Method of producing peptides

专利摘要:
There is disclosed a novel polypeptide, a process for preparing the same, a pharmaceutical composition containing the same as well as the use thereof. The novel polypeptide which is useful as an analgesic corresponds to the formula given below: …<CHEM>… wherein the meaning of R<1> and R<2>, A, B, C, D, E, and F is the same defined in the claims.
公开号:SU1433415A3
申请号:SU3974503
申请日:1985-11-06
公开日:1988-10-23
发明作者:Есино Хироси；Цутия Ютака；Канеко Такеру；Наказава Такахиро；Икеда Масухиро；Араки Син；Ямацу Киеми；Татибана Синро；Аракава Есихиро
申请人:Эйсай Ко.,Лтд (Фирма)；
IPC主号:

专利说明:


s
The invention relates to a method for producing peptides — new biologically active compounds that can be used in medicine,
The purpose of the invention is to obtain new peptides with higher analgesic activity.
PRI me R 1, Synthesis of CH-Tir-1: Gly-Pli-Fen-Leu-Arg-CH Arg-Le-Lei-.
A. Dissolve in 200 ml of tetrahydrofuran. 25 g of BOC-B-Leu-OI-H O. The solution is cooled before and added
11 ml of N-methylmorpholine and
h
9.56 ml of ethyl ester of chloroholic acid 1. After 5 minutes, 12.9 g of a 70% aqueous solution of ethylamine are added to the solution, and the mixture is stirred at approximately -5 ° C for 2 hours. After concentration, the residue is ethyl acetate and washed with an aqueous solution of sodium hydrogencarbonate and water successively. After concentration to dryness, 24.5 g of BOC-D-Leu-ShS Nd, T.L. UZ-106 ° C are obtained.
Thin layer chromatography (TLC): Rf value 0.77 (ethyl acetate). Optical rotation: (a). P +20.0 (, methanol).
 Calculated,%: C 60.44; H 10.14; N 10.84. , C ,.
Found,%: C 60.42; H 10.33; N 10.86.
B. Synthesis of Mr. CH5Arg (Tos) -D-LeuNHC HC. Dissolve in 15 ml of tetrahydrofu; 1.43 g of Z-CHjApr (Tos) -OH, (a) g: -15, dimethylformamide, synthesized from H-Arg (Tos) -OH, is dissolved. After cooling to -30 ° C, 0.33 m of N-methyl morpholine and 0.29 MP of chloroholic acid ethyl ester are added to the solution. After 5 minutes, a solution of 817 mg of H-D-Leu-NHC was added to the mixture (synthesized by treatment of BOC-D-Leu-HHC Nd trifluoroacetic acid
lot in the presence of anisole) and 0.83 ml of triethylamine in 7 ml of tetrahydrofuran and the mixture is stirred at about -5 ° C for 2 hours. After concentration, the residue is dissolved in ethyl acetate, washed with aqueous sodium hydrogen carbonate solution and water, sequentially and concentrated dry to obtain 1.58 g of glassy Z- (Tos) -D-Leu-ShS.GN5.
,
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.-
about ,
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TLC: Rf value of 0.68 (methanol / / chloroform 1/7).
Calculated,%: C 58.42; H 7.19; N 13.63,
SzoN44 0 3
Found,%: C 58.29; H 7.19; N 13.40.
B. Sintez BOK-Arg (Tos). (Tos) - -D-Leu-MHC N.
Dissolved in 4 ml of dimethylformamide 1.1 g. H, Arg (Tos) -D-Leu-ShS, H5, obtained by catalytic reduction of g-, C.N.Arg (Tos) -D-Ley-ShS.N5 in Pd / C catalyst, 983 mg. BOK-Arg (Tos) -OH and 372 mg N-hydroxybenzotriazole. Under ice-cooling, 520 mg of dicyclohexylcarbodiimide was added to this solution, and the mixture was stirred in a refrigerator for 24 hours and then at room temperature for 24 hours. The precipitates thus obtained are filtered off and the filtrate is concentrated. The concentrated residue is purified by chromatography on a column of silica gel (eluent: methanol / CHS 1/15) to obtain 1.2 g of the glassy product BOC-Arg (Tos) -SN3- Arg (Tos) -D-Lei -YNS N.
TLC: Rf 0.64 (methanol / chloroform 1/7). Optical rotation:. (A) d -20.6 (, methanol).
Calculated,%: C 52.72; H 7.30; N 15.37.
C oH64N,
Found,%: C 52.82; H 7.22; N 15.06.
