Allicin in Blood.Pdf

European Journal of Medicinal Chemistry 45 (2010) 1912–1918

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European Journal of Medicinal Chemistry

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Original article Reaction mechanisms of allicin and allyl-mixed disulﬁdes with proteins and small thiol molecules

Talia Miron a,*, Irving Listowsky b, Meir Wilchek a a Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel b Department of Biochemistry, Albert-Einstein College of Medicine, Bronx, NY, USA article info abstract

Article history: Allylsulfides from garlic are chemopreventive agents. Entering cells they are expected to initially interact Received 15 October 2009 with glutathione. Accordingly, reaction mechanisms of the product, S-allylthio-glutathione, with model Received in revised form proteins and thiols were analyzed in cell free systems. With glutathionyl, cysteinyl or captopril repre- 14 January 2010 senting S-allyl aliphatic adducts, the reaction with sulfhydryl groups resulted in mixed disulfide Accepted 15 January 2010 mixtures, formed by both, S-allyl and aliphatic moieties. Available online 21 January 2010 To improve conventional prodrug treatment of blood pressure, cancer and intestinal inflammation S-allylthio prodrugs, such as S-allylthio-6-mercaptopurine and S-allylthio-captopril were synthesized. Keywords: Allicin Synergistic activities of the 2 constituents, as well as increased cell permeability allow for efficient in vivo Glutathione activity. Upon reaction of these derivatives with glutathione, S-allylthio-glutathione is formed, while S-Allylthio-mixed disulfide 6-mercaptopurine is the leaving group. Excess cellular glutathione enables several cycles of sulfhydryl- Prodrug disulfide exchange reactions to occur, extending the hybrid drug’s pharmacodynamics. Mechanism of action Ó 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction and participates in many important biological processes, including maintenance of a reducing intracellular environment Allicin, diallyl thiosulfinate, is the major biologically active [5] and detoxification of oxidants and electrophiles [6].GSHalso compound derived from garlic. It is produced by the interaction of participates in cellular redox reactions and mixed disulfide the enzyme alliinase (alliin lyase; EC 4.4.1.4) with its substrate, formation, which leads to the production of S-glutathiolated alliin (S-allyl-L-cysteine sulfoxide) (Scheme 1) [1]. proteins [7–10]. Allicin is a short-lived compound which easily diffuses through The mechanisms by which the allylsulfides reduce the risk of cell membranes (diffusion coefficient 5 108 cm2 s1) [2] and diseases may be rationalized on the basis of their chemistry [11]. exerts its biological effects by rapidly reacting with intracellular Thus, they could affect GSH levels and cellular redox status, or free thiols, such as reduced glutathione (GSH), cysteine and sulf- react directly with key proteins involved in various physiological hydryl groups of proteins. The reaction of the allylthio group with processes. However, details of the functional course of action of those cellular components constitutes the major beneficial effects allylsulfides are obscure. Since the initial cellular products are of allicin. The first product is most likely that of the S-allylthio- likely to be GSH adducts, this study was designed to determine mixed disulfide (AS-SX) with GSH as depicted in Scheme 2 below. the outcome and reaction mechanisms of S-allylthio-glutathione Intracellular GSH is the major low molecular weight thiol that (GSSA) with model proteins and low molecular weight thiols. is present at millimolar concentrations in many cell types [3,4] The putative products, the mixed disulfide, can in turn, be involved in further exchange reactions with free thiols, potentially modulating various physiological processes in the cell Abbreviations: ASH, allylmercaptan; AS-SX, S-allylthio-mixed disulfide; CPSH, [12–14]. captopril; CPSSA, S-allylthio-captopril; DTNB, 5,50-dithio-bis (2-nitrobenzoic acid); G3PDH, glyceraldehyde 3-phosphate dehydrogenase GSSA, S-allylthio-glutathione; Several S-allylthio-mixed disulfide compounds (AS-SX) were GSH, Reduced glutathione; GSSG, Glutathione oxidized; GS-S-CP, S-glutathionyl- prepared (including drugs containing free thiol groups) and their thiocaptopril: NTB, 2-nitro-5-thiobenzoate; PTP1B, Protein tyrosine phosphatase disulfide exchange reactions with GSH and proteins containing free 1B; SA-6MP, S-allylthio-6-mercaptopurine; SA-6MPR, S-allylthio-6-mercaptopurine sulfhydryl groups were studied, in order to follow the formation of riboside. the various intermediates and final products. These substances * Corresponding author. Tel.: þ972 8 9343627; fax: þ972 8 9468256. E-mail addresses: [email protected] (T. Miron), [email protected]. could shed light on the reaction mechanisms of S-allylthio-mixed aecom.yu.edu (I. Listowsky), [email protected] (M. Wilchek). disulfide (AS-SX) with cellular thiols.

