Synthesis and evaluation of 9,9-dimethylxanthene tricyclics against trypanothione reductase, Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani

Article abstract of DOI:10.1016/S0960-894X(00)00154-2

Derivatives of 9,9-dimethylxanthene was synthesised and evaluated against trypanothione reductase (TR) and in vitro against parasitic trypanosomes and leishmania. High in vitro antiparasitic activity was observed for some derivatives with one compound showing high activity against all three parasites (ED₅₀ values of 0.02, 0.48 and 0.32 μM, for Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani, respectively). The lack of correlation between inhibitory activity against TR and ED₅₀ values suggests that TR is not the target. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00154-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1147±1150
Synthesis and Evaluation of 9,9-Dimethylxanthene Tricyclics
Against Trypanothione Reductase, Trypanosoma brucei,
Trypanosoma cruzi and Leishmania donovani
Kelly Chibale, a,* Mark Visser, a Vanessa Yardley, b Simon L. Croft b
and Alan H. Fairlamb c
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDepartmentofInfectiousandTropicalDiseases,LondonSchoolofHygieneandTropicalMedicine,KeppelStreet,LondonWC1E7HT,UK
cDepartment of Biochemistry, The Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Received 19 November 1999; accepted 28 February 2000
AbstractÐDerivatives of 9,9-dimethylxanthene were synthesised and evaluated against trypanothione reductase (TR) and in vitro
against parasitic trypanosomes and leishmania. High in vitro antiparasitic activity was observed for some derivatives with one
compound showing high activity against all three parasites (ED50 values of 0.02, 0.48 and 0.32 mM, for Trypanosoma brucei, Try-
panosoma cruzi, and Leishmania donovani, respectively). The lack of correlation between inhibitory activity against TR and ED50
values suggests that TR is not the target. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
found in other tricyclic compounds already reported as
competitive inhibitors of TR, where the tricyclic moiety
binds in the hydrophobic pocket involved in the recog-
nition of the spermidine moiety of trypanothione dis-
ul®de, the substrate for TR.6,7 Second, the chemically
reactive 2,7 and 4,5 positions of the xanthene moiety
provide potential multiple sites for introducing chemical
diversity.8,9 This would ultimately aid analogue syn-
thesis and exploration of structure±activity relationships
within this class of compounds. Third, a terminal ter-
tiary amino group (exempli®ed by the dimethylamino
group) was incorporated into compounds 1±3 to pro-
vide a positive charge which has been shown to favor TR
over glutathione reductase (GR), the closest related host
enzyme.10 Intermediary functional groups (amides,
amines and thioureas) were incorporated to improve
solubility properties.
Tricyclic compounds exempli®ed by acridines, benzoa-
zepines, phenothiazines, and pyridoquinolines have pro-
vided drug leads in the area of chemotherapy of
trypanosomiasis and leishmaniasis.1 4 Inhibition of the
parasite enzyme trypanothione reductase (TR) is one
possible mechanism of action of these compounds. TR is
a vulnerable target for drugs that disrupt the natural
redox defence systems of the parasitic protozoa Trypa-
nosoma and Leishmania, the causative agents of human
African trypanosomiasis (T. brucei sspp), Chagas disease
(T. cruzi), and leishmaniasis (Leishmania sspp).5
A literature search revealed that tricyclics based on the
9,9-dimethylxanthene moiety have not been previously
investigated as potential TR inhibitors. Thus we decided
to conduct a preliminary investigation into the potential
of the subject compounds exempli®ed by 1±3 (Fig. 1). The
rationale behind the choice of the subject compounds was
as follows: First, being an aromatic hydrophobic tricyclic
moiety, the 9,9-dimethylxanthene moiety bears resem-
blance to the aromatic hydrophobic tricyclic moieties
Chemistry
The synthesis of derivatives 1±3 is depicted in Schemes 1
and 2. The starting materials 4b and 5 are commercially
available while 4a was synthesised from 5 by adapting a
literature method.11 Compound 1a was synthesised in
moderate yield in a one-pot reaction from 4a while 1c was
prepared via the corresponding acid chloride derivative of
4b (Scheme 1).
*Corresponding author. Tel.: +27-21-650-2553; fax: +27-21-689-
7499; e-mail: chibale@psipsy.uct.ac.za
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00154-2

1148
K. Chibale et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1147±1150
Figure 1. Structures of 9,9-dimethylxanthene tricyclics with two side chains.
