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1.  Substituted 2-Phenyl-Imidazopyridines: A New Class of Drug Leads for Human African Trypanosomiasis 
Journal of medicinal chemistry  2014;57(3):828-835.
A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl) oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogues were prepared, which led to the identification of 64, a substituted 2-(3-aminophenyl) imidazopyridine. This compound showed antiparasitic activity in vitro with an EC50 of 2 nM and displayed reasonable drug-like properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 64 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent anti-parasitic properties and its ease of synthesis, compound 64 represents a new lead for the development of drugs to treat human African trypanosomiasis.
PMCID: PMC3962778  PMID: 24354316
2.  Chemotherapy of Second Stage Human African Trypanosomiasis: Comparison between the Parenteral Diamidine DB829 and Its Oral Prodrug DB868 in Vervet Monkeys 
PLoS Neglected Tropical Diseases  2015;9(2):e0003409.
Human African trypanosomiasis (HAT, sleeping sickness) ranks among the most neglected tropical diseases based on limited availability of drugs that are safe and efficacious, particularly against the second stage (central nervous system [CNS]) of infection. In response to this largely unmet need for new treatments, the Consortium for Parasitic Drug Development developed novel parenteral diamidines and corresponding oral prodrugs that have shown cure of a murine model of second stage HAT. As a rationale for selection of one of these compounds for further development, the pharmacokinetics and efficacy of intramuscular (IM) active diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and oral prodrug2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) were compared in the vervet monkey model of second stage HAT. Treatment was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. Results showed that IM DB829 at 5 mg/kg/day for 5 consecutive days, 5 mg/kg/day every other day for 5 doses, or 2.5 mg/kg/day for 5 consecutive days cured all monkeys (5/5). Oral DB868 was less successful, with no cures (0/2) at 3 mg/kg/day for 10 days and cure rates of 1/4 at 10 mg/kg/day for 10 days and 20 mg/kg/day for 10 days; in total, only 2/10 monkeys were cured with DB868 dose regimens. The geometric mean plasma Cmax of IM DB829 at 5 mg/kg following the last of 5 doses was 25-fold greater than that after 10 daily oral doses of DB868 at 20 mg/kg. These data suggest that the active diamidine DB829, administered IM, should be considered for further development as a potential new treatment for second stage HAT.
Author Summary
Treatment of human African trypanosomiasis (HAT, sleeping sickness) suffers from a shortage of medicines that are both effective, especially against the second (late) stage of the disease, and safe for patients. The development of new HAT medicines also has been significantly influenced by the perceived need for easily administered oral medicines to reduce the need for hospitalization of patients in resource-poor settings where HAT typically occurs. However, the clinical status of second stage patients is likely to dictate the need for their hospitalization, thus both oral and parenterally administered medicines would be utilised effectively. Therefore, in an effort to develop new medicines that meet efficacy and safety requirements, we evaluated a novel injectable diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and its oral prodrug formulation 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) in the vervet monkey model of second stage HAT. Treatment with either compound was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. DB829 was dosed at 5 mg/kg/day for 5 consecutive days, 5 mg/kg/day every other day for 5 doses or 2.5 mg/kg/day for 5 consecutive days intramuscularly (IM) while DB868 was administered at 20, 10 or 3 mg/kg/day for 10 consecutive days orally. Clinical and parasitological monitoring was carried out for at least 300 days before the monkeys were declared cured. All IM DB829 and oral DB868 dose regimens were well tolerated. In addition, all monkeys (5/5) treated with IM DB829 were confirmed cured. In contrast, oral DB868 cured only 1/4 monkeys at either 10 or 20 mg/kg and did not cure any monkey when dosed at 3 mg/kg. These results indicate that IM DB829 is a suitable compound for further development as treatment for second stage HAT.
PMCID: PMC4318582  PMID: 25654243
3.  In Vitro and In Vivo Evaluation of 28DAP010, a Novel Diamidine for Treatment of Second-Stage African Sleeping Sickness 
African sleeping sickness is a neglected tropical disease transmitted by tsetse flies. New and better drugs are still needed especially for its second stage, which is fatal if untreated. 28DAP010, a dipyridylbenzene analogue of DB829, is the second simple diamidine found to cure mice with central nervous system infections by a parenteral route of administration. 28DAP010 showed efficacy similar to that of DB829 in dose-response studies in mouse models of first- and second-stage African sleeping sickness. The in vitro time to kill, determined by microcalorimetry, and the parasite clearance time in mice were shorter for 28DAP010 than for DB829. No cross-resistance was observed between 28DAP010 and pentamidine on the tested Trypanosoma brucei gambiense isolates from melarsoprol-refractory patients. 28DAP010 is the second promising preclinical candidate among the diamidines for the treatment of second-stage African sleeping sickness.
