Synthesis, characterization, antiamoebic activity and cytotoxicity of new pyrazolo[3, 4-d]pyrimidine-6-one derivatives

Abstract

A new series of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a–2j) were prepared by using the Biginelli multicomponent cyclocondensation of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (1a), different aromatic aldehydes, and urea with a catalytic amount of HCl at reflux temperature. These compounds were characterized by IR, ¹H NMR, ¹³C NMR, and Mass spectral data. In vitro antiamoebic activity was performed against HM1:IMSS strain of Entamoeba histolytica. The results showed that the compounds 2b, 2i, and 2j with IC₅₀ values of 0.37 µM, 0.04 µM, and 0.06 µM, respectively, exhibited better antiamoebic activity than the standard drug metronidazole (IC₅₀ = 1.33 µM). The toxicological studies of these compounds on human breast cancer MCF-7 cell line showed that the compounds 2b, 2i, and 2j exhibited >80% viability at the concentration range of 1.56–50 µM.

Keywords::

• 3-Methyl-1-phenyl-1H-pyrazol-5(4H)-one
• Pyrazolo[3;4-d]pyrimidine-6-one derivatives
• Entamoeba histolytica
• MTT assay

Introduction

Parasitic infections constitute one of the most widespread human health problems, and most of them occur through contaminated food or water. The human intestine is a major target of these ingested pathogenic microorganisms, resulting in severe infections, of which amoebiasis (potential life-threatening dysentery) is one. Amoebiasis is the second leading cause of death from a protozoan parasite, Entamoeba histolytica, and remains as a major health problem in Third World countries (1). It affects more than 10% of the world’s population, and untreated infection may lead to severe complications including hepatic amoebiasis and intestinal tissue destruction (2). Globally, amoebiasis accounts for 50 million clinical cases and is responsible for approximately 110,000 deaths annually (3,4). Metronidazole (MNZ) is known to be highly effective amoebicide and is considered to be the drug of choice for the treatment of amoebiasis, but recent studies have shown that this drug have several toxic effects such as genotoxicity, gastric mucus irritation, and spermatozoid damage (5,6). Furthermore, failures in the treatment of several intestinal protozoan parasites may result from drug resistant to parasites (7,8). According to the International Agency for Research on Cancer (IARC), MNZ is classified in the 2B group, that is, potentially carcinogenic to humans and proved to be carcinogenic to animals (9). This prompted us to search for new antiamoebic agents.

Pyrimidines are of great importance in fundamental metabolism, being an integral part of DNA and RNA and found as core structure in a large verity of compounds that exhibited important biological activities such as anticancer, antimicrobial, antioxidant, and antiviral activities (10–14). In the recent years, a lot of attention has been drawn by the pyrimidines derivatives due to their diverse range of activities, especially calcium channel blocker property (15). Moreover, several alkaloids containing the dihydropyrimidine core unit that have been isolated from marine sources also showed interesting biological properties. In particular, the batzelladine alkaloids have been found to be potent HIV gp-120-CD₄ inhibitors (16,17). According to the literature, pyrazolones are also used as starting materials for the synthesis of biologically active pyrazolo[3,4-d]pyrimidine derivatives (18,19).

In view of the number of pharmacological significances of pyrimidine derivatives and as a part of our continuous efforts toward the development of more potent amoebicidal agents, we herein report the synthesis, characterization, in vitro antiamoebic activity, and cytotoxicity of a new series of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2b–2j), based on modification of the classical one-pot Biginelli reaction (18,20).

Materials and methods

All the required chemicals were purchased from Merck and Aldrich Chemical Company (USA). Precoated aluminum sheets (silica gel 60 F₂₅₄, Merck Germany) were used for thin-layer chromatography (TLC) and spots were visualized under UV light. Elemental analysis was carried out on CHNS Elementar (Vario EL-III), and the results were within ±0.3% of the theoretical values. IR spectra were recorded on Perkin-Elmer model 1600 FT-IR RX1 spectrophotometer as KBr discs. ¹H NMR and ¹³C NMR spectra were recorded on Bruker Spectrospin DPX 300 MHz and Bruker Spectrospin DPX 75 MHz spectrometer, respectively, using CDCl₃ and DMSO-d₆ as a solvent and trimethylsilane (TMS) as an internal standard. Splitting patterns are designated as follows; s, singlet; d, doublet; t, triplet; m, multiplet. Chemical shift values are given in ppm. The FAB mass spectra of the compounds were recorded on JEOL SX 102/DA-6000 mass spectrometer using Argon/Xenon 6KV (10 mA) as the FAB gas, and m-nitrobenzyl alcohol (NBA) was used as the matrix.

