Synthetic and pharmacological studies on some 1-isonicotinoyl-3- methyl-4-(4-substituted phenyl)-3a,4-dihydro pyrazolo [3,4-c]pyrazoles and their ethoxyphthalimide derivatives

Isoniazid reacted with ethylacetoacetate in absolute ethanol to give 2-isonicotinoyl-5-methyl-2,4-dihydro-3 H -pyrazol-3-one (1) , which upon condensation with various aldehydes (2a-d) afforded the related arylidene derivatives (3a-d) . 1-Isonicotinoyl-3-methyl-4-(4-substitutedphenyl)-3a,4-dihydropyrazolo[3,4-c ]pyrazoles (4a-d) were obtained as the result of cyclocondensation reaction between 3a-d and hydrazine hydrate. Subsequently compounds 4a-d were converted to corresponding 2-N -ethoxyphthalimido-6-isonicotinoyl-4-methyl-3-(4-substituted phenyl)-3,3a-dihydro pyrazolo[3,4-c ]pyrazoles 6a-d by the treatment with phthalimidoxyethyl bromide (5) . Structures of synthesized compounds were elucidated by means of IR, 1 H NMR and mass spectral data. Final compounds were screened for four biological activities namely “antibacterial, antifungal, antiviral and anticancer”.


Introduction
2][3] Isoniazid is one of the primary drugs used in combination with ethanbutol, rifampin, streptomycin and pyrazinamide to treat tuberculosis. 4Despite the large number of compounds containing the isoniazid moiety which have already been synthesized and tested, there is still a need for new compounds of this kind, 5 due to the increasing resistance of bacterial strains of certain type of antibiotics. 6The efficiency of pyrazole as chemotherapeutic agent is well established and their chemistry has been extensively studied.][9][10][11] Pyrazoles are an interesting group of compounds many of which possess broad spectrum pharmacological properties, such as analgesic, antipyretic, antidepressant and antirheumatic 12,13 and are also well known for their pronounced anti-inflammatory activity 14 as well they are used as potent antidiabetic agents 15 .][18][19] Alkoxyphthalimides are a class of compounds well known for a long time and still continue the object of considerable interest, mainly due to their applications in different fields particularly as pharmaceuticals.Mainly these are used as potent anticancer 20 and anticonvulsant [21][22] agents.In addition, presence of pyridine ring is conducive to improvements of the biological activity.These valid observations regarding the pharmacological importance of the above mentioned moieties and in connection with our on going programme of synthesizing alkoxyphthalimide derivatives [23][24][25] of certain heterocyclic systems led us to undertake the synthesis of some new combinational molecules, incorporating above moieties in them with the hope of augmentation in biological activities

Results and Discussion
To prepare 2-isonicotinoyl-5-methyl-2,4-dihydro-3H-pyrazol-3-one (1), isoniazid (isonicotinic acid hydrazide) was treated with ethylacetoacetate in absolute alcohol.Alternatively, (1) was prepared by the reaction of isoniazid and ethylacetoacetate in presence of sodium ethoxide solution in ethyl alcohol.Presence of base reduces the reaction time but yields are not satisfactory.So the base free method is preferred, although it takes a longer time.Formation of (1) is confirmed by the disappearance of bands near 3300-3400 cm -1 for NH 2 functionality and presence of a band near 2973 cm -1 for methylene and methyl group.Analysis of its 1 H NMR spectrum revealed the signals of aromatic proton near δ 7.99-8.79,which confirmed the presence of pyridine nucleus in it.Transformation of 2-isonicotinoyl-5-methyl-2,4-dihydro-3H-pyrazol-3one (1) to its corresponding arylidene derivatives (3a-d) was achieved by its treatment with various 4-substituted benzaldehydes (2a-d).Structure of these compounds was established spectroscopically, i.e. presence of a singlet at δ 6.5 (3a) for methine proton.
Compounds (3a-d) when were subjected to reaction with hydrazine hydrate, a cyclocondensation reaction afforded 4-(4-chlorophenyl/4-methoxyphenyl/4-N,Ndimethylaminophenyl/phenyl)-1-isonicotinoyl-3-methyl-3a,4-dihydropyrazolo [3,4- c]-pyrazole  (4a-d).The structures of these compounds were determined from their analytical and spectral data.The IR absorptions due to the N-H function appeared at 3430 cm -1 .The absorption bands associated with other functionalities present all appeared in the expected regions.The 1 H NMR spectra of the compound (4a) exhibited a sharp singlet at δ 6.23 corresponding to the NH proton of the pyrazole ring.The N-H proton of (4a-d) was replaced from ethoxyphthalimide moiety by the reaction with phthalimidoxyethyl bromide (5).
The resultant product was identified as 2-N-ethoxyphthalimido-6-isonicotinoyl-4-methyl-3-(4-substituted phenyl)-3,3a-dihydro pyrazolo [3,4-c]pyrazoles (6a-d).The IR spectra of (6a) display strong absorption band for CO-N-CO group at around 1789-1728 cm -1 , while N-O and C-O bond give relatively weak absorption bands at 1493 and 1079 cm -1 respectively.Disappearance of NH stretching band around 3460 cm -1 also confirmed the replacement of hydrogen of pyrazole NH by ethoxyphthalimide, which was present in its precursor.Additional proof for the proposed structure of (6a-d) was provided by close observation of 1 H NMR spectra, which showed disappearance of NH signal at δ 6.

