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Antifungal potential of binary and mixed-ligand complexes of N,2′-diphenyl acetohydroxamic acid
Antifungal Potential of Binary and Mixed-Ligand Complexes of N,2’-Diphenyl Acetohydroxamic Acid M. Janardhan Rao Department of Chemistry, University College of Engineering, Osmania University, Hyderabad-7, India
ABSTRACT Chelating potential of N,2’-DPAHA with 3d metal ions such as 01(n), Ni(II), Co(B), Zn(l.9, and Cd@) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligaad complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(H), Zn@l), and Cd(B) is presumably due to ~-bonding effects. In the stabiliion of the Co(n) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to ?r-bonding effects. Rmgicidal activity of N,Z’-DPAHA and its binary complexes with Cu(II), Ni(Il), and Co@) was examined against Fusariumoxysporumusing theinhibition zone technique. Binary complexes of Zn@) and Cd(B) with N,Z’-DPAHA and mixed-liiand complexes M(II)-Gly or Phen-N,2’-DPAHA, where M(H) = Cu(JI), Ni@), Zn(II), Co(B), and Cd(B) were screened against Alternariualfernataby slide germination technique.All mixed-Ii@ complexes exhibited fongicidal activity but did not improve signilicantly compared to binary complexes. Synergestic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:l) of the secondary ligand (N.FDPAHA), and the fungicidal potential of the mixed-ligand complex involving N,Z’-DPAHA as secondary ligand was not increased.
ABBREWIATIONS Gly, Glycine; Phen, 1.1~pbenanthrolimq N,Z’-DPAHA, N,2’-diphenyl acetohydroxamic acid; M(II), Metal nitrate ion; M, metal ion; A, primary ligand; L, secondary &and.
INTRODUCTION Wide-range biological activity of hydroxamic acids and various aspects of hydroxamic acid chemistry has inspired chemists to study the ligation behavior of these
Address reprint requests and comzspondence to Dr. M. Janardhsn Baa, Deparhmnt of Medicine, Division of Hematology, U921; AECOM, 1300 Morris Park Ave., Bronx, NY 10461 (1992). U.S.A. Journoi of [norgonic Biochemiptry, 46.207-214 (1992)
0 1992 Elscvier Science PublishingCo., Inc., 655 Avenue of the Americas, NY, NY 10010
247 0162-0134/92/$5.00
208
M. J. Rao
ligands toward various transition metal ions [l- 111. Detailed knowledge of underlying principles in the formation of mixed-ligand complexes in biological fluids strengthens their importance and, it is worthwhile to study their formation and stability. The formation of mixed ligand-complexes in solution occurs through the coordination of ligands to the metal ions either in two distinct pH regions or in overlapping pH regions, a reflection of pK of the ligating molecule. With an overall objective of investigating the nature of mixed-ligand complex formation involving NJ’-DPAHA and other ligands and their stability and fungicidal potential, a detailed study of various transition metal complexes with O-O (NJ’-DPAHA), O-N (Gly), N-N (Phen) donor ligands has now been undertaken. Our previous studies suggested that the photo-stabilization of polyolefins by binary complexes of hydroxamic acids [12] and fungicidal activity of binary metal complexes involving hydroxamic acid [13] increase with the stability of the complex. To establish a relation between structural aspects of the binary as well as mixed-ligand complexes and their biological function, we have selected two test organisms, AIternaria alternata and Fusarium oxyspomm, in the present investigation. Binary and mixed-ligand complexes of 3d metal ions such as Cu(II), Ni(II), Co@), Zn(II), and Cd(II) were synthesized and their structural stabilities and fungicidal potential were estimated. MATERIALS AND METHODS NJ’diphenylacetohydroxamic acid was prepared as described earlier and characterized by its melting point and elemental analysis [14]. The purity of the ligand was determined by thin layer chromatography. All chemicals used were of AnalaR quality (BDH, India). Metal nitrate solutions were prepared and estimated by standard methods [15]; pH measurements with an accuracy of ztO.01 pH units were recorded on a systronic pH meter-324 fitted with glass and saturated calomel electrodes. For determination of the formation constants of mixed-ligand complexes, MAL, MA’L, the following sets of solutions (50 cc) were prepared and titrated against standard KOH (50mM); 1) HNO, (2mM), 2) HNO, (2mM) + A or A’ (HIM), 3) HNO, (2mM) + L (lmM), 4) HNO, (2mM) + 1:l mixtureof A or A’ and metal nitrate (In&I), 5) HNOs(2n-M) + 1:l mixture of L and metal nitrate (In&I), 6) HNO, (2mM) + 1: 1: 1 A or A’, L and me&l nitrate (1mM) where A = Phen, A’ = Gly, and L = N,2’-DPAHA. Titrations of each set were carried out twice to check the reproducibility of the data. Titrations were carried out in water-ethanol (50% (v/v) medium at 303 K. Potassium nitrate was used for adjusting the ionic strength. The pH correction for aquo-organic solvent was carried out by the method of Van Uittert and Hass [16]. Binary and mixed-ligand complexes were prepared by refluxing the methanolic solutions of metal nitrates and ligand solutions in the ratio of 1:20 and 1: 10: 10, respectively. Metal estimations of the complexes have been carried out as reported earlier [ 131. Fuugicidal Screening Medium, Potato Sucrose Agar (PSA) was prepared as reported earlier [13]. It was autoclaved at 15 lb and the pH was adjusted to 5.0 using acetic acid. Various concentrations of 50, 100,200,400, and 500 ppM of hydroxamic acid and its binary or mixed-ligand complexes were prepared in water-methanol (50% v/v). Spore
suspension of each fungus, Alternaria alternata isolated from onion leaves, Fusarium oxysporium, which is a pathogenic on chickpeas was prepared in redistilled water from lOday-old culture. The concentration of spore suspension was adjusted to appear 80-90 per low power field ( x 100). Three drops of spore suspension, hydroxamic acid or its binary or mixed-ligand complexes, were pipetted onto the slide. Each treatment was conducted in triplicate and controls were maintained simultaneously. Germination counts were recorded in about 300 conidia (spores) after 48 hr incubation and compared with control. RESULTS AND DISCUSSION The mixed-ligand complex formation depends on the chelating ability of two ligands A and L when they are in equilibrium with a metal ion (Eqs. (l), (2), and (4)): M2++ AH “$MA-‘“-2’+nH+, MA-(“-2) + LH ,=MAL-
(1)
(n+m-Z) + mH+,
Pi& = [MALI/[MA][L] M’++AH n +LH m=MAL-(“+‘“-2)
9
- ,H++ mH+,
BMMAL = [MALI /[Ml[Al[Ll9
logS:;, =logP& - logKM,, , AlogK = log &_
(2)
- 1ogK ML.
