Squalamine analogues as potential anti-trypanosomal and anti-leishmanial compounds

Article abstract of DOI:10.1016/S0960-894X(00)00196-7

This paper concerns the synthesis of various simplified analogues of the novel anti-microbial agent, squalamine. The compounds were then investigated for activity against Trypanosoma brucei, the causative agent of African trypanosomiasis, Trypanosoma cruz

Full text of DOI:10.1016/S0960-894X(00)00196-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1237±1239
Squalamine Analogues as Potential Anti-Trypanosomal and
Anti-Leishmanial Compounds
a
a
b
b
Soghra Khabnadideh, Choon Leei Tan, Simon L. Croft, Howard Kendrick,
b
a,
Vanessa Yardley and Ian H. Gilbert *
aWelsh School of Pharmacy, Cardi€ University, Redwood Building, King Edward VII Avenue, Cardi€, CF10 3XF, UK
bDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London, WC1E 7HT, UK
Received 1 December 1999; accepted 29 March 2000
AbstractÐThis paper concerns the synthesis of various simpli®ed analogues of the novel anti-microbial agent, squalamine. The
compounds were then investigated for activity against Trypanosoma brucei, the causative agent of African trypanosomiasis, Try-
panosoma cruzi, the causative agent of Chagas disease and Leishmania donovani, the causative agent of visceral leishmaniasis. Sev-
eral compounds showed in vitro activity, especially against T. brucei and L. donovani. However, one compound showed poor in vivo
activity. # 2000 Elsevier Science Ltd. All rights reserved.
8
Introduction
et al. Some of these analogues showed similar anti-
bacterial activity to the parent compound squalamine.
Variations in the structure of the analogues led to
changes in the spectrum of activity against a variety of
bacteria and yeasts. A number of conclusions can be
drawn from these studies.
Squalamine (1, Fig. 1) was ®rst isolated in 1993 from the
1
dog®sh shark, Squalus acanthias. It was shown to have
potent anti-microbial activity against Gram positive and
Gram negative bacteria and fungi. In addition, squala-
mine has been shown to have anti-angiogenic properties
2
,3
.
.
.
The precise structure of the polyamine is not
important.
The sulphate group can be replaced by a carboxylate
or even removed altogether.
The structure of the steroid can also be varied.
in several experimental tumour models. The toxicity
1
of squalamine has also been investigated. It has been
shown to cause haemolysis of red blood cells, although
this occurs at higher levels than that required for anti-
microbial activity, suggesting that there is a therapeutic
window.
The mode of action of squalamine and its derivatives
has not yet been determined, although a number of
Squalamine itself can only be obtained in small quan-
tities from the dog®sh shark. Although it has been pre-
7,8
suggestions have been made,
including membrane
4
,5
9
pared synthetically,
the synthesis is long and not
disruption. Squalamine may achieve this by acting as
an ionophore.10 It has been shown experimentally that
squalamine recognises negatively charged phospholipid
viable for detailed structure activity studies. Therefore a
number of simpli®ed analogues of squalamine have
6
11
been prepared, for example by Sadownik et al. who
membranes, and also has been implicated in disrup-
+
+
12
placed the sulphate group on the 3-position of the ster-
oid ring and the polyamine on the steroid side chain to
give compound 2 (Fig. 1), which was prepared in just
three synthetic steps. This compound retained some of
the anti-microbial activity of squalamine. A further set
tion of a Na /H antiport. Squalamine shows a
novel mechanism of action and it is important to assess
the extent of its potential as an antimicrobial agent.
Squalamine and its analogues have not been tested
against the parasitic protozoa responsible for leishma-
niasis, Chagas disease and African trypanosomiasis. As
part of a programme to derive novel agents against
these diseases we prepared a number of analogues of
squalamine to test against the causative parasites of the
7
of analogues were prepared by Jones et al. and Kikuchi
*
4
Corresponding author. Tel.: +44-29-2087-5800; fax: +44-29-2087-
149; e-mail: gilbertih@cf.ac.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00196-7

1
238
S. Khabnadideh et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1237±1239
Figure 1.
diseases: Leishmania donovani, Trypanosoma cruzi and
Trypanosoma brucei respectively. We were interested to
determine the role of the sulphate group, so compounds
were prepared with and without this group. We were also
interested to see the role of the amines in the polyamine
side chain, so protected and un-protected compounds
were prepared and tested.
was then reacted with di-tert-butyl dicarbonate to give
the fully protected compound 4b. The acetate on the 3-
position was then removed using lithium hydroxide to
give 5, followed by sulphation with pyridine±sulphur
trioxide complex to give 6 and then removal of the
BOC protecting groups with tri¯uoroacetic acid to give
7 and 8.
Chemistry
Biological assays
The squalamine analogues were prepared by a mod-
Compounds were investigated for activity against the
clinically relevant forms of T. brucei (bloodstream try-
pomastigote form), T. cruzi (amastigote form cultured
in macrophages) and L. donovani (amastigote form cul-
tured in macrophages) using established protocols.
The data is presented in Table 1.
6
,8
i®cation of the literature methods (Scheme 1). The
starting steroid, 3b-acetoxybisnor-5-cholenic acid, was
activated as the p-nitrophenol ester and then reacted
either with BOC-protected spermidine or unprotected
spermine. In the case of the latter, the reaction mixture
1
3
Scheme 1.

