Synthesis , characterization and antimicrobial activity of novel benzofuran-and thiophene-containing diketoxime derivatives

2,2'-Thiobis[1-(2-benzofuranyl)ethanone] (1). Yield: 80 %, 5.6 g; m.p.: 144– 145 °C; Anal. Calcd. for C20H14O4S: C, 68.56; H, 4.03 %. Found: C, 68.59; H, 4.01 %; FTIR (KBr, cm–1): 1680 & 1663 (C=O); 1H-NMR (400 MHz, DMSO-d6, δ / ppm): 8.00 (2H, s, H-3), 7.83 (2H, d, J = 7.8 Hz, H4), 7.70 (2H, d, J = 8.4 Hz, H7), 7.55 (2H, dd, J = 7.2 Hz & 8.40 Hz, H6), 7.38 (2H, dd, J =7.1 & 7.5 Hz, H5), 4.11 (4H, s, CH2); 13C-NMR (100 MHz, DMSO-d6, δ / ppm): 185.48 (C=O), 155.58 (C8), 151.37 (C2), 129.21 (C9), 127.20 (C6), 124.65 (C5), 124.26 (C5), 115.59 (C7), 112.74 (C3), 37.66 (CH2). 1,1′-(2,5-Thiophenediyl)bis[1-(2-benzofuranyl)methanone] (2). Yield: 60 %, 0.89 g; m.p.: 223–224 °C; Anal. Calcd. for C22H12O4S: C, 70.96; H, 3.25 %. Found: C, 70.92; H, 3.24 %; FTIR (KBr, cm–1) : 1618 (C=O), 1185 (C–O–C); 1H-NMR (400 MHz, CDCl3, δ / ppm): 8.37 (2H, s, H3), 7.84 (2H, s, H12), 7.79 (2H, d, J = = 7.6 Hz, H4), 7.69 (2H, d, J = 8.4 Hz, H7), 7.56 (2H, dd, J = 7.6 & 8.0 Hz, H6), 7.39 (2H, t, J =7.6 Hz, H5); 13C-NMR (100 MHz, CDCl3, δ / ppm): 174.84 (C=O), 155.86 (C8), 151.94 (C2), 147.17 (C11), 133.94 (C12), 128.63 (C9), 126.75 (C6), 124.15 (C5), 123.34 (C4), 115.57 (C7), 112.39 (C3). 1,1′-(2,5-Thiophenediyl)bis[1-(2-benzofuranyl)methanone], 1,1′-dioxime (3). Yield: 90 %, 0.97 g; m.p.: 272–274 °C; Anal. Calcd. for C22H14N2O4S: C, 65.66; H, 3.51; N, 6.96 %. Found: C, 65.63; H, 3.55; N, 6.95 %; FTIR (KBr, cm–1): 3250–3000 (OH), 1583 (C=N), 1030 (N–O); 1H-NMR (400 MHz, DMSO-d6, δ / / ppm): 13.1 (2H, s, OH E,E-isomer), 12.8 (2H, s, OH Z,Z-isomer, 7.9–7.29 (24H, m, benzofuran & thiophene rings H); 13C-NMR (100 MHz, DMSO-d6, δ / / ppm): 154.78, 153.43, 151.54, 145.12, 142.09, 141.86, 141.15, 133.76, 131.72,


INTRODUCTION
Benzofuran derivatives are an interesting class of heterocyclic compounds.Many benzofuran derivatives find application in several fields, including as antioxidants, brightening agents, a fluorescent sensor 1 and other fields of chemistry. 2enzofuran derivatives form a number of natural products, such as bergapten, nodakenetin, xanthotoxin and khellin (Fig. 1).Thiophene and its derivatives exhibit interesting biological activities and considerable electroluminescent properties. 3,4These compounds have been used as fluorescent markers and sensors in energy transfer and light-harvesting systems. 5he diketoxime derivatives are very important model systems in applied chemistry.In general, they are used in medicine, photography, agriculture, textiles, dye chemistry and the manufacture of semiconductors.4][15][16][17][18][19][20] Various heterocyclic-substituted oxime ethers were extensively studied due to their diverse biological properties. 21xime ethers are used as anti-inflammatory drugs, 22,23 β-adrenergics, 24 mild inhibitory active compounds in poultry science 25 and as various materials with steroidal effects. 26The biological activities of the oxime ether pharmacophore -C=N-O-R is also well documented. 27However, studies in the literature concerning bisbenzofuran-containing oxime compounds are limited.
Due to the applications of benzofuran, thiophene and diketoxime derivatives mentioned above, it was decided to design new benzofuran-and thiophene-based systems consisting of functionalized diketoxime.
The aim of this study was the synthesis of 1,1′-(2,5-thiophenediyl)bis[1-(2--benzofuranyl)methanone], its dioxime derivative, and new reaction products of the dioxime derivative with various acyl and alkyl halides.In addition, the antimicrobial behavior of all the synthesized compounds was investigated.

