Int J Pharm Pharm Sci, Vol 8, Issue 1, 247-254Original Article

NOVEL PYRAZOLINES: SYNTHESIS AND EVALUATION OF THEIR DERIVATIVES WITH ANTICANCER AND ANTI-INFLAMMATORY ACTIVITIES

L. SIVA SANKER REDDY*¹, M. BHAGAVAN RAJU², C. SRIDHAR³

¹Dept. of Pharmaceutical Chemistry, Creative Educational Society’s College of Pharmacy, N. H-7, Chinnatekur, Kurnool, Andhra Pradesh 518218, ²Gland Institute of Pharmaceutical Sciences, Kothapet, Shivampet, Medak, Telangana 502313, ³Sri Padmavathi School of Pharmaceutical Sciences, Tirupathi, Andhra Pradesh 517503
Email: shiva_s_rl@yahoo.co.in

Received: 01 Jul 2015 Revised and Accepted: 25 Nov 2015

ABSTRACT

Objective: Synthesis of novel pyrazolines (P2-P4 & P7-P9) from the chalcones (C2-C10) obtained by condensing different aldehydes with 2-acetyl- 5-bromothiophene and evaluates them for in vitro anticancer and anti-inflammatory activities.

Methods: The synthesized pyrazolines and chalcones were screened for anticancer activity against human breast cancer cell lines-MCF-7 and MDA-MB-468 in the range of 100 nm to 100 µm. Inhibition of bovine albumin denaturation and heat-induced hemolysis in vitro methods were followed to screen for anti-inflammatory activity. The structures of synthesized compounds were confirmed based on the IR, ¹H NMR and mass spectral data.

Results: Among the synthesized compounds, methoxy trisubstituted pyrazoline derivative (P6) exhibited an interesting profile of anticancer activity against MCF-7 cell line with GI₅₀<0.1 μ M. similar to that of the standard drug doxorubicin. Compounds C8, P8, P3 have moderate anti-inflammatory activity in bovine denaturation and heat induced hemolytic method.

Conclusion: Novel pyrazolines and chalcones were synthesized and evaluated for anticancer and anti-inflammatory activity. The methoxy containing compounds one of which P6 found to be active against MCF-7 breast cancer cell line. The chloro-substituted compounds found to show anti-inflammatory activity.

Keywords: Pyrazoline, Chalcone, MCF-7, MDA-MB-468, Anti-inflammatory, Anticancer.

INTRODUCTION

For centuries, cancer has been prevailing as most serious disease and its incidence is rising day-to-day in the world. Cancer treatment usually falls into the category of surgery, radiation and chemotherapy. Despite of all these treatments, cancer is still continuing as uncontrollable disease and exploring for new approaches in anticancer therapy. Chemotherapy is generally used to treat cancer that has spread or metastasized because the medicines travel throughout the entire body. Recently, several substituted thiophenes and pyrazoles have been reported for anticancer activity. [1-4]. Pyrazoles substituted with another heterocyclic compound such as thiophene resulted in compounds with improved anti-proliferative activity against a number of solid and hematological tumors. [5] Even some prescribed drugs like omeprazole (proton pump inhibitor), eprosartan (angiotensin II receptor antagonist) and lore diplons (anxiolytic agent) have pyrazole ring connected with another heterocyclic moiety. Thiophene nucleus is also an important heterocyclic ring which is part of some of the drugs like raloxifene (osteoporosis), olanzapine (antipsychotic), and clopidogrel (antiplatelet agent). Thus, we were interested in synthesizing thiophene substituted pyrazolines and look for their anti-proliferative activity.

As a part of our research work, we synthesized a series of 1-(5-bromothiophen-2-yl)-3-(phenyl) prop-2-en-1-one and 1-(3-(5-bromo-thiophene-2-yl)-5-(aryl)-4,5-dihydropyrazole-1-yl) ethanone and tested biologically. The method followed for the synthesis of the final compounds is in accordance with the literature [6]. Later, the anticancer activity of compounds was reported by screening against human breast cancer cell lines MCF-7 and MDA-MB-468 and in vitro anti-inflammatory activity was done by inhibition of bovine albumin denaturation method and heat induced hemolytic method.

MATERIALS AND METHODS

Chemistry

Melting points of the compounds were determined using open capillary melting point apparatus and were reported uncorrected. Ultraviolet, visible spectroscopic analysis has been carried out in UV-visible double beam spectrophotometer (LAB INDIA 3000+), IR spectra was recorded by a KBr pellet method using a bruker FTIR ALPHA transmission mode spectrophotometer. The ¹H NMR spectra were recorded in DMSO-d6 by NMR 300MHZ spectrometers using tetramethyl silane as an internal standard. All the chemicals and solvents used in this study were of analytical grade (S. D. FINE Chem. Limited, Mumbai). Reaction progress was checked by TLC in a solvent-vapor-saturated chamber on glass plates coated with Silica Gel GF₂₅₄ followed by visualization under UV light (254 nm). The solvent system used for thin layer chromatography was n-hexane: ethyl acetate (8:2).

