JPS60105697A - Peptide, manufacture and medicine - 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 (Field of Industrial Application) This invention relates to a novel natriuretic factor obtained from the atrium of rats, which has vascular hypotonia activity, hypotensive activity, and renovascular hypertension in mammals. diuretic activity of very high order, along with antihypertensive activity in
It has diuretic activity.

This atrial natriuretic factor, later referred to as ANF, is
W1 has the characteristic of a peptide consisting of 26 to 103 amino acid residues and probably plays a role in the innate maintenance of water and electrolyte balance in an endocrine-like capacity. Furthermore, the present invention provides a method for producing the above-mentioned ANF'i from a natural isolated source and producing this natural ANF'i sheath, and also provides a method for producing the ANF'i from a natural isolated source, and a method for producing the ANF'i from the C-terminus by total synthesis. It relates to a process for its preparation in the form of free carboxylic acids and in the form of carboxylic acid amides, and to intermediates in this synthesis. The invention further provides a medicament containing said natural or synthetic ANF in its free form or in the form of a salt with a pharmaceutically acceptable acid, and as a diuretic and/or natriuretic for this medicament. It relates to the use as a vasorelaxant and as an antihypertensive agent in mammals with eg renal intubular hypertension.

BACKGROUND OF THE INVENTION Rat atria are known to contain a toxic white matter active factor that can induce strong and moderate diuretic and natriuretic responses when injected intravenously in rats. C de Bold ship.

Life Sc1. ．． 28.89-94 (1981))
.

atrial natriuretic factor
This substance, called uretic factor (ANF), is a type of uretic factor (ANF).
ardiocyte) secretory machines/granules (5ecrot
ory-1ike granule) (de Bold, A. J., Can, J., Phys.
iol.

Pbarmacol, 60, 324-330 (198
2); Carcla et al.

Expertja,, 38, 1071-10
73 (1982)].

During this period, efforts have been made to purify and chemically characterize ANF in the cardiac muscle of rats and other mammals (Trlppodo et al.).

Proc, Soc, Exp, Blol, i', 'led
, 170, 502-508 (1982): P
o1lock et al., Proc, Fed.

Amer, Soc, Exp, 131o1.41,990
A (1982); Klernert et al., Physi
ologist, 25, 289A (1982)]
. However, in the study of Koori et al., IC was compromised due to the small amount of material obtained from the atrial homogenate and the apparent heterogeneity of the biologically active factors.
Trippodo et al., Proc.

Soc, Exp, B1n1. 84ed, 170,
502-508 (1982): Th1bault
etc., l-1ypertonslon.

5 (5upp1.I) 75-80 (1983); 1'
rlppodo et al. (1983) l1yperte
sion.

5 (Suppl, I) 81-88; Currie
9.

5science, 221, 71-73 (198
3) ].

The first characterization of active ANF showed that it was a mound-based pezoted with approximately 5000 molecules Fa (
Th1bault etc., Hypertension,
5 (Snppl.

I) 75-80 (1983); de 1old et al.

Life Sci,, 33. '297-302 (19
83): de 13o1d, A, J,, Proe, So
c,Expt,BioloMed,.

170.133-138 (1982)]. On the other hand, the existence of a peptide with a larger molecular weight that has natriuretic activity was also reported [formerly vault A? '＋
Hypertension, 5 (Suppl, l) 7
5-80 (1983): Trippodo et al., (1
983) Hypertenslon, 5 (Supp
I, I) 81-88: Currle et al., 5cie
nce, 221, 71-73 (1983):
da Bold et al., Life Set, 33゜2
97-302 (1983)), and more recently, deBol
d, etc. (Llfe Sc1..33, 297-302 (1
(983) published the purification of low-molecular-weight zotedate, which has diuretic and natriuretic effects, by high-pressure liquid chromatography. Cardionathrin-1 (cardio
This peptide, termed natrin-1), was isolated from rat atrial extract. This thing de↓ is about 5000
and appears to contain 49 amino acids. Recently, the amino acid sequence of Flynn and de Bold cardionathrin-1 fBiochem,
l31ophys, Res, Comm.

Vol. 117, pp. 859-865 (1983). Furthermore, Grammer et al. [(1983) Bio
cben+, Biophys, Res, Comm, ]
]↓6,696-703 has vasorelaxant activity)
Isolation of a peptide from rat atrium that has both IJ and diuretic activity and a molecular weight of about 3800 and f
? 7ml was written. Furthermore, Th1bault et al. (1,
983) FEBSL@tter 164, 286-29
0 is a heartbeat) A gave me 11! 37! It was described that Q-made sodium urinary tezotides of class I and these peptides were apparently composed of 26 (decoy) and 31 and 33 amino acid residues.Finally, Cur
ria et al.

(1984) Science223. A7-69. is a bioactive atrial peptide [atriobedetin (atrio
peptin], and two of them were completely determined by determining their respective amino acid sequences. This amino acid sequence is derived from the stiff atriopeptene.

It was shown to consist of 21 and 23 amino acid residues.

(Problem 8A seeks to solve) Natural ANF from rat atrium The process of this invention, which does not require complete production, produces the compounds of this invention in substantially pure form in reasonably good yields. and for this, the characterization of the compounds of this invention by amino acid analysis,
and has the advantage that the entire structure of each of these compounds can be clearly determined by sequence determination.

Accurate knowledge about the structure of the compound of this invention B':2
G-tFurthermore, -Is this compound all one? Shuttle standard methods of detide synthesis, e.g. Llng'5srSold Phas
e5ynthesis of Scmatogtat
in-28J Biochem, Biophys, Re
s, Comm, 95.94.5-951 (1980). By quoting this National Instruments Court, this decision was rejected.
(7) However, in the synthetic method of this invention, the classical method and in-phase method of peptide synthesis, which can be used for the production of all highly bioactive compounds of this invention, as described below. I like the corny introduction.
Something interesting was discovered. In other words, is the synthesis method of this invention economically viable? It has the advantage of being provided in sufficient quantities for use as a therapeutic agent in research and medical practice.

In addition, these fi';17j [units] can be synthesized and used to study the synthesis and activation gates.

The knowledge about the amino acid sequence of the compound of these authors also allows for the production of the peptide by recombinant DNA techniques. For example, appropriate NA sequences are prepared according to standard methods, and the iL et al. sequences are appropriately introduced into a plasmid. This plasmid is transferred to bacteria such as E. coli (E, c.
oli), followed by bacterial fermentation and expression of 4guted. For example, Villa-Komaroff et al., A Bacterial Clone Synthe
tizing Proinsulln, Proc.
Natl.

Acad, Set, U, S, A,, 75: 37
27-3731 (1978) e-san J [Gi. This description is incorporated by reference into this Specification 1.

In this J specification, the symbol representing the amine sequence is Δ
reh, Blochem, J3iophyg, 115
I UPAC-I published in , 1-12 (1966)
The UB firing method is followed, but the following single-letter symbols may be used in conjunction with the appropriate symbols for convenience.

L=Leu=ClIsine I=Ile-Isoleucine A=A1m=Alanine K=Lyg=Lysine G=Gl
y = Nibrisin E = Glu = Glutamine P = Pro = Proline D = Asp = Asno elagic acid R = Ar g = Arginine Q = G 1 n = Glutamine V = Va1 - Valine N = Aen - Asunoya line 5=Sar=Serine Y=Tyr=Tyrosine C=Cy
s=cystine W=Trp÷tryptophan P=Ph
e=phenylalanine T=Thr=threonine The symbol of the protecting group used in the synthesis method is 5chroeder
and Lubker The Peptidea J Academic Press, New York and London.

It was described in 1965, such as Boe:tert.
-butyloxycarbonyl', Bzl:benzyl:Ac
m: acetamidomethyl. Other abbreviations used are, for example: TFA: )lifluoroacetic acid; DMF: dimethylformamide; PTH: phenylthiohydantoin; HBT: ]-hydroxybenzyltriazole; HPLC: high performance liquid chromatography:
TLC: Thin layer chromatography; other symbols and/or abbreviations are commonly used in peptide chemistry and will be readily understood by those skilled in the art.

The following words used in this specification are registered trademarks or trade names. C48 Sezo Ichino? ,ri(5ep-Pr+k)
:Blo-Ge1 P-10:CM Bimegle (13io-Gel) A:Mono
)S! rR515: CNμ Dendanokkutsuku (Bon
dapak): Cl8tt 7F'ndano4 tsuku; Beckman W 34M-
January; Dionex D C5
A 1ljJl'1i :Blo-8il
) TSK IEX-530CM ; y”? rib 1
/n (Polybrene)”, Quadrol (Q
uadro l): Sephz mayto (Sequem
at ) P-6 Autono 9-ta (Autono
nverter)”, Sefat Tux (5eph
adex) ”, and speedno 4, yuri (5pe
edVac).

(Means for Solving the Problems) This invention generally provides the following amino acid sequence: (wherein, Y is -
Asn, -Asn-8er, -Asn-8er
-Phe, -Asn-8er-Phe-Arg,
and -Asn-8ar-Phe-Arg-Tyr; 2 is 011, or NH2; and X is H, 5er-.

5er-8er-, Arg-8e'r-8er-, A
rg-Arg-8er-8br-, Leu-Arg-A
rg-8er-8er, 5er-Leu-Δrg-
Arg-8er-3sr-, Arg-8er-Leu-
Arg-Arg-8er-8er-, Pro-Arg-
8er-Leu-Arg-Arg-8er-8er-.

Gly-Pro-Δrg-8er-L+eu-Arg-
Arg-8er-8er-.

8la-Gly-Pro-Arg-8er-Leu-A
rg-Arg-3er-8er-+ and T, *u-Al
a-Gly-Pro-Arg-8er-Leu-Arg
-Arg-8er-8er-,
) concerning a novel peptide expressed by

The peptides described above are harvested substantially pure, as shown by amino acid sequence and analysis, and chromatographic data below. Knead invention is -P in its pure form! The term "substantially pure" as used herein means substantially free of peptides having an amino acid terminus that differs from that of the peptide. However, the term "substantially pure form" precludes the possibility of a combination of the two clauses of this invention, each of which is in substantially pure form.

As shown later, the most active natural ANF is 33
It is a low-molecular-weight peptide consisting of 32, 31, and 26 amine FIs, and the later 38i bezoted V133
It was found that the amino terminal side of the intramolecular region of ANF, which contains 50 amino acid residues, corresponds to the excised portion. 1. For convenience, the eftide with 33 amino acid residues is denoted as ANF(1-33), and its amine-terminated somatic sounds ANF(2-33) and ANF(
3-33)-1 and ANF(8-33).

The rat atrium extract further contains an intermediate, high molecular weight A
NF, which was identified as an amino-terminal extension of ANF(1-33). These extended F bodies are highly active ANF with 33 to 26 amino acid residues.
are the biological precursors of
It is called F. Their diuretic and natriuretic activities are also somewhat lower than those of the most active hypoalgesics mentioned above: ANF. One such probe ANF is a peptide consisting of 73 amino acids and designated ANF-Hl, and the other probe ANF consists of up to 103 amino acid residues and is cumulatively designated ANF-H2. be done. Both ANF-Hl and ANF-H2 are ANF (
1-33), with ANF in the 33 amino acid residues in which each cal precedes the xy-terminus.
It has the same amino acid sequence as (1-33). moreover,
p, NF-H2) is an amino-terminal extension of dANF-Hl.

The ANF and Bro ANF of this invention obtained by the method described below are characterized by amino acid analysis. +J and their structure determined by sequencing. Because it is not possible to distinguish between the C-terminal amino acid ii (-COOH) and the corresponding acid amide (-CONH2) depending on the amine sequence, in the following formulas, the C-terminus of each peptide is
-Indicates the designation (OH, or NF2) at the terminal. However, fx shows later that both the free acid form and the acid amide form of the ANF of this invention exhibit substantially the same level of activity, so that the above-mentioned ambiguity is not important. This invention is
The next low molecule A produced from the low molecular region at the molecular sieve stage
This can be exemplified by NF K.

Namely: ANF(1-33) II-Leu-Ala-G1 y-Pro-Arrt
-8a r-Lets-Arg-Arg-8er-Ph
e-Arg=ryr-((H or Nll2)ANF(2
-33) H-A la <ly-Pro-Arg-8er-L
eu-Arg-Arg-8er-Phe-Arg-Ty
r-(αI or NF2)ANF(3-33) H-Gly-Pro-Arg-8s r-Leu-A
rg-Arg-8er-Phe-Arg-Tyr-(O
H or -H2) and the following margin ANF (8-33) H-Arg-Arg- &r-Phe-Arg-'l'yr-(OH or NH,
, ).

As mentioned above, the intermediate molecules at the molecular sieve stage 1. Two types of ANF-jNawajiAN obtained from the region and the high molecular weight region
F-Hl and ANF-H2 are included within the scope of this invention and are amino terminal extensions of ANF(1-33).

ANF (1-33) consisting of 73 amino acid residues is preceded by a peptide chain of 40 amino acid residues in i1; contains amino acid residues and is an amino-terminal extension of ANF-Hl.

ANF-Hl is expressed by the following formula, and the arrow in the formula represents ANF(
]-33) amino acid sequence, ``jnawaji residue A41 ~
73 and the preceding 40 amino acid residues are shown.

1 1 -+ d bb aa ho -α -Φ -I-11-+ r mouth -to recommendation -α η (Specific description) ANF<8-33) ? Is the xy-terminus a free acid or cal? A is a gizamide
Synthesis of NF'(8-33) is carried out as follows.0 ANF(8-33)Y If desired to be obtained as the free acid, the octapeptide fragment containing amino acid residues 1626-33 is Using cross-linked Merrifield resin,
Then, it is bonded to this resin as Boc-Tyr(Bzl)-0-■ and is produced by a solid phase method starting from 33 fc residues. Appropriately preserved amino acids corresponding to residues A32 to 26 are linked to the above starting materials one after another according to the following method to obtain a preserved octapeptide resin, which is then deprotected. Set [Cyr (Acrn)
(7) The octapeptide was cleaved from the resin support to form the octapeptide with the following formula, II-Leu-Gly-Cys.
(Acm)-Asn-8er-Phe-Arg-Tyr
Obtained as the hrJR salt represented by -OH-HOΔc (2).

A tetrapeptide fragment containing all residues 7622-25,
It is produced using a solid phase method according to the general method described in Section 2.

Starting from Boa-Gly-0-■, this starting material was added to residues 24-A2271 according to method (A) below.
A 2" L detrabebuted lipid was obtained by sequentially linking the polymers, which was then cleaved from 41% by treatment with MeOH-TEA to obtain all the corresponding protected methyl esters. 8J. This is a medical examination without paying.
Then, with hydrazine, the corresponding B
ge -buto2 peptide hydrazide Boc-Ala-G
lu-8e r -G ly -NfINH2 (5 Neuhiiqru.

A hegizapeptide h piece containing amino acid residues A 16-21 is prepared as follows. A trifluoropeptide fragment containing residues 1.9-21 was prepared in a one-phase manner as described above, cleaved from the support resin with MeOH-TEA, and trifluoro1. '1 acid (T
FA) by treatment with the corresponding methyl ester t
TFA salt f(-Arg(1'JO2)(Ie-Gly
-OCH3-TFA (,9>).Next 1c
% J' 115 ] 8 [Roc-A31) (
TI7.1)-0H], &17 [Boc-11
e-OH・S/3H20], and JFLl 6
[as Boc-8r g (R), , )-OH] was successively ligated by the classical method VC to form the above triceptate (
9) is obtained. The resulting retained hexapeptide methyl ester was decaptured and treated with hydrazine to form the corresponding Boa-hexaizotet 9 hydrazide'.
f! : Boc-Arg-I 1 e -As
p-Arg-I1e-G1y-NHNH
2.2[10A4Ci]

The octapeptide fragment containing the amino acid residues 1 (&, 8-15) is prepared by the solid-phase method in the general method described above. Starting from Boc-Gly-0@), according to method (A) below. This starting material was successively linked to the appropriately spaced amino acid gold corresponding to residue A14-8 to yield the corresponding 13oc-mectate, which fMeO
Cleavage of the corresponding Boa-octapeptide methyl ester from the resin support by treatment with H-TEA gives the acetate salt Boc-Arg-Arg by treatment with hydrazine.
-8er-8er-Cys(Acm)-Phe-Gly
-G lyNHNH2·2HOΔactJ'J'fr is obtained.

Amino-protected amino acid or peptide hydrazide J-,:
++'+: It's a combination with the right eggplant 1 or 1
This method, commonly referred to as the ``azide method,'' can be used under refrigeration conditions.4. Are you familiar with Nitride (Nitrite J Stel)? :(convert each hydrazine to the corresponding acyl azide using tcrt-buternitritone 1''i isoamyl nitride.

In this way, a solution of acyl azide was added to the solution of acyl azide. In addition to the ``jin group ((13) and the card d ? The preferred right-hand nitride is isoamyl nitride and the preferred organic base is N,N-noisozolopylethylamine.

ANF (8-33) No synthesis K Oi'T-upper k Norr
7))'' u゛JJ, by linking the Roe-tetra(deterhydrazide) obtained above to the octawave ptedoacetic acid carrier (3) under the conditions of the at method, the corresponding 13oc - Obtain the dodecapeptide and then engineer it by TFA - J-K Yone vs. ':1-S-P dener?detide acetate H-Ala-Gln-8er-
Gly-Leu-Gly-Cys(Acm)-Asn-
8er-Pbe-Arg-Tyr-OLI・2HOAc
Get 0. Next, the corresponding Boc-octadecapeptide was obtained by linking the Boc-hexa-4-butide hydrazide obtained by the above method to the above dodecapeptide acetate e) under the conditions of the azide method. , which is then treated with TFA in the presence of a small amount of 1,2-ethanedithiol to obtain the corresponding octadecapeptide K TFA salt, i.e. (TFA) I-Arg-11e-Asp.
-Arg-I 1e-Gly-Ala-Gln-8er
-Gly-Leu-Gly-Cya(Acm)-Asn
-8er-Phe-Arg-Tyr-OH1. Next, the R6cm octapeptide hydrazide ayu obtained as described above was linked to the above-mentioned Okukude force wave ptedo TFA salt) under the conditions of the azide method.
The corresponding Boe-peptide is obtained, consisting of 6 amino acid residues and having the same amino acid sequence as ANF'(8-33). Protected Ac on cysteine residues at positions 12 and 28
Removal of the m group and formation of a disulfide bridge between Cya and Cy,s by treatment with iodine in aqueous HOAe, followed by treatment with TFA, resulted in l:,j7,ANF' (8-33) Th 20,
! ::(>H-Arg-Arg-3er-8er-8
er-Phe-Arg-Tyr-OfI・6 HOAc
''C-J fl, obtained as vinegar aρ salt.

ANF(8-33) i If desired as an amide, the amino acid residue IFi, 26-33'ff:
The octazepted fragment containing 1-bridged p-methylbenzhydracylamine (84BIIA) Cedar, January'T
'= is one point [iu,], and is produced by the IT-1 method. Starting from Boc-Tyr(Bz,)-NH-MBIjJρ and constructing residue J kettle 32 in a manner similar to that described above,
Appropriately retained a corresponding to -26 is ligated with noic acid gold one after another to obtain a retained color Boa-octapeptide-NH-MBH@. Protection No. 11E[ey
e(Acm)] by treatment with HF, thereby cleaving the peptide from the resin support, to form the corresponding Boa-i cuquiptedamide formula H-Leu-Gly- Cys(Aem) -As
n-8er-Phe-Arg-Tyr-NH2”2H
OAc ql) I'j7: Obtained as A2 tomb. ANF
(8-33) The corresponding Boa-dodecapeptide amide was obtained by linking Boa-tetrapeptide hydrazide ■ to octapeptide amide (III) using the above-mentioned "azide method" for the production of the amide, Next, change this to T
By treatment with FA, the corresponding dodeca-4zotide amide gold formula H-Ala-Gln-8er-Gly-Le
u-Gly-Cys(Acm)-Asn-8er-Ph
Obtained as the acetate salt represented by e-Arg-Tyr-NH2.2HOAc.

The Boc-hexapeptide hydrazide tttp is linked to the dodecapeptide amide fragment by the azide method, the resulting Boc-octadecapeptide is purified by gel filtration over denatured dextran, and treated with TFA. Two pieces of octadecapeptide amide, H-A
rg-11e-Asp-Arg-11e-Gly-Al
a-Gln-8er-Gly-Leu-Gly-Cys
(Acm)-Asn-8er-Phe-Δrg-Ty
Obtained as TFA ii of r-NII2.4 TFA.

In the final step, BOC-octapeptide hydrazide 0**, nuctadecar 2-ph6-F under the conditions of the azide method
The corresponding Boa-2 butide amide is formed from 26 amino acid groups and has the same sequence as ANF (8-33) by fast ligation to the -pT protein.

obtain. Boa-ANF (8-3
3) An amide is obtained and treated with TFA to obtain an ri'++ acid base represented by the formula NH2.6 HOAe.

