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Synthesis and Antiproliferative Activity of 1-(4-(1H-Indol-3-Yl)-6-(4-Methoxyphenyl)Pyrimidin-2-yl)Hydrazine and Its Pyrazolo Pyrimidine Derivatives | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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Synthesis and Antiproliferative Activity of 1-(4-(1H-Indol-3-Yl)-6-(4-Methoxyphenyl)Pyrimidin-2-yl)Hydrazine and Its Pyrazolo Pyrimidine Derivatives

Ekhlass Nassar1*, Yaser A El-Badry2,3, Afaf MM Eltoukhy1 and Rezek R Ayyad4,5

1Organic Chemistry Department, Faculty of Women’s for Arts, Science and Education, Ain Shams University, Cairo, Egypt

2Organic Chemistry Lab, Faculty of Specific Education, Ain Shams University, 11566 Abbasseya, Cairo, Egypt

3Organic Chemistry Lab, Faculty of Science, Taif University, Khurma, Kingdom of Saudi Arabia

4Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt

5Medicinal Chemistry Department, Faculty of Pharmacy, Delta University, Gamasa Ad-dakahlia, Egypt

*Corresponding Author:
Ekhlass Nassar
Organic Chemistry Department
Faculty of Women’s for Arts
Science and Education, Ain Shams University, Cairo, Egypt
E-mail: [email protected]

Received date: March 17, 2016; Accepted date: April 07, 2016; Published date: April 14, 2016

Citation: Nassar E, El-Badry YA, Eltoukhy AMM, Ayyad RR (2016) Synthesis and Antiproliferative Activity of 1-(4-(1H-Indol-3-Yl)-6-(4-Methoxyphenyl)Pyrimidin-2-yl)Hydrazine and Its Pyrazolo Pyrimidine Derivatives. Med chem (Los Angeles) 6:224-233. doi:10.4172/2161-0444.1000350

Copyright: © 2016 Nassar E, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The target compounds 1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)hydrazine (5) was synthesized by reacting 6-(1H-Indol-3-yl)-4-(4-methoxyphenyl)pyrimidine-2(1H)-thione 4 with hydrazine hydrate. Compound 5 was used as a precursor for the synthesis of new pyrazolo pyrimidine derivatives 6-9. Moreover, the 5-amino-1H-pyrazole-4-carbonitrile derivative 6 was then converted into another set of novel compounds 10-14. On the other hand a series of transformations were carried out using the newly synthesized 1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl) pyrimidin-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (11) to afford the pyrazolo pyrimidine derivatives 15-18. Antiproliferatve activities for some of the newly synthesized compounds were evaluated.

Keywords

Pyrazolopyrimidine; Indole aldehyde; Chalcone; Antiproliferative activity; Triazin-4-one: 5-aminopyrazole

Introduction

Chalcones constitute an important group of natural products and are the precursors of flavonoids, isoflavonoids and many synthetic heterocycles of different biological activities including pyrimidines, pyrazolines, pyrazolopyrimidines and diazepines [1-5].

Chemically, chalcones are characterized by their easy synthesis by Claisen-Schmidt condensation in good yields and the presence of the α,β unsaturated carbonyl system linked to two aromatic rings which encourage the chalcones cyclization through Michael addition to give interesting heterocyclic compounds which exhibit antitumor [6,7], antimitotic [8], antimutagenic [9] antibacterial [10], antiviral [11], anti-inflammatory [12], antiulcerative [13] and hepatoprotective [14]. In the present work we have been synthesized a series of novel heterocyclic compounds derived from a previous synthesized chalcone compound [1]. The new compounds were screened for their antiproliferative activities.

Experimental

General

Melting points were measured on a digital Electrothermal 9100 apparatus (Kleinfeld, Gehrden, Germany) and are uncorrected. FTIR spectra (KBr) were obtained on a Nicolet 205 spectrophotometer (Nicolet, Madison, WI, USA). The 1H NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer (Varian inc., Palo Alto, CA, USA). Chemical shifts are expressed in δ values. The mass spectra were recorded on a Shimadzu GCMS-QP-1000EX mass spectrometer (Shimadzu, Kyoto, Japan) at 70 eV. Elemental analysis were carried out at the Micro-analytical Center of Cairo University. All reactions were routinely followed by TLC.

3-(2-hydrazinyl-6-(4-methoxyphenyl)pyrimidin-4-yl)-1H-indole (5)

The hydrazino-pyrimidine derivative 5 was prepared by reacting compound 4 [1] (10.0 mmol) with hydrazine hydrate (10.0 mmol) catalyzed by acetic acid (5 drops) in refluxing ethanol for 6 h. Evaporation of alcohol and recrystallization with ethanol gave compound 5 as pale brown crystals mp 160-2°C, yield 80% (element test for the product confirmed the disappearance of sulfur). IR: νmax/cm-1 3212 (NH2), 3184 (NH), 3109 (NH), 1618 (C=N), 1576 (C=N). 1H MR (DMSO-d6): δ 3.78 (s, 3H, OCH3), 7.05-7.3 (m, 4H, Ar H’s), 7.52 (d, 2H, J=8.8 Hz, Ar-H), 7.94 (s, 1H, pyrimidin), 8.4 (d, 2H, J=8.8 Hz, Ar-H's), 8.78 (s, 1H, indole), 8.93-8.95 (brs, 3H, D2O Exch., NH2, NH), 11.65 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 331 [M+, 5.08]. Anal. Calcd. For C19H17N5O (331.37): C, 68.87; H, 5.17; N, 21.13. Found: C, 68.76; H, 5.35; N, 21.30.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)-5-amino-1H-pyrazole-4-carbonitrile (6)

