Chapter IV

116

5.0 CHAPTER –IV BIS (PYRAZOLYL) METHANES

5.1 INTRODUCTION

Pyrazole nucleus has pronounced pharmacological applications as anti- anxiety

(Haufel and Breitmaier, 1974; Wustrow et al., 1998), antipyretic, analgesic and anti-

inflammatory drugs (Eid et al., 1978; Menozzi et al., 1997). Certain alkyl pyrazoles show

significant bacteriostatic, bactericidal and fungicidal activities (Rich and Horsfall, 1952;

Potts, 1986). 1H- Pyrazole -3-carboxylic acid esters incorporated pyrazole nucleosides

have shown potent and selective anti-viral/anti-tumor activity (Manfredini et al., 1992,

1996).

5.1.1 SYNTHETIC APPROACHES

5.1.1.1 Bisheterocycles

Since the discovery of the triphenylmethyl radical by Gomberg in 1900, triaryl and

triheteroarylmethanes have attracted much attention of organic chemists and many such

compounds have found widespread applications in synthetic, medicinal and industrial

chemistry (Duxbery, 1993; Shchepinov and Korshun, 2003). Inter alia, the triarylmethyl

derivatives are useful as protective groups, photochromic agents and dyes (Rys and

Zollinger, 1972). Ring hydroxylated triaryl methanes have been reported to exhibit anti-

tumor and antioxidant activities (Mibu and Sumoto, 2000). Also, bisheteroarylmethanes are

of interest to the food industry as natural components of certain food and beverage items as

well as flavor agents in coffee (Katritzky et al., 1993). Most of the methods available for

the syntheses of triarylmethanes are multistep processes and/or require harsh reaction

conditions (Katritzky and Toader, 1997; Riad et al., 1989).

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117

5.1.1.2 Bis(indolyl)methanes

The indole moiety (Houlihan et al., 1992) is probably the most common and

important feature of a variety of natural products and medicinal agents with significant

biological activities including antimicrobial, antiviral and anti-tumor (Wu et al., 1988;

Merino et al., 1993; Ramirez and Garcio-Rubio, 2003). Among the various indole

derivatives, bis(indolyl)methanes constitute an important group of bioactive metabolites of

terrestrial and marine group origin.

Ji et al. (2004) reported a simple method for the syntheses of bis(indolyl)methanes

catalyzed by iodine under solvent-free condition.

I2, rt

NH

R ArCHO

NH

ArNH

R

R

solvent-free+

5.1.1.3 Bis(4-hydroxycoumarin)

Coumarin derivatives have recently revealed new biological activities with

interesting potential therapeutic applications besides their traditional employment as

anticoagulant-vitamin K (Stahman et al., 1947) and sustaining agents-photosensitizing

action of furocoumarin (Murray et al., 1982). They find applications as antibiotics-

novobiocin and analogs (Hinman et al., 1956) and anti-tumor drug-geiparvarin (Chen et al.,

1999).

Molecular iodine has been used as an efficient catalyst for the one-pot syntheses of

3,3’-arylmethylene bis (4-hydroxycoumarin) in water (Kidwai et al., 2007).

Chapter IV

118

O O

OH

ArCHOO

ArOH

OO O

OH

+I2 (10 mol%)

H2O, 100oC, 1 atm

5.1.1.4 Di(uracilyl)arylmethanes

Pyrimidines represent a broad class of compounds which have received

considerable attention due to wide range of biological activities (Melik-Ogan Zhanyan et

al., 1985). Several patents have been reported on the synthesis of these heterocycles.

Pyrimidine derivatives find applications as bronchodilators (Coates, 1990), vasodilators

(Figueroa-villar et al., 1992) antiallergic, antihypertensive (Raddatz and Bergmann, 1988)

and anticancer agents (Ramsey, 1974).

Di(uracilyl)aryl methanes and their homologues were synthesized through HBr-

acetic acid catalyzed condensation of uracil derivatives with readily available

arylaldehydes (Kumar et al., 2006).

N

NO

OR'

R

N

NO

OR'

R

N

N

OR'

O

R

Ar

ArCHO+HBr-CH3CO2H

120oC

5.1.1.5 Di(pyrrolyl)methanes

The pyrrole ring system (Jones and Bean, 1977) is an useful structural element in

medicinal chemistry and has found broad application in drug development as antibacterial,

antiviral, anti-inflammatory, anti-tumor and antioxidant activities (Furstner, 2003).

5-Substituted di(pyrrolyl)methanes were synthesized by the reaction of N-

tosylimines with excess pyrrole in the presence of metal triflates (Temelli and Unalerogolu,

2006).

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119

NH

H

NTs

R NH

NH

R

+ M(OTf)x (10 mol%)

5.1.1.6 Bis(furfuryl)methanes

Among the various skeletal features in natural products, furans are not only the key

subunits but are also important chemicals of commerce in the form of furfural,

tetrahydrofuran and their derivatives. These heterocycles have found applications in

pharmaceuticals, fragrances and dyes (Hou et al., 1998). Due to the medicinal importance

of these compounds, there has been a longstanding interest in the development of simple

and stereo selective methods to synthesize the furan ring efficiently.

Nair et al. (2005) have synthesized bis(furfuryl)methanes by the condensation of

furan derivatives with various aldehydes in the presence of gold(III)chloride/acetonitrile

under argon atmosphere.

