JPH0393796A - Ghrh analogous compound - Google Patents

Abstract

NEW MATERIAL: A compound of formula I [Q¹ is 3-carboxypropyl, 2- carboxybenzamidomethyl or an ω- or α,ω-substituted alkyl of formula II (X is OH; Y is H or OH; m is 1 or 2; n is 0, 1 or 2); R₂ is Ala; R₃ is Asp; R₈ is Asn; R₁₀ is Try; R₁₂ is Lys; R₁₃ is Val; R₁₄ and R₁₇ are each Leu; R₁₅ is Ala; R₁₈ is Tyr; R₂₁ is Lys; R₂₂ is Leu; R₂₃ is Leu; R₂₄ is Gln; R₂₅ is Asp; R₂₇ is Met or Ala; R₂₈ is Asn; Q² is formula III (p is 2-6)].

USE: To stimulate the release of pituitary growth hormone.

PROCESS: A t-butoxycarbonylating agent is reacted with NH-Q² in the presence of a base and then the product is reacted with an arylsulfonyl halide in the presence of an aq. alkali solution. The product is coupled with a supporting carrier using a di(lower alkyl)carbodiimide.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to peptides that affect pituitary function in humans or other animals. In particular, the present invention relates to peptides that promote growth hormone release in the hypophysis.

Physiologists have long known that the hypothalamus produces special substances that stimulate or inhibit the secretion of respective pituitary hormones, and that it also controls the secretory functions of the adenohypophysis. admitted to doing so. Hypothalamic releasing factors include the pituitary hormone thyrotropin (tripeptide TRF), the pituitary gonadotropin progesterone and follicle-stimulating hormone (decapeptide LRFSLH-RH or GnRH), and the pituitary hormone and adrenocorticotropin (41 It is characterized by the number amino acid polypeptide CRF). An inhibitor called somatostatin is also characterized by a tetradepotent peptide form that suppresses growth hormone (GW) secretion. GH-releasing prisoners (GRF'S) have been isolated from rat, pig, cow, sheep, goat and human hypothalamus as well as human pancreatic tumors.

With the exception of rat, all have characterized GRFS containing 44 amino acids with an amidated carboxy terminus. Each of these pituitary-tropic prisoners was reproduced by total synthesis. Natural structural analogues can be used to elucidate structure-activity relationships and ultimately provide synthetic congoners with improved properties such as enhanced hormonal activity and/or metabolic stability. It was also synthesized for

Research on synthetic human growth hormone releasing factor (hGRF) and its similar compounds [(N. Ring et al., Biochemical and Biophysical Research Communications, 1984, Vol. 122, 304-310)
Pages: Volume 123, pp. 854-861; Bui. A. Lance et al., Biochemical and Biophysical Research Communications 1984 No. u9a
pp. 165-272 (N. Ling. . at al.
．． Blochem. Blol) hys. Res.
COauaun- (1984). vol. 122
, f) p. 304-310: vol. 123.
pp. 854-861; V. A. Lance
．． et al. ．． Biochem. Blophy
s. Res. COma+un. ．． 1984. v
ol. 119. pp. 165-272)] revealed the following facts a) and b).

a) Removal of the NH2 single terminal tyrosine residue of hGRF reduces its activity to 0. Reduce to 1%. N-acetylation of this residue or a change that replaces L with the D isomer results in hGRF
The in vitro bioactivity of (1-40)-OH was 2-3.
% and increase the in vivo potency of hGRF(1-29)-NH2 by 10-12 times: these findings demonstrate that the presence of such residues is essential for conferring hGRF molecules with high bioactivity. .

b) have at least the first 29 residues and, for example, hGRF(1-29)-NH2 and hGRF(1-37
) - The fragment ending with the amino acid NH2 is hG
Has at least 50% of the efficacy of RF. Removal of the C-terminal amide group or deletion of amino acids results in a significant decrease in bioactivity, even if the C-terminus is amidated. For example, hGRF (1-29)OH, hGRF
(1-27)-NH2 and hGRF (1-23)-NH
2 are 12.6% and 1.12% of hGRF potency, respectively.
5% and 0.24% are shown. These findings demonstrate that arginine amide, -Ar at position 29 of hGRF
It shows the importance of g-NH2.

Deamidation of the C-terminal rupoxiamide function, e.g. hG
RF(1-29)NH2 to hGRF(1-29)O
Regarding the transformation to H, which occurs readily under physiological conditions with the action of hydrolytic enzymes and is accompanied by a substantial loss of bioactivity, agmatine (A
In view of our previous observation that the 4-guanidinobutylamino group, termed the gm) residue, plays the role of an arginine residue in some tribeptide inhibitors of tribucin-like enzymes and confers resistance to enzymatic degradation. Peptides, 1982 (K. Prague and P. Mellon, ID.S.), Walter Gruyter, Berlin-New York, 1983, 64.
pp. 3-647 (S. Bajusz. et al..
in: PEPTIDES, 1982. (K.B.
raha and P. Melon. cds)lWa
ltcr Gruytcr, Berlin-New
York. 1983. pp. 643-647).

] Classical (liquid phase) methods have been used to date for agmatine peptide preparation.

Preferably, peptide synthesis techniques that have proven suitable for the preparation of GRF8 and its analogues are employed to provide a methodology for solid phase peptide synthesis.

A novel series of syntheses that, in view of the provision of an omega-guanidino lower alkyl group at the terminal 28 position of the peptide, highly stimulates the release of GH in animals, including humans, that resists enzymatic degradation in the body. A peptide is provided. Similarly, a method for attaching said omega-guanidinoalkyl group to a support phase;
By solid phase synthesis? Methods of synthesizing the peptide and cleaving the peptide from the support phase are provided.

The final product of the invention is a peptide (abbreviated form [PeP]) having the following sequence:

A polypeptide having the structure represented by the following formula I and a pharmaceutically acceptable salt adduct thereof having a pharmaceutically acceptable organic or inorganic base and an organic or inorganic acid.

Q'-CO-R2-R3-Ala4-110!
) -Phea-1"hr7-1?a-SOr9 -R
to-Argt■-R12-Rti-R14-Rls-
Gln16-l? 17-R1a-A l al9-Ar
gzo-R21-R2 2-R2 B-R24-R2
s-l 1 e2 6-R27-R2g-NH-Q 2
(I) [However, Q1 is 3-carboxypropyl, 2-carboxybenzamidomethyl, or the general formula ■ (However, X=OH, Y=H, OH, NH2 or CH3 CONH, m=1 or 2 n=0. 1 or 2), R2 is Ala or D-Ala, R3 is Asp, D-Asp, Glu or D-Glu, R8 is Asn, D-Asn, Ser or D-Ser
, R10 is Tyr or D-Tyr, RB is Lys, D-Lys, Arg, Orn, D-Ar
g or D-Orn, R13 is Val or lie, R14 is Leu or D-Leu, R15 is Gly or Ala, R17 is Leu or D-Leu, R18 is Tyr or Ser, R21 is Lys, D-Lys, Orn, D-Orn,A
rg or D-Arg, R22 is Leu, D-Leu or Ala, R23 is L
eu or D-Leu, R24 is Gin or His, R2, is As p', D-As p, G 1 u or D-Glu, R27 is Met, D-Me t, A 1 a,
N1e, Ile, Val, NVa or Leu, R28 is Asn or Set, Q2 is -(CH2)p Nil-C-NH2
(n[) represents a low 11 N+1 class omega guanidinoalkyl group (in the formula, p=2 to 6). ] The peptide [P e I'J of the present invention is synthesized according to the following method.

The first method for the NH2-Q2 moiety is to combine a t-butoxycarbonylating agent with Nil- in the presence of a base.
Q2 to form Boc-NH-Q2(rV) and the Boc-Nil-Q2
and the following formula 11al so2 −<O> −0
-CIl2CO-CH2-COOH (V)
(However, in the formula, Hat represents chlorine or bromine, and <O
> represents (throughout the specification) an unsubstituted benzene ring.

) is reacted with the arylsulfonyl halide represented by ), and the support phase [
SP] couple to Boc-Nil-Q"-SO
2-(0)-0-CH2-CO-[SP
] (■) (similar, in the formula, ,Q2 = -(C
H2)l)-NH-C(N112)-N-(■). ), yielding the corresponding Boc -Nil-Q ” -SO2 - <
0>-OCII 2 -CO-CH2-COOH
It consists of a series of steps to form (Vl).

