DD216924A1 - PROCESS FOR THE PREPARATION OF S-ALKOXYCARBONYLTHIODERIVATES OF THIOLS, CYSTEIN AND CYSTEIN-CONTAINING PEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of S-alkoxycarbonylthio derivatives of thiols and cysteine-containing peptides, which are used as stable activated intermediates for the preparation of unsymmetrical disulfides or cysteine peptides. The S-alkoxycarbonylthio derivatives are prepared by the process according to the invention via N-alkoxycarbonylthio-imides which are obtainable from corresponding sulfenyl chlorides and imides in high yield and purity. S-Acylamidomethyl-protected thiol groups can be selectively activated against S-trityl-protected. Already existing disulfide bonds are not attacked by the reagents, so they are particularly suitable for the synthesis of molecules with multiple unsymmetrical disulfide bonds.

Description

Process for the preparation of S-alkoxycarbonylthio derivatives of thiols, cysteine and cysteine-containing peptides

·. ^. - · Field of application of the invention

The invention relates to a process for the preparation of S-alkoxycarbonylthio derivatives of thiols, cysteine and cysteine-containing peptides, which can be converted advantageously into asymmetrical disulphides or cystine peptides by known processes. Bas's process is particularly useful for ^: synthesis of unsymmetrical disulphide structures biologically active compounds, including peptides of pharmaceutical interest, such as insulin, oxytocin, vasopressin, calcitonin, prolactin, growth hormone and analogues thereof,

Char akteristik the known technical solutions

In the known methods for targeted knotting unsymmetrical disulfide bonds respectively a thiol of ^v w are any of free or S-protected form activated, and the resulting, more or less stable intermediate then with the eweiten thiol principle, again in the Porm a free SH Vefbindung or a suitably protected derivative, implemented in a targeted manner. Among the various known methods, the method of activating the thiol compounds as S-alkoxycarbonylthio derivatives (J · Amer «Chenu Soc · 92,729 (1971) J DE 2231775; HeIv. Chim. Acta 56j., 1370 (1973)) has an egg series of merits, since the activated intermediate compounds can be prepared both from the free and from acylamidomethyl- and trityl-protected thiol derivatives by reaction with an alkoxycarbonylsulfenyl chloride in a crystallizable and readily storable form free of symmetrical disulfides. Subsequently, .. -, ... , , - - - '· ⁽ 2

, '' ·. · ''' ²

\ '....' '"'. '. Are the S-alkoxycarbylthio derivatives selectively converted to unsymmetrical disulfides with free thiols in good yields?

+ Cl-S-CO-OR ¹ - * RSS-CO-OR · + (X-Cl) RSS-CO-OR «+ .Η ·· -8Η-- ♦ RSSR ^si + COS + R * -OH

I ¹ for the use of this method in the peptide chemistry speaks mainly the stability of the "S-Alkoxyearbonylthioeysteinderivate under the usual peptide synthetic reaction Very activities, the handling of the" activated Zwischenverbind ung ⁿ as S-protected compounds permits ₀ As a disadvantage of the method, particularly in the manufacture asymmetric cystine peptides, however, is the introduction of the Alkoxycarbonylthio groups on the Alkoxycarbonylsulfenylchloride to see because they are not sufficiently selective react due to their high reactivity * Thus, it _s not possible side by side present S-Acylamidomethyl- and S-trityl-protected cysteine selectively activated individually » Already existing disulfide bonds and amino acids containing nucleophilic centers are attacked by the alkoxycarbonylsulfenyl chlorides.

It is known that Alkoxycarbonylsulfenylchloride can be implemented with other nueleophilen reagents except with thiols, including with üHÜTucleophilen as Äminen (Z ₀ Org, "Khim _a Ί5 ^ 1004 (1979" and amides (Angew · 'Chem. Qg _x 63 (I970 )) _e drawback proves to be the formation of hydrogen chloride in the activation step, which can verursaohen acid-labile protecting groups due to its high acidity KTebenreaktionen as esterification and transesterification reactions in alcoholic, (solvents Jo Orge Chenu 4Oj. 950 (1975)) and spin-offs _e

