Synthesis of erythro- B13 enantiomers and stereospecific action of full set of B13-isomers in MCF7 breast carcinoma cells: Cellular metabolism and effects on sphingolipids

Article abstract of DOI:10.1016/j.bmc.2021.116011

B13 is an acid ceramidase (ACDase) inhibitor. The two chiral centers of this aromatic amido alcohol lead to four stereoisomers, yet we have little knowledge about its erythro- enantiomers, (1R, 2S) and (1S, 2R). In this paper, for the first time, the synthesis of two erythro- enantiomers is described, and the compounds are evaluated along with two threo- enantiomers, (1R, 2R) and (1S, 2S). The key metabolites and sphingolipid (SL) profile of the full set of B13 stereoisomers in MCF7 breast carcinoma cells are presented. The results demonstrated that the erythro- enantiomers were more effective than the threo- enantiomers on growth inhibition in MCF7 cells, although there were no statistically significant differences within the threo- and erythro- series. Measurement of intracellular levels of the compounds indicated that the erythro- seemed a little more cell permeable than the threo- enantiomers; also, the (1R, 2S) isomer with the same stereo structure as natural ceramide (Cer) could be hydrolyzed and phosphorylated in MCF7 cells. Furthermore, we also observed the formation of C16 homologs from the full set of B13 isomers within the cells, indicating the occurrence of de-acylation and re-acylation of the amino group of the aromatic alcohol. Moreover, the decrease in the Cer/Sph ratio suggests that the growth inhibition from (1R, 2S) isomer is not because of the inhibition of ceramidases. Taken together, (1R, 2S) could be developed as a substitute of natural Cer.

Full text of DOI:10.1016/j.bmc.2021.116011

Bioorg. Med. Chem. 32 (2021) 116011
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of erythro- B13 enantiomers and stereospecific action of full set of
B13-isomers in MCF7 breast carcinoma cells: Cellular metabolism and
effects on sphingolipids
,
,
,
,*
Aiping Baia b, Jacek Bielawskia b, Alicja Bielawskaa b, Yusuf A. Hannunc
a Department of Biochemistry & Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA
b Lipidomics Shared Resources, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425, USA
c Departments of Medicine and Biochemistry & 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
Keywords:
B13
B13 is an acid ceramidase (ACDase) inhibitor. The two chiral centers of this aromatic amido alcohol lead to four
stereoisomers, yet we have little knowledge about its erythro- enantiomers, (1R, 2S) and (1S, 2R). In this paper,
for the first time, the synthesis of two erythro- enantiomers is described, and the compounds are evaluated along
with two threo- enantiomers, (1R, 2R) and (1S, 2S). The key metabolites and sphingolipid (SL) profile of the full
set of B13 stereoisomers in MCF7 breast carcinoma cells are presented. The results demonstrated that the erythro-
enantiomers were more effective than the threo- enantiomers on growth inhibition in MCF7 cells, although there
were no statistically significant differences within the threo- and erythro- series. Measurement of intracellular
levels of the compounds indicated that the erythro- seemed a little more cell permeable than the threo- enan-
tiomers; also, the (1R, 2S) isomer with the same stereo structure as natural ceramide (Cer) could be hydrolyzed
and phosphorylated in MCF7 cells. Furthermore, we also observed the formation of C16 homologs from the full
set of B13 isomers within the cells, indicating the occurrence of de-acylation and re-acylation of the amino group
of the aromatic alcohol. Moreover, the decrease in the Cer/Sph ratio suggests that the growth inhibition from
(1R, 2S) isomer is not because of the inhibition of ceramidases. Taken together, (1R, 2S) could be developed as a
substitute of natural Cer.
