Synthesis and immunomodulating activity of new analogues of fingolimod

Article abstract of DOI:10.1002/ardp.201100092

Five new immunomodulators 1a-1e by using a trans-4-alkyl-substituted cyclohexane to replace the flexible C8 alkyl chain of Fingolimod were synthesized. For in-vitro test, the compounds were dissolved in DMSO as a stock solution and diluted to a desired co

Full text of DOI:10.1002/ardp.201100092

Arch. Pharm. Chem. Life Sci. 2012, 345, 93–100
93
Full Paper
Synthesis and Immunomodulating Activity of New
Analogues of Fingolimod
Xiang-Jun Feng1*, Xiao-Ying Yang2*, Yu Luo1, Xin Li2, Wei Tang2, Jian-Ping Zuo2, and Wei Lu1,3
1 Department of Chemistry, East China Normal University, Shanghai, P. R. China
2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai, P. R. China
3 Institute of Drug Discovery and Development, East China Normal University, Shanghai, P. R. China
Five new immunomodulators 1a–1e by using a trans-4-alkyl-substituted cyclohexane to replace the
flexible C8 alkyl chain of Fingolimod were synthesized. For in-vitro test, the compounds were dissolved
in DMSO as a stock solution and diluted to a desired concentration with RPMI 1640 nutrient solution.
For in-vivo test, the compounds were prepared in pathogen-free saline containing 0.5% DMSO. These
new immunomodulators displayed more potent immunoinhibitory activities in vitro and moderate
immunomodulating activities in vivo. They show therapeutic effects on DNFB-induced DTH reaction
and inhibitory effects on the antigen-specific T-cell proliferation.
Keywords: Antigen-specific T-cell proliferation / Fingolimod / Immunomodulators
Received: March 10, 2011; Revised: May 24, 2011; Accepted: June 10, 2011
DOI 10.1002/ardp.201100092
Introduction
lymphocytes and inhibit lymphocyte egress out of lymphoid
organs. As a consequence, the immunoreactive T-cells are
depleted in the peripheral tissue [14, 15]. Only the phosphory-
lated molecule (S)-Fingolimod-phosphate, which binds to four
of the five known sphingosine-1-phosphate receptors (S1P1,3-5
but not S1P2), acts as a S1P receptor agonist, while the
enantiomer (R)-Fingolimod-phosphate shows weak or no
agonistic activity on these receptors [16–18].
Immunomodulators play important clinical roles in organ
transplantation and the treatment of autoimmune diseases
[1]. To date, they have progressed through four generations.
The first and second generations include the antiproliferative
agents and the lymphocyte depletion agents, separately.
The application of calcineurin antagonists was recognized as
the third generation resulting in revolutionary changes in the
development of immunomodulators. The fourth generation
includes the cell proliferation signal inhibitors [2]. At present,
both cyclosporin A (CsA) [3] and tacrolimus (FK506) [4, 5] are the
established immunomodulators and widely used in organ
transplantation.
Additionally, Graeb et al. reported that Fingolimod was able
to prevent tumor growth and metastasis. It showed a strong
antiangiogenic effect by abrogating the stimulating effect of
vascular endothelial growth factor (VEGF 20 ng/mL) in a
human umbilical vein endothelial cell spheroid model [19].
Structure-activity relationship (SAR) studies of Fingolimod
analogues showed that the 2-aminopropane-1,3-diol headgroup
was critical for their immunomodulating activities. The phenyl
ring was also important for the potent activities of these analogues
by modifying their pharmacological properties [1, 20–22].
In order to develop new immunomodulators, we decided to
design and synthesize Fingolimod analogues. To design new
Fingolimod analogues, we decided to keep the 2-aminopropane-
1,3-diol moiety and the phenyl ring, and plan to increase the
conformational rigidity and sterical restriction of the flexible
C8 alkyl chain of Fingolimod when conformational flexibility
Fingolimod (2-amino-2-(2-(4-octylphenyl)ethyl)propane-1,3-
diol hydrochloride, FTY720), a novel synthetic analogue of
sphingosine ISP-1 (myriocin, Fig. 1) currently in phase III
clinical trials for the treatment of relapsing-remitting
multiple sclerosis meanwhile approved in several countries
including USA, has displayed favorable immunomodulating
properties in numerous investigations [6–13]. It can form the
active principle (S)-Fingolimod-phosphate by stereoselective
phosphorylation in vivo to induce apoptosis of activated
Correspondence: Prof. Wei Lu, East China Normal University, Shanghai
200062, P.R. China.
E-mail: wlu@chem.ecnu.edu.cn
Fax: þ86 21 62602475
Additional correspondence: Prof. Jian-Ping Zuo, jpzuo@mail.shcnc.ac.cn
*These two authors contributed equally to this paper.
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X.-J. Feng et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 93–100
OH
OH
NH2 HCl
OH
HOOC
HO
R
NH2 OH
O
myriocin
1a- 1e
HO
OH
NH2 HCl
1a: R = H; 1b: R = Et; 1c: R = n-Pr
1d: R = n-Bu; 1e: R = n-Pen
Figure 2. The structures of new immunomodulators.
Fingolimod
Results and discussion
O
OH
P
OH
HO
O
Chemistry
NH2
(S)-Fingolimod-P
The synthetic effort towards these new immunomodulators
was carried out as shown in Scheme 1. First, the hydrophilic
headgroup and the lipophilic hydrocarbon chain were syn-
thesized separately. The aldehyde moiety (2) was obtained
from tris-(hydroxymethyl)aminomethane (TRIS) in three
steps according to our earlier described method [24].
The lipophilic hydrocarbon chains can be synthesized from
their corresponding acids 3a–3e which are easily available
starting materials. Reduction of ester which can be obtained
from corresponding acids by conventional esterification, with
KBH4/LiCl gave corresponding alcohols 5a–5e. These alcohols
were treated with PBr3 in toluene to afford the corresponding
Figure 1. Myriocin, Fingolimod and (S)-Fingolimod-phosphate.
could be a complicating factor in drug development [23]. As can
be seen in Fig. 2, we selected trans-4-alkyl-substituted cyclohex-
ane to achieve effective rigidity and sterical restriction.
