Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory activities against Trypanosoma cruzi

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3576–3579

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Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory activities against Trypanosoma cruzi Bessy Gutiérrez a, Christian Muñoz a, Luis Osorio a, Krisztina Fehér b, Tünde-Zita Illyés c, Zsuzsa Papp c, Ambati Ashok Kumar c, Katalin E. Kövér c, Hernán Sagua a, Jorge E. Araya a, Patricio Morales d, László Szilágyi c,⇑, Jorge González a,⇑ a

Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile Department of Organic Chemistry, University of Ghent, 9000 Ghent, Belgium Department of Chemistry, University of Debrecen, P.O. Box 20, H-4010 Debrecen, Hungary d Biomedical Department, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile b c

a r t i c l e

i n f o

Article history: Received 24 January 2013 Revised 10 April 2013 Accepted 12 April 2013 Available online 22 April 2013 Keywords: Trypanosoma cruzi Chagas disease Disulfide Carbohydrate

a b s t r a c t Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(b-D-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50 values ranging from 4 to 11 lM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well. Ó 2013 Elsevier Ltd. All rights reserved.

Trypanosoma cruzi is the etiologic agent of Chagas’s disease, an endemic parasitosis in Latin America with 12–14 million people infected.1 Acute infections are usually asymptomatic, but the ensuing chronic T. cruzi infections have been associated with high rates of morbidity and mortality. Recently, Chagas’s disease has also been recognized as an opportunistic disease in HIV-infected individuals2,3 and reports appeared about worldwide spreading due to international migration.4,5 A number of drugs were reported to be effective against T. cruzi in vitro or in animal models, but none proved to be completely satisfactory for clinical use. Similarly, there are no adequate chemoprophylactic drugs to be used to eliminate the parasite from the blood of serologically positive donors in order to prevent transfusion-associated Chagas’s disease. Currently only two drugs, nifurtimox (a nitrofuran derivative, Nif) and benznidazole (a nitroimidazole derivative, Bz) introduced in the 1960s and 1970s, are available for the treatment of chagasic patients. These drugs are effective for acute infections, but their use for chronic patients re-

⇑ Corresponding authors. Tel.: +36 52 512900x22589; fax: +36 52 512 747x23744 (L.S.); tel.: +56 55 637376; fax: +56 55 637108 (J.G.). E-mail addresses: [email protected] (L. Szilágyi), [email protected] (J. González). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.04.030

mains controversial.6 Furthermore, studies of the mechanisms of action indicated that their antiparasitic activity is inextricably linked to mammalian host toxicity.7 The efficacy of these drugs also depends on the susceptibility of T. cruzi strains and resistance to benznidazol has been reported.8,9 Due to these inadequacies research programs were initiated to find alternative drugs for the treatment of chagasic patients.7,10 A large variety of synthetic compounds have recently been tested for antitrypanosomal activity in high-throughput screening campaigns, encompassing 100 00011 and more than 300 000 structures10, respectively. Targets and patented drugs have been reviewed.12 Posaconazole recently emerged as a promising candidate in mice13 and humans.14 However, new compounds showing higher potency and selectivity in both the acute and chronic stages of Chagas’s disease and/or better tolerability are still needed.15,16 Of the large number of the molecules tested so far for antitrypanosomal activity just a few contained disulfide bonds.17,18 Glycosyl disulfides were shown to exert inhibitory activities against endogenous lectins in vivo on human tumor cell lines and it was suggested that ‘glycosyl disulfides have potential as platform for inhibitor design’.19,20 We have recently described specific binding of oligovalent aromatic mannosyl disulfide derivatives to a lectin21 and explored the conformational features of diglycosyl

