Bioorganic & Medicinal Chemistry Letters 24 (2014) 298–301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Triazino indole–quinoline hybrid: A novel approach to antileishmanial agents Rashmi Sharma a, Anand Kumar Pandey a, Rahul Shivahare b, Khushboo Srivastava b, Suman Gupta b, Prem M. S. Chauhan a,⇑ a b
Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226 031, India Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226 031, India
a r t i c l e
i n f o
Article history: Received 13 August 2013 Revised 28 October 2013 Accepted 9 November 2013 Available online 17 November 2013 Keywords: Antileishmanial Hybrid In vitro Leishmania donovani Quinoline Triazino-indole
a b s t r a c t A novel series of 1,2,4-triazino-[5,6b]indole-3-thione covalently linked to 7-chloro-4-aminoquinoline have been synthesized and evaluated for their in vitro activity against extracellular promastigote and intracellular amastigote form of Leishmania donovani. Among all tested compounds, compounds 7a and 7b were found to be the most active with IC50 values 1.11, 0.36 lM and selectivity index (SI) values 67, >1111, respectively, against amastigote form of L. donovani which is several folds more potent than the standard drugs, miltefosine (IC50 = 8.10 lM, SI = 7) and sodium stibo-gluconate (IC50 = 54.60 lM, SI P 7). Ó 2013 Published by Elsevier Ltd.
Even after intensive research activity, not many medical alternatives are available for patients afflicted by tropical illnesses in developing countries.1 Affecting 88 countries of which 72 are classified as developing countries, including 13 of the least developed countries in the world and with a population of 350 million at risk, leishmaniasis, is still widespread and devastating human disease.2 The disease is caused by obligate intra-macrophage protozoa of the genus Leishmania and transmitted to humans by 30 different species of phlebotomine sandflies.3 Increase in travel and migration is allowing leishmaniasis to become an emerging disease worldwide. Visceral leishmaniasis (VL),4 which accounts for most of the 70,000 leishmaniasis related deaths per year, is strongly linked to poverty and 90% of the cases are in the poorest areas of India, Bangladesh, Ethiopia, Brazil, Nepal and Sudan. Since a large number of cases are typically unreported in third world countries it is safe to assume that the case-load would be much higher than official reports. This disease is fatal if left untreated. Moreover, co-infection with HIV/AIDS has worsened the whole scenario of the disease. It is currently reported for 2–9% of all VL cases and is likely to increase.5 Synthesis of a low cost drug for leishmaniasis with acceptable side effects has been a significant challenge in drug research.6 No new clinical options are available for the
⇑ Corresponding author. Fax: +91 (522)2771941. E-mail addresses: [email protected], [email protected] (P.M.S. Chauhan). 0960-894X/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmcl.2013.11.018
treatment except miltefosine, a liposome formulation of amphotericin B and a topical formulation of paromomycin.7 Quinoline derivatives,8 the basis of chemotherapy against malaria9 for more than 60 years, posses a wide range of biological activities including antileishmanial,10 antibacterial,11 antifungal,12 anti-HIV,13 antitumor,14 anti-alzheimer15 etc., Leishmania spp. share many common biochemical characteristics with plasmodium genera and hence quinoline based chemotherapeutic agents are under considerable interest as anti-leishmanial agents.16 An orally active 8-aminoquinoline analogue WR6026 (A) (Fig. 1), developed by the Walter Reed Army Institute in collaboration with Glaxo Smith Kline was recently in clinical trials.17 On the other hand triazino[5,6-b]indole scaffold is known to posses convincing biological activities such as antiviral,18 antimalarial19 and
A
B
Figure 1. (A) WR6026, (B) 3-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-ylthio)-5methyl-5H-[1,2,4]triazino[5,6-b]indole.
