Structure-Based Discovery and Development of Highly Potent Dihydroorotate Dehydrogenase Inhibitors for Malaria Chemoprevention

Abstract

Malaria remains a serious global health challenge, yet treatment and control programs are threatened by drug resistance. Dihydroorotate dehydrogenase (DHODH) was clinically validated as a target for treatment and prevention of malaria through human studies with DSM265, but currently no drugs against this target are in clinical use. We used structure-based computational tools including free energy perturbation (FEP+) to discover highly ligand efficient, potent, and selective pyrazole-based Plasmodium DHODH inhibitors through a scaffold hop from a pyrrole-based series. Optimized pyrazole-based compounds were identified with low nM-to-pM Plasmodium falciparum cell potency and oral activity in a humanized SCID mouse malaria infection model. The lead compound DSM1465 is more potent and has improved absorption, distribution, metabolism and excretion/pharmacokinetic (ADME/PK) properties compared to DSM265 that support the potential for once-monthly chemoprevention at a low dose. This compound meets the objective of identifying compounds with potential to be used for monthly chemoprevention in Africa to support malaria elimination efforts.

Figure 1

Previously described P. falciparum DHODH inhibitors.

Figure 2

Stepwise design of pyrazolopyridinone series from the previously reported pyrrole-based PfDHODH inhibitor series.

Figure 3

Cocrystal structures of inhibitors in complex with PfDHODH. Panels display select residues within the 4 Å shell of the inhibitor in the binding sites. Key H-bonds are shown by dashed lines with distances in Å. (A) Binding mode comparison of pyrazoles 4 (tan) and S3 (green) aligned to the structure of pyrrole 2 (pink; pdb 7KZ4²⁷). (B) Comparison of 13 (raspberry; FMN in yellow) with 4 (tan). (C) Comparison of 40 (blue-gray) to 65 (DHODH amino acid carbons in teal; 65 in magenta; FMN in yellow). (D) Rotated view of the 65 binding site showing bridge NH₂ interactions with V532 and L531 backbone carbonyls. Colors as defined in C. Refinement statistics are presented in Table S2.

Figure 4

Structures of late-lead compounds.

Figure 5

(A) Correlation between FEP+ predicted and measured PfDHODH pIC₅₀ values. Mean unsigned error = 0.55 pIC₅₀. (B) Correlation between PfDHODH IC₅₀ and Pf3D7 EC₅₀ across the three subseries. Linear regression R² = 0.79. All data are taken from Tables 1–9 and supplemental Tables S1 and S3.

Figure 6

Representative compounds for metabolite identification studies following incubation with either hLM (S8, Table S3; 46, Table 7) or hCH (45 and 47, Tables 6 and 7; S9 and S10, Table S3). Each of the top row compounds underwent N-dealkylation (blue shaded) forming a product with −28 amu. S8 also showed evidence of oxidation of the bridge alcohol to a ketone (−2 amu, green shaded) and a single hydroxylation product (+16 amu) which was not confirmed but suspected to be on the pyridinone methyl (orange shaded). No metabolites could be detected for 46 and 47 under the incubation conditions utilized.

Figure 7

Plasma concentration versus time profiles for selected compounds following administration to male Sprague–Dawley rats. (A) IV dosing (1 mg/kg) with a 10 min infusion. (B) PO dosing (3 mg/kg) by gavage of a suspension formulation. Data represent the mean ± SD (n = 3 rats). (C) Unbound volume of distribution and (D) unbound intrinsic clearance in rats following IV administration (1 mg/kg) as a function of Log D_7.4. Unbound CL_int was calculated using eq 3 (Methods section). Late lead compounds are shown as red symbols where numbers represent compound numbers. Data for C and D are shown in Table S6.

