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Synthesis, anti-HIV and antitubercular activities of lamivudine prodrugs
European Journal of Medicinal Chemistry 40 (2005) 1373–1376 www.elsevier.com/locate/ejmech
Short communication
Synthesis, anti-HIV and antitubercular activities of lamivudine prodrugs Dharmarajan Sriram *, Perumal Yogeeswari, Gayatri Gopal Medicinal Chemistry Research Laboratory, Pharmacy Group Birla Institute of Technology and Science, Pilani-333031, India Received 29 December 2004; received in revised form 20 July 2005; accepted 25 July 2005 Available online 29 August 2005
Abstract The synthesis of a novel series of lamivudine prodrugs involving N4- substitution with isatin derivatives is described. The in-vitro antiretroviral activities indicated that compound 3b was found to be equipotent to lamivudine with EC50 of 0.0742 ± 0.04 µM. Lamivudine prodrugs bearing fluoroquinoles antibacterial showed 92–100% inhibition against Mycobacterium tuberculosis strain H37Rv at 6.25 µg ml–1. At pH 7.4, 37 °C, the hydrolytic t1/2 ranged between 120 and 240 min. © 2005 Published by Elsevier SAS. Keywords: Lamivudine prodrug; Anti-HIV activity; Antimycobacterial activity
1. Introduction
2. Synthesis
Acquired immunodeficiency syndrome (AIDS) is caused by the retrovirus, human immuno deficiency virus (HIV) [1]. The HIV infection, which targets the monocytes expressing surface CD4 receptors, eventually produces profound defects in cell-mediated immunity [2]. Overtime infection leads to severe depletion of CD4 T-lymphocytes (T-cells) resulting in opportunistic infection (OIs) like tuberculosis (TB), fungal, viral, protozoal and neoplastic diseases and ultimately death. TB is the most common OI in people with AIDS and it is the leading killer of people living with HIV/AIDS. The development of active TB accelerates the progression of HIV disease towards full-blown AIDS, accompanied by enhanced HIVreplication. The majority of people infected with the HIV virus develop TB as the first manifestation of AIDS. Through logic and orderly thinking, it appears that an ideal drug for HIV/AIDS patients should suppress HIV-replication thereby acting as anti-HIV drug and also should treat OI like TB [3–5]. As a result we undertook a study to prepare and evaluate lamivudine prodrugs in an effort to identify compounds, which could suppress HIV-replication and also inhibit the M. tuberculosis.
Esterification of the 5’-hydroxyl group of anti-HIV 2’,3’dideoxynucleosides has been used extensively in attempts to improve brain uptake and in vivo efficacy [6]. In this paper we modified in the 4-amino group of 3’-thia-2’, 3’-dideoxycytidine (lamivudine). The synthesis of lamivudine prodrugs proceeded smoothly by condensing parent drug (2) with an equimolar ratio of isatin and its 5-substituted derivatives (1) in presence of glacial acetic acid to form Schiff’s bases (3). The N-Mannich bases (4a–h) of the above Schiff’s bases were prepared by condensing acidic imino group of isatin with formaldehyde and various aryl piperazines (fluoroquinolone antibacterials) with 72–85% yield (Table 1). The purity of the synthesized compounds was checked by TLC and by elemental analyses; and the compounds of this study were identified by spectral data. In general, IR spectra showed C=N (azomethine) peak at 1640 cm−1 and CH2 (Mannich methylene) peak at 2860 and 2840 cm−1. In the 1H-NMR spectra the signals of the respective protons of the prepared prodrugs were verified on the basis of their chemical shifts, multiplicities and coupling constants. The spectra showed a singlet at d 4.8–5.1 ppm corresponding to –NCH2N– group; multiplet at d 3.8–4.1 ppm for piperazine proton; and triplet at d 5.2 ppm for OH group. The elemental analysis results were within ± 0.4% of the theoretical values Scheme 1.
