Synthesis of FTY720 (Fingolimod) Derivatives Containing Serine Structure

Full text of DOI:10.1002/bkcs.11370

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DOI: 10.1002/bkcs.11370
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Y. S. Oh et al.
KOREAN CHEMICAL SOCIETY
Synthesis of FTY720 (Fingolimod) Derivatives Containing Serine
Structure
Yoon Sin Oh,†,‡‡ Taeho Lee,‡,‡‡ Sang Mi Shin,§ Jitendra Shrestha,¶ Doohyun Lee,‡
¶,
Eun-Young Park,¶, and Dong Jae Baek
*
*
†Department of Food and Nutrition, Eulji University, Seongnam 13135, South Korea
‡College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University,
Daegu 41566, South Korea
§College of Pharmacy, Chosun University, Gwangju 61452, South Korea
¶College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Jeonnam,
58554, South Korea. *E-mail: parkey@mokpo.ac.kr; dbaek@mokpo.ac.kr
Received October 26, 2017, Accepted December 8, 2017
Keywords: FTY720, Fingolimod, Anticancer, Sphingosine, Serine
FTY720 (Fingolimod, Gilenya) is a fungal metabolite
derived from the Chinese herb Iscaria sinclarii. It is struc-
turally and biologically similar to sphingosine. FTY720 is
phosphorylated (FTY720-P) by sphingosine kinase 2 (SK2)
and shows immunosuppressive effects. FTY720-P binds to
sphingosine-1-phosphate (S1P) on the lymphocyte surface
for decomposition, isolation of lymphocytes from lymph
nodes and prevents their release into blood.1 The mecha-
nism has an immunosuppressive effect in patients with mul-
tiple sclerosis.2 Multiple sclerosis is a major neurological
disorder affecting 2.3 million patients worldwide, and
occurs mainly in young women.3 FTY720 is an oral treat-
ment for multiple sclerosis first approved by the US FDA
in 2010. In addition, FTY720 activates protein phosphatase
2A (PP2A), which plays a tumor suppressor role, resulting
in anticancer effects in cancers of blood, liver, bladder,
colon and rectum showing reduced cellular PP2A
activity.4–7 FTY720 stimulates beta cell regeneration in
mice and normalizes hyperglycemia.8,9 Biologically active
lipids, such as sphingolipids have been studied for a long
time in various disease models. In this study, we investi-
gated the role of FTY720, which is structurally and biologi-
cally similar to sphingolipid. A definitive therapeutic agent
is unavailable for multiple sclerosis (MS) underscoring the
need for discovery and development of selective com-
pounds and derivatives with therapeutic activity. FTY720
carries a polar head group, an aromatic backbone, and an
eight-carbon chain. Analysis of the structure–activity rela-
tionship of FTY720 derivatives synthesized and reported
thus far revealed significant activity associated with the
head group.10,11 Structurally, however, the two hydroxyl
groups of FTY720 and the linked tert-amine prevent syn-
thesis of various derivatives.12–14 The polar head group
developed so far shows unique physiological activities that
differ between FTY720 and modified FTY720 derivatives
in stereoselectivity. For example, ROME ((R)-FTY720
methyl ether)11 is known to selectively inhibit SK2. How-
ever, FTY720 derivatives in the chiral head group are diffi-
cult to synthesize and involve multiple synthetic steps.
FTY720 derivatives incorporating serine in the head group
yield chiral products in simple synthetic steps. As shown in
Scheme 2, compounds 1 and 2, which are FTY720 deriva-
tives with a known serine structure, show inhibitory effects
with IC50 values of 5.0 and 4.3 μM for SK1, respectively.15
Serine-FTY720 derivatives exhibiting such SK1 inhibitory
effects display potential anticancer properties. Thus, this
study introduced serine, which is commercially available at
a low price, to synthesize additional derivatives for antican-
cer research (Figure 1).
By introducing serine into aniline, we obtained com-
pounds 1 and 2 from compounds 19 and 20. We obtained
compounds 3 and 4 by using Sonogashira coupling to intro-
duce 1-octyne into commercially available 4-iodobenzyla-
mine. The purified product 9 was reduced via hydrogenation
using palladium to obtain compound 10 and the method was
used to obtain compounds 3 and 4 via EDCI coupling and
Boc deprotection. The known compounds 11 and 12 were
synthesized15–17 and mesyl and azide were introduced with-
out purification to obtain compounds 5–8. Similarly, the
compounds 15 and 16 so obtained were hydrogenated and
reduced to amines. The compounds 17 and 18 were used to
obtain the final FTY720 derivatives 5–8 similarly
(Schemes 1 and 2).