G. Sintez BOK-Ley-Arg (Tos) -CH5Arg (Tos) -D-Ley-YNS2N5.
1.645 g of WOK-Leu is dissolved in 12 ml of dimethylformamide. The solution is cooled before and 0.726 ml of N-methylmorpholine and 0.631 ml of chloroic acid ethyl ester are added to it. After 5 minutes, a solution of 4.986 g of CP5COOH was added to the solution. H-Arg (Tos) - (Tos) -D-Leu-No.1C, E55, synthesized by treatment with BOC-Arg (Tos) -SnzArg.Thos) -D-Lei -ShS2H5 with trifluoroacetic acid in the presence of anisole, and 0.726 ml of N-methylmorpholine in 12 ml of dimethylformamide, and the mixture is stirred for approximately -5 seconds for 2 hours. After concentration and subsequent solidification with methanol / ether, 5.283 g of BOC-Leu Arg (Tos) -CHj Apr (Toe) -D-Ley-. M.p. 120-125 With (decomposition).
TLC: Rf 0.66 (methanol / chloroform 1/7). Optical rotation: (a). 25.8 (, methanol).
Calculated,%: C 54.36; H 7.67; N 14.84.
,, 0, oS ,,.
Found,%: C 54.49; H 7.63; N 14.62.
D. Synthesis of BOC-Fen-Ley-Arg (Tos) - (Tos) -D-Leu-ShS, H5.
1.465 g of BOC-Phen-OH is dissolved in 12 MP of dimethylformamide. The solution is cooled to -3 ° C and 0.608 ml of N-metsh1morfolin and 0.528 ml of ethyl chloroforic acid are added to it. After 5 minutes, 4.691 g of H-Phen-Leu-ArgSTos) - -CHjApr (Toc) -fl- neft-NHC H5, synthesized by treatment of BOC-Fen-Leu-Arg (Tos) -CH, Arg (T (s) -D-Leu-pC H5 with trifluoroacetic acid in the presence of anisole, and 0.608 ml of N-methylmorpholine in 12 ml dimethylformamide, and the mixture is stirred at approximately -5 ° C for 2 hours. After concentration, the residue is dissolved in ethyl acetate, and the solution is washed with an aqueous solution of sodium bicarbonate and water after sequence. After concentration followed by solidification under the action of a mixture of methanol / / ether gave 5.072 g of Boc-Phe-Leu -Arg {Tos) (Tos) -D-Leu-NNS2N5-. M.p. 127-132-C (decomposition).
TLC: Rf 0.66 (methanol / chloroform 1/7). Optical rotation: (a) jj -25.4 (, methanol).
Calculated,%: C H 7.48; N 14.18.
C55H64NiiO VSj CH, OH.
Found,%: C 56.64; H 7.33; N 13.86.
E. Synthesis of BOC-CH, Tyr (C1 Bzl) -Gly- -Gly-OH.
Dissolve 9.09 g of BOC-CH Tyr- (C1 Bzl) OH, (a). P -49 ° (, ethanol) in 150 ml of tetrahydrofuran, synthesized according to C.T. Cheung and 2.53 g of N - oxysuccinimide. After cooling with ice, 4.12 g of dicyclohexylcarbodiimide was added to the solution, and the mixture was stirred in a refrigerator overnight. The white crystals thus obtained are filtered and 2.91 g of H-Gly-Gly-OH and 38 ml of an aqueous solution of 1.888 g of sodium bicarbonate are added to the filtrate. The mixture is stirred at room temperature for
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2 days and then concentrated. A dilute aqueous solution of citric acid and ethyl acetate are added to the mixture and the ethyl acetate layer is separated. After washing with water followed by concentration, the product is purified by chromatography on a column of silica gel with an eluted mixture of methanol / chloroform 1/30 and solidified with ether / n-hexane to give 9.23 g of BOC -CHСTyrS Bzl) Gly-Gly-OH . T.Sh1.70- (decomposition).
TLC: Rf value 0.79 (methanol / / acetic acid / chloroform 4/1/12). Optical rotation: (a) -17 (C-1, methanol).
Calculated,%: C 55,54; H 5.99; N 6.94.
, N, 0, C1 OjSCjHjOC Hs.
Found,%: C 55.45; H 5.81; N 6.89.
J. Synthesis of BOK-CH-Tyr (C12Bzl) -Gly- -Gly-Fen-Ley-Arg (Tos) -CH Apr (Toe) -D- Lei-ShS Well.
682 ml of BOC-CH, Tyr (C12Bzl) -Gly-Gly--OH and 195 mg of N-hydroxybenzstriazole are dissolved in 4 ml of dimethylformamide. While cooling this solution with ice, 272 mg of dicyclohexylcarbodiimide was added to it. After stirring for 2 hours, a solution of 1.167 g of CF, COOH.H-Phen-Leu-Arp (Toc) (Toc) -D-Leu-NHCJHy synthesized by BOC-Fen-Leu-Arg (Toc ) SNzArg (Tos) -D-Ley-Sh.N. trifluoroacetic acid in the presence of anisole, and 0.132 ml of N-methiLmorpholine in 8 ml of dimethylformamide, and the mixture is stirred in the refrigerator overnight. The precipitate thus formed is filtered off and the filtrate is concentrated and purified by chromatography on a column of silica gel (eluent: methanol / chloroform 1/20). After curing with methanol / ether, 1.391 g of BOC- -CHJTip - () - Gly-Gly-Phen-Leu-Arg (Tos) -SNzArg (Tos) -D-Leu-MNS H5 are obtained. M.p. 130-135 ° C (decomposition).
