Discovery of novel phosphonic acid derivatives as new chemical leads for inhibitors of TNF-α production

Article abstract of DOI:10.1016/S0968-0896(02)00343-7

2-(Acylamino)benzylphosphonic acid 6 derived from an artificial substrate of sphingomyelinase was found to show inhibitory activity of TNF-α production. Structural optimization was started with the chemical modification of 6. The discovery of another chem

Full text of DOI:10.1016/S0968-0896(02)00343-7

Bioorganic & Medicinal Chemistry 10 (2002) 3807–3815
Discovery of Novel Phosphonic Acid Derivatives as New Chemical
Leads for Inhibitors of TNF-ꢀ Production
Toshiaki Matsui,a,* Shinya Takahashi,b Naoki Matsunaga,b Kazunori Nakamura,a
Nagashige Omawari,c Masaru Sakai,b Wataru Kamoshima,b Kouichiro Terai,c
Hiroyuki Ohno,b Takaaki Obata,b Hisao Nakaib and Masaaki Todab
aFukui Research Institute, Ono Pharmaceutical Co., Ltd., Yamagishi, Mikuni, Sakai, Fukui 913-8638, Japan
bMinase Research Institute, Ono Pharmaceutical Co., Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
cHeadquarters, Ono Pharmaceutical Co., Ltd., Doshoumachi, Chuou, Osaka 541-8526, Japan
Received 9January 2002; accepted 5 August 2002
Abstract—2-(Acylamino)benzylphosphonic acid 6 derived from an artificial substrate of sphingomyelinase was found to show
inhibitory activity of TNF-a production. Structural optimization was started with the chemical modification of 6. The discovery of
another chemical leads 7, 8, 10 and 16 for the development of structurally new inhibitors of TNF-a production is reported.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
of the phosphate moiety of 16,7 followed by conforma-
tional analysis led to the discovery of the highly potent
inhibitors 2 and 3, which exhibited efficacy in animal
models of disease (in LPS-induced shock model of mice
or in d-(+)-galactosamine/LPS-induced hepatitis model
of rats).7 These compounds are expected to be clinically
useful although no specialized uses are yet intended.
Because of their two asymmetric centers, they are pre-
dicted to be problematic from the point of view of syn-
thetic cost. Screening was further continued to identify
more cost-effective molecules as drug candidates. We
performed rapid screening at the dose of 10 mg/kg, iv,
in mice because we could not detect any inhibitory
activity in vitro assay systems in phosphonic acid series
as well as 2-(acylamino)-2-phenylethyl disodium phos-
phates as described above. In the process, compound 6,
derived from an artificial substrate8 of sphingomyeli-
nase9 (Fig. 2), was found to show moderate activity to
inhibit the production of TNF-a. This information
prompted us to carry out the molecular design of new
chemical leads as shown in Figure 3. We here report the
discovery of 2-(acylamino)benzylphosphonic acids 7, 8,
10 and 16, as more cost-effective chemical lead.
The inhibition of TNF-a production has emerged as a
target in the treatment of a multitude of disorders
including rheumatoid arthritis (RA), multiple sclerosis,
cachexia, sepsis, ulcerative colitis, congestive heart fail-
ure and Crohn’s disease because of its therapeutic
potential.1 Particular attention has been paid to small
molecules possessing activity to inhibit the production
of TNF-a.2 As shown in Figure 1, we reported the dis-
covery of a new class of compound, 2-(acylamino)-2-
phenylethyl disodium phosphate,3,4 as a chemical lead.
Structural optimization was started with the chemical
modification of the chemical lead 1 which inhibits LPS-
induced TNF-a expression in the liver and spleen of
mice.5 However, this series of compounds, 2-(acyl-
amino)-2-phenylethyl disodium phosphates, does not
show any inhibitory activity in vitro according to our
biological evaluations although they exhibited very
potent inhibitory activity in vivo. For example, they did
not exhibit the inhibitory activity in LPS-induced TNF-
a production in vitro: (i) in whole blood from rat,
mouse or human; (ii) in peripheral blood mononuclear
cells (PBMC) from rat, mouse or human; and (iii) in
spleen cells from rat or mouse. Metabolic stabilization
Chemistry
The synthesis of all the biologically evaluated com-
pounds 7–19 described in Tables 1 and 2 is outlined in
Scheme 1. Appropriately substituted 2-nitrobenzyl
*Corresponding author. Tel.: +81-756-82-6161; fax: +81-756-82-
8420; e-mail: to.matsui@ono.co.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00343-7

3808
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
Figure 1. Discovery of new inhibitors 7, 8, 10 and 16.
Figure 2. Discovery of a new chemical lead 6.
Figure 3. Molecular design of cost-effective molecules.
Table 1. Biological evaluation of 2-(octanoylamino)benzylphosphonic
acids 7–16
halides 20a–j13ꢀ16 were subjected to Arbuzov reaction to
afford the corresponding diethyl phosphonates 21a–j,
reduction of the nitro group of which provided 22a–j.
Acylation of the anilines 22a–j with octanoyl chloride
followed by dealkylation of the diethyl phosphonates of
23a–j provided the phosphonic acids 7–16. Difluoro-
methylation was successfully carried out by a known
procedure10 to afford 24. Catalytic hydrogenation of 24
followed by the acylation of 25 provided 26. Dealkylation
of the phosphonate 26 afforded 17. Saturated derivatives
18 and 19 were prepared by the catalytic reduction of 23a
as described in Scheme 1. Chemical yields of all the reac-
tions described above are given in Table 3.
Compd
X
Inhibition of TNF-a
productiona ID50 (mg/kg, iv) in mice
7
8
9
10
11
12
13
14
15
16
H
3-Me
4-Me
5-Me
6-Me
2.8
1.5
(59)b
0.9
(50)b
(36)b
(39)b
6.8
3-OMe
4-OMe
5-OMe
6-OMe
4,5-OCH2O–
Results and Discussion
(ꢀ21)b
1.6
In the course of further screening for inhibitors of TNF-
a production, 2-(hexadecanoylamino) benzylphosphonic
acid 6, which was derived from an artificial substrate of
aSee Experimental.
bInhibition (%) at 10mg/kg, iv.

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
3809
Table 2. Biological evaluation of the miscellaneous compounds 17–19
Compd
Inhibition of TNF-a production in micea (% inhibition at 10 mg/kg, iv)
17
18 (racemic)
19 (tacemic)
47
25
16
aSee Experimental.
.
