Assessment of a pretomanid analogue library for African trypanosomiasis: Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides.

A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.

Alkanediamide-Linked Bisbenzamidines Are Promising Antiparasitic Agents.

A series of 15 alkanediamide-linked bisbenzamidines and related analogs was synthesized and tested in vitro against two Trypanosoma brucei (T.b.) subspecies: T.b. brucei and T.b. rhodesiense, Trypanosoma cruzi, Leishmania donovani and two Plasmodium falciparum subspecies: a chloroquine-sensitive strain (NF54) and a chloroquine-resistant strain (K1). The in vitro cytotoxicity was determined against rat myoblast cells (L6). Seven compounds (5, 6, 10, 11, 12, 14, 15) showed high potency against both strains of T. brucei and P. falciparum with the inhibitory concentrations for 50% (IC50) in the nanomolar range (IC50 = 1-96 nM). None of the tested derivatives was significantly active against T. cruzi or L. donovani. Three of the more potent compounds (5, 6, 11) were evaluated in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant (KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds 5 and 6 were highly effective in curing mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538, but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies indicate AT-rich DNA sequences as possible binding sites for these compounds. Several of the tested compounds are suitable leads for the development of improved antiparasitic agents.

Dibenzosuberyl substituted polyamines and analogs of clomipramine as effective inhibitors of trypanothione reductase; molecular docking, and assessment of trypanocidal activities.

Trypanothione reductase (TR) is an enzyme critical to the maintenance of the thiol redox balance in trypanosomatids, including the genera Trypanosoma and Leishmania that are parasites responsible for several serious diseases. Analogs of clomipramine were prepared since clomipramine is reported to inhibit TR and cure mice infected with trypanosomes, however its psychotropic activity precludes its use as an anti-trypanosomal therapeutic. The clomipramine analogs contained a tricyclic dibenzosuberyl moiety. Additionally a series of polyamines with N-dibenzosuberyl substituents were prepared. All compounds studied were competitive inhibitors of TR and showed trypanocidal activities against Trypanosoma brucei in vitro. The analogs of clomipramine were poor inhibitors of TR, whereas the polyamine derivatives were effective TR inhibitors with the most potent compound, N(4),N(8)-bis(dibenzosuberyl)spermine (7), having a Ki value of 0.26µM. However, compound (7) did not prolong the lives of mice infected with trypanosomes. Analysis of docking studies indicated: the tricyclic groups of inhibitors bind at four distinct hydrophobic regions in the active site of TR; the importance of the chlorine substituent of clomipramine in binding to TR; and binding of the dibenzosuberyl groups of (7) occur at separate and distinct hydrophobic regions within the active site of TR.

Benzoxaboroles: a new class of potential drugs for human African trypanosomiasis.

Human African trypanosomiasis, caused by the kinetoplastid parasite Trypanosoma brucei, affects thousands of people across sub-Saharan Africa, and is fatal if left untreated. Treatment options for this disease, particularly stage 2 disease, which occurs after parasites have infected brain tissue, are limited due to inadequate efficacy, toxicity and the complexity of treatment regimens. We have discovered and optimized a series of benzoxaborole-6-carboxamides to provide trypanocidal compounds that are orally active in murine models of human African trypanosomiasis. A key feature of this series is the presence of a boron atom in the heterocyclic core structure, which is essential to the observed trypanocidal activity. We also report the in vivo pharmacokinetic properties of lead compounds from the series and selection of SCYX-7158 as a preclinical candidate.

SCYX-7158, an orally-active benzoxaborole for the treatment of stage 2 human African trypanosomiasis.

BACKGROUND: Human African trypanosomiasis (HAT) is an important public health problem in sub-Saharan Africa, affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. We have discovered and optimized a novel class of small-molecule boron-containing compounds, benzoxaboroles, to identify SCYX-7158 as an effective, safe and orally active treatment for HAT. METHODOLOGY/PRINCIPAL FINDINGS: A drug discovery project employing integrated biological screening, medicinal chemistry and pharmacokinetic characterization identified SCYX-7158 as an optimized analog, as it is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense, is efficacious in both stage 1 and stage 2 murine HAT models and has physicochemical and in vitro absorption, distribution, metabolism, elimination and toxicology (ADMET) properties consistent with the compound being orally available, metabolically stable and CNS permeable. In a murine stage 2 study, SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD×7 days). In vivo pharmacokinetic characterization of SCYX-7158 demonstrates that the compound is highly bioavailable in rodents and non-human primates, has low intravenous plasma clearance and has a 24-h elimination half-life and a volume of distribution that indicate good tissue distribution. Most importantly, in rodents brain exposure of SCYX-7158 is high, with C(max) >10 µg/mL and AUC(0-24 hr) >100 µg*h/mL following a 25 mg/kg oral dose. Furthermore, SCYX-7158 readily distributes into cerebrospinal fluid to achieve therapeutically relevant concentrations in this compartment. CONCLUSIONS/SIGNIFICANCE: The biological and pharmacokinetic properties of SCYX-7158 suggest that this compound will be efficacious and safe to treat stage 2 HAT. SCYX-7158 has been selected to enter preclinical studies, with expected progression to phase 1 clinical trials in 2011.

