Synthesis of novel FTY720 analogs with anticancer activity through PP2A activation

Article abstract of DOI:10.3390/molecules23112750

FTY720 inhibits various cancers through PP2A activation. The structure of FTY720 is also used as a basic structure for the design of sphingosine kinase (SK) inhibitors. We have synthesized derivatives using an amide chain in FTY720 with a phenyl backbone, and then compounds were screened by an MTT cell viability assay. The PP2A activity of compound 7 was examined. The phosphorylation levels of AKT and ERK, downstream targets of PP2A, in the presence of compound 7, were determined. Compound 7 may exhibit anticancer effects through PP2A activation rather than the mechanism by inhibition of SK1 in cancer cells. In the docking study of compound 7 and PP2A, the amide chain of compound 7 showed an interaction with Asn61 that was different from FTY720, which is expected to affect the activity of the compound.

Full text of DOI:10.3390/molecules23112750

molecules
Article
Synthesis of Novel FTY720 Analogs with Anticancer
Activity through PP2A Activation
Jitendra Shrestha 1,†, Sung Hwan Ki 2,† , Sang Mi Shin 2, Seon Woong Kim 1, Joo-Youn Lee 3,4
,
Hee-Sook Jun 5,6 , Taeho Lee 7, Sanghee Kim 3 , Dong Jae Baek 1,* and Eun-Young Park 1,
*
1
College of Pharmacy and Natural Medicine Research Institute, Mokpo National University,
Jeonnam 58554, Korea; shresthasimon2011@mokpo.ac.kr (J.S.); tjsdnd123@mokpo.ac.kr (S.W.K.)
College of Pharmacy, Chosun University, Gwangju, 61452, Korea; shki@chosun.ac.kr (S.H.K.);
2
smshin@chosun.ac.kr (S.M.S.)
College of Pharmacy, Seoul National University, Seoul 08826, Korea; leejy@krict.re.kr (J.-Y.L.);
3
pennkim@snu.ac.kr (S.K.)
Korea Chemical Bank, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea
Lee Gil Ya Cancer and Diabetes Institute, Department of Molecular Medicine, Gachon University,
4
5
Incheon 21999, Korea; hsjun@gachon.ac.kr
College of Pharmacy and Gachon Institute of Pharmaceutical Science, Gachon University,
6
Incheon 21936, Korea
7
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu
41566, Korea; tlee@knu.ac.kr
*
Correspondence: dbaek@mokpo.ac.kr (D.J.B.); parkey@mokpo.ac.kr (E.-Y.P.);
Tel.: +82-61-450-2705 (D.J.B.); +82-61-450-2706 (E.-Y.P.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 August 2018; Accepted: 22 October 2018; Published: 24 October 2018
Abstract: FTY720 inhibits various cancers through PP2A activation. The structure of FTY720 is also
used as a basic structure for the design of sphingosine kinase (SK) inhibitors. We have synthesized
derivatives using an amide chain in FTY720 with a phenyl backbone, and then compounds
were screened by an MTT cell viability assay. The PP2A activity of compound
The phosphorylation levels of AKT and ERK, downstream targets of PP2A, in the presence of
compound , were determined. Compound may exhibit anticancer effects through PP2A activation
rather than the mechanism by inhibition of SK1 in cancer cells. In the docking study of compound
and PP2A, the amide chain of compound showed an interaction with Asn61 that was different from
7 was examined.
7
7
7
7
FTY720, which is expected to affect the activity of the compound.
Keywords: FTY720; protein phosphatase 2A; sphingosine kinase; colorectal cancer; derivative
1. Introduction
Sphingolipids such as sphingosines, ceramides, glycolipids and sphingosine-1-phosphate (S1P)
are important cell signaling lipids that control cellular processes. Sphingolipids play an essential
role in cell death and survival, proliferation, recognition and migration [
of five S1P-specific G protein-coupled receptors (GPCRs; named S1P1-5) and regulates the action of
various intracellular proteins [ ]. Sphingosine kinase (SK) has two isoforms: SK1 and 2, and catalyzes
sphingosine to S1P. Increased cellular SK and S1P cause cell survival, proliferation and migration [ ].
SK inhibitors with various anticancer activities have been developed [ ]. However, the relationship
1,2]. S1P binds to a family
3,4
5
6
between SK2 and cancer is less evident than that of SK1, and it is necessary to develop selective
inhibitors for SK1 and SK2. Ceramides modulate protein kinase activities and promote apoptosis
through various pathways [7]. Some glycolipids activate natural killer T cells (NKT cells), and they
Molecules 2018, 23, 2750; doi:10.3390/molecules23112750
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2750
2 of 12
induce various cytokines to suppress cancer or regulate the immune response [
8
]. Regulation of
various sphingolipids is medically available, but there is a continuous requirement for regulators of
sphingolipids that are selective for the disease.
