5.0 chapter –iv bis (pyrazolyl)...
TRANSCRIPT
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).
Chapter IV
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).
Chapter IV
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
Chapter IV
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
Chapter IV
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.
Chapter IV
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
Chapter IV
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.
Chapter IV
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
5.7 REFERENCES
Abdel-Rahman, A.H., Kesh, E.M., Hanna, M.A., El-Bady, Sh.M. Bioorg. & Med. Chem.
2004, 12, 2483.
Amr,A.E.,Abdel-Latif,N.A. and Abdalla,M.M.Bioorg .Med.Chem.2006,14,373.
Bansal, E., Srivastava, V.K. and Asok kumar, Eur. J. Med. Chem., 2001, 36, 81.
Buckland, R., Giraudon, P. and Wild, F. J. Gen. Virol. 1989, 70, 435.
Chen, Y. L.; Wang, T. C.; Tzeng, C. C.; Cheang, N. C. Helv. Chim. Acta 1999, 82, 191.
Coates, W. J. Eur. Patent 351058, 1990.
Cruikshank, R., Dugid, J. P., Marmion, B.P. and Swain, R.H.A., Medical Microbiology,
12th ed.; Vol. II, Churchill Livingston, New York, 1975, 196.
Duxbury, D. F. Chem. Rev. 1993, 93, 381.
Eid, A.I., Kira, M.A. and Fahmy, H.H. J. Pharm. Belg., 1978, 33, 303.
Figueroa-Villar, J. D.; Carneiro, C. L.; Cruz, E. R. Heterocycles 1992, 34, 891.
Francis, D. and Rita, L. J. Immunol. Methods, 1986, 89, 271.
Furstner, A. Angew. Chem. Int. Ed. 2003, 42, 3528.
Gomberg, M. J. Am. Chem. Soc. 1900, 22, 757.
Haufel, J. and Breitmaier, E., Angrew. Chem., 1974, 13, 604.
Hinman, J. W., Hoeksema, H., Caron, E. L. and Jackson, W. G. J. Am. Chem. Soc. 1956,
78, 1072.
Hou, X. L., Cheung, H. Y., Hon, T. U., Kwan, P. L., Lo, T. H., Tong, S. Y. and Wong, H.
N. C. Tetrahedron 1998, 54, 1955.
Houlihan, W. J., Remers, W. A. and Brown, R. K. Indoles: Part I, Wiley: New York, NY,
1992.
Chapter IV
Hu, J.M. and Hsiung, G.T. Antiviral Res. 1989, 11,217.
Ismail, T.M., Yamanaka, N.K., Saliki, J.T., El-Kholy, A., Mebus, C. and Yilma, T., Virol.,
1995, 208, 776.
Ji, S. –J., Wang, S. –Y., Zhang, Y. and Loh, T. –P. Tetrahedron 2004, 60, 2051.
Jones, R.A. and Bean, G.P. The Chemistry of Pyrroles, Academic, London, 1977.
Katritzky, A. R. and Toader, D. J. Org. Chem. 1997, 62, 4137.
Katritzky, A. R. Xie, L. and Fan, W.-Q. J. Org. Chem. 1993, 58, 4376.
Kidwai, M.; Bansal, V.; Mothsra, P.; Saxena, S.; Somvanshi, R. K.; Dey, S.; Singh, T. P. J.
Mol. Cat. A: Chemical 2007, 268, 76.
Korgaokar, S.S., Patel, P.H., Shah, M.J. and Parekh, H.H. Indian J. Pharm. Sci. 1996, 58,
222.
Kumar, S., Vaishalli, M., Kaur, N. and Kaur, K. Tetrahedron Lett. 2006, 47, 8483
Lamb, R. A. and Kolakfsy, D., Paramyxoviridae: The viruses and their replication. In
Fields Virology; Fields, B.N., Knipe, D.M., Howley, P.M. et al., Eds., 3rd ed.;
Lippincott-Raven publishers, Philadelphia, 1996, 1178.
Limo, M. and Yilma, T., Virology, 1990, 175, 323.
Maddiralla, S.J., Gokak, V.S. and Basanagoudar, L.D. Indian J. Chem. 2004, 43B, 2410.
Manfredini, S., Bazzanini, R., Baraldi, P.G., Bonora, M., Marangoni, M., Simoni, D., Pani,
A., Scintu, F., Pinna, E., Pisano, L. and Colla, P.L. Anti-Cancer drug Des. 1996, 11,
193.
