Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

Article abstract of DOI:10.1016/j.bmc.2006.01.071

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. T

Full text of DOI:10.1016/j.bmc.2006.01.071

Bioorganic & Medicinal Chemistry 14 (2006) 4182–4192
Preparation of antibodies against a novel immunosuppressant,
FTY720, and development of an enzyme immunoassay for FTY720
Norimasa Matsumoto,a,* Takeyuki Kohno,a Shuji Uchida,a Takaaki Shimizu,a
Haruko Kusumoto,a Ryoji Hirose,b Kazuo Yanada,a Wasako Kurio,a Shoji Yamaguchi,a
Kazuhito Watabea and Tetsuro Fujitaa,ꢀ
aFaculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
bResearch Laboratory, Taito Co., Ltd, 1-26 Higashi Shiriike-Shinmachi, Nagata-ku, Kobe 653-0023, Japan
Received 26 October 2005; revised 26 January 2006; accepted 27 January 2006
Available online 28 February 2006
Abstract—FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin).
To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA)
was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized
with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)pro-
pane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of
quantification by the EIA was 0.06–10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration
in serum after oral administration of 1 (1 mg/kg) to rats.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
stage of Cordyceps sinclairii and is parasitic on cicada
larvae. It has been reported that ISP-I inhibits serine
palmitoyltransferase of an interleukin-2 (IL-2)-depen-
dent mouse cytotoxic T cell line, CTLL-2, at picomolar
concentrations.13 His group simplified the structure of
ISP-I in order to reduce its toxicity, to improve its phys-
icochemical properties, and to identify the essential
structure for immunosuppressive activity14 and obtained
a candidate compound, FTY720 (1: 2-amino-2-(2-(4-
octylphenyl)ethyl)propane-1,3-diol hydrochloride),15,16
which does not inhibit serine palmitoyltransferase.
Compound 1 has an entirely different action mechanism
from established drugs such as CsA and FK506. It does
not affect IL-2 production from mitogen-stimulated rat
spleen cells17 and suppresses the immune response by
sequestering lymphocytes from peripheral blood and
spleen to the secondary lymphoid tissues (lymph nodes,
Peyer’s patches, and so on).18–20 Recently, it was report-
ed by Mandala et al.21 and Brinkmann et al.22 that the
phosphorylated form of 1 acts as an agonist of sphingo-
sine-1-phosphate (S1P). Like sphingosine, 1 is converted
by sphingosine kinase to FTY720 monophosphate,
which is the active form of the drug. FTY720 mono-
phosphate signals through four of the five known S1P
receptors, which belong to the endothelial-differentia-
tion-gene (EDG) family of G-protein-coupled receptors.
Immunosuppressants have important clinical roles in or-
gan transplantation and the treatment of autoimmune
diseases. At present, the established immunosuppres-
sants, cyclosporin
A
(CsA),1 and tacrolimus
(FK506),2–4 are widely used in organ transplantation.
However, both drugs, which have similar mechanisms
of action,5 have severe side effects, notably renal toxici-
ty.6,7 Therefore, it would be desirable to develop a novel
immunosuppressant with less toxicity and an entirely
different mechanism of action.
In the late 1980s, Fujita et al. of our group isolated a
unique immunosuppressant, ISP-I8 (2: myriocin9,10 and
thermozymocidin11,12), from the fermentation broth of
Isaria sinclairii (ATCC24400), which is the imperfect
Keywords: FTY720; Immunosuppressant; Hapten; Enzyme
immunoassay.
*
Corresponding author at present address: Taito Bio Industry
Research Corporation, 50-18 Fukaehama-machi, Higashinada-ku,
Kobe 658-0023, Japan. Tel.: +81 78 435 0821; fax: +81 78 413
3847; e-mail: Norimasa.Matsumoto@mitsui-sugar.co.jp
ꢀ
Research Institute for Production Development, 15 Shimogamo
Morimoto-cho, Sakyo-ku, Kyoto 606-0805, Japan.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.071

N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
4183
Very recently, Matloubian et al.23 established, by using
1, that S1P receptor 1 is essential for lymphocyte recir-
culation and regulates egress from both thymus and
peripheral lymphoid organs. It has been shown that 1
has a more potent immunosuppressive activity than
CsA and FK506 in rat skin and cardiac allograft mod-
els.17,18,24 Furthermore, combination therapy with the
other drugs, CsA and FK506, showed a remarkable syn-
ergistic effect in prolonging skin, cardiac, and renal allo-
graft survival.17,18,25–27 Compound 1 is also expected to
be useful for the treatment of other diseases, including
multiple sclerosis,22 rheumatoid arthritis,28 spontaneous
dermatitis,29 and myasthenia gravis,30 based on findings
in animal models.
The Wittig condensation product obtained from the
aldehyde 11 with (3-(ethoxycarbonyl)-2-propenyl)tri-
phenylphosphonium bromide was hydrogenated to af-
ford an ester 12. The ester 12 was reduced with
lithium aluminum hydride to give an alcohol 13. The
alcohols, 10 and 13, were treated with tosyl chloride to
give tosylates, 14 and 15, respectively. Compounds 14
and 15 were treated with potassium thioacetate to give
thioacetates 16 and 17, and then acid hydrolysis yielded
the desired haptens, 3 and 4, respectively (Chart 1).
Recently, a clinical trial of 1 in renal transplantation pa-
tients has been performed.31 Quantitative analysis of 1
in the blood was accomplished by HPLC/MS/MS.31
The instrumentation is expensive and requires skill to
operate. On the other hand, immunoassay is a common
laboratory method, which is suitable to screen large
numbers of samples. Therefore, the development of
immunoassay technology for 1 would be of great value.
We have succeeded in preparing the first specific anti-
bodies against 1. A part of this work was reported in
our preliminary communication.32 Here, we present full
details of the synthesis of two FTY720 analogues,
2-amino-2-(2-(4-(4-mercaptobutyl)phenyl)ethyl)propane-
1,3-diol hydrochloride (3) and 2-amino-2-(2-(4-(8-mer-
captooctyl)phenyl)ethyl)propane-1,3-diol hydrochloride
(4), as haptens, the preparation of hapten-carrier pro-
teins and their use to raise antibodies, the specificity of
the antibodies, and the development of an EIA for 1.
2. Results
2.1. Synthesis of haptens and preparation of hapten-
carrier proteins
To prepare antibodies in animals against a low-molecu-
lar compound such as 1, conjugation to a carrier protein
such as ovalbumin (OVA) is generally required. We de-
signed compounds 3 and 4 as haptens containing the
characteristic structure, 2-aminopropane-1,3-diol and a
side chain including a phenyl group, of 1. The thiol
group of the haptens was expected to undergo
Michael-type addition to maleimide groups of a carrier
protein bearing suitable groups, such as e-maleimido-
caproic acid N-hydroxysuccinimide ester (EMCS).33
The haptens 3 and 4 were synthesized as follows. Ethyl
4-phenylbutyrate 534 was bromoacetylated to give 6,
which was reduced with triethylsilane in trifluoroacetic
acid to afford 7.35 The bromide 7 was condensed with
diethyl acetamidomalonate to give the triethylester 8.
Reduction of 8 with sodium borohydride, followed by
treatment with 2,2-dimethoxypropane, afforded a pro-
tected monoester 9 as a major product and an alcohol
10 as a minor product. The ester 9 was reduced with lith-
ium aluminum hydride to give the alcohol 10, which was
oxidized with oxalyl chloride to afford an aldehyde 11.
Chart 1. Structures of FTY720 and related compounds.

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N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
Antigens were prepared as shown in Scheme 1. OVA
was selected as a carrier protein for the immunogen.
Maleimide groups were introduced into OVA by using
EMCS to give maleimide-OVA. 3-OVA conjugate and
4-OVA conjugate were prepared by Michael-type addi-
tion to the maleimide-OVA of thiols 3 and 4, respective-
ly. The average numbers of 3 and 4 molecules
introduced per OVA molecule were 6.8 and 2.4, respec-
tively, as calculated from the decrease in the number of
maleimide groups. The residual maleimide moieties were
protected with 2-mercaptoethylamine hydrochloride
and purified by gel filtration on a column to give the
3- and 4-OVA conjugates, respectively.
and 4, but also with 1, as shown in Table 1. Compounds
21–27, which have a phenyl ring at a variety of positions
within the side chain of 1, but with a constant chain
length (m + n = 10), were slightly cross-reactive with
anti-3-OVA IgG, but did not cross-react at all with
anti-4-OVA IgG. The non-aromatic 20 was not cross-
reactive at all with either of the antibodies. The
2-aminoalcohol 18 and N-acetate 19 were slightly
cross-reactive with anti-3-OVA IgG, but were not
To develop an enzyme immunoassay (EIA) for 1 and an
enzyme-linked immunosorbent assay (ELISA) for anti-3
and anti-4 IgGs, we selected bovine serum albumin
(BSA) as a carrier protein for the antigen. Horseradish
peroxidase (HRP) was selected as a label. BSA conju-
gates and HRP conjugates of 3 or 4 were prepared in
a similar manner to that used for the preparation of
3-OVA.
