Design and synthesis of potent antileishmanial cycloalkylidene-substituted ether phospholipid derivatives

Article abstract of DOI:10.1021/jm701166b

Two series of novel ether phospholipids (EPs) have been synthesized. The first includes cyclodecylidene-or cyclopentadecylidene-substituted EPs carrying N,N,N-trimethylammonium or N-methylpiperidino or N-methylmorpholino head groups. The second series enc

Full text of DOI:10.1021/jm701166b

J. Med. Chem. 2008, 51, 897–908
897
Design and Synthesis of Potent Antileishmanial Cycloalkylidene-Substituted Ether Phospholipid
Derivatives
Theodora Calogeropoulou,*,† Panagiotis Angelou,† Anastasia Detsi,†,‡ Irene Fragiadaki,§ and Effie Scoulica*,§
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vassileos Constantinou AVenue,
11635 Athens, Greece, and Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine,
UniVersity of Crete, 71409 Heraklion, Greece
ReceiVed September 18, 2007
Two series of novel ether phospholipids (EPs) have been synthesized. The first includes cyclodecylidene-
or cyclopentadecylidene-substituted EPs carrying N,N,N-trimethylammonium or N-methylpiperidino or
N-methylmorpholino head groups. The second series encompasses more rigid head groups in combination
with cycloalkylidene moieties in the lipid portion. In addition, hydrogenated derivatives were obtained. All
the new analogues, except 33, were 1.5- to 62-fold more potent than miltefosine against the intracellular L.
infantum, and the most active ones were also less cytotoxic against the human monocytic cell line THP1
and less hemolytic than miltefosine. The analogues that combine high potency with low cytotoxicity and
hemolytic activity were 19, 37, 21 23, 38, 39, and 40. Cyclopentadecylpentylphosphocholine (38) possesses
an IC50 of 0.7 µM against L. infantum amastigotes and is the least cytotoxic analogue, since it does not
present toxicity against THP1 macrophages, even at a concentration that is 800-fold the antiparasitic IC50
value, and does not present significant hemolytic activity.
Introduction
antimonials (sodium stibogluconate and meglumine antimonate),
pentamidine, and amphotericin B.2
Leishmaniasis, one of the most neglected tropical diseases,
is a parasitic disease that is transmitted by the bite of the infected
phlebotomine sandfly and constitutes a major public health
problem especially in the tropical and subtropical regions of
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. The
parasite exists in two different forms: the flagellated one in the
gut of the sandfly vector and the amastigote in the mammalian
host that is the cause of the acute desease.
The high resistance developed to pentavalent antimonials,
high-dose regimens, and long treatment courses using parenteral
administration, as well as, renal and cardiac toxicity, are major
drawbacks. On the other hand, the high toxicity and declining
efficacy of pentamidine have restricted its use. Amphotericin
B and its lipid formulation proved to be very effective in the
treatment of leishmaniasis, but the cost is still prohibitively high.
Paromomycin and sitamaquine are two compounds currently
undergoing clinical trials.3,4
The serious problems associated with the treatment of
leishmania infections, such as development of drug resistance,
the need for low cost drugs, and the limited funding available
for parasitic diseases, have recently led many research groups
to design and synthesize novel antileishmanial compounds as
well as screen natural products for their antileishmanial activity.
Most recent examples of synthetic compounds include nitrogen
heterocycles, such as quinoline,5,6 dihydropyridopyrimidine,7
and pyrazolopyridine8 derivatives as well as nitrogen-containing
biphosphonates9 and arylanthranilodinitriles.10 Moreover, screen-
ing of natural products has identified several promising leads
and has provided new scaffolds for chemical derivatization.11–15
In 1987, the “serendipitous” discovery of the antileishmanial
activity of hexadecylphosphocholine (miltefosine, HePC),16 an
alkyllysophospholipid initially developed as an antitumor agent,
constituted a major breakthrough in antileishmanial chemo-
therapy. 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).17 According
to the latest clinical studies in Colombia, cure rates of 91-100%
were obtained with a dose of 2.5 (mg/kg)/day for 28 days, with
mild side effects (mostly gastrointestinal) in 35–60% of the
patients.18
Visceral leishmaniasis (VLa, or “kala-azar”), mucocutaneous
leishmaniasis (MCL, ulceration of the skin and hyperdevelop-
ment of the mucous membranes), and cutaneous and diffuse
cutaneous leishmaniasis (CL and diffuse CL) are the distin-
guishable forms of the disease, which includes several clinical
syndromes and, if untreated, can have devastating consequences.
Establishment 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
coinfection is now considered an extremely serious new disease
with severe clinical, diagnostic, chemotherapeutic, epidemio-
logical, and economic implications.1
Even though chemotherapy is currently the only way to treat
the various forms of leishmaniasis, since no vaccine is yet
available, the arsenal of drugs against the disease is still limited.
Today, first line antileishmanial drugs include pentavalent
* To whom correspondence should be addressed. For T.C.: phone, +3010
7273833; fax, +3010 7273818; e-mail, tcalog@eie.gr. For E.S.: phone/
fax, +302810 394576; e-mail, scoulica@med.uoc.gr.
†
National Hellenic Research Foundation.
Current Address: Laboratory of Organic Chemistry, School of Chemical
‡
Engineering, National Technical University of Athens, Heroon Polytechniou
9, Zografou Campus, 15773 Athens, Greece.
§
University of Crete.
Despite its advantages, miltefosine has a long half-life
(100–200 h) in humans and a low therapeutic ratio, character-
istics that could encourage development of resistance, especially
a Abbreviations: HePC, hexadecylphosphocholine; HIV, human immu-
nodeficiency virus; VL, visceral leishmaniasis; CL, cutaneous leishmaniasis;
GPI, glycosylphosphatidylinositol; PI, propidium iodide.
10.1021/jm701166b CCC: $40.75
2008 American Chemical Society
Published on Web 01/26/2008

898 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Calogeropoulou et al.
Figure 1. Structures of ether phospholipids I-VII.
in India where VL is an anthroponosis. Moreover, it is not
suitable for pregnant women because it has been shown to cause
teratogenesis in animals19 and it did not give satisfactory results
when administered to HIV-coinfected patients, since most of
them relapsed.20 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 have to be addressed.21
pecially against the strain L. infantum, which causes the AIDS
associated coinfection in Europe. In particular, the presence of
a 2-(4-alkylidenecyclohexyloxy)ethyl moiety renders the result-
ing compounds more active against L. infantum promastigotes
while the presence of ω-cyclohexylidenealkyl or ω-adamantyl-
idenealkyl groups results in enhanced activity against L.
donoVani promastigotes. The most active analogues against both
L. donoVani and L. infantum promastigotes were 2-(4-dode-
cylidenecyclohexyloxy)ethylphosphocholine (I), 11-cyclohex-
ylideneundecylphosphocholine (II), 11-adamantylideneundecyl-
phosphocholine (III), their hydrogenated analogues (IV-VI),
and 2-(4-tetradecylidenecyclohexyloxy)ethylphosphocholine
(VII), which were all more potent than miltefosine (Figure 1).
Moreover, preliminary evaluation of the cytotoxicity of the most
active ether phospholipids on the human monocytic cell line
THP1 revealed that the new compounds 11-cyclohexylidene-
undecylphosphocholine and 11-adamantylideneundecylphos-
phocholine were not only more potent antileishmanial agents
but also less cytotoxic than miltefosine.31
The mode of action of miltefosine is not yet fully understood.
Several molecular targets in trypanosmatids have been sug-
gested, including alkyl lipid metabolism and phospholipid
biosynthesis22 of the parasite, glycosylphosphatidylinositol (GPI)
anchor biosynthesis, signal transduction23 and induction of
apoptosis,24,25 and inhibition of the glycosome-located alkyl-
specific acyl-CoA acyltransferase, an enzyme involved in lipid
remodeling.22 Recently, it has been demonstrated that milte-
fosine is inserted into the membrane by miscibility and interacts
with sterols.26 Miltefosine and the phospholipids of analogous
structure edelfosine and perifosine as well as fluorescent
phospholipid derivatives have been shown to be taken up across
the plasma membrane by a specific protein-dependent translo-
cation step that requires a miltefosine transporter and a protein
factor, both recently identified and characterized. Inactivation
of this inward-directed translocation activity causes resistance
to miltefosine in vitro against both the promastigote and
amastigote stages of the parasite and influences the parasite
burden in the liver of infected mice.27 Laboratory-induced
resistance to miltefosine has also been shown to be related to
two mutations on the aminophospholipid translocase.28a Re-
cently, it has been reported that alkylphosphocholines can be
recognized and actively transported out of the leishmania cell
by an ABCG-like transporter, which belongs to the ATP-binding
cassette transporters, suggesting that the parasite can employ
multiple resistance mechanisms.28b
The current knowledge on the structure-antiparasitic actitvity
relationships of miltefosine analogues is limited.29,30 In addition,
miltefosine is not the most active drug among compounds of
the phospholipid structural family because some alkyllysophos-
pholipid analogues that have been synthesized and tested for
their antileishmanial activity have exhibited higher acti-
vity.16,17,29,30 With the issues of resistance, side effects and
production cost still requiring special attention and the mech-
anism of action still requiring elucidation, the field of design
and synthesis of novel antileishmanial phospholipid derivatives
is an ongoing challenge.
