Discovery, synthesis and biological evaluation of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel sphingomyelin synthase 1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.07.060

Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC₅₀ value of 5.2 μM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found to be the most potent SMS1 inhibitor with an IC₅₀ value of 2.1 μM in in vitro assay. The molecular docking studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphingomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would advance the development of more effective SMS1 inhibitors.

Full text of DOI:10.1016/j.bmc.2015.07.060

Bioorganic & Medicinal Chemistry 23 (2015) 6173–6184
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery, synthesis and biological evaluation of 2-(4-(N-
phenethylsulfamoyl)phenoxy)acetamides (SAPAs) as novel
sphingomyelin synthase 1 inhibitors
Ya-li Li a, Xiang-yu Qi a, Hui Jiang b, Xiao-dong Deng a, Yan-ping Dong d, Ting-bo Ding a, Lu Zhou a,
Peng Men a, Yong Chu a, Ren-xiao Wang c, , Xian-cheng Jiang a,b, , De-yong Ye a,
⇑
⇑
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, No. 826, Zhangheng Rd., Shanghai 201203, China
b State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA
c State Key Lab of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
d Department of Food and Pharmaceutical Engineering, Suihua University, Suihua 152061, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingomyelin synthase (SMS) has been proved to be a potential drug target for the treatment of
atherosclerosis. However, few SMS inhibitors have been reported. In this paper, structure-based virtual
screening was performed on hSMS1. SAPA 1a was discovered as a novel SMS1 inhibitor with an IC50 value
Received 26 June 2015
Revised 26 July 2015
Accepted 27 July 2015
Available online 30 July 2015
of 5.2
synthesized and their biological activities toward SMS1 were evaluated. Among them, SAPA 1j was found
to be the most potent SMS1 inhibitor with an IC50 value of 2.1 M in in vitro assay. The molecular docking
lM in enzymatic assay. A series of 2-(4-(N-phenethylsulfamoyl)phenoxy)acetamides (SAPAs) were
l
Keywords:
Sphingomyelin synthase
Inhibitor
Structure-based virtual screening
Molecular docking
Point mutagenesis
studies suggested the interaction modes of SMS1 inhibitors and PC with the active site of SMS1. Site-
directed mutagenesis validated the involvement of residues Arg342 and Tyr338 in enzymatic sphin-
gomyelin production. The discovery of SAPA derivatives as a novel class of SMS1 inhibitors would
advance the development of more effective SMS1 inhibitors.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
polar lipoprotein lipids.3 Furthermore, plasma SM level has been
proved as an independent risk factor for coronary artery disease
Sphingomyelin (SM) is the most abundant sphingolipid in
plasma membrane. It also accounts for approximately 20 percent
of the phospholipids in human plasma, but 63–75 percent of
plasma SM exists in atherogenic low density lipoprotein (LDL)
and very low density lipoprotein (VLDL).1,2 Epidemic investigations
have revealed that patients with type II hyperlipidemia have
increased plasma SM levels and increased proportions of SM in
in humans.4,5
Sphingomyelin synthase (SMS) is the last enzyme in SM
biosynthetic pathway. It transfers the phosphocholine head group
of phosphatidylcholine (PC) onto ceramide and thus releases SM
and diacylglycerol (DAG).6 As a SMS isoform, SMS1 was expressed
ubiquitously in all the tested tissues. However, SMS1 is the major
SMS in macrophages. SMS1 is primarily localized to the trans
Golgi7,8, and is a key factor in the control of SM level in the cells
and on the cell plasma membrane.6,9 Over-expression of SMS1 sig-
nificantly increased the level of intracellular SM, atherogenic
potential and subsequent atherosclerotic lesions in mice.10,11 Con-
trarily, SMS1 deficiency could reduce plasma and cell membrane
SM levels and attenuate atherogenesis in mice.12,13 Thus SMS1
might serve as a potential therapeutic target of atherosclerosis.
SMS1 inhibitors would be potent drugs for the treatment of
atherosclerosis.14
Abbreviations: SM, sphingomyelin; SMS, sphingomyelin synthase; PC,
phosphatidylcholine; DAG, diacylglycerol; TM, transmembrane; hSMS1, human
sphingomyelin synthase 1; His, histidine; Asp, aspartic acid; SAPA, 2-(4-(N-phenethyl-
sulfamoyl)phenoxy)acetamide; SAR, structure–activity relationships; Phe, phenylala-
nine; Leu, leucine; Ala, alanine; Arg, arginine; Trp, tryptophane; Tyr, tyrosine;
C6-NBD-Cer,
6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)-sphin-
gosine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; C6-NBD-SM, N-(N-(7-
nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl)sphingosyl
choline; HPLC, high performance liquid chromatography; IC50
concentration.
phosphoryl
50% inhibitory
,
There has been no report of SMS1 purified in its active form so
far. However, human SMS1 has been cloned more than ten years
ago.15,16 SMS1 has an N-terminal Sterile Alpha Motif (SAM) which
has been proved to have no effect on SMS activity.7,17 Except the
⇑
Corresponding authors. Tel.: +86 21 54925128 (R.W.); tel.: +1 718 270 6701
(X.J.); tel./fax: +86 21 51980125 (D.Y.).
E-mail addresses: wangrx@mail.sioc.ac.cn (R.-x. Wang), xjiang@downstate.edu
(X.-c. Jiang), dyye@shmu.edu.cn (D.-y. Ye).
http://dx.doi.org/10.1016/j.bmc.2015.07.060
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
SAM domain, the six transmembrane (TM1–TM6) and four highly
conserved motifs (D1–D4) of hSMS1 reside on the sequence of
M130-Q353 amino acids.15,16 Two conserved motifs D3 (C277-
T286 aa) and D4 (H328-H348 aa) localize at TM4 and TM6, respec-
tively. Especially, motif D4 (H328-H348 aa) at TM6 is critical for
the catalytic function of SMS1.18 For the discovery of SMS1
inhibitor, a three-dimensional structure of human sphingomyelin
synthase 1 (hSMS1, M130-Q353 aa) has been built by homology
modeling, optimized through molecular dynamics and proved the
rationality.19 Moreover, a domain locating on motifs D3 and D4
of hSMS1 was discovered to be the active site for SM production.15
Three residues His285, His328 and Asp332 in motifs D3 and D4,
forming a HHD triad, have been proved to be key amino acids
involved in SM production by site-directed mutagenesis.17 How-
ever, the roles of other amino acids in the active site as well as
the catalytic mechanism involved in SM production were still
under investigation.
library (http://www.specs.net) was updated and contained over
220,000 compounds which were to be screened through molecu-
lar docking. The reported structural model of hSMS119 was energy
optimized before being employed in the virtual screening. The
molecular docking was conducted by targeting the validated
active site of hSMS1 and performed on the GOLD software and
the GLIDE software sequentially. A total of 95 compounds were
finally selected among the top-ranked candidates after visual
examination. The screening procedure was fully detailed in
Section 4.
Samples of these 95 compounds were purchased from SPECS Inc
and tested for their biological activities without further purifica-
tion. The SMS1 inhibitory activities of these compounds were
evaluated in an in vitro enzymatic assay. Among them, 2-(4-(N-
phenethylsulfamoyl)phenoxy)acetamide (SAPA) derivative 1a
(Fig. 2), the only active compound, exhibited dose-dependent inhi-
bition toward SMS1 overexpressed Hela cell lysate with an IC50
Up to now, very few SMS inhibitors have been reported in the
literatures. Potassium tricyclo[5.2.1.0(2,6)]-decan-8-yl-dithiocar-
bonate D609 (Fig. 1), which was firstly found as a cytotoxic anti-
virus and anti-tumor reagent,20,21 was reported with a weak SMS
inhibitory effect in numerous enzyme or cell types in vitro
value of 5.2
potent than D609 which showed modest SMS1 inhibitory activity
with an IC50 value of 219 M (Fig. 3b). Therefore SAPA 1a, as a
novel scaffold of SMS1 inhibitors, was considered to be the lead
compound for further structural modification.
lM (Fig. 3a). It was approximately forty fold more
l
(IC50 = 177–600 l
M).6,22–24 But the highly instable structure of
D609 (t1/2 = 19.5 min in saline solution at 24 °C) hindered its fur-
ther development to become an effective SMS inhibitor. Although
the prodrug modification of D609 could improve its chemical sta-
bility,23 the inhibitory activity toward SMS should be enhanced.
2.2. Chemistry
SAPA derivatives were designed and synthesized following the
general route described in Scheme 1. The N-substituted-2-
chloroacetamides 3a–3r were synthesized by the nucleophilic sub-
stitution reaction of various substituted aniline or benzylamine
2a–2r with chloracetyl chloride in the presence of pyridine.
Besides the key intermediate, 4-hydroxybenzene-1-sulfonyl chlo-
ride 5 was obtained from chloroformylation of compound 4 by
thionyl chloride with catalytic amount of DMF. Then compounds
7a–7d were prepared by the reaction of the intermediate 5 with
different amines 6a–6d in moderate yields after recrystallization.
Finally the target compounds SAPA 1a and 1d–1w were achieved
through the general electrophilic substitution of compounds 7a–
7d by 2-chloroacetamides 3a–3r. The yields were in the range of
30–80% and the unfavorable attacking of nitrogen atom on sulfon-
amide group could be avoided by controlling temperature and ratio
of substrates.