PMCID: PMC4136055  PMID: 24867978
4.  Pharmacokinetic Comparison To Determine the Mechanisms Underlying the Differential Efficacies of Cationic Diamidines against First- and Second-Stage Human African Trypanosomiasis 
Human African trypanosomiasis (HAT), a neglected tropical disease, is fatal without treatment. Pentamidine, a cationic diamidine, has been used to treat first-stage (hemolymphatic) HAT since the 1940s, but it is ineffective against second-stage (meningoencephalitic, or central nervous system [CNS]) infection. Novel diamidines (DB75, DB820, and DB829) have shown promising efficacy in both mouse and monkey models of first-stage HAT. However, only DB829 cured animals with second-stage infection. In this study, we aimed to determine the mechanisms underlying the differential efficacies of these diamidines against HAT by conducting a comprehensive pharmacokinetic characterization. This included the determination of metabolic stability in liver microsomes, permeability across MDCK and MDR1-MDCK cell monolayers, interaction with the efflux transporter MDR1 (P-glycoprotein 1 or P-gp), drug binding in plasma and brain, and plasma and brain concentration-time profiles after a single dose in mice. The results showed that DB829, an azadiamidine, had the highest systemic exposure and brain-to-plasma ratio, whereas pentamidine and DB75 had the lowest. None of these diamidines was a P-gp substrate, and the binding of each to plasma proteins and brain differed greatly. The brain-to-plasma ratio best predicted the relative efficacies of these diamidines in mice with second-stage infection. In conclusion, pharmacokinetics and CNS penetration influenced the in vivo efficacies of cationic diamidines against first- and second-stage HAT and should be considered when developing CNS-active antitrypanosomal diamidines.
PMCID: PMC4068519  PMID: 24798280
5.  Identification and Optimization of an Aminoalcohol-Carbazole Series with Antimalarial Properties 
ACS Medicinal Chemistry Letters  2013;4(11):1037-1041.
Recent observations on the emergence of artemisinin resistant parasites have highlighted the need for new antimalarial treatments. An HTS campaign led to the identification of the 1-(1-aminopropan-2-ol)carbazole analogues as potent hits against Plasmodium falciparum K1 strain. The SAR study and optimization of early ADME and physicochemical properties direct us to the selection of a late lead compound that shows good efficacy when orally administrated in the in vivo P. berghei mouse model.
PMCID: PMC4027556  PMID: 24900603
Malaria; WHO; SAR; carbazole; Plasmodium falciparum; Plasmodium berghei; IC50; hERG
6.  Synthesis and Antiprotozoal Activity of Dicationic 2, 6-Diphenylpyrazines and Aza-Analogues 
Bioorganic & medicinal chemistry  2013;21(21):10.1016/j.bmc.2013.08.006.
Dicationic 2,6-diphenylpyrazines, aza-analogues and prodrugs were synthesized; evaluated for DNA affinity, activity against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) in vitro, efficacy in T. b. r. STIB900 acute and T. b. brucei GVR35 CNS mouse models. Most diamidines gave poly(dA-dT)2 ΔTm values greater than pentamidine, IC50 values: T. b. r. (4.8 to 37 nM) and P. f. (10 to 52 nM). Most diamidines and prodrugs gave cures for STIB900 model (11, 19a and 24b 4/4 cures); 12 3/4 cures for GVR35 model. Metabolic stability half-life values for O-methylamidoxime prodrugs did not correlate with STIB900 results.
