Synthesis and bioevaluation of ω-N-amino analogs of B13

Article abstract of DOI:10.1016/j.bmc.2009.01.057

Novel ω-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204

Full text of DOI:10.1016/j.bmc.2009.01.057

Bioorganic & Medicinal Chemistry 17 (2009) 1840–1848
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioevaluation of
x-N-amino analogs of B13
Aiping Bai a, Zdzislaw M. Szulc a, Jacek Bielawski a, Nalini Mayroo a, Xiang Liu b, James Norris b,
Yusuf A. Hannun a, Alicja Bielawska a,
*
a Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, PO Box 250509, Charleston, SC 29425, USA
b Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel
x-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid cer-
Received 14 November 2008
Revised 21 January 2009
Accepted 24 January 2009
Available online 31 January 2009
amidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and
ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.;
Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015].
Representative analog LCL464, (1R,2R)-2-N-(120-N,N-dimethylaminododecanoyl amino)-1-(400-nitro-
phenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than
LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of
sphingosine and specific increase of C14- and C16-ceramide. LCL464 did not induce lysosomal destabiliza-
tion nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity
in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activa-
tion. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of
ACDase.
Keywords:
Ceramide
Acid ceramidase
Inhibitors
Lysosomes
B13
LCL204
LCL464
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and confirmed that the active analogs increased endogenous Cer and
inhibited CDases.8–12 B13, a potent and specific ACDase inhibitor in vi-
The stimulus-controlled pathways of sphingolipid (SPL) metabo-
lism provide a rich network of bioactive molecules with pivotal roles
in the regulation of diverse cell functions. SPL metabolites, namely
ceramide (Cer) and sphingosine 1-phosphate (S1P), are increasingly
being recognized for their important role as signaling molecules in-
volved in regulation of survival, proliferation and cell death.1 Cer is
known to be a key modulator of cancer cell growth and apoptosis.
Conversely, S1P acts as an anti-apoptotic tumour protective agent.2
Acid ceramidase (ACDase), a lysosomal enzyme, causes degradation
of Cer into sphingosine (Sph) and free fatty acids at pH optimum
ꢀ4.5, which distinguish it from other CDases. ACDase represents a
newtarget forcancertherapybecauseof its importancein regulating
the Cer–Sph–S1P inter-metabolism.3,4 Elevation of ACDase was
shown in many tumour cell lines when compared to normal tissue.5
Recently we showed that inhibition of ACDase by B13, (1R,2R)-2-N-
(tetradecanoylamino)-1-(40-nitrophenyl)-1,3-propandiol, and its
analogs induced apoptotic cell death.3 These and other data on AC-
Dase inhibition indicate that it is a promising target in anticancer
therapy because many analogs of B13 act as chemotherapeutic
agents for several type of cancers.6,7
tro, has been shown to induce apoptosis in many cancer cell lines.
3,10–12
Tumour growth was prevented when nude mice were treated
with B13 in two different aggressive human colon cancer cell lines that
had metastasized to the liver without any toxicity to normal cells.11
B13 has also been used in combination with other therapies to achieve
tumours shrink in animal studies.12 However, the inhibitory effect of
B13 on ACDase cellular activity is questionable because B13, as a neu-
tral lipophylic molecule may not specifically and efficiently reach and
accumulate in the acidic compartment where its potential target is
present. To improve B13 cellular targeting properties, we designed
and synthesized several B13 lysosomotropic analogs with enhanced
anticancer activity.7 LCL204, (1R,2R)-2-N-(tetradecylamino)-1-(40-
nitrophenyl)-1,3-propandiol hydrochloride, represented its closest
analog. LCL204 specifically targeted lysosomes; however, it also
caused induction of lysosomal destabilization and proteolytic degra-
dation of ACDase very early in the process3,13 It can be assumed that
this analog is also toxic to the normal tissues (data not published).
Similar destructive effects on lysosomes and cellular ACDase were ob-
served for D-e-Sph (data not shown).
To avoid these unwanted side effects, which were caused by the di-
rect replacement of the N-amido group in the structure of B13 into the
N-amino group in LCL204 (representing analog of N-myristyl-Sph), we
developed a novel series of lysosomotropic inhibitors of ACDase with-
out causing neither lysosomal destabilization nor degradation of
ACDase.
Previously, we synthesized a set of lipophilic N-acyl-amino-phenyl
alcohols (aromatic analogs of Cer) acting as potent anticancer agents
* Corresponding author. Tel.: +1 843 792 0273; fax: +1 843 792 4322.
E-mail address: bielawsk@musc.edu (A. Bielawska).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.057

A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
1841
OH
OH
HN
O2N
5
O
B13
Targeted ceramide analogs
OH
Multimodal sphingosine analog
OH
OH
OH
HN
N
O2N
O2N
H
NR1R2
5
Cl
n
H
O
LCL 204
Class E analogs: LCL433, 449, 463,464, 488, 506
Scheme 1. Design of the new lysosomotropic analogs of B13 and LCL204: Class E analogs.
Based on the structures of the leading compounds, B13 and
LCL204, and by combining structural elements of B13 (the amide
bond) and LCL204 (the varied amino functions), we designed and
synthesized several new analogs: LCL 433, 449, 463, 464, 488
and LCL506 (Scheme 1).
E analogs inhibited ACDase efficiently with LCL464 showing the
highest inhibition (55%).
