Synthesis and biophysical characterization of chlorambucil anticancer ether lipid prodrugs

Article abstract of DOI:10.1021/jm900091h

The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol he

Full text of DOI:10.1021/jm900091h

3408
J. Med. Chem. 2009, 52, 3408–3415
Synthesis and Biophysical Characterization of Chlorambucil Anticancer Ether Lipid Prodrugs
Palle J. Pedersen,† Mikkel S. Christensen,† Tristan Ruysschaert,‡ Lars Linderoth,† Thomas L. Andresen,§ Fredrik Melander,|
Ole G. Mouritsen,‡ Robert Madsen,† and Mads H. Clausen*,†
Department of Chemistry, Technical UniVersity of Denmark, KemitorVet, Building 201, DK-2800 Kgs. Lyngby, Denmark, Department of
Physics and Chemistry, MEMPHYSsCenter for Biomembrane Physics, UniVersity of Southern Denmark, CampusVej 55,
DK-5230 Odense M, Denmark, Department of Micro- and Nanotechnology, Technical UniVersity of Denmark, DK-4000 Roskilde, Denmark,
and LiPlasome Pharma A/S, Technical UniVersity of Denmark, KemitorVet, Building 207, DK-2800 Kgs. Lyngby, Denmark
ReceiVed September 11, 2008
The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described.
The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with
phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar
liposomes (86-125 nm) and are hydrolyzed by phospholipase A2, resulting in chlorambucil release. Liposomal
formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in
the presence of phospholipase A2, with IC50 values in the 8-36 µM range.
Introduction
Ever since Gregoriadis et al.1 suggested liposomes as drug
carriers in 1974, serious efforts have been put into the
development of liposomes as efficient drug delivery systems
for the treatment of cancer. The discovery that liposomes
accumulate to a high degree in tumor tissue2 if their surface is
covered with poly(ethylene glycol) was a major improvement
over earlier formulations and made these nanoparticles ap-
plicable as drug carriers of chemotherapeutics to tumor tissue.
Such liposomal drug delivery systems based on the enhanced
permeation and retention (EPRa) effect were utilized in the
commercially successful liposomal formulation of doxorubicin.
However, it is now apparent that this formulation is not generally
useful for the majority of potentially interesting drug candidates
Figure 1. Four target chlorambucil prodrug ether lipids. Prodrugs 1a
and 1b have a phosphatidylcholine headgroup with a C16 and a C18
because of the lack of a controlled drug release.3 An optimal
drug delivery formulation should be able to retain and stabilize
the carried drug during blood circulation and effectively release
the drug in the target tissue.3 This calls for the utilization of
site specific release mechanisms, and several have been
investigated, e.g., enzymatic release4 and pH,5 light,6 and heat
sensitive liposomes.7
Liposomal drug delivery has mainly relied on the encapsula-
tion of hydrophilic drugs in the aqueous core3,8 or on trapping
hydrophobic molecules in the lipid bilayer.9 Although this
approach is successful, it does suffer from some limitations such
as the potential for leakage and the fact that the release of the
active drug is not directly coupled to the mechanism activating
the carrier. One strategy that addresses both these issues is the
formulation of a lipid-prodrug conjugate that is susceptible to
selective degradation by endogenous enzymes in the target
tissue, serving to simultaneously degrade the carrier and release
ether chain, respectively. Target compounds 2a and 2b have the
negatively charged phosphatidylglycerol headgroup.
the drug.3 By covalent incorporation of the active chemothera-
peutic agent in the delivery system, the problem with premature
leakage is effectively circumvented. Herein, we describe the
synthesis and characterization of prodrugs (1 and 2, Figure 1)
that are suitable for liposomal delivery to cancerous tissue and
susceptible to secretory phospholipase A2 activation.
Secretory phospholipase A2 (sPLA2) is overexpressed in
cancer tissue,10 and has previously been exploited in liposomal
drug delivery.11 Subtype sPLA2 IIA has been identified in
several human tumors including breast,12 stomach,13 colorectal,14
pancreatic,15 prostate,10a,16 and liver cancer.17 The prodrugs are
based on covalently attaching an anticancer drug in the sn-2
position of sn-1 ether phospholipids. The principle is illustrated
in Figure 2: sPLA2 will hydrolyze the sn-2 ester bond, releasing
both the anticancer drug bound to the sn-2 position and an
anticancer ether lipid (AEL). Ether lipids were chosen because
of their higher stability and because of the cytotoxicity of the
lyso-lipids released upon sPLA2 hydrolysis.11 Furthermore, the
lyso-ether lipids have potential to attenuate the toxicity of
chlorambucil by increasing the cellular uptake of the drug,3 and
thus, the two molecules released by sPLA2 hydrolysis will work
in unison against cancer cells.
* To whom correspondence should be addressed. Phone: +45 45252131.
Fax: +45 45933968. E-mail: mhc@kemi.dtu.dk.
†
Department of Chemistry, Technical University of Denmark.
University of Southern Denmark.
Department of Micro- and Nanotechnology, Technical University of
‡
§
Denmark.
|
LiPlasome Pharma A/S, Technical University of Denmark.
a Abbrevations: EPR, enhanced permeation and retention; sPLA2, secre-
tory phospholipase A2; AEL, anticancer ether lipid; PMB, p-methoxybenzyl;
DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DBU, 1,8-diazabicyclo-
[5.4.0]undec-7-ene; DLS, dynamic light scattering; SUV, small unilaminar
vesicles.