Our lead discovery of the new antileishmanial ring-substituted
ether phospholipid derivatives urged more in-depth assessments
of the structural requirements for enhanced antileishmanial
activity and minimal cytotoxicity for this class of compounds.
Thus, we have designed and synthesized two series of novel
compounds. In the first series we increased the ring size of the
cycloalkane functionality in the lipid portion by incorporating
cyclodecylidene or cyclopentadecylidene groups linked to the
phosphate polar headgroup by an oligomethylene bridge of 5
or 11 carbons, as previously reported.30 In addition, we have
varied the polar headgroup, using N,N,N-trimethylammonium,
N-methylpiperidino, or N-methylmorpholino moieties. In the
second series, in order to explore the influence of the flexibility
of the headgroup on the activity of the compounds, we
incorporated more rigid head groups, namely, the trans-2-
hydroxy-N,N,N-trimethylcyclopentanamino or the trans-2-hy-
droxy-N,N,N-trimethylcyclohexanamino group, the 3-(2-hydroxy-
ethylidene)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane group and
(4-hydroxy-but-2-ynyl)trimethylammonium group in combina-
tion with cyclopentadecylidene-, cyclohexylidene-, and ada-
mantylidene-substituted lipid portions. Finally, we synthesized
hydrogenated analogues of the first series in order to explore
the effect of the double bond on the activity and the toxicity of
the compounds.
In this context, we recently reported30,31 the synthesis of a
series of ring-substituted ether phospholipids, incorporating
4-alkylidenecyclohexyloxylethyl, cyclohexylidene, or adaman-
tylidene groups in the lipid portion and polar head groups other
than choline in order to evaluate how these structural changes
affect the antileishmanial activity of the compounds. Our results
indicated that introduction of cycloalkane rings in alkylphos-
phocholines provides compounds with enhanced activity, es-
The antileishmanial activity of the new analogues was
evaluated in vitro against the promastigote forms of L. infantum
and L. donoVani and against the intracellular amastigote form
of L. infantum. The cytotoxicity of the new compounds was
assessed on the human monocytic cell line THP1. Moreover,
since hemolysis is a problem associated with alkylphosphocho-

Ether Phospholipid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 899
Scheme 1a
a Reagents and conditions: (a) [(CH3)3Si]2NK, THF; (b) MeOH, H+, 40 °C; (c) LiAlH4, THF.
Scheme 2a
lines, preventing their use in injectable form, we studied the
hemolytic properties of the new compounds on human red blood
cells.
Chemistry
The synthetic strategy followed for the preparation of the ether
a Reagents and conditions: (a) (CH3)2NH, 40% in H2O; (b) methyl p-to-
phospholipids 19–36 is depicted in Schemes 5 and 6 and
involves phosphorylation of the appropriate alcohols using
POCl3 and subsequent attachment of the desired head groups.
For the synthesis of the required starting alcohols 5a,b (Scheme
1) a Wittig reaction between the cyclodecanone (1a) or
cyclopentadecanone (1b) and 4-carboxybutyl triphenylphos-
phonium bromide (2) was employed. The resulting unsaturated
acids 3a,b were transformed to the corresponding methyl esters
4a,b which, after treatment with LiAlH4 in THF, produced the
alcohols 5a,b in high yields (82%). Alcohols 8a,b were obtained
in a similar manner (Scheme 1); however, in this case 10-
methoxycarbonyldecyltriphenylphosphonium bromide (6)30 was
employed in the Wittig reaction, and as a result, the corre-
sponding unsaturated esters 7a,b were obtained directly. Reduc-
tion of esters 7a,b afforded the desired alcohols 8a,b in almost
quantitative yields (91–100%). Alcohols 8c and 8d were
prepared as previously described.30
The required head groups were obtained after quaternization
of N-methylpiperidine or N-methylmorpholine to N-(2-hydroxy-
ethyl)-N-methylpiperidinium bromide or N-(2-hydroxyethyl)-
N-methylmorpholinium bromide, respectively, which was ef-
fected upon treatment with 1-bromoethanol, as previously
described.30 The synthesis of the sterically constrained trans-
2-hydroxy-N,N,N-trimethylcyclohexanamino and trans-2-hy-
droxy-N,N,N-trimethylcyclopentanamino head groups is depicted
in Scheme 2 and involves aminolysis of the epoxide ring of 9a
or 9b, with aqueous dimethylamine to provide the corresponding
trans-amino alcohols 10a and 10b.33 N-Methylation of 10a,b
by p-toluenesulfonate yields the respective quaternized amines
11a and 11b, isolated as the p-toluenesulfonate salts after
recrystallization from acetone.
luenesulfonate, Et2O, room temp.
Scheme 3a
a Reagents and conditions: (a) [(CH3)3Si]2NK, (C2H5O)2P(O)CH2C
(O)C2H5, THF, 80 °C; (b) LiAlH4, THF; (c) methyl p-toluenesulfonate,
Et2O, room temp.
derived ester to the allylic alcohol 14 and methylation of the
amino group to the corresponding p-toluenesulfonate salt 15
(Scheme 3).
The variety of sterically constrained head groups that we used
for our structure–activity relationship studies was further
enriched with compound 18, which bears a carbon-carbon triple
bond. The synthesis of this compound is shown in Scheme 4
and involves hydroxymethylation of the terminal acetylenic
carbon of 3-(dimethylamino)-1-propyne (16) followed by quat-
ernization to the p-toluenesulfonate salt.
The synthesis of the desired ether phospholipids was affected
via two methods. Method A (Scheme 5), which we have
previously employed to synthesize other phospholipid deriva-
tives,30,34 involves phosphorylation of the alcohols with phos-
In addition, we prepared the bicyclic headgroup 15 by
performing a Wadsworth-Emmons reaction between tropinone
(12) and triethyl phosphonoacetate followed by reduction of the

900 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Calogeropoulou et al.
Scheme 4a
Scheme 6a
a Reagents and conditions: (a) n-BuLi, HCHO, THF; (b) methyl p-tolu-
enesulfonate, Et2O, room temp.
Scheme 5a
a Reagents and conditions. Method B: (a) (1) P(O)Cl3, Et3N, THF; (2)
11a, 11b, 15, or 18, pyridine,CHCl3; (b) H2O, 2-propanol.
a Reagents and conditions: (a) (1) P(O)Cl3, Et3N, THF; (2) H2O, 2-pro-
panol; (b) pyridine, 40 °C; (c) pyridine, MSNT, HOCH2CH2N+R1R2R3,
40 °C.
the miltefosine analogues 35 and 36, incorporating the
sterically hindered trans-2-hydroxy-N,N,N-trimethylcyclo-
pentanamino and trans-2-hydroxy-N,N,N-trimethylcyclohex-
anamino head groups.
phorus oxychloride in the presence of triethylamine in THF,
followed by hydrolysis and treatment with pyridine in order to
form the corresponding pyridinium salts. These were in turn
coupled to the appropriate choline, N-methylpiperidino or
N-methylmorpholino head groups in the presence of 1-(mesi-
tylenesulfonyl)-3-nitro-1,2,4-triazolide (MSNT) as a condensing
agent. This method was successfully used for the synthesis of
compounds 19–26, but it did not prove to be efficient in the
synthesis of compounds 27–36 substituted by the constrained
head groups, probably because of steric hindrance. As a result,
the more direct one-pot phosphorylation method B (Scheme 6)
was employed in which the appropriate alcohols 8a-d were
treated with POCl3, and to the dichlorophosphate ester formed
in situ was added a pyridine/chloroform solution of the
p-toluenesulfonate salt of the corresponding quaternized amino
alcohol (11a, 11b, 15, and 18) to afford after hydrolysis the
desired phospholipids 27–36.
The lipid portion of phospholipids 27–32 was selected on
the basis of the highest activity and minimum toxicity exhibited
by the novel ether lipids 19-26 and our previous findings on
analogous lead compounds.30 Thus, from 5-(cyclopentade-
cylidene)pentanol (5b) we prepared compounds 27 and 28 and
from 11-cyclohexylideneundecanol (8c) and 11-adamantylide-
neundecanol (8d) (prepared as described previously30) we
obtained compounds 29–34. In addition, we have synthesized
Finally, in order to investigate the effect of the double bond
present in the lipid portion of our compounds, we synthesized
the saturated analogues of selected ether phospholipids (Scheme
7). Thus, catalytic hydrogenation of compounds 19, 21, 22, and
23 using 10% Pd/C in methanol provided compounds 37, 38,
39, and 40, respectively.
Biological Evaluation
Two in vitro models were employed for the evaluation of
antileishmanial activity, as described in the Experimental
Section. Thus, the new analogues 19–40 (Schemes 5-7) were
evaluated in vitro against promastigote cultures of Leishmania
donoVani MON 703 and Leishmania infantum MON 235 (Table
1). Furthermore, the antiparasitic activity of derivatives 19–40
was assessed using the intracellular amastigote form of Leish-
mania infantum MON 235 in infected human monocytic THP-1
cells (Table 1).
