JPS61243100A - Anp derivative - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Object of the Invention The present invention relates to an ANP derivative having an antihypertensive diuretic effect, and more specifically to an atrial natriuretic hormone (αtr) useful for the treatment of heart disease or essential hypertension.
iαlnatriwrgtic polypeptide
e<ANP).

Prior Art Recently, Kangetsu, Matsuo et al. have developed a polypeptide [α-h1Lman At
realNatriuratic PoLypapti
da = α-hANP] and its structure was identified as the following formula consisting of 28 amino acids: In animal studies, furosemide (fb), which is currently frequently used as an antihypertensive diuretic,
It has been reported that it is about 100 times more powerful per weight compared to (Bioehgtn, Bi
ophys, Rgs.

Coma, 118.181 (1984)].

In addition, Ishii et al. conducted an animal study using rats regarding the effects of α-hANP on blood pressure and electron excretion, and found a dose-dependent decrease in blood pressure and an increase in urine volume, urinary sodium, and urinary potassium. (27th Japanese Society of Nephrology p-18)
5, November 16, 1980), α-h
Pharmaceutical products using ANP or its derivatives have not yet been found on the market, and the development of an ARP#Jj conductor that is chemically and enzymatically stable, long-lasting, and more powerful than α-hANP is expected.

Conventionally, hypertension has been broadly classified into hypertension with a clear cause and hypertension with no clear cause. The former is classified as secondary hypertension, and includes those caused mainly by renal disorders, endocrine disorders, pregnancy intoxication, aortic coarctation, and central nervous system disorders. On the other hand, the latter type of hypertension whose etiology is unclear is classified as essential hypertension, and 80 to 909 of all cases of hypertension have been included in this classification.

In the case of secondary hypertension, hypertension symptoms can be improved by treating the underlying disease, but in the case of primary malignant hypertension of unknown cause, treatment for the disease may include antihypertensive diuretics, The problem has been solved by administering vasodilators and the like. However, recently it has been suggested that the above-mentioned α-5ARP is one of the substances related to the pathogenesis of heart disease or essential hypertension, and research on essential hypertension involving lα-hANP has suddenly come into focus. .

The present inventors aimed to use this α-5ANP derivative as a therapeutic agent for heart disease or malignant hypertension. As a result of intensive studies to obtain a novel α-hARP derivative having the following properties, a compound superior to α-hANP was discovered, and the present invention was completed.

The present invention relates to the above-mentioned α-hANP-related novel polypeptide or a salt thereof, and an antihypertensive diuretic containing the same as an active ingredient. The polypeptide of the present invention is represented by the following general formula 1.

However, in the formula, R1 represents D-Met%L-Law, Il- or N1m, or -NH(Cfft)B C (where n is 1 to
10, preferably an integer from 4 to 10).

Constituent amino acids in general formula I are type 1 unless otherwise specified, and their abbreviations are as follows (IUPAC (Inte
rnα-tionaL Union of Pure
and Applied Chgmi-stry) -
1UB (1%tgrtsational Union
of Bio-chamistry).

Ala: Alanine Arg: Arginine Aap: Aspartic acid ・Cyanidistine Gln: Glutamine Glν: Glycine ILa: Isoleucine Las: Leucine Mat: Methionine NLg: Norleucine? A#: Phenylalanine S# De: Serine The polypeptide of the present invention can be synthesized by condensing amino acids one by one and extending a peptide chain according to the usual peptide synthesis method, or by condensing two or several amino acids. It can be produced by a method of coupling fragments consisting of amino acids, or by a combination thereof.

The condensation of two amino acids, the condensation of amino acids and peptides, or the condensation of peptides with peptides can be carried out using conventional condensation methods, such as the azide method, mixed acid anhydride method, DCC (dicyclohexylcarbodiimide) method, and active ester method (p- Nitrophenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), Woodward reagent method, carbonyldiimidazole method, redox method, DCC-HOBt method, etc. be able to.

These condensation reactions may be carried out by either a solution method or a solid phase synthesis method. When a peptide chain is stretched by a solid phase method, the C-terminal amino acid is bound to an insoluble carrier. As the insoluble carrier, any material can be used as long as it reacts with the carboxy group of the C-bed amino acid to form a detachable bond, such as halomethyl resins such as chloromethyl resins and bromomethyl resins, hydroxymethyl resins, Aminomethyl resin, benzhydrylamine resin, t-alkyloxycarbonyl hydrazide resin, etc. can be used.

