Suzuki coupling based synthesis and in vitro cytotoxic evaluation of Fingolimod and analogues

Article abstract of DOI:10.1016/j.tet.2013.02.030

A concise synthesis of Fingolimod (FTY720) and its analogues was described. The key step involved Suzuki coupling of arylboronate 11 with different bromides. In addition, the newly synthesized Fingolimod analogues (5a-i and 6a-c) were evaluated for their in vitro cytotoxicity against SMCC-7721 and HL-60 cell lines. The preliminary SAR was discussed.

Full text of DOI:10.1016/j.tet.2013.02.030

Tetrahedron 69 (2013) 2927e2932
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Suzuki coupling based synthesis and in vitro cytotoxic evaluation
of Fingolimod and analogues
,
a
b
,
c
Tie-Wen Mei a, Yu Luo a , Xiang-Jun Feng , Wei Lu , Bo Yang
*
*
a Department of Chemistry, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
b Institute of Drug Discovery and Development, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
c College of Pharmaceutical Science, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A concise synthesis of Fingolimod (FTY720) and its analogues was described. The key step involved
Suzuki coupling of arylboronate 11 with different bromides. In addition, the newly synthesized Fingo-
limod analogues (5aei and 6aec) were evaluated for their in vitro cytotoxicity against SMCC-7721 and
HL-60 cell lines. The preliminary SAR was discussed.
Received 25 December 2012
Received in revised form 28 January 2013
Accepted 4 February 2013
Available online 14 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Concise synthesis
Key step
Suzuki coupling
Cytotoxicity
1. Introduction
(SphKs) and sphingosine-1-phosphate receptors (S1PRs).2 SphKs
are oncogenic lipid kinases, because it can catalyze the conversion
Sphingolipids, major components of membrane lipids, have
emerged as key signaling molecules involved in the regulation of
the physiological and pathophysiological functions.1 Sphingolipid
metabolites, such as ceramide, sphingosine (SP, 1, Fig. 1) and
sphingosine-1-phosphate (S1P, 2, Fig. 1) play an important role in
the regulation of cell proliferation, survival, and cell death. They are
interconvertible by the different metabolic enzymes in a complex
lipid signaling system. It was confirmed that in this lipid signaling
system there are two important drug targets: sphingosine kinase
of the pro-apoptotic sphingosine to the pro-growth product S1P.3
Thus, SphKs inhibitors are deemed to have potential antitumor
activity. On the other hand, S1PRs are involved in the immune
regulation. As a result, S1PRs modulators are considered to have the
potential for treatment of multiple sclerosis (MS) and the rejection
in organ transplant.
Fingolimod (FTY720, 3, Fig. 1), an orally active immunomodu-
latory drug, was approved by FDA in 2010 as a first-line treatment
for MS.4 It was developed from the myriocin (ISP-1, 4, Fig. 1),
a fungal metabolite of the Chinese herb Isaria sinclairii.5 Fingolimod
is phosphorylated in vivo by sphingosine kinase type 2 (SphK2) to
form biologically active (S)-FTY720-phosphate, which is a potent
OH
OH
NH2
H2N
C13H27
OH
agonist for four sphingosine-1-phosphate receptors (S1PR1,3,4,5
)
and prevents the release of lymphocytes from lymphoid tissue.6e8
Recently, it was reported that FTY720 and its derivatives exhibited
antitumor activity by acting as an SphKs inhibitor. Pchejetski and
co-workers reported that Fingolimod acted as a sphingosine kinase
type 1 (SphK1) inhibitor and induced prostate cancer cell apoptosis
in a manner independent of S1PRs.9 In addition, Lim and co-
workers reported that (R)-FTY720 methyl ether exhibited antitu-
mor activity against breast cancer cells by acting as a specific SphK2
inhibitor.10 These reports prompted us to further explore FTY720
and its analogues as potential antitumor agents.
OH
SP, 1
Fingolimod (FTY720), 3
OH
NH2
O
HO2C
HO
C13H27
O
P
OH
OH
O
OH
S1P, 2
OH
NH2
ISP-I (myriocin), 4
Fig. 1. FTY720 and related molecules.
Several research groups have pointed out the importance of the
lipophilic tail region of FTY720 analogues to their biological
* Corresponding authors. Tel./fax: þ86 21 6260 2475; e-mail addresses: yluo@
chem.ecnu.edu.cn (Y. Luo), wlu@chem.ecnu.edu.cn (W. Lu).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.02.030

2928
T.-W. Mei et al. / Tetrahedron 69 (2013) 2927e2932
activity.11e13 Therefore, we decided to focus our efforts on the li-
pophilic region and study its antitumor activity.
Table 1
Screening the Suzuki coupling reaction conditionsa
NHBoc
There are several synthetic methods for the construction of this
lipophilic moiety. These include the classical Fried-Craft acylation,
Weinreb amide based nucleophilic substitution, Fe(acac)3-cata-
lyzed cross-coupling, Sonogashira coupling and Wittig reac-
tion.14e17 Here we report a versatile synthetic route for Fingolimod
analogues 5aei and 6aec (Fig. 2) based on Suzuki coupling re-
action, which allows for convenient structural modification of the
lipophilic side-chain. Furthermore, the newly prepared analogues
were evaluated for their in vitro anti-proliferative ability against
two tumor cell lines (SMCC-7721 and HL-60) and preliminary SAR
was discussed.
