Highly potent inhibitors of TNF-α production. Part II: Metabolic stabilization of a newly found chemical lead and conformational analysis of an active diastereoisomer

Article abstract of DOI:10.1016/S0968-0896(02)00380-2

Design and synthesis of metabolically stabilized inhibitors of TNF-α production, which could be new drug candidates, are reported. Conformational analysis of an active diastereoisomer was performed based on biological evaluations of the conformationally f

Full text of DOI:10.1016/S0968-0896(02)00380-2

Bioorganic & Medicinal Chemistry 10 (2002) 3787–3805
Highly Potent Inhibitors of TNF-ꢀ Production. Part II:
Metabolic Stabilization of a Newly Found Chemical Lead and
Conformational Analysis of an Active Diastereoisomer
Toshiaki Matsui,a,* Takashi Kondo,b Yoshitaka Nishita,a Satoshi Itadani,b
Hiroshi Tsuruta,b Setsuko Fujita,b Nagashige Omawari,c Masaru Sakai,b
Shuichi Nakazawa,c Akihito Ogata,b Hideaki Mori,a Wataru Kamoshima,b
Kouichiro Terai,b Hiroyuki Ohno,b Takaaki Obata,b Hisao Nakaib and Masaaki Todab
aFukui Research Institute, Ono Pharmaceutical Co., Ltd., Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8638, Japan
bMinase Research Institute, Ono Pharmaceutical Co., Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
cHeadquarter, Ono Pharmaceutical Co., Ltd., Doshoumachi, Chuou, Osaka 541-8526, Japan
Received 25 June 2002; accepted 5 August 2002
Abstract—Design and synthesis of metabolically stabilized inhibitors of TNF-a production, which could be new drug candidates,
are reported. Conformational analysis of an active diastereoisomer was performed based on biological evaluations of the con-
formationally fixed indane derivatives 17 and 18. Structure–activity relationships (SARs) based on biological evaluations of the
optically active derivatives are also discussed. Full details including chemistry are reported.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
TNF-a plays a central role in the initiation and main-
tenance of many inflammatory and autoimmune dis-
eases such as rheumatoid arthritis (RA), multiple
sclerosis, sepsis, ulcerative colitis, congestive heart fail-
ure, inflammatory bowel disease and Crohn’s dis-
ease.1a,b In the preceding paper,2aÀc we reported on the
discovery of a new chemical lead followed by its opti-
mization. Since the final goal of our project is to identify
and develop an inhibitor of TNF-a production which is
a clinically useful drug candidate, we next focused our
attention on the metabolic stabilization of the newly
discovered chemical leads without loss of their highly
potent activity because one of the crucial drawbacks of
them as a drug candidate was found to be the rapid
metabolic hydrolysis of their phosphate moiety as found
in our in vitro study (Fig. 1 and Scheme 1). Reported in
this publication are the full details of the discovery
*Corresponding author. Tel.: +81-756-82-6161; fax: +81-756-82-
8420; e-mail: to.matsui@ono.co.jp
Figure 1. Design of the metabolically stabilized molecules.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00380-2

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Scheme 1. Metabolic hydrolysis of the phosphates 12 and 21.
Scheme 2. Synthesis of compounds 2, 5, 10 and 11. Reagents and conditions; (a) see ref 3a,b (b) n-C7H15COCl, NaHCO3aq, dioxane; Method
i
A(c, e, f): (c) (i) n-BuLi, THF; (ii) (BnO)2P(O)Cl; Method B (d–f): (d) (i) Pr2NP(OBn)2, tetrazole, CH3CN; (ii) m-CPBA, CH2Cl2; (e) H2, Pd–
C, MeOH; (f) NaOHaq, EtOH; (g) (Boc)2O, 1 N-NaOH, dioxane; Method C (h–k, f): (h) (Cl3CCH2O)2P(O)Cl, DMAP, Py; (i) 4 N-HCl, dioxane; (j)
n-C7H15COCl, Py, CH2Cl2; (k) Zn, Py, AcOH; (l) l-lysine, EtOH, H2O.
Scheme 3. Synthesis of compounds 8 and 6. Reagents and conditions: (a) (Boc)2O, 1 N-NaOH, dioxane; (b) PhCOOH, DEAD, Ph3P, THF; (c)
NaOMe, MeOH; Method C (d–h): (d) (Cl3CCH2O)2P(O)Cl, DMAP, Py; (e) 4 N-HCl, dioxane; (f) n-C7H15COCl, Py, CH2Cl2; (g) Zn, Py, AcOH; (h)
NaOHaq, EtOH.

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3789
Scheme 4. Synthesis of compounds 9 and 7. Reagents and conditions: (a) PhCOOH, DEAD, Ph3P, THF; (b) NaOHaq, THF, MeOH; (c) 4 N-
HCl, dioxane; (d) n-C7H15COCl, NaHCO3aq, THF; Method B (e–g): (e) (i) i-Pr2NP(OBn)2, tetrazole, CH3CN; (ii) m-CPBA, CH2Cl2; (f) H2, Pd–C,
MeOH; (g) NaOHaq, EtOH.
Scheme 5. Synthesis of compounds 3 and 4. Reagents and conditions; (a) n-C7H15COCl, NaHCO3aq, THF; (b) i-PrI, K2CO3, acetone; (c) n-
C6H13OCOCl, NaHCO3aq, THF; Method A(d, f, g): (d) (i) n-BuLi, THF; (ii) (BnO)2P(O)Cl; Method B (e–g): (e) (i) i-Pr2NP(OBn)2, tetrazole,
CH3CN; (ii) m-CPBA, CH2Cl2; (f) H2, Pd-C, MeOH; (g) NaOHaq, EtOH; (h) tris(hydroxymethyl)aminomethane, EtOH, H2O.
Scheme 6. Synthesis of compound 13. Reagents and conditions: (a) (Boc)2O, CHCl3; (b) MeI, K2CO3, DMF; (c) MeMgBr, THF; (d) 4 N-
HCl/dioxane; (e) n-C7H15COCl, NaHCO3aq, dioxane; Method B (f–i): (f) i-Pr2NP(OBn)2, tetrazole, CH3CN; (g) m-CPBA, CH2Cl2; (h) H2, Pd–C,
MeOH; (i) NaOHaq, EtOH.
Scheme 7. Synthesis of compound 14. Reagents and conditions: (a) TMSCN, TMSOTf, CH2Cl2; (b) PhMgI, Et2O; (c) NaBH4, MeOH; (d) (Boc)2O;
Method C (e–h): (e) (Cl3CCH2O)2P(O)Cl, DMAP, LiI, Py; (f) 4 N-HCl/dioxane; (g) n-C7H15COCl, Py, CH2Cl2; (h) Zn, Py, AcOH.

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T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
Scheme 8. Synthesis of compound 15. Reagents and conditions: (a) NaN3, NH4Cl, dioxane, H2O; (b) H2, Pd–C, 6 N-HCl, EtOH; (c) n-C7H15COCl,
NaHCO3aq, THF; (d) TBDPSCl, imidazole, DMF; Method B (e–i): (e) i-Pr2NP(OBn)2, tetrazole, CH3CN; (f) m-CBPA, CH2Cl2; (g) H2, Pd–C,
MeOH; (h) TBAF, THF; (i) NaOHaq, EtOH.
Scheme 9. Synthesis of compound 16. Reagents and conditions: (a) n-C7H15COCl, NaHCO3aq, dioxane; Method D (b–e): (b) i-Pr2NP(OCH2CH2CN)2,
tetrazole, CH3CN, CH2Cl2; (c) m-CPBA, CH2Cl2; (d) 50%Me2NHaq, EtOH; (e) NaOHaq, EtOH.
Scheme 10. Synthesis of compounds 17 and 18. Reagents and conditions: (a) n-C7H15COCl, NaHCO3aq, dioxane; (b) (Cl3CCH2O)2P(O)Cl, DMAP,
Py; (c) Zn, AcOH, Py; (d) NaOHaq, EtOH; Method E (e–d): (e) (BnO)2P(O)OH, DEAD, Ph3P, THF; (f) H2, Pd–C, MeOH.
Scheme 11. Synthesis of compound 19. Reagents and conditions: (a) EtOCOCl, NaHCO3aq, dioxane; (b) LiALH4, THF; (c) 4 N-HCl, dioxane; (d)
n-C7H15COCl, NaHCO3aq, dioxane; Method A(e–g): (e) (i) n-BuLi, THF; (ii) (BnO) 2P(O)Cl; (f) H2, Pd–C, MeOH; (g) NaOHaq, EtOH.
process for the metabolically stabilized derivatives as
well as conformational analysis of an active diastereoi-
somer which was based on the SARs from the con-
formationally fixed indane derivatives that possessed
spatially fixed functional groups.
Chemistry
Synthesis of all the compounds listed in the Tables 2–4
are described in Schemes 2–11. As outlined in Scheme
2, compounds 5 and 10 were prepared from 22a, while

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3791
2 and 11 were prepared from 22b. Styrene derivatives
22a, b were converted to 23a, b according to the
reported procedure.3a,b N-Acylation of 23a, b afforded
24a, b, which were converted to 25a, b, respectively
according to the following successive procedures: (1)
phosphorylation;4aÀc and (2) deprotection by hydro-
genolysis. Compounds 25a, b were converted to their
corresponding disodium salts 5 and 2, respectively
according to the usual procedure. Compounds 26a, b
were also prepared from 22a, b according to the
reported procedure.3a,b Compound 26a could be con-
verted to 10 via 29 and 30 according to the following
successive procedures: (1) N-tert-butoxycarbonylation;
(2) phosphorylation;5,6 (3) acidic deprotection; (4)
N-acylation; (5) reductive deprotection;5,6 and 6) treat-
ment with NaOH aq. Compound 26b was transformed
to 11 via 27 and 28 according to the same procedures
as described above. The l-lysine salt of 25b was pro-
was prepared from the aminoalcohol 56 via 57. N-Acy-
lation of 56 gave 57, which was converted to the dis-
odium salt 16 according to the usual procedure.
Synthesis of conformationally fixed isomers 17 and 18 is
outlined in Scheme 10. N-Acylation of the aminoalcohol
58 with octanoyl chloride afforded 59, bis(2-tri-
chloroethoxy)phosphorylation5,6 of which provided 60.
Reductive deprotection5,6 of 60 followed by the treat-
ment with sodium hydroxide afforded 17. Dibenzyl
phosphorylation of the hydroxy group of 59 with a
SN2 inversion gave 61,11 deprotection of which affor-
ded 62. The free acid form 62 was converted to 18 by
the usual method. The N-methyl derivative 19 was
prepared as described in Scheme 11. Compound 64 was
prepared from (2R)-2-amino-2-phenylethanol 63 by the
following successive reactions: (1) N-ethoxycarbonyla-
tion; (2) reduction with LiAlH4; (3) treatment with acid;
and (4) N-acylation with octanoyl chloride. Compound 19
was prepared from 64 according to the usual procedure.
.
duced as the crystalline compound 25b Lys. As descri-
bed in Scheme 3, compound 31 and 33 were
prepared from 23a and 26a, respectively according to
the following successive reactions: (1) N-tert-butox-
ycarbonylation; (2) Mitsunobu reaction;7 and (3) alka-
line hydrolysis. Compounds 8 and 6 were prepared from
31 and 33, respectively according to the same proce-
dures as described for the preparation of 10 from 29 via
30. As described in Scheme 4, compounds 9 and 7 were
prepared from 36 and 38 respectively according to the
same procedures as described for the preparation of 2
from 24b. Compounds 36 and 38 were prepared from 35
and 37, respectively according to the following succes-
sive reactions: (1) Mitsunobu reaction;7 (2) alkaline
hydrolysis; (3) acidic deprotection; and (4) N-acylation.
As described in Scheme 5, compounds 3 and 4 were also
prepared from 23c and 23b, respectively according to
essentially the same procedures as described for the
preparation of 5 and 2 from 23a and 23b, respectively.
The diammonium salt of 40b was produced as the
crystalline bis[tris(hydroxymethyl)aminomethane] salt
Results and Discussion
To identify an inhibitor of TNF-a production which
could be a clinically useful drug candidate, the design
and synthesis of metabolically stable derivatives was
carried out and their ability to inhibit TNF-a produc-
tion was evaluated biologically. Plasma TNF-a produc-
tion and the potency of the test compounds were
evaluated according to the same procedures as described
in the preceding papers.2aÀc Apharmacological evalua-
tion of the optimized derivatives 2–4 in the animal dis-
ease models was also carried out.
Conformational analysis of the optically active diaster-
eoisomers 2 and 9 was performed based on the informa-
tion obtained from the SARs of the conformationally
fixed indane derivatives 17 and 18.
.
(40b TRIS). Synthesis of compound 13 is described in
According to our in vitro experimental data regarding
the metabolism of the test compounds, the newly dis-
covered inhibitor 12 was very sensitive to metabolic
hydrolysis with tissue homogenates as described in
Scheme 1 and Table 1. The formed metabolite 20 did not
show any inhibitory activity as shown in Table 4. The
metabolic inactivation of 12 with tissue homogenates
from the small intestine and liver was very fast as descri-
bed in Table 1 while with the plasma it was relatively
slow. As such, it was thought that the metabolic stabili-
zation of the chemical lead 1 could be accomplished by
blocking the rapid hydrolysis of the phosphate moiety
in 1. In fact, 1-methyl-2-octanoylamino-2-phenylethyl
disodium phosphate 21 consisting of four isomers 5, 6, 8
and 10 was found to be stable after its treatment with tis-
sue homogenates as described in Scheme 1 and Table 1.
Scheme 6. Protection of the amino group of 418 fol-
lowed by O-alkylation provided 42, dimethylation of
which with methyl magnesium bromide afforded 43.
Deprotection followed by N-acylation provided 44,
which was transformed to 13 by the above-described
method. Synthesis of 14 is described in Scheme 7.
Cyclobutanone 45 was converted to 46 by the
conventional method.9 Compound 46 was converted to
47 by the following successive reactions: (1) treatment
with phenyl magnesium iodide followed by sodium
borohydride reduction;10 and (2) protection of the
amino group with tert-butoxycarbonyl group. Com-
pound 47 was converted to 14 via 48 according to the
usual procedures.5,6 Synthesis of the hydroxymethyl
derivative 15 is outlined in Scheme 8. Aring opening
reaction of the epoxide 49 with sodium azide afforded
50, catalytic hydrogenation of which gave the amino-
alcohol 51. N-Acylation of 51 followed by the selective
monosilylation of the diol 52 provided 53, which was
converted to 54 by the usual procedure. Catalytic
hydrogenation of 54 followed by the treatment with
TBAF provided 55, which was converted to 15 by the
usual method. As described in Scheme 9, compound 16
Based on the above-described results, the design and
synthesis of the metabolically stabilized inhibitors was
started with the introduction of a methyl group into the
optically active backbone. As shown in Table 2, two
classes of four isomers 5, 6, 8 and 10 as well as 2, 7, 9
and 11 were synthesized and biologically evaluated
individually. According to our intact data, the meta-

