JPS62190198A - Peptide derivative - Google Patents

Abstract

NEW MATERIAL:An analogue (GHRH-4) of growth hormone-releasing hormone (GHRH) expressed by the formula [Agm represents agmatine or residue of the formula H2N(CH2)4NHC(=NH)NH2]. USE:A remedy and diagnosticum for puerilism or auxetic for an animal. PREPARATION:For example, plural fragments of intermediate for GHRH-A are synthesized by step-wire elongation method of peptide and bonded with each other. First the formula Boc-Ser(Bzl)-OSu (Boc represents t-butoxycarbonyl; Bzl represents benzyl; OSu represents N-hydroxysuccinimide ester) is reacted with Agm.H2SO4, and amino acids are successively bonded according to a peptide sequence. On the other hand, a compound expressed by the formula Boc-Gln-OH is used as a raw material and amino acids are successively bonded therewith according to the peptide sequence to obtain an intermediate. Then these intermediates are bonded to obtain the compound expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a peptide derivative that is effective as a therapeutic agent for pediatric diseases, a gli agent for diagnosing pediatric diseases, and a growth agent for animals.

Prior Art Since the structure of growth hormone-releasing hormone (GHRH) was elucidated, various G)-IR) (analogues with stronger activity or, conversely, antagonistic effects) have been synthesized. , conventional GHRH is human GHRH (1-44)-NH2 represented by the following structural formula: Tyrl -A 1a-ASD-A la -11e
-Phe -Thr-ASn-Ser -Tyr-A
ro-Lys-Val-1eu-Gly-Gln-l
eu-8er-Ala-ArO-iys-1-eu −
1-eu-Q In-ASp-11e-Met-3
er-A rg-GIn-GIn-(:, ly
-Glu -Ser-A sn-G ln-G ln
-A rQ-G l'/-A Ia-A rO-A l
As typified by a-ArlJ-Leu44-NH2, it has a long peptide chain, so the synthesis method is complicated and requires a lot of time and effort. In addition, in terms of GH release activity, no material with sufficiently satisfactory activity has been obtained.

The problem that the invention aims to solve is that the peptide chain is short and can be synthesized relatively easily.
It is desired to develop a human GHRH analog with high GH release activity and excellent stability and sustainability.

Means for Solving the Problems In view of the above-mentioned problems, the inventors of the present invention have conducted intensive research and found that the peptide chain is shorter than that of human GHRH, the G)-1 releasing activity is high, and the stability and sustainability are improved. human GH with
An RH(1-,29) analog was discovered. That is,
Human GHRH (1-29
) analog (hereinafter referred to as GHRH-A) is represented by the following structural formula.

Tyrl-A 1a-ASD-A la -1le -
Phe -Thr-8sn-Ser-Tyr-
A r(1-L ys -V at -1eu -G
ly -G In -1eu -3er -A Ia
-A rg -Lys-Leu -L eu-Gln-
ASD-11e-Nle-3er-A G111
29 In the formula, AQmG; IL Agmadine H2N (CH2)4
NH (=NH) represents an NH2 residue.

Amino acids constituting GHRH-A of the present invention include glycine (Gly), alanine (Ala), serine (Ser),
Threonine (Thr>, Valine (Vat>), Leucine (
Chain amino acids such as LeU) and isoleucine (I le), aromatic amino acids such as phenylalan (Phe) and tyrosine (Tyr), acidic amino acids such as aspartic acid (ASn) and glutamine (Gln), and arginine (Arg).
, lysine (Lys), etc., as well as norleucine (Nle), an amino acid analog, and agmatine (Nle), a carboxyl form of arginine.
Aam). Among the amino acids that make up the above peptide, A! It is desirable that a2 is D-integrated.

To synthesize the human GHRH-A of the present invention, the stepwise elongation method commonly used in conventional peptide synthesis as well as the protection and removal of the generally used amine LL carboxyl group and side chain functional group,
This can be easily carried out by using a fragment condensation method or by appropriately combining these methods.

Protecting groups for amino groups and side chain functional groups used in the present invention include trityl (Trt-), benzoyl (
BZ-), benzyl (BZI-), tosyl (TOS-)
, benzyloxycarbonyl (Z-), t-butoxycarbonyl (3oc-), o-chlorobenzyloxycarbonyl<Ct Z-), 0-bromobenzyloxycarbonyl (Br Z-) and other commonly used protective groups. However, it is preferable to use 3oc-1Z- and the like because it is easy to introduce and remove a protecting group from an amino group. In addition, as protecting groups for side chain functional groups, 3er, Tyr, A
Preferably, BZI-1Br Z-1Tos-5CI Z-m is used as the protecting group for the side chain functional groups of rg and LyS, respectively.

In addition, the carboxyl group protection methods used in the present invention include t-butyl ester (-OBu), benzyl ester (-0Bzl), ethyl ester (-〇Et), D-nitrophenyl ester (-ONI)).
, phenacyl ester (-〇pac), N-hydroxysuccinimide ester (-0SII>, etc.), and other commonly used protecting groups. -0Bzl
and -ester formation with PacJ3 is preferred.

The method of protection using these protecting groups or the method of removing the protecting group can be carried out using conventional means commonly used in the synthesis of peptides.

Thus, for example, intermediate synthesis B of GHRH-A of the invention
Synthesis of oc-Gln16-Agm29 (Tos)
Le1. : t, t, Boc-3er (BZI)-0S
Using u as a starting material, the guanidino group of soc-ser-Agm obtained by reaction with A(IIHzSO4) was protected with TosCl, and then the α-amino group and, if necessary, the side chain functional group were protected. 13oc-N le-DC
HA, Ile.

, A, 5p(OBZI) with these active esters or H
It is reacted with a condensing agent such as dicyclohexylcarbodiimide, 1·-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC■) in the presence of OBt to form a fragment (A>BOC-ASI) (0BZI)-Ile- Nle-
3er(Bzl) -AgIIl(TOS> ・
...(Increase A> by 1.

On the other hand, using Boc-131n-Ql-1 as a raw material, paC
After reaction with -3r to form Boc-Gln-OPac,
Sequentially, 3oc-1eu-, 1eu-, L VS(CI
Z).