NHBoc
O
CH Br
O
O
O
O
B
O
12a
11
13a
Entry
Catalyst
Base
Solvent
Yieldb
1
2
3
4
5
6
7
8
PdCl2(dppf)
K2CO3
K2CO3
K2CO3
NaOH
CsF
KOAc
K2CO3
K2CO3
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane/H2O
Toluene
39%
30%
87%
82%
34%
23%
78%
26%
PdCl2(PPh3)2
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
OH
OH
H2N
a
Reagents and conditions: 11 (1 equiv), 12a (1.5 equiv), catalyst (0.05 equiv), base
(2 equiv), solvent, N2, reflux 12 h.
OH
OH
H2N
R
b
Isolated yields.
R
R=CnH2n+1
n=0-8
R=CnH2n+1
n=0, 6 and
8
NHBoc
NHBoc
6a-c
5a-i
O
R
CH2Br
R
O
O
O
12a-i
O
Fig. 2. Designed FTY720 analogues.
B
O
a
11
13a-i
2. Results and discussion
OH
OH
H2N
R
a: R = H;
b: R = Me;
c: R = Et;
2.1. Synthesis of key arylboronate 11
d: R = n-Pr; e: R = n-Bu; f: R = n-Pen
g: R = n-Hex; h: R = n-Hep; i: R = n-Oct
b
5 a-i
As shown in Scheme 1, the starting material 7 and 8 were pre-
pared according to the reported methods.18 Treatment of 7 and 8
with K2CO3 gave a mixture of E/Z geometrical isomers (compound
9). Complete reduction of the double bond and the nitro group
afforded amine 10 in a good yield. Compound 10 was treated with
BPO, B2(pin)2, and t-BuONO to afford the desired arylboronate 11 in
a moderate yield.19
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, dioxane, N2, reflux. (b) 6 M
HCl, MeOH, 50 ꢀC.
2.3. Synthesis of analogues 6aec
The second subseries of analogues 6aec was synthesized by
coupling of 11 with alkyl-substituted styrene bromides. However,
the application of the former reaction conditions to these sub-
strates resulted in very low yields. Thus, we had to screen the re-
action conditions again. The coupling of arylboronate 11 with
styrene bromide 14a was chosen as the test substrates. As illus-
trated in Table 2, the solvent played an important role in this re-
action. A mixture of toluene and dioxane was the most effective,
while a single solvent resulted in poor yields (entries 1e3 and 5e7).
In contrast to the former coupling conditions, PdCl2(dppf) served as
a more effective catalyst than Pd(PPh3)4 and NaOH provided
a higher yield than K2CO3 in this Suzuki coupling.
NHBoc
O
PPh3Br
O
OHC
O2N
O
O
+
NHBoc
a
b
O2N
9
7
8
NHBoc
NHBoc
O
O
O
O
c
O
B
H2N
O
10
11
Scheme 1. Reagents and conditions: (a) K2CO3, THF/DMF (3:1), reflux. (b) H2, Pd/C
(10 wt %), MeOH, rt. (c) B2(pin)2, BPO, t-BuONO, CH3CN, rt.
Table 2
Screening the Suzuki coupling reaction conditionsa
2.2. Synthesis of analogues 5aei
Br
NHBoc
NHBoc
O
O
O
The first subseries of analogues 5aei was prepared by coupling
of arylboronate 11 with different alkyl-substituted benzyl bro-
mides. To screen for suitable coupling conditions, we chose to focus
on the coupling of arylboronate 11 with benzyl bromide 12a as the
test substrates. As shown in Table 1, among the three catalysts
tested, Pd(PPh3)4 was the most effective in accomplishing the re-
action. Through screening the four bases, it was found that K2CO3
was more effective than other bases. In addition, dioxane as the
solvent gave a much better yield than toluene.
Under these optimal conditions, arylboronate 11 was coupled
with a variety of alkyl-substituted benzyl bromide 12aei20,21 to
give compounds 13aei in good to moderate yields. Subsequently,
hydrolysis of these intermediates proceeded smoothly under acidic
conditions to afford FTY720 analogues 5aei (Scheme 2).
O
14a
O
B
O
15a
11
Entry
Catalyst
Base
Solvent
Yield
1
2
3
4
5
6
7
8
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
PdCl2(dppf)
PdCl2(dppf)
PdCl2(dppf)
PdCl2(dppf)
K2CO3
K2CO3
K2CO3
NaOH
NaOH
NaOH
NaOH
K2CO3
Dioxane
Toluene
Trace
Trace
68%
63%
76%
25%
Trace
60%
Toluene/dioxane
Toluene/dioxane
Toluene/dioxane
Dioxane
Toluene
Toluene/dioxane
a
Reagents and conditions: 11 (1 equiv), 14a (1.5 equiv), catalyst (0.05 equiv), base
(2 equiv), solvent, N2, reflux 12 h.

T.-W. Mei et al. / Tetrahedron 69 (2013) 2927e2932
2929
Using this protocol, boronate 11 was reacted with alkyl-
substituted styrene bromines22,23 (Scheme 3). The resulting in-
termediates 15aec were further hydrolyzed to give FTY720 ana-
logues 6aec in good yields.