Figure 8

In vivo efficacy of 82 in the PfalcHuMouse model. (A) Parasitemia versus day and dose. The graph shows the % infected human erythrocytes versus time from treatment initiation (Day 1). Mice were dosed with vehicle (n = 2 mice), chloroquine (CQ, n = 1 mouse), or 82 at 4 dose levels (1, 3, 10, and 30 mg/kg) (n = 1 mouse per dose level). Only the parasite clearance phase of the study is shown. Recrudescence of parasitemia occurred as follows: CQ (day 8); 82 1 mg/kg (day 17), 10 mg/kg (>19 days), 30 mg/kg (day 22), and 3 mg/kg (>60 days, recrudescence not observed). (B) PK data after the day 1 and day 6 doses showing blood levels of 82. Trough concentrations at 0, 72, and 120 h were determined prior to the next dose. Days of treatment are indicated by black (82) or orange (CQ) arrows. The human predicted plasma MIC (Table 12) was converted to a micromolar blood concentration assuming B/P = 0.68, which was measured in wild-type mice. LQ, limit of quantitation of parasitemia.

Scheme 1. Synthesis of Compounds 5–11

Reagents and conditions: (a) 4,4′-Ditert-butyl-2,2′-dipyridyl, (1,5-cyclooctadiene)(methoxy) iridium(l) dimer, pinacolborane, tetrahydrofuran (THF), pentane, rt, 24 h; (b) 5-{Bromomethyl)-2-(trifluoromethyl) pyridine, Pd(dppf)Cl₂, N,N-diisopropylethylamine (DIPEA), toluene/xylene, water, 130 °C, 20–30 min, MW; (c) LiOH·H₂O, EtOH:H₂O, rt, 2 h; {d) 1,1-Diethoxy-3-(alkylamino)propan-2-one, HATU, Et₃N, CH₂Cl₂, 3 h; (e) p-Toluene sulfonic acid, toluene, 125 °C, 2 h.

Scheme 2. Synthesis of Compounds 12–17

Reagenls and conditions: (a) N-Bromosuccinimide (NBS), CH₃CN, rt, 2 h; (b) Methyl boronic acid, Cs₂CO₃,1,4-dioxane, rt, Pd(dppf)Cl₂; (c) Cyclopropylboronic acid, Cs₂CO₃, Pd(dppf)Cl₂-dichloromethane (DCM), dioxane/water, 110 °C, 2 h, MW; (d) N-Chlorosuccinimide (NCS), CH₃CN, rt, 16 h.

Scheme 3. Synthesis of Compounds 21–23

Reagents and conditions: (a) 5-(Bromomethyl)-2-chloropyridine, Pd(dppf)Cl₂, DIPEA, toluene/xylene, water, 130 °C, 20–30 min, MW; (b) LiOH·H₂O, EtOH:H₂O, rt, 2 h; (c) 2,2-Diethoxy-N-ethylethanamine, HATU, Et₃N, CH₂Cl₂, 3 h; (d) p-toluene sulfonic acid, toluene, 125 °C, 2 h; (e) NCS, CH₃CN, rt, 16 h; (f) piperidine/1-methylpiperazine/Morpholine, sodium tert-butoxide, XPhos, Pd₂(dba)₃, 1,4-dioxane, 90 °C, 30 min, MW.

Scheme 4. Synthesis of Compounds 26–31

Reagents and conditions: (a) POBr₃, CH₃CN, 80 °C; (b) LiOH-H₂0, THF, H₂0, rt; (c) HATU, Et₃N, CH₂Cl₂, rt; (d) Trifluoroacetic acid (TFA), CH₃CN, 80 °C; (e) Pd(dppf)Cl₂, Et₃N, CO (100 psi), MeOH, 100 °C; (f) LiBH₄, THF; (g) NBS, CH₃CN; (h) Potassium cyclopropyltrifluoroborate, Pd(OAc)₂, RuPhos, K₂C0₃, toluene-water, 100 °C; (i) MsCI, Et₃N, CH₂Cl₂, 0 °C; (j) Alkylamines, Et₃N, CH₂Cl₂, rt.

