DD215537A1 - PROCESS FOR SYNTHESIS OF ANGIOTENSIN I - Google Patents

Abstract

The invention relates to a method for producing angiotensin I (ATI) / 1 / Asp-Arg-Val-Tyr-IIe-His-Pro-Phe-His-Leu / 1 /. The new method of preparation consists in the linking of the N-terminal tetrapeptide with the C-terminal hexapeptide. The tetrapeptide used is the Z-Asp (OBzl) -Arg (NO deep 2) Val-Tyr-OH and as hexapeptide H-Ile-His-Pro-Phe-His-Leu-ONb. Field of application of the invention is the pharmaceutical industry.

Description

The invention relates to a method for the production of

Angiotensine (AT I),

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Iieu / 1 /

which is suitable for use in RIA kit · Application of the invention is the pharmaceutical industry ·· ^";.. '* *...'". · .. ^; . ''·

ι ''. '·' '' ·, '' '. , . '

Characteristic of the known technical solutions The production of angiotensin I has already been described several times. Both pest phase methods (M.C. Khosla et al., Med. Chemistry 20, (2) 315 (1977)) and classical methods were used in solution (Ή · Aoyagi, Bull. ChenuSoc · Japan 41, 433 (1968)) · H · Aoyagi synthesized AT I from two fragments 5 +5, using the azide method for the condensation of both fragments · Both histidine residues were replaced by benzyl groups in the Imidazole side chain protected ·

Both methods inherit a number of disadvantages that make an application for industrial synthesis advantageous (expensive purification and often difficult reaction) ·

Object of the invention

The object of the invention is to synthesize a decapeptide of the formula 1 which is suitable for use in the RIA technique, which has a high purity and whose synthesis avoids the disadvantages of the stated processes.

Explanation of the essence of the invention

The object of the present invention is to develop an advantageous process for the production of angiotensin I. The object was achieved by constructing angiotensin I from two fragments 1 to 4 and 5 to 10 according to the following scheme:

Asp

bad

Val

OBzI -OPfp H

OBzl

ÖBzl

UO,

Tyr

lie

Boo Boo

OH H

OH

DCei / HOHB

His

Per phe

OH

Bo c Bo c

His

OH

Bo c

Bo c H

H-

Bo c

^

blip

Buc / '

leu

H -j-leu OB

ON b

ONb ONb

ONb ONb ONb

ON b

ONb ONb

Ciir ornat ο gr aphi β

The N-terminal tetrapeptide is thereby synthesized stepwise, the C-terminal hexapeptide by combining fragments 5-6, 7-1-8 and 9-10. In this case, the C-terminal dipeptide is synthesized according to the invention by the active ester coupling of Boc ₂ -His-ONp leucine p'-nitrobenzyl ester (prepared according to Mc Laren, Austr. Jv Chem. 31, Cl978) 1865) in good yields. After cleavage of the Boo protective groups, the dipeptide is synthesized either by means of complex ¹¹ P ' ¹ (J * Kovacs and L · Kisfaludy, J · Amer. Chem · Sbc · JJi ₄ (1967) 183) or using DCCI / HQNB with the dipeptide Boc-Pro-Phe-OH to form the tetrapeptide Boc-Pro-Phe-His-Ieu-OiJb The Boc group cleavage with HCl in glacial acetic acid gave the thin-layer chromatographically uniform tetrapeptide dihydrochloride, which was subsequently extended with Boc-Ile-His-QH by applying DC CI / HÖliB to He xape pt id · For the condensation of the two main fragments Z-Asp- Arg (NO ₂ ) -Val-Tyr-OH and H-Ile-His-ProPhe ^ His-ieu-OIib wi dicyclohexylcarbodiimide (DCCI) and N-hydroxyphenylorbornene dicarboximide (HOWB) in a ratio of 1: 3 to minimize racemization · By the choice of protecting groups, terminally benzyloxycarbonylj, C-terminal p-nitrobenzyl ester and nitro- for the arginine side chain a problem-free splitting off of all protective groups is made possible. The crude product thus obtained already has a high degree of purity and can be further purified by chromatography on carboxymethyl cellulose.

