Synthesis, Crystal Structural Characterization and Biological Properties of Thiosemicarbazones of Schiff Bases Derived from 4-Acyl-2-pyrazoline-5-one

: A novel synthesis, single crystal and biological activity of 4-acylthiosemicarbazone-3-methyl-1-(4`-methylphenyl)-2-pyrazolin-5-one by condensation of 4-acyl-3-methyl-1-(4`-methylphenyl)-2-pyrazolin-5-one with thiosemicarbazide was carried out. The compounds were characterized on the basis of elemental analysis, IR, 1 H NMR, Mass, DSC and 13 C NMR spectral data. The compounds were tested for their antibacterial activity against various gram +ve and -ve bacteria. The results were compared with the marketed drugs. The crystal structure was determined by single x-ray diffraction. 4-Acetyl thiosemicarbazone-3-methyl-1-(4`-methylphenyl)-2-pyrazolin-5-one(AcPTMP-ths) crystallizes in the monoclinic system, space group P21/n with a=6.0828(7)Å, b=29.547(4)Å, c=7.9101(15)Å, α =90°, β =95.602(15)°, γ =90°, V=1414.9(4) Å 3 , Z=4, D c =1.429 mg/m 3 and 4-Propionylthiosemicarbazone-3-methyl-1-(4`-methylphenyl)-2-pyrazolin-5-one ( PropPTMP-ths) crystallizes in the monoclinic system, space group P21/c with a=13.5622(10)Å, b=13.3671(12)Å, c=22.151(2)Å, α =90°, β =93.13(7)°, γ =90°, V=4010.1(6) Å 3 , Z=8, D c =1.310 mg/m 3 . The compounds were screened for antibacterial properties and exhibited potential activity. -4-methylthi-osemicarbazide or 4,4-dimethyl thiosemicarbazide or 4,4-diphenyl thiosemicarbazide (l.1 mol) was taken and to this reaction mass catalytic amount of acetic acid (~15-20 mL) was added and refluxed for 3 to 5 h. Cooled to room temperature. Solid light yellow to brown product was filtered and washed with methanol or ethanol. 4-Propionyl-thiosemicarbazone-3-methyl-1-(4`-methylphenyl)-2-pyrazolin-5-one PropPTMP-ths) was light yellow powder and its yield was 80%. Crystals of the compound was obtained by slow diffusion from DMF/ methanol solution and was stable in the air for long time. Attempt to grow crystals of the compound in many different solvents was unsuccessful. of compound in


Introduction
The chemistry of thiosemicarbazones has received considerable attention in view of their variable bonding modes, promising biological implications and structural properties [1][2][3] .
The structure diversity of thiosemicarbazide based compounds is considerably increased not only due to the condensation of the different carbonyls but also due to the alkylation of the different part of the thiosemicarbazide 4 .Thiosemicarbazone usually act as chelating ligands with transition metal ion, bonding through the sulphur and hydrazine nitrogen atom.Thiosemicarbazones and their complexes have received considerable attention because of their pharmacological activities 5 .The pyrazolone derivatives have attracted much attention because of their interesting structural properties and application [6][7][8][9][10][11] .
Thiosemicarbazones can coordinate to metal as neutral molecules or after deprotonation, as anionic ligands and can adopt variety different coordination modes.The possibility of their being able to transmit electronic effects between a reduce unit and metal centre is suggested by the delocalization of the π bonds in the thiosemicarbazone chain 12 .Thiosemicarbazone compounds can be converted in to complexes by reaction with metal ions and the reaction product has very important uses 13,6 .
The new compound of pyrazolone thiosemicarbazone (Figure 1) reported in our earlier paper was synthesized 13 .As an extension of this work, we herein report the synthesis, structural characterization and antibacterial activity of compound AcPTMP-ths and PropPTMP-ths.Reaction formulas are as follow in Scheme 1 and the reaction 4-acyl-2-pyrazoline-5-one with thiosemicarbazide produce Schiff base 11,[21][22][23] was carried out in alcohol such as ethanol or methanol at reflux temperature and crystallization by aqueous alcohol for purification as shown Scheme 2. Reaction formulas are as follow in scheme 2 A yellow prismatic crystals of the titled compounds obtained.

