Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
New heterocyclic compounds: Synthesis and antitrypanosomal properties S. Pomel a, F. Dubar b, D. Forge c, , P. M. Loiseau a, C. Biot b,⇑ a
Univ. Paris-Sud, Faculté de Pharmacie, UMR 8076 CNRS, Chimiothérapie Antiparasitaire, 92290 Châtenay-Malabry, France Univ. Lille 1, UGSF, UMR 8576 CNRS, 59650 Villeneuve d’Ascq, France c Laboratory of Organic Chemistry, Faculty of Sciences, University of Mons-UMONS, Mons, Belgium b
a r t i c l e
i n f o
Article history: Received 16 December 2014 Revised 9 March 2015 Accepted 10 March 2015 Available online xxxx Keywords: Quinoline Quinolone Benzimidazole Ferroquine Trypanosoma Sleeping sickness
a b s t r a c t Three new series of quinoline, quinolone, and benzimidazole derivatives were synthesized and evaluated in vitro against Trypanosoma brucei gambiense. In the quinoline series, the metallo antimalarial drug candidate (ferroquine, FQ) and its ruthenium analogue (ruthenoquine, RQ, compound 13) showed the highest in vitro activities with IC50 values around 0.1 lM. Unfortunately, both compounds failed to cure Trypanosoma brucei brucei infected mice in vivo. The other heterocyclic compounds were active in vitro with IC50 values varying from 0.8 to 34 lM. One of the most interesting results was a fluoroquinolone derivative (compound 2) that was able to offer a survival time of 8 days after a treatment at the single dose of 100 lmol/kg by intraperitoneal route. Although no clear-cut structure–activity relationships emerged, further pharmacomodulations are worth to be developed in this series. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Human African trypanosomiasis or sleeping sickness is a parasitic disease transmitted by the tsetse fly, the causative agent being Trypanosoma brucei gambiense in West and Central Africa and Trypanosoma brucei rhodesiense in East Africa.1,2 Cattle are also infected by Trypanosoma species which are responsible for the loss of protein for human nutrition in sub-Saharian areas.3 The current drugs used in Humans for the second-stage of the disease such as nifurtimox-eflornithine combination or melarsoprol are toxic, expensive, and able to generate drug resistance justifying the need to develop new anti-trypanosomal drugs.4 Due to the attractiveness of heterocyclic derivatives and well known drugs as sources for anti-trypanosomal agents, we designed three different scaffold structures including quinoline, quinolone and benzimidazole rings.5–7 In the quinoline series, we focused on the organometallic drug candidate ferroquine (FQ, SSR97193; Fig. 1). Indeed, FQ is currently in Phase IIB clinical trial in combination with OZ439 for the treatment of uncomplicated Plasmodium falciparum malaria.8–10 (http:// www.mmv.org/research-development/project-portfolio/oz439fq website). FQ has a specific parasiticidal effect against Plasmodium ⇑ Corresponding author. Tel.: +33 (0)3 2043 4884; fax: +33 (0)3 2043 6555.
E-mail address: [email protected] (C. Biot). Present position: Sodexo, Avenue Charles Lemaire 1, 1160 Bruxelles, Belgium.