2.2. Preparation and specificity of antibodies
Three rabbits were intracutaneously immunized three
times with 3- or 4-OVA conjugate in Freund’s complete
adjuvant at three-week intervals. Antibody IgG against
3 or 4 could be detected in sera from the corresponding
rabbits by ELISA (data not shown), in which a 3-BSA-
or 4-BSA-coated solid phase was incubated with test
sera and then, after washing, with HRP-labeled goat
(anti-rabbit IgG) Fab0.
To examine the specificity of anti-3-OVA IgG and anti-
4-OVA IgG, their epitopes were analyzed by a compet-
itive EIA, using FTY720 (1), the derivatives (18–27),14,16
and the metabolites (28–31)36 in Chart 2 with HRP-la-
beled 3 or HRP-labeled 4 (Fig. 1, method A). The results
clearly indicate that the antibodies bind not only with 3
Chart 2. Structures of derivatives (18–27) and metabolites (28–31) of 1.
Scheme 1. Preparation of hapten-carrier protein.

N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
4185
cross-reactive with anti-4-OVA IgG. These results indi-
cate that both antibodies recognize the 2-amino-2-(2-
phenylethyl)propane-1,3-diol moiety.
The metabolites 28–31, having 2-amino-2-(2-phenyleth-
yl)propane-1,3-diol and carboxylic acid groups, were
reactive with anti-3-OVA IgG and anti-4-OVA IgG, be-
cause the antibodies recognized the 2-amino-2-(2-phen-
ylethyl)propane-1,3-diol moiety.
2.3. Development of an EIA for FTY720
An EIA for FTY720 (1) based on a competitive format
(Fig. 1, method B) was conducted to examine the appli-
cability of anti-3-OVA IgG for the assay of 1. A calibra-
tion curve for 1 obtained by this method is shown in
Figure 2 (open circles). We developed a specific EIA
for 1 using this method, since the sensitivity by this
method was higher than that by the method A.
To develop a specific EIA for 1, it was necessary to re-
move the acidic metabolites 28–31 from samples which
contain the metabolites, that is, serum and plasma.
For this purpose, alkaline-chloroform extraction was
performed before measuring 1 by this method. The acid-
ic metabolites 28–31, which cross-reacted with anti-3-
OVA antibody, were efficiently eliminated (Fig. 3).
The recovery rate of 1 in the presence of serum was
extremely low, probably due to the matrix effect of the
serum proteins. Alkaline-chloroform extraction also im-
proved the recovery rate of 1 in serum. The calibration
curve for 1 in the presence of serum was almost identical
with that in the absence of serum (Fig. 2). The range of
quantification was 0.06–10 ng/mL in the presence of
serum.
Figure 1. Procedure of epitope analysis of anti-3-OVA IgG and anti-4-
OVA IgG by method A, and a competitive EIA for 1 by method B.
Method A: FTY720 (or related compounds 18–31) and HRP-labeled 3
(or HRP-labeled 4) are mixed and trapped on an anti-3-OVA IgG-
coated solid phase (or anti-4-OVA IgG-coated solid phase). The solid
phase is washed, and the bound HRP activity is detected. Method B:
compound 1 is incubated with HRP-labeled anti-3-OVA Fab0, and
then free HRP-labeled anti-3-OVA Fab0 is trapped on a 3-BSA-coated
solid phase. The solid phase is washed, and the bound HRP activity is
detected.
To examine the applicability of the specific EIA, the
concentration of 1 in serum after oral administration
Table 1. Epitope analysis and cross-reactivity of derivatives (18–27)
and metabolites (28–31) of 1
Compound
Reactivity (%)a
Anti-3-OVA IgG Anti-4-OVA IgG
1
3
100
345
178
100
300
231
0
4
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0.6
14
0
0
0
5.9
0
0.4
3.7
8.9
0
0
0
13
0
15
19
0
0
178
141
326
668
173
79
300
363
Epitope and cross-reactivity were examined by using a competitive
EIA (method A). The concentration (C) of the compounds was cal-
Figure 2. Calibration curves for 1. Compound 1 was diluted with
buffer and subjected to competitive EIA (open circle). Compound 1
was diluted with buffer (closed circle) and with rat control serum
(closed triangle), then subjected to alkaline-chloroform extraction,
followed by competitive EIA.
culated by curve fitting using
(%) = C · 100/actual concentration.
a Binding reactivity of the antibodies for 1 is designated as 100.
1 as a standard. The reactivity

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N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
but also for patients with immunological disorders, such
as multiple sclerosis,22 rheumatoid arthritis,28 dermati-
tis,29 and myasthenia gravis.30 The present immunoas-
say will be useful to assess the safety and efficacy of 1
in the clinical setting.
4. Conclusion
Initially, the hapten 3 was synthesized and coupled with
OVA, and antibody against the 3-OVA was prepared in
rabbits. The obtained anti-3-OVA antibody binds with
FTY720 (1), and recognizes the 2-amino-2-(2-phenyleth-
yl)propane-1,3-diol moiety, but is highly cross-reactive
with the metabolites (28–31) of 1. To obtain an antibody
specific for the alkyl side chain moiety of 1, the hapten 4,
which has the same length of alkyl side chain as 1, was
synthesized and coupled with OVA, and antibody
against the 4-OVA was prepared. The specificity of the
anti-4-OVA antibody for the 2-amino-2-(2-phenyleth-
yl)propane-1,3-diol moiety is slightly higher than that
of the anti-3-OVA antibody, but the specificity for the al-
kyl side chain moiety is similar to that of the anti-3-OVA.
The solubility of 3 in buffer is better than that of 4, and
the conjugate of 3 could be easily prepared. Therefore,
we selected anti-3-OVA antibody to develop a competi-
tive EIA for 1. Consequently, the acidic metabolites of
1 had to be removed by alkaline-chloroform extraction
prior to quantification of 1. The range of quantification
of the EIA was 0.06–10 ng/mL. The developed EIA
should be suitable to measure blood levels of 1 in large
numbers of samples from patients in the clinical setting.
Figure 3. Dose–response curves for 1 and the metabolites (28–31)
before and after alkaline-chloroform extraction. Open symbols, before
extraction; closed symbols, after extraction. Compound 1 (circle), 28
(cross), 29 (triangle), 30 (square), and 31 (diamond) were subjected to
EIA for 1 before and after alkaline-chloroform extraction.
of 1 to rats (1 mg/kg) was measured. A time–course
curve of 1 is shown in Figure 4. The concentration of
1 reached a maximum at 12 h (Cmax: 33 ng/mL) and
decreased with a half life of approximately 60 h (t1/2).
AUC0–96h was 3100 ng h/mL.
3. Discussion
Very recently, it was clarified that the active form of
FTY720 (1) is the monophosphate.21,22 Therefore, it will
be important to examine the pharmacokinetics of intra-
cellular FTY720 monophosphate. The pharmacokinet-
ics of intracellular triphosphorylated compounds,37
such as the active forms of the antiviral agents lamivu-
dine and zidovudine, has been investigated with
HPLC/MS/MS, and this approach is also likely to be
applicable to FTY720 monophosphate.
5. Experimental
5.1. Chemistry
5.1.1. General information. Melting points were deter-
mined on a Yanagimoto micro melting point apparatus
(MP-S3) without correction. IR spectra were taken on a
Nihon Bunkou A-102 infrared spectrophotometer. UV
spectra were taken on a Hitachi U-2000 spectrometer.
Mass spectra (EI-MS and FAB-MS) were taken on a
Compound 1 is expected to be widely applied in clinical
treatment not only for organ transplantation patients,
1
JEOL JMS-DX300 spectrometer. H NMR (400 MHz,
CDCl3) spectra were taken on a JEOL JNM-GX400
spectrometer with TMS as an internal standard; chemi-
cal shifts are reported in parts per million. TLC was per-
formed on aluminum seated silica gel 60 F254 (Merck).
For column chromatography, silica gel (70–230 mesh,
Merck) was used. Organic solvent extracts were dried
over anhydrous magnesium sulfate, and evaporation
of solvents was performed under reduced pressure.
5.1.2. Ethyl 4-(4-(bromoacetyl)phenyl)butyrate (6). To a
stirred solution of 5 (20.0 g, 1.0· 10ꢀ1 mol) and bromo-
acetyl chloride (10.4 mL, 1.3· 10ꢀ1 mol) in dried CHCl3
(250 mL) was added portionwise AlCl3 (30.6 g,
2.3· 10ꢀ1 mol) over a period of 15 min at ꢀ10 ꢁC, and
the mixture was stirred at room temperature for 2 h.
