Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti-Trypanosoma brucei Agents.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Synthesis, Anti-Proliferative Evaluation and Mechanism of 4-Trifluoro Methoxy Proguanil Derivatives with Various Carbon Chain Length.

Among the known biguanide drugs, proguanil has the best antiproliferative activity. In contrast, newly synthesized biguanide derivatives containing fluorine atoms have excellent biological activity, among which trifluoromethoxy compounds show the strongest ability. Preliminary work in our laboratory exhibited that n-heptyl containing proguanil derivatives on one alkyl chain side have better biological activity than those with a shorter carbon chain. However, the relationship between the length of the carbon chain and the activity of the compounds is unknown. In this study, we synthesized 10 new trifluoromethoxy-containing proguanil derivatives with various carbon chain lengths. The phenyl side is fixed as the trifluoromethoxy group with change of carbon chain length in alkyl chain side. It was found that the anti-cancer abilities of 5C-8C with n-pentyl to n-octyl groups was significantly better than that of proguanil in the five human cancer cell lines. The colony formation assay demonstrated that 6C-8C at 0.5 to 1.0 µM significantly inhibited the colony formation of human cancer cell lines, much stronger than that of proguanil. Pharmacologically, 8C activates AMPK, leading to inactivation of the mTOR/p70S6K/4EBP1 pathway. Thus, these novel compounds have a great potential for developing new anti-cancer candidates.

Biguanides: Species with versatile therapeutic applications.

Biguanides are compounds in which two guanidine moieties are fused to form a highly conjugated system. Biguanides are highly basic and hence they are available as salts mostly hydrochloride salts, these cationic species have been found to exhibit many therapeutic properties. This review covers the research and development carried out on biguanides and accounts the various therapeutic applications of drugs containing biguanide group-such as antimalarial, antidiabetic, antiviral, anticancer, antibacterial, antifungal, anti-tubercular, antifilarial, anti-HIV, as well as other biological activities. The aim of this review is to compile all the medicinal chemistry applications of this class of compounds so as to pave way for the accelerated efforts in finding the drug action mechanisms associated with this class of compounds. Importance has been given to the organic chemistry of these biguanide derivatives also.

Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening.

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 µM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.

Synthesis, biological evaluation and anti-proliferative mechanism of fluorine-containing proguanil derivatives.

Proguanil, a member of biguanide family, has excellent anti-proliferative activities. Fluorine-containing compounds have been demonstrated to have super biological activities including enhanced binding interactions, metabolic stability, and reduced toxicity. In this study, based on the intermediate derivatization methods, we synthesized 13 new fluorine-containing proguanil derivatives, and found that 7a,7d and 8e had much lower IC50 than proguanil in 5 human cancerous cell lines. The results of clonogenic and scratch wound healing assays revealed that the inhibitory effects of derivatives 7a,7d and 8e on proliferation and migration of human cancer cell lines were much better than proguanil as well. Mechanistic study based on representative derivative 7a indicated that this compound up-regulates AMPK signal pathway and downregulates mTOR/4EBP1/p70S6K. In conclusion, these new fluorine-containing derivatives show potential for the development of cancer chemotherapeutic drugs.

Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone.

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil's action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo.

Structural Insights into the Development of Cycloguanil Derivatives as Trypanosoma brucei Pteridine-Reductase-1 Inhibitors.

Cycloguanil is a known dihydrofolate-reductase (DHFR) inhibitor, but there is no evidence of its activity on pteridine reductase (PTR), the main metabolic bypass to DHFR inhibition in trypanosomatid parasites. Here, we provide experimental evidence of cycloguanil as an inhibitor of Trypanosoma brucei PTR1 (TbPTR1). A small library of cycloguanil derivatives was developed, resulting in 1 and 2a having IC50 values of 692 and 186 nM, respectively, toward TbPTR1. Structural analysis revealed that the increased potency of 1 and 2a is due to the combined contributions of hydrophobic interactions, H-bonds, and halogen bonds. Moreover, in vitro cell-growth-inhibition tests indicated that 2a is also effective on T. brucei. The simultaneous inhibition of DHFR and PTR1 activity in T. brucei is a promising new strategy for the treatment of human African trypanosomiasis. For this purpose, 1,6-dihydrotriazines represent new molecular tools to develop potent dual PTR and DHFR inhibitors.

Application of molecular docking and PSO-SVR intelligent approaches in antimalarial activity prediction of enantiomeric cycloguanil analogues.

