JPS59152357A - Tetrapeptide derivative - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R<1> is H or amino-protecting group; R<2> is omega-carboxyl-protecting group; R<3> is alpha-carboxyl-protecting group). USE:Synthetic intermediate of decapeptide, useful as a substance for investigating carcinogenic mechanism. It has antagonistic inhibiting activity against the carcinogenic protein produced by carcinogenic gene of avian sarcoma virus. PREPARATION:The compound of formula I can be prepared by reacting the glutamic acid derivative of formula II (R<1>' is amino-protecting group) with the tripeptide derivative of formula III in the presence of a condensation agent (e.g. dicyclohexylcarbodiimide, Woodward reagent, etc.) in a solvent such as hexamethylphosphoramide, tetrahydrofuran, etc. at 0-50 deg.C. If necessary, the protecting group of the alpha-amino group is removed.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the general formula (wherein R1 is a hydrogen atom or a protecting group for an amino group, 2 is a protecting group for an ω-carboxyl group, hb, and fL3 is a protecting group for an α-carboxyl group. It relates to a tetrapeptide derivative represented by

In the present invention, the protecting group for the amine group of R1 of the tetrapeptide represented by the general formula [I] is acetyl, benzoyl, trifluoroacetyl, phthalyl, tosyl,
Acyl-type protecting groups such as 0-nitrophenylsulfenyl,
Urethane-type protecting groups such as t-butoxycarbonyl group (BOC), t-amyloxycarbonyl group, benzyloxycarbonyl group (Z), substituted benzyloxycarbonyl group,
Protecting groups for carboxyl groups of fL2 and R3 include methoxy group, ethoxy group, benzyloxy group, (OBzA)
, substituted benzyloxy group, t-butoxy group, diphenylmethoxy group, etc., -0CI(2CH
2SC) R3,0-phenacyl group [-0CH2COC,
, I(,, -(OPAC)), -0CH2-C51(4
N etc. can be exemplified.

The tetrapeptide derivative represented by the general formula CI) has a competitive inhibitory effect on the oncoprotein produced by the avian sarcoma virus oncogene (srC gene);
In addition, the decapeptide (H-Leu-Ilu-CMu-A
sp -Asn-G7u-Tyr-Thr-Al
It is useful as an intermediate for the synthesis of a-Ar g-OH) (see the reference examples below).

The tetrapeptide derivative represented by the general formula [I] of the present invention can be produced according to the reaction formula shown below.

R2 几2 几2 CID CIn) (wherein, Rs
b A protecting group for an amino group, R2 is a protecting group for an ω-carboxyl group, and R3 is a protective group for an α-carboxyl group. ) [First step] In this step Fi, in the presence of the combination agent, the glutamic acid derivative represented by the general formula [ID] and the tripeptide derivative represented by the general formula [■] are reacted to form the general formula CI
The compound represented by -a) is produced.

The glutamic acid derivative represented by the above general formula [II], which is a raw material for this step, is an industrially easily available compound. (See reference side).

H-Asn -G#u -0PAC-HCIJ↓ This step must be carried out in the presence of a condensing agent, and the condensing agent is dicyclohexylcarbodiimide (DCCI
), Woodward reagent, 1-ethoxycarbonyl-2
-Ethoxy-1,2-dihydroquinoline (EHDQ)
, ethoxyacetylene, etc. can be used. still,
Usually, when using I) CCI-i, N-
Hydroxysuccinoimide, 1-hydroxybenztriazole, N-hydroxy-5-norbornene-2,3
- It is preferable to add carboxyimide or the like in an equimolar to slightly excess amount because the reaction is accelerated.

This step is preferably carried out in a solvent, for example, an amide thread solvent such as hexamethylphosphoramide, dimethylformamide, dimethylacetamide, tetrahydrofuran,
Ether solvents such as dioxane and ester solvents such as ethyl acetate and butyl acetate can be used. In order to carry out the reaction efficiently, it is preferable to use an ether solvent.

When the tripegtide fatty conductor, which is a raw material, is used in the form of a salt such as salt in this process, for example, an organic base such as triethylamine, N-methylmorpholine, an inorganic base such as sodium hydroxide, potassium hydroxide, ammonia, etc. There is a necessity to perform in the presence of.

