Synthesis, characterization and biological evaluation of novel 1-N-substituted thiocarbomoyl-3-ferrocenyl-2-pyrazoline derivatives

Article abstract of DOI:10.14233/ajchem.2016.19851

Some novel 1-N-substituted thiocarbomoyl-3-ferrocenyl-2-pyrazoline derivatives were synthesized and evaluated for in vitro antiamoebic activity against HM1:IMSS strain of Entamoeba histolytica. The results showed that most of the compounds exhibited promising activity (IC₅₀ values in the range of 0.050-1.683 μM) than the reference drug metronidazole (IC₅₀ = 1.78 μM). Active compounds were further screened for cytotoxicity against human embryonic kidney-293 (HEK-293) normal cell lines to ensure their toxic effect and the results revealed that active compounds were least toxic in the concentration range of 2.5-50 μM for 48 h and 2.5-25 μM for 72 h. At 100 μM for 48 h and at 50 μM for 72 h only four compounds 2c, 2h, 2k and 2l showed maximum viability and least cytotoxicity, respectively, concluding that all the screened compounds were least cytotoxic against human embryonic kidney-293 (HEK-293) normal cell lines in the concentration range of 2.5-50 and 2.5-25 μM.

Full text of DOI:10.14233/ajchem.2016.19851

Asian Journal of Chemistry; Vol. 28, No. 8 (2016), 1835-1840
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19851
Synthesis, Characterization and Biological Evaluation of Novel
-N-Substituted Thiocarbomoyl-3-ferrocenyl-2-pyrazoline Derivatives
1
1
,*
1
2
HUMAIRA PARVEEN , RAEDAH AIYED SULIMAN ALATAWI , SALMAN AHMAD KHAN ,
1
1
3
1,4
MOHAMMED ISSA AL-AHMDI , SAYEED MUKHTAR , AMIR AZAM and NADIA H. ELSAYED
1
2
3
4
Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk, Kingdom of Saudi Arabia
Department of Chemistry, Faculty of Science, King Abdul Aziz University, Jeddah, Kingdom of Saudi Arabia
Department of Chemistry, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi-110 025, India
Department of Polymers and Pigments, National Research Center, Dokki, Cairo 12311, Egypt
*Corresponding author: Tel: +966 14423/7227; E-mail: humaira_chem@yahoo.co.in
Received: 22 January 2016; Accepted: 2 March 2016; Published online: 30 April 2016;
AJC-17902
Some novel 1-N-substituted thiocarbomoyl-3-ferrocenyl-2-pyrazoline derivatives were synthesized and evaluated for in vitro antiamoebic
activity against HM1:IMSS strain of Entamoeba histolytica. The results showed that most of the compounds exhibited promising activity
(
IC50 values in the range of 0.050-1.683 µM) than the reference drug metronidazole (IC50 = 1.78 µM). Active compounds were further
screened for cytotoxicity against human embryonic kidney-293 (HEK-293) normal cell lines to ensure their toxic effect and the results
revealed that active compounds were least toxic in the concentration range of 2.5-50 µM for 48 h and 2.5-25 µM for 72 h. At 100 µM for
48 h and at 50 µM for 72 h only four compounds 2c, 2h, 2k and 2l showed maximum viability and least cytotoxicity, respectively,
concluding that all the screened compounds were least cytotoxic against human embryonic kidney-293 (HEK-293) normal cell lines in
the concentration range of 2.5-50 and 2.5-25 µM.
Keywords: Novel ferrocenyl pyrazolines, Entamoeba histolytica, Cytotoxicity.
compounds which play an important role in the diverse
biological activities such as antifungal [15], anticonvulsant
INTRODUCTION
Amoebiasis, also known as entamoebiasis is an infection
[
16], anticancer [17], antibacterial [18], antitumor [19],
caused by Entamoeba histolytica. Symptoms may include
abdominal pain, mild diarrhoea, bloody diarrhoea or severe
colitis with tissue death [1]. Presently, more than 50 million
people are affected by this disease, causing 100,000 fatalities
worldwide annually [2]. This infection also causes abscess in
other body organs viz. liver [2] and brain [3].
Nitroimidazole such as metronidazole is the highly
recommended drug for the treatment of anaerobic protozoan
infections [4], but clinical resistance to nitroimidazole based
drugs has been observed [5] and metronidazole also has been
found carcinogenic to rodents [6]. Therefore, new therapeutic
agents with least toxicity are urgently required.
antimicrobial [20-24], antiamoebic [25,26], antidepressant
[
27,28] and anti-inflammatory [29-34].
In view of these observations and as a part of our ongoing
program devoted to the synthesis of biologically active hetero-
cycles [35-38], we report herein in vitro antiamoebic activity
and toxicity of novel 1-N-substituted thiocarbomoyl-3-
ferrocenyl-2-pyrazoline derivatives.
