Antiprotozoal and cytotoxicity evaluation of sulfonamide and urea analogues of quinacrine

Article abstract of DOI:10.1016/S0960-894X(01)00528-5

Sulfonamide and urea derivatives of quinacrine with varying methylene spacer lengths were synthesised and tested for inhibition of trypanothione reductase (TryR) and for activity in vitro against strains of the parasitic protozoa Trypanosoma, Leishmania, and Plasmodium. These derivatives are superior inhibitors of TryR relative to quinacrine with the best compound being 40 times more potent. Urea derivatives generally displayed good in vitro activity against all parasites.

Full text of DOI:10.1016/S0960-894X(01)00528-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2655–2657
Antiprotozoal and Cytotoxicity Evaluation of Sulfonamide and
Urea Analogues of Quinacrine
Kelly Chibale,a,* Hayley Haupt,a Howard Kendrick,b Vanessa Yardley,b
Ahilan Saravanamuthu,c Alan H. Fairlambc and Simon L. Croftb
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDepartment of Infectious and TropicalDiseases, London Schoolof Hygiene & TropicalMedicine, KeppelStreet,
London WC1E 7HT, UK
cDivision of Biological Chemistry & Molecular Microbiology, School of Life Sciences, Wellcome Trust Biocentre,
University of Dundee, Dundee DD1 5EH, UK
Received 31 May 2001; accepted 27 July 2001
Abstract—Sulfonamide and urea derivatives of quinacrine with varying methylene spacer lengths were synthesised and tested for
inhibition of trypanothione reductase (TryR) and for activity in vitro against strains of the parasitic protozoa Trypanosoma,
Leishmania, and Plasmodium. These derivatives are superior inhibitors of TryR relative to quinacrine with the best compound being
40 times more potent. Urea derivatives generally displayed good in vitro activity against all parasites. # 2001 Elsevier Science Ltd.
All rights reserved.
We have become interested in utilising the acridine
moiety of the antiprotozoal drug quinacrine 1 (also
known as mepacrine) as a potential template for the
design and discovery of antileishmanial, antigiardial,
antimalarial, and antitrypanosomal agents. The use of 1
in the treatment of cutaneous leishmaniasis and its in
vitro activity against Trypanosoma cruzi are known.1,2 It
is noteworthy that acridine derivatives have been con-
sidered for the treatment of several protozoan infec-
tions.3 Recently, a number of 9-anilinoacridines and
bis(9-amino-6-chloro-2-methoxyacridines) with anti-
malarial, antileishmanial and antitrypanosomal activ-
ities have been reported.4À6 Various modes of action of
quinacrine and derivatives have previously been pro-
posed, including inhibition of trypanothione reductase
(TryR),7 intercalation,8 binding to haem,9 and inhibition
of the enzyme topoisomerase II.6
Within the context of inhibition of TryR by quinacrine,
two binding modes for 1 have been predicted.10 The
tricyclic moiety of quinacrine binds in two hydrophobic
pockets of the enzyme. We became interested in testing
this model by incorporating methylene spacers between
the tricyclic moiety of 1 and a second hydrophobic
aromatic moiety. This led to the preliminary design of
sulfonamides 2 and ureas 3 (Fig. 1). The sulfonamide
Figure 1. Chemical structures of quinacrine 1 and sulfonamide 2, and urea derivatives 3.
*Corresponding author. Tel.: +27-21-650-2553; fax: +27-21-687-7499; e-mail: chibale@science.uct.ac.za
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00528-5