PMCID: PMC3846175  PMID: 24012380
7.  In Silico Prediction and Experimental Evaluation of Furanoheliangolide Sesquiterpene Lactones as Potent Agents against Trypanosoma brucei rhodesiense 
As a continuation of our earlier study on the in vitro antiprotozoal activity of 40 natural sesquiterpene lactones (STLs), we extended the set of tested compounds from our laboratories to 59. On the basis of this extended data set, further enriched by literature data for 10 compounds tested under the same conditions, our quantitative structure-activity relationship (QSAR) analyses for activity against T. brucei rhodesiense (etiologic agent of human African trypanosomiasis, or sleeping sickness) were continued, and the QSAR model thus obtained with 69 structures was used to predict the activity of a virtual library of 1,750 STL structures. As a major result from these calculations, furanoheliangolide-type compounds, a subclass of STLs hitherto untested against T. brucei rhodesiense, were predicted to have an exceptionally high level of in vitro activity. Four representative compounds of this type, goyazensolide, 4,5-dihydro-2′,3′-epoxy-15-deoxygoyazensolide, budlein A, and 4,15-isoatriplicolide tiglate, were therefore tested. They displayed 50% inhibitory concentrations (IC50s) of 0.07, 0.20, 0.07, and 0.015 μM, respectively, so that the in silico prediction was experimentally confirmed. 4,15-Isoatriplicolide tiglate is the most potent STL against T. b. rhodesiense found. Furanoheliangolide STLs were thus identified as interesting leads against this parasite which deserve more detailed investigations.
PMCID: PMC3910805  PMID: 24165182
8.  New Promising Compounds with in Vitro Nanomolar Activity against Trypanosoma cruzi 
ACS Medicinal Chemistry Letters  2013;4(6):538-541.
The antiparasitic activity of azole and new 4-aminopyridine derivatives has been investigated. The imidazoles 1 and 3–5 showed a potent in vitro antichagasic activity with IC50 values in the low nanomolar concentration range. The (S)-1, (S)-3, and (S)-5 enantiomers showed (up to) a thousand-fold higher activity than the reference drug benznidazole and furthermore low cytotoxicity on rat myogenic L6 cells.
PMCID: PMC4027138  PMID: 24900706
Trypanosoma cruzi; CYP51; antiparasitic activity; Chagas disease; azole; aminopyridine
9.  Activities of Psilostachyin A and Cynaropicrin against Trypanosoma cruzi In Vitro and In Vivo 
Antimicrobial Agents and Chemotherapy  2013;57(11):5307-5314.
In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection.
PMCID: PMC3811247  PMID: 23939901
10.  Pharmacokinetics, Trypanosoma brucei gambiense Efficacy, and Time of Drug Action of DB829, a Preclinical Candidate for Treatment of Second-Stage Human African Trypanosomiasis 
Antimicrobial Agents and Chemotherapy  2013;57(11):5330-5343.
Human African trypanosomiasis (HAT, also called sleeping sickness), a neglected tropical disease endemic to sub-Saharan Africa, is caused by the parasites Trypanosoma brucei gambiense and T. brucei rhodesiense. Current drugs against this disease have significant limitations, including toxicity, increasing resistance, and/or a complicated parenteral treatment regimen. DB829 is a novel aza-diamidine that demonstrated excellent efficacy in mice infected with T. b. rhodesiense or T. b. brucei parasites. The current study examined the pharmacokinetics, in vitro and in vivo activity against T. b. gambiense, and time of drug action of DB829 in comparison to pentamidine. DB829 showed outstanding in vivo efficacy in mice infected with parasites of T. b. gambiense strains, despite having higher in vitro 50% inhibitory concentrations (IC50s) than against T. b. rhodesiense strain STIB900. A single dose of DB829 administered intraperitoneally (5 mg/kg of body weight) cured all mice infected with different T. b. gambiense strains. No cross-resistance was observed between DB829 and pentamidine in T. b. gambiense strains isolated from melarsoprol-refractory patients. Compared to pentamidine, DB829 showed a greater systemic exposure when administered intraperitoneally, partially contributing to its improved efficacy. Isothermal microcalorimetry and in vivo time-to-kill studies revealed that DB829 is a slower-acting trypanocidal compound than pentamidine. A single dose of DB829 (20 mg/kg) administered intraperitoneally clears parasites from mouse blood within 2 to 5 days. In summary, DB829 is a promising preclinical candidate for the treatment of first- and second-stage HAT caused by both Trypanosoma brucei subspecies.
PMCID: PMC3811327  PMID: 23959303
11.  In vitro and in vivo characterization of the antimalarial lead compound SSJ-183 in Plasmodium models 
The objective of this work was to characterize the in vitro (Plasmodium falciparum) and in vivo (Plasmodium berghei) activity profile of the recently discovered lead compound SSJ-183. The molecule showed in vitro a fast and strong inhibitory effect on growth of all P. falciparum blood stages, with a tendency to a more pronounced stage-specific action on ring forms at low concentrations. Furthermore, the compound appeared to be equally efficacious on drug-resistant and drug-sensitive parasite strains. In vivo, SSJ-183 showed a rapid onset of action, comparable to that seen for the antimalarial drug artesunate. SSJ-183 exhibited a half-life of about 10 hours and no significant differences in absorption or exposure between noninfected and infected mice. SSJ-183 appears to be a promising new lead compound with an attractive antimalarial profile.
