Targeting (cellular) lysosomal acid ceramidase by B13: Design, synthesis and evaluation of novel DMG-B13 ester prodrugs

Article abstract of DOI:10.1016/j.bmc.2014.10.025

Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1 h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.

Full text of DOI:10.1016/j.bmc.2014.10.025

Bioorganic & Medicinal Chemistry 22 (2014) 6933–6944
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Targeting (cellular) lysosomal acid ceramidase by B13: Design,
synthesis and evaluation of novel DMG-B13 ester prodrugs
Aiping Bai a,b, Zdzislaw M. Szulc a,b, Jacek Bielawski a,b, Jason S. Pierce a,b, Barbara Rembiesa a,b
,
Silva Terzieva a,b, Cungui Mao d, Ruijuan Xu d, Bill Wu a, Christopher J. Clarke d, Benjamin Newcomb d,
a,b,
Xiang Liu c, James Norris c, Yusuf A. Hannun d, , Alicja Bielawska
⇑
⇑
a Department of Biochemistry & Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA
b Lipidomics Facility, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA
c Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA
d Department of Medicine and the Stony Brook Cancer Center at Stony Brook University, Stony Brook, NY 11794, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a
moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its
N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13,
LCL522 (3-O-DMG-B13ꢀHCl) and LCL596 (1-O-DMG-B13ꢀHCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-
B13ꢀ2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification
of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The
early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their
lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80–97% Control/1 h) by
DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.
Received 9 January 2014
Revised 6 October 2014
Accepted 15 October 2014
Available online 22 October 2014
Keywords:
B13
DMG-B13 prodrugs
Inhibitors
Acid ceramidase
Lysosomes
Cancer
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
lysosomes.3,4 The role of this enzyme is to hydrolyze lysosomal
Cers, thus providing Sph, which in turn can serve as a substrate
Bioactive sphingolipid (SPL) metabolites, specifically ceramide
species (Cn-Cer), sphingosine (Sph), and sphingosine 1-phosphate
(S1P), are increasingly recognized for their important roles as sig-
naling molecules involved in regulation of survival, proliferation,
and cell death.1,2 Acid ceramidase (ACDase, ASAH1) is a 55 kDa
polypeptide that is processed, under the normal growth conditions,
of Sph kinases to synthesize S1P or as a substrate for ceramide
synthases for the regeneration of Cers. Nonetheless, a cell type
specific nuclear expression of ASAH1 has been reported recently
identifying its key role for nuclear lipid metabolism in regulating
gene transcription.5 Thus, ACDase represents a new target for
cancer therapy due to its importance in regulating Cer-Sph-S1P
inter-metabolism and nuclear gene transcription.5–13 Moreover,
elevation of ACDase has been observed in many cancer cell lines
and tumor tissues, particularly in the late stage of tumors, as
compared to normal conditions.8–10,12 Inhibition of cellular ACDase
sensitizes tumors to therapies and advances tumor cell killing. It
may therefore represent an efficient and promising strategy for
cancer treatment.
Over the last decade, our group has been focusing on develop-
ment of ACDase inhibitors based on the structural and stereochem-
ical modifications of ceramide. We have synthesized a set of
lipophilic phenyl-N-acyl-amino-alcohols (aromatic analogs of
Cer) that acts as anticancer agents, and we have demonstrated that
the active analogs increased endogenous Cer and decreased
into the
a-and b-subunits (a-ACDase and b-ACDase) within the
Abbreviations: B13, (1R,2R)-2-(tetradecanoylamino)-1-(40-nitrophenyl)-1,3-
propandiol; DMG, N,N-dimethyl glycine; LCL521, 1,3-O,O-DMG-B13 dihydrochlo-
ride; LCL522, 3-O-DMG-B13 hydrochloride; LCL596, 1-O-DMG-B13 hydrochloride;
CX, stable mono-DMG-B13; ACDase, acid ceramidase; NCDase, neutral ceramidase;
AlkCDase, alkaline ceramidase; ASM, acid sphingomyelinase; Sph, sphingosine; Cer,
ceramide; Cn-Cer, N-acyl-ceramide species; S1P, sphingosine 1-phosphate; dhSph,
dihydrosphingosine; dhCer, dihydroceramide; DCI, 1,10-carbonyldiimidazole; DCM,
dichloromethane; TEA, triethyl amine; DIPEA, diisopropylethylamine.
⇑
Corresponding authors. Tel.: +1 631 444 8067; fax: +1 631 444 1719 (Y.A.H.);
tel.: +1 843 792 0273; fax: +1 843 792 1627 (A.B.).
E-mail addresses: yusuf.hannun@abumed.org (Y.A. Hannun), bielawsk@musc.
edu (A. Bielawska).
http://dx.doi.org/10.1016/j.bmc.2014.10.025
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

6934
A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
endogenous Sph via inhibition of CDases.14–16 Among them, B13,
(1R,2R)-2-(tetradecanoylamino)-1-(40-nitrophenyl-1,3-propandiol),
which induces apoptosis in many cancer cell lines, functions as an
ACDase inhibitor.16 This discovery made B13 a potential key
compound to develop ACDase-related cancer research.11,16–19
However, B13, as a neutral lipophilic molecule, may not be able
to specifically and efficiently reach and accumulate in the
lysosomes where mainly ACDase resides.3,4 Our previously pub-
lished results displayed a dual action of B13 on cellular Cer and
Sph, with an increase of Cer and decrease of Sph at high doses,
and with no effect on Cer and no effect or a small increase of Sph
especially during early treatments with low doses.19 These results
suggest the possible effects of B13 on unidentified proteins that
may transport B13 to the lysosomes; action of B13 on ACDase in
compartments other than lysosomes, perhaps in the nucleus where
the presence of this enzyme and its inhibition by B13 has been
reported;5 or possible B13 effects on other enzymes, perhaps on
other CDases, ceramide syntheses (CerSs) or acyl-transferases
(ATases), that regulate Cer and Sph levels independently of ACDase,
as previously suggested.19
nuclear compartment where ASAH1 was detected at low levels
only.5 Results are compared to the action of B13.
2. Results and discussion
2.1. Evaluation of B13 as ACDase inhibitor
Our in vitro results showed a dose-dependent effect of B13 on
ACDase activity when an acidic MCF7 cell lysate was used as an
enzymatic source (Y = 2.267–0.02047X, R2 = 0.9627), IC50 27.7
l
M,
M B13 almost completely inhibited activity of this
enzyme (95%).
Fig. 1A). 50
l
NCDase and AlkCDase activity results showed no inhibitory
effects of B13 on those enzymes at concentrations ranging from
1 to 50
to 250
l
M (Fig. 1B). In addition, our results indicate that B13, up
l
M, did not inhibit those enzymes (not shown), thus
indicating the specificity of B13 to ACDase and corroborating our
previously published results.16,27 B13 action on NCDase seems to
contradict recently published results26 that show almost equal
inhibitory effects of B13 on the acid and the neutral CDases
Several structurally varied analogs of B13 have been developed
so far.11–13,19–26 Our first choice for lysosomal analogs of B13
was based on its amide group modifications that resulted in
(1R,2R)-2-(N-tetradecylamino)-1-(40-nitrophenyl)-1,3-propandiol
hydrochloride (LCL204), and (1R,2R)-2-N-(120-N,N-dimethylami-
nododecanoylamino)-1-(400-nitrophenyl)-1,3-propandiol (LCL464),
Scheme 1.11,13,19 These analogs increased cytotoxicity of B13,
decreased cellular Sph and specifically increased C14-, C16-, and
(ꢁ30%/80
In MCF7 cells, there was little change in the cellular activity of
ACDase except at high concentrations (ꢁ50% C inhibition/50 M/
5 h and 20–30% inhibition/50 M, 24 h), as shown in Figure 1C.
These results were paralleled by decreased level of -ACDase
protein (ꢁ40% C/50 M/5 h, Western blot, not shown) suggesting
that B13 may affect ACDase stability non-specifically.
lM).
l
l
a
l
Functionally, there was a moderate effect of B13 on cellular Sph
(Fig. 1D). A decrease in Sph levels was observed only for the high
C18-Cer. Unfortunately, LCL204 also caused induction of lysosomal
destabilization, resulting in a proteolytic degradation of ACDase
very early in the process.12,19 Additional structural modifications
toward B13 have been carried out by others.25,26 Some analogs
showed improved effects on ACDase inhibition in an in vitro
assay as compared to B13. Interestingly, in vitro results of B13
concentration (30 lM or higher). If the bulk of Sph derives from
the action of ACDase, these results indicate relatively poor
effectiveness of B13 on ACDase in MCF7 cells. Furthermore, the
strongest effects on Sph by B13 at 5 h were partially recovered
after
inhibition/5 h to 40% inhibition/24 h as observed for 50
B13 applied at a low concentration had no effects on cellular Cer
a
prolonged incubation with this compound (70%
action on ACDase and neutral CDase (NCDase) showed
a
lM B13).
similar and low inhibition on both isoforms of CDase.26 Thus,
the current work also reevaluates B13 specificity to ACDase
inhibition.
whereas 50 lM B13 increased cellular Cer to 140% after 1 h of
treatment (data not shown) and to ꢁ270% after 24 h.19 However,
this high dose treatment was beyond the lethal dose for 24 h
treatments.11
The goal of the present study is the ‘lysosomal delivery’ of B13
via its DMG-modified prodrugs, Scheme 2, which are expected to
cause B13 accumulation in lysosomes, given the ionizable amines,
and inhibition of lysosomal ACDase. This approach represents a
novel concept for targeted drug release into a specific cellular
compartment.
In this manuscript we discuss design and present synthesis of
DMG-B13 prodrugs. Cellular metabolism of DMG-B13 prodrugs,
specificity to cellular ACDase, and early effects on Sph-Cer balance
and lysosomal targeting are studied in MCF7 cells where ASAH1
has a predominant cytoplasmic localization in contrary to the
In summary, B13 inhibited ACDase activity both in vitro and in
cells but only at the high concentrations, affecting both Sph and
Cer. Moreover, the effects of 50 lM B13 on cellular Cer were not
specific as to the Cer species, contrary to the effects of LCL204
and LCL464, our previously reported lysosomotropic inhibitors of
ACDase.12,19
Thus, facts of a high expression of the lysosomal ACDase versus
relatively low expression of the nuclear ACDase5 and high levels of
B13 in the nuclear fraction of MCF7 cells (data not shown) may
Scheme 1. Chemical structures of previously developed lysosomotropic B13 analogs.