ArCHOO O

Ar

OAuCl3

CH3CN, rt, Ar+

R

NR' CO2Et Sc(OTf)3

toluene/reflux

NR'

RCO2EtEtO2C

+

Chapter IV

120

5.1.2 Biological importance

Rani et al. (2002) reported the anti-inflammatory activity of azopyrazolinyl

derivatives against carrageenan induced oedema in rats at 50 mg/kg orally. The compounds

have shown promising anti-inflammatory activity. Patil et al. (1995) reported the analgesic

activity of phenyl pyrazolyl sydnones by acetic acid induced writhing method in mice at

300 mg/kg orally one compound gave maximum protection (62 %).

Saundane et al. (2005) reported the antimicrobial activity of some indole

derivatives containing pyrazoline system against S. aureus, E. coli, A. fluvus, and A. niger

at 1 mg/ml concentration by cup plate method. Some of the compounds exhibited moderate

activity against S. aureus and one compound exhibited a comparable antifungal activity.

Shivarama et al. (2000) reported the antibacterial properties of arylfuryl pyrazolines. Some

selected pyrazolines were screened against E. coli, S. aureus, B. subtilis and P. aeruginosa.

Maddirala et al. (2004) reported the antimicrobial activity of formyl pyrazolyl phenyl

indoles against B. cirroflagellosus, E. coli and S. aureus.

The antimicrobial activities of 1H-pyrazole carboxylates were evaluated by Sridhar

et al. (2004) against E. coli (ATCC 25922), S. aureus (ATCC 29213), P. aeruginosa

(ATCC 27853) and Enterobacter faecalis, Fusarium oxysporum, Curvularia lunata,

Alternaria alternata. All the compounds exerted inhibitory effects against all human

pathogenic bacteria and plant pathogenic fungi. Korgaokar et al. (1996) reported

antimicrobial activity of pyrazolines bearing chlorophenyl sulphonamido phenyl moiety.

The compounds were screened against B. megaterium, S. citrus, E. coli, Salmonella

typhosa, A. niger. Some of the compounds showed significant activity. Amr et al. (2006)

reported about the synthesis and anti-androgenic activity of some androstano[17,16-

Chapter IV

121

c]pyrazolines. Some of the compounds exhibited better anti-androgenic activity at 0.3 mg

dose level daily subcutaneous injection for 12 days compared to that of Cyproterone.

Sivaprasad et al. (2006) reported the synthesis and antimicrobial activity of

pyrazolylbisindoles at a concentration of 1 mM. The antimicrobial activities were evaluated

against Candida albicans, Staphylococcus epidermidis and Pseudomonas aeruginosa by

agar diffusion method. Antifungal activity of these compounds was evaluated by Poison

plate technique against two plant pathogenic fungi viz Rhizoctonia solani and Curvularia

lunata under in vitro condition. Most of the compounds exhibited activity.

Bansal et al. (2001) reported the synthesis and anti-inflammatory activity of aryl-3-

(β-aminonaphthyl)-2- pyrazolines at 50 mg/kg dose level by rat paw oedema method. Some

of the compounds exhibited promising anti-inflammatory with a lower ulcerogenic liability

than the standard drugs phenyl butazone and indomethacin.

In this study, modification of the 4,4’-arylmethylene-bis(5-hydroxypyrazoles)

through newer routes of synthesis using commercially available starting material, low cost

catalyst and solvent along with the lines of green chemistry and microwave assisted

reactions are to be explored to get an enhanced biological profile.

Para influenza, mumps, measles, peste des petits ruminants (PPRV), rinderpest and

human respiratory syncytial viruses are examples of members of the family

Paramyxoviridae. They can infect human beings and animals (Lamb and Kolakfsky, 1996).

PPRV is classified as the member of the morbilli virus, subgroup of the family

Paramyxoviridae which includes measles, rinderpest, canine distemper virus, phocine

distemper virus and dolphin distemper virus (Limo and Yilma, 1990). Among these

measles virus is a ubiquitous pathogen responsible for both acute and persistent infection

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122

(Buckland et al., 1989). It continues to be a major problem in developing countries with an

estimated 49 million cases reported in 1989, resulting in the death of 1.5 million children

worldwide (WHO, 1990).

Rinderpest- like disease observed in sheep in Tamil Nadu during 1989, turned out to

be the first report of PPR in India (Shaila et al., 1989). It causes disease in sheep and goats

and other small ruminants. The disease is characterized by erosive stomatitis, enteritis and

pneumonia. The disease has high morbidity and mortality rate and effective control of this

disease is of economic importance in endemic areas (Ismail et al., 1995).

In the present study, for the first time, screening results of the antimicrobial and

antiviral activity of bispyrazoles were reported.

Chapter IV

123

5.2 OBJECTIVES

In the quest to develop a mild and practical protocol for the synthesis of

bis(pyrazolyl)methanes, it was speculated that potassium hydrogen sulphate might be

ideal for effecting the condensation of aldehydes and pyrazolones. Pyrazolone moieties

possess a wide range of biological applications and this has motivated us

To synthesize and characterize a few bis(pyrazolyl)methanes

To study the possible antibacterial, antifungal and antiviral activities of the

synthesized compounds

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124

5.3 EXPERIMENTAL

5.3.1 Materials and methods

Potassium hydrogen sulphate was obtained from Aldrich. All melting points were

uncorrected. IR spectra were recorded on a Perkin Elmer FT-IR spectrophotometer. 1H and

13C NMR spectra were recorded in DMSO-d6 and CDCl3 using TMS as an internal

standard on a JEOL spectrometer and Bruker spectrometer at 500 MHz and 125 MHz and

300 MHz and 75 MHz respectively. Mass spectra were recorded on a JEOL DX 303 HF

spectrometer. Elemental analyses were recorded using a Thermo Finnigan FLASH EA

1112 CHN analyzer. Column chromatography was performed on silica gel (200-400 mesh,

SRL, India). Analytical TLC was performed on precoated plastic sheets of silica gel G/UV-

254 of 0.2 mm thickness (Macherey-Nagel, Germany).

