Synthesis and antifungal activities of miltefosine analogs

Article abstract of DOI:10.1016/j.bmcl.2013.06.096

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycose

Full text of DOI:10.1016/j.bmcl.2013.06.096

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4828–4831
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal activities of miltefosine analogs
Ranga Rao Ravu a, Ying-Lien Chen b, Melissa R. Jacob a, Xuewen Pan c, Ameeta K. Agarwal a,
Shabana I. Khan a,d, Joseph Heitman b, Alice M. Clark a,d, Xing-Cong Li a,d,
⇑
a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi, MS 38677, USA
b Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA
c Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
d Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and lim-
ited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of
compounds for the treatment of systemic mycoses, nine analogs (3aÀ3i) were synthesized by modify-
ing the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of
these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida
krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hex-
adecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-
{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-
dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum
Received 24 May 2013
Revised 19 June 2013
Accepted 27 June 2013
Available online 6 July 2013
Keywords:
Alkylphosphocholine
Miltefosine
Antifungal
Cryptococcosis
Candidiasis
inhibitory concentrations (MIC) of 2.5–5.0
lg/mL against all tested pathogens, when compared to mil-
tefosine with MICs of 2.5–3.3 g/mL. Compound 3a showed low in vitro cytotoxicity against three mam-
l
malian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse
model of systemic infection did not demonstrate efficacy. The results of this study indicate that further
investigation will be required to determine the potential usefulness of the alkylphosphocholines in the
treatment of invasive fungal infections.
Ó 2013 Elsevier Ltd. All rights reserved.
Miltefosine (hexadecylphosphocholine) is
a
synthetic
and maturation.11 Mechanistic studies indicated that miltefosine
alkylphosphocholine that belongs to the class of phospholipids. It
was initially developed as an antineoplastic agent,1,2 and was later
discovered to possess antileishmanial properties and registered as
the first oral drug for the treatment of visceral leishmaniasis in In-
dia and Germany, and for the treatment of cutaneous leishmaniasis
in Colombia.3 Pharmacokinetic studies indicate that miltefosine
has good bioavailability and a long half-life in patients with leish-
mania (7 days for the first elimination and 31 days for the terminal
elimination).4 This may be attributable to its improved in vivo
antileishmanial activity relative to analogs with even more potent
in vitro activities.3 Miltefosine also possesses antibacterial,5 anti-
protozoal,6 and antiviral activities.7
Miltefosine was demonstrated to be active against Candida
albicans and Cryptococcus neoformans in 1999.8 In recent years, mil-
tefosine was found to exhibit broad-spectrum antifungal activities
against clinically important fungal pathogens9 and dermato-
phytes10 in addition to inhibiting C. albicans biofilm formation
inhibits cytochrome c oxidase in the model organism Saccharomy-
ces cerevisiae and phospholipase
B in the fungal pathogen
C. neoformans,9,12 while in human cells it inhibits activation of
the protein kinase B pathway as well as phosphatidylcholine syn-
thesis.13 However, none of these targets is essential for the survival
of fungal cells according to what is known in S. cerevisiae. There-
fore, miltefosine and analogs remain to be an intriguing class of
compounds in terms of their precise antifungal target.
Miltefosine gained particular interest in antifungal therapy due
to the reported in vivo efficacy in a mouse model of cryptococco-
sis.9 In a more recent study aimed at further evaluating its
in vivo efficacy in mouse models of cryptococcal meningoenceph-
alitis and disseminated cryptococcosis, miltefosine demonstrated
limited effects in mice that were challenged with a low infecting
inoculum.14 Meningoencephalitis requires the drug to cross the
blood brain barrier to exert its action. Given that miltefosine has
a higher distribution in the lung and kidney of mice than in brain,15
we hypothesized that it might be more active in vivo against sys-
temic mycoses rather than infections in the brain. With chemical
synthesis, new analogs could be prepared and included for testing
this hypothesis. Therefore, we designed and synthesized several
⇑
Corresponding author. Address: National Center for Natural Products Research,
School of Pharmacy, The University of Mississippi, University, MS 38677, USA. Tel.:
+1 662 915 6742; fax: +1 662 915 7989.