* Corresponding author. Tel.: +91 1596 24 4684; fax: +91 1596 24 4183. E-mail address: [email protected] (D. Sriram). 0223-5234/$ - see front matter © 2005 Published by Elsevier SAS. doi:10.1016/j.ejmech.2005.07.006
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Table 1 Biological properties, lipophilicity and stability of various prodrugs of Lamivudine #
R
Yield M.P. (%) (°C)
3a 3b 4a 4b 4c 4d 4e 4f 4g 4h Lamivudine
H F H Cl F H Cl Cl Cl F –
84 73 80 78 84 85 82 77 76 82
#
214 207 232 212 189 236 231 219 222 213
Anti-HIV activity (µM) EC50a 2.11 ± 0.89 0.0742 ± 0.04 > 134.0 > 11.3 1.11 ± 0.52 49.0 ± 2.33 > 12.5 > 4.73 > 10.5 1.16 ± 0.11 0.1 ± 0.05
CC50b > 200 > 200 134.0 ± 11.1 11.3 ± 1.23 123.0 ± 14.3 121.0 ± 10.8 12.5 ± 1.33 4.73 ± 0.35 10.5 ± 1.55 136 ± 12.2 > 200
SIc > 95 > 2100 – – 110 2.47 – – – 117.24 > 2000
Antimycobacterial activityd (% inhibition) 56 82 92 96 100 98 100 98 100 100 0
log Pe
1.14 1.42 –0.95 –0.91 –0.08 –0.72 –0.75 –0.92 –0.76 –0.53 –1.14
In-vitro hydrolysis t1/2 (min)f 150 120 ND ND 150 240 ND ND ND 120 –
Compounds tested. a Effective concentration of compound achieving 50% protection in CEM cell lines against the cytopathic effect of HIV-1. b Cytotoxic concentration of compound required to reduce the viability of mock infected CEM cells by 50%. c Selectivity index or ratio of CC50/EC50. d At the concentration of 6.25 µg ml–1. e Calculated using SciLogP program. f At pH 7.4 at 37 °C and “ND” indicates not determined.
Scheme 1. (i) Glacial acetic acid/Ethanol, Microwave irradiation at 80% intensity; (ii) HCHO, aryl piperazines, ethanol.
3. Result and discussion The antiviral activity of lamivudine and its prodrugs against HIV-1 was determined in-vitro in T4 lymphocytes (CEM cell
line) [7] (Table 1). Compound 3b was found to be equipotent to lamivudine with EC50 of 0.0742 ± 0.04 µM, CC50 of > 200 µM and selective index (SI) of > 2100. The introduction of fluorine group at the 5th position of isatin enhanced
D. Sriram et al. / European Journal of Medicinal Chemistry 40 (2005) 1373–1376
the activity (3b, 4c, and 4h), whereas the introduction of 5-chloro group (4b,e,f, and 4g) increased the toxicity to CEM cell line. The Mannich bases (4a–h) were less active, when compared to Schiff’s bases (3a–b), whose calculated logP was found to be greater than 1. All compounds were screened against Mycobacterium tuberculosis strain H 37 Rv at a single concentration, 6.25 µg ml–1 in BACTEC 12B medium using the BACTEC 460 radiometric system [8]. All the Mannich bases (4a–h) were found to be most active with 92–100% inhibition. Among the Schiff’s bases compound 3b showed 82% inhibition. Parent drug lamivudine did not show any activity at 6.25 µg ml–1. The usefulness of the prodrugs of lamivudine should depend not only on the stability of the prodrug for its transport across the cell membrane but also upon its reversion to the parent compound intracellularly, especially in the virally infected cells. The half-lives (t1/2) of hydrolysis of the prodrugs were therefore determined at pH 7.4, 37 °C [9]. The data in Table 1 indicated that the various prodrugs of lamivudine were susceptible to hydrolysis with t1/2 in the range of 120–240 min. In the present study we identified that Schiff’s base 3b was found to show equipotent anti-HIV activity to parent compound, lamivudine. Compound 4c inhibited both HIV1 replication and shows 100% inhibition against M. tuberculosis in the preliminary screening. Thus these prodrugs would be beneficial for the effective treatment of HIV/AIDS. 4. Experimental protocols 4.1. Chemistry Melting points were determined in one end open capillary tubes on a Büchi 530 melting point apparatus and are uncorrected. Infrared (IR) and proton nuclear magnetic resonance (1H-NMR) spectra were recorded for the compounds on Jasco IR Report 100 (KBr) and Brucker Avance (300 MHz) instruments, respectively. Chemical shifts are reported in parts per million (ppm) using tetramethyl silane (TMS) as an internal standard. Elemental analyses (C, H, and N) were undertaken with Perkin–Elmer model 240C analyzer. The homogeneity of the compounds was monitored, by ascending thin layer chromatography (TLC) on silicagel-G (Merck) coated aluminum plates, visualized by iodine vapor. Developing solvents were chloroform–methanol (9:1). 4.1.1. General procedure for the preparation of Schiff’s bases Equimolar quantities (0.01 mol) of (sub)isatin and lamivudine were dissolved in warm ethanol containing 1 ml of glacial acetic acid. The reaction mixture was irradiated in an unmodified domestic microwave oven at 80% intensity with 30 s per cycle for 3 min and set aside. The resultant solid was washed with dilute ethanol dried and recrystallized from ethanol–chloroform mixture. Yield: 73–84%.