In order to investigate the role of sulfonyl group on 1a, SAPA
analogues 1b and 1c were designed and synthesized following
the route described in Scheme 2. 4-Hydroxybenzoic acid 8 was
treated with thionyl chloride in methanol under reflux condition
to afford the corresponding methyl ester 9. Then the intermediate
10 was afforded by the reaction of compound 9 with N-substi-
tuted-2-chloroacetamides 3a. Subsequently the hydrolysis of com-
pound 10 was carried out to give compound 11, which was treated
respectively with amines 6a and 6e to afford the corresponding
SAPA analogues 1b and 1c.
Furthermore,
a-aminonitrile derivatives have been identified as
the first series of SMS inhibitors discovered through rational
design. Among them, the representative compound D2 (Fig. 1)
exhibited more potent SMS inhibitory activity than D609.25,26
However, the molecule of compound D2 contains
a-aminonitrile
group, which is regarded as a potential toxigenic structure. There-
fore, more effective SMS1 inhibitors should be discovered to
understand the role of SMS1 in cell functions and animal pheno-
types as well as to meet the further study of SMS1 inhibitors as
potent therapeutical drugs for the treatment of atherosclerosis.
In this study, structure-based virtual screening was performed
to discover novel SMS1 inhibitors. A series of SAPA derivatives
were designed and synthesized through structure sectionalized
modification for in vitro enzymatic assay and SAR studies. Fortu-
nately, six of them showed potent SMS1 inhibitory activities with
IC50 values lower than 10 lM in enzymatic assay. The binding
modes of SMS1 inhibitors and substrate PC with hSMS1 were
investigated. The involvement of residues Arg342 and Tyr338 at
the active site of hSMS1 in SM production were evaluated by
site-directed mutagenesis.
2. Results and discussion
2.1. Structure-based virtual screening of the SPECS library
The structures of all new compounds were elucidated from
their analytical and spectroscopic data which were collected in
the Section 4.
A multi-step structure-based virtual screening was employed
to discover potential small molecule SMS1 inhibitors. The SPECS
CN
O
S
O
R
O
S-K+
N
H
S
O
S
O
R = -CH3
or R = -CH2CH2CH3
or R = -C(CH3)3
D609
D2
IC50 = 25 µM on SMS
IC50 = 177-600 µM on SMS
prodrugsof D609
Figure 1. Chemical structures of reported sphingomyelin synthase inhibitors.

Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
6175
2.4. Structure–activity relationships (SAR) study
O
O
N
H
In order to explore the interesting enzymatic profile, a prelimi-
nary structure–activity relationship of SAPA derivatives was ana-
lyzed based on the results of in vitro SMS1 enzymatic assays.
Firstly, the substitution of sulfonyl group (ASO2A, 1a) by car-
bonyl group (AC@OA, 1b) in SAPA derivatives could eliminate
SMS1 inhibitory activities remarkably. It was indicated that the
carbonyl group would diminish the interaction of SMS1 with sul-
fonyl group which might be vital for SMS1 inhibition. However,
it should also be noticed that the sulfonyl group would not be
the sole contributor to the SMS1 inhibitory activities of SAPA
derivatives. The optimal size of chain between the sulfonamide
(ASO2NHA) with hydrophobic G group (1a vs 1d–1f) would also
be necessary for SMS1 inhibitory activities. Moreover, SMS1 inhibi-
tory activity was dramatically attenuated by the two methylene
chain linking A ring with the amide (ACONHA, 1g vs 1h). It sug-
gested that compound with longer chain linking A ring with the
amide would not be accommodated by the limited domain of
SMS1. Furthermore, the substituent R group attached to the A ring
was also important for maintaining SMS1 inhibitory activities of
SAPA derivatives. Both the steric and electronic effect of R group
could contribute to the inhibitory activities of SAPA derivatives
against SMS1. The introducing of substituent R group on para posi-
tion of A ring (1h vs 1j) would be favorable for improving the inhi-
bitory activity when the A ring was linked with the amide
(ACONHA) by a methylene chain.
O
S
Cl
N
O
H
SAPA 1a
IC50 = 5.2 µM on SMS1
Figure 2. The chemical structure and SMS1 inhibitory activity of SAPA 1a.
2.3. Biological evaluation
All the newly prepared SAPA derivatives were evaluated for
their SMS1 inhibitory activities with the publicly known inhibitor
D609 as the positive control. SMS1 overexpressed Hela cell lysate
was prepared according to a method described in Section 4. The
Hela cell lysate was pre-incubated with the corresponding SAPA
derivatives at 37 °C for 30 min in the presence of test buffer.
C6-NBD-Cer and DMPC were added as substrates into the biolog-
ical test system. After the mixture was incubated at 37 °C for an
additional 2 h, the resulting mixture was quenched and cen-
trifuged. The amount of C6-NBD-Cer and C6-NBD-SM in the
supernatant liquid were quantified by the fluorescent HPLC-based
analysis.27 Finally, the inhibitory activities of SAPA derivatives
were evaluated by the decrease of the product C6-NBD-SM com-
pared with control groups. The SMS1 inhibitory activities of SAPA
derivatives were showed in Table 1. The IC50 values represent the
compound concentration at which 50 percent of SMS1 activity
was inhibited.
The structure–activity relationships achieved by SAPA deriva-
tives would be helpful for developing more potent SMS1 inhibitors.
2.5. Molecular docking study
The results of biological evaluation exhibited that half of the
SAPA derivatives could inhibit SMS1 activity with IC50 values lower
Molecular docking study was firstly performed using the GOLD
software to investigate the interaction of SMS1 inhibitors with
hSMS1. The binding mode of SAPA 1a with hSMS1 was showed
in Figure 5a and b. As it was shown, SAPA 1a located at the active
site of hSMS1 which was responsible for SM production and thus
blocked the entry of PC as a substrate molecule. The segment of
the amide (ACONHA) with 4-chlorobenzyl group of SAPA 1a
reached into the interior of the domain where His285, His328
and Asp332 formed a triad, and was enveloped by hydrophobic
residues Phe173, Phe177, Leu281 and Ala325. Meanwhile, the
than 25
potent SMS1 inhibitory activities with IC50 values lower than
10 M. The SMS1 inhibitory activities of SAPA 1j, 1k, 1n and 1v
were shown in Figure 4a–d respectively. Especially, SAPA 1j with
the IC50 value of 2.1 M on SMS1 inhibition was approximately
lM. Among them, SAPA 1a, 1i, 1j, 1k, 1n and 1v showed
l
l
one hundred fold more potent than D609. Therefore, SAPA 1j
became the most potent SMS1 inhibitor to the best of our
knowledge.
Figure 3. The SMS1 inhibitory activities of SAPA 1a and D609. The dose-dependent inhibition by three concentrations of SAPA 1a (2.5, 10 and 50
lM, respectively) against
SMS1 is shown in Figure 3a. The modest inhibition by three concentrations of D609 (50, 250 and 500 M, respectively) against SMS1 is shown in Figure 3b. The results are
l
expressed as percent of SMS1 activity in control groups in which there is no SMS1 inhibitor. The shown values are the means of triplicate assays. Values are shown in
means SD.

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Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
O
Cl
NH2
O
n
a
N
H
n
R
+
R
Cl
Cl
Cl
2a-2r
3a-3r
n = 0~2
n = 0~2
H
N
O
O
O
Na+O-
G
O
S
S
S
m
O
N
G
m
d
S
c
b
O
O
H
N
H
R
O
O
3a-3r
n
G
NH2
m
HO
O
HO
OH
7a-7d
4
5
6a-6d
1a, 1d-1w
n = 0~2
m = 0~3
6a m = 2, G = phenyl
6b m = 3, G = phenyl
6c m = 1, G = 4-chlorophenyl
6d m = 0, G = phenyl
Scheme 1. General synthesis route for SAPA derivatives 1a and 1d–1w. Reagents and conditions: (a) H2O/pyridine; (b) SOCl2, DMF; (c) CH2Cl2, pyridine; (d) K2CO3, KI,
acetone, 60 °C.
O
O
O
Cl
a
OH
b
O
O
c
H
N
O
HO
O
HO
8
9
Cl
O
N
H
10
Cl
3a
O
O
Cl
Cl
OH
d
G
N
m
H
N
H
N
H
O
O
G
NH2
m
O
O
1b, 1c
11
6a, 6e
6a m = 2, G = phenyl
6e m = 0, G = 4-pyridyl
Scheme 2. Synthesis route for SAPA analogues 1b and 1c. Reagents and conditions: (a) SOCl2, CH3OH; (b) K2CO3, CH3CN; (c) LiOH, H2O/THF; (d) EDCI/HOBt, CH2Cl2.
sulfonamide (ASO2NHA) group on SAPA 1a interacted with residue
Arg342 in motif D4 by forming hydrogen bond (2.4 Å) which would
fix SAPA 1a into the binding pocket. The unique interactions with
residue Arg342 of hSMS1 were not detected in the reported bind-
SAPA 1b had no interaction with residue Arg342 in motif D4. The
carbonyl group (AC@OA) of SAPA 1b demolished the hydrogen
bonds formed between the sulfonamide (ASO2NHA) group of SAPA
1a and Arg342. The p–p interaction was also weakened by a dihe-
ing mode of compound D2 with hSMS1.25 Furthermore, a
p
–p
dral angle formed by the middle benzene ring of SAPA 1b and the
imidazole ring of residue His285. The disadvantageous conforma-
tion of SAPA 1b in the active site of hSMS1 might account for its
remarkable decrease in SMS1 inhibitory activity. It also reinforced
the hypothesis that the interaction with residue Arg342 in motif
D4 might be important for SAPA derivatives and D609 to hold their
SMS1 inhibitory activities.