Preparation of 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (1a)

A mixture of phenyl hydrazine (0.1 mol) and ethyl acetoacetate (0.1 mol) in 50 ml ethanol was heated under reflux for 5 h. After cooling, the reaction mixture was poured into 200 ml of ice water. The crude product was filtered, dried, and recrystallized with 95% ethanol.

Yield 75%; Solid; m.p.: 123°C Anal. calc. for C₁₀H₁₀N₂O: C 68.95, H 5.79, N 16.08%; found: C 68.88, H 5.73, N 18.03%; IR ν_max (cm⁻¹): 3044 (Ar-H), 2976 (C-H), 1709 (C=O), 1588 (C=N), 1340 (C-N); ¹H NMR (CDCl₃) δ(ppm): 7.86 (d, 2H, J = 7.8Hz, Ar-H), 7.40–7.35 (t, 2H, J = 7.8 Hz, Ar-H), 7.19 (t, 1H, J = 7.2Hz, Ar-H), 3.39 (s, 2H, -CH₂ pyrazolone ring), 2.16 (s, 3H, CH₃ pyrazolone ring), ¹³C NMR (CDCl₃) δ(ppm): 170.48 (C=O), 147.1 (pyrazole), 139.5, 134.7, 131.7, 129.5, 124.5, (Ar-C), 13.6 (CH₃ pyrazolopyrimidine ring); FAB-MS (m/z): [M⁺+1] 175.19.

Synthesis of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j)

General procedures

A mixture of urea (0.01 mol), aromatic aldehyde (0.01 mol) and 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (0.01 mol) in absolute ethanol (30 ml) contain 2–3 drops of 37% HCl as catalyst was refluxed for 5 h. The crude product which precipitated on cooling was filtered and washed with cold ethanol and then recrystallized from appropriate solvent.

4, 5-Dihydro-3-methyl-1,4-diphenyl-1 H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2a)

Yield 35%; Solid; (Ethanol) mp: 167°C; Anal. calc. for C₁₈H₁₆N₄O: C 71.04, H 5.30, N 18.41%; found: C 71.02, H 5.23, N 18.36%; IR ν_max (cm⁻¹): 3233 (N-H), 3064 (Ar-H), 2923 (C-H), 1689 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 8.22 (s, 1H, NH pyrimidine), 8.14 (s, 1H, NH pyrimidine), 7.37–7.08 (m, 10H, Ar-H), 5.13 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ(ppm): 162.5 (C=O), 151.8 (pyrazolopyrimidine ring), 147.1 (pyrazole ring), 139.5, 134.7, 131.7, 129.5, 124.5 (Ar-C), 117.8 (pyrazolopyrimidine ring), 50.1 (-CH pyrimidine), 13.6 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 305.44.

4-(4-Chlorophenyl)-4,5-dihydro-3-methyl-1-phenyl-1H-pyrazolo[3,4d]pyrimidin-6 (7H)-one (2b)

Yield 37%; Solid; (Ethanol) mp: 237°C; Anal. calc. for C₁₈H₁₅N₄OCl: C 63.81, H 4.46, N 16.54%; found: C 63.72, H 4.39, N 16.48%; IR ν_max (cm⁻¹): 3255 (N-H), 3088 (Ar-H), 2846 (C-H), 1678 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 8.24 (s, 1H, NH pyrimidine), 8.01(s, 1H, NH pyrimidine), 7.65 (d, 2H, J = 7.8 Hz, Ar-H), 7.47–7.42 (t, 2H, J = 7.8 Hz, Ar-H), 7.33–7.21 (m, 5H, Ar-H) 4.99 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ(ppm): 161.8 (C=O), 152.4 (pyrazolopyrimidine ring), 148.8 (pyrazole ring), 139.5, 135.7, 130.2, 122.4 (Ar-C), 113.2 (pyrazolopyrimidine ring), 52.65 (-CH pyrimidine), 14.8 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 339.38.

