JPS59216859A - Synthesis of somatoclinine in liquid phase and intermediate peptide - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION The invention relates to the synthesis of hpGRF (somatocrinin) in the liquid phase and the intermediate bezoti r.

hpGRF (human pancreatic growth hormone releasing factor) or somatocrinin is a peptide made up of 44 amino acids. Its amino acid sequence is as follows.

H-Tyr-Ala-Aap-Ata-Ite-Ph
e-Thr-Asn-8er-Tyr-Arg-LyI
! -Vat-Leu-G outside-Gtn-Leu-8er-
Amu A2r'g-Gtn-GLy-Gtu-8e r
-A s n -Gtn-Gtu-Ar g-Gty
-Ata-0 Arg-Ala-Arg-Leu-NH2 This was discovered from an extract of pancreatic cancer by A. Gilmen et al. (Science 218°585-587 (1982)).

Both in vitro and in vivo, the peptide with
It has particularly high activity in promoting the release of growth hormone (GH). Especially in vitro, its effectiveness is several fentmoles/m7! (ED5o=15 fentmol/-). The therapeutic interest of this substance in human medicine therefore lies in the treatment of dwarfism and growth defects in pediatrics. Other uses include repairing anabolic protein defects (stress ulcers, cartilage cracks or wounds, excessive burns (
It is possible to treat anabolic phase (during the anabolic phase), skin repair, and osteoporosis).

In the field of veterinary medicine, the main interest of this compound is in livestock (
It is clear that this is due to increases in body weight and milk production (cows, sheep, pigs, chickens, etc.).

The industrial development of j(D, j?) Oftide requires the synthesis of large quantities of this substance. By conventional synthesis in a solid phase, the amount of active substance synthesized in a short period of time is Since the amount is small and the cost is extremely high, it is not suitable for large-scale drug development.

The present invention provides a liquid phase synthesis method that can be carried out on a commercial scale and has high yields and high purity of active substances. The principle of this method is based on fragment-by-fragment synthesis.

The method of the present invention is characterized in that fragments having the following characteristics are successively joined.

a) The acid function of the side chain of Asi4ragic acid and glutamic acid and the side chain amine function of lysine form a BoC group (tartiobutoxycarbonyl).
Protected with a protecting group that is stable under the deprotection environment of

b) The guanidine function of arginine is protected by protonation.

e) The amino group of the N-terminal amino acid is protected by a BoC group.

d) selectively removing the BoC group of the N-terminal amine in an elongation step by hydrolysis with trifluoroacetic acid;
The coupling is carried out in a neutral solvent and all retaining groups at the end of the sequence are removed by hydrolysis in a trifluoroacetic acid solution of methanesulfonic acid or trifluoromethanesulfonic acid of the 01-free LIM.

The method of this invention was discovered by Earl Gilmen and is as active as hpGRF-1-44;
hpGRF- which can be used for similar purposes.
It can also be applied to the synthesis of 1-40.

The GRF synthesis method is further characterized by the following features.

Protection of monofunctional side chains is minimal.

- Temporary preservation of side chain guanidine functionality of arginine by nitro group. Arginine is introduced into the sequence in the form of nitroguanidine. The nitro functionality is then removed as quickly as possible by catalytic hydrogenation with gaseous hydrogen from Pd/activated carbon or a hydrogen generator such as formic acid or ammonium formate. Thus, in the synthesized fragments with arginine in the sequence, at the end of the synthesis the guanidine function is protected only by protonation with the aid of a strong acid (eg hydrochloric acid).

The side chains of monoglutamic acid and al/l-line acid can also be prepared by catalytic hydrogenation (T(2/Pd/activated charcoal)) with hametanesulfonic acid (0,5M)-trifluoroacetic acid or methanesulphonic acid (0,5M)-trifluoroacetic acid (0,5M)-trifluoroacetic acid. protected by a group that can be cleaved in a fluoroacetic acid mixture. In this invention, benzyl (0Bzl ) or 2,
6 dichloropendyl ester is recommended. These protecting groups are stable in the intermediate deprotection environment of alpha amines (removal of the t-butyroxycarnyl (BoC) group with trifluoroacetic acid).

- The amine function of the side chain of lysine is protected by a group that is cleavable under the same conditions as described above. In this invention, benzyloxycarbonyl (z), 2-chloro or 2-promopenzyloxycardenyl (2-
C1 straddle 2-BrZ) is recommended.

The hydroxyl functions present in threonine, serine and tyrosine are unprotected.

One from the synthesized fragments! 'Notide elongation occurs in the presence of 1-hydroxypenzotriazole, a binding agent,
benzotriacylyloxyphos hexafluorophosphinium (BOP) by using dicyclohexylcarbodiimide, or by the method using oxyazide (Curtius), dimethylformamide or dimethylforpoxide. It is carried out in a suitable solvent such as. Separation of the product from the reaction solution can be accomplished by adding a third solvent (ether, ethyl acetate, etc.) that insolubilizes and precipitates the iptide.

Finally, combining a solid-phase water-wash type purification into the binding step is limited, using a suitable solvent to remove impurities introduced by the slight excess of fragments and binding agent that are finally combined.

Each fragment coupling operation is followed by a Boc (tertio-butoxycarnyl) on the amine on which the next coupling takes place.
Intermediate decolonization of the phloem base is performed. Deprotection is thus carried out using trifluoroacetic acid (50150) dissolved in methylene chloride.
(volume ratio).

Deprotection of the side chain at the end of zezotide elongation is catalyzed (
For example, in the presence of Pd/C), slightly pressurized (
1-5V4) by hydrogenation with gaseous hydrogen or hydrogen generators such as formic acid or ammonium formate.

This type of retention group can be removed by strong acids such as methanesulfonic acid in trifluoroacetic acid (0.5M) or trifluoromethanesulfonic acid in trifluoroacetic acid (0.5M).

After the final depreservation, the product is purified by filtration over Sephadex Glu G with 30% acetic acid. GRF
The fractions enriched in -1-44 are collected and chromatographed on a carboxy type ion exchange resin (cation) with a gradient of increasing ionic power depending on the type of ion exchange resin used. The highest purity fractions are collected and purified by partition chromatography on a suitable support of type Sephadex G50 or Biorol P10, or by self-flow partitioning.

Fractions with acceptable purity titers by analytical HPLC were collected. Others were reused.

A variation of this method replaces the partition chromatography or countercurrent partition method with HPLCK for h'A.

The fragments used in the synthesis of hpGRF-1-44 are defined based on chemical considerations with particular regard to minimizing the risk of racemization during the fragment ligation step.

Each fragment is then synthesized slowly in the liquid phase according to a tailored strategy.

The scheme ■ shown below shows one of the synthetic strategies that can be used to synthesize hpGRF-1-44.

Vertical arrows indicate the boundaries of the fragments used in the synthesis, each of which is designated by the following letters:

A: Fragment 4O-44 B: Fragment 33-39 C: Fragment 28-32 D: Fragment 25-27 E: Fragment 2l-24 F: Fragment 16-20 G: Fragment 12-15 I: Fragment 5-11 : Fragment 1-4 Furthermore, the side chain protecting groups are symbolized as follows.

X, = ester of benzyl type (0Bzl) X2 = carbamate protecting group of benzyloxycarginyl (z) type Tables 17Th to 2 below show the synthesis methods for fragments A to I.

17- Table ■ (Fragment work) Ty r -A l a -A sp -A L
ahpGRF-1-44 can also be produced by a method using the following fragment.

20-44 Peptide 5-19 -<Petit J 1-4　　　　-<Putide I These are combined in the following order:

5-19 + 20-44 5-441-4 +
5-44 1-44 Peptide J and peptides are synthesized by the fragment method.

Peptide 5-19 is synthesized from three fragments and peptide 20-44 from seven fragments.

Schemes I and ■ shown below illustrate the manufacturing strategy for peptides J and K. The method for making 4 petidoes is already shown in Scheme I.

Schematic ■Kakeputite, J and K are combined to form hpGRF
1-44 is shown.

Tables X through X■ show methods of synthesizing fragments not shown in Scheme I below.

Table X (subfragment B,) G tu , A r g −□GLy Table X
(Sub-fragment B2) H,: OBzt ::: El a2El 397- Finally in all cases it is sufficient to reduce the fragment to alanine amide in order to obtain hpGRF 1-40.

The scope of this invention will be more easily understood by the following examples.

Use the following abbreviations.

Amino acid nomenclature is represented by symbols recommended by the IUPAC-IUB Biochemistry Department, Nomenclature Committee.

Ata: Alanine Arg: Arginine Asn: Asunogragin Arg: Arginine Gtn: Glutamine GLu: Glutamic acid aty nigericin 14e: Isoleucine L@u: Leucine Lyg: Lysine Met: methionine Phe: Phenylalanine Ser: Serin Thr: Threonine Tyr: Tyrosine Vat: Valin All of these are Lr except for glycine.

CCM: Thin layer chromatography 0Bzt: Penzyl ester z: Penzyloxycarponyl (carbamate) Boa: Tertioptyrosoxycarponyl (carbamate) CH3 ■ CH, -C-0-C I II TC30 DMF Nidimethylformamide NEM : N-Methylmorpholine TFA : Trifluoroacetic acid MA : Coupling method with mixed anhydride 0NSU : N-Hydroquine succinimide activated ester ON+) : Orthonitrophenol Troc : )! J Chloroethoxylic? Nil (
Carbanate) 1 cct3- CH2-0-C 0But: Ester CI with tertiobutanol (3 0Tcp: L3s5-trichlorophenol activated ester 0HBT2N-hydroxybenzotriazole oNb
: N-hydroxy 5-nor? Runene 2゜3-dical is ximide 1 BOP: hexafluorophosphate of hengetriazolyloxyphosphonium DCHa: dicyclohegycylamine r) CU dicyclohexyl urinary accumulation DCC straddle DCC'I dicyclohexylcal Positive imide TA: room temperature DIPEA diisopropylethylamine EPP:
Polypeptide purity TFMSA: ) Lifluoromethanesulfonic acid AAA
Amino Acid Analysis HPLC: High Performance Liquid Chromatography Chromatographic media are indicated by volume.

BEW, :Butanol, AcOT(,,I(2072
/7/21By2: Butanol, AeOHXH2
067/10/23BPEW,: Butanol, pyridine, AcOH% H2O30/12/12/25 EPAW: Ac0Et, pyridine, HCO2
H, H2O63/21/10/6 BPEW2: Butanol, pyridine, AeOH% H2
O42/24/4/30 41 Example 1 1 (-Ata-Arg-Ata-Arg-Leu -N
Synthesis of I(2 (Fragment A) 1, No2 H-A r g -LEU-NH2 130 g of leucine amide (H-Leu-NH2) was dissolved in 1.51 DMF at room temperature. 333 g of Boc
-Arg(No2)-OHs and then 500g BO
Added P. Using pl (test paper (small sample diluted with water)), - was adjusted to 7 with N-methylmorpholine (NEM). Stir the solution and check the progress of the reaction by TLC.
I checked it. The reaction was stopped after 4 hours. 2 liquid
Evaporate to dryness in vacuo at 5°C. Take the residue in 14 water,
The obtained solid was dissolved in water, 5% NaHCOx aqueous solution, water,
Washed with ethyl acetate and finally dried in air. I checked this on TLC.

The above solid was dissolved in 50% of trifluoroacetic acid and methylene chloride.
150 (by volume) added to mixture 21.

The solution was stirred for 10 minutes at room temperature and evaporated to dryness at room temperature in a 42-degree vacuum. Take this residue in ether and wash it.
Examined with TLC and N rope.

Yield: 339 g (90 parts) (expressed as white solid trifluoroacetate) 2. H-kla-Ar g (No 2 ) L
e 1+ -NH2443g H-Arg(No2)L
eu-NT (2 trifluoroacetate to 21 DM
Dissolved in F. 200g BoC-Ata-OH was added followed by 500g BOP. pl (using a test paper (taking a small amount of the reaction solution), -( was adjusted to 7 with NEM).

The solution was stirred and the progress of the reaction was checked by TLC. The reaction was stopped after 4 hours. The residue was taken up in 21 parts of water and 21 parts of ethyl acetate. The organic phase was washed with 5-NaT (aq. CO3, water) and evaporated to dryness. The tribezotide was recrystallized in ethyl acetate/ether and finally dried in vacuo. This was checked by TLC and NMR.

The product was dried and treated under the same conditions as in Example 1-1.
Treated with a 50-50 (body 2&) mixture of FA/CH2C42.

Yield: 412 g expressed as white powder trifluoroacetate (Part B) (checked with TLC and N plate) 3. Arg(NO2)-AmuArg(No2)-
According to the operating conditions of LeU-NH2 Example 1-1, TFV
By treatment with CH2C42 mixture, 2゜51
515 g of H-Ata-Ar g (
No2)-Leu-NH2 and 333g of BoC
-Arg(No2)-OH and 500! ? (7) B.O.
607 g (85 V) of white solid was obtained, which was checked by TLC and gate.

4, Z-A Mu Arg (No2)-Ata-Arg
-Leu-NT (T' of 41 in the same manner as in Example 1-2 (but TFA/CH2C42 treatment was not performed))
715g of E-Arg(No2)Ata- dissolved in MF
Arg(No2)-Leu-NH,.

of trifluoroacetate and 233 g of 2-Ata-
OT (and 500g of BOP, 612g of Z-A
ta-Arg(No2)-Ata-Arg(No2)L
Eu-NH2 (76%) was obtained, which was recrystallized from a DMF-ether mixture and the resulting white powder was checked by TI, C and NMR.