Compounds 5feh exhibited the most potent cytotoxicity, while
other compounds with either longer or shorter chains showed
much weaker activity.
3. Conclusion
Br
NHBoc
O
NHBoc
O
In summary, in order to explore the biological and chemical
space of Fingolimod, a convergent synthesis of FTY720 and its an-
alogues was developed by employing Suzuki coupling reaction. In
this convergent synthetic method, arylboronate 11 served as the
key intermediate. In addition, the prepared new analogues were
evaluated for their in vitro cytotoxicity against SMCC-7721 and HL-
60 cell lines and the preliminary SAR was discussed.
O
R
O
14a-c
O
B
a
O
15a-c
11
R
OH
OH
H2N
b
a: R = H;
b: R = n-Hex;
c: R = n-Oct
6a-c
R
Scheme 3. Reagents and conditions: (a) PdCl2(dppf), NaOH, toluene/dioxane, N2, re-
flux. (b) 6 M HCl, MeOH, 50 ꢀC.
4. Experimental section
4.1. General experimental
2.4. Synthesis of FTY720
1H NMR spectra were recorded on a Bruker DRX-500 (500 MHz).
13C NMR spectra were obtained on a JNM-EX400 (100 MHz). Mass
spectra were determined on a Finnigan MAT-95 mass spectrometer.
All reagents were used directly as obtained commercially, unless
otherwise noted.
Similarly, FTY720 was synthesized as shown in Scheme 4. Iodide
1624 was coupled with compound 11 to provide intermediate 17 in
a good yield. Hydrogenation of 17 furnished a known compound 18
in a quantitative yield, which was further transformed into FTY720
according to references.
4.2. Synthesis of 10
NHBoc
O
NHBoc
O
O
I
4.2.1. tert-Butyl 5-(4-aminophenethyl)-2,2-dimethyl-1,3-dioxan-5-
ylcarbamate (10). A suspension of aldehyde 8 (5.50 g, 21.29 mmol),
phosphonium salt 7 (12.24 g, 25.54 mmol), and K2CO3 (8.3 g,
59.61 mmol) in a mixture of THF (75 mL) and DMF (25 mL) was
heated under reflux for 6 h. After completion of the reaction, THF was
evaporated and the reaction mixture was quenched with water and
then extracted with ethyl acetate (3ꢁ150 mL). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated to give a residue, which was purified by silica gel flash
chromatography (ether:ethyl acetate¼7:1) to give a mixture of E/Z
isomer alkene 9. Thereafter, to a mixture of 9 and methanol (150 mL)
was added 10% Pd/C (0.8 g) and hydrogenated for 8 h at room tem-
perature. The reaction mixture was filtered through Celite and the
solvent was concentrated to give compound 10 (5.68 g, 76% yield
from 8) as a white solid; mp 160e163 ꢀC; 1H NMR (500 MHz, CDCl3):
O
5
O
B
O
16
n-C6H13
11
a
17
OH
OH
H2N
NHBoc
O
O
n-C8H17
n-C8H17
b
c
18
FTY720
Scheme 4. Reagents and conditions: (a) PdCl2(dppf), NaOH, toluene/dioxane, N2, re-
flux. (b) H2, Pd/C (10 wt %), MeOH, rt. (c) 6 M HCl, MeOH, 50 ꢀC.
2.5. Biological results and discussion
With these FTY720 analogues in hand, the in vitro anti-
proliferative activity was evaluated in SMCC-7721 and HL-60 cell
lines. The results were summarized in Table 3. The cytotoxicity of
each compound was expressed was the concentration of killing 50%
of the tumor cells. As shown in Table 3, the cytotoxicity of most
compounds were comparable to that of FTY720. It was interesting
d
1.41 (s, 3H), 1.43 (s, 3H), 1.47 (s, 9H), 1.92 (t, J¼8 Hz, 2H), 2.44e2.48
(m, 2H), 3.55 (s, 2H), 3.66 (d, J¼12 Hz, 2H), 3.88 (d, J¼11 Hz, 2H), 4.98
(s,1H), 6.61 (d, J¼8 Hz, 2H), 6.96 (d, J¼8 Hz, 2H); HRMS (EI): m/z calcd
for C19H30N2O4 [M]þ: 350.2206, found: 350.2203.
that the length of the lipophilic tail played
a
critical role.
4.3. Synthesis of 11
Table 3
4.3.1. tert-Butyl-2,2-dimethyl-5-(4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
In vitro cytotoxicity against two cell lines
borolan-2-yl)phenethyl)-1,3-dioxan-5-ylcarbamate
(11). B2(pin)2
(3.63 g, 14.27 mmol) and BPO (71 mg, 0.29 mmol) were weighed in
a 100 mL round-bottom flask. MeCN (50 mL), arylamine 10 (5.0 g,
14.27 mmol), and t-BuONO (2.20 g, 21.40 mmol) were then added in
succession. The resulting reaction mixture was allowed to stir for
5 h at room temperature. The solution was then concentrated under
reduced pressure to give a residue, which was purified by silica gel
flash chromatography (ether:ethyl acetate¼12:1) to give com-
pound 11 (3.84 g, 58%) as a white solid; mp 145e148 ꢀC; 1H NMR
Compounds
IC50 (mM)
SMCC-7721
HL-60
5a
5b
5c
5d
5e
5f
5g
5h
5i
6b
6c
FTY720
2.9
6.6
10.3
14.6
2.5
0.2
0.8
0.1
8.8
20.1
5.7
10.1
11.8
3.5
1.3
1.1
1.8
19.0
13.8
13.8
7.9
(500 MHz, CDCl3):
d 1.33 (s, 12H), 1.41 (s, 3H), 1.43 (s, 3H), 1.47 (s,
9H), 1.98 (s, 2 H), 2.56e2.60 (m, 2H), 3.66 (d, J¼12 Hz, 2H), 3.88 (d,
J¼11 Hz, 2H), 4.98 (s, 1H), 7.19 (d, J¼8 Hz, 2H), 7.71 (d, J¼8 Hz, 2H);
HRMS (EI): m/z calcd for C25H40BNO6 [M]þ: 461.2949, found:
461.2950.