TLC: Rf value of 0.64 (methanol / / chloroform 1/7). Optical rotation: (a). P -35.3 (, methanol).
Calculated,%: C 55.92; H 6.77; N 12.70.
CT6 "" .., - CH,
Found,%: C 55.06; H 6.49; N 12.52.
S143
3. Synthesis of CH Tir-Gly-Gly-Fen-Ley-Arg-CH Arg-D-Lei-Shs Nr,
Dissolve in 10 ml of hydrogen fluoride in the presence of 0.2 ml of anisole in a pressure tight reaction vessel for HF at -5 ° C 220. mg BOC-CH Tyr (ICl Bur) -Gly-Gly-Fen-Leu-Arg (Tos) - - | СНзАрг (Тос) -Д-Лей-ШС Н5. The solution was stirred for 1 hour and then the HiF was distilled off from the reaction mixture. The residue is washed with ether and dissolved in water. The solution is treated with ion-with Amberlite 1PA-93 resin of benzyl resin (ukS 52.73; H 7.50;
Calculated,%: N15.37,
CrHH4oNbOb81 „.Н ,, 0
Found,%; C, 52.77; H 7.60; N 15.14.
B. Synthesis (Tos) -D-Lay-Apr (Toc) -NH.
3.336 g of g-CH3 Arg (Tos) -OH is dissolved in 30 ml of tetrahydrofuran. The solution is cooled to -20 ° C and 0.77 ml of N-methylmorpholine and 0.67 ml of chloroic acid ethyl ester are added to it. After 5 minutes, the solution is added to the mixture.
Ausulphate type) and dried from the dead; 15 3.882 g of CF COOH ND-Leu-Arg (Toc) -NH,
living. The crude peptide thus obtained (120 mg) was purified by high-performance liquid chromatography (Nucleosil phase 5c 18, column diameter 2, length 25 cm, eluent 0.1% HCl in a solution of water: acetonitrile 81:91) —and then freeze-dried, to receive 70 mg of CH Tir-Gli-Gli-Fen-Ley-Arg-CH, - -Arg-D-Lei-NNS H y.
TLC: Rf value of 0.70 (butanol / ./acetic acid / pyridine / water 15/5/5/8). Optical rotation: Ca), -21.8 ° (, 4, 0.01 N. HCl) . acc-spec (FAB): 1036 (M + H). Amino acid analysis: Gly 1.87 (2); Lei 1.96 (2); Hair dryer 1.00 (1); Apr 0.95 (1) (pshs, corresponding to CHj and, not calculated). : PRI and e p 2. Synthesis of Thir-DCH ns-1 Li-Fen-Zis-Arg-CH5 Arg-D-Ley-Arg-Sh,
A. Dissolve 2.493 g of BOC-D-Leu-OH-H O. O. Rast-Yaor in 10 ml of dimethylforma-Mamid, cool before and add 1.1 ml of N-methylmorpholine and 0.96 MP of chlorogolic acid ethyl ester. After 5 min to the mixture of the added - H) t solution 4,414 g of CFjCOOH.H-Apr (Toc) - H and 1.65 ml of N-methylmorpholine in 20 ml
synthesized by treating BOC-D-Leu-Apr (Toc) -NH with trifluoroacetic acid in the presence of anisole, and 1.17 ml of triethylamine in 30 ml of tetra-2Q hydrofuran, and the mixture is stirred at approximately -5 ° C for .2 hours. After concentration followed by curing with methanol / water yields 6.14 g of 2-SisArg (Tos) - 25 D-Leu-Ap g (Tos) -Sh ,. M.p. 100-113 ° C (decomposition).
TLC: Rf 0.44 (methanol / chloroform 1/7), Optical rotation: (a) -3.4 ° (, methanol).
Calculated,%: C 54.18; H 6.71; N 15.04,
C4iH58N, oO, Sj CH OH
Found,%: C 54.12; H 6, B2; N 14.85.