Chiral center
Sphingolipid
Metabolites
Ceramide
1. Introduction
with (1R, 2R) while bearing aggressive human colon cancer cells that
had already metastasized to the liver.4 Also, in other animal studies, (1R,
B13, N-((1R, 2R)-1,3-dihydroxy-1-(4-nitrophenyl)-propan-2-yl)tet-
radecanamide, also named D-NMAPPD, was first synthesized in 1992,
and it was later found to inhibit acid ceramidase (ACDase).1–2 B13 is a
typical aromatic sphingolipid (SL) analog, contains all features that
natural SL contains, except that the two chiral centers are (1R, 2R)
[threo- isomer], which differs from natural ceramide [Cer, (2S, 3R),
erythro- isomer]. (1R, 2R) is an ACDase inhibitor, and its in vitro IC50 can
2R) has been used in combination with other therapies to achieve better
tumor shrinkage. Since then, one publication mentioned chiral centers
of B13,5 however, that study only examined two threo- enantiomers, and
found that (1R, 2R) was more effective in LNCaP whereas (1S, 2S) was
more potent in PC3 prostate cancer cells. LNCaP cells are androgen-
sensitive prostate adenocarcinoma cells and have low metastatic po-
tential, whereas PC3 does not respond to androgen deprivation, gluco-
corticoids or fibroblast growth factors, and has high metastatic
potential. Therefore (1S, 2S) seems a more preferable option to shrink a
highly metastatic prostate tumor. Indeed, that paper also presented
preliminary in vivo studies, showing that (1S, 2S) sensitized tumors to
achieve 20 μM when the cell lysate is used as enzyme source under acidic
assay conditions.3 Notably, (1R, 2R) has been shown to induce apoptosis
in many cancer cell lines. Moreover, in vivo studies have shown that
tumor growth was completely prevented when nude mice were treated
Abbreviations: B13, N-((1R, 2R)-1,3-dihydroxy-1-(4-nitrophenyl)-propan-2-yl) tetra-decanamide; ACDase, acid ceramidase; CDase, ceramidase; SL, sphingolipid;
PCC, pyridinium chlorochromate; Cer, ceramide; Sph, sphingosine; UHPLC-MS/MS, ultra performance liquid chromatography - tandem mass spectrometer; D-e-
MAPP, N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)tetradecanamide; DES, dihydroceramide desaturase; TLC, thin layer chromatography; ESI, electrospray ioniza-
tion; MRM, multiple reaction monitoring.
* Corresponding author.
E-mail address: yusuf.hannun@stonybrookmedicine.edu (Y.A. Hannun).
https://doi.org/10.1016/j.bmc.2021.116011
Received 30 October 2020; Received in revised form 22 December 2020; Accepted 28 December 2020
Available online 8 January 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

A. Bai et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116011
radiation in a xenografted PC3 mouse model, resulting in reduction of
both tumor volume and tumor weight.
Our group and others have made various structural modifications on
B13;6–8; nevertheless, changes were all made based on the B13′s threo-
enantiomers: (1R, 2R) or (1S, 2S). Therefore, in this paper, we focus on
the synthesis of B13′s erythro- enantiomers: (1R, 2S) and (1S, 2R), and
the evaluation of full set of B13 stereoisomers’ SL profile and cytotox-
icity in MCF7 cells.
2. Results and discussion
2.1. Synthesis of erythro- B13
The synthesis of threo- B13 was published previously.1 The general
approach for synthesis of erythro- B13 starts from threo- B13. Therefore,
(1R, 2S) B13 was synthesized from (1S, 2S) B13, and (1S, 2R) B13 was
synthesized from (1R, 2R) B13 (1, Scheme 1). In detail, trityl chloride
was used to protect the primary hydroxyl group. Then (1R, 2R)-3-O-tri-
tyl B13 (2a) was oxidized by pyridinium chlorochromate (PCC) to pro-
duce (1R, 2R)-3-O-trityl-1-oxo-B13 (3a). Subsequently, (1R, 2R)-3-O-
trityl-1-oxo-B13 (3a) was reduced by sodium borohydride to generate
(1R, 2R)-3-O-trityl-B13 (2a) and (1S, 2R)-3-O-trityl-B13 (4a). After flash
column chromatography separation, purified (1S, 2R)-3-O-trityl B13
(4a) was de-protected under acidic condition to yield (1S, 2R) B13 (5a).
NMR, optical rotation, and MS data were used to confirm all newly
synthesized isomers. After 2 times flash column chromatography sepa-
ration, (1S, 2R) and (1R, 2S) B13 isomers were purified as waxy white
solids, with a total yield of 11.3% and 12.3% and with no additional
synthetic optimization.
Fig. 1. Cytotoxicity of full set of B13 isomers in MCF7 viable cell analysis at 48
h. MCF7 cells were treated with vehicle, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50,
and 100uM of different B13 isomers for 48 h and then MTT assays were per-
formed. The results are expressed as percentage relative to untreated cells and
are presented as means ± st dev. of single experiment with 5 time replicates.
Table 1
IC50 values (in µM) of full set of B13 isomers at 24, 48 and 72 h.