SAR of 2-amino and 2,4-diamino pyrimidines with in vivo efficacy against Trypanosoma brucei.

A series of 2,4-diaminopyrimidines was investigated and compounds were found to have in vivo efficacy against Trypanosoma brucei in an acute mouse model. However, in vitro permeability data suggested the 2,4-diaminopyrimidenes would have poor permeability through the blood brain barrier. Consequently a series of 4-desamino analogs were synthesized and found to have improved in vitro permeability.

Discovery of novel benzoxaborole-based potent antitrypanosomal agents.

We report the discovery of benzoxaborole antitrypanosomal agents and their structure-activity relationships on central linkage groups and different substitution patterns in the sulfur-linked series. The compounds showed in vitro growth inhibition IC50 values as low as 0.02 µg/mL and in vivo efficacy in acute murine infection models against Tryapnosoma brucei.

Synthesis and SAR of alkanediamide-linked bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity.

A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystiscarinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibitors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were the most potent compounds against P. carinii. N,N'-Bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4 displayed potent inhibition (IC50=2-3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the amidine groups in 4 were moved from the para to the meta positions or replaced with amides.

Chemotherapy of human african trypanosomiasis.

Human Africa trypanosomiasis is a centuries-old disease which has disrupted sub-Saharan Africa in both physical suffering and economic loss. This article presents an update of classic chemotherapeutic agents, in use for >50 years and the recent development of promising non-toxic combination chemotherapy suitable for use in rural clinics.

Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor.

Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

Novel S-adenosylmethionine decarboxylase inhibitors for the treatment of human African trypanosomiasis.

Trypanosomiasis remains a significant disease across the sub-Saharan African continent, with 50,000 to 70,000 individuals infected. The utility of current therapies is limited by issues of toxicity and the need to administer compounds intravenously. We have begun a program to pursue lead optimization around MDL 73811, an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMetDC). This compound is potent but in previous studies cleared rapidly from the blood of rats (T. L. Byers, T. L. Bush, P. P. McCann, and A. J. Bitonti, Biochem. J. 274:527-533). One of the analogs synthesized (Genz-644131) was shown to be highly active against Trypanosoma brucei rhodesiense in vitro (50% inhibitory concentration, 400 pg/ml). Enzyme kinetic studies showed Genz-644131 to be approximately fivefold more potent than MDL 73811 against the T. brucei brucei AdoMetDC-prozyme complex. This compound was stable in vitro in rat and human liver microsomal and hepatocyte assays, was stable in rat whole-blood assays, did not significantly inhibit human cytochrome P450 enzymes, had no measurable efflux in CaCo-2 cells, and was only 41% bound by serum proteins. Pharmacokinetic studies of mice following intraperitoneal dosing showed that the half-life of Genz-644131 was threefold greater than that of MDL 73811 (7.4 h versus 2.5 h). Furthermore, brain penetration of Genz-644131 was 4.3-fold higher than that of MDL 73811. Finally, in vivo efficacy studies of T. b. brucei strain STIB 795-infected mice showed that Genz-644131 significantly extended survival (from 6.75 days for controls to >30 days for treated animals) and cured animals infected with T. b. brucei strain LAB 110 EATRO. Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration.

Metabolism of an alkyl polyamine analog by a polyamine oxidase from the microsporidian Encephalitozoon cuniculi.

Encephalitozoon cuniculi is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{N-[o-(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an E. cuniculi amine oxidase activity. The primary natural substrate for this oxidase activity was N'-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and K(m) of 2 microM. In the presence of N'-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of (13)C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of (13)C-labeled BW-1 to E. cuniculi preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with E. cuniculi. The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.

Novel trypanocidal analogs of 5'-(methylthio)-adenosine.

The purine nucleoside 5'-deoxy-5'-(hydroxyethylthio)-adenosine (HETA) is an analog of the polyamine pathway metabolite 5'-deoxy-5'-(methylthio)-adenosine (MTA). HETA is a lead structure for the ongoing development of selectively targeted trypanocidal agents. Thirteen novel HETA analogs were synthesized and examined for their in vitro trypanocidal activities against bloodstream forms of Trypanosoma brucei brucei LAB 110 EATRO and at least one drug-resistant Trypanosoma brucei rhodesiense clinical isolate. New compounds were also assessed in a cell-free assay for their activities as substrates of trypanosome MTA phosphorylase. The most potent analog in this group was 5'-deoxy-5'-(hydroxyethylthio)-tubercidin, whose in vitro cytotoxicity (50% inhibitory concentration [IC50], 10 nM) is 45 times greater than that of HETA (IC50, 450 nM) against pentamidine-resistant clinical isolate KETRI 269. Structure-activity analyses indicate that the enzymatic cleavage of HETA analogs by trypanosome MTA phosphorylase is not an absolute requirement for trypanocidal activity. This suggests that additional biochemical mechanisms are associated with the trypanocidal effects of HETA and its analogs.

