CC BY ND NC 4.0 · SynOpen 2018; 02(02): 0114-0121
DOI: 10.1055/s-0036-1591980
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Copyright with the author

Synthesis and Cytotoxic Evaluation of 3-(4-Fluorophenyl)-4,5-dihydro-5-(3,4,5-trimethoxy/4-nitrophenyl)-N-(substituted-phenyl)pyrazole-1-carboxamide Analogues

Department of Pharmaceutical Chemistry, Maharishi Arvind College of Pharmacy, Ambabari Circle, Jaipur, Rajasthan 302 039, India   Email: jawedpharma@gmail.com
,
Bhawani S. Kumawat
Department of Pharmaceutical Chemistry, Maharishi Arvind College of Pharmacy, Ambabari Circle, Jaipur, Rajasthan 302 039, India   Email: jawedpharma@gmail.com
,
Sonu Kumawat
Department of Pharmaceutical Chemistry, Maharishi Arvind College of Pharmacy, Ambabari Circle, Jaipur, Rajasthan 302 039, India   Email: jawedpharma@gmail.com
,
Piush Sharma
Department of Pharmaceutical Chemistry, Maharishi Arvind College of Pharmacy, Ambabari Circle, Jaipur, Rajasthan 302 039, India   Email: jawedpharma@gmail.com
,
Mohammad A. Bakht
Department of Chemistry, College of Science & Humanities, Prince Sattam Bin Abdulaziz University, P.O. Box 11323, Saudi Arabia
,
Mohd Z. Hassan
Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha 62529, Saudi Arabia
,
Afzal Hussain
Department of Pharmaceutical Science & Technology, Birla Institute of Science & Technology, Mesra, Ranchi, Jharkhand 835 215, India
,
Pankaj Saraswat
Department of Pharmaceutical Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301 030, India
,
Habibullah Khalilullah
Department of Pharmaceutical Chemistry, Unaizah College of Pharmacy, Qassim University, Al-Qassim 51911, Kingdom of Saudi Arabia
› Author Affiliations
Further Information

Publication History

Received: 24 January 2018

Accepted after revision: 14 March 2018

Publication Date:
23 April 2018 (online)

 

Abstract

A novel series of 3-(4-fluorophenyl)-4,5-dihydro-5-(3,4,5-trimethoxy/4-nitro phenyl)-N-(substituted-phenyl)pyrazole-1-carboxamide analogues 4an was synthesized in two steps from 4-fluoroacetophenone. The pyrazoline analogues were evaluated for cytotoxicity against two breast cancer cell lines (MCF-7 and MBA-MD-231) by the sulforhodamine B (SRB) assay. N-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4b) showed the most promising cytotoxicity among the series, with GI50 <0.1 and 45.8 μM against the cancer cell lines, MCF-7 and MDA-MB-231, respectively. The anticancer activity of 4b was found to be comparable to that of the standard drug adriamycin (GI50 <0.1) against the MCF-7 cancer cell line. Structure activity relationships (SAR) are also considered.


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In 2015, nearly 8.8 million cancer related deaths were reported. In India, every year over 0.7 million new cancer patients are registered and 0.5 million cancer related death are reported,[1] [2] and it is expected that new cases of cancer will amount to 19.3 million per annum worldwide by 2025.[3] Chemotherapy is an important approach to cancer treatment, but it has drawbacks of toxicity, resistance, and genotoxicity.[4] Today, we need to focus on drug discovery programmes to develop effective and safer anticancer agents for cancer treatment.

Five-membered pyrazoline rings have received much attention because of their diverse biological potential. Some of the pyrazoline incorporated compounds that have promising biological activities are shown in Figure [1]. Pyrazoloacridine (I) is a new anticancer agent in Phase II clinical trial.[5] [6] [7] 3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) (II) is a hypoxia-inducible factor (HIF)-1 inhibitor.[8,9] Axitinib (AG013736) (III) is an endothelial growth factor receptor (VEGFR) inhibitor in clinical practice.[10] [11] 4-(4-Chlorophenyl)-2-(3-(3,4-dimethylphenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole (IV) is an EGFR TK inhibitor (IC50 = 0.06 μM).[12] 5-Bromo-3-{2-[5-(4-methoxyphenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-yl]-4-oxo-4,5-dihydro-1,3-thiazol-5-ylidene}-2,3-dihydro-1H-indol-2-one (V) is a potent anticancer agent with promising activity against HOP-92 (GI50 < 0.01 μM), HCT-116 (GI50 = 0.018 μM), SNB-75 (GI50 = 0.0159 μM), RXF 393 (GI50 = 0.0197 μM), and NCI/ADR-RES (GI50 = 0.0169 μM).[13] 3-Benzofuran-2-yl-5-(4-dimethylaminonaphthalen-1-yl)-4,5-dihydropyrazole-1-carbothioic acid amide (VI) was found to possess anticancer activity against various cancer cell lines, including HT-29 (IC50 = 1.3±0.4), PC-3 (IC50 = 0.9±0.5), MCF-7 (IC50 = 1.3±0.4), H-460 (IC50 = 1.4±0.8), A-549 (IC50 = 1.10±0.5), PaCa-2 (IC50 = 0.9±0.8), and Panc-1 (IC50 = 1.2±0.2) targeting tyrosinase.[14] All compounds IVI contain the pyrazoline nucleus (Figure [1]). Furthermore, reports of other biological activities of pyrazoline analogues include antitubercular,[15] anticonvulsant,[16] antimicrobial,[17] [18] selective HER inhibition,[19] anti-inflammation,[20] anti-HIV,[17] carbonic anhydrase inhibition,[21] antiproliferatin[22] [23] and tyrosinase inhibition.[14] Hence, we have synthesized some novel pyrazoline analogues and report herein their cytotoxicity evaluation. The 4-fluorophenyl pyrazoline pharmacophore was chosen because the same pharmacophore is present in IV, which shows excellent anti-EGFR TK inhibition.

