For correspondence:-  Mohd Imran   Email: mran_inderlok@yahoo.co.in   Tel:+966535129629

Received: 26 September 2015        Accepted: 9 January 2016        Published: 28 February 2016

Citation: Imran M, A, Alsalman AJ. Synthesis and evaluation of antimicrobial activity of some 2-morpholinomethylamino-4-(7-unsubstituted/substituted coumarin-3-yl)-6-chlorosubstitutedphenyl pyrimidines. Trop J Pharm Res 2016; 15(2):393-404 doi: 10.4314/tjpr.v15i2.24

© 2016 The authors.
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Microbial infections have been creating problems for mankind since centuries and scientists have also developed a large number of antimicrobial agents for the treatment of these infections. According to one new report, about 40 new microbial diseases have been identified since 1970s and more than 2 million Americans are suffering from antibiotic resistance, of which about 23,000 die each year [1]. Because of the development of antibiotic resistance and emergence of new microbial diseases, there is a need to develop new antimicrobial agents for the treatment of microbial infections.

Pyrimidine derivatives have an important place in medicinal chemistry as these are associated with a broad range of biological activities [2-4] including antioxidant activity [5-7] and antimicrobial activity [8-13]. The clinical importance of pyrimidine nucleus is also evident by the marketing of clinically used pyrimidine derivatives as well as fused pyrimidine derivatives, for example, as antineoplastic agent (Tegafur), as vasodilator (Dipyridamole), as expectorant (Tasuldine) and as antibacterial agent (Trimethoprim, Piromidic Acid, Tetroxoprim, Metioprim), as antifungal agent (Flucytosine), and as antiviral agent (Broxuridine, Idoxuridine) [14]. Recently, the significance and biological importance of pyrimidine derivatives including their clinical applications in the microbial world has been reviewed [15]. The antimicrobial activity of pyrimidine derivatives against broad range of microbes makes it an important skeleton in medicinal chemistry and drug development against microbes. Morpholine nucleus is an integral part of linezolid, a clinically used drug for the treatment of infections caused by gram-positive bacteria [16]. A number of morpholine containing chemical compounds have also been reported as antimicrobial agents [17-22]. Encouraged by these observations and also in continuation of our search for potent antimicrobial agents [23,24] including antimicrobial agents having coumarin moiety [25,26], we decided to prepare some 2-morpholinomethylamino-4-(7-unsubstituted/substitutedcoumarin-3-yl)-6-chlorosubstitutedphenyl pyrimidines, herein after the title compounds (1a-18a), as antimicrobial agents.

General

Melting points were measured in open capillary tubes and are uncorrected. IR (KBr) spectra were recorded on a Nicolet, 5PC FT-IR spectrometer (Browser Morner, USA) and ¹H-NMR spectra on a Bruker DRX-300 FT NMR (Bruker, Germany) spectrophotometer using TMS as internal reference (chemical shift in δ ppm). Mass spectra were recorded on a Jeol-JMS-D-300 mass spectrometer (70 eV) (Jeol, Japan). Satisfactory analysis for C, H, and N was obtained for the compounds within ± 0.4 % of the theoretical values. Purity of the compounds was checked on silica gel G plates using iodine vapours as visualizing agent. R_f value of the compounds was determined by using a mixture of benzene and acetone (9:1). All reagents used in the present work were of analytical grade. The synthetic pathway for the preparation of the title compounds (1a-18a) is provided in .

The 2-amino-4-(7-substituted/unsubstituted coumarin-3-yl)-6-(chlorosubstitutedphenyl) pyrimidines (1-18) prepared according to our previous report [25] were reacted with morpholine and formaldehyde in absolute ethanol to provide the title compounds (1a-18a).

General method for the synthesis (1a-18a)

A mixture of 2-amino-4-(7-unsubstituted/ substituted coumarin-3-yl)-6-chlorosubstituted phenyl pyrimidines (0.01 mole), morpholine (0.01 mole) and formaldehyde (0.015 moles) was refluxed in absolute ethanol for 6 to 10 h. The reaction mixture was reduced to half of its volume and poured on crushed ice. The solid separated was filtered, washed with water repeatedly, dried and recrystallized from ethanol.

Evaluation of antimicrobial activity

The title compounds (1a-18a) were tested for their in vitro antimicrobial activity by serial plate dilution method [27,28] against Gram-positive bacteria, Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633) and Bacillus cereus (ATCC 9946); Gram-negative bacteria, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), Bordetella bronchiseptica (ATCC 4617) and Proteus vulgaris (ATCC 9920); fungi, Candida albicans (ATCC 2091), Aspergillus niger (MTCC 281), Aspergillus flavus (MTCC 277), Monascus purpureous (MTCC 369) and Penicillium citrinum (NCIM 768).

The microorganisms were obtained from the Department of Microbiology, Majeedia Hospital, New Delhi, India. The Department of Microbiology of Majeedia Hospital obtained some of these microorganisms from the Institute of Genomics and Integrative Biology, New Delhi, India.

Nutrient agar medium and Sabouraud dextrose medium were used for antibacterial activity and antifungal activity, respectively. The compounds were tested at concentrations of 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL. The reference or standard antibiotics, ofloxacin and ketoconazole, were used at 50, 25 and 12.5 μg/mL concentrations for antibacterial activity and antifungal activity, respectively. Sterile dimethyl sulfoxide (DMSO) was used for the preparation of desired concentrations of the synthesized compounds and standard antibiotics.  Sterile dimethyl sulfoxide without the synthesized compounds and standard antibiotics served as control group. The minimum inhibitory concentrations (MICs) values of the synthesized compounds, ofloxacin and ketoconazole, were also determined. The minimum inhibitory concentration (MIC) has been defined as the lowest concentration of a compound that inhibited visible growth of microorganisms on the plate.

Statistical analysis

All antimicrobial activity data are presented as mean ± SEM (n = 6). The data were analyzed by one-way analysis of variance (ANOVA) with Dunnett's Multiple Comparison Test with respect to control group and standard groups using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA). The results were considered significantly different at p < 0.05.

The title compounds (1a-18a) were prepared according to the method outlined in . The characterization data of the intermediates, (C), (D) and (1-18) of the were in line with our previously published data [25,26]. The structures of the title compounds (1a-18a) were confirmed on the basis of their IR, ¹H-NMR, Mass and elemental analysis data. The appearance of the IR absorption peaks from 3290 cm^-1 to 3280 cm^-1 confirmed the stretching vibration of N-H group of –NH-CH₂- moiety; from 1710 cm^-1 to 1705 cm^-1 confirmed the stretching vibration of C=O group of the coumarin moiety; from 1611 cm^-1 to 1604 cm^-1 confirmed the stretching vibration of C=N group of the pyrimidine ring; from 1545 cm^-1 to 1540 cm^-1 confirmed the stretching vibration of C=C group of aromatic C=C bond; and from 1135 cm^-1 to 1130 cm^-1 confirmed the stretching vibration of C-O-C group of coumarin moiety and morpholine moieties present in the title compounds (1a-18a). The appearance of the signals in the ¹H-NMR spectra of the title compounds (1a-18a) at δ (ppm) values from 2.63 to 2.72 confirmed four protons of –CH₂-N-CH₂- portion of the morpholine moiety; from 3.50 to 3.58 confirmed four protons of –CH₂-O-CH₂- portion of the morpholin moiety; from 4.27 to 4.42 confirmed two methylene protons of –NH-CH₂- moiety; from 6.90 to 7.75 confirmed the number of aromatic protons; and from 7.80 to 789 confirmed the secondary amino group (exchangeable with D₂O) of –NH-CH₂- moiety of the title compounds (1a-18a). The elemental analysis and molecular ion peaks of the title compounds (1a-18a) were also consistent with the assigned structures. The detailed physical constants, FTIR, ¹H-NMR, mass and elemental analysis data of the title compounds (1a-18a) are presented as follows.

2-(Morpholinomethylamino)-4-(coumarin-3-yl)-6-(4-chlorophenyl) pyrimidine (1a)

Yield: 55 %; m.p.: 147-149 ^oC; R_f: 0.76; IR (KBr) cm^-1: 3289 (N-H), 1706 (C=O), 1607 (C=N), 1544 (C=C), 1131 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.66 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.52 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.27 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.91-7.68 (m, 10H, Ar-H), 7.86 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₁N₄O₃Cl), Found% (Calculated%): C, 64.20 (64.21); H, 4.72 (4.72); N, 12.47 (12.48); Mass (m/z): 448 (M⁺, C₂₄H₂₁N₄O₃Cl), 449 (M⁺+1), 164 (100%, C₉H₇NCl).

2-(Morpholinomethylamino)-4-(coumarin-3-yl)-6-(2,6-dichlorophenyl)pyrimidine (2a)

Yield: 55%; m.p.: 140-142 ^oC; R_f: 0.74; IR (KBr) cm^-1: 3287 (N-H), 1707 (C=O), 1611 (C=N), 1543 (C=C), 1133 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.69 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.57 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.32 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.93-7.73 (m, 9H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₀N₄O₃Cl₂), Found% (Calculated%): C, 59.62 (59.64); H, 4.17 (4.17); N, 11.58 (11.59).

2-(Morpholinomethylamino)-4-(coumarin-3-yl)-6-(2,4-dichlorophenyl)pyrimidine (3a)

Yield: 50 %; m.p.: 155-157 ^oC; R_f: 0.71; IR (KBr) cm^-1: 3287 (N-H), 1706 (C=O), 1607 (C=N), 1542 (C=C), 1130 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.66 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.50 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.02-7.75 (m, 9H, Ar-H), 7.84 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₀N₄O₃Cl₂), Found% (Calculated%): C, 59.63 (59.64); H, 4.17 (4.17); N, 11.57 (11.59); Mass (m/z): 483 (M⁺, C₂₄H₂₀N₄O₃Cl₂), 484 (M⁺+1), 199 (100%, C₉H₆NCl₂).

2-(Morpholinomethylamino)-4-(coumarin-3-yl)-6-(2-chlorophenyl)pyrimidine (4a)

Yield: 55 %; m.p.: 147-149 ^oC; R_f: 0.77; IR (KBr) cm^-1: 3286 (N-H), 1706 (C=O), 1605 (C=N), 1545 (C=C), 1133 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.68 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.55 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.02-7.74 (m, 10H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₁N₄O₃Cl), Found% (Calculated%): C, 64.20 (64.21); H, 4.71 (4.72); N, 12.47 (12.48).

2-(Morpholinomethylamino)-4-(7-chlorocou-marin-3-yl)-6-(4-chlorophenyl)pyrimidine (5a)

Yield: 55 %; m.p.: 172-174 ^oC; R_f: 0.69; IR (KBr) cm^-1: 3280 (N-H), 1705 (C=O), 1607 (C=N), 1544 (C=C), 1130 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.69 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.53 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.90-7.71 (m, 9H, Ar-H), 7.85 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₀N₄O₃Cl₂), Found% (Calculated%): C, 59.62 (59.64); H, 4.16 (4.17); N, 11.57 (11.59); Mass (m/z): 483 (M⁺, C₂₄H₂₀N₄O₃Cl₂), 484 (M⁺+1), 164 (100%, C₉H₇NCl).

2-(Morpholinomethylamino)-4-(7-bromocou-marin-3-yl)-6-(4-chlorophenyl)pyrimidine (6a)

Yield: 60 %; m.p.: 177-179 ^oC; R_f: 0.66; IR (KBr) cm^-1: 3287 (N-H), 1710 (C=O), 1608 (C=N), 1542 (C=C), 1132 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.70 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.54 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.39 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.90-7.69 (m, 9H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₀N₄O₃ClBr), Found % (Calculated%): C, 54.60 (54.62); H, 3.82 (3.82); N, 10.60 (10.62).

2-(Morpholinomethylamino)-4-(7-chlorocou-mrin-3-yl)-6-(2,6-dichlorophenyl)pyrimidine (7a)

Yield: 50 %; m.p.: 185-187 ^oC; R_f: 0.69; IR (KBr) cm^-1: 3290 (N-H), 1708 (C=O), 1608 (C=N), 1544 (C=C), 1131 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.71 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.53 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.42 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.03-7.73 (m, 8H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₃), Found % (Calculated%): C, 55.66 (55.67); H, 3.70 (3.70); N, 10.81 (10.82).

2-(Morpholinomethylamino)-4-(7-bromocou-marin-3-yl)-6-(2,6-dichlorophenyl)pyrimidine (8a)

Yield: 55 %; m.p.: 171-173 ^oC; R_f: 0.66; IR (KBr) cm^-1: 3287 (N-H), 1705 (C=O), 1610 (C=N), 1543 (C=C), 1132 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.72 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.50 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.99-7.69 (m, 8H, Ar-H), 7.86 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₂Br), Found % (Calculated%): C, 51.25 (51.27); H, 3.40 (3.41); N, 9.95 (9.96); Mass (m/z): 562 (M⁺, C₂₄H₁₉N₄O₃Cl₂Br), 564 (M⁺+2), 199 (100%, C₉H₆NCl₂).

2-(Morpholinomethylamino)-4-(7-chlorocou-marin-3-yl)-6-(2,4-dichlorophenyl)pyrimidine (9a)

Yield: 55 %; m.p.: 173-175 ^oC; R_f: 0.72; IR (KBr) cm^-1: 3287 (N-H), 1706 (C=O), 1604 (C=N), 1544 (C=C), 1130 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.70 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.51 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.03-7.74 (m, 8H, Ar-H), 7.89 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₃), Found % (Calculated%): C, 55.66 (55.67); H, 3.70 (3.70); N, 10.82 (10.82).

2-(Morpholinomethylamino)-4-(7-bromocou-marin-3-yl)-6-(2,4-dichlorophenyl)pyrimidine (10a)

Yield: 60 %; m.p.: 176-178 ^oC; R_f: 0.71; IR (KBr) cm^-1: 3280 (N-H), 1705 (C=O), 1605 (C=N), 1540 (C=C), 1133 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.71 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.55 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.42 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.04-7.70 (m, 8H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₂Br), Found % (Calculated%): C, 51.25 (51.27); H, 3.40 (3.41); N, 9.95 (9.96).

2-(Morpholinomethylamino)-4-(7-chlorocou-marin-3-yl)-6-(2-chlorophenyl)pyrimidine (11a)

Yield: 50 %; m.p.: 185-187 ^oC; R_f: 0.72; IR (KBr) cm^-1: 3280 (N-H), 1705 (C=O), 1605 (C=N), 1542 (C=C), 1130 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.69 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.58 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.37 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.01-7.74 (m, 9H, Ar-H), 7.86 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₀N₄O₃Cl₂), Found % (Calculated %): C, 59.63 (59.64); H, 4.16 (4.17); N, 11.58 (11.59).

2-(Morpholinomethylamino)-4-(7-bromocou-marin-3-yl)-6-(2-chlorophenyl)pyrimidine (12a)

Yield: 55 %; m.p.: 176-178 ^oC; R_f: 0.76; IR (KBr) cm^-1: 3280 (N-H), 1706 (C=O), 1606 (C=N), 1544 (C=C), 1130 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.70 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.56 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 7.02-7.69 (m, 9H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₂₀N₄O₃ClBr), Found % (Calculated %): C, 54.61 (54.62); H, 3.81 (3.82); N, 10.60 (10.62); Mass (m/z): 527 (M⁺, C₂₄H₂₀N₄O₃ClBr), 529 (M⁺+2), 164 (100 %, C₉H₇NCl).

2-(Morpholinomethylamino)-4-(7-chlorocou-marin-3-yl)-6-(2,5-dichlorophenyl)pyrimidine (13a)

Yield: 45 %; m.p.: 190-192 ^oC; R_f: 0.82; IR (KBr) cm^-1: 3288 (N-H), 1707 (C=O), 1609 (C=N), 1541 (C=C), 1132 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.68 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.55 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.95-7.70 (m, 8H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₃), Found % (Calculated%): C, 55.66 (55.67); H, 3.68 (3.70); N, 10.81 (10.82).

2-(Morpholinomethylamino)-4-(7-bromocou-marin-3-yl)-6-(2,5-dichlorophenyl)pyrimidine (14a)

Yield: 50 %; m.p.: 210-212 ^oC; R_f: 0.75; IR (KBr) cm^-1: 3283 (N-H), 1709 (C=O), 1611 (C=N), 1544 (C=C), 1134 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.65 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.51 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.29 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.99-7.72 (m, 8H, Ar-H), 7.82 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₂Br), Found% (Calculated%): C, 51.26 (51.27); H, 3.40 (3.41); N, 9.95 (9.96).

2-(Morpholinomethylamino)-4-(7-chlorocoumarin-3-yl)-6-(3,5-dichlorophenyl)pyrimidine (15a)

Yield: 40 %; m.p.: 185-187 ^oC; R_f: 0.68; IR (KBr) cm^-1: 3285 (N-H), 1705 (C=O), 1607 (C=N), 1541 (C=C), 1133 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.63 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.54 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.39 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.97-7.68 (m, 8H, Ar-H), 7.80 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₃), Found% (Calculated%): C, 55.65 (55.67); H, 3.69 (3.70); N, 10.80 (10.82); Mass (m/z): 517 (M⁺, C₂₄H₁₉N₄O₃Cl₃), 518 (M⁺+1), 199 (100%, C₉H₆NCl₂).

2-(Morpholinomethylamino)-4-(7-bromocoumarin-3-yl)-6-(3,5-dichlorophenyl)pyrimidine (16a)

Yield: 55 %; m.p.: 173-175 ^oC; R_f: 0.73; IR (KBr) cm^-1: 3281 (N-H), 1707 (C=O), 1610 (C=N), 1543 (C=C), 1130 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.64 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.51 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.27 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.96-7.70 (m, 8H, Ar-H), 7.83 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₂Br), Found % (Calculated %): C, 51.26 (51.27); H, 3.40 (3.41); N, 9.95 (9.96).

2-(Morpholinomethylamino)-4-(7-chlorocoumarin-3-yl)-6-(3,4-dichlorophenyl)pyrimidine (17a)

Yield: 45 %; m.p.: 160-162 ^oC; R_f: 0.71; IR (KBr) cm^-1: 3286 (N-H), 1705 (C=O), 1606 (C=N), 1541 (C=C), 1132 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.68 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.57 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.33 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.95-7.72 (m, 8H, Ar-H), 7.85 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₃), Found % (Calculated %): C, 55.66 (55.67); H, 3.69 (3.70); N, 10.81 (10.82).

2-(Morpholinomethylamino)-4-(7-bromocoumarin-3-yl)-6-(3,4-dichlorophenyl)pyrimidine (18a)

Yield: 45 %; m.p.: 191-193 ^oC; R_f: 0.82; IR (KBr) cm^-1: 3289 (N-H), 1709 (C=O), 1605 (C=N), 1544 (C=C), 1135 (C-O-C); ¹H-NMR (CDCl₃, DMSO-d₆) d ppm: 2.66 (t, J = 8Hz, 4H, -CH₂-N-CH₂-), 3.56 (t, J = 8Hz, 4H, -CH₂-O-CH₂-), 4.35 (d, J = 12Hz, 2H, -NH-CH₂-N-), 6.95-7.73 (m, 8H, Ar-H), 7.88 (s, 1H, NH, exchangable with D₂O); Elemental Analysis (C₂₄H₁₉N₄O₃Cl₂Br), Found % (Calculated %): C, 51.26 (51.27); H, 3.40 (3.41); N, 9.95 (9.96); Mass (m/z): 562 (M⁺, C₂₄H₁₉N₄O₃Cl₂Br), 564 (M⁺+2), 199 (100%, C₉H₆NCl₂).

Antimicrobial activity

The antimicrobial activity data of the title compounds (1a-18a) at different concentrations against Gram positive bacteria, Gram negative bacteria and fungi is provided in , and , respectively. For the explanation of the antimicrobial activity result this discussion, the zone of inhibition produced by the MIC of standard drugs, ofloxacin and ketoconazole, has been considered as 100 % for comparing the antibacterial activity and antifungal activity data of the title compounds (1a-18a), respectively.

The antibacterial activity of ofloxacin against Gram positive bacteria revealed that it has a MIC value of 25 μg/mL against S. aureus, E. faecalis and S. epidermidis; and it has a MIC value of 12.5 μg/mL against B. subtilis and B. cereus. The antibacterial activity of the title compounds (1a-18a) with respect to ofloxacin revealed that the compound 4a (MIC = 100 μg/mL) displayed highest activity of about 93.98 % with p < 0.05 against S. aureus; the compound 15a (MIC = 125 μg/mL) displayed highest activity of about 83.60 % with p < 0.0001 against E. faecalis; the compound 9a (MIC = 100 μg/mL) displayed highest activity of about 91.03 % (p < 0.0001) against S. epidermidis; the compound 14a (MIC = 150 μg/mL) displayed highest activity of about 88.5 % with p < 0.0001 against B. subtilis; and the compound 18a (MIC = 100 μg/mL) displayed highest activity of about 91.45 % with p < 0.0001 against B. cereus.

Some compounds exhibited good but statistically non-significant antibacterial activity results (p > 0.05) with respect to ofloxacin, for example, the compound 13a (MIC = 100 μg/mL) displayed about 99.88 % activity against S. aureus; compound 18a (MIC = 125 μg/mL) displayed about 94.75 % activity against S. aureus; and compounds 15a (MIC = 175 μg/mL) displayed about 98 % activity against S. epidermidis. The antibacterial activity produced by the compounds 4a, 9a, 13a, 14a, 15a and 18a was lower than the antibacterial activity produced by ofloxacin and they also had a higher MIC values than ofloxacin. Other compounds did not produced comparable antibacterial activity against Gram positive bacteria even at higher concentrations with respect to ofloxacin.

The antibacterial activity of ofloxacin against Gram negative bacteria revealed that it has a MIC value of 12.5 μg/mL against E. coli; P. aeruginosa, K. pneumonia and P. vulgaris; and it has a MIC value of 25 μg/mL against B. bronchiseptica. The antibacterial activity of the title compounds (1a-18a) with respect to ofloxacin revealed that the compound 4a (MIC = 75 μg/mL) displayed the highest activity of about 90.83 % with p < 0.0001 against E. coli; the compounds 4a (MIC = 50 μg/mL) and 9a (MIC = 100 μg/mL) displayed highest activity of about 88.20 % and 88.46 %, respectively, with p < 0.0001 against P. aeruginosa; the compound 18a (MIC = 100 μg/mL) displayed highest activity of about 91.03 % with p < 0.05 against K. pneumonia; the compound 10a (MIC = 100 μg/mL) displayed highest activity of about 85.75 % (p < 0.0001); and the compound 9a (MIC = 75 μg/mL) displayed highest activity of about 92.72 % (p < 0.0001) against P. vulgaris. The antibacterial activity produced by the compounds 4a, 9a, 10a and 18a was lower than the antibacterial activity produced by ofloxacin and they also had a higher MIC value than ofloxacin. Other compounds did not produced comparable antibacterial activity against Gram negative bacteria even at higher concentrations with respect to ofloxacin.

The antifungal activity of ketoconazole against fungi revealed that it has a MIC value of 12.5 μg/mL against C. albicans, A. niger and M. purpureous; and it has a MIC value of 25 μg/mL against A. flavus and P. citrinum. The antifungal activity of the title compounds (1a-18a) with respect to ketoconazole revealed that the compound 10a (MIC = 100 μg/mL) displayed highest activity of about 86 % with p < 0.0001 against C. albicans; the compounds 5a (MIC = 100 μg/mL) and 16a (MIC = 100 μg/mL) displayed highest activity of about 94.35 % and 94.18, respectively, with p < 0.05 against A. niger; the compound 17a (MIC = 125 μg/mL) displayed highest activity of about 86.59 % with p < 0.05 against A. flavus; the compounds 5a (MIC = 50 μg/mL), 7a (MIC = 75 μg/mL), and 10a (MIC = 100 μg/mL) displayed highest activity of about 86.83, 84.95, and 87.24 %, respectively, with p < 0.0001 against M. purpureous; and the compounds 3a (MIC = 75 μg/mL) and 15a (MIC = 125 μg/mL) displayed highest activity of about 107.90 and 108.05 %, respectively, against P. citrinum. The antifungal activity produced by the compounds 3a and 15a was more than ketoconazole and statistically significant also. However, their MIC was also high. Some compounds exhibited good but statistically non-significant antifungal activity (p > 0.05) with respect to ketoconazole, for example, the compound 9a (MIC = 100 μg/mL) and compound 17a (MIC = 100 μg/mL), respectively, displayed about 95.81 and 95.05 % activity against A. niger; the compounds 3a (MIC = 75 μg/mL), 7a (MIC = 75 μg/mL), 9a (MIC = 75 μg/mL), 11a (MIC = 100 μg/mL), and 16a (MIC = 75 μg/mL), respectively, displayed activity of about 92.59, 93.67, 91.27%, 97.17, and 96.17 % against A. flavus. The antifungal activity produced by the compounds 3a, 5a, 7a, 9a, 10a, 11a, 16a, and 17a was lower than the antifungal activity produced by ketoconazole and they also had a higher MIC value than ketoconazole. Other compounds did not produced comparable antifungal activity against fungi even at higher concentrations with respect to ketoconazole.

A total of eighteen new compounds (1a-18a) were synthesized, and their structures were confirmed on the basis of their IR, ¹H-NMR, Mass and elemental analysis data. The characteristic peaks in ¹H-NMR spectra that confirmed the formation of the compounds (1a-18a) from the compounds (1-18) [25] were the appearance of the signals at δ (ppm) values from 2.63 to 2.72 for the four protons of –CH₂-N-CH₂- portion of the morpholine moiety; from 3.50 to 3.58 for the four protons of –CH₂-O-CH₂- portion of the morpholin moiety; and from 4.27 to 4.42 for the two methylene protons of –NH-CH₂- moiety. The compounds (1a-18a) were tested for their in vitro antimicrobial activity by serial plate dilution method [27,28] against five Gram-positive bacteria; five Gram-negative bacteria; and five fungi.

Compound 3a (MIC = 75 µg/mL; p < 0.0001) and 15a (MIC = 125 µg/mL; p < 0.001) produced superior antifungal activity than the standard drug, ketoconazole (MIC = 25 µg/mL; p < 0.0001) against P. citrinum. Compound 4a (MIC = 100 μg/mL; p < 0.05) displayed highest but moderate activity against S. aureus with respect to the standard drug, ofloxacin (MIC = 25 µg/mL; p < 0.0001). Compound 4a (MIC = 75 μg/mL; p < 0.0001) also exhibited highest but moderate activity against E. coli with respect to the standard drug, ofloxacin (MIC = 12.5 µg/mL; p < 0.0001). Further, compound 4a showed less activity than ofloxacin and also had higher MIC values than the standard drug ofloxacin. It is evident from the antimicrobial activity data mentioned in , , and that the title compounds are better antifungal agents than antibacterial agents. This is contrary to our earlier reported work [25]. This shows that the addition of the morpholine moiety to these type of compounds [25] increases their antifungal activity. Accordingly, there is a possibility that the replacement of the morpholine moiety by its bioisosteres, for example piperidine moiety, in these compounds may also produce promising antifungal compounds.  Accordingly, this study may be extended to acquire more information about the structure activity relationships of these type of compounds. It is also believed that the synthesized compounds might be inhibiting the growth of all tested microorganism by same mechanism as earlier reported pyrimidine moiety containing drugs [15].

The structure activity relationship study of the title compounds (1a-18a) revealed that the chloro substitution with respect to positions 2, 3 and 5 of the phenyl ring along with a chloro/bromo substituted coumarin moiety are critical for activity against Gram positive bacteria; the chlorosubstitution with respect to positions 2, 3, and 4 of the phenyl ring along with a chloro/bromo substituted coumarin moiety are critical for activity against Gram negative bacteria; the chlorosubstitution with respect to positions 2, 4, and 6 of the phenyl ring along with a chloro substituted coumarin moiety are critical for activity against fungi; the chloro substitution at position 2 of the phenyl ring along with a chloro/bromo substituted coumarin moiety is relatively more critical for activity against Gram positive bacteria, Gram negative bacteria and fungi.

It is evident from the antimicrobial activity data of the title compounds (1a-18a) that the compounds 3a and 15a produced higher antifungal activity than standard drug ketoconazole against P. citrinum. However, these compounds produced superior effect at higher concentration, and therefore, are considered to be less potent than ketoconazole. Some of the compounds displayed promising antibacterial activity at higher concentrations. It is also evident that the title compounds are better antifungal agents than as antibacterial agents. These compounds may be modified to achieve more potent antimicrobial activity. Accordingly, further studies to acquire more information about structure activity relationships are in progress in our laboratory.