Convenient route synthesis of some new benzothiazole derivatives and their pharmacological screening as antimicrobial agents

Cyanoacetylation of uracils or their derivatives [1-4], has been reported to be successfully achieved by heating the respective substrate with a mixture of acetic anhydride and cyanoacetic acid as a cyano-acetylating mixture. The structure of the reactive species formed in this case has been assigned as a cyano-ketene. The use of this cyano-acetylating mixture (cyanoacetic acid and acetic anhydride) has somehow been forgotten and on the other hand less convenient reagents like the pyrazole derivative 1 has been used [5] (Figure 1). The phenolic ester 2 of cyanoacetic acid is also suggested for cyano-acetylation since it generates cyano-ketene when heated [6]. In the last two decades, we have been involved in a program aiming to develop new simple procedures or novel precursors for the synthesis of heterocyclic compounds of biological interest to be evaluated as biodegradable agrochemicals [7-9]. In continuation with this program some heterocyclic compounds containing the benzothiazole nucleus were required for biological activity studies. Therefore, 2-(benzo]d[thiazol-2-yl)3-oxopentanedinitrile (4) was obtained for the irst time by us via cyanoacetylation of 2-cyanomethylbenzothiazole (3) seemed to be a versatile candidate to ful ill this objective (Figure 1).


Introduction
Thiazoles and benzothiazole derivatives represent a well known important group of heterocylic compounds due to their biological and pharmaceutical activities. Benzothiazoles with cyanomethyl group at position-2 have been the subject of extensive study in the recent past. Numerous reports have appeared in the literature, which highlight their chemistry and uses.
Cyanoacetylation of uracils or their derivatives [1][2][3][4], has been reported to be successfully achieved by heating the respective substrate with a mixture of acetic anhydride and cyanoacetic acid as a cyano-acetylating mixture. The structure of the reactive species formed in this case has been assigned as a cyano-ketene. The use of this cyano-acetylating mixture (cyanoacetic acid and acetic anhydride) has somehow been forgotten and on the other hand less convenient reagents like the pyrazole derivative 1 has been used [5] (Figure 1). The phenolic ester 2 of cyanoacetic acid is also suggested for cyano-acetylation since it generates cyano-ketene when heated [6]. In the last two decades, we have been involved in a program aiming to develop new simple procedures or novel precursors for the synthesis of heterocyclic compounds of biological interest to be evaluated as biodegradable agrochemicals [7][8][9]. In continuation with this program some heterocyclic compounds containing the benzothiazole nucleus were required for biological activity studies. Therefore, 2-(benzo]d[thiazol-2-yl)-3-oxopentanedinitrile (4) was obtained for the irst time by us via cyanoacetylation of 2-cyanomethylbenzothiazole (3) seemed to be a versatile candidate to ful ill this objective ( Figure 1). found that compound 3 will lead to an excellent building block for the synthesis of target compounds. Thus, when 2-cyanomethylbenzothiazole (3) was treated with cyanoactic acid in presence of acetic anhydride, it afforded the corresponding compound 4, as only one isolable product which indicated by TLC control tested ( Figure 1). Synthesis of this compound was reported for the irst time by us   [10].
Moreover, benzothiazole derivative 4 has latent functional substituent which renders it to be a versatile starting substrate for further chemical transformations that open new routes for the preparation of substituted benzothiazole derivatives with possible biological activity. Now, we have extended our synthetic program to the synthesis of otherwise inaccessible heterocyclic ring system utilizing compound 4 as the key starting material. Thus, a mixture of 4 and DMF/DMA in dry xylene was re luxed for 5-6 hours to yield a product formulated as 5 ( Figure 2). Structure 5 was illustrated based on its correct elemental and spectral analyses. The structure of compound 5 was con irmed by the 1 H NMR spectrum which revealed a singlet signal at δ 2.47 ppm due to two methyl protons and a singlet signal at δ 7.60 ppm due to ole inic CH proton. Also, the mass spectrum showed a molecular ion peak at m/z 296 (M + , 80%) corresponding to the molecular formula C 15 H 12 N 4 OS.
Reaction of this type has not been previously reported, however it was found to give products in excellent yield under very mild conditions. Compounds containing a pyrimidine ring have been reported to exhibit antimicrobial as well as anti-HIV activity [15,18]. On the other hand, pyran ring is known to have a broad spectrum of biological activity. Combination of the two above ring systems may be lead to very active bioactive compounds.