In this paper, we describe the design and synthesis of five
new Fingolimod analogues with restricted conformational
flexibility. We also report the immunomodulating activities
of these novel molecules.
b
a
R
CH2OH
R
COOMe
R
COOH
4a-4e
3a-3e
5a-5e
CHO
O
O
Br
c
NHCbz
PPh3 Br
d
R
2
R
e
6a-6e
7a-7e
O
O
g
O
O
f
R
R
R
HN
H2N
Cbz
9a-9e
8a-8e
3a-9a: R = H,
3b-9b: R = Et,
3c-9c: R = n-Pr,
OH
NH2 HCl
OH
3d-9d: R = n-Bu,
3e-9e: R = n-Pen
1a-1e
Reagents and conditions: (a) MeOH, H2SO4, reflux, 10 h; (b) KBH4, LiCl, THF, reflux, 8 h; (c) PBr3,
toluene, r.t., 4 h; (d) PPh3, toluene, reflux, 6 h; (e) compound 2, K2CO3, THF/DMF (3:1), reflux, 12 h;
(f) H2, Pd/C (10 wt%), MeOH, r.t., 3 h; (g) aq. HCl (1 M), MeOH, 50°C, 3 h.
Scheme 1. Synthesis of compound 1a–1e.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 93–100
Synthesis and Immunomodulating Activity of New Analogues of Fingolimod
95
Table 1. Inhibitory activities of Fingolimod and compounds 1a-1e
on ConA- and alloantigen-induced splenocyte proliferation.
of the control value. The data showed that all the new analogues
displayed potent immunosuppressive activities in vitro and their
activities were basically the same as that of Fingolimod.
IC50 (mM)
ConA
MLR
Inhibitory effect on DNFB-induced DTH response
To test the immunosuppressive activities of these compounds
in vivo, we used the DNFB-induced DTH model which is based
on a cell-mediated pathologic response involved with CD4þ T
cell activation. All the compounds, by oral administration,
could inhibit DTH reaction, reduced the ear swelling and ear
weight. As shown in Fig. 3, compounds 1a and 1b displayed
slightly worse activities, but the inhibitory activities of com-
pounds 1c–1e were basic agreement with Fingolimod.
Fingolimod
0.67 ꢀ 0.46
1.42 ꢀ 0.39
0.81 ꢀ 0.15
0.68 ꢀ 0.10
0.51 ꢀ 0.11
0.57 ꢀ 0.08
0.64 ꢀ 0.27
0.15 ꢀ 0.01
0.31 ꢀ 0.17
0.19 ꢀ 0.01
0.24 ꢀ 0.01
0.39 ꢀ 0.18
1a
1b
1c
1d
1e
ConA- and alloantigen-induced proliferation assays were carried
out for 48 h and 96 h, respectively. The results were expressed as
mean þ standard error.
Inhibitory effect on OVA-specific T-cell response
Since compounds 1c and 1e showed more potent activities, they
were further tested and confirmed for the immunosuppressive
properties on antigen-specific lymphocyte proliferative response
in OVA-immunized mice. As shown in Fig. 4, compared with
lymphocytes from na¨yve mice, lymphocytes from OVA-immu-
nized mice exhibited a prominent antigen-specific recall response
to OVA. Oral treatment with Fingolimod, compounds 1c and 1e
(3 mg/(kg day)) significantly suppressed the OVA-specific lympho-
cyte proliferation. Both compounds 1c and 1e displayed basically
same potent inhibitory activities with Fingolimod in vivo.
In summary, five new immunomodulators 1a–1e by using
a trans-4-alkyl-substituted cyclohexane to replace the flexible
C8 alkyl chain of Fingolimod were synthesized. They show
therapeutic effects on DNFB-induced DTH reaction and
inhibitory effects on the antigen-specific T-cell proliferation.
bromides 6a–6e. Reaction of bromide with PPh3 provided
corresponding Wittig salts 7a–7e in excellent yields.
Then, heating of these Wittig salts 7a–7e with the aldehyde
2 in the presence of K2CO3 furnished the desired alkenes 8a–
8e as an E/Z mixture. Simple hydrogenation removed Cbz
group and reduced the double bond in one step to give
corresponding alkanes 9a–9e. Removal of the acetonide
groups of alkanes afforded compounds 1a–1e.
In-vitro and in-vivo studies
For in-vitro test, the compounds were dissolved in DMSO as a
stock solution and diluted to a desired concentration with
RPMI 1640 nutrient solution. For in-vivo test, the compounds
were prepared in pathogen-free saline containing 0.5% DMSO.
Inhibitory effect on murine splenocyte proliferation
The activities of compounds 1a–1e are evaluated on splenocyte
proliferation induced by Concanavalin A (ConA) and mixed
lymphocyte culture reaction (MLR) in vitro. The results of these
compounds are summarized in Table 1. The immunosuppres-
sive activity of each compound was expressed as the concen-
tration of compound that reduced cell proliferation to 50% (IC50)
Experimental
Chemistry
1H NMR spectra were recorded on a Bruker DRX-500 (500 MHz).
13C-NMR spectra were obtained on a JNM-EX400 (100 MHz). MS
spectra (MS) were determined on a Finnigan MAT-95 mass spec-
trometer. Chemical shifts (d) are reported in parts per million
Figure 3. Compounds 1a–1e inhibit DNFB-induced DTH reaction with oral administration.
