Synthesis, Antimicrobial and Anticancer Evaluations of Novel Thiazoles Incorporated Diphenyl Sulfone Moiety

Abstract A novel series of thiazoles incorporated phenylsulfonyl group have been designed and synthesized from the treatment of N-(1-(4-(phenylsulfonyl)phenyl)ethylidene)hydrazine carbothioamide with various types of α-halocarbonyl compounds in one-pot reaction strategy under microwaves irradiations. Studying all possible spectral data for the prepared compounds were proved their structure as well as the suggested mechanism of their reactions. The results of antimicrobial activity of some selected derivatives proved their potency in which some derivatives showed activity exceeded the activity of the reference drugs. In addition, selected derivatives were investigated for their anticancer activities against HepG-2 and HCT-116 cell lines to shed some light on the anticancer activity of the newly synthesized thiazole derivatives. One of the tested thiazole derivatives showed good activity in comparison with doxorubicin reference. Graphical Abstract


Introduction
With tremendous progress in the field of medicine and the treatment, humans are suffering from a lack of effective and safe medicines to treat cancer and microbial diseases. This drives scientists to work continuously to discover new anticancer and antimicrobial agents. 1 Cancer is considered as one of the leading causes of death worldwide after the cardiovascular diseases. Globally, cancer deaths account for 17% of all human deaths. 2 Moreover, continuous resistance of microbes to known antibiotics is the reason for these researches. 3 The rapid emergence of bacterial resistance worldwide has recently been observed, clearly affecting human health and often threatening life. Therefore, effective antimicrobial agents must belong to new categories, because the structural change of the existing drugs that have already developed resistance seldom provides a main solution. 4 Thiazole and phenyl sulfone moieties are important two pharmacological scaffolds which responsible for the potent activities in their heterocyclic compounds as antitumor, 5,6 antimicrobial, 7,8 anti-HCV 9,10 and anti-inflammatory. 11,12 The valuable starting synthons for heterocyclic compounds having S and N atoms are thiosemicarbazones. [13][14][15][16] Their diverse synthetic and biological applications have been concerned by researchers for a long time. 17,18 One of the most successful ways to obtain novel biologically active drugs is to hybridize different drugs in one structure. 19,20 In addition, using the microwave irradiations in the synthesis of new bioactive heterocyclic compounds are well known to enhance the yield of the compounds and reduce the reaction time. 13 As a continuation of our research process in the field of synthesis of heterocyclic compounds that are biologically active, [21][22][23][24][25][26] and based on all of the aforementioned, we aim in this research project to synthesize heterocyclic system combined from both thiazole with phenyl sulfone moiety. This will be achieved via the reactivity of 4-acetyl diphenyl sulfone thiosemicarbazone toward various derivatives of halogenated compounds. The structure of all products and their antimicrobial activity as well as the antitumor activity of selected derivatives will be investigated and discussed.

