Scaffold hybridization strategy towards potent hydroxamate-based inhibitors of Flaviviridae viruses and Trypanosoma species

Abstract

Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats. Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while dengue has no clinically approved therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern. Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid scaffold attached by an acetohydroxamic acid group (CH₂CONHOH), aiming at derivatives with dual activity. The novel spiro-carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC₅₀ values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group.

Fig. 1. Representative chemical structures of clinically approved DAAs and their respective EC₅₀s against HCV genotype 1b.

Fig. 2. Currently available marketed drugs for treatment of HAT and Chagas disease. Highlighted regions are reported to play a major role in the mechanism of action. Chemical structures of potential drug candidates fexinidazole and acoziborole.

Fig. 3. Indole–flutimide analogue (I) with antiviral activity, cycloheptane spiro-substituted 2,6-DKP analogue (II) with trypanocidal activity and novel synthesized analogues reported in this study. Metal binding groups are indicated in plum elliptical shape.

Scheme 1. A) Starting ketones B) synthesis of the target acetohydroxamic acid analogues 16–18, 16a–18a and 16b–18b. Reagents and conditions: (a) method A: NaCN (2.27 eq.), (NH₄)₂CO₃ (6 eq.), EtOH/H₂O (1.1 : 1), reflux, 8 h–11 days; | method B: KCN (1.3 eq.), (NH₄)₂CO₃ (5 eq.), EtOH/H₂O (1 : 3), MW (100 W), 120 °C, 25 min–1 h; (b) (i) [(CH₃)₃Si]₂NK (1.02 eq.), dry THF (0–5 °C) and then r.t., 20 min–1 h, argon; (ii) BrCH₂COOCH₂Ph (1.05 eq.), dry DMF, 35–38 °C, 48 h, argon; (c) (i) NaH (1.2 eq.), dry DMF, (0–5 °C) and then r.t., 15 min, argon; (ii) PhCH₂Br (1.2 eq.), 60 °C, 7 days, argon; (d) H₂, Pd/C 10%, EtOH/AcOEt 3 : 1, 50–55 psi, 40–45 °C, 3 h; (e) method A: EDCI·HCl (1.2 eq.), HOBt (1.2 eq.), PhCH₂ONH₂·HCl (1.2 eq.), TEA (5.8 eq.), dry CH₂Cl₂/dry DMF, 30–35 °C, 40 h, argon; | method B: (i) CDI (1.2 eq.), dry THF, 28–30 °C, 1 h, argon; (ii) PhCH₂ONH₂·HCl (1.2 eq.), TEA (1.82 eq.), 28–30 °C, 24 h, argon; (f) H₂, Pd/C 10%, EtOH/AcOEt 3 : 1, 50–55 psi, 40–45 °C, 3 h. C) Methyl esters obtained because of unexpected transesterification reaction (pink frames).

Fig. 4. A) Screening of compounds against HCV genotype 1b replication. Huh5-2 cells, harboring HCV genotype 1b (Con1) subgenomic replicon, were seeded at 30% confluency and treated for 72 h with the compounds at 3 μM. B) Activity of compound 18, against HCV genotype 1b, 3a and 4a RNA replication, using dose–response curve analysis. Huh5-2, Huh7.5-3a and Huh7.5-4a cells, harboring subgenomic replicons of HCV genotype 1b (Con1), 3a (S52) or 4a (ED43), respectively, were seeded at 30% confluency and treated for 72 h with serial dilutions of 18. In both A and B, as a marker of viral RNA replication, firefly luciferase activity was determined and calculated as relative light units (RLU) per μg of total protein. Values obtained with compound-treated cells were expressed as percentage of those obtained with cells treated with the solvent DMSO (control). Bars represent mean values obtained from three separate experiments in triplicate. Error bars represent standard deviation (SD).

Fig. 5. Inhibitory effect of compounds on HCV RNA levels and protein expression in a subgenomic HCV Con1 replicon assay. A) Quantification of (+) strand HCV RNA by RT-qPCR in Huh7.5-4a replicon cells treated with serial dilutions of compounds 17a, 18 or the solvent DMSO. Values from compound-treated cells are expressed as percentage of those obtained with cells that received the solvent (control). mRNA levels of the housekeeping gene YWHAZ were used for normalization. B) Indirect immunofluorescence for NS5A (left panels) in Huh7.5-4a replicon cells, treated with serial dilutions of compound 18 or the solvent DMSO. Nuclei were stained with propidium iodide (PI; middle panels). Merged images are shown at the right. Bar, 50 μm.