Scheme 1. Enzymatic production of allicin.

2. Chemistry activity. The maximum extent of S-glutathionylation of the non- denatured enzyme under these conditions is 2-per tetramer. PTP1B All of the S-allylthio-derivatives were synthesized by coupling has 6 cysteine residues including a very reactive sulfhydryl at the allicin to the indicated sulfhydryl-derivatives at pH 6.5, using the active site. Two major reaction products were obtained in the ratio of allicin: SH-derivatives 1:1.8 (Scheme 3). reaction of PTP1B and GSSA. These included a 37 453 Da component indicating the addition of 2 allylmercapto moieties, and a 37 758 Da 3. Results component indicative of the addition of 2 allylmercapto moieties as well as a single S-glutathionylated group linked to the protein. 3.1. Reactions of S-allylthio-glutathione (GSSA) with sulfhydryl groups of proteins 3.2. Reaction between allicin and GSH

Papain was incubated with GSSA to identify mixed disulfide All of the S-allylthio-derivatives were prepared using an excess derivatives formed during the reaction. Previously we showed that of allicin. The reaction was carried out at room temperature, pH 6.5. modification of papain with GSSA abolishes its enzymatic activity Under these conditions the product is stable and can be readily in a concentration dependent manner [15]. GSSA modification of isolated. When allicin (5 mM) reacted with excess of GSH (50 mM), papain in this study caused a decrease in catalytic activity in the first product formed rapidly was GSSA, which ultimately was a biphasic manner, reaching a total loss of activity after 18 h. These converted back to GSH, while releasing allylmercaptan (ASH). Only results are consistent with the formation of a papain-S-SX deriva- traces of ASH were detected by HPLC (due to its high volatility). tive. Using the tritiated allyl moiety ([3H]GSSA) enabled tracking the formation of modified papain products. Gel permeation chro- 3.3. Reaction between S-allylthio-captopril (CPSSA) and GSH matography of the reaction mixture using a PD-10 column, sug- gested that a major product is [3H] allyl-S-S papain since the CPSSA [18] and GSH were reacted at a molar ratio of 1:1. The radioactivity and protein peaks overlap (Fig. 1). In the 4th–5th ml data in Table 2A show molecular masses of the various reactants, peak, the ratio is 0.7–0.9 mCi labeled allyl group per mmole protein, intermediates and products, as established by ESI-MS and HPLC implying that at least 70% of the protein is S-allylthio-papain. retention times (Rt). However, to determine whether other derivatives, such as The time course of product formation in the reaction mixture of glutathionyl-S-S-papain were also formed papain was modified S-allylthio-captopril (CPSSA) and GSH at room temperature, pH 6.5 with unlabeled GSSA, and protein mixed disulfide formation was is shown in Table 2B. analyzed by ESI-MS. Unmodified papain showed the presence of ESI-MS analysis of the reaction mixture HPLC peaks indicated two major species [16] with molecular