Chemistry
Melting points of phenyl sulfone derivatives were determined in 0.5 mm (o.d.) glass capillaries by using a Stuart SMP3 melting point apparatus. All IR spectra for the synthesized phenyl sulfone derivatives were recorded in KBr disks Shimadzu FTIR 8101 PC infrared spectrophotometer. NMR (nuclear magnetic resonance) spectra have been determined in deuterated solvent (DMSOd 6 ) utilizing a Bruker Avance 500 instrument at 400 or 850 MHz for 1 H NMR or 125 MHz for 13 C NMR in DMSO-d 6 solutions. The measuring units for d and J are ppm and Hz, respectively. Also, elemental analyses were measured on Elementarvario-LIII C-H-N-S analyzer which was made in Germany. The MARS system of CEM was utilized for emitted microwave irradiation using Teflon HP-500 vessels. All chemicals used in this research were obtained from Sigma-Aldrich and were utilized without any further purification. Compounds 13, 15 and 17 were synthesized following the previously described procedures. 27,28 Organic synthesis N-(1-(4-(phenylsulfonyl) phenyl)ethylidene)hydrazine carbothioamide (3) Method A (thermal method). Equivalent amounts of acetyl sulfone derivative 1 (2.6 g, 0.01 mole) and thiosemicarbazide 2 (0.91 g, 0.01 mole) in 30 mL EtOH were refluxed, after boiling and dissolving the solid ingredients, drops of hydrochloric acid were added and reflux was continued to complete the reaction. The solid product was formed on cooling, filtrated and washed with MeOH. The crude product was recrystallized using ethanol to afford phenylsulfonyl thiosemicarbazone derivative 3.
Method B (microwaves method). Equivalent amounts of acetyl sulfone derivative 1 (2.6 g, 0.01 mole) and thiosemicarbazide 2 (0.91 g, 0.01 mole) were mixed in 30 mL EtOH containing drops of hydrochloric acid then mixed in a HP-500 reaction vessel. The vessel was capped tightly and irradiated by microwaves (800 W power) using pressurized conditions at 70 C for a period of time as depicted in Table 1. The solid product was formed on cooling, filtrated and washed with MeOH. The crude product was recrystallized using ethanol to afford the phenylsulfonyl thiosemicarbazone derivative 3 as white crystals, m. Reaction of phenylsulfonyl thiosemicarbazone 3 with x-bromoacetophenone derivatives 4a-c, ethyl chloroacetate (6), and chloroacetonitrile (8) General procedure Method A (thermal method). Equimolar amounts of phenylsulfonyl thiosemicarbazone 3 (0.83 g, 2.5 mmol), appropriate: x-bromoacetophenone derivatives 4a-c or ethyl chloroacetate 6 or chloroacetonitrile 8 (2.5 mmol) in 15 mL of dioxane containing catalytic amount of triethylamine (TEA) were heated under reflux for a period of appropriate time as illustrated in Table 1. The solid products were formed on cooling, filtrated and washed with MeOH. The crude products were recrystallized using ethanol to afford the corresponding thiazole derivatives 5a-c, 7 and 9, respectively.
Method B (microwaves method). Equimolar amounts of phenylsulfonyl thiosemicarbazone 3 (0.83 g, 2.5 mmol), appropriate: x-bromoacetophenone derivatives 4a-c or ethyl chloroacetate 6 or chloroacetonitrile 8 (2.5 mmol) in 15 mL of dioxane containing catalytic amount of TEA were mixed in a HP-500 reaction vessel. The vessel was capped tightly and irradiated by microwaves (800 W power) using pressurized conditions at 110 C for a period of time as depicted in Table 1. The solid products were formed on cooling, filtrated and washed with MeOH. The crude products were recrystallized using ethanol to afford the corresponding thiazole derivatives 5a-c, 7 and 9, respectively.    Reaction of thiosemicarbazone derivative 3 with the hydrazonoyl halides 13a-e, 15a,b and 17 General procedure Method A (thermal method). Equimolar amounts of phenylsulfonyl thiosemicarbazone 3 (0.83 g, 2.5 mmol), appropriate hydrazonoyl halides (13a-e or 15a,b or 17) (2.5 mmol) in 15 mL of dioxane containing catalytic amount of TEA were heated under reflux for a period of appropriate time as illustrated in Table 2. The solid products were formed on cooling, filtrated and washed with MeOH. The crude products were recrystallized using ethanol/dimethylformamide mixture as a recrystallization solvent to afford the corresponding thiazole derivatives 14a-e or 16a,b or 18, respectively.
Method B (microwaves method). Equimolar amounts of phenylsulfonyl thiosemicarbazone 3 (0.83 g, 2.5 mmol), appropriate hydrazonoyl halides (13a-e or 15a,b or 17) (2.5 mmol) in 15 mL of dioxane containing catalytic amount of TEA were mixed in a HP-500 reaction vessel. The vessel was capped tightly and irradiated by microwaves (800 W power) using pressurized conditions at 110 C for a period of time as depicted in Table 2. The solid products were formed on cooling, filtrated and washed with MeOH. The crude products were recrystallized using ethanol to afford the corresponding thiazole derivatives 14a-e or 16a,b or 18, respectively.