Zoom Image
Figure 1 Pyrazoline incorporated compounds and their biological activities

In the initial step, (2E)-1-(4-fluorophenyl)-3-(substituted-phenyl)prop-2-en-1-one derivatives 3a and 3b were synthesized from 4-fluoroacetophenone by Claisen–Schmidt condensation. 4-Fluroacetophenone 1 (0.05 mol, 6.07 mL) and aromatic aldehyde 2a or 2b (0.05 mol) were dissolved in absolute ethanol (50 mL) and 30% NaOH solution was added dropwise with continuous stirring at room temperature for 4 h.[15] [19] The reaction mixture was kept standing overnight and further poured into the crushed ice to obtain a pale-yellow precipitate of (2E)-1-(4-fluorophenyl)-3-(substituted-phenyl)prop-2-en-1-one derivatives 3a and 3b. In the subsequent step an equimolar mixture of 3a or 3b and substituted phenyl semicarbazide was heated at reflux in glacial acetic acid for 12 h to obtain the 3-(4-fluorophenyl)-4,5-dihydro-5-(3,4,5-trimethoxy/4-nitrophenyl)-N-(substituted phenyl)pyrazole-1-carboxamide analogues 4an. The substituted phenyl semicarbazides were synthesized as per the reported method.[21] The reaction was monitored throughout by thin-layer chromatography (TLC silica gel 60 F254; mobile phase benzene/acetone, 8:2). The title compounds 4an were further recrystallized from ethanol to give the pure compounds in yields of 65–81%. The synthetic protocol is summarized in Scheme [1].

Zoom Image
Scheme 1 Synthetic protocol for the synthesis of analogues 4an

Analogues 4an were further characterized based on their IR, NMR (1H and 13C) and MS data. The prototype compound 4b showed amide stretching band at 3302 cm–1 for NH, and 1682 cm–1 for the carbonyl function (C=O) in the IR spectra. Other prominent absorption peaks were found at 1522, 810, and 690 cm–1 for C=N (pyrazoline), C–F and C–Cl, respectively. In the 1H NMR spectrum, two doublet peaks at δ = 3.16 and 3.78 ppm corresponding to the two protons of pyrazoline ring (Ha and Hb), a triplet at δ = 5.19 ppm corresponding to the other proton of the pyrazoline ring (Hc); a multiplet δ = 7.01–7.21 ppm was observed for the corresponding four aromatic protons (4-fluorophenyl); four doublet δ = 7.25, 7.68, 7.74, and 8.11 ppm for eight aromatic protons. The CONH peak was obtained as a singlet at δ = 8.36 ppm. The 13C NMR spectrum showed peaks at δ = 166.01, 165.19, 151.83, 149.63, 146.29, 134.02, 130.82, 129.61, 129.11, 127.92, 123.09, 120.91, 115.61, 61.31, and 39.43 ppm. The mass spectral data showed the [M+H]+ signal at m/z 439, corresponding to the molecular formula C22H16ClFN4O3.

Similarly, the prototype compound 4i showed amide stretching band at 3312 cm–1 for NH, and 1685 cm–1 for the carbonyl function (C=O) in the IR spectra. Other prominent absorption peaks were found at 1523, 812 and 694 cm–1 for C=N (pyrazoline), C–F and C–Cl, respectively. In the 1H NMR spectrum, two doublet peaks at δ = 3.42 and 3.58 ppm corresponding to the two of pyrazoline ring (Ha and Hb), a triplet at δ = 5.41 ppm corresponding to the another proton of pyrazoline ring (Hc); a singlet peak at δ = 3.84 ppm corresponding to the six methoxy protons (OCH3); a singlet peak at δ = 3.93 ppm corresponding to the three methoxy protons (OCH3); a singlet peak at δ = 7.23 ppm corresponding to the two aromatic protons (3,4,5-trimethoxyphenyl); a multiplet δ = 7.32–7.42 ppm was observed for the corresponding four aromatic proton (4-fluorophenyl); two doublet δ = 7.70 and 7.89 ppm for four aromatic protons (4-chlorophenyl). The CONH peak was obtained as singlet δ = 8.26 ppm. The 13C NMR showed peaks at δ = 166.71, 165.02, 151.83, 150.63, 137.89, 137.23, 134.02, 130.81, 129.99, 129.63, 129.11, 123.01, 115.66, 104.33, 61.95, 56.56, 56.24, and 39.41 ppm. The mass spectral data showed the [M+H]+ signal at m/z 484, corresponding to the molecular formula C25H23ClFN3O4. The purity was checked by microanalysis (C, H and N analysis).