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T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
Table 1. Results of the in vitro metabolism of 12 and 21d
Remaining% after incubation (30 min)a
Tissue homogenates
12
21
Mouse liver
N.D.b
93
97
95
95
97
91
b
Mouse small intestine
Mouse plasma
Rat liver
Rat small intestine
Rat plasma
N.D.
89
N.D.b
N.T.c
74
aIn vitro stability of compounds 12 and 21 was analyzed by HPLC after treatment with tissue homogenates. To a solution of the test compound in
PBS (À) (15 mg/100 mL) 1% of tissue homogenate was added. The mixture was incubated at 37 ꢀC for 30 min, and the reaction quenched with
MeOH (2 mL). [iso-Butyl paraben (10 mg/mL) was used as an internal standard for 12. iso-Amyl paraben (10 mg/mL) was used as an internal stan-
dard for 21]. After centrifugation at 3000 rpm for 10 min, the organic layer was evaporated. A solution of the residue in CH3CN/H2O (1/1, 200 mL)
was analyzed by HPLC [column: Inertsil ODS-3, 4.6Â150 mm (GL Science Inc.); detection: UV at 210 nm; Eluent: A=20 mM KH2PO4 (pH2) /
B=CH3CN, (gradient)d; Flow rate: 1.0 mL/min; 25 ꢀC; Sample: 50 mL was injected].
bND: Not Detected.
cNT : Not Tested.
dGradient for analyzing 12.
gradiant for analyzing 21.
time (min)
A/B
Time (min)
A/B
0
60/40
60/40
20/80
20/80
60/40
60/40
0
60/40
60/40
50/50
50/50
60/40
60/40
20.0
20.5
35.5
36.0
46.0
10
10.1
25.0
25.1
35.0
Table 2. Biological evaluation of the metabolically stabilized derivatives
Compd
R1
R2
R3
R4
R5
Inhibition of TNF-a productiona
ID50 (mg/kg, iv) rats
5
2
6
7
8
9
10
11
3
Me
Me
Me
Me
H
H
H
H
Me
Me
H
H
H
–NHCO–n-C7H15
–NHCO-n-C7H15
H
H
OMe
H
OMe
H
OMe
H
OMe
OiPr
OMe
4.5
0.03
(17)c
3.7
H
–NHCO–n-C7H15
–NHCO–n-C7H15
H
H
–NHCO–n-C7H15
–NHCO–n-C7H15
H
H
H
Me
Me
Me
Me
H
–NHCO–n-C7H15
–NHCO–n-C7H15
H
(23)c
(À13)b
(5)c
H
(38)b
0.05
0.02
–NHCO–n-C7H15
–NHCOO–n-C6H13
H
H
4
H
aSee Experimental.
bInhibition (%) at 10 mg/kg, iv.
cInhibition (%) at 30 mg/kg, iv.
bolically stabilized compound 5 exhibited more potent
activity than 12 although their ID50 values in rats
appeared to be close to each other (5: 4.5 mg/kg, iv; 12:
3.0 mg/kg, iv). For example, intravenous administration
of 5 (3 mg/kg) demonstrated a more than 50% inhibi-
tion of TNF-a production (58%) while that of 12 (3 mg/
kg) demonstrated less than 50% inhibition (43%). Also
in their evaluation in mice, 5 exhibited a more potent
ID50 value (0.4 mg/kg, iv) than 12 (0.8 mg/kg, iv). Thus,
metabolic stabilization of 5 was found to be effective at
increasing its activity.
This successful results of the metabolic stabilization was
thought to be beneficial for the discovery of the highly
potent inhibitors 2–4 described in Table 5.
As shown in Table 2, the (S)-configuration of the newly
introduced methyl group and (R)-configuration of the
N-acyl moiety were needed for potent inhibitory activity
as illustrated in compounds 5, 2 and 7. Introduction of a
meta-methoxy group into the phenyl moiety of 5 affor-
ded 2 with a marked increase in activity. The same ten-
dency was observed upon the chemical modification of 6

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3793
Table 3. Attempted study to search for a substitute for the (1S)-
methyl group
acceptable instead of the (1S)-methyl group. The syn-
thesized compounds 13–16 were evaluated biologically
and their potentials compared with the chemical leads
12 and 5. Introduction of another methyl group at the
geminal position of 2 afforded 13 with a marked reduc-
tion in activity presumably because of the bulkiness of
the newly constructed 2,2-dimethyl moiety, while the
less hindered 2,2-trimethylene derivative 14, which was
biologically evaluated as a dl-mixture, had an ID50
value of 9.5 mg/kg, iv in mice. Replacement of the
methyl group of 12 with a hydroxymethyl group pro-
vided 15 with a decrease in activity. Introduction of a
phenyl moiety to 5 instead of a methyl group afforded
16 with a marked reduction in activity. Thus, it was
concluded that (1S)-methyl is the optimized partial
structure.
Inhibition of TNF-a productiona
ID50 (mg/kg, iv) rats
Compd
R1
R2
R3
12
5
13 (dl)
H
Me
Me
H
H
H
H
3.0
4.5
Me OMe
(12)b
9.5c
(42)b
14 (dl) –CH2-CH2-CH2—
H
H
15
16
CH2OH
H
H
H
(3)b
To elucidate the three dimensional active conformation,
compounds 17–19, in which free rotations are restricted
and/or blocked, were synthesized as the optically active
forms and evaluated biologically as described in Table
4. Interestingly, the trans-isomer 18 was much more
potent than the cis-isomer 17. The trans-isomer 18 was
estimated to occupy a three dimensional structure close to
the real active conformer. This SAR strongly suggested an
active conformation of the optimized compounds 2–4.
aSee Experimental.
bInhibition (%) at 10 mg/kg, iv.
cTested in mice.
to 7 while 6 exhibited relatively weak inhibitory activity.
Amarked reduction of inhibitory activity was observed
in all the (R)-methyl derivatives 8–11. Therefore, the
configuration of the methyl group was thought to have
a dominant effect on the ability to inhibit TNF-a pro-
duction. The configuration of the N-acyl moiety also
plays an important role, although it dose not seem to be
as critical as that of the methyl group (R1), as illustrated
in 5 and 7. Introduction of a meta-methoxy group also
increased the inhibitory activity as observed upon the
chemical modification of 5 to 2 and 6 to 7. Replacement
of the meta-methoxy group of 2 with a meta-iso-
propyloxy group provided 3 which retained the highly
potent activity. Replacement of the N-octanoyl moiety
of 2 with a N-hexyloxycarbonyl moiety afforded 4 also
with strong activity to inhibit the TNF-a production.
Thus, the (1S,2R)-configuration was found to be a
structural requirement for the potent inhibitory activity
in this series of compounds.
As illustrated in Fig. 2, the more active compound 2 is
able to occupy the favored conformation more easily
than the less active compound 9 because of a less hin-
dered intramolecular repulsion between the methyl group
and the ortho-hydrogen of the phenyl moiety. The
N-methyl derivative 19 retained quite a good activity as
shown in Table 4. The N-methyl group did not appear to
prevent 19 from occupying the favored conformation.
As shown in Fig. 3, increased production of the plasma
TNF-a after the intravenous administration of LPS (30
mg/kg) was significantly suppressed by administration of
compounds 2–4 in a dose-dependent manner. The ID50
values of 2–4 are 0.03, 0.05 and 0.02, respectively.
As shown in Table 3, further chemical modification was
continued to identify another substituent which was
The efficacy in disease models12aÀe and safety of 2–4
were evaluated.
Table 4. Analysis of the structural requirements for the inhibitory activity
Compd
R
Inhibition of TNF-a productiona
ID50 (mg/kg, iv) rats
17
18
19
20
C7H15
C7H15
C7H15
C7H15
(34)b
2.4
9.8
(14)c
aSee Experimental.
bInhibition (%) at 10 mg/kg, iv.
cInhibition (%) at 10 mg/kg, iv in mice.

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T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
Figure 2. Proposed active conformation based on the more active trans-isomer 18.
Figure 3. Effect of compound 2, 3, and 4 on LPS-induced TNF-a production in rats. Test compound was administered intravenously just before an
intravenous injection of LPS. After 90 min of LPS injection, heparinized blood was obtained. Plasma TNF-a concentration was determined by
ELISA. The data were expressed as the meanÆSEM (n=5; ***, significantly different from LPS control, P<0.001, ANOVA-Dunnett’s t-test).
Table 5. Biological evaluation of 2–4
Compd
LPS-induced shock
model in mice
d-(+)-Galactosamine/LPS-induced
hepatitis model in rats
(n=18)b
Safety in rats
(n=3) MLDd
mg/kg, iv
(n=18 or 20)a
MEDc mg/kg, iv
(survival rate)
MEDc mg/kg, iv
(mortality rate)
(survival rate)
2
3
4
0.1 (10/18)
0.3 (10/18)
0.1 (14/20)
0.3 (7/18)
0.3 (8/18)
0.1 (6/18)
>100 (0/3)
100 (1/3)
>100 (0/3)
aSee Experimental.
bSee Experimental.
cMED (minimum effective dose): survival rates are described in parentheses.
dMLD (minimum lethal dose): at least one of the tested animals died at this dose. Mortality rates are described in parentheses.

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3795
Figure 4. Effect of compound 4 on LPS-induced shock model in mice. BALB/c mice were injected intravenously with compound 4 and then
immediately given an intraperitoneal injection of LPS (20 mg/kg). The survival rate of the mice was evaluated after 96 h (n=5–20; *, significantly
different from LPS control, P<0.01; N.S., not significant, Mantel–Cox test).
Figure 5. Effect of compound 4 on d-(+)-galactosamine/LPS-induced hepatitis model in rats. SD rats were injected intravenously with compound 4
and then immediately given an intraperitoneal injection of d-(+)-galactosamine/LPS (1 g/7.5 mg/kg). The survival rate was evaluated after 96 h
(n=18/group; *, significantly different from d-(+)-galactosamine/LPS control, P<0.01; N.S., not significant, Mantel–Cox test).