masses of 23 428 Da (a) and that it contained S-glutathionyl-captopril (GS-S-CP: mw 522, Rt: 23 458 Da (b). Modified papain revealed the formation of 2 distinct 4.6 min), S-allylthio-glutathione (GSSA: mw 379, Rt: 5.2 min) free products derived from each respective form. Since papain has only captopril: (CPSH: mw 217, Rt:13.0 min) as well as the starting one free sulfhydryl group, the modification results in the formation materials (Table 2B). The products that appear initially are GSSA of either S-allylthio-papain (additional mass of 73 Da, with or and CPSH. At a later stage GS-S-CP, GSSG and allylmercaptan (ASH) without Naþ) or S-glutathionyl papain (additional mass of 306 Da, are formed. In this reaction, reduced glutathione reacted with both with or without Naþ)(Table 1). moieties of S-allylthio-captopril to yield at first, S-allylthio-gluta- Papain was also reacted with S-allylthio-captopril (CPSSA). Mass thione (GSSA) and captopril (CPSH). Only at later stages do these spectra of the products indicated that two distinct modifications intermediates react with one another to yield the final product, had occurred; S-captropril-papain (23 643 kDa, additional mass of S-glutathionyl-captopril (GS-S-CP). 215 Da) and S-allylthio-papain (23 506, 23 531 kDa, additional mass The reaction is pH dependent and at pH 8.4 the maximal yield of of 73 Da). GS-S-CP, GSSA and CPSH were observed after 25 min at room Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) is temperature. To obtain more precise values of reaction rates and a protein containing a reactive sulfhydryl group and known to intermediate analyses, the reaction was performed at pH 6.5. The undergo S-glutathionylation under conditions of oxidative stress overall reaction is shown in Scheme 4. [17]. After incubation of the enzyme with a ten-fold molar excess of The second step of the above reaction was deduced from the GSSA at pH 7.4, the products were analyzed by HPLC and ESI-MS. kinetics of product formation at pH 6.5 as shown in Fig. 2. The presence of roughly equivalent amounts of unmodified G3PDH As CPSH reacted with GSSA (at equimolar ratios of approxi- (35 778 Da) as well as modified S-glutathionylated G3PDH mately 9 mM, pH 6.5 at room temperature), the products GSH and (36 084 Da) were obtained without substantial loss of enzymatic GS-S-CP formed after 2 min and their amounts increased for 80 min, attaining steady state levels for up to 20 h. About 60% of the starting material (GSSA) was converted to GSH after 80 min, which indicates that S-glutathionyl is the preferred leaving group. The formation of CPSSA was observed after 20 min and increased with time, reaching its maximal amount after 2.5 h. After 20 h, CPSH and GSSA (the starting materials) could not be detected and the reac- Scheme 2. The reaction of allicin with GSH. tion mixture contained GSH (6.3 mM), GS-S-CP (2.8 mM) and CPSSA 1914 T. Miron et al. / European Journal of Medicinal Chemistry 45 (2010) 1912–1918

Scheme 3. General synthesis of S-allylthio-derivatives.

