Synthesis and Antimicrobial, Antiproliferative Evaluation of Novel Quinolone and Conazole Analogues via Conventional and Microwave Techniques

1,2,4-Triazole-3-one (3), acquired from cinnemaldehyde was converted to the corresponding carbox(thio)amides via several steps (6a-c). Their reaction with sodium hydroxide gave the 1,2,4-triazole derivatives (7a-c). Compound 3 treatment with 2-bromo-1-(4-chlorophenyl) ethanone or 2-chloro-1-(2,4-dichlorophenyl)ethanone afforded the compounds 8a,b and by reducing these compounds reduction products were obtained (9a,b). The synthesis of (10a-e) was carried out by the reaction compounds 9a,b with different benzyl chlorides. Then oxadiazol derivative (12) was obtained by ring closure from hydrazide compound 5. Subsequently compounds 3, 7a-c and 12 were treated with various amines in the presence of formaldehyde to yield Mannich bases (11a-e, 14a-e, 13a,b). Microwave-assisted and conventional techniques were utilized for the syntheses. The structures of newly synthesized compounds were illuminated by spectroscopic methods. Their antimicrobial (MIC method), and anticancer activities (Abay’s method) were examined. Results showed that most of the compounds exhibited good antimicrobial activities. Especially compounds 14a-e which is a mannich base showed very good antitubercular activity against Mycobacterium smegmatis compared with Streptomycin standard drug. Also compounds 8a and 9b have been found to have strong antiproliferative effects on the HeLa cervical cancer cells and also these compounds did not have cytotoxic effect on normal cell.


Introduction
Cancer, is the result of uncontrolled growth of cells a major health concern and amongst the most important reasons of death worldwide [1][2][3]. The World Health Organization's cancer agency warns that there will be 22 million new cases of cancer every year within the next two decades [4]. Hence, cancer treatment is the primary task of today's medical research [5]. Despite the invention of several chemotherapeutic agents, still treatment of cancer is a major challenge, because of multi-drug resistance, toxicity or poor bioavailability. The effectiveness of many anticancer drugs is limited by acquired resistance to drugs, side effects due to their toxicity to normal cells because of their inability to differentiate between normal and cancerous cells. In this context, there is need to design and synthesize new, effective and less toxic anticancer agents throwing a challenge to medicinal chemists all over the World [6,7].
Infectious diseases continue to be a leading threat to human health, and the rapid development of bacterial resistance to current antibiotic chemotherapies has rendered lots of therapy weapons less effective [8]. It is anticipated that antibiotic resistance is going to cause more than 10,000,000 deaths per year by the year 2050, posing a formidable challenge for disease treatment as pathogens become resistant to clinical drugs [9]. The World Health Organization has launched a global action plan calling on all countries to take measures towards drug-resistant microbes, and the discovery of e cacious and safer antimicrobials with new or multiple mechanisms of action has been an urgent need to combat resistant strains [10,11].
One of the main strategies for the discovery of new drugs is combining two or more pharmacophoric moieties in a single molecule to obtain the synergistic effect or to obtain antitumor agents that have a novel mode of action. For this purpose, various quinolone and conazole derivatives with biological activity were synthesized in this study. In a round-bottom ask, a mixture of 1,2,4-triazol-3-one (2) (10 mmol) and cinnemaldehyde (10 mmol) was heated on oil bath at 110-120 o C for 2 hours. The solid obtained by cooling the mixture to room temperature was crystallized from the ethanol: water (1: 3) mixture. The crystals obtained were puri ed by crystallizing several more times from the same solvent and after drying in vacuo, it was identi ed as compound 3.     and 10d) or 2,6-dichlorobenzyl chloride (for 10b) (3 mmol) was added and MW irradiation was maintained for additional 12 min under the same conditions. After evaporating the solvent under reduced pressure, an oily product appeared. Water was added into it and extracted with 15 mL of ethyl acetate three times in the presence of K 2 CO 3 . The organic layer was dried on Na 2 SO 4 and solvent was evaporated under reduced pressure. The crude product was puri ed by column chromatography on silicagel (nhexane/ethyl acetate, 3:7).           in sterile culture asks was transferred to the waste container. 10 mL of trypsin-EDTA was added to the culture ask containing the cells. The ask's lid was closed and incubated for 1-2 minutes at 37°C in a CO 2 incubator (5% CO 2 ). Thus, the cells adhering to the surface were removed from the surface. After Antiproliferative activity studies were carried out according to the method used by Abay [37]. Samples

Chemistry
The primary target of this study was to develop antimicrobial hybrid substances covering various pharmacophore structures. Reactions of last and intermediate compounds were achieved as pictorial in Scheme 1, Scheme 2, Scheme 3, and Scheme 4. The synthesis was carried out by utilizing microwaveassisted and conventional methods. The nishing of the synthesis was observed via the thin-layer chromatography (TLC) process. All product structures were based on the foundation of spectral and physicochemical data.

