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Journal of Enzyme Inhibition and Medicinal Chemistry Volume 27, 2012 - Issue 6
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Research Article

Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase

Michael H. DuyzendDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Christopher T. ClarkDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Shayna L. SimmonsDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Wade B. JohnsonDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Anna M. LarsonDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Aaron M. LeconteDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Andrew W. WillsDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Matthew Ginder-VogelDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
April K. WilhelmDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
,
Josephine A. CzechowiczDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
&
David G. AlbergDepartment of Chemistry, Carleton College, Northfield, Minnesota 55057, USACorrespondence[email protected]
 show all
Pages 784-794 | Received 01 Jun 2011, Accepted 07 Jul 2011, Published online: 15 Nov 2011

Abstract

Trypanothione reductase (TR) is found in the trypanosomatid parasites, where it catalyses the NADPH-dependent reduction of the glutathione analogue, trypanothione, and is a key player in the parasite’s defenses against oxidative stress. TR is a promising target for the development of antitrypanosomal drugs; here, we report our synthesis and evaluation of compounds 3–5 as low micromolar Trypanosoma cruzi TR inhibitors. Although 4 and 5 were designed as potential irreversible inhibitors, these compounds, as well as 3, displayed reversible competitive inhibition. Compound 3 proved to be the most potent inhibitor, with a Ki = 2 µM.

Introduction

The trypanosomatid parasites, trypanosomes and leishmania, are responsible for several serious diseases of humans and domestic animals, including African sleeping sickness, Chagas’ disease, Nagana cattle disease, and a complex of leishmanial infectionsCitation1. These parasites possess an unusual thiol metabolism based on the NADPH-dependent flavoenzyme trypanothione reductase (TR)Citation2. TR is a crucial player in sustaining the parasite’s redox balance by maintaining the antioxidant trypanothione (N1,N8-bis(glutathionyl)spermidine, 1)Citation3 in its reduced dithiol form ()Citation2, Citation4. In fact, the trypanothione/TR couple replaces the ubiquitous and closely related glutathione (2)/glutathione reductase (GR) system found in host organismsCitation5. Genetic studies indicate that the parasites require TR for growth and virulence, relying heavily on the trypanothione/TR system to combat oxidative stressCitation6–8. Moreover, despite the analogous reactions catalysed by TR and GR and the significant homology the enzymes share, TR and GR possess nearly complete specificity for their respective substratesCitation4. This specificity, combined with the parasite’s reliance on a functioning trypanothione-TR system, has focused considerable attention on TR as a target for the development of antiparasitic drugsCitation9–13. Here, we disclose our recent work on inhibitors of TR from Trypanosoma cruzi, the causative agent of Chagas’ disease, with three new competitive reversible inhibitors 3–5 ().

Figure 1.  The reactions catalysed by trypanothione reductase and glutathione reductase.

Figure 1.  The reactions catalysed by trypanothione reductase and glutathione reductase.

Figure 2.  Inhibitors of trypanothione reductase.

Figure 2.  Inhibitors of trypanothione reductase.

Our interest in Fmoc-containing diaminosuberic acid derivative 3 follows from our previous reports of TR inhibition by related Cbz-containing diaminosuberic acid derivatives, such as 6 (Ki = 48 µM)Citation14, and by macrocyclic substrate analogues (7–9) ()Citation15. With trypanothione analogues 7–9, we were intrigued to find that the enzyme binds deazatrypanothione 7 (Ki = 826 µM) rather poorly despite its close structural similarity to the natural substrate. On the other hand, binding was improved with 8 (Ki = 145 µM), and even more so with 9 (Ki = 16 µM), where hydrophobic Cbz and Fmoc groups, respectively, replace the charged γ-glutamyl residue of 7 and trypanothione. It is well-established that TR has a predilection to bind hydrophobic compounds and that it will accept a Cbz group in the place of trypanothione’s γ-glutamyl residues in alternate substratesCitation16 and substrate analogue inhibitorsCitation17. Indeed, based on structural studies of TR, Douglas and co-workersCitation18 proposed that the enzyme possesses a hydrophobic pocket, dubbed the Z-site, not utilised in binding trypanothione, but which may accommodate the Cbz group in Cbz-containing inhibitors and substratesCitation19. If the Cbz groups of our inhibitors are exploiting the Z-site, our results with macrocycles 7–9 suggested that the Z-site may accommodate the larger Fmoc group, as well, and we thought the roughly 10-fold increase in affinity shown for the Fmoc macrocycle 9 over the Cbz counterpart 8 may be generalisable. To test this hypothesis, we prepared 3, the Fmoc analogue of our previously reported Cbz-containing inhibitor 6.

Dibromide 4 and ester 5 were designed as potential covalent TR inhibitors. In order to reduce its disulfide substrate, TR utilises a pair of active site cysteine residues (Cys53 and Cys58), which undergo a series of disulfide exchanges with oxidised trypanothione, ultimately releasing reduced substrate and leaving the active site cysteine pair oxidised as the disulfideCitation20, Citation21. In turn, NAPDH provides the reducing power to return the enzyme to its reduced state. In the course of substrate reduction, Cys53 reacts with the substrate disulfide, transiently forming a covalent enzyme-substrate disulfide adduct. We reasoned that dibromide 4 might act as an irreversible inactivator, wherein one of its bromine moieties could be displaced by the enzyme’s nucleophilic cysteine residue, resulting in an irreversible covalent adduct. In the case of ester 5, we surmised that the electrophilic bridging ester moiety, formed from the side chains of a serine and an aspartic acid residue, might provide a favourable interaction with the enzyme’s nucleophilic Cys53−perhaps forming a tetrahedral covalent adduct with the enzyme.

Materials and methods

All reagents purchased from commercial suppliers were used without further purification. Tetrahydrofuran (THF), dichloromethane (CH2Cl2), diisopropylethylamine (DIEA), and triethylamine (TEA) were distilled from calcium hydride prior to use. Flash chromatography was performed by the method of Still, Kahn, and MitraCitation22 using the indicated eluting solvent. Preparative HPLC was performed using an ISCO Model 2350 HPLC pump with an ISCO V4 Variable Wavelength Detector. Infrared spectra were recorded on a Mattson 4020 Galaxy Series FTIR spectrometer via thin film or a Thermo Scientific Nicolet iS10 FTIR Spectrometer using the Attenuated Total Reflectance (ATR) method on a ZnSe ATR crystal. NMR spectra were obtained in the indicated solvent on a Varian Unity-plus 400 MHz spectrometer. 1H NMR data are reported in the following manner: chemical shift (δ) in ppm, downfield from internal tetramethylsilane (multiplicity, integrated intensity, coupling constants in hertz). For spectra recorded in methanol-d4, the chemical shifts are reported relative to residual CD2HOD at 3.31 ppm. 13C NMR spectra were obtained at 100 MHz using broad-band decoupling. Chemical shifts are reported relative to TMS or CDCl3 at 77.2 ppm (downfield positive). DEPT spectra provided carbon multiplicities where indicated in tabulated 13C NMR peak data. Enzyme assays were carried out on a Hewlett-Packard 8453 Diode Array UV–visible spectrophotometer. High resolution mass spectral data were recorded at the University of Minnesota Mass Spectrometry Service Laboratory.

