Synthesis and antifungal activity of some 2-benzothiazolylthioacetyl amino acid and peptide derivatives 1

A series of benzothiazolylthioacetyl amino acid and peptide derivatives including glycoside, hydrazide and hydrazone moieties was synthesized with the aim of evaluating their antifungal activity. Their chemical structures were confirmed by 1 H-NMR, IR, mass spectrometry and elemental analyses. Out of the thirty two tested compounds, three derivatives have high activity as fluconazole at 100 ppm and six at 1000 ppm toward Candida albicans , whereas they were inactive toward Aspergillus flavus .


Introduction
[13][14] [B011] These observations prompted us to undertake systematic study of the synthesis and antifungal evaluation of some mercaptobenzothiazolyl amino acid and peptide derivatives.The 2-mercaptobenzothiazole derivatives were synthesized as shown in schemes 1 and 2. The selection of the amino acids L-serine, L-tyrosine and DL-threonine is based on their presence as major constituents of the antifungal antibiotics iturins A and D, bacillomycins L, D and F and mycosubtilin. 11In addition L-hydroxyproline is found in the antifungal antibiotic echinocandin B 15 whereas L-methionine is one of the constituent amino acids of the antifungal compound AK-3, isolated from Synechocystis sp. 16

Chemistry
In the present study, 2-benzothiazolylthioacetyl amino acid ester derivatives 2a-d and 3 were prepared from hydrazide 1 17 via the racemization-free azide coupling method. 18,19 drazide 1 was treated with nitrous acid (NaNO 2 / HCl) in strong acid medium at low temperature.The resulting azide is unstable at higher temperature, so it was extracted by cold ethyl acetate, neutralized and washed also at low temperature.
The azide solution in ethyl acetate reacted with amino acid methyl esters hydrochloride, previously treated with triethylamine in ethyl acetate at low temperature, to produce 2-benzothiazolylthioacetyl amino acid ester derivatives 2a-d and 3 in good yields and their chemical structures were confirmed by 1 H NMR and elemental analysis.The 1 H NMR spectra showed, doublet signal at 8.3 ppm for the NH proton of the peptide bond (except the hydroxyproline derivative 3), multiplet signals between 6.8 and 8.0 ppm for the four aromatic protons, multiplet signal at 4.5-5.0ppm for α-CH proton of the amino acids and singlet signal at 3.6 ppm for the three protons of OCH 3 of the ester groups.Also, the geminal coupling between the two protons of the thioacetyl group SCH 2 CO appears between 3.8 -4.3 ppm as two doublet signals.The other signals for the amino acid side chains are reported in the experimental part.
The dipeptide derivatives 6a-g and 7a, b were prepared from their corresponding amino acid ester derivatives 2a-d and 3 after conversion to hydrazides 4a-d and 5 by boiling with excess hydrazine hydrate in methanol.The 1 H NMR, IR spectra and elemental analyses of 4a-d and 5 confirmed their structures as shown from the data reported in the experimental part.The dipeptide derivatives 6a-g and 7a, b were obtained by the azide-coupling method in 30-43 % yields (Scheme 1).The 1 H NMR spectra revealed, two doublets in the range 7.5 -8.5 ppm for the two NH protons of the peptide bonds, other two multiplets in the range 4.0 -5.0 ppm for the α-CH protons of the two amino acids, in addition to other several signals corresponding to protons of the individual side chains.
Scheme 1: Synthesis of benzothiazolylthioacetyl amino acid and peptide derivatives.
Acetylation of the hydroxyl group of the amino acid derivatives 2a, b, and d with acetic anhydride in the presence of pyridine failed to give pure products.However, exceptionally compound 3 gave the acetyl derivative 8 in good yield, which was easily purified by crystallization from ethyl acetate / petroleum ether.The 1 H NMR spectrum of 8 showed a singlet signal at 2.02 ppm for the three protons of the acetyl group COCH 3 .
Next, our target was the synthesis of the glycopeptides 10a,b by the procedure reported by Schmidt et al. 20 Glycosylation of 2d and 3 as an alcohol-acceptor precursor, with O-(2:3,5:6-di-O-isopropylidene-α-D-mannofuranose)trichloroacetimidate (9) as donor precursor in the presence of catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTF) as Lewis acid afforded 10a,b in 35.8 % and 28 % yield respectively.The O-glycofuranosyl trichloroacetimidate 9 is characterized by its stability at room temperature for long periods and gives the α-anomer derivatives (Scheme 2).The structures of 10a,b were identified from the 1 H NMR and elemental analysis.In the 1 H NMR spectra the sugar protons appeared as singlet at 5.34 ppm for H-1 of 10a and at 5.09 ppm for H-1 of 10b, confirming the α-anomer of the glycosides 10a,b.The doublet at 4.59 ppm (J = 5.8 Hz) was attributed to H-2, whereas multiplet at 4.78-4.71ppm assigned for H-3.The singlet at 3.68 ppm and the four singlet signals at 1.41, 1.33, 1.29 and 1.22 ppm were assigned to OCH 3 and 4 CH 3 of isopropylidene groups respectively.
Reaction of the hydrazide 4c with diethyl malonate led to the formation of the linear hydrazino ethyl acetate derivatives 12. 1 H NMR spectra of the product showed singlet signal at 3.25 ppm for (-CO-CH 2 -CO-), multiplet signal at 4.12-4.06ppm and triplet signal at 1.16 ppm for OCH 2 CH 3 group indicative of the formation of linear product 12 rather than the cyclic one 11.Trials to obtain the corresponding pyrazolidinedione derivative 11 either by fusion of 12 or by heating in DMF were unsuccesful.
Hydrazone derivatives have gained importance due to their application in pharmaceutical chemistry.The biological activity associated with these compounds was attributed to the presence of the (-CONHN=CH-) moiety. 21In the present study, some hydrazones have been synthesized by refluxing the hydrazides 4c,d with different carbonyl compounds such as isatin, p-nitrobenzaldehyde, anisaldehyde, vanillin and benzene-1,4-dicarbaldehyde.The structures of the hydrazones 13a,b, 14a-f and 15 were investigated by IR, 1 H NMR, mass spectrometry and elemental analyses.(See the experimental part).The 1 H NMR spectra of hydrazones 14a, 14b and 14c (Table 1) revealed the existence of a mixture from E-and Z-stereoisomers (Fig. 1) in which the Eform predominates.This conclusion is supported by a previously published data on similar compounds. 22,23 Fig.

