succeeding steps being as set out above, the 
peptide as it is assembled being attached to said synthetic resin 
substrate; followed by a step of removing the peptide from said synthetic 
resin substrate by transesterification with methanol to give the methyl 
ester of AA I, followed by hydrolysis and cyclization as recited above; 
removal of sidechain protecting groups being accomplished either before or 
after removal of the peptide from said synthetic resin substrate; the 
steps of cyclization and formation of the A substituent in said method 
being accomplished at any time and in any order during preparation of 
peptides of different linear extent, after preparation of the minimal 
quadripeptide as recited above. 
A preferred method involves preparation of the peptide of desired linear 
extent and desired A substituent by solid phase sequential synthesis, 
which is then removed by transesterification to give the linear, protected 
(N-terminus) methyl ester. The N-terminus protecting group, preferably 
benzyloxycarbonyl or chlorobenzyloxycarbonyl, is removed by catalytic 
hydrogenation, followed by hydrolysis of the methyl ester using potassium 
hydroxide in water and dioxane. Cyclization is then effected using 
diphenylphosphorylazide in dimethylformamide, using triethylamine, 
diisopropylethylamine, or sodium bicarbonate as the base additive. 
Purification is accomplished by silica gel and/or sephadex gel 
chromatography. 
The phenyl analog of statine, (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic 
acid (AHPPA) can be prepared in accordance with the procedure described by 
Rich et al., J. Med. Chem. 23: 27-33 (1980). 
Other analogs of statine may be prepared in a straightforward manner. For 
example, the cyclohexylalanine analog of statine, 
(3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA) ca be 
prepared by catalytic hydrogenation (using H.sub.2 /Rh on alumina, or 
other suitable catalyst) of the BOC-AHPPA, prepared as described in the 
paragraph immediately above. Alternatively, this and similar statine 
analogs can be prepared in accordance with the procedure described for 
statine, where the BOC-Leu starting material is replaced with the amino 
acid containing the desired side chain. Thus, BOC-ACHPA can also be 
prepared starting from BOC-L-cyclohexylalanine, itself prepared, for 
example, by catalytic reduction of BOC-Phe, in the same manner as 
described for BOC-AHPPA. 
The novel inhibitory peptides of the present invention are prepared by 
using the solid phase sequential synthesis technique. 
In the following description several abbreviated designations are used for 
the amino acid components, certain preferred protecting groups, reagents 
and solvents. The meanings of such abbreviated designations are given 
below in Table I. 
TABLE I 
______________________________________ 
Abbreviated 
Designation 
______________________________________ 
Amino Acid 
AHPPA (3S,4S)-4-amino-3-hydroxy- 
5-phenylpentanoic acid 
ACHPA (3S,4S)-4-amino-5-cyclo- 
hexyl-3-hydroxypentanoic 
acid 
Ala L-alanine 
Arg L-arginine 
DAB 2-S--amino-4-aminobutyric acid 
Gly L-glycine 
His D or L-histidine 
HLys homolysine, 2S--amino-6-amino- 
heptanoic acid 
Ile L-isoleucine 
Leu L-leucine 
Lys L-lysine 
Met L-methionine 
Orn L-ornithine 
Phe L-phenylalanine 
Ser L-serine 
Sar L-sarcosine (N--methylglycine) 
Sta (3S,4S)-statine 
Thr L-threonine 
Trp L-tryptophan 
Tyr L-tyrosine 
Val L-valine 
Protecting 
Groups 
BOC tert-butyloxycarbonyl 
CBZ benzyloxycarbonyl 
2-Cl--CBZ 2-chlorobenzyloxycarbonyl 
IBU iso-butyryl 
IVA iso-valeryl 
DNP dinitrophenyl 
OMe methyl ester 
Activating 
Groups 
HBT 1-hydroxybenzotriazole 
Condensing 
Agents 
DCCI dicyclohexylcarbodiimide 
DPPA diphenylphosphorylazide 
Reagents 
TEA triethylamine 
TFA trifluoroacetic acid 
Solvents 
A ammonium hydroxide (conc.) 
