3-Ketone-4,6-diene ceramide analogs exclusively induce apoptosis in chemo-resistant cancer cells

Article abstract of DOI:10.1016/j.bmc.2013.12.065

Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with different backbones, in order to test the hypothesis that extending the conjugated system in ceramide analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy. The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylceramide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain intrinsic properties, ceramide analogs' pro-apoptotic activity is not disrupted by the normal drug-resistance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.

Full text of DOI:10.1016/j.bmc.2013.12.065

Bioorganic & Medicinal Chemistry 22 (2014) 1412–1420
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3-Ketone-4,6-diene ceramide analogs exclusively induce apoptosis
in chemo-resistant cancer cells
Adharsh P. Ponnapakam a, Jiawang Liu b, Kaustubh N. Bhinge c, Barbara A. Drew a, Tony L. Wang a,
James W. Antoon a, Thong T. Nguyen b, Patrick S. Dupart b, Yuji Wang d, Ming Zhao d, Yong-Yu Liu c,
a
Maryam Foroozesh b, , Barbara S. Beckman
⇑
a Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, United States
b Department of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, United States
c College of Pharmacy Basic Pharmaceutical Sciences, University of Louisiana at Monroe, 1800 Bienville, Monroe, LA 71209, United States
d College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence
shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under
the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with
different backbones, in order to test the hypothesis that extending the conjugated system in ceramide
analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer
cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy.
The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell
lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-
8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair
of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations
indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial
pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylcer-
amide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain
intrinsic properties, ceramide analogs’ pro-apoptotic activity is not disrupted by the normal drug-resis-
tance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 19 October 2013
Revised 16 December 2013
Accepted 26 December 2013
Available online 8 January 2014
Keywords:
Ceramide
Glucosylceramide synthase (GCS)
P-glycoprotein
Multidrug resistance
Anti-cancer drugs
1. Introduction
MDR1, the P-gp-encoding gene.6 Furthermore, drug-resistant cancer
cells exposed to GCS inhibitors become sensitive to anticancer
Ceramides, known as ‘tumor suppressor lipids’, regulate the anti-
cancersignalsimplicatedinapoptosis, cellcyclearrest, andautopha-
gic responses.1,2 Exposure to chemotherapeutic agents and radiation
therapy lead to increased levels of endogenous ceramide, thereby
inducing apoptosis through mitochondrial or non-mitochondrial
pathways.3 Similarly, synthetic ceramide analogs have been shown
toimitate ceramide action on its downstream targets, leading to
apoptosis and cell cycle arrest.2 However, ceramide-metabolizing
enzymes can decrease cellular ceramide levels, resulting in cell pro-
liferation, migration, and survival.4,5 One such enzyme, glucosylcer-
amide synthase (GCS), converts ceramide into glucosylceramide on
the cytosolic surface of the Golgi apparatus. Evidence shows a close
relationship between GCS and P-glycoprotein (P-gp), an important
multidrug-resistance (MDR) protein. GCS inhibitor treatment in
multidrug-resistant cancer cells down-regulates the expression of
agents.6–8 Ceramide analogs have the potential to act as inhibitors
of these ceramide-metabolizing enzymes and thus maintain cellular
ceramide levels necessary to induce cell death.9 Therefore, ceramide
analogs can exert their anti-cancer activities through two main ap-
proaches: (1) acting on ceramide downstream targets to promote
cell death, and (2) inhibiting ceramide-metabolizing enzymes to in-
crease cellular ceramide levels.
Due to the crucial role of ceramide in cell death regulation, hun-
dreds of anticancer ceramide analogs have been synthesized and
investigated in recent years.10–16 Our previous work and that of
many other research groups have shown that certain ceramide
analogs preferentially inhibit the growth of chemo-resistant cancer
cells in comparison to regular cancer cells.17–22 Because of the rela-
tionship between GCS and multi-drug resistance, a hypothesis was
formed that manipulation of glucosylceramide levels by inhibiting
GCS is a useful way of inducing preferential killing of MDR cells.18
In this work, we have tested this hypothesis by determining GCS
activity in the chemo-sensitive and -resistant cancer cells.
⇑
Corresponding author. Tel.: +1 504 520 5078; fax: +1 504 520 7942.
E-mail address: mforooze@xula.edu (M. Foroozesh).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.065

A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
1413
In this study, we designed 3-ketone-4,6-diene-containing cera-
2.2. Isomerism and NMR spectral properties of compound 3
mide analogs to determine whether extending the conjugated
system in the backbone of the analogs would lead to an increase
in the anticancer activity and selectivity towards chemo-resistant
cancer cells. This design was based on the lead compounds 4,6-
diene-ceramide and analog 3 as previously reported (Fig. 1).22,23
As mentioned above, compound 3 was obtained as a mixture
of 3-up and 3-down diastereomers. However, we could not assign
R or S designations to the two. Each compound shows complex
NMR spectral behaviors, which are fully displayed in the Supple-
mentary Materials. A section of the 13C NMR spectra for these dia-
stereomersis is shown in Figure 2. Generally, two sets of peaks are
seen for each single pure compound. The ratio of these two sets of
peaks is 0.55:0.45 for both 3-up and 3-down. It is obvious that
the two sets of peaks represent two relatively stable conforma-
tions of each compound. The hypothesized models of conforma-
tional isomerism are shown in Figure 3. The sp2 hybridization of
the nitrogen atom in the carbamate group results in a planar car-
bamate functional group (pink residue). Due to the presence of a
bulky group (–COCH2SOPh) on the carbon next to the N in the
five-membered oxazoline ring, the steric effects lead to the –
COCH2SOPh group locating in the space above or below the carba-
mate plane. Since these two poses cannot freely interchange, two
relatively stable conformations are formed which are reflected by
the two sets of signals in the NMR spectra. Figure 3 shows the two
possible conformations of 3 in half-chair and envelope forms,
The
a-ketone–diene on the ceramide backbone and phenylacetyl
functional group on the amide side chain are expected to increase
the molecular rigidity of analog 406, which in turn is expected to
enhance its interaction with ceramide downstream targets and
enhance cell death. We also seek to elucidate the mechanism of
the cell death elicited by our compounds.
