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Synthesis, spectroscopic and antibacterial studies of some schiff bases of 4-(4-bromophenyl)-6-(4-chlorophenyl)-2-aminopyrimidine
⁎Corresponding author. prasadpharmachem@gmail.com (Sunnapu Prasad)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
The chalcone of p-bromoacetophenone and p-chlorobenzaldehyde was prepared and converted into aminopyrimidine by treating with guanidine nitrate. The aminopyrimidines so obtained were converted into Schiff bases by treating with different substituted benzaldehydes. The structures of synthesized compounds were confirmed by physical parameters and spectral studies such as UV, IR, and PMR. The synthesized Schiff bases were screened with different bacterial species for the antibacterial activity by cup plate method and serial dilution technique. The confirmation of the biological activity of the final compounds was established by bioautography. The mono and dihydroxy amino pyrimidines (I-g, I-j) of the novel series were found to be potential against bacteria.
Keywords
Chalcone
2-Aminopyrimidine
Schiff bases
Antibacterial activity
Bioautography
Spectral studies
1 Introduction
Antibacterial agents inhibit the growth and survival of microorganism without any serious toxicity to the host. The life-threatening infectious diseases caused by multidrug-resistant bacteria have been increased day by day throughout the world. The spread of antibiotic resistance has become a major problem for managing infectious diseases. But for most of the antibacterial agents in clinical practice, resistance was reported for at least one bacterial pathogen.
However, because of the development of resistance by the organism toward some bacteria-antibiotic combinations, their clinical utility was severely declined from the market within a 5-year time span. The need of antibiotics is closely tied to the rapidity and proliferation of resistance developed by the organism and a number of other drugs used for the same therapeutic indication. The recent flurry of research activity is to develop drugs for treating multidrug resistant (MDR) gram positive bacteria, including MRSA (Methicillin resistant Staphylococcus aureus), Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp., Enterococcus faecium, Enterobacteriaceae and Mycobacterium tuberculosis.
There are always some unpredictable factors influencing the antimicrobial drug discovery such as the appearance of new diseases, or newly recognized association of established diseases with infectious agents. Examples of previously unrecognized infectious diseases are Legionnaire’s disease that was found to be associated with Legionella pneumophila (Bush, 2004). Chalcones show various activities such as anti-inflammatory, antibacterial and antifungal activities (Kalirajan et al., 2009). Aminopyrimidines (2-,4-,5-,6-aminopyrimidines) were reported for having various antibacterial (Rashinkar et al., 2009) antifungal and antituberculosis (Patel et al., 2006) activities against different microorganisms. Based on the above observations and in continuation of our research on antimicrobial agents an attempt was made here to synthesis the Schiff’s bases of amino pyrimidines (Gulcan et al., 2012).
2 Materials and methods
All the chemicals used in this study were ordered and purchased from sigma Aldrich chemicals. Melting point is carried out in open capillary tube by using melting point apparatus LAB INDIA MR-VIS Scientific. The purity of the compounds was confirmed by TLC (pre-coated) SiO2 gel aluminum plates (E MERK) using toluene: methanol: ethyl acetate (6:3:1) and visualized by iodine chamber technique. IR spectra were recorded on JASCO FT/IR-140 spectrophotometer. UV spectra were recorded on JASCO V-530 UV/VIS spectrophotometer. 1H NMR spectra were recorded on BRUKER ULTRA SHIELDED NMR-300 MHZ.
2.1 Step 1: Synthesis of 3-(4-bromophenyl)-1-(4-chlorophenyl) prop-2-en-1-one (I)
The compound (I) was synthesized by reported method (Vogel’s text book 5th edition). A solution of 22 g of sodium hydroxide in 200 ml of water and 100 ml of rectified spirit in a 500 ml, bolt-head flask provided with a mechanical stirrer was immersed in an ice bath (0.02 mole) of freshly distilled p-bromoacetophenone. It was stirred and then 0.01 mole of p-chlorobenzaldehyde was added. The temperature of the mixture was maintained at 25 °C and stirred vigorously until the mixture was so thick that stirring was no longer effective. Then the stirrer was removed and the reaction mixture was left in an ice chest or refrigerator overnight. The product was filtered with suction on a Buchner funnel, washed with cold water until the washings are neutral to litmus and then again washed with 20 ml of ice-cold rectified spirit. The crude chalcone, after dried in the air, was recrystallized from rectified spirit.