Chemistry

(9S,14S)-9,14-bis((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5,8,15,18-tetraoxo-N1,N22-dipropyl-4,7,16,19-tetraazadocosane-1,22-diaminium 2,2,2-trifluoroacetate (3). A solution of 62 mg (0.053 mmol) of 26 in 1 mL of trifluoroacetic acid was stirred at room temperature. After 2.5 h, the trifluoroacetic acid was removed under reduced pressure and the residue was purified by reverse phase HPLC. Purification was achieved on a Rainin Dynamax-60A C18 reverse phase column (25 cm × 21.4 mm (id)), eluting with 70:30 CH3OH/H2O with 0.1% TFA at a flow rate of 12 mL/min (eluant was monitored at 265 nm, Rt ≈ 20 min). Solvent removal under reduced pressure afforded 47 mg (74% yield) of 3 as the trifluoroacetate salt: IR (ATR, ZnSe crystal) ν 3411, 3041, 2941, 2860, 2458, 2402, 1667, 1452, 1427, 1351, 1202, 1178, 1133, 833, 799, 760, 741, 721 cm−1; 1H NMR (400 MHz, CD3OD) δ 7.81 (d, 4H, J = 7.5 Hz), 7.67 (dd, 4H, J = 3.8,7.3 Hz), 7.40 (app t, 4H, J = 7.5 Hz), 7.31 (app t, 4H, J = 7.4 Hz), 4.46 (dd, 2H, J = 6.8, 10.5 Hz), 4.34 (dd, 2H, J = 7.0, 10.5 Hz), 4.23 (dd, 2H, J = 6.8, 7.0 Hz), 4.00 (dd, 2H, J = 5.9, 8.4 Hz), 3.88 (d, 2H, J = 16.8 Hz), 3.77 (d, 2H, J = 16.8 Hz), 3.31-3.20 (m, 4H, partially obscured by CD2HOD peak), 2.92 (t, 4H, J = 7.4 Hz), 2.85 (t, 4H, J = 7.5 Hz), 1.88-1.73 (m, 6H), 1.73-1.61 (m, 2H), 1.64 (sex, 4H, J = 7.5 Hz), 1.49-1.35 (m, 4H), 0.96 (t, 6H, J = 7.5 Hz) ppm; 13C NMR (100 MHz, CD3OD) δ 175.8 (C), 172.6 (C), 158.9 (C), 145.17 (C), 145.15 (C), 142.59 (C), 142.58 (C), 128.9 (CH), 128.20 (CH), 128.19 (CH), 126.2 (CH), 126.1 (CH), 120.99 (CH), 120.97 (CH), 68.0 (CH2), 56.9 (CH), 50.6 (CH2), 48.4 (CH), 46.1 (CH2), 43.7 (CH2), 36.8 (CH2), 32.3 (CH2), 27.4 (CH2), 26.4 (CH2), 20.6 (CH2), 11.2 (CH3) ppm; HRMS (ESI) m/z calcd for C54H71N8O8 [M+H]+ 959.5389, found 959.5417.

(9S, 14S)-9, 14-bis(((Benzyloxy)carbonyl)amino)-11, 12-dibromo-5,8,15,18-tetraoxo-N1,N22-dipropyl-7,16-dioxa-4,19-diazadocosane-1,22-diaminium 2,2,2-trifluoroacetate (mixture of the 11R,12S and 11S,12R diastereomers) (4). A mixture of 104 mg (0.0910 mmol) of dibromide 28 in 1.5 mL of trifluoroacetic acid was stirred at room temperature for 1 h. The trifluoroacetic acid was removed under reduced pressure and the residue was purified by reverse phase HPLC on a Rainin Dynamax-60A C18 reverse phase column (25 cm x 21.4 mm (id)), eluting with 60:40 CH3OH/H2O with 0.1% TFA at a flow rate of 12 mL/min (eluant was monitored at 254 nm, Rt ≈ 15 min). Solvent removal under reduced pressure afforded 77 mg (72% yield) of 4 as the trifluoroacetate salt: 1H NMR (400 MHz, 10% CD3OD/CDCl3) δ 8.91-8.62 (m, 1H, CONH), 8.19-8.01 (m, 0.6H CONH), 7.41-7.24 (m, 10H), 7.06 (d, 0.3H, J = 7.7 Hz, CONH) (low integral values for the amide protons due to partial solvent exchange), 5.18-5.05 (m, 4H), 4.76-4.45 (m, 7H), 4.40-4.31 (m, 1H), 3.34-3.21 (m, 4H), 2.99-2.75 (m, 8H), 2.73-2.59 (m, 1H), 2.58-2.40 (m, 2H), 2.40-2.27 (m, 1H), 1.94-1.80 (m, 4H), 1.69 (sex 4H, J = 7.3 Hz), 0.98 (t, 3H, J = 7.3 Hz), 0.97 (t, 3H, J = 7.3 Hz) ppm; 13C NMR (100 MHz, 10% CD3OD/CDCl3) δ 171.3 (C), 171.0 (C), 157.4 (C), 157.3 (C), 156.7 (C), 136.3 (CH), 136.1 (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.3 (CH), 127.8 (CH), 127.6 (CH), 67.3 (CH2), 67.2 (CH2), 63.43 (CH2), 63.37 (CH2), 54.8 (CH), 53.1 (CH), 53.0 (CH), 52.7 (CH), 52.6 (CH), 49.8 (CH2), 49.7 (CH2), 44.84 (CH2), 44.78 (CH2), 38.7 (CH2), 37.0 (CH2), 35.57 (CH2), 35.52 (CH2), 35.46 (CH2), 35.41 (CH2), 26.0 (CH2), 19.5 (CH2), 10.9 (CH3) ppm; HRMS (ESI) m/z calcd for C40H59Br2N6O10 [M+H]+ 941.2654, found 941.2662.

