JPH0363558B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel peptides that inhibit renin. The present invention also provides pharmaceutical compositions containing the novel peptides of the present invention as active ingredients, methods of treating renin-related hypertension and hyperaldosteronism, diagnostic methods utilizing the novel peptides of the present invention, and novel peptides of the present invention. The present invention relates to a method for producing a peptide. Renin is a proteolytic enzyme with a molecular weight of approximately 40,000 that is produced and secreted by the kidneys. It acts on plasma substrates to separate the deropeptide angiotensin, which is secreted by glomerular cells and converted to the potent pressor agent angiotensin. The renin-angiotensin system therefore plays an important role in normal cardiovascular homeostasis and in some types of hypertension. Previous attempts to modulate or manipulate the renin-angiotensin system have been successful with the use of inhibitors of angiotensin converting enzyme. In light of this success, it seems reasonable to conclude that the action of renin on the substrate, a specific inhibitor of the limiting enzyme step that ultimately regulates the production of angiotensin, will be at least uniformly successful. Therefore, effective inhibitors of renin have long been sought as therapeutic agents and research tools. There has been substantial interest in the synthesis of useful renin inhibitors for several decades and the following table lists the main classes of renin inhibitors that have been studied as well as their inhibition constants. Type Ki (M) renin antibody Probably 10 ^-6 pepstatin 10 ^-6 ~10 ^-7 phospholipid 10 ^-3 substrate analog Tetrapeptide 10 ^-3 otator ~ tridecapeptide 10 ^-5 ~10 ^-6 J. Antibiot ( Tokyo) Volume 23, pp. 259-262 1970
In 2007, Umezawa et al. reported the isolation of a peptide from Actinomyces (actinomycosis) that is an inhibitor of aspartyl proteases such as pepsin, cathepsin D, and renin. This peptide, known as pepstatin, was discovered by Gross et al., Science, Vol. 175, p. 656, 1971, and reduced blood pressure in vivo after injection of porcine renin into nephrectomized rats. However, pepstatin has not found wide application as an experimental drug due to its limited solubility and inhibition of various other acid proteases besides renin. The structure of pepstatin is shown below. To date, many efforts have been made to produce specific renin inhibitors based on substrate analogy. Because the human renin substrate has only recently become clear (Tewksbury et al., Circulation 56, 60).
(Supplemented page 132, October 1979), until now substrate analogs were based on the known porcine renin substrate. While human and porcine renin substrates are not identical, a substrate analog based on porcine renin has always been considered useful in the art as a predictor of human renin inhibitory effects due to the closely related actions of the two renins. Ta. Thus, while porcine renin does not cleave human renin substrates, on the other hand human renin cleaves porcine renin substrates. Poulsen et al.
Biochim.Biophys.ActaMed.Vol. 106, No. 439~
See page 453, 1957. For example, using porcine renin analogy, an octapeptide sequence extending from histidine-6 to 4 rosine-13 was found to have kinetic parameters substantially the same as those of the complete tetradecapeptide renin substrate. The amino acid sequence of the porcine renin substrate octapeptide is as follows. −His6−Pro7−Phe8−His9−Leu10
-Leu11-Val12-Tyr13- Renin cleaves this substrate between Leu ¹⁰ and Leu ¹¹ . The renin-inhibiting peptides of the invention are also based on human renin substrate similarity. The human renin octapeptide sequence related to the porcine renin substrate octapeptide is as follows. −His6−Pro7−Phe8−His9−Leu10
−Val11−Ile12−His13− Similar to the pig renin substrate, human renin is Leu ¹⁰
cleave this substrate between and Val ¹¹ . Kokubu et al., Biochem. Pharmacol. Volume 22, No.
pp. 3217-3223, 1973 is the remainder of the porcine renin substrate.
We have synthesized a number of tetrapeptide analogs found between 10 and 13, but while they can show inhibition,
The suppression factor was only about 10 ^-3 M. Analogues of even larger segments of the renin substrate have also been synthesized: Breton et al., Biochemistry
14, pp. 3892-3898, 1975 and Poulsen et al., Biochemistry, vol. 12, pp. 3877-3882,
1973. Two of the main obstacles that had to be overcome to obtain effective renin inhibitors useful in vivo were poor solubility and poor binding (large inhibition constants). Modifications that increased solubility were soon established; the inhibitory properties of peptides depended significantly on the hydrophobicity of the various amino acid residues, and solubility could be increased by replacing lipophilic amino acids with hydrophilic isosteric residues. The opposite will occur. Other methods for increasing solubility have had limited success. Various modifications designed to increase binding to renin have also been made, but again with limited success. Haver and Burton for a more detailed description of past efforts to produce effective inhibitors of renin.