PMCID: PMC3832383  PMID: 24255594
antimalarial studies; cross-resistance; stage-specificity; Plasmodium falciparum
12.  Novel linear triaryl guanidines, N-substituted guanidines and potential prodrugs as antiprotozoal agents 
European journal of medicinal chemistry  2008;43(12):10.1016/j.ejmech.2008.02.008.
A series of triaryl guanidines and N-substituted guanidines designed to target the minor groove of DNA were synthesized and evaluated as antiprotozoal agents. Selected carbamate prodrugs of these guanidines were assayed for their oral efficacy. The linear triaryl bis-guanidines 6a,b were prepared from their corresponding diamines 4a,b through the intermediate BOC protected bis-guanidines 5a,b followed by acid catalyzed deprotection. The N-substituted guanidino analogues 9c–f were obtained in three steps starting by reacting the diamines 4a,b with ethyl iso-thiocyanatoformate to give the carbamoyl thioureas 7a,b. Subsequent condensation of 7a,b with various amines in the presence of EDCI provided the carbamoyl N-substituted guanidine intermediates 8a–f which can also be regarded as potential prodrugs for the guanidino derivatives. Compounds 9c–f were obtained via the base catalyzed decarbamoylation of 8a–f. The DNA binding affinities for the target dicationic bis-guanidines were assessed by ΔTm values. In vitro antiprotozoal screening of the new compounds showed that derivatives 6a, 9c and 9e possess high to moderate activity against Trypanosoma brucei rhodesiense (T.b.r.) and Plasmodium falciparum (P.f.). While the prodrugs did not yield cures upon oral administration in the antitrypanosomal STIB900 mouse model, compounds 8a and 8c prolonged the survival of the treated mice.
PMCID: PMC3815585  PMID: 18455271
Antiprotozoan; Bis-guanidines; DNA binding; Triaryl guanidines
13.  Synthesis and activity of azaterphenyl diamidines against Trypanosoma brucei rhodesiense and Plasmodium falciparum 
Bioorganic & medicinal chemistry  2009;17(18):10.1016/j.bmc.2009.07.080.
A series of azaterphenyl diamidines has been synthesized and evaluated for in vitro antiprotozoal activity against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) and in vivo efficacy in the STIB900 acute mouse model for T. b. r. Six of the 13 compounds showed IC50 values less than 7 nM against T. b. r. Twelve of those exhibited IC50 values less than 6 nM against P. f. and six of those showed IC50 values ≤0.6 nM, which are more than 25-fold as potent as furamidine. Moreover, two of them showed more than 40-fold selectivity for P. f. versus T. b. r. Three compounds 15b, 19d and 19e exhibited in vivo efficacy against T. b. r. much superior to furamidine, and equivalent to or better than azafuramidine. The antiparasitic activity of these diamidines depends on the ring nitrogen atom(s) location relative to the amidine groups and generally correlates with DNA binding affinity.
PMCID: PMC3813006  PMID: 19699098
Diamidines; Antiprotozoal agents; DNA binding affinity; Azaterphenyl
14.  Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol 
The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.
Author Summary
Human African Trypanosomiasis, or sleeping sickness, is a fatal disease restricted to sub-Saharan Africa, caused by Trypanosoma brucei gambiense and T. b. rhodesiense. The treatment relies on chemotherapy exclusively. Drug resistance in T. brucei was investigated mainly in laboratory-selected lines and found to be linked to mutations in transporters. The adenosine transporter TbAT1 and the aquaglyceroporin TbAQP2 have been implicated in sensitivity to melarsoprol and pentamidine. Mutations in these transporters rendered trypanosomes less susceptible to either drug. Here we analyze T. brucei isolates from the field, focusing on isolates from patients where melarsoprol treatment has failed. We genotype those isolates to test for mutations in TbAQP2 or TbAT1, and phenotype for sensitivity to pentamidine and melarsoprol. Six T. b. gambiense isolates were found to carry mutations in TbAQP2. These isolates stemmed from relapse patients and exhibited significantly reduced sensitivity to pentamidine and melarsoprol as determined in cell culture. These findings indicate that mutations in TbAQP2 are present in the field, correlate with loss of sensitivity to pentamidine and melarsoprol, and might be responsible for melarsoprol treatment failures.