As a conventional method for peptide synthesis, side chain amino groups and carboxyl groups at the α and ω positions of amino acids are protected/protected as necessary.
Need to be deprotected. Applicable protecting groups for amino groups include, for example, benzyloxycarbonyl (abbreviated as Z),
0-chlorobenzyloxycarbonyl (K(2-CI)
), F-nitrobenzyloxycarbonyl (J?(NO
t)),'I)-methoxybenzyloxycarbonyl (
Z (OMg)), t-butoxycarbonyl (Boc),
t-amyloxycarbonyl (Aoe), inbornyloxycarbonyl, adamantyloxycarbonyl, 2
-(4-biphenyl)-2-7'ropyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonylethoxycarbonyl (Mac), trifluoroacetyl, phthalyl, formyl, 2-nitrophenyl Examples include sulfenyl (NFS), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl<Mpt).

Examples of protecting groups for carboxyl groups include benzyl ester (OBzl), 4-nitrobenzyl ester (OBz
l(NO, )), t-butyl ester (OBxsu, 4-
Examples include pyridyl methyl ester (0?sc).

During the synthesis of the peptide of the present invention, it is desirable to protect specific amino acids having functional groups other than amino and carboxyl groups in their side chains, such as arginine, cysteine, and serine, with an appropriate protecting group as necessary. For example, the guanidino group of arginine is nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2,6-simethylbenzenesulfonyl (Mda).
, l + 3 p 6-trimethylphenylsulfonyl (Arts) The thiol group of cysteine is benzyl, p-methoxybenzyl, triphenylmethyl, acetylaminomethyl, ethylcar/<moyl, 4-methylbenzyl, 2#4 ,6-)+7 methylpenzyl (Tmb)
etc., and the hydroxyl group of serine is benzyl, t-butyl,
You can protect it with acetyl, tetrahydropyranyl, etc.
-0 Next, as an example of the peptide of the present invention, cyclonathrin-
54 (FIG. 1) is shown.

The protective groups for the functional groups retained until the final target product is obtained can be removed all at once or stepwise in the final step.

For example, as shown in the examples below, Asp's Bzl, A
Z (OMg) of la, Tmh of Cys, and Mta of Arg can be eliminated by reacting hydrogen fluoride or trifluoromethanesulfone@ in the presence of dimethyl sulfide.

The reaction products and final target products in each of the above steps can be obtained using conventional separation methods for peptides, such as extraction, recrystallization, Chromadog2F (gel filtration, ion exchange, distribution, adsorption, reverse phase), electrophoresis, AC distribution, etc. It can be isolated and purified by

Although the above reaction scheme is shown for cyclonathrin-54, other peptides can also be produced according to the above methods.

In addition, as the salt of the peptide represented by the general formula I, appropriate pharmacologically acceptable salts such as hydrochloride, hydrochloride, nitrate, sulfate, acetate, malate, formate, lactate can be used. , tartrates, succinates, citrates, inorganic or organic acidic salts such as p-toluenesulfonic acid, or salts), oligosaccharides such as IJium salts, potassium salts, calcium salts, ammonium salts, triethylamine salts Ammonium salts or amine salts, etc., are exemplified and prepared by conventional methods.

C. Effects of the Invention The novel polypeptide of the present invention and its salts are chemically stable and long-lasting, and have stronger antihypertensive and diuretic effects than the known US-known α-hANP, and are effective as an antihypertensive diuretic. used for.

Therefore, the present invention further relates to an antihypertensive diuretic agent containing the novel polypeptide represented by the general formula 1 or a salt thereof as an active ingredient.

The administration method and dosage of the antihypertensive diuretic of the present invention can be appropriately determined by those skilled in the art depending on the symptoms, etc., but it is generally administered parenterally (intravenously, subcutaneously). Depending on the case, it may also be administered orally.

Examples The following are production examples and pharmacological test examples of the compounds of the present invention, but these are illustrative and do not limit the present invention.The abbreviations of protecting groups used in the following examples are @ Z (QMm): p-methoxybenzyloxycarbonyl ONp: p-nitrophenoxy0w1s nisuximidoxy Bzl: benzyl 2: penzyloxycarbonyl Mts: 1.3.6-)9 methoxybenzyloxycarbonyl ( Dimethylenesulfonyl) Tmb: 2.4.6-trimethylbenzyl Below A means Dissolve the sample in ethyl acetate, 596 citric acid, 5XNαHC
After washing with saturated brine, it was dried over sulfuric acid and concentrated. A method in which the residue is recrystallized from an appropriate solvent.

The solvent system used for M9 and TLC is as shown below.

Rfl: Chloroform-methanol-water (8:3:1)
Rf2: Chloroform-methanol (10:1) Rf
3: Butanol-vinegar-water (3:l:1) Rf4: Chloroform-methanol (30:1) Rf5: Chloroform-methanol-water (18:2:l) Production example Nisole 2.14 mg, and trifluoroacetic acid 98. 5
- were added in this order, and after stirring for 1 hour under water cooling, excess trifluoroacetic acid was distilled off under reduced pressure at 8M temperature. Hexane was added to the residue, which was washed twice, and then evaporated again under reduced pressure. The obtained nine oily substances were dissolved in 15 m of dimethylformamide (DMJ') and neutralized with triethylamine under ice cooling.

