Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase

Article abstract of DOI:10.3109/14756366.2011.604319

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 K_i=2 μM.

Full text of DOI:10.3109/14756366.2011.604319

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011, 1–11, Early Online
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.604319
ReseaRch aRtIcle
Synthesis and evaluation of substrate analogue inhibitors of
trypanothione reductase
Michael H. Duyzend, Christopher T. Clark, Shayna L. Simmons, Wade B. Johnson, Anna M. Larson,
Aaron M. Leconte, Andrew W. Wills, Matthew Ginder-Vogel, April K. Wilhelm, Josephine A.
Czechowicz, and David G. Alberg
Department of Chemistry, Carleton College, Northfield, Minnesota 55057, USA
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.
Keywords: Trypanothione reductase, inhibitors, Trypanosoma cruzi, trypanosome
Introduction
functioning trypanothione-TR system, has focused con-
e trypanosomatid parasites, trypanosomes and
leishmania, are responsible for several serious diseases
of humans and domestic animals, including African
sleeping sickness, Chagas’ disease, Nagana cattle dis-
ease, and a complex of leishmanial infections1. ese
parasites possess an unusual thiol metabolism based
on the NADPH-dependent flavoenzyme trypanothione
reductase (TR)2. TR is a crucial player in sustaining the
parasite’s redox balance by maintaining the antioxidant
trypanothione (N1,N8-bis(glutathionyl)spermidine, 1)3 in
its reduced dithiol form (Figure 1)2,4. In fact, the trypano-
thione/TR couple replaces the ubiquitous and closely
related glutathione (2)/glutathione reductase (GR) sys-
tem found in host organisms5. Genetic studies indicate
that the parasites require TR for growth and virulence,
relying heavily on the trypanothione/TR system to com-
bat oxidative stress6–8. 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 substrates4. is
specificity, combined with the parasite’s reliance on a
siderable attention on TR as a target for the development
of antiparasitic drugs9–13. Here, we disclose our recent
work on inhibitors of TR from Trypanosoma cruzi, the
causative agent of Chagas’ disease, with three new com-
petitive reversible inhibitors 3–5 (Figure 2).
Our interest in Fmoc-containing diaminosuberic acid
derivative 3 follows from our previous reports of TR inhi-
bition by related Cbz-containing diaminosuberic acid
derivatives, such as 6 (Ki = 48 µM)14, and by macrocyclic
substrate analogues (7–9) (Figure 2)15. With trypanothi-
one 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 trypano-
thione. 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 substrates16 and substrate analogue
Address for Correspondence: David G. Alberg, PhD, Department of Chemistry, Carleton College, One North College Street, Northfield,
Minnesota 55057, USA. Tel.: +1 507-222-4410; Fax: +1 507-222-4400. E-mail: dalberg@carleton.edu.
(Received 01 June 2011; revised 06 July 2011; accepted 07 July 2011)
1

2
M.H. Duyzend et al.
inhibitors17. Indeed, based on structural studies of TR,
Douglas and co-workers18 proposed that the enzyme
possesses a hydrophobic pocket, dubbed the Z-site, not
utilised in binding trypanothione, but which may accom-
modate the Cbz group in Cbz-containing inhibitors and
substrates19. If the Cbz groups of our inhibitors are exploit-
ing 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 hypoth-
esis, we prepared 3, the Fmoc analogue of our previously
reported Cbz-containing inhibitor 6.
substrate, TR utilises a pair of active site cysteine resi-
dues (Cys53 and Cys58), which undergo a series of dis-
ulfide exchanges with oxidised trypanothione, ultimately
releasing reduced substrate and leaving the active site
cysteine pair oxidised as the disulfide20,21. 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
covalentenzyme-substratedisulfideadduct.Wereasoned
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
Dibromide 4 and ester 5 were designed as potential
covalent TR inhibitors. In order to reduce its disulfide
Figure 1. e reactions catalysed by trypanothione reductase and glutathione reductase.
Figure 2. Inhibitors of trypanothione reductase.
Journal of Enzyme Inhibition and Medicinal Chemistry
GENZ 604319