The support phase [SP] has the following formula 112 N -CH2 -<
O>“[Pm] (IX)” (However, in the formula, [Pm
] is the polymerization substrate. ) is preferable.

This method predicts the construction of peptide sequences according to the Boc protocol, which will be detailed later.

Other embodiments in which the guaninoalkyl group is attached to the support phase are also available. This method involves reacting N-pendyloxycarbonyl chloride with NH2-Q2 in the presence of a base to form Cbz-NH-Q2, and reacting the above Cbz-NH-Q2 with the following formula in the presence of a strong aqueous base. 11al -SO2 - <0> - 0-CII2
-00011 (X)Ms (similar, in the formula, Mal is chlorine or bromine, M is 1 carbon number
-5 lower alkyl or lower alkoxy, and S represents 1-3. ) with a substituted arylsulfonyl chloride, and the Cbz group was removed by a catalytic hydrogenation reaction to form II 2 N-Q ” -SO2 − <
0>O-CH2-CO-CH2-COOH (X
I)/' . . -NH-Q ” -So 2 -<O
>-OCII2-CO-CH2-COOH
It consists of a series of steps in which (XI I)/Ms is obtained and this product is reacted with the support layer [SP] to form Ms.

As mentioned above, this method is based on the Fm
Preferred if oc brotocorol is to be used.

Utilizing the aforementioned protected amino-alkyl-guanidino-sulfophenoxyacetyl-terminated support phase, a given bebutide itself can be synthesized conventionally by solid phase synthesis utilizing the protecting groups and specific methods detailed below. Formed by law.

When the synthesis is complete, the guanidinoalkyl end of the peptide becomes
It is cleaved from the attached sulfonyl group by one or two methods. If Boc brotocol is utilized, the cleavage agent is anhydrous hydrofluoric acid, while in the case of Fmoc brotocol, the cleavage agent is trifluoroacetic acid.

Pharmaceutical compositions according to the invention contain a peptide of formula (I) or a non-toxic salt of any of them, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier. Such pharmaceutical compositions can be used for both human and veterinary clinical medicine and diagnostic purposes as therapeutic administration. Additionally, these pharmaceutical compositions improve food utilization and increase lean/fat ratios favorable for muscle gain in fat prices, while promoting growth in mixed-breed animals and increasing milk production in lactating cows. It can be used for

Effect: The nomenclature used to define peptides is IUPAC-IUB, with the N-terminal amino group on the left and the C-terminal carboxyl group on the right.
Commission on Biochemical Nomenkrehecher (IUPAC-IUB COma+ission
on B1ochemical Noa+encla
ture) [European Journal of Biochemistry-1984, Vol. 188, pp. 9-37 (
European J. 81ochca+. 198
4. 138. 9-37)].

Gly, Ala, Val, Leu by natural amino acids
, I le, Ser, Thr, Lys, Arg, Asp
, Asn, Glu, Gln, Cys, Met, Phe,
Represented is one of the common naturally occurring amino acids found in proteins consisting of Tyr, Pro, Trp and His. Nle represents norleucine, Nva represents norvaline, and MeA1a represents N-methyl-alanine. When an amino acid residue has isomeric forms, it is the L form of the amino acid unless otherwise indicated.

Other contractions used are:

AcOH Acetic acid AcOEt Ethyl acetate Ac20 Acetic anhydride Boc-t - BfOicicarbonyl~DIC
Diisobrobylcal f diimide A
gm Agmatine DCM Dichloromethane Chx Cyclohexyl DIEA Diisobrobylethylamine DMF Dimethyl Honlumamide HOBt l - and Droxybenzene Triazole Hydrate HPLC
A-type U-body chromatograph I-Fmoc
- 7 fluorenyl haxycarbonyl M e
O H Methyl alcohol TEA
}Lethylamine DCC Summer Dicyclohesylcarbodiimide 2-Clcbz 2-Chloro Benjilotei cyclocarbonyl D C 8
2.6 - Dichlorobenzyl T
os p - toluenesulnenyl TFA trifluoroacetic acid Cbz benzyloxycarbonyl The process of the present invention is carried out using a number of support phases. These support phases include aminomethyl resin (preferably 1% crosslinked),
penzhydrylamine resin (preferably 25 crosslinked), p
- amino or hydroxy resins such as methylbenzhydrylamine resins (preferably 25 crosslinked).

As starting materials for this method, many amino lower alkylguanidines having 2 to 6, preferably 3 to 5 carbon atoms in the alkyl moiety can be used. However, particularly preferred are agmatine or 4-guanidinobutylamine.

The key to the success of this invention is to provide a stable but easily cleavable bridge group between the guanidino moiety and the support. It has been found that such a bridge is easily provided by a sulfonyl phenoxy acetyl moiety.

In one embodiment of the synthesis example, the main amino group of agmatine is protected with a t-butoxycarbonylating agent, preferably di-t-butyl dicarbonate, preferably by reaction at room temperature in the presence of a base, and the sulfonyl
Aryl that is chloride or sulfonyl bromide
Provides a protected substance (V ) that is reacted in a similar solvent system as the sulfonyl halide (V). Although the final synthetic sequence follows the Boc protocol, the aromatic ring of the arylsulfonyl halide is important because such a situation allows the use of trifluoroacetic acid as the intermediate deprotecting agent and hydrogen fluoride as the final cleavage agent from the resin support phase. ,
Not replaced. The protected aminoalkylguanidinose sulfonylphenoxyacetic acid (Vl) thus obtained is then coupled to the support phase. In a preferred method, coupling is performed using any known dialkylcarbodiimide coupling method.

For example, l − in dimethylformamide at room temperature.
Diisobrobyl carbodiimide is utilized in the presence of hydroxybenzene triazole hydrate. Also, N. N' dicyclohexyllupodiimide is also used. In this method, the BOC-aminoalkylguanidino sulfonylphenoxyacetic acid (Vl) can be prepared by:
carbodiimide in a molar ratio of 1, N. N'
The dicyclohexyl urea is filtered and the resulting solution is added to the support phase, preferably an aminomethyl resin.

As mentioned above, if the protecting group is Boc, the aryl moiety of the sulfonamide bridge is not substituted. However, Fm
If it is preferred to proceed under the oc protocol, the phenyl moiety may be substituted with up to three methyl or methoxy groups, as these weaken the sulfonamide groups and allow trifluoroacetic acid cleavage. is preferred.

In a second embodiment, which is an alternative to utilizing Boc for the protection of amino alkyl guanidino components, N-pendyloxycarbonyl chloride is dissolved in the presence of an aqueous alkali, preferably sodium hydroxide, with stirring at about 4N. and cooled to about 5 to about 15°C. Upon acidification, the protected product precipitates and is then reacted with arylsulfonyl chloride (X) as described above. However, as mentioned above, the arylsulfonyl chloride (X) in this case should be 1-3 substituted, being alkyl or lower alkoxy, preferably methyl or methoxy. This reaction is carried out in the presence of a strong but dilute aqueous base, preferably about IN sodium hydroxide. The reaction mixture is made acidic and the product is extracted with a water-immiscible spinal solvent, preferably ethyl acetate. The extract is then concentrated and hydrogenated in a reducing inert solvent in the presence of a catalyst such as palladium on activated carbon, preferably at ambient temperature and pressure. When ethyl acetate is used as an extractant, it is also used as a hydrogenation solvent. Cbz
After removal of the group, product (XI) is converted to Fmo in the presence of base.
c-C! J to produce the corresponding Fmoc-protected aminoalkylguazinosulfonyl aryl oxyacetic acid (XII), which is subsequently coupled to the support phase in the manner described above.

Peptides are synthesized by any suitable method such as solid phase techniques, partial solid phase techniques, fragment enrichment or classical liquid phase synthesis methods. Using recently developed recombinant DNA technology,
Portions of analogous compounds containing only natural amino acid residues can be prepared. For example, dedicated solid phase synthesis techniques are presented in the following textbook ["Solid Face Peptide Synthesis" G.I. M. Stewart and J. Dee. Young, Beers Chemical Company. Rockford, 111. , 1984 (2nd edition), Jeevalany and R. B. Merrifield "Zabebutidos" C.I. Page 1-285, 1979, Academic Press, Incorporated and M. Bodansuzky “Principles Obeptid Synthesis”
Springfield Lark 1984 ("Sol1
d Phase Peptide Synthe
sis'. J. }i. Stcwart and
J. D. Young. P1crec
Chew. CON+pany. Roekford
．． Ill. ．． 1984 (2nd. cd.).
G. Barany and I? ．． B. Mcrri
ficld. “The Peptides.Ch.
1. 1-285, pp. 1979. Acad
emic Press. Inc. ．． and M.
Bodanszky-”Princ1plcs of
PcptidcSynthcsis”. Sprin
gcr-Vcrlag. 1984. )] The old solution synthesis method is described in detail in the following paper:
Vuunsier (editor) (1974) George Schieme Verlag, Stuttgart West Germany ("Mcthoden dcr Organ1sch
cn Chcmic (lloubcn-Weyl):
Synthcsa won Pcptidcn.
E. Wunsch (editor) (1974
) Georg Thica+e Verlag, St.
u17gart, W. Germany. )].