To a solution of compound 5 (10 mmol) in ethanol (30 mL), ethoxymethylene malononitrile (10 mmol), was added. The reaction mixture was heated under reflux condition for 8 h. Then the formed precipitate was filtered and recrystallized from ethanol to give pale brown crystals of compound 6 mp 174-6°C, yield 76% IR: νmax/cm-1 3356 (NH2), 3164 (NH), 2224 (C≡N).1H NMR (DMSO-d6): δ 3.49 (s, 3H, OCH3), 7.01-7.27 (m, 4H, Ar H’s), 7.46 (d, 2H, J=8.6 Hz, Ar-H), 7.87 (s, 1H, pyrazole), 8.28 (s, 1H, indole), 8.30 (d, 2H, J=8.6 Hz, Ar-H's), 8.56 (s, 1H, pyrimidin), 8.85 (s, 2H, D2O Exch., NH2), 11.66 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 407 [M+, 0.39]. Anal. Calcd. For C23H17N7O (407.43): C, 67.80; H, 4.21; N, 24.06. Found: C, 67.66; H, 4.38; N, 24.25.

3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(4-methoxyphenyl)pyrimidin-4-yl)-1H-indole (7)

A solution of compound 5 (10 mmol) in absolute ethanol and acetylacetone (10 mmol) was heated at reflux temperature for 5 h. The mixture was evaporated under reduced pressure, and the obtained product was recrystallized from ethanol to afford pale brown crystals of pyrazolo pyrimidine derivative 7 mp 187-9°C, yield 70% IR: νmax/cm-1 3170 (NH), 3109 (CH-Ar.), 1634 (C=N), 1600 (C=N), 1574 (C=N). 1H NMR (DMSO-d6): δ 2.3 (s, 3H, CH3), 2.58 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 6.99 (s,1H, pyrazole), 7.17-7.26 (m, 4H, Ar-H’s), 7.53 (d, 2H, J=8.6 Hz, Ar-H's), 7.94 (s, 1H, indole), 8.38 (d, 2H, J=8.6 Hz, Ar-H's), 8.94 (s, 1H, pyrimidine), 11.7 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 395 [M+, 2.0]. Anal. Calcd. For C24H21N5O (395.46): C, 72.89; H, 5.35; N, 17.71. Found: C, 72.70; H, 5.52; N, 17.90.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 3-methyl-1H-pyrazol-5(4H)-one (8)

Compound 5 (10 mmol) and ethyl acetoacetate (10 mmol) in acetic acid (30 mL) was heated at reflux temperature for 6 h. The mixture was poured into ice cold water and the obtained product washed with ice cold water, dried and recrystallized from ethanol to afford pale brown crystals of 8 mp 210-2°C, yield 72% IR: νmax/cm-1 3216 (NH), 1718 (C=O). 1H NMR (DMSO-d6): δ 1.22 (s, 3H, CH3), 2.24 (s, 2H, CH2, pyrazol), 3.83 (s, 3H, OCH3), 7.02-7.23 (m, 4H, Ar H’s), 7.52 (d, 2H, J=8.6 Hz, Ar-H's), 7.9 (s, 1H, indole), 8.4 (d, 2H, J=8.6 Hz, Ar-H's), 8.95 (s, 1H, pyrimidin), 11.72 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 397 [M+, 54]. Anal. Calcd. For C23H19N5O2 (397.43): C, 69.51; H, 4.82; N, 17.62. Found: C, 69.66; H, 4.64; N, 17.80.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 3,4-dimethylpyrano[2,3-c]pyrazol-6(1H)-one (9)

Compound 5 (10 mmol) and ethyl acetoacetate in excess (30 mL) was heated at reflux temperature for 6 h. The mixture was poured into ice cold water and the obtained product washed with water, dried and recrystallized from ethanol to give pale brown crystals of the pyrazolone compound 9 mp 188-0°C, yield 72% IR: νmax/cm-1 3367 (NH), 1715 (C=O). 1H NMR (DMSO-d6): δ 2.25 (s, 3H, CH3), 2.32 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 5.60 (s, 1H, pyranone), 6.99-7.26 (m, 4H, Ar H’s), 7.38 (d, 2H, J=8.8 Hz, Ar-H's), 7.42 (d, 2H, J=8.8 Hz, Ar-H's), 8.11 (s, 1H, indole), 8.70 (s, 1H, pyrimidin), 10.41 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 463 [M+, 8.6]. Anal. Calcd. For C27H21N5O3 (463.49): C, 69.97; H, 4.57; N, 15.11. Found: C, 69.78; H, 4.72; N, 15.30.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 1H-pyrazolo[3,4-d]pyrimidin-4-amine (10)

A mixture of compound 6 (10 mmol) and formamide (30 ml) was heated at 150°C for 3 h. The reaction mixture was cooled and poured into water. The formed solid was filtered off, dried and recrystallized from ethanol to afford the pyrazolo pyrimidine derivative 10 as pale brown crystals 152-4°C, yield 68% IR: νmax/cm-1 3389 (NH2), 3190 (NH). 1H NMR (DMSO-d6): δ 3.89 (s, 3H, OCH3), 6.93-7.13 (m, 4H, Ar H’s), 7.23 (d, 2H, J=8.7 Hz, Ar-H's), 7.35 (d, 2H, J=8.7 Hz, Ar-H's), 7.93 (s,1H, D2O Exch., NH2), 8.01 (s, 1H, prazole), 8.07 (s, 1H, pyrimidine), 8.12 (s, 1H, indole), 8.2 (s, 1H, pyrimidin), 10.82 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 434 [M+, 24.1]. Anal. Calcd. For C24H18N8O (434.45): C, 66.35; H, 4.18; N, 25.79. Found: C, 66.50; H, 4.32; N, 25.61.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (11)

Synthesis of compound (11):

Method A: Compound 6 (10 mmol) was heated under reflux conditions in formic acid (30 ml, 85%) for 8 h. The reaction mixture was cooled and poured into ice cold water. The formed solid was filtered off, dried, and recrystallized from dimethylformamide to give 11.