Finally, the interaction of substrate PC with hSMS1 was
explored and illustrated in Figure 5g and h. The phosphocholine
head group of PC reached into the HHD triad (His285, His328
and Asp332) formed domain, and was surrounded by a hydropho-
bic patch encircled by residues Phe173, Phe177, Leu281 and
Ala325. In addition, substrate PC interacted with residue Arg342
in motif D4 by forming two hydrogen bonds (1.7 Å and 2.1 Å)
which were more effective than that of SAPA 1a with residue
Arg342. The hydrogen bond formed between PC and residue
Arg342 with the help of residue Tyr338 as its partner was favorable
for locking the conformation of PC to facilitate the catalytic reac-
tion. The two carbon chain tails of PC lay in the groove on the sur-
face of hSMS1 (Fig. 5h). It should be noticed that residue Arg342
interaction was found between the middle benzene ring of SAPA
1a and the imidazole ring of residue His285. Finally, the
phenylethylamino group connecting with sulfonyl group lay fitly
at a hydrophobic site encircled by residues Leu289 and Trp346.
The binding mode of D609 with hSMS1 was also illustrated
(Fig. 5c and d) and compared with the mode of SAPA 1a with
hSMS1. As it was reported, the negative charged sulfur atom on
D609 formed
a strong salt-bridge interaction with residue
Arg342 under the support of residue Tyr338 which partly mim-
icked the hydrogen bonds between SAPA 1a and hSMS1. It should
be noted that there is no interaction formed between D609 with
the interior of the active site as SAPA 1a did. The weak interaction
of D609 with hSMS1 may be the reason that it was less effective
than SAPA 1a toward SMS1 inhibition.
The interaction of SAPA 1b with hSMS1 was further studied and
revealed in Figure 5e and f. As the mode of SAPA 1a with hSMS1,
SAPA 1b located at the active site of hSMS1. The segment of the
amide (ACONHA) with 4-chlorobenzyl group of SAPA 1b reached
into the interior of the domain. However, as shown in Figure 5e,

Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
6177
Table 1
shown in Figure 6, Y338A and R342A significantly reduced their
SMS1 activity. Notably, these experimental results fit very well
with a previous virtual mutation analysis showing that R342A or
Y338A causes a significant loss of binding energy in comparison
with wild type.19 The result indicated that both Arg342 and
Tyr338 have an effect on the catalytic activity of SMS1 and thus
affect the SM production. It should be noted that, unlike H285A,
H328A and D332A, Y338A and R342A do not eliminate the SMS1
activities utterly. Therefore, Arg342 and Tyr338 might play sup-
portive roles in the catalytic SM production. The involvement of
residues Arg342 and Tyr338 in SM production was helpful for
understanding the mysterious catalytic mechanism of the SM pro-
duction and for guiding the design of SMS1 inhibitors.
SMS1 inhibitory activities of SAPA derivatives 1a–1w
H
G
N
Z
m
H
N
R
A
B
O
n
O
Compound
Z
G
m
n
R
SMS1
IC50
(lM)
a
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
Sulfonyl
Carbonyl
Carbonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Sulfonyl
Phenyl
Phenyl
2
2
0
3
1
0
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
2
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
-H
5.2
>100
>100
>100
>50
>100
>50
23.1
5.9
2.1
Pyridin-4-yl
Phenyl
4-Chlorophenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
3. Conclusion
In summary, we have discovered SAPA 1a as a novel SMS1 inhi-
bitor through structure-based virtual screening. Twenty three
SAPA derivatives were synthesized and half of them exhibited
more potent SMS1 inhibitory activity than D609. Among them,
-H
4-OCH3
4-Br
4-F
3-F
-H
4-Cl
8.1
15.6
14.7
6.6
10.5
>50
14.2
>100
>100
>100
>100
9.2
SAPA 1j is the most potent SMS1 inhibitor (IC50 = 2.1 lM). The
interaction modes of SMS1 inhibitors with hSMS1 were disclosed
through molecular docking studies. The involvement of residues
Arg342 and Tyr338 in the catalytic SM production were evaluated
through point mutagenesis for the first time. Our study would pro-
vide a foundation for the further study of SMS1 catalytic mecha-
nism and for the development of more potent SMS1 inhibitors.
4-F
4-OCH3
2-OCH3
2-CH3
2-NO2
2-CN
2-OCF3
3-OCH3
2-OCH3, 4-F
1s
1t
1u
1v
1w
D609
4. Experimental protocols
>100
219b
4.1. Structure-based virtual screening
a
IC50 values are the means of three separate determinations on SMS1 overex-
pressed Hela cell lysate and were determined by more than five concentrations of
each inhibitor.
The compound D609 was used as a positive control in the biological assay.
Statistical calculation of IC50 values was performed on GraphPad Prism 5.02
(GraphPad Software, Inc.).
A multi-step structure-based virtual screening was employed to
discover potential small molecule SMS inhibitors. Firstly, the entire
SPECS library (ꢀ220,000 compounds, from http://www.specs.net)
was filtered by drug-likeness rules, which required qualified candi-
dates to have molecular weight lower than 1000 daltons, number
of hydrogen bond acceptors and donors between 2 and 10, number
of non-hydrogen atoms between 9 and 50, and no more than 10
rotatable single bonds. In this step we selected out approximately
80,000 qualified molecules. Then, these molecules were docked
into the active site of hSMS1 using the GOLD software suite
(version 5.0, released by CCDC). The hSMS1 structure was kept
fixed during the entire docking process. Key parameters used in
this docking task were set as follows: Island number = 5; popula-
tion size on each island = 100; total genetic algorithm opera-
tions = 30,000; mutation rate = 95%; crossover rate = 95%;
migration rate = 10%; scoring function = ChemScore. Up to twenty
binding poses were generated for each docked molecule. After this
step, 20,000 molecules with the highest binding scores were
selected out.
b
was involved in the interaction of hSMS1 not only with SMS1 inhi-
bitors but also with substrate PC. Meanwhile, the cooperation
between residues Arg342 and Tyr338 on the same motif D4 includ-
ing a hydrogen bond (1.9 Å) and
p–cation interaction were also
detected in the docking modes of hSMS1 with SMS1 inhibitors as
well as substrate PC.
The docking studies could illuminate the SMS1 inhibitory activ-
ity and the SAR of SAPA derivatives. The predicted role of residue
Arg342 in the binding of hSMS1 with substrate PC or SMS1 inhibi-
tors as well as its interaction with Tyr338 aroused our curiosity to
investigate the unclear function of these residues in the catalytic
reaction of SM production by SMS1.
2.6. Point mutagenesis
These molecules selected by GOLD were further screened by the
Glide module implemented in the Schrödinger software (version
2008, released by the Schrödinger Inc.) at the ‘extra-precision’
(XP) mode. Default parameters were employed in molecular dock-
ing. The hSMS1 structure was also kept fixed during this process.
Then, the top-ranked 4000 molecules were subjected to similarity
analysis to remove structurally redundant ones. Similarity matrix
based on molecular fingerprint ECFP4 was generated by using
Pipeline Pilot (released by Accelrys Inc.). A total of 500 clusters
were obtained based on the similarity matrix. The compound with
the highest Glide binding score was selected from each cluster.
These 500 compounds were visually examined to eliminate (i)
those in unreasonable binding poses, and (ii) those containing
undesired chemical moieties or not suitable for further chemical
According to the putative five-step catalytic mechanism
involved in SM production, the initiation of the reaction is involved
in the binding of a two-chain choline phospholipid PC to the active
site. Subsequently, His328 and Asp332 act coordinately to
mediate a nucleophilic attack on phosphomonoester bond.15 How-
ever, the catalytic triad, His285, His328 and Asp332, in the center
of the enzyme, needs support of other amino acid residues.
To evaluate the role of residues Arg342 and Tyr338 which are in
motif D4 and the active site of SMS1, we replaced them with
alanine and prepared Y338A and R342A respectively, using
site-directed mutagenesis approach. All the methods were per-
formed and modified as the study published by Yeang et.al.17 As

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Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
Figure 4. The SMS1 inhibitory activities of SAPA derivatives. The dose-dependent inhibition by different concentrations of SAPA 1j, 1k, 1n and 1v (2.5, 10 and 50 lM) against
SMS1 are shown respectively in Figure 4a–d. The results are expressed as percent of SMS1 activity in control groups in which there is no SMS1 inhibitor. The shown values are
the means of triplicate assays. Values are shown in means SD.
modification. Finally, a total of 95 compounds were selected as
candidates. Samples of these compounds were purchased from
the SPECS Inc. for subsequent biological evaluations.
quaternary pump, a G1329B ALS and a G4212B DAD detector.
The HPLC method consisted of the following: Agilent C18 RP col-
umn (250 mm  4.6 mm, 5
lm); column temperature 25 °C; inject
volume 20 l; HPLC solvent H2O/CH3OH (0.1% TFA) = 30/70 (v/v);
l
4.2. Chemistry
flow rate of 1.0 mL/min; detector wavelength of 254 nm. Retention
times (tR) were in minutes. Melting points were determined by a
SGW X-4 thermometer and were uncorrected (slide method). The
yield was not optimized.
