Hit-to-lead optimization of a benzene sulfonamide series for potential antileishmanial agents†

A series of benzene sulphonamides with good potency and selectivity against Leishmania spp. intracellular amastigotes was identified by high-throughput screening. Approximately 200 compounds were synthesized as part of a hit-to-lead optimization program. The potency of the series appears to be strongly dependent on lipophilicity, making the identification of suitable orally available candidates challenging due to poor pharmacokinetics. Despite not identifying a clinical candidate, a likely solvent exposed area was found, best exemplified in compound 29. Ongoing detailed mode-of-action studies may provide an opportunity to use target-based medicinal chemistry to overcome the issues with the current series.


Parasitology assays
For leishmaniasis assays, 3×10 4 PMM were seeded in each well of a 96-well plate.
After 24h, 5×10 5 amastigotes/well were added and incubated for 2h at 37°C. After this period, compound dilutions were added, and the plates were further incubated for 5 days at 37°C and 5% CO 2 . Total parasite burdens were microscopically assessed after Giemsa staining. In treated wells with high amastigote burdens, an overall estimate of the total burden per well was made without discrimination between the number of infected macrophages and the number of amastigotes per infected cell. In treated wells with low burdens, exact counting was performed.
The results were expressed as % reduction in parasite burdens compared to control wells and an IC 50 was calculated. Miltefosine was included as the reference drug (IC 50 ~5 μM).
Parasite burdens were assessed after adding the substrate CPRG (chlorophenol red β-D-galactopyranoside): 50 μL/well of a stock solution containing 15.2 mg CPRG + 250 μL Nonidet in 100 mL PBS. The change in color was measured spectrophotometrically at 540 nm after 4 hours incubation at 37°C. The results were expressed as % reduction in parasite burdens compared to control wells and an IC 50 (50% inhibitory concentration) was calculated. Benznidazole was included as the reference drug (IC 50 ~2-5 μM).
For human African trypanosomiasis, assays were performed in 96-well plates, each well containing 10 µL of the compound dilution and 190 µL of the parasite suspension (1.5×10 4 trypomastigotes/well for T. b. brucei and 4×10 3 for T. b. rhodesiense). Parasite growth was assessed after 72h at 37°C by adding resazurin.
After 6h (T. b. rhodesiense) or 24h (T. b. brucei), fluorescence was measured (λex 550 nm, λem 590 nm). The results were expressed as % reduction in parasite growth/viability compared to control wells and an IC 50 was calculated. Suramin was included as the reference drug (IC 50 ~0.05 μM).

ADME assays
For the experimental determination of LogD, test compounds were prepared at a physiologically relevant pH of 7.4 at 200nM and 2% DMSO in a 50/50 mix of S6 mobile phase A (5% methanol in 10mM ammonium acetate and adjusted to pH 7.4) and B (100% methanol adjusted to pH 7.4) with an appropriate Internal Standard at 4nM, and injected onto an Ascentis Express RP Amide column, and retention times are compared to a standard curve of 9 commercial drugs covering a LogD range of -1.86 to 6.1. The retention times of each of the 9 standards is plotted against their literature LogD values. The resulting equation for this line, y = mx + b, is used to calculate the LogD values for the test compounds. The retention time in minutes of each test compound is substituted for "x" in the equation, and the resultant "y" is the experimental LogD value for that test compound.
For the experimental stability determination of test compounds in liver microsomes in the presence of NADPH a clearance rate is determined. Assay conditions were 0.25 mg/mL liver microsomal protein from the species of interest (mouse and human), 0.5 uM test compound, at pH 7.4 and 37°C. The reagent was purchased commercially, and the work did not involve the use of animals or humans. Samples were taken at 0, 5, 10, 15, 20 and 30 mins in singlet. The reaction was started after the T0 is taken with the addition of NADPH at 0.5uM. Reaction is stopped by addition of 95% acetonitrile/5% methanol containing an internal standard. Time point samples are combined in compound groups of six that have been pre-sorted by mono molecular weight and analyzed by LC/MS/MS. Peak area ratios (analyte peak area/internal standard peak area) are converted to % remaining using the area ratio at time 0 as 100%. Half-life (t½ = ln(2)/k) and intrinsic clearance (Cl int = k x 1000/(0.25)) in µL/min/mg were calculated from % S7 remaining versus incubation time. From this plot, the slope (k) was determined.
Data was qualified if t½>4X the last time point.
The reaction was stirred for 1 h and then quenched with 1M HCl (5 mL). The mixture was extracted with EtOAc (10 mL), dried (MgSO 4 ) and concentrated in vacuo to give crude product which was purified by column chromatography (50-