Although the reaction can be carried out at OC to .50C, the reaction at room temperature is preferred because the operation is simple.

In addition to condensation methods other than those mentioned above, this step should be carried out using condensation methods normally used in this type of field, such as the AND method, mixed acid anhydride method, active ester method, and enzyme method [Second Step] Said general formula CI-a) obtained in one step
The compound represented by the general formula CI-b is produced by carrying out a deprotection reaction of the α-amine group of the compound represented by the formula CI-b.

The deprotecting group reaction in this step can be carried out by acid decomposition. - Examples of the acid decomposition method include a method using a hydrogen chloride-ethyl acetate solution, a method using hydrogen bromide-acetic acid, a method using hydrofluoric acid, and a method using trifluoroacetic acid. Among these methods, the method using a hydrogen chloride-ethyl acetate solution is preferred because the reaction proceeds in a short time under mild conditions.

This step is preferably carried out in a solvent; for example, an ether solvent such as tetrahydrofuran or dioxane, or an ester solvent such as ethyl acetate can be used.

The reaction proceeds smoothly by performing it at the lowest temperature possible.

Incidentally, in order to convert the tetrapeptide derivative of the present invention into a useful decapeptide etc., it is necessary to remove the protecting group from the C-terminus to the N-terminus.

For example, as shown in the second step, a useful decapeptide can be obtained by removing the N-terminal binding group and sequentially condensing it with other peptide fragments. As protecting groups suitable for this purpose, it is preferable to select a urethane-type protective group for type R1, an alkoxy-protecting group for R2, and an O-phenacyl group for type 3.

Hereinafter, the present invention will be explained in more detail with reference to Reference Examples, Examples, and Test Examples.

ζExample 1 Literature description (J, Chem, Sac, '(C) 19
67, 1830) mmoA)kN,N-Dimethylformamide 170d and triethylamine 7.
3 (52,5 mmoA) and BOC-Asn-08
Add u18.1g (55mmol) and 1 at 25υ
Stirred for 8 hours. The amphoteric triethylamine hydrochloride was separated and concentrated under reduced pressure, and water was added to the residue to obtain 25 g of a crude product as a crystalline substance. This was recrystallized with MeOf (21.5 g (yield 75.5%)).

Melting point 180-182C2 [α: 1D -15,6°
(0,5,DMF).

Elemental analysis value Measured value; C60,90・H6,01°N7
．． 31 chi.

Calculated value as CzeH3sN30e; C61,15°H6,19,N 7.38%, 几fO,81(8
8N), 0.85 (BAW).

Reference Example 2 I 20.0g (35.0mmo)) acetic acid x chill 90 ml PI! = 90 g of 6N salt solution/ethyl acetate was added to the turbid liquid, and the mixture was stirred at 25C for 20 minutes. After concentration under reduced pressure, 200 d of dry ether was added to the residue to give 1 as colorless crystals.
7.7 g (yield 9 s, o %) of and was obtained. Melting point 168-173C, [α]D-13,0'(0,5,
'DMF).

Elemental analysis value Measured value; C56,68, H5,67, N
8.22゜C16,84%; C24H2□N30
7・HC7・1/2H20, teno calculation value; C56
,97,H5,58,N 8.30° (77,01%,
几f 0.34 (SBN), 0.21 (BA
W).

Reference Example 3 0PAC-HCjl(II) 13.3g (41.0mmoA) was dissolved in 100d of N,N-dimethylformamide, and to this was added 4.1yJ (37.4mmoA) of N-methylmorpholyph (37.4mmoA) under water cooling.
1-Hydroxybenztriazole 4.8g (37,
7mmo7) and dicyclo+7/l/7.7g (37.4mmol) of carbodiimide were added, and the mixture was stirred for 1 hour under water cooling and then for 18 hours at 25C.

The by-produced dicyclohexyl urea was removed by distillation, the filtrate was concentrated, and water was added to the residue to obtain a crude product as a crystalline substance. This was washed with hydrochloric acid and water, and then recrystallized with methanol to obtain 5.3 g (yield: 96.1 g) of z.

Melting point 164-16sc, (α]D-16.1°(1
, 0, DMF), elemental analysis value Measured value; C61, 85,
)l 6.07. N 7.16%.

