SU593659A3 - Method of preparing l-leucine-13-motiline - Google Patents

Description

one

The invention relates to a method for producing a new, previously unknown pripeptid-H ' -peucin 13-motipin, which can be used in medicine.

A known method for producing L-verpeucine 13-motilin ij by condensing individual peptide fragments with a modified carbodiimide method 2 based on diaoicodexipcarbodiimide-N-oxysuccinimide and removing protective groups with trifluoroacetic acid. The following protected fragments were used for the synthesis of L-norpeycin 13-methyline: g

T. Arginyl (bromohydrate) asparagInyl-N-tert.h-butyloxycarbonyl-lysyl-glycyl-gputamin-tr-er. -Butyltin ether (amino acids 18-22);

:P. N -Gputamyl- (Y-tert-butyl afcr) -glutamyl- (U-tert-butyl ether) ČTs-tert, -butypoxycarbonyl-lysyl-glutamic acid- Jf-rTpeT-. Butyl ether (amino acids 14-17) j

11L N (benzyloxycarbonip) -argyyl-norlecyl (amino acids l2-13) j

iV. M-Benzyloxycarbonyl-glu Tamil

(Y-tert., Butyl ether) -leuciputine (amino acids 9-11);

Y. O-tert-Butyl-threonyl-O-tert-butid-oz; 1-glycine (amino acids O-8);

Vi. N is tert.-Voutiloxycarbopip-fennpapanil-valyl-propyl-isoleucyl-4) enipapat1n (amino acids 1-5).

The purpose of the invention is the synthesis of new biologically active motilin derivatives.

The goal is achieved by the proposed method of obtaining b -peicin-13-motilina formula

lis 6 those 9 10

H-Phe-Vae-Pro-JEe-Pbe-Thf-T n-Qty-G.eu-Leui 2. -fi 14 15 16 iT 8 19 th -Gen-Ar0 - beo-G | eu-C, &; u-Lv5-Gtu-Arpf-A ", h-LfS2f 2l.

-G, Ev-G, En-OH

that enclosed kontstatsii protected fragments:

tert-yutyl. eff1tr arginip (hydrobromide) -sp ar agini ln N -t-bugyloxycarbo, nyl lysyl-glycyl-glutamine (18-22.);