299
R. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 298–301
antileishmanial20 etc., Recently, our group reported 3-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-ylthio)-5-methyl-5H-[1,2,4]triazino[5,6-b] indole (B) (Fig. 1) against L. donovani with IC50 = 4.01 lM.20 As part of our continuing research20–23 which approaches pharmacophore hybridization to develop novel chemotherapeutic agents against L. donovani, we developed a series of 7-chloro-N(2-(5-alkyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)alkyl)quinolin4-amine 7 and evaluated their antileishmanial activity as well as their cytotoxicity. The route used to synthesize representative compounds of this class is depicted in Scheme 1. N-Alkylation of the isatin was achieved with commercially available alkyl halides in the presence of K2CO3 base in N,N-dimethylformamide (DMF) and were
Cl
HN
N
Cl
Cl
N 2
O
R
N R'
R
N R' 6
N
NH Cl
N
N R'
N 7
N N
O
n
S R
d
c
N N
e
2a; n = 2 2b; n = 3 2c; n = 5
O
R R R R
N 3
O
N H
n Br
b
1
R
HN
n OH
a Cl
cyclized to the 1,2,4-triazino-[5,6b]indole-3-thione 6 in aqueous K2CO3 with thiosemicarbazide at reflux condition.19 The second series of pharmacophore 3 was prepared via a classical nucleophilic aromatic substitution reaction between the appropriate aminoalkyl alcohol (2-aminoethanol, 3-aminopropan-1-ol, 5-aminopentan-1-ol) and 4,7-dichloroquinoline followed by subsequent bromination with H2SO4/HBr at 140 °C.24 Interestingly, increasing the number of carbon atoms in the alkyl alcohol chain (n = 2, 3, 5) attached with 7-chloroquinoline was found to disfavor bromination 3, but the synthesis of (2c) was nevertheless achieved using the same route as for 3a and 3b, albeit in a lower yield. Further nucleophilic substitution to displace the bromide of the 4-(b-bromoalkyl)-7-chloroquinoline 3 with
SH
5 4 = -H, R' = -H, -Me, -Et, -Pr, -i Pr, -Bu, - i Bu, -allyl, -benzyl = -5Br, R' = -H, -Me, -Et = -5NO 2, R' = -Me = -7CF3 , R' = -Me
Scheme 1. Reagents and conditions: (a) aminoalkyl alcohol, neat, 80–120 °C, 4–6 h; (b) H2SO4/HBr, 140 °C, 3–4 h; (c) alkyl halide. K2CO3, DMF, rt, 4–16 h; (d) thiosemicarbazide, K2CO3, H2O, reflux, 5–8 h; (e) K2CO3, DMF, rt, 0.5–4 h.
Table 1 Synthesis of triazino indole–quinoline hybrid 7a–s via nucleophilic substitution
HN
n Br
N N
N N Cl
N
R
3
a b
6
N R'
N
S
SH N R'
R
n
NH Cl
N N 7
Entry
Reaction conditiona
Reaction time (min)
Yieldb (%)
7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s
K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3,
90 120 90 60 65 80 75 110 85 45 30 40 170 240 210 190 215 185 135
86 89 85 81 86 87 83 78 85 88 83 89 83 79 82 74 79 86 88
DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF,
rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt
Reaction conditions: 3 (1 equiv), 6 (1 equiv), K2CO3 (1.3 equiv), DMF as solvent at room temperature. Isolated yield.
300
R. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 298–301
alkyl chain length
N N S Br, NO2 CF3
R
N R'
n
NH Cl
N N
H, Me, Et, Pr, iPr Bu, iBu, allyl, benzyl Figure 2. Structural parameters considered in the SAR analysis.