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disulfides.22,23 Cytotoxic activities of symmetrical 1,10 -glycosyl disulfides against human tumor cell lines have been recorded.24,25 In view of these findings we have envisioned that it might be worth to test the anti-trypanosomal activities of such structures. The compounds selected for the tests are characterized by the attachment of one or more monosaccharide sugars to an aromatic core (1b, 2b, 3, 4b, 5, 6, 7b, 8) or to another monosaccharide (10 and 11) by disulfide linkages. Compound 9, of similar structure but containing a simple sulfide bond, rather than disulfide, was added for comparison. The chemical structures of the compounds tested in this study are shown in Scheme 1. Aromatic glycosyl disulfides 3, 5, 6, 821 and sulfide 926 have been previously described. Diglycosyl disulfides 10 and 11 were also prepared as published.27,28 1b, 2b, 4b, and 7b were synthesized as shown in Scheme 2. Aromatic methanethiosulfonates I, II, III, and IV21 react smoothly with 1-thio-2,3,4,6-tetra-O-acetyl-b-D-galactopyranose29 at low temperatures to provide per-O-acetylated (b-D-galactopyranosyldithiomethylene)benzenes 1a, 2a, 4a, and 7a. Deacetylation with lithium hydroxide (LiOHxH2O) in methanol furnished 1b, 2b, 4b, and 7b, with free sugar hydroxyls. The structures of the new compounds have been established by standard NMR and MS spectroscopy. Synthetic procedures are described in the Supplementary material. Since various strains of T. cruzi exhibit different levels of resistance to Nif and Bz, and this variability may partially explain the observed differences in effectiveness of chemotherapy, in vitro tests were conducted to compare the activities of our molecules against Bz-susceptible (CL), -moderate (Y), and resistant (H510 a Colombian clone) T. cruzi strains. Furthermore, in order to assess the potential use of these compounds for the prophylaxis of banked blood, assays evaluating the effect of the drugs against trypomastigotes were performed at 4 °C in the presence of blood. The results are summarized in Table 1 and Fig. S1. The direct effect of the glycosyl disulfides on tissue culture derived trypomastigotes was evaluated after 18 h of treatment at 37 °C. 2b and 4b were found to be the most active compounds with IC50 values ranging from 4.2 ± 1.2 to 10.9 ± 1.2 lM against the tested strains. These compounds also retained their activities, at

S

S

Glyc

S

somewhat lower level, against the Y strain at 4 °C in the presence of blood constituents, this is promising with regard to the potential use in blood banks. Further molecules, 1b, 3, 5 and 7b displayed moderate antitrypanosomal activities against one of the strains at least (Table 1). Cytotoxicities of all compounds were evaluated after 18 h of incubation with confluent HeLa, Vero and peritoneal macrophage cell cultures. All compounds, displayed low cytotoxicities with cytotoxic concentrations (CC50) ranging from moderate (240 lM) to high (>2000 lM) levels (Table 1). Currently marketed drugs display limited efficacy against the chronic form of the Chagas disease which is often diagnosed late when the disease has already progressed and caused irreversible damage in the heart and in other organs.7 It is therefore important to find compounds that are effective against the intracellular forms of T. cruzi. Therefore, we evaluated the effects of the glycosyl disulfides on parasite release from T. cruzi infected HeLa cell cultures after 18 h of treatment at 37 °C. Several molecules of the panel we tested, such as 1b, 3, 4b, 5, 6 and 11, were able to inhibit intracellular proliferation or parasite differentiation when the number of parasites released by T. cruzi infected HeLa cells was measured, see Figure 1. This effect is comparable to that observed for benznidazol (Fig. 1 and Refs. 30,31). Although no significant correlation could be observed between activity against cell derived trypomastigotes and amastigotes, it should be taken into account that amastigotes and trypomastigotes are two morphologically different cells that live in two different environments, the bloodstream in the case of the trypomastigotes, and the cell cytoplasm in the case of the amastigotes. Inspecting the results of our inhibition tests listed in Table 1 (see also Fig. S1), the following observations can be made. First, sizeable inhibitory activities, on one T. cruzi strain at least, were observed only for aromatic disulfide derivatives (1b, 2b, 3, 4b, 5, 7b). Second, the presence of both the aromatic ring and the disulfide linkage appears to be essential. This is clearly demonstrated by the much lower activities of 10 and 11 (no aromatic rings) on the one hand, and by the comparison of 1b to 9, on the other. While the disulfide 1b is a moderate inhibitor of T. cruzi growth (at 37 °C), 9, lacking just one S-atom in comparison with 1b, is much less active

S

S S

S

S

Glyc

S

2b, 3 S

Glyc

S

Glyc

S

Glyc

S

S

O HO

OH

Glyc = α-D-manno-

O OH

OH

1b, 2b, 4b, 7b, 9

OH OH OH

Glyc

Glyc

S

7b, 8

6

HO

Glyc

S 4b, 5

S

Glyc

Glyc = β-D-galacto-

Glyc

Glyc

S 1b

S

S

S

9 HO HO

OH O

HO S

S

OH

O HO

OH OH

10

3, 5, 6, 8 AcO AcO

OAc O O S

AcO S

OAc 11

Scheme 1. Chemical structures of the molecules tested.

O AcO

OAc OAc

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CH2SSGal(OR)4 I. i) 1a R = Ac 1b R = H

SSO2CH3

SSO2CH3

OAc OAc O AcO

II., III. SH

i)

OAc

ii)

SSO2CH3

i)

CH2SSGal(OR)4 CH2SSGal(OR)4 2a, 4a R =Ac 2b, 4b R = H

ii)

SSO2CH3

CH3SO2S IV.