The adverse effects of the new derivatives were evaluated in
two systems. In the first system we assessed the apoptosis effect
of the compounds against the THP-1 host cell line, and in the
second system we evaluated the compounds’ toxic effect on

Ether Phospholipid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 901
Table 2. In Vitro Cytotoxicitya in the Human Monocytic Cell Line
THP1 and Hemolytic Activity in Human Erythrocytes
Scheme 7a
cytotoxicity
IC50
hemolysis hemolysis
at 100 at 500
>50 (µM) HC50 (µM) µM (%) µM (%)
cytotoxicity hemolysis
compd
19
(µM)
>50
37.9 ( 2
>50
>50
>50
37.7 ( 2
>50
36.2 ( 3.1
>50
>50
30.7 ( 3.2
28.7 ( 3.2
>50
39.7 ( 0.3
>50
>50
349.1
>100
>100
>100
68.4 ( 2.8
>100
100
2.1
25.5
38.0
71.6
45.4
17.7
36.0
16.9
80.5
60.7
66.4
81.6
30.8
66.8
82.3
41.8
43.0
57.4
100.0
88.3
100.0
51.4
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
55.4
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
153.6
272.2
372.3
72.61 >100
>100
44.3 ( 1.9
75.4
88.54 76.7 ( 2.9
76.3 ( 4.7
59.8 ( 2
74.95 >100
66.6 ( 5.3
147.3
a Reagents and conditions: (a) H2, 10% Pd/C, MeOH, 1 atm.
61.3 ( 3.6
75.04 ∼100
Table 1. In Vitro Antileishmanial Activitya against the Promastigote
Forms of L. infantum and L. donoVani and the Intracellular Amastigote
Form of L. infantum
34.1 ( 0.4
21.3 ( 3.2
>50
43.1 ( 3.6
100.0
20.4
1.7
>100
363.3
>500
332.8
243.8
>100
100.0
100.0
80.8
81.1
100.0
intracellular amastigote
potency promastigotes IC50 (µM)
>50
>100
8.7
form L. infantum
>50
>100
28.7
34.8
96.1
L. infantum
MON 235
L. donoVani
MON 703
MON 235IC50
>50
>100
compd
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
(µM)
miltefosine 28.6 ( 2.5
38.3 ( 2.8
76.8
<3.2
3.3 ( 0.6
0.11 ( 0.02
3.8 ( 0.8
4.1 ( 0.9
4.4 ( 0.5
0.75 ( 0.04
3.6 ( 0.3
0.9 ( 0.1
0.2 ( 0.02
1.6 ( 0.4
1.1 ( 0.2
1.1 ( 0.22
0.30 ( 0.2
0.8 ( 0.1
9.1 ( 0.5
5.2 ( 1
3.3 ( 1.2
1.7 ( 0.5
2.2 ( 0.2
0.7 ( 0.4
1 ( 0.4
0.4 ( 0.1
6.7 ( 1
a Results are expressed as the mean ( SEM of three independent exper-
iments.
2.9 ( 0.3
2.2 ( 0.7
1.7 ( 0.5
1.8 ( 0.5
100
1 ( 0.24
2.3 ( 0.7
2 ( 0.9
1.94 ( 0.5
68.2
cyclopentadecylidene series, analogues 21–26, a more detailed
SAR was performed, varying not only the oligomethylene bridge
but the headgroup as well. The 11-cyclopentadecylideneundecyl
derivatives 24–26 show moderate to very low activity against
both promastigote strains with IC50 values ranging from 41.5
to 100 µM, with the choline substituted analogue 24 being the
least active and the N-methylmorpholino derivative 26 the most
potent of the three. Replacement of the undecyl chain by a pentyl
group, compounds 21–23, results in high activity against both
promastigote leishmania strains, which is not influenced by the
nature of the headgroup (IC50 values in the range of 2 µM,
Table 1).
In the second series, compounds 27–34, we opted to use the
5-cyclopentadecylidenepentyl, 11-cyclohexylideneundecyl, and
11-adamantylideneundecyl groups as lipid portions on the basis
of the in vitro results of the present report and our previous
work.30,31 In addition, two new straight-chain derivatives bearing
a hexadecyl group, as miltefosine, were synthesized in order to
get an insight on the effect on activity of cyclic moieties in the
lipid portion and the headgroup of alkylphosphocholines. All
but three compounds (27, 31, 34) were inactive against the
promastigote form of L. infantum, IC50 values greater than 100
µM, while the activities against the promastigote L. donoVani
were moderate to satisfactory, IC50 values between 3.1 and 71.9
µM.
The intracellular amastigote form is the more relevant
parasitic stage for compound biological evaluation, and we were
very pleased to find that all new analogues, except compound
33, were more potent than miltefosine (IC50 ) 6.7 µM) against
the intracellular amastigote L. infantum (Table 1). A more
detailed SAR is now described. In the first series, compounds
19–26, the 11-cyclodecylideneundecyltrimethylammonium de-
rivative 20 is the most potent with an IC50 value of 0.1 µM.
The activity decreases by 30-fold for the 5-cyclodecylidene-
pentyltrimethylammonium ether phospholipid 19 (IC50 ) 3.3
µM) in which the distance between the cyclodecylidene moiety
and the phosphate group is shorter. The 5-cyclopentadecylidene-
pentyl-substituted ether phospholipids 21–23 possess similar
84.5
50.2
65.9
41.5
<3.1
6 ( 2.1
8.6 ( 1.4
36.6/16.1
3.9 ( 0.8
16.6
<3.1
>100
>100
>100
38 ( 2.5
>100
>100
44.3/95.9
>100
>100
21.7
1.4 ( 0.2
2.8 ( 0.3
1.9 ( 0.3
23.9 ( 4.2
41.6
41.1 />100
54.3/>100
71.9/>100
<3.13
0.9 ( 0.1
1 ( 0.1
1 ( 0.2
8.7 ( 0.7
35
36
37
38
39
40
miltefosine
a Results are expressed as the mean ( SEM of three independent exper-
iments.
human red blood cells (Table 2). All experiments were per-
formed in three independent experiments.
Results and Discussion
The first screening of the new analogues 19–40 was per-
formed on the promastigote form of the parasite and provided
a general comparative evaluation of the compounds against the
two leishmania species employed in this study. In general, all
analogues showed increased activity against the promastigote
form of L. donoVani than against L. infantum (Table 1). More
specifically, in the first series the 5-cyclodecylidenepentylcho-
line-substituted ether phospholipid 19 was marginally active
against L. infantum (IC50 )76.8 µM) while it possessed good
activity against L. donoVani (IC50 < 3.2 µM). Introduction of
an 11-carbon spacer between the phosphate group and the 10-
membered ring, compound 20, led to an increased activity
against both promastigote strains with IC50 values of 2.9 µM
against L. infantum and 1 µM against L. donoVani. In the

902 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Calogeropoulou et al.
activity with IC50 values of 3.8, 4.1, and 4.4 µM, respectively.
Replacement of the pentyl group by an undecyl in the cyclo-
pentadecylidene series resulted in a marked increase of activity
against the intracellular amastigotes of L. infantum, which was
influenced by the headgroup. The trimethylammonium derivative
24 and the N-methylmorpholino analogue 26 possessed similar
activity (IC50 ) 0.8 and 0.9 µM, respectively) while, the
N-methylpiperidino-substituted ether phospholipid 25 was the
least potent of the three (IC50 ) 3.6 µM).
decyl-substituted analogues are less cytotoxic than the 11-
cyclohexylideneundecyl- or hexadecyl-substituted ether phos-
pholipids. With respect to headgroup SAR, the trans-2-hydroxy-
N,N,N-trimethylcyclopentanamino headgroup is preferred over
the trans-2-hydroxy-N,N,N-trimethylcyclohexanamino head-
group with respect to toxicity against THP1 macrophages.
Furthermore, the more constrained tropane-derived headgroup
renders the corresponding ether phospholipid 33 the least
cytotoxic of the second series (IC50 value of 147.3 µM).
The second series, compounds 27–36, comprises ether
phospholipids bearing constrained head groups and more
specifically trans-2-hydroxy-N,N,N-trimethylcyclohexanamino
or trans-2-hydroxy-N,N,N-trimethylcyclopentanamino groups.
Within the trans-2-hydroxy-N,N,N-trimethylcyclopentanamino-
containing ether phospholipids 27, 29, 31, and 35 the most active
are the 5-cyclopentadecylidenepentyl-substituted analogue 27
Since hemolysis is a problem associated with alkylphospho-
cholines, which prevents their use in injectable form, it was
considered important to study the hemolytic properties of the
new compounds. Thus, all the new analogues were evaluated
for their hemolytic activity against red blood cells (Table 2).