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T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
Table 6. Oral activity of the representative compounds
spectra were taken on a Varian Gemini-200, VXR-200s
or Mercury 300 spectrometer. MS spectra were obtained
on a Hitachi M1200H, JMS-DX303HF or PerSeptive
Voyager Elite spectrometer. The matrix assisted laser
desorption ionization-time of flight high-resolution
mass spectra (MALDI-TOF, HRMS) were obtained on
a PerSeptive Voyager Elite spectrometer. IR spectra
were measured on a Perkin–Elmer FT-IR 1760X or
Jasco FT/IR-430 spectrometer. Elemental analyses for
carbon, hydrogen and nitrogen were carried out by the
Analytical Section of ONO Pharmaceutical Co., Ltd. on
a Perkin–Elmer PE2400 SeriesII CHNS/O analyzer.
Melting points were uncorrected. Optical rotations were
measured on a Jasco DIP-1000 polarimeter. Column
chromatography was carried out on silica gel (Merck
silica gel 60 (0.063–0.200 mm) or Fuji Silysia FL60D).
Thin layer chromatography was performed on silica gel
(Merck TLC plate, silica gel 60 F254). The following
abbreviations for solvents and reagents are used: THF,
tetrahydrofuran; EtOAc, ethyl acetate; MeOH, methanol;
EtOH, ethanol; DMF, dimethylformamide; CH2Cl2, di-
Compd
Inhibition of TNF-a production
ID50 (mg/kg, rats, n=5 )
iv
po
Fed
Fasted
12
2
3
3.0
>300
53
63
104
16
9
0.03
0.05
0.02
4
20
4
As described in Table 5, the minimum effective doses
(MEDs) of compounds 2–4 in the LPS-induced shock
model of mice were 0.1, 0.3 and 0.1 mg/kg, iv, respec-
tively. The minimum effective doses (MEDs) of com-
pounds 2–4 in the d-(+)-galactosamine/LPS-induced
hepatitis model of rats were 0.3, 0.3 and 0.1 mg/kg, iv,
respectively. Among the tested, compound 4 was the
most hopeful one. As shown in Figs 4 and 5, compound
4 demonstrated an efficacy equivalent to predonisolone
(10 mg/kg, iv), which was used as the positive control, at
the dose of 0.3 mg/kg, iv, significantly13 in both of the
above described disease models.
.
chloromethane; CHCl3, chloroform; EDC HCl, 1-[3-
(dimethylamino)-propyl]-3-ethylcarbodiimide hydro-
.
chloride; HOBt H2O, N-hydroxybenzotriazole hydrate;
m-CPBA, meta-chloroperbenzoic acid; DEAD, diethyl
azodicarboxylate; TBDPSCl, tert-butylchlorodiphenyl-
silane; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TBAF,
tetrabutylammonium fluoride; DMAP, N,N-dimethyl-
pyridin-4-amine
With respect to the safety concern, the minimum lethal
dose (MLD) of 3 in rats was 100 mg/kg, iv, while the
MLDs of 2 and 4 were more than 100 mg/kg, iv,
respectively. All three compounds, 2–4, demonstrated a
sufficient margin of safety for pharmacological
evaluation.
N-[(1R,2S)-2-Hydroxy-1-(3-methoxyphenyl)propyl]octa-
namide (24b). (1R,2S)-1-Amino-1-(3-methoxyphenyl)
propan-2-ol hydrochloride 23b was prepared from
1-methoxy-3-[(1E)-prop-1-enyl]benzene 22b by follow-
ing the known procedure:3a,b 23b: white solid; TLC
Rf=0.47 (CHCl3/MeOH/H2O, 10/5/1); MS (MALDI-
TOF, Pos.) m/z 182 (M+H)+; 1H NMR (300 MHz,
CDCl3) d 8.53 (brs, 3H), 7.23 (t, J=8.1 Hz, 1H), 7.06 (s,
1H), 6.97 (d, J=7.8 Hz, 1H), 6.83 (dd, J=8.4, 2.4 Hz,
1H), 4.85–4.60 (br, 1H), 4.46–4.32 (m, 2H), 3.70 (s, 3H),
0.99 (d, J=6.0 Hz, 3H). To a stirred mixture of 23b
(20.0 g, 92.0 mmol) and NaHCO3 (23.2 g, 275.9 mmol)
in THF (92 mL)–H2O (92 mL) was added dropwise
octanoyl chloride (15.9 mL, 92.9 mmol) at 0 ꢀC and
stirring was continued at that temperature for 7 h. The
reaction mixture was poured into H2O and extracted
with EtOAc. The organic layer was successively washed
with H2O and brine before being dried over MgSO4.
Removal of the solvent by evaporation afforded a white
solid, which was washed with n-hexane and dried under
reduced pressure to obtain 24b as a white powder (24.7
g, 87%): TLC Rf=0.32 (EtOAc/toluene, 2/1); MS
(APCI, Pos. 20eV) m/z 308 (M+H)+, 290; IR (KBr)
The representative compounds 12 and 2–4 were also
evaluated for their oral activity. As shown in Table 6,
much higher dosages than expected were needed to
reproduce the same extent of ID50 values as those
obtained after their intravenous administration. These
results were ascribed to their presumed poor oral
absorption. Three to seven-fold ID50 values were
obtained in fed rats compared to fasted rats. Their site
of action was also estimated to be in the liver and spleen
according to the experimental results reported in the
preceding paper.2c
Conclusion
In summary, through the design and synthesis of meta-
bolically stabilized inhibitors of TNF-a production, we
have discovered the drug candidates 2–4 for use in the
treatment of diseases caused by the overexpression of
TNF-a. These three compounds demonstrated efficacy in
animal models of disease and are expected to be clinically
useful although no specialized uses are yet intended.
3308, 2927, 1645, 1604, 1543, 1261 cmÀ1 1H NMR
;
(300 MHz, CDCl3) d 7.27 (t, J=7.8 Hz, 1H), 6.89–6.82
(m, 3H), 6.33 (brd, J=7.8 Hz, 1H), 4.90 (dd, J=7.8, 3.9
Hz, 1H), 4.11–4.03 (m, 1H), 3.80 (s, 3H), 2.24–2.19 (m,
2H), 1.90 (brs, 1H), 1.68–1.57 (m, 2H), 1.31–1.24 (m,
8H), 1.09 (d, J=6.3 Hz, 3H), 0.89–0.85 (m, 3H).
Experimental
General directions
Analytical samples were homogeneous as confirmed by
TLC, and the spectroscopic results obtained were
consistent with the assigned structures. All 1H NMR
N-[(1R,2S)-2-Hydroxy-1-phenylpropyl]octanamide (24a).
(1R,2S)-1-Amino-1-phenylpropan-2-ol hydrochloride
23a was prepared from (1E)-prop-1-enylbenzene 22a

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3797
according to the known procedure.3a,b The title com-
pound 24a was prepared from 23a according to essen-
tially the same procedure as described for the
preparation of 24b from 23b: white powder; TLC
Rf=0.40 (n-hexane/EtOAc, 1/2); MS (MALDI, Pos.) m/
z 300 (M+Na)+, 278 (M+H)+;IR (KBr) 3305, 1644,
colorless oil (4.82 g, 80% yield): TLC Rf=0.56 (EtOAc/
toluene, 2/1); MS (APCI, Pos. 20eV) m/z 336 (M+H)+;
1
IR (KBr) 3307, 2928, 1641, 1544, 1258, 1118 cmÀ1; H
NMR; (300 MHz, CDCl3) d 7.24 (dd, J=8.7, 7.8 Hz,
1H), 6.85–6.80 (m, 3H), 6.29 (d, J=7.8 Hz, 1H), 4.90
(dd, J=7.8, 3.9 Hz, 1H), 4.58–4.50 (m, 1H), 4.12–4.01
(m, 1H), 2.24–2.19 (m, 2H), 2.13 (brd, J=6.6 Hz, 1H),
1.68–1.58 (m, 2H), 1.33 (d, J=6.0 Hz, 3H), 1.33 (d,
J=6.0 Hz, 3H), 1.32–1.23 (m, 8H), 1.10 (d, J=6.3 Hz,
3H), 0.89–0.85 (m, 3H).
1
1549, 1454, 1123 cmÀ1; H NMR (300 MHz, CDCl3) d
7.40–7.23 (m, 5H), 6.28 (d, J=8.0 Hz, 1H), 4.95 (dd,
J=8.0, 3.6 Hz, 1H), 4.09 (m, 1H), 2.23 (t, J=6.6 Hz,
2H), 1.95 (d, J=7.5 Hz, 1H), 1.63 (m, 2H), 1.27 (m,
8H), 1.09 (d, J=6.3 Hz, 3H), 0.87 (t, J=6.6 Hz, 3H).
General method A
Hexyl
(1R,2S)-2-hydroxy-1-(3-methoxyphenyl)propyl-
carbamate (39b). To a stirred mixture of 23b (14.5 g,
66.7 mmol) and NaHCO3 (16.8 g, 200 mmol) in THF/
H2O (70 mL/70 mL) was added hexyl chlor-
idocarbonate (11.0 mL, 67.3 mmol) at 0 ꢀC. Stirring was
continued at that temperature for 30 min and at room
temperature for 30 min. The reaction mixture was
poured into ice-water and extracted with EtOAc. After
the organic layer was successively washed with H2O and
brine, it was dried over MgSO4. Removal of the solvent
by evaporation gave a solid which was washed with n-
hexane to afford 39b as a white solid (19.7 g, 96% yield):
TLC Rf=0.28 (n-hexane/EtOAc, 1/1); MS (MALDI-
(1S,2R)-2-(3-Isopropyloxyphenyl)-1-methyl-2-(octanoyla-
mino)ethyl disodium phosphate (3). To a stirred solution
of 39c (4.0 g, 11.9 mmol) in THF (100 mL) was added
dropwise n-butyllithium in n-hexane (1.53 M, 21.0 mL,
32.1 mmol) at À78 ꢀC under an argon atmosphere.
Dibenzylphosphorochloridate in THF4a (1 M, 35.7 mL,
35.7 mmol) was added to the resulting mixture at that
temperature and stirring was continued for 10 min at
À20 ꢀC. The reaction was quenched with 1M NaOH
and the resulting mixture was warmed to room tem-
perature with stirring. After the reaction mixture was
diluted with EtOAc, the organic layer was successively
washed with saturated NaHCO3 aq and brine, it was
dried over MgSO4. Removal of the solvent by evapora-
tion gave a residue which was purified by column chro-
matography on silica gel (Merck 7734, n-hexane/EtOAc,
4/1) to afford dibenzyl (1S,2R)-2-(3-isopropyloxyphe-
nyl)-1-methyl-2-(octanoylamino)ethyl phosphate as a
colorless oil (4.54 g, 64% yield): TLC Rf=0.58
(EtOAc/toluene, 1/1). A mixture of dibenzyl (1S,2R)-2-(3-
isopropyloxyphenyl) - 1 - methyl - 2 - (octanoylamino)ethyl
phosphate (4.54 g) and 10% Pd-C (400 mg) in MeOH
was stirred at room temperature under an atmospheric
pressure of hydrogen for 1 h. Removal of the catalyst by
filtration through a pad of Celite followed by evapora-
tion gave an oily residue which was dissolved in satu-
rated NaHCO3 aq and extracted with EtOAc. The
aqueous layer was acidified by 1 M HCl and then
extracted with EtOAc. The organic layer was succes-
sively washed with H2O and brine before being dried
over MgSO4. Removal of the solvent by evaporation
gave 40c as a white amorphous powder (2.56 g, 81%).
To a stirred solution of 40c (2.24 g, 5.4 mmol) in EtOH
(50 mL) was added 1 M NaOH (10.8 mL, 10.8 mmol) at
room temperature. Removal of the solvent by evapora-
tion followed by the dissolution of the residue in EtOH
was repeated several times to remove the H2O azeo-
tropically. The addition of Et2O followed by evapora-
tion afforded 3 as a white amorphous powder: TLC
Rf=0.33 (CHCl3/MeOH/H2O, 65/35/8); IR (KBr) 3423,
1
TOF, Pos.) m/z 310 (M+H)+; H NMR (300 MHz,
CDCl3) d 7.32–7.22 (m, 1H), 6.92–6.81 (m, 3H), 5.50
(brd, J=6.9 Hz, 1H), 4.67–4.56 (br, 1H), 4.13–3.95 (m,
3H), 3.81 (s, 3H), 1.80–1.47 (m, 2H), 1.42–1.20 (m, 6H),
1.11 (d, J=6.3 Hz, 3H), 0.88 (t, J=6.9 Hz, 3H).
N-[(1R,2S)-2-Hydroxy-1-(3-isopropoxyphenyl)propyl]oc-
tanamide
(39c).
3-[(1R,2S)-1-Amino-2-hydroxy-
propyl]phenol hydrochloride 23c was prepared from 1-
(benzyloxy)-3-[(1E)-prop-1-enyl]benzene by following
the known procedure.3a,b 23c: TLC Rf=0.45 (CHCl3/
1
MeOH/H2O, 65/35/8); H NMR (300 MHz, CDCl3) d
7.23 (t, J=8.4 Hz, 1H), 6.92–6.88 (m, 2H), 6.84–6.81
(m, 1H), 4.20–4.12 (m, 2H), 1.04 (d, J=6.0 Hz, 3H).
N-[(1R,2S)-2-hydroxy-1-(3-hydroxyphenyl)propyl]octan-
amide was prepared from 23c according to essentially
the same procedure as described for the preparation of
24b from 23b. Purification was performed by column
chromatography on silica gel (Merck 7734, n-hexane/
EtOAc, 1/1) to give N-[(1R,2S)-2-Hydroxy-1-(3-hydroxy-
phenyl)propyl]octanamide as a colorless viscous oil
(80% yield): TLC Rf=0.43 (EtOAc/toluene, 2/1); 1H
NMR (300 MHz, CDCl3) d 8.05 (brs, 1H), 7.14 (t,
J=7.5 Hz, 1H), 6.78–6.70 (m, 3H), 6.65 (d, J=8.1 Hz,
1H), 4.81 (dd, J=8.1, 4.2 Hz, 1H), 4.08–3.98 (m, 1H),
2.85 (brs, 1H), 2.20–2.15 (m, 2H), 1.65–1.52 (m, 2H),
1.30–1.20 (m, 8H), 1.06 (d, J=6.6 Hz, 3H), 0.90–0.80
(m, 3H). To a stirred mixture of N-[(1R,2S)-2-hydroxy-
1-(3-hydroxyphenyl)propyl]octanamide (5.28 g, 18.0
mmol) and K2CO3 (6.22 g, 45.0 mmol) in acetone (40
mL) was added 2-iodopropane (2.70 mL, 27.0 mmol).
Stirring was continued under reflux for 12 h. The reac-
tion mixture was poured into ice–water and extracted
with Et2O. The organic layer was successively washed
with H2O and brine before being dried over Na2SO4.
Removal of the solvent by evaporation gave a residue
which was purified by column chromatography on silica
gel (Merck 7734, n-hexane/EtOAc, 1/2) to give 39c as a
1
1637, 1089, 984 cmÀ1; H NMR (300 MHz, CD3OD) ꢀ
7.12 (t, J=7.8 Hz, 1H), 6.99 (brd, J=7.8 Hz, 1H), 6.96
(brs, 1H), 6.73–6.69 (m, 1H), 4.64–4.54 (m, 3H), 2.35–
2.19 (m, 2H), 1.61–1.49 (m, 2H), 1.29 (d, J=6.0 Hz,
3H), 1.26 (d, J=6.0 Hz, 3H), 1.30–1.20 (m, 8H), 1.06
(d, J=6.3 Hz, 3H), 0.89–0.84 (m, 3H); optical rotation
[a]2D4 À53.3 (c 1.05, MeOH); MS (FAB, Pos.) m/z 460
(M+H)+, 438, 416; HRMS (MALDI-TOF, Pos.)
+
.
calcd for C20H32NO6P 2Na+H : 460.1841; found:
460.1805.