Reagents were purchased from commercial sources and used
without further purification except special case. Flash column
chromatography was carried out at medium pressure using silica
gel (200–300 mesh) purchased from Qingdao Haiyang Chemical
Co. Ltd. All the reactions were monitored by thin layer chromatog-
raphy (TLC) on silica gel. Mass spectra data were given with electro
spray ionization (ESI) produced by a Finnigan MAT-95, LCQ-DECA
spectrometer and IonSpec 4.7 T. MS. High resolution mass spectra
data were given by AB 5600+ Q TOF. 1H NMR and 13C NMR spectra
data were obtained on Varian Mercury Plus 400 spectrometers
working at 400 MHz or Bruker Ascend™ 600 spectrometers work-
ing at 600 MHz. Chemical shifts (d) were reported in parts per mil-
lion (ppm) relative to internal tetramethylsilane (TMS) and J values
were reported in Hertz. Peak multiplicity were described as singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br),
broad singlet (br s), and broad multiplet (br m). The purities of
all tested SAPA derivatives were higher than 95% by HPLC, which
were performed on an Agilent 1260 HPLC system with a G1311B
4.2.1. The preparation of 2-(4-(N-phenethylsulfamoyl)pheno-
xy)acetamides (SAPA 1a and 1d–1w)
4.2.1.1. 4-Hydroxybenzene-1-sulfonyl chloride (5).
A mix-
ture of sodium 4-hydroxybenzenesulfonate 4 (1.96 g, 10 mmol)
in thionyl chloride (3.7 ml) and catalytic DMF (0.06 ml) was stirred
at 60 °C for 3.5 h. After cooling to ambient temperature, the reac-
tion mixture was poured into ice water (10 ml) and extracted by
dichloromethane, washed with brine, dried over Na2SO4. The
organic solvent was evaporated and compound 5 was obtained
as 1.93 g colorless oil. ESI-MS m/z 192.9 (M+H)+.
4.2.1.2.
(7a–7d).
4-Hydroxy-N-substituted-benzenesulfonamides
General procedure: To solution of amine
a
(10 mmol) in dichloromethane (20 ml) and pyridine (1.6 ml) was
added the solution of 4-hydroxybenzene-1-sulfonyl chloride 5

Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
6179
Figure 5. Docking studies of SMS1 inhibitors and PC at the active site of hSMS1. The docking studies were performed on the GOLD software suite (version 5.0, released by
CCDC). The interaction of SAPA 1a, D609, SAPA 1b and substrate PC with hSMS1 displayed by solid ribbons are shown in Figure 5a, c, e and g. The interaction of SAPA 1a, D609,
SAPA 1b and substrate PC with hSMS1 displayed the protein surface are shown respectively in Figure 5b, d, f and h. The ribbons in colors stand for the six transmembrane
(TM1-TM6) of hSMS1. The active site of hSMS1 composed of motifs D3 (C277-T286 aa) and D4 (H328-H348 aa) localizes at TM4 (ribbon in brownish red) and TM6 (ribbon in
red) respectively. Black dashed lines stand for hydrogen bonds or salt bridges. The length of hydrogen bonds or salt bridges is shown by characters in blue with the unit Å. The
protein surface of hSMS1 is shown in electrostatic surface with transparence. The yellow arrows on the protein in Figure 5b, f and h point to the interior of hSMS1 that SAPA
1a, SAPA 1b and substrate PC reached.
Figure 6. The activities and protein levels of wild type and point mutated SMS1. The SMS1 activities were performed on wild type (WT) or mutant SMS1 (Y338A and R342A)
and results are shown in Figuer 6a. The protein levels of each enzyme are shown in a western blot in Figure 6a. The quantitative comparison of wild type SMS1 (WT) with
point mutated SMS1 (Y338A and R342A) on SMS1 activity is shown in Figure 6b. The shown values are the means of triplicate assays. Values are means SD.
(1.93 g, 10 mmol) in dichloromethane (10 ml) slowly at 0 °C. The
reaction mixture was stirred at room temperature for 1.5 h,
washed with 1 mol/L HCl (aq) and brine, and then dried over Na2-
SO4. After the organic solvent was evaporated, the crude product
was recrystallized in dichloromethane and 4-hydroxy-N-substi-
tuted-benzenesulfonamide 7a–7d were obtained as light yellow
solid (two steps yield 30–40%). 7a mp 137.1–139.8 °C. ESI-MS
m/z 276.1 (MÀH)À. 1H NMR (400 MHz, DMSO-d6) d 10.37 (s, 1H),
7.61 (d, J = 8.6 Hz, 2H), 7.44 (t, J = 5.7 Hz, 1H), 7.26 (t, J = 7.3 Hz,
2H), 7.19 (d, J = 6.7 Hz, 1H), 7.14 (d, J = 7.4 Hz, 2H), 6.90 (d,
J = 8.6 Hz, 2H), 2.90 (dd, J = 14.2, 6.7 Hz, 2H), 2.66 (t, J = 7.5 Hz,
2H). 13C NMR (151 MHz, DMSO-d6) d 161.3, 139.3, 130.8, 129.3,
129.1, 128.8, 126.7, 116.0, 44.6, 35.7.
4.2.1.3. N-Substituted-2-chloroacetamides (3a–3r).
General
procedure: To a solution of aniline or benzylamine (10 mmol) in
water (20 ml) and pyridine (1.6 ml) was added chloracetyl chloride
(1.69 g, 15 mmol) slowly at 0 °C. The reaction mixture was stirred
at 0 °C for 1.0 h and then diluted with water (50 ml). The precipi-
tate was collected by filtration, washed with water and dried under
vacuum. N-substituted-2-chloroacetamides 3a–3r were obtained
as solid (yield 50–80%).
4.2.1.4.
N-(4-Chlorobenzyl)-2-(4-(N-phenethylsulfamoyl)phe-
mixture of 2-chloro-N-(4-
noxy)acetamide (SAPA 1a).
A
chlorobenzyl)acetamide 3a (0.22 g, 1.0 mmol), 4-hydroxy-N-phe-
nethyl-benzenesulfonamide 7a (0.42 g, 1.5 mmol), K2CO3 (0.21 g,

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Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
1.5 mmol) and KI (33 mg, 0.2 mmol) in acetone (20 ml) was stirred
at 60 °C for 7.0 h. After the organic solvent was evaporated, the
residual was diluted with water (20 ml), extracted with dichloro-
methane, washed with brine and then dried over Na2SO4. After fil-
tration and condensation, the crude product was obtained and
recrystallized in ethyl acetate/hexane (1:1, v: v) to afford 1a as
white solid (0.19 g, yield 42%): mp 151.4–153.3 °C. ESI-MS m/z
459.2 (M+H)+. HRMS (ESI) of C23H23ClN2O4S (M+H)+ calcd,
459.1140; found, 459.1145. 1H NMR (400 MHz, DMSO-d6) d 8.74
(t, J = 6.1 Hz, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.58 (t, J = 5.5 Hz, 1H),
7.34 (d, J = 8.3 Hz, 2H), 7.24 (dd, J = 7.9, 6.1 Hz, 4H), 7.17 (d,
J = 7.3 Hz, 1H), 7.11 (t, J = 7.9 Hz, 4H), 4.65 (s, 2H), 4.30 (d,
J = 6.1 Hz, 2H), 2.89 (dd, J = 13.2, 7.1 Hz, 2H), 2.65 (t, J = 7.5 Hz,
2H). 13C NMR (101 MHz, DMSO-d6) d 167.2, 160.4, 138.6, 138.2,
132.6, 131.3, 129.0, 128.6, 128.3, 128.1, 126.2, 115.1, 66.9, 44.0,
41.1, 35.1. HPLC purity: tR = 10.742, 98.5%.
J = 7.1 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 4.55 (s, 2H), 3.35 (s, 2H),
2.90 (dd, J = 13.4, 7.1 Hz, 2H), 2.72 (t, J = 7.3 Hz, 2H), 2.65 (t,
J = 7.5 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 166.8, 160.4,
139.1, 138.6, 132.5, 128.5, 128.2, 126.1, 125.9, 114.9, 66.8, 43.9,
39.8, 35.1, 34.9. HPLC purity: tR = 8.995, 98.0%.
4.2.1.9.
etamide (SAPA 1h).
N-Benzyl-2-(4-(N-phenethylsulfamoyl)phenoxy)ac-
SAPA 1h was prepared by the reaction
of N-benzyl-2-chloroacetamide 3c with 7a according to the proce-
dure described for SAPA 1a. Yield 62%; mp 118.8–121.4 °C. ESI-MS
m/z 425.2 (M+H)+. HRMS (ESI) of C23H24N2O4S (M+H)+ calcd,
425.1530; found, 425.1526. 1H NMR (400 MHz, DMSO-d6) d 8.71
(t, J = 6.1 Hz, 1H), 7.75–7.69 (m, 2H), 7.58 (t, J = 5.8 Hz, 1H), 7.33–
7.18 (m, 8H), 7.18–7.10 (m, 4H), 4.67 (s, 2H), 4.34 (d, J = 6.1 Hz,
2H), 2.96–2.88 (q, J = 7.5 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) d 167.1, 160.4, 139.1, 138.6, 132.5, 128.6,
128.5, 128.3, 128.2, 127.1, 126.7, 126.2, 115.0, 66.9, 44.0, 41.7,
35.2. HPLC purity: tR = 7.358, 100%.
4.2.1.5.
