Structure, Antibacterial Activity and Theoretical Study of the Forming Mechanism of Compound [(5-Methyl- 3-oxo-2-phenyl-1H-pyrazol-4-yl)-thiophen- 2-yl-methylene] Thiosemicarbazide①

ZHU Hu-Ling ZHU Jin-Hu HUANG Zhi-Qing LV Li-Jun WEI Zhen

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Structure, Antibacterial Activity and Theoretical Study of the Forming Mechanism of Compound [(5-Methyl- 3-oxo-2-phenyl-1H-pyrazol-4-yl)-thiophen- 2-yl-methylene] Thiosemicarbazide①

ZHU Hua-Linga②ZHU Jin-HuabHUANG Zhi-Qianga②LV Li-JuanaWEI Zhena

a(300384)b(030002)

A new thiosemicarbazone compound derived from 1-phenyl-3-methyl-4-(2-thenoyl) pyrazolone-5 (HPMTP) and thiosemicarbazide has been synthesized and characterized by IR, H－ NMR, MS, elemental analysis, UV and single-crystal X-ray diffraction. The compound crystallizes in monoclinic system,space group with= 7.5925(8),= 20.263(2),= 11.4669(13) ?,= 107.825(8)°,= 1679.5(3) ?3,= 4,= 0.0316 and= 0.0687.The results of antibacterial activity test against Escherichia coli and Bacillus subtilis indicate that the compound possesses the same antibacterial activity as the contrast (Norfloxacin). Theoretical study of the forming mechanism to the title compound at the RHF/6-31G(d) level shows that there are two steps. The distal amino group of thiosemicarbazide is added to the 4-carbonyl group of HPMTP which forms TM. Then a dehydration reaction occurs in TM and generates a stable product PC.

synthesis, structure, antibacterial activity,forming mechanism

1 INTRODUCTION

Thiosemicarbazones and their derivatives are studied widely for their special properties such as antibacterial[1, 2], anti-tuberculosis[3], antiviral[4, 5], anticancer[6-8], antimalarial[9, 10]activities and lumi- nescence[11, 12]. 4-acylpyrazolone-thiosemicarbazo- nes are also widely synthesized and researched for their antibacterial activity, photochromism and anticancer activity[13-18], but these studies are mainly focused on the 4-acylpyrazolone-thiosemicarbazones in which 4-acyl is benzoyl, substituted benzoyl, alkyl-acyl, furoyl,. The studies of 4-thenoylpyra- zolone-thiosemicarbazones are few. Here we report the synthesis, crystal structure, antibacterial activity and the formation mechanism of compound 1-phe- nyl-3-methyl-4-(2-thenoyl)pyrazolone-5-thiosemi-carbazide.

2 EXPERIMENTAL

2. 1 Reagents and instruments

Compound1-phenyl-3-methyl-4-(2-thenoyl)-pyrazolone-5 was synthesized according to the litera- ture[19]. Other reagents with AR grade were com- mercially available and used without further purifi- cation. A Carlo-Erba 1106 Elemental Analyzer was utilized for elemental analysis. IR spectra (KBr disks) were measured on an IRAffinity-1 spectrometer. Melting points were measured on a WRS-1B melting point apparatus and uncorrected. Crystal structure determination was carried out on a Rigaku Saturn724 CCD diffractometer. UV-Vis spectra were obtained on a UV-250IPC spectrometer.MS spectra were recorded on a TQD HPLC-MS (Agricultural analysis laboratory of Tianjin Agricultural Univer- sity).1H NMR spectra were recorded on a Bruker Avance 400 spectrometer with TMS as an internal standard in DMF-7.

2. 2 Synthesis of the title compound

The title compound was synthesized by refluxing the mixture of 1-phenyl-3-methyl-4-(2-thenoyl) py- razolone-5 (HPMTP, 15 mmol) and thiosemicarba- zide (15 mmol) in ethanol (100 mL) over a steam bath for about 8 h, then the solution was cooled down to room temperature. After five days, colorless prism was obtained and dried in air. The product was recrystallized from ethanol, which afforded colorless crystals (m.p. 234～236 ℃) suitable for X-ray ana- lysis. Elemental analysis calculated for C16H15N5OS2: C, 53.78; H, 4.20; N, 19.61; S, 17.92%. Found: C, 53.68; H, 4.23; N, 19.81; S, 17.67%. Selected FT-IR data (KBr, cm-1)max: 3429(w, NH2), 3336(s, NH), 1637(m, C=N), 1571(m, phenyl), 1476(m), 1410(s), 1376(s), 1277(s), 848(s), 745(s), 716(s).UV, 242(1.6), 260(2.1), 282(2.9). ESI-MS,/(%): 358 ([M+H]+, 100).1H NMR (DMF-7, 400 Hz),: 8.70 (s, 2H, NH2), 8.45 (s, 1H, NH), 6.94～8.00 (m, 8H), 2.54 (s, 1H, NH), 1.93 (s, 3H, CH3).

