Synthesis, Characterization, and Microbiocidal Activity of &kthyl-( 2-Thiophenomethylene) Aryloxyacetic Acid, Hydrazides and Their Metal Complexes R@sh Malhotra, Mange1 S. Mallk, Jai P. Shgh, and Kuldlp S. Dhlndsa Department of Chemistry and Biochemistty, India

Haryana Agricultural University, Hisar,

ABSTRACT Complexes of o~methyl-(2-thiophenomethylene) aryloxyacetic acid hydrazides with Cu(II) and Zn(II) metal salts were synthesized aod characW& by elemental analysis, molecular weight detemkation, molar conductance, and magnetic moment and spe&W+c techni-. In these complexes, the ligands form a conjugate O-N-S tridenate system, thus coordinating with metal through oxygen of the carbonyl group, nitrogen of axomethine, and sulphur of thiophenemoiety. Octahedral geometry is proposed for all the complexes. Antifungal activity of the lii and their Cu(II) and Zn(II) complexes was determkd against plant pathogenic fungi viz. Altemaria alternata, Rhiwctonia soiani, Colletotrichum capsicum, and Glomemtla cinguiata at 2g’C. Antibwterial activity of ligands and their metal complexes was determined on gram positive Bacillus subtik andgram negativeEwherichio coli bwteria at 37’C by the serial dilution method. In some cases an increase in biocidai activity of the ligands on coordination with metal ions was observed in terms of minimum inhibitory concentration (MC) values.

INTROiXJCTION Organic ligands containing nitrogen, oxygen, or sulphur are mported to possess a wide range of biological activities [l-3] possibly due to the involvement of the donor sites for big with biological receptors. Acid hydrazides having nitrogen and oxygen as donor sites are amkated with mtimicrobial [4], antituberkular[5], and

Address reprint reqesta to: Dr. Kuldip S. Dhindsa, Department of Chem&y Haryana Agricultural University, Hisar-125 004, Indii.

and Biochemistry,

Journal of Inorganic Biochemistry, 45,269-275 (1992) 269 @ 1992 Elscvier sCicncc Publishing Co., Inc., 655 Avenue of the Amwicru, NY, NY 10010 01624134/92/$5.00


R. Malhotra et al.

















antiinflammatoryactivities [6]. The microbial activity of these compounds is due to the fact that the nitrogen and oxygen of amino and carbonyl groups are present in the free state and therefore can combine easily with fungi cells to check their growth [7]. The literature reports reveal that the microbiocidal activity of a potential biologically active ligand can be altered several times on being coordinated with suitable metal ions [8- 111. Keeping this in view, the synthesis and bioevaluation of some new ligands having an O-N-S tridentate system and their Cu(II) and Zn(II) complexes were undertaken to explore the possibility of their use as biocidal agents against some microorganisms. The selection of bacteria and fungi was based upon their economic importance.

EXPERIMENTAL All chemicals used were of A.R. grade and the solvents were distilled prior to use. The aryloxyacetic acid hydrazides were synthesized by the reported method [ 121. Synthesis of Ligands cr-Methyl-(24hiophenomethylene) aryloxyacetic acid hydrazides were synthesized by refluxing substituted phenoxyacetic acid hydrazides with 2-acetylthiophene in 1: 1 molar ratio in alcohol for about 3 hr. The reaction mixture was allowed to cool overnight at room temperature. The crystals obtained were filtered, washed with ether, and dried. Synthesis of Metal Complexes A mixture of metal salt and ligand (1:2) in ethanolic medium (pH = 10) was refluxed for 2 hr. The solid thus separated was filtered, washed well with ethanol, and dried under vacuum. Microbiocidal Assay The ligands and their complexes were tested against plant pathogenic fungi viz. Aiternaria alternata, Rhizoctonia solani, Colletrotrichum capsicum, and Glomeralla cingulata; and bacteria viz. Escherichia coli and Bacillrcs subtilis. The proper temperature, pH, necessary nutrients, and growth media free from other microorganisms were employed for the preparation of cultures of fungi and bacteria,



