Indian Journal of Chemistry Vol. 43A, January 2004, pp. 83-87

Synthesis and reactivity studies of mononuclear zinc hydroxo complex

Shalu Tyagi, Chokhe Lal Sharma, Sukh Mahendra Singh# & Udai P Singh*

Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India

#School of Biotechnology, Banaras Hindu University, Varanasi 221005, India

Received 4 December 2002; revised 8 August 2003

The synthesis of mononuclear zinc hydroxo, Zn(OH)(HB(3-Bu'-5-Pripzh) 2 using sterically hindered pyrazolylborate ligand i.e. hydrotri s(3-tert-butyl-5-i sopropyl-I-pyrazolyl)borate ligand has been described. The structu re of 2 is very similar to the active site of the enzyme carbonic anhydrase. The complex 2 is found to stabilize zinc complexes of urac il and its halo derivatives where uracil and its halo derivatives are bound as monodentate ligand via its deprotonated N I. The halogenated uracil complexes have been injected in Dalton' s Lymphoma tumour system in mice and it is found that Zn[HB(3-Bu'-5-Pripz)3](5-0uorouracilate) exhibits significant antitumour activity. The complex 2 is a very reactive species and is used as catalyst in ester hydrolysis. The complex 2 is also found to promote the hydrolysis of various esters and the max imum rate of hydrolysis is observed wi th p-nitro­phenyl acetate.

The metal, zinc ions plays a very important role in many biological processes and is involved in many metalloenzymes I. The unusual variability and co­ordination flexibility of Zn(Il) is well-established and could be an important determinant in the specificity of this cation in a number of zinc metalloenzymes. The most important zinc-containing enzyme is carbonic anhydrase (CA) whose ligation with the zinc ion by three histidine imidazoles can be modelled by tridentate ligands and several zinc hydroxo complexes have been already reported in literature2

-4. Although tetrahedral geometry is usual for zinc complexes, a co-ordination number of 5 as a trigonal-bipyramidal or square-pyramidal arrangement is also commons. The square-pyramidal structure is catalytically unimportant but trigonal-bipyramidal pentacoordinate complexes are deemed to be essential for the catalytic action of zinc metalloenzymes. It has been suggested that, in carbonic anhydrase (CA), carboxypeptidase (C PA) and alcohol dehydrogenase (ADH), the formation of a pentacoordinated zinc intermediate

may take place in their catalytic cycle6. Zinc is also present in various enzymes along the biosynthetic pathways of the nucleobases or for phosphate transfers during nucleotide interconversions 7-9.

Significant contributions to the zinc complex chemistry of nucleobases, nucleosides, sugar-derived phosphates and nucleotides have been made by the

. h 8 10- 14 vanous researc groups· . The metal coordination of nucleobases, their

precursors, derivatives and of nucleosides, nucleotides is a very active area of research . Nucleobases i.e. uracil and its halo-derivatives are in focus through studies on zinc complexes with 5-fluorouracil , a potent anticancer drug. During these studies it became obvious that the deprotonation of mildly acidic NH functions is facilitated in the presence of zinc ions and the encapsulation of zinc by sterically demanding tripodal ligands facilitates complexation of the corres­ponding anionic species. The various substituted pyrazolylborate ligands (Tp) are found to stabilize a wide range of monoanionic species X by protecting them in the hydrophobic pocket of the neutral complexes Tp-Zn-X ls.

The present paper describes the synthesis and characterization of mononuclear zinc hydroxo complex and its reaction products with uracil and its halo derivatives. The isolated uracil and halouracil complexes have been tested for their anti tumour activity against Dalton's Lymphoma tumour system. The zinc-hydroxo complex is also used as catalyst in various ester hydrolysis.