5. Ata-Arg-Ata-Arg-Leu-N
H2*3 HCL200g Z-Ata-Arg(
No,,,)Ata-Arg(No,,)-Leu-
NH2 (0.25 mol) was suspended in 271! methanol containing 0.8 mol T(CL). 40 g Pd/C
(10% Pd) was added and this was stirred for 24 hours under a pressure of 1.2 pars under a hydrogen atmosphere. Thereafter, the completion of the reaction was checked using TI and C.

The catalyst was filtered off and the solvent was evaporated in vacuo at room temperature.

The solid residue was purified by chromatography on silica dal. The eluent was 50:12:12:25 (butanol, pyridine, HO2CCH3, H2O).
volume) mixture was used.

Fractions containing pure Hiko product were collected, evaporated and lyophilized.

Yield: 119g' (69cm) of white powder Check: NMR, TLC Amino acid analysis: Leul, 03 (1) Arg 1.92 (2) Ata 1.95 (2) 45 Example ■ RoC-(, tu( OBzt)-Arg-Gty-OT
(, Synthesis of HCt (fragment Bl) 1, Z-Arg(No2)-Gty-OBztH-
3.37 g (0.01 mol) of tosylate of Gty-OBzt was dissolved in 15 ml of DMF' and 1 equivalent of NEM (
Added 1.3 ml. This solution was mixed with 1 equivalent of NEM (
3, dissolved in 15 ml of DMF containing 1,,3 ml)
53 g (0.01 mol) of Z-A r g (No2
)-0T(added to).

4.5 g (0.01 mol) of ROP was added to the reaction mixture, the pH was adjusted to 7 by adding NEM, and the mixture was incubated for 2 h at room temperature.
The mixture was stirred for 0 minutes (completion of the reaction was confirmed by TLC: chloroform-methanol # (3/1)).

The reaction mixture was evaporated to dryness under reduced pressure (0,1,)l at a temperature below 30°C. The residue was taken up in 100 ml of ethyl acetate. This solution was strongly stirred with 120 g of ice water.
Pour into m1. After stirring several times, a precipitate was obtained, which was left overnight in the refrigerator.

The solid with the following solid dimensions is continuous 46- I washed it properly.

2 times with 100 ml of IIium aqueous solution
5% So HK-8o4 with 10101 liters of 2 aqueous solution
The mixture was washed twice with 100 ml of water and twice with 100 ml of ether and then dried in vacuum (0.1 Torr) to constant weight.

Yield: 4g (80cm) Koffler melting point: 149°C TLC: Chloroform-methanol (3/1) Rf: 0
．． Check with 73NMR 2, HC/--T(-Arg-Qty-OH600
100 g (0.2 mol) of Z-Arg (NOz )-Gty-OBzt were suspended in a mixture of ml of water, 600 ml of N-hydrochloric acid and 100 ml of tetrahydrofuran. 60g of Pd/C (Pd10) containing 50g of water was added. This mixture was heated at room temperature under a pressure of 15 Torr for 4 hours.
Hydrogenation was carried out for 8 hours. Filter the catalyst, add Amberly) I R4541 fat (OH type) to the aqueous solution, and add PI
I was set to 6.5. Washed the resin. The pH of this solution is
Fresh resin was added to 85 ml and stirred in a rotavapor for 20 minutes under vacuum (25 ml). After washing the resin and checking the presence of ammonium chloride in the aqueous solution by Nessler's test, it was evaporated to dryness under reduced pressure (0,1) at a temperature below 30°C. A gummy residue was obtained, which was dried overnight in a desiccator containing phosphoric anhydride.

Yield: 43.19g (80.6g) TLC: Ethyl acetate-pyridine-formic acid-water (40-2
1-10-6) RBo, x Saka rotist positive (red spot) Check by NMR 3, Hoe-Gtu (OBzt)-Arg (HCt
)-Gty-OH41,59 T(CL-H-Arg-
Gt3'-0T ((0,155 mol), 400 ml
of DMF and 130 ml of water. - was adjusted to 7 by adding N hydrochloric acid.

The following were simultaneously added to this solution:

・68 in a mixture of 100 ml of DMF and 40 ml of water
．． 8g of Hoe-Gtu(OBzt)-ONP (0,
150ml) dissolved in a mixture of 100ml DMF and 40ml water.
．． 7 g of hydroxybenzotriazole (0,150 mol) dissolved in 37.8 ml of NEM in 100 ml of T') MF.
(2XO, 150 mol) was dissolved at room temperature. 6) was checked.

After 1 hour of reaction, 13.76 g of Boa-Gtu (OBz
t)-ONP was added.

After 2 hours of reaction, 13.76.9 T3oc-Gtu(O
Bzt)-ONP was added.

After 3 hours of reaction, 7 g of Boc-Gtu(OBzt)-O
NPs were added and the reaction continued for 1 hour.

The reaction mixture was then evaporated to dryness under reduced pressure (0.1 Torr) and at a temperature below 30°C. A thick oil was obtained which was dissolved in 500 rnl of ethyl acetate.

Addition of ether 11 gave a thick oil.

This was left in the refrigerator for 24 hours and the liquid phase was decanted.

The remaining oil was taken up in 500 ml of methanol.

Add 500g of silica (70-230 mesh) to this solution.
was added and the solvent was evaporated using rotor paper. The resulting powder was mixed with an 80:20 mixture of chloroform and methanol and the resulting glue was placed on top of a column of silica dal (200 m high, 85 tran diameter) packed in an 80:20 mixture of chloroform-methanol. I put it in.

The product was eluted using:

・80:20 mixture of chloroform and methanol: 25
1. Chloroform and methanol 50:50? JL compound:
101 ・Methanol: 301 Purity checked by TLC (& lysine-ethyl acetate e-water, 2L
-40-10-6) was used to check 50-.

Two fractions were collected.

・Fraction A29.80.9 TLC: pyridine-ethyl acetate-formate-water.

21-40-10-6: Rf: Check with 0.75N HpLc-gpp: 96.9 schiAAA: G
tu 1.01- Gty 1.01- Arg O,
98. Fraction B 36.82 g TLC: pyridine-ethyl acetate-water.

21-40-10-6: Rf: 0.7511 (N-plate Suictor compatible HPLC-EPP: 99.61 Section AAA: GLu 0.93- GLy 0.99-
Arg 1.08 Harunai 1 Progress” Boa-Gtu (OBzt)-8er-Asn-Gtn
-OT((fragment B2) synthesis 1, Boc-Asn-Gtn-OBztTFA,
500 m of H-Gtn-OBzt (0,445 mol)
1 in DMF.

The following were successively added to this solution:

11103.3. Boa-Asn-OH (0,4
45 moles)・62.3mA! of NEM · 217.8 g of BOP (0.5 mol).

Next, NEM was added to bring the solution back to -7.

The mixture was stirred at room temperature and NEM was added as needed to maintain -7. The reaction was checked by TLC (chloroform:methanol=3:1). After 1 hour, the reaction was stopped and 1.51 g of ethyl acetate was added to precipitate the product. After stirring this for 1 hour, it was left in the refrigerator overnight.

Drain the solid and wash it three times with 500ml of ethyl acetate.
Washed with ether (11) and dried.

Yield: 108.13g (53.9g) TLC: Chloroform-methanol (3:1):Rf
: 0.63 2, TFA, T(-Agn-Gtn-OBzt
102I Boe-Asn-Gtn-OBzt(0,2
66 mol) in 500 ml of T cooled in a water bath.
Dissolved in FA. The aquarium was removed and stirred at room temperature for 15 minutes. Light insoluble material was discarded and stirring continued for 15 minutes. The reaction solution was concentrated to 1/4 under reduced pressure (25 torr) and taken up in 300 ml of ether.

Repeat this operation twice (2x300ml of ether)
, residual solvent was removed in vacuo (25 Torr).

A hygroscopic white solid was obtained.

Yield: 107g TLC: Chloroform-methanol = 3:1:Rr:o
, os 3, Boa-8er-Asn-G/, n-0BzA
0.226 mol TFA, H-Asn-Gtn-O
Bzt was dissolved in 1.51 DMF. The following were added sequentially to this solution:

-48,4I! BoC-8or-OH, 1/2 H2
O (0,266 mol) ・34.8 ml NEM (0.27 mol) ・111 g
of BOP (0.25 mol) then NEM was added to bring the solution back to -7.

The mixture was stirred at room temperature, adding NEM if necessary to keep the temperature at 7. The progress of the reaction is T
LC (chloroform-methanol-acetic acid 9-2-0.5
) was checked. After 2 hours, the reaction was stopped. Light insoluble materials were filtered and the reaction solution was purified under reduced pressure (0.1 Torr).
It was evaporated to dryness at a temperature below 30°C.

The residual oil was taken up in 806 ml of ethyl acetate.

After stirring this for 1 hour, it was left in the refrigerator overnight.

Drain the solid and wash with ethyl acetate (2 x 200 ml
), then x-chill, (200rnl) wash,
Dry in vacuo.

Yield: 108.32g (89%) TLC: Chloroform-methanol-acetic acid, 9-2-0
．． 5Rf:0.42 4, TFA*H-8er-Asn-Gtn-O
BztO 12 mol TFA + T(-8er-Asn
-Gtn-OBzt was dissolved in 11 water. 22.49+7) Pd/C (Pb 10%) containing 50 g of water
was added to this solution. The mixture was stirred at room temperature under 15 torr for 24 hours with stirring. The catalyst was filtered and dried under reduced pressure (0
, 1) and evaporated to dryness at a temperature below 30°C. The residue was taken up in ether and the solid was drained, washed several times with ether and dried.

Yield: 139°C TLC: Tetrahydrofuran-bilinone-formic acid-water = 6
0-20-10-6 :Rf:0.81 5, TFA + )(-8er-Asn-Gtn-
OT(0, 2mofiV TFA r T(-8er-A
Dissolve sn-Gtn-OBzt in 11 water]-7. 5
22.4. Contains 0% water. ! 9 Pd/C (Pd 1
．． 0) was added to this solution. The mixture with
Hydrogenation was carried out under a pressure of Torr for 24 hours with stirring power. The catalyst was removed and evaporated to dryness under reduced pressure (0,1) liters at a temperature below 30°C. The residue was dried overnight in a desiccator containing phosphoric anhydride.

Yield: 93.66g TLC: Tetrahydrofuran-gylysine-formic acid-water,
60-20-10-6: Rf: 0.45 6, Bee-Gtu(OBzt)-8er-As
n-GAn-OH0.2 mol of TFA-T(-8e r
-Asn-G)', n-OH with 100 ml of water and 60
Dissolved in a mixture with 0 ml DMF'.

Adjust the pH of the solution to 7 by adding NEM to 17.87.11 g
A solution of Boc-Gtu(OBzt)-ONP (0.19 mol) in 600 ml DMF was added followed by 29.59 HOBT (0.19 mol). N
EM was added to bring the mixture back to -7 and stirred at room temperature. After stirring for 1 hour, 2.7 g of BoC-G7u (O
Bzt)-ONP was added.

The reaction was analyzed by TLC (tetrahydrofuran-pyridine-formic acid-
O checked with water = 60-20-10-6 and chloroform-methanol-acetic acid = 95-5-3) After 30 minutes of the last addition, the light insoluble material was filtered and under reduced pressure (0.1 Torr) It was evaporated to dryness at a temperature below 30°C. The resulting oil was taken up in 800 ml of ethyl acetate and a slightly gelatinous precipitate formed. It was removed and washed with ethyl acetate (2 x 40 lll), then ether and dried.

Yield: 114.3 g (90%) The product has to be washed again with ethyl acetate (1,21), stirring for 4 hours at room temperature. The solid was removed, washed with ether, and dried.

Yield: 95.85g (71.9g) TLC: Tetrahydrofuran-pyridine-formic acid-water = 6
0-20-10-6Rf:0.62n-butanol-pyridine-acetic acid-water=50-12-12-5Rf:0.46IT (NMR compatible Hpbc-gpp: s 7.s IAAA:
Asn(Asp): 1.01-Ssr: 0.
89-Gtu (Gtn): 2.05 Melting point (Kofler): 134°C Decomposition example ■ Boa-8or-Arg-Gtn-Gtn-Gty-O
Synthesis of H*HCL (Fragment C) 57-1, 75.36 g of HCl H-Gty-OMs (
168.2 g of Z
-Gtn-OH (0,6), 256.41 g of BOP (
0.7 M) and 165.4 ml of 'pJ-x Chi/l/
-1=/l/folin (1,3M) was added to this.

After reacting for 20 hours at room temperature, the solution was concentrated under reduced pressure and
The residue was taken up in ethyl acetate. This organic solution was washed with:

eSodium bicarbonate solution ・Sodium chloride solution ・KH804/2S04 solution ・Sodium chloride solution The organic solution was dried over MgSO4 and filtered.

After standing for 20 hours, crystals precipitated. After filtration, the crystalline fractions in aqueous medium were collected, washed with water saturated AeOEt, filtered and dried in vacuo to yield 7.78 g of product (m++pe 158-159 C).

The crystal fraction in AeOEt was converted to water-saturated Ac0Et.
After washing with 58° C., filtration and drying in vacuo, 61.89 of the product (m, p, : 150-154° C.) was obtained.

Further crystals were obtained from the aqueous and organic phases.