Common to such synthetic methods is the protection of the reactive side chain functional groups of many amino acid moieties with suitable protecting groups that protect them from chemical reactions occurring at that site until the group is finally removed. It is.

Similarly, what is common is to protect the fragment during the entire reaction at the α-amino or alkoxy group of the amino acid, followed by selective removal of the α-amino protecting group and subsequent reactions at that position. The purpose is to make things happen. It is therefore common during the synthetic steps to produce intermediate compounds containing each of the amino acid residues located in a given sequence of the peptide chain with a side chain protecting group linked to the appropriate residue.

Intermediates of the following formula are considered to be within the scope of this invention.

That is, [PR] [PeP] is attached to a support phase [S P] selected from the group consisting of amino resins, said combination having the following structure, [PR] [:PeP Co802 -<0>
O CII2 CO [Spl/Ms [However, in the formula, M is hydrogen, lower alkyl or lower alkoxy having 1 to 5 carbon atoms, preferably methyl or methoxy, and S is 0, 1. 2 or 3, [SP] preferably represents an amino type resin. ] [PR][PeP] has the following structure X'-Q'-CO-R2-R3(X3)-
Ala4-11cb-Phca-Thr7(X'
)-R a (X7or H)-Ser9(X7)-R 1o(X") -AI'g1t(X1]) -1?
l2(X”.X 12) -Rl3-Rl4-R1s
-GIntc+-Rt7-Rta (X7or X 10
) -Ala15-Argz.

(X11)-R21(X12)-R22-R23-
R24(II Or X”)-R25(X3)-
lie 26-R27-R28 (II or X 7)
-NH-Q 2'IA [wherein, X1 represents either a hydroxyl or amino protecting group or nil, and depends on the meaning of Y in structure (1).

X3 is a suitable ester-forming protecting group for the carboxyl group of Asp or Glu. Cyclohexyl ester is preferred in the Boc protocol (t-butyl ester is preferred in the Fmoc protocol).

X7 is a suitable protecting group for the hydroxyl group of Thr or Ser, such as t-butyl, Bzl and 2.6-
It is dichlorobenzyl. Preferred protecting groups are Bzl for the Boc protocol and t- for the FmoC protocol.
Butyl.

X10 is a suitable protecting group for the phenolic hydroxyl group of Tyr, such as t-butyl, Bzl, 4Br-Cb
z and 2,6-dicoobenzyl (DCB).

Suitable protecting groups are 2,6-dichlorobenzyl for the Boc protocol and t-butyl for the Fmoc protocol.

Xl1 is a suitable protecting group for the guanidino group of Arg, such as nitro, Tos, 4-methoxy-2.3.8-trimethylbenzenesulfonyl. Tos is a preferred group in the Boc protocol, and especially 4-methoxy-2.3.6-}limethylbenzenesulfonyl in the Fmoc protocol.

XI2 is a suitable protecting group for the side chain amino group of Lys. Examples of preferred side chain amino protecting groups include 2-chloropendyloxycarbonyl (2(1-Z), pendyloxycarbonyl, and in the Boc protocol 2
-Chloropendyloxycarbonyl is a preferred protecting group, and Boc is also utilized in this manner in the Fmoc protocolor.

X24 is hydrogen or a protecting group for the imidazole nitrogen of His, such as Tos.

The choice of side complex amino protecting group is not critical, except for the selection of some common ones that are removed during deprotection of the α-amino group during synthesis.

The following general rules are given in the selection of specific side chain protecting groups to be used in peptide synthesis.

(a) The protecting group preferably has a protective property of -a,
They are not separated under coupling conditions. (b) The protecting group is
It should be stable to the reagents and preferably under the reaction conditions chosen for removal of the α-amino protecting group at each step of the synthesis.

and (C) the side chain protecting group must be removable upon completion of the synthesis containing the given amino acid sequence under reaction conditions that will not alter the peptide chain.

Peptides can be synthesized using the Merrifield [Journal of American Chemical
Society, Volume 85, Page 2149, 1963 (M
errifield, J. Am. CheIl.
Soc. ．． 85. P. 2149 (1963) )]
Preferably, they are prepared using solid phase synthesis methods, as generally indicated by .

Boc- or Pmoc-NH-Q”-80 2
-(0>-0-Cll2/Ms-CO-[SP] is prepared as described above and constitutes the starting material for the synthetic sequence.

After removal of the α-amino protecting group, the remaining α-amino and side-chain protected amino acids are added in a stepwise order to obtain the intermediate compounds defined herein, or alternatively each amino acid is added synthetically separately. and some are coupled together before being added to the solid phase reactor. Selection of appropriate coupling reagents is within the skill of the art.

In particular, as a coupling agent, N. N'-diisopropylene diimide (DIC) is preferred.

Each protected amino acid or amino acid sequence is introduced into a solid phase reactor in approximately 3-fold excess, and the coupling is carried out in a solvent or in dimethylformamide (DMF): CH2Cn
2 (1:1) or DMF or CH2CU2
Performed in a single solution. If the reaction is incomplete, the coupling step is repeated before removing the α-amino protecting group prior to the next amino acid coupling. The good results of the coupling reactions at each step of the synthetic method are preferably confirmed by ninhydrin reactions as shown in the literature by E. Kaiser et al. [E. Kaiser et al. Analytical Biochemistry Volume 34 Page 595 197
Year 0 (P. Kaiscr, at al.. Ana
l. B1ochci. +34 595 (1970)
].

After completion of a given amino acid sequence, the intermediate peptide not only cleaves the peptide from the resin but also removes all remaining side chain protecting groups X3 X7 XIO ) (II, ) and similarly α-amino or hydroxyl
It may be removed from the resin support by treatment with reagents such as liquid hydrogen fluoride or trifluoroacetic acid, which also cleaves the group X1. As mentioned above, these groups can be Boc or Fmoc.
It has different value depending on whether the protocol is used or not, ensuring a given free form of the peptide.

The products of the present invention are utilized to increase milk production in milk-producing dairy animals, preferably goats and cows, most preferably female cows. The compounds of the invention are administered to a subject intravenously (1.v), subcutaneously (S.C.) or intramuscularly (1.m.), preferably subcutaneously or intramuscularly. saline is also acceptable, other carriers known to those skilled in the art may also be used, and the dosage per injection is:
A dosage range of 0.2 to 2 μg/kg body weight is preferred. Administration is 1 to 4 times a day. It is convenient to inject the animal when expressing milk. Other equivalent modes of administration are within the scope of this invention. That is, continuous instillation, depot injection, time-release mode such as microcapsules, etc.

EXAMPLE The following example demonstrates a sophisticated method for peptide synthesis by solid phase technology.

Example I Synthesis of Boc-Agmatine (V) Agmatine sulfate (19.5
6g, 85.51M) and 25ml triethyleamine (
Di-tert-butyl dicarbonate 2 was added to a stirred suspension in 50% aqueous tetrahydrofuran containing
Added 5g. After 4 hours, additional dicarbonate 12.
5g was added, and after another 4 hours, 12.5g was added to bring the total amount to 50. 0g (229mM) was obtained. The mixture was stirred overnight and then filtered through a Puchner funnel to remove insoluble matter. The oral liquid was transferred to a separatory funnel, the bottom aqueous layer was drawn off, and this one was extracted with 100 moles of diethyl ether and then freeze-dried. The residue was dissolved in 200 mJ of water. To this solution, 15 g of sodium hydrogen carbonate was added, and the mixture was sufficiently stirred and pressed until a solid substance started to be formed. After cooling in a water bath, a dry solid substance was collected. 15.8 g of product 1 melting point 126-128°C (
Disassembly).