(4)
(5) (6) (7)
The evidence for the mixed-ligand complex formation was obtained by constructing a composite curve, out of curves M(H)-primary ligand and M(H)-secondary ligand. It was assumed that there is no interaction between M(H)-primary ligand and a secondary ligand. The composite curve was placed significantly above the 1: 1: 1 experimental titration curve pointing to the interaction between M(n)-primary ligand and secondary ligand which results in the formation of the mixed-ligand complex. Further evidence for the mixed-ligand complex formation was derived from the observation that insoluble precipitates obtained during the titration of M@)-N,2’DPAHA binary system were absent during the titration at M(H)-primary ligandsecondary ligand. Stepwise formation of M(H)-Phen-N,2’-DPAHA was confirmed based on the analysis of titrations of M(lI)-Phen and M(II)-Phen-NJ’-DPAHA. Titration curves (not shown) of M(H)-Phen-N,ZDPAHA drifts away from M(H)Phen from pH 6.2 but coincides with M(H)-Phen in the low pH region, 4 to 6.2, indicating that the N,2’-DPAHA ligate in stepwise to form mixed-ligand complex. The nature of the complex equilibrium, whether stepwise or simultaneous, was also concluded based on the method suggested by Martell and Smith [ 171. The step-wise mixed-ligand stability constants (log 8) for the interaction of M(H)-Phen and N,2’DPAHA were calculated as reported earlier [ 181 and were accurate within f 0.08 log units. A study of the log /3 values (Table 1) reveal that the stability constants of mixed-ligand complexes [M(H)-Phen-NJ’-DPAHA] were significantly greater than log K, of 1:2 binary complexes [M(H)-(N,2’-DPAHA),] but are close to log K,
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TABLE 1. Statdity Constants (Iog K ,and log K, ) of Binary Complexes’ [M@)-N,2’-DPAHA] and Step-Wise Stability Constants (log 8) of Mixed-Ligand Complexesb [MO-Phcn-N,2’-DPAHA] Metal Ion ww Ni(II) Co(u) Zn(n) ‘.X-Q
log B
logK,
1ogKz
AlogKC
9.42 6.12 5.04 6.39 4.22
9.20 6.30 5.24 6.55 4.50
6.70 4.30 3.82 4.50 3.34
- 0.22 0.18 0.20 0.16 0.28
*Data from Rae et al., 1981 [23]. b log 0 is calcukd for the interaction of M(U)-Phen and NJ’-DPAHA as a step-wise mixed-&and stability constant using Irving and Rossotti’s method [18] in 50% water-ethanol mixtures (v/v) at 303 K and 0.1 M ionic strength @NOa). ‘Alog K is ehnated from the difference of logK, of M(H)NJ’-DPAHA and log fl of mixed-ligand complex [M(II)Phen-N,2’-DPAHA] .