AcOH acetic acid 
C chloroform 
DMF dimethylformamide 
E ethyl acetate 
M methanol 
P pyridine 
THF tetrahydrofuran 
W water 
______________________________________ 
The synthesis of the peptides of the present invention by the solid phase 
technique is conducted in a stepwise manner on chloromethylated resin. The 
resin is composed of fine beads (20-70 microns in diameter) of a synthetic 
resin prepared by copolymerization of styrene with 1-2 percent 
divinylbenzene. The benzene rings in the resin are chloromethylated in a 
Friedel-Crafts reaction with chloromethyl methyl ether and stannic 
chloride. The Friedel-Crafts reaction is continued until the resin 
contains 0.5 to 5 mmoles of chlorine per gram of resin. 
The amino acid selected to be the C-terminal amino acid of the linear 
peptide is converted to its amino protected derivative. The carboxyl group 
of the selected C-terminal amino acid is bound covalently to the insoluble 
polymeric resin support, as for example, as the carboxylic ester of the 
resin-bonded benzyl chloride present in chloromethyl-substituted 
polystyrene-divinylbenzene resin. After the amino protecting group is 
removed, the amino protected derivative of the next amino acid in the 
sequence is added along with a coupling agent, such as 
dicyclohexylcarbodiimide. The amino acid reactant may be employed in the 
form of a carboxyl-activated amino acid such as ONP ester, an amino acid 
azide, and the like. Deprotection and addition of successive amino acids 
is performed until the desired linear peptide is formed. 
The selection of protecting groups is, in part, dictated by particular 
coupling conditions, in part by the amino acid and peptide components 
involved in the reaction. 
Amino-protecting groups ordinarily employed include those which are well 
known in the art, for example, urethane protecting substituents such as 
benzyloxy-carbonyl (carbobenzoxy), p-methoxycarbobenzoxy, 
p-nitrocarbobenzoxy, t-butyoxycarbonyl, and the like. It is preferred to 
utilize t-butyloxycarbonyl (BOC) for protecting the .alpha.-amino group in 
the amino acids undergoing reaction at the carboxyl end of said amino 
acid. The BOC protecting group is readily removed following such coupling 
reaction and prior to the subsequent step by the relatively mild action of 
acids (i.e. trifluoroacetic acid, or hydrogen chloride in ethyl acetate). 
The OH group of Thr and Ser can be protected by the Bzl group and the 
-amino group of Lys can be protected by the INOC group or the 
2-chlorobenzyloxycarbonyl (2-Cl-CBZ) group. Neither group is affected by 
TFA, used for removing BOC protecting groups. After the peptide is formed, 
the protective groups, such as 2-Cl-CBZ and Bzl, can be removed by 
treatment with HF or by catalytic hydrogenation. 
After the peptide has been formed on the solid phase resin, it may be 
removed from the resin by a variety of methods which are well known in the 
art. For example the peptide may be cleaved from the resin with hydrazine, 
by ammonia in methanol, or by methanol plus a suitable base. 
Preparation of the novel inhibitory peptides of the present invention 
utilizing the solid phase technique is illustrated in the following 
examples, which however, are not intended to be any limitation of the 
present invention. 
EXAMPLE 1 
##STR20## 
The title peptide, where the bracket beneath the name indicates the points 
of cyclization, from the terminal zeta-amino group of the L-Homolysyl 
residue to the carboxyl group of the L-Phenylalanine residue by an amide 
link, was prepared by a combination of solid phase and solution methods. 
The unavailability of L-Homolysine necessitated the synthesis of a 
suitably protected DL-compound, which was incorporated into the peptide to 
produce a mixture of two diastereomers as final products, which could be 
separated and identified as to D- or L- (the L- being preferred) at the 
final stage. Alternatively, separation of amino acid isomers of 
DL-homolysine could be accomplished by well-known methods to give the 
L-isomer, which could be incorporated above into the growing peptide. When 
the L-isomer is available (as in the case of the analogous L-lysine 
analog) the incorporation of the single isomer is to be preferred. 
The synthesis of .alpha.-BOC-zeta-CBZ-homolysine (DL), using several 
modifications of techniques described by other workers (S. Takagi and K. 