N-(4-Chlorobenzyl)-2-(4-(N-(3-phenylpropyl)sul-
SAPA 1d was pre-
famoyl)phenoxy)acetamide (SAPA 1d).
pared by the reaction of 3a with 4-hydroxy-N-(3-phenylpropyl)-
benzenesulfonamide 7b according to the procedure described for
SAPA 1a. Yield 35%; mp 153.2–155.9 °C. ESI-MS m/z 473.3
(M+H)+. HRMS (ESI) of C24H25ClN2O4S (M+H)+ calcd, 473.1296;
found, 473.1302. 1H NMR (400 MHz, DMSO-d6) d 8.75 (s, 1H),
7.70 (d, J = 8.5 Hz, 2H), 7.52 (s, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.23
(dd, J = 12.7, 7.7 Hz, 4H), 7.18–7.06 (m, 5H), 4.66 (s, 2H), 4.32 (d,
J = 5.5 Hz, 2H), 2.69 (d, J = 5.9 Hz, 2H), 2.52 (d, J = 7.6 Hz, 2H),
1.70–1.55 (m, 2H). 13C NMR (151 MHz, DMSO-d6) d 167.8, 160.9,
141.8, 138.8, 133.4, 131.8, 129.6, 129.0, 128.7, 128.6, 126.2,
115.6, 67.5, 42.5, 41.7, 32.6, 31.3. HPLC purity: tR = 13.728, 98.5%.
4.2.1.10. N-(4-Methoxybenzyl)-2-(4-(N-phenethylsulfamoyl)ph-
enoxy)acetamide (SAPA 1i).
SAPA 1i was prepared by the
reaction of 2-chloro-N-(4-methoxybenzyl)acetamide 3d with 7a
according to the procedure described for SAPA 1a. Yield 53%; mp
129.8–131.7 °C. ESI-MS m/z 455.2 (M+H)+. HRMS (ESI) of C24H26N2-
O5S (M+H)+ calcd, 455.1635; found, 455.1639. 1H NMR (400 MHz,
DMSO-d6) d 8.64 (t, J = 6.0 Hz, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.58 (t,
J = 5.6 Hz, 1H), 7.25 (t, J = 7.3 Hz, 2H), 7.20–7.08 (m, 7H), 6.84 (d,
J = 8.6 Hz, 2H), 4.62 (s, 2H), 4.25 (d, J = 6.0 Hz, 2H), 3.70 (s, 3H),
2.90 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) d 167.0, 160.5, 158.2, 138.7, 132.6, 131.1, 128.6, 128.3,
126.2, 115.1, 113.6, 66.9, 55.0, 44.1, 41.2, 35.2. HPLC purity:
tR = 7.059, 97.4%.
4.2.1.6. N-(4-Chlorobenzyl)-2-(4-(N-(4-chlorobenzyl)sulfamo-
yl)phenoxy)acetamide (SAPA 1e).
SAPA 1e was prepared by
the reaction of 3a with N-(4-chlorobenzyl)-4-hydroxybenzenesul-
fonamide 7c according to the procedure described for SAPA 1a.
Yield 41%; mp 144.2–146.0 °C. ESI-MS m/z 479.0 (M+H)+. HRMS
(ESI) of C22H20Cl2N2O4S (M+H)+ calcd, 479.0594; found, 479.0592.
1H NMR (400 MHz, DMSO-d6) d 8.75 (s, 1H), 8.07 (s, 1H), 7.72 (d,
J = 8.7 Hz, 2H), 7.39–7.29 (m, 4H), 7.25 (t, J = 8.5 Hz, 4H), 7.11 (d,
J = 8.7 Hz, 2H), 4.66 (s, 2H), 4.32 (d, J = 5.9 Hz, 2H), 3.92 (d,
J = 3.6 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 167.8, 161.0,
138.8, 137.4, 133.5, 132.2, 131.9, 129.9, 129.6, 129.1, 128.6,
115.6, 67.5, 45.8, 41.7. HPLC purity: tR = 12.924, 99.2%.
4.2.1.11. N-(4-Bromobenzyl)-2-(4-(N-phenethylsulfamoyl)phe-
noxy)acetamide (SAPA 1j).
SAPA 1j was prepared by the
reaction of N-(4-bromobenzyl)-2-chloroacetamide 3e with 7a
according to the procedure described for SAPA 1a. Yield 60%; mp
152.6–154.7 °C. ESI-MS m/z 503.1 (M+H)+. HRMS (ESI) of C23H23
-
BrN2O4S (M+H)+ calcd, 503.0635; found, 503.0639. 1H NMR
(400 MHz, DMSO-d6) d 8.74 (t, J = 5.8 Hz, 1H), 7.71 (d, J = 8.6 Hz,
2H), 7.58 (t, J = 5.7 Hz, 1H), 7.48 (d, J = 8.2 Hz, 2H), 7.25 (t,
J = 7.3 Hz, 2H), 7.18 (d, J = 8.2 Hz, 3H), 7.12 (t, J = 8.2 Hz, 4H), 4.65
(s, 2H), 4.29 (d, J = 5.9 Hz, 2H), 2.90 (q, J = 13.7, 6.8 Hz, 2H), 2.65
(t, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d 167.5, 160.6,
138.8, 132.8, 131.2, 129.6, 128.8, 128.5, 126.4, 120.0, 115.3, 67.1,
44.3, 41.4, 35.3. HPLC purity: tR = 11.923, 98.7%.
4.2.1.7.
N-(4-Chlorobenzyl)-2-(4-(N-phenylsulfamoyl)phe-
SAPA 1f was prepared by the
noxy)acetamide (SAPA 1f).
reaction of 3a with 4-hydroxy-N-phenylbenzenesulfonamide 7d
according to the procedure described for SAPA 1a. Yield 39%; mp
192.0–193.7 °C. ESI-MS m/z 431.1 (M+H)+. HRMS (ESI) of
C21H19ClN2O4S (M+H)+ calcd, 431.0827; found, 431.0831. 1H NMR
(400 MHz, DMSO-d6) d 10.17 (s, 1H), 8.70 (s, 1H), 7.69 (d,
J = 8.8 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 7.20 (dd, J = 12.2, 8.2 Hz,
4H), 7.10–7.03 (m, 4H), 6.99 (t, J = 7.3 Hz, 1H), 4.61 (s, 2H), 4.28
(d, J = 5.9 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 167.7, 161.3,
138.7, 138.4, 132.4, 131.8, 129.6, 129.5, 129.3, 128.6, 124.3,
120.3, 115.6, 67.4, 41.6. HPLC purity: tR = 7.806, 98.4%.
4.2.1.12. N-(4-Fluorobenzyl)-2-(4-(N-phenethylsulfamoyl)phe-
noxy)acetamide (SAPA 1k).
SAPA 1k was prepared by the
reaction of 2-chloro-N-(4-fluorobenzyl)acetamide 3f with 7a
according to the procedure described for SAPA 1a. Yield 61%; mp
148.0–150.6 °C. ESI-MS m/z 443.2 (M+H)+. HRMS (ESI) of C23H23
-
FN2O4S (M+H)+ calcd, 443.1435; found, 443.1439. 1H NMR
(400 MHz, DMSO-d6) d 8.72 (t, J = 6.1 Hz, 1H), 7.70 (d, J = 8.8 Hz,
2H), 7.58 (t, J = 5.8 Hz, 1H), 7.29–7.21 (m, 4H), 7.18 (d, J = 7.2 Hz,
1H), 7.11 (m, J = 8.6, 4.3 Hz, 6H), 4.64 (s, 2H), 4.30 (d, J = 6.0 Hz,
2H), 2.90 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) d 167.2, 160.4, 138.7, 135.4, 132.6, 129.2,
128.6, 128.3, 126.2, 115.1, 115.0, 114.8, 66.9, 44.1, 41.1, 35.2. HPLC
purity: tR = 7.652, 98.2%.
4.2.1.8. N-Phenethyl-2-(4-(N-phenethylsulfamoyl)phenoxy)ac-
etamide (SAPA 1g).
SAPA 1g was prepared by the reaction
of 2-chloro-N-phenethylacetamide 3b with 7a according to the
procedure described for SAPA 1a. Yield 37%; mp 127.6–129.8 °C.
ESI-MS m/z 439.2 (M+H)+. HRMS (ESI) of C24H26N2O4S (M+H)+
calcd, 439.1686; found, 439.1690. 1H NMR (400 MHz, DMSO-d6)
d 8.22 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.58 (t, J = 5.6 Hz, 1H),
7.29–7.21 (m, 4H), 7.17 (dd, J = 6.3, 4.3 Hz, 4H), 7.13 (d,
4.2.1.13. N-(3-Fluorobenzyl)-2-(4-(N-phenethylsulfamoyl)phe-
noxy)acetamide (SAPA 1l).