due to its specific accumulation in parasite digestive vacuole in which induced oxidative stress results in parasite death.11–16 The spectrum of action of FQ has already been investigated against other protozoan parasites.17 We have previously shown that FQ was strongly active in vitro against African trypanosomes (IC50 100 lM. The in vitro activity was similar between ruthenoquine (RQ, compound 13) and FQ suggesting similar mechanism of action without redox activation.16 The presence of a methyl group on the nitrogen in the position 4 of the quinoline ring (compound 12) was responsible for a ten-fold decrease activity in comparison with the parent molecules (FQ or RQ). Thus, the ruthenocenyl compound 14 exhibited similar activity as the ferrocenyl compound 12. Therefore, the presence of this methyl group, which prevents the formation of intramolecular hydrogen bonds,27 has a significant deleterious effect on antitrypanosomal activity. The intramolecular H-bond was thought to be involved in membranes crossing and delivery of the compound close to its active site. Concerning the benzimidazole series, the presence of a dimethylaminomethyl group is clearly deleterious for the activity as observed for compounds 8 and 9 comparatively to compounds 4 and 5, respectively. However, the fluoro substitution on the benzimidazole slightly enhanced the antitrypanosomal activity as observed for compounds 5 and 9 versus 4 and 8, respectively. When the ferrocene moiety was substituted by two benzimidazole rings, there was no added-value in the activity in comparison with ferrocene substituted by only one benzimidazole ring. In addition, the influence of fluoro atom was negligible. The dual compound resulting from the covalent linking of the 4-aminoquinoline with benzimidazole scaffolds (compound 6 and 7) led to active compounds. These results suggest a synergic effect between the benzimidazole moiety and the 4-aminoquinoline group. Moreover, it appears that the redox properties of ferrocene have no role in the mechanism of action of this series. Indeed, the compounds with 4-aminoquinoline group have demonstrated a better activity compared to the ferrocene derivatives.
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S. Pomel et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx Table 1 In vitro activity of 14 compounds on T. b. gambiense MW (g mol1)
Trypanosoma brucei gambiense IC50 (lM) ± SD
Cytotoxicity CC50 (lM) ± SD
SI*
1
359
26.00 ± 0.72
>30⁄⁄
1.2
2
449
6.03 ± 0.28
>30⁄⁄
5
3
521
23.08 ± 2.29
27.80 ± 3.70⁄⁄
1.2
4
302
10.99 ± 1.53
17.48 ± 0.67⁄⁄⁄
1.6
5
370
6.80 ± 1.52
17.79 ± 1.49⁄⁄⁄
2.6
6
388
1.98 ± 0.02
10.56 ± 1.29⁄⁄⁄
5.3
7
320
3.79 ± 0.21
15.48 ± 1.88⁄⁄⁄
4.1
8
359
>100
3.32 ± 0.97⁄⁄⁄
0
9
377
34.15 ± 1.11
>200⁄⁄⁄
5.9
10
418
9.24 ± 0.36
15.42 ± 0.83⁄⁄⁄
1.7
11
454
10.12 ± 0.32
35.12 ± 3.71⁄⁄⁄
3.5
Compounds
Formulas
(continued on next page)
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S. Pomel et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1 (continued) MW (g mol1)
Trypanosoma brucei gambiense IC50 (lM) ± SD
Cytotoxicity CC50 (lM) ± SD
SI*
12
447
1.02 ± 0.08
8.58 ± 0.07⁄⁄⁄
8.4
13
479
0.10 ± 0.05
1.49 ± 0.08⁄⁄⁄
14.9
14
493
0.87 ± 0.04
11.55 ± 0.06⁄⁄⁄
13.3
Ferroquine
433
0.13 ± 0.04
1.06 ± 0.01⁄⁄⁄
8.2
Pentamidine
596
0.0049 ± 0.0018
ND
ND
Compounds
Formulas
CC50: Cytotoxicity 50%. Cytotoxicity assays were performed either on 3T6 cells (⁄⁄) or on LLC-MK2 cells (⁄⁄⁄). ND: not determined. * Selectivity index (SI) = CC50 on 3T6/IC50 on T. b. gambiense.