Hayashi, Chem. Pharm. Bull. 7, 183 (1959); R. Goudry, Can. J. Chem. 31 
1060 (1953); A. Paquet, Can. J. Chem. 54 733 (1976), is briefly outlined 
in the accompanying scheme: 
##STR21## 
Step A: 
N-isobutyryl-L-Histidyl-L-Prolyl-L-Phenylalanyl-DL-(zeta-CBZ)-Homolysyl-(3 
S,4S)-Statyl-L-Leucyl-L-Phenylalanyl-O-Resin 
The title peptide resin was prepared by standard solid phase methodology, 
as described in Erickson & Merrifield, Proteins, 3rd. ed., 2, 257-527, 
1971, using a Beckman 990B peptide synthesizer to carry out the operations 
according to the attached programs. The starting polymer was BOC-Phe 
esterified to 2% cross-linked polystyrene-divinylbenzene (6 mmol, 5.0 g). 
The N.sup..alpha. -BOC derivatives of His-(DNP), Pro, Phe, and Leu were 
coupled using dicyclohexylcarbodiimide with an equivalent of the additive 
1-hydroxybenzotriazole hydrate. The Sta was prepared in accordance with 
Rich, et al., J. Org. Chem. 43 3624, 1978. The BOC-group was removed with 
40% trifluoroacetic acid. A coupling of 60 minutes followed by a 
recoupling of 120 minutes (2.5 equivalents each time of BOC-amino acid 
were used for each amino acid except Sta and zeta-CBZ-homolysine). In 
order to conserve the amounts of the latter two, an initial coupling of 
1.25 equivalents of BOC-amino acid (in 1:1 CH.sub.2 Cl.sub.2 /DMF), with 
DCC and 1 equivalent of HBT.H.sub.2 O) for 18 hours was followed by steps 
1-3 of the recouple program 2 and an additional coupling of 18 hours using 
the original (saved) coupling solution. This effected &gt;99% complete 
reaction of the residues, preserving their supply. The N-terminal 
isobutyryl group was coupled for 30 minutes as a symmetrical anhydride 
generated in situ from 5.0 equivalents of isobutyric acid and 2.5 
equivalents of DCC. This was followed by a recoupling also using the 
symmetrical anhydride. The DNP-protecting group on His was removed in the 
final program using two 25-minute treatments with 10% thiophenol in DMF. 
The finished resin was dried in vacuo. 
Step B: 
N-isobutyryl-L-Histidyl-L-Prolyl-L-Phenylalanyl-DL-Homolysyl-(3S,4S)-Staty 
l-L-Leucyl-L-Phenylalanine 
A one-quarter portion (nominally 1.5 mmole) of the above (A) resin peptide, 
approximately 3.5 g, was suspended in 50 ml methanol under nitrogen, to 
which 5 ml diisopropylethylamine (DIPEA) was added. The suspension was 
stirred for 3.5 hours, filtered and the residue resuspended as above and 
stirred for 3 hours, filtered, and resuspended as above for a final 18 
hours and filtered. The combined filtrates were evaporated to give 2.07 g 
crude Ibu-His-Pro-Phe-DL-(zeta-CBZ)-Homolys-Sta-Leu-Phe-OCH.sub.3. This 
crude was dissolved in 10 ml ethyl acetate and washed three times with 30 
ml water. The ethyl acetate was dried over Na.sub.2 SO.sub.4, filtered, 
and the solution evaporated giving 1.45 g of a yellow powder, which was 
revealed to be predominantly one compound by HPLC and TLC analyses. At 
this stage the two expected diastereomers, due to use of DL-homolysine, do 
not separate. 
The crude material was hydrogenated in 40 ml ethanol containing 3 ml acetic 
acid and 3 ml water using 800 mg 10% Pd/C under 50 lbs. pressure of 
hydrogen in a Parr apparatus, for 3.5 hours. The solution was filtered to 
remove catalyst and evaporated to give 1.92 g of the crude free amine, 
Ibu-His-Pro-Phe-DL-Homolys-Sta-Leu-Phe-OCH.sub.3, as its diacetic acid 
hydrated salt. 