IC50
(μ
M) isomers
24 h
48 h
72 h
(1R, 2R)
(1S, 2S)
(1S, 2R)
(1R, 2S)
42.27 ± 1.05
54.08 ± 1.04
17.96 ± 1.12
10.23 ± 0.57
35.59 ± 0.79
43.65 ± 1.07
13.06 ± 0.36
8.08 ± 0.19
37.01 ± 0.91
46.63 ± 1.30
13.51 ± 2.49
9.39 ± 0.40
2.2. Inhibitory effects of the full set of B13 isomers on MCF7 cell growth
First, we determined the profile of B13 isomers’ cytotoxicity in MCF7
cells. The results indicated that there was little difference within threo-
[(1R, 2R) vs (1S, 2S)] and erythro- [(1S, 2R) vs (1R, 2S)] enantiomers; but
they did show 3–5 fold differences between threo- and erythro- enan-
tiomers, with erythro- isomers being more effective than threo- isomers
on MCF7 cells growth inhibition (MTT assay, Fig. 1 and Table 1). The
sequence of IC50 values in MCF7 cells is (1S, 2S) > (1R, 2R) ≫ (1S, 2R)>
(1R, 2S).
difference in their cell permeability. Our results showed that after 1 h
incubation, the cellular level of all isomers increased in a dose depen-
dent pattern, especially for erythro- enantiomers at the high dose
[Fig. 2A]. Interestingly, when we prolonged the incubation time (24 h),
three of the four isomers were maintained at a level similar to 1 h, yet the
cellular level of (1R, 2S) B13 dropped dramatically [Fig. 2B].
Because (1R, 2S) B13 has a stereo-structure comparable to Cer
[Fig. 3A and B], we then explored whether (1R, 2S) B13 also functioned
as a substrate for ceramidases (CDases) and therefore could be hydro-
lyzed and further metabolized in cells. This pathway has been demon-
strated with exogenous C6-Cer in A549 cells.9 In MCF7 cells, treatment
with 17C6-Cer results in the formation of the free sphingoid base, 17Sph,
indicating the operation of CDase. This is also accompanied by
2.3. Cell uptake and key metabolites of full set of B13 isomers in MCF 7
cells
Next, we measured the cellular level of full set of B13 isomers to
investigate whether the difference in cytotoxicity was related to the
Scheme 1. Synthetic outline for erythro B13. Reagents and conditions: (i) trityl chloride, DMAP, Et3N, CH2Cl2, rt; (ii) CrO3.Py, CH2Cl2, 4 ◦C to rt; (iii) NaBH4,
CH3OH, 4 ◦C to rt; (iv) acetic acid, rt.
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Bioorganic & Medicinal Chemistry 32 (2021) 116011
Fig. 2. Intracellular level of full set of B13 isomers after 1 h (A) and 24 h (B) incubation in MCF7 cells. Cells were treated with 1, 5 and 10uM of different B13 isomers
for indicated time before cell pellets were isolated. Cellular levels of each isomer were then quantified by UHPLC-MS/MS analysis of lipid extract from cell pellets.
Results are presented as means ± st dev. of triplicates. (*p < 0.05 (vs (1R, 2R)); ** p < 0.05 (vs (1S, 2S)).
Fig. 3. Structure of (1R, 2S) B13 (A) and natural Ceramide (B).
formation of Cer with various N-acyl species on the 17 sphingoid
backbone, with 17C16, 17C24 and 17C24:1-Cer being the three major
metabolites produced within 24 h [Fig. 4]. This then illustrates the re-
acylation of the 17Sph by the various Cer synthases.10
We therefore examined further the metabolism of the full set of B13
isomers using UHPLC-MS/MS. The results showed that after 24 h incu-
bation, compared to the standards, mainly C16 homologs and a very
small amount of the C24:1 homologs were observed for all four isomers
[Suppl Fig 1 and Table 1]. UHPLC-MS/MS conditions were the same as
previously reported.11
To confirm our findings, we also quantified each metabolite (free
base, B13-homologs with C16 and C24:1 fatty acyl chain length)
[Fig. 5]. Unexpectedly, the results indicated that all isomers could be
hydrolyzed and more or less metabolized to form homologs with C16
and C24:1 fatty acyl chain length, but C16- were 10 times more preva-
lent than C24:1 homologs [Fig. 5B-C]. Surprisingly, it was not (1R, 2S)
but (1S, 2S) showing relatively high level of these two metabolites.
Moreover, we only detected trace amount of the free base hydrolyzed
from the parent B13 isomers, and there were no statistical differences
among the isomers [Fig. 5A], suggesting rapid re-acylation, possibly
through the action of Cer synthases.
Since de-acylation/re-acylation appeared to act on all 4 isomers, we
searched for additional metabolites, especially for (1R, 2S) that could
explain its declining levels in cells, which suggest metabolism to some
other compounds. When measuring the phosphorylation of the aromatic
SLs, we found that phosphorylated free-base was specific for (1R, 2S)
isomer, resulting in the highest phosphorylation among the full set of
B13 isomers [Fig. 5D]. The data also suggest that free base that released
from (1R, 2S) isomer can be a substrate of kinases.
