I

,,

Ligational Behavior ;of WSubs&uted Acid Hydrazides Towards Transition Metals and Potentiation of Their Microbiocidal .Activity Rajesh Malhotra, Jai Pal Singh, Mamta Dudeja, and Kuldip Singh Dhindsa Department of Chemistry and Biochemistry, Haryana Agricultural University, Hisar, India

ABSTRACT New complexes of Cu(n), Ni(II), and Co(E) with 3-benzoyl-1-[2-N-(substituted-2’-thienyl methyhm%hylene/methylene)] prop-2-ene-l-oic acid hydrazides have been synthesized and characterized by elemental analysis, molecular weight determination, molar conductance, and magnetic moment and spectnrscopc techniques. Conductaace measurements h&ate the nonionic nature of the complexes. From the spectroscopic studies, it h&s been concluded that the N-substituted acid hydraxkk act as tridentate ligands forming an O-N-S coqjugate system and coordinating with metal ions through oxygen of carbonyl group, nitrogen of m, and sulphur of thiophene moiety. mgeometryh@ been pqosed for all the complexes. The ligands and theii complexes were tested for in vitro growth inhibitory activity against phytopathogenic fungi viz. Altemoria aiternata, Coiletotrichum cap&urn, Fusarium orysporum, and Rhizoctonio soiani at 28’C; and backria viz. pm positive Bacillus subtik sod pm qative Ekherichio coli at 37’C by a two-fold serial dilution technique. In some casesan increaseinthebiocidalactivityoftheligandsasaconsequenceofcoordinationwithmecalions was observed in term8 of minimum inhibitory conccW&on (ME) values. The trend of growth inhibitionintbecolnplcxeswasfoundtobein~order: Cu>Ni>Co.

INTRODUCTION Aromatic acid hydmides have evoked considerable intemt of coordination chemists andbiochemistsbecauseof~~esversetilecbelatingagentstowards various metal ions [l-3]. Acid hydmzides exhibit a broad spcchum of biological activities which incl& bacterkidal[4], fungicidal [S], nemticidal[6], antislar{73,aad~~~].Tbcactirityof~af(hepotentiellybidogically

~repriattequesEst~:P~~f~~rK.S.Dl~i~W,Deparrmant~fQlemieeydBiochemistry, Hazyma Agridtud University.Hisar-125004, India.

Journalof InorganicBiachemisny,46,119- 127(1992) 0 1992Elscvii SciaKsPub-

co.. Inc..655Avalle oftk Ammk.88, NY, NY 10010 01624m4/92,$5~:

l;ag R. MMhotm et al.

-AL ZQIcety~, 2-acetyl-s-chloro-tbioRhene, and 2uuboxyaldehyde thiophene weru promued from Aldrich. AU other chemicals used were of A.R. grade and s&en& were d&iikd prior to use. 3-Renxoyl-prop2-ene-l-oic acid hydraxides were ayll&&& by the reported method [13].

3-Substi~-~yl-~2~~-l~ic acid hydra&de (0.01 mol) was refluxed on a steam bath with ~-2~I/~xy~~y& thiophene (0.01 mol) in ethanol (25 ml) for 3 hr. The & mixture was allowed to cool at room temperature and the sepam&d solid was filtered and crystallixed from ethauol to give the required @and of the following general 5trnMure: Ligand

~+N”N,c$iij

Fi;:

H

H

j;

i H H Cl

mvll

ZS-W-Q

H

=-WI

&54CH,l,

a3

HL,

2,5-(C&),

CH3

SCHEME

1.

Synthesisof MeteI Complexes Metal salt and ligand were taken in 1:2 molar ratio in ethanolic medinm @H = 10) and refluxed for about 2 hr. The solid thus obtained was fIltered, washed well with ethanol, and dried under reduced pressure.

All the synthesixed ligands and their correspondiug complexes were tested for in vitro growth inhibitory activity against ~~~oge~c fungi viz. Afternaria alternata, Coilefotrichum cap&urn, Fusarium oxysponrin , and Rhizoctonia solani; and bacteria viz. Bacillus ,subtilis and Ekherichia coli. Proper temperature, pH, necessary nutrients, and grov+th media free from other mjcroorganisms were employed for the ~0~ of cultures of mngi and ba&eria, using aseptic te&n.iques [14]. The stock solutions were prepared by dissolving the compounds in dimethylsulpboxide and testing was carried out using a two-fold serial dilution technique [15, 161 to determined MIC values. Dimethylsulphoxide was taken as control. The i~~on period for fungi and bacteria was 72 hr at 28°C and 24 hr at