250 ml of this material was dissolved in 2.5 ml of warm water, 2.5 ml of saturated sodium bicarbonate solution was added, and the mixture was kept in a refrigerator overnight. 160 ml of material was obtained. Melting point 134-136
°C (decomposition).

15 g of crude Boc-agmatine was added to 50 ml of hot water, cooled to room temperature, and the product was sprinkled and kept in the refrigerator overnight.

Yield 10.4g, melting point 134-135°C (decomposition), second
Yield ffl [2.4 g, melting point 134-135°C (decomposition)]
was obtained by adding an equal volume of saturated sodium bicarbonate solution. Total yield m12.9go Example 2 Synthesis method of 4-chlorosulfonylphenoxyacetic acid Phenoxyacetic acid 50n+M (7.6g) was added to 75ml of chloroform
25 m of chloroform dissolved in
chlorosulfonic acid (16.5 ml) was added dropwise over 10-15 minutes. The temperature was held at 0° C. for 20 minutes, followed by an additional 30 minutes at room temperature. The two-phase liquid was poured into a beaker containing approximately 300 g of crushed ice. The separated aqueous phase was extracted twice with 50 ml of ether, and then the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the residual material was recrystallized from an ether-hexane mixture. yield. 9.75g (77.8%), melting point 155-157
°C (uncorrected) TLC, Sigma F2,4 analysis plate detection, UV
254 nm, 355 nm Eluent A) Ethyl acetate acetic acid (9: lv/v) Eluent B) Ether-hexane (2:1 v/v) with 1 drop of acetic acid added to 10 ml of eluent System A ．． 0
．． 85 System B, 0.34 R,,4-chlorosulfonyl phenoxyacetic acid System A, 0. 77 System B. If the above method is followed using 2.3.5-}limethylphenoxyacetic acid and 2.3.6-}limethylphenoxyacetic acid instead of 0.17 phenoxyacetic acid, the corresponding 4chlorosulfonyl- 2.3.5-Trimethylphenoxyacetic acid is obtained (Rf value, ether-hexane-acetic acid mixture (8:4) on a Sigma F 254 TLC plate.
: 0. 0 5 (v/v)) and 0.7 respectively.
8 and 0.75).

Example 3 Boc-Agmatine-[SPA Co (Vll')
Boc-agmatine (4.29 g. 10 mM) was added to a mixture of acetone (50 ml) and IN sodium hydroxide (50 ml) with stirring, cooled in a water bath, and 4-
Chlorosulfonylphenoxyacetic acid (3.8g. 1
5. mM) was added to the solution. After stirring for 3 hours, the pH of the reaction mixture was adjusted to 11~11 while adding 4N sodium hydroxide.
Hold at 12. The reaction mixture is acidified with 1.0% citric acid solution, concentrated to half its volume under reduced pressure and extracted with ethyl acetate (3×50o+1). The combined extracts are washed with saturated sodium chloride solution, dried over sodium sulfate and evaporated under reduced pressure. Boc-agmatine-sulfonylphenoxyacetic acid (abbreviation Boc-agmatine [
SPA]) is obtained as a thick oil (3.6g.81
%). 0.5 with a 9=1 (v/v) mixture of ethyl acetate acetic acid on a Sigma F254 TLC plate.
.

Cbz-agmatine instead of Boc-agmatine and 4-chlorosulfonyl-2.3.5-trimethylphenoxyacetic acid or 4-chlorosulfonyl-2,3.6-trimethyl instead of 4-chlorosulfonylphenoxyacetic acid. If the above method is followed using phenoxyacetic acid, the corresponding Cb
z-Agmatine-2.3.5-trimethyl- [SPA
] and Cbz-agmatine-2.3.8-}remethyl-[SPA] are obtained.

Example 4 Coupling of Boc-Agmatine-[SPA] to the support phase Boc-Agmatine-[SPA] in 50 ml DMF
3. 11 g (7mM) in H for 30 minutes at room temperature.
OBt 1. 89g (14a+M) and D I C
1. The active ester solution produced in this way was added to 5 g of aminomethyl resin (0.7 m/g capacity) submerged in dichloromethane in a 10% diisopropylethylamine chloroform mixture. Deprotonated and washed twice with DMF. The reaction was complete in 90 minutes and was wiped with ninhydrin reaction. Finally, the coupled resin was washed with DMF, dichloromethane, and again with DMF.

Example 5 Synthesis of Cbz-Agmatine Agmatine sulfate (2.28g, 10o
+M) and sodium hydrogen carbonate (2.52g.30mM>
was dissolved in a mixture of 20 ml of water and 20 ml of dioxane. The mixed solution was cooled on a water bath, and pendyloxycarbonyl chloride (1.8 5 ml,
13mM) was added dropwise over 15 minutes. Stirred at 0° C. for 1 hour and at room temperature overnight. The reaction mixture was dried to dryness, the residual pellets were mixed with 2×50 ml of chloroform, and the solution was evaporated under reduced pressure. The oil compound was used without further purification. Rl, Sigma,
,,0.66 with a mixture of ethyl acetate pyridine and acetic acid monohydrate (45:20:6:11 (v/v)) on a 4TLC plate.

Detection, under UV radiation Sakaguchi reagent Example 6 5 (or 2,3.6) monotrimethylphenoxyacetic acid was dissolved in ethanol (50 ml) and hydrogenated over palladium activated carbon catalyst (0.5 g). . The catalyst was filtered off and the solvent was removed under reduced pressure. The residue was recrystallized from an ethanol-water mixture to give agmatine-sulfonyl-2.3.5 (or 2.3.6)-}rimethylphenoxyacetic acid (2.70 r, 70% and 2 .78g, 7
25).

The Rt values of both compounds are the same, Sigma F254TL
On a C plate, a mixture of n-butanol-acetic acid monohydrate (4
: 1 : 1 (v/v)) and is 0.28.

Example 7 Cbz-agmatine (2.85 g, 10 mM) and 4-chlorosulfonyl-2.3.5-trimethylphenoxyacetic acid (or 4-chlorosulfonyl-2.3.6-trimethylphenoxyacetic acid) ( 4.39g.15mM) were reacted as described in Example 3.

The obtained Cbz-agmatine-sulfonyl-2,3, agmatine-sulfonyl-2.3.5 (or 2,3
．． 6) Monotrimethylphenoxy 61: acid (2.70g.
7mM) to sodium carbonate} (18.6mM)
dioxane (llml) dissolved in a 10% solution of
added. Stir the solution and cool it in an ice-water bath. 9
-Fluorenyl methyl cyclocarbonate (1.82
g, 701M) was added. Stirring was continued for 4 hours at water bath temperature and for a further 8 hours at room temperature. Add the mixture to 400 ml of water
ml and extracted with 2×100 ml of ether. The aqueous phase was acidified with l:1 hydrochloric acid solution and ethyl acetate 3X
Extracted with 100ml.

After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure. The obtained Fmoc-agmatine-sulfonyl-2.3
．． 5 (or 2.3.6)-1-limethylphenoxyacetic acid was dissolved in DMF (50ml) and coupled to 5g of aminomethyl resin as described in Example 4.

Example 8 A method for synthesizing hGHRH-like compounds of the following formula.

3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3-Ala4-1105-Phea-T
hr7-AsnB-Ser9-Tyr+o-Arg
++-LyS+z-Valtv-LOu14-Gll+
5-Glntb-Lcu I 7-Set s-A
Ia l 9-Arg2o-LVSz 1-Lcu2
1-Leu2q-G1n+4-Asp25-11Q2b
-N102r-SOr2s-NH-<Cll2)
4NH-C-(NI12)-N-SO2-(0
>-OCH2-CO-[SP] is converted to an appropriate Boc-NH-(CII2)4-NH-C according to the methods shown in Schedule A and Schedule B below.
-(Nil2)-N-SO2-(0>-O
Beckman 99 for the first time from CII2-CO-[SP]
It was performed in a stepwise manner with 0 synthesizers.

a) Unblocking and attachment De-blocking is performed according to Schedule A below.

Schedule A l. Deprol: 5 in DCM
0% TFA2. Iho'M: 50%T in DCM
FA3. DCMIi clean 4. 2-panol Il#5. medium flI: lO%TEA6 in DcM. Me
OH cleaning7. 10% TEA in neutralized nDCM 8. MsOH Shizu9. Neutralization: 10% TEA in DCM 10. MeOH & Purification 11. DCMIi purification (3 giants) It is recommended that the coupling be performed according to the following schedule B.

l. Coupling. D to Boc-amino acids in DCM or ltDMF, depending on the solubility of the amino acids.
Add IC MeOH (or MeO} [YoQDMF) & Clean DCM Wash MeOH Wash DCM Wash MeOH Wash DCM Wash Briefly, BOC is used for α-amino acid protection. Benzyl ether is used as a hydroxyl side chain protecting group for Ser and Thr.