Object of the invention

The aim of the invention is a process for the preparation of the known S-alkoxycarbonylthio derivatives of thiols, cysteine and cysteine-containing peptides, which are used for the synthesis of unsymmetrical disulfides, to a more selective ₉ particularly when used in the field of peptide synthesis less lovable reactions * " '.'. ''. '' 3. '

presentation; the essence of the invention

The invention has for its object to provide a process for the preparation of the known, advantageously used for the synthesis of unsymmetrical disulfides S-alkoxycarbonylthio derivatives of thiols, cysteine and cysteine peptides, which avoids the known disadvantages of the preparation with the highly reactive alkoxycarbonylsulfenylChloriden «This Nachtelle are particularly in electrophilic weaving reactions, in the relatively low selectivity of the activating reagents to optionally present together differently protected thiol functions and in the release of the highly acidic hydrogen chloride, which can also cause side reactions ₀

While avoiding these disadvantages, the object of the invention is to develop activating reagents for introducing the alkoxycarbonylthio groups into thiols, cysteine and cysteine peptides which have a lower, but still sufficient, S-electrophilicity, in order to obtain the advantages of the alkoxycarbonyl sulfenyl chlorides. to activate both free and S-acylamidomethyl- and S-trityl-protected thiols. The envisaged activating reagents, unlike the alkoxycarbonyl sulphenyl chlorides, should have leaving groups * that are well-stabilized anions of a less strong acid in order to limit these side reactions too.

According to the invention, the preparation of the S-alkoxyo-carbonylthio derivatives of thiols, of cysteine or of cysteine-containing peptides with N-alkoxycarbonylthio-imides, which consists of alkoxycarbonylsulfenyl chlorides and imides, especially cyclic imides, in the presence of bases, especially tertiary amines and in good yields:

RO-CO-S-Cl

E = alkyl, aralkyl, cycloalkyl, alkoxyalkyl

Y = ^ C = O; ^ StT <and other carbonyl ana · * »

delete groups.

For example, Jff-methoxycarbonylthio-imides of succinimide (CH-O-CO-S-lTSu), phthalimides (CH ₃ -O-CO-S-IIHIt) and saccharin (CH ₃ O-CO-S-ITSaC) or the 2-methoxy-ethyloxycarbonylthio-imide of succinimide (leO-CHpCH 0-C0 ~ S-NSu) are prepared as well crystallizing, in most organic solvents readily soluble, storable over months reagents in over 80 # iger yield

It has now been found that such N ~ alkoxycarbonylthioimides convert free, S-acylamidomethyl- and S-urityl-protected thiol compounds, such as the alkoxycarbonylsulfenyl chlorides, into activated S-alkoxycarbonylthio derivatives, but at a reduced rate owing to the lower reactivity. "Surprisingly, the activation rates differ Thiolderivaten so strong that reactions with the reagents according to the invention for the selective selective activation of several differently protected Thiolfunktionen in this manner can be carried out sequentially in a molecule, which is not possible with the corresponding sulfenyl chlorides.

For example, at equimolar conversion of CH.sub.2 O-COS-liSac in chloroform / methanol (2: 1), the quantitative S <»activation at room temperature for SH-free Boc-Cys-OH required less than 60 minutes, for Boc.sup.-Cys (B. Acm) -OH at least 4 hours and at Boc-Cys (Trt) -OH for about 30 days.

Boc-Cys-OH

CH ₃ O-CO-S-HSAC

BoC-Cys (Acm) -OH-] - ^: > Boc-Cys (Mct) -OH + H-NSac

Boc-Cys (Trt) -OH

Acm = acetamidomethyl

Trt = trityl

Met = methoxycarbonylthio

Boo s tert -butyloxycarbonyl

Bs, it is then for the first time possible to selectively activate S-acetamidomethyl-protected cysteines in addition to S-trityl-protected cysteines, which substantially extends the strategic possibilities in syntheses of peptides with a number of disulfide bridges, eg, _e insulin.

In this sense, it is also noteworthy that disulfide protecting groups such as tert-butylthio, i-propylthio or ethylthio groups and already existing cystine disulfide bridges are unexpectedly unaffected by N-alkoxycarbonylthio-imides, in contrast to reactions with sulfenyl chlorides.

