REACTION OF SCHIFF BASES WITH THIOGLYCOLIC ACID: SYNTHESIS OF THIAZEPINE-1(2H)-ONE AND THIAZOLIDINE-4-ONE COMPOUNDS

Reaction of Schiff bases with thioglycolic acid was set up with the Dean stark apparatus in toluene. The Schiff bases of aniline (1a) and p-bromoaniline (2a) gave 2-(4hydroxy-3-methoxy-5-nitrophenyl)-3-phenylthiazolidine-4-one (1b) and 3-(4-bromophenyl)2-(4-hydroxy-3-methoxy-5-nitrophenyl)thiazolidine-4-one (2b) only; 1-naphthyl amine (3a) gave a mixture of 2-(4-hydroxy-3-methoxy-5-nitrophenyl)-1,2-dihydronaphtho[1,2d][1,3]thiazepine-5(4H)-one (3b) and 2-(4-hydroxy-3-methoxy-5-nitrophenyl)-3-(naphthalen1-yl)thiazolidine-4-one (3b’) compounds; 2-naphthyl amine (4a) gave 4-(4-hydroxy-3methoxy-5-nitrophenyl)-4,5-dihydronaphtho[2,1-d][1,3]thiazepine-1(2H)-one (4b). The yields of the reaction were moderate to high. The structures of these compounds were elucidated using 1H-NMR and 13C-NMR and mass spectral analysis. The biological test showed that Schiff base 4a was not active on any bacteria. Thiazolidine-4-one compound 1b was not active on bacteria and fungi but was active against cancer cell line KB with IC50 21.33 g/mL. Thiazepine compound 4b exhibited an activity on Staphyloccccus aureus bacterium with IC50 64.00 g/mL and KB with IC50 11.52 g/mL.

According to the literature, the synthesis of the thiazepine-1(2H)-one ring was not reported.In this paper, we reported the initial study on using the reaction of a Schiff base with thioglycolic acid to obtain both thiazolidine-4-one ring and thiazepine-1(2H)-one ring (Scheme 1).

Experimental section
Solvents and other chemicals were purchased from Sigma-Aldrich, Merck and were used as received, unless indicated.The 1 H-NMR and 13 C-NMR spectra were recorded on the Bruker Avance 500 NMR spectrometer in DMSO-d6.The chemical-shift data for each signal were reported in ppm units.Mass spectra were obtained from Mass Spectrometry Facility of Vietnam Academy of Science and Technology on an LC-MSD-Trap-SL spectrometer.
The mixture was washed with a 3% NaHCO3 solution (3 × 10 mL) and brine.The organic layer was dried over Na2SO4 then concentrated in vacuo.The products were re-crystallized in ethanol.

Bioactivity test
The bioactivity tests were performed using the Broth dilution method [8].Compounds 1b, 4a, and 4b were selected for the bacterial test including Gram (+) (Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum), Gram (-) (Salmonella enteric, Escherichia coli, Pseudomonas aeruginosa), fungal test (Candida albicans), and cancer cell line KB.All tests were screened in the Laboratory of Applied Biochemistry of Vietnam Academy of Science and Technology.

Synthesis
The synthesis of the Schiff bases was conducted following the procedure reported by our group [9].The Schiff bases reacted with thioglycolic acid in the same conditions.The formation of thiazolidine-4-one was explained by Islooret al. [4] in which the attack of the N-nucleophile to the carbonyl group of the carboxylic acid is the key step to form the thiazolidine-4-one ring.In the cases of Schiff bases 1a and 2a, only the formation of the thiazolidine-4-one ring was observed.Meanwhile, the formation of both thiazolidine-4-one and thiazepine-1-one was observed in the case of Schiff base 3a.In contrast, in the case of Schiff base 4a, only thiazepine-1one was formed.
This was the results of different nucleophiles attacking the carbonyl groups as an electrophilic center (E + ) (Fig. 1).If nitrogen attacks the carbonyl group, the reaction will form the thiazolidine-4-one ring (cases a and b).On the other hand, if C (case b) or C (case c) attack the carbonyl group, the reaction will form the thiazepine ring.The mechanism of the formation of thiazepine-1-one 4b is shown in Scheme 2. The >C=Ngroup was first activated by a proton of thioglycolic acid to form intermediate I that was then attacked by the nucleophilic sulfide to form intermediate II.Next, the electrophilic substitution at C of the naphthyl group took place to form intermediate III, followed by the substitution of a proton at C, and finally, a molecule of water was eliminated to form thiazepine-1-one 4b.
According to the observations above, therefore, the nucleophility of carbons at the ortho position of aniline and vanillin parts might be weaker than that of nitrogen in the cases of 1a and 2a (Picture a in Fig. 1).So, the nucleophile nitrogen attacking the carbonyl group was preferred to form thiazolidine-4-one only.In the case of 3a, the mixture of products was recorded by 1 H-NMR spectrum since all the purification methods failed.It was found that the ratio of thiazepine/thiazolidine was 2.5/1.0 from the signals of H8 in 3b and 3b' (Fig. 2).This result revealed that the formation of thiazepine was more favourable than that of thiazolidine, but both could be formed.In the case of Schiff base 4a, the C was the best nucleophile [10], hence, the attack to the carbonyl group was the easiest way to form thiazepine product 4b (Fig. 1(c) and Scheme 2).