The phenolic hydroxyl group of Tyr is protected with DCB and the side chain carboxyl group of Asp is protected in the cyclohexyl ester form. Tos is used to protect the guanidino group of Arg, and 2-C I-Z is used as a protecting group for the Lys side chain.

Three equivalents of BOC protecting group amino acid and three equivalents of DIC are commonly used in DCM. Boc-Arg(Tos)
or when Boc-Lys (2-c 1-2) is coupled, 50% DMF/DCM is used.
B oc-A 1 a SB oc-lie%B
oc-Leu and Boc-Nle are coupled via a symmetric anhydride, and the HOBt ester method
Used for coupling c-Gln and Boc-Asn.

The following compound is obtained.

3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3(X3)-Ala,s-11a5-
Phca-Thry (X')-Asn s-Sc
r9(X')-Tyr to(X10)-Arg+
+(X”)-Lys 12 (X 12)-Val +
i-LOu+4-Gly+5-Gln+6-LOu17
-SOr+a(X')-Aja 19-Arg2o(
X")-L)'S 21 (X 12)-Lcu 22
-Lcu23-Gln24-ASI)2s (X 3)
-110 26-NIO27-Scr2a(X')-
Ni1-<CH2) 4-NH-C (NH2
)-N-802-(0>OCII 2-
OCII 2 -CO- [SP CO. [Similarly, in the formula,
X3 represents 0-cyclohexyl ester, X7 represents 0-benzyl ether, XIO represents o-2.6-ClllBz ether, Xll represents tosyl and Xl2 represents 2-Cj)-z. Ko.

To cleave and deprotect the protected peptide-resin, 18 m-
It was treated with 1 ml of cresol and 10 ml of hydrogen fluoride (HF). After removing the HF under high vacuum, the peptide-resin residue is washed with dry diethyl ether and ethyl acetate. Then, the peptides were extracted with 50% acetic acid water.
The resin is filtered off and lyophilized.

The crude peptide is manufactured by Beckmann Brepp-35021 H.
PLC system (Beckman Type 163 with variable wavelength UV detector) or Beckman HPLC
System (Beckman 420 gradient controller, 24114M solvent delivery modulus, 1
65 variable wavelength detector and a Kip and Sonnen BD41 recorder). In the preparation system, the separation was carried out using spherical C18 silica gel (300A
41.5X150 filled with pore size, 12μm particle size)
A mm DYNAMAX column (Lynin Inc., Hoburn, Mass.) was used.

In a semi-preparative system, purification is performed using VYDAC (vYDAC).
) C18 reverse phase column (10X250mm, 300A pore size, 5μm particle size).

A gradient eluent is used. Solvent A is 0.1% aqueous TFA. and solvent B consisted of 0.1% TFA in 70% aqueous acetonitrile. Column elution is 220nm
and monitored at 280 nm.

Veptide is Hewlett-Packard HP-1090
Substantially (>95%) purity is determined using i&body chromatography. Peptide is 4.6X25
A gradient (40- to 30-
70%B) A mixture of solvent A and solvent B was added to 1. 2ml/
The mixture was chromatographed at a speed of 1 minute. hGHRH
A similar compound has an HPLC retention time of 20.1 minutes.

Example 9 A method for synthesizing an hGHRH analog compound represented by the following formula.

H-Tyr-D-Ala2-Asp3-Al
a,s -I1c5 -Phc6 -1"hr7 -A
snB-Scrg-Tyr1o-Arg1t-Ly
stz-Val+s-Lcut 4-GIVt s-G
ln16-Lcut 7-Scr l. -Ala19
-Arg2u-LyS2 1-Lcu2 2-LOu2
3-G I n24-ASII) 2s-1 102 b
-NIOz7-S(3r28-NH-(Cll2
) 4-NH-C(NH2)-NH to the appropriate Pmoc-NH- according to the methods in Schedules C and D below.
(Cll2)4-Nil-C(NII
2)-N-SO2-<0>(Ms)-0CII
It was synthesized stepwise from 2-CO-[SP] using a Beckman 990 synthesizer.

Deprotection is carried out according to Schedule C below.

DCM washing (2 times) DMF washing (2@) Deprotection 3: 20% viveridine in DMF i:
20% Biperidine in DMF DMF washes (2x) Dioxane/water (2:1) washes (2@) DMF washes (2x)
@) Coupling will be performed according to the following Schedule D.

l. Force/1 ring: DCM or DCM/DMF 11) Fmoc-7 mi/1 (4-6 equivalent) to DIC (2
-3 equivalent l) 2. DMF washing 1) (2@) 3. MeOH & Purification (2nd item) 4. 0CM wash (3@) Simply put, 4-6 equivalents of Fmoc-protected amino acids and 2
-3 equivalents of DIC will react in DCM or DCM/DMF, depending on the solubility of the particular protected amino acid.

T-butyl ether is used as a hydroxyl side chain protecting group for Ser, Thr and Tyr. BOC is
used as a protecting group for the Lys side chain and the amino group of Tyr, 4-Me o-2.3.6-tribenzenesulfonyl was used for the protection of the guanidino group of Arg,
The Asp side chain carboxy group is protected with t-butyl ester. Tyr is optionally protected with t-butyl ether.

The following compound is obtained.

X1-Tyr+ (XIO)-D-Ala2-As
p3(X3)-Ala4-1105-Phca-
Thr7(X7)-Asna(X')-Scr9
(X7)-Tyr to(X")-Arg t+(X
”)-Lys 12(X 12)-Val 13-LO
U+4-Gly+s-GIn+6-LOut7-SOr
t8(X')-Ala t9-Arg2o(X")-
LVS 21 (X 12)-Lcu 22-LOu2
3-Gln24-ASI)25(X3)-11c2
6-NIO27-Scrzs(X')-NH-(C
ll2) 4-NH-C-(NH2)
-N-802 -(0)(Ms)-0CII 2
-CO-[Sp] [carrier, where X3 is t-butyl ester, X7 is t-butyl ether, X10 is t-butyl ester, x'' is 4-methoxy-2.3.6-trimethylbenzylsulfonyl, And X1 and Xl2 are B
Represents oc. ] The protected peptide produced as described above is used as a starting material. Cleavage of the bebutide from the solid support and removal of the side chain protecting groups was carried out simultaneously by reaction of the protected peptide resin with TFA-thioanisole (5%) at a temperature of 25-30 °C for 2-3 hours (resin 10 per duram in the valleys
ml of liquid was used). After the reaction was completed, the mulch was removed under vacuum at room temperature. The residual material was first extracted with ether (to remove non-polar by-products) and the crude bebutide was extracted with 50%
The filter cake was extracted with acetic acid and the filtrate was lyophilized.

Example 10 Synthesis of the following hGHRH-like compound 3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3-Ala4-lie5-Phe6-Th
ry-Asnl3-Serg-TYrs a-Ar
g 1I-D-LYS Iz-Val! q-Ll3
ul4-Alat 9-Gin l 6-Lcut t
-Scr s s-A fat 9-Arg2o-Ly
s21-Lcu22-Leu2t-GInz4-A
Sp25-1 1(3z6-N1027-Ser2s-
NH-(Cll2) 4NH-C-(NH2)-N
II is Bo instead of Boc-Lys (2-cl-Z)
A protected bebutide resin of the following formula was obtained by following the method of Example 8, except that c-D-Lys (2-c 1-Z) was volatilized to the 21st position.

3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3(OChx)-Ala4-11c)-
Phca-1'hr7(Bzl)-ASl18-Sc
rg (B!1) −'ryr+o(DCB) −Ar
g++(Tos) 1D- Lys 1z(2-CI-
Z)-Va113-Lcu14-Ala15-Glnt
c, -Lcut7-Sorts (B!1) 1^1a19
-Arg2o(Tos) -Lys2, (2-cl-1
)-LOu2z-LOu23-G I n24-As
p2s (Och X )-l 1 026-N 1
027-Ser2s(Bxl) -N}I-(Cll
2) 4-NH-C-(NH2)-N-[8
1)A]-{81)] which is then converted to the desired peptide according to the method of Example 9 in its backward fashion. hGHR obtained in this way
The HPLC retention time of the H analogue compound is 19.2 minutes.

