Polyamines with N-(3-phenylpropyl) substituents are effective competitive inhibitors of trypanothione reductase and trypanocidal agents

Article abstract of DOI:10.1016/S0960-894X(00)00643-0

Several N-(3-phenylpropyl)-substituted spermidine and spermine derivatives were prepared and found to be potent competitive inhibitors of Trypanosoma cruzi trypanothione reductase (seven compounds with K_i values <5 μM are described). The most e

Full text of DOI:10.1016/S0960-894X(00)00643-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 251±254
Polyamines with N-(3-Phenylpropyl) Substituents are E€ective
Competitive Inhibitors of Trypanothione Reductase and
Trypanocidal Agents
Zhili Li,a Michael W. Fennie,b Bruce Ganem,c Matthew T. Hancock,a Muris Kobaslija,b
Donna Rattendi,d Cyrus J. Bacchid and Mary C. O'Sullivanb,*
aDepartment of Chemistry, Indiana State University, Terre Haute, IN 47809, USA
bDepartment of Chemistry and Biochemistry, Canisius College, 2001 Main Street, Bu€alo, NY 14208, USA
cDepartment of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853, USA
dHaskins Laboratories and Department of Biology, Pace University, New York, NY 10038, USA
Received 6 September 2000; accepted 9 November 2000
AbstractÐSeveral N-(3-phenylpropyl)-substituted spermidine and spermine derivatives were prepared and found to be potent
competitive inhibitors of Trypanosoma cruzi trypanothione reductase (seven compounds with Ki values <5 mM are described). The
most e€ective inhibitor studied was compound 12 with a Ki value of 0.151 mM. Six of the compounds described are also e€ective
trypanocides with IC50 values <1 mM. # 2001 Elsevier Science Ltd. All rights reserved.
Several members of the family Trypanosomatidae are the
causative agents of human and livestock diseases that
include African sleeping sickness (Trypanosoma brucei
rhodesiense and T. b. gambiense), Chagas' disease (Try-
panosoma cruzi) and visceral leishmaniasis or kala-azar
(Leishmania donovani). Diseases caused by trypano-
somes are a serious world problem. For example, Cha-
gas' disease is endemic in 21 Central and South
American countries with an estimated 16±18 million
people infected.1 Also, a resurgence of African sleeping
sickness is devastating populations in southern Sudan,
the Congo, and other sub-Saharan African countries.2
Current treatments for these diseases and other forms of
trypanosomiasis and leishmaniasis are often unsatisfac-
tory. Problems associated with anti-trypanosomal drugs
include their limited ecacies, human toxicity, high cost,
and the emergence of drug-resistant trypanosome
strains.3 One strategy for the development of novel anti-
trypanosomal agents is to target a unique feature of the
thiol metabolism of these parasites. In most organisms,
glutathione is an important antioxidant and levels of
reduced glutathione are maintained by glutathione reduc-
tase (GR). However, Trypanosomatidae do not contain
GR, instead they have a unique enzyme, trypanothione
reductase (TR) (EC 1.6.4.8). TR catalyzes the reduction of
the disul®de of a glutathione±spermidine conjugate
named trypanothione (N1,N8-bis(glutathionyl)spermi-
dine). The mechanism of action of TR is essentially
identical to that of GR. The two enzymes have struc-
tural similarities; however, TR and GR have di€erent
substrate speci®cities.4 TR is crucial to the antioxidant
defenses of Trypanosomatidae. Targeted gene replacement
studies have indicated that TR is essential for survival of
leishmania and trypanosoma.5 Consequently, inhibitors of
TR have potential as e€ective, anti-trypanosomal drugs.6
Inhibitors of TR are structurally diverse, but all reversible
inhibitors contain amino and hydrophobic groups.7,8
Several of the most e€ective inhibitors are polyamine
derivatives with aromatic substituents.9 11 We have
been investigating the inhibition pro®les of structurally-
simple polyamine derivatives and recently reported that
N4,N8-bis(3-phenylpropyl)spermine (1) is a potent com-
petitive inhibitor of T. cruzi TR (Ki 3.48 mM) and an
e€ective trypanocide (IC50 values of 0.58±0.79 mM
against T. brucei strains).10,11 However, compound 1 did
not prolong the lifetimes of mice infected with T. brucei
and hence does not show anti-trypanosomal activity in
vivo.10 Since, compound 1 is such an e€ective trypano-
cide in vitro, and given that no other N-(3-phenylpro-
pyl)-polyamine derivatives have been investigated, we
*Corresponding author. Tel.: +1-716-888-2352; fax: +1-716-888-3112;
e-mail: osulliv1@canisius.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00643-0

252
Z. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 251±254
felt compelled to study the inhibiting and trypanocidal
activities of this class of compounds since we envisaged
that the substitution pattern of the 3-phenylpropyl moiety
may profoundly a€ect the biological activities of these
compounds.