Preparation of chalcones

0.01 Mol (2.05g) of 2-acetyl-5-bromothiophene taken in a 100 ml round bottom flask containing 20 ml of ethanol, to that equimolar quantity of substituted benzaldehydes added. The contents of the flask were stirred continuously using a magnetic stirrer, and the temperature was maintained below 20 ° C. Then 0.1 ml of 40% KOH was added drop by drop to the flask. The reaction was monitored by using a precoated TLC plate. After completion of the reaction, the contents of the flask were neutralized with dilute HCl to get precipitates of chalcones & filtered, washed with cold ethanol, dried and recrystallized from ethanol.

Preparation of 2-pyrazolines

0.002 moles of chalcone, 0.008 mole of hydrazine hydrate were taken in a 100 ml round bottom flask containing 30 ml of glacial acetic acid and refluxed for 70 h at 140 °C. The reaction mixture was monitored by using a precoated TLC plate. After completion of the reaction the content of the flask was poured into the crushed ice to get brown precipitate. The precipitate was dried and purified by column chromatography. Different gradients of ethyl acetate: petroleum ether, i.e. 2%, 4%, 6%, 8%, 10% and 12% was used to elute the pure compound successively. The eluent containing the compound was collected separately and evaporated to get the pure compound.

Table 1: List of synthesized compounds

Sample code	Chalcone and pyrazoline derivative
C2	(2E)-1-(5-bromothiophen-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one
C3	(2E)-1-(5-bromothiophen-2-yl)-3-(4-chlorophenyl)prop-2-en-1-one
C4	(2E)-1-(5-bromothiophen-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-one
C5	(2E,4E)-1-(5-bromothiophen-2-yl)-5-phenylpenta-2,4-dien-1-one
C6	(2E)-1-(5-bromothiophen-2-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
C7	(2E)-1-(5-bromothiophen-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one
C8	(2E)-1-(5-bromothiophen-2-yl)-3-(2-chlorophenyl)prop-2-en-1-one
C9	(2E)-1-(5-bromothiophen-2-yl)-3-phenylprop-2-en-1-one
C10	(2E)-1-(5-bromothiophen-2-yl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one
P2	1-[3-(5-bromothiophen-2-yl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone
P3	1-[3-(5-bromothiophen-2-yl)-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone
P4	1-[3-(5-bromothiophen-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone
P6	1-[3-(5-bromothiophen-2-yl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone
P7	1-[3-(5-bromothiophen-2-yl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone
P8	1-[3-(5-bromothiophen-2-yl)-5-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone
P9	1-[3-(5-bromothiophen-2-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone

In vitro anticancer activity by SRB assay [18]

The effect of synthesized compounds on cell growth was determined on two human tumor cells MCF-7 & MDA-MB-468. The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (Wt/Vol) trichloroacetic acid and stained for 30 min with 0.4% (Wt/Vol) SRB dissolved in 1% acetic acid after which the excess dye was removed by repeatedly washing with 1% (Vol/Vol) acetic acid. The protein-bound dye was dissolved in 10 mM tris base solution for OD determination at 564 nm using a microplate reader.

Appropriate positive controls were run in each experiment, and each experiment was repeated thrice.
Results are in terms of

GI₅₀ (concentration of the compound that produces 50% inhibition of the cells),

TGI (concentration of the compound that produces total inhibition of the cells) and

LC₅₀ (concentration of the compound that kills 50% of the cells)

Compounds with GI₅₀ ≤ 1µM was considered as active ones.
Doxorubicin was taken as a positive control.

In vitro anti-inflammatory activity [19-21]

Inhibition of bovine albumin denaturation method

To 2 ml of various concentrations of test or standard solutions, 2.8 ml of normal saline (pH=7.4) and 0.2 ml of 1% bovine albumin solution was added. Simultaneously blank samples were prepared for each concentration without the addition of 1% bovine albumin solution and an equal volume of normal saline (pH 7.4) was added to each blank sample. To 4.8 ml of normal saline (pH 7.4), 0.2 ml of 1% bovine albumin solution was added and used as a control. The test/standard samples were incubated for 15 min at 70 ° C. Then the tubes were cooled under running tap water and then absorbance was recorded at 660 nm. % inhibition of denaturation of bovine albumin was calculated using the formula,

Where A=absorbance of the control,

A1= absorbance of the test/standard

Heat-induced hemolytic method

To 1 ml of various concentrations of test or standard solutions, 1 ml of 1% RBC’s suspension was added. Simultaneously blank samples were prepared for each concentration without the addition of 1% RBC’s solution and an equal amount of normal saline was added to each blank sample. An equal amount of 1% RBC’s solution and normal saline was added and was used as a control.

All these samples were taken into centrifuge tubes and incubated in a water bath at 56 °C for 30 min. The tubes were cooled under running tap water and then centrifuged at 2500 rpm for 15 min and absorbance of the supernatant was taken at 560 nm. % inhibition was calculated using formula:

Where A=absorbance of the control, A1= absorbance of the test/standard.