2. Results and discussion
2.1. Synthesis of ceramide analogs 401, 402, 403, 404, and 406
The syntheses of analogs 401–406 were achieved according to
Chun’s method as reported previously.24 Condensation of serine
methyl ester derivative 1 with methyl phenyl sulfoxide gave b-
ketosulfoxide 3. In Chun’s paper, a diastereomeric mixture of 3
was mentioned. Since the sulfur atom of the sulfinyl functional
group is a chiral center, theoretically, compound 3 consists of
two diastereomers, 3R and 3S (Scheme 1). These two isomers (3-
up and 3-down, a higher spot and a lower spot observed by TLC)
were successfully separated in this work through silica column
chromatograph with petroleum ether/ethyl acetate 1.5:1 as the
eluent. The ratio of 3-up and 3-down depends on the starting
material (methyl phenyl sulfoxide) rather than the reaction. We
could not designate the absolute configurations of 3-up and 3-
down, because each compound has a very complex NMR spectrum,
suggesting presence of conformational isomerism, which will be
discussed in the followed section. Fortunately, as synthetic inter-
mediates, 3R and 3S possess the same reactivity with the allylic
bromide 2, which was derived from the allylic alcohol ((E)-tri-
dec-2-en-1-ol), to give 3-keto-4,6-diene 401, thus isolation of 3R
and 3S was not necessary in the actual synthetic procedure.
Employing mild reaction conditions (K2CO3 in dimethylformamide
(DMF) at room temperature), an all-trans diene 401 was produced
executively. The all-trans configuration could not be directly con-
firmed by the 1H NMR spectrum of 401 since the H-signals overlap
in 6.2–6.1 ppm region. However, in its derivatives, 1H NMR, COSY,
and NOE spectra can determine the all-trans configuration. Selec-
tive reduction of 3-keto-4,6-diene (401) by diisobutylaluminum
hydride (DIBAL-H) in tetrahydrofuran (THF) gave (S)-3-hydroxy-
4,6-diene (402) as the predominant product in 50% yield. After
treatment of 401 with trifluoroacetic acid in dry dichloromethane
respectively. In both conformations the
a-H (in blue) is axial,
which is consistent with the observations in 1H NMR spectra.
The starting material 1, and ceramide analogs 401, and 402 all
show the same dualistic signals in NMR spectra with different ra-
tios. Once the five-membered ring is opened, the compounds pro-
duced (405 and 406) lose this characteristic, suggesting that the
ring is critical in restricting the conformational interchange. In
our previous work, we observed similar dualistic signals in the
six-membered ring compounds, N-Boc-carboline-3-carboxylic
acid and its derivatives.25 These observations indicate that the
in-ring nitrogen amidation and the presence of a bulky group
on the neighboring ring carbon play important roles in the forma-
tion of the two restricted conformations.
2.3. Ceramide analogs inhibiting cellular viability and
clonogenic survival in MCF-7, MDA-MB-231, and MCF-7TN-R
cells
In order to determine the cytotoxic effects of the ceramide ana-
logs on MCF-7, MDA-MB-231, and MCF-7TN-R cells, MTT cell via-
bility assays were performed using various concentrations of
each analog ranging from 1 to 100
ble 1. Compounds 401 and 406 were the most effective compounds
across all cell lines with IC50 values of 4.05 1.30 and
4.26 1.48 M, respectively, in the chemo-resistant MCF-7TN-R
lM. The results are shown in Ta-
lM
l
(DCM), 3-keto-4,6-diene-sphingosine 405 was obtained. D-Sphin-
cell line. Interestingly, IC50 values for all analogs except analog
401 were lower in the chemo-resistant MCF-7TN-R cells compared
to the sensitive MCF-7 cells, indicating that these compounds exhi-
bit increased therapeutic potential in drug-resistant cancers
(Table 1).
gosine (purchased from CNH Technologies, Inc., Woburn, MA),
(S)-2-amino-3-hydroxy-N-tetradecylpropanamide (synthesis was
described previously23), and compound 405 were amidated with
phenyl acetyl chloride to give the ceramide analogs, 403, 404,
and 406, respectively.
OH
OH
O
O
HO
O
C13H27 HO
O
C11H23 HO
N
H
C13H27 HO
O
C10H21
NH
NH
N
NH
C8-Ceramide (C8-Cer)
4,6-Diene-ceramide
Analog 3
Analog 406
Figure 1. The structure of C8-ceramide, 4,6-diene-ceramide, analogs 3 and 406. C8-ceramide is a short-chain ceramide widely used as a positive control in the studies of
ceramide analogs.

1414
A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
HO
Br
C10H21
C10H21
O
S
O
S
O
O
O
b
H
N
H
N
H
N
a
OCH3
O
O
O
Boc
Boc
Boc
2
1
3S
3R
c
O
OH
H
N
H
d
f
C10H21
C10H21
O
O
N
Boc
Boc
O
401
402
e
HO
O
C10H21
O
NH
HO
C10H21
NH2 CF3COOH
406
405
O
N
OH
C14H29
O
OH
HO
O
HO
O
C13H27
H
f
f
C14H29
NH
NH
HO
N
H
HO
C13H27
NH2 D-Sphingosine
NH2
403
404
trans-D-erythro-2-amino-4-
(S)-2-amino-3-hydroxy
-N-tetradecylpropanamide
octadecene-1,3-diol
Scheme 1. Synthetic routes for analogs 401, 402, 403, 404, and 406. Reagents and conditions: (a) methyl phenyl sulfoxide, LDA/THF; (b) triphenylphosphine, NBS/CH2Cl2; (c)
K2CO3/DMF; (d) DIBAL-H/THF; (e) trifluoroacetic acid/CH2Cl2; (f) phenylacetyl chloride, NaHCO3/CH2Cl2.
Figure 2. 13C NMR spectrum sections for the two diastereomers3-up and 3-down. In each spectrum, two sets of carbon peaks are observed, suggesting the presence of two
conformational isomers for each diastereomer. The complete spectral data are provided in the Supplementary Materials.
There is a debate in the literature as to the reliability of short-
term viability assays in predicting clinical response. Therefore,
we performed long-term clonogenic survival assays to better
determine the efficacy of these analogs. As shown in Table 2, com-
pounds 401 and 406 were again the most effective with IC50 values
When comparing analogs with different backbones, it is obvious
that analog 406 with 3-ketone-4,6-diene moiety shows higher effi-
cacy than its counterparts with 3-hydroxy-4-ene (403) or serine
amide (404) backbones in both short-term and long-term assays.