Measurments
The FT-IR spectra were recorded on a Perkin-Elmer infrared spectrophotometer Model 2000 in the range of 4000-400 cm -1 by using KBr pellets (Vaibhav Analytical, Ahmedabad, India).Elemental analysis (C, H, N and S) was performed on a Perkin-Elmer Model-2400 elemental analyzer (SCICAR, vallabhvidhyanagar, Gujarat (India)).Nuclear magnetic resonance spectra ( 1 H NMR and 13 C-NMR) were recorded on a Bruker -400 MHz, spectrometer using DMSO-d6 as a solvent (CSMCRI Bhavnagar, India).Single crystal x-ray powder diffraction analysis done at IIT, Powai, Mumbai.

Results and Discussion
The new Schiff base 4-acyl thiosemicarbazone-3-methyl-1-(4`-methylphenyl)-2-pyrazolin-5-one would be good precursors for metal complexes like lanthanide series and others metals like Co, Cr, Mn, Mg, Pd, Ni, Fe, Zn, Ca, Hg and As in bidentet or tridentate respectively.We disclosed analytical data like elemental analysis, 1 H NMR, 13 C NMR, IR, Mass spectra, DSC and x-ray powder diffraction for new Schiff base ligands formation confirmation.The single crystal preparation and biological activity of metal complexes synthesized from above ligands is in progress.

Structural determination
Chemical shift of hydrogen in the IR spectrum, the υ(N-H) spectral region is broad, showing the existence of N-H-O hydrogen bond.The band for υ(C=O) at 1628 cm -1 is at lower wave number than that usually found for carbonyls, reflecting the involvement in hydrogen bonding with NH of the thiosemicarbazone and pyrazol-5-one compound.In 1 H NMR N(1) is easily distinguished from that of N(2) because the latter (7.161 ppm) appears as a doublet due to that the two hydrogen atom on N(2) are nonequivalent because one of them is involved in hydrogen bonding, which is consistent with the crystal structure analysis.The single peak at 8.3 ppm is assignable to the N(1)-H.The single of 10.2 ppm is not a unit because N(5) share the same hydrogen with O on the neighboring molecule.
Suitable x-ray quality crystals of the compounds AcPTMP-ths & PropPTMP-ths were grown from dimethylsulfoxide(DMSO) and methanol solvent mixture at room temperature, and x-ray crystallographic data are recorded by mounting a single-crystal of compound PropPTMP-ths (0.23x0.21x0.18)mm 3 and compound AcPTMP-ths (0.26x0.19x0.15)mm 3 on glass fibers.Oxford diffraction XCALIBUR-S CCD area detector diffractometer equipped with an LN-2 low-temperature attachment was used for the cell determination and intensity data collection.Appropriate empirical absorption corrections were applied using multi-scan programs.Monochromated Mo Kα radiation (λ = 0.71073 Å) was used for the measurements.The crystal structures were solved by direct methods and refined by full matrix least squares SHELXL-97.Crystallographic data are presented in Table 1 where as bond length and bond angles are summarized in Table 2.A computational program PLATON 24 was used to study the involvement of weak interactions and corresponding distances are summarized in Table 3.The molecular structure along with the crystallographic numbering scheme of PropPTMP-ths is given in Figure 2. The compound crystallizes in monoclinic crystal system with space group P2 1 /c.Each unit consists of two asymmetric molecules of ligand along with two molecules of co-crystallized solvent DMSO molecule.The pyrazolone ring (N5-N4-C6-C5-C7) and phenyl (C9-C10-C11-C12-C14-C15) rings do not lie in the same plane and deviate with dihedral angle of 27.34°.The bond length of C6-O1 1.246(6)Å and S(1)-C(1) 1.682(6)Å is in good agreement with a carbon-oxygen and carbon-sulphur double bond respectively.The bond length between C9-C10 = 1.398 (7)Å is quite close to that obtained for aromatic ring system whereas, N(3)-C(2) 1.346(7)Å and N(5)-C(7) 1.316 (7)  consists well with a carbon-nitrogen double bond.Pertinent bond lengths and angles for compound PTMPP-THSC are listed in Table 2 and are lying in the reported range.