(4.1 mM), that is 70%, 31% and 46% from each of the starting reac- isosbestic points revealed the presence of two distinct compounds tants, respectively (Fig. 2). The oxidized forms, GSSG and captopril in solution without any indication of intermediate products. disulfide (CPSSCP) were also observed in the reaction mixture at Further evidence was obtained from ESI-MS analysis, which indi- room temperature after 20 h at pH 8.4. cated the presence of only 6-MPR and GSSA. SA-6MP [19] reacted with GSH in a similar manner, the only products being GSSA and 6-MP, but this reaction occurred at 3.4. Reaction of S-allylthio-6-mercaptopurine riboside (SA-6MPR) a faster rate. In both cases the leaving groups are 6-MPR or 6-MP and S-allylthio-6-mercaptopurine with GSH and the mixed disulfide formed is GSSA. Scheme 5 describes the products formed by the reaction of SA-6MP with GSH. GSH and SA-6MPR [19]were mixed at equimolar concentrations (7 mM, in 50 mM phosphate buffer, pH 7.2 at room temperature). The reaction was very rapid and after 10 min, SA-6MPR dis- 3.5. Reaction of S-allylthio-6-mercaptopurine (SA-6MP) and 2- appeared. The products were identified by HPLC and ESI-MS anal- nitro-5-thiobenzoate (NTB) ysis. Optical spectra of the reaction mixture showed a shift of the absorption maximum from 284 nm (SA-6MPR) to 324 nm (6-MPR) The high activity of SA-6MPR and SA-6MP was also demon- (Fig. 3). In order to decrease the reaction rates, the reaction was strated by their reaction with 2-nitro-5-thiobenzoate (NTB) [20].In performed at pH 6.0. Samples from the reaction mixture were taken this case the mercaptopurine is the leaving group and S-allylthio- at different time points, diluted with 50% ethanol and spectra were NTB is formed (Scheme 6). measured. Based on the absorbance decrease of NTB in this reaction, we After 30 min, a peak of absorbance at 324 nm indicated the developed a spectrophotometric assay to determine the concen- 1 1 presence of 6-MPR, whereas no SA-6MPR could be detected. The tration of SA-6MP and SA-6MPR (e412 14 150 M cm ) (unpub- lished data).

4. Discussion

Allicin reacts with free thiol groups of proteins and GSH. During uptake by cells some of it reacts with thiol containing membrane proteins [21] but the major product is GSSA. The life span of allicin in cells is short due to its volatility and instability. To bypass this problem we devised cell penetrable S-allylthio disulﬁde-derivatives

Table 1 ESI-MS data of papain modiﬁed with GSSA and with CPSSA.

Papain Delta MW Exp MW MS observed Papain (SH)a 0 23 428 Papain (SH)b 0 23 458

Papain/GSSA S-allylthio-papain (a) 73 23 501 23 506 S-allylthio-papain (b) 73 23 531 23 531 S-allylthio-papain (b)/Naþ 96 23 554 23 554 S-glutathionyl papain (b) 305 23 763 23 763 S-glutathionyl papain (b)/Naþ 328 23 786 23 789

Papain/CPSSA S-allylthio-papain (a) 73 23 501 23 506 Fig. 1. Chromatographic pattern of Radioactivity (C) and protein concentration (B)of S-allylthio-papain (b) 73 23 531 23 531 modiﬁed papain. Chromatography was carried out on PD-10 column equilibrated with S-captopril-papain (a) 215 23 643 23 643 50 mM Na acetate, 2 mM EDTA pH 6.2. Fractions volume 0.8 mL. T. Miron et al. / European Journal of Medicinal Chemistry 45 (2010) 1912–1918 1915

Table 2A increasing with the increase in the basicity and the nucleophilic Mass and retention time of various glutathionyl and captopril derivatives. capacity of the thiolate anion. The nature of the leaving thio moiety Compound GSSG GSH GS-S-CP GSSA CPSH CPSSA depends on the substituent involved. MS (Da) 612 307 522 379 217 289 Analysis of the reactions between S-allylthio-derivatives with HPLC Rt (min) 4.1 4.3 4.6 5.2 5.8 13.0 either reduced glutathione or with model proteins containing a reactive sulfhydryl group provided information on the process of intermediate and ﬁnal product formation. Table 2B The S-allylthio-mixed disulﬁdes used contained an S-allyl group Time-scale evolution of products in the reaction of GSH with CPSSA (mM). and one S-aliphatic or S-aromatic moiety. In the case of gluta-