The reaction mixture was poured into ice water
(200 mL) and extracted with CHCl3 (100 mL). The ex-
tract was washed with saturated aqueous NaHCO3
Figure 4. Concentration of 1 in serum after oral administration of 1
(1 mg/kg) to rats (n = 4–6). Each value indicates the mean, and vertical
bars indicate SD.

N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
4187
(200 mL) and then brine (50 mL), dried, and evaporat-
ed. The residue was chromatographed on silica gel
with n-hexane–AcOEt (9:1) to give 6 (28.8 g, 88%) as a
colorless oil: bp 188–192 ꢁC (0.1 mmHg ); IR mmax
4.4· 10ꢀ3 mol) in EtOH (7 mL) was added sodium boro-
hydride (0.80 g, 2.1· 10ꢀ2 mol) under ice cooling and the
mixture was stirred at room temperature for 7 h. The
reaction mixture was poured into a mixture of ice water
(10 mL) and AcOEt (50 mL), and the aqueous layer was
acidified to pH 3 with 1 N HCl. The extracted organic
solution was washed with brine (10 mL), dried, and
evaporated. The residue (1.6 g) was dissolved in 2,2-
dimethoxypropane (9.0 mL, 7.3· 10ꢀ2 mol) and to this
stirred solution was added p-toluenesulfonic acid mono-
hydrate (82 mg, 4.3· 10ꢀ4 mol). The mixture was stirred
at room temperature for 18 h and concentrated. To the
residue was added ice water (10 mL), followed by extrac-
tion with AcOEt (30 mL). The extract was washed with
brine (5 mL), dried, and evaporated. The residue was
chromatographed on silica gel with n-hexane–AcOEt
(2:1) to give 9 (0.84 g, 49%) and with n-hexane–AcOEt
(1:2) to give 10 (0.23 g, 15%).
1
(CCl4) cmꢀ1: 1740 (COOEt, BrCH2CO); H NMR d:
1.24 (3H, t, J = 7.0 Hz, CO2CH2CH3), 1.98 (2H,
quintet, J = 7.5 Hz, CH2CH2CH2CO2Et), 2.33 (2H, t,
J = 7.5 Hz, CH2CO2Et), 2.73 (2H, t, J = 7.5 Hz,
PhCH2), 4.15 (2H, q, J = 7.0 Hz, CO2CH2CH3), 4.43
(2H, s, BrCH2CO), 7.30, 7.91 (each 2H, d, J = 8.0 Hz,
ArH); EI-MS m/z: 313 ([M]+).
5.1.3. Ethyl 4-(4-(2-bromoethyl)phenyl)butyrate (7). To a
stirred solution of 6 (7.9 g, 2.5· 10ꢀ2 mol) in trifluoroace-
tic acid (25 mL) was added triethylsilane (8.9 mL,
5.6· 10ꢀ2 mol) under ice cooling and the mixture was stir-
red at room temperature for 4 h. The reaction mixture was
poured into a mixture of ice water (150 mL) and AcOEt
(200 mL), and NaHCO3 (27 g) was added. The extracted
organic solution was washed with saturated aqueous
NaHCO3 (20 mL) and brine (2· 15 mL), then dried, and
evaporated. The residue was chromatographed on silica
gel with n-hexane–AcOEt (40:1) to give 7 (5.1 g, 68%) as
Compound 9: colorless needles (recrystallized from n-
:
hexane–Et2O); mp 52–53 ꢁC; IR mmax (CCl4) cmꢀ1
1
1735 (COOEt), 1680 (CONH); H NMR d: 1.25 (3H, t,
J = 7.0 Hz, CO2CH2CH3), 1.42 (6H, s, C(CH3)2), 1.92
(2H, quintet, J = 7.5 Hz, PhCH2CH2CH2CO2Et), 2.01
(3H, s, NHCOCH3), 2.03–2.07 (2H, m, PhCH2CH2C),
2.30 (2H, t, J = 7.5 Hz, Ph(CH2)2CH2CO2Et), 2.49–
2.53 (2H, m, PhCH2CH2C), 2.60 (2H, t, J = 7.5 Hz,
PhCH2(CH2)2CO2Et), 3.67, 3.97 (each 2H, d,
1
a colorless oil: IR mmax (CCl4) cmꢀ1: 1740 (COOEt); H
NMR d: 1.25 (3H, t, J = 7.0 Hz, CO2CH2CH3), 1.94
(2H, quintet, J = 7.5 Hz, CH2CH2CH2CO2Et), 2.31
(2H, t, J = 7.5 Hz, CH2CO2Et), 2.63 (2H, t, J = 7.5 Hz,
PhCH2(CH2)2CO2Et), 3.13 (2H, t, J = 7.5 Hz,
BrCH2CH2), 3.54 (2H, t, J = 7.5 Hz, BrCH2CH2), 4.12
(2H, q, J = 7.0 Hz, CO2CH2CH3), 7.13 (4H, br s, ArH);
EI-MS m/z: 298 ([M]+).
J = 12.0 Hz,
(CH2O)2C(CH3)2),
4.12
(2H,
q,
J = 7.0 Hz, CO2CH2CH3), 5.71 (1H, br s, NH), 7.08
(4H, br s, ArH); EI-MS m/z: 391 ([M]+); Anal. Calcd
for C22H33NO5: C, 67.49; H, 8.50; N, 3.58. Found: C,
67.45; H, 8.40; N, 3.63.
5.1.4. Diethyl 2-(acetylamino)-2-(2-(4-(3-(ethoxycarbon-
yl)propyl)phenyl)ethyl)propane-1,3-dioate (8). To a solu-
tion
of
diethyl
(acetylamino)malonate
(4.8 g,
Compound 10: colorless plates (recrystallized from ben-
zene); mp 107–108 ꢁC; IR mmax (KBr) cmꢀ1: 3440 (OH),
1675 (CONH); 1H NMR d: 1.42 (6H, s, C(CH3)2),
1.57–1.63 (2H, m, PhCH2CH2(CH2)2OH), 1.63–1.69
(2H, m, Ph(CH2)2CH2CH2OH), 2.01 (3H, s,
NHCOCH3), 2.02–2.07 (2H, m, PhCH2CH2C), 2.49–
2.53 (2H, m, PhCH2CH2C), 2.60 (2H, t, J = 7.5 Hz,
2.2· 10ꢀ2 mol) in dried DMF (10 mL) was added 60%
sodium hydride (0.74 g, 1.9· 10ꢀ2 mol) under an argon
atmosphere, and the mixture was stirred at 90 ꢁC. To
the mixture were added dried THF (10 mL) and a solution
of 7 (2.2 g, 7.4· 10ꢀ3 mol) in dried THF (5 mL). The mix-
ture was refluxed for 2 h and concentrated. To the residue
was added ice water (20 mL), and the whole mixture was
extracted with Et2O (80 mL). The extract was washed
with brine (2· 20 mL), dried, and evaporated. The residue
was chromatographed on silica gel with n-hexane–AcOEt
(2:1) to give 8 (2.6 g, 81%) as a colorless oil: bp 200–230 ꢁC
(oil bath temp, 0.1 mmHg); IR mmax (CCl4) cmꢀ1: 1740
PhCH2(CH2)3OH),
3.65
(2H,
t,
J = 7.4 Hz,
Ph(CH2)3CH2OH), 3.66, 3.97 (each 2H, d, J = 12.0 Hz,
(CH2O)2C(CH3)2), 5.74 (1H, br s, NH), 7.08 (4H, br s,
ArH); 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.
1
(COOEt), 1690 (CONH); H NMR d: 1.12–1.27 (9H,
CO2CH2CH3 3·), 1.92 (2H, quintet, J = 7.5 Hz,
CH2CH2CH2CO2Et), 1.97 (3H, s, NHCOCH3), 2.30
(2H, t, J = 7.5 Hz, Ph(CH2)2CH2CO2Et), 2.43–2.47
(2H, m, PhCH2CH2C), 2.60 (2H, t, J = 7.5 Hz,
PhCH2(CH2)2CO2Et), 2.65–2.69 (2H, m, PhCH2CH2C),
4.12 (2H, q, J = 7.0 Hz, Ph(CH2)3CO2CH2CH3), 4.18–
5.1.6. N-(5-(2-(4-(4-Hydroxybutyl)phenyl)ethyl)-2,2-di-
methyl-1,3-dioxan-5-yl)ethanamide (10). To a stirred
solution of 9 (1.3 g, 3.3· 10ꢀ3 mol) in dried Et2O
(80 mL) was added lithium aluminum hydride (0.38 g,
1.0· 10ꢀ2 mol) under ice cooling, and the reaction mix-
ture was stirred at room temperature for 2 h. Saturated
aqueous Rochelle salt (KNaC4H4O6Æ4H2O) was added
to the mixture under ice cooling, and the organic solu-
tion was decanted, dried, and evaporated. The residue
was recrystallized from benzene to give 10 (0.78 g,
67%).