thione 6 was achieved using K2CO3 in DMF at ambient temperature. After completion of reaction (shown by TLC), ice cold water was added in the reaction mixture and the precipitate obtained was collected by filtration. Simple washing with water followed by methanol was sufficient to achieve the series of representative compound in good to excellent yield for spectroscopic analysis without any further purification (Table 1). All the synthesized compounds were characterized by spectroscopic methods such as NMR (1H, 13C), Mass and IR. All synthesized compounds were simultaneously evaluated against extracellular promastigote and intracellular amastigote stage of the parasite and standard antileishmanials, miltefosine and SSG were included in the study as control drugs. As the intracellular amastigote form is clinically relevant parasitic stage for biological evaluation, we were pleased to find that synthesized analogues which were inactive against promastigote nevertheless showed poor to excellent activity against amastigote form of L. donovani, which is perhaps due to their immunostimulatory nature. Since a large set of the compounds showed significant antileishmanial activity, we were able to establish a detailed structure-activity relationship. Chain length joining the two
pharmacophores, alkyl group at nitrogen atom of triazino indole (R0 ) and substitution on the aromatic C(8)/C(6) position (R) were considered as structural parameters in the SAR analysis (Fig. 2). Analysis of the antileishmanial activity in relation to the chain length joining the two pharmacophores revealed that compounds with two carbon atom chain were moderate to quite active (IC50 = 7.10–0.36 lM) with significant selectivity index (SI P 1111–7) (entry 7a–7c), however, lengthening the carbon chain (3 and 5 carbon atoms) resulted in the loss of activity against intracellular form of parasite (entry 7d–7h) which suggests 2 carbon chain as the most promising chain length joining two pharmacophores (Table 2). Also it was found that, unlike for amastigotes all the above mentioned analogues (entry 7a–7h) were found good to moderately active (IC50 = 24.14–2.66 lM) in case of antipromastigote assay. The possibility of increase in lipophilicity of compounds by substituting R0 = H with R0 = Me enhanced activity up to threefold and significantly improved the selectivity (SI = >1111) (compare entry 7a–7b) but further increase in the number of carbon atom in straight chain at R0 led to decrease in activity against intracellular stage of parasite and increase in cytotoxicity. However, branching at substitution (R0 = iPr and iBu) resulted in major improvement in activity corresponding to their straight chain compounds against both forms of parasite (7i and 7j), with a slight improvement in selectivity (entry 7k and 7l). Unsaturation at R0 group also led to the increase in activity against both form of parasite (compare entry 7i and 7m). Bulkier benzyl group was introduced to investigate the effect of enhanced aromatic character but it was found to be toxic to cells in antiamastigote assay and moderately active in antipromastigote assay (entry 7n). Furthermore, substitution on the aromatic C(8) position with a bromo or nitro led to reduced activity or toxicity in all four derivatives (entry 7o–7r) against amastigote form, however nitro at aromatic C(8) position (entry 7r) was found to be quite active against extracellular strain of the parasite. Functionalization at position C(6) with trifluoro-
Table 2 In vitro antileishmanial activity against extracellular promastigotes and intracellular amastigotes and their cytotoxicity
The gray shaded values represented the most potent compound in present series. MF (miltefosine) and SSG (sodium stibo-gluconate): standard antileishmanial drugs. IC50 > 15 lM = inactive; IC50 5–15 lM = moderately active; IC50 < 5 lM = active compounds. a SI for each compound was calculated as ratio between, CC50 in vero cells and IC50 against Leishmania amastigotes. b Compounds were toxic for J-774A.1 macrophage cells. c ND = Not done. d NA = Not available.
R. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 298–301
methyl was found to be potent with moderate activity and selectivity against both extra and intracellular form of L. donovani (entry 7s). In summary, a new series of triazino indole–quinoline hybrid has been developed as potent antileishmanial chemotherapeutic agent against L. donovani amastigotes and promastigotes. The synthetic efficiency of the methodology is demonstrated in its high yield and easy purification process clubbed with the advantage of low cost starting material. Out of the synthesized 19 derivatives in the present series, most of the compounds were found active with IC50 in the range of 0.36–29.48 lM against intracellular amastigote strain of parasite with significant selectivity. On the basis of biological evaluation and SAR study of the series, triazino indole– quinoline hybrids seem to be promising antileishmanial chemotherapeutic agents to be explored further in future. Acknowledgments The authors thank the Council of Scientific and Industrial Research, India for financial support, for the award of senior research fellowship and the S.A.I.F. division, CDRI, Lucknow, for providing spectroscopic data. Discussions regarding NMR with Dr. Sanjeev Shukla were helpful. CDRI communication number is 8565. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11. 018. References and notes 1. 2. 3. 4.
Enserink, M. Science 2006, 314, 1373. Desjeux, P. Nat. Rev. Microbiol. 2004, 2, 692. Kobets, T.; Grekov, I.; Lipoldova, M. Curr. Med. Chem. 2012, 19, 1443. Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R. W.; Alvar, J.; Boelaert, M. Nat. Rev. Microbiol. 2007, 5, 873.