Substrate analogue inhibitors of trypanothione reductase
3
acid residue, might provide a favourable interaction with
the enzyme’s nucleophilic Cys53−perhaps forming a tet-
rahedral covalent adduct with the enzyme.
(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.
Materials and methods
All reagents purchased from commercial suppliers
were used without further purification. Tetrahydrofuran
(THF), dichloromethane (CH2Cl2), diisopropylethylam-
ine (DIEA), and triethylamine (TEA) were distilled from
calcium hydride prior to use. Flash chromatography
was performed by the method of Still, Kahn, and Mitra22
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 ermo 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 chemi-
cal shifts are reported relative to residual CD2HOD at
3.31 ppm. 13C NMR spectra were obtained at 100MHz
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.
(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,12Sand 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. e 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
1
salt: H 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.
Chemistry
(9S,14S)-9,14-bis((((9H-Fluoren-9-yl)methoxy)
carbonyl)amino)-5,8,15,18-tetraoxo-N1,N22-dipro-
pyl-4,7,16,19-tetraazadocosane-1,22-diaminium 2,2,2-
trifluoroacetate (3). A solution of 62mg (0.053 mmol) of
26 in 1 mL of trifluoroacetic acid was stirred at room tem-
perature. 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,
( 9 S , 1 4 S ) - 9 , 1 4 - b i s ( ( ( B e n z y l o x y ) c a r b o n y l )
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.
e mixture was allowed to stir under an N2 atmosphere
for 2 h. e 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%
1
1133, 833, 799, 760, 741, 721 cm−1; H 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
© 2011 Informa UK, Ltd.
GENZ 604319

4
M.H. Duyzend et al.
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.
4-oxobutanoic acid 18. Following the procedure of
Kunz and co-workers25, 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). is mix-
ture was allowed to stir under an N2 atmosphere at room
temperature for 1.5 h. e solvent was removed under
reduced pressure and the residue was taken up in 50 mL
of ethyl acetate. e 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.
(S)-4-(Allyloxy)-3-(((benzyloxy)carbonyl)amino)-4-
oxobutanoic acid 15. A mixture of 5.72 g (20.7 mmol) of
the known oxazolidinone 1423 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. e allyl alcohol was removed under
reduced pressure and the residue partitioned between
75 mL of ether and 50 mL of water. e organic layer was
washed twice with 10% KHSO4, once with brine, and
was dried over Na2SO4. e 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. e spectral data for
15 match that reported in the literature24.
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. e 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)-1-Allyl
4-((S)-3-(allyloxy)-2-(((benzyloxy)car-
bonyl)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 1625, 576 mg (1.85
mmol) of Cbz-Asp-α-allyl ester 15, and 430 mg (2.24
mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiim-
ide hydrochloride (EDC) in 2.0 mL of CH2Cl2 was added
23 mg (0.19 mmol) of DMAP. After stirring at 0°C over-
night, the mixture was diluted with ethyl acetate, washed
with 10% KHSO4, sat. aq. NaHCO3, and brine, and dried
over MgSO4. e solvents were removed under reduced
pressure and the residue was purified by flash chromatog-
raphy (50% EtOAc/hexane) to afford 778 mg (76 % yield)
of 17 as a clear oil: IR (film) δ 3350, 3065, 3034, 2953, 1725,
1
1525, 1455, 1337, 1206, 1060, 989, 937, 741, 699cm−1; H
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)-4-((S)-14-(((Benzyloxy)carbonyl)amino)-2,
2-dimethyl-4,10,13-trioxo-5-propyl-3,12-dioxa-5,9-diaz-
apentadecan-15-yl) 1-(2-((3-((tert-butoxycarbonyl)(pro-
pyl)amino)propyl)amino)-2-oxoethyl) 2-(((benzyloxy)
carbonyl)amino)succinate 20. To a mixture of 429mg
(1.56 mmol) of glycolate 19, 249 mg (0.510 mmol) of
diacid 18, and 360 μL (2.07 mmol) of DIEA in 4.0mL of
THF was added 614 mg (2.05 mmol) of DEPBT26. After
stirring overnight, the mixture was diluted with 30mL of
ethyl acetate and washed with 10 mL portions of the fol-
lowing: 10% citric acid, water, sat. aq. NaHCO3, and brine.
e organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure. e residue was
purified by flash column chromatography (80% EtOAc/
(S)-2-(((Benzyloxy)carbonyl)amino)-4-((S)-2-
(((benzyloxy)carbonyl)amino)-2-carboxyethoxy)-
GENZ 604319
Journal of Enzyme Inhibition and Medicinal Chemistry