It is also one of the advantages of the process according to the invention that the much more selectively reacting IT-alkoxycarbonylthio-imides on easily electrophilic attacking amino acids such as tryptophan, methionine, tyrosine and histidine no side reactions · Thus, for example, the particularly sensitive tryptophan, the N-Bo "Derivative of the sulfenyl halides in a few hours" quantitatively, probably by sulfenylations, is changed, even after several days by CELO-CO-SN "Sac not attacked" The resulting in the reactions with the N-alkoxycarbonylthio-imides, only weakly Acidic imides can be easily removed from the reaction mixtures by recrystallization, chromatographic methods or, in favorable cases, by extraction with aqueous alkali metal bicarbonate solutions. Their low acidity protects sensitive structures of the thiol compounds from acid-catalyzed weaving reactions as evidenced by the action of chlorine are known when the alkoxyearbonylsulfenyl chlorides are used

Thereafter arise as advantages of erfindungsgeraäßen method of preparing the usable for the formation of unsymmetrical disulfides S-alkoxycarbonylthio derivatives of thiols, cysteine and cysteine-containing peptides with N-AIkoxycarbonylthio-imides

the easy accessibility of N-alkoxycarbonylthio-imides in good yields from aoxycarbonylsulfenyl chlorides and their high storage stability,

the quantitative formation of S-alkoxycarbonylthio derivatives from free S-acylamidomethyl- and S-trityl-protected thiol compounds,

the distinctly different reaction rate between S-acetamidomethyl- and S-trityl-protected thiols, which allows the selective activation of S-acetamido methyl-deuterated cysteine in addition to S-trityl-protected cysteines,

the stability of disulfide bonds in cystine peptides or S-alkylthio-protected cysteines to the action of L-alkoxycarbonylthio-imidene; avoidance of lifting reactions on easily electrophilically vulnerable amino acids during the activation step...

are when using the corresponding sulfenyl halides caused by the high acidity of the hydrogen chloride formed the prevention of lifting reactions _s "

Execution examples

The invention is described in the examples which follow. "The ¹ R" values refer to thin layer chromatography on silica gel plates:

System AB: acetone-benzene (1: 1)

System BDA: Benzen-dioxane-glacial acetic acid (45: 30: 4) System BWA: Ethanol-water-sisessig (4: 1: 1)

The following abbreviations are used:

OMe - methyl ester.

OtBu - tert, butyl ester. .

Boc- tert-butyloxycarbonyl

Bpoc - 2- (p-biphenylyl) -2-propyloxycarbonyl

Acm - acetylaminomethyl "

Trt - trityl (Tr iphenylraethyl)

Met - Methyloxycarbonylthio

Meet - 2- (methyloxy) -ethyloxycarbonylthio

Mcs-Cl - Methyloxycarbonyl sulfenyl chloride

Mecs-Cl - 2- (methyloxy) -ethyloxycarbonylsulfenyl chloride

TEA - triethylamine ~

DCHA - dicyclohexylamine

THP - tetrahydrofuran

DMP - dimethylformamide -. , /

PE petroleum ether (bp 30 - 60 ^° C.)

EE, - ethyl acetate

1 · Preparation of H-alkoxycarbonylthio-imides Example 1

If-Meth.vloxycarbonylthio-sucoinimid (Mct-NSu) 0.991 g (1OnMoI) succinimide and 1.123 g (11 mmol) of TEA THP are introduced under stirring at 20 ⁰ C in 40 ml * Then, 1.266 g (10 mmol) Mcs-Cl in 20 ml of THP, rapidly giving a white precipitate of TEA.HCl.