301
5. Alvar, J.; Aparicio, P.; Aseffa, A.; Den Boer, M.; Cañavate, C.; Dedet, J.-P.; Gradoni, L.; Ter Horst, R.; López-Vélez, R.; Moreno, J. Clin. Microbiol. Rev. 2008, 21, 334. 6. Santos, D. O.; Coutinho, C. E. R.; Madeira, M. F.; Bottino, C. G.; Vieira, R. T.; Nascimento, S. B.; Bernardino, A.; Bourguignon, S. C.; Corte-Real, S.; Pinho, R. T.; Rodrigues, C. R.; Castro, H. C. Parasitol. Res. 2008, 103, 1. 7. Freitas-Junior, L. H.; Chatelain, E.; Kim, H. A.; Siqueira-Neto, J. L. Int. J. Parasitology: Drugs Drug Resistance 2012, 2, 11. 8. Marella, A.; Tanwar, O. P.; Saha, R.; Ali, M. R.; Srivastava, S.; Akhter, M.; Shaquiquzzaman, M.; Alam, M. M. Saudi Pharm. J. 2013, 21, 1. 9. Teguh, S. C.; Klonis, N.; Duffy, S.; Lucantoni, L.; Avery, V. M.; Hutton, C. a; Baell, J. B.; Tilley, L. J. Med. Chem. 2013. doi: org/10.1021/jm400656s. 10. Coimbra, E. S.; Carvalhaes, R.; Grazul, R. M.; Machado, P. A.; De Souza, M. V. N.; Da Silva, A. D. Chem. Biol. Drug Des. 2010, 75, 628. 11. Eswaran, S.; Adhikari, A. V.; Chowdhury, I. H.; Pal, N. K.; Thomas, K. D. Eur. J. Med. Chem. 2010, 45, 3374. 12. Vandekerckhove, S.; Tran, H. G.; Desmet, T.; D’hooghe, M. Bioorg. Med. Chem. Lett. 2013, 23, 4641. 13. Ahmed, N.; Brahmbhatt, K. G.; Sabde, S.; Mitra, D.; Singh, I. P.; Bhutani, K. K. Bioorg. Med. Chem. 2010, 18, 2872. 14. Lilienkampf, A.; Mao, J.; Wan, B.; Wang, Y.; Franzblau, S. G.; Kozikowski, A. P. J. Med. Chem. 2009, 52, 2109. 15. Camps, P.; Formosa, X.; Galdeano, C.; Muñoz-Torrero, D.; Ramírez, L.; Gómez, E.; Isambert, N.; Lavilla, R.; Badia, A.; Clos, M. V.; Bartolini, M.; Mancini, F.; Andrisano, V.; Arce, M. P.; Rodríguez-Franco, M. I.; Huertas, O.; Dafni, T.; Luque, F. J. J. Med. Chem. 2009, 52, 5365. 16. Liu, H.; Nolan, L. Antileishmanial mode of action of derivatives of the quinoline alkloid (QUININE) from the bark of Cinchona Ledgeriana; 1996. 17. Dietze, R.; Carvalho, S. F.; Valli, L. C.; Berman, J.; Brewer, T.; Milhous, W.; Sanchez, J.; Schuster, B.; Grogl, M. Am. J. Trop. Med. Hyg. 2001, 65, 685. 18. Agents, A.; Gladych, J. M. Z.; Hornby, R.; Hunt, J. H.; Jack, D.; Boyle, J. J.; Ferlauto, R. J.; Haff, R. F.; Kormendy, C. G.; Stanfield, F. J.; Stewart, R. C. J. Med. Chem. 1972, 15, 277. 19. Kgokong, J. L.; Smith, P. P.; Matsabisa, G. M. Bioorg. Med. Chem. 2005, 13, 2935. 20. Gupta, L.; Sunduru, N.; Verma, A.; Srivastava, S.; Gupta, S.; Goyal, N.; Chauhan, P. M. S. Eur. J. Med. Chem. 2010, 45, 2359. 21. Pandey, A. K.; Sharma, R.; Shivahare, R.; Arora, A.; Rastogi, N.; Gupta, S.; Chauhan, P. M. S. J. Org. Chem. 2013, 78, 1534. 22. Sharma, M.; Chauhan, K.; Shivahare, R.; Vishwakarma, P.; Suthar, M. K.; Sharma, A.; Gupta, S.; Saxena, J. K.; Lal, J.; Chandra, P.; Kumar, B.; Chauhan, P. M. S. J. Med. Chem. 2013, 56, 4374. 23. Chauhan, S. S.; Gupta, L.; Mittal, M.; Vishwakarma, P.; Gupta, S.; Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2010, 20, 6191. 24. Elderfield, R. C.; Gensler, W. J.; Birstein, O.; Kreysa, F. J.; Maynard, J. T.; Galbreath, J. J. Am. Chem. Soc. 1946, 68, 1250.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Triazino indole–quinoline hybrid: A novel approach to antileishmanial agents Rashmi Sharma a, Anand Kumar Pandey a, Rahul Shivahare b, Khushboo Srivastava b, Suman Gupta b, Prem M. S. Chauhan a,⇑ a b
Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226 031, India Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226 031, India
a r t i c l e
i n f o
Article history: Received 13 August 2013 Revised 28 October 2013 Accepted 9 November 2013 Available online 17 November 2013 Keywords: Antileishmanial Hybrid In vitro Leishmania donovani Quinoline Triazino-indole
a b s t r a c t A novel series of 1,2,4-triazino-[5,6b]indole-3-thione covalently linked to 7-chloro-4-aminoquinoline have been synthesized and evaluated for their in vitro activity against extracellular promastigote and intracellular amastigote form of Leishmania donovani. Among all tested compounds, compounds 7a and 7b were found to be the most active with IC50 values 1.11, 0.36 lM and selectivity index (SI) values 67, >1111, respectively, against amastigote form of L. donovani which is several folds more potent than the standard drugs, miltefosine (IC50 = 8.10 lM, SI = 7) and sodium stibo-gluconate (IC50 = 54.60 lM, SI P 7). Ó 2013 Published by Elsevier Ltd.
