Synthesis, Crystal Structure, and In Vitro Cytotoxic, Antitumor And Antimicrobial Evaluation of a New Pyrazole Derivative N-3,5-Trimethyl-Nphenyl-1-H-Pyrazole-1-Carbothioamide

Article abstract of DOI:10.1007/s11094-021-02324-0

Anew pyrazole molecule of N-3,5-trimethyl-N-phenyl-1-H-pyrazole-1-carbothioamide (MePhPyC) has been synthesized and characterized by elemental analysis, single crystal x-ray diffraction (XRD), and mass, ¹H NMR, ultraviolet-visible (UV-Vis), and

Full text of DOI:10.1007/s11094-021-02324-0

DOI 10.1007/s11094-021-02324-0
Pharmaceutical Chemistry Journal, Vol. 54, No. 10, January, 2021 (Russian Original Vol. 54, No. 10, October, 2020)
SYNTHESIS, CRYSTAL STRUCTURE, AND IN VITRO CYTOTOXIC,
ANTITUMOR AND ANTIMICROBIAL EVALUATION
OF A NEW PYRAZOLE DERIVATIVE N-3,5-TRIMETHYL-N-
PHENYL-1-H-PYRAZOLE-1-CARBOTHIOAMIDE
K. Subin Kumar,1,* N. P. Priya,2 and K. K. Aravindakshan2
Original article submitted February 11, 2020.
A new pyrazole molecule of N-3,5-trimethyl-N-phenyl-1-H-pyrazole-1-carbothioamide (MePhPyC) has been
1
synthesized and characterized by elemental analysis, single crystal x-ray diffraction (XRD), and mass, H
NMR, ultraviolet-visible (UV-Vis), and IR spectroscopic techniques. The XRD data show that MePhPyC mol-
ecule crystallizes in the monoclinic system, P21/C space group, a = 13.0473(7) Å, b = 12.6191(6) Å,
c = 8.1871(4) Å, a = 90°, b = 106.288(2)°, n = 900 and V =1293.86(11) Å3. The antibacterial activity of
MePhPyC against test bacteria and antifungal activity against test fungi were investigated. The compound was
found to possess a broad spectrum of biological activities. The synthesized molecule of MePhPyC showed
highest cytotoxicity with IC50 = 49 mg/mL. In the present study, MePhPyC was found to be efficient against
EAC-induced ascites tumor. A dose of 5 mg/kg body weight (bwt) was more effective than the other two con-
centrations (15 and 10 mg/kg bwt). In both cases of in vitro cytotoxicity and in vivo ascites tumor treatment,
MePhPyC suggests its potential use as an anticancer agent.
Key words: N-3,5-trimethyl-N-phenyl-1-H-pyrazole-1-carbothioamide; antibacterial activity; antifungal ac-
tivity; in vitro cytotoxic activity; anticancer agent; zone of inhibition.
1. INTRODUCTION
line derivatives are important compounds constituting the
basic framework of drugs such as celecoxib and deracoxib
well recognized for their multifaceted pharmacological and
medicinal applications [13]. They have wide application in
heterocyclic synthesis and medicine [14]. The best-described
property of almost every group of pyrazoles is in the treat-
ment of inflammation and inflammation associated disor-
ders, such as arthritis [15]. Pyrazolines have been reported to
show a wide spectrum of biological activity, including anti-
bacterial [16], antifungal [17], anti-inflammatory [18], anti-
amoebic [19], and antidepressant properties [20, 21].
Heterocyclic compounds bearing heteroatoms such as ni-
trogen, oxygen, and sulfur constitute the core structures of a
number of biologically interesting compounds. Where these
molecules are linked with each other, the resultant molecule
generally has increased potency. Pyrazoles have five-mem-
bered heterocyclic ring; a compound having some structural
features with two nitrogen atoms in adjacent position are
known as azoles [1]. Recently, pyrazole derivatives have
been found in nature [1], in particular, b-[1-pyrazolyl]ala-
mine was isolated from the seeds of water melon (Citurllus
lanatus). Pyrazole derivatives are recognized in the literature
as important, biologically active heterocyclic compounds.