Arg(Tos), Ala, 3er(Bzl)
, leu and Qln were reacted, and Zn/ACOH
The fragments as carboxyl components (
B) 13oc-Gln-1eu-3er-(Bzl)
-AIa -Aro-(Tos) -Lys(CI
Z) -Leu-Leu -Gln-OH...
(B) Obtain.

The resulting protected peptide, i.e. fragment (A
) was deprotected by treatment with trifluoroacetic acid (TFA)/HCI, and then combined with fragment (B) in the presence of a condensing agent to form 3oc-Q 1n1-1eu17-
----3er28([3zl) -Aom2" (
Tos) 'E: Combined R.

When using an active ester in the above reaction, p-nitrophenyl ester, N-hydroxysuccinimide ester, etc. can be cited as examples. In addition, as a solvent, dioxane, dimethylformamide (DMF
>, butanol (Bu OH), methanol (Me
Those commonly used for peptide synthesis, such as OH), can be suitably used. The reaction proceeds at v temperature, but the reaction can be accelerated by heating if desired.

To isolate GHRH-A from the reaction mixture, for example, the reaction mixture is evaporated to dryness, the residue is purified by column chromatography, and then freeze-dried.

Note that GHRH-A may be in the form of an acid addition salt, and the acid in this case may be a mineral acid such as hydrochloric acid or sulfuric acid, or an organic acid such as acetic acid or toluenesulfonic acid. To obtain its free form, the acid addition salt may be neutralized with an alkali.

GHRH-A of the present invention has higher GH release activity than human GHRH (1-44), has excellent stability and sustainability, and is a therapeutic agent for pediatric disease, a diagnostic agent for pediatric disease, and a growth promoter for animals (livestock). 9) j.

Example Next, the present invention will be explained in detail using examples.

Example 1 ◇Boc-8er(Bzl)-A(1111(TO3
): Boc-8er (Bzl) -0Su 19. h
(50mM) Agll1-H25o411,4g
(501M) water 301 + dioxane 30m++NEt
Suspended to 37111 (1eQ), 2.5 at air temperature
Stir for days. After distilling off the solvent, dissolve in 500 ml of ACOEt, wash with Na2CO3 saturated with salt, and saturated brine.
Dry with a2SO4. Leave ACOEt
t /E t Repeat 20 decants B QC-S
An oily substance (assuming that 1511M was obtained) of er-A gln was obtained. Dissolve in 50ml of DMF, cool, and T
Add O8C+ 4.3(1(1,5ec+) and lNNa
22.5 ml (1.5 eQ) of OH2 was added dropwise. Although an oily substance precipitated, the mixture was kept stirring and adjusted to pH=9 with 1N NaOH.
After 2 and 3 hours, add TosCl 1. IC
I(0,38eq), 1,4(1(0,49
eq) was added.

After 5 hours, the pH was adjusted to -7 with INHCl and the solvent was distilled off. Add 8 COE t 200ml, INHC+, water.

Wash with 5% NaHCO3, water, dry with Na2SO4, then AC
OEt was distilled off and powdered from n-hexane. This crude powder was diluted with CHCl 3:1yle OH:AcOH (9
It was purified by silica gel chromatography eluting with 8:2:1) and the main fraction was collected by TLC check. After distilling off the solvent, dissolve in 100 ml of AcOEt and add 5% NaHC.
After washing with O3 and water and dehydrating with a luene flash, powder from n-hexane was obtained, and the target products 2 and 3 (+ (8,2%)
I got it.

Elemental analysis: C27H3906N5S ・1/2CeH
Measurement value C, 58, 07 as z 4.1/2H20
; H, 7,56; N, 11.68% analysis value C, 5
8,01: H, 7,30: N, 11.74%◇B
oc-Nle-3er(Bzl) -Aom(Tos)
:Boc-8er(Bzl)-A(1111(TO
3) 1.12(](221M was treated with TFA 5mlr cooling for 10 minutes, IT-25 minutes, 3.5N HCl
/dioxane 0.7 go1 < 1.2 eQ) and then the solvent was distilled off. TI! Et20 was added to the solution to form a powder, which was then dried under reduced pressure over NaOH for 3.5 hours. B
oc -N le-D CHA 990n+g(1,
The oil obtained by desalting 2eq) in a conventional manner and HOB
CH2C with t 310 mg (1,15 eq)
It was dissolved in 225 ml of I + 5 ml of DMF, 42 ml (1,15 eq) of cold WsC was added dropwise under 1) H4), and the mixture was stirred overnight. After confirming the completion of the reaction by TLC and flam, CH2Cl2 was distilled. Ac0
Add 100 ml of Et, 1N HCI, water, 5%
After washing with NaHCO3 and water and dehydrating with a toluene flash, ACOEt was distilled and powdered from ACOEt/n-hexane to obtain target products 1 and 13 (1 (83.7%)).

◇Boc -11e-N Ie-3er (Bzl) -
AOII (TO3) 13 oc-N le -A 01
11 (Tos > (Hereinafter, -$1 represents the abbreviation of the amino acid residue sandwiched between the N-terminal and C-terminal amino acid residues. > 1,09a (1,62mM) was added to TFA5m
After cooling with l for 10 minutes and at air temperature for 25 minutes, excess acid was distilled off, Et20 was added to the residue to form a powder, and after filtering, Na
Dry under vacuum over OH for 3.5 hours. Dissolve in DMFloml, add NEt30.23n+1 (1 eq) under cooling, then 559 mg of Boc-11e-03u
(1,05 eq) was added, and the mixture was stirred for 35 hours while occasionally adjusting the pH with NEt3. After confirming the completion of the reaction with TLC and flam, 100 ml of ΔCOEt was added, and I
A powder precipitated out upon washing with N HCI. Wash as it is with water, 5% NaHCO3, and water, dehydrate with toluene flash, powder from CHCl3+MeOH/Et20, reprecipitate from the same system, and obtain the desired product 1.06 (1 (82,8
%) was obtained.