The effects of the LCL compounds on the cellular activity of AC-
Dase were tested from the cells treated with the 10 lM LCL analogs
for 2 h (listed in Section 4). Results showed that all tested com-
pounds had a higher potential for ACDase inhibition (Fig. 2) com-
pared to the in vitro data (Fig. 1). The highest inhibition (65%)
was detected for LCL464. LCL204 caused ꢀ35% inhibition. Interest-
ingly, B13, the most potent inhibitor in vitro, did not show ACDase
inhibition at the cellular level.
2. Results and discussion
2.1. Structure design and chemistry
Because the three-dimensional structure of ACDase is not avail-
able, we developed a new class of compounds (Scheme 1) based on
our extensive knowledge of molecular recognition of Cers and its
analogs by CDases as their substrates.7,14–16 We proposed to synthe-
size a hybrid set of B13 and LCL204 analogs with the combined key
structural elements of the N-amide group and the N-amino function,
critical for effective molecular recognition by ACDase and targeting
to lysosomal compartment.10,13 These analogs represent (1R,2R) iso-
mers and function differently from the primary, secondary and ter-
tiary amino groups introduced into the aliphatic, cyclic and
heterocyclic systems: LCL463 with NH2 group representing primary
amine; LCL506 and LCL488 with N-methyl and N-octyl groups,
respectively, representing secondary amines; and LCL464 with
N,N-dimethyl group or LCL433 and LCL449 with N-imidazyl and N-
morpholinyl groups, respectively, representing tertiary amines.
Synthetic outline for the preparation of Class E analogs is shown
in Scheme 2. All synthesized model compounds were characterized
by NMR, MS, confirming their structural homogeneity (listed in
Section 4).
2.3. Corresponding effects of selected analogs on endogenous
Cer species and Sph
To connect the observed effects of compounds of interest on the
cellular activity of ACDase to the cellular SPLs, we investigated the
effects on endogenous Cer species (Cn-Cer) and Sph (a substrate
and product of ACDase). The cells were treated with 10 lM Class
E analogs for 2 h and showed only small effect on the total Cer
(98–104%, not shown). However, these analogs caused an expected
decrease in Sph (50–72%) and specific increase in C14-Cer (200–
400%) and C16-Cer (120–150%), Figure 3A.
The effects of 10 lM LCL464, LCL204 and B13 treated for 2 h on
cellular C14, C16, C18, C24, C24:1-Cer and Sph (Fig. 3B) showed that
LCL464 increased C14-Cer to 300%, C16-Cer to 143%, and decreased
Sph to 50% of the control; and the amount of increase on these two
Cers was balanced by a small decrease of C24- and C24:1-Cers, the
major Cer component of MCF7 cells. 10 lM LCL204 increased C14,
C16 and C18-Cer to 220%, 200% and 160%, respectively, without
changes for C24- and C24:1-Cers, and decreased Sph to 30% of the
2.2. Inhibitory effects on ACDase in vitro and at cellular level
control. 10 lM B13 increased all Cn-Cer to 135–200% range; inter-
estingly, it also increased cellular level of Sph to ꢀ140%.
2.4. Effects of selected analogs on lysosomal stability
Lysosomal stability of selected compounds was shown in Figure
The inhibitory effects of all new analogs as compared to the ac-
tion of B13 and LCL204 are shown in Figures 1 and 2. Acidic MCF7
cell lysate was used as an enzymatic source of ACDase for the
in vitro assays (listed in Section 4). When in vitro ACDase inhibi-
tion was tested with 50 lM LCL compounds (Fig. 1), the highest ef-
4, only 10
its stability to ꢀ90% when compared to the control. 10
had very little effect (ꢀ10%) on the early treatment and recovery as
note in 5 h treatment. 10 M B13 did not cause lysosomal dysfunc-
l
M LCL204 caused lysosomal destabilization lowering
fect was for unmodified B13 (ꢀ90% inhibition). Under identical
conditions, LCL449 and LCL488 representing the new Class E ana-
logs similar to LCL204, showed only ꢀ5% of inhibition. Other Class
l
M LCL464
l

1842
A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
OH
H
ii
OH
OH
N
O2N
NH2
OH
5
O
OH
OH
LCL463
NH2
1
iii
O2N
N
N
O2N
H
NHCH3
5
O
i
LCL506
OH
iv
OH
OH
OH
H
O2N
H
N(CH3)2
5
OH
O
O
LCL464
N
O
OH
O2N
Br
n
v
LCL429: n = 7
LCL509: n = 1
N
N
O2N
H
N
H
LCL488
OH
vi
OH
OH
O2N
H
N
N
5
O
LCL 433
OH
vii
N
O2N
H
N
5
O
O
LCL 449
Scheme 2. Synthetic outline for the preparation of Class E analogs. Reagents and conditions: (i)
x-Br-acyl chlorides, DIPEA, THF, rt; (ii) 2 M ammonium in ethanol, 1.5 N
NaOH, rt; (iii) methylamine, 33% in absolute ethanol, 1.5 N NaOH, rt; (iv) 2 M N,N-dimethylamine in THF, 1.5 N NaOH, rt; (v) octylamine, THF, 1.5 N NaOH, rt; (vi) imidazol,
THF, 1.5 N NaOH, rt; (vii) morpholine, THF, 1.5 N NaOH, rt.