Calculation IK as 040H46N4012; C62,
01, H5-98°N7.23%, f 0.90
(BAAW), 0.88 (BAAW).

Reference example 4 PsUCASp ASn (j[u UrAA
; u limb) --i-m 15.5g (20mm
14. in the same manner as in Diagnosis Example 2 using ol) as the starting material.
2 g (yield 100%) of ■ was obtained. Melting point 89-96
C, (α)D-7,8° (0,5.

(DMF), elemental analysis value Measured value; C58, 50°H5
,56,N 7.80. C14, 74%.

035H38N4010-HQ!・1/2H20 Toshiichi? 1-(7) Calculated value; C58,37, H5,60,
N 7.78. Cno 4,92chi.

几f 0.33 (BAW), 0.60 (BAA
W).

Example 1 0PAC-H (J (rV) 4.0 g (12.0 mm0 l) was injected into tetrahuman o7
Suspend in 3 ooml of run and add 1.2 rttl (11,0 mm o A!) of N-methylmorpholine under water cooling.
), 1-hydroxybenztriazole 1.4g (1
0.5 mmo/) and 2.3 g (11.0 mmo, d)f of dicyclohexylcarbonimide were added, and the mixture was stirred at oC for 1 hour and then at 25C for 24 hours. The precipitated crude product was filtered, washed with water, hydrochloric acid, and water, and then crystallized from MeOH to obtain 9.7 g (yield: 97.2). Melting point 178-182C2 [α) D-13.6° (0.2
, DMf'), Elemental analysis value Measured value; C62,51゜1 (5,84, N 7.01%l C5□H59N
Calculated value as 5015; C62,83,H5,98
, N 7.05%.

几f O, 93 (BAW), 0.91 (BAAW
)-V 15. Depart Og (15mmoA')'1l
13.1 g (
VI was obtained with a yield of 94.0%. Melting point 213-21
7c, [α]D -11,oO (0,2,DMF), elemental analysis value Measured value; C60,18°H5,65,N
7.48. C73,81%l C4□H5,N50□
3.HCA-1/4H20: Toshi” IC (7) calculated value;
C60, 38° H5, 66, N 7.49. +J
3.79%, Rf O,65(BAW), 0.72
(BAAW).

reference? 115 V'I 11.2 g (12,0 mmo, /) and literature description [He l v.

Chim, Acta・54413 (19'71>
) BOC-Leu-I7e-() prepared by method
H5.
Methylmorpholine 1.5 m (13.2 mmol), 1-hydroxytriazole 2.0 g (1
4,4 mmo J) and dicyclohexylcarbodiimide 3.0 g (14,4 mmo J) were added, and 1 oc.
After that, the reaction was carried out at 10C for 32 hours. 1 of the reaction solution
The dicyclohexyl urea produced as a by-product was separated from each other and further concentrated, and water was added to crystallize it. This was dried, washed with 1, hydrochloric acid, and water, and then recrystallized from 1,4-dioxane to obtain 112 g (yield: 76.9%) of 2. Melting point 228-230C9(α)D-
36,00 (0,2, ACOH).

Elemental analysis value Measured value; C62,66, H6,23,N
7.699L C64H81N7017 (D
ttN value; C62,92°H6,37,N 7.59
%, B, f 0.84 (BAAW), 0.70
(AW).

Reference example 6 Z-Leu -41e -OH+ VI 3.3g (3.5mmol) and Z-Le
u-I 71 e -0R1,6g (4,2mmo)
) was used as a starting material and the reaction was carried out in the same manner as in Reference Example 5. 1,4-dioxane ψ / M connection 6 t, Te 3. Og (yield 68.7%)
*XVlIl was obtained.

Melting point 237-240C, Lα] D measurement failure + 7C
Cumulative analysis value Measured value; C64, 15, H6, 57, N
7.88 chi.

Calculated value as C67H7, N70□7; C64,15
゜H6,35,N 7.84g, Rf O,
83 (BAAW).

0.69 (AW).

Reference example 7 ■ 6-Og (4,9mmo, /) in 90% acetic acid 12
Add 8.0g of activated zinc powder dissolved at 0M and heat at 25C.
The mixture was stirred vigorously for 2 hours. After distributing the zinc powder from house to house, water was added to the solution, and crude bottom matter (2) was precipitated. p wave collection, 1,4-
Recrystallization from dioxane gave 4.4 g (yield 81.9%) of . Melting point 238-24 IC.