◇Boc-Asp(OBzl) -I 1e-Nle-
8er (Bzl) -Agm (Tos): 3oc -I le-Aom (TO3) 98511
IQ (1.25mM) was added to 5ml of TFA under cooling.
After treatment at air temperature for 25 minutes, 0.43 ml (1.2 eq) of 3.5N)-10I/dioxane was added, and the solvent was distilled. Ei 20 was added to the residue to form a powder, which was filtered and dried under reduced pressure over NaOH for 2.5 hours. Carboxyl component 445mg (1,1eq), ) (Q3t 186
D M F 12m1 with mg (1,1eQ)
Dissolve in WSCI 0.25ml (1,
1 eq) was added dropwise to pH 4, s) and stirred overnight. After confirming the completion of the reaction by TLC and flam, water was added under cooling to form a powder, and after filtering, water, n-hexane,
Wash with Et2○, dissolve in CHCl3+ MeOH, dehydrate with toluene flush, and then remove CHCl3. MeO)-
Powder twice from 1/Et20, target 1.1! +
(88.7%) was obtained.

AAA (6N HCI, 1○8℃, 22 hours) ASp (1,00), 5er (0,93>, 1
le(0,92>.

N1e(1,00).

Elemental analysis: Calculated value as C30H72011N9S C, 60, 46: H, 7, 31: N, 11.28
% analysis value C, 60, 28; H, 7, 44: N, 1
1.27%◇Boc-Gln-OPac: Boc-Gln-OH24.6g (0.IM)
and PaCBr20Q (0,1M>) in DMF (80m
Dissolve in 1) and cool under NE t :s 14ml
(0, IM) Rough m lower shim m% EEIW. Process the reaction solution with -ACOEt (300m!), 1NH
Washed with CI, H20, 0.5% NaHCC), H20 and dried over anhydrous magnesium sulfate. AcoE℃ was distilled into a powder by adding WEt20, and M e OH.

Recrystallize from Ac0Et, /Et20 to obtain target object 3
2flJ <87.8%) is 19.

◇3oc-leu-Gln-Qpac: Boc-Gln
-OPac 23,7q (65mM) was added to TFA (60mM).
ml) for 10 minutes under cooling and 30 minutes at room temperature, and
．． 95N HCI/dioxane 11.2ml
After adding (78mM), the solvent was distilled and the remaining solution was filled with Et.
20 was added to form a powder, which was collected by filtration and dried over NaOH for 4 hours. This powder was suspended in DMF (150a+l) and
oc-1eu-OH [monohydrate 16.2g (65mM
) and HOBt 9°71J (72111M) were dissolved, and while cooling, 2ml of WSC113° (72aM) was added dropwise and stirred overnight. After storing DMF, A c OE t (500 m
1) Add 1NHCl, H20, 5% NaHCC)
3. Washed with H20r and dried with anhydrous magnesium sulfate. A
After COEt was distilled, n-hexane was added to crystallize it, and ACO
Recrystallization from Et/Et2Q and n-hexane yielded 26 g (83.8%) of the desired product.

◇Boc-Leu-Leu-Gln-OPac: Boc
-Leu-Gln-OPac 22.5 (1(47
mM) with TFA (80 ml) for 10 minutes under cooling, then 3 minutes at air temperature.
Treated for 0 minutes, 3.5N HC+/dioxane 151
After adding (52.5ml 1vl), the solvent was distilled under reduced pressure,
EtpO was added to the residue to form a powder, which was collected by filtration and dried over NaOH for 3 hours. Add this powder to DMF (10,0ml
), [3 oc -L eu -Q day [1 water m
13g (oil obtained by dehydrating >52mM with toluene flush),!: HOBt 7 (1 (52mM
) and cooled with WSC19.5ml (52mM
) was added dropwise and stirred overnight. ACOEt (5000+
1) Add IN t-1cI, H2O.

5% NaHCC)3. After washing with H20T' (gel between the rows was precipitated), ACOEt was ground by adding standing water, collected by filtration, and washed with water and EtpO to obtain 25 g (90%) of the target product.

◇Boc-Lys (CI Z) -Leu-Leu-
Gln-Qpac: 3oc-Leu-1-eu-Gln-OPac24.2
(1 (41mM) was treated with TFA (100ml) for 10 minutes under cooling and 40 minutes at room temperature, and then treated with 6.9N HC+/
After adding 7 ml of dioxane (49 mM), the solvent was distilled under reduced pressure, and Ej20 was added to the residue to form a powder, which was collected by filtration and Na0
) 1 and dried for 3 hours. This powder was mixed with DMF (300 m
Boc-Lys(CIZ)-0
)-1-TBA24c+ (49mM) was desalted in a conventional manner and the oil obtained was combined with HOB t 6.6a (49
) Whole mash, WSC 19ml under cooling (49mM
) was added dropwise and stirred overnight to become a homogeneous solution. Ice-cooled 0.5N) (Pour into CI to make powder, collect by filtration, wash with water and EtzO, CHCl 3. MeOH/E tp
Re-precipitate twice from o to obtain the target 32.5! l] (89
, 3%).

◇Boc-Arg(Tos)-Lys(CIZ)
-Leu -Leu-Gln-OPac: Boc -Lys (CIZ) -G In-0P
ac 32.1g (36mM) in TFA (150ml
) T: Treated for 10 minutes under cooling and 40 minutes at room temperature; 3.
5N HC+/dioxane 12ml (42mM
), the solvent was distilled under reduced pressure, and 21-20 was added to the residue to form a powder, which was collected by filtration and dried over NaOH for 3 hours. 3oc-Arg(TO3)-0H18.5g of this powder
(43.2mM), HOBt 5°8 g (43.2mM)
a+M) was dissolved in DMF (2501), WS C17.9 ml (43.2 m~1) was added dropwise under cooling, and the mixture was stirred overnight. Pour into ice-cold 2.5% NaHCO3 water to make a powder, collect by filtration, wash with water and EtzO, and add CHCI
3. It was reprecipitated twice from MeOH/E t20 to obtain the desired product 42.5 (1 (98.6%)).

◇Boa-A Ia-Aro (Tos) -Lys(
CI Z ) -Seu -Leu -G In -
OPac: Boc-Arg(Tos)-Gin-OPa
c 420 (35,11M) to TFA (150m
+ ) for 10 minutes under cooling and at room temperature for 40 minutes, 3.5
After adding 12 ml of N HC+/dioxane (>42 mM), the solvent was distilled under reduced pressure, and EtpO was added to the residue to form a powder, which was filtered and dried over NaOH for 3 hours.
oc-Ala-0)-17(+ (36,9w+M)
, HOBt 5o (37m M> with: DM
Dissolve in F (250 ml) and WSCI 6 under cooling.
．． 8n+1 (37mM) was added dropwise and stirred overnight. Pour into ice-cold water to make a powder, collect by filtration, and wash with water and EtpO.
CHCI 3. It was reprecipitated twice from ~1eOH/Et 2Q to obtain the desired product 44.30 (99.6%).