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
B13
LCL204 LCL433 LCL449 LCL463 LCL464 LCL488 LCL506
B13
LCL204 LCL433 LCL449 LCL463 LCL464 LCL488 LCL506
Figure 1. Effects of 50 lM Class E in comparison to B13 and LCL204 on ACDase activity in vitro. Results are presented as % Control (each dose was duplicated).
tion. Although, as note for the higher concentration, LCL464 has the
potential to destabilize the lysosomes; however, this dysfunction
was recovered by the extended incubation time.
did not degrade ACDase in 5 h treatments. This effect was observed
only for LCL204 as previously reported.3 Furthermore, even if the
concentration of Class E analogs was increased up to 50 lM, the ana-
logs still did not show any effects on ACDase degradation.
2.5. Effects of selected analogs on acid ceramidase degradation
2.6. Apoptotic effect of LCL464
Effects of Class E analogs on ACDase expression in comparison to
B13 and LCL204 were determined by Western blot (Fig. 5A) and were
normalized by Actin (Fig. 5B). 10 lM Class E analogs, similar to B13,
Apoptotic effect of LCL464 was investigated in MCF7 cells via its
effect on Caspase 3/7, a group of cysteine proteases that mediate

A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
1843
400.0
350.0
300.0
250.0
200.0
150.0
100.0
50.0
AC
C14Cer
C16Cer
Sph
Actin
Ctr B13 204 433 449 463 464
10uM
AC
Actin
Ctrl B13 433 449 463 464
30uM
0.0
LCL433
LCL449
LCL463
LCL464
Figure 2. Effects of 10 lM Class E, B13 and LCL204 on cellular activity of ACDase at
2 h of treatments. Results are presented as % Control (each dose was duplicated).
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
10uM
30uM
350.0
LCL464
LCL204
B13
300.0
250.0
200.0
150.0
100.0
50.0
ctrl
B13
LCL204
LCL433
LCL449
LCL463
LCL464
Figure 5. Effects of selected analogs on ACDase stability. (A) Western blots of
10 M and 30 M treatments at 1 h and 5 h. (B) Normalization by Actin.
l
l
0.0
C14-Cer
C16-Cer
C18-Cer
C24-Cer
C24:1-Cer
Sph
2.7. Cellular concentration of the selected analogs
Figure 3. Early effects of 10
lM Class E analogs on Cer–Sph balance. (A) Up-
regulation of C14- and C16-Cer and down-regulation of Sph at 2 h. (B) Effect of 10
lM
LCL464, LCL204 and B13 on endogenous Cer species and Sph in MCF7 cells for 2 h
treatment. Results are presented as % Control (each dose was duplicated).
Cellular levels of the representative analogs in MCF7 cells were
established by high-performance liquid chromatography–mass
spectrometry (HPLC–MS) analysis.
In general, cellular level of LCL464 represented only ꢀ0.1% of
140.0
the treatment applied and was much lower than the corresponding
B13 and LCL204. Comparison results for 10 lM treatments at 2 h
1hour
120.0
showed that the level of B13 was ꢀ10ꢁ higher than LCL464, and
level of LCL204 was ꢀ25ꢁ higher than LCL464 (Fig. 7). These differ-
ences were even more dramatic for the extended treatments when
the level of LCL464 was decreased. Decrease on the cellular level of
LCL464 from ꢀ2–5 h of treatments following its fast uptake was
observed for all Class E analogs, indicating on their strong binding
to some receptors or, perhaps, metabolism. Some analogs, for
5hour
100.0
80.0
60.0
40.0
20.0
0.0
example, LCL463 containing the free NH2 group at
x-position,
were present at a much lower levels than the corresponding
LCL464 (data not shown). These results suggest that potency of
LCL464 will be increased after improving its cellular delivery.
2.8. Inhibitory effects on cell growth
LCL464
B13
LCL204
Figure 4. Effect of 10 lM LCL464, B13 and LCL204 on lysosomal stability for 1 and
5 h treatments.
Effects of Class E analogs were examined in MCF7 breast carci-
noma cells and compared to the activity of B13 and LCL204. IC50
values represent a drug concentration that reduces the cell number
by 50% during incubation for 48 h, signifying the growth inhibitory
power of the agents.18 All tested analogs showed concentration-
dependent cell growth inhibition shown in Figure 8A. Almost all
Class E analogs, except LCL449, were more potent than B13 in tu-
mour cells killing, but in general were less effective than LCL204.
execution.19 Caspase 3/7 can be activated by apoptotic signals from
both death receptors and intracellular/mitochondrial pathways.20
LCL464 caused a dose dependent induction of apoptosis for 24 h
incubation, even increasing Caspase 3/7 activity to ꢀ175% in the
10 lM treatment (Fig. 6).

1844
A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
300.0
250.0
200.0
150.0
100.0
50.0
70.0
60.0
50.0
40.0
30.0
20.0
48hrs
72hrs
10.0
0.0
0.0
10.0
20.0
40.0
50.0
Concentration (uM)
0.0
SCC14a
Du145
MCF7
J3RD4
A549
PPC1
Figure 6. Effects of LCL464 on Caspase 3/7 activity at 24 h.
Figure 9. Inhibitory effects of LCL464 on different cancer cell lines. IC50 profile (48
and 72 h).