Scheme 1. Synthesis of 7–19. Reagents: (a) P(OEt)3, toluene, reflux; (b) H2, Pd-C, EtOH or SnCl2 2H2O, EtOH; (c) n-C7H15COCl, Et3N, CH2Cl2;
(d) TMSBr, CHCl3 or TMSI, CHCl3; (e) NaHMDS, (PhSO2)2NF, THF; (f) H2, Pd-C, MeOH; (g) H2, PtO2, AcOH, separation, cis (42%), trans
(15%); Chemical yields of the above-described reactions (unless noted in parentheses) are listed in Table 3.
sphingomyelinase as shown in Figure 2, was found to
exhibit moderate inhibitory activity (ID50=6.3 mg/kg, iv
in mice). Based on this information, the molecular
design of a cost-effective inhibitor of TNF-a production
was started. As described in one of our full papers,7 oral
dosing of phosphate analogues exhibited much less
potency relative to their iv dosing. Based on their
structural similarity, phosphonic acid analogues were
also considered to show much more activity by their iv
dosing rather than by their oral dosing. Rapid screening
was performed in duplicate or more at a dose of 10 mg/
kg, iv, only (n=5). The values of ID50 determination were
then carried out for the compounds which showed more
than 50% inhibition at the dose of 10 mg/kg, iv. When all
experiments for the values of ID50 determination of com-
pounds were performed, chemical lead 1 was used as a
positive control (ID50=0.8 mg/kg, iv, in mice).
According to the process described in Figure 3, com-
pounds 7–19, in which the metabolically labile phos-
phate moiety is replaced with a presumed metabolically
stable phosphonate moiety11 and the two asymmetric
centers are removed, were synthesized and evaluated as
to their ability to inhibit TNF-a production in mice
(Tables 1 and 2).
Table 3. Chemical yields of the reactions described in Scheme 1
Compd
Yield (%)
20a–j!21a–j 21a–j!22a–j 22a–j!23a–j 23a–j!7–16
The outcome of the biological evaluation of compounds
7–16 is described in Table 1. Based on the information
described in the preceding papers,3,4 N-octanoyl was
favored in the chemical modification of the phenyl
moiety. The compound 7 was more than twice as active
as the chemical lead 6. Introduction of a 3-methyl group
into the phenyl moiety of 7 afforded 8 with enhanced
activity. 4-Methyl derivative 9 also retained the activity.
7
8
9
10
11
12
13
14
15
16
70
Quantity
82
30
Quantity
83
Quantity
Quantity
97
Quantity
99
99
85
94
8
97
60
Quantity
85
89
45
76
88
9
70
94
59
96
59
53
6988
65
9
94
61
67
9
9
2
9
3
5-Methyl derivative 10 was the most potent inhibitor
among this class of compounds 7–16 while 6-methyl
99
Quantity

3810
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
derivative 11 was the least active of all the methyl-sub-
stituted derivatives 8–11. Introduction of a methoxy
group into the phenyl moiety of 7 was predicted to
optimize the inhibitory activity because of the excellent
potential shown by 1–3. Consequently, compounds 12–
15 were synthesized and evaluated biologically. Unex-
pectedly, 12–15 were less potent than the corresponding
methyl derivatives 8–11, respectively. In both cases, the
5-substituted derivatives 10 and 14 were the most active
compounds in each class. 4,5-Methylenedioxy derivative
16 demonstrated more potent activity than either of the
corresponding monosubstituted compounds 13 and 14.
The formation of a five-membered ring fused to posi-
tion-4 and-5 of the phenyl moiety of 7 was effective in
increasing the inhibitory activity. As a result, introduc-
tion of a substituent into position-6, which is the ortho-
position of the methylphosphonic acid moiety, mark-
edly reduced the activity whichever substituent, lipo-
philic or hydrophilic, was introduced as illustrated in 11
and 15. The introduction of a relatively hydrophilic
substituent such as a methoxy group into position-6 was
particularly deleterious, more so than that of a lipo-
philic substituent such as a 6-methyl group.
with proteins other than the target. As described in one
of the preceding papers,4 the three dimensional
arrangement of the acidic phosphate moiety and N-acyl
moiety was considered to be critical to the activity.
Based on this information, two geometrical isomers of
2-(acylamino)cyclohexylmethylphosphonic acid, 18 and
19, were synthesized as racemates and evaluated biolo-
gically. Both compounds showed less than 50% inhibi-
tion of TNF-a production at 10 mg/kg, iv in mice. Thus,
the aromatic moiety was thought to be indispensable to
the biological activity of this class of derivatives.
As shown in Figure 4a and b, increased production of
the plasma TNF-a after the intraperitoneal (ip) admin-
istration of LPS (5 mg/kg) was significantly suppressed
by administration of compounds 1 (positive control), 8,
16 and 10 in a dose-dependent manner. The ID50 values
of 8, 16 and 10 are 1.5, 1.6 and 0.9, mg/kg, iv, respec-
tively. These data showed the possibility of the newly
discovered phosphonic acid derivatives as a more cost-
effective chemical lead for the inhibitors of TNF-a pro-
duction. Regarding the toxicity or side-effects of these
compounds, hypotension activity, which was observed
in some of the phosphate analogues,12 was not observed
after iv administration of 16 up to 10 mg/kg in rats. All
the test compounds exhibited no in vitro TNF-a inhibi-
tion in the known assay systems. Mechanism of their
action remains to be clarified. According to our experi-
mental data, compound 1 was found to inhibit LPS-
induced increase of TNF-a mRNA expression in mouse
liver and spleen.5 As a result, these phosphonate analo-
gues was speculated to show TNF-a inhibition by the
same mechanism of action as that of 1 because of their
structural similarity.
Miscellaneous modifications of 7 produced 17–19, the
biological evaluations of which are described in Table 2.
Replacement of the methylene moiety attached directly
to the phosphonic acid group of 7 with a difluoromethyl
moiety afforded 17 with a significant reduction in inhi-
bitory activity. The increased acidity of 17 caused by the
conversion of the methylene moiety to a more electron-
attracting difluoromethylene moiety was estimated to be
one of the factors responsible for the reduced activity
because of the greater chance of nonspecific interactions
Figure 4. Effect of compound 1, 8, 16 and 10 on LPS-induced TNF-a production in mice. Compound was administrated intraperitonealy just before
an intraperitoneal injection of LPS. After 90 minuets of LPS injection, heparinized blood was obtained. Plasma TNF-a concentration was deter-
mined by ELISA. The data were expressed as the meanꢁSEM (n=5/group; **, significantly different from LPS control, P<0.01, *, P<0.05,
ANOVA-dunnett’s t-test).

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
3811
Conclusion
(200 MHz, CDCI3) d 7.16–7.00 (m, 2H), 6.81–6.74 (m,
2H), 4.30 (brs, 2H), 4.18–3.92 (m, 411), 3.16 (d, J=23.0
Hz, 2H), 1.25 (t, J=7.2 Hz, 611).
In summary, further screenings of a chemical lead fol-
lowed by optimization were attempted in an effort to
obtain more cost-effective inhibitors of TNF-a produc-
tion and identify drug candidate. Based on the mole-
cular design described in Figure 3, we have produced
several chemical leads, most notably 7, 8, 10 and 16,
with the potential for further optimization. An increase
in the acidity of the phosphonic acid moiety and the
saturation of the aromatic moiety to the cyclohexyl
moiety reduced the inhibitory activity as illustrated in
17, 18 and 19. Mechanism of their action remains to be
clarified. Further optimization of these chemical leads is
in progress in our laboratory.