A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
6935
Scheme 2. Synthetic outline for the preparation of DMG-B13 prodrugs Reagents and conditions: (i) N,N-dimethyl glycine: 1,10-carbonyldiimidazole: B13 = 1:1:1.1, CH2Cl2, rt;
(ii) N,N-dimethyl glycine: 1,10-carbonyldiimidazole: B13 = 10:10:1, CH2Cl2, rt; (iii) B13: trityl chloride = 1:0.9, DMAP (Cat.), CH2Cl2, rt; (iv) N,N-dimethyl glycine: 1,10-
carbonyldiimidazole: trityl-B13 = 1:1:0.9, rt; (v) 80% acetic acid or BF3 in MeOH solution, CH2Cl2, rt; (vi) 2 M HCl in ethyl ether, 4 °C.
160.0
140.0
NCDase
A
B
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
AlkCDase
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
*
*
10
20
30
40
50
0.0
2.0
B13 (μM)
10.0
50.0
B13 (μM)
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
1h
5h
24h
2h
5h
24h
D
C
*
*
*
*
*
*
*
*
1.0
5.0
10.0
20.0
30.0
40.0
50.0
2.5
10
25
50
B13 (μM)
B13 (μM)
Figure 1. Effects of B13 on CDases’ activities in an in vitro assays and at the cellular levels in MCF7 cells. All results are presented as mean SD of duplicates. ⁄p value <0.05 (vs
Control). (A) ACDase activity in an in vitro assay. Results are expressed as pmol of released [3H] palmitic acid/mg protein/h, and are presented as % Control. (B) NCDase and
AlkCDase activities, in an in vitro assays. NCDase results are expressed as a rate of the conversion of the fluorescence released from NBD-C12-Cer to NBD-dodecanoic acid, and
are presented as % Control. AlkCDase results are expressed as pmol of cellular Sph released from D-e-C18:1-Cer using microsomes of HeLa cells overexpressing ACER2, and are
presented as % Control. (C) Cellular ACDase activity assay. After treatment with B13 or vehicle control, as was described in the text, cells were isolated and ACDase activity was
measured by in vitro assay as described under A. Results are expressed as pmol of released [3H] palmitic acid/mg protein/h, and are presented as % Control. (D) Release of
cellular Sph by B13. Results are expressed as Sph (pmol)/nmol Lipid Phosphate, and are presented as % Control.

6936
A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
suggest that B13 may rather cause a low efficiency effect on the
nuclear form of this enzyme.
Therefore, all these results prompted us to improve the cellular
action of B13, that is, increase its delivery and compartmental
targeting to the lysosomes that may intensify B13’s efficacy.
of B13 from LCL522 and 596 was observed up to 2–5 h when the
released B13 had reached a plateau (Fig. 2A). Reciprocally, the
mono-DMG B13 prodrugs were decreased in a time-dependent
manner starting from 0.5 h and were almost completely eliminated
after 5–10 h of treatment.
Metabolism of di-DMG-B13, LCL521 was different from that of
the mono-DMG analogs, as shown in Fig. 2B. LCL521 (10 lM)
2.2. Principles of design and synthesis of the lysosomal B13-
prodrugs through DMG modification
was partially and slowly metabolized to B13 and unexpectedly to
a mono-DMG intermediate, a metabolite dubbed CX (30%/15 min,
of the total mixture), whose presence was noticed even for the
longer treatments (Fig. 21). The level of cellular LCL521 was almost
stable up to 2 h, followed by its slow decrease; its presence was
noticed for 24 h of treatment (27% of total mixture). B13 was
increased slowly in a time-dependent manner, reaching 32% of
The prodrug approach is commonly used to improve the phys-
icochemical and targeting properties of the parent drug. In general,
a ‘prodrug’ should represent a pharmacologically inactive chemical
that can be converted into an active drug through metabolizing
enzymes in vivo. Addition of a metabolically labile group should
ultimately result in improvement of pharmacokinetics, safety,
and therapeutic efficacy of the active compound.28–32
Knowing that the whole B13 structure is essential for its inhib-
itory function, we decided to apply dimethyl glycine (DMG) as a
lysosomotropic modifier (Scheme 2). The DMG-approach, previ-
ously reported for the lysosomal modification of other classes of
bioactive alcohols, should release B13 from its hydrolysable DMG
esters, thus improve B13 lysosomal targeting.33,34
total mixture/10 h. CX represented
a stable mono-DMG-B13
metabolite that was at plateau over the treatment time
(50–70 units/0.25–10 h)). LCL521 was a major component of the
mixture up to 5 h of treatment. For 10 h of treatment, all compo-
nents of the mixture (LCL521, CX and B13) were at similar levels
(ꢁ70–90 unit). CX represented
a major metabolite of 1 lM
LCL521 over the treatment time up to 10 h (ꢁ80% of total mixture,
Fig. 2B2).
DMG is a non-protein amino acid naturally found in animal and
plant cell, which is produced as an intermediate in the metabolism
of choline to glycine. DMG is formed from trimethylglycine
(betaine) and is absorbed by the small intestine and transported
by portal circulation to the liver. DMG is not toxic except at very
high doses; the LD50 of its hydrochloride salt is 3.9 g/kg in rats.
DMG has demonstrated the ability to enhance immune system
function and to advance neurological function, as well as antiviral
and antibacterial defenses.35,36
In addition, DMG can form a hydrochloride salt, thus improving
B13’s solubility and its prodrug’s stability. Scheme 2 depicts the
synthetic approach and chemical structures of DMG-B13 prodrugs.
The B13 molecule contains two hydroxyl groups, the primary
(3-OH) and the secondary (1-OH), providing possibilities for the
formation of three distinct esters. Three different reaction
sequences were then developed, and the following hydrochloride
salts of B13 prodrugs were efficiently synthesized: 1,3-O,
O-DMG-B13 (LCL521), 3-O-DMG-B13 (LCL522) and 1-O-DMG B13
(LCL596). We produced pure LCL521 in 60% yields by coupling of
B13 with excess of DMG in the presence of DCI and TEA or DIPEA
in anhydrous DCM, after the treatment of the formed free base of
1,3-O, O-DMG-B13 with HCl. LCL522 was prepared in a similar
fashion in 52% yield, but the proportions of key reagents were
modified to drive esterification only on the primary hydroxyl
group. In order to synthesize LCL596, a three-step procedure was
developed. First, the 3-OH group of B13 was protected by the trityl
group; then the DMG pro moiety was introduced into the 1-OH
group. The protected 3-O-trityl mono ester of B13 was subjected
to BF3 ether solution in anhydrous methanol to give a free base
of LCL596. Final treatment of the formed 1-O-DMG-B13 base with
HCl produced LCL596 in 36% overall yield.
2.3.2. Total cellular uptake of LCL521 as compared to the mono-
DMG-B13 prodrugs
Total cellular uptake of 10 lM LCL522, 596 and 521, represent-
ing the sum of all cellular components, is shown in Fig. 2C. All pro-
drugs were very quickly taken up by the cells, with similar levels
for the mono-DMG prodrugs but much higher levels for LCL521,
the di-DMG-B13. Cellular uptake of LCL522 and LCL596 was at a
plateau over the experimental time (15 min-24 h); LCL521 showed
a time-dependent small increase in its cellular uptake up to 2 h,
remained at plateau up to 10 h, and then decreased at 24 h of treat-
ment. Those results may indicate stronger cellular permeability
and stability for LCL521, as well as greater stability of its major
metabolite CX.
As shown in Figure 2D, the cellular level of B13 released from all
10 lM DMG- B13 prodrugs was at a comparable level of 10 lM B13
treatment. During early treatments (1–2 h), cellular levels of B13
from direct deliveries were higher than levels generated from
prodrugs (e.g., B13/B13 (LCL521)/B13 (LCL522/B13 (LCL596);
50:25:25:25/0.25 h). During later treatments (5 and 10 h), cellular
levels of B13 from direct delivery and B13 released from LCL521
were at comparable levels.
The results indicate that B13 modification by introduction of
DMG ester moieties did not improve the overall cellular delivery
of B13; however the following findings suggest that cellular (lyso-
somal) effectiveness of B13 was markedly improved.
2.3.3. Cellular transformation of LCL521 to metabolite CX
Comparative LC–MS/MS results of metabolite CX and the
mono-DMG-B13 compounds, LCL522 and LCL596, showed
identical fragmentation patterns recorded under the same applied
conditions (mass transition: 508.1–405.1; loss of the DMG moiety,
as well as chromatographic retention time: 2.53 min), which
suggests that the cellular CX may represent a mixture of the
mono-DMG-B13 regioisomers.
Although, as shown in Figure 2A, both LCL522 and LCL596 were
quickly and almost equally metabolized to B13 (ꢁ90–95% for 5 h of
treatment), the formation of a three-component mixture (LCL521,
CX and B13) was always observed for LCL521 treatments with
metabolite CX being present even for 24 h time point (Fig. 2B1
and 2B2).
As expected, the introduction of the DMG moiety into B13
markedly improved B13’s water solubility. For instance, the
solubility of LCL521 was ꢁ430 mg/mL at 25 °C versus only
8 lg/mL for B13 under the same conditions.
2.3. Metabolism and uptake of DMG-B13 prodrugs in MCF7 cells
2.3.1. Cellular metabolism of the mono- and di-DMG-B13
prodrugs
Identification of the major and stable CX metabolite of LCL521
will require further investigation.
Next, the ability of LCL521, 522 and 596 to be metabolized to
B13 was evaluated (Fig. 2A and Fig. 2B). A time-dependent increase