J -t. -butylBoyl ester N -benzyloxy carbonyl-gputamyl (-t-. -butted  ether) glutamip (-tert. -butyl ether) -M -t. - 6 utipoxycarbonylpizip-G71utaminovy kspoty {14-17); t N - tris. -benzopoxic 1 Erbonip-L-aprHHHn-, ii-peicin (12-13); -Beneipoxycarbonipgpugamip (if -tert. -butyl ether) -pazipugpugamina {9-11); O-tert. -butiptreonip-O-Treg. butyro tyrosylgpicine (6-8) and N -t-tert. -butipoxycarbonipfenipapanip-wapip-propip-isopeucine-phenipapanine (1-5) with N, N -dicyclohexylcarbodiimide-N -oxy-succinimide, remove the triangle with trifluoroacetic acid with acetic acid.    .  FIG.  .  1 shows the schemes. and obtaining fragment i; in fig.  2 - the same, fragment II; in fig.  3 - the same, fragments of FIG.  4 shows the production scheme (fragment III, the combination of the fragments in FIG.  5 - scheme.  ma getting $ g of fragment 1Y, combining fragments 0 and (G-I1). , U1 and V, f-TV and Y-YI.  : -,.     .  ,,, S & The following examples of abbreviations and symbols common in peptide chemistry are used in the scheme and in the examples: b — configuration pointer, 2 — benzyl xycarbonyl residue, second — tert. - butyloxycarbonic current axis,; BCC1) rM, M-dicyclohexylcarbodiimide method,. , DCCD / HQ; & V. -N, N-dicyclohexipkar.  bodyimide-H-oxysuccinimide method.  : DCCD / HQBT-N, N Dicyclohexylcarbodiimide-N-hydroxybenzotriazole method.  PAM is a phosphosphoicogrammethod; SV is an oxysuccinimide residue; NP t is a p-trophy or a residue, tBu.  - hot. -butyl residue, DBSI dibenzosulfimide as so leo. form, tel,.  .  AL is a methyl residue, TPE is trifluoroacetic acid.   P p: and measure 1.  Obtaining fragment I (part of the sequence: impersonal 18-22) H-it-Oi B. U (22 b) is obtained from Z-GEn OH (22 d).  esterification of trebutyl.  Acetic ester. ACIDS when using sulfuric acid. acid as catalysis toro and col.  benzyloxy removal.  of the sore protective group by hydrogens.  HGEn -OtBu is combined with Z -QEv -OSV. (21), as a result, receive the ether ben, zilodsikarb: onil-dipeptide (21-22a).  N -. The protecting groups are removed by rt-gdrogenolysis, the resulting H1Qfv-QEn -OtBu (21-22b) P1odust condensate with Z-Asn-b (s (BQC) -OH (19-2O) N, N -carbodiimide-N-oxy-curved) In this way, in Rezupta - those of which is formed tert. β-butyl ether of benzylcarboxylate tetrapeptinda (19-22a).  Dipepgid (19-2O) is prepared by aminoacylation of H-Lvs (BOC) -OH (20) with Z-Abn-ONP (19).  From compound Z-A3p.  - Lvs (V. OS) -Qtv -Qtn -Ot Bu (19-22a) benzylxycarbonyl protection The 1st group was removed by catalytic hydrogenation, resulting from the ester derivative of tetrapeptide (19-22) condensing with (cP. iw-Zf) -ON P (18), receive, - 2- Arg (d ,. ) -A5n - bvs (BOci-QCv -Gen-OiBO ri8-22a). : A.  Dibenzenesulfimide salt tert. β-jty glutamine ester.  103 g of tert. -benzyloxycarbonylglutamine-butyl ester in 2 liters of methyl alcohol is subjected to: atomic hydrogenation: (papladium black) while 91 g of dibenzenesulfimide in 500 ppm of methyl alcohol is added dropwise at pH 3.5.  The precipitate is filtered off. The filtrate is evaporated in vacuo, complete with azeotropic distillation with benzene. A solution of the oily residue in ethyl acetic ester is shifted with diethyl ether. and the product crystallizes.  For a more complete purification, the product is recrystallized from methyl alcohol and diethyl ether, t.  square  l35-136 ° G, fctI + 10.78 (c 1.0; in methyl alcohol).  Output 146g (97% of the theoretically calculated value. cheni).  B. ; tert -Benzyloxycarbonyl-glicyl-b-butyl-butyl ester.  A suspension of 115 g of dibenzopuls. fist salt salt tert-. butyl ester of L-glutamine in -800 ml of dichirrmethane is mixed with 32.2 MP of triethylamine and then with 70.5 g of benzyloxy carbonylglycine-N oxychroicine ether ether.  The move continues. within 24 hours at room temperature, then evaporated in vacuo ,. The oily residue is diluted with ethyl acetate, the solution is washed with aqueous solutions of pymonic acid, potassium biconate, and grits, dissolved in sodium sulfate and evaporated in vacuo.  A masse product is obtained, yield 87 g (93% of the theoretically calculated value) B.  Dibenzenesulfimide salt tert. - BUTIPEL ester glidip-L-glutamine.  