Analog 406 is even more potent than 4,6-diene-ceramide (struc-
ture shown in Fig. 1), activity of which was previously reported
of 1.85 1.22 lM and 1.81 1.18 lM, respectively, in the MCF-
7TN-R cell line. Similar to what was observed in the short-term
viability assays, analogs 401 and 406 exhibited the most potent
anti-survival effects in the drug-resistant cell lines. Taken together,
these results provide proof of principle that these analogs exhibit
anti-cancer properties.
in the chemo-resistant MCF-7TN-R cell line (IC50 11.3 lM in cell
viability assay).22 The fact that 3-ketone-4,6-diene backbone is
more effective than 3-hydroxy-4,6-diene backbone in killing
cancer cells was also proven through comparing the activities of
analogs 401 and 402, indicating that rigid modification of ceramide

A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
1415
2.4. Ceramide analogs 401 and 406 induce apoptosis through
the intrinsic cell death pathway
The mechanism of ceramide-induced cell death through the
induction of apoptosis is well-established.21,22 In this work, we
were interested in determining whether our analogs inhibited cell
survival through enhancement of programmed cell death. Given
that analogs 401 and 406 were the most effective in the viability
and clonogenic survival assays, we further determined the ability
of these analogs in inducing apoptosis in chemo-resistant breast
cancer cells. Fragmented oligonucleotides were determined as a
measure of programmed cell death. As seen in Figure 4A, treatment
with analog 406 resulted in a 4.30 1.10 fold (p <0.05) increase in
apoptosis compared to the control. Similarly, analog 401 increased
programmed cell death by 3.09 0.56 fold (p <0.05). Both analogs
exhibited increased apoptotic activity compared to parental C8-Cer
(structure shown in Fig. 1).
Apoptosis is initiated through either the extrinsic or intrinsic
cell death pathways. We further determined whether these ana-
logs utilized the intrinsic pathway through the determination of
cellular caspase-9 levels. Caspase-9 is known to be activated in
breast cancer cells exclusively in the intrinsic cell death. As shown
in Figure 4B, analog 406 increased caspase-9 activity 3.59 0.45
fold (p <0.05), while analog 401 induced caspase-9 activity
1.86 0.75 folds compared to the vehicle control. These results
were greater than parental C8-Cer (structure included in Fig. 1),
which demonstrated only a 1.18 0.09 fold (p <0.05) increase in
caspase-9 activity, thus correlating with our apoptosis findings.
Figure 3. Hypothesized conformations for the conformational isomers of com-
pound 3. For easy observation two methyl groups in 2-position of the oxazolidine-
are not shown up in the projections. The bulky group –COCH2SOPh is located above
the carbamate group plane in the half-chair form, while below the amide plane in
the envelope form. In both conformations a-H is in an axial position.
2.5. Resistant cancer cells NCI/ADR-RES and MCF-7/Doxsensitive
to analog 406
Table 1
IC50 values for ceramide analogs in MTT viability assay (
l
M)
MDA-MB-231
IC50 M)
Compound
MCF-7
MCF-7TN-R
To clarify the capability of analog 406 for selectively killing che-
mo-resistant cancer cell lines, anti-viability activities of analog 406
were evaluated independently in pairs of sensitive-resistant lines,
OVCAR-8 to NCI/ADR-RES ovarian cancer cells and MCF-7 to
MCF-7/Dox breast cancer cells. As it was observed above, analog
(l
401
402
403
404
406
3.91 1.51
26.44 1.00
37.28 2.45
233.6 4.87
22.03 1.52
17.23 0.94
75.15 0.88
51.23 0.76
N.D.
4.05 1.30
9.91 0.65
4.74 1.07
28.96 0.78
4.26 1.48
406 exhibits a lower IC50 (4.92
tant NCI/ADR-RES cells than towards chemo-sensitive OVCAR-8
cells (7.82 M, Fig. 5A), indicating its preferentially killing of che-
lM, Fig. 5B) towards chemo-resis-
161.4 2.28
MCF-7, MDA-MB-231, and MCF-7TN-R cells were treated with increasing concen-
trations of the analogs and studied for 24 h. The values are the mean SE of three
independent experiments. N.D., not determined.
l
mo-resistant cells. On the other hand, analog 406 equally inhibits
the viability of MCF-7 and MCF-7/Dox cells (Fig. 5C and D), sug-
gesting that the selectivity towards chemo-resistant cells varied
in different cell lines developed by different drugs. Nevertheless,
chemo-resistant MCF-7/Dox cells are still sensitive to analog 406
at the same degree as chemo-sensitive MCF-7 cells, proving that
analog 406’s activity is not interrupted by multi-drug resistance
mechanism.
Table 2
IC50 values of ceramide analogs in clonogenic survival assay (lM)
Compound
MCF-7
MDA-MB-231
IC50 M)
MCF-7TN-R
(l
401
402
403
404
406
5.07 0.17
5.69 0.65
4.18 0.52
10.16 0.14
3.40 0.37
1.45 0.06
3.17 0.10
1.59 0.10
17.55 0.10
1.40 0.13
1.85 1.22
5.19 0.11
5.62 0.44
10.05 0.59
1.81 1.18
2.6. Effect of analog 406 on glucosylceramide synthase (GCS)
Since glucosylceramide synthase (GCS) is an important target
for inhibiting P-gp and consequently reversing or overcoming mul-
ti-drug resistance, the effect of analog 406 on glucosylceramide
synthase (GCS) was studied in both OVCAR-8 and NCI/ADR-RES cell
lines. Based on modification of a previously described protocol, the
activity of GCS in cells was determined using the ratio of glucosyl-
ceramide to ceramide concentrations.26 The ratio of glucosylcera-
mide to ceramide spots’ intensity as observed on thin layer
chromatography (TLC) plates (Fig. 6) shows that the reference ana-
log 3 has a weak inhibitory activity toward GCS, while analog 406
significantly reduces GCS activity in chemo-sensitive OVCAR-8
cells but does not influence GCS activity in chemo-resistant
NCI/ADR-RES cells, suggesting that sensitivity of resistant cells to
analog 406 is not attributed to GCS inhibition. Thus, the capacity to
sensitize resistant cells is expected to be a common characteristic
MCF-7, MDA-MB-231, and MCF-7TN-R cells were treated with increasing concen-
trations of the analogs studied and allowed to grow until colony formation was
noted (generally 10–12 days). The values are the mean SE of three independent
experiments.
backbone improves anti-cancer activities. On the other hand, 3-ke-
tone-4,6-diene backbone is also a reactive residue, a so-called Mi-
chael acceptor, which can react with endogenous nucleophiles,
suggesting that these compounds may be a pro-drug in inducing
cell death. The selective killing of chemo-resistant cells was ob-
served with certain analogs, especially analog 406. Thus, more cell
lines were studied to evaluate the effectiveness of analog 406 to-
wards chemo-resistant cells as described in the following sections.