values of 1: 1 binary complexes [M(II)-N,2’-DPAHA]. It was evident from the stability constant data that the Cu(II)-mixed-ligand system was more stable than the 1: 1 binary system of M(H)-N,2’-DPAHA. The A 1ogK values for mixed-ligand complexes which were less than the statistically expected value (0.6) indicates that the mixed-ligand complexes are significantly stabilized over the binary complexes [M(II)-(N,2’-DPAHA),]. The stabilization of the mixed-ligand complexes could be understood by considering that Phen not only induces a strong ligand field to metal u-bonding but also acts as a powerful x-acceptor. The remarkable stability observed in the mixed-ligand complexes of Cu(II) cannot be ascribed to ~-bonding effects alone because the difference in the xdonor capacity of Cum) and the other metal ions was not like the difference in the stabilization of the mixed-ligand complexes. Ligation of Phen or N,2’-DPAHA to Cu(II) (aquo) results in a stronger distortion of an already distorted octahedral configuration in inducing a sign&ant gain in stabilization energy. The ligation of NJ’-DPAHA (secondary l&and) to Cu(II) (Phen) is energetically favored over the ligation of NJ’-DPAHA to Cu(II) (aquo) because Cu(Il) (Phen) already possesses sufficiently distorted octahedral configuration, a geometry which is ideal for the ligation of NJ’-DPAHA. Thus the stabilization effected by the stereochemical favoring of the ligation of N,2’-DPAHA to Cu(II) (Phen) coupled with that induced by &onding effects over-weights the destabilization due to steak and statistical effects and leads to the observed negative A 1ogK value. This also suggests that ligands containing oxygen as donor ligands (N,2’-DPAHA) form more stable complexes with Cu(II) @hen) than with Cu(II) (aqua), and the equilibrium of the typeMA+ML=MAL+Mliesfartotheright.Thestabilizationofthemixedligand complex over 1:2 [M(II)-(N,2’-DPAHA),] binary complex of secondary ligand could also be justified by considering that the coordination of Phen, a nitrogen donor, to Cu(II) (Phen) results in a loss of Jahn-Tellar stabilization energy [ 191, but such a loss of stabilization energy is .minimum, when N,2’-DPAHA, an oxygen donor is ligated to Cu@)(Pben). The enhanced stabiiion of M(U)-Phen-N,2’-
BINARY AND MIXED-LIGAND COMPLEXES
211
DPAHA over 1:2 binary complexes of M(H)-@I$?‘-DPAHA), is presumably due to neutralktion of charge in mixed-ligand complex MA2’++ ML,‘-= 2MAL. It was suggested that when a mixed-ligand complex forms from neutral and negative ligands both enthalpy and entropy effects favor the mixed-ligand complex over the disproportion products [20, 211. For the M(H)-Gly-NJ’-DPAHA system, the overall stability constant (log 8’) was calculated from plot of pL vs log(1 - n/n) as log /3’ = pL 0.5 + pL 1.5 [18]. The log/I’ values for M(H)-Gly-N,2’-DPAHA where M(H) = Cu(II), Ni(II), Co(H), and Zn(II) complexes are 17.9 k0.12, 12.5st0.14, 10.3iO.18, and 13.2k0.12, respectively. It was clear that log @ values for the mixed-ligand complexes were greater than the corresponding binary complexes for all the metal ions studied. The results suggest that bii complexes containing two similar ligands are not as much favored as those containing two dissimilar ligands as in mixed-ligand complexes. Since Gly (0,N donor) is a better u-donor than NJ’-DPAHA (0,O donor) (see Fig. l), the electron density accumulated at the metal ion due to the L + M u-bonding in M(II)-Gly-N,2’-DPAHA complex would be more than that in M(II)(N,2’-DPAEIA), . The observed log /3’ values of mixed-ligand complexes revealed that the electronic repulsion induced by u-bonding is not affecting the stability of the mixed-ligand complex. The enhanced stabilization of M(II)-Gly-N,2’-DPAIIA may also be due to less steak eflbct by the presence of only one bulky molecule, i.e., N,2’-DPAHA as compared to that in M(H) (N,2’-DPAHA),. In the (M(@-Gly-N,2’-DPAIIA) mixedligand complex, the metal to N,2’-DPAHA bonds are weaker as compared to metal to glycine. This is due to change in the donor atoms from bii to ternary complex. The log /3 or log 8’ values of mixed-ligand complexes follow the order Cu@) > Zn(II) > Ni(lI) > Co(H) > Cd(H). The stability constants of both binary and mixedligand complexes of Zn(II) were more than those of Ni(II). It is known that the ligand containing the nitrogen atom as donor forms a stable complex with a ligand-sensitive metal ion like Ni(II) while oxygen donor ligand forms a stable complex with the ligand field-insensitive metal ion like Zn(n). In conclusion, the
c
FIGURH 1. (A) M@) (phen) (N,2’-DPAHA)), 2H20, M(H) = Cu(ll); (B) M(H) (Gly) (N,2’DPAHA), SH,O, M(H) = Cu(TI); (C) M(H) (N,2’DPAHA),, 2H,O, M(H) = Cu(Il)
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M. J. Rao