FTY720 (Fingolimod, Gilenya, Novartis) is synthesized based on the structure of myriocine,
a fungal metabolite of the Chinese herb Iscaria sinclarii. It is structurally and biologically similar
to sphingosine. In 2010, FTY720 became the first oral multiple sclerosis treatment approved by the
US FDA. FTY720 also activates protein phosphatase 2A (PP2A) and inhibits the growth of various
cancer cells (blood cancer, liver cancer, bladder cancer and colorectal cancer) [9–12]. FTY720 is
also known to inhibit SK1 activity by 50% [13]. In its structure, FTY720 has an aromatic backbone,
an aliphatic chain and a polar head group. This basic structure of FTY720 has been used as
a model on which to design materials for controlling sphingolipids. So far, significant changes in
biological activity have been observed in derivatives of the polar head group of FTY720. For example,
FTY720 has an inhibitory effect on SK1, but ROME ((R)-FTY720 methyl ether) [14], in which the
methoxy group is substituted for the hydroxyl group, shows selective inhibition to SK2. However,
in the case of ROME, to enantioselectively introduce the methoxy group, 11 synthetic steps are
required [15]. In contrast, RB-042 and compound 51 with a pyrrolidine head group are relatively easy
to synthesize and yield stereoselective compounds (Figure 1). These compounds exhibit biologically
unique activity changes. RB-005 shows selective inhibition of SK1, but RB-042 with (R)-(
−
)-prolinol
inhibits both SK1 and SK2 [16 17]. These biological studies provide structural information useful
,
for the development of SK inhibitors. RB-012 with a quaternary ammonium salt inhibited human
A549 lung cancer cells, and showed reduced xenograft growth in mice through 14-3-3 protein
regulation [18]. Biologically useful results have been observed in the derivatives of the aromatic
part of the FTY720 structure. For example, RB-065 is a triazole compound introduced by the click
reaction. This compound has the same 4-hydroxypiperidine head structure as RB-005, but it does
not inhibit SK2, and it has higher selectivity than RB-005 [17]. Triazole is known as a bioisostere of
amide compounds and is widely used in medicinal chemistry [19]. Compound 51 has a structure
containing oxadiazole, and it has an IC50 of 0.058 µM in SK1 inhibitory activity. The oxadiazole
structure exhibited a much higher inhibitory effect than other heterocycle compounds [20].
K145 (3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione), which is
a backbone structure of phenyl ether, selectively inhibits only SK2 Ki of 6.4 µM. K145 decreases
cellular S1P without altering ceramide levels. K145 was found to be orally available and inhibited
growth and S1P levels in U937 xenograft tumors in nude mice [21]. The relationship between this
structure and activity shows that the backbone structure containing heteroatoms is a useful structure
for the design of FTY720 analogues. Although the structure of FTY720 has been studied and applied to
various targets, FTY720 derivatives having selectivity to target for pharmaceutical development are
required. We synthesized derivatives of FTY720 containing amide chain and investigated whether
these derivatives show selectivity to the target.
Figure 1. FTY720 (1) and known biologically active compounds.

Molecules 2018, 23, 2750
3 of 12
2. Results and Discussion
2.1. Chemical Synthesis
4-Aminophenethyl alcohol was used as a starting material to obtain compound 11 in which
an acyl chain was introduced by EDCI coupling. Various amines were introduced into the tosylated
compound 12 to obtain the final compounds 2–10 (Scheme 1).
Scheme 1. Synthesis of FTY720 derivatives
DMAP, CH2Cl2, rt, 12 h; (b) TsCl, NEt3, CH2Cl2, rt, 6 h; (c) amines, MeCN, 50 C, 12 h.
2
–
10. Reagents and conditions; (a◦) heptanoic acid, EDCI,
2.2. Compound 7 Inhibits the Growth of Human Cancer Cell Lines In Vitro
We performed an MTT assay on human colorectal cancer line HT-29 in order to investigate the
cell cytotoxic effects of newly synthesized compounds
effects were observed in compound (Figure 2A). In order to observe the cytotoxic effect of compound
in various cancer cell lines, we investigated the cell viability assay in human colorectal cancer lines
HT-29 and HCT-116, human gastric cancer cell line AGS and human prostate cancer cell line PC-3 in
the concentration range of 2.5, 5, 10, 20 and 40 M along with 20 M FTY720. The result of the MTT
assays shows that compound significantly inhibits the growth of all human cancer cells in vitro in
a dose- and time-dependent manner (Figure 2B–E). We estimated the IC50 of compound in cancer
cell lines (Table 1). To examine whether the cytotoxic effects in the compound -treated cell might be
a result of the increased apoptosis, we performed an annexin V-FITC cell apoptosis assay. We found
that incubation of HT-29 cells with compound for 24 h resulted in a significant increase in the number
of apoptotic cells relative to the number of untreated control cells (Figure 2F).
2–10. We found that the greatest cell death
7
7
µ
µ
7
7
7
7
Table 1. Cytotoxic activity of compound
7
. IC50 values for each cell line were calculated by using
GraphPad Prism 7.
IC50 (µM) 24 h
Compound
HT-29
HCT-116
AGS
PC-3
Compound 7
10.39
7.335
11.04
7.456

Molecules 2018, 23, 2750
4 of 12
Figure 2. Biological activities of FTY720 derivatives; (
at 40 M for 24 h (FTY720 ( ) 20 M), and cell viability was measured by EZ-CYTOX kit (n = 8).
Cytotoxic effects of compound and FTY720 in different cancer cells. The 3000 cells/well were plated
in a 96-well plate and treated with compound in the concentrations of 2.5, 5, 10, 20 or 40 M, and
FTY720 20 M in HT-29 cells ( ), HCT-116 cells ( ), AGS cells ( ), and PC-3 cells ( ); ( ) annexin V-FITC
SD from three independent experiments.
A) HT-29 cells were treated with compounds 2–10
µ
1
µ
7
7
µ
µ
B
C
D
E
F
assays were performed in HT-29 cells. Data are means
* p < 0.05, ** p < 0.01 compared with control cells.