A solution of 80 g Tert. - butyl benyloxycarbonylglycyl-b-glutamine in 9OOmp ethyl alcohol, by analogy with sposobom, in paragraph A, is subjected to hydrogeolytic deacylirovia (60 g of dibenolsulfimide) and processing.  May get the shape of the residue, which cristapes on when rubbed with petroleum ether t. square   (with ), (p1. , (c - 1.0 in methyl alcohol).  Yield 110 g (98% of theoretically calculated value).  G.  Benzyloxycarbonip-lj-asparaginyl-N -t. -butyloxycarbonyl-Ij-lysine A solution of benzIltriethylammonium soya obtained from 51 g of N-tert. -butipoxycar bonip-1l-lysine in 7OO ml of dimethylformamide is stirred for 48 hours at 20 ° C with 81 g of 4-nitrophenipoyl benzyloxycarbonip-L-asparagine and 1. pyridine equivalent.  The reaction mixture is evaporated in vacuo, the residue is treated with ethyl acetate and a solution of potassium sulfate.  The friable precipitate formed is filtered off and then recrystallized from a mixture of ethyl alcohol and methyl ether, t.  square  product 174-175 ° C (with decomp. ) ,. e ° +.  (from 1, 0, in pyridine).  Yield 70 g (71% of theoretically calculated value).  ; D.  tert -Butyloxy ester of benzyloxycarbonyl-1 "-asparaginyl-tert. -butyloxycarbonyl-1 -lysiplitzip- U -glutamine.  Raspvor 61,5g dibenzbpsupfimidnoy sopi tert-butyl ester of glycyl-U-glutamine and 54.5 g of benzyloxycarbonyl-C-aspar ginyl-N. -tr. -butyloxycarbonyl-Cg-lysine in 7 ° C liter of dimethylformamide is mixed at 0 ° C. first with 15.5 ml of triethypamine and then after 15 minutes with 13. g N-oxnsuccinimide and 23 g N, N-dicyclohexylcarbodiimide.  The reaction mass is stirred at OS for 3 hours,. then the temperature is raised to room temperature over the next 24 hours, stirring is continued at room temperature, H, N-dicyc. The inil is moistened with inu, filtered, and the filtrate is evaporated in vacuo.  According to the scientific residue, it is crystallized from ethyl acetate.  The product is purified by recrystallization from a mixture of methyl alcohol, water and isopropyl alcohol and ethyl acetate, t.  square  Product 155-156 With LC. J-JJS--2O, 8 (s-1.0, in ethyl alcohol).  Output 63 g (78% of the theoretically calculated value), E.  Gd-tert chloro-t-butyl ester 1d -asparagikyl-N -t. -butyloxycarbonyl-b-pizipgpipits-1, -glutamine. .  47, S g yells. -butyloxy benzyloxycarbonyl-L -asparaginez-N-tert. -butyloxy-carbonyl-U-lysylglycyl-b -glutamine: Promoted according to D) in 8OO ml of methyl alcohol is hydrogenated under the conditions described above.  The pI value is maintained constant (5.5) with 25.2 ml of a 2.5N solution of hydrogen chloride in methyl alcohol.  The precipitate is filtered, the filtrate.  The product formed at the same time, when the product under conditions of growth with danepine ether, crystallizes, yielding a besigzepy liopeini-like substance, t, pl.  EZ, (°, 5 (s - 1, O,; in methyl alcohol).  B) 4Oi loop (97% of theoretically calculated dipcha ™ ° l & J.  tert - Butter ester of N, N, M-grns-benzyloxy. Arbonip-b-arginip-ssgragragnsffln-N -t. -butipoxycarbonip-b-pyzipgpiIIP- C -glutamine, 31.9 g of tert hydrochloride. α-asparaginyl-N -butyl ester. -butyloxy | sarbosh-C-lysylglycyl-b -gputamine (obtained, in accordance with paragraph E) and 33.68 g of N-oxysuccinimide ester N, NN Grisbenzyloxycarbonyl-b-argism in 1 l of dimethylformamide mixed with 7 ml of triethyl amine; .  After 24 hours of stirring at room temperature, the reaction mixture is evaporated in vacuo, the residue is precipitated from methyl alcohol and water and then recrystallized from methyl alcohol to give a colorless, porous product, dl-j) of -7.6 Cc 1.0, in glacial acetic acid . acid).  Yield 46.5 g (8O% of the theoretically calculated value), -3.  Hydrobromide dihydrate tert. β-butyl ester of β-argin1dl- (hydrobromide) -b-asparaginyl-N -t-. -butyloxycarbonip 1 (-lysylglyl-b-glutamine. .  45.5 g tert. -butyl ester of N jK N -tris-benzipoxparbonyl-b-are. gynil-b-asparaginyl-N -t. -butyloxycarbonip-. L-lysylglycyl-U-glutamine (obtained in accordance with paragraph F) -in 1.5 l. mixtures of dimethylformamide and methyl alcohol (2: 1) are subjected to hydrogenolytic deactivation while maintaining a constant pH value (4.5) with 8O ml of 1N pacD solution of hydrobromic acid.  