Antimicrobial Activity
All newly products were tested for their antimicrobial properties utilizing the minimal inhibition concentration method (MIC) and the outcomes for active molecules are illustrated in Table 1. The antimicrobial activities of compounds (2-9) were tested against 4 bacteria and 3 yeasts.  Ca  Sc   3  --500  ----500  -5  -------125  -6c  ----250  ----7a 125 No antimicrobial effects were found against to the test microorganisms in the studied concentration ranges of the 4, 6a,b, 8a and 10c compounds. Therefore, these results are not included in the table.

Anticancer Activity
The antiproliferative activity potentials of the molecules were examined by Abay's [37] method against HeLa cells. Three different doses of the samples showed different antiproliferative activity against the cells ( Fig. 1 and Fig. 2). High Cell Index (CI) values (red line) were obtained from the wells without the molecules samples. Lower CI values were obtained from the wells in which the molecules were added.
Only the medium (DMEM) was added to the nal three wells. These three wells were used to obtain a baseline. No impedance change was observed in these wells containing only DMEM. Therefore, the Cell Dose effect investigations are performed by considering terms such as a dose dependent effect, dosedependent reverse effect, hormesis and inverse hormesis [38][39][40]. Antiproliferative effects of these molecules against HeLa cells are shown in Fig. 1 and Fig. 2. CI values obtained from wells with no cells increased rapidly (red line). Different doses of DMSO were added to the wells with HeLa cells and no DMSO-induced antiproliferative activity was observed (Fig. 3). The effect of all doses of 5FU used as a positive control was strong, and CI values were very close to each other. None of the lower doses of the molecules didn't showed antiproliferative effect (10 µg mL -1 , turquoise line). All of the low doses of the molecules produced the same CI as the control group. Only the low dose of compound 8a partially dissociated from the control group towards the end of the experiment (after 35 hours). However, this situation cannot be considered as a net antiproliferative effect. On the 5FU graph (Fig. 4)  The dose effect differences of these molecules were most clearly seen in 8a and 9b. These effects of molecules with strong antiproliferative effects are due to their structural forms. The antiproliferative activity potentials of the molecules that didn't show any effect or have a weak effect against to HeLa cells should be examined against other cancer cell lines and their effects should be investigated.