A series of antifolate compounds, i.e. 1-(4-chlorophenyl)-6,6-dimethyl-1,3,5-triazine-2,4-diamine, or cycloguanil analogues, have shown effective inhibiting properties against Plasmodium falciparum dihydrofolate reductase (PfDHFR). In this work, the stereoselectivity of PfDHFR to the R and S enantiomer of cycloguanil analogues was obtained from molecular docking calculations and integrated into QSAR study to obtain a more accurate prediction model. Results indicate that PfDHFR can bind to cycloguanil analogues in the R and S enantiomers. Cycloguanil analogues with alkyl chain substituent prefer the R enantiomer over S because they do not experience steric hindrance with the Phe58 side chain, while cycloguanil analogues with phenol chain substituent prefer the S enantiomer over R because they do not experience steric hindrance with Leu46 and Met55 side chains. Particle swarm optimization and support vector regression were used to select relevant descriptors and generate the effective prediction model, with a high statistical significance level (r2training = 0.941; r2test = 0.884).

Biowaiver Monographs for Immediate Release Solid Oral Dosage Forms: Proguanil Hydrochloride.

Literature data relevant to the decision to waive in vivo bioequivalence testing for the approval of generic immediate release solid oral dosage forms of proguanil hydrochloride are reviewed. To elucidate the Biopharmaceutics Classification System (BCS) classification, experimental solubility and dissolution studies were also carried out. The antimalarial proguanil hydrochloride, effective via the parent compound proguanil and the metabolite cycloguanil, is not considered to be a narrow therapeutic index drug. Proguanil hydrochloride salt was shown to be highly soluble according to the U.S. Food and Drug Administration, World Health Organization, and European Medicines Agency guidelines, but data for permeability are inconclusive. Therefore, proguanil hydrochloride is conservatively classified as a BCS class 3 substance. In view of this information and the assessment of risks associated with a false positive decision, a BCS-based biowaiver approval procedure can be recommended for orally administered solid immediate release products containing proguanil hydrochloride, provided well-known excipients are used in usual amounts and provided the in vitro dissolution of the test and reference products is very rapid (85% or more are dissolved in 15 min at pH 1.2, 4.5, and 6.8) and is performed according to the current requirements for BCS-based biowaivers.

Atovaquone oral bioavailability enhancement using electrospraying technology.

Atovaquone in combination with proguanil hydrochloride, marketed as Malarone® tablets by GlaxoSmithKline (GSK), is prescribed for the treatment of malaria. High dose and poor bioavailability are the main hurdles associated with atovaquone oral therapy. The present study reports development of atovaquone nanoparticles, using in house designed and fabricated electrospraying equipment, and the assessment of bioavailability and therapeutic efficacy of the nanoparticles after oral administration. Solid nanoparticles of atovaquone were successfully produced by electrospraying and were characterized for particle size and flow properties. Differential Scanning Calorimetry, X-ray Diffraction, Fourier Transform Infrared Spectroscopy studies were also carried out. Atovaquone nanoparticles along with proguanil hydrochloride and a suitable wetting agent were filled in size 2 hard gelatin capsules. The formulation was compared with Malarone® tablets (GSK) and Mepron® suspension (GSK) in terms of in vitro release profile and in vivo pharmacokinetic studies. It showed 2.9-fold and 1.8-fold improved bioavailability in rats compared to Malarone® tablets and Mepron® suspension respectively. Therapeutic efficacy of the formulation was determined using modified Peter's 4-day suppressive tests and clinical simulation studies using Plasmodium berghei ANKA infected Swiss mice and compared to Malarone®. The developed formulation showed a 128-fold dose reduction in the modified Peter's 4-day suppressive tests and 32-fold dose reduction in clinical simulation studies. Given that only one capsule a day of developed formulation is required to be administered orally compared to 4 Malarone® tablets once a day and that too at a significantly reduced dose, this nanoparticle formulation will definitely reduce the side-effects of the treatment and is also likely to increase patient compliance.

Host dihydrofolate reductase (DHFR)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus.

We have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza A and B virus and respiratory syncytial virus (RSV) via targeting of the host dihydrofolate reductase (DHFR) enzyme. Most analogues proved active against influenza B virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (EC50 = 0.060 µM), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. Activity was also observed for two influenza A strains, including a virus with the S31N mutant form of M2 proton channel, which is the most prevalent resistance mutation for amantadine. Importantly, the compounds displayed nanomolar activity against RSV and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (EC50 ∼0.008 µM), far surpassing the potency and safety profile of the licensed drug ribavirin (EC50 = 5.8 µM, SI > 43).

Bioavailability enhancement of atovaquone using hot melt extrusion technology.