Fed.Proc.Fed.Am.Soc.Exp.Biol. Volume 38, No. 2768
See p. ~2773, 1979. For other papers describing the efforts that existed to invent renin inhibitors, see Marshall, Federation Proc. Volume 35, pp. 2494-2501;
1976, Burton et al., Proc. Natl. Acad. Sci. USA.
Vol. 77, pp. 5476-5479, September 1980, Suketa et al., Biochemistry Vol. 14, pp. 3188, 1975,
Swarus, Pharmac. Ther. Volume 7, Nos. 173-201
Page, 1979, Kokub et al., Nature vol. 217, no. 456
~457 pages, February 3, 1968, Matsushita et al., J.
Antibiotics Vol. 28, pp. 1016-1018 1975 12
Tsuki, Lazar et al., Biochem.Pharma. Volume 23, No.
pp. 2776-2778, 1974, Miller et al., Biochem.
Pharma. Vol. 21, pp. 2941-2944, 1972, Haber, Clinical Science Vol. 59, pp. 7S-19S,
1980 and Ritzi et al., J.Org.Chem. vol. 43, no.
3624 pages, 1978 and J.Med.Chem. Volume 23, No. 27
See p. 1980. According to the invention the formula: [In the formula, A is hydrogen, lower alkylcarbonyl, benzyloxycarbonyl, phenoxy lower alkylcarbonyl, cyclopentylcarbonyl, or lower alkyloxycarbonyl, B-B is a single bond, His or Sar, D is a single bond or Pro, and R ² is phenyl , R ¹ is 4-imidazolyl R ³ is isopropyl, -NH-CHR ⁴ -CO- is Leu, Ile, Gly, Ala,
Val, cyclohexyl Ala or Phe, -NH-CHR ⁵ -CO- is His, Phe, leu, Tyr,
Ala or Val, B-E is _NH2 , Gly-OH, Gly-OMe,
Phe- _NH2 or Lys- _NH2 ] and pharmaceutically usable salts thereof. In the above definitions, the term "alkyl" is meant to include both branched and straight chain hydrocarbon groups having the indicated number of carbon atoms. The novel renin inhibitory peptides of the present invention also generally have amino acid components and their closely related analogs according to the following formula: A-B-B-D-F-G-Sta-H-I-B-E (). It can be described by the body.
The ABD and E components correspond to the same part of the equation. In the formula, Sta represents the less commonly used amino acid statine and its closely related analogues, the presence of which constitutes a unique feature of the renin-inhibitory peptides of the present invention. Statins are named as 4(S)-amino-3(S)-hydroxy-6-methylpeptanoic acid and have the following formula: can be represented by As shown in the above formula, the natural Staken delta substituent is essentially an isopropyl or leucine side chain. R ³ is as shown in the formula by the substituents, isopropyl groups are higher alkyl groups of up to 6 carbon atoms, cycloalkyl groups containing 3 to 7 carbon atoms, phenyl and methyl, trifluoromethyl, hydroxy , methoxy, fluoro, chloro, bromo and iodo. Particularly preferred are phenyl substituents. These modifications of the native Staken structure are in accordance with the hydrophobicity deemed necessary to maintain the inhibitory effect of the entire peptide. The remaining general amino acid components of the formula are as follows. A has the same meaning as above in the formula. BB is a single bond, His or Sar. D either does not exist or is Pro. F is Phe. G is His. H can be Phe, Leu, He, Gly, Ala, Val, cyclohexyl Ala. I is His, Phe, Leu, Tyr, Ala, or Val
and B-B has the same meaning as above in the formula. Closely related analogs of the common amino acids listed above, such as alpha-aminobutyric acid (Abu)