Antimicrobial activity
Antimicrobial activity assay All the microbial strains used in the current study have been supplied from Al-Azhar University in Cairo, Egypt from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB). The method used for recording the antimicrobial activity according to the literature method. 29 In vitro cytotoxic efficiency The HepG-2 and HCT-116 (human liver and human colon cancer cell line, respectively) cell cultures used in the current study have been supplied from the American Type Culture Collection (Rockville, MD) and were kept in Dulbecco's modified Eagle's medium, supplemented by 10% of fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. Cells grew at 37 C in a humid atmosphere of 5% CO 2 .
MTT cytotoxicity assay 3-[4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was profited in order to assess the cytotoxicity of thiazole carrying morpholine derivatives versus HepG-2 (human liver cancer cell line) and HCT-116 (colon cancer cell line). This method is dependent on the dissent of the salt of tetrazole by mitochondrial dehydrogenases in the cells. 30 The method used for MTT cytotoxicity assay was described in detailed in the previous report. 14 Scheme 1. Synthesis of thiosemicarbazone derivative 3.
The structure of the phenylsulfonyl thiosemicarbazone 3 was assured using the results of spectral and elemental analyses. For example, the molecular weight of thiosemicarbazone 3 was consistent with molecular ion peak appeared in its mass spectrum. The infrared spectrum of 3 revealed absorption bands assigned to the NH 2 and NH functionalities at 3446, 3336 cm À1 and the absorption band for C ¼ N at 1592 cm À1 , respectively. The spectrum of 1 H NMR of thiosemicarbazone 3 revealed three singlet signals at d 10.40, 8.40 and 8.10 ppm assigned to the three NH protons, respectively, in addition to signals in the region of d 7.62-8.14 and 2.30 ppm characterized for nine aromatic rings protons and the CH 3 protons, respectively ( Figure 1). Surprisingly, there are three singlet signals for three NH protons in 1 H NMR of compound 3, but these signals have been interpreted due to the presence of intramolecular-hydrogen bond between one of the protons of NH 2 function with the nitrogen atom of N ¼ C group as indicated in Figure 1. 13,14,31 Also, the 13  Reaction between N-(1-(4-(phenylsulfonyl)phenyl)ethylidene)hydrazine carbothioamide (3) and a-bromocarbonyl derivatives 4a-c under two types of heating microwaves irradiation or thermal conditions and in the presence of Et 3 N gave the corresponding phenylsulfonyl-thiazole derivatives 5a-c as depicted in Scheme 2. The suggested structure for such products has been assured through the results extracted from their spectra and elemental analysis. For instance, the IR spectrum of phenylsulfonyl-thiazole derivative 5a was characterized by the presence of a broad absorption band for NH at 3444 cm À1 . Moreover, its 1 H NMR spectrum has a set of remarkable singlet signals in the regions d 2.33, 7.45 and 11.53 ppm assigned to the methyl, thiazole-H and NH groups in addition to the multiplet signals of the aromatic protons in the region of d 7.47-8.0 ppm. The presence of thiazole-H at d ¼ 7.30-7.45 ppm for all derivatives 5a-c rather than CH 2 group proved structure 5A and excluded the existence of isomer 5B (Figure 2). For all phenylsulfonyl-thiazole derivatives 5a-c, their mass spectra revealed the molecular ion peaks at the expected m/z values.
On the other hand, reaction of N-(1-(4-(phenylsulfonyl) phenyl) ethylidene)hydrazine carbothioamide (3) with ethyl chloroacetate 6 or a-chloroacetonitrile 8 in dioxane and in the presence of trimethylamine under thermal or microwaves irradiation for a period of time as illustrated in Scheme 3 and Table 1, gave thiazole derivatives 7 and 9, respectively. The two structures 7 and 9 were assured based on their spectral and elemental analysis. For instance, the IR spectrum of thiazole 7 was characterized by the existence of carbonyl C ¼ O absorption band at 1701 cm À1 in addition to the NH absorption band at 3444 cm À1 . Moreover, the 1 H NMR spectra of thiazoles 7 and 9 showed all the expected aliphatic and aromatic proton signals, as in case of 1 H NMR of derivative 9 revealed four singlet signals at d 2.35, 3.58, 8.42 and 10.43 ppm for CH 3 , NH 2 , thiazole-H and NH protons.
Interestingly, when the reactions were exposed to microwaves irradiation for mins, the products were collected in high yield, but longer reaction time was needed to obtain lower yields under ordinary thermal heating conditions ( Table 1). The important points of these previous reaction processes include shorter reaction times and higher yields than the conventional methods.
The two Schemes 4 and 5 illustrate the proposed mechanism for the reaction of phenyl sulfone-thiosemicarbazone with a-haloketone and a-chloroacetonitrile. The two mechanisms started with the nucleophilic substitution reaction to form the intermediates 10 and 12, respectively. Intermediate 10 tend to cyclization with the elimination of water or ethanol molecule through the non-isolable intermediate 11 to isolate the final product 5 or 7, respectively (Scheme 4), while intermediate 12 followed Michel type addition to give the final product 9 (Scheme 5).
Condensation of phenylsulfonyl-carbothioamide derivative 3 with 2-oxo-N-arylpropane-hydrazonoyl chloride 13a-e in dioxane under reflux or microwaves irradiation and in the presence of a base catalyst (Et 3 N) afforded in all cases only one product as monitored by TLC analysis of the crude products. These products were suggested to be phenysulfonyl-thiazole derivatives 14a-e (Scheme 6) according to their spectral and elemental analysis results. All IR spectra of derivatives 14a-e were characterized by the disappearance of absorption bands of NH 2 of the thiosemicarbazone or the carbonyl absorption band of the hydrazonoyl chloride. The 1 H NMR spectrum of thiazole derivative 14d characterized by the presence of all expected signals for 20 protons as illustrated in Figure 2. It should be noted that the reaction between hydrazonoyl chlorides 13 and the phenyl sulfone-thiosemicarbazone derivatives 3 exposing to microwaves irradiation is much favorable than normal heating in the time of the reaction and the percentage yield in some cases as tabulated in Table 2. The reaction proceeds steadily in all cases with hydrazonoyl chlorides 13a-e having an EDG (electron-donating group) or an EWG (electron-withdrawing group) substituent on the aromatic ring.
From the investigation of the tautomeric structures in Figure 3, compounds 14a-e would be present in one or more of different tautomeric forms 14A-C. The electronic absorption spectra of phenylsulfonyl-thiazoles 14a-e were investigated in dioxane and revealed in each case three absorption bands in the regions 464-452, 391-318, 299-261 nm ( Table 3). The large value of absorption band 464-452 nm is in the same range as that reported for the (N ¼ N) azochromophore. [31][32][33] This result indicated that the thiazole derivatives 14a-e found only in one of the two azo form 14A or 14B in the solution phase, and the other hydrazone form 14C was excluded. [34][35][36] To choose the correct tautomeric structure of phenylsulfonyl thiazole derivatives 14 from the two forms 14A or 14B, the two NOE difference experiments for derivative 14d were screened which revealed that the double irradiation at the two CH 3 groups each at d ¼ 2.09 and 2.64 ppm has not enhanced the NH signal in each test for such derivative. This result is compatible with the tautomeric form 14A as there isn't any electronic effect between any of the two CH 3 and the NH signal in such compound.