2. 3 X-ray crystallography

A colorless prism with dimensions of 0.12mm ×0.10mm ×0.08mm was selected for X-ray analyses. All X-ray data were collected at 113(2) K on a Rigaku Saturn724 CCD diffractometer equipped with a multilayer-monochromatized Mo-radia- tion (= 0.71073 ?) by using ascan mode in the range of 2.01≤≤27.88o. The crystal is of monoclinic system, space groupwith= 7.5925(8),= 20.263(2),= 11.4669(13) ?,= 107.825(8)°,= 1679.5(3)?3,= 4,D= 1.414 g/cm3,= 0.71073 ?,(Mo) = 0.331 mm-1,(000) = 744,= 0.947, the final= 0.0316 and= 0.0687. The structure was solved by direct methods and refined on2by full-matrix least- squares methods with SHELXS-97[20]. The structure was further refined by full-matrix least-squares method on2with SHELXL-97[21]. During refine- ment, all H atoms were geometrically positioned and treated as riding on their parent atoms, with C–H = 0.93 ? for the aromatic, 0.96 ? for the methyl and N–H= 0.86 ? withiso(H) = 1.2eq(Caromatic, N) or 1.5eq(Cmethyl).

2. 4 Antimicrobial test

Preliminary in vitro tests for antibacterial activity of the compound were carried out by disk diffusion method against Escherichia coli and Bacillus subtilis at the concentration of 1, 0.5 and 0.25 mg/mL, respectively, with the solvent of DMF. The disks with tested compounds and the blank (solvent) were added onto the Petri dishes inoculated with the tested bacterial strains. After cultivation for 24 h at 37 ℃, diameters of the inhibition zone were measured and DMF was inactive under the applied conditions. Norfloxacin was used as a reference.

2. 5 Forming mechanism of the compound

To gain a better understanding of the compound, we further studied the forming mechanism and some chemical thermodynamic properties. To the possible addition reaction and dehydration reaction during the formation of the title compound, the RHF/6- 31G(d) level was adopted to study the optimization configuration and harmonic frequency of the reac- tants, products, intermediates and transition states. The possible forming route of the title compound is shown in Fig. 1. All the quantum chemical calcula- tions were performed using Gamess[22]. The graphics program used to prepare figures is Macmolplt 7.4.2[23].

Fig. 1. Possible forming route of the title compound

3 RESULTS AND DISCUSSION

3. 1 Spectroscopic characterization

IR spectrum of the compound exhibits a strong and sharp band at 1571 cm-1assigned to the phenyl ring,thebands at 3336 and 3429 cm-1are attributed to N–H, and that at 1637 cm-1to C=N, which suggest the formation of the Schiff base. In the1HNMR spectrum, the signals at 7.00～8.00 ppm are assigned to the protons of aromatic ring. The singlets at 8.70 and 8.45 ppm are attributed to the proton of S=C–NH2and S=C–NHgroups, respectively. The signal of NH proton in the pyrazol ring is observed at 2.54 ppm. In the UV data of the compound, three absorption peaks at 242, 260 and 282 nm belong to thetransition of phenyl group,transition andtransition of the conjugated system, respec- tively, which shows a large conjugated system in the molecule of the compound. The elemental analysis data are in good agreement with the formu- lae proposed for the compound. All the data of infrared, ultraviolet, MS,1H NMR and elemental analysis suggest that the assigned structure of the compound is in consistent with the expected one.

3. 2 X-ray crystallography

The perspective view of the title compound is shown in Fig. 2. The selected hydrogen bonding interactions are listed in Table 1. In the compound, the N(4), H(4), N(3), C(11), C(8), C(7) and O(1) atoms of the carbonyl group bonded to the pyrazole ring form a nonplanar seven-membered ring which adopts an envelope conformation via intramolecular N(4)–H(4)···O(1) hydrogen bond. The coplanar atoms O(1), C(7), C(8) and C(11) (with the largest deviation of –0.0146(20) ? for atom C(7)) form the mean plane of the envelope. The mean plane and the bonded pyrazole ring are essentially coplanar, with the dihedral angle to be 3.72(8)°. The N3–N4–H4 plane forms the up-warping part of the envelope, making a dihedral angle of 49.27(92)° with the mean plane. The pyrazole ring of the molecule makes dihedral angles of 32.64(7) and 60.24(7)° with the benzene and thiophen rings, respectively. An intermolecular N(2)–H(2)···O(1) hydrogen bond is observed in the structure of the compound (Table 1, Fig. 3), which links the molecules into a one-dimen- sional chain structure. Weak N(5)–H(5)A···O(1) links the one-dimensional chains into a two-dimen- sional structure.