8.7
2.9
4.0

2930
T.-W. Mei et al. / Tetrahedron 69 (2013) 2927e2932
4.4. General procedure for the synthesis of 13aei and 15aec
1.97 (d, J¼7.7 Hz, 2H), 2.51e2.57 (m, 4H), 3.67 (d, J¼12 Hz, 2H),
3.88e3.90 (m, 4H), 4.98 (s, 1H), 7.08e7.09 (m, 8); HRMS (EI): m/z
calcd for C32H47NO4 [M]þ: 509.3505, found: 509.3502.
Compound 12aei (0.26 mmol) or 14aec (0.26 mmol), arylbor-
onic ester 11 (0.17 mmol) were carefully dissolved in 10 mL corre-
sponding solvent, then corresponding base (0.35 mmol) were
added to the solution. After carefully degassing the mixture, cor-
responding catalyst (0.009 mmol) was added. The mixture was
refluxed under nitrogen for 12 h. After cooling, water was added,
the mixture was extracted with ethyl acetate (3ꢁ20 mL), and the
combined organic layers were dried over anhydrous Na2SO4 and
evaporated to dryness. The residue was purified by silica gel flash
chromatography (ether:ethyl acetate¼10:1) to give 13aei, 15aec,
respectively.
4.4.8. tert-Butyl-5-(4-(4-heptylbenzyl)phenethyl)-2,2-dimethyl-1,3-
dioxan-5-ylcarbamate (13h). Yield 71%; White solid; mp 87e89 ꢀC;
1H NMR (500 MHz, CDCl3):
d
0.87 (t, J¼7 Hz, 3H), 1.26e1.30 (m, 8H),
1.40e1.43 (m, 6H), 1.46 (s, 9H), 1.56e1.59 (m, 2H), 1.96 (d, J¼7.7 Hz,
2H), 2.50e2.56 (m, 4H), 3.66 (d, J¼12 Hz, 2H), 3.89 (s, 4H), 4.99 (s,
1H), 7.06e7.09 (m, 8H); HRMS (ESI): m/z calcd for C33H49NO4Na
[MþNa]þ: 546.3559, found: 546.3540.
4.4.9. tert-Butyl 2,2-dimethyl-5-(4-(4-octylbenzyl)phenethyl)-1,3-
dioxan-5-ylcarbamate (13i). Yield 70%; White solid; mp
4.4.1. tert-Butyl 5-(4-benzylphenethyl)-2,2-dimethyl-1,3-dioxan-5-
77e79 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.87 (t, J¼7 Hz, 3H),
ylcarbamate (13a). Yield 87%; White solid; mp 81e83 ꢀC; 1H
1.26e1.29 (m, 10H), 1.40e1.43 (m, 6H), 1.46 (s, 9H), 1.56e1.59 (m,
2H), 1.96 (d, J¼7.8 Hz, 2H), 2.50e2.56 (m, 4H), 3.66 (d, J¼12 Hz, 2H),
3.89 (s, 4H), 5.01 (s, 1H), 7.07e7.08 (m, 8H); HRMS (ESI): m/z calcd
for C34H51NO4Na [MþNa]þ: 560.3716, found: 560.3724.
NMR (500 MHz, CDCl3):
d 1.41 (s, 3H), 1.43 (s, 3H), 1.47 (s, 9H), 1.97
(t, J¼7.8 Hz, 2H), 2.52e2.55 (m, 2H), 3.67 (d, J¼12 Hz, 2H), 3.89 (d,
J¼12 Hz, 2H), 3.94 (s, 2H), 4.96 (s, 1H), 7.09 (s, 4H), 7.17e7.20 (m,
3H), 7.26e7.29 (m, 2H); HRMS (ESI): m/z calcd for C26H35NO4Na
[MþNa]þ: 448.2464, found: 448.2469.
4.4.10. (E)-tert-Butyl 2,2-dimethyl-5-(4-styrylphenethyl)-1,3-dioxan-
5-ylcarbamate (15a). Yield 76%; White solid; mp 51e53 ꢀC; 1H NMR
4.4.2. tert-Butyl 2,2-dimethyl-5-(4-(4-methylbenzyl)phenethyl)-1,3-
(400 MHz, CDCl3):
d
1.43 (d, J¼8 Hz, 6H), 1.48 (s, 9H), 1.99e2.05 (m,
dioxan-5-ylcarbamate (13b). Yield 83%; White solid; mp
2 H), 2.56e2.60 (t, J¼8 Hz, 2H), 3.69 (d, J¼12 Hz, 2H), 3.90 (d,
J¼12 Hz, 2H), 5.00 (s, 1H), 7.07 (s, 2H), 7.18 (d, J¼8 Hz, 3H), 7.35 (t,
J¼8 Hz, 2 H), 7.43 (d, J¼8 Hz, 2H), 7.50 (d, J¼8 Hz, 2H); HRMS (EI):
m/z calcd for C27H35NO4 [M]þ: 437.2566, found: 437.2560.