Diethyl 2-(octanoylamino)benzylphosphonate (23a). To a
stirred solution of 22a (1.53 g, 4.94 mmol) and Et3N
(1.76 mL, 12.6 mmol) in CH2C12 (20 mL)ꢂwas added
octanoyl chloride (1.29mL, 8.56 mmol) at 0 C. Stirring
was continued at that temperature for 30 min and at
room temperature for 2 h. The reaction mixture was
poured into ice-cold 1 M HCl and extracted with
EtOAc. The organic layer was successively washed with
H2O saturated NaHCO3 aq, brine and dried over
MgSO4. 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 23a as a colorless oil (2.26 g, 97% yield): TLC
Rf=0.41 (n-hexane/EtOAc, 1/1); MS (APCI, Pos.) m/z
370 (M+H)+; 1H NMR (200 MHz, CDC13) d 9.44
(brs, IH), 7.78 (d, J=7.8 Hz, 1H), 7.29(m, 1H), 7.18–
7.02 (m, 2H), 4.18–3.84 (m, 4H), 3.12 (d, J=23.0 Hz,
2H), 1.42–1.20 (m, 14H), 0.88 (t, J=6.2 Hz, 3H).
Experimental
General directions
Analytical samples were homogeneous as confirmed by
TLC, and afforded spectroscopic results consistent with
the assigned structures. All 1H NMR spectra were taken
on a Varian Gemini-200, VXR-200s, Unity-INOVA 500
or Mercury 300 spectrometer. MS spectra were obtained
on a Hitachi M1200H, JMS-DX303HF or PerSeptive
Voyager Elite spectrometer. Matrix assisted laser deso-
rption 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. Column chromatography was
carried out on silica gel (Merck silica gel 60 (0.063–
0.200 mm) or Fuji Silysia FL60D). Thin layer chroma-
tography was performed on silica gel (Merck TLC plate,
silica gel 60 F254). The following abbreviations for sol-
vents and reagents are used: THF, tetrahydrofuran;
EtOAc, ethyl acetate; MeOH, methanol; EtOH, etha-
nol; CH2Cl2, dichloromethane; CHCl3, chloroform.
2-(Octanoylamino)benzylphosphonic acid (7). To a stir-
red solution of 23a (2.25 g, 6.1 mmol) in CHCl3 (10 mL)
was added bromotrimethylsilane (2.42 mL. 18.3 ml) at
room temperature and stirring was continued for 20 h at
that temperature. Removal of the solvent by evapora-
tion gave a residue, which was purified by column
chromatography on silica gel (Merck 7734, CHCl3/
MeOH, 20/1) to afford an oily residue, which was soli-
dified by Et2O to obtain 7 as white powder: 70% yield;
TLC Rf=0.31 (CHCl3/MeOH/AcOH, 8/1/1); IR (KBr)
3266, 2926, 2855, 1658, 1589, 1534, 1452, 1418, 1294,
1269, 1195, 1087 cmꢀ1; 1H NMR (200 MHz, DMSO-d6)
d 10.2 (s, 1H), 7.63 (d, J=7.5 Hz, 1H), 7.21–6.98 (m,
3H), 2.96 (d, J=21.0 Hz, 2H), 2.27 (t, J=7.5 Hz, 2H),
1.61 (m, 2H), 1.40–1.18 (m, 8H), 0.87 (t, J=6.5 Hz, 3H);
MS (FAB, Pos) m/z 352 (M+K)+, 314 (M+H)+, 296;
HRMS
C15H24NO4P+Na: 366.1341; found: 366.1354.
(MALDI-TOF,
Pos.)
calcd
for
General procedure A. Diethyl 2-nitrobenzylphosphonate
(21a). To a stirred solution of 1-(bromomethyl)-2-nitro-
benzene 20a (6.48 g, 30 mmol) in toluene (60 mL) was
added triethyl phosphite (5.14 mL, 30 mmol) and stir-
ring was continued under reflux for 5 h. Removal of the
solvent by evaporation gave a residue, which was pur-
ified by column chromatography on silica gel (Merck
7734, n-hexane/EtOAc, 1/1–1/4) to afford 21a as a pale
yellow oil (5.76 g, 70% yield): TLC Rf=0.26 (n-hexane/
Preparation of 9–11 and 13–16. These compounds were
prepared according to the essentially same procedures
as described for the preparation of 7 from 20a.
4-Methyl-2-(octanoylamino)benzylphosphonic acid (9).
The title compound 9 was prepared from 1-(bromo-
methyl)-4-methyl-2-nitrobenzene 20c according to the
same procedure as described for the preparation of 7
from 20a. 21c: 82% yield; TLC Rf=0.30 (n-hexane/
EtOAc, 1/3); 1H NMR (200 MHz, CDCl3) d 7.77 (s,
1H), 7.35 (s, 2H), 4.12–3.96 (m, 4H), 3.65 (d, J=23.0
Hz, 2H), 2.41 (d, J=2.0 Hz, 3H), 1.24 (t, J=7.0 Hz,
6H). 22c: 97% yield; TLC Rf=0.24 (CHCl3/MeOH, 50/
1
EtOAc, 1/4); MS (APCI, Pos.) m/z 274 (M+H)+; H
NMR (200 MHz, CDCl3) d 7.96 (d, J=7.8 Hz, 1H),
7.50–7.36 (m, 3H), 4.10–3.96 (m, 4H), 3.71 (d, J=23.0
Hz, 2H), 1.24 (t, J=7.2 Hz, 6H).
1
Diethyl 2-aminobenzylphosphonate (22a). A mixture of
21a (5.75 g, 21 mmol) in EtOH (30 mL) and 5% Pd-C
(500 mg) was stirred at room temperature under an
atmospheric pressure of hydrogen for 6 h. Removal of
the catalyst by filtration through a pad of Celite fol-
lowed by evaporation afforded 22a quantitatively as a
pale yellow oil (5.12 g): TLC Rf=0.48 (CHCl3/MeOH,
1); H NMR (200 MHz, CDCl3) d 6.92 (dd, J=8.0. 3.0
Hz, 1H), 6.63–6.55 (m, 2H), 4.43–4.25 (m, 2H), 4.10–
3.89(m, 4H), 3.11 (d, J=21.0 Hz, 2H), 2.25 (d, J=3.0
Hz, 3H), 1.25 (t, J=7.0 Hz, 6H). 23c: quant.; TLC
1
Rf=0.43 (n-hexane/EtOAc, 1/1); H NMR (200 MHz,
CDCl3) d 9.39 (s, 1H), 7.61 (s, 1H), 7.02 (dd, J=8.0, 3.0
Hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 4.16–3.86 (m, 4H),
3.08 (d, J=21.0 Hz, 2H), 2.41 (t, J=7.0 Hz, 2H), 2.33
1
1/4); MS (APCI, Pos.) m/z 244 (M+H)+; H NMR

3812
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
(d, J=3.0 Hz, 3H), 1.82–1.65 (m, 2H), 1.50–1.20 (m, 8H),
1.25 (t, J=8.0 Hz, 6H), 0.88 (t, J=7.0 Hz, 3H). 9: 59%
yield; white powder; TLC Rf=0.80 (CHCl3/MeOH/
AcOH, 5/1/1); IR (KBr) 3265, 2927, 1654, 1538 cmꢀ1; 1H
NMR (200MHz, CD3OD) d 7.33 (s, 1H), 7.18 (dd, J=3,8
Hz, 1H), 6.98 (d, J=8 Hz, 1H), 3.08 (d, J=21.0 Hz, 2H),
2.40 (t, J=8.0 Hz, 2H), 2.30 (d, J=2.0 Hz, 3H), 1.81–
1.62 (m, 2H), 1.59–1.16 (m, 8H), 0.91 (t, J=7.0 Hz, 3H);
MS (MALDI-TOF, Pos.) m/z 366(M+K)+, 350
(M+Na)+, 328 (M+H)+; HRMS (MALDI-TOF,
Pos.) calcd for HRMS (MALDI-TOF, Pos.) calcd for
C16H26NO4P+Na: 350.1497; found: 350.1493.