EXPERIMENTAL
All the chemicals were purchased fromAldrich Chemical
Company (USA). Precoated aluminum sheets (silica gel 60
Ferrocenyl derivatives with marked biologically activities
have long been targets of synthetic investigations [7-10]. It has
been found that the introduction of a ferrocenyl moiety into a
drug molecule has been recognized as a useful approach for
the development of more effective therapeutic agents [11-14].
Moreover, pyrazoles and their reduced forms, pyrazolines
are an important class of nitrogen containing heterocyclic
F254,Merck Germany) were used for thin-layer chromatography
(TLC) and spots were visualized under UV light. Elemental
analyses were performed on Heraeus Vario EL III analyzer.
The results were within ± 0.3 ꢀ of the theoretical values.
Melting points were determined on MEL-TEMP capillary
melting point apparatus and are uncorrected. IR spectra were
recorded on Perkin-Elmer model 1600 FT-IR RX1 spectro-

1
836 Parveen et al.
Asian J. Chem.
1
13
S
photometer as KBr discs. H NMR and C NMR spectra
were recorded on Bruker AVANCE 400 spectrometer using
KOH +
S
C
+ HR
CDCl
3
or DMSO-d as solvent with TMS as internal standard.
6
Chemical shift values are given in ppm. ESI-MS was recorded
on a MICROMASS QUATTRO II triple quadrupole mass
spectrometer.
Fe
ClCH COONa
2
CH
3
(HCHO)n
+
S
O
General procedure for the preparation of Mannich
base (1): A mixture of acetyl ferrocene (0.06 mol), paraformal-
dehyde (0.12 mol), dimethylamine hydrochloride (0.12 mol),
absolute ethanol (150 mL) and 0.5 mL of conc. hydrochloric
acid was refluxed for 3 h under nitrogen atmosphere. The
resulting solution was allowed to cool and an additional 0.12
mol of paraformaldehyde was added. The mixture was then
refluxed for another 3 h and allowed to cool overnight. On
cooling in an ice salt bath, the red solution deposited brown
crystals, which were collected and recrystallized from a
minimum quantity of isopropyl alcohol to give 9.0 g (47 ꢀ)
of orange yellow salt (Mannich base 1) [39].
NaOOCCH2
HOOCCH2
S
C
R
HCl
Ethanol, HCl
(CH ) NH.HCl
3 2
S
S
C
R
NH -NH .H O
2
2
2
Fe
CH3
N
.HCl
CH3
S
C
O
1)
(
NH -NH
R
2
General procedure for the synthesis of compounds (1a-
(
1a-1l
)
1
l) thiosemicarbazones: All the N-4-substituted thiosemi-
carbazones (1a-1l) were prepared according to literature proce-
dure [40], using a two-step synthetic route.
Synthesis of 1-N-substituted thiocarbamoyl-3-ferrocenyl-
Methanol, NaOH
2
-pyrazolines (2a-2l): To a mixture of ethanol (8 mL) acetic
acid (5 mL) and water (2 mL), appropriate thiosemicarbazide
1.2 mmol) and ferrocenyl Mannich base (1 mmol) was added
(
N
R
and refluxed under nitrogen for 10-20 h. The solvent was
removed from the reaction mixture under reduced pressure.
Water was added and the aqueous phase was extracted with
ethyl acetate. The organic phase was dried over anhydrous
sodium sulphate, filtered and concentrated (Scheme-I). The
crude dark red residue was purified by column chromatography
on silica gel (2 ꢀ ethyl acetate in chloroform) to afford the
pure compound.
N
Fe
S
(
2a-2l
)
where R= Cyclic and aliphatic amines
Scheme-I: General synthesis of 1-N-substituted thiocarbomoyl-3-ferrocenyl-
2-pyrazoline derivatives (2a-2l)
3
-Ferrocenyl-2-pyrazoline-1-thiocarboxamide (2a):
Yield 30 ꢀ; m.p.: 160-162 °C; orange solid. Anal. calc. for
SFe: C 53.67, H 4.79, N 11.41 ꢀ. Found: C 53.39, H
69.5 (meta-C
45.6 (2 × CH
5
H
2
4
), 68.6, 68.32 (C
5
H
5
), 67.2, 66.4 (ortho-C
5
H ),
4
+
), 13.7 (2 × CH ); ESI-MS m/z: [M +1] 370.