Structure determination
The mass spectrometry data of compounds 1b, 2b and 4b agreed with the expected structures.For example, +MS of compound 1b had a peak at m/z 347 indicating that the molecular formula of 1b must be C16H14N2O5S with a molecular weight of 346 g/mol.+MS of compound 2b also gave pseudo molecular peaks at m/z 425 au and m/z 427 audue to isotopes 79 Br and 81 Br that agreed with molecular formula C16H13BrN2O5S.+MS of compound 4b showed a base peak at m/z 397 au and -MS showed a base peak at m/z 395 au indicating that the molecular weight of compound 4b was 396 g/mol matching molecular formula C20H16N2O5S (see experimental section).
The IR spectra of all compounds showed the vibration of O-H bond at about 3200 cm -1 (br), C-H bond in the region of3100-2800 cm -1 ; >C=O bonds at about 1669-1675 cm -1 and >C=C<,>C=N-bonds in the region of 1500-1610 cm -1 .Especially, the IR spectrum of compound 4b had a vibration at 3231 cm -1 indicating the stretching vibration of the N-H bond which was different from that of thiazolidine compounds 1b and 2b.

Fig. 3. Comparisonof H8 and H9 of 1b and 4b
The 1 H-NMR and 13 C-NMR spectra of compound 4b did not have H8 and H9 peaks as in 1b (Fig. 3).They moved into a stronger field: H8 was a singlet peak at 5.83 ppm that had a cross peak with C6 at 115.9 ppm, C2 at 116.6 ppm, C10' at 125.5 ppm, C1 at 128.1 ppm, C1' at 130.7 ppm, C2' at 132.9 ppm on the HMBC correlation spectrum.Two protons H9 were at 3.00 ppm (d, J = 12.0 Hz) and at 2.96 ppm (d, J = 12.0 Hz) that had the cross peaks with C8 at 46.6 ppm and C10 (>C=O) at 169.0 ppm.In addition, there were 8 aromatic protons belonging to the benzene and naphthalene rings in 4b compared with in 4a.This indicated that the electrophilic aromatic substitution happened at C2 (C) of the naphthyl group where the richest electron area was and made a seven-member thiazepine ring.Another peak at 9.9 ppm (s, 1H) did not attach to any carbons (no cross peak in HSQC), but it had a cross peak with C9 and C1', therefore, it had to be the proton on a nitrogen.The other protons and carbons were assigned and shown in Table 1 and Fig. 4. For example, H2 was at 7.36 ppm as a singlet peak correlating with C6 at 115.9 ppm, and so on.

Bioactivity evaluation
The bioactivity test results are shown in Table 2. Schiff base 4a was not active on any bacteria.Thiazolidine compound 1b was not active on bacteria and fungi but was active against cancer cell line KB with IC50 21.33 g/mL.Thiazepine compound 4b exhibited an activity on Staphyloccocus aureus bacteriumwithIC50 64.00 g/mL and KB with IC50 11.52 g/mL.

Fig. 4 .
Fig. 4.NMR analysis of compound 4b: a part of HSQC spectrum (left); a part ofHMBC spectrum (middle) and a part of MS spectrum (right)