Synthesis, Characterization, In Vitro and In Vivo Screening of Unsymmetrical Borole Complexes of 2-Hydroxy-N-Phenylbenzamide and its Derivatives

Biochemical aspects, synthesis and characterization of some boron complexes of 2-hydroxy-N-phenylbenzamide (HOPhBenz) and its semicarbazone (HOPhBenz.SCZH) and thiosemicarbazone (HOPhBenz.TSCZH) are described. These derivatives were prepared by the reactions of 2-isopropoxy-4-methyl-1,3,2-dioxaborolane, and 2-isopropoxy-4-methyl-1,3,2-dioxaborinane with 2-hydroxy-N-phenylbenzamide, 1-[(2-hydroxyphenyl)-1-N-phenylamino]hydrazinecarboxamide (HOPhBenz.SCZH), and 1-[(2-hydroxyphenyl)-1-N-phenylamino]hydrazinecarbothioamide(HOPhBenz.TSCZH) in a 1:1 molar ratio. In order to assess the increase of the inhibitor potency, (HOPhBenz), (HOPhBenz.SCZH), (HOPhBenz.TSCZH) and their boron complexes have been tested in vitro against a number of pathogenic fungi and bacteria at different concentrations and were found to possess remarkable fungicidal and bactericidal properties. The testicular sperm density, testicular sperm morphology, sperm motility, density of cauda epididymal spermatozoa and fertility in mating trials and biochemical parameters of reproductive organs are discussed.


INTRODUCTION
Boron chemistry has continued to provide the most novel developments in the field of coordination chemistry. Inorganic chemistry of boron is much more diverse and complex than that of any other element in the Periodic Table and this is due to the elucidation of tremendous range of bondin intractions. Several organoboranes find promising applications in the synthesis of insect pheromones'. Boron azomethine complexes of various ligands have been found to possess conspicuous biocidal activity2. Boron-nitrogen compounds act as antivirals, fungicides, pesticides and even traces in medicine3. Bicyclic boron compounds have cytotoxic activity 46 and are used for the treatment of certain brain tumors7. The borohydride complexes of benzothiazepinole derivatives were found to inhibit P388 Leukemia cell in mice8. There is still a sufficient scope for studies on N and S/O donor systems regarding their biochemical significance. Recently, a variety of biologically important nitrogen and sulfur/oxygen donor ligands. Keeping all these uses in mind we have synthesized, characterized and screened some unsymmetrical borole complexes, the results of which are discussed in the present paper.

RESULTS AND DISCUSSION
Reactions of unsymmetrical boroles with HOPhBenz, HOPhBenz.SCZH and HOPhBenz.TSCZH have been carried out in a 1:1 molar ratio in dry benzene. These reactions proceed with the liberation of the isopropanol/benzene azeotrope as B(R)(OPri)+ HOPhBenz Benzene B(R)(OPhBenz)+P?OH complexes these bands remain unaltered. The nitrogen atom of the >C=N group does not take part in the coordination as evidenced by the appearance of this band in the same position.

IR Spectra
On the basis of IR spectral studies an intramolecular hydrogen bonded structure for HOPhBenz has been established. In the case of HOPhBenz.SCZH the coordination can takes place either through the -NH phenyl group or through oxygen of the >C=O group. The carbonyl streching frequency (1685 cm) shifts towards the lower frequency side, indicating that coordination has taken place through the carbonyl oxygen. The presence of a benzene ring at the nitrogen atom makes it less available for the coordination to the boron ion. HOPhBenz behaves purely as a bidentate chelating agent. In the IR spectra of conjugated bases broad bands at 3400-3200 cmare assigned to vOH due to the phenolic OH. This band disappears in the case of complexes indicating the possible loss of proton on complexation and subsequent formation of B-O bond due to the presence of new band in the region of 1345-1360 cm frequencies, respectively.