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A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
Figure 4. Effects of ceramide analogs on breast cancer intrinsic cell death. MCF-7TN-R cells were treated with double IC50 concentrations (the IC50 values determined from
MTT viability assay) for 24 h. (A) Treatment with analog 406 induced a 4.30 1.10 fold (⁄p <0.05) increase in apoptosis compared to vehicle control. (B) Treatment with analog
406 induced a 3.59 0.45 fold (⁄p <0.05) increase in caspase-9 activity compared to vehicle control. DMSO, vehicle control; Taxol and C8-Cer, positive control. The values are
the mean SE of three independent experiments.
Figure 5. Ceramide analog 406 effectively eliminates drug-resistant cancer cells in ovarian and breast cancers. Error bars represent the standard errors of three independent
experiments. Cells were treated with ceramide analogs for 72 h. ⁄p <0.01 compared with in cells treated with analog 3. The IC50 values of analogs in each cell line are indicated.
(A) Drug-sensitive OVCAR-8 human ovarian cancer cells. (B) Drug-resistant NCI/ADR-RES human ovarian cancer cells. (C) Drug-sensitive MCF-7 human breast cancer cells. (D)
Drug-resistant MCF-7/Dox human breast cancer cells.
of ceramide analogs regardless of the action on GCS/P-gp pathway.
A possible explanation is that due to their lipid solubility, ceramide
analogs can freely pass through the plasma membrane. Another
explanation given by Shirahama et al. is that P-glycoprotein acts
as a transporter for most chemotherapeutic agents but not for cer-
amide analogs.20 Therefore, ceramide analogs ignore the high
expression of MDR proteins in resistant cancer cells. While more
data on ceramide derivatives and their anti-cancer activities are
needed to provide clarification, ceramide analogs should continue
to be studied as potential chemotherapeutic agents for overcoming
cancer multi-drug resistance.
3. Conclusions
For many cancer patients, resistance to chemo and endocrine
therapy (especially multidrug-resistance) is a major cause of

A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
1417
Figure 6. Fluorescent chromatograms of lipid components in OVCAR-8 (A) and NCI/ADR-RES (B) cells. Cells were grown for 24 h in 6-well plates with a 10% FBS RPMI-1640
medium, and then exposed to blank control (DMSO), analog 3, or analog 406 in 5% FBS RPMI-1640 medium for 4 h at 37 °C. Cells were switched to 1% BSA RPMI-1640 medium
containing NBD C6-Cer complexed to BSA. After 2 h of incubation at 37 °C, lipids were extracted using chloroform. Fluorescent chromatograms were captured by the
AlphaImager, and were inverted using the Adobe Photoshop CS2 Program. Cer, ceramide; GluCer, glucosylceramide.
treatment failure. We have observed that certain ceramide ana-
logs have the capability to selectively kill resistant cancer cells
in comparison to regular cancer cells. Discovering chemothera-
peutic agents capable of reversing or ignoring resistance is a valid
and important approach for prolonging the lives of advance stage
cancer patients. Thus, for the studies presented here, we designed
and synthesized five new ceramide analogs, and evaluated their
activity on various sensitive and resistant cancer cell lines.
Among all of the ceramide analogs with 3-hydroxy-4-ene, 3-hy-
droxy-4,6-diene, 3-ketone-4,6-diene, and serine amide back-
bones, which we have designed, synthesized, and studied in this
and previous work, analogs 401 and 406 with the 3-ketone-4,6-
diene backbone are the most effective in the chemo-resistant
MCF-7TN-R cell line. Analog 406 selectively kills TNF-induced
resistant breast cancer cells MCF-7TN-R and doxorubicin-induced
resistant ovarian cancer cells NCI/ADR-RES, compared with
regular MCF-7 and OVCAR-8 cells, respectively. However, this
compound has comparable inhibitory activity towards MCF-7
and its resistant cell line MCF-7/Dox. Mechanism investigations
show that analogs 401 and 406 can induce cell apoptosis via
the intrinsic pathway. These findings suggest that these
compounds activate ceramide downstream effectors, supporting
our hypothesis that prolonging the conjugated system of the
ceramide backbone increases the pro-apoptotic effects. Cellular
GCS assays have shown that analog 406 does not interfere with
the activity of glucosylceramide synthase in chemo-resistant can-
cer cells. This indicates that the selectivity of analog 406 towards
resistant cancer cells does not correlate with GCS inhibition.
Together, our results show that pharmacological intervention
via alteration of ceramide signaling and metabolism has promis-
ing therapeutic potential for the treatment of chemo-resistant
cancers.
2D NMR experiments were performed on a Bruker Avance III
400 MHz NMR spectrometer, and the data was processed using
the MestReNova NMR software (School of Chemistry, University
of Bristol, Bristol, UK). All of the tested compounds were confirmed
to be >95% pure via RP-HPLC method. RP-HPLC was performed on
an hp Hewlett Packard Series 1050 (Column: Phenomenex Gemini-
NX 5u C18 110A). The HPLC conditions were methanol/water
(85:15) at a 1.0 mL/min flow rate with a UV detector (Sig = 254.4
nm, Ref = 360.1 nm). Mass spectral data were obtained using an
Agilent 6890 GC with a 5973 MS. High-resolution mass spectra
(HRMS) were performed on a Bruker Spectrospin APEX TM 47e
FT-IRC instrument.