E-mail address: xcli7@olemiss.edu (X.-C. Li).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.096

R. R. Ravu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4828–4831
4829
new miltefosine analogs and evaluated their antifungal activities
in vitro and in vivo in a candidiasis mouse model.
Me
N
Me
N
O
P
R1
OH
R1
O
OR2
Me
OH
b
a
N
The available structure–antifungal activity relationship (SAR)
information on alkylphosphocholines was the basis for designing
new compounds in this study. A hydrophobic chain in the miltefo-
sine analogs with 16–18 carbon atoms is necessary for antifungal
activity.16 Reduction of the alkyl chain length to 12 carbon
atoms,16,17 increasing the chain length to 22 carbon atoms,8 or
insertion of ester/amide functionalities in the middle of this
chain16 significantly reduces the antifungal activity. Structurally
more complex alkylglycerophosphocholines exhibit moderate
activities against C. albicans and C. neoformans, when compared
with alkylphosphocholine derivatives.16 Extensive modification of
the N-substitution and the C2 unit of the choline moiety (head
group) resulted in a large number of compounds,17–19 some of
which showed activities more potent than erucylphosphocholine19
that is eight-fold less potent than miltefosine.16 Within this class,
octadecylphosphocholine demonstrates as much as a four-fold in-
crease in in vitro potency against C. albicans when compared to
miltefosine.16 It appears that the intact head group or the presence
of at least two small N-methyl groups plays a key role for anti-
fungal activity. Based on the above SAR information, we decided
to synthesize compounds by slightly modifying the structure of
miltefosine (Fig. 1).
We first designed compound 3a with a benzyl group replacing
one methyl group of the choline structural moiety in miltefosine,
taking into consideration the strong antimicrobial activity of ben-
zalkonium chloride that possesses the benzyldimethylammonio
structural moiety.20 However, the zwitterion nature of 3a makes
it distinctly different from the cationic surface-acting benzalkoni-
um chloride. While this design allows the compound to retain most
of the structural features required for antifungal activity within the
class, it also increases lipophilicity due to the introduction of an
aromatic ring, as indicated by the calculated octane-water parti-
tion coefficient (clogP) from 1.80 for miltefosine to 3.80 for 3a,21
which may improve antifungal properties. The synthetic method
for the preparation of 3a is an adaption of the reported proce-
dures18,22 and is depicted in Scheme 1. Quaternization of
N,N-dimethylaminoethanol (1) with benzyl bromide afforded the
quaternary ammonium salt 2, which was subject to phosphoryla-
tion reaction of n-hexadecanol with POCl3 followed by hydrolysis
to afford the target compound.23
Me
1
Me
2
Me
O
3
R1
R2
c LogP
3a
3b
3c
3d
3e
3f
n-C16H33
n-C14H29
n-C18H37
n-C16H33
n-C16H33
n-C16H33
n-C16H33
n-C16H33
3.80
CH2
2.97
4.49
3.69
3.77
4.29
4.39
4.21
CH2
CH2
O2N
MeO
Cl
CH2
CH2
CH2
3g
3h
Br
CH2
CH2
CH2
3i
n-C16H33
2.38
Scheme 1. Reagents and conditions: (a) R1X, CH3CN, room temperature, 1–3 h; (b)
(1) R2OH, POCl3, Et3N, CHCl3, 0 °C, room temperature, 2 h, (2) pyridine, 2a–2i, 0 °C,
room temperature, 12 h, (3) H2O, room temperature, 1 h.
when compared with 3a (Table 1), and compound 3c was only ac-
tive against C. glabrata with an MIC/MFC of 4.2/4.2 lg/mL, indicat-
ing that C16 is an optimal alkyl chain length.
Keeping a constant C16 alkyl chain, we next synthesized six
analogs (3dÀ3i) with different head groups by replacing one
methyl group of the choline moiety in miltefosine with p-nitroben-
zyl, p-chlorobenzyl, p-bromobenzyl, p-methoxybenzyl, cinnamyl,
and allyl groups. Among these, compound 3d with an N-4-nitro-
benzyl substitution produced the best in vitro activity profiles,
exhibiting slightly improved potency against C. glabrata and A.
