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Title:
PROCESS FOR THE PREPARATION OF S-CARBAMOYL CYSTEINE DERIVATIVES
Document Type and Number:
WIPO Patent Application WO/2006/056233
Kind Code:
A1
Abstract:
The present invention relates to a process for the preparation of S-carbamoyl cysteine derivatives of formula (I) starting from a cysteine alkyl ester, comprising few steps, which do not include the use of harmful reagents, and allow to obtain in high yields the desired products of formula (I).

Inventors:
ATTOLINO EMANUELE (IT)
NATIVI CRISTINA (IT)
Application Number:
PCT/EP2004/053075
Publication Date:
June 01, 2006
Filing Date:
November 24, 2004
Export Citation:
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Assignee:
PROTERA S R L (IT)
ATTOLINO EMANUELE (IT)
NATIVI CRISTINA (IT)
International Classes:
C07C329/06; C07C333/04
Other References:
JAROSLAV MRA'Z: "GAS CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF N-ACETYL-S-(N-METHYLCARBAMOYL)CYSTEINE, A METABOLITE OF N,N-DIMETHYLFORMAMIDE AND N-METHYLFORMAMIDE, IN HUMAN URINE", JOURNAL OF CHROMATOGRAPHY 431, 1988, pages 361 - 368, XP002347359
JAMES P. HUBBELL ET AL.,: "METABOLIC FATE OF THE N,N-DIALKYLCARBAMOYL MOIETY OF THIOCARBAMATE HERBICIDES IN RATS AND CORN", J. AGRIC. FOOD CHEM.,, vol. 25, no. 2, 1977, pages 404 - 413, XP009053774
XIANGMING GUAN ET AL.,: "IDENTIFICATION OF S-(n-BUTYLCARBAMOYL)GLUTATHIONE, A REACTIVE CARBAMOYLATING AGENT, AS A BILIARY METABOLITE OF BENOMYL IN THE RAT", J. AGRIC. FOOD CHEM. 1994, vol. 42, 1994, pages 2953 - 2957, XP009053755
J. A. MACLAREN: "2-OXOTHIAZOLIDINE-4-CARBOXYLIC ACID AND RELATED SUBSTANCES", AUST. J. CHEM.,, vol. 21, 1968, pages 1891 - 1896, XP009053752
Attorney, Agent or Firm:
Gervasi, Gemma (Corso di Porta Vittoria 9, Milan, IT)
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Claims:
CLAIMS
1. A process for the preparation of Scarbamoyl cysteine derivatives of general formula (I) (I) wherein R is selected from between H and alkyl groups, linear or branched, having from 1 to 5 carbon atoms, R' is an alkyl group, linear or branched, having from 1 to 5 carbon atoms, possibly substituted with one or more halogen atoms, R" is selected from between H and an acetyl group, or pharmaceutically acceptable salts thereof, said process comprising the following steps: i) reaction of cysteine ester of formula (II) with a phenylchloroformiate of formula (III) in an organic solvent, to obtain the Sphenoxycarbonyl cysteine ester of formula (IV) (II) (III) (IV) wherein Ri, equal or different from R, is an alkyl group, linear or branched, having from 1 to 5 carbon atoms, R'", equal or different from R", is selected from acetyl and amino protecting groups, and R2, R3, R4, R5 and Re, equal or different from each other, are chosen from between H and electron withdrawing groups, provided that at least one amongst R2, R3, R4, R5 and Re is different from H; ii) reaction of the Sphenoxycarbonyl cysteine ester of formula (IV) coming from step i) with R1NH2 or a salt thereof in an organic solvent in the presence of a base, to obtain the Scarbamoyl cysteine of formula (V) (TV) (V) wherein R', Ri, R2, R3, R4, R5 and Re are defined as above, so that when R'" is equal to R" and Ri is equal to R, the compound of formula (V) corresponds to the desired Scarbamoyl cysteine derivative of formula (I), whereas, when R"' is different from R" and/or Ri is different from R, the above compound of formula (V) is subjected to deprotection from the group R'" and/or acetylation if the S carbamoyl cysteine derivative of formula (I) in which R" is acetyl is desired, and/or to hydrolysis of the ester COORi if the compound of formula (I) in which R is H is desired.
2. The process according to claim 1 , wherein said organic solvent in step i) is methylene chloride.