◇Boc-8er (3zl) -A la-Arg
(Tos) -Lys(CI Z)-leu-leu
-Gln-OPac: Boc-A, rg(Tos)-G
ln-OPac41 (34,7mM') in TFA (1
50 ml) under cooling for 10 minutes and at room temperature for 40 minutes,
3.5N) IcI/dioxane 12ml (42m
After adding M), the solvent was distilled under reduced pressure, and EixO was added to the residue to form a powder, which was filtered and dried on Na0) for 113 hours.

This powder was mixed with 3oc-8er(BZI')-OH10,
BQ (36,411M), HOBt 5Q (
37mM) in DMF (250ml) and cooled with 6.8ml of WS CI (37IIIM).
) was added dropwise and stirred overnight. Pour into ice-cold water to make powder, collect by filtration, wash with water, EtpO, CHCl:s, MeOH
/Re-precipitated twice from Et20 to obtain the target first 47111 (93
, 7%).

◇Boc-Leu-8er (Bzl) -Ala-△r
c+ (,Tos>-LyS(CIZ)-Le
u-Leu-Gln-OPac:Boc-Se
r (B zl ) -G In-○P aC4G, 3
0 (32mM) in TEA (150ml) under cooling.
After adding 12 ml of 3.5N HC+/dioxane (42mM), the solvent was distilled under reduced pressure, and Ej20 was added to the residue to form a powder. After filtering, N
a.

It was dried on H for 3 hours. Add this powder to 3oc-1eu-OH
[Boc-1eu-OH-H208,4(1(33,
Oil obtained by dehydrating HOBt 4.70 (6mM) with traene flash] DM with HOBt 4.70 (35mM)
Dissolved in F (3001) and cooled WS CI 6.8
ml (37mM) was added dropwise and stirred overnight. Pour into ice-cold water to make a powder, collect by filtration, wash with water and EtpO, and CHC
I:s, reprecipitation was carried out twice from MeOH/EtQ○ to obtain 49 g (98.2%) of the desired product.

◇3oc-Gln-1-eu-8er (BZI) -A
la-Arg(Tos)-Lys(CIZ)
-Leu-Leu-Gln -○Pac: 3oc -1eu-GIn -OPac 48g (
30.8mM) was treated with TFA (150ml) for 10 minutes under cooling and 50 minutes at room temperature, and then treated with 3.5N HC.
+/v with dioxane 101 (35111fVI) added
1L of the solvent was distilled under reduced pressure, and Ei20 was added to the residue to form a powder, which was filtered and dried over NaOH for 3 hours. Bo
c-Gln-OH8゜4a (34n+M), HOBt
D fvl F with 4.6o (34i+M)
(250ml) and 6.2ml of WSCI under cooling.
(34mM) was added dropwise and stirred overnight. Pour into ice-cold water to make a powder, collect by filtration, wash with water and EtpO, and DMF/E.
After reprecipitating twice from t20, the target product 47 (1 (90.4%
) was obtained.

AAA (6N HCI/Ph OH, 108°C,
22 hours) Lys(1,08), NH3(1,1
2x2).

, A, rg(1,00) , 5er(0,90) ,
Glu(1,02x2), Ala(1,00)
, Leu(1,09x3)◇13oc-Gln-
Leu-8er(Bzl)-Ala-Arg(Tos
) ” Lys (CI Z ) -Leu-Leu-G
ln-OH: Boc-Gln-Gin-OPac 25.3g (15
mfvl) was dissolved in ACOH (600ml), 491;l (75011M) of zinc powder was added, and the mixture was reacted on a water bath at 47°C. Check the reaction by HPLC. Add 24.5 liters of zinc powder at 35 minutes and 45 minutes, respectively. Sa (375m
M), 12.5 (1 (191111M) added 50
Stir for 1 minute. After filtering out the zinc powder, ACOH was distilled and water was added to the residue to make powder. After filtering, it was washed with water and Ei20, and DM
Target compound 23(+ (97.7%)) was obtained from F/MeOH, E tqo to TIN)Sm.

AAA (6N l-10I /Ph OH
, 108°C, 22 hours) Lys(1,03), N
H3(1,10X2).

Arg (0,96), 5er (0,94), Qlu (1
,01x2) ,Ala(1,00) ,
L eu (1,01x3)◇[3oc-1eu
-Gly-OEt :Boc-Leu-OH[Bo
c-Leu -0H-H2024,9p (1001
1 M) and 15.5 g of G ly-OE t -HCl (II
OIIM) was dissolved in CH2Cl2 (150 ml) and N E t 315.4 ml (1101
M) and then DCC22! ] (107
A solution of +aM) in CH2Cl2 (25 ml) was added dropwise and stirred overnight. After confirming the completion of the reaction by TLC, CH2
Cl2 was distilled, and the residue was added with ACOEt (500ml
) was added, washed with INHC+, water, 5% NaHCO3, and water, and dried over anhydrous sodium sulfate. After distilling ACOEt, it is powdered from Ac0Et/n-hexane, and ACO
Recrystallize from Et/n-hexane to obtain 27.5 g of target product.
(87%).

mp87-88°.

◇Boc-1,eu-G+y-OH:Boc-Leu
-Gly-OEt 27.5(] (87mM>@M
e Dissolve in QH (100 ml), under cooling, 1 NN
aOH90111 was added dropwise, and the mixture was allowed to return to air temperature and stirred for 1 hour. After neutralization with 6N HCl, the solvent was distilled and 5%N
Dissolve in aHCC) 3, wash with EtpO, and 2NHCI
1) LAcOEt (500 ml) was released. After washing with water, it was dried over anhydrous magnesium sulfate, and after distilling ACOEt, n-hexane was added to form a powder. After being collected by filtration, it was recrystallized from ACOEt/n-hexane to obtain the target product 24 (+ (96%)).