Collect these crystals, wash them as described above, and... 9 products of similar nature were obtained.

Overall yield: 107.63 g - checked by NMR and TLC 2, HCL, H-Gtn-Gty-OMe70
61. in 4 ml DMF and 880 ml methanol.
8g Z-Gtn-Gty-OMe (175,9mM
) in a solution of 176 m while cooling in a water tank.
1 N HCl solution and then 3.19 pa/c (
10% Pd) was added. After hydrogenation under pressure of 35 Crn mercury for 3 hours and 30 minutes, the catalyst was filtered over Celite and the solution was concentrated under reduced pressure. The residue was sifted and used in the next step.

3. The Boa-Gtn-Gtn-Gty-OMe residue obtained in the above step (theoretically 175.9 mM) was dissolved in 500 ml of DMF and cooled in a 1-1 water bath.
7.69 Boc-GLn-ONPs (211,1mM
), 26.4 g of hydroxybenzotriazole (2
11,1 mM), and N-x tylmorpholine was added until the voice was 7. After reacting for 200 hours at room temperature, the solution was solidified in one piece. Addition of ethyl acetate resulted in a precipitate, which was washed with AcOEt and dried in vacuo to give 64.27 g of product (yield: 82 g).

I checked with Todoroki M and TLC.

4. TFA = H-Gtn-Gtn-Gty
-OMe 98.83 g of Boc-Gtn-GLn-GLy-OMe in 300 mJ of dichloromethane, 400 ml of TFA in a cooled suspension in a water bath.
added.

After reacting in a water bath for 30 minutes and at room temperature for 1 hour, it was concentrated under reduced pressure to half the volume, and the residue was poured into stirred ether. After filtration, washing and drying in vacuo, 119 g □ of product were obtained.

5. Bee-Arg(No2)-Gl-n-G1.
119 g of the above n-Gty-OMe product was dissolved in 11.90 mJ of DMF' cooled in a water bath, and 7
゛7.91g of Hoe-Arg(NO2)-0H(24
4mM), 97.51g BOP (266,2mM
), and N-ethylmorpholine was added until the pH reached 7. After reacting for 200 hours at room temperature, the solution was poured into 81 ml of ethyl acetate. The obtained precipitate was filtered and Ac0Et
Wash and dry in vacuum 1. Ta. 153.7 g of product was obtained. Checked by NMR and TLC.

6, TFA, H-Arg(No2)-Gtn-
Gtn-Gty-OMe 600 cooled in water tank
153.7 g Roe-Arg in ml dichloromethane
(NO2)-Gtn-Gtn-Gty-OMe (theoretically 221.8 mM) was suspended at 750 m
1 of TFA was added. After leaving for 1 hour at room temperature 250
ml of TFA was added, and after 30 minutes, an additional 25 (1/ml of TFA) was added.
Added A. After reacting for 1 hour, the solution was concentrated to 1/3 of the volume, and the residue was poured into 31 ether with stirring. The precipitate was removed, washed with ether and dried in vacuo.

167 g of product were obtained.

61-7, Boa-8er-Arg(No2)-Gtn-
1,51 cooled in Gl,n-GLy-OMe water bath
1679 TFA in DMF l)! -Arg(NO2
)-Gtn-Gtn-GtY-OMe (theoretically 2
21.8mM) dissolved in 97.52jj of B
oc-8er-ONb (266,2?FIM), 33
．． 31 g of hydroxybenzotriazole (266,2
(mM), and N-ethylmorpholine was added at pH 7. After reacting for 3 hours at room temperature, 1 of DMF
The remaining solution was poured into ethyl acetate with stirring. The precipitate was filtered, washed with ethyl acetate and dried in vacuo. 153.3 g of product was obtained (yield 94
．． 2%). Checked by NMR and TLC.

8, Boc-8er-Arg-Gtn-Gtn-G
ly-OMet AcOT (11 methanol, 1
153.3 g of Boc- in 1 part of water and 500 ml of acetic acid
8er-Arg(NO2)-Gtn-Gtn-Gty-
A solution containing OMe (208.9mM) was mixed with Pd/
Hydrogenated in the presence of 10 g of C (10 pd) for 20 hours. After filtering off the catalyst and concentrating the solution, the residue was taken up in water and lyophilized. 153 g (yield: 62-97.8) of product was obtained.

9. Boe-8er-Arg-Gtn-Gin-G
104.4g in DMF of ty-OIT + HCl21
Boc-8er-Arg-Gln-Gtn-Gty-
A clear solution containing OMe (139.4mM) was added with 11 g of water, then 27.88 g of Na0T (in 50 ml of water) was added.
(697 mM) was added to the solution after cooling in a water bath. After reacting at around 15℃ for 15 minutes, the pH of the liquid
At the age of 6.5, it was neutralized with N-hydrochloric acid. After evaporating,
The residue was triturated in ethyl acetate. Take the obtained precipitate,
Washing with AcOEt, drying in vacuo L and then drying in air gave 132.79 of the product.

Purification: 109g Roe-3er-Arg-GLn
-Gln-GLy-OT (, HCl was purified by countercurrent partitioning in a n-butanol-methanol-water (4:1:5) mixture.

After 700 transfer L/L, the product was divided into three fractions, evaporated and lyophilized, yielding 27.5 g from one fraction and 23.7 g from each of the two fractions.
9 g and 16.90 g of product were obtained, the latter two fractions being repurified. Checked by NMR and TLC.

AAA: Ser O,91-Arg: 0.97
-Gtn(G/!, u): 2, 05- a, ty:
1.01 Example V uoe-Asp(onzz)-rze-Met-NH-
Synthesis of NH2 (fragment D) 1, Hoe-Met-NH-NTT Troc50
4.31 to ml of ethyl acetate,! i' (10mM)
A solution of Boa-Met-OHe DCHa dissolved in 2
Treated with a saturated solution of potassium hydrogen sulfate in the presence of 0 ml of water at -4 to 3. The aqueous phase was extracted several times with ethyl acetate and the extracts were dried over magnesium sulfate. 2.28g (11mM) of H2N-NT (-T
roc (Troc = 2.2+2-)lichloroethoxycardenyl. This is Ya Tuma Chem, Pha
rm.

Bull, 1,971.19,420) was added.

After cooling in a water bath, dissolve in 10 ml of ethyl acetate.
．． 379 (11 mM) of 97% DDC was added. - After the night, the product gradually returned to room temperature, the dicyclohexyl urea was filtered and dried (2.04 g), and the organic solution was washed successively with the following aqueous solutions.

5 sulfuric acid 4 times - hydrogen sulfate 2 times, NaC12M2x5 sodium bicarbonate 2 times, NaCl2M 2 times, and finally water 2 times.

After drying with magnesium sulfate and concentrating until substantially dry, hexane was added to the point where it became thick and the product was allowed to stand overnight at 4°C. The precipitate was filtered, washed with a 4:1 mixture of hexane and ethyl acetate, and dried in vacuo. 346 g (79 g) of product was obtained. m, p, -92 TLC: Chloroform-methanol-AcOH (95:
5:3) Rf:0.45 2, TFA, H-Met-N)T-NHTro
c 43.9 g (0.1 M) Roc-Met in 200 ml dichloromethane and 20 rnl ethanedithiol
The M solution in which -NH-NH-Troc was dissolved was treated with 200 ml of trifluoro-65-acetic acid in a water tank while stirring under a nitrogen atmosphere, the water tank was removed, and the product was incubated for 1 hour or i.
-4 points. The volatile reagents were first removed under a pressure of 20 Torr and then 0.1 Torr, and the remaining oil was washed twice with 75 ml of InsolonoP-Nol, evaporated in vacuo and 75 ml!
The product was separated by washing twice with hexane and decanting, and dried overnight in vacuo in the presence of potassium hydroxide until it became hard and sweet. ! Used in 1 or less steps.

3. Boc-Ite-Met-NH-NH-Tro
The above oil (approximately 0.1 M) of cTFA, H-Met-NT (-NHTroc) was dissolved in 6500 ml of ethyl acetate, cooled in a water bath, and this was dissolved in 12.7 ml (0.1 M
) of N-x tylmorpholine, then 14.85 g (
0,M) of HOBt・■H20, then 34.35g(
0,08M) 130cm1te-O3u z then 1
Treated with 2.7 ml (0.1 M) of N-ethylmorpholine (NEM).

After 30 minutes, the aquarium was removed and NEM continued to be added periodically to maintain -17.

After 20 hours, the reaction was complete and the next aqueous solution I washed it continuously and cleaned it every minute.

5-Ti salt - Potassium bicarbonate 3 times, water 3 times, 5-Ti sodium bicarbonate 3 times, and finally water until neutral.

After drying over magnesium sulfate and evaporating the solvent in vacuo, 64. A gum of q was obtained and chromatographed on a silica column of dichloromethane containing 0-1.5 parts methanol. T(PL,C purity is 98.9
30 g (68 t) of product were obtained which had the expected structure according to the NMR spectral structure. m, p, 88
~92℃4, TFA, H-I7e-Met-N
l(-NH-Troc140ml of dichloromethane and 1
27.69 (50mM) in 4ml ethanedithiol
Boc-I/,e-Met-NH-N)T-Tro
The solution of c was cooled in a water bath, stirred under nitrogen atmosphere, and then 140 ml of trifluoroacetic acid was added thereto over 5-6 minutes. Remove the aquarium and after 45 minutes,
Evaporate the reagent to the maximum and remove the residual oil at 2X60mA! The product was isolated by taking up in isozolopanol, evaporating and finally washing twice with 60 ml of 400 ml of sodium chloride. After drying over potassium hydroxide in vacuo overnight, a glassy oil was obtained that was used in the next step (3 og).

5. BoC-Asp(OBzt)-Dingte-M
et-NH-NT(-Troc 250 g of the above oil
ml of THF', 5.4 ml (50mM) of NEM and 18.9g (45mM) of Roe-Asp.
(OBzt) Treated with O8u. NEM was continuously added to maintain pi at 6-7. After 4 hours, the T liver was evaporated, taken up in 400 ml of ethyl acetate and the product was separated by washing as described in (3) above. After drying and evaporating the residue (36,5 g), it was mixed with 0-1.5%
It was purified by chromatography on a silica column using dichloromethane containing methanol as the solvent.

25 showing an NMR spectrum consistent with the expected structure.
g (66'l of material was thus obtained. m,
p, =96-100°C 6. BoC-Asp(OBzt)-Ite-Met
-NH-NH2DMF and Ac0T (7.57fl (10mM) in 80ml of an 80:20 mixture of Roe-As
p(OBzt)-I/, e-Me t-NT(-N
The solution containing Troe was treated with stirred powdered zinc 6,54...9 for 30 minutes.The zinc was filtered and the filtrate was treated with 800 g of ice until the ice melted. The product was isolated by filtration and repeated washing of the filtrate with water. The white solid was dried first in air and then in high vacuum in the presence of potassium hydroxide and phosphoric anhydride. TLC and filtration. 5, with a purity of about 95% according to
60 g (96%) of product was obtained.

m, p, 180-185℃, checked by TLC and HPLC Example ■ Boe-Arg-Lys(Z)-Leu-Leu-OH
(Cross section E1) 1, Boa-Leu-Leu-OMe
18.4. ! 5 containing NEM (0,16M) of il
In 00 ml of dioxane, 9.059 H-Leu-
OMe, HCL (0.05M), 7.4g HOB
t(0,055M) and 18g of BoC-Leu-ON
Su (0,055 M) was added continuously. The product was stirred at room temperature for 18 hours and the pH was adjusted to 6-7 during the reaction using PII test paper and adding NEM if necessary. The solvent was evaporated, the residue was dissolved in Ac0Et, and the organic solution was washed continuously with 5% K
T(SO2-, washed twice with SO2, NaCt/water, 5t NaHCO3, NaCl/water. The product was washed with MgS
Dry over O4 and evaporate the solvent. The residue was dissolved in a minimum amount of ether and the product was precipitated with a sieve, which was taken up and dried.

Yield: 15g (83%) TLC: Chloroform-methanol-acetic acid = 5-5-3 AeOEt-hexane 25/75m, p,: 131~
136°C Silicadal 6o Merck (70
-230 mesh) and eluting with the same solvent.

2. H-Leu-Leu-OMe + TFA27
, 5 g of Boc-Leu-Leu-OMe (0,0
76M) was covered with 1 gQmz TFA at room temperature.

He developed a slight fever. After 30 minutes, the solvent was evaporated and the residue -
70 = Take up oil in ether and scratch product. A white precipitate was obtained. Intane was added and the product was taken and dried.

Yield: 2s, 2g (loochi) TI, C: Chloroform-methanol-acetic acid 9515/
3 Chloroform-methanol-acetic acid 80/1515 3, Boe-LylI(Z)-Leu-Leu-O
The following were added sequentially to 600 ml of AcOEt cooled in a Me water bath.

-28.2g of H-Leu-Leu-OMe, TF
A (0,076M)・26.22g of NEM (0,
0228M')・11.23g of HOBt (0,0
83M)-46,4g of Hoe-LyII(Z)OT
cp (0,083M) The product was stirred and the water bath was removed.

If necessary, -1 was adjusted to 6-7 by NEM.

After 18 h, the organic solution was continuously diluted with 5 tKT (SO2-2So4, NaCt/water, 5'lr NaHCO3,
Washed with NaCt/water. The product was dried over MgSO4.