Data are expressed as mean W S.D. Three independent experiments were performed with similar results. ꢁp < 0.05, ꢁꢁp < 0.01, versus vehicle
group; n ¼ 10 mice/group.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 93–100
4-((1r,4r)-4-Amylcyclohexyl) benzoic acid methyl ester 4e
White solid, yield: 86%, m.p.: 49–518C; 1H-NMR (CDCl3) d: 7.95
(d, J ¼ 7 Hz, 2 H), 7.27 (d, J ¼ 7 Hz, 2 H), 3.90 (s, 3 H), 2.50–2.52
(m, 1 H), 1.88–1.90 (m, 4 H), 1.45–1.47 (m, 2 H), 1.22–1.33 (m, 9 H),
1.04–1.08 (m, 2 H), 0.90 (t, 3 H).
General procedure for the synthesis of compounds 5a–5e
A mixture of 4a–4e (0.09 mol), KBH4 (0.11 mol) and LiCl
(0.11 mol) in THF (100 mL) was refluxed for 8 h, then cooled
to r. t. and quenched with saturated NH4Cl solution (60 mL).
After stirring for 2 h, the mixture was extracted with
ethyl acetate; the organic phase was washed with brine,
dried with Na2SO4, and concentrated to give crude 5a–5e,
respectively.
4-Cyclohexylbenzyl alcohol 5a
Figure 4. Compounds 1c and 1e suppressed OVA-specific T-cell
response.
White solid, yield: 89%, m.p.: 35–378C; 1H-NMR (CDCl3) d: 7.28
(d, J ¼ 8 Hz, 2 H), 7.20 (d, J ¼ 8 Hz, 2 H), 4.64 (s, 2 H), 2.50–2.52
(m, 1 H), 1.83–1.88 (m, 4 H), 1.76–1.77 (m, 1 H), 1.38–1.43 (m, 4 H),
1.26–1.27 (m, 1 H); 13C-NMR (CDCl3) d: 26.1, 26.8, 34.4, 44.3, 65.2,
127.0, 127.1, 138.3, 147.6. EI-MS: (m/z) 190 [M]þ.
The results were expressed as mean ꢀ S.E.M. of three samples.
ꢁ
ꢁꢁ
P < 0.01, versus vehicle group. Two independent
P < 0.05,
experiments were performed that gave similar results.
(ppm). Data are reported as follows: chemical shift, multiplicity
(br s ¼ broad singlet, d ¼ doublet, dd ¼ doublet of doublet,
dt ¼ doublet of triplet, m ¼ multiple, s ¼ singlet and t ¼ triplet),
coupling constants (Hz), integration. All reagents were used
directly as obtained commercially, unless otherwise noted.
4-((1r,4r)-4-Ethylcyclohexyl) benzyl alcohol 5b
White solid, yield: 98%, m.p.: 38–398C; 1H-NMR (CDCl3) d: 7.29
(d, J ¼ 8 Hz, 2 H), 7.21 (d, J ¼ 8 Hz, 2 H), 4.65 (s, 2 H), 2.44–2.49
(m, 1 H), 1.87–1.89 (m, 4 H), 1.57 (s, 1 H), 1.41–1.46 (m, 2 H), 1.20–
1.28 (m, 3 H), 1.03–1.08 (m, 2 H), 0.90 (t, 3 H); 13C-NMR (CDCl3)
d: 11.5, 29.9, 33.2, 34.3, 39.1, 44.4, 65.3, 127.0, 127.1, 138.3, 147.5.
EI-MS (m/z): 218 [M]þ.
General procedure for the synthesis of compounds 4a–4e
A mixture of 3a–3e (0.1 mol), anhydrous MeOH (200 mL), and
conc. H2SO4 (0.5 mL) was refluxed for 10 h and cooled to r.t. After
removal of MeOH under reduced pressure, the residue was diluted
with H2O and extracted with ethyl acetate. The combined organic
extract was washed with saturated Na2CO3 solution and brine, then
dried over Na2SO4 and evaporated to give 4a–4e, respectively.
4-((1r,4r)-4-Propylcyclohexyl) benzyl alcohol 5c
White solid, yield: 92%, m.p.: 48–498C; 1H-NMR (CDCl3) d: 7.31
(d, J ¼ 8 Hz, 2 H), 7.23 (d, J ¼ 8 Hz, 2 H), 4.67 (s, 2 H), 2.48–2.51
(m, 1 H), 1.89–1.91 (m, 4 H), 1.46–1.48 (m, 2 H), 1.34–1.37 (m, 3 H),
1.22–1.25 (m, 2 H), 1.07–1.08 (m, 2 H), 0.92 (t, 3 H); 13C-NMR
(CDCl3) d: 14.4, 20.0, 33.6, 34.4, 37.0, 39.7, 44.4, 65.3, 127.0, 127.1,
138.3, 147.5. EI-MS (m/z): 232 [M]þ.
4-Cyclohexylbenzoic acid methyl ester 4a
White solid, yield: 90%, m.p.: 41–438C; 1H-NMR (CDCl3) d: 7.94
(d, J ¼ 8 Hz, 2 H), 7.25 (d, J ¼ 8 Hz, 2 H), 3.88 (s, 3 H), 2.54–2.55
(m, 1 H), 1.84–1.88 (m, 4 H), 1.74–1.76 (m, 1 H), 1.34–1.45 (m, 4 H),
1.24–1.29 (m, 1 H).
4-((1r,4r)-4-Butylcyclohexyl) benzyl alcohol 5d
White solid, yield: 90%, m.p.: 47–488C; 1H-NMR (CDCl3) d: 7.29 (d,
J ¼ 9 Hz, 2 H), 7.21 (d, J ¼ 9 Hz, 2 H), 4.66 (s, 2 H), 2.48–2.50 (m,
1 H), 1.86–1.90 (m, 4 H), 1.46–1.48 (m, 2 H), 1.24–1.32 (m, 7 H),
1.07–1.08 (m, 2 H), 0.92 (t, 3 H); 13C-NMR (CDCl3) d: 14.1, 23.0, 29.2,
33.6, 34.4, 37.1, 37.3, 44.4, 65.2, 127.0, 127.1, 138.3, 147.4. EI-MS
(m/z): 246 [M]þ.