91e93 ꢀC; 1H NMR (500 MHz, CDCl3):
d 1.45 (s, 3H), 1.48 (s, 3H), 1.51
(s, 9H), 2.00 (t, J¼7.8 Hz, 2H), 2.34 (s, 3H), 2.55e2.59 (m, 2H), 3.71
(d, J¼12 Hz, 2H), 3.93e3.95 (m, 4H), 5.04 (s, 1H), 7.09e7.15 (m, 8H);
HRMS (EI): m/z calcd for C27H37NO4 [M]þ: 439.2723, found:
439.2718.
4.4.11. (E)-tert-Butyl-5-(4-(4-hexylstyryl)phenethyl)-2,2-dimethyl-
1,3-dioxan-5-ylcarbamate (15b). Yield 54%; White solid; mp
4.4.3. tert-Butyl 5-(4-(4-ethylbenzyl)phenethyl)-2,2-dimethyl-1,3-
57e59 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.88 (t, J¼6.5 Hz, 3H),
dioxan-5-ylcarbamate (13c). Yield 70%; White solid; mp
1.30e1.34 (m, 6H), 1.42e1.44 (m, 6H), 1.48 (s, 9H), 1.60e1.62 (m,
2H), 2.00e2.02 (m, 2H), 2.55e2.63 (m 4H), 3.68e3.71 (m, 2H),
3.91e3.93 (m, 2H), 5.01 (s, 1H), 7.04 (s, 2H), 7.15e7.17 (m, 4H),
7.40e7.44 (m, 4H); HRMS (ESI): m/z calcd for C33H47NO4Na
[MþNa]þ: 544.3404, found: 544.3405.
97e99 ꢀC; 1H NMR (500 MHz, CDCl3):
d
1.25 (t, J¼7.6 Hz, 3H),
1.45e1.48 (m, 6H), 1.51 (s, 9H), 2.00 (t, J¼8 Hz, 2H), 2.55e2.58 (m,
2H), 2.62e2.67 (m, 2H), 3.70 (d, J¼12 Hz, 2H), 3.92e3.94 (m, 4H),
5.03 (s, 1H), 7.11e7.15 (m, 8H); HRMS (EI): m/z calcd for C28H39NO4
[M]þ: 453.2879, found: 453.2881.
4.4.12. (E)-tert-Butyl-2,2-dimethyl-5-(4-(4-octylstyryl)phenethyl)-
4.4.4. tert-Butyl-2,2-dimethyl-5-(4-(4-propylbenzyl)phenethyl)-1,3-
1,3-dioxan-5-ylcarbamate (15c). Yield 51%; White solid; mp
dioxan-5-ylcarbamate (13d). Yield 80%; White solid; mp
62e64 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.89 (t, J¼7 Hz, 3H),
106e108 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.96 (t, J¼7.3 Hz, 3H),
1.28e1.32 (m, 10H), 1.43e1.45 (m, 6H), 1.49 (s, 9H), 1.61e1.63 (m,
2H), 1.99e2.01 (m, 2H), 2.56e2.62 (m 4H), 3.68e3.70 (m, 2H),
3.90e3.92 (m, 2H), 5.00 (s, 1H), 7.04 (s, 2H), 7.16e7.18 (m, 4H),
7.41e7.43 (m, 4H); HRMS (ESI): m/z calcd for C35H51NO4Na
[MþNa]þ: 572.3716, found: 572.3709.
1.44e1.46 (m, 6H), 1.50 (s, 9H), 1.62e1.67 (m, 2H), 2.00 (t, J¼8 Hz,
2H), 2.54e2.59 (m, 4H), 3.69 (d, J¼12 Hz, 2H), 3.93 (s, 4H), 5.02 (s,
1H), 7.11e7.12 (m, 8H); HRMS (EI):m/z calcd for C29H41NO4 [M]þ:
467.3036, found: 467.3040.
4.4.5. tert-Butyl 5-(4-(4-butylbenzyl)phenethyl)-2,2-dimethyl-1,3-
4.5. Synthesis of 18
dioxan-5-ylcarbamate (13e). Yield 64%; White solid; mp
83e85 ꢀC; 1H NMR (400 MHz, CD3OD):
d
0.94 (t, J¼8 Hz, 3H), 1.35
4.5.1. tert-Butyl 2,2-dimethyl-5-(4-octylphenethyl)-1,3-dioxan-5-
ylcarbamate (18). According the general procedure, the com-
pound 16 was reacted with 11 to give compound 17 in E/Z mixture.