CH2SSGal(OR)4

CH2SSGal(OR)4

(RO)4GalSSH2C 7a R = Ac 7b R = H

ii)

i) -50°C, DMF, ii) LiOHxH2O / CH3OH

Scheme 2. Syntheses of the compounds tested.

Table 1 Trypanocidal activities and cytotoxic effects of the tested compounds Compounds

1b 2b 3 4b 5 6 7b 8 9 10 11 Benzni-dazol * **

Activity against different T. cruzi tripomastigote strains: IC50 (lM)* ± S.D.

Cytotoxicity CC50 (lM)** ± S.D.

4 °C

37 °C

Cell lines

Y

Y

H510

CL

VERO

HeLA

Macrophage

139.9 ± 2.3 30.5 ± 1.2 38.8 ± 1.1 10.4 ± 1.2 116.8 ± 1.3 225.1 ± 3.2 210.9 ± 1.7 260.6 ± 1.8 247.0 ± 2.8 243.7 ± 1.6 153.4 ± 1.3 P500

52.2 ± 1.2 4.8 ± 1.2 14.2 ± 1.2 8.7 ± 1.2 42.8 ± 1.3 98.4 ± 1.2 14.5 ± 1.2 233.3 ± 1.4 238.2 ± 1.4 241.4 ± 1.4 111.8 ± 1.3 25.6 ± 1.5

49.7 ± 1.2 4.2 ± 1.2 19.7 ± 1.5 5.7 ± 2.7 11.9 ± 1.9 109.0 ± 1.3 57.3 ± 1.2 263.4 ± 1.4 247.2 ± 1.4 271.5 ± 1.4 123.7 ± 1.3 ND

40.5 ± 1.6 10.9 ± 1.2 26.4 ± 1.3 4.4 ± 1.2 58.1 ± 1.5 120.0 ± 1.4 73.5 ± 1.6 276.2 ± 1.4 283.1 ± 1.4 287.2 ± 1.4 138.5 ± 1.3 ND

>2000 247.0 ± 1.4 >2000 251.3 ± 1.4 500.8 ± 1.5 >2000 >2000 >2000 >2000 >2000 256.0 ± 1.4 ND

>2000 238.9 ± 1.5 >2000 242.3 ± 1.2 487.7 ± 1.4 >2000 >2000 >2000 >2000 >2000 248.6 ± 1.3 ND

>2000 241.4 ± 1.4 >2000 248.4 ± 1.2 495.7 ± 1.3 >2000 >2000 >2000 >2000 >2000 252.1 ± 1.5 ND

The IC50 is defined as the concentration required to reduce the fluorescence of the control (without the test compound) by 50%. The CC50 is defined as the concentration needed to reduce the viability of HeLa cells by 50%.

Fig. 1. Effects on the intracellular development of T. cruzi. Infected HeLa cell cultures were incubated during 18 h with 25 lM solutions of the compounds listed on the x-axis. The activity against intracellular development was evaluated by counting the number of released trypomastigotes per mL. Experiments were performed in triplicate and values are expressed as the media ± 1S.D. Testing procedures are described in the Supplementary material.

(Table 1). Another correlation bears evidence for the importance of the sugar configurations attached to the aromatic ring: in tests conducted at 37 °C the b-galactosyl derivative 7b is significantly

more active than a-mannosyl-substituted 8 and, similarly, 4b (a b-galactosyl derivative) is more active than 5 (an a-mannosyl derivative). In terms of the IC50 values molecules 2b and 4b clearly