S. Khabnadideh et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1237±1239
1239
Table 1. ED50 (mM) values for activity of compounds against trypa-
nosomes and leishmania
Conclusion
A series of analogues of squalamine have been prepared.
Several showed signi®cant in vitro activity against T.
brucei and L. donovani and little activity against T. cruzi.
At this stage it is unclear why there is such a marked dif-
ference in activity between the species. This may have
something to do with the nature of the membrane.
Interestingly three compounds were active against L.
donovani which is found in the phagolysosomal vacuole
in the macrophage, whereas all were inactive against T.
cruzi amastigotes in the macrophage cytoplasm.
Compound
L. donovanib
T. cruzib
T. bruceib Toxicity
(KB cells)
Spermidine series
c
4a
5a
6a
7a
8a
17.3
25.7
4.9
44.7
39.9
>42
2.6, 0.55
111
65
>15 (toxic)a 4.7, 1.5
c
>36 (toxic)
>63 (toxic) 1.9, 0.63
13.5 >53
>36
>360
3.3
>526
c
Spermine series
4b
5b
6b
7b
8b
4.7
12.6
>30
5.0
20.5
>34 (toxic) 1.1, 0.57c
31
27
1.5
15
<0.47
>36
>30
0.60
5.4
In general the presence of the BOC or acetate protecting
groups had little e€ect on activity, whilst the presence of
the sulphate group appeared to decrease activity.
c
>57 (toxic) 0.56, 1.1
>48 >48
a
Toxic=toxic e€ects seen on the macrophages.
Standard Drugs: L. donovani, Pentostam, ED50=6.16 mgSb /ml; T.
b
V
cruzi, benznidazole ED50=7.45 mg/ml; T. brucei, pentamidine=100%
inhibition at 1 mg/mL.
Results of two experiments.
Acknowledgements
c
Trypanosoma brucei
We wish to acknowledge the support of the Royal
Society, and the World Health Organisation Special
Programme for Training and Research in Tropical Dis-
eases for ®nancial support. We also wish to acknowl-
edge the EPSRC National Mass Spectrometry Service
Centre.
Compounds show the greatest activity against T. brucei,
with ®ve compounds showing ED50 values at or around
1
mM. A number of points can be made.
.
.
The spermidine and spermine series show similar
activity.
The presence of the BOC protection on the poly-
amine or the acetate on the hydroxyl seemed to
have minimal e€ect on the activity (compare com-
pounds 4 and 5 with 7).
References and Notes
.
The sulphated compounds (6 and 8) were the least
active compounds. This may be due to poor
uptake into the parasites.
1. Moore, K. S.; Wehrli, S.; Roder, H.; Rogers, M.; Forrest, J.
N.; McCrimmon, D.; Zaslo€, M. Proc. Natl. Acad. Sci. USA
1993, 90, 1354.
. Sills, A. K.; Williams, J. I.; Tyler, B. M.; Epstein, D. S.;
2
Leishmania donovani
Sipos, E. P.; Davis, J. D.; McLane, M. P.; Pitchford, S.; Che-
shire, K.; Cannon, F. H.; Kinney, W. A.; Chao, T. L.; Dono-
witz, M.; Laterra, J.; Zaslo€, M.; Brem, H. Cancer Research
Compounds 4b, 6a and 7b showed signi®cant activity
against L. donovani. In general the spermine series (b)
was more active than the spermidine series (a).
1
998, 58, 2784.
. Teicher, B. A.; Williams, J. I.; Takeuchi, H.; Ara, G.;
3
Herbst, R. S.; Buxton, D. Anticancer Research 1998, 18, 2567.
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rahedron Letts. 1994, 35, 8103.
5. Pechulis, A. D.; Bellevue, F. H.; Cio, C. L.; Trapp, S. G.;
Fojtik, J. P.; McKitty, A. A.; Kinney, W. A.; Frye, L. L. J.
Org. Chem. 1995, 60, 5121.
.
Again the fully protected compounds (4) showed
similar or greater activity than the partially (5) or
fully deprotected (7) compounds.
Sulphation reduced the activity of compounds (6
and 8) with the exception of 6a which is also BOC
protected.
4
.
6. Sadownik, A.; Deng, G.; Janout, V.; Regen, S. L. J. Am.
Chem. Soc. 1995, 117, 6138.
Trypanosoma cruzi
7. Jones, S. R.; Kinney, W. A.; Zhang, X.; Jones, L. M.;
Selinsky, B. S. Steroids 1996, 61, 565.
In general compounds showed much poorer activity
against T. cruzi with higher concentrations giving rise to
toxic side e€ects on the host macrophages in which the
T. cruzi was cultured.
8
. Kikuchi, K.; Bernard, E. M.; Sadownik, A.; Regen, S. L.;
Armstrong, D. Antimicrob. Agents Chemother. 1997, 41, 1433.
9. Selinksy, B. S.; Zhou, Z.; Fojtik, K. G.; Jones, S. R.; Dol-
lahon, N. R.; Shinnar, A. E. Biochim. Biophys. Acta Ð Mem-
branes 1998, 1370, 218.
1
0. Deng, G.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1996,
118, 8975.
1. Merritt, M.; Lanier, M.; Deng, G.; Regen, S. L. J. Am.
Chem. Soc. 1998, 120, 8494.
2. Akhter, S.; Nath, S. K.; Tse, C. M.; Williams, J.; Zaslo€,
M.; Donowitz, M. American Journal of Physiology Ð Cell
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13. Croft, S. L.; Snowden, D.; Yardley, V. J. Antimicrobial
Chemotherapy 1996, 38, 1041.
In vivo tests
1
Compound 7b was also investigated against rodent mod-
els of African trypanosomiasis and leishmaniasis. There
was no e€ect against mice infected with T. b. rhodesiense
1
(
dosed at 50 mg/kg for 4 days intraperitonally) and only a
6% reduction in parasite load of mice infected with L.
donovani (dosed at 50 mg/kg for 5 days intraperitonally).
1