2.4. Effects of the full set of B13 isomers on endogenous SL profile in
MCF7 cells
Fig. 4. Major metabolites (>10% of total 17Cn-Cer) of 5uM 17C6-Cer in MCF7
cells at 24 h. Cells were treated with 5uM 17C6-Cer for 24 h before cell pellets
were isolated, lipids were extracted and cellular level of 17Cn-Cer were
measured by UHPLC-MS/MS approach. Results are expressed as pmol/nmol
lipid phosphate, and are presented as % total 17Cn-Cer. All results are pre-
sented as means ± st dev. of duplicates.
Last, by using the UHPLC-MS/MS, we also measured the key SL
profile of the full set of B13 isomers. First, we compared the Cn-Cer
profile of different isomers after 10uM treatment [Fig. 6A-B].
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Bioorganic & Medicinal Chemistry 32 (2021) 116011
Fig. 5. Intracellular levels of the full set of B13 isomers’ key metabolites in MCF7 cells (24 h). Cells were treated with each of 10uM B13 isomer for 24 h before
cell pellets were isolated, lipids were extracted, and cellular levels of key metabolites (free base, C16 and C24:1 analogs) were measured by UHPLC-MS/MS approach.
Results are expressed as pmol/nmol lipid phosphate, and are presented as means ± st dev. of triplicates. A. Free base; B. C16 analogs; C. C24:1 analogs; D.
Phosphorylated free base. (* p < 0.01 (vs (1S, 2S)); ** p < 0.05 (vs (1S, 2S)).
Interestingly, at 1 h, the elevation of Cn-Cer was only observed for (1R,
2S) B13. Remarkably, the ACDase inhibitor, (1R, 2R) B13, at 10uM
which is significantly below its IC50 in MCF7 cells growth, did show
specific increases in C14 and 16-Cer, although the decrease was also
significant in C24, C26:1 and C26-Cer very long chain Cn-Cer. When we
prolonged the incubation time, at 24 h, (1R, 2R) exhibited a similar Cn-
Cer profile as (1R, 2S), which clearly elevated long chain Cn-Cer (≤C18),
however (1S, 2S) was the only isomer that kept decreasing almost all Cer
species, and no specific changes were found with (1S, 2R) isomer. Our
results also imply that Cn-Cer showed a response prior to cell killing.
As for the Cn-dhCer profile, after 1 h incubation, the only highly
elevated Cn-dhCer was also from (1R, 2S) B13, in particular, the very
long fatty acyl chain length were all enhanced [n ≥ 22, Fig. 6C]. Yet
when we prolonged the incubation time (24 h), the response of Cn-dhCer
went to the opposite way. Although both threo- isomers showed distinct
increase of Cn-dhCer, in particular dhC14, dhC16, as well as all the very
long chain Cn-dhCer (n ≥ 22), the responses from erythro- isomers were
all moved to the control level, in particular (1R, 2S) [Fig. 6D]. Our re-
sults also suggest that rather than CDases, (1S, 2S) may have inhibitory
effects on dihydroceramide desaturase (DES), due to its ability to in-
crease of Cn-dhCer and decrease of Cn-Cer at 24 h.
and 10uM). However, when we calculated the Cer/Sph ratio [Fig. 7B],
up to the 10uM concentration, only (1R, 2R) B13 showed a slightly
elevated ratio that did not achieve statistical significance compared to
the control, while other isomers decreased this ratio, thus, indicating
much more complex effects on Cer and Sph metabolism, with Cer either
being decreased or Sph being increased, or both.
3. Summary and conclusion
Aromatic analogs of SL have been designed and pre-clinically vali-
dated as inhibitors of enzymes of SL metabolism because of their
structural similarity to natural SL. Moreover, aromatic analogs of SL are
UV active, thus are easy to monitor.
To search for novel structures that can mimic the action of natural SL
or inhibit the formation of SL, so as to further investigate SL metabolic
pathways, our group has developed various sets of aromatic analogs of
SL with a structural feature containing phenyl-amino-alcohol. Among
them, B13 [N-((1R, 2R)-1,3-dihydroxy-1-(4-nitrophenyl)-propan-2-yl)
tetra-decanamide] and D-e-MAPP [N-((1S, 2R)-1-hydroxy-1-phenylpro-
pan-2-yl)tetra-decanamide] were both synthesized by A. Bielawska et al
in 1992. Later, B13 was found to inhibit ACDase, while D-e-MAPP in-
hibits alkaline CDase13. Moreover, our group also discovered that L-e-
MAPP (1R, 2S), the enantiomer of D-e-MAPP, which has the comparable
stereo-structure to natural Cer, was a substrate of alkaline CDase, and it
was metabolized by alkaline CDases to an extent similar to what
Our previous study showed that 10uM (1R, 2R) B13 (ACDase in-
hibitor) increased Cer and somewhat decreased Sph at 24 h in the same
cell line, which indicated its inhibitory effects on cellular ACDase.12
Thus, by using the UHPLC-MS/MS, we also measured endogenous Sph
and calculated the Cer/Sph ratio to briefly explore whether we could
evaluate a similar or better inhibitory response among the full set of B13
isomers. The results [Fig. 7A] showed that except for 10uM (1R, 2S)
treatment, all other isomers displayed a dose dependent decrease of
endogenous Sph, in particular (1S, 2R) B13, which led to a significant
decrease in the level of endogenous Sph at all doses that we selected (1, 5
occurred with C16-Cer13
.