Figure 2. Molecular structure of compound PropPTMP-ths
Weak force studies reveal that fourteen hydrogen bonds are formed in the molecular packing of this compound.Six hydrogen bonds are conventional hydrogen bonds whereas remaining eight bonds are non-conventional hydrogen bonds.The non-conventional hydrogen bonds involve C-H unit as donor groups, whereas, acceptors are oxygen, nitrogen and sulphur atoms (Table 3).
Crystal packing of compound PropPTMP-ths involving six intermolecular hydrogen bonding is as shown in Figure 3 which consists of 18-membered pseudo cavity formed by hydrogen binding.

Figure 3. Packing of PropPTMP-ths forming cavity
The molecular structure along with the crystallographic numbering scheme of AcPTMP-ths is depicted in Figure 4.The compound crystallizes in monoclinic crystal system with space group P2 1 /n.Each unit consists of four molecules of the compouns.The molecule is almost coplanar and the central pyrazolone ring (N5-N4-C5-C4-C6) slightly deviate from phenyl ring with dihedral angle of 2.65°.Carbon-oxygen and carbon-sulphur double are found to be C5-O1 1.249(5)Å and S(1)-C(1) 1.672(5)Å respectively which are in good agreement with the reported literature values.Carbon-nitrogen double bond N(3)-C(2) 1.341(7)Å and N(5)-C(6) 1.305(5) consists well and the bond length between C14-C13 = 1.382(6)Å is quite close to that obtained for aromatic ring system.All other bond lengths and angles for compound AcPTMP-ths are listed in Table 2 and are lying in the reported range.

Figure 4. Molecular structure of compound AcPTMP-ths
Weak force studies reveal the presence of seven hydrogen bonds in the molecular packing of this compound.Four hydrogen bonds are conventional hydrogen bonds whereas remaining three bonds are non-conventional hydrogen bonds.The non-conventional hydrogen bonds involve C-H unit as donor groups, whereas, acceptors are oxygen, nitrogen and atoms (Table 3).
Chemical shift of hydrogen in the IR spectrum, the υ(N-H) spectral region is broad, showing the existence of N-H-O hydrogen bond.The band for υ(C=O) at 1628 cm -1 is at lower wave number than that usually found for carbonyls, reflecting the involvement in hydrogen bonding with NH of the thiosemicarbazone and pyrazol-5-one compound.In 1 H NMR N( 1) is easily distinguished from that of N(2) because the latter (7.161 ppm) appears as a doublet due to that the two hydrogen atom on N(2) are nonequivalent because one of them is involved in hydrogen bonding, which is consistent with the crystal structure analysis.The single peak at 8.3 ppm is assignable to the N(1)-H.The single of 10.2 ppm is not a unit because N(5) share the same hydrogen with O on the neighboring molecule.
The x-ray structural investigation of AcPTMP-ths shows that in this compound has a stair form packing arrangement.An ORTEP plot along with the atom numbering scheme of AcPTMP-ths is given in Figure 4 and 5.The figure also describes arrangement of the molecules in the unit cell with intermolecular content through atoms with other atoms of AcPTMP-ths.The bond lengths and angles for compound AcPTMP-ths are listed in Table 2.