(9S,14S)-9,14-bis(((Benzyloxy)carbonyl)amino)-5,8,12,15,18-pentaoxo-N1,N22-dipropyl-7,11,16-trioxa-4,19-diazadocosane-1,22-diaminium 2,2,2-trifluoroacetate (5). To a solution of 176 mg (0.176 mmol) of 20 in 1 mL of CH2Cl2 was added 2 mL of TFA. The mixture was allowed to stir under an N2 atmosphere for 2 h. The reaction mixture was concentrated under reduced pressure and purified by reverse phase HPLC on a Rainin Dynamax-60A C18 reverse phase column (25 cm × 21.4 mm (id)). Diamine 5 was eluted with 60:40 CH3OH/H2O with 0.1% TFA at a flow rate of 12 mL/min (eluant was monitored at 254 nm, Rt ≈ 8 min). Solvent removal under reduced pressure afforded 130 mg (72% yield) of 5 as the trifluoroacetate salt: 1H NMR (400 MHz, 10% CD3OD/CDCl3) δ 7.37-7.27 (m, 10H), 5.15-5.05 (m, 4H), 4.75-4.51 (m, 7H), 4.44 (dd, 1H, J = 3.8, 11.5 Hz), 3.34-3.24 (m, 4H), 3.05-2.81 (m, 10H), 1.93-1.81 (m, 4H), 1.69 (sex, 4H, J = 7.5 Hz), 0.98 (t, 6H, J = 7.5 Hz) ppm; 13C NMR (100 MHz, 10% CD3OD/CDCl3) δ 170.6 (C), 169.1 (C), 156.9 (C), 136.2 (C), 128.6 (CH), 128.4 (CH), 127.93 (CH), 127.88 (CH), 67.4 (CH2), 67.3 (CH2), 64.5 (CH2), 63.5 (CH2), 53.3 (CH), 50.6 (CH), 49.7 (CH2), 44.9 (CH2), 36.4 (CH2), 35.66 (CH2), 35.62 (CH2), 26.0 (CH2), 19.5 (CH2), 10.9 (CH3) ppm; HRMS (FAB) calcd for C39H57N6O12 (M+H)+ 801.4034, Found 801.4043.

(S)-4-(Allyloxy)-3-(((benzyloxy)carbonyl)amino)-4-oxobutanoic acid 15. A mixture of 5.72 g (20.7 mmol) of the known oxazolidinone 14Citation23 and 8.92 g (27.8 mmol) of caesium carbonate in 280 mL of allyl alcohol was stirred at room temperature for 28 h. To this mixture was added 9.2 mL of 6 M HCl. The allyl alcohol was removed under reduced pressure and the residue partitioned between 75 mL of ether and 50 mL of water. The organic layer was washed twice with 10% KHSO4, once with brine, and was dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (1% acetic acid in EtOAc) to afford 4.09 g (65% yield) of 15 as a pale yellow oil. The spectral data for 15 match that reported in the literatureCitation24.

(S)-1-Allyl 4-((S)-3-(allyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropyl) 2-(((benzyloxy)carbonyl)amino)succinate 17. To a cold (0°C) solution of 543 mg (2.07 mmol) of Cbz-Ser-allyl ester 16Citation25, 576 mg (1.85 mmol) of Cbz-Asp-α-allyl ester 15, and 430 mg (2.24 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 2.0 mL of CH2Cl2 was added 23 mg (0.19 mmol) of DMAP. After stirring at 0°C overnight, the mixture was diluted with ethyl acetate, washed with 10% KHSO4, sat. aq. NaHCO3, and brine, and dried over MgSO4. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (50% EtOAc/hexane) to afford 778 mg (76 % yield) of 17 as a clear oil: IR (film) δ 3350, 3065, 3034, 2953, 1725, 1525, 1455, 1337, 1206, 1060, 989, 937, 741, 699 cm−1; 1H NMR (400 MHz, CDCl3) ν 7.32-7.29 (m, 10 H), 5.96-5.77 (m, 4 H), 5.32-5.19 (m, 4 H), 5.12-5.07 (m, 4 H), 4.71-4.59 (m, 6H), 4.50 (dd, 1H, J = 3.5, 11.4 Hz), 4.34 (dd, 1H, J = 3.2, 11.3 Hz), 2.97 (dd, 1H, J = 5.1, 17.0 Hz), 2.87 (dd, 1h, J = 4.6, 17.0 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 170.5 (C), 169.7 (C), 169.2 (C), 156.0 (C), 155.9 (C), 136.2 (C), 131.4 (CH), 131.3 (CH), 128.60 (CH), 128.57 (CH), 128.28 (CH), 128.24 (CH), 128.18 (CH), 128.14 (CH), 119.3 (CH2), 119.1 (CH2), 67.28 (CH2), 67.24 (CH2), 66.66 (CH2), 66.58 (CH2), 64.9 (CH2), 53.3 (CH), 50.5 (CH2), 37.1 (CH2) ppm; HRMS (FAB) m/z calcd for C29H33N2O10 (M+H)+ 569.2130, Found 569.2135.

(S)-2-(((Benzyloxy)carbonyl)amino)-4-((S)-2-(((benzyloxy)carbonyl)amino)-2-carboxyethoxy)-4-oxobutanoic acid 18. Following the procedure of Kunz and co-workersCitation25, to a solution of 1.05 g (1.85 mmol) of diallyl ester 17 in 10 mL of THF was added 1.61 mL (18.5 mmol) of morpholine and 10 mg of tetrakis(triphenylphosphine)palladium (0). This mixture was allowed to stir under an N2 atmosphere at room temperature for 1.5 h. The solvent was removed under reduced pressure and the residue was taken up in 50 mL of ethyl acetate. The organic layer was washed with four 20 mL portions of 2 M HCl, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford 0.891 g of a sticky white powder (98% crude yield). Crude 18 was used directly in the subsequent transformation: 1H NMR (400 MHz, CDCl3) δ 11.28 (bs, 2 H), 7.39-7.00 (m, 11H), 6.25-5.95 (m, 1H), 5.15-4.95 (m, 4H), 4.68-4.18 (m, 4H), 3.13-2.69 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 174.0, 172.5, 170.4, 156.5, 156.4, 135.88, 135.86, 128.49, 128.45, 128.20, 128.17, 128.10, 128.04, 67.39, 67.37, 64.7, 53.1 50.2, 36.5 ppm.

tert-Butyl (3-(2-hydroxyacetamido)propyl)(propyl)carbamate 19. A solution of 0.560 mL (7.24 mmol) of methyl glycolate and 1.00 mL (7.24 mmol) of N-propyl-1,3-propanediamine in 7.5 mL of toluene was refluxed for 3.5 h. After the mixture was allowed to cool, the solvent was removed under reduced pressure. The residue was dissolved in 7.5 mL of CH2Cl2 and 1.58 g (7.24 mmol) of di-tert-butyldicarbonate and 1.01 mL (7.24 mmol) of TEA were added. After allowing the mixture to stir for 17 h, the solvent was removed under reduced pressure and the residue was purified by flash chromatography (40% acetone/hexane) to provide 1.62 g (81 % yield) of 19 as a yellow oil: IR (film) ν 3350, 2970, 2930, 2865, 1670, 1530, 1480, 1420, 1370, 1305, 1250, 1150, 1075, 975, 905, 865, 760 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.33 (bs, 1H), 4.10 (s, 2H), 3.33-3.25 (m, 4H), 3.10 (t, 2H, J = 6.9 Hz), 1.76-1.65 (m, 2H), 1.54 (sex, 2 H, J = 7.4 Hz), 1.45 (s, 9H), 0.88 (t, 3H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ ppm 172.5 (C); 156.3 (C); 79.7 (C); 62.1 (CH2); 48.8 (CH2); 43.9 (CH2); 35.8 (CH2); 28.4 (CH3); 28.0 (CH2); 21.8 (CH2); 11.3 (CH3) ppm; MS (EI) m/z 274 (M+), 218, 201, 187, 173, 145, 143, 116, 98, 88, 72, 57, 44, 41, 31, 30; HRMS (ESI) m/z calcd for C13H26N2O4Na (M+Na)+ 297.1790, Found 297.1785.

