JPS5867656A - Synthetic tuftisin analog and manufacture - 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 present invention provides a synthetic pegtide, and more specifically, a synthetic pegtide.
Technique #1K relates to a method for producing ltr-active analogs of natural beta-re7tisine and ζ0 synthetic tuffdecine analogs with higher potent stimulant activity compared to uftlsln).

Announcer of the above evening 7 news! 2r is useful in IN therapy as a broad-spectrum immunostimulant.

A natural large-effect stimulus (gutide (ri7decine)
It consists of H chain o7 dament. Jin no te, trape l? Cyt@bios exhibits a stimulant effect on many immune responses both in vivo and in vitro; therefore, it substantially stimulates leukocyte and tumor cI77-yO* action activity (Cyt@bios, Ma.1. 6. Mu21-2
';l, 1972; mu, Com5
+tsmtvpoulos sV, A, NaJJar
” Tuftlmln, a nat@ral
andg@n@ral phagoeytosis
stimlatlag p@pt1dsaff@
@t1ng ma*rophag*s and p@1
Useful as a medicinal compound to replace tr-globulin in certain pancreatic disorders with considerable loss of organic resistance to ymorph*nlear infection and in splenic removal* It is known from the literature that (J.

P*d1at # vol, 80 em 4, 1972
Year: A.

C5astantopoul*s e V, enter N
aJjar e J,W.

&alth ” Tuftlsln d@fl@l*
ney; a mowaymdrova* vl
th d@f@et1ve phagoayteal
m”.

564-567 members).

In this field, a large number of synthetic tisine analogs are known, which are produced by converting one or two a() acid components in the molecule (Int, J, P*ptld Prot).
ein R@m, vol, 9 e N 1, 197
7 years; D.

D@nop1nskm, E, Navr@@
ka, I, Z, 81@I! 1101°8,
81opsk, St, 8syman1*e
e E, Kl@m*wska@Partial m
*qw@tx@mm of k1st@n*s wit
htsIfHiln aot1vity' a 7
1-7 pp. 7, lloeklm.

111ophys, mueta s vol, 496 e
Nl, t*yy year; M, Fr1dkin e Y
, 8 tabinsky, V., Zakmtk,
Z#aplr@r @Tuftls1m and
some analogs.

1i1ysth*sim and int@rae
tlon vlth humanpkolymorp
honul@ar l*ykocytv* = , 2
03-211).

However, these converted metutitan analogs have only insufficient potent stimulant activity compared to natural metuticine.

The present invention is directed to providing synthetic tisine analogs with increased potency stimulatory activity and which are economically useful.

This objective is achieved by coupling the synthetic metsutitan analogue, dalicyl-lysil-fa-lyluardinine.

According to the present invention, DARISILURISIRU! The method for producing lolirufurginine is to react the p-2) C10x nyl ester of benzoloxycal-12-glorin with fulginine to produce lolirufurginine. Lolyruarginine was obtained (the Baozhao group was removed from the resulting dipeptide to obtain the Lolyrufluzenin, which was converted into N1-benzyloxycarbyl in t@rt-butylo).
t@rt-butyloxycar-nyl-N'-benzyloxycar is reacted with pentasulfo-phenyl ester of lysine to obtain nyl-lysyl-glolyl-arginine,
The resulting tripeptide's N-terminal (a) group was removed by turokking ps, and the procinder-removed tripeptide was combined with benzyloxycar-nyl-glycine ink fluorophenyl nistert Jli1 to form a benzyloxycar-nyl- Glycyl-penzoloxycar is nyl-lysyl-! Lolyarginine is obtained, and then the preserved tetrapeptide is purified by hydrogenation, followed by isolation of the desired raw IIL product. Glycyl-lycyl-glycyl-arginine (Gly
-Lye-Pro-mrt; ) has the formula:

This material decomposes at temperatures above 110°C, and at certain rotations [α] 20° -62.3° (@0.48.5
11CH, Coon) is a white amorphous lyophilized product.

Amino acid analysis data: Gly-1,1e Lym-0,9a Pro-
1,0@mu rg -1,0m electrophoresis analysis data Komi 1IhO mobility at 900vo voltage on paper FN-16 (G
For the new compounds according to the present invention, "mim is 1" measured for histidine in 1N-iD acid using
．． 100! (-2, 4).