factors such as a) neutralization of charges in mixed-ligand complexes, b) electronic effects and formation of a-bonds, c) stereochemical effect, and d) steak hinderance play a vital role in enhancing the stability of the mixed-ligand complex. Fungi&al Potential of the Binary and Mixed-Ligand Complexes The antifungal potential of N,2’-DPAHA and binary complexes of Cu(II), Ni(II), and Co(n) were tested against Fusarium oxysporum using the inhibition zone technique [22]. The structure of these complexes ‘were established previously and were given the empirical formulas as Cu(II)-(NJ’-DPAHA),. 2H,O (see Fig. l), Ni(II)(N,2’-DPAHA),, and Co@)-(N,2’-DPAHA), [13]. The fungicidal potential of N,2’-DPAHA against Fusarium oxysporum has increased nearly four-fold upon chelation with CL@. A linear relation between fungitoxicity and stability of binary complexes of Cu(II), Ni(II), and Co(H) was observed (Tables 2 and 3). The testing against Alternaria alternata was carried out by the slide germination technique [22]. All mixed-ligand complexes exhibited inhibitory effect at the given concentration level. Binary, Cu(II)-N,2’-DPAHA, and mixed-ligand complex Cu(II)-Phen-NJ’-DPAHA exhibited 97% and 98% inhibition of fungal growth of Alternaria alternata at 500 ppM, respectively. NJ’-DPAHA, when used as a fungicide, has less activity at the same concentration, and its fungitoxicity nature was increased by chelation to metal ion. But fungicidal potential was not significantly
TABLE 2. Fungicidal Activity” of NJ’-DPAHA, its Binary [M(U)-NJ’-DPAHA] and Mixed-Ligand Complexes [M(H)-Phen-NJ’-DPAHA] Against Altemaria Alternata Concentration Levels in ppM Compound
50
loo
200
N,2’-DPAHAb Cum-N,2’-DPAHAb Ni(lI)-N,2’-DPAHAb Co@)-NJ’-DPAHAb Zn@)-NJ’-DPAHA Cd@)-N,Z’-DPAHA Cu(II)-Phen-NJ’-DPAHA Ni(II)-Phen-NJ’-DPAHA Co@)-Phen-NJ’-DPAHA Zn(II)-Phen-N,2’-DPAHA Cd@)-Phen-N,Z’-DPAHA Cu(II)-Gly-N,2’-DPAHA Ni@)-Gly-N,2’-DPAHA Zn(II)-Gly-N,2’-DPAHA Cd@)-Gly-N,2’-DPAHA
48 25 29 38 27 42 21 27 35 24 38 26 28 30 35
45 22 21 36 24 39 18 21 21 17 29 20 22 24 28
31 17 23 30 20 34 12 18 20 12 20 16 18 16 24
400
500
25 5 8 16 7 20 8 12 15 8 16 10 12 9 18
20 3 5 12 4 17 2 5 8 4 12 2 4 4 10
‘Germinating medium was PSA, pH 5.0 at room temp. Pungicidai activity is expressed as average percent of spore germination after 48 hr incubation. All experiments were carried out in triplicate and results were agreed with in 2% variation. b Data from Rao et al., 1985 [13].
BINARY AND MIXED-LIGAND COMPLEXES 213
TABLB3. Fungi&al Potential* of N,Z’-DPAHA and its Binary Complexes of Cu(B), Ni(H), andCo(II) Against Fusarium Oq&porum
Compound N,Z’-DPAHA Cu(Il)-NJ-DPAHA Ni(II)-N,2’-DPAHA Co@)-N-2’-DPAHA
Concentration Levels ’ inppM 50 100 200 400 500 0.11 0.55 0.42 0.38
0.28 0.85 0.73 0.63
0.45 1.35 0.98 0.86
0.72 2.41 1.81 1.55
0.91 3.52 2.53 2.15
’ Fungicidal potential is expressed as inhibition area in mm after 48 hr incubation. Culture medium was PSA, pH 5.0 at room temperature. All experiments were carried out three times and results were consistent with the variation of 0.05 mm inhibition areas.
improved when N,2’-DPAHA was used as a secondary ligand in mixed-ligand complexes. The beneficial effects of mixed-ligand complexes were expected. The most obvious interpretation is that the synergistic effect of two ligands has increased the stability of the mixed-ligand complex but not its fungicidal potential. The author irs grateful to Professor T. Navaneeth Rao and Professor B. Sethuram, Department of Chemistry, Osmania University, Hyderabad, India, for their constant encouragement and the facilities extended.
REFERENCES 1. Chemistry and Biology of Hydroxamic Acids, H. Keh and S. Karger, Eds., New York, 1982. 2. J. B. Neilands, Science 156, 1433 (1%7). 3. N. R. Gevirtz, D. Tendler, G. Lurinsky, and R. L. Wasserman, N. Engl. J. Med. 273, 95 (1%5). 4. E. A. Kaczka, C. 0. Gitterman, E. L. Dulaney, and K. Falkers, Biochemistry 1, 340 (1%2). 5. H. C. J. Chtenheijm and J. D. M. Herscheid, Chem. Rev. 86, 697 (1986). 6. M. J. Miller, Chem. Rev. 89, 1563 (1989). 7. E. Lipczynska-Kochany, Chem. Rev. 91,477 (1991). 8. E. Farkas, J. Szoke, T. Kiss, H. Kozlowski, and W. Bal, .Z. Chem. Sot. Dalton Trans. 2247 (1989). 9. B. Kurzak, H. Kozlowski, and P. Decoek, 1. Znorg. Biochem. 41, 71 (1991). 10. M. J. Rao, B. Sethuram, and T. N. Rao, Indian .Z. Chem. 19, 379 (1980). 11. M. J. Rao, B. Sethuram, and T. N. Rao, .Z. Zndiun. Chem. Sot. 63, 663 (1986). 12. M. J. Rao, B. Sethuram, and T. N. Rao, Indian J. Chem. 22, 516 (1983). 13. M. J. Rao, B. Sethurarn, and T. N. Rao, J. Znorg. Biochem. 24, 155 (1985). 14. U. Priyadarshini and S. G. Tondon, J. Chem. Eng. Data. 12, 143 (1967). 15. A. Vogal, A textbook of Quantitative Inorganic Analysis, Longmans, Green, London, 1961, pp. 358,434,443-444, and 480.