4-(3-Chlorophenyl)-4,5-dihydro-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6 (7H)-one (2c)

Yield 40%; Solid; (Ethanol:Methanol) mp: 133°C; Anal. calc. for C₁₈H₁₅N₄OCl: C 63.81, H 4.46, N 16.54%; found: C 63.74, H 4.41, N 16.47 %; IR ν_max (cm⁻¹): 3257 (N-H), 3041 (Ar-H), 2853 (C-H), 1653 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 9.19 (s, 1H, NH pyrimidine), 8.22 (s, 1H, NH pyrimidine), 7.69 (d, 2H, J = 7.8 Hz, Ar-H), 7.49–7.19 (m, 7H, Ar-H), 4.97 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ(ppm): 162.3 (C=O), 151.7 (pyrazolopyrimidine ring), 147.6 (pyrazole ring), 135.7, 132.2, 131.1, 130.3, 122.4 (Ar-C), 118.4 (pyrazolopyrimidine ring), 50.97 (-CH pyrimidine), 14.04 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 339.21.

4, 5-Dihydro-3-methyl-1-phenyl- 4-p-tolyl-1H-pyrazolo [3,4-d] pyrimidin-6(7H)-one (2d)

Yield 45%; Solid; (Ethanol) m.p: 188°C; Anal. calc. for C₁₉H₁₈N₄O: C 71.68, H 5.70, N 17.60%; found: C 71.59, H 5.62, N 17.54%; IR ν_max (cm⁻¹): 3250 (N-H), 3086 (Ar-H), 2857 (C-H), 1698 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 8.11 (s, 1H, NH pyrimidine), 8.01 (s, 1H, NH pyrimidine), 7.65 (d, 2H, J = 7.8 Hz, Ar-H), 7.49 (t, 2H, J = 7.5 Hz, Ar-H), 7.33–7.05 (m, 5H, Ar-H), 4.99 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring), 2.37 (s, 3H, CH₃ phenyl); ¹³C NMR (DMSO-d₆) δ(ppm): 162.6 (C=O), 155.3 (pyrazolopyrimidine ring), 148.3 (pyrazole ring), 140.5, 139.7, 132.4, 130.3, 124.3 (Ar-C), 113.4 (pyrazolopyrimidine ring), 50.07 (-CH pyrimidine), 21.5 (CH₃ Aromatic), 14.1 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 319.54.

4,5-Dihydro-4-(4-isopropylphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2e)

Yield 48%; Solid; (Ethylacetate) m.p: 192°C; Anal. calc. for C₂₁H₂₂N₄O: C 72.81, H 6.40, N 16.17%; found: C 72.76, H 6.33, N 16.19%; IR ν_max (cm⁻¹): 3293 (N-H), 3031 (Ar-H), 2921 (C-H), 1698 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 8.02 (s, 1H, NH pyrimidine), 7.97 (s, 1H, NH pyrimidine), 7.86 (d, 2H, J = 8.1 Hz, Ar-H), 7.79 (s, 1H, J = 8.1 Hz, Ar-H), 7.46–7.35 (m, 3H, Ar-H), 7.21–6.90 (m, 3H, Ar-H), 5.17 (s, 1H, -CH pyrimidine), 3.77 (m, 1H, -CH (CH₃)₂), 2.48 (s, 3H, CH₃ pyrazole ring), 2.16 (s, 3H, CH₃ isopropylphenyl), 2.11 (s, 3H, CH₃ isopropylphenyl); ¹³C NMR (DMSO-d₆) δ(ppm): 161.5 (C=O), 153.8 (pyrazolopyrimidine ring), 148.8 (pyrazole ring), 138.6, 135.3, 130.8, 123.4 (Ar-C), 118.8 (pyrazolopyrimidine ring), 51.47 (-CH pyrimidine), 13.8 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 347.56.

4,5-Dihydro-4-(3-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2f)

Yield 35%; Solid; (Ethanol) m.p: 176–177°C; Anal. calc. for C₁₈H₁₆N₄O₂: C 67.49, H 5.03, N 17.49%; found: C 67.43, H 5.05, N 17.42%; IR ν_max (cm⁻¹): 3408 (O-H), 3192 (N-H) 3071 (Ar-H), 2917 (C-H), 1665 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 10.2 (s, 1H, NH pyrimidine), 10.0 (s, 1H, Ar-OH), 9.74 (s, 1H, NH pyrimidine), 7.65–6.52 (m, 9H, Ar-H), 5.11 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ(ppm): 162.5 (C=O), 159.3 (C-OH), 154.9 (pyrazolopyrimidine ring), 147.8 (pyrazole ring), 139.3, 137.1, 135.42, 130.8, 122.4, (Ar-C), 112.4 (pyrazolopyrimidine ring), 52.44 (-CH pyrimidine), 13.9 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 321.11.