Trypanocidal activity of piperazine-linked bisbenzamidines and bisbenzamidoxime, an orally active prodrug.

A series of 32 piperazine-linked bisbenzamidines (and related analogues) were analysed for their in vitro and in vivo trypanocidal activity against a drug-sensitive strain of Trypanosoma brucei brucei and a drug-resistant strain of Trypanosoma brucei rhodesiense. The compounds showed similar potencies against both strains. The most potent compounds were bisbenzamidines substituted at the amidinium nitrogens with a linear pentyl group (8, inhibitory concentration for 50% (IC(50))=1.7-3.0 nM) or cyclic octyl group (17, IC(50)=2.3-4.6 nM). Replacement of the diamidine groups with diamidoxime groups resulted in a prodrug (22) that was effective orally against T. b. brucei-infected mice. Three compounds (7, 11 and 15) provided 100% cure when administered parenterally. The results indicate that the nature of the substituents at the amidinium nitrogens of bisbenzamidines strongly influence their trypanocidal activity.

Novel alkylpolyaminoguanidines and alkylpolyaminobiguanides with potent antitrypanosomal activity.

A series of polyaminoguanidines and polyaminobiguanides were synthesized and evaluated as potential antitrypanosomal agents. These analogues inhibit trypanothione reductase (TR) with IC50 values as low as 0.95 microM, but do not inhibit the closely related human enzyme glutathione reductase (GR). The most effective analogues, 7a, 7b and 8d, inhibited parasitic growth in vitro with IC50 values of 0.18, 0.09 and 0.18 microM, respectively. These agents represent a promising new class of potential antitrypanosomal agents.

Multiple triclosan targets in Trypanosoma brucei.

Trypanosoma brucei genes encoding putative fatty acid synthesis enzymes are homologous to those encoding type II enzymes found in bacteria and organelles such as chloroplasts and mitochondria. It was therefore not surprising that triclosan, an inhibitor of type II enoyl-acyl carrier protein (enoyl-ACP) reductase, killed both procyclic forms and bloodstream forms of T. brucei in culture with 50% effective concentrations (EC(50)s) of 10 and 13 microM, respectively. Triclosan also inhibited cell-free fatty acid synthesis, though much higher concentrations were required (EC(50)s of 100 to 200 microM). Unexpectedly, 100 microM triclosan did not affect the elongation of [(3)H]laurate (C(12:0)) to myristate (C(14:0)) in cultured bloodstream form parasites, suggesting that triclosan killing of trypanosomes may not be through specific inhibition of enoyl-ACP reductase but through some other mechanism. Interestingly, 100 microM triclosan did reduce the level of incorporation of [(3)H]myristate into glycosyl phosphatidylinositol species (GPIs). Furthermore, we found that triclosan inhibited fatty acid remodeling in a cell-free assay in the same concentration range required for killing T. brucei in culture. In addition, we found that a similar concentration of triclosan also inhibited the myristate exchange pathway, which resides in a distinct subcellular compartment. However, GPI myristoylation and myristate exchange are specific to the bloodstream form parasite, yet triclosan kills both the bloodstream and procyclic forms. Therefore, triclosan killing may be due to a nonspecific perturbation of subcellular membrane structure leading to dysfunction in sensitive membrane-resident biochemical pathways.

Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues.

The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials.

Synthesis and evaluation of analogues of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine as inhibitors of tumor cell growth, trypanosomal growth, and HIV-1 infectivity.

A well-defined series of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine analogues was designed and synthesized in order to further ascertain the optimal structural requirements for S-adenosylmethionine decarboxylase inhibition and potentially to augment and perhaps separate their antiproliferative and antitrypanosomal activities. Most structural modifications had a deleterious affect on both the antitrypanosomal and antineoplastic activity of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine. However, di-O-acetylation of the parent compound produced a potential prodrug that caused markedly pronounced inhibition of trypanosomal and neoplastic cell growth and viability. Moreover, the acetylated derivative of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine did inhibit HIV-1 growth and infectivity, whereas the parent compound did not.

Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin.

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors.

Novel synthetic polyamines are effective in the treatment of experimental microsporidiosis, an opportunistic AIDS-associated infection.

Microsporidia are eukaryotic obligate intracellular protists that are emerging pathogens in immunocompromised hosts, such as patients with AIDS or patients who have undergone organ transplantation. We have demonstrated in vitro and in vivo that synthetic polyamine analogs are effective antimicrosporidial agents with a broad therapeutic window. CD8-knockout mice or nude mice infected with the microsporidian Encephalitozoon cuniculi were cured when they were treated with four different novel polyamine analogs at doses ranging from 1.25 to 5 mg/kg of body weight/day for a total of 10 days. Cured animals demonstrated no evidence of parasitemia by either PCR or histologic staining of tissues 30 days after untreated control animals died.