[α) D -18,5° (0-2, DMF), elemental analysis value Measured value; C60,87, H6,78, N 8.
86%.

Calculated value as C56H75N 70□6; C61,0
2゜)i 6.86. N 8.90%, , 1l
(lf O, 79 (BAAW).

0.84 (AW).

Reference Example 8 XVIII 2.8 g (2.2 mmo Ale Starting material) A reaction was carried out in the same manner as in Reference Example 7. Recrystallization from acetic acid gave 2.2 g (yield 87.2%). XIX
I got it.

Melting point 218-221r:, [(1)D-23,
0° (0,2.

Ac0H), elemental analysis value Measured value; C62,21,H
6,68N8.61%, C59H73N70 engineering. Calculated value as; C62,37, H6,48, N 8.6
3%, RfO, 77 (BAAW), 0.83 (AW)
- Reference example 9 O2 BOC-Ala -0NP +H-Arg-OBzll
.

TosOH O2 one+BOC-AA! a -Ar, g' -0Bzll
(IX) BOC-Aha-0NF 31.0g
(0,10moA) and NO2H-Arg-OBzl-2TosOH65,4g (0
, 10moA) iN,N-dimethylformamide 400
Dissolve in water and add 29.3 ml of triethylamine under water cooling! (
0.21 ml) was added thereto, and the mixture was stirred for 1 hour under OC and then for 12 hours under 25U. After the reaction mixture was concentrated under reduced pressure, water was added and extracted with ethyl acetate, and the extract was washed with hydrochloric acid and water. After condensation, the resulting oil was tritylated with petroleum ether to give 4 as a crystalline material.
5.6 g (yield 95.0%) of IX was obtained.

Melting point 112~1i3C, (α, 1D-30.6° (0
,5.

Etog, 7c elementary analysis value Measured value; C52, 38°1 (
6,91,N 17.43%, C21H3□N60
Total 1 as □--Lfl 花E;)l 52.49.
H6,71,N 17°49%.

About f 0.81 (8BN), 0.83 (BAW
).

NO□ BOC-41a-Arg-OBzl (IX) ->
O2 ■ H-AA' a -Ar g -0Bz l -HC4
(X) IX 38.4 g (80 mmo, d) was mixed with the starting material and 31 #) 3z, 4 g (#) 3z, 4 g (
xvi* with a yield of 97.2%). Melting point 136-145
C2[α]D-11,20(0,5,MeOH), Elemental analysis value Measured value; C45,87°H6,18,N
19.92. C18, 37%.

Calculated value as C16H24N605・HC,d; C
46,10°) 16.04. N 20.16. C7
8,50%, Rf O, 33 (SBN), 0.3
2 (BAW)・Reference Example 11 zlNO2 I HOC-Thr-Of(+)(-Ala-Arg-.

0Bzl(X) → z1 X 25. Og (60,0 mmol) and BQC-T
h r-OH22.3g (72.0mmoA) kN,
Dissolved in 300 g of N-dimethylformamide and added O
N-methylmorpholyy7.9g (72.0mmo
7), 1-hydroxy gamma henttriazole 8.2 g
(61,0 mmoA) and dicyclohexylcarbodiimide 13.0 g (66,0 mmol) were added, and OC
The mixture was stirred for 1 hour at 25C for 12 hours. The by-produced dicyclohexyl urea was removed from the P side, the P solution was concentrated, and the residue was extracted with ethyl acetate. After washing with heavy water, hydrochloric acid and water, it was reduced to 11 and crystallized with petroleum ether to give 39.7.
g (yield 98.5%) of XI was obtained. Melting point: 93~
105C,(α)D-22,5°(1,0,MeOL(
),, elemental analysis (i measurement value; C57,00,146,
87, N 14.53%.

Calculated value as C32H451”709; C57,22
゜)L 6.75. N 14.60% f O,
69 (SB, N).

0.89 (B person W).

Reference example 12 BOC-Thr-Ala-Arg-OBzl (XI) →
Starting material, 33-6 g (50 mmol) of XI,!
: L'' [8 considerations, 2 zs, Og (yield 89.5%) was obtained by the same method as Example 2. Melting point 118-12qc, (
α) D-10,00(1,0,MeOH), Elemental analysis value Measured value; C51,88°H6,20,N 15.
67, C16,01%.