These compounds are readily synthesized via the condensa-
tion reaction between 1,3-dicarbonyl compounds (or their
equivalents) with hydrazine [2 – 12]. N-substituted pyrazo-
In the present study, a new compound of N-3,5-trime-
thyl-N-phenyl-1-H-pyrazole-1-carbothioamide (MePhPyC)
was synthesized and its structure was determined using sin-
gle-crystal x-ray diffraction (XRD) method and character-
ized by elemental analysis and IR, 1H NMR, and mass spec-
troscopy techniques. The anti-bacterial activity of MePhPyC
pyrazole against some bacteria and anti-fungal activity
against some fungi has been investigated. It is hoped that this
compound will be more suitable for use in pharmaceutical
fields than the other known substituted pyrazole molecules.
The structure of MePhPyC is shown in Fig. 1.
1
Department of Chemistry, Govt. College Madappally, Kozhikode, Kerala,
673102 India.
2
Department of Chemistry, University of Calicut, Malappuram, Kerala,
673635 India.
*
e-mail: subinnhc@gmail.com
1079
0091-150X/21/5410-1079 © 2021 Springer Science+Business Media, LLC

1080
K. Subin Kumar et al.
pellets. The electronic absorption spectrum in the range of
200 – 700 nm was measured with Jasco UV-Vis spectropho-
tometer. The 1H NMR spectrum in dimethyl sulfoxide
(DMSO-d ) was recorded on 500-MHz Brucker AV 500 FT
6
NMR spectrometer, and the mass spectrum was recorded us-
ing Schimadzu-5050A model GC-MS instrument.
2.2. Synthesis of N-3,5-trimethyl-N-phenyl-1H-pyrazole-
1-carbothioamide (MePhPyC)
Fig. 1. The structure of N-3,5-trimethyl-N-phenyl-1-H-pyrazole-1-
carbothioamide (MePhPyC).
A mixture of 12.0 mL (0.20 mol) of carbon disulfide and
12.6 mL (0.20 mol) of N-methylaniline was treated with
250 mL 1N NaOH solution and stirred at room temperature
for about two hours until obtaining pale orange colored ho-
mogenous solution. To this solution was added a small por-
tion of about 23.3 g (0.20 mol) sodium acetate with stirring
continued for 6 – 7 h. To the resultant pale golden yellow so-
lution was added 25 mL of concentrated HCl and the result-
ing golden yellow precipitate was filtered, and washed with
cold alcohol. Finally, diethylether was doubly recrystallized
from methanol. The product (I) was found to be TLC pure in
7:3 mixture of methanol and chloroform.
2. EXPERIMENTAL
2.1. Materials and Methods
Carbondisulphide, N-methylaniline, hydrzenehydrate
and acetylacetone were purchased from Sigma-Aldrich and
used without further purification. Organic solvents and all
other chemical substances were purchased from Merck. Re-
actions were monitored by thin-layer chromatography (TLC)
The resulting solid crystals of 17.70 g (0.0733 mol) car-
boxymethyl-N-methyl-N-phenyl dithiocarbamate (I) in
20 mL of 98% hydrazine hydrate and 10 mL of water were
heated on water bath for about 10 min. When colorless crys-
tal began to appear, the heating was continued for another
5 min and the crystals were filtered, washed, and dried under
vacuum. Then, the crude product of N-methyl-N-phenyl
thiosemicarbazide (II) was recrystallized from a mixture of
on pre-coated silica gel F plates from Merck and the spots
254
were visualized by keeping in iodine chamber and exposure
to UV light. Chromatographic columns of 70 – 230 mesh sil-
ica gel were used for separation. The melting point was de-
termined using a Fisher-Johns apparatus and remained un-
corrected. Elemental analysis for C, H, N and S was carried
out using Vario EL III CHNS analyzer. The IR spectra were
recorded on Jasco FTIR-4100 spectrophotometer using KBr
Scheme 1. Preparation of N-3,5-trimethyl-N-phenyl-1-H-pyrazole-1-carbothioamide (MePhPyC).

Synthesis, Crystal Structure, and In Vitro Cytotoxic, Antitumor and Antimicrobial Evaluation
1081
ethanol and water to obtain colorless triclinic crystals. The
product II was found to be TLC pure in 7:3 mixture of etha-
nol and chloroform.
CH ), 3.3 (3H, s, CH ), 3.5 (3H, s, CH ), 7.3 (1H, CH),
3 3 3
7.5 – 7.3 (5H, m, Ar-H); mass spectrum: m/z = 150.
