INTRODUCTION:

The word pyrimidine was coined by Pinner in 1884 from a combination of the words ‘Pyridine’ and ‘amidine’ as it has the structural similarity to those compounds. Since these initial exploration hundreds of pyrimidine containing compounds have been emerged in medicinal field. Tremendous modifications upon this scaffold and its significance in nature make it an attractive area of study. Pyrimidine substituted with another heterocycle is widely used in the design and discovery of anticancer drugs. Gefitinib¹, Erlotinib and lapatinib² like anticancer drugs that act as tyrosine kinase inhibitors contains aminopyrimidine as their core moiety and has gained market approval worldwide.

The literature survey revealed that pyrimidine derivatives have been found to possess biological activities like Anti HIV³, Antiviral⁴, Antimicrobial⁵, Herbicidal⁶, Antitubercular⁷, Antitumor⁸, Antiinflammatory⁹, Anticonvulsant¹⁰, Antibacterial¹¹, Antimalarial¹² and Antithyroid¹³. Extensive studies on Pyrimidine derivatives are still in focus because of their diverse chemical reactivity, accessibility and wide range of biological activities. Pyrrole^14,15is widely known as a biologically active scaffold which possesses a diverse nature of activities. The attachment of different pharmacophores in a pyrrole ring system has led to the formation of more active compounds. Pyrrole containing analogs are a potential source of biologically active compounds that contains a significant set of advantageous properties and can be found in many natural products. The marketed drugs containing a pyrrole ring system are known to have many biological properties such as antipsychotic, β-adrenergic antagonist, anxiolytic, anticancer (leukemia, lymphoma and myelofibrosis etc.), antibacterial, antifungal, antiprotozoal, antimalarial and many more. Due to the diversity of these analogs in the therapeutic response profile, many researchers have been working to explore this skeleton to its maximum potential against several diseases or disorders.

The widespread properties of pyrrole and pyrimidine have prompted us to synthesize them in single molecular framework in order to study their pharmacological activity. Hence, the present investigation was undertaken to synthesize and to evaluate the anticancer activity of pyrimidine derivatives containing pyrrole nucleus.

MATERIALS AND METHODS:

Chemicals, Reagents and Cell Lines:

All the chemicals used were of analytical grade and procured from Sigma- Aldrich, Loba chemie Pvt Ltd, India and Sisco Research Laboratories Pvt. Ltd., India. o-Toluidine, Sulphuric acid, Nitric acid, Sodium hydroxide, 50% Ethanol, n- Butanol, 50% aqueous solution of cyanamide, Methanol, Diethyl ether, N, N-Dimethylformamide dimethyl acetal, Cyclohexane, Stannous chloride dihydrate, Hydrochloric acid, Ethyl acetate, Sodium sulfate, Dichloromethane, Thionyl chloride, Tetra Hydro Furan (THF), Tri Ethyl Amine (TEA), Propyl amine, Ammonia, and 2-Acetyl Pyrrole. Human lung cancer cell line A549 was purchased from National Centre for Cell Science. A549 cells were preserved in cryoperservation medium, which conatins 10% DMSO, 30% Fetal Bovine Serum and 60% DMEM.

Instrumentation:

Melting points was measured in open capillaries and were uncorrected. All compounds were checked by TLC with silica gel glass plates and viewed under UV light at 254 nm. The FT-IR spectrum was recorded in the range of 4000-450cm^-1 using Perkin Elmer Spectrum one FT-IR instrument at a resolution of 1.0cm^-1. H¹ NMR spectra were recorded in CDCl₃ on a Bruker Avance^TM III 500MHz NMR Spectrometer. Chemical shifts (δ) were reported in parts per million (ppm). Mass spectra for the newly synthesized compounds were obtained from the Q-Time of Flight Mass Spectrometer (Micromass).

