New aspects of the formation of 2-substituted thiazolidine-4-carboxylic acids and their thiohydantoin derivatives

Aromatic aldehydes reacted readily with ( R )-cysteine in boiling acidified methanol to give diastereomeric mixtures of the corresponding 2-(aryl substituted) thiazolidine-4-carboxylic acids. 4-Nitrobenzaldehyde under similar conditions afforded one isomer of 2-(4-nitrophenyl)- thiazolidine-4-carboxylic acid, which epimerized in the NMR solvents into a diastereomeric mixture. 2-Nitrobenzaldehyde reacted with ( R )-cysteine to afford 3,5-bis-(2-nitrophenyl)- tetrahydro-1 H -thiazolo[3,4-c ]oxazol-1-one as the sole product, which collapsed in the NMR solvent into a diastereomeric mixture of the thiazolidine-4-carboxylic acids. The thiazolidine derivatives reacted smoothly with phenyl isothiocyanate to give single isomers of the corresponding thiohydantoins.


Introduction
3][4][5][6][7] TCDs exhibit a broad spectrum of anticancer activities [8][9][10][11][12] against, e.g.liver, 13,14 breast, [13][14][15] colon, 13,16 prostate, 17 endometrial 13 and melanoma 17 cell lines.In the same vein, TCDs are used as, e.g.antimalarial, 18,19 selective Na + /Ca 2+ exchange inhibitors, 20 immunostimulating agents, 21 tyrosinase inhibitors, 22 influenza A neuraminidase inhibitors, 23 antitubercular, 24 HIV-1 protease inhibitors 2,25 and ACE inhibitors. 26Furthermore, TCDs were introduced as versatile scaffolds in the syntheses of natural products, i.e. (+)-biotin, 27,28 (+)-lyngbyabellin M, 29 (+)-hyalodendrin, 30 prepiscibactin 31 and dehydroluciferin. 32Glutathione is a tripeptide works as a cellular protective agent and cannot be used as a drug because it does not enter the living cells. 33The vital amino acid building block in the in vivo synthesis of glutathione is (R)-cysteine, which cannot be supplemented directly due to toxicity and instability problems. 33,34TCDs work as (R)-cysteine prodrugs 33,35 which metabolize intracellularly to release (R)-cysteine to stimulate glutathione synthesis.Straightforward formation of TCDs opens the door for this process to be used for detection of cysteine amino acid and Nterminal cysteines 36 and also for estimation/binding of aldehydes. 37he aim of this work is to use acidified methanol as a solvent in the preparation of some TCDs and to shed some light on the mechanism of their formation, and their conversion into the corresponding diastereospecific thiohydantoins.

Results and Discussion
The reaction of (R)-cysteine (1), salicylaldehyde (2a) and N-phenylmaleimide (3) (NPM, a reactive dipolarophile) in acidified methanol under reflux failed to give the expected cycloadduct 6, 38 and instead gave a 98% yield of the well known (2RS)-(2-hydroxyphenyl)thiazolidine-(4R)carboxylic acids (7a) and (8a) in a 1.5:1 ratio, respectively.It is worth mentioning that using acidified methanol gave a better yield and an opposite diastereomeric ratio compared to the previously reported results 39 (Table, entry a).This result encouraged us to study the diastereoselectivity in the formation of 2-(substituted)thiazolidine-4-carboxylic acids by using our conditions.