In this study, we now report on the synthesis of erythro- B13 isomers
and determine the action of the full set of B13 isomers on MCF7 breast
carcinoma cells. Cell cytotoxicity showed that the erythro- pair was more
effective than the threo- pair on MCF7 cells growth. Interestingly, each of
the B13 isomers could be metabolized in the cells forming mainly their
4

A. Bai et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116011
Fig. 6. Cn-Cer profile (A. 1 h; B. 24 h) and Cn-dhCer profile (C. 1 h; D. 24 h) of full set of B13 isomers at 10uM. Cells were treated with each of 10uM B13 isomer for
the indicated time before cell pellets were isolated, lipids were extracted, and cellular level of Cn-Cer and Cn-dhCer were measured by UHPLC-MS/MS approach.
Results are expressed as pmol/nmol lipid phosphate, and are presented as means ± st dev. of triplicates (*p < 0.05, vs Control).
C16-homologs and a small amount of C24:1-homologs thus acting
similarly to 17C6-Cer cellular metabolism. Furthermore, (1R, 2S) B13,
with the comparable stereo-structure to the natural Cer, could be hy-
drolyzed and phosphorylated, and was the most potent on MCF7 cell
growth inhibition [Fig. 8]. At this point, we have not identified the ki-
nase that acts on the (1R, 2S) B13. Based on these cumulative findings,
we conclude that the (1R, 2S) isomer could be developed as a substitute
of natural Cer for cell studies.
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Bioorganic & Medicinal Chemistry 32 (2021) 116011
Fig. 7. Dose dependent response of full set of B13 isomers on Sph (A) and Cer/Sph ratio (B) at 24 h. Cells were treated with 1, 5 and 10uM of each B13 isomer for 24
h before cell pellets were isolated, lipids were extracted and cellular level of Sph was measured by UHPLC-MS/MS approach. Results are expressed as pmol/nmol
lipid phosphate, and are presented as means ± st dev. of triplicates (*p < 0.05, vs Control).
Fig. 8. (1R, 2S) B13′s possible metabolic pathway in MCF7 cells.
4. Methods
2H), 1.560 (s, 2H), 1.460 (m, 2H), 1.127–1.3 (m, 18H), 0.864 (t, J = 7.2,
3H). ESI-MS (CH3OH, relative intensity, %): m/z 687.13 ([MNa]+, 100).
Calcd. for C42H52N2O5 m/z 664.39 [M]; C42H52N2NaO5, m/z 687.38
[MNa].
4.1. Chemistry
All solvents and general reagents were purchased from Sigma-
Aldrich and VWR. Synthetic outline for the preparation of erythro B13
was presented in scheme 1. Thin layer chromatography (TLC), 1H NMR,
optical rotation, and MS confirmed purity of the synthesized com-
pounds. Detection was performed under ultraviolet light (254 nm). Flash
chromatography was performed using EM Silica Gel 60 (230–400 mesh)
with the indicated solvent systems. 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 δ scale from
the internal standard of residual chloroform (7.27 ppm). Optical rota-
tion data were determined with a JASCO P-1010 polarimeter. Mass
spectral data were reported in a positive ion electrospray ionization
(ESI) mode on Thermo Finnigan TSQ 7000 triple quadrupole mass
spectrometer. Samples were infused in methanol with an ESI voltage of
4.0 kV and capillary temperature of 200 ◦C. Specific structural features
were established by fragmentation pattern upon electrospray (ESI/MS/
MS) conditions, specific for each of the compound studied.
4.1.3. (2R)-N-(1-(4-nitrophenyl)-1-oxo-3-(trityloxy)propan-2-yl)tet-
radecanamide (3a) was prepared from 2a, crude product 3a was purified
by flush column chromatography at ethyl acetate-hexane, 1:2 (v/v) in
yield of 58% as colorless oil. Rf 0.47; 1H NMR (400 MHz, CDCl3) 8.185
(d, J = 8.8, 2H), 7.917 (d, J = 9.2, 2H), 7.147 (m, 15H), 6.739 (d, J =
7.2, 1H), 5.599 (m, 1H), 3.572 (dd, J = 5.2, 3.6, 1H), 3.365 (dd, J = 4.0,
4.0, 1H), 2.296 (t, J = 7.6, 2H), 1.661 (m, 2H), 1.242 (m, 20H), 0.865 (t,
J = 6.8, 3H). ESI-MS (CH3OH, relative intensity, %): m/z 685.17
([MNa]+, 100). Calcd. for C42H50N2O5 m/z 662.37 [M];
C42H50N2NaO5, m/z 685.36 [MNa].