That is, the synthesis of ANF(8-33) amide is A'NF
(s-a3) Similar to face synthesis. In addition, the following J
: Sea urchin, these two compounds differ little in their respective natriuretic activities.

The diuretic activity and natriuretic activity of biological
GarcLaFu, (1982)Experient
la 38.

1071-1073 methods, and Th1bault f
i.

(1983) Hypertension 5, (
5uppl.

1) Entaparbital (6
Anesthetize with 0fn9/kP 1-P-) and perform a bladder photocath
Female Sderada Dawloy with chill and cervical fi'l/intravenous catheter (5pra, gue-Dawloy)
) Measured in rats (180-200y). The animal is injected with 5 g of glucose (3?zl/hr) during 45 min and 1 hr. Ru.

Fundamentals IIy Collection” between T12, ff1lj constant f @A
NF 2, pH 7, 4Q') Krebs
1 ml of the solution was injected as a halus through the jugular catheter, and urine was collected at 20 minute intervals.
Then, the concentration of sodium is measured by full-length optical analysis. The results are expressed as μEq of Na+ (ΔttEq
Na+/20 min) and specific activity as Δμ
Defined as Eq Na/20 min/~ stria white matter. Protein is measured by the Bradford assay (Speetor).

T. (1978) Δnal, Biochem, 86
, 142-146 No? About the season]. In this method, bovine albumin is used instead of substantially pure ANF as a standard, and the peptide identity is determined from an amino sequencer. Activity in the picomolar range has been shown for pure ANF on the actual 5'f, and o, 15-0.75
After administration of nanomoles, the amount of urine excreted in rats increased tenfold, and the amount of urine excreted increased by 20 times.
increase twice.

Hereinafter, the vasorelaxant activity of the ANF of the present invention is measured in vitro by a method involving the use of IJ horns in mammalian vessels. Briefly, healthy rabbits were anesthetized with bentoparbital sodium (30 m9/9 j-v-);
The renal artery is quickly excised, fat and connective tissue removed, and the arterial tissue cut into spirals. The helical strip thus obtained was placed between a fixed substrate and a force transducer, immersed in a bath of continuously oxygenated Claras solution at 37°C, pH 7.5, and the strip of arterial tissue A tension of 500-700 m9 is applied. Add L-/lupinegrine bitaltrate (NE) to the bath
The concentration of 3.9 X IF' ~ 1.6 X 100
-2n, the NE-induced contractions are recorded on the +9 graph and the cumulative dose-response curve is obtained.

If the standard curve is reproducible, 0.25 μl of each ANF is added to the bath and this is repeated 5 times. AN
It was found that the presence of F reduced the response to pNE.

The results are then expressed as the standard fIA error (SEM) expressed as 9-cents of the maximum response, and the covariance analysis and Doune (
Comparisons between different curves are made using the Dunnett test.

Decreased response to NEK induced by ANF K (
Displacement of the curve to the left, i.e. to higher levels of NE concentration'! Statistically significant with iP<0.01.

The blood pressure lowering activity or antihypertensive activity of ANF of this invention is as follows:
It is measured using female rats as follows. In the first group of animals, the right kidney was tightened and the left renal artery was constricted to obtain 2-kidney 1-hypertension.

In the second group of animals.

By constricting the left renal artery and resecting the right kidney, we obtain 1-Kidney 1-Krig hypertension. Sham-operated rats are used as controls in the first group, and single nephrectomized rats are used as controls in the second group. Animals in groups 1 and 2 were classified as hypertensive if their cardiac systolic blood pressure was consistently 150 msHg or higher (measured indirectly with a til cuff) for 3 weeks before the experiment. Ideally, on the day of the experiment, these rats and rome 2
Two paired groups of animals are anesthetized with pentabarbital (60 m97 to 9 jp,) and fitted with a bladder catheter, a jugular intravenous catheter, and a carotid intraarterial catheter.

Animals were injected with VC 5% glucose (2,1 m/!/h) for 30 minutes before measurements and during the evaluation period, and blood pressure was continuously monitored directly by an intra-arterial catheter-linked blood pressure transducer, and the blood pressure was monitored continuously with an intra-arterial catheter-linked blood pressure transducer. Record it in a regraph.

After a 20 min basal urine collection period, 1 μl of each ANF dissolved in % 1 m/ml of Clairass solution was added once.
(US) through the jugular catheter, and urine is collected for two periods of 20 minutes each, and blood pressure is monitored. In both the first and second experimental groups, a significant decrease in blood pressure that continued for 30 to 40 minutes or more was observed, indicating the antihypertensive effect of ANF in mammals with renovascular hypertension. is proven.

The reduction in blood pressure observed in the two normotensive control groups is similarly significant and lasts for over 20 minutes, confirming the blood pressure lowering activity of the ANF of this invention.

The ANF of this invention is a powerful diuretic and natriuretic.

It is a vascular hypotonia, blood pressure lowering, and antihypertensive agent.

and is useful in pathological conditions associated with water and/or electrolyte imbalances, and in the treatment of hypertension, particularly renovascular hypertension. The ANF of this invention is a pegutide that is normally produced by the mammalian body under physiological conditions and is therefore substantially non-toxic.

and have the advantage of providing the desired effect at very low dosage levels. The compound itself is water-soluble at the very low concentrations in which it is normally used, but it is free to dissolve in water with pharmaceutically acceptable acids such as acetic acid, citric acid, malic acid, or succinic acid. Acetate salts, which are preferably used in the form of acid addition salts, are particularly advantageous since they are easily obtained as products of the synthetic methods described below.

Such freely water-soluble salts can be used as desired.
issonas etc., He1v, Chim, Acta 4
3, 1349 (1960) by treatment with a suitable ion exchange resin, such as salts with pharmaceutically acceptable acids. Suitable ion exchange resins include strongly basic anion exchange resins such as Greenstein and Win.
itxrChemistry of the Am1n
o Ac1d+ JJohn Wiley and 5
ons, Inc., = New York and London 196 L
, Vot2., page 1456.

Basicly substituted crosslinked polyethylene resins such as Amberly IRA-400 or IRA-410 are preferred. The freely water-soluble salts of the vegutides of this invention can further be prepared by treatment with a poorly water-soluble pharmaceutically acceptable acid, such as tannic acid or monic acid. Can be converted to salt. Generally, the vinegar addition salts of vegutides and pharmaceutically acceptable acids of this invention are completely biologically equivalent to the pegutides themselves.

When the benotide of this invention or its acid addition salt with a pharmaceutically acceptable acid is used as a medicine.

They are administered systemically, by intravenous, intraperitoneal or intramuscular injection, or by sublingual or intranasal administration, as a composition in combination with a pharmaceutically acceptable carrier. For administration by injection or via the nose, K may be prepared in a sterile aqueous solution containing other solutes, such as laxatives or preservatives, and sufficient salts or glucose to make the solution isotonic. Preferably, a pegtide is used as the medium. Additionally, when the above composition is used as an intranasal spray, a small amount of a pharmaceutically acceptable interface is added to the composition to ensure that the pegutide is rapidly absorbed through the nasal mucosa. An activator may also be included. For sublingual administration, the benotide of this invention is preferably formulated as a rapidly dissolving tablet with a solid carrier or excipient such as lactose. Examples of such excipients or excipients can be found in standard pharmaceutical texts, such as Remington's Pharmaceutieal.
Science, Matsukunoya Pritzink Co., Easton, Pennsylvania, 1970. Intranasal or sublingual administration is not as strict as intravenous injection, but
Convenient for treatment. When it is desired to administer the peptide of the present invention in order to obtain a diuretic effect, a natriuretic effect, a vascular tone-reducing effect, a blood pressure lowering effect, or an antihypertensive effect, for example, in the treatment of hypertension and congestive heart failure,
The dosage depends on the species, age, body weight, sex and condition of the patient, and the chosen mode of administration.

Generally, one begins with a dosage substantially less than the optimal dosage of each peptide. Thereafter, the dosage is increased in small increments until the optimal effect in a given situation is achieved. General K, the peptides of this invention are most preferably administered at a dosage level that provides an effective concentration of the respective peptide in the patient's blood without causing harmful side effects, and Preferably, the range is from about 0.3μ to about 20 fill weight, although it will vary as mentioned above; however, for effective results, from about 1.4μ to about 14μ /
Most preferred is a dosage in the range of 1 to 9 liters of body weight.

In this invention, Niputide (is a long-acting form, a slow-release form,
or depot doIIage
Continuous feeding over an extended period of time is often preferred. Such dosage forms include 1 pharmaceutically acceptable salt forms with low solubility in body fluids.

For example, bezoti in the form of any of the above salts 1. Alternatively, it will contain the peptide in a water-soluble salt form in combination with a protective carrier that prevents rapid release. In the latter case, for example, the glutide may be formulated with non-antigenic partially decomposed gelatin in the form of a viscous liquid; or the 1:J:-E'gutide may be formulated in a pharmaceutically acceptable solid carrier, such as zinc hydroxide. Let it absorb,
Administer as a suspension in a medically acceptable liquid; Pyrrolidone, sodium alginate, gelatin, ligalacturonic acid, such as pectin, or certain mucopolysaccharides, together with an acceptable liquid carrier, preservative, or surfactant as an aqueous or non-aqueous pharmaceutical agent. Formulate into a suspension.

Examples C1 of these formulations, standard mulberry texts, e.g. Remtngton's Pharmaceut cited earlier.
described in lcalSciences. This invention-! ) Long-acting, slow-release formulations of 0tide may be further coated with pharmaceutically acceptable coating materials such as gelatin; It can also be obtained by microcassellation into ribinyl alcohol or ethyl cellulose.

Further examples of coating materials for this method used for microencapsulation are J. A. Herbjy.

Encyclopedia of Chemical
Technology.

Vo113, 2nd printing, Riley, New York, 1967
It is described on pages 436-456. Such formulations and suspensions of salts of peptides that are sparingly soluble in body fluids, c.
．． 02 μg to approximately 20 μg of peptide to 1 kll + 1 body weight
It is designed for daily release and is preferably administered by intramuscular injection. Alternatively, any of the solid dosage forms previously described, or a water-soluble salt of the peptide,
or solid carrier dispersions or solid Japanese adsorbents, such as ethylene glycol methacrylate or U.S. Pat.
Neutral hydrodarue dispersions of polymers of similar cross-linked monomers, such as those described in US Pat. The peptides of this invention are also useful as intermediates in the synthesis of other peptides with valuable biological activity.

For example, ANF (8-33) can be synthesized by standard methods for -276tide synthesis, and in a general manner similar to that described below for the synthesis of ANF (8-33).
3-33), ANF' (2-33) or ANF (1
-33) can be used as a starting material for producing.

Amino Acid Analysis Except for the amino acid sequence analysis of ANF (a-33), carboxymethylation of the peptides of this invention was performed as part of the amino acid analysis and sequencing process.

Crestf 1eld Kasa, (196
3) J, BIol, Cbem, 238.622-627
Reduction using dithiothreitol according to the method of .

The reduced cysteine was then dialkylated with iodoacetic acid. The peptide, C18μ Bondano J? Desalted on a Kuku column using 01% trifluoroacetic acid-0,191S trifluoroacetic acid and acetonitrile.

For amino acid analysis, 5 μg of each carboxymethylated penotide was hydrolyzed in 5.7N HCl and O1 mercanotoethanol at 105° C. for 22 hours. Separation of amino acids was carried out on a modified 120C Nickman Amino Acid Analyzer using a Peckman W3 column (8 micron beads, cross-linked sulfonated polystyrene resin) or a Dionex DC5 column (7 micron diameter, 8-way cross-linked,
M. using a crosslinked sulfonated polystyrenic resin).

Fauconnet et al., Anal, Biochem
, 91. 430-408 (1978).

Sequencing Determination of the amino acid sequence of the reduced and carboxymethylated peptides of this invention was performed using a state-of-the-art 890C Peckman sequencer.

Sequencing d, 3mg in a cup? Librene [hexadimethrine
bromlde), N, N, N', N'-tetramethyl-1, with 1,3-nobromonolonogun
6-hexanediamine s91)mer] and 100 nmol of dibenotide (Leu-Val), and after drying 0.3 M quatrol (QaarOl).
Four to six complete sequencing cycles were performed using Pi-valent solution (N,N,N',N'-tetrakis(bitroxyprovir)-ethylenesoamine).

All conversions to PT)T (phenylthiohydantoin)-amino acids were performed after the cleavage step with Cephemat (Seq
uemat) P-6 auto converter (Auto
converter) and mof) L/CS-510
PTH amino acid derivatives were analyzed automatically using a controller and a Vista 402 Plotter-I integrator.
Varia y (Vari
an) Using 5500 liquid chroma dog 27, L
axure et al., Can, J., Biochem, Ce1
1. Blol, 6 ], ] 287-2921983
) was quantified by high-pressure liquid chromatography.

Atria from female or male rats, preferably i+11'
Atria from only sex rats were treated with 1-5 mM El) TA, 1
Homogenized in IM acetic acid (10 m/!/g) containing mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo.) and effective concentrations of other enzyme inhibitors, preferably 12.5 μM pezostatin. do. The homogenate is centrifuged, the supernatant is frozen, thawed, and centrifuged again. The final seminal fluid is passed through a bed of octadecylsilane, preferably C48 Seguno or Cucartoli (Waters Associates, 72 g atrium), and the active substance is transferred to a column of polyacrylamide beads with a molecular weight cut-off of 20,000. , preferably Bio-Gel
) p-10 (Bio-Ra Laboratories, Mississauga, Ontario, Canada). Elution with 0.1 M acetic acid and bioassaying of the eluate yielded three distinct regions of activity, which were divided into a low molecular weight region (4000-6000 daltons), an intermediate molecular weight region (4000-6000 daltons), The active fractions from each region obtained by the above operation were treated with a 9-molecular sieve, referred to as the 6000-10,000 dalton) region and the high molecular weight (10,000-15,000 dalton) region, respectively. J-1
For example, cross-linked agarose beads with carboxymethyl groups% jlL
By applying it to a column of 0M Bio-Rad A (Bio-Ra Laboratories) and eluting with a linear gradient of 0.01~], OM ammonium acetate (p! 15.0 > oml, -, 7y). , purified separately.

Combine the active fractions and apply to a column of a suitable corner ion exchanger, such as Mono 5HR515 (Pharmacia Fine Chemicals), PretLll, Parian Model 5060, prepared for use in liquid chromatography; and pH 6.5 odor 0.02~i, OM
cD vinegar fi! )! Elute with a linear gradient with J-fr-ruamine. In the case where the active fraction from the low molecular weight region obtained by molecular sieving operation is used to initiate the above steps, the most active fraction is approximately 0.75 M−0.
From the fractions thus obtained, appropriate fractions were selected, eluted in . Therefore, good-mashi< is 0
0°C in acetonitrile. ．． 1 (V/V) %T
Reverse-phase HpLclcjt on a CNμ Bondano 4T (Waters Associates) using FA, further K[
(See Figures 2 and 4). The active fraction thus obtained is purified by reverse-phase HPLCK on finely ground (preferably 10μ) octadecylsilane using 0.1% T, F'A and acetonitrile at room temperature.
Further purification is by reverse phase HPLC on a 48μ Bondapak (Waters Associates). Finally, the thus obtained sample containing room temperature lcoi-C, 0,13(
v/v) Reversed phase H on C18μ bonder using hebutafluorobutyric acid (HFBA) and acetonitrile
PLC (for example, see Figure 3). However, if the active substance appears as a single symmetrical peak in the subsequent two chromatographic steps using 0.1% TFA and acetonitrile, low molecular weight ANF is produced in substantially pure form. Therefore, K is 0.13 (V
/V) %0)) The final purification step described above using IFBA and acetonitrile is not necessary. On the other hand, glow AN
F, i.e., the amino-terminal extension of ANF described above, a method similar to that described above is used, however.

The intermediate molecular weight region and polymer a: region obtained by the above molecular sieve operation are used in the subsequent 'FW production step,
This purification step ensures that each of the four-A
To obtain NF, K includes a final purification step by reversed phase HPLC on a C18μ Bondano with 0.13 (V/V)% hegtafluorobutyric acid and acetonitrile.

As an alternative to the above-mentioned method, although not advantageous, the active fraction obtained from molecular sieve 1 and those obtained by combining the molecular weight region and the high molecular weight region (6,000 to 15,000 daltons) with Biosil TSK IEK-530CM (ion exchanger manufactured by Bio-Rad Laboratories) was passed through a column of 0.008 M/min using a linear gradient of 15-thiacetonitrile containing ammonium FA acids from 0.02 M to 0.4 M. and can also be eluted at a flow rate of 0.7 rnl 7 min. The active peak produced by Y〆I in 0.3M ammonium formate was finally purified in reverse phase on a CNμ bonder d'pack using a linear gradient of 0.1% TFA and 20% to 50% acetonitrile at 0°C. Purify by HPLC. Several peaks with natriuretic activity are found and two major peaks are identified as ANF (8-33) and ANF (1-33), respectively. Similar results were obtained when using a mono S HR5/5 column as described above, the active material was eluted in approximately 0.8 M triethylamine acetate and was subjected to final purification with a CN1t bondano. , and each ANF (8-
33) and ANF (1-33) are obtained (see e.g. Figure 4).5 In general, in the above method, all operations are performed at 4°C unless otherwise specified. . At the end of each chromatographic step,! Lyophilize the material at 7.

It is then stored at -20°C until use. Insoluble UI (
< 6 ml), use a Sapantas bead pack concentrator to incubate polysterene tubes (1,5
Freeze-drying is carried out in n). The large volume sample in the first stage is freeze-dried in a glass flask. For the next step, the /fl was dissolved in 1-31 Ll of 0.01 M ammonium acetate (pH 5,0) and an aliquot was taken for biological assay (Garcia et al., (1999)
82) Experience 38.1071-1
073, cited earlier) is repeated I7.

When using as starting material the combined fractions exhibiting natriuretic activity obtained from the low molecular weight region in the molecular sieving operation and carrying out the next purification step as described above, four distinct benotide, namely ANF (1-33), ANF (2-33), and ANF, respectively.
(3-33) and ANF (8=33) are obtained,
Amino acid analysis reveals the υ characteristic. The following 1st article includes amino acid analysis of comparative synthetic ANF (8-33).
The results are shown in the table.

This result indicates that the above-mentioned four unique vegutides are closely related to each other. As previously described in this specification 11), the mammalian heart 11j is
It contains several sodium utilization factors in both the low molecular weight region, the intermediate molecular weight S region, and the high molecular weight region. The results of ANFO fractionation from the low molecular weight region with a Mono5t (R515 column) show the diversity of active fractions obtained at that stage, as shown in Figure 1. Further purification was performed as described previously. The selection of the particular fractions to be performed is such that one of the tetrapyramidal pegutides described immediately above is ultimately obtained.

The structure of the above ANF peptide is established by sequencing. Two of the carboxymethylated, reduced, and alkylated peptides ANF (1-33) and ANF (8-33)
The complete sequence of ANF (8-33) was established by amino-terminal Edman digestion in 0 μl/ml, and all amino acid residues were identified (initial yield of 5.78 nmol, average repeat yield of 90 .8%, see Figure 6), the results are in complete agreement with the results of the amino acid analysis in Table 1. In sequencing ANF'(1-33), all amino acid residues except those at positions 24 and 25 were identified.

The initial yield is 1.92 nmol and the average repeat yield is 92.9% (see Figure 7).

However, the results of the amino acid analysis shown in Table 1 show that the sequences of the amino acid residues 8-23 and the amino acid residues 26-33 are ANF.
Coinciding with the fact that the case is the same as in (8-33), it was determined that the &24 residue is serine and the -25 residue is glycine, and ANF (s -33
) is an amino-terminal deletion of ANF(1-33),
It is shown that the first seven amino acids of the latter are missing in the former. In the sequencing of native, i.e., non-carboxymethylated, ANF (3-33), only 23 amino acid residues were identified, the initial yield was approximately 2.52 nmol, and the average repeat yield was The rate is 90.6. However, the identified t or residue is A3-5.7-11 of ANF (1-33). and 1
3-27, and they appear in the same sequence order in both venotides. This fact is contrary to the results of amino acid analysis shown in Table 1.ANF (3-33
) has the structure as an amine-terminal deletion product of ANF(1-33), and the former lacks the first amino acid residue of the latter. Table 2 below shows the above results.

The following is a complete list of the natural ANs prepared and identified as described above.
The structure of the F-<gutide is shown below, these bezotides contain a disulfide bridge between CyS12 and Cy828, and where XX is selected from OH and Ni12. ANF-<putide (XX=OH) with a free carboxylic acid as the carboxy terminus is replaced by ANF bezotide with a carboxamide as the carboxy terminus (XX=N
H2) is biologically equivalent.