SAPA 1l was prepared by the
reaction of 2-chloro-N-(3-fluorobenzyl)acetamide 3g with 7a
according to the procedure described for SAPA 1a. Yield 52%; mp

Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
6181
139.4–141.6 °C. ESI-MS m/z 443.2 (M+H)+. HRMS (ESI) of C23H23
-
4.2.1.18. N-(2-Methoxyphenyl)-2-(4-(N-phenethylsulfamoyl)ph-
enoxy)acetamide (SAPA 1q). SAPA 1q was prepared by the
reaction of 2-chloro-N-(2-methoxyphenyl)acetamide 3l with 7a
according to the procedure described for SAPA 1a. Yield 76%; mp
104.2–106.3 °C. ESI-MS m/z 441.3 (M+H)+. HRMS (ESI) of C23H24N2-
O5S (M+H)+ calcd, 441.1479; found, 441.1483. 1H NMR (400 MHz,
FN2O4S (M+H)+ calcd, 443.1435; found, 443.1440. 1H NMR
(400 MHz, DMSO-d6) d 8.76 (t, J = 5.9 Hz, 1H), 7.71 (d, J = 8.5 Hz,
2H), 7.58 (t, J = 5.8 Hz, 1H), 7.32 (dd, J = 13.9, 7.7 Hz, 1H), 7.25 (t,
J = 7.1 Hz, 2H), 7.18 (d, J = 6.7 Hz, 1H), 7.12 (t, J = 6.7 Hz, 4H), 7.04
(dd, J = 17.6, 7.8 Hz, 3H), 4.67 (s, 2H), 4.34 (d, J = 5.7 Hz, 2H), 2.89
(q, J = 6.8 Hz, 2H), 2.65 (t, J = 7.3 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) d 167.4, 163.4, 161.0, 160.5, 142.3, 142.2, 138.7, 132.6,
130.2, 128.7, 128.4, 126.3, 123.2, 115.1, 113.9, 113.7, 113.5, 66.9,
44.1, 41.3, 35.2. HPLC purity: tR = 7.603, 98.2%.
DMSO-d6)
d 9.29 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.72 (d,
J = 8.8 Hz, 2H), 7.59 (t, J = 5.8 Hz, 1H), 7.24 (t, J = 7.3 Hz, 2H),
7.20–7.02 (m, 7H), 6.91 (t, J = 7.5 Hz, 1H), 4.86 (s, 2H), 3.83 (s,
3H), 2.91 (q, J = 13.6, 7.2 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H). 13C NMR
(101 MHz, DMSO-d6) d 166.0, 160.5, 149.5, 138.8, 132.9, 128.8,
128.5, 126.4, 125.1, 120.5, 115.2, 111.3, 67.1, 55.9, 44.2, 35.3. HPLC
purity: tR = 10.389, 100%.
4.2.1.14.
etamide (SAPA 1m).
2-(4-(N-Phenethylsulfamoyl)phenoxy)-N-phenylac-
SAPA 1m was prepared by the reaction
of 2-chloro-N-phenylacetamide 3h with 7a according to the proce-
dure described for SAPA 1a. Yield 65%; mp 140.4–143.2 °C. ESI-MS
m/z 411.2 (M+H)+. HRMS (ESI) of C22H22N2O4S (M+H)+ calcd,
411.1373; found, 411.1374. 1H NMR (400 MHz, DMSO-d6) d 10.15
(s, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.61 (d, J = 7.7 Hz, 2H), 7.57 (t,
J = 5.8 Hz, 1H), 7.31 (t, J = 7.9 Hz, 2H), 7.24 (t, J = 7.3 Hz, 2H),
7.20–7.10 (m, 5H), 7.07 (t, J = 7.4 Hz, 1H), 4.80 (s, 2H), 2.96–2.86
(q, J = 7.6 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) d 165.9, 160.6, 138.7, 138.3, 132.6, 128.7, 128.6, 128.3,
126.2, 123.7, 119.6, 115.0, 67.0, 44.1, 35.2. HPLC purity:
tR = 7.652, 100%.
4.2.1.19. 2-(4-(N-Phenethylsulfamoyl)phenoxy)-N-(o-tolyl)ac-
etamide (SAPA 1r).
SAPA 1r was prepared by the reaction
of 2-chloro-N-(o-tolyl)acetamide 3m with 7a according to the pro-
cedure described for SAPA 1a. Yield 66%; mp 112.8–114.3 °C. ESI-
MS m/z 425.2 (M+H)+. HRMS (ESI) of C23H24N2O4S (M+H)+ calcd,
425.1530; found, 425.1527. 1H NMR (400 MHz, DMSO-d6) d 9.57
(s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.59 (t, J = 5.8 Hz, 1H), 7.37 (d,
J = 7.6 Hz, 1H), 7.25 (t, J = 7.3 Hz, 2H), 7.22–7.07 (m, 8H), 4.84 (s,
2H), 2.91 (q, J = 7.2 Hz, 2H), 2.66 (t, J = 7.5 Hz, 2H), 2.12 (s, 3H).
13C NMR (101 MHz, DMSO-d6) d 166.1, 160.6, 138.8, 135.5, 132.7,
132.3, 130.4, 128.7, 128.4, 126.3, 126.1, 125.8, 125.4, 115.2, 67.1,
44.2, 35.3, 17.7. HPLC purity: tR = 7.623, 97.2%.
4.2.1.15. N-(4-Chlorophenyl)-2-(4-(N-phenethylsulfamoyl)phe-
noxy)acetamide (SAPA 1n).
SAPA 1n was prepared by the
reaction of 2-chloro-N-(4-chlorophenyl)acetamide 3i with 7a
according to the procedure described for SAPA 1a. Yield 68%; mp
4.2.1.20.
noxy)acetamide (SAPA 1s).
N-(2-Nitrophenyl)-2-(4-(N-phenethylsulfamoyl)phe-
SAPA 1s was prepared by the
179.8–181.6 °C. ESI-MS m/z 445.2 (M+H)+. HRMS (ESI) of C22H21
-
reaction of 2-chloro-N-(2-nitrophenyl)acetamide 3n with 7a
according to the procedure described for SAPA 1a. Yield 59%; mp
182.0–185.1 °C. ESI-MS m/z 456.2 (M+H)+. HRMS (ESI) of C22H21N3-
O6S (M+H)+ calcd, 456.1224; found, 456.1227. 1H NMR (400 MHz,
DMSO-d6) d 10.83 (s, 1H), 8.07 (dd, J = 8.3, 1.3 Hz, 2H), 7.78–7.72
(m, 3H), 7.60 (t, J = 5.5 Hz, 1H), 7.41–7.34 (m, 1H), 7.27–7.15 (m,
5H), 7.12 (d, J = 6.9 Hz, 2H), 4.87 (s, 2H), 2.91 (dd, J = 13.0, 7.2 Hz,
2H), 2.64 (t, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d
166.6, 159.9, 140.3, 138.6, 134.8, 133.1, 131.4, 128.7, 128.6,
128.3, 126.2, 125.3, 125.2, 124.1, 115.1, 67.0, 44.0, 35.1. HPLC pur-
ity: tR = 13.421, 98.0%.
ClN2O4S (M+H)+ calcd, 445.0983; found, 445.0979. 1H NMR
(400 MHz, DMSO-d6) d 10.29 (s, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.65
(d, J = 8.7 Hz, 2H), 7.58 (t, J = 5.7 Hz, 1H), 7.37 (d, J = 8.8 Hz, 2H),
7.24 (t, J = 7.3 Hz, 2H), 7.17 (d, J = 7.1 Hz, 1H), 7.13 (dd, J = 7.6,
5.4 Hz, 4H), 4.81 (s, 2H), 2.91 (q, J = 7.0 Hz, 2H), 2.65 (t, J = 7.5 Hz,
2H). 13C NMR (101 MHz, DMSO-d6) d 166.2, 160.6, 138.7, 137.3,
132.7, 128.7, 128.3, 127.4, 126.3, 121.2, 115.1, 67.0, 44.1, 35.2.
HPLC purity: tR = 13.132, 99.1%.
4.2.1.16. N-(4-Fluorophenyl)-2-(4-(N-phenethylsulfamoyl)phe-
noxy)acetamide (SAPA 1o).
SAPA 1o was prepared by the
reaction of 2-chloro-N-(4-fluorophenyl)acetamide 3j with 7a
according to the procedure described for SAPA 1a. Yield 61%; mp
4.2.1.21. N-(2-Cyanophenyl)-2-(4-(N-phenethylsulfamoyl)phe-
noxy)acetamide (SAPA 1t).
SAPA 1t was prepared by the
137.6–139.7 °C. ESI-MS m/z 429.2 (M+H)+. HRMS (ESI) of C22H21
-
reaction of 2-chloro-N-(2-cyanophenyl)acetamide 3o with 7a
according to the procedure described for SAPA 1a. Yield 36%; mp
169.4–172.4 °C. ESI-MS m/z 436.2 (M+H)+. HRMS (ESI) of C23H21N3-
O4S (M+H)+ calcd, 436.1326; found, 436.1321. 1H NMR (400 MHz,
DMSO-d6) d 10.44 (s, 1H), 7.83 (dd, J = 7.8, 1.1 Hz, 1H), 7.73 (d,
J = 8.8 Hz, 2H), 7.71–7.67 (m, 1H), 7.62 (t, J = 6.8 Hz, 2H), 7.38
(dd, J = 11.8, 4.3 Hz, 1H), 7.24 (t, J = 7.3 Hz, 2H), 7.17 (d, J = 8.6 Hz,
3H), 7.15–7.11 (m, 2H), 4.90 (s, 2H), 2.91 (q, J = 7.2 Hz, 2H), 2.65
(t, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d 166.8, 160.4,
139.3, 138.7, 133.9, 133.2, 132.8, 128.6, 128.3, 126.2, 125.7,
116.7, 115.1, 107.8, 66.8, 44.1, 35.2. HPLC purity: tR = 6.016, 98.1%.