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16. L. G. Van Uittert and C. G. Hasa, J. Am. Chem. Sot. 75,451 (1953). 17. A. E. Martell and R. M. Smith, Critical Stability Con&m& Plenum Press, New York, vols. 1 ad 2, 1974 and 1975. 18. H. M. Irvin8 and H. S. Rossotti, .I. Chem. Sot. 2904 (1954). 19. H. A. Jabn and E. Teller, Proc. Roy. Sot. (London) A164,220(1937). 20. P. V. Sbelvraj and M. *tappa, J. Inorg. Nucl. Chem. 38, 837 (1976). 21. D. N. Sbdke, Inorg. Chim. Acta. 32, LA5 (1979). 22. Y. L. Nene, Fungicides in Plant D&ease Control, IBH Publishing Company, Oxford, 1971, p. 287. 23. M. J. Rao, B. Setburam, and T. N. Rab, Indian J. Chem. 20, 1136 (1981).
Received November 18, 1991; accepfed December 4, 1991
ABSTRACT Chelating potential of N,2’-DPAHA with 3d metal ions such as 01(n), Ni(II), Co(B), Zn(l.9, and Cd@) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligaad complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(H), Zn@l), and Cd(B) is presumably due to ~-bonding effects. In the stabiliion of the Co(n) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to ?r-bonding effects. Rmgicidal activity of N,Z’-DPAHA and its binary complexes with Cu(II), Ni(Il), and Co@) was examined against Fusariumoxysporumusing theinhibition zone technique. Binary complexes of Zn@) and Cd(B) with N,Z’-DPAHA and mixed-liiand complexes M(II)-Gly or Phen-N,2’-DPAHA, where M(H) = Cu(JI), Ni@), Zn(II), Co(B), and Cd(B) were screened against Alternariualfernataby slide germination technique.All mixed-Ii@ complexes exhibited fongicidal activity but did not improve signilicantly compared to binary complexes. Synergestic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:l) of the secondary ligand (N.FDPAHA), and the fungicidal potential of the mixed-ligand complex involving N,Z’-DPAHA as secondary ligand was not increased.
ABBREWIATIONS Gly, Glycine; Phen, 1.1~pbenanthrolimq N,Z’-DPAHA, N,2’-diphenyl acetohydroxamic acid; M(II), Metal nitrate ion; M, metal ion; A, primary ligand; L, secondary &and.
INTRODUCTION Wide-range biological activity of hydroxamic acids and various aspects of hydroxamic acid chemistry has inspired chemists to study the ligation behavior of these
Address reprint requests and comzspondence to Dr. M. Janardhsn Baa, Deparhmnt of Medicine, Division of Hematology, U921; AECOM, 1300 Morris Park Ave., Bronx, NY 10461 (1992). U.S.A. Journoi of [norgonic Biochemiptry, 46.207-214 (1992)
0 1992 Elscvier Science PublishingCo., Inc., 655 Avenue of the Americas, NY, NY 10010
247 0162-0134/92/$5.00
208
M. J. Rao
ligands toward various transition metal ions [l- 111. Detailed knowledge of underlying principles in the formation of mixed-ligand complexes in biological fluids strengthens their importance and, it is worthwhile to study their formation and stability. The formation of mixed ligand-complexes in solution occurs through the coordination of ligands to the metal ions either in two distinct pH regions or in overlapping pH regions, a reflection of pK of the ligating molecule. With an overall objective of investigating the nature of mixed-ligand complex formation involving NJ’-DPAHA and other ligands and their stability and fungicidal potential, a detailed study of various transition metal complexes with O-O (NJ’-DPAHA), O-N (Gly), N-N (Phen) donor ligands has now been undertaken. Our previous studies suggested that the photo-stabilization of polyolefins by binary complexes of hydroxamic acids [12] and fungicidal activity of binary metal complexes involving hydroxamic acid [13] increase with the stability of the complex. To establish a relation between structural aspects of the binary as well as mixed-ligand complexes and their biological function, we have selected two test organisms, AIternaria alternata and Fusarium oxyspomm, in the present investigation. Binary and mixed-ligand complexes of 3d metal ions such as Cu(II), Ni(II), Co@), Zn(II), and Cd(II) were synthesized and their structural stabilities and fungicidal potential were estimated. MATERIALS AND METHODS NJ’diphenylacetohydroxamic acid was prepared as described earlier and characterized by its melting point and elemental analysis [14]. The purity of the ligand was determined by thin layer chromatography. All chemicals used were of AnalaR quality (BDH, India). Metal nitrate solutions were prepared and estimated by standard methods [15]; pH measurements with an accuracy of ztO.01 pH units were recorded on a systronic pH meter-324 fitted with glass and saturated calomel electrodes. For determination of the formation constants of mixed-ligand complexes, MAL, MA’L, the following sets of solutions (50 cc) were prepared and titrated against standard KOH (50mM); 1) HNO, (2mM), 2) HNO, (2mM) + A or A’ (HIM), 3) HNO, (2mM) + L (lmM), 4) HNO, (2mM) + 1:l mixtureof A or A’ and metal nitrate (In&I), 5) HNOs(2n-M) + 1:l mixture of L and metal nitrate (In&I), 6) HNO, (2mM) + 1: 1: 1 A or A’, L and me&l nitrate (1mM) where A = Phen, A’ = Gly, and L = N,2’-DPAHA. Titrations of each set were carried out twice to check the reproducibility of the data. Titrations were carried out in water-ethanol (50% (v/v) medium at 303 K. Potassium nitrate was used for adjusting the ionic strength. The pH correction for aquo-organic solvent was carried out by the method of Van Uittert and Hass [16]. Binary and mixed-ligand complexes were prepared by refluxing the methanolic solutions of metal nitrates and ligand solutions in the ratio of 1:20 and 1: 10: 10, respectively. Metal estimations of the complexes have been carried out as reported earlier [ 131. Fuugicidal Screening Medium, Potato Sucrose Agar (PSA) was prepared as reported earlier [13]. It was autoclaved at 15 lb and the pH was adjusted to 5.0 using acetic acid. Various concentrations of 50, 100,200,400, and 500 ppM of hydroxamic acid and its binary or mixed-ligand complexes were prepared in water-methanol (50% v/v). Spore
suspension of each fungus, Alternaria alternata isolated from onion leaves, Fusarium oxysporium, which is a pathogenic on chickpeas was prepared in redistilled water from lOday-old culture. The concentration of spore suspension was adjusted to appear 80-90 per low power field ( x 100). Three drops of spore suspension, hydroxamic acid or its binary or mixed-ligand complexes, were pipetted onto the slide. Each treatment was conducted in triplicate and controls were maintained simultaneously. Germination counts were recorded in about 300 conidia (spores) after 48 hr incubation and compared with control. RESULTS AND DISCUSSION The mixed-ligand complex formation depends on the chelating ability of two ligands A and L when they are in equilibrium with a metal ion (Eqs. (l), (2), and (4)): M2++ AH “$MA-‘“-2’+nH+, MA-(“-2) + LH ,=MAL-
(1)
(n+m-Z) + mH+,
Pi& = [MALI/[MA][L] M’++AH n +LH m=MAL-(“+‘“-2)
9
- ,H++ mH+,
BMMAL = [MALI /[Ml[Al[Ll9
logS:;, =logP& - logKM,, , AlogK = log &_
(2)
- 1ogK ML.
(4)
(5) (6) (7)
The evidence for the mixed-ligand complex formation was obtained by constructing a composite curve, out of curves M(H)-primary ligand and M(H)-secondary ligand. It was assumed that there is no interaction between M(H)-primary ligand and a secondary ligand. The composite curve was placed significantly above the 1: 1: 1 experimental titration curve pointing to the interaction between M(n)-primary ligand and secondary ligand which results in the formation of the mixed-ligand complex. Further evidence for the mixed-ligand complex formation was derived from the observation that insoluble precipitates obtained during the titration of M@)-N,2’DPAHA binary system were absent during the titration at M(H)-primary ligandsecondary ligand. Stepwise formation of M(H)-Phen-N,2’-DPAHA was confirmed based on the analysis of titrations of M(lI)-Phen and M(II)-Phen-NJ’-DPAHA. Titration curves (not shown) of M(H)-Phen-N,ZDPAHA drifts away from M(H)Phen from pH 6.2 but coincides with M(H)-Phen in the low pH region, 4 to 6.2, indicating that the N,2’-DPAHA ligate in stepwise to form mixed-ligand complex. The nature of the complex equilibrium, whether stepwise or simultaneous, was also concluded based on the method suggested by Martell and Smith [ 171. The step-wise mixed-ligand stability constants (log 8) for the interaction of M(H)-Phen and N,2’DPAHA were calculated as reported earlier [ 181 and were accurate within f 0.08 log units. A study of the log /3 values (Table 1) reveal that the stability constants of mixed-ligand complexes [M(H)-Phen-NJ’-DPAHA] were significantly greater than log K, of 1:2 binary complexes [M(H)-(N,2’-DPAHA),] but are close to log K,
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M. J. Rao
TABLE 1. Statdity Constants (Iog K ,and log K, ) of Binary Complexes’ [M@)-N,2’-DPAHA] and Step-Wise Stability Constants (log 8) of Mixed-Ligand Complexesb [MO-Phcn-N,2’-DPAHA] Metal Ion ww Ni(II) Co(u) Zn(n) ‘.X-Q
log B
logK,
1ogKz
AlogKC
9.42 6.12 5.04 6.39 4.22
9.20 6.30 5.24 6.55 4.50
6.70 4.30 3.82 4.50 3.34
- 0.22 0.18 0.20 0.16 0.28
*Data from Rae et al., 1981 [23]. b log 0 is calcukd for the interaction of M(U)-Phen and NJ’-DPAHA as a step-wise mixed-&and stability constant using Irving and Rossotti’s method [18] in 50% water-ethanol mixtures (v/v) at 303 K and 0.1 M ionic strength @NOa). ‘Alog K is ehnated from the difference of logK, of M(H)NJ’-DPAHA and log fl of mixed-ligand complex [M(II)Phen-N,2’-DPAHA] .