C27H37N702・) Calculated value as IC1H20; C51,79, H6,44, N 15.66, Q!
5.66%.

f(,f O,58(SBN),0.36(BAW
) - Reference Example 13 0BzA! -HCl(X[) Bzl N02 (1 ->BOC-Tyr-Thr-Ala-Arg-OBz
l(XI[I)XII 15.7 g (25,0mm
o7) and BOC-Tyr-0H8,4g (30
, 0 mmol) was used as a starting material and reacted in the same manner as in Reference Example 11. The crude product was obtained as a crystalline substance and was recrystallized from MeOH to give 16.2 g (yield 70.3
%) (obtained Dll. Melting point 178-182U, Cα]
D 16.2° (1,0, MeOH), elemental analysis value
Measured value: C64,28, H6,07, N 12.34
%.

Calculated value as C48H54N801□; C62,73
・) f 5.92. N 12.19chi, I O,8
4 (SBN).

0.89 (BAAW).

Reference example 14 Bzl NO2 I BOC-Tyr-Thr-AJa-Arg-OBzl(
XII[)XIl[12,4g (13,5mmo])
In the same manner as in Example 2, O12,
0 g (100% yield) of XIV was obtained. li8 points 1
43~xssc, [α9-11,60(1,0,'
MeOH), original Tf-analytical value measured value; C55,5
1, H6, 16, N 14.14. C74, 70%.

036H46N8011 ・HCII-1/2H20
t, -'co calculation (i; C55,41,H6-20
, N 14.36. (J 4.54%.

几f　O, 56 (BAAW), 0.21 (CMA).

Reference example 15? zノ? 0□ H-Tyr-Th r-Aza-Arg-OBzl3-
NO7(XIV) ->(zlu-'17r-'l'hr-Aha-Arg-U
BZJ! ! (XV)■ 2-3 g (2.1 mmol) and 1.6 g (2.1 mmol) of Venttoria sole 0.29 g (2.1m
mol) and zinclohexylcarphodiimide 0.
45 g (2.2 mrrto7) was added, and the reaction was carried out for 1 hour under water cooling and for 40 hours at 25C. The reaction mixture was cooled on ice, and the precipitated gel-like substance was washed with hydrochloric acid and water, and then recrystallized from acetic acid to give 2.4 g (yield: 63.0%).
') +7)XV 'e got. Melting point 237-240
C.

[α:] p 23.50 (0, 2,, Ac0H)
+ Elemental analysis value Measured value; C60,53,)16-7
5. N11.50%r C92H119N150□
Calculated value as 4; C60,75,H6,59°N
11.55%, Rf O, 80 (BAAW),
0.81 CAW) - Reference Example 16 Bz/ 0H' (XIX) + H-Tyr-Thr -Ah
a-XIX 1.8g (L6mmol) and XIV
A reaction was carried out in the same manner as in Reference Example 15 using 1.3 g (1.6 mmo/) as a starting material. Recrystallize with acetic acid to obtain 1
．． 6g" (yield 56.0%) of Xx was obtained. Melting point 24
4-247C.

[α) D -20,0° (0,2, Ac0H), elemental analysis value measurement; C61,56, H6,56, N 1
0.99%.

C05H1□7N+50□4 and L teno calculation i p C6
'1.58゜H6-36, N 11.34%, Rf
O, 79 (BAAW).

0.80 (AW).

Reference example 17 0Bzll -TFA (XVI) XV 600'H! (0.33 mmol) and anisole 0.12#LA! and trifluoroacetic acid 15IFL
li was added, and the mixture was stirred at 25C for 30 minutes. After vacuum concentration,
Addition of dry ether gives 598 as a colorless crystalline substance.
A crude product of η was obtained. This was used in the next reaction without purification or identification.

Reference example 18 Tyr-Thr-Aha-Arg-OBzlj -TF
A (XVI)→H-Leu-Iノe-G, du-A
sp -Asn-(iJu-Tyr-Thr-A7a
-Arg-()H-AcOH(□) XVI 500mf was suspended in 250d of 80% acetic acid,
10%T on this! Add d-61, Og and dry with hydrogen for 42 hours to remove any loss. After the catalyst was separated, it was concentrated under reduced pressure, water was added, and the mixture was washed with ether. The aqueous layer was lyophilized to yield crude product 380~ of free decapeptide (XVII). This was purified with 8ephadex LH-20 (90% acetic acid) and Amberlite
)'s X■■ was obtained.