A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
6937
160.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
LCL521
B13(LCL521)
CX
A
B1
120.0
80.0
LCL522
40.0
B13(LCL522)
LCL596
B13(LCL596)
0.0
0.0
5.0
10.0
Time (h)
15.0
20.0
25.0
0.0
5.0
10.0
Time (h)
15.0
20.0
25.0
400.0
320.0
240.0
160.0
80.0
20.0
16.0
12.0
8.0
LCL521
LCL521
B2
C
LCL522
LCL596
B13
B13(LCL521)
CX
4.0
0.0
0.0
0.0
5.0
10.0
15.0
20.0
25.0
0.0
5.0
10.0
15.0
20.0
25.0
Time (h)
Time (h)
100.0
D
80.0
60.0
40.0
20.0
0.0
*
B13
B13(LCL521)
B13(LCL522)
B13(LCL596)
0.0
5.0
10.0
15.0
20.0
25.0
Time (h)
Figure 2. Metabolism and uptake of DMG-B13 prodrugs in MCF7 cells, time course. MCF7 cells were treated with either vehicle or 10
and 24 h. Cellular levels of each compound were quantified by LC–MS/MS analysis of lipid extracts from cell pellets. Results are presented as mean SD of duplicates. (A)
Metabolism of 10 M mono-DMG B13-prodrugs, LCL522 and LCL596. Results are expressed as pmol of cellular LCL522, LCl596 and B13/ nmol of Lipid Phosphate. (B)
Metabolism of di-DMG B13-prodrug, LCL521. B1. 10 M LCL521; B2. 1 M LCL521. Results are expressed as pmol of LCL521, CX and B13/nmol Lipid Phosphate. (C) Total
cellular uptake of 10 M DMG-B13 prodrugs. Results represent the sum of all cellular components of DMG-B13 prodrugs or B13 itself and are expressed as levels of
lM inhibitors for 15 min, 1, 2, 5, 10, 15
l
l
l
l
compounds (pmol)/nmol Lipid Phosphate. (D) Comparison between the cellular levels of B13 released from 10
Results are presented as level of B13 (pmol)/nmol Lipid Phosphate.
lM DMG-B13 prodrugs and 10 lM B13 applied directly.
2.4. Evaluation of DMG-B13 prodrugs as ACDase inhibitors
All 10 lM DMG-B13 prodrugs caused early (1 h) inhibitory
effects on the cellular ACDase activity as shown in Figure 3B.
LCL521 acted as the most potent inhibitor, followed by LCL522
and LCL596, whereas B13 had only a small inhibitory effect.
2.4.1. Effects of DMG-B13 prodrugs on ACDases activities
The in vitro assay was performed with the free bases of 20 lM
and 50
drugs showed significantly lower inhibitory effects as compared to
B13, especially for 50 M concentration demonstrating an impor-
tance of the free hydroxyl groups of B13 for enzymatic inhibition
(Fig. 3A and Fig. 1A). In summary, the highest inhibition was
l
M LCL596, LCL522, and LCL521 (Fig. 3A). As expected, pro-
Interestingly, LCL521 applied at a lower concentration, 1
showed an effect similar to 10 M treatment, suggesting a high
efficiency on this enzyme inhibition.
The early effects of 10 M DMG-B13 prodrugs on ACDase (as
detected by the Western blot via its cleaved -subunit, -ACDase)
were investigated next (Fig. 3C). Results were compared to the
effects of 10 M B13 and 10 M LCL204, our previous lysosomo-
tropic inhibitor (Scheme 1). LCL521 caused a time-dependent
decrease of the -ACDase form, whereas LCL522 and B13 had no
effects, or rather slightly increased -ACDase levels. LCL204 caused
also a time-dependent decrease of the -form, however, this effect
lM,
l
l
l
a
a
observed for B13 (IC50/ꢁ30
lM, Fig. 1A), whereas the other com-
pounds had moderate effects only.
l
l
2.4.2. Effects of DMG-B13 prodrugs on cellular ACDase activity
We evaluated effects of these compounds on the cellular activity
of ACDase (Fig. 3B) and ACDase protein (Fig. 3C) in MCF7 cells.
a
a
a