Acyl component: Z (Okh)-Law-Gly-
Dissolve 40 g of NHNH in DMF16-, and add 6.5 g of 4N hydrogen chloride-dimethylformamide at -30C with stirring.
4 mg and 1.74 mg of isoamyl nitrite were added in this order, and after stirring at -10°C for 10 minutes, it was confirmed that the hydrazine reagent was negative. The mixture was further cooled to -40°C and neutralized with triethylamine.

Triethylamine was added to the amine component in order to neutralize hydrogen azide that reacted with the reaction, and the amyl component was quickly added to the amine component, followed by stirring in the refrigerator overnight.

After checking that the reaction was completed by TLC, DMF was distilled off under reduced pressure, and water was added to the residue for crystallization. After drying the crystals under reduced pressure, reprecipitate them from DMF/ether, take the crystals in a furnace, and dry them under reduced pressure to obtain the desired product (4.79).
p(82,3X) was obtained. (Compound 1) R, f, : 0J3 (8:3:l=dichlororum:methanol:water) under ice cooling with anisole 0.74 sd,) refluoro vinegar 11
After stirring for 1 hour under water cooling, excess trifluoroacetic acid was distilled off under reduced pressure at room temperature, and the residue was washed with hexane. The obtained product was again distilled off under reduced pressure at room temperature, and the residue was dissolved in DMFlo- and neutralized with triethylamine under cooling.

Acyl component (b): Z (OMe)-Ala-G
in-Bar-Gly-NHNH,% dimethylformamide 1911t, dissolved in dimethyl sulfoxide 8-4N aqueous chloride/DMF 2.05m at -30°C with stirring.
After adding 0.54 sd of isoamyl nitrite,
The mixture was stirred at -10°C for 10 minutes and confirmed to be negative for the hydrazine reagent. The mixture was further neutralized with calatriethylamine at -40°C with vigorous stirring. To neutralize hydrogen azide produced by the reaction, 0.57m-) of triethylamine is added to the amine component (α), and <b
y was added and stirred overnight in the refrigerator. After confirming the completion of the reaction by TLC, the solvent was concentrated under reduced pressure, and water was added to the residue for crystallization.

After removing the crystals and drying them, DMF + dimethyl sulfoxide (
It was reprecipitated from DMSO)/ethanol. F was removed and dried under reduced pressure to obtain the desired product 2.14. ? (67,5X) was obtained. (compound normal, p,: 250-252℃ R, f, 20, 64 (8: 3:
l )-L a w-G ly-fi y 5-N
H,1 amine component (α): Compound (2) 2.011 was added with anisole 0.47 yd, l-! under water cooling. After adding 10 wt of J fluoroacetic acid in this order and stirring for 1 hour under ice cooling,
Excess trifluoroacetic acid was distilled off under reduced pressure at room temperature. Ether was added to the residue, and the precipitated crystals were separated and dried under reduced pressure. This crystal was dissolved in DMF14- and neutralized with triethylamine 0.33- while cooling with water.

ts acyl group @b): Z (OMg)-Arg-I
Dissolve L g-G Ly-NHNH2 in DMFIOydK, add 4N hydrogen chloride/DMF1.68m at -30°C, then add 0.45-isoamyl nitrite, and -1
The mixture was stirred at 0°C for 10 minutes, and the hydrazine reagent was confirmed to be negative. It was cooled to -40°C and neutralized with triethylamine. Triethylamine 0,47- is added to the amine component (a) to neutralize hydrogen azide generated by the reaction, and (G)
After quickly adding (6) to the mixture, the mixture was stirred in the refrigerator overnight. After confirming the completion of the reaction by TLC, the solvent was distilled off under reduced pressure, and the residue was crystallized from water. DMF after drying
Reprecipitation from 10 DMSO/ethanol (EtoH). The crystals were separated and dried under reduced pressure to obtain the desired product 2.669 (85,69). (Compound 3) p,: 250-252°C R, f,: 0.54 (8: 3: 1) Ami 4 paper order): imitation (a) Anisole 36-, trifluoro under cooling to 2p41 After adding 10-acetic acid in this order and stirring for 1 hour under cooling, the trifluoroacetic acid was distilled off at room temperature, and the crystals precipitated with ether were dried in a tank T.
FA salt was added to DMF20m and neutralized with EtsN.