(S)-4-((S)-14-(((Benzyloxy)carbonyl)amino)-2,2-dimethyl-4,10,13-trioxo-5-propyl-3,12-dioxa-5,9-diazapentadecan-15-yl) 1-(2-((3-((tert-butoxycarbonyl)(propyl)amino)propyl)amino)-2-oxoethyl) 2-(((benzyloxy)carbonyl)amino)succinate 20. To a mixture of 429 mg (1.56 mmol) of glycolate 19, 249 mg (0.510 mmol) of diacid 18, and 360 μL (2.07 mmol) of DIEA in 4.0 mL of THF was added 614 mg (2.05 mmol) of DEPBTCitation26. After stirring overnight, the mixture was diluted with 30 mL of ethyl acetate and washed with 10 mL portions of the following: 10% citric acid, water, sat. aq. NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (80% EtOAc/hexane) to afford 201 mg (39 % yield) of 20 as viscous yellow oil: IR (film) ν 3320, 2970, 2935, 2876, 1751, 1724, 1676, 1668, 1540, 1421, 1366, 1249, 1154, 1062, 912, 734, 697 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.76 (bs, 1H), 7.63 (bs, 1H), 7.37-7.25 (m, 10H), 6.66 (d, 1H, J = 8.0 Hz), 6.53 (d, 1H, J = 7.7 Hz), 5.15-5.05 (m, 4H), 4.92-4.42 (m, 8H), 3.33-2.92 (m, 14H), 1.64-1.44 (m, 8H), 1.40 (s, 9H), 1.39 (s, 9H), 0.86 (t, 3H, J = 7.4), 0.85 (t, 3H, J = 7.4) ppm; 13C NMR (100 MHz, CDCl3) δ 169.9 (C), 168.4 (C), 166.5 (C), 166.3 (C), 157.0 (C), 156.5 (C), 156.4 (C), 136.2 (C), 128.58 (CH), 128.54 (CH), 128.28 (CH), 128.24 (CH), 128.14 (CH), 128.08 (CH), 78.0 (C), 67.25 (CH2), 67.21 (CH2), 64.5 (CH2), 63.79 (CH2), 63.76 (CH2), 53.4 (CH), 50.8 (CH), 48.9 (CH2), 42.9 (CH2), 36.8 (CH2), 34.7 (CH2), 28.4 (CH3), 27.3 (CH2), 21.9 (CH2), 11.4 (CH3) ppm; HRMS (FAB) m/z calcd for C49H72N6O16Na (M+Na)+ 1023.4903, Found 1023.4990.

tert-Butyl (3-(2-(((benzyloxy)carbonyl)amino)acetamido)propyl)(propyl)carbamate 21. To a cold (0°C) stirring solution of Cbz-glycine N-hydroxysuccinimide ester (3.75 g, 12.2 mmol, Novabiochem) and a catalytic amount of DMAP in 30 mL of CH2Cl2 was added N-propyl-1,3-propanediamine (1.71 mL, 12.4 mmol, Aldrich). This mixture was allowed to warm to room temperature and was stirred overnight. After a total of 13 h, the reaction mixture was washed with 30 mL of sat. aq. NaHCO3. Solid NaCl was added to the aqueous layer, it was extracted with CH2Cl2 (4 × 20 mL), and the combined organic layers were dried over MgSO4 and filtered. The solvents were removed under reduced pressure to give N-(benzyloxycarbonyl)glycine 3-(propylamino)propylamide as a yellow viscous oil (3.66 g, 96%): IR (ATR, ZnSe crystal) ν 3293, 3069, 3034, 2931, 2873, 1712, 1652, 1530, 1454, 1243, 1156, 1047, 989, 737, 697 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.60 (bs. 1H), 7.35-7.27 (m. 5H), 5.44 (bs, 1H), 5.10 (s, 2H), 3.81 (d, 2H, J = 5.5 Hz), 3.35 (q, 2H, J = 5.6 Hz), 2.71 (t, 2H, J = 5.5 Hz), 2.53 (t, 2H, J = 7.1 Hz), 1.81 (bs, 1H), 1.66 (quint, 2H, J = 5.6 Hz), 1.48 (sex, 2H, J = 7.4 Hz), 0.89 (t, 3H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 169.2 (C), 156.7 (C), 136.3 (C), 128.5 (CH), 128.1 (CH), 128.0 (CH), 66.9 (CH2), 51.3 (CH2), 47.8 (CH2), 44.5 (CH2), 38.6 (CH2), 28.1 (CH2), 22.5 (CH2), 11.7 (CH3) ppm; HRMS (ESI) calcd for C16H26N3O3 (M+H)+ 308.1974, Found 308.1964.

To a cold (0°C) solution of the crude N-(benzyloxycarbonyl)glycine 3-(propylamino)propylamide (3.66 g, 12.2 mmol) and 2.69 g (12.3 mmol) of di-tert-butyldicarbonate in 18 mL of CH2Cl2, was added 1.72 mL (12.3 mmol) of triethylamine. The mixture was stirred for 10 h, concentrated under reduced pressure, and the residue was purified by flash chromatography (25% acetone/chloroform) to afford 21 as a light yellow viscous oil (3.97 g, 80%): TLC Rf 0.70 (25% acetone/chloroform); IR (film) ν 3318, 3098, 3070, 3038, 2975, 2936, 2880, 1726, 1680, 1536, 1480, 1459, 1420, 1368, 1252, 1157, 1055, 910, 738, 696 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.42 (bs, 0.8H, major rotamer), 7.37-7.28 (m, 5H), 6.68 (bs, 0.2H, minor rotamer), 6.00 (bs, 0.2H, minor rotamer), 5.87 (bs, 0.8H, major rotamer), 5.11 (s, 2H), 3.85 (d, 2H, J = 4.4 Hz), 3.28-3.15 (m, 4H), 3.06 (t, 2H, J = 7.4 Hz), 1.71-1.56 (m, 2H), 1.52 (sex, 2H, J = 7.4 Hz), 1.43 (s, 9H), 0.86 (t, 3H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 169.0 (C), 156.6 (C), 136.4 (C), 128.4 (CH), 128.1 (CH), 128.0 (CH), 79.6 (C), 67.0 (CH2), 48.7 (CH2), 44.5 (CH2), 43.3 (CH2), 35.7 (CH2), 28.4 (CH3), 27.6 (CH2), 21.8 (CH2), 11.3 (CH3) ppm; HRMS (ESI) calcd for C21H33N3O5Na (M+Na)+ 430.2318, Found 430.2316.