2.3. X-ray Crystal Structure Determination
N-Methyl-N-phenyl thiosemicarbazide II (0.01 mol) was
dissolved in 30 mL of methanol was refluxed for 4 h on a
water bath after adding 1.12 mL (0.01 mol) of acetylacetone.
The resulting solution was allowed standing for one day
(Scheme 1). The precipitated pale yellow color solid was
washed with petroleum ether and dried in a desiccator. The
product III with a structure of N-3,5-trimethyl-N-phenyl-1-
H-pyrazole-1-carbothioamide (MePhPyC) was recrystallized
from hot methanol and as pale yellow single crystals suitable
for x-ray structural analysis: yield, 2.01 g (61%); Anal. Calc.
for C H N S: C, 63.58; H, 6.11; N, 17.11; S, 13.04; Found:
XRD measurements were made on Brucker AXS Kappa
APEX II CCD diffractometer with graphite monochromated
Mo Ka radiation. Pale Yellow block crystals with dimension
0.40 ´ 0.35 ´ 0.30 mm mounted on a glass fiber and used for
data collection. Cell constant and an orientation matrix for
data collection obtained by least square refinement of the dif-
fraction data from 3189 unique reflection for MePhPyC.
Data collected at a temperature of 296 (2) K to a maximum
2q value of 28.30°. The structure resolved by direct method
and refined anisotropically using the least-squares technique
implemented in the SHELX-97 package [22, 23]. All hydro-
gen atoms were added according to theoretical models. The
unit cell parameters and experimental details are given in Ta-
bles 1 and 2.
13 15
3
C, 63.45; H, 6.09; N, 17.10; S, 13.08; mp > 92°C; color: pale
yellow. UV/Vis, l (e): 238 nm, 344 nm; FT-IR (KBr; n
max
max,
cm-1): 1493 (Ar-C=C), 1478 (C=C), 1593 (C=S), 1250
(N-N), 1558 (N=C), 1644 (N-CH), 3065 (=C-H), 2921 (C-H
1
asym.); H NMR (500 MHz, DMSO; d, ppm): 2.5 (3H, s,
TABLE. 2. Selected Bond Angles and Bond Lengths in MePhPy
TABLE 1. Crystal Structure Data for Compound MePhPy
Parameter
1 (MePhPy)
Bond
S(1)-C(8)
Bond length, Å
1.642(2)
1.333(2)
1.440(2)
1.464(3)
1.359(2)
1.370(2)
1.421(2)
1.327(3)
1.377(3)
1.378(3)
1.365(3)
1.485(3)
1.395(3)
1.498(3)
1.372(3)
1.371(3)
1.376(3)
1.375(3)
Bond
Bond angle, deg
122.37 (15)
120.11 (17)
116.35 (16)
112.95 (15)
128.45 (17)
118.58 (15)
103.65 (16)
120.45 (19)
120.33 (18)
119.11 (18)
105.17 (18)
123.50 (18)
131.27 (19)
113.44 (17)
126.04 (15)
120.48 (14)
111.59 (18)
120.0 (2)
Formula
C13H15N3S
245.34
C(8)-N(1)-C(6)
C(8)-N(1)-C(7)
C(6)-N(1)-C(7)
C(12)-N(2)-N(3)
C(12)-N(2)-C(8)
N(3)-N(2)-C(8)
C(9)-N(3)-N(2)
C(1)-C(6)-C(5)
C(1)-C(6)-N(1)
C(5)-C(6)-N(1)
N(2)-C(12)-C(11)
N(2)-C(12)-C(13)
C(11)-C(12)-C(13)
N(1)-C(8)-N(2)
N(1)-C(8)-S(1)
N(2)-C(8)-S(1)
N(3)-C(9)-C(11)
N(3)-C(9)-C(10)
C(11)-C(9)-C(10)
C(2)-C(1)-C(6)
C(3)-C(2)-C(1)
C(12)-C(11)-C(9)
C(5)-C(4)-C(3)
C(4)-C(5)-C(6)
C(2)-C(3)-C(4)
Formula weight
N(1)-C(8)
N(1)-C(6)
N(1)-C(7)
N(2)-C(12)
N(2)-N(3)
N(2)-C(8)
N(3)-C(9)
C(6)-C(1)
C(6)-C(5)
C(12)-C(11)
C(12)-C(13)
C(9)-C(11)
C(9)-C(10)
C(1)-C(2)
C(2)-C(3)
C(4)-C(5)
C(4)-C(3)
Temperature (K)
296(2)
System
Monoclinic
P21/c
Space group
a (Å)
13.0473(7)
12.6191(6)
8.1871(4)
(90)
b (Å)
c (Å)
a (°)
106.288(2)°
(90)
b (°)
g (°)
V (Å 3)
1293.86(11)
4
Z
Dcalc (mg m-3)
m (mm-1)
1.63
0.232
F (0 0 0)
520
Crystal size (mm)
Total reflection
Independence reflections
Rint
0.40 ´ 0.35 ´ 0.30
22491
3189
128.4 (2)
0.0362
119.2 (2)
3189
Collected reflections [I > 2s(I)]
R1, wR2 (all data)
120.9 (2)
R1 = 0.0514
wR2 = 0.1622
R1 = 0.0725
wR2 = 0.1882
106.63 (18)
120.2 (2)
119.6 (2)
R1, wR2 [I > 2s(I)]
119.6 (2)