IC₅₀ values

IC₅₀ was calculated using GraphPad prism software

Statistical analysis

All the data were expressed as mean±SEM. Statistical significance was tested by using one-way ANOVA followed by the Turkey’s test using a computer-based fitness program (Graph pad prism 5)

RESULTS AND DISCUSSION

Synthesis

Claisen-Schmidt condensation reaction between 2-acetyl-5-bromothiophene and different substituted aldehydes catalysed by 40% KOH gave chalcones (C2-C10). The obtained chalcones were cyclised in the presence of glacial acetic acid to give 2-pyrazoliones (P2-P4 & P7-P9) (Scheme-I). All the synthesized compounds were characterized by FT IR, ¹NMR and mass spectroscopic data.

In the IR spectra of 1-(5-bromothiophen-2-yl)-3-(phenyl)prop-2-en-1-one (C2-C10) characteristic absorption due to a carbonyl group appears in the range of 1655-1630 cm^-1. The olefinic double (C=C) appears in the range of1593-1522 cm^-1. The ¹HNMR spectra, the two olefinic protons (CH=CH) appears as a doublet in the region of δ 6.9-7.9 ppm and trans type of geometrical isomerism can be confirmed due to J>14. The thiophene protons seen as doublets were distinguished from other aromatic protons based on the J value which is 4. Aromatic protons of the benzene appear as a complex multiple in the range of δ 6.8-8.3 ppm.

The IR spectra of 1-(3-(5-bromothiophene-2-yl)-5-(aryl)-4, 5-dihydropyrazole-1-yl) ethanone (P2-P4 &P6-P9), the characteristic absorption due to a carbonyl group appears in the range of 1673-1645 cm^-1. The absorption of C-Br stretching appears in the range of 580-591 cm^-1. ¹H NMR data showed Ha, Hb, Hx type of coupling due to spin coupling of CH₂ protons with CH proton of the pyrazoline nucleus with doublet of doublets around δ 3.099 ppm (1H, dd, Ha), δ 3.811 ppm (1H, dd, Hb), and δ 5.646 ppm (1H, dd, Hx) respectively, with coupling constants(J_ab=17.6, J_ax=5.2, J_bx=11.6). The thiophene protons were in the range of δ 7.03 to 6.9 ppm as a doublet with J value of 4. The phenyl protons were lying in the region of δ 6.3 to 8.2 ppm with J values 7-9 depending on the type of substitution on the phenyl ring. The acetyl protons on the pyrazoline nucleus were present as a single and had δ value of 2.3 to 2.4 ppm. Thus, all the protons of pyrazoline compounds were accounted.

Spectral data of compounds

Compound C 2:(2E)-1-(5-bromothiophen-2-yl)-3-(4-nitro-phenyl)prop-2-en-1-one

IR (KBr)V_max in cm^-1:C=O str =1649.16, C=C str =1586.86, Ar-H str =3076.73, =C-H str = 2924.13,Ar-N-O str =1516.82, C-Br str = 670.70; ¹H NMR (CDCl₃) in δ (ppm): 7.197 (d, 1H, C4 of thiopheneJ=4), 7.638 (d, 1H, C3 of thiophene J=4), 7.39 & 7.868 (d, 2H,-CH=CH-trans J=15.6), 7.79 & 8.29 (d, 4H,Ar-HJ=8.8& J=8.4).

Compound C 3:(2E)-1-(5-bromothiophen-2-yl)-3-(4-chloro-phenyl) prop-2-en-1-one

IR (KBr)V_max in cm^-1: C=O str =1650.08, C=C str =1593.94, Ar-H str =3072.28 cm^-1, Ar-C=C str =1491.88, =C-H str = 2924.94, C-Clstr = 771.88; ¹H NMR (CDCl₃) in δ (ppm): 7.16 (d, 1H, C4 of thiopheneJ=4), 7.30 (d, 1H, C3 of thiophene), 7.805 (d, 1H,-CH=CH-trans J=15.6), 7.6 (m, 1H,-CH=CH-trans J=17.6), 7.42 (d, 2H, orthoAr-H J=8.4) & 7.56 (d, 2H Meta Ar-H J=8.4)

Compound C 4:(2E)-1-(5-bromothiophen-2-yl)-3-(4-methoxy-phenyl)prop-2-en-1-one

IR (KBr)V_max in cm^-1: Aliphatic C-H str = 2838.82, C=O str =1645.48, C=C str =1587.27, Ar-H str =3084.58, Ar-C=C str =1510.30, =C-H str = 3004.19, C-O-C str = 1302.44, 1031.93;¹H NMR (CDCl₃) in δ (ppm): 7.15 (d, 1H, C4 of thiophene J=4),7.575 (d, 1H, C3 of thiophene), 7.83 (d, 1H,-CH=CH-trans J=15.6), 7.218 (d 1H,-CH=CH-trans J=15.6), 7.606 (m, 2H,orthoAr-H J=8.8) & 6.952 (d, 2H, meta Ar-H J=8.4) 3.86(s, 3H,para Ar-OCH₃).