Table 2 In vivo antitrypanosomal evaluation of compounds 2, 5, 6 and 13 on the Trypanosoma b. brucei/mice model and in vivo toxicity Compound
Dose
lmol/
Parasitemia (number of parasites/ll blood) ± SD at
mg/kg
Day 2
Day 3
Day 4
Day 30
50.60 25.30 51.5 59.6 /
0 0 20.8 ± 5.7 24.1 ± 4.9 0 13.7 ± 2.2 25.8 ± 4.2 24.1 ± 4.9 0 0 23.7 ± 3.1 0 23.4 ± 3.9
0 0 380 ± 53 410 ± 25 97 ± 18 117 ± 11 420 ± 68 401 ± 33 0 0 498 ± 32 0 444 ± 46
0 7 36,024 ± 3100 41,520 ± 3300 31,590 ± 2190 28,317 ± 2456 38,900 ± 4100 40,200 ± 3400 24.6 ± 4.8 804 ± 51 42,607 ± 3915 0 41,824 ± 3823
/ / / / / / / / / / / 0 /
kg 2 5 6 13 Ferroquine Chloroquine diphosphate Pentamidine di-isethionate Excipient
100 50 100 50 100 50 100 50 100 50 100 100 /
Finally, among the fluoroquinolone series, compound 2 has the best activity comparatively to compounds 1 and 3, where the benzyl group is replaced by a piperazinyl one. Related compounds were found to be active in vitro against Trypanosoma brucei and Trypanosoma cruzi.28,29 The antitrypanosomal activities of fluoroquinolones of this series are in the same range than those previously described, confirming the interest of this scaffold as a source of interesting antitrypanosomal agents.28 In the study by Nenortas et al.28, ciprofloxacin exhibited an IC50 value at 52 lM against T. brucei whereas compound 1 in the present study, as ciprofloxacin ethyl ester, showed an IC50 value at 26 lM. Thus, the fluoroquinolonemediated inhibition of type II DNA topoisomerase activity could supply new antitrypanosomal agents in the future. However,
Mean survival time/controls (Days ± SD)
Number of mice cured/ total number of mice
Cure rate (%)
Toxicity: number of dead mice [day of death posttreatment]
8.2 ± 1.6 6.1 ± 1.2 0 0 1.2 ± 0.8 1.1 ± 0.7 0 0 4.5 ± 1.8 3.8 ± 1.4 0 >30 0
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/10 10/10 0/10
0 0 0 0 0 0 0 0 0 0 0 100 0
No toxicity No toxicity No toxicity No toxicity No toxicity No toxicity No toxicity No toxicity 1[Day 3]; 1[Day 4] No toxicity No toxicity No toxicity No toxicity
Hiltensperger et al.30 described new quinolone derivatives exhibiting IC50 values of 47 nM on T. brucei but affecting the mitochondrial morphology. Therefore, quinolones could exhibit at least two different antitrypanosomal mechanisms of action as a function of chemical groups substituted on the scaffold. Further pharmacomodulations studies are necessary to understand the structure–activity relationships responsible for each of these mechanisms of action. One representative of each three chemical series, as fluoroquinolones, 4-aminoquinoline-benzimidazoles, benzimidazomono- and di-substituted ferrocenes and ferroquine derivatives, were interesting enough to be studied in vivo. We have therefore selected the compounds exhibiting the best in vitro activity that were compounds 2, 5, 6 and 13. These compounds exhibited a
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S. Pomel et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