H NMR Spectra
The proton magnetic resonance spectral data of HOPhBenz, HOPhBenz.SCZH, HOPhBenz.TSCZH and their boron complexes have been recorded in DMSO-d 6. The following structural inferences have been drawn by compairing the spectra of conjugated bases with those of corresponding boron complexes (Table I). (7.5Hz) (7.5Hz) (7.8Hz) (7.3Hz) (7.8Hz) (7.3Hz) The disappearance of the -OH proton signal in case of complexes indicates the deprotonation and complexation through the functional group. The signal due to the NH protons attached to the phenyl ring remain unchanged in the complexes. The disappearance of the NH resonance signals of HOPhBenz.SCZH and HOPhBenz.TSCZH in the case of the complexes provides evidence for the complexation through the functional group. The-NH protons remain almost unchanged.
Taruna Pandey and R. V. Singh Metal-Based Drugs' 1" C NMR Spectra The q3C NMR spectral data have been recorded for HOPhBenz, HOPhBenz.SCZH, HOPhBenz.TSCZH and their boron complexes. The shifts of the carbon attached to O and S indicates the involvement of these atoms in coordination (Table II).  Tables III and IV. 11 B NMR Spectra The B Nuclear magnetic resonance were observed between i17.24 and 35.23 ppm (Table I) and which clearly supports a tricoordinated environment around the boron atom.
On the basis of the spectral evidences, the boron derivatives have been assigned the following structure with tricoordinated boron atom. Taruna Pandey and R. K Singh Metal-Based Drugs BIOCHEMICAL ASPECTS All the conjugated bases and their corresponding boron complexes were tested for the in vitro growth inhibitory activity against pathogenic fungi viz. Fusarium oxysporum, Alternaria alternata, Rhizoctonia bataticola and bacteria viz. Staphylococcus aureus and Xanthomonas compestris. Proper temperature, necessary nutrient and growth media free from other microorganism were employed for the preparation of cultures of fungi and bacteria using aseptic techniques. The antifungal activity of the complexes was evaluated by the Agar Plate Technique and antibacterial activity was evaluated by Paper Disc Plate Method 7. The data of fungicidal and bactericidal activities of conjugated bases and their respective boron complexes against pathogenic fungi and bacteria were recorded in Tables V and VI.   Fourty two male albino rats were used in present study. Rats having weight between 175 to 200 g were divided into seven groups of six animals each. Animals were housed in polypropylene cages measuring 12"x10"x8" under controlled environmental conditions and fed with rat feed pellets and free acess to water.  for the same period. These animals were screened for fertility test and autopsied for detail biochemical changes. Reproductive organs were excised, blotted free of blood, weighed and were frozen for biochemical estimations. The sperm motility and density of cauda epididymal spermatozoa, total cholestrol, total protein, sialic acid, fructose and acid phosphatase were determined by Standard Laboratory Techniques.   Group C compared with Group B Groups D and F compared with Group B Groups E and G compared with Group C (i) Body and Organ Weights Body weight of rats were not affected "after HOPhBenz, HOPhBenz.TSCZH and their boron complexes administration, However, the weight of testes, epididymis, seminal vesicle and ventral prostate were significantly described (Table VIII). (ii) Fertility Test The slugish motile spermatazoa were unable to fertilize normal cyclic females. The test was 71 to 93% negative in rats treated with these compounds. (iii) Sperm Motility The sperm motility declined significantly after treatment with the compounds. Synthesis, Characterization, In Vitro and In Vivo Screening of Unsymmetrical 9orole Complexes Of 2-Hydroxy-N-Phenylbenzamide and its Derivatives (iv) Sperm Density The sperm density in testes and cauda epididymis were declined significantly after treatment (Table IX). EXPERIMENTAL All the chemicals and solvents were dried and purified by standard methods. The reactions were carried out under anhydrous conditions.

Preparation of Hydrazinecarboxamide and Hydrazinecarbothioamide
Hydrazinecarboxamide and Hydrazinecarbothioamide of 2-hydroxy-N-phenyl-benzamide were prepared by the condensation of (HOPhBenz) with semicarbazide hydrochloride (in presence of sodium acetate) and thiosemicarbazide in a l:l molar ratio in alcoholic medium. The mixture was refluxed for 2-3 hours. On cooling, crystals were separated out which were recrystallized in the same solvent and finally dried in vacuum. The physical properties of these moieties are as follows The unimolar reactions of unsymmetrical boroles with 2-hydroxy-N-phenylbenzamide and its derivatives were carried out in dry benzene. The reaction mixture was refluxed for 10-12h on a fractionating column and the progress of the reaction was monitored by the liberation of azeotropic isopropanol/benzene. After the completion of the reaction, the excess of the solvent was distilled off and the product was dried in vacuo. It was repeatedly washed with dry cyclohexane and again dried for 3-4h. These complexes were recrystalized in (1:1) solution of benzene and ether. The important physical properties and analytical data are given in Table X. Fe-Kc target and operated at 30 KV and 40mA. The diffraction data were refined using a least-squares refinment computer programme to obtain structural parameters. The programme essentially uses the least squares method of fitting of dhk values for the different reflections. The intensity of Fe-Kot radiation diffracted from the powder specimen was detected by a solid state detector and recorded as function of 20, where 0 is the angle of incidence.

Biochemical Changes
Total protein and sialic acid contents of testes, epididymis, ventral prostate and seminal vesicle were depleted significantly after treatment with starting materials and their boron complexes. The acid phosphatase levels of testes epididymis and ventral prostate were also reduced significantly. A significant decrease in seminal vascular fructose contents was also noticed whereas the testicular cholestrol contents was increased significantly after the treatment with various compounds (Tables XI and XII). The present study revealed that administration of the starting materials, HOPhBenz and HOPhBenz.TSCZH and their boron complexes caused a significant reduction in the weights of testes and other sex accessory glands. The development and function of male sex accessory glands the prostate and seminal vesicle are a well documented androgen dependent process, z The reduction in the weights of these organs could be due to reduced androgen level 2. Sperm motility is considered as an important parameters in evaluating the fertility potential. HOPhBenz, HOPhBenz.TSCZH and their boron complexes significantly reduced the fertility of male rats. Since a number of androgen sensitive parameters (protein, sialic acid, fructose, acid phosphatase and total cholestrol) in target organs were found to be altered by these compounds, it is expected that the structure and function of epididymis and other sex accessory organs are changed. MOzt of the activities of HOPhBenz.TSCZH, and its effect on fertility is due to the presence of thio group Earlier it has been reported that boron exposure results in infertility, oligespernia and decreased libido in occupationally exposed men and rats. Our finding further suggested that addition of the boron moiety to the starting materials HOPhBenz or HOPhBenz.TSCZH increased their activity, and the boron complexes of HOPhBenz.TSCZH being indeed more effective fertility inhibitors.
Synthesis, Characterization, In Vitro and In Vivo Screening of Unsymmetrical Borole Complexes Of 2-Hydroxy-N-Phenylbenzamide and its Derivatives