Compound C 5:(2E, 4E)-1-(5-bromothiophen-2-yl)-5-phenyl-penta-2, 4-dien-1-one

IR (KBr)V_max in cm^-1: C=O str =1634.88, C=C str =1572.08, Ar-H str =3105.88, Ar-C=C str =1572.08, 1445.91, =C-H str = 3025.17, C-Br str = 686.29;¹H NMR (CDCl₃) in δ (ppm): 7.13 (d, 1H, C4 of thiopheneJ=4),7.52 (m, 1H C3 of thiophene), 6.906 (d, 1H,-CH=CH-trans J=14.8), 7.65 (m, 1H,-CH=CH-trans J=15.48), 6.98 (m, 1H,-CH=CH-trans), 7.03 (m, 1H,-CH=CH-trans) 7.516 (m, 2H,orthoAr-H J=8.4) & 7.314 to 7.402 (m, 3H, meta & paraAr-H).

Compound C 6:(2E)-1-(5-bromothiophen-2-yl)-3-(3, 4, 5-tri-methoxyphenyl) prop-2-en-1-one

IR (KBr) Vmax in cm^-1: Aliphatic C-H str = 2938.37,C=O str =1645.24, C=C str =1522.50, Ar-H str =3113.22, Ar-C=C str =1499.61, =C-H str = 3004.74, C-O-C str = 1214.49, C-Br str = 681.35;¹H NMR (CDCl₃) in δ (ppm):7.2 (m,1H, C4 of thiopheneJ=4),7.618 (d, 1H, C3 of thiophene), 7.15 (m, 1H,-CH=CH-), 7.79 (d, 1H,-CH=CH-trans J=15.6), 6.851 (s, 2H,orthoAr-H), 3.926 (s, 6H, meta (Ar-OCH₃)₂), 3.906 (s, 3H, meta Ar-OCH₃).

Compound C 7:(2E)-1-(5-bromothiophen-2-yl)-3-(3,4-di-methoxyphenyl) prop-2-en-1-one

IR (KBr) V_max in cm^-1: Aliphatic C-H str = 2834.62, C=O str =1639.66, C=C str =1570.53, Ar-H str =3079.72, Ar-C=C str =1511.08, =C-H str = 2933.22, C-O-C str = 1254.11, C-Br str = 591.60 cm^-1; ¹H NMR (CDCl₃) in δ (ppm): 7.197 (s, 1H, C4 of thiopheneJ=4), 7.6 (d, 1H, C3 of thiophene), 7.245 (m, 1H,-CH=CH-), 7.815 (d, 1H,-CH=CH-trans J=15.2), 6.89 (d, 1H, meta Ar-H), 3.95 (s, 6H, meta, para (Ar-OCH₃)₂).

Compound C 8:(2E)-1-(5-bromothiophen-2-yl)-3-(2-chloro-phenyl) prop-2-en-1-one

IR (KBr) V_max in cm^-1: C=O str =1655.14, C=C str =1593.94, Ar-H str =3092.02, Ar-C=C str =1471.55, =C-H str = 2622.64, C-Clstr = 775.32, C-Br str = 689.31; ¹H NMR (CDCl₃) in δ (ppm):7.167 d (1H C4 of thiopheneJ=4),7.601 d (1H C3 of thiophene), 7.089 m (1H,-CH=CH-), 8.236 (d, 1H,-CH=CH-trans J=15.6), 7.735 (dd, 1H orthoAr-H),7.466 (dd, 1H, meta Ar-H), 7.319 to 7. 372 (m, 2H, meta, paraAr-H).

Compound C 9: (2E)-1-(5-bromothiophen-2-yl)-3-phenylprop-2-en-1-one

IR (KBr) V_max in cm^-1: C=O str =1649.12, C=C str =1593.26, Ar-H str =3074.07, Ar-C=C str =1521.14, =C-H str = 3027.33, C-Br str = 685.59;¹H NMR (CDCl₃) in δ (ppm): 7.161 (d, 1H, C4 of thiopheneJ=4),7.60 (d, 1H, C3 of thiopheneJ=4), 7.06 (m, 1H,-CH=CH-), 7.863 (d, 1H,-CH=CH-trans J=15.6), 7.646 (m, 2H,orthoAr-H), 7.419 to 7. 434 (m, 3H, 2 meta, 1paraAr-H).