4.1.4 (2S)-N-(1-(4-nitrophenyl)-1-oxo-3-(trityloxy)propan-2-yl)tet-
radecanamide (3b) was prepared from 2b, crude product 3b was purified
by flush column chromatography at ethyl acetate-hexane, 1:3 (v/v) in
yield of 45.4% as colorless oil. Rf 0.30; 1H NMR (400 MHz, CDCl3) 8.176
(d, J = 7.2, 2H), 7.893 (d, J = 7.2, 2H), 7.145 (m, 15H), 6.774 (d, J =
7.6, 1H), 5.591(m, 1H), 3.574 (dd, J = 3.6, 3.2, 1H), 3.355 (dd, J = 4.0,
3.6, 1H), 2.301 (t, J = 7.2, 2H), 1.665 (m, 2H), 1.243 (m, 20H), 0.865 (t,
J = 6.8, 3H). ESI-MS (CH3OH, relative intensity, %): m/z 685.03
([MNa]+, 100). Calcd. for C42H50N2O5 m/z 662.37 [M];
C42H50N2NaO5, m/z 685.36 [MNa].
4.1.1. N-((1R, 2R)-1-hydroxy-1-(4-nitrophenyl)-3-(trityloxy)propan-
2-yl) tetra-decanamide (2a) was prepared as previously described.3
4.1.2. N-((1S, 2S)-1-hydroxy-1-(4-nitrophenyl)-3-(trityloxy)propan-
2-yl) tetra-decanamide (2b).
4.1.5.
N-((1S,
2R)-1-hydroxyl-1-(4-nitrophenyl)-3-(trityloxy)
Crude compound 2b was purified by flush column chromatography
propan-2-yl)tetra-decanamide (4a) was prepared from 3a, crude prod-
at ethyl acetate-hexane, 1:2 (v/v) in yield of 81.8%. Rf 0.34; [α D22.3
+
]
uct 4a was purified by flush column chromatography at ethyl acetate-
hexane, 1:4 (v/v) in yield of 37% as waxy oil. Rf 0.16; [α D24.6
ꢀ 9.5 (C
]
27.0 (C 1.0, CH3OH); 1H NMR (400 MHz, CDCl3) 8.109 (d, J = 8.8, 2H),
7.444 (d, J = 8.8, 2H), 7.245–7.394 (m, 15H), 5.965 (d, J = 8.4, 1H),
5.064(d, J = 3.2, 1H), 4.198 (m, 1H), 3.949 (d, J = 3.6, 1H), 3.435 (dd,
J = 4.8, 4.8, 1H), 3.328 (dd, J = 3.6, 4.0, 1H), 2.063 (td, J = 7.2, 1.6,
1, CH3OH), 1H NMR (400 MHz, CDCl3) 8.02 (dd, J = 6.8, 2.4, 2H), 7.307
(m, 17H), 5.952 (d, J = 8.0, 1H), 5.038(dd, J = 3.2, 3.2, 1H), 4.456 (d, J
= 7.6, 1H), 4.379 (m, 1H), 3.267 (dd, J = 4.8, 4.8, 1H), 3.097 (dd, J =
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Bioorganic & Medicinal Chemistry 32 (2021) 116011
4.0, 3.2, 1H), 2.207 (t, J = 7.6, 2H), 1.622 (m, 2H), 1.242 (m, 20H),
0.863 (t, J = 6.8, 3H). ESI-MS (CH3OH, relative intensity, %): m/z
687.20 ([MNa]+, 100). Calcd. for C42H52N2O5 m/z 664.39, [M];
C42H52N2NaO5, m/z 687.38 [MNa].
4.2.4. Statistical analysis
Where indicated, data are represented as mean ± SD. Statistical
analysis was performed using two-sided t test, with p-value < 0.05
considered statistically significant.
4.1.6 N-((1R, 2S)-1-hydroxyl-1-(4-nitrophenyl)-3-(trityloxy)propan-
2-yl)tetra-decanamide (4b) was prepared from 3b, crude product 4b
was purified by flush column chromatography at ethyl acetate-hexane,
Declaration of Competing Interest
1:4 (v/v) in yield of 39.3% as waxy oil. Rf 0.16; [α D
]
22.4+8.0 (C 1,
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CH3OH); 1H NMR (400 MHz, CDCl3) 8.017 (dd, J = 7.2, 2.0, 2H),
7.332–7.237 (m, 17H), 5.964 (d, J = 8.0, 1H), 5.041(m, 1H), 4.46 (d, J
= 7.6, 1H), 4.388 (m, 1H), 3.263 (dd, J = 5.2, 4.8, 1H), 3.101 (dd, J =
4.0, 4.0, 1H), 2.204 (t, J = 6.8, 2H), 1.63–1.242 (m, 22H), 0.863 (t, J =
6.8, 3H). ESI-MS (CH3OH, relative intensity, %): m/z 687.13 ([MNa]+,
100). Calcd. for C42H52N2O5 m/z 664.39 [M]; C42H52N2NaO5 m/z
687.38 [MNa].