4.1.1. (E)-1-Bromotridec-2-ene (2)
At 0 °C, to a solution of 1.89 g (10 mmol) of (E)-tridec-2-en-1-ol
and 2.75 g (10.5 mmol) of triphenylphosphine (Ph3P) in 30 mL of
dry CH2Cl2 (DCM), 1.96 g (11 mmol) of N-bromosuccinimide
(NBS) was added. The reaction mixture was stirred at 0 °C for
1 h, and then stirred at room temperature for 1 h. The mixture
was diluted with 120 mL of hexane and passed through a pad of sil-
ica gel (2 cm). The filtrate was concentrated to give 2.06 g (79%) of
(E)-1-bromotridec-2-ene as
a
colorless oil. 1H NMR (CDCl3,
300 MHz) d 6.11 (m, 2H), 4.37 (d, J = 7.5 Hz, 2H), 1.96 (m, 2H),
1.29 (br, 16H), 0.96 (t, J = 6.0 Hz, 3H).13C NMR (CDCl3, 75 MHz) d
138.79, 129.72, 35.34, 35.07, 32.78, 32.64, 32.52, 32.27, 32.01,
25.80, 17.12.
4.1.2. (4S)-tert-Butyl 2,2-dimethyl-4-(2-
(phenylsulfinyl)acetyl)oxazolidine-3-carboxylate (3)
At ꢀ15 °C, to a solution of 1.00 g of methyl phenyl sulfoxide
(7.1 mmol) in 5 mL of anhydrous tetrahydrofuran (THF), 4.0 mL
of lithium diisopropylamide (LDA, 2 M solution in THF/n-hep-
tane/ethylbenzene, 8.0 mmol) was added. The mixture was stirred
at ꢀ15 °C for 30 min and then chilled to ꢀ78 °C. A solution of 1.00 g
of (S)-3-tert-butyl 4-methyl 2,2-dimethyloxazolidine-3,4-dicar-
boxylate (3.8 mmol) in 5 mL of THF was added dropwise. The solu-
tion was stirred at ꢀ78 °C for 2 h and allowed to warm to room
temperature overnight. After saturated aqueous NH4Cl (15 mL)
solution was added, the product was extracted with EtOAc, washed
with brine, and dried over with MgSO4. Purification by column
chromatography (petroleum ether/EtOAc, 1.5:1) gave 1.20 g (86%)
4. Experimental section
4.1. Chemistry
All chemicals were purchased from Sigma–Aldrich Corporation
(St. Louis, MO) and Fisher Scientific International Inc (Hampton,
NH), except D-sphingosine/HCl, which was purchased from CNH
Technologies Inc (Woburn, MA). 1H NMR and 13C NMR spectra
were recorded on a Bruker Fourier 300 MHz FT-NMR spectrometer.

1418
A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
of (4S)-tert-butyl 2,2-dimethyl-4-(2-(phenylsulfinyl)acetyl)oxazol-
idine-3-carboxylate as a colorless oil. This is a mixture of two dia-
stereomers. After a further column chromatography (50 g of silica
gel) with petroleum ether/EtOAc (1.5:1), two pure diastereomers
(3-up as a colorless oil and 3-down as colorless crystals) were
isolated.
32.62, 31.90, 29.61, 29.50, 29.33, 29.21, 29.17, 28.36, 26.33,
24.54, 22.67, 14.11. HRMS calcd for C25H45NNaO4 (M+Na)+,
446.3246; found, 446.3247.
4.1.5. (S,4E,6E)-2-Amino-1-hydroxyheptadeca-4,6-dien-3-one
trifluoroacetate (405)
3-up 1H NMR (CDCl3, 300 MHz) d 7.69 (m, 2H), 7.53 (m, 3H),
4.52 (m, 0.45H), 4.41 (m, 0.55H), 4.16–4.03 (m, 2.55H), 3.94 (dd,
J = 3.5 Hz, J = 9.8 Hz, 1H), 3.80 (d, J = 14.7 Hz, 0.45H), 1.65–1.35
(m, 15H). 13C NMR (CDCl3, 75 MHz) d 200.04, 152.53 (151.19),
143.56 (143.26), 131.77 (131.60), 129.56 (129.40), 124.15, 95.43
(94.72), 81.52 (81.33), 66.17 (65.99), 65.86 (65.29), 64.38 (64.14),
28.23, 26.11 (25.56), 24.87 (23.49).ESI-MS (m/e) 390 [M+Na]+,
406 [M+K]+, 575 [2M+Na]+.
At 0 °C, to a solution of 0.25 g (0.59 mmol) of (S)-tert-butyl 2,2-
dimethyl-4-((2E,4E)-pentadeca-2,4-dienoyl)oxazolidine-3-carbox-
ylate in 5 mL of dichloromethane (DCM), 0.5 mL of trifluoroacetic
acid (TFA) was added. After the mixture was stirred at 0 °C for
4 h, the solvent was removed under vacuum, and the residue was
crystallized in diethyl ether and petroleum ether to give 0.15 g
(64%) of (S,4E,6E)-2-amino-1-hydroxyheptadeca-4,6-dien-3-one
trifluoroacetate as
a
colorless crystal. 1H NMR (DMSO-d6,
3-down mp 115–117 °C. 1H NMR (CDCl3, 300 MHz) d 7.71 (m,
2H), 7.53 (m, 3H), 4.41 (m, 0.55H), 4.30 (m, 0.45H), 4.19–3.70 (m,
4H), 1.64–1.40 (m, 15H).13C NMR (CDCl3, 75 MHz) d 200.82
(199.94), 152.55 (151.14), 143.67 (143.58), 131.78 (131.69),
129.49 (129.41), 124.32, 95.48 (94.80), 81.58, 66.81 (66.08),
65.62, 64.50 (63.97), 28.29, 26.17 (25.62), 24.80 (23.34).ESI-MS
(m/e) 390 [M+Na]+, 406 [M+K]+, 575 [2 M+Na]+.
300 MHz) d 8.18 (br, 1H), 7.36 (dd, J = 10.5 Hz, J = 15.6 Hz, 1H),
6.44–6.30 (m, 3H), 5.51 (br, 1H), 4.39 (s, 1H), 3.86 (m, 2H), 2.17
(q, J = 6.6 Hz, 2H), 1.39–1.23 (m, 16H), 0.84 (t, J = 6.3 Hz, 3H).13
C
NMR (DMSO-d6, 75 MHz)
d 194.05, 148.79, 146.01, 129.44,
124.22, 60.28, 59.61, 33.21, 31.95, 29.63, 29.47, 29.38, 29.26,
28.73, 22.73, 14.58. HRMS calcd for
282.2433; found, 282.2437.