FN2O4S (M+H)+ calcd, 429.1279; found, 429.1283. 1H NMR
(400 MHz, DMSO-d6) d 10.20 (s, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.63
(dd, J = 8.7, 5.1 Hz, 2H), 7.57 (s, 1H), 7.24 (t, J = 7.3 Hz, 2H), 7.20–
7.10 (m, 7H), 4.79 (s, 2H), 2.91 (d, J = 6.0 Hz, 2H), 2.64 (t,
J = 7.4 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 166.4, 161.1,
159.6, 158.0, 139.2, 135.1, 133.2, 129.1, 128.8, 126.7, 122.1,
115.9, 115.7, 115.5, 67.5, 44.5, 35.7. HPLC purity: tR = 8.388, 100%.
4.2.1.17. N-(4-Methoxyphenyl)-2-(4-(N-phenethylsulfamoyl)ph-
enoxy)acetamide (SAPA 1p).
SAPA 1p was prepared by the
reaction of 2-chloro-N-(4-methoxyphenyl)acetamide 3k with 7a
according to the procedure described for SAPA 1a. Yield 79%; mp
172.7–174.4 °C. ESI-MS m/z 441.2 (M+H)+. HRMS (ESI) of
4.2.1.22.
2-(4-(N-Phenethylsulfamoyl)phenoxy)-N-(2-(trifluo-
SAPA 1u was pre-
romethoxy)phenyl)acetamide (SAPA 1u).
C
23H24N2O5S (M+H)+ calcd, 441.1479; found, 441.1478. 1H NMR
pared by the reaction of 2-chloro-N-(2-(trifluoromethoxy)-
phenyl)acetamide 3p with 7a according to the procedure described
for SAPA 1a. Yield 55%; mp 124.3–126.6 °C. ESI-MS m/z 495.2
(M+H)+. HRMS (ESI) of C23H21F3N2O5S (M+H)+ calcd, 495.1196;
found, 495.1193. 1H NMR (400 MHz, DMSO-d6) d 9.92 (s, 1H),
7.85 (dd, J = 8.0, 1.4 Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.59 (t,
J = 5.6 Hz, 1H), 7.44–7.38 (m, 1H), 7.36 (dd, J = 7.9, 1.4 Hz, 1H),
7.29 (dd, J = 7.8, 1.5 Hz, 1H), 7.27–7.21 (m, 2H), 7.17 (d,
J = 7.2 Hz, 1H), 7.14–7.10 (m, 4H), 4.88 (s, 2H), 2.90 (q, J = 7.2 Hz,
(400 MHz, DMSO-d6) d 10.00 (s, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.57
(t, J = 5.2 Hz, 1H), 7.51 (d, J = 8.9 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H),
7.17 (d, J = 7.3 Hz, 1H), 7.16–7.10 (m, 4H), 6.88 (d, J = 8.9 Hz, 2H),
4.76 (s, 2H), 3.70 (s, 3H), 2.91 (dd, J = 13.1, 6.9 Hz, 2H),
2.65 (t, J = 7.5 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 166.0,
161.2, 156.1, 139.2, 133.2, 131.8, 129.1, 128.8, 126.7,
121.9, 115.5, 114.3, 67.6, 55.7, 44.6, 35.7. HPLC purity: tR = 7.121,
100%.

6182
Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
2H), 2.65 (t, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d 166.5,
160.4, 140.4, 138.7, 132.7, 129.9, 128.6, 128.3, 127.7, 126.2,
125.5, 121.4, 118.8, 114.9, 66.8, 44.1, 35.2. HPLC purity:
tR = 14.231, 100%.
J = 8.6 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 8.9 Hz, 2H),
4.61 (s, 2H), 4.32 (d, J = 5.8 Hz, 2H).
4.2.2.4. 4-(2-((4-Chlorobenzyl)amino)-2-oxoethoxy)-N-phenet-
hylbenzamide (SAPA 1b).
To a mixture of 11 (0.13 g,
4.2.1.23. N-(3-Methoxyphenyl)-2-(4-(N-phenethylsulfamoyl)ph-
0.41 mmol) and (3-(dimethylamino)propyl)ethylcarbodiimide
hydrochloride (EDC, 94 mg, 0.49 mmol) in CH2Cl2 (10 ml) was
added 1,2,3-benzotriazol-1-ol monohydrate (HOBt, 69 mg,
0.45 mmol). A clear solution was observed after stirring for
30 min, and 6a (49 mg, 0.41 mmol) was added to the reaction
solution, which was stirred at ambient temperature overnight
(12 h). The reaction solution was washed with water and brine,
dried over Na2SO4 and subjected to flash chromatograph eluting
with ethyl acetate/heptanes (3:1) to provide 1b as white solid
(0.10 g, yield 58%): mp 203.0–204.5 °C. ESI-MS m/z 423.2 (M+H)+.
HRMS (ESI) of C24H23ClN2O3 (M+H)+ calcd, 423.1470; found,
423.1475. 1H NMR (400 MHz, DMSO-d6) d 8.72 (s, 1H), 8.44 (s,
1H), 7.79 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 7.9 Hz, 2H), 7.31–7.15 (m,
7H), 7.00 (d, J = 8.4 Hz, 2H), 4.61 (s, 2H), 4.31 (d, J = 5.5 Hz, 2H),
3.45 (d, J = 5.8 Hz, 2H), 2.82 (t, J = 6.8 Hz, 2H). 13C NMR
(151 MHz, DMSO-d6) d 168.0, 166.0, 160.3, 140.1, 138.8, 131.8,
129.6, 129.3, 129.1, 128.8, 128.6, 128.0, 126.5, 114.7, 67.4, 41.7,
41.3, 35.7. HPLC purity: tR = 11.315, 99.0%.
enoxy)acetamide (SAPA 1v).
SAPA 1v was prepared by the
reaction of 2-chloro-N-(3-methoxyphenyl)acetamide 3q with 7a
according to the procedure described for SAPA 1a. Yield 46%; mp
107.0–108.0 °C. ESI-MS m/z 441.3 (M+H)+. HRMS (ESI) of C23H24N2-
O5S (M+H)+ calcd, 441.1479; found, 441.1479. 1H NMR (400 MHz,
DMSO-d6) d 10.13 (s, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.57 (s, 1H),
7.30 (s, 1H), 7.28–7.10 (m, 9H), 6.65 (d, J = 7.9 Hz, 1H), 4.79 (s,
2H), 3.70 (s, 3H), 2.91 (s, 2H), 2.65 (t, J = 7.5 Hz, 2H). 13C NMR
(151 MHz, DMSO-d6) d 166.4, 161.1, 160.0, 140.0, 139.2, 133.2,
130.1, 129.1, 128.8, 126.7, 115.5, 112.4, 109.7, 105.9, 67.5, 55.5,
44.6, 35.7. HPLC purity: tR = 8.100, 98.9%.
4.2.1.24. N-(4-Fluoro-2-methoxyphenyl)-2-(4-(N-phenethylsul-
famoyl)phenoxy)acetamide (SAPA 1w).
SAPA 1w was pre-
pared by the reaction of 2-chloro-N-(4-fluoro-2-methoxyphenyl)-
acetamide 3r with 7a according to the procedure described for
SAPA 1a. Yield 74%; mp 161.3–163.5 °C. ESI-MS m/z 459.2
(M+H)+. HRMS (ESI) of C23H23FN2O5S (M+H)+ calcd, 459.1384;
found, 459.1386. 1H NMR (400 MHz, DMSO-d6) d 9.33 (s, 1H),
7.92–7.84 (m, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.58 (s, 1H), 7.24 (t,
J = 7.2 Hz, 2H), 7.15 (dd, J = 17.4, 8.9 Hz, 5H), 7.00 (d, J = 10.9 Hz,
1H), 6.74 (t, J = 8.5 Hz, 1H), 4.84 (s, 2H), 3.83 (s, 3H), 2.90 (s, 2H),
2.65 (t, J = 7.5 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d 166.4,
160.9, 160.7, 159.1, 151.8, 139.2, 133.3, 129.1, 128.8, 126.7,
123.4, 123.2, 115.6, 106.6, 106.5, 100.4, 100.3, 67.5, 56.8, 44.6,
35.7. HPLC purity: tR = 10.451, 99.4%.
4.2.2.5. 4-(2-((4-Chlorobenzyl)amino)-2-oxoethoxy)-N-(pyridin-
4-yl)benzamide (SAPA 1c).
SAPA 1c was prepared by the
reaction of 11 with pyridin-4-amine 6e according to the procedure
as described for SAPA 1b. To a solution of 1c (0.40 g, 1.0 mmol) in
dry ethyl acetate (10 ml) was added HCl (g)/ethyl acetate (1.2 ml,
1.5 mmol, c = 1.25 mol/L) dropwise at 0 °C. Then the solid was fil-
tered and dried in vacuum. The hydrochloride of 1c was given as
white solid (0.34 g, yield 79%): mp 193.0–197.0 °C. ESI-MS m/z
396.1 (M+H)+. HRMS (ESI) of C21H18ClN3O3 (M+H)+ calcd,
396.1109; found, 396.1116. 1H NMR (400 MHz, DMSO-d6) d 10.47
(s, 1H), 8.76 (s, 1H), 8.46 (s, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.78 (d,
J = 4.8 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H),
7.11 (d, J = 8.4 Hz, 2H), 4.66 (s, 2H), 4.32 (d, J = 5.5 Hz, 2H). 13C
NMR (151 MHz, DMSO-d6) d 167.9, 166.2, 161.2, 150.5, 146.8,
138.8, 131.9, 130.3, 129.6, 128.7, 127.3, 115.0, 114.5, 67.5, 41.7.
HPLC purity: tR = 2.541, 98.8%.