It is crucial for the prodrug strategy that suitable drug
candidates are available. Since the incorporated drug will be
part of the lipophilic membrane, a hydrophobic nature is an
10.1021/jm900091h CCC: $40.75
2009 American Chemical Society
Published on Web 04/29/2009

Chlorambucil Anticancer Ether Lipid Prodrugs
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3409
Figure 2. Schematic overview of the drug delivery concept. The AEL prodrugs are formulated as liposomes. Because of the EPR effect, the
liposomes will accumulate in cancer tissues and sPLA2, which is up-regulated in cancer tissue, will hydrolyze the AEL prodrug lipids, releasing
two anticancer drugs.
Scheme 1. Synthesis of Lipid Precursors 6a and 6ba
temperature in order to obtain homogeneous reaction mixtures
and achieve full conversion in the transformations.
The choline headgroup was attached to the primary alcohols
6a and 6b by reaction with phosphorus oxychloride and NEt3,
followed by addition of choline tosylate, pyridine, and finally
H2O (Scheme 2).11a,29 Excess choline tosylate was removed on
a Reagents: (a) C16H33OH or C18H37OH, BF3 ·OEt2, CH2Cl2; (b) (i)
an MB-3 ion-exchange column, and after purification by flash
column chromatography the PMB-group was removed with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in an 18:1 mix-
ture of CH2Cl2 and H2O,30 which resulted in full conversion
within 3 h with isolated yields of 99% and 79%. The final
attachment of chlorambucil to the lipid was achieved via a
Steglich esterification with DCC and a catalytic amount of
DMAP.31 When the acylation of 7a was performed in ethanol-
free chloroform at 20 °C or CH2Cl2 in the temperature range
from 0 °C to reflux, we did not observe any incorporation of
chlorambucil, but when the conditions were changed to reflux
in 1,2-dichloroethane, the acylation occurred in a 75% yield,
albeit only after adding 5 equiv of chlorambucil and DCC in
portions over 31 h. The acylation of 7b led to an isolated yield
of 76% in refluxing chloroform, and that was not improved by
using 1,2-dichlorethane as the solvent. Changing the coupling
reagents to EDCI and DMAP32 did not improve the conversion
of the AELs.
The phosphoramidite 11 needed for the installment of the
glycerol headgroup was synthesized in four steps from allyl
p-methoxybenzoate (8) (Scheme 3). The key step was a
Sharpless asymmetric dihydroxylation33 of 8, which occurred
with excellent enantioselectivity (97% ee, chiral HPLC, and
Mosher ester analysis; see Supporting Information). TBDMS
protection and reduction of the p-methoxybenzoate with DIBAL-H
at -78 °C afforded the TBDMS-protected glycerol 10. The
coupling between 10 and the commercially available phospho-
rylating agent (i-Pr)2NPClO(CH2)2CN resulted in the desired
phosphoramidite 11 in a very satisfactory yield, isolated as a
1:1 diastereomeric mixture as evident from 31P NMR.
PMBTCA, La(OTf)3, toluene; (ii) CsOAc, DMSO, DMF; (iii) NaOMe,
MeOH.
obvious requirement and, furthermore, a carboxylic acid moiety
is needed for the attachment of the drug to the AEL backbone.
We have identified a number of candidates such as chlorambucil
(3),18 all-trans retinoic acid,19 and prostaglandins.20 In the
present study prodrugs made from chlorambucil are investigated.
Chlorambucil (3) is a chemotherapeutic agent of the mustard
gas type,21 and it was originally synthesized by Everett et al. in
1953.18a It is used clinically for the treatment of lymphocytic
leukemia22 typically in combination with other drugs. Chloram-
bucil is orally administrated but undergoes rapid metabolism,
and as a result, the stability in aqueous environments is low
and 3 has an elimination half-life of 1.5 h.23 The prodrug
formulation could remedy this, since this system will shield
chlorambucil from degradation through the incorporation in the
lipophilic part of the liposomal membrane and deliver it directly
to the tumor, decreasing metabolism compared to the oral
administration route. To investigate the effect of sn-1 ether chain
length and headgroup charge on enzymatic activity, prodrugs 1
and 2 were prepared with both C16 and C18 ether chains and
a choline and a glycerol phosphate headgroup, respectively.
Biophysical and biological characterization of the synthesized
chlorambucil prodrugs (1, 2) is included, with focus on liposome
formulation, particle size determination, and in particular sPLA2
activity. Proof-of-principle of the strategy is demonstrated in
three cancer cell lines, providing the first successful example
of this prodrug approach to liposomal drug delivery.
The glycerol headgroup was attached to the lipid backbone
(6a and 6b) via reaction with 11 under activation of tetrazole
and successive oxidation with tBuOOH (Scheme 4). Deprotec-
tion of the PMB-group was achieved with DDQ in moist
CH2Cl2. Acylation with chlorambucil in CH2Cl2 followed by
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) mediated deprotec-
tion of the cyanoethylene group34 afforded the lipids 12a and
12b in good yields over five steps. Finally, removal of the
TBDMS protection groups was achieved by treatment with HF
in MeCN/H2O, providing 2a and 2b.