It is understood that in addition to Ala, Val, Leu and Ile, substituted phenyl derivatives of aliphatic amino acids and Phe are encompassed by the general description of the novel inhibitory peptides of the present invention as represented by the formula and its definition. Peptides having the formula and definition thereof that encompass derivatives of natural statins represented by the definition of the R ³ substituent in the formula therefore represent preferred peptides of the invention. Particularly preferred inhibitory peptides of the invention are: Isobutyryl-His-Pro-Phe-His-Sta-Val
−His.Gly− _{NH2isobutyryl} −His−Pro−Phe−His−Sta−Ile
-His-NH ₂ tert-butyloxycarbonyl-Phe-His-Sta
-Ila-His- _{NH2benzyloxycarbonyl} -Phe-His-Sta-
Ile−His−NH _2isovaleryl −His−Pro−Phe−His−Sta−Ile
-His- _{NH2isovaleryl} -His-Pro-Phe-His-Sta-Leu
-His- _NH2 His-Pro-Phe-His-Sta-Leu-Phe- _{NH2isovaleryl} -His-Pro-Phe-His-Sta-Leu
−Phe− _NH2acetyl −Pro−Phe−His−Sta−Leu−Phe−
_NH2acetyl -Phe-His-Sta-Leu-Phe- _NH2tert -butyloxycarbonyl-Phe-His-Sta
−Leu−Phe− _{NH2isobutyryl} −His−Pro−Phe−His−Sta−Ala
-Phe- _{NH2isobutyryl} -His-Pro-Phe-His-Sta{cyclohexylAla-Phe- _NH2tert -butyloxycarbonyl-His-Pro-Phe
-His-Sta-Leu-Leu-OCH ₃ tert-butyloxycarbonyl-His-Pro-Phe
-His-Sta-Leu-Tyr- _{NH2isobutyryl} -His-Pro-Phe-His-Sta-Leu
−Phe−Lys− _{NH2isovaleryl} −His−Pro−Phe−His−Sta−Leu
-Val-Phe- _NH2 The inhibitory peptide of the present invention can be further correctly recognized by substrate analogues from the following illustration of the formula along with the octapeptide of some porcine renin substrates, renin being Leu ¹⁰ and Leu Cut between ¹¹ . As can be seen, a unique aspect and substantial feature of the present invention is the substitution of a single statin amino acid moiety with a dual amino acid sequence: Leu ¹⁰ -Leu ¹¹ in a unique porcine renin substrate. Substituting statins with both leucine amino acids rather than just one leucine appears to result in an improved substrate analog due to the greater linear range as compared to a single leucine moiety. Statins therefore more closely approximate the linear range, thereby providing a better "match" to the renin enzyme. The inhibitory peptides of the present invention can also be further recognized by substrate analogs from the following illustration of the formula along with the octapeptide sequence of some human renin substrates, where renin is cleaved between Leu ¹⁰ and Val ¹¹ . do. As can be seen, a unique aspect and substantial feature of the present invention is the substitution of a single statin amino acid component with a dual amino acid sequence: Leu ¹⁰ -Val ¹¹ in a unique human renin substrate. Substituting statins with both leucine and valine amino acids, rather than just one leucine, appears to result in an improved substrate analogue for the larger linear range of statins as compared to a single leucine component. It will be done. Therefore, statins have a linear range of αLeu−
It more closely approximates Val, thereby providing a good "match" to the human renin enzyme. It is also suitable to substitute Val ¹² for Leu and Tyr ¹³ for Phe in order to increase the inhibitory effect of the resulting peptide on the native substrate. Therefore, the most preferred inhibitory peptide of the present invention is His-Pro-Phe-His-
It is Sta-Leu-Phe- _NH2 . Other particularly preferred peptides of the invention are the following, suitably described by structural changes at different positions of the overall peptide sequence represented by the formula and numbered positions.