(d, J=7.0 Hz, 1H), 7.22–7.03 (m, 2H), 3.17 (d, J=22.0
Hz, 2H), 2.41 (t, J=8.0 Hz, 2H), 2.40 (s, 3H), 1.82–1.64
(m, 2H), 1.50–1.18 (m, 8H), 0.91 (t, J=7.0 Hz, 3H); MS
(MALDI-TOF, Pos.) m/z 350 (M+Na)+; HRMS
(MALDI-TOF, Pos.) calcd for C16H26NO4P+Na:
350.1497; found: 350.1470.
4-Methoxy-2-(octanoylamino)benzylphosphonic acid (13).
The title compound 13 was prepared from 1-(bromo-
methyl)-4-methoxy-2-nitrobenzene 20g according to the
same procedure as described for the preparation of 7
1
from 20a. 21g: 69% yield; TLC Rf=0.44 (EtOAc); H
NMR (200 MHz, CDCl3) d 7.48 (d, J=3.0 Hz, 1H),
7.37 (dd, J=9.0, 3.0 Hz, 1H), 7.10 (ddd, J=9.0, 3.0, 1.0
Hz, 1H), 4.03 (dq, J=8.0, 7.0 Hz, 4H), 3.86 (s, 3H), 3.62
(d, J=22.0 Hz, 2H), 1.24 (t, J=7.0 Hz, 6H). 22g: 88%
yield; TLC Rf=0.28 (EtOAc); MS (MALDI-TOF, Pos.)
m/z 296 (M+Na)+, 274 (M+H)+; 1H NMR (200 MHz,
CDCl3) d 6.93 (dd, J=8.0, 3.0 Hz, 1H), 6.36–6.25 (m,
2H), 4.44–4.18 (m, 2H), 4.14–3.91 (m, 4H), 3.75 (s, 3H),
3.05 (d, J=20.0 Hz, 2H), 1.25 (t, J=7.0 Hz, 6H). 23g:
85% yield; TLC Rf=0.75 (EtOAc); 1H NMR
(200 MHz, CDCl3) d 9.51 (s, 1H), 7.02 (dd, J=8.0, 2.0
Hz, 1H), 6.66 (dd, J=8.0, 3.0 Hz, 1H), 4.17–3.89(m,
4H), 3.80 (s, 3H), 3.06 (d, J=21.0 Hz, 2H), 2.42 (t,
J=8.0 Hz, 2H), 1.83–1.06 (m, 10H), 1.25 (t, J=7.0 Hz,
6H), 0.88 (t, J=7.0 Hz, 3H). 13: 76% yield; white pow-
der; TLC Rf=0.18 (CHCl3/MeOH/AcOH, 8/1/1); IR
5-Methyl-2-(octanoylamino)benzylphosphonic acid (10).
The title compound 10 was prepared from 1-(bromo-
methyl)-5-methyl-2-nitrobenzene 20d according to the
same procedure as described for the preparation of 7
from 20a. 21d: 30% yield; TLC Rf=0.20 (n-hexane/
1
EtOAc, 1/3); H NMR (200 MHz, CDCl3) d 7.89(d,
J=8.0 Hz, 1H), 7.27–7.23 (m, 2H), 4.03 (dq, J=8.0. 7.0
Hz, 4H), 3.69(d, J=23.0 Hz, 2H), 2.41 (s, 3H), 1.23 (t,
J=7.0 Hz, 6H). 22d: quant.; TLC Rf=0.62 (CHCl3/
1
MeOH, 9/1); H NMR (200 MHz, CDCl3) d 6.87–6.83
(m, 2H), 6.63 (d, J=8.0 Hz, 1H), 4.26–3.88 (m, 4H),
3.10 (d, J=21.0 Hz, 2H), 2.22 (s, 3H), 1.25 (t, J=7.0
1
Hz, 6H). 23d: 85% yield; TLC Rf=0.73 (EtOAc); H
NMR (200 MHz, CDCl3) d 9. 27 (s, 1H), 7.63 (d, J=8.0
Hz, 1H), 7.13–7.07 (m, 1H), 6.95 (s, 1H), 4.12–3.90 (m,
4H), 3.08 (d, J=21.0 Hz, 2H), 2.40 (t, J=7.0 Hz, 2H),
2.29(s, 3H), 1.83–1.64 (m, 2H), 1.47–1.20 (m, 8H), 1.25
(t, J=8.0 Hz, 6H), 0.88 (t, J=6.0 Hz, 3H). 10: 9 6%
yield; white powder; TLC Rf=0.31 (CHCl3/MeOH/
AcOH, 8/1/1); IR (KBr) 3263, 2927, 2856, 1652, 1532
(KBr) 3257, 2931, 1651, 1537, 1293, 1057 cmꢀ1 1H
;
NMR (200 MHz, CD3OD) d 7.21–7.13 (m, 2H), 6.74
(dd, J=3.0, 8.0 Hz, 1H), 3.76 (3H,S), 3.05 (d, J=21.0
Hz, 2H), 2.40 (t, J=8.0 Hz, 2H), 1.80–1.62 (m, 2H),
1.48–1.20 (m, 8H), 0.90 (t, J=7.0 Hz, 3H); MS (APCI,
Neg.) m/z 342 (M–H)ꢀ; HRMS (MALDI-TOF, Pos.)
calcd for C16H26NO5P+Na: 366.1446; found: 366.1399.
1
cmꢀ1; H NMR (200 MHz, CD3OD) d 7.36 (d, J=8.0
Hz, 1H), 7.16–7.03 (m, 2H), 3.08 (d, J=21.0 Hz, 2H),
2.39(t, J=8.0 Hz, 2H), 2.30 (s, 3H), 1.80–1.62 (m, 2H),
1.44–1.18 (m, 8H), 0.91 (t, J=7.0 Hz, 3H); MS
(MALDI-TOF, Pos.) m/z 350 (M+Na)+; HRMS
(MALDI-TOF, Pos.) calcd for C16H26NO4P+Na:
350.1497; found: 350.1481.