All the new compounds exhibit lower hemolytic activity than
miltefosine (HC50 ) 38.3 µM). Specifically, from the first series,
analogues 19–21 and 23–26 possess HC50 of >100 µM while
compound 22 has HC50 of 68.4 µM.
and the 11-adamantylidene-substituted derivative 31 (IC50
)
0.2 µM and 0.3 µM, respectively). The activity against L.
infantum amastigotes was decreased for the 11-cyclohexylide-
neundecyl analogue 29 (IC50 ) 1.1 µM), the least potent being
the straight-chain derivative 35 (IC50 ) 3.3 µM). The order of
activity against the intracellular amastigote L. infantum is slightly
different in the case of the trans-2-hydroxy-N,N,N-trimethyl-
cyclohexanamino headgroup, analogues 28, 30, 32, and 36. The
most potent is compound 32 (IC50 ) 0.8 µM), which contains
an 11-adamantylideneundecyl moiety followed by the 11-
cyclohexylideneundecyl analogue 30 (IC50 ) 1.1 µM) and the
5-cyclopentadecylidenepentyl-substituted ether phospholipid 28
In the second series, compounds 27–36, the 5-cyclopentade-
cylidenepentyl-substituted derivatives 27 and 28 possess HC50
values of 44.3 and 76.7 µM, respectively, and the 11-cyclo-
hexylideneundecyl-substituted ether phospholipids 29 and 30
possess HC50 values of 76.3 and 59.8 µM, respectively. Among
the 11-adamantylideneundecyl derivatives 31–34, analogue 31
bearing the trans-2-hydroxy-N,N,N-trimethylcyclopentanamino
headgroup possess HC50 of >100 µM and analogue 34 bearing
the alkynyl headgroup exhibits HC50 of ∼100 µM, while 32
and 33 are more hemolytic with HC50 values of 66.6 and 61.3
µM, respectively. Concerning the straight-chain analogues 35
and 36, the nature of the headgroup influences the hemolytic
activity to a great extent with the trans-2-hydroxy-N,N,N-
trimethylcyclopentanamino group rendering 35 hemolytic (HC50
) 43.1 µM) and the trans-2-hydroxy-N,N,N-trimethylcyclohex-
anamino group rendering 36 nonhemolytic (HC50 > 100 µM).
By comparision of the percent hemolysis at 100 and 500 µM
(Table 2) of compounds 19–21 and 23–26, there are differences
that depend on both the lipid portion and the headgroup. It seems
that choline is the headgroup of choice followed by the
N-methylmorpholino and finally the N-methylpiperidino head
groups. However, there is no clear trend as to which combination
of structural features is the optimum.
(IC50 ) 1.6 µM) and the straight chain analogue 36 (IC50
)
1.7 µM). Since the 11-adamantylideneundecyl derivatives 31
and 32 exhibited high activity, two additional adamantylidene
compounds were synthesized, 33 and 34, which contained a
tropane-derived headgroup and an alkynyl headgroup, respec-
tively. Unfortunately, both 33 and 34 were less active against
the amastigote L. infantum than the other two adamantylidene
derivatives 31 and 32; moreover, they were the least active in
this series with IC50 values of 9.1 and 5.2 µM, respectively.
Since human monocytic THP-1 cells infected with the
appropriate Leishmania species are used for the evaluation of
the leishmanicidal activity of compounds against the intracellular
amastigote stages of the parasite, we assessed the cytotoxicity
of all new analogues against THP-1 cells (Table 2), and the
majority of them was found to be less cytotoxic than miltefosine
(IC50 ) 28.6 µM).
More specifically, in the first series the presence of the
undecyl group confers toxicity to the ether phospholipids 20,
24, 25, and 26 with IC50 values of 37.9, 37.7, 72.6, and 36.2
µM, respectively. Conversely, the pentyl-substituted derivatives
19, 21, 22, and 23 are not cytotoxic with IC50 values 349.1,
153.6, 272.2, and 372.3 µM, respectively. The 5-cyclode-
cylidenepentylcholine-substituted 19 is 2-fold less cytotoxic than
the 5-cyclopentadecylidenepentylcholine derivative 21. Within
the 5-cyclopentadecylidenepentyl derivatives 21–23 the head-
group influences toxicity, which decreases in the order of
choline-substituted 21, N-methylpiperidino-substituted 22, and
N-methylmorpholino-substituted derivative 23, which is the least
cytotoxic of this series.
The influence of the double bond on antileishmanial activity
was explored by comparing ether phospholipids 19 and 21-23,
which are the least cytotoxic against THP1 monocytes and
possess hemolytic activity higher than 100 µM and better
antileishmanial activity against the intracellular amastigotes than
miltefosine, with their saturated congeners 37–40, which were
obtained by catalytic hydrogenation. We were delighted to find
out that antileishmanial activity was improved and toxicity was
reduced. More specifically, the absence of the double bond in
the cyclodecadecylidene-substituted choline derivative increased
antileishmanial activity against the amastigotes L. infantum by
1.5-fold (IC50 ) 2.2 µM) while slightly reducing cytotoxicity
against THP-1 monocytes (IC50 ) 363.3 µM) and hemolysis
of human erythrocytes (HC50 > 100 µM, hemolysis of 1.7% at
100 µM). The absence of a double bond in the cyclodecapen-
tylidene-substituted ether phospholipids was more dramatic
especially for the choline derivative 38 whose antileismanial
activity against intracellular L. infantum increased 5.5-fold (IC50
) 0.7 µM), being 9-fold more active than HePC; moreover, 38
is noncytotoxic (IC50 > 500 µM), has hemolytic activity (HC50
>100 µM), and undergoes hemolysis (8.7% at 100 µM). The
same trend was observed for the N-methylpiperidino-substituted
derivatve 39 whose antileismanial activity against intracellular
The analogues of the second series 29, 30, 32, 35, and 36
exhibit toxicity comparable to miltefosine with IC50 values of
30.7, 28.7, 39.7, 34.1, and 21.3 µM, respectively. Conversely,
derivatives 27, 28, 31, 33, and 34 are less cytotoxic than
miltefosine with IC50 values of 75.4, 88.54, 74.95, 147.3, and
75.04 µM, respectively. With respect to the lipid portion SAR,
the 5-cyclopentadecylidenepentyl- and 11-adamantylideneun-

Ether Phospholipid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 903
Figure 2. Comparative assessment of the effect of miltefosine (20 µM) and compound 38 (20 µM) on L. infantum (B, C) and L. donoVani (E, F)
promastigotes and after treatment of THP-1 cells with 50 µM miltefosine (H) or 50 µM compound 38 (I). Cells were double-stained with PI and
SYBR-14, and dead/alive ratios were sorted by flow cytometry. Population stained with SYBR14 is sorted in the area S, and PI stained population
is sorted in the area P. Quantification of dead/alive cells, after measuring 10 000 events per sample is depicted in the histograms. Gray bars represent
the SYBR 14 stained alive cells, and slashed bars represent the PI stained dead cells.
L. infantum increased 4-fold (IC50 ) 1. µM) (6.5-fold more
active than HePC) and its cytotoxicity (IC50 ) 332.8 µM) and
hemolytic activity decreased (HC50 > 100 µM, hemolysis of
28.7% at 100 µM). Finally, for the N-methylmorpholino-
substituted derivative 40, hydrogenation of the double bond led
to an increased antiparasitic activity against intracellular L.
infantum (10.6-fold, IC50 ) 0.41 µM), rendering 40 16-fold more
active than HePC. However, its cytotoxicity increased slightly
(IC50 ) 243.8 µM) while its hemolytic activity decreased (HC50
> 100 µM, hemolysis of 34.8% at 100 µM).
It has been previously observed that both promastigote and
amastigote stages of L. donoVani, L. major, L. tropica, L.
aethiopica, L.mexicana, and L. panamensis vary in their in vitro
sensitivity. In all assays L. donoVani was the most sensitive
species.35 The difference in activity of the various ether
phospholipids of this study against L. infantum and L. donoVani
promastigotes may be due to dissimilarities in both membrane
sterol36,37 and lipid content38 of the two Leishmania species.
Miltefosine and the new ether phospholipids of the present study,
except analogues 21–23, were more active against the intracel-
lular L. infantum than the promastigote form of the parasite.
Although the in vitro test systems for amastigotes and prom-
astigotes are not directly comparable, alkylphosphocholines
appear to be more active against the intracellular amastigotes,
and this could be partially explained by their ability to activate
macrophages.39
compromised patients generally have a poor response to
antimonials, and failure or relapse rates of 25% within 1-36
months are commonly reported.40b Thus, VL is considered a
major contributor to a fatal outcome in coinfected patients.41
All new analogues, except compound 33, were 1.5- to 62-fold
more potent than miltefosine against the intracellular amastigote
L. infantum, and the most active ones were also less cytotoxic
and hemolytic than HePC. These data, taken together, may
indicate that ring-substituted ether phospholipids may be useful
in the treatment of Leishmania/HIV coinfection. In addition,
the length of the alkyl chain of the most active ether phospho-
lipids of the present study varies from 5 to 11 carbon atoms,
and this could be advantageous for their metabolic clearance.
Furthermore, all new analogues are less hemolytic than milte-
fosine, which indicates that some of the new compounds could
be administered intravenously, and thus, they may show reduced
gastrointestinal toxicity and higher plasma concentrations in
vivo.
Compound 38 is the least cytotoxic analogue of the present
study, while its antileishmanial activity is in the submicro-
molar range, being 9-fold more active than miltefosine. A
comparative flow cytometric assessment of the cytotoxicity
of 38 and miltefosine on THP-1 cells, as well as their effect
against leishmania promastigotes, is shown in Figure 2. We
observe a notable difference in the ability of compound 38
(20 µM) to kill L. infantum (Figure 2C) and L. donoVani
promastigotes (Figure 2F) compared to miltefosine (20 µM)
(parts B and E of Figure 2, respectively). Moreover, the
comparison of cytotoxicity of HePC and 38 is striking and
worth mentioning. Miltefosine at 50 µM is very cytotoxic to
THP1 macrophages (Figure 2H) while, after treatment with
compound 38 (50 µM) (Figure 2I), the THP1 monocytes are
There has been increasing numbers of patients with L.
infantum and HIV coinfection, especially in Mediterranean
countries during the past decade,40 with clinical, diagnostic,
chemotherapeutic, epidemiological, and economic implications.