4.22 (4H, m, C(CO2CH2CH3) ), 6.79 (1H, br s, NH),
2
7.07 (4H, br s, ArH); EI-MS m/z: 435 ([M]+); Anal. Calcd
for C23H33NO7: C, 63.43; H, 7.64; N, 3.22. Found: C,
63.43; H, 7.41; N, 3.07.
5.1.5. Ethyl 4-(4-(2-(5-(acetylamino)-2,2-dimethyl-1,3-di-
oxan-5-yl)ethyl)phenyl)butanoate (9) and N-(5-(2-(4-(4-
hydroxybutyl)phenyl)ethyl)-2,2-dimethyl-1,3-dioxan-5-
yl)ethanamide (10). To a stirred solution of 8 (1.9 g,
5.1.7. N-(2,2-Dimethyl-5-(2-(4-(4-oxobutyl)phenyl)ethyl)-
1,3-dioxan-5-yl)ethanamide (11). To a stirred solution of
oxalyl chloride (88.3 lL, 1.0· 10ꢀ3 mol) in dried CH2Cl2

4188
N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
(7 mL) was added dried DMSO (158 lL, 2.2· 10ꢀ3 mol)
in CH2Cl2 (7 mL) at ꢀ70 ꢁC under an argon atmo-
sphere, and the solution was stirred for 10 min. To the
PhCH2CH2C), 2.60 (2H, t, J = 7.4 Hz, PhCH2(CH2)6–
), 3.66, 3.96 (each 2H, d, J = 12.0 Hz, (CH2O)2C(CH3)2),
4.12 (2H, q, J = 7.1 Hz, CO2CH2CH3), 5.75 (1H, br s,
NH), 7.07 (4H, br s, ArH); EI-MS m/z: 447 ([M]+);
HR-MS: Anal. Calcd for C26H41NO5: 447.2985. Found:
447.2984.
solution was added
a solution of 10 (300 mg,
8.6· 10ꢀ4 mol) in CH2Cl2 (7 mL). The mixture was stir-
red for 10 min and then dried Et3N (0.60 mL,
4.3· 10ꢀ3 mol) was added. The reaction mixture was
poured into ice water (10 mL). The organic layer was
washed with brine (2· 5 mL), dried, and evaporated.
The residue was chromatographed on silica gel with
n-hexane–AcOEt (3:2) to give 11 (0.28 g, 93%): colorless
needles (recrystallized from Et2O); mp 102–104 ꢁC; IR
mmax (KBr) cmꢀ1: 1720 (CHO), 1680 (CONH); 1H
NMR d: 1.42 (6H, s, C(CH3)2), 1.94 (2H, quintet,
J = 7.4 Hz, PhCH2CH2CH2CHO), 2.02 (3H, s,
NHCOCH3), 2.02 (2H, m, PhCH2CH2C), 2.44 (2H, dt,
J = 7.4 Hz and 2.0 Hz, Ph(CH2)2CH2CHO), 2.49–2.53
(2H, m, PhCH2CH2C), 2.63 (2H, t, J = 7.4 Hz,
PhCH2(CH2)2CHO), 3.69, 3.97 (each 2H, d,
J = 12.0 Hz, (CH2O)2C(CH3)2), 5.69 (1H, br s, NH),
7.07, 7.10 (each 2H, d, J = 8.3 Hz, ArH), 9.74 (1H, t,
J = 2.0 Hz, CHO); EI-MS m/z: 347 ([M]+); Anal. Calcd
for C20H29NO4: C, 69.14; H, 8.41; N, 4.03. Found: C,
68.88; H, 8.22; N, 3.81.
5.1.9. N-(5-(2-(4-(8-Hydroxyoctyl)phenyl)ethyl)-2,2-di-
methyl-1,3-dioxan-5-yl)ethanamide (13). To a stirred
solution of 12 (0.26 g, 5.8· 10ꢀ4 mol) in dried Et2O
(80 mL) was added lithium aluminum hydride (72 mg,
1.9· 10ꢀ3 mol) at room temperature for 2 h. The reac-
tion mixture was treated in a similar manner to that used
for the preparation of 10 to give 13 (0.17 g, 72%): color-
less needles (recrystallized from n-hexene–Et2O); mp 86–
1
88 ꢁC; IR mmax (CHCl3) cmꢀ1: 1680 (CONH). H NMR
d: 1.29–1.35 (8H, m, Ph(CH2)2(CH2)4(CH2)2OH),
1.42 (6H, s, C(CH3)2), 1.53–1.60 (4H, m,
PhCH2CH2(CH2)4CH2CH2–), 2.01 (3H, s, NHCOCH3),
2.03–2.07 (2H, m, PhCH2CH2C), 2.49–2.53 (2H, m,
PhCH2CH2C), 2.54 (2H, t, J = 7.4 Hz, PhCH2(CH2)7–),
3.62–3.65 (2H, m, Ph(CH2)7CH2O H), 3.67, 3.98 (each
2H, d, J = 12.0 Hz, (CH2O)2C(CH3)2), 5.71 (1H, br s,
NH), 7.07 (4H, br s, ArH); EI-MS m/z: 405 ([M]+); Anal.
Calcd for C20H31NO4: C, 71.07; H, 9.69; N, 3.45. Found:
C, 70.89; H, 9.40; N, 3.26.
5.1.8. Ethyl 8-(4-(2-(5-(acetylamino)-2,2-dimethyl-1,3-di-
oxan-5-yl)ethyl)phenyl)octanoate (12). To a stirred solu-
tion of triphenylphosphine (5.0 g, 1.9· 10ꢀ2 mol) in
dried benzene (5 mL) was added ethyl 4-bromocroto-
nate (4.83 mL, 2.6· 10ꢀ2 mol) under an argon atmo-
sphere, and the mixture was stirred at room
temperature for 4 h. The precipitate was washed with
dried benzene (2· 10 mL) followed with CHCl3
(2· 10 mL) and recrystallized from CH3CN to give
(3-(ethoxycarbonyl)-2-propenyl)triphenylphosphonium
bromide (7.2 g, 83%) as a white powder. To a suspen-
sion of 60% sodium hydride (24 mg, 5.8· 10ꢀ4 mol) in
dried DMF (6 mL) was added (3-ethoxycarbonylallyl-
5.1.10. 4-(4-(2-(5-(Acetylamino)-2,2-dimethyl-1,3-dioxan-
5-yl)ethyl)phenyl)butyl 4-methylbenzenesulfonate (14).
To a stirred solution of 10 (0.45 g, 1.3· 10ꢀ3 mol) in
dried CH2Cl2 (5 mL) and dried Et3N (0.36 mL,
2.6· 10ꢀ3 mol) was added p-toluenesulfonyl chloride
(0.30 g, 1.6· 10ꢀ3 mol) under ice cooling. The reaction
mixture was stirred at room temperature for 13 h and
concentrated. To the residue was added ice water
(5 mL), and the whole mixture was extracted with
AcOEt (20 mL). The extract was washed with brine
(1 mL), dried, and evaporated. The residue was chro-
matographed on silica gel with n-hexane–AcOEt (1:3)
to give 14 (0.49 g, 75%) as a white powder: mp 123–
124 ꢁC; IR mmax (CHCl3) cmꢀ1: 1675 (CONH); 1H
NMR d: 1.42 (6H, s, C(CH3)2), 1.60–1.65 (4H, m,
PhCH2(CH2)2CH2–), 2.02 (3H, s, NHCOCH3), 2.02–
2.07 (2H, m, PhCH2CH2C), 2.44 (3H, s, PhCH3),
2.48–2.53 (2H, m, PhCH2CH2C), 2.60 (2H, t,
J = 7.5 Hz, PhCH2(CH2)3–), 3.67, 3.97 (each 2H, d,
J = 12.0 Hz, (CH2O)2C(CH3)2), 4.02 (2H, t, J = 6.0 Hz,
CH2OTs), 5.73 (1H, br s, NH), 7.00, 7.07 (each 2H, d,
J = 8.0 Hz, ArH), 7.33, 7.78 (each 2H, d, J = 8.0 Hz,
ArH); EI-MS m/z: 503 ([M]+).