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4.1.2. 3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2dihydropyrimidin-4-yl}imino)-1,3-dihydro-2H-indol-2-one (3a) Yield: 84%; m.p.: 214 °C; IR (KBr): 3200, 1730, 1654, 1640 1153, 1129 cm−1; 1H-NMR (CDCl3) d (ppm): 3.26 (dd, 2H, 2″-CH2), 3.66 (dd, 2H, 5″-CH2), 5.35 (t, 1H, 4″-CH), 6.33 (t, 1H, 1″-CH), 6.83–7.32 (m, 4H, Ar–H), 7.47 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 10.4 (s, 1H, NH); Anal. for C16H16N4O3S: . 4.1.3. General procedure for the preparation of Mannich bases To a suspension of appropriate Schiff bases (0.02 mol) in ethanol were added appropriate aryl piperazines (0.02 mol) and 37% formaldehyde (0.5 ml) and irradiated in a microwave oven at an intensity of 80% with 30 s per cycle. The number of cycle in turn depended on the completion of the reaction, which was checked by TLC. The reaction timing varied from 1.5 to 3 min. The solution obtained after the completion of the reaction was kept at 0 °C for 30 min and the resulting precipitate was recrystallized from a mixture of DMF and water. 4.1.4. 3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2dihydropyrimidin-4-yl}imino)-1-[(1-Ethyl-6-fluoro-1,4dihydro-4-oxo-3-quinoline carboxylic acid–7piperazinyl)methyl]-1,3-dihydro-2H-indol-2-one (4a) Yield: 80%; m.p.: 232 °C; IR (KBr): 3320, 2860, 2840, 1730, 1654, 1640, 1153, 1129 cm−1; 1H-NMR (CDCl3) d (ppm): 1.28 (t, 3H, CH3 of C2H5), 3.24 (dd, 2H, 2″-CH2), 3.66 (dd, 2H, 5″-CH2), 3.7–4.1 (m, 8H, –piperazine–H), 4.25 (q, 2H, CH2 of C2H5), 5.1 (s, 2H, –NCH2N), 5.34 (t, 1H, 4″-CH), 6.30 (t, 1H, 1″-CH), 6.58–7.3 (m, 6H, Ar–H), 7.52 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 8.66 (s, 1H, C2–H); Anal. for C33H34FN7O6S: . 4.1.5. 3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2dihydropyrimidin-4-yl}imino)-1-[(1-cyclopropyl-6-fluoro1,4-dihydro-4-oxo-3-quinoline carboxylic acid–7piperazinyl)methyl]-1,3-dihydro-2H-indol-2-one (4d) Yield: 85%; m.p.: 236 °C; IR (KBr): 3326, 2860, 2840, 1718, 1730, 1693, 1640, 1153, 1129 cm−1 ; 1H-NMR (CDCl3) d (ppm): 0.88–1.1 (m, 4H, cyclopropyl-H), 3.24 (dd, 2H, 2″-CH2), 3.5 (m, 1H, cyclopropyl-H), 3.66 (dd, 2H, 5″-CH2), 3.7–4.1 (m, 8H, –piperazine–H), 5.1 (s, 2H, –NCH2N), 5.34 (t, 1H, 4″-CH), 6.30 (t, 1H, 1″-CH), 6.68–7.2 (m, 6H, Ar–H), 7.52 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 8.6 (s, 1H, C2–H); Anal. for C34H34FN7O6S: . 4.1.6. 5-chloro-3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5yl]-1,2-dihydropyrimidin-4-yl}imino)-1-[(1-cyclopropyl-6fluoro-8-methoxy-1,4-dihydro-4-oxo-3-quinoline carboxylic acid–7-(3-methyl)piperazinyl)methyl]-1,3-dihydro-2Hindol-2-one (4g) Yield: 76%; m.p.: 222 °C; IR (KBr): 3326, 2860, 2840, 1730, 1652, 1640, 1153, 1129 cm−1 ; 1H-NMR (CDCl3) d