fumigatus when compared to miltefosine (Table 1). Compound 3e
with an N-4-methoxybenzyl substitution also showed good activi-
ties similar to 3a and 3d (Table 1). Compounds 3f and 3g with a
halogen-substituted aromatic ring were only active against C. glab-
rata and A. fumigatus, and 3i with an N-allylic substitution, the only
compound without an aromatic ring in this series, was only active
As shown in Table 1, in vitro antifungal testing by the method
described previously24 indicated that compound 3a showed potent
activities with minimum inhibitory concentrations (MICs) ranging
from 2.5 to 5.0 lg/mL against the opportunistic fungal pathogens
C. albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus,
and C. neoformans. The compound was also fungicidal against all
tested fungal pathogens with minimum fungicidal concentrations
(MFCs)25 from 2.5 to 15.0
l
g/mL. Its antifungal potency is similar
to that of miltefosine with MICs and MFCs of 2.1–3.3 and 2.1–
9.2 g/mL, respectively, against the aforementioned pathogens.
To investigate the influence of the chain length on the anti-
against C. glabrata with an MIC/MFC of 16.6/16.6 lg/mL. It appears
that among the five tested fungal species, C. glabrata is most sus-
ceptible to this series of compounds. Evidently, the minor struc-
tural differences of these compounds, especially for compounds
3a and 3dÀ3g, have a significant effect on their activity profiles.
In addition, the permeability of the compounds towards different
fungal cells, which may be associated with their lipophilicities,
may play a role in the observed activities. Coincidentally, the three
compounds 3a, 3d, and 3e with close chemical structures that
showed excellent activity profiles have similar calculated logp val-
ues ranging from 3.69 to 3.80 (Scheme 1).
The in vitro antifungal activity data of miltefosine obtained in
this study (Table 1) are similar to those reported in the literature.9
The potent activities of the three synthetic analogs (3a, 3d, and 3e)
are further evident by comparison with the ‘gold standard’ clinical
drug amphotericin B. Compounds 3a and 3d that showed strong
l
fungal activity within this series, analogs 3b and 3c with the same
head group but an alkyl chain length of C14 and C18, respectively,
were prepared by a synthetic method similar for 3a. However,
compound 3b showed decreased activity against C. albicans, C.
glabrata, C. krusei, and A. fumigatus in terms of MICs and MFCs
Me
N
O
P
R1
O
O
Me
O
Figure 1. Miltefosine (R1 = Me) based synthetic template.

4830
R. R. Ravu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4828–4831
Table 1
In vitro antifungal activity and cytotoxicity of miltefosine and compounds 3a–3ia
Antifungal activityb (MIC/MFC,
l
g/mL)
Cytotoxicityc
(IC50 g/mL)
,
l
C. albicans ATCC
90028
C. glabrata ATCC
90030
C. krusei ATCC
6258
A. fumigatus ATCC
90906
C. neoformans ATCC
90113
Vero HepG2 LLC-
PK11
Miltefosine
3a
3b
3c
3d
3e
3f
3g
2.5/2.5
2.5/2.5
6.7/8.3
—/—d
2.5/2.5
4.2/4.2
—/—
—/—
—/—
—/—
0.9/1.0
3.3/4.2
3.3/3.3
8.3/8.3
4.2/4.2
2.5/2.5
2.5/3.3
11.2/11.2
10.8/10.8
2.5/3.3
16.6/16.6
1.3/1.5
2.5/3.3
5.0/15.0
10.0/10.0
—/—
2.5/3.3
2.0/3.3
—/—
—/—
3.3/3.3
—/—
2.9/9.2
2.5/2.5
10.0/–
—/—
2.5/7.5
2.5/7.5
12.5/12.5
20.0/20.0
2.5/5.0
—/—
2.1/2.1
3.3/3.3
3.3/3.3
—/—
2.5/2.5
4.2/4.2
—/—
—/—
—/—
—/—
0.5/0.5
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
13
11.5
12.9
21
21.3
19.6
13.2
2.7
4.8
10.8
4.7
1.6
1.9
3.2
5.5
2.1
4.1
3h
3i
Amphotericin B
Doxorubicin
1.7/1.7
2.5/5.0
>5
0.9
0.7
a
Both antifungal and cytotoxicity data are mean values based on three independent experiments except for the antifungal data of compound 3f with mean values from two
independent experiments.
b
MIC: minimum inhibitory concentration (lowest concentration that allows no detectable growth). MFC: minimum fungicidal concentration (the lowest concentration
that kills the fungus), which was determined by removing 5
l
L from each assay well with no visible growth, transferring to fresh media and incubating at the appropriate
g/mL; the highest test concentration for amphotericin B was 5 g/mL.