In the same manner, compound 5 was reacted with 1,3-indandione in boiling glacial acetic acid to give the corresponding indenopyranone derivative 8 ( Figure 3). 1 H NMR revealed two singlet signals at δ 4.64 and 8.47 ppm due to methine proton and C 4 -H pyran ring. The appearance of signal of C 4 -H pyranone ring in this low magnetic ield (δ 8.47 ppm) is obviously due to the deshielding effect by the adjacent carbonyl group on the indinone, supporting its correct structure 8, unambiguously. Also, the mass spectrum showed the molecular ion peak at m/z (%) 398 (M + ,70) corresponding to the molecular formula C 22 H 10 N 2 O 4 S. Figure 4 showed that 2-(benzo]d[thiazole-2-yl)-4-((dimethylamino)methylene)-3-oxopentanedinitrile) (5) reacted with barbituric acid X=O to give compound 6 and with thiobarbituric acid X=S to give compound 7. To account for the formation of igure (4), it is suggested that the reaction start with Michael-type addition of the CH 2 group in barbituric and thiobarbituric acids to the activated double bond of compound 5 followed by elimination of dimethylamine and addition cyclization of the OH group to the nitrile group. In the manner, Figure 5 showed that compound 5 reacted with 1,3-indandione as nucleophilic attack to CN group followed by cyclization to give indenopyranone derivative 8.
It was found that compound 5 was reacted with some activated nitriles to give benzothiazoloazepine and (benzothiazolyl) pyridine derivatives. Therefore, when 5 was reacted with malononitrile in re luxing ethanol containing a catalytic amount of piperidine it afforded the corresponding derivative 9 ( Figure 6). The structure of compound 9 was con irmed by the 1 H NMR spectrum which revealed a singlet signal at δ 6.49 ppm due to NH 2 protons and a singlet signal at δ 8.00 ppm due to CH proton. Also, the mass spectrum showed the molecular ion peak at m/z (%) 317 (M + , 100) corresponding to the molecular formula C 16 H 7 N 5 OS.
Similarly, compound 5 was reacted with thiocyanoacetamide in re luxing DMF containing a catalytic amount of TEA for 6 hours. It afforded the corresponding pyridine derivative 10 ( Figure 6). 1 H NMR revealed spectrum singlet signal at δ 4.64 due to methine proton, doublet at δ 5.20 ppm due to C 5 -H pyridine ring and doublet signal at δ 7.86 ppm due to C 4 -H pyridine ring, singlet signal at δ 10.1 ppm due to NH proton. Also, the mass spectrum showed the molecular ion peak at m/z (%) 352 (M + , 40) corresponding to the molecular formula C 16   In a similar reaction mechanism to that of compound 9, compound 5 reacted with cyanoacetamide to afford compound 11 ( Figure 6). The mass spectrum showed the molecular ion peak at m/z (%) 335 (M + ,50) corresponding to the molecular formula C 16 H 9 N 5 O 2 S.
On the other hand, compound 5 was reacted with (2-cyanomethyl) benzothiazole in re luxing ethanol containing a catalytic amount of piperidine, it afforded the corresponding benzothiazolopyridine derivative 12 ( Figure 6). The 1 H NMR spectrum showed two singlet signals at δ 4.65 and 7.86 ppm attributable to methine and C 4 -H pyridine protons, respectively. Also, the mass spectrum showed the molecular ion peak at m/z (%) 426 (M + , 50) corresponding to the molecular formula C 22 H 10 N 4 O 2 S 2 .
In a similar reaction mechanism to that of compound 9, compound 5 was reacted with ethyl cyanoacetate in re luxing ethanol containing a catalytic amount of piperidine for 8 hours. It afforded derivative 13 ( Figure 6). The 1 H NMR spectrum showed triplet  signal at δ 1.38 ppm due to CH 3 protons, quartet signal at δ 4.35 ppm due to CH 2 protons and a singlet signal at δ 6.50 ppm due to NH 2 protons.
In addition, heating 5 with an equimolar amount of α-naphthol in re luxing glacial acetic acid gave the corresponding benzocoumarine derivative 14 ( Figure 7). The 1 H NMR spectrum showed a singlet signal at δ 4.65 ppm due to CH proton, doublet signal at δ 7.21 ppm due to aromatic protons, multiplet signal at δ 7.35-8.23 ppm due to aromatic protons and a singlet signal at δ 8.45 ppm due to C 4 -H pyran. The mass spectrum showed the molecular ion peak at m/z (%) 396 (M + ,50) corresponding to the molecular formula C 23 H 12 N 2 O 3 S.