Cytotoxicity

The cytotoxicity of analogues 4an was tested against two breast cancer cell lines (MCF-7 and MDA-MB-231) according to the sulforhodamine B (SRB) assay.[24] The percent growth control was recorded for each drug at four different drug molar concentrations (10–7, 10–6, 10–5, and 10–4M). Most of the pyrazoline analogues showed significant cytotoxicity at higher dose of 10–4M. The cytotoxicity of compounds 4d (growth percent (GP) = –55.1%) and 4m (GP = –54.6%) was found to be comparable to the standard drug adriamycin (GP = –57.8%) at the higher dose concentration (10–4 M). The results of percent growth control at different molar concentrations are summarized in Table [1]. The plotted growth curve is shown in Figure [2a] (MCF-7) and Figure [2b] (MDA-MB-231).

Zoom Image
Figure 2 (a) Growth curve of analogues 4an against (a) MCF-7 and (b) MDA-MB-231

Table 1 Anticancer Activity (% Control Growth) of 4an against Breast Cancer Cell Lines (MCF-7 and MDA-MB-231) at Four Molar Concentrations

Comp.

Molar Drug Concentrations (M)

MCF-7

MDA-MB-231

10–7

10–6

10–5

10–4

10–7

10–6

10–5

10–4

4a

78.9

78.2

13.4

–12.4

103.5

84.9

75.6

33.3

4b

–8.4

–12.5

–15.9

–34.9

92.4

62.0

63.4

21.3

4c

88.3

101.9

74.3

–31.0

112.8

104.3

98.9

31.9

4d

81.7

88.4

50.1

–55.1

107.2

100.0

82.3

30.7

4e

100.8

106.7

42.7

1.3

100.7

106.4

78.6

35.2

4f

89.8

103.3

52.3

–18.7

115.1

98.1

99.1

39.1

4g

115.9

105.0

32.6

2.1

108.9

106.6

81.9

37.1

4h

74.3

77.4

18.0

–5.3

118.8

127.7

113.6

38.5

4i

53.4

77.9

–8.2

–24.1

102.4

101.4

86.8

41.9

4j

86.5

95.1

7.8

–4.0

125.8

125.3

106.7

94.2

4k

79.1

74.1

13.8

–16.0

107.7

104.0

89.2

173.3

4l

92.4

88.0

16.6

–6.7

133.0

145.3

124.0

68.4

4m

77.2

89.6

27.7

–54.6

102.5

95.7

72.1

41.1

4n

86.1

91.8

18.8

–8.1

105.7

99.0

94.0

32.0

ADRa

5.5

–24.1

–61.3

–57.8

31.8

39.8

–19.7

–15.7

a ADR = Adriamycin

Three further dose-related parameters, LC50, GI50, and TGI, were also recorded for each compound; these are given in Table [2]. The LC50 was found to be >100 μM for each of the pyrazoline analogues 4an against both the breast cancer cell lines (MCF-7 and MDA-MB-231). The TGI was found to be between <0.1 and 97.3 μM against MCF-7 cancer cell lines and >100 μM against MDA-MB-231 cancer cell line. The pyrazoline analogues 4an showed promising cytotoxicity against the MCF-7 (GI50 = <0.1 to 42.9 μM), whereas the cytotoxicity was found to be moderate or low against MDA-MB-231 (GI50 = 45.8 to >100 μM). Eight compounds, 4d, 4j, 4n, 4l, 4f, 4c, 4e, and 4g showed less cytotoxicity against MCF-7 cancer cell line, with GI50 ranging between 21.3 and 42.9 μM. Five compounds, 4i (GI50 = 6.6 μM), 4k (GI50 = 12.4 μM), 4a (GI50 = 14.2 μM), 4h (GI50 = 14.9 μM) and 4m (GI50 = 16.0 μM) showed moderate cytotoxicity against MCF-7. N-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4b) showed the highest cytotoxicity among the series, with GI50 of <0.1 μM against the cancer cell line MCF-7. The cytotoxicity of compound 4b and the standard drug adriamycin (GI50 = <0.1 μM) was found to be equal against the MCF-7 cancer cell line. The images of growth for some of the pyrazoline having significant cytotoxicity against MCF-7 cancer cell line are shown in Figure [3].

Zoom Image
Figure 3 Images of growth control against MCF-7 cancer cell line

Table 2 LC50, TGI, and GI50 of 4an against MCF-7 and MDA-MB-231 cancer cell lines

Comp.

Drug concentrations calculated from graph (μM)

MCF-7

MDA-MB-231

LC50

TGI

GI50

LC50

TGI

GI50

4a

>100

79.0

14.2

>100

>100

69.6

4b

>100

<0.1

<0.1

>100

>100

45.8

4c

>100

74.7

34.5

>100

>100

76.1

4d

>100

58.3

21.3

>100

>100

70.9

4e

>100

97.7

42.1

>100

>100

75.0

4f

>100

80.6

34.0

>100

>100

83.7

4g

>100

97.3

42.9

>100

>100

78.5

4h

>100

87.5

14.9

>100

>100

>100

4i

>100

61.5

6.6

>100

>100

85.0

4j

>100

88.4

23.0

>100

>100

>100

4k

>100

75.3

12.4

>100

>100

NEa

4l

>100

86.4

24.8

>100

>100

>100

4m

>100

55.1

16.0

>100

>100

80.4

4n

>100

85.2

24.3

>100

>100

>100

ADRb

82.9

2.7

<0.1

>100

50.9

<0.1

a NE = Not evaluated

b ADR = Adriamycin.

All the pyrazoline analogues showed moderate or low cytotoxicity against the MDA-MB-231 cancer cell line, and compound 4b showed the highest cytotoxicity among the series of pyrazoles 4an, with GI50 of 45.8 μM. Images of growth for some of the pyrazoline with significant anticancer activity against MDA-MB-231 cancer cell line are shown in Figure [4].