B. Synthesis of BrK-Arg (Tos) -SndLrg (Tos) - D-Leu-Arg (Tos) -Sh,
In 17 ml of dimethylformamide, 4.744 g of CH Arg (Tos) -D-Leu-Arg (Tos) -NHj prepared by catalytic recovery by (Thos) -D-Leu-Arg {Tos) -Sh are dissolved in 17 ml. in the presence of Pd / C, 2.918 g BOC-Arg (Tos) OH and 1.1 N-hydroxybenzotriazole. Under cooling with ice, 1.543 g are added to this solution.
thirty
dimethylformamide and the mixture is stirred dicyclohexylcarbodiimide, and the mixture
- ft
After concentration, the residue is dissolved in ethyl acetate and washed successively with a solution of sodium bicarbonate and water. After concentrating, Ether is added to the residue to cause the product to harden. Thus, 4.96 g of BOC-D-Leu-Arg (Tos) -Sh2 are obtained. M.p. 110 - 120 C (decomposition),
TLC: Rf 0.49 (methanol / chloroform 1/7). Optical rotation: (a). P: p +13.0 (, methanol).
50
stirred in the refrigerator for a day and then at room temperature for a day. The precipitate thus formed is filtered off and the filtrate is concentrated. The residue is purified by chromatography on a silica gel column (eluent: methanol / / chloroform 1/15) and solidified with ether to obtain 4.917 g of BOC-Arg (Tos) - gg -CH, Arg (Tos) -D-Leu-Arg (Tos) -MN ,. M.p. 131-13b with (decomposition).
TLC: Rf 0.44 (methanol / chlorofor 1/7). Optical rotation: (a) j, -16.7 ° (, methanol).
synthesized by treating BOC-D-Leu-Apr (Toc) -NH with trifluoroacetic acid in the presence of anisole, and 1.17 ml of triethylamine in 30 ml of tetra-Q hydrofuran, and the mixture is stirred at approximately -5 ° C for .2 hours. After concentration followed by curing with methanol / water yields 6.14 g of 2-SisArg (Tos) - 5 D-Leu-Ap g (Tos) -Sh ,. M.p. 100-113 ° C (decomposition).
TLC: Rf 0.44 (methanol / chloroform 1/7), Optical rotation: (a) -3.4 ° (, methanol).
Calculated,%: C 54.18; H 6.71; N 15.04,
C4iH58N, oO, Sj CH OH
Found,%: C 54.12; H 6, B2; N 14.85.
B. Synthesis of BrK-Arg (Tos) -SndLrg (Tos) - D-Leu-Arg (Tos) -Sh,
In 17 ml of dimethylformamide, 4.744 g of CH Arg (Tos) -D-Leu-Arg (Tos) -NHj prepared by catalytic recovery by (Thos) -D-Leu-Arg {Tos) -Sh are dissolved in 17 ml. in the presence of Pd / C, 2.918 g BOC-Arg (Tos) OH and 1.1 N-hydroxybenzotriazole. Under cooling with ice, 1.543 g are added to this solution.
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stirred in the refrigerator for a day and then at room temperature for a day. The precipitate thus formed is filtered off and the filtrate is concentrated. The residue is purified by chromatography on a column of silica gel (eluent: methanol / / chloroform 1/15) and solidified with ether to obtain 4.917 g of BOC-Arg (Tos) - -CH, Arg (Tos) -D-Leu-Arg (Tos) - MN M.p. 131-13b with (decomposition).
TLC: Rf 0.44 (methanol / chlorofor 1/7). Optical rotation: (a) j, -16.7 ° (, methanol).
Calculated,%: C, 51.32; H 6.76; N 16.43.
Csi itsNuOnS, -
Found,%: C 51.15; H 6.54; 48
G. Synthesis of BOC-Cys (SNZBzl) -Arg (Tos.) 7CH5Arg (Tos) -D-Leu-Arg (Tos) -Nk
V Dissolve in 4 ml of dimethylformamide 747 mg of WOK-Cys- (CH, Bzl) -OH, the solution is cooled to -20 ° C and 0.254 MP of N-methylmorpholine and 0.221 ml of ethyl chloroformate are added to it. acid. After 5 minutes, a solution of 2.497 g Arg (Tos) -CH Arg (Tos) -D-Leu-Arg (Tos) - -NHij, synthesized by BOC-Arg- (Tos) -CH, Arg (Tos), is added to the solution. -D-Leu- Apr (Toc) -NH, j, trifluoroacetic acid in the presence of anisole and O, 277mp N-methyl morpholine in 6 ml of dimethylformamide, and the mixture is stirred for 2 hours at approximately -5 ° C. After concentration, the mixture is dissolved in ethyl acetate and then washed with a solution of sodium hydrogen carbonate and water. consistently. After additional concentration, the product is cured with a mixture of methanol and ether to obtain 2.548 g of BOC-Cys (CH5Bzl) -Arg (Tos) - -CH, Arg (Tos) -D-Leu-Arg (Tos) -Sh2.T. pl. 126-132 0 (decomposition).
TLC: Rf 0.51 (methanol / chloroform 1/7), Optical rotation: (a). -20.6 (, methanol).