The process according to the invention is explained below by way of examples:

embodiments

Example 1:. '<''; , ..,; .. ^; ·. BOC ₂ HiS-IeU-ONb (MW 603,658)

2.47 g H-leu-ONb'HCl (8 mmol) are dissolved in 15 ml of DMF there are added 0.4 ml of NMM and cooled to +5 ⁰ C. Then add 3.9 g BOC ₂ HiS-ONp and stir overnight * Work-up:

The DMF is evaporated off and the oil is taken up in 30 ml of ethyl acetate. The organic phase is up to Enffärbung with

: "'.."'; , , '..., Λ. 5-, ././ν

The mixture is then shaken with NaCl / H ₂ O ₂ × and the organic phase is dried over MgSO 4. dried, Essigester is spun off, and the remaining oily residue triturated several times with hexane · The semi-solid product is washed with little ^ether: added and the white gel-like product was triturated with hexane · · Pp: 92-94 ⁰ C a white powder SSJ ^ f = -. 7.59 °

Yield: 4 g »83 % c = 1 methanol -

Example 2; '',: '''''''.'['....:'.' - ^; '

BOC-Pro-Phe-His-Leu-ONb (MW: 747,858)

1.73 g (4.78 mmol) of BOC-Pro-Phe-OH were dissolved in 15 ml of THP, 0.88 g (4.9 mmol) of HONB was added, cooled to 0 to -5 ⁰ C, 1.1 g The precipitated dicyclohexylurea was separated off, washed several times with THP and the filtrate combined with the washings was concentrated on a rotary evaporator. The resulting yellowish oil was evaporated in a rotary evaporator 10 ml of DMP and dissolved in 15 ml of DMP dissolved 2.19 g (4.59 mmol) of H-His ^ Leu-ONb: from the dihydrochloride - with 1.51 ml (13.6 mmol) of N-methylmorpholine - added The reaction time was 15 h at room temperature. The reaction solution was filtered and concentrated. The resulting oil was taken up in ethyl acetate and extracted successively with sodium bicarbonate, NaCl-saturated KHSO 2 solution and NaCl-saturated water. The ethyl acetate phase was dried over magnesium sulfate , concentrated and the remaining oil with H exan solid, ground · Yield: 3 g 88.2 # pp .: 129 - 32 ⁰ C focj |. ³ = - 38.06

(c = 1, DMP) DC: (A) S3 uniform R _p - 0.71,

SBN: Rj ₁ = 0.71

BAW: Hj ₁ = 0.66 uniform

Example 3; ,. '. ;; -, ·:; ..' · ':'} ': . ^; , \ - ',; ^: ':'^; . '^; BOC-IIe-HiS ^ PrO-PhB-HiS-LeU-ONb (MW: 998.124)

530 mg (1.5 mmol) BOC-Ile-His ^ OH and 225 mg (1 mmol 65 ₅₎

^: · .. ^; -: ' ^: . - '' ·. - · ^; · ,. - '',. ,

HOWB were dissolved in 10 ml ΦΗΙ ¹ , cooled to -5 ⁰ C, treated with 309 ing (1.5 tDMol) l) GCI and after 45 min * at this temperature nachgergerrt · After separation and repeated washing of the urea were Concentrated washing solutions combined with the filtrate and the remaining oil was taken up in 10 ml of XlIiF and treated with 872 mg (1> 35 mmol) of H-Pro-Phe-His-LeU-ONb x 2HCl and 0.45 ml (4 mmol) N Stirring at room temperature, the reaction solution was concentrated on a rotary evaporator, the remaining oil was taken up in chloroform and shaken out in succession with NaCl-saturated EHSO., WaHCO ₃ solution and water saturated with CH ₂ Cl - awake over MgSO 4. was concentrated and the residual oil with hexane rubbed firmly · Röhausbeute: 1.09 g (74 #) Pp .: 160 - ß 63 ⁰ C * J | = ^4> 47.58

The substance showed in the DC (B) in addition to a strong main spot two more weave stains ·

Example 4: '_:; , , · · . , ... , , ; ·. Ile-His-ro-Phe-His-Leu-Oilb χ 2HCl (m.w.: 1007.405) 889 mg (0.89 mmol) of BÖC hexapeptide were in; Dissolved 10 ml 3 W HCl / AcOH and stirred for 40 min. · Wach 35 min · was the BOC cleavage according to TLC in S 3 complete. The reaction solution was poured into 40 ml of iSther, the precipitated flocculent precipitate was separated off and triturated several times with ether on the proteo. The residue was removed by vacuum drying over EOH and P ₃ O ₅ in vacuo.