ANF (1-33) is the following formula, H-Leu-Ala-Gly-Pro-Arg-3er
-Leu-Arg-Arg-; ANF (2-33) is represented by the following formula, 1l-Ala-Gly-Pro-Arg-8er-
Leu-Arg-Arg-8er-Phe-Arg-T
Represented by yr-XX; ANF (3-a 3 ) is the following formula, H-Gly-Pro-Arg-8e r-Leu-Ar
g, -Arg-8er-Phe-Arg-Tyr-X
It is represented by X; and ANF (s −33) is the following formula.

H-Arg-Arg- It is represented by 8er-Phe-Arg-Tyr-XX.

Glow! of ANF is an amine terminal extension of ANF (1-33) and is also called Glow ANF. ! Guchido's manufacturing capabilities? P: is substantially the same as the method described above for producing ANF having 33 to 26 amino acids in each il molecule. However, the atrium should be homogenized for 5 m.
The active fraction obtained from the intermediate molecular weight region in the molecular sieving operation was used for subsequent purification steps, and the final purification was carried out in the presence of 0.1 (V/V) EDTA.
% trifluoroacetic acid and acetonitrile, and 0.13 (V/V)% tetrafluorobutyric acid (HFBA) in acetonitrile.
LC)? : I'll do it. Briefly, rat atria were treated with 5 mM EDTA, 1 mM phenylmethylsulfonyluene, and 12.s μM NOS.
Homogenize in IM acetic acid containing 7'statin (10 yni/I atrium), centrifuge, and freeze the supernatant fluid.
~, thawed, centrifuged again, and supernatant 111
was passed through a bed of octadecylsilane (C18 Segupak cartridge) and passed through a column of polyacrylamide with a molecular weight cutoff of 20,000, 0.1
．． A molecular sieve operation was performed using acetic acid of
, and high molecular weight region (Hl, 000-15.0 (10 Daltons). The active fraction obtained from the intermediate molecular weight region is further subjected to IQJQ by the same series of steps as described above. Chromatographed from cross-linked agarose beads with carboxymethyl groups (Biodal-A) K using 0.01-1.0M ammonium acetate as a linear gradient with 0.02-10M triethylamine acetate, H6,5K. Cation exchanger 2 body (mono 5) IR515 for liquid chromatography using elution
), in this case select the appropriate fraction, and convert it into ．． Micronized (10μ) cyanonorobylsilane (CNμ Bong・
The active fractions were subjected to reversed-phase HPLC at 0° C. and linearly eluted with acetonitrile at 0.degree.
Min'jf9j crushed L7C (10μ) octadecylsilane (
Reverse phase on C48μ Bondano and Tsuku] (subject to PLC,
and eluted with a linear gradient of acetonitrile at ambient temperature, and the active fraction thus obtained was o
, 1a (vAr)% henotafluorobutyric acid (HF
BA) was used to finely pulverize and perform reverse phase HPLC on fc (10μ) octadecylsilane (Cf8μ bonder/base).
Marked with K, and the surroundings? ! Final purification by elution with a linear gradient of acetonitrile at iW degrees yields a substantially pure gulocaptide (or glo ANF) designated ANF'-II 1 (see Figure 8).

Perform the same method as above, except that
The active fraction from the high molecular weight region obtained in the molecular sieving step using a column is used and further '! It is important to make W-made products.

Substantially pure globegutide (or glow ANF), designated ANF-f(2), is obtained.

globegutide ANF-H] and ANF-H2 1.
Reversed phase HPLC on an octadecylsilane (C18μ Bondapak) column using 0,1% TFA and acetonitrile elutes at approximately 35% acetonitrile, while low molecular weight ANF (1-33), ANF
(2-33), ANF (3-33), and ANF (
It is noted that 8-33) elutes at about 22% to 26% acetonitrile. Additionally, 0.13% H
In the final purification step using FBA and acetonitrile, ANF-Hl and ANF-H2 were purified from an octadecylsilane (C18μ Bondapak) column, eluting in about 4 (l) of acetonitrile. H
Amino acid analysis of 1 and ANF-H2 was performed at 5.7 N H
After hydrolysis at 180°C for 24 hours in vacuum in C1,
Amino acids are separated and quantified in duplicate as described above. The following table 3 [Sara K for comparison of companies in this table.

Based on each determined sequence (ANF-H1 and AN
F (1-33) containing the amino acid composition).

In this regard, ANF-Hl calculated from amino acid analysis
It is noted that the amino acid composition of is in reasonably good agreement with data obtained from subsequent sequencing. AN
Amino acid analysis of F-H2 showed the presence of at least 95 amino acid residues, excluding Trp, and
The ratios of Ile, Tyr and Phe in 2 are the same as in ANF-H1 and ANF (1-33), and ANIi'-H2 is the amino-terminal extension of ANF'-H1. This strongly suggests that. Evidence is accumulating for the putative ANF-H1, which is composed of 41 molecules of 103 amino acid residues and has an 11q structure of ANF-H1, which is extended by 30 additional amino acids at the amino terminus. It is being done.

The following is all white Table 3 Amino acid composition of ANF-sgutide Amino acids ANF-fll ANF-HI ANF'-
H2ANFl-33SEQ” Aax 6.42 (Ci) 6 8.32 (8) 2
1'hr 1.05 (1) ], 1.09 (1)
08er 8.42 (9) 9 8.95 (9)
5Glx 4.42 (4) 4 11.12 (11
) IPro 6.69 (6-7) 6 9.02 (
9) IGly 10.69 (11) 11 ], 0
．． 38 (10) 6Ala 5.49 (5) 5
8.89 (9) 2Val 2.05 (2) 2
3.98 (4) OMet O(0) 0 1.55
(2) 011e 1.91 (2) 2 2.00
(2) 2Leu 8.19(8) 8 11.99(
12) 3Tyr 0.91 (1) 1 1.08
(1) IPhe 2.13 (2) 2 2.34
(2) 2) Tis O,17(0) OO,32(n
) 0Lys 1.53 (1-2) 2 3.19
(3) OArg 9.45. (9-10) 10 1
0.44 (Hl) 6Cys” Undetermined 2 2.2
(2) 2Trp Undecided 2 Undecided 0 Total 67-70"" 73 95*'￥*33
Table 3 Notes in the margin below: *: ANF-HI SEQ means the amino acid composition of ANF-H1 calculated based on the determined sequence. This also applies to the ANF (1
-33) can also be applied.

**For ANF-H2, Cys content was quantified as carboxymethyl cysteine.

On the other hand, for ANF-Hl, there were 51 Cys and 1 pupil and 1 mouth because it was carried out using natural guado.

*** This is a prefix for n1-calculated amino r1;2, excluding Trp and Cys for ANF-Hl, and excluding Trp and Cys for ANF-H2.
are excluded. Numbers within 0 represent the nearest integer.

Reduced IL and carboxymethylated Noto8NF-
Sequencing of H1 was performed by the method described above and
The sequence of amino acid residues 1-58 identified in the figure is shown. Repeat yield and initial yield were 94.2 and 4.97, respectively.
The correlation coefficient was 0.959. The above array is expressed by the following formula.

In the above formula, the amino acid residue sequence A41-58 is ANF
It is noted that amino acid residue A 1-18 of (1-33) is the same as that of amino acid residue A 1-18. In addition, natural (i.e.
(not reduced and carboxymethylated) ANF-
Amino acid analysis of H1 excluded Trp and Cys (67
−70 amino acid residues, and Ile, Ty
It shows that the ratio of r and Phe is the same as ANF (1-33) (see Table 3). These data are
Coupled with the fact that the single tyrosine residue shown to be present in F-H1 is also found as the carboxy-terminal tyrosine of ANF(1-33), the sequence of ANF-HI is similar to that of ANF(1-33). 40 amino acid residues 1-40 are ANF (1-33
), it is established that there are a total of 73 amino acid residues in K ANF-Hl as shown in FIG. In Figure 10, the arrows indicate the first 40 amino acid residues and ANF (1-,3'3)
and the disulfide bond between Cys and CyS63 [between CyS12 and cyS28 of ANF (1-33)] is shown. This sequencing method uses carboxy-terminal free acid (-COO
H) and the corresponding acid amide (-CONH2) cannot be distinguished, so the following formula, 0 H-Glu-Va 1-Pro-Pro-Trp-T
hr-Gly-Gl u-Val -Asn-0 Pro-8er-Gln-Arg-Asp-Gly-G
ly-Ala-Leu-Gly-A 1 a-Leu
-Leu -Lys -8e r-Lys-Leu-A
rg-Al a-Le u-4550 Le u-Ala-Gly-Pro-Arg
-8s r -Le u-Arg-Arg-8e r
- (wherein XX is selected from OH and NH2) represents the structure of ANF-H1.

The sodium urinary activity of ANF-H1 was determined in a manner similar to that described above, and the synthetic ANF (8-33
). Figure 11 shows that the natriuretic activity of ANF-H1 at the dose levels tested is slightly lower than that of synthetic ANF (8-33) and exhibits activity in the picomolar range. .

The values shown are on the average of at least three measurements,
E, M.

Synthesis of ANF (s -33) acid or amide Briefly, ANF (s -33) acid or amide is synthesized by ligation of four fragments as follows
(8-33) Producing an acid or amide. ANF (8-3
3) When attempting to produce an acid, an octabegutide fragment containing amino acid residues A26 to 33 of ANF (8-33) is synthesized by a solid phase method using 2% cross-linked Merrifield resin, and A tetrahectide fragment containing amino acid residues 1 (x22 to 25) is also prepared by cleaving and deprotecting the protein (to obtain the phantom octabegutide). The amino acid residue is synthesized and cleaved from the resin support by transesterification using MeOH to yield the corresponding methyl ester, which is converted to the corresponding l1oc-tetrabegutide hydrazide of formula (dan). The hexabezotide fragment containing Me 16-21 is produced in multiple steps: synthesized by an in-phase method;
The corresponding 13oe was cut from the resin support using OH.
- Obtaining tripegtide methyl ester Starting from the tripeptide prepared by K., adding appropriately protected residues 18, 17 and 16 in the appropriate order by classical ligation methods, The obtained hexabegutide methester is then deprotected and converted to the corresponding Boc-hexabegutide hydrazide of formula (16). Octabezotide 1j'Ii containing amino acid residues 8-15
There was even a piece of moss. In-phase synthesis using 2% cross-linked Merrifield resin, cleavage from the resin support by transesterification using MeOH, and the resulting methyl ester of formula (1
9) to the corresponding Boc-octapeptide hydrazide. Boa-tetrabegutide hydrazide (dan) is linked to dioctapeptide acid (2) by the above-mentioned "azide method" and then the amine-terminated Boc group is removed to obtain the corresponding dodecamigtide of formula (21). Get acid. Boc-hexabegutide hydrondide (
16) is linked to the above dodecabezotide acid (21) by the "azide method" and the amine-terminated Boa group is removed 5th to obtain the corresponding octadecabezotide acid of formula (main 1). The Boc-octapeptide aladazide (19) is linked to the above octagutidate acid (Kul) by the “azide method”, the protecting group is removed, and a disulfide bridge is established between Cys and Cys. Especially the shi style (26)
ANF(8-33) acid is obtained.

When it is desired to obtain ANF (8-33) amide as the final product, octa-''2f tide containing amino acid residues 26 to 33 is used as a support, and p-methylbenzhydrylamine resin (MBH■) is used. The corresponding octapeptide amide of formula (28) is obtained by cleavage from the resin support.The rest of the process is completely similar to the method described above. That is, tetrapanotide hydrazide (ω) is linked to the above octapeptide amide (28) by the “azide method” and then the amino-terminal Boc
The corresponding dodecapeptide amide (30) was obtained by removing the - group: The Boc-hexapeptide hydrazide (16) was linked to the above dodecapeptide amide (30) by the "azide method" and then Remove the amine-terminal Boc group to generate the corresponding octapeptide amide (4'):
The BoC-octadegutide hydrazide (19) is linked to the above octadecagutide amide (32) by the “azide method”, then the retaining group is removed, and the Cys and Cys
By establishing a disulfide bridge between
5) ANF(8-33) amide is obtained.

The following is a general method for in-phase synthesis of the fragments used in the production of ANF (8-33) # and ANF (8-33) 7mid.

In-phase synthesis Grotocol A: CH2Cl2.2 min (3
x); 33% TFA-CI (2CA2.2 min and 25
Minutes; CH2C42, 2 minutes (Knee 3X); 1:
9 (■ B) NEt3- CH2Cl2.1 min and 3 min; CH2C62, 2 min (5X); BoC-amino acid (1,5 eq.), minimum volume of CH2C42 or DMF
-2 minutes in CH2C62: 1. DCCI of OM (1,
5 eq.) in Cl12C12 for 15-20 min; CH2
Cl, 2 minutes (5x).

Iterative connection Noroto':j)'', l:9 (v/v
) N]Dt3-CH2C12, 2 min (1x), CH2
C42, 2 min (5X); Boc-amino acids (10-1°5 eq.) 100 MF I411.2 eq. HOBt, 2
min; 1.0 M DCCI in CH2C42 (1,0-1
．． 5 equivalents), 15-120 minutes: DMF, 2 minutes (lX
): CH2Cl2.2 min (5x). Fragment hardness 1
Synthesis is carried out using a manually operated shaker, except in the case of carboxy-terminal octapeptide (1) and amide (and), which uses a Bare 2 Fufu 990 synthesizer. 1%
A double-crosslinked Merrifield resin is used in all syntheses, except for the synthesis of octapeptide amide (27), which uses crosslinked p-methylbenzhydrylamine 4i'i, 1 fat. All washes are carried out at a ratio of 10 ml/I resin.

More specifically, ANF(8-33) acid is synthesized by the following method.

H.Leu-Gly-Cys(Acm)-Asn-8e
r(Bxl)-Phe-Arg(NO2)-Tyr(
Bxl)-0■(]).

BoC-Tyr (Bx
1) Starting from −〇■ [7 and manufacturing by the in-phase method.

Synthesis is performed on a Peckman 990 Begtide Synthesizer. The ligation operation is repeated twice for each amino acid using an amino acid whose α-amino group is protected by a BoC group. The second protection of arginine is carried out by a nitro group, for serine by a benzyl group, and for cysteine by an acetamidomethyl (Acm) group. For the first connection Phe, S6v, G
ly and Leu are dissolved in methylene chloride. Any first linkage and all second linkages are obtained in dimethylformamide by adding 1 equivalent of hydroxypenztriazole hydrate, while for Asn, 2 equivalents of HBT are added in both linkages. It is done by adding.

Common synthetic glotcols are as follows (all volumes are 80 g unless otherwise noted): (1
) CH2Cl2.5 x 2 minutes; (2) 40% TFA/C
H2Cl2.2X2 minutes: (3) 40chi TFA/CH
2Ct2.25 minutes: (4) CH2Cl2.3 x 2 minutes: (5) 10th section TEA/CH2C422 x 10 minutes: (
6) CH2Cl2.5 x 2 min: (7) BoC-amino acid, (2.5 equivalents in i Q ml of solvent (if necessary)
(8) IM of DCCI in CH2C42
20ml of solution! , 1 hour; (9) DMF, 2X
2 minutes: (10) Menu, 1 x 2 minutes; (11)
CH2C42, ■ × 2 minutes: (12) MeoH11 × minutes: (13) CH2C42, 2 × 2 minutes.

Repeat steps (4)-(13) for the second concatenation operation. However, stage 8 will be extended to 4 hours. If starting with Cys, add % CI (2% Ct2 of 50% dithiol in Ct2) to all TFA solutions immediately before its addition.

The final Boa removal is steps (1) to (4), (6)
, and (10) to (13).

After drying the Beguchito bell 1 fat overnight, it is used directly for the subsequent steps shown below.

H・Leu-Gly-Cys (Asm)-As
n-8e r-Phe-Ary-Tyr-OH-HOA
c (2).

A mixture of resin-bound benotide (1), m-cresol and dimethyl sulfide was added to a Kel
Cool to -78°C in a -F vessel. Approximately 1 volume of anhydrous H
After condensing F into the container*:, l'+% compound is 0
Stir for 20 minutes at <RTIgt;C,</RTI> and then concentrate under reduced pressure to constant volume. The residue is triturated with ether, filtered and the filtrate is washed several times with ether. Crude peptide, 1
Elute from the resin with % HOAc and lyophilize to give a gummy solid. This material was purified on silica gel with EtOAc-pyridine-HoAc-H2O (10
-5-12).

Next, chromatography is performed using (10-5-1-3). The center cut was evaporated to dryness under reduced pressure and the residue was lyophilized from 1 inch of HOAe.

Octapenotide (dan) is obtained as a colorless solid of acetate salt.

Boc-Ala-Gln-8er(By, 1)-Gly
-0■ (3).

The glutinated resin (Dan) is prepared from Boc-Gly-0 using in-phase synthesis Grotocol A. For the introduction of alanine, two religation operations are necessary to obtain a satisfactory Kaiger test result. Removal of the BoC group at the tribenotide stage was performed using 33V of TF.
Performed using 4N lIC1/dioxane instead of Δ-CH2C12.

Boa-Ala-Gln-8er (BZI)-Gly
-OMe (4).

Resin-bound tetrabezotide Boc-Ala-Gin-8e
r(BZ l )-Gty-o■ as a catalyst in TEA
Transesterify with MeOH using 1-2 at room temperature
After stirring for a period of time, the liquid is withered and dried under reduced pressure. EtOAc the residue
Susibusushi with C 2. The gelatinous solid was collected by filtration, washed with EtOAc, and dried. MeOI
After ultrasonic treatment with I at 40° C. 11 times, the solid was filtered with water and dried to obtain the title compound (4).

Boc-Ala-Gl n-n-8er-Gly-O (
5).

etori Medium Hoe-Ala-Gin-8er (Bz
)-Gly-084e (3) A suspension of 10% Pd/C in 50% aqueous HOAc was treated with a suspension of 10% Pd/C in 50% aqueous HOAc. After incubating for 2 hours under an atmosphere of ice and nitrogen, the mixture was treated with F on a previously washed SueCel mat, and the P solution was evaporated under offshore pressure. After adding an additional portion of gton and ■■20, evaporate to a small volume. The residue was diluted with rDtOH-H2O and lyophilized to obtain the compound.

Roc-Ala-Gln-8er-Gly-NIINI
(2(6).

Boc-Ala-Gin-8er-Gly- in MeOH
Add NI (2NH2) to the solution of OMe.
After the time device, mat 3? t; rr:T'' te stem, Jj'-l-)h A-stop frh So'I is dissolved in H,0 and i·ζ is released, and this is turned twice.

Triturate the residue with ether and centrifuge.

Wash EtoAclCJ and dry on anhydrous KOII for 48 hours to obtain 1loc-tetravegutiPhydracyto.

Boa-Arg(No2)-i)e-Gly-0@(
7).

t, 1 lipid-bound tripenotide (7), 0.93 mmol/
Starting from Boc-Gly-0■ loaded with
oc-I 1 e-OH and Boa-Arg (No2)
-OH is prepared by introducing grotocol AK. However, based on the Kaiser test after one connection operation, repeating the connection operation is not required.

Boc-Arg(N02)-11e-Gly-OCH5
(8).

Sandals of resin-bound tribegutide (7) are suspended in methanol [118] and treated with triethylamine.

Stir the mixture for 25 hours. 1st and 2nd hour U
, V, analysis shows that the removal is complete within 1 hour (λ<=2=268 nm).

After separating the cisbent resin by filtration, the raw material is separated by evaporating the solution under reduced pressure and triturating the residue with EtOAc to obtain a crystalline solid.

The title compound is isolated as a colorless solid by filtration, washing with EtOAc, and drying in vacuo.

(TFA) H-Ar g (No 2)-I le
-G 1 y-OCf(s (9) -CH2C22
(8)
Treat with 100q61 RiflumeroFit acid without stirring. Everything dissolves within 1 minute. After 30 minutes, the reaction mixture is poured into ether, which has been cooled to -20 DEG C., with stirring. The precipitated TFA salt was separated by filtration and washed with ether.

Then, by vacuum drying, the title compound 9 is obtained as a colorless solid.

Boa-Asp(Bzl)-Arg(No2)-11
e-Gly-OCH3 (10).

A solution of t-Bee-asnocragic acid β-pencyl ester in FJt08C was cooled to -5°C under nitrogen.
and N-methylmorpholine followed by ingtyl chloroformate. After 8 minutes, DMFI"iff
1) Add a solution of -2gutide ester together with N-methylmorpholine, then add more N-methylmorpholine in portions. A viscous precipitate formed after 1 hour.

Add EtOAc and N20. The mixture is completely in solution (two phases) after one run at 20 uK. This was separated and the EtOAc phase was mixed with 0.3M citric acid, lT2O
, saturated NaHCO3, N20, and 50% saturated NaC
Wash with step 4. Precipitation of the product occurs over a period of 24 hours. The solid was separated by filtration and washed with EtOAc.
It is then washed with lI20 and dried under vacuum to obtain the title compound as a white solid. Separate P solution and E
The tOAc phase is treated as described above to provide an additional amount of the title compound (10).

(HCt)H-Asp([3zl)-Arg(No
□)-Ile-Gly-OCH3 (11).