Emerging parasite resistance and poor oral bioavailability of anti-malarials are the two cardinal issues which hinder the clinical success of malaria chemotherapy. Atovaquone-Proguanil is a WHO approved fixed dose combination used to tackle the problem of emerging resistance. However, Atovaquone is a highly lipophilic drug having poor aqueous solubility (less than 0.2 µg/ml) thus reducing its oral bioavailability. The aim of the present investigation was to explore hot melt extrusion (HME) as a solvent-free technique to enhance solubility and oral bioavailability of Atovaquone and to develop an oral dosage form for Atovaquone-Proguanil combination. Solid dispersion of Atovaquone was successfully developed using HME. The solid dispersion was characterized for DSC, FTIR, XRD, SEM, and flow properties. It was filled in size 2 hard gelatin capsules. The formulation showed better release as compared to Malarone® tablets, and 3.2-fold and 4.6-fold higher bioavailability as compared to Malarone® tablets and Atovaquone respectively. The enhanced bioavailability also resulted in 100% anti-malarial activity in murine infection model at 1/8(th) therapeutic dose. Thus the developed methodology shows promising potential to solve the problems associated with Atovaquone therapy, namely its high cost and poor oral bioavailability, resulting in increased therapeutic efficacy of Atovaquone.

Importance of cytochromes in cyclization reactions: quantum chemical study on a model reaction of proguanil to cycloguanil.

Proguanil, an anti-malarial prodrug, undergoes cytochrome P450 catalyzed biotransformation to the pharmacologically active triazine metabolite (cycloguanil), which inhibits plasmodial dihydrofolate reductase. This cyclization is catalyzed by CYP2C19 and many anti-malarial lead compounds are being designed and synthesized to exploit this pathway. Quantum chemical calculations were performed using the model species (Cpd I for active species of cytochrome and N4-isopropyl-N6-methylbiguanide for proguanil) to elucidate the mechanism of the cyclization pathway. The overall reaction involves the loss of a water molecule, and is exothermic by approximately 55 kcal/mol, and involves a barrier of approximately 17 kcal/mol. The plausible reaction pathway involves the initial H-radical abstraction from the isopropyl group by Cpd I, followed by two alternative paths- (i) oxygen rebound to provide hydroxyl derivative and (ii) loss of additional H-radical to yield 1,3,5-triazatriene, which undergoes cyclization. This study helped in understanding the role of the active species of cytochromes in this important cyclization reaction.

Mycobacterium tuberculosis dihydrofolate reductase reveals two conformational states and a possible low affinity mechanism to antifolate drugs.

Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.

Quinolone-3-diarylethers: a new class of antimalarial drug.

The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite's life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite's mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

Delivery of atovaquone and proguanil across sublingual membranes, in vitro.

Malarone(™), a combination of atovaquone (AT) and proguanil (PR), is indicated for the prophylaxis and treatment of uncomplicated Plasmodium falciparum malaria. This study aimed to determine in vitro the feasibility of delivering the combination of AT and PR as a spray formulation via the sublingual route, using Franz diffusion cells incorporating porcine sublingual mucosa. Firstly, 1 mg mL(-1) of each drug in 20% 1,8-Cineole in ethanol was used; and secondly, 5 mg mL(-1) AT and 1 mg mL(-1) PR in 20% 1-methyl-2-pyrrolidone in ethanol was examined, dosed every 2 h over a 12-h period and receptor phase samples were analyzed by HPLC. From the first study, mean fluxes for AT and PR were 12.89 ± 1.2 and 5.88 ± 0.9 µg cm(-2) h(-1) respectively; pharmacokinetic calculations indicated that these fluxes were insufficient to achieve the target plasma concentrations for AT and PR of 1.4 µg mL(-1) and 200 ng mL(-1) respectively, in the treatment of falciparum malaria. However, in the second study, the fluxes of AT and PR increased to 50.92 ± 20.8 and 12.01 ± 1.5 µg cm(-2) h(-1) respectively, and pharmacokinetic calculations indicated that therapeutic plasma concentrations are attainable for pediatric application.

Exploring QSAR, pharmacophore mapping and docking studies and virtual library generation for cycloguanil derivatives as PfDHFR-TS inhibitors.

Resistance of available antimalarial drugs against Plasmodium species is one of the major problems of malaria control in the developing world. In the present study, we have performed QSAR, pharmacophore mapping and molecular docking studies of cycloguanil derivatives as Plasmodium falciparum dihydrofolate reductase thymidylate synthase (PfDHFR-TS) inhibitors to explore essential features required for the antimalarial activity and important interaction patterns between the enzyme and ligands for the design of new potent PfDHFR-TS inhibitors. The QSAR studies have been carried out using topological parameters along with thermodynamic and structural descriptors. Acceptable values of internal and external validation parameters for the developed QSAR models confirm acceptability of the models. Pharmacophore mapping revealed that two hydrogen bond donor (HBD) features and a hydrophobic feature (HYD) are important parameters for PfDHFR-TS inhibitory activity. The docking studies suggest that the PfDHFR-TS inhibitors interact with Asp54, Ile14, Ile164, ser108, Ser111, Tyr170, Met55, Ala16, Thr185, Leu46, Cys15, Phe58, Ile112, Trp48, Tyr57 and Leu119 amino acid residues. The QSAR, pharmacophore and docking studies inferred that i) branching of the substituents at R1 and R2 positions should be less (small alkyl chain substituents are favored); ii) the electronegativity of the molecules should be high but within some limit; iii) the size and volume of the molecules should be high; iv) molecules should be flexible enough; v) R configuration at C6 position of the triazine ring favors the inhibitory binding affinity; vi) the substituents of the phenyl ring at 3, 4 and 5 position of the phenyl ring should be small hydrophobic groups. Based on these studies, we have designed a library of cycloguanil derivatives with good in silico predicted PfDHFR-TS inhibitory activity.