compounds 1, 3, and 6 was surprisingly problematic.
Standard reductive amination reaction conditions (i.e.,
formation of imine followed by reduction with sodium
borohydride, or one-step reductive amination using
sodium cyanoborohydride) produced the desired pro-
ducts in negligible yield. However, reaction of 1, 3, or 6
with 2 mol equiv of hydrocinnamaldehyde and sodium
triacetoxyborohydride13 produced mixtures of mono-
and higher alkylated products that were separable by
¯ash chromatography on silica gel (using a gradient sol-
vent system ranging from 0.3% NH4OH and 5.0%
CH3OH in CH2Cl2 to 1.0% NH4OH and 20% CH3OH
in CH2Cl2). Therefore, this strategy enabled the rapid
preparation of a range of N-(3-phenylpropyl) sub-
stituted spermidine and spermine analogues.14
In this paper, we report the preliminary results of a
structure±activity study of N-(3-phenylpropyl)-sub-
stituted spermine and spermidine derivatives against
recombinant T. cruzi TR. Additionally, we report the in
vitro activities of several of these compounds against four
T. brucei strains. As expected, the quantity and location of
the 3-phenylpropyl moieties dramatically in¯uences the
inhibition and trypanocidal pro®les of compounds. Sev-
eral of the compounds described are potent competitive
inhibitors of TR, with the most e€ective compound
being N1,N1,N4,N8,N12-penta(3-phenylpropyl)spermine
(12) (Ki value of 0.151 mM). Compound 12 is one of the
most e€ective competitive inhibitors of TR described to
date. Furthermore, none of the compounds studied
inhibited GR, indicating that they show speci®city for
TR. Compounds that were e€ective TR inhibitors were
also e€ective trypanocides in vitro.
Trypanothione reductase from T. cruzi was puri®ed
following the procedure of Walsh et al.15 from an SG5
Escherichia coli strain (a glutathione reductase deletion
mutant) containing the expression vector pIBITczTR
The synthetic strategies used to prepare spermidine and
spermine analogues are detailed in Schemes 1 and 2,
respectively. The initial steps of both syntheses relied on
the one-step selective tri¯uoroacetylation of the primary
amino groups of polyamines as described previously.12
Compounds 1 and 6 were prepared as described pre-
viously.10 Alkylation of the primary amino groups of
Scheme 1. (i) PhCH2CH2CH2Br, Et3N, CH3CN, re¯ux; (ii) NH4OH,
MeOH, re¯ux; (iii) PhCH2CH2CHO, NaBH(OAc)3, CH2ClCH2Cl.
Scheme 2. (i) PhCH2CH2CHO, NaBH(OAc)3, CH2ClCH2Cl; (ii) H2,
Pd/C, EtOH.

Z. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 251±254
253
described by Sullivan and Walsh.16 The e€ects of synthetic
compounds on the rate of reduction of trypanothione by
T. cruzi TR were assayed spectrophotometrically by
monitoring the oxidation of NADPH at 340 nm.17
Enzyme activity was measured at 23 ꢀC in HEPES buf-
fer (100 mM, pH 7.25) containing EDTA (1 mM),
NADPH (0.18 mM) and oxidized trypanothione, with a
TR concentration of 2.2 mg/mL. For each compound
assayed, the velocity of the TR-catalyzed reaction was
measured at ®ve trypanothione concentrations (15±
75 mM) and ®ve inhibitor concentrations.