propyl)triphenylphosphonium
bromide
(0.28 g,
6.3· 10ꢀ4 mol) at ꢀ25 ꢁC under an argon atmosphere
and the mixture was stirred for 10 min. To the mixture
was added a solution of 11 (0.20 g, 5.8· 10ꢀ4 mol) in
dried DMF (5 mL) and the mixture was stirred at the
same temperature for 0.5 h. The reaction mixture was
poured into ice water (20 mL) and extracted with Et2O
(30 mL). The extract was washed with brine (3 mL),
dried, and evaporated. The residue was chromato-
graphed on silica gel with Et2O to give ethyl 8-(4-
(2-(5-(acetylamino)-2,2-dimethyl-1,3-dioxan-5-yl)ethyl)-
phenyl)-2,4-octadienoate (0.13 g, 52%) as a yellow oil. A
solution of ethyl 8-(4-(2-(5-(acetylamino)-2,2-dimethyl-
1,3-dioxan-5-yl)ethyl)phenyl)-2,4-octadienoate (0.51 g,
1.1· 10ꢀ3 mol) in dried AcOEt (4 mL) was stirred under
a hydrogen atmosphere in the presence of 10% Pd–C
(0.10 g) for 2 h. The catalyst was removed by filtration
and the filtrate was evaporated to give 12 (0.49 g,
99%) as a pale yellow oil: IR mmax (CCl4) cmꢀ1: 1720
(COOEt), 1675 (CONH); 1H NMR d: 1.25 (3H, t,
J = 7.1 Hz, CO2CH2CH3), 1.24–1.31 (8H, m,
Ph(CH2)2(CH2)4CH2–), 1.42 (6H, s, C(CH3)2), 1.94
(2H, quintet, J = 7.4 Hz, PhCH2CH2(CH2)5–), 2.01
(3H, s, NHCOCH3), 2.03–2.07 (2H, m, PhCH2CH2C),
2.20 (2H, t, J = 7.4 Hz, CH2CO2Et), 2.48–2.53 (2H, m,
5.1.11. 8-(4-(2-(5-(Acetylamino)-2,2-dimethyl-1,3-dioxan-
5-yl)ethyl)phenyl)octyl 4-methylbenzenesulfonate (15). To
a stirred solution of 13 (0.16 g, 3.9· 10ꢀ4 mol) in dried
CH2Cl2 (2.5 mL) and dried Et3N (0.11 mL,
1.9· 10ꢀ4 mol) was added p-toluenesulfonyl chloride
(89 mg, 4.5· 10ꢀ4 mol) under ice cooling, and the reac-
tion mixture was stirred at room temperature for 15 h.
The reaction mixture was treated in a similar manner
to that used for the preparation of 14 to give 15
(0.18 g, 81%): mp 61–63 ꢁC; IR mmax (CHCl3) cmꢀ1
:
1180, 1190 (SO2), 1680 (CONH); 1H NMR d:
1.20–1.32 (8H, m, Ph(CH2)2(CH2)4(CH2)2OTs),

N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
4189
1.42 (6H, s, C(CH3)2), 1.52–1.66 (4H, m,
PhCH2CH2(CH2)4CH2CH2–), 2.01 (3H, s, NHCOCH3),
2.03–2.07 (2H, m, PhCH2CH2C), 2.44 (3H, s, PhCH3),
2.49–2.53 (2H, m, PhCH2CH2C), 2.53 (2H, t,
J = 7.4 Hz, PhCH2(CH2)7–), 3.67, 3.98 (each 2H, d,
J = 12.0 Hz, (CH2O)2C(CH3)2), 4.01 (2H, t, J = 6.4 Hz,
CH2OTs), 5.71 (1H, br s, NH), 7.06, 7.08 (each 2H, d,
J = 8.0 Hz, ArH), 7.34, 7.79 (each 2H, d, J = 8.0 Hz,
ArH); EI-MS m/z: 559 ([M]+); HR-MS: Anal. Calcd
for C31H45NO6S: 559.2967. Found: 559.2967.
2.23 (1H, t, J = 8.0 Hz, SH), 2.46–2.51 (4H, m,
PhCH2(CH2)2CH2SH), 2.53–2.59 (2H, m,
PhCH2CH2C), 3.52 (4H, d, J = 5.0 Hz, CH2OH 2·),
5.36 (2H, t, J = 5.0 Hz, CH2OH 2·), 7.11 (4H, br s,
ArH), 7.82 (3H, br s, NH3+); FAB-MS (positive) m/z:
284 ([M+HꢀHCl]+); Anal. Calcd for C15H26NO2SCl:
C, 56.32; H, 8.19; N, 4.38. Found: C, 56.37; H, 8.09;
N, 4.49.
5.1.15.
2-Amino-2-(2-(4-(8-mercaptooctyl)phenyl)eth-
yl)propane-1,3-diol hydrochloride (4). To a solution of
17 (60 mg, 1.3· 10ꢀ4 mol) in EtOH (2 mL) was added
10% HCl (1.0 mL) under an argon atmosphere and the
mixture was refluxed for 5 h. The reaction mixture was
concentrated to give 4 (31 mg, 62%) as a powder: white
powder (recrystallized from MeOH–Et2O); mp 97–
5.1.12. S-(4-(4-(2-(5-(Acetylamino)-2,2-dimethyl-1,3-di-
oxan-5-yl)ethyl)phenyl)butyl)ethanethioate (16). To a
solution of 14 (0.45 g, 8.9· 10ꢀ4 mol) in dried EtOH
(10 mL) was added 90% potassium thioacetate (0.13 g,
1.0· 10ꢀ3 mol) under an argon atmosphere. The mixture
was refluxed for 1 h and concentrated. To the residue
was added ice water (5 mL) and the whole mixture
was extracted with AcOEt (20 mL). The extract was
washed with brine (1 mL), dried, and evaporated. The
residue was chromatographed on silica gel with n-hex-
ane–AcOEt (1:1) to give 16 (0.29 g, 80%) as a white
powder: mp 77.5–79 ꢁC; 1H NMR d: 1.42 (6H, s,
C(CH3)2), 1.58–1.66 (4H, m, PhCH2(CH2)2CH2–), 2.01
(3H, s, NHCOCH3), 2.03–2.07 (2H, m, PhCH2CH2C),
2.32 (3H, s, SCOCH3), 2.49–2.53 (2H, m, PhCH2CH2C),
2.57 (2H, t, J = 7.5 Hz, PhCH2(CH2)3–), 2.88 (2H, t,
J = 7.0 Hz, CH2SAc), 3.67, 3.98 (each 2H, d,
J = 11.0 Hz, (CH2O)2C(CH3)2), 5.71 (1H, br s, NH),
7.05, 7.08 (each 2H, d, J = 8.5 Hz, ArH); EI-MS m/z:
407 ([M]+).
1
104 ꢁC (dec); H NMR (DMSO-d6) d: 1.23–1.35 (8H,
m, Ph(CH2)2(CH2)4(CH2)2SH), 1.48–1.56 (4H, m,
PhCH2CH2(CH2)4CH2CH2–), 1.75–1.80 (2H, m,
PhCH2CH2C), 2.20 (1H, t, J = 7.4 Hz, SH), 2.45 (2H,
q, J = 7.4 Hz, Ph(CH2)7CH2SH), 2.50–2.54 (2H, m,
PhCH2(CH2)7–), 2.55–2.60 (2H, m, PhCH2CH2C), 3.52
(4H, d, J = 5.0 Hz, CH2OH 2·), 5.37 (2H, t,
J = 5.0 Hz, CH2OH 2·), 7.10 (4H, br s, ArH), 7.83
(3H, br s, NH3+); FAB-MS (positive) m/z: 340
([M+HꢀHCl]+).
5.2. Biological procedures
5.2.1. Buffers. The regularly used buffers were 10 mmol/
sodium phosphate buffer, pH 7.0, containing
L
0.1 mmol/L NaCl (buffer A); buffer A containing 1 g/L
bovine serum albumin (BSA, Nacalai Tesque, Kyoto,
Japan) (buffer B); 0.1 mol/L sodium phosphate buffer,
pH 6.0, containing 5 mmol/L EDTA (buffer C).
5.1.13. S-(8-(4-(2-(5-(Acetylamino)-2,2-dimethyl-1,3-di-
oxan-5-yl)ethyl)phenyl)octyl)ethanethioate (17). To a
solution of 15 (0.18 g, 3.2· 10ꢀ4 mol) in dried EtOH
(10 mL) was added 90% potassium thioacetate (44 mg,
9.5· 10ꢀ5 mol) under an argon atmosphere. The reac-
tion mixture was treated in a similar manner to that used
for the preparation of 16 to give 17 (0.14 g, 94%): mp
33–35 ꢁC; IR mmax (CHCl3) cmꢀ1: 1690, 1680 (CONH,
5.2.2. Measurement of thiol groups38. The concentration
of thiol groups was calculated from the absorbance at
324 nm (e: 19,800) of pyridine-4-thione, which is the
reduction product of 4,40-dithiodipyridine.
COS).
1H
NMR
d:
1.23–1.35
(8H,
m,
Ph(CH2)2(CH2)4(CH2)2SAc), 1.42 (6H, s, C(CH3)2),
1.52–1.59 (4H, m, PhCH2CH2(CH2)4CH2CH2–), 2.01
(3H, s, NHCOCH3), 2.03–2.07 (2H, m, PhCH2CH2C),
2.32 (3H, s, SCOCH3), 2.49–2.53 (2H, m, PhCH2CH2C),
2.54 (2H, t, J = 7.4 Hz, PhCH2(CH2)7–), 2.86 (2H, t,
J = 7.4 Hz, CH2SAc), 3.67, 3.98 (each 2H, d,
J = 12.0 Hz, (CH2O)2C(CH3)2), 5.71 (1H, br s, NH),
7.08 (4H, br s, ArH); EI-MS m/z: 463 ([M]+); HR-MS:
Anal. Calcd for C26H41NO4S: 463.2756. Found:
463.2762.