5.3.2 General procedure for the synthesis of 4,4’-arylmethylene-bis (5-

hydroxypyrazoles) (3a-j)

To the round bottomed flask containing 1-phenyl-3-methyl-pyrazol-5-one (2

mmol) and aromatic aldehyde (1 mmol) in water, KHSO4 (20 mol %) was added and

stirred at room temperature. After the completion of the reaction, the solid product obtained

was filtered and dried. The pure product was obtained by recrystallisation from ethanol.

5.3.3 Antibacterial activity Antibacterial study was carried out for the synthesized bis(pyrazolyl)methanes 3a-

j by disc diffusion method against ATCC gram positive and gram negative bacterial strains

at 1000 µg, 500 µg and 100 µg concentrations.

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125

5.3.3.1 Materials requirement:

• The gram positive organism used for this study was Staphylococcus aureus and the

gram negative organism was and Klebsiella pneumoniae (The strains were received

from Department of Veterinary Microbiology, Madras Veterinary College,

Chennai-600 007).

• The medium Tryptose Soy Agar (TSA) powder (HiMedia, Mumbai) was used at 4 g

/100 ml to prepare solid agar plates and was used for both Gram positive and Gram

negative bacteria.

5.3.3.2 Method (Cruickshank et al., 1975)

The same procedure was followed as given in chapter -1.Students‘t’ test was used for

statistical analysis. P values < 0.001 and <0.01 were considered to be statistically

significant.

5.3.4 Antifungal activity

The in vitro anti-fungal activity of the bis(pyrazolyl)methanes 3a-j were studied

against Candida albicans using disc diffusion method at 1000 µg, 500 µg and 100 µg

concentrations.

5.3.4.1 Materials requirement

• The ATCC strain of Candida albicans was used for the anti-fungal study. (The

strain was received from Department of Veterinary Microbiology, Madras

Veterinary College, Chennai-600 007)

• The medium of Sauboraud’s Dextrose Agar (HiMedia, Mumbai) was used at 6.5

g/100 ml concentration for preparing solid agar plates.

Chapter IV

126

5.3.4.2 Method

The same procedure was followed as given in chapter -3.

5.3.5 In vitro antiviral study

5.3.5.1 Introduction

Virus is an ultramicroscopic infectious parasite responsible for significant

morbidity and mortality in populations worldwide (Sharma and Sharma, 2007).

Many bacterial infections can now be successfully controlled by chemotherapeutic agents.

Satisfactory treatment of viral infections however still remains difficult. Viruses unlike

bacteria are obligate parasites as they are active only within the host cells. Virus outside the

host cells is inert as it cannot replicate independently. They have to use energy generating,

RNA or DNA replicating, and protein synthesizing machinery of the host cells for their

growth. They not only replicate in host cells but direct them to make new viral particles.

Therefore, it is very difficult to find an antiviral drug that would selectively inhibit or kill

the virus without being toxic to the host (Satoskar and Bhandarkar, 1991). Several anti-

viral drugs are currently licensed for use. All are of use in only a limited number of

situations and may be toxic to the host. Ideal antiviral agents remain to be developed.

Viral diseases can be controlled by vaccination or by antiviral drug therapy or by

stimulating host defense mechanisms. Vaccines are available to prevent measles, small

pox, chicken pox, rubella, mumps, poliomyelitis, yellow fever and hepatitis-B. However,

for HIV and rhinovirus (common cold) infections and their utility is very limited. Further

the vaccines cannot prevent the spread of active infection within the host; hence they are of

no use once the infection has occurred. Nevertheless, passive immunity can be provided

with human immunoglobulin to assist the body’s own defense mechanisms. Intravenously

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127

administered immunoglobulin can provide immediate passive immunity, while with

intramuscular immunoglobulin; the occurrence of peak plasma concentration may take 2 -

3 days.

For an antiviral agent to be optimally active, the patient must have a competent host

immune system that can eliminate or effectively halt virus replication.

Immunocompromised patients are more prone to frequent viral replications that may recur

when antiviral drugs are stopped. The chemotherapy of viral infections may involve

inhibition of any of the steps in viral attachment, uncoating, penetration, replication,

growth and release of progeny virions.

5.3.5.2 Materials requirement

• Vero cell line (Received from Animal Biotechnology, Madras Veterinary College,

Chennai-7)

• 3-(4,5-dimehtyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye

(HiMedia, Mumbai)

• DMSO (Sigma, USA)

• Eagle’s Minimum Essential Medium (HiMedia, Mumbai), Fetal calf serum

(Invitrogen, USA), Trypsin Versene Glucose solution (HiMedia, Mumbai),

penicillin- 100 IU and streptomycin-100 μg per ml of medium (HiMedia, Mumbai)

• PPR virus with 106.5 TCID50/ml titre (Received from Animal Biotechnology,

Madras Veterinary College, Chennai-7)

• Haematoxylin and Eosin stains, Carnoy’s fixative

• Bio Tek ELISA reader

• Nikon inverted binocular microscope

Chapter IV

128

• 96 well microtitre plates, 25 cm2 tissue culture flask (Nunc, USA)

5.3.5.3 Method

5.3.5.3.1 Cytotoxic assay (Francis and Rita, 1986)

The end point of microtitration assay is usually an estimate of the number of cells.