2.2 Step 2: Synthesis of 4-(4-bromophenyl)-6-(4-chlorophenyl)-2-aminopyrimidine (I-1)
The compound (I-1) was synthesized by reported method (Kumar et al., 2011). The chalcone (0.01 mole) was dissolved in alcohol (25 ml) and guanidine nitrate (0.01 mole) was added to it. Then a solution of potassium hydroxide (5 ml of 40%) was added to the reaction mixture and refluxed for 10 h. The reaction mixture was then cooled, poured into crushed ice and product separated out was filtered, washed with water, dried and recrystallized from alcohol.
2.3 Step 3: Synthesis of Schiff base (I-a–I-h)
The compounds (I-a–I-h) were synthesized by reported method (Kadhim et al., 2014). A mixture of 4-(4-bromophenyl)-6-(4-chlorophenyl)-2-aminopyrimidine (0.01 mole) and substituted benzaldehyde (0.01 mole) in ethanol (25 ml) with a few drops of glacial acetic acid were refluxed for a period of 4 h and kept for overnight until the precipitate is obtained. The solid separated was filtered, dried and recrystallized from ethanol. The purity of the product was established by single spot on the TLC plate. The solvent system used was toluene: methanol: ethyl acetate (6:3:1).
2.3.1 3-(4-Bromophenyl)-1-(4-chlorophenyl) prop-2-en-1-one (I)
Yield: 80%. M.p: 168.2 °C. IR (νmax, cm−1): 3419.17 (N—H), 1655.59 (—C⚌O), 1602.56 (Ar·C—C), 1561.09 (N—O), 833.098 (C—Cl), 538.042 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 8.192–8.289 (m, 8H, Ar—CH), 7.554 (d, 1H, α-H, Olefin H), 7.755 (d, 1H, β-H, Olefin H). UV (λmax, DMSO): 320.
2.3.2 4-(4-Bromophenyl)-6-(4-chlorophenyl) 2-aminopyrimidine (I-1)
Yield: 70%. M.p: 177.7 °C. IR (νmax, cm−1): 3463.53 (N—H), 1639.2 (—C⚌O), 1486.85 (Ar·C—C), 1579.41 (N—H), 804.171 (C—Cl) 492.723 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 8.191–8.289 (m, 8H, Ar—CH), 7.755 (s, 1H, Pyrimidine-H), 3.483 (s, 2H, —NH). UV (λmax, DMSO): 269.
2.3.3 [4-(4-Bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(4-methoxyphenyl) methanimine (I-a)
Yield: 64%. M.p: 187.5 °C. IR (νmax, cm−1): 3464.49 (N—H), 1639.2 (—C⚌O), 1594.84 (N—H), 1578.45 (Ar·C—C), 726.068 (C—Cl), 659.536 (C—Br) 1H NMR: (DMSO-d6, δ, ppm): 8.192–8.289 (m, 12H, Ar C—H), 7.587 (s, H, Pyrimidine-H) 9.855 (s, 1H, N⚌CH) 3.603 (s, 3H, —OCH3). UV (λmax, DMSO): 268.
2.3.4 [4-(4-Bromophenyll)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(4-fluorophenyl) metanimine(I-b)
Yield: 60%. M.p: 183.8 °C. IR (νmax, cm−1): 3466.42 (N—H), 3306.36 (O—H), 3190.65 (C—H), 1640.16 (—C⚌O), 1577.49 (Ar·C—C), 1456.96 (C—H), 1360.53 (Ar·C—N), 968.09 (O—H), 828.277 (C—Cl), 583.361 (C—Br) 1H NMR: (DMSO-d6, δ, ppm): 7.321–8.289 (m, 12H, Ar—H, s,1H, HC⚌C), 2.521 (s, 1H, HC⚌N). UV (λmax, DMSO): 264.
2.3.5 [4-(4-Bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(3,4-dimethoxyphenyl) methanimine (I-c)
Yield: 61%. M.p: 180.4 °C. IR (νmax, cm−1): 3466.42 (N—H), 2935.13 (C—H), 2719.14 (C⚌O), 1680.66 (Ar·C—C), 1386.57 (Ar·C—N) 1134.9 (C—O), 808.028 (C—Cl), 583.361 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.588–8.288 (m, 11H, Ar—H, s, 1H, HC⚌C), 2.522 (s, 1H, HC⚌N), 3.394 (s, 3H, OCH3). UV (λmax, DMSO): 258.