selectivity index (SI) as follows: for compounds 2 and 6, SI values were higher than 5 and for compound 5, SI value was higher than 2. The less cytotoxic product was compound 13 with a SI value close to 15. Table 2 gathers results of in vivo activity of these compounds on the T. b. brucei Swiss mice model. Ferroquine was the single compound being toxic at 100 lmol/kg, whereas no toxicity was found at the same dose for all other compounds. Compound 2 was the most active, clearing the trypanosomes from the blood for at least 4 days and allowing the survival time of 8 days. However, such an activity was too slight to consider this compound as promising. The in vivo trial was very stringent since it consisted in one single treatment, selecting the very powerful compounds only. Further pharmacomodulations will be developed on the fluoroquinolones series in order to optimize this interesting antitrypanosomal series. Acknowledgments The authors would like to thank P. Grellier and P. Vincendeau who kindly provided the strain FéoITMAP/1893 of Trypanosma brucei gambiense. The authors would like to thank the Programme Hubert Currien Tournesol, Egide (Project PVB/AD/FR/ad/SOR/2011/34676: ‘Mise au point de protocoles expérimentaux respectueux de l’environnement pour la préparation de nouveaux antipaludiques métallocéniques’) to support the chemical part of this project. References and notes 1. Franco, J. R.; Simarro, P. P.; Diarra, A.; Jannin, J. G. Clin. Epidemiol. 2014, 6, 257. 2. Simarro, P. P.; Diarra, A.; Ruiz-Postigo, J. A.; Franco, J. R.; Jannin, J. G. PLoS Negl. Trop. Dis. 2011, 5, e1007. 3. Selby, R.; Bardosh, K.; Picozzi, K.; Waiswa, C.; Welburn, S. C. Parasit. Vectors 2013, 6, 281. 4. Babokhov, P.; Sanyaolu, A. O.; Oyibo, W. A.; Fagbenro-Beyioku, A. F.; Iriemenam, N. C. Pathog. Glob. Health 2013, 107, 242. 5. Seixas, J. D.; Luengo-Arratta, S. A.; Diaz, R.; Saldivia, M.; Rojas-Barros, D. I.; Manzano, P.; Gonzalez, S.; Berlanga, M.; Smith, T. K.; Navarro, M.; Pollastri, M. P. J. Med. Chem. 2014, 57, 4834.
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6. Wube, A.; Hüfner, A.; Seebacher, W.; Kaiser, M.; Brun, R.; Bauer, R.; Bucar, F. Molecules 2014, 19, 14204. 7. Loiseau, P. M.; Mettey, Y.; Vierfond, J. M. Int. J. Parasitol. 1996, 26, 1115. 8. Biot, C.; Taramelli, D.; Forfar-Bares, I.; Maciejewski, L. A.; Boyce, M.; Nowogrocki, G.; Brocard, J. S.; Basilico, N.; Olliaro, P.; Egan, T. J. Mol. Pharm. 2005, 2, 185. 9. Mombo-Ngoma, G.; Supan, C.; Dal-Bianco, M. P.; Missinou, M. A.; Matsiegui, P. B.; Ospina-Salazar, C. L.; Issifou, S.; Ter-Minassian, D.; Ramharter, M.; Kombila, M.; Kremsner, P. G.; Lell, B. Malar. J. 2011, 10, 53. 10. Supan, C.; Mombo-Ngoma, G.; Dal-Bianco, M. P.; Ospina-Salazar, C. L.; Issifou, S.; Mazuir, F.; Filali-Ansary, A.; Biot, C.; Ter-Minassian, D.; Ramharter, M.; Kremsner, P. G.; Lell, B. Antimicrob. Agents Chemother. 2012, 56, 3165. 11. Henry, M.; Briolant, S.; Fontaine, A.; Mosnier, J.; Baret, E.; Amalvict, R.; Fusaï, T.; Fraisse, L.; Rogier, C.; Pradines, B. Antimicrob. Agents Chemother. 2008, 52, 2755. 12. Dubar, F.; Khalife, J.; Brocard, J.; Dive, D.; Biot, C. Molecules 2008, 13, 2900. 13. Dubar, F.; Bohic, S.; Dive, D.; Guérardel, Y.; Cloetens, P.; Khalife, J.; Biot, C. ACS Med. Chem. Lett. 2012, 3, 480. 14. Dubar, F.; Bohic, S.; Slomianny, C.; Morin, J. C.; Thomas, P.; Kalamou, H.; Guérardel, Y.; Cloetens, P.; Khalife, J.; Biot, C. Chem. Commun. (Camb.) 2012, 910. 15. Dive, D.; Biot, C. Curr. Top. Med. Chem. 2014, 14, 1684. 16. Dubar, F.; Slomianny, C.; Kahlife, J.; Dive, D.; Kalamou, H.; Guérardel, Y.; Grellier, P.; Biot, C. Angew. Chem., Int. Ed. 2013, 52, 7690. 17. Pomel, S.; Biot, C.; Bories, C.; Loiseau, P. M. Parasitol. Res. 2013, 112, 665. 