2656
K. Chibale et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2655–2657
Scheme 1. Reagents and conditions: (a) 32.0 equiv of H2N(CH2)nNH2, phenol, 90 ꢀC, 4 h, 90–95%; (b) 1.2 equiv of 2-naphthylsulfonyl chloride, 2.0
equiv of Et3N, DMF, 43–94%; (c) 1.1 equiv of benzyl isocyanate, CH2Cl2, 25 ꢀC, h, 60–96%.
and urea moieties were incorporated with a view to
improving solubility properties of the molecules. We
were also interested in the in vitro antiparasitic activities
of these compounds in view of previous aforementioned
literature reports on related compounds.4À6
explain this loss in selectivity.12 The three- and four-
carbon methylene spacer length appears optimum for
inhibition of TryR within this series of compounds.
Compounds were tested in vitro for activity against
amastigote forms of Leishmania donovani (cultured in
murine macrophages), amastigote forms of Trypano-
soma cruzi (cultured in murine macrophages), and the
bloodstream form trypomastigote Trypanosoma brucei
rhodesiense. Experimental details have previously been
described.13 For Plasmodium falciparum, all compounds
were tested against the chloroquine-sensitive strain (3-
D7) and the most potent also tested against a chloro-
quine-resistant strain (K1). The whole cell inhibition
assay of Plasmodium falciparum growth in human red
blood cells was carried out in a 48 h [3H]-hypoxanthine
incorporation assay.14,15 Potential toxicity of the quin-
acrine derivatives was determined against the KB cell
line in comparison to podophyllotoxin. The data are
presented in Table 2. Data for standard control drugs
are included for comparative purposes.
The synthesis of the target molecules was simple and
straightforward and is depicted in Scheme 1. The amine
scaffolds 5 were synthesised by reacting 6,9-dichloro-2-
methoxyacridine 4 with a large excess of diamines in
phenol. The resulting products were converted to the
respective sulfonamide and urea products by standard
reaction with the respective acid chloride and isocya-
1
nate. All new compounds gave H NMR, FABMS and,
in relevant cases, microanalysis data consistent with
their structures.
The enzyme assays were performed essentially as
described before.11 Recombinant T. cruzi TryR (128
mU) was assayed using a Beckman DU640 spectro-
photometer in 40 mM HEPES, pH 7.5, 1 mM EDTA
and 200 mM NADPH at 25 ꢀC followed by the addition
of 100 mM Try[SH]2. Human glutathione reductase,
purified from human erythrocytes (42.3 mU), was ana-
lysed in a similar manner and under identical conditions
followed by addition of glutathione disulfide (100 mM).
Enzyme mixtures were preincubated with NADPH (10
min at 25 ꢀC) before the addition of varying concentra-
tions of inhibitor added in DMSO (1% v/v final con-
centration).
Compounds generally showed significant activity
against L. donovani with compounds 2b, 3b, and 3c
being the most superior. These data correlate well with
the data for inhibition of TryR in Table 1. With the
exception of urea compounds 3c and 3d, which showed
good activity against T. cruzi, the rest of the compounds
displayed weak to no activity at the test concentrations.
Like with inhibition of TryR and activity against L.
donovani, compounds 2b, 3b, and 3c showed the best
activity against T. brucei. The same picture is more or
less true for P. falciparum where the compounds showed
the greatest activity, compound 3b being most note-
worthy. This compound was 4 and 10 times more active
than chloroquine against 3D7 and K1 strains, respec-
tively. The fact that 3b is less active against the K1 than
the 3D7 strain suggests haem polymerization as a target,
like the 4-aminoquinolines.9 This, in turn, suggests cross-
resistance with chloroquine. The compounds that were
active against L. donovani and T. cruzi were also toxic to
As can be seen from the data in Table 1, compounds 2
and 3 were generally superior to quinacrine in inhibiting
TryR. Sulfonamides 2 were generally superior to ureas
3. This may be due to a more hydrophobic naphthalene
moiety in 2 compared to the benzyl moiety in 3. How-
ever, despite the superior inhibitory activities of the
derivatives, selectivity against the equivalent human
enzyme, glutathione reductase, was lost. The loss of a
positive charge (from the terminal tertiary amino group)
in moving from quinacrine to the new derivatives could
Table 1. Inhibition of trypanothione reductase and glutathione reductase by quinacrine and analogues
Compound
Number of
methylene groups (n)
Trypanothione
reductase IC50 (mM)
Glutathione reductase
IC50 (mM)
Selectivity
indexa
Quinacrine 1
N/A
133Æ11
5.9Æ0.6
3.3Æ0.3
5.0Æ0.2
19.3Æ1.0
13.1Æ0.7
15.5Æ0.8
11.4Æ0.7
>1000
9.9Æ0.4
>7.5
1.7
8.2
2.8
1.4
3.4
3.6
1.5
2a
2b
2c
3a
3b
3c
3d
2
3
4
2
3
4
6
27.2Æ0.6
13.9Æ1.3
27.2Æ0.6
44.7Æ4.4
55.8Æ 2.6
17.1Æ 3.1
aRatio IC50 GR/IC50 TryR.