A sample of tetrabegutide (ig) suspended in EtOAc was cooled to -40°C under a gentle stream of nitrogen and saturated with HCt gas, while keeping the temperature from rising above -110°C. do. After 5 minutes of saturation, the solution is purged with nitrogen for 0 minutes.

8@m7! The title compound (
11) precipitates as a white solid. This is washed with ether and dried under vacuum.

Boc-11e-Asp(B*l)-Arg(NO2)
-11e-Gly-OCH, (12).

A sample of HCt salt (11) and a 1 molar excess of Boc-
Ile-0H-1/2H20 in DMF with N-
Mixed with methylmorpholine and hydroxybenztriazole, the solution was mixed with 1-(3-dimethylaminoglovir)-3-ethylcarbodiimide hydrochloride (EDC
). After 3 hours, the DMF was removed in vacuo, and N20 was added to disperse the P-1 product formed by shaking, followed by crushing, filtering, and washing with N20. After washing with ether and drying under vacuum, the product (12) is obtained.

(HCt)H-11e-Agp(Bzl)-Arg(
A sample of NO2)-11e-Gly-OCH3(i3)s pentapenotide (L2) was suspended in EtOAc and treated in exactly the same manner as described above for the preparation of tetrapenotide HC4 salt (11) to yield the product ( 13
).

Boc-Arg(NO2)-11e-Asp(Bzl)
-Arg(NO2)-Ile-Gly-OCH5(14
) l.

Sandal of HCl salt (13) and about 1.2 molar quotient of B
oc-Arg(NO2)-0H was mixed with N-methylmorpholine and hydroxybenztriazole in DMF, the solution was then treated with EDC, and after 3 hours the reaction mixture was converted to the mixture described for pentapegtide (12). After further treatment in exactly the same manner as described above, compound (!) is obtained as a white solid.

Boc-Arg-11e-Asp-Arg-11e-G
ly-OCH, 2) IOAc (15).

Sandals of the protected bexapegtide ester (14) were suspended in methanol 150% HOAc (4:IV/V) with 10% Pd/charcoal in the presence of nitrogen, replacing the nitrogen with hydrogen. , and the mixture is stirred for 2 hours (the peptide is all dissolved after 1.5 hours). The catalyst is stripped off through Celite and the solvent is removed in vacuo/the product is separated by lyophilization after loading. This phase material was purified on silica dal as % eluent F;tOAc-
Birinone-HoAc-1120 (10-5-1-1)
The compound (Dan) is obtained by preparing In by chromatography using .

Boc-Arg-11e-Asp-Arg-11e-G
ly-NHNII2·2HOAc (16).

A sample of hexapeptide ester (15) is treated with hydrazine/methanol (1:2 V/'V) in solution for 5 minutes. The solvent was removed in vacuo and the residue was evaporated with two portions of ethanol for 7 hours.
It is then evaporated in the same manner along with 3 parts of n-butyl alcohol. The hydrazide is obtained as a colorless solid by lyophilization.

Boc-Arg-Arg-8er-8er-Cys(A
cm)-Phe-cty-ciy-o (17).

Pegtide resin (17), Boc-Gly-0■ 1
．． Starting from 19 molar Gly/Ii resin, GIY t P he * and Cys
(Acm). For the final four residues, activation with DC (J-TiOB t) is used. For the introduction of two Ser residues, one ligation operation for K is sufficient. Satisfactory Kaiser test results Two ligation operations are required for the first Arg residue and three ligation sweeps are required for the later residues to obtain the 4-notide resin. and used directly to prepare octaOftide methyl ester (18).

Boc-Arg-Arg-8or-8er-Cys(A
etn)-Phe-Gly-Gly-OMe・2HOA
c (18).

A mixture of pegutide resin (υ) in triethylamine-MeOH (1:9) is stirred magnetically for 1 hour.

The mixture is filtered and the P liquid is evaporated in vacuo to a glass. The crude product was purified with EtOAc-viridiy-
Using silica dal 60 (230-400 mesh) packed in ll0Ac-H2O (12-5-1-3),
EtOAc-pyridine-HoAc-)I20
Chromatograph by elution with (1,2-5-1-3), (11-5-1-3), and (10-5=1-3). Rf'0.25(1(1-5-1")
3) Combine the fractions containing the product with K and evaporate to dryness.
, l'3 gives the title product with a purity of 96%.

A second crop of 89% purity from the sideband? ? ).

The structure of the product was determined by FABMS (M=10 s
3).

Confirmation is then performed by sequencing after removing the Boc group.

Boc-Arg-Arg-8er-8er-Cys(A
cm) Phe-Gly-Gly-N) INH2・2HO
Ae (19).

A mixture of off-p-"g 5-1' methyl ester (18) in MeOH/Nll2NH2 (2:IV/V) is held at 25 °C for 5 min, at which point a total solution is obtained; remove the solvent; j'l: Vapor evaporate, and the residue is I(2
Freeze-dry from 0 to 2 pieces. Residual hydrazine is removed by rinsing the product from the dimethodol in ether (3x). After drying over anhydrous KOH for 16 hours, the title compound is obtained with a purity of 96% by HPLCK.

During hydrazinolysis, no degradation of arginine to ornithine is observed.

Boc-Aln-Gln-8er-Gly-Leu-G
ly-Cys(Aem)-Asn-8@r-Phe-A
rg-Tyr-OH-HOAC (20) A solution of hydrazide (6) in eDMF is cooled to −25° C. under nitrogen and dissolved in 4.18 M (approximately 2.4 molar equivalents) HCl in tetrahydrofuran. Iso-amyl nitrite (approximately 1
．． 2 molar equivalents) over 30 minutes. -25℃
Stirring is continued for 1 hour. A weak positive reaction is observed in the test with wet starch/KI paper. This solution of acyl azide was cooled to -40°C and octabenotide (
1) (approximately 0.83 molar equivalents) and N,N-diisonorobyl ethylamine was added in portions to form the final PII.
7.5 (determined by applying an aliquot to wet narrow range P+1 test paper), the suspension was heated to -25°C.
Stir with a magnet for 48 hours, dilute with DMF, and heat at 5°C.
for 108 hours and evaporated in vacuo to an oily residue. Product to EtOAc-HO8 -
Grinding with HzO(10-5-32,5)K
Purification followed by lyophilization from 7% I (OAc) gives the title compound as a colorless solid with a purity of 97.1% on HPLC.

H-Ala-Gl n-8er-Gly-Leu-Gl
y-Acm(Cyg)-Asnn-8er-Gly-L
eu-Gly-A 2HOAc 5alt(21):+
Trifluoroacetic acid is added to the test tube containing the (tough) sandals, and the test tube is agitated to dissolve the 4-gutide. After 4 minutes of complete dissolution, add the dissolved solution to 20 volumes of cold ether. The precipitate was concentrated by filtration to obtain a solid, which was chromatographed on a column of C-25 finely divided Cendex using 2M 10AC, the fractions were concentrated and lyophilized, and then chromatographed at 99% by HPLC. The title compound is obtained with a purity of .3%.

The following is all white Boe-Arg-11e-Asp-Arg-11e-G
ly-Ala-Gln-8er-Gly-Leu-Gl
y-Cys(Acm)-Asn-8er-Phe-Ar
g-Tyr-OH·3flOAc (22).

A sandal of hydrazide (16) in DMF was cooled to -25 °C under N2 and cooled to about 5 °C in tetrahydrofuran.
Add molar equivalents of 4.2 M rlct. Iso-amyl nitrite (approximately 1.5 molar equivalents) is added over 4 hours. Stirring was continued for 1 hour at −25° C., and a weak positive reaction was obtained during the test using starch starch/KI paper. This solution of acyl azide was cooled to -40°C and ca.
Add 66 molar equivalents of (21) and add N,N-diisonorobyl ethylamine in portions to bring the final P11 to 7
．． 0 (measured by applying an aliquot to a wet narrow range PII test strip). The suspension was heated to -25°C for 3
and magnetic M1 press at 5° C. for 1 day, followed by vacuum drying to obtain an oily residue. This product was mixed with 50% H
After purification by gel filtration through Sephadex G-50 (fine) in OAc, removal of the solvent and lyophilization, the title product is obtained with a purity of 94% by IIPLC.

(TFA) H-Arg-I'le-Asp-Arg-
11e-Gly-Ala-Gln-8er-Gly-L
eu-Gly-Cys(Acm)-Asn-8er-P
he-Arg-Tyr-OH (23).

Octapenotide (ban) suspended in C■■2Ct2,
Treat with 1,2-ethanedithiol and then 100% TFA with stirring for 20 minutes (all dissolves within 2 minutes). The reaction mixture was quenched by transferring to about 20 volumes of cold (-10°C) ether, the precipitated solid was allowed to stand for 10 minutes, and the product was separated by filtration, washed with ether, and dried in vacuo. Then, HPLC
The title compound of 90% purity is obtained.

Boc-Arg-Arg-8e r-8e r-Cy
s (Acm)-Phe-Gly-Gly-ArH
-I 1 e-AI! 1p-ArH-I 1 e-Gl
y-Al a-Gl n -8e r-Gly-Leu
-Gly-Cys (Acm)-Asn-8e r-
Phe-Arg-Tyr-OH・6HOAc (24)
.

A solution of 9) chloride in DMF was cooled to -25 °C under N2 and 2.5 °C in tetrahydrofuran.
Add molar quotient of 5.38 M HCl. In-
Add the amyl nidrites over 60 minutes.

Stirring is continued for 1 hour at -25°C and a weak positive reaction is obtained in the wet starch/ZK paper test. This solution of acyl azide was cooled to -40°C and about 1 molar equivalent of (23)
and N,N-diisoglobylethylamine in portions to a final pH of 7.2 (wet narrow range P
I (measured by applying test paper button aliquots). The suspension is magnetically stirred at -25°C for 72 hours and at 5°C for 16 hours and then evaporated in vacuo to give an oily residue. Sephadex G-50 (fine)
and 50'16 HOA
Fi\ is produced by filtration through a dull solution eluted with an aqueous solution. HP
Evaporation of the fractions containing the product with a different elution time than the LCK starting material gives the title compound with a purity of 92% by HpLC.

From F margin Gly-Cys-Agn-8er-Phe-ArH-T
yr-OH5HOAc(25) e [BocANF
(8-33) acid].

Boa-bis-Acm-Cys in 50% HOAC
12I28 derivative (24), glacial acetic acid/■I20 (4:
IV/v) solution of iodine (concentration 2.25~/1n1.
).・Complete conversion to fusulfide requires 2 hours; at this point, remove excess iodine by treating with zinc powder for 1 to 2 minutes, remove zinc by centrifugation, and drain the supernatant liquid. evaporated and the residue was applied to a Sephadex G-50 (fine) column and 50%
Elute with HOAc. Fraction containing the product (HPL
C) are combined and lyophilized after evaporation of the HOAc to give the title compound with a purity of 90% by HPLC. Zinc is not detected by X-ray microanalysis.

H-Arg-Arg-8er-8er-eys-Phe
-Gly-Gly-Arg-11e-Asp-Arg-
11e-Gly-Ala-Gln-8er-Gly-L
eu-Gly-Cys-Asn-8er-Phe-Ar
g-Tyr-OH・6HOAc (26) [ANF(
8-33) acid].

BocANF (8-3
The ZanFol from 3) is treated with 100% TFA, then further treated with the same volume of 100% TFA, and rapidly dissolved after 3 minutes. After 20 minutes, add approximately 10 volumes of the solution to a refrigerator (-10°C).
) In addition to the ether, collect the precipitated solids by filtration.
and add to Sephadex G-5o (ea>, 5
Elute with 0% HOAc to obtain a single peak of correct molecular weight.

The combined fractions were evaporated and lyophilized to give the general product (2G) with a purity of <94% by Ellman analysis by f(PLC). is shown. Sequencing data, α-
The presence of the expected residues in the correct order, including the asbartylpezotide linkage, is confirmed. Purification to 100% (HPLC) is performed using preparative HPLC.

More specifically, NNF(8-33)amide is produced as follows.

H'Leu-Gly-Cys(Acm)-Asn-8e
r(Bzl)-Phe-Arg(NO2)-Tyr (
Bzl)-NH-MBH@ (27).

The synthetic glotocol used is an ester-bonded resin (1
) is substantially the same as that of World, Boc-Ty
r (2; 6-dichlorobenzyl) with p-methylbenzhydrylamine resin (Biochemical Co., USA).

1q6 crosslinking, 0.414 mmol amine/I4U4 fat), and after this, the remaining amino groups were removed by CH2C6
Capping by acetylation with an acid anhydride/pyridine solution in 2 (15 equivalents of 1M solution) yields the octabegutide resin described above.

11-Leu-Gly-CylI(Aem)-Asn-
8er-Phe-Arg-Tyr-NH2・2JIOA
c (28).

A mixture of resin-bound Begted, m-cresol and trimethyl sulfide was prepared in a Kel
Cool to -78°C in a -F vessel.

Approximately 0.2 volumes of anhydrous HF are condensed into the vessel and the stirred mixture is heated at 0° C. for 2 hours. The mixture is concentrated to constant volume under reduced pressure and recooled to -78°C. Approximately 1 volume of anhydrous HF is condensed in the vessel and the mixture is stirred at 0° C. for 45 minutes and then concentrated to constant volume under reduced pressure. The residue is triturated with ether, filtered and the filtrate is washed several times with ether. The crude peptide was eluted from the resin with 2M I(OAc) and evaporated to dryness to give a gummy residue, which was purified on a silica dal with EtOAc-pyridine-HoAc-[20(
10-5-1-1).

The fractions containing the desired product are evaporated to dryness and rechromatographed in two patches on a Sephadex G-25 (fine) column with 2M HOAc as eluent. The fractions are collected, evaporated to dryness and lyophilized from N20 to give the title product (28) with a purity of 98.7% by HPLC.

Boc-Ala-Gln-8e'r-Gly-Leu-
Gly-Cyg(Acm)-Asn-8er-Phe-
Arg-Tyr-Ni2·HOAc (29).

A sandal solution of hydrazide (6) in D is cooled to -25°C under N2 and cooled in a tetrahydrone run at ~30°C.
Add molar equivalents of 9.1 MHC/. Iso-amyl nitrite (approximately 1.1 molar equivalents) is added over 30 minutes. Stirring is continued for 1.2 hours at -25°C and a weak positive reaction is obtained in the wet starch/KI paper test. This solution of acyl azide was cooled to -40 °C and (28) (
Add approximately 0.755 molar equivalents.

N,N-acylazide diisoglobylethylamine is then added in portions to give a final -7.0 (wet narrow range pi (determined by applying an aliquot to a test strip)). Magnetically stir at 25°C for 48 hours and at 5°C for 40 hours.

The reaction mixture was diluted with methanol and the product was ca.
Precipitate from a volume of ether. The solids were filtered and washed several times with ether to give 80% by tPLc.
．． The title compound with 4% purity is obtained as a colorless solid.

H-Ala-Gln-8er-Gly-Leu-Gly
-Acm(Cya)-A an-8er-Phe-Ar
g-Tyr-NH22HOAc-salt (30)
.

~ In a flask equipped with a magnetic stirrer (29)
sandal and about 10 parts of trifluoroacetic acid.

The solution is stirred for 2 minutes and then about 10 volumes of cold ether are added. Rinse the flask with additional TFA and add this to the ether mixture.

A solid was obtained by collecting the precipitate by filtration, which was chromatographed on a G-25 microsephadex with 2 M HOAc, concentrating the fractions, and lyophilizing by bbnpLc. The title compound is obtained with a purity of 96.7%.

13oc-Arg-11e-Δsp-Arg-I 1e
-Gly-Ala-Gln-8er-Gly-Leu-
Gly-Cys(Acm)-Ann-8er-Phe-
Arg-Tyr-Ni2.5HOAc (31).

DMF medium hydracito (], 6) sandals N2
Cool to -120°C and add about 100 molar equivalents of 5.4M HC/= in tetrahydrofuran.

Iso-amyl nitrite (approximately 1.3 molar equivalents) is added over 60 minutes. Stirring is continued for 3 hours at -25°C and a weak positive reaction is obtained in the wet starch/KI paper test. This solution of acyl azide was cooled to -40°C, dodecapegtide (30) (approximately 0.6 molar equivalents) was added, and N,N-diisoglobylethylamine was added in portions until the ti was 7. .5 (wet narrow range pt
+Measure by applying an aliquot to the test strip), Jl! ! ! The suspension was stirred magnetically for 44 hours at -25°C and then evaporated in vacuo to give an oily residue. The product was purified using Sephadex G-50 (fine).
Dull filtration was carried out by elution with 0% HOAc, then the combined fractions were filtered by filtration using a 77" tube G-25 (fine) and eluting with 2 N HOAc. Purify the pure product (HPLC
) are combined and lyophilized to give the title compound as a colorless solid with 94% purity by IIPLC.

H4rg-11e-Asp-Arg-11e-Gly-
Ala-Gln-8er-Gly-Leu-Gly-C
yg(Acm)-Asn-3er-Phe-Arg-T
yr-Ni2·ltTFA (32).

A mixture of (31) in TFA (approximately 20 parts) is stirred at 25° C. for 4 minutes (after 2 minutes, everything is dissolved).

Cold ether (about 6 volumes) and petroleum ether (about 10 volumes) are added and the precipitated solids are collected by centrifugation. After trituration with ether, the product is dissolved in anhydrous KO
Drying on a H pellet gives the title compound as an amorphous solid.

Boc-ArIT-Arg-8er-3er-Cys(
Acm)-Phe-Gly-Gly-Arg-11e-
Asp-Arg-11e-Gly-Ala-Gln-8
er-Gly-Leu-Gly-Cys(Acm)-A
sn-8er-Phe-Ary-Tyr-NH2・6H
OAc (33).

A sample of hydrazide (19) in DMF was cooled to -25°C under N2 and cooled in tetrahydrofuran (ca.
Add 0 molar equivalents of 5.12M HCl. Iso-
Add amyl nidrito for 30 minutes.

Stirring was continued for 0.5 hours at -25°C, and the wet starch ZKI
A weak positive reaction is obtained in the paper test.

This solution of acyl azide was cooled to -40 °C, (32)
(approximately 0.6 molar equivalents) and N,N-diisoglobylethylamine was added in portions to bring the final PII to 7.
．． 2 (determined by applying an aliquot to a wet narrow range test strip). The suspension was incubated at -25°C for 48 hours.
An oily residue is obtained by stirring for 5 hours and at 5° C. for 5 minutes and then evaporating in vacuo. The product was purified using a column packed with Sephadex G-50 (fine).
Purify by dull filtration by eluting with 0% aqueous HOAc.

The product-containing fractions were combined, the solvent was evaporated in vacuo, and the residue was purified by lyophilization from I20.
The title compound is obtained with a purity of 98.3% according to , , npt, c.

Boc-Arg-Arg-8or-8er-Cys-
Phe-Gly-Gly-Arg-11e-Asp-A
, rg-11e-Gly-Ala-Gln-8er-G
ly-Leu-Gly-Cys-Agn-8er-Ph
e-Arg-Tyr-NH25)iOAc salt (34).

Add the solution of (2) in 180% HOAc to the solution of (33) in 50% HOAe. After thorough stirring, flask I/
Passed through CN2 and kept in the dark at 26°C for 1 hour 50 minutes. The reaction is stopped by shaking with zinc powder, removing the zinc dust and washing twice with N20. The P solution was concentrated under reduced pressure, and the residue was converted into G-50'
(Fine) Chromatographed using 50% HOAc on a column of Sephadex.

The fractions were concentrated and lyophilized using HPLC.
The title product is obtained with a purity of 98%.

H-A, rg-Arg-8er-8er-Cys-Ph
e-Gly-Gly-Arg-11e-Asp-Arg
-11e-Gly-Ala-Gln-8sr-Gly-
Leu-Gly-Cys-Asn-8er-Phe-A
rg-Tyr-NIT26HOAc salt (35).

Approximately 25 parts of T are added to the test tube containing the sample of (34).
Add FA. Stir the tube and add the solution to about 3 volumes of cold ether after 2 minutes for the solids to dissolve. The ether mixture is diluted with an equal volume of pet ether, and the precipitate is collected by centrifugation and washed with petroleum ether. The crude product was purified by HPLCK, the fractions were concentrated,
and after lyophilization from 2M HOAc, HPLC
The title compound with a purity of 97% by K, i.e. AN
F (8-33) is obtained.

Resin-bound vegutide (1), (3), (7),
All of the above compounds except (17) were characterized by physical constants, and their compositions were confirmed by amino acid analysis.

Synthetic ANF (8-33) acid and ANF(s -3,3
) The sodium utilization activity of the amide was measured by the method described in ^Q, and natural ANF (2-33) and natural A
Compare with NF (3-33). Figure 212 shows the results obtained. Each dose was confirmed by quantitative amino acid analysis, and each was the average of at least 4 separate measurements (±S
, E, M, ) 1 synthetic ANF (8-33) acid and ANF (8-33) amide are both natural AN
It is shown that it has a natriuretic activity comparable to that of F (3-33), and that the activity of native ANF (2-33) appears to be somewhat higher than that of the above compounds. However, all of the above peguteds have high activity in the picomolar range.

