Abstract
Several substituted phenoxy acetic acid derived pyrazolines were synthesized by the reaction between 2-{4-[3-(2,4-dihydroxyphenyl)-3-oxo-1-propenyl]-2-methoxyphenoxy} acetic acid and substituted acid hydrazides and were tested for their in vitro cytotoxicity and antiviral activity. None of the compounds showed any specific antiviral activity [50% antivirally effective concentration (EC50) ≥ 5-fold lower than minimum cytotoxic concentration]. The most cytotoxic of the series was 2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-hydroxybenzoyl-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3j), with a minimum cytotoxic concentration of 0.16 μg/mL in human embryonic lung (HEL) cells.
Keywords::
Introduction
While great progress has been made in the development of antibiotics for the treatment of bacterial infections, there are only a limited number of antiviral drugs that can be used clinically. The strength of anti-viral therapy raises a number of public health concerns regarding the optimal use of drugs. Specificity against virus replication is the key issue in antiviral chemotherapy. Because of the close interaction between virus replication and normal cellular metabolism, it was originally too difficult to interrupt the virus replicative cycle without adversely affecting the host cell metabolism. It is now clear, however, that several events in the virus replicative cycle either do not occur in normal uninfected cells or are controlled by virus-specified enzymes that differ structurally and functionally from the corresponding host cell enzymes.
The intrinsic property of viruses as obligate intracellular parasites has been major stumbling block in developing antiviral drugs because chemicals that inhibit viral replication also inhibit host cellular functions that leads to drug toxicity. The expanding list of emerging or re-emerging viral infections that requires treatment has invoked significant attention; in addition, resistance to antiviral drugs has also become a serious concern.
Earlier we have reported pyrazoline derivatives as anti-mycobacterial [Citation1,Citation2], antiproliferative and cytotoxic Citation3,Citation4,Citation5,Citation6,Citation7 activity. Others have reported on the wide variety of biological activities of pyrazoline derivatives Citation8,Citation9,Citation10,Citation11. Here, we report on the synthesis and in vitro antiviral activity evaluation of a series of novel phenoxy acetic acid derivatives.
Materials
All the chemicals used were laboratory grade and procured from E. Merck (Germany) and S.D. Fine Chemicals (India). Melting points were determined by the open tube capillary method and are uncorrected. The thin layer chromatography (TLC) plates (silica gel G) were used to confirm the purity of commercial reagents used, compounds synthesized and to monitor the reactions as well. Two different solvent systems: toluene: ethyl acetate: formic acid (5:4:1) and petroleum ether: toluene: acetic acid (5:4:1), were used to run the TLC and spots were located under iodine vapors/UV light. IR spectra were obtained on a Perkin-Elmer 1720 FT-IR spectrometer (KBr Pellets). 1H-NMR spectra were recorded on a Bruker AC 300 MHz, spectrometer using TMS as internal standard in DMSO-d6.
Methods
Chemistry
2-{4-[3-(2,4-Dihydroxyphenyl)-3-oxo-1-propenyl]-2-methoxyphenoxy} acetic acid (2)
A mixture of 2-(4-formyl-2-methoxyphenoxy) acetic acid (1) (0.01 mole) and 1-(2,4-dihydroxyphenyl) ethanone (0.01 mole) in oxygen-free methanol were stirred at room temperature in the presence of base (aqueous solution of potassium hydroxide 30%; 5 mL) till completion of the reaction.The reaction mixture was allowed to stand overnight and then poured into ice-cold water followed by neutralization with HCl. The solid separated was filtered, dried and crystallized from ethanol. The purity of the chalcone was checked by TLC.
IR: (KBr) cm− 1: 3033 (COOH), 1682 (C = O), 1572 (C = C); 1H-NMR (DMSO-d6) ppm: 10.1 (1H, s, COOH), 9.2 (2H, s, 2 × OH), 6.5-8.3 (6H, m, aromatic), 6.1-6.5 (2d, 1H each, J = 8.4, 6.9Hz, CH = CH), 3.6(3H, s, OCH3), m/z: 345(M+1); Anal.Calcd. for C18H16O7: C, 62.79; H, 4.68; O, 32.53%. Found: C, 62.80; H, 4.67; O, 32.54%.
General procedure for synthesis of compound (3a-m)
A solution of 2-{4-[3-(2,4-dihydroxyphenyl)-3-oxo-1-propenyl]-2-methoxyphenoxy} acetic acid (0.001 mole) in 15 mL of glacial acetic acid and the appropriate acid hydrazides (0.001 mole) was refluxed for 12 h. Excess of solvent was removed under reduced pressure and the reaction mixture was poured onto crushed ice (20 gm). The product so obtained was filtered, washed with water and recrystalized from methanol.