3-Ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)(pyrrolidin-
1-yl)methanethione (2c): Yield 38 ꢀ; m.p.: 197-199 °C;
3
C
14
H
15
N
3
-1
4
.82, N 11.27 ꢀ. IR (KBr, νmax, cm ): 3355 (N–H), 2926 (C-
1
H), 1545 (C=N), 1447 (C=C), 1282 (C–N), 1077 (C=S); H
NMR (DMSO-d ) δ (ppm) 8.64 (s, 2H, NH ), 4.66 (s, 1H,
ferrocene), 4.52 (s, 1H, ferrocene), 4.43 (t, 2H, J = 9.7 Hz, H-
orange powder. Anal. calc. for C18
H
21
N SFe: C 58.85, H 5.72,
3
N 11.44 ꢀ. Found: C 58.62, H 5.81; N 11.24 ꢀ. IR (KBr,
max, cm ): 2930 (C-H), 1542 (C=N), 1437 (C=C), 1153
(C–N), 1072 (C=S); H NMR (DMSO-d ) δ (ppm) 4.68 (s,
1H, ferrocene), 4.54 (s, 1H, ferrocene), 4.46 (t, 2H, J = 8.7 Hz,
H-5 pyrazoline), 4.41 (s, 2H, ferrocene), 4.07 (s, 5H, ferrocene),
3.30 (t, 2H, J = 9.3 Hz, H-4 pyrazoline), 2.68-3.11 (m, 4H,
6
2
-
1
ν
1
5
3
d
pyrazoline), 4.39 (s, 2H, ferrocene), 4.06 (s, 5H, ferrocene),
6
13
.29 (t, 2H, J = 9.7 Hz, H-4, pyrazoline); C NMR (DMSO-
) δ (ppm): 178.2 (C=S pyrimidine), 154.9, 68.6, 42.2
), 69.8, 69.6 (meta-C ), 68.9,
), 67.7, 66.2 (ortho-C ), ESI-MS m/z: [M +1]
6
(
pyrazoline) 76.0 (ipso-C
H
5 4
5
H
4
+
13
6
3
8.7 (C
14.11.
5
H
5
H
5 4
-CH
NMR (DMSO-d
(pyrazoline) 75.6 (ipso-C
69.0 (C ), 67.4, 66.8 (ortho-C
2
pyrrolidine), 1.79-1.89 (m, 4H, -CH
2
pyrrolidine);
C
6
) δ (ppm): 176.8 (C=S), 155.7, 68.4, 41.6
), 69.9, 69.8 (meta-C ), 69.2,
), 45.2 (2 × CH pyrro-
pyrrolidine). ESI-MS m/z: [M +1]
3
-Ferrocenyl-2-pyrazoline-1-(N,N-diethyl)thiocar-
boxamide (2b): Yield 28 ꢀ; m.p.: 154-157 °C; yellow powder.
Anal. calc. for. C18 SFe: C 58.53, H 5.23, N 11.38 ꢀ:
Found: C 58.81, H 6.37, N 11.27 ꢀ. IR νmax cm : 2968 (C-H),
5
H
4
5
H
4
H
5 5
5
H
4
2
+
H
23
N
3
lidine), 31.2 (2 × CH
368.02.
2
-1
1
1
(
541 (C=N), 1445 (C=C), 1225 (C–N), 1081 (C=S); H NMR
DMSO-d ) δ (ppm) 4.65 (s, 1H, ferrocene), 4.53 (s, 1H,
ferrocene), 4.46 (t, 2H, J = 9.5 Hz, H-5 pyrazoline), 4.36 (s,
H, ferrocene), 4.08 (s, 5H, ferrocene), 3.28 (t, 2H, J = 9.6 Hz,
H-4, pyrazoline), 3.72 (q, 4H, J = 6.4 Hz, 2 × CH ), 1.68 (t, 6H,
) δ (ppm): 177.0
C=S), 155.8, 69.1, 42.2 (pyrazoline) 74.9 (ipso-C ), 69.8,
3-Ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)(N-
cyclopentyl)carbothioamide (2d): Yield 26 ꢀ; m.p.: 148-
150 °C; red powder. Anal. calc. for. C19
6.03, N 11.02 ꢀ Found: C 59.59, H 6.17, N 11.32 ꢀ. IR (KBr,
νmax, cm ): 3335 (N-H), 2931 (C-H), 1566 (C=N), 1457 (C=C),
1173 (C–N), 1074 (C=S); H NMR (DMSO-d
(s, 1H, N-H), 4.69 (s, 1H, ferrocene), 4.55 (s, 1H, ferrocene),
6
H
23
N SFe: C 59.84, H
3
2
-1
2
13
1
J = 7.4 Hz, 2 × CH
(
3
); C NMR (DMSO-d
6
6
) δ (ppm) 8.68
5
H
4

Vol. 28, No. 8 (2016) Synthesis and Biological Evaluation of Novel 1-N-Substituted Thiocarbomoyl-3-ferrocenyl-2-pyrazoline Derivatives 1837
4
4
9
.42 (t, 2H, J = 9.7 Hz, H-5 pyrazoline), 4.39 (s, 2H, ferrocene),
ferrocene), 3.46-3.35 (m, 4H, -NCH
Hz, H-4 pyrazoline), 1.81-1.73 (m, 4H, -CH
1.34 (m, 1H, -CH, piperidine), 1.22 (s, 3H, -CH
2
), 3.24 (t, 2H, J = 10.4
piperidine), 1.49-
piperidine);
) δ (ppm): 176.0 (C=S), 156.3, 68.5, 41.5
(pyrazoline) 76.2 (ipso-C ), 69.8, 69.6 (meta-C ), 69.1,
68.4 (C ), 67.8, 66.5 (ortho-C ), 46.7 (2 × CH piperidine),
30.9 (2 × CH piperidine), 45.2 (CH), 18.9 (-CH ). ESI-MS
m/z: [M +1] 396.25.