K. Chibale et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2655–2657
Table 2. In vitro sensitivity of parasites to quinacrine analogues 2 and 3
2657
Compound
ED50 (mg/mL)
Toxicity ED50 (mg/mL)
L. donovani
T. cruzi
T. brucei
P. falciparum
3D7a
P. falciparum
K1b
Podophyllotoxin
Pentostam
0.008
8.9
Benznidazole
Pentamidine
Chloroquine
2a
2b
2c
3a
3b
3c
3d
12.4
0.0002
0.002
0.025
0.010
0.033
0.069
0.0005
0.0013
0.14
0.15
ndc
nd
nd
nd
0.015
nd
nd
5.8
1.9
3.3
10.7
1.9
1.9
5.8
>30
23.9
>30
>30
22.0
<1
0.47
2.7
0.4
0.4
4.1
0.7
0.8
4.3
0.078
0.12
0.42
0.083
0.043
0.46
6.8
aChloroquine-sensitive strain.
bChloroquine-resistant strain.
cNot determined.
References and Notes
KB cells at the same concentrations, indicating a lack of
selectivity against these parasites.
1. Neal, R. A. In The Leishmaniasis; Peters, W., Killick-Ken-
drick, R., Eds.; Academic: London, 1987; pp 793–845.
2. Hammond, D. J.; Cover, B.; Gutteridge, E. W. Trans. R.
Soc. Trop. Med. Hyg. 1984, 78, 91.
3. Obexer, W.; Schmid, C.; Barbe, J.; Galy, J. P.; Brun, R.
Trop. Med. Parasitol. 1995, 46, 49.
4. Girault, S.; Grellier, P.; Berecibar, A.; Maes, L.; Mouray,
E.; Lemiere, P.; Debreu, M.-A.; Davioud-Charvet, E.; Ser-
gheraert, C. J. Med. Chem. 2000, 43, 2646.
5. Gamage, S. A.; Figgitt, D. P.; Wojcik, S. J.; Ralph, R. K.;
Ransijn, A.; Mauel, J.; Yardley, V.; Snowdon, D.; Croft, S. L.;
Denny, W. A. J. Med. Chem. 1997, 40, 2634.
6. Gamage, S. A.; Tepsiri, N.; Wilairat, P.; Wojcik, S. J.;
Figgitt, D. P.; Ralph, R. K.; Denny, W. A. J. Med. Chem.
1994, 37, 1486.
7. Bonse, S.; Santelli-Rouvier, C.; Barbe, J.; Krauth-Siegel,
R. L. J. Med. Chem. 1999, 42, 5448.
8. Chen, T. K.; Fico, R.; Canellakis, E. S. J. Med. Chem.
1978, 21, 868.
9. Dorn, A.; Vippagunta, S. R.; Matile, H.; Jaquet, C.; Ven-
nerstrom, J. L.; Ridley, R. G. Biochem. Pharmacol. 1998, 55,
727.
A series of sulfonamide and urea analogues of quina-
crine generally show superior activity (relative to
quinacrine) against TryR. Although sulfonamide
derivatives were more active than ureas in inhibiting
TryR, this trend did not correlate with the in vitro
activities against L. donovani, T. cruzi, and T. brucei.
Urea compounds in particular showed significant activ-
ity in vitro against all four parasites. Although T. cruzi
appears to be the least sensitive comparared to L.
donovani, it is noteworthy that compounds display
activity against these intracellular parasites where the
drug must cross the macrophage to reach the amasti-
gote. The mode of action of these compounds is not
clear and merits further investigation. Clearly, if a
common mechanism were operative, then inhibition of
TryR7 and polymerisation of haem into haemozoin9 can
be excluded as mechanisms, since malaria parasites do
not possess TryR and kinetoplastids do not polymerise
haem.
10. Horvath, D. J. Med. Chem. 1997, 40, 2412.
11. Borges, A.; Cunningham, M. L.; Tovar, J.; Fairlamb,
A. H. Eur. J. Biochem. 1995, 228, 745.
12. 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.
13. Chibale, K.; Visser, M.; Yardley, V.; Croft, S. L.; Fair-
lamb, A. H. Bioorg. Med. Chem. Lett. 2000, 10, 1147.
14. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay,
J. D. Antimicrob. Agents Chemother. 1979, 16, 710.
15. O’Neill, M. J.; Bray, D. H.; Boardman, P.; Phillipson,
J. D.; Warhurst, D. C. Planta Med. 1985, 394.
Acknowledgements
Financial support was through Grants 052075/Z/97
and 2039478 from the Wellcome Trust and National
Research Foundation of South Africa respectively (KC),
and UNDP/World Bank/WHO Special Programme for
Research and Training in Tropical Diseases (HK, VY,
SLC). AHF is supported by the Wellcome Trust.