2-{4-[1-benzoyl-3-(2,4-dihydroxyphenyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxy phenoxy} acetic acid (3a )
IR: (KBr) cm− 1: 3245(COOH), 1682(C = O), 1563(C = N), 1318(C–N); 1H-NMR (DMSO-d6) ppm: 11.4(1H, s, COOH), 8.8(2H, s, 2 × OH), 7.4–7.9 (11H, m, aromatic), 4.6(1H, t, CH), 3.9(3H, s, OCH3), 2.5(2H, d, CH2), m/z: 463 (M+1); Anal. Calcd. for C25H22N2O7: C, 64.93; H, 4.80; N, 6.06; O, 24.22%. Found: C, 64.92; H, 4.81; N, 6.05; O, 24.24%.
2-{4-[1-(2-chlorobenzoyl)-3-(2,4-dihydroxyphenyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3b)
IR: (KBr) cm− 1: 3190(COOH), 1685(C = O), 1545(C = N), 1325(C–N); 1H-NMR (DMSO-d6) ppm: 10.2(1H, s, COOH), 9.4(2H, s, 2 × OH), 6.8–8.1(10H, m, aromatic), 4.8(1H, t, CH), 3.6(3H, s, OCH3), 2.5(2H, d, CH2), m/z: 497 (M+1); Anal.Calcd. for C25H21ClN2O7: C, 60.43; H, 4.26; Cl, 7.13; N, 5.64; O, 22.54%. Found: C, 60.44; H, 4.27; Cl, 7.12; N, 5.62; O, 22.53%.
2-{4-[1-(4-chlorobenzoyl)-3-(2,4-dihydroxyphenyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3c)
IR: (KBr) cm− 1: 3048(COOH), 1714(C = O), 1555(C = N), 1315(C–N); 1H-NMR (DMSO-d6) ppm: 10.3(1H, s, COOH), 9.9(2H, s, 2 × OH), 7.7–7.8(10H, m, aromatic), 4.5(1H, t, CH),3.3(3H, s, OCH3), 2.5(2H, d, CH2), m/z: 497 (M+1); Anal.Calcd. for C25H21ClN2O7: C, 60.43; H, 4.26; Cl, 7.13; N, 5.64; O, 22.54%. Found: C, 60.45; H, 4.27; Cl, 7.12; N, 5.62; O, 22.55%.
2-{4-[1-(4-bromobenzoyl)-3-(2,4-dihydroxyphenyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3d)
IR: (KBr) cm− 1: 3173(COOH), 1654(C = O), 1560(C = N), 1317(C–N); 1H-NMR (DMSO-d6) ppm: 11.4(1H, s, COOH), 9.7(2H, s, 2 × OH), 6.7–8.1(10H, m, aromatic), 4.6(1H, t, CH),3.8 (3H, s, OCH3), 2.5(2H, d, CH2), m/z: 542 (M+1); Anal.Calcd. for C25H21BrN2O7: C, 55.47; H, 3.91; Br, 14.76; N, 5.17; O, 20.69%. Found: C, 55.49; H, 3.93; Br, 14.78; N, 5.18; O, 20.72%.
2-{4-[1-(4-nitrobenzoyl)-3-(2,4-dihydroxyphenyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3e)
IR: (KBr) cm− 1: 3219(COOH), 1740(C = O), 1551(C = N), 1381(C–N); 1H-NMR (DMSO-d6) ppm: 10.4(1H, s, COOH), 9.8(2H, s, 2 × OH), 8.0–8.03(10H, m, aromatic), 4.7(1H, t, CH), 3.9 (3H, s, OCH3), 2.5(2H, d, CH2), m/z: 508 (M+1); Anal.Calcd. for C25H21N3O9: C, 59.17; H, 4.17; N, 8.28; O, 28.38%. Found: C, 59.16; H, 4.18; N, 8.27; O, 28.40%.
2-{4-[3-(2,4-dihydroxyphenyl)-1-(4-methylbenzoyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3f)
IR: (KBr) cm− 1: 3255(COOH), 1654(C = O), 1541(C = N), 1324(C–N), 1H-NMR (DMSO-d6) ppm: 10.1(1H, s, COOH),9.0 (2H, s, 2 × OH), 6.8–8.0(10H, m, aromatic), 5.71 (1H, dd, J = 6.6, 6.3Hz), 4.42(1H, dd, J = 12.0, 12.0Hz), 3.92(1H, dd, J = 6.3, 6.3Hz), 3.8 (3H, s, OCH3), m/z: 477 (M+1); Anal.Calcd. for C26H24N2O7: C, 65.54; H, 5.08; N, 5.88; O, 23.50%. Found: C, 65.53; H, 5.09; N, 5.90; O, 23.49%.
