Design and synthesis of new adamantyl-substituted antileishmanial ether phospholipids

Article abstract of DOI:10.1016/j.bmcl.2010.07.078

A series of new 2-[3-(2-alkyloxy-ethyl)-adamantan-1-yl]-ethoxy substituted ether phospholipids was synthesized and their antileishmanial activity was evaluated against Leishmania infantum amastigotes. The majority of the new analogues were significantly l

Full text of DOI:10.1016/j.bmcl.2010.07.078

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5484–5487
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of new adamantyl-substituted antileishmanial
ether phospholipids
a
b
b
c
c
Ioannis Papanastasiou , Kyriakos C. Prousis , Kalliopi Georgikopoulou , Theofilos Pavlidis , Effie Scoulica ,
a
b,
*
Nicolas Kolocouris , Theodora Calogeropoulou
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Panepistimioupoli-Zografou, Athens 15771, Greece
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, Athens 11635, Greece
Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine, University of Crete, Heraklion 71409, Greece
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 2-[3-(2-alkyloxy-ethyl)-adamantan-1-yl]-ethoxy substituted ether phospholipids was
synthesized and their antileishmanial activity was evaluated against Leishmania infantum amastigotes.
The majority of the new analogues were significantly less cytotoxic than miltefosine while, antiparasitic
activity depended on the length of the 2-alkyloxy substituent. The most potent compounds were
Received 24 May 2010
Revised 16 July 2010
Accepted 18 July 2010
Available online 22 July 2010
{2-[[[3-(2-hexyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-N,N,N-trimethyl-ammonium
inner salt (5b) and {2-[[[3-(2-octyloxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-N,N,
N-trimethyl-ammonium inner salt (5c).
Keywords:
Leishmania infantum
Ether phospholipids
Miltefosine
Ó 2010 Elsevier Ltd. All rights reserved.
Antiparasitic
Leishmaniasis, is a parasitic disease which constitutes a major
public health problem especially in the tropical and subtropical re-
gions of the world. The causative agent is the protozoan parasite of
the genus Leishmania and depending on the species, the infection
leads to a large spectrum of clinical manifestations including cuta-
neous, mucocutaneous, and visceral forms. It is estimated that it
causes 70,000 deaths annually, a rate surpassed only by malaria
among other parasitic diseases.1 The parasite is transmitted by
the bite of the infected phlebotomine sandfly and even though most
forms of the disease are transmissible only among animals, human
leishmaniasis is increasingly spreading throughout the world. It is
currently endemic in 88 countries on five continents (Africa, Asia,
Europe, and North and South America), and the population at risk
reaches 350 million people. According to WHO, 12 million people
sidered an extremely serious new disease with severe clinical, diag-
nostic, chemotherapeutic, epidemiological, and economic
implications. To date, co-infection with leishmaniasis and HIV has
been reported in 34 countries in Africa, Asia, Europe, and South
3
America. Chemotherapy is currently the only way to treat the var-
ious forms of leishmaniasis, since no vaccine is yet available, how-
ever, the arsenal of drugs against the disease is still limited. Today,
first line antileishmanial drugs are pentavalent antimonials (so-
4
dium stibogluconate and meglumine antimonate), which are
5
slowly being replaced by liposomal amphotericin B, pentamidine
or the first oral drug against the visceral form of the disease, mil-
6
tefosine. However, all the aforementioned drugs have serious
drawbacks such as toxicity, poor efficacy or high cost. In addition,
the emergence of drug resistant parasites has complicated the cur-
rent chemotherapeutic strategies and thus, the invention of more
2
are infected worldwide, with 2 million new cases per year. The dis-
tinguishable forms of the disease include visceral leishmaniasis
7
effective and less toxic drugs is highly desirable. This has led many
(
VLa, or ‘kala-azar’), mucocutaneous leishmaniasis (MCL, ulceration
research groups including our to design and synthesize novel
antileishmanial compounds. Miltefosine (hexadecylphosphocho-
line) constituted a major breakthrough in antileishmanial chemo-
therapy, since this compound is effective against both visceral
and cutaneous leishmaniasis, displays good bioavailability, and is
currently registered as an oral drug for the treatment of the disease
in India (in 2002) and Colombia (in 2005). Despite its advantages,
miltefosine has a long half-life (100–200 h) in humans and a low
therapeutic ratio, characteristics that could encourage develop-
ment of resistance, especially in India where VL is an anthroponosis.