Method B: Compound 12 (10 mmol) was heated under reflux temperature in triethyl orthoformate (30 ml) for 10 h. The product which separated on cooling was filtered off, washed with ethanol, dried and recrystallized from dimethylformamide to give pale brown crystals of compound 11 mp 190-2°C, yield 65% IR: νmax/cm-1 3398 (NH), 3177 (NH), 1665 (C=O). 1H NMR (DMSO-d6): δ 3.87 (s, 3H, OCH3), 7.10-7.15 (m, 4H, Ar- H’s), 7.35 (d, 2H, J=8.6 Hz, Ar-H's), 7.40 (d, 2H, J=8.6 Hz, Ar- H's), 7.45 (s,1H, D2O Exch., NH), 7.50 (s, 1H, prazole), 8.11 (s, 1H, indole), 8.18 (s, 1H, pyrimidine), 8.21 (s, 1H, pyrimidin), 10.99 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 435 [M+, 63]. Anal. Calcd. For C24H17N7O2 (435.44): C, 66.20; H, 3.94; N, 22.52. Found: C, 66.38; H, 3.76; N, 22.71.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 5-amino-1H-pyrazole-4-carboxamide (12)

To (10 m mol) of compound 6, cold concentrated sulphuric acid (10 ml), was added portion wise with ice bath cooling and stirring. After 10 minutes, the ice bath was removed and the mixture was stirred for an additional 15 minutes. The resulting pale yellow solution was carefully poured onto crushed ice and the resulting precipitate was collected, washed with water, dried and recrystallized from dioxane to give yellow crystals of the pyrazolo carboxamide 12 mp 167-9°C, yield 65% IR: νmax/cm-1 3398 (NH2), 3267 (NH2), 3114 (NH), 1640 (C=O). 1H NMR (DMSO-d6): δ 3.82 (s, 3H, OCH3), 7.25-7.29 (m, 4H, Ar-H’s), 7.49 (d, 2H, J=8.8 Hz, Ar-H's), 8.05 (d, 2H, J=8.8 Hz, Ar-H's), 8.29 (s, 2H, D2O Exch., NH2), 8.41 (s, 1H, prazole), 8.57 (s, 1H, indole), 8.69 (s, 1H, pyrimidin), 8.74 (s, 2H, D2O Exch., NH2), 12.72 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 425 [M+, 1.03]. Anal. Calcd. For C23H19N7O2 (425.44): C, 64.93; H, 4.50; N, 23.05. Found: C, 64.70; H, 4.70; N, 23.26.

7-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 3H-pyrazolo[3,4-d][1,2,3]triazin-4(7H)-one (13)

To a suspended solution of compound 12 (10 m mol) in concentrated hydrochloric acid (30 ml) at 0-5°C, a solution of sodium nitrite (3.00 g) in water (5 ml) was added over 20 minutes. After 2 h of stirring at room temperature the foamy mixture was filtered off. The resulting solid was washed with ice cold water, dried and recrystallized from ethanol to give pale brown crystals of compound 13 mp 148-0°C, yield 64% IR: νmax/cm-1 3230 (NH), 3118 (NH), 1685 (C=O).1H NMR (DMSO-d6): δ 3.84 (s, 3H, OCH3), 7.15-7.26 (m, 4H, Ar H’s), 7.31 (d, 2H, J=8.6 Hz, Ar-H's), 7.65 (d, 2H, J=8.6 Hz, Ar-H's), 8.21 (s, 1H, prazole), 8.49 (s, 1H, indole), 8.63 (s, 1H, pyrimidine), 8.99 (s, 1H, D2O Exch., NH), 11.82 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 436 [M+, 0.04]. Anal. Calcd. For C23H16N8O2 (436.40): C, 63.30; H, 3.70; N, 25.68. Found: C, 63.45; H, 3.90; N, 25.50.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 6-thioxo-6,7-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4(5H)- one (14)

To a solution of compound 12 (10 m mol) in dimethylformamide (30 ml), 20% potassium hydroxide solution (potassium hydroxide 1.68 g, water 7 ml) and carbon disulfide (5 ml) were added. The reaction mixture was heated under reflux for 15 h, then poured into water and filtered off. The filtrate was precipitated with HCl (0.1N) and the solid product was collected, washed with water, dried, and recrystallized from ethanol/dimethylformamide to give pale brown crystals of compound 14 mp 198-0°C, yield 68% IR: νmax/cm-1 3431 (NH), 3216 (NH), 3148 (NH), 1727 (C=O).1H NMR (DMSO-d6): δ 3.84 (s, 3H, OCH3), 7.14-7.25 (m, 4H, Ar H’s), 7.48 (d, 2H, J=8.6 Hz, Ar-H's), 8.26 (s, 1H, prazole), 8.28 (d, 2H, J=8.6 Hz, Ar-H's), 8.32 (s, 1H, indole), 8.35 (s, 1H, pyrimidin), 8.90 (s, 1H, D2O Exch., NH), 9.93 (s, 1H, D2O Exch., NH), 11.67 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 467[M+, 5]. Anal. Calcd. For C24H17N7O2S (467.50): C, 61.66; H, 3.67; N, 20.97. Found: C, 61.48; H, 3.82; N, 20.80.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 4-chloro-1H-pyrazolo[3,4-]pyrimidine (15)

A mixture of compound 11 (10 m mol) was heated in phosphorus oxychloride (20 mL) for 10 h. The solution was cooled and poured into ice-water. The solid was filtered off, dried and purified on silica gel using chloroform /n-hexane (1:1) as eluent to give pale brown crystals mp 270-2°C, yield 64% IR: νmax/cm-1 3433 (NH). 1H NMR (DMSO-d6): δ 3.81 (s, 3H, OCH3), 7.09-7.26 (m, 4H, Ar H’s), 7.43 (d, 2H, J=8.7 Hz, Ar-H's), 8.37 (d, 2H, J=8.7 Hz, Ar-H's), 8.41 (s, 1H, prazole), 8.58 (s, 1H, indole), 8.63 (s, 1H, pyrimidin), 8.87 (s, 1H, pyrimidin), 11.67 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 453 [M+, 2.0]. Anal. Calcd. For C24H16ClN7O (453.88): C, 63.51; H, 3.55; N, 21.60. Found: C, 63.35; H, 3.72; N, 21.40.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 1H-pyrazolo[3,4-d]pyrimidine-4-thiol (16)

A solution of compound 15 (10 m mol) and thiourea (10 m mol) in dioxane (40 ml) was heated at reflux for 4 h. The formed precipitate was collected and dissolved in NaOH (20 ml, 10%). Then the mixture was filtered off and the filtrate was precipitated with HCl (0.1 N). The solid product was collected and recrystallized from ethanol to give pale brown crystals mp 157-9 °C, yield 60% IR: νmax/cm-1 3285 (NH), 3138 (NH). 1H NMR (DMSO-d6): δ 3.80 (s, 3H, OCH3), 7.29-7.33 (m, 4H, Ar H’s), 7.39 (d, 2H, J=8.8 Hz, Ar-H's), 7.48 (d, 2H, J=8.8 Hz, Ar-H's), 7.99 (s, 1H, prazole), 8.19 (s, 1H, pyrimidin), 8.23 (s, 1H, indole), 8.29 (s, 1H, pyrimidin), 11.34 (s, 1H, D2O Exch., indole NH), 12.16 (s, 1H, D2O Exch., indole SH). MS: m/z (%) 451 [M+, 18.0]. Anal. Calcd. For C24H17N7OS (451.50): C, 63.84; H, 3.80; N, 21.72. Found: C, 63.55; H, 3.72; N, 21.56.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 4-(methylthio)-1H-pyrazolo[3,4-d]pyrimidine (17)

Compound 16 (0.005 mol.) in ethanol (20 ml), and 10% sodium hydroxide solution (5 ml) were treated with methyl iodide (0.005 mol.). The reaction mixture was refluxed for 6 h, then cooled and poured into ice cold water. The separated solid was filtered off, dried and recrystallized from dioxane to give pale brown crystals mp 178-0°C, yield 68% IR: νmax/cm-1 3285 (NH). 1H NMR (DMSO-d6): δ 1.23 (s, 3H, SCH3), 3.89 (s, 3H, OCH3), 7.03-7.27 (m, 4H, Ar H’s), 7.39 (d, 2H, J=8.6 Hz, Ar-H's), 7.80 (s, 1H, prazole), 8.11 (d, 2H, J=8.6 Hz, Ar-H's), 8.17 (s, 1H, indole), 8.26 (s, 1H, pyrimidin), 8.95 (s, 1H, pyrimidin), 9.90 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 465 [M+, 18.0]. Anal. Calcd. For C25H19N7OS (465.5): C, 64.50; H, 4.11; N, 21.06. Found: C, 64.65; H, 3.97; N, 21.26.

1-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)- 4-hydrazinyl-1H-pyrazolo[3,4-d]pyrimidine (18)

Synthesis of compound (18):

Method A: To a solution of compound 15 (1.67 g, 0.005 mol) in dioxane (30 ml), hydrazine hydrate (2 ml) was added and the mixture was heated at reflux for 4 h. The reaction mixture was cooled and poured into water and the separated solid was filtered off, dried and recrystallized from dioxane.

Method B: Compound 17 (0.005 mol) was heated in hydrazine hydrate (20 ml) for 2 h, cooled then the reaction mixture was poured into water, neutralized with HCl, filtered, dried and purified with column chromatography (silica gel) using chloroform: methanol (9:1), as eluent to give pale brown crystals mp 165-6°C, yield 60% IR: νmax/cm-1 3456 (NH2), 3385 (NH), 3224 (NH). 1H NMR (DMSO-d6): δ 3.83 (s, 3H, OCH3), 7.02-7.16 (m, 4H, Ar H’s), 7.21 (d, 2H, J=8.8 Hz, Ar-H's), 7.32 (d, 2H, J=8.8 Hz, Ar-H's), 7.91 (s, 1H, prazole), 7.98 (s, 1H, pyrimidin), 8.24 (s, 1H, indole), 8.29 (s, 1H, pyrimidin), 8.99-918 (brs, 3H, D2O Exch., NH2, NH), 11.42 (s, 1H, D2O Exch., indole NH). MS: m/z (%) 449 [M+, 1.63]. Anal. Calcd. For C24H19N9O (449.47): C, 64.13; H, 4.26; N, 28.05. Found: C, 63.95; H, 4.45; N, 28.23.

Results and Discussion

Through several years of research and cross referencing other related journals on the preparation of chalcones and their derivatives, chalcone compound 3 and thiopyrimidinone 4 were prepared as reported in literature [1].

The hydrazino-pyrimidine derivative 5 was synthesized by reacting the thiopyrimidinone 4 with hydrazine hydrate in refluxing alcohol, the structure of compound 5 was confirmed by IR, 1H NMR, MS spectra and elemental analysis, where its IR revealed the absorption bands at νmax=3212 for the NH2 and 3184, 3109 cm-1 for the two NH groups, 1H NMR spectrum gave the signals at δ=8.93-8.95 as a broad singlet for NH2, hydrazine NH and at 11.65 ppm., for the indole NH respectively. MS spectrum substantiated it's exact molecular weight (cf. Scheme 1 and Experimental section).

medicinal-chemistry

Scheme 1

The hydrazino-pyrimidine derivative 5 was used as an assorted precursor for the synthesis of some biologically active heterocycles, where the amino pyrazole carbonitrile compound 6 was synthesized by refluxing a 1:1 mixture of compound 5 in ethanol with ethoxymethylene malononitrile for 4 h. The structure of compound 6 was confirmed by IR, MS, 1H NMR and elemental analysis, where its IR spectrum gave the absorption bands at νmax=3312 for NH2, 3175 for NH Indole and 2226 cm-1 for the CN group respectively, it’s 1H NMR substantiated an NH2 singlet at 8.86 and a singlet at δ=11.66 ppm for NH indole.