3. The process according to claim 1 , wherein said reaction in step i) is carried out at room temperature for 2.5 hours.
4. The process according to claim 1 , wherein said reaction in step i) is carried out in the presence of pyridine.
5. The process according to claim 1, wherein said organic solvent in step ii) is tetrahydrofuran.
6. The process according to claim 1 , wherein said base in step ii) is triethylamine.
7. The process according to claim 1 , wherein said reaction in step ii) is carried out at room temperature for 30 minutes.
8. The process according to claim 1 , wherein said hydrolysis is carried out dissolving the compound having the ester group COORi in HCI, thus obtaining the corresponding compound having the free carboxylic group COOH.
9. The process according to claim 1 , further comprising the purification of the crude Scarbamoyl cysteine derivative of formula (I) by crystallisation in a solvent.
10. The process according to claim 1 , wherein R and R1 , equal or different from each other, are selected from between methyl and ethyl.
11. The process according to claim 1 , wherein R' is selected from between methyl and ethyl, possibly substituted with a chlorine atom.
12. The process according to claim 1 , wherein R2 = R3 = R5 = R6 = H and R4 is an electron withdrawing group.
13. The process according to claims 1 or 12, wherein said electron withdrawing group is selected from the group consisting of NO2, CN, NO, SO3R7, COOR7, COR7 and CONR7R8, wherein R7 and R8, equal or different from each other, are chosen from between H and alkyl groups, linear or branched, having from 1 to 5 carbon atoms.
14. The process according to claim 13, wherein said electron withdrawing group is NO2.
15. The process according to claim 1 , wherein said amino protecting group is selected from the group consisting of tbutoxycarbonyl (tBoc), 9 fluorenylmethoxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz).
16. The process according to claim 1 , wherein said group R'" is acetyl.
17. The process according to claim 1 , wherein R", equal to R'", is acetyl; Ri, equal to R, is methyl; R2 = R3 = R5 = R6 = H and R4 is NO2.
18. Compounds of formula (IV) (IV) wherein R'" is selected from between acetyl and amino protecting groups, Ri is an alkyl group having from 1 to 5 carbon atoms, and R2, R3, R4, R5 and R6, equal or different from each other, are chosen from between H and electron withdrawing groups, provided that at least one amongst R2, R3, R4, R5 and R6 is different from H.
19. Compounds of formula (IV) according to claim 18, wherein Ri is selected from methyl and ethyl.
20. Compounds of formula (IV) according to claim 18, wherein Ri is methyl.
21. Compounds of formula (IV) according to claim 18, wherein said amino protecting group is selected from the group consisting of tbutoxycarbonyl (tBoc), 9fluorenylmethoxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz).
22. Compounds of formula (IV) according to claim 18, wherein R1" is acetyl.
23. Compounds of formula (IV) according to claim 18, wherein R2 = R3 = R5 = Re = H and R4 is an electron withdrawing group.
24. Compounds of formula (IV) according to claims 18 or 23, wherein said electron withdrawing group is selected from the group consisting of NO2, CN, NO, SOeRr, COOR7, COR7 and CONR7R8, wherein R7 and R8, equal or different from each other, are chosen from between H and alkyl groups, linear or branched, having from 1 to 5 carbon atoms.
25. Compounds of formula (IV) according to claim 24, wherein said electron withdrawing group is NO2.
26. Compound of formula (IV) according to claim 18, wherein R'" is acetyl, Ri is methyl, R2 = R3 = R5 = Re = H and R4 is NO2.
Description:
Process for the preparation of S-carbamoyl cysteine derivatives