To a solution of 17 (0.35 mmol) in 10 mL methanol was added
20 mg 10% Pd/C. The reaction mixture was hydrogenated for 5 h at
room temperature. The mixture was then filtered. The filtrate was
concentrated to give compound 18 (yield 78% from 16) as a white
(m, 2H), 1.41 (d, J¼5 Hz, 6H), 1.48 (s, 9H), 1.56e1.58 (m, 2H), 1.97 (t,
J¼8 Hz, 2H), 2.50e2.59 (m, 4H), 3.77 (d, J¼8 Hz, 2H), 3.85e3.97 (m,
4H), 7.08 (m, 8H); HRMS (EI): m/z calcd for C30H43NO4 [M]þ:
481.3192, found: 481.3195.
4.4.6. tert-Butyl-2,2-dimethyl-5-(4-(4-pentylbenzyl)phenethyl)-1,3-
dioxan-5-ylcarbamate (13f). Yield 74%; White solid; mp
solid; mp 62e64 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.89 (t, J¼6.8 Hz,
100e102 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.91 (t, J¼7 Hz, 3H),
3H), 1.21 (m, 10H), 1.43 (m, 6H), 1.47 (s, 9H), 1.58 (m, 2H), 1.97 (m,
2H), 2.52e2.57 (m 4H), 3.67 (d, J¼12.0 Hz, 2H), 3.91 (d, J¼12.0 Hz,
2H), 4.98 (s, 1H), 7.08 (s, 4H).
1.33e1.36 (m, 4H), 1.44e1.46 (m, 6H), 1.49 (s, 9H), 1.60e1.63 (m,
2H), 1.97 (t, J¼8 Hz, 2H), 2.54e2.60 (m, 4H), 3.69 (d, J¼12 Hz, 2H),
3.91e3.92 (m, 4H), 5.01 (s, 1H), 7.10e7.12 (m, 8H); HRMS (EI): m/z
calcd for C31H45NO4 [M]þ: 495.3349, found: 495.3348.
4.6. General procedure for the synthesis of compounds 5aei,
6aec and Fingolimod
4.4.7. tert-Butyl 5-(4-(4-hexylbenzyl)phenethyl)-2,2-dimethyl-1,3-
dioxan-5-ylcarbamate (13g). Yield 69%; White solid; mp
A solution of 13aei, 15aec or 18 (0.11 mmol) in 10 mL methanol
and 0.2 mL aqueous HCl (6 M) was heated at 50 ꢀC for 3 h. The
solvent was then evaporated to dryness to give crude compound,
116e118 ꢀC; 1H NMR (500 MHz, CDCl3):
d
0.88 (t, J¼6 Hz, 3H),
1.26e1.34 (m, 6H), 1.41e1.43 (m, 6H), 1.47 (s, 9H), 1.55e1.59 (m, 2H),

T.-W. Mei et al. / Tetrahedron 69 (2013) 2927e2932
2931
which was treated with aq NaOH solution (2.5 mol/L) until
pH¼8e9, the mixture was extracted with ethyl acetate (3ꢁ20 mL),
and the combined organic layers were dried over anhydrous
Na2SO4 and evaporated to dryness. The residue was purified by
crystallization (CH2Cl2:MeOH¼15:1) to give pure 5aei, 6aec and
Fingolimod, respectively.
128.67, 128.90, 128.93, 138.85, 139.12, 140.22, 141.30; HRMS (ESI):
m/z calcd for C24H36NO2 [MþH]þ: 370.2746, found: 370.2759.
4.6.8. 2-Amino-2-(4-(4-heptylbenzyl)phenethyl)propane-1,3-diol
(5h). Yield 68%; White solid; mp 133e135 ꢀC; 1H NMR (500 MHz,
DMSO-d6):
(m, 4H), 2.48e2.56 (m, 4H), 3.21e3.28 (m, 4H), 3.82 (s, 2H), 4.45 (s,
2H), 7.06e7.09 (m, 8H); 13C NMR (100 MHz, DMSO-d6):
13.92,
d
0.84 (t, J¼6.7 Hz, 3H), 1.23e1.25 (m, 8H), 1.47e1.53
4.6.1. 2-Amino-2-(4-benzylphenethyl)propane-1,3-diol (5a). Yield
61%; White solid; mp 126e128 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
22.07, 28.50, 28.53, 28.65, 31.05, 31.24, 34.75, 36.75, 40.39, 55.57,
65.20, 128.19, 128.23, 128.46, 128.49, 138.42, 138.67, 139.78, 140.84;
HRMS (ESI): m/z calcd for C25H38NO2 [MþH]þ: 384.2903, found:
384.2897.
d
1.45e1.49 (m, 2H), 2.50e2.55 (m, 2H), 3.20e3.29 (m, 4H), 3.88 (s,
2H), 4.39 (s, 2H), 7.08e7.12 (m, 4H), 7.15e7.21 (m, 3H), 7.26e7.29
(m, 2H); 13C NMR (100 MHz, DMSO-d6):
28.48, 36.84, 40.70, 55.37,
d
65.35, 125.82, 128.19, 128.33, 128.50, 128.58, 138.18, 140.95, 141.51;
HRMS (EI): m/z calcd for C18H23NO2 [M]þ: 285.1729, found:
285.1727.