Synthetic ANF (8-33) acid (26), and ANF (
8-33) The vasorelaxant activity of the amide (35) was measured in vitro by the method described above, and the vasorelaxant activity of the natural ANF (3-33)
). Figure 13 shows the average ± S of the obtained results.

All three pegutides have vasorelaxant effects, with a high degree of statistical significance (P<o, oi), denoted as E and M.
With this, a reactive cloth for norepinepurine (NE) K,
This replaces higher dosage levels of NE. Moreover, no differences are observed between natural ANF and synthetic ANF.

Below, the hypotensive and anti-hypertensive activities of the synthetic ANF (8-3s) acid (E1) are determined in rats by the method previously described in detail. Briefly, the left renal artery was clamped with a clip in the first group of rats, and the right kidney was left intact ([2-kidney, 1
-Clip") developed hypertension and used a group of jam-operated rats as a control; and in the second group:
The left renal artery is clamped as described above, and the right kidney is surgically removed ("1-kidney, 1-clip"), and animals with single nephrectomy are used as subjects.

Blood pressure is monitored at regular intervals and animals are considered hypertensive if their systolic blood pressure is consistently 150 ramHg or higher over the 3 weeks prior to the start of the experiment. Synthetic ANF (8-3'3) acid (1 μy in 1 ml of Claylag solution) is then administered intravenously as a bolus injection. A significant decrease in blood pressure is observed in all groups. The effect can be seen in hypertensive animals, e.g.
Renal, most pronounced in the 1-clip group, where the decrease in blood pressure amounted to about 33% of the initial value, and 4
Continues for a long time from 0 minutes. Similarly, high blood pressure “1-Kidney,
The effect was slightly lower in the ``1-ri17 Tsubu''group;
In this case, the decrease in blood pressure is approximately 25 degrees below the initial value and continues for 30 minutes. In the two virtually normotensive groups, the drop in blood pressure was approximately 22 to 2 of the initial value.
4% and lasts for 20 minutes.

The invention will now be described in more detail by way of example. However, this does not limit the scope of the invention.

Female and male Sprague
pawley) atria from rats were treated with 1 mM EPTA.
, homogenized in IM acetic acid containing 1 mM phenylmethylnulfonyl fluoride and 12.5 μM begstatin. 20 minutes pal at 30.000XP
Method 1', A, Reference 1+m-t clears were frozen overnight at 20°C, thawed, and centrifuged again at 100.000X for 30 minutes. The final supernatant was converted to C,8SEP-
PAK (octadecylsilane, obtained from Waters, Inc. 75 Shima) was passed through a cartridge (2 atria per cartridge) and the active substance was loaded onto a Bioglu P-10 column (2,
5X90 crn) (polyacrylamide beads, molecular weight cutoff 20,000, manufactured by Viola Dora 7 If Rad 1 Nose) and eluted with 0.1 M acetic acid. The activity of successive elution fractions was measured. molecular weight is 4,
000 to 6,000 samples, and 6. OOO
Samples showed a strength of activity in the range of ~15,000(7). Select each sample within the above molecular weight range and −
We did it together. - Each of the two combined samples was further purified separately.

Next, each sample was applied to a CM Bio-Glu A column (1,5X
30 cm) (cation exchanger, cross-linked agarose beads with carboxymethyl groups, Bio-Rad Laboratories) and was applied by a linear gradient using 1450 ml of 001-10 M ammonium acetate, p!(5,O). The ANF was then loaded onto a MonoS HR515 column (cation exchanger,
(manufactured by Pharmacia Fine Chemicals).

A gradient of 0.002 to 1.0 M triethylamine acetate, pH 6.5, was used.

Two minute fractions were collected. For the low molecular weight region, most of the active material eluted at approximately 0.9M triethylamine acetate. However, for high molecular weight materials, most of the active material eluted at approximately 0.8M triethylamine acetate.

The low molecular weight IJ diuretic factor was further purified by reverse phase HPLCIC using 93 columns by the following sequential steps. CNμ Pondapak (cyanoglobilsilane 10μ, Waters) (0.4 x 30 cm)
, 0.1 (v/v) Titrifluoroacetic acid-0,1 (V
/V)% trifluoroacetic acid and acetonitrile, 0°C
; C48μ bonder (octadecylsilane 10μ,
Waters) (0.4 x 30cm), 0.1 (v
/v)% trifluoroacetic acid - 0,1 (v/v) trifluoroacetic acid and acetonitrile, room temperature; C18μ Potudano! Tsuku (0.4X30crn), 0.13 (v/v
) Tidrifluoroacetic acid-0,13 (v/v) % heptafluorobutyric acid and acetoni) IJ/pe room temperature. The purified material is referred to as ANF(3-aa) (see second barley).

High molecular weight natriuretic factor is Blo-8i 1TS
K IEX-530CM column (cation exchanger, manufactured by Viola Laboratories) (0,4X 30c+++)
and containing 15% acetonitrile.
A linear gradient of 0.02 M to 0.4 M ammonium acetate of 5,0 with a gradient of 0.008 M/min and 0
It was eluted at a flow rate of 7 ml/Z min. The active peak was finally purified using a C Nμ Gindapack column (0.4×30
cm) It was purified by soil reverse phase method. Two major activity peaks were observed and were designated ANF(8-33) and ANF(3-33) (see Table 2).

Generally, in the above-mentioned ¥11 manufacturing process, all operations were carried out at 4°C unless otherwise noted. The material was lyophilized at the end of each chromatographic step and stored at -20°C until use. Small capacity ((6ml)
) for Servant's Beadback (Savan
Freeze-drying was carried out in polystyrene tubes (1,5X7 crn) using a concentrator (1,5 peed Vae) and for large volumes in the first stage in glass flasks. For subsequent steps, sample 1-3
rnl of 0.01M ammonium acetate pH 5,Q. Biological measurements [Gareia et al. (1982)
Expertia 38.1071-1073)
An aliquot was taken for this purpose, and this measurement was performed in duplicate.

Proteins were determined using the Brandford method [5peetor T. (1978) Anal,
Biochem.

86.142-146] using bovine albumin as a standard. However, in the final step, the amount of peptide was calculated using an amino acid sequencer.

Amino Acid Analysis and Sequencing Except for the amino acid sequence analysis of ANF-1,
Calxymethylation of 1, ANF-II, and ANF-m was performed as part of the amino acid analysis method. synutein residues, Crestfield et al.
A,N,,More S,and5te4nW,H,(
1963) T, Blol, Chem, 238.622
-627) using dithiothreitol. The reduced cysteine was then dialkylated with iodoacetic acid. 4Ptide on a C48μ Dendano or Tsuku column with 0.1% trifluoroacetic acid-0.1%
) Desalted using lifluoroacetic acid and acetonitrile.

Each Cal 7+ of 5μ? 5.7N of oxymethylated pezotide
I(C) and 01 thymercazotoethanol at 105°C
After hydrolysis for 22 hours, amino acid analysis was performed.

Separation of amino acids by M. Fauconnet and J.

Rochemont (Anal, Biochem,
, 91.403-409 (1978)) using a Beckman W3 column (8 micron beads, cross-linked sulfonated polystyrene-based resin) or a Dionex DC5A column (5 micron diameter, 8% cross-linked,
A modified 120C-Peckman amino acid analyzer was performed using a cross-4-linked sulfonated polystyrene-based resin).

Amino acid sequencing of all three separated substances was
Performed using the latest 890C Peckman sequencer,
It was. Sequencing was carried out using 3 my polybrene and 1
This was done after loading 00 nmol of dipeptide (Leu-Val). After drying, add 0.3M Quadro buffer (
Four to six complete cycles of sequencing were performed using Quadrol buffer (N,N,N',N'-tetrakis(hydroxypropyl)ethylene-diamine). P.T.
All conversions to H(phenylthiohydantoin)-amino acids were performed in a Sequemat P-6 Autoconveter after the cleavage step.
0Lazure etc. [
Lazure C,, 5eldah N, G,, Chr
etien M., La1lier R., and St.
Pierre S., Can, J.

Bioehem, Ce1l Biol, 61.28
7-292 (1983)), and the amino acid visiting conductor was analyzed and quantified by high pressure liquid chromatography.

Results For this example, 27F atria from 320 rats were used as starting material and processed according to the general method described above. The initial homogenate was determined to have Na + 840.00 excreted in 20 minutes in the Pior and Sei measurements.
It contained a total activity equivalent to 0 μEq and had a specific activity of 360 μEq of excreted Na20 min/my protein.

At the Biodal P-10 stage, approximately half of the active substances are low molecular weight substances.
000). The elution pattern of low molecular weight natriuretic factor in the Mono S column is shown in FIG. Active substance is found in almost all fractions, indicating product heterogeneity, but most activity is found in the 40-44 minute fraction. At this stage the yield was less than 5% and ANF(3-33) was 131,000 μEq.
It has a specific activity of excreted Na20 min/1n9. Reversed phase I (PLO) was used for further purification.
ANF (3-33) in CNμ Denderpack column
shows the elution of Although a peak with natriuretic activity was identified, the specific activity of the recovered material was very low compared to the previous step ((84,000 μEq excreted Na/20 min/
IIv), the peptide was shown to be partially inactivated. Next, the peptide ANF (3-33) was applied to a C18μ bondocolumn, 0.1% trifle, and vinegar [ff-0.1%)! Elute with J fluoroacetic acid and acetonitrile (0.2%/min, 1 rlLl/min) and finally apply to the same column, this time with 0.13% butafluorobutyric acid-0.13% butafluorobutyric acid and It was eluted with acetonitrile (Figure 3). In the two final stages, the peptide showed a single peak. ANF(3-
The final yield of 33) was 120 μl.

High molecular weight natriuretic factor eluted at approximately 0.8M on the Mono S column. Elution no J? Turns showed large heterogeneity, similar to small molecule factors. Only the purest fractions were combined. Biosil (Bi
On the o-8il) column, this material eluted at approximately 0.3M ammonium chloride. CN at the last stage
In the μ bonding column, two main peaks were observed, and ANF(8-33) and ANF(1-3
3), and each of these final yields is 3
0μ and 20μ, and specific activity of 450. OOO
and U 297. OO011Eq Excretion Na”/209
1/QH2 (see Figure 4).

ANF (3-3) obtained by purification of biologically active material
The amino acid compositions of three forms of ANF, designated ANF(8-33) and ANF(1-33), are shown in Table 1.

From the amino acid composition data, all three forms are Thr
It is observed that it lacks , Val, Met, S and Lys, but contains Arg, Set and cxy in high proportions. Considering the low content of acidic amino acids, this composition is consistent with the expected basicity of ANF' already mentioned. These data also
This suggests that the amino acids in the species peptides are mostly hydrophobic. Furthermore, A N F (8-33) and A N
From the composition of F (1-33), it can be inferred that two cysteine residues are present in the molecule, and therefore one intramolecular disulfide bridge is probably formed.

Finally, a comparison of the compositions of the three species shows great similarities between the contents of many amino acids, suggesting very close homology.

Figures 5.6 and 7 show the yield of each PT)I-amino acid plotted against cycle number. Natural (not carboxymethylated) ANF (3-33) (20
Automated Edman degradation of μF) allowed positive identification of 23 out of 25 residues (Figure 5). Identification of the other two residues is ANF(8-33) and ANF(
1-33). No different sequences were found, which was very important. Although the initial yield was low (calculated to be about 2.52 nmol), 4, which corresponds to positions 6 and 12 of ANF (1-33)
Only the amino acid occupying position 1 and 10 could not be identified with certainty. Selected stable PTH-
The average repeat yield calculated from the amino acid yield is 90.9
%Met. To identify unidentified residues in ANF (3-33), reduced and alkylated AN
F(8-33) and ANF(1-33) were also sequenced using the same method. AN fully consistent with amino acid composition
The predicted sequence of F(8-33) was obtained and is shown in Table 2.

Furthermore, the primary structure of ANF(8-33) is ANF(3-3
3) Check the sequence. Why Riki et al., ANF (8-33)
26 amino acids present in ANF (3-33)
This is because they can be arranged in the same order in the second
(see table). From the amino acid composition, ANF (8-33
) is recognized as an incomplete form of ANF (3-33).

ANF(8-33) consists of the first N-terminal 5 residues Gly-Pro-ArH-X found in ANF(3-33).
This is because it does not contain -Leu. On the other hand, ANF(8-
Sequencing of CANF(3-33) occupying position 6
] and 12 of ANF (1-33)
The residue corresponding to the position was allowed to be positively identified as cysteine, and the amino acid sequence present at the C-terminus of ANF(8-33) was shown. The average repeat sequence and initial yield for the ANF(8-33) sequence were 90.8% each.
and 5.78 nmol (see Figure 6). lastly,
The amino acid composition of ANF(1-33) is
3) or ANF (8-33), clearly indicating that it is an extended form. Indeed, as shown in Table 2, the amino acid sequence of ANF(1-33) revealed that in addition to those present in the sequences of ANF(3-33) and ANF(8-33), there are A long vegutide consisting of 33 amino acids containing 2 and 7 residues was sequenced. ANF (1-33) 6th place CANF (
Corresponding to position 4 of 3-33)] can be positively identified. ANF (3-33) and ANF (8-
From the results obtained for 33), ANF(1-33)
It is concluded that position 24 is occupied by the Ser residue and position 25 is occupied by the G17 residue.

The repeat yield and initial yield for the ANF-1 sequence were 92.9% and 1.92 nmol, respectively.

The amino acid composition is 3 belonging to the sequence ANF (1-33).
Completely consistent with the identification of 3 amino acids.

The results obtained from the sequences of the three ANF forms are shown in Table 2, along with the complete amino acid sequence of the long ANF, (1-33) peptide, consisting of 33 amino acids. This result shows that ANF (
This suggests the possibility that ANF(3-33) and ANF(8-33) originate from ANF(1-33) as a result of the extraction and purification procedures used, e.g. by proteolytic digestion in the homogenate. ANF(8-33) and ANF(1-33) purified from the high molecular weight region are ANF
The fact that IJ A has approximately the same molecular weight as (3-33)
WTt*1O-02)uy(AND/1-QQ)-AI
This would indicate an artifact based on adsorption to other proteins in the crystals. However, in subsequent experiments (see, e.g., Example 2), ANF(8-33) and ANF(1-33) are also obtained from the low molecular weight region.

ANF (3-33), ANF (8-33) and ANF
The peptide designated (1-33) has the following amino acid sequence.

rANF(3-33) ) H-Gly-Pro-Arg-8er-Leu-Arg
-Arg-8er-8er-Cys-Phe-Gly-
Gly-Arg-11e-Asp-Arg-11e-G
ly-Ala-Gln-8er-Gly-Leu-Gl
y-Cys-Asn-3e r-Phe-Arg
-Tyr -0R(ANF(8-33)) H-Arg-Arg- 8er -Ser -Cys -Phe -Gly -
Gly-Arg-11e-Asp-Arg-1
1e-Gly-Ala-Gin-8er-Gly-Le
u-Gly-Cys-Aan-8er-Phe-Arg
-Tyr-OH and below (ANF (1-33)) H-Lau -Ala -Gly -Pro -Arg
-Ser -Leu -Arg -Arg -8er
-Ser -Cys -Phe -Gly -Gly
-Arg, -11e -Asp -Arg -11e
-Gly-Ala-Gln-Ser -Gly-Leu
-Gly-Cys -Asn-8ar-Phe-Ar
To determine whether g-Tyr-OHANF(1-33) is related to other known peptides or proteins, we used Dayhoff's Mutition Data Matrix SEARCH method (Dayhoff M, D, et al., Protein 5equence Date Ba
5e.

At1as of Protein 5equence
and 5structure SNationalB
iochemical Re5earch Foundr
ration, Washington, D.C., June 1, 1983). When compared with the 2,222 registrations found, 7 were submitted.
There were no peptides or protein segments that contained sequences significantly (>30%) homologous to that of cANF-III, confirming a novel feature of this sequence. ANF (1
-33) and the amino acid composition of the 33 residues of da Bold and Flynn (Life Sci.

33.297-302 (1982)) for cicardionatrine I.
), all residues of ANF(1-33) are shown to be found in cardionathrin 1.

However, the relationship between these peptides cannot be determined because the sequence of cardionathrin 1 is not available for comparison.

Although each of the above purified peptides is biologically active, the shortest ANF (8-33) is ANF (3'
-33) or ANF(1-33), suggesting that some parts of the amino acid sequence of ANF(3-33) and ANF(1-33) are not essential for activity. be done.

Preparation and Purification of ANF(2-33) The preparation and purification of ANF(2-33) was carried out according to the general method described in Example 1 with minor modifications.

Atria from 650 striped rats (48
F) was homogenized (100 ml/
9) Shita. After centrifugation for 20 minutes at 30,000× jE, the supernatant was frozen overnight at 20° C., thawed, and centrifuged again at 100,000× for 30 minutes. The final supernatant was transferred to a C4B 5ep-Pak cartridge (octadecylsilane, manufactured by Waters).
(1 cartridge per 29 atria) and the active substance was passed through a Bioglu P-10 column (2,5X906n,
polyacrylamide beads, cut-off molecules g2o,o
oo, Viola Dora (Radleys)) and eluted with 0.1M acetic acid. The activity of sequentially eluted fractions was measured and samples ranging in molecular weight from 4.000 to 6,000 were combined.

The low molecular weight substance thus obtained was then applied to a 0M Bio-Glu A column (1,5X 30 cm, cation exchanger, cross-linked agarose beads with carboxymethyl groups, made by Biola Radera Tories) and 0.01~
1.0M ammonium acetate P115.0 was eluted with a linear gradient using 450ml. Next, ANF,
Mon for use on a liquid chromatograph (Gurian Model 5060).

Purification was performed using an S 111515 column (cation exchanger, manufactured by Pharmacia Fine Chemicals).

(A gradient of 102 to 1,0 M triethylamine acetate pH 6,5 was used. ANF (2-33) was approximately 0.
Eluted in 75M acetic acid) ethylamine.

The ANF was then further purified by reverse phase HPLC using two columns in the following order. CNμ is 0.1 (v/v) (cyanopropylsilane 10μ, manufactured by Waters) (0.4
) Titrifluoroacetic acid - using 0.1 (v/'v)% trifluoroacetic acid and acetonitrile, 0°C; v)%
Using trifluoroacetic acid and acetonitrile, room temperature.

The purified material was analyzed by amino acid analysis.

Amino acid analysis was performed on 5μ of carboxymethylated pezotide after hydrolysis at 105°C for 22 hours in 5.7NHC and 0.1% Mercatoethanol. The carboxymethyl group of cysteine residue is Crest
field etc. (Crestfield A, N, Mo
reS, and 5teinW, H, (1968) J.
Biol, Chem, 235:622-627)
Reduction using dithiothretyl (dithiothretyl) and alkylation with iodoacetic acid were performed. Separation of amino acids after hydrolysis is described by Fauconnet and Roch
emont (Fauconnet M, and Roeh
emont J, (1978) Anal, Bioc
hem, 91:403-409).
Peckman W3 (8μ beads, cross-linked sulfonated polystyrene based resin) column or Dioneknu (
12 modified using DC5A, 7 μ beads, 8 cross-linked sulfonated polynutylene-based resin) column.
The analysis was performed using an OC-Peckman amino acid analyzer. The separated product was pure since a single symmetrical peak was obtained after the two final chromatographic steps. The final yield was approximately 40 microns. Amino acid composition showed that this peptide consisted of 32 amino acids. The composition is 1
A
It was the same as NF (1-33) (see Table 1)
.

This product has the following amino acid sequence: II-Ala-Gly-Pro-Arg-8er-Le
u-Arg-Arg-8er-6er-Cys-Phe
-Gly-Gly-Arg-11e-Asp-Arg-
11e-Gly-Ala-Gln-8er-Gly-L
cu-Gly-Cys-Asn-8er-Phe-Ar
It was identified as ANF(2-33) with g-Tyr-OH.

ANF (1-33), AN
F(a-a3) and ANF(8-33) are also obtained.

Biological measurements of the natriuretic activity of native ANF(2-33) and ANF(3-33) and synthetic ANF(8-33) acid and amide forms (Garcia R, Ca
ntin R, Th1bault G, OngH,
and Genest J. (1982) Experien.
tia 38: 1071-1073: Th1b
ault G, ,Garcia R, ,Cantin
M., and Genest J. (1983) Hypert.
ensfon 5 (5upp1.1): 75-
83) (both cited above). Female Sprague-Dawley rats were used as bioassay animals (
180~zoop) was used. They were anesthetized with Vent Nol Biter # (60m97kFl ip,) and fitted with bladder and jugular catheters. Animals were given 5 (w/
v,)% glucone injection (2.1 ml 1 hour) was performed. Urine was collected into pre-weighed vials at 20 minute intervals. After the basal collection period, ANF peptide [natural AN
F(2-33) or (3-33), or synthetic (8-
33) Acid or amide form; 15-1350 pmol] was injected by delsu injection into 1 ml of Clarus solution PH 7.4. At least 4 rats were used for each dose. n) The IJ concentration was measured using a flame photometer. The results were expressed as change in excreted Na+/20 min SEM.