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of S-carbamoyl cysteine derivatives of formula (I) hereinafter reported.

STATE OF THE ART

Many S-carbamoyl cysteine derivatives of interest are known for different uses. N- acetyl-S-(N-methylcarbamoyl)cysteine for example is a human metabolite of N, N- dimethylformamide (DMF), and it is necessary to have available a large amount of this product when analyses for searching this metabolite in people exposed to

DMF have to be carried out.

DMF is an organic solvent widely used in manufacture of electronic components, synthetic textiles and leather, of chemicals and pharmaceuticals; it has a high solubility in water and low vapour pressure, and it can be readily absorbed by exposed workers through respiratory apparatus as well as through the skin.

This organic solvent is known to be toxic to the liver, and liver damages were observed in exposed workers; DMF is also known to cause skin problems and alcohol intolerance. Moreover, even if there are no conclusive evidences at this moment, it seems that exposure to DMF increases the risks of developing cancer.

Biological monitoring is therefore recommended for all workers exposed to DMF, and it is usually performed by collecting urine and analysing it for several metabolites of DMF.

The biological marker currently used to estimate exposure to DMF during the working day is urinary N-methylformamide, measured by gas chromatography.

Nevertheless, the amount of N-methylformamide in urine only accounts for a minimal quantity of the absorbed DMF.

N-acetyl-S-(N-methylcarbamoyl)cysteine, commonly named with the acronym

AMCC, is another human metabolite of DMF. As DMF biomarker N-acetyl-S-(N- methylcarbamoyl)cysteine accounts for a higher quantity of the absorbed dose of

DMF when compared with N-methylformamide. Thus many Governmental and

Intergovernmental Health Organisations have recently acknowledged AMCC as the biomarker for evaluating exposure to DMF, since it accumulates and can be found in high concentrations even in urine of workers exposed to low doses of

DMF. Besides being very sensitive, AMCC as biomarker of DMF is moreover endowed with a very high specificity: in fact, it is always found in workers exposed to DMF, but it is not found at all in non-exposed people.

AMCC is moreover a metabolite of methyl isocyanate, an extremely noxious reagent, used in the manufacture of carbamate pesticides, such as metham and dazomet, and in manufacture of polyurethane foams.

Many methods for the determination of AMCC in urine are therefore known, wherein the amount of the metabolite is measured by gas chromatography or liquid chromatography (see for example Kafferlein H. U. et al. J. of

Chromatography B., 734 (1999) 285-298). Standardisation and calibration of these methods with standard solutions of AMCC are necessary before accomplishing the measurements of samples to be analysed, and this forces to the need of large amounts of AMCC.

But AMCC is not commercially available, because the only known process for preparing AMCC include the reaction of N-acetylcysteine with methyl isocyanate, a very expensive, noxious, reactive and carcinogenic volatile liquid, which have to be handled with a lot of precautions when used in laboratory so that the process for preparing AMCC is not scalable up to industrial scale.

Processes including the above said reaction with methyl isocyanate have been described for example by Mraz J. in J. of Chromatography, 431 (1988) 361-368, and by Slatter J.G. et al. in Chem. Res. Toxicol. 4 (1991) 157-161.

Moreover, S-carbamoyl-L-cysteine derivatives having antitumoural activity on human tumour models are disclosed for example in European Patent Application

No. 139 005. These compounds have been extensively studied and disclosed in the scientific literature too, wherein their chemotherapeutic effect is confirmed (see for example Nemeth L. et al. Arzneim. Forsh. 28(7) (1978) 1119-23). As reported by European Patent Application No. 139 005, such compounds can be prepared allowing a suitable L-cysteine derivative to react with alkyl isocyanate.

As far as the Applicant is aware of, no other process for preparing the S- carbamoyl cysteine derivatives of interest has been disclosed, different from processes including the use of alkyl isocyanate.

There is therefore a strong need of a profitable process for the preparation of

AMCC and other S-carbamoyl cysteine derivatives of interest, not having the drawbacks of the above said preparation processes known in the art.

SUMMARY OF THE INVENTION

The Applicant has now found a novel synthetic pathway that, starting from commercially available products, or readily obtainable from commercially available products, allows to obtain in high yields the desired S-carbamoyl cysteine derivatives of formula (I) herein below reported having high purity, without using methyl isocyanate or similar very noxious reagents.

Subject of the present invention is therefore a process for the preparation of S- carbamoyl cysteine derivatives of general formula (I)

(I) wherein:

R is selected from between H and alkyl groups, linear or branched, having from 1 to 5 carbon atoms,

R' is an alkyl group, linear or branched, having from 1 to 5 carbon atoms, possibly substituted with one or more halogen atoms,

R" is selected from between H and an acetyl group, or pharmaceutically acceptable salts thereof, said process comprising the following steps: i) reaction of cysteine ester of formula (II) with a phenyl-chloroformiate of formula