2.3.6 [4-(4-Bromophenyll)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(3,4,5-trimethoxyphenyl)metanimine (I-d)
Yield: 55%. M.p: 176.6 °C. IR (νmax, cm−1): 3466.42 (N—H), 3306.36 (O—H), 3188.72 (C—H), 1640.16 (—C⚌O), 1577.49 (Ar·C—C), 1457.92 (C—H) 1360.53 (Ar·C—N), 804 (C—Cl), 583.361 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.525–8.128 (m, 10H, Ar—H, s, 1H, HC⚌C), 2.514 (s, 1H, HC⚌N), 3.376 (s, 3H, OCH3). UV (λmax, DMSO): 269.
2.3.7 [4-(4-Bromophenyll)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(3-nitrophenyl)metanimine (I-e)
Yield: 52%. M.p: 174.8 °C. IR (νmax, cm−1): 3467.38 (N—H), 1640.16 (—C⚌O), 1578.45 (Ar·N—H), 1535.06 (Ar·C—C), 1384.64 (Ar·C—N), 1376.53 (N—O), 805.135 (C—Cl), 585.29 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.586–8.286 (m, 12H, Ar—H, s-1H, HC⚌C) 2.517 (s, 1H, HC⚌N). UV (λmax, DMSO): 267.
2.3.8 [4-(4-Bromophenyll)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(4-nitrophenyl)metanimine (I-f)
Yield: 50%. M.p: 178.6 °C. IR (νmax, cm−1): 3434.6 (N—H), 1487.81 (C—H), 1583.27 (Ar·C—C), 816.706 (C—Cl), 603.61 (C—Br), 1675.84 (Ar·N—H), 1345.11 (Ar·C—N), 1267.97 (C—N). 1H NMR: (DMSO-d6, δ, ppm): 7.586–8.283 (m, 12H, Ar—H, s, 1H, HC⚌C), 2.510–2.515 (s, 1H, HC⚌N). UV (λmax, DMSO): 266.
2.3.9 4-[(E) {[4-(4-bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]imino} methyl]phenol(I-g)
Yield: 62%. M.p: 194.6 °C. IR (νmax, cm−1): 3467.38 (N—H), 3308.29 (O—H) 1640.16 (C⚌O), 1578.45 (Ar·N—H) 1535.06 (Ar·C—C), 1384.64 (Ar·C—N), 1376.53 (N—O), 805.135 (C—Cl), 585.29 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.295–8.288 (m, 12H, Ar—H, s, 1H, HC⚌C), 2.505–2.515 (s, 1H, HC⚌N). UV (λmax, DMSO): 269.
2.3.10 [4-(4-Bromophenyll)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-(2-chlorophenyl)methanimine (I-h)
Yield: 60%. M.p: 191.8 °C. IR (νmax, cm−1): 3467.38 (N—H), 3307.32 (O—H), 3188.72 (C—H), 1640.16 (C⚌O), 1577.49 (Ar·N—H), 1536.02 (Ar·C—C), 1486.85 (C—H) 1386.57 (Ar·C—N), 1360.53 (N—O) 968.09 (O—H), 804.171 (C—Cl), 584.325 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.586–8.284 (m, 12H, Ar—H, s, 1H, HC⚌C), 2.505–2.516 (s, 1H, HC⚌N). UV (λmax, DMSO): 266.
2.3.11 4-[(E)-{[4-(4-bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]limino}]-1-phenyl dimethyl amine (I-i)
Yield: 58%. M.p: 191.8 °C. IR (νmax, cm−1): 3465.46 (N—H), 1640.16 (C⚌O), 1594.84 (Ar·N—H), 1536.02 (Ar·C—C), 1486.85 (C—H), 1383.68 (Ar·C—N), 1229.4 (C—N), 805.135 (C—Cl), 584.325 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.587–8.286 (m, 12H, Ar—H, s, 1H, HC⚌C), 2.500–2.511 (s, 1H, HC⚌N). UV (λmax, DMSO): 270.