For the synthesis of compounds 1b, 2a, 2b, and 3, the
key intermediates were dialdehyde 6 and a,b-unsatu-
rated diester 7. Compound 6 was obtained via ortho
lithiation while Wittig two-carbon homologation of 6
gave 7. Compound 1b was synthesised in a one-pot
reaction from dialdehyde 6 and subsequent in situ
reduction of the imine formed using polymer-supported
borohydride while compound 2a was obtained in four
steps involving catalytic hydrogenation (Pd-C), hydro-
lysis, activation of the acid via acid chloride formation
and coupling to the appropriate amine. Compounds 2b
and 3 were synthesised from a common intermediate 9
as shown. While the two-step sequence to 2b proceeded
in satisfactory yield with the exception of the ®nal step,
low yields were a feature of reactions leading to 3
(Scheme 2). All new compounds gave 1H NMR and
FABMS consistent with their structures.
Results and Discussion
The data in Table 1 shows the activity of derivatives 1±3
against TR, L. donovani amastigotes, T. cruzi amasti-
gotes as well as T. brucei bloodstream form trypomasti-
gotes.12 The data for standard control drugs is included
for comparison purposes. As far as the inhibition of TR
is concerned compounds 1a±1c bearing either one (1b)
or no methylene spacer (1a and 1c) between the tricyclic
moiety and the secondary nitrogen atom generally show
weaker inhibition of TR compared to derivatives with a
two or three carbon methylene spacer (2a, 2b, and 3).
For in vitro antiparasitic activity, the rapidly dividing
extracellular T. brucei bloodstream form trypomastigotes
showed the highest in vitro sensitivity to derivatives 1±3
with ED50 values below 5 mg/mL in all cases while the
Scheme 1. Reagents and conditions: (a) 2.5 equiv of (PhO)2P(O)N3, 2.0
equiv of Et3N, 2.0 equiv of H2N(CH2)3NMe2, PhMe, 85 ꢀC, 18 h, 65%;
(b) 3.0 equiv of (COCl)2, DMF (cat.), CH2Cl2, re¯ux, 3 h; then (c) 2.0
equiv of H2N(CH2)3NMe2, 2.0 equiv Et3N, CH2Cl2, 25 ꢀC, 12 h, 91%.
Scheme 2. Reagents and conditions: (a) 3.0 equiv of BuLi, 3.0 equiv of TMEDA; then (b) 3.0 equiv of DMF, Et2O, 25 ꢀC, 18 h, 72%; (c) 1.8 equiv of
H2N(CH2)3NMe2, MeOH, 25 ꢀC, 15 h; (d) 4.0 equiv of Amberlite IRA±400 borohydride resin, 25 ꢀC, 18 h, 100%; (e) 3.0 equiv of Ph3P=CHCO2Et,
CH2Cl2, re¯ux, 1 h, 93%; (f) H2, Pd-C, EtOAc, 25 ꢀC, 18 h, 100%; (g) 3.0 equiv of LiOH.H2O, THF:H2O (10:1), 25 ꢀC, 12 h, 93%; (h) 3.0 equiv of
(COCl)2, DMF (cat.), CH2Cl2, re¯ux 3 h, then 2.0 equiv of H2N(CH2)3NMe2, 2.0 equiv Et3N, CH2Cl2, 25 ꢀC, 12 h, 65%; (i) 2.0 equiv of LiAlH4,
Et2O, 0 ꢀC, 0.5 h, 97%; (j) 3.0 equiv of I2, 3.0 equiv of Ph3P, 3.0 equiv of imidazole, Et2O:CH3CN (3:1), 25 ꢀC, 5 h, 72%; (k) 16.0 equiv of
H2N(CH2)3NMe2, THF, re¯ux, 1 h, 68%.; (l) 2.0 equiv of Ph3P, 2.0 equiv of DIAD, 2.0 equiv of (PhO)2P(O)N3, THF, 15 to 25 ꢀC, 4 h, 57%; (m)
14.0 equiv of carbon CS2, 2.0 equiv of DCC, THF, 25 ꢀC, 18 h, 20%; (n) 2.6 equiv of H2N(CH2)3NMe2, CH2Cl2, 25 ꢀC, 18 h, 100%.