tert-Butyl (3-(2-aminoacetamido)propyl)(propyl)carbamate 22. To a flask containing 3.80 g (9.33 mmol) of 21 was added 50 mg of 10% palladium on carbon, followed by 30 mL of methanol. The reaction vessel was evacuated, charged with H2, and the mixture was stirred vigorously overnight. The reaction mixture was filtered through Celite to remove the catalyst and the solvent was removed under reduced pressure to afford 2.48 g (97% yield) of 22 as a yellow viscous oil: IR (ATR, ZnSe crystal) ν 3307, 3075, 2966, 2916, 2874, 2848, 1659, 1536, 1478, 1417, 1365, 1246, 1150, 909, 771, 730 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.66 (bs, 0.7H, amide N-H, major rotamer), 7.41 (bs, 0.3H, amide N-H, minor rotamer), 3.38 (s, 2H), 3.31-2.20 (m, 4H), 3.13-3.03 (m, 2H), 2.25 (bs, 2H), 1.75-1.60 (m, 2H), 1.50 (tq, 2H, J = 7.4, 7.4 Hz), 1.43 (s, 9H), 0.84 (t, 3H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 166.5 (C), 156.1 (C), 79.6 (C, major rotamer), 79.4 (C, minor rotamer), 49.2 (CH2, major rotamer), 49.0 (CH2, minor rotamer), 45.2 (CH2, minor rotamer), 44.8 (CH2, major rotamer), 41.5 (CH2), 37.9 (CH2, minor rotamer), 37.4 (CH2, major rotamer), 28.6 (CH3), 28.0 (CH2), 22.0 (CH2, major rotamer), 21.6 (CH2, minor rotamer), 11.5 (CH3) ppm; HRMS (FAB) m/z calcd for C13H28N3O3 [MH]+ 274.2132, found 274.2125.

Dibenzyl ((14S,19S,E)-2,2,31,31-tetramethyl-4,10,13,20,23,29-hexaoxo-5,28-dipropyl-3,30-dioxa-5,9,12,21,24,28-hexaazadotriacont-16-ene-14,19-diyl)dicarbamate 24. To a cold (0°C) solution of 166 mg (0.340 mmol) of diacid 23Citation14 and 218 mg (0.797 mmol) of amine 22 in 4 mL of CH2Cl2 was added 250 µL (1.43 mmol) of DIEA and 510 mg (0.98 mmol) of PyBOPCitation27. After stirring for 22 h, the mixture was diluted with 25 mL of ethyl acetate and washed with 10 mL portions of 5% citric acid, 50% brine, sat. aq. NaHCO3, 50% brine, and brine, and then dried over Na2SO4. After filtration and removal of the solvent under reduced pressure, the residue was purified by flash chromatography (gradient of 50→80% acetone/hexanes) to afford 320 mg (95% yield) of 24 as a clear viscous oil: IR (film) δ 3295, 2968, 2933, 2875, 1665, 1536, 1418, 1366, 1248, 1153, 1027, 739, 698 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.36-7.29 (m, 12H), 7.28-7.18 (bs, 2H), 6.27-6.10 (bs, 2H), 5.60-5.45 (m, 2H), 5.15-5.05 (m, 4H), 4.40-4.20 (m, 2H), 3.97-3.73 (m, 4H), 3.32-3.11 (m, 8H), 3.11-2.98 (m, 4H), 2.56-2.36 (m, 4H), 1.73-1.54 (m, 4H), 1.51 (sex, 4H, J = 7.4 Hz), 1.42 (s, 18H), 0.86 (t, 6H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 171.8, 168.8, 156.6, 156.3, 136.2, 129.3, 128.5, 128.21, 128.16, 79.7, 67.1, 55.2, 48.8, 43.4, 43.0, 35.8, 35.6, 28.4, 27.6, 21.8, 11.3 ppm; HRMS (ESI) m/z calcd for C50H76N8O12Na [M+Na]+ 1003.5480, found 1003.5597 and m/z calcd for C50H76N8O12Na2 [M+2Na]+2 513.2690, found 513.2665.

bis((9H-Fluoren-9-yl)methyl) ((14S,19S)-2,2,31,31-Tetramethyl-4,10,13,20,23,29-hexaoxo-5,28-dipropyl-3,30-dioxa-5,9,12,21,24,28-hexaazadotriacontane-14,19-diyl)dicarbamate 26. To a flask containing 320 mg (0.33 mmol) of 24 was added 100 mg of 10% palladium on carbon, followed by 10 mL of ethanol. The reaction vessel was evacuated, fitted with a balloon, and then charged with H2. The mixture was stirred vigorously for 3 d. The reaction mixture was filtered through Celite and the solvent was removed under reduced pressure to afford 219 mg of crude diamine 25. The crude diamine was dissolved in 5 mL of CH2Cl2, cooled to 0°C, and 222 mg (0.658 mmol) of 9-fluorenylmethyl N-succinimidyl carbonate was added, followed by 90 µL (0.65 mmol) of TEA. After 30 min, the mixture was diluted with 65 mL of ethyl acetate and washed with 20 mL portions of 5% citric acid, 50% brine, and dried over Na2SO4. After filtration and removal of solvents under reduced pressure, the residue was purified by flash chromatography (gradient of 20→80% acetone/CHCl3). A second purification by flash chromatography (gradient of 20→50% acetone/CHCl3) was required to afford 104 mg (28% yield) of 26: TLC (40% acetone/CHCl3, Rf = 0.2); IR (film) ν 3304, 3283, 2964, 2932, 2873, 1689, 1664 1643, 1545, 1478, 1450, 1418, 1365, 1247, 1152, 739 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.67 (d, 4H, J = 7.5 Hz), 7.50 (d, 4H, J = 7.3 Hz), 7.31 (app t, 4H, J = 7.5 Hz), 7.26-7.19 (bs, 2H), 7.21 (app t, 4H, J = 7.4 Hz), 7.16-7.05 (bs, 2H), 5.90-5.78 (bs, 2H), 4.33 (d, 4H, J = 6.5 Hz), 4.25-4.15 (m, 2H), 4.11 (t, 2H, J = 6.5 Hz), 3.96-3.75 (m, 4H), 3.24-3.04 (m, 8H), 3.02-2.89 (m, 4H), 1.73-1.54 (m, 4H), 1.90-1.71 (m, 2H), 1.66-1.47 (m, 6H), 1.43 (sex, 4H, J = 7.4 Hz), 1.37-1.32 (m, 4H), 1.33 (s, 18H), 0.76 (t, 6H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 172.4 (C), 168.9 (C), 156.8 (C), 156.6 (C), 144.01 (C), 143.96 (C), 141.51 (C), 141.49 (C), 127.9 (CH), 127.3 (CH), 125.3 (CH), 120.2 (CH), 79.9 (C), 67.1 (CH2), 54.7 (CH), 49.0 (CH2), 47.4 (CH), 43.3 (CH2), 35.9 (CH2), 32.1 (CH2), 29.9 (CH2), 28.6 (CH3), 27.8 (CH2), 24.7 (CH2), 22.0 (CH2), 11.5 (CH3), ppm; HRMS (ESI) m/z calcd for C64H86N8O12Na [M+Na]+ 1181.6257, found 1181.6212.