Even after intensive research activity, not many medical alternatives are available for patients afflicted by tropical illnesses in developing countries.1 Affecting 88 countries of which 72 are classified as developing countries, including 13 of the least developed countries in the world and with a population of 350 million at risk, leishmaniasis, is still widespread and devastating human disease.2 The disease is caused by obligate intra-macrophage protozoa of the genus Leishmania and transmitted to humans by 30 different species of phlebotomine sandflies.3 Increase in travel and migration is allowing leishmaniasis to become an emerging disease worldwide. Visceral leishmaniasis (VL),4 which accounts for most of the 70,000 leishmaniasis related deaths per year, is strongly linked to poverty and 90% of the cases are in the poorest areas of India, Bangladesh, Ethiopia, Brazil, Nepal and Sudan. Since a large number of cases are typically unreported in third world countries it is safe to assume that the case-load would be much higher than official reports. This disease is fatal if left untreated. Moreover, co-infection with HIV/AIDS has worsened the whole scenario of the disease. It is currently reported for 2–9% of all VL cases and is likely to increase.5 Synthesis of a low cost drug for leishmaniasis with acceptable side effects has been a significant challenge in drug research.6 No new clinical options are available for the
⇑ Corresponding author. Fax: +91 (522)2771941. E-mail addresses: [email protected], [email protected] (P.M.S. Chauhan). 0960-894X/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmcl.2013.11.018
treatment except miltefosine, a liposome formulation of amphotericin B and a topical formulation of paromomycin.7 Quinoline derivatives,8 the basis of chemotherapy against malaria9 for more than 60 years, posses a wide range of biological activities including antileishmanial,10 antibacterial,11 antifungal,12 anti-HIV,13 antitumor,14 anti-alzheimer15 etc., Leishmania spp. share many common biochemical characteristics with plasmodium genera and hence quinoline based chemotherapeutic agents are under considerable interest as anti-leishmanial agents.16 An orally active 8-aminoquinoline analogue WR6026 (A) (Fig. 1), developed by the Walter Reed Army Institute in collaboration with Glaxo Smith Kline was recently in clinical trials.17 On the other hand triazino[5,6-b]indole scaffold is known to posses convincing biological activities such as antiviral,18 antimalarial19 and
A
B
Figure 1. (A) WR6026, (B) 3-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-ylthio)-5methyl-5H-[1,2,4]triazino[5,6-b]indole.