C
17H32NO2 (M+H)+,
4.1.3. (S)-tert-Butyl 2,2-dimethyl-4-((2E,4E)-pentadeca-2,4-
dienoyl)oxazolidine-3-carboxylate (401)
4.1.6. N-((S,4E,6E)-1-Hydroxy-3-oxoheptadeca-4,6-dien-2-yl)-2-
phenylacetamide (406)
To a solution of 0.60 g of (4S)-tert-butyl 2,2-dimethyl-4-(2-
(phenylsulfinyl)acetyl) oxazolidine-3-carboxylate (1.6 mmol) in
5 mL of DMF, K2CO3 (170 mg, 1.2 mmol) was added. After the mix-
ture was stirred at room temperature for 1 h under nitrogen, a
solution of 0.42 g of (E)-1-bromotridec-2-ene (1.6 mmol) in 4 mL
of DMF was added. The mixture was stirred for 4-5 days. Deionized
water (5 mL) was added, and the product was extracted with
diethyl ether (3 ꢁ 15 mL), washed with brine (3 ꢁ 15 mL) and
water (3 ꢁ 15 mL), and dried over with MgSO4. Concentration
and purification by flash column chromatography (petroleum
ether/EtOAc 10:1) gave 0.34 g (50%) of (S)-tert-butyl 2,2-di-
methyl-4-((2E,4E)-pentadeca-2,4-dienoyl)oxazolidine-3-carboxyl-
ate as a colorless oil. 1H NMR (CDCl3, 300 MHz) d 7.28 (m, 1H),
6.29–6.14 (m, 3H), 4.67 (dd, J = 3.0 Hz, J = 7.6 Hz, 0.36H), 4.47
(dd, J = 3.8 Hz, J = 7.7 Hz, 0.64H), 4.16 (m, 1H), 3.96 (dd, J = 3.0 Hz,
J = 9.3 Hz, 0.36H), 3.91 (dd, J = 3.9 Hz, J = 9.3 Hz, 0.64H), 2.18 (q,
J = 6.6 Hz, 2H), 1.71–1.25 (m, 31H), 0.87 (t, J = 6.3 Hz, 3H). 13C
NMR (CDCl3, 75 MHz) d 197.21 (196.33), 151.51 (152.29), 147.60
(147.23), 144.91, 128.76 (128.90), 122.34 (123.30), 95.09 (94.45),
80.53 (80.79), 66.03 (65.62), 64.46 (64.06), 33.22, 31.90, 29.58,
29.55, 29.42, 29.32, 29.19, 28.62, 28.37, 28.24, 25.29 (26.08),
At 0 °C, to a solution of 0.10 g (0.25 mmol) of (S,4E,6E)-2-amino-
1-hydroxyheptadeca-4,6-dien-3-one trifluoroacetate in 5 mL of
DCM, 5 mL of saturated NaHCO3 was added. The mixture was stir-
red vigorously for 5 min before the addition of 80 lL (0.50 mmol)
phenylacetyl chloride. The reaction mixture was then left to stir
at room temperature for 16 h. The organic phase was removed un-
der vacuum, and a white precipitate was formed. 50 mL of EtOAc
was then added, and the organic phase was washed with KHSO4
(5%), saturated NaCO3, and saturated NaCl, and dried over by
MgSO4. After removal of the solvent, the residue was recrystallized
using petroleum ether and ethyl ether to give 70 mg (70%) of N-
((S,4E,6E)-1-hydroxy-3-oxoheptadeca-4,6-dien-2-yl)-2-phenyl-
acetamide as a colorless powder. 1H NMR (CDCl3, 300 MHz) d 7.32–
7.26 (m, 6H), 6.81 (d, J = 6.0 Hz, 1H), 6.28 (dt, J = 6.5 Hz, J = 15.0 Hz,
1H), 6.19 (d, J = 15.6 Hz, 1H), 6.18 (dd, J = 9.9 Hz, J = 15.0 Hz, 1H),
4.85 (m, 1H), 3.92 (d, J = 11.4 Hz, 1H), 3.79 (dd, J = 5.0 Hz,
J = 11.4 Hz, 1H), 3.64 (s, 2H), 2.19 (q, J = 6.8 Hz, 2H), 1.43 (m, 2H),
1.26 (br, 14H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d
195.17, 172.15, 148.94, 146.36, 134.33, 129.30, 129.04, 128.56,
127.49, 123.43, 64.51, 60.02, 43.56, 33.30, 31.90, 29.59, 29.55,
29.42, 29.32, 29.20, 28.54, 22.69, 14.13. HRMS calcd for C25H37-
NNaO3 (M+Na)+, 422.2671; found, 422.2676.
24.11 (25.14), 22.68, 14.12. HRMS calcd for
C25H43NNaO4
(M+Na)+, 444.3090; found, 444.3088.