The coexistence of these diseases produces cumulative defi-
ciency of the immune response because Leishmania parasites
and HIV destroy the same cells, leading to an exponential
increase of disease severity and consequences. These immuno-

904 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Calogeropoulou et al.
pyridine (7 mL) was added dropwise, with cooling, 1-(mesitylen-
2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) (593 mg, 2 mmol)
followed by the addition of choline chloride (205 mg, 1.5 mmol)
or N-(2-hydroxyethyl)-N-methylpiperidinium bromide (448 mg, 1.5
mmol) or N-(2-hydroxyethyl)-N-methylmorpholinium bromide (452
mg, 1.5 mmol), and the mixture was stirred at 40 °C for 48 h.
After cooling, the mixture was hydrolyzed by the addition of
2-propanol/H2O, 7:2, and stirred for 0.5 h at ambient temperature.
The solvents were evaporated in vacuo and the residue was
subjected to flash column chromatography (CH2Cl2/MeOH, 60:50,
and then CH2Cl2/MeOH/25% NH4OH, 60:50:5) to afford the desired
ether phospholipids.
Method B. To a solution of phosphorus oxychloride (0.09 mL,
1 mmol) in dry THF (5 mL) was added at 0 °C dry triethylamine
(0.25 mL, 1.8 mmol) and subsequently a solution of the corre-
sponding alcohol (1 mmol) in dry THF (18 mL). The mixture was
stirred at ambient temperature for 2 h. Subsequently, choline
p-toluenesulfonate (358 mg, 1.3 mmol) or toluene-4-sulfonate(2-
hydroxycyclopentyl)trimethylammonium (11a) (410 mg, 1.3 mmol)
or toluene-4-sulfonate(2-hydroxycyclohexyl)trimethylammonium
(11b) (428 mg, 1.3 mmol) or toluene-4-sulfonate[3-(2-hydroxy-
ethylidene)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane] (15) (459
mg, 1.3 mmol) or toluene-4-sulfonate(4-hydroxybut-2-ynyl)trim-
ethylammonium (18) (388 mg, 1.3 mmol), dry pyridine (12 mL),
and dry CHCl3 (10 mL) were added and the reaction mixture was
stirred at ambient temperature for 48 h. Hydrolysis and purification,
according to method A, afforded the desired ether phospholipids.
{2-[(5-Cyclodecylidenepentyloxy)hydroxyphosphinyloxy]ethyl}-
N,N,N-trimethylammonium Inner Salt (19). Method A described
above using 5-(cyclodecylidene)pentanol (5a) (224.3 mg, 1 mmol)
and choline chloride (205 mg, 1.5 mmol) led to the formation of
compound 19 as a yellowish, gummy solid (67 mg, 17%). 1H NMR
(δ) 5.08 (t, J ) 6.71 Hz, 1H), 4.22 (bs, 2H), 3.75 (bs, 4H), 3.32 (s,
9H), 2.16–1.91 (m, 6H), 1.60–1.20 (m, 18 H); 31P NMR (δ) -1.04;
13C NMR (δ) 138.5, 126.0, 66.2, 66.1, 65.6, 65.5, 59.2, 54.2, 35.6,
35.2, 30.9, 30.8, 27.8, 27.5, 26.5, 26.2, 26, 25.9, 25.8, 25.7, 24.7,
24.5, 23.2, 20.8, 20.5. Anal. (C20H40NO4P) C, H,N.
{2-[(5-Cyclodecylideneundecyloxy)hydroxyphosphinyloxy]ethyl}-
N,N,N-trimethylammonium Inner Salt (20). Following method
A described above using 11-(cyclodecylidene)undecanol (8a) and
choline chloride, compound 20 was obtained as a yellowish, gummy
solid (246 mg, 52%). 1H NMR (δ) 5.13 (t, J ) 6.71 Hz, 1H), 4.25
(bs, 2H), 3.76 (bs, 4H), 3.38 (s, 9H), 2.14–1.94 (m, 4H), 1.65–1.22
(m, 32H); 31P NMR (δ) -1.22; 13C NMR (δ) 137.9, 126.6, 66.1,
65.8, 59.2, 54.3, 35.1, 31.9, 31.0, 30.8, 29.9, 29.6, 29.4, 28.2, 28.1,
26.0, 25.7, 24.8, 24.7, 24.5, 23.1, 22.3. Anal. (C26H52NO4P) C,
H, N.
{2-[(5-Cyclopentadecylidenepentyloxy)hydroxyphosphinyloxy]-
ethyl}-N,N,N-trimethylammonium Inner Salt (21). Following
method A described above using 5-(cyclopentadecylidene)pentanol
(5b) and choline chloride, compound 21 was afforded as a
yellowish, gummy solid (152 mg, 33%). 1H NMR (δ) 5.03 (t, J )
6.71 Hz, 1H), 4.19 (bs, 2H), 3.73 (bs, 4H), 3.26 (s, 9H), 1.93 (bs,
6H), 1.55–1.28 (m, 28H); 31P NMR (δ) -1.07; 13C NMR (δ) 140.3,
124.6, 66.2, 66.1, 65.7, 65.6, 59.2, 54.2, 37.6, 30.7, 29.9, 27.8, 27.7,
27.6, 27.4, 27.1, 26.7, 26.6, 26.5, 26.4. Anal. (C25H50NO4P.3H2O)
C, H, N.
similar to the control (untreated THP1 monocytes) (control
in Figure 2G).
Conclusions
To date, there is no vaccine against leishmaniasis and
chemotherapy is the main weapon in our arsenal. There is an
urgent need for better drugs against Leishmania. Teratogenicity,
potential of resistance, and low therapeutic window pose
limitations on miltefosine, and it is desirable to optimize its
structure with regard to activity and/or overcoming these
drawbacks. We have elucidated some of the structural features
required for antileishmanial activity of alkylphosphocholines and
shown that the hemolytic activity and cytotoxicity of this class
of compounds can be substantially decreased by introducing
cycloalkanes into the lipid portion while activity is increased.
The series of 22 new ring-substituted ether phospholipids
reported in the present study allows us to further refine the
pharmacophoric elements of this class of antileishmanial
compounds. All new analogues, except compound 33, are 1.5-
to 62-fold more potent than miltefosine against the intracellular
amastigote L. infantum. The presence of constrained head groups
is beneficial to activity; however, it results in increased toxicity
and hemolytic activity in comparison to the choline-substituted
congeners. The analogues that combine high potency and low
cytotoxicity and hemolytic activity are 5-cyclodecylidenepen-
tylphosphocholine (19) and its hydrogenated derivative 37,
5-cyclopentadecylidenepentylphosphocholine (21) and its N-
methylmorpholino congener 23, and the hydrogenated deriva-
tives 38-40. All the hydrogenated derivatives 37–40 are not
cytotoxic, being more potent than their unsaturated counterparts.
5-Cyclopentadecylpentylphosphocholine (38) is the least cyto-
toxic analogue (toxicity against THP1 macrophages of >500
µM), possessing very low hemolytic activity (HC50 > 100 µM)
and high potency against L. infantum amastigotes IC50 ) 0.7
µM. These findings make compound 38 an interesting and
suitable candidate for further studies in vivo as well as against
trypanosmatidae.
Experimental Section
Chemistry. All reactions were carried out under scrupulously
dry conditions. NMR spectra of all new compounds were recorded
in CDCl3, unless stated otherwise, using a Bruker AC 300
1
spectrometer operating at 300 MHz for H, 75.43 MHz for 13C,
1
and 121.44 MHz for 31P. H NMR spectra are reported as δ with
CHCl3 resonance at 7.24 ppm used as the chemical shift resonance.
13C NMR shifts are expressed in units relative to CDCl3 at 77.00
ppm, while 31P NMR spectra are reported as δ relative to 85%
H3PO4 used as an external standard. Silica gel plates (Merck F254
)
were used for thin-layer chromatography. Chromatographic puri-
fication was performed with silica gel (200–400 mesh). Analyses
of the elements were carried out by the microanalytical section of
the Institute of Organic and Pharmaceutical Chemistry of the
National Hellenic Research Foundation.
1-{2-[(5-Cyclopentadecylidenepentyloxy)hydroxyphosphinyloxy]-
ethyl}-1-methylpiperidinium Inner Salt (22). Following method
A described above using 5-(cyclopentadecylidene)pentanol (5b) and
N-(2-hydroxyethyl)-N-methylpiperidinium bromide, compound 22
was formed as a yellowish, gummy solid (214 mg, 43%). 1H NMR
(δ) 5.04 (t, J ) 6.71 Hz, 1H), 4.27 (bs, 2H), 3,81–3,53 (m, 8H),
3.30 (s, 3H), 1.95–1.50 (m, 14H), 1.30–1.22 (m, 26H); 31P NMR
(δ) -0.50; 13C NMR (δ) 140.1, 124.6, 65.4, 65.3, 63.3, 61.9, 61.7,
61.6, 61.5, 61.4, 61.3, 61.0, 58.7, 58.5, 58.4, 55, 48.5, 37.5, 29.9,
27.7, 27.5, 27.3, 27.1, 26.7, 26.6, 26.4, 20.8, 20.1. Anal.