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(ppm): 0.92–1.10 (m, 4H, cyclopropyl-H), 1.14 (d, 3H, CH3 of pip), 3.24 (dd, 2H, 2″-CH2), 3.46 (s, 3H, –OCH3), 3.6– 4.18 (m, 10H, cyclopropyl-H, –piperazine–H, 5″-CH2), 5.2 (s, 2H, –NCH2N), 5.34 (t, 1H, 4″-CH), 6.30 (t, 1H, 1″-CH), 6.68–8.02 (m, 4H, Ar–H), 7.52 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 8.6 (s, 1H, C2–H) Anal. for C36H37ClFN7O7S: . 4.2. Anti-HIV screening [7] Candidate agents were dissolved in dimethylsulfoxide, and then diluted 1:100 in cell culture medium before preparing serial half- log10 dilutions. T4 lymphocytes (CEM cell-line) were added and after a brief interval HIV-1 was added, resulting in a 1:200 final dilution of the compound. Uninfected cells with the compound served as a toxicity control, and infected and uninfected cells without the compound served as basic controls. Cultures were incubated at 37 °C in a 5% carbon dioxide atmosphere for 6 days. The tetrazolium salt, XTT was added to all the wells, and cultures were incubated to allow formazan color development by viable cells. Individual wells were analyzed spectrophotometrically to quantitative formazan production, and in addition were viewed microscopically for detection of viable cells and confirmation of protective activity. 4.3. Antimycobacterial activity Primary screening was conducted at 6.25 µg ml–1 against M. tuberculosis strain H37Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the microplate Alamar Blue Assay (MABA) [8].
ethyl sulfoxide) and the mixture was incubated at 37 °C in a water bath. At various time intervals (0–4 h), 100 µl of the samples were withdrawn and added immediately to ice-cold methanol (400 µl). The samples were centrifuged and the supernatants were filtered through nylon 66 filters (0.45 pm) and analyzed by spectrophotometrically [9]. From the observed optical density changes, at various wavelengths, the half-lives of the analogues were calculated. Acknowledgements The authors deeply acknowledge the financial support of the Council of Industrial and Scientific Research, New Delhi (No. 01(1979)/05/EMR-II). The authors also thank Dr. Edward Sausville, National Cancer Institute, USA and Dr. S. Ananthan from the Southern Research Institute, Birmingham, Alabama, USA, for support in biological testing. References [1] [2] [3] [4] [5] [6] [7]
4.4. In-vitro stability studies To 990 µl of phosphate buffer (0.2 M, pH 7.4) was added 10 µl of a solution of appropriate drugs (10 mg ml–1 in dim-
[8] [9]
S. Broder, R.C. Gallo, N. Engl, J. Med. 311 (1984) 1292–1297. D.L. Bowen, H.C. Hane, A.C. Fauci, Ann. Intern. Med. 163 (1985) 704–709. D. Sriram, P. Yogeeswari, Curr. Med. Chem. 10 (2003) 1909–1915. D. Sriram, P. Yogeeeswari, N. Srichakravarthy, T.R. Bal, Bioorg. Med. Chem. Lett. 14 (2004) 1085–1087. D. Sriram, T.R. Bal, P. Yogeeswari, Bioorg. Chem. 12 (2004) 5865– 5873. O.W. Weislow, R. Kiser, D. Fine, J. Bader, R.H. Shoemaker, M.R. Boyd, J. Natl. Cancer Inst. 81 (1989) 577–586. L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997) 1004–1009. S.K. Agarwal, S.R. Gogu, S.R.S. Rangan, K.C. Agrawal, J. Med. Chem. 33 (1990) 1505–1509. S.G. Kerr, T.I. Kalman, J. Med. Chem. 35 (1992) 1996–2001.