using aseptic techniques [ 131. All the apparatus used were sterilized by reported procedure [ 141. The stock solutions of synthesized compounds were prepared in DMSO and tested according to the serial dilution method [16, 171 to determine the MIC values. DMSO was taken as control. The incubating period for bacteria was 24 hr at 37°C and for fungi, 72 hr at 28°C. The conventional fungicide 2-(methoxycarbamoyl) benzimidazole (bavistin) and streptopencillin as bactericide were used as standards. The plots (cont. vs compounds) were drawn for comparing the fungicidal activity results and are given in Figure 1. Physical Methods Infrared spectra of the compounds were recorded on at Perkin-Elmer spectrophotometer, model 1850, range 4000-200 cm-’ in nujol mull. ‘H NMR spectra were recorded on a Varian EM 390-90 MH, spectrophotometer, using TMS as internal standard in DMSOd, or TFA. Electronic spectra of the metal complexes were recorded on a Hitachi 330 spectrophotometer using DMSO or DMF as reference. Molecular weights of the complexes were determined by cryoscopic methods. Conductance measurements were carried out in dry DMSO or DMF at low3 M on a Digital Conductivity Meter, model NDC 732. Magnetic measurements were carried out at 30°C by Guoy’s method using Hg [Co(CNS),] as the calibrant. All ligands and complexes were analyzed for C, H, and N on an automatic elemental analyzer, model 1106. Copper, zinc, and sulphur, in the compounds were estimated gravimetrically [ 171.

RESULTS AND DISCUSSION Refluxing of aryloxyacetic acid hydrazides with 2-acetylthiophene in ethanol resulted in the formation of title ligands. The homogeneity of these ligands was checked regularly by thin layer chromatography (TLC) over silica gel. The compounds were characterized by spectroscopic techniques and elemental analyses (Table 1). In the ‘H NMR spectra, singlets at 5.1 and 3.2 ppm were assign for OCH_, and -CH_, protons, respectively. The protons of thiophene moiety appeared at 7.3 and 7.7 ppm and the protons of aryloxy moiety appeared as multiplet at 8.6-8.1 ppm. The integral proton ratio of various groups in the spectrum of each ligand was well in agreement with the proposed structure. Complexes of ar-methyl-(2-thiophenomethylene) aryloxyacetic acid hydrazides with Cu(II) and Zn(II) salts were prepared and characterized by elemental analysis (Table 2) and molecular weight determination. The complexes were found to possess general formula ML, where HL is the ligand. The molecular conductance values in dry DMF/DMSO at 10e3 M showed that complexes of both Cu(II) and Zn(II) were nonionic in nature. Infrared Spectra The important infrared frequencies along with their assignments are given in Table 3. The infrared spectra of free ligands (HL) exhibit Y NH absorption bands at 3230-3210 cm-’ but the expected v OH frequency at 3350-3500 cm-’ was not observed, which indicates that in solid state the ligands exist in ketonic form (I). However, in solution and in the presence of metal ions these ligands may form an equilibrium mixture of tautomeric forms I and II.














FIGURE 1. Antifungal activity of ligands @IL,_,) and complexes (Cu(L,_,& and Zn(L,_,), Rhizoctonia Solani (X ), Colletotrichum Capsicum (O), and Giomerella Cinguiata (A).




Zn(LN)2 Zn(Lv?J -(WI

against Alternuriu Altemutu






AnalyticalDataofLigands Analytical data (96) Found (Rqd.)



M.P. (‘C)

Yield (96)





180 207 205 224 211 235

75 80 85 75 80 80

61.0 (61.3) 54.6 (54.4) 54.0 (54.4) 52.3 (52.6) 61.1 (60.7) 44.2 (44.5)

4.9 (5.1) 4.1 (4.2) 4.2 (4.2) 4.0 (4.1) 4.9 (5.1) 3.1 (2.9)

10.3 (10.2) 9.0 (9.1) 8.9 (9.1) 12.8 (13.2) 9.2 (8.9) 7.3 (7.4)

11.5 (11.7) 10.6 (10.4) 10.1 (10.4) 10.2 (10.0) 9.7 (10.1) 8.6 (8.5)

TABLE 2. Analytical and Magnetic Data of Cu(lT)and Zn(II) Complexes Analytical Data (46) hund (Reqd.) compound WL,),

~Wz WL,), Whh w&)2 CM.


Zn(W, Zn(W2 Zn(hl)2 zn(LlV)2 Zn(L








peff B.M.