mp 121- 123゜◇Boc-Leu-G1y-QPac:Boa-Leu
-Gly-OH20,2a (70mM) and Pac-
B r 13.4 (] (]70mM) in DMF (
50 ml>, under cooling, N E j :s 9
．． 8111 (70 m~1) was added dropwise and stirred overnight. A COE t (500ml) was added to the reaction solution, and 1
Washed with N HCI, water, 5% Na HCO 3 ° water, dried over anhydrous magnesium sulfate, and after distilling ACOEt, n-
Add hexane to powder, collect by filtration, ACOEt/n
-Recrystallized from hexane to obtain the target compound 25(+ (87,
7%).

mp114-115゜◇13oc-Val-1eu-a+y-opac:Bo
c -Leu -G ly -OPac 180 (4
4.3mM) in TFA (70ml under cooling for 10 minutes, v
After treatment at room temperature for 45 minutes, 6.8N HCI/dioxane (1.81 < 53.2 mM) was added, the solvent was distilled under reduced pressure, and EtpO was added to the residue to form a powder. After filtering, NaO
It was dried under reduced pressure for 3 hours on H. 5oc-vat-OH10,
1(1(46,5mM), HOBt 6,30(4
6.5mM) in DMF (60ml),
While cooling, 18.5 ml of WSC (46.5 mM) was added dropwise and stirred overnight. It was poured into ice-cold water to form a powder, and after filtering,
The desired product 21.30 (95.1%) was obtained by reprecipitation twice from COEt/n-hexane.

◇Boc-Lys (CI Z) -Vat-Leu-Q
ly-0Pac: Boc-Vat-Leu-Gly-OPacl 5.
20 (3Off1M) was treated with TFA (45ml) for 10 minutes under cooling and 25 minutes at room temperature, and 6.95N
After adding 5.2 ml (36 mM) of HCI/dioxane, the solvent was distilled off, and Et20 and n-hexane were added to the residue to make a powder. After stripping, it was dried under reduced pressure over NaOH for 3 hours. 3oc-Lys(CIZ)-OH[Boc-Ly
s(CI Z) - 0 days・TB A 16.8g (
34.5mM> was dissolved in DMF (60ml) with 4.46g of HOBt (33lIIM) and WS01 under cooling.
6 ml (33 tnM) was added dropwise and stirred overnight. Pour into ice-cold water to make a powder bed, and after filtering, CHCI:s, MeOH/
Re-precipitate twice from Ej20 to obtain 20°5g of the target material (85%
) was obtained.

◇Boc-Arg(Tos)-Lys(CIZ)
-Vat-Seu-G1y-OPac: Boc-Lys(CIZ)-Gly-○Pac1G.

(201M) in TFA (40ml) T: 10 under cooling
25 min at variable temperature, 6.95 N HCI
/Dioxane 3.5ml (241111VI>) was added, the solvent was distilled off, and Ei20 was added to the residue to form a powder.
After filtering, it was dried under reduced pressure over NaOH for 3 hours. 13oc -
Arg (Tos) -OH9,9(1 (23mM)
, HOBt 3.1g<23mM) with D
Dissolve in M F (30ml) and cool under WS01 4.
2 ml (23111M) was added dropwise and stirred overnight. Pour into ice-cold water to make a powder, filter and collect CHCI 3. MeO
It was reprecipitated from H/Ei20 and then purified by heating with MeOH to obtain the desired product 17.60 (79.3%).

◇Boc-Tyr(Br Z) -Arg(Tos)
-Lys(CI Z) -Vat-Leu-Gly-O
Pac:Boc-Aro(T03)-Gl
y-OPac 16.7g (15a+M) T E
A (401111) for 10 minutes under cooling and 50 minutes at room temperature, 6.95N HCI/dioxane 2.6m
After adding E.l (18mM), the solvent was distilled off and the residue was
t20 was added to form a powder, which was collected by filtration and dried under reduced pressure over NaOH for 3 hours. Boc-Tyr(BrZ)-0H8,
5o (18mM), HOBt 2.3g (1
8mM> was dissolved in CDM F (40ml), WSC13.2ml <18a+M) was added dropwise under cooling, and the mixture was stirred overnight. Pour into ice-cold water to make a powder, filter it out, and
HCI 3. Reprecipitated from Me○H/Et20 and then purified by heating with MeOH to obtain the target product 21.2+11
(95.1%) was obtained.

0Boc-3er(Bzl)-Tyr(Br Z)-A
r.

(Tos)-LyS(CI Z)-Vat-Leu-
Gly-OPac: Boc-Tyr(BrZ)-GIV-
OPac 19.4g (1311M>
ml) for 10 minutes under cooling and 35 minutes at room temperature, 6.9
5 N HCI/dioxane 2.3 ml (15,
6,mM), the solvent was distilled off, and the residue was diluted with Et20.
was added to form a powder, which was then dried under reduced pressure over NaOH for 3 hours after drying. Boc-8er (Bzl) -0) 14.2g
(14.3mM), dissolved in DtvlF (45ml) together with HOBt 2p (15mM), under cooling, WS
C12, 7ml (15mM) trW4 lower tf
t stir.

Pour into ice-cold water to make powder, collect by filtration, and add CHCl 3゜M.
It was reprecipitated from eOH/Et20 and purified with NIeOH humidification to obtain the target product 211 (96.8%).

◇ BOC-△ 5n-8er (Bzl) -Tyr
(Br Z) -Arg(Tos) -LVS (
CI Z ) -Val -L eu -Gly-O
Pac: Boc-Ser (Bzl)-G 1y-OPac
3.5g (2.1ml 1vl > T F A < 1
0ml> for 10 minutes under cooling and 30 minutes at air temperature, 3.
5N HC+/dioxane 0.72m1 (2,5
After adding 10 mL of the solvent, Ei20 was added to the residue to form a powder, which was collected by filtration and dried under fd pressure over NaOH for 3 hours. B oc -A sn -OH536, 5 mg (2
,3mM), HOBt 326,41H] (2
,4mM) Steller'bni DMF <17m1) Nimkashi, frIfJI, WSCI O,44m1 (
2.4 mM) was added dropwise and stirred overnight. It was poured into ice-cold water to form a powder, collected by filtration, reprecipitated from CHCl3/MeOH, and then purified by heating with MeOH to obtain the desired product 3.35 (]
(I got 889.6%.

AAA (6N HCI/Ph OH, 108°C, 2
2 hours) 1-ys(1,00), Arc+(0,
96), Asp(i,ol), 5et(0,88
), Glv[1,02).