EXPERIMENTAL
All the chemicals were supplied from Sigma-Aldrich and used without further purification.Melting points were measured using a differential scanning calorimeter (Shimadzu DSC-50) and are uncorrected.The NMR spectra were determined on a Bruker AC 400 (400 MHz) spectrometer, with tetramethylsilane (TMS) as the internal standard in DMSO-d 6 or CDCl 3 as solvents.The FT-IR spectra were recorded as KBr pellets on a Perkin-Elmer Spectrum One FTIR spectrometer.
The synthesis scheme to compounds 1-4 is presented in Fig. 2, whereas analytical and spectral data are given in Supplementary material to this paper.

Synthesis of compounds 1-3
The required starting material 2-acetylbenzofuran was obtained by cyclocondensation reaction.Currently, several general methods are known for the preparation of 2-acetylbenzofuran. 28-(Benzofuran-2-yl)-2-bromoethanone was obtained by reaction of 2-acetylbenzofuran with bromine in acetic acid, according to the general procedure described in the literature, 29 with slight modifications.

Synthesis of 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone] (2).
A solution of glyoxal was prepared by refluxing a mixture of 0.34 g (1.60 mmol) glyoxal trimer dihydrate and 10 mL of methanol and 30 mL of dioxane for 1 h under magnetic stirring.The keto sulfide (1.40 g, 4 mmol) was dissolved in the solution.A solution of sodium methoxide (0.10 g of sodium dissolved in 10 mL of methanol) was added dropwise to this solution at 40 °C.A precipitate then began to form.The mixture was stirred for 30 min.The formed solid was filtered, washed with methanol and dried.

General procedure for the synthesis of 4a-d
Triethylamine (0.07 mL, 0.5 mmol) was added to a stirred solution of 3 (0.10 g, 0.25 mmol) in 5 mL of tetrahydrofuran.The mixture was stirred at room temperature for 10 min.The appropriate acyl chloride (0.5 mmol) was added dropwise at 0 °C and the reaction mixture was stirred at room temperature for 24 h.The mixture was poured into water and the formed precipitate was filtered, dried in air and recrystallized from ethanol.