In order to determine the anticancer properties of the
newly synthesized sphingosine-serine hybrid compound,
we analyzed the degree of cell death in HT29 cells (human
colorectal adenocarcinoma cell). Significant cell death was
observed with all compounds when tested at a concentra-
tion of 40 μM. The most lethal effects were observed in
compound 8 (Figure 2(a)). In addition, compound 4 carrying
L-serine showed weak anticancer properties (Figure 2(a)).
To determine whether the cell death induced by FTY720
‡‡These authors contributed equally to this work.
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Figure 1. Synthesized FTY720 derivatives.
Figure 2. Effects of FTY720 derivatives on HT29 cells viability.
(a) HT29 cells were treated with compounds 1–8 at 40 μM for
24 h (FTY720 20 μM), and cell viability was measured by EZ-
CYTOX Kit (n = 11). (b) Cells were stained with annexin-V-
FITC. Apoptotic cells were counted by cytometer. *p < 0.05,
**p < 0.01 compared with control cells.
chemical shifts are reported in 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 (Santa Clara, CA, USA) using electrospray
ionization (ESI). All compounds were ≥95% pure as deter-
mined by examining their HRMS and 1H NMR spectra.
Scheme 1. Synthesis of compounds 10, 17, and 18. Reagents and
conditions: (a) 1-octyne, Pd(PPh3)4, CuI, NEt3, 60 ꢀC, 12 h;
(b) 5% Pd/C, H2, EtOAc, rt., 12 h; (c) MsCl, NEt3, CH2Cl2, rt.,
6 h; (d) NaN3, DMF, 80 ꢀC, 12 h.
(R)-2-Amino-3-Hydroxy-N-(4-Octylphenyl)Propanamide
Hydrochloride 1.
To a solution of 19 (100 mg,
ꢀ
0.25 mmol) in THF (5 mL) at 0 C was added 1 M HCl
(1 mL). After being stirred at room temperature for 12 h,
the reaction mixture was evaporated and was washed with
EtOAc/hexane (1/1), evaporated, and dried. Compound
1
1 (77 mg, 92%) was obtained as a white solid. H NMR
(400 MHz, CDCl3:CD3OD = 3:1[v/v]) δ 10.34 (s, NH),
8.43 (s, NH), 7.59 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.2 Hz,
2H), 4.30 (d, J = 4.1 Hz, 1H), 4.19 (dd, J = 12.0, 3.9 Hz,
1H), 4.12 (s, OH), 4.03 (dd, J = 11.8, 6.7 Hz, 1H), 2.66 (t,
J = 7.6 Hz, 2H), 1.66 (d, J = 6.2 Hz, 2H), 1.49–1.28 (m,
10H), 0.97 (t, J = 6.3 Hz, 3H); 13C NMR (100 MHz,
CDCl3:CD3OD = 3:1[v/v]) δ 166.6, 141.3, 136.8, 130.5,
121.9, 62.5, 57.3, 37.0, 33.6, 33.2, 31.1, 30.9, 24.3, 15.4;
ESI-HRMS (M + H)+ m/z calcd for C17H29N2O2 293.2229,
found 293.2218.
Scheme 2. Synthesis of compounds 1–8. Reagents and conditions:
(a) Boc-D-serine or Boc-L-serine, EDCI, DMAP, CH2Cl2, rt.,
12 h; (b) 1 M HCl, THF, rt., 12 h.
derivatives was a result of increased apoptosis, we used
annexin-V staining. The number of annexin-V stained cells
was significantly increased in HT29 cells treated with com-
pound 8 for 24 h (Figure 2(b)). This result showed that the
L and D forms of serine exhibit distinct anticancer proper-
ties. Our findings highlight the relationship of carbon num-
bers in serine and phenyl groups with anticancer properties.