Discussion
Schiff base (3) was obtained from the reaction of 3-alkyl-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-one (2) compound with cinnamaldehyde, which was obtained from the reaction of ester ethoxycarbonylhydrazone (1) and hydrazine hydrate [34]. The object was to combine the 1,2,4-triazole nucleus to the cinnamaldehyde since it is known that more e cacious antimicrobial substances can be discovered by adding two biologically effective components together into a single molecular structure [41,42]. Thereafter product (3) was transformed to the corresponding hydrazide derivative (5)  The intramolecular cyclization of products (6a-c) at basic media afforded in the transformation of carbox(thio)amide function alter to 5-oxo(mercapto)-1,2,4-triazole derivatives and so molecules 7a-c were synthesized. These compounds were characterized by the presence of a signal at 13.70 and 14.02 ppm in the 1 H NMR data as a D 2 O exchangeable singlet con rming the existence of a -SH function (7a,b) and 10.77 ppm -NH function (for 7c). The stretching band derived from this groups appeared at 2929 and 2932 cm − 1 , and C = 0 (for 7c) observed at 1626 cm − 1 at the FT-IR data of these molecules (Scheme 1).
Alkylation of products 3, viaa2-bromo-1-(4-chlorophenyl)-ethanone or 2-chloro-1-(2,4-dichlorophenyl)ethanone in ethanol performed compound 8a,b. These reactions, which took place in 1440 minute in the conventional method, took place in 6 minute in the microwave irradiated method. Studies related to microwave synthesis which support organic synthesis in a shorter time and higher e ciency are available in the literature [43][44][45]. NH proton attached to the triazole group disappeared for compound 3 at thea 1 H NMR spectra. New aromatic peaks were resonated in the region 7.27-7.67 ppm. In 13 C NMR datas of molecules, the carbon atom (C = O) were observed between 192.56 and 194.43 ppm for the newly added carbonyl group.
Compounds 9a,b was obtained with the reduction of the carbonyl structure of products 8a,b with sodium borohydride utilizing both classical heating and MWairradiation. When we compare the traditional and microwave method MW irradiation reduced the reaction time froma960 min. to 8amin. and increased the yields. Looking at compound number 9a,b, the carbonyl group peak has evanesced at the 1 H NMR and 13 C NMR datas and OH peak added between 5.41 and 6.44 ppm in the 1 H NMR. The spreading band obtained this group (OH) appeared between 3374 and 3251 cm − 1 , in the FT-IR data of molecules.
Reactions of molecules 10a-e, were afforded reaction of molecule 9a,b and benzyl chlorides, such as 2,4dichloro-, 2,6-dichloro-and 4-chlorobenzyl,chlorides in a ambiance with of NaH via MW sythesis method at 80°C and 100 W for 5 min. In both FT-IR and 1 H NMR datas of the molecules, the peaks due to the -OH group have disappeared. Another peaks approving molecule structures were displayed at the concerned chemical ranges in the 1 H NMR and 13 C NMR spectra. Moreover, [M + 1] ion signals were appeared at the concerned m/z ranges auxiliaring the offered structures of molecules 10a-e (Scheme 2).
Oxadiazole compound (12) was obtained as a result of ring closure reaction of hydrazide compound (5) with CS 2 in basic media. The NH and NH 2 peaks resulting from the hydrazide compound disappeared in both the FT-IR and 1 H NMR data of the oxadiazol derivative compound. Instead of those peaks, SH peaks were added at 14.66 ppm in 1 H NMR and 2749 cm − 1 in the FT-IR. In addition synthesized molecules con rmed 13 C NMR and Mass spectral data and elemental analysis results consistent with the assigned sutructures (Scheme 3).
Mannich reaction is a three-component condensation reaction involving active hydrogen containing compound, formaldehyde and a secondary amine. The amino alkylation of aromatic substrates by Mannich reaction is of considerable importance for the synthesis and modi cation of biologically active compounds [46]. Mannich bases found numerous practical applications in the eld of medicinal chemistry, it could be responsible for enhancing physicochemical properties [47]. Mannich bases linked 1,2,4-triazole derivatives as containing a signi cant biological activity that has been reported in the literature [48,49]. Furthermore, several Mannich bases of triazole derivatives including piperazine, thiomorpholine, or morpholine moiety were synthesized as antimicrobial agents in our laboratory [50,51].
Fluoroquinolones are known as the most broadly utilized synthetic antimicrobial substances; privileged with wide spectrum antibacterial property, relatively low occurrence of toxic and adverse effects along with an perfect safety pro le [47].
Considering these facts in this research, the aminoalkylation of structures 3, 7a-c, and 12 with different amines, such as nor oxacin (for 11a, 13a, 14a, 14b), cipro oxacin (for 11b, 13b, 14c, 14d, 14e), morpholine (for 11c), thiomorpholine (11d), and 4-phenylpiperazine (for 11e) in an ambiance with formaldehyde was performed using the MW-assisted Mannich synthesis reactions (Scheme 2, Scheme 3 and Scheme 4). No signal symbolizing the presence of the NH band exists on the 1 HNMR and FT-IR spectra of products (11a-e, 13a,b, 14a-e ) and in the 1 H NMR and 13 C NMR spectra of molecules extra signals originated from amine moieties were observed at the concerned chemical ranges. These molecules displayed mass spectral datum records reasonable with their constructions.
The use of microwave (MW) irradiation method consequences in very in uential and clean results with notable developments compared to classical processes. The process via MW irradiation ensured the more helpful road with developed synthesis yields and shorter synthesis times [54]. Green Chemistry would like the high yield of synthetic processes, the use of less toxic solvents, and the decrease in phases of synthetic schemes [53].

Conclusions
This study statements the synthesis of novel compounds having various bioactive units via microwave irridation and conventional techniques. Microwave method provided more e cient way the synthesis of desired compounds. Also, antibacterial, and antiproliferative activity of the synthesized molecules were determined. Among the synthesized compounds, the best antimicrobial activities were found to show compounds 14a-e which are Mannich bases. Especially these compounds showed very good antitubercular activity against M. smegmatis compared with Streptomycin standard drug. And also they exhibited better activity against to E. coli than ampicillin used as standard drug. Middle and high doses of compound 8a and 9b were found to have strong anticancer activity on the HeLa cervical cancer cells.  Adding different doses of DMSO to HeLa cell wells (The concentration unit is µg/mL).