5.2.3. Measurement of maleimide groups38. Samples
including maleimide group were incubated with a
known amount of 2-mercaptoethylamine hydrochloride
and the remaining thiol groups was measured as above.
The amount of maleimide groups was calculated from
the difference between the former and the latter.
5.2.4. 3-Protein conjugate and 4-protein conjugate
5.2.4.1. Maleimide-protein. Maleimide groups were
introduced into ovalbumin (OVA, Nacalai Tesque,
Kyoto, Japan), BSA, or horseradish peroxidase (HRP,
Boehringer Mannheim GmbH, Mannheim, Germany)
using e-maleimidocaproic acid N-hydroxysuccinimide
ester (EMCS, Dojindo Laboratories, Kumamoto,
Japan). The protein solution (10 mg/mL, 0.6 mL) in
0.1 mol/L sodium phosphate buffer, pH 7.0, was incu-
bated with EMCS (30–80 mmol/L, 0.06 mL) in DMF
at 30 ꢁC for 30 min. The reaction mixture was subjected
to gel filtration on a column of Sephadex G-25 (Pharma-
cia LKB Biotechnology, Uppsala, Sweden) with buffer C
or centrifuged column procedure using Sephadex G-50
(Pharmacia LKB Biotechnology) with buffer C to give
5.1.14.
2-Amino-2-(2-(4-(4-mercaptobutyl)phenyl)eth-
yl)propane-1,3-diol hydrochloride (3). To a solution of
16 (77 mg, 1.9· 10ꢀ4 mol) in EtOH (2 mL) was added
10% HCl (1.5 mL) under an argon atmosphere and the
mixture was refluxed for 8 h. The reaction mixture was
concentrated to give 3 (60 mg, 99%): colorless plates
(recrystallized from MeOH–Et2O); mp 105–113 ꢁC
(dec); IR mmax (KBr) cmꢀ1: 3550–3150 (OH), 3150–
2400 ðNH3þÞ; 1H NMR (DMSO-d6) d: 1.51–1.55
(2H, m, Ph(CH2)2CH2CH2–), 1.61–1.63 (2H, m,
PhCH2CH2(CH2)2–), 1.76–1.79 (2H, m, PhCH2CH2C),

4190
N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
maleimide-protein. The concentration of maleimide-
protein was calculated from the absorbance at 280 nm
(OVA: extinction coefficient, 0.74 gꢀ1 L cmꢀ1, Mr (rela-
tive molecular mass), 45,000, BSA: extinction coeffi-
cient, 0.63 gꢀ1 L cmꢀ1, Mr, 66,200) or 403 nm (HRP:
extinction coefficient, 2.275 gꢀ1 L cmꢀ1, Mr, 40,000).
5.2.4.5. 4-BSA conjugate. Compound 4 (1.4 mg) was
dissolved in MeOH (0.37 mL). The maleimide-BSA
(0.25 mL, 26.5 nmol), in which the average number of
maleimide groups introduced per BSA molecule had
been 2.0, was diluted with a mixture of ice MeOH
(0.32 mL) and buffer C (0.58 mL). The diluted malei-
mide-BSA was incubated with an aliquot (32.6 lL) of
the 4 solution (134 nmol as thiol group) at 4 ꢁC over-
night. The reaction mixture was centrifuged at
3000 rpm for 10 min. The supernatant (0.89 mL) was
incubated with 0.01 mol/L 2-mercaptoethylamine
hydrochloride in buffer C (87.4 lL) at 30 ꢁC for 15 min
and then with 0.01 mol/L N-ethylmaleimide in buffer C
(184.1 lL) at 30 ꢁC for 15 min. The mixture was dia-
lyzed against buffer A (1 L) overnight to give 4-BSA
conjugate. The average number of 4 moieties conjugated
per BSA molecule was 2.0, as calculated from the de-
crease in the number of maleimide groups. The amount
of the 4-BSA conjugate obtained was 0.54 mg.
5.2.4.2. 3-OVA conjugate. Compound 3 (1.2 mg) was
dissolved in buffer C (3.0 mL) and passed through a Mil-
lipore filter (0.45 lm). The maleimide-OVA (0.484 mL,
66.8 nmol), in which the average number of maleimide
groups introduced per OVA molecule had been 8.4,
was incubated with an aliquot (1.48 mL) of the 3 solu-
tion (666 nmol as thiol group) at 4 ꢁC for 20 h. The reac-
tion mixture was further incubated with 0.1 mol/L
2-mercaptoethylamine hydrochloride in buffer
C
(18.6 lL) at 30 ꢁC for 15 min and subjected to gel filtra-
tion on a column (1· 30 cm) of Sephadex G-25 with
buffer A to give 3-OVA conjugate. The average number
of 3 moieties conjugated per OVA molecule was 6.8, as
calculated from the decrease in the number of maleimide
groups. The amount of the 3-OVA conjugate obtained
was 2.27 mg.
5.2.4.6. HRP-labeled 3. Compound 3 (0.5 mg) was
dissolved in water (0.5 mL). The maleimide-HRP
(0.566 mL, 79.8 nmol), in which the average number of
maleimide groups introduced per HRP molecule had
been 0.93, was incubated with an aliquot (1.47 mL) of
the 3 solution (516 nmol as thiol group) at room temper-
ature for 1 h. After incubation, the reaction mixture
(0.6 mL) was subjected to the centrifuged column proce-
dure using Sephadex G-50 and buffer C. The filtrate
(0.784 mL) was incubated with 0.1 mol/L 2-mercapto-
ethylamine hydrochloride in buffer C (7.84 lL) at room
temperature for 15 min and dialyzed against buffer A
(500 mL) for two days. The average number of 3 moie-
ties conjugated per HRP molecule was 1.0, as calculated
from the decrease in the number of maleimide groups.
The amount of the HRP-labeled 3 obtained was
1.93 mg.
5.2.4.3. 4-OVA conjugate. Compound 4 (1.6 mg) was
dissolved in MeOH (0.43 mL). The maleimide-OVA
(3.3 mL, 347 nmol), in which the average number of
maleimide groups introduced per OVA molecule had
been 4.8, was diluted with a mixture of ice MeOH
(10.0 mL) and buffer C (20.5 mL). The diluted malei-
mide-OVA was incubated with an aliquot (0.23 mL) of
the 4 solution (690 nmol as thiol group) at 4 ꢁC over-
night. The reaction mixture was centrifuged at
7000 rpm for 10 min. The supernatant (30.0 mL) was
incubated with 0.25 mmol/L 2-mercaptoethylamine
hydrochloride in buffer C (80.0 lL) at 30 ꢁC for 15 min
and then with 0.5 mmol/L N-ethylmaleimide in buffer
C (0.16 mL) at 30 ꢁC for 15 min. The reaction mixture
was dialyzed against buffer A (1 L) overnight and sub-
jected to gel filtration on a column (1· 30 cm) of Sepha-
dex G-25 with buffer A to give 4-OVA conjugate. The
4-OVA conjugate was concentrated in an ultrafiltration
apparatus (Amicon 8010) using a Diaflo Ultrafiltration
Membrane PM-10 (Amicon, Inc., Bervery, MA, USA).
The average number of 4 moieties conjugated per
OVA molecule was 2.4, as calculated from the decrease
in the number of maleimide groups. The amount of the
4-OVA conjugate obtained was 3.20 mg.
5.2.4.7. HRP-labeled 4. Compound 4 (0.4 mg) was
dissolved in 4 mmol/L b-cyclodextrin (Wako Pure
Chemical Industries, Osaka, Japan) in buffer
C
(0.5 mL). The maleimide-HRP (0.20 mL, 48 nmol), in
which the average number of maleimide groups intro-
duced per HRP molecule had been 0.66, was incubated
with an aliquot (0.718 mL) of the 4 solution (240 nmol
as thiol group) at 4 ꢁC for two days. The reaction mix-
ture was incubated with 0.1 mol/L 2-mercaptoethyl-
amine hydrochloride in buffer C (19 lL) at 30 ꢁC for
15 min and then with 0.1 mol/L N-ethylmaleimide in
buffer C (38 lL) at 30 ꢁC for 15 min. The mixture was
subjected to gel filtration on a column (1· 30 cm) of
Sephadex G-25 with buffer A to give HRP-labeled 4.
The average number of 4 moieties conjugated per
HRP molecule was 0.66, as calculated from the decrease
in the number of maleimide groups. The amount of the
HRP-labeled 4 obtained was 1.40 mg.