PMCID: PMC3794916  PMID: 24130910
15.  Synthesis, DNA binding and antileishmanial activity of low molecular weight bis-arylimidamides 
The effects of reducing the molecular weight of the antileishmanial compound DB766 on DNA binding affinity, antileishmanial activity and cytotoxicity are reported. The bis-arylimidamides were prepared by the coupling of aryl S-(2-naphthylmethyl)thioimidates with the corresponding amines. Specifically, we have prepared new series of bis-arylimidamides which include 3a, 3b, 6, 9a, 9b, 9c, 13, and 18. Three compounds 9a, 9c, and 18 bind to DNA with similar or moderately lower affinity to that of DB766, the rest of these compounds either show quite weak binding or no binding at all to DNA. Compounds 9a, 9c, and 13 were the most active against L. amazonensis showing IC50 values of less than 1 µM, so they were screened against intracellular L. donovani showing outstanding activity with IC50.values of 25–79 nM. Despite exhibiting little in vitro cytotoxicity these three compounds were quite toxic to mice.
PMCID: PMC3560421  PMID: 22840696
Leishmaniasis; arylimidamides; DB766; antileishmanial agents
16.  Safety, Pharmacokinetic, and Efficacy Studies of Oral DB868 in a First Stage Vervet Monkey Model of Human African Trypanosomiasis 
There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD). In this study, we investigated the safety, pharmacokinetics, and efficacy of a new orally administered CPDD diamidine prodrug, 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868; CPD-007-10), in the vervet monkey model of first stage HAT. DB868 was well tolerated at a dose up to 30 mg/kg/day for 10 days, a cumulative dose of 300 mg/kg. Mean plasma levels of biomarkers indicative of liver injury (alanine aminotransferase, aspartate aminotransferase) were not significantly altered by drug administration. In addition, no kidney-mediated alterations in creatinine and urea concentrations were detected. Pharmacokinetic analysis of plasma confirmed that DB868 was orally available and was converted to the active compound DB829 in both uninfected and infected monkeys. Treatment of infected monkeys with DB868 began 7 days post-infection. In the infected monkeys, DB829 attained a median Cmax (dosing regimen) that was 12-fold (3 mg/kg/day for 7 days), 15-fold (10 mg/kg/day for 7 days), and 31-fold (20 mg/kg/day for 5 days) greater than the IC50 (14 nmol/L) against T. b. rhodesiense STIB900. DB868 cured all infected monkeys, even at the lowest dose tested. In conclusion, oral DB868 cured monkeys with first stage HAT at a cumulative dose 14-fold lower than the maximum tolerated dose and should be considered a lead preclinical candidate in efforts to develop a safe, short course (5–7 days), oral regimen for first stage HAT.
Author Summary
Development of orally administered medicines for human African trypanosomiasis (HAT) would potentially reduce the need for patient hospitalization, thus lowering healthcare costs. In this study, we investigated the potential of a novel diamidine prodrug, DB868 (CPD-007-10), as an oral treatment for first stage HAT. When administered to uninfected monkeys by oral gavage, DB868 was well tolerated up to a maximum dose of 30 mg/kg/day for 10 days (cumulative dose [CD] = 300 mg/kg). DB868 was absorbed into the systemic circulation and was converted to the active compound DB829 in concentrations that were potentially therapeutic for blood trypanosomes. Subsequently, DB868 was evaluated for efficacy in the first stage vervet monkey model of HAT in which treatment was initiated at 7 days post-infection with T. b. rhodesiense KETRI 2537. All infected monkeys were cured, even at the lowest of the three dose regimens tested: 3 mg/kg/day for 7 days (CD = 21 mg/kg), 10 mg/kg/day for 7 days (CD = 70 mg/kg) and 20 mg/kg/day for 5 days (CD = 100 mg/kg). DB868 conversion to DB829 was comparable between uninfected and infected monkeys. In view of its favourable safety and oral efficacy profile, we conclude that DB868 is a suitable candidate for development as a new treatment for first stage HAT.
PMCID: PMC3674995  PMID: 23755309
17.  Synthesis, Biological Evaluation and Structure-Activity Relationships of N-Benzoyl-2-hydroxybenzamides as Agents Active against P. falciparum (K1 strain), Trypanosomes, and Leishmania 
Journal of Medicinal Chemistry  2012;55(7):3088-3100.