2-{4-(3-(2,4-dihydroxyphenyl)-1-[2-(2-methylphenoxy)acetyl]-4,5-dihydro-1H-5-pyrazolyl(-2-methoxyphenoxy}acetic acid (3g)
IR: (KBr) cm− 1: 3304(COOH), 1685(C = O), 1540(C = N), 1382(C–N); 1H-NMR (DMSO-d6) ppm: 10.8(1H, s, COOH), 9.4(2H, s, 2 × OH), 6.7–8.2(10H, m, aromatic), 4.8(1H, t, CH),3.4(3H, s, OCH3), 2.5(2H, d, CH2), m/z: 507 (M+1); Anal.Calcd. for C27H26N2O8: C, 64.02; H, 5.17; N, 5.53; O, 25.27%. Found: C, 64.03; H, 5.17; N, 5.54; O, 25.27%.
2-{4-(3-(2,4-dihydroxyphenyl)-1-[2-(4-methylphenoxy)acetyl]-4,5-dihydro-1H-5-pyrazolyl(-2-methoxyphenoxy}acetic acid (3h)
IR: (KBr) cm− 1: 3220(COOH), 1680(C = O), 1559(C = N), 1378(C–N); 1H-NMR (DMSO-d6) ppm: 10.1(1H, s, COOH), 9.0(2H, s, 2 × OH), 6.5–8.4(10H, m, aromatic), 4.9(1H, t, CH), 3.3(3H, s, OCH3), 2.5(2H, d,CH2), m/z: 507(M+1); Anal.Calcd. for C27H26N2O8: C, 64.02; H, 5.17; N, 5.53; O, 25.27%. Found: C, 64.02; H, 5.17; N, 5.53; O, 25.27%.
2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-phenoxyacetyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3i )
IR: (KBr) cm− 1: 3180(COOH), 1692(C = O), 1550(C = N), 1342(C–N); 1H-NMR (DMSO-d6) ppm: 10.3(1H, s, COOH), 8.7(2H, s, 2 × OH), 6.6–8.03 (10H, m, aromatic), 4.6 (1H, t,CH), 3.5 (3H, s, OCH3), 2.5(2H, d, CH2); m/z: 493 (M+1); Anal.Calcd. for C26H24N2O8: C, 63.41; H, 4.91; N, 5.69; O, 25.99%. Found: C,63.40; H, 4.91; N, 5.70; O, 25.97%.
2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-hydroxybenzoyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3j)
IR: (KBr) cm− 1: 3260(COOH), 1740(C = O), 1559(C = N), 1386(C–N); 1H-NMR (DMSO-d6) ppm: 10.1(1H, s, COOH), 9.3(2H, s, 2 × OH), 6.8–8.1(10H, m, aromatic), 4.6(1H, t, CH),3.4(3H, s, OCH3), 2.5(2H, d, CH2), m/z: 479(M+1); Anal.Calcd. for C25H22N2O8: C, 62.76; H, 4.63; N, 5.86; O, 26.75%. Found: C, 62.77; H, 4.64; N, 5.86; O, 26.77%.
2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-phenylacetyl)-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3k)
IR: (KBr) cm− 1: 3199(COOH), 1654(C = O), 1560(C = N), 1317 (C–N); 1H-NMR (DMSO-d6) ppm: 10.5(1H, s, COOH), 8.9(2H, s, 2 × OH), 6.8–8.3(10H, m, aromatic), 5.82(1H, dd, J = 5.7, 5.7 Hz), 4.71 (1H, dd, J = 12.3, 12.3 Hz), 3.91 (1H, dd, J = 6.0, 5.7 Hz), 3.6(3H, s, OCH3), m/z: 477(M+1); Anal.Calcd. for C26H24N2O7: C, 65.54; H, 5.08; N, 5.88; O, 23.50%. Found: C, 65.54; H, 5.09; N, 5.88; O, 23.51%.