Synthesis of Chlorambucil AEL Prodrugs
Anticancer ether lipids have previously been synthesized via
different routes, e.g., starting from D-mannitol24 or glycidols.25
Commercially available (R)-glycidyl tosylate (4) served as our
starting material, and the aliphatic ether chain was introduced
by ring-opening of the epoxide under Lewis acid catalysis,26
resulting in yields of 89% and 97% for 5a and 5b, respectively
(Scheme 1). The p-methoxybenzyl (PMB) group was chosen
for protection of the secondary alcohol27 and introduced by using
p-methoxybenzyl trichloroacetimidate with La(OTf)3 catalysis.28
The resulting tosylate was converted to the acetate with CsOAc
in a 9:1 mixture of DMSO and DMF and the ester hydrolyzed
with NaOMe in MeOH at 40 °C, yielding the primary alcohols
6a and 6b in overall yields of 70% and 78%, respectively, over
three steps. It was essential to carry out the hydrolysis at elevated
Biophysical and Biological Data
The chlorambucil AEL prodrugs (1, 2) were formulated as
liposomes by extrusion in HEPES buffer using the dry lipid
film technique.35 The lipid solutions were analyzed by dynamic

3410 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Scheme 2. Synthesis of Chlorambucil AEL Prodrugs 1a
Pedersen et al.
a Reagents: (a) (i) POCl3, Et3N, CH2Cl2; (ii) choline tosylate, pyridine; (iii) H2O; (iv) DDQ, CH2Cl2, H2O; (b) 3, DCC, DMAP, CHCl3 or 1,2-dichloroethane.
Scheme 3. Synthesis of the Phosphoramidite 11a
bucil was detected, proving that the liposomal formulation
enhances chlorambucil stability significantly. These findings
were further supported by the 4-nitrobenzylpyridine alkylating
assay,38 which showed that alkylation by chlorambucil occurred
when liposomes of 1b and 2b were subjected to sPLA2, whereas
no alkylation of 1b and 2b was detected in the absence of sPLA2
(see Supporting Information).
To demonstrate the sPLA2-dependent cytotoxicity of the
chlorambucil AEL prodrugs, we investigated the activity of 1b
against HT-29 and MT-3 cancer cells for 24 h and 2b against
a Reagents: (a) K2OsO4 ·2H2O, (DHQD)2PHAL, K3Fe(CN)6, K2CO3,
the same two cell lines in addition to ES-2 cells (Table 2). None
tBuOH, H2O; (b) (i) TBDMSOTf, DIPEA, CH2Cl2; (ii) DIBAL-H, CH2Cl2;
of these cells secrete sPLA2, which is an advantage in these
(c) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2.
studies because it enables us to control the presence or ab-
sence of the enzyme in each experiment. Before addition of
snake (Agkistrodon pisciVorus pisciVorus) venom sPLA2, all
liposome formulations have IC50 values significantly higher than
light scattering (DLS) in order to investigate the particle size,
and DLS analysis revealed that 1 and 2 form particles in the
liposome size region (Table 1) and with a low polydispersity,
indicating formation of small unilaminar vesicles (SUVs). Initial
that of free chlorambucil (entries 1-3), demonstrating that
cytotoxicity is associated with the prodrug activation by sPLA2.
confirmation of enzymatic hydrolysis was obtained by treating
Upon addition of the enzyme, both prodrugs display IC50 values
the liposome solutions with snake (Naja mossambica mossa-
below that of chlorambucil itself for all three tested cell lines
mbica) venom sPLA2 for 24 h at 37 °C. Snake venom sPLA2 is
(entries 4 and 5), suggesting a cooperative effect of the two
a convenient model enzyme, since it is not sensitive to the charge
cytotoxic compounds released, chlorambucil and AELs. The
of the interfacial region, unlike human sPLA2, but shows the
lyso-ether lipid 7b has activity in the same range as the prodrugs
same substrate specificity.10d,36 DLS measurements of the
with added sPLA2 (compare entries 4-5 and entry 6). Earlier
resulting solutions confirmed that the liposomes had been
studies of sn-1 ether lipids with a fatty acid in the sn-2 position
degraded, as only particles with a diameter of less than 5 nm
have shown that AEL alone is more cytotoxic than the liposome
were present. Incubation of the liposomes for 24 h without
formulation,11a lending further support to our belief that the two
enzyme did not result in a change in particle size as measured
active components work in unison to kill the cancer cells.
by DLS (data not shown).
Phospholipase A2 alone has no effect on cell viability (entry
In order to investigate the hydrolysis on a molecular level,
we applied MALDI-TOF MS and HPLC. MALDI-TOF MS has
7). Taken together, the data in Table 2 clearly show the potential
of this prodrug strategy for sPLA2 mediated degradation of
recently been exploited as a very fast and sensitive technique
liposomes consisting of sn-1 ether lipids with an anticancer drug
for detection of lipids,37 and we decided to study the enzyme
activity with this method in order to verify that the lipids were
covalently bound in the sn-2 position.
consumed and the anticancer drugs released. Figure 3 shows
Conclusion
the digestion of the chlorambucil AEL prodrugs 1a and 2a and
the release of AELs catalyzed by snake (Agkistrodon pisciVorus
pisciVorus) venom sPLA2. The spectra show the disappearance
over time of the prodrugs signals (M + H+ and M + Na+) and
the emergence of the expected AEL signals (M + H+ and M +
Na+). From the spectra it is also possible to get information
about the conversion rate, and whereas 2a is almost fully
consumed after 2 h, 1a needs more than 24 h for full digestion
by sPLA2. The MALDI-TOF MS analysis of 1b and 2b (see
Supporting Information) revealed that full degradation is
obtained in 2-6 h. These results were verified by HPLC (Figure
4 and Supporting Information). Neither HPLC nor MALDI-
TOF MS was capable of detecting the released chlorambucil,
but that was not surprising given the low stability of free
chlorambucil in an aqueous environment.23 Chatterji et al. report
15 min as the half-life of chlorambucil in a buffer like the
HEPES buffer at 37 °C.23b MALDI-TOF analysis (see Sup-
porting Information) of liposome solutions of 1b and 2b stored
for 6 weeks at 20 °C showed that the prodrug lipids were intact.