Acknowledgement
Financial support was provided in part by the National Cancer
Institute [PO1-CA097132 (YAH)], and National Institutes of Health-
National Center for Advancing Translational Sciences [UL1TR001450
Voucher Pilot Program (APB)]. Research was supported in part by the
Lipidomics Shared Resource, Hollings Cancer Center, Medical Univer-
sity of South Carolina [P30 CA138313] and the Lipidomics and Patho-
biology COBRE, Department Biochemistry, MUSC [P20 RR017677]. We
especially acknowledge the National Center for Research Resources and
the Office of the Director of the National Institutes of Health for the
funding [C06 RR018823], which provided laboratory space for Lip-
idomics Facility in MUSC’s Children’s Research Institute.
4.1.7. N-((1S, 2R)-1,3-dihydroxy-1-(4′-nitrophenyl)-propan-2-yl)
tetradecanamide ((1S, 2R) B13; 5a) was prepared from 4a, crude
product 5a was purified by flush column chromatography at chlor-
oform–methanol, 4:1 (v/v) in yield of 61.4% as waxy solid. Rf 0.31;
[
α 2D2.7
]
ꢀ 6.5 (C 0.25, CH3OH); 1H NMR (400 MHz, CDCl3) 8.229 (d, J =
8.8, 2H), 7.603 (d, J = 8.8, 2H), 6.312 (d, J = 8.0, 1H), 5.161(d, J = 3.2,
1H), 4.105 (m, 1H), 3.790 (dd, J = 3.2, 3.2, 1H), 3.646 (dd, J = 3.6, 3.6,
1H), 2.241 (t, J = 7.2, 2H), 1.239 (m, 20H), 0.862 (t, J = 6.8, 3H). ESI-
MS (CH3OH, relative intensity, %), m/z 423.18 ([MH]+, 100). Calcd. for
C23H38N2O5, m/z 422.28 [M]; C, 65.38; H, 9.06; N, 6.63. Found: C,
63.72; H, 9.06; N, 6.43.
4.1.8. N-((1R, 2S)-1,3-dihydroxy-1-(4′-nitrophenyl)-propan-2-yl)
tetradecanamide ((1R, 2S) B13; 5b) was prepared from 4b, crude
product 5b was purified by flush column chromatography at chlor-
oform–methanol, 4:1 (v/v) in yield of yield 84.2% as waxy solid. Rf 0.31;
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116011.
[
α 2D2.6
]
+5.1 (C 0.235, CH3OH); 1H NMR (400 MHz, CDCl3) 8.222 (d, J =
References
8.4, 2H), 7.598 (d, J = 8.4, 2H), 6.337 (d, J = 7.6, 1H), 5.161(d, J = 4.0,
1H), 4.098 (m, 1H), 3.780 (dd, J = 3.2, 3.2, 1H), 3.638 (dd, J = 3.6, 3.6,
1H), 2.232 (t, J = 7.2, 2H), 1.238 (m, 20H), 0.861 (t, J = 6.8, 3H). ESI-
MS (CH3OH, relative intensity, %), m/z 423.11 ([MH]+, 100). Calcd. for
C23H38N2O5, m/z 422.28 [M]; C, 65.38; H, 9.06; N, 6.63. Found: C,
64.74; H, 9.23; N, 6.51.
[1] Bielawska A, Linardic CM, Hannun YA. Ceramide-mediated biology. J Biol Chem.
1992;267(26):18493–18497. PMID: 1526986.
[2] Raisova M, Goltz G, Bektas M, Bielawska A, Riebeling C, Hossini AM, et al. Bcl-2
overexpression prevents apoptosis induced by ceramidase inhibitors in malignant
melanoma and HaCaT keratinocytes. FEBS Lett. 2002;516(1–3):47–52. PMID:
11959101.
[3] Bai A, Suzlc Z, Bielawski J, Pierce J, Rembisa B, Terzieva S. etal Targeting (cellular)
lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-
B13 ester prodrugs. Bioorg Med Chem. 2014;22(24):6933–6944. PMID: 25456083.
[4] Selzner M, Bielawska A, Morse MA, Rudiger HA, Sindram D, Hannun YA, et al.