4-((1r,4r)-4-Ethylcyclohexyl) benzoic acid methyl ester 4b
White solid, yield: 95%, m.p.: 43–448C; 1H-NMR (CDCl3) d: 7.95
(d, J ¼ 9 Hz, 2 H), 7.27 (d, J ¼ 9 Hz, 2 H), 3.90 (s, 3 H), 2.50–2.55
(m, 1 H), 1.89–1.91 (m, 4 H), 1.43–1.50 (m, 2 H), 1.25–1.90 (m, 2 H),
1.19–1.21 (m, 1 H), 1.04–1.08 (m, 2 H), 0.91 (t, 3 H).
4-((1r,4r)-4-Amylcyclohexyl) benzyl alcohol 5e
White solid, yield: 86%, m.p.: 48–508C; 1H-NMR (CDCl3) d: 7.29 (d,
J ¼ 7 Hz, 2 H), 7.21 (d, J ¼ 7 Hz, 2 H), 4.66 (s, 2 H), 2.45–2.47 (m,
1 H), 1.87–1.88 (m, 4 H), 1.46–1.48 (m, 2 H), 1.23–1.33 (m, 9 H),
1.07–1.08 (m, 2 H), 0.90 (t, 3 H); 13C-NMR (CDCl3) d: 14.1, 22.7, 26.6,
32.2, 33.6, 34.3, 37.3, 37.4, 44.4, 65.2, 127.0, 127.1, 138.3, 147.5.
EI-MS (m/z): 260 [M]þ.
4-((1r,4r)-4-Propylcyclohexyl) benzoic acid methyl ester 4c
White solid, yield: 84%, m.p.: 42–438C; 1H-NMR (CDCl3) d: 0.90
(t, 3 H), 1.06–1.08 (m, 2 H), 1.21–1.24 (m, 2 H), 1.33–1.36 (m, 3 H),
1.47–1.48 (m, 2 H), 1.87–1.90 (m, 4 H), 2.51–2.53 (m, 1 H), 3.90 (s, 3
H), 7.27 (d, J ¼ 9 Hz, 2 H), 7.95 (d, J ¼ 9 Hz, 2 H).
General procedure for the synthesis of compounds 6a–6e
To a solution of compound 5a–5e (0.07 mol) in toluene (60 mL) at
ꢂ108C was added PBr3 (0.028 mol) dropwise over a period of
20 min. Then, the mixture was stirred for 4 h at room tempera-
ture and poured into ice water. The solution was extracted with
4-((1r,4r)-4-Butylcyclohexyl) benzoic acid methyl ester 4d
White solid, yield: 88%, m.p.: 39–408C; 1H-NMR (CDCl3) d: 7.95
(d, J ¼ 8 Hz, 2 H), 7.27 (d, J ¼ 8 Hz, 2 H), 3.90 (s, 3 H), 2.50–2.53
(m, 1 H), 1.87–1.90 (m, 4 H), 1.44–1.47 (m, 2 H), 1.30–1.31 (m, 5 H),
1.23–1.25 (m, 2 H), 1.04–1.08 (m, 2 H), 0.90 (t, 3 H).
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Arch. Pharm. Chem. Life Sci. 2012, 345, 93–100
Synthesis and Immunomodulating Activity of New Analogues of Fingolimod
97
ethyl acetate, washed with brine, dried over Na2SO4, and con-
centrated to give 6a–6e, respectively.
(d, J ¼ 9 Hz, 2 H), 2.34–2.39 (m, 1 H), 1.80–1.85 (m, 4 H), 1.32–
1.35 (m, 2 H), 1.20–1.26 (m, 3 H), 0.98–1.01 (m, 2 H), 0.90 (t, 3 H).
1-Bromomethyl-4-cyclohexylbenzene 6a
4-((1r,4r)-4-Propylcyclohexyl)benzyl(triphenyl)-
Colorless oil, yield: 98%; 1H-NMR (CDCl3) d: 7.31 (d, J ¼ 8 Hz, 2 H),
7.20 (d, J ¼ 8 Hz, 2 H), 4.51 (s, 2 H), 2.50–2.53 (m, 1 H), 1.85–1.89
(m, 4 H), 1.75–1.78 (m, 1 H), 1.36–1.46 (m, 4 H), 1.25–1.28 (m, 1 H);
13C-NMR (CDCl3) d: 26.0, 26.8, 33.7, 44.3, 44.2, 127.2, 128.9, 135.0,
148.4. EI-MS (m/z): 252 [M]þ.
phosphonium bromide 7c
White solid, yield: 96%, m.p.: 238–2398C; 1H-NMR (CDCl3) d: 7.69–
7.78 (m, 9 H), 0.89 (t, 3 H), 7.60–7.64 (m, 6 H), 6.94–6.99 (m, 4 H),
5.32 (d, J ¼ 9 Hz, 2 H), 2.35–2.37 (m, 1 H), 1.77–1.84 (m, 4 H), 1.29–
1.35 (m, 4 H), 1.18–1.21 (m, 3 H), 0.99–1.01 (m, 2 H).
1-Bromomethyl-4-((1r,4r)-4-ethylcyclohexyl)benzene 6b
Colorless oil, yield: 74%; 1H-NMR (CDCl3) d: 7.34 (d, J ¼ 8 Hz, 2 H),
7.22 (d, J ¼ 8 Hz, 2 H), 4.52 (s, 2 H), 2.48–2.53 (m, 1 H), 1.91–1.94
(m, 4 H), 1.44–1.51 (m, 2 H), 1.25–1.33 (m, 3 H), 1.08–1.12 (m, 2 H),
0.96 (t, 3 H); 13C-NMR (CDCl3) d: 11.5, 29.9, 33.1, 33.7, 34.2, 39.0,
44.4, 127.2, 129.0, 148.3. EI-MS (m/z): 280 [M]þ.