Example 11 A method for synthesizing an hGHRH analogue compound of the following formula.

3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3-Alas-11a5-Phca-Th
ry -Asns -Ser9-T)' r 1t+-
Arg 11-LyS 12-Val l 3-Lcu
14-AIa+ 5-Glnt b-L. 00 1y-
Sar l s-A fat q-Arg2o-D-L
ys21-Lcu22-LOLJ2 1-Gln24-
ASp25-1 10z6-Nle27-SOr28-
Nil-(Cll2)4-N11-C-(N
il 2 )-NH is Boc-Lys (2-c 1 -Z) at position 21
Boc -D-Lys (2-c 1-Z) instead of
, was carried out according to the method of Example 8, except for inserting ,
A protected peptide resin of the following formula was obtained.

3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3 (OChx)-A1a4-11e5
-Phea -Thr7 -(Bzl)-Asna-S
er9 (B!1) -Tyrto (DCB) -Arg
t, (Tos) 1 t, YS12 (2-c I- 1
)-Va I 1x-LeU14-Alat s-G
lnt b-L(3u17-Serts(Bxl)-
Alat, -Arg2o(Tos)-D-Lys2t
(2-cl-2)-Leuz z-Leu23-G l
n24-AS p2 s (OChx)-1 IC!
2 b-N I e27-Ser2s(Bzl) -N
i1-(Cl+2)4-NH-C(NI12
).NIl-CSPA]-[S] This is then similarly converted to the given bebutide according to the method of Example 8. hG thus obtained}{
What is the HPLC retention time of RHII-like compounds? It is 9.6 minutes.

Example 12 A method for synthesizing an hGHRH analogue compound of the following formula.

3-(4-hydroxyphenyl)probionyl-Ala
2-ASp3-AIa4-1105-Phc6-
1'hr7 -AsrtB -Scrg-Tyr l
o-Arg11-D-Lys t 2-Val t 3
-LOu14-Alat s-Glnt b-Lcu
l ■-Scr L 8-A l a 15-Arg2
o-D-Lys 2 , -LOuz z-LOuz 3
-Gln24-ASp2s-1 10■6-. N102
7-SOr2g-(CII2)4-NH1C-(
NH2)-NH is Boc-Lys (2-cl
-Z) instead of Boc -D-Lys (2 -cl
-Z) was synthesized according to the method of Example 8, except that -Z) was inserted, and a protected peptide resin of the following formula was obtained.

3-(4-hydroxyphenyl)probionyl-Al1
2-ASp3 (OChx)-AIa4-11e5-
Phca-Thr7(Bzl)-Asn a-Scr9
(Bzl)-Tyrto(DCB)-Arg t+(T
os)-D-Lys +2(2-el-Z)-Val+
3-LOu14-Ala15-Glnt6-LCU17
-Scru+(Bzl)-Ala +9-Argzo(
Tos)-D-Lys. (2-cl-z)-Lcu2
2-Lcu2 3-G. l nz4-Asp29 (O
Chx)-1 jc26-N 102 7-SOr2a
(Bzl)-Nil-(Cll2)4-Nil-C
(NI+2)-N-[SPA]-[SI)] This is then similarly converted to the desired peptide according to the method of Example 8. The HPLC retention time of the obtained hGHRH analog compound is 17.9 minutes.

Example 13 A method for synthesizing the following hGHRH-like compound.

3-(4-hydroxyphenyl)propionyl-Al8
2-ASI)3-Ala,s-1105-Ph
ea -Thrv -Asns -Scr9-Tyr
l o-Arg t t-D-Lys 12-Val
13-LOut a-A fat s-GIn+ b-
Lcul 7-Scr l s-A Ia 19-A
rg2o=Lys21-D-Lcu22-LOuz
31 Gln24-^Sp2s-l 1026-N10
27-SOr2a-NH-(Cll2)4-N
il-C-(NH)2-NH is Boc at position 22
-Boc-D-Leu was inserted instead of Leu, and Boc-D-Lys (2-c 1-Z) was inserted instead of Boc-Lys (2-CI-Z) at position 12. , was synthesized according to Example 8 to obtain a protected peptide-resin of the following formula.

3-(4-hydroxyphenyl)propionyl-Ala
2-ASI)3 (OChx)-Ala 4-1
105-Phca-Thr7(Bzl)-Asne
-Scr9(Bzl)-Tyr to(DCB)-Ar
g 17(Tos)-D-Lys +z(2-CI-
Z)-Vall3-Lcu14-Ala+5-Glnt
6-LOu+7-Scrts(Bzl)-Ala 19
-Arg2o(1'os)-L)'S 2, (2-el
-z)-D-Lcuz 2-Leu2 3-G Inz
4-ASp25(OChx)-+102 b-N 10
27-Ser28(Bzl)-Nil-(Cll2)
4-NH-C(NH2)-N-[S1)Aco[Sl)] This is then similarly converted to the given bebutide according to the method of Example 8. The HPLC retention time of the obtained hGHRH-like compound is 17.9 minutes. .

Example 14 A method for synthesizing the following hGHRH-like compound.

3-(4-hydroxyphenyl)propionyl-Ala
2-Asp3-AIa4-11cb-Phca-Th
r7-Asna-Ser9-Tyr 1o-Argt
t-D-Lys l z-Vat I q-Lcu1
4-A1a+ q-GIn+ 6-Lcul t-S
cr t s-A l a1 9-Arg2o-Lys
2 t-Lcu2 z-D-Lcu21-Gln24
-ASp2s-1 102s-N1027-SOr2a
-Nil-(Cll2)4-NH-C-(NH
) 2 -Ni1? , insert Boc-D-Leu in place of Boc-Leu at position 23, and insert Boc-D-Leu at position 12.
Boc-D instead of oc-LVS (2-CI-Z)
-Lys (2-c 1-Z) was synthesized according to Example 8, except that (2-c 1-Z) was suppressed to obtain a protected peptide resin represented by the following formula.

3-(4-hydroxyphenyl)propionyl-Ala
2. -Asp3(OChx)-Ala 4 -1105
-Phca-Thr7(Bzl)-Asn a-Sc
r9(Bzl)-'l'yr to(DCB)-Arg
17(Tos)-D-Lys +z(2-CI-2)
-Val13-Lcu14-Alat, -Glnt6-
Lcut7-Scrta(Bzl)-Ala l9-A
rgzo(Tos)-Lys zt(2-el-Z)-
LOU2 ■-D-LOu23-G l 12 4-A
SI) 2 5 (OChx)-l 102 6-N
1021-SOr2s(BZI)-Nil-(Cll
2) 4-Nil-C(Nl+)2-N-[S
PA]-[SI)] This is then similarly converted to the given bebutide according to the method of Example 8. The HPLC retention time of the obtained hGHRH analog compound is 17.6 minutes.

Example 15 A method for synthesizing the following hGHRH-like compound.

? -Carboxybenzoyl-Gly-Ala2-Asp
3-Ala4-1105-Phca-Thr7-A
sna-Scr9-Tyrto-Arg++-D-Ly
s t 2-Val 1 3-LOuta-Alat
5-Gln16-Lout 7-Scr l s-A
Ial9-Argzo-D-LyS2, -LO
(72 2-LO(72 3-G I n24-ASp
25-I fe26-NI027-Serzs-NH-
(Cll2)4-NH-C-(NH)2
-Nl1 is Boc-Lys (2-Z 1c
Boc-D-Lys (2-Z -cl
), and then Boc-Gly replaces 3-(4-hydroxyphenyl)propionyl with the N-terminus and Gl)
/ residue was synthesized according to Example 8, except that it was reacted with phthalic anhydride after Boc group removal, to obtain a protected peptide resin represented by the following formula.

2-Carboxybenzoyl-Gly-AIa2-Asp
:+ (QChx)-Ala 4 -1105 -P
hce-Thr7(Bzl)-Asna-Scr9
(Bzl)-Tyr to (DCB)-Arg 1t
(Tos)-D-Lys 12(2-CI-z)-Va
l s 3-Lcu+ a-Ala+ s-Gln+
6-Lcu12-Scr l 8 (Bzl)-Ala
t9=Argzo(Tos)-D-Lys 2t(2
-cl-z)-Lcu22-LOu23-Gl n24
-ASI)25 (OChx)-1 1026-N 1
02 7-SOr2s (Bzl?-Nil-(CI+
2) 4-Nil-C(Ni1)2
-N-[SPAco[SP] This is then similarly converted to the predetermined peptide according to the method of Example 8. The HPLC retention time of the obtained hGHRH-like compound was 15
, 5 minutes.