The solvent was evaporated and the residual oil was dissolved in chloroform and silica gel 60 Merck (70-2
30 mesh) column (height 90 m, diameter 5 cm)
) placed at the top. The product was eluted with chloroform and fractionated. The fractions containing pure product were evaporated and the resulting foam was redissolved in ether and evaporated to dryness. The residue was ground in an inthane, drained and dried.

Yield: 42.4g (91g) TLC: AeOEt/hexane 1/2 chloroform-MeO! (-Checked with AeOH95/5/3 NMR. HPLC-gPP: 98.7 CH4, B
oC-Arg-Lys(Z)-Leu-Leu-OMe
15.5g Boc-Lys(Z)-Leu-Leu
-OMe (0,025M) was added to Zooml T at room temperature.
Covered with FA. After 30 minutes, with occasional stirring, the solvent was completely evaporated. Dissolve the residual oil in 150 ml of dioxane and use DIPEA to measure 6 to 7 using test paper.
I made it. The product was cooled in a water bath and 7.21 g of Boe
-Arg-OH, H2O, HCL (0,025M)
, 13.44 gnoBOP (0,03M) and 10
．． 32. ! 9 of IMPEA (0.08M) was added. This was stirred, the water bath was removed, and the necessary PL was adjusted to 6-7 with DIPEA. After 18 hours, the dioxane was evaporated and the residual oil was dissolved in Ac0Et to a concentration of 1-.51.
NaT(COs, washed successively with NaC4/water. The product was dried over MgSO4, the solvent was evaporated, the oil was dissolved in a 5:95 mixture of MeOH and chloroform saturated with water, and this same mixture was A column of 6° Merck (70-230 mesh) silica gel as a solvent (height 85 cf.
n1 (4 cm in diameter). The product was eluted with:

5:95 mixture of 1,51 MeOT(/chloroform) saturated with water - 7.5:92.5 mixture of 1,11 MeOT(/OH/chloroform) saturated with water - 2,57 MeOT(/chloroform) The product was fractionated and the pure fractions were evaporated, taken up in ether, completely evaporated in vacuo and extracted.

-73 = Yield: 16.8g (87g) TLC: Chloroform/MeOT(/AcOH95
: 5:3 Chloroform/MeOT(/AcOH80:15
: 5 Check with NMR IF (PI, C-EPP 91.
8 Section 5, Boa-Arg-Lys(Z)-Leu
-Leu-OH13.98g of Boc-Arg-Lys
(Z)-Leu-Leu-OMe (0,018M) 2
Dissolved in 50 ml of methanol. Add 40ml of water and then add 5.67g of crushed parita (0,
018M) was added.

The product was stirred at room temperature for 2 hours and 30 minutes. The end point of the reaction was confirmed by TLC. CO2 was bubbled through until -6 and the product was filtered. The filtrate was evaporated, the residue was dissolved in isoblockol and filtered, the alcohol was concentrated and the product was precipitated with ether.

Yield: 12.31g (89g) TLC: Riooform-MeOH-AeOH90/1
55 74- 2-Butanol-AcOH-Water 72/7/21 Check by NMR npLc-Epp: 95.8
s-Example ■ Boc-Gtn-Leu-8er-Ata-(')Me
(Fragment F1) 1, Boa-8er-Ata
-OMe25g H-Ata-OMe, HCl and 6
69 Boc-8er-ONb in 500 ml DMF
It was dissolved in The product was cooled in a water bath and 25 ml of NE
24 g of N-hydroxypenzotriazole was added while stirring with a magnetic stirrer, and NEM was added to adjust the pH to 6.5-7. The product was heated at room temperature to 18
I stirred the time. After checking by TLC, the solvent was
The mixture was evaporated at 1 (0.1 μl, 35° C.). The resulting oil was dissolved in 1500 ml of chloroform and the product was saturated with chloride).
5 times). The solution was dried over Na2SO4 and evaporated to dryness. An oil is obtained, which is dried by sieving to a constant weight (30° C., 1 Torr).

Yield: 53.78゜q -TI, confirmed by C2, H
-8er-Ata-OMe, TF'A52.1
9 of Boc-8er-Ata-OMe was added to 100 ml of methylene chloride chilled in an ice bath while stirring with a magnetic stirrer. 250 ml of cold TFA was added and the product was stirred at room temperature for 20 minutes and filtered. The filtrate was evaporated to dryness (35°C water jet pump). Addition of 21 ether resulted in a white solid precipitate. It was drained, washed three times with ether, and dried to constant weight (30° C., 0.1 Torr).

Yield: 40.82,93, Boa-Leu-8er-Ata-OMe4
0.9 H-8er-Ata-OMe + TFA and 3
2.9g of Boc-Leu-OHe H,,O in 40
Dissolved in 0 ml DMF.

The product was cooled in a water bath, and from the magnetic stalate, 14.4 ml of NEM and 65.8 g of B were added.
Added OP and enough NFM to keep - between 6.5 and 7. The product was stirred at room temperature for 2 hours. After checking by TLC, the product was evaporated to dryness. )
The resulting oil was dissolved in 1500 mJ of chloroform, washed twice with saturated NaCt solution, three times with 5-thiosulfate/potassium bicarbonate, three times with saturated sodium bicarbonate, and dried over Na2SO4. , evaporated to dryness (30°C
- water jet pump). b1) The resulting oil was solidified in ether. Addition of fragment A = 299 hexane resulted in a second jet B, which was identified by TLC. B=1
3.7g Yield: 42.7g, checked by N plate and TLC 4, H
-Leu-8or-Ata-OMe, TFA29
g of Boe-Leu-8er-Ata-OMe was suspended in 50 me of methylene chloride, which was chilled in a water bath.

100mA! After adding and dissolving the cold TFA, the product was stirred at room temperature for 30 minutes and filtered. The filtrate was evaporated to dryness (35°C, water jet pump).

The resulting oil was solidified in ether (with several changes of solvent). The product was dried to constant weight (30°C
, 0.1 Torr) 77- Yield: 30.2. ! i' 5, Boe-GLn-Leu-3er-Ata-
OMe30g T(-Leu-8er-Ata-OMe
, TFA and 25.5.9 Boe-Gtn-ONP
300 while stirring ≠λ with a magnetic stirrer.
ml of DP/yF. The product was neutralized with 9.7 ml of NEM. Add 1 eg OHB t, NEM
was added and stirred at room temperature for 2 hours while maintaining the pH at 6.5 to 7.

After checking with TLC, concentrate the liquid to 90% and reduce to 0.1
), 35°C), and 10,100O. The product precipitated in the form of a glue. Take the product and wash it, 5
Extracted twice with 00ml of chloroform and three times with ether. The product was dried in a constant weight trench (0.1 Torr, 30°C).

Yield: 32.1g TLC: Chloroform/MeOH/AcOH9/
210.5 Rt: 0.81 HPLC: EPP = 91ch AAA: Ser 0.86- Gtu 1.02-
Ata 0.99-Leu 0.9 9 78- Example ■ Boc-Tyr-Arg-Lys(Z)-Vat-Le
u-Gty-OT(, T(C6 (fragment G1) 1, Z-Leu-Gty-OCH30, 22 mo/l
/ (7) DM Z-Leu-OH for 500 rll
Dissolved in F. The following were added sequentially to this solution:

・25.11g of HC/! , , H-Gty-OCH
3 (0.2 mol) · 28 ml of NEM · 969 BOP (0.22 mol) Then, NEM was added to adjust - to 7.

The reaction mixture was stirred at room temperature, keeping its −7 by adding NEM if necessary. The reaction was analyzed by TLC (chloroform/methanol/acetic acid = 9515/9). After 2 hours the reaction was complete.

The reaction mixture was evaporated to dryness under reduced pressure (0,2) liters at a temperature below 30°C. The residue was taken up in 1/2 ethyl acetate.

The solution was washed successively with:

・Hydrogen carbonate) IJum aqueous solution (4X200m/)-
5o4HK-8o4 to 2 aqueous solution (4X 200 ml
)・Chloride) IJium saturated aqueous solution (2X200mA'
) Dry LJ over magnesium sulfate and evaporate to dryness under reduced pressure. The residue was taken up in ether (500 m/s), triturated, drained and dried.

Yield: 56.7g (84.28g) TLC: Chloroform-methanol-acetic acid 9515/3 Rf: 0.44 2, HCL-H-Leu-Gty-OCH3569
(0,166 mol) of Z-Leu-Gty-OCIF (
.

was dissolved in 900 ml of 0123N hydrochloric acid methanol. This solution contains P d/C (10
%Pd) 69 was added. Hydropower generation was carried out at room temperature and atmospheric pressure for 24 hours. Filter the catalyst and remove the solution under reduced pressure.
It was evaporated to dryness at a temperature below 30°C. Pour the residue into ether (
2×50 ml), decanted and the product was dried in a desiccator in the presence of phosphoric anhydride. A white powder was obtained.

Yield: 33.53g (84.4g) TLC: Chloroform/methanol/acetic acid = 90/2
0/9 Rf: 0.15 Confirmed by NMR 3, Hoe-Vat-Lsu-Gty-OCH32
8,24,F Boc-Vat-OH (0,13 mol)
400 m of tetrahydrofuran cooled to -10°C
Dissolved in l. 14.45 ml (0.13 mol) of isobutyl chloroformate was added.
Stir vigorously for 15 minutes at ℃. This mixture contains -10
31.5 g of HClH-Leu-GLy
-OCH3 (0.13 mol) in advance 14.45 m/
of tetrahydrofuran neutralized with N-methylmorpholine was added. The reaction mixture was stirred in a water bath for 2 hours and then at room temperature for 2 hours. The reaction mixture was removed under reduced pressure.
It was evaporated to dryness at a temperature below 30°C. The residue was taken up in a 500M-500mg mixture of ethyl acetate and water. The aqueous phase was decanted and the organic phase was washed successively with:

・Hydrogen carbonate) IJium aqueous solution (2X50ml)-S
o4! (K-8o4 to 2 (pH2) aqueous solution (2X50m
/) 81- - Sodium chloride aqueous solution (50 mA, dried over magnesium sulfate and evaporated to dryness under reduced pressure at a temperature below 30°C. The residue was taken up in pentane (50 m7), drained and dried in vacuo. (0.1) le).

Yield: 39.7g (76g) TLC: Chloroform/acetone = 75/25Rf =
0.46 Confirmed by NMR 4, TFA, H-Vat-Leu-Gty-O
CR339g Boc-Vat-Leu-Gty-OC
H, (0,097 mol) in 190 ml cooled in a water bath.
d in TFA. After dissolution, the water bath was removed and the mixture was stirred at room temperature for 30 minutes. . The product was evaporated to dryness under reduced pressure and the residue was taken up in 100+/ml ether.
Triturate and decant the ether. This operation was repeated twice and the last traces of solvent were removed under reduced pressure (0,1) liters.

Yield: 44.8g TLC: Chloroform/methanol/acetic acid = 82-90/20/3 Rf: 0.36 5, Boc-Lys(Z)-Vat-Leu-Gt
y-OCH325,55g TFA, H-Vat-
Leu-Gty-OCT (3 (0,0615 mol))
00 ml of DMF. NEM was added to bring the pI of the solution to 7. 32.47 g of Bo
c-Lys(Z)-0TCP (0,058 mol) and 9
g of HOBT (0,059 mol) was added.

The pI of the reaction mixture was brought back to 7 by adding NEM.

The mixture was stirred at room temperature while maintaining PI-1 at 7 by adding NEM as necessary. The reaction was analyzed by TLC (chloroform/methanol = 90/10; chloroform/methanol/acetic acid =
90/20/3 (2 solvents)).

After 1 hour, 1.96 g of Hoe-Lys(Z)-0TC
P (0,0035 mol) and 0.61. ! 7) (OB
Added T. NEM was added to bring the pH of the solution back to 7.

After 2 hours, the completion of the reaction was checked by TLC.

The reaction mixture was evaporated to dryness under reduced pressure (0,1) at a temperature below 30°C. The residue was taken up in 500 ml of water and triturated. The resulting solid was drained and washed with:

- 2S04 aqueous solution (pH 2) in 500ml of HKSO4-
)・500 ml saturated aqueous sodium bicarbonate solution・2
00 ml of water'' ether (200 ml x 2) Dry.

The impure product was chromatographed on a silica column (5m×150,). Eluted with a 80:20 mixture of chloroform/vinegar, ethyl acid (rate 600ml/
hr).

Fractions that were confirmed to be good by TLC were collected and evaporated to dryness under reduced pressure. The residue was taken up in ether and triturated, and the resulting solid was drained.

White powder 35.14g (86cm) TLC:Chloroform/methanol 90:10 Rf
:0.62 Chloroform/methanol/acetic acid s 7.7 : 9.4 : 2.8 Rt : 0
．． Check with 52NMR, AAA: ACGty 1. OO-Vat 0
．． 9 6-Leul, 0 6-Lys
O,986, TFA-HT, yg(Z)-Vat-L
Boa-Lys (Z) of eu-Gty-OCH 315g
-Vat-Leu-Gty-OCH3 (22,6mM)
75ml of TFA/methylene chloride 1/75+++A! The mixture was dissolved and evaporated to dryness under reduced pressure at a temperature below 30°C. Ether containing 20 grams of hexane (100 ml)
The residue was removed. The solid produced was drained and dried.

14.51 g of white solid was obtained.