±
2.3. Effect of PF-543 and Compound 7 on SK1 Activity
In order to investigate whether the cytotoxic effect of compound
activity, the SK1 inhibitory effect was tested. As a result, compound
15% compared with control, whereas the conventional SK1 inhibitor PF-543 [22] inhibited SK1 activity
7
mediates the inhibition of SK1
7
inhibited SK1 activity by about

Molecules 2018, 23, 2750
5 of 12
by about 45%. These results suggest that compound
7
induces antitumor activity through a mechanism
other than SK1 activity inhibition (Supplementary Data Figure S1).
2.4. Compoud 7 Effectively Activates PP2A and Decreases the Phosphorylation of ERK and AKT
Protein phosphatase 2A (PP2A) is a main protein serine/threonine phosphatase, maintains the
signaling homeostasis of cells through dephosphorylation of several signaling proteins, and inhibits
tumor growth [23]. In colorectal cancer, the activity of PP2A is inhibited, and FTY720 effectively
activates the functions of PP2A [24]. In this study, we performed the PP2A activity assay in HT-29 cells
treated with compound 7. The PP2A activity was more significantly increased in compound 7-treated
cell lysate than the untreated control groups (Figure 3A). Furthermore, we performed the Western
blotting of phosphorylation of ERK and AKT, which are known to be dephosphorylated by PP2A.
Compared to the control, the phosphorylation of ERK and AKT was significantly decreased in the
compound
7-treated group without affecting the total expression level (Figure 3B,C). These results
suggest that compound 7 may exhibit anticancer effect through PP2A activation in cancer cells.
Figure 3. PP2A activity of compound
7
7
in HT-29 cells. (
and compound
and FTY720 for 12 h, protein extracted, separated, and probed with
A
1
) PP2A activity was measured in HT-29 cells
treated with the 20
with or without 20
µ
M of compound
(FTY720) for 12 h. ( ) Cells were treated
B,C
µ
M compound
7
pERK1/2, ERK1/2, pAKT and AKT. The band was analyzed by UVITEC imaging software, and data
were plotted as means
with control cells.
±
SD for three replicates of the experiments. * p < 0.05, ** p < 0.01 compared
2.5. Molecular Modeling Studies of FTY720 and Compound 7 Against the Human I2PP2A/SET
A docking study was conducted to determine if compound effectively binds to PP2A (PDB code
2E50) [25]. As the hydroxy group of FTY720 interacts with Glu206 and Lys209, the methoxyphenyl
group, which is the head group of compound , interacts with the Lys209 (cation-pi interaction).
7
7

Molecules 2018, 23, 2750
6 of 12
The secondary amine group of compound
7
interacts with Asp210, and the amide linker interacts with
Asn61. The terminal acyl chain of compound
7 showed interactions with Phe68, as FTY720 (Figure 4).
Figure 4. Docking model of FTY720 (1, gray, ball and stick) and compound 7 (purple, ball and stick)
in the I2PP2A/SET. The X-ray structure of IPP2A/SET was obtained from the Protein Data Bank
(PDB code 2E50). The I2PP2A/SET is represented by a blue ribbon model. The hydrogen bonds
are shown as a green dashed line, and an orange dashed line represents electrostatic interactions.
Also, the hydrophobic interactions are shown as a pink dashed line.
3. Experimental Section
3.1. Synthesis in General
All chemicals were reagent grade and used as purchased. Reactions were run under nitrogen
and were monitored by TLC using silica gel 60 F254 aluminum-backed plates. Flash column
chromatography was performed on silica gel grade 60 (230
quality and were used as received. A Bruker Avance I spectrometer was used to record H NMR
and 13C NMR spectra, and chemical shifts were reported in
−
400 mesh). All solvents were of anhydrous
1
δ units relative to deuterated solvents,
which served as internal references, at 400 and 100 MHz, respectively. High-resolution mass spectra
were recorded on an Agilent Technologies G6520A Q-TOF mass spectrometer using electrospray
ionization (ESI). By examining their HRMS and 1H NMR spectra, all compounds were determined to
be ≥95% pure.
3.2. Synthesis
N-(4-(2-Hydroxyethyl)phenyl)heptanamide (11) EDCI (1.7 g, 0.0087 mol), DMAP (45 mg, 0.36 mmol),
and heptanoic acid (0.95 g, 0.0073 mol) were added to a stirred solution of 4-aminophenethyl
alcohol (1.0 g, 0.0073 mol) in CH2Cl2 (15 mL). The reaction mixture was stirred under nitrogen
for 12 h. The solvent was removed in vacuo and the resulting residue was purified by flash column
1
chromatography to afford the desired products 11 (1.6 g, 90%). H NMR (400 MHz, CDCl3)
δ 7.30
(d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H), 3.61 (t, J = 7.0 Hz, 2H), 2.65 (t, J = 7.0 Hz, 2H), 2.19 (t, J = 7.6
Hz, 2H), 1.61–1.45 (m, 2H), 1.26–1.09 (m, 6H), 0.74 (t, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ
173.4, 136.8, 135.0, 129.6(2C), 120.8(2C), 63.6, 38.9, 37.6, 31.9, 29.3, 26.1, 22.9, 14.3; ESI-HRMS (M + H)+
m/z calcd. for C15H24NO2 250.1807, found 250.1844.