299
R. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 298–301
antileishmanial20 etc., Recently, our group reported 3-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-ylthio)-5-methyl-5H-[1,2,4]triazino[5,6-b] indole (B) (Fig. 1) against L. donovani with IC50 = 4.01 lM.20 As part of our continuing research20–23 which approaches pharmacophore hybridization to develop novel chemotherapeutic agents against L. donovani, we developed a series of 7-chloro-N(2-(5-alkyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)alkyl)quinolin4-amine 7 and evaluated their antileishmanial activity as well as their cytotoxicity. The route used to synthesize representative compounds of this class is depicted in Scheme 1. N-Alkylation of the isatin was achieved with commercially available alkyl halides in the presence of K2CO3 base in N,N-dimethylformamide (DMF) and were
Cl
HN
N
Cl
Cl
N 2
O
R
N R'
R
N R' 6
N
NH Cl
N
N R'
N 7
N N
O
n
S R
d
c
N N
e
2a; n = 2 2b; n = 3 2c; n = 5
O
R R R R
N 3
O
N H
n Br
b
1
R
HN
n OH
a Cl
cyclized to the 1,2,4-triazino-[5,6b]indole-3-thione 6 in aqueous K2CO3 with thiosemicarbazide at reflux condition.19 The second series of pharmacophore 3 was prepared via a classical nucleophilic aromatic substitution reaction between the appropriate aminoalkyl alcohol (2-aminoethanol, 3-aminopropan-1-ol, 5-aminopentan-1-ol) and 4,7-dichloroquinoline followed by subsequent bromination with H2SO4/HBr at 140 °C.24 Interestingly, increasing the number of carbon atoms in the alkyl alcohol chain (n = 2, 3, 5) attached with 7-chloroquinoline was found to disfavor bromination 3, but the synthesis of (2c) was nevertheless achieved using the same route as for 3a and 3b, albeit in a lower yield. Further nucleophilic substitution to displace the bromide of the 4-(b-bromoalkyl)-7-chloroquinoline 3 with
SH
5 4 = -H, R' = -H, -Me, -Et, -Pr, -i Pr, -Bu, - i Bu, -allyl, -benzyl = -5Br, R' = -H, -Me, -Et = -5NO 2, R' = -Me = -7CF3 , R' = -Me
Scheme 1. Reagents and conditions: (a) aminoalkyl alcohol, neat, 80–120 °C, 4–6 h; (b) H2SO4/HBr, 140 °C, 3–4 h; (c) alkyl halide. K2CO3, DMF, rt, 4–16 h; (d) thiosemicarbazide, K2CO3, H2O, reflux, 5–8 h; (e) K2CO3, DMF, rt, 0.5–4 h.
Table 1 Synthesis of triazino indole–quinoline hybrid 7a–s via nucleophilic substitution
HN
n Br
N N
N N Cl
N
R
3
a b
6
N R'
N
S
SH N R'
R
n
NH Cl
N N 7
Entry
Reaction conditiona
Reaction time (min)
Yieldb (%)
7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s
K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3, K2CO3,
90 120 90 60 65 80 75 110 85 45 30 40 170 240 210 190 215 185 135
86 89 85 81 86 87 83 78 85 88 83 89 83 79 82 74 79 86 88
DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF, DMF,
rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt rt
Reaction conditions: 3 (1 equiv), 6 (1 equiv), K2CO3 (1.3 equiv), DMF as solvent at room temperature. Isolated yield.
300
R. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 298–301
alkyl chain length
N N S Br, NO2 CF3
R
N R'
n
NH Cl
N N
H, Me, Et, Pr, iPr Bu, iBu, allyl, benzyl Figure 2. Structural parameters considered in the SAR analysis.
thione 6 was achieved using K2CO3 in DMF at ambient temperature. After completion of reaction (shown by TLC), ice cold water was added in the reaction mixture and the precipitate obtained was collected by filtration. Simple washing with water followed by methanol was sufficient to achieve the series of representative compound in good to excellent yield for spectroscopic analysis without any further purification (Table 1). All the synthesized compounds were characterized by spectroscopic methods such as NMR (1H, 13C), Mass and IR. All synthesized compounds were simultaneously evaluated against extracellular promastigote and intracellular amastigote stage of the parasite and standard antileishmanials, miltefosine and SSG were included in the study as control drugs. As the intracellular amastigote form is clinically relevant parasitic stage for biological evaluation, we were pleased to find that synthesized analogues which were inactive against promastigote nevertheless showed poor to excellent activity against amastigote form of L. donovani, which is perhaps due to their immunostimulatory nature. Since a large set of the compounds showed significant antileishmanial activity, we were able to establish a detailed structure-activity relationship. Chain length joining the two