Chromatography analysis data: Chromatography mobility R, -0,16 (3
0:2:6:240 ratio *Oa-tutanol-pyridine-
The biological activity of leuarginine with respect to water-acetic acid) was tested in vitro.For this purpose, the effect of this compound on the phagocytic activity of segmented neutrophils in rat blood was tested in vitro. Nirite is known to stimulate phagocytosis! I(Illtmouthut@
of Organ amount e 11℃mtk*s1s
ofLatvlan Aead@my of 8e1*
Noes 1IllllL Memorial Day 7 Chishin (Thy-Ly
s-Pro-mrg) ・2CH,C0OH-5H2
The effect was compared with the effect of 0). Phagocytic capacity of segmented neutrophils Ir1B, 8. Measured according to the method of Gost Ma (Pro@sed1ngs of AMN
8Bfm eV@presy PitanIJa
@Rol@of Prot@1m Quantity LNm
tr1tion' a vol, 13 e5ms, 2
m 1950-Moscow: V, 8. Go
st*v, M, N, P@tryash1na
mg, A, Popovklna a A, K, 8
aakov” Phago@ytosls＊Compl
@m@nt and Ble@d Coagul
atlon atPr@t@in D@fiai*no
y in 1 Food” *pp. 110-116).

For testing, rat blood diluted with sodium 2-titanate in a ratio of 1=1 was mixed with the physiological solution of synthetic titanate according to the invention according to the method of Ren. Then, agglutination-positive strains (soaguloposi) were cultured for 1 day.
tlv life 5traln) Staphylococcus aureus 1-medium 1bl
Added at nO#R degrees. Mixture on thermostat
The temperature was maintained at ℃ for 30 minutes.

Then fix the smear (-Peng・ar) with M[L, memenol fix)% Romanovsky-G
Count the colored segmented neutrophils according to ymmaloo, and calculate the number of segmented neutrophils walling cutilia (one arm per active phagocyte) and the number of segmented neutrophils within one segmented neutrophil. The characteristics of zero phagocytosis for which the average number (phagocytosis-a) was determined are shown in the table below.

As you can see from the margin table below! Lysyl-lysyl-prolyl-arginine is similar to touchin itself, but more pt.
Activates the negative effects of repetition.

7'' a Reru Aldenha is manufactured by the following method.

P-Nitroa of Penjiruo Ishikar I Nirup Shirin!
The nyl ester is reacted with arginine to yield di(petidopenzyloxycal dlx-prolyl-arginine).

Next, the resulting compound was dissolved in glacial acetic acid and evacuated with hydrogen bromide O glacial acetic acid solution! Obtain Loliru Arginine.

The tripeptide (t@rt- Butyloxycar #N1-pencylcal is nyl-lysyl-
fQ lylu-arginine) is produced and treated with a 70-aqueous solution of trifluoro-clf# acid for 6 times to give N1-benzyloxycarnylysilyl-glolyl-arginine.

Next, benzyloxycar-nyl-glycine 6-pentafluorophenyl ester and tripeptide ++ He-penzyloxycar-nyl-lysyl-deropi were reacted with monofluinine to form benzyloxycal-nyl-glycyl-N1. -benzyloxycar is nyl-lysyl-! Loli Lou Arginine Teru.

The obtained preserved tetrapegtide is converted into hydrogen 1121
Prokinda is released at 0 to swell glycyl-lysyl-7''olylu-arginine.

The general formula for synthesis is schematically shown in Figure 111.

A new joint broadcasting program (Darishirulishirou!)
It has high phagocytosis-stimulating activity at a much lower dose than tisine.

Furthermore, since the synthesis of tisine uses a rather expensive threonine, the use of synthetic tisine analogues according to the present invention can substantially reduce manufacturing costs.

Further details of the present invention will now be described with reference to the following specific examples.

In the synthesis, amino acids and their #conductors available from %R@ama1 (Hungary M) were used.

All amino acids in glycine were in the L form.

The value of the melting point measured in the open capillary was used without correction.・Specific OvMIi is a 5P glycyl polarimeter Perkin E1m
*Measured with r141. Electrophoretic analysis was performed using the IN (S method).
4) And! ! N (4-=m1.4) OrIP Sunchu paper F
It was carried out using N-16 (GDR). electric wire 1111
The zero mobility was determined for histidine (EIIt), and the chromatographic mobility values were determined for the system, a-tanol-ethanol-acid-water, 80:10:5:30 (M); -acetic acid-water, 30:20
:6:24(11): plate 811uf*l,
Eastman and N.rek. Ion exchange chromatography uses α-cellulose Wkatm
Performed using amCM-32;
#Okisu to get the formal internal gradient-Ulfr・gram
(KB, Sweden) was used. Evaporation was carried out in a vacuum convertible evaporator at a residual pressure of 1510-1 and a temperature of 30° C. (1) p-nide of benzyloxycar-nylpyrroline c17
To a solution of znylester 37f (0.1 mol) in nosoxane 360111t was added with vigorous stirring a solution of arginine 16fP (0.09 mol) in water 160-. The solvent was then evaporated for 2 hours and the precipitate was dissolved in 100 mg of dimethylformat. The solution was stirred at room temperature for 72 hours and then poured into a glass of ethyl acetate.