Short communication
Synthesis, anti-HIV and antitubercular activities of lamivudine prodrugs Dharmarajan Sriram *, Perumal Yogeeswari, Gayatri Gopal Medicinal Chemistry Research Laboratory, Pharmacy Group Birla Institute of Technology and Science, Pilani-333031, India Received 29 December 2004; received in revised form 20 July 2005; accepted 25 July 2005 Available online 29 August 2005
Abstract The synthesis of a novel series of lamivudine prodrugs involving N4- substitution with isatin derivatives is described. The in-vitro antiretroviral activities indicated that compound 3b was found to be equipotent to lamivudine with EC50 of 0.0742 ± 0.04 µM. Lamivudine prodrugs bearing fluoroquinoles antibacterial showed 92–100% inhibition against Mycobacterium tuberculosis strain H37Rv at 6.25 µg ml–1. At pH 7.4, 37 °C, the hydrolytic t1/2 ranged between 120 and 240 min. © 2005 Published by Elsevier SAS. Keywords: Lamivudine prodrug; Anti-HIV activity; Antimycobacterial activity
1. Introduction
2. Synthesis
Acquired immunodeficiency syndrome (AIDS) is caused by the retrovirus, human immuno deficiency virus (HIV) [1]. The HIV infection, which targets the monocytes expressing surface CD4 receptors, eventually produces profound defects in cell-mediated immunity [2]. Overtime infection leads to severe depletion of CD4 T-lymphocytes (T-cells) resulting in opportunistic infection (OIs) like tuberculosis (TB), fungal, viral, protozoal and neoplastic diseases and ultimately death. TB is the most common OI in people with AIDS and it is the leading killer of people living with HIV/AIDS. The development of active TB accelerates the progression of HIV disease towards full-blown AIDS, accompanied by enhanced HIVreplication. The majority of people infected with the HIV virus develop TB as the first manifestation of AIDS. Through logic and orderly thinking, it appears that an ideal drug for HIV/AIDS patients should suppress HIV-replication thereby acting as anti-HIV drug and also should treat OI like TB [3–5]. As a result we undertook a study to prepare and evaluate lamivudine prodrugs in an effort to identify compounds, which could suppress HIV-replication and also inhibit the M. tuberculosis.
Esterification of the 5’-hydroxyl group of anti-HIV 2’,3’dideoxynucleosides has been used extensively in attempts to improve brain uptake and in vivo efficacy [6]. In this paper we modified in the 4-amino group of 3’-thia-2’, 3’-dideoxycytidine (lamivudine). The synthesis of lamivudine prodrugs proceeded smoothly by condensing parent drug (2) with an equimolar ratio of isatin and its 5-substituted derivatives (1) in presence of glacial acetic acid to form Schiff’s bases (3). The N-Mannich bases (4a–h) of the above Schiff’s bases were prepared by condensing acidic imino group of isatin with formaldehyde and various aryl piperazines (fluoroquinolone antibacterials) with 72–85% yield (Table 1). The purity of the synthesized compounds was checked by TLC and by elemental analyses; and the compounds of this study were identified by spectral data. In general, IR spectra showed C=N (azomethine) peak at 1640 cm−1 and CH2 (Mannich methylene) peak at 2860 and 2840 cm−1. In the 1H-NMR spectra the signals of the respective protons of the prepared prodrugs were verified on the basis of their chemical shifts, multiplicities and coupling constants. The spectra showed a singlet at d 4.8–5.1 ppm corresponding to –NCH2N– group; multiplet at d 3.8–4.1 ppm for piperazine proton; and triplet at d 5.2 ppm for OH group. The elemental analysis results were within ± 0.4% of the theoretical values Scheme 1.