6938
A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
120.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
20μM
A
B
100.0
80.0
60.0
40.0
20.0
0.0
50μM
*
*
*
*
*
*
*, **
*, **
140.0
1h
5h
LCL521
Ctrl 1h 5h
LCL522
1h 5h 1h 5h 1h 5h
B13
LCL204
C2
C1
α−ACDase
120.0
100.0
80.0
60.0
40.0
20.0
0.0
Actin
Figure 3. Effects of DMG-B13 prodrugs on ACDase activity, in an in vitro assay, and in MCF7 cells. (A) ACDase activity in an in vitro assay. Results are expressed as pmol of
released [3H] palmitic acid/mg protein/h, and presented as % Control. Results are presented as mean SD of duplicates; ⁄p value <0.05 (vs control). (B) Cellular ACDase activity
assay. After treatment with inhibitors or vehicle control for 1 h, as was described in the text, cells were isolated and ACDase activity was measured through in vitro assay as
described under A. Results are expressed as pmol of released [3H] palmitic acid/mg protein/h, and presented as % Control. Results are presented as mean SD of
quadruplicates; ⁄p value <0.05 (vs control), ⁄⁄p value <0.05 (vs B13). (C) ACDase protein level. MCF7 cells were treated with either 10
for 1 h and 5 h. ACDase protein expression was visualized by Western blot as described. The density of the
were expressed as % Control. C1. Western blots C2. Fluorescence densities normalized to the actin ⁄p value <0.05 (vs Control).
l
M DMG-B13 prodrugs, B13 or LCL204
a
-ACDase protein bends was normalized to the actin, and results
was much lower and delayed comparing to the action of LCL521.
Interestingly, 1 M LCL521 did not affect cellular ACDase protein
(not shown). These results suggest differences among the cellular
action of B13, the mono- and the di- DMG-B13 prodrugs and
an accelerated response of ACDase to LCL521 in comparison to
LCL204.
LCL522 and 596 (ꢁ4–6 pmols) was accompanied by an expected
small increase of Cer, (ꢁ10 pmol). Whereas a fast and almost com-
plete loss of cellular Sph by 10 M LCL521 (9 pmol) accompanied
l
l
by increase of Cer (40 pmol) suggested a more profound action
on cellular ACDase, it can also suggest a possible additional effect
on other Cer related enzymes. Our results on the regulation of
cellular key SPLs, Sph and Cer, showed a higher effect of the di-
DMG-B13 prodrug over the mono-DMG-B13 prodrugs, which
may require a higher concentration to reach efficiency of LCL521.
Most importantly, these prodrugs showed an appropriate correla-
tion on the cellular Sph-Cer balance.
In summary, corresponding results on the inhibition of cellular
ACDase activity and decrease of Sph proved inhibitory effects of
DMG-B13 prodrugs on cellular ACDase. LCL521 applied at a higher
concentration, similarly to LCL204, affected also ACDase protein
Importantly,
cellular ACDase activity, whereas 10
decreased affect on the -form of this enzyme.
1
lM
LCL521 acted as
a potent inhibitor of
l
M LCL521 had an additional,
a
2.4.3. Effects of DMG-B13 prodrugs on the cellular Sph-Cer
balance
The early effects (1 h) on cellular Sph-Cer balance are shown in
Fig. 4A and B. Unlike B13, a very strong decrease of Sph (ꢁ97–60%
C) was observed after treatments with all DMG-B13 prodrugs.
LCL521 was the most effective compound on decreasing cellular
(decrease of the cleaved a- form of this enzyme).
Sph (97% C/10 lM and 80% C/1 lM), Figure 4A. However, cellular
Sph was almost completely recovered (5 h) after treatments with
the mono-DMG-B13 prodrugs, whereas LCL521 maintained
2.4.4. Specificity of DMG-B13 prodrugs to the cellular ACDase
Results presented in the previous section showed the DMG-B13
prodrugs’ action on the cellular ACDase but did not prove their
specificity to this enzyme. For this reason, effects of a DMG-B13
prodrug, especially LCL521, and B13 on the cellular neutral and
alkaline CDases, in addition to ACDase, were investigated as well.
The increase of the cellular levels of Sph in response to the overex-
pression of NCDase (ASAH2) or AlkCDase (ACER2, one of ACERs)
was used as the readout of cellular activity of these two enzymes.
To over express ASAH2 or ACER2, we used an inducible expression
system (TET-ON) in which overexpression is ideally induced by
tetracycline (Tet), but not by its vehicle ethanol (Et) as described.42
suppression (95% C/10
The mono-DMG-B13 prodrugs (10
increase (100% C-105% C) in cellular Cer (Fig. 4B), nor effects on
specific Cn-Cer species (not shown). On the other hand, 10
l
M and 50%/1
lM), results not shown.
l
M) showed almost no
lM
LCL521 caused an increase in cellular Cer (130%/1 h) with the pref-
erence to C16-Cer (not shown). Furthermore, a correlation between
decrease of Sph and increase of Cer can be found if results are
expressed in absolute values (pmols). Control samples (1 ꢂ 10*6
MCF7 cells) usually contain ꢁ10 times more Cer (140 pmols) than
Sph (ꢁ10 pmols). Summarizing results, a decrease of Sph by

A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
6939
120.0
100.0
80.0
60.0
40.0
20.0
0.0
0.6
Vector
ASAH2
ACER2
A
A
0.5
0.4
*,**
0.3
0.2
0.1
0.0
*
*,**
*,**
l
1
1
3
o
2
2
5
1
B
n
5
a
L
L
h
C
C
L
M
μ
t
L
E
0
M
M
μ
1
μ
1
0
1
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
50.0
40.0
30.0
20.0
10.0
0.0
B
ASAH2
ACER2
B
l
1
1
3
o
2
2
5
1
B
n
5
a
L
L
C
h
t
C
L
L
μM
E
0
M
M
μ
1
μ
1
0
1
Figure 4. Effects of DMG-B13 prodrugs on the cellular Sph-Cer balance. MCF7 cells
were treated with B13, B13 prodrugs or vehicle control for 1 h as was described in
the text. Cell pellets were isolated, lipids were extracted and cellular levels of Sph
and Cer were measured by LC–MS/MS approach. (A) Effect on cellular Sph. Results
are expressed as pmol Sph/nmol Lipid Phosphate, and are presented as % Control.
All results are presented as mean SD of duplicates, ⁄p value <0.001 (vs Control);
⁄⁄p value <0.001 (vs B13). (B) Effect on cellular Cer. Results are expressed as pmol
total Cer/nmol Lipid Phosphate, and are presented as % Control. All results are
presented as mean SD of duplicates, ⁄p value <0.05 (vs Control).
Figure 5. Specificity of DMG-B13 prodrugs to the cellular ACDase. Effects on the
AlkCDase and the NCDase activities. The increase in the cellular levels of Sph in
response to the overexpression of the NCDase or AlkCDase in HeLa cells: ASAH2-
HeLa or ACER2-HeLa, respectively, was used as the readout of cellular activity of
these two enzymes, as was described in the text. After treatments for 1 h, cell
pellets were isolated, lipids were extracted and cellular levels of Sph and Cer were
measured by LC–MS/MS approach. (A) Effects on the cellular Sph. Results are
expressed as pmol Sph/nmol Lipid Phosphate. (B) Effects on the ratio of the cellular
Sph in ACER2-HeLa or ASAH2-HeLa cells versus Vector-HeLa cells. Results are
expressed as a ratio of the levels of Sph (pmol) normalized to Lipid Phosphate
(nmol).
We used HeLa cell lines that were stably transfected with the
ASAH2- or ACER2-expressing plasmid or empty vector. Effects of
the overexpression of those enzymes in HeLa cell lines: HeLa-
ASAH2 and HeLa-ACER2 in comparison to HeLa-Vector cells, and
effects of the treatments with B13, LCL521 and vehicle (Et) on
the cellular Sph are shown in Figure 5.
Results showed that HeLa-ACER2 cells had a much higher levels
of Sph (110 pmoles or 0.05/0.44 pmole/nmole Lipid Phosphate),
Figure 5A, and lower level of total Cer, (200 pmoles, not shown)
than HeLa-Vector cells. These results suggest that overexpression
of ACER2 increased the generation of Sph by hydrolyzing Cer in
cells. Therefore, Vector and ACER2 cell lines were appropriate for
testing if compounds inhibit ACER2 cellular activity.
In contrast, HeLa-ASAH2 cells had slightly lower levels of Sph
than vector cells (Fig. 5A) whereas total Cer was increased (ꢁ1.7
fold, not shown), suggesting that NCDase may not contribute to
the generation of Sph in HeLa cells. Therefore, HeLa-ASAH2 cells
were not appropriate for testing our compounds. To test effects
of DMG-B13 prodrugs and B13 on the cellular NCDase, we applied
an approach similar to the cellular ACDase activity assay, for exam-
ple, using MCF7 cell neutral lysate for the enzymatic reaction
(Fig. 6).
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
Figure 6. Effect of DMG-B13 prodrugs on the cellular activity of NCDase in MCF7
cells. Cellular NCDase activity assay. After MCF7 cell treatment with inhibitors or
vehicle control for 1 h, as was described in the text, cells were isolated and NCDase
activity was measured by in vitro assay as described in the text. Results are
expressed as pmol of released [3H] palmitic acid/mg protein/h, and presented as %
Control. Results are presented as mean SD of duplicates.
2.4.5. Effects on the cellular AlkCDase in HeLa-ACER2 cells
As shown in Figure 5A, treatment with B13 did not affect the
Sph increase by ACER2 overexpression, suggesting that this