The resulting residue was separated, washed with acetic acid on a filtrate, and precipitated from an ethanol-ethyl acetate mixture. The yield of product (1) was 35) (96);
The product had the following properties: melting point 190°C, [α]
2'-46.6℃; (s 1.0 = R20);
R1”0.53 (Mu I811mf@l) R0゜66
(B # Eastmin): li:jI,,R
9,56(yi(R2,4).

t@rt-butyloxy7cal-N1-benzyloxycal is nyl-lysyl-! loliruarginine a)
-4 ndyloxycar ~-f lolilual pxy (
1) 35F (0,086*le) 12N-11r/CM
250 mg of ICOOH was added for 1 hour. The resulting mixture was evaporated to dryness and the residue was triturated with dry diethyl ether.

I can get it! ! Trade goods are sent door to door, dried in a vacuum, and washed with 500 yen of water.
mgK dissolved. Add the solution to 7 nionite (amlsnlt)
・) causes a negative reaction with bromide ions (m@gat1vs
r@aet1om) I6 was placed in j, and it was dried by nuclear power. The yield of f-lolyluardiene) was quantified (23
1. The product is 2R,-α00 (A
, 811ufol ): E, , mQ, 95 (
PHHI34).

b) frs relua ja4=/(2) 23 P (0,
086 mol) was dissolved in 100 m++ gK of water and stirred in this #I cabinet.
Added a solution of N1-pencyloxycargeni, luricin bentafluoro2 enyl ester 53f (α1%) in Nan 300-, 70 Wk 2 hours ss+;
was evaporated and the residue was dissolved in dimethipformamide 150- and stirred at room temperature (20° C.) for 48 hours.

Next, evaporate the dimethylformand and use 1/1/2 of its volume.
3 and the solution was poured into 117 μl of ether.The resulting residue was separated, washed with ether on a filter, and dried in vacuo.

Ethyl acetate-nithanol-ether mixture 6 (3:1:3
After reprecipitation from 0), a product (3) 47 with the following properties:
? (74L) was obtained: melting point 90°C.

(α3.-39.0°Ce 1.0, 10'jlCH
, Cool): RfexO, 51 (A, Eastm
an), 0.82 (B alcamtmail);
E, B, = 0.34 (pH = 2-4) @ Zenin (5) a) t・rt-Butyloxycal is nyl-N1-pencyloxycal is nyl-lysyl-! lolirou fulginine (
a) a, a3? (10-ken remol) with trifluoro vinegar al
The reaction mixture was tin-salted with i07-aqueous solution for 1.5 hours, and the progress of the reaction was controlled by thin layer chromatography. The solution was evaporated to dryness and the residue was worked up under ether, separated, dried in vacuo, then dissolved in 200 ml of water and concentrated with anionite. The S solution was evaporated to dryness. Dry in vacuum @ N#-penzyloxycar-Nirur J & ldf
Nino (4) 5.6F (Analysis IN) was obtained, which has the following properties and 92 Rf ■ 0.3 0 (A e Eas
tman ) w α6 g (B , Eastman
)* E
% Benzyloxycarm; pentafluorophenyl ester of rutaricin 1.24 t into the solution with stirring.
A solution of diochitin (3.6 mmol) in 30-mg was added and after 2 hours the solution was evaporated to dryness and the residue was dissolved in dimethylformand 8-0. The reaction mixture was stirred for 72 hours and acetic acid Combine with ethyl 200- and put it in the refrigerator for 12 minutes.
Saved time.

The resulting precipitate was separated and washed with ethyl acetate and ether. The yield of vacuum-dried benzyloxycar is 2.31P (965
Compound (5) had the following properties: melting point 175°C; [α]2'=-31.5°゛←a 1. O
: M*OH); R1m 0.40 (A t M@
r@k ) a' 0-74 (B e M@rek
) ; "11'* = 0.36 (T" = 2.4).

Chrysyl-lysyl-7"a lylu-arginine (6) Benzyloxycar-nyl-glycyl-N"-pezyloxycal I-lylycyl-lylyl-arginine (5) 1
．． 0? (1,39 t remol) was hydrogenated under atmospheric pressure for 10 hours in a solution of all vinegar and methanol 20 in the presence of palladium black. The product was purified on a column of α-cellulose (2,5×15 ag), Rimformally increasing ionic strength (from 0.01 to 0.01, yJ (from 4.5 to 0.01)
1M, to pl'iW6.4, then 0.2M.