* Corresponding author. Tel.: +91 1596 24 4684; fax: +91 1596 24 4183. E-mail address: [email protected] (D. Sriram). 0223-5234/$ - see front matter © 2005 Published by Elsevier SAS. doi:10.1016/j.ejmech.2005.07.006
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Table 1 Biological properties, lipophilicity and stability of various prodrugs of Lamivudine #
R
Yield M.P. (%) (°C)
3a 3b 4a 4b 4c 4d 4e 4f 4g 4h Lamivudine
H F H Cl F H Cl Cl Cl F –
84 73 80 78 84 85 82 77 76 82
#
214 207 232 212 189 236 231 219 222 213
Anti-HIV activity (µM) EC50a 2.11 ± 0.89 0.0742 ± 0.04 > 134.0 > 11.3 1.11 ± 0.52 49.0 ± 2.33 > 12.5 > 4.73 > 10.5 1.16 ± 0.11 0.1 ± 0.05
CC50b > 200 > 200 134.0 ± 11.1 11.3 ± 1.23 123.0 ± 14.3 121.0 ± 10.8 12.5 ± 1.33 4.73 ± 0.35 10.5 ± 1.55 136 ± 12.2 > 200
SIc > 95 > 2100 – – 110 2.47 – – – 117.24 > 2000
Antimycobacterial activityd (% inhibition) 56 82 92 96 100 98 100 98 100 100 0
log Pe
1.14 1.42 –0.95 –0.91 –0.08 –0.72 –0.75 –0.92 –0.76 –0.53 –1.14
In-vitro hydrolysis t1/2 (min)f 150 120 ND ND 150 240 ND ND ND 120 –
Compounds tested. a Effective concentration of compound achieving 50% protection in CEM cell lines against the cytopathic effect of HIV-1. b Cytotoxic concentration of compound required to reduce the viability of mock infected CEM cells by 50%. c Selectivity index or ratio of CC50/EC50. d At the concentration of 6.25 µg ml–1. e Calculated using SciLogP program. f At pH 7.4 at 37 °C and “ND” indicates not determined.
Scheme 1. (i) Glacial acetic acid/Ethanol, Microwave irradiation at 80% intensity; (ii) HCHO, aryl piperazines, ethanol.
3. Result and discussion The antiviral activity of lamivudine and its prodrugs against HIV-1 was determined in-vitro in T4 lymphocytes (CEM cell
line) [7] (Table 1). Compound 3b was found to be equipotent to lamivudine with EC50 of 0.0742 ± 0.04 µM, CC50 of > 200 µM and selective index (SI) of > 2100. The introduction of fluorine group at the 5th position of isatin enhanced
D. Sriram et al. / European Journal of Medicinal Chemistry 40 (2005) 1373–1376
the activity (3b, 4c, and 4h), whereas the introduction of 5-chloro group (4b,e,f, and 4g) increased the toxicity to CEM cell line. The Mannich bases (4a–h) were less active, when compared to Schiff’s bases (3a–b), whose calculated logP was found to be greater than 1. All compounds were screened against Mycobacterium tuberculosis strain H 37 Rv at a single concentration, 6.25 µg ml–1 in BACTEC 12B medium using the BACTEC 460 radiometric system [8]. All the Mannich bases (4a–h) were found to be most active with 92–100% inhibition. Among the Schiff’s bases compound 3b showed 82% inhibition. Parent drug lamivudine did not show any activity at 6.25 µg ml–1. The usefulness of the prodrugs of lamivudine should depend not only on the stability of the prodrug for its transport across the cell membrane but also upon its reversion to the parent compound intracellularly, especially in the virally infected cells. The half-lives (t1/2) of hydrolysis of the prodrugs were therefore determined at pH 7.4, 37 °C [9]. The data in Table 1 indicated that the various prodrugs of lamivudine were susceptible to hydrolysis with t1/2 in the range of 120–240 min. In the present study we identified that Schiff’s base 3b was found to show equipotent anti-HIV activity to parent compound, lamivudine. Compound 4c inhibited