values of 1: 1 binary complexes [M(II)-N,2’-DPAHA]. It was evident from the stability constant data that the Cu(II)-mixed-ligand system was more stable than the 1: 1 binary system of M(H)-N,2’-DPAHA. The A 1ogK values for mixed-ligand complexes which were less than the statistically expected value (0.6) indicates that the mixed-ligand complexes are significantly stabilized over the binary complexes [M(II)-(N,2’-DPAHA),]. The stabilization of the mixed-ligand complexes could be understood by considering that Phen not only induces a strong ligand field to metal u-bonding but also acts as a powerful x-acceptor. The remarkable stability observed in the mixed-ligand complexes of Cu(II) cannot be ascribed to ~-bonding effects alone because the difference in the xdonor capacity of Cum) and the other metal ions was not like the difference in the stabilization of the mixed-ligand complexes. Ligation of Phen or N,2’-DPAHA to Cu(II) (aquo) results in a stronger distortion of an already distorted octahedral configuration in inducing a sign&ant gain in stabilization energy. The ligation of NJ’-DPAHA (secondary l&and) to Cu(II) (Phen) is energetically favored over the ligation of NJ’-DPAHA to Cu(II) (aquo) because Cu(Il) (Phen) already possesses sufficiently distorted octahedral configuration, a geometry which is ideal for the ligation of NJ’-DPAHA. Thus the stabilization effected by the stereochemical favoring of the ligation of N,2’-DPAHA to Cu(II) (Phen) coupled with that induced by &onding effects over-weights the destabilization due to steak and statistical effects and leads to the observed negative A 1ogK value. This also suggests that ligands containing oxygen as donor ligands (N,2’-DPAHA) form more stable complexes with Cu(II) @hen) than with Cu(II) (aqua), and the equilibrium of the typeMA+ML=MAL+Mliesfartotheright.Thestabilizationofthemixedligand complex over 1:2 [M(II)-(N,2’-DPAHA),] binary complex of secondary ligand could also be justified by considering that the coordination of Phen, a nitrogen donor, to Cu(II) (Phen) results in a loss of Jahn-Tellar stabilization energy [ 191, but such a loss of stabilization energy is .minimum, when N,2’-DPAHA, an oxygen donor is ligated to Cu@)(Pben). The enhanced stabiiion of M(U)-Phen-N,2’-
BINARY AND MIXED-LIGAND COMPLEXES
211
DPAHA over 1:2 binary complexes of M(H)-@I$?‘-DPAHA), is presumably due to neutralktion of charge in mixed-ligand complex MA2’++ ML,‘-= 2MAL. It was suggested that when a mixed-ligand complex forms from neutral and negative ligands both enthalpy and entropy effects favor the mixed-ligand complex over the disproportion products [20, 211. For the M(H)-Gly-NJ’-DPAHA system, the overall stability constant (log 8’) was calculated from plot of pL vs log(1 - n/n) as log /3’ = pL 0.5 + pL 1.5 [18]. The log/I’ values for M(H)-Gly-N,2’-DPAHA where M(H) = Cu(II), Ni(II), Co(H), and Zn(II) complexes are 17.9 k0.12, 12.5st0.14, 10.3iO.18, and 13.2k0.12, respectively. It was clear that log @ values for the mixed-ligand complexes were greater than the corresponding binary complexes for all the metal ions studied. The results suggest that bii complexes containing two similar ligands are not as much favored as those containing two dissimilar ligands as in mixed-ligand complexes. Since Gly (0,N donor) is a better u-donor than NJ’-DPAHA (0,O donor) (see Fig. l), the electron density accumulated at the metal ion due to the L + M u-bonding in M(II)-Gly-N,2’-DPAHA complex would be more than that in M(II)(N,2’-DPAEIA), . The observed log /3’ values of mixed-ligand complexes revealed that the electronic repulsion induced by u-bonding is not affecting the stability of the mixed-ligand complex. The enhanced stabilization of M(II)-Gly-N,2’-DPAIIA may also be due to less steak eflbct by the presence of only one bulky molecule, i.e., N,2’-DPAHA as compared to that in M(H) (N,2’-DPAHA),. In the (M(@-Gly-N,2’-DPAIIA) mixedligand complex, the metal to N,2’-DPAHA bonds are weaker as compared to metal to glycine. This is due to change in the donor atoms from bii to ternary complex. The log /3 or log 8’ values of mixed-ligand complexes follow the order Cu@) > Zn(II) > Ni(lI) > Co(H) > Cd(H). The stability constants of both binary and mixedligand complexes of Zn(II) were more than those of Ni(II). It is known that the ligand containing the nitrogen atom as donor forms a stable complex with a ligand-sensitive metal ion like Ni(II) while oxygen donor ligand forms a stable complex with the ligand field-insensitive metal ion like Zn(n). In conclusion, the