(S)-2-Amino-3-Hydroxy-N-(4-Octylphenyl)Propanamide
Hydrochloride 2. Compound 2 was prepared from 20
according to the same reaction procedure to that described
1
for 1. Yield = 94%; H NMR (400 MHz, CDCl3:CD3OD =
Experimental
3:1[v/v]) δ 10.20 (s, NH), 8.38 (s, NH), 7.46 (d,
J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 4.11 (dt,
J = 7.1, 3.5 Hz, 1H), 4.03 (dd, J = 11.9, 4.2 Hz, 1H), 3.88
(dd, J = 11.8, 6.8 Hz, 1H), 2.59 (t, J = 7.2 Hz, 2H),
1.62–1.46 (m, 2H), 1.20–1.12 (m, 10H), 0.84 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 165.5, 140.2, 135.7, 129.4, 120.8, 61.3, 58.0,
56.2, 35.9, 32.5, 32.2, 30.0, 29.9, 29.8, 23.2, 14.3; ESI-
HRMS (M + H)+ m/z calcd for C17H29N2O2 293.2229,
found 293.2299.
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 (230–400 mesh). All
solvents were of anhydrous quality and were used as
received. H NMR and 13C NMR spectra were recorded on
a Bruker Avance I spectrometer (Billerica, MA, USA), and
1
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(R)-2-Amino-3-Hydroxy-N-(4-Octylbenzyl)Propanamide
Hydrochloride 3. Compound 3 was prepared from 21
according to the same reaction procedure to that described
CDCl3:CD3OD = 3:1[v/v]) δ 7.08 (d, J = 4.3 Hz, 4H),
3.93 (dd, J = 7.7, 4.4 Hz, 2H), 3.87–3.76 (m, 1H), 3.25
(t, J = 7.0 Hz, 2H), 2.62 (dd, J = 14.4, 7.0 Hz, 2H), 2.55
(t, J = 7.6 Hz, 2H), 1.83 (dt, J = 14.6, 7.2 Hz, 2H),
1.63–1.53 (m, 2H), 1.34–1.26 (m, 10H), 0.89 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 168.1, 141.6, 140.0, 129.5, 129.3, 61.8, 56.3,
40.3, 36.5, 33.7, 33.1, 32.8, 32.2, 30.6, 30.4, 23.7, 14.5;
ESI-HRMS (M + H)+ m/z calcd for C20H35N2O2
335.2699, found 335.2621.
(S)-2-Amino-3-Hydroxy-N-(3-(4-Octylphenyl)Propyl)
Propanamide Hydrochloride 8. Compound 8 was pre-
pared from 26 according to the same reaction procedure to
that described for 1. Yield = 84%; 1H NMR (400 MHz,
CDCl3:CD3OD = 3:1[v/v]) δ 7.00 (s, 4H), 4.01–3.93 (m,
1H), 3.90 (dd, J = 11.9, 4.0 Hz, 1H), 3.70 (dd, J = 11.8,
7.0 Hz, 1H), 3.17 (t, J = 7.1 Hz, 2H), 2.53 (t, J = 7.2 Hz,
2H), 2.46 (t, J = 7.6 Hz, 2H), 1.82–1.70 (m, 2H), 1.48 (dd,
J = 14.8, 7.3 Hz, 2H), 1.24–1.13 (m, 10H), 0.79 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 166.6, 140.5, 138.2, 128.3, 128.0, 60.7, 55.0,
39.3, 35.4, 32.4, 31.7, 31.4, 29.4, 29.3, 29.0, 22.4, 13.4;
ESI-HRMS (M + H)+ m/z calcd for C20H35N2O2 335.2699,
found 335.2662.
1
for 1. Yield = 88%; H NMR (400 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 7.00 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz,
2H), 4.20 (s, 2H), 3.92–3.88 (m, 1H), 3.82 (d, J = 12.0 Hz,
1H), 3.63 (dd, J = 11.8, 6.8 Hz, 1H), 2.38 (t, J = 7.6 Hz,
2H), 1.45–1.31 (m, 2H), 1.17–0.99 (m, 10H), 0.69 (t,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 142.7, 134.9, 128.0, 127.9, 61.1, 55.4, 43.7,
35.9, 32.2, 31.9, 29.8, 29.5, 22.9, 14.3; ESI-HRMS
(M + H)+ m/z calcd for C18H31N2O2 307.2386, found
307.2324.