4,5-Dihydro-4-(3,4-dimethoxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6 (7H)-one (2g)

Yield 30%; Solid; (Ethanol:Methanol) m.p: 183°C; Anal. calc. for C₂₀H₂₀N₄O₃: C 5.92, H 5.53, N 15.38%; found: C 5.87, H 5.47, N 15.33%; IR ν_max (cm⁻¹): 3263 (N-H), 3076 (Ar-H), 2853 (C-H), 1663 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 8.81 (s, 1H, NH pyrimidine), 8.04 (s, 1H, NH pyrimidine), 7.90 (d, 2H, J = 8.1 Hz, Ar-H), 7.90 (d, 2H, Ar-H), 7.69 (s, 1H, Ar-H), 7.43–7.11 (m, 3H, Ar-H), 5.10 (s, 1H, -CH pyrimidine), 3.86 (s, 6H, 2xOCH₃), 2.48 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ(ppm): 161.9 (C=O), 152.9 (pyrazolopyrimidine ring), 147.2 (pyrazole ring), 140.7, 135.4, 131.6, 130.3, 122.4 (Ar-C), 116.3 (pyrazolopyrimidine ring), 52.08 (-CH pyrimidine), 14.64 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 365.17.

4,5-Dihydro-4-(1H-indol-3-yl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2h)

Yield 45%; Solid; (Ethylacetate) m.p: 238°C; Anal. calc. for C₂₀H₁₇N₅O: C 69.96, H 4.99, N 20.40%; found: C 69.91, H 4.93, N 20.32%; IR ν_max (cm⁻¹): 3249 (N-H), 3067 (Ar-H), 2891 (C-H), 1653 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 12.66 (s, 1H, NH, Indole), 9.80 (s, 1H, NH pyrimidine), 8.16 (s, 1H, NH pyrimidine), 7.97 (d, 2H, J = 7.8 Hz, Ar-H), 7.59–7.12 (m, 8H, Ar-H), 5.22 (s, 1H, -CH pyrimidine), 2.47 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ (ppm): 162.5 (C=O), 153.6 (pyrazolopyrimidine ring), 148.9 (pyrazole ring), 140.1, 134.7, 132.2, 131.6, 130.8, 123.4, (Ar-C), 115.3 (pyrazolopyrimidine ring), 50.34 (-CH pyrimidine), 13.84 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 344.25.

4-(2-Chloroquinoline-3-yl)-4,5-dihydro-3-methyl-1-phenyl-1H-pyrazolo[3,4-d] pyrimidine-6(7H)-one (2i)

Yield 28%; Solid; (Ethanol) m.p: 168°C; Anal. calc. for C₂₁H₁₆N₅OCl: C 64.70, H 4.14, N 7.96%; found: C 64.63, H 4.08, N 7.87%; IR ν_max (cm⁻¹): 3298 (N-H), 3061 (Ar-H), 2955 (C-H), 1668 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 12.3 (s, 1H, NH pyrimidine), 10.0 (s, 1H, NH pyrimidine), 8.49 (s, 1H, quinoline), 7.97–7.17 (m, 9H, Ar-H & quinoline), 5.13 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring); ¹³C NMR (DMSO-d₆) δ(ppm): 161.6 (C=O), 152.3 (pyrazolopyrimidine ring), 147.3 (pyrazole ring), 139.6, 133.7, 132.2, 131.6, 128.3, 121.4 (Ar-C), 115.3 (pyrazolopyrimidine ring), 52.80 (-CH pyrimidine), 14.67 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 390.37.

4-(2-Chloro-7-methylquinoline-3-yl)-4, 5-dihydro-3-methyl-1-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2j)

Yield 35%; Solid; (Ethanol) m.p: 194°C Anal. calc. for C₂₂H₁₈N₅OCl: C 65.43, H 4.49, N 17.34%; found: C 65.38, H 4.43, N 17.27%; IR ν_max (cm⁻¹): 3296, (N-H) 3048 (Ar-H), 2977 (C-H), 1665 (C=O); ¹H NMR (DMSO-d₆) δ(ppm): 12.2 (s, 1H, NH pyrimidine), 10.1 (s, 1H, NH pyrimidine), 8.40 (s, 1H, quinoline), 8.13 (s, 1H, quinolne), 7.67 (d, 1H, J= 8.1 Hz, quinoline), 7.59–7.20 (m, 6H, Ar-H & quinoline), 5.10 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH₃ pyrazole ring), 2.38 (s, 3H, quinoline); ¹³C NMR (DMSO-d₆) δ(ppm): 162.4 (C=O), 153.6 (pyrazolopyrimidine ring), 148.4 (pyrazole ring), 140.6, 133.7, 132.1, 131.9, 129.3, 123.1 (Ar-C), 114.3 (pyrazolopyrimidine ring), 50.13 (-CH pyrimidine), 14.12 (CH₃ pyrazole ring); FAB-MS (m/z): [M⁺+1] 404.42.

Pharmacology

All the pyrazolo[3, 4-d]pyrimidine-6-one derivatives (2a–2j) were screened in vitro against HM1:IMSS strain of E. histolytica by microdilution method (21). All the experiments were carried out in triplicate at each concentration level and repeated thrice. Cytotoxicity of active compounds has been studied by MTT assay on breast cancer MCF-7 cell line. The results of biological activity and cytotoxicity are summarized in Table 1.

Table 1.  In vitro antiamoebic activity of pyrazolo[3,4-d] pyrimidine-6-one derivatives (2a–2j) against HM1:IMSS strain of Entamoeba histolytica and cytotoxicity profile of compounds 2b, 2i, 2j and metronidazole.

Compound	R	Antiamoebic activity		Cytotoxicity profile
		IC50 (μM)	SD^a	IC50 (μM)	SD^a
2a		4.24	0.18	ND	ND
2b		0.37	0.13	>100	0.18
2c		9.23	0.34	ND	ND
2d		7.25	0.16	ND	ND
2e		15.5	0.20	ND	ND
2f		7.99	0.26	ND	ND
2g		14.3	0.33	ND	ND
2h		8.51	0.16	ND	ND
2i		0.04	0.21	68.49	0.16
2j		0.06	0.19	>100	ND
Metronidazole		1.33	0.11	>100	0.28

^aStandard deviation.

ND, not done.

In vitro antiamoebic assay

All the compounds (2a–2j) were screened in vitro for antiamoebic activity against HM1:IMSS strain of E. histolytica by microdilution method (21). E. histolytica trophozoites were cultured in wells of 96-well microtiter plate by using Diamond TYIS-33 growth medium (22). The test compounds (1 mg) were dissolved in 40 μl DMSO and the final solutions in each well contained 0.1% DMSO or less, which did not affect viability (23,24). The stock solutions of the compounds were prepared freshly before use at a concentration of 1 mg/ml. Twofold serial dilutions were made in the wells of 96-well microtiter plate (costar). Each test includes MNZ as a standard amoebicidal drug, control wells (culture medium plus amoebae) and a blank (culture medium only). All the experiments were carried out in triplicate at each concentration level and repeated thrice. The amoeba suspension was prepared from a confluent culture by pouring off the medium at 37°C and adding 5 ml of fresh medium, chilling the culture tube on ice to detach the organisms from the side of the flask. The number of amoeba/ml was estimated with a haemocytometer, using trypan blue exclusion to confirm the viability. The suspension was diluted to 10⁵ organism/ml by adding fresh medium and 170 μl of this suspension was added to the test and control wells in the plate so that the wells were completely filled (total volume, 340 μl). An inoculum of 1.7 × 10⁴ organisms/well was chosen so that confluent, but not excessive growth, took place in control wells. Plates were sealed and gassed for 10 min with nitrogen before incubation at 37°C for 72 h. After incubation, the growth of amoeba in the plate was checked with a low-power microscope. The culture medium was removed by inverting the plate and shaking gently. Plate was then immediately washed with sodium chloride solution (0.9%) at 37°C. This procedure was completed quickly and the plate was not allowed to cool to prevent the detachment of amoebae. The plate was allowed to dry at room temperature, and the amoebae were fixed with methanol and when dried, stained with (0.5%) aqueous eosin for 15 min. The stained plate was washed once with tap water, then twice with distilled water and allowed to dry. A 200-μl portion of 0.1N sodium hydroxide solution was added to each well to dissolve the protein and release the dye. The optical density of the resulting solution in each well was determined at 490 nm with a microplate reader. The percent inhibition of amoebal growth was calculated from the optical densities of the control and test wells and plotted against the logarithm of the dose of the drug tested. Linear regression analysis was used to determine the best fitting line from which the IC₅₀ value was found. The IC₅₀ values in μM are reported in Table 1.

MTT assay

MCF-7 cells were cultured and maintained as monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal calf serum (FCS), antibiotics: 100 IU/ml of penicillin and 100 μg/ml of streptomycin. All cells were cultured in flasks at 37°C in the 100%-humidity atmosphere and 5% of CO₂ (25). Only viable cells were used in the assay. Exponentially growing cells were plated at 1.2 × 10⁴ cells per well into 96-well plates and incubated for 48 h before the addition of drugs. Stock solutions of compounds were initially dissolved in 20% (v/v) DMSO and further diluted with fresh complete medium. The growth-inhibitory effects of the compounds were measured using standard tetrazolium MTT assay. After 48 h of incubation at 37°C, the medium was removed and 25µl of MTT reagent (5 mg/ml) in serum-free medium was added to each well. The plates were incubated at 37°C for 4 h. At the end of the incubation period, the medium was removed and pure DMSO (100 µl) was added to each well. The metabolized MTT product dissolved in DMSO was quantified by reading the absorbance at 570 nm. All assays were performed in triplicate. Percent viability was defined as the relative absorbance of treated versus untreated control cells. Plates were analyzed in an ELISA plate reader (Labsystems Multiskan RC, Helsinki, Finland) at 570 nm with a reference wavelength of 655 nm.

Results and Discussion

Chemistry

The synthesis of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a–2j) was performed in a manner as outlined in Scheme 1. The reaction of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one, urea and various aromatic aldehydes in absolute ethanol using HCl as a catalyst and refluxed for 5 h gave pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a–2j). The compounds were stable in solid state and were soluble in methanol and DMSO. Melting points were recorded on KSW melting point apparatus and are uncorrected.

Scheme 1.  General method for the synthesis of pyrimidine derivatives (2a–2j). Reagent and conditions: (i) C₂H₅OH, 78°C reflux 5–6 h, (ii) Urea, HCl (2–3 drops), different aromatic aldehydes, where R = aryl group of different aldehydes.

Synthesis

In the present work, Biginelli-type cyclocondensation reaction was studied for the preparation pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j). The classical three-components urea, aromatic aldehyde, and 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one were carried out in alcoholic solution containing a few drops of concentrated hydrochloric as catalyst, and refluxed for 5 h, as shown in Scheme 1. All the compounds (2a–2j) were recrystallized from appropriate solvents and the yield was in the range of 36–48%. All the compounds were highly soluble in methanol and DMSO and were characterized by IR, ¹H and ¹³C NMR, and Mass spectra. The purity of the compounds was confirmed by elemental analysis and data were found in accordance with ±0.3%.

Characteristics IR bands provides significance indication for the formation of 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (1a). The appearance of absorption bands at 1709, 1588, and 1340 cm⁻¹ corresponding to the C=O, C=N, C-N stretching vibrations, confirmed the formation of cyclized product (1a). In cases of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j), a sharp band was observed for NH-stretching vibrations in the range 3233–3298 cm⁻¹ showed the formation of pyrazolo[3,4-d]pyrimidine ring in all the compounds (2a–2j). The appearance of strong band in the range of 1653–1698 cm⁻¹ due C=O stretching vibration also confirmed the formation of desired compounds (2a–2j) containing pyrazolo[3,4-d]pyrimidine ring.

Moreover the structure of 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (1a) was further confirmed by ¹H NMR spectra. The appearance of singlet for two protons at 3.39 due to CH₂ group showed the formation of desired pyrazolon ring in the structure (1a). The appearance of singlet at 2.16 ppm corresponds to the methyl group of pyrazolon nucleus also confirmed the formation of cyclized structure (1a). The structure of 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (1a) was further supported by ¹³C NMR spectra. The appearance of signal at 170 ppm due to (C=O) group of desire pyrazolon nucleus confirmed the formation of 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one.