Antibacterial assays
Bacterial cultures were obtained in Mueller-Hinton broth (Difco) for Escherichia coli (ATCC 25922) Salmonella enterica subsp.enterica serovar Typhimurium (NRRL 4420) and Staphylococcus aureus (ATCC 6538) after 24 h of incubation at 37±0.1 °C.Testing for the bacteria was realized in Mueller-Hinton broth.The final inoculum size for all bacteria was 10 5 CFU mL -1 .The test compounds were dissolved in DMSO at an initial concentration of 1024 μg mL -1 and then serially diluted to 1 μg mL -1 in culture medium.A set of tubes containing only inoculated broth served as the control.Antibacterial activity was determined after incubation for 24 h at 37 °C.The minimum inhibitory concentration (MIC) and defined as the lowest concentration of the compounds that inhibited the visible growth of a microorganism.All experiments were replicated two times to define the MIC values.
Reaction of 3 with appropriate acyl chlorides in THF in the presence of triethylamine gave the oxime ester compounds 4a-d.Furthermore, reaction of compound 3 with the appropriate alkyl chloride in acetone in the presence of K 2 CO 3 , gave the oxime ether derivative 4e.
All new compounds were characterized by 1 H-NMR, 13 C-NMR and FT-IR spectroscopy and elemental analysis.These data are given in the Supplementary material to this paper.
In FT-IR spectrum, the most important evidence of the formation 1 was the observation of two stretching vibrations at about 1663 and 1680 cm -1 , belonging to the two C=O groups.Additionally, in the 1 H-NMR spectrum, a signal at 4.1 ppm as a singlet belonging to -SCH 2 -protons and in the 13 C-NMR spectrum, a signal at 185.48 ppm belonging to the C=O group proved the formation of compound 1.
In the FTIR spectrum of compound 2, the C=O stretching vibration was observed at 1618 cm -1 .Due to conjugation, this stretching vibration shifted to a lower wave number.In the 1 H-NMR spectrum of this compound, the disappearance of the singlet signal at 4.1 ppm, belonging to -SCH 2 protons, was evidence for the formation of compound 2.
In the IR spectrum of 3, while OH band of the oxime was displayed between 3000-3250 cm -1 , the C=N stretching band and the N-O stretching band were seen at 1583 and at 1030 cm -1 , respectively.As is known, there are three geometrical isomers of the dioximes due to the two oxime groups including the anti- -(E,E), amphi-(E,Z) and syn-(Z,Z) forms, which are shown in Fig. 3. 31 It was expected that 3 would show two different isomeric structures.When the 1 H-NMR spectrum for compound 3 was investigated, the N-OH resonances appeared as two singlets at 13.1 ppm (for the E,E isomer) and at 12.8 ppm (for the Z,Z isomer).][34][35][36] In the 13 C-NMR spectrum of dioxime 3, twenty-four carbon signals were seen, supporting a syn-and anti-isomer mixture of compound 3.In the 13 C-NMR spectrum of compound 3, the C=N signal was displayed at 153 ppm while the C=O signal had disappeared.Dioxime 3 was not expected to be in the amphi form due to different integral values belonging to the N-OH protons at 13.1 and 12.8 ppm.The different integral values belonging to the two N-OH protons confirmed that compound 3 consisted of a syn-and anti-isomer mixture.Moreover, if the amphi-isomer were present in the mixture, more carbon peaks could be expected in the 13 C-NMR spectrum.Due to molecular balancing, the molecules in the amphi-isomer structure break down.
Additionally, a TLC run of a sample of the crystalline dioxime 3 was performed and doubled spots on the plate were observed.Therefore, it was concluded that the dioxime 3 consisted of a mixture of EE/ZZ isomers.
Reactions of dioxime 3 with appropriate alkyl and acyl chlorides gave isomer mixtures that consisted of dioxime ether and dioxime ester type compounds, 4a-e.The disappearance of strong broad -OH band in the 3250-3000 cm -1 region in the FT-IR spectrum of 4a-d and the appearance of strong bands at around 1755-1740 and 1250-1050 cm -1 , which were due to C=O and C-O stretching vibrations, respectively, indicated the formation of ester products.
The ester derivatives of the dioxime 4b (Fig. 4) and 4c (Fig. 5) were obtained as a mixture of anti-and amphi-forms.In the 1 H-NMR spectrum of 4b, the signals of thiophene ring protons (H a 1 and H a ) in the ester unit of the amphi--form of 4b appeared as two doublets at 7.32 (J = 3.5 and 5.1 Hz) and 7.14 (J = = 3.6 Hz and 5.8 Hz) ppm.In the 1 H-NMR spectrum of 4b, the signals of the thiophene ring protons of the anti-form of 4b are probably among multiple signals at 7.33-8.15ppm.In the 1 H-NMR spectrum of compound 4c, both resonances of the furan ring proton (H b ) belonging to the anti-isomer of 4c appeared as a singlet signal at 6.87 ppm.Signals of the H a 1 and H a protons belonging to amphi-isomer of 4c appeared as two singlets at 6.82 and 6.63 ppm.For a symmetrical dioxime in the anti-form, only a single peak was expected. 37,38These three values might correspond to a mixture of anti-and amphi-forms. 39,40 Amphi-(E,Z)-and b) anti-(E,E)-isomers of 4c.
The amounts of the syn-, anti-and amphi-isomers of 3 and 4c-e were calculated from the integrated heights in the 1 H-NMR spectra and these values are given in Table I.The ester derivatives of the dioxime 4a, 4d and 4e (Fig. 6) were obtained as a mixture of anti-and syn-forms.In the 1 H-NMR spectrum of 4d, signal of the OCH 3 protons belonging to the anti-isomer of 4d was observed as a singlet signal at 3.87 ppm.In addition, the signal of the OCH 3 protons belonging to the syn--isomer of 4d appeared as a singlet at 3.75 ppm.In the 1 H-NMR spectrum of 4e, signals of the OCH 2 protons belonging to the anti-and syn-isomer were observed as two singlets at 5.47 and 5.40 ppm, respectively.In the 13 C-NMR spectra of 4b and 4c, the 48 carbon signals prove that these dioxime derivatives consisted of anti-and amphi-isomer mixtures.Moreover, in the 13 C-NMR spectra of 4a, 4d and 4e, the 34, 38 and 40 carbon signals, respectively, proved that these dioxime derivatives consisted of anti-and syn-isomer mixtures.
The OCH 3 proton signals at 3.87 ppm (anti-isomer) and 3.75 ppm (syn-isomer) also have good correlation with the quaternary carbon signal at 162.72 ppm in HMBC spectrum (Fig. S-7c of the Supplementary material).The 13 C-signals at 56.17 ppm (anti-isomer) and 56.02 ppm (syn-isomer) give cross peaks with two OCH 3 protons belonging to the anti-and syn-isomers at 3.87 and 3.75 ppm, res-pectively, in the HSQC spectrum (Fig. S-7d of the Supplementary material).It is clear that the signals at 56.17 and 56.02 ppm belong to the aliphatic OCH 3 carbons of anti-and syn-isomers of 4d.All these data obtained from HSQC and HMBC spectra showed that the signal at 162.72 ppm is the quaternary carbon (C-17) on the disubstituted phenyl ring.The 13