Example 16 A method for synthesizing the following hGHRH-like compound.

Glutaryl-D-Ala2-Asp3-AIa4-11
cb-Phca-1”hr7-Asna-Ser9-
Tyr+o-Argt+-D-Lyst2-Val13
-LOU14-A fat q-G ln 1c+-
Lcut ■-Scr t a-A fat ,-A
rgz*-D-Lys 2 1-Leu2z-Leuz
3-G I n z4-ASp2s-1+02 b-
N1027-Serzs-NH-(Cll2)
4-NH-C-(NH)2-Ni1 was prepared by inserting Boc-D-Ala in place of Boc-Ala at the 2-position, removing the BOC group, and then reacting with glutaric anhydride. A protected peptide resin represented by the following formula was synthesized according to Example 8.

glutaryruo-AIa2-ASp3 (OChx
)-Ala4-lie5-Phca-Thr7(
Bzl)-Asn B-Scrg (Bzl)-Ty
r to(DCB)-Arg t+(Tos)-D-L
ys t2(2-CI-z)-Val+q-Lcu+4
-AIa+q-Gln+6-Leu+7-Ser+s(
BZI)-All +9-Arg2t+(Tos)-D
-Lys 2+ (2-cl-z)-Leu22-Lcu
z3-Gln24-Asp25(OChX)-Ilc2
6-Nlcz7-Scr2s(Bzl)-Nil-(C
II2) 4-NH-C(Nl1)2”N-
CSPA]-[SP is then similarly converted to the desired peptide according to the method of Example 8. Obtained h
The HPLC retention time of the GHRH analog is 19.4 minutes.

Testing Methods Compounds of the invention were tested both in vitro and in vivo. In vitro growth hormone releasing activity is summarized in Table 1. The in vitro effects of the compounds on GH release in rats after intravenous (i.v.) and subcutaneous (SC) injections were determined in the second phase, respectively. 3. 4. It is shown in Table 5.

Example 17 In vitro growth hormone-releasing activity was assayed using a hyperfused rat hypogonial somatic cell line (S. Vighand
A. V. Sehally. Pcptidcs 5. Su
ppl. :241-347.1984) 2 each peptide,
hGH-RH (1-29)NH2 (as a control) and hGHRH analogues of the invention were administered over 3 minutes (1 ml perfusion) at various concentrations as described below. The GH content of the collected lml fractions was determined by RIA.

Table 1 h for GH release in hyperfused rat pituitary cell line
In vitro effects of GHRH-like compounds Peptide test conditions hGIIRII (1-29)N
I21 potency against 2 (nM) (-1) *hG111? II (1-29) NH22 and io (control) Example 8 0.02 0.1 (sample) Example 10 0.1 0.5 (sample) Example 11 0.1 0.5 (sample) Example 12 0.1 0.5 (sample) Example 14 0.2 1.0 (sample) *. 1.0 by 4-point test method 254 73.5 33.3 34.2 34.8 Example 18 Using adult female rats, bentobarbital (5
mg/100 g. b. w. ) anesthesia, i.e. intraperitoneal injection, and 30 min after bentobarbital injection, blood samples were taken from the jugular vein (pre-treated levels) and immediately hG H - R H (1-29)N
I12 (as a control) or hGH-R
H(1-29)NII2 analogue compound was injected intravenously.

Blood samples were taken from the jugular vein 5,15.30 minutes after injection. Blood samples were centrifuged to remove plasma and GH levels were measured by RIA.

The results are shown in Table 2 and summarized in Table 3.

Table 2 hGH on GH release in rats after intravenous (iv) injection
In Vivo Effects of RH Analog Compounds Table 3 hGI calculated from data in Table 2 by 4-point assay
II? II(1-29) Efficacy of hGHRH analogs of Examples 8.10 and 11 against NH2(-1) hGIIRl1(1-29) 2.5 88+12
．． INI+2 (control) 1.0 70±11.
9421+29.9 225±29.8 112+IO. 8 103±13.4 60+8.6 79±9.3 Example 8 0.25 72±11.0 643
±88.6 241±21.0 108±18.9
(Sincerity fee) 0.183±11.3 103±38.0
146±24.3 92±11.0 Example 10
0.25 76±10.4 599±81.5
180±13.5 71+lO. 6hGIIRI
1 (1-29) 2.5 79±16.5Nlb
(Control > 1.0 56±l1.2274±23
．． 6 164±14.6 58±10.7 23±4.4 4l±7.5 23±U. t Example 110.560±9.0 485±70.4
121±29.6 26±3.8hGllRI
1 (1-29) 2.5 84±15NII2 (control) 1.0 57±12.2269±32.4 154±7.4 87±25.2 42±l3 85±19.8 73±36 Example 12 0.25 61±8.5 377
±50.2 73±8.440±16.2 hGIIRII Ifl compound 11! i hour (minutes later)
Efficacy (mln.) 5 15.16 15 20.96 5 13.4g 15 213.92 5 10.62 15 12.44 5 16.68 Example 8 (Sample) Example 10 (Sample) Example 11 (Metal Material) Example 12 (Wipe material) *Reliability limit effectiveness ratio* (10.52-21.87) (12.74-33.46) (10.38-17.51) (8.50-85.21 ) (5.92-19.07) (3.51-44.06) (11.40-24.40) *Mean ± SEM Example 19 Adult female rats were used and bentobarbital (5 m
g/100 g. b. v. ) anesthesia, i.e. intraperitoneal injection, and 30 min after bentobarbital injection, blood samples were taken from the jugular vein (pre-treated levels) and immediately hGH-RH(1-29)NI
12 (as a control) or hGH-RH analogues were injected intravenously. Blood samples were taken at 15.30 post-injection.
Samples were taken from the condylar vein at 60 minutes. Blood samples were centrifuged to remove plasma and GH levels were determined by RIA.
was measured.

The mucilage is shown in Table 4 and summarized in Table 5.

Claims (23)