(S)-2-Amino-3-Hydroxy-N-(4-Octylbenzyl)Propanamide
Hydrochloride 4. Compound 4 was prepared from 22
according to the same reaction procedure to that described
1
for 1. Yield = 89%; H NMR (400 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 7.00 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz,
2H), 4.17 (s, 2H), 3.90–3.87 (m, 1H), 3.85 (d, J = 12.7 Hz,
1H), 3.65 (d, J = 7.5 Hz, 1H), 2.37 (t, J = 7.3 Hz, 2H),
1.43–1.34 (m, 2H), 1.19–1.02 (m, 10H), 0.68 (t,
J = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 142.7, 134.8, 129.0, 128.0, 61.2, 55.5, 43.7,
35.8, 32.2, 31.8, 29.7, 29.5, 22.9, 14.2; ESI-HRMS
(M + H)+ m/z calcd for C18H31N2O2 307.2386, found
307.2372.
Cell Culture and Proliferation Assays. HT29 cells were
maintained in Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal bovine serum, 100 U/mL penicillin,
(R)-2-Amino-3-Hydroxy-N-(4-Octylphenethyl)Propana-
mide Hydrochloride 5. Compound 5 was prepared from
23 according to the same reaction procedure to that
ꢀ
and 100 μg/mL streptomycin at 37 C in a humidified 5%
CO2/95% air atmosphere. HT29 cells were seeded in 96-
well plates at a density of 3 × 103 cells/100 μL/well and
incubated for 24 h. The cells were then incubated in culture
medium containing synthetic compounds (40 μM). Follow-
ing 24 h of incubation, the cell viability was determined
using a EZ-CYTOX kit according to the manufacturer’s
protocol.
1
described for 1. Yield = 92%; H NMR (400 MHz, CDCl3:
CD3OD = 3:1[v/v]) δ 7.11 (d, J = 3.0 Hz, 4H), 3.87–3.80
(m, 2H), 3.71 (dd, J = 12.4, 7.9 Hz, 1H), 3.56–3.37 (m,
2H), 2.86–2.73 (m, 2H), 2.56 (t, J = 7.6 Hz, 2H),
1.62–1.54 (m, 2H), 1.37–1.23 (m, 10H), 0.89 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 168.0, 142.2, 137.3, 129.7, 129.6, 61.8, 56.3,
42.3, 36.5, 36.0, 33.1, 30.6, 30.4, 23.7, 14.4; ESI-HRMS
(M + H)+ m/z calcd for C19H33N2O2 321.2542, found
321.2533.
Annexin-V Staining. Apoptosis was determined using an
annexin-V FITC apoptosis detection kit, according to the
manufacturer’s instructions. Briefly, the cells were washed
with ice-cold PBS and resuspended in binding buffer. The
cell suspension was incubated with annexin-V-FITC and
propidium iodide at room temperature. After incubation,
stained cells were analyzed by Arthur™ Image Based Cell
Analyzer.
(S)-2-Amino-3-Hydroxy-N-(4-Octylphenethyl)Propana-
mide Hydrochloride 6. Compound 6 was prepared from
24 according to the same reaction procedure to that
1
described for 1. Yield = 95%; H NMR (400 MHz, CDCl3:
CD3OD = 3:1[v/v]) δ 7.11 (d, J = 3.5 Hz, 4H), 3.85 (dt,
J = 7.0, 4.2 Hz, 2H), 3.71 (dd, J = 12.5, 8.0 Hz, 1H),
3.56–3.37 (m, 2H), 2.87–2.75 (m, 2H), 2.56 (t, J = 7.6 Jz,
2H), 1.63–1.54 (m, 2H), 1.35–1.27 (m, 10H), 0.89 (t,
J = 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3:CD3OD =
3:1[v/v]) δ 168.0, 142.2, 137.5, 129.8, 129.6, 61.8, 56.3,
42.3, 36.6, 36.0, 33.0, 32.8, 30.6, 30.4, 23.7, 14.4; ESI-
HRMS (M + H)+ m/z calcd for C19H33N2O2 321.2542,
found 321.2502.
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 (NRF-2015R1C1A1A01
053302) and (NRF-2017R1A1A1A05001129).