(III) in an organic solvent, to obtain the S-phenoxycarbonyl cysteine ester of formula (IV)

wherein Ri, equal or different from R, is an alkyl group, linear or branched, having

from 1 to 5 carbon atoms, R"', equal or different from R", is selected from acetyl and amino protecting groups, and R 2 , R 3 , R 4 , Rs and R 6 , equal or different from each other, are chosen from between H and electron withdrawing groups, provided that at least one amongst R2, R3, R4, R 5 and Re is different from H; ii) reaction of the S-phenoxycarbonyl cysteine ester of formula (IV) coming from step i) with R'NH 2 or a salt thereof in an organic solvent in the presence of a base, to obtain the S-carbamoyl cysteine of formula (V)

(IV) (V) wherein R, R 1 , R 2 , R3, R4, R 5 and R 6 are defined as above, so that when R'" is equal to R" and Ri is equal to R, the compound of formula (V) corresponds to the desired S-carbamoyl cysteine derivative of formula (I), whereas, when R" is different from R" and/or Ri is different from R, the above compound of formula (V) is subjected to deprotection from the group R'" and/or acetylation if the S- carbamoyl cysteine derivative of formula (I) in which R" is acetyl is desired, and/or to hydrolysis of the ester COORi if the compound of formula (I) in which R is H is desired.

Further subject of the invention are the novel intermediate compounds of formula

(IV) above reported.

Features and advantages of the present invention will be illustrated in details in the following description.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention with the expression "amino protecting group" any suitable group typically used as protecting group for the amino functionality in peptide chemistry is meant, such as t-butoxycarbonyl (t-Boc), 9- fluorenylmethoxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz).

With the expression "electron withdrawing groups" groups selected from amongst

NO 2 , CN, NO, SO3R7, COOR 7 , COR 7 and CONR 7 R 8 are preferably meant,

wherein R 7 and Rs, equal or different from each other, are chosen from between H and alkyl groups, linear or branched, having from 1 to 5 carbon atoms. More preferred are those compounds in which the electron withdrawing group is NO 2 .

Examples of alkyl groups, linear or branched, having from 1 to 5 carbon atoms according to the present invention are methyl, ethyl, propyl, /-propyl, n-butyl, /-butyl and f-butyl. Methyl and ethyl are preferred.

The starting compounds of formula (II) of the present process are commercially available products or they can be prepared by means of procedures widely used and well-known to any person skilled in art. Starting compounds of formula (II) having the stereogenic centre of either L or D configuration may be used, even if the L-configurated compounds (II) are preferred. The configuration of the starting compounds is then maintained in the present process, from which a S-carbamoyl cysteine derivative of formula (I) is obtained having the stereogenic centre in the same configuration as the starting compounds.

According to a preferred embodiment of the present process, the process is applied to the preparation of S-carbamoyl cysteine derivatives of formula (I) wherein R is H, R' is methyl, and R" is acetyl starting from compounds of formula

(II) wherein R"' is acetyl and Ri is methyl. In this case, the process comprises therefore the steps i) and ii) above illustrated, from which a compound of formula

(V) is obtained wherein R'" = R" = acetyl, R' is methyl and Ri is methyl, followed by hydrolysis of the ester COORi, finally obtaining the desired compound of formula (I) wherein R" is acetyl, R' is methyl and R is H.

Preferred compounds of formula (III) are those wherein R 2 = R 3 = R 5 = Re = H and

R4 is an electron withdrawing group; preferably R 4 is NO 2 .

The reaction in step i) of the present process is typically carried out using methylene chloride as organic solvent, preferably at room temperature for 2.5 hours. Pyridine can be added to the reaction mixture.

In step ii) of the present process tetrahydrofuran can be used for example as the organic solvent, and triethylamine as the base. The reaction in step ii) is carried out at room temperature for 30 minutes.

The reagent R 1 NH 2 used in step ii) is selected depending on the desired compound of formula (I). As above defined, R' is an alkyl group, linear or

branched, having from 1 to 5 carbon atoms, possibly substituted with one or more halogen atoms; preferably, R 1 is selected from between methyl and ethyl, possibly substituted with Cl.

Deprotection and/or N-acetylation and/or hydrolysis may be carried out according to the commonly used procedures known to any skilled person. For example, the hydrolysis of the ester group COORi may be carried out dissolving the ester in

HCI, thus obtaining the corresponding compound having a free carboxylic group

COOH.