In our efforts to identify novel chemical scaffolds for the development of new antiprotozoal drugs, a compound library was screened against T. gondii tachyzoites with activity discovered for N-(4-ethylbenzoyl)-2-hydroxybenzamide 1a against T. gondii as described elsewhere.1 Synthesis of a compound set was guided by T. gondii SAR with 1r found to be superior for T. gondii, also active against Thai and Sierra Leone strains of P. falciparum, and with superior ADMET properties as described elsewhere.1 Herein, synthesis methods and details of the chemical analysis of the compounds in this series are described. Further, this series of N-benzoyl-2-hydroxybenzamides was re-purposed for testing against four other protozoan parasites: T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum (K1 isolate). Structure-activity analyses led to the identification of compounds in this set with excellent anti-leishmanial activity (compound 1d). Overall, compound 1r was the best and had activity 21-fold superior to that of the standard anti-malarial drug chloroquine against the K1 P. falciparum isolate.
PMCID: PMC3330251  PMID: 22352841
18.  Anti-protozoal activity of aporphine and protoberberine alkaloids from Annickia kummeriae (Engl. & Diels) Setten & Maas (Annonaceae) 
Malaria, trypanosomiasis and leishmaniasis have an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armory of drugs for the treatment of malaria, trypanosomiasis and leishmaniasis. This underscores the continuing need for the discovery and development of new anti-protozoal drugs. Consequently, there is an urgent need for research aimed at the discovery and development of new effective and safe anti-plasmodial, anti-trypanosomal and anti-leishmanial drugs.
Bioassay-guided chromatographic fractionation was employed for the isolation and purification of antiprotozoal alkaloids.
The methanol extract from the leaves of Annickia kummeriae from Tanzania exhibited a strong anti-plasmodial activity against the multi-drug resistant Plasmodium falciparum K1 strain (IC50 0.12 ± 0.01 μg/ml, selectivity index (SI) of 250, moderate activity against Trypanosoma brucei rhodesiense STIB 900 strain (IC50 2.50 ± 0.19 μg/ml, SI 12) and mild activity against Leishmania donovani axenic MHOM-ET-67/82 strain (IC50 9.25 ± 0.54 μg/ml, SI 3.2). Bioassay-guided chromatographic fractionation led to the isolation of four pure alkaloids, lysicamine (1), trivalvone (2), palmatine (3), jatrorrhizine (4) and two sets of mixtures of jatrorrhizine (4) with columbamine (5) and palmatine (3) with (−)-tetrahydropalmatine (6). The alkaloids showed low cytotoxicity activity (CC50 30 - >90 μg/ml), strong to moderate anti-plasmodial activity (IC50 0.08 ± 0.001 - 2.4 ± 0.642 μg/ml, SI 1.5-1,154), moderate to weak anti-trypanosomal (IC50 2.80 ± 0.001 – 14.3 ± 0.001 μg/ml, SI 2.3-28.1) and anti-leishmanial activity IC50 2.7 ± 0.001 – 20.4 ± 0.003 μg/ml, SI 1.7-15.6).
The strong anti-plasmodial activity makes these alkaloids good lead structures for drug development programs.
PMCID: PMC3599822  PMID: 23445637
Annickia kummeriae; Enantia kummeriae; Annonaceae; Alkaloids; Aporphine; Protoberberine; Antiplasmodial; Antitrypanosomal; Antileishmanial; Cytotoxicity
19.  In vitro interaction of artemisinin derivatives or the fully synthetic peroxidic anti-malarial OZ277 with thapsigargin in Plasmodium falciparum strains 
Malaria Journal  2013;12:43.
Semi-synthetic artemisinin derivatives are powerful peroxidic drugs in artemisinin-based combination therapy (ACT) recommended as first-line treatment of Plasmodium falciparum malaria in disease-endemic countries. Studies by Eckstein-Ludwig and co-workers showed both thapsigargin and artemisinin specifically inhibit the sarcoplasmic reticulum Ca2+−ATPase of Plasmodium falciparum (PfATP6). In the present study the type of interaction between thapsigargin and artemisinin derivatives as well as the ozonide OZ277 (RBx11160 or arterolane) was evaluated in parasite cultures. The latter compound is an adamantane-based peroxide and the first fully synthetic clinical candidate recently registered in India by Ranbaxy Laboratories Ltd. for anti-malarial combination therapy.
Drug interaction studies were performed using a previously described fixed ratio method and anti-malarial activity measured using the [3H] hypoxanthine incorporation assay.