Molecules 2018, 23, 2750
7 of 12
4-Heptanamidophenethyl 4-methylbenzenesulfonate (12) To a solution of 11 (1.35 g, 0.0054 mol) and
triethylamine (3.77 mL, 0.027 mol) in CH2Cl2 (30 mL) at 0 ◦C was added p-toluenesulfonyl chloride
(1.54 g, 0.0082 mol). After being stirred at room temperature for 6 h, the reaction mixture was
evaporated, diluted with water, and the product was extracted with EtOAc. The extract was washed
with brine, dried, and evaporated. Flash column chromatography with hexane/EtOAc (10:1) as the
1
eluent gave compound 12 (2.0 g, 94%). H NMR (400 MHz, CDCl3)
δ 7.65 (d, J = 8.3 Hz, 2H), 7.39
(d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 4.14 (t, J = 7.0 Hz, 2H), 2.87 (t, J = 7.0
Hz, 2H), 2.40 (s, 3H), 2.35–2.29 (m, 2H), 1.72–1.62 (m, 2H), 1.38–1.22 (m, 6H), 0.85 (t, J = 6.9 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) δ 172.2, 145.2, 137.3, 133.2, 132.4, 130.3(2C), 129.8(2C), 128.2(2C), 120.5(2C),
71.1, 38.1, 35.1, 32.0, 29.3, 26.1, 22.9, 22.0, 14.5; ESI-HRMS (M + H)+ m/z calcd. for C22H30NO4S
404.1896, found 404.1858.
N-(4-(2-(4-Hydroxypiperidin-1-yl)ethyl)phenyl)heptanamide, 4-methylbenzenesulfonate salt (2) To a solution
of 12 (50 mg, 0.12 mmol) in 7 mL of acetonitrile was added 4-hydroxypiperidine (38 mg, 0.37 mmol).
The reaction mixture was stirred at 50 ◦C for 12 h and concentrated. Purification by silica
gel chromatography, eluting with CH2Cl2/MeOH (10:1), gave 44 mg (73%) of compound
2 as
1
a slightly yellow waxy solid. H NMR (400 MHz, MeOD/CDCl3 = 3/1)
δ 7.52 (d, J = 8.2 Hz, 2H),
7.28 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 3.57 (s, OH), 2.89 (t, J = 7.8
Hz, 2H), 2.70–2.63 (m, 5H), 2.53–2.47 (m, 2H), 2.15 (s, 3H), 2.13 (t, J = 7.6 Hz, 2H), 1.86–1.73 (m, 2H),
1.60–1.41 (m, 4H), 1.19–1.01 (m, 6H), 0.67 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ
175.0, 143.1, 142.6, 138.9, 130.7(4C), 127.5(2C), 122.2(2C), 61.0, 51.3, 38.9, 33.7, 33.3, 33.0, 30.7, 27.5,
24.2, 22.9, 15.6; ESI-HRMS (M+H)+ m/z calcd. for C20H33N2O2 333.2542, found 333.2575.
(R)-N-(4-(2-(2-(Hydroxymethyl)pyrrolidin-1-yl)ethyl)phenyl)heptanamide, 4-methylbenzenesulfonate salt (3)
Compound
as that described for
3
was prepared from 12 and (R)-(
−
)-prolinol according to the same reaction procedure
1
. Yield = 76%; 1H NMR (400 MHz, MeOD/CDCl3 = 3/1)
δ
7.54 (d, J = 8.2 Hz,
2H), 7.30 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 3.63 (qd, J = 12.5, 5.3 Hz,
2H), 3.54–3.46 (m, 1H), 3.39 (td, J = 11.9, 5.5 Hz, 1H), 3.31 (dd, J = 10.5, 4.2 Hz, 1H), 2.97 (td, J = 11.3,
5.6 Hz, 1H), 2.82 (dddd, J = 20.8, 18.8, 13.2, 5.4 Hz, 3H), 2.16 (s, 3H), 2.15 (t, J = 8.0 Hz, 2H), 2.03–1.91
(m, 1H), 1.85 (dd, J = 13.7, 7.5 Hz, 2H), 1.67 (td, J = 13.5, 6.8 Hz, 1H), 1.49 (dt, J = 15.3, 7.6 Hz, 2H),
1.22–1.04 (m, 6H), 0.68 (t, J = 6.7 Hz, 3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ 173.6, 141.8,
141.0, 137.9, 132.0, 129.3(4C), 126.1(2C), 120.9(2C), 60.8, 55.1, 37.5, 31.9, 31.8, 29.3, 26.6, 26.1, 23.2, 22.8,
21.4, 14.2; ESI-HRMS (M+H)+ m/z calcd. for C20H33N2O2 333.2542, found 333.2517.
N-(4-(2-(4-(Hydroxymethyl)piperidin-1-yl)ethyl)phenyl)heptanamide, 4-methylbenzenesulfonate salt (
Compound was prepared from 12 and 4-piperidinemethanol according to the same reaction
procedure as that described for . Yield = 82%; H NMR (400 MHz, MeOD/CDCl3 = 3/1) δ 7.61
4)
4
1
1
(d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.1 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 3.30
(d, J = 6.0 Hz, 4H), 2.94–2.87 (m, 2H), 2.87–2.78 (m, 2H), 2.52 (t, J = 11.1 Hz, 2H), 2.23 (s, 3H), 2.22–2.17
(m, 2H), 1.76 (d, J = 12.6 Hz, 2H), 1.60–1.37 (m, 5H), 1.25–1.10 (m, 6H), 0.75 (t, J = 6.7 Hz, 3H); 13
C
NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ 174.8, 143.2, 142.5, 139.1, 130.8(4C), 127.5(2C), 122.2(2C), 67.4,
54.7, 39.0, 38.3, 33.3, 32.2, 30.7, 27.8, 24.3, 23.0, 15.7; ESI-HRMS (M + H)+ m/z calcd. for C21H35N2O2
347.2699, found 347.2629.