Substrate analogue inhibitors of trypanothione reductase
5
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.
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. e reaction vessel was
evacuated, charged with H2, and the mixture was stirred
vigorously overnight. e 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,
tert-Butyl (3-(2-(((benzyloxy)carbonyl)amino)acet-
amido)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). is
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. e 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.
1
1365, 1246, 1150, 909, 771, 730 cm−1; H 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,
2 0 , 2 3 , 2 9 - h e x a ox o - 5 , 2 8 - d i p ro py l - 3 , 3 0 - d i ox a -
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 2314 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 PyBOP27. 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,
To
a
cold (0°C) solution of the crude
3-(propylamino)pro-
N-(benzyloxycarbonyl)glycine
pylamide (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. e mixture was
stirredfor10 h,concentratedunderreducedpressure,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) δ7.42 (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-
© 2011 Informa UK, Ltd.
GENZ 604319

6
M.H. Duyzend et al.
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.
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,
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. e reaction
vessel was evacuated, fitted with a balloon, and then
charged with H2. e mixture was stirred vigorously for
3 d. e reaction mixture was filtered through Celite
and the solvent was removed under reduced pres-
sure to afford 219 mg of crude diamine 25. e 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.
13
J = 7.4 Hz), 1.40 (s, 18H), 0.85 (t, 6H, J = 7.4 Hz) ppm;
C 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 pyri-
dinium tribromide (tech., 90%). e reaction mixture
was allowed to warm to room temperature and was
stirred for a total of 16 h. e mixture was diluted with
ethyl acetate, washed with 5% aq. citric acid, water, and
brine, and dried over MgSO4. e 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,
1
1543, 1422, 1366, 1251, 1154, 1051, 738, 698 cm−1; H
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.
(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 2314, 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 DEPBT26. 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
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
GENZ 604319
Journal of Enzyme Inhibition and Medicinal Chemistry

Substrate analogue inhibitors of trypanothione reductase
7
expression vector pIBITczTR, as described by Walsh,
and co-workers28,29. Enzyme activity was followed using
disulfide 2930 as TR substrate and by monitoring the oxi-
dation of NADPH spectrophotometrically at 340 nm29.
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. e Ki
values for each inhibitor were determined by measur-
ing 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. us, all final assay solu-
tions involving inhibitors 3 and 4 were adjusted to 2%
methanol (inhibitor stock solutions in 20% aq. metha-
nol); 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. e data were fit to the competitive inhibi-
tion model (Y = Vmax*[S]/((Km(1+[I]/Ki)+S)) using the
nonlinear least-squares method of Cleland with the pro-
gram COMP31. 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 programs31,
either failed to give a fit or gave a fit significantly worse
than that obtained with the competitive model. e Km
value for assay substrate 29 was 6.7 µM, an average of
three separate determinations.
indication of time-dependent or irreversible inhibition.
To confirm that inhibitors 4 and 5 did not slowly inactiva-
tion TR, we compared assays run under the standard con-
ditions 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 inhibi-
tion compared to standard assays with no pre-incuba-
tion. 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 inhibi-
tion than that observed in control assays conducted with
no preincubation.
Glutathione reductase assays
e 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 µM32), 150 µM
NADPH, and ~0.2 µg/mL GR. All assay solutions were
also adjusted to 2% methanol for assays involving com-
pounds 3 and 4, and 0.5% methanol for 5, since inhibitor
stock solutions required methanol for complete dissolu-
tion 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 con-
centration of 725 µM of this compound.
Results and discussion
e data for all three inhibitors were consistent
with simple reversible competitive inhibition, with no
Our synthetic efforts toward ester 5 originally began with
an intent to prepare analogue 13, which more closely
mimics trypanothione than does 5 (Scheme 1). 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 Scheme 1. We readily
prepared both the seryl portion (12) and methyl-ester-
protected aspartyl subunit (10) via standard peptide syn-
thesis 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 amino-
succinimidesunderbothbasicandacidicconditions33.e
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 1034. In order to avoid
this complication, we chose to substitute less nucleophilic
glycolate groups in place of the glycyl residues.
Our synthesis of ester 5 is shown in Scheme 2.
Treatment of l-aspartic acid-derived oxazolidinone 1423
Scheme 1. Unsuccessful route to ester 13.
© 2011 Informa UK, Ltd.
GENZ 604319