54.8 (55.1) 49.4 (49.3) 49.2 (49.3) 47.8 (47.9) 54.8 (55.2) 41.3 (41.0) 54.8 (55.0) 49.6 (49.2) 49.1 (49.2) 48.2 (47.9) 54.8 (55.1) 41.2 (40.9)

4.1 (4.3) 3.7 (3.5) 3.2 (3.5) 3.6 (3.4) 4.1 (4.3) 2.6 (2.4) 4.3 (4.25) 3.7 (3.5) 3.2 (3.5) 3.1 (3.4) 4.2 (4.3) 2.8 (2.4)

8.9 (9.2) 8.7 (8.2) 8.4 (8.2) 9.8 (10.0) 7.8 (8.0) 6.4 (6.8) 9.4 (9.2) 8.0 (8.2) 8.2 (8.2) 9.6 (9.9) 7.8 (8.0) 6.5 (6.8)

10.2 (10.5) 9.6 (9.4) 9.1 (9.4) 8.9 (9.1) 8.9 (9.2) 8.1 (7.8) 10.2 (10.5) 9.2 (9.4) 9.6 (9.4) 9.2 (9.1) 8.9 (9.2) 7.8 (7.8)

10.3 (10.4) 9.1 (9.3) 9.4 (9.3) 8.7 (9.0) 9.2 (9.1) 7.5 (7.7) 10.5 (10.7) 9.5 (9.6) 9.6 (9.6) 9.0 (9.3) 9.1 (9.4) 8.3 (7.9)

1.89 1.77 1.80 1.79 1.87 1.90


TABLE 3. ImportantItbred Absorption with Assignments of Ligands and Their Complexes Y (C=N) COlIlpOUIIdS =I WI HI-m HLIV HLv HJ-VI WLI), ~(W2 WL


Wh)2 WL


cuu-,)2 a, Zn(W2 W-lll)2 WLlV)2 WLv)2 Zn(L


1610 1605 1600 1610 1605 16fXl 1580 1580 1570 1585 1580 1575 1580 1575 1570 1580 1580 1580




(N-N) 1080 1080 1070 1075 1080 1070 1040 1040 1045 1045 1040 1050 1050 1040 1045 1030 1035 1040



450 455 460 460 460 450 440 440 445 450 440 445



390 380 380 370 375 380 360 365 360 375 365 360



280 280 280 290 300 295 280 300 305 300 290 290


R. Malhotra et al.

Since all the complexes were nonelectrolyte in nature and were prepared at high pH values, the ligands possibly exist in enolic form (II) in which deprotonation easily takes place [ 181. This was further confirmed by the absence of Y (C=O) frequencies expected at 1680-1640 cm-’ in the spectra of the complexes. The coordination of oxygen atom to the metal was supported by the appearance of Y (C-O) frequencies at 1120-1090 cm-‘. The coordination of azomethine nitrogen atom to the metal ions was indicated by the displacement of the band assigned to v (C=N) stretch. The complexes showed a negative spectral shift of v (C=N) vibration from 1610-1600 Cm-’ in free ligands to 1580-75 cm-’ in the complexes. This indicates that the contribution of C=N stretching has been reduced as the nitrogen atom is involved in bond formation with the metal ion [ 191. The participation of azometbine nitrogen atom in coordination was further confirmed by the shifting of v (N-N) stretching from 1080 cm- ’ in the ligands to 1050-1040 cm-’ in the complexes [20]. The coordination through sulphur of thiophene moiety is confirmed by splitting and shifting of the ring frequencies which are. observed at 570 and 490 cm- ’ in the ligand [21]. In the far IR region the metal complexes showed bands at 460-430 cm-‘, 390-360 cm-‘, and 300-280 cm-’ which were assigned to M-O, M-N, and M-S vibrations, respectively, [22]. These observations confirm the tridentate nature of the ligands. Magnetic and Electronic Spectra The magnetic moment value of Cu(II) complexes have been observed in the range 1.77- 1.90 B.M. (Table 1) which correspond to one unpaired electron [23] and sp3d2 hybridization with octahedral geometry. A broad band in the region of 14000- 15000 - ’ in the absorption spectra of Cu(II) complexes is probably due to *Es-*T,, rrzsitions, thereby suggesting the distorted octahedral configuration [24]. The Zn(II) complexes have been found to be diamagnetic in nature. On the basis of elemental analysis, infrared spectra, molecular weight determination, and molar conductance studies, octahedral structures are proposed for Zn(II) complexes. Microbiocidal Activity Antibacterial Activity. The ligands and complexes were tested against E. coli. and B. subtilis. All the ligands and complexes were found to exhibit complete growth inhibition against both organisms at 50 ppm. Except for complexes Zn(L,),, Cu(L,),, and Zn(L,),, all ligands and complexes inhibited the growth of bacteria at 25 ppm. The ligands and Cu(L,),, Zn(L,),, and Cu(L,,), complexes exhibited activity at 12.5 ppm. Among the complexes only Cu(L,), could show growth inhibition at 6.25 ppm. Streptopenicilline, used as a standard, was found to be much more effective than the ligands and complexes. Antifungal Activity. The ligands and their Cu(II) and Zn(II) complexes were tested against various plant pathogenic fungi viz. Alternaria alternata, Rhizoctonia