Yield: 760 mg (84 #)

Pp, j 168 - 70 ° pxj ψ = - 35,03 (c = 1,

, - ·. '. -; - ^:. · '., · Λ ...'. MeOH)

DC: (B) BAW Rj ₁ = 0.64

Example 5:.

Z-Asp (0Bzl) -Arg (NO ₂ ) -Val-Tyr-0H (MWs 820.83) 1.43 g (4 mmol) of Z-Asp (0Bzl) -OH are dissolved in 15 ml of OJHP * at 920 mg (5 raM oil) pentafluorophenol and cooled to - 5 ⁰ C · wax the addition of 825 mg (4 mmol) DCCI was ·

The resulting urea is separated off, washed, and the washing solutions are combined with the filtrate and concentrated. The resulting oil was taken up in 20 ml of DMF and admixed with 2.75 g (5 mmol) H-Arg (NO ₂ ) -Val-Tyr-0H x 1.16 HBr in 10 ml of DMP with addition of 1.35 ml (2.2 mmol) of N-methylmorpholine and stirred for 5 hrs · at room temperature , (The pH must at pH 7 - 8.) · the solution is filtered and to approx · ^1/3 of original volume concentrated and iii ice-cold 1 N HCl (HACL saturated) was dropped · The resulting white precipitate is separated off, multiple washed with water, in the air and then dried in vacuo over KOH and ^ ₂ ⁰ S ·

Yield: 3.09 g (94%)

DC: (A) uniform in S3 & J ψ = - 3.74

Example 6:

Z ^ Asp (OBzl) ATg (HO ^ Val-Tyr-Ile-Hi s-Pro-Phe-His-Leu-OHb

(MG: 1700,895)

780 mg (0.95 mmol) of Z-Asp (OBzl) -Arg (HO ₂₎ -Val-Tyr-OH and 292 mg (1.35 mmol) of HONB were dissolved in 5 ml of DMP at - 5 ⁰ C cooled and 247 Then the mixture was warmed to room temperature and stirred for 4 hours. Then, 850 mg (0.84 mmol) of H-Ile-His-Pro-Phe-His-leu-ONb χ 3HCl were added Add 0.36 ml (3.36 mmol) of N-methylmorpholine in 5 ml of DMP and stir overnight. The solution was concentrated a little on a rotary evaporator and poured into ethyl acetate, the precipitate was separated off and washed with K ₂ CO solution. The somewhat greasy pesticide was recrystallized from ethanol.

Yield: 1.22 g (86 #)

Pp «: 163 - 65 ⁰ C

· Pp: 178-80 ⁰ C (after ümkristallisation) DC: (A) before the Ümkristallisation of ethanol, BAW Py Rp = 0.81 uniform

· - ^; , ''. ' . , · ·:, '' · '. · ·; . ' 8th

;. , ;. ·; ^>; ..:. ;;. ·.. · ·. /. β

BAW R-, S 0,51 = main fie ck, R-, = 0,8 = secondary spot

SBJJ '. Rji ~ 0.56 = major spot, Rj, = 0.32 = minor spot staining with Pauly's reagent and TDM

Yield after recrystallization: 1.07 g (75 g)

Example 7: /. . ''':;'['''''. .. ^·:.. '.. · H-Asp-Arg-Val-Ty ^-Ile-His-Pro-Phe-His-Leu-OH χ X HOAc χ

YH ₂ O (MW: 1296.97);

760 mg (0.45 in mol) protected Dekapeptid was dissolved in 10 ml of hydrogenation solution (MeOH / AcOH / HgO; 3: 3i1), with 135mg freshly prepared Pd black catalyst and stirred under the hydrogen stream (Ratio contactor: catalyst amount 100 mg After 24 hours, the catalyst was centrifuged off and the reaction solution hydrogenated for a further 21 hours with fresh catalyst. After separation of the catalyst, the solution was concentrated and the oily residue was rubbed with ethanol and recrystallized from methanol.