Zoom Image
Figure 4 Images of growth control against MDA-MB-231 cancer cell line

Structure activity relationship studies (SAR) were established with the anticancer data (GI50) and the results are summarized in Figure [5].

Zoom Image
Figure 5 Structure activity relationship studies on analogues 4an

Pyrazolines with a 4-nitrophenyl substituent at position 5 of the pyrazoline showed higher anticancer activity than those with 3,4,5-trimethoxyphenyl substitution. Furthermore, substrates with a 4-chloro substitution at the N-phenyl group showed the maximum anticancer activity. The order of anticancer activity was found to be 4-Cl>4-F>2-Cl>4-Br>4-OCH3>4-CH3>2,6-(CH3)2. The anticancer results showed that electron-withdrawing substituents at the N-phenyl group were found to be more significant than electron-releasing substitutions.

In conclusion, novel pyrazolines have been synthesized in satisfactory yield. All the pyrazoline analogues were evaluated for cytotoxicity against two breast cancer cell lines (MCF-7 and MDA-MB-231). N-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4b) showed the most promising anticancer activity among the series. Further structure activity relationships are also presented. Pyrazolines with 4-nitrophenyl substitution at position 5 of the pyrazoline showed higher anticancer activity and 4-chloro-substitution on the N-phenyl group led to maximum anticancer activity.


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Preparation of 3-(4-Fluorophenyl)-4,5-dihydro-5-(3,4,5-trimethoxy/4-nitro phenyl)-N-(substituted-phenyl)pyrazole-1-carboxamide Analogues 4a–n; General Procedure

An equimolar mixture of (2E)-1-(4-fluorophenyl)-3-(substituted phenyl)prop-2-en-1-one 3a or 3b (0.0015 mol) and substituted phenyl semicarbazides (0.0015 mol) was heated at reflux in glacial acetic acid for 12 h. The reaction mixture was further concentrated and excess of solvents was removed under vacuum and the reaction mixture was poured onto crushed ice, filtered, dried and washed with water to obtain analogues 4an. The title compounds were further recrystallized from ethanol. Thin-layer chromatography (benzene/acetone, 8:2) was used to monitor the reaction.


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N,3-Bis(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4a)

Yield: 0.513 g (81%); brown solid; m.p. 180–182 °C.

IR (KBr): 3322 (NH), 1685 (C=O), 1522 (C=N), 810 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6):δ = 3.29 (dd, J = 4.1, 13.1 Hz, 1 H, Ha), 3.63 (dd, J = 4.1, 13.1 Hz, 1 H, Hb), 5.19 (t, 1 H, Hc), 7.01–7.62 (m, 8 H, ArH), 7.64 (d J = 8.1 Hz, 2 H, ArH), 8.12 (d, J = 8.1 Hz, 2 H, ArH), 8.26 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.01, 165.21, 158.41, 151.81, 149.61, 146.21, 131.52, 130.81, 129.63, 127.91, 123.21, 120.92, 115.79, 115.61, 61.13, 39.41.

LC-MS: m/z = 423 [M + H]+.

Anal. calcd.: C, 62.56; H, 3.82; N, 13.26; found: C, 62.54; H, 3.85; N, 13.22.


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N-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4b)

Yield: 0.494 g (75%); pale-brown solid; m.p. 178–180 °C.

IR (KBr): 3322 (NH), 1680 (C=O), 1522 (C=N), 810 (C-F), 690 (C-Cl) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.16 (dd, J = 4.1, 13.1 Hz, 1 H, Ha), 3.78 (dd, J = 4.1, 13.1 Hz, 1 H, Hb), 5.19 (t, 1 H, Hc), 7.01–7.21 (m, 4 H, ArH), 7.25 (d J = 8.0 Hz, 2 H, ArH), 7.68 (d, J = 8.1 Hz, 2 H, ArH), 7.74 (d, J = 8.1 Hz, 2 H, ArH), 8.11 (d, J = 8.0 Hz, 2 H, ArH), 8.14 (d, J = 8.1 Hz, 2 H, ArH), 8.36 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.01, 165.19, 151.83, 149.63, 146.29, 134.02, 130.82, 129.61, 129.11, 127.92, 123.09, 120.91, 115.61, 61.31, 39.43.

LC-MS: m/z = 439 [M + H]+.

Anal. calcd.: C, 60.21; H, 3.67; N, 12.77; found: C, 60.24; H, 3.69; N, 12.74.


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N-(4-Bromophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4c)

Yield: 0.536 g (74%); creamy solid; m.p. 150–152 °C.

IR (KBr): 3302 (NH), 1682 (C=O), 1519 (C=N), 811 (C-F), 598 (C-Br) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.19 (dd, J = 4.1, 13.1 Hz, 1 H, Ha), 3.63 (dd, J = 4.1, 13.1 Hz, 1 H, Hb), 5.21 (t, 1 H, Hc), 7.01–7.19 (m, 4 H, ArH), 7.35 (d, J = 8.0 Hz, 2 H, ArH), 7.41 (d, J = 7.9 Hz, 2 H, ArH), 7.54 (d, J = 7.9 Hz, 2 H, ArH), 8.14 (d, J = 8.0 Hz, 2 H, ArH), 8.29 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.91, 165.09, 151.89, 149.61, 146.49, 134.91, 131.91, 130.81, 129.61, 127.93, 123.87, 120.95, 115.69, 61.37, 39.49.