1082
K. Subin Kumar et al.
Antifungal activity Determination by filter paper disc
method. Disc diffusion method [26, 27] was used to deter-
mine antifungal activity of the sample. The potato dextrose
agar was used as basal medium for testing fungi. It is pre-
pared by taking yeast extract (3 gm/lit), peptone (10 gm/lit),
dextrose (20 gm/lit), agar (15 gm/lit), and distilled water (1
lit) at pH (6) were plated into Petri dishes and kept to solidi-
fication. The potato dextrose plates were inoculated with
each fungal culture (10 days old) by point inoculation. The
filter paper disc (5 mm in diameter) impregnated with
100 mL and 50 mL concentrations of the extracts were placed
on test organism-seeded plates. Blank disc impregnated with
solvent DMSO followed by drying off was used as negative
control and Nystatin (10 mg) was used as positive control.
The activity was determined after 70 h of incubation at 27°C.
The diameters of the inhibition zones were measured in mm.
Fig. 2. ORTEP-Diagram of MePhPyC Structure with Ellipsoids at
50% Probability.
2.5. Anticancer Studies
2.4. Antimicrobial Activity Assay
Evaluation of MePhPyC anticancer potential. To con-
duct in vitro cytotoxicity studies, 50 mg of N-3,5-trimethyl-
N-phenyl-1H-pyrazole-1-carbothioamide (MePhPyC) was
dissolved in 1 mL of DMSO. For in vivo ascites and solid tu-
mor study, 50 mg of MePhPyC was first mixed with 1 mL
DMSO and further diluted to desired concentration using
distilled water. ACI (Adayar Cancer Institute) Chennai sup-
plied essential Ehrlich ascites carcinoma (EAC) cell lines
and propagated as transplantable tumors in the intraperito-
neal cavity of BALB/C mice. L929 (mouse lung fibroblast)
cell line was supplied by NCCS (National Centre for Cell
Sciences) Pune. Studies were carried out on laboratory fe-
male mice (Swiss albino mice having 20 – 25g body weight)
and were collected from the Small Animal Breeding Station,
Mannuthy, Thrissur, Kerala. Animals were kept in cages un-
der standard conditions of temperature and humidity of
Amala Cancer Research Centre, Trissur Kerala, and fed with
standard mouse chow (Sai Durga Feeds and Foods,
Bangalore, India) and water ad libitum. All experiments on
mice in this study were carried out with the prior approval
and permission of Institutional Animal Ethics Committee
(IAEC) and were conducted strictly according to the guide-
lines of CPCSEA constituted by the Animal Welfare Divi-
sion, Government of India. Mouse lung fibroblast (L929
cells) were cultured in DMEM medium supplemented with
FBS (10% v/v), streptomycin (100 mg/mL) and penicillin
Preparation of inoculum. Gram positive and Gram-ne-
gative bacteria were pre-cultured in nutrient broth overnight
in a rotary shaker at 37°C and centrifuged at 10,000 rpm for
5 min. The pellet was suspended in doubly distilled water
and the cell density was standardized spectrophotometrically
(A, nm). The fungal inoculums (Penicillium, Fusarium, and
Aspergillus) were prepared from 5 to 10 day old cultures
grown on Potato dextrose agar medium. Petri dishes were
flooded with 10 mL of distilled water and sterile spatula was
used for scraping conidia. The spectrophotometer (A, nm)
was used to adjust the spore density for each fungus to obtain
a final concentration of approximately 105 spores/mL.