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
(R)-2-Amino-3-Hydroxy-N-(3-(4-Octylphenyl)Propyl)
Propanamide Hydrochloride 7. Compound 7 was pre-
pared from 25 according to the same reaction procedure
References
1. J. Ingwersen, O. Aktas, P. Kuery, B. Kieseier, A. Boyko,
1
to that described for 1. Yield = 87%; H NMR (400 MHz,
H. P. Hartung, Clin. Immunol. 2012, 142, 15.
Bull. Korean Chem. Soc. 2018
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BULLETIN OF THE
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2. J. Chun, H. P. Hartung, Clin. Neuropharmacol. 2010, 33, 91.
3. E. Leray, T. Moreau, A. Fromont, G. Edan, Rev. Neurol.
2015, 172, 3.
4. J. J. Oaks, R. Santhanam, C. J. Walker, S. Roof, J. G. Harb,
G. Ferenchak, A.-K. Eisfeld, J. R. Van Brocklyn, R. Briesewitz,
S. A. Saddoughi, K. Nagata, R. Bittman, M. A. Caligiuri,
O. Abdel-Wahab, R. Levine, R. B. Arlinghaus, A. Quintas-
Cardama, J. M. Goldman, J. Apperley, A. Reid, D. Milojkovic,
M. K. Ziolo, G. Marcucci, B. Ogretmen, P. Neviani, D. Perrotti,
Blood 2013, 122, 1923.
5. K. T. NG, K. Man, J. W. Ho, C. K. Sun, T. K. Lee, Y. Zhao,
C. M. Lo, R. T. Poon, S. T. Fan, Int. J. Oncol. 2007, 30, 375.
6. H. Azuma, S. Takahara, S. Horie, S. Muto, Y. Otsuki,
Y. J. Katsuoka, J. Urol. 2003, 169, 2372.
7. L. Zhang, H. D. Wang, X. J. Ji, Z. X. Cong, J. H. Zhu,
Y. Zhou, Oncol. Rep. 2013, 30, 2571.
S. A. Barr, S. M. Kim, S. G. Roy, G. Liu, D. Fallegger,
L. Sernissi, C. Brandt, N. Moitessier, A. J. Snider, S. Clare,
M. Müschen, A. Huwiler, M. T. Kleinman, S. Hanessian,
A. L. Edinger, Leukemia 2017, 31, 669.
11. D. G. Watson, F. Tonelli, M. Alossaimi, L. Williamson,
E. Chan, I. Gorshkova, E. Berdyshev, R. Bittman, N. J. Pyne,
S. Pyne, Cell. Signal. 2013, 25, 1011.
12. K. Hinterding, S. Cottens, R. Albert, F. Zecri, P. Buehlmayer,
C. Spanka, V. Brinkmann, P. Nussbaumer, P. Ettmayer,
K. Hoegenauer, N. Gray, S. Pan, Synthesis 2013, 11, 1667.
13. S. Kim, H. Lee, M. Lee, T. Lee, Synthesis 2006, 5, 753.
14. K. G. Lim, C. Sun, R. Bittman, N. J. Pyne, S. Pyne, Cell.
Signal. 2011, 23, 1590.
15. Y. Xiang, G. Asmussen, M. Booker, B. Hirth, J. L. Kane Jr,
J. Liao, K. D. Noson, C. Yee, Bioorg. Med. Chem. Lett.
2009, 19, 6119.
8. Z. Zhao, J. Choi, C. Zhao, Z. A. Ma, J. Biol. Chem. 2012,
287, 5562.
9. H. Moon, J. Chon, J. Joo, D. Kim, J. In, H. Lee, J. Park,
J. Choi, Diabetes Metab. Res. Rev. 2013, 29, 19.
10. A. N. McCracken, R. J. McMonigle, J. Tessier, R. Fransson,
M. S. Perryman, B. Chen, A. Keebaugh, E. Selwan,
16. D. J. Baek, N. MacRitchie, N. G. Anthony, S. P. Mackay,
S. Pyne, N. J. Pyne, R. Bittman, J. Med. Chem. 2013,
56, 9310.
17. D. J. Baek, N. MacRitchie, N. J. Pyne, S. Pyne, R. Bittman,
Chem. Commun. 2013, 49, 2136.
Bull. Korean Chem. Soc. 2018
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