LC-MS: m/z = 484 [M + H]+.

Anal. calcd.: C, 54.67; H, 3.34; N, 11.59; found: C, 54.64; H, 3.37; N, 11.57.


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N-(2-Chlorophenyl)-3-(4-fluorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4d)

Yield: 0.526 g (80%); pale-brown solid; m.p. 112–114 °C.

IR (KBr): 3321 (NH), 1678 (C=O), 1523 (C=N), 812 (C-F), 694 (C-Cl) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.21 (dd, J = 4.3, 11.1 Hz, 1 H, Ha), 3.53 (dd, J = 3.1, 12.1 Hz, 1 H, Hb), 5.23 (t, 1 H, Hc), 6.94–7.52 (m, 8 H, ArH), 7.55 (d, J = 7.9 Hz, 2 H, ArH), 8.14 (d, J = 7.9 Hz, 2 H, ArH), 8.26 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.92, 165.06, 151.83, 149.65, 146.51, 134.95, 130.83, 130.51, 129.64, 129.10, 127.91, 127.11, 125.85, 123.01, 120.91, 115.61, 61.36, 39.43.

LC-MS: m/z = 439 [M + H]+.

Anal. calcd.: C, 60.21; H, 3.67; N, 12.77; found: C, 60.25; H, 3.65; N, 12.75.


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3-(4-Fluorophenyl)-N-(4-methylphenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4e)

Yield: 452 g (72%); pale-brown solid; m.p. 184–186 °C.

IR (KBr): 3319 (NH), 1681 (C=O), 1516 (C=N), 812 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 2.34 (s, 3 H, CH3), 3.19 (dd, J = 3.1, 10.1 Hz, 1 H, Ha), 3.51 (dd, J = 3.2, 11.1 Hz, 1 H, Hb), 5.23 (t, 1 H, Hc), 7.01 (d, J = 7.8 Hz, 2 H, ArH), 7.04–7.22 (m, 4 H, ArH), 7.38 (d, J = 7.9 Hz, 2 H, ArH), 7.52 (d, J = 7.8 Hz, 2 H, ArH), 8.14 (d, J = 7.9 Hz, 2 H, ArH), 8.29 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.90, 165.11, 151.85, 149.61, 146.41, 134.05, 132.93, 130.81, 129.63, 129.39, 127.93, 121.55, 120.95, 115.61, 61.39, 39.45, 21.35.

LC-MS: m/z = 419 [M + H]+.

Anal. calcd.: C, 66.02; H, 4.58; N, 13.39; found: C, 60.05; H, 4.55; N, 13.37.


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3-(4-Fluorophenyl)-N-(4-methoxyphenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4f)

Yield: 0.424 g (65%); pale-brown solid; m.p. 136–138 °C.

IR (KBr): 3309 (NH), 1684 (C=O), 1514 (C=N), 814 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.21 (dd, J = 5.6, 12.1 Hz, 1 H, Ha), 3.53 (dd, J = 5.3, 12.1 Hz, 1 H, Hb), 3.73 (s, 3 H, OCH3), 5.26 (t, 1 H, Hc), 6.77 (d, J = 7.9 Hz, 2 H, ArH), 7.02–7.19 (m, 4 H, ArH), 7.39 (d, J = 8.1 Hz, 2 H, ArH), 7.53 (d, J = 7.9 Hz, 2 H, ArH), 8.14 (d, J = 8.1 Hz, 2 H, ArH), 8.26 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.90, 165.11, 156.33, 151.81, 149.63, 146.40, 130.88, 129.63, 128.21, 127.93, 122.66, 120.91, 115.55, 114.51, 61.31, 55.93, 39.43.

LC-MS: m/z = 435 [M + H]+.

Anal. calcd.: C, 63.59; H, 4.41; N, 12.90; found: C, 63.56; H, 4.45; N, 12.88.


#

3-(4-Fluorophenyl)-N-(2,6-methylphenyl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4g)

Yield: 0.486 g (75%); pale-brown solid; m.p. 190–192 °C.

IR (KBr): 3309 (NH), 1684 (C=O), 1514 (C=N), 814 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 2.21 (s, 6 H, CH3), 3.14 (dd, J = 6.1, 7.8 Hz, 1 H, Ha), 3.75 (dd, J = 6.1, 11.1 Hz, 1 H, Hb), 5.18 (t, 1 H, Hc), 6.84 (s, 1 H, ArH), 6.77–719 (m, 7 H, ArH), 7.39 (d, J = 8.0 Hz, 2 H, ArH), 8.14 (d, J = 8.0 Hz, 2 H, ArH), 8.16 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.29, 165.14, 151.81, 149.63, 146.41, 134.69, 134.20, 130.81, 129.61, 127.91, 126.31, 124.21, 120.93, 115.61, 61.39, 39.46, 15.56.

LC-MS: m/z = 433 [M + H]+.

Anal. calcd.: C, 66.66; H, 4.89; N, 12.96; found: C, 66.63; H, 4.87; N, 12.99.


#

N,3-Bis(4-Fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4h)

Yield: 0.546 g (78%); creamy solid; m.p. 170–172 °C.