Similarly, compound 5 reacted with β-naphthol to give the corresponding benzocoumarine derivative 15 (Figure 7). The IR and 1 H NMR spectra showed a similar picture to that of compound 14. Heating compound 5 with resorcinol in glacial acetic acid afforded the corresponding 7-hydroxycoumarine derivative 16 (Figure 7). The 1 HNMR spectrum showed four singlet signal at δ 4.65, 5.00, 7.10 and 8.27 ppm due to C-H (methine proton), OH, C 4 -H and C 8 -H coumarine ring, respectively. Besides two doublets at δ 6.55 and 7.90 ppm due to aromatic protons and multiplet signals at δ 7.55-8.23 ppm due to the other aromatic protons. The mass spectrum showed the molecular ion peak at m/z (%) 362 (M + ,40) corresponding to molecular formula C 19 H 10 N 2 O 4 S.
Moreover, it has been studied the reaction of compound 5 with some nitrogen nucleophiles which afforded a product of nucleophilic substitution reactions with N(CH 3 ) 2 moiety .Thus, it has been found that reaction with hydrazine hydrate and/or phenylhydrazine afforded the corresponding pyrazolyl derivatives 17a,b, respectively ( Figure 8). The IR spectrum, in general, showed stretching frequencies at 3420, 3350, 2220, 1700 and 1640 cm -1 due to NH 2 , NH, CN, C=O and C=N, respectively. 1 HNMR spectrum (in general) showed singlet signals at δ 4.62, 5.60 and 10.20 ppm due to CH, NH 2 and NH protons while the aromatic protons appeared as multiplet at δ 7.40-7.80 ppm. The mass spectrum of 17a showed the molecular ion peak at m/z (%) 283 (M + ,50) corresponding to molecular formula C 13 H 9 N 5 OS, while the mass spectrum of 17b showed the molecular ion peak at m/z (%) 359 (M + ,60) corresponding to molecular formula C 19 H 13 N 5 OS. Moreover, reaction of 5 with some aromatic amines (namely, p-nitroaniline, p-toluidine and 2-aminopyridine) in re luxing ethanol in the presence of a catalytic amount of TEA afforded the corresponding (benzothiazolyl) pyridine derivatives 18ac ( Figure 8). Structures of 18a-c were based on their correct analytical and spectral analyses. The mass spectrum of 18a showed the molecular ion peak at m/z (%) 389 (M + ,80) corresponding to molecular formula C 19 H 11 N 5 O 3 S. The mass spectrum of 18b showed the molecular ion peak at m/z (%) 358 (M + ,40) corresponding to molecular formula C 20 H 14 N 4 OS. The mass spectrum of 18c showed the molecular ion peak at m/z (%) 345 (M + ,70) corresponding to molecular formula C 18 H 11 N 5 OS.
On the other hand, re luxing of compound 5 with mercaptoethanol in glacial acetic acid gives the cyclo addition product 19 (Figure 8). The 1 H NMR spectrum revealed two methyl protons at δ 2.27 ppm as singlet signal, two triplets at δ 3.68 and 3.70 ppm due to S-CH 2 and O-CH 2 protons, respectively, two doublets at 2.76 and 2.86 ppm due to S-CH and C-CH protons, respectively. The mass spectrum of 19 showed the molecular ion peak at m/z (%) 356 (M + ,70) corresponding to molecular formula C 17 H 16 N 4 OS 2 .

Biological Activity
Antimicrobial Evaluation: Nineteen of the newly synthesized targeted compounds were evaluated for their in vitro antimicrobial activity against Bacillus subtilis and Bacillus thuringiensis as example of Gram positive bacteria and Escherichia coli y and Pseudomonas aeruginosa as example of Gram negative bacteria. They were also evaluated for their in vitro antifungal potential against Fusarium oxysporum and Botrytis fabae fungal strains.
Agar-diffusion method [19], was used for the determination of the antibacterial and antifungal activity. Chloramphenicol, Cephalothin and Cycloheximide and Ampicillin were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the disks in mm. The minimum inhibitory concentration (MIC) measurement was determined for compounds showed signi icant growth inhibition zones (>14 mm) using two fold serial dilution method [20].