c
FIGURH 1. (A) M@) (phen) (N,2’-DPAHA)), 2H20, M(H) = Cu(ll); (B) M(H) (Gly) (N,2’DPAHA), SH,O, M(H) = Cu(TI); (C) M(H) (N,2’DPAHA),, 2H,O, M(H) = Cu(Il)
212
M. J. Rao
factors such as a) neutralization of charges in mixed-ligand complexes, b) electronic effects and formation of a-bonds, c) stereochemical effect, and d) steak hinderance play a vital role in enhancing the stability of the mixed-ligand complex. Fungi&al Potential of the Binary and Mixed-Ligand Complexes The antifungal potential of N,2’-DPAHA and binary complexes of Cu(II), Ni(II), and Co(n) were tested against Fusarium oxysporum using the inhibition zone technique [22]. The structure of these complexes ‘were established previously and were given the empirical formulas as Cu(II)-(NJ’-DPAHA),. 2H,O (see Fig. l), Ni(II)(N,2’-DPAHA),, and Co@)-(N,2’-DPAHA), [13]. The fungicidal potential of N,2’-DPAHA against Fusarium oxysporum has increased nearly four-fold upon chelation with CL@. A linear relation between fungitoxicity and stability of binary complexes of Cu(II), Ni(II), and Co(H) was observed (Tables 2 and 3). The testing against Alternaria alternata was carried out by the slide germination technique [22]. All mixed-ligand complexes exhibited inhibitory effect at the given concentration level. Binary, Cu(II)-N,2’-DPAHA, and mixed-ligand complex Cu(II)-Phen-NJ’-DPAHA exhibited 97% and 98% inhibition of fungal growth of Alternaria alternata at 500 ppM, respectively. NJ’-DPAHA, when used as a fungicide, has less activity at the same concentration, and its fungitoxicity nature was increased by chelation to metal ion. But fungicidal potential was not significantly
TABLE 2. Fungicidal Activity” of NJ’-DPAHA, its Binary [M(U)-NJ’-DPAHA] and Mixed-Ligand Complexes [M(H)-Phen-NJ’-DPAHA] Against Altemaria Alternata Concentration Levels in ppM Compound
50
loo
200
N,2’-DPAHAb Cum-N,2’-DPAHAb Ni(lI)-N,2’-DPAHAb Co@)-NJ’-DPAHAb Zn@)-NJ’-DPAHA Cd@)-N,Z’-DPAHA Cu(II)-Phen-NJ’-DPAHA Ni(II)-Phen-NJ’-DPAHA Co@)-Phen-NJ’-DPAHA Zn(II)-Phen-N,2’-DPAHA Cd@)-Phen-N,Z’-DPAHA Cu(II)-Gly-N,2’-DPAHA Ni@)-Gly-N,2’-DPAHA Zn(II)-Gly-N,2’-DPAHA Cd@)-Gly-N,2’-DPAHA
48 25 29 38 27 42 21 27 35 24 38 26 28 30 35
45 22 21 36 24 39 18 21 21 17 29 20 22 24 28
31 17 23 30 20 34 12 18 20 12 20 16 18 16 24
400
500
25 5 8 16 7 20 8 12 15 8 16 10 12 9 18
20 3 5 12 4 17 2 5 8 4 12 2 4 4 10
‘Germinating medium was PSA, pH 5.0 at room temp. Pungicidai activity is expressed as average percent of spore germination after 48 hr incubation. All experiments were carried out in triplicate and results were agreed with in 2% variation. b Data from Rao et al., 1985 [13].
BINARY AND MIXED-LIGAND COMPLEXES 213
TABLB3. Fungi&al Potential* of N,Z’-DPAHA and its Binary Complexes of Cu(B), Ni(H), andCo(II) Against Fusarium Oq&porum
Compound N,Z’-DPAHA Cu(Il)-NJ-DPAHA Ni(II)-N,2’-DPAHA Co@)-N-2’-DPAHA
Concentration Levels ’ inppM 50 100 200 400 500 0.11 0.55 0.42 0.38
0.28 0.85 0.73 0.63
0.45 1.35 0.98 0.86
0.72 2.41 1.81 1.55
0.91 3.52 2.53 2.15
’ Fungicidal potential is expressed as inhibition area in mm after 48 hr incubation. Culture medium was PSA, pH 5.0 at room temperature. All experiments were carried out three times and results were consistent with the variation of 0.05 mm inhibition areas.
improved when N,2’-DPAHA was used as a secondary ligand in mixed-ligand complexes. The beneficial effects of mixed-ligand complexes were expected. The most obvious interpretation is that the synergistic effect of two ligands has increased the stability of the mixed-ligand complex but not its fungicidal potential. The author irs grateful to Professor T. Navaneeth Rao and Professor B. Sethuram, Department of Chemistry, Osmania University, Hyderabad, India, for their constant encouragement and the facilities extended.
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16. L. G. Van Uittert and C. G. Hasa, J. Am. Chem. Sot. 75,451 (1953). 17. A. E. Martell and R. M. Smith, Critical Stability Con&m& Plenum Press, New York, vols. 1 ad 2, 1974 and 1975. 18. H. M. Irvin8 and H. S. Rossotti, .I. Chem. Sot. 2904 (1954). 19. H. A. Jabn and E. Teller, Proc. Roy. Sot. (London) A164,220(1937). 20. P. V. Sbelvraj and M. *tappa, J. Inorg. Nucl. Chem. 38, 837 (1976). 21. D. N. Sbdke, Inorg. Chim. Acta. 32, LA5 (1979). 22. Y. L. Nene, Fungicides in Plant D&ease Control, IBH Publishing Company, Oxford, 1971, p. 287. 23. M. J. Rao, B. Setburam, and T. N. Rab, Indian J. Chem. 20, 1136 (1981).
Received November 18, 1991; accepfed December 4, 1991