The viability of cells is done directly by cell count. Cell viability is measured by MTT [3-

(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide] reduction. MTT is a

yellow water soluble tetrazolium dye that is reduced by live cells. Water soluble

tetrazolium dye is reduced by live cells to an insoluble purple formazan product.

The test compounds were dissolved in DMSO at 10 mg/100 µl concentration and from this

stock solution 1000 µg, 750 µg, 500 µg, 250 µg, 100 µg, 50 µg, 25 µg and 12.5 µg

concentrations were used to assess the cytotoxicity. A monolayer formed vero cells in 25

cm2 flask were trypsinised and seeded into 96 well microtitre plates at 10,000cells/well.

After getting a confluent monolayer, the growth medium was discarded and fresh

maintenance medium containing different concentration of the different test compounds

and 2 wells for each concentration were used. The plates were incubated at 37ºC with 5 %

CO2 in an incubator for 72 h. At the end of incubation, the microtitre plates with test

compounds were washed with fresh minimum essential medium (MEM) two times and

then the MTT dye at 5 mg/ml concentration were used. The plates were incubated at 37ºC

with 5 % CO2 for 3-8 h and then 100 µl/ well DMSO were added. The readings were taken

at 570 nm in Bio Tek ELISA reader. The results were compared with controls without test

compounds and non-toxic concentration of the test compounds was derived by calculating

the concentration of the test compounds required to reduce the viability by 50 %.

Chapter IV

129

5.3.5.3.2 Antiviral assay (Hu and Hsiung, 1989)

The test compounds with non-toxic concentration were prepared and kept ready.

The Vero cells in flask were seeded onto 96 well microtitre plates at 10,000 cells/ well. The

non-toxic concentration of test compounds in 100 µl of 100 TCID50 PPRV was allowed to

react at 37ºC for 1 h. Then the mixture was layered onto the preformed Vero cells after

discarding the growth medium. Controls like cell control, virus control were also made.

The plates were incubated at 37ºC with 5% CO2 for 5 days to get the complete viral

cytopathic changes. At every 24 h, the cells were observed under microscope to note down

the antiviral effect. At the end of incubation, the plates were washed with fresh MEM

(minimum essential medium) and fixed with carnoy’s fixative, stained with Haematoxylin

and Eosin. The readings were recorded by observing under microscope and antiviral effect

of the test compounds was calculated.

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130

5.4 SPECTRAL DATA

4,4’-Phenylmethylene-bis(3-methyl-5-hydroxypyrazole) 3a

Pale yellow solid. mp: 171-173°C. υmax (KBr): 3430, 2920, 1615, 1489, 1402, 1290, 1140

cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.30 (s, 6H), 3.97 (br s, 2H, OH, D2O

exchangeable), 4.95 (s, 1H), 7.12 (m, 1H), 7.21 (m, 6H), 7.39 (t, 4H, J = 7.6 Hz), 7.66 (d,

4H, J = 8.4 Hz). 13C NMR (DMSO-d6, 125 MHz): δ 12.0, 33.5, 121.3, 126.4, 126.5, 127.7,

128.7, 129.5, 137.4, 142.5, 146.8. MS (m/z): 436 (M+). Anal. Calcd for C27H24N4O2: C,

74.29; H, 5.54; N, 12.83. Found: C, 74.22; H, 5.48; N, 12.78.

4,4’-(3-Methylphenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3b

White solid. mp: 203-205°C. υmax (KBr): 3430, 3063, 1612, 1560, 1499, 1401, 1366, 1307,

1108 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.22 (s, 9H), 3.40 (br s, 2H, OH, D2O

exchangeable), 4.90 (s, 1H), 7.06 (m, 3H), 7.19 (t, 2H, J = 7.6 Hz), 7.38 (t, 4H, J = 7.65

Hz), 7.48 (s, 1H), 7.67 (d, 4H, J = 7.65 Hz). 13C NMR (DMSO-d6, 125 MHz): δ 12.3, 20.3,

31.9, 120.9, 122.4, 124.7, 126.0, 126.7, 128.6, 129.5, 131.0, 135.8, 140.4, 146.2. MS (m/z):

450 (M+). Anal. Calcd for C28H26N4O2: C, 74.65; H, 5.82; N, 12.44. Found: C, 74.57; H,

5.78; N, 12.39.

4,4’-(4-Methylphenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3c

White solid. mp: 203-204°C. υmax (KBr): 3432, 2921, 1600, 1501, 1408, 1294, 1026 cm-1.

1H NMR (DMSO-d6, 500 MHz): δ 2.21 (s, 3H), 2.28 (s, 6H), 3.36 (br s, 2H, OH, D2O

exchangeable), 4.87 (s, 1H), 7.03 (d, 2H, J = 8.4 Hz), 7.10 (d, 2H, J = 7.65 Hz), 7.19 (t,

2H, J = 7.65 Hz), 7.39 (t, 4H, J = 7.65 Hz), 7.67 (d, 4H, J = 7.65 Hz). 13C NMR (DMSO-

d6, 125 MHz): δ 12.2, 21.1, 33.3, 121.0, 122.8, 124.7, 125.1, 126.1, 127.6, 129.2, 129.5,

Chapter IV

131

135.3, 139.7, 146.8. MS (m/z): 450 (M+). Anal. Calcd for C28H26N4O2: C, 74.65; H, 5.82;

N, 12.44 . Found: C, 74.53; H, 5.75; N, 12.38.