BzL acyl component (6): Z((Wg)-Asp-OR0,
849 was dissolved in dimethylformamide 8- and heated to -15°C.
Add triethylamine:/(Et,N)0.36-,
followed by imptyl chloroformate (IBCF) 0.
Add 31- and continue stirring at -15°C for 15 minutes, (
Add (6) to α). After 1 hour, it was confirmed by TLC that the reaction was complete, and water was added to cause crystallization. After drying, reprecipitation was performed using DMF/DMSO/ethyl acetate. Separate the crystals and dry them under reduced pressure to obtain a target product of 2.52p (9
L856) was obtained. (Compound 4) m, p. :238-241℃ R,f, : 0.57 (8:3:l) [α
) D: +x & 1 (c = α535, solvent DMF) Amine component (a): Compound (4) 1.1, Aniso-# (124t
It.

10 Mt of trifluoroacetic acid was added in this order under water cooling, and after stirring for 1 hour, TFA was distilled off, crystallized with ether, and F
Remove and dry. Add 8ml of DMF + 8ml of DNSO and dissolve.
Neutralized with EtsN.

ts Acyl component (6): Z(OMg)-Arrj→(
Dissolve 0.73 g of t-NHNH in DMF6dIIC and add 4 NHCl, 4 TO, 6 in accordance with the usual method.
Add 6 mg of inamyl nitrite and 0.18 mg of inamyl nitrite, and neutralize with Et and N. A neutralizing amount of Et3N0.18- was added to the amine component in advance, and (b) was added to (α), overnight.
Stir in the refrigerator.

After confirming the completion of the reaction by TLC, water was added for crystallization, and the mixture was dried several times. DMf acetic acid N (AcOH)
The crystals were separated and dried under reduced pressure to obtain the desired product 1.1Op (71-4N). (Compound 5) m,
p, : 236-238°C R,f, : 0.63 (8:3:l)-I
l g-G ly-A l a-G j x-8e
r-Gly-L g u-Gly=T normal b Cvs -NH2 Amine component (a): 0.15 d of anisole and 6 td of TFA were added to 1 g of compound (5) as described above, and after stirring for 1 hour,
process. Crystallize with ether, filter and dry. DMF16
- and a neutralizing amount of EtsN was added.

7-/A/Component (b): Z (OMg)-Cylt
(Ttnb)-Pha-Gly-Gly-NIIN Island o
1492 was dissolved in DMF8-, 4NHC1/DMF
O,426wt, lAN O,11- was added according to a conventional method, and neutralized with Et and N.

Et, NO, 12- to neutralize hydrogen azide was added in advance, 8 of (b) was added to (α), and the mixture was stirred overnight in a refrigerator. After confirming the completion of the reaction by TLC, water was added to crystallize the mixture, and the crystals were separated and dried. Reprecipitated from DMF/HtOH, collected several crystals, and dried under reduced pressure to obtain the desired product, 0.832
g(67,6X) was obtained.

(Compound 6) m, p, : 244°R,f. : 0 (82 3 : 1) [α)
D: +11.2 (c=0.445.5oLvfnt
DMSO) Amino Ac1d Ratios:A
spO,97,Ear O,83,Gjul,02,G
IY5.18, AjcLl, 03, Cysα84, Me
t O, 83, IJal, 00, L a s 1-0
0sPha O,98, Arg 2.04 (Rec
, ofZ+g%89%)7 α-hANP (7-23
)-HE, deprotection, purified protected α-hANP (7-23
) -NHt 200 ~ (76,6 μtnol) m
-Frezy k 241.4 fig 12.30 mm
oL), IMTFMSA-Thio7nisole/TFA
9.19 d;frC(7) were added in this order, and immediately after replacing with Ar, the mixture was stirred for 90-n under cooling with water. Add ether, crystallize, centrifuge, discard the ether layer, wash again with ether, and centrifuge. After drying the crystals, 50
% AcOH (approximately 511 t) and Amber-1
Add ite IRA-400 (approx. 1) and cook for 30 min.
Stir ・After removing Re5i% from F, dissolve F solution at 500% with purified water.
dilute to m and make pET with CNH. Furthermore, dilute iodine tincture was added until the pale yellow color did not disappear, and the mixture was slowly stirred at room temperature for 1 hour. After freeze-drying, the crystals are immersed in Yuha's 1NAcO.
Combine with H, 5epha-der; G-2551L
per /ssg (φ2 x 68aa, LNAc
Gel filtration with OH6m/fraction), 260
Absorbance was measured at 5 m. Based on the graph, the peak fraction was lyophilized and the purity was verified by reverse phase EPLC.