ν falls »

Tray 3 hours is filtered, the solvent i.Y, withdrawn and the residue crystallized from a little hot methanol After filtration, washing rait PE and

  , · - 8th ' "

Drying i * V "PPO ₁ - remain 1.240 g of analytically pure colorless ladein with BV = 94 ⁰ C (yield 79 *?) · '

Example 2:

0.991 g (10 mmol) Succinimi'd and 1.123 g (11 mlol) TEA are dissolved in 40 ml of THP tint he stirring at 20 ⁰ C vorgelegt® Then, 1.706 g (10 mmol) Mecs-Cl are added dropwise in 20 ml of THP, wherein quickly a white precipitate of TEA »HC1 precipitates« After 4 hours it is filtered, the solvent i ©? _e evaporated and the residue from hot methanol crystallized "After filtration _s washing with PE and drying i _e V" over P ^ O ^ remaining 1,910 g of analytically pure colorless needles with mp = 97-98 ⁰ C (yield: 82%) ·

Example 3i

N ~ Methyloxycarbonylthio ~ phthalimide (Mot-NPht) 2,943 g (20 mmol) of phthalimide and 2.246 g (22 mmol) of TEA 'are placed in 200 ml of EA under stirring at 20 ⁰ C then be 3.037 g (24 mmol) of Mcs-Cl in 50 ml of EA, slowly precipitating a white precipitate of TEA "HCl". After 48 hours, filter, evaporate the solvent ".V" and extract the residue with 100 ml of PE. The remaining substance is dissolved in 100 ml of ethyl acetate and extracted with 3 x 20 ml of water * After drying the EE phase over Na ₃ SO. and filtering the solution i * W concentrated at 40 ⁰ C and to turbidity with PE offset * After filtration and washing the crystals with PE and drying i * Υ © PPO _s remaining 3.94 g of analytically pure crystals of P '= 138 C (yield based on phthalimide: 83 »·

Example 4:

.N-Methyloxycarbonylthio-saccharoharin (Mct-NSac) -

9,160 g (50 mmole) of saccharin, and 5.566 g (55 mmol) of TEA in 100 ml of EA presented with stirring at 20 ⁰ C. Then 7.593 g (60 mmol) Mcs-Cl are added dropwise in 20 ml of EA, and slowly and a white precipitate of TEA ₉ HCl precipitates ^

Wax 24 hours is filtered, '' the .Lösungsmittel i _e V. evaporated and the residue. 100 ml PE'.extrahierte The remaining solid is dissolved in 100 ml Essigester with 3 χ 20 ml of water extracted · ^v After drying the EA phase over _Na? S0 ^ and filtering becomes the solution ieV #. concentrated at 40 ⁰ C and to turbidity with PE offset * After filtration and washing the crystals with PE and drying i "V" PPO ₁ - remain 10.68 g of analytically pure crystals with F · = 134 based ⁰ C (yield based on saccharin : 88 $ &) · - '

2 Preparation of S- alkyloxycarbonylthiolethiol compounds

Example 5: S-Methylox.yoarbonylthio-cysteaniin hydrochloride

1.1.35 g (10 mmol of cysteamine hydrochloride and 2.733 g (10 mM mol) of Mct-NSac are stirred in 100 ml of methylene chloride-methanol (2: 1) at 20 ° C. for one hour.

After stripping off the solvent mixture, fractional crystallisation from methanol with ether gives 1.67 g of analytically pure colorless flakes with F = 106-107 ⁰ C (yield: 82 %).

R _f (EWA) = 0.76. -

Example 6:

2 ~ _(i S-Methyloxycarbonylthio) - 'mercaptoT3ropion.Yl glyoin-1} 632 g (10 mmol) ^ - mercaptopropionyl-glycine and 1.892 g (10 mmol) of MCT FSU in 100 ml of chloroform-methanol (2: 1) stirred at 20 ⁰ C for two hours.

After removal of the solvent mixture is obtained on extraction of the residue with ether and cases from this ether phase with PE 2.1 g of analytically pure crystals

F. = 124 ⁰ C (Yield: 83 #) »

R _f (EWA) = 0.77.