2-{4-(3-(2,4-dihydroxyphenyl)-1-[2-(1-naphthyloxy)acetyl]-4,5-dihydro-1H-5-pyrazolyl(-2-methoxyphenoxy}acetic acid (3l)
IR: (KBr) cm− 1: 3304(COOH), 1760(C = O), 1560(C = N), 1380(C–N); 1H-NMR (DMSO-d6) ppm: 10.1(1H, s, COOH), 8.2(2H, s, 2 × OH), 7.1–7.8(13H, m, aromatic), 4.8(1H, t, CH) 3.4(3H, s, OCH3), 2.5(2H, d, CH2); m/z: 543(M+1); Anal.Calcd. for C30H26N2O8: C, 66.41; H, 4.83; N, 5.16; O, 23.59%. Found: C, 66.42; H, 4.83; N, 5.17; O, 23.60%.
2-{4-(3-(2,4-dihydroxyphenyl)-1-[2-(2-naphthyloxy)acetyl]-4,5-dihydro-1H-5-pyrazolyl(-2-methoxyphenoxy}acetic acid (3m)
IR: (KBr) cm− 1: 3304(COOH), 1760 (C = O), 1560(C = N), 1380(C–N); 1H-NMR (DMSO-d6) ppm: 10.1(1H, s, COOH), 8.2(2H, s, 2 × OH), 7.1–7.8(13H, m, aromatic), 4.8(1H,t,CH); 3.4(3H, s, OCH3), 2.5(2H, d, CH2); m/z: 543 (M+1); Anal.Calcd. for C30H26N2O8: C, 66.41; H, 4.83; N, 5.16; O, 23.59%. Found: C, 66.41; H, 4.83; N, 5.17; O, 23.59%.
Antiviral activity assay
Confluent cell cultures in micro titer trays were inoculated with 100 CCID50 (1 CCID50 corresponding to the virus stock dilution that proved infective for 50% of the cell cultures). After 1 h of virus adsorption to the cells, residual virus was removed and replaced by cell culture medium (Eagle's minimal essential medium) containing 3% foetal calf serum and various concentrations of the test compounds. Viral cytopathogenicity was recorded as soon as it reached completion in the untreated virus-infected cell cultures, i.e., at 1 to 2 days for vesicular stomatitis; at 2 days for Coxsackie and herpes simplex virus types 1 and 2 and Sindbis virus; and 6 to 7 days for reo- and parainfluenza viruses. The antiviral activity of the compounds is expressed as the concentration required to inhibit viral cytopathogenicity by 50%.
Cytotoxicity assays
Cytotoxicity was monitored by direct microscopical inspection of the cell monolayers which had not been infected but were treated by the compounds at the same concentration as used in the antiviral activity assays [Citation12].
Results and discussion
Chemistry
The physical constants of pyrazoline derivatives (3a–m) are shown in , whereas results obtained from the antiviral testing is described in this study are shown in ,, and a reaction sequence for the preparation is outlined in . The desired chalcone was obtained, by Claisen-Schmidt condensation, by reacting 2-(4-formyl-2-methoxyphenoxy) acetic acid with 1-(2,4-dihydroxyphenyl)ethanone in the presence of a base. Reaction between chalcone, 2-{4-[3-(2,4-dihydroxyphenyl)-3-oxoprop-1-enyl]-2-methoxyphenoxy} acetic acid and the appropriate acid hydrazide in the presence of glacial acetic acid, afforded after a reaction time varying from 6–12 h, the pyrazolines (3a-m) in 78–89% yield. TLC and elemental analyses was done to confirm the purity of the compounds. Analytical and spectral data [IR & 1H-NMR] of all the synthesized compounds were found in full agreement with the proposed structures, which was further confirmed by mass fragmentation of the selected compounds. The IR spectra of chalcone revealed C = O, C = C, stretching at 1682, 1572 cm− 1 where as in general pyrazoline (3b) revealed C = N, C–N and C = O band at 1545, 1325 and 1685 cm− 1, respectively. In the 1H-NMR spectra the signals of the respective protons of the compounds were verified on the basis of their chemical shifts, multiplicities and coupling constants. The spectra showed a singlet at δ10.2 ppm corresponding to the COOH group. The pyrazoline ring formation showed a characteristic doublet and triplet for C-4 methylene and C-5 protons at a shift at 2.5 and 4.8 respectively, a singlet at δ3.6 ppm for methoxy group, a multiplet at δ6.8–8.1 ppm for aromatic protons and a singlet at δ9.4 ppm for OH proton. It was found interesting that only two compound of the series 3f and 3k have shown usual pyrazoline splitting.
Table I. Physical constants of the synthesized compounds.
Table II. Cytotoxicity and antiviral activity of compounds in Crandell-Rees Feline Kidney (CRFK) cell cultures.
Table III. Cytotoxicity and antiviral activity of compounds in human embryonic lung (HEL) cell cultures.
Table IV. Cytotoxicity and antiviral activity of compounds in HeLa cell cultures.