Vat (1,00), Leu (0,97),
'ryr (0,9 elemental analysis: C34H105021N
Calculated value C as t3SCIBr-H2C, 56,10: )l, 6.00: N
, 10.13% analysis 1iiIC, 56, 19: H,
5,96: N, 10.05%◇Boc-Asn-
Ser (BZI) -Tyr (Br Z) -A
r! ] (Tos) -Lys (CIZ) -Va
l-Leu-Gly-OH: Boc-Asn-Gly-OPac 1,7!11
(0.95mM) was dissolved in ACOH (20ml),
1.25g (19mM) of zinc powder was added and reacted on a water bath at 43°C. After 1 hour and 2.5 hours, zinc powder was administered at 1,250 (19mM) and 625mc+, respectively.
(9.5mM) was added and stirred for 3 hours and 15 minutes. After filtering off the zinc dust, the ACOH was distilled off, water was added to the residue to form a powder, and after filtering, CHCl:s was obtained.

Re-precipitate from MeOH/E t 20 to obtain the target product 1.4
4 (1 (91,1%)) was obtained.

AAA (6N HCI/Ph OH, 108°C,
22 hours) Lys(1,01), Arg(1,0
0), Asp(1,01), 5er(0,86
), Gly(1,00).

Val (1,03), 1eu (0,99),
Tyr(1,02).

Elemental analysis: C76H9902ON13SCI Br-1
Calculated value as ,5H20 C,54,04; H,6,09: N, 10
．． 78% analysis value C, 53,88: H, 6,17: N
, 10,59%◇Boc-Phe-Thr (Bzl
) -0Pac:Boc-Thr(Bzl)-0Pac
[[3oc-Thr(Bzl)-0H21,65g(7
0mM> and pac-3r13.4g (701M)
[Oil obtained by conventional synthesis from TFA701]
10 minutes under cooling and 35 minutes at air temperature.
After adding HCI/dioxane 221 (84mM), the solvent was distilled off and Et20 was added to the precipitated powder and ground.
After filtering, it was dried under reduced pressure over NaOH for 2 hours. This powder is D
MF (80-1) and CH2CI 42 (20111)
18.6 g of 3oc-Phe-OH (
70mM) and HOB t Lag (701M)?
17')'L7. : After cooling, WS C112, 8m
1 (70mM) was added dropwise and stirred overnight. After distilling off CH2Cl2, add A COE t (800ml) and
N HCl, l-120, 5% NaHCO3, H2
It was washed with C and dried over anhydrous sodium sulfate. ACOE;
t was distilled in-hexane was added to crystallize, and MeOH was added.

After dissolving in Ac0Et and treating with activated carbon, Ac0Et /n-
Recrystallized twice from hexane to obtain 29.63 g (7
4.1%) was obtained.

◇Boc -11e -Phe -Thr (Bzl)
-0Pac:Boc-Phe-Thr (Bzl)
-0Pac 17.2 g (30mM > TEA4
01 for 10 minutes under cooling and 30 minutes at air temperature, 3.8N
HCI/dioxane 9.5ml (36mM)
After adding , the solvent was distilled off, EtQO and n-hexane were added to the residue to form an oil, and the decantation process was repeated, followed by NaOH.
The mixture was dried under reduced pressure for 2 hours. Dissolve in DMF (35 ml),
Boa-11e-O8u after neutralization with NE t3 under cooling
Add 10.3 (+ <31.5 mM>, pH!A
The reaction was carried out for 45 hours under normal conditions (D because gel precipitated during the process)
MF and CH2Cl2 were added to make a homogeneous solution). CH
After distilling 2Cl2, Ac0Et (700ml) was added, 1NHC+, and water.

5% Na HCO:s, washed with water anhydrous 1iAi9v
Dried with gnesium. After storing Ac0Et, CHCl
3. Powder twice from ~1eOH/Et20 and object 16
g (77,6%) is 19.

◇Boa -A la -11e -Phe -Thr
(3zl) -Q pac : Boc -11e -Phe-Thr (Bzl) -
0-Pac 15°8g (23mM > T E A
After treatment for 10 minutes under cooling of 40ml and 35 minutes at v temperature, excess acid was distilled off, Et20 was added to the residue to form a powder, and after filtering, N
Dry under vacuum over aOH for 3 hours. Dissolved in a mixed solvent of DMF (40Bt> and CH2CH2Cl2 (10) and neutralized with NEt:s under cooling 'IBoc-Ala-O8u7,
2 (1(25,3m~1) was added and reacted overnight.CH
2Cl2 was distilled and mixed with water to form a powder, and after filtering, CHCl
3. It was powdered twice from MeOt-1/Et20 to obtain 15.98 g (90.8%) of the desired product.

◇Boc -A Stl (Oct(eX') -A
la -1! e -P he -Thr(Bzl)-0
Pac: Boc-A 1a-Thr (Bzl) -0 Pacl
2g (15.8mM) was treated with TFA (501) for 10 minutes under cooling and 40 minutes at room temperature, and then treated with 6.9N H
After adding 3.4 ml (23.7 mM) of C+/dioxane, the solvent was distilled off, and Et20 was added to the residue to form a powder.
After removal, it was dried under reduced pressure over NaOH for 3 hours. 3 oc-
A 5p(Qc)lex)OH5,5(1(17,4m
M), HOBt 2,35! + (17,4III
Dissolved in DMF (40Bt) with W
SCI 3.2n+l (17.4mM) was added dropwise and stirred for 4 hours. The powder was poured into ice-cold water, collected by filtration, and recrystallized twice from MeOH to obtain the target product 14 (1 (92,
7%).

◇BOC-D-AIa-ASp (OCI-1eX) -
Ala-11e-Phe-Thr(Bzl)-〇P a
c: Boc -A sp (□cHex) -Thr
(Bzl) -OP ac 11,2o (11,7
>mM> was treated with TFA (35n+1) for 10 minutes under cooling and at room temperature for 40 minutes, and after adding 2.55+nl (17.6mM) of 6.9N HC+/dioxane, the solvent was distilled off, and the residue was diluted with Et20. was added to form a powder, collected by filtration, and dried under reduced pressure over NaOH for 3 hours. 3oc-D-Ala
-OH2,55+11 (13,5aM), t-10
Bt 1°82 (1 (13,51M) with DMF
(50Bt) and cooled, WS C12, 48m
1 (13.5mM>'Fr) was added dropwise and stirred overnight. Ice-cold water was poured into the solidified reaction solution to form a powder, and the powder was collected by filtration.
tvl e OH reflux followed by CHCl 3.
The target product 11 (1 (9
1.7%).