pharmacophores, alkyl group at nitrogen atom of triazino indole (R0 ) and substitution on the aromatic C(8)/C(6) position (R) were considered as structural parameters in the SAR analysis (Fig. 2). Analysis of the antileishmanial activity in relation to the chain length joining the two pharmacophores revealed that compounds with two carbon atom chain were moderate to quite active (IC50 = 7.10–0.36 lM) with significant selectivity index (SI P 1111–7) (entry 7a–7c), however, lengthening the carbon chain (3 and 5 carbon atoms) resulted in the loss of activity against intracellular form of parasite (entry 7d–7h) which suggests 2 carbon chain as the most promising chain length joining two pharmacophores (Table 2). Also it was found that, unlike for amastigotes all the above mentioned analogues (entry 7a–7h) were found good to moderately active (IC50 = 24.14–2.66 lM) in case of antipromastigote assay. The possibility of increase in lipophilicity of compounds by substituting R0 = H with R0 = Me enhanced activity up to threefold and significantly improved the selectivity (SI = >1111) (compare entry 7a–7b) but further increase in the number of carbon atom in straight chain at R0 led to decrease in activity against intracellular stage of parasite and increase in cytotoxicity. However, branching at substitution (R0 = iPr and iBu) resulted in major improvement in activity corresponding to their straight chain compounds against both forms of parasite (7i and 7j), with a slight improvement in selectivity (entry 7k and 7l). Unsaturation at R0 group also led to the increase in activity against both form of parasite (compare entry 7i and 7m). Bulkier benzyl group was introduced to investigate the effect of enhanced aromatic character but it was found to be toxic to cells in antiamastigote assay and moderately active in antipromastigote assay (entry 7n). Furthermore, substitution on the aromatic C(8) position with a bromo or nitro led to reduced activity or toxicity in all four derivatives (entry 7o–7r) against amastigote form, however nitro at aromatic C(8) position (entry 7r) was found to be quite active against extracellular strain of the parasite. Functionalization at position C(6) with trifluoro-
Table 2 In vitro antileishmanial activity against extracellular promastigotes and intracellular amastigotes and their cytotoxicity
The gray shaded values represented the most potent compound in present series. MF (miltefosine) and SSG (sodium stibo-gluconate): standard antileishmanial drugs. IC50 > 15 lM = inactive; IC50 5–15 lM = moderately active; IC50 < 5 lM = active compounds. a SI for each compound was calculated as ratio between, CC50 in vero cells and IC50 against Leishmania amastigotes. b Compounds were toxic for J-774A.1 macrophage cells. c ND = Not done. d NA = Not available.
R. Sharma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 298–301
methyl was found to be potent with moderate activity and selectivity against both extra and intracellular form of L. donovani (entry 7s). In summary, a new series of triazino indole–quinoline hybrid has been developed as potent antileishmanial chemotherapeutic agent against L. donovani amastigotes and promastigotes. The synthetic efficiency of the methodology is demonstrated in its high yield and easy purification process clubbed with the advantage of low cost starting material. Out of the synthesized 19 derivatives in the present series, most of the compounds were found active with IC50 in the range of 0.36–29.48 lM against intracellular amastigote strain of parasite with significant selectivity. On the basis of biological evaluation and SAR study of the series, triazino indole– quinoline hybrids seem to be promising antileishmanial chemotherapeutic agents to be explored further in future. Acknowledgments The authors thank the Council of Scientific and Industrial Research, India for financial support, for the award of senior research fellowship and the S.A.I.F. division, CDRI, Lucknow, for providing spectroscopic data. Discussions regarding NMR with Dr. Sanjeev Shukla were helpful. CDRI communication number is 8565. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11. 018. References and notes 1. 2. 3. 4.
Enserink, M. Science 2006, 314, 1373. Desjeux, P. Nat. Rev. Microbiol. 2004, 2, 692. Kobets, T.; Grekov, I.; Lipoldova, M. Curr. Med. Chem. 2012, 19, 1443. Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R. W.; Alvar, J.; Boelaert, M. Nat. Rev. Microbiol. 2007, 5, 873.
