PRIMACINS, N-cinnamoyl-primaquine conjugates, with improved liver-stage antimalarial activity

PRIMACINS, N-cinnamoyl-primaquine conjugates, with improved liver-stage antimalarial activity† Bianca P erez, a C atia Teixeira, ab In^ es S. Albuquerque, c Jiri Gut, d Philip J. Rosenthal, d Miguel Prud^ encio* c and Paula Gomes* a Received 2nd May 2012, Accepted 16th July 2012 DOI: 10.1039/c2md20113e Novel primaquine derivatives, obtained by conjugation of the drug’s aliphatic amine with different cinnamic acids, resulted in increased in vitro activity, compared to primaquine, against liver-stage malarial parasites. The compounds were non-cytotoxic to human hepatoma cells, suggesting that they are a promising new class of agents for the treatment and prevention of malaria. Effective and safe antimalarial drugs active against both liver and erythrocytic parasite stages will be valuable components of malaria eradication strategies. 1 Mammalian infection by malarial parasites is initiated by an infected mosquito bite, followed by a clinically silent liver-stage infection, during which thousands of merozoites are formed to be released into the bloodstream and trigger the clinically relevant erythrocytic stage of the life cycle. 2 Drugs able to act on the liver stage are highly relevant, as they can be used in chemoprophylaxis to prevent all clinical manifestations of malaria. 3 Moreover, such drugs are particularly relevant in P. vivax and P. ovale infections, as in these species latent forms that are called hypnozoites are not eliminated by most available therapies, persist in the liver for long periods, and subsequently cause malaria relapses. At present, drugs acting against parasite liver forms are scarce, and primaquine (PQ, 1in Scheme 1) remains the only drug in clinical use that acts against liver stages of all Plasmodium species, 4 including P. vivax and P. ovale hypnozoites. 5 Unfortunately, PQ’s low oral bioavailability and high hemotoxicity hold back its clinical use, especially in vulnerable populations including pregnant women, infants and the elderly. In addition, PQ causes hemolysis in patients with congenital deficiency of glucose-6-phosphate dehydrogenase, 5 complicating its use to treat P. vivax and P. ovale malaria and limiting its use in chemoprophylaxis. For the past decade, we have been working on chemical approaches to mask the PQ aliphatic amine in order to obtain analogues resistant to oxidative deamination, the major metabolic pathway underlying the drug’s low oral bioavailability (Scheme 1) by conversion into an inactive metabolite, carboxyprimaquine (2in Scheme 1). 5 To this end, we have successfully developed imidazoquines (3in Scheme 1) 6,7 and primacenes (4in Scheme 1), 8,9 peptidomimetic and organometallic derivatives of PQ with promising antimalarial properties, respectively. Now, we disclose a new family of PQ derivatives, the PRIMACINS (5 in Scheme 1), obtained by coupling PQ to cinnamic acids, as the latter was formerly reported to possess interesting antimalarial properties. 10 PRIMACINS were found to have higher in vitro activity than PQ against the liver-stage of the rodent malarial parasite P. berghei (Fig. 1). PRIMACINS were tested against liver stages of the rodent parasite P. berghei and against erythrocytic stages of the human parasite P. falciparum (Table 1). Compounds 5were twoor more-fold more potent than PQ against liver stage P. berghei and were non-toxic to Huh7 human hepatoma cells in vitro, as shown by cell confluency analysis (Fig. 1). The activities present a relatively short activity range (1.38–2.39 mM), so any discussion on structure–activity relationships based on the IC 50 values obtained would be of no significance. Furthermore, given that the mechanisms of action of PQ against liver-stage parasites are still poorly understood, 5 it is always risky to attempt a justification for activity trends observed, if any. Anyway, lipophilicity may have had some influence on compound’s activity, as the two most active compounds, 5h and 5c, were also the ones presenting higher log Pvalues, 5.77 and 6.15, 12 respectively. Regarding erythrocytic-stage activity, 5c was the only compound that displayed an IC 50 below 10 mM of the eleven compounds 5, which was nevertheless one order of magnitude higher than that of the reference erythrocytic-stage drug, artemisinin. The broad inactivity of compounds 5against erythrocytic-stage parasites was not particularly surprising, as (i) PQ is itself a modest blood-schizontocidal 5 and (ii) compounds 5lack a a Centro de Investigac¸~ ao em Qu ımica da Universidade do Porto, Departamento de Qu ımica e Bioqu ımica, Faculdade de Ci^ encias, Universidade do Porto, R. do Campo Alegre, 687, P-4169-007 Porto, Portugal. E-mail: [email protected]; Fax: +351 220402659; Tel: +351 220402563 b CICECO, Departamento de Qu ımica, Universidade de Aveiro, Campus Universit ario de Santiago, 3810-193 Aveiro, Portugal c Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. E-mail: [email protected] d Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143-0811, USA † Electronic supplementary information (ESI) available: Detailed synthetic procedures, spectroscopic data and biological evaluation regarding compounds 5. See DOI: 10.1039/c2md20113e 1170 | Med. Chem. Commun., 2012, 3, 1170–1172 This journal is ªThe Royal Society of Chemistry 2012 Dynamic Article LinksC < MedChemComm Cite this: Med. Chem. Commun., 2012, 3, 1170 www.rsc.org/medchemcomm CONCISE ARTICLE basic amine: the relevance of a basic amino group linked to the 8aminoquinoline core through a chain of two to six carbons for the activity of PQ against erythrocytic Plasmodia was established three decades ago. 5,13,14 In fact, more recent well-known PQ derivatives displaying some erythrocytic-stage activity have the PQ’s basic aliphatic chain either conserved (e.g., tafenoquine) or modified in a way that reasonably preserves its basicity (e.g., bulaquine and sitamaquine). 