(C28H54NO4P) C, H, N.
General Procedure for the Preparation of Ether Phospho-
lipids. Method A. To a solution of phosphorus oxychloride (0.09
mL, 1 mmol) in dry THF (5 mL) was added at 0 °C dry
triethylamine (0.25 mL, 1.8 mmol) and subsequently a solution of
the corresponding alcohol (1 mmol) in dry THF (7 mL). The
mixture was stirred at ambient temperature for 2 h, and then H2O
(5 mL) was added and stirring was continued for 1 h. The aqueous
layer was extracted first with EtOAc and then with CH2Cl2. The
combined organic extracts were dried over anhydrous Na2SO4 and
the solvent was evaporated in vacuo to afford the corresponding
phosphoric acid derivative, which was transformed to the pyridinium
salt by the addition of 5 mL of anhydrous pyridine and stirring for
12 h at 40 °C. After the mixture was cooled, the solvent was
evaporated in vacuo. To a solution of the above salt (1 mmol) in
4-{2-[(5-Cyclopentadecylidenepentyloxy)hydroxyphosphinyloxy]-
ethyl}-4-methylmorpholinium Inner Salt (23). Following method
A described above using 5-(cyclopentadecylidene)pentanol (5b) and

Ether Phospholipid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 905
N-(2-hydroxyethyl)-N-methylmorpholinium bromide, compound 23
was formed as a yellowish, gummy solid (121 mg, 24%). 1H NMR
(δ) 5.04 (t, J ) 6.71 Hz, 1H), 4.28 (bs, 2H), 3.99–3.71 (m, 12 H),
3.45 (s, 3H), 1.94 (bs, 6H), 1.56–1.29 (m, 28H); 31P NMR (δ)
-0.57; 13C NMR (δ) 138.5, 126, 66.2, 66.1, 65.6, 59.2, 54.2, 35.6,
35.2, 30.9, 30.8, 27.9, 27.5, 26.5, 26.2, 26.0, 25.9, 25.8, 25.7, 24.7,
24.5, 23.2, 20.8, 20.5. Anal. (C27H52NO5P·2.5H2O) C,H,N.
{2-[(11-Cyclopentadecylideneundecyloxy)hydroxyphosphinyl-
oxy]ethyl}-N,N,N-trimethylammonium Inner Salt (24). Follow-
ing method A described above using 11-(cyclopentadecylidene)
undecanol (8b) and choline bromide, compound 24 was afforded
as a yellowish, gummy solid (281 mg, 52%). 1H NMR (δ) 5.04 (t,
J ) 6.71 Hz, 1H), 4.17 (bs, 2H), 3.71 (bs, 4H), 3.31 (s, 9H), 1.89
(bs, 6H), 1.55–1.28 (m, 40H); 31P NMR (δ) -0.69; 13C NMR (δ)
139.7, 125.2, 66.1, 66.0, 65.5, 59.1. 54.1, 37.5, 31.1, 31.0, 30.0,
29.9, 29.7, 29.5, 29.4, 27.8, 27.5, 27.4, 27.1, 26.8, 26.7, 26.4, 25.9,
25.7. Anal. (C31H62NO4P·3H2O) C, H, N.
1-{2-[(11-Cyclopentadecylideneundecyloxy)hydroxyphosphinyl-
oxy]ethyl}-1-methylpiperidinium Inner Salt (25). Following
method A described above using 11-(cyclopentadecylidene)unde-
canol (8b) and N-(2-hydroxyethyl)-N-methylpiperidinium bromide,
compound 25 was obtained as a yellowish, gummy solid (291 mg,
50%). 1H NMR (δ) 4.99 (t, J ) 6.71 Hz, 1H), 4.17 (bs, 2H),
3,76–3,50 (m, 8H), 3.23 (s, 3H), 1.95–1.50 (m, 14H), 1.30–1.22
(m, 38H); 31P NMR (δ) -0.47; 13C NMR (δ) 139.6, 125.2, 65.4,
63.3, 61.7, 58.5, 49.6, 48.5, 37.4, 31.0, 30.9, 30.1, 29.9, 29.6, 29.4,
29.3, 27.7, 27.4, 27.3, 27.1, 26.7, 26.6, 26.4, 26.3, 25.8, 20.8, 20.1.
Anal. (C34H66NO4P·2H2O) C, H, N.
1H), 4.86 (bs, 1H), 3.97–3.65 (m, 3H), 3,36 (s, 9H), 2.09–1.90 (m,
8H), 1.73 (bs, 4 H), 1.48 (bs, 8H), 1.21 (bs, 14H); 31P NMR (δ)
-1.09; 13C NMR (δ) 139.3, 121.4, 79.8, 79.7, 74.3, 74.2, 65.3,
65.2, 52.0, 37.1, 32.2, 31.0, 30.9, 30.2, 29.6, 29.5, 28.6, 27.8, 27.1,
27.0, 26.9, 25.9, 24.6, 19.8. Anal. (C25H48NO4P·2H2O) C, H, N
trans-{2-[(11-Cyclohexylideneundecyloxy)hydroxyphosphinyl-
oxy]}-N,N,N-trimethylcyclohexaneammonium Inner Salt (30).
Following method B described above using 11-(cyclohexylidene)
undecanol (8c) and trans-(2-hydroxycyclohexyl)trimethylam-
monium toluene-4-sulfonate (11b) gave compound 30 as a white,
1
gummy solid (90 mg, 19%). H NMR (δ) 5.01 (t, J ) 6.71 Hz,
1H), 4.41 (bs, 1H), 3.83–3.74 (bs, 3H), 3.36 (s, 9H), 2.36 (bs, 1H),
2.22 (bs, 1H), 2.08–1.83 (m, 7H), 1.64–1.21 (m, 27H); 31P NMR
(δ) -2.10; 13C NMR (δ) 139.3, 121.4, 73.7, 73.6, 53.3, 50.0, 37.1,
34.0, 30.9, 30.8, 30.2, 29.6, 29.5, 29.4, 29.3, 29.1, 29.0, 28.8, 28.6,
28.5, 27.8, 27.1, 27.0, 26.9, 25.9, 24.9, 23.5. Anal. (C26H50NO4P)
C, H, N.
trans-{2-[[11-(Adamantylidene)undecyloxy]hydroxyphosphinyl-
oxy]}-N,N,N-trimethylcyclopentaneammonium Inner Salt (31).
Following method B described above using 11-(adamantylidene)
undecanol (8d) and trans-(2-hydroxycyclopentyl)trimethylam-
monium toluene-4-sulfonate (11a), compound 31 was obtained as
1
a white, gummy solid (90 mg, 18%). H NMR (δ) 4.99 (t, J )
6.71 Hz, 1H), 4.88 (bs, 1H), 3.95–3.72 (m, 3H), 3,35 (s, 9H), 2.78
(s, 1H), 2.28 (s, 1H), 2.14 (bs, 2 H), 1.91–1.53 (m, 18H), 1.23 (bs,
16H); 31P NMR (δ) -1.16; 13C NMR (δ) 147.3, 116.3, 81.1, 80.0,
79.9, 74.4, 74.3, 71.0, 65.4, 65.3, 52.2, 45.9, 40.5, 39.9, 38.9, 37.3,
32.3, 32.0, 31.0, 30.9, 30.4, 29.7, 29.6, 29.3, 28.7, 26.5, 25.9, 24.7,
19.9. Anal. (C29H52NO4P) C, H, N.
4-{2-[(11-Cyclopentadecylideneundecyloxy)hydroxyphosphinyl-
oxy]ethyl}-4-methylmorpholinium Inner Salt (26). Following
method A described above using 11-(cyclopentadecylidene)unde-
canol (8b) and N-(2-hydroxyethyl)-N-methylmorpholinium bromide
afforded compound 26 as a yellowish, gummy solid (215 mg, 37%).
1H NMR (δ) 5.05 (t, J ) 6.71 Hz, 1H), 4.24 (bs, 2H), 3.96–3.70
(m, 12 H), 3.44 (s, 3H), 1.94 (bs, 6H), 1.53–1.19 (m, 40H); 31P
NMR (δ) -0.63; 13C NMR (δ) 139.7, 125.2, 65.6, 65.0, 64.2, 60.7,
58.5, 48.1, 37.5, 31.1, 31.0, 30.2, 30.1, 30.0, 29.9, 29.8, 29.6, 29.5,
29.4, 29.3, 29.0, 27.8, 27.5, 27.4, 27.1, 26.8, 26.7, 26.4, 25.9. Anal.
(C33H64NO5P) C, H, N.
trans-{2-[[11-(Adamantylidene)undecyloxy]hydroxyphosphinyl-
oxy]}-N,N,N-trimethylcyclohexaneammonium Inner Salt (32).