◇Boc-Try(BrZ)-Ala-Asp(O
cHex)-A la -11e-Phe-Thr (
Bzl) -0Pac:Boc-D-A Ia-Thr
(Bzl)-0Pac 11° (10.7mM)
was treated with TFA (40Bt) for 10 minutes under cooling and 40 minutes at room temperature, and treated with 6.9N HC+/dioxane 2.3+al
(161M) was added, the solvent was distilled off, and the residue was Et
20 was added to form a powder, which was collected by filtration and dried under reduced pressure over NaOH for 3 hours. Boa-Tyr (Br Z) 0) I S, 83
Q (11,81aM), HOBt 1.6 Q
(11,8111M) in DMF (50Bt), cooled, and WS C12.2ml (11.8mM
) was added dropwise and stirred overnight. Pour ice-cold water into the reaction solution to make a powder, collect by filtration, and add MeOH/Et20 followed by MeO.
Reprecipitation was performed from only H to obtain the desired product 14.70 (98%).

AAA (6N HCI/Ph OH, 108°C,
22 hours> Asp(1,04), Thr(0,9
7), Ala(1,0Ox2), l1e(1
,00), Tyr(,1,01>.

phe(1,01) Elemental analysis: Calculated value as C72H88017N7Br C, 61, 62: H, '6.32: N,
6.99% analysis value C, 61,45: H, 6,45:
N, 6.99%◇Boc-Tyr (Br Z)
-D-Ala-Asp(OcHex) -Ala-
I Ie -Phe-Thr (Bzl) -O
H: Boc-Tyr(BrZ)-Thr(Bzl)
-0Pac 10! ;I (7,13mM) as Ac0
)-1 (150Bt) and zinc powder 8(+ (
12.2mM) was added thereto and stirred for 50 minutes on a 43°C water bath. After filtering out the zinc powder, distill off AC○to1, add water to the residue to make a powder, and after filtering, MeOH/Ei! From No. 20, 8.49 (91.7%) of the desired product was obtained by reprecipitation.

AAA <6N HCI/Ph OH, 108°C,
22 hours) AS+) (1,01), Thr(0,
96), Ala(1,0Ox2), l1e
(0,96), Tyr(0,97)Phe(0,9
6> Elemental analysis: Calculated value C,5 as C64H82N7Br
9,81: H, 6,43: N, 7.63% analysis value (:,, 60.09: H, 6,63: N,
7.50%◇3oc-Gln-1-eu-8er(
Bzl) -Ala-Arg(Tos) -L ys(
CI Z) -L eu-L eu-Gln -Asp
(OBzl) -1le -N Ie-8er (B
zl) -1+m(Tos) : 13oc-Asp (OB Zl)-Aom (Tos
) 497Il+g (0:511M>) was treated with TEA5n+l for 10 minutes under cooling and 25 minutes at room temperature to give 5.9N M
After adding 0.1 ml of CI/dioxane (1,2 eQ), the solvent was distilled off. Et20 was added to the residue to form a powder, which was filtered and dried over Na01-1 overnight. Carboxyl component 7
85 mg < 1 eQ).

D with HOBt 711f (1 (1,05eq)
Dissolve in 7 ml of MF + 8 ml of NtvlP and add to WSC under cooling.
Drop I 96 μm (1,05 eq) 1) H5
) reacted. 5° After 5 hours carboxyl component 39 mQ
(0,05eQ), )-108t 10mg(0,1
5eQ), WSCI-HCl3 mg (0,0
5eQ) was added and stirred overnight. After confirming the completion of the reaction by TLC and flam, water was added under cooling to form a powder, collected by filtration, washed with water, n-hexane, and Et20, and
After dehydration with HCl 3 + MeOHk-W or L toluene flush, powder was obtained from CHCI:s and MeOH/Et20 to obtain the desired product at 1.05° (8L 1%).

AAA (6N l-1cI, 108°C, 2
2 hours) Lys(1,03), NH3(1,12X2
), Ar(1(0,97), Asp(0,9
5) 5ep (0,92x2), Glu
(0,99x2) Ala(1,00),
IIe(0,88).

l eu (1,06x3), N Ie
(0,86) Elemental analysis: C118H17S! 027N2
Calculated value as 3S2CI 2H20 0.57,14; )-(,7,15: N
, 12.99% analysis value C,57,07: H7,16
; N, 12.97%◇Boc-Asn-8er
(Bzl) -Tyr (BrZ) -Aro
(Tos)-Lys<cl z> -Val-Leu-
Gly-Gln-Leu-8er(Bzl)-Al
a-Arg(Tos)-Lys(CIZ)-L
eu-Leu-Qln -Asp (OBzl) -1
1e-N 1e-8er (Bzl) -ACIll
(TO3) : Boc-Gln- to gffl (Tos> 10 (
0.41mM) in TFA 10n+1 under cooling.
After treatment at air temperature for 35 minutes, 5.9NHC+/dioxane 80μ+ (1.2eq) was added, and the solvent was distilled off. Ei20 was added to the residue to make a powder, and after filtering, NaOH
The mixture was dried under reduced pressure for 3 hours. Carboxyl component 716mg
(1,05eQ).

DMF7+ with HOBt 61mg (1,1eQ)
183μl of WSC (1,1eQ>
was added dropwise (pH 5) to react. After 3 hours, it became a homogeneous solution, and after 4 hours, 201 g of carboxyl component (0,0
3eQ).

HOBt 311(+(0,05eQ), WSC
2.4 mQ (0.03 eq) of l-HCl was added and stirred overnight. After confirming the completion of the reaction by TLC and flam, water was added under cooling to form a powder, which was collected by filtration and water, n-
hexane.

Washed with EtpOr, CHCl3+ MeOHk: Dissolved and dehydrated with toluene flash, powdered from ACOEt Suspend/Et 20, and the target material 1,594J (
97%).

AAA (6N HCI, 108°C 22 hours) LIS (1,04x2), NH3 (1,13X
3), Arg(0°97x2), ASD(
1,0OX2), 5er(0,92x3)Gl
u(0,97X2>, Gly(1,06),
Ala (0°99), Val (1,02), l
1e(0,84>.