The precipitate is filtered, filter. the atoms are evaporated in vacuo, the residue is reprecipitated from a mixture of ethyl alcohol and diethyl ether, an amorphous powdery substance is obtained with -4.8 (with 3.0, in 8O% acetic acid).  The yield of the product is 37 g (98% of the theoretically calculated value).  Yield at all stages of the% 3% based on the initial H-QEn - OtBu (22b).  2o - -7.6t, respectively cAlj jj.  -9.4 (s - 1, in acetic acid).  Chist-n product tested chromatographically in the systems: n-butanop-ped tksh.  acetic acid — Loda 3; 1: 1 and Ti — r. enTaif-tr. Essential butyl alcohol ped 1 and acetic acid 5: 1: 1.  Calculated / 1fold,%: C 57/98 / 57.70 j H 6.69 / 6,504 N 13.29 / 13.29; O 22.02 / V22; 45.  Beneipoxycarbonyl protective groups. Probes are removed by catalytic hydrogenolysis by adding two equivalents of hydrogen bromide.  Fragment I is obtained, namely H-Afg (HBr) -Aftn -LvS (Bocj-qe-qen-ot sin.  ii8-22b).  Output 98%, -4 ,, respectively, tdl5 -6, O 5 (with "1, in 8O% acetic acid).  The purity was checked by chromatography in the following systems: n-butyl alcohol ice on acetic acid-water 3: 1: 1 and tertiary amyl / pyrimidine-water alcohol 35:: 35: 30. : Calculated / found,%: C 4O, 21 / 4O, 46i H 6.85 / 6.93} N 16.12 / 15.89; BP: 16.72 / 16.51.  PRI me R 2.  Preparation of the And agent (partial sequence 14-17).  The benzyloxycarbonyl dinpeptide C1617a ester) is obtained by condensation -7- - bsie (BOC) -OSV (16) and (Ot Vi) -OMe (17).  The subsequent alkaline saponification of ether and the subsequent catalytic diacylization through a dipeptide derivative (1617 t) results in H-Lvs (BOC) -Qbu (Ot Bu) -OH (16-17c). The product is condensed twice in succession with L-QBs- (O-b Wee) -OSV (15, respectively 14), each time using a glutamic acid derivative as the main component.  In this way receive. I, namely Z-Q6u- (OtBu | -qcu (Ot Vi) -uje (Boc) -Qeu (ot) -OH (14-17a).  Named # 1e products obtained by the method described in example 1.  Output 48% based on the original H-QBo (Ot BU.  ) -OMe (17); t.  square  product 147149 ° C.   - -9.8. 1 1 °, respectively.  -12.3 ° (c 1, in dimgylformamide). The purity is checked by chromatography in cyclohexane-chloroform-ice systems for acetic acid 45:45:10.   Calculated / found,%: C 59.02 / 58.70; H 7.86 / 8.04; N7.48 / 7.72.  PRIMER 3.  Receipt fragment III (partial sequence 12-13).  For the preparation of N N, N Tris-benzyloxycarboxylic-U-arginyl-L-leucine 30 g Z. -Argr (SU, W Z ::) - OSV (12) and 11.6 g of leucine (13) are administered into. interaction with mixing for.  18 hours in a mixture of dioxane and water (1000: 500 ml) after adding 89 ml 1 n.  sodium hydroxide solution.  Acidification 89 ml 1 n.  hydrochloric acid, the reaction mixture is treated with ethyl acetate, the solution is washed with dilute hydrochloric acid and water, dried over sodium sulfate, the solvent is prepared in vacuo.  The residue was recovered from a mixture of water-methanol and ethyl acetate-acetic acid-diethyl ether-petroleum ether.  Yield 26.6 g (86.6% of the theoretically calculated value); t.  square  126-128 ° C; d, g -5.8 + 1, respectively 54 -7.1 (s - 1, and acetic acid).  The purity of the product is checked by chromatography in the system: n-heptane-tertiary butyl alcohol-acetic acid (5:: 1: 1).  Calculated / found,%: C 62.6 / 62. 59: Mr. 6.28 / 6.41; N 10.15 / 9.94.  Amino acid analysis: ARSG 1, OO; Leu 1.00.  Example 4  Getting a fragment. j (partial sequence 9-11).  The dipeptide -OH, is reacted with 2 -Geu (Ot Bu) -OsV (9).  .  as a result, a fragment of W is obtained with a yield of 74%, namely, Z-Geu (ot Bz) -iei-qen-one (9-11).  The conditions of the experiment, as in the example.  1, t.  square  product 146-148 ° e, EAli) - 31.6. ± 1, respectively, D54b 5 -. 38.1 (with "1, in methyl alcohol).  The purity of the product is checked by chromatography in the following systems: n-heptane-tertiary butyl alcohol-ice for acetic acid (5: 1: 1) and butyl alcohol-ice for acetic acid-water (3: 1: 1).  Calculated / found,%: C 58.12 / 58.05; H, 24; N 9.68 / 9.48.  P r and m  e 5.  Obtaining a fragment of N (partial sequence 6-8).  Compound (tBu) -QCa-OH (7g.  -8) condense with Z-TtiK (tBu) -OSV (6), get benzyloxycarboxylate tripeptide (6-8a), which is catalytic as a result. go hydrogenolysis gave the desired fragment; 1, namely H-TtiK tBU) -Tug (iBu) (6-81)).  