4.2.2. The preparation of 4-(2-((4-chlorobenzyl)amino)-2-oxoe-
thoxy)-N-substituted-benzamide (SAPA 1b,1c)
4.2.2.1. Methyl 4-hydroxybenzoate (9).
To a solution of 8
(10.00 g, 72 mmol) in methanol (20 ml) was added SOCl2 (6.8 ml)
dropwise at 0 °C under N2. The reaction mixture was stirred at
70 °C for 2 h and then cooled to room temperature. After the
organic solvent was evaporated, the residue was extracted by ethyl
acetate, washed with brine and dried over Na2SO4. After filtration
and evaporation, 9 was obtained as white solid (10.00 g, yield
91%) and was used in next step without further purification. ESI-
MS m/z 153.1 (M+H)+.
4.3. Biological evaluation
4.3.1. Materials
6-((N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)-sph-
ingosine (C6-NBD-Cer) and 1,2-dimyristoyl-sn-glycero-3-phospho-
choline (DMPC) were purchased from Santa Cruz Inc (USA). D609,
the public SMS inhibitor, was purchased from MedChem Express
Inc. Milli-Q deionized water from a Millipore water purification sys-
tem (Bedford, MA, USA) was used throughout the study. All other
reagents were of analytical grade. Buffer 1 (0.25 M sucrose, 50 mM
TrisÁHCl, pH 7.4, 1 mM EDTA) was prepared for the storage of SMS1
overexpressing Hela cell lysate. Buffer 2 (100 mM HEPES, pH 7.4,
30 mM MnCl2, 3% BSA) was prepared for the assay of SMS1 inhibitory
activities.
4.2.2.2. Methyl 4-(2-((4-chlorobenzyl)amino)-2-oxoethoxy)ben-
zoate (10).
To a solution of 9 (3.49 g, 22.9 mmol) and 3a
(5.00 g, 22.9 mmol) in CH3CN (100 ml) was added K2CO3 (6.32 g,
45.8 mmol). The reaction mixture was heated at 68 °C for 2 h and
then cooled to ambient temperature. After the organic solvent
was evaporated, the residual was diluted with ethyl acetate/water.
The organic layer was washed with brine, dried over Na2SO4 and
condensed. 10 was afforded as yellow solid (7.23 g, yield 94%)
and used without further purification. ESI-MS m/z 334.1 (M+H)+.
4.2.2.3.
acid (11).
4-(2-((4-Chlorobenzyl)amino)-2-oxoethoxy)benzoic
An Agilent 1260 HPLC system with a G1311B quaternary pump,
a G1329B ALS and a G1321B FLD detector was used in our study.
To a solution of 10 (4.00 g, 12 mmol) in THF
(12 ml) and H2O (12 ml) was added LiOHÁH2O (1.16 g, 48 mmol).
The reaction mixture was stirred at 40 °C for 2 h and then the
organic solvent was evaporated. The residue was poured into HCl
(aq, 2 mol/L). The solid was filtered, washed with water and dried
in vacuum. 11 was obtained as white solid (3.60 g, yield 94%): mp
214.1–216.5 °C. ESI-MS m/z 318.1 (MÀH)À. 1H NMR (400 MHz,
An Agilent C18 RP column (250 mm  4.6 mm,
5 lm) was
employed in the chromatographic separations.
4.3.2. SMS1 overexpressed Hela cell lysate
Hela cells were purchased from the ATCC and maintained in
DMEM high glucose medium with 10% fetal bovine serum at
37 °C in 5% CO2. Hela cells overexpressed SMS1 was established
DMSO-d6)
d 8.74 (s, 1H), 7.87 (d, J = 8.9 Hz, 2H), 7.34 (d,

Y.-l. Li et al. / Bioorg. Med. Chem. 23 (2015) 6173–6184
6183
by transfecting with pcDNA3.1 vector containing SMS1 and
maintained in DMEM, 10% fetal bovine serum with G418 (final
concentration 500 lg/mL) for every generation thereafter. Cells
overexpressed SMS1 were then subcultured and siRNA for SMS2
were transfected when cells were 60% confluence. Three days after
siRNA SMS2 treatment, cells were washed with PBS three times
and suspended in SMS activity assay buffer (0.25 M sucrose,
4.5.2. Cell culture and transfection
Hela cells were cultured in the 5% CO2, 37 °C water bath with
DMEM, 10% (v/v) FBS, 100 U/ml penicillin and streptomycin, and
2 mM glutamine. Plasmids were transfected into Hela cells with
LipofectamineÒ 2000 (Invitrogen).
4.5.3. Immunoprecipitation
50 mM TrisÁHCl pH 7.4, 1 mM EDTA, 100
l
g/ml phenylmethylsul-
Cells expressing SMS1-Flag were lysed with CelLytic™ M buffer
(Sigma). After cell debris were cleared at 8200 g for 10 min, super-
fonyl fluoride, 1 g/ml aprotinin and 1
l
lM leupeptin) after being
scraped from dishes. SMS1 overexpressed Hela cells were homoge-
nated by glass bead homogenizer. The cell lysates were centrifuged
at 700 g for 10 min at 4 °C. The postnuclear supernatant was col-
lected as the source of enzyme for determining the SMS1 inhibitory
activities of SAPA derivatives.28
natants were collected and blocked with 30
Agarose Beads for 30 min. Beads were centrifuged 30 s at 8,200 g
and supernatant was then incubated with 30 l EZview™ Red
ll Protein A/G PLUS-
l
ANTI-FLAG M2 Affinity Gel (Sigma) for overnight.
4.5.4. Western blot
After immune precipitation, SDS PAGE loading buffer was added
into ANTI-FLAG M2 Affinity Gel and the mixture was boiled at
95 °C for 5 min. Eluted protein was used, separated on the SDS
page and then immunoblotted with an anti-Flag HRP (Sigma) for
1 h. Then protein was detected by the chemilluminescence method
(Pierce).
4.3.3. SMS1 inhibitory activities assay in vitro
294
Hela cell lysate (equivalent to 0.5 mg total protein) in buffer 1,
10 l DMSO solution of the corresponding SAPA derivatives, 30
buffer 2 and 226 l water, was pre-incubated at 37 °C for 30 min.
Then 3 l C6-NBD-Cer in DMSO (1.0 mM) and 3 l DMPC in DMSO
(40 mM) were added as substrates into the biological test system.
Subsequently, the total 300 l mixture was incubated at 37 °C for
an additional 2.0 h. The test system was quenched by 600 l etha-
ll test mixture, which contained 28 ll SMS1 overexpressed
l
ll
l
l
l
4.5.5. SMS1 activity
l
After immune precipitation, ANTI-FLAG M2 Affinity Gel bound
with SMS1-Flag were incubated with 50 mM TrisÁHCl (pH 7.4),
l
nol and centrifuged at 10000 rpm for 10 min. The supernatant was
collected and the amount of C6-NBD-Cer and C6-NBD-SM in the
supernatant were quantified by a fluorescent HPLC-based analytic
25 mMKCl, C6-NBD-Ceramide (0.1 lg/lL), andphosphatidylcholine
(0.01 lg/lL) at 37 °C for 1 h. Lipids were extracted by chloro-
method.27 20
ll extracts from the supernatant were analyzed for
form/methanol (2:1/v:v). Then lipids were separated on Thin Layer
Chromatography Plates (Whatman) with CHCl3/methanol/ammo-
niac hydroxide (14:6:1).
the formation of C6-NBD-SM by HPLC using the reversed-phase
C18 column and an isocratic elution with methanol/water/trifluo-
roacetic acid (89:11:0.1 (v/v)). The flow rate of the mobile phase
was 1.0 mL/min. Detection was performed with a fluorescence
spectrophotometer (kexcitation = 475 nm, kemission = 525 nm). Assays
were accomplished at least in triplicate. Statistical analysis was
performed on Prism 5.02 (GraphPad Software, Inc.).
Acknowledgments
We gratefully acknowledge the financial support from the Chi-
nese National Natural Science Foundation (Grant No. 30973641),
the Key Scientific and Technological Foundation of Shanghai
Science and Technology Committee (Grant No. 11431920102),
the 12th Graduate Innovation Foundation of Fudan University,
and the open grant of the State Key Laboratory of Bio-organic
and Natural Products Chemistry, CAS.
4.4. Molecular docking
The reported three-dimensional structure of human sphin-
gomyelin synthase 1 was employed and loaded into the GOLD soft-
ware suite (version 5.0, released by CCDC). Small molecules for
docking studies were docked into the active site of hSMS1. The
three-dimensional structures of substrate PC and SMS1 inhibitors
including SAPA 1a, 1b and D609 were constructed and energy opti-
mized. Then the optimized structures of these molecules were
selected as the docking ligands. The GA runs for each ligand were
set 30 and therefore thirty binding poses were generated for each
docked molecule. The scoring function in the docking process was
set as GoldScore. The genetic algorithm (GA) search options were
set as preset with 100,000 operations. The pose with the highest
binding score of each docked ligand was selected out and studied
for the interaction with hSMS1.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.060.
References and notes
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2006, 3, 5.
4.5. Point mutagenesis
6. Li, Z.; Hailemariam, T. K.; Zhou, H.; Li, Y.; Duckworth, D. C.; Peake, D. A.; Zhang,
Y.; Kuo, M.-S.; Cao, G.; Jiang, X.-C. Biochimic. Biophys. Acta (BBA)-Mol. Cell Biol.
Lipids 2007, 1771, 1186.
All the methods were performed and modified as previous
paper.17 Details are described as followed.
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