.04 (s, 5H, ferrocene), 3.61 (m, 1H, -CH), 3.30 (t, 2H, J =
2
13
.3 Hz, H-4, pyrazoline), 1.54-2.03 (m, 8H, -CH
) δ (ppm): 172.9 (C=S), 155.6, 68.9, 42.0
pyrazoline) 75.8 (ipso-C ), 69.7, 69.5 (meta-C ), 68.2,
8.1 (C ), 67.0, 66.3 (ortho-C ), 50.3 (CH), 31.2 (2 ×
CH ), 24.5 (2 × CH ). ESI-MS m/z: [M +1] 380.22.
-Ferrocenyl-2-pyrazoline-1-(N,N-dipropyl)thiocar-
boxamide (2e): Yield: 27 ꢀ; m.p.: 174-175 °C; red powder.
Anal. calc. for C20 SFe: C 60.45, H 6.80, N 10.57 ꢀ
Found: C 60.72, H 6.45, N 10.26 ꢀ. IR (KBr, νmax, cm ): 2958
C-H), 1540 (C=N), 1445 (C=C), 1226 (C–N), 1080 (C=S);
2
); C NMR
3
13
(
(
DMSO-d
6
C NMR (DMSO-d
6
H
5 4
5
H
4
H
5 4
5
H
4
6
H
5 5
H
5 4
H
5 5
H
5 4
2
+
2
2
2
3
+
3
3-Ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)(N-cyclo-
hexyl)carbothioamide (2i): Yield 28 ꢀ; m.p.: 203-205 °C;
H
27
N
3
-1
orange solid. Anal. calc. for C20
H
25
3
N SFe: C 60.75, H 6.32; N
(
10.63 ꢀ. Found: C 60.55, H 6.41, N 10.50 ꢀ. IR (KBr, νmax
,
1
-1
H NMR (DMSO-d
H, ferrocene), 4.45 (t, 2H, J = 8.8 Hz, H-5 pyrazoline), 4.32
s, 2H, ferrocene), 4.05 (s, 5H, ferrocene), 3.67 (t, 4H, J = 5.3
Hz, 2 × CH ), 3.28 (t, 2H, J = 10.7 Hz, H-4, pyrazoline), 1.69-
.95 (m, 4H, 2 × CH
NMR (DMSO-d ) δ (ppm): 176.9 (C=S), 156.2, 69.1, 41.9
pyrazoline) 74.3 (ipso-C ), 69.8, 69.6 (meta-C ), 68.5,
8.3 (C ), 67.7, 66.4 (ortho-C ), 52.6 (2 × CH ), 32.8 (2
), 13.7 (2 × CH ); ESI-MS m/z: [M +1] 398.
-Ferrocenyl-2-pyrazoline-1-(N-propyl)thiocarboxa-
6
) δ (ppm) 4.68 (s, 1H, ferrocene), 4.54 (s,
cm ): 3328 (N-H), 2954 (C-H), 1568 (C=N),1455 (C=C), 1233
1
1
(
(C–N), 1076 (C=S); H NMR (DMSO-d ) δ (ppm) 8.57 (s,
6
1H, N-H), 4.67 (s, 1H, ferrocene), 4.54 (s, 1H, ferrocene),
4.43 (t, 2H, J = 9.7 Hz, H-5 pyrazoline), 4.41 (s, 2H, ferrocene),
4.03 (s, 5H, ferrocene), 3.72-3.61 (m, 1H, -CH), 3.29 (t, 2H,
2
13
1
2
), 1.05 (t, 6H, J = 6.6 Hz, 2 × CH ); C
3
13
6
8.6 Hz, H-4 pyrazoline), 1.78-2.24 (m, 10H, -CH
(DMSO-d ) δ (ppm): 178.1 (C=S), 155.2, 69.1, 41.2
(pyrazoline) 76.4 (ipso-C ), 69.8, 697 (meta-C ), 68.3,
), 67.4, 67.1 (ortho-C ), 53.2 (CH), 31.5, 26.3,
). ESI-MS m/z: [M +1] 396.0.