Synthesis of Novel 4-acylthiosemicarbazon-2-pyrazoline-5-one
The reaction of 3-methyl-1-(4`-methylphenyl)-2-pyrazoline-5-one (PTMP) with acetyl chloride or propionyl chloride were carried out under the ordinary condition.The reaction 4-acyl-2-pyrazoline-5-one with thiosemicarbazide produce Schiff base as carried out in alcohol such as ethanol or methanol at reflux temperature and crystallization by aqueous alcohol for purification.A yellow prismatic crystal of the titled compounds obtained.The formation of 4-acylthiosemicarbazon-2-pyrazoline-5-one was confirmed by spectral data viz. 1 H NMR, 13 C NMR and FTIR.

Antibacterial activity investigation
In the present study, the antibacterial activities of the AcPTMP-ths, PropPTMP-ths and standard drugs were tested by the agar-cup method in DMF solvent (at concentration of 5000 µg/mL as well as 2500 µg/mL) and were checked against Gram negative bacteria Escherichia coli MTCC-739), Pseudomonas aeruginosa (MTCC-741), Salmonella typhi (MTCC-733), Proteus vulgaris (MTCC-1771) and Klebsiella pneumoniae (MTCC-109) and Gram positive bacteria Staphylococcus aureus (MTCC-96) and Bacillus subtilis (MTCC-619).Table 3 shows that AcPTMP-ths and PropPTMP-ths compounds have good antibacterial activities against these bacteria.The results (Table 4) showed that almost all compounds showed nearly better activity.It is interesting to note that due to the presence of pyrazolone group, these compounds were found to be efficient antibacterial agents.Use of these ligands system now covers a full of areas ranging from general consideration of metal-sulphur bonding and electron delocalization in transition metal complexes to potential biological activity work is under analysis.

Method
The antibacterial activities of AcPTMP-ths and PropPTMP-ths were studied by standard cupplate-agar diffusion method using Mueller-Hinton agar (Himedia, Bombay) 25 .The measured quantity of the fresh culture of the respective test organism (0.5 mL) was added to each heated (nearly < 40 •C) agar media tubes.The tubes were shaken well and the inoculated media were poured in the sterilized petri dishes (10 cm diameter) and then allowed to set for 30 min.The test solutions of 5000 µg/mL and 2500 µg/mL dilutions of the respective AcPTMP-ths and PropPTMP-ths (5000 µg/mL and 2500 µg/mL) were prepared in Dimethyl-formamide (DMF) .The concentrations of tested compounds were decided by MIC (Minimal Inhibitory concentration) method and selection of test cultures was decided by performing ditch method.Four cups of 5 mm diameter were cut in the culture media on the petridishes.A 50 micro liter (µL) solution of particular diluted compound (5000 µg/mL or 2500 µg/mL) was filled with micropipette in the four cups of one of the Petri dishes.All the petri dishes were allowed to remain in the refrigerator maintained at < 10 •C for < 1 h to allow diffusion of the respective solutions.The petridishes were then transferred to an incubator maintained at < 37 o C and kept for nearly 24 h.The zones of inhibition appeared were measured with vernier calipers.The control of DMF showed slight activity.The activities of all compounds are represented by size of the diameter of zone of inhibition in millimeter.
The antibacterial activity of standard antibiotics was performed by following standard Kirby-Bayer method (combi-disc method, purchased from Pathoteq Biological Laboratories (India)).The antibacterial activity of all known antibiotics is represented by size of the diameter of zone of inhibition in millimeter.

Conclusion
A number of multi-functionalized 4-acylthiosemicarbazone-2-pyrazoline-5-ones were conveniently prepared by literature method.Investigation of biological activities of this type of ligands and their metal complexes have identified them as promising potential antibacterial agent against Gram-positive as well as Gram-negative bacteria.They have broad-spectrum activity and were found as competitive as marketed known antibiotics drugs.

3 Figure 1 .
Figure 1.Pyrazolone thiosemicarbazone Many 4-acyl derivatives of 1-phenyl-3-methyl-2-pyrazoline-5-one have been prepared and applied for the metal complexes.The reaction pyrazolone with acetyl chloride and propionyl chloride were carried out under the ordinary condition mentioned below.

Table 3 .
Selected parameters for weak interactions in complexes 1 & 2