5.2.4.4. 3-BSA conjugate. The maleimide-BSA
(0.45 mL, 45.5 nmol), in which the average number of
maleimide groups introduced per BSA molecule had
been 10.1, was incubated with an aliquot (1.01 mL) of
the 3 solution (666 nmol as thiol group) used in Section
5.2.4.2 at 4 ꢁC for 20 h. The reaction mixture was fur-
ther incubated with 0.1 mol/L 2-mercaptoethylamine
hydrochloride in buffer C (14.6 lL) at 30 ꢁC for 15 min
and subjected to gel filtration on a column (1· 30 cm)
of Sephadex G-25 using buffer A. The average number
of 3 moieties conjugated per BSA molecule was 9.6, as
calculated from the decrease in the number of maleimide
groups. The amount of the 3-BSA conjugate obtained
was 2.09 mg.
5.2.5. Antisera. Three rabbits (Japanese White, 2.7–
2.8 kg, female) were intracutaneously injected 3 times
with the 3-OVA conjugate (0.1 mg) or the 4-OVA conju-
gate (0.1 mg) in Freund’s complete adjuvant at three-
week intervals. Blood was collected a week after the last
injection, and the antisera were stored at ꢀ20 ꢁC.

N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
4191
5.2.6. Rabbit (anti-3-OVA) IgG, rabbit (anti-4-OVA)
IgG, and rabbit (anti-3-OVA) Fab0. IgG, F(ab0)2 and
Fab0 were prepared by Ishikawa’s method.38 The
amounts of rabbit (anti-3-OVA) IgG and rabbit (anti-
4-OVA) IgG obtained were 23.1 and 25.9 mg, respec-
tively, from 10 mL of the corresponding serum. The
amount of rabbit (anti-3-OVA) Fab0 obtained was
1.5 mg from 5.0 mg of the corresponding IgG.
5.2.9. Epitope analysis and test for cross-reactivity using a
competitive EIA (method A). Rabbit (anti-3-OVA) IgG
(0.1 mg/mL) in 0.1 mol/L sodium phosphate buffer, pH
7.5, containing 1 g/L NaN3 was diluted 100-fold with
normal rabbit IgG (0.1 mg/mL) in the same buffer.
The diluted rabbit (anti-3-OVA) IgG (0.15 mL) was
added to a microtiter plate and incubated at room tem-
perature for 2 h. After incubation, the diluted rabbit
(anti-3-OVA) IgG was removed and the plate was
washed four times with buffer A (0.2 mL). The washed
plate was incubated with buffer B (0.2 mL) at room tem-
perature for 2 h. An aliquot (0.075 mL) of HRP-labeled
3 (0.2 pmol) in buffer B was mixed with compound 1
(0.1, 0.3, 1, 3, and 10 pmol), 3, 4 or 18–31 (0.3, 3, or
30 pmol) in buffer B (0.075 mL), and the mixture was
added to the plate and incubated at room temperature
for 3 h. After incubation, the mixture was removed
and the plate was washed four times with ice-cold buffer
A (0.25 mL). The HRP activity bound to the plate was
assayed as described in Section 5.2.8.1. Epitope analysis
and test for cross-reactivity of rabbit (anti-4-OVA) IgG
were performed in a similar manner to that used for rab-
bit (anti-3-OVA) IgG.
5.2.7. HRP-labeled rabbit (anti-3-OVA) Fab0. The malei-
mide-HRP (0.398 mL, 32.6 nmol), in which the average
number of maleimide groups introduced per HRP mol-
ecule had been 1.85, was incubated with rabbit (anti-3-
OVA) Fab0 (32.9 nmol) in buffer C (0.54 mL) at 4 ꢁC
overnight. The reaction mixture was subjected to gel fil-
tration on a column (1.5· 45 cm) of Ultrogel AcA 44
(Bio Sepra S.A. Villeneuve la Garenne Cedex, France)
using 0.1 mol/L sodium phosphate buffer, pH 7.0, to
give HRP-labeled rabbit (anti-3-OVA) Fab0. The aver-
age number of rabbit (anti-3-OVA) Fab0 molecules con-
jugated per HRP molecule was 0.82, as calculated from
the absorbance at 280 and at 403 nm.38 The amount of
the HRP-labeled rabbit (anti-3-OVA) Fab0 obtained
was 1.8 mg.
5.2.10. Competitive EIA for 1
5.2.8. Antibody response
5.2.10.1. Alkaline-chloroform extraction of 1 from
serum. The sample (0.4 mL) containing 1 was mixed
with 1 N NaOH (0.1 mL) and water (0.5 mL). CHCl3
(5 mL) was added to the mixture and the whole mixture
was shaken for 10 min. After shaking, the mixture was
centrifuged at 3000 rpm for 10 min. The CHCl3 layer
was transferred to two glass test tubes (1.5 mL each).
The extract was evaporated to dryness at 40 ꢁC using
a centrifugal concentrator (Taitec Co., Saitama, Japan).
The residue was subjected to a competitive EIA as de-
scribed in Section 5.2.10.2.
5.2.8.1. Anti-3 IgG. Before and after (7, 10, 22, 26, 31,
and 41 days) administration of 3-OVA conjugate, blood
samples were collected from an ear into plastic tubes,
and the sera (approx. 0.1 mL) were stored at ꢀ20 ꢁC.
The 3-BSA conjugate (0.01 mg/mL) in 0.1 mol/L sodium
phosphate buffer, pH 7.5, containing 1 g/L NaN3
(0.15 mL) was added to a microtiter plate (Sanko Jun-
yaku, Tokyo, Japan) and incubated at 4 ꢁC overnight.
The 3-BSA conjugate was removed by aspiration and
the plate was washed four times with buffer A (0.2 mL).
The washed plate was incubated with buffer B containing
1 mmol/L MgCl2 and 1 g/L NaN3 (0.2 mL) at 37 ꢁC for
2 h, and the buffer was removed. The serum samples
(0.15 mL), which had been diluted 105-fold with buffer
B, were added to the plate and incubated at 37 ꢁC for
2 h. The serum samples were removed and the plate was
washed four times with buffer A (0.2 mL). To the plate
was added HRP-labeled goat (anti-rabbit IgG) Fab0
(5 ng) in buffer B (0.15 mL), and incubation was contin-
ued at 37 ꢁC for 1 h. After incubation, HRP-labeled goat
(anti-rabbit IgG) Fab0 was removed and the plate was
washed four times with buffer A (0.25 mL). HRP activity
bound to the plate was measured by incubating the plate
with 7.5 mmol/L o-phenylenediamine (Nacalai Tesque,
Kyoto, Japan) in 50 mmol/L sodium acetate buffer, pH
5.0, containing 0.25 mg/mL BSA, 5 mmol/L EDTA,
and 0.025% hydrogen peroxide (0.1 mL) at 37 ꢁC for
10 min. The enzyme reaction was terminated by addition
of 1.2 mol/L H2SO4 containing 2.4 g/L Na2SO3
(0.05 mL), and the absorbance at 490 nm was measured
using a microplate reader.
5.2.10.2. Competitive EIA (method B). 3-BSA conju-
gate (0.01 mg/mL) in 0.1 mol/L sodium phosphate buff-
er, pH 7.5, containing 1 g/L NaN3 was diluted 300-fold
with BSA (0.01 mg/mL) in the same buffer. The diluted
3-BSA conjugate (0.15 mL) was added to a microtiter
plate and incubated at 4 ꢁC overnight. The diluted 3-
BSA conjugate was removed and the plate was washed
four times with buffer A (0.2 mL). The washed plate
was incubated with buffer B (0.2 mL) at room tempera-
ture for 2 h. Buffer B was removed from the plate prior
to use. The residue containing 1 was dissolved in
10 mmol/L sodium phosphate buffer, pH 8.5, containing
0.1 mol/L NaCl and 1 g/L BSA (0.12 mL), and incubated
with an aliquot (0.06 mL) of the HRP-labeled rabbit
(anti-3-OVA) Fab0 (60 fmol) in the same buffer at 4 ꢁC
overnight. After incubation, an aliquot (0.15 mL) of
the reaction mixture was added to the plate and was incu-
bated at 4 ꢁC for 6 h. The reaction mixture was removed
and the plate was washed four times with ice-cold
10 mmol/L sodium phosphate buffer, pH 8.5, containing
0.1 mol/L NaCl (0.25 mL). The HRP activity bound to
the plate was assayed as described in Section 5.2.8.1.
5.2.8.2. Anti-4 IgG. Before and after (7, 14, 22, 35, and
43 days) administration of 4-OVA conjugate, blood sam-
ples were collected, and anti-4 IgG in serum was mea-
sured in a similar manner to that used for anti-3 IgG.
5.2.11. Measurement of bioavailability. Wistar rats
(10-weeks-old, male, Japan SLC, Inc., Shizuoka, Japan)
were fasted, but allowed free access to water overnight

4192
N. Matsumoto et al. / Bioorg. Med. Chem. 14 (2006) 4182–4192
17. Chiba, K.; Hoshino, Y.; Suzuki, C.; Masubuchi, Y.;
Yanagawa, Y.; Ohtsuki, M.; Sasaki, S.; Fujita, T. Trans-
plant. Proc. 1996, 28, 1056–1059.