Leu (1,05x4), N1e (0,86)
, TVr (0°97).

Elemental analysis: Cx 89H261044N36S3CI
28r ・3.5H20 and T(7) Calculated value C, 56,
03: H, 6°67: N, 12.45% Analysis value C, 56,05; H, 6,64: N, 12.3
9%◇Boc-Tyr(Br Z)-D-Ala-A
sp(OcHex)-A la-I le-Phe-T
hr (Bzl)-Asn-8er(Bzl)-T
yr(SrZ)-Arg(Tos)-Lys(CI
Z) -Vat-Leu-Gly-Gln-Leu-
5er(Bzl)-Ala-Arg(Tos)Lys(
CI Z) -Leu-Leu-Gin-Asp(
OBzl) -11e-N 1e-8er(Bzl)
-Aom (TO3): Boa-A 5n-AOII (Tos) 598u
(0.15mM) in TFA51111 for 10 hours under cooling.
After treatment at room temperature for 40 minutes, 0.1 ml (large excess) of 5.9N HC+/dioxane was added and the solvent was distilled off. Ei20 was added to the residue to form a powder, which was filtered and dried over NaOH overnight.

Carboxyl component 222mo (1,15eq),
DM with HOBt24111(+ (1,2eq)
Dissolve in 8 ml of F and add WSC [33 μl (1,
2 eq) was added dropwise to react IIH4.5). 8
Carboxyl component 29mp (0.15eq) after Sukema
, HOBt 3+nl (0,15eq).

Added WSCI4μm (0,15eq) at night. After confirming the completion of the reaction with TLC and flam,
Add water while cooling to make a powder, collect by filtration, wash with water, n-hexane, and Et20 in that order, dissolve in CHCl:s+MeOH, dehydrate with toluene flash, and then remove CHCl 3. M
eOH/Et! 20 to powder, then M e O
Purify with H heating suspension to obtain 470 mcl (6
0.8%) I got it.

AAA (6N HCl, 108°C, 22 hours) Lys(1,0Ox2), NH3(1,08
X3>, Aro(0°92x2), As1
)(1,0Ox3), Thr(0,95>.

5er (0,88x3), Glu(0,92x2
), Gly(1,06), Ala(1,01
x3), Vat(0,95).

11e(0,86x2), Leu(1,0Ox4
) , N1e(0,80> , Tyr(1,02
x2), Phe(0,89) elemental analysis: C248
H333057N43S3C12Br 2/3H20
Calculated value as C, 57, 18; H, 6, 56: N, 11
．． 56% analysis value C, 57, 17: l-1, 6, 37:
N, 11.50%◇'rry-D-Ala-Asp-
Ala-11e-Phe-Thr-A sn-
S er -T yr -A rg-L ys-V a
l-L eu -GIy -Q In -1eu -S
er -A la -A rg -1ys -L e
u −1eu −GIn −A sp −11e −N
le-8er-Agm: 232 mg (45 μM) of protected peptide was heated at −1° C. in the presence of 0.61 anisole and 5.41 μl of hydrogen fluoride.

After treatment for 60 minutes, excess acid was distilled off. After dissolving in 2N AcOH and washing three times with Et20, DOWeX1X2
(AcOform, ca 40ml) was passed through (eluted with water) and dried. The obtained crude product was CM-
C, DIA ION HP-20,5ephade
The product was purified sequentially using x LH-20 to obtain 30 mg of the target product.

AAA (6N HCl, 108 °C, 22 hours) Lys (1,02 x 2'), NH3 (102
4X3), Arg(1,12x2), Asp
(1,06x3>, Thr(0,90>.

5er(0,96x3 > , Glu(1,00x2
), Gly(1032), Ala(1,0O
x3), Vat (0,87).

1 le(0,96x2), Leu(1,01x
4), Nle(0,83), TVr(
0,86x2), Phe(0,97) elemental analysis: C149H247041N43・4AcOH・1
Calculated value as 3,5H20 C, 49, 88: H, 7, 73; N, 15
．． 93% analysis value C, 49, 85: H, 7, 44: N
, 15.94% Example 2; GH release activity test 1 n ViVOt' is the coexisting endogenous GHRH, 5
Due to RIF, the GH release activity of exogenously administered human GHRH is modified, making accurate evaluation impossible in the past.

Therefore, in this study, an SD platform weighing 350 to 450 g without anesthesia and without restraint with a cannula chronically placed in the right atrium was used.
) against rat GHRHγ globulin (AGG> and anti-5
By administering RIFγ globulin (ASG) (11), human GHRH that does not cross with AGG was found to be able to eliminate the effects of endogenous GHRH1SRIF.
(1-44)-NH2, human GHRH (1-29)-N
H2, human GHRH(27-44)-NH2, [D-T
hr7]-human GHRH(1-44)-NH2(GHR
H-■) and GHRH-A of the present invention were randomly administered intravenously at 1 hour intervals. ! The UGH reaction was observed.

As a result, the pulsatile GH secretion pattern disappeared in rats administered ASG1AGG. When the same m m of human GHRH-A was administered to these rats every hour, the same level of plasma GH increasing response was reproduced. Also, GHRH-A
A dose-response relationship was also observed between 0.025 and 0.2 nmol/KO.

Further, the results when using an amount of Q and 1 nmol/Ko are shown in the table below.

As is clear from the table, human GHRH(1-29)-N
H2, human GHRH(1-44)-NH2, and the GHRH-A of the present invention all caused a clear increase in blood clot GH, and their GH release activities were in the ratio of 1:2:3, respectively, and the GHR
H-A was the most potent.

On the other hand, human GHRH(27-44)-NH2 and GHRH
-T did not show any GH release activity in this case.

Effects of the Invention As is clear from the above, the human GHRH analogue, which is a peptide derivative of the present invention, has the effect of the conventional human GHRH (1-4
Compared to 4>, it has a shorter peptide chain and higher GH release activity, and is expected to be used as a pediatric drug.

Claims (2)

[Claims] (1) A peptide derivative represented by the following structural formula. [There is a gene sequence] In the formula, Agm is agmatine, H_2N(CH_2)_4
NHC (=NH) represents the NH_2 residue. (2) The peptide derivative according to claim 1, wherein Ala^2 is D-form.