Manfredini, S., Bazzanini, R., Baraldi, P.G., Guarneri, M., Simoni, D., Marongiu, M.E.,
Pani, A., Tramontano, E. And Colla, P.L. J. Med. Chem. 1992, 35, 917.
Melik-Ogan Zhanyan, R. G., Khachatryan, V. E. and Gapoyan, A. S. Russ. Chem. Rev.
Chapter IV
1985, 54, 262.
Menozzi, G., Mosti, L., Fossa, P., Mattioli, F. and Ghia, M. J. Heterocycl. Chem. 1997, 34,
963.
Merino, A., Madden, K. R., Lane, W. S., Champoux, J. J. and Deinberg, D. Nature 1993,
365, 227.
Mibu, N. and Sumoto, K. Chem. Pharm. Bull. 2000, 48, 1810.
Murray, R. D. H., Mendez, J. and Brown, S. A. The Natural Coumarins, Wiley, Chichester,
UK, 1982.
Nair, V., Abhilash, K. G. and Vidya, N. Org. Lett. 2005, 7, 5857.
Patil, B.M., Badami, B.V. and Puranik, G.S. Indian Drugs, 1995, 32, 493.
Potts, K.C.H. In Comprehensive Heterocyclic Chemistry, Pergamon: Oxford, 1986, Vol. 5,
part 4A.
Raddatz, P. and Bergmann, R. Ger. Patent 360731, 1988.
Ramirez, A. and Garcia-Rubio, S. Curr. Med. Chem. 2003, 10, 1891.
Ramsey, A. A. U.S. Patent 3830812, 1974, FMC Corp.
Rani, P., Srivastava, V.K and Kumar, A. Indian J. Pharm. Sci. 2002, 64, 535.
Riad, A., Mouloungui, Z., Delmas, M. and Gaset, A. Synth. Commun. 1989, 19, 3169.
Rich, S. and Horsfall, J.G. Phytopathology, 1952, 42, 457.
Rys, P. and Zollinger, H. Fundamentals of the Chemistry and Application of Dyes; Wiley-
Interscience: New York, 1972.
Satoskar, R. S. and Bhandarkar, S. D., Pharmacology and Pharmacotherapeutics. , 12th ed.,
Popular Prakashan, Mumbai, 1991, 687.
Saundane, A.R., Jaishree, B. and Veeresha Sharma, P.M., Indian J. Heterocyclic Chem.,
Chapter IV
2005, 14, 331.
Shaila, M.S., Purushothaman, V., Bhavasar, D., Venugopal, K. and Venkatesan, R.A., Vet.
Rec. 1989, 125, 602.
Sharma, H.L. and Sharma, K.K. Principles of pharmacology, First Edn., Paras Medical
publisher, Hyderabad, 2007, 798.
Shchepinov, M. S. and Korshun, V. A. Chem. Soc. Rev. 2003, 32, 170.
Shivarama Holla, B., Akber Ali, P.M. and Shivananda, M.K. Farmaco, 2000, 55, 256.
Sivaprasad, G., Perumal, P.T., Prabavathy, V.R. and Mathivanan, N., Bioorg. Med. Chem.
Lett., 2006, 16, 6302.
Sridhar, R., Perumal, P.T., Etti, S., Shanmugam, G., Ponnuswamy, M.N., Prabavathy, V.R.
and Mathivanan, N. Bioorg. Med. Chem. Lett. 2004, 14, 6035.
Stahman, A., Ikawa, M. and Link, K. P. U.S. Patent 2427578, 1947.
Temelli, B. and Unalerogolu, C. Tetrahedron, 2006, 62, 10130.
WHO, 1990, Global estimate for health situation assessment and projections. Division of
epidemiological surveillance, health situation and trend assessment, WHO, Geneva.
Wu, H. Y., Shyy, S. H., Wang, J. C. and Liu, L. F. Cell, 1988, 53, 433.
Wustrow, D.J., Capiris, T., Rubin, R., Knobelsdorf, J.A., Akunne, H., Davis, M.D.,
MacKenzie, R., Pugsley, T.A., Zoski, K.T., Heffner, T.G. and Wise, L.D., Bioorg.
Med. Chem. Lett., 1998, 8, 2067.
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