2.3.12 4-[(E) {[4-(4-bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]im no} methyl]benzene-1,3-diol (I-j)
Yield: 52%. M.p: 198.6 °C. IR (νmax, cm−1): 3466.42 (N—H), 3306.36 (O—H), 1640.16 (C⚌O), 1592.91 (Ar N—H), 1536.99 (Ar·C—C), 1487.81 (C—H), 1388.5 (Ar·C—N), 1360.53 (N—O), 1228.43 (C—N), 969.055 (O—H), 848.525 (C—Cl), 550.577 (C—Br). 1H NMR: (DMSO-d6, δ, ppm): 7.586–8.283 (m, 11H, Ar—H), 2.511–2.516 (s, 1H, HC⚌N), 6.865 (s, 1H, OH). UV (λmax, DMSO): 264.
2.3.13 [4-(4-Bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]-1-phenylmethanimine(I-k)
Yield: 48%. M.p: 173.6 °C. IR (νmax, cm−1): 3467.38 (N—H), 1640.16 (C⚌O), 1536.99 (Ar C—C), 804.171 (C—Cl), 584.325 (C—Br), 1577.49 (ArN—H), 1385.6 (Ar C—N), 1228.43 (C—N), 3307.32 (O—H), 969.055 (O—H), 1487.81 (C—H), 1361.5 (N—O), 829.241 (⚌C—H). 1H NMR: (DMSO-d6, δ, ppm): 7.587–8.286 (m, 13H, Ar—H), 2.515–2.520 (s, 1H, HC⚌N). UV (λmax, DMSO): 265.
2.3.14 [4-(4-Bromophenyl)-6-(4-chlorophenyl) pyrimidin-2-yl]imino}methyl]-2-methoxyphenol (I-L)
Yield: 51%. M.p: 174.6 °C. IR (νmax, cm−1): 3466.42 (N—H), 1639.2 (C⚌O), 1536.99 (Ar C—C), 804.171 (C—Cl), 620.002 (C—Br), 1579.41 (Ar N—H), 1385.6 (Ar C—N), 1227.47 (C—N), 1537.95 (C—C), 1486.85 (C—H), 1360.53 (N—O), 726.068 (C—H). 1H NMR: (DMSO-d6, δ, ppm): 7.587–8.286 (m, 11H, Ar—H), 2.502–2.514 (s, 1H, HC⚌N), 6.862 (s, 1H, OH), 3.366 (s, 3H, OCH3). UV (λmax, DMSO): 255.
3 Antibacterial activity
The newly synthesized compounds were screened for antibacterial activity. For antibacterial studies microorganisms such as S. aureus NCIM 2079, Bacillus subtilis NCIM 2063, Escherichia coli NCIM 2918, P. aeruginosa NCIM 2036 were used (Table 1), and assayed by minimum inhibitory concentration (MIC) by serial dilution technique. To determine MIC the compounds were dissolved in dimethyl sulfoxide. Ciprofloxacin was used as the standard and incubated for 24 h at 37 °C (Table 2).
| Compound code | Diameter of zone of inhibition (mm) | |||
|---|---|---|---|---|
| Gram positive bacteria | Gram negative bacteria | |||
| SA | BS | EC | PA | |
| I | – | – | 15 mm | 12 mm |
| I-1 | 17 mm | 18 mm | 18 mm | 15 mm |
| I-a | 15 mm | 15 mm | 17 mm | 13 mm |
| I-b | 13 mm | 12 mm | 15 mm | 13 mm |
| I-c | 15 mm | 15 mm | 12 mm | 16 mm |
| I-d | 11 mm | 14 mm | 12 mm | – |
| I-e | 14 mm | 14 mm | – | 13 mm |
| I-f | 14 mm | 15 mm | – | 14 mm |
| I-g | 16 mm | 20 mm | 18 mm | 19 mm |
| I-h | 11 mm | 16 mm | 18 mm | 18 mm |
| I-i | 14 mm | 16 mm | 18 mm | 16 mm |
| I-j | 19 mm | 18 mm | 18 mm | 18 mm |
| I-k | 14 mm | 15 mm | 18 mm | 19 mm |
| I-L | 15 mm | 14 mm | 16 mm | 16 mm |
| Control | – | – | – | – |
| Ciprofloxacin | 25 mm | 26 mm | 30 mm | 26 mm |
(–) Indicates no noticeable zone of inhibition.
