Antibody against a novel, myriocin (ISP-I)-based immunosuppressant, FTY720

Article abstract of DOI:10.1016/S0960-894X(99)00695-2

An antibody was prepared by immunizing rabbits with an ovalbumin conjugate of 2-amino-2-(2-(4-(4-mercaptobutyl)-phenyl)ethyl)propane-1,3-diol HCl (AMPD-4), which contains the essential structure of the novel immunosuppressant FTY720. As the antibody react

Full text of DOI:10.1016/S0960-894X(99)00695-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 337±339
Antibody Against a Novel, Myriocin (ISP-I)-Based
Immunosuppressant, FTY720
Tetsuro Fujita, a,* Norimasa Matsumoto, a Shuji Uchida, a Takeyuki Kohno a,
Takaaki Shimizu, a Ryoji Hirose, b Kazuo Yanada, a Wasako Kurio a
and Kazuhito Watabe a
aFaculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge Hirakata, Osaka 573-0101, Japan
bResearch Laboratory, Taito Co., Ltd, 1-26 Higashi Shiriike-Shinmachi, Nagata-ku, Kobe, 653-0023, Japan
Received 4 November 1999; accepted 10 December 1999
AbstractÐAn antibody was prepared by immunizing rabbits with an ovalbumin conjugate of 2-amino-2-(2-(4-(4-mercaptobutyl)-
phenyl)ethyl)propane-1,3-diol HCl (AMPD-4), which contains the essential structure of the novel immunosuppressant FTY720. As
the antibody reacted to not only AMPD-4, but also FTY720, it should be useful for immunoassay of FTY720 in body ¯uids, tissues
and cells. # 2000 Elsevier Science Ltd. All rights reserved.
FTY720 (1: 2-amino-2-(2-(4-octylphenyl)ethyl)propane-
1,3-diol HCl),1 a novel immunosuppressant, was dis-
covered by chemical modi®cation based on the structure±
activity relationships2±4 of ISP-I (2),5±8 a metabolite of the
fungus Isaria sinclairii. Compound 1 has more potent
activity than established drugs such as cyclosporin A and
FK506.9±11 Furthermore, combination therapy with the
other drugs showed a remarkable synergistic e€ect in
prolonging skin, cardiac, and renal allograft survi-
val.9,10,12±14 This is considered to be because 1 has an
entirely di€erent action mechanism from the other
drugs.9 In spite of the established therapeutic e€ective-
ness of 1, little clinical and experimental knowledge is
available about its pharmacokinetic properties, dis-
position, and so on, due to the lack of an antibody
against 1.
Synthesis of 2-amino-2-(2-(4-(4-mercaptobutyl)phenyl)
ethyl)propane-1,3-diol HCl (AMPD-4)
AMPD-4 (3) was synthesized according to Scheme 1.15
An ester 4,16 which was prepared from 4-phenylbutyric
acid, was bromoacetylated to give 5. Compound 5 was
reduced with triethylsilane in tri¯uoroacetic acid to a€ord
6,17 which was converted to 7. The iodide 7 was condensed
with diethyl acetamidomalonate to give the triethylester 8.
Reduction of triester 8 with sodium borohydride, fol-
lowed by treatment with 2,2-dimethoxypropane, a€orded
a protected monoester 9 as a major product and an
alcohol 10 as a minor product. The ester 9 was reduced
with lithium aluminum hydride to give the alcohol 10,
which was converted to a tosylate (11). Compound 11 was
treated with potassium thioacetate to give the thioacetate
12, and then acid hydrolysis yielded AMPD-4 (3).
The aims of this study were (1) to synthesize 2-amino-2-
(2-(4-(4-mercaptobutyl)phenyl)ethyl)propane-1,3-diol
HCl, AMPD-4 (3), which contains the essential struc-
ture of 14 and a thiol group that can be used to con-
jugate the molecule to a maleimide-modi®ed carrier
protein, (2) to prepare AMPD-4-carrier protein con-
jugate, and (3) to examine the availability of the
AMPD-4-carrier protein conjugate as an antigen to
prepare an antibody against 1.
Preparation of AMPD-4-protein conjugates (Scheme 2)
Maleimide groups were introduced into ovalbumin (OVA)
and bovine serum albumin (BSA) by using e-male-
imidocaproic acid N-hydroxysuccimide ester (EMCS),18
and the average numbers19 per protein molecule were 8.4
and 10.1, respectively. AMPD-4-protein conjugates were
prepared by Michael-type addition of the thiol group of 3
with the maleimide moieties. The average numbers of
molecules of 3 introduced per protein molecule were 6.8
and 9.6, respectively, which were calculated from the
*Corresponding author. Tel.: +81-72-866-3143; fax: +81-72-866-
3146; e-mail: fujita@pharm.setsunan.ac.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00695-2