A 1.85 g portion of this material was dissolved in 40 ml 1:1 dioxane:water 
into which was added over 3 hours approximately 38 ml 0.1N HCl at a rate 
necessary to keep the "pH" between 10.5 and 11.5 on a pH meter adjusted to 
"pH 10" with a 1:1 mixture of pH 10 buffer and dioxane. The hydrolysis 
reaction can be followed by TLC, 60:40:3:6 
chloroform:methanol:water:ammonium hydroxide, with the starting material 
at R.sub.f =0.8 going to two equal spots at R.sub.f 's 0.55 and 0.48. The 
dioxane was stripped from the solution and the pH adjusted to 6.5 with 
0.1N HCl. The solution was extracted 2.times.120 ml with n-butanol and the 
butanol was evaporated. The residue was triturated with ether and dried to 
give 0.850 g of a white solid, predominantly two spots on TLC and two 
peaks on HPLC representing the two expected diastereomeric products: 
Ibu-His-Pro-Phe-D- or L-Homolys-Sta-Leu-Phe. 
Step C: 
N-isobutyryl-L-Histidyl-L-Prolyl-L-Phenylalanyl-L-Homolysyl-(3S,4S)-Statyl 
-L-Leucyl-L-Phenylalanyl 
The linear material above (B) was cyclized by dissolving a 0.425 g portion 
in 50 ml dimethylformamide to which was added 21 mg (approximately 1 
equivalent) LiN.sub.3, 0.0715 ml (approximately 1 equivalent) 
diisopropylethylamine, and 0.208 g (approximately 6 equivalents) 
NaHCO.sub.3. The solution over N.sub.2 was cooled to 0.degree. C. 0.356 ml 
(approximately 4 equivalents) diphenylphosphorylazide was added, and the 
solution was stirred at 0.degree. C. for 72 hours. Examination by TLC 
revealed loss of starting material and two new product spots at R.sub.f 's 
0.48 and 0.40 in 60:40:3:6, chloroform:methanol:water:ammonium hydroxide. 
The reaction was evaporated and the residue partitioned between 200 ml 
n-butanol and 50 ml water. The n-butanol layer was washed 3 times with 25 
ml 5% NaHCO.sub.3, once with 50 ml water and stripped of solvent to give 
0.72 g of crude cyclic material. 
A 0.250 g portion of this crude was applied to a silica gel column 
(particle size 0.040 to 0.063 mm, 2.5.times.55 cm) packed in 
100:15:1.5:1.0, chloroform:methanol:water:acetic acid and eluted with the 
same solvent. The pure faster running spot was isolated as 73.2 mg, 
designated Isomer A, and slower running diastereomer was obtained as 54.1 
mg, designated Isomer B. Both could be precipitated from ethyl 
acetate/ether. The use of 300 MHz 'H NMR unambiguously identified Isomer A 
as the desired diastereomer, 
##STR22## 
by comparison with similar cyclic compounds containing an L-amino acid, 
such as L-Lysine, in place of homolysine, the synthesis of which was begun 
from the pure L-Lysine isomer. Satisfactory elemental analysis and amino 
acid analyses were obtained for each isomer, and HPLC and TLC showed high 
purity of a single compound: HPLC of Isomer A 88.9%. The identity of the 
cyclic product as the monomeric product was confirmed by fast atom 
bombardment mass spectrometry, which showed the expected parent ion peak 
due to a compound of molecular weight 1011 as predicted. A small impurity 
in the HPLC suspected to be dimeric material can be removed easily by 
chromatography on Sephadex G-25 eluting in 50% acetic acid. 
EXAMPLE 2 
##STR23## 
The title peptide was prepared by standard solid phase methodology as 
described in Erickson and Merrifield, Proteins, 3rd ed., 2: 257-527, 1976, 
using a Beckman Model 990B peptide synthesizer to carry out the operations 
according to the attached programs. Cyclization of the de-protected linear 
peptide was effected using diphenylphosphoryl azide in DMF containing an 
excess of sodium bicarbonate. 
Step A: Isobutyryl-L-Histidyl-L-Prolyl-L-Phenylalanyl-(N.sup..epsilon. 
-2-chloro-benzyloxycarbonyl)-L-Lysyl-(3S,4S)-Statyl-L-Leucyl-L-Phenylalany 
l-O-Resin 
The starting polymer resin was BOC-Phe esterified to 2% cross-linking 
polystyrene-divinylbenzene (2 mmol, 1.65 g). The N.sup..alpha. 