Table V. Cytotoxicity and antiviral activity of compounds in Vero cell cultures.
Scheme 1. Synthesis of pyrazolines.
Antiviral activity
Antiviral activity was tested against Feline Corona Virus (FIPV) and Feline Herpes Virus (in Crandell-Rees Feline Kidney (CRFK) cell cultures), herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus, vesicular stomatitis virus, herpes simplex virus-1 TK-KOS ACVr (in HEL cell cultures); vesicular stomatitis virus, Coxsackie virus B4, Respiratory syncytial virus (in HeLa cell cultures); parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus (in Vero cell cultures). For each compound, the Minimum Cytotoxic Concentration (MCC) [or 50% cytotoxic concentration (CC50) for CRFK] was determined. Ribavirin, Brivudin, Acyclovir, Ganciclovir, Dextran sulfate (DS-5000) and (S)-DHPA were used as the reference compounds.
The results of the antiviral assays are shown in ,. Cytotoxicity towards uninfected host cells was determined under same conditions as antiviral activity i.e microscopic evaluation of the cell morphology of the confluent cell monolayer which either had or had not been inoculated with virus. As the criterion for specific antiviral activity was taken the inhibition of virus-induced cytopathogenicity at a concentration that was at least 5-fold lower than the concentration required to alter the morphology of the uninfected host cells.
As per this criterion none of the compounds tested (3a-3m) showed any specific antiviral activity (,). The most cytotoxic of the series was 3j, with a minimum cytotoxic concentration of 0.8 μg/mL in Vero cell cultures and 0.16 μg/mL in HEL (human embryonic lung) cells.
Acknowledgements
The authors thank Mrs. Leentje Persoons (Rega Institute for Medical Research) for excellent technical assistance with the antiviral assays and Dr. Ashraf Ali (Research Scientist, Chemical Research Division, Ranbaxy Laboratories, Gurgaon, India) for valuable help and suggestions.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- M Shahar Yar, AA Siddiqui, MA Ali. Synthesis and evaluation of phenoxy acetic acid derivatives as a antimycobacterial agents. Bioorg Med Chem Lett 2006;16:4571–4574.
- M Shahar Yar, AA Siddiqui, MA Ali, D Sriram, P Yogeshwari. Synthesis and in vitro antimycobacterial activity of N1-nicotinoyl-3-(4′-hydroxy-3′-methylphenyl)-5-[(sub)phenyl]- 2-pyrazolines. Bioorg Med Chem Lett 2006;16:3947–3949.
- M Shahar Yar, AA Siddiqui, MA Ali, V Murugan, C Raghu. Synthesis and Cytotoxic activity of novel pyrazoline derivatives against human lung tumor cell line (A549). J Chin Chem Soc 2007;54:81–86.
- M Shahar Yar, MA Ali, E De Clercq. Synthesis of 5-(4-hydroxy-3-methylphenyl)-5-(substituted phenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity. J Enzyme Inhib Med Chem 2007;22 (6):702–708.
- E De Clercq. Antiviral and antimetabolic activities of neplanocins. Antimicrob Agents Chemother 1985;28:84–89.
- E De Clercq. Antiviral therapy of HIV infections. Clin Microbiol Rev 1995;8:200–239.
- CS Crumpacker. Molecular targets of antiviral therapy. N Engl J Med 1989;321:163–172.
- B Kalluraya, JK John, BS Holla. Studies on the synthesis and biological activity of some 4-(5-aryl-2-furfurylidene)-1,3-disubstituted-2-pyrazolin-5-ones. J Indian Chem Soc 1995;72:99–101.
- Y Rajendra Prasad, A Lakshmana Rao, L Prasoona, K Murali, P Ravi Kumar. Synthesis and antidepressant activity of some 1,3,5-triphenyl-2-pyrazolines and 3-(2″-hydroxy napthalen-1″-yl)-1,5-diphenyl-2-pyrazolines. Bioorg Med Chem Lett 2005;15:5030–5034.
- RH Udupi, AS Kushnoor, AR Bhat. Synthesis and biological evaluation of certain pyrazoline derivatives of 2-[6-methoxy napthyl]-propionic acid (Naproxen). Indian J Het Chem 1998;8:63–66.
- V Harinadha Babu, Ch Sridevi, A Joseph, KK Srinivasan. Synthesis and biological evaluation of some novel pyrazolines. Ind J Pharm Sci 2007;69 (3):470–473.
- R Pauwels, J Balzarini, M Baba, R Snoeck, D Schols, P Herdewijn, J Desmyter. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods 1988;20:309–321.