SA = Staphylococcus aureus NCIM 2079, BS = Bacillus subtilis NCIM 2063, EC = Escherichia coli NCIM 2911, PA = Pseudomonas aeruginosa NCIM 2036.
| Minimal inhibitory concentration (μg/ml) | ||||
|---|---|---|---|---|
| Compound codes | Gram positive bacteria | Gram negative bacteria | ||
| SA | BS | ES | PA | |
| I | 1000 | 1000 | 1000 | 2000 |
| I-1 | 2000 | 2000 | 2000 | 2000 |
| I-a | 1000 | 2000 | 1000 | 1000 |
| I-b | 2000 | 2000 | 1000 | 2000 |
| I-c | 1000 | 2000 | 2000 | 2000 |
| I-d | 1000 | 1000 | 1000 | 1000 |
| I-e | 1000 | 1000 | 2000 | 1000 |
| I-f | 1000 | 1000 | 500 | 1000 |
| I-g | 1000 | 1000 | 1000 | 1000 |
| I-h | 1000 | 1000 | 1000 | 1000 |
| I-i | 1000 | 1000 | 1000 | 1000 |
| I-j | 500 | 1000 | 500 | 1000 |
| I-k | 1000 | 1000 | 1000 | 1000 |
| I-L | 500 | 1000 | 500 | 500 |
| Dilutions for MIC | ||||||||||
| No of tubes for MIC | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Concentration of drug (μg/ml) | 4000 | 2000 | 1000 | 500 | 250 | 125 | 62.5 | 31.25 | 15.625 | 7.812 |
SA = Staphylococcus aureus NCIM 2079, BS = Bacillus subtilis NCIM 2063, EC = Escherichia coli NCIM 2911, PA = Pseudomonas aeruginosa NCIM 2036.
4 Bio-autography
Bio-autography is carried out for all synthesized compounds. This method is used quantitatively to determine low concentration of the synthesized compounds. The major concern for quantization via bio-autography has been the evaluation of the zone of the inhibition of the bio-autograph resulting from the eluted TLC spot on precoated TLC plates by using toluene:methanol:ethylacetate (6:3:1) (Table 3).
| Compound code | Concentration (μg/ml) | Rf value | Zone of inhibition(mm) |
|---|---|---|---|
| I | 500 | 0.833 | 10 |
| I-1 | 500 | 0.675 | 7 |
| I-a | 500 | 0.723 | 9 |
| I-b | 500 | 0.583 | 8 |
| I-c | 500 | 0.512 | 10 |
| I-d | 500 | 0.575 | 7 |
| I-e | 500 | 0.567 | 7 |
| I-f | 500 | 0.786 | 8 |
| I-g | 500 | 0.675 | 7 |
| I-h | 500 | 0.587 | 7 |
| I-i | 500 | 0.498 | 6 |
| I-j | 500 | 0.678 | 6 |
| I-k | 500 | 0.498 | 8 |
| I-L | 500 | 0.678 | 8 |
5 Results and discussion
The structures of all the synthesized compounds were determined on the basis of the FT/IR and 1H NMR data. The spectral data of FT/IR and 1H NMR, confirm the structures of newly synthesized compounds. The antibacterial activity of the newly synthesized compounds (I-1, I-g, I-j) showed a good activity against gram positive organisms (S. aureus, B. subtilis), while other compounds show significant to moderate activity. And the compounds (I-g, I-h, I-i, I-j, I-k) showed good activity against gram negative organisms (P. aeruginosa, E. coli), other compounds show significant to moderate activity. All the synthesized compounds are subjected to Bioautography. Compounds are elucidated on TLC plates with the solvent system and kept for antimicrobial activity on the media. All the compounds got clear zone of inhibition and coincided with the Rf values of TLC, this confirms that all the pure forms of the synthesized compounds only have the antimicrobial activity (see Scheme 1).
6 Conclusion
Finally, the new aminopyrimidine Schiff bases derivatives were successfully synthesized and evaluated for their antibacterial activity and bioautography with significant results. The study states that the synthesized compounds having potential antibacterial activity and bioautography show the pure synthesized compounds only showing the activity.
Acknowledgement
The authors are greatly thankful to SASTRA UNIVERSITY, Thanjavur, for carrying out the spectral analysis. Our sincere gratitude toward management and staffs of College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Tamil Nadu, India, for carrying out the synthetic work for the present study.
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