Calculated,%: C, 52.81; H 6.82; N 14.66.
C q, N 150, e S4 CH OH H2.0
Found,%: C 52.78; H 6.43; N 14.29.
D. Synthesis of BOK-Fen-Zis (SNBl) - Apr (Tos) -CH, Arg (Tos) -D-Ley-Arg (Tos) -NH ..
467 mg of BOC-Fen-OH is dissolved in dimethylformamide. The solution is cooled before and 0.194 ml of N-methylmorpholine and 0.168 ml of ethyl chloroformate are added to it. After 5 minutes, a solution of 2.234 g of CP, COpH-H-Fen-Cys (CH3Bzl) - Apr (Tos) -CH, Arg (Tos) -D-Leu-Arg (Tos) - -NHj, synthesized by treating BOC-Fen-Cis (CH, Bzl) -Arg (Tos) - (Toe) -D-Leu-Apr (Toe) -NH with trifluoroacetic acid in the presence of anisole, and ml of N-methylmorpholine in 5 MP of dimethylformamide, and the mixture is stirred in for .2 h at -5 s. After the wasp drain
The cake is dissolved in ethyl acetate, and the solution is washed with an aqueous solution of sodium hydrogencarbonate and water successively. After additional concentration, the product is cured by adding a mixture of methanol and ether to obtain 2.126 g of BOC-Fen-Cys (CH5Bzl) - Arg (Shh) -CH Apr (Toe) -D-Lay-Apr (-Sh. T, pl. 124-130 C (decomposition).
TLC: Rf value of 0.56 (methanol / / chloroform 1/7). Optical rotation: (a) j5 -18.9 (, methanol).
Calculated,%: C 54., 83; H 6.89; N.
, 60i4Sv 5 / 2CH, OH
Found.,%: C 54.62; H 6.34; N 13.64.
E. Synthesis of BOC-D-Cys- (CH Bzl) -Gly- -OC, lH5.
3.233 g of BOC-D-Cys (CH, Bzl) -OH is dissolved in 15 ml of dimethylformamide. The solution is cooled before and 1.1 ml of N-methylmorpholine and 0.956 ml of ethyl chloroformate are added to it. After 5 minutes, a solution of 1.396 g of NSG H-Gly-OS Well and 1.1 ml of N-methylmorpholine in 20 ml of dimethylformamide is added to this solution, and the mixture is stirred for 2 hours at a temperature of about -5 s. After concentration, the residue was dissolved in ethyl acetate, and the solution was washed successively with an aqueous solution of sodium hydrogencarbonate and water. After concentration, n-hexane was added to the residue to cause the product to solidify. Thus, 3.6 g of BOC-D-Cys-Gly-OS KS- are obtained. M.p. 80-82 C.
TLC: Rf value of 0.74 (chloroform / / ethyl acetate 2/1). Optical rotation: (a) jj +30.2 (, methanol).
Calculated,%: C 58.51; H 7.37; N 6.82.
Found,%: C 58.35; H 7.23} N 6.69.
J. Synthesis of Boc-Tyr () - D-Cys - (CH, Bzl) -Gly-OCjH 5-.
Dissolve 2.068 g of Boc-Tyr () -OH in 20 ml of tetrahydrofuran. The solution is cooled to -20 ° C and 0.517 ml of N-methylmorpholine and 0.45 ml of ethyl chloroformate are added to it. After 5 minutes, a solution of 1.94 g of CFjCOOH-H-D-Cys (CH, Bzl) - Gly-OS Well, synthesized by treatment with BOC-D-Cys (CH, Bzl) -Gly-OC4H, trifluoroacetic acid in the presence of anisole, and 1 ml of triethyl atgane in 20 ml of tetrahydrofuran, and the mixture is stirred for 2 hours at a temperature of about -5 ° C. After concentrating s Water is added to the residue to form a precipitate, which is filtered ™ E | ay and dissolved in a mixture of methanol and loroform. After concentrating, ether is added to the residue to cause solidification of the product. Thus, 2.611 g of C1, Bel) -D-Cys (CH, Hazl) -Gly-OC H5 are obtained.
15
149-150 C.
TLC: Rf value of 0.63 (chloroform / ethyl palladate 2/1), Optical rotation: (a). P +17, (, dimethylforma- .. amide),
Calculated,%: C 59.01; Ы 5,91; 5.73,
.
Found,%: C 58.94; H 5.75; 5.62.
3. Synthesis of wok-tyr () - D-IjUac (CH3, Val) -Gly-OH.
In 30 ml of tetrahydrofuran-ija, 2.345 g of WOK-Tyr (C15. Bzl) -D-15s () -Gly-OC H5 is dissolved and then added
solution 3j2 MP 1 and, sodium hydroxide solution. The mixture was stirred at 1 |: with an ambient temperature for 1 h, 3.2 ml of 1N was added to it. salt 1 | sislota. The resulting mixture was concentrated. Water is added to the residue to solidify. Thus, 1.899 g of BOC-Tyr () - D-Cys () - Gly-OH is obtained. So pl., 133-138 ° C (decomposition).