30.0
noma, epithelial-like cell lines) J3DR4 (human melanoma), A549
(human alveolar epithelial cell), PPC1 (human prostate carcinoma)
were compared to MCF7 cells (Fig. 9), LCL464 showed the highest
25.0
20.0
15.0
10.0
5.0
effect on growth of MCF7 cells (10.2 lM and 7.2 lM, respectively)
for 48 and 72 h treatments. Coincidently, these effects corre-
sponded to the levels of ACDase with the MCF7 cells having the
highest ACDase protein expression detected in the same amount
of the total protein (data not shown).
Based on the in vitro and the cellular level results, LCL464 was
selected as the most potent inhibitor of ACDase, a potent antican-
cer agent.
0.0
LCL464
B13
LCL204
3. Summary and conclusion
Figure 7. Cellular levels of 10 lM LCL464, B13 and LCL204 at 2 h.
Results on B13 presented in this manuscript prove its high inhib-
itoryeffect onACDaseinvitro;however, at the cellularlevel, itdid not
inhibit this enzyme. These results correspond to our hypothesis that
B13 as a neutral molecule was not able to reach lysosomal ACDase. It
also corroborates the conclusion we made previously that B13 in
MCF7 cells may act on some N-acyl-transferase enzymes if used at
a low concentrations especially in early treatments, or as a non Cer
species specific inhibitor of other CDases, similar to D-e-MAPP, a
known as inhibitor of the alkaline CDase.3 Both compounds increase
all Cer species and decrease Sph, if used at a high concentration.3
The most active analogs from Class E were LCL463 followed by
LCL464. The IC50 values of Class E analogs ranged from ꢀ6.0 to
15.5 lM, except for LCL449 (49.9 lM), Figure 8B. The comparable
results for LCL204 and B13 showed the following IC50 values:
4.3 M and 20.0 M, respectively.
Anti-proliferate effect of LCL464 was also investigated in differ-
l
l
ent tumour cell lines. When IC50 values for SCC14a (human head
and neck squamous cell carcinoma) Du145 (human prostate carci-
60.0
140.0
B13
LCL204
49.90
120.0
LCL433
LCL449
LCL463
50.0
LCL464
LCL488
LCL506
100.0
40.0
80.0
60.0
40.0
20.0
0.0
30.0
19.60
20.0
14.80
15.50
10.20
12.10
10.0
0.0
6.43
4.29
B13
LCL204 LCL433 LCL449 LCL463 LCL464 LCL488 LCL506
0.1
10
10.0
100.0
Concentration (µM)
Figure 8. Inhibitory effects of Class E analogs in comparison to B13 and LCL204 on MCF7 cell growth. (A) Results from MTT assay at 48 h. (B) IC50 values at 48 h.

A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
1845
To improve B13 targeting to the lysosome, previously we de-
4. Experimental
4.1. Chemistry
signed and synthesized LCL204 representing Class C analogs.3
Chemical structures of these two compounds differ only in the
attachment of the C14-carbon chain to the amino function of
(1R,2R) 1-(40-nitrophenyl)-2-amino-1,3-propandiol via the tetra-
decanoyl-linkage in B13 or tetradecyl-linkage in LCL204. Thus,
B13 represents an aromatic analog of Cer while LCL204 represent
an aromatic analog of Sph. LCL204 was able to reach ACDase, its
cellular target, even after 15 min incubation, causing induction of
All solvents, general reagents (Scheme 2) were purchased from
Aldrich–Sigma and Fluka. [9,10-3H] Palmitic acid was purchased
from American Radiolabeled Chemical, Inc. (St. Louis, MO).
(2S,3R,4E) [N–3H]-C16-ceramide (specific activity: 4 ꢁ 106 cpm/
nmol) and (2S,3R,4E) C16-ceramide were synthesized by the Lipi-
domic Core, Medical University of South Carolina.21–23 B13 and
LCL204 were synthesized as described previously.7 Synthetic out-
line for the preparation of Class E analogs is presented here. TLC,
1H NMR and MS confirmed purity of all synthesized compounds.
Reaction progress was monitored with analytical normal and re-
verse-phase thin layer chromatography (NP TLC or RP TLC) using
aluminum sheets with 0.25 mm Silica Gel 60-F254 (Merck) or
0.150 mm C18-silica gel (Sorbent Technologies). Detection was
performed by UV (254 nm), the PMA reagent (ammonium hepta-
molybdate tetrahydrate cerium sulfate, 5:2, g/g) in 125 mL of 10%
H2SO4) and the Dragendorff reagent (Fluka) following heating of
the TLC plates at 170 °C. Flash chromatography was performed
using EM Silica Gel 60 (230–400 mesh) with the indicated eluent
systems. Melting points were determined in open capillaries on
an Electrothermal IA 9200 melting point apparatus and were
reported uncorrected. 1H NMR spectra were recorded on Bruker
AVANCE 400 MHz spectrometer equipped with Oxford Narrow
Bore Magnet. Chemical shifts were reported in ppm on the d scale
from the internal standard of residual chloroform (7.27 ppm) and
methanol (4.87 ppm). Mass spectral data were recorded in a
positive ion electrospray ionization (ESI) mode on Thermo Finni-
gan TSQ 7000 triple quadrupole mass spectrometer. Samples
were infused in a methanol solution with an ESI voltage of
4.5 kV and capillary temperature of 200 °C. Specific structural
features were established by fragmentation pattern upon electro-
spray (ESI/MS/MS) conditions, specific for each of the compound
studied.
cell death later with the IC50 value of 5 l
M/24 h in MCF7 cells.3
However, LCL204 also caused lysosomal destabilization and prote-
olytic degradation of lysosomal enzymes,3 which could be the rea-
son for its cyto-toxic effect on normal cell lines (not shown).