4.6.9. 2-Amino-2-(4-(4-octylbenzyl)phenethyl)propane-1,3-diol
(5i). Yield 70%; White solid; mp 130e132 ꢀC; 1H NMR (500 MHz,
DMSO-d6):
(m, 4H), 2.48e2.56 (m, 4H), 3.18e3.30 (m, 4H), 3.82 (s, 2H),
7.06e7.08 (m, 8H); 13C NMR (100 MHz, DMSO-d6):
13.71, 21.88,
d
0.84 (t, J¼6.7 Hz, 3H), 1.23e1.26 (m, 10H), 1.52e1.53
4.6.2. 2-Amino-2-(4-(4-methylbenzyl)phenethyl)propane-1,3-diol
(5b). Yield 72%; White solid; mp 134e136 ꢀC; 1H NMR (500 MHz,
d
DMSO-d6):
3.19e3.25 (m, 4H), 3.82 (s, 2H), 4.47 (s, 2H), 7.08 (s, 8H); 13C NMR
(100 MHz, DMSO-d6): 21.06, 29.00, 37.32, 40.83, 55.95, 65.81,
d
1.44e1.47 (m, 2H), 2.24 (s, 3H), 2.50e2.54 (m, 2H),
28.37, 28.45, 28.51, 28.64, 30.78, 31.09, 34.61, 36.44, 40.29, 55.72,
65.03, 128.02, 128.06, 128.32, 128.36, 138.28, 138.48, 139.65, 140.60;
HRMS (ESI): m/z calcd for C26H40NO2 [MþH]þ: 398.3059, found:
398.3054.
d
128.66, 128.95, 128.99, 129.40, 135.23, 138.90, 138.93, 141.34; HRMS
(EI): m/z calcd for C19H25NO2 [M]þ: 299.1885, found: 299.1879.
4.6.10. (E)-2-Amino-2-(4-styrylphenethyl)propane-1,3-diol
4.6.3. 2-Amino-2-(4-(4-ethylbenzyl)phenethyl)propane-1,3-diol
(6a). Yield 63%; White solid; mp 185e187 ꢀC; 1H NMR (400 MHz,
(5c). Yield 76%; White solid; mp 135e137 ꢀC; 1H NMR (500 MHz,
CD3OD):
d
1.95 (d, J¼16 Hz, 2H), 2.65e2.69 (m, 2H), 3.68 (s, 4 H),
DMSO-d6):
4H), 3.19e3.25 (m, 4H), 3.83 (s, 2H), 4.45 (s, 2H), 7.08e7.10 (m, 8H);
13C NMR (100 MHz, DMSO-d6):
15.61, 27.69, 28.46, 36.73, 40.34,
d
1.14 (t, J¼7.6 Hz, 3H), 1.44e1.47 (m, 2H), 2.50e2.56 (m,
7.14 (s, 2H), 7.24 (d, J¼8 Hz, 3H), 7.34 (m, 2H), 7.51 (m, 4H); 13C NMR
(100 MHz, CD3OD):
d 28.65, 35.89, 56.04, 64.70, 126.40, 126.57,
d
126.65, 128.40, 128.46, 138.65, 141.00, 141.70; HRMS (EI): m/z calcd
55.50, 65.22, 125.68, 128.14, 128.45, 128.49, 138.41, 138.68, 140.79,
141.14; HRMS (ESI): m/z calcd for C20H28NO2 [MþH]þ: 314.2120,
found: 314.2115.
for C19H23NO2 [M]þ: 297.1729, found: 297.1718.
4.6.11. (E)-2-Amino-2-(4-(4-hexylstyryl)phenethyl)propane-1,3-diol
(6b). Yield 67%; White solid; mp 189e191 ꢀC; 1H NMR (500 MHz,
4.6.4. 2-Amino-2-(4-(4-propylbenzyl)phenethyl)propane-1,3-diol
DMSO-d6):
4H), 2.55e2.63 (m, 4H), 3.32e3.36 (m, 4H), 7.13 (s, 2 H), 7.17e7.20
(m, 4H), 7.47e7.48 (m, 4H); 13C NMR (100 MHz, DMSO-d6):
13.94,
22.05, 28.30, 28.58, 30.83, 31.09, 34.88, 35.74, 56.61, 64.27, 126.27,
126.32, 127.36, 128.53, 128.61, 134.55, 134.61, 141.78, 142.45; HRMS
(ESI): m/z calcd for C25H36NO2 [MþH]þ: 382.2746, found: 382.2740.
d
0.86 (t, J¼6.5 Hz, 3H), 1.24e1.28 (m, 6H), 1.56e1.62 (m,
(5d). Yield 69%; White solid; mp 133e136 ꢀC; 1H NMR (500 MHz,
DMSO-d6):
4H), 3.21e3.27 (m, 4H), 3.83 (s, 2H), 4.45 (s, 2H), 7.09 (s, 8H); 13C
NMR (100 MHz, DMSO-d6): 13.64, 24.12, 28.48, 36.76, 36.85, 40.37,
d
0.87 (t, J¼7.2 Hz, 3H), 1.48e1.56 (m, 4H), 2.47e2.56 (m,
d
d
55.51, 65.21, 128.18, 128.29, 128.43, 128.49, 138.42, 138.73, 139.54,
140.83; HRMS (ESI): m/z calcd for C21H30NO2 [MþH]þ: 328.2277,
found: 328.2271.