-BOC-derivatives of Leu, Sta, N.sup..epsilon. -2-Cl-CBZ-Lys, Phe, Pro, and 
His-DNP were coupled using dicyclohexylcarbodiimide with an equivalent of 
the additive 1-hydroxybenzotriazole hydrate. The Sta was prepared 
according to Rich, et al., J. Org. Chem. 43: 3624, 1978. The BOC-group was 
removed with 40% trifluoroacetic acid. A coupling of 30 minutes followed 
by a recoupling of 60 minutes (2.5 equivalents each time of BOC-amino 
acid) were used for each amino acid, except for Sta. These coupling times 
had been demonstrated previously to give complete coupling (as judged by 
the method of Kaiser) in this sequence. An additional recoupling of His 
was performed following Pro. In order to conserve the amounts of Sta 
employed an initial coupling using 1.25 equivalents of BOC-Sta plus equal 
amounts of HBT and DCCI were stirred in the coupling step in 18 ml 1:1 
DMF/CH.sub.2 Cl.sub.2, for 6 hours, followed by a recouple of 6 hours 
using the same saved coupling solution, without the addition of more DCCI. 
This was found to give complete coupling. The N-terminal isobutyryl group 
was coupled for 30 minutes as the symmetrical anhydride formed in situ 
from 5.0 equivalents of isobutyric acid and 2.5 equivalents of DCCI (no 
HBT). This was followed by a recoupling similarly. The DNP protecting 
group on His was removed in the final program using two 25-minute 
treatments with 10% thiophenol in DMF. The finished resin peptide (3.2 g) 
was dried and suspended in 40 ml of dry methanol. 
Step B: 
Isobutyryl-L-Histidyl-L-Prolyl-L-Phenylalanyl-L-Lysyl-(3S,4S)-Statyl-L-Leu 
cyl-L-Phenylalanine 
To the suspension prepared in (A) above was added 10 ml 
diisopropylethylamine, and the reaction mixture was stirred under dry 
nitrogen for 18 hours. The mixture was then filtered and the resin beads 
washed with methanol and CH.sub.2 Cl.sub.2. The yellow solution (combined 
all filtrates) was evaporated under reduced pressure to give 2.4 g of 
crude methyl ester. This crude product was dissolved in 50 ml of methylene 
chloride containing 5 ml methanol and washed with water. The organic lower 
layer was dried over sodium sulfate and evaporated to give 2.0 g of a 
yellow powder. This crude material could be purified on silica gel to give 
Ibu-His-Pro-Phe-(Cl-CBZ)-Lys-Sta-Leu-Phe-OCH.sub.3, but was most 
conveniently carried on to the next step without purification. Hydrolysis 
of the methyl ester was effected in 100 ml 1:1 dioxane (peroxide free), 
water, using 1N NaOH dripped in slowly over 3 hours. Evaporation of the 
dioxane and extraction of the aqueous layer with CH.sub.2 Cl.sub.2 removed 
some impurities and yellow color from the aqueous layer, containing the 
peptide. Neutralization of the aqueous layer with an equivalent of 1N HCl, 
gave an oily precipitate, which was extracted into ethyl acetate, dried 
and evaporated to give 1.7 g of the free acid. This material was dissolved 
in 30 ml ethanol containing 2 ml water and 1 ml acetic acid and 
hydrogenated on a Parr apparatus at 40 lbs. H.sub.2 pressure using 0.2 g 
Pd/C catalyst for 5 hours. After TLC revealed the complete removal of the 
Cl-CBZ group, the solution was filtered through a Celite pad, and 
evaporated. The residue was dissolved in water (30 ml) and the pH adjusted 
to pH 6.5 with 0.1N NaOH, causing some precipitate to form. At this pH, 
the approximate isoelectric point for the Zwitterion product (the mean of 
the pKa's of the Lys-amine and Phe-carboxyl), the product could be cleanly 
extracted into n-butanol, which could be washed with water, and the 
n-butanol evaporated to give 1.4 g of crude 
Ibu-His-Pro-Phe-Lys-Sta-Leu-Phe, B. 
##STR24## 
A 0.5 g portion (nominally 0.5 mmol) of the crude linear peptide, described 
in (B) was dissolved in 50 ml dry, degassed DMF in which 0.42 g (10 
equivalents) of sodium bicarbonate was suspended. The solution was cooled 
to 0.degree. C. and stirred. To this solution was added 0.41 g (0.323 mL) 
diphenylphosphoryl azide (3 equivalents) and the stirring continued for 48 
hours. The solution was then evaporated, and the residue suspended in 
ethyl acetate and washed with water. The ethyl acetate layer was dried and 
evaporated to give the crude product residue which was dissolved in 50% 
acetic acid and applied to a Sephadex G-25 sodium packed in 50% acetic 
acid. Fractionation, primarily by molecular size, on this column gave a 
major peptide-containing peak, detected by ultraviolet spectroscopy at 210 
nm, which proved to be monomeric material. This peak was collected and 
evaporated to give 0.40 g of a moderately pure material as judged by TLC. 