Here we showed that LCL204 acted as poor inhibitor of ACDase
in vitro, causing activation of this enzyme at concentration lower
than 40 lM (data not shown). At the cellular level its inhibitory ef-
fect on ACDase was moderate because this enzyme was partially
degraded by LCL204. For extended treatments, its efficacy was in-
creased probably due to the partial recovery from lysosomal desta-
bilization. These two parent compounds, B13 and LCL204, showed
opposite effects on ACDase in vitro and on its cellular activity.
Described here, Class E analogs, a hybrid set of B13 and LCL204,
having the combined key structural elements of the amide group
and the N-alkylamino function, are necessary for effective molecu-
lar recognition by ACDase and targeting the lysosomal compart-
ment, and were designed with the assumption that they would
inhibit cellular ACDase without affecting lysosomal dysfunction.
All Class E analogs showed inhibitory effects (moderate to low)
on ACDase in vitro; however, they were less potent than B13. Class
E analogs also affected cellular ACDase activity, with LCL464, 463
and LCL 488 representing the most potent inhibitors, the opposite
effect of B13. However, their potency was decreased with extended
treatments different from the action of LCL204. Time-dependent
decrease of the hydrolytic activities of ACDase by Class E analogs
may be related to the observed time-dependent increase in pH val-
ues of the lysosomes data not shown. The new analogs had differ-
ent effects on the lysosomal dysfunction compared to the action of
LCL204, causing interruption during short treatments only or in the
high concentrations, however, without degradation of ACDase. In
conclusion, Class E analogs represent inhibitors of ACDase acting
both in vitro and on cellular levels. These analogs also cause paral-
lel changes in cellular SPLs, decrease in Sph, increase in C14- and
C16-Cers and also some decrease in C24- and C24:1-Cers. The same
profile was shown previously for LCL204 and its analogs.3
Syntheses of Class E analogs were performed as shown in
Scheme 1 via their
x
-Br-analogs LCL429 and LCL509, obtained
from
1 and corresponding bromoacylchlorides as previously
described.7
4.1.1. Preparation of
x-Br-analogs LCL429 and LCL509
4.1.1.1. (1R,2R)-2-N-(120-Bromododecanoylamino)-1-(400-nitro-
phenyl)-1,3-propandiol (LCL429).
(a) Preparation of 12-
bromododecanoyl chloride. 12-Bromododecanoic acid (0.3 g,
1.1 mmol) was dissolved in dry cyclohexane (4 mL) by stirring at
45 °C for 20 min. To this well-stirred and cooled down to rt mix-
ture one drop of dry pyridine and oxalyl chloride (0.145 mL,
1.65 mmol) were added over 1 min. After the addition was com-
pleted, the reaction mixture was heated to 50 °C for 25 min. After
stirring for an additional 30 min. at rt, the reaction mixture was
evaporated to dryness by purging dry nitrogen gas into the reac-
tion flask and dried under the high vacuum. The freshly prepared
12-bromododecanoyl chloride was dissolved in 3 mL anhydrous
THF and taken directly to the next step.
(b) Synthesis of LCL429. To the well-stirred mixture of (1R,2R)-
2-amino-1-(40-nitrophenyl)-1,3-propandiol (1 mmol) in THF
(10 mL) and 50% aqueous solution of sodium acetate (5 mL), the
freshly prepared 12-bromododecanoyl chloride (1.1 mmol) was
added dropwise over 1 min. After the addition was completed,
the reaction mixture was stirred for another 30 min at rt. The or-
ganic layer was separated and the aqueous layer was extracted
with ethyl acetate (3 ꢁ 15 mL). The combined organic extracts
was dried over anhydrous MgSO4, filtered and evaporated to dry-
ness under reduced pressure to give crude product. This material
was purified by flash column chromatography (CHCl3/MeOH,
Class E analogs showed a concentration dependent MCF7 cell
growth inhibition with IC50 values ranging from ꢀ6.0 to 15.5
lM,
except for LCL449 (49.9 M). This compound, the -morpholino-
l
x
analog, showed a different profile on Sph–Cer changes for the ex-
tended treatments, causing decrease of C16-Cer and increase of
Sph data not shown. Class E analogs enhanced the tumour cell kill-
ing potency of B13 (IC50 = 20
LCL204 (IC50 = 4.3 M). The most active analogs from Class E were
LCL463 (IC50 = 6.4 M), followed by LCL464 (10.2 M). Results
lM) but were less effective than
l
l
l
comparing the intracellular levels of B13, LCL204 and LCL464
showed that LCL464 would be much more potent if structure mod-
ification is done in order to improve its cellular delivery and to
avoid its binding to the cellular protein. Synthetic work on the
modified prodrugs of LCL464 is under development.
In conclusion, LCL464, a selected compound from Class E, inhib-
ited ACDase in vitro, suggesting its recognition by the enzyme,
caused neither lysosomal dysfunction nor degradation of ACDase,
and showed a high and early inhibitory effect on the cellular AC-
Dase. This effect generated proapoptotic C14-and C16-Cers resulting
in cell death via apoptosis. All these results suggest that LCL464,
and its close congeners, may act as novel chemotherapeutic agents
through site-specific ACDase inhibition.