No significant hydrolysis of the chloroethyl groups of chloram-
In the present study we have synthesized a series of novel
prodrugs and shown that they form small unilamellar vesicles
that are stable in size over time. It was found that sPLA2 can
hydrolyze all the prodrugs of this type, showing how diverse
sPLA2 substrates can be, which makes sPLA2 an excellent target
for future prodrug strategies. The approach described here is a
new application of prodrugs in liposomal formulations, and we
believe it has significant advantages over conventional liposomal
drug delivery system, where hydrophilic drugs are encapsulated.
Problems with leakage during circulation are circumvented
because of the covalent attachment of the active compound to
the phospholipids. Furthermore, since the drugs are contained
in the liposome membrane until activated by the enzyme, our
strategy opens up for the use of lipophilic compounds that would
otherwise be too toxic if employed systemically because of their
affinity for biological membranes. Lastly, the use of prodrug
strategies where the drug is protected in the membrane may
open new possibilities with respect to drugs with low stability

Chlorambucil Anticancer Ether Lipid Prodrugs
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3411
Scheme 4. Synthesis of Chlorambucil AEL Prodrugs 2a
a Reagents: (a) (i) 11, tetrazole, CH2Cl2, MeCN; (ii) tBuOOH; (iii) DDQ, CH2Cl2, H2O; (iv) 3, EDCI, DMAP, CH2Cl2; (v) DBU, CH2Cl2; (b) HF, MeCN,
H2O.
Table 1. DLS Analysis of Chlorambucil AEL Prodrugsa
DLS. The DLS measurements were obtained using a BI-200SM
goniometer from Brookhaven Instruments (New York), applying a
fixed scattering angle of 90° with a 632.8 nm HeNe laser.
sPLA2 Activity Measurements Monitored by DLS. The
chlorambucil AEL prodrugs (2 mL, 0.05 mM), formulated in an
aqueous buffer (0.15 M NaCl, 5 mM CaCl2, 2.5 mM HEPES, pH
7.4), were incubated at 37 °C with snake venom sPLA2 from Naja
mossambica mossambica (1.93 nmol) for 24 h. The sPLA2 from
Naja mossambica mossambica was purchased from Sigma-Aldrich
Chemical Co. The resulting solutions were analyzed by DLS as
described above.
before sPLA2 addition
after sPLA2 addition,
prodrug
diameter (nm)
polydispersity
diameter (nm)
1a
1b
2a
2b
124
125
104
113
0.12
0.22
0.08
0.05
<5
<5
<5
<5
a Determined before and after addition of snake venom (Naja mossambica
mossambica) sPLA2.
in a biological environment, as evident from the stability of
chlorambucil in the liposomal formulation.
sPLA2 Activity Measurements Monitored by HPLC and
MALDI-TOF MS. The chlorambucil AEL prodrugs (2 mM) were
hydrated in an aqueous buffer (0.15 M KCl, 30 µM CaCl2, 10 µM
EDTA, 10 mM HEPES, pH 7.5) for 1 h at 60 °C and then sonicated
for 1 h at 60 °C, providing a clear solution. The formulated
chlorambucil AEL prodrugs (0.40 mL, 2 mM) were diluted in an
aqueous buffer (2.1 mL, 0.15 M KCl, 30 µM CaCl2, 10 µM EDTA,
10 µM HEPES, pH 7.5), and the mixture was stirred at 37 °C in a
container protected from light. The catalytic reaction was initiated
by addition of snake (Agkistrodon pisciVorus pisciVorus) venom
sPLA2 (20 µL, 42 µM). The purified snake venom sPLA2 was
donated by Dr. R. L. Biltonen (University of Virginia). Sampling
was done after 0, 2, 6, 8, 20, 24, and 90 h by collecting 100 µL of
the reaction mixture and rapidly mixing it with a solution of CHCl3/
MeOH/H2O/AcOH 4:8:1:1 (0.5 mL) in order to stop the reaction.
The mixture was washed with water (0.5 mL), and the organic phase
was isolated by extraction. For HPLC 30-75 µL of the organic
phase was injected on a 5 µm diol column and eluted with an
isocratic eluent (CHCl3/MeOH/H2O 730:230:30 for 1a and 2a;
CHCl3/MeOH/25% aqueous NH3 800:195:5 for 2a and 2b). An
evaporative light scattering detector was used for detection. For
the MALDI-TOF MS measurements 9 µL of the organic phase was
mixed with 3 µL of 2,5-dihydroxybenzoic acid (DHB) matrix (0.5
M DHB, 2 mM CF3COONa in MeOH), and 0.5 µL of this mixture
was used for the MS analysis.
Cytotoxicity. Colon cancer HT-29 and ovarian cancer ES-2 cells
were cultured in McCoy’s 5A medium in the presence of 10% FCS
and 1% Pen-Strep (InVitrogen). Breast cancer MT-3 cells were
cultured in RPMI medium. Cells were plated in 96-well plates at
a density of 1 × 104 cells per well 24 h prior to addition of the
tested compound. Chlorambucil (3) was solubilized in DMSO and
water (final DMSO concentration max of 0.5%). Liposomes were
diluted in PBS, and initial lipid concentration in the liposome
solutions was determined by phosphorus analysis.40 After 24 h of
incubation, the substances were removed and the cells were washed
and incubated in complete medium for another 48 h. Cytotoxic
activity was assessed using a standard 3-(4,5-dimethylthiazolyl)-
2,5-diphenyltetrazolium bromide (MTT) assay (Cell Proliferation
Kit I, Roche, Germany).41 Cell viability is expressed as percentage
reduction of incorporated MTT.