Example 7:

Boc ~ Cys (Mct) ~ OH ',

a) 0.585 g (2 mmol) of Boc-Cys (Acm) -OH, and 0.605 g (2.2 mmol) MCT NSAC in 20 ml of chloroform-methanol (2: 1) for 24 hours at 20 ⁰ C stirred. After concentration of the solution

The main fraction, after removal of the solvent mixture, gives 0.605 g of analytically pure oily product. After uptake in ether and addition of 0.365 g of DCHA, 0.925 g of Boc-Cys (Mct) are added with the addition of PE. -OH.DCHA with F. "145'- 146 ⁰ C (total Ausys related to Cys derivative: 94%) · R _f (BDA) = 0.75 ·

b) 0.927 g (2 mmol) of Boc-Cys (Trt) -OH and O, 605 g (2.2 mmol) MCT NSAC in 50 ml of EE-glacial acetic acid (1: 1) for 7 days at 20 ⁰ C stirred · Work-up as under a) gives the same results

Example 8 Bo c-Cy s CM et) -Asn-OtBu

1 * 390 g (3 mmol) Boc-Cys (Acm) -Asn-0tBu and 1.09 g (4 mmol) of Mct-NSac are stirred in 100 ml of DMP for one hour at 20 ⁰ C * After removal of the solvent is the Residue taken up in 100 ml of EA and extracted with 3 x 20 ml of 0.1 N aqueous NaHCO., - Solution and 2 χ 20 ml of water After drying the EE phase over Na ₂ SO ^, piledrilling and removal of the solvent remains an oily Residue which, when precipitated from chloroform with PE, gives 1.37 g of analytically pure substance with F * = 101-103 ⁰ C (yield based on Cys derivative: 95 ft). R ^ (AB) = 0.48; R ^ (BDA) = 0.69. .

Example 9s

2.22 g (3.6 mmol) of Bpoc-Leu-Cys (Acm) -Gy-OMe and 1.09 g (4 mmol) of Mct-NSac are stirred in 25 ml of methanol for 2 hours at 20 ⁰ C · After stripping of the solvent, the residue is chromatographed on LH 20 in chloroform-methanol (2: 1). The main fraction, after stripping off the solvent mixture, gives 2.19 g of analytically pure S-Mct-tripeptide derivative with I ₉ = 81-82 ⁰ C (yield based on Cys Derivative: 96 90 × R- (AB) = 0.69 ×

^x 11

Claims (3)

Erfindun ^ sanspruch 1 » Process for the preparation of S-alkoxycarbonylthioderivates of thiols of the general formulas I or II, HO-CO-SSR ₁ (I)
R ₂ - (LiH-CHR ₃ -CO) _n -IiH-CH-CO- (LiH-CHR ₃ -CO) _m -R ₄ (II) H-O-CO-S-S .. ':' where R is an alkyl, aralkyl, cycloalkyl or a alkoxyalkyl
R _{1 is} an alkyl, cycloalkyl or aryl group or a heterocyclic radical Rp denotes a hydrogen atom or one in the peptide cheraie known amino protecting group Ro same or different of the known, optionally protected amino acid side chains R. an OH group, an alkoxy group or a Amino group and .. η and m are zero or any integer numbers, characterized in that a free or protected thiol compound of the general formulas III or IV, R ₁ -SX. ^ (III)
R ₂ - (IrH-CHR ₃ -CO) _n -IrH-CH-CO- (XiH-CHR ₃ -CO) _m -R ₄ (IV) Γ, X. where R ₁ , R _? , R ', R ,, η and m have the abovementioned meaning and X represents a hydrogen atom, an acylamidomethyl or an aralkyl group, with an H-alkoxycarbonylthio-imide of the general formula (V) in which R has the meaning given above and Y is for ^ C = O or a carbonylahalogroup such as ^ S0 _? . is implemented,  2 "" Process according to item 1, characterized in that N- 'alkoxycarbonylthio-imides of general formula V are used which are obtained from imides, in particular cyclic imides and alkoxycarbonylsulfenyl halides » 3 * Process according to item 1 and 2, characterized in that it is used for the introduction of Alkoxycarbonylthio «group preferably the N-alkoxycarbonylthio-imides of succinimides or saccharin, which is alkoxy-, carbonylsulfenylchloriden and the imides in the presence of a tertiary base produced The method of item 1, characterized in that the same performs the introduction of alkoxycarbonylthio group in a Kohlenwasserstoffj chlorinated hydrocarbon, alcohol, ether, Carbonsäureester, carboxylic acid amide or mixtures at temperatures of about -30 ⁰ C to + ^60/0 C «*.