4-((1r,4r)-4-Butylcyclohexyl)benzyl(triphenyl)phosphonium
bromide 7d
White solid, yield: 97%, m.p.: 240–2418C; 1H-NMR (CDCl3) d: 7.66–
7.77 (m, 9 H), 7.59–7.63 (m, 6 H), 6.93–6.97 (m, 4 H), 4.26
(d, J ¼ 11 Hz, 2 H), 2.32–2.37 (m, 1 H), 1.76–1.83 (m, 4 H),
1.20–1.33 (m, 9 H), 0.98–1.02 (m, 2 H), 0.88 (t, 3 H).
1-Bromomethyl-4-((1r,4r)-4-propylcyclohexyl)benzene 6c
White solid, yield: 79%, m.p.: 35–378C; 1H-NMR (CDCl3) d: 7.29 (d,
J ¼ 8 Hz, 2 H), 7.16 (d, J ¼ 8 Hz, 2 H), 4.47 (s, 2 H), 2.44–2.45 (m,
1 H), 1.84–1.86 (m, 4 H), 1.40–1.44 (m, 2 H), 1.31–1.34 (m, 3 H),
1.18–1.22 (m, 2 H), 1.02–1.04 (m, 2 H), 0.88 (t, 3 H); 13C-NMR
(CDCl3) d: 14.4, 20.0, 33.5, 33.8, 34.2, 37.0, 39.7, 44.4, 127.3, 129.0,
135.1, 148.3. EI-MS (m/z): 294 [M]þ.
4-((1r,4r)-4-Amylcyclohexyl)benzyl(triphenyl)phosphonium
bromide 7e
White solid, yield: 94%, m.p.: 242–2438C; 1H-NMR (CDCl3) d: 7.70–
7.78 (m, 9 H), 7.61–7.65 (m, 6 H), 6.95–6.99 (m, 4 H), 5.33
(d, J ¼ 9 Hz, 2 H), 2.34–2.39 (m, 1 H), 1.68–1.85 (m, 4 H), 1.19–
1.35 (m, 11 H), 1.00–1.03 (m, 2 H), 0.89 (t, 3 H).
General procedure for the synthesis of compounds 9a–9e
A suspension of aldehyde 2 (0.01 mol), Wittig salt 7a–7e (0.01 mol),
and K2CO3 (0.03 mol) in a mixture of THF (60 mL) and DMF (20 mL)
was refluxed for 12 h. After completion of the reaction, THF was
evaporated and the reaction mixture was quenched with water
and then extracted with ethyl acetate. The combined organic
layers were washed with brine, dried with Na2SO4, and concen-
trated to give a residue, which was purified by silica gel flash
chromatography to give alkenes 8a – 8e in E/Z mixture. To a
solution of the mixture 8a – 8e in methanol (30 mL) was added
10% Pd/C (10%). The reaction mixture was hydrogenated for 3 h at
room temperature. Then the mixture was filtered. The filtration
was concentrated to give compound 9a–9e, respectively.
1-Bromomethyl-4-((1r,4r)-4-butylcyclohexyl)benzene 6d
White solid, yield: 96%, m.p.: 33–358C; 1H-NMR (CDCl3) d: 7.33 (d,
J ¼ 9 Hz, 2 H), 7.20 (d, J ¼ 9 Hz, 2 H), 4.51 (s, 2 H), 2.48–2.49 (m,
1 H), 1.88–1.90 (m, 4 H), 1.44–1.46 (m, 2 H), 1.25–1.34 (m, 7 H),
1.05–1.08 (m, 2 H), 0.92 (t, 3 H); 13C-NMR (CDCl3) d: 14.1, 23.0, 29.2,
33.6, 33.7, 34.2, 37.1, 37.3, 44.4, 127.3, 129.0, 135.1, 148.3. EI-MS
(m/z): 308 [M]þ.
1-Bromomethyl-4-((1r,4r)-4-amylcyclohexyl)benzene 6e
White solid, yield: 81%, m.p.: 31–328C; 1H-NMR (CDCl3) d: 7.28 (d,
J ¼ 7 Hz, 2 H), 7.15 (d, J ¼ 7 Hz, 2 H), 4.45 (s, 2 H), 2.42–2.45 (m,
1 H), 1.85–1.87 (m, 4 H), 1.42–1.45 (m, 2 H), 1.21–1.32 (m, 9 H),
1.03–1.05 (m, 2 H), 0.89 (t, 3 H); 13C-NMR (CDCl3) d: 14.1, 22.7, 26.6,
32.2, 33.5, 34.2, 37.2, 37.3, 44.3, 127.2, 128.9, 135.1, 148.2. EI-MS
(m/z): 322 [M]þ.
5-Amino-5-[2-4-cyclohexylbenzene)ethyl]-2,2-dimethyl-
1,3-dioxane 9a
1
White solid, yield: 73% from 7a, m.p.: 60–638C; H-NMR (CDCl3)
General procedure for the synthesis of compounds 7a–7e
A mixture of 6a–6e (67 mmol) and PPh3 (67 mmol) in toluene
(100 mL) was refluxed under a nitrogen atmosphere for 6 h.
Then, the reaction mixture was cooled. The white solid which
precipitated, was collected and washed with toluene (30 mL) and
dried to give pure 7a–7e, respectively.
d: 7.10–7.14 (m, 4 H), 3.79 (d, J ¼ 11 Hz, 2 H), 3.48 (d, J ¼ 11 Hz, 2
H), 2.61–2.65 (m, 2 H), 2.46 (s, 1 H), 1.82–1.84 (m, 7 H), 1.72–1.75
(m, 1 H), 1.60–1.63 (m, 2 H), 1.36–1.45 (m, 10 H); 13C-NMR (CDCl3)
d: 26.1, 26.9, 27.4, 28.2, 29.7, 34.5, 37.6, 44.1, 48.7, 70.1, 98.2,
126.9, 128.1, 139.2, 146.8. HRMS (EI): Calcd. for C20H31NO2 [M]þ
317.2355, found: 317.2355.