4,4’-(4-Methoxyphenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3d

White solid. mp: 222-224°C. υmax (KBr): 3499, 2971, 1605, 1505, 1405, 1364, 1247, 1168

cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.29 (s, 6H), 3.41 (br s, 2H, OH, D2O

exchangeable), 3.67 (s, 3H), 4.88 (s, 1H), 6.80 (d, 2H, J = 8.4 Hz), 7.14 (d, 2H, J = 8.4 Hz),

7.19 ( t, 2H, J = 6.9 Hz), 7.39 (t, 4H, J = 7.65 Hz), 7.68 (d, 4H, J = 8.4 Hz). 13C NMR

(DMSO-d6, 125 MHz): δ 12.1, 32.9, 55.5, 114.0, 120.4, 121.0, 123.5, 124.8, 126.1, 128.7,

129.5, 134.5, 146.8, 158.0. MS (m/z): 466 (M+). Anal. Calcd for C28H26N4O3: C, 72.09; H,

5.62; N, 12.01. Found: C, 72.0; H, 5.57; N, 12.10.

4,4’-(4-Fluorophenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3e

Pale yellow solid. mp: 166-168°C. υmax (KBr): 3400, 2922, 1599, 1510, 1410, 1340, 1176

cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.26 (s, 6H), 3.39 (br s, 2H, OH, D2O

exchangeable), 5.09 (s, 1H), 7.07 (d, 2H, J = 6.9 Hz), 7.20 (t, 3H, J = 7.65 Hz), 7.39 (m,

5H), 7.66 (d, 4H, J = 8.4 Hz). 13C NMR (DMSO-d6, 125 MHz): δ 12.1, 27.8, 115.6, 115.8,

121.3, 124.6, 126.2, 128.7, 129.5, 129.8, 129.9, 146.4. MS (m/z): 456 (M+). Anal. Calcd for

C27H23FN4O2: C, 71.35; H, 5.10; N, 12.33. Found: C, 71.22; H, 5.06; N, 12.27.

4,4’-(4-Nitrophenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3f

Pale brown solid. mp: 230-232°C. υmax (KBr): 3430, 2922, 1599, 1502, 1413, 1347, 1186

cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.32 (s, 6H), 3.46 (br s, 2H, OH, D2O

exchangeable), 5.10 (s, 1H), 7.20 (t, 2H, J = 7.45 Hz), 7.39 (t, 4H, J = 8.0 Hz), 7.48 (d, 2H,

J = 9.15 Hz), 7.67 (d, 4H, J = 8.0 Hz), 8.13 (d, 2H, J = 8.55 Hz). 13C NMR (DMSO-d6, 125

MHz): δ 12.1, 33.7, 121.1, 123.9, 126.3, 129.2, 129.5, 146.5, 146.8, 150.9. MS (m/z): 481

Chapter IV

132

(M+). Anal. Calcd for C27H23N5O4: C, 67.35; H, 4.81; N, 14.54. Found: C, 67.20; H, 4.77;

N, 14.45.

4,4’-(3,4-Dimethoxyphenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3g

Pale yellow solid. mp: 200-202°C. υmax (KBr): 3450, 3430, 2920, 1615, 1489, 1402, 1290,

1140 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.29 (s, 6H), 3.51 (br s, 2H, OH, D2O

exchangeable), 3.63 (s, 3H), 3.67 (s, 3H), 4.84 (s, 1H), 6.80 (t, 2H, J = 8.4 Hz), 6.86 (s,

1H), 7.19 (t, 2H, J = 7.65 Hz), 7.39 (t, 4H, J = 7.65 Hz), 7.67 (d, 4H, J = 7.6 Hz). 13C NMR

(DMSO-d6, 125 MHz): δ 12.2, 33.5, 55.9, 56.0, 112.1, 119.8, 121.1, 124.6, 125.8, 126.1,

129.5, 135.6, 138.0, 146.7, 147.7, 148.9. MS (m/z): 496 (M+). Anal. Calcd for C29H28N4O4:

C, 70.15; H, 5.68; N, 11.28. Found: C, 70.04; H, 5.63; N, 11.38.

4,4’-(4-Hydroxy-3-methoxyphenyl)methylene-bis(3-methyl-5-hydroxypyrazole) 3h

Pale pink solid. mp: 200-202°C. υmax (KBr): 3450, 3430, 2920, 1615, 1489, 1402, 1290,

1140 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ 2.28 (s, 6H), 3.39 (br s, 2H, OH), 3.64 (s,

3H), 4.82 (s, 1H), 6.64 (m, 2H), 6.83 (s, 1H), 7.19 (t, 2H, J = 6.9 Hz), 7.39 (t, 4H, J = 7.65

Hz), 7.67 (d, 4H, J = 7.65 Hz), 8.77 (br s, 1H). 13C NMR (DMSO-d6, 125 MHz): δ 12.2,

33.4, 56.2, 112.4, 115.7, 120.2, 121.1, 123.5, 124.6, 126.1, 129.5, 133.8, 145.4, 146.7,

147.7. MS (m/z): 482 (M+). Anal. Calcd for C28H26N4O4: C, 69.70; H, 5.43; N, 11.61.

Found: C, 69.58; H, 5.38; N, 11.54.