Scheme 1. The competition between cycloaddition and cyclization.
It seems that the Schiff's base 4 undergoes cyclization in such case much faster than the generation of azomethine ylide 5 and this could be mainly attributed to the bigger size and softer nature of the sulfur atom.The reaction starts by the formation of imine 4 which suffers an intramolecular nucleophilic attack by the thiol group from either side to furnish a diastereomeric mixture of 2-(substituted)thiazolidine-4-carboxylic acids. 40,41The isomerization at C-4 of the thiazolidine via deprotonation/protonation is ruled out based on spectroscopic data (vide infra).It is believed that, under our conditions, the generation of azomethine ylide has not been formed and hence the chirality at C-4 remained unchanged.) 100:0 iii (1.9:1, 42 1:19 43 ) 1 H-NMR spectra were recorded in: (i) CDCl3/TFA, (ii) DMSO-d6, (iii) CDCl3/CD3OD Furthermore, refluxing an equimolar mixture of (R)-cysteine (1) and benzaldehyde (2c) as the carbonyl component in acidified methanol gave an 88% yield of 2-phenylthiazolidine-4-carboxylic acid (7c) and (8c) as an isomeric mixture in a 2:1 ratio, respectively (Entry c).The pure configuration of both diastereomers 7c and 8c was assigned on the basis of the experimental NOE data which supported the relationship between the substituents at C-2 and C-4 to be cis-7c and trans-8c.Thus, in the case of 7c, irradiation of 2-H gave 3.8 and 3.7% enhancement for 4-H and 5-Ha protons, respectively.Also, irradiation of 4-H showed 3.1 and 1.4% enhancement of 2-H and 5-Ha protons, respectively.However, in the case of 8c, irradiation of 2-H and 4-H protons gave no effect across the ring.
Aryl aldehydes 2d-g reacted with (R)-cysteine (1) under the same conditions to produce diastereomeric mixtures of 2-substituted thiazolidine-4-carboxylic acids 7d-g and 8d-g in 75-93% yield (Entries d-g).It is worth noting that the diastereomeric ratio depends on the NMR solvent.Thus, the 1 H-NMR spectrum of the isomeric mixture 7f and 8f in CDCl3/CD3OD showed a 2.5:1 ratio, respectively, whereas in CDCl3/TFA, the isomeric ratio of 7f and 8f was 1.4:1, respectively.This result also confirmed that the epimerization process at C-2 is solvent and pH dependent. 44,45he recorded NOE data of 7f and 8f, yet again, supported the cis-relationship in the case of the major isomer 7f and trans-relationship of the minor isomer 8f (see experimental).Surprisingly, the use of 4-nitrobenzaldehyde (2h) as the carbonyl component lead to the formation of 2-(4-nitrophenyl)thiazolidine-4-carboxylic acid (7h) as the only product in an 89% yield (Entry h).The stereochemistry of 7h was established on the basis of its spectral data (see experimental and chart 1a).Standing compound 7h in the NMR solvent (CDCl3/CD3OD) for 20min, 7h and 24h at room temperature gave an isomeric mixture of 7h and 8h in 21:1, 3:1 and 1.4:1 ratios, respectively (Chart 1b-1d).Extending the time for 48h (Chart 1e) under the same conditions afforded a 1:1 ratio and no further change was noticed even after one week.It is believed that the obtained isomer 7h in solution would be in equilibrium with Schiff's base 4h, which suffers a nucleophilic attack by the thiol group from the opposite side to give the other cyclization product 8h (Scheme 2).Repeating the 1 H-NMR experiment for compound 7h in a different solvent (CDCl3/TFA) showed immediately an isomeric mixture of 7h and 8h in a 2:1 ratio, respectively.It is obvious that the presence of TFA accelerates the epimerization rate, and the unshared pair of electrons on the thiazolidine nitrogen drives the epimerization process.On the other hand, (R)-cysteine (1) reacted with 2-nitrobenzaldehyde (2i) as the carbonyl component under the same conditions to give a 46% yield of 3,5-bis(2-nitrophenyl)tetrahydro-1Hthiazolo [3,4-c]oxazol-1-one (9) as the sole product (Scheme 3).The obtained bicyclic compound is attributed to the high reactivity of the aldehyde and the steric congestion, which facilitate the formation of the second oxazolone ring.
0][41][42][43] It seems that using our optimal conditions in the synthesis of 2-(substituted)thiazolidine-4-carboxylic acids affects both the experimental yield and diastereomeric ratio (Table ).Thus, using such conditions afforded diastereomeric mixtures of 7 and 8 in 75-98% yields, and the cis-diastereomer 7 was obtained as the major product in all cases.Interestingly, the cis-diastereomer 7h was obtained as the sole product, and it was also lucky for us to separate thiazolo [3,4-c]oxazol-1-one 9 as the only product with no trace of the thiazolidine derivatives.Thiohydantoins are common skeletons in organic synthesis and have a broad spectrum of biological applications. 46,47Annulation of hydantoin ring to thiazolidine derivatives showed anticancer activity (modulator of P53 activity) 11 and assumed to treat Alzheimer via interaction with amyloid β peptide (Aβ 25-35). 48It seemed to us that annulation of thiohydantoin moiety to thiazolidines might have biological applications.
Lalezari 49 and Balalaie 50 have reported two different methods for obtaining 6-phenyl-5thioxotetrahydroimidazo[1,5-c]thiazol-7-one (12b) in 86 and 97% yields, respectively.However, the same product was formed in 95% yield by using our simple and convenient procedure.Thus, stirring an equimolar mixture of thiazolidine-4-carboxylic acid (7b) and phenyl isothiocyanate (11)  in acidified methanol at room temperature gave 12b (Scheme 5).The stereochemistry of 12b was assigned according to its spectral data and by comparison with the previously reported data. 50heme 5. Formation of thiohydantoins 12.  Similarly, the reaction of an isomeric mixture of 7c and 8c (R = Ph) with 11 gave 3,6-diphenyl-5-thioxo-tetrahydro-imidazo[1,5-c]thiazol-7-one 12c as the only product in a 97% yield.However, the diastereomeric mixtures of 7d-h and 8d-h reacted in the same manner with 11 to afford thiohydantoins 12d-h as the only products in excellent yields (91-99%).The stereochemistry of these thiohydantoins 12 was confirmed based on the NOE data of 12c,f (see experimental).Thiohydantoins 12 are stable and no sign of isomerization at C-3 was observed and this could be mainly attributed to the presence of thioxo-group adjacent to N-4.Our results comply with the literature work, which showed that the N-protection of thiazolidine-4-carboxylic acid prevented the epimerization at C-2. 36 The possible mechanism for thiohydantoins 12 started through the preferential attack of the thiazolidine nitrogen of 7 on phenyl isothiocyanate (11) from the opposite face to give the favourable thiourea derivatives 13 (K1>>K2) (Scheme 6). 40,51However, such conformation of 13 prevents the formation of cis-thiohydantoins 14 and alternatively, the intermediate 15 would consequently lead to the trans-thiohydantoins 12.
Györgydeák et al., 52 reported a different mechanism for the synthesis of thiohydantoin-fused thiazolidines, assuming a break-down of C-2-N-3 bond in the thiourea intermediate to produce a ring open intermediate with a positively charged sulfur in order to change the stereochemistry at C-2.However, the same authors have refused a similar mechanism suggested by referees during their early work on N-acetylthiazolidines. 41We believe that our proposed mechanism (vide supra) would serve well to rationalise the stereochemistry at C-3 in the obtained thiohydantoins.Scheme 6. Plausible mechanism for thiohydantoins 12.