TLC: Rf value of 0.25 (methanol / / chloroform 1/7). Optical rotation: (a) j, +3 5.8 ° (, methanol).
Calculated-eHOs%: C 57.95; H.5.58; N 5.96.
..
Found,%: C 57.81; H 5.33; N 5.92,
And „Synthesis of BOK-Tyr () - D-1b1S (CH | Bzl) -Gli-Fen-Cis (CH, Bzl) -Apr (Toe) CHj Apr (Tos) -D-Ley - Arg (Tos) -Shg .
 983 mg of BOC-Tyr () -D-Cys (CH3B3) -Gly-OH and 226 mg of N-hydroxybenzotriazole are dissolved in 5 ml of dimethylformamide. While cooling with ice, 316 mg of dicyclohexyl-: carbodiimide is added to the solution. -After stirring for 2 hours, a solution of 1.94 g is added to this solution. H-Fen-Cys, (CH5Bzl) -Arg (Tos) (Tos) -D - Ley20
35
40
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BOC-Phen-Cis (CH3V3) -Arg (Trs) -D-Leu-Apr (Toc) -Sh2 trifluoro-succinic acid treatment in the presence of anisole S and 0.167 MP of N-methylmorpholine in 10 ml of dimethylformamide, and the mixture is stirred in a refrigerator overnight, the precipitate thus obtained is filtered. After chromatographic purification on a column of silica gel (eluted with methanol / chloroform 1/20), followed by curing with methanol / ether, 2.0 g BOC-Tyr () -D-Cys (CH, Bzl) -Gly-Phen -Cis (SNzBzl) -Arg (Tos) -CH5Arg (Tos) -D-Leu-Arg (Tos) -Sh2., Tpl, 123-130 s (decomposition),
TLC: Rf value 0.63 (methanol / / chloroform 1/7), Optical rotation: (a) and -17j5 ° (, dimethylformamide).
 Calculated,%: C 56.60; —H 6.52; N 11.89.
C ,, 2, N ,, 0 gSyCLi-CjHjOCjH53 / 2CK Found,%: C 56.38; H 6.18; N 11. ,, 72,
K, Sintez Tyr-D-Tsis-Gli-Fen-TsisArg-SN rg-D-Ley-Arg-MN.
515 mg BOC-Tyr () - D-Cys (CH3) -Gly-Fen-Cys (CH 3 Bs) - Arg (Tos) -C% Apr (Toe) -D-Lay-Apr (Toe) - -NHj are dissolved 20 ml of hydrofluoric acid in the presence of 5 ml of anisole at-5 ° C in a reaction vessel 5 sealed for hydrogen fluoride (HF). The solution is stirred for 2 hours, and then distilled off by hydrogen HF (HF), the solution is stirred for 2 hours, and then the HF is distilled off from the reaction system. The residue is washed with ether and dissolved in water, the solution is treated with Amberlite 1PA-93 resin (acetic acid type) and then freeze dried. 320 mg of the dried product are dissolved in 1.3 liters of water and the solution is made alkaline to pH 8 with aqueous ammonia. Air is then introduced into the solution with stirring for 2 days, and the mixture is acidified to pH 6 and. then freeze-dried. The crude peptide thus obtained is purified by high performance liquid chromatography (Nucleosil phase 5 C 18, column diameter and length 2 and 25 cm, eluent: 0505% HCl solution in water / / acetonitrile 88/12) and dried by living, obtaining 140 mg of Tir-D CysApr (Toc) -NH, synthesized by Gly-Fen-Cys-Arg-CH3Arg-D-Lei-Arg-NN,
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BOC-Phen-Cis (CH3V3) -Arg (Trs) -D-Leu-Apr (Toc) -Sh2 trifluoro-succinic acid treatment in the presence of anisole S and 0.167 MP of N-methylmorpholine in 10 ml of dimethylformamide, and the mixture is stirred in a refrigerator overnight, the precipitate thus obtained is filtered. After chromatographic purification on a column of silica gel (eluted with methanol / chloroform 1/20), followed by curing with methanol / ether, 2.0 g BOC-Tyr () -D-Cys (CH, Bzl) -Gly-Phen -Cis (SNzBzl) -Arg (Tos) -CH5Arg (Tos) -D-Leu-Arg (Tos) -Sh2., Tpl, 123-130 s (decomposition),
TLC: Rf value 0.63 (methanol / / chloroform 1/7), Optical rotation: (a) and -17j5 ° (, dimethylformamide).