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Table: The compounds of the formula can be used in the form of salts derived from inorganic or organic acids and bases. Included among such acid addition salts are: acetates, adipates, alginates, aspartates,
Benzoate, benzenesulfonate, hydrogen sulfate, butyrate, citrate, sulfonate, sulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumar Acid salt, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfone acid salt, 2-naphthalenesulfonate, nicokenate, oxalate, pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate salts, tartrates, thiocyanates, tosylates and undecanoates. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, dicyclohexylamine salts, salts with organic bases such as N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine. Also basic nitrogen-containing groups are lower alkyl halides such as methyl, ethyl, propyl and butyl chloride, bromide and iodide; dialkyl sulfates such as dimethyl, diethyl, dibutyl; and diamyl sulfate, decyl, lauryl, myristyl. and can be tetratomized with agents such as long chain halides such as stearyl chloride, bromide and iodide, aralkyl halides such as benzyl and phenethyl bromide, and the like. Water- or oil-soluble or dispersible products are thereby obtained. The novel peptides of the present invention have an excellent degree of action in the treatment of renin-associated hypertension and hyperaldosteronism. For these purposes, the compounds of the invention may be administered parenterally, by inhalation spray or rectally in dosage unit formulations containing the usual non-toxic pharmaceutically acceptable carriers, adjuvants and excipients. can do. As used herein, the term parenteral includes subcutaneous injection, 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 this invention are useful in the treatment of humans. The pharmaceutical compositions can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Possible excipients and solvents that may be employed include 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 injectables. The peptides of the invention can also be administered in the form of suppositories for rectal administration of the drug. These compositions are prepared by mixing the drug with suitable non-irritating excipients that are solid at normal temperatures but liquid at rectal temperatures and therefore melt to release the drug in the rectum. be able to. Such materials are cocoa butter and polyethylene glycols. Dosage levels of about 2 to 35 grams per day are useful in treating the symptoms indicated above. For example, renin-associated hypertension and hyperaldosteronism are effectively treated by administering 30 mg to 0.5 g of the compound per kg body weight per day. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. However, any particular dosage level for any particular patient will depend on the activity of the particular compound used, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combinations, and the specifics being treated. It is understood that the severity of the disease depends on a variety of factors, including the severity of the disease. The novel renin-inhibiting peptides of the present invention can also be utilized in diagnostic methods for the purpose of establishing the significance of renin as a cause or contributor to hypertension or hyperaldosteronism in a particular patient. For this purpose, the novel peptide of the present invention has a body weight of 1
It can be administered in a single dose of 0.1 to 10 mg per kg. Both in vivo and in vitro methods can be used. For in vivo methods, parenteral administration of the novel peptides of the invention as a single dose at hypotensive dose levels is also suitable, but preferably the novel peptides of the invention are administered to the patient by intravenous injection, resulting in an increase in blood pressure. Can cause excessive depression. This decrease in blood pressure, when occurring, indicates plasma renin levels above normal. An in vitro method that can be used involves incubating body fluids, preferably plasma, with the novel peptides of the invention, deproteinizing them, and then measuring the amount of angiotensin produced in nephrectomized pentonium-treated rats. include. Other in vitro methods involve mixing plasma or other body fluids with the novel peptides of the invention and injecting the mixture into a test animal. The difference in pressor response with and without added peptide is the amount of renin content in the plasma. Pepstatin can be used in the methods described above as an activity control. For illustrations of the use of pepstatin in this type of diagnostic method, see US Pat.
See No. 3873681. The novel peptides of the invention can be made by procedures known for making peptides from their constituent amino acids, which are described in more detail below. Statins, which are less commonly used amino acids, are described by Ritzi et al., J.Org.Chem. vol. 43, p. 3624 (1978).
It can be manufactured by the operation described in 2003). The novel inhibitory peptides of the present invention are produced using solid phase continuous synthesis techniques. In the following description, respective abbreviations are used for amino acid moieties, certain suitable protecting groups, reagents, and solvents. The meanings of such abbreviations are shown in Table 1 below.

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Table: The peptide synthesis of the invention by solid phase technology is carried out in a stepwise manner on chloromethylated resins. The resin consists of fine beads (20-70 microns in diameter) of synthetic resin made by copolymerization of styrene and 1-2% divinylbenzene. The benzene ring of the resin is chloromethylated using chloromethyl methyl ether and stannic chloride in a Friedel-Crafts reaction. The Friedel-Crafts reaction is continued until the resin contains 0.5 to 5 mmol 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 covalently bonded to an insoluble polymeric resin support, such as the carboxylic acid ester of resin-bound benzyl chloride present in chloromethyl-substituted polystyrene-divinylbenzene resin. After removing the amino protecting group, the amino protected derivative of the next amino acid in the sequence is added along with a coupling agent such as dicyclohexylcarbodiimide. Amino acid reactants can be used in the form of carboxyl-activated amino acids such as ONp esters, amino acid azides, and the like. Deprotection and addition of successive amino acids are performed until the desired linear peptide is formed. The choice of protecting group is dictated in part by the amino acid and peptide moieties involved in the reaction, and in part by the specific coupling conditions. Commonly used amino protecting groups are, for example, benzyloxy-carbonyl (carbobenzoxy), p-
Included are those well known in the art such as urethane protecting substituents such as methoxycarbobenzoxy, p-nitrocarbobenzoxy, t-butyoxycarbonyl, and the like. It is preferred to utilize t-butyloxycarbonyl (BOC) to protect the α-amino group of the amino acid that undergoes the reaction at the carboxyl terminus of the amino acid. The BOC protecting group is easily removed by the next coupling reaction and the action of a relatively mild acid (ie, trifluoroacetic acid or hydrogen chloride in ethyl acetate) prior to the next step. The DH groups of The and Ser are protected by Bzl groups, and the amino group of Lyo is an INOC group or 2-chlorobenzyloxy-carbonyl (2-CL-CBZ).