N-(4-(2-(4-(2-Hydroxyethyl)piperazin-1-yl)ethyl)phenyl)heptanamide, 4-methylbenzenesulfonate salt (
Compound was prepared from 12 and 1-(2-hydroxyethyl)piperazine according to the same reaction
procedure as that described for . Yield = 62%; H NMR (400 MHz, MeOD/CDCl3 = 3/1) δ 7.59
5)
5
1
1
(d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 3.52
(t, J = 5.6 Hz, 2H), 2.63 (dd, J = 10.3, 6.1 Hz, 2H), 2.56–2.38 (m, 12H), 2.23 (s, 3H), 2.22–2.17 (m, 2H),
1.59–1.49 (m, 2H), 1.26–1.12 (m, 6H), 0.75 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)

Molecules 2018, 23, 2750
8 of 12
δ
174.9, 143.4, 142.5, 138.5, 137.1, 130.7(4C), 127.5(2C), 122.1(2C), 60.0, 38.9, 34.3, 33.4, 30.7, 27.5, 24.3,
22.9, 15.7; ESI-HRMS (M + H)+ m/z calcd. for C21H36N3O2 362.2808, found 362.2833.
N-(4-(2-(4-Phenyl-1H-imidazol-1-yl)ethyl)phenyl)heptanamide, 4-methylbenzenesulfonate salt (
6) Compound
6
was prepared from 12 and 4-phenylimidazole according to the same reaction procedure as
that described for 8.69 (s, NH),
1
. Yield = 54%; 1H NMR (400 MHz, MeOD/CDCl3 = 3/1)
δ
7.49 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 5.6 Hz, 1H), 7.27 (d, J = 4.4 Hz, 1H), 7.23 (d, J = 7.5 Hz, 2H),
7.16 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 7.3 Hz, 2H), 6.83–6.79 (m, 1H),
6.76 (d, J = 8.2 Hz, 2H), 6.46 (d, J = 8.2 Hz, 1H), 4.18 (t, J = 5.9 Hz, 1H), 4.09 (t, J = 5.4 Hz, 1H),
2.83 (t, J = 5.5 Hz, 1H), 2.55 (t, J = 6.0 Hz, 1H), 2.15–2.07 (m, 2H), 2.11 (s, 3H), 1.51–1.36 (m, 2H),
1.16–0.97 (m, 6H), 0.63 (t, J = 6.2 Hz, 3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ 175.2, 143.2,
142.4, 139.6, 138.2, 137.2, 133.0, 132.6, 132.5, 131.2, 131.1, 130.8, 130.6, 127.5, 126.4, 122.4, 122.3, 121.6,
52.8, 50.4, 38.9, 37.3, 37.0, 33.3, 30.7, 27.5, 24.2, 22.8; ESI-HRMS (M + H)+ m/z calcd. for C24H30N3O
376.2389, found 376.2391.
N-(4-(2-((4-Methoxybenzyl)amino)ethyl)phenyl)heptanamide (7) Compound 7 was prepared from 12 and
4-methoxybenzylamine according to the same reaction procedure as that described for
1
. Yield =
1
72%; H NMR (400 MHz, MeOD/CDCl3 = 3/1)
δ 7.39 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H),
7.03 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 3.71 (s, 3H), 3.64 (s, 2H), 2.81–2.73 (m, 2H), 2.70 (dd,
J = 10.9, 4.3 Hz, 2H), 2.26 (t, J = 7.6 Hz, 2H), 1.66–1.55 (m, 2H), 1.34–1.18 (m, 6H), 0.80 (t, J = 6.8 Hz,
3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ 174.9, 160.7, 138.6, 136.5, 132.2, 131.5(2C), 130.7(2C),
122.1(2C), 115.7(2C), 56.9, 54.4, 51.5, 38.9, 36.5, 33.3, 30.7, 27.5, 24.2, 15.6; ESI-HRMS (M + H)+ m/z
calcd. for C23H33N2O2 369.2542, found 369.2538.
N-Benzyl-2-(4-heptanamidophenyl)-N,N-dimethylethan-1-aminium 4-methylbenzenesulfonate (
8) Compound
8
was prepared from 12 and N-benzyldimethylamine according to the same reaction procedure as
1
that described for 1. Yield = 66%; H NMR (400 MHz, MeOD/CDCl3 = 3/1) δ 7.71 (d, J = 8.2 Hz,
2H), 7.52 (d, J = 8.5 Hz, 2H), 7.49–7.40 (m, 5H), 7.15 (t, J = 8.2 Hz, 4H), 4.54 (s, 2H), 3.50–3.41 (m, 2H),
3.11–3.01 (m, 2H), 3.08 (s, 6H), 2.37–2.24 (m, 2H), 2.32 (s, 3H), 1.72–1.60 (m, 2H), 1.39–1.21 (m, 6H),
0.87 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ 175.0, 146.9, 144.1, 142.0, 139.7,
139.1, 134.6, 134.2, 133.5, 132.7, 131.7, 131.1, 131.0, 130.5, 129.5, 128.7, 127.4, 122.4, 122.0, 72.8, 70.0, 66.7,
38.9, 36.4, 33.3, 30.6, 30.2, 27.4, 24.2, 22.8, 15.6; ESI-HRMS (M + H)+ m/z calcd. for C24H36N2O 368.2822,
found 368.2809.