Moreover, it is not suitable for pregnant women because it has been
of the skin and hyper development of the mucous membranes), and
cutaneous and diffuse cutaneous leishmaniasis (CL and diffuse CL)
and, if untreated, can have devastating consequences. Establish-
ment of the infection and progression of the disease are favored
by the compromised immune system of patients, like those infected
with HIV, and as a result, leishmania/HIV co-infection is now con-
8
,9
*
Corresponding author.
E-mail address: tcalog@eie.gr (T. Calogeropoulou).
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.078

I. Papanastasiou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5484–5487
5485
shown to cause teratogenesis in animals10 and it did not give
Table 1
In vitro antileishmanial activitya against the intracellular amastigote form of L.
infantum
satisfactory results when administered to HIV-coinfected patients,
since most of them relapsed.11 The efficacy of miltefosine against
other non-Indian VL and the efficacy against the wide range of CL
syndromes in the new and the old World are also major issues that
need to be addressed. With the issues of resistance, side effects
and production cost still requiring special attention and the
mechanism of action still requiring elucidation, the field of design
and synthesis of novel antileishmanial phospholipid derivatives
is an ongoing challenge, especially against the strain Leishmania
Compound
Intracellular amastigote
form L. infantum
Cytotoxicity
IC50 M)
Selectivity
index
(l
1
2
13
MON 235 IC50 (lM)
I
II
III
IV
V
VI
VII
0.031 ± 0.01
5.69 ± 0.93
1.34 ± 0.56
5.2 ± 1.0
0.30 ± 0.2
0.8 ± 0.1
59.50 ± 2.11
168.7
37.45 ± 0.69
75.04
74.95
39.7 ± 0.3
147.3
1919.3
29.6
27.9
14.5
249.8
49.6
16.2
8.7
infantum, which causes the AIDS-associated co-infection in
Europe. In this context, we recently reported1
4–16
9.1 ± 0.5
6.7 ± 1
the synthesis of
Miltefosine (control)
28.6 ± 2.5
series of ring-substituted ether phospholipids, incorporating
-alkylidenecyclohexyloxylethyl, cyclohexylidene, cyclodecylidene
a
4
Results are expressed as mean ± SEM of three independent experiments.
cyclopentadecylidene or adamantylidene groups in the lipid
portion linked to the phosphate polar head-group by an oligometh-
ylene bridge of 5 or 11 carbons. In addition, we have varied the
polar head-group, using N,N,N-trimethylammonium, N-methylpi-
peridino, or N-methylmorpholino moieties, or we introduced more
rigid head groups, namely the trans-2-hydroxy-N,N,N-trimethyl-
cyclopentanamino or the trans-2-hydroxy-N,N,N-trimethylcyclo-
hexanamino group, the 3-(2-hydroxy-ethylidene)-8,8-dimethyl-8-
azonia-bicyclo[3.2.1]octane group and (4-hydroxy-but-2-ynyl)-
trimethylammonium group.
thus, resulting in more constrained derivatives. In addition, the
distance between the adamantane moiety and the head group
would be a short ethylene spacer, instead of an oligomethylene
1
4,16
spacer of 11 or 5 carbon atoms,
thus, giving us the opportunity
to investigate the optimum spacer length. In our design we have
maintained choline as head group on the basis of our previous find-
1
6
ings which indicated that this group confers to the corresponding
ether phospholipids reduced cytotoxicity while, activity remained
high. Our aim was also to investigate the effect of the alkoxyethyl-
ene group on activity and toxicity and to this end we varied the
length or the nature of the corresponding alkyl group (butyl, hexyl,
octyl, decyl, dodecyl and benzyl).
Our results indicated that introduction of cycloalkane rings in
alkylphosphocholines provides compounds with enhanced activity,
against L. infantum, and reduced cytotoxicity on the human
monocytic cell line THP-1.16 Among the ring-substituted ether
phospholipid derivatives that we synthesized, those containing an
adamantylidene moiety (Fig. 1) exhibited high activity against
L. infantum amastigotes and low cytotoxicity against THP-1 macro-
The synthetic strategy followed for the preparation of the new
ether phospholipids 5a–f is depicted in Scheme 2 and involves
phosphorylation of the appropriate alcohols 4a–f, followed by
hydrolysis and formation of the corresponding pyridinium salts,
which were in turn coupled to choline p-toluenesufonate.19 The
1
6,17
phages (Table 1).