Reaction GSSG GSH GS-S-CP GSSA CPSH CPSSA thionyl, cysteinyl or captopril as the S-aliphatic moieties, the time (h) reaction with sulfhydryl groups of proteins resulted in mixed 0 10.0 0.3 9.5 0.4 disulfide mixtures that are formed by both the S-allyl as well as by 0.1 9.3 0.3 0.5 0.1 0.4 0.2 9.0 0.4 the aliphatic moieties, as exemplified by the reaction of GSSA with 0.3 7.5 0.4 1.2 0.1 0.9 0.1 7.2 0.1 papain and PTP1B. The reaction between GSSA and papain yielded 0.6 7.1 0.2 1.5 0.3 1.1 0.1 1.3 0.1 6.7 0.1 S-allylthio-papain and Glutathionyl-S-S-papain. The modification 1.0 7.0 0.2 2.0 0.3 2.3 0.1 1.9 0.1 5.5 0.2 (loss of enzyme activity) takes several hours. Both products, ASS- 3.0 6.8 0.2 3.5 0.2 2.5 0.1 2.3 0.2 3.1 0.1 20.0 0.3 0.1 6.3 0.2 3.5 0.2 2.1 0.2 2.1 0.1 2.8 0.1 protein and GSS-protein are stable in a cell free system, whereas in cells the reaction is probably reversible due to intervention of Results represent means S.D. of three independent experiments. various enzymes such as thioredoxins and glutaredoxins [22]. The S-allyl mixed disulfide exchange reactions may be repre- (AS-SX) to react with GSH via sulfhydryl-disulfide exchange reac- sented by Scheme 7. tions. Several mercapto containing prodrugs were employed to When S-allyl mercaptocaptopril (CPSSA) reacted with reduced prepare AS-SX derivatives and their interaction with GSH and glutathione (GSH) (equimolar concentrations), all the possible proteins was analyzed in cell free systems. Attempts were made to product combinations (GSSA, CPSH and GS-S-CP) were detected. improve the pharmacological performance of the prodrugs and to The early appearance of GSSA and CPSH indicated that captopril in identify GSSA reaction products, thus enabling the evaluation of the mixed disulfide is a better leaving group than the S-allylthio their potential as new hybrid prodrugs. The poor cell penetrability moiety. Upon longer incubation, GS-S-CP appeared in the reaction of the original drugs was overcome by the addition of a hydro- mixture, reaching its maximal level at 3 h, and staying at that level phobic allylmercapto group. Upon reaction with excess intracellular for up to 20 h. It was also found that GSSA and CPSH, both formed in GSH the desired intracellular GSSA would be formed, while this reaction, proceed to react with, each other, to yield GS-S-CP, releasing the original prodrug inside the cells, thus obtaining which is a stable mixed disulfide in the cell free system. While stage a synergistic effect of the original prodrug and a series of beneficial 1 of the reaction (Scheme 8) showed full mass conservation of the redox reactions. glutathione and the allyl moieties, the recovery of the S-allylthio Reactions between S-allylthio-mixed disulfides and free sulf- moiety at stage 2 was only 50%. The explanation for this loss was hydryl groups are non-enzymatic thiol-disulfide exchange reac- deduced from the strong odor emitted by allylmercaptan, a volatile tions. They usually occur spontaneously at pH > 5.5, reactivity compound formed by this leaving group.

Scheme 4. The reaction of GSH with CPSSA. 1916 T. Miron et al. / European Journal of Medicinal Chemistry 45 (2010) 1912–1918

Fig. 2. Kinetics of product formation in the reaction mixture of GSSA and CPSH at ambient temperature pH 6.5. A. The reactant GSSA (B) and the reaction products GSH (C) and GS- S-CP (-). B. The starting reactant captopril (CPSH (B)) and the reaction products of GS-S-CP (-) and CPSSA (C).