1846
A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
10:1, v/v) to give pure product in 78% yield as a white microcrys-
talline powder, mp 53.8–55.0 °C; TLC (CHCl3/MeOH, 15:1, v/v),
Rf = 0.2; 1H NMR (400 MHz , CDCl3) d 8.21 (d, J = 8.4 Hz, 2H), 7.57
(d, J = 8.4 Hz, 2H), 6.13 (d, J = 8.0 Hz, 1H), 5.24 (s, 1H), 4.17 (m,
1H), 3.91 (s, 2H), 3.42 (t, J = 7.2 Hz, 2H), 2.12 (m, 2H), 1.86 (p,
J = 7.2 Hz, 2H), 1.46 (m, 4H), 1.10–1.30 (m, 12H).
2.79 (t, J = 7.2 Hz, 2H), 2.04 (dt, J = 7.6, 2.8 Hz, 2H), 1.56 (m, 2H),
1.00–1.40 (m, 14H). ESI-MS (CH3OH, relative intensity, %) m/z
410.3 ([M+H]+, 100). Calcd for C21H35N3O5, m/z 409.2 [M].
4.1.2.4. (1R,2R)-2-N-(120-N,N-Dimethylaminododecanoylami-
no)-1-(400-nitrophenyl)-1,3-propandiol (LCL464).
Prepared
from LCL429 and 2 M dimethyl amine in THF solution in 95% yield.
TLC (CHCl3/MeOH/NH4OH, 10:1:0.01, v/v/v), Rf = 0.12; 1H NMR
(CDCl3, 400 MHz) 8.20 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H),
6.34 (d, J = 8.4 Hz, 1H), 5.21 (d, J = 3.2 Hz, 1H), 4.19 (m, 1H), 3.86
(m, 2H), 2.40 (t, J = 7.6 Hz, 2H), 2.31 (s, 6H), 2.12 (t, J = 7.2 Hz,
2H), 1.20–1.50 (m, 18H). ESI-MS (CH3OH, relative intensity, %) m/
z 438.4 ([M+H]+, 100). Calcd for C23H39N3O5, m/z 437.3 [M].
4.1.1.2. (1R,2R)-2-N-(60-Bromohexanoylamino)-1-(400-nitro-
phenyl)-1,3-propandiol (LCL509).
Prepared from 6-bromo-
hexanoyl chloride and (1R,2R)-2-amino-1(40-nitrophenyl)-1,3-
propandiol as shown for LCL429. The crude product was purified
by flash column chromatography (CHCl3/MeOH, 7:1, v/v) to give
the pure product as white microcrystalline powder in 84% yield,
mp 87.1–87.5 °C. TLC (CHCl3/MeOH, 7:1, v/v), Rf = 0.17, 1H NMR
(400 MHz, CDCl3) d 8.17 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H),
6.31 (d, J = 8.8 Hz, 1H), 5.20 (s, 1H), 4.16 (dt, J = 4.8, 3.2 Hz, 1H),
3.80 (d, J = 4.0 Hz, 2H), 3.35 (t, J = 6.8 Hz, 2H), 2.12 (dt, J = 7.2,
6.8 Hz, 2H), 1.77 (p, J = 7.2 Hz, 2H), 1.48 (p, J = 7.6 Hz, 2H), 1.29
(h, J = 7.6 Hz, 2H).
4.1.2.5. (1R,2R)-2-N-[60-(N0-Octylamino)hexanoylamino]-1-(400-
nitrophenyl)-1,3-propandiol
(LCL488).
Prepared
from
LCL509 and octylamine in 32% yield. TLC (CHCl3/MeOH/NH4OH,
4:1:0.01, v/v/v), Rf = 0.23; 1H NMR (CDCl3, 400 MHz) 8.12 (d,
J = 8.4 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 7.8 Hz, 1H),
5.17 (d, J = 3.2 Hz, 1H), 3.79 (m, 1H), 3.68 (m, 2H), 2.84 (m, 4H),
2.15 (t, J = 7.6 Hz, 2H), 1.20–1.80 (m, 18H), 0.87 (t, J = 7.2 Hz, 3H).
ESI-MS (CH3OH, relative intensity, %) m/z 438.3 ([M+H]+, 100).
Calcd for C23H39N3O5, m/z 437.2 [M].
4.1.2. General procedure for the preparation of Class E analogs
Prepared from LCL429 or LCL509 and corresponding amines:
ammonia, methylamine, dimethylamine, imidazol, morpholine or
octylamine and purified by flash column chromatography. Briefly,
to a well-stirred solution of LCL429 or LCL509 (0.156 mmol) in a
mixture of 1.5 N NaOH (4 mL) and THF (8 mL), the corresponding
amine was added at room temperature. The reaction mixture
was kept at rt or under reflux until the reaction was completed
as monitored by TLC (CHCl3/MeOH, 10:1, v/v). The organic layer
was separated, and the aqueous layer was extracted twice with
THF (2 ꢁ 5 mL). The combined organic extracts were dried over
anhydrous Na2SO4 and evaporated under a reduced pressure to
dryness to give crude product. This material was purified by col-
umn chromatography using the suitable solvent systems to give
a pure target compounds as a light yellow oils.