Furthermore, we recently reported on the
trypanocidal activity of new 1-alkyl-2-aminoadamantanes and the
corresponding guanyl hydrazones and congeneric thiosemi-
synthesis of the required alcohols 4a–f was effected by esterifica-
1
8
20
carbazones.
The promising results of the new
tion of 1,3-bis(carboxymethyl)adamantane (1) followed by
4
reduction of 1,3-adamantanediacetate (2) by LiAlH to the corre-
x
-adamantylidene-substi-
tuted ether phospholipid derivatives, urged us to explore further
the substitution pattern of the adamantane moiety for enhanced
antileishmanial activity and reduced cytotoxicity. Herein, we de-
scribe the synthesis and biological evaluation of 2-[3-(2-alkyloxy-
ethyl)-adamantan-1-yl]-ethoxy substituted ether phospholipids.
Our aim was to introduce an ethylene spacer between the adaman-
tane tricyclic system and the phosphate group, while, at position 3
an alkoxyethylene moiety would be attached. In this manner the
adamantane system would be inserted within the lipid portion
sponding symmetric diol 3, which was in turn monoalkylated to af-
ford the desired alcohols 4a–f, as shown in Scheme 1.
The antiparasitic activity of derivatives 5a–f was assessed using
the intracellular amastigote form of L. infantum field strain MK-1 in
infected human monocytic THP-1 cells (Table 2, Supplementary
data). THP-1 cells infected with the appropriate Leishmania species
are used for the evaluation of the leishmanicidal activity of com-
pounds against the intracellular amastigote stages of the parasite,
and therefore we assessed the cytotoxicity of all new analogues
O
P
N
N+
O
O
O
7
O
I
P
O
O
O
7
V
O
O
P
N
N+
O
O
7
O
O
II
P
O
O
N+
7
P
N
O
VI
O
O
O
O
P
7
O
III
O
O
O
P
7
O
N
VII
O
O
7
O
IV
Figure 1. Structures of 11-adamantylideneundecyl-substituted ether phospholipids (I–VII).

5
486
I. Papanastasiou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5484–5487
CH COOH
2
CH COOEt
CH CH OH
2
2
2
a
b
CH COOH
2
CH COOEt
CH CH OH
2
2
2
1
2
3
CH CH OH
2
2
c
CH CH OR
2
2
4
4
4
4
4
4
a: R = C H
(34.7%)
(43.6%)
(43.0%)
(36.6%)
(55.7%)
4
6
9
13
b: R = C H
c: R = C H
8
1
17
d: R = C
H
0
21
25
6 5
e: R = C
H
2
1
f: R = CH C H (29%)
2
2
Scheme 1. Reagents and conditions: (a) (1) SOCl /reflux overnight; (2) EtOH, reflux 2 h, 100%; (b) LAH, THF, reflux 2 h, 87%; (c) (1) NaH, DMF, rt, 2 h; (2) RX, DMF, rt, six days.
O
P
a,b,c
N
ROH
+
POCl
3
O
RO
O
R
R
Yield
4
a
5a
5b
66%
69%
(
CH ) OC H
(CH ) OC H
2
2
4
9
2 2
4
9
4b
(
CH ) OC H
(CH
2
)
2
OC
6
H
13
2
2
6
13
4
c
5c
5d
5e
67%
65%
35%
(
CH ) OC H
(CH ) OC H
2 2 8 17
2
2
8
17
4d
(
CH ) OC10H21
(CH ) OC10H21
2 2
2
2
4e
(
CH ) OC12H25
(CH ) OC12H
2 2 25
2
2
4f
5f
54%
(
CH ) OBn
(CH ) OBn
2 2
2
2
Scheme 2. Reagents and conditions: (a) (1) P(O)Cl
3 3 2
, Et N, THF; (2) H O, 2-propanol; (b) pyridine, 40 °C; (c) pyridine, TIPS-Cl, choline p-toluenesulfonate.
against the human monocytic THP-1 host cell line (Table 2 and
Fig. 2, Supplementary data).
The most active analogues are the 3-hexyloxyethyl-adamantyl-
substituted analogue 5b and the 3-octyloxyethyl-adamantyl-
We examined the antileishmanial activity only against the
intracellular amastigote stage of the parasite for two reasons.