Following method B described above using 11-(adamantylidene)
undecanol (8d) and trans-(2-hydroxycyclohexyl)trimethylam-
monium toluene-4-sulfonate (11b) led to the formation of compound
1
32 as a white, gummy solid (172 mg, 33%). H NMR (δ) 4.98 (t,
J ) 6.71 Hz, 1H), 4.41 (m, 2H), 3.80–3.78 (bs, 2H), 3.33 (s, 9H),
2.76 (s, 1H), 2.33–2.27 (m, 3H), 1.81–1.41 (m, 20H), 1.23 (bs,
16H); 31P NMR (δ) -2.67; 13C NMR (δ) 147.3, 116.3, 74.0, 73.9,
65.4, 65.3, 53.4, 40.5, 40.4, 39.9, 39.8, 39.7, 39.6, 38.9, 37.3, 37.2,
33.9, 32.0, 31.0, 30.9, 30.4, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 29.0,
28.9, 28.8, 27.7, 28.6, 28.5, 28.4, 26.6, 26.5, 26.4, 25.9, 25.7, 24.9,
23.5. Anal. (C30H54NO4P) C, H, N.
trans-{2-[(5-Cyclopentadecylidenepentyloxy)hydroxyphosphinyl-
oxy]}-N,N,N-trimethylcyclopentaneammonium Inner Salt (27).
Following method B described above using 5-(cyclopentade-
cylidene)pentanol (5b) and trans-(2-hydroxycyclopentyl)trimethyl-
ammonium toluene-4-sulfonate (11a), compound 27 was obtained
8,8-Dimethyl 3-{2-[11-[adamantylidene)undecyloxy]hydro-
xyphosphinyloxy]ethylidene}-8-azoniabicyclo[3.2.1]octane In-
ner Salt (33). Following method B described above using 11-
(adamantylidene)undecanol (8d) and 3-(2-hydroxyethylidene)-8,8-
dimethyl-8-azoniabicyclo[3.2.1]octane, p-toluenesulfonate (15) led
to the formation of compound 33 as a white, gummy solid (55 mg,
10%). 1H NMR (δ) 5.74 (bs, 1H), 4.98 (t, J ) 7.32 Hz, 1H),
4.32–4.27 (m, 3H), 4.08 (bs, 1H) 3.76–3.74 (bs, 2H), 3.44–3.07
(m, 7H), 2.76 (bs, 3H), 2.36–2.25 (m, 4H), 1.94–1.54 (m, 16H),
1.23–1.18 (m, 16H); 31P NMR (δ) -0.41; 13C NMR (δ) 147.3,
129.6,127.8, 116.3, 74.0, 69.4, 69.0, 65.4, 65.3, 60.3, 50.9, 43.9,
40.5, 39.8, 38.9, 37.3, 36.7, 32.0, 31.0, 30.9, 30.5, 30.3, 29.7, 29.6,
29.5, 29.2, 28.6, 26.5, 25.9. Anal. (C32H54NO4P) C, H, N.
{4-[(11-Adamantylideneundecyloxy)hydroxyphosphinyloxy]-
but-2-ynyl}-N,N,N-trimethylammonium Inner Salt (34). Fol-
lowing method B described above using 11-(adamantylidene)-
undecanol (8d) and toluene-4-sulfonate(4-hydroxybut-2-ynyl)
trimethylammonium (18) gave compound 34 as a gummy solid (291
1
as a white, gummy solid (11 mg, 22%). H NMR (δ) 5.05 (t, J )
6.71 Hz, 1H), 4.85 (bs, 1H), 3.93–3.70 (m, 3H), 3.73 (s, 9H),
2.15–2.09 (m, 2 H), 1.96 (bs, 6H), 1.75–1.71 (m, 4 H), 1.57–1.30
(m, 28H); 31P NMR (δ) -1.60; 13C NMR (δ) 139.4, 124.7, 80.1,
80.0, 74.6, 74.5, 65.8, 65.3, 65.0, 52.1, 50.2, 37.5, 37.1, 35.5, 32.4,
30.9, 30.6, 29.9, 29.5, 29.3, 29.2, 28.6, 28.2, 27.8, 27.7, 27.6, 27.4,
27.3, 27.2, 26.9, 26.8, 26.7, 26.6, 26.5, 26.0, 25.8, 25.6, 24.6, 20.3.
Anal. (C28H54NO4P) C, H, N.
trans-{2-[(5-Cyclopentadecylidenepentyloxy)hydroxyphos-
phinyloxy]}-N,N,N-trimethylcyclohexaneammonium Inner Salt
(28). Following method B described above using 5-(cyclopenta-
decylidene)pentanol (5b) and trans-(2-hydroxy-cyclohexyl)trim-
ethylammonium toluene-4-sulfonate (11b), compound 28 was
1
afforded as a white, gummy solid (117 mg, 23%). H NMR (δ)
5.05 (t, J ) 6.71 Hz, 1H), 4.38 (bs, 1H), 3.83–3.76 (m, 3H), 3.31
(s, 9H), 2.39–2.36 (bs, 1H), 2.19 (bs, 1H), 1.95–1.88 (m, 7H),
1.68–1.51 (m, 3 H), 1.27 (bs, 31H); 31P NMR (δ) -2.26; 13C NMR
(δ) 138.8, 125.0, 73.9, 73.8, 65.5, 65.4, 53.3, 37.5, 37.1, 35.5, 33.9,
30.8, 30.7, 30.5, 30.4, 29.9, 29.5, 29.3, 29.1, 28.5, 28.1, 28.0, 27.8,
27.7, 27.5, 27.4, 27.1, 26.9, 26.6, 26.5, 26.4, 26.0, 25.9, 25.7, 25.5,
24.6, 23.4. Anal. (C29H56NO4P) C, H, N.
1
mg, 50%). H NMR (δ) 4.98 (t, J ) 7.32 Hz, 1H), 4.49 (bs, 2H),
4.47 (bs, 2H), 3.74–3.72 (m, 2H), 3.36 (s, 9H), 2.76 (bs, 1H), 2.26
(bs, 1H), 1.89–1.54 (m, 16H), 1.23–1.20 (m, 14H); 31P NMR (δ)
-0.50; 13C NMR (δ) 147.2, 116.3, 89.4, 76.5, 65.5, 64.9, 56.1,
52.6, 40.5, 39.8, 39.0, 38.9, 37.3, 32.0, 31.9, 30.9, 29.7, 29.6, 29.5,
29.2, 28.9, 28.6, 28.5, 28.4, 28.3, 26.4, 25.9. Anal. (C28H48
NO4P·H2O) C, H, N.
trans-{2-[(11-Cyclohexylideneundecyloxy)hydroxyphosphinyl-
oxy]}-N,N,N-trimethylcyclopentaneammonium Inner Salt (29).
Following method B described above using 11-(cyclohexylidene)
undecanol (8c) and trans-(2-hydroxycyclopentyl)trimethylam-
monium toluene-4-sulfonate (11a) gave compound 29 as a white,
trans-{2-[Hexadecyloxy]hydroxyphosphinyloxy]}-N,N,N-tri-
methylcyclopentaneammonium Inner Salt (35). Following method
B described above using hexadecanol (8e) and trans-(2-hydroxy-
cyclopentyl)trimethylammonium toluene-4-sulfonate (11a) afforded
1
gummy solid (128 mg, 28%). H NMR (δ) 5.01 (t, J ) 6.71 Hz,

906 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Calogeropoulou et al.
compound 35 as a gummy solid (204 mg, 40%). 1H NMR (δ) 4.80
(bs, 1H), 3.88–3.62 (m, 3H), 3.24 (s, 9H), 2.09–2.03 (m, 2H),
1.73–1.66 (m, 4H), 1.53–1.49 (m, 2H), 1.19–1.17 (m, 26H), 0.79
(t, J ) 7.0 Hz, 3H); 31P NMR (δ) -1.07; 13C NMR (δ) 79.94,
79.9, 74.3, 74.2, 65.4, 65.3, 51.9, 32.2, 31.9, 31.8, 30.9, 30.8, 30.2,
29.6, 29.5, 29.4, 29.3, 25.8, 24.5, 22.6, 19.8, 14.0. Anal.
(C24H50NO4P) C, H, N.
trans-{2-[(Hexadecyloxy)hydroxyphosphinyloxy]}-N,N,N-tri-
methylcyclohexaneammonium Inner Salt (36). Method B de-
scribed above using hexadecanol (8e) and trans-(2-hydroxycyclo-
hexyl)trimethylammonium toluene-4-sulfonate (11b) afforded
compound 36 as a gummy solid (286 mg, 50%). 1H NMR (δ)
4.39–4.37 (m, 1H), 3.89–3.75 (m, 3H), 3.24 (s, 9H), 2.65–2.51 (m,
4H), 2.43–2.39 (m, 1H), 2.19–2.15 (m, 1H), 1.86–1.21 (m, 30H),
0.83 (t, J ) 6.7 Hz, 3H); 31P NMR (δ) -1.88; 13C NMR (δ) 73.8,
73.7, 65.7, 65.6, 53.4, 33.9, 31.9, 30.7, 29.6, 29.3, 25.7, 24.9, 23.2,
22.6, 14.0. Anal. (C25H52NO4P·H2O) C, H, N.
{2-[(5-Cyclodecylpentyloxy)hydroxyphosphinyloxy]ethyl}-
N,N,N-trimethylammonium Inner Salt (37). To a solution of
compound 19 (20 mg, 0.05 mmol) in MeOH (1 mL) was added a
catalytic amount of Pd/C (10% w/w). The suspension was stirred
under 1 atm of hydrogen gas for 24 h. The reaction mixture was
filtered through Celite and the filtrate was concentrated in vacuo
to afford compound 37 as a yellowish, gummy solid (20 mg, 100%).
1H NMR (δ) 4.08 (bs, 2H), 3.68 (bs, 2H), 3.46 (bs, 2H), 3.05 (s,
9H), 1.45–1.0 (m, 27 H); 31P NMR (δ) -0.53; 13C NMR (δ) 66.1,
58.9, 53.9, 36.9, 35.4, 30.5, 30.4, 30.3, 27.1, 25.9, 25.4, 25.3, 24.7,
23.5. Anal. (C20H42NO4P) C, H, N.
Determination of in Vitro Antileishmanial Activity in Prom-
astigote Cultures. All compounds were dissolved in DMSO/
ethanol, 50/50 v/v, to a final concentration of 65 mM, and linear
5-fold dilutions ranging from 100 to 0.8 µM were done in the culture
medium. An amount of 200 µL of promastigote culture at 2 × 106
cells/mL was incubated with 200 µL of the appropriate compound
concentration in a 24-well tissue culture plate (CELLSTAR,
Greiner) at 26 °C. After 72 h, cultures were washed in PBS, were
resuspended in 300 µL of Hepes buffered solution, and were stained
with propidium iodide (PI) and SYBR-14 using the LIVE/DEAD
viability kit (Molecular Probes) according to the manufacturer
recommendations. Cell samples were analyzed by flow cytometry
as described below. Calculation of IC50 values was done according
to Hills et al.42,43
Determination of in Vitro Antileishmanial Activity against
the Intracellular L. infantum. THP-1 cells were differentiated with
1 mM retinoic acid (Sigma) for 3 days at 37 °C and 5% CO2.
Infection of THP-1 cells with promastigotes was achieved by mixing
the THP-1 and the parasite cultures at a ratio of 1:4 and incubation
at 37 °C and 5% CO2. The next day cells were washed by
centrifugation at 400g for 10 min, the pellet was resuspended in
RPMI medium, and the suspension was overlaid with an equal
volume of Histopaque 1077 (Invitrogen). The free promastigotes
were removed by centrifugation at 1000g for 20 min. The cell layer
was washed with PBS twice and resuspended in RPMI at 4 × 105
cells/mL. Infection rate was assessed microscopically by Giemsa
staining of the infected THP-1 suspension. An amoutn of 200 µL
of infected THP-1 was plated in a 24-well tissue culture plate
(Cellstar, Greiner) and mixed with an equal volume of the
appropriate compound concentration. Incubation proceeded for 72 h
at 37 °C and 5% CO2, and the percentage of infected cells was
monitored microscopically after Giemsa staining. The IC50 was
calculated as described for the promastigotes above.
Assessment of Hemolytic Potential. EDTA-preserved peripheral
blood from healthy volunteers was centrifuged in order to remove
serum, and red blood cells were washed thrice in PBS. After the
final wash, cells were distributed in 96-well microplates (100 µL/
well) and an equal volume of the appropriate compound concentra-
tion was added. The tested compounds were diluted in PBS in
concentrations ranging from 100 to 6.25 µM. Incubation proceeded
at 37 °C for 1 h, and red blood cells were then centrifuged at 800g
for 10 min. Absorbance of the supernatants were measured at 550
nm with reference filter at 625 nm. The percentage of hemolytic
activity of each drug at different concentrations was estimated using
[(A - A0)/(Amax - A0)] × 100, where A0 is the background
hemolysis obtained by incubation with PBS and Amax is the 100%
hemolysis achieved after incubation in H2O.
Assessement of Cytotoxicity in THP-1 Monocytic Cells. As a
quantitative measurement of the cell damage after incubation with
different concentrations of drugs, dual staining with SYBR-14 and
PI (Molecular Probes, The Neatherlands) was used. THP-1 cell
cultures were incubated at 1 × 106 cells/mL with different
concentrations of the compounds ranging from 50 to 1.56 µM. After
an incubation period of 72 h approximately 4 × 106 cells were
stained with PI and SYBR-14 as described for the promastigote
staining. The ratio alive/dead cells was assessed by flow cytometry
as described below.
Flow Cytometric Analysis. Cell samples were analyzed on an
Epics Elite model flow cytometer (Coulter, Miami, FL). Excitation
of both dyes was done at 488 nm. Differential monitoring of the
dyes was achieved by reading the green fluorescence of SYBR-14
at 545 nm and the red fluorescence of PI at 645 nm. At least 10 000
cells were analyzed per sample, and each staining experiment was
repeated thrice. Data analysis was performed on fluorescence
intensities that excluded cell autofluorescence and cell debris.
{2-[(5-Cyclopentadecylpentyloxy)hydroxyphosphinyloxy]ethyl}-
N,N,N-trimethylammonium Inner Salt (38). 38 was prepared
following the procedure described for compound 37, starting from
compound 21 (139 mg, 0.3 mmol) and giving a yellowish, gummy
1
solid (130 mg, 93%). H NMR (δ) 4.20 (bs, 2H), 3.72 (bs, 4H),
3.29 (s, 9H), 1.53–1.22 (m, 37H); 31P NMR (δ) -1.00; 13C NMR
(δ) 66.1, 65.8, 59.2, 54.2, 50.0, 44.9, 36.6, 35.3, 32.4, 31.0, 27.7,
27.2, 27.0, 26.8, 26.6, 26.5, 26.3, 24.6. Anal. (C25H52NO4P) C,
H, N.
4-{2-[(5-Cyclopentadecylpentyloxy)hydroxyphosphinyloxy]-
ethyl}-1-methylpiperidinium Inner Salt (39). 39 was prepared
following the procedure described for compound 37, starting from
compound 22 (195 mg, 0.39 mmol) and giving a yellowish, gummy
1
solid (190 mg, 97%). H NMR (δ) 4.18 (bs, 2H), 3.76–3.46 (m,
8H), 3.24 (s, 3H), 1.77 (bs, 4H), 1.59–1.47 (bs, 4H), 1.21–1.15
(m, 35H); 31P NMR (δ) -0.53; 13C NMR (δ) 65.5, 65.4, 63.2,
61.7, 58.5, 48.6, 36.4, 35.2, 32.4, 31.1, 31, 27.6, 27, 26.9, 26.7,
26.6, 26.4, 26.3, 24.6, 20.8, 20.1. Anal. (C28H56NO4P) C, H, N.
4-{2-[(5-Cyclopentadecylpentyloxy)hydroxyphosphinyloxy]-
ethyl}-4-methylmorpholinium Inner Salt (40). 40 was prepared
following the procedure described for compound 37, starting from
compound 23 (110 mg, 0.22 mmol) and giving a yellowish, gummy
1
solid (110 mg, 100%). H NMR (δ) 4.30 (bs, 2H), 4.03–3.74 (m,
12 H), 3.48 (s, 3H), 1.56–1.29 (m, 37H); 31P NMR (δ) -1.07; 13
C
NMR (δ) 66.1, 65, 60.7, 58.6, 48.3, 45, 36.6, 35.3, 32.4, 31, 27.7,
27.2, 26.9, 26.8, 26.7, 26.6, 26.5, 26.4, 25.9, 24.6. Anal.
(C27H54NO5P) C, H, N.
Evaluation of in Vitro Antiparasitic Activity. Cell Culture
and Parasite Strains. The human monocytic cell line THP-1 was
used as a host cell for the leishmania parasites. Infection rate of
this cell line ranged between 35% and 45% and was significantly
lower than the infection rate achieved with mouse peritoneal
macrophages (85–95%). However, the use of THP-1 as the parasite
host cell for the evaluation of the compound antileishamnial activity
against the intracellular parasite proved to be highly reproducible
and convenient for the large number of compounds to be tested in
three independent experiments. THP-1 cells are maintained in
culture in RPMI 1640 supplemented with 10% FCS, L. glutamine,
and antibiotics at 37 °C/5% CO2. Promastigotes of Leishmania
infantum MHOM/TN/80/IPT1/LEM 235 and Leishmania donoVani
MHOM/IN/80/DD8/LEM 703 were grown in RPMI 1640 supple-
mented with 10% FCS, L. glutamine, and antibiotics at 26 °C.
Acknowledgment. The authors thank Helen Koutala for the
flow cytometry measurements. This work was supported by the
Greek General Secretariat for Research and Technology (Grant
EPAN YB39).

Ether Phospholipid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 907
(21) Berman, J.; Bryceson, A. D. M.; Croft, S.; Engel, J.; Gutteridge, W.;
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metabolism, GPI anchor biosynthesis and signal transduction are
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Supporting Information Available: Elemental analysis data for
compounds 5a,b, 8a,b, 11a,b, 15, 18–40 and experimental proce-
dures and spectroscopic data for compounds 3a,b, 4a,b, 5a,b, 7a,b,
8a,b, 10a,b, 11a,b, 13-15, 17, and 18. This material is available
free of charge via the Internet at http://pubs.acs.org.
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