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stand out as the most potent inhibitors of the tested panel being active against all three of the tested T. cruzi strains. The antitrypanosoma activities of both compounds exceed that of benznidazol in these in vitro tests against the Y strain (Table 1). In recent years it has been recognised that the binding energy per heteroatom or ligand efficiency (LE)32,33 is a useful parameter to characterise the potency of an inhibitor molecule with respect to its molecular weight. We have calculated LE values and another score, the size-compensated LE or Fit Quality (FQ) proposed by Reynolds et al.34 (see Table S2 and Fig. S2, Supplementary material). In general, comparison based on ligand efficiencies allows highlighting smaller, weaker affinity compounds. Indeed, the monosubstitued disulfide derivative, 1b displays the highest LE values (0.19–0.22) comparable to that of benznidazole (0.24). On the other hand, the highest size-compensated FQ scores were obtained for 2b and 4b (0.50) again similar to that of benznidazole (0.50). Lipophilic ligand efficiency35,36 is another parameter* linking activity and lipophilicity in an attempt to assess druglikeness.35–37 Calculated LLE values listed in Table S2 (Supplementary material) are close to five for 2b and 4b which is promising as a value of five or above is considered optimal for a drug candidate.35,36 Remarkably, 2b and 4b display similar high values for all three T. cruzi strains; this is exceeding the values for the reference drug Bz especially at 4 °C (Table S2). Further improvement of the activity in the series is therefore conceivable by attachment of lipophilic moieties during a follow-up optimisation process. Antichagasic drugs display not only limited efficacy but are also cytotoxic.7 The cytotoxicities of our compounds were measured on confluent HeLa, Vero and peritoneal macrophage cell cultures as noted above. The Selectivity Index (SI) measures the activity against cytotoxicity of the tested molecule; SI for benznidazol has been reported being under 1.38,39 Calculated values for our molecules show outstanding selectivities for 3 and 7b and high selectivities for 1b, 2b and 4b (Table S3 and Fig S3, Supplementary material). In summary, our studies have shown that oligovalent aromatic glycosyl disulfides display trypanocidal activities against trypomastigotes, the infective stage of T. cruzi from different strains. 2b and 4b proved to be the most active compounds against all three of the tested strains at 37 °C and these activities were maintained, although somewhat lowered, at 4 °C. This is indicating a potential for these compounds to be used to prevent T. cruzi infection in blood banks. Cytotoxic concentrations ranged from moderate to high levels resulting in selectivity indices of 30 to 60 for the most active molecules as contrasted to a value of one for the reference drug benznidazole. Furthermore, it is remarkable that several members of the tested panel, like 1b, 3, 4b, 5, 6 and 11, are cellpermeable and were effective against amastigotes, the intracellular stage of the parasite, as well. Work aimed at optimizing the chemical structures of glycosyl disulfides and other related analogues is currently being pursued in our laboratories. Acknowledgments The skillful technical assistance of Sára Balla (Debrecen) is greatly appreciated. We thank TÁMOP-4.2.2.A-11/1/KONV-20120025 and OTKA K 105459 Grants (to K.E.K.) in Hungary as well as CODEI Grant No. 5381 from Antofagasta University for financial support.

⁄

We thank a referee to draw our attention to this point.