338
T. Fujita et al. / Bioorg. Med. Chem. Lett. 10 (2000) 337±339
decrease in the number of maleimide groups.19 The resi-
dual maleimide moieties were protected with 2-mercap-
toethylamine and puri®ed by gel ®ltration on a column
of Sephadex G-25 to give AMPD-4-protein conjugates.
in Freund's complete adjuvant three times at three-week
intervals. As shown in Figure 1, anti-AMPD-4 IgG
antibody could be detected in sera from every rabbit by
enzyme-linked immunosorbent assay (ELISA), in which
an AMPD-4-BSA-coated solid phase was incubated
with test sera and, after washing, with horseradish per-
oxidase (HRP)-labelled goat (anti-rabbit IgG) Fab'.
This result showed that AMPD-4-OVA conjugate was
e€ective as an antigen.
Immunization of rabbits with AMPD-4-OVA conjugate
Three rabbits (Japanese White, 2.7±2.8 kg) were intracu-
taneously injected with AMPD-4-OVA conjugate (0.1 mg)
Scheme 1. (a) BrCH2COCl, AlCl3, CHCl3; (b) Et3SiH, TFA; (c) Nal. 2-butanone; (d) NaH, DMF-THF; (e) NaBH4, MeOH; (f) (CH3)2C(OCH3)2,
TsOH; (g) LiAlH4, Et2O; (h) TsCl, Et3N, CH2Cl2; (i) KSCOCH3, EtOH; (j) 10% HCl (aq), EtOH.
Scheme 2.

T. Fujita et al. / Bioorg. Med. Chem. Lett. 10 (2000) 337±339
339
2. Sasaki, S.; Hashimoto, R.; Kiuchi, M.; Inoue, K.; Ikumoto,
T.; Hirose, R.; Chiba, K.; Hoshino, Y.; Okumoto, T.; Fujita,
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3. Fujita, T.; Hamamichi, N.; Kiuchi, M.; Matsuzaki, T.;
Kitao, Y.; Inoue, K.; Hirose, R.; Yoneta, M.; Sasaki, S.;
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Kiuchi, M.; Hirase, S.; Chiba, K.; Sakamoto, H.; Arita, M. J.
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Figure 1. Three rabbits (*, Á, &) were inoculated with AMPD-4-
OVA. Anti-AMPD-4 IgG was measured by ELISA using AMPD-4-
BSA as an antigen.
9. Chiba, K.; Hoshino, Y.; Suzuki, C.; Masubuchi, Y.; Yana-
gawa, Y.; Ohtsuki, M.; Sasaki, S.; Fujita, T. Transplant. Proc.
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Amano, Y.; Chiba, K. Transplant. Proc. 1996, 28, 1060.
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Higashi, H.; Kataoka, H.; Ohtsuki, M.; Teshima, K.; Chiba,
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Figure 2. Standard curve for FTY720 (1) in competitive enzyme
immunoassay using HRP-labelled anti-AMPD-4-OVA Fab'.
15. All new compounds in this communication gave analytical
and spectroscopic data in full accord with their assigned
structures. Compound 3: colorless plates. Mp 105±113 ꢀC
(dec). IR nmax (CHCl3) cm 1: 3550±3150, 3150±2400. 1H
NMR (DMSO-d6) d 1.53 (2H, m), 1.62 (2H, m), 1.76 (2H, m),
2.23 (1H, t, J=8.0 Hz), 2.46±2.55 (4H, m), 2.59 (2H, m), 3.52
(each 2H, d, J=5.0 Hz), 5.36 (2H, t, J=5.0 Hz), 7.11 (4H, br
s), 7.82 (3H, br s). FAB-MS (negative) m/z: 318, 320 (M±1)+.
Anal. calcd for C15H26NO2SCl: C, 56.32; H, 8.19; N, 4.38.
Found: C, 56.37; H, 8.09; N, 4.49. Compound 10: colorless
Speci®city of the antibody
An enzyme immunoassay for 1 based on a competitive
format was conducted to examine the applicability of
the antibody for the assay of 1. In brief, HRP-labelled
anti-AMPD-4-OVA Fab' was incubated with 1 and
trapped on an AMPD-4-BSA-coated solid phase. The
solid phase was washed, and the bound HRP activity
was detected. A standard curve for 1 obtained by this
method is shown in Figure 2. This result clearly indi-
cates that the antibody prepared, reacts not only with 3,
but also with 1. Therefore, it should be applicable to
study the pharmacokinetic properties and disposition of
1. The enzyme immunoassay method for 1 containing
the speci®city will be reported in detail elsewhere.
needles. Mp 106±108 ꢀC. IR nmax (KBr) cm 1: 3440, 1680. H
1
NMR (CDCl3) d: 1.42 (6H, s), 1.55 (2H, m), 1.65 (2H, m),
2.01 (3H, s), 2.05 (2H, m), 2.51 (2H, m), 2.63 (2H, t, J=7.4
Hz), 3.66, 3.96 (each 2H, d, J=12.0 Hz), 5.69 (1H, br s), 7.07
(4H, m). EI-MS m/z: 349 (M+). Anal. calcd for C20H31NO4:
C, 68.74; H, 8.94; N, 4.01. Found: C, 68.66; H, 8.66; N, 3.83.
16. Hwa, J. C. H.; Fleming, W. A. J. Org. Chem. 1957, 22,
1106.
17. Momose, D.; Satoh, M.; Nonaka, Y.; Isawa, H.; Yanagi,
N.; Takehana, Y. Japan. Patent 93-155995 (Chem. Abstr.
1995, 123, 198841).
References and Notes
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1. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.;
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