Yield: 94.7% TLC: Chloroform/methanol/acetic acid = 9515/
3 Rf: 0.12 7, Boa-Arg(J(C4)-Lys(Z)-
Vat-Leu-Gty-OCH314,1,! i+ TFA-H-Lys (Z)-Vat-L@u-Gty
-OCH.

(21?FIM) was dissolved in 50 ml of DMF. NEM was added to the Kono solution to make -6 to 7. 6.57 g of Bee-Arg(T(Ct)-0H'1H20 (20 m
M) and 10.6I of BOP (24mM) were then added to this solution.

- of the reaction solution was returned to 6-85-7 by adding NEM. The mixture was stirred at room temperature while maintaining the pH at 6 to 7 by adding NEM as necessary. The reaction was followed by TLC (chloroform/methanol-80/20).

After 5 hours the reaction was complete. The reaction mixture was heated under reduced pressure (0,
1) It was evaporated to dryness at a temperature below 30°C. The oil obtained was taken up in 300 rnl of ethyl acetate. The resulting solid was drained and washed with ether (first black clay (
jet')). Ether was added to the above filtrate. A solid precipitate was obtained, which was drained (second black soil).
. These two black soils were identical on TLC. These were mixed with 200 ml of ethyl acetate saturated with water and 6 ml of water.
The solid was filtered by stirring with 0 ml of the mixture for 1 hour. The solid was drained, washed with 60 ml of water and 300 ml of ether and dried.

Yield near 8.7 (13,49,!9) TLC: Chloroform/methanol (20/20) Rf=0.32 Confirmed by NMR 8. TFA=H-Arg(TTC4)-Lys(Z
)-Vat-Leu-Gty-CH3 1, 3, 3 g of Boc-Arg(HCt)-Lys (
Z)-Vat-T, eu-Gty-OC)(3(15,
5mM) in 66 ml of TFA/methylene chloride
Dissolved in 760 ml mixture. The product was stirred at room temperature for 40 minutes and then evaporated to dryness under reduced pressure at a temperature below 30°C. The residue was taken up in 100 ml of an 80/20 mixture of ether/hexane. The resulting solid was drained, washed with hexane, and dried (weight 1.5.13 g).

TLC: Chloroform/methanol-3/IRf: 0.
35 9, Boa-Tyr-Arg(HCt)-Lyg(
Z)-Vat-Lau-Gty-OCT(3 15, 5mM TFA-T(-Arg(HCt)-Ly
s(Z)-Vat-Leu-Gty-OCH, 50m
1 in DMF. NEM was added to bring the solution to -6. Next, add 8.209 Boe-Try-O to this solution.
TCP (15,5X 10 mM) and 2.41. !
i+ HOBT (15,5X 10 mM) was added.

NEM was added to set - back to 6. The reaction mixture was stirred at room temperature while maintaining −6 by adding NEM as needed. The reaction was carried out by TT, C (chloroform/methanol = 3/
1). After 3 hours, the reaction was complete. The reaction mixture was evaporated to dryness under reduced pressure (0,11-l) and at a temperature below 30°C. The residual oil was taken up in ethyl acetate (300++l). The resulting gelatinous solid was drained and mixed with ethyl acetate (100 m/) and ethyl acetate/ether (1/1:
100 ml), ether (100 ml) and finally hexane (100 mA). The product was dried to constant weight under reduced pressure.

Yield: 96 g (15.14 g) TLC: Chloroform/methanol (3/1) Rf: 0
．． 47 Confirmed by NMR AAA: AC: Gty 0.95- Vato,
98-Leul, 02-Lys 1.01-Arg
-1,01-Tyr 1.05) IPLC: 93.
81 CH Epp 10, Boa-Tyr-Arg(HCt)-Lys
(Z)-Vat-Leu-Gty-OH 7g of Ro e-Tyr-Arg(T(C4)-Lys
(Z)-Van-Leu-Gty-OCH, (6,8
6mM) in a mixture of 7oml dioxane and 35mJ water. 6.4 ml of 4N sodium hydroxide (25.6 mM, 3.7 equivalents) was added to this solution and stirred at room temperature for 30 minutes.

Cholera was diluted with 200 ml of water and 800 ml of ethyl acetate. Add (N)T(Ct) to this mixture and PH
I made it 3. Decant the ethyl acetate and drain the solids!
-da (first bad ball). Ethyl acetate was evaporated to dryness. Second
A black earth-like substance was obtained. These two black earth-like substances are T
Identical on LC, they were collected, washed with ether and dried.

Yield near 4.7% (5.16g) TLC: Chloroform/methanol (2/1) Rf: 0
．． 45 [Checked by NMR AAA: Ac:Gtyl, 0O-Vato, 97-
Leul, 04-Tyr O, 95-Lys O, 99
-Arg 1.05T (PLC-EPP 91.52chi89 one example - Boc-■te-Phe-Thr-Asn-8er-N
H-NH2 (fragment H4) 1, BoC-Asn-8
er-OMe 23.329 BoC-Asn-0H (0,1,M) and 19.7 g in 250 ml DMF
22.69 g of DCCI was added to a cooled solution of NbOH (0.11 M) in a water bath. After reacting for 3 hours at room temperature, the DCU was filtered, cooled in a water bath, and 15.56 g of HCt-T(-8et-OMe was added.HEM was added to adjust the pH to 7.4 at room temperature. After 20 hours at +4° C., the solution was concentrated under reduced pressure. The residue was taken up in a 50150 mixture of AeOEt/n-sitanol. The organic solution was washed successively with:

・Saturated hydrogen carbonate solution) ・Saturated sodium chloride solution ・Saturated sodium chloride solution ・Saturated sodium chloride solution ・Saturated sodium chloride solution ・Sat. crystallized inside. The product was filtered, washed with ether/hexane for 90 minutes and dried in vacuo to give 26.9 g of product.

Yield: 80.8% immediately confirmed by TLC 2, TFA, H-Asn-8er-OMel 3.5
9 Boc-Asn-8er-OMe (40.5mM
) was dissolved in 54 mA' of dichloromethane, and 81 ml of TFA was added to a solution chilled in a water bath. After reacting for 1 hour at room temperature, the solution was concentrated under reduced pressure and the residue was taken up in ether. A thick oil was obtained which was used in the next step.

3. To a solution of the crude product obtained on Boc-Thr-Asn-8er-OMa dissolved in 300 m of DMF and cooled in a water bath, 14.09 g of Boc-Thr-O was added.
NSu (44,44mM), 558g I(OBt
(41.3mM) and NEM was added until the pH was 7. After reacting for 200 h at room temperature, the solution was concentrated under reduced pressure and the residue was taken up in Ac0Et/n-butanol mixture. The organic solution was successively washed with the following: 2S04 aqueous solution, saturated sodium chloride solution, 15 g of KH804/', saturated aqueous sodium bicarbonate solution, saturated aqueous sodium chloride solution)
After drying over aqueous MgSO4, the solution was concentrated under reduced pressure and the residue was crystallized in an ether/hexane mixture. 8.2
8 g of product was obtained.

Yield: 47 grams Immediately confirmed by TLC 4, TFA+H-Thr-Asn-8er-OMe
8 g Boc-Thr-A in 32 ml dichloromethane
To the cooled solution of sn-8er-OMe in a water bath was added 54 m/ml of TFA. After reacting for 1 hour at room temperature, the solution was concentrated under reduced pressure and the residue was precipitated with ether. After filtration, this was used in the following steps.

5. Boa-Phe-Thr-Asn-8er-O
7.82 g of Boc-Phe-ONP was added to a solution of the precipitate obtained on Ma dissolved in 100 ml of DMF.
(20,24mM), HOBt of 2.539 (18,
71 ml) and NEM until −7 was added with cooling in an ice bath.

After 4 hours, the solution was concentrated under reduced pressure and the residue was taken up in AeOEt. The organic solution was washed with:

・Saturated sodium bicarbonate solution ・Saturated sodium chloride aqueous solution ・5 tKH8O4/kC2SO4 aqueous solution ・Saturated chloride)
After drying over IJum solution MgSO4 and concentrating the organic phase, the residue was crystallized in ether. 10 g of product was obtained.

Yield: 93.5% Confirmed by TLC and Todoroki 6, TFA, H-Phe-Thr-Asn-8er
-OMs 10g Boe in 4Qml dichloromethane-
Phe-Thr-Asn-8er”OMe (17,1
9mM) dissolved in the solution for 60m while cooling in a water bath.
/ of TFA was added. After reacting for 1 hour at room temperature, the product was precipitated with ether. After drying, an imme-like substance was obtained.

Yield: 5.14 g product 93- Yield: 48.5 chia, Boc-Ite-Phe-Thr-Asn-
6er-OMel 00 at DMF, 5.14 g TFA, H-Phe-Thr-A8n-OMa (8,
78ynM) dissolved in a cooled solution in a water bath.
．． 17g BoC-Ite-O8u (9.66ml
), HORt of 1.309 (9,62mM),
Pl (NEM and NEM were added until Pl became 7.200 at room temperature
After reacting for an hour, the solution was concentrated under reduced pressure and the residue was taken up in ether to give a precipitate, which was filtered and dried in vacuo. 5.24 g of product was obtained.

Yield: 85.9% and T (confirmed by PLC 8, Boa-Ite-Phe-Thr-AI!In
In-8or-NH-NH240 in DMF and 200 m
l of methanol and 5 g of Hoe-Ite-Phe-T
A cooled solution of hr-Asn-8sr-OMe (7.2mM) in a water bath was charged with 3.5mA! 80 of
Added hydrazine dihydrogen. After 21 hours of reaction at room temperature, 0.9 ml of the same hydrogenated hydrazine solution was added. After a further 3 hours -94= The resulting barrel was filtered, washed with methanol and dried in vacuo. 3.949 products were obtained.

Yield near 8.8% Confirmed by Chi and TI, C Example X Roe-Tyr-AmuAsp(OBzt)-Ata-
OH (@ Kata ■) 1, Boa-Asp (QRzt) -
Amu-0But tertiary butylalaninate hydrochloride 3
．． 62g (0.02 mol) of 30TnIl! of acetonitrile. Add 2.52 ml of NEM, then add 8.4.9 (0.02 mol) of Hoe-Asp (O
Bzt)-ONSu was added. Add NEM to pH 6
It was kept at ~6.5. Stir for 18 hours. After checking by TLC, the liquid was evaporated to dryness and the oil was poured into 50 ml of ACOE.
Wash twice with KH8O4/2SO4 solution,
It was then washed with brine, dried over Na2SO4 and evaporated to dryness. The resulting oil was dissolved in hexane, a small amount of solid was filtered off, and the filtrate was evaporated to dryness. The oil obtained was used in the trench.

TLC: C1 (C15/acetone/ACO) (=
80/1515Rf = 0.8 2, Boc-AAa-Asp(OBzt)-At
The crude oil obtained on a-OH was dissolved in 5Qm/c of cold TFA and stirred for 1 hour and 30 minutes. As much TFA as possible was evaporated and the residual oil was taken up three times in anhydrous ether and decanted. This is dessicated in vacuum in the presence of inthane.
It dried inside. A solid foam was obtained, which was ground and dried.

9.4g of crude product was obtained, which was used as Part 1! , was used in the bonding process.

The above TF'A salt was mixed with 20 ml of DMF and 18 ml (7)
Dissolved in a mixture with distilled water. NEM was added until -7 was reached (using a PH meter). 16m1 DMF
4 g (0,014 mol) of Boc-Ata-ONSu
was added to the solution. Add NEM and adjust pH to 6.5~
I kept it at 7. After 5 hours and 30 minutes, the reaction was completed (TL
Check with C). The solvent was evaporated as much as possible and the residue was taken up in a mixture of water and ethyl acetate. The organic phase was dissolved in NaHCO
s solution, washed with brine, dried over Na 2 SO 4 and evaporated to dryness. The residue was dissolved in ether and precipitated with hexane to give a gummy material which, after grinding, crystallized.

The solid was drained and dried in air.

Yield: 5.05g TLC: CHCL3-acetone-ACOH=80/
15/! 5Rf = 0.28 3, Z-Tyr-Ata-Asp'(OBzt)-
Ata-OH1g (2mmol) of Boe-Ata-A
sp(OBzt)-Ata-OH was added to 10 ml of stirred, ice-cooled TFA.

After stirring for 30 minutes at room temperature, as much TFA as possible was evaporated and the residue was taken up twice in anhydrous ether, decanted and dried in a vacuum desiccator in the presence of potassium hydroxide. A solid foamy material was obtained.

This crude product was dissolved in a mixture of 4 ml DMF and 2 ml water. NEM was added to bring the - to 7-7.5.

1,! i' (2,4 mmol) of 1-Tyr-O
NSu was added and - maintained at 7-7.5 by NEM (pH meter). After 2 hours and 30 minutes, the solvent was evaporated as much as possible at 40° C. after checking by TLC.

The residue was taken up in ethyl acetate and washed with water. A small amount of solid was removed and the ethyl acetate was washed with a mixture of sulfate/bisulfate and brine. If the product is left for several hours, a precipitate will form,
This was drained, washed with ether and dried in the air.

500 μm were thus obtained. A second, slightly more impure black earth material, 350 ml, was obtained by concentrating the ethyl acetate.