3798
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
(1S,2R)-1-Methyl-2-octanoylamino-2-phenylethyl diso-
diumphosphate (5). The title compound 5 was prepared
from 24a according to the general method A: TLC
Rf=0.24 (CHCl3/MeOH/H2O, 65/25/4); IR (KBr) 3336,
1637, 1535, 1454, 1385, 1089 cmÀ1; 1H NMR (300 MHz,
CD3OD) d 7.42 (brd, J=6.6 Hz, 2H), 7.25–7.14 (m,
3H), 4.62–4.57 (m, 2H), 2.33–2.18 (m, 2H), 1.60–1.50
(m, 2H), 1.35–1.15 (m, 8H), 1.03 (d, J=6.6 Hz, 3H),
0.86 (brt, J=6.6 Hz, 3H); optical rotation [a]2D5À64.5 (c
0.96, MeOH); MS (FAB, Pos.) m/z 424 (M+Na)+, 402
(M+H)+; HRMS (MALDI-TOF, Pos.) calcd for the
free acid form C17H28NO5P+Na+: 380.1603; found:
380.1599.
1.65–1.55 (m, 2H), 1.30–1.25 (m, 8H), 1.21 (d, J=6.3
Hz, 3H), 0.90–0.85 (m, 3H). To a stirred solution of 25b
(2.13 g, 5.5 mmol) in EtOH (50 mL) was added 1 M
NaOH (11 mL, 11 mmol) at 0 ꢀC. Removal of the sol-
vent by evaporation followed by the dissolution of the
residue in EtOH was repeated several times to remove
the H2O azeotropically. Then addition of Et2O followed
by evaporation afforded 2 as a white amorphous pow-
der (5.18 g, 98%): TLC Rf=0.28 (CHCl3/MeOH/H2O,
65/35/8); IR (KBr) 3389, 1638, 1534, 1087, 984 cmÀ1
;
1H NMR (300 MHz, CD3OD) d 7.14 (t, J=8.4 Hz, 1H),
7.01–6.99 (m, 2H), 6.74 (ddd, J=8.4, 2.7, 1.2 Hz, 1H),
4.65–4.53 (m, 2H), 3.78 (s, 3H), 2.35–2.19 (m, 2H),
1.61–.49 (m, 2H), 1.30–1.20 (m, 8H), 1.05 (d, J=6.3 Hz,
3H), 0.88–0.84 (m, 3H); optical rotation [a]2D4 À63.3 (c
1.02, MeOH); MS (FAB, Pos.) m/z 432 (M+H)+, 454,
General method B
(1S,2R)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-
no)ethyl disodiumphosphate (2). To a stirred mixture of
24b (25.5 g, 83 mmol) and tetrazole (11.64 g, 166 mmol)
in CH3CN (330 mL) was added dibenzyl diisopropyl-
phosphoramidite (33.5 mL, 99.7 mmol) at room tem-
perature and stirring was continued at that temperature
for 3 h. The reaction mixture was poured into satu-
rated NaHCO3 aq and the resultant precipitates were
removed by filtration. The filtrate was then diluted
with EtOAc and washed with brine before being dried
over Na2SO4. Removal of the solvent by evaporation
gave dibenzyl (1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-
(octanoylamino)ethyl phosphite which was used for the
next reaction without further purification (52.0 g,
>100%, pale yellow oil): TLC Rf=0.60 (n-hexane/
EtOAc, 1/2). To a stirred solution of dibenzyl (1S,2R)-1
-methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl
phosphite (52.0 g) in CH2Cl2 (415 mL) was added m-
410; HRMS (MALDI-TOF, Pos.) calcd for
+
.
C18H28NO6P 2Na+H : 432.1528; found: 432.1524.
.
L-Lysine salt of compound 2 (25 Lys). Compound 2 was
one of the best inhibitors for TNF-a production, so an
alternative salt was prepared. To a stirred solution of
25b (15.6 g, 40.2 mml) in EtOH/H2O (20/1, 130 mL)
was added l-lysine (5.94 g, 40.6 mmol). Stirring was
continued under reflux until the reaction mixture chan-
ged from a suspension to a clear solution. After cooling
to room temperature, the resultant precipitates were
collected by filtration and dried under reduced pressure
.
to give 25 Lys as a white powder (17.7 g, 82%): TLC
Rf=0.22 (CHCl3/MeOH/H2O, 10/5/1); MS (FAB, Pos.)
m/z 534 (M+H)+, 426, 410, 388, 290, 147; IR (KBr)
3296, 2927, 2856, 1643, 1602, 1545, 1467, 1416, 1379,
1346, 1258, 1165, 1124, 1049 cmÀ1; 1H NMR (300 MHz,
CD3OD) d 7.18 (t, J=8.1 Hz, 1H), 6.99–6.92 (m, 2H),
6.82–6.76 (m, 1H), 4.84 (d, J=3.3 Hz, 1H), 4.64–4.51
(m, 1H), 3.78 (s, 3H), 3.56 (t, J=6.0 Hz, 1H), 2.92 (t,
J=7.5 Hz, 2H), 2.34–2.17 (m, 2H), 1.95–1.75 (m, 2H),
1.75–1.41 (m, 6H), 1.38–1.17 (m, 8H), 1.12 (d, J=6.6
Hz, 3H), 0.87 (t, J=6.9 Hz, 3H); mp 194.5–196. 5 ꢀC;
optical rotation [a]2D6 À45.99 (c 1.0, MeOH); Anal calcd
ꢀ
CPBA(77%, 20.5 g, 91.4 mmol) at 0 C and stirring was
continued at that temperature for 1 h. The reaction
mixture was treated with saturated Na2S2O3 aq and
extracted with CH2Cl2. The organic layer was succes-
sively washed with H2O, saturated NaHCO3 aq and
brine before being dried over Na2SO4. The m-chlor-
obenzoic acid was removed by passing the mixture
through a pad of Al2O3 (100 g). Removal of the solvent
by evaporation gave dibenzyl (1S,2R)-1-methyl-2-(3-
.
for C18H30NO6P C6H14N2: C, 54.02; H, 8.31; N, 7.87;
Found: C, 54.04; H, 8.37; N, 7.79.
methoxyphenyl)-2-(octanoylamino)ethyl
phosphate
(1S,2R)-1-Methyl -2-[(hexyloxycarbonyl)amino]-2-(3-
methoxyphenyl) ethyl disodium phosphate (4). The title
compound 4 was prepared from 39b according to the
general method B: TLC Rf=0.30 (CHCl3/MeOH/H2O,
65/35/4); IR (KBr) 3239, 2932, 1699, 1602, 1491, 1456,
which was used for the next reaction without further
purification (50.6 g, >100%, colorless oil): TLC
Rf=0.35 (n-hexane/EtOAc, 1/2). A mixture of dibenzyl
(1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylamino)
ethyl phosphate (50.6 g) and 10% Pd-C (50% wet, 10 g)
in MeOH (850 mL) was vigorously stirred at room
temperature under an atmospheric pressure of hydrogen
for 5.5 h. Removal of the catalyst by filtration through a
pad of Celite followed by evaporation afforded an oily
residue which was dissolved in saturated NaHCO3 aq and
extracted with EtOAc. The aqueous layer was acidified
by 1 M HCl and extracted with EtOAc. The organic
layer was washed with brine and dried over Na2SO4.
Removal of the solvent by evaporation gave 25b (23.5 g,
73% in 3 steps, beige amorphous powder): TLC
Rf=0.28 (CHCl3/MeOH/H2O, 65/35/8); 1H NMR
(300 MHz, CD3OD) d 7.22 (t, J=8.1 Hz, 1H), 6.96–6.90
(m, 2H), 6.84–6.81 (m, 1H), 5.01 (d, J=4.2 Hz, 1H),
4.71–4.60 (m, 1H), 3.78 (s, 3H), 2.26 (t, J=7.2 Hz, 2H),
1436, 1343, 1255, 1105 cmÀ1 1H NMR (300 MHz,
;
CD3OD) d 7.12 (t, J=8 Hz, 1H), 7.04–6.94 (m, 2H),
6.78–6.72 (m, 1H), 4.66–4.52 (m, 1H), 4.49 (m) and 4.38
(brs) total 1H, 3.93 (t, J=6.6 Hz, 2H), 3.78 (s, 3H),
1.64–1.48 (m, 2H), 1.40–1.20 (m, 6H), 1.05 (d, J=6.6
Hz, 3H), 0.88 (m, 3H); optical rotation [a]2D6 À48.83 (c
1.2, MeOH); mp >300 ꢀC (decomposed); MS (FAB,
Pos.) m/z 434 (M+H)+, 412, 390; HRMS (MALDI-
+
.
TOF, Pos.) calcd for C17H26NO7P 2Na+H : 434.1321;
found: 434.1363.
Bis[tris(hydroxymethyl)aminomethane] salt of compound
.
40b (40b TRIS). Compound 4 was one of the best inhi-
bitors for TNF-a production, so an alternative salt was
prepared. Amixture of 40b (21.8 g, 56.2 mmol) and