Reverse phase HPLCretention time 4.8 minutes Example 2. (Lgz)-(X-hANP(7-2
3)-NH,0) Production-G l? L-8e r-G
ly-Law-Gly-4:ya-NH2 amine component): Compound (4) 1.2. Anisole 0.2 to p
4d, add TFA911t, after TFA treatment, crystallize with ether, dissolve in DMF4d + DMSO10-,
Neutralized with Et and N.

g was dissolved in DMF8ydTIC and 4NHCl/DMF
0.66 d, 0.18 mg of 1AN was added, and finally neutralized with Et and N as described above. In order to neutralize hydrogen azide generated in the reaction (cL), Et, NO, 184- was added,
(b) was quickly added to (α), stirred overnight in a refrigerator, completion of the reaction was confirmed by Asahi TLC, and water was added for crystallization.

F was removed, dried, and reprecipitated with DMF/ethyl acetate.

The crystals were collected and dried to obtain the desired product 1.109 (72,19).

(Compound 7) P: 233-236°C R, f: 0.69 (8: 3: 1) Amine component (b) Compound (?) 1 & K anisole 0.1
6m, added TFAlosg, 0°C, 1hTFA treatment, crystallized with ether, dissolved in DMF20d, Et3N
It was neutralized.

Dissolve 0.50p in DMF8-, 4NHCI/DMF
α432-1l AN O,115-added and finally I:
Neutralized with t and N.

A neutralizing amount of E t3N O is added to the amine component (a) in advance.
, 12- were added, and (6) was added to (α). Stir in the refrigerator overnight. Completion of the reaction was confirmed by TLC, water was added thereto for crystallization, and reprecipitation was performed from DMF+DMSO/F: ton to obtain the desired product 772cm (623%). (Compound 8) Inert, p,: 235-238°C R, f, :Ot, 8:3:1) 14° (α), , +7.32 (c=0.41. so
lwnt DMSO) Amiso Ac1d Rat
ios: Aspl, 04, SADE 0.84, GLsL
OT, GLy6.42, ALa 1.09, Cysl,
01. ILm LO3,1aa2.0G,? AmB9
9, Arg 2.23 (Rac, oflaas 62
%)&cLa1&”]-(thANPC1-23
)-Nik (1) Deprotection, purification and maintenance (Lgs”)-αh
243 pl of m-cresol and IM TFMSA-thioanilin/TF19.26- were added in this order to ANP (723) NHt 200II9, stirred for 90 minutes under water cooling, and crystallized with ether. This is separated by centrifugation, the ether layer is discarded, washed again with ether, and separated by centrifugation. The ether layer was discarded and the solution was dried under reduced pressure. Dissolve the crystals in approximately 211 tons of 5ON acetic acid and add Amber 2ite IRA.
Add approximately 2y of -400 and stir for 30 minutes. Rasin
After 1gp, dilute solution F with purified water to 50G-
The pH was adjusted to 7 with H. Oxidation was carried out with dilute iodine tincture until the pale yellow color disappeared. The mixture was slowly stirred at room temperature for 1 hour and freeze-dried. After freeze-drying, the crystals were dissolved in a small amount of water, filtered, and the P solution was dissolved in 5 aphadaz G-
255upper fins (42890cm.

1N AcOH* 5sJ/fraction%), and the absorption was measured at O, D, 260am. The least absorbed fractions 31-39 were collected and lyophilized. The crystals were converted to %BrbOH:AcOH:H, 0=4:
1: Dissolve in a small amount of the upper layer of 5, 5 ephadaz
G-25(M) φ2.8 x 34cyn, 4
wl/no°ration, folin-Low
Lyophilize fraction %4'd-T'l, which had a high absorption measured by O-D and 760% m by the RY method, dissolve the obtained crystals in a small amount of water, filter, and then dilute the F solution with CH30N.
269i%0.3X TFA 3d/ms, 254
am, Aha: Purity was assayed using reverse phase HPLC at α08. Ilo de bar; BP-ODSφ7.9
X 25C111 The target product obtained was filtered with 28# (21X).

Example a Production of cyclonathrin-54 L ff
DM (OMg)-Let-Gly-OBZIp-methoxybenzyloxycarbonyl-leucine 7.509
Dissolved in Flowt and cooled to -15°C, 4.2 m of triethylamine, 3.3 m of isobutyl chloroformate.
were added in this order. After 1 to 2 minutes, a DMF solution of glycine benzyl ester, paratoluene sulfonate 9.8.9 and triethylamine 4.2 was added. 30 at -15℃
After reacting at room temperature for 60 minutes, the mixture was concentrated under reduced pressure and purified by Method A. Recrystallize from methanol and ether to obtain the desired product 7.2
Got 8p. (Compound l) Temperature, p, 103 to 105°C 10- and reacted for 1 hour. After concentrating under reduced pressure at room temperature, it was washed with hexane and dried. (Compound 2) a BoC-Gly-La? It was dissolved in Gly-OHt-butoxycarbonylglycine 46Tf/fDMF60- and cooled to -15°C. 3.0-mer of triethylamine and 2.55-mer of inbutyl chloroformate were added in this order.