The 2 step reaction of GSH with CPSSA is presented in Scheme 8. the amount of GSH in cells is in large excess (mM range) in relation Thus, the aliphatic moiety of S-allyl mixed disulfides generates to allicin or S-allylmercapto-drugs (mM or nM). various mixed disulfides in the thiol-disulfide exchange reactions, Furthermore, the S-allyl moiety will form intracellular GSSA that whereas the aromatic moiety does not (Scheme 7). In the reactions may continue to modify reactive sulfhydryl groups, yielding various of SA-6MP or SA-6MPR, with an aliphatic free SH such as GSH, products, either small or high-molecular weight mixed disulfides, a very fast release of 6-MP or 6-MPR occurred. The same situation further subjected to mixed disulfide exchange reactions. Addition- applies to the free thiol of the aromatic NTB. 6-MPR and 6MP are ally, the allylmercaptan (ASH) moiety reacts with metalloproteins. In not sufficiently nucleophilic to react with GSSA. the case of histone deacetylase (HDAC), for instance, inhibition of SA-6MP and SA-6MPR are potential anti-cancer prodrugs. The activity was observed due to its binding to zinc in the active site [23]. mode of action of these compounds with free sulfhydryl groups The rest of the allylmercaptan is released by evaporation (Scheme 9). suggests that these promising lipophilic prodrugs, upon entering The fact that allicin has never been detected in mammalian the living cells will promptly react with GSH and release the purine blood, urine or stool even following a short period after adminis- moiety inside the cells, where it can act as a purine analogue and tering/consuming large amounts of the purified compound or garlic interfere with DNA synthesis. The allyl moiety contributes to the in its raw form [24,25] can be explained by its immediate conver- lipophilicity of these compounds, and hence to their increased sion into the above described cellular mixed disulfides, GSSA, capacity for cell membrane penetration. The S-allyl modified Protein-S-SA or Protein-S-SG. However, the discovery of active disulfide activity lasts longer as compared to the parent molecule intermediates, even 20 h after reacting allyl derivatives with free (allicin). While this study was performed at molar 1:1 ratios of thiol bearing molecules, point out the promising prolonged activity reactants in order to determine all the intermediates and products, of the hybrid S-allylthio prodrugs in vivo. Not only has the time scale, and the membrane penetrability improved significantly, but the multiple intermediates formed suggest a variety of cellular targets to be affected by these prodrugs.

5. Experimental

5.1. General

Papain (EC 3.4.22.2) was obtained from Worthington (Freehold, NJ). Protein tyrosine phosphatase 1B (PTP1B, (residues 1–321) EC 3.1.3.48) was a gift from Dr. Zhong-Yin Zhang [26]. Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (G3PDH, EC 1.2.1.12), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s Reagent), 6-mercaptopurine (6-MP), captopril, L-cysteine and reduced glutathione (GSH) were purchased from Sigma (St. Louis, MO). Porapak Q (100–120 mesh) was obtained from Waters Associates, (Milford, MA, USA); PD-10 (pre-packed Sephadex G-25) from Pharmacia LKB, Biotechnology, Uppsala, Sweden. Allicin was produced as previously described [27]. Free sulfhydryl groups were 1 1 Fig. 3. Optical spectra of the reaction mixture of GSH and SA-6MPR at pH 6.0. Spectra determined with DTNB [28] by using EM 14 150 M cm at 412 nm were measured at time 0 min (a); 10 min (b); 15 min (c) and 30 min (d). according to Riddles et al. [29]. T. Miron et al. / European Journal of Medicinal Chemistry 45 (2010) 1912–1918 1917

Scheme 5. The reaction of S-allylthio-6-mercaptopurine (SA-6MP) with GSH.

Scheme 6. The reaction of SA-6MP with 2-nitro-5-thiobenzoate (NTB).