Antibacterial activity determination by agar cup
method. The antibacterial activity of pyrazole derivatives
was studied by agar cup method [24, 25]. Glass Petri dishes
were sterilized and nutrient broth was used as basal medium
for bacterial testing. The medium (nutrient broth) was pre-
pared by taking Beet extract (1 gm/lit), Yeast extract
(2 gm/lit), peptone (5 gm/lit), NaCl (5 gm/lit), and agar
(15 gm/lit) with pH (7) were plated into Petri dishes and al-
lowed to solidify. The selected bacterial culture as single col-
ony was inoculated into the broth medium and kept over-
night at 25°C. The overnight bacterial culture was spread
evenly over the entire surface and left undisturbed for a few
minutes to percolate the culture. Wells (4 mm) were created
using a sterile borer in the solidified agar medium. The se-
(100 U/mL) and kept at 37°C in incubator with 5% CO .
2
Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites car-
cinoma (EAC) cells maintained in the intraperitoneal cavity
of mouse were used for the study according to [28, 29].
Trypan blue exclusion method for in vitro cytotoxicity
studies. The synthesized compound MePhPyC was investi-
gated for short-term in vitro cytotoxicity using DLA cells.
The tumor cells collected from the peritoneal cavity of tu-
mor-bearing mice were washed three times with PBS and
normal saline. Trypan blue dye exclusion test is used to de-
lected compounds were added to each well (500 mL) at pe-
riphery and a reference compound was added at the centre.
Thus prepared plates were incubated at 25°C (room tempera-
ture) for about 3 to 5 days. After incubation period, the plates
were collected and the inhibition zone was recorded in mm.
Dimethyl sulfoxide was used as solvent to prepare stock so-
lutions (5 mg in 0.5 mL) of the compound initially and main-
tain the proper control.

Synthesis, Crystal Structure, and In Vitro Cytotoxic, Antitumor and Antimicrobial Evaluation
1083
Fig. 3. The structure of MePhPyC ligand.
Fig. 4. Percentage in vitro cytotoxicity of MePhPyC.
termine the number of viable cells present in cell suspension.
It is based on the principle that live cells possess intact cell
membranes that exclude trypan blue dye, whereas dead cells
do not. A cell suspension is simply mixed with the trypan
blue dye and then visually examined to determine whether
cells take up or exclude dye.
3. RESULTS AND DISCUSSION
3.1. Synthesis of N-3,5-trimethyl-N-phenyl-H-
pyrazole-1-carbothioamide)
MePhPyC in vivo toxicity studies. 24 Swiss albino
mice were divided into 4 groups (6 animals/group) and
treated at 5, 10, 15, and 20 mg/kg in groups 1, 2 3, and 4, re-
spectively. The drug was administered once daily (i.p.) and
then monitored for 5 – 6 weeks to determine their mortality
rate.
The formation of pyrazole compound MePhPyC is
shown in Scheme 1. Condensation of N-methyl aniline and
carbon disulphide results in the formation of methyl(phenyl)-
carbamodithioic acid (I). Compound I reacts with hydrazine
hydrate to form thiosemicarbazide (II). Finally, we put
thiosemicarbazide and acetyl acetone under reflux for about
3 h to convert the mixture into single solid product N-3,5-tri-
methyl-N-phenyl-H-pyrazole-1-carbothioamide (III).
Effect of MePhPyC treatment on the survival rate of
ascites tumor bearing mice. Female mice (6–8 weeks old,
weighing 26–30 g) were divided into 5 groups of 6 animals
each. Viable EAC cells 106 in 0.1 mL of phosphate buffered
saline (PBS) were injected into the peritoneal cavity. Group
1: control; group 2: 5 mg/kg, treated; group 3: 10 mg/kg,
treated; group 4: 15 mg/kg, treated; and group 5: standard
drug (cyclophosphamide), treated. Drug and cyclophospha-
mide were introduced by intraperitoneal injection from the
first day of tumor induction. The pattern of animal loss
caused by tumor burden was recorded and the percentage in-
crease in life span was calculated as, % ILS = [(T—C)/C]
×100, where T and C are the values of mean survival in
treated and control mice, respectively.