2
); C NMR
(
6
×
H
5 4
5
H
4
6
H
5 5
H
5 4
2
H
5 4
H
5 4
+
CH
2
3
68.0 (C
22.5 (-CH
5
H
2
5
H
5 4
+
3
mide (2f): Yield 27 ꢀ; m.p.: 176-177 °C; orange solid. Anal.
calc. for. C17 SFe: C 57.46, H 5.91, N 11.83 ꢀ Found: C
3-Ferrocenyl-2-pyrazoline-1-(N-butyl methyl)thiocar-
boxamide (2j): Yield 27 ꢀ; m.p.: 166-168 °C; purple powder;
H
21
N
3
-1
5
2
7.22, H 5.61, N 11.95 ꢀ. IR (KBr, νmax, cm ): 3345 (N–H),
998 (C-H), 1555 (C=N), 1446 (C=C), 1226 (C–N), 1075
Anal. calc. for C19
Found: C 59.82, H 6.22, N 10.76 ꢀ. IR (KBr, νmax, cm ): 2954
H
25
N SFe: C 59.53, H 6.52, N 10.96 ꢀ.
3
-1
1
(
(
C=S); H NMR (DMSO-d
6 2
) δ (ppm) 8.06 (s, 2H, NH ), 4.66
(C-H), 1545 (C=N), 1436 (C=C), 1224 (C–N), 1084 (C=S);
1
s, 1H, ferrocene), 4.53 (s, 1H, ferrocene), 4.42 (t, 2H, J = 9.7
H NMR (DMSO-d ) δ (ppm) 4.68 (s, 1H, ferrocene), 4.53 (s,
6
Hz, H-5 pyrazoline), 4.38 (s, 2H, ferrocene), 4.02 (s, 5H,
ferrocene), 3.29 (t, 2H, J = 9.7 Hz, H-4, pyrazoline), 3.16 (m,
1H, ferrocene), 4.41 (t, 2H, J = 9.3 Hz, H-5 pyrazoline), 4.37
(t, 2H, J = 7.9 Hz, CH ), 4.32 (s, 2H, ferrocene), 4.04 (s, 5H,
ferrocene), 3.42 (t, 2H, J = 9.4 Hz, CH ), 3.28 (t, 2H, J = 9.8
Hz, H-4, pyrazoline), 1.65-1.84 (m, 2H, CH ), 1.53 (s, 3H,
CH ), 1.22-1.35 (m, 2H, CH ), 1.02 (t, 3H, J = 6.5 Hz, CH );
C NMR (DMSO-d ) δ (ppm): 177.5 (C=S), 156.5, 69.2, 41.2
(pyrazoline) 74.2 (ipso-C ), 69.9, 69.7 (meta-C ), 68.6,
68.4 (C ), 67.6, 67.2 (ortho-C ), 46.3 (CH ), 32.8 (CH ),
31.6 (CH ), 23.1 (CH ), 13.3 (CH ); ESI-MS m/z: [M +1] 384.
2
2
H, CH
2
), 1.62-1.74 (m, 2H, CH
2
), 1.02 (t, 3H, J = 6.15 Hz,
) δ (ppm): 174.5 (C=S), 155.7,
7.9, 38.2 (pyrazoline) 77.2 (ipso-C ), 69.7, 69.5 (meta-
), 68.2, 68.1 (C ), 67.5, 67.3 (ortho-C ), 48.8, 23.9
), 12.8 (CH ); ESI-MS m/z: [M +1] 356.
-Ferrocenyl-2-pyrazoline-1-(N-butyl)thiocarboxamide
2g): Yield 36 ꢀ; m.p.: 179-180 °C; yellow powder, Anal.
SFe: C 58.53, H 6.23, N 11.38 ꢀ Found: C
2
13
-CH
3
); C NMR (DMSO-d
6
2
4
C
H
5 4
3
2
3
13
5
H
4
H
5 5
5
H
4
6
+
(CH
2
3
5
H
4
5
H
4
3
5
H
2
5
5
H
3
4
2
3
+
(
2
calc. for. C18
5
2
H
23
N
3
3-Ferrocenyl-2-pyrazoline-1-(N-isopropyl)thiocar-
boxamide (2k): Yield 22 ꢀ; m.p.: 192-194 °C;Yellow solid.
