Bisguanidine, Bis(2-aminoimidazoline), and Polyamine Derivatives as Potent and Selective Chemotherapeutic Agents against Trypanosoma brucei rhodesiense. Synthesis and in Vitro Evaluation

The in vitro screening for trypanocidal activity against Trypanosoma brucei rhodesiense of an in-house library of 62 compounds [i.e. alkane, diphenyl, and azaalkane bisguanidines and bis-(2-aminoimidazolines)], which were chosen for their structural similarity to the trypanocidal agents synthalin (1,10-decanediguanidine) and 4,4′-diguanidinodiphenylmethane and the polyamine N1-(3-amino-propyl)propane-1,3-diamine, respectively, is reported. The original synthetic procedure for the preparation of 21 of these compounds is also reported. Most compounds displayed low micromolar antitrypanosomal activity, with five of them presenting a nanomolar inhibitory action on the parasite: 1,9-nonanediguanidine (1c), 1,12-dodecanediguanidine (1d), 4,4′ -bis[1,3-bis(tert-butoxycarbonyl)-2-imidazolidinylimino]diphenylamine (28a), 4,4′-bis(4,5-dihydro-1H-2-imidazolylamino)diphenylamine (28b), and 4,4′-diguanidino-diphenylamine (32b). Those molecules that showed an excellent in vitro activity as well as high selectivity for the parasite [e.g. 1c (IC50 = 49 nM; SI > 5294), 28b (IC50 = 69 nM; SI = 3072), 32b (IC50 = 22 nM; SI = 29.5), 41b (IC50 = 118 nM; SI = 881)] represent new antitrypanosomal lead compounds.Peer Reviewe

Recent advances in antitrypanosomal chemotherapy: Patent literature 2002-2004

Sleeping sickness and Chagas disease (African and American trypanosomiases, respectively) are protozoan parasitic diseases threatening millions of people in sub-Saharan Africa and Latin America. Trypanosomiases are among the most neglected diseases in the world, desperately lacking financial support for investigation. The current chemotherapy of both diseases is poor and suffers from intolerable side effects and low efficacy in many cases. A review of the patent literature from 2002 to early 2005 claiming molecules with antitrypanosomal activity afforded 36 entries, equally shared between industry and acadaemia. Among the targets validated against trypanosomes, patents dealing with protease inhibitors were the most represented (16 patents). Other targets claimed in the patent literature included membrane architecture (sterol biosynthesis inhibitors, protein farnesyltransferase inhibitors), DNA (DNA binders, tubulin inhibitors) and pyrimidine metabolism (cytidine triphosphate [CTP] synthetase inhibitors). Natural products were also a great source of trypanocidal lead compounds (9 patents). A few patents claiming compounds with antitrypanosomal activity, but disclosing no specific target, were also encountered. © 2005 Ashley Publications Ltd.Peer Reviewe

Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of T. brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in Trypanosoma brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase

Bisimidazoline arylamides binding to the DNA minor groove: N1-hydroxylation enhances binding affinity and selectivity to AATT sites

Bisimidazoline arylamides and related compounds are high affinity DNA minor groove binders with a preference for AT over GC-rich DNA. However, further selectivity towards different classes of AT-sites (e.g., CGAATTCG, CATATATAT) is not always observed with these series. In this work, we wanted to understand the effect of imidazoline ring N-substitution on binding to DNA AT-sites. The structure¿affinity relationships of a series of structurally related bisimidazoline compounds were studied by UV titrations and surface plasmon resonance (SPR) experiments using fish sperm DNA and different hairpin oligonucleotides. We found that in this series, the presence of N1¿OH groups enhances the binding affinity to dsDNA CGAATTCG oligonucleotide, resulting in a higher selectivity for dsDNA containing AATT over (AT)4 sequences. The docking models showed that the N-hydroxy derivatives bind in a more planar conformation to the CGAATTCG DNA sequence, display more favorable van der Waals interactions, and show additional H-bonds with the bases and the sugar-phosphate backbone.C. R. was a recipient of a PhD fellowship from the govern- ment of Panama (SENACYT grant BIDP-2008-030). The assis- tance of Silvia Soto Alvarez with the SPR experiments and Eden Gebreselassie with the UV experiments is gratefully acknowledged. We thank Dr. N. Jagerovic, Dr. P. Goya, and Dr. Alkorta for logistical collaboration. We acknowledge sup- port of the publication fee by the CSIC Open Access Publica- tion Support Initiative through its Unit of Information Resources for Research (URICI)Peer Reviewe

New bis(2-aminoimidazoline) and bisguanidine DNA minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity

A series of 75 guanidine and 2-aminoimidazoline analogue molecules were assayed in vitro against Trypanosoma brucei rhodesiense STIB900 and Plasmodium falciparum K1. The dicationic diphenyl compounds exhibited the best activities with IC50 values against T. b. rhodesiense and P. falciparum in the nanomolar range. Five compounds (7b, 9a, 9b, 10b, and 14b) cured 100% of treated mice upon ip administration at 20 mg/kg in the difficult to cure T. b. rhodesiense STIB900 mouse model. Overall, the compounds that bear the 2-aminoimidazoline cations benefit from better safety profiles than the guanidine counterparts. The observation of a correlation between DNA binding affinity at AT sites and trypanocidal activity for three series of compounds supported the view of a mechanism of antitrypanosomal action due in part to the formation of a DNA complex. No correlation between antiplasmodial activity and in vitro inhibition of ferriprotoporphyrin IX biomineralisation was observed, suggesting that additional mechanism of action is likely to be involved. © 2008 American Chemical Society.Peer Reviewe