 Calculated,%: C 56.60; —H 6.52; N 11.89.
C ,, 2, N ,, 0 gSyCLi-CjHjOCjH53 / 2CK Found,%: C 56.38; H 6.18; N 11. ,, 72,
K, Sintez Tyr-D-Tsis-Gli-Fen-TsisArg-SN rg-D-Ley-Arg-MN.
515 mg BOC-Tyr () - D-Cys (CH3) -Gly-Fen-Cys (CH 3 Bs) - Arg (Tos) -C% Apr (Toe) -D-Lay-Apr (Toe) - -NHj are dissolved 20 ml of hydrofluoric acid in the presence of 5 ml of anisole at-5 ° C in a reaction vessel 5 sealed for hydrogen fluoride (HF). The solution is stirred for 2 hours and then HF is distilled off with hydrogen (HF). The solution is stirred for 2 hours, and then HF is distilled off from the reaction system. The residue is washed with ether and dissolved in water, the solution is treated with Amberlite 1PA-93 resin (acetic acid type) and then freeze dried. 320 mg of the dried product are dissolved in 1.3 liters of water and the solution is made alkaline to pH 8 with aqueous ammonia. Air is then introduced into the solution with stirring for 2 days, and the mixture is acidified to pH 6 and. then freeze-dried. The crude peptide thus obtained is purified by high performance liquid chromatography (Nucleosil phase 5 C 18, column diameter and length 2 and 25 cm, eluent: 0505% HCl solution in water / / acetonitrile 88/12) and dried by living, obtaining 140 mg Tir-D Cys, 1A3
TLC: Rf value 0.56 (butanol / / acetic acid / pyridine / water 15/5/5/8). Optical rotation; (a) j, -29 (, 4, 0.01 N. HCl). Mass Spectrum (FAB): 1183 (M + H). Amino Acid Analysis: Gly 1.02 (1); Cis 1.83 (2); Lei 1.04 (1); Tyr 0.80 (1); Hair dryer 1.00 (1); Apr 2.66 (2); Cys, conditioned CH, Arg, was not calculated.
Examples 3-29. Given in Table. 1 synthesized compounds in a similar conventional liquid-phase manner as in Examples 1 and 2. In these experiments, dinorphin derivatives modified at positions 1-3 (Tyr-Gly-Gly), at positions 4-7 (Fen-Ley Arg-Arg) , in positions 4-8 (Fen-Ley-Arg-Ilei) and in positions 4-9
Example
CH TyrCH, TyrCH, TyrCH Tyr-CH5Tyr-CH, Tyr-CH, Tyr-Tyr-L - CH, TyrCHjTHp-
AT
Gly-Gly-Phen-Gly-Gly-Phen-Thly-Gly-Fen-Thly-Gly-Fen-Thly-Gly-Fen-Thly-Gly-Fen -Hairdryer
Thly-Gly-Fen {Cis-Gly-Fen-Gly-Gly-Fen-Thly-Gly-FenA
Aphi-Gly-Phen-Gly-Gly-Phen5. 2
(Fen-Leu-Arg-Arg-Leu-Arg), synthesized in a stepwise manner, starting from the C-terminal group in each peptide. Then, these derivatives at positions 1-3 and those at positions 4-7, 4-8, or 4-9 are condensed together according to the method of dicyclohexylcarbodiimides or the mixed anhydride method. All protecting groups are removed with hydrogen fluoride (HF) and the product is purified by preparative high performance liquid chromatography using a reversible phase carrier. In Example 13, all protecting groups were removed by KF, and the compound was oxidized with air and then purified by preparative high performance liquid chromatography.
Table 1
Compound
F
NH
-D-Lei-ON -D-Leu-OS Well -D-Lei-Ш-Д-Lei-ШС Н5.
Recounted
Lei-Apr-Apr
-Ley-Arg-Arg-Ley-Arg-Arg-Ley-Arg-Arg-Ley-Arg-Apr (napa-NO) -Ley-Arg-D-Ley-Sh. -Ley-CH-Arg-Arg-D-Ley-Sh, -Lay-Arg-CH-Arg-D-Ley-Sh
(para-SH4) -Ley-Arg-CHzArg-D-Lei-ShS.GN-Ley-Lees-SN-Arg-D-Ley-Sh, with Arg-CH, Arg-D-Ley-ShS N-Ley-Arg -Arg-D-Ley-Arg-Sh-Ley-Arg-CH-Arg-D-Ley-Arg-ShS N
Sun D E:
- (napa-NO, j) -Ley-Arg-OH-Arg-D-Leu-Arg-S-Met (0) -Arg-CH5Arg-D-Lei-Arg-HH2
Tret-Ley: (CH), DOS. (PL) COOH
Homo Apr: N Н4С (“МН) ШСН СН4СН СН С (Ш4) CОН
Hairdryer (para-III) N02- -CH2CH (NH2) COOH
Sar:
In tab. 2 lists the compounds, composition, optical angle values obtained under the proposed 15 rotation and Rf values. method, indicating amino acids
... ".