(2S,7S,E)-bis(2-((3-((tert-Butoxycarbonyl)(propyl)amino)propyl)amino)-2-oxoethyl) 2,7-bis(((benzyloxy)carbonyl)amino)oct-4-enedioate 27. To a mixture of 201 mg (0.0427 mmol) of diacid 23Citation14, 317 mg (1.16 mmol) of glycolate 19, and 305 µL (1.75 mmol) of DIEA in 5 mL of THF was added 524 mg (1.75 mmol) of DEPBTCitation26. After stirring at room temperature for 15 h, the mixture was diluted with 30 mL of ethyl acetate and washed with 10 mL portions of 5% aq. citric acid, water, sat. aq. NaHCO3, and brine, and then dried over MgSO4. After filtration and removal of the solvents under reduced pressure, the crude product was purified by flash chromatography (80% ethyl acetate/hexane) to afford 260 mg (62% yield) of 27 as a yellow oil: IR (film) ν 3324, 3065, 3035, 2967, 2934, 2875, 1754, 1689, 1674, 1538, 1421, 1366, 1250, 1154, 1054, 744, 740, 698 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.79-7.67 (m, 2H), (7.37-7.27 (m, 10H), 6.32-6.17 (m, 2H), 5.60-5.43 (m, 2H), 5.16-5.02 (m, 4H), 4.69 (d, 2H, J = 15.6 Hz), 4.58-4.48 (m, 2H), 4.49 (d, 2H, J = 15.6 Hz), 3.37-3.22 (m, 4H), 3.21-2.96 (m, 8H), 2.68-2.48 (m, 4H), 1.66-1.53 (m, 4H), 1.49 (sex, 4H, J = 7.4 Hz), 1.40 (s, 18H), 0.85 (t, 6H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 170.3 (C), 166.5 (C), 156.8 (C), 156.3 (C), 136.3 (C), 129.0 (CH), 128.5 (CH), 128.1 (CH), 128.0 (CH), 67.0 (CH2), 63.4 (CH2), 54.0 (CH), 48.8 (CH2), 43.0 (CH2), 35.0 (CH2), 34.7 (CH2), 28.4 (CH3), 27.4 (CH2), 21.8 (CH2), 11.3 (CH3), ppm; HRMS (ESI) m/z calcd for C50H74N6O14Na [M+Na]+ 1005.5155, found 1005.5156.

(2S,7S)-bis(2-((3-((tert-Butoxycarbonyl)(propyl)amino)propyl)amino)-2-oxoethyl) 2,7-bis(((benzyloxy)carbonyl)amino)-4,5-dibromooctanedioate (mixture of the 4R,5S and 4S,5R diastereoisomers) 28. To a cold (0°C) solution of 47 mg (0.048 mmol) of alkene 27 in 1.2 mL of CH2Cl2 was added 69 mg (1.9 mmol) of pyridinium tribromide (tech., 90%). The reaction mixture was allowed to warm to room temperature and was stirred for a total of 16 h. The mixture was diluted with ethyl acetate, washed with 5% aq. citric acid, water, and brine, and dried over MgSO4. The dried solution was filtered, the solvents removed under reduced pressure, and the residue was purified by flash chromatography to afford 46 mg (84% yield) of 28 as a mixture of the 4R,5S and 4S,5R dibromide diastereoisomers: IR (film) ν 3320, 3064, 3035, 2969, 2935, 2876, 1753, 1688, 1665, 1543, 1422, 1366, 1251, 1154, 1051, 738, 698 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.72 (t, 1H, J = 6.0 Hz), 7.65 (t, 1H, J = 6.0 Hz), 7.42-7.24 (m, 10H), 6.81 (d, 1H, J = 8.6 Hz), 6.74 (d, 1H, J = 9.1 Hz), 5.19-5.06 (m, 4H), 4.91-4.62 (m, 4H), 4.54-4.32 (m, 3H), 4.30-4.19 (m, 1H), 3.45-3.32 (m, 1H), 3.32-2.83 (m, 12H), 2.77-2.63 (m, 1H), 2.54-2.38 (m, 1H), 2.34-2.18 (m, 1H), 1.58-1.34 (m, 8H), 1.40 (s, 9H), 1.38 (s, 9H), 0.85 (t, 3H, J = 7.4 Hz), 0.84 (t, 3H, J = 7.4 Hz) ppm; 13C NMR (100 MHz, CDCl3) δ 170.3 (C), 170.1 (C), 166.5 (C), 166.3 (C), 157.1 (C), 156.9 (C), 156.2 (C), 136.2 (C), 128.6 (CH), 128.2 (CH), 128.1 (CH), 80.2 (C), 67.2 (CH2), 67.1 (CH2), 63.53 (CH2), 63.46 (CH2), 55.1 (CH), 54.2 (CH), 53.2 (CH), 52.6 (CH), 48.74 (CH2), 48.71 (CH2), 42.64 (CH2), 42.58 (CH2), 40.1 (CH2), 39.0 (CH2), 34.32 (CH2), 34.23 (CH2), 28.48 (CH3), 28.43 (CH3), 27.1 (CH2), 21.7 (CH2), 11.32 (CH3), 11.3 (CH3) ppm; HRMS (ESI) m/z calcd for C50H74Br2N6O14Na [M+Na]+ 1163.3522, found 1163.3535.