both HIV1 replication and shows 100% inhibition against M. tuberculosis in the preliminary screening. Thus these prodrugs would be beneficial for the effective treatment of HIV/AIDS. 4. Experimental protocols 4.1. Chemistry Melting points were determined in one end open capillary tubes on a Büchi 530 melting point apparatus and are uncorrected. Infrared (IR) and proton nuclear magnetic resonance (1H-NMR) spectra were recorded for the compounds on Jasco IR Report 100 (KBr) and Brucker Avance (300 MHz) instruments, respectively. Chemical shifts are reported in parts per million (ppm) using tetramethyl silane (TMS) as an internal standard. Elemental analyses (C, H, and N) were undertaken with Perkin–Elmer model 240C analyzer. The homogeneity of the compounds was monitored, by ascending thin layer chromatography (TLC) on silicagel-G (Merck) coated aluminum plates, visualized by iodine vapor. Developing solvents were chloroform–methanol (9:1). 4.1.1. General procedure for the preparation of Schiff’s bases Equimolar quantities (0.01 mol) of (sub)isatin and lamivudine were dissolved in warm ethanol containing 1 ml of glacial acetic acid. The reaction mixture was irradiated in an unmodified domestic microwave oven at 80% intensity with 30 s per cycle for 3 min and set aside. The resultant solid was washed with dilute ethanol dried and recrystallized from ethanol–chloroform mixture. Yield: 73–84%.
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4.1.2. 3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2dihydropyrimidin-4-yl}imino)-1,3-dihydro-2H-indol-2-one (3a) Yield: 84%; m.p.: 214 °C; IR (KBr): 3200, 1730, 1654, 1640 1153, 1129 cm−1; 1H-NMR (CDCl3) d (ppm): 3.26 (dd, 2H, 2″-CH2), 3.66 (dd, 2H, 5″-CH2), 5.35 (t, 1H, 4″-CH), 6.33 (t, 1H, 1″-CH), 6.83–7.32 (m, 4H, Ar–H), 7.47 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 10.4 (s, 1H, NH); Anal. for C16H16N4O3S: . 4.1.3. General procedure for the preparation of Mannich bases To a suspension of appropriate Schiff bases (0.02 mol) in ethanol were added appropriate aryl piperazines (0.02 mol) and 37% formaldehyde (0.5 ml) and irradiated in a microwave oven at an intensity of 80% with 30 s per cycle. The number of cycle in turn depended on the completion of the reaction, which was checked by TLC. The reaction timing varied from 1.5 to 3 min. The solution obtained after the completion of the reaction was kept at 0 °C for 30 min and the resulting precipitate was recrystallized from a mixture of DMF and water. 4.1.4. 3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2dihydropyrimidin-4-yl}imino)-1-[(1-Ethyl-6-fluoro-1,4dihydro-4-oxo-3-quinoline carboxylic acid–7piperazinyl)methyl]-1,3-dihydro-2H-indol-2-one (4a) Yield: 80%; m.p.: 232 °C; IR (KBr): 3320, 2860, 2840, 1730, 1654, 1640, 1153, 1129 cm−1; 1H-NMR (CDCl3) d (ppm): 1.28 (t, 3H, CH3 of C2H5), 3.24 (dd, 2H, 2″-CH2), 3.66 (dd, 2H, 5″-CH2), 3.7–4.1 (m, 8H, –piperazine–H), 4.25 (q, 2H, CH2 of C2H5), 5.1 (s, 2H, –NCH2N), 5.34 (t, 1H, 4″-CH), 6.30 (t, 1H, 1″-CH), 6.58–7.3 (m, 6H, Ar–H), 7.52 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 8.66 (s, 1H, C2–H); Anal. for C33H34FN7O6S: . 4.1.5. 