After 1-2 minutes, compound (1) and triethylamine 2.27- dissolved in M2O- were added. -15℃ for 30 minutes,
After reacting at room temperature for 60 minutes, the mixture was concentrated under reduced pressure and purified by Method A. Rf1=0.66 This oily substance was dissolved in 50 methanol and subjected to catalytic reduction using about 100 ~ palladium black as a catalyst.

Completion of the reaction was confirmed using a thin layer Chroma Dog 2F, and 4.509 of an oily substance was obtained. (Compound 3) R/1=0.4
04 EaC-8et<Egl)-GLy-Law-
Gly-OR compound 3 4.509 and anisole 1.4
11t was dissolved in TFA4- and reacted with OC for 1 hour. TFA was distilled off under reduced pressure at room temperature, washed with hexane, and dried. The resulting oil was dissolved in 20% DMF, triethylamine 6.4xd, t-butoxycarbonyl-O
-Benzyl-L-serine-N-hydroxysuccinimide ester (6.12p) was added, and after reacting overnight at room temperature, the mixture was neutralized with acetic acid to pH 7.0. After the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with IN hydrochloric acid and saturated brine, dried over sodium sulfate, and ethyl acetate was distilled off under reduced pressure. The resulting oily substance 5! L, chloroform:methanol (9
: silica gel chromatography using l) as eluent (
5X15α). The target product 5.7351 was obtained.

(Compound 4) R/l = 0.67 Rf5 = 0.33 Eh z(OMg)-Gin-8er(Bzl)-G
Lll-Law-Gly compound 4 5.73& and anisole 1,2 were dissolved in TFA lodl and reacted at 0°C for 1 hour. TFA was distilled off under reduced pressure at room temperature, washed with hexane, and dried. The obtained oily substance was mixed with DMSO:DMF (1:
1) Triethylamine 4.59m, p dissolved in 50-
-Methoxybenzyloxycarbonyl-L-glutamine-p-nitrophenyl ester (5.2298 g) was added and reacted overnight at room temperature. After neutralizing with acetic acid to pH 7, the solvent was distilled off under reduced pressure. IN hydrochloric acid was added to the residue, and the precipitated solid was collected, washed with water, dried, and reprecipitated from DMF and ethyl acetate to obtain 5.54p of the target product. (Compound 5) m, p,
:148 150℃ Rfl: 0.36 a Z(OMtt)-Ala-Gin-8ar(Ez
l) -Gly-Law-GlyOH Compound 5 5.5411 and anisole 2.3 tMt were dissolved in TFAlod and reacted at 0°C for 1 hour. T at room temperature
FA was distilled off under reduced pressure, ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure. C(7) powder;'gDMso
:DMF (1:l) 50s/IC dissolved, p-methoxybenzyloxycarbonyl-L-arania-p-
Add 3.50 g of nitrophenyl ester, 3.0-
of triethylamine was slowly added dropwise, and the mixture was allowed to react overnight at room temperature. After neutralizing the reaction solution with acetic acid, the solvent was distilled off under reduced pressure, IN hydrochloric acid and ice were added, and the precipitated solid was collected as P.

After washing with water and drying, the product was reprecipitated from DMSO:DMF(2)/ethanol to obtain the desired product 5.20.9. (Compound 6
) C, p,: 220℃ Rfl: 0.31? Z<OMg)-Ary(Mts)-11a-Gl
rAla-GLn-8ay(j?gj)-GJy-L#
5-Gjy-ORp-methoxybenzyloxycarbonyl-No-2-J sitylenesulfonyl-L-arginyl-L-ynleucylglycine hydrazide 5.6 g was dissolved in DMFLO- at room temperature and cooled to -50°C with stirring. 4
N-hydrochloric acid-DMF4.16- and inamyl nitrite1.
27- were added in this order, and then stirred at -1θ°C for 20 minutes, then further cooled to -60°C, and 1.8% of triethylamine was added.
Neutralized with 411t. On the other hand, 2.9 tdTFA l O- of anisole were added in this order to 15.2 g of Compound V under ice-cooling, and after stirring for 1 hour under water-cooling, TFA was distilled off under reduced pressure at room temperature. Diethyl ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure. This substance was mixed into DMSO-DMF (
l:1) lOOtd, and 3.16-triethylamine was added under water cooling.