.Table 2
Note. In amino acid analysis, the ratio is calculated only
amino acids: Gly, Ley, Fen, Apr, Ala, Tyr, Liz, Ser, Tsis and Glu.
go - definition of C "0.4 n, HC1. Determination of Rf value in TLC: butanol / acetic acid / pnrndine / water- - 15/5/5/8.
The animal test results illustrate the effectiveness of the compounds as anesthetic agents.
15 . 143
Test 1. Test for anesthesia. The test compound is dissolved in physiological saline and administered intravenously or subcutaneously to male ddy men (body weight 20-27 g, when studied, usually divided into groups of 8). The analgesic activity is measured in a test.
Tania with pinching tail.
In this test, the pinching of the tail at the base of the tail, including the anal mucosa, is placed a clamp that develops a pressure of about 300 g, and the latency of biting of this clamp is measured. Before the experiment, animals were selected for nociceptivity of the tail clamp, and those mice that did not bite the clamp within 3 s were excluded from the experiment. A latency of more than 6 s was used as the criterion for anesthesia.
EjtjQ values (dose, 50% anesthetizing) are calculated according to the method of Litchfield and Wilcoxon. The results are shown in Table. 3 (with intravenous injection) and in table. 4 (with subcutaneous injection).
Table 3
sixteen
Continuation of table 3
-
14
15
sixteen
17
Dinorphin (1-13)
1.8 0.7 0.7 2.0 more than 25.0
15
Table 4
soci
Pinching of the tail EDo, mg / kg (subcutaneously)
25
five
0
five
0
five
Test 2. Opioid activity. The opioid activities of the compounds are determined by the proposed method using the rabbit seed channel. In this trial, adult male rabbits are killed by injecting air into the ear vein. Immediately after death, laparotomy is performed and the right and left seed canals are removed. This seed is pushed out of the channels into the Ringer solution. Each channel is cut into pieces (2.5 cm long from the end of the prostate gland). These pieces of the seed channel are suspended with a screw in a 6 ml glass cell at a constant temperature and stimulated with an electrical stimulator having platinum electrodes under the following conditions: frequency 0.1 Hz, 1 mlc and 90 V. Reduction caused by electrical stimulation , write through the converter.
Opioid activity is determined based on the inhibition of contraction caused by electrical stimulation.
The results are presented in table. 5 in units of 50% inhibition concentration (YKud).
Table 5
From tab. 5, it is obvious that the compounds of the proposed method show a very large activity compared to dynorphin A. In addition, they have a greater activity in inhibiting the contraction of the ileal longitudinal muscles of the guinea pig and seminal ducts caused by electrical stimulation.
Peptide compounds obtained under the conditions of the proposed method have opioid activities similar to those of dinorfin, and their effect is very strong, significant analgesic effects are manifested by intravenous injection or subcutaneous injection.
It is very valuable that the compounds obtained by the proposed method show a strong analgesic effect when administered systematically by intravenous or subcutaneous injection, while dinorphin and its derivatives have a barely noticeable analgesic effect by intravenous injection, since these compounds are unstable in the blood.
The ratio between the toxicity (minimum lethal dose) of the peptide compounds obtained in Examples 1 and 2 and the effective dose is shown in Table. 6
Table 6
Thus, the compounds obtained under the conditions of the proposed method can be classified as moderate toxic and have a higher analgesic activity than dinorphin.

权利要求:
Claims (1)
[1]
Invention Formula
The method of producing peptides of the general formula
Rf
g
X
LTyr-А-Uly - В - С - D-Е-Т
where R, g is hydrogen or one of R, and R is hydrogen, the other is C -C-alkyl; A - Gly D-Cys, bonded
C C, if C - Cys; B - Phe, (n-N02) Phe; C is Leu, Met (O), Suz, associated with A, if A is D Cys;
D is Arg, CH, Arg, Lys; E - Arg, CHjArg; F - NH, D-.eu-X, where X is OH, NHj,., Or D-Leu-Arg-X, where X is Shd,,
TAKING OFF the fact that they are gradually increasing the peptide chain, starting from the BFB of the protected C-terminal amino acid, using the carbodiimide method and the mixed anhydride method to activate the carboxyl groups, and removing the protected groups from the obtained protected peptide.

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法律状态:
2005-01-10| REG| Reference to a code of a succession state|Ref country code: RU Ref legal event code: MM4A Effective date: 20021107 |

优先权:

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申请号 | 申请日 | 专利标题

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JP59236076A|JPH0680079B2|1984-11-09|1984-11-09|Polypeptide|

---