N-(4-Heptanamidophenethyl)-N,N-dimethylcyclohexanaminium 4-methylbenzenesulfonate (
was prepared from 12 and N,N-dimethylcyclohexylamine according to the same reaction procedure as
that described for 7.49 (d, J = 8.2 Hz, 2H),
. Yield = 63%; 1H NMR (400 MHz, MeOD/CDCl3 = 3/1)
9) Compound 9
1
δ
7.31 (d, J = 8.5 Hz, 2H), 6.95 (dd, J = 8.1, 5.4 Hz, 4H), 3.93 (t, J = 6.8 Hz, 1H), 3.27–3.19 (m, 2H), 2.84
(s, 6H), 2.81–2.73 (m, 2H), 2.65 (t, J = 6.8 Hz, 1H), 2.11 (s, 3H), 2.13–2.09 (m, 3H), 1.94 (d, J = 10.5 Hz,
2H), 1.77 (d, J = 13.4 Hz, 2H), 1.54–1.40 (m, 4H), 1.29–1.05 (m, 8H), 0.65 (t, J = 6.8 Hz, 3H); 13C NMR
(100 MHz, MeOD/CDCl3 = 3/1)
δ 174.9, 146.8, 144.0, 141.8, 139.7, 130.4(4C), 127.4(2C), 122.4(2C), 74.5,
65.1, 38.8, 33.2, 30.6, 29.9, 27.8, 27.4, 27.0, 26.3, 24.1, 23.0, 22.7, 15.5; ESI-HRMS (M + H)+ m/z calcd. for
C23H40N2O 360.3135, found 360.3123.
1-(4-Heptanamidophenethyl)-1-methylpiperidin-1-ium 4-methylbenzenesulfonate (10) Compound 10 was
prepared from 12 and 1-methylpiperidine according to the same reaction procedure as that
. Yield = 90%; 1H NMR (400 MHz, MeOD/CDCl3 = 3/1)
described for 1 δ 7.54 (d, J = 8.1 Hz,
2H), 7.36 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 6.6 Hz, 4H), 3.98 (t, J = 6.8 Hz, 1H), 3.37–3.25 (m, 1H),
3.21 (t, J = 5.7 Hz, 2H), 2.93 (s, 3H), 2.81 (dd, J = 10.1, 6.7 Hz, 1H), 2.70 (t, J = 6.8 Hz, 1H), 2.19–2.13 (m,
2H), 2.17 (s, 3H), 1.67 (dd, J = 10.7, 5.6 Hz, 4H), 1.59–1.42 (m, 4H), 1.24–1.06 (m, 8H), 0.70 (t, J = 6.8 Hz,
3H); 13C NMR (100 MHz, MeOD/CDCl3 = 3/1)
δ 175.0, 146.9, 142.0, 139.7, 139.1, 130.6(2C), 129.5(2C),

Molecules 2018, 23, 2750
9 of 12
127.5(2C), 122.4(2C), 63.0, 38.9, 36.4, 33.3, 30.6, 29.4, 27.4(2C), 24.2, 23.2, 22.8, 22.5, 21.7, 15.6; ESI-HRMS
(M + H)+ m/z calcd. for C21H36N2O 332.2822, found 332.2871.
3.3. Chemicals, Reagents and Antibodies
DMEM, RPMI, fetal bovine serum (FBS), penicillin–streptomycin and trypsin 0.25% were
purchased from GE Healthcare Life Sciences HyClone Laboratories (Pittsburgh, PA, USA). EZ-CYTOX
was obtained from DoGenBio Co. Ltd. (Seoul, Korea). ApoScanTM annexin V-FITC apoptosis detection
kit was received from Biobud (Cat. No.: LS-02-100, Gyeonggi-do, Korea). PP2A activity assay kit was
acquired from Millipore Corporation (Billerica, MA, USA). SK1 assay kit was received from Echelon
Corporation (Santa Clara, CA, USA). AKT antibody was purchased from Cell Signaling Technology
(Danvers, MA, USA). Anti-ERK, anti-pERK, anti-pAKT, anti-β-actin, and anti-HRP conjugated
anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology
(Dallas, TX, USA). Protein marker was received from Thermo Fisher Scientific (Waltham, MA, USA)
Western blotting ECL solution was obtained from Millipore Corporation (Burlington, MA, USA)
3.4. Cancer Cell Line and Culture
Colorectal cancer cell lines (HT-29 and HCT-116), gastric cancer cell line (AGS) and prostate cancer
cell line (PC-3) were purchased from Korean Cell Line Bank. Cells were cultured in RPMI/DMEM
medium supplied with 10% FBS, streptomycin (100 U/mL) and penicillin (100 U/mL) in 95%
humidified, 5% CO2 incubator at 37 ◦C.
3.5. MTT Cell Viability Assay
Cancer cell viability assay was performed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium
bromide (MTT) assay. Briefly, subsequent 3000 cells/well were plated in 96-well plates, after
24/48 h treatment of derivatives and FTY720, EZ-CYTOX 10 µL was added and incubated for 90
min. Absorbance was recorded using Thermo Scientific Multiskan GO (Waltham, MA, USA) at
450 nm wavelengths.
3.6. Annexin V Assay
Cell apoptosis assay was determined using annexin V-FITC and propidium iodide (PI) staining as
described in the manufacturer’s instructions. Briefly, HT-29 cells (5
×
105/well) were seeded in 12-well
plates. Cells were treated with compound for 24 h and cell apoptosis was analyzed by annexin
7
V-FITC staining. Data analysis was performed using an Arthur image-based cytometer.
3.7. PP2A Activity Assay
PP2A activity assay was a PP2A immunoprecipitation phosphatase assay kit operated according
to the manufacturer’s instruction. Briefly, prepared protein aliquot was incubated with anti-PP2A
subunit C-antibody and protein A agarose slurry at 4 ◦C for 2–3 h, and then phosphopeptide was
added. Samples were recorded by Thermo Scientific Multiskan GO.