8
M.H. Duyzend et al.
Scheme 2. e synthesis of inhibitor 5.
Scheme 3. e synthesis of inhibitors 3 and 4.
GENZ 604319
Journal of Enzyme Inhibition and Medicinal Chemistry

Substrate analogue inhibitors of trypanothione reductase
9
with allyl alcohol under basic conditions provided the
known Cbz-Asp-OAllyl (15)24,35. e EDC-mediated ester-
ification of acid 15 and Cbz-Ser-OAllyl (16)25 afforded
doubly allyl protected 17 in 76% yield. e removal of the
allyl protecting groups and coupling the resulting diacid
18 with 2 equiv of glycolate 19 using the coupling reagent
DEPBT26, 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.
Both inhibitors 3 and 4 were prepared from diamino-
suberic acid derivative 23, which we have previously
reported (Scheme 3)14,36. Fmoc inhibitor 3 retains the
glycyl residues of the natural substrate; however, dibro-
mide 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 bro-
mides by glycyl nitrogen atoms. In addition to the afore-
mentioned 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 moi-
ety in the position of the bromides of 414.
e synthesis of Fmoc-containing inhibitor 3 is shown
in Scheme 3. PyBop-mediated27 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 pal-
ladium affected both the removal of the Cbz groups and
the reduction of the alkene. e 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-mediated26 coupling of 23 with 2 equiv of gly-
colate 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 dibro-
mide diastereomers 4 in 72% yield.
We evaluated the inhibitory activity of compounds 3–5
against T. cruzi TR28, using 29 as the disulfide substrate−a
known and readily prepared alternative to the costly try-
panothione (Figure 3)30. e rate of reduction of 29 was
determined spectrophotometrically by following the con-
comitant oxidation of NADPH at 340nm29. e kinetic con-
stants were determined by nonlinear least-squares fit of
the data using Cleland’s COMP program31 and are given in
Table 1. e inhibition kinetics observed for all three inhib-
itors was fully consistent with simple reversible competi-
tive inhibition, with no indication of any time-dependent
behaviour (Figure 4). 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.
Figure 3. TR assay substrate 29.
Table 1. Inhibition of TR by compounds 3–5 and by previously
reported inhibitors 6–9a
Compound
Ki (µM)
3
4
5
2.0 0.2
15.9 1.5
8.9 0.9
6 14
48 3
7 15
826 76
145 24
8 15
9 15
16 2
aAll assays were run using 29 as the TR substrate30. Our
determination of Km for 29 was 6.7 µM (cf. trypanothione
K
m =50 µM28). Kinetic constants were calculated using Cleland’s
nonlinear least-squares program COMP31.
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. us, 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 TR37,38 and GR39 are modi-
fied 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-ni-
trosourea (BCNU)40,41. Ajoene, a natural product derived
fromgarlic, hasalsobeenfoundtocovalentlymodifyboth
GR and TR42. e natural product lunarine selectively
inhibits TR over GR, reacting with the parasite enzyme’s
Cys53 via a Michael-like addition reaction43 and, more
recently, vinyl ketones have also been shown to undergo
conjugate addition reactions with TR44. us, 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 nucleo-
philic attack on their electrophilic moieties. It seems
likely that molecules containing electrophilic groups
that are more conformationally accommodating may
We also assessed the abilities of 3–5 to inhibit yeast glu-
tathione reductase (GR). Treating GR with up to 725 µM
© 2011 Informa UK, Ltd.
GENZ 604319

10 M.H. Duyzend et al.
Figure 4. Competitive inhibition of TR by 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 31
.
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 acknowledg-
ment is made to the donors of e 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.
Figure 5. Potential electrophilic irreversible TR inhibitors.
be more successful covalent inhibitors. For instance,
structurally simpler derivatives of our inhibitors that
incorporate various electrophilic side chains, such as in
30–32 (Figure 5), may more easily adopt a conformation
amenable to reaction with the Cys53 thiol.
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