IC50: 50% growth inhibition. The highest test concentration for compounds 3a–3i and miltefosine was 25 g/mL; the highest test concentration for doxorubicin was 5 g/
temperature for 2–3 days. The highest test concentration for compounds 3a–3i and miltefosine was 20
l
l
c
l
l
mL.
d
Not active at 20 lg/mL.
activity against C. albicans with the same MIC/MFC of 2.5/2.5
mL are equally potent as miltefosine, and are about two to three
fold less potent than amphotericin B which has an MIC/MFC of
0.9/1.0 lg/mL (Table 1).
The in vitro cytotoxicity testing of compounds 3aÀ3i and mil-
tefosine were performed by the method described previously26
against HepG2 (human hepatic carcinoma), Vero (African green
monkey kidney fibroblast) and LLC-PK11 (pig kidney epithelial)
cells in comparison with the cytotoxic drug doxorubicin. These
compounds were not cytotoxic against Vero cells up to the highest
l
g/
of demonstrating in vivo antifungal efficacy in our candidiasis
mouse model cannot be attributed to its chemical instability with-
in bloodstream and infection sites. The previous study by Wieder-
hold et al.14 suggested that protein binding could be associated
with the ineffectiveness of miltefosine in the mouse model of dis-
(A)
PBS
3a
5 mg/kg, ip
3a 10 mg/kg, ip
MTF 5 mg/kg, ip
MTF 10 mg/kg, ip
tested concentration of 25
icity towards HepG2 cells with IC50 values in the range of 11.5 to
>25 g/mL and LLC-PK11 cells with IC50 values ranging from 1.6
to 10.8 g/mL (Table 1). It is important to note that the in vitro
lg/mL. However, they showed cytotox-
l
l
antifungal activity of these compounds was not correlated with
the in vitro cytotoxicity. For example, the antifungal activity of
compounds 3c and 3h was less potent than compound 3a but they
were more cytotoxic than 3a (Table 1). Among the three potent
antifungal compounds (3a, 3d, and 3e), 3a was least cytotoxic,
which was similar to miltefosine. Therefore, compound 3a was se-
lected, along with miltefosine, for in vivo efficacy studies in a
mouse model of candidiasis.
Taking advantage of the reported dosing values of miltefosine
used in the mouse model of cryptococcosis (1.8–45 mg/kg
orally)9,14 and the 50% effective dose of miltefosine produced in
the mouse model of leishamniasis (7.72 mg/kg intraperitoneally),3
the mice were treated with 3a and miltefosine at 5 and 10 mg/kg
via intraperitoneal administration at 4, 24, 48, 72, and 96 h post
inoculation with 1 Â 106 cells of C. albicans SC5314 via tail-vein
injection. The results showed that all compound-treated mice died
on or before day 12, which is similar to the control mice treated
with phosphate buffered saline (PBS) (Fig. 2A). In a separate exper-
iment with reduced dosing for 3a at 1 and 5 mg/kg and miltefosine
at 5 mg/kg via the same treatment, the mice died on or before day
8, same as the control group treated with PBS. Oral administration
of compound 3a at 5 and 25 mg/kg also did not enhance the sur-
vival rate of mice when compared with the control mice treated
with PBS (Fig. 2B). This indicated that miltefosine and compound
3a did not exhibit in vivo efficacy against C. albicans in a mouse
model of systemic infection.
Days post infection
(B)
PBS
3a
3a 25 mg/kg, oral
5 mg/kg, oral
Days post infection
Figure 2. In vivo antifungal efficacy of compound 3a and miltefosine (MTF) against
Candida albicans infection. Five male CD1 mice (6–7 weeks old) per group were
infected with 1 Â 106 cells of C. albicans SC5314, followed by treatment with
phosphate buffered saline (PBS) or compound by intraperitoneal injection (panel A)
and by oral route (panel B) at 4, 24, 48, 72, and 96 h post inoculation. Survival of the
animals was monitored for 12 days. For the intraperitoneal administration exper-
iment, P values evaluated by a Log-rank test are as follows: 0.8758 for PBS versus 3a
5 mg/kg, 0.0179 for PBS versus MTF 5 mg/kg, 0.1021 for PBS vs MTF 10 mg/kg, and
0.1822 for 3a 5 mg/kg vs MTF 5 mg/kg.