Compound C 10: (2E)-1-(5-bromothiophen-2-yl)-3-[4-(propan-2-yl) phenyl] prop-2-en-1-one

IR (KBr) V_max in cm^-1: Aliphatic C-H str = 2868.78, C=O str =1640.69, C=C str =1588.90, Ar-H str =3078.10, =C-H str = 2957.41, CH₃ bending= 1412.45;¹H NMR (CDCl₃) in δ (ppm): 7.157 (d, 1H, C4 of thiopheneJ=4),7.585 (m, 1H, C3 of thiophene), 7.275(m, 1H,-CH=CH-), 7.853 (d, 1H,-CH=CH-trans J=15.6), 7.559 (m, 2H, orthoAr-H), 7.297 (m, 2H, meta Ar-H), 2.95 (septet, 1H, CH of isopropyl), 1.282 & 1.265 (6H–(CH₃)₂).

Compound P 2: 1-[3-(5-bromothiophen-2-yl)-5-(4-nitrophenyl)-4, 5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1: Aliphatic C-H str = 2925.85, C=O str =1666.37, Ar-C=C str =1605.79 cm^-1, 1514.99, Ar-H str =3107.29, Ar-N-O str =1514.99,1216.40, C-Br str = 587.26; ¹H NMR (CDCl₃) in δ (ppm): 2.37(s, 3H,-CH₃,), 3.099 (dd, 1H, Ha), 3.811 (dd, 1H, Hb), 5.646 (dd, 1H, Hx), 8.21 (d, 2H,Ar-H, J=8.4 HZ), 7.40 (d, 2H, Ar-H, J=8.4), 7.036 to 6.928 (dd, 2H, thiopheneH), (J_ab=17.6, J_ax=5.2, J_bx=11.6)

Table 2: Physico-chemical characterization data of synthesized chalcones of scheme I

Code	Molecular formula	Molecular weight	Melting point (ºC)	% yield (%)	R_fvalue*	Colour
C2	C₁₃H₈BrNO₃S	338.17	193-196	72	0.62	Yellow flakes
C3	C₁₃H₈BrClOS	327.62	135-138	67	0.64	Creamish white Amorphous
C4	C₁₄H₁₁BrO₂S	323.20	128-130	82	0.70	Cream
C5	C₁₅H₁₁BrOS	319.21	128-130	86	0.68	Light yellow Crystalline needles
C6	C₁₆H₁₅BrO₄S	383.25	140-143	82	0.59	Yellow; Crystalline needles
C7	C₁₅H₁₃BrO₃S	353.23	103-106	64	0.62	Dark Yellow; Crystalline needles
C8	C₁₃H₈BrClOS	327.62	123-126	60	0.67	Cream; Amorphous
C9	C₁₃H₉BrOS	293.17	70-73	63	0.58	Light yellow; Crystals
C10	C₁₆H₁₅BrOS	335.25	78-80	60	0.62	Cream; Amorphous
P2	C₁₅H₁₂BrN₃O₃S	394.24	143-145	71	0.56	Creamy crystals
P3	C₁₅H₁₂BrClN₂OS	383.69	188-189	68	0.58	White crystals
P4	C₁₆H₁₅BrN₂O₂S	379.27	159-160	95	0.60	Creamy crystals
P6	C₁₈H₁₉BrN₂O₄S	439.32	90-92	78	0.64	Yellow crystals
P7	C₁₇H₁₇BrN₂O₃S	409.29	116-118	79	0.58	Creamy crystals
P8	C₁₅H₁₂BrClN₂OS	383.69	180-183	86	0.56	Light brown crystals
P9	C₁₅H₁₃BrN₂OS	349.24	200-202	76	0.58	Dark brown crystals

*n-Hexane: Ethyl acetate (8:2)

Compound P 3: 1-[3-(5-bromothiophen-2-yl)-5-(4-chloro-phenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1: Ali C-H str = 2929.09, C=O str =1645.67, Ar-C=C str =1524.49, Ar-H str =3060.76, C-Br str = 580.79, C-Clstr = 626.89; ¹H NMR (CDCl₃) in δ (ppm): 2.35(s, 3H,-CH₃,), 3.07 (dd, 1H, Ha), 3.73 (dd, 1H, Hb), 5.55 (dd, 1H,Hx), 7.30 (d, 2H, Ar-H, J=8.4 HZ), 7.16 (d, 2H, Ar-H, J=8.4), 6.921 (s, 1H, C-2 H of thiophene), 7.024 (s, 1H, C-3 H of thiophene),(J_ab=17.6, J_ax=4.8, J_bx=11.6); ESI-MS 385 (M+H)⁺.

Compound P 4: 1-[3-(5-bromothiophen-2-yl)-5-(4-methoxy-phenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1: Aliphatic C-H str = 2840.59, C=O str =1657.18, Ar-C=C str =1514.32, Ar-H str =3064.73, C-Br str = 582.28, Ar-C-O-C str = 1248.50, 1028.05 cm^-1; ¹H NMR (CDCl₃) in δ (ppm): 2.34(s, 3H,-CH₃,), 3.1 (dd, 1H, Ha), 3.703 (dd, 1H, Hb), 3.77 (s, 3H, p-Ar-OCH₃) 5.55 (dd, 1H, Hx), 7.154 (d, 2H, Ar-H, J=8.4), 6.855 (d, 2H, Ar-H, J=8.4), 6.921 (s, 1H, C-2 H of thiophene), 7.019 (s, 1H, C-3 H of thiophene), (J_ab=17.6, J_ax=4.8, J_bx=11.6); ESI-MS 381 (M+H)⁺.