(morpholinomethyl)benzenesulfonamide (24)
Based on a literature procedure, to a solution of 2-methoxy-5methylbenzenesulfonyl chloride (1.00 g, 4.53 mmol) in CCl 4 (15 mL) was added AIBN (150 mg, 0.91 mmol) and N-bromosuccinimide (887 mg, 4.98 mmol). The mixture was bubbled with N 2 for 15 min and then heated to reflux and stirred for 16 h under an N 2 atmosphere. The reaction was then concentrated in vacuo and purified by column chromatography (1-20% EtOAc in hexanes) to give a 4:1 mixture of desired bromo SI04 and bis-bromo products (471 mg). The mixture was dissolved in DCM (3 mL) and cooled to 0 o C. Triethylamine (280 µL, 2.03 mmol) was added followed by a dropwise addition of (4-fluorophenyl)methanamine (150 µL, 1.35 mmol). The reaction was stirred for The reaction was stirred at rt for 1 h and then partitioned with sat. NH 4 Cl (5 mL) and separated. The organic phase was dried (MgSO 4 ) and concentrated in vacuo to give crude sulfonamide mixture which was purified by column chromatography (10-60% EtOAc in hexanes) to S28 give pure mono-bromo sulfonamide SI05 (402 mg, 1.04 mmol, 23% over two steps).
Sulfonamide SI05 (136 mg, 0.350 mmol) was dissolved in MeCN (2 mL) and morpholine (61 µL, 0.701 mmol) was added. The reaction was stirred for 2 h and then concentrated in vacuo. The residue was partitioned between EtOAc (5 mL) and water (5 mL), separated and the organic phase was dried (MgSO 4 ) and concentrated in vacuo to give crude morpholine SI06.
To a solution of morpholine SI06 (80 mg, 0.20 mmol) in DMF (1 mL) at 0 o C was added NaH (60 wt%, 12 mg, 0.30 mmol) and TBAI (7.5 mg, 0.02 mmol). The mixture was stirred at 0 o C for 1 h and then 4-fluorobenzyl bromide (28 µL, 0.22 mmol) was added. The reaction was allowed to warm to rt and stirred for 2 h.
Water (5 mL) was added and the reaction was extracted with EtOAc (2 x 5 mL).
To a solution of SI11 (550 mg, 1,540 mmol) in ordinary acetone (20 mL) was added solid potassium carbonate (1.51 g, 11.0 mmol), 18-crown-6 ether (10 mg), and benzyl bromide (290 mg, 1.70 mmol). The slurry was vigorously stirred at room temperature for 12 hours. Acetone was evaporated in vacuum and the evaporation residue was partitioned between ethyl acetate (40 mL) and water (15 S33 mL). After phase separation, the organic layer was washed with water (3 x 10 mL) and brine. The organic phase was dried over magnesium sulphate, filtered, and concentrated to dryness in vacuum to give pure SI12 (690 mg, 1.54 mmol, 100%) as an off-white, crystalline solid.
One-pot procedure: Dry tetrahydrofurane (15 mL  residue was partitioned between ethyl acetate (40 mL) and water (10 mL). After phase separation, the organic layer was washed with water (3 x 10 mL) and brine.
To a solution of SI17 (1.10 g, 2.850 mmol) in acetone (30 mL) was added solid potassium carbonate (4.00 g, 28.9 mmol), 18-crown-6 ether (10.0 mg, 0.037 mmol), and benzyl bromide (1.71 g, 10.0 mmol). The slurry was vigorously stirred at room temperature for 12 hours. The reaction mixture was concentrated in vacuum and the evaporation residue was partitioned between ethyl acetate (40 mL) and water (10 mL). After phase separation, the organic layer was washed with water (3 x 10 mL) and brine. The organic phase was dried over magnesium sulfate, filtered, and concentrate to in vacuum. The crude product was purified by column chromatography (EtOAc/hexanes 1:2) to give pure SI18 (540 mg, 0.820 mmol, 29%).
To a solution of SI18 (530 mg, 0.810 mmol) in dry THF (10 mL) was added methanol (1.0 mL) and potassium tert-butoxide (90.0 mg, 0.810 mmol). The mixture was stirred at room temperature for 3h. The reaction mixture was concentrated in vacuum and the product was extracted with ethyl acetate. The crude product was purified by column chromatography (CHCl 3 /methanol 30:1) to S37 give SI19 as a tacky solid. The solid was rinsed with diethyl ether and dried in vacuum to give pure SI19 (500 mg, 0.760 mmol, 95%) as an off-white solid.
SI19 (480 mg, 0.740 mmol) was dissolved in a blend of tetrahydrofuran (10 mL) and methanol (10mL and the reaction was heated to reflux and stirred for 16 h. The reaction was then concentrated in vacuo and partitioned between EtOAc (10 mL) and water (10 mL).
The mixture was separated, and the organic phase was dried (MgSO 4 ) and concentrated in vacuo. The crude product was purified by column chromatography  Following the General procedure for Sulfonamide formation of 2-chloropyridine-3-sulfonyl chloride SI21 (500 mg, 2.36 mmol) and bis(4-fluorobenzyl)amine (500 mg, 2.14 mmol) were reacted for 16 h and purified by column chromatography (   To a solution of 4-fluoroaniline (50 mg, 0.450 mmol) in CH 2 Cl 2 (2 mL) was added pyridine (150 µL, 1.80 mmol) followed by 2,5-dimethoxybenzenesulfonyl chloride (128 mg, 0.540 mmol) and the reaction was stirred at rt for 16 h. The reaction was concentrated in vacuo and then re-dissolve with EtOAc (10 mL) and