Table 1. Ki values for the competitive inhibition by compounds of
trypanothione disul®de reduction by recombinant TR from T. cruzi
Compound
Ki (mM)ÆSD
3 R1=R2=H
14.1Æ0.9
4 Mixture of two isomers: R1=H, R2=
PhCH2CH2CH2 and R1=PhCH2CH2CH2, R2=H
5 R1=R2=PhCH2CH2CH2
4.67Æ1.10
3.50Æ0.34
Inhibition type was assessed by the patterns of three clas-
ses of plots: 1/v against 1/[So] for various [I]; 1/v against [I]
for various [So]; and [So]/v against [I] at various [So]. All
compounds tested showed linear competitive inhibition
against TR reduction of trypanothione. For each inhi-
bitor concentration, Km(obs) (with standard deviation)
and Vmax were determined from a nonlinear regression
analysis of the plot of v against [So] using the Michaelis±
Menten equation. The Ki value was then determined from
a weighted regression analysis of the plot of Km(obs)
against [I] using the equation below (weighting factors
used were 1/(SD of Km(obs))):
1 R1=R2=R3=R4=H
3.48Æ0.38
1.38Æ0.30
0.614Æ0.147
0.151Æ0.032
10.3Æ4.7
10 R1=PhCH2CH2CH2, R2=R3=R4=H
11 R1=R3=PhCH2CH2CH2, R2=R4=H
12 R1=R2=R3=PhCH2CH2CH2, R4=H
13 R1=R2=R3=R4=PhCH2CH2CH2
Kmꢀobs† ˆ ꢀKm‰IŠ=Ki† ‡ Km
The Ki values for each compound and their standard
deviations are given in Table 1. None of the compounds
tested caused the TR-mediated oxidation of NADPH in
the absence of trypanothione. Thus, as expected, none of
the compounds tested were TR substrates. At concentra-
tions of 250 mM, compounds did not inhibit the reduction
of glutathione by yeast GR (EC 1.6.4.2), assayed using
similar conditions to those described with TR (due to the
limited solubilities of 12 and 13 in water, the e€ects of
these compounds against GR were measured at 20 mM).
Hence, inhibitors show speci®city for TR with respect to
GR.
14 R1=R2=H
37.6Æ6.8
12.3Æ1.7
3.62Æ1.10
15 R1=H, R2=PhCH2CH2CH2
16 R1=R2=PhCH2CH2CH2
Table 2. Trypanocidal activities of compounds against four T. brucei
ssp. strains in vitro
IC50 (mM)
Compounda
Lab 110b
K 243c
K 269c
K 243-
As-10-3d
1
4
5
10
11
12
13
15
16
0.66
0.31
0.22
0.27
0.12
3.05
22
0.79
0.53
0.36
0.16
0.14
3.35
35
0.58
0.38
0.54
0.16
0.16
2.2
0.66
0.57
0.46
0.16
0.15
5.5
24
2.2
0.53
The in vitro trypanocidal activities of compounds on
bloodstream forms of clinically isolated strains of T. b.
brucei ssp. were examined using a standard growth
screen.18 The trypanosome strains used were T. b. brucei
Lab EATRO and three clinical isolates of T. b. rhodesiense.
The in vitro trypanocidal activities are given in Table 2.
30.5
1.8
0.81
0.40
1.5
0.16
0.27
aThe hydrochloride salts of compounds were used (the tri¯uoroacetate
salt of 1 was used as previously reported10).