Compound P 6: 1-[3-(5-bromothiophen-2-yl)-5-(3, 4, 5-trimethoxyphenyl)-4, 5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1:Aliphatic C-H str = 2826.52, C=O str =1664.80, Ar-C=C str =1506.84, Ar-H str =3085.12, C-Br str = 591.83, Ar-C-O-C str = 1237.66, 1006.90; ¹H NMR (CDCl₃) in δ (ppm): 2.38(s, 3H,-CH₃,), 3.1 (dd, 1H, Ha), 3.7 (dd, 1H, Hb), 3.80 (s, 3H, p-Ar-OCH₃), 3.826 (s, 6H, m-Ar-(OCH₃)₂), 5.521 (dd, 1H, Hx), 7.154 (d, 2H, Ar-H, J=8.4 HZ), 6.399 (d, 2H, Ar-H, J=8.4), 6.927 (s, 1H, C-2 H of thiophene), 7.027 (s, 1H, C-3 H of thiophene), (J_ab=17.6, J_ax=4.8, J_bx=11.6); ESI-MS 441 (M+H)⁺.

Compound P 7: 1-[3-(5-bromothiophen-2-yl)-5-(3,4-dimethoxy-phenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1: Aliphatic C-H str = 2834.71, C=O str =1657.64, Ar-C=C str =1518.62, Ar-H str =2999.84, C-Br str = 578.98, Ar-C-O-C str = 1258.43, 1025.12; ¹H NMR (CDCl₃) in δ (ppm): 2.36(s, 3H,-CH₃,), 3.11 (dd, 1H, Ha), 3.71 (dd, 1H, Hb), 3.83 (s, 3H, p-Ar-OCH₃), 3.85 (s, 3H, m-Ar-(OCH₃), 5.54 (dd, 1H, Hx), 6.76 (d, 2H, Ar-H, J=9.6 HZ), 6.81 (d, 1H, Ar-H, J=8), 6.92 (s, 1H, C-2 H of thiophene), 7.025 (s, 1H, C-3 H of thiophene), (J_ab=17.6, J_ax=4.4, J_bx=11.6); ESI-MS 411 (M+H)⁺.

Compound P 8: 1-[3-(5-bromothiophen-2-yl)-5-(3-chloro-phenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1: Aliphatic C-H str = 2927.73, C=O str =1673.45, Ar-C=C str =1530.85, Ar-H str =3325.17, C-Clstr = 635.24; ¹H NMR (CDCl₃) in δ (ppm): 2.42(s, 3H,-CH₃,), 3.01 (dd, 1H, Ha), 3.83 (dd, 1H, Hb), 5.92 (dd, 1H, Hx), 7.22 (m, 2H, Ar-H, J=4.4 HZ), 7.05 (m,1H, p, Ar-H, J=9.2), 7.406 (m, 1H,meta, Ar-H, J=9.2), 7.003 to 6.909 (dd, 2H, thiopheneH), (J_ab=17.6, J_ax=4.8, J_bx=11.6); ESI-MS 385 (M+H)⁺.

Compound P 9: 1-[3-(5-bromothiophen-2-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone

IR (KBr) V_max in cm^-1: C=O str =1659.68, Ar-C=C str =1452.02, Ar-H str =3085.56, C-Br str = 577.14; ¹H NMR (CDCl₃) in δ (ppm):2.36(s, 3H,-CH₃,), 3.11 (dd, 1H, Ha), 3.73 (dd, 1H, Hb), 5.59 (dd, 1H, Hx), 6.91 (s, 1H, C-2 H of thiophene), 7.016 (s, 1H, C-3 H of thiophene), (J_ab=17.6, J_ax=4.8, J_bx=11.6), 7.2 (s,1H, p-Ar-H,), 7.21 (d, 2H, ortho-Ar-H, J=7.2), 7.3 (2H, m, meta-Ar-H, J=7.2); ESI-MS 351 (M+H)⁺.

Biological activities

The synthesized compounds were evaluated for their anticancer activity in selected human breast cancer cell lines MCF-7 and MDA-MB-468. IC₅₀values, defined as the concentration corresponding to 50% growth inhibition were based on concentration and exponential cell growth curves as shown in fig. 1-4. The compounds that exhibit GI₅₀ ≤ 1 µM are considered to be active. All the compounds exhibited signiﬁcant anticancer activity with GI₅₀ values ranging from<0.1 to>100 µM, while the positive control doxorubicin demonstrated the GI₅₀<0.1µM in the cell lines employed. Compound P6 exhibits an interesting profile of anticancer activity with MCF-7 cell line with GI₅₀<0.1 µM. Even the activity of P6 in MDA-MB-468 cell line was also found to be GI₅₀ = 36.6 µM, which is the best activity when compared to all other compounds.