protected by the group. The group is unaffected by TFA used to remove the BOC protecting group. After generating the peptide, protecting groups such as 2-Cl-CBZ can be removed by treatment with HF or catalytic hydrogenation. After the peptide is produced on the solid phase resin, it can be removed from the resin by various methods known in the art. For example, peptides can be cleaved from a hydrazine-containing resin with ammonia in methanol or with methanol and a suitable base. The production of the novel inhibitory peptides of the invention using solid phase technology is illustrated in the following examples. Example 1 N-isobutyryl-L-histidyl-L-prolyl-L-phenylalanyl-L-histidyl-(3S,4S)-statyl-L-valyl-L-histidyl-L-glycylamide The title compound was synthesized from Ericsson and Marifield. , Proteins, 3rd Edition, Volume 2, Pages 257-527, 1976, using a Beckman Model 990B Peptide Synthesizer, performing the operations according to the attached program. Manufacture. The starting polymer was 2% cross-linked polystyrene-BOC-esterified to divinylbenzene.
Gly (6 mmol, 5.00 g). His-DNP,
Val, Sta, His−DNP, Phe and Pro N〓−
The BOC derivative is coupled using dicyclohexylcarbodiimide with an equivalent addition of 1-hydroxybenzotriazole hydrate. Sta is prepared according to Rittsch et al., J.Org.Chem. vol. 43, p. 3624, 1978. Remove the BOC group with 40% trifluoroacetic acid. Each amino acid except Sta is coupled for 60 minutes and then recoupled for 120 minutes (BOC - 2.5 equivalents of amino acids each time). These coupling times were previously shown and result in perfect coupling (as determined by the Kaiser method) with this arrangement. To maintain the amount of Sta used, N〓−BOC−Sta(CH ₂ Cl ₂ /
A first coupling using 1.08 equivalents (in 20 ml of DMF 1:1) results in approximately 95% complete reaction. Further addition of an amount of DCC equivalent to 0.12 equivalents of N-BOC-Sta to the stirred suspension results in complete coupling in a further 18 hours. The N-terminal isobutyryl group is attached for 60 minutes such that a symmetrical anhydride is generated at that position from 5.0 equivalents of isobutyric acid and 2.5 equivalents of DCC. This is then recoupled with 2.5 equivalents of isobutyric acid, HBT and DCC for 120 minutes. His DNP
Protecting groups are removed in the final program by two 25 min treatments with 10% thiophenol in DMF. Final resin
100ml containing dry peptide and magnetic stirring bar
Suspend in 30 ml of methanol containing 1 g of ammonium acetate in a pressure bottle. The suspension was incubated at −20°C under nitrogen.