Experimental All solvents used were purified by literature

methods l6 and treated under nitrogen prior to use. The reagents of the highest grade commercially available were used without further purification. The esters, tris(4-nitrophenyl)phosphate (trisNPP) and bis(4-nitrophenyl)phosphate hydrate (bisNPP) were purchased from Fluka Chemie AG, Switzerland whereas 4-nitrophenyl disodiumorthophosphate (monoNPP) and 4-nitrophenylacetate (PNPA) were purchased from s. d. fine - CHEM LTD (India). The preparations of the complexes were performed under nitrogen by standard Schlenk techniques. KHB(3-Bu'-5-Pripzh was prepared by the method described

84 INDIAN J CHEM, SEC A, JANUARY 2004

previouslyl7. 'H-NMR spectra were recorded on a

JEOL-GX-270 (270 MHz) spectrometer at 25°C. The

chemical shifts are reported as values (8, ppm) downshifted from the internal standard Me4Si. IR measurements were carried out in KBr using a Perkin­Elmer model 1600 FT-lR spectrometer. FO-MS spectra were recorded on a Hitachi M-80 spectro­meter. The X-ray data collection were performed on a

PW 1710 Philips diffractometer using CuKa radiation.

Zn(N03){HB(3-BlI'-5-P";pzh} 1

A mixture of Zn(N03h.6H20 (0.2974 g, 1.0 mmol) and 0.5078 g (l.0 mmol) of KHB(3-Bu'-5-P/pzh was stirred in a mixture of dichloromethane (25.0 ml) and acetone (5.0 ml) for 3 h. After removal of salt by filtration, thc solvcnt was evaporated under vacuum. The resulting colourless crystalline compound (1) was obtained in 51 % yield (0.320 g). Anal. calcd. for C30Hs2N70 3BZn: C, 56.75; H, 8.25; N, 15.44: Found C, 56.35; H, 8.17 ; N, 15.41. IR (KBr, cm" ): 2566 (1)

BH). 'H-NMR (C60 6, 8 ppm): 1.18 (d, 1=7Hz, 18 H, CHMe2), 1.51 (s, 27H, CMe3), 3.16 (sept, 1=7Hz, 18H, CHMe2), 6.00 (s, 3H, pz ). FO-MS(m/z): 633.

Zn(OH){HB(3-BlI'-5-P/pzh} 2

A toluene solution of 1 (0.30 g, 0.47 mmol) in 20.0 ml was stirred with 10.0 ml of 1.0 N aqueous NaOH for 30 min. Toluene phase was separated and dried over sodium sulphate for 2 h. Sodium sulphate was removed by filtration and the solvent was dried under vacuum to give pure sample of 2 as a white crystalline solid in 76% yield (0.22 g) . Anal. ca lcd. for C30H 53N60BZn: C, 61 .07; H, 9.05; N, 14.24: Found C, 61.35; H, 9.27; N, 14.55 . IR (KBr, cn'-'):

3686 (1) OH), 2566 (1) BH). 'H-NMR (C60 6, 8 ppm): 1.12 (d, J=7Hz, 18 H, CHMe2), 1.57 (s, 27H, CMe3), 3.53 (sept, 1=7Hz, 3H, CHMe2), 5.98 (s, 3H, pz). FO­MS (m/z): 588.

[Zn{ H B( 3-Btt'-5-P,JpzhJ h(C03) 3

0.0072 g (0.012 mmol) of 2 was dissolved in 5.0 ml toluene and was allowed to react with pure CO2 for 10 h. The solvent was evaporated to dryness under vacuum and the white solid compound was obtained in 34% yield (0.005 g). Anal. ealcd. for C6IHI 04 NI 20 JB2Zn2: C, 61.41; H, 8.79; N, 14.09: Found C, 61.60; H, 8.71; N, 14.31. lR (KBr, cn'-'): 2564 (1) BH), 1607 (1) CO). FO-MS(mlz): 1193.

Zn{ HB( 3-Bll'-5-P/pzh}(lIracilate) 4 A suspension of uracil (0.087 g, 0.78 mmol) in

10.0 ml of methanol was added to a solution of 2 (0.458 g, 0.78 mmol) in 15.0 ml of dichloromethane. After overnight stirring, the solution was filtered and filtrate was evaporated to dryness by using an oil pump vacuum. Yield: 79% (0.419 g). Anal. calcd. for C34 HssNs0 2BZn: C, 59.60; H, 8.10; N, 16.38: Found C, 60.04; H, 8.25; N, 16.61. IR (KE r, cm"): 2566 (1)

BH); 3122 (1) NH); 1716 (1) C2=O); 1666 (1) C4=O).