The promising results shown by compound P6 on both cell lines suggest that it has potent broad-spectrum anticancer activity. However, compounds P2-P4 and P7-P9 also expressed significant activity values of GI₅₀<100 µM in MCF-7 breast cancer cell lines. Whereas, in anticancer activity assay against MDA-MB-468 cell lines compound P8 shown GI₅₀ value 57.2 µM and rest all shown GI₅₀>100 µM. C6 and P6 having tri-methoxy substitution showed GI₅₀(µ molar concentration of drug/compound causing 50% inhibition of the cell growth) value of 28.6 µM and<0.1 µM respectively against MCF-7 breast cancer cell lines. C7 having dimethoxy substitution and P6 having tri-methoxy substitution has GI₅₀ value of 16.7 µM and 36.6 µM respectively against MDA-MB-468 breast cancer cell lines. Doxorubicin was the standard drug which has GI₅₀ value of<0.1 µM. The other compounds have GI₅₀ value greater than 16 µM. Concentrations of the test material used were 10, 20, 40 and 80µg/ml.

The in vitro anti-inflammatory activity was performed by inhibition of bovine albumin denaturation method and heat induced hemolytic method. The inhibitory activity of the compounds was compared with the control and the significance factor “p” was less than 0.001 for all the compounds. The compound with chloro group as a substituent showed the highest inhibition activity suggesting that electron donating groups may aid the activity (table 3-6). The inhibitory activity of the compounds was compared with the control and the significance factor “p” was less than 0.001 for all the compounds.

Fig. 1: % Control growth in human breast cancer cell line MCF-7

Fig. 2:% Control growth in human breast cancer cell line MDA-MB-468

Table 3: Bovine albumin denaturation method

Conc. (μg/ml)

% Inhibition±SEM*

Diclofenac sodium

C10

75.3±

0.364

31.9±0.190

31.2±

0.503

41.1±

0.049

33.9±

0.607

40.1±

0.429

37.1±

0.283

44.8±

0.024

40.2±

0.301

30.8±

0.239

81.4±

0.234

34.0±

0.424

47.0±

0.432

41.9±

0.291

49.0±

0.602

42.6±

0.793

31.0±

0.541

64.7±

0.023

43.8±

0.207

32.9±

0.266

86.0±

0.321

44.1±

0.560

40.1±

0.233

32.9±

0.502

22.5±

0.103

32.2±

0.670

21.4±

0.580

32.6±

0.435

33.2±

0.328

33.7±

0.024

94.0±

0.423

35.3±

0.457

42.6±

0.342

55.3±

0.649

44.2±

0.547

42.4±

0.368

22.8±

0.798

44.60±

0.672

41.7±

0.721

24.8±

0.640

100

96.4±

0.624

59.2±

0.628

54.7±

0.235

51.8±

0.129

45.4±

0.694

60.3±

0.117

30.7±

0.402

69.5±

0.264

45.5±

0.024

40.7±

0.452

120

98.0±

0.245

35.2±

0.610

45.2±

0.166

53.3±

0.712

37.5±

0.590

35.2±

0.064

40.5±

0.142

62.6±

0.264

39.9±

0.429

32.1±

0.126

IC₅₀(μg/ml)

7.873

76.40

68.0

62.80

156.62

46.28

241.6

46.249

98.0

78.92

*All the values are average of three readings, mean±SEM, SEM = Standard Error Mean IC₅₀= Half maximal inhibitory concentration.

Table 4: Bovine albumin denaturation method

Conc. (μg/ml)

% Inhibition±SEM*

Diclofenac sodium

75.3±

0.364

42.8±

0.439

36.2±

0.563

41.8±

0.299

40.9±

0.767

49.7±

0.024

33.1±

0.238

58.3±

0.444

81.4±

0.234

48.0±

0.004

39.0±

0.420

45.1±

0.216

41.8±

0.627

44.7±

0.730

43.0±

0.121

56.0±

0.043

86.0±

0.321

40.2±

0.830

30.2±

0.038

31.5±

0.215

33.2±

0.359

39.1±

0.525

25.5±

0.668

40.9±

0.534

94.0±

0.423

42.0±

0.968

46.0±

0.890

39.0±

0.846

30.6±

0.642

39.8±

0.25

52.20±

0.684

54.0±

0.842

100

96.4±

0.624

32.2±

0.558

32.2±

0.558

57.8±

0.129

45.4±

0.694

60.3±

0.117

30.7±

0.402

69.5±

0.264

120

98.0±

0.245

56.6±

0.665

56.6±

0.665

50.3±

0.672

39.5±

0.580

36.5±

0.054

42.5±

0.342

60.6±

0.284

IC₅₀

(μg/ml)

7.873

63.89

48.69

59.90

189.50

65.24

39.42

43.89

*All the values are the average of three readings, mean±SEM, SEM = Standard Error Mean, IC₅₀= Half maximal inhibitory concentration.