4,4’-Furfurylmethylene-bis(3-methyl-5-hydroxypyrazole) 3i

White solid. mp: 189-191°C. υmax (KBr): 3430, 2923, 1604, 1499, 1408, 1282, 756 cm-1.

1H NMR (DMSO-d6, 500 MHz): δ 2.27 (s, 6H), 3.46 (br s, 2H, OH, D2O exchangeable),

4.95 (s, 1H), 6.09 (s, 1H), 6.31 (s, 1H), 7.20 (t, 2H, J = 7.45 Hz), 7.39 (t, 4H, J = 8.0 Hz),

7.47 (s, 1H), 7.67 (d, 4H, J = 8.0 Hz). 13C NMR (DMSO-d6, 125 MHz): δ 12.1, 28.8,

Chapter IV

133

106.7, 110.9, 121.1, 126.2, 129.5, 142.1, 146.5, 154.7. MS (m/z): 426 (M+). Anal. Calcd for

C25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 70.31; H, 5.16; N, 13.07.

4,4’-Pyridylmethylene-bis(3-methyl-5-hydroxypyrazole) 3j

Pale yellow solid. mp: 232-234°C. υmax (KBr): 3428, 2915, 1599,1499, 1420, 1289 cm-1.

1H NMR (DMSO-d6, 500 MHz): δ 2.31 (s, 6H), 3.60 (br s, 2H, OH, D2O exchangeable),

5.02 (s, 1H), 7.19 (t, 2H, J = 7.6 Hz), 7.33 (m, 1H), 7.39 (t, 4H, J = 8.4 Hz), 7.67 (d, 5H, J

= 7.65 Hz), 8.38 (m, 2H). 13C NMR (DMSO-d6, 125 MHz): δ 12.2, 31.5, 104.3, 121.1,

123.9, 126.2, 129.5, 136.0, 137.8, 138.6, 146.7, 147.2, 148.8. MS (m/z): 437 (M+). Anal.

Calcd for C26H23N5O2: C, 71.38; H, 5.30; N, 16.01. Found: C, 71.27; H, 5.25; N, 15.95.

Chapter IV

134

5.5 RESULTS AND DISCUSSION

5.5.1 Synthesis of 4,4’-arylmethylene-bis(5-hydroxypyrazoles)

Among various nitrogen heterocycles, the pyrazoline nucleus has been reported to

possess great importance in the field of medicine and biochemistry. In continuation with the

search for simple non-hazardous methods for the transformations in organic synthesis, a

highly versatile and efficient synthesis of bis(pyrazolyl)methanes from aryl aldehydes,

pyrazolone and catalytic amounts of potassium hydrogen sulphate was developed.

Over the past few years, potassium hydrogen sulphate has emerged as powerful

catalyst in various organic transformations. Owing to several advantages such as

inexpensive, non-toxic and eco-friendly, potassium hydrogen sulphate affords the desired

product in good to excellent range yields with high selectivity. The present work shows

that potassium hydrogen sulphate proves to be an excellent catalyst in aqueous reaction

medium.

The synthetic pathway employed in the preparation of 4,4’-arylmethylene-bis(5-

hydroxypyrazoles) is outlined in Scheme 4. Pyrazolone (2 mmol), aldehyde (1 mmol) and

potassium hydrogen sulfate (20 mol %) in water (20 ml) were added to a flask and stirred

at room temperature for about 24 hr. After completion of the reaction, the precipitated solid

was filtered and dried. The crude product was purified by recrystallization from ethanol (70

%). .

Chapter IV

135

Scheme 4

A range of aromatic and heteroaromatic aldehydes were subjected to react with 3-

methyl-5-pyrazolones in the presence of 20 mol % of KHSO4 and water as solvent (Table

4.1). It was found that both aromatic and heteroaromatic aldehydes reacted equally good to

afford 4,4’-arylmethylene-bis(5-hydroxypyrazoles) in excellent yields.

NN

CH3

ON

N

R

NN

OHOH

CH3

CH3

+ RCHO2H2O, rt

1

2

3a-j

KHSO4(20 mol%)

Chapter IV

136

Table 4.1 Synthesis of 4,4’-arylmethylene-bis(5-hydroxypyrazoles) in water

NN

NNCH3

CH3

OHOH

Ph

Ph

NN

NNCH3

CH3

OHOH

Ph

Ph

CH3

NN

NNCH3

CH3

OHOH

Ph

Ph

CH3

NN

NNCH3

CH3

OHOH

Ph

Ph

MeO

NN

NN

CH3

CH3

OHOH

Ph

Ph

F

Entry Time (min) Yielda (%)

1

2

3

15

20

20

92

90

90

4

5

20 90

15 91

Product (3)

3b

3c

3d

3a

3e

Chapter IV

137

NN

NNCH3

CH3

OHOH

Ph

Ph

O2N

NN

NNCH3

CH3

OHOH

Ph

Ph

MeO OMe

NN

NNCH3

CH3

OHOH

Ph

Ph

OH OMe

NN

NNCH3

CH3

OHOH

Ph

Ph

O

NN

NN

NCH3

CH3

OHOH

Ph

Ph

6

7

8

10

25

25

94

88

88

9

10

15 94

15 94

3f

3g

3h

3i

3j

aIsolated yield.