Inhibition studies

Trypanothione reductase assays

T. cruzi trypanothione reductase (TR) was purified from an overproducing strain of Escherichia coli SG5, a glutathione reductase deletion mutant containing the expression vector pIBITczTR, as described by Walsh, and co-workersCitation28, Citation29. Enzyme activity was followed using disulfide 29Citation30 as TR substrate and by monitoring the oxidation of NADPH spectrophotometrically at 340 nmCitation29. All assays were carried out at 25°C in 100 mM HEPES (pH 7.8) containing 1 mM EDTA, 150 µM NADPH, with TR at approximately 7 nM for assays of 3 and 5, or 24 nM for assays of 4. Each assay was performed in at least duplicate, many in triplicate or quadruplicate. The Ki values for each inhibitor were determined by measuring the initial reaction rates at five different substrate concentrations ranging from 4 µM to 70 µM and with the following inhibitor concentrations: [3] = 0, 2.2, 5.1, 10.2, 15.3, 19.7 µM; or [4] = 0, 18.7, 62.5, 109.3; or [5] = 0, 15.3, 61.1, 107.0, 152.9 µM. Aqueous inhibitor stock solutions contained methanol, required for complete dissolution of the compounds. Thus, all final assay solutions involving inhibitors 3 and 4 were adjusted to 2% methanol (inhibitor stock solutions in 20% aq. methanol); for the more soluble ester 5 (stock solution in 10% aq. methanol), all final assay solutions were adjusted to 0.5 % methanol. Control experiments indicated that 2% methanol had no inhibitory effect on TR over the course of an assay. The data were fit to the competitive inhibition model (Y = Vmax*[S]/((Km(1+[I]/Ki)+S)) using the nonlinear least-squares method of Cleland with the program COMPCitation31. Attempts to fit the data for each inhibitor to the uncompetitive and noncompetitive inhibition models (equations Y = Vmax*[S]/(Km+[S](1+[I]/Ki)) and Y = Vmax*[S]/(Km(1+[I]/Kis) + S(1+[I]/Kii)), respectively), using Cleland’s UNCOMP and NCOMP programsCitation31, either failed to give a fit or gave a fit significantly worse than that obtained with the competitive model. The Km value for assay substrate 29 was 6.7 µM, an average of three separate determinations.

The data for all three inhibitors were consistent with simple reversible competitive inhibition, with no indication of time-dependent or irreversible inhibition. To confirm that inhibitors 4 and 5 did not slowly inactivation TR, we compared assays run under the standard conditions to those in which the inhibitor was pre-incubated with the enzyme. Under the assay conditions described above, preincubating 49 µM of dibromide 4 with TR (~7 nM) for up to 2.3 h prior to initiating the reaction with 18 µM of substrate 29, resulted in no greater TR inhibition compared to standard assays with no pre-incubation. Likewise, preincubating 153 µM of ester 5 with TR (~7 nM) for up to 17.5 min prior to initiating the reaction with 50 µM of substrate 29 resulted in no greater inhibition than that observed in control assays conducted with no preincubation.

Glutathione reductase assays

The inhibitory effects of compounds 3–5 on the rate of reduction of glutathione by yeast glutathione reductase (Sigma) were determined by following the oxidation of NADPH spectrophotometrically at 340 nm. Assays were run at 25°C in 100 mM HEPES (pH 7.8) containing 1 mM EDTA, 30 µM glutathione (Km = 55 µMCitation32), 150 µM NADPH, and ~0.2 µg/mL GR. All assay solutions were also adjusted to 2% methanol for assays involving compounds 3 and 4, and 0.5% methanol for 5, since inhibitor stock solutions required methanol for complete dissolution of these compounds. Control experiments indicated that 2% methanol had no inhibitory effect on GR. For 3 and 4, no inhibition of GR was observed up to 58 µM of 3, or 130 µM of 4, the solubility limits of these compounds. For ester 5, no inhibition of GR was observed up to a concentration of 725 µM of this compound.

Results and discussion

Our synthetic efforts toward ester 5 originally began with an intent to prepare analogue 13, which more closely mimics trypanothione than does 5 (). Esters 5 and 13 differ in that 5 contains glycolate residues in place of the glycyl resides found in 13 and the natural substrate. Our plan for the synthesis of 13 was to couple the aspartyl and seryl derivatives, 11 and 12, through an ester linkage of their side chains, as shown in . We readily prepared both the seryl portion (12) and methyl-ester-protected aspartyl subunit (10) via standard peptide synthesis methods (data not shown); however, our attempts to hydrolyse the methyl ester of 10 resulted in undesired side reactions. It is well-established that aspartyl β-ester-containing peptides undergo facile ring closing to aminosuccinimides under both basic and acidic conditionsCitation33. The cyclic imide intermediates, in turn, can open to provide both α-aspartyl and β-aspartyl peptides. Furthermore, these undesired reactions are particularly pronounced for the asp-gly sequence, as found in 10Citation34. In order to avoid this complication, we chose to substitute less nucleophilic glycolate groups in place of the glycyl residues.

Scheme 1.  Unsuccessful route to ester 13.

Scheme 1.  Unsuccessful route to ester 13.

Our synthesis of ester 5 is shown in . Treatment of l-aspartic acid-derived oxazolidinone 14Citation23 with allyl alcohol under basic conditions provided the known Cbz-Asp-OAllyl (15)Citation24,Citation35. The EDC-mediated esterification of acid 15 and Cbz-Ser-OAllyl (16)Citation25 afforded doubly allyl protected 17 in 76% yield. The removal of the allyl protecting groups and coupling the resulting diacid 18 with 2 equiv of glycolate 19 using the coupling reagent DEPBTCitation26, provided 20 in 39% yield. Finally, removal of the Boc protecting groups provided ester 5 in 72% yield after purification by preparative reverse phase HPLC.

Scheme 2.  The synthesis of inhibitor 5.

Scheme 2.  The synthesis of inhibitor 5.

Both inhibitors 3 and 4 were prepared from diaminosuberic acid derivative 23, which we have previously reported ()Citation14, Citation36. Fmoc inhibitor 3 retains the glycyl residues of the natural substrate; however, dibromide 4, like ester 5, incorporates glycolate moieties in the place of glycine resides. Again, this change was made in order to avoid unwanted intramolecular reactions, in this case, the possible nucleophilic displacement of the bromides by glycyl nitrogen atoms. In addition to the aforementioned aspartyl rearrangement, we had previously observed a similar reaction of a glycyl amide N atom, in an attempt to prepare an inhibitor with an epoxide moiety in the position of the bromides of 4Citation14.

Scheme 3.  The synthesis of inhibitors 3 and 4.

Scheme 3.  The synthesis of inhibitors 3 and 4.

The synthesis of Fmoc-containing inhibitor 3 is shown in . PyBop-mediatedCitation27 coupling of diacid 23 with 2 equiv of glycine derivative 22 afforded alkene 24 in 95% yield. Treatment of 24 with H2 in the presence of palladium affected both the removal of the Cbz groups and the reduction of the alkene. The resulting crude diamine 25 was converted to its bis-Fmoc derivative 26, and removal of the Boc groups of 26 under acidic conditions provided 3 in 74% yield, after purification by preparative reverse phase HPLC.