Table 5: Heat-induced hemolytic method

Conc. (μg/ml)

% Inhibition±SEM*

Diclofenac sodium

C10

74.8±

0.282

34.0±

0.424

33.2±

0.303

45.1±

0.949

38.4±

0.670

44.6±

0.480

34.8±

0.203

46.8±

0.824

42.2±

0.310

36.6±

0.294

78.2±

0.644

44.0±

0.544

57.6±

0.430

51.9±

0.491

48.8±

0.260

44.8±

0.736

35.0±

0.468

54.8±

0.823

48.6±

0.446

36.2±

0.863

89.0±

0.482

43.3±

0.260

44.1±

0.346

36.8±

0.684

32.8±

0.468

38.8±

0.127

25.7±

0.452

34.6±

0.428

38.8±

0.645

34.2±

0.465

91.2±

0.514

38.6±

0.46

44.9±

0.264

55.3±

0.399

46.8±

0.647

43.8±

0.640

42.1±

0.579

54.90±

0.820

48.2±

0.530

44.6±

0.602

100

93.2±

0.321

57.6±

0.268

59.8±

0.548

52.3±

0.560

42.8±

0.680

50.6±

0.946

40.7±

0.682

62.6±

0.246

42.6±

0.240

48.6±

0.252

120

94.4±

0.821

36.7±

0.262

44.2±

0.257

54.5±

0.625

37.4±

0.856

38.2±

0.664

46.±6

0.220

52.4±

0.462

36.3±

0.626

38.6±

0.582

IC₅₀(μg/ml)

8.624

62.46

67.24

68.63

166.20

66.68

221.8

42.86

110.0

76.46

*All the values are the average of three readings, mean±SEM, SEM = Standard Error Mean IC₅₀= Half maximal inhibitory concentration

Table 6: Heat-induced hemolytic method

Conc. (μg/ml)

% Inhibition±SEM*

Diclofenac sodium

74.8±

0.282

48.4±

0.494

46.2±

0.425

51.4±

0.299

48.6±

0.434

48.6±

0.243

38.8±

0.224

54.5±

0.484

78.2±

0.644

46.8±

0.404

49.2±

0.402

42.1±

0.616

47.8±

0.060

48.3±

0.630

44.8±

0.251

52.0±

0.482

89.0±

0.482

46.6±

0.80

38.2±

0.083

30.6±

0.624

38.9±

0.569

36.4±

0.456

35.8±

0.688

44.6±

0.584

91.2±

0.514

46.0±

0.680

45.0±

0.870

39.0±

0.044

38.6±

0.240

38.8±

0.425

32.80±

0.824

58.8±

0.242

100

93.2±

0.321

36.4±

0.588

36.8±

0.458

52.0±

0.290

48.3±

0.734

58.4±

0.868

44.6±

0.259

59.2±

0.242

120

94.4±

0.821

52.6±

0.550

52.6±

0.465

56.3±

0.58

38.5±

0.880

39.0±

0.654

46.9±

0.802

64.2±

0.740

IC₅₀(μg/ml)

8.624

64.90

56.60

68. 0

141.0

59.20

176.04

68.38

*All the values are average of three readings, mean±SEM, SEM = Standard Error Mean IC₅₀= Half maximal inhibitory concentration

CONCLUSION

The synthesis of chalcones and condensing them to form pyrazolines was done according to the reported methods. The synthesized chalcones and pyrazolines were screened for anti-tumor activity against human breast cancer cell lines-MCF-7 and MDA-MB-468. Compound P6 was found to be an active agent against human breast cancer cell lines-MCF7. The same compound showed anticancer activity against human breast cancer cell lines MDA-MB-468 but not as active as against cancer cell lines-MCF. The anti-inflammatory activity also established in all the synthesized compounds shown significant inhibition.

The compounds C8, P3 and P8 shown moderate anti-inflammatory activity. It is found that the compounds with chloro substitution have in vitro anti-inflammatory activity.

ACKNOWLEDGEMENT

The authors are thankful to the head of the department of chemistry, Creative educational society’s college of pharmacy, Kurnool, India, for providing laboratory facilities to carry out the research work. We also thank LailaImpex, R&D center, Vijayawada for analysing the compounds for ¹H NMR and Mass. We are greatful to ACTREC-Mumbai for performing the anti-cancer activity.

CONFLICT OF INTERESTS

Declared none

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Int J Pharm Pharm Sci, Vol 8, Issue 1, 247-254Original Article

NOVEL PYRAZOLINES: SYNTHESIS AND EVALUATION OF THEIR DERIVATIVES WITH ANTICANCER AND ANTI-INFLAMMATORY ACTIVITIES

L. SIVA SANKER REDDY*1, M. BHAGAVAN RAJU2, C. SRIDHAR3

L. SIVA SANKER REDDY*¹, M. BHAGAVAN RAJU², C. SRIDHAR³