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3799
Tris(hydroxymethyl)aminomethane (13.6 g, 112 mmol)
in EtOH/H2O (20/1, 200 mL) was heated under reflux
until the reaction mixture changed from a suspension to
a clear solution. After cooling to room temperature, the
resultant precipitates were collected by filtration and
tallization from EtOAc/n-hexane to afford 33 as a white
crystal (1.09 g, 88% yield): TLC Rf=0.33 (n-hexane/
EtOAc, 2/1); H NMR (300 MHz, CDCl3) d 7.40–7.25
(m, 5H), 5.32 (d, J=8.1 Hz, 1H), 4.62–4.50 (m, 1H),
4.10–3.95 (m, 1H), 2.10–1.95 (br, 1H), 1.43 (s, 9H), 1.23
(d, J=6.3 Hz, 3H).
1
.
dried under reduced pressure to give 40b TRIS as a
white powder (24.7 g, 70% yield): TLC Rf=0.20
(CHCl3/MeOH/H2O, 65/25/4); MS (FAB, Pos.) m/z
511, 412, 390, 122; IR (KBr) 2931, 1691, 1611, 1541,
1491, 1466, 1077 cmÀ1; 1H NMR (300 MHz, CD3OD) d
7.18 (t, J=8.0 Hz, 1H), 6.69 (s) and 6.95 (d, J=8.0 Hz)
total 2H, 6.80–6.75 (m, 1H), 4.66–4.52 (m, 2H), 3.96 (t,
J=6.6 Hz, 2H), 3.78 (s, 3H), 3.58 (s, 12H), 1.64–1.50
(m, 2H), 1.42–1.24 (m, 6H), 1.08 (d, J=6.3 Hz, 3H),
0.94–0.84 (m, 3H); optical rotation [a]2D6 À32.16 (c 1.0,
MeOH); mp 130–133 ꢀC; Anal calcd for C17H28NO7
General method C
(1S,2S)-1-Methyl-2-octanoylamino-2-phenylethyl diso-
diumphosphate (6). To a stirred mixture of 33 (873 mg,
3.48 mmol) and DMAP (636 mg, 5.22 mmol) in pyri-
dine (10 mL) was added bis(2,2,2-trichloroethyl) chlor-
idophosphate (1.98 g, 5.22 mmol) at 0 ꢀC. Stirring was
continued at room temperature for 18 h. The reaction
mixture was diluted with EtOAc. The organic layer was
successively washed with 1 M HCl, H2O and brine
before being dried over Na2SO4. Removal of the solvent
by evaporation gave 34 as a colorless oil (3.15 g, quant)
which was used for the next reaction without further
purification: TLC Rf=0.50 (n-hexane/EtOAc, 2/1). A
solution of 34 (3.15 g, 3.48 mmol) in 4M HCl/dioxane
(20 mL) was stirred at room temperature for 2 h.
Removal of the solvent by evaporation gave a residue
which was solidified by Et2O to afford (1S,2S)-2-amino-
1-methyl-2-phenylethyl bis(2,2,2-trichloroethyl) phos-
phate hydrochloride as a white powder (1.73 g, 94%
yield): TLC Rf=0.50 (CHCl3/MeOH); 1H NMR
(200 MHz, CDCl3+CD3OD) d 7.62–7.50 (m, 2H), 7.50–
7.38 (m, 3H), 5.25–5.05 (m, 1H), 4.82–4.55 (m, 4H), 4.40
(d, J=8.8 Hz, 1H), 1.40 (d, J=6.2 Hz, 3H). To a stirred
suspension of (1S,2S)-2-amino-1-methyl-2-phenylethyl
bis(2,2,2-trichloroethyl) phosphate hydrochloride (1.72
g, 3.24 mmol) in CH2Cl2 (16 mL) were added octanoyl
chloride (631 mg, 3.89 mmol) and pyridine (1.31 mL,
16.2 mmol) at 0 ꢀC. Stirring was continued at room
temperature for 45 min. Removal of the solvent by
evaporation gave (1S,2S)-1-methyl-2-phenyl-2-(octa-
noylamino)ethyl bis(2,2,2-trichloroethyl) phosphate
which was used for the next reaction without further
purification. To a stirred solution of (1S,2S)-1-methyl-2-
phenyl-2-(octanoylamino)ethyl bis(2,2,2-trichloroethyl)
phosphate (1.96 g, 3.24 mmol) in pyridine (15 mL)/
AcOH (3 mL) was added Zn powder (1.90 g, 29.2
mmol) at 0 ꢀC. Stirring was continued at room tem-
perature for 2 h. Removal of the Zn powder by filtration
through a pad of Celite followed by evaporation affor-
ded an oily residue which was dissolved in 5M NaOH
and extracted with EtOAc. The aqueous layer was
acidified by 2M HCl and extracted with EtOAc. The
organic layer was successively washed with H2O and
brine before being dried over Na2SO4. Removal of the
solvent by evaporation gave a residue, which was pur-
ified by column chromatography on silica gel (Merck
7734, CHCl3/MeOH/NH4OH, 65/25/2-CHCl3/MeOH/
H2O, 65/25/4) to afford a solid. The solid was dissolved
in 2M HCl/EtOAc. After the organic layer was succes-
sively washed with H2O and brine, it was dried over
Na2SO4. Removal of the solvent by evaporation gave a
residue, which was solidified by n-hexane to obtain
(1S,2S)-1-methyl-2-octanoylamino-2-phenylethyl dihy-
drogen phosphate as a white powder (697 mg, 60%
yield from 34). The title compound 6 was prepared from
.
.
P 2C4H11NO3 H2O: C, 46.22; H, 8.07; N, 6.47; Found:
C, 46.41; H, 8.19; N, 6.49.
tert-Butyl (1S,2S)-2-hydroxy-1-phenylpropylcarbamate
(33). (1S,2R)-1-Amino-1-phenylpropan-2-ol hydrochlo-
ride 26a was prepared from (1E)-prop-1-enylbenzene
22a according to the known procedure:3a,b 26a: white
powder; TLC Rf=0.50 (EtOAc/AcOH/H2O 3/1/1); MS
(APCI, Pos, 20eV) m/z 152, 135, 106; 1H NMR
(200 MHz, CDCl3+CD3OD) d 7.50–7.30 (m, 5H), 4.43–
4.28 (m, 1H), 4.21 (d, J=3.6 Hz, 1H), 1.04 (d, J=6.6
Hz, 3H). To a stirred solution of 26a (1.26 g, 6.72
mmol) in dioxane (35 mL) were added 2 M NaOH (6.8
mL, 13.4 mmol) and di-tert-butyl dicarbonate (1.76 g,
8.06 mmol) at 0 ꢀC and stirring was continued at that
temperature for 3 h. The reaction mixture was diluted
with EtOAc. After the organic layer was successively
washed with H2O and brine, it was dried over Na2SO4.
Removal of the solvent by evaporation gave a residue
which was solidified with n-hexane to afford tert-butyl
(1S,2R)-2-hydroxy-1-phenylpropylcarbamate 29 as a
white powder (92% yield): TLC Rf=0.53 (n-hexane/
1
EtOAc, 1/1); H NMR (300 MHz, CDCl3) d 7.40–7.25
(m, 5H), 5.45–5.30 (br, 1H), 4.70–4.52 (br, 1H), 4.15–
4.00 (m, 1H), 1.90–1.70 (br, 1H), 1.41 (s, 9H), 1.09 (d,
J=6.3 Hz, 3H). To a stirred mixture of 29 (1.54 g, 6.12
mmol), PPh3 (1.60 g, 6.12 mmol) and benzoic acid (747
mg, 6.12 mmol) in THF (60 mL) was added DEAD
(2.3 M in toluene, 2.7 mL, 6.12 mmol) at 0 ꢀC and stir-
ring was continued at that temperature for 2 h.
Removal of the solvent by evaporation gave a residue
which was purified by column chromatography on silica
gel (Merck 7734, n-hexane/EtOAc, 3/1) to afford an oil,
which was solidified by n-hexane to obtain (1S, 2S)-2-
[(tert-butyloxycarbonyl)amino]-1-methyl-2-phenylethyl
benzoate as a white powder (83% yield): TLC Rf=0.58
1
(n-hexane/EtOAc, 3/1); H NMR (200 MHz, CDCl3) d
8.08–7.98 (m, 2H), 7.62–7.20 (m, 8H), 5.50–5.35 (m,
1H), 5.20 (d, J=9.2 Hz, 1H), 4.98–4.80 (m, 1H), 1.40–
1.20 (m, 12H). To a stirred solution of (1S,2S)-2-[(tert-
butyloxycarbonyl)amino]-1-methyl-2-phenylethyl
benzoate (1.74 g, 4.9 mmol) in MeOH (50 mL) was
added NaOMe (53 mg, 0.98 mmol) at room temperature
and stirring was continued at that temperature for 20 h.
Removal of the solvent by evaporation gave a residue
which was purified by column chromatography on silica
gel (Merck 7734, n-hexane/EtOAc, 3/1–1/1) and recrys-

3800
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
(1S,2S)-1-methyl-2-octanoylamino-2-phenylethyl dihy-
drogen phosphate according to the same procedure as
described for the preparation of 2 from 25b: white
powder; TLC Rf=0.43 (CHCl3/MeOH/H2O, 65/35/8);
IR (KBr) 3399, 2928, 2857, 2359, 1637, 1549, 1496,
1455, 1378, 1301, 1204, 1090 cmÀ1; 1H NMR (300 MHz,
CD3OD) d 7.40–7.30 (m, 2H), 7.30–7.15 (m, 3H), 4.41–
4.25 (m, 2H), 2.38–2.15 (m, 2H), 1.63–1.45 (m, 2H),
1.38–1.12 (m, 8H), 1.06 (d, J=6.0 Hz, 3H), 0.87 (t,
J=6.8 Hz, 3H); optical rotation [a]2D5 +17.3 (c 1.0,
MeOH); MS (FAB, Pos.) m/z 424 (M+Na)+, 402
2/1) to obtain tert-butyl (1S,2S)-2-hydroxy-1-(3-meth-
oxyphenyl)propylcarbamate (1.36 g, 51% yield): TLC
Rf=0.52 (n-hexane/EtOAc, 1/1); H NMR (300 MHz,
1
CD3OD) d 7.28 (dd, J=9.0, 7.2 Hz, 1H), 6.87–6.79 (m,
3H), 5.30 (brs, 1H), 4.53 (brs, 1H), 4.03 (brs, 1H), 3.80
(s, 3H), 1.43 (brs, 9H), 1.22 (d, J=6.3 Hz, 3H). Asolu-
tion of tert-butyl (1S,2S)-2-hydroxy-1-(3-methox-
yphenyl)propylcarbamate (1.34 g, 6.72 mmol) in 4 M
HCl/dioxane (20 mL) was stirred at 0 ꢀC for 2 h.
Removal of the solvent by evaporation followed by the
dissolution of the residue in toluene was repeated sev-
eral times to give (1S,2S)-1-amino-1-(3-methox-
yphenyl)propan-2-ol hydrochloride as an oil. To a
stirred mixture of (1S,2S)-1-amino-1-(3-methox-
yphenyl)propan-2-ol hydrochloride in THF (50 mL)
and 0.5M NaHCO3 (50 mL) was added octanoyl chlo-
ride (1.1 g, 6.79 mmol) in dioxane (5 mL) at 0 ꢀC and
stirring was continued at that temperature for 4 h. The
reaction mixture was diluted with EtOAc. The organic
layer was successively washed with 1M HCl and brine
before being dried over MgSO4. Removal of the solvent
by evaporation afforded a residue, which was purified
by column chromatography on silica gel (Merck 7734,
n-hexane/EtOAc, 2/1–1/1) to afford 38 (1.53 g, 100%
(M+H)+, 380; HRMS (MALDI-TOF, Pos.) calcd for
+
.
C17H26NO5P 2Na+ H : 402.1422; found: 402.1437.
(1R,2R)-1-Methyl-2-octanoylamino-2-phenylethyl diso-
diumphosphate (8). The title compound 8 was prepared
from 23a according to the general method C; TLC
Rf=0.24 (CHCl3/MeOH/H2O, 65/25/4); IR (KBr) 3408,
1636, 1455, 1091, 985 cmÀ1 1H NMR (300 MHz,
;
CD3OD) d 7.35 (brd, J=7.2 Hz, 2H), 7.26 (brt, J=7.2
Hz, 2H), 7.18 (m, 1H), 4.40–4.24 (m, 2H), 2.38–2.10 (m,
2H), 1.55 (m, 2H), 1.35–1.10 (m, 8H), 1.05 (d, J=5.7 Hz,
3H), 0.87 (t, J=6.6 Hz, 3H); optical rotation [a]2D4 À14.9
(c 1.0, MeOH); MS (FAB, Pos.) m/z 424 (M+Na)+, 402
(M+H)+, 380; HRMS (MALDI-TOF, Pos.) calcd for
1
yield): TLC Rf=0.22 (n-hexane/EtOAc, 1/1); H NMR
+
.
C17H26NO5P 2Na+H : 402.1422; found: 402.1428.
(200 MHz, CD3OD) d 7.26 (dd, J=8.4, 8.4 Hz, 1H),
6.87–6.78 (m, 3H), 6.29 (brd, J=8.4 Hz, 1H), 4.87 (dd,
J=8.4, 4.0 Hz, 1H), 4.09 (m, 1H), 3.80 (s, 3H), 2.26
(brt, J=8.2 Hz, 2H), 1.70–1.60 (m, 2H), 1.40–1.25 (m,
8H), 1.20 (d, J=6.6 Hz, 3H), 0.87 (brt, J=6.6 Hz, 3H).
(1R,2S)-1-Methyl-2-octanoylamino-2-phenylethyl diso-
diumphosphate (10). The title compound 10 was pre-
pared from 29 according to the general method C; off-
white powder; TLC Rf=0.23 (CHCl3/MeOH/H2O, 65/
1
25/4); IR (KBr) 3346, 1639, 1538, 1454, 1093 cmÀ1; H
(1S,2S)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-
no)ethyl disodiumphosphate (7). The title compound 7
was prepared from compound 38 according to the Gen-
eral Method B: 94% yield; TLC Rf=0.22 (CHCl3/MeOH/
H2O, 65/35/4); IR (KBr) 3412, 2929, 1638, 1457, 1266,
1092, 984, 801 cmÀ1; 1H NMR (200MHz, CD3OD) d 7.16
(dd, J=8.0, 8.0 Hz, 1H), 6.92 (m, 2H), 6.74 (ddd, J=8.0,
2.4, 1.2 Hz, 1H), 4.35–4.29 (m, 2H), 3.76 (s, 3H), 2.38–2.12
(m, 2H), 1.53 (m, 2H), 1.30–1.20 (m, 8H), 1.05 (d, J=6.0
Hz, 3H), 0.86 (brt, J=6.6 Hz, 3H); optical rotation [a]D25
+17.0 (c 1.0, MeOH); MS (FAB, Pos.) m/z 454
NMR (300 MHz, CD3OD) d 7.44 (d, J=6.9 Hz, 2H),
7.28–7.12 (m, 3H), 4.61 (m, 2H), 2.35–2.18 (m, 2H), 1.55
(m, 2H), 1.25 (m, 8H), 1.04 (d, J=6.3 Hz, 3H), 0.87 (t,
J=6.3 Hz, 3H); optical rotation [a]2D5 +60.9 (c 1.25,
MeOH); MS (FAB, Pos.) m/z 402 (M+H)+, 380, 358;
.
HRMS (MALDI-TOF, Pos.) calcd for C17H26NO5P 2-
Na+H+: 402.1422; found: 402.1416.
N-[(1S,2S)-2-Hydroxy-1-(3-methoxyphenyl)propyl]octa-
namide (38). tert-Butyl (1S,2R)-2-hydroxy-1-(3-methox-
yphenyl)propylcarbamate 37 was prepared from 26b
according to the same procedure as described for the
preparation of tert-butyl (1S,2R)-2-hydroxy-1-phenyl-
propylcarbamate from 26a. (1S,2R)-2-[(tert-Butyloxy-
carbonyl)amino]-1-methyl-2-(3-methoxyphenyl)ethyl
benzoate was prepared from 37 according to the same
procedure as described for the preparation of (1S,2S)-2-
[(tert-butyloxycarbonyl)amino]-1-methyl-2-phenylethyl
benzoate from tert-butyl (1S,2R)-2-hydroxy-1-phenyl-
propylcarbamate (96% yield). To a stirred solution of
(1S,2R)-2-[(tert-butyloxycarbonyl)amino]-1-methyl-2-
(3-methoxyphenyl)ethyl benzoate (3.48 g, 9.5 mmol) in
THF (50 mL)/MeOH (20 mL) was added 1 M NaOH
(20 mL, 20 mmol) at room temperature and stirring was
continued at that temperature for 20 h. Removal of the
solvent by evaporation gave a reside, which was extrac-
ted with EtOAc. The organic layer was successively
washed with 1 M NaOH and brine before being dried
over MgSO4. Removal of the solvent by evaporation
afforded a residue which was purified by column chro-
matography on silica gel (Merck 7734, n-hexane/EtOAc,
(M+Na)+, 432 (M+H)+; HRMS (MA LDI-TOF, Pos.)
+
.
calcd for C18H28NO6P 2Na+H : 432.1528; found:
432.1524.
(1R,2R)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-
no)ethyl disodiumphosphate (9). The title compound 9
was prepared from 36 according to the general method
B: white powder; TLC Rf=0.28 (CHCl3/MeOH/H2O,
65/25/4); IR (KBr) 3328, 2929, 1638, 1457, 1265, 1091,
801 cmÀ1 1H NMR (200MHz, CD3OD) d 7.16 (dd,
;
J=8.0, 8.0 Hz, 1H), 6.92 (m, 2H), 6.74 (ddd, J=8.0, 2.4,
1.2 Hz, 1H), 4.35–4.29 (m, 2H), 3.76 (s, 3H), 2.38–2.12 (m,
2H), 1.53 (m, 2H), 1.30–1.20 (m, 8H), 1.05 (d, J=6.0 Hz,
3H), 0.86 (brt, J=6.6 Hz, 3H); optical rotation [a]2D5 À16.6
(c 1.0, MeOH); MS (FAB, Pos.) m/z 454 (M+Na)+, 432
(M+H)+; HRMS (MALDI-TOF, Pos.) calcd for
+
.
C18H28NO6P 2Na+H : 432.1528; found: 432.1568.
(1R,2S)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-
no)ethyl disodiumphosphate (11). The title compound 11
was prepared from 27 according to the General Method