18. Valdez, J.; Cedillo, R.; Hernandez-Campos, A.; Yepez, L.; Hernandez-Luis, F.; Navarrete-Vazquez, G.; Tapia, A.; Cortes, R.; Hernandez, M.; Castillo, R. Bioorg. Med. Chem. Lett. 2002, 12, 2221. 19. Perez-Molina, J. A.; Perez-Ayala, A.; Moreno, S.; Fernandez-Gonzalez, M. C.; Zamora, J.; Lopez-Velez, R. J. Antimicrob. Chemother. 2009, 64, 1139. 20. Oh, S.; Kim, S.; Kong, S.; Yang, G.; Lee, N.; Han, D.; Goo, J.; Siqueira-Neto, J. L.; Freitas-Junior, L. H.; Song, R. Eur. J. Med. Chem. 2014, 84, 395. 21. Loiseau, P. M.; Lubert, P.; Wolf, J. G. Antimicrob. Agents Chemother. 2000, 44, 2954. 22. Mosmann, T. J. Immunol. Methods 1983, 65, 55. 23. Dubar, F.; Wintjens, R.; Martins-Duarte, E. S.; Vommaro, R. C.; de Souza, W.; Dive, D.; Pierrot, C.; Pradines, B.; Wohlkonig, A.; Khalife, J.; Biot, C. Med. Chem. Commun. 2011, 2, 430. 24. Mueller-Westerhoff, U. J. Organomet. Chem. 1993, 463, 163. 25. Picart-Goetgheluck, S.; Delacroix, O.; Maciejewski, L.; Brocard, J. Synthesis 2000, 2000, 1421. 26. Naeimi, H.; Alishahi, N. J. Ind. Eng. Chem. 2014, 20, 2543. 27. Biot, C.; Chavain, N.; Dubar, F.; Pradines, B.; Trivelli, X.; Brocard, J.; Forfar, I.; Dive, D. J. Organomet. Chem. 2009, 694, 845. 28. Nenortas, E.; Kulikowicz, T.; Burri, C.; Shapiro, T. A. Antimicrob. Agents Chemother. 2003, 47, 3015. 29. Ma, X.; Zhou, W.; Brun, R. Bioorg. Med. Chem. Lett. 2009, 19, 986. 30. Hiltensperger, G.; Jones, N. G.; Niedermeier, S.; Stich, A.; Kaiser, M.; Jung, J.; Puhl, S.; Damme, A.; Braunschweig, H.; Meinel, L.; Engstler, M.; Holzgrabe, U. J. Med. Chem. 2012, 55, 2538.
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Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
New heterocyclic compounds: Synthesis and antitrypanosomal properties S. Pomel a, F. Dubar b, D. Forge c, , P. M. Loiseau a, C. Biot b,⇑ a
Univ. Paris-Sud, Faculté de Pharmacie, UMR 8076 CNRS, Chimiothérapie Antiparasitaire, 92290 Châtenay-Malabry, France Univ. Lille 1, UGSF, UMR 8576 CNRS, 59650 Villeneuve d’Ascq, France c Laboratory of Organic Chemistry, Faculty of Sciences, University of Mons-UMONS, Mons, Belgium b
a r t i c l e
i n f o
Article history: Received 16 December 2014 Revised 9 March 2015 Accepted 10 March 2015 Available online xxxx Keywords: Quinoline Quinolone Benzimidazole Ferroquine Trypanosoma Sleeping sickness
a b s t r a c t Three new series of quinoline, quinolone, and benzimidazole derivatives were synthesized and evaluated in vitro against Trypanosoma brucei gambiense. In the quinoline series, the metallo antimalarial drug candidate (ferroquine, FQ) and its ruthenium analogue (ruthenoquine, RQ, compound 13) showed the highest in vitro activities with IC50 values around 0.1 lM. Unfortunately, both compounds failed to cure Trypanosoma brucei brucei infected mice in vivo. The other heterocyclic compounds were active in vitro with IC50 values varying from 0.8 to 34 lM. One of the most interesting results was a fluoroquinolone derivative (compound 2) that was able to offer a survival time of 8 days after a treatment at the single dose of 100 lmol/kg by intraperitoneal route. Although no clear-cut structure–activity relationships emerged, further pharmacomodulations are worth to be developed in this series. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Human African trypanosomiasis or sleeping sickness is a parasitic disease transmitted by the tsetse fly, the causative agent being Trypanosoma brucei gambiense in West and Central Africa and Trypanosoma brucei rhodesiense in East Africa.1,2 Cattle are also infected by Trypanosoma species which are responsible for the loss of protein for human nutrition in sub-Saharian areas.3 The current drugs used in Humans for the second-stage of the disease such as nifurtimox-eflornithine combination or melarsoprol are toxic, expensive, and able to generate drug resistance justifying the need to develop new anti-trypanosomal drugs.4 Due to the attractiveness of heterocyclic derivatives and well known drugs as sources for anti-trypanosomal agents, we designed three different scaffold structures including quinoline, quinolone and benzimidazole rings.5–7 In the quinoline series, we focused on the organometallic drug candidate ferroquine (FQ, SSR97193; Fig. 1). Indeed, FQ is currently in Phase IIB clinical trial in combination with OZ439 for the treatment of uncomplicated Plasmodium falciparum malaria.8–10 (http:// www.mmv.org/research-development/project-portfolio/oz439fq website). FQ has a specific parasiticidal effect against Plasmodium ⇑ Corresponding author. Tel.: +33 (0)3 2043 4884; fax: +33 (0)3 2043 6555.
E-mail address: [email protected] (C. Biot). Present position: Sodexo, Avenue Charles Lemaire 1, 1160 Bruxelles, Belgium.
due to its specific accumulation in parasite digestive vacuole in which induced oxidative stress results in parasite death.11–16 The spectrum of action of FQ has already been investigated against other protozoan parasites.17 We have previously shown that FQ was strongly active in vitro against African trypanosomes (IC50 100 lM. The in vitro activity was similar between ruthenoquine (RQ, compound 13) and FQ suggesting similar mechanism of action without redox activation.16 The presence of a methyl group on the nitrogen in the position 4 of the quinoline ring (compound 12) was responsible for a ten-fold decrease activity in comparison with the parent molecules (FQ or RQ). Thus, the ruthenocenyl compound 14 exhibited similar activity as the ferrocenyl compound 12. Therefore, the presence of this methyl group, which prevents the formation of intramolecular hydrogen bonds,27 has a significant deleterious effect on antitrypanosomal activity. The intramolecular H-bond was thought to be involved in membranes crossing and delivery of the compound close to its active site. Concerning the benzimidazole series, the presence of a dimethylaminomethyl group is clearly deleterious for the activity as observed for compounds 8 and 9 comparatively to compounds 4 and 5, respectively. However, the fluoro substitution on the benzimidazole slightly enhanced the antitrypanosomal activity as observed for compounds 5 and 9 versus 4 and 8, respectively. When the ferrocene moiety was substituted by two benzimidazole rings, there was no added-value in the activity in comparison with ferrocene substituted by only one benzimidazole ring. In addition, the influence of fluoro atom was negligible. The dual compound resulting from the covalent linking of the 4-aminoquinoline with benzimidazole scaffolds (compound 6 and 7) led to active compounds. These results suggest a synergic effect between the benzimidazole moiety and the 4-aminoquinoline group. Moreover, it appears that the redox properties of ferrocene have no role in the mechanism of action of this series. Indeed, the compounds with 4-aminoquinoline group have demonstrated a better activity compared to the ferrocene derivatives.