'H-NMR (COCl3, 8 ppm): 1.09 (d, J=7Hz, I8H, CHMe2), 1.54 (s, 27H, CMe3), 3.50 ( sept, 1=7Hz, 3H, CHMe2), 5.01 (d, 31=7.2Hz, I H, CH (Uracil», 5.49 (d, 31=7.2Hz, IH, CH (Uracil), 5.96 (s, 3H, pz), 6.48 (s, 1 H, NH (Uracil)).

ZIl{ HB( 3-Bll'-5-Pripzh}(5-jluorouracilate) 5 A suspension of 5-flourourac il (0.148 g,

1.14 mmol) in 10.0 ml of methanol was added to a solution of 2 (0.674 g, 1.14 mmol) in 15 .0 ml of dichloromethane. After overnight stirring, a clear solution was formed. The solution was filtered and evaporation of solvent produced a white coloured compollnd which was dried under vacuum. Yield: 76% (0.610 g). Anal. calcd . for C3-lHs4Ns0 2BZnF: C, 58.17; H, 7.75; N, 15.96: Found C, 57.99; H, 7.83; N,

15.82. IR (KBr, em"): 2555 (1) BH); 3112 (1) NH);

1727 (1) C2=O); 1655 (1) C4=O).

ZIl[H B( 3-Bu' -5-P";pzh]( 5-clzlorollracilate) 6 A sllspension of 5-chlorou racil (0 .1 25 g,

0.85 mmol) in 10.0 ml of methanol was added to a solution of 2 (0.458 g, 0.78 mmo:; ) in 15 .0 ml of dichloromethane. After overnight sti ::Ting, the reaction mixture was filtered and the evapcration of solvent under vacuum gave a white coloured compound in 8 1 % (0.453 g) yield. Anal. calcd. for C34Hs4Ns0 2BZnCI: C, 56.83; H, 7 .57; N, 15.59: Found C, 56.70; H, 7.69; N, 15 .45 . lR (KBr, cm-'):

2567 (1) BH); 3157 (1) NH); 1725 (1) C2=O); 1676 (1) C4=O).

Zn[ H B( 3 -Bl/ -5-p/pzhH 5-brolllollracilate) 7 A suspension of 5-bromouracil (0.261 g,

1.37 mmol) in 10.0 ml of methanol was added to a solution of 2 (0.806 g, 1.37 mmoJ) in 15.0 mJ of dichloromethane. After overnight stirring solution was filtered and the filtrate was evaporated to dryness on vacuum pump. Yield: 89% (0 .959 g) . Anal. calcd. for C 34 Hs4Ns0 2BZnBr: C, 53.52; H, 7.13; N, 14.68:

NOTES 85

Found C, 53.28; H, 7.27; N, 14.42. IR (KBr, cm-I):

2562 (u BH); 3156 (u NH); 1723 (u C2=O); 1674 (u C4=O).

Dalton 's Lymphoma (DL) cells were used as tumour target cells in this study. DL is a trans­plantable tumour cell lymphoma (H2

d) of spontaneous

origin, obtained from Tata Memorial Cancer Centre (Mumbai , India) and maintained in laboratory in culture ill vitro, as well as in ascites, by serial transplantation into BALB/C(H2

d) mice via intra­

peri tonial injecti on of 5x 105 cells/mice. DL cells were also maintained in a cryopreserved state as a standard reference stock. Estimation o f cell viabilit/ 8

, effect of compounds on DNA replication of DL cells ill vitro l9

and morphological evaluation of apoptotic DL cells20

were evaluated according to the procedure described earlier21.

Kinetic measurements for the cleavage of ester were monitored at 25°C with a Perkin-Elmer UY­visible Lambda 35 spectrophotometer by monitoring either the rate of disappearance of ester for bi sNPP

(Amax = 290 nm ) and monoNPP «Amax = 305 nm) or rate of appearance of p-nitrophenolate for PNPA and

trisNPP (Amax = 402 nm) . The spectra were recorded immediately after mixing the toluene solutions of substrate and es ters and then the reaction was followed until no change in spectra was observed22

.