As shown in Figure 12, natural ANr(34s) and synthetic ANF(8-33) acid or amide forms have comparable natriuretic activity. Interestingly, ANF (2-
The activity of 33) appears to be somewhat higher than that of other forms.

In any case, both of these peptides are highly active in the picomolar range.

Example 4. Natural ANF (3-33) and synthetic ANF (8
-33) Male New Sealant Rabbit (18-2.0
kg) were fed Prlna rabbit food and had free access to drinking water. Sodium bendoparbital anesthesia (30m to Neptano)
Under 9Agi, v,), the renal artery was quickly excised, excess fat and connective tissue was gently removed, and the arterial tissue was cut into spirals. Each arterial strip 7' (1 question x 15 to 20 Wn) was tested in a continuously oxygenated Clairus solution (02-5 section of 95 cm, CO2, 37°C, pH
7, 4) in a 20 ml tissue bath.

Strip fixed base and force transducer (glass, FT
-03C). Contractions were recorded on a Model 7 Granupolygraph.

A tension of 500-750 m9 was applied to each strip.

The tension was adjusted and the bath fluid was changed every 15 minutes.

The 7 trips were equilibrated for 2 hours before starting the experiment.

The solution used in this study was NaC: 119: KC: 4.7: KH2PO4: 1.8: MgS
O4-7H20:1.17: CaCノ2''6H20:
2.5: NaTlC0: 25.0: and Glucone: 5.5 (both mmol/)).

For each arterial strip, the cumulative dose-response curve to NE (L-norepinephrine bitaltrate, manufactured by Sigma Chemical) was determined from 3.9X10 to 1.6X10
Obtained in the mM range. Once the standard curve is reproducible, add 0.25μ of synthetic ANF(8-33) acid, synthetic ANF(8-33) amide or pure natural ANF(3) to the bath.
-33), and then measurements were performed five times. N
The contractions induced for each dose of E were expressed as the maximal response.

Results are expressed as mean above S]Ji. Covariance analysis and Du
The various curves were compared using the net test.

Figure 13 shows that both synthetic ANF and natural ANF shifted the dose response curve for NE to the right (P<0.01
)It is shown that. This effect was more pronounced at lower NE levels. The critical value of NE response is ANF
rose in the presence of. There was no difference between natural ANF and synthetic ANF.

Example 5. Preparation and purification of ANF-H1 and ANF-H2 A 48F atrium (65
0 rat) was homogenized, 9 ANF was extracted using a Cl8SepPak cartridge, and Bioglu P
A Siglute filtration was carried out at -10. However, the hogenization buffer contained 5 mM EDTA instead of 1 mM.

Three consecutive regions of different molecular weights containing active substances were collected from the Bioglu P-10 column.

These regions are converted into low molecular weight forms (4,000 to 6,000
da 1 ), intermediate molecular weight form (6,000-10.00
0 dal), and high molecular weight forms (10,000-15,
000 dal).

Each region was then separated separately using a CM-Bioglu A column, a Mo
Further purification was performed as described in Example 1 using a no S HR515 2mm and CN μm Bondapak column and then a 018 μm Dendapack column. Final purification was performed using a C18 μm Bondano or Tsuku column.
．． Elution was performed with 13 (v/v)% heptafluorobutyric acid and acetonitrile.

Amino acid analysis of native-('gutide, or reduced and carboxymethylated peptides was carried out twice after hydrolysis at 180° C. for 24 hours in vacuo in 5.7 NHCJ. Separation and quantification of amino acids was carried out in Example 1. This was carried out using the method described in .

Reduced and carboxymethylated extended form of AN
Amino-terminal Edman degradation of F is performed using Seqemat (Seq
uemat) P-6 autoconverter and model 5C
- Beckman 890C with 510 controller installed
In Sixenter, 0.3M Quadrol (Quadrol)
drol) program and 3■ polybrene (Pol
ybrene) (Aldorich
) ) was used. Phenylthiohydantoin (
PTH) wo, Vista 402 Protsuta 1/
Varian with inch grater
Identification and quantification by HPLC using a 5500 liquid chromatograph.

By treating the intermediate and high molecular weight regions as described above, seven extended forms of ANF were detected. Most of these peptides were recovered in too small a quantity to allow further sequence characterization, and only two peptides obtained with yields of 50-100 μy were further analyzed. The long form of ANF was analyzed by reverse phase HP on a C18 μm Bondapak column using a trifluoroacetic acid/acetonitrile system.
ANF(1-33), ANF(1-33), ANF(1-33), ANF(1-33),
(2-33), ANF (3-33), and ANF (8-
33) eluted at 22-26% acetonitrile. Final purification of the long form of ANF was performed on a 018 μm Bondapak column and eluted using a hezotafluorobutyric acid/acetonitrile system.

The two peptides thus purified are referred to as ANF-Hl and ANF-I (2). Figure 8 shows this final n?,!
The state of elution of ANF-Hl in each step is shown. It is known that in this elution system, basic peptides are eluted at higher acetonitrile concentrations than in the trifluorodiacetic acid system.

Table 3 shows the amino acid composition of ANF-Hl and ANF-H2 along with that of ANF(1-33). See also Table 1 and Example 1. First, it is recognized that ANF-Hl and ANF-H2 are composed of approximately 70 and 95 amino acids, respectively. Furthermore, A.N.F.
Ile 5Try and Phe in the same ratio as (1-33)
The presence of ANF suggests that these peptides may be extended forms of ANF.

Figure 9 shows the various PTs obtained in the sequence of ANF-Ml.
Identification and yield of H-amino acids are shown. The repeat yield and initial yield of this sequence calculated from the linear regression line are 9.
4.2% and 4.97 nmol, correlation coefficient is 095
It was 9. Obviously, this sequence u, ANF-Hl is A
It has been shown to be an N-terminal extension of NF. This means that residue 4 of ANF-Hl, as shown in FIG.
The 18 amino acids corresponding to the sequence starting from 1 are ANF (
It is proven that the first 18 amino acids of 1-33) are the same.

The known presence of one C-terminal Tyr in ANF (1-33) and the sequence shown in Figure 9 suggest that ANF-H
Based on the amino acid composition of 1 and ANF(1-33), it is concluded that 9-H1 contains the previously described sequence of ANF(1-33) (see Figure 10). Therefore,
When ANF(1-33) is obtained from ANF-I(1), cleavage between Leu and Leu4 is involved. Such 0 Leu cleavage also occurs within ANF(1-33), and The deletion products ANF(2-33) and ANF(8-3
It has already been observed that 3) is obtained (see also Examples 1 and 2).

Preliminary sequence data for ANF-H2 indicate that this peptide consists of a 30 amino acid extension at the N-terminus of ANF-Hl, for a total of 103 amino acids, thus indicating that ANF-H2 and ANF(1 -33)). The results of B agree reasonably well with the amino acid composition of ANF-H2 (see Table 3).

In order to clarify the relationship between the sequence of ANF-Hl, which consists of 73 amino acids, and the sequences of known peptides or proteins and their segments, Natlonal Biomed
ical Foundation Mutation
data MatrIx7' program and 5equen
A computerized data puncture search was performed using the CE Data Bank, Georgetown University, Washington, DC. No significant homology (>30%) was found when compared to 2700 proteins in the databank. This confirms a novel feature of the determined sequence, similar to that observed for ANF(1-33) (see Example 1).

In Figure 11, the natriuretic activity of the 73 amino acid ANF-Hl is compared to synthetic ANF (8-33). As expected, ANF(8-33) is 350μ
Eq Na gives a dose response curve showing a critical value of approximately 800 pmol to produce a 20 minute natriuretic response. For comparison, two doses of ANF-Hl
only was used. This is because there was less material. ANF-
The observed natriuretic activity of Hl appears to be slightly lower than that of ANF(8-33) for the doses used.

By tightening the left renal artery with a silver flang with a 0.20 m internal gear and leaving the contralateral kidney intact,
Female Sedge Rag-Dawley Rat (180-2ooy)
2-renal, 1-clip hypertension occurred. 1-Kidney,
1-clip hypertensive rats were prepared similarly. However, the contralateral kidney was removed by surgery. Two additional groups of rats were used as control animals. The first group includes jam (s
ham) surgery was performed to expose the left kidney and detach the renal artery from surrounding tissue, and the second group underwent right nephrectomy.

Blood pressure was measured indirectly under soft ether anesthesia using a tail cuff twice a week, and a glass model 7 fitted with a 7P8 preamplifier and a model 1010 glass crystal microphone as a mother pulse detector. recorded on a vorigraph. Rats were considered hyperactive if their systolic blood pressure was once 150 mm Hg or higher for 3 weeks before starting the experiment.

On the day of the examination, the animals were treated with pentaparbital (60rn9/
The animal was anesthetized with ACgi, p-), and a bladder catheter, jugular intraforaminal catheter, and carotid artery catheter were inserted. 5 tiglucose (2
, 1 m/h) was injected into the animal. The intra-arterial catheter was connected to a blood pressure converter (Gould Statham P23ID).
Direct blood pressure was continuously monitored by connecting to a model 7 glass polygraph and recording on a model 7 glass polygraph. After 20 min of basal urine collection, 1 μl of synthetic ANF (8-33)
was administered as a pornu infusion through a jugular intravenous catheter. Prior to injection, ANF was diluted in clarus solution of lae. Post-infusion beds were collected and blood pressure was monitored over two more periods of 20 minutes each. The results are shown in Table 4.

As is clear from Table 4 in the margin below, a significant decrease in blood pressure was observed in all groups, and was even more remarkable in hypertensive animals. This effect lasts for more than 20 minutes, 2-renal, 1
-Longer duration was observed in the CLIP hypertension group.

-General: In-phase synthesis protocol A: CH2C for 2.2 minutes (3xC
33% TFA-CI (2Ci□, 2 min and 25 min; CH
2Cl2.2 minutes (3X): 1:9 (v/v
) NEt3-CH2C 2.1 min and 3 min: CH2
C 2.2 minutes (5x): small amount of CH2C12 or DMF
-CH2Cノ, medium Boa-amino acid (1,5 equivalents), 2
1.0 M DCCI (1.5 eq.) in CH2C, 15-20 min: CH,C 2.2 min (5x).

Repetitive linkage glotocol: 1:9 (v/v) NE
t5-CH2C-2.2 minutes (1x): CH2C-2
．． 2 min (5x): BoC-amino acids (10-15 eq) + 1.2 eq HOBt in DMF, 2 min; CH2C
1.0 M DCCI (1.0-15 equivalents) in No. 2, 1
5-120 min: DMF, 2 min (1x); CH2C12,
2 minutes (5x). This was done by solid incorporation of the fragments or using a manual shaker. However, in the case of carboxy-terminal octapeptide acids and amides, a Peckfu 990 synthesizer was used.

In all syntheses, Boc-amino acids coupled to 2-crosslinked Merrifield resin were used. A ratio of 10 ml/y resin was used for all washes.

(a) H*Leu-Gly-Cys(Acm)-As
m-8er(Bzl)-Phe-Arg(NO2)-T
yr(Bzl)-0■(↓) Octapeptide resin (1)
is chloromethyl resin (Labsy Nuthemes, 2% cross-linked,
1 prepared by the Cs base method from 13 mmol C4 cold resin)
0.0y (8 mmol) of Boc-Tyr (Bz l )
Prepared starting from -0■.

Synthesis was carried out on a Pecman 990 Pezotide Synthesizer. A double linkage procedure was used for each amino acid whose α-amino group was protected by a Boc group. A second capture was performed using the nitro group for arginine, the benzyl group for serine, and the acetamidomethyl (Acm) group for cysteine. For the first coupling operation, Phe, Ser, Gly and T, eu
was dissolved in methylene chloride. Other first ligations and all second ligations were carried out 1 in dimethylformamide.
An equivalent amount of hydroxypenztriazole hydrate was added. However, for Asn, 2 is used in both concatenation operations.
An equivalent amount of HBT was added.

The general synthesis protocol is as follows (all volumes are f3Qrul unless otherwise noted).

(1) CH2C-2, 5 x 2 minutes: (2) 40% TF
A/CH2C12, 2 x 2 minutes: (3) 40% TFA/C
H2C 2.25 minutes; (4) CH2C 2.3 x 2 minutes;
(5) 10chi TFA/C) I2C 2.2 x 10 minutes:
(6) CH2C 2.5 x 2 minutes: (7) B o
e-amino acid, 2.5 equivalents in 60 ml of solvent (add !7HBT if necessary): (8) DCCI, CH2
Cj! 20ml of IM solution in 1° for 1 hour: (9) DM
F, 2 X 2 min; α* MeOH, 182 min: (I
I) CH2C 2.1 x 2 min; α4 MeOH, 18
2 minutes; α-CH2C1□, 2×2 minutes.

Steps 4-13 were repeated for the second ligation operation. However, stage 8 was extended to 4 hours. If you start with Cys,
8 ml of 50% ethanedithiol in CH2C was added to all TFA solutions immediately before their use.

Final Boa removal was completed by performing steps 1-4.6 and 10-13.

- After drying overnight, the weight of the peptide resin was 17.8F.

(b) HIILeu-Gly-Cys(Acm)-A
sn-8er-Phe-Arg-Tyr-OH・HOA
c(2) 5.57 F (2.4 mmol) of resin-bound peptide (1
), 2 ml m-cresol and 131111 (D
) The mixture of H/l/S/l/phyto was cooled to -78°C in a Kel-F vessel equipped with a magnetic stirring frame. Approximately 5 mJ of anhydrous HF was condensed in a container,
The stirred mixture was then warmed to 0° C. for 2 hours. The mixture was concentrated in vacuo to constant volume and recooled to -78°C.

After condensing 401 rLl of anhydrous HF into the vessel, the mixture was stirred at 0° C. for 20 minutes and then concentrated under reduced pressure to constant volume. The residue was triturated with ether, filtered, and the filtrate was washed several times with ether. The crude product was eluted from the resin with 100% HOAc and
Then, a gummy solid of 4.OF was obtained by freeze-drying. This material was coated on 300 ml of silica gel with E.
tOAc-pyridine-HoAc-I20 (10-5-1
-2) and then chromatographed using (10-5-1-3). 91.302 (0.89 mmol) (3
7%) of a colorless solid was obtained.

Rf = 0.4.7, BuOII-e'lysine-H
oAc-H20(60-20-6-24) :M+I
I=1031 S IIPLC purity 97.1.

(a) Boc-Ala-Gin-8er (By, 1)
-Gly-0■(3) Peptide-resin (3) was synthesized using solid phase synthesis protocol A.
19 mmol mol y/y-resin). Introduction of alanine required two reconsolidation four-leaf crops to obtain satisfactory Kaiser test results. Removal of the Boa group at the tripeptide stage was carried out using 33% TFA-CH2C (4N HC7/dioxane).

Gly-0 (14F, 11.5 mmol) was treated with MeO (70 mmol) using TEA (50 ml) as a catalyst.
0 ml). 1.75 at room temperature
After stirring for an hour, the mixture was filtered to remove the resin and the p-liquid was evaporated to dryness in vacuo. Triturate the residue with EtOAc, collect the gelatinous solid by filtration, and
Washed with tOAe and dried (4.xy). 40
After sonication with 40 °C of MeOH, the solids were collected by filtration and dried.
A product of F (yield: 41 cm) was obtained. Purity 94.6% as determined by HPLC: Rf=0.4, CIICJ5-M
eOH-I20 (90-10-1). Elemental analysis, calculated value (C26H3, N509): C55,21, H6
, 95, N12.38: Measured value: C54,73, H6,
8B, N12.95°) INMR (DMSO-d6) was consistent with the structural formula.

(c) Boa-Ala-Gln-8er-Gly-O
Me (5) EtOH (Boe-Ala in 50QmA)
-Gln-8er(Bzl)-Gly-OMe(3)(
A suspension of 1,4F) was dissolved in a 50% aqueous HOAc solution (
A suspension of 10% pd/c (o, 7 y) in 25 m/c) was used. After the device was in a hydrogen atmosphere for 2 hours,
The mixture was filtered through a previously cleaned suit f-cell mat and the filtrate was evaporated in vacuo to 100 g. Additional EtOH and N20 were added and then evaporated to a small volume. The residue was diluted to a volume of 130 g and lyophilized to give a 1.2 F (100%) product. Purity by HPLC: 94.5%: 1
1f=0.35, CHCノ2-Me 0H-H20(
85-15-1,5).

(d) Boa-Ala-Gin-8er-Gly-N
I(Nl(2(6)BoC-A in MeOH (10ml)
la-Gin-8er-Gly-OMe(A) (0,
l rul of NH2NH2 was added to the solution of I P ).

After incubating for 2 hours at 23°C, the solvent was evaporated in vacuo.

The residual oil was dissolved in n2o(iom/) and evaporated twice. The residue was triturated with ether, centrifuged, washed with EtOAc, and anhydrous K
Drying over OH for 48 hours gave 93 ready product. HP
Purity by LC 94q6:l=0.2, CHCj! 3-
MeOH-N20 (75-25-2,5).

(16) (a) Boa-Arg(No2)-Its-Gly-
0 ■ (7,) resin-bound tripeptide ('L), 0.9
From Boa-Gly-0■ loaded at 3 mmol/litre,
Boc-11e-OH and Bo according to Grotocol A
It was prepared by introducing a-Arg(NO2)-0H. However, based on the Kaiser test after one ligation operation, repeated ligation operations were not necessary.

(b) Boa-Arg(N02)-11e-Gly-
OCH3(8)100+++/! Resin-bound tripeptide (7) suspended in methanol of 8.2 y (5,0
A sample (equivalent to mmol) was treated with 1Qml of triethylamine. The molten metal was stirred for 2.5 hours. Analysis of U and V at 1 and 2 hours showed complete removal within 1 hour (λN0-””268
nm). After separation of the cispent resin by filtration, the p-liquid was evaporated under reduced pressure and the product was separated by trituration of the residue with 50 m/ml of EtOAc to obtain a crystalline solid.

Li by filtration, washing with EtOAc, and vacuum drying:
, 7p (yield 100%) of white solid was obtained. TLC:
Rf = 0.7, CH(I5-MeOH-N20 (
, 80-20-2): Purity 99.6 by HPLC
%.

(c) (TFA) H-Arg(No2)-11e-
Gly-OCH, (9) 2.55 F (5.06 mmol) of the tripeptide suspended in 15 mi of CH2C-2
sample of 15ml of 1001) while stirring.
Treated with lifluoroacetic acid. Everything dissolved within 1 minute. After 30 minutes, the reaction mixture was preheated to -20°C.
The mixture was poured into 200 g of ether, with stirring, which had been cooled to 200 g.

The precipitated TFA salt was separated by filtration, washed with ether, and dried in vacuo to give 2.525 m (96% yield) of a white solid. TLC: Rf = 0.3, CH
Cノ, -MeOH-)120 (80-20-2)
: Purity 99.7% by HPLC.

(d) Hoe-Asp(Bzl)-Arg(No□)
-11e-Gly-OCH3(10) , Do\Q A solution of BoC-asnoligic acid β-pendylgo, ster was cooled to -5°C under nitrogen, and C0.55ml 1t
7) Treated with N-methylmorpholine, then 0.65 m
1. Treated with isobutyl chloroformate. After 8 min, a solution of 2.4051 (4, (3 mmol) of zotide ester (9,) in 10 ml of DMF was added to 0
．． Add with 40 ml of N-methylmorpholine and add in portions 25 mz of N-methylmorpholine. After 1 hour, a viscous precipitate formed and 10 ml of E
Add tOAe and 20 ml of 1I20. After one unit at 20°C, the mixture was completely dissolved (two phases).

Separate this and add 0.3M citric acid to the EtOAc phase. , N20, saturated NaIC0s, I (20, and 50% saturated NaC). Precipitation of the product occurred over a period of 24 hours. Separate the solids by filtration,
A white solid of 1.362 was obtained by washing with EtOAc, then N20, and drying in vacuo. p
The liquid was separated and the E t OA e phase was treated as described above at 1.57 PCs, with a total yield of 2.93F (89%).
] TLC: Rt = o, 4 (cacJ
3-MeoH-n2o(90-1O-1), 0.5 [
EtOAc-HOAc-isooctane-I20(12-
2-2-10) upper layer): HPI, purity 94 in C
%: PMR (360MHz-CD5OD), φ
CH20-(5,14s), Arg, Asp″CH
(4,48t, 4.43t), 11e"CH(4
, 26d), Guy “CH2 (3,98d, 3.8
8d), As P 5C) I2 (2,93,2,8
8d/d; 2.77.2.27 d/d).