In case of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j), NH protons were appeared in the range of 7.97–12.6 ppm only as a singlet due to poor resolution of its signal revealed the presence of pyrimidines nucleus in all the compounds. A sharp peak representing the methine proton of the pyrimidine was observed in the range of 4.97–5.22 ppm also confirming the formation of the pyrazolo[3,4-d]pyrimidine nucleus in all the synthesized compounds (2a–2j) (18). The appearance of signals at 8.11–8.13 and 8.40–8.49 ppm were found in compounds (2i) and (2j) for chloroquinoline rings, a singlet at 10.0 ppm for OH proton was observed in compound (2f). For methyl group of pyrazol ring, a singlet at 2.47–2.48 ppm was found in all the compounds (2a–2j), whereas for compounds (2e) and (2j), the appearance of singlet for methyl group was found in the range of 2.11–2.38 ppm. A singlet at 3.86 ppm for two OCH₃ protons was found in compound (2g).The structure of the compounds (2a–2j) was further supported by ¹³C NMR spectra. The appearance of signals in the range of 161.5–162.8 ppm and 50.1–52.8 ppm due to C=O and methine carbon atom (pyrimidine nucleus) confirmed the formation of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j) (Figure 1). The signal due to aromatic and aliphatic carbons resonates at their usual positions and the data shown in the experimental section.

Figure 1.  Pyrazolo[3,4-d]pyrimidine-6- one derivatives (2a–2j).

Antiamoebic activity

Preliminary experiments were carried out to determine the in vitro antiamoebic activity of all the compounds (2a–2j) by microdilution method using HM1:IMSS strain of E. histolytica. The results are summarized in Table 1. The data are present in terms of percent growth inhibition relative to untreated controls and plotted as probit values as a function of drug concentration. The antiamoebic effect was compared with the most widely used antiamoebic medication MNZ which had a 50% inhibitory concentration (IC₅₀) of 1.33 μM in our experiments. The results showed that the synthesized pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a–2j) and their structural activity relationship (SAR) established on the basis of substituted and unsubstituted aromatic ring attached with the methine carbon atom of pyrimidine nucleus. The pyrazolo[3,4-d]pyrimidine-6-one derivatives with substituted and unsubstituted phenyl ring (2a–2h) and quinoline ring (2i, 2j) attached with the pyrimidine ring showed IC₅₀ for antiamoebic activity in the range of 0.04–14.3 μM. Therefore out of ten compounds screened in vitro antiamoebic activity, three compounds (2b, 2i, and 2j) were found to be more active against E. histolytica as compared with reference drug MNZ (IC₅₀ = 1.33 μM). Among these three compounds, two compounds 2i (IC₅₀ = 0.04 µM) and 2j (IC₅₀ = 0.06 µM) substituted by chloroquinoline rings showed excellent antiamoebic activity and the rest one compounds 2b (IC₅₀ = 0.37 µM) substituted by hydroxyl phenyl showed moderatively active than the standard drug MNZ.

Cytotoxicity profile

To examine the effect of antiamoebic compounds 2b, 2i, 2j and MNZ on cell proliferation, we ensured their cytotoxicity on human breast cancer MCF-7 cell line. A subconfluent population of MCF-7 cells was treated with increasing concentrations of compounds, and the number of viable cells was measured after 48 h by MTT cell viability assay. The concentration range for all the compounds 2b, 2i, 2j and MNZ was 1.56–100 µM. Figure 2 depicts the compounds 2b, 2i, 2j and MNZ exhibited >80% viability at the concentration range of 1.56–50 µM. On increasing the concentration range up to 100 µM, only compound 2i (viability 27%) showed cytotoxicity against the human breast cancer MCF-7 cell line. The cytotoxicity IC₅₀ values along with the standard deviation values of 2b, 2i, 2j and MNZ are given in Table 1.

Figure 2.  Percentage of viable cells after 48 h. pre-treatment of human breast cancer MCF-7 cell line with compounds 2b, 2i, 2j and metronidazole (MNZ), evaluated by MTT assay.

Conclusion

A new series of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a–2j) was prepared by simple one-pot Biginelli method. Compounds 2b, 2i, and 2j with IC₅₀ values of 0.37 µM, 0.04 µM, and 0.06 µM, respectively, exhibited higher antiamoebic activity than the standard drug MNZ (IC₅₀ = 1.33 µM). Cytotoxicity studies were performed on human breast cancer MCF-7 cell line and results showed that the compounds 2b, 2i, 2j and MNZ offered remarkable viability (>80% at 50 μM).

Declaration of interest

This work was supported by Council of Scientific and Industrial Research [grant no. 01(2278)/08/EMR-II New Delhi, India]. F.H. is thankful to the University Grant commission (UGC), India for the financial support.