5-Methoxy-2-(octanoylamino)benzylphosphonic acid (14).
The title compound 14 was prepared from 1-(bromo-
methyl)-5-methoxy-2-nitrobenzene 20 h according to the
same procedure as described for the preparation of 7
from 20a. 21h; 65% yield; TLC Rf=0.29( n-hexane/
1
6-Methyl-2-(octanoylamino)benzylphosphonic acid (11).
The title compound 11 was prepared from 1-(bromo-
methyl)-6-methyl-2-nitrobenzene 20e according to the
same procedure as described for the preparation of 7
from 20a. 21e: quant.; TLC Rf=0.24 (n-hexane/EtOAc,
EtOAc, 1/4); H NMR (200 MHz, CDCl3) d 8.06 (d,
J=9.0 Hz, 1H), 6.96–6.82 (m, 2H), 4.07 (dq, J=8.0, 7.0
Hz, 4H), 3.89(s, 3H), 3.76 (d, J=23.0 Hz, 2H), 1.25 (t,
J=7.0 Hz, 6H). 23h: 88% yield; TLC Rf=0.61 (n-hex-
ane/EtOAc, 1/3); 1H NMR (200 MHz, CDCl3) d 9.17 (s,
1H), 7.62 (d, J=9.0 Hz, 1H), 6.84 (dt, J=9.0, 3.0 Hz,
1H), 6.70 (t, J=3.0 Hz, 1H), 4.16–3.88 (m, 4H), 3.78 (s,
3H), 3.09(d, J=21.0 Hz, 2H), 2.40 (t, J=8.0 Hz, 2H),
1.83–1.66 (m, 2H), 1.51–1.20 (m, 8H), 1.26 (t, J=7.0
Hz, 6H), 0.88 (t, J=6.0 Hz, 3H). 14: 61% yield; white
powder; TLC Rf=0.48 (CHCl3/MeOH/AcOH, 8/1/1);
IR (KBr) 3270, 2927, 2854, 1651, 1537, 1239, 1057
1
1/3); H NMR (200 MHz, CDCl3) d 7.71(d, J=8.0 Hz,
1H), 7.44 (d, J=8.0 Hz, 1H), 7.28 (dt, J=8.0, 2.0 Hz,
1H), 4.01 (dq, J =8.0, 7.0 Hz, 4H), 3.77 (d, J=23.0 Hz,
2H), 2.53 (d, J=2.0 Hz, 3H), 1.24 (t, J=7.0 Hz, 6H).
1
22e: 99% yield; TLC Rf=0.69(CHCl /MeOH, 9/1); H
3
NMR (200 MHz, CDCl3) d 6.96 (dt, J=8.0, 3.0 Hz,
1H), 6.69–6.56 (m, 2H), 4.10–3.91 (m, 4H), 3.19 (d,
J=21.0 Hz, 2H), 2.31 (d, J=2.0 Hz, 3H), 1.25 (t, J=7.0
1
cmꢀ1; H NMR (200 MHz, CD3OD) d 7.32 (d, J=9.0
1
Hz, 6H). 23e: 89% yield; TLC Rf=0.80 (EtOAc); H
Hz, 1H), 6.89(t, J=3.0 Hz, 1H), 6.82 (dt, J=9.0, 3.0
Hz, 1H), 3.77 (s, 3H), 3.08 (d, J=22.0 Hz, 2H), 2.38 (t,
J=8.0 Hz, 2H), 1.82–1.63 (m, 2H), 1.49–1.20 (m, 8H),
0.90 (t, J=7.0 Hz, 3H); MS (APCI, Neg.) m/z 342 (M–
H)ꢀ; HRMS (MALDI-TOF, Pos.) calcd for
C16H26NO5P+Na: 366.1446; found: 366.1414.
NMR (200 MHz, CDCl3) d 9.39 (s, 1H), 7.57 (d, J=8.0
Hz, 1H), 7.18 (d, J=2.8 Hz, 1H), 7.00 (d, J=8.0 Hz,
1H), 4.16–3.72 (m, 4H), 3.17 (d, J=21.0 Hz, 2H), 2.42
(t, J=8.0 Hz, 2H), 2.34 (d, J=1.0 Hz, 3H), 1.84–1.18
(m, 10H), 1.25 (t, J=7.0 Hz, 6H), 0.88 (t, J=7.0 Hz,
3H). 11: 59% yield; white powder; TLC Rf=0.47
(CHCl3/MeOH/AcOH, 8/1/1); IR (KBr) 3269, 2856,
6-Methoxy-2-(octanoylamino)benzylphosphonic acid (15).
The title compound 15 was prepared from 1-(bromo-
1
1651, 1536 cmꢀ1; H NMR (200 MHz, CD3OD) d 7.36

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
3813
methyl)-6-methoxy-2-nitrobenzene 20i according to the
same procedure as described for the preparation of 7
from 20a. 21i: 98% yield; TLC Rf=0.23 (EtOAc); H
reaction mixture was neutralized with 5M NaOH and
the resulting precipitates were removed by filtration.
The filtrate was extracted with EtOAc. The organic
layer was washed with brine and dried over MgSO4.
Removal of the solvent by evaporation gave 22b, which
was used for the next reaction without further purifica-
tion: TLC Rf=0.45 (CHCl3/MeOH, 20/1); MS
(MALDI-TOF, Pos.) m/z 280 (M+Na)+, 258
(M+H)+; 1H NMR (200 MHz, CDCl3) d 7.01–6.88 (m,
2H), 6.65 (t, J=7.0 Hz, 1H), 4.13–3.93 (m, 2H), 3.14 (d,
J=21.0 Hz, 2H), 2.20 (s, 3H), 1.26 (t, J=7.0 Hz, H).