4.1.7. N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-2-yl)-2-
phenylacetamide (403)
4.1.4. (S)-tert-Butyl-4-((R,2E,4E)-1-hydroxypentadeca-2,4-
dienyl)-2,2-dimethyloxazolidine-3-carboxylate (402)
To a solution of 0.10 g (0.29 mmol) of sphingosine HCl in 5 mL
of DCM, 5 mL of saturated NaHCO3 was added under argon atmo-
sphere at 0 °C. The mixture was stirred vigorously for 5 min before
At ꢀ15 °C, to
a
solution of (S)-tert-butyl 2,2-dimethyl-4-
oxazolidine-3-carboxylate
((2E,4E)-pentadeca-2,4-dienoyl)
(0.25 g, 0.59 mmol) in 10 mL of dry THF, diisobutylaluminium hy-
dride (DIBAL-H, 0.70 mmol) was added. The temperature was grad-
ually raised to 0 °C. After 2 h, the reaction solution was quenched
with 20 mL of water, extracted with diethyl ether (3 ꢁ 25 mL),
washed with brine, and dried over with MgSO4. Concentration
and purification by column chromatography (petroleum ether/
EtOAc 2:1) gave 180 mg (72%) of (S)-tert-butyl 4-((R,2E,4E)-1-
hydroxypentadeca-2,4-dienyl)-2,2-dimethyloxazolidine-3-carbox-
ylate as a colorless oil. 1H NMR (CDCl3, 300 MHz) d 6.25 (dd,
J = 10.5 Hz, J = 14.7 Hz, 1H), 6.01 (dd, J = 10.2 Hz, J = 15.0 Hz, 1H),
5.66 (m, 1H), 5.53 (dd, J = 6.0 Hz, J = 15.0 Hz, 1H), 4.28–3.86 (m,
5H), 2.04 (q, J = 6.9 Hz, 2H), 1.48–1.46 (m, 15H), 1.24 (br, 16H),
the addition of 80 lL (0.50 mmol) phenylacetyl chloride. After stir-
ring at room temperature for 16 h, 25 mL of water was added. The
organic phase was removed under vacuum, and white precipitate
was formed. After filtration, the precipitate was dried, and washed
with a mixture solvent (petroleum ether/EtOAc, 4:1, 20 mL) to give
70 mg (58%) N-((2S,3R,E)-1,3-dihydroxyoctadec-4-en-2-yl)-2-
phenylacetamide as a colorless powder. GC/MS (m/e) 399 [MꢀH2-
O]+, 381 [Mꢀ2H2O]+.1H NMR (CDCl3, 300 MHz) d 7.34–7.27 (m,
5H), 6.24 (d, J = 7.0 Hz, 1H), 5.70 (dt, J = 6.8 Hz, J = 15.4 Hz, 1H),
5.44 (dd, J = 6.4 Hz, J = 15.4 Hz, 1H), 4.24 (br, 1H), 3.86 (m, 2H),
3.66 (d, J = 8.7 Hz, 1H), 3.60 (s, 2H), 2.98 (br, 1H), 2.88 (br, 1H),
1.99 (q, J = 6.6 Hz, 1H), 1.27 (br, 22H), 0.89 (t, J = 6.8 Hz, 3H). 13C
NMR (CDCl3, 75 MHz) d 171.83, 134.65, 134.35, 129.33, 128.98,
128.49, 127.39, 74.07, 62.31, 54.93, 43.77, 32.24, 31.93, 29.70,
0.86 (t, J = 6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz)
135.41, 131.93, 129.45, 128.94, 94.43, 81.06, 73.80, 64.87, 62.33,
d 154.12,

A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
1419
29.67, 29.64, 29.50, 29.37, 29.25, 29.06, 22.70, 14.14. HRMS calcd
for C26H43NNaO3 (M+Na)+, 440.3141; found, 440.3146.
4.2.3. Clonogenic survival assays
Colony formation assays were performed as previously
described.21,23 MCF-7, MDA-MB-231, and MCF-7TN-R cells were
plated in 6-well plates at 1000 cells per well in 2 mL of 10% DMEM.
After allowing 24 h for cell adhesion, treatment with gradient dilu-
tions of analogs was initiated. Colony growth was assessed each
day until suitable colony formation was noted (generally
10–12 days after treatment). Cells were incubated with 3% glutar-
aldehyde for 30 min in order to fix cells to the plate. Media was
then removed in a water bath, and plates were allowed to air
dry. After fixation, plates were treated with a 0.2% solution of crys-
tal violet and 20% methanol in deionized water for a period of
40 min. Plates were then washed in distilled water and again air
dried. Colonies were hand counted on a light plate with formations
of greater than 30 cells quantifying as sufficient size for a positive
colony count. Clonogenic survival was normalized to non-analog-
treated DMSO control.
4.1.8. (S)-3-Hydroxy-2-(2-phenylacetamido)-N-
tetradecylpropanamide (404)
To a solution of 0.20 g (0.48 mmol) of (S)-2-amino-3-hydroxy-
N-tetradecylpropanamide in 15 mL of DCM, 12 mL of saturated
NaHCO3 was added under argon atmosphere at 0 °C. The mixture
was stirred vigorously for 5 min before the addition of 160 lL
(1.00 mmol) phenylacetyl chloride. After stirring at room tempera-
ture for 16 h, 40 mL of water was added. The organic phase was re-
moved under vacuum, and a white precipitate was formed. After
filtration, the precipitate was dried, and washed with a mixture
solvent (petroleum ether/EtOAc, 4:1, 20 mL) to give 165 mg (82%)
(S)-3-hydroxy-2-(2-phenylacetamido)-N-tetradecylpropanamide
as
a
colorless powder. GC/MS (m/e) 399 [MꢀH2OꢀH]+, 442
[M+Na+H]+.1H NMR (CDCl3, 300 MHz) d 7.36–7.24 (m, 5H), 6.91
(t, J = 5.1 Hz, 1H), 6.85 (d, J = 6.9 Hz, 1H), 4.41 (m, 1H), 4.00 (dd,
J = 2.5 Hz, J = 11.0 Hz, 1H), 3.83 (br, 1H), 3.59 (s, 2H), 3.53 (m,
1H), 3.16 (q, J = 6.8, 2H), 1.41 (m, 2H), 1.25 (br, 22H), 0.88 (t,
4.2.4. Apoptosis assays
As previously described, MCF-7TN-R cells were plated at 7500–
10,000 cells in 200 lL of media per well in 96-well plates using
J = 6.8 Hz, 3H). 13C NMR (CDCl3, 75 MHz)
d
172.41, 170.85,
134.53, 129.44, 129.25, 127.74, 63.08, 54.18, 43.65, 39.82, 32.19,
29.93, 29.89, 29.82, 29.64, 29.58, 29.54, 27.11, 22.97, 14.40. HRMS
calcd for C25H42N2NaO3 (M+Na)+, 441.3093; found, 441.3110.
phenol-free DMEM.32,33 Cells were incubated and given 24 h to ad-
here to plates. Cells were then treated with twice the IC50 concen-
trations of ceramide analogs obtained through MTT viability assay,
and incubated for an additional 24-hour period to allow prolifera-
tion. Plates were analyzed using a cell death detection ELISA kit
purchased from Roche Applied Sciences. Assays were performed
in duplicate on three separate occasions at least 24 h apart. For
analysis, values were normalized to an MTT viability assay per-
formed on the same plate.