Both solutions were combined and stirred at 4°C for 48 hours, then the solvent was distilled off under reduced pressure, water was added to the residue, the precipitated solid was collected and diluted with DMF.
- Perform reprecipitation from DMSO (1:1)/methanol,
730 g of the target product was obtained. (Compound 7) 9.224°C Rfl=0.22 &Z(OAfs)-Asp(OBzL)-Arg(
Mts)-1La-GLy-Ala-Gln-8ttr
(Bzl)-GLy-Las-Gly-OB compound 7
Anisole 2.44-1TFAL was added to 7.3 g under water cooling in this order, and after stirring for 1 hour under water cooling, TFA was distilled off under reduced pressure at room temperature. Diethyl ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure. Add this substance to DMSO
-DMF (1:1) 100- was dissolved, and 2.35 yd of triethylamine and 344 g of p-methoxybenzyloxycarbonyl-O-benzyl-L-aspartic acid p-nitrophenyl ester were added, and the mixture was stirred for 14 hours. Water and IN hydrochloric acid were added to the reaction solution, and the precipitated solid was taken out and dried. Reprecipitation was performed from DMSO-DMFC1:1)/methanol to obtain a target compound 4.6811. (Compound 8) m, p, 22e C R/1:0.21 a If(OMg)-ArgCMta)-Mat(
0)-Asp(OEzl)-Arg(Mts)-Jig
-Gly-Ala-Gin-8et(Bcl)-Gj
y-Lew-GLy-OR Compound 8 4.68jI with ice, imperial anisole 1.7-
17-1TFAloo was added in this order, and after stirring for 1 hour under water cooling, TFA was distilled off under reduced pressure at room temperature. Diethyl ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure.

This substance was dissolved in DMSO-DMF (L: 1) 50mK
The mixture was dissolved and 1.91 d of triethylamine was added under ice cooling.

On the other hand, p-methoxybenzyloxycarbonyl-? -Medilenesulfonyl-L-arginyl-L-methionine sulfoxide hydrazide 4.25.9') DMF at room temperature
Dissolved in IO-, cooled to -50°C with stirring, and diluted with 4N hydrochloric acid-
DMF3. T4- and isoamyl nitrite LOOd were added in this order, and then stirred at -1θ°C for 20 minutes.
The mixture was cooled to 60°C and neutralized with 2.07-triethylamine.

Combine both solutions and react in the same manner as in 7 to obtain 5.22g of the target product.
I got it. (Compound 9) m-p・225°C Rfl:0.31 1α Z(OMg)-Phg-Gly-GLll-Ar
Q(Mts)-Met(0)-Aap(OBzL)-
Arg(Mts)-11e-Gly-ALa-01%-
Bar(BzL)-Gly-Law-GLy-OHp
-Methoxybenzyloxycarbonyl-L-7 enylalanyl glycyl hydrazide 924rn9 in DMF at room temperature.
Dissolve 5ydK, cool to -50°C with stirring, and add 4Nm acid-
DMF 1.21- and isoamyl nitrite 0.322-
were added in this order, and then stirred at -10°C for 20 minutes, then further cooled to -60°C, and triethylamine 0.674-
It was neutralized. On the other hand, 1.1 dTFA5- of anisole was added to compound [2,1 under water cooling in this order, and after stirring for 1 hour under water cooling, TFA was distilled off under reduced pressure at room temperature. Diethyl ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure. This material was dissolved in DMSO-DMF (1:1) 20-
Triethylamine 0.61 under water cooling? - was added.

Both solutions were combined and stirred at 4°C for 48 hours, then the solvent was distilled off under reduced pressure, 17V hydrochloric acid-water was added to the residue, the precipitated solid was separated, and reprecipitated from DMSO-DMF (1: l)/methanol. The target product 1.969 was obtained. (Compound 10
) Normally, 9.242°C R/l: 0.19 IL II (OMg)-NH-(CHt)y-C0
-Ph e-Gly-Gly-Arg (Mt
s )-Arg t (0)-Asp (OBz l
)-Arg<Jlts)-11e-Gly-A
l a-Gl n-8sr (Bz l )-Gly
-Leu-GlyOH Compound 10 1.9611 under ice cooling Anisole 0.94
sd.