[α) D −34,0° (0,5,20% AcOH),
Elemental analysis value Measured value; C47,11, H6,61
, N 13.47%.

C5□H,,□N 1,02o-2AcOH-4H20
Calculated value as; C47,52,H6,98,N
13.85%, amino acid analysis value Asp 2.15
．． Thr 1.00. GA! u 2-18゜AI
! a O,98,Ile 1.04. Leu 1.0
0. Tyr O,94゜ArgO,92,Rf O
, 43 (BAAW), 0.84 (CMW).

Mass spectrometry value (M-1-H)+1223. NM 几(D2
0); δ0.82~0.98 (12H+ CH31
dfLe,up-Iie)tδ1.2
8 (3H2C)L3of Ma), δ2.05
(6H, CH3of Ac0H) + δ3.90~
4.55 (10H, α-CHofAmino Ac
id) r δ6.98 (4H, C=eHof T
yr).

Test Example 1 [Antibody production ability of decapeptide] Decapeptide and hexapeptide were administered to rabbits using a carrier (KLH), and the obtained antibodies were tested by radioimmunoassay. The results are shown in FIG. 1. The vertical axis of the figure shows the weight of the labeled antigen-antibody conjugate as a percentage of the total amount, and the horizontal axis shows the added unlabeled antigen in molar concentration. For an antigen-antibody complex in dynamic equilibrium,
In the case of decapeptide, the labeled antigen and unlabeled antigen can easily replace each other at a concentration as low as 0:93 pmol, while in the case of hexapeptide, the concentration is as high as 20 mmol, indicating that decapeptide specifically binds antibodies. I can see that you are making it. This specific antibody creation ability of decapeptide is
This is thought to be based on the presence of Tyr, and therefore it is highly likely that it acts as a specific antibody against oncogenic proteins as well.

Test Example 2 [Decapeptide inhibitor (inhibition)]
Activity] 5RI-A+ Y-73+ Fujinami strain [Avian sarcoma virus (avian sarcoma virus)
Oncogenic proteins are collected by adding antiserum to Lysate (Aysate) of cells made cancerous with oma virus), and phosphorylated with P32-ATP in the presence of decapeptide (1nhibitor). After removing the serum, the obtained phosphorylated protein CIAQiI+to4factoryβ,/phospFlocLted-
Table 1 shows the results of fractionation and purification of the sample using acrylamide gel and measurement using radioautography.

Table 1 Strain Decapeptide CPM Residual Activity -819
2 S L (-A 70.
1+ 5746 - 2356 y-7388, 6 + 2088 - 1431 It can be seen that the decapeptide inhibits both cases.

This indicates that the decapeptide is in competition with the active part of the oncogenic protein; in other words, the synthetic decapeptide has the ability to suppress the action of the oncogenic protein.

[Brief explanation of drawings]

FIG. 1 is a diagram for explaining the effect of decapeptide on its ability to produce antibodies. Patent Applicant Procedures Amendment @ (Voluntary) January 3, 1980 ■ Japan Patent Office Commissioner Kazuo Wakasugi 1゜ Indication of the case 1982 Can Patent Application No. 22811 2o Name of the invention Tetrapeptide derivative 3゜ Amendment Column 5 of “Detailed Description of the Invention” of the applicant’s specification, contents of amendment (1) “Benzyloxy group,
” Delete “,”. (3) On the bottom line of page 35, “20 mmol J is
Correct to nmolJ. that's all

Claims (2)

[Claims] (1) Tetrapeptide derivatives represented by the general formula % (in the formula, R1 is a hydrogen atom or a protecting group for an amino group, R2 is a protecting group for an ω-carboxyl group, and 几3 is a protecting group for an α-carboxyl group). It is a protecting group.) (2) R'' is a hydrogen atom or an amino group, a urethane-type protective group, 1 (2 is an alkoxy-type protecting group of a carboxyl group, R3 is an O-phenacyl group, Claim No. 1) Compounds described in Section.