Formation and identification of a degradant in chlorproguanil-dapsone-artesunate (Dacart™) tablets.

Chlorproguanil hydrochloride, dapsone and artesunate are three compounds with anti-malarial properties developed as a triple combination drug product (Dacart™) for the treatment of malarial infections. During long-term stability studies, a degradant was observed which increased with time and had the potential to limit the shelf-life of the product. Through a combination of HPLC and spectroscopic analyses, the structure of the degradant was identified to be an adduct of a fragment of artesunate with dapsone. The response factor was determined to allow an accurate assessment of its levels in drug product. The likely mechanism for its formation is postulated to be via the water-mediated degradation of artesunate to give succinic acid followed by reaction of the liberated succinic acid with dapsone. The formation of this degradant demonstrates a potential stability risk for future combination therapies incorporating artesunate. These risks are particularly pertinent to products of this type given the climatic conditions which prevail in countries where such therapies are likely to be employed.

Quantitative structure-activity relationships for cycloguanil analogs as PfDHFR inhibitors using mathematical molecular descriptors.

Computed molecular descriptors were used to develop quantitative structure-activity relationships (QSARs) for binding affinities (K(i)) for a set of 58 cycloguanil (2,4-diamino-1,6-dihydro-1,3,5-triazine) analogues for dihydrofolate reductase (DHFR) enzyme extracted from wild and A16V+S108T mutant type (a double mutation) malaria parasite Plasmodium falciparum (Pf). High-quality models were obtained in both cases. The results of statistical analyses show that ridge regression (RR) outperformed the two other modelling methods, principal component regression (PCR) and partial least squares (PLS). For both enzymes, recognition of the inhibitors was based on four broad categories of descriptors encoding information on: (1) the electronic character of the various atoms in the molecule, (2) the size and shape of the structure, (3) the degree of branching in the molecular skeleton, and (4) two to five atom molecular fragments with aliphatic carbon at one end and aliphatic or aromatic carbon or nitrogen at the other end. The subsets of influential descriptors underlying the QSARs for the wild versus the mutant DHFR are quite non-overlapping. This indicates that the two enzymes recognize the inhibitor molecules on the basis of mutually distinct structural attributes. Such differential QSARs can be useful in the design of novel drugs active against malaria parasites which are growing in resistant to existing chemotherapeutic agents.

3D-QSAR analysis of cycloguanil derivatives as inhibitors of A16V + S108T mutant Plasmodium falciparum dihydrofolate reductase enzyme.

A three-dimensional quantitative structure-activity relationship (3D-QSAR) study was carried out on cycloguanil derivatives which are reported as growth inhibitors of Plasmodium falciparum clone (T9/94 RC17) which harbors A16V+S108T mutant dihydrofolate reductase (DHFR) enzyme. Comparative of Molecular Field Analysis (CoMFA) and Comparative of Molecular Similarity Indices Analysis (CoMSIA) were carried out to investigate the structural requirements for the activities of these compounds and to derive predictive models that may be used for designing novel PfDHFR enzyme inhibitors. The global minimum energy (within the search space) conformation of the most active compound (38) was obtained by using simulated annealing, and was subsequently used as a template to build the structures of the rest molecules used in the study. The CoMFA model gave statistically significant results with r(cv)(2) and r(ncv)(2) values of 0.654 and 0.951, respectively. The combination of steric, electrostatic and hydrophobic fields resulted in the best CoMSIA model with r(cv)(2) and r(ncv)(2) values of 0.669 and 0.907, respectively. The predictive abilities of the CoMFA and CoMSIA models were also evaluated using test compounds which gave r(pred)(2) values of 0.735 and 0.557, respectively. The results of bootstrapping analyses also confirmed that the generated models are robust and reliable. The models were graphically interpreted using CoMFA and CoMSIA contour plots. The structural regions responsible for the differences in anti-plasmodial activities were analyzed with respect to their electrostatic, steric and hydrophobic nature. The results obtained from this study could be used for rational design of potent inhibitors against A16V+S108T mutant PfDHFR enzyme.