Purification by silica gel chromatography (500 g silica; 0.04-0.063 mm 
particle size; 80:15:0.75:0.75, chloroform:methanol:water:acetic acid) 
gave 0.35 g of a pure product after evaporation and precipitation from 
CH.sub.2 Cl.sub.2 /ether and drying. TLC: 80:20:2:1, 
chloroform:methanol:water:acetic acid, R.sub.f =0.35. HPLC: 99.8% pure. 
Amino acid analysis: Lys.sub.1.01 His.sub.1.01 Pro.sub.1.01 Leu.sub.1.04 
Phe.sub.1.98 Sta.sub.0.97. 'H NMR (360 MHz): spectrum was consistent with 
structure. Fast atom bombardment mass spectrometry confirmed MW 997 as 
expected for the cyclic monomeric product. 
______________________________________ 
SCHEDULE OF STEPS FOR 6 MMOL RUN 
Step Solvent/Reagent Vol. (ml) Mix time (min) 
______________________________________ 
Coupling Program 1 
1 CH.sub.2 Cl.sub.2 
6 .times. 60 
2 
2 40% TFA in CH.sub.2 Cl.sub.2 
1 .times. 60 
2 
3 40% TFA in CH.sub.2 Cl.sub.2 
1 .times. 60 
25 
4 CH.sub.2 Cl.sub.2 
3 .times. 60 
2 
5 10% TEA in CH.sub.2 Cl.sub.2 
2 .times. 60 
5 
6 CH.sub.2 Cl.sub.2 
3 .times. 60 
2 
7 BOC-amino acid, HBT 
40 5 
in 1:1 DMF/CH.sub.2 Cl.sub.2 
8 1.0M DCCI in CH.sub.2 Cl.sub.2 
15 60 
9 DMF 1 .times. 60 
2 
10 MeOH 2 .times. 60 
2 
11 CH.sub.2 Cl.sub.2 
1 .times. 60 
2 
Re-Coupling Program 2 
1 CH.sub.2 Cl.sub.2 
1 .times. 60 
2 
2 10% TEA in CH.sub.2 Cl.sub.2 
2 .times. 60 
5 
3 CH.sub.2 Cl.sub.2 
3 .times. 60 
2 
4 BOC-amino acid, HBT 
40 5 
in 1:1 DMF/CH.sub. 2 Cl.sub.2 
5 1.0M DCCI in CH.sub.2 Cl.sub.2 
15 120 
6 DMF 1 .times. 60 
2 
7 MeOH 2 .times. 60 
2 
8 CH.sub.2 Cl.sub.2 
5 .times. 60 
2 
Program 3 (DNP removal) 
1 CH.sub.2 Cl.sub.2 
1 .times. 60 
2 
2 DMF 2 .times. 60 
2 
3 10% phenylthiol in DMF 
1 .times. 60 
25 
4 DMF 1 .times. 60 
2 
5 10% TEA in CH.sub.2 Cl.sub.2 
1 .times. 60 
2 
6 DMF 2 .times. 60 
2 
7 10% phenylthiol in DMF 
1 .times. 60 
25 
8 DMF 3 .times. 60 
2 
9 MeOH 2 .times. 60 
2 
10 CH.sub.2 Cl.sub.2 
2 .times. 60 
2 
11 MeOH 2 .times. 60 
2 
12 CH.sub.2 Cl.sub.2 
2 .times. 60 
2 
13 MeOH 2 .times. 60 
2 
______________________________________ 
EXAMPLE 3-9 
Following the standard solid phase methodology described above in Example 
1, additional inhibitory peptides of the present invention were prepared. 
The peptides prepared are set out in the following table. Satisfactory 
amino acid analyses were obtained by Spinco method for each listed 
peptide. 
______________________________________ 
Exm. 
No. Peptide 
______________________________________ 
##STR25## 
4. 