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3801
B: white powder; TLC Rf=0.20 (CHCl3/MeOH/H2O,
65/25/4); IR (KBr) 3333, 2928, 1638, 1535, 1256, 1088,
(m, 3H), 6.43 (d, J=8.7 Hz, 1H), 3.79 (s, 3H), 2.24–2.19
(m, 2H), 1.68–1.58 (m, 2H), 1.30–1.07 (m, 8H), 0.86 (t,
J=6.6 Hz, 3H).
1
984 cmÀ1; H NMR (300 MHz, CD3OD) d 7.13 (dd,
J=8.1, 8.1 Hz, 1H), 7.01–6.99 (m, 2H), 6.73 (ddd,
J=8.1, 2.4, 1.2 Hz, 1H), 4.63–4.57 (m, 2H), 3.77 (s, 3H),
2.36–2.18 (m, 2H), 1.55 (m, 2H), 1.35–1.20 (m, 8H), 1.05
1,1-Dimethyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl
disodiumphosphate (13). The title compound 13 was
prepared from 44 according to the general method B:
TLC Rf=0.31 (CHCl3/MeOH/H2O, 65/25/4); IR (KBr)
3321, 2928, 1621, 1538, 1490, 1455, 1388, 1368, 1243,
(d, J=6.3 Hz, 3H), 0.86 (brt, J=6.6 Hz, 3H); optical
25
rotation [a]
+65.4 (c 1.0, MeOH); MS (FAB, Pos.)
D
m/z 432 (M+H)+; HRMS (MALDI-TOF, Pos.) calcd
+
for C18H28NO6P 2Na+H : 432.1528; found: 432.1505.
1088 cmÀ1 1H NMR (300 MHz, CD3OD) d 7.13 (t,
;
.
J=7.8 Hz, 1H), 7.09–7.05 (m, 2H), 6.73–6.69 (m, 1H),
4.49 (d, J=1.5 Hz, 1H), 3.78 (s, 3H), 2.27 (t, J=7.2 Hz,
2H), 1.66 (s, 3H), 1.62–1.53 (m, 2H), 1.32–1.23 (m, 8H),
1.16 (s, 3H), 0.86 (t, J=6.3 Hz, 3H); MS (FAB, Pos.) m/
z 446 (M+H)+; HRMS (MALDI-TOF, Pos.) calcd for
Methyl [(tert-butoxycarbonyl)amino](3-methoxypheny-
l)acetate (42). Methyl amino(3-hydroxyphenyl)acetate
41 was prepared from 3-(benzyloxy)benzaldehyde
according to the same procedure as described in the
preceding paper.2c,8 To a stirred solution of 41 (5.0 g,
27.6 mmol) in CHCl3 (150 mL) was added dropwise a
solution of di-tert-butyl dicarbonate (5.96 g, 27.3 mmol)
in CHCl3 (20 mL) at room temperature and stirring was
continued at that temperature for 5 h. The reaction was
quenched with NH4OH and extracted with CHCl3. The
organic layer was successively washed with saturated
NaHCO3, H2O and brine before being dried over
Na2SO4. Removal of the solvent by evaporation gave
methyl [(tert-butoxycarbonyl)amino](3-hydroxyphenyl)-
acetate which was used for the next reaction without
further purification: TLC Rf=0.39 (n-hexane/EtOAc, 1/1).
+
.
C19H30NO6P 2Na+H : 446.1684; found: 446.1651.
tert-Butyl (1-hydroxycyclobutyl)(phenyl)methylcarbamate
(47). To a stirred mixture of cyclobutanone (5.0 g, 71
mmol) and trimethylsilyl cyanide (7.03 g, 71 mmol) in
CH2Cl2 (100 mL) was added dropwise a solution of tri-
methylsilyl trifluoromethanesulfonate (1.27 mL, 7.1
mmol) in CH2Cl2 (50 mL) at À78 ꢀC and stirring was
continued at that temperature for 2.5 h. The reaction
was quenched with a few drops of pyridine. The result-
ing mixture was poured into saturated NaHCO3 aq and
extracted with CH2Cl2. The organic layer was succes-
sively washed with H2O and brine before being dried
over MgSO4. Removal of the solvent by evaporation
To
a
stirred mixture of methyl [(tert-butoxy-
carbonyl)amino](3-hydroxyphenyl)acetate (27.6 mmol)
and K2CO3 (11.5 g, 82.9 mmol) in DMF (54 mL) was
added iodomethane (5.16 mL, 82.9 mol) at room tem-
perature and stirring was continued at that temperature
for 2 h. The reaction mixture was treated with H2O and
then extracted with EtOAc. After the organic layer was
successively washed with 1 M HCl, H2O and brine, it
was dried over Na2SO4. Removal of the solvent by
evaporation gave 42 (7.91 g, 97% yield in 2 steps): TLC
1
gave 46 as a colorless oil (7.08 g, 59% yield): H NMR
(300 MHz, CDCl3) d 3.09 (t, J=8.1 Hz, 2H), 2.63–2.58
(m, 1H), 2.40–2.23 (m, 1H), 2.03–1.80 (m, 2H), 0.22 (s,
6H), 0.15 (s, 3H). To a stirred solution of PhMgI in
Et2O (2M, 23.1 mL, 46.1 mmol) was added dropwise a
solution of 46 (7.08 g, 41.9 mmol) in Et2O (50 mL) at
0 ꢀC. Stirring was continued at room temperature for 2
days. To this resulting mixture a solution of NaBH4
(1.71 g, 45.3 mmol) in MeOH (50 mL) was added at
0 ꢀC and stirring was continued at room temperature for
4.5 h. Next H2O (25 mL) and 1 M HCl (100 mL) were
added to the stirred reaction mixture. Stirring was con-
tinued at room temperature for 1 h. After removal of
the organic layer, the aqueous layer was adjusted to pH
9 by 1 M NaOH and extracted with CHCl3. The organic
layer was dried over K2CO3. Removal of the solvent by
evaporation gave 1-[amino(phenyl)methyl]cyclobutanol
which was used for the next reaction without further
purification. The title compound 47 was prepared from
1-[amino(phenyl)methyl]cyclobutanol according to
essentially the same procedure as described for the pre-
paration of methyl [(tert-butoxycarbonyl)amino](3-
hydroxyphenyl)acetate from 41: 13% yield in 3 steps;
off-white solid; TLC Rf=0.37 (n-hexane/EtOAc, 3/1),
1
Rf=0.63 (n-hexane/EtOAc, 1/1); H NMR (200 MHz,
CDCl3) d 7.31–7.22 (m, 1H), 6.95–6.82 (m, 3H), 5.52–
5.48 (m, 1H), 5.28 (d, J=7.0 Hz, 1H), 3.79 (s, 3H), 3.71
(s, 3H), 1.42 (s, 9H).
tert-Butyl 2-hydroxy-1-(3-methoxyphenyl)-2-methylpro-
pylcarbamate (43). To a stirred solution of 42 (4.07 g,
13.8 mmol) in THF (64 mL) was added dropwise
methylmagnesium bromide (0.84 M in Et2O 63.9 mL,
53.4 mmol) at 0 ꢀC. Stirring was continued at room
temperature for 2 h. The reaction was quenched with
1 M HCl and extracted with EtOAc, and the organic
layer was washed with brine before being dried over
Na2SO4. Removal of the solvent by evaporation gave
43: TLC Rf=0.50 (n-hexane/EtOAc, 1/1); 1H NMR
(300 MHz, CDCl3) d 7.27–7.22 (m, 1H), 7.11–6.79 (m,
3H), 5.50 (d, J=6.9 Hz, 1H), 4.48 (d, J=6.9 Hz, 1H),
3.83 (s, 3H), 1.40 (s, 9H), 1.32 (s, 3H), 1.06 (s, 3H).
1
MS (APCI, Pos. 40eV) m/z 278 (M+H)+, 178, 161; H
NMR (300 MHz, CDCl3) d 7.40–7.20 (m, 5H), 5.51 (br,
1H), 4.76 (d, J=8.4 Hz, 1H), 2.40–2.23 (m, 1H), 2.20–
2.00 (m, 2H), 1.93–1.63 (m, 3H), 1.43 (s, 9H).
N-[2-hydroxy-1-(3-methoxyphenyl)-2-methylpropyl]oc-
tanamide (44). The title compound 44 was prepared
from 43 according to essentially the same procedures as
described for the preparation of 38 from tert-butyl
(1S,2S)-2-hydroxy-1-(3-methoxyphenyl)propylcarbamate:
TLC Rf=0.48 (n-hexane/EtOAc, 1/2); 1H NMR
(300 MHz, CDCl3) d 7.24 (t, J=7.2 Hz, 1H), 6.88–6.79
1-(Phenylmethyl)-1-(octanoylamino)cyclobutyl dihydrogen
phosphate (14). The title compound 14 was prepared
from 47 according to the general method C: TLC
Rf=0.44 (CHCl3/MeOH/H2O, 65/25/4); IR (KBr) 3252,
2930, 2858, 1557, 1456, 1010 cmÀ1; 1H NMR (300 MHz,