Cool to and bubble anhydrous ammonia through the suspension until saturated. Then seal the pressure bottle and let it warm to room temperature. The suspension is stirred for 72 hours, after which the pressure valve is carefully opened and the ammonia is allowed to escape. Strain the bead suspension in an orange-red solution;
Wash the beads. evaporate the liquid and washing liquid,
Dry the solid. This crude product is then mixed with water (200ml)
and ethyl acetate (200ml). Wash the ethyl acetate layer three times with 100 ml of 1% citric acid solution. The product and acid layer are neutralized with solid sodium bicarbonate. A yellow oil precipitates from water when it becomes basic. The basic aqueous solution and precipitated oil are then extracted four times with 100 ml of dichloromethane, and the organic layer is dried and evaporated to yield 6.4 g of a crude yellow solid. This solid was mixed with chloroform/methanol/water 8:20:2.5 50
Silica column (E. Merck N.9385 silica, particle size
0.040~0.063mm8.9×47cm (silica gel approx. 1500g)
to be loaded. The column is eluted with the same solvent at 18 ml/min, after which 2700 ml fractions are collected (27 ml each). The pure product is fixed by TLC of the fractions. These fractions are combined and evaporated to a light yellow oil. Dissolve the oil in 300ml of water. The solution is filtered (10μ) and lyophilized to produce the final product. TLC: 50:
40:10 C/M/A R _f =0.66 No impurities are detected at the 2% level for all of the above, ie the product is more than 99% pure. HPLC: >99% Single Peak Spinco: His 2.02 Pro 1.00 Phe 0.99 Val 0.99 Gly 0.99

[Table] Example 2-6 Following the standard solid phase method described in Example 1 above, equivalent amounts of the appropriate
The inhibitory peptides of the invention are further produced by substituting a BOC-amino acid and generating an N-terminal group to replace an isobutyryl group, if necessary, according to procedures well established in the art. The produced peptides are shown in the following table, and the values listed below for each amino acid indicate the results of spincoamino acid analysis.

[Table] Example 7 N-isovaleryl-L-histidyl-L-prolyl-L-phenylalanyl-L-histidyl-(3S,4S)-statyl-L-leucyl-L-
Phenylalaninamide Ericsson and Merrifield, Proteins, using a Beckman 990B peptide synthesizer operating according to the attached program.
The title peptide was prepared by standard solid phase methods as described in 3rd Edition, Volume 2, Pages 257-527, 1976. The starting polymer was BOC-Phe esterified with 2% cross-linked polystyrene-divinylbenzene.
(6 mmol, 5.00 g). Leu, Sta,
N〓-BOC derivatives of His-DNP, Phe and Pro were coupled using dicyclohexylcarbodiimide with equivalent addition of 1-hydroxybenzotrizole hydrate. Rittsch et al., J.Org.Chem. No.
43, p. 3624, 1978.
The BOC group was removed with 40% trifluoroacetic acid. Katsupring for 60 minutes, then katsupring again for 120 minutes.
min (BOC-2.5 equivalents of amino acid each time) was used for each amino acid except Sta. These coupling times were previously shown and indicated complete coupling (as determined by Kaiser method) for this sequence. 1.08 equivalents of N〓−BOC−Sta to maintain the amount of Sta used
Initial coupling for 72 hours with (CH ₂ Cl ₂ /DMF 1:1 in 20 ml) showed about 95% complete reaction. Furthermore, 0.12 equivalents of N〓−BOC−Sta and the same amount of
Addition of DCC to the stirred suspension showed complete coupling after a further 18 hours. The N-terminal isovaleryl group was attached for 60 minutes so that the symmetrical anhydride was generated in situ from 5.0 equivalents of isovaleric acid and 2.5 equivalents of DCC. This was then combined with 2.5 equivalents of isovaleric acid, HBT and DCC.
Recoupled for 120 minutes. The DNP protecting group on His was removed in the final program using two 25 min treatments with 10% thiophenol in DMF. Dry the completed resin-peptide and mix with a magnetic stirring bar for 100 min.
Suspended in 30 ml of methanol containing 1 g of ammonium acetate in a ml pressure bottle. The suspension was incubated at −20°C under nitrogen.
and anhydrous ammonia was bubbled into the suspension until saturated. The pressure bottle was then sealed and allowed to warm to room temperature. The suspension was stirred for 72 hours and the ammonia was allowed to escape after carefully opening the pressure valve. The suspension of beads in an orange-red solution was filtered to wash the beads. The liquid and washings were evaporated and the solid was dried. This crude product was then mixed with water (200 ml) and ethyl acetate (200 ml).
ml). The ethyl acetate layer was washed three times with 100 ml of 1% citric acid solution. The product and acid layer were neutralized with solid sodium bicarbonate. A yellow oil precipitated from the water when it became basic. The basic aqueous solution and precipitated oil were then extracted four times with 100 ml of dichloromethane, and the organic layer was dried and evaporated to form a crude yellow solid.