The sum 50% and 90% fractional inhibitory concentration (∑FIC50, 90) of the interaction of thapsigargin with OZ277, artemether or artesunate, against NF54 and K1 strains of P. falciparum ranged from 0.9 to 1.4.
The interaction of thapsigargin with OZ277, artesunate or artemether was additive, data consistent with previous observations indicating that activity of anti-malarial peroxides does not derive from reversible interactions with parasite targets.
PMCID: PMC3566918  PMID: 23368889
Thapsigargin; Artesunate; Artemether; OZ277; Plasmodium falciparum; Interaction study; Isobolograms
20.  Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism 
Background and Methodology
Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach.
Principal Findings
A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity.
Conclusions and Significance
Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.
Author Summary
The multitarget approach can represent a promising strategy for the discovery of innovative drug candidates against neglected tropical diseases. However, multitarget drug discovery can be very demanding, because of the highly time-consuming step related to the fine balancing of the biological activities against selected targets. An innovative workflow for discovering multitarget drugs can be envisioned: i) design and synthesis of natural-like compounds; ii) test them using phenotypic cell-based assays; iii) fishing potential targets by means of chemical proteomics. This workflow might rapidly provide new hit candidates that can be further progressed to the hit-to-lead and lead optimization steps of the drug discovery process. The two latter steps can benefit from information on the molecular target(s), which may be identified by chemical proteomics. Herein, we report on the elucidation of the mode of action of a new series of anti-trypanosomal naphthoquinone compounds, previously tested using cell-based assays, by means of chemical proteomics, classical biochemistry, molecular and system biology.
PMCID: PMC3547856  PMID: 23350008
21.  Novel 3-Nitro-1H-1,2,4-triazole-based Aliphatic and Aromatic Amines as anti-Chagasic Agents 
Journal of Medicinal Chemistry  2011;54(23):8214-8223.
A series of novel 2-nitro-1H-imidazole- and 3-nitro-1H-1,2,4-triazole-based aromatic and aliphatic amines were screened for anti-trypanosomal activity and mammalian cytotoxicity by the Drugs for Neglected Diseases initiative (DNDi). Out of 42 compounds tested, eighteen 3-nitro-1,2,4-triazoles and one 2-nitroimidazole displayed significant growth inhibitory properties against T. cruzi amastigotes (IC50 ranging from 40 nM to 1.97 μM), without concomitant toxicity towards the host cells (L6 cells), having selectivity indices (SI) 44 to 1320. Most (16) of these active compounds were up to 33.8-fold more potent than the reference drug benznidazole, tested in parallel. Five novel 3-nitro-1,2,4-triazoles were active against bloodstream form (BSF) T. b. rhodesiense trypomastigotes (IC50 at nM levels and SI 220 to 993). An NADH-dependent nitroreductase (TbNTR) plays a role in the anti-parasitic activity, since BSF T. b. brucei trypomastigotes with elevated TbNTR levels were hypersensitive to tested compounds. Therefore, a novel class of affordable 3-nitro-1,2,4-triazole-based compounds with antitrypanosomal activity has been identified.
PMCID: PMC3258117  PMID: 22023653
nitrotriazoles; amines; T. cruzi; Chagas disease; antitrypanosomal agents
22.  Agrochemicals against Malaria, Sleeping Sickness, Leishmaniasis and Chagas Disease 
In tropical regions, protozoan parasites can cause severe diseases with malaria, leishmaniasis, sleeping sickness, and Chagas disease standing in the forefront. Many of the drugs currently being used to treat these diseases have been developed more than 50 years ago and can cause severe adverse effects. Above all, resistance to existing drugs is widespread and has become a serious problem threatening the success of control measures. In order to identify new antiprotozoal agents, more than 600 commercial agrochemicals have been tested on the pathogens causing the above mentioned diseases. For all of the pathogens, compounds were identified with similar or even higher activities than the currently used drugs in applied in vitro assays. Furthermore, in vivo activity was observed for the fungicide/oomyceticide azoxystrobin, and the insecticide hydramethylnon in the Plasmodium berghei mouse model, and for the oomyceticide zoxamide in the Trypanosoma brucei rhodesiense STIB900 mouse model, respectively.
Author Summary
Even though agrochemistry and infectious disease control have the same principle goal – the suppression of harmful organisms without harming human health and the environment – there have been only very limited activities to exploit this overlap for the development of new antiinfectious drugs so far. In this study and for the first time, over 600 commercial agrochemicals were systematically screened against the infectious pathogens causing malaria, sleeping sickness, Chagas disease and leishmaniasis. Many highly active compounds with known low mammalian toxicity were identified in cell based assays, and the activity of some of them could even be confirmed in first animal model studies. Further expansion of this concept to other pathogens and the examination of analogues of the identified hits, potentially available from agrochemical companies, would allow for a very efficient source of novel drug candidates.