4.6.12. (E)-2-Amino-2-(4-(4-octylstyryl)phenethyl)propane-1,3-diol
(6c). Yield 67%; White solid; mp 189e191 ꢀC; 1H NMR (500 MHz,
4.6.5. 2-Amino-2-(4-(4-butylbenzyl)phenethyl)propane-1,3-diol
DMSO-d6):
(m, 4H), 3.26e3.32 (m, 4H), 7.14 (s, 2H), 7.16e7.19 (m, 4H), 7.47e7.48
(m, 4H); 13C NMR (100 MHz, DMSO-d6):
14.41, 22.54, 29.12, 29.29,
31.33, 31.73, 35.35, 36.63, 39.51, 39.68, 56.58, 65.28, 126.74, 126.78,
127.79,127.88,129.00,134.99,135.12,142.25,143.13; HRMS (ESI): m/
z calcd for C27H0NO2 [MþH]þ: 410.3059, found: 410.3066.
d 0.85 (m, 3H),1.23e1.27 (m,10H),1.55 (s, 4H), 2.54e2.61
(5e). Yield 66%; White solid; mp 134e136 ꢀC; 1H NMR (500 MHz,
DMSO-d6):
4H), 2.49e2.54 (m, 4H), 3.20e3.27 (m, 4H), 3.82 (s, 2H), 4.45 (s, 2H),
7.08 (s, 8H); 13C NMR (100 MHz, DMSO-d6):
13.74, 21.71, 28.46,
d
0.86 (t, J¼7.2 Hz, 3H), 1.24e1.29 (m, 2H), 1.45e1.52 (m,
d
d
33.17, 34.38, 36.65, 40.36, 55.64, 65.10, 128.17, 128.23, 128.44,
128.49, 138.43, 138.67, 139.72, 140.78; HRMS (ESI): m/z calcd for
C22H32NO2 [MþH]þ: 342.2433, found: 342.2428.
4.6.13. Fingolimod (3). Yield 69%; White solid; mp: 122e124 ꢀC; 1H
NMR (500 MHz, MeOD):
1.55 (m, 2H), 1.84e1.87 (m, 2H), 2.52e2.61 (m, 4H), 3.59e3.65 (m,
4H), 7.05e7.11 (m, 4H).
d
0.86 (t, J¼7 Hz, 3H), 1.25e1.28 (m, 10H),
4.6.6. 2-Amino-2-(4-(4-pentylbenzyl)phenethyl)propane-1,3-diol
(5f). Yield 68%; White solid; mp 133e135 ꢀC; 1H NMR (500 MHz,
DMSO-d6):
2.50e2.55 (m, 4H), 3.21e3.27 (m, 4H), 3.83 (s, 2H), 4.45 (s, 2H), 7.08
(s, 8H); 13C NMR (100 MHz, DMSO-d6):
13.86, 21.90, 28.47, 30.67,
d
0.84 (t, J¼7.1 Hz, 3H), 1.26 (s, 4H), 1.47e1.52 (m, 4H),
4.7. In vitro antitumor activity assay
d
30.87, 34.68, 36.69, 40.37, 55.61, 65.16, 128.17, 128.22, 128.44,
128.48, 138.41, 138.66, 139.76, 140.80; HRMS (ESI): m/z calcd for
C23H34NO2 [MþH]þ: 356.2590, found: 356.2584.
Compounds 5aei and 6b,c were studied for their in vitro anti-
cancer activities against SMCC-7721 and HL-60 cells by MTT-based
assay. Cells were maintained as a suspension in RPMI-1640 me-
dium supplemented with 10% fetal calf serum at 37 ꢀC, in a hu-
midified atmosphere containing 5% CO2. Then the cells were
planted onto standard 96-well plates at the concentration 104 cells
per well and allowed to proliferate for 24 h under the above con-
ditions. The compounds were added in six threefold dilutions
4.6.7. 2-Amino-2-(4-(4-hexylbenzyl)phenethyl)propane-1,3-diol
(5g). Yield 67%; White solid; mp 134e136 ꢀC; 1H NMR (500 MHz,
DMSO-d6):
4H), 2.46e2.54 (m, 4H), 3.19e3.25 (m, 4H), 3.80 (s, 2H), 4.45 (s, 2H),
7.04e7.06 (m, 8H); 13C NMR (100 MHz, DMSO-d6):
14.37, 22.49,
28.79, 28.95, 31.45, 31.54, 35.20, 37.26, 40.83, 55.92, 65.73, 128.63,
d
0.82 (t, J¼6.4 Hz, 3H), 1.22e1.23 (m, 6H), 1.44e1.51 (m,
d
(10
mg/mL to 0.04
mg/mL) with Fingolimod co-assayed as a positive
control. After 72 h exposure period, then 15
mL of 5 mg/mL MTT

2932
T.-W. Mei et al. / Tetrahedron 69 (2013) 2927e2932
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were added to each well and the plates were incubated for 4 h at
37 ꢀC. The medium was then aspirated and the formazan product
was solubilized by 100 mL DMSO. The absorbance was measured
at 570 nm using an ELISA Reader. Each assay was carried out at
least three times, and the results of the experiment were summa-
rized in Table 3.
Acknowledgements
We gratefully acknowledge the financial support for new cen-
tury excellent talents in university (NCET). And we also thank Lab of
Organic Functional Molecules, the Sino-French Institute of ECNU for
supports.
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Supplementary data
14. Adachi, K.; Kohara, T.; Nakao, N.; Afita, M.; Chiba, K.; Mishina, T.; Sasaki, S.;
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.02.030.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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