The MIC (μg/mL) and inhibition zone diameters values are recorded in table 1. The results depicted in table 1 revealed that the most of tested compounds displayed variable inhibitory effects on the growth of the tested Gram-positive and Gramnegative bacterial strains, and also against anti-fungal strain.
In general, most of the tested compounds revealed better activity against the Grampositive rather than the Gram-negative bacteria. It would be also noticed that compound belonging to pyrimidine and pyridine thione derivatives exhibited better antimicrobial potentialities than parent 3 and members of coumarin, benzocoumarin and pyrazole series. Regarding the structure activity relationship (SAR) of the newly heterocyclic derivatives against Gram-positive bacteria, the results revealed that compounds 7,10 and 12 exhibited broad spectrum antibacterial pro ile against the tested organisms. Compounds containing and sulfur atoms and contains electron withdrawing groups such as C 6 H 4 NO 2 -p, C=O and C=S recorded higher activity. In this view, compounds 18a, 18b and 18c showed good activity. Also, compounds 7,10 and 12 were equipotent to Chloramphenicol and Ampicillin in inhibiting the growth of Bacillus subtilis (MIC 3.125 μg/mL), while its activity was 50%, lower than of Chloramphenicol against Bacillus thuringiensis. Moreover, compounds 8 and 16 were about 25% of the activity of Chloramphenicol and Ampicillin and 50% of cephalothin against Bacillus subtilis and its activity was 25% lower than of Chloramphenicol and 50% lower than Cephalothin against Bacillus thuringiensis.
On the other hand, compounds 5, 9,11,14,15 exhibited moderate growth inhibitory against Gram-positive bacteria as revealed from MIC values (25μg/mL). Compound 17a showed relatively good growth inhibitory pro ile against Bacillus subtilis was about 25% of the activity of Chloramphenicol and Ampicillin and 50% of Cephalothin against the same organism. Also, compound 17b showed weak growth inhibitory against Bacillus subtilis (MIC 50μg/mL). Regarding to the activity of the newly synthesized compounds, against antifungal strains, the results revealed that compounds 10 and 7 were 50% lower than Cycloheximide in inhibitory the growth of B. fabae and F. oxysporum (MIC 6.25μg/mL). While the activity of compound 8 and 16 were 25% lower than Cycloheximide against F. oxysporum (MIC 12.5μg/mL). It is worth mentioning that the incorporation of coumarin or benzocoumarin moiety to benzothiazole nucleus decreased the antimicrobial activity. On the other hand, conversion of compound 3 to pyridine and pyrimidine thione enhanced the antimicrobial activity. In conclusion, the objective of the present study was to synthesize and investigate the antimicrobial activities of some new functionalized benzothiazole derivatives with the hope of discovering new structure leads serving as antimicrobial agents. Our aim has been veri ied by the synthesis of different groups of structure hybrids comprising basically the benzothiazole moiety.

Experimental
All melting points are recorded on Gellenkamp electric melting point apparatus. The spectra ν (cm -1 ) (KBr) were recorded on a Perkin Elmer Infrared Spectrophotometer Model 157. The 1 H NMR spectra were obtained on a Varian Spectrometer at 200 MHz, using TMS as an internal reference and DMSO-d 6 as solvent. The 13 C NMR spectra were recorded on JEOL-ECA500 (National Research Center, Egypt). The mass spectra were recorded at 70 eV with kratos MS equipment and/or a Varian MAT 311 A Spectrometer. Elemental analysis (C, H and N) were carried out at the Micro analytical Center of Cairo University, Giza, Egypt.

Minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) measurements [21].
The microdilution susceptibility test in Muller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, chloramphenicol, cephalothin and cycloheximide were prepared in DMF at concentration of 1000 μg/ mL. Each stock solution was diluted with standard method broth (Difco) to prepare serial two fold dilutions in the range of 500-3125 μg/mL 10 mL of broth containing about 10 6 CFU/Ml of test bacteria was added to each well of 96-well microtiter plate. The sealed microplates were incubated at 37°C for 24h for antibacterial activity and at 37°C for 48h for antifungal activity in a humid chamber. At the end of incubation period, the minimal inhibitory concentration (MIC S ) values were recorded at the lowest concentration of the substance that had no visible turbidity. Control experiments with DMF and uninoculated media were run parallel to the test compounds under the same conditions, as shown in tables 1 and 2.