(e) (HC))H-Asp(Bzl)-Arg(
NO2)-11e-Gly-OCH3 (see) 1,452 (2,05
A sample of tetrapezotide (mmol) was cooled to −40° C. under a stream of nitrogen and saturated with HC2lf while maintaining the temperature not to rise above 110° C. After 5 minutes of saturation, the solution is purged with nitrogen for 60 minutes. Addition of 80 m/ml of ether precipitated a white solid, which was collected by filtration, washed with ether, and dried in vacuo. Yield 1.26F (94chi)
: TLC:Rf=0.7, CHCJ, -MeOH-
I20 (80-20-2): Purity 96% by HPLC.

(f) Doc-11e-Asp(Bzl)-Arg(
NO□)-11e-Gly-0CH3 (see) IC of 9011Q (1,39 mmol)! salt (11) and 428 mg (1.7 mmol) of Boc-11
e-OH-1+/! H20 45m7! 0 in DMF of
．． Mixed with 18 ml of N-methylmorpholine and 324 ml of hydroxybenztriazole, the solution was reduced to 326 ml.
1-(3-dimethylaminozorobyl)-3-ethyl car day? Treated with diimide hydrochloride (EDC). After 3 hours, remove the DMF in vacuo and add 30 d of I20,
The resulting solids were then shaken to disperse, then ground, filtered, and washed with I20, then ether, and vacuum dried to yield a 1.07 F product (94 g).
12) was obtained. TLC: Rt = 0.6 (CHC
).

-MeOH-H□O (85-15-1,5)), 0.
7 (EtOAc-HOAc-isooctane-I20(
12-2-2-10), upper layer]; by HPLC) purity 9
5%.

(g) (HC))H-11e-Asp(Bzl)-A
rg(NO2)-11e-Gly-OCH3(g) 1,025'(1,2
4 mmol) of pentabegutide (12) was treated exactly as described for the preparation of pentapezotide/S++ H (J (11)) to give 0.94.9' (yield 10
0chi) product was obtained. TLC: Rf =
0.7, C) IC! , -MeoH-H2o(80-20
-2): Purity 97% by HPLC.

(h) Boc-Arg(NO2)-11e-Asp(
Bzl)-Arg(NO2)-Ile-Gly
-OCTT, (Q) 0.84F (1.11 mmol)
of H (44 m(13) sl) J sol and 433 m9 (1,36 mmol) of BoC-A
rg(No2)-OH in 34 ml DMF at 0.0.
15mA! of N-methylmorpholine and 256 m of hydroxybenz) IJ azole, the solution was then treated with 266 m of EDC and after 3 hours the reaction mixture was purified exactly as described for pentapezotide (12). By processing 1.03PC yield 91%)
A white solid was obtained. TLC: Rf = 0.5 (
C.H.C.J.

-MeOH-I20(85-15-1,5)), 0.5
(EtOAe-pyridine-HoAc-HzO(50
-5-1-1), 0.3 (upper layer of EtOAc-HOAc-isooctane-I20 (12-2-2-10));
I (96 CH by PLO.

(i) Boa-Arg-11e-Asp-11s-G
A sample of the protected hexapegtide ester (14) of ly-OCH3.2HOAc (15) ~0.99F (0.95 mmol) was heated in 130 mA' of methanol/50% HOAe (4:1 v/v).
091 with 10% Pd-charcoal under nitrogen, the nitrogen was replaced with hydrogen, and the mixture was stirred for 2 hours (all pezotide dissolved after 05 hours).
. After the catalyst was filtered off through Celite and the solvent removed in vacuo, the crude product was isolated by lyophilization. Yield 0.95 P: TLC: Rf=0.5, EtOAc-
Pyridine-HoAe-I20(10-5-1-3) (
(5-10% impurities are calculated in the low Rf part): HP
Purity by LC: 93cm (4% impurities). This material was extracted with EtOAc-pyridine-HoAc-I20 as eluent.
Purification by chromatography on silica gel using (10-5-1-1) gave a final product of 0.839 (corresponding to a yield of 60% for pezotide). I (97.5% purity by PLC: FAB-M
e843 (M+H).

(j) Boc-Arg-11e-Asp-Arg-1
1e-Gly-NHNII2.2H,OAc (16) 782 m9 (0.54 mmol) of the hexapeptide ester (15) was dissolved in 32 m1 of hydrazine/methanol (1:2 v/v) as a solution. Processed for 5 minutes. The solvent was removed in vacuo and the residue was dried for 7 hours and evaporated twice with ethanol and then similarly evaporated three times with n-butanol. Dihydrazide was separated by freeze-drying and o6sp (100% yield)
) was obtained as a white solid. TLC: Rf = 0.6
[EtOAc-pyridine-HoAc-I(20(7-
5-1-3) ), 0.5 (BuOH-HOAc-N
20 (10-2-6) ): PMR (360MHz
5CDsOD) As p Cl12(2,85,2
,81d/d: 2.55.2.51 d/u), Ar
g, Asp"CH (4,52t, 4.35m, 4.
2 env), Ile″CH (4,22d.

4.08 d), c, ty″CH2 (3,97 d,
3.76 d).

NN2H2・2HOAc (Q) (a) Boe-Arg-Arg-8or-8er-C
ys(Acm)-Phe-Gly-Guy-00(17
) The peptide resin (17) was converted into Boc-Gly-0 (1,
Starting from 19 mm Gly/P resin), Gly', Phe, and C
It was prepared by introducing ys(Acm). Activation with DCCI (IOBt) was used for the last four residues. One ligation operation was sufficient for the introduction of two Ser residues. , two ligation operations were required for the first arginine residue, and three ligation operations were required for the final residue.The peptide resin (Tone 6) was air-dried;
and was used directly to prepare octapeptide methyl ester (18).

(b) Boc-Arg-Arg-8er-8er-C
ys(Acm)-Phe-Gly-Gly-OMe-2
HOAc (18) Trimethylamine-MeOH (1:
9.3501nl medium bezotide resin (17) (6fi
) was magnetically stirred for 1 hour. The mixture was filtered and the p-liquid was evaporated in vacuo to form an f2 sliver (5,
453') was obtained. The crude product was purified using silica glue 60 (230-400 meth) packed in EtOAc-pyridine-HoAc-N20 (12-5-1-3) and EtOAc-pyridine-HOAc-N20.
(12-5-1-3) (1)), (11-5-1-
3) (1,5)) and (10-5-1-3) were eluted and subjected to cyclomatograph treatment. Rf=0.
25 (10-5-1-3) were combined and evaporated to dryness to HP
A product t, 4tp (23%) with a purity of 96% as determined by LC was obtained. Site No Gundoka Tsuta et al. 8
A second crop of 2.2F with 9% purity was obtained. The structure of the product was confirmed by FABMS (M=1053) and sequencing after removal of the Boc group.

(c) Boc-Arg-Arg-8er-8er-C
ys(Acm)Phe-Gl)r-Gly-NHNH2
・2HOAc, (19) MeOH:NH2NH2(2
: 1 v/v: of octapezotide methyl ester (18) (100 m9) in 1.5 mQ? 25℃
Hold it for 5 minutes, and at this time use I'I] for total strength! It became a solution. The solvent was evaporated in vacuo and the residue was lyophilized from N20. Residual hydrazine was removed by precipitating the product from methanol with ether.

After drying on anhydrous KOT for 16 hours, 85 mg of product was obtained, which had a purity of 96% by HPLC, Rf = 0.2 (EtOAc-Pyridine-■■oAc-
n2o(1o-s-1-3)). During hydrazinolysis, no degradation of arginine to ornithine occurred.

(a) Boc-Ala-Gln-8er-Gly-L
eu-Gly-Cys (Acm)-1L 5ml DMF
The medium hydrazide (6) (9.099 mmol) was cooled to -25° C. under N2 and 4.18 M (2.42 mmol) HCl in tetrahydrofuran was added.
added. Iso-amyl nitrite (0.16 ml, 1
．． 19 mmol) was added over 30 minutes. -25
C. Enoki stirring was continued for 1 hour. A weak positive reaction was obtained in the wet starch/KI paper test. This solution of acyl azide was cooled to -40°C and (A) (1,21P
, 0.83 m% Le) @ addition, and N, N-
Diisoglobylethylamine (0.35 m) was added in portions to give a final - of 7.5 (wet narrow range, determined by applying an aliquot to H test paper). The suspension was magnetically stirred at -25°C for 48 hours, and DM
Trituration at 5° C. for 108 hours and evaporation in vacuo gave an oily residue. The product was converted into EtOAc-HU
A colorless solid was obtained by mashing with Ac-n2o (10-5-1-2,5) and then lyophilization from 7%) (OAc. Rf = (1,61, EtOA
c-pyridine-HoAc-N20 (8-5-1-4);
Purity 97.1% by HPLC.

(b) H.Ala-Gln-8er-Gly-Leu
-Gly-Acm(Cys)-Asn-3er-Phe
-Arg-Tyr-OH2HOAc 5alt(21) 7.4 ml of trifluoroacetic acid was added to a test tube containing 541"2 (0.29 mmol) of (20) and the test tube was stirred to dissolve the penotide. It was dissolved.

After 4 minutes of complete dissolution, the resulting solution was added to 150-cold ether. The precipitate was collected by filtration to obtain a solid, which was chromatographed on a 5×100 column of G-25 fine Sephadex using 2M HOAc, and the fractions were concentrated and lyophilized. 3. '75
A product of 1 (0.22 mmol) (75.0 mmol) was obtained.

Purity 99.3% by HPLC.

(a) Boc-Arg-11e-Asp-Arg-1
1e-Gly-Ala-Gln-8er-Gly-Le
u-Gly-Cys(Acm)-Asn-8er-Ph
e-Arg-Tyr-OH.3110Ac (22) to 2, hydrazide (16) in 5 ml DMF (19,
A sample of 21.5 μmol) was cooled to -25° C. under N2 and diluted with 4.2 M I in tetrahydrofuran.
IC, 2.5 μJ (105 μmol) of N was added. (5.4μ), 30 μmol) was added over 4 hours. A weak positive reaction was shown in a test using wet starch/KI test paper. This solution of acyl azide was cooled to -40°C and (21) (26,4 Da, 14.5
rn mol) and N,N-diyne 76I:I
Add bilethylamine (19μA) little by little to final P.
H was determined to be 7.0 (measured by applying an aliquot to wet narrow range P11 test paper). This solution was heated at -25°C for 3 days.
and 5° C. with magnetic stirring for 1 day and evaporated in vacuo until an oily residue was obtained. The product was mixed with 50% HOA
After purification by gel filtration through Sephadex G-50 (fine) in c, removal of solvent and lyophilization, 2
3. A product of sy+9 (71% yield) was obtained. TLC:
Rf 20.4 (BuOH-HOAc-HzO-
10-2-6) (94% purity by HPLC).

(b) (TFA) H-Arg-11e-Asp-A
rg-11e-Gly-Ala-Gln-8er-Gl
y-Leu-Gly-Cys(Acm)-Asn-8e
r-Phe-Arg-Tyr-OH (23) 0, 20
Octabenotide (22) suspended in ml of CH2C
(21.7 mg, 9.4 μmol) was mixed with 10 μg of 1,2-ethanedithiol, then 0.0 μl of 1,2-ethanedithiol, and then 0.5 μm of 1,2-ethanedithiol with stirring for 20 minutes.
Treated with 22 ml of 100% TFA (all dissolved within 2 minutes). The reaction was stopped by transferring to 10 μm of cold (−10° C.) ether, the precipitated solid was allowed to stand for 10 minutes, and the product was collected by filtration, washed with ether, and dried under vacuum for 20 minutes. 1 m? (yield 8
0%) of product was obtained. TLC:Rf = 0.4 (
BuOII-ITOAc-N20=10-2-6)
() Purity 90% by IPLC).

Example 13. ANF(8-33) acid (26)H-Arg-
Arg-8er-8er-Cyn-Phe-Gly-G
ly-Arg-11e-Asp-Arg-11e-Gl
y-Ala-Gln-8er-Gly-Leu-Gly
-Cys-Asn-8e r-Phe-Arg-Tyr
-On'6HOAc (26) (ANF(8-33) acid) (a) Boc-Arg-Arg-8er-8er-
Cys(Acm)-Phe-Gly-Gly-Arg-
11e-Asp-Arg-11e-Gly-Ala-G
ln-8er-Gly-Leu-Gly-Cys(Ac
Hydrazide (19) (15,3 m9) in 0.6 ml DMF was exposed to 6HOAc (24) under N2.
℃ and 5.38MHCj! in tetrahydrofuran.
Added 20μ. Isoamyl alcohol (3,4μ)
) was added over 60 minutes. Stir 1 at -25℃
Lasted for an hour. A positive reaction was shown in a test using wet starch/K I test paper. This solution of acyl azide
Cool to 0°C, add (23) (20■), and N,
N-diisopropylethylamine (26μ) was added in portions to give a final pH of 7.2 (determined by applying an aliquot to a test strip). Magnetically stirred for 72 hours at 5°C and 16 hours at 5°C and then evaporated in vacuo until an oily residue was obtained.
The sample was prepared using a 5×10t Crn column packed with 0 (fine) and eluted with 50% HOAc. Rf = 0.1 (EtOAc-pyridine-H
oAc-II20 =8-5-1-3) and Rf
= 0.2 (BuOH-IIOAc-H2O== 5
-1-2) and a product with a different elution time than the starting material on HPLC, the product of 13.3. A purity of 92% was obtained.

(b) Boa-Arg-Arg-8er-8er-C
s-Phe-Gly-Gly-Arg-11e-Asp
-Arg-11e-Gly-Ala-Gln-8er-
Gly-Leu-Gly-CylI-Asn-8e
r-Phe-Arg-Tyr-OH fist5HOAc(2
5) (Boa ANF (8-33) acid) Q, 3 m
50'16 of l 9.7m9 C2,9μ in HOAc
A sample of 12.28 Boa-e7-Acm-Cys u conductor (24) in glacial acetic acid/H2O (4:1 v/v)
Medium iodine solution (concentration 2.25 m97 ml) 4.
Add 9 ml to 7'C.

Two hours are required for complete conversion to diflufide, at which point the excess iodine is removed by treatment with 120 μl of zinc powder for 1-2 minutes, and the zinc is removed by centrifugation and the supernatant is removed. Evaporate the supernatant and apply the residue to a Sephadex G-50 (fine) column,
It was then eluted with 50 HOAc. Both parts containing the product (identified in npr, c) are combined and ll0A
After evaporating C, freeze-dry it to 7.7m? (yield 80
%) of product was obtained. TLC: Rf = 0.5 (
BuOH-pyridine-HoAc-H20=40-2
0-6-24), Rf = 0.2 (BuOH-H
OAc-H20=5-1-2), purity 90 by HPLC.

Zinc was not detected by X&J microanalysis.

All white below (c) H-Arg-Arg-8er-8er-Cyi
-Phe-Gly-Gly-Arg-11e-Asp-
Arg-11e-Gly-Ala-Gln-8er-G
ly-Leu-Gly-Cys-Asn-8er-Ph
e-Arg-Tyr-OH・6HOAc (26) (A
NF(8-33) acid) ~7.5 B moistened with 0.1 ml methylene chloride
A sample of oa ANF(8-33) acid (25) was
．． The solution was treated with 1 r/Ll of 100% TFA and further rapidly dissolved by adding 0.14 10 (ITFA) after 3 minutes. After 20 minutes, the solution was diluted with 5H
of cold (-10°C) ether and the precipitated solids were collected by filtration and added to Cairdex G-50.
(fine) and eluted with 50% HOAc. This resulted in a single peak of the correct molecular weight. - The combined fractions were evaporated and lyophilized to give about 6 μm of the final product (26). TLC: Rf
=0.6 (BuOH-Pyridine-HoAc-H20=
30-20-6-24), Rf = 0.1 (BuO
H-HOAc-820=10-2-6). HP L
Purity by C: 90% (impurities 6, 4, 1, 3%). According to Ellmin analysis, 〈4〉 free S
It showed R. Sequencing confirmed the presence of the expected residues in the correct order, including the α-asbartylpezotide linkage. Purification to 100% (as determined by HPLC) is achieved at a later date by applying the sample to preparative HPLC.

(a) IleLeu-Gly-Cya(Acm')-
Asn-8er(Bzl)-Phe-Arg(NO□)
-Tyr(Bzl)-NH-MBIll13) (27
) The synthesis method is essentially the same as that of the ester-bonded resin (Y). However, Boc-Tyr (2,6-dichlorobenzyl) is substituted with p-methylbenzhydramine resin (U, S
, Biochemical Corp., 1% crosslinked, 0.41
The remaining amino groups were then acetylated with acetic anhydride/pyridine (15 molar IM solution) in CH2C.

Starting from '99-3F (8 moles) of MBHA resin, 2
A 7.5 F octapegtide resin (27) was obtained.

l＼ノ(b) HIILeu-Gly-Cys(Acm)-A
sn-8er-Phe-Arg-Tyr-N)12・2
HOAc (28) 6.8 jp (2 mmol) of resin-bound peptide (27), 3d m-cresol and 19.5
mA! of dimethyl sulfoxide was cooled to -78°C in a Kel-F vessel equipped with a magnetic stirrer.

Approximately 7.54 g of anhydrous HF was condensed into the vessel and the stirred mixture was warmed to 0° C. over 2 hours. The mixture was concentrated under reduced pressure to constant volume and −7
Recooled to 8°C. Approximately 301 nI! After condensing anhydrous HF into the vessel, the mixture was stirred at 0° C. for 45 minutes and concentrated under reduced pressure to constant volume.

The residue was triturated with ether, filtered and the residue was washed several times with ether. Crude pezotide was added to 2 M H
Elution from the resin with OAc and evaporation to dryness gave a gummy residue, which was dissolved in EtOAe-pyridine-HoA
Chromatographed on silica gel 200P using e-N20 (10-5-1-1). Both aliquots containing the desired product were evaporated to dryness and 2M
Sephadex G in two doses using HOAc.
-25 (fine) column 5 x 100 cn1 soil. Both parts were combined, evaporated to dryness, and then evaporated to dryness from lI20 to a total of 396 cm (0.28 m
mol) (14%) of product was obtained. Rf = 0.32
(EPAW=10-5-1-1): M+H=1029
: Purity 98.7 by HPLC.

(a) Boc-Ala-Gln-3er-Gly-I
au-Gly-Cys(Acm)-Asn-8or-P
he-Arg-Tyr-NH2・HOAc (29)5
hydrazide (dan) (150In9.0,
33 mmol) was cooled to -25° C. under N2 and 99.1 mmoles of tetrahydrofuran was added. Inamylnitrile (o.9 5ml
.

0.38 mmol) was added over 30 minutes.

Stirring was continued for 1-2 hours at -25°C. Wet starch/K
It showed a weak positive reaction on the I test paper. This solution of acylazyl r was cooled to -40°C, ('28) ~ (354m9, 0.025 mmol) was added and N,N-diisoglobylethylamine (0.15-)
was added in small portions to give a final pi of 7.0 (wet narrow range, determined by applying aliquots to H test paper). The suspension was heated to -25°C for 48 hours and
The mixture was stirred with a magnet at ℃ for 40 hours.

The reaction mixture was diluted with 6 ml of methanol and the product was precipitated from 75me ether. The solid was filtered off and washed several times with ether to yield 451 m9 of a colorless solid. ) Purity 80, Section 4 as measured by IPLC.

(b) H.Ala-Gln-8er-Gly-Leu
-Gly-Cys(Acm)-Asn-8or-Phe
-Arg-Tyr-NH2 2HOAc aalt (
30) 11-/ 10d circle with magnetic nutala and 5.
0 ml of trifluoroacetic acid was added. Add this solution to 2
Stirred for a minute and then added to 50ml of cold ethanol. The flask was rinsed with an additional 1.5 drops of TFA, which was then added to the ether mixture. The precipitate was evaporated to obtain a solid, which was chromatographed on a column of Sephadex G-25 (fine powder) 5 x 100 cJn using 2M HOAe, both fractions were concentrated and 235 m9 after lyophilization. (0.14 mmol) (yield 58.0%) of the product was obtained. Purity 96.7% as determined by HPLC:
M+H=1372.

(a) Boc-Arg-11e-Asp-Arg-1
1e-Gly-Ala-Gln-8er-Gly −
Leu-Gly-C)rs(Acm)-As
n-Ser-Phe-Arg-Tyr-N
H2 @5HOAc (31) ~ hydrazide (16) in 20 m1 of DMF (202 m9,
A sample of 163 μmol) was cooled to −25° C. under N2 and dissolved in 5.4 M HCJo. 3
m1 (16.2 mmol) was added. Inamyl nitrite (30 μm, 210 μmol) was added over 60 minutes. Stirring was continued for 3 hours at -25°C. Wet starch/K
The test paper showed a weak positive reaction.

The solution of acyl azide was cooled to -40°C, dodecapezotide (30) (181 m 9.107 μmol) was added,
N,N-diisosolvylethylamine was then added in portions to give a final PI4 of 7.5 (measured by applying an aliquot to wet narrow range pH test paper).