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S. Pomel et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx Table 1 In vitro activity of 14 compounds on T. b. gambiense MW (g mol1)
Trypanosoma brucei gambiense IC50 (lM) ± SD
Cytotoxicity CC50 (lM) ± SD
SI*
1
359
26.00 ± 0.72
>30⁄⁄
1.2
2
449
6.03 ± 0.28
>30⁄⁄
5
3
521
23.08 ± 2.29
27.80 ± 3.70⁄⁄
1.2
4
302
10.99 ± 1.53
17.48 ± 0.67⁄⁄⁄
1.6
5
370
6.80 ± 1.52
17.79 ± 1.49⁄⁄⁄
2.6
6
388
1.98 ± 0.02
10.56 ± 1.29⁄⁄⁄
5.3
7
320
3.79 ± 0.21
15.48 ± 1.88⁄⁄⁄
4.1
8
359
>100
3.32 ± 0.97⁄⁄⁄
0
9
377
34.15 ± 1.11
>200⁄⁄⁄
5.9
10
418
9.24 ± 0.36
15.42 ± 0.83⁄⁄⁄
1.7
11
454
10.12 ± 0.32
35.12 ± 3.71⁄⁄⁄
3.5
Compounds
Formulas
(continued on next page)
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S. Pomel et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1 (continued) MW (g mol1)
Trypanosoma brucei gambiense IC50 (lM) ± SD
Cytotoxicity CC50 (lM) ± SD
SI*
12
447
1.02 ± 0.08
8.58 ± 0.07⁄⁄⁄
8.4
13
479
0.10 ± 0.05
1.49 ± 0.08⁄⁄⁄
14.9
14
493
0.87 ± 0.04
11.55 ± 0.06⁄⁄⁄
13.3
Ferroquine
433
0.13 ± 0.04
1.06 ± 0.01⁄⁄⁄
8.2
Pentamidine
596
0.0049 ± 0.0018
ND
ND
Compounds
Formulas
CC50: Cytotoxicity 50%. Cytotoxicity assays were performed either on 3T6 cells (⁄⁄) or on LLC-MK2 cells (⁄⁄⁄). ND: not determined. * Selectivity index (SI) = CC50 on 3T6/IC50 on T. b. gambiense.
Table 2 In vivo antitrypanosomal evaluation of compounds 2, 5, 6 and 13 on the Trypanosoma b. brucei/mice model and in vivo toxicity Compound
Dose
lmol/
Parasitemia (number of parasites/ll blood) ± SD at
mg/kg
Day 2
Day 3
Day 4
Day 30
50.60 25.30 51.5 59.6 /
0 0 20.8 ± 5.7 24.1 ± 4.9 0 13.7 ± 2.2 25.8 ± 4.2 24.1 ± 4.9 0 0 23.7 ± 3.1 0 23.4 ± 3.9
0 0 380 ± 53 410 ± 25 97 ± 18 117 ± 11 420 ± 68 401 ± 33 0 0 498 ± 32 0 444 ± 46
0 7 36,024 ± 3100 41,520 ± 3300 31,590 ± 2190 28,317 ± 2456 38,900 ± 4100 40,200 ± 3400 24.6 ± 4.8 804 ± 51 42,607 ± 3915 0 41,824 ± 3823
/ / / / / / / / / / / 0 /
kg 2 5 6 13 Ferroquine Chloroquine diphosphate Pentamidine di-isethionate Excipient
100 50 100 50 100 50 100 50 100 50 100 100 /
Finally, among the fluoroquinolone series, compound 2 has the best activity comparatively to compounds 1 and 3, where the benzyl group is replaced by a piperazinyl one. Related compounds were found to be active in vitro against Trypanosoma brucei and Trypanosoma cruzi.28,29 The antitrypanosomal activities of fluoroquinolones of this series are in the same range than those previously described, confirming the interest of this scaffold as a source of interesting antitrypanosomal agents.28 In the study by Nenortas et al.28, ciprofloxacin exhibited an IC50 value at 52 lM against T. brucei whereas compound 1 in the present study, as ciprofloxacin ethyl ester, showed an IC50 value at 26 lM. Thus, the fluoroquinolonemediated inhibition of type II DNA topoisomerase activity could supply new antitrypanosomal agents in the future. However,
Mean survival time/controls (Days ± SD)
Number of mice cured/ total number of mice
Cure rate (%)
Toxicity: number of dead mice [day of death posttreatment]
8.2 ± 1.6 6.1 ± 1.2 0 0 1.2 ± 0.8 1.1 ± 0.7 0 0 4.5 ± 1.8 3.8 ± 1.4 0 >30 0
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/10 10/10 0/10