Chapter IV

138

The structures of the compounds 3a-j were confirmed by IR, 1H and 13C NMR

spectroscopy, mass spectrometry and elemental analysis. The mass spectrum of 3i

displayed the molecular ion (M+) peak at m/z 426. The IR spectrum (Spectrum 4.3)

showed –OH stretching at 3430 cm-1. The 1H NMR spectrum (Spectrum 4.1) of 3i showed

singlets at δ 2.27 (-CH3) and δ 4.95 (-CH). Aromatic protons were seen in the range δ 6.09

– 7.67 and a broad singlet at δ 3.46 due to –OH (D2O exchangeable). Resonances at δ 12.1

(methyl group), δ 28.8 (-CH) and δ 106.7-154.7 (aromatic carbons) were observed in the

13C NMR spectrum (Spectrum 4.2).

5.5.2 Antimicrobial activities

Molecular modification involves the chemical alteration of a known and previously

characterized organic compound for the purpose of enhancing its usefulness as a drug.

This may enhance its specificity for a particular body target site, increased potency,

improved rate, extent of absorption, modify its pharmacokinetics in the body, reduced

toxicity or change other physico-chemical properties. Knowledge of chemical structure–

pharmacologic activity relationships plays an important role in designing new molecules.

Saundane et al. (2005) reported the antimicrobial activity of some indole derivatives

containing pyrazoline system against S. aureus, E. coli, A. fluvus, and A. niger at 1 mg/ml

concentration by cup plate method. Compounds with methyl and bromo or phenyl and

methoxy substituent exhibited moderate activity against S. aureus and a compound with

phenyl and methoxy exhibited a comparable antifungal activity against A. niger and A.

flavus. The antimicrobial activities of 1H-pyrazole carboxylates were evaluated by Sridhar

et al. (2004) against E. coli (ATCC 25922), S. aureus (ATCC 29213), P. aeruginosa

(ATCC 27853) and Enterobacter faecalis, Fusarium oxysporum, Curvularia lunata,

Chapter IV

139

Alternaria alternate at 0.1 and 0.5 mg/ml concentration. All the compounds exerted

inhibitory effects against all human pathogenic bacteria and plant pathogenic fungi. Among

the compounds tested, formyl and methyl substituents significantly inhibited bacterial

growth from 25-97 % and 51-91 % respectively. Formyl substituents exhibited highly

significant antifungal activity (24-76 %).

Korgaokar et al. (1996) reported the antimicrobial activity of pyrazolines bearing

chlorophenyl sulphonamido phenyl moiety. The compounds were screened against B.

megaterium, S. citrus, E. coli, Salmonella typhosa, A. niger and displayed moderate

activity and significant activity against E. coli. Simple pyrazoline derivatives bearing

chloro, methoxy, and nitro groups exhibited maximum activity. Shivarama et al. (2000)

reported the antibacterial properties of arylfuryl pyrazolines. Some selected pyrazolines

were screened against E. coli, S. aureus, B. subtilis and P. aeruginosa. The compounds

carrying nitro, bromo and chlorophenyl furyl groups showed a similar degree of activities

against E. coli, and S. aureus compared with the standard drug furacin. MIC values were

determined by serial dilution method. The MIC values were found between 3.3 – 25

mg/ml. Maddirala et al. (2004) reported the antimicrobial activity of formyl pyrazolyl

phenyl indoles against B. cirroflagellosus, E. coli and S. aureus at 1000 μg/ml

concentration. Some of the compounds showed potent activity against E. coli and S.

aureus.

Sivaprasad et al. (2006) reported the synthesis and antimicrobial activity of

pyrazolylbisindoles at a concentration of 1 mM. The antimicrobial activities were evaluated

against Candida albicans, Staphylococcus epidermidis and Pseudomonas aeruginosa by

agar diffusion method. Anti-fungal activity of these compounds was evaluated by Poison

Chapter IV

140

plate technique against two plant pathogenic fungi viz Rhizoctonia solani and Curvularia

lunata under in vitro condition. Most of the compounds having chloro and bromo, methoxy

and bromo, dibromo, bromo, chloro substituents in the phenyl or indole portion of the

compound exhibited activity. Compound with methoxy and nitro moiety did not show any

antimicrobial activity and a compound with nitro moiety did not show any antifungal

activity.

5.5.2.1 Antibacterial activity

Abdel Rehman et al. (2004) reported the antibacterial and antifungal activities of

some new spiroindoline based heterocycles against Bacillus subtilis, Bacillus megatherium,

Escherichia coli, Aspergillus niger and Aspergillus oryzae. The results revealed that the

prepared spiro 3 H-indoles -3, 4’-pyrano(3’,2’-d) oxazole derivative showed comparable

anti-bacterial activity, spiro 3H-indole-3,4’-pyrazolo(3’,4’-b) pyrano(3’,2’-d) oxazole

derivatives revealed very high anti-bacterial activity and this might be as a result of the

presence of the extended fused pyrazole moiety in their structure. On the other hand, all the

compounds exhibited an interesting high antifungal activity.

In the present study, the synthesized compounds (3a-j) were tested against S. aureus,

K. pneumoniae at 100, 500 and 1000 µg concentration. Compound 3c and 3f having methyl

and nitro substituent exhibited significant antibacterial activity against K. pneumoniae.

Compound 3c also showed significant activity against S. aureus (Table 4.2). Compound 3a

without any substitution in the phenyl portion did not have any activity. Change of 4-

methyl (3c) by 4-methoxy (3d), 3-methyl (3b), 2-fluoro (3e), 3,4-dimethoxy (3g) and 3-

methoxy 4-hydroxy (3h) substitutions in the phenyl portion of bis(pyrazolyl)methanes did

not have any activity. Change of phenyl portion (3a) by furfuryl (3i) and 3-pyridyl (3j) also

did not have any activity.