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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. 04.030. These data include MOL files and InChiKeys of the most important compounds described in this article. References and notes 1. Pinto Dias, J. C. Cadernos de Saude Publica 2007, 23, S13. 2. de Almeida, E. A.; Silva, E. L.; Guariento, M. E.; Aoki, F. H.; Pedro, R. D. Ann. Trop. Med. Parasitol. 2009, 103, 471. 3. Almeida, E. A.; Lima, J. N.; Lages-Silva, E.; Guariento, M. E.; Aoki, F. H.; TorresMorales, A. E.; Pedro, R. J. Trans. R. Soc. Trop. Med. Hyg. 2010, 104, 447. 4. Coura, J. R.; Vinas, P. A. Nature 2010, 465, S6. 5. Norman, F. F.; de Ayala, A. P.; Perez-Molina, J. A.; Monge-Maillo, B.; Zamarron, P.; Lopez-Velez, R. Plos Negl. Trop. Dis. 2010, 4. 6. Croft, S. L.; Barrett, M. P.; Urbina, J. A. Trends Parasitol. 2005, 21, 508. 7. Urbina, J. A. Acta Tropica 2010, 115, 55. 8. Filardi, L. S.; Brener, Z. Trans. R. Soc. Trop. Med. Hyg. 1987, 81, 755. 9. Murta, S. M. F.; Gazzinelli, R. T.; Brener, Z.; Romanha, A. J. Mol. Biochem. Parasitol. 1998, 93, 203. 10. Andriani, G.; Chessler, A. D. C.; Courtemanche, G.; Burleigh, B. A.; Rodriguez, A. Plos Negl. Trop. Dis. 2011, 5. 11. Holloway, G. A.; Charman, W. N.; Fairlamb, A. H.; Brun, R.; Kaiser, M.; Kostewicz, E.; Novello, P. M.; Parisot, J. P.; Richardson, J.; Street, I. P.; Watson, K. G.; Baell, J. B. Antimicrob. Agents Chemother. 2009, 53, 2824. 12. Duschak, V. G.; Couto, A. S. Recent Patents Anti-Infect. Drug Disc. 2007, 2, 19. 13. Olivieri, B. P.; Molina, J. T.; de Castro, S. L.; Pereira, M. C.; Calvet, C. M.; Urbina, J. A.; Araujo-Jorge, T. C. Int. J. Antimicrob. Agents 2010, 36, 79. 14. Pinazo, M. J.; Espinosa, G.; Gallego, M.; Lopez-Chejade, P. L.; Urbina, J. A.; Gascon, J. Am. J. Trop. Med. Hyg. 2010, 82, 583. 15. Guedes, P. M. M.; Silva, G. K.; Gutierrez, F. R. S.; Silva, J. S. Exp. Rev. Anti-Infect. Ther. 2011, 9, 609. 16. Munoz, M.; Murcia, L.; Segovia, M. Exp. Rev. Anti-Infect. Ther. 2011, 9, 5. 17. Elwaer, A.; Douglas, K. T.; Smith, K.; Fairlamb, A. H. Anal. Biochem. 1991, 198, 212. 18. Gallwitz, H.; Bonse, S.; Martinez-Cruz, A.; Schlichting, I.; Schumacher, K.; Krauth-Siegel, R. L. J. Med. Chem. 1999, 42, 364. 19. André, S.; Pei, Z. C.; Siebert, H. C.; Ramstrom, O.; Gabius, H. J. Bioorg. Med. Chem. 2006, 14, 6314. 20. Martin-Santamaria, S.; Andre, S.; Buzamet, E.; Caraballo, R.; FernandezCureses, G.; Morando, M.; Ribeiro, J. P.; Ramirez-Gualito, K.; de PascualTeresa, B.; Canada, F. J.; Menendez, M.; Ramstrom, O.; Jimenez-Barbero, J.; Solis, D.; Gabius, H. J. Org. Biomol. Chem. 2011, 9, 5445. 21. Murthy, B. N.; Sinha, S.; Surolia, A.; Jayaraman, N.; Szilágyi, L.; Szabó, I.; Kövér, K. E. Carbohydr. Res. 2009, 344, 1758. 22. Fehér, K.; Matthews, R. P.; Kövér, K. E.; Naidoo, K. J.; Szilágyi, L. Carbohydr. Res. 2012, 352, 223 (Erratum to Carbohydr. Res. 2011, 346, 2612-2621). 23. Fehér, K.; Matthews, R. P.; Kövér, K. E.; Naidoo, K. J.; Szilágyi, L. Carbohydr. Res. 2011, 346, 2612. 24. Adinolfi, M.; Capasso, D.; Di Gaetano, S.; Iadonisi, A.; Leone, L.; Pastore, A. Org. Biomol. Chem. 2011, 9, 6278. 25. Caraballo, R.; Sakulsombat, M.; Ramström, O. Chem. Commun. 2010, 46, 8469. 26. Brito, I.; Szilágyi, L.; Kumar, A. A.; Albanez, J.; Bolte, M. Acta Crystallogr. 2011, E67, o2308. 27. Bell, R. H.; Horton, D.; Miller, M. J. Carbohydr. Res. 1969, 9, 201. 28. Szilágyi, L.; Illyés, T. Z.; Herczegh, P. Tetrahedron Lett. 2001, 42, 3901. ˇ erny´, M.; Staneˇk, J.; Pacák, J. Monatsh. Chem. 1963, 94, 290. 29. C 30. Petray, P. B.; Morilla, M. J.; Corral, R. S.; Romero, E. L. Mem. Inst. Oswaldo Cruz 2004, 99, 233. 31. Da Silva, C. F.; Junqueira, A.; Lima, M. M.; Romanha, A. J.; Sales, P. A.; Stephens, C. E.; Som, P.; Boykin, D. W.; Soeiro, M. D. C. J. Antimicrob. Chemother. 2011, 66, 1295. 32. Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9997. 33. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430. 34. Reynolds, C. H.; Tounge, B. A.; Bembenek, S. D. J. Med. Chem. 2008, 51, 2432. 35. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881. 36. Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.; Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Bioorg. Med. Chem. Lett. 2009, 19, 4406. 37. ChemAxon 2013, Marvin Sketch5.12.0 (). 38. Sanchez-Moreno, M.; Sanz, A. M.; Gomez-Contreras, F.; Navarro, P.; Marin, C.; Ramirez-Macias, I.; Jose Rosales, M.; Olmo, F.; Garcia-Aranda, I.; Campayo, L.; Cano, C.; Arrebola, F.; Yunta, M. J. R. J. Med. Chem. 2011, 54, 970. 39. Sanchez-Moreno, M.; Marin, C.; Navarro, P.; Lamarque, L.; Garcia-Espana, E.; Miranda, C.; Huertas, O.; Olmo, F.; Gomez-Contreras, F.; Pitarch, J.; Arrebola, F. J. Med. Chem. 2012, 55, 4231.