N-S-

ACID HYJJRAZIDES

121

A Perkin-Ehner Model 1850, i&Wed spectropbotometer WaSusedtOobtaint8e spectra of the ligands and complexes, using both Nujol and KBr techniques. ‘H NMRspectrawe~recordedonaVarianEM390_90MHzspectrophdameter,usiag TMS as internal standard in DMSOd, or TFA. Electronic spectra of metal complexes were nxorded on a Hitachi 330 specttqhotometer using DMSO or DMF as a reference. Molecular weights of the complexes were determined by cryoscopic methods. conductance values were determined in dry DMSC or DMF at 10d3 M cont. on a Digital Conductivity Meter, NDC 732. Magnetic measurements were carried out at room temperature by Guoy’s method using mercury (II)tetmthiocyanatocobaltate (II) as the calibrant. All the ligands and complexes were analyzed for C, H, and N on automatic elemental analyser Model 1106. Metals (01, Ni, and Co) and sulphur in the compounds were estimated gravimetrically [17]. RESULTS AND DISCUSSION N-substituted acid hydraxides on refluxing with substituted-2-acetyl/carboxyal&hyde thiophene in ethanol resulted in the formation of title ligands in good yields. The ligands were characterixedby specuosqic techniques and elemental analysis (Table 1). These ligands were assigned trans coniiguration on the basis of coupling constants (16 Hz) between two downfield olefinic protons, which appeamd at 6.8 and 8.1 ppm as doublets. The protons of thiophene moiety and benxoyl group appeamd as multiplet at 7.0-8.0 ppm. CH, group attached to thiophene or phenyl group appeared at 2.7 or 2.3 ppm, respectively. The integral proton ratio of various groups in the spectrum of each ligand was well in agreement with the proposed structure (Scheme 1). Complexes of title ligands with Cu(lI), Ni(Il), and Co@) salts were prepared and characterixed by elemental analysis (Table 2), molecular weight determination, magnetic susceptibility (Table 2), molar conductance, and spectroscopic studies. The complexes were found to possess the general formula ML, where M is metal and HL is ligand. The low molar conductance values (0.5-5.0 ohm-’ cm* mol-‘) indicated their nonelectrolytic nature due to charge neutralization of the metal ion with the ligand.

infrared spectra of free ligands @IL) exhibited bands for Y NH around 3300-3260 cm-’ which disappeared in the complexes suggesting enolixation of the keto group followed by the formation of complexes through deprotonation. Since all the complexes were nonelectrolyte in nature and were pmpared at high pH values, the ligands possibly exist in enolic form in which deprotonation can easily take place [18]. This was further confirmed by the absence of NH-C=0 frequencies in the complexes, which appeamd at 1640-30 cm-’ in the parent ligands. The band at 1120-10 cm-’ due to Y C-O frequencies suggested the coordination of oxygen atom of carbonyl group to the metal.

122

R. ‘Malhotra kt al.

N-SUB!STlTUTEDACID HYDRAZIDES 123

124 R, Malhotra et al.

TABLE 1. Analytical and Spectral Data of Ligands

LigatldS

MP

Yield

(“C)

(A)

C

149

78

63.0 (63.4)

4.3 (4.2)

142

72

64.1 (64.4)

138

75

AMlytiCaldata%, Found (Reqd.) H N

S

NMR

10.1 (9.8)

11.1 (11.3)

4.5 (4.7)

9.2 (9.4)

10.9 (10.7)

57.5 (57.7)

4.2 (3.9)

(8.4)

(9’::)

6.8 (d, 1 H, olefinic H, J = 16Hz) 8.1 (d, 1 H, olelinic H, J = 16Hz) 7.0-8.0 (m, 8H, Ar-H and thienyl-H) 8.8 (8, 1 H, N = CH) 2.7 (s, 3 H, thienyl-CHB) 6.8 and 8.1 (d, 2 H, J = 16Hz 7.0-7.9 (m, 7 H, Ar-H, and thietlyl-H) 7.0-8.0 (m, 6 H, ArH, and thienyl-H) RestissameasinHL, 2.3 (s, 3 H, Ar-CH,) 7.0-8.0 (m, 7 H, Ar-H, and tbienyl-H) RestissameasinHL, 2.4 (s, 3 H, Ar-CH & 2.7 (s, 3 H, thienyl-CH,) 6.9 and 8.2 (d, 2 H, olefinic H, J = 16Hz 7.2-8.0 (m. 6 H, Ar-H, end thienyl-H) 6.9-8.0 (m, 5 H, Ar-H, and thienyl-H) RestissameasinHLV 2.4 (s, 6 H, Ar-(CH,), 6.9-7.6 (m, 6 H, Ar-H, and thienyl H) 8.8 (s, 1 H, N = CH) Olefinic protons as usual position 2.4 (s, 6 H, Ar-(CH,), 2.7 (s, 3 H, thienyl-CH,) 7.0-7.9 (m, 6 H, Ar-H, and thienyl-H) 7.2-8.0 (m, 5 H, Ar-H, end tJ3ienyl H) RestissameasHLw,