For each compound studied, inhibition of TR increased
with increasing numbers of 3-phenylpropyl substituents,
with N1,N1,N4,N8,N12 - penta(3 - phenylpropyl)spermine
(12) being the most e€ective inhibitor studied. However,
the hexa-substituted derivative (13) was 1.5 orders of
magnitude less e€ective than 12, suggesting that the TR
active site cannot e€ectively accommodate the steric
bulk of 13. The location of the substituents also impac-
ted the inhibiting activities of compounds. Compounds
with substituents at N1 and N12 were less e€ective inhi-
bitors than corresponding analogues with N4 and N8
substituents, for example, compound 15 was not as
e€ective as 1. The inhibition pro®les of the spermidine
analogues studied were similar to those of spermine
analogues with the corresponding number of 3-phenyl-
propyl substituents, that is compounds 4 and 1 have
bT. b. brucei Lab 110 EATRO is a drug-sensitive strain.
cKETRI 243 and KETRI 269 are uncloned clinical isolates of T. b.
rhodesiense.
dKETRI 243-As-10-3 is a pentamidine and melarsoprol-resistant clone
of T. b. rhodesiense KETRI 243.
similar Ki values. Therefore, for this class of compounds,
the number of hydrophobic substituents has a greater
in¯uence on the binding of inhibitors to the active site of
TR than the net charge of the inhibitor. Although, TR
and GR have similarities, the compounds studied did
not inhibit GR. These observations can be rationalized
by comparing the electrostatic and steric characteristics
of the active sites of TR and GR. The active site of TR
is large, contains a glutamate residue (E19) and a

254
Z. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 251±254
hydrophobic area, whereas GR has a smaller active site
containing several cationic residues (R37, R38, and
R347) that are not present in TR.6,19 The active site of
TR can therefore accommodate large, positively
charged compounds. The charge of inhibitors is known
to be a major factor in their selectivity for TR versus
GR.8 However, charge is not the sole factor involved in
the binding of compounds to TR, since spermine does
not inhibit TR.10 The crystal structures of the trypa-
nothione±TR complex and that of the mepacrine±TR
complex indicate that hydrophobic interactions between
ligands and a hydrophobic wall in the active site (L18,
W22, Y111, and M114) play an important role in ligand
binding.19,20 Hence, the hydrophobicity of the 3-phenyl-
propyl moiety and possibly its ability to participate in
cation±p interactions21 are important contributing factors
to the binding of N-(3-phenylpropyl)-substituted poly-
amines to the active site of TR.
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Most of the compounds studied also displayed in vitro
trypanocidal activities. These activities correlated
approximately with each compound's ability to inhibit
TR. One factor that possibly may have contributed to
discrepancies in the correlation between Ki and IC50
values is the presence of polyamine oxidase which occurs
in fetal bovine serum (this is required in the culture med-
ium for the blood form of T. brucei). However, since
polyamine oxidase catalyzes the oxidation of primary
amines, it is extremely unlikely that polyamine oxidase
would degrade compound 12. Compound 12 was the
major exception to the observed overall correlation, being
the most e€ective inhibitor of TR but one of the less
e€ective trypanocides studied. Another factor that
could contribute to disparities in the observed correla-
tion is the possibility that the compounds studied may
cross the trypanosomal cell membrane at di€ering rates.
In conclusion, this paper describes a class of novel,
e€ective TR inhibitors that also display signi®cant try-
panocidal activity. Ecient, selective syntheses of the
compounds in this study are being developed in order to
produce the quantities of compounds needed for in vivo
trypanocidal studies.
10. O'Sullivan, M. C.; Zhou, Q.; Li, Z.; Durham, T.; Rattendi,
D.; Lane, S.; Bacchi, C. J. Bioorg. Med. Chem. 1997, 5, 2145.
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Acknowledgements
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14. The 1H, 13C NMR, and mass spectra of all compounds
were consistent with the assigned structures.
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We are extremely thankful to Drs. Christopher Walsh and
Kari Nadeau (Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School),
David Alberg (Chemistry Department, Carleton College)
and Christopher Mehlin (Department of Pathobiology,
University of Washington) for providing the SG5 E. coli
strain containing pIBITczTR. This investigation received
®nancial support from the UNDP/World Bank/WHO
Special Programme for Research and Training in Tropical
Diseases (M. C. O. grant #920173 and C. J. B. 950504).
18. Sufrin, J. R.; Rattendi, D.; Spiess, A. J.; Lane, S.; Mar-
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