The pyrazolo pyrimidine compound 7 was synthesized by heating an alcoholic solution of compound 5 (10.0 mmol.) with acetylacetone (10.0 mmol.) at reflux temperature for 5 h. The FTIR spectrum of compound 7 disclosed the absorption bands at 3169 for NH indole, 1635 and 1575 cm-1 for C=N groups respectively. 1H NMR revealed NH indole singlet at δ=10.41, singlet pyrimidine H at δ=8.95 ppm and two singlets for the two CH3 protons respectively.

The pyrimidine pyrazolone compound 8 was obtained by refluxing a solution of compound 5 and ethyl acetoacetate in acetic acid for 6 h. The structure of compound 8 was deduced from its analytical and spectral data, which were in full agreement with the proposed structure (cf. Experimental section).

Furthermore, the hydrazino-pyrimidine derivative 5 was reacted with ethyl acetoacetate in excess to afford compound 9. IR spectrum of compound 9 revealed the absorption bands at νmax=3367 and 1715 cm-1 characteristic for NH indole and C=O groups respectively, 1H NMR exhibited the NH indole singlet at δ=10.82, two singlets at δ=1.23 and 2.43 for 2 CH3 protons and a singlet at δ=5.6 ppm for pyranone H respectively (Scheme 2).

medicinal-chemistry

Scheme 2

The 5-amino-1H-pyrazole-4-carbonitrile compound 6 was found to be an adequate, key starting for the synthesis of some other new heterocyclic compounds, where it was heated in formamide for 3 h., to produce compound 10. The structure of compound 10 was confirmed by its spectral and analytical data.

The pyrazolo pyrimidinone derivative 11 was obtained on one hand by refluxing compound 6 in excess formic acid and on the other hand by heating compound 12 under reflux condition in triethyl orthoformate. The IR spectrum of compound 11 substantiated the absorption bands of indole NH, pyrimidinone NH and C=O respectively at their prospective values. 1H NMR, MS and elemental analysis gave the confirmatory data for compound 11 (cf. Scheme 3 and Experimental section).

medicinal-chemistry

Scheme 3

Treatment of compound 6 with cold concentrated sulphuric acid portion wise and sterilizing on an ice bath for about an hour. afforded the pyrazolo carboxamide derivative 12. IR spectrum of compound 12 substantiated absorption bands at 3398 for pyrazole NH2, 3267 for amide NH2, 3114 for indole NH and 1640 for the amide C=O, it's 1H NMR displayed three singlets at δ 8.48, 9.14 and 11.42 ppm for (D2O exchangeable) pyrazole NH2, amide NH2 and indole NH respectively.

Continuing a series of synthesis the pyrazolo carboxamide derivative 12 was transformed into the pyrazolo triazinone 13 by the addition of sodium nitrite solution to a suspended solution of 12 in concentrated hydrochloric acid at 0-5°C with stirring at room temperature.

Structure of compound 13 was approved by IR, 1H NMR, MS measurements and elemental analysis (cf. Scheme 3 and Experimental section).

Refluxing a solution of compound 12 in dimethylformamide with 20% potassium hydroxide solution and carbon disulfide (5 ml) afforded the thioxopyrazolo pyrimidinone derivative 14, which structure was deduced from its analytical and spectral data, (cf. Experimental section).

The pyrimidinone derivative compound 11 was heated in phosphorus oxychloride for 10 h. to give compound 15 which in turn was transformed by reaction with thiourea into the pyrimidin-4-thiol compound 16.

Compound 16 in ethanol and 10% sodium hydroxide solution was treated with methyl iodide then the reaction mixture was subjected to reflux affording the methylthiopyrazolo pyrimidine derivative 17.

Compound 18 could be synthesized by reacting both of compounds 15 and 17 with hydrazine hydrate. Structures 15, 16, 17, and 18 were based on their correct elemental analysis and spectral data (cf. Scheme 4 and Experimental section).

medicinal-chemistry

Scheme 4

Anti-proliferative activity

The newly synthesized compounds 5, 7, 8, 9, 11, 12, 14 and 18 were tested for their in vitro antiproliferative activities in the National Cancer Institute (NCI), where a single dose (10 μM) of the test compounds was used against 60 cell lines panel assay [15-19].

The data were reported as mean-graph of the percent of growth of the treated cells, and presented as percentage growth inhibition (GI%) caused by the tested compounds (Table 1).

%Growth inhibition (GI%)
Subpanel tumor cell lines 5 7 8 9 11 12 14 18
Leukemia  
CCRF-CEM - nt nt 26 nt 22 nt -
HL-60(TB) - nt nt - nt - nt -
K-562 - nt nt 19 nt 33 nt 23
MOLT-4 - nt nt 31 nt 40 nt -
PRMI-8226 - nt nt 44 nt 58 nt 17
SR nt nt nt nt nt 34 nt -
Non-small cell lung cancer  
A549/ATCC - 13 33 28 34 41 72 58
EKVX Nt 11 17 nt 16 nt 25 nt
HOP-62 13 75 - - 55 43 NT 91
NCI-H226 20 45 54 nt L 69 55 73
HOP-92 - 10 nt 46 76 nt nt nt
NCI-H23 - - 24 11 26 - 31 -
NCI-H322M - 11 18 25 37 17 35 29
NCI-H460 - - 36 - 43 19 32 52
NCI-H522 11 30 43 96 69 L nt 35
Colon Cancer  
Colo 205 - - 17 - 12 - - -
HCC-2998 - - 10 - 25 - 23 -
HCT-116 - 14 49 17 57 48 48 66
HCT-15 - - 12 - 16 28 10 24
HT29 - - 44 - 30 24 39 22
KM12 - - 49 13 43 nt 23 nt
SW-620 - - 19 - 19 14 - 16
CNS Cancer  
SF-268 - 14 41 19 31 - 61 60
SF-295 - - 16 25 21 34 55 41
SF-539 - 46 L 28 89 74 94 67
SNB-19 - - 12 16 11 - 37 27
SNB-75 28 23 47 45 L 74 L L
U251 - 25 45 20 43 30 94 56
Melanoma  
LOX IMVI - - 26 18 39 - 32 29
MALME-3M - 17 39 19 51 16 55 67
M14 - 22 27 - 35 25 61 13
MDA-MB-435 - - 22 11 33 12 24 -
SK-MEL-2 - - 23 16 43 27 55 48
SK-MEL-28 - - 22 - 25 30 40 27
SK-MEL-5 - - 55 13 61 26 33 10
UACC-257 - - 31 11 39 39 80 43
UACC-62 - - 57 34 47 47 24 15
Ovarian Cancer  
IGROVI - - 16 - 26 - 25 -
OVCAR-3 - - 23 12 57 - 31 33
OVCAR-4 11 - 19 37 77 39 52 L
OVCAR-5 - - 15 - - 12 - -
OVCAR-8 - 55 32 21 59 27 L 71
NCI/ADR-RES - - 33 18 34 16 nt 17
SK-OV-3 - 28 28 - 61 19 53 70
Renal Cancer  
786-o - - 42 17 57 - L 28
A498 - 17 89 36 86 65 L 33
ACHN - - 43 22 63 55 34 47
CAKI-1 - 12 24 28 18 37 16 35
RXF 393 - 29 70 - 76 43 64 64
SN12C - - 12 18 17 21 31 11
TK-10 - - 28 - 45 - L -
UO-31 - 23 38 55 29 26 52 29
Prostate Cancer  
PC-3 10 - 29 34 26 57 65 17
DU-145 - - 28 - 20 - 46 17
Breast Cancer  
MCF7 21 - 30 16 30 30 18 23
MDA-MB-231/TCC - 15 56 36 65 49 59 48
HS 578T - 45 53 23 56 60 76 94
BT-549 - 28 69 12 47 27 87 -
T-47D - - 45 22 88 36 27 65
MDA-MB-468 - nt nt - nt - nt 32

Table 1: Percentage growth inhibitor (GI%) of in vitro subpanel tumor cell lines at 10 UM conc.

It was found that all tested compounds displayed significant activities against the tested cell lines using 10 μM concentration with positive cytotoxic effects (PCE) of 7/58, 23/53, 53/53, 52/53, /49/49, 41/57, 46/57 and 46/58 respectively (Table 2).

60 cell lines assay in one dose 10.0UM conc.
Compd No. MGI% PCE
5 0 7/58
7 10 23/53
8 35 53/53
9 19 41/57
11 44 52/53
12 29 46/57
14 50 49/49
18 34 46/58

Table 2: Mean growth inhibition percentage and positive cytotoxic effects of new compounds.

Compounds 7, 8, 9, 11, 12, 14 and 18 showed cytotoxic effects on the most of the cancer cell lines with mean growth inhibition percentage (MGI%) of 10%, 35%, 44%, 50%, 19%, 29% and 34% (Table 2) and compound 5 showed slight selective activity against HOP-62, NCI-H226, NCI- H522, SNB-75, OVCAR-4, PC-3 and MCF7 cancer cell lines with GI values of 13%, 20%, 11%, 28%, 11%, 11% and 21% respectively (Table 1).

With respect to broad spectrum antitumor activity, close examination of the data presented in Table 1 revealed that compounds 11, 14, and 18 are the most active derivatives showing effectiveness towards numerous cell lines belong to different tumor subpanels. Consequently, compounds 11, 14, and 18 were selected and tested against a panel of 60 different tumor cell lines at a 5 – log dose range [1-5].

These response parameters GI50, TGI, and LC50 were calculated for each cell line, using the known drug 5 – Fluorouracil (5 – FU) as a positive control.

Compounds 11, 14 and 18 exhibited remarkable growth inhibitory activity pattern against renal cancer (GI50=4.26, 3.43 and 12.30, μM) non-small cell lung cancer (GI50=5.65, 3.92 and 7.83 μM) breast cancer (GGI50I50=4.16, 4.16 and 6.16 μM) ovarian cancer GI50 9.54, 6.3 μM and melanoma cancer (GI50=9.58, 4.24 and 3.70 μM) CNS (GI50=5.43, 2.50 and 3.38 μM) prostate cancer GI 9.58, 4.24 and 3.7 (GI50=27.22, 4.89 and 18.8 μM) respectively

It was found also that compounds 11, 14 and are about 1.5-3.0 fold more active than the positive control 5 FU, with GI50 (10.47, 7.24 and 14.12 μM) TGI (58.8, 36.30 and 60.25 μM) and LC50 (> 100, 87.09 and 95.49 μM) value respectively (Table 3).

Subpanel tumor cell lines
Compd No. Activity Leukemia NSC lung cancer Colon cancer CNS Cancer Melanoma Ovarian cancer Renal cancer Prostate cancer Breast cancer MG-MIDa
11 GI50 84.5 5.65 51.9 5.43 6.56 9.54 4.26 27.22 4.16 10.47
  TGI b 42.65 b 33.65 19.85 60.25 39.81 b 67.09 58.8
LC50 b b b 97.72
2.5
b b b b b b
14 GI50 86.09 3.92 36.43 84.7 4.24 6.3 3.47 4.89 4.16 7.24
  TGI b 35.89 72.21 56.75 45.47 26.0 26.76 b 27.28 36.30
LC50 b 71.03 93.32 3.38 b 93.11 98.0 b 70.79 87.9
18 GI50 b 7.83 63.09 18.28 3.7 10.23 12.3 18.8 6.16 14.12
  TGI b 40.36 b 69.50 79.03 87.09 45.49 b 57.32 60.25
LC0 b 94.40 b b b b 97.72 b b 95.49
5-FU GI50 15.1 b 8.4 72.1 70.6 61.4 45.6 22.7 76.4 22.6
  TGI b b b b b b b b b b
LC50 B b b b b b b b b b

Table 3: Compounds 11, 14 and 18 mediam growth inhibitory (GI50 UM) total growth inhibitory (TGI, UM) and mediam lethal (LC50, UM) concentration of in vitro subpanel cell lines.

Comparing the antitumor activities of compounds 11, 14 and 18 with those of Gefitinib and Erlotinib showed that compounds 11, 14 and 18 (Table 4) possess activities almost equal to or higher than those of Gefitinib and Erlotinib against most cell lines except non-small lung (EKVX and NCI – H522), melanoma (SK – MEL – 28), ovarian cancer (IGROVI and SK – OV – 3) renal cancer (ACHN and TK- 10) and breast cancer (MDA-MB468) on the other hand compounds 8 and 12 showed selective activities toward CNS, renal and breast cancer cell lines, compound 9 gave selective activities toward leukemia cell lines, where compound 7 displayed moderate antitumor activities.

GI50(UM)
Subpanel tumor cell lines 11 14 18 Erlotinib Gefitinib
Non-small cell lung cancer
A549/ATCC 10.5 5.15 8.08 7.94 7.94
EKVX 8.59 5.12 nt 0.05 0.05
HOP-62 2.30 2.03 2.94 12.58 10.00
NCI-H226 3.20 2.66 2.66 6.30 15.84
NCI-H23 8.30 4.17 26.6 19.95 15.84
NCI-H460 7.24 6.72 nt 5.01 6.30
NCI-H522 4.63 3.65 11.10 1.00 6.30
HOP-92 nt nt 1.9 6.30 7.94
NCI-H322M nt nt 70.4 0.05 0.08
NCI-H460 nt nt 5.31 5.01 6.30
Colon Cancer
Colo 205 >100 15.3 >100 31.62 6.30
HCC-2998 >100 50.6 >100 79.34 10.00
HCT-116 3.38 6.74 4.35 5.01 7.94
HT29 >100 16.6 >100 50.11 3.98
KM12 29.9 >100 >100 63.09 7.94
CNS Cancer
SF-268 6.91 3.57 4.21 19.95 7.94
SF-295 7.01 1.94 nt 15.84 1.99
SF-539 3.46 2.37 2.5 12.58 10.00
SNB-19 21.4 2.93 3.17 3.98 12.58
SNB-75 1.82 1.57 1.9 12.58 6.30
U251 3.93 3.18 7.06 19.95 10.00
Melanoma
LOX IMVI 55.0 4.71 6.4 5.01 7.94
MALME-3M 4.07 3.3 4.73 5.01 3.16
M14 8.38 16.1 24.4 6.30 5.01
MDA-MB-435 9.55 4.91 >100 15.84 3.16
SK-MEL-2 3.38 3.41 4.40 12.58 12.58
SK-MEL-28 7.04 3.02 6.30 31.62 0.31
SK-MEL-5 3.67 - 32.4 15.84 3.98
UACC-257 4.86 3.55 4.96 100.00 6.30
UACC-62 2.93 2.35 15.7 1.25 5.01
Ovarian Cancer
IGROVI 32.5 7.34 27.7 0.25 0.20
OVCAR-3 9.36 7.68 5.05 3.16 5.01
OVCAR-4 3.41 2.11 3.95 19.95 7.94
OVCAR-5 58.0 >100 >100 19.95 10.00
OVCAR-8 4.42 3.46 5.01 7.94 10.00
NCI/ADR-RES 8.97 3.30 9.25 6.30 12.58
SK-OV-3 3.06 2.91 4.75 0.39 0.63
Renal Cancer
786-O 3.2 3.51 4.17 5.01 7.94
A498 1.01 1.75 15.8 1.58 0.4
ACHN 3.49 4.63 3.72 0.15 0.2
CAKI-1 5.08 3.05 >100 0.10 0.16
RXF 393 2.24 2.55 3.48 6.3 5.01
SN12C 5.24 4.70 78.0 6.3 6.3
TK-10 5.58 4.06 7.43 0.10 0.10
UO-31 5.97 1.95 11.6 1.99 1.25
Breast Cancer
MCF7 9.98 7.93 32.0 10.0 10.0
MDA-MB-231/ATCC 3.42 3.73 4.2 1.99 12.58
HS 578T 3.23 2.46 2.18 6.3 10.0
BT-549 2.98 2.02 5.02 39.81 7.94
T-47D 4.01 6.57 7.04 3. 16 6.3
MDA-MB-468 4.11 5.72 5.39 0.2 0.01

Table 4: GI50 values (UM) of compounds 11, 14 and 18 Gefitinib and Erlotinib over the most cell lines of non-small lung cancer, colon cancer, CNS cancer, Melanoma, Ovarian cancer, Renal cancer and Breast cancer.

Regarding the activity toward individual cell lines, compounds 9, 12 showed selective activity against leukemia cell lines CCRF-CEM, K – 562, MOLT -4 and PRMI – 8226 with GI values of 26%, 22%, 19%, 23%, 31%, 40% and 44%, 58%, respectively.

Compound 12 revealed selective activities towards SR leukemia cell lines with GI values of 34% whereas compound 18 disclosed weak activity against k-562 and PRMI-8226 cell lines with GI values of 23% and 17%.

Non-small lung A549/ATCC cell line proved to be selectively sensitive to 8, 9, 11, 12, 14 and 18 with GI values of 33% 34%, 72%, 28%, 41 and 58%. In addition compounds 7, 12, and 18 proved to be susceptible to the HOP-62, NCI-H226 and NCI/H522 cell lines with GI values of 33%, 34%, 91%, 45%, 69%, 73%, 30% lethal and 35%, where compounds 8, 11 and 14 showed activities against NCI-H226 and NCI-H22, NCI-H323M and NCI-H460 cell lines with GIS values of 54%, lethal 55%, 24%, 26%, 31%, 18%, 37%, 35%, 36%, 43%, and 32%.

Compounds 9 and 11 showed strong activities against HOP-92 and NCI-H522 cell lines in 76%, 46%, 69%, and 96%, Meanwhile, compounds 12 and 18 showed certain activity against NCI-H3222-M and NCI-H460 cell lines in 17%, 29%, 19% and 52% respectively, compounds 9, 11, and 14 showed activities in 55%, 25% and 25% against HOP-62, EKVX and NCI-H322M cancer cells.

For the different cell lines of colon cancer, compounds 8, 11, 12, 14 and 18, showed GI values of 49%, 57%, 48%, 48%, 66% against colon HCT-122 and 44%, 30%, 39%, 24%, 22% with colon HT29 cell lines, while compounds 11 and 14 demonstrated moderate activities against HCC- 2998 cancer cell lines with GI values 25% and 23% respectively, on the other hand compounds 12 and 18 verified sensitivity in 28% and 24% to colon HCT-15 cancer cell, compounds 8, 11, and 14 displayed modest activities against KM12 cancer cell with GI value 94%, 43% and 23% respectively.

On screening the activities toward CNS cancer cell lines, compounds 7, 8, 9, 11, 14, 12, 18 showed strong potency against CNS cancers, SF-539, SNB-75 and U251 with GI values of 46%, 89%, 94%, 28%, 74%, 67%, 23%, 47% lethal 74% lethal, 25%, 45%, 43%, 94%, 20%, 30% and 56%, Compounds 9, 11, 14 and 18 showed GI values of 31%, 61%, 19%, 60%, 21% 55%, 25% and 41% to CNS cancer SF-268 and SF- 295 cell lines, respectively. CNS cancer SNB-19 proved to be sensitive to compounds 14 and 18 with GI values of 37% and 27% while compounds 8 and 12 showed certain potency to SF-268 and SF-295 cancer cells with GI values of 41% and 34% respectively.

On screening of the activities of tested compounds towards melanoma it was found that compounds 8, 11 and 14 have the values of GI ranging from 22% to 80% against LOX, IMVI, MALME-3M, M14, MDA-MB-435, MDA-MB-435, SK-MEL-2, SK-MEL 28, SK- MEL-5 UACC-257 and UACC-62 cell lines respectively.

Compound 7 showed moderate activities against M14 cell line, whilst compounds 9 and 12 possessed activity against UACC-62 cell line with GI values of 34% and 47%, Melanoma SK-MEL-2, SK-MEL-28 UACC-257 cell lines were found to be sensitive to compounds 12 and 18 in as shown in Table 1, at the same time compound 18 was active towards LOX IMVI and MALME-3M cell lines GI, 29% and 67%, and compound 12 has slight activity against SK-MEL-5 cell line with GI value 26%.

The results obtained toward ovarian cancer cell lines revealed that compounds 7, 8 and 11 showed moderate activities against ovarian OVCAR-8 and SK-OV-3 cell lines, compounds 8, 11 and 14 were effective towards OVCAR-3 and OVCAR-4 cell lines while compounds 9 and 12 showed different activities against OVCR-8 OVCAR-4 cell lines respectively (cf, Table 1).

The reactivity of compounds 8, 11, 14 and 18 against Ovarian NCI3/ADR-RES, IGR-OVI, OVCAR-8 and OVCAR-4, OVCAR-3 and SK-OV-3 cell lines were also measured and recorded in Table 1.

In case of renal cancer, compounds 8, 11, 14 and 18 substantiated different activities against 786-O, A498, ACHN, CAKI-1, RXF393, SNI2C, TK-10 and UO-31 cell lines with GI values from 24%-lethal. Renal A498, ACHN, CAKI-I, SN12C and UO-31 cell lines were found to be sensitive to compounds 9 and 12 with GI values of 36%, 65%, 22%, 55%, 28%, 37%, 18%, 21%, 55% and 26% respectively, whereas RXF393 cell line was affected by compounds 7 and 12 with GI values of 29% and 43% respectively.

Prostate PC-3 and DU-145 cell lines were proved to be selectively sensitive to compounds 8, 11 and 14 with GI values of 29%, 26%, 65%, 28%, 20% and 46% respectively, while compounds 9 and 12 possessed selective activity towards PC-3 cell line with GI values of 34% and 57% respectively.

Regarding the breast cancer; MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D cell lines were found to possess convincing response to compounds 8, 9, 11, 12 and 18 with GI values ranging from 23% to 94%, compound 7 showed GI effectiveness against breast HS 578T and BT-549 cell lines with values of 45% and 28%, additionally compound 18 gave selective activity against breast MDA-MB-468 cell line with GI values of 32%.

Conclusion

The authors herein endeavored to design and synthesize new pyrazolopyrimidine derivatives and screen some of these compounds for their anti-proliferative activity.

Acknowledgements

The authors would like to express their great appreciation to the staff at Micro-analytical Center of Cairo University and the National Cancer Institute (NCI) in Egypt for their assistance with the collection of data.

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Review summary

  1. Joodi
    Posted on Sep 29 2016 at 9:09 pm
    Authors were well demonstrated about the synthesis of analogs; some compounds (compound 5, 6, and 11) were re-elaborated into various analogs, interesting heterocyclic derivatives with wide spectrum of biological activity, which they were mentioned in introduction.

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