The observed rate constants Kobsd. (S- I) were calculated from the decay slope2g

.23 .

Results and discussion The reaction of Zn(N03h.6 H20 with K[HB(3-Bu'-

5-PripzhJ results in the formation of complex 1. The complex 1 is five coordinate species with N30 2 donor set. The presence of uas(N03) at 1530 cm-I and

us(N03) at 1261 cm- I in the IR spectrum suggests the bidentate character of nitrate group coordinated to zinc. The reaction of 1 (toluene solution) with l.0 N NaOH results in the formation of 2. The presence of u OH at 3686 cm-I suggests the presence of hydroxo group. The crystal structure of 2 could not be determined but on the basis of other analogous monomeric zinc hydroxo complexes with a hindered tris(pyrazolyl)borate ligand available in the litera­tures2a.3.4 and also on the basis of mass determination it can be infered that 2 is also monomeric species . Based on the spectral data (including JR , NMR and mass spectra) in present study, the proposed coordination environment of 2 is very similar to the active site of carbonic anhydrase where zinc center is

bound to three histidine imidazole groups and a water molecule [His3-Zn-OH2J2+ (His = histidine) and the initial states of the catalytic cycle are considered to involve initial deprotonation of the coordinated water to give the active zinc hydroxide derivative [His3-Zn­OHt, followed by reaction with CO2 to give a zinc bicarbonate derivative [His3-Zn-OC02Ht. The complex 2 is very reactive and its reactivity studies towards CO2, uracil, 5-halogenated uracils and as catalyst in various esters hydrolysis have been reported in present study.

Tn a typical reaction, the complex 2 reacts with CO2 and results in the formation of carbonato complex 3 (Scheme 1). The formation of 3 was confirmed by spectroscopic methods including mass measurement. Similar reaction of mononuclear cobalt hydroxo complex with CO2 has been reported where X-ray study established unsymmetrical binding of CO2

group24. Efforts are still going onto get single crystal of the complex 3.

In de novo sy nthesis, the pyrimidine nucleotides originate from amino acids via heterocyclic carboxylic acids. The decarboxylation of the pyrimidine nucleotides after attachment of the sugar and phosphate units results in formation of uridine monophosphate, which is the precursor of the other pyrimidine nucleotides25

. Zinc enzymes are involved in several steps of this sequence including the formation of oligonucleotides and polymerase activity. Al so several metal complexes of zinc, have been found to act as drugs. Uracil causes tumour growth whereas 5-halogenated uracils specially 5-fluorouracil act as antitumour and now-a-days frequently injected to the patient in combination of cis-platin for the treatment of gastric cancer. It therefore seems justified to prepare HB[3-Bu'-5-Pr'pzhJZnX (where X is uarcil, 5-f1uorouracil , 5-chlorouracil and 5-bromoruracil) type complexes by the reaction of 2 and uracil/halogenated uracils and to screen for their antitumour properties . In a typical experiment when 2 was allowed to react with uracil

2+ CO,~ E~Zn/~-o-Zn/<~ / "0/ ,

N 'N

Scheme 1

86 INDIAN J CHEM, SEC A, JANUARY 2004

and its derivatives, the reaction resulted in the formation of HB[3-Bu'-5-PripzhlZn-uracil/halo­genated uracils by deprotonation of the NH groups and Zn-N coordinated complexes were formed . The compositions of the complexes were derived from their spectral data and coordination modes of uracil and its derivatives have been proposed on the basis of IR and NMR data. Uracil and its derivatives are bound to zinc via Nl. This can be explained on the basis of acid base properties of NH functions: while the monoanion of uracil exists as a 1: 1 tautomeric mixture of the N 1- and N3- deprotonated species, the most acidic NH function of uracil is located at N 1. Based on above observations the molecular structure of HB[3-Bu'-5-PripzhlZn-uracillhalogenated uracils may be proposed as shown in Scheme 2.