TLC: Chloroform/ACOH=3/I Rf
: 0.4butanol/ACOHA (20=7277
/2Rf: 0.85 Chloroform-acetone-ACOH = 80/1515 Rf: 0.1) (PLC: Polybezotide purity Confirmed at 90.9% per year Example X T(-Arg-Lys(Z)-Leu-Leu- Gtn
-Asp(Bzt)-Ite-Me t-8e r-A
r g-GLn-Gin-Gty-Gtu (OBzt
) -8e r-Agn -Gtn-Gtu(OBzt
)-Arg-Gty-Ata-Arg-Ata-Arg
-Leu-98- NI(2 (Peptide K) Synthesis 1. Boc-Gtu(OBz/!,)-Arg-G
ty-Ata-Arg-Ata-Arg-Leu-NI
2,3HC4 34,479 H-Ata-Arg-Ata-Arg-
Leu-NI(,,,.

3HC4 (57mM) was dissolved in 230ml DMF.

Add 28g ROP (63mM), then 33.5g (57mM) in 190+++l DMF within 1 hour.
M) Boa-Gtu(OBzt)-Arg-Gty-
A solution of OH was added. The pH of the reaction mixture was maintained at 6 by adding NEM. After 4 hours, the reaction mixture was concentrated to half volume in vacuo and the residue was poured into 800 ml of ethyl acetate. The resulting precipitate was filtered, washed with ethyl acetate, washed with ether and dried in vacuo.

56.8 g of product was obtained with a yield of 80 g.

Identification 2 IH Immediate AAA: Gtu 0.98- Gty 1.01-
AAA 1.97-Leu 1.05-Arg 2
．． TLC in 97 HPLC Reverse Phase EPP 95 CH EPAW 40/20/10/6: Rf
=O6552, H-Gtu(OBzt)-Arg-Gt
y-Ata-Arg-Ata-Arg-Le u-1■
25.3 g of 2+ TFA + HCl (250 ml of the above product) was stirred for 30 minutes in a mixture of 2 ct2 and 250 m, 1 of TFA. The reaction mixture was concentrated to half volume in vacuo and poured into 1200 ml of ice-cold ether. The resulting precipitate was filtered, washed with ether and dried over potassium hydroxide in vacuo.

Yield: 100も3, Boc-Gtu(OBzt)-8er-Asn
-Gtn-Gtu(OBzt)-Arg-Gty-AmuArg-Ata-Arg-Leu-NI2. HC4 TFA salt (45mM) 400ml (DD
22.2 in a solution dissolved in MF and neutralized with NEM.
Add 9 BOP (50mM) and within 1 hour, 3
30g (45yy+M) Bo in 00ml DMF
c -GLu (OBzt)-8er-Asn-Gt
A solution of n-OH was added. Part of the reaction mixture! 6
I kept it. After 6 hours, the liquid was concentrated to half its volume in vacuo at 30°C and heated at 12oC.

ml of ethyl acetate. Filter the formed precipitate
□, washed with ethyl acetate, washed with ether and dried in vacuo.

Yield: 60.5 g (75 g) Product was confirmed by M and AAA. 'AAA:Asp
1.0O-Sar0.8l-GLu2.7l-G7y
O,89-Ata 1.82-Leu O,96-A
rg 3.02 T (PI, C reverse phase EPP 70% TLC: EPAW30/20/10/6 Rf:
0.354, )I-Gtu(ORzt)-8er
-Asn-Gtn-Gtu(OBzt)-Arg-Gt
y-Ata-Arg-Ata-Arg-Leu-NI2
．． TFAyC1 53.3 g (30 mM) of the above product were stirred in a mixture of 250 ml dichloromethane and 250 ml TFA for 35 minutes. After concentrating to half volume in vacuo, the residue was poured into 1300 ml of water-cooled ether. The precipitate formed was filtered, washed with ether and dried over potassium hydroxide in vacuo.

5. Boc-8er-Arg-Gtn-Gtn-G
ty-Glu(OBzt)-8er-Asn-Gtn-
Gtu(OBzt)-Arg-Gty-AmuArg-
Ata-Arg-Leu-NI2. HC1101- Dissolve the above TFA salt in 300+++/DMF and add NE
Neutralized with M. 14.8 g of BOP (33 mM) was added, followed by 21.4 g of BoP (30 mM).
c-8er-Arg-Gln-Gtn-Gty-OH was added in portions within 1 hour.

NEM was added to keep the - of the reaction mixture around 6.

After 7 hours, the liquid was poured into 21 ACOEts. The resulting precipitate was filtered, washed with ethyl acetate, washed with ether and dried in vacuo.

Yield: 57g (80%) Immediate product identification AAA: Ser 1.7, Arg 4.1, Gt
u 4.7, aty 2.05, Asp 1. O01A
ta 1.91, Leu 0197 TLC: BPEWI Rf: 0.356,
H-8er-Arg-Gtn-G7n-Gty-Gtu
(OBzt)-8er-Asn-Gtn-Gtu(OB
zt)-Arg-Gty-Ata-Arg-AmuA
r g-Le u-? 'JH2# TFA r HC
The above product with tl 4.219 (6mM) was added to 80 m
A solution suspended in 1 liter of dichloromethane was diluted with 1 liter under a nitrogen atmosphere.
The cells were treated with 120 ml of trifluoroacetic acid for 0 min. After stirring at room temperature for 45 minutes, concentrate in vacuo to 1/3 of the original volume, stir and cool (+4
℃) Pour into 750 ml of ether.

The precipitate was filtered, washed with ether and dried over potassium hydroxide and then over phosphoric anhydride in vacuo.

13.9 g (97%) of product was obtained, which was
It was used for reactions below t.

7. Roc-Asp-(OBzt)-Ite-Me
t-8er-Arg-Gtn-Gtn-Gty-Gtu
(ORzt)-8er-Asn-Gtn-Gtu(OB
zt)-Arg-Gty-Ata-Arg-Ata-A
rg-Leu-NT (2, HCt 76, 5.82 g (10 mM) in 6 ml DMF
Boc-Asp(OBzt)-Ite-Met→m-
A solution containing NT (2) was heated to 6.66 ml (40 mM) of gaseous 6.ON HCl at -20°C to -25°C.
/dioxane, 1.4% in 5 ml DMF
It was treated with a solution containing 5 ml (12 mM) of terthiobutyl nitrite. After 1 hour at -20°C to -25°C, add 8 ml of diisozolobyl ethylamine (DIPEA).
) to neutralize it so that the apparent - is 6 to 7,
When the total volume is 100 ml with a small amount of DMF, 0.1
A M azide solution was obtained (which was always stored at about -20°C).

13.84 g (approximately 5.8 mM) of the above TF'A salt was mixed with 58 ml (1) DMSO and 87 ml DMSO.
The apparent pH of the solution dissolved in the mixture with F was measured with DTPEA.
The solution obtained in this way was heated to -20°C to -25°C with 2.5 ml of tare (
(approximately 1.25 equivalents) of the above azide solution was added over approximately 10 minutes.

Adjust the pH to 6-7 with a small amount of DIPEA and bring the solution to 1-1.
Stored at 5°C. After 16 hours, an additional 14.5 m/(0
, 25 equivalents) of the above azide solution was added.

When checked by TLC, it was found that the reaction was completed after 24 hours, but after 40 hours, this solution was
It was stopped by pouring into 750 ml of stirred water-cooled ethyl acetate.

The precipitate obtained as above was filtered, washed several times with the same solvent and dried in vacuo in the presence of phosphoric anhydride. 14
．． 35 g was thus obtained and used as is in the following steps.

8, H-Agp(OBzt)-1te-Met-8
er-Arg-Gtn-Gtn-GLy-Gtu (O
BZ)',)-8er-Asn-Gtn-Gti(OB
zt)-Arg-Gty-Ata-Arg-AmuAr
g-Leu-NH2+TFA.

14 g of HCl above product was added to 80 ml of CH2Ct2+]J
Q+++l TFA + 1.0 ml ■S (CH2
) Stirred in 2SH for 35 minutes. After concentrating to half volume in vacuo, the reaction mixture was poured into 800 ml of water-cooled ether. The resulting precipitate was filtered, washed with ether and dried over potassium hydroxide in vacuo.

Yield: 100 Ti9, Boe-Gtn-Asp(OFlzt)-It
e-Met-8er-Arg-Gtn-Gtn-Gty
-Gtu(OBzt)-8er-Asn-Gtn-G/
, u(OBzt)-Arg-Gty-A/-a-Arg
-Ata-Arg-Leu-NT (2!TFA salt (4,9mM) obtained on HCl was added to 120ml of D
The solution in a mixture of MF and 30 ml DMSO was heated with N
Neutralize with EM, 740Tn9 HOBt (5,5mM
) and then add 2.02,9 B o c in small portions.
-Gtn-0Np (5.5mM) was added and the -4 of the reaction mixture was kept near 6 by adding NEM. After 6 hours, the reaction mixture was poured onto 800 ml of AcOEt. The resulting precipitate was filtered, washed with ethyl acetate then ether and dried in vacuo.

Weight: t4. sg Yield: 100% The product was identified by AAA.

Asp 1. , 97.5er2.01Gtu 6.12
, aty2.06, Met O,55, ILe O,9
3, LauO,99, Arg 3.81 TLC: Rf = 0.45 BPEIWllo,
)I-Gtn-Asp(OBzt)-1te-Me
t-8or-Arg-Gtn-Gtn-Gty-Gtu
(OBzt)-8er-Agn-Gtn-Gtu(OB
zt)-Arg-Gty-AmuArg-AmuArg
-Leu-NT (2, TFA, HCl 14.79 of the above product was added to 80 m/CH2C42+1
0ml H8(CFI2)2SH+ 120ml T
Stir in FA for 35 minutes. After concentrating to half volume in vacuo, the reaction mixture was poured into 600 ml of water-cooled ether. The resulting precipitate was filtered, washed with ether and dried over potassium hydroxide in vacuo.

Weight: 14.5g 11, Roe-Arg-Lys(Z)-Leu-L
eu-Gtn-Asp(OBzA)-Ite-Met-
8er-Arg-Gln-Gtn-Gly-Glu(O
Bzt)-8er-Asn-GAn-G/=u(OBz
t)-Arg-Gty-Ata-Arg-Ata-Ar
g-Leu-NH2,T (TFA salt obtained on Ct (
4°9 mM) in 120 ml DMF and 401 nl
2.44 g of BOP (5.5 mM) and 4.2
Boc-Arg-Lys(Z)-Leu-Leu- of g
OFI, HCl (5.5mM) was added in small portions over 30 minutes. NEM was added to keep the - of the reaction mixture around 6. After 23 hours, the reaction mixture was diluted with 11 AeOEt
Poured on top. The precipitate formed was filtered, washed with AeOEtN then Et20 and dried in vacuo.

M: 16.5g Yield: 93 chi The product was identified by NMR and AAA.

Asp 2.10, Ser 1.86, Gtu 6.2
, aty2.0 0, ata 2.0 0. Me
tO,55, IteO980, Leu 2.8
9, Lys 0.8 7, Arg4.67 TLC: Rf = 0.40 (BPI□1) Example sequence Boc-Lte-Phe-Asn-Thr-8er-T
yr-Arg-Lys(Z)-Val-Leu-Gty
-Gtn-Leu-8er-Ata-OH,HCl(A
! Synthesis of peptide J) 1, H-Gtn-Leu-8er-Ata-0C
I (3109 ml dichloromethane and 100 ml T
10.3 g (18,8 mM) of Boe-Gtn-Leu-8er-Ata-(')CH in a mixture with FA
3 was stirred for 35 minutes.

The reaction mixture was concentrated in vacuo to approximately 50 rnt and poured onto 300 ml of ether. Drain the precipitate,
Washed with ether and dried in vacuo.

2. Boa-Tyr-Arg(Z)-Vat-Le
u-Gty-Gtn-Leu -8e r Ata -
The TFA salt obtained on 0CH5+ HCl was 200 m
18.99 (18.99) dissolved in dimethyl ) formamide and neutralized with N-ethylmorpholine.
Boc-Tyr-Arg-Lys (Z
)-Vat-Leu-Gty-OH, then 9.29 of BOP (21 mM), then NEM to bring the pl The resulting precipitate was filtered and washed three times with 100 ml of Ac0Et and 3 times with 100 ml of Et.
Washed 3 times with 20°C and dried in vacuo.

24.169 products were obtained.

Yield: 90 TLC: Rf = 0.20 (B1'WI) αT
) = -17,6° (Cc: DMF containing 1-5'% AcOH) The product was identified by IHNMR and AAA.

Ser 1.01, G/, u O, 99, Gty 1
．． 06, Atal, 01, VatO, 98, Leu
1.97,Tyrl,0O1Lys O,9B,A
rg 0.99T (PLC reverse phase (EPP: 92T)
3. T(-Tyr-Arg-Lys(Z)-Vat
-Leu-Gty-Gtn-Leu-8er-Arg-
OCH3, HCt, TFA8.66, ji' (6,
1mM) of the above product in 37ml dichloromethane +
35 in 3.5 ml anisole + 40 ml TFA
I stirred it for a minute. After concentrating in vacuo to half a volume, the resulting white precipitate was filtered and
Washed with ether and dried in vacuo over potassium hydroxide.

Weight: 8.68g Yield: 100 Ti 4, Boc-Its-Phe-Thr-Asn-8
er-N 35.94 g (8.55mM) of Boe-
Ile-Phe-Thr-Asn-8e r-NH-N
H2 was dissolved in 27 m/m DMSO + 37 m7 DMF'. After cooling to -25°C, 6. t ml of 5
．． 6N HCl/dioxane solution, then preheated to -20°C.
1, pre-diluted in 5 ml of D
23 at of terthiobutyl nitrite (1.2 eq.) was added. After stirring for 1 hour and 15 minutes at -20°C to -25°C, 5.1 m of diisopropylethylamine (DIPEA)
) to adjust - to around 6, then add 3 ml of D
MF was added to bring the volume of the solution to 85 TrLl and the solution was heated to -25°C.
Saved with.