Firstly, this is the disease relevant form and secondly because we
and others have observed that alkylphosphocholines appear to be
more active against the intracellular amastigotes than the promas-
tigote stage, an effect which can be partially explained by their
ability to activate macrophages.21
substituted analogue 5c with IC50 values of 17.1 ± 1.8
16.2 ± 2.1 M, respectively. Moreover, these compounds do not ex-
hibit cytotoxicity (IC50>50 M). Decreasing the length of the alkyl
chain of the alkyloxyethyl substituent at position C3 of the adaman-
tane moiety to butyl, compound 5a, results in dramatic decrease of
lM and
l
l
activity (IC50>50
lM) while cytotoxicity remains unaffected
(IC50>50 M). Conversely, increasing the alkyl chain to decyl or
l

I. Papanastasiou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5484–5487
5487
Table 2
N,N,N-trimethyl-ammonium inner salt (5c) which were not
cytotoxic.
a
In vitro antileishmanial activity against the intracellular amastigote form of L.
infantum
Compound
Intracellular amastigote
form L. infantum field
Cytotoxicity
IC50 (lM)
Selectivity
index
Acknowledgements
strain MK-1 IC50 (lM)
The authors would like to thank Helen Koutala for the flow
cytometry measurements. K.C. Prousis is grateful to the State
Scholarships Foundation of Greece for financial support. This work
was supported in part by the European Commission Marie-Curie
Transfer of Knowledge Programme (MTKD-CT-2004-014399)
5
5
5
5
5
5
a
b
c
d
e
f
>50
>50
>50
>50
18.4 ± 2.5
21.4 ± 2.8
>50
NA
NA
NA
NA
NA
NA
11.2
17.1 ± 1.8
16.2 ± 2.1
>18.4
>21.4
41.8 ± 2.5
2.56 ± 1.2
‘
SOPHOLIDES’.
Miltefosine (control)
28.6 ± 2.5
NA: not applicable.
Supplementary data
a
Results are expressed as mean ± SEM of three independent experiments.
Experimental procedures, spectroscopic and analytical data for
compounds 2, 3, 4a–f and 5a–f. Evaluation of in vitro antiparasitic
activity and cytotoxicity of compounds 5a–f. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.07.078.
References and notes
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Croft, S. L.; Seifert, K.; Yardley, V. Indian J. Med. Res. 2006, 123, 339.
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C.; Russo, R.; Sundar, S.; Alvar, J. Clin. Infect. Dis. 2006, 43, 917.
Croft, S. L.; Engel, J. Trans. R. Soc. Trop. Med. Hyg. 2006, 100, S4.
Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R. W.; Alvar, J.;
Boelaert, M. Nat. Rev. Microbiol. 2007, 5, 873.
Figure 2. Cell viability (%) of THP-1 macrophages at 50 lM compound
concentration.
6
7
.
.
8
9
.
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Croft, S. L.; Barrett, M. P.; Urbina, J. A. Trends Parasitol. 2005, 21, 508.
Soto, J.; Soto, P. Expert Rev. Anti Infect. Ther. 2006, 4, 177.
dodecyl, compounds 5d and 5e, respectively, increases cytotoxicity
IC50 = 18.4 ± 2.5 M and 21.4 ± 2.8 M, respectively). However,
antileishmanial activity of compounds 5d and 5e is higher than
the cytotoxicity (IC50>18.4 M and >21.4 M, respectively) and as
a result, we cannot distinguish between specific antiparasitic activ-
ity and general cytotoxicity for these compounds. Finally the ben-
1
1
0. Herwaldt, B. N. Eng. J. Med. 1999, 341, 1840.
1. Sindermann, H.; Engel, K. R.; Fischer, C.; Bommer, W. Clin. Infect. Dis. 2004, 39,
(
l
l
1520.
l
l
12. Berman, J.; Bryceson, A. D. M.; Croft, S.; Engel, J.; Gutteridge, W.; Karbwang, J.;
Sindermann, H.; Soto, J.; Sundar, S.; Urbina, J. A. Trans. R. Soc. Trop. Med. Hyg.
2006, 100S, S41.
13. Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol. Rev. 2006, 19, 111.
zyl-substituted derivative 5f is not cytotoxic (IC50>50
possesses moderate antileishmanial activity IC50 = 41.8 ± 2.5
In general, four of the new derivatives are less cytotoxic than
miltefosine (IC50 = 28.6 ± 2.5 M), which is in accordance with our
previous findings that the presence or cycloalkane rings in the lipid
portion of alkylphosphocholines reduces cytotoxicity. Our results
underscore certain structural features modulating the leishmani-
cidal activity of the adamantane-containing alkylphosphocholines.