8.1

HLN

220

78

64.1 (64.4)

5.0 (4.7)

9.4 (9.4)

11.1 (10.7)

HLv

142

70

65.8 (65.4)

4.7 (5.1)

9.2 (8.9)

(1::;)

59.2 (58.9)

4.4 (4.3)

7.8

3.9

(8.1)

(4.2)

J%l

H&II

122

80

182

74

65.1 (65.4)

5.1 (5.1)

8.7 (8.9)

10.4 (10.2)

123

75

65.9 (66.2)

5.9 (6.1)

8.9

10.2

(8.6)

(9.8)

60.2 (59.9)

4.9 (5.2)

8.0

9.1

(7.8)

(8.9)

130

82

A sharp band around 1620-1595 cm-’ was diagnostic of the azine chromophore (-C=N-N=C-) [19]. The coordination through nitrogen of azomethine group was indicated by negative spectral shift of v C=N vibration from 1620-1600 cm-’ in the free ligands to 1580-70 cm-’ in the complexes. This indicates that the contributionof C-N stretching has been reduced as nitrogen atom is involved in the bond formation with metal ion. The participationof nitrogen was further cotirmed by shifting of v N-N stretching from 107040 cm-’ to 1030-20 cm-’ [20]. Splittingand shifting of ring frequencies of thiophene moiety which were observed at

N-SUBSTlTUTED

ACID HYDBAZIDBS

125

TABLE 2. Analytical and Magnetic Data of Cu(IQ, Ni(II), and Co(n) Complexes Compwnd

H

C

Analytical Data 96Found (Reqcl.) N S Metal

56.9 (57.2) 58.8 (58.4) 53.1 (52.8) 58.2 (58.4) 59.5 (59.5) 53.8 (54.1) 59.2 (59.5) 60.4 (60.5) 54.9 (55.2) 57.2 (57.6) 59.1 (58.8) 52.9 (53.2)

3.7 4.2 3.0 3.6 4.6 3.7 4.8 4.6 3.8 3.6 3.8 3.2

(3.5) (3.9) (3.3) (3.9) (4.4) (3.7) (4.4) (4.7) (4.1) (3.5) (4.0) (3.3)

W-d2

58.6 (58.8) 60.2 (59.9) 54.4 (54.4) 60.0 (59.9) 61.1 (60.9) 55.2 (55.5) 58.0 (57.6) 58.6 (58.8) 53.4 (53.2) 59.2 (58.8) 59.6 (59.9) 54.2 (54.4) 60.3 (59.9) 61.2 (60.9)

WJqx),

55.3 (55.5)

WWZ (3u(Lld2 cw-,I, cw,), cw-“12 wbl)2 WL,,), wblI)2 W&x), ww2 WL,), WL,), WL,), CM-V), WJ-VI)2 wLu)2 w&m)2 whd2 W-d2 WLd2 NW-

d2

Ni@w)2 WLv)2 W-d2 W-,)2

(10.1) (9.6) (8.7) (9.7) (9.3) (8.4) (9.3) (8.9) (8.1) (9.4) (9.0) (8.2)

CleffBM

8.0 (8.9) 8.3 (8.5) 7.8 (7.7) 8.9 (8.5) 8.6 (8.2) 7.0 (7.4) 8.0 (8.2) 7.4 (7.8) 7.4 (7.1) 8.7 (9.0) 8.8 (8.6) 7.9 (7.7)

10.2 (10.1) 10.0 (9.7) 8.9 (8.8) 10.0 (9.7) 8.9 (9.3) 8.6 (8.5) 9.0 (9.3) 8.9 (9.0) 8.3 (8.2) 9.9 (10.2) 10.0 (9.8) 8.7 (8.7)

9.8 9.9 8.9 9.6 9.6 8.4 9.0 9.1 7.8 9.1 9.3 7.9

4.2 (4.0) 4.6 (4.4) 3.5 (3.7) 4.2 (4.4) 5.0 (4.8) 3.9 (4.1) 3.3 (3.5) 4.2 (4.0) 3.6 (3.3) 3.8 (4.0) 4.2 (4.4) 3.9 (3.7) 4.6 (4.4) 4.9 (4.9)

8.3 (8.6) 8.0 (8.2) 7.6 (7.4) 8.4 (8.2) 8.1 (7.9) 6.9 (7.2) 9.1 (9.0) 8.3 (8.6) 7.4 (7.7) 8.7 (8.6) 8.0 (8.2) 7.7 (7.4) 8.4 (8.2) 8.2 (7.9)

9.6 (9.8) 9.6 (9.4) 8.3 (8.5) 9.1 (9.4) 8.9 (9.0) 8.1 (8.2) 10.3 (10.2) 9.6 (9.8) 8.6 (8.7) 9.7 (9.8) 9.2 (9.4) 8.6 (8.5) 9.6 (9.4) 8.7 (9.0)