3.8. Immunoblotting
Cell extracts from HT-29 cells treated with compound
quantification, protein samples (20–30 g/lane) were separated by electrophoresis and then transferred
to Immobilon-P PVDF membranes. The membranes were incubated with anti- -actin, anti-ERK,
7 and FTY720 were prepared. After protein
µ
β
anti-pERK, anti-AKT and anti-pAKT antibodies. The primary antibodies were detected using
horseradish peroxidase-conjugated anti-mouse IgG with UVITEC or X-ray film.

Molecules 2018, 23, 2750
10 of 12
3.9. Molecular Modeling Studies
Molecular modeling studies of FTY720 and compound
7
against the human I2PP2A/SET
were performed using Schrödinger Suite 2017-2 (Schrödinger, LLC, http://www.schrodinger.com).
The X-ray crystal structure was obtained from the Protein Data Bank (http://www.rcsb.org/pdb,
PDB code: 2E50). The protein preparation was revised using Protein Preparation Wizard in Maestro
v.11.2 and the receptor grid box was generated as 25
×
25
×
25 Å cubic size centered on complexed
ligand. The ligands were minimized using OPLS_2005 force field with a dielectric constant value
80.0 in MacreoModel v.11.6. Flexible dockings were performed using the Glide v.7.5 program with
standard precision method. The docking models of ligands were visualized using Discovery Studio
2016 (http://www.biovia.com).
3.10. Statistical Analysis
The results were analyzed using GraphPad Prism 7 with three independent experiments
(mean ± SD). One-way ANOVA with post-hoc analysis using the Newman-Keuls multiple comparison
test was used to analyze parametric data. Results were accepted as significant for p-values < 0.05.
4. Conclusions
We have synthesized new materials with very few synthetic steps, unlike the synthesis of FTY720
and its derivatives. Among the newly synthesized compounds, compound
7 is considered to have the
most effective anticancer property, and the activity of this property was thought to be due to PP2A
activation rather than the mechanism leading to inhibition of SK1. Docking studies in PP2A of FTY720
and compound
7
showed that the aromatic head group of compound
7
could effectively replace the
is
complex structure of the head group of FTY720. In addition, the amide linker of compound
7
expected to show good activity results by interacting with Asn61, which does not interact with FTY720.
These biological results and the structure of the compounds provide important information for the
design of new sphingolipid-based compounds targeting PP2A.
Supplementary Materials: The following are available online: Effect of PF-543 and compound
7 on SK1 activity,
1
cell cytotoxicity of compound
spectra for compounds.
7
in cancer and normal cell line and copies of H NMR, 13C NMR and HRMS
Author Contributions: T.L. and S.K. designed the compounds, J.-Y.L. did docking studies, D.J.B. synthesized
compounds, J.S., S.H.K., S.M.S., S.W.K., and E.-Y.P. did the biological studies, H.-S.J., E.-Y.P. and D.J.B. wrote
this paper.
Acknowledgments: This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning
(2015R1C1A1A01053302).
Conflicts of Interest: All authors declare no financial/commercial conflict of interests.
References
1.
2.
3.
Pitson, S.M. Regulation of sphingosine kinase and sphingolipid signaling. Trends Biochem. Sci. 2011, 36, 97–107.
[CrossRef] [PubMed]
Ogretmen, B. Sphingolipid metabolism in cancer signalling and therapy. Nat. Rev. Cancer 2018, 18, 33–50.
[CrossRef] [PubMed]
Chun, J.; Goetzl, E.J.; Hla, T.; Igarashi, Y.; Lynch, K.R.; Moolenaar, W.; Pyne, S.; Tigyi, G. International Union
of Pharmacology. XXXIV. Lysophospholipid receptor nomenclature. Pharmacol. Rev. 2002, 54, 265–269.
[CrossRef] [PubMed]
4.
5.
Maceyka, M.; Harikumar, K.B.; Milstien, S.; Spiegel, S. Sphingosine-1-phosphate signaling and its role in
disease. Trends Cell Biol. 2012, 22, 50–60. [CrossRef] [PubMed]
Pyne, N.J.; McNaughton, M.; Boomkamp, S.; MacRitchie, N.; Evangelisti, C.; Martelli, A.M.; Jiang, H.R.;
Ubhi, S.; Pyne, S. Role of sphingosine 1-phosphate receptors, sphingosine kinases and sphingosine in cancer
and inflammation. Adv. Biol. Regul. 2016, 151–159. [CrossRef] [PubMed]

Molecules 2018, 23, 2750
11 of 12
6.
7.
8.
9.
Pitman, M.R.; Costabile, M.; Pitson, S.M. Recent advances in the development of sphingosine kinase
inhibitors. Cell. Signal. 2016, 28, 1349–1363. [CrossRef] [PubMed]
Newton, J.; Lima, S.; Maceyka, M.; Spiegel, S. Revisiting the sphingolipid rheostat: Evolving concepts in
cancer therapy. Exp. Cell Res. 2015, 333, 195–200. [CrossRef] [PubMed]
Rossjohn, J.; Pellicci, D.G.; Patel, O.; Gapin, L.; Godfrey, D.I. Recognition of CD1d-restricted antigens by
natural killer T cells. Nat. Rev. Immunol. 2012, 12, 845–857. [CrossRef] [PubMed]
Oaks, J.J.; Santhanam, R.; Walker, C.J.; Roof, S.; Harb, J.G.; Ferenchak, G.; Eisfeld, A.K.; Van Brocklyn, J.R.;
Briesewitz, R.; Saddoughi, S.A.; et al. Antagonistic activities of the immunomodulator and PP2A-activating
drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies. Blood 2013, 122, 1923–1934.