6940
A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
200.0
compound does not inhibit ACER2 cellular activity. This is consis-
tent with the in vitro results (Fig. 1B). 1 M LCL521 slightly
enhanced Sph increase, suggesting that at a low concentration,
LCL521 does not inhibit ACER2 cellular activity. 10 M LCL521,
however, slightly decreased the Sph level, suggesting that this
compound at a high concentration may inhibit ACER2 activity. To
further investigate this point, data are also expressed as a ratio of
CDase/Vector (Fig. 5B) based on the fact that ACDase is present
in both vector and Hela-ACER2 cells and that strong and unique
inhibitory effects of LCL521 on ACDase could be unexploited
properly. And indeed, results showed the most increase of Sph
*
1h
5h
A
l
160.0
120.0
80.0
40.0
0.0
l
*
*
*
from LCL521 treatment (2 folds/1
lM and 5 folds/10 lM) whereas
B13 caused a slight decrease of Sph B13 (0.85 fold).
These results suggest that LCL521 did not inhibit cellular
AlkCDase whereas B13 may have a very small inhibitory effect
on this cellular enzyme.
200.0
160.0
120.0
80.0
40.0
0.0
B
2.4.6. Effects on the cellular NCDase in MCF7 cells
*
We used MCF7 cells and evaluated effects of 10 lM DMG-B13
prodrugs on the cellular activity of NCDase after 1 h of treatment
(Fig. 6). LCL521 did not show inhibition of NCDase activity,
whereas B13, LCL522 and LCL596 had a small, ꢁ10–20% inhibitory
effects on this enzyme.
In summary, LCL521 acted as a specific inhibitor of the cellular
ACDase, whereas the mono DMG-B13 prodrugs, similarly as B13,
affected also the cellular neutral and alkaline ceramidases, but
*
with
a low efficiency. These results may also suggest that
metabolism of LCL522 and LCL596 to B13 may partially occur
before they reach the lysosomes.
2.5. Lysosomal action of DMG-B13 prodrugs
Figure 7. Lysosomal action of DMG-B13 prodrugs. (A) Effect on the lysosomal pH
changes. MCF7 cells were treated with either inhibitors or vehicle control for 1 and
5 h before incubation with Lysotracker Red (LTR, tracking of acidic organelles) for
additional 30 min. Fluorescence relative intensity was measured by FACS analysis
(564–606 nm). Decrease in the fluorescence intensity corresponded to the increase
in the lysosomal pH, and a minimum of 10,000 events were scored for each sample.
Each dose was triplicated. Results were expressed as % Control, ⁄p value <0.05 (vs
control). (B) Effect on the lysosomal ASMase activity. MCF7 cells were treated with
either inhibitors or vehicle control for 1 h, cell pellets were isolated, cell lysates
were prepared and used to measure activity of the cellular ASMase by release of
[methyl-14C] choline from choline-[methyl-14C] SM as was described in the text.
Results are expressed as formed nmol Cer /mg protein/h, and presented as %
Control. Results are presented as mean SD of triplicates, ⁄p value <0.05 (vs
control).
DMG-B13 prodrugs, representing lipophilic amines, can accu-
mulate in the lysosomes, a process known as lysosomal trapping.37
They may affect ASMase whose lysosomal location is generally
accepted.38 To prove lysosomal action of DMG-B13 prodrugs, their
cellular effects on the lysosomal pH changes and the lysosomal
ASMase activity were investigated as well.
2.5.1. Effect on the lysosomal stability
The effects of 10
were examined by Lysotracker Red (LTR) uptake, and results were
also compared to the action of 10 M B13 and 10 M LCL204, a
lM DMG-B13-prodrugs on lysosomal stability
l
l
previously published lysosomal inhibitor of ACDase (Fig. 7A). The
early, 1 h treatments with LCL521 and LCL204 caused decrease of
LTR uptakes (ꢁ80–70% C, respectfully), whereas B13 and LCL522
had no decreasing effects. Extended, 5 h treatments caused a com-
plete recovery in LTR uptake by LCL521, small increasing tendency
for LCL204 but below the control level, increase for LCL522 (to
ꢁ180% C), and no change for B13.
Moreover, LCL521 acted as inhibitor of the lysosomal ACDase
and ASMase whereas LCL522 and LCL596 may be specific to the
lysosomal ACDase.
3. Summary and conclusions
Here, we report development of a compartmentally-targeted
B13 prodrugs that lack efficacy on ACDase inhibition in vitro but
are converted to the active metabolite in cells with significantly
enhanced effectiveness on this enzyme.
These results suggest a lysosomal action of LCL521 and LCL522.
Importantly, a lysosomal destabilization effect of LCL521 was not
permanent in contrast to LCL204.
Our results indicate that B13 is a specific inhibitor of ACDase
2.5.2. Effect on the lysosomal ASMase activity
showing almost complete inhibition of this enzyme at 50 lM in
The early effects, 1 h treatments, with DMG-B13 prodrugs on
ASMase activity in MCF7 cells are shown in Figure 7B. Results
an in vitro assay. We want to further emphasize the needs for
the enzymatic assays standardization (enzymatic substrates and
sources, and assay conditions) in order to provide reliable and
comparable results.26 The inhibitory effect of B13 on ACDase in
MCF7 cells was noticed only at the high concentrations, which
suggest that B13 is recognizable and can reach cellular ACDase.
Future investigation of the compartmental effects of B13 on
lysosomal versus nuclear ACDase is needed.
showed inhibition only by LCL521 (85% C/10
C/1 M, respectfully). These effects are comparable to the action
of LCL204 (95% C/10 M). 10 treatments with the
lM and 30%
l
l
lM
mono-DMG-B13 analogs: LCL522, 596 as well as B13 did not
inhibit activity of this enzyme.
These results suggest a lysosomal delivery of LCL521 whereas
LCL522 and LCL596 may require applications at higher concentra-
tions or a longer treatment time.
Application of the DMG prodrugs resulted in significant libera-
tion of B13. There was a fast and complete release of B13 from the