Chapter IV

141

5.5.2.2 Antifungal activity

In the present study, the synthesized compounds (3a-j) were tested against

C. albicans at 100, 500 and 1000 µg concentration. None of the compounds showed any

activity against C. albicans for the three concentrations tested (Table 4.2).

5.5.2.3 Antiviral activity of 4, 4’-arylmethylene-bis (5-hydroxypyrazoles)

Antiviral evaluation of the synthesized bis(pyrazolyl)methanes 3a-j were carried

out in vero cell line using peste des petits ruminants virus (PPRV) which is a RNA virus of

the Morbilli virus genus and as the members are serologically related, the antiviral effect of

this compound against PPRV could very well be applied to other viruses also. Before

studying the antiviral effect of the test compounds, it is mandatory to assess the cytotoxic

concentration of the test compounds under in vitro condition using cell line.

The antiviral activity of the synthesized BPMs (3a-j) was evaluated against PPRV

by CPE inhibition assay. All the compounds were tested for cytotoxic activity in vero cell

line by MTT assay method (Francis and Rita, 1986). The cytotoxic concentration (CC50) of

the compounds was between 6.25 – 250 µg/100 µl. The concentrations that were non-toxic

to the vero cell culture were selected for anti-viral screening (Hu and Hsiung, 1989).

Among the tested compounds, compounds 3i and 3j having furfuryl and pyridyl ring

exhibited potent activity against PPRV with 100 % CPE inhibition. Compound 3a without

any substitution in the phenyl ring showed 75 % CPE inhibition, compound 3b showed 50

% CPE inhibition and compounds 3c, 3d, 3e, 3f, 3g and 3h showed lesser than 50 % CPE

inhibition (Fig. 4.1a-e and 4.2a-d). The results are given in Table 4.3

Chapter IV

142

5.6 SUMMARY

In conclusion, we have developed a facile and simple method for the synthesis of 4,

4’-arylmethylene-bis(5-hydroxypyrazoles) and were screened for antimicrobial and

antiviral activity .The compounds 3c and 3f showed significant antibacterial activity

against K. pneumoniae. Compound 3c showed significant antibacterial activity against S.

aureus. The compounds (3a-3j) did not have any activity against C. albicans. The

cytotoxic concentration (CC50) of the compounds was between 6.25 – 250 µg/100 µl.

Compound 3a and compound 3b showed 75 % and 50 % CPE inhibition and compounds

3i and 3j exhibited potent activity against PPRV with 100 % CPE inhibition and found to

be more potent than the standard drug used. Further biological evaluation to delineate the

mode of action as well as study of animal models to assess the full potential of

bis(pyrazolyl)methanes is warranted.

Chapter IV

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Chapter IV

Spectrum 4.1 1H NMR spectrum of Compound 3i

Chapter IV

Spectrum 4.2 13C NMR spectrum of Compound 3i

Spectrum 4.3 IR spectrum of Compound 3i

4000.0 3000 2000 1500 1000 400.00.0

10

20

30

40

50

60

70

80

90

100.0

cm-1

%T

3430.16

2921.59

1603.79

1499.191407.86

1281.61

1188.72 1010.48

755.61

690.94

596.22

499.78

443.41

Chapter IV

Table 4.2 Antibacterial and anti fungal activities of BPMs

* P<0.01, ** P<0.001 when compared with control (6 mm) – student’s t - test

Compound

No

Zone of inhibition of different conc. of compounds for different organisms in mm

Klebsiella pneumoniae Staphylococcus aureus Candida

albicans 1000 µg 500 µg 100 µg 1000 µg 500 µg 100 µg

3a - - - - - -

All the three

concentrations of

different compounds

did not have any

activity against this

yeast organism

3b - - - - - -

3c 8** 7.5** 7* 8** 8** 7*

3d - - - - - -

3e - - - - - -

3f 8** 7* 7* - - -

3g - - - - - -

3h - - - - - -

3i - - - - - -

3j - - - - - -

Standard Gentamycin-32** (10 µg) Ciprofloxacin-35** (5 µg) Nystatin-25**

(100 units)

Chapter IV

Fig. 4.1a-e Normal and treated Vero cells

(Phase contrast photomicrograph, 40x)

a) b) c)

Normal Vero cells

d) e)

Compound 3i & 3j treated Vero cells showing 100% CPE inhibition

Chapter IV

Fig. 4.2a-d CPE of PPRV in Vero cells

(Phase contrast photomicrograph, 40 x & 200 x)

a) b)

48 h PI 96 h PI

CPE of PPRV in Vero cells

(Phase contrast photomicrograph, 200x)

c) d)

Multinucleated giant cells in compound 3c & 3f treated cells- 96 h PI

Chapter IV

Table 4.3 Antiviral activity of 4, 4’-arylmethylene-bis (5-hydroxypyrazoles) 3a-j

Compounds

Cytotoxic

concentration

(μg/100 μl)

Effective

concentration

(μg/100 μl)

CPE inhibition

(%)

3a 12.5 6.25 75

3b 25.0 12.5 50

3c 6.25 - <50

3d 6.25 - <50

3e 6.25 - <50

3f 6.25 - <50

3g 25.0 - <50

3h 12.5 - <50

3i 12.5 6.25 100

3j 250 125 100

Ribavirin 25 90