4.1.2.6. (1R,2R)-2-N-(120-N-Methylaminododecanoylamino)-1-
(400-nitrophenyl)-1,3-propandiol (LCL506).
Prepared from
LCL429 and 2 M methylamine in THF solution in 90% yield. TLC
(CHCl3/MeOH/NH4OH, 4:1:0.01, v/v/v), Rf = 0.12; 1H NMR (CD3OD,
400 MHz) 8.18 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 5.17 (d,
J = 3.2 Hz, 1H), 4.19 (m, 1H), 3.76 (dd, J = 10.8, 7.6 Hz, 1H), 3.62
(dd, J = 10.8, 5.6 Hz, 1H), 2.91 (t, J = 8.0 Hz, 2H), 2.65 (s, 3H), 2.09
(t, J = 7.2 Hz, 2H), 1.68 (m, 2H), 1.00–1.42 (m, 16H). ESI-MS
(CH3OH, relative intensity, %) m/z 424.2 ([M+H]+, 100). Calcd for
C22H37N3O5, m/z 423.17 [M].
4.2. Biology
4.1.2.1.
(4000-nitrophenyl)-1,3-propandiol (LCL433).
(1R,2R)-2-N-[120-(100-Imidazol)dodecanoylamino]-1-
Prepared from
4.2.1. Cell culture
LCL429 and imidazol in 33% yield. TLC (CHCl3/MeOH/concd
NH4OH, 10:1:0.01, v/v/v), Rf = 0.15; 1H NMR (400 MHz, CDCl3) d
8.18 (d, J = 8.4 Hz, 2H), 7.78 (s, 1H, Imidazol-H), 7.59 (d,
J = 8.4 Hz, 2H), 7.10 (s, 1H, Imidazol-H), 6.99 (s, 1H, Imidazol-H),
6.50 (d, J = 10 Hz, 1H), 5.26 (d, J = 3.2 Hz, 1H), 4.22 (m, 1H), 4.02
(t, J = 6.8 Hz, 2H), 3.88 (d, J = 4.4 Hz, 2H), 2.08 (t, J = 7.6 Hz, 2H),
1.81 (p, J = 6.8 Hz, 2H), 1.46 (p, J = 7.6 Hz, 2H), 1.08–1.40 (m,
14H). ESI-MS (CH3OH, relative intensity, %) m/z 461.3 ([M+H]+,
100). Calcd for C24H36N4O5, m/z 460.27 [M].
MCF7 cells (breast adenocarcinoma, pleural effusion) were pur-
chased from American Type Culture Collection (ATCC, Rockville,
MD, USA) and grown in RPMI 1640 media (Life Technologies,
Inc.), supplemented with 10% FBS, 100 unit/mL penicillin, and
100 lg/mL streptomycin and maintained under standard incubator
conditions (humidified atmosphere 95% air, 5% CO2 at 37 °C). Cells
in the exponential growth phase were harvested from the cultures
and used in all of experiments.
4.2.2. In vitro acid ceramidase activity assay
4.1.2.2. (1R,2R)-2-N-(120-(100-Morpholine)dodecanoylamino]-1-
Detection of the total cellular ACDase enzymatic activity was
performed according to the literature.8,24 Briefly, MCF7 cells
were lysed under acidic condition [pH 4.5, contained 50 mM so-
dium acetate, 5 mM Magnesium chloride, 1 mM EDTA and
0.5%Triton X-100] ; and protein level was determined using
BCA protein assay kit (Pierce, Rockford, IL). This cell lysate was
used as ACDase enzyme resource. Equal amounts of N-
[9,10-3H] D-e-C16 Cer, enzymatic substrate, were mixed with
(4000-nitrophenyl)-1,3-propandiol (LCL449).
Prepared from
LCL429 and morpholine in 60% yield. TLC (CHCl3/MeOH/concd
NH4OH, 10:1:0.01, v/v/v). Rf = 0.12, 1H NMR (CDCl3, 400 MHz)
8.20 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 6.21 (d, J = 8.4 Hz,
1H), 5.22 (d, J = 3.2 Hz, 1H), 4.18 (m, 1H), 3.85 (t, J = 4.8 Hz, 2H),
3.72 (t, J = 4.8 Hz, 4H), 2.46 (abroad, 6H), 2.32 (t, J = 8.0 Hz, 2H),
2.10 (dt, J = 7.2, 4.0 Hz, 2H), 0.92–1.50 (m, 16H). ESI-MS (CH3OH,
relative intensity, %) m/z 480.3 ([M+H]+, 100). Calcd for
C25H41N3O6, m/z 479.3 [M].
100 lL 0.2% Triton X-100 and 100 lL 0.4% Cholate and dried
down under the nitrogen condition. All the testing compounds
were dissolved in absolute ethanol to prepare a stock solution.
Calculated amount of the compounds were added to the sub-
strate in the buffer to get a certain concentration in the total
4.1.2.3. (1R,2R)-2-N-(120-Aminododecanoylamino)-1-(400-nitro-
phenyl)-1,3-propandiol (LCL463).