Compound 23b was prepared from 22b according to the
same procedure as described for the preparation of 23a
from 22a. 23b: 60% yield; white powder; TLC Rf=0.71
(EtOAc); MS (MALDI-TOF, Pos.) m/z 422 (M+K)+,
406 (M+Na)+, 384 (M+H)+; 1H NMR (200 MHz,
CDCl3) d 9.02 (s, 1H), 7.21–7.13 (m, 1H), 7.11 (t, J=8.0
Hz, 1H), 7.06–6.91 (m, 1H), 4.10–3.83 (m, 4H), 3.11 (d,
J=21.0 Hz, 2H), 2.43 (t, J=8.0 Hz, 2H), 2.25 (s, 3H),
1.84–1.61 (m, 2H), 1.48–1.16 (m, 14H), 0.89(t, J=7.0
Hz, 3H). The title compound 8 was prepared from 23b
according to the same procedure as described for the
preparation of 7 from 23a. 8: 94% yield; white powder;
TLC Rf=0.28 (AcOEt/AcOH/H2O, 8/1/1); IR (KBr)
1
NMR (200 MHz, CDCl3) d 7.50 (d, J=8.0 Hz, 1H),
7.35 (dt, J=8.0, 2.2 Hz, 1H), 7.12 (d, J=8.0 Hz, 1H),
4.15–3.90 (m, 7H), 3.84 (d, J=23.2 Hz, 2H), 1.27–1.18
(m, 6H). 22i: The product was used for the next reaction
without further purification; 99% yield; TLC Rf=0.57
(CHCl3/MeOH, 10/1). 23i: 92% yield; TLC Rf=0.33 (n-
hexane/EtOAc, 1/1); 1H NMR (200 MHz, CDCl3) d
9.51 (s, 1H), 7.43 (d, J=8.0 Hz, 1H), 7.24 (dt, J=8.0,
2.6 Hz, 1H), 6.70 (d, J=8.0 Hz, 1H), 4.14–3.93 (m, 4H),
3.83 (s, 3H), 3.26 (d, J=21.4 Hz, 2H), 2.41 (t, J=7.2
Hz, 2H), 1.85–1.60 (m, 2H), 1.50–1.21 (m, 14H), 0.91–
0.85 (m, 3H). 15: 93% yield; white powder; TLC
Rf=0.31 (CHCl3/MeOH/AcOH, 8/1/1); IR (KBr) 3263,
2929, 2857, 1656, 1594, 1537, 1477, 1441, 1414, 1314,
1265, 1197, 1148, 1077, 1012 cmꢀ1; 1H NMR (200 MHz,
DMSO-d6) d 10.3 (s, 1H), 7.31 (d, J=8.0 Hz, 1H), 7.15
(dt, J=8.0, 2.2 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H), 3.77 (s,
3H), 3.04 (d, J=21.2 Hz, 2H), 2.26 (t, J=7.4 Hz, 2H),
1.65–1.59(m, 2H), 1.28 (brs, 8H), 0.90–0.83 (m, 3H);
MS (MADLI-TOF, Pos.) m/z 382 (M+K)+, 366
(M+Na)+, 344 (M+H)+; HRMS (MALDI-TOF,
Pos.) calcd for C16H26NO5P+Na: 366.1446; found:
366.1440.
3259, 2927, 2857, 1655, 1529, 1469 cmꢀ1 1H NMR
;
(200 MHz, CD3OD) d 7.23–7.11 (m, 3H), 3.08 (d,
J=22.0 Hz, 2H), 2.45 (t, J=8.0 Hz, 2H), 2.21 (s, 3H),
1.85–1.63 (m, 2H), 1.58–1.19(m, 8H), 0.91 (t, J=7.0
Hz, 3H); MS (FAB, Pos.) m/z 328 (M+H)+; HRMS
(MALDI-TOF, Pos.) calcd for C16H26NO4P+Na:
350.1497; found: 350.1540.
[6-(Octanoylamino)-1,3-benzodioxol-5-yl]methylphospho-
nic acid (16). The title compound 16 was prepared from
5-(iodomethyl)-6-nitro-1,3-benzodioxole 20j according
to the same procedure as described for the preparation
of 7 from 20a. 21j: 94% yield; 1H NMR (200 MHz,
CDCl3) d 7.51 (s, 1H), 6.86 (d, J =2.4 Hz, 1H), 6.11 (s,
2H), 4.10–4.00 (m, 4H), 3.67 (d, J=22.5 Hz, 2H), 1.26
(t, J=6.6 Hz, 6H). 23j: quant.; TLC Rf=0.55 (n-hex-
ane/EtOAc, 1/3); 1H NMR (200 MHz, CDCl3) d 9.26 (s,
1H), 7.23 (s, 1H), 6.75 (d, J=2.0 Hz, 1H), 5.95 (s, 2H),
4.20–3.91 (m, 4H), 3.01 (d, J=21.0 Hz, 2H), 2.39(t,
J=8.0 Hz, 2H), 1.81–1.52 (m, 2H), 1.44–1.18 (m, 8H),
1.27 (t, J=7.0 Hz, 6H), 0.88 (t, J=7.0 Hz, 3H). 16: 67%
yield; white powder; TLC Rf=0.35 (CHCl3/MeOH/
H2O, 65/25/4); IR (KBr) 3253, 2927, 1650, 1536, 1506,
3-Methoxy-2-(octanoylamino)benzylphosphonic acid (12).
The title compound 12 was prepared from 1-(bromo-
methyl)-3-methoxy-2-nitrobenzene 20f according to the
same procedure as described for the preparation of 8
1
from 20b. 21f: 83% yield; TLC Rf=0.56 (EtOAc); H
NMR (200 MHz, CDCl3) d 7.39(d, J=8.0 Hz, 1H),
7.12 (ddd, J=8.0, 2.0, 1.0 Hz, 1H), 6.96 (dd, J=8.0, 2.0
Hz, 1H), 4.04 (dq, J=8.0, 7.0 Hz, 4H), 3.89(s, 3H), 3.20
(d, J=22.0 Hz, 2H), 1.27 (t, J=7.0 Hz, 6H). 22f: 9 9 %
yield; TLC Rf=0.45 (EtOAc); 1H NMR (200 MHz,
CDCl3) d 6.78–6.62 (m, 3H), 4.58–4.32 (m, 2H), 4.22–
3.91 (m, 4H), 3.84 (s, 3H), 3.14 (d, J=21.0 Hz, 2H),
1.25 (t, J=7.0 Hz, 6H). 23f: 45% yield; TLC Rf=0.25
1
1488, 1038 cmꢀ1; H NMR (300 MHz, CD3OD) d 6.95
(s, 1H), 6.78 (d, J=2.1 Hz, 1H), 5.94 (s, 2H), 3.02 (d,
J=21.3 Hz, 2H), 2.38 (t, J=7.0 Hz, 2H), 1.72 (q, J=7.0
Hz, 2H), 1.45–1.20 (m, 8H), 0.91 (t, J=6.9Hz, 3H); MS
(MALDI-TOF, Pos) m/z 380 (M+Na)+, 358
(M+H)+; HRMS (MALDI-TOF, Pos.) calcd for
C16H26NO6P+Na: 380.1239; found: 380.1256.
1
(n-hexane/EtOAc, 1/3); H NMR (200 MHz, CDCl3) d
8.32 (s, 1H), 7.15 (d, J=8.0 Hz, 1H), 6.19–6.77 (m, 2H),
4.10–3.90 (m, 4H), 3.83 (s, 3H), 3.13 (d, J=21.0 Hz,
2H), 2.42 (t, J=8.0 Hz, 2H), 1.82–1.60 (m, 2H), 1.52–
1.20 (m, 8H), 1.24 (t, J=7.0H, 6H), 0.89(t, J=7.0 Hz,
3H). 12: 53% yield; white powder; TLC Rf=0.31
(CHCl3/MeOH/AcOH, 8/1/1); IR (KBr) 3257, 2927,
General Procedure B. 3-Methyl-2-(octanoylamino)ben-
zylphosphonic acid (8). Compound 21b was prepared
from 1-(bromomethyl)-3-methyl-2-nitrobenzene 20b
according to the essentially same procedures as descri-
bed for the preparation of 21a from 20a. quant.; MS
(MALDI-TOF, Pos.) m/z 310 (M+Na)+, 288
(M+H)+; 1H NMR (200 MHz, CDCl3) d 7.43–7.13 (m,
3H), 4.08 (dq, J=22.0, 7.0 Hz, 4H), 3.25 (d, J=22.0 Hz,
2H), 2.34 (s, 3H), 1.26 (t, J=7.0 Hz, 6H). To a stirred
solution of 21b (6.2 g, 21.6 mmol) and concd HCl (18.8
1
2856, 1636, 1531, 267, 1006 cmꢀ1; H NMR (200 MHz,
CD3OD) d 7.23 (t, J=8.0 Hz, 1H), 7.03–6.90 (m, 2H),
3.80 (s, 3H), 3.08 (d, J=21.0 Hz, 2H), 2.42 (t, J=8.0
Hz, 2H), 1.81–1.64 (m, 2H), 1.51–1.19(m, 8H), 0.91 (t,
J=7.0 Hz, 3H); MS (MALDI-TOF, Pos.) m/z 382
(M+K)+, 366 (M+Na)+, 344 (M+H)+; HRMS
(MALDI-TOF, Pos.) calcd for C16H26NO5P+Na:
366.1446; found: 366.1472.