Fig. 2. Molecular structure of the title compound with atomic numbering scheme

Fig. 3. Chain formed by the intermolecular N–H···O hydrogen bond (shown in dashed lines) in the title compound

Table 1. Hydrogen Bond Lengths (?) and Bond Angles (°)

Symmetry codes: (a)–1/2, –+3/2,–1/2; (b)–1/2, –+3/2,+1/2.

3. 3 Antimicrobial activity

The average diameter data of inhibition zone are listed in Table 2. The result shows that the title compound nearly has no ability to inhibit the growth of E. coli (gram-negative), but it has some ability to inhibit the growth of bacillus subtilis (gram-positive), and the inhibition effect is the same as the contrast (Norfloxacin).

Table 2. Average Diameter Data (mm) of Inhibition Zone and Inhibition Rate（%）of the Compound

3. 4 Forming mechanism study of the compound

The configurations of reactants, products, inter- mediates and transition states optimized at the RHF/6-3lg(d) level are shown in Fig. 4.Potential energies of the intermediates and transition states in the reaction are depicted in Fig. 5.

Fig. 4. Optimized configurations of reactants, products, intermediates and transition states at the RHF/6-3lg(d) level

Fig. 5. Potential energy of the intermediates and transition state in the reaction

The formation mechanism of the title compound is as follows:

There are two steps in the reaction of HPMTP and thiosemicarbazide. Firstly, the distal amino group of thiosemicarbazide is added to the 4-carbonyl group of HPMTP, undergos transition state TS1, then forms the stable intermediate TM. The calculated activation energy of this step is 313.86 kJ/mol. To form TS1, the bond lengths of N(34)–H(17) and C(26)=O(35) of the reactants become longer from 1.002, 1.198 ? to 1.201, 1.326 ?, respectively. With the leaving of atom H(7), atom N(34) moves towards atom C(26) and finally forms intermediate TM. It is proved by the IRC calculation that transi- tion state TS1 is really bonded to the reactant and the intermediate TM. The Δof the reaction is 121.80 kJ/mol, which shows this is an endothermic reaction. The Δof the reaction is 139.94 kJ/mol, so it is not spontaneous at 298 K. Secondly, a dehydra- tion reaction occurs in TM undergoing transition state TS2, and finally the stable product PC is generated. The calculated activation energy of this step is 204.756 kJ/mol. The bond length of N(34)– C(26) in TM becomes shorter from 1.473 to 1.446 ? to form TS2. With the departure of water molecule, the final product PC is formed. It is proved by the IRC calculation that transition state TS2 is really bonded to the product PC and the intermediate TM. The Δof the reaction is –54.986 kJ/mol, sugges- ting it is an exothermic reaction. The Δof the reaction is –73.096 kJ/mol, so this reaction is spontaneous at 298 K.

(1) Patil, S. A.; Naik, V. H.; Kulkarni, A. D.; Kamble, U.; Bagihalli, G. B.; Badami, P. S. DNA cleavage, in vitro antimicrobial and electrochemical studies of Co(II), Ni(II), and Cu(II) complexes with-substituted thiosemicarbazide Schiff bases.2010, 4, 688-699.

(2) Pahontu, E.;Fala, V.; Gulea, A.; Poirier, D.; Tapcov, V.; Rosu, T. Synthesis and characterization of some new Cu(II), Ni(II) and Zn(II) complexes with salicylidene thiosemicarbazones: antibacterial, antifungal and in vitro antileukemia activity.2013, 8, 8812-8836.

(3) Klayman, D. L.; Bartosevich, J. F.; Griffin, T. S.; Mason, C. J.; Scovill, J. P. 2-Acetylpyridine thiosemicarbazones. 1. a new class of potential antimalarial agents.1979, 7, 855-862.

(4) Andrei, G. M.; Pieroni, O. I.; Gatica, H. S.; Coto, C. E. Substituted benzaldehyde thiosemicarbazone with antiviral activity against poliovirus.1984, 12, 869-873.

(5) Shipman, J. C.; Smith, S. H.; Drach, J. C.; Klayman, D. L. Antiviral activity of 2-acetylpyridine thiosemicarbazones against herpes simplex virus.1981, 4, 682-685.

(6) Hu, W. X.; Zhou, W.; Xia, C. N.; Wen, X. Synthesis and anticancer activity of thiosemicarbazones.2006, 8, 2213-2218.