3.2. UV-Vis and IR spectra
The UV-Vis spectra of MePhPyC have been obtained in
10 – 2 M chloroform at room temperature. This molecule ex-
hibit the absorption peak due to the localized p–p* transition
at 238 mm. The intense shoulder peak in 344 mm is attrib-
uted to the n–p* transition in imino group. A strong absorp-
tion band at 1644 cm-1 in IR spectra of complex is assigned
to azomethine group, n(CH–N) mode [24]. The weak band of
TABLE. 4. Antibacterial Activity of MePhPyC
Zone of Inhibition
Si No.
Organism
(mm)
1
TABLE 3. H NMR Spectral Data for MePhPyC Pyrazole
1
2
3
4
5
6
7
Klebsiella pneumonia
Eschericia coli
9
Assignment
Chemical shift (d, ppm)
0
2.5
3.3
(3H, s, CH3)
(3H, s, CH3)
(3H, s, CH3)
(1H, CH)
Salmonella typhi
Serratia marcescences
Pseudomonas
12
8
3.5
11
14
7
7.3
aeruginosa
7.5 – 7.3
(5H, m, Ar-H)
Proteus vulgaris

1084
K. Subin Kumar et al.
st
Fig. 5. 5 mg/kg drug treated mice at 1 day.
th
Fig. 6. 5 mg/kg drug treated mice at 10 day.
the aromatic n(=C–H) stretching frequency of pyrazole was
observed at 3065 cm-1. Furthermore, the n(C=C) and n(C=S)
stretching vibrations were observed at 1478 and 1593 cm-1,
respectively.
studied in vitro at a concentration of 10 mL against 7 bacterial
stains. The screening results indicated that all compounds ex-
hibited antibacterial properties against all tested bacteria.
The compound showed better inhibitory activity against Pro-
teus vulgaris and Salmonella typhi and greater inhibitory ac-
tivity against Aeruginosa strain. Data on the inhibition zone
diameter (in mm) for MePhPyC are presented in Table.4. The
confidence interval (95%) of antibacterial activities of
3.3. 1H NMR Spectrum
The chemical shifts of various types of protons in the 1H
NMR spectrum of MePhPyC are listed in Table 3.
MePhPyC at a concentration of 10 mL against seven bacterial
stains is given in Table 5.
3.4. X-ray crystal structure
The XRD measurements were made on a STOE IPDSIIT
Data on the antifungal activity of MePhPyC studied in
diffractometer with graphite monochromated MoK radia-
a
vitro at a concentration of 10 mL against three fungal stains
are given in Table 6. The screening results indicated that the
sample exhibited antifungal activity to the tested fungi. It
was noted that the MePhPyC showed a greater inhibitory ac-
tivity against Aspergillus. Data on the confidence interval of
inhibition zone diameter (in mm) for MePhPyC are pre-
sented in Table 7.
tion. Pale yellow block crystals of MePhPyC with dimen-
sions 0.40 ´ 0.35 ´ 0.30 mm were mounted on a glass fiber
and used for data collection. The ORTEP-3 view of the
MePhPyC with atom numbering scheme is shown in Figs. 2
and 3.
3.5. Antimicrobial Activity
MePhPyC was tested for its antibacterial and antifungal
activities in the laboratory by two conventional techniques,
i.e. agar cup method and filter paper disc method, respec-
tively. The antibacterial activities of pyrazole derivative were
3.6. Cytotoxicity and Anticancer Activity Studies
Short-term in vitro cytotoxicity analysis. The pyrazole
derivative, MePhPyC showed potential cytotoxic activity for
DLA cell line (Table 1). The 200 mg/ml exhibited maximum
activity and the concentration required for 50% death (IC )
50
was found to be 48 mg/ml. (Table.8 and Fig. 4).
TABLE 5. Confidence Interval for Inhibition Zone Diameter (mm)
In vivo toxicity of MePhPyC. To conduct in vivo toxic-
ity studies, we grouped the animal into four groups, each
group having 4 Swiss albino female mice. The results of tox-
icity studies of MePhPyC on 24 Swiss albino mice, 4 groups,
at four concentrations (20, 15, 10 and 5 mg/kg) showed that
20 mg/kg was slightly toxic to the animals. Therefore, this
concentration was avoided and 15, 10 and 5 mg/kg were only
of MePhPyC against Bacterial Strains
Zone
Confidence
iinterval (95%)
[LCL-UCL]
of inhibition
(mm) ± SDa
Si No.