Prepared from LCL429 and
ammonia in 40% yield as a white microcrystalline powder, mp 46–
48 °C. TLC (CHCl3/MeOH/concd NH4OH, 1:1:0.01, v/v/v), Rf = 0.12;
of 200
lowed by addition of 30
and sonication to get homogenous solution and was continue
by the addition of 100 L acidic assay buffer containing 0.2 M
acetic acid, 0.2 M sodium acetate and 0.5% Triton X-100, fol-
l
L volume. The mixture was dried under nitrogen fol-
lL of DI water to each tube, vortexing
1
H NMR (CD3OD, 400 MHz) 8.13 (d, J = 8.8 Hz, 2H), 7.61 (d,
J = 8.8 Hz, 2H), 5.10 (d, J = 2.8 Hz, 1H), 4.15 (m, 1H), 3.73 (dd,
J = 10.8, 7.6 Hz, 1H), 3.53 (dd, J = 10.8, 6.0 Hz, 1H), 3.27 (m, 2H),
l

A. Bai et al. / Bioorg. Med. Chem. 17 (2009) 1840–1848
1847
lowed by equal amount of MCF7 cell lysate and lysate buffer to
bring up the total reaction volume to 200 L. The final reaction
4.2.7. Apoptotic assay
l
Apoptotic cell death was established by the Caspase 3/7 activity.
pH value was around 4.2. The reaction mixture was kept at
37 °C for an hour and stopped by adding Dole’s alkaline. Extrac-
tion of the released [3H]palmitic acid, the product of the hydro-
lytic activity of ACDase, proceeded as described.8,24 Quantitation
of the radioactivity was performed on LS 6500 multi-purpose
scintillation counter (Beckman Coulter Inc. Fullerton, CA). Results
were calculated from the duplicated experiments and shown as
% of control.
Briefly, MCF7 cells were seeded at density of 5 ꢁ 103/50
lL/well
overnight in 96 well plates (Corning, Acton, MA, USA). Then cells
were treated with vehicle or diluted LCL464 at indicated concen-
trations in another 50 lL medium. After 8 h and 24 h incubation,
Caspases 3/7 activities were measured using Apo-ONE Homoge-
neous Caspase 3/7 Assay Kit according to the manufacturer’s
instructions (Promega). Fluorescence was measured by a Fluostar
dual Xuorescence/absorbance plate reader (BMG Laboratories,
Durham, NC, USA) with 485 nm excitation and 520 nm Emission.
Data represent mean SD.
4.2.3. Cellular activity of acid ceramidase
MCF7 cells were seeded in 100 mm dishes overnight before
being treated with the drugs at the indicated concentrations. Cells
were then lifted by a gently scraping and lysed using acidic lyses
buffer; the same operation was conducted as in vitro assay. Protein
concentration was qualified using BCA protein assay kit (Pierce,
Rockford, IL). The measurement of ACDase activity was performed
as described under Section 4.2.4. Results were calculated from the
duplicated experiments and shown as % of control.
4.2.8. Cell viability assay
The MTT assay was used to quantify viable MCF7 cells
according to the literature.18 Briefly, MCF7 cells were seeded at
a density of 5 ꢁ 103 cells/100
lL/well on the 96 well-plate, and
cultured overnight to let the cell fully attached before the assay.
Then, 100
of either DMSO (control) or various concentrations (0.78–
100 M) of the test compounds in DMSO solution (<0.1%) were
lL of complete medium containing the same amount
l
4.2.4. LC–MS analysis of endogenous Cn-Cer and Sph
added to the cells. After 48-h incubation, the supernatants were
removed, and the MTT assay was performed according to the
protocol. Cell viability was expressed as a percentage of control
calculated by the formula Ad/Ac ꢁ 100, where Ad and Ac represent
the absorbance of drug-treated and untreated control cells,
respectively. For this assay, each dose was processed with 5 time
replicates, and the bar graphs shown two independent experi-
ments. Results are expressed as % of control cells. The IC50 rep-
resents the drug concentration resulting in 50% growth
inhibition compared to the control cells.
Advanced analyses of Cer species and Sph were performed by
the Lipidomics Core at MUSC on a Thermo Finnigan TSQ 7000, tri-
ple-stage quadrupole mass spectrometer operating in a Multiple
Reaction Monitoring (MRM) positive ionization mode as de-
scribed.3,17 Final results were expressed as the level of the particu-
lar SPLs/phospholipids (Pi) determined from the Bligh and Dyer
lipid extract and expressed as SPLs/Pi (pmol/nmol).
4.2.5. Lysosomal stability assay
Lysosomal stability was measured using the Lysotracker Red
dye DND-99 (Molecular probes, Oregon).13 Briefly, MCF7 cells were
seeded 5 ꢁ 105/well in 6 well-plate overnight. Media were re-
moved completely, and a fresh 2% FBS media containing the same
amount of either DMSO or the test compounds in DMSO solution
were added and incubated at 37 °C for 1 and 5 h before adding
200 nM LTR for 30 min at 37 °C. After treatments, cells were lifted
with trypsin, washed twice with PBS, and resuspended in 0.5 mL
PBS. LTR fluorescence was measured using FACS analysis (564–
606 nm). Decrease in the fluorescence intensity corresponded to
the increase in the lysosomal pH value, and a minimum of
10,000 events were scored for each sample. Each dose was tripli-
cated. Results were expressed as % Control.
Acknowledgements
Financial support was provided by NCI Grant No. IPO1-
CA097132. Special acknowledgement is for NCRR Grant No.
CO6RR018823 providing laboratory space for Lipidomics Shared
Resource in the CRI building of MUSC. We thank Dr. Jennie Ariail
for editorial assistance.
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