IR (KBr): 3319 (NH), 1682 (C=O), 1553 (C=N), 810 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.41 (dd, J = 6.04, 11.2 Hz, 1 H, Ha), 3.58 (dd, J = 6.4, 10.1 Hz, 1 H, Hb), 3.83 (s, 6 H, OCH3), 3.91 (s, 3 H, OCH3), 5.42 (t, 1 H, Hc), 7.23 (s, 2 H, ArH), 7.32–7.62 (m, 8 H, ArH), 8.26 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.91, 165.12, 158.66, 151.83, 150.66, 137.89, 137.21, 131.51, 130.83, 129.63, 123.21, 115.71, 115.60, 104.93, 61.93, 56.56, 56.25, 39.46.

LC-MS: m/z = 468 [M + H]+.

Anal. calcd.: C, 64.23; H, 4.96; N, 8.99; found: C, 64.25; H, 4.99; N, 8.96.


#

N-(4-Chlorophenyl)-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4i)

Yield: 0.537 g (74%); yellow solid; m.p. 164–166 °C.

IR (KBr): 3312 (NH), 1685 (C=O), 1523 (C=N), 812 (C-F), 694 (C-Cl) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.42 (dd, J = 4.0, 11.2 Hz, 1 H, Ha), 3.58 (dd, J = 13.2, 10.0 Hz, 1 H, Hb), 3.85 (s, 6 H, OCH3), 3.93 (s, 3 H, OCH3), 5.41 (t, 1 H, Hc), 7.23 (s, 2 H, ArH), 7.32–7.42 (m, 4 H, ArH), 7.70 (d, J = 15.2 Hz, 2 H, ArH), 7.89 (d, J = 15.6 Hz, 2 H, ArH), 8.26 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.71, 165.02, 151.83, 150.63, 137.89, 137.23, 134.02, 130.81, 129.99, 129.63, 129.11, 123.01, 115.66, 104.33, 61.95, 56.56, 56.24, 39.41.

LC-MS: m/z = 484 [M + H]+.

Anal. calcd.: C, 62.05; H, 4.79; N, 8.68; found: C, 62.05; H, 4.81; N, 8.65.


#

N-(4-Bromophenyl)-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4j)

Yield: 0.515 g (65%); pale-brown solid; m.p. 170–172 °C.

IR (KBr): 3318 (NH), 1680 (C=O), 1543 (C=N), 810 (C-F), 598 (C-Br) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.41 (dd, J = 4.1, 13.1 Hz, 1 H, Ha), 3.61 (dd, J = 4.2, 12.1 Hz, 1 H, Hb), 3.85 (s, 6 H, OCH3), 3.91 (s, 3 H, OCH3), 5.39 (t, 1 H, Hc), 7.22 (s, 2 H, ArH), 7.24–7.39 (m, 4 H, ArH), 7.41 (d, J = 8.0 Hz, 2 H, ArH), 7.53 (d, J = 8.0 Hz, 2 H, ArH), 8.24 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.92, 165.12, 151.89, 150.61, 137.81, 137.21, 134.92, 131.89, 130.89, 129.61, 123.83, 118.71, 115.61, 104.36, 61.92, 56.56, 56.21, 39.43.

LC-MS: m/z = 528 [M + H]+.

Anal. calcd.: C, 56.83; H, 4.39; N, 7.95; found: C, 56.85; H, 4.41; N, 7.92.


#

N-(2-Chlorophenyl)-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4k)

Yield: 0.522 g (72%); pale-yellow solid; m.p. 174–176 °C.

IR (KBr): 3321 (NH), 1680 (C=O), 1551 (C=N), 812 (C-F), 698 (C-Cl) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.34 (dd, J = 3.1, 10.1 Hz, 1 H, Ha), 3.59 (dd, J = 3.1, 10.2 Hz, 1 H, Hb), 3.83 (s, 6 H, OCH3), 3.91 (s, 3 H, OCH3), 5.38 (t, 1 H, Hc), 7.12 (s, 2 H, ArH), 7.14–7.42 (m, 8 H, ArH), 8.22 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d6 ): δ = 166.80, 165.09, 151.81, 150.69, 137.85, 137.21, 134.92, 130.81, 130.51, 129.61, 129.10, 127.11, 125.86, 123.02, 115.61, 104.37, 61.92, 56.55, 56.26, 39.49.

LC-MS: m/z = 484 [M + H]+.

Anal. calcd.: C, 62.05; H, 4.79; N, 8.68; found: C, 62.05; H, 4.81; N, 8.65.


#

N-(4-Methylphenyl)-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4l)

Yield: 0.486 g (70%); pale-yellow solid; m.p. 176–178 °C.

IR (KBr): 3319 (NH), 1688 (C=O), 1549 (C=N), 811 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 2.34 (s, 3 H, CH3), 3.35 (dd, J = 4.3, 12.1 Hz, 1 H, Ha), 3.61 (dd, J = 3.1, 11.1 Hz, 1 H, Ha), 3.85 (s, 6 H, OCH3), 3.92 (s, 3 H, OCH3), 5.33 (t, 1 H, Hc), 6.98 (d, J = 7.9 Hz, 2 H, ArH), 7.13 (s, 2 H, ArH), 7.14–7.25 (m, 4 H, ArH), 7.53 (d, J = 7.9 Hz, 2 H, ArH), 8.23 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.81, 165.09, 151.83, 150.63, 137.82, 134.05, 132.91, 130.81, 130.21, 129.61, 129.30, 121.56, 115.62, 104.32, 61.91, 56.56, 56.22, 39.49, 23.31.

LC-MS: m/z = 463 [M+], 464 [M + H]+.

Anal. calcd.: C, 67.37; H, 5.65; N, 9.07; found: C, 67.35; H, 5.67; N, 9.05.