Dibromide 4 was prepared in a similar fashion. DEPBT-mediatedCitation26 coupling of 23 with 2 equiv of glycolate 19 gave alkene 27 in 62% yield. Bromination of alkene 27 using pyridinium tribromide provided 28, as an inseparable mixture of the 4R,5S and 4S,5R dibromide diastereoisomers. Removal of the Boc groups, followed by purification by reverse phase HPLC provided dibromide diastereomers 4 in 72% yield.

We evaluated the inhibitory activity of compounds 3–5 against T. cruzi TRCitation28, using 29 as the disulfide substrate−a known and readily prepared alternative to the costly trypanothione ()Citation30. The rate of reduction of 29 was determined spectrophotometrically by following the concomitant oxidation of NADPH at 340 nmCitation29. The kinetic constants were determined by nonlinear least-squares fit of the data using Cleland’s COMP programCitation31 and are given in . The inhibition kinetics observed for all three inhibitors was fully consistent with simple reversible competitive inhibition, with no indication of any time-dependent behaviour (). Moreover, prolonged pre-incubation of dibromide 4 or ester 5 with TR did not result in enzyme inactivation, or any increase in inhibition compared to assays run without pre-incubation, suggesting that these inhibitors do not react covalently with the enzyme.

Table 1.  Inhibition of TR by compounds 3–5 and by previously reported inhibitors 6–9a

Figure 3.  TR assay substrate 29.

Figure 3.  TR assay substrate 29.

Figure 4.  Competitive inhibition of TR y 3–5. (a) Lineweaver–Burk (L–B) plot for TR inhibition in the absence (•) and presence of 3 at 2.2 µM (□), 5.1 µM (▴), 10.2 µM (◊), 15.3 µM (▪), and 19.7 µM (○). (b) L–B plot for TR inhibition in the absence (•) and presence of 4 at 18.7 µM (□), 62.5 µM (▴), and 109.3 µM. (c) L–B plot for TR inhibition in the absence (•) and presence of 5 at 15.3 µM (□), 61.1 µM (▴), 107.0 µM (◊), and 152.9 µM (▪). Each point represents the average of two to four experimental determinations; the lines are based on theoretical values derived from the nonlinear least-squares fit of the data sets using the COMP program Citation31.

Figure 4.  Competitive inhibition of TR y 3–5. (a) Lineweaver–Burk (L–B) plot for TR inhibition in the absence (•) and presence of 3 at 2.2 µM (□), 5.1 µM (▴), 10.2 µM (◊), 15.3 µM (▪), and 19.7 µM (○). (b) L–B plot for TR inhibition in the absence (•) and presence of 4 at 18.7 µM (□), 62.5 µM (▴), and 109.3 µM. (c) L–B plot for TR inhibition in the absence (•) and presence of 5 at 15.3 µM (□), 61.1 µM (▴), 107.0 µM (◊), and 152.9 µM (▪). Each point represents the average of two to four experimental determinations; the lines are based on theoretical values derived from the nonlinear least-squares fit of the data sets using the COMP program Citation31.

We also assessed the abilities of 3–5 to inhibit yeast glutathione reductase (GR). Treating GR with up to 725 µM of ester 5 resulted in no detectable inhibition. Likewise concentrations up to 58 µM of 3 and 130 µM of dibromide 4, the solubility limits of these compounds, also failed to inhibit GR. Thus, all three inhibitors display significant specificity for the parasite enzyme.

We were pleased to learn that the Fmoc inhibitor 3 does bind to TR more than an order of magnitude more tightly than its Cbz-containing analogue 614 (2 µM versus 48 µM), confirming our hypothesis that substituting Cbz for Fmoc groups in our diaminosuberic acid-derived inhibitors would increase TR binding by about a factor of 10. On the other hand, we were disappointed to find that neither dibromide 4 nor ester 5 displayed irreversible TR inhibition.

It is well-established that TRCitation37,Citation38 and GRCitation39 are modified specifically at their redox active Cys residues (Cys53 in TR) by the thiol-specific reagent iodoacetamide, and by nitrosourea drugs such as 1-bis(2-chloroethyl)-1-nitrosourea (BCNU)Citation40, Citation41. Ajoene, a natural product derived from garlic, has also been found to covalently modify both GR and TRCitation42. The natural product lunarine selectively inhibits TR over GR, reacting with the parasite enzyme’s Cys53 via a Michael-like addition reactionCitation43 and, more recently, vinyl ketones have also been shown to undergo conjugate addition reactions with TRCitation44. Thus, there is abundant evidence for the reactivity of the enzyme’s catalytic cysteine thiol group with electrophiles. We suspect the failure of 4 and 5 to covalently modify TR is not a function of their reactivity, but is more likely due to their binding in a conformation that precludes a nucleophilic attack on their electrophilic moieties. It seems likely that molecules containing electrophilic groups that are more conformationally accommodating may be more successful covalent inhibitors. For instance, structurally simpler derivatives of our inhibitors that incorporate various electrophilic side chains, such as in 30–32 (), may more easily adopt a conformation amenable to reaction with the Cys53 thiol.

Figure 5.  Potential electrophilic irreversible TR inhibitors.

Figure 5.  Potential electrophilic irreversible TR inhibitors.

In conclusion, we have prepared three new substrate analogue inhibitors, 3–5, of TR. The 2 µM binding of Fmoc inhibitor 3 underscores TR’s affinity for hydrophobic ligands. Compounds 4 and 5 also bind with low micromolar affinity; however, despite their potential to irreversibly inhibit TR, these compounds, like 3, behave as reversible competitive inhibitors. Encouragingly, TR does appear to be quite accommodating of large substituents, like the two bromine moieties of 4, in the region of our inhibitors that presumably binds near the catalytic cysteine residues. This steric tolerance suggests that even fairly large thiol-reactive groups, such as the maleimide moiety in 32, may function as effective electrophiles in potential irreversible inhibitors.

Acknowledgements

We are grateful to Professor Christopher Walsh and Kari Nadeau (Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School) for providing the SG5 E. coli strain containing TR expression vector pIBITczTR. We also thank Dogan Sahin and Brian N. Cook for the preliminary synthetic work outlined in Scheme 1, and Jack Rousseau for preparing a sample of intermediate 20. Finally, we dedicate this article in memory of our co-author, Wade B. Johnson (1985-2009), a true scholar with an adventurous spirit.

Declaration of interest

We thank the US National Institutes of Health (Grant 1 R15 AI053113-01) and the Howard Hughes Medical Institute for their generous financial support, and acknowledgment is made to the donors of The Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. We also thank the 3M Foundation for helping to fund the purchase of a 400 MHz NMR spectrometer.

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