4-Cyclohexylbenzyl (triphenyl) phosphonium bromide 7a
White solid, yield: 97%, m.p.: 237–2398C; 1H-NMR (CDCl3) d: 7.68–
7.77 (m, 9 H), 7.60–7.63 (m, 6 H), 6.94–6.98 (m, 4 H), 5.29
(d, J ¼ 11 Hz, 2 H), 2.35–2.41 (m, 1 H), 1.70–1.81 (m, 5 H),
1.18–1.36 (m, 5 H).
5-Amino-5-{2-[4-((1r,4r)-4-ethylcyclohexyl)benzene]-
ethyl}-2,2-dimethyl-1,3-dioxane 9b
White solid, yield: 71% from 7b, m.p.: 78–798C; 1H-NMR (CDCl3) d:
7.01–7.14 (m, 4 H), 3.78 (d, J ¼ 10 Hz, 2 H), 3.47 (d, J ¼ 10 Hz, 2 H),
2.61–2.65 (m, 2 H), 2.48–2.53 (m, 1 H), 1.86–1.88 (m, 4 H), 1.60–
1.63 (m, 5 H), 1.42–1.44 (m, 8 H), 1.25–1.28 (m, 2 H), 1.02–1.05 (m,
2 H), 0.90 (t, 3 H); 13C-NMR (CDCl3) d: 11.5, 19.8, 27.4, 28.2, 30.0,
33.2, 34.3, 37.6, 39.1, 44.2, 48.6, 70.1, 98.2, 127.0, 128.1, 139.2,
145.6. HRMS (EI): Calcd. for C22H35NO2 [M]þ 345.2668, found:
345.2667.
4-((1r,4r)-4-Ethylcyclohexyl)benzyl(triphenyl)phosphonium
bromide 7b
White solid, yield: 96%, m.p.: 243–2458C; 1H-NMR (CDCl3) d: 7.69–
7.78 (m, 9 H), 7.60–7.64 (m, 6 H), 6.94–6.97 (m, 4 H), 5.31
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2.54–2.57 (m, 2 H), 2.40–2.43 (m, 1 H), 1.75–1.83 (m, 6 H), 1.38–
1.42 (m, 2 H), 1.22–1.25 (m, 3 H), 1.00–1.03 (m, 2 H), 0.88 (t, 3 H);
13C-NMR (CDCl3) d: 11.3, 27.9, 29.4, 32.6, 33.2, 33.8, 38.4, 38.9,
60.2, 60.9, 126.5, 127.9, 140.0, 144.8; anal. calcd. (C19H32ClNO2):
C, 66.74; H, 9.43; N, 4.10. Found: C, 66.49; H, 9.18; N, 3.98.
NMR for the free base of 1b: white solid, m.p.: 166–1688C.
1H-NMR (CDCl3) d: 7.06–7.10 (m, 4 H), 4.42 (s, 2 H), 3.31–3.24 (m,
4 H), 2.50–2.54 (m, 2 H), 2.42–2.47 (m, 1 H), 1.76–1.82 (m, 4 H), 1.44–
1.48 (m, 2 H), 1.40–1.41 (m, 2 H), 1.22–1.26 (m, 3 H), 1.01–1.04 (m,
2 H), 0.88 (t, 3 H); 13C-NMR (CDCl3) d: 11.3, 28.4, 29.4, 32.6, 33.9,
36.9, 38.4, 43.4, 55.3, 65.4, 126.4, 127.9, 140.7, 144.2. HRMS (ESI):
Calcd. for C19H31NO2 [M]þ: 305.2355, found: 306.2440 (MþþH).
5-Amino-5-{2-[4-((1r,4r)-4-propylcyclohexyl)benzene]-
ethyl}-2,2-dimethyl-1,3-dioxane 9c
White solid, yield: 67% from 7c, m.p.: 74–768C; 1H-NMR (CDCl3) d:
7.09–7.13 (m, 4 H), 3.79 (d, J ¼ 10 Hz, 2 H), 3.48 (d, J ¼ 10 Hz, 2 H),
2.61–2.64 (m, 2 H), 2.44–2.45 (m, 1 H), 1.84–1.86 (m, 8 H), 1.60–
1.63 (m, 2 H), 1.42–1.44 (m, 7 H), 1.32–1.35 (m, 2 H), 1.19–1.21 (m,
2 H), 1.02–1.04 (m, 2 H), 0.89 (t, 3 H); 13C-NMR (CDCl3) d: 14.4, 19.9,
27.5, 28.2, 33.6, 34.4, 37.0, 37.5, 39.7, 44.2, 48.7, 70.0, 98.2, 127.0,
128.1, 139.2, 145.6. HRMS (EI): Calcd. for C23H37NO2 [M]þ
359.2824, found: 359.2819.
5-Amino-5-{2-[4-((1r,4r)-4-butylcyclohexyl)benzene]-
ethyl}-2,2-dimethyl-1,3-dioxane 9d
2-Amino-2-{2-[4-((1r,4r)-4-propylcyclohexyl)phenyl]-
White solid, yield: 69% from 7d, m.p.: 78–808C; 1H-NMR (CDCl3) d:
7.09–7.13 (m, 4 H), 3.79 (d, J ¼ 10 Hz, 2 H), 3.47 (d, J ¼ 10 Hz, 2 H),
2.61–2.65 (m, 2 H), 2.44–2.45 (m, 1 H), 1.85–1.86 (m, 4 H), 1.60–
1.63 (m, 2 H), 1.41–1.44 (m, 8 H), 1.23–1.31 (m, 7 H), 1.05–1.08 (m,
2 H), 0.90 (t, 3 H); 13C-NMR (CDCl3) d: 14.1, 19.9, 23.0, 27.3, 28.3,
29.2, 33.6, 34.4, 37.1, 37.3, 37.6, 44.2, 48.7, 70.1, 98.2, 126.9,
128.1, 139.2, 145.6. HRMS (EI): Calcd. for C24H39NO2 [M]þ
373.2981, found: 373.2982.
ethyl}propane-1,3-diol hydrochloride 1c
White solid, yield: 76%, m.p.: 186–1888C; 1H-NMR (CDCl3) d: 7.85
(s, 3 H), 7.09–7.14 (m, 4 H), 5.37 (t, 2 H), 3.51–3.52 (m, 4 H), 2.54–
2.57 (m, 2 H), 2.49–2.50 (m, 1 H), 1.75–1.81 (m, 6 H), 1.39–1.42 (m,
2 H), 1.30–1.33 (m, 3 H), 1.18–1.19 (m, 2 H), 1.01–1.04 (m, 2 H),
0.87 (t, 3 H); 13C-NMR (CDCl3) d: 14.2, 19.4, 27.9, 28.4, 33.0, 33.2,
33.8, 36.3, 39.2, 43.4, 60.2, 60.9, 126.5, 127.9, 140.0, 144.8; anal.
calcd. (C20H34ClNO2): C, 67.49; H, 9.63; N, 3.94. Found: C, 67.37; H,
9.66; N, 4.13.