Organism
1
2
3
4
5
6
7
Klebsiella pneumonia
Eschericia coli
[7.87 – 10.13]
[0 – 0]
9 ± 1
0 ± 0
Salmonella typhi
Serratia marcescences
Pseudomonas
[10.87 – 13.13]
[6.87 – 9.13]
[9.87 – 12.13]
[12.87 – 15.13]
[5.87 – 8.13]
12 ± 1
8 ± 1
11 ± 1
14 ± 1
7 ± 1
TABLE 6. Antifungal Activity of MePhPyC
aeruginosa
Zone of inhibition
Si No.
Organism
Penicillium
(mm)
Proteus vulgaris
1
2
3
9
SD = Standard Deviation, LCL = Lower Confidence Limit;
a
UCL = Upper Confidence Limit; Values are mean ±SD of three
Fusarium
8
Aspergillus
12
replicates.

Synthesis, Crystal Structure, and In Vitro Cytotoxic, Antitumor and Antimicrobial Evaluation
1085
Fig. 7. Effect of MePhPyC of survival rate of Ascitis tumour endur-
ing mic.
Fig. 8. Effect of MePhPyC on the life span rate of ascitis tumor in
mice.
selected for in vivo studies, as they were nontoxic to the ani-
mals.
erage life span of the animals by 38.7, 28.9 and 21.7 %, re-
spectively (Fig. 5 & Fig. 6), at 5, 10 and 15 mg/kg doses
(Tables 9 and 10, Figs. 7 and 8).
Effect of MePhPyC on ascites tumor development.
The animals of the tumour control group survived for a pe-
riod of 14.8 d. Those treated with cyclophosphamide sur-
vived for 26.1 d. The MePhPyC treated at 15, 10 and
5 mg/kg increased the survival rate of animals by 18.6, 20.0
and 22.1 d respectively (Table. 4). Thus, the pyrazole deriva-
tive MePhPyC was found to be effective in increasing the av-
Our antitumor studies of MePhPyC gave fascinating and
promising results due to the N-N-S donor system being a
common feature or MePhPyC with carcinostatic potency. In
vitro cytotoxicity studies on MePhPyC at various concentra-
TABLE 9. Effect of MePhPyC of survival rate of Ascites tumour
enduring mice
TABLE 7. Confidence Interval of inhibition zone diameter (in mm)
for MePhPyC against Fungal Strains[10.87 – 13.13]
Treatment (mg/kg)
Survival rate (days)
Zone
Confidence
interval (95%)
[LCL-UCL]
Control
15
14.8
18.6
20.0
22.1
26.1
of inhibition
(mm) ± SD
Si No.
Organism
10
1
2
3
Penicillium
Fusarium
[7.87 – 10.13]
[6.87 – 9.13]
9 ± 1
8 ± 1
05
Standard*
Aspergillus
[10.87 – 13.13]
12 ± 1
* Cyclophosphamide (10 mg/kg).
SD = Standard Deviation, LCL = Lower Confidence Limits,
a
UCL = Upper Confidence Limits; Values are mean ±SD of three
replicates
TABLE 10. Effect of MePhPyC on the life span rate of ascites tu-
mor enduring mice
Treatment (mg/kg)
Increase in Life span (%)
TABLE 8. In Vitro Percentage Cytotoxicity of MePhPyC
Control
15
–
Percentage cytotoxicity of MePhPyC
Concentration (mg/mL)
25.6
35
200
100
50
89
72
58
39
28
10
05
49.3
76.3
Standard*
20
10
* Cyclophosphamide (10 mg/Kg)

1086
K. Subin Kumar et al.
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synthesized compound MePhPyC exhibited maximum
15. M. A. Berghot and E. B. Moawad, Eur. J. Pharm. Sci., 20, 173
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cytotoxicity with IC = 49 mg/mL. From the present study,
50
we conclude the MePhPyC is efficient against EAC-induced
ascites tumor. A dose of 5 mg/kg bwt was more effective
than the other two concentrations (15 and 10 mg/kg bwt). In
both cases of in vitro cytotoxicity and in vivo ascites tumor
treatment, MePhPyC suggests its potential use as an
anticancer agent.
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