Experimental Section
General. Starting materials, reagents, and solvents were pur-
chased from Sigma-Aldrich Chemical Co. and used without further
purification. Reactions involving air or moisture sensitive reagents
were carried out under N2, and flasks were dried by flame heating
under reduced pressure. DMF, DMSO, MeCN, CH2Cl2, CHCl3, 1,2-
dichloroethane, and toluene were dried over 4 Å molecular sieves.
Pyridine and NEt3 were dried over KOH. Evaporation of solvents
was done under reduced pressure (in vacuo). TLC was performed
on Merck aluminum sheets precoated with silica gel 60 F254 plates.
Compounds were visualized by charring after dipping in a solution
of p-anisaldehyde (10 mL of H2SO4 and 10 mL of p-anisaldehyde
in 200 mL of 95% EtOH), KMnO4 (1.5 g of KMnO4, 10 g of
K2CO3, and 2.5 mL of 5% NaOH in 150 mL of H2O), or Cemol
(6.25 g of (NH4)6Mo7O24 and 1.5 g of Ce(SO4)2 in 250 mL of 10%
aqueous H2SO4). Flash column chromatography was performed
using Matrex 60 Å silica gel. Purity of all compounds was found
to be equal to or greater than 95% by elemental analysis or HPLC
(see below).
NMR spectra were recorded using a Varian Mercury 300 MHz
spectrometer or a Varian Unity Inova 500 MHz spectrometer.
Chemical shifts were measured in ppm and coupling constants in
Hz, and the field is indicated in each case. The solvent peaks from
1
CDCl3 (7.26 ppm in H NMR and 77.16 ppm in 13C NMR) or
acetone-d6 (2.05 ppm in 1H NMR) were used as standards.39 HPLC
was performed on a Waters Alliance HPLC equipped with a DAD,
using a LiChrospher Si 60 column and eluting with water/
isopropanol/heptane mixtures. Elemental analyses were obtained
from H. Kolbe, Mikroanalytisches Laboratorium, Mu¨lheim/Ruhr,
Germany. IR analysis was carried out on a Perkin-Elmer 1600 series
FTIR spectrometer, as KBr pills or neat between AgCl plates.
Melting points were measured by a Buch & Holm melting point
apparatus and given in degrees Celsius (°C) uncorrected. HRMS
was recorded on an Ionspec Ultima Fourier transform mass
spectrometer.
Liposome Preparation and Particle Size Determination. The
chlorambucil AEL prodrugs were dissolved in CHCl3 in a glass
tube, dried under a stream of N2, and then placed under vacuum
for 3-15 h to form a thin film. The film was solubilized by addition
of aqueous buffer (0.15 M NaCl, 2.5 mM HEPES, pH 7.4) and
vortexed periodically over 1 h at 20 °C. Subsequently, the solutions
were extruded through a 100 nm polycarbonate cutoff membrane
using a Hamilton syringe extruder (Avanti Polar Lipids, Birming-
ham, AL), yielding unilamellar vesicles with a diameter ranging
from 86 to 125 nm and with a low polydispersity as measured by
1-O-Hexadecyl-2-(4-(4-(bis-(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-phosphocholine (1a). Compound 7a (67 mg, 0.139
mmol) was dissolved in anhydrous 1,2-dichloroethane (3.5 mL),
and the mixture was heated to reflux under an atmosphere of N2.
DMAP (10 mg, 0.082 mmol), chlorambucil (3) (64 mg, 0.209
mmol), and DCC (1 M in CH2Cl2, 0.2 mL, 0.2 mmol) were added,
and after 5 and 19 h additional portions of chlorambucil (3) (64
mg, 0.209 mmol) and DCC (1 M in CH2Cl2, 0.2 mL, 0.2 mmol)

3412 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Pedersen et al.
Figure 3. MALDI-TOF MS monitoring of snake (Agkistrodon pisciVorus pisciVorus) venom sPLA2 activity on chlorambucil AEL prodrug 1a
(top) and 2a (bottom). The spectra demonstrate that the prodrugs (right) are consumed and the AELs (left) are released.
Figure 4. HPLC chromatogram for the snake (Agkistrodon pisciVorus pisciVorus) venom sPLA2 hydrolysis experiment on chlorambucil AEL
prodrug 1a showing the amount of prodrug (left) and AEL (right) before the addition of the enzyme and after 2, 4, 6, and 24 h.
Table 2. IC50 Values (µM) of Chlorambucil (3), a Lyso-Ether Lipid
(7b), and the Prodrugs 1b and 2b in the Presence and Absence of
sPLA2
7.6 Hz, 2H), 2.36 (t, J ) 7.6 Hz, 2H), 1.90 (p, J ) 7.6 Hz, 2H),
1.54 (p, J ) 6.8 Hz, 2H), 1.32-1.26 (m, 26H), 0.88 (t, J ) 6.8
Hz, 3H). 13C NMR (75 MHz, 4:1 CDCl3/CD3OD) δ 174.1, 144.8,
130.8, 130.0 (2C), 112.6 (2C), 72.2 (d, J ) 8.2 Hz), 72.1, 69.6,
66.8, 64.4 (d, J ) 5.2 Hz), 59.2 (d, J ) 5.2 Hz), 54.5, 54.4, 54.4,
53.9 (2C), 40.9 (2C), 34.2, 34.0, 32.3, 30.1, 30.0, 29.9, 29.9, 29.7,
a
entry
compd
IC50 HT-29
IC50 MT-3
IC50 ES-2
1
2
3
4
5
6
7
3
1b
2b
70 ( 10
>200
>200
32 ( 2
10 ( 1
18 ( 5
b
95 ( 21
>200
>200
36 ( 4
36 ( 4
35 ( 1
b
34 ( 3
nd
97 ( 2
nd
8 ( 0,5
30 ( 1
b
27.2, 26.4, 23.0, 14.3. IR (neat) 2923, 2852, 2366, 1734, 1091 cm-1
.
m/z (M + H+) 767.43.