#

N-(4-Methoxyphenyl)-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4m)

Yield: 0.503 g (70%); pale-yellow solid; m.p. 176–178 °C.

IR (KBr): 3312 (NH), 1682 (C=O), 1523 (C=N), 812 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 3.41 (dd, J = 3.6, 12.8 Hz, 1 H, Ha), 3.62 (dd, J = 2.0, 16.8 Hz, 1 H, Hb), 3.70 (s, 6 H, OCH3), 3.72 (s, 6 H, OCH3), 5.22 (t, 1 H, Hc), 7.23 (s, 2 H, ArH), 7.24–7.42 (m, 4 H, ArH), 7.69 (d, J = 8.0 Hz, 2 H, ArH), 7.88 (d, J = 8.0 Hz, 2 H, ArH), 8.25 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.91, 165.19, 156.31, 151.81, 150.61, 137.81, 130.81, 130.23, 129.61, 128.21, 122.66, 115.61, 114.05, 104.38, 61.97, 56.56, 56.26, 39.41.

LC-MS: m/z = 480 [M + H]+.

Anal. calcd.: C, 65.13; H, 5.47; N, 8.76; found: C, 65.15; H, 5.49; N, 8.73.


#

N-(2,6-Dimethylphenyl)-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (4n)

Yield: 0.537 g (75%); creamy solid; m.p. 182–184 °C.

IR (KBr): 3288 (NH), 1678 (C=O), 1541 (C=N), 812 (C-F) cm–1.

1H NMR (400 MHz, DMSO-d 6): δ = 2.49 (s, 3 H, CH3), 3.56 (dd, J = 11.6, 13.2 Hz, 1 H, Ha), 3.63 (dd, J = 7.2, 7.2 Hz, 1 H, Hb), 3.85 (s, 6 H, OCH3), 3.93 (s, 3 H, OCH3), 5.19 (t, 1 H, Hc), 7.23 (s, 2 H, ArH), 7.32–7.42 (m, 4 H, ArH), 7.43–7.71 (m, 4 H, ArH), 8.25 (s, 1 H, CONH).

13C NMR (100 MHz, DMSO-d 6): δ = 166.91, 165.19, 151.81, 150.61, 137.81, 137.24, 134.61, 134.23, 130.81, 129.61, 126.31, 124.21, 115.63, 104.31, 61.91, 56.56, 56.24, 39.41, 15.51.

LC-MS: m/z = 478 [M + H]+.

Anal. calcd.: C, 67.91; H, 5.91; N, 8.80; found: C, 67.95; H, 5.93; N, 8.78.


#
#

Acknowledgment

The Management of Maharishi Arvind College of Pharmacy is acknowledged for providing the necessary research facilities. The authors are also grateful to Dr. Reddy Lifescience, Hyderabad and Anticancer Drug Screening Facility (ACTREC), Navi Mumbai, India.

  • References

  • 1 Siegel RL. Miller KD. Jemal A. CA: Cancer J. Clin. 2017; 67: 7
  • 2 Nandakumar A. National Cancer Registry Programme. Indian Council for Medical Research, Consolidated Report of the Population Based Cancer Registries 1990-96. New Delhi: Indian Council of Medical Research. 2009;
  • 3 Cancer fact sheet February 2017: http://www.who.int/mediacentre/factsheets/fs297/en/
  • 4 Aydemir N. Bilaloglu R. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2003; 537: 43
  • 5 Dees EC. Rowinsky EK. Noe DA. O’Reilly S. Adjei AA. Elza-Brown K. Donehower RC. Invest. New Drugs 2003; 21: 75
  • 6 Berg SL. Blaney SM. Sullivan J. Bernstein M. Dubowy R. Harris MB. J. Pediatr. Hematol./Oncol. 2000; 22: 506
  • 7 Ramaswamy B. Mrozek E. Kuebler JP. Bekaii-Saab T. Kraut EH. Invest. New Drugs 2011; 29: 347
  • 8 Na JI. Na JY. Choi WY. Lee MC. Park MS. Choi KH. Lee JK. Kim KT. Park JT. Kim HS. Am. J. Transl. Res. 2015; 7: 751
  • 9 Chang LC. Lin HY. Tsai MT. Chou RH. Lee FY. Teng CM. Hsieh MT. Hung HY. Huang LJ. Yu YL. Kyo HC. Br. J. Pharmacol. 2014; 171: 4010
  • 10 George DJ. Clin. Cancer Res. 2007; 13: 753
  • 11 Rini B. Wilding G. Hudes G. Stadler WM. Kim S. Tarazi J. Bycott P. Liau K. Dutcher J. EJC Suppl. 2007; 5: 300
  • 12 Lv PC. Li DD. Li QS. Lu X. Xiao ZP. Zhu HL. Bioorg. Med. Chem. Lett. 2011; 21: 5374
  • 13 Havrylyuk D. Zimenkovsky B. Vasylenko O. Gzella A. Lesyk R. J. Med. Chem. 2012; 55: 8630
  • 14 Qin H. Shang Z. Jantan I. Tan OU. Hussain MA. Sher M. Bukhari SN. A. RSC Adv. 2015; 5: 463330
  • 15 Ahsan MJ. Samy GJ. Habibullah K. Bakht MA. Hassan MZ. Eur. J. Med. Chem. 2011; 46: 5694
  • 16 Ahsan MJ. Samy GJ. Khalilullah H. Moham G. Stables JP. Bioorg. Med. Chem. Lett. 2012; 22: 7029
  • 17 Shamsuzzaman, Khanam H. Dar AM. Siddiqui N. Rehman S. J. Saudi Chem. Soc. 2016; 20: 7
  • 18 Yang B. Yang YS. Yang N. Li G. Zhu HL. Sci. Rep. 2016; 6: 27571
  • 19 Elbayaa RY. Badr MH. Khalil AA. Abdelhadi M. Drug Res. (Stuttgart, Ger.) 2013; 63: 271
  • 20 Ahsan MJ. Samy GJ. Stables JP. Med. Chem. Res. 2013; 22: 2746
  • 21 Gul HI. Yamali C. Sakagami H. Angeli A. Leitans J. Kazaks A. Tars K. Ozgun DO. Supuran CT. Bioorg. Chem. 2018; 77: 411
  • 22 Yakaiah S. Kumar PS. V. Rani PB. Prasad KD. Aparna P. Bioorg. Med. Chem. Lett. 2018; 28: 630
  • 23 Ahsan MJ. Lett. Drug Des. Discovery 2012; 9: 823
  • 24 Vichai V. Kirtikara K. Nat. Protoc. 2006; 1: 1112

  • References

  • 1 Siegel RL. Miller KD. Jemal A. CA: Cancer J. Clin. 2017; 67: 7
  • 2 Nandakumar A. National Cancer Registry Programme. Indian Council for Medical Research, Consolidated Report of the Population Based Cancer Registries 1990-96. New Delhi: Indian Council of Medical Research. 2009;
  • 3 Cancer fact sheet February 2017: http://www.who.int/mediacentre/factsheets/fs297/en/
  • 4 Aydemir N. Bilaloglu R. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2003; 537: 43
  • 5 Dees EC. Rowinsky EK. Noe DA. O’Reilly S. Adjei AA. Elza-Brown K. Donehower RC. Invest. New Drugs 2003; 21: 75
  • 6 Berg SL. Blaney SM. Sullivan J. Bernstein M. Dubowy R. Harris MB. J. Pediatr. Hematol./Oncol. 2000; 22: 506
  • 7 Ramaswamy B. Mrozek E. Kuebler JP. Bekaii-Saab T. Kraut EH. Invest. New Drugs 2011; 29: 347
  • 8 Na JI. Na JY. Choi WY. Lee MC. Park MS. Choi KH. Lee JK. Kim KT. Park JT. Kim HS. Am. J. Transl. Res. 2015; 7: 751
  • 9 Chang LC. Lin HY. Tsai MT. Chou RH. Lee FY. Teng CM. Hsieh MT. Hung HY. Huang LJ. Yu YL. Kyo HC. Br. J. Pharmacol. 2014; 171: 4010
  • 10 George DJ. Clin. Cancer Res. 2007; 13: 753
  • 11 Rini B. Wilding G. Hudes G. Stadler WM. Kim S. Tarazi J. Bycott P. Liau K. Dutcher J. EJC Suppl. 2007; 5: 300
  • 12 Lv PC. Li DD. Li QS. Lu X. Xiao ZP. Zhu HL. Bioorg. Med. Chem. Lett. 2011; 21: 5374
  • 13 Havrylyuk D. Zimenkovsky B. Vasylenko O. Gzella A. Lesyk R. J. Med. Chem. 2012; 55: 8630
  • 14 Qin H. Shang Z. Jantan I. Tan OU. Hussain MA. Sher M. Bukhari SN. A. RSC Adv. 2015; 5: 463330
  • 15 Ahsan MJ. Samy GJ. Habibullah K. Bakht MA. Hassan MZ. Eur. J. Med. Chem. 2011; 46: 5694
  • 16 Ahsan MJ. Samy GJ. Khalilullah H. Moham G. Stables JP. Bioorg. Med. Chem. Lett. 2012; 22: 7029
  • 17 Shamsuzzaman, Khanam H. Dar AM. Siddiqui N. Rehman S. J. Saudi Chem. Soc. 2016; 20: 7
  • 18 Yang B. Yang YS. Yang N. Li G. Zhu HL. Sci. Rep. 2016; 6: 27571
  • 19 Elbayaa RY. Badr MH. Khalil AA. Abdelhadi M. Drug Res. (Stuttgart, Ger.) 2013; 63: 271
  • 20 Ahsan MJ. Samy GJ. Stables JP. Med. Chem. Res. 2013; 22: 2746
  • 21 Gul HI. Yamali C. Sakagami H. Angeli A. Leitans J. Kazaks A. Tars K. Ozgun DO. Supuran CT. Bioorg. Chem. 2018; 77: 411
  • 22 Yakaiah S. Kumar PS. V. Rani PB. Prasad KD. Aparna P. Bioorg. Med. Chem. Lett. 2018; 28: 630
  • 23 Ahsan MJ. Lett. Drug Des. Discovery 2012; 9: 823
  • 24 Vichai V. Kirtikara K. Nat. Protoc. 2006; 1: 1112

Zoom Image
Figure 1 Pyrazoline incorporated compounds and their biological activities
Zoom Image
Scheme 1 Synthetic protocol for the synthesis of analogues 4an
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Figure 2 (a) Growth curve of analogues 4an against (a) MCF-7 and (b) MDA-MB-231
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Figure 3 Images of growth control against MCF-7 cancer cell line
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Figure 4 Images of growth control against MDA-MB-231 cancer cell line
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Figure 5 Structure activity relationship studies on analogues 4an