##STR26## 
5. 
##STR27## 
6. 
##STR28## 
7. 
##STR29## 
8. 
##STR30## 
9. 
##STR31## 
______________________________________ 
For the peptides prepared above, various analytical methods were carried 
out to verify the structure of the peptide products. The following table 
indicates which methods were employed and summarizes the results where 
practicable. 
______________________________________ 
Analytical Method 
TLC.sup.1 
Example 
(No. of 
No. systems) HPLC.sup.2 
AA.sup.3 
EA.sup.4 
NMR.sup.5 
FAB.sup.6 
______________________________________ 
3 98+ 95.5% X X X X 
4 95+ 97.0% X X X X 
5 90+ 89.1% X X X X 
6 98+ 99.4% X X X X 
7 98+ 98.8% X X X -- 
8 98+ 97.0% X X X X 
9 95+ 95.0% X X X X 
______________________________________ 
.sup.1 TLC = thin layer chromatography on silica gel; visualization by 
reagents which tend to detect peptides; % refers to estimated purity. 
.sup.2 HPLC = high pressure liquid chromatography; detection by 
ultraviolet absorption at 240 nm or 210 nm; chromatography is reverse 
phase, values should be 1.00 .+-. 0.03. 
.sup.3 AA = amino acid analysis; peptides are hydrolized to their 
component amino acids, which are then quantitatively measured; values 
should be 1.00 .+-. 0.03. 
.sup.4 EA = elemental analysis 
.sup.5 NMR = nuclear magnetic resonance spectroscopy at 360 MHz for 
protons; X = spectrum consistent with structure; -- = not performed. 
.sup.6 FAB = fast atom bombardment mass spectrum; confirms molecular 
weight expected for monomeric cycle; X = spectrum consistent with 
structure; -- = not performed. 
EXAMPLE 10 
Hog Renin Inhibition 
An assay was carried out in order to determine the inhibitory potency of 
the peptides of the present invention. The assay measured the inhibition 
of hog kidney renin, and was in accordance with the procedure described in 
Rich et al., J. Med. Chem. 23:27, 1980, except that a pH of 7.3 was used. 
The results of the assay, illustrated in the table below, are expressed as 
I.sub.50 values, which refers to the concentration of peptide inhibitor 
necessary to produce 50% inhibition of renin activity. This I.sub.50 value 
is obtained typically by plotting data from four inhibitor concentrations. 
Pepstatin was used as an active control. 
__________________________________________________________________________ 
Peptide I.sub.50 (M) 
__________________________________________________________________________ 
##STR32## 2.3 .times. 10.sup.-7 
##STR33## 2.4 .times. 10.sup.-5 
##STR34## 7.1 .times. 10.sup.-6 
##STR35## 1.7 .times. 10.sup.-6 
##STR36## 2.8 .times. 10.sup.-6 
##STR37## 1.7 .times. 10.sup.-6 
##STR38## 4.5 .times. 10.sup.-7 
##STR39## 2.6 .times. 10.sup.-8 
__________________________________________________________________________ 
EXAMPLE 11 
Human Renin Inhibition 
An assay was carried out in order to determine the inhibitory potency of 
the peptides of the present invention. The assay measured the inhibition 
of human kidney renin purified as described in Bangham, D. R., Robertson, 
I., Robinson, J. I. S., Robinson, C. J., and Tree, M., Clinical Science 
and Molecular Medicine, 48 (Supp. 2): 136s-159s (1975), and further 
purified by affinity chromatography on pepstatin-aminohexyl-Sepharase as 
described in Poe, M., Wu., J. K., Florance, J. R. Radkey, J. A., Bennett, 
C. D., and Hoagsteen, K., J. Biol. Chem. (1982, in press). The assay was 
also in accordance with Poe et al. cited above. Results are expressed as 
K.sub.I values, which refer to the dissociation constant of the inhibited 
enzyme-inhibitor complex. This K.sub.I value was obtained as described 
above. Pepstatin was used as an active control. The results are set out in 
the table below. 
__________________________________________________________________________ 
Peptide K.sub.I (M) 
__________________________________________________________________________ 
##STR40## 5.5 .times. 10.sup.-8 
##STR41## 6.4 .times. 10.sup.-8 
##STR42## 1.9 .times. 10.sup.-7 
__________________________________________________________________________