-
1
8.76, H 6.49, N 11.12 ꢀ. IR (KBr, νmax, cm ): 3348 (N–H),
968 (C-H), 1557 (C=N), 1446 (C=C), 1223 (C–N), 1076
Anal. calc. for. C17
Found: C 57.42, H 5.93, N 11.85 ꢀ. IR (KBr, νmax, cm ): 3345
H
21
N SFe: C 57.47, H 5.91, N 11.82 ꢀ
3
1
-1
(
(
C=S); H NMR (DMSO-d
s, 1H, ferrocene), 4.55 (s, 1H, ferrocene), 4.43 (t, 2H, J = 8.7
6
) δ (ppm) 8.15 (s, 1H, NH), 4.67
(N–H), 2993 (C-H), 1558 (C=N), 1447 (C=C), 1229 (C–N),
1
Hz, H-5 pyrazoline), 4.39 (s, 2H, ferrocene), 4.08 (s, 5H,
1075 (C=S); H NMR (DMSO-d
6
) δ (ppm) 8.08 (s, 2H, NH ),
2
ferrocene), 3.30 (t, 2H, J = 9.6 Hz, H-4, pyrazoline), 3.26 (t,
4.67 (s, 1H, ferrocene), 4.50-4.56 (m, 1H, CH), 4.58 (s, 1H,
ferrocene), 4.41 (t, 2H, J = 9.7 Hz, H-5 pyrazoline), 4.38 (s,
2H, ferrocene), 4.04 (s, 5H, ferrocene), 3.27 (t, 2H, J = 9.7
2
2
d
H, J = 6.7 Hz, CH
H, CH ), 1.03 (t, 3H, J = 5.4 Hz, -CH
) δ (ppm): 176.8 (C=S), 156.3, 69.2, 41.5 (pyrazoline) 74.2
), 69.6, 69.3 (meta-C ), 68.2, 68.0 (C ), 67.3,
6.5 (ortho-C ), 48.6, 33.4, 22.6, (CH ), 13.7 (CH ); ESI-
2
), 1.74-1.82 (m, 2H, CH
2
), 1.43-1.65 (m,
13
2
3
); C NMR (DMSO-
13
6
Hz, H-4, pyrazoline), 1.32 (t, 6H, J = 6.15 Hz, -2CH
NMR (DMSO-d ) δ (ppm): 174.2 (C=S), 155.8, 47.3, 38.4
(pyrazoline) 77.1 (ipso-C ), 69.4, 69.2 (meta-C ), 68.3
68.1 (C ), 67.5, 67.3 (ortho-C ), 38.1 (CH), 14.8 (2CH );
ESI-MS m/z: [M +1] 356.
3
); C
(
ipso-C
5
H
4
H
5 4
H
5 5
6
6
H
5 4
2
3
H
5 4
5
H
4
+
MS m/z: [M +1] 370.
-Ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)(4-methyl-
piperidin-1-yl)methanethione (2h): Yield 32 ꢀ; m.p.: 193-
94 °C; yellow powder. Anal. calc. for C20 SFe: C 60.75,
H 6.32, N 10.63 ꢀ. Found: C 60.61, H 6.67, N 10.39 ꢀ. IR
H
5 5
5
H
4
3
+
3
3-Ferrocenyl-2-pyrazoline-1-(N-isobutyl)thiocarboxa-
mide (2l): Yield 29 ꢀ; m.p.: 122-124 °C; yellow powder,Anal.
1
H
25
N
3
calc. for. C18
H
23
N SFe: C 57.56, H 6.25, N 11.33 ꢀ Found: C
3
-
1
-1
(
(
1
KBr, νmax, cm ): 2934 (C-H), 1555 (C=N), 1463 (C=C), 1241
57.59, H 6.21, N 11.28 ꢀ. IR (KBr, νmax, cm ): 3342 (N–H),
1
C–N), 1075 (C=S); H NMR (DMSO-d
H, ferrocene), 4.52 (s, 1H, ferrocene), 4.47 (t, 2H, J = 10.7
Hz, H-5 pyrazoline), 4.42 (s, 2H, ferrocene), 4.05 (s, 5H,
6
) δ (ppm) 4.66 (s,
2968 (C-H), 1556 (C=N), 1444 (C=C), 1229 (C–N), 1072
1
(C=S); H NMR (DMSO-d
6
) δ (ppm) 8.17 (s, 1H, NH), 4.66
(s, 1H, ferrocene), 4.52 (s, 1H, ferrocene), 4.41 (t, 2H, J = 8.7

1
838 Parveen et al.
Asian J. Chem.