3802
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
CD3OD) d 7.45–7.38 (m, 2H), 7.35–7.20 (m, 3H), 5.15
(d, J=2.4 Hz, 1H), 2.88 (q, J=12 Hz, 1H), 2.45 (q,
J=12 Hz, 1H), 2.32–2.14 (m) and 2.27 (t, J=6 Hz) total
4H, 1.94–1.80 (m, 1H), 1.78–1.52 (m, 3H), 1.40–1.16 (m,
8H), 0.88 (t, J=6.6 Hz, 3H); MS (FAB, Pos.) m/z 406
(M+Na)+, 384 (M+H)+; HRMS (MALDI-TOF,
Pos.) calcd for C19H30NO5P+Na+: 406.1759; found:
406.1731.
prepared from 53 according to the same procedure as
described for the preparation of dibenzyl (1S,2R)-1-
methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl
phosphate from 24b: colorless oil; 70% yield from 53.
1
54: H NMR (300 MHz, CDCl3) d 7.63–7.58 (m, 5H),
7.40–7.20 (m, 20H), 5.49 (dd, J=8.1, 3.0 Hz, 1H), 5.20–
4.85 (m, 5H), 4.65–4.55 (m, 1H), 3.65–3.0 (m, 2H), 2.20–
2.10 (m, 2H), 1.65–1.50 (m, 2H), 1.38–1.20 (m, 8H), 1.07
(s, 9H), 0.85 (m, 3H). (1R,2R)-1-tert-Butyldiphenylsily-
loxymethyl-2-octanonyamino-2-phenylethyl dihydrogen
phosphate was prepared from 54 according to the same
procedure as described for the preparation of 25b
from dibenzyl (1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-
(octanoylamino)ethyl phosphate: TLC Rf=0.50
(CHCl3/MeOH/H2O, 65/25/4); 1H NMR (300 MHz,
CDCl3) d 7.70–7.60 (m, 4H), 7.45–7.20 (m, 1H), 5.50 (d,
J=3.3 Hz, 1H), 4.63–4.58 (m, 1H), 3.74 (dd, J=10.5,
4.2 Hz, 1H), 3.42 (dd, J=10.5, 7.8 Hz, 1H), 2.24 (t,
J=7.2 Hz, 2H), 1.65–1.53 (m, 2H), 1.38–1.20 (m, 8H),
1.09 (s, 9H), 0.89 (t, J=6.3 Hz, 3H). To a stirred solu-
tion of (1R,2R)-1-tert-butyldiphenylsilyloxymethyl-2-
octanolyamino-2-phenylethyl dihydrogen phosphate
(1.8 g, 3 mmol) in THF (20 mL) was added TBAF (1 M
in THF, 7.5 mL, 7.5 mmol) at room temperature and
stirring was continued at that temperature for 15 min.
The reaction was quenched with H2O and the pH
adjusted to 12 with 2 M NaOH before the organic layer
was extracted with Et2O. The aqueous layer was acid-
ified with 2 M HCl and extracted with EtOAc. Next, the
organic layer was successively washed with H2O and
brine before being dried over Na2SO4. Removal of the
solvent by evaporation gave 55 as an amorphous pow-
der (700 mg, 63% yield): TLC Rf=0.23 (CHCl3/MeOH/
H2O, 65/25/4); 1H NMR (300 MHz, CDCl3) d 7.24–7.20
(m, 5H), 5.28 (d, J=5.0 Hz, 1H), 4.60–4.43 (m, 1H),
3.60–3.40 (m, 2H), 2.28 (t, J=7.5 Hz, 2H), 1.60 (m, 2H),
1.40–1.20 (m, 8H), 0.88 (m, 3H). 15: TLC Rf=0.23
(CHCl3/MeOH/H2O, 65/25/4); IR (KBr) 3418, 2928,
2856, 1644, 1539, 1455, 1094 cmÀ1; 1H NMR (300 MHz,
CD3OD) d 7.44 (d, J=7.2 Hz, 2H), 7.26–7.14 (m, 3H),
4.93 (d, J=2.7 Hz, 1H), 4.48 (m, 1H), 3.38–3.25 (m,
2H), 2.36–2.18 (m, 2H), 1.55 (m, 2H), 1.38–1.18 (m,
8H),0.86 (t, J=6.3 Hz, 3H); MS (FAB, Pos.) m/z 418
(2R,3R)-3-Amino-3-phenylpropane-1,2-diol hydrochloride
(51). Amixture of [(2 S,3S)-3-phenyloxiran-2-yl]metha-
nol 49 (5.6 g, 37.3 mmol), NaN3 (4.86 g, 74.6 mmol) and
NH4Cl (991 mg, 17.8 mmol) in dioxane (50 mL)/H2O (5
mL) was heated at 80 ꢀC for 2 days with stirring. After
cooling, the reaction mixture was diluted with EtOAc.
The organic layer was successively washed with H2O
and brine before being dried over Na2SO4. Removal of
the solvent by evaporation gave a residue which was
purified by column chromatography on silica gel
(Merck 7734, n-hexane/EtOAc, 3/1–1/1) to afford 50 as
a colorless oil (4.28 g, 59% yield): TLC Rf=0.38 (n-
hexane/EtOAc, 1/1); 1H NMR (300 MHz, CDCl3) d
7.50–7.35 (m, 5H), 4.65 (d, J=6.9 Hz, 1H), 3.60 (m,
1H), 3.80–3.65 (m, 2H), 2.19 (br, 1H), 1.97 (br, 1H). A
mixture of 50 (4.28 g, 22 mmol) and 10% Pd-C (430 mg)
in EtOH (60 mL)/6 M HCl (3.7 mL) was stirred at room
temperature under an atmospheric pressure of hydrogen
for 14 h. Removal of the catalyst by filtration through a
pad of Celite followed by evaporation afforded 51 as a
1
white solid: H NMR (300 MHz, CD3OD) d 7.54–7.46
(m, 2H), 7.45–7.39 (m, 3H), 4.44 (d, J=3.9 Hz, 1H),
4.08–4.00 (m, 1H), 3.40 (dd, J=11.0, 5.5 Hz, 1H), 3.28
(dd, J=11.0, 6.0 Hz, 1H).
N-[(1R,2R)-2,3-dihydroxy-1-phenylpropyl]octanamide
(52). The title compound 52 was prepared from 51
according to essentially the same procedure as described
for the preparation of 24b from 23b: white solid; 83%
yield; TLC Rf=0.55 (EtOAc); 1H NMR (300 MHz,
CDCl3) ꢀ 7.43–7.25 (m, 5H), 6.16 (d, J=7.2 Hz, 1H),
5.01 (t, J=7.8 Hz, 1H), 3.86 (dt, J=7.8, 3.0 Hz, 1H),
3.66 (dq, J=12.6, 3.0 Hz, 2H), 2.21 (t, J=7.5 Hz, 2H),
1.62 (m, 2H), 1.40–1.10 (m, 8H), 0.87 (m, 3H).
(M+H)+, 396, 374; HRMS (MALDI-TOF, Pos.) calcd
+
.
for C17H26NO6P 2Na+H : 418.1371; found: 418.1407.
(1R,2R)-1-Hydroxymethyl-2-octanolyamino-2-phenylethyl
disodiumphosphate (15). The title compound 15 was
prepared from 55 according to the general method B: to
a stirred mixture of 52 (1.3 g, 4.3 mmol) and imidazole
(589 mg, 8.7 mmol) in DMF (20 mL) was added
TBDPSCl (1.4 g, 5.2 mmol) at room temperature and
stirring was continued at that temperature for 18 h. The
reaction mixture was diluted with Et2O. The organic
layer was successively washed with saturated NaHCO3
aq, H2O and brine before being dried over Na2SO4.
Removal of the solvent by evaporation gave 53 quanti-
tatively as a colorless oil (2.3 g): TLC Rf=0.35 (n-hex-
N-[(1R,2S)-2-Hydroxy-1,2-dipenylethyl]octanamide (57).
The title compound 57 was prepared from (1S,2R)-2-
amino-1,2-diphenylethanol 56 according to essentially
the same procedure as described for the preparation of
24b from 23b: white powder; 84% yield; 1H NMR
(300 MHz, CDCl3) d 8.11 (d, J=9.6 Hz, 1H), 7.35–7.10
(m, 10H), 5.35 (d, J=4.8 Hz, 1H), 4.94 (dd, J=9.4, 7.8
Hz, 1H), 4.70 (m, 1H), 1.93 (t, J=7.0 Hz, 2H), 1.34–
0.90 (m, 10H), 0.85 (t, J=6.2 Hz, 3H).
1
ane/EtOAc, 2/1); H NMR (300 MHz, CDCl3) d 7.73
Method D
(m, 1H), 7.60 (m, 3H), 7.45–7.30 (m, 6H), 7.28–7.20 (m,
5H), 6.70 (d, J=8.1 Hz, 1H), 5.19 (dd, J=8.1, 4.2 Hz,
1H), 3.99 (dd, J=9.6, 4.2 Hz, 1H), 3.59 (dd, J=10.8,
4.2 Hz, 1H), 3.48 (dd, J=10.8, 5.7 Hz, 1H), 2.18 (t,
J=7.2 Hz, 2H), 1.60 (m, 2H), 1.38–1.15 (m, 8H), 1.09
(s, 9H), 0.87 (t, J=6.6 Hz, 3H). The compound 54 was
(1S,2R)-1,2-Diphenyl-2-octanoylaminoethyl disodium phos-
phate (16). To a stirred mixture of 57 (770 mg, 2.3
mmol) and tetrazole (318 mg, 4.5 mmol) in CH3CN (25
mL)–CH2Cl2 (25 mL) was added a solution of bis(2-
cyanoethyl)diisopropylamidophosphite (678 mg, 2.5