(7) Kalaivani, P.; Prabhakaran, R.; Ramachandran, E.; Dallemer, F.; Paramaguru, G.; Renganathan, R.; Poormima, P.; Vijaya Padma, V.; Natarajan, K. Influence of terminal substitution on structural, DNA, protein binding, anticancer and antibacterial activities of palladium(II) complexes containing 3-methoxy salicylaldehyde-4 (N) substituted thiosemicarbazones.2012, 8, 2486-2499.

(8) Su, W.; Qian, Q.; Li, P.; Lei, X.; Xiao, Q.; Huang, S.; Huang, C.; Cui, J. Synthesis, characterization, and anticancer activity of a series of ketone-N4-substituted thiosemicarbazones and their ruthenium(II) arene complexes.2013, 21, 12440-12449.

(9) Klayman, D. L.; Scovill, J. P.; Bartosevich, J. F.; Mason, C. J. 2-Acetylpyridine thiosemicarbazones. 2. N4, N4-disubstituted derivatives as potential antimalarial agents.1979, 11, 1367-1373.

(10) Scovill, J. P.; Klayman, D. L.; Franchino, C. F. 2-Acetylpyridine thiosemicarbazones. 4. complexes with transition metals as antimalarial and antileukemic agents.1982, 10, 1261-1264.

(11) Sun, Q. Z.; Liu, H.; Chai, L. Y. Crystal structure of a hexanuclear silver(I) compound [Ag6L66]·4DMF and luminescence discussion of a series of silver (I) clusters.2013, 5, 667-672.

(12) Sun, Q. Z.; Liao, S. Y.; Chai, L. Y.; Xu, X. W.; Yao, J. J.; Fang, Q. J. L. Synthesis, structure and comparison of luminescent property between two hexanuclear silver (I) thiosemicarbazone clusters.2012, 9, 1229-1234.

(13) Rana, A.; Parekh, N.; Dabhi, H.; Bhoi, D.; Kumari, N. Synthesis, crystal structural characterization and biological properties of thiosemi-carbazones of schiff bases derived from 4-acyl-2-pyrazoline-5-one.2011, 4, 1820-1831.

(14) Guo, M.; Guo, J.; Jia, D.; Liu, H.; Liu, L.; Liu, A.; Li, F. Synthesis, properties, and mechanism of two novel photoisomerization pyrazolones in the solid state.2013, 1035, 271-276.

(15) Vyas, K. M.; Jadeja, R. N.; Patel, D.; Devkar, R. V.; Gupta, V. K. A new pyrazolone based ternary Cu(II) complex: synthesis, characterization, crystal structure, DNA binding, protein binding and anti-cancer activity towards A549 human lung carcinoma cells with a minimum cytotoxicity to non-cancerous cells.2013, 65, 262-274.

(16) Liu, G.; Liu, L.; Hu, X.; Jia, D.; Yu, K. Synthesis and crystal structural characterization of a thiosemicarbozone derived from 4-acylpyrazolone.2006, 10, 1233-1237.

(17) Hu, J.; Liu, L.; Jia, D.; Guo, J.; Xie, X.; Wu, D.; Sheng, R. Crystal structures and photoisomerization behaviors of five novel pyrazolone derivatives containing furoyl group.2011, 1, 117-124.

(18) Li, J. Z.; Sha, J. Q.; An, Y. M.; Yu, W. J. Synthesis, characterization and bioactivity of Ni(Ⅱ), Co(III) and Cr(III) complexes of acylpyrazolone- thiosemicarbazne.2004, 1, 23-26.

(19) Jensen, B. S. The synthesis of 1-phenyl-3-methyl-4-acyl-pyrazolones-5.1959, 8, 1668-1670.

(20) Sheldrick, G. M. A short history of SHELX.2007, 1, 112-122.

(21) Sheldrick, G. M.. University of G?ttingen, Germany 1997.

(22) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S. J.; Windus, T. L.; Dupuis , M.; Montgomery, J. A. General atomic and molecular electronic structure system.1993, 11, 1347-1363.

(23) Bode, B. M.; Gordon, M. S. MacMolPlt: a graphical user interface for GAMESS.1998, 3, 133-138.

18 February 2014;

27 May 2014 (CCDC 986237)

① This project was supported by the National Natural Science Foundation of China (No. 81303306), Natural Science Foundation of Tianjin (No. 14JCQNJC06300) and Science Development Foundation of Tianjin Agricultural College (No. 2011N06)

Born in 1975, associate professor. E-mail: zhuhualing2004@126.com; Huang Zhi-Qiang, Born in 1979, lecturer.