A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
6941
mono DMG prodrugs and a significantly slower release of B13 from
di-DMG prodrug with persistent accumulation. Importantly, the
generation of B13, which we assume occurs in the lysosome due
to the ionizable amine on the prodrugs, was accompanied by a
markedly improved cellular action of B13 that was observed from
all DMG-B13 prodrugs, especially LCL521. Considering the cellular
CDases, LCL521 showed specificity to the ACDase whereas the
mono DMG-B13 prodrugs, in addition to the inhibition of the
ACDase, had also a small effect on the NCDase, similar to B13,
suggesting that their partial metabolism may occur before they
reached the lysosomes. All DMG-B13 prodrugs caused an early
decrease of cellular Sph, as opposed to the action of B13 itself.
Our previous results specified that inhibition of ACDase by
LCL204 and desipramine also resulted in very early and profound
drop in the cellular Sph,19,39 thus, early effects on Sph may be
partially taken as a measure of cellular inhibition of ACDase,
presumably the lysosomal ACDase, based on fact that LCL521
inhibited also the lysosomal ASMase. We also predict that cellular
B13, generated from DMG-B13 prodrugs, could be trapped in the
lysosomes, nonetheless more effectively for LCL521. Alternatively,
coupled with the unique detection of a metabolite CX, a stable
mono DMG-B13, LCL521 may be involved in the formation of
strong complexes with certain lysosomal proteins to play an
important role in the early and persistent inhibition of ACDase.
Therefore, appropriate validation of LCL521, a ‘lysosomal mixture’
(B13 + CX + LCL521), is necessary to tune and further optimize the
described drug targeting approach. Cell fractionation studies
toward separation of lysosomes or labeling experiments with the
appropriate fluorescent LCL521 and B13 analogs should be consid-
ered for the further exploration of the cellular action of LCL521.
Importantly, our recently published collaborative results in a
xenograft mice model of prostate cancer showed that LCL521 com-
pletely prevented tumor relapse after combination with ionization
radiation.40
reported in ppm on the d scale from the internal standard of resid-
ual chloroform (7.27 ppm) and methanol (4.87 ppm). Mass spectral
data were recorded in a positive ion electrospray ionization (ESI)
multiple reaction monitoring (MRM) mode on Thermo Fisher TSQ
Quantum/Accela LC/MS/MS liquid chromatography/ triple quadru-
pole mass spectrometry system. Samples in a methanol solution
were injected through Accela autosampler. ESI voltage of 4.5 kV
and capillary temperature of 200 °C were applied throughout the
analyses. Specific structural features were established by fragmen-
tation pattern upon electrospray (ESI/MS/MS) conditions, specific
for each of the compound studied.
Synthetic compounds: B13, LCL521, 522 and 596 used in the
study were synthesized by the Lipidomics Facility, MUSC (Scheme 2)
4.1.1.2. Synthesis and characterization of DMG-B13 ester pro-
drugs. 4.1.1.2.1. (1R,2R)-2-N-(Tetradecanoylamino)-1-(40-nitrophe
nyl)-propyl-1,3-O,O-di-(N,N-dimethylamino) acetate dihydrochloride
(1,3-di-DMG-B13, LCL521). The synthesis was carried out accord-
ing to established methods with small modification.41 Briefly, N,N-
dimethyl glycine (103 mg, 1 mmol) and 1,10-carbonyldiimidazole
(162 mg, 1 mmol) were mixed in anhydrous CH2Cl2 (10 mL) and
stirred for 2 h before adding B13 (42.3 mg, 0.1 mmol) followed
by a small amount of either triethyl amine (TEA) or diisopropyleth-
ylamine (DIPEA). The reaction mixture was stirred at room temper-
ature until there was no trace of starting material. The product was
evaporated to dryness, and the residue was purified through col-
umn chromatography using chloroform-methanol-ammonium
hydroxide concd (15:1:0.025; v/v/v) to isolate wax solid freebase.
Then, at 4 °C, 2 M hydrochloride ether solution was added drop-
wise, with mixing, into the obtained freebase ethyl acetate solu-
tion. The reaction mixture was maintained at 4 °C until TLC
showed no trace of starting material and kept at the same condi-
tion for another 30 min before evaporation to dryness. The residue
was further dried down under high vacuum. Target hydrochloride
salt was recrystallized from ethyl acetate to give 40 mg (60%) of
Taken together, DMG-B13 prodrug modification to the
DMG-B13 prodrugs, especially to LCL521, defines a novel approach
to target enzymes of sphingolipid metabolism in a compartment-
specific manner.
LCL521 as a light yellow microcrystalline powder. TLC (CHCl3-CH3-
23.6
OH–NH3ꢀH2O concd, 10:1:0.1, v/v/v), Rf = 0.63; [
a]
ꢃ5.12 (c 0.5,
D
CH3OH), 1H NMR (400 MHz, CD3OD); 8.29 (d, J = 8.8 Hz, 2H, ArH),
7.70 (d, J = 8.8 Hz, 2H, ArH), 6.27 (d, J = 4.0 Hz, 1H, CONH), 4.80
(m, 1H, CH), 4.59 (dd, J = 5.2, 7.2 Hz, 1H, CH), 4.44 (s, 2H), 4.22
(dd, J = 17.0, 8.1 Hz, 4H, COCH2N), 3.01 (s, 12H, 2 N(CH3)2), 2.21
(m, 2H, CH2), 1.48 (m, 2H, CH2), 1.00–1.40 (m, 20H), 0.93 (t,
J = 6.6 Hz, CH3); 13C NMR (150.9 MHz, CD3OD) d 175.15, 165.61,
164.94, 148.00, 143.38, 127.60, 123.31, 75.45, 64.63, 56.94, 56.79,
51.26, 43.41, 35.47, 31.68, 29.39,29.36, 29.25, 29.14, 29.07, 28.88,
25.49, 22.34, 13.05; ESI-MS (CH3OH, relative intensity,%): m/z
calcd. for C31H53N4O7, 593.34. Found: 593.40 ([MH+ꢃ2HCl], 60).
Anal. Calcd for C31H54Cl2N4O.72H2O (701.71) C, 53.06; H, 8.33; N,
7.98; Cl, 10.10. Found: C, 52.52; H, 8.35; N, 7.76; Cl, 10.95.
4. Experimental
4.1. Material and methods
4.1.1. Chemistry
4.1.1.1. Chemicals.
All solvents and general reagents were pur-
chased from Sigma–Aldrich and Fluka. [9,10-3H] Palmitic acid was
purchased from American Radiolabeled Chemical, Inc. (St. Louis,
MO). (2S, 3R, 4E) [3H] C16-Cer (specific activity: 4 ꢂ 106 cpm/nmol)
and (2S,3R,4E) C16-Cer were synthesized in the Lipidomic Facility,
Medical University of South Carolina. B13 and LCL204 were syn-
thesized by Lipidomics Facility Synthetic Unit, MUSC, as described
before.11,14 DMG-B13 prodrugs, LCL521, 522 and 596, used in the
study were synthesized by the Lipidomics Facility, Synthetic Unit,
MUSC. Synthetic outline for the preparation of B13 prodrugs is
shown in Scheme 2.
4.1.1.2.2.
(1R,2R)-2-N-(Tetradecanoylamino)-1-hydroxyl-1-(40-
nitrophenyl)propyl-3-O-(N,N-dimethylamino) acetate hydrochloride
(3-DMG-B13, LCL522). This compound was prepared using the
same method as LCL521, except the ratio of N,N-dimethyl glycine
to B13 was 1.0: 1.1. Pure LCL522 was obtained from its correspond-
ing free base in overall 52% yield after crystallization from n-hex-
ane–ethyl acetate (1:4; v/v) as a light yellow waxy solid. TLC
TLC, 1H NMR, EA and MS confirmed purity and conformation of
the synthesized compounds. Reaction progress was monitored by
analytical normal phase thin layer chromatography (NP TLC) using
aluminum sheets coated with 0.25 mm Silica Gel 60-F254 (Merck).
Detection was performed by fluorescence quenching at 254 nm
with the PMA reagent (ammonium heptamolybdate tetrahydrate
to cerium sulfate, 5:2, w/w in 125 mL of 10% H2SO4). Flash chroma-
tography was performed using EM Silica Gel 60 (230–400 mesh) in
combination with the indicated solvent systems. NMR spectra
were recorded on Bruker AVANCE 400 or 600 MHz spectrometer
equipped with Oxford Narrow Bore Magnet. Chemical shifts were
(chloroform-methanol-ammonium hydroxide conc., 10:1:0.1; v/v/
22.9
v), Rf = 0.39; [
a]
D
ꢃ10.7 (c 0.5, CH3OH) 1H NMR (400 MHz, CD3
OD) d 8.22 (d, J = 8.8 Hz, 2H, ArH), 7.68 (d, J = 8.8 Hz, 2H, ArH),
5.11 (d, J = 2.5 Hz, 1H, CH), 4.67 (dd, J = 5.2, 5.2 Hz, 1H, CH), 4.54
(m, 1H, CH), 4.33 (dd, J = 8.3, 8.3 Hz, 1H, CH), 4.14 (Q, J = 17 Hz,
2H, COCH2N), 2.99 (s, 6H, N(CH3)2), 2.95 (t, J = 7.5 Hz, CH2), 2.10
(m, 2H, CH2), 1.10–1.40 (m, 20H), 0.93 (t, J = 7.0 Hz, CH3); 13C
NMR (150.9 MHz, CD3OD)
d 176.71, 167.37, 151.27, 148.80,
128.43, 124.26, 71.94, 67.55, 58.16, 54.36, 44.66, 36.89, 33.18,
30.88, 30.82, 30.68, 30.60, 30.57, 30.14, 27.05, 23.84, 14.53;