Bis(2-aminoimidazolinium)diphenyl Compounds as DNA Minor Groove Binders with in Vivo Antitrypanosomal and Antimalarial Activity: the cation is important

Abstract of oral communication presented in the "2nd World Conference on Magic Bullets (Ehrlich II)" October 3-5, 2008 Nürnberg, GermanyBackground: A pragmatic approach to the discovery of new drugs for neglected diseases is the “recycling” of available compounds. We have successfully applied this strategy during the last years with the (re)discovery of an attractive class of compounds (i.e., 2-aminoimidazolinium derivatives) showing excellent in vivo activity against T. brucei rhodesiense and P. falciparum, the ethiological agents of rhodesiense sleeping sickness and severe malaria, respectively. Methods: Based on their structural similarity with known antitrypanosomal and antimalarial agents, several series of dicationic compounds as well as their monocationic and neutral analogues were screened in vitro against T. b. rhodesiense, P. falciparum, and rat skeletal myoblast L6-cells as control for cytotoxicity. The compounds showing the highest activity and acceptable selectivity were assayed in vivo in models of acute and chronic T. brucei infections (STIB900 and GVR35 strains, respectively), and murine malaria (P. berghei). Their interaction with the DNA minor groove was also measured by thermal melting curves (Tm) and SPR experiments on AT sequence DNA polymers. Results: Several dicationic leads with nM in vitro activity and excellent selectivity against T. b. rhodesiense and P. falciparum were identified. A number of compounds cured 100% of the mice infected with T. b. rhodesiense and 4 compounds reduced the parasitemia in mice infected with P. berghei. A correlation between DNA binding affinity and trypanocidal activity was observed, indicating that DNA binding may be part of their mechanism of action. Most importantly, we found that the 2-aminoimidazoline cation afforded molecules with superior safety profile compared with its guanidine counterpart. Conclusion: 1) The rational screening of in-house libraries of compounds is a validated approach to find new drug leads for neglected diseases. 2) Bis(2-aminoimidazoline) derivatives represent a very promising class of DNA minor groove binding agents that have already demonstrated their antiprotozoal potential in vivo.UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR)(RB); CSIC–I3 program (PIE200680I121); Spanish Ministerio de Educación y Ciencia (SAF2006–04698); Consejería de Educación Cultura y Deporte de la Comunidad Autónoma de La Rioja (FR); Research on DNA interactions is supported by National Institutes of Health grant AI064200 (W.D.W.).Peer reviewe

Mitochondrion targeted trypanosome alternative oxidase inhibitors as chemotherapeutic agents against T. brucei

Trabajo presentado en el XII SEQT Mini Symposium. IIIrd Spanish/Portuguese/Brazilian Meeting, celebrado en Madrid del 17 al 18 de noviembre de 2016.During their life-cycle, trypanosomes adapt their energy metabolism to the availability of nutrients in their environment. Hence, procyclic forms of T. brucei have a fully functional respiratory chain and synthesize ATP by oxidative phosphorylation in the mitochondrion. In contrast, respiration of bloodstream forms (BSF) of T. brucei (i.e. the human-infective form) relies exclusively on glycolysis for energy production. The trypanosome alternative oxidase (TAO) is the sole terminal oxidase enzyme to re-oxidize NADH accumulated during glycolysis. It is a cyanide-resistant and cytochrome-independent ubiquinol oxidase which is sensitive to the specific inhibitors salicylhydroxamic acid (SHAM) and ascofuranone. This enzyme which is essential to the viability of BSF trypanosomes and has no counterpart in the mammalian host is a potential target for chemotherapy. To boost the activity of TAO inhibitors against T. brucei, we investigated a chemical strategy consisting in the conjugation of the inhibitor with lipophilic cations (LC) that can cross lipid bilayers by non-carrier mediated transport, and thus accumulate specifically into the mitochondrion, driven by the plasma and mitochondrial transmembrane potentials (negative inside). This design afforded several LC¿TAO inhibitor conjugates active in the submicromolar to low nanomolar range against wild type and resistant strains of African trypanosomes (T. b. brucei, T. congolense). Selectivity over human cells was >500. Studies of the effects on purified TAO, parasite respiration, mitochondrial membrane potential (¿m), and cell cycle suggest that TAO is a likely target of the compounds in vivo

Synthesis and structure-activity analysis of new phosphonium salts with potent activity against African trypanosomes

A series of 73 bisphosphonium salts and 10 monophosphonium salt derivatives were synthesized and tested in vitro against several wild type and resistant lines of Trypanosoma brucei (T. b. rhodesiense STIB900, T. b. brucei strain 427, TbAT1-KO, and TbB48). More than half of the compounds tested showed a submicromolar EC 50 against these parasites. The compounds did not display any cross-resistance to existing diamidine therapies, such as pentamidine. In most cases, the compounds displayed a good selectivity index versus human cell lines. None of the known T. b. brucei drug transporters were required for trypanocidal activity, although some of the bisphosphonium compounds inhibited the low affinity pentamidine transporter. It was found that phosphonium drugs act slowly to clear a trypanosome population but that only a short exposure time is needed for irreversible damage to the cells. A comparative molecular field analysis model (CoMFA) was generated to gain insights into the SAR of this class of compounds, identifying key features for trypanocidal activity. © 2012 American Chemical Society.Peer Reviewe