0 0 0 0 0 0 0 0 0 0 0 100 0
No toxicity No toxicity No toxicity No toxicity No toxicity No toxicity No toxicity No toxicity 1[Day 3]; 1[Day 4] No toxicity No toxicity No toxicity No toxicity
Hiltensperger et al.30 described new quinolone derivatives exhibiting IC50 values of 47 nM on T. brucei but affecting the mitochondrial morphology. Therefore, quinolones could exhibit at least two different antitrypanosomal mechanisms of action as a function of chemical groups substituted on the scaffold. Further pharmacomodulations studies are necessary to understand the structure–activity relationships responsible for each of these mechanisms of action. One representative of each three chemical series, as fluoroquinolones, 4-aminoquinoline-benzimidazoles, benzimidazomono- and di-substituted ferrocenes and ferroquine derivatives, were interesting enough to be studied in vivo. We have therefore selected the compounds exhibiting the best in vitro activity that were compounds 2, 5, 6 and 13. These compounds exhibited a
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selectivity index (SI) as follows: for compounds 2 and 6, SI values were higher than 5 and for compound 5, SI value was higher than 2. The less cytotoxic product was compound 13 with a SI value close to 15. Table 2 gathers results of in vivo activity of these compounds on the T. b. brucei Swiss mice model. Ferroquine was the single compound being toxic at 100 lmol/kg, whereas no toxicity was found at the same dose for all other compounds. Compound 2 was the most active, clearing the trypanosomes from the blood for at least 4 days and allowing the survival time of 8 days. However, such an activity was too slight to consider this compound as promising. The in vivo trial was very stringent since it consisted in one single treatment, selecting the very powerful compounds only. Further pharmacomodulations will be developed on the fluoroquinolones series in order to optimize this interesting antitrypanosomal series. Acknowledgments The authors would like to thank P. Grellier and P. Vincendeau who kindly provided the strain FéoITMAP/1893 of Trypanosma brucei gambiense. The authors would like to thank the Programme Hubert Currien Tournesol, Egide (Project PVB/AD/FR/ad/SOR/2011/34676: ‘Mise au point de protocoles expérimentaux respectueux de l’environnement pour la préparation de nouveaux antipaludiques métallocéniques’) to support the chemical part of this project. References and notes 1. Franco, J. R.; Simarro, P. P.; Diarra, A.; Jannin, J. G. Clin. Epidemiol. 2014, 6, 257. 2. Simarro, P. P.; Diarra, A.; Ruiz-Postigo, J. A.; Franco, J. R.; Jannin, J. G. PLoS Negl. Trop. Dis. 2011, 5, e1007. 3. Selby, R.; Bardosh, K.; Picozzi, K.; Waiswa, C.; Welburn, S. C. Parasit. Vectors 2013, 6, 281. 4. Babokhov, P.; Sanyaolu, A. O.; Oyibo, W. A.; Fagbenro-Beyioku, A. F.; Iriemenam, N. C. Pathog. Glob. Health 2013, 107, 242. 5. Seixas, J. D.; Luengo-Arratta, S. A.; Diaz, R.; Saldivia, M.; Rojas-Barros, D. I.; Manzano, P.; Gonzalez, S.; Berlanga, M.; Smith, T. K.; Navarro, M.; Pollastri, M. P. J. Med. Chem. 2014, 57, 4834.
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