3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2dihydropyrimidin-4-yl}imino)-1-[(1-cyclopropyl-6-fluoro1,4-dihydro-4-oxo-3-quinoline carboxylic acid–7piperazinyl)methyl]-1,3-dihydro-2H-indol-2-one (4d) Yield: 85%; m.p.: 236 °C; IR (KBr): 3326, 2860, 2840, 1718, 1730, 1693, 1640, 1153, 1129 cm−1 ; 1H-NMR (CDCl3) d (ppm): 0.88–1.1 (m, 4H, cyclopropyl-H), 3.24 (dd, 2H, 2″-CH2), 3.5 (m, 1H, cyclopropyl-H), 3.66 (dd, 2H, 5″-CH2), 3.7–4.1 (m, 8H, –piperazine–H), 5.1 (s, 2H, –NCH2N), 5.34 (t, 1H, 4″-CH), 6.30 (t, 1H, 1″-CH), 6.68–7.2 (m, 6H, Ar–H), 7.52 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 8.6 (s, 1H, C2–H); Anal. for C34H34FN7O6S: . 4.1.6. 5-chloro-3-({1-[2-(hydroxymethyl)-1,3-oxathiolan-5yl]-1,2-dihydropyrimidin-4-yl}imino)-1-[(1-cyclopropyl-6fluoro-8-methoxy-1,4-dihydro-4-oxo-3-quinoline carboxylic acid–7-(3-methyl)piperazinyl)methyl]-1,3-dihydro-2Hindol-2-one (4g) Yield: 76%; m.p.: 222 °C; IR (KBr): 3326, 2860, 2840, 1730, 1652, 1640, 1153, 1129 cm−1 ; 1H-NMR (CDCl3) d
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(ppm): 0.92–1.10 (m, 4H, cyclopropyl-H), 1.14 (d, 3H, CH3 of pip), 3.24 (dd, 2H, 2″-CH2), 3.46 (s, 3H, –OCH3), 3.6– 4.18 (m, 10H, cyclopropyl-H, –piperazine–H, 5″-CH2), 5.2 (s, 2H, –NCH2N), 5.34 (t, 1H, 4″-CH), 6.30 (t, 1H, 1″-CH), 6.68–8.02 (m, 4H, Ar–H), 7.52 (d, 1H, 5′-H), 8.48 (d, 1H, 6′-H), 8.6 (s, 1H, C2–H) Anal. for C36H37ClFN7O7S: . 4.2. Anti-HIV screening [7] Candidate agents were dissolved in dimethylsulfoxide, and then diluted 1:100 in cell culture medium before preparing serial half- log10 dilutions. T4 lymphocytes (CEM cell-line) were added and after a brief interval HIV-1 was added, resulting in a 1:200 final dilution of the compound. Uninfected cells with the compound served as a toxicity control, and infected and uninfected cells without the compound served as basic controls. Cultures were incubated at 37 °C in a 5% carbon dioxide atmosphere for 6 days. The tetrazolium salt, XTT was added to all the wells, and cultures were incubated to allow formazan color development by viable cells. Individual wells were analyzed spectrophotometrically to quantitative formazan production, and in addition were viewed microscopically for detection of viable cells and confirmation of protective activity. 4.3. Antimycobacterial activity Primary screening was conducted at 6.25 µg ml–1 against M. tuberculosis strain H37Rv (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the microplate Alamar Blue Assay (MABA) [8].
ethyl sulfoxide) and the mixture was incubated at 37 °C in a water bath. At various time intervals (0–4 h), 100 µl of the samples were withdrawn and added immediately to ice-cold methanol (400 µl). The samples were centrifuged and the supernatants were filtered through nylon 66 filters (0.45 pm) and analyzed by spectrophotometrically [9]. From the observed optical density changes, at various wavelengths, the half-lives of the analogues were calculated. Acknowledgements The authors deeply acknowledge the financial support of the Council of Industrial and Scientific Research, New Delhi (No. 01(1979)/05/EMR-II). The authors also thank Dr. Edward Sausville, National Cancer Institute, USA and Dr. S. Ananthan from the Southern Research Institute, Birmingham, Alabama, USA, for support in biological testing. References [1] [2] [3] [4] [5] [6] [7]
4.4. In-vitro stability studies To 990 µl of phosphate buffer (0.2 M, pH 7.4) was added 10 µl of a solution of appropriate drugs (10 mg ml–1 in dim-
[8] [9]
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