.
mL) in EtOH (100 mL) was added SnCl2 H2O (14.2 g,
62.9mmol) in EtOH (20 mL) at room temperature and
stirring was continued for 2 h at that temperature. The
Difluoro[2-(octanoylamino)phenyl]methfr1phosphonic acid
a
(17). To
stirred solution of sodium bis(-

3814
T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
trimethylsilyl)amide (17.6 mL, 1M in THF, 17,6 mmol)
in THF (20 mL) was added dropwise a solution of 21a
(2.18 g, 7.99 mmol) in TAF (20 mL) at ꢀ78 ꢂC and
stirring was continued for l h at that temperature. To
the reaction mixture was added dropwise a solution of
N-fluorobenzenesulfonimide (6.31 g, 20 mmol) in THF
(20 mL) at that temperature. Stirring was continued at
that temperature for 2 h and then at ꢀ20 ꢂC for 1 h. The
reaction mixture was quenched with ice-cold 1 M HCl
and extracted with EtOAc. The organic layer was suc-
cessively washed with H2O, saturated NaHCO3, brine
and dried over MgSO4. Removal of the solvent by eva-
poration gave a residue, which was purified by column
chromatography on silica gel (Merck 7734, n-hexane/
EtOAc, 1/2) to afford diethyl difluoro(2-nitrophe-
nyl)methylphosphonate 24 (2.03 g). The product was
used for the next reaction without further purification:
TLC Rf=0.55 (n-hexane/EtOAc, 1/4); 1H NMR
(200 MHz, CDC13) d 7.90–7.50 (m, 4H), 4.40–4.20 (m,
4H), 1.40–1.30 (m, 6H). Diethyl (2-aminophenyl)-
difluoromethylphosphonate 25 was prepared from 24
according to the same procedure as described for the
preparation of 22a from 21a (51% yield from 21a).
Diethyl difluoro[2-(octanoylamino)phenyl]methylphos-
phonate 26 was prepared from 25 according to the same
procedure as described for the preparation of 23a from
22a. 26: 88% yield; TLC Rf=0.31 (n-hexane/EtOAc, 2/
2H), 1.82 (ddd, J=21.0, 17.0, 8.0 Hz, 1H), 1.78–1.70
(m, 2H), 1.66–1.56 (m, 2H), 1.49(ddd, J=21.0, 18.0, 7.0
Hz, 1H), 1.45–1.16 (m, 14H), 1.04 (t, J=8.0 Hz, 6H),
0.85 (t, J=8.0 Hz, 3H). trans-isomer: 15% yield; TLC
Rf=0.39(EtOAc); 1H NMR (500 MHz, CDCl3) d 6.23
(d, J=10.0 Hz, 1H), 4.16–4.00 (m, 4H), 3.52–3.46 (rn,
1H), 2.18 (t, J=8.0 Hz, 2H), 2.08–1.99 (m, 2H), 1.90
(ddd, J=21.0, 16.0, 6.0 Hz, 1H), 1.78–1.60 (m, 2H),
1.51 (ddd, J=21.0, 16.0, 5.0 Hz, lH), 1.39–1.18 (m,
18H), 1.17–1.06 (m, 1H), 0.85 (t, J=8.0 Hz, 3H). The
title compound 18 was prepared from cis-diethyl[2-
(octanoylamino)cyclohexyl]methylphosphonate accord-
ing to the same procedure as described for the prepara-
tion of 7 from 23a: 24% yield; beige powder; TLC
Rf=0.51 (CHCl3/MeOH/AcOH, 8/1/1); IR (KBr) 3296,
2929, 2857, 1643, 1543, 1465, 1138 cmꢀ1 1H NMR
;
(200 MHz, CD3OD) 84.13–4.02 (br, 1H), 2.24 (t, J=8.0
Hz, 2H), 2.24–2.01 (m, 1H), 1.81–1.14 (m, 20H), 0.89(t,
J=7.0 Hz, 3H); MS (MALDI-TOF, Pos.) m/z 364
(M+2Na–H)+, 342 (M+Na)+, 320 (M+H)+; HRMS
(MALDI-TOF, Pos.) calcd for C15H30NO4P+Na:
342.1810; found: 342.1795.
trans-12-(Octanoylamino)cyclohexyljmethylphosphonic
acid (19). The title compound 19 was prepared from
trans-diethyl [2-(octanoylamino)cyclohexyl]methylphos-
phonate according to the same procedure as described
for the preparation of 7 from 23a: 12% yield; white
amorphous powder; TLC Rf=0.17 (CHCl3/MeOH/
AcOH, 20/1/1); IR (KBr) 3434, 2932, 2857, 1638, 1545,
1
1); H NMR (200 MHz, CDCl3) d 9.36 (brs, 1H), 8.04
(d, J=8.5 Hz, 1H), 7.53–7.45 (m, 2H), 7.20 (t, J=7.6
Hz, 1H), 4.50–4.02 (m, 4H), 2.39(t, J=7.4 Hz, 2H),
1.74 (m, 2H), 1.48–1.20 (m, 14H), 0.88 (t, J=6.6 Hz,
3H). The title compound 17 was prepared from 26
according to the essentially same procedure as described
for the preparation of 7 from 23a using iodo-
trimethylsilane instead of bromotrimethylsilane: 85%
yield; white powder; TLC Rf=0.36 (CHCl3/MeOH/
AcOH, 5/1/1); IR (KBr) 3301, 2929, 2858, 1646, 1590,
1
1450, 1123 cmꢀ1; H NMR (200 MHz, CD3OD) 83.51–
3.33 (m, 1H), 2.32–2.10 (m, 1H), 2.18 (t, J=7.0 Hz, 2H),
2.06–0.97 (m, 20H), 0.89 (t, J=7.0 Hz, 3H); MS
(MALDI-TOF, Pos.) m/z 342 (M+Na)+, 320
(M+H)+; HRMS (MALDI-TOF, Pos.) calcd for
C15H30NO4P+Na: 342.1810; found: 342.1842.