TFA 5. After adding Om in this order and stirring for 1 hour under water cooling, TFA was distilled off under reduced pressure at room temperature. Diethyl ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure. This material was dissolved in DMSO-DMF (1: l) for 20 yd.
Dissolved in triethylamine 0.364 m, 8-p-
Methoxybenzyloxycarbonylaminooctanoic acid
p-nitrophenyl ester 7'13m9 was added and stirred at room temperature for 14 hours. Water and 1N hydrochloric acid were added to the reaction mixture, and the precipitated solid was separated and dried. DMEiO-DMF (1:
1)/re-precipitation from methanol to obtain the target product 1.
I got 689. (Compound 11) Normal, p, 242°C A'/1:0.18 IZ z(QMg) NH(CHt)t' Co
Pn-Gly-Gly-Arg (Mt s )
-Met(0)-Asp(OEzl)-Ar
g(Mts)-ILa-GLy-Ala-Gln
-8arCBzl)-(1;jy-=Lgs-Gtl-
ODNp Compound 11 500! n9 with DMF-DMSO (1:
1) Dissolve the tc, add 1 g of 2,4-dinitrophenol 1711I and dicyclohexylcarbodiimide (hereinafter abbreviated as DCC) 1921M1 in this order under ice-cooling, and dissolve at room temperature for 1 g.
React for 4 hours. The precipitated solids were counted, and the solvent of the mother liquor was distilled off under reduced pressure. Diethyl ether was added to the residue, and the precipitated solid was dried several times to obtain the target product 4801rMi. (
Compound 12) R/l: 0. O 1a Cyc jo (NH-(CH2)?-C0-
Phe-Gly-Gly-Arg(Mta)→1
et(0)-Asp<0Bzl)-Arg(Mta)
-rim-Gly-Ala-Gln-8er(Bzl)
-Gjy-Lgx-Gly) Compound 12 3 di of anisole was added in this order under ice cooling, and the mixture was stirred for 1 hour under water cooling, and then TFA was distilled off under reduced pressure at room temperature. Diethyl ether was added to the residue, and the precipitated powder was separated and dried under reduced pressure. Dissolve this material in DMF60-
Triethylamine 52μ! , hydroxybenztriazole 126ay, and Dcc192rnti were added and reacted at 25°C for 14 hours. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitated powder was collected in F and dried to obtain 240 mg% of the desired product. (Compound 13) 14 C11c lo (NH-(CHx)?-C
0-Phe-GL y-Gly-Arg→/s t-
Asp-Arg-II e-Gly-Ala-Gin-
8er-Gly-Las-Gly) Compound 13 IIg to m-cresol 741 μE1 dimethyl sulfide 501 μA! was added, and 4 st of hydrofluoric acid was added under ice-cooling, and the mixture was stirred at 0°C for 1 hour. Hydrogen fluoride was distilled off under reduced pressure, diethyl ether was added to the residue, and the precipitated solid was centrifuged and dried. This was dissolved in 1N acetic acid, adjusted to pH 8 with 5S aqueous ammonia, and stirred for 30 minutes. Sephadex G25 superfine column (2
x90cIIL) using 1N acetic acid as eluent. Fractionation was carried out in 3 fractions, and the 38th to 66th fractions were freeze-dried to obtain the desired product 22.

(Compound 14) Reverse phase HPLCrotation timg 19.4
min, zorbazB P-OD8147.19W11
X 25cm, 20%-40XCM in 0.396TFA
, CM.

Pharmacological test example L 1) Test compound: α-hANP (723) -NHt
2) Test animals: 3 urethane-anesthetized rats/% dose 3) Administration method: α-hANP(7-23)-NH,
0.9 after injection of 10μ3130μy and 100μy
Instill X saline solution at a rate of 1.8 m/hour/rat.

4) Results: The rate of increase/decrease (56) between the pre-administration value and the maximum responise value for each pharmacological parameter is shown in the table below.

Example 1) Test compound: cyclonatron-54 (Figure 1) 2
) Test animals: 3 urethane-anesthetized rats/each dose 3) Administration method: 1013 doses of cyclonatron-54 into the femoral vein.
After injection of 0 and 100 μy, 0.9% saline was injected 1.
Infusion at a rate of 8 mg/hour/rat 4) Results For the 2% pharmacological evaluation parameters, the rate of increase/decrease (9) between the pre-administration value and the maximum responise value is shown in the table below.

All of the test compounds exhibited a strong dose-dependent antihypertensive diuretic effect. In particular, cyclonathrine-54, which was used as a test compound in Example 2, is a chemically stable antihypertensive/diuretic with a novel mechanism of action. It is highly expected that this will become the case.

[Brief explanation of drawings]

FIG. 1 shows the three-dimensional structure of cyclonatron-54. Figure 1 Procedural amendment May 22, 1985 Commissioner of the Patent Office Manabu Shiga 1, Indication of the case 1985 Patent Application No. 82692 2, Name of the invention ANP derivative 3, Person making the amendment Relationship with the case Name of patent applicant: Abbott Laboratories 4, Agent 5, Subject of amendment 6, Contents of amendment (1) Insert the following sentence between lines 8 and 9 on page 13 of the specification.

Claims (2)

[Claims] (1) A polypeptide or its salt represented by the general formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼. [However, in the formula, R_1 represents D-Met, L-Leu, Il
Represents e or Nle, and R_2 is ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (Here, n is 1
~10). ] (2) An antihypertensive diuretic whose active ingredient is a polypeptide represented by the general formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or a salt thereof. [However, in the formula, R_1 is D-Met, L-Leu, Ile
- or Nle, R_2 is ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (Here, n is 1
~10). ]