[Claims] (1) A polypeptide having a structure represented by the following formula I and a pharmaceutically acceptable salt adduct thereof having a pharmaceutically acceptable organic or inorganic base and an organic or inorganic acid. Q^1-CO-R_2-R_3-Ala_4-Ile_
5-Phe_6-Thr_7-R_8-Ser_9-R
_1_0-Arg_1_-R_1_2-R_1_3-R
_1_4-R_1_5-Gln_1_6-R_1_7-
R_1_8-Ala_1_9-Arg_2_0-R_2
_1-R_2_2-R_2_3-R_2_4-R_2_
5-Ile_2_6-R_2_7-R_2_8-NH-
Q^2(I) [However, Q^1 is 3-carboxypropyl, 2-carboxybenzamidomethyl, or general formula II ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (However, X=OH, Y= H, OH, NH_2 or CH_3CONH, m=1 or 2 n=0, 1 or 2) Omega or α-omega substituted alkyl, R_2
is Ala or D-Ala, R_3 is Asp, D-Asp, Glu or D-Glu
, R_8 is Asn, D-Asn, Ser or D-Ser
, R_1_0 is Tyr or D-Tyr, R_1_2 is Lys, D-Lys, Arg, Orn, D
-Arg or D-Orn, R_1_3 is Val or Hele, R_1_4 is Leu or D-Leu, R_1_5 is Ala, R_1_7 is Leu or D-Leu, R_1_8 is Tyr or Ser, R_2_1 is Lys, D-Lys, Orn, D-Orn
, Arg or D-Arg, R_2_1 is Leu, D-Leu or Ala, R_2
_3 is Leu or D-Leu, R_2_4 is Gln or His, R_2_5 is Asp, D-Asp, Glu or D-G
lu, R_2_7 is Met, D-Met, Ala, Nle, I
le, Val, NVa or Leu, R_2_8 is Asn or Ser, Q^2 is a lower omega-guanidino-alkyl group having ▲mathematical formula, chemical formula, table, etc.▼(III) (however, in the formula, p=2~ 6). ] (2) The compound according to claim 1, wherein p=4. (3) Q^1 is 2-(4-hydroxyphenyl)ethyl, R_2 and R_1_5 are Ala, and R_2_7
The compound according to claim 2, wherein is Nle. (4) The compound according to claim 2, wherein R_1_2 is D-Lys. (5) The compound according to claim 3, wherein R_1 and R_2_1 are D-Lys. (6) The compound according to claim 4, wherein R_2_2 is D-Leu. (7) The compound according to claim 4, wherein R_2_3 is D-Leu. (8) The compound according to claim 5, wherein Q^1 is 2-carboxybenzamidomethyl. (9) Claim 1 consisting of the following series of steps:
A method for immobilizing the Q^2 segment of the NH_2-Q^2 moiety defined in Section 1 on a support phase [SP] made of an amino resin,
react with Q^2 to form Boc-NH-Q^2,
and -5 in water-soluble ether in the presence of aqueous alkali.
At a temperature of ~10°C, the Boc-NH-Q^2 and the following formula Hal-SO_2-<O>-O-CH_2-COO
H (However, in the formula, Hal represents chlorine or bromine.)
The support phase [S] is reacted with the arylsulfonyl halide represented by
Boc-NH-Q^2-SO_2-<O>-O-CH_
The corresponding Boc-NH-Q^2-SO_2-, which forms 2-CO-[SP] (wherein Q^2 represents ▲mathematical formula, chemical formula, table, etc.▼(IV)). <O>-OCH_2
-forming COOH. (10) Support phase [SP] is Boc-NH-Q^2-SO_2-<O>-O-CH_
In order to generate 2-CO-NH-CH_2-<O>-[Pm] (wherein, [Pm] represents the polymerized part of the support phase), the following formula H_2N-CH_2-<O> -[Pm] The method according to claim 9, wherein the method is selected from the following amino resins. (11) A method for immobilizing the -NH-Q^2 moiety defined in claim 1, comprising the following series of steps, on a support phase [SP] consisting of an amino resin: - In the presence of a base, Benzyloxycarbonyl chloride and NH_2-Q^2
・In the presence of a strong aqueous base, the above-mentioned Cbz-NH-Q^2 and the following formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the formula , Hal is chlorine or bromine, M is 1 carbon number
-5 lower alkyl or lower alkoxy, and S=
Represents 1-3. ) is reacted with substituted arylsulfonyl chloride, and the Cbz group is removed by catalytic hydrogenation reaction to produce ▲Numerical formula, chemical formula, table, etc.▼, and in the presence of a base, this product and 9-fluorenyl By reacting with methoxycarbonyl chloride, ▲mathematical formula, chemical formula,
There are tables, etc. ▼ (However, in the formula, S = 1-3) is produced, and - This product is reacted with the support phase [SP] ▲ There are mathematical formulas, chemical formulas, tables, etc. To generate ▼. (12) Support phase [SP] has ▲ mathematical formula, chemical formula, table, etc. ▼ (However, in the formula, [Pm] represents the polymerization part of the support phase.) In order to generate the following formula H_2N- 12. The method according to claim 11, wherein the amino resin is CH_2-<O>-[Pm]. (13) Next structure ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, in the formula, [PeP] is the next structure Q^1-CO-R_2-R_3-Ala_4-Ile_
5-Phe_6-Thr_7-R_8-Ser_9-R
_1_0-Arg_1_1-R_1_2-R_1_3-
R_1_4-R_1_5-Gln_1_6-R_1_7
-R_1_8-Ala_1_9-Arg_2_0-R_
2_1-R_2_2-R_2_3-R_2_4-R_2
_5-Ile_2_6-R_2_7-R_2_8-NH
-Q^2 M is methyl or methoxy, S is 1-3, [PR] is t
- Represents a side chain carboxyl group protected by a butyl ester group, the phenolic and alcoholic side chains are trifluoroacetic acid sensitive groups selected from t-butyl ether, the amino group is t-butoxycarbonyl, and the guanidino side The chain is protected by a substituted benzenesphonyl group, Q^1 is a 3-carboxypropyl or 2-carboxybenzamidomethyl group, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [However, X=OH, Y=H, OH, NH_2 or CH_3CONH, m=1 or 2 n=0, 1 or 2. ), omega or α-omega substituted alkyl of the structure, R_2
is Ala or D-Ala, R_3 is Asp, D-Asp, Glu or D-Glu
, R_8 is Asn, D-Asn, Ser or D-Ser
, R_1_0 is Tyr or D-Tyr, R_1_2 is Lys, D-Lys, Arg, Orn, D
-Arg or D-Orn, R_1_3 is Val or Hele, R_1_4 is Leu or D-Leu, R_1_5 is Ala, R_1_7 is Leu or D-Leu, R_1_8 is Tyr or Ser, R_2_1 is Lys, D-Lys, Orn. , D-Orn
, Arg or D-Arg, R_2_2 is Leu, D-Leu or Ala, R_2
_3 is Leu or D-Leu, R_2_4 is Gln or His, R_2_5 is Asp, D-Asp, Glu or D-G
lu, R_2_7 is Nle, R_2_8 is Asn or Ser, and Q^2' is a lower omega-guanidino having the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, in the formula, p is 2 to 6.) - represents an alkyl group. ) A support phase consisting of an amino resin [S
A method of cleaving the protected peptide [PR][PeP] attached to [P]. (14) The support phase [SP] is selected from amino resins of the following formula H_2N-CH_2-<O>-[Pm] (wherein, [Pm] represents the polymerized part of the support phase). 14. The method according to claim 13. (15) The method according to claim 13, wherein P is 4. (16) Next structure ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (However, in the formula, [PeP] is the next structure Q^1-CO-R_2-R_3-Ala_4-Ile_
5-Phe_6-Thr_7-R_8-Ser_9-R
_1_0-Arg_1_1-R_1_2-R_1_3-
R_1_4-R_1_5-Gln_1_6-R_1_7
-R_1_8-Ala_1_9-Arg_2_0-R_
2_1-R_2_2-R_2_3-R_2_4-R_2
_5-Ile_2_6-R_2_7-R_2_8-NH
-Q^2 M is hydrogen, [PR] represents a side chain carboxyl group protected with a benzylic ester group or a cycloalkyl ester group, and its phenolic and alcoholic side chains are a group consisting of benzylic or substituted benzylic ethers. trifluoroacetic acid resistant group selected from, the amino side group is protected with a benzyloxycarbonyl or halobenzyloxycarbonyl group, the guanidino side chain is protected with a p-toluenesulfonyl group, and Q_1 is 3-carboxypropyl or 2-carboxybenzamide. Methyl group, following formula ▲ mathematical formula,
There are chemical formulas, tables, etc.▼ [However, X=OH, Y=H, OH, NH_2 or CH_3CONH, m=1 or 2, n=0, 1 or 2. ) Omega or α-omega substituted alkyl of the structure R_2
is Ala or D-Ala, R_3 is Asp, D-Asp, Glu or D-Glu
, R_8 is Asn, D-Asn, Ser or D-Ser
, R_1_0 is Tyr or D-Tyr, R_1_2 is Lys, D-Lys, Arg, Orn, D
-Arg or D-Orn, R_1_3 is Val or He, R_1_4 is Leu or D-Leu, R_1_5 is N-methyl-Gly or Ala, R_1
_7 is Leu or D-Leu, R_1_8 is Tyr or Ser, R_2_1 is Lys, D-Lys, Orn, D-Orn
, Arg or D-Arg, R_2_2 is Leu, D-Leu or Ala, R_2
_3 is Leu or D-Leu, R_2_4 is Gln or His, R_2_5 is Asp, D-Asp, Glu or D-G
lu, R_2_7 is Nle, R_2_8 is Asn or Ser, and Q^2' is a lower omega guanidinoalkyl having the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, in the formula, p is 2 to 6.) represents a group. ) A support phase selected from amino resins [S
A method of cleaving the protected peptide [PR][PeP] attached to [P]. (17) The method according to claim 16, wherein P is 4. (18) A method for increasing milk production in a female milk-producing mammal, comprising administering to the female milk-producing mammal a production-increasing dose of the compound according to claim 1 in a physiologically acceptable carrier. . (19) Claim 18, wherein the form of administration is injection.
The method described in section. (20) The method according to claim 19, wherein the mode of administration is S, C or i, m injection. (21) The method according to claim 19, wherein the frequency of administration is 1 to 4 injections per day. (22) The dosage is 0.2 to 2.0% per kg of body weight per injection.
19. The method according to claim 18, wherein the amount is 0 μg. (23) The method according to claim 18, wherein the mammal is a cow.