[CrossRef] [PubMed]
10. Ng, K.T.; Man, K.; Ho, J.W.; Sun, C.K.; Lee, T.K.; Zhao, Y.; Lo, C.M.; Poon, R.T.; Fan, S.T. Marked suppression
of tumor growth by FTY720 in a rat liver tumor model: The significance of down-regulation of cell survival
Akt pathway. Int. J. Oncol. 2007, 30, 375–380. [PubMed]
11. Azuma, H.; Takahara, S.; Horie, S.; Muto, S.; Otsuki, Y.; Katsuoka, Y. Induction of apoptosis in human bladder
cancer cells in vitro and in vivo caused by FTY720 treatment. J. Urol. 2003, 169, 2372–2377. [CrossRef]
[PubMed]
12. Zhang, L.; Wang, H.D.; Ji, X.J.; Cong, Z.X.; Zhu, J.H.; Zhou, Y. FTY720 for cancer therapy (Review). Oncol. Rep.
2013, 30, 2571–2578. [CrossRef] [PubMed]
13. Tonelli, F.; Lim, K.G.; Loveridge, C.; Long, J.; Pitson, S.M.; Tigyi, G.; Bittman, R.; Pyne, S.; Pyne, N.J. FTY720
and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in
human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells.
Cell. Signal. 2010, 22, 1536–1542. [CrossRef] [PubMed]
14. Watson, D.G.; Tonelli, F.; Alossaimi, M.; Williamson, L.; Chan, E.; Gorshkova, I.; Berdyshev, E.; Bittman, R.;
Pyne, N.J.; Pyne, S. The roles of sphingosine kinases 1 and 2 in regulating the Warburg effect in prostate
cancer cells. Cell. Signal. 2013, 25, 1011–1017. [CrossRef] [PubMed]
15. Lu, X.; Sun, C.; Valentine, W.J.; Shuyu, E.; Liu, J.; Tigyi, G.; Bittman, R. Chiral vinylphosphonate and
phosphonate analogues of the immunosuppressive agent FTY720. J. Org. Chem. 2009, 74, 3192–3195.
[CrossRef] [PubMed]
16. Baek, D.J.; MacRitchie, N.; Pyne, N.J.; Pyne, S.; Bittman, R. Synthesis of selective inhibitors of sphingosine
kinase 1. Chem. Commun. 2013, 49, 2136–2138. [CrossRef] [PubMed]
17. Baek, D.J.; MacRitchie, N.; Anthony, N.G.; Mackay, S.P.; Pyne, S.; Pyne, N.J.; Bittman, R. Structure-activity
relationships and molecular modeling of sphingosine kinase inhibitors. J. Med. Chem. 2013, 56, 9310–9327.
[CrossRef] [PubMed]
18. Woodcock, J.M.; Coolen, C.; Goodwin, K.L.; Baek, D.J.; Bittman, R.; Samuel, M.S.; Pitson, S.M.; Lopez, A.F.
Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics. Oncotarget
2015, 6, 14522–14536. [CrossRef] [PubMed]
19. Bonandi, E.; Christodoulou, M.S.; Fumagalli, G.; Perdicchia, D.; Rastelli, G.; Passarella, D. The 1,2,3-triazole
ring as a bioisostere in medicinal chemistry. Drug Discov. Today 2017, 22, 1572–1581. [CrossRef] [PubMed]
20. Xiang, Y.; Hirth, B.; Kane, J.L., Jr.; Liao, J.; Noson, K.D.; Yee, C.; Asmussen, G.; Fitzgerald, M.; Klaus, C.;
Booker, M. Discovery of novel sphingosine kinase-1 inhibitors. Part 2. Bioorg. Med. Chem. Lett.
2010, 20, 4550–4554. [CrossRef] [PubMed]
21. Liu, K.; Guo, T.L.; Hait, N.C.; Allegood, J.; Parikh, H.I.; Xu, W.; Kellogg, G.E.; Grant, S.; Spiegel, S.; Zhang, S.
Biological characterization of 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione
(K145) as a selective sphingosine kinase-2 inhibitor and anticancer agent. PLoS ONE 2013, 8, e56471.
[CrossRef] [PubMed]
22. Schnute, M.E.; McReynolds, M.D.; Kasten, T.; Yates, M.; Jerome, G.; Rains, J.W.; Hall, T.; Chrencik, J.;
Kraus, M.; Cronin, C.N.; et al. Modulation of cellular S1P levels with a novel, potent and specific inhibitor of
sphingosine kinase-1. Biochem. J. 2012, 444, 79–88. [CrossRef] [PubMed]
23. Kaur, A.; Westermarck, J. Regulation of protein phosphatase 2A (PP2A) tumor suppressor function by PME-1.
Biochem. Soc. Trans. 2016, 44, 1683–1693. [CrossRef] [PubMed]

Molecules 2018, 23, 2750
12 of 12
24. Cristóbal, I.; Manso, R.; Rincón, R.; Caramés, C.; Senin, C.; Borrero, A.; Martínez-Useros, J.; Rodriguez, M.;
Zazo, S.; Aguilera, O. PP2A inhibition is a common event in colorectal cancer and its restoration using
FTY720 shows promising therapeutic potential. Mol. Cancer Ther. 2014, 13, 938–947. [CrossRef] [PubMed]
25. Muto, S.; Senda, M.; Akai, Y.; Sato, L.; Suzuki, T.; Nagai, R.; Senda, T.; Horikoshi, M. Relationship between
the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity. Proc. Natl. Acad. Sci. USA
2007, 104, 4285–4290. [CrossRef] [PubMed]
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).