Antimicrobial screening
Antimicrobial activity, i.e. antibacterial and antifungal was screened by the Well or Cup method 27 in nutrient agar and dextrose agar medium.The agar medium was sterilized by autoclaving at 15 psi and 121 0 C for twenty minutes.The medium was poured in Petri dishes and left to solidify.These Petri dishes were inoculated with 0.2 mL suspension of organism by spread plate method 28 .Three or four wells of 11 mm diameter were made in the medium with the help of a sterile borer and filled with 500 ppm solution of testing compound in DMF.Similarly other wells were made for standard drugs and filled with standard concentration.These Petri plates were incubated at 37 0 C in an incubator.The Petri dishes were examined for zone of inhibition after 24-48 hrs.For the present investigation; one gram positive-B.subtilis and three gram negative strains P. mirabilis, E. coli and K. pneumoniae were used.Two standard drugs were used for comparative study viz.ciprofloxacin and roxithromycin.Candida albicans (MTCC227) and Aspergillus fumigatus (MTCC2550) were used as the testing fungal strains.Amphotericin B and flucanazole were used as standard drugs.Zone of inhibition is measured in mm.Activity index of all the synthesized compounds was also calculated against all the standard drugs.
Compounds (4a-d) and their ethoxyphthalimide derivatives (6a-d) were assayed for antimicrobial activity.Screening results of the compounds (4a-d) established that the (4b) showed comparable activity against all the tested micro-organisms as compared to the standard drugs used, while (4a) exhibited good activity and (4c) & (4d) were either inactive or weakly active against all the four bacterial and two fungal strains.Overall activity profile of compounds (4a-d) was found to be moderate to poor.Ciprofloxacin was found to be stronger drug than roxithromycin.
Therefore in the present study, we attempted to increase antimicrobial activities by fusing the ethoxyphthalimide moiety with the pyrazolo[3,4-c]pyrazole ring system.Compound (6b) was found to be the strongest amongst all.Compounds have more comprehensive fungus-inhibiting properties than that of the bacterial.Even three to four folds antifungal activity was observed than standards.Compound (6a) and (6b) showed strong activity against P. mirabilis, B. subtilis, C. albicans and A. fumigatus while moderate to good activity against K. pneumoniae and E. coli.The effect of incorporation of ethoxyphthalimide moiety to the pyrazole ring system was much pronounced on their activity.As far as the relation between structure and activity are concerned the chloro and methoxy substituted compounds were found to show better activity than the others.

Antiviral testing
Four final compounds were screened for antiviral activity against influenza A (H1N1 and H3N2 subtypes) and influenza B viruses and cytotoxicity in MDCK (Madin Darby Canine Kidney) cells.Results have been expressed in the form of EC 50 values (Effective concentration or concentration producing 50% inhibition of virus-induced cytopathic effects as determined by visual scoring of the CPE or by measuring the cell viability with the colorimetric formazan based MTS assay).
The reference compounds oseltavimir carboxylate and ribavirin were active against influenza virus; their EC 50 values are clearly lower than their MCC value (concentrations causing minimal toxicity).For amantadin and rimantidin, the best activity was seen with the H3N2 strain.These compounds are known to be inactive against influenza B. Also the H1N1 A/PR/8/34 strain that is used in tests is known to be less sensitive to amantadin and rimantadin.Among the range of four compounds tested, none was able to inhibit the cytopathic effects of influenza A or B at subtoxic concentration or at the highest concentration tested (100 µg/mL).

Experimental Section
General Procedures.Melting points were taken in open capillary tubes and are therefore uncorrected.Purity of the compounds was checked on silica gel G TLC plates of 2 mm thickness using n-hexane and ethylacetate as solvent system.The visualization of spot was carried out in an iodine chamber.The IR spectra of the compounds were recorded in the 4000-450 cm -1 ranges using KBr discs on FTIR IR RX1 Perkin Elmer spectrophotometers and 1 H NMR were recorded on a Bruker DRX-300 MHz spectrometer (CDCl 3 ) using TMS as an internal standard.The ESI-MS were recorded on a MICROMASS QUATTRO II triple quadrupole mass spectrometer having a JASCOPU-980 HPLC pump connected to it.
Antiviral assay in HEL, HeLa and Vero cells with the four compounds (6a-d) have been observed on Herpes simplex virus-1 & 2, Vaccinia virus, Vesicular stomatitis virus, Coxsackie virus B4, Respiratory syncytical virus, Para-influenza-3 virus, Reovirus-1 and Puntatoro virus.No specific antiviral effects (i.e.minimal antivirally effective concentration) were noted for any of the compounds evaluated against any of the viruses.

3b-d) were
prepared with some change in reflux time and reaction work up.Their characteristic spectral and analytical data are given below: