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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 262-269

Synthesis of 4-phenyl-4,5-dihydropyranopyrazolone derivatives with activated potassium carbonate: Evaluation of anticancer activity on cancer cell lines and apoptosis mechanism


1 Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran

Date of Web Publication30-Oct-2019

Correspondence Address:
Dr. Hadi Adibi
Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah.
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrptps.JRPTPS_82_19

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  Abstract 

A green and efficient one-pot, four-component synthesis of 4-phenyl-4,5-dihydropyranopyrazolone derivatives 5a–5l is described in ethanol-water with activated potassium carbonate and evaluation of anticancer activity on cancer cell lines and apoptosis mechanism is also investigated. This method provides several advantages such as environmental friendliness, shorter reaction time, excellent yields, and simple workup procedure. The in vitro cytotoxic activity of the synthesized compounds was investigated against cancer cell lines (PC-3, MCF-7, and A-2780) in comparison with doxorubicin, a well-known anticancer drug, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric assay. Also apoptosis studies were investigated by caspase-3 and caspase-9 enzymes and mitochondrial membrane potential. Our compounds showed acceptable and moderate cytotoxicity compared with doxorubicin in the studied cell lines. The compounds 5g in PC3 cell line (half maximal inhibitory concentration (IC50= 104 µM)), 5g and 5i in MCF7 cell line (IC50 = 23 and 87 µM, respectively), and 5g–5i in A2780 cell line (IC50 = 60, 50, and 31 µM, respectively) showed the best results close to the control drug doxorubicin. The compound 5h showed significant result in the activation of caspase-3 and caspase-9 enzymes in comparison with the control. Only the 5g in MCF7 cells and the 5g–5i derivatives in A2780 cells caused increased mitochondrial membrane potential compared to the control group.

Keywords: Caspase, cytotoxic activity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, synthesis, pyranopyrazolone


How to cite this article:
Hosseinzadeh L, Mahmoudian N, Ahmadi F, Adibi H. Synthesis of 4-phenyl-4,5-dihydropyranopyrazolone derivatives with activated potassium carbonate: Evaluation of anticancer activity on cancer cell lines and apoptosis mechanism. J Rep Pharma Sci 2019;8:262-9

How to cite this URL:
Hosseinzadeh L, Mahmoudian N, Ahmadi F, Adibi H. Synthesis of 4-phenyl-4,5-dihydropyranopyrazolone derivatives with activated potassium carbonate: Evaluation of anticancer activity on cancer cell lines and apoptosis mechanism. J Rep Pharma Sci [serial online] 2019 [cited 2019 Nov 16];8:262-9. Available from: http://www.jrpsjournal.com/text.asp?2019/8/2/262/269956




  Introduction Top


Cancer is a disease of worldwide importance and its incidence is rising. According to information from the World Health Organization, more than 11 million people are diagnosed with cancer, and also more than 13% of overall deaths are directly caused by cancer every year worldwide.[1] Cancer cells are characterized by unlimited replicative potential, self-sufficiency ingrowth signals and insensitivity to antigrowth signals, sustained angiogenesis, metastasis, and evasion of apoptosis.[2] Apoptosis or programmed cell death is an important phenomenon for remission of damaged cells. Apoptosis can be activated by two major pathways: the extrinsic pathway, in which specific cell death receptors located on the cell surface membrane are activated by specific ligands, and the intrinsic pathway, where primarily mitochondria are involved.[3] Apoptosis plays a vital role in normal embryonic development as well as in adult life, such as elimination of dispensable or excess cells. It has been known that defects in the apoptosis pathways and the ability to evade cell death is one of the hallmarks of cancers, which results in uncontrollable tumor cell growth, as well as tumor resistance to chemotherapeutic agents.[4] Therefore, finding of new therapeutic agents for neoplastic diseases with focus on the apoptosis pathways is one of the top subjects in this area of research. It has been well documented that many of the clinically useful cytotoxic agents induce apoptosis in cancer cells. The proapoptotic chemotherapeutic agents that target tubulin polymerization such as Taxol and vinca alkaloids, including vincristine, vinblastine, and vinorelbine, are among the most potent and commonly prescribed antineoplastic agents. The development of chemoresistance as well as dose-limiting neurologic and bone marrow toxicity, however, has limited the use of tubulin-targeting agents. This clearly highlights the urgent need for novel chemotherapeutic agents for more effective treatment of cancer.[5] Pyranopyrazoles are the fused heterocyclic compounds that show bactericidal,[6] molluscicidal,[7],[8] and analgesic activity,[9] and act as hypoglycemic and anticancer agents.[10],[11] They are also potential inhibitors of human Chk1 kinase.[12] Celecoxib (1), sulfaphenazole (2), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB or mGluR5 Ligand) (3), linazolac (4), mepiprazole (5), and rimonabant (6) are some of the pyrazole-based drugs available today in the market [Figure 1].[13] Owing to their biological significance, there has been considerable interest in developing synthetic methods for the preparation of pyranopyrazole derivatives.[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24]
Figure 1: Structures of pyrazole derivatives used as biologically active compounds

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Green chemistry techniques continue to grow in importance. Alternative processes help to conserve resources and to reduce costs. The replacement of conventional solvents with water, which is harmless to health and is available in large quantities, is an interesting basic approach along these lines.[25],[26],[27] One of the tools used to combine economic aspects with the environmental ones is the multicomponent reaction strategy, the process consists of two or more synthetic steps, which are carried out without isolation of any intermediate, thus reducing time, saving money, energy, and raw materials.[28] As a part of our program aimed at developing new selective and environmentally friendly methodologies for the preparation of fine chemicals and developing anticancer agents,[29] we performed the synthesis of 4-phenyl-4,5-dihydropyranopyrazolone derivatives 5a–5l through a four-component reaction with activated potassium carbonate as catalyst [Scheme 1]. Then, the in vitro cytotoxic activity of the synthesized compounds 5a–5l was investigated against PC3, MCF7, and A2780 cancer cell lines in comparison with doxorubicin, a well-known anticancer drug, using MTT colorimetric assay. Considering the importance of apoptotic cell death as a key factor of a potential antitumor drug, in the next set of experiments, the apoptotic potentials of the most potent compounds were investigated.
Scheme 1: Reagents and conditions for the synthesis of pyranopyrazolones 5a–5l

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  Materials and Methods Top


Chemistry

All starting materials, reagents, and solvents were purchased from Merck and Sigma-Aldrich Chemical Companies, Kermanshah University of Medical Sciences, Kermanshah, Iran. All yields referred to isolated yield. The structure of compounds was characterized by Infrared (IR), Nuclear Magnetic Resonance (1H NMR) spectra, and mass spectrometry (MS). Merck silica gel 60 F254 plates were used for Thin layer chromatography (TLC). 1H-NMR spectra were recorded using a Bruker 500 spectrometer, and chemical shifts were expressed as (ppm) with tetramethylsilane as internal standard using Deuterated chloroform (CDCl3) or dimethyl sulfoxide (DMSO)-d6 as solvent. The IR spectra were obtained on a Shimadzu 470 spectrophotometer (potassium bromide disks). The melting point was taken on a Kofler hot stage apparatus and was uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer at 70eV, Kermanshah University of Medical Sciences, Kermanshah, Iran.

General procedure for the synthesis of pyranopyrazolones 5a–5l with activated potassium carbonate as the catalyst in water-ethanol

Pyranopyrazole derivatives 5a–5l were synthesized by the one-pot four-component condensation of an aromatic aldehyde (1.0 mmol, 2a–2l), methyl acetoacetate (1.0 mmol, 4), hydrazine hydrate (1.0 mmol, 3), Meldrum’s acid (1.0 mmol, 1), and activated potassium carbonate (10 mol%) as the catalyst, in water-ethanol (5mL, 1:1) for a designated time listed in [Table 1] (120–240min). After completion of the reaction followed by TLC analysis, the reaction mixture was cooled and the precipitated solid was filtered and washed several times with cool water. The obtained products (5a–5l) were crystallized in appropriate solvent (ethanol) and the corresponding pyranopyrazolones 5a–5l were obtained in 59%–85% yields [Scheme 1], [Table 1].[29] The spectral data of the compounds 5a–5j are as following:
Table 1: Synthesis of pyranopyrazolones using activated potassium carbonate in water-ethanol and the corresponding IC50 (μM) values against different carcinoma cell lines

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4-(3-Methoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5a)

Chemical formula: C14H14N2O3; molecular weight: 258.27 (g/mol); yield: 66%; melting point: 105°C; Fourier-transform infrared spectroscopy (FT-IR) (KBr, cm−1): [INLINE 1] 3448 (stretch NH), 3012 (stretch CH, aromatic), 2935 (stretch CH, aliphatic), 1604 (stretch C=O ring), 1573 (stretch C=C), 1477 (stretch C=C), 1435 (binding CH); 1265 (stretch C–O), 1045 (stretch C–O), 786 (binding CH, OOP), 690 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 11.5 (s, 1H, NH), 7.50 (t, 2H, J = 8.5 Hz, H5aromatic), 7.05 (s, 1H, H2aromatic), 6.8 (m, 2H, H4,6aromatic), 4.70 (t, 1H, J = 7.5 Hz, CH), 3.86 (s, 3H, OCH3), 2.91 (m, 2H, CH2), 2.05 (s, 3H, CH3); MS (m/z, %): 258 (M), 217 (M-41, 100), 161 (M-97, 71.7), 135 (M-123, 76.9), 91 (M-167, 27.3), 71 (M-181,48,7).

4-(4-Methoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5b)

Chemical formula: C14H14N2O3; molecular weight: 258.27 (g/mol); yield: 65%; melting point: 145°C; FT-IR (KBr, cm−1): [INLINE 2] 3367 (stretch NH), 2931 (stretch CH, aliphatic), 1600 (stretch C=O ring), 1508 (stretch C=C), 1462 (stretch C=C), 1369 (binding CH); 1249 (stretch C–O), 1026 (stretch C–O), 833 (binding CH, OOP), 675 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 11 (s, 1H, NH), 7.80 (d, 2H, J = 10 Hz, H2,6aromatic), 7.05 (d, 2H, J = 10 Hz, H3,5aromatic), 4.10 (t, 1H, J = 7.5 Hz, CH), 3.68 (s, 3H, OCH3); 2.90 (s, 3H, CH3), 2.03 (m, 2H, CH2); MS(m/z, %): 258 (M), 217 (M-41, 100), 185 (M-73, 31.3), 161 (M-97, 41.5), 91 (M-167, 16.9), 77 (M-181, 17.7).

4-(2,3-Dimethoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5c)

Chemical formula: C15H16N2O4; molecular weight: 288.30 (g/mol); yield: 60%; melting point: 150°C; FT-IR (KBr, cm−1): [INLINE 3] 3448 (stretch NH), 3047 (stretch CH, aromatic), 2943 (stretch CH, aliphatic), 1624 (stretch C=O ring), 1593 (stretch C=C), 1489 (stretch C=C), 1438 (binding CH); 1265 (stretch C–O), 1087 (stretch C–O), 821 (binding CH, OOP), 678 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 8.67 (s, 1H, NH), 7.85 (d, 1H, J = 7.9 Hz, H4aromatic), 7.55 (d, 1H, J = 8.2 Hz, H6aromatic), 4.65 (t, 1H, J = 7.5 Hz, CH), 3.54 (s, 3H, OCH3); 3.30 (s, 3H, OCH3), 3.28 (s, 3H, CH3), 2.14 (m, 2H, CH2); MS (m/z, %): 288 (M), 222 (M-66, 100), 204 (M-84, 100), 194 (M-94, 50.8), 94 (M-194, 17.7), 77 (M-211, 11.8).

4-(2-Hydroxy-4-methoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5d)

Chemical formula: C14H14N2O4; molecular weight: 274.27 (g/mol); yield: 80%; melting point: 164°C; FT-IR (KBr, cm−1): [INLINE 4] 3417 (stretch NH), 3066 (stretch OH), 2939 (stretch CH, aliphatic), 1612 (stretch C=O ring), 1566 (stretch C=C), 1462 (stretch C=C), 1431 (binding CH); 1257 (stretch C–O), 1018 (stretch C–O), 802 (binding CH, OOP), 640 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 11.5 (s, 1H, NH), 7.77 (d, 1H, J = 8.5 Hz, H6aromatic), 6.95 (d, 1H, J = 8.5 Hz, H5aromatic), 6.77 (s, 1H, H3aromatic), 6.47 (s, 1H, OH), 4.70 (t, 1H, J = 7.5 Hz, CH), 3.84 (s, 3H, OCH3), 2.68 (m, 2H, CH2), 1.95 (s, 3H, CH3); MS (m/z, %): 274 (M), 271 (M-3, 27), 171 (M-103, 38.9), 135 (M-139, 96), 127 (M-147, 51.5), 92 (M-182, 70.3), 77 (M-197, 100).

4-(2,4-Dihydroxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5e)

Chemical formula: C13H12N2O4; molecular weight: 260.25 (g/mol); yield: 85%; melting point: 270°C (decomposition); FT-IR (KBr, cm−1): [INLINE 5] 3514 (stretch NH), 3468 (stretch OH), 3217 (stretch OH), 1616 (stretch C=O ring), 1558 (stretch C=C), 1504 (stretch C=C), 1454 (binding CH); 1257 (stretch C–O), 848 (binding CH, OOP), 648 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 11.36 (s, 1H, NH), 8.71 (s, 1H, H3aromatic), 7.69 (d, 1H, J = 10 Hz, H6aromatic), 7.36 (d, 1H, J = 10 Hz, H5aromatic), 6.70 (s, 1H, OH), 6.29 (s, 1H, OH), 4.55 (t, 1H, J = 7.5 Hz, CH), 2.73 (m, 2H, CH2), 2.05 (s, 3H, CH3); MS: (m/z, %): 260 (M), 255 (M-5, 51.6), 206 (M-54, 76.2), 162 (M-98, 100), 94 (M-166, 15.2), 76 (M-184, 12.7).

4-(2,4-Dimethoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5f)

Chemical formula: C15H16N2O4; molecular weight: 288.30 (g/mol); yield: 68%; melting point: 201°C; FT-IR (KBr, cm−1): [INLINE 6] 3443 (stretch NH), 3008 (stretch CH, aromatic), 2943 (stretch CH, aliphatic), 1604 (stretch C=O ring), 1577 (stretch C=C), 1500 (stretch C=C), 1419 (binding CH); 1269 (stretch C–O), 1026 (stretch C–O), 829 (binding CH, OOP), 678 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 8.77 (s, 1H, NH), 7.85 (s, 1H, H3aromatic), 6.60 (m, 2H, H5.6Aromatic), 4.60 (t, 1H, J = 7.0 Hz, CH), 3.82 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 2.77 (m, 2H, CH2), 2.11 (s, 3H, CH3); MS (m/z, %): 288 (M), 222 (M-66, 100), 194 (M-94, 50), 94 (M-194, 17), 77 (M-211, 11).

4-(2,5-Dimethoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5g)

Chemical formula: C15H16N2O4; molecular weight: 288.30 (g/mol); yield: 74%; melting point: 145°C; FT-IR (KBr, cm−1): [INLINE 7] 3444 (stretch NH), 3001 (stretch CH, aromatic), 2943 (stretch CH, aliphatic), 1620 (stretch C=O ring), 1581 (stretch C=C), 1492 (stretch C=C), 1423 (binding CH), 1261 (stretch C–O), 1045 (stretch C–O), 813 (binding CH, OOP), 667 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 10.77 (s, 1H, NH), 8.86 (s, 1H, H6aromatic), 7.44 (d, 1H, J = 7.2 Hz, H4aromatic), 7.05 (s, 1H, J = 7.2 Hz, H3Aromatic), 3.79 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 2.28 (m, 2H, CH2), 1.95 (s, 3H, CH3); MS (m/z, %): 288 (M), 222 (M-66, 100), 204 (M-84, 80), 194 (M-94, 51), 94 (M-194, 18), 77 (M-211, 12).

4-(3,4-Dimethoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5h)

Chemical formula: C15H16N2O4; molecular weight: 288.30 (g/mol); yield: 75%; melting point: 175°C; FT-IR (KBr, cm−1): [INLINE 8] 3429 (stretch NH), 2958 (stretch CH, aliphatic), 1624 (stretch C=O ring), 1581 (stretch C=C), 1462 (binding CH); 1261 (stretch C–O), 1141 (stretch C–O), 813 (binding CH, OOP), 678 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 8.57 (s, 1H, NH), 8.23 (s, 1H, H2aromatic), 7.30 (d, 1H, J = 6.5 Hz, H6aromatic), 7.01 (d, 1H, J = 7.00 Hz, H5aromatic), 4.50 (t, 1H, J = 7.1 Hz, CH), 3.78 (s, 6H, 2(OCH3), 2.93 (m, 2H, CH2), 2.12 (s, 3H, CH3); MS (m/z, %): 288 (M), 286 (M-2, 57.6), 247 (M-41, 29.6), 191 (M-97, 100), 164 (M-124, 34.7), 92 (M-196, 33), 77 (M-211, 43.2).

4-(3,5-Dimethoxyphenyl)-3-methyl-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5i)

Chemical formula: C15H16N2O4; molecular weight: 288.30 (g/mol); yield: 70%; melting point: 140°C; FT-IR (KBr, cm−1): [INLINE 9] 3448 (stretch NH), 3005 (stretch CH, aromatic), 2947 (stretch CH, aliphatic), 1681 (stretch C=O ring), 1597 (stretch C=C), 1454 (binding CH); 1292 (stretch C–O), 1064 (stretch C–O), 844 (binding CH, OOP), 678 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 8.59 (s, 1H, NH), 7.00 (s, 2H, H2.6aromatic), 6.61 (s, 1H, H4aromatic), 4.70 (t, 1H, J = 7 Hz, CH), 3.75 (s, 6H, 2(OCH3), 2.99 (m, 2H, CH2), 2.1 (s, 3H, CH3); MS:(m/z, %): 288 (M), 269 (M-19, 25.4), 285 (M-3, 16.94), 205 (M-83, 70.3), 191 (M-97, 100), 164 (M-124, 72.8), 92 (M-196, 26.2), 77 (M-211, 47.4).

3-Methyl-4-(2, 3, 4-trihydroxyphenyl)-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5j)

Chemical formula: C13H12N2O5; molecular weight: 276.24 (g/mol); yield: 80%; melting point: 280°C; FT-IR (KBr, cm−1): [INLINE 10] 3414 (stretch NH), 3271 (stretch OH), 3078 (stretch CH, aromatic), 2924 (stretch CH, aliphatic), 1612 (stretch C=O ring), 1585 (stretch C=C), 1496 (stretch C=C), 1411 (binding CH); 1296 (stretch C–O), 1056 (stretch C–O), 821 (binding CH, OOP), 663 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 11.01 (s, 1H, NH), 8.60 (s, 3H, OH), 7.21 (d, 1H, J = 8.5 Hz, H5aromatic), 6.81 (d, 1H, J = 8.5 Hz, H6aromatic), 4.55 (t, 1H, J = 7.1 Hz, CH), 3.32 (m, 2H, CH2), 2.46 (s, 3H, CH3); MS (m/z, %): 276 (M), 248 (M-28, 100), 231 (M-45, 42.3), 105 (M-171, 98.3), 77 (M-199, 57.6).

3-Methyl-4-(2, 3, 4-trimethoxyphenyl)-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5k)

Chemical formula: C16H18N2O5; molecular weight: 318.32 (g/mol); yield: 60%; melting point: 186°C; FT-IR (KBr, cm−1): [INLINE 11] 3448 (stretch NH), 3016 (stretch CH, aromatic), 2939 (stretch CH, aliphatic), 1612 (stretch C=O ring), 1589 (stretch C=C), 1492 (stretch C=C), 1411 (binding CH); 1288 (stretch C–O), 1091 (stretch C–O), 810 (binding CH, OOP), 671 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 8.71 (s, 1H, NH), 7.70 (d, 1H, J = 7 Hz, H6aromatic), 6.89 (d, 1H, J = 10 Hz, H5aromatic), 3.84 (s, 6H, 2(OCH3)), 4.68 (t, 1H, J = 7.2 Hz, CH), 3.74 (s, 3H, OCH3), 2.92 (m, 2H, CH2), 2.16 (s, 3H, CH3); MS (m/z, %): 318 (M), 311 (M-7, 11.8), 210 (M-108, 31.3), 179 (M-139, 100), 151 (M-167, 91.5), 94 (M-224, 22.8), 77 (M-241, 44.9).

3-Methyl-4-(3, 4, 5-trimethoxyphenyl)-4,5-dihydropyrano [2,3-c]pyrazol-6(2H)-one (5l)

Chemical formula: C16H18N2O5; molecular weight: 318.32 (g/mol); yield: 59%; melting point: 184°C; FT-IR (KBr, cm−1): [INLINE 12] 3437 (stretch NH), 2935 (stretch CH, aliphatic), 1620 (stretch C=O ring), 1577 (stretch C=C), 1504 (stretch C=C), 1415 (binding CH); 1234 (stretch C–O), 813 (binding CH, OOP), 621 (binding NH, OOP); 1H-NMR (DMSO-d6, 500 MHz): δ (ppm) 8.61 (s, 1H, NH), 7.17 (s, 2H, H2.6aromatic), 4.58 (t, 1H, J = 7.5 Hz, CH), 3.80 (s, 6H, 2(OCH3)), 3.69 (s, 3H, OCH3), 2.82 (m, 2H, CH2), 2.30 (s, 3H, CH3); MS:(m/z, %): 318 (M), 315 (M-3, 23.7), 299 (M-19, 36.4), 221 (M-97, 100), 194 (M-124, 72.8), 91 (M-227, 54.2).

Cell culture conditions

PC3, MCF7, and A2780 cell lines were purchased from Pasteur Institute of Iran, Tehran, Iran. Cell lines were grown and maintained in a humidified incubator at 37°C with 5% CO2 atmosphere. Cells were cultured in DMEM-F12 (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% (V/V) heat inactivated fetal bovine serum, and antibiotics (100 IU/mL penicillin and 100 µL/mL streptomycin) at 37°C in 95% CO2 humidified incubator. Doxorubicin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), penicillin, and streptomycin were purchased from Sigma-Aldrich. The test compounds were dissolved in DMSO, diluted with media, and stored as the stock solutions with a concentration of 1.0 mg/mL at –20°C (the concentration of DMSO was less than 1%).[30],[31],[32]

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cytotoxicity assay

Cytotoxic effects of the compounds 5a–5l were studied in MCF7, PC3, and A2780 cell lines evaluated by MTT assay. Exponentially growing mammalian cells (on 96-well plates) were exposed for 24h to different concentrations of compounds. Control cells were kept in medium containing 0.1% DMSO. After 24h of incubation, the medium was removed and 0.1 mg/well of MTT was added to the cells, and the plates were further incubated for 3h at 37°C. The formazan crystals were solubilized in 0.1mL of DMSO and the optical density (OD570) was measured using a microplate reader (BioTek Instruments, Kermanshah University of Medical Sciences, Kermanshah, Iran). Half maximal inhibitory concentration (IC50) values were calculated by plotting the log10 of percent cell viability versus drug concentrations. After 24h, the cells were washed, trypsinized, stained by trypan blue (0.4%), and the number of viable and dead cells was scored.[30],[31],[32]

Measurement of mitochondrial membrane potential

In this study, mitochondrial membrane potential (MMP) was evaluated by rhodamine 123 fluorescent dye. Depolarization of MMP during cell apoptosis results in the loss of rhodamine 123 from the mitochondria and a decrease in intracellular fluorescence intensity. After treatment, cells were incubated with rhodamine 123 for 30min at 37°C. The florescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 520 nm using a florescence microplate reader (BioTek, H1M).[30],[31],[32]

Determination of caspase-3 and 9 activities

The caspase-3 and 9 activities were measured by commercial caspase assay kit (Sigma-Aldrich), according to the manufacturer’s protocol. The kit is based on the hydrolysis of the peptide substrate Ac-DEVD-pNA by caspase-3, AcDEVD-pNA (for caspase-3), and Ac-LEHD-pNA (for caspase-9), resulting in the release of the p-nitroaniline (pNA) moiety. Briefly, 1 × 106 cells were collected and lysed with 50 μL of chilled lysis buffer and incubated on ice for 10min. Cell lysates were centrifuged at maximum speed for 5min at 4°C. Then, 10 μL of cell lysate was combined with an equal amount of substrate reaction buffer containing caspase-3 and caspase-9 colorimetric substrates. After 2-h incubation at 37°C, the pNA light emission was quantified using a microplate reader at 405 nm (BioTek, H1M). Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allowed determination of the fold increase in caspase-3 and caspase-9 activities. The protein content was determined by the Bradford method using the bovine serum albumin as a standard.[30],[31],[32]


  Results and Discussion Top


Chemistry

Although the reported methods for the synthesis of pyranopyrazolone derivatives are effective, they are confronted with certain drawbacks of environment compatibility by the use of toxic and expensive catalysts. In this approach, we used water-ethanol instead of large amounts of organic solvents, workup was simplified, and the reaction times were considerably decreased. In this reaction, the one-pot synthesis was optimized by a mixture of methyl acetoacetate (4) (1.0 mmol), hydrazine hydrate (3) (1.0 mmol), Meldrum’s acid (1) (1.0 mmol), and aromatic aldehydes (2a–2l) (1.0 mmol) in the presence of activated potassium carbonate (10 mol%) in water-ethanol (5mL, 1:1) and was stirred at reflux for the time listed in [Table 1]. The experimental procedure is very simple and convenient, and under the reaction conditions used, it can tolerate a variety of functional groups. Simply by adding catalyst to a mixture of reactants, rapid and convenient condensation is achieved at reflux in a mixture of water-ethanol. After the completion of the reaction (monitored by TLC), the residue was filtered and washed with cooled water, then it was purified by recrystallization from ethanol to produce the desired solid and it was confirmed by physical and spectral data.

Cytotoxic and apoptosis mechanism

The in vitro cytotoxic activity of pyranopyrazolone derivatives 5a–5l was evaluated by MTT assay on PC3, MCF7, and A2780 cell lines [Table 1]. The dose–effect curves were generated and the sensitivity to pyranopyrazolone derivatives was expressed as a drug concentration that caused 50% growth inhibition. Pyranopyrazolone derivatives efficiently inhibited all cell growth tested in a dose-dependent manner. The data for doxorubicin as a positive control were included for comparison. The most potent of the compounds was 5g against PC3 cell line (IC50 = 104 µM), 5f, 5g, and 5i against MCF7 cell line (IC50 = 101, 87, and 23 µM, respectively), 5e, 5g, 5h, and 5i against A2780 cell line (IC50 = 75, 60, 50, and 31 µM, respectively), which seems to be the best ones relative to the control drug. It seems electron-donating substituents (methoxy and hydroxy) are effective on the potency of the test compounds so that the best results were obtained for these groups. All of the compounds 5a–5l have less cytotoxic activity compared to the control drug. The compounds 5g–5i proved to be outstandingly potent cytotoxic agents, especially against A2780 and MCF7 cells. Therefore, in subsequent experiments, 5g–5i were adopted for the identification of mechanisms of action on A2780 and MCF7 cell lines.

The apoptosis induction capacity instead of necrotic induction is considered to be the important factor of a potential anticancer drug. Therefore, the mechanism involved in cytotoxic effects of selected compounds was evaluated using well-characterized apoptosis markers. Activation of caspase cascade is critical in the initiation of apoptosis in various biological systems. A member of this family, caspase-3 and caspase-9 enzymes, has been identified as being a key mediator of apoptosis. Our results showed that 24-h treatment with IC50 concentration of 5h increased caspase-3 and caspase-9 activation in A2780 cell line [Figure 2]. In addition, the compound 5g increased caspase-9 activity significantly in MCF7 cell line [Figure 3].
Figure 2: The effect of the compounds 5g–5i on mitochondrial membrane potential in A2780 cell line. Data are expressed as the mean ± standard error of mean (SEM) of three separate experiments (n = 3). *** = p < 0.001

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Figure 3: The effect of the compounds 5g–5i on caspase-3 and caspase-9 activity in A2780 cell line.

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Mitochondria are double-membrane-bound organelles that play significant roles in activating apoptosis through intrinsic pathway in mammalian cell. During apoptosis, the MMP decreases. A decrease in MMP leads to matrix condensation and the release of cytochrome c from the mitochondrial intermembrane space. The release of cytochrome c triggers the activation of caspase-9, the apical proteases in intrinsic pathway of apoptosis, which goes on to activate the effector caspases. The result indicated that 5g was able to significantly decrease MMP and also increase caspase-9 activity in MCF7 cell line, thus implying that mitochondrial pathway of apoptosis is involved in apoptotic induction of 5g [Figure 4]. In the A2780 cells, the compound 5h could decrease MMP significantly [Figure 5]. In addition, this compound was able to increase caspase-9 activity significantly in A2780 cells, indicating that 5h induces apoptosis via mitochondrial pathway.
Figure 4: The effect of the compounds 5g and 5i on caspase-3 and caspase-9 activity in MCF7 cell line.

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,
Figure 5: The effect of the compounds 5g and 5i on mitochondrial membrane potential in MCF7 cell line. Data are expressed as the mean ± standard error of mean (SEM) of three separate experiments (n = 3). * = p < 0.05

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  Conclusion Top


In conclusion, we have been able to introduce an efficient and environmentally friendly approach for the synthesis of pyranopyrazolones via a four-component condensation, using activated potassium carbonate as a solid base catalyst in heterogeneous conditions. The reactions are characterized by noncorrosiveness, safety, low cost and waste, ease of separation, high yields, and short reaction times. Furthermore, cytotoxic activity of the test compounds was evaluated against a panel of cancer cell lines in vitro comparable to doxorubicin. Some of the pyranopyrazolones showed good tumor-specific cytotoxicity, indicating a new drug candidate for cancer chemotherapy.

Acknowledgement

We gratefully acknowledge Vice Chancellor for Research and Technology, Kermanshah University of Medical Sciences for the financial support (Grant No. 93325). This work was performed in partial fulfillment of the requirements for PharmD of Navid Mahmoudian, in the Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Financial support and sponsorship

This work was financially supported by Kermanshah University of Medical Sciences (Grant No. 93325).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Anand P, Kunnumakkara AB, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan ST, et al. Cancer is a preventable disease that requires major lifestyle changes. Pharm Res 2008;25:2097-116.  Back to cited text no. 1
    
2.
Hanahan D, Weinberg RA The hallmarks of cancer. Cell 2000;100:57-70.  Back to cited text no. 2
    
3.
Zhang HZ, Kasibhatla S, Wang Y, Herich J, Guastella J, Tseng B, et al. Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay. Bioorg Med Chem 2004;12:309-17.  Back to cited text no. 3
    
4.
Zhang HZ, Drewe J, Tseng B, Kasibhatla S, Cai SX Discovery and SAR of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based HTS assay. Bioorg Med Chem 2004;12:3649-55.  Back to cited text no. 4
    
5.
Broxterman HJ, Georgopapadakou NH Anticancer therapeutics: “Addictive” targets, multi-targeted drugs, new drug combinations. Drug Resist Updat 2005;8:183-97.  Back to cited text no. 5
    
6.
Nasr MN, Gineinah MM Pyrido[2, 3-d]pyrimidines and pyrimido[5’,4’:5, 6]pyrido[2,3-d]pyrimidines as new antiviral agents: Synthesis and biological activity. Arch Pharm (Weinheim) 2002;335:289-95.  Back to cited text no. 6
    
7.
Abdelrazek FM, Metz P, Metwally NH, El-Mahrouky SF Synthesis and molluscicidal activity of new cinnoline and pyrano[2,3-c]pyrazole derivatives. Arch Pharm (Weinheim) 2006;339:456-60.  Back to cited text no. 7
    
8.
Abdelrazek FM, Metz P, Kataeva O, Jäger A, El-Mahrouky SF Synthesis and molluscicidal activity of new chromene and pyrano[2,3-c]pyrazole derivatives. Arch Pharm (Weinheim) 2007;340:543-8.  Back to cited text no. 8
    
9.
Kuo SC, Huang LJ, Nakamura H Studies on heterocyclic compounds. 6. Synthesis and analgesic and anti-inflammatory activities of 3,4-dimethylpyrano[2,3-c]pyrazol-6-one derivatives. J Med Chem 1984;27:539-44.  Back to cited text no. 9
    
10.
Nadia MR, Nahed YK, Fahmyb A, El-Sayeda AAF Facile synthesis of fused nitrogen containing heterocycles as anticancer agents. Pharma Chem 2010;2:400-6.  Back to cited text no. 10
    
11.
Wang JL, Liu D, Zhang ZJ, Shan S, Han X, Srinivasula SM, et al. Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci U S A 2000;97:7124-9.  Back to cited text no. 11
    
12.
Foloppe N, Fisher LM, Howes R, Potter A, Robertson AG, Surgenor AE Identification of chemically diverse Chk1 inhibitors by receptor-based virtual screening. Bioorg Med Chem 2006;14:4792-802.  Back to cited text no. 12
    
13.
El-Arab EE, Synthesis and cytotoxicity of novel pyrazole derivatives derived from 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one. Egypt J Chem 2015;58:741-53.  Back to cited text no. 13
    
14.
Kanagaraj K, Pitchumani K Solvent-free multicomponent synthesis of pyranopyrazoles: Per-6-amino-β-cyclodextrin as a remarkable catalyst and host. Tetrahedron Lett 2010;51:3312.  Back to cited text no. 14
    
15.
Shaabani A, Sarvary A, Rezayan AH, Keshipour S Synthesis of fully substituted pyrano[2,3-c]pyrazole derivatives via a multicomponent reaction of isocyanides. Tetrahedron 2009;65:3492.  Back to cited text no. 15
    
16.
Litvinov YM, Shestopalov AA, Rodinovskaya LA, Shestopalov AM New convenient four-component synthesis of 6-amino-2,4-dihydropyrano[2,3-c]pyrazol-5-carbonitriles and one-pot synthesis of 6’-aminospiro[(3H)-indol-3,4’-pyrano[2,3-c]pyrazol]-(1H)-2-on-5’-carbonitriles. J Comb Chem 2009;11:914-9.  Back to cited text no. 16
    
17.
Mecadon H, Rohman MDR, Kharbangar I, Laloo BM, Kharkongor I, Rajbangshi M, et al. L-Proline as an efficient catalyst for the multi-component synthesis of 6-amino-4-alkyl/aryl-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles in water. Tetrahedron Lett 2011;52:3228.  Back to cited text no. 17
    
18.
Dyachenko VD, Rusanov EB Novel approaches to synthesis of 4-alkyl-6-amino-5-cyano-3-methyl(propyl,phenyl)-2H,4H-pyrano[2,3-c]pyrazoles. Chem Heterocycl Compd 2004;40:231.  Back to cited text no. 18
    
19.
Al-Matar HM, Khalil KD, Adam AY, Elnagdi MH Green one pot solvent-free synthesis of pyrano[2,3-c]-pyrazoles and pyrazolo[1,5-a]pyrimidines. Molecules 2010;15:6619-29.  Back to cited text no. 19
    
20.
Litvinov YM, Rodinovskaya LA, Shestopalov AM A new convenient four-component synthesis of 6-amino-2H, 4H-pyrano[2,3-c]pyrazole-5-carbonitriles and one-pot synthesis of 6′-amino-5-cyano-1,2-dihydrospiro-[(3H)-indole-3,4′-(4′H)-pyrano[2,3-c]pyrazol]-2-ones. Russ Chem Bull Int Ed 2009;58:2362.  Back to cited text no. 20
    
21.
Vasuki G, Kumaravel K Rapid four-component reactions in water: Synthesis of pyranopyrazoles. Tetrahedron Lett 2008;49:5636.  Back to cited text no. 21
    
22.
Shestopalov AM, Emeliyanova YM, Shestopalov AA, Rodinovskaya LA, Niazimbetova ZI, Evans DH Cross-condensation of derivatives of cyanoacetic acid and carbonyl compounds. Part 1: Single-stage synthesis of 1′-substituted 6-amino-spiro-4-(piperidine-4′)-2H,4H-pyrano[2,3-c]pyrazole-5-carbonitriles. Tetrahedron 2003;59:7491.  Back to cited text no. 22
    
23.
Gogoi S, Zhao CG Organocatalyzed enantioselective synthesis of 6-amino-5-cyanodihydropyrano[2,3-c]pyrazoles. Tetrahedron Lett 2009;50:2252-5.  Back to cited text no. 23
    
24.
Siddekha A, Nizam A, Pasha MA An efficient and simple approach for the synthesis of pyranopyrazoles using imidazole (catalytic) in aqueous medium, and the vibrational spectroscopic studies on 6-amino-4-(4’-methoxyphenyl)-5-cyano-3-methyl-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazole using density functional theory. Spectrochim Acta A Mol Biomol Spectrosc 2011;81:431-40.  Back to cited text no. 24
    
25.
Li CJ, Chan TH Comprehensive Organic Reactions in Aqueous Media. 2nd edn,Weinheim: John Wiley, 2007.  Back to cited text no. 25
    
26.
Chanda A, Fokin VV Organic synthesis “on water.” Chem Rev 2009;109:725-48.  Back to cited text no. 26
    
27.
Breslow R Hydrophobic effects on simple organic reactions in water. Acc Chem Res 1991;24:159.  Back to cited text no. 27
    
28.
Domling A, Herdtweck E, Ugi I Multi-component reactions V: The seven-component reaction. Acta Chem Scand 1998;52:107.  Back to cited text no. 28
    
29.
Adibi H, Hosseinzadeh L, Farhadi S, Ahmadi F Synthesis and cytotoxic evaluation of 6-amino-4-aryl-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-carbonitrile derivatives using borax with potential anticancer effects. J Rep Pharm Sci 2013;2: 27-35.  Back to cited text no. 29
    
30.
Adibi H, Majnooni MB, Mostafaie A, Mansouri K, Mohammadi M Synthesis, and in-vitro cytotoxicity studies of a series of triazene derivatives on human cancer cell lines. Iranian J Pharm Res 2013;12:695-703.  Back to cited text no. 30
    
31.
Rajabalian S, Foroumadi A, Shafiee A, Emami S Functionalized N-(2-oxyiminoethyl) piperazinyl quinolones as new cytotoxic agents. J Pharm Pharm Sci 2007;10:153-8.  Back to cited text no. 31
    
32.
Hosseinzadeh L, Behravan J, Mosaffa F, Bahrami G, Bahrami A, Karimi G Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species. Food Chem Toxicol 2011;49:1102-9.  Back to cited text no. 32
    


    Figures

  [Figure 1], [Scheme 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]



 

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