Biological evaluation
The antifungal activities of the synthesized compounds were measured using the well technique of the agar diffusion method with comparison to fluconazole and 2mercaptobenzothiazole MBT.All the tested compounds were screened for antifungal activities in vitro against Aspergillus flavus as a representative of mold fungi and Candida albicans as a representative of yeasts.The recorded data, [Tables 2 & 3], lead to the following conclusions: − None of the synthesized and tested compounds had comparable activity to fluconazole towards both Aspergillus flavus and Candida albicans.− All the synthesized and tested benzothiazolyl derivatives were devoid of antifungal activity (except 10a) against the mold Aspergillus flavus.− It is evident that Candida albicans is more sensitive to benzothiazolyl amino acid and dipeptide derivatives than Aspergillus flavus.− It is clear that out of the thirty two tested compounds three derivatives have enhanced activity as fluconazole at 100 ppm and six at 1000 ppm toward Candida albicans.
Table2: The antifungal effects of the synthesized compounds against mold (Aspergillus flavus) and yeast (Candida albicans) fungi indicated by the diameter of the inhibition zones (well method). Compd.

Aspergillus flavus
Candida albicans − The tyrosine and O-acetyl hydroxyproline methyl ester derivatives 2a and 8 respectively were the most potent compounds against Candida albicans among the tested benzothiazolyl derivatives.− Conversion of the benzothiazolyl amino acid ester derivatives to the corresponding hydrazides does not improve appreciably their antifungal activity with exception of the methionine derivative 4c.− Hydrazone derivatives showed higher activity than the corresponding hydrazides especially these of isatin 13a, b and methionine 14a, c. − In general all the tested dipeptide derivatives were actually inactive with exception of 7a, b, which showed slight activity against both Candida albicans and Aspergillus flavus.
1: E-and Z-Forms of Compounds 14a-c Table1: Data of 1 H NMR Spectra of Compounds 14a-c