K. Chibale et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1147±1150
1149
Table 1. Inhibition of TR and in vitro antiprotozoal activity of control drugs and compounds 1±312
Compound
% Inhibition of TR
ED50 (mg/mL) (mM)
Cytotoxicitya MIC (mg/mL)
L. donovani
T. cruzi
T. brucei
Pentostam
Benznidazole
Pentamidine
1a
1b
1c
2a
2b
3
Ð
Ð
Ð
12.5 (102.7)b
Ð
Ð
>30
>30
0.32 (0.55)
17.03 (32.6)
>30
Ð
8.5 (32.7)
Ð
>30
>30
0.28 (0.48)
>30
>30
Ð
Ð
0.01 (0.03)
4.5 (9.66)
3.32 (7.58)
0.01 (0.02)
4.3 (8.24)
0.07 (0.14)
0.06 (0.098)
40
>300
>300
>30
>300
>30
>30
13.8
28.4
58
63
52
>30
>30
aCytotoxicity is derived from the host macrophages used to culture the amastigote stages of T. cruzi and L. donovani.
bPentostam is sodium stibogluconate and the ED50 value is expressed as mg Sbv/mL (mM Sbv).
intracellular slow dividing amastigote stages of L.
donovani and T. cruzi were the least sensitive. Com-
pounds 1c, 2b, and 3 showed the highest in vitro
potency against T. brucei. However, the most notable
compound is 1c, which on a molar basis, showed high
activity against all three parasites with ED50 values
either comparable (T. brucei) or superior (L. donovani
and T. cruzi) to the standard control drugs.
Rowland for help with the antiparasitic in vitro assays.
Financial support from the Wellcome Trust (Interna-
tional Research Development grant to KC, grant no.
052075/Z/97). Additional support from the University of
Cape Town and Foundation for Research Development
(KC) is gratefully acknowledged. This investigation
received ®nancial support from the UNDP/World
Bank/WHO Special Programme for Research and
Training in Tropical Diseases (TDR) (SLC and VY).
AHF is supported by the Wellcome Trust.
Although compound 2a shows comparable activity
against TR to 2b and 3 it does not possess comparable
high in vitro activity against T. brucei. Coupled with the
lack of activity of 2b and 3 against L. donovani and T.
cruzi but high activity against T. brucei, this suggests
that TR inhibition is not totally responsible for the
observed in vitro activities of these compounds. Other
factors such as cell penetration and metabolism, etc.,
may be playing a crucial role. It is noteworthy that for
the intracellular L. donovani and T. cruzi amastigotes,
the drug needs to pass through the macrophage to reach
the amastigote. Hence achieving selective toxicity is a
greater challenge in L. donovani and T. cruzi than in T.
brucei. For compound 1c, which showed high activity
against all three parasites but even weaker inhibitory
activity against TR, it is apparent that TR is not the tar-
get. Nevertheless it is encouraging that the more potent
compounds 1c, 2b and 3 are not overtly toxic to the
mammalian macrophages at or below a concentration of
30 mg/mL (estimated microscopically).
References and Notes
1. Benson, T. J.; McKie, J. H.; Garforth, J.; Borges, A.; Fair-
lamb, A. H.; Douglas, K. T. Biochem. J. 1992, 286, 9.
2. Chan, C.; Yin, H.; Garforth, J.; McKie, J. H.; Jaouhari, R.;
Speers, P.; Douglas, K. T.; Rock, P. J.; Yardley, V.; Croft, S.
L.; Fairlamb, A. H. J. Med. Chem. 1998, 41, 148.
3. Krauth-Siegel, R. L.; Lohrer, H.; Bucheler, U. S. In Bio-
chemica Protozoology; Coombs, G. H; North, M. J. (Eds.);
London: Taylor and Francis, 1991, pp 493±503.
4. Krauth-Siegel, R. L.; Jacoby, E. M.; Jockers-Scherubl, M.
C.; Schlichting, I.; Barbe, J. T. In Flavins and Flavoproteins;
Stevenson, K. J., Massey, V.; Williams, C. H., Jr. Eds.; Uni-
versity of Calgary: Canada, 1996; pp 35±44.
5. Fairlamb, A. H.; Blackburn, P.; Ulrich, P.; Chait, B. T.;
Cerami, A. Science 1985, 227, 1485.
6. Jacoby, E. M.; Schlichting, I.; Lantwin, C. B.; Kabsch, W.;
Krauth-Siegel, R. L. Proteins 1996, 24, 73.
7. Bond, C. S.; Zhang, Y.; Berriman, M.; Cunningham, M. L.;
Fairlamb, A. H.; Hunter, W. N. Structure 1999, 7, 81.
8. Carell, T.; Wintner, E. A.; Rebek, J. J. Angew. Chem., Int.
Ed. Engl. 1994, 33, 2061.
9. Carell, T.; Wintner, E. A.; Bashir-Hashemi, A.; Rebek, J.
Angew. Chem., Int. Ed. Engl. 1994, 33, 2059.