carbonyl)benzenesulfonamide (42)
Following the General procedure for Sulfonamide formation 2-methoxy-5-  h and then allowed to warm to rt and stirred overnight. The reaction was concentrated in vacuo and then partitioned between Et 2 O (10 mL) and water (10 mL). The mixture was separated and the organic phase was washed with sat. brine   To a solution of isobutylamine (50 µL, 0.503 mmol) in CH 2 Cl 2 (1 mL) at 0 o C was added 2-methoxy-5-(morpholine-4-carbonyl)benzenesulfonyl chloride SI35 (50 mg, 0.156 mmol) and the reaction was stirred for 1 h. The reaction was diluted with EtOAc (5 mL) and washed with 1 M HCl (2 mL) and then sat. brine (5 mL).

(trifluoromethyl)pyrimidin-5-yl)methyl)benzenesulfonamide (47)
The reaction was cooled to 0 °C and MeOH (3 mL) was added cautiously. The reaction was allowed to warm to rt and stirred for 30 min and then concentrated.

S58
The reaction was stirred at 0 °C for 5 min and then allowed to warm to rt and stirred for 2 h. Quenched with sat. NaHCO 3 (5 mL), separated and re-extracted with more DCM (2 x 5 mL). The combined organics were dried (MgSO 4 ) and concentrated in vacuo to give crude product. The organic phase was dried (MgSO 4 ) and concentrated in vacuo to give a 1:1 mixture of (2-(trifluoromethyl)pyrimidin-5yl)methyl methanesulfonate SI43 and 5-(chloromethyl)-2-(trifluoromethyl) pyrimidine SI44 (105 mg, ~0.45 mmol) which was used in the next step without further purification.
To a suspension of sodium hydride (60wt%, 12 mg, 0.313 mmol) in DMF (0.7 mL) at 0 °C was added N-benzyl-2-methoxy-5-(morpholine-4-carbonyl)benzene sulfonamide SI45 (60 mg, 0.154 mmol) and the reaction was stirred for 15 min at 0 °C and a further 45 min at rt. The reaction was then cooled to 0 °C again and a solution of the mesylate/chloride mixture of SI43 and SI44 (1:1, 40 mg, 0.20 mmol) in DMF (0.3 mL) was added in one portion. The reaction was stirred at 0 °C for 15 min and then allowed to warm to rt and stirred for a further 1 h. The reaction was cooled to 0 °C again and water (10 mL) added. The mixture was extracted with EtOAc (2 x 5 mL). The combined organics were washed with sat.