The experiment was carried out under the conditions described in Example 1. ; The product yield is 81% in two stages. in the calculation of the applied dipeptide (7-8) j t. square  126-127 ° C, t e (, + 7.9. ± 1 °, respectively, dtj l / +8.9 (s 1, in methylated alcohol).  Chromatographic control of the purity of the product is carried out in a mixture of tertiary amio-specific alcohol, pyridine and water, taken in a ratio of 35:35:30, Calculated / found,%: C 60.88 / 60.69; H 8.30 / 8.31; N 8.88 / 8.91, calculated on d1speptide 1/4 mol.  crystallization ethyl ester) of acetic acid ester.  Example 6.  The preparation of fragment H (partial consistency 1-5), by the Z-ZBe-OH (4) carbodiimide method, is combined with H-Phe-OtBu (5).  In the subsequent catalytic hydrogenopia of the benepipoxycarbonyl dipeptide intermediate ester (4-5a), the H Ifilje-Ptie-Oi Bi (4-5lj) is isolated in a yield of 9O%.  At the same time, phosphorus is azo-; the BOC-VaE -OH (2а) and H-Pro-OHM (For) condense with the aid, resulting in the formation of tert-butyloxycarbonyl dipeptide ester (2-For).  Subsequent alkaline saponification of the ester results in the use of more than 70% BOC-Vo (e; -Pj 0-, OH 12-3 b).  It is simpler and with a higher yield (83%) dipeptdes (2-313) is obtained by condensing B00-Vol & 05 V (2b) with two equivalents of proline (3b).  Both dipeptide derivatives are combined using the carbodiimide method to form the Boc-vae-Pro-3e-he-ot Bu (2-5a.  Impact  trt}). torus acetic acid at (2-5a).  results in the free tetrapeptide Hr-VoiE-Pi o-Be Phe-OH) 2-5D), to which BOC-PTie -OSV - (1) is attached in a conventional manner and in a known manner with the formation of BOC- - oB-gPTo-Sse - PJie-O (1-5a) (fragment 41). .  Experimental conditions are described in Example 1.  The product yield is 63% in the three last stages per (), t.  square  218%; sp SC XI - 64.2 i l, respectively {сА,. . g -75.82 (with 1 ,.  in acetic acid), the chromatographic purity of the product was tested in a mixture of n-butypic alcohol, ice from the scientific research institute of acetic acid and water, taken in a ratio of 3: 1: 1, and in a mixture of tertiary amyl alcohol, pyridine and water, taken in the ratio of 35:35:30.  Calculated / found,%: C 64.88 / 64.72; H 7.64 / 7.701 N 9.70 / 9.61.  Example 7.  Condensation of fragments I - X (full hypotheses 1-22). Fragment I (18-22), containing a red carboxyl group, is combined with fragment I (14-17a) by the carbodiimide method and the resulting tert. -butyl ester of N-benzyloxycarbonylnonapeptide (14-22U) is converted by catalytic hydrogenolysis to H-KuCOt Bu) -Gieu {ot BU).  (BOC) -Geu (0-t Bu) - (HB) -ASn - Lvs (BOC) -, G &) - G | ett-0t Bu (14-221).  ; Connection with the fragment W using this method leads to the formation of I Arfl - ((f-sh- Z2)-Lei-0, eu (01 Bu) -GP. U (Ot Vo) -Hpc (BOC) -eiEu (Ot Bu) (HBf) -Asn-bs (s (BOC) -G, Ev-GEnГ 0t B and (12-22a).  The catalytic hydrogenation of undecapeptide (12-22a) removes the glue and basyloxycarbonyl protecting groups, and as a result of the ipapitalization of the resulting free guanidine functional groups, hydrobromic acid produces (HBl) leu-G & u. (ot Wee) -Gibu (0t Wee) -Lss (BOC) -G, eu (ot Bij) -Arg- (HBr) -Asn-bvs. (BOC) -G, ev-G, en-Ot Bu (1. 2-22 b) in the form of hydrobromide.  The japepeptide is concentrated with an IN (9-11) fragment by the indicated dicyclo-hexylcarbodiimide-M-oxybenzogriazopic method and the resulting H-b yr-ya-ya-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-yc-y-xyloxygenezinecarboxylic acid; GiEu-Argr- (HBl-) -Leu-Geu (Qt Bu) -CiEu (Ot Bu) -L4S- (BOC) (ot Bu) -Arg- (HB) -Asn-Lvs (BOC) -G, e ( -Gren-OtBu (). .  In the process of this combination, fragments of t (6-801) and N1 (1-5a), the last. after translating it into N-oxy-succinimide (1-5), however, pure BOC-Plie-VoiE-Pro-aee-Phe-Tht-Avi) -g (tBu) (1-8) cannot be obtained, Amino acid analysis showed the presence of impurities.  in the amount of approximately 1% 1O (1-5a) or (1-513).  Attempts to separate this mixture were unsuccessful.  