5. Boc-Te-Phe-Thr-Agn-8
or-Tyr-Lys(Z)-Vat-Leu-G
ty-Gtn-Lsu-8or-Ata-O
CH3t HC1 The TF'A salt obtained in Example M-3 was dissolved in 70 ml of DMF, and after neutralization with DIPEA,
This was added to 70 ml of the solution obtained at 110°C at -20°C.

DTPEA was added to adjust the pH to 7, stirred at -20°C for 1 hour, and the solution was kept at -12°C. 22 hours later, 5
ml of azide solution was added and brought to -7 with DIPEA.

After 45 hours, the reaction solution was heated to 1200mA! of ethyl acetate. The resulting white precipitate was filtered, washed with ethyl acetate then ether and dried in vacuo.

Weight: 10.46. ! 9 Yield: 86% A second stale material was obtained by concentrating the mother liquor and reprecipitating with an ethyl acetate/ether mixture, giving an overall yield of 95%. The products were pinned together and identified by IHNMR and AAA.

AIIp 1.02, Thr 1.03, Ser 1.
91, GtuO497, Gty 1.06, Ats
1.06, Vatl, OONite 0.94, Le
u 1.98, Tyr 1.04, Phel, 02, L
ys O,94, Arg 1.02TLC: Rf =
0.20 (BEwT) HPLC reverse phase (EPP:
90) Example X ■ Z-Tyr-Ata-Asp(OBzt)-Ata-I
te-Phe-Thr-Agn-8e r-Tyr-A
rg-Lys(Z)-Vat-Leu-Gty-Gtn
-Leu-8er-Ata-Arg-Lys(Z)-L
eu-Leu-Gtn-Asp(OBzt)-Ite
-Me t-8e r-Arg-Gtn-Gtn-Gt
y(OBzt)-8e r-Asn-Gtn-Gtu(
OBzt)-Arg-Gty-Ata-Arg-Ata
-Arg-Leu-NH2,T(C4(GRF 1-4
4 protection) synthesis 1, H-Arg-Lys(Z)-L
eu-Leu-Gtn-Asp(OBzt)-Ite-
Me t-8e r-Arg-Gtu-Gtn-Gty
-Gtu(OBz/)-8er-Asn-Gln-Gt
u(OBzt)-Arg-Gty-Ata-Arg-A
ta-Arg-Leu-NH2, HCl, TFA Example 1.4 g of the product obtained in side 11 was added to 60 ml of CH
2C42 + 70 ml (D TFA + 7 ml ethanedithiol mixture) was stirred for 35 min. After concentration in vacuo to half volume, the residue was poured into 500 ml water-cooled ether. The resulting precipitate was filtered, washed with ether,
Dry in vacuo over potassium hydroxide (weight nia, 44
g).

6. Boc-Ite-Phe-Thr-Asn-8
er-Tyr-Arg-Lys(Z)-Val-Gty
-Gin-Leu-Gty-Gln-Leu-8er-
The product obtained on 5-3 g (2.7 mM) of Ata-OH, HCl was
0 ml of DMSO-) dissolved in 5 ml of water. 10m
/(7) IN sodium hydroxide was added one portion at a time, stirred for 30 minutes, and then neutralized with 10 ml of IN hydrochloric acid. The reaction solution was poured onto 11 portions of ethyl acetate. The resulting precipitate was filtered, washed four times with ethyl acetate, twice with water, and four times with ether, and dried in vacuo over P2O5-H.

Weight 4.56g Yield 86chi The product was identified by 1) (NMR and AAA).

TLC: Rf = 0.5 BEWI2, Ho
e-4te-Phe-Thr-Asn-8er-Tyr
-Arg-Lya(Z)-Vat-Leu-Gty-G
tn-Leu-8er-Ata-Arg-Lys(Z)
-Leu-Leu-Gtn-Asp(OBzj)-It
s-Me t-Ss r-Arg-Gtn-Gtn-G
ty-GAu(OBzt)-8e t-Asn-Gtn
-Gtu(OBzt)-Arg-Gty-AmuArg
-A Mu Arg-Leu-NH2, HCl6.6.1i
+ above TFA salt (1.9mM) was added to 36ml of 113
- 1.0 g nop (2,3 mM) dissolved in DMSO + 90 ml DMF and neutralized with NEM
Then, 3.8 g of Example M-6 (1.9 mM) of Ipti-P was added, and the - of the reaction solution was adjusted to 6 with NEM. After 19 hours, 1.15 mM BOP was added. 26 hours later,
The reaction was poured onto 600 TLl of AcOEt. The resulting precipitate was filtered, washed with Ac0Et, filtered and dried in vacuo.

Yield: 88chi (9.2g) The product was identified by bleaching and AAA.

3. H-Ite-Phe-Thr-Asn-8er-
Tyr-Arg-Lys(Z)-Vat-Leu-Gt
y-Gtn-Leu-8er-Ata-Arg-Lys
(Z)-Leu-Leu-Gtn-A8p(OBzt)
-Ite -Me t-8e r-Arg-Gtn-G
Ln-GLy-GLu(OBzt)-8er-Asn-
Gtn-Gtu(OBzt)-Arg-Gty-Ata
-Arg-Ata-Ar(H-Leu-NH2, HCt
, TFA 9.1 g of the above product was added to 100 ml of TF
Stir in A+5 ml ethanedithiol mixture for 35 minutes. The reaction solution was poured onto 500 ml of water-cooled ether. The resulting precipitate was filtered and washed with Et 20, 114
- Dry in vacuo over potassium hydroxide.

Yield: 100g of the above TFA salt (1,66mM) of the protected GRF-1-44 (5r)r
Example IX was dissolved in a mixture of nl DMF + 50 Tnl DMSO and neutralized by NEM.
-3 obtained 1, 18,! i' (1,78mM
) peptide and 0.799 BOP (1,78mM
) was added. - was kept around 6 by NEM. After 23 hours at room temperature, the reaction solution was diluted with 600 ml of AeO.
Pour into Et. The precipitate formed was filtered, washed with ethyl acetate, then ether and dried in vacuo.

Yield: 8.54g (84%) Confirmed by weight and TLC AAA: Asp 4.2, Thr O192,8e
r 4.02, GLu 7.37 (7), GLy 3
．． 14 (3), Ata 5.11, Vatl, 01
, Met 0.61, Ite 1.84, Leu 4
．． 98, Tyr 1.87, Phe O, 95, Arg
5.90 Example
of TFA + 0 in a TFMSA mixture of 19.8+++l
Stir for 1 hour at ℃, add 27 ml of thioanisole and 1
1 ml of metacresol was added to this. Then 1t
of ether was added to this. The resulting precipitate was filtered, washed with ether and dried in vacuo in the presence of potassium hydroxide for 1 hour.

The product was redissolved in 200 ml of water and cation exchange resin Amberlite 1R45 (
acetate form) was added. The resin was filtered, washed with water, and the filtrate was concentrated to 50 m/ml and lyophilized.

Yield: 3.85 g It was immediately confirmed that protecting group 2 and 0Bzt had disappeared.

TLC (BEPWI): Main stream 4. (7) Rf=0.3
8 Example Xv Purification of GRFI-44 The deprotected wavezotide obtained in Example

Fractions containing the expected peptide were pooled, evaporated, and lyophilized.

The lyophilized product thus obtained was treated with 0.1 M (pT (4,,5) to 0.4 M (pH
6,5) using the linear gradient of CM-32 Cal? It was purified by chromatography on a cation exchange resin of the oxymethylcellulose (Whatman) type. For example, if 1 g of peptide is to be purified in one chromatography, the floor space is approximately 50 m1 and the height is 20 c1n.
A chromatography column is used.

Bedecyl with a purity of 80% () IPLC) or higher
fractions were collected and lyophilized to a constant weight (
to remove the CH3C02NH4 buffer).

Finally, the lyophilized product was subjected to partition chromatography using the following 117-solvent system using fine Sephadex G50 as a stationary liquid phase support.

n-Butanol/ethanol/pyridine 10.2N acetic acid = 4/1/1/7 (volume ratio) Chromatography fractions were checked by HPLC, and those with a purity of 95% or higher were collected and freeze-dried.

The product was finally analyzed for amino acids.

Tyr: 1.91 (2) Ata: 4.88 (5) Asn, Asp: 3.86 (4) Ite: 1
．． 96 (2) Phe: 0.93 (1) Thr: 1.02 (1) Ssr: 3.88 (4) Arg: 5.89 (6) t, ya: x, 9B (2) Vat: 1. 01 (1) Leu: 5.10 (5) GLy: 2.98 (3) GLn, Gtu: 6.82 (7) 118- Met: 0.91 (1) Example X■ Roe-G, u -3er-Asn-Gtn-Gtu-
Synthesis of Arg-Gty (Fragment B) 1, Z-Arg(No2)-Gty-OBz/.

glycine benzyl ester) (H-Gty
-0-Bzt sylate) 353g was dissolved in 21 DMF. 115 g of NEM followed by 353 g of Z −
Arg(No,,)-OH and finally 500g of BOP were added. A sample of the reaction solution was taken, diluted with water, checked with pH test paper, and adjusted to -7 by adding NEM.

The reaction solution was stirred and the progress of the reaction was followed by TLC. The reaction was completed after 4 hours, and the mixture was evaporated to dryness at 35°C. The residue was taken up in 21 parts of ethyl acetate and 21 parts of water. Under these conditions, a solid is obtained, which is drained and continuously, while still solid,
5% aqueous solution of 2S04 in HKSO4+, pure water, NaHC
Washed with 5 portions of O3 aqueous solution and finally with water.

The product was dried in air and confirmed by NMR and TLC.

Yield: 410g (82g) 2. H-Gtu(OBzt)-Arg-Gty-O
500 g of the above compound is converted into 100 g of Pd/C (10
A solution containing 31% of HCtN-DMF solution was hydrogenated under 1.5 par hydrogen pressure.

After 24 hours, the reaction was complete (monitored by TLC).

After removing the catalyst, a portion of the solution was taken as a sample, diluted with water, and checked with pH test paper, and N-ethylmorpholine was added to adjust the pH to 7.

458g of BoC-Gtu(OBzt)-ONp is 11
of the solution in DMF was added. The reaction solution was diluted with 11 parts of water, 135 g of 0HBT was added, and enough parts of NEM were continuously added to maintain Pl at 7.

The solution was stirred at room temperature for 4 hours. After confirming that the reaction was complete, the solvent was evaporated in vacuo at 35°C. The residue was taken up in ethyl acetate. Under these conditions the product became a solid. This was drained and chloroform/methanol (
70730 volume ratio) as eluent. Fractions containing pure product were evaporated and lyophilized to yield 412 g (75%) of white powder compound. This was confirmed by TLC and NMR.

The above product was mixed with TFA,! at room temperature. : CH2Cl2
50150 was dissolved in the mixture 31. Bring this solution to room temperature for 1
The mixture was kept for 5 minutes and evaporated to dryness in vacuo while being kept at 20°C. The residue was taken up in ethyl ether, and the resulting solid was drained and dried. The yield was 4239 (100 Ti) in the form of trifluoroacetate.

3. H-Gtn-Gtu(OBzt)-Arg-G
ty-OH564g of H-Gtu(OBzt)-A
rg-Gty-OH·)lifluorocascetate was dissolved in 31 dimethylformamide. A portion of the liquid was diluted with water, tested with a test paper, and adjusted to -7 by adding NEM.

367 g of B o c -Gtn-ONp in 11 DM
Dissolved in F. The reaction solution was diluted with 11 parts of water. 135g
of 0 HBT and a sufficient amount of NEM to maintain -7.

121-The reaction mixture was stirred at room temperature for 4 hours. The end point of the reaction is T
Monitored by LC, then the solvent was evaporated in high vacuum at 35°C. The evaporation residue was triturated in ethyl acetate. Under these conditions a hygroscopic solid is obtained, which can be used as is! 21 with a trifluoroacetic acid-CH2Cl2 (50150 volume ratio) mixture.

The liquid was kept at room temperature for 10 minutes and finally evaporated in vacuo at 20°C. When the evaporation residue is triturated in ethyl ether, 5
25 g (76%) of H-Gtn-Gtu(OBzt)-
Arg-GAy-OH (confirmed by NMR and TLC) was obtained.

4. H-Asn-Gtn-Gtu(OBzt)-A
Using strictly the conditions of rg-Gty-OH execution male side l-2, H-Gtn-Gtu(OBzt)-Arg-Gty-
OH・)Lifluoroacetate) (691g) and 393g
of Boa-Aan-ONb, 685g (85%)
A white solid product was obtained, confirmed by miniaturization and TLC.

5. H-8-or-Asn-Gtn-Gtu (OB
H-Asn-Gtn-Gtu(OBzt)-Arg- of 806g122- under the same conditions as in the male side-2
From Gty-OH and 366 g of Hoe-8er-ONb, 688 g (77%) of the product was obtained in the form of trifluoroacetate, confirmed by Mation and TLC.