Since miltefosine as an antileishmanial drug has proven favor-
able pharmacokinetic profiles in mice15 and human,4 the failure

R. R. Ravu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4828–4831
4831
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seminated cryptococcosis. While the reasons for the lack of the
in vivo antifungal activity of miltefosine and analog 3a may be
worth investigating, we should not exclude the possibility that
chemical optimization may afford novel miltefosine-based deriva-
tives that are highly selective and permeable to fungal cells and
ultimately exert in vivo efficacy. Another potential therapeutic area
for this class of compounds is the treatment of fungal biofilm-re-
lated infections on medical devices such as catheters, given that
miltefosine can inhibit C. albicans biofilm formation and matura-
tion.11 Thus, further work will be required to determine the poten-
tial usefulness of miltefosine and its analogs in the treatment of
invasive or device-related fungal infections.
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Acknowledgements
The authors thank Ms. Marsha Wright for in vitro antifungal
testing, Mr. John Trott for in vitro cytotoxicity testing, and Dr. Bhar-
thi Avula for high resolution mass spectrometry analyses. This
work was supported by the NIH, NIAID, Division of AIDS, Grant
No. AI 27094, the USDA Agricultural Research Service Specific
Cooperative Agreement No. 58-6408-2-0009, the Duke University
Center for AIDS Research (CFAR) with assistance of the NIH funded
program (2P30 AI064518-06 to Y.-L.C.) and NIH/NIAID R01 grant
AI50438 (J.H.), and NIH/NHGRI R01 grant HG004840 (X.P.).
ˇ
´
17. Lukác, M.; Mrva, M.; Fischer-Fodor, E.; Lacko, I.; Bukovsky, M.; Miklášová, N.;
Ondriska, F.; Devínsky, F. Bioorg. Med. Chem. Lett. 2009, 19, 6346.
ˇ
ˇ
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18. Lukác, M.; Mojziš, J.; Mojzišová, G.; Mrva, M.; Ondriska, F.; Valentová, J.; Lacko,
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I.; Bukovsky, M.; Devínsky, F.; Karlovská, J. Eur. J. Med. Chem. 2009, 44, 4970.
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Toronto, ON, Canada. http://www.acdlabs.com/products/pc_admet/physchem/
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Supplementary data
22. Salmon, A.; Jutzi, P. J. Organomet. Chem. 2001, 637–639, 595.
Supplementary data (experimental procedures and spectro-
scopic data for compounds 3a–3i) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2013.06.096.
23. Spectroscopic
data
for
N-benzyl-N,
N-dimethyl-2-
{[(hexadecyloxy)hydroxyphosphinyl]oxy}-ethanaminium inner salt (3a): 1H NMR
(400 MHz, CD3OD) d 7.59 (d, 2H, J = 7.2 Hz, aromatic H), 7.51 (m, 3H, aromatic
H), 4.62 (br s, 2H, N–CH2-Ph), 4.30 (br s, 2H, N–CH2CH2–O), 3.85 (q, 2H, J = 6.4
Hz, –OCH2–), 3.62 (m, 2H, N–CH2CH2–O), 3.11 (s, 6H, 2ÂN–CH3), 1.57 (m, 2H, –
CH2), 1.39À1.20 (m, 26H, 13ÂCH2), 0.86 (t, 3H, J = 6.7 Hz, CH3) (the assignment
of the 1H NMR resonances was facilitated by 2D NMR experiments of HMQC
and HMBC); 13C NMR (100 MHz, CD3OD) d 134.4 (2C), 131.8, 130.2 (2C), 128.8,
70.2, 66.9 (d), 65.3 (m), 60.1 (d), 51.2 (t, 2C), 33.1, 31.9 (d), 30.8 (6C), 30.8 (2C),
30.5 (2C), 26.9, 23.7, 14.6; HRESIMS m/z 482.3395 (calcd for [C27H50NO4P–H]À,
482.3405).
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25. Minimum fungicidal concentration (MFC) was determined by removing 5 lL
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The MFC was defined as the lowest test concentration that allows no growth of
the organism on agar.
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