6942
A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
ESI-MS (CH3OH, relative intensity, %): m/z calcd for C27H46N3O6,
508.34. Found: 508.40 ([MH+ꢃHCl], 70). Anal. Calcd for C27H46ClN3-
O6H2O (562.1) C, 57.7; H, 8.6; N, 7.48; Cl, 6.31. Found: C, 56.7; H,
8.14; N, 7.00; Cl, 7.70.
N(CH3)2), 2.11 (m, 2H, CH2), 1.00–1.40 (m, 20H), 0.93 (t,
J = 7.0 Hz, 3H, CH3); 13C NMR (150.9 MHz, CD3OD) d 175.17,
165.7, 149.82, 147.25, 126.99, 122.75, 70.38, 65.85, 56.61, 52.87,
43.19, 43.17, 35.41, 31.69, 29.40, 29.37, 29.35, 29.33, 29.19,
29.11, 29.08, 28.66, 25.56, 22.35, 13.07; ESI-MS (CH3OH, relative
intensity, %): m/z calcd for C27H46N3O6, 508.34. Found: 508.35
([MH+-HCl], 70); Anal. Calcd for C27H46ClN3O6 (544.1) C, 59.6; H,
8.52; N, 7.72; Cl, 6.52. Found: C, 58.2; H, 8.31; N, 7.24; Cl, 6.98.
4.1.1.3. (1R,2R)-2-N-(Tetradecanoylamino)-1-(40-nitrophenyl)-3-
hydroxylpropyl-1-O-(N,N-dimethylamino) acetate hydrochloride
(1-DMG-B13, LCL596). 4.1.1.3.1. (1R,2R)-2-N-(Tetradecanoylami-
no)-1-(40-nitrophenyl)-1-hydroxylpropyl-3-O-trityl (3-Tr-B13). To
the stirred solution of B13 (422.5 mg, 1 mmol) in anhydrous CH2Cl2
(100 mL), we added trityl chloride (250.9 mg, 0.9 mmol) and a
catalytic amount of DMAP. The reaction was stopped when trace
amount of trityl chloride was observed. After evaporation, the
residue was purified by flush column chromatography (ethyl ace-
tate–hexane, 1:2; v/v) to obtain 600.1 mg (90.3%) of pure 3-Tr-B13
4.2. Biology
4.2.1. Cell culture
MCF7 human breast adenocarcinoma cell line was obtained from
ATCC (Manassas, VA), cultured in complete medium (RPMI 1640
medium supplemented with 10% FBS and 100 lg/mL Normocin),
as light yellow oil. Rf = 0.35; [a D20.9
]
ꢃ24.7 (c 0.5, CH3OH). 1H NMR
and maintained under standard incubator condition (humidified
atmosphere with 5% CO2 at 37 °C). Cells in the exponential growth
phase were harvested from the culture and used in all experiments.
HeLa T-Rex cells stably expressing human alkCDase 2 (ACER2), or
NCDase (ASAH2) or empty vector, which previously generated in
the lab, were maintained in MEM medium as described.42
(400 MHz, CDCl3) d 8.11 (d, J = 8.8 Hz, 2H, ArH), 7.44 (d, J = 8.4 Hz,
2H, ArH), 7.25–7.40 (m, 15H, ArH), 5.97 (d, J = 8.4 Hz, 1H, CONH),
5.06 (t, J = 3.6, 4.0 Hz, 1H, CH), 4.20 (m, 1H, CH), 3.96 (d, J = 3.6 Hz,
1H, OH), 3.43 (dd, J = 4.8, 4.8 Hz, 1H, CH), 3.33 (dd, J = 3.6, 3.6 Hz,
1H, CH), 2.06 (td, J = 7.6, 2.0 Hz, 2H, CH2), 1.13–1.48 (m, 20H), 0.86
(t, J = 7.2, 6.4 Hz, 3H, CH3).
4.2.2. In vitro acid ceramidase (ACDase) activity assay
4.1.1.4. (1R,2R)-2-N-(Tetradecanoylamino)-1-(40-nitrophenyl)-
3-O-trityl-propyl-1-O-(N,N-dimethylamino)acetate (1-DMG-3-
The ACDase activity assay was processed under acidic condi-
tions (MCF7 cells lysed, pH 4.5) as previously described.13 Briefly,
protein concentration was determined using the BCA protein assay
kit (Thermo Scientific, Rockford, IL), equal amounts of [9,10-3H] D-
e-C16-Cer and test compound were mixed with 0.2%TritonX-100
and 0.4% cholate and dried down under nitrogen followed by addi-
tions of equal amount of the protein and assay buffer (0.2 M ace-
tate buffer, 0.5% Triton X-100, pH 4.5). Assay was implemented
at 37 °C for an hour and terminated by addition of Dole’s alkaline
solution. [9,10-3H] palmitic acid, a hydrolytic product of ACDase
was extracted and counted to calculate the enzyme activity. Radio-
activity was quantified using LS 6500 multipurpose scintillation
counter (Beckman Coulter).
Tr-B13).
N,N-Dimethyl glycine (148.5 mg, 1.44 mmol) and
1,10-carbonyldiimidazole (233.5 mg, 1.44 mmol) were mixed in
anhydrous CH2Cl2 (30 mL) and stirred for 2 h under the exclusion
of moisture before adding 3-Tr-B13 (320 mg, 0.48 mmol), followed
by a small amount of either TEA or DIPEA. The reaction was
stopped when a trace amount of starting material was observed
(min 24 h). The solvent was evaporated to dryness, residue was
purified by the column chromatography (ethyl acetate–hexane–
TEA, 1:1:0.04; v/v/v) to get pure waxy solid of 1-DMG-3-Tr-B13
221.7 mg (61.4%). 1H NMR (400 MHz, CD3OD) d 8.05 (d, J = 8.8 Hz,
2H, ArH), 7.38 (d, J = 8.8 Hz, 2H, ArH), 7.23–7.36 (m, 15H, ArH),
6.28 (d, J = 7.2 Hz, 1H, CONH), 6.06(d, J = 9.4 Hz, 1H, CH), 4.53 (m,
1H, CH), 3.25 (dd, J = 4.8, 4.8 Hz, 1H, CH), 3.16 (d, J = 1.3 Hz, 2H,
CH2), 2.88 (dd, J = 3.2, 3.2 Hz, 1H, CH), 2.30 (s, 6H, N(CH3)2), 2.14
(dt, J = 7.4, 2.1 Hz, 2H, CH2), 1.56 (m, 2H, CH2), 1.25 (m, 22H),
0.88 (t, J = 6.6 Hz, 3H, CH3).
4.2.3. In vitro neutral ceramidase (NCDase) activity assay
The NCDase activity assay was processed as previously
described with a minor modification.27,43 Briefly, enzyme activity
was measured using D-e-C12-NBD-Cer at a final concentration of
50
ton-X100, pH 7.5). The enzymatic reaction was started by addition
of 10 g protein of HEK293 cells that previously overexpressed
NCDase. Reaction mixtures were incubated at 37 °C for 1 h before
terminated by the addition of 300 L chloroform-methanol (1:1,
v/v). Organic phase was spotted on TLC plate and then developed
with chloroform–methanol–25% ammonia (90:20:0.5, v/v/v) to
separate NBD-C12-Cer from NBD-dodecanoic acid. Rate of conver-
sion of the fluorescent-labeled substrate and product was analyzed
and quantified with a PhosphorImager Storm 860) system. Results
were quantified by densitometric analysis using Image Quant
software.
lM in a total volume of 100 lL (25 mM Tris buffer, 0.3% Tri-
4.1.1.5. (1R,2R)-2-N-(Tetradecanoylamino)-1-(40-nitrophenyl)-
3-hydroxylpropyl-1-O-(N,N-dimethylamino)acetate hydrochlo-
l
ride (1-DMG-B13, LCL596).
+4 °C solution of 1-DMG-3-Tr-B13 (51 mg, 0.07 mmol) in dry
CH2Cl2 (10 mL), Et2O.BF3 (15
L) was added. The formed reaction
To a stirred and cooled up to
l
l
mixture was stirred for 1 h at rt. The solvent was evaporated to
dryness, and the obtained residue was purified by column chroma-
tography (chloroform–methanol–ammonium hydroxide concd,
10:1:0.1, v/v/v) to give 31 mg (91%) of pure 1-DMG-B13 free base
as a pale yellow waxy solid. TLC (ethyl acetate–methanol, 11:1,
25
v/v); Rf = 0.22; [
a
]
ꢃ23.9 (c 0.5, CHCl3). This material was dis-
D
solved in anhydrous ethyl acetate (4 mL), and 2 M HCl solution in
a dry diethyl ether (0.3 mL) was added drop-wise at +4 °C. The
reaction mixture was stirred at +4 °C for an additional 30 min.
The mixture was evaporated under reduced pressure to dryness.
Residue was dried down under high vacuum. The crude hydrochlo-
ride was recrystallized from dry ethyl acetate–hexane (3:1, v/v) to
give 24 mg (72%) of LCL-596 as a pale yellow microcrystalline
4.2.4. In vitro alkaline ceramidase (AlkCDase) activity assay
The AlkCDase activity assay was processed as described previ-
ously.42 Briefly, microsomes of HeLa cells overexpressing ACER2
were incubated with D-e-C18:1-Cer substrate (150 lM) and the
various amount of test compound in the assay buffer (25 mM
Tris–HCl, 5 mM CaCl2 and MgCl2, 0.15% Nonidet P-40, pH 8.5) in
powder. TLC (chloroform–methanol–ammonium hydroxide concd,
a total volume of 40 lL. The reaction mixtures were incubated at
+1.26 (c 0.55, CH3OH) 1H NMR
37 °C for 30 min and stopped by extraction with chloroform-meth-
anol (2:1, v/v). To determine the enzyme activity, the level of Sph
released from Cer by the AlkCDase action was measured in lipid
extract by HPLC after OPA derivatization using 17C-D-e-Sph as
an internal standard.
22.3
10:1:0.1, v/v/v), Rf = 0.33; [
(400 MHz, CD3OD)
a
]
D
d
8.22 (d, J = 8.8 Hz, 2H, ArH), 7.69
(d, J = 8.8 Hz, 2H, ArH), 5.13 (d, J = 2.6 Hz, 1H, CH), 4.64 (dd,
J = 5.4, 5.4 Hz, 1H, CH), 4.54 (m, 1H, CH), 4.33 (dd, J = 2.8, 7.9 Hz,
1H, CH), 4.20 (dd, J = 17.0, 13.4 Hz, 2H, NCH2), 3.02 (s, 6H,