6. Boe-Gtu (OBzt)-8er-Asn
-Gtn-Gtu(OBzt)-Arg-Gty-OH
(Fragment B) 894 g of H-8e r-Aan-Gtn-Gtu(O
Bzt)-Arg-czy-ou trifluoroacetate and 458 g of Boe-Gtu(OBzt)-ONp
824 g (80%) of the expected product were obtained by crystallization and purification in DMF-ether (50150 vol.).

The product was confirmed by NMRITLC and AAA.

Gtn-Gtu: 2.92 (3) Ser: 0
．． 97 (1) Asn: 1.03 (1) Arg: 0.97 (1) Gty: 0.96 (1) Strategies listed in Tables V, V+, ■ and ■, and Example ■
According to the methods described in 1 to 2 and W, the following compounds can be obtained.

Hoe-Ly@(Z)-Leu-Leu-Gtn-OH
(Fragment E) Boc-Gtn-Leu-8e r-Ata
-Arg-OH (Fragment F) Hoe-Lys (Z)-Va
A-Leu-Gty-OH (Fragment G) Boc-Ite-
Phe-Thr-Aan-8er-Tyr-Arg-O
H (fragment H) Example layer Boa-G/-u (OBzt)-8er-Asn-Gt
n-Gtu(OBzt)-Arg-Gty-Ata-
Arg-Ata-Arg-Leu-NH2 (fragment B-A
) 69.2g of Fragment A hydrochloride was dissolved in 5007d of DMF. One part of the liquid was diluted with water, checked with a test paper, and NEM was added to adjust the - to 7. 11
7 g of Fragment B and 55 g of BOP were added, the pH was adjusted to 7 again by adding NEM, and the solution was stirred at room temperature for 14 hours. After confirming the end point of the reaction by TLC, two portions of ethyl ether were added to the reaction solution. A white powder precipitate formed under these conditions. This was recrystallized in DMF-ethyl acetate.
□Set. The drained product was washed in solid form with ethyl acetate and dried.

149,! 9 (81%) white product was obtained, TLC
This was confirmed by NNfFt.

Amino acid analysis: Gtn-Gtu: 2.92 (3) Ser: 1.0
2 (1) Asn: 0.97 (1) Arg: 2.89 (3) GAy: 1.01 (1) Ata: 1.98 (2) Leu: 0.93 (1) Deprotection of N-terminal nitrogen The product obtained in the above example (184 g) was added to a mixture of 750 m/m TFA and 750 ml CH2Cl2 with stirring at room temperature. After dissolution, the mixture was kept at the same temperature for 15 minutes and evaporated in vacuo at 25°C. Trituration of the evaporation residue in ether gave a white powder, which was dried in vacuo and confirmed by TLC and NMR.

According to the method of Example Layer, different stages of peptide elongation,
The following compounds can be obtained.

125-Fragment C-B-A Boe-8er-Arg-Gtn-Gtn-Gty-G
tu(OBzt)-3er-Asn-Gtn-Gtu-
(H) Arg-Gty-Ata-Arg-Ata-Arg-L
eu-NH2 fragment D-C-B-A OBzt Boc-Asp-Ite-Met-8er-Arg-G
An-Gtn-Gty-Gtu(OBzt)-8et-
(H) Asn-Gtn-Glu(OBzt)-Arg-Gty
-Ata-Arg-Ata-Arg-Leu -NH2 In this case the fragment is introduced using activation of azide (-CN3)1.

During selective deprotection (removal of Boe), as soon as methionine enters the sequence, TFA-CH2C4
Using impurity removal agents in different treatments according to 2. In this invention, a 5-TFA-CH2Cl2 solution of thioanisole is recommended as an agent that protects methionine from oxidation.

Fragment E-D-C-B-A Boc-Lys(Z)-Leu-Leu-Gtn-As
p(OBzt)-Ite--12 each-Me t-8e r-Arg-Gtn(H)Gtn-G
ty-Gtu(OBzt)-8er-Asn-Gtn-
Gth(OBzt)-Arg-Gty-Ata-Arg
-Ata-Arg-Leu-NH2 fragment F-E-D-C
-B-A Boc-Gtn-Leu-8er-Ata-Arg-L
ys(Z)-Leu-Leu-Gtn-Agp(OBz
l) (H) Tte-Met-8er-Arg-Gtn-Gtn-G
tu(OBzt)-8er-Asn-Gtn-Gtu(
OBzt)-Arg-Gty-Ata-Arg-Ata
-Arg-Leu -M■2 Fragment G-F-E-D-C-B-A Boe-Lys(Z)-Vat-Leu-G/-y-G
tn-Leu-8er-Ata-Arg-Lys(Z)
-Leu-Leu-8er-Asp(OBzt)-It
e-Met-8er-Arg-Gtn-Gtn-Gty
-Gtu(OBzt)-8er-Asn-Gtn-GL
u (OBzt) -Ar g-Gty-Ata-Ar
g-Ata-Ar g-Leu-NH2 fragment H-G
-F -E -D -C-B -ABoc-Ite-
Phe-Thr-Aan-8er-Tyr-Arg-L
ys(Z)-Vat-Leu-(H) Gty-Gtn-Leu-8ar-A2a-Arg-L
ys(Z)-Leu-Leu-Gtn-As p(OB
zt) -Ite -Me t-8e r-Ar g-
Gtn-Gtn-Gty-GAu(OBzt)-8er
-Aen-Gtn-Gtu(OBzt)-Arg-Gt
y-Ata-Arg-Ata-Arg-Leu diNH2
7-8er-Tyr-Arg-Lys(Z)-V7-8er
-Tyr-Ar/-n-Leu-8er-Ata -A
r g-Lys (Z) -Le u -Le 1l
-Gtn-As p (OBzt) -I4e -Me
t -8e r-Ar g-Gln -Gtn-GA
y-Glu (OBzt)-8e r-As n -G
tn-Gtu(OBzt)-Arg-GAy-Ata-
Deprotection and purification of Arg-Ata-Atg-Leu-H2 hpGRF is carried out as described in Examples X■ and X■.

Applicant's agent Patent attorney Takehiko Suzue 1.5♀・6
・%4B Commissioner of the Japan Patent Office Kazuo Wakasugi 1, Indication of the case Japanese Patent Application No. 1983-031335 2, Title of the invention 3, Person making the amendment Relationship with the case Patent applicant Sanofu
A4. Agent

Claims (1)

[Scope of Claims] (1) A method for synthesizing hpGRF 1-44 and hpGRF 1-40 using fragments in a liquid phase, including the following steps a) In the sequence order of GRF, the following a), b), C)
Combine 'ljr pieces with 4e[ of 'ljr' pieces one after another; a)
The acid function of the side chains of aspartic acid and glutamic acid and the amine function of the side chain of lysine are protected by protecting groups that are stable in the environment of Boc group deprotection; b) The guanidine function of arginine is protected by protonation. c) The amine group of the N-terminal amino acid is preserved by the Boc group. During elongation, the Boc group is selectively removed from the N-terminal amino acid of the peptide by hydrolysis with trifluoroacetic acid. The above bonding is carried out in a neutral/polar solvent;
and c) After completion of the sequence, the product is separated from the reaction solution by precipitation using a valve plate solvent after binding of methanesulfonic acid or trifluoromethanesulfonic acid (2) at a concentration of 01 to IM, respectively. The method described in Scope 1. (3) The method of claim 1, wherein the crude reaction product is purified by countercurrent partitioning and permeation chromatography using a sill. (4) The method for synthesizing hpGRF 1-44 according to any one of claims 1 to 3, wherein the following nine 4-peptide fragments are successively linked in this order. φH-Ata-Arg-Ata-Arg-Leu-NH
2(40-44) (Fragment A) @ Boc-Gtu(OBzt)-8er-Asn −
Gtn-Gtu(OBzt)-p, rg-Gty-OR
(33-39) (Fragment B)・BoC-8er-Ar
g-Gtn-Gtn-Gty-OH(28-32) (Fragment C) ・BoC-Asp(OBzt)-11e-Met-N
H-NH,, (25-27) (Fragment D) e Boc-Lys(Z)-Leu-Leu-Gtn
-OH(21-24) (Fragment E) a Boc-Gtn-I, eu-8er -Ata-A
rg -OH(16-20) (Fragment F) IIBoa-Lys(Z)-Vat-Leu-Gty-
OH(12-15) (Fragment G) @ Boe-Ite-Phe-Thr-Asn-8e
r-Tyr-Arg-OH (5-11) (fragment H) Z-Tyr-A Asp (OBzt)-Ala-O
H(1-4) (Fragment I) (5) hpGR using alanine amide in place of Fragment A
A method according to any one of claims 1 to 4 for obtaining F 1-40. (6) The method for synthesizing hpGRF 1-44 according to any one of claims 1 to 3, wherein the following three fragments are successively linked in this order. *H-Arg-T, ya(Z)-Leu-Leu-G
ln-Asp(OBzt)-I le -Me t-8
e r-Arg-Gtn-Gtn-Gly-Gtu (
OBzt)-8er-Asn-Gtn-Gtu(OBz
t)-Arg-Gty-Ata-Arg-AmuArg
-Leu-NH2 (20-44 sequence of GRF, peptide K) *Boe-Ite-Phs-Thr-Asn-8er
-Tyr-Arg-Lys(Z)-Vat-Leu-
Gty-Gtn-Leu-8er-Ata-OH(
5-19 sequence of GRF, peptide J) IIZ-Tyr-
A1. a-A, 5p(OBzt)-Ata-OH(GR
1-4 sequence of F, peptide ■) (7) The method according to claim 6, in which hpGRF 1-40 is obtained using a peptide fragment corresponding to the 20-40 sequence of GRF in place of peptide KK. (8) Coupling of penzotriazolyloxyphonium (BOP) to hexafluorofluoride in the presence of 1-hydroxypenzotriazole (binding agent) in a suitable solvent such as dimethylformamide or dimethylsulfoxide. Phosphate or dicyclohexylcal? 8. A method according to any one of claims 1 to 7, which is carried out by using diimide or by activating oxiazide. (9) Any one of claims 1 to 8
Intermediate bezotide fragment used in the method described in Section 0 01 The following sub-fragments having the characteristics of a) and bL a) below are combined continuously in a liquid phase according to the sequence order of GRF. The intermediate peptide according to claim 9, which is a peptide obtained by. @ H-Ata-Arg-Ata-Arg-Leu -
NH2(40-44) (Fragment A) ・Boa-Gtu(OBzt)-Arg-Gty-OH
(37-39) (Fragment Bl) e Boa-Gtu(OBz t)-8er-Asn
-GLn-OH(33-36) (Fragment B2) e Iloc-8er-Arg-GLn -Gtn-G
ty-OH(28-32) (Fragment C) *Boc-Asp(OBzt)-11e-Met-N
)I-NT■2(25-27) (Fragment D) Boc-Arg-Lys(Z)-Leu-Lsu-
OT ((20-23) (Fragment E,) a) The acid functionality of the side chains of asnocragic acid and glutamic acid and the amine functionality of the side chain of lysine are protected by a holding group that is stable in the deprotection environment of the BoC group. b) the guanidine functionality of arginine is preserved by protonation; C) the amino group of the N-terminal amino acid of fragments B1, B2, CXD, and El is hydrated with trifluoroacetic acid during peptide elongation. B can be selectively removed by decomposition
It is secured by the oC group, and the bonding takes place in a neutral polar solvent. 01) A peptide having the 20-40 sequence of GRF obtained by sequentially combining the following subfragments having the following &), b), and e) in the liquid phase according to the sequence order of GRF. An intermediate peptide according to claim 9 which is a fragment. 'H-Ata-N)j2-Boc-Gtu(OBzt)-Arg-Gty
-OH(37-39) (Fragment Bl) m Boc-Gtu(OBzt)-8er-Asn-G
tn -OH(33-36) (Fragment B2) *Bee-8er-Arg-Gtn -Gtn -G
ty-OH(28-32) (Fragment C) -Boc-xsp(OBzt)-Ite-Met-NH
-NH2(25-27) (Fragment D) *Boa-Arg-Lys(Z)-Leu-Leu
-OR(20-23) (Fragment E,) a) The acid function of the side chain of asno, lysic acid and glutamic acid and the amine function of the side chain of lysine are protected by a protecting group that is stable in the deprotection environment of the Bee group. b) the guanidine function of arginine is protected by protonation; C) the amino groups of the N-terminal amino acids of fragments B1, B2, C, D, E are converted to trifluoroacetic acid during peptide elongation. B can be selectively removed by hydrolysis by
It is protected by an ee group and the coupling is carried out in a neutral polar solvent. (1) The claimed invention is 127'' J obtained by sequentially combining the following sub-fragments having the following characteristics a), b) and c) in a liquid phase according to the sequence order of GRF. Intermediate peptide according to scope item 9. a) the acid function of the side chain of the lysine of fragment G is protected by a protecting group that is stable in the deprotection environment of the Bee group, b) the guanidine of fragments 10-15 the functionality is protected by protonation; C) the amino group of the N-terminal amino acid of fragments G and H is protected by a Bee group that can be selectively removed by hydrolysis with trifluoroacetic acid during peptide elongation; The bonding is carried out in a neutral polar solvent. The intermediate wave zotide according to claim 9, which is zotide. a) The acid function of the side chain of aspartic acid is protected by a protecting group that is stable in the deprotection environment of the Bee group, b) The amino group of the N-terminal alpha amino acid is protected by the deprotection of the side chain protecting group. Protected by a group that is cleavable under the environment.