10. Faerman, C. H.; Savvides, S. N.; Strickland, C.; Brei-
denbach, M. A.; Ponasik, J. A.; Ganem, B.; Ripoll, D.;
Krauth-Siegel, R. L.; Karplus, P. A. Bioorg. Med. Chem. 1996,
4, 1247.
In conclusion within this series of compounds: (i) there
is no clear correlation between potency as inhibitors of
TR and the in vitro antiparasitic activities, and (ii) there
is no apparent single structural feature controlling in
vitro antiparasitic activities. These observations are con-
sistent with conclusions of previous ®ndings.2,16,17 In the
case of compound 1c which shows pronounced activity
against all three parasites, TR is clearly not the target.
The mechanism of action of the promising derivatives
1c, 2b, and 3 needs investigation if they are to serve as
useful leads for rational drug design.
11. Nowick, J. S.; Ballester, P.; Ebmeyer, F.; Rebek, J., Jr. J.
Am. Chem. Soc. 1990, 112, 8902.
12. TR assays: These were performed essentially as described
before13 by equilibrating a mixture of the assay bu€er (40 mM
Hepes, pH 7.5, 1 mM EDTA), 150 mM NADPH and 100 mM
test compound at 26.5 ꢀC for 15±20 min. After addition of TR
(T. cruzi recombinant puri®ed from Escherichia coli) the mix-
ture was pre-incubated for 2 min at 26.5 ꢀC. To initiate the
reaction 100 mM trypanothione disul®de (Bachem) was added.
Triplicate assays were performed. In vitro antiparasitic activity:
Acknowledgements
We thank Ahilan Saravanamuthu and Dr. David Parkin
for help with enzyme assays, Peter J. Rock and Diane

1150
K. Chibale et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1147±1150
L. donovani (MHOM/ET/67/L82) and T. cruzi (MHOM/BR/
00/Y): Peritoneal macrophages were harvested from female
CD1 mice (Charles River Ltd., Margate, UK) by peritoneal
lavage 24 h after starch (Merk Ltd., Leics., UK) induced
recruitment. After two washes in medium the exudate cells
were dispensed into 16-well Lab-tektm tissue culture slides
(Nunc Inc., IL, USA) at 4 Â 104/well in a volume of 200 mL of
RPMI-1640 medium (Sigma-Aldrich Company Ltd., Dorset,
UK) and 10% heat inactivated foetal calf serum (Harlan-Sea-
Lab Ltd, Crawley, UK). After 24 h, macrophages were infec-
UK) and nifurtimox (Bayer, UK) or benznidazole were used
as the respective control drugs. T. brucei (S427): Compounds
were tested in triplicate in a 3-fold dilution series from a top
concentration of 30 mM. Parasites were diluted to 2 Â 105/mL
and added in equal volumes to the test compounds in 96-well,
¯at bottom Microtest III tissue culture plates (Becton Dick-
inson and Company, NJ, USA). Appropriate controls with
pentamidine isethionate (Rhone-Poulonc-Rorer) as the stan-
dard were set up in parallel. Plates were maintained for 3 days
at 37 ꢀC in a 5% CO2/air mixture. Compound activity was
determined by the use of a tetrazolium salt colorimetric
assay15 on day 3.
13. Borges, A.; Cunningham, M. L.; Tovar, J.; Fairlamb, A.
H. Eur. J. Biochem. 1995, 228, 745.
14. Neal, R. A.; Croft, S. L. J. Antimicrob. Chemother. 1984,
14, 312.
15. Ellis, J. A.; Fish, W. R.; Sileghem, M.; McOdimba, F. Vet.
Parasitol. 1993, 50, 143.
16. Evans, A. T.; Croft, S. L. Biochem. Pharmacol. 1994, 48,
613.
ted at
a
ratio of 10:1 (4 Â 105/well) with L. donovani
amastigotes freshly isolated from hamster spleen or at a ratio
of 5:1 (2 Â 105/well) with T. cruzi trypomastigotes derived
from the overlay of MDCK ®broblasts. Infected macrophages
were then maintained in the presence of drug in a 3-fold dilu-
tion series, with quadruplicate cultures at each concentration,
for 5 days for L. donovani cultures and 3 days for T. cruzi cul-
tures. After these periods of drug exposure slides were metha-
nol ®xed and Giemsa stained and drug activity determined by
counting the percentage of macrophages cleared of amasti-
gotes in treated cultures in comparison to untreated cultures14
Sodium stibogluconate (NaSbv) (Glaxo-Wellcome, Dartford,
17. Hammond, D. J.; Hogg, J.; Gutteridge, W. E. Exp. Para-
sitol. 1985, 60, 32.