Yield: 401 mg (70 ^) TLC: (A) SBET R ^ = 0.24 Main spot j

r_β 0.32 and 0.36 two weak minor spots BAWPy R _x . , 0.76 no secondary spots

Example 8:

Purchase of the chromatography

Approximately 1 g of crude product was dissolved in 5 ml of starting buffer and insolubles were centrifuged off. After application, elution was carried out with a linear gradient of 0.01 to 0.1 N NH.OAc at pH 6. Substance was precipitated at an ionic strength of about 0.08 N NH .OAc eluted · The extinction of the eluate is monitored at 252 nm via a üvioord · · The appropriate fractions were monitored by TLC and selected. After two Lyophllisierung (to completely remove the NH.OAc) were isolated from various approaches 500 to 700 mg of AT I * It was a column 2,8 χ 40 em used. The running speed was 50 ml / h ·

Rechromatographic control over CMtJ (250 mg) gave a uniform symmetrical peak and 230 mg AT I.

Substance characteristics: -

H-Asp-Arg-Val-Tyr-Ile ^ HiB-Pro-Phe-His-Leu • X GH ₃ COOH ·

X = about 2 Y = 5-8

Mo! Masse 1,296.97 for 10Q # owned peptide

white lyophilisate, odorless, slightly soluble in water,

a 0.1 M solution is colorless melting range 248> 258 ⁰ G 7

Optical rotation ß <J | ³ == 46.6 ° c = 1 in 0.05 M acetic acid

.-. ·. '''^; - -''.''''''''Acid''^; DS. Chromatography uniform in "SBN Rf = 0.2 -0.3 - ^y '- ^: - \:" / "' BAW Py Rf = 0.7 - 0.8

used abbreviations

AT I Angiotensin I BAW Butanol / Elsessig / water Boc / BOC tert # -Buty! oxyearbonyl CMC carboxymethylcellulose BCCI Dlcyclöhexylcarbodiimid DMP. '. , ^: dimethylformamide HOHB LII-hydroxy-5-norbornendicarboximid MM N ~ methylmorpholine -ONI) - Litrobenzylester < -ONp ^: ., '- Jfitroptienylester THP - tetrahydrofuran SBlT - sec »Butanol / 3 ¹ ^ Ammonia Z. .: ' BenzyloxyoarbOnyl

Claims (5)

1. A process for the preparation of angiotensin I / 1 /, characterized in that the N-terminal tetrapeptide Asp-Arg-Val-Tyr is condensed with the hexapeptide Ile-His-Pro-Phe-His-leu. Asp-Arg-Val-Tyr-Ile-His ^ Pro-Phe-His-Leu / 1 / 2. The method according to item 1, characterized in that the tetrapeptide Z-Asp (OBzI) -Arg (NO ₂ ) -Val-Tyr-OH and as hexapeptide the H-Ile-His-Pro-Phe-His-Leu ONb is used. ; ·. , .-. ^: .. "', ..' .. .. '' .. '\'" .. · .; 3 Method according to items 1 and 2, characterized in that a hexapeptide is used which is synthesized by combining the fragments His-LeU, Pro-Phe and Ile-His in such a way that H-His-Leu-OWb and Boc-Pro Phe-OH to BOc-Pro-Phe-His-Leu-ONb be implemented and that * out after splitting off the Boc group, the C-terminal hexapeptide is obtained by linking with Boc-Ile-His-OH. 4. Method according to item 3, characterized in that a C-terminal dipeptide His-Leu is used, which is formed from Bocg-His-olip and leucine p-nitrobenzyl ester, preferably by Aktivesterkupplung. 5 · Process according to item 1 and 2, characterized in that the fragments 1 - 4 and 5 - 10 are condensed using dicyclohexylcarbodiimide and JJ-hydroxynorbornendicarboximide in a ratio of 1: 3 and then the protective groups are split off.