The resulting crude region (1-8) is combined with the dicyclohexipcarbrdiimide-N by the oxysuccinimide method with the region indicated above (9-22), as a result of FTO, the BOS-Phe -VaE-Pro-UEe-Phe-Tht-- (iBu) -Tyr (- t-Bu) -Q y-QEu (OiBu) -Leu-QPu- -Ang (HBr) -Leu-agu (OlBu) -Q2u (OiBu) -Lye,; (BOC) -qEu - ((HBh) -AbH-L, yu (BOC) rQ y-QEu-OlBU (i-22a).  After cleavage of all protecting groups with anhydrous trifluoroacetic acid and the subsequent replacement of trifluoroacetate bromine ions by ion-exchange chromatography on a weakly basic, Dowex 44 ion-exchange resin (acetate form), crude leucine-13-motilin (l-22b) is obtained, which results in no. The litchi of the crude section (1-8) had an erroneous sequence, which was also confirmed during the cleaning operation.  The condensation of the fragments was carried out under the following conditions, -.   .  BUT.  Condensation I with | 9.2 g of 4regiment 1 (in accordance with Example 1 of 3), 9.36 g of fragment C (obtained in accordance with 1-measure 2) and 1.4 mp of triethylamine in 200 ml of dimethylformamide are mixed at 0 ° C with 2.3 g of K - immediately after that with 3.1 g of N, N -dicyclohexylcarbodiimide.  The reaction mixture is stirred for 24 hours at 5 ° C and then for 4 days at room temperature, the precipitate is filtered off and the filtrate is evaporated in vacuo.  The residue obtained is treated several times with water and finally twice precipitated from methyl and ethyl (HN) acetic acid ester.  Received in the form of an amorphous powdered substance tert. -butyl ether benzyloxyc bonip-glutames S 3 -t. -butyl ether) -i-glutamyl (2 - “ret. butyl ether new-M - ret.  butyloxycarbonyl-L glossyl-C -4 lutamyl {jf -t. -butyl ether) - C vrginil (hydrobromide} - L-vsparaginil-N tert. -butyloxycarbonyl- L -chpisylglycylglutamine.  C che -14, (with 1, in dimethylformamide).  Yield 14.8 g 184% of the theoretically calculated value).  A solution of 12.3 g of the obtained compound in 8OO ml of methyl alcohol with the support of a scientific research institute of constant pH is subjected to catalytic hydrogenation in the usual way (pH 5.5, 7 ml of 1N).  hydrobromic acid).  Phyptrat. evaporated under vacuum and the residue obtained is reprecipitated twice from ethyl alcohol and ethyl acetate ethyl acetate. 1 As a result, hydrobromide is obtained as the desired condensation product (14-22b).  hot -butylbone ether b -glut1i1ip (: -tert. -butyl ether) -l-glutamylST-tert-butyl ether) -, - tert. -butyloxycarbovip- -pisil-b -glutapsU-ter. -butyl ether) -b-arginip (hydrobromide) -b-aspa raginyl N -t. -butipoxycarbonip-j-pizipgitsilglutamina ,,.  21.5 (s.  in methyl alcohol. ).  .   Yield 11.4 g (96% of theoretically calculated value). i.  B.  Condensation (I - 5) with W, Solution 1.7 g of peptide (14-22 b), Is4 g 2-Ar-g- (b, sh-22) -Ley-OH (12-13), 0.3 g N-oxySuccinimide and 0.14 ml of triethypamine in 1OO ml of dimethylformamide are mixed at -1 ° C with 0.412 g of N, N-dicyclohexylcarbodiimide and the reaction mixture is stirred for 24 hours at 4 ° C, then for another 24 hours at room temperature.  Separating dicyclohexoylurea is filtered off, the filtrate is evaporated in vacuo and the residue, after evaporation, evaporated from methyl alcohol and ethyl ester of ethyl acetate 59 after evaporation.  The resulting product is carefully treated with water, filtered and dried, the result is tert. -butyl ester of N, N °, H-tris- (benzyloxycarbonyl-C-argynyl-U-lecyl 1 | -g utamyl-1T -t-t. butnlovy 9fir) -b-glutamylG-ret-butyl ether) - M -t. butyloxycarbo nip-Li-lisch-C-glutamyl (c) -tret. -butyl ether) -l-arginyl (hydrobromide) -U. -ac spine-m-gtret. -by tyloxycarbonyl-.  Cpisylglycyl-L-glutamine {12-22a).  Yield 1.94 g (85% of theoretically calculated value).  The chromatographic purity of the product is checked by chromatography in a mixture of n-heptane, tertiary butyl alcohol and acetic acid, taken in a ratio of 3: 2: 1, and in a mixture of n-butyl alcohol, acetic acid and water, taken in a ratio 3: 1: 1.  Amino acid analysis data:, 2.02, Agog2.01, Asp.  1.00, eii 3.98, qe: a 1.01, Ueu.  1.00.  1.8 g of the peptide in 50 O ml of methyl alcohol are deacylated by catalytic hydrogenation with dropwise addition of 15j7O ml of 0.1 N.  hydrobromic acid, with a pH value of 4.5.  The catalyst is filtered off.  The filtrate is evaporated in vacuo, the residue obtained is reprecipitated from methyl alcohol and ethyl acetic acid.  