X-ray powder diffraction studies

The X-ray powder diffrac tion data (data not shown) of the complexes have been indexed according to the Ito's method26

. The indexing pattern yield the lattice constants, a = b = 17.05, c = 70.71 A for 4; a = b = 13.17, c = 31.62 A for 5; a = b = 15 .05, c = 31.62 A for 6; a = b = 14.12, c = 33.3 A for 7, indicating tetragonal symmetry for all these complexes. Similar structure for Zn-uracil complexes has been proposed by Vahrenkamp et at. with different pyrazolylborate ligand27

. The isolated uracil and 5-halogenated uracil complexes were tested for their antitumour activity against Dalton 's Lymphoma (DL) tumour system.

Biological studies

Cell death caused by induction of apoptosis or necrosis by the synthesized metal complexes were tested against the DL tumour system in vitro and are given in Table 1. Apoptosis (programmed cell death) is a well-documented mode of cell death under a number of physiological conditions28

. Treatment of DL cells with some compounds result in a decrease of cell viabi lity, indicating cell death . It was also determined if the cell death was due to the induction of apoptosis in DL cells. Indeed with some of the test compounds, which decreased cell viabili ty, an increase in % of apoptosis cells was observed. However, this correlation was not observed with all the test compounds. The test compound, which decreased cell viability also induced an inhibition of DNA synthesis as indicated by decreased 3H_ thymidine incorporation. This indicates that one of the

X - H. F. CI. Br

Scheme 2

Table I-Effect of metal complexes on the viability, 3H-thymidine incorporation and induction of apoptosis

in tumour cells

Complexes

Medium alone

4

5 6

7

% Viability of target cells after

6 h incubation

82.3±4.2 52.2±3.6 80.8±2.9"

59.4±3.9" 65.4±4.4"

% Inh ibition % Apoptotic of DNA cells after 2 h synthesis incubation

16.6±O.7 31.2±1.1 21.3±3.9 72.6±2.7 58.3±3.7" 46.2±2.2 46.6±4. 1" 52.9±B.1 59.8±4.5"

Values are mean Standard Deviation of a representative experiment conducted in triplicate. In other 2 experiments similar results were obtained. "Significantly different (P<O.05) from the val ues of DL cells incubated in medium alone. % inhibition of DNA synthesis was measured by 3H-thymidine incorporation as descri bed in the Experimental Section. Values are calc~.Ia ted vs. samples incubated in medium alone.

mechanism by which these compounds manifest their antitumour activity is by inhibiting DNA synthesis, which is a fundamental feature of cell growth . However, it remains to be establi shed if these compounds directly bind to DNA modulating its repl icative potential or indirectly by binding to some DNA binding proteins.

The complex 2 was also used as catalyst in various esters hydrolysis and the course of ester's cleavage was monitored spectrophotometrically either by the rate of disappearance of ester or by the appearance of 4-nitrophenolate anion . The rate constants were calculated using the plots between 10g(Ain,-A,) versus time and found 1.69x104

S·I for PNPA (0.0168 mmol of 2 in toluene and 0.0056 mmol of ester in acetoni trile), 1.97x103

S·I for mono-NPP (0.0 15 mmol of 2 in toluene and 0.015 mmol of ester in methanol), 1.51x103

S·I for bis-NPP (0.0134 mmol of2 in toluene ancl 0.0134 mmol of ester in methanol) and 1.38x 103

S· I for tris-NPP (0.0168 mmol of 2 in toluene and 0.0168 mmol of ester in acetoni trile). The above results provide estimates of the pseudo-first order rate constants. The rate of the generation of 4-nitro-

NOTES 87

phenolate (at 402 nm) is found to increase with time. A prolonged (time vary from ester to ester) reaction at room temperature did not change the spectra, indicating that no more hydrolysis occurs. Similar type of ester hydrolysis using metal-bound hydroxo species has been performed by several workers using different metal ions29

-34 earlier but well characterized

mononuclear hydroxo zinc complexes were not used in similar reaction till date. From the present study it is clear that the rate of ester hydrolysis vary from ester to ester and is found to be fast with PNPA in comparison with other esters used.

Acknowledgements Authors are thankful to UGC, New Delhi, for

financial support in the form of major research scheme [No. F-12-7418 (SR-l)].

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