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3803
mmol) in CH3CN (5 mL) at room temperature and
stirring was continued at that temperature for 1.5 h. The
reaction mixture was poured into saturated NaHCO3 aq
and extracted with EtOAc. The organic layer was
washed with brine and dried over Na2SO4. Removal of
the solvent by evaporation gave bis(2-cyanoethyl)
(1S,2R)-1,2-diphenyl-2-(octanoylamino)ethyl phosphite
as a white powder which was used for the next reaction
without further purification (1.2 g, quant): TLC
Rf=0.43 (n-hexane/EtOAc, 1/1). To a stirred solution
of bis(2-cyanoethyl) (1S,2R)-1,2-diphenyl-2-(octanoyl-
amino)ethyl phosphite (1.2 g) in CH2Cl2 (30 mL) was
added m-CPBA(57%, 685 mg, 2.3 mmol) at 0 ꢀC and
stirring was continued at that temperature for 30 min.
To the reaction mixture saturated Na2S2O3 aq was
added and extracted with CH2Cl2. The organic layer
was successively washed with H2O, saturated NaHCO3
aq and brine before being dried over Na2SO4. The m-
chlorobenzoic acid was removed by passing the solution
through a pad of Al2O3. Removal of the solvent by
evaporation gave bis(2-cyanoethyl) (1S,2R)-1,2-diphe-
nyl-2-(octanoylamino)ethyl phosphate as a white pow-
der which was used for the next reaction without further
purification (1.0 g, 84% yield): TLC Rf=0.20 (CH2Cl2/
EtOAc, 1/1). To a stirred solution of bis(2-cyanoethyl)
(1S,2R)-1,2-diphenyl-2-(octanoylamino)ethyl phosphate
(1.0 g, 1.9 mmol) in EtOH (20 mL) was added 50%
Me2NH aq and stirring was continued under reflux for 8
h. After cooling, removal of the solvent by evaporation
gave a residue, which was dissolved in 1 M NaOH and
extracted with Et2O. The aqueous layer was acidified
with 1 M HCl and extracted with EtOAc. The organic
layer was successively washed with H2O and brine
before being dried over Na2SO4. Removal of the solvent
by evaporation gave (1S,2R)-1,2-diphenyl-2-(octanoyla-
mino)ethyl dihydrogen phosphate as a white solid (641
mg, 80% yield), which was converted to the disodium
salt 16 according to the same procedure as descried for
the preparation of 2 from 25b: TLC Rf=0.41 (CHCl3/
MeOH/H2O, 65/25/4); IR (KBr) 3062, 2927, 1652, 1520,
1453, 1114 cmÀ1; 1H NMR (300 MHz, CD3OD) d 7.20–
7.05 (m) and 7.00 (m) total 10H, 5.68 (dd, J=10.2, 2.4
Hz, 1H), 4.96 (d, J=2.4 Hz, 1H), 2.40–2.30 (m, 2H),
1.65–1.55 (m, 2H), 1.40–1.20 (m, 8H), 0.88 (t, J=6.3
Hz, 3H); MS (FAB, Pos.) m/z 486 (M+Na)+, 464
the same procedure as described for the preparation of
1
34 from 33: 50% yield; H NMR (200 MHz, CDCl3) d
7.30–7.20 (m, 4H), 6.06 (brd, J=9.0 Hz, 1H), 5.75–5.67
(m, 1H), 5.40–5.33 (m, 1H), 4.61 (d. J=7.0 Hz, 2H),
4.59 (d, J=7.0 Hz, 2H), 3.38 (dd, J=17.6, 1.6 Hz, 1H),
3.24 (dd, J=17.6, 4.4 Hz, 1H), 2.32 (dd, J=7.8, 7.2 Hz,
2H), 1.80–1.65 (m, 2H), 1.50–1.20 (m, 8H), 0.89 (t,
J=6.6 Hz, 3H).
(1R,2S)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl
disodiumphosphate (17). The title compound 17 was
prepared from 60 according to the same procedures as
described for the preparation of 6 from (1S,2S)-1-
methyl-2-phenyl-2-(octanoylamino)ethyl bis(2,2,2-tri-
chloroethyl) phosphate: 60% yield; TLC Rf=0.29
(CHCl3/MeOH/H2O, 65/25/4); IR (KBr) 3305, 1626,
1542, 1459, 1106, 986 cmÀ1 1H NMR (300 MHz,
;
CD3OD) d 7.25–7.05 (m, 4H), 5.27 (d, J=4.8 Hz, 1H),
5.01 (m, 1H), 3.35 (m, 1H), 3.03 (dd, J=16.4, 4.4 Hz,
1H), 2.50–2.25 (m, 2H), 1.70 (m, 2H), 1.50–1.20 (m, 8H),
0.90 (m, 3H).; MS (FAB, Pos.) m/z 422 (M+Na)+, 400
(M+H)+; HRMS (MALDI-TOF, Pos.) calcd for
+
.
C17H24NO5P 2Na+H : 400.1266; found: 400.1301.
Method E
Dibenzyl (1S,2R)-1-(octanoylamino)-2,3-dihydro-1H-in-
den-2-yl phosphate (61). To a stirred mixture of 59 (1.1
g, 4.0 mmol), Ph3P (1.3 g, 4.8 mmol) and dibenzyl
hydrogen phosphate (1.3 g, 4.8 mmol) in THF (12 mL)
was added DEAD (40% in toluene, 2.1 mL, 4.8 mmol)
at 0 ꢀC. Stirring was continued at room temperature for
24 h. Removal of the solvent by evaporation gave a
residue which was purified by column chromatography
on silica gel (FL60D, n-hexane/EtOAc, 5/1–4/1) twice to
afford 61 (260 mg, 12%): TLC Rf=0.55 (CH2Cl2/
1
MeOH, 19/1); H NMR (300 MHz, CDCl3) d 7.40–7.15
(m, 14H), 5.89 (d, J=8.1 Hz, 1H), 5.53 (dd, J=8.1, 7.5
Hz, 1H), 5.10–5.01 (m, 4H), 4.92 (m, 1H), 3.22 (dd,
J=15.6, 6.9 Hz, 1H), 3.01 (dd, J=15.6, 7.8 Hz, 1H),
2.17 (dd, J=8.1, 7.2 Hz, 2H), 1.80–1.55 (m, 2H), 1.40–
1.20 (m, 8H), 0.87 (t, J=6.9 Hz, 3H).
(1S,2R)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl
dihydrogen phosphate (62). The title compound 62 was
prepared from 61 according to the same procedure as
described for the preparation of 25b from dibenzyl
(1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylami-
no)ethyl phosphate: 83% yield; 1H NMR (200 MHz,
CD3OD) d 7.30–7.10 (m, 4H), 5.39 (brd, J=5.8 Hz,
1H), 4.85 (m, 1H), 3.40 (dd, J=16.0, 7.0 Hz, 1H), 3.04 (dd,
J=16.0, 6.6 Hz, 1H), 2.26 (brt, J=7.4 Hz, 2H), 1.70–1.55
(m, 2H), 1.45–1.20 (m, 8H), 0.90 (brt, J=6.6 Hz, 3H).
(M+H)+, 442; HRMS (MALDI-TOF, Pos.) calcd for
+
.
C22H28NO5P 2Na+H : 464.1579; found: 464.1557.
N-[(1R,2S)-2-Hydroxy-2,3-dihydro-1H-inden-1-yl]octana-
mide (59). The title compound 59 was prepared from
(1R,2S)-1-aminoindan-2-ol 58 according to essentially
the same procedure as described for the preparation of
24b from 23b: 86% yield; TLC Rf=0.13 (n-hexane/
1
EtOAc, 3/2); H NMR (300 MHz, CDCl3) d 7.30–7.10
(m, 4H), 6.20 (brd, J=8.4 Hz, 1H), 5.37 (dd, J=8.4, 5.1
Hz, 1H), 4.61 (ddd, J=5.1, 5.1, 2.4 Hz, 1H), 3.16 (dd,
J=16.5, 5.1 Hz, 1H), 2.93 (dd, J=16.5, 2.4 Hz, 1H),
2.31–2.26 (m, 2H), 1.70–1.50 (m, 2H), 1.40–1.20 (m,
8H), 0.88 (brt, J=6.9 Hz, 3H).
(1S,2R)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl
disodiumphosphate (18). The title compound 18 was
prepared from 62 according to the same procedure as
described for the preparation of 2 from 25b: 87% yield;
TLC Rf=0.29 (CHCl3/MeOH/H2O, 65/25/4); IR (KBr)
3430, 1631, 1543, 1461, 1096, 987 cmÀ1 1H NMR
;
(1R,2S)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl
bis(2,2,2-trichloroethyl) phosphate (60). The title com-
pound 60 was prepared from 59 according to essentially
(200 MHz, CD3OD) d 7.14 (m, 4H), 5.16 (d, J=7.6 Hz,
1H), 4.67 (ddd, J=8.0, 7.6, 7.0 Hz, 1H), 3.41 (dd,
J=15.8, 7.0 Hz, 1H), 2.94 (dd, J=15.8, 8.0 Hz, 1H),

3804
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
2.25 (m, 2H), 1.68 (m, 2H), 1.45–1.20 (m, 8H), 0.89 (m,
3H); MS (FAB, Pos.) m/z 422 (M+Na)+, 400
(M+H)+; HRMS (MALDI-TOF, Pos.) calcd for
% Inhibition=100À(CÀS)/(LÀS)Â100
C: Plasma TNF-a concentration of LPS-treated ani-
mals pretreated with a test compound.
L: Plasma TNF-a concentration of LPS-treated ani-
mals pretreated with saline.
+
.
C17H24NO5P 2Na+H : 400.1266; found: 400.1275.
N-[(1R)-2-hydroxy-1-phenylethyl]-N-methyloctanamide
(64). Ethyl (1R)-2-hydroxy-1-phenylethylcarbamate was
prepared from (2R)-2-amino-2-phenylethanol 63
according to essentially the same procedures as descri-
bed for the preparation of 24b from 23b. To the stirred
suspension of LiAlH4 (760 mg, 20 mmol) in THF (50
mL) was added dropwise a solution of ethyl (1R)-2-
hydroxy-1-phenylethylcarbamate (2.1 g, 10 mmol) in
THF (10 mL) under reflux. Stirring was continued for
11 h under reflux. To this stirred reaction mixture H2O
(0.76 mL), 15% NaOH aq (0.76 mL) and H2O (2.2 mL)
were successively added at 0 ꢀC and the vigorous stirring
was continued. The precipitates were removed by filtra-
tion and the filtrate was concentrated to give a residue,
which was dissolved in dioxane (5 mL). To the mixture
was added 4 M HCl in dioxane (3 mL) and removal of the
solvent by evaporation gave a residue, which was solidified
by Et2O/EtOH to afford (2R)-2-(methylamino)-2-phenyl-
ethanol (665 mg, 36% yield): TLC Rf=0.19 (CH2Cl2/
MeOH/AcOH, 3/1/1). The title compound 64 was pre-
S: Plasma TNF-a concentration of saline-treated ani-
mals also pretreated with saline.
Inhibition of LPS-induced plasma TNF- ꢀ production in
rats (po). The experiments were performed using male
Sprague–Dawley (CD (IGS)) rats, 6–8 weeks of age,
purchased from Charles River Breeding Laboratories
(Shizuoka, Japan). The animals were given access to
food and water ad libitum and were maintained on 12 h
light/dark cycle at 22–23 ꢀC. All experimental proce-
dures were conformed to the Animal Care and Use
Committee protocols filed at ONO Pharmaceutical Co.,
Ltd. (Osaka, Japan). In experiments under fasted con-
dition, rats were fasted for about 16 h. Test compounds
and LPS from Escherichia coli strain 055 B5 (DIFCO
LABORATORIES, Detroit, MI, USA) were dissolved
in saline. Compounds were administrated orally (1–100
mg/kg) to rats 30 min prior to intravenous injection of
LPS at the dose of 30 mg/kg. After 90 min of LPS
injection, heparinized blood was obtained. Blood was
centrifuged and plasma samples were kept frozen at
À80 ꢀC. Plasma TNF-a concentration was determined
by enzyme-linked immunosorbent assay using a com-
mercial kit (BIOSOURCE international inc., CA, USA)
according to the manufacturer’s instructions. The data
were expressed as the meanÆSEM of 5 animals per
group or ID50 values. ID50 values, which describe the
effective dose with 50% inhibition of TNF-a produc-
tion, were determined by log-linear regression analysis
(3–4 doses per compound).
pared
from
(2R)-2-(methylamino)-2-phenylethanol
according to essentially the same procedure as described
for the preparation of 24b from 23b: 67% yield: TLC
Rf=0.26 (n-hexane/EtOAc, 1/1); 1H NMR (200MHz,
CDCl3) d 7.40–7.15 (m, 5H), 5.85, 5.14 (dd, J=9.4, 5.0
Hz, 1H), 4.23–4.00 (m, 2H), 2.75 (s, 3H), 2.56–2.35 (m,
3H), 1.75–1.60 (m, 2H), 1.45–1.20 (m, 8H), 0.85 (m, 3H).
(2R)-2-[hexanoyl(methyl)amino]-2-phenylethyl dihydrogen
phosphate (19). The title compound 19 was prepared
from 64 according to the general method A: 17% yield;
TLC Rf=0.23 (CHCl3/MeOH/H2O, 65/25/4); IR (KBr)
3399, 1618, 1451, 1406, 1096, 987 cmÀ1
;
1H NMR
Inhibition of LPS-induced plasma TNF-ꢀ production in
mice (iv). Male BALB/c mice aged 8 weeks (n=5) were
injected intravenously with the test compounds (0.01–
0.1 mg/ 10 mL/kg), and then immediately given an
intraperitoneal injection of LPS (5 mg/ 10 mL/kg).
Plasma TNF-a production was determined by ELISA
90 min after the LPS challenge using a commercial kit
(GENZYME). ID50=The dosage required to inhibit
plasma TNF-a production by 50%. The data were
expressed as the meanÆSEM of 5 animals per group or
ID50 values.
(300 MHz, CD3OD, 2 rotamers) d 7.34–7.20 (m, 5H),
5.94 (dd, J=9.0, 6.0 Hz, 0.55H), 5.32 (dd, J=9.0, 5.1
Hz, 0.45H), 4.41–4.13 (m, 2H), 2.93 (s, 1.65H), 2.88 (s,
1.35H), 2.60–2.35 (m, 2H), 1.60 (m, 2H), 1.45–1.20 (m,
8H), 0.89 (m, 3H); MS (FAB, Pos.) m/z 402 (M+H)+;
.
HRMS (MALDI-TOF, Pos.) calcd for C17H26NO5P 2-
Na+H+: 402.1422; found: 402.1430.
Biological assay method
Inhibition of LPS-induced plasma TNF-ꢀ production in
rats (iv). LPS from the Escherichia coli strain 055 B5
(Difco laboratories) and the test compounds were all
dissolved in saline. Male Sprague–Dawley (CD)/IGS
rats (Charles River Inc., Japan) aged 6–8 weeks (n=5)
were injected intravenously with the test compounds
(0.01–0.1 mg/ 10 mL/kg), and then immediately given
an intraperitoneal injection of LPS (30 mg/kg). Plasma
TNF-a production was determined by ELISA90 min
after the LPS challenge using a commercial kit (R&D
Systems). ID50 Values were determined by log-linear
regression analysis (3–4 doses per compound).
ID50=The dosage required to inhibit plasma TNF-a
production by 50%. The data were expressed as the
meanÆSEM of 5 animals per group or ID50 values.
% Inhibition=100À(CÀS)/(LÀS)Â100
C: Plasma TNF-a concentration of LPS-treated ani-
mals pretreated with a test compound.
L: Plasma TNF-a concentration of LPS-treated ani-
mals pretreated with saline.
S: Plasma TNF-a concentration of saline-treated ani-
mals also pretreated with saline.
LPS-induced shock model in mice. LPS from Escherichia
coli strain 055 B5 (Difco Laboratories, Detroit, MI,
USA) and the test compounds were all dissolved in saline.
Female BALB/c mice (Charles River Inc., Shizuoka,
Japan), 7 weeks of age, were injected intravenously with
the test compounds and then immediately given an

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3787–3805
3805
intraperitoneal injection of LPS (20 mg/kg). The survi-
val rate of the mice was evaluated after 96 h. Pre-
donisolone (Shionogi Pharmaceutical Co., Ltd., 10 mg/
kg, iv), which was used as the positive control, demon-
strated an efficacy (survival rate: 13/20) equivalent to 4
(survival rate 15/20 at 0.3 mg/kg, iv) in this model. The
survival rate of the controls which were given saline was
2/20. Results are expressed as the meanÆSEM. Para-
meters were analyzed with ANOVA-Dunnett’s t-test,
but survival was analyzed with the Mantel-Cox test.
M.; Nakazawa, S.; Ogata, A.; Mori, H.; Ohno, H.; Obata, T.;
Nakai, H.; Toda, M. Bioorg. Med. Chem. Lett. 2002, 12, 907.
(c) Matsui, T.; Kondo, T.; Nishita, Y.; Itadani, S.; Nakatani,
S.; Omawari, N.; Sakai, M.; Nakazawa, S.; Ogata, A.; Mori,
H.; Terai, K.; Kamoshima, W.; Ohno, H.; Obata, T.; Nakai,
H.; Toda, M. Bioorg. Med. Chem. Accepted for publication.
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¨
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