W do. hydrobromide dihydrate t, -.  butyl ether 1 | -arginip (hydrobromide) / - b-lecyl-Iri -glutamine- (2p -t. -butyl ether): 1 -gputamil (-tert, -butyl ether) -, d -th. -butyloxycarbonyl-i. lisl -1 -J -putamip (W-tert. -butyp epic) -argi, nipShydrobromide) -asparaginip-S -tert, -buttylo shsarbrynil-c1 -lyslipiaip-, 1 -gputamine (12-22) ,, Output 1.61 g (98% of theoretically calculated value) :: Calculated / found,%: C 47.12 / 47.20; I 7.43 / 7.50; N 14.07 / 14.00 V, Condensation (I-D-ffl) with G-.  1.5 g peptide (12-22 b).  and 0.87 g of peptide (9-il) in 1OO ml of dimethylformamide is condensed under the conditions described in paragraph B by the method, with the addition of O, 1O5 ml of triethylamine with 0.210 g of li-oxibevzotriazole and 0.320 g of dicyclohexylcarbodiimide. .  .  :,. . . . -. .  Get tert dihydrate. -butyl ether N -benzyloxy-carbonip-l -glutamin (-t. -butiprya efyr) g U -peicyl-U-glutaminyl-. L-fingyl (hydrobromide) - 1. - lions number-. gameplay butyl ether) -M.  -trvt. butyloxyted) bonil-L-lysyl-, L-glut / h I / mep -tret. -util ether) -arginyle (gndg robromide) -c-parajinip-N -t. -butypoxycarbonip-b -pizipgipits-1 -gpugamin (9-22a).  Yield 1, b g (83% of theoretically calculated value).  Calculated / found,%: C 51.41 / 51.28; H 7.57 / 7.55; N 13.63 / 13.57; Bf 6.22 / / 6.15, 1.6 g of peptide (9-22a) in 6OO ml of methyl alcohol are decapitated by means of a catapic gdrirvan, maintaining {) a constant pH value, by analogy with that described in paragraph B, the catalyst is filtered the filtrate is evaporated in vacuo and the resulting residue is precipitated from methyl alcohol and ethyl acetate. The tert-hydrobromide tetrahydrate, U -glutamine ester, is obtained (. -butyl ether) -l-peynyl-It-rnyTaMmnw-; -argini l (hydrobrom id) - U-peicil b -glutamyl (T-tert. -butyl ether) -b-glutamyl (, / Y-tert. - butyl ether) - - tert. -butyloxycarbonyl-lysyl-tb-glutamyl {-trit.  -butyl ether) -l-arginylShydrobromide) -. L-asparaginyl-M-tert. -butyloxycarbonyl-. 1 lysylglycyl-14-glutamine {9-22b).  & 1 APP 1.5 g (96% of theoretically calculated, value), -5, and 3 -7.2 (with O, 7, in methyl alcohol. The chromatographic homogeneity of the product is shown in a mixture of n-butyl alcohol, acetic acid and water (3 : 1: 1). amino acid analysis:. liVS.  2.04, A) rg-2.01j ASja 0.98, GKu 6.05, Stv l, lo ,, Ueu 2.00.   G.  Condensation S J D 2. 1.6 g of the fragment. 41 and 6.9 g of N-oxysuccinimide in 4OO ml of dimethylformamide are mixed at -5 ° C with 6.3 g / W, N -dicyclohexylcarbodiimide.  The reaction mixture is stirred for 2 hours at OS and then overnight at room temperature.  After filtration, the filtrate is evaporated in vacuo.  The oily product obtained is crystallized from the alcohol izogfopilovogo alcohol,. .  The N-oxysuccinimide ester is obtained. butyloxycarbonir- UF nipalanil-b-wapi- B-prolyl-isoisole-W-phenylapanine.  Exit 21. 3 g (88% of theoretically calculated value), t.  square .  190-192 С + 59.28 ° (с 1, О, in dioxane).  12.2 g of the L / and 3.8 ml of triethylamine in 400 ml of dimethylformamide are mixed with 14.8 g of the above succinimide residue obtained from the VJ fragment After stirring in reHeiffle 24 hours at room temperature, the reaction mixture is evaporated in vacuo and the resulting meloidous residue is partitioned between ethyl acetate and ethyl acetate. , acid G solution of citric acid, i acTBOp of the product in ethyl effect with acetic acid wash / dry and evaporate in vacuo.  From a mixture of ethyl ester of acetic acid to petroleum ether, tert is obtained as a colorless crystalline substance. -butyl sicarbonyl L-phenipalanil-1 -vapip-b -prolyl-b-isoleuc-Li -phenylUganyl-O-tert, -butyl-L -treonyl-O-tert. -butyl- -tyroskyglycine (1-8), Yield 18i2 g (87% of theoretically).  read value), dl 1 - -44.0 ° (with ag in ethyl alcohol), / D, Condensation (1 - AND -IM-W) with (V - Nj), 1.28 g of peptide (9-22 b ), in cooTBercTBtffl with clause B, 1.17 g of yeppt-, yes (1-8), obtained in accordance with clause D, O, O7 ml of triethylamine and O, and. 5g N-oxysuccinimide (or c. another experience 0,2O g N-oxybenzotriazopa) and 1OO. ml of dimega formimide are mixed at a temperature of -1 O with 0.206 g of H, f-di shkloheks 1p carbodiimide.  