6.4g was produced. This solid was dissolved in 50 ml of chloroform/methanol/water/acetic acid 80:20:2.5:1, and a silica column (E. Merck No. 9385 silica, particle size 0.040-0.063
mm) 8.9 x 47 cm (approximately 1500 g of silica gel). The column was eluted with the same solvent at 18 ml/min and 2700 ml tail fractions were collected (27 ml each). Pure product was found in cuts 70-110. These fractions were combined and evaporated to a pale yellow oil. Acetic acid oil 20
ml and added 300 ml of water. The solution was filtered (10μ) and lyophilized to yield 4.45g of a pale yellow powder.

Table: No impurities were detected at the 1% level for all of the above, ie the product was more than 99% pure. HPLC: 99% or more Single peak spinco: His 1.96
Peptide 89.4% based on molecular weight 1037.3 Pro 1.01 100% based on molecular weight 1157.4 Phe 2.00 Sta 1.02 Leu 1.02 _NH3 1.25 ¹ HNMR: 300MHZ Spectrum is consistent with structure Recommendation: No unexpected peaks.

【table】

[Table] Example 8-43 Following standard solid phase methods as described in Example 7 above, equivalent amounts of the appropriate
The inhibitory peptides of the present invention were prepared by substituting BOC-amino acids and generating the N-terminal group to replace the isovaleryl group, if necessary, according to well-established procedures in the art. The produced peptides are listed in the table below, and the numbers listed below for each amino acid indicate the results of spincoamino acid analysis.

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[Table] Various analytical methods were performed on the peptides produced above to assay the structure of the peptide products. The following table indicates the methods used and summarizes the results where applicable.

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[Table] Example 44 Renin Suppression in Pig An assay was conducted to quantify the inhibitory efficacy of the peptides of the present invention. The assay quantified the inhibition of porcine renal renin and used PH7.3 as described by Ritzi et al., J.Med.
Chem. Vol. 23, p. 27, 1980. The assay results illustrated in the table below are expressed as I ₅₀ values, which refer to the concentration of peptide inhibitor required to produce 50% inhibition of renin action. This I ₅₀ value is typically obtained by plotting data from four inhibitor concentrations. Pepstatin was used as an activity control.

[Table] Example 45 Porcine renin and human renin inhibition To demonstrate the correlation between porcine renin inhibition and human renin inhibition by a single peptide unit, the porcine renin inhibition described in Example 44 above. Four of the peptide inhibitors evaluated in the assay are further described by Haber et al., J. Clin. Endocrinol. Vol. 29, p. 1349.
It was evaluated using the human renin assay based on the 1969 method. This method uses radioimmunoassay techniques to quantify the amount of angiotensin product of renin cleavage of the substrate. Hino plasma (lyophilized) was used as the human substrate and source of human renin. I ₅₀ values were obtained by plotting the data at several inhibitor concentrations. The comparison results are shown below. Pepstatin was used as an activity control.

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Claims (1)

[Claims] 1 Formula: [Wherein A is hydrogen, lower alkylcarbonyl, benzyloxycarbonyl, phenoxy lower alkylcarbonyl, cyclopentylcarbonyl or lower alkyloxycarbonyl, B-B is a single bond, His or Sar, D is a single bond or Pro, R ² is phenyl , R ¹ is 4-imidazolyl R ³ is isopropyl, -NH-CHR ⁴ -CO- is Leu, Ile, Gly, Ala,
Val, cyclohexyl Ala or Phe, -NH-CHR ⁵ -CO- is His, Phe, leu, Tyr,
Ala, or Val, B-E is NH ₂ , Gly-OH, Gly-OMe,
Phe- _NH2 or Lys- _NH2 ] and pharmaceutically usable salts thereof. 2 The peptide is isobutyryl-His-Pro-Phe
-His-Sta-Val-His-Gly- _NH2 . The peptide according to claim 1, which is -His-Sta-Val-His-Gly-NH2. 3 The peptide is isobutyryl-His-Pro-Phe
-His-Sta-Ile-His- _NH2 . The peptide according to claim 1, which is -His-Sta-Ile-His-NH2. 4 The peptide is tert-butyloxycarbonyl-
The peptide according to claim 1, which is Phe-His-Sta-Ile-His- _NH2 . 5 Peptide is benzyloxycarbonyl-Phe
-His-Sta-Ile-His- _NH2 . The peptide according to claim 1, which is -His-Sta-Ile-His-NH2.