1b + sPLA2
2b + sPLA2
7b
1-O-Octadecyl-2-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-phosphocholine (1b). Compound 7b (67 mg, 0.131
mmol) was dissolved in anhydrous CHCl3 (4 mL), and the mixture
was heated to reflux under an atmosphere of N2. DMAP (10 mg,
0.082 mmol), chlorambucil (3) (40 mg, 0.131 mmol), and DCC (1
M in CH2Cl2, 130 µL, 0.130 mmol) were added, and after 3, 6.5,
23.5, 26.5, and 31 h additional portions of chlorambucil (3) (40
mg, 0.131 mmol) and DCC (1 M in CH2Cl2, 130 µL, 0.130 mmol)
were added. After 48 h the mixture was concentrated in vacuo and
purified by column chromatography (CH2Cl2/MeOH 4:1, then
CH2Cl2/MeOH/H2O 30:20:1) to give 79 mg (76%) of 1b as an oil.
Rf ) 0.15 (CH2Cl2/MeOH/H2O 30:20:1). 1H NMR (500 MHz, 4:1
CDCl3/CD3OD) δ 7.07 (d, J ) 8.5 Hz, 2H), 6.65 (d, J ) 8.5 Hz,
2H), 5.16 (p, J ) 5.8 Hz, 1H), 4.22 (s, 2H), 4.06-3.94 (m, 2H),
3.72 (t, J ) 6.8 Hz, 4H), 3.64 (t, J ) 6.8 Hz, 4H), 3.63-3.59 (m,
2H), 3.56 (s, 2H), 3.48-3.40 (m, 2H), 3.20 (s, 9H), 2.56 (t, J )
sPLA2
a Cytotoxicity was measured using the MTT assay as cell viability 48 h
after incubation with the indicated substances for 24 h and shown by mean
( SD (n ) 3); nd ) not determined. Snake (Agkistrodon pisciVorus
pisciVorus) venom sPLA2 was added to a final concentration of 5 nM. b No
change in cell viability was observed after 24 h.
were added. After 27 h the mixture was concentrated in vacuo and
purified by column chromatography (CH2Cl2/MeOH 4:1; then
CH2Cl2/MeOH/H2O 30:20:1) to give 80 mg (75%) of 1a as an oil.
Rf ) 0.18 (CH2Cl2/MeOH/H2O 30:20:1). 1H NMR (500 MHz, 4:1
CDCl3/CD3OD) δ 7.08 (d, J ) 8.6 Hz, 2H), 6.65 (d, J ) 8.6 Hz,
2H), 5.16 (p, J ) 5.9 Hz, 1H), 4.22 (s, 2H), 4.03-3.99 (m, 2H),
3.72 (t, J ) 7.1 Hz, 4H), 3.64 (t, J ) 7.1 Hz, 4H), 3.63-3.59 (m,
2H), 3.56 (s, 2H), 3.49-3.39 (m, 2H), 3.19 (s, 9H), 2.56 (t, J )

Chlorambucil Anticancer Ether Lipid Prodrugs
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3413
7.5 Hz, 2H), 2.36 (t, J ) 7.5 Hz, 2H), 1.90 (p, J ) 7.5 Hz, 2H),
1.54 (m, 2H), 1.32-1.26 (m, 30H), 0.88 (t, J ) 6.9 Hz, 3H). 13C
NMR (75 MHz, 4:1 CDCl3/CD3OD) δ 174.1, 144.9, 130.8, 130.1
(2C), 112.6 (2C), 72.3 (d, J ) 8.6 Hz), 72.1, 69.6, 66.9, 64.5 (d, J
) 5.2 Hz), 59.4 (d, J ) 5.2 Hz), 54.5, 54.4, 54.4, 53.9 (2C), 40.9
(2C), 34.3, 34.0, 32.3, 30.1, 30.0, 29.9, 29.9, 29.8, 27.3, 26.4, 23.1,
14.3. IR (neat) 2923, 2852, 2366, 1734, 1518, 1247, 1088, 750
cm-1. m/z (M + Na+) 817.44.
(S)-(2,3-Di-O-tert-butyldimethylsilyl)glyceryl 2-Cyanoethyl-N,N-
diisopropylphosphoramidite (11). Alcohol 10 (904 mg, 2.82 mmol)
and diisopropylethylamine (1.0 mL, 5.92 mmol) were dissolved in
anhydrous CH2Cl2 (10 mL) under an atmosphere of N2. 2-Cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (1.0 g, 4.22 mmol) was
added dropwise, and the mixture was stirred at 20 °C for 1.5 h,
after which EtOAc (20 mL) and saturated NaHCO3 (50 mL) were
added and the organic layer was isolated by extraction with EtOAc
(2 × 50 mL). The combined organic phases were concentrated in
vacuo, and the residue was purified by column chromatography
(EtOAc) to give 1352 mg (92%) of 11 (two diastereoisomers, 1:1)
as a colorless oil. Rf ) 1.0 (EtOAc). 1H NMR (300 MHz, CDCl3):
δ 3.87-3.46 (m, 5H), 2.67-2.61 (m, 2H), 1.20-1.17 (m, 12H),
0.90 (s, 9H), 0.89 (s, 9H), 0.09-0.06 (m, 12H). 13C NMR (75 MHz,
CDCl3): δ 117.7, 73.3, 65.0, 58.6, 43.1 (2C), 26.1 (3C), 26.0 (3C),
24.7 (4C), 20.5, 18.5, 18.3, -4.4, -4.5, -5.2, -5.3. 31P NMR (202
MHz, CDCl3): δ 149.0, 148.5. IR (neat): 2958, 2929, 2883, 2857
cm-1. m/z (M + Na+) 543.32.