A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
6943
4.2.5. Cellular activity of acid ceramidase (ACDase) assay
The cellular ACDase activity was measured as previously
described. 1312 Briefly, MCF7 cells were seeded in 100 mm dishes
overnight before being treated accordingly. Cells were then
washed with cold PBS and lifted by gently scraping followed by
the in vitro assay protocol described under Section 4.2.2.
proteins per sample were separated on NuPAGE 4–12% Bis–Tris
gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose
membranes (Bio-Rad, Hercules, CA). Membranes were blocked for
one hour at room temperature in TBS containing 0.1% Tween-20
and 5% non-fat dry milk, and incubated overnight at 4 °C with
the primary antibody mouse monoclonal anti-ACDase (Pharmin-
gen, San Diego, CA). After washing 3 times with TBS-T, membranes
were incubated
1 hour with the secondary antibody goat
4.2.6. Cellular activity of neutral ceramidase (NCDase) assay
Briefly, MCF7 cells were seeded in 100 mm dishes overnight
before being treated accordingly. Cells were then washed with cold
PBS and lifted by gently scraping followed by cell pellet lysed
under the neutral condition, and protein concentration was deter-
mined using the BCA protein assay kit (Thermo Scientific, Rockford,
IL). The NCDase activity assay was processed using equal amounts
of [9,10-3H] D-e-C16-Cer mixed with 0.3%TritonX-100, and dried
down under nitrogen. Lipid film was then homogenized with
water, followed by additions of equal amount of the protein and
assay buffer (50 mM Tris–HCl, 0.3% Triton X100, 1 mM CaCl2, pH
7.1). Reaction was incubated at 37 °C for an hour and terminated
by Dole’s alkaline solution. [9,10-3H] palmitic acid, a hydrolytic
product of NCDase, was extracted and counted to calculate the
enzyme activity. Radioactivity was quantified using LC6500 multi-
purpose scintillation counter (Beckman Coulter).43
anti-mouse IgG-HRP conjugate (Sigma, St. Louis, MO). Membranes
were then washed 3 times with TBS-T, and protein bands were
visualized with the super signal HRP substrate (Pierce, Rockford,
IL). For each sample, the density of ACDase protein bands were
normalized by the actin band, and results were expressed as %
Control.
4.2.10. Lysosomal stability assay
Lysosomal stability was measured using the Lysotracker Red
(LTR) dye DND-99 (Molecular probes, Oregon).8 Briefly, MCF7 cells
were seeded 5 ꢂ 105/well in 6 well-plate overnight. Media were
then replaced with 2% FBS which contained the same amount of
either ethanol or the test compounds in ethanol solution (ethanol,
60.1%) and incubated at 37 °C for 1 and 5 h before adding 200 nM
LTR for 30 mins at 37 °C. Then, cells were lifted with trypsin,
washed twice with PBS, and re-suspended 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 done in triplicate. Results
were expressed as % Control.
4.2.7. Cellular activity of alkaline ceramidase (ACER2) or neutral
ceramidase (ASAH2) assays
HeLa cells overexpressing ASAH2, ACER2, or empty vector were
grown to a 60% confluence before they were treated with tetracy-
cline (5 ng/mL) overnight. Next day, the cells were then treated
with B13 (10 lM), LCL521 (1 or 10 lM), or ethanol (the vehicle
control for both B13 and LCL521) for 1 h before they were har-
vested and processed as described in the previous studies.42 Sphin-
golipids were extracted from these cell samples and were analyzed
by LC–MS/MS.
4.2.11. HPLC–MS/MS analysis
Advanced analyses of endogenous Sph and Cers and intracellu-
lar B13 and DMG-B13 prodrugs were performed on ThermoFisher
TSQ Quantum liquid chromatography/triple-stage quadrupole
mass spectrometer (LC/MS/MS) system, operating in a multiple
reaction monitoring (MRM) positive ionization mode, as previously
described.45 Briefly, after the treatment, culture media was
removed and cells were washed two times with cold PBS before
gently scraping the plate. Cell pellets were fortified with the
4.2.8. Cellular activity of acid sphingomyelinase (ASMase) assay
The assay was processed as described previously using MCF7
cell lysates.44 Briefly, following treatment, cells were washed twice
in cold PBS and scraped in ice-cold lysis buffer (0.2% Triton X-100,
50 mm Tris–HCl, pH 7.4, 1:200 phosphatase inhibitor mixtures 1
and 2, 1:200 protease inhibitor mixture, and 1.0 mM EDTA).
Lysates were sonicated briefly (2 ꢂ 10 s pulses), and cellular debris
and unbroken cells were pelleted by centrifugation at 1000ꢂg for
1 min at 4 °C. Equal amounts of clarified lysate (in a total volume
internal standards ISs: 17C base
17S1P, 17C16-Cer, 17C24:1-Cer, 13C base
13C16-Cer and 18C base -erythro-sphingosine: 18C17-Cer, then
D
-erythro-sphingosine: 17Sph,
D-erythro-sphingosine:
D
extracted into a one-phase solvent system with ethyl acetate/
iso-propanol/water (60/30/10% v/v). Lipid extract was divided into
two portions and used for MS analysis and for determination of the
level of Lipid Phosphate after further re-extraction by Bligh and
Dyer method (used for MS data normalization).46 Original lipid
extracts, after evaporation and reconstitution in 100 lL of acidified
(0.2% formic acid) methanol, were injected on the Accela/TSQ
Quantum LC/MS/MS system and gradient-eluted from the BDS
of 100
containing 100
lL lysis buffer) were added to 100 lL of reaction mixture
l
M porcine brain SM and 1 ꢂ 105 cpm of choline-
[methyl-14C] SM (specific radioactivity = 1.5 ꢂ 105 cpm/
lL) pre-
sented in micelles containing 0.2% Triton X-100 in sodium acetate
buffer (250 mM, pH 5.0) supplemented with 1.0 mM EDTA. Assays
were run for 30 min at 37 °C, and the reaction was stopped by add-
ing 1.5 mL of chloroform-methanol (2:1, v/v) followed by addition
of 0.4 mL water (Folch extraction). Phases were separated by cen-
Hypersil C8, 150 ꢂ 3.2 mm, 3-
lm particle size column, with
1.0 mM methanolic ammonium formate/2 mM aqueous ammo-
nium formate mobile phase system. Peaks corresponding to the
target analytes and IS were collected and processed using the
Xcalibur software system.
trifugation at 2000g for 5 min at room temperature. 800 lL of
upper (aqueous) phase was counted by scintillation. The assay
was linear with respect to time and protein/volume, and substrate
hydrolysis was less than 10%.
Quantitative analyses were based on the calibration curves gen-
erated by spiking an artificial matrix with known amounts of the
target synthetic standards and an equal amount of the internal
standard. The target analyte peak area ratio from the samples were
similarly normalized to their respective IS and compared to the cal-
ibration curves using a linear regression model. Final results were
expressed as the levels of the particular compound (pmols) nor-
malized to cellular Lipid Phosphate, Pi (nmols). Changes were
expressed in comparison to the control samples representing
treatments with the equal amount of ethanol only.
4.2.9. Acid ceramidase protein expression by Western Blot
ACDase protein expression was performed as previously
described.13 Briefly, MCF7 cells were seeded in 100 mm dishes
overnight and treated accordingly. Then cells were washed with
cold PBS and lifted by gently scraping the plates. Cells were then
lysed with regular lysate buffer containing protease inhibitors. Pro-
tein concentration was determined using the BCA protein assay kit
(Pierce, Rockford, IL). Cell lysates corresponding to 35 lg of

6944
A. Bai et al. / Bioorg. Med. Chem. 22 (2014) 6933–6944
15. Bielawska, A.; Greenberg, M. S.; Perry, D.; Jayadev, S.; Shayman, J. A.; McKay, C.;
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Financial support was provided by the NIH-NCI: PO1-CA097132
(YAH, AB), R41CA139637 (AB, SphingoGene Inc. subcontract to
MUSC) and P30 CA138313 (AB, Lipidomics Shared Resource, HCC,
MUSC); NIH: NCRR-UL1RR029882 (APB, Voucher Pilot Program),
P20 RR017677 (AB, Lipidomics Core, SC Lipidomics and Pathobiol-
ogy COBRE, Department Biochemistry, MUSC) and 1R01 CA163825
(CM). We especially acknowledge the National Center for Research
Resources – United States, Grant C06 RR018823, providing labora-
tory space for Lipidomics Facility in MUSC’s Children’s Research
Institute. We acknowledge also the Atlantic Microlab, Inc. for the
compounds’ elemental analysis. We sincerely appreciate Dr. David
Perry of Charleston Southern University for editorial assistance of
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