4.2. Bioassays
4.2.1. Cell culture
MCF-7, MDA-MB-231, MCF-7TN-R, MCF-7/Dox, OVCAR-8, and
NCI/ADR-RES cells were cultured as previously described.27–30
Briefly, cells were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) (Invitrogen, Carlsbad, CA) enriched with 10% fetal bovine
serum (FBS), 1% MEM amino acids, 1% MEM non-essential amino
acids, 1% Anti/Anti, human recombinant insulin, 1% sodium pyru-
vate and plasmocin (10% DMEM) (all from Life Technologies, Gai-
thersburg, MD). MCF-7/Dox, OVCAR-8, and NCI/ADR-RES cells
were culture in RPMI-1640 medium (Invitrogen, Carlsbad, CA) sup-
4.2.5. Caspase-9 assays
Caspase activity assays were performed as previously
described.33 Briefly, MCF-7TN-R cells were plated at 7500–10,000
cells in 200 lL of media per well in 96-well plates using phenol-
free DMEM. Cells were incubated and given 24 h to adhere to
plates. Cells were then treated with twice the IC50 concentrations
of ceramide analogs obtained by means of MTT viability assays,
and incubated for an additional 24-hour period to allow prolifera-
tion. Plates were then analyzed using a Caspase Glo 9 assay pur-
chased from Promega. Assays were performed in triplicate on
three separate occasions at least 24 h apart.
plemented with 10% FBS, 100 units/mL penicillin, 100 lg/mL strep-
tomycin and 584 mg/liter L-glutamine. Cells were cultured in
75 cm2 and 125 cm2 tissue culture flasks (Sarsdedt, Newton, North
Carolina) in 37 °C humidified atmosphere of 5% CO2 and 95% air.
4.2.2. Viability assays
Viability assays in MCF-7, MDA-MB-231, and MCF-7TN-R cells
4.2.6. Statistical analysis
Statistical analysis of IC50 values were calculated from dose–re-
sponse curves using GraphPad Prism 5.0 (GraphPad Software),
were performed according to previously published protocols.27,28
Cell lines were plated at 7500–10,000 cells in 200 lL of media
per well in 96-well plates using phenol-free DMEM. Cells were
incubated and given 24 h to adhere to plates. Cells were then trea-
ted with gradient concentrations of ceramide analogs, and incu-
bated for an additional 24-hour period to allow proliferation.
using
the
equation:
IC50
Y ¼ Bottom þ ðTop ꢀ bottomÞ=½1 þ 10ðLog
ꢀXÞꢂ, assuming a stan-
dard slope, where the response goes from 10% to 90% of maximal
as X increases over two log units. Differences in IC50 and other
activity data were compared using Student’s unpaired t-test with
p <0.05 as the limit of statistical significance. Experiments compar-
ing multiple concentrations to the control were tested with one-
way ANOVA with Bonferroni post-test to compare individual
concentrations.
MTT (20 lL) was added to each well and allowed to incubate for
3–4 h. Cells were then suspended in 20% SDS buffer and 50%
DMF for 24 h. The plates’ absorbance values were read using a
spectrophotometer at 550 nm. For analysis, viability values were
set to percent control of dimethyl sulfoxide (DMSO) vehicle, and
carried out in quadruplicate on three separate occasions at least
24 h apart.
Cell viability of MCF-7, MCF-7/Dox, OVCAR-8, and NCI/ADR-RES
was determined by quantitation of ATP, an indicator of live cells,
using the CellTiter-Glo luminescent cell viability assay (Promega,
Madison, WI) kit, as described previously.31 Briefly, cells
(4000 cells/well) were grown in 96-well plates with 10% FBS
RPMI-1640 medium overnight. Cells were treated with ceramide
analogs for 72 h. Cell viability was determined by the measurement
of luminescent ATP in a Synergy HT microplate reader (BioTek, Win-
nooski, VT, USA) following incubation with CellTiter-GloReagent.
4.2.7. Cellular GCS assay
Cells (1 ꢁ 106 cells/35-mm dish) were grown in 10% FBS RPMI-
1640 medium for 24 h, and then treated with ceramide analog at
its IC50 concentration for additional 24 h in 5% RPMI-1640 medium.
Cells were switched to 1% BSA RPMI-1640 medium containing
50 lM NBD C6-ceramide complexed to BSA. After 2 h of incubation
at 37 °C, lipids were extracted, and resolved on high performance
TLC plates (Partisil, Piscataway, NJ, USA) in a solvent system
containing chloroform/methanol/3.5 N ammonium hydroxide
(85:15:1, v/v/v), as described previously.26,34 NBD C6-glucosylcera-

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A. P. Ponnapakam et al. / Bioorg. Med. Chem. 22 (2014) 1412–1420
10. Szulc, Z. M.; Bai, A.; Bielawski, J.; Mayroo, N.; Miller, D. E.; Gracz, H.; Hannun, Y.
A.; Bielawska, A. Bioorg. Med. Chem. 2010, 18, 7565.
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AlphaImager HP imaging system (Alpha Innotech, Santa Clara,
CA). For quantitation of GCS activity, ratio of fluorescence of GlcCer
to fluorescence of Cer was determined using Synergy HT multi-
detection microplate reader (BioTek, Winooski, VT, USA). NBD C6-
ceramide (Invitrogen, Carlsbad, CA, USA) and NBD C6-glucosylcer-
amide (N-hexanol-NBD-glucosylceramide; Matreya LLC, Pleasant
Gap, PA) were used as standards.
16. Liu, J.; Antoon, J. W.; Ponnapakkam, A.; Beckman, B. S.; Foroozesh, M. Bioorg.
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We acknowledge NIH AREA grant number 1R15CA159059-01
(Multi-Target, Mechanism-Based Drug Design for the Treatment
of Breast Cancer: Synthesis, Screening, and Mechanism Investiga-
tion of a Ceramide Analogue Library), and DoD Breast Cancer
Research Award number W81XWH-11-1-0105 (BC102922) (A
Drug Discovery Partnership for Personalized Breast Cancer Ther-
apy). We also like to acknowledge the support of Louisiana Cancer
Research Consortium and NIH-RCMI (grant number G12RR026260)
for support of the Core Facilities at Xavier University of Louisiana.
Supplementary data
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Supplementary data (1H and 13C NMR spectra, 2D NMR spectra,
and activity data of all the tested compounds) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2013.12.065.
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