Elute with ammonium acetate solution at pH-6,9).

At an elution rate of ti 6 Gsg/hour, the fractional volume of 0 is rarely 10-
150-2200 aliquots were collected, evaporated and lyophilized first with water and then with ammonium acetate. The yield of desired product (6) was 0.6 PC6g. The product has the following properties: melting point 110°C <accompanied by decomposition>: ca]:'-e 2.3° (・0.48
# 511 CH, Cool): l, α16 (
B.

M@rek) *Ell m layer 1-10
(f wealth 2-4) *

[Brief explanation of the drawing]

Figure 1 shows a schematic diagram of the synthesis of dalicylurisil-7"Ellyruarginine. Patent applicant Institute Oinicheskopsinte de Akadekui Naukratvi Iskoi Nisnisar Patent attorney Mitsuki Minoru Patent attorney Kazuyuki Shiryodate, patent attorney Nobuo Uchida, patent attorney Akiyuki Yamaguchi Figure 1 procedural amendment (voluntary) May 1980 λρ Commissioner of the Patent Office Shima 1) Haruki Tono 1, Indication of the case 1988 Patent application No. 161103 2, Name of the invention Synthetic tuffden analog and its manufacturing method (new name) 3,
Relationship with the case of the person making the amendment Name of the patent applicant Institut Organit Escogosynteza Academy Naukratvi Iskoi Nisnisar 4, Agent 5, Target of amendment) “Title of the invention” column (2) of the specification "Claims" column (3) of the specification "Detailed Description of the Invention" column 6, Contents of amendment (21 Amend - in the claims of the specification as shown in the attached sheet 0 +3 ) In the detailed description of the invention in the specification, (
b) “Touch Shin (tuf)” in the second to third lines from the bottom of the second page
tisin) J as ``tough day (tuftsin)
) Corrected to J. −) All the terms “touchin” e in the following passages → 3rd
Correct r tuftisiu J on page 13, page 4, line 4, page 4, line 14, and page 17 to j tuftsinJ. ) Correct r ponopinska J in line 11 of the 4th sentence to r Konopinska J. (e) r polymorphon on page 3, line 13
polym
Correct to orphonuclear 1 eukocytesJ. (to) page 8, line 2 +7) r Tb'fJ to rThr
JK, correct. ζTo1 rKBJ on page 1813, line 18 r LKB
Correct to J. Ifi$15 page 3rd line r2N-BrJ is r2N-
Correct to HBrJ. C11815 pages! @Lines 9-10 “Negative reaction for bromine ion”
! "treated with" is changed to "negative reaction (nega
tive reaction), that is, until no bromide ions are detected in the supernatant. 7. Claims as amended by the attached documents 1 copy 2. Claims] Formula 1: A synthetic tuftene analog characterized in that it is glycyl-lysyl-prolyl-arginine with formula %. 2. React p-nitroester of benzyloxycarbonyl-proline with arginine to produce cetehenzyloxycarbonylprolyl-arginine, remove the protecting group from the resulting dipeptide to obtain prolyl-arginine,
It was reacted with the pentafluorophenyl ester of tert-butyl-oxycarbonyl-N6-penzyloxycarbonyl-lysine and
f1J luarginine was obtained, and the N of the resulting tripeptide was
unblocking the terminal aino group, and subjecting the unblocked tripeptide to pentafluoronisterte condensation of benzyloxycarbonyl-glycine to produce benzyloxycarbonyl-glycyl-Nl-benzyloxycarbonyl-lysyl-prolyl-arginine; Thereafter, the preserved t-tetrapepdide is deblocked by hydrogenation and the desired product is isolated. A method for producing a synthetic tuften analog with the formula: glycyl-lysyl-prolyl-arginine.

Claims (1)

[Claims] 1. A synthetic tactile arginine containing formula: % formula %). 2. The benzoloxycar-12-glolin-p-2) tetraester is converted into a trimethyloxycarp to form a nylprolyl-aldfxn, and the resulting di(butide group is removed to form a derivative). It was obtained by reacting it with I@rt-butyl-oxycar-nyl-penzyloxycar-benzyl-lysine (04-ntafluoron phenyl ester) to give CalMnyl-lysyl-7'H-lyrufurginine was obtained, the resulting tree was removed, and the deactivated tripeptide was converted to benzyloxycarmine; the pentafluoronister of rhudalysin. The benzyloxycar is condensed to form nyl-glycyl-N1-benzyloxycar-nyl-lysyl-fa lyluarinine, and then the preserved tetra(gt) is pro- and de-kindred by hydrogenation, and in places A method for producing a synthetic tisine analog with the formula: