An Efficient Synthesis of Chalcone, Acetyl Pyrazzoline and Amino Pyrimidine Derivatives Incorporate Indole Nucleus as Antimycobacterial and Antimicrobial Agents

Antimicrobial diseases, caused by microbial species, are one of the most important diseases worldwide [1]. Tuberculosis (TB) is one of the most important chronic communicable bacterial diseases caused by Mycobacterium tuberculosis. TB is one of the major causes of morbidity and mortality throughout the world. Approximately 32% of the world‘s population is currently living with this infectious disease. It is a fatal disease and one of the leading causes of the death all over the world especially in the developing countries like India. The incidences of failure in the treatment of microbial infections have increased because of the emergence of multidrug-resistant strains due to misuse of antimicrobial drugs. Therefore, the synthesis of effective, novel antimicrobial compounds has become extremely important [2]. Chalcones (1,3-diaryl-2-propene-1-ones) and other biogenetically-related compounds belonging to the flavonoid family are natural substances found in a number of plants or prepared synthetically. They consist of two aromatic rings joined by a three-carbon α,β-unsaturated carbonyl system [3]. Chalcones have been found to exhibit many pharmacological activities, including anti-microbial [4] and anti-tuberculosis [5], anticancer [6], anti-inflammatory [7], antioxidant [8] etc activities. Pyrazolines are an important class of heterocyclic compounds containing two nitrogen atoms in the five membered ring. Pyrazoline derivatives are the electron rich nitrogen heterocycles which play an important role in the diverse biological activities. These heterocyclic compounds widely occur in nature in the form of alkaloids, vitamins, pigments and as constituents of plant and animal cell. Considerable attention has been focused on the pyrazolines and substituted pyrazolines due to their

[12] etc. Pyrimidines are the heterocyclic aromatic compounds similar to benzene and pyridine containing two nitrogen atoms at positions 1 and 3 of the six membered rings. Heterocycles containing pyrimidine moiety are of great interest because they constitute an important class of natural and nonnatural products, many of which exhibit useful biological activities and clinical applications [13]. The pyrimidines represent one of the most active classes of compounds possessing wide spectrum of biological activities like significant in vitro activity against antimicrobial [14], antileishmanial [15], anti-inflammatory [16], analgesic [17], antimycobacterial [18] etc. In view of the above mentioned knowledge of different pharmacophores, we have designed and synthesized some new chalcones and converted into its analogues acetyl pyrazolines and amino pyrimidine having indole scaffold. Compounds were subjected to evaluation of their antimicrobial and antimycobacterial potency against various strains.

The reagents and solvents used for reaction were of analytical reagent (AR) grade. Melting points were determined in open capillary method and are uncorrected. IR spectra were recorded on Shimadzu FTIR 8401 spectrophotometer using potassium bromide pellets. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 F (MHz) spectrometer (Bruker Scientific Corporation Ltd., Switzerland) using CDCl3 as a solvent and TMS as an internal standard at 400 MHz frequency respectively. Chemical shifts are reported in parts per million (ppm) and coupling constant (J) are reported in Hertz. Elemental analysis was carried out by Perkin-Elmer 2400 series-II elemental analyser (Perkin-Elmer, USA). Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan). TLC was run on E-Merck pre-coated 60 F254 plates and the spots were rendered visible by exposing to UV light or iodine chamber. Reference drugs antimicrobial and antitubercular activity are Ampicillin, Chloramphenicol, Ciprofloxacin, Griseofulvin, Nystatin, Rifampicin and Isoniazid used of commercial grade.

5-Methoxy-1H-indole-3-carbaldehyde (I) (0.01 mol), benzyl chloride (II) (0.01 mol) and anhydrous K2CO3 in dimethylformamide (DMF) were charged in a 100 ml round bottomed flask, fitted with a reflux condenser. The reaction mixture was heated under reflux temperature for 5-6 hours. After completion of the reaction as monitored by TLC, the reaction mixture was cooled, and poured onto water⋅ The precipitated solid was filtered off, washed with water, dried and recrystallized from ethanol gives 1-benzyl-5-methoxy-1H-indole-3-carbaldehyde (III). FTIR (KBr, vmax, cm-1): O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 3.8 (s, 3H, -OCH3), 3.9 (s, 2H, -CH2), 10.5 (s, 1H, -CHO), 6.5 to 8.6 (m, 9H, 08 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm) : 51.2 (CH2), 54.2 (OCH3), 103.4 (CH), 105.6 (CH), 112.4 (CH), 114.5 (CH), 118.1 (CH), 126.3 (CH),131.4 (CH), 132.5 (C), 136.0 (CH), 139.1 (CH), 133.2 (CH), 137.2 (CH), 141.8 (C), 143.2 (C), 151.2 (C), 152.4 (C-N), 175.2 (CO); LCMS (m/z): 266.1 (M+1). General method for the preparation of 3-(1-benzyl-5-methoxy-1H-indol-3-yl)-1-(substitutedphenyl)prop-2-en-1-one (Va-e) Substituted acetophenone (IVa-e) (0.01 mol) and 1-benzyl-5-methoxy-1H-indole-3-carbaldehyde (0.01 mol) (III) dissolved in isopropyl alcohol was taken in a 100 ml conical flask. The reaction proceed by applying classical Claisen-Schmidt condensation reaction i.e. To make it alkaline, solution of 40% KOH (5ml) was added in it. Then the reaction mixture was stirred for 24 hours on a magnetic stirrer at room temperature. The progress of reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into crushed ice, neutralized with dilute hydrochloric acid and the mixture was agitated for 4 hours a yellow solid was obtained. Finally, the product was isolated by filtration, crystallized from ethanol gives product 3-(1-benzyl-5-methoxy-1H-indol-3-yl)-(1-substitutedphenyl)prop-2-en-1-one (Va-e). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(2,5-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (Va) FTIR (KBr, vmax, cm-1): 3015 (aromatic =CH streching), 2970 (C-H streching of alkane), 1640 (C=O streching), 1550 (CH=CH streching), 1512 (aromatic C=C streching), 1416 (-OCH3 streching), 1262 (C-N streching), 1225 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 3.8-4.0 (m, 9H, -OCH3), 4.2 (s, 2H, -CH2), 5.8 (1H, d, CO-CH=, J = 6.2 Hz), 6.9 to 8.3 (m, 12H, 11 Ar-H and 1-CH of indole moiety), 8.2 (1H, d, Ar-CH=, J = 6.1Hz); 13C NMR (400 MHz, CDCl3, δ ppm) : 50.0 (CH2), 54.2, (OCH3), 111.4 (CH), 113.0 (CH), 115.2 (CH), 116.7 (CH), 118.4 (CH), 121.3 (=CH), 120.5 (CH), 121.4 (C), 130.2 (C), 136.7 (CH), 138.9 (CH), 140.5 (CH), 142.3 (CH), 144.2 (=CH), 151.8 (C), 150.3 (C), 154.9 (C), 161.3 (C-N), 178.2 (CO); LCMS (m/z): 398.9 (M+1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(2,4-dihydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (Vb) FTIR (KBr, vmax, cm-1): 3506 (-OH streching), 3026 (aromatic =CH streching), 2925 (C-H streching of

streching), 1518 (aromatic C=C streching), 1412 (-OCH3 streching), 1260 (C-N streching), 1220 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 3.9 (m, 3H, -OCH3), 5.3 (m, 3H, -OH), 5.5 (m, 3H, -OH), 4.9 (s, 2H, -CH2), 6.1 (1H, d, CO-CH=, J = 8.1 Hz), 6.9 to 8.3 (m, 12H, 11 Ar-H and 1-CH of indole moiety), 8.2 (1H, d, Ar-CH=, J = 8.3 Hz); 13C NMR (400 MHz, CDCl3, δ ppm) : 48.2 (CH2), 55.1, (OCH3), 112.3 (CH), 114.5 (CH), 116.3 (CH), 118.3 (CH), 119.3 (CH), 120.2 (=CH), 122.1 (CH), 123.7 (C), 128.8 (C), 131.4 (CH), 133.2 (CH), 139.3 (CH), 143.5 (CH), 145.8 (=CH), 150.2 (C), 157.1 (C), 159.4 (C), 162.5 (C-N), 172.5 (CO); LCMS (m/z): 400.5 (M+1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (Vc) FTIR (KBr, vmax, cm-1): 3069 (aromatic =CH streching), 2978 (C-H streching of alkane), 1630 (C=O streching), 1563 (CH=CH streching), 1532 (aromatic C=C streching), 1420 (-OCH3 streching), 1259 (C-N streching), 1212 (asymmetric C-O-C streching of ether linkage), 786 (C-Cl streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.8 (m, 3H, -OCH3), 4.3 (s, 2H, -CH2), 6.3 (1H, d, CO-CH=, J = 7.5 Hz), 6.8 to 8.0 (m, 12H, 11 Ar-H and 1-CH of indole moiety), 8.3 (1H, d, Ar-CH=, J = 7.6 Hz); 13C NMR (400 MHz, CDCl3, δ ppm) : 39.1 (CH2), 56.5, (OCH3), 109.4 (CH), 112.1 (CH), 114.8 (CH), 116.5 (CH), 117.4 (CH), 119.8 (=CH), 121.0 (CH), 122.9 (C), 125.7 (C), 130.0 (CH), 132.5 (CH), 134.6 (CH), 140.8 (CH), 142.8 (=CH), 152.3 (C), 155.0 (C), 160.6 (C-N), 169.3 (CO); LCMS (m/z): 434.5 (M-1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (Vd) FTIR (KBr, vmax, cm-1): 3025 (aromatic =CH streching), 2956 (C-H streching of alkane), 1649 (C=O streching), 1563 (CH=CH streching), 1518 (aromatic C=C streching), 1420 (-OCH3 streching), 1260 (C-N streching), 1221 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 3.6-4.0 (m, 12H, -OCH3), 4.6 (s, 2H, -CH2), 5.1 (1H, d, CO-CH=, J = 5.9 Hz), 7.0 to 8.1 (m, 11H, 10 Ar-H and 1-CH of indole moiety), 8.3 (1H, d, Ar-CH=, J = 5.8 Hz); 13C NMR (400 MHz, CDCl3, δ ppm) : 39.4 (CH2), 55.6, (OCH3), 110.5 (CH), 112.5 (CH), 114.7 (CH), 116.8 (CH), 117.0 (CH), 123.5 (=CH), 124.0 (CH), 126.7 (C), 131.2 (C), 134.6 (CH), 137.8 (CH), 142.6 (CH), 143.0 (CH), 145.7 (=CH), 150.5 (C), 152.8 (C), 153.0 (C), 160.8 (C-N), 169.0 (CO); LCMS (m/z): 458.2 (M+1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(3-chloro-4-pyridine)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (Ve) FTIR (KBr, vmax, cm-1): 3069 (aromatic =CH streching), 2967 (C-H streching of alkane), 1643 (C=O streching), streching), 1410 (-OCH3 streching), 1260 (C-N streching), 1232 (asymmetric C-O-C streching of ether linkage), 769 (C-Cl streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.8 (m, 3H, -OCH3), 3.9 (s, 2H, -CH2), 6.3 (1H, d, CO-CH=, J = 5.7 Hz), 6.5 to 7.9 (m, 12H, 11 Ar-H, CH of indole and CH of pyridine moiety), 8.0 (1H, d, Ar-CH=, J = 5.6 Hz); 13C NMR (400 MHz, CDCl3, δ ppm) : 36.5 (CH2), 57.2, (OCH3), 108.3 (CH), 111.2 (CH), 113.4 (CH), 115.2 (CH), 117.9 (CH), 120.5 (=CH), 122.4 (CH), 123.7 (C), 126.7 (C), 132.0 (CH), 134.7 (CH), 138.3 (CH), 141.9 (CH), 143.1 (=CH), 149.0 (C), 151.4 (C), 153.2 (C), 159.0 (C-N), 170.1 (CO); LCMS (m/z): 401.0 (M-1) General method for the preparation of 1-(3-(1-benzyl-5-methoxy-1H-indol-3-yl)-5-(substitutedphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (VIa-e) An appropriate chalcone (Va-e) (0.01 mol) and hydrazine hydrate (0.015 mol) was charged in a 100 ml round bottomed flask, fitted with a reflux condenser. To make the mixture acidic catalytic amount of glacial acetic acid (5 ml) was added. The reaction mixture was heated under reflux temperature for 5-6 hours. The progress of the reaction was investigated by TLC using toluene: methanol (12:6 v/v) eluent as mobile phase. After completion of the reaction, the mixture was cooled to room temperature then poured into crushed ice and neutralised with Na2CO3. The solid mass separated was collected by filtration, washed well with hot water and recrystallised from ethanol gives product (VIa-e) in good yield. 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(2,5-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (VIa) FTIR (KBr, vmax, cm-1): 3012 (aromatic =CH streching), 2925 (C-H streching of pyrazoline moiety), 1663 & 1576 (C=O and C=N streching of pyrazoline moiety), 1512 (aromatic C=C streching), 1354 (CH3 streching of pyrazoline moiety), 1248 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 2.6 (s, 3H, -COCH3), 3.2 (dd, 1H, -CHx-CH, J = 11.3 & 14.1 Hz), 3.6 (dd, 1H, -CHy-CH, J = 11.4 & 13.9 Hz), 4.8 (dd, 1H, -CH-CH2-Ar, J = 5.2 & 13.9 Hz), 3.8-3.9 (m, 9H, OCH3), 7.2 to 8.2 (m, 12H, 11 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 22.5 (CH3, pyrazoline moiety), 37.5 (CH2, methylene, pyrazoline moiety), 41.3 (CH3), 55.0 (OCH3), 61.0 (CH-Ar), 110.5 (CH), 114.5 (CH), 115.2 (CH), 118.3 (CH), 120.0 (CH), 122.7 (CH), 125.9 (CH), 128.3 (CH), 130.1 (C), 132.4 (CH), 133.2 (C), 143.4 (C), 150.2 (C), 151.7 (C-OCH3), 160.2 (C=N), 169.0 (CO pyrazoline moiety); LCMS (m/z): 482.5 (M-1).

FTIR (KBr, vmax, cm-1): 3405 (-OH streching), 3016 (aromatic =CH streching), 2963 (C-H streching of pyrazoline moiety), 1659 & 1571 (C=O and C=N streching of pyrazoline moiety), 1510 (aromatic C=C streching), 1350 (CH3 streching of pyrazoline moiety), 1224 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 2.2 (s, 3H, -COCH3), 3.5 (dd, 1H, -CHx-CH, J = 10.4 & 14.5 Hz), 3.7 (dd, 1H, -CHy-CH, J = 10.5 & 13.9 Hz), 5.0 (dd, 1H, -CH-CH2-Ar, J = 6.2 & 13.8 Hz), 3.8 (s, 3H, OCH3), 5.2 (s, 2H, OH), 7.0 to 8.3 (m, 12H, 11 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 19.2 (CH3, pyrazoline moiety), 36.3 (CH2, methylene, pyrazoline moiety), 38.1 (CH3), 50.2 (OCH3), 62.4 (CH-Ar), 111.6 (CH), 113.0 (CH), 116.7 (CH), 118.5 (CH), 122.4 (CH), 123.8 (CH), 124.1 (CH), 127.5 (CH), 129.2 (C), 131.2 (CH), 132.7 (C), 140.5 (C), 151.0 (C), 155.8 (C-OCH3), 158.2 (C=N), 167.2 (CO pyrazoline moiety); LCMS (m/z): 456.1 (M+1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-( 2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (VIc) FTIR (KBr, vmax, cm-1): 3110 (aromatic =CH streching), 2925 (C-H streching of pyrazoline moiety), 1669 & 1546 (C=O and C=N streching of pyrazoline moiety), 1512 (aromatic C=C streching), 1359 (CH3 streching of pyrazoline moiety), 1229 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 1.9 (s, 3H, -COCH3), 3.0 (dd, 1H, -CHx-CH, J = 9.8 & 12.1 Hz), 3.4 (dd, 1H, -CHy-CH, J = 9.7 & 12.4 Hz), 5.3 (dd, 1H, -CH-CH2-Ar, J = 9.6 & 12.4 Hz), 3.9 (s, 3H, OCH3), 6.9 to 8.2 (m, 12H, 11 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 18.6 (CH3, pyrazoline moiety), 31.0 (CH2, methylene, pyrazoline moiety), 39.4 (CH3), 58.2 (OCH3), 68.2 (CH-Ar), 112.5 (CH), 113.3 (CH), 116.2 (CH), 118.4 (CH), 121.3 (CH), 125.2 (CH), 129.0 (CH), 130.1 (CH), 132.4 (C), 133.0 (CH), 135.0 (C), 137.5 (C), 143.4 (C), 151.3 (C-OCH3), 156.1 (C=N), 160.8 (CO pyrazoline moiety); LCMS (m/z): 492.6 (M+1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (VId) FTIR (KBr, vmax, cm-1): 3026 (aromatic =CH streching), 2910 (C-H streching of pyrazoline moiety), 1665 & 1570 (C=O and C=N streching of pyrazoline moiety), 1508 (aromatic C=C streching), 1356 (CH3 streching of pyrazoline moiety), 1242 (asymmetric C-O-C streching of ether linkage); 1H NMR (400 MHz, CDCl3, δ ppm): 2.1 (s, 3H, -COCH3), 3.1 (dd, 1H, -CHx-CH, J = 11.5 & 13.2 Hz), 3.3 (dd, 1H, -CHy-CH, J = 11.4 & 13.2 Hz), 4.2 (dd, 1H, -CH-CH2-Ar, J = 5.9 & 13.9 Hz), 3.7-3.9 (m, 16H, OCH3), 6.5 to 8.1 (m, 11H, 10 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 22.5 (CH3, pyrazoline moiety), 37.5 (CH2, methylene, (C), 134.8 (CH), 135.1 (C), 145.3 (C), 151.0 (C), 156.7 (C-OCH3), 161.8 (C=N), 168.2 (CO pyrazoline moiety); LCMS (m/z): 483.2 (M+1). 1-(3-(1-Benzyl-5-methoxy-1H-indol-3-yl)-5-(3-chloro-4-pyridine)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (VIe) FTIR (KBr, vmax, cm-1): 3020 (aromatic =CH streching), 2960 (C-H streching of pyrazoline moiety), 1645 & 1525 (C=O and C=N streching of pyrazoline moiety), 1512 (aromatic C=C streching), 1356 (CH3 streching of pyrazoline moiety), 1220 (asymmetric C-O-C streching of ether linkage), 789 (C-Cl streching); 1H NMR (400 MHz, CDCl3, δ ppm): 2.6 (s, 3H, -COCH3), 3.5 (dd, 1H, -CHx-CH, J = 10.4 & 14.5 Hz), 3.6 (dd, 1H, -CHy-CH, J = 10.6 & 13.8 Hz), 5.2 (dd, 1H, -CH-CH2-Ar, J = 6.2 & 13.7 Hz), 3.9 (s, 3H, OCH3), 7.2 to 8.1 (m, 13H, 12 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 18.4 (CH3, pyrazoline moiety), 34.2 (CH2, methylene, pyrazoline moiety), 39.3 (CH3), 51.6 (OCH3), 63.3 (CH-Ar), 112.4 (CH), 114.5 (CH), 115.2 (CH), 116.4 (CH), 119.5 (CH), 121.3 (CH), 122.5 (CH), 128.4 (CH), 129.1 (C), 131.2 (CH), 132.7 (C), 142.7 (C), 150.1 (C), 156.8 (C-OCH3), 159.3 (C=N), 168.0 (CO pyrazoline moiety); LCMS (m/z): 429.2 (M+1). General method for the preparation of 4-(1-benzyl-5-methoxy-1H-indol-3-yl)-6-(substitutedphenyl)pyrimidin-2-amine (VIIa-e) Compound (Va-e) (0.01 mol) condensed with guanidine hydrochloride (0.01mol) in the presence of alkaline medium (5 ml 40% KOH) in ethanol at refluxed temperature for 5-6 hours in 100 ml round bottomed flask. The progress of the reaction was monitored by TLC using toluene: methanol (10:3 v/v) eluent as mobile phase. After completion of the reaction, the reaction mixture was poured into crushed ice and neutralised with dilute HCl. Finally, the product was filtered, washed with water, dried and recrystallised in acetone gives product (VIIa-e) with good yield. 4-(1-Benzyl-5-methoxy-1H-indol-3-yl)-6-(2,5-dimethoxyphenyl)pyrimidin-2-amine (VIIa) FTIR (KBr, vmax, cm-1): 3426 (NH2 str. 10 amine of pyrimidine moiety), 3061 (aromatic =CH streching), 2975 (C-H streching of pyrimidine moiety), 1652 (C=N streching of pyrimidine moiety), 1529 (aromatic C=C streching), 1221 (asymmetric C-O-C streching of ether linkage), 1129 (OCH3 streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.7-4.0 (m, 9H, OCH3), 5.2 (s, 2H,-NH2), 6.7 - 8.2 (m, 14H, 13 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 19.4 (CH2), 59.2 (OCH3), 103.2 (CH, pyrimidine moiety), 110.2 (CH), 113.5 (CH), 118.3 (CH), 120.4 (CH), 121.2 (CH), 130.8 (CH), 133.7 (CH), 137.0 (CH), 137.2 (C), 139.5 (C), 142.3 (C),

LCMS (m/z): 467.2 (M+1). 4-(1-Benzyl-5-methoxy-1H-indol-3-yl)-6-(2,4-dihydroxyphenyl)pyrimidin-2-amine (VIIb) FTIR (KBr, vmax, cm-1): 3526 (OH str.), 3426 (NH2 str. 10 amine of pyrimidine moiety), 1121 (OCH3 streching) 3054 (aromatic =CH streching), 2965 (C-H streching of pyrimidine moiety), 1640 (C=N streching of pyrimidine moiety), 1525 (aromatic C=C streching), 1221 (asymmetric C-O-C streching of ether linkage), 1129 (OCH3 streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.8-3.9 (m, 3H, OCH3), 5.4 (s, 2H,-NH2), 5.6-5.7 (s, 4H,-OH), 6.9 - 8.1 (m, 14H, 13 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 21.2 (CH2), 56.5 (OCH3), 104.7 (CH, pyrimidine moiety), 108.7 (CH), 112.0 (CH), 114.5 (CH), 119.7 (CH), 123.0 (CH), 131.7 (CH), 134.0 (CH), 136.8 (CH), 139.0 (C), 140.0 (C), 143.8 (C), 150.8 (C), 151.0, 152.4, 156.7 (C, pyrimidine moiety); LCMS (m/z): 409.5 (M+1). 4-(1-Benzyl-5-methoxy-1H-indol-3-yl)-6-(2,4-dichlorophenyl)pyrimidin-2-amine (VIIc) FTIR (KBr, vmax, cm-1): 3356 (NH2 str. 10 amine of pyrimidine moiety), 3019 (aromatic =CH streching), 2969 (C-H streching of pyrimidine moiety), 1620 (C=N streching of pyrimidine moiety), 1556 (aromatic C=C streching), 1241 (asymmetric C-O-C streching of ether linkage), 1076 (C-F streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.9 (s, 3H, OCH3), 5.6 (s, 2H,-NH2), 6.9 - 8.3 (m, 13H, 12 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 32.5 (CH2), 59.7 (OCH3), 99.5 (CH, pyrimidine moiety), 110.7 (CH), 113.5 (CH), 115.7 (CH), 117.0 (CH), 119.8 (CH), 123.3 (CH), 130.7 (CH), 132.8 (CH), 134.9 (C), 136.8 (C), 139.7 (C), 153.7 (C), 155.4, 156.7, 161.2 (C, pyrimidine moiety); LCMS (m/z): 446.3 (M+1). 4-(1-Benzyl-5-methoxy-1H-indol-3-yl)-6-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine (VIId) 3427 (NH2 str. 10 amine of pyrimidine moiety), 3066 (aromatic =CH streching), 2971 (C-H streching of pyrimidine moiety), 1663 (C=N streching of pyrimidine moiety), 1531 (aromatic C=C streching), 1236 (asymmetric C-O-C streching of ether linkage), 1136 (OCH3 streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.8-4.1 (m, 12H, OCH3), 5.3 (s, 2H,-NH2), 6.7 - 8.1 (m, 14H, 13 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 19.6 (CH2), 59.2 (OCH3), 104.5 (CH, pyrimidine moiety), 111.3 (CH), 112.3 (CH), 114.1 (CH), 119.2 (CH), 122.3 (CH), 125.4 (CH), 130.6 (CH), 135.3 (CH), 138.8 (C), 139.1 (C), 143.0 (C), 153.5 (C), 154.5, 155.2, 158.5 (C, pyrimidine moiety); LCMS (m/z): 437.2 (M+1). 4-(1-Benzyl-5-methoxy-1H-indol-3-yl)-6-(3-chloro-4-pyridine)pyrimidin-2-amine (VIIe) pyrimidine moiety), 3010 (aromatic =CH streching), 2953 (C-H streching of pyrimidine moiety), 1654 (C=N streching of pyrimidine moiety), 1524 (aromatic C=C streching), 123 (asymmetric C-O-C streching of ether linkage), 1123 (OCH3 streching),789 (OCH3 streching); 1H NMR (400 MHz, CDCl3, δ ppm): 3.9 (m, 3H, OCH3), 5.4 (s, 2H,-NH2), 6.9 - 8.2 (m, 14H, 13 Ar-H and 1-CH of indole moiety); 13C NMR (400 MHz, CDCl3, δ ppm): 19.2 (CH2), 56.1 (OCH3), 102.5 (CH, pyrimidine moiety), 110.2 (CH), 112.1 (CH), 115.3 (CH), 116.2 (CH), 123.7 (CH), 129.5 (CH), 134.0 (CH), 136.5 (CH), 138.8 (C), 139.5 (C), 141.2 (C), 153.4 (C), 150.2, 153.2, 156.8 (C, pyrimidine moiety); LCMS (m/z): 416.7 (M+1).

RESULT AND DISCUSSION

The reaction sequence for title the starting precursors (III) and chalcones (Va-e) are depicted in Scheme 1. The key intermediate chalcone (Va-e) is subjected to a cycloaddition condensation reaction with hydrazine hydrate and guanidine hydrochloride gives corresponding 1-acetylpyrazoline and 2-aminopyrimidine derivatives respectively as depicted synthetic path in Scheme 2. The formation of all these new heterocyclic derivatives were fully characterised by means of spectroscopic techniques such as FT-IR, 1H NMR, 13C NMR and LCMS. As an example, in the IR spectrum of compound Va, a strong absorption band is observed at 1550 and 1640 cm-1 which corresponds to the stretching vibration of the CH = CH and C=O functionality of α, β- unsaturated carbonyl group of chalcone moiety. The C=C functionality of aromatic ring was observed at 1512 cm-1 respectively.

Scheme 1. Methodical synthetic route for the target compounds (Va-g), (VIa-g) and (VIIa-g)

unsaturated carbonyl group protons. The other remaining eleven aromatic and indole protons appeared as a multiplet signal at δ 6.9-8.3 ppm. Finally, the 13C NMR spectra of the compound Va was recorded in CDCl3 and the spectral signals were in good agreement with the proposed structure. In the 13C NMR spectrum of compound Va, the most deshielded signal that appeared at δ 178.2 ppm was assigned to the carbonyl carbon of the chalcone moiety. The signal for CH = CH functionality of α, β- unsaturated carbonyl group was appeared at δ 121.3 and 144.2 ppm. The signals for aromatic carbons appeared between at δ 111.4-154.9 ppm in the 13C spectrum. In the IR spectrum of compound VIa, a strong absorption band is observed at 1663 cm-1 which corresponds to the stretching vibration of the C=O functionality of acetyl group attached at N1 position in pyrazoline ring. A broad stretching band for the C=N functionality of pyrazoline unit and C=C functionality of aromatic ring is observed at 1576 and 1512 cm-1 respectively. The C4''-H stretching of pyrazoline ring was observed at 2925 cm-1. A strong absorption band was observed at 1354 cm-1 due to the presence of the CH3 group. The 1H NMR spectrum of compound VIa showed a singlet at δ 2.6 ppm for the COCH3 protons. The pro-chiral methylene protons C4''-H of pyrazoline appeared as two distinct doublets of a doublet at δ 3.2 ppm (J = 11.3 & 14.1 Hz) and at δ 3.6 ppm (J = 11.4 & 13.9 Hz) for the CHx-CH and CHy-CH protons, thereby indicating that both the protons are magnetically non-equivalent and diastereotopic while the chiral C5''-H proton of pyrazoline appeared as a doublets of a doublet at δ 4.8 ppm (J = 5.2 & 13.9 Hz) due to CH-CH2-Ar proton. The other remaining twelve aromatic protons appeared as a multiplet signal at δ 7.2 - 8.2 ppm. Finally, the 13C NMR spectra of the cyclised product were recorded in CDCl3 and the spectral signals were in good agreement with the proposed structures. In the 13C NMR spectrum of compound VIa, the shielded signal at δ 37.5 and 41.3 ppm was assigned to the methylene and methyl carbon of pyrazoline ring. The most deshielded signal that appeared at δ 169.0 ppm was assigned to the carbonyl carbon of the acetyl group attached with the pyrazoline unit. The signals for aromatic carbons appeared between δ 110.5-151.7 ppm in the 13C spectrum. The IR spectrum of compound VIIa showed a strong characteristic band at 1652 cm-1 and 3426 cm-1 due to the C=N and NH2 group of pyrimidine ring. The C5''-H stretching of pyrimidine ring was observed at 2975 cm-1. The aromatic C=C stretching was observed at 1529 cm-1 respectively. The 1H NMR spectrum of compound VIIa showed a sharp singlet at δ 5.2 due to the NH2 protons, and it also showed a sharp singlet at δ 7.3 due to HC=C, which confirmed the cyclisation of the chalcone into a pyrimidine ring. The other remaining CH carbon of pyrimidine ring and signal at δ 152.4, 154.3 and 159.6 ppm assigned to the C=N carbon of pyrimidine ring which assigned the pyrimidine unit. The signals for aromatic carbons appeared between δ 113.4-156.5.0 ppm in the 13C spectrum. The obtained elemental analysis values are in good agreement with theoretical data. Further, mass spectra of all the title compounds showed molecular ion peak M+ corresponding to their exact mass which is in agreement with its proposed structure.

Antimicrobial activity [19] was screened against Staphylococcus aureus (MTCC 96) Streptococcus pyogenes (MTCC 442), Escherichia coli MTCC 443, Pseudomonas aeruginosa (MTCC 441) by using ampicillin, chloramphenicol and ciprofloxacin as the standard antibacterial drugs. Antifungal activity was screened against three fungal species Candida albicans (MTCC 227), Aspergillus niger (MTCC 282) and Aspergillus clavatus (MTCC 1323) by using griseofulvin and nystatin were used as the standard antifungal drugs. The minimal inhibitory concentration (MIC) of all the synthesised compounds was determined by the broth micro dilution method according to National Committee for Clinical Laboratory Standards (NCCLS) [20]. All the synthesised compounds (Va-e), (VIa-e) and (VIIa-e) were screened for their antibacterial and antifungal activities in three sets against bacteria and fungi used in the present protocol. The results are summarised in Table 2. Antimicrobial screening data of compounds chalcone (Va-e), 1- acetyl pyrazoline (VIa-e) and 2-amino pyrimidine derivatives (VIIa-e) shows that compound Vc and VIe showed an outstanding inhibitory effect i.e. MIC = 62.5 and 50 µg/ml against Staphylococcus aureus as compared ampicillin (MIC = 250 µg/ml) and moderate to chloramphenicol and ciprofloxacin

and VIIe (MIC = 100 µg/ml) showed better activity compared to ampicillin (MIC = 250 µg/ml) and poor to chloramphenicol and ciprofloxacin (MIC = 50 µg/ml) against Staphylococcus aureus. In the case of pathogenic Streptococcus pyogenes, compound Ve and Vic (MIC = 62.5 µg/ml) showed an outstanding inhibitory effect whereas compound Vb, Vc, Vd, VIb, VID, VIe, VIIa, VIIb and VIId (MIC = 100 µg/ml) were found to be comparable to ampicillin (MIC = 100 µg/ml) and moderate to chloramphenicol and ciprofloxacin (MIC = 50 µg/ml). Against Gram negative bacteria, compound Vc (MIC = 62.5 µg/ml) showed maximum activity against Escherichia coli as compared to ampicillin while compounds Va, Vb, Vd, VIc, VIe, VIIb and VIIc (MIC = 100 µg/ml) showed similar activity against Escherichia coli upon comparison with the standard drug ampicillin and lowest to chloramphenicol (MIC = 50 µg/ml) and ciprofloxacin (MIC = 25 µg/ml). Compound VIIc (MIC = 50 µg/ml) and Va and VIe (MIC = 62.5 µg/ml) showed excellent activity to ampicillin (MIC = 100 µg/ml) and modest to chloramphenicol (MIC = 50 µg/ml) and ciprofloxacin (MIC = 25 µg/ml) against Pseudomonas aeruginosa. The remaining compounds showed moderate to good activity to inhibit the growth of bacterial pathogens and were found less effective than the employed standard drugs. The antibacterial results revealed that most of the prepared compounds showed improved activity against the Gram-positive bacteria rather than Gram-negative bacteria. From in vitro antifungal activity data, it is found that compounds Vc, VIc and VIe (MIC = 100 µg/ml) displayed highest antifungal activity against Candida albicans as compared to griseofulvin (MIC = 500 µg/ml) and equivalent to nystatin (MIC = 100 µg/ml). Compounds Ve, VIa, VId, VIIa, VIIb, VIIc and VIId showed the same potency as griseofulvin (MIC = 500 µg/ml) against Candida albicans. Compound Vc, VIc and VIIc (MIC = 100 µg/ml) showed equipotent to griseofulvin (MIC = 100 µg/ml) and nystatin (MIC = 100 µg/ml) against Aspergillus niger. While compound Ve and VIe (MIC = 100 µg/ml) were found to be active against the fungal pathogen Aspergillus clavatus.

S. a.: Staphylococcus aureus, S. p.: Streptococcus pyogenes, E. c.: Escherichia coli, P. a.: Pseudomonas aeruginosa, C. a.: Candida albicans, A. n.: Aspergillus niger, A. c.: Aspergillus clavatus. Ampi: Ampicillin, Chlo.: Chloramphenicol, Cipr.: Ciprofloxacin, Gris.: Greseofulvin, Nyst.: Nystatin. ‗-‗: not tested.

The in vitro antitubercular activity of all the newly synthesized compounds were determined by using Lowenstein-Jensen medium (conventional method) against Mycobacterial tuberculosis H37Rv strain [20]. The observed results are presented in Table 3 in the form of inhibition (%), relative to that of standard antitubercular drugs isoniazid and rifampicin. Compounds demonstrating more than 90% inhibition in the primary screening were retested at lower concentration (MIC) in a Lowenstein–Jensen medium and evaluated for their MIC values. Among the compounds screened for antitubercular activity, compounds Vb (MIC = 50 µg/ml), Vc (MIC = 62.5 µg/ml), VIb (MIC = 50 µg/ml) VId (MIC = 100 µg/ml), VIIa (MIC = 62.5 µg/ml), VIIb (MIC = 100 µg/ml) and VIIe (MIC = 62.5 µg/ml) were found to possess the greatest potency against Mycobacterium tuberculosis with 82, 86, 84, 90, 96, 83 and 99 % inhibition respectively (Table 3). Other derivatives showed moderate to poor antitubercular activity.

CONCLUSION

A new class of chalcone and its derivatives, as a novel class of ant tubercular and antimicrobial agents was synthesized. The newly synthesized novel heterocyclic showed good ant tubercular and antimicrobial activities against both drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis as well as antimicrobial species. These results make new indole clubbed chalcone, pyrazoline and pyrimidine derivatives interesting lead molecules for further synthetic and biological evaluation.

The authors are grateful thankful to RSIC Punjab University for the FTIR analysis, 1H NMR, and 13C NMR spectral analysis as well as elemental analysis

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Department of Chemistry, Veer Bahadur Singh Purvanchal University, Jaunpur jayesh77777@yahoo.co.in