301
5. Alvar, J.; Aparicio, P.; Aseffa, A.; Den Boer, M.; Cañavate, C.; Dedet, J.-P.; Gradoni, L.; Ter Horst, R.; López-Vélez, R.; Moreno, J. Clin. Microbiol. Rev. 2008, 21, 334. 6. Santos, D. O.; Coutinho, C. E. R.; Madeira, M. F.; Bottino, C. G.; Vieira, R. T.; Nascimento, S. B.; Bernardino, A.; Bourguignon, S. C.; Corte-Real, S.; Pinho, R. T.; Rodrigues, C. R.; Castro, H. C. Parasitol. Res. 2008, 103, 1. 7. Freitas-Junior, L. H.; Chatelain, E.; Kim, H. A.; Siqueira-Neto, J. L. Int. J. Parasitology: Drugs Drug Resistance 2012, 2, 11. 8. Marella, A.; Tanwar, O. P.; Saha, R.; Ali, M. R.; Srivastava, S.; Akhter, M.; Shaquiquzzaman, M.; Alam, M. M. Saudi Pharm. J. 2013, 21, 1. 9. Teguh, S. C.; Klonis, N.; Duffy, S.; Lucantoni, L.; Avery, V. M.; Hutton, C. a; Baell, J. B.; Tilley, L. J. Med. Chem. 2013. doi: org/10.1021/jm400656s. 10. Coimbra, E. S.; Carvalhaes, R.; Grazul, R. M.; Machado, P. A.; De Souza, M. V. N.; Da Silva, A. D. Chem. Biol. Drug Des. 2010, 75, 628. 11. Eswaran, S.; Adhikari, A. V.; Chowdhury, I. H.; Pal, N. K.; Thomas, K. D. Eur. J. Med. Chem. 2010, 45, 3374. 12. Vandekerckhove, S.; Tran, H. G.; Desmet, T.; D’hooghe, M. Bioorg. Med. Chem. Lett. 2013, 23, 4641. 13. Ahmed, N.; Brahmbhatt, K. G.; Sabde, S.; Mitra, D.; Singh, I. P.; Bhutani, K. K. Bioorg. Med. Chem. 2010, 18, 2872. 14. Lilienkampf, A.; Mao, J.; Wan, B.; Wang, Y.; Franzblau, S. G.; Kozikowski, A. P. J. Med. Chem. 2009, 52, 2109. 15. Camps, P.; Formosa, X.; Galdeano, C.; Muñoz-Torrero, D.; Ramírez, L.; Gómez, E.; Isambert, N.; Lavilla, R.; Badia, A.; Clos, M. V.; Bartolini, M.; Mancini, F.; Andrisano, V.; Arce, M. P.; Rodríguez-Franco, M. I.; Huertas, O.; Dafni, T.; Luque, F. J. J. Med. Chem. 2009, 52, 5365. 16. Liu, H.; Nolan, L. Antileishmanial mode of action of derivatives of the quinoline alkloid (QUININE) from the bark of Cinchona Ledgeriana; 1996. 17. Dietze, R.; Carvalho, S. F.; Valli, L. C.; Berman, J.; Brewer, T.; Milhous, W.; Sanchez, J.; Schuster, B.; Grogl, M. Am. J. Trop. Med. Hyg. 2001, 65, 685. 18. Agents, A.; Gladych, J. M. Z.; Hornby, R.; Hunt, J. H.; Jack, D.; Boyle, J. J.; Ferlauto, R. J.; Haff, R. F.; Kormendy, C. G.; Stanfield, F. J.; Stewart, R. C. J. Med. Chem. 1972, 15, 277. 19. Kgokong, J. L.; Smith, P. P.; Matsabisa, G. M. Bioorg. Med. Chem. 2005, 13, 2935. 20. Gupta, L.; Sunduru, N.; Verma, A.; Srivastava, S.; Gupta, S.; Goyal, N.; Chauhan, P. M. S. Eur. J. Med. Chem. 2010, 45, 2359. 21. Pandey, A. K.; Sharma, R.; Shivahare, R.; Arora, A.; Rastogi, N.; Gupta, S.; Chauhan, P. M. S. J. Org. Chem. 2013, 78, 1534. 22. Sharma, M.; Chauhan, K.; Shivahare, R.; Vishwakarma, P.; Suthar, M. K.; Sharma, A.; Gupta, S.; Saxena, J. K.; Lal, J.; Chandra, P.; Kumar, B.; Chauhan, P. M. S. J. Med. Chem. 2013, 56, 4374. 23. Chauhan, S. S.; Gupta, L.; Mittal, M.; Vishwakarma, P.; Gupta, S.; Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2010, 20, 6191. 24. Elderfield, R. C.; Gensler, W. J.; Birstein, O.; Kreysa, F. J.; Maynard, J. T.; Galbreath, J. J. Am. Chem. Soc. 1946, 68, 1250.