Antibacterial activity
The minimal inhibitory concentration (MIC) of the synthesized compounds was determined against S. aureus, E. coli and S. enterica subsp.enterica serovar Typhimurium using a standard broth dilution technique.All the MIC results are presented in Table II.The obtained data showed that the compounds were able to inhibit the growth of the selected microorganisms in vitro with MIC values between 32 and 128 µg mL -1 .
Among the synthesized compounds, 4e exhibited good activity against S. aureus and E. coli and 1 against S. enterica subsp.enterica serovar Typhimurium with an MIC value of 32 µg mL -1 .The other compounds showed moderate activity against the tested microorganisms.

SUPPLEMENTARY MATERIAL
Analytical and spectral data, as well as the NMR spectra, of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
Acknowledgment.The authors thank Firat University for financial support of this work.
C-signals at 132.59 and 132.35 ppm have a cross peak with the proton resonances around 8.14-8.04ppm (anti--isomer) and 7.94-7.92ppm (syn-isomer) in the HSQC spectrum.These proton signals have good β-correlation with the carbon signals at 164.35 and 164.17 ppm in the HMBC spectrum.It is clear that the signals at 164.35 and 164.17 ppm belong to the carbonyl carbon (C-13) of anti-and syn-isomers of 4d.In the HSQC spectrum, the signals around 7.83-7.77ppm have a cross peak with the 13 C-signals at 112.38 and 112.31 ppm.In the HMBC spectrum, the same proton signals are in correlation with the quaternary carbon (C-4) signals at 127.54 ppm (anti-isomer) and 127.46 ppm (syn-isomer).These data show that the signals around 7.83-7.77ppm belong to the olefinic protons attached to the olefinic carbons (C-3) on benzofuran, which give signals at 112.38 and 112.31 ppm.The correlations in the HMBC spectrum show that the carbons giving the signals at 155.49 and 154.81 ppm, 148.81 and 148.51 ppm and 127.54 and 127.46 ppm belong to C-9, C-2 and C-4 of the anti-and syn-isomers, respectively.The signals around 7.83-7.94ppm have cross peaks with the 13 C-signals at 123.64 ppm (anti-isomer) and 122.99 ppm (syn-isomer) in the HSQC spectrum and have α-correlation with the quaternary carbon signals at 155.49 and 154.81 ppm and 127.54 and 127.46 ppm in the HMBC spectrum.The protons giving the signals around 7.83-7.94ppm are attached to C-5, which resonate at 123.64 ppm (anti--isomer) and 122.99 ppm (syn-isomer).The quaternary carbon signals at 155.49 and 154.81 ppm and 148.81 and 148.51 ppm have α-correlation with the signals at 7.65-7.59ppm in the HMBC spectrum, and these proton signals have cross peak with the 13 C-signal at 113.48 ppm (anti-isomer) and 113.36 ppm (syn-isomer) in the HSQC spectrum.These data show that the signals around 7.65-7.59ppm are due to the protons attached to C-8.