(CH2Cl2/MeOH/H2O 65:25:4. 1H NMR (500 MHz, CDCl3/CD3OD
3:1): δ 6.96 (d, J ) 8.7 Hz, 2H), 6.53 (d, J ) 8.7 Hz, 2H), 5.08 (q,
J ) 5.4 Hz, 1H), 3.82-3.75 (m, 2H), 3.70-3.58 (m, 3H),
3.54-3.51 (m, 4H), 3.46-3.36 (m, 8H), 3.29-3.22 (m, 2H), 2.45
(t, J ) 7.7 Hz, 2H), 2.24 (J ) 7.7 Hz, 2H), 1.77 (q, J ) 7.4 Hz,
2H), 1.42 (m, 2H), 1.15 (s, 30H), 0.78 (s, 18H), -0.01 (s, 6H),
-0.05 (s, 6H). 13C NMR (75 MHz, CDCl3/CD3OD 3:1): δ 173.2,
144.1, 130.2, 129.4, 111.8, 72.4 (d, J ) 9.6 Hz), 71.5, 71.5 (d, J )
7.8 Hz), 68.8, 66.6 (d, J ) 5.5 Hz), 64.6, 63.6 (d, J ) 5.4 Hz),
53.3, 40.2, 33.6, 33.3, 31.6, 29.4, 29.4, 29.3, 29.2, 29.1, 26.5, 25.7,
25.6, 25.5, 22.4, 18.0, 17.8, 13.8, -4.9, -5.0, -5.7, -5.7. 31P NMR
(202 MHz, CDCl3/CD3OD 3:1): δ -2.00. IR (neat): 3448, 2926,
2854, 1735, 1617, 1519, 1464, 1360, 1252, 1108 cm-1. m/z (M +
Na+) 1034.57.
1-O-Hexadecyl-2-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-phospho-(S)-glycerol (2a). Compound 12a (0.42 g, 0.43
mmol) was dissolved in MeCN (9 mL) and cooled to 0 °C. Then
40% aqueous HF (1 mL) was added, and the mixture was allowed
to reach 20 °C while being stirred vigorously for 2 h. The reaction
mixture was then poured into saturated aqueous NaHCO3 (20 mL)
and extracted with CH2Cl2 (3 × 10 mL) and EtOAc (10 mL). The
combined organic extracts were dried over Na2SO4 and concentrated
in vacuo and the residue was purified by column chromatography
(CH2Cl2/MeOH/H2O 65:25:4) to afford 2a (0.19 g, 59%). Rf ) 0.56
(CH2Cl2/MeOH/H2O 65:25:4). 1H NMR (500 MHz, CDCl3:CD3OD
3:1): δ 6.88 (d, J ) 8.6 Hz, 2H), 6.45 (d, J ) 8.6 Hz, 2H), 4.98 (q,
J ) 5 Hz, 1H), 3.84-3.71 (m, 3H), 3.60 (q, J ) 5 Hz, 1H),
3.54-3.51 (m, 4H), 3.46-3.39 (m, 8H), 3.30-3.20 (m, 2H), 2.37
(t, J ) 7.5 Hz, 2H), 2.17 (t, J ) 7.6 Hz, 2H), 1.71 (q, J ) 7.3 Hz,
2H), 1.35 (t, J ) 6.6 Hz, 2H), 1.07 (s, 26H), 0.69 (t, J ) 6.8 Hz,
3H). 13C NMR (75 MHz, CDCl3/CD3OD 3:1): δ 173.2, 144.0,
129.9, 129.2, 111.7, 71.4 (d, J ) 5.4 Hz), 71.3, 70.4 (d, J ) 4.8
Hz), 68.7, 66.1 (d, J ) 4.9 Hz), 63.8 (d, J ) 4.8), 61.8, 53.1, 40.1,
33.5, 33.4, 33.2, 33.0, 31.5, 29.3, 29.2, 29.1, 29.1, 28.9, 26.4, 25.6,
22.2, 13.5. 31P NMR (202 MHz, CDCl3/CD3OD 3:1): δ -0.99. IR
(neat): 3345, 2923, 2853, 1732, 1616, 1519, 1466, 1356, 1248, 1115,
1002 cm-1. m/z (M + H+) 778.36.
1-O-Octadecyl-2-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-phospho-(S)-glycerol (2b). The synthesis was per-
formed as for 2a, starting from 12b (334 mg, 0.33 mmol) and
affording 2b (83 mg, 32%). Rf ) 0.56 (CH2Cl2/MeOH/H2O 65:
25:4). 1H NMR (500 MHz, CDCl3/CD3OD 3:1): δ 6.89 (d, J ) 8.5
Hz, 2H), 6.46 (d, J ) 8.2 Hz, 2H), 4.99 (q, J ) 5 Hz, 1H),
3.84-3.72 (m, 3H), 3.60 (q, J ) 5 Hz, 1H), 3.53 (m, 4H),
3.47-3.41 (m, 8H), 3.30-3.20 (m, 2H), 2.37 (t, J ) 7.5 Hz, 2H),
2.17 (t, J ) 7.5 Hz, 2H), 1.71 (q, J ) 7.5 Hz, 2H), 1.35 (t, J ) 6.5
Hz, 2H), 1.07 (s, 30H), 0.70 (t, J ) 6.8 Hz, 3H). 13C NMR (75
MHz, CDCl3/CD3OD 3:1): δ 173.3, 144.0, 130.0, 129.2, 111.8,
71.5, 71.4 (d, J ) 6.3 Hz), 70.5 (d, J ) 4.8 Hz), 68.7, 66.1, 63.8
(d, J ) 5.6 Hz), 61.9, 53.2, 40.1, 33.5, 33.2, 31.5, 29.3, 29.3, 29.2,
29.1, 29.0, 26.4, 25.6, 22.3, 13.6. 31P NMR (202 MHz, CDCl3/
CD3OD 3:1): δ -0.31. IR (neat): 3332, 2923, 2853, 1733, 1616,
1519, 1466, 1355, 1236, 1115, 1002 cm-1. m/z (M + Na+) 828.37.
1-O-Hexadecyl-2-(4-(4-(bis-(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-(2-cyanoethylphospho)-(S)-2,3-di-O-tert-butyldimeth-
ylsilylglycerol (12a). To a solution of 6a (0.79 g, 1.8 mmol) and 11
(1.3 g, 2.5 mmol) in CH2Cl2 (10 mL) was added molecular sieves
(3 Å). After the mixture was stirred for 30 min, 1H-tetrazole in
acetonitrile (5.5 mL, 0.45 M, 2.5 mmol) was added and the mixture
stirred for another 30 min before 5.5 M tert-butyl hydroperoxide
in decane (0.50 mL, 2.8 mmol) was added and the mixture stirred
for 1 h before being concentrated in vacuo. The residue was purified
by column chromatography (EtOAc/heptane 1:1) to yield 1.63 g
of an oil. 31P NMR showed two signals at -0.66 and -0.82 in the
1:1 ratio set by the amidite. The product was subsequently treated
with DDQ (0.45 g, 2.2 mmol) in CH2Cl2 (10 mL) and water (0.6
mL) for 2 h before Na2SO3 was added and the mixture diluted with
CH2Cl2. The mixture was filtered and concentrated in vacuo before
purification by column chromatography (CH2Cl2, then EtOAc/
heptane 1:1) afforded 1.16 g. 31P NMR showed two signals at -0.10
and -0.18 ppm. The deprotected compound was dissolved in
CH2Cl2 (12 mL) together with chlorambucil (0.70 g, 2.3 mmol),
and EDCI (0.59 g, 3.1 mmol) and DMAP (0.38 g, 3.1 mmol) were
added. After being stirred for 2 h, the mixture was concentrated in
vacuo and purified by column chromatography (EtOAc/heptane 1:1)
and the resulting 1.36 g product dissolved in CH2Cl2 (10 mL) and
treated with DBU (0.20 mL, 1.3 mmol) for 30 min. The reaction
mixture was concentrated in vacuo and the residue was purified by
column chromatography (CH2Cl2/MeOH 9:1) to give the phospho-
1
lipid (0.96 g, 54%). Rf ) 0.73 (CH2Cl2/MeOH/H2O 65:25:4). H
NMR (500 MHz, CDCl3/CD3OD 3:1): δ 6.90 (d, J ) 8.5 Hz, 2H),
6.47 (d, J ) 8.5 Hz, 2H), 5.00 (q, J ) 5 Hz, 1H), 4.08 (m, 1H),
3.83-3.76 (m, 2H), 3.71-3.59 (m, 3H), 3.55-3.53 (m,
4H), 3.47-3.38 (m, 8H), 3.30-3.21(m, 2H), 2.38 (t, J ) 7.5 Hz,
2H), 2.19 (t, J ) 7.5 Hz, 2H), 1.73 (q, J ) 7.3 Hz, 2H), 1.36 (m,
2H), 1.09 (s, 26 H), 0.72 (s, 18H), -0.06 (s, 6H), -0.11 (s, 6H).
13C NMR (75 MHz, CDCl3/CD3OD 3:1): δ 173.1, 144.0, 130.0,
129.3, 111.8, 72.4 (d, J ) 9.5 Hz), 71.5, 71.4 (d, J ) 6.3 Hz),
68.8, 66.5 (d, J ) 5.6 Hz), 64.6, 63.5 (d, J ) 5.2 Hz), 53.2, 40.1,
33.5, 33.3, 31.5, 29.3, 29.3, 29.2, 29.1, 29.0, 26.4, 25.6, 25.5, 25.4,
22.3, 17.9, 17.7, 13.6, -5.1, -5.1, -5.8, -5.8. 31P NMR (202 MHz,
CDCl3/CD3OD 3:1): δ -1.82. IR (neat): 3450, 2922, 1732, 1616,
1519, 1463, 1360, 1252, 1102 cm-1. m/z (M + Na+) 1006.53.
1-O-Octadecyl-2-(4-(4-(bis-(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-(2-cyanoethylphospho)-(S)-2,3-di-O-tert-butyldimeth-
ylsilylglycerol (12b). The synthesis was performed as for 12a,
starting from 6b (650 mg, 1.40 mmol) and 11 (1.0 g, 1.9 mmol),
affording 810 mg (57%) of 12b as a colorless oil. Rf ) 0.73
Acknowledgment. We thank the Danish Council for Stra-
tegic Research (NABIIT Program) for financial support.
MEMPHYSsCenter for Biomembrane Physics is supported by
the Danish National Research Foundation.
Supporting Information Available: Analytical and spectral data
for all synthesized compounds, experimental procedures for the
synthesis of 5a, 5b, 6a, 6b, 7a, 7b, 9, and 10, prodrugs stability
data, Mosher ester analysis data of 10, further MALDI-TOF MS
and HPLC data for sPLA2 degradation experiments, and alkylating
assay data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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