5-Amino-5-{2-[4-((1r,4r)-4-amylcyclohexyl)benzene]-
NMR for the free base of 1c: white solid, m.p.: 173–1748C.
1H-NMR (CDCl3) d: 7.06–7.10 (m, 4 H), 4.44 (s, 2 H), 3.20–3.32
(m, 4 H), 2.50–2.54 (m, 2 H), 2.44–2.46 (m, 1 H), 1.75–1.81 (m, 4 H),
1.45–1.48 (m, 2 H), 1.38–1.41 (m, 2 H), 1.30–1.33 (m, 3 H), 1.17–
1.19 (m, 2 H), 1.00–1.02 (m, 2 H), 0.87 (t, 3 H); 13C-NMR (CDCl3) d:
14.2, 19.4, 27.9, 28.4, 33.0, 33.2, 33.9, 36.3, 36.9, 43.4, 55.3, 65.4,
126.4, 127.9, 140.7, 144.2. HRMS (ESI): Calcd. for C20H33NO2 [M]þ:
319.2511, found: 320.2585 (MþþH).
ethyl}-2,2-dimethyl-1,3-dioxane 9e
White solid, yield: 67% from 7e, m.p.: 76–778C; 1H-NMR (CDCl3) d:
7.09–7.13 (m, 4 H), 3.78 (d, J ¼ 10 Hz, 2 H), 3.47 (d, J ¼ 10 Hz, 2 H),
2.61–2.64 (m, 2 H), 2.44–2.45 (m, 1 H), 1.84–1.86 (m, 4 H), 1.60–
1.64 (m, 2 H), 1.42–1.44 (m, 8 H), 1.22–1.32 (m, 9 H), 1.05–1.08 (m,
2 H), 0.89 (t, 3 H); 13C-NMR (CDCl3) d: 14.1, 19.7, 26.6, 27.5, 28.2,
32.2, 33.6, 34.4, 37.3, 37.4, 37.6, 44.2, 48.7, 70.0, 98.2, 126.9,
128.1, 139.2, 145.6. HRMS (EI): Calcd. for C25H41NO2 [M]þ
387.3137, found: 387.3139.
2-Amino-2-{2-[4-((1r,4r)-4-butylcyclohexyl)phenyl]-
General procedure for the synthesis of compounds 1a–1e
A solution of 9a–9e (3.1 mmol) in methanol (2 mL) and aqueous
HCl (4.0 mL) was heated at 508C for 3 h. Then, the solvent was
evaporated to dryness to give crude compound 1a–1e, which was
crystallized from CH2Cl2/MeOH (15:1).
ethyl}propane-1,3-diol hydrochloride 1d
White solid, yield: 80%, m.p.: 185–1878C; 1H-NMR (CDCl3) d: 7.92
(s, 3H), 7.09–7.13 (m,4H), 5.39 (t, 3H), 3.51–3.54 (m, 4H), 2.55–2.58
(m, 2H), 2.48–2.52 (m, 1H), 1.75–1.81 (m, 6H), 1.38–1.41 (m, 2H),
1.27–1.28 (m, 5H), 1.21–1.22 (m, 2H), 1.00–1.04 (m, 2H), 0.87 (t, 3H).
13C-NMR (CDCl3) d: 13.9, 22.4, 27.9, 28.6, 33.1, 33.2, 33.9, 36.5, 36.6,
43.4, 60.3, 60.9, 126.5, 127.9, 139.0, 144.7; anal. calcd. (C21H36ClNO2):
C, 68.17; H, 9.81; N, 3.79. Found: C, 68.20; H, 9.61; N, 3.93.
NMR for the free base of 1d: white solid, m.p.: 174–1758C.
1H-NMR (CDCl3) d: 7.06–7.10 (m, 4 H), 4.44 (s, 2 H), 3.21–3.24 (m,
4 H), 2.50–2.54 (m, 2 H), 2.44–2.46 (m, 1 H), 1.75–1.81 (m, 4 H),
1.46–1.49 (m, 2 H), 1.38–1.41 (m, 2 H), 1.27–1.28 (m, 5 H), 1.21–
1.22 (m, 2 H), 1.00–1.04 (m, 2 H), 0.88 (t, 3 H); 13C-NMR (CDCl3) d:
13.9, 22.4, 28.4, 28.6, 33.1, 33.9, 36.5, 36.6, 36.9, 39.3, 43.4, 55.3,
65.4, 126.4, 127.9, 140.8, 144.2. HRMS (ESI): Calcd. for C21H35NO2
[M]þ: 333.2668, found: 334.2763 (MþþH).
2-Amino-2-[2-(4-cyclohexylphenyl)ethyl]propane-1,3-diol
hydrochloride 1a
White solid, yield: 81%, m.p.: 191–1938C; 1H-NMR (CDCl3) d: 7.90
(s, 3 H), 7.10–7.14 (m, 4 H), 5.36–5.38 (t, 2 H), 3.50–3.54 (m, 4 H),
2.55–2.58 (m, 4 H), 2.43–2.45 (m, 1 H), 1.74–1.79 (m, 4 H), 1.68–
1.70 (m, 1 H), 1.33–1.38 (m, 4 H), 1.21–1.24 (m, 1 H); 13C-NMR
(CDCl3) d: 25.6, 26.4, 27.9, 33.3, 34.1, 43.4, 60.2, 61.0, 126.6, 128.1,
139.0, 145.1; anal. calcd. (C17H28ClNO2): C, 65.05; H, 8.99; N, 4.46.