Firstly, the two carbon spacer between the adamantane moiety and
the phosphate head group is not optimal since all the new com-
l
M), and
14. Avlonitis, N.; Lekka, E.; Detsi, A.; Koufaki, M.; Calogeropoulou, T.; Scoulika, E.;
Siapi, E.; Kyrikou, I.; Mavromoustakos, T.; Tsotinis, A.; Makriyannis, A. J. Med.
Chem. 2003, 46, 755.
l
M.
15. Kapou, A.; Benetis, N. P.; Avlonitis, N.; Calogeropoulou, T.; Koufaki, M.;
l
Scoulica, E.; Nikolaropoulos, S. S.; Mavromoustakos, T. Bioorg. Med. Chem. 2007,
1
5, 1252.
16. Calogeropoulou, T.; Angelou, P.; Detsi, A.; Fragiadaki, I.; Scoulica, E. J. Med.
Chem. 2008, 51, 897.
17. The activity of compound
I (Fig. 1) against intracellular amastigotes of
Leishmania infantum was determined but not previously published.
8. Papanastasiou, I.; Tsotinis, A.; Kolokouris, N.; Prahalingam, S. R.; Kelly, J. M. J.
Med. Chem. 2008, 51, 1496.
1
19. General procedure for the preparation of ether phospholipids (5a–f) . To a
solution of phosphorus oxychloride (0.12 mL, 1.3 mmol) in dry THF (5 mL) was
added at À10 to À5 °C, a mixture of the corresponding alcohol 4a–f (1 mmol)
and dry triethylamine (0.25 mL, 1.8 mmol) in dry THF (7 mL). The reaction
mixture was stirred at this temperature for 15 min and for additional 30 min at
pounds are less active than the
tives I–VII (Fig. 1), previously synthesized by us.
x-adamantylideneundecyl deriva-
14,16
Secondly, the
alkyl substituent of the alkyloxyethyl group at C3 modulates the
relation between activity and cytotoxicity against THP-1 macro-
phages. Short alkyl chains (butyl to octyl) are preferred for non-tox-
icity versus longer (decyl to dodecyl), maybe due to detergent
effects to cellular membranes.
In conclusion, we have synthesized a series of new 2-[3-(2-
alkyloxy-ethyl)-adamantan-1-yl]-ethoxy substituted ether phos-
pholipids. The majority of the new analogues were significantly
less cytotoxic than miltefosine while, antiparasitic activity against
L. infantum amastigotes depended on the length of the 2-alkyloxy
substituent. The most potent compounds were {2-[[[3-(2-hexyl-
oxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-
N,N,N-trimethyl-ammonium inner salt (5b) and {2-[[[3-(2-octyl-
oxy-ethyl)-adamant-1-yl]-ethoxy]hydroxyphosphinyloxy]ethyl}-
rt. Then H
aqueous layer was extracted with EtOAc and then with CH
organic extracts were dried over anhydrous Na SO
2
O (5 mL) was added and stirring was continued for 30 min. The
Cl . The combined
and the solvent
2
2
2
4
was evaporated in vacuo to afford the corresponding phosphoric acid
derivative, which was converted to the pyridinium salt by addition of 5 mL
of anhydrous pyridine, stirring for 2 h at 40 °C and evaporation of the solvent in
vacuo. To a solution of the above salt (1 mmol) in pyridine (7 mL) were
sequentially added choline p-toluenesulfonate (0.413 g, 1.5 mmol) and 2,4,6-
triisopropylbenzenesulfonyl chloride (TIPS-Cl) (0.455 g, 1.5 mmol) and the
mixture was stirred at ambient temperature for 72 h. Subsequently, the
mixture was hydrolyzed at °C by addition of 2-propanol/H
stirring for 0.5 h, the solvent was evaporated in vacuo and the residue was
purified by flash column chromatography (CH Cl /MeOH, 60:50/50:60/30:70)
2
O (7:2) 9 mL, and
2
2
to afford the desired ether phospholipid derivatives 5a–f.
0. Bott, K.; Hellmann, H. Angew. Chem., Int. Ed. Engl. 1966, 5, 870.
1. Croft, S. L.; Neal, R. A.; Pendergast, W.; Chang, J. H. Biochem. Pharmacol. 1987,
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2
2