Hz, H-5 pyrazoline), 4.32 (s, 2H, ferrocene), 4.05 (s, 5H, ferro-
cene), 3.55 (t, 2H, J = 6.08, CH ), 3.38 (t, 2H, J = 9.6 Hz, H-
, pyrazoline), 3.26 (t, 2H, J = 6.7 Hz, CH ), 1.92-2.02 (m,
H, CH, J-= 7.26), 1.05 (t, 6H, J = 6.08 Hz, -CH
) δ (ppm): 176.5 (C=S), 157.2, 69.5, 41.8 (pyra-
zoline) 74.4 (ipso-C ), 69.5, 69.1 (meta-C ), 68.4, 68.2
), 67.4, 66.2 (ortho-C ), 47.8, (CH ), 38.9 (CH), 14.6
); ESI-MS m/z: [M +1] 369.
in vitro Antiamoebic assay: The newly synthesized
was dissolved in 100 µL of DMSO and then the absorbance
(A) value was measured at 570 nm by a multi-scanner auto
reader. The following formula was used for the calculation of
the percentage of cell viability (CV):
2
4
1
2
13
3
); C NMR
(DMSO-d
6
Absorbance of experimental samples
CV (ꢀ) =
×100
H
5 4
H
5 4
Absorbance of the control
(C
5
H
5
H
5 4
2
+
(2CH
3
RESULTS AND DISCUSSION
The chemical structures of all the compounds (2a-2l) were
compounds 2a-2l were tested for in vitro antiamoebic activity
against HM1:IMSS strain of E. histolytica by microdilution
1
13
characterized by IR, H NMR, C NMR and ESI-MS studies.
Assignment of selected characteristic IR bands provides signi-
ficant indications for the formation of the cyclized ferrocenyl
pyrazoline analogues (2a-2l). All the compounds showed
,
method according to reported procedure [36]. The IC50 values
in µM are reported in Table-1.
-1
intense bands in the region 1084-1072 cm due to the (C=S)
stretch of the thiocarboxamide group. All compounds showed
TABLE-1
ANTIAMOEBIC ACTIVITY OF THE COMPOUNDS (2a-2l)
AGAINST HM1:IMSS STRAIN OF Entamoeba histolytica
-1
(C=N) stretch at 1568-1540 cm because of the ring closure.
-1
Antiamoebic activity
In addition, the absorption at 1241-1153 cm was attributed to
the (C-N) stretch vibrations, which also confirms the formation
of desired pyrazoline ring in all the compounds. The com-
pounds (2a, 2d, 2f, 2g, 2k and 2l) showed additional sharp
S. No.
R
a
IC (µm)
S.D. (±)
5
0
2
a
–NH
–N(C
2
8.289
1.09
0.98
2b
2
H
5
)
2
19.643
-1
band in the region 3348-3328 cm due to the NH stretch. The
2
c
N
1.675
0.504
0.34
0.23
structures of all compounds (2a-2l) were further supported
by H NMR spectra. The pyrazolines protons at C-4 and C-5
1
H
carbons appeared as triplets at δ 3.24-3.30 and δ 4.41-4.46
ppm, respectively. The NH protons of thiocarboxamide group
of the compounds (2a, 2d, 2f, 2g, 2k and 2l) showed a singlet
at δ 8.04-8.68 ppm. The protons belonging to the ferrocenyl
group and the other aliphatic groups were observed with the
expected chemical shifts and integral values. The structures
2d
N
2e
–N(C
–NH–(CH
–NH– (CH
3
H
7
)
2
7.746
9.153
2.980
0.53
0.96
0.39
2
f
) CH
2 2 3
2g
2
)
3
CH
3
13
of all these compounds were further confirmed by C NMR
spectra. The C-4 and C-5 carbons of the pyrazoline ring
resonate at δ 41.2-42.0 and 68.0-69.2 ppm, respectively.All the
compounds showed at signal at 154.9-156.5 ppm attributed to
azomethine carbon of pyrazoline ring.All compounds displayed
a signal at 176.0-178.2 ppm assigned to thiocarboxamide
carbon (C=S). The carbons at ferrocenyl group and 1-N substi-
tuted groups resonate at their usual positions.
2
h
N
CH3
1.130
0.65
H
2
i
0.128
0.41
N
2
j
–N–C
–NH–CH(CH
–NH–CH CH(CH
Metronidazole
4
H
9
(CH
3
)
1.360
0.050
1.683
1.780
0.77
1.07
0.82
0.19
2k
)
3 2
2
l
2
)
3 2
Antiamoebic activities: Preliminary experiments were
carried out to determine in vitro antiamoebic activity of all
the compounds (2a-2l) by micro dilution method using
HM1:IMSS strain of E. histolytica. The results are summarized
in Table-1. The data are present in terms of percent growth
inhibition relative to untreated controls and plotted as probit
values as a function of drug concentration. The antiamoebic
effect was compared with the most widely used antiamoebic
medication metronidazole which had a 50 ꢀ inhibitory
concentration (IC50) of 1.78 µM in our experiments. The results
showed that most of the compounds (2c, 2d and 2h-2l) exhi-
bited promising activity than he reference drug metronidazole.