8.9 (9.0) 8.7 (8.6) 8.2 (7.8) 8.4 (8.6) 8.6 (8.3) 7.9 (7.6) 9.6 (9.4) 8.8 (9.0) 8.0 (8.2) 9.2 (9.0) 8.4 (8.6) 8.0 (7.8) 8.7 (8.6) 8.3 (8.3)

4.10 4.52 4.12 4.50 4.20 4.20 3.27 3.05 3.02 3.06 3.30 3.14 3.35 3.42

4.4 (4.1)

7.4 (7.2)

8.0 (8.2)

7.2 (7.6)

3.17

1.76 1.79 1.81 1.74 1.84 1.77 1.74 1.86 1.79 4.72 4.60 4.35

590 and 510 cm- ’ in the ligands indicated the coordination of sulphur of thiophene ring to the metal [2 11. In the far IB region the bands at 450-420,410-390, and 300-270 cm-’ in the complexes were assigned to M-O, M-N, and M-S vibration, respectively P21. Magnetic and JZkctrouic Spectra The magnetic moments of Cu(II), Ni(B), and Co(n) complexes calculated from corrected magnetic susceptibility are given in Table 2. The magnetic moment values of Cu(H) complexes have been observed in the range 1.74- 1.86 BM, indicating spin-only value with octahedral geometry [23]. This was further congrmed by the electronic spectra of the Cu(II) complexes in which the only band observed in the region 14OtKL13CUUIcm-’ was probably due to *Es-*T2s transitions suggesting the distorted octahedral geometry [24]. All the Ni(H) complexes were proposed to have octahedral geometry on the basis of their magnetic moments (3.0-3.5 BM) [25]. This was also supported by electronic spectra in which three bands around 10500-l 1300, 17300- 19200, and 26500-27700 cm-’ were probably due to the three spin allowed transitions from 3A2s- ‘T2, (v,), ‘A2s- 3T,, (v,), and 3A2s-3T,g (v,), respectively. The energy ratio v2 /v, for these complexes was found with in the range as requimd for octahedral complexes [24, 261. The effective magnetic moments of all the Co(H) complexes were found in the range of

126 R. Malhotra et al. 4.1-4.7 BM, which suggested o&kdral

geometq of the complexes [25]. Thme bands observed in the region 7500-8100, 14400-16000, and 19480-21ooO cm-‘, could be assigned to 4T,,-4T2s (v,), 4T,,-4A2g (+), and 4T,,-4T,s transitions, respectively. The ratio Y~/Y~ for the complexes found in the range 1.9-2.2 also supported an octahedral geometry [27, 281. The above studies show that the ligand functions as neutral tridentate (ONS) donor coordinating through enolic oxygen, axomethine nitrogen, and sulphur of thiophene moiety.

Microbiocidal Activity The ligands and their metal complexes were tested against phytopathogenic fungi viz. Altemaria aitemata, Colletotrichum cap&urn, Fusarium oxysporum, and Rhizoctonia solani; and bacteria Bacillus subtilis and Escherichia coli. A perusal of the data (Figs. 1 and 2) indicatesthat most of the compounds exhibited promising activity against all the tested microorganisms. Colletotrichum cap&urn was the least effected organism by the ligands but the activity increased on complexation with metal ions and the complexes of Cu(II) were most toxic. Ligands showed more activity against Rhizoctonia solani, than the correspondingcomplexes of Ni(II) and Co@). Among the Cu(II) complexes, only Cu(Lm), and Cu(L& showed MIC values at 3.125 ppm against this organism (Fig. l(d)). Fusarium oxysporum was moderately effected organism by li8ands but their complexes were quite active. Not much variation has been observed in the activity of ligands and their Ni(II) and Co@) complexes against Aitemaria aiternata but corresponding Cu(II) complexes were more toxic. Bavistin, used as a standaid, was more effective than both the ligands and complexes. Most of the ligands showed complete inhibition growth at more than 100 ppm against E. coii, but their corresponding metal complexes were more toxic (Fig. 2(a)). Among the complexes Cu(L,), exhibited growth inhibition up to 6.25 ppm. The toxicity of ligands and complexes against B. subtilis varied between 12.5-50 ppm. All the ligands and complexes were less toxic than stre@pencillin used as a standard for the comparison of results. It is clear from the fungicidal and bactericidal screening data that some of the complexes were more toxic in comparison to the parent ligand. The incmase in the activity of the complexes may be due to the effect of metal ions on the normal cell process. Cu(II) complexes were found to be more potent than Ni(lI) and Co(n) complexes and the general trend of growth inhibition against both fungi and bacteria was found to be in the order Cu > Ni > Co.

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N-SUBSTITUTED ACID HYDRAZIDES 127

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Received November 13, 1991; accepted November 23, 1991