Experimental Section
General technical data Thin layer chromatography (TLC) was carried out on aluminium plates pre-coated with silica gel 60 F254 (Merck), and were visualised using ultraviolet light and/or aqueous KMnO4/I2.Proton nuclear magnetic resonance spectra were recorded at 300MHz on Bruker DPX300 and Oxford NMR instruments.Chemical shifts (δ) are reported in parts per million relative to tetramethylsilane (δ = 0.00) and coupling constants are given in hertz (Hz).The following abbreviations are used: s = singlet, d = doublet, br = broad, dd = doublet of doublets, dt = doublet of triplets, m = multiplet, t = triplet, td = triplet of doublets. 13C-NMR spectra were recorded at 75 MHz on a Bruker DPX300 instrument and chemical shifts are reported in parts per million (ppm). 1 H-NMR peak assignments are mainly based on DEPT135, COSY, HMQC and HMBC spectral data.Accurate masses were obtained using a Bruker Daltonics micrOTOF spectrometer.Mass spectra were recorded at 70ev using Shimadzu GCMS-QP1000EX mass spectrometer.The IR spectra were measured on Shimadzu IR instrument.Melting points (m.p.) were determined on a Kofler hot-stage apparatus and are uncorrected.All compounds are named according to the IUPAC system using the ChemBioDraw Ultra 12.0 program.

Method (A): Synthesis of 2-substituted thiazolidine-4-carboxylic acids
Stirring with heating under reflux an equimolar mixture (0.01 mol) of the carbonyl component 2 and (R)-cysteine 1 in acidified methanol (10:0.1 v/v MeOH/AcOH, 10 mL) for an appropriate time.The corresponding thiazolidine derivatives precipitated out of hot solution during the reflux.The solvent was cooled, concentrated and filtered to afford the crude product which was crystallized from aqueous ethanol to give colourless amorphous solid.