solani, Colletotrichum

caps&m, and Giomeralla cingulata. A perusal of the activity data reveals that G. cingulata was the most affected and R. solani the least affected organism by the ligands. Among the ligands, HL, and HL, were the most active. In general, Cu(II) and Zn(II) complexes were more active than the corresponding ligands and Cu(II) complexes showed more toxicity than Zn(II) complexes. R. solani was least effected by the ligands but it was most effected by the complexes. A. alternata was moderately effected by ligands and complexes. Interestingly, the ligands HL v and HL,, showed good growth inhibition for G. cingulata which decreased on complexation with Cu(II). No definite change of activity was observed with the change of structure of ligands and complexes. All the ligands and complexes were less active than bavistin, used as a standard for the comparison of results.

REFERENCES 1. K. Dey, J. Sci. Ind. Res. 33, 76 (1974). 2. K. K. Chatnrvedi and R. Kansal, Znd. J. Phurm. 37, 85 (1975). 3. C. 0. Wilson, 0. Gisvold, and R. E. Dcerge, Text Book of Organic Medicinal and Pharmaceutical Chemistry, 7th Ed., Lippincott, Philadelphia, 1977. 4. N. K. Thimmaiah, L. Loyd, D. W. Chandrappa, and T. Gujjarahalli, Transition Met. Chem. 10,99 (1985). 5. H. M. Mokhtar, Pharm. Zie. 39, 150 (1979). 6. T. M. Aminabhavi, N. S. Biradar, and W. E. Rudziuki, Znorg. Chim. Actu. 78, 107 (1983).

7. S. P. Mittal, S. K. Sharma, R. V. Singh, and J. P. Tandon, Curr. Sci. 50, 483 (1981). 8. J. R. J. Sorenson, J. Med. Chem. 19, 135 (1976). 9. R. C. Sharma, R. S. Shanna, and S. P. Tripathi, Curr. Sci. 52, 410 (1983). 10. S. Ghosh, T. K. Bandyopadhyay, P. K. Ray, and M. S. Misra, J. Znorg. Biochem. 20, 79 (1984). 11. Y. Pin and 2. Xiaoping, J. Inorg. Biochem. 37, 61 (1989). 12. H. Otzalesi, J. Castel, P. Fulcra& P. Chevallet, D. Soulas, and A. M. Noel, Tran. Sot. Pharm. Montpelier. 33, 623 (1974). 13. E. R. Rawlins, Bentley’s Text Book of Pharmaceutics, 8th Ed., Boiiiere Tindall, London, 1977. 14. R. Cruichank et al., Medical Microbioiogy . The Practice of Microbiology, 12th Ed., Churchill Livingstone, Edinburgh, 1975. 15. C. G. Donald and A. R. Williams, Assay Methods of Antibiotics. A Laboratory Manual, Medical Encyclopedia, 1955. 16. J. C. Gould, Brit. Med. Bull. 16, 29 (1960). 17. A. I. Vogel, QuantitativeInorganic Analysts, Longmans, London, 1977. 18. L. S. Sacconi, J. Amer. Chem. Sot. 74, 4503 (1952). 19. M. Mashima, Bull. Chem. Sot. Japan 37, 974 (1964). 20. M. Mohan, P. Shanna, and N. K. Jha, Znorg. Chim. Actu 106,117 (1985). 21. G. Socrates, Infrared Characteris& Group Frequencies, John Wiley, (1980). 22. J. R. Ferrarro, Low Frequency Vibrations of Inorganic and Coordination Compounds, Plenum, New York, 1971. 23. R. L. Dutta and M. M. Hossain, Indian J. Chem. 23A, 30 (1984). 24. M. Kelton, A. B. P. Lever, and B. S. Rarnaswamy, Can. J. Chem. 48, 3185 (1984).

Received July 15, 1991; accepted September 27, 1991

Synthesis, characterization, and microbiocidal activity of alpha-methyl-(2-thiophenomethylene) aryloxyacetic acid hydrazides and their metal complexes.

Complexes of alpha-methyl-(2-thiophenomethylene) aryloxyacetic acid hydrazides with Cu(II) and Zn(II) metal salts were synthesized and characterized b...
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