Found: C, 64.78; H, 8.70; N, 4.59.
NMR for the free base of 1a: white solid, m.p.: 163–1658C.
1H-NMR (CDCl3) d: 7.08 (m, 4 H), 4.53 (d, 2 H), 3.20–3.30 (m,
4 H), 2.50–2.55 (m, 4 H), 2.43 (s, 1 H), 1.68–1.76 (m, 4 H), 1.60–
1.65 (m, 1 H), 1.35–1.41 (m, 2 H), 1.29–1.31 (m, 4 H), 1.21–1.22 (m,
1 H); 13C-NMR (CDCl3) d: 25.6, 26.4, 28.5, 34.1, 36.9, 55.4, 65.3,
126.4, 128.0, 140.7, 144.5. HRMS (ESI): Calcd. for C17H27NO2 [M]þ:
277.2042, found: 278.2112 (MþþH).
2-Amino-2-{2-[4-((1r,4r)-4-amylcyclohexyl)phenyl]-
ethyl}propane-1,3-diol hydrochloride 1e
White solid, yield: 75%, m.p.: 180–1828C; 1H-NMR (CDCl3) d: 7.87
(s, 3 H), 7.09–7.14 (m, 4 H), 5.34 (t, 3 H), 3.51–3.53 (m, 4 H), 2.53–
2.56 (m, 2 H), 2.45–2.47 (m, 1 H), 1.76–1.82 (m, 4 H), 1.41–1.44 (m,
2 H), 1.19–1.30 (m, 9 H), 1.05–1.06 (m, 2 H), 0.87 (t, 3 H); 13C-NMR
(CDCl3) d: 13.9, 22.1, 26.0, 27.9, 31.6, 33.1, 33.3, 33.9, 36.6, 36.9,
44.5, 60.3, 60.9, 126.6, 128.0, 139.1, 144.8; anal. calcd.
(C22H38ClNO2): C, 68.81; H, 9.97; N, 3.65. Found: C, 68.97; H,
9.85; N, 3.79.
2-Amino-2-{2-[4-((1r,4r)-4-ethylcyclohexyl)phenyl]-
ethyl}propane-1,3-diol hydrochloride 1b
White solid, yield: 78%, m.p.: 184–1868C; 1H-NMR (CDCl3) d: 7.83
(s, 3 H), 7.09–7.14 (m, 4 H), 3.36–3.38 (t, 2 H), 3.32–3.52 (m, 4 H),
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Synthesis and Immunomodulating Activity of New Analogues of Fingolimod
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NMR for the free base of 1e: white solid, m.p.: 174–1768C.
1H-NMR (CDCl3) d: 7.06–7.10 (m, 4 H), 4.45 (s, 2 H), 3.20–3.26
(m, 4 H), 2.50–2.54 (m, 2 H), 2.45–2.47 (m, 1 H), 1.75–1.81 (m, 4 H),
1.47–1.48 (m, 2 H), 1.40–1.43 (m, 2 H), 1.20–1.29 (m, 9 H), 1.05–
1.06 (m, 2 H), 0.87 (t, 3 H); 13C-NMR (CDCl3) d: 13.9, 22.1, 26.0, 28.5,
31.6, 33.1, 33.3, 33.9, 36.6, 36.8, 44.4, 55.5, 65.2, 126.4, 128.0,
138.3, 139.0, 144.3. HRMS (ESI): Calcd. for C22H37NO2 [M]þ:
347.2824, found: 348.2830 (MþþH).
swelling was expressed as the increase in ear thickness and
ear patch weight between left (DNFB treated) and right
(untreated) ear 24 h after challenge.
OVA-specific T cell response
OVA at 2 mg/mL in phosphate-buffered saline (PBS) was emul-
sified in an equal volume of complete Freund’s adjuvant. The
emulsion (100 mL containing 100 mg OVA) was injected sub-
cutaneously into the backs of the C57BL/6 mice on day 1. OVA-
immunized mice were administered orally with vehicle or
compounds once daily for 14 days. On day 14, splenocytes
(4 ꢃ 105 cells/well) from each group were stimulated with or
without OVA (100 mg/mL) in 96-well flat-bottom plates. Cells
were pulsed with 0.5 mCi/well [3H]thymidine for 8 h before
harvesting and assessed for [3H]thymidine incorporation at
48 h.
Biological assays
Female BALB/c and C57BL/6 mice (6- to 8-week-old) were
obtained from Shanghai Laboratory Animal Center of the
Chinese Academy of Sciences. The mice were housed under
specific pathogen-free conditions. The environment was
maintained at 22 ꢀ 18C with a 12 h light and dark cycle.
All mice were allowed to acclimatize in our facility for 1 week
before any experiments were started. Experiments were car-
ried out according to the National Institutes of Health Guide
for Care and Use of Laboratory Animals and were approved by
the Bioethics Committee of the Shanghai Institute of Materia
Medica.
We gratefully acknowledge the financial support for new century excellent
talents in university (NCET). And we also thank Lab of Organic Functional
Molecules, the Sino-French Institute of ECNU for support. This work was
supported by National Science Fair Committee (NSFC): 1072652, National
Science & Technology Major Project 2009ZX09103-070, 2009ZX09102-019
and 2009ZX09301-001.
Splenocyte preparation
Mice were sacrificed, and spleens were removed aseptically. A
single cell suspension was prepared after cell debris, and
clumps were removed. Erythrocytes were depleted with
ammonium chloride buffer solution. Lymphocytes were
washed and resuspended in RPMI 1640 medium containing
10% FBS.
The authors have declared no conflict of interest.
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