Scheme 5. Synthesis of Compounds 32–40

Reagents and conditions: (a) iPrMgCl·LiCl, THF, 0 °C, 15 min; then N-methoxy-N-methyl-6-(trifluoromethyl)nicotinamide or 5-chloro-N-methoxy-N-methyl-6-(trifluorometriyl)nicotinamide, THF, 30 min; (b) NaBH₄, MeOH, 0 °C to rt, 2 h; (c) NBS, CH₃CN, rt, 2 h; (d) Pd(PPh₃)₄, (MeBO)₃, K₂C0₃, dioxane/H₂0, 110 °C, 3–16 h; (e) Pd(OAc)₂, RuPhos, cPrBF₃K, K₂C0₃, toluene/H₂0, 110 °C, 12–16 h; (f) SOCl₂, CH₂Cl₂, 0 °C to rt, 1–2 h; (g) NH₃, MeOH, 70 °C, 6–16 h.

Scheme 6. Synthesis of Compounds 41–45

Reagents and conditions: (a) R₁MgBr, THF, −5–0 °C, 30 min.

Scheme 7. Synthesis of Compounds 46–56

Reagents and conditions: (a) N-(2,2-Diethoxyethyl)cyclopropanamine, HATU, Et₃N, CH₂Cl₂, 3–12 h; (b) TFA, CH₃CN, 80 °C, 16 h; (c) iPrMgCl·LiCl, THF, 0 °C, 15 min; then N-methoxy-N-methyl-6-(trifluoromethyl)nicotinamide or 5-chloro-N-methoxy-N-methyl-6-(trifluoromethyl)nicotinamide, THF, 30 min; (d) R₁MgBr, THF, −5–0 °C, 30 min; (e) iPrMgCl·LiCl, THF, 0 °C, 15 min; then aryl aldehyde, THF, 30 min; (f) CuCN, DMF, 140–150 °C, 10–14 h; (g) MnO₂, CH₂Cl₂, rt, 12–16 h.

Scheme 8. Synthesis of A. Pyrazolopyridazinone and B. Pyrazolopyrimidinone Key Intermediates

Reagents and conditions: (a) POBr₃, DMF, DCE, 90 °C, 16 h; (b) Cyclopropylhydrazine hydrochloride, EtOH, 100 °C, 3 h; (c) iPrMg·LiCI, THF, 0 °C, 15 min; then 5-Chloro-N-methoxy-N-methyl-6-(trifluoromethyl)nicotinamide, THF, 30 min; (d) iPrMg·LiCl, THF, 0 °C, 15 min; then 5-Chloro-6-trifluoromethylbenzaldehyde, THF, 30 min. (e) Mn0₂, CH₂Cl₂, rt, 12–16 h; (f) Zn, NH₄Cl, THF/H₂0, rt, 4 h; (g) Formamidine acetate, DIPEA, nBuOH, 110 °C, 1 h; (h) Br₂, AcOH, 95 °C, 16 h; (i) Cyclopropylboronic acid, Cu(OAc)₂, Na₂CO₃, pyridine, 1,4-dioxane, 70 °C; (j) iPrMgCl·LiCl, THF, 0 °C, 15 min; then 5-Chloro-N-metoxy-N-methyl-6-(trifluoromethyl)nicotinamide, THF, 30 min; (k) iPrMg·LiCl, THF, 0 °C, 15 min; then 5-Chloro-6-trifluoromethylbenzaldehyde, THF, 30 min; (l) MnO₂, CH₂Cl₂, rt, 12–16 h.

Scheme 9. Synthesis of Compounds 57–70

Reagents and conditions: (a) tert-Butanesurfinamide, Ti(OiPr)₄, THF, reflux, overnight; (b) Alkylmagnesium bromide, THF, −5–0 °C, 0.5 h; (c) HCl, MeOH, rt, 1–2 h; (d) Alkylmagnesium bromide, THF, −5–0 °C, 0.5 h.

Scheme 10. Synthesis of Compounds 71–84

Reagents and conditions: (a) TMSCF₃, CsF, DME, 0 °C, then HCl, rt; (b) tert-Butanesulfinamide, Ti(OPr)₄, THF, 80 °C; (c) TMSCF₃, TBAT, THF, −55 °C; (d) 4 M HCl in methanol, rt.