All the pyrazoline derivatives substituted with cyclic amines
(2c, 2d, 2h and 2i) showed good activity (IC50 values in the
range of 0.504-1.675 µM), whereas, among pyrazoline
derivatives substituted with aliphatic amines only compounds
(2j, 2k and 2l), exhibited increased activity (IC50 values in the
range of 0.050-1.68 µM). Alkyl substituted pyrazolines (2a,
2b and 2e-2g) had no effect on the activity. Compound 2k
was emerged as most active compound with the IC50 value
0.050 µM among all pyrazoline derivatives. Therefore, out of
a
Standard Deviation. The compounds with bold font IC50 values are
more active than metronidazole.
MTT assay protocol: The human embryonic kidney-293
(
HEK-293) cells were obtained from NCCS (Pune, India). The
cells were cultured in RPMI (Sigma) with 10 ꢀ foetal bovine
serum and 1 ꢀ penicillin-streptomycin-neomycin. The effect
of compounds 2c, 2d, 2h-2l and the standard drug metronidazole
on cell proliferation was measured by using an MTT-based
assay [41]. Briefly, the cells (5,000/well) were incubated in
triplicate in a 96-well plate in the presence of various concen-
trations of compounds 2c, 2d, 2h-2l as well as metronidazole
or vehicle (DMSO) alone in a final volume of 100 µL at 37 °C
and 5 ꢀ CO in and humidified atmosphere chamber for 48 h
and 72 h. At the end of this time period, 20 µL of 3-(4,5-
dimethylthiozole-2-yl)-2,5-diphenyl tetrazolium bromide
2
(
MTT) solution (5 mg/mL in PBS) was added to each well and
the cells were incubated at 37 °C in a humidified atmosphere
chamber for 4 h. After 4 h, the supernatant was removed from
each well. The coloured formazan crystal produced from MTT

Vol. 28, No. 8 (2016) Synthesis and Biological Evaluation of Novel 1-N-Substituted Thiocarbomoyl-3-ferrocenyl-2-pyrazoline Derivatives 1839
1
2 compounds screened for in vitro antiamoebic activity, 7
by the reaction of β-dimethylaminopropionyl ferrocene
hydrochloride (1) with N-4-substituted thiosemicarbazides
(1a-1l). in vitro Antiamoebic activity was examined using
HM1:IMSS strain of E. histolytica and results showed that
most of the compounds exhibited higher antiamoebic activity
(IC50 = 0.050-1.680 µM) than the reference drug metronidazole
(IC50=1.78 µM). The MTT assay revealed that active compounds
and metronidazole were least toxic in the concentration range
of 2.5-50 µM for 48 h and 2.5-25 µM for 72 h. At 100 µM for
48 h and at 50 µM for 72 h only four compounds 2c, 2h, 2k
and 2l showed maximum viability and least cytotoxicity,
respectively, concluding that all the screened compounds were
least cytotoxic against human embryonic kidney-293 (HEK-
293) normal cell lines in the concentration range of 2.5-50
and 2.5-25 µM. These results identified that these pyrazoline
derivatives are new leads in antiamoebic chemotherapy. This
study suggests that the preliminary activity of these compounds
may further be explored for the development of new targets
for amoebiasis.
compounds (2c, 2d and 2h-2l) were found more active than
the reference drug metronidazole, suggesting the bene-ficial
potential of these leads that need to be further explored in
order to discover and develop better and yet safer therapeutic
agents for amoebiasis.
Cytotoxicity: In the present study, only active compounds
(
2c, 2d and 2h-2l) were chosen for their cytotoxicity against
human embryonic kidney-293 (HEK-293) normal cell lines
to ensure their toxic effect.A confluent population of HEK-293
cells was treated with increasing concentrations of compounds
and the number of viable cells was measured after 48 h and
72 h by MTT cell viability assay based on mitochondrial reduc-
tion of yellow MTT tetrazolium dye to a highly coloured blue
formazan product which, after solubilization, can be measured
spectrophotometrically. This assay usually shows high corre-
lation with number of live cells and cell proliferation. The
concentration range for all the compounds (2c, 2d, 2h-2l and
MNZ) is mentioned in Fig. 1, which illustrates that all the
active compounds and metronidazole were least-cytotoxic in
the concentration range of 2.5-50 µM for 48 h and 2.5-25 µM
for 72 h. At 100 µM for 48 h and 50 µM for 72 h, only four
compounds, 2c, 2h, 2k and 2l showed maximum viability and
least cytotoxicity, respectively. Therefore, it can be concluded
that the cytotoxicity of all the compounds (2c, 2d and 2h-2l)
was found to be concentration-dependent and all the screened
compounds were least-cytotoxic against the human embryonic
kidney-293 (HEK-293) normal cell lines in the concentration
range of 2.5-50 µM and 2.5-25 µM.
ACKNOWLEDGEMENTS
The authors thank the Deanship of Scientific Research,
University ofTabuk, Tabuk, SaudiArabia for the financial support
for this research work via Grant No. (S-1436-0064).
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