Induction of apoptotic cell death and prevention of tumor growth by ceramide
analogues metastatic human colon cancer. Can. Res. 2001;61(3):1233–1240. PMID:
11221856.
4.2. Biology
4.2.1. Cell culture
The MCF7 human breast adenocarcinoma cell line, originally ob-
tained from American Type Culture Collection (ATCC, Manassas, VA)
and was cultured in complete medium (RPMI 1640 medium supple-
mented with 10% FBS, 2 mM L-glutamine and 100 µg/ml Normocin
(VWR)) and maintained under standard incubator condition (humidi-
fied atmosphere with 5% CO2 at 37 ◦C). Cells in the exponential growth
phase are harvested from the culture and used in all experiments.
[5] Samsel L, Zaidel G, Drumgoole HM, Jelovac D, Drachenberg C, Rhee JG, et al. The
ceramide analog, B13, induces apoptosis in prostate cancer cell lines and inhibits
tumor growth in prostate cancer xenografts. Prostate. 2004;58(4):382–393. PMID:
14968439.
[6] Szulc Z, Mayroo N, Bai A, Bielawski J, Liu X, Norris J, et al. Novel analogs of D-e-
MAPP and B13. Part1: synthesis and evaluation as potential anticancer agents.
Bioorg Med Chem. 2008;16(7):1015–1031. PMID: 17869115.
[7] Bai A, Szulc Z, Bielawski J, Mayroo N, Liu X, Norris J, et al. Synthesis and bio-
evaluation of omega-N-amino analogs of B13. Bioorg Med Chem. 2009;17(5):
1840–1848. PMID: 19217788.
4.2.2. Cell viability assay
The MTT assay was used to quantify viable cells according to the
literature.14 Results are expressed as % of control viable MCF7 cells,
with means ± st dev. of 5x replicates.
[8] Bhabak KP, Kleuser B, Huwiler A, Arenz C. Effective inhibition of acid and neutral
ceramidases by novel B13 and LCL464 analogues. Bioorg Med Chem. 2013;21(4):
874–882. PMID: 23312611.
[9] Ogretmen B, Pettus BJ, Rossi MJ, Wood R, Usta J, et al. Biochemical mechanisms of
the generation of endogenous long chain ceramide in response to exogenous short
chain ceramide in the A549 human lung adenocarcinoma cell line. Role for
endogenous ceramide in mediating the action of exogenous ceramide. J Biol Chem.
2002;277(15):12960–12969. PMID: 11815611.
4.2.3. Lipid extract preparation and UHPLC-MS/MS analysis for cellular
SL and B13 isomers
Advanced analysis of Cer species, Sph and endogenous B13 isomers
were performed on a Thermo Finnigan TSQ 7000, triple stage quadruple
mass spectrometer operating in a multiple reaction monitoring (MRM)
positive ionization mode as described.15 Final results were presented as
the level of SL or chemical (pmol) per phosphate (Pi); lipid phosphate
was determined from the Bligh and Dyer lipid extraction16 and
expressed as total phospholipid phosphate (nmol).
[10] Mullen TD, Spassieva S, Jenkins RW, et al. Selective knockdown of ceramide
synthases reveals complex inter-regulation of sphingolipid metabolism. J Lipid Res.
2011;52(1):68–77. PMID: 20940143.
[11] Bielawski J, Szulc Z, Hannun YA, Bielawska A. Simultaneous quantitative analysis
of bioactive sphingolipids by high-performance liquid chromatography-tandem
mass spectrometry. Methods. 2006;39(2):82–91. PMID: 16828308.
[12] Bielawska A, Bielawski J, Szulc Z, Mayroo N, Liu X, Bai A, Elojeimy S, et al. Novel
analogs of D-e-Mapp and B13. Part2. Signature effects on bioactive sphingolipids.
Bioorg Med Chem. 2008;16(2):1032–1045. PMID: 17881234.
[13] Bielawska A, Greenberg MS, Perry D, et al. (1S, 2R)-D-erythro-2-(N-myristoyla-
mino)-1-phenyl-1-propanol as an inhibitor of ceramidase. J Biol Chem. 1996;271
(21):12646–12654. PMID: 8647877.
7

A. Bai et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116011
[14] Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a
precise and rapid dye method for measuring cell growth/cell kill. J Immunol
Methods. 1989;119(2):203–210. PMID: 2471825.
[15] Bielawski J, Pierce JS, Snider J, Rembiesa B, Szulc Z, Bielawska A. Sphingolipids
analysis by high performance liquid chromatography-tandem mass spectrometry
(HPLC-MS/MS). Adv Exp Med Biol. 2010;688:46–59. PMID: 20919645.
[16] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J
Biochem Physiol. 1959;37(8):911–917. PMID: 13671378.
8