18. Chiba, K.; Yanagawa, Y.; Masubuchi, Y.; Kataoka, H.;
Kawaguchi, T.; Ohtsuki, M.; Hoshino, Y. J. Immunol.
1998, 160, 5037–5044.
19. Chiba, K.; Yanagawa, Y.; Masubuchi, Y.; Kataoka, H.;
Kawaguchi, T.; Ohtsuki, M.; Hoshino, Y. J. Immunol.
1998, 160, 5493–5499.
20. Pinschewer, D. D.; Ochsenbein, A. F.; Odermatt, B.;
Brinkmann, V.; Hengartner, H.; Zinkernagel, R. M.
J. Immunol. 2000, 164, 5761–5770.
before all experiments. Compound 1 (1 mg/mL) in water
was administered orally at a dose of 1 mg/kg without
anesthesia. Before and after (2, 4, 6, 8, 10, 12, 24, 48,
and 96 h) administration, blood samples (approx.
0.3 mL) were collected from a tail vein through a hepa-
rinized needle into plastic tubes (1.5 mL), and the sera
(approx. 0.1 mL) were stored at ꢀ20 ꢁC. The sera were
diluted 8-fold with water and subjected to the competi-
tive EIA for 1.
21. Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.;
Xie, J.; Milligan, J.; Thornton, R.; Shei, G.-J.; Card, D.;
Keohane, C.; Rosenbach, M.; Hale, J.; Lynch, C. L.;
Rupprecht, K.; Parsons, W.; Rosen, H. Science 2002, 296,
346–349.
22. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.;
Cottens, S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker,
T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K.
R. J. Biol. Chem. 2002, 277, 21453–21457.
Acknowledgments
This work was supported in part by a Grant-in Aid for
Scientific Research (No. 13672331) from the Ministry of
Education, Culture, Sports, Science, and Technology,
Japan. The authors are grateful to Yoshitomi Pharma-
ceutical Industries, Ltd. (present name: Mitsubishi Phar-
ma Corporation) for the gift of compounds 21–27.
23. Matloubian, M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.;
Xu, Y.; Brinkmann, V.; Allende, M. L.; Proia, R. L.;
Cyster, J. G. Nature 2004, 427, 355–360.
References and notes
24. Hoshino, Y.; Suzuki, C.; Ohtsuki, M.; Masubuchi, Y.;
Amano, Y.; Chiba, K. Transplant. Proc. 1996, 28, 1060–
1061.
25. Kawaguchi, T.; Hoshino, Y.; Rahman, F.; Amano, Y.;
Higashi, H.; Kataoka, H.; Ohtsuki, M.; Teshima, K.;
Chiba, K.; Kakefuda, T.; Suzuki, S. Transplant. Proc.
1996, 28, 1062–1063.
26. Suzuki, S.; Enosawa, S.; Kakefuda, T.; Amemiya, H.;
Hoshino, Y.; Chiba, K. Transplant. Proc. 1996, 28, 1375–
1376.
27. Hoshino, Y.; Yanagawa, Y.; Ohtsuki, M.; Nakayama, S.;
Hashimoto, T.; Chiba, K. Transplant. Proc. 1999, 31,
1224–1226.
28. Matsuura, M.; Imayoshi, T.; Okumoto, T. Int. J. Immu-
nopharmacol. 2000, 22, 323–331.
29. Kohno, T.; Tsuji, T.; Hirayama, K.; Watabe, K.; Mat-
sumoto, A.; Kohno, T.; Fujita, T. Biol. Pharm. Bull. 2004,
27, 1392–1396.
30. Kohno, T.; Tsuji, T.; Hirayama, K.; Iwatsuki, R.; Hirose,
M.; Watabe, K.; Yoshikawa, H.; Kohno, T.; Matsumoto,
A.; Fujita, T.; Hayashi, M. Biol. Pharm. Bull. 2005, 28,
736–739.
1. Borel, J. F. Pharmacol. Rev. 1990, 41, 259–371.
2. Kino, T.; Hatanaka, H.; Hashimoto, M.; Nishiyama, M.;
Goto, T.; Okuhara, M.; Aoki, H.; Imanaka, H. J.
Antibiot. 1987, 40, 1249–1255.
3. Tanaka, H.; Kuroda, A.; Marusawa, H.; Hatanaka, H.;
Kino, T.; Goto, T.; Hashimoto, M.; Taga, T. J. Am.
Chem. Soc. 1987, 109, 5031–5033.
4. Peters, D. H.; Fitton, A.; Plosker, G. L.; Faulds, D. Drugs
1993, 46, 746–794.
5. Schreiber, S. L.; Liu, J.; Albers, M. W.; Karmacharya, R.;
Koh, E.; Martin, P. K.; Rosen, M. K.; Standaert, R. F.;
Wandless, T. J. Transplant. Proc. 1991, 23, 2839–2844.
6. Ryffel, B.; Donatsch, P.; Madorin, M.; Matter, B. E.;
Ruttimann, G.; Schon, H.; Stoll, R.; Wilson, J. Arch.
Toxicol. 1983, 53, 107–141.
7. McCauley, J. Curr. Opin. Nephrol. Hypertens. 1993, 2,
662–669.
8. Fujita, T.; Inoue, K.; Yamamoto, S.; Ikumoto, T.; Sasaki,
S.; Toyama, R.; Chiba, K.; Hoshino, Y.; Okumoto, T.
J. Antibiot. 1994, 47, 208–215.
9. Kluepfel, D.; Bagli, J.; Baker, H.; Charest, M.-P.; Kudel-
ski, A.; Sehgal, S. N.; Vezina, C. J. Antibiot. 1972, 25, 109–
115.
10. Bagli, J. F.; Kluepfel, D.; St-Jacques, M. J. Org. Chem.
1973, 38, 1253–1260.
31. Budde, K.; Schmouder, R. L.; Brunkhorst, R.; Nashan,
B.; Lucker, P. W.; Mayer, T.; Choudhury, S.; Skerjanec,
¨
A.; Kraus, G.; Neumayer, H. H. J. Am. Soc. Nephrol.
2002, 13, 1073–1083.
32. Fujita, T.; Matsumoto, N.; Uchida, S.; Kohno, T.;
Shimizu, T.; Hirose, R.; Yanada, K.; Kurio, W.; Watabe,
K. Bioorg. Med. Chem. Lett. 2000, 10, 337–339.
33. Hashida, S.; Imagawa, M.; Inoue, S.; Ruan, K.-h.;
Ishikawa, E. J. Appl. Biochem. 1984, 6, 56–63.
34. Haw, J. C. H.; Fleming, W. A. J. Org. Chem. 1957, 22,
1106–1107.
35. Momose, D.; Satoh, M.; Nonaka, Y.; Isawa, H.; Yanagi,
N.; Takehana, Y. Jpn. Patent 93-155995; Chem. Abstr.
1995, 123, 198841.
36. Funakoshi, T.; Etou, K.; Nishimine, H.; Hirose, R.; Miura,
S.; Matsuki, H. Jpn. Pharmacol. Ther. 1998, 26, 287–303.
37. Moore, J. D.; Valette, G.; Darque, A.; Zhou, X. J.;
Sommadossi, J. P. J. Am. Soc. Mass Spectrom. 2000, 11,
1134–1143.
38. Ishikawa, E.; Imagawa, M.; Hashida, S.; Yoshitake, S.;
Hamaguchi, Y.; Ueno, T. J. Immunoassay 1983, 4, 209–
327.
11. Craveri, R.; Manachini, P. L.; Aragozzini, F. Experientia
1972, 28, 867–868.
12. Aragozzini, F.; Manachini, P. L.; Craveri, R.; Rindone,
B.; Scolastico, C. Tetrahedron 1972, 28, 5493–5498.
13. Miyake, Y.; Kozutsumi, Y.; Nakamura, S.; Fujita, T.;
Kawasaki, T. Biochem. Biophys. Res. Commun. 1995, 211,
396–403.
14. Fujita, T.; Hirose, R.; Yoneta, M.; Sasaki, S.; Inoue, K.;
Kiuchi, M.; Hirase, S.; Chiba, K.; Sakamoto, H.; Arita,
M. J. Med. Chem. 1996, 39, 4451–4459.
15. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.;
Mishina, T.; Sasaki, S.; Fujita, T. Bioorg. Med. Chem.
Lett. 1995, 5, 853–856.
16. Kiuchi, M.; Adachi, K.; Kohara, T.; Minoguchi, M.;
Hanano, T.; Aoki, Y.; Mishina, T.; Arita, M.; Nakao, N.;
Ohtsuki, M.; Hoshino, Y.; Teshima, K.; Chiba, K.;
Sasaki, S.; Fujita, T. J. Med. Chem. 2000, 43, 2946–2961.