The present process for preparing products of formula (I) as defined above, may therefore comprise the following steps: i) reaction of cysteine ester of formula (II) with a phenyl-chloroformiate of formula

(III) in an organic solvent, to obtain the S-phenoxycarbonyl cysteine ester of formula (IV)

wherein Ri is an alkyl group, linear or branched, having from 1 to 5 carbon atoms, R'", equal or different from R", is selected from between acetyl and amino protecting groups, preferably chosen from t-Boc, Fmoc and Cbz, and R2, Rs, R 4 , R5 and Re, equal or different from each other, are chosen from between H and electron withdrawing groups, provided that at least one amongst R2, R3, R4, R5 and R 6 is different from H; ii) reaction of the S-phenoxycarbonyl cysteine ester of formula (IV) coming from step i) with R 1 NH 2 or a salt thereof in an organic solvent in the presence of a base, to obtain: ii-a) when R'", different from R", is an amino protecting group, the S-carbamoyl cysteine of formula (V)

OV) (V) wherein R', R 1 , R 2 , R 3 , R4, R 5 and Re are defined as above, or ii-b) when R"', equal to R", is acetyl, and Ri is different from R, the S-carbamoyl cysteine of formula (Vl) in which R" is acetyl

OV) (VI) wherein R', Ri, R 2 , R 3 , R4, R 5 and Re are defined as above, and R" is acetyl; ii-c) when R'", equal to R", is acetyl, and Ri is equal to R, the S-carbamoyl cysteine of formula (I) in which R" is acetyl and R is an alkyl group

(N) 0) wherein R J , R 1 , R 2 , R 3 , R 4 , R 5 and Re are defined as above, R is an alkyl group and

R" is acetyl; iii) possible deprotection of the S-carbamoyl cysteine of formula (V) coming from step ii-a), to obtain: iii-a) when R 1 is different from R, the S-carbamoyl cysteine of formula (VII)

(V) (VII) wherein R, Ri and R' are defined as above, and R" is H; iii-b) when Ri is equal to R, the S-carbamoyl cysteine of formula (I) in which R is an alkyl group

(V) (I) wherein Ri and R' are defined as above, R is an alkyl group and R" is H; iv) possible N-acetylation of the S-carbamoyl cysteine of formula (Vl) in which R" is H coming from step iii-a) to obtain the corresponding compound of formula (Vl) in which R" is acetyl; v) possible hydrolysis of: v-a) the S-carbamoyl cysteine of formula (Vl) above reported coming from step H- b) or from step iv) to obtain the S-carbamoyl cysteine of formula (I) in which R is H and R" is acetyl; v-b) the S-carbamoyl cysteine of formula (VII) above reported coming from step iii- a), to obtain the S-carbamoyl cysteine of formula (I) in which R and R" are H. According to a particular embodiment of the present process, the crude S- carbamoyl cysteine derivative of formula (I) may be subjected to a purification step, for example by crystallisation in a solvent.

The Applicant has found that the advantages offered from the process herein described are considerable. As a matter of fact, thanks to the present process, a substantial improvement over the state of the art has been made, due to the fact that the use of methyl isocyanate is avoided, and the desired S-carbamoyl cysteine derivatives of formula (I) are obtained in high yields and purity. The following examples are reported as a non-limiting illustration of the invention.

EXAMPLE 1

Preparation of N-acetyl-S-^-nitro-phenoxycarbonvDcvsteine methyl ester

To a solution of 3.54 g (0.020 mol) of N-acetyl-cysteine methyl ester and pyridine

(2.4 ml, 0.030 mol) in 80 ml of CH 2 CI 2 , 4-nitrophenyl-chloroformiate (5.24 g, 0.026 mol) was added under stirring at O 0 C. The reaction mixture was stirred at room temperature for 2.5 h and then HCI 0.2 M (50 ml) was added. The separated organic phase was washed with 50 ml of NaOH 0.1 M and of a saturated solution of NaCI, in that order, then dried over Na 2 SO- J . The solvent was evaporated under reduced pressure affording the title compound as crude product. A small amount of this crude product was purified by column chromatography carried out using silica Kieselgel 60 Merck (70-230 mesh) for the stationary phase and ethyl acetate as the eluent, thus affording the pure compound of the title as a white solid.

Mp 129-130 0 C.

[α] D +62 (C i 1 CHCI 3 ).