This suspension was magnetically stirred at -25°C for 44 hours,
It was then evaporated in vacuo until an oily residue was obtained.

The product was filtered through Sephadex G-50 (fine) eluting with 50% HOAc, then the combined product fractions were filtered through Sephadex G-25 (fine) eluting with 2N HOAc. ) I have completed the above steps. Fractions containing pure product (confirmed by HPLC) were collected and lyophilized to yield 220m9 of a white solid. HPLC
Purity: 94% by FAB-MS: 2184 (
M+H).

Margin below (b) )t*Arg-11e-Asp-Arg-11
e-Gly-Ala-Gln-8et-Gly-Leu
-Gly-Cys(Acm) -Asn -Ser
-Phe -Arg-'l')rr-TFA in NIT2
(Sardines) A mixture of (31) (140 m9) in TFA (2,5 ml) was stirred at 25°C for 4 min (gold dissolved after 2 min) in cold ether (15 ml') and petroleum. Ether (25 ml) was added and the precipitated solid was collected by centrifugation. After crushing it with the ether (
2x20ml) and the product was dried over anhydrous KOH sifter to give 148m9 of an amorphous solid.

Example 17. ANF(8-33)amide(35)H-Ar
g-Arg-8er-8er-Cys-Phe-Gly
-Gly-Arg-11e-Asp-Arg-11e-
Gly-Ala-Gln-8er-Gly-Ws-A
sn -Se r -I'he -k, g -Tyr
-N1126■IOAc5alt(35) (a) Boc-Arg-Arg-8er-8er-C
ys(Acm)-Phe-Gly-Gly-Arg-
11e -Asp -Arg -11e -Gly-A
la -Gln -Ser -Gly-Leu-Gly
-Cys(Aem)-Asn-8er-Phe-Ary
-Tyr-NN2・6flOAe (33) ~ 3,3vri o Dhry (hydrodihydrazide) <
961v) was cooled to -25°C under N2 and diluted with 20.2 m of 5.12 M HCl in tetrahydrofuran.
Added l. Inamylni) (13μ)) was added over a period of 30 minutes. Stirring was continued for 0.5 hour at -25°C. Wet starch/KI paper gave a weak positive reaction. This solution of acyl azide was cooled to -40°C and (
32) (x4om9) and N,N-diisosolvylethylamine (0.2ml) was added in portions to a final pH of 7.2 (by applying an aliquot to wet narrow range IJI test paper). measurement). The suspension was magnetically stirred at -25°C for 48 hours and at 5°C for 5 hours and then evaporated in vacuo to give an oily residue.

The product was filtered through a column packed with Sephadex G-50 (fine), 5 x 1006n, and 50% H
Eluted with OAc aqueous solution. Rf value is 0, 38 (Bu
OH-pyridine-)10Ac-N20''60-2
Fractions containing product 0-6-24) were combined, the solvent was evaporated in vacuo, and the residue was lyophilized from N20 to give product 1 with 98.3% purity (by HPLC).
Obtained 20m9.

(b) Boc-1℃g-kg-8er-8er-Cy
s-Phe-Gly-Gly-Arg-11e-Asp
-Arg-11e-Gly-Ala-Gln-3er-
Gly-Leu-Katsutes -Asn -Ser -Ph
e -Arg -Tyr -NH,, 5HOAc salt (3
4) 64 mg (0,017
mmol) of (33) in 80% HOAc.
30 ml of a solution of 8 m9c 0.27 mmol) was added. After thorough stirring, the flask was flushed with N2 and kept in the dark at 26°C for 1 hour and 50 minutes. reaction,
It was stopped by shaking with 242 m9 (37 mmol) of zinc powder and washed twice with 25 Fd each of water. The p solution was concentrated under reduced pressure and the residue was reduced to 50%
Sephadex G-50 (fine) using HOAc
57Tn! (0,01
5 mmol) of product was obtained. Purity 98 by HPLC
%: Rf = 0.42 (BuOH-pyridine-
110Ac-HzO=60-20-6-24).

(c) H.Arg-Arg-8er-8er-Cy+
s-Phe-Gly-Gly-kg-11e-Asp-
Arg-11e-Gly-Ala-Gln-8er-6
1y-Leu-Gly-Cys-Asn-8er-Ph
e-Arg-Tyr-NH26)10Ac salt (35) 0.24 rnl of TFA was added to a test tube containing 10.2 µmol (2.2 μmol) of (34). After stirring the test tube and dissolving the solids for 2 minutes, 0.2 ml
of cold ether. The ether mixture was diluted with 9.8 ml of petroleum ether and the precipitate was collected by centrifugation and washed with petroleum ether. HPL the crude solids
Purify with C, concentrate both fractions, and add 3.5 ml of 2 M
After freeze-drying from HOAc, 7.2 m? (1,8
μmol) of product was obtained. Purity determined by HPLC was 97%.

The structures of the compounds prepared by the methods described in Examples 7-17 were established by amino acid analysis.

The results are shown in Table 5.

ν, bottom margin

[Brief explanation of drawings]

FIG. 1 is a four-turn elution of low molecular weight CM Biooglu active on a MonoS HR515 column using triethylamine acetate (Me16.5). Flow speed: 1rn
l! /min, and 2 minute fractions were collected. Figure 2 shows the ANF (
3-33) is shown. The 40-44 minute fraction of the Mono S column was transferred to a CNμ Bondapack column (0.4 x 30 m).
applied to. Elution is 0.1%) using 20% to 50% acetonitrile in lifluoroacetic acid (0.25%/min).
The flow rate was 1 d/min. Figure 3 shows the C of the peptide ANF (3-33) prepared in 1 blue.
18μ bonderpack chromatography is shown. Elution was carried out using 20% to 50% a7tonitrile in 0.13% butafluorobutyric acid (0.2%/min) at 11 rLl.
/min. Figure 4 shows the combination of the intermediate molecular weight region and high molecular weight region after mono-S chromatography using CNμ bonder/4.
'Tsuku chromatography is shown. Elution was performed using 20% to 50% a7tonitrile in 0.1% trifluoroacetic acid (0,3
75 cm/min) using 1. The flow rate was 67 m. The diagonal part shows a peak with natriuretic activity,
Two of them are ANF (8-33) and ANF, respectively.
(1-33). Nos. 5.6 and 12 represent native (non-carboxymethylated) ANF(3-33) and reduced and alkylated ANF(8-33) and ANF, respectively.
Automatic amino-terminal decomposition of (1-33) is shown. PTH-N
The yield of PTII-amino acids corrected for the intrale IS standard is shown as a function of residue number. Slopes (indicating repeat yields) and intercepts (indicating initial sequence yields) were obtained by linear regression analysis for selected stable PTI (-amino acids). - Shows the chromatography of I11. Elution is 0
．． Acetonitrile #30 in 13% Hebutafluorobutyric Acid
% to 50% (0.2%/min) at a flow rate of 17 min. FIG. 9 shows automated NlI 2-terminal cleavage of the ANF-Hl peptide. The yield of PTH-amino acids normalized to the PTH-norleucine internal standard is expressed as a function of residue number. Calculate the slope and intercept with a selected stable PTH-
were obtained by linear regression analysis for amino acids, which yielded replicate and initial yields, respectively. Figure 10 shows the complete amino acid sequence of the ANF-Hl peptide. Arrows indicate the first 40 amino acids and ANF (1-33
) and the beginning of the array ANF(1-3
Leu4o-Leu4 necessary to produce 0)
1 cleavage is shown. The sulfide bridge between the cysteines occupying sites 52 and 62 is also shown. Figure 11 shows synthetic ANF (8-33) and ANF-H.
Figure 2 shows a dose response curve for natriuresis of 1. The total amount is the average of at least three measurements (±S, E, M,)
It is. Figure 12 shows natural ANF (2-33), natural ANF (3
-33) Synthetic ANF (8-33) and Synthetic ANF (8
-33) Dose-response curves obtained from biological tests for IJ diuretic effects of amides are shown. Figure 13 shows natural ANF (3-33), synthetic ANF (8
-33) Constriction produced in a mammalian vascular strip (Rabbit Arterial Nutrif) by adding norepinepurine (NE) to the bath before and after the addition of cereals and synthetic ANF(8-33) amide. The in vitro response curve of FIG. Patent Applicant: Intuitiet Dou Lucierche Clinic Dou Montreal Patent Application Agent Patent Attorney Akira Aoki Patent Attorney Kazuyuki Nishidate Patent Attorney (1) Yukio Patent Attorney Akira Yamaguchi Patent Attorney Masaya Nishiyama Purification of Drawings (II: (No change in content) (--H%l N:>c+. (Hu'u(HIJ% mA (--HυNricHrif-11111tIQIZI r#Ha cycle 4k
-l Wakoji - Lwog hassle (Katsu X Me 5 H Continuing priority claim on page 1
CA) [Phase] 449640 @ Inventor Marta Cantin Quebecois, Canada 1560 0 Inventor Nabil Seyda Quebecois, Canada
274 0 Inventor Claude L'Azure, Quebec, Canada 4678 0 Inventor Michiel Krechen Canada, Quebec, Roudock, Hautremont, Pernat-Avec, Ile des
Sur, Coro-Streak, Talard de Ormoyareik, Montréal, Court St. Power 1, Appartement 1404 Procedural amendment (method) % formula % 1, Indication of the case 1982 Patent application No. 177613 2, Name of the invention Peptide and its Manufacturing method and medicine containing the peptide 3, Relationship with the case of the person making the amendment Patent applicant name Institut de Recherche Clinic de Montréal 4, Agent 6, Subject of amendment (1) Zuso Takume 7, Amendment Contents (1) Drawings (2) Separate 1 8. List of attached documents (1) Engraved drawings 1 copy (2) Roast annotations 1 copy each

Claims (1)

[Claims] The tenth amino acid sequence N-z (wherein, Y is -Asn, -Asn-8er, -
Aan-8er-Phe, -Asn-8er-Ph
e-Arg, and -Asn-8er-Phe-Δarg
-Tyr; 2 is OH. or NH2; and X is H, 5ar-, 5er-
8er-, Arg-8er-Leu-Arg-Arg-
8er-8er-. Pro-Arg-3er-Leu-Arg-Arg-8
er-8er-. Gly-Pro-Arg-8er-Leu-Arg-A
rg-8er-8er-. Ala-Gly-Pro-Arg-8er-Leu-A
rg-Ar, g-8er-8or-. Leu-Ala-Gly-Pro-Arg-8er-L
su-Arg-Arg-8er-3er-, and Glu
-Val-I'ro-Trp-Thr-Gly-Glu
-Val-Asn-Pro-8ar-Gln-Arg-
Asp-Gly-Gly-Ala-Leu-Gly-A
rg-Gly-Pro-Trp-Asp-Asp-Pr
o-8er-Asp-Arg-8er-Ala-Leu
-Lys-8pr-Lys-Leu-Arg-Ala-
Leu-Leu-Ala-Gly-Pro-Arg-8
A peptide represented by ) selected from the group consisting of er-Leu-Arg-Arg-8er-8er-. 2. 4f TEDO according to claim 1, wherein Y is -Asn-8er-Phe-Arg-Tyr. 3. A peptide represented by the following formula, namely ANF(1
-33) H-Leu-Ala-Gly-Pro-Arg-8er
-Leu-Arg-Arg-8er-Phe-Arg-Tyr-Z. Below is the margin ANF (2-33) Ser-Pbe-Arg-Tyr-Z. ANF (3-33) Ser-Phe-Arg-Tyr-Z jANF (s
-33) (In these formulas, 2 is OH or NH2.) Bezotide according to claim 1. 4. ANF-[-TI expressed by the following formula. The zotide according to claim 1, wherein 2 is OH1 or NFI2. 5. The following amino acid sequence: (wherein, Y is -Asn, -Asn-8er-, -Ag
n-8er-Phe, -Asn-8er-Pha-
Arg and -Asn-8er-Phe-Arg-1'y
selected from the group consisting of r; Z u OH1 or NH2
and X1dH, 5er-, 5er-8er-
. Arg-8er-8er, Arg-Arg-8er-
8er-, Leu-Arg-Arg-3erSer-,
5er-Leu-Δrg-Arg-8er-8er-. Arg-8er-Leu-Arg-Arg-8sr-8
Er-. Pro-Arg-8er-Leu-Arg-Arg-8
er-8er-. Gly-I'ro-Δrg-8er-Leu-Arg-
Arg-8er-8er-. Al a -Gly-Pro-Arg-8e r-L
eu-Arg-Arg-8er-8er-. Leu-Ala-Gly-Pro-Arg-8er-L
eu-Arg-Δarg-8er-8er, and Glu
-Val -Pro-Trp -TbrmGly-Gl
u-Va 1-Asn-Pro-8et-Gl
n-Arg-Asp-Gly-GIy-Ala-L
eu-Gly-Arg-Gly-Pro'rrp-A
sp-Ala-Lou-Leu-Ala-Gly-Pr
o-Arg-8er-Leu-Δrg-Arg-8er
diuretic, natriuretic, comprising an active deposit of a peptide represented by ) selected from the group consisting of
Vascular hypotonia, hypoactivity, or antihypertensive agents. 6. A method for producing a peptide ANF(8-33) acid represented by the following formula, Ser-Phe-Arg-Tyr-OH, which comprises a tetrapeptide hydrazide Boa-Ala-Gln-8e.
By treatment with HCt in r-Gly-NI-INH2f solution and with isoamyl nitride, the corresponding acyl azide was obtained as a whole solution: to which the octapeptide H-Leu-Gly-Cys(Acm)-Asn -8
er-Phe-Arg-Tyr-OH1 and sufficient amount of N
, N-diisozolobyl ethylamine is added to form a basic mixture, the solvent is evaporated, and the residue is lyophilized from HOA c; The dodecapeptide H-Ala-Gln-8er-Gly-Le was prepared by adding ether to form a precipitate, preparing it by chromatography, and evaporating the eluate.
u-Gly-Cys(Acm)-Asn-8er-Ph
e-Arg-Tyr-OH was obtained: hexabutide hydrazide Boc-Arg-11e-A
sp-Arg-I le -Gly-NHNH2f By treatment with 11C1I/C in a bath solution and then with isoamyl nitride, a solution of the corresponding acyl azide was obtained; A sufficient amount of N,N-diisopropylethylamine is used to form a dibasic mixture, the solvent is evaporated, the residue is purified by dull filtration, the eluate is evaporated, and 1,2-ethane dithemer is added. Evening in the presence of”: 7'i'
100% TFA; add ether to form a precipitate, then filter and dry to obtain octadeca-coated H-Arg-I le -Allr.
)-Arg-I 1e-Gly-Ala-Gin-8e
r-Gly-Leu-Gly-Cys(Aem)-As
n-8er-Phe-Arg-Tyr-OHf TFA
Obtained as salt: Octapeptide hydrazide Boc-Arg-Arg-
8e r -8e r -Cys (Acm) -rh
o -Gly-Gly-NI(NII2) was treated with 1U of the corresponding acyl azide by treatment with J((JC) and then with inamyl nitride.
; To this, the octadecapeptide obtained as above and a sufficient amount of N,N-diisozolobyl ethylamine are added to form a basic mixture, the solvent is evaporated, and the residue is purified by gel filtration. The eluate was collected, iodine solution was added, and the mixture was stirred 1:1 until complete conversion to disulfide was achieved; evaporated, the solvent was purified by gel filtration, and the eluate was evaporated; the residue thus obtained was treated with 100% TFA;
A method characterized in that ANF(8-33)f2' of the above formula is obtained by forming a precipitate with ether, purifying the precipitate by passing through a gel i15, and then freeze-drying the entire eluate. 7. A method for producing a peptide ANF(8-33) amide represented by the following formula, Ser-Phe-Δrg-Tyr(2), wherein:
-8cr-Gly-NrlNH2”f: T in solution
V treatment S'l by IICj! L, then treatment with iso-amyl nitride 1 gives the corresponding acylat as a solution; by adding all of the LK71 soderobyl ethylamine! 1lJi, a ternary mixture is formed, the solvent is evaporated, and the residue is frozen from HOAc.
+ Treat until completely dissolved, and add ether'ff:
The zotideamide H-Ala-Gin-8e was added to the cytodeca to form a precipitate and Mk, purified by chromatography, and the eluate was completely evaporated.
r-Gly-Leu-Gly-Cys(Acm)-As
n-8er-Phe-Arg-Tyr-NH2 was obtained; hexapeptide hydrazide Boc-Arg-41a-A
, 1p-Ar g -I 1 e -G ly -N
HNH2'ft: A solution of the corresponding acyl azide is obtained by treatment with HCl in solution and then with in-amyl nitride; By adding ethylamine! 11 basic mixture is formed,
Evaporate the solvent, purify the residue by gel filtration, evaporate the eluate, and treat the residue with TFA; add ether to form a precipitate, then filter and heat dry. Octadecapeptide amide H-Arg
-11e-Asp-Arg-11e-Gly-Ala-
Gln-ser-Gly-Leu-Gly-Cyg(A
cm)-Asn-8er-P'he-Arg-T y
rNH2k Obtained as TFA salt: Octapeptide hydrazide Boc-Arg-Arg-8er-8er-Cy
Treatment with 11C1 in s(Acm)-Phe-Gly-Gly-NHNH2 total solution followed by treatment with iso-amyl nitride gave a solution of the corresponding acyl azide; Adding mectadecazotedamide and a sufficient amount of N,N-diisopropylethylamine to form a basic mixture, evaporating the solvent, purifying the residue by gel filtration, and collecting the eluate; Iodine solution is added and the mixture is heated for 2 hours until complete conversion to disulfide is achieved.
Stir: add an excess of 1 t) v/) powder, then remove the excess zinc, evaporate the solvent with AI, purify the residue by gel filtration, and evaporate the eluate; thus The obtained residue was treated with TFA, precipitated with ether,
A method characterized in that ANF(8-33) amide of the above formula is obtained by preparing the precipitate by gel filtration and freeze-drying the eluate. 8. Any method for producing butide represented by the following formula %)))) (In these formulas, 2 is OH or NH2),
Rat atria were treated with 1-5 mPA EDTA, 1 m
Homogenize in a liquor and drinking solution containing M phenylmethylsulfonyl fluoride and pepstatin, centrifuge, separate the supernatant and freeze, M freeze and centrifuge the latter, The solution was passed through a bed of octadecylsilane, the active substance was stored in a 20,000 mol.
The heat is divided into the intermediate molecular weight region and the polymer molecular weight region; the active fraction obtained from the low molecular weight region is called Cal? A cross-linker containing all oxymethyl groups was applied to the loin beads at a temperature of 0.01 M to 1.0 M in ammonium acetate at pH 5.9.
M out with a linear gradient of 0 M; active fraction collection,
This was applied to a cation exchanger manufactured by L11 for use in liquid chromatography, and P
0 of triethylamine acetate at ll 6.5
．． Elute with a linear gradient from 0.02 to 1.0 M; select the appropriate activity fraction and incubate at 6.0°C.
The active product thus obtained was further purified by reverse phase HPLC on finely ground cyanolobylfuran with TFA and acetonitrile kumquats at a concentration of 0.1 (V/V) at room temperature. V) TFA in charge
&υ” Acetonitrile was purified by reverse phase 1 (PLO) on finely ground octadecylsilane; and in some cases, the active ingredient thus obtained was purified at 0.13 (V/V)% at room temperature. 1) ANF(1-33), ANF(2-33), AN
F(3-33), or ANF(8-33) meeting all features 9, the following formula, ゛1. :IeaWbo-to ■ In this method, rat atria were treated with acetic acid containing 5mM EDTA, 1mM phenylmethylsulfonyl fluoride, and an effective amount of pepstatin. Homogenize in aqueous solution,
Centrifuge, separate supernatant, freeze, thaw,
The latter is then centrifuged and passed through a bed of ometadecylsilane to remove the active substance with a cut-off molecular weight of 20,000 ft.
0 pa? The active fraction was pooled on lyacrylamide, eluted with 0.1 M acetic acid, and the active substance was separated into low, medium and high molecular weight regions; Apply to a column of cross-linked agarose beads with methyl groups and pl
(Elute with a linear gradient of 0.01 M to 1.0 M in 5,0 ammonium acetate; collect the active fraction and apply all this to a cation exchanger prepared for use in liquid chromatography. , and eluted with 0.02 M to 1.0 M of triethylamine acedate at p[16,5; 0.1 (V/V)% at
Reversed phase HP on finely ground cyanopropylsilane using an rα gradient of TFA and acetonitrile.
Purification by LC; the active product thus obtained was further purified by reverse phase HPLC on finely ground octadecylsilane using 0.1 (V/V)% TFA and acetonitrile D-line dalasiendo at room temperature. and optionally the active ingredient thus obtained using a linear gradient B- of 0.13 (V/V)% bebutafluorobutyric acid and acetonitrile at room temperature.
A method characterized in that υANF-H1f is obtained by performing b81q purification by applying it to finely ground octadecylsilane.