1452, 1296, 1140, 1030 cmꢀ1
;
1H NMR (200 MHz,
Biological assay method. Inhibition of LPS-induced
Plasma TNF-ꢀ production in mice. The experiments
were performed using male BALB/c mice, 8 weeks of
age, purchased from Charles River Breeding Labora-
tories (Shizuoka, Japan). Animals were given access to
food and water ad libitum and were maintained on 12 h
light/dark cycle at 22–23 ꢂC. All experimental proce-
dures were conformed to the Animal Care and Use
Committee protocols filed at ONO Pharmaceutical Co.,
Ltd. (Osaka, Japan). Test compounds and LPS from
Escherichia coli strain 055 B5 (DIFCO LABORA-
TORIES, Detroit, MI, USA) were dissolved in saline.
Compounds were injected intravenously (0.01–0.1 mg/
10 mL/kg) to mice, and then immediately given an
intraperitoneal injection of LPS (5 mg/10 mL/kg). After
90 min of LPS injection, heparinized blood was
obtained. Blood was centrifuged and plasma samples
were kept frozen at ꢀ80 ꢂC. Plasma TNF-a concentra-
tion was determined by enzyme-linked immunosorbent
assay (ELISA) using a commercial kit (GENZYME,
USA) according to the manufacturer’s instructions. The
data were expressed as the meanꢁSEM 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).
DMSO-d6) d 7.86 (d, J=8.0 Hz, 1H), 7.40 (d, J=8.0
Hz, 1H), 7.30 (t, J=8.0 Hz, 1H), 7.06 (t, J=8.0 Hz,
1H), 2.24 (m, 2H), 1.56 (m, 2H), 1.40–1.20 (m, 8H), 0.86
(m, 3H); MS (FAB, Neg.) m/z 348 (M–H)ꢀ; HRMS
(MALDI-TOF, Pos.) calcd for C15H22F2NO4P+Na:
372.1152; found: 372.1145.
cis-[2-(Octanoylamino)cyclohexyl]methylphosphonic acid
(18). A mixture of 23a (970 mg, 2.63 mmol) in AcOH
.
(10 mL) and PtO2 xH2O (268 mg) was stirred at room
temperature under an atmospheric pressure of hydrogen
for 4 days. Removal of the catalyst by filtration through
a pad of Celite and the filtrate was diluted with EtOAc.
The organic layer was successively washed with H2O,
saturated NaHCO3, brine and dried over MgSO4.
Removal of the solvent by evaporation gave a residue,
which was purified by column chromatography on silica
gel (Merck 7734, n-hexane/EtOAc, 1/1–0/1) to afford
cis-diethyl [2-(octanoylamino)cyclohexyl]methylphos-
phonate and trans-diethyl [2-(octanoylamino)cyclohex-
yl]methylphosphonate. Stereo chemistry of them was
determined by NMR technique (NOE). cis-isomer: 42%
yield; TLC Rf=0.21 (EtOAc); 1H NMR (500 MHz,
CDCl3) d 6.76–6.64 (brs, 1H), 4.26–4.19(m, 1H), 3.95–
3.80 (m, 4H), 2.27–2.19(brs, 1H), 2.15 (t, J=8.0 Hz,

T. Matsui et al. / Bioorg. Med. Chem. 10 (2002) 3807–3815
3815
7. Matsui, T.; Kondo, T.; Nishita, Y.; Itadani, S.; Tsuruta,
H.; Fujita, S.; Omawari, N.; Sakai, M.; Nakazawa, S.;
Ogata, A.; Mori, H.; Kamoshima, W.; Terai, K.; Ohno, H.;
Obata, T.; Nakai, H.; Toda, M. Bioorg. Med. Chem., in
press.
8. 2-(N-Hexadecanoylamino)-4-nitorophenylphosphocholine
(HNP), a water-soluble synthetic substrate for sphingomyeli-
nase, was purchased from Sigma Chemical Co., Ltd.
9. Claus, R.; Russwurm, S.; Meisner, M.; Kinscherf, R.;
Deigner, H. P. Cerr. Drug Targets 2000, 1, 185.
10. Taylor, S. D.; Dinaut, A. N.; Thadani, A. N.; uang, Z.
Tetrahedron Lett. 1996, 37, 8089.
11. A series of phosphate analogues were rapidly metabolized
to afford the corresponding alcohol as described in one of our
previous papers.7 Design and synthesis of phosphonic acid
analogues instead of phosphate analogues were quite reason-
able way of thinking in the medicinal chemistry because
phosphonic acid can not be metabolically hydrolyzed although
we have no experimental data of the improved metabolic sta-
bility.
% Inhibition ¼ 100 À ðC À SÞ=ðL À SÞ ꢀ 100
C: Plasma TNF-a concentration of LPS-treated animals
pretreated with a test compound. L: Plasma TNF-a
concentration of LPS-treated animals pretreated with
saline. S: Plasma TNF-a concentration of saline-treated
animals also pretreated with saline.
References and Notes
1. Tracey, K. J.; Cerami, A. Annu. Rev. Med. 1994, 45, 491.
2. Marriott, J. B.; Westry, M.; Dalgleish, A. G. Drug Disc.
Today 1997, 2, 273.
3. Matsui, T.; Kondo, T.; Nishita, Y.; Itadani, S.; Nakatani,
S.; Omawari, N.; Sakai, M.; Nakazawa, S.; Ogata, A.; Ohno,
H.; Obata, T.; Nakai, H.; Toda, M. Bioorg. Med. Chem. Lett.
2002, 12, 903.
4. Matsui, T.; Kondo, T.; Nishita, Y.; Itadani, S.; Tsuruta,
H.; Fujita, S.; Omawari, N.; Sakai, M.; Nakazawa, S.; Ogata,
A.; Mori, H.; Ohno, H.; Obata, T.; Nakai, H.; Toda, M.
Bioorg. Med. Chem. Lett. 2002, 12, 907.
12. For example, compound 1 lowered the blood pressure by
15 mmHg at the dose of 4.2 mg/kg, iv in rats.
13. Bromides 20a, b, d and e were prepared from their corre-
sponding carboxylic acids according to conventional proce-
dures.
14. According to the conventional procedure bromide 20c was
prepared from 4-methyl-2-nitro benzoic acid which was in
turn prepared from 4-chloro-3-nitro toluene: i) CuCN in
DMF, ii) H2SO4, H2O.
5. 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., in press.
15. Bromides 20f–i were prepared from their corresponding
ortho-nitro toluenes by bromination with NBS and benzoyl
peroxide in CCl4.
16. Iodide 20j was prepared by the iodination of the com-
mercially available alcohol according to a known procedure
(I2, Ph3P, imidazole, Et2O–CH3CN).
6. We focused our attention on the metabolic stabilization of
the newly discovered chemical lead 1 without loss of its TNF-a
inhibitory activity because one of the critical drawbacks of it
as a drug candidate was found to be the rapid metabolic
hydrolysis of its phosphate moiety as found in our in vitro
study. See ref 7.