Modified papain was subjected to chromatography on a PD-10 Mass spectra of small molecules were analyzed by using column. HPLC fractionation of PTP1B and glyceraldehyde 3-phos- Micromass Platform LCZ 4000, Micromass, Manchester, UK. phate dehydrogenase was done on Vydac C4 reversed-phase Ionization Mode: ESI-ElectroSpray ionization. column (1 250 mm). Protein peaks were eluted using increasing linear gradients of acetonitrile/0.1% TFA (solvent B) at a flow rate of 5.2. General synthesis procedures 0.05 mL/min, and were subjected to ESI-MS analysis. HPLC analyses of low molecular weight compounds were per- Sulfhydryl-compounds (1 mmol) were added to an allicin solu- formed on a LiChrosorb RP-18 (7 250 mm) column, using tion (0.55 mmol, in phosphate buffer, pH 6.5). The reaction was methanol (60%) in water containing 0.01% trifluoroacetic acid, at carried out at room temperature in 50% ethanol for several hours. a flow rate of 0.55 mL/min, and absorbance was recorded at 210 nm. Excess allicin was removed by ether extraction. After solvent removal 1HNMR spectra were measured on a Bruker Avance-500 spec- under reduced pressure, the product was isolated from water. trometer (Bruker, Bremen, Germany). Mass spectra of proteins were analyzed by using ion electrospray ESI-MS. MALDI-TOF data were 5.2.1. Allicin [27](compound 1) 1 collected on a Bruker Reflex IIIÔ MALDI-TOF mass spectrometer H NMR (400 MHz, D2O) d in ppm: 6.07 (m, 1H), 6.03 (m, 1H), (Bruker, Bremen, Germany) equipped with a delayed extraction ion 5.55 (dq, 2H), 5.37 (dq, 2H), 3.97 (dq, 2H), 3.85 (m, 2H). source, a reflector and a 337 nm nitrogen laser, and on an API Q-STAR Pulsari Electrospray-Quadrupole TOF tandem mass spec- 5.2.2. S-allylthio-glutathione (GSSA) [2,15] (compound 2) 1 trometer (MDS-Sciex, Canada, ABI) equipped with a nano- H NMR (400 MHz, D2O) d in ppm: 5.88 (m, 2H), 5.88 (m, 1H), 5.2 electrospray source (MDS Proteomics, Odense, Denmark). (m, 2H), 4.72 (dd. 1H), 3.76 (d, 2H), 3.74 (t, 1H), 3.36 (dd, 2H), 2.52 (q, 2H), 2.14 (q, 2H). MS (EI): [M þ 1] at m/z 380.

5.2.3. S-allylthio-captopril (CPSSA)[18] (compound 3) 1 H NMR (300 MHz, CDCL3) d in ppm: 5.80 (m, 1H), 5.15 (m, 2H), 4.55 (m, 1H), 3.64 (t, 2H), 3.29 (d, 2H), 3.03 (m, 2H), 2.64 (m, 1H), 2.44, 2.20 (m, 4H), 1.18 (d, 3H). MS (EI): [M þ 1] at m/z 290.

5.2.4. S-allylthio-6-mercaptopurine riboside (SA-6MPR) [19] (compound 4) 1 H NMR (500 MHz, CDCL3) d in ppm: 8.87 (s, 1H), 8.10 (s, 1H), 5.88 (d, 1H) 5.87 (m, 1H), 5.12 (m, 2H), 5.12 (m, 1H), 4.55 (d, 1H), 4.39 (s, 1H), 390, (dd, 2H) 3.56 (d, 2H), MS (EI): [M þ 1] at m/z 357.

5.2.5. S-allylthio-6-mercaptopurine (SA-6MP) [19](compound 5) 1 H NMR (500 MHz, CDCL3) d in ppm: 12.19 (s, 1H), 8.96 (s, 1H), Scheme 7. Hypothetical products of the reaction between S-allylthio-mixed disulﬁde 8.30 (s,1H), 5.91 (m,1H), 5.14 (m, 2H), 3.61 (d, 2H). MS (EI): [M þ 1] and free SH compounds. at m/z 225. 1918 T. Miron et al. / European Journal of Medicinal Chemistry 45 (2010) 1912–1918

Scheme 8. The S-allyl mixed disulﬁde exchange reactions (a 2 step general scheme).

quantitatively by HPLC separation at different time periods and their molecular weight was determined by (ESI-MS).

Acknowledgement

This work was supported by grants from La Foundation Raphael et Regina Levy and the Atran Foundation.

References

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