The reaction mixture is stirred for 2 days at 0 ° C and for 3 hours. days at room temperature.  The residue obtained after removing the solvent in vacuo is thoroughly treated with heated ethyl acetic acid, a resinous product of the first stage. Methyl alcohol and ethyl acetic acid are dried and, after trituration with ethyl acetate, dried.  Then for 5 hours the product is thoroughly treated with water and repotted from methyl cniipra and water.  Get hexahydrate trep-butyl, spnr ,. and W is tert. -butyloxyncarbonyl- b - (|) enip apanil-M-valip-1--prolyl-из -isole-ф -phenylLalanil-O tert, -butyl-C-greonyl-O -t-t. - butyl i-tyrosine glycyl-Li-glutamga (-t. - butyrate ester) - - leucnp-b -gputaminyl-L-arginyl (hydrobromide) -b- gsicyl, 1 «- umyl (W-treg. butyl eff1f) - - gputamil (| -tret. - butyl ether) -N -.  -tr. -butyloxycarbonyl- (. 4 | -lysil- U g / utamil (. -tr. -butyl and ether) - b-argin11l (guide: robromide) -. 1: -asparaginyl-H- -tret. -bughyloxycarbonyl-L-lysylglyyl- ij. -glutamine (1-22a), Yield 1.65 g (91% of theoretically calculated value), Calculate. but / found,%: C 54.03 / 54.13; H 7.82 / 7.80; - 12.68 / 12.67; Bf 4.38 / / 4.6. ; 1 g of the protected peptide () s half of the skin in accordance with paragraph D, is mixed with 40 ml of ice-cooled trifluoroacetic acid and the mixture is kept for 2 hours at room temperature.  Excess trifluoroacetic acid is dropped in a vacuum at the lowest possible temperature, the resulting residue is dissolved in dilute acetic acid pot, the solution is treated twice with 4 g of a weakly basic anion exchanger in an acetate form (for example, Dowex 44), after which the eluate obtained after ion exchange is freeze-dried.  Get 1 -phenylalanyl-14 - valyl-g, -prolyl- L - isoleucipat-U -phenylalanil-: L -tre onyl-igr-tyrosylglycyl-c1 -glutamyl-L -leucip- 1 -glutaminyl-b - ginip-b -leucyl b -glutamyl- u -glutamyl-. and -lysil-1 | -gluten-C-orginil-L -asparaginip-lysylglycyl-U glutamine.  The product yield of 0.7 4 g  PRI me R 8.  Preparation of the pure product and amino acid analysis.  Purification is carried out using ion exchange chromatography using a strongly basic anion exchange resin, for example, in the acetate form and known as OAU-Sefa. dex A-25 resins based on modified dextran with diethyl- (2-oxypropyl) -aminoethyl residues as functional rpyntt, and then when using a strongly acidic cation-exchange resin, for example, which is ammonium form and is known as 3-Sephadex 0 -25 cm 1l based on modified dextran with sulfopropyl residues as functional groups.  The amino acid number 1 analysis was carried out after acid hydrolysis (6 n.  salt on the acid) peptide, and it turned out that almost the same values are achieved when carrying out the hydrolysis within 2O and 72 hours.  The results are presented below.  Calculated Found Out of 2.00 2 2 1 1 6 1 2 ArgAsp Thr.   1 59 9 1,942 0,931 1,822 Briopogic activity of freshly dried ti-leucine-13-motilin is almost equal to the activity; li-norleucine-13 motilin, thus, does not differ much from the activity of natural motilin.  Formula and 30 B et e and L-leucine-13-mr or How to obtain formulas 1 34 567 b5 H-Ptie-yae-Pfo-ee-Phe-T-hr-T r-CiE-Qeil10 And 12 1Z 4 Y5 16 P 16 -beu- (en-Argr-Leu-Qeu-QEu4v5-Gieo-Artr 920 gG Z2 -Aen-b (sC, e: -QCn-OH about tl and h ay i and so; ; that subject to condensation in any sensitivity, protected 4 agents: tert. -butyl ester arginyl {hydrobromide) -aeparaginyl- | (-ipeT. -butyloxycarbonylisyl-glicyl-gputamina (18-22), U -t. N-benzyloxycarbonyl-glutamyl-butyl ester (Jjj-TpieT. -butyl ether) -glutamyl (. -butyl ether) -M-tert. -butyloxycarbonylisyl glutamic acid (14-17), K, N -trp-benzyloxycarbonyl-1 -argynyl-b-leucine (12-13), N -benzyloxycarbonylglutamylSTtert-β-butyl ester) -leucylglutamine (9-11), O -tr. -buttirone-Or-TpeTibutyltyrosyl-glycine (6-8); and K-tert. -butyloxycarbonyphenylapanil-valylg-prolyl-iso-leucyl-phenylalanine (1-5) with, N-dicyclohexylcarbodiimide- {1 -oxysuccinimide and removal of the protective groups with trifluoroacetic acid.  Sources of information taken into account during the examination: 1. Copyright certificate for the application number 2139J, 8/23-O4, cl.  C O7 C 1OS / 52, 1974, 2, Schroeder E. , Lyubke K.  Peptides, h.  I, M. World, 1967, p.  116.

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