1 H NMR (200 MHz, CDCI 3 , δ/ppm): 2.06 (s, 3H, CH 3 CO); 3.37 (dd, 1H, Cys-β-

CH 2 ); 3.58 (dd, 1H, Cys-β-CH 2 ); 3.78 (s, 3H, CH 3 O); 4.95 (m, 1H, Cys-α-CH); 6.55

(d, 1H, CH 3 CONH); 7.35 (d, 2H, aromatics); 8.28 (d, 2H, aromatics).

13 C NMR (50 MHz, CDCI 3 , δ/ppm): 22.9 (CH 3 CO); 33.3 (CyS-CH 2 ); 51.5 (Cys-CH);

52.9 (CH 3 O); 121.8, 125.3, 145.5 and 155.3 (aromatics); 168.9, 170.0 and 170.4

(carbonylics).

EXAMPLE 2

Preparation of N-acetyl-S-(N-methylcarbamoyl)cysteine methyl ester

To the stirred solution of crude N-acetyl-S-(4-nitro-phenoxycarbonyl)cysteine methyl ester prepared as described above in Example 1 , triethylamine (6.7 ml,

0.048 mol) and MeNH 2 -HCI (1.62 g, 0.024 mol) in 60 ml of THF, water was added dropwise till complete dissolution of solid MeNH 2 -HCI. The reaction mixture was stirred at room temperature for 30 min. The solvents were evaporated under reduced pressure and the residue was dissolved in EtOAc, washed with HCI 0.5M

(30 ml) and extracted with EtOAc (5 x 50 ml), then dried over Na 2 4 . The solvent was evaporated under reduced pressure affording the crude title compound, which was then purified by flash chromatography carried out using Kieselgel 60 (230-400 mesh) for the stationary phase and ethyl acetate as the eluent. The pure

compound of the title was obtained (4.14 g) as white solid (yield = 88%).

Mp 81-83°C.

[<X]D +20 (c 1.2, CHCI 3 ).

1 H NMR (200 MHz 1 CDCI 3 , δ/ppm): 2.03 (s, 3H, CH 3 CO); 2.86 (d, 3H, CH 3 N); 3.33

(d, 1H, Cys-β-CH 2 ); 3.35 (d, 1H, Cys-β-CH 2 ); 3.75 (s, 3H, CH 3 O); 4.72 (ddd, 1H,

Cys-α-CH); 6.44 (br s, 1 H, CH 3 NHCO); 7.13 (d, 1 H, CH 3 CONH).

13 C NMR (50 MHz, CDCI 3 , δ/ppm): 22.8 (CH 3 CO); 28.0 (CH 3 N); 31.1 (CyS-CH 2 );

52.5 (CH 3 O); 53.3 (Cys-CH); 167.2, 170.5 and 170.7 (carbonylics).

EXAMPLE 3

Preparation of N-acetyl-S-fN-methylcarbamovDcvsteine

A solution of N-acetyl-S-(N-methylcarbamoyl)cysteine methyl ester prepared as described above in Example 2 (4.0 g, 0.017mol) in 50 ml of aqueous HCI 36% was stirred at room temperature for 4 days. The reaction mixture was concentrated under reduced pressure, the residue was diluted with CH 3 CN and dried over

Na 2 SO 4 . After filtration and evaporation the crude title compound was obtained as a solid foam. Purification by crystallisation (CH 3 CN-EtOAc) gave the compound of the title as a white solid (3.38 g, yield = 81%).

Mp 155-157 0 C (Lett. M p = 156-157 0 C reported by Mraz J. in J. of Chromatography,

431 (1988) 361-368)

[αb -12.5 (c 0.9, H 2 O).

1 H NMR (200 MHz, D 2 O, δ/ppm): 1.80 (s, 3H, CH 3 CO); 2.56 (s, 3H, CH 3 N); 3.02

(dd, 1 H, Cys-β-CH 2 ); 3.25 (dd, 1 H, Cys-β-CH 2 ); 4.41 (dd, 1 H, Cys-α-CH).

13 C NMR (50 MHz, D 2 O, δ/ppm): 21.9 (CH 3 CO); 27.6 (CH 3 N); 30.6 (CyS-CH 2 );

53.3 (Cys-CH); 169.2, 173.6 and 174.4 (carbonylics).