PMCID: PMC3493374  PMID: 23145187
23.  Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis 
Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-yl]-nicotinamide (DB820), exhibiting plasma Cmax values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.
Author Summary
New drugs are needed to treat sleeping sickness, especially the second stage of the disease, which is characterised by the presence of parasites (trypanosomes) in the brain. The purpose of this work was to determine whether DB844, a new drug that is converted to the active form (DB820) after oral administration, has the potential to treat second stage sleeping sickness. Two dosing regimens of DB844 were evaluated in two groups of vervet monkeys that were infected experimentally with trypanosomes. Treatment was initiated four weeks after infection, when the monkeys were in second stage sleeping sickness, as confirmed by the presence of trypanosomes in brain fluid. Orally administered DB844 was well absorbed, tolerated and resulted in a decrease of trypanosomes in both the blood and brain fluid. However, some monkeys relapsed after treatment, with an overall cure rate of approximately 40% in both study groups. For at least two days after last dosing, the active drug, DB820, achieved blood concentrations known to be at least 19 times more than the minimum concentration that has been shown to be effective against a stringent human infective trypanosome isolate (STIB 900). These results represent an advance in efforts to develop new related compounds as oral treatments for sleeping sickness.
PMCID: PMC3404106  PMID: 22848769
24.  Isothermal Microcalorimetry, a New Tool to Monitor Drug Action against Trypanosoma brucei and Plasmodium falciparum 
Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.
Author Summary
Microcalorimetry is a technology developed to record minute changes in temperature as a result of physical, chemical or biological reactions over time. The method has been applied to bacterial and eukaryotic cells and it was found that the metabolic activity of living cells in a culture medium produces enough heat flow to be measured. Protozoan parasites, some of which cause tropical diseases such as African sleeping sickness or malaria, are larger cells than bacteria and are metabolically very active. We explored the applicability of heat flow measurement to follow the growth of a parasite population and to study the effect of drugs. We first established optimal parameters for obtaining heat flow curves of a growing parasite culture. Then we added antiparasitic drugs at two concentrations and followed the heat flow curves over several days. Thus we could determine the time of onset of drug action and the time until all parasites stopped producing heat (time to kill). The microcalorimeter measurements once per second allowed a continuous monitoring of changes in the parasite population. This novel tool is accurate and simple to use, and will certainly prove to be of great value for the discovery and development of new drugs for protozoan parasites.
PMCID: PMC3367992  PMID: 22679520
25.  Antitrypanosomal Activity of Fexinidazole, a New Oral Nitroimidazole Drug Candidate for Treatment of Sleeping Sickness ▿  
Antimicrobial Agents and Chemotherapy  2011;55(12):5602-5608.
Fexinidazole is a 5-nitroimidazole drug currently in clinical development for the treatment of human sleeping sickness (human African trypanosomiasis [HAT]), caused by infection with species of the protozoan parasite Trypanosoma brucei. The compound and its two principal metabolites, sulfoxide and sulfone, have been assessed for their ability to kill a range of T. brucei parasite strains in vitro and to cure both acute and chronic HAT disease models in the mouse. The parent molecule and both metabolites have shown trypanocidal activity in vitro in the 0.7-to-3.3 μM (0.2-to-0.9 μg/ml) range against all parasite strains tested. In vivo, fexinidazole is orally effective in curing both acute and chronic diseases in the mouse at doses of 100 mg/kg of body weight/day for 4 days and 200 mg/kg/day for 5 days, respectively. Pharmacokinetic data indicate that it is likely that the sulfoxide and sulfone metabolites provide most, if not all, of the in vivo killing activity. Fexinidazole and its metabolites require up to 48 h exposure in order to induce maximal trypanocidal efficacy in vitro. The parent drug and its metabolites show no in vitro cross-reactivity in terms of trypanocidal activity with either themselves or other known trypanocidal drugs in use in humans. The in vitro and in vivo antitrypanosomal activities of fexinidazole and its two principal metabolites provide evidence that the compound has the potential to be an effective oral treatment for both the T. b. gambiense and T. b. rhodesiense forms of human sleeping sickness and both stages of the disease.
PMCID: PMC3232772  PMID: 21911566

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