Synthesis:

2-Methyl-5-nitroaniline (2)

51.1mL of o-toluidine (1) was added dropwise to 122.3mL of sulphuric acid cooled to ice cold condition with vigorous stirring. Mixture of 8.9mL of 65% nitric acid and 27.2mL of sulphuric acid was cooled to ice cold condition and added^{16, 17}to the above mixturefor a period of 2h. Finally, it was poured into crushed ice. It was made alkaline, with aqueous sodium hydroxide. The product formed was filtered and dried in air. Recrystallization was done using 50% ethanol.

N-(2-Methyl-5-nitrophenyl)guanidinium nitrate (3)

2-methyl-5-nitroaniline (2, 25g) in n-butanol (120mL) was taken. 65% aqueous nitric acid (10.5mL) was added dropwise. 50% aqueous solution of cyanamide (22.7mL) was added to the above mixture. The mixture was refluxed for 12h and subsequently it was cooled to 0^ºC. The precipitate formed was collected by filtration and washed with an ice cooled solution of 50% ethanol and diethyl ether taken in equal quantity. The product was dried in air for further use.

4-(Chloromethyl) benzoyl Chloride (10)

4-(hydroxyl methyl)-benzoic acid (9, 15.2g), dichloromethane (50mL), and thionyl chloride (50 mL) was added into a round bottomed flask and it was refluxed for 5h. The excess thionyl chloride and dichloromethane were removed. The residue was cooled to ice cold condition for 12h. White crystals. mp 30-32^°C

Scheme I

3-Dimethylamino-1-(1H-pyrrol-2-yl)prop-2-en-1-one (6a)

A mixture of 2-acetyl pyrrole (4a, 24.21g) and N, N-dimethylformamide dimethyl acetal (5, 34.4mL) were refluxed for 16h and then concentrated to get residue. Cyclohexane (100mL) was added to the residue and it was cooled to ice cold condition. The precipitate formed was filtered and dried in air. Yellow solid. mp 81- 82^ºC.

N-(2-methyl-5-nitrophenyl)-4-(1H-pyrrol-2-yl)pyrimidin-2-amine (7a)

To a mixture of 3-Dimethylamino-1-(1H-pyrrol-2-yl)prop-2-en-1-one (6a, 26.96g) and N-(2-methyl-5-nitrophenyl)guanidinium nitrate (3, 51.40g) in n-butanol (200mL), sodium hydroxide (8.63g) was added in solid form and it was refluxed for 16h. After the completion of 16h it was cooled in ice. The precipitation formed was filtered. It was washed with ethanol and diethyl ether. The product was dried in air for further use. Yellow powder. mp 197-199^ºC.

4-methyl-N-[4-(1H-pyrrol-2-yl)pyrimidin-2-yl]benzene-1,3-diamine (8a)

Stannous chloride di hydrate (11.29 g in 30mL of hydrochloric acid) was cooled in ice. N-(2-methyl-5-nitrophenyl)-4-(1H-pyrrol-2-yl)pyrimidin-2-amine (7a, 3.69g) in portions was added to the above mixture and stirred vigorously for 6h. The mixture was poured into crushed ice and it was made alkaline using solid sodium hydroxide. Extracted three times with ethyl acetate (100mL). The combined organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. Yellow crystals. mp 141-143^ͦC

4- (chloromethyl)–N-(4-methyl-3-{[4-(1H-pyrrol-2-yl) pyrimidin-2-yl]amino}phenyl) benzamide (11a)

4-methyl-N-[4-(1H-pyrrol-2-yl)pyrimidin-2-yl]benzene-1,3-diamine (0.277g), Tetra hydro furan (5mL), and Triethylamine (0.29mL) were refluxed. The reaction mixture was cooled in ice and maintained for 10 min. 4-(chloromethyl)benzoyl chloride (10, 0.217g) in Tetra Hydro Furan (2mL) was added dropwise to the above mixture within 10min. TLC of reaction mass indicated the absence of starting compound. After stirring the mixture at ice cold condition for 3h, 15mL of water was added dropwise. The resultant precipitate was collected by filtration and washed with 100mL of water. The product was dried at 75-80^°C. Yellow crystals. mp 267-269^°C

Compound 13f

4-[aminomethyl]-N-(4-methyl-3-{[4-(1H-pyrrol-2-yl)pyrimidin-2-yl]amino}phenyl) benzamide

4-(chloromethyl)-N-(4-methyl-3-{[4-(1H-pyrrol-2-yl)pyrimidin-2-yl]amino}phenyl)benzamide (0.429g) and ammonia (12f, 11.1mL) were added in a round bottomed flask and refluxed for 3h. The reaction mass was checked for the absence of compound (11a). The resultant mixture was cooled to room temperature and 10mL of water was added to it. The precipitate was collected by filtration and washed with 100mL of water. The product was dried at 75-80^C and recrystallized using methanol. White crystals. Yield 94.71%, Melting range 207-209^C; IR (KBr) cm^-13455.67 (Ar N-H str), 3077.18(Ar C-H str), 1580.45(-C=C-, -C=N- ring str), 2897.09 (-C-H-str), 2787.05 (N-H str), 1687(amide -C=O str), 1200.11(-C-C-str), 1008.43(C-Nstr), 806.45(N-H wag), 756.11 (C-H out of plane bending), 699.91(N-H out of plane bend); ¹H NMR (CDCl₃, δ, ppm) 8.64 (s, 2H), 8.08 (s, 1H), 7.87 – 7.64 (m, 3H), 7.45 – 6.84 (m, 7H), 6.49 – 6.29 (m, 1H), 4.79 (m, 1H), 4.10 (s, 2H), 2.29 (m, 3H), 1.35 (s, 2H). ms: m/z 399.4 [M+1].

Compound 13h

4-[ ( propylamino ) methyl] - N- ( 4- methyl - 3 - { [ 4- ( 1H- pyrrol- 2- yl) pyrimidin- 2 -yl] amino} phenyl)benzamide

4-(chloromethyl)-N-(4-methyl-3-{[4-(1H-pyrrol-2-yl)pyrimidin-2-yl]amino} phenyl) benzamide (0.429g) and propylamine (12h, 11.1mL) were added into a round bottomed flask and refluxed for 3h. The reaction mass was checked for the absence of compound (11a). The resultant mixture was cooled to room temperature and 10mL of water was added to it. The precipitate was collected by filtration and washed with 100mL of water. The product was dried at 75-80^C. and recrystallized using methanol. White crystals. Yield 97.40%, Melting range 218-220^C; IR (KBr) cm^-3457.09 (Ar N-H str), 2861.69 (-C-H-str), 2799.32 (N-H str), 1650.41(amide -C=O str), 1586.09 (-C=C-, -C=N- ring str), 1200.10 (-C-C-str), 1056.09 (C-N str), 700.61 (C-H out of plane bending), 805.09 (N-H wag), 700.61 (N-H out of plane bend); ¹H NMR (CDCl₃, δ, ppm) 8.64 (s, 2H), 8.13 – 7.74 (m, 4H), 7.44 – 6.86 (m, 8H), 6.39 (m, 1H), 4.79 (m, 1H), 2.87 – 2.60 (m, 4H), 2.29 (m, 3H), 1.95(s, 2H), 1.27(m, 3H). ms: m/z 441.2 [M+1], 442.9 [M+2]

Invitro anticancer activity

3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay

To determine cell viability and metabolizing cells present in a cell population, the colorimetric MTT metabolic assay [18-24]was used. The yellow coloured 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) along with NADH and NADPH reduces water soluble tetrazolium salts into a purple coloured and insoluble formazan which precipitates out of solution. Living cells in the population is proportional to the formation of the formazan crystals.

Method:

Fifty thousand Human lung cancer cells A549 were seeded in 96 well plates and incubated under standard culture conditions (37ºC) for 24h. After 24h, the medium was removed. Fresh medium containing synthesized compounds 13f, 13h and standard sunitinib in the concentration ranging from 5µM to 75µM were added to the cells and further incubated for 24 and 48h. After particular timeperiod the medium with the synthesized compounds 13f, 13h and standard sunitinib were removed. Cells were washed with 1X Phosphate Buffered Saline (1X PBS). After washing, 100µL of MTT dye was added to the cells, from the solution of 5mg/mL. The cells with MTT dye were incubated for 3h. After 3h, the medium was carefully removed without disturbing the formazan crystals. The formazan crystals formed by the activity of viable cells, were dissolved in 100µL of acidified isopropanol. The purple color solution was read at 570 nm as reference. The percentage of viable cells was calculated with the optical density. The Optical Density (OD) for control cells was assumed to be 100% viable and the corresponding viability in treated wells was calculated with respect to control cells.

Abs _Sample – Abs _Blank

% Viable cells = --------------------------------- X 100

Abs _Control – Abs _Blank

RESULTS AND DISCUSSION:

The designed compounds 13f and 13h were prepared by conventional method. These Compounds were characterized using IR, ¹H-NMR and Mass Spectroscopy. The IR spectrum of the synthesized compounds revealed the presence of Aromatic N-H stretching, -C-H stretching, -C-C stretching and –C=O amide stretching. In ¹H-NMR spectra, Alkyl protons and Aromatic protons are found in their appropriate δ values. Base peak, [M+1] peak and [M+2] peak were observed in mass spectra of the synthesized compounds.

The synthesized compounds 13f, 13h and standard drug sunitinib were tested against Human Lung cancer cell lines (A549) for their inhibitory effect on it. Various doses of synthesized compounds and the standard drug were added to cells for 24h and 48h of incubation. The curve of cell growth was determined by means of a MTT assay. In this assay, if the absorbance value of the control cell is higher compared to the absorbance value of the cells treated with anticancer drug than it indicates a reduction in the rate of cell proliferation.

Compound 13f and 13h showed a strong cytotoxic effect of 48.48% and 48.8% compared to the standard exhibiting 35.20% at the concentration of 25µM for 24h. At 48h compounds 13f and 13h showed 53.19% and 50.53% of strong inhibitory effect at 25µM concentration. The IC₅₀ value calculated using the obtained results was found to be 25µM.

Table 1: Percentage of scavenging activity at 24h

Compound Code	5µM	25µM	50µM	75µM
13f	20.16	48.48	51.84	55.84
13h	24.00	48.80	65.28	67.84
Standard	26.08	35.20	44.48	59.20

Table 2: Percentage of scavenging activity at 48h

Compound Code	5µM	25µM	50µM	75µM
13f	21.99	53.19	57.27	58.51
13h	42.11	50.53	64.45	64.63
Standard	27.84	29.79	53.90	67.02

CONCLUSION:

The present study comprises the synthesis and spectral studies of compounds 4-[aminomethyl]-N-(4-methyl-3-{[4-(1H-pyrrol-2-yl)pyrimidin-2-yl]amino}phenyl) benzamide (13f) and 4-[ ( propylamino ) methyl] - N- ( 4- methyl - 3 - { [ 4- ( 1H- pyrrol- 2- yl) pyrimidin- 2 -yl] amino} phenyl)benzamide (13h). The compounds were synthesized with good percentage of yield. Spectral data’s of the synthesized compounds were consistent with the assigned structure. Invitro studies conducted on Human Lung cancer cell lines (A549) using MTT assay method reported 48.48%, 48.80% and 35.20% of strong cytotoxic effect at a concentration of 25µM for 24h. In vitro anticancer activity revealed that 13f and 13h showed more potent anticancer activity as compared to the standard drug sunitinib. To conclude, the above findings clearly demonstrated that the compound 13f and 13h is a good anticancer agent that can be developed as a potential anticancer agent

ACKNOWLEDGEMENT:

The authors are grateful to the Founder Chairperson Dr. K. Veeramani of Periyar College of Pharmaceutical Sciences, Trichy-21, for extending necessary facilities to carry out this work.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

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Received on 23.11.2019 Modified on 06.02.2020

Research J. Pharm. and Tech. 2020; 13(12):6243-6247.

DOI: 10.5958/0974-360X.2020.01088.4