Antimicrobial activity
The novel synthesized phenyl sulfone derivatives 3, 5a-c, 7, 9, 14a-e, 16a,b and 18 were screened in vitro for their antimicrobial activity against two fungi species, four Gram Positive Bacteria and four Gram negative Bacteria as listed in Tables 4-6. The microorganisms were tested against the activity of solutions of concentration 10 mg/mL of each compound and using an inhibition zone diameter (IZD) in mm/mg sample as criterion for the antimicrobial activity. The fungicide Amphotericin B and the bactericides Ampicillin and Gentamycin were used as references to evaluate the potency of the tested phenyl sulfone derivatives under the same conditions. The tested derivatives contain the starting thiosemicarbazone 3 and derivative 16 and 18 which didn't contain thiazole ring to compare the activity of compounds incorporated thiazole moiety 5a-c, 7, 9 and 14a-e with them. The results of antifungal activity are depicted in Table 4 indicated that all investigated derivatives showed promising activity against the two fungi species. The antifungal activity of starting thiosemicarbazone derivative 3 against the species was found effective and near that for the reference drug Amphotericin B as shown in Table 4. Conversion of the carbothioamide derivative 3 to thiazole moiety as in derivatives 5a, 5c, 7 and 14a enhanced their activity as illustrated in Table 4. The most antifungal active derivatives 5a, 5c and 14a were found to have halogen atoms (Cl or Br) at the para-position of the phenyl group on the thiazole ring. The   Table 5. These data indicated that all tested derivatives have no antifungal activity against Streptococcus pyogenes. While all Table 4. Preliminary anti-fungi activity for compounds 3, 5a-c, 7, 9, 14a-e, 16a,b and 18.

Sample
Aspergillus niger Geotrichum candidum In case of Bacillus subtilis, the starting thiosemicarbazone derivative 3 was found the most reactive derivative from the investigated series. Table 6 contains the results of anti-gram negative bacterial activity of the tested series. No activity was observed against Pseudomonas aeruginosa, on the contrary, we found that all derivatives showed good activity in comparison with the reference drug for Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. The most reactive derivatives against these three bacteria are 14e, 9, and 14b, respectively. From all the previously discussed antimicrobial results, we can highlight the importance of the synthesized series of phenyl sulfone derivatives in this study as they are effective antimicrobials and the biological study can be completed on them to produce an effective drug for one of the known microbes.

Antitumor activity
The in vitro tumor inhibitory activity of selected five phenyl sulfone derivatives 3, 7, 14a,b and 16b was studied against the liver cancer cell line (HepG-2) and human colon cancer cell line (HCT-116) in contrast with the familiar Doxorubicin reference drug under the same conditions using colorimetric MTT analysis. By comparing the results recorded in Table 6 with that for the reference drug, we noticed that the most active phenyl sulfone derivative is compound 14b which showed IC 50 ¼ 7.97 mg/mL (HepG-2) and 5.44 mg/mL (HCT-116). The activity of thiazole derivative 14b exceeded the activity of the reference drug (Doxorubicin) as illustrated in Table 7. This result proves that the hybridization of the two active scaffolds (phenyl sulfone and thiazole moieties) in one compound produce an active drug.

Disclosure statement
There are no conflicts of interest.