5 Compared to other cinnamic derivatives, which present low mM activities against Pf Dd2 and W2 strains, 10,15 combining the cinnamic moiety with PQ clearly led to a loss of erythrocytic-stage activity. This suggests that the lack of compounds 5activity is due to the presence of the 8aminoquinoline pharmacophore and supports that the absence of the aforesaid aliphatic amine is detrimental for inhibitory erythrocytic activity of PQ derivatives. As shown here, the absence of the aforementioned aliphatic amine did not preclude the significant activity of PRIMACINS 5 against liver-stage Plasmodia. This result agrees with our previous findings on PQ derivatives 4, as these organometallic compounds were also highly active against liver-stage parasites, regardless of whether a basic aliphatic amine group was present. Yet, compounds 4which lacked a basic aliphatic amine group Scheme 1 Chemical structures of primaquine (1), its main metabolite, carboxyprimaquine (2), PQ derivatives formerly developed by us, namely, imidazoquines (3) 6,7 and primacenes (4), 8,9 and PRIMACINS herein reported (5): (i) PQ was coupled to cinnamic acids in the presence of 1.1 molar equivalents (eq.) of TBTU and 2 eq. of N-ethyl-N,N-diisopropylamine, at room temperature for 24 h, using N,N-dimethylformamide as solvent. Fig. 1 Activity of PRIMACINS 5against P. berghei liver stages. Anti-infective activity (infection scale, bars) and toxicity to hepatoma cells (cell confluency scale, circles) are shown. Primaquine (1)at10mM was included for comparison. Infection loads of Huh7 cells, a human hepatoma cell line, were determined by bioluminescence measurements of cell lysates 48 h after infection with luciferase-expressing P. berghei parasites. 11 This journal is ªThe Royal Society of Chemistry 2012 Med. Chem. Commun., 2012, 3, 1170–1172 | 1171 were inactive both against erythrocytic-stage parasites and as transmission-blocking agents. 8,9 Concluding remarks Novel primaquine derivatives, PRIMACINS 5, have been synthesized through a cheap and simple one-step condensation of the parent antimalarial drug with a series of cinnamic acids. The compounds were screened in vitro for their activity against two major stages of malarial infection in the mammalian host, the liverand the erythrocytic-stage, and found to have about twoor more-fold higher potency than the parent drug against liver parasites while non-toxic to human hepatoma cells. Masking the aliphatic amine group of primaquine through conjugation with the cinnamic moiety impairs the metabolic oxidative deamination that underlies its low oral bioavailability, which in turn requires administration of high doses of this hemotoxic antimalarial drug. Thus, PRIMACINS represent a new class of compounds toward the development of more effective and potentially safer liver-stage active primaquine derivatives. To the best of our knowledge, this is an unprecedented disclosure of primaquine–cinnamic acid conjugates as antimalarial leads. Acknowledgements This work was mainly supported by Fundac¸~ ao para a Ci^ encia e a Tecnologia (FCT, Portugal) through project PTDC/QUI-QUI/ 116864/2010. Funding to CIQUP through the strategic project PEst-C/QUI/UI0081/2011 is also acknowledged to FCT. CT thanks FCT for post-doctoral grant SFRH/BPD/62967/2009. MP acknowledges FCT for grant PTDC/SAU-MII/099118/2008. ISA is hired under FCT grant PTDC/SAU-GMG/100313/2008. Thanks are due to FCT also for both the LC-MS and NMR facilities, respectively funded through projects CONC-REEQ/ 275/QUI and REDE/1517/RMN/2005. Ant onio M. Mendes is acknowledged for his help with data analysis and presentation. Notes and references 1J.A.N  ajera, M. Gonz alez-Silva and P. L. Alonso, PLoS Med., 2011, 8, e1000412. 2 M. Prud^ encio, A. Rodriguez and M. M. Mota, Nat. Rev. Microbiol., 2006, 4, 849. 3 T. N. C. Wells, J. N. Burrows and J. K. Baird, Trends Parasitol., 2010, 26, 145. 4 T. Rodrigues, M. Prud^ encio, R. Moreira, M. M. Mota and F. Lopes, J. 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Table 1 Antiplasmodial activities of compounds 5 Compound R IC 50 SD (mM) against liver-stage P. berghei IC 50 (mM) against erythrocytic-stage P. falciparum a Reference drug b — 7.50 (PQ) 5.64 nM (ART) 5a H 2.01 0.27 $10 5b p-Me 2.13 0.58 $10 5c pi Pr 1.54 0.35 4.84 5d p-OMe 2.35 0.19 $10 5e m-F 1.63 0.16 $10 5f p-F 2.33 0.47 $10 5g p-Cl 2.39 0.19 $10 5h p-Br 1.38 0.33 $10 5i o-NO 2 2.36 0.58 $10 5j m-NO 2 2.12 0.49 $10 5k p-NO 2 2.24 0.27 $10 a Chloroquine-resistant strain W2. b PQ for liver-stage activity assays; artemisinin (ART) for erythrocytic-stage activity assays. 1172 | Med. Chem. Commun., 2012, 3, 1170–1172 This journal is ªThe Royal Society of Chemistry 2012