Discovery of a new chemical lead for a matrix metalloproteinase inhibitor

Article abstract of DOI:10.1016/j.bmc.2006.01.059

A series of N-benzoyl γ-aminobutyric hydroxamic acids were synthesized and evaluated as matrix metalloproteinase inhibitors. First, we focused on chemical modification of the N-benzoyl residue. Introduction of electron-rich para-substituents was effective

Full text of DOI:10.1016/j.bmc.2006.01.059

Bioorganic & Medicinal Chemistry 14 (2006) 4241–4252
Discovery of a new chemical lead for a matrix
metalloproteinase inhibitor
Masahiro Ikura,* Shingo Nakatani, Shingo Yamamoto, Hiromu Habashita,
Tsuneyuki Sugiura, Kanji Takahashi, Koji Ogawa, Hiroyuki Ohno,
Hisao Nakai and Masaaki Toda
Minase Research Institute, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
Received 24 October 2005; revised 24 January 2006; accepted 25 January 2006
Available online 14 February 2006
Abstract—A series of N-benzoyl c-aminobutyric hydroxamic acids were synthesized and evaluated as matrix metalloproteinase
inhibitors. First, we focused on chemical modification of the N-benzoyl residue. Introduction of electron-rich para-substituents
was effective to increase the inhibitory activity. Especially, some of the analogs with relatively more planar N-acyl residues, such
as 10 and 11, demonstrated more potent activity. Second, chemical modification of the c-aminobutyric hydroxamic acid moiety
was carried out to optimize the three-dimensional arrangement of the two pharmacophores (hydroxamic acid and N-acyl residues).
Among the tested, the c-aminobutyric hydroxamic acid moiety was found to be the best spacer for connecting the above-mentioned
two pharmacophores. Synthesis and structure–activity relationships are discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tioned types of diseases. A large number of synthetic
MMP inhibitors (MMPIs) have been reported. Depend-
ing upon their zinc-binding, MMPIs can be assigned to
several classes, such as the carboxylic acids, hydroxamic
acids, thiols, phosphorus-based ligands, or sulfodii-
mines, among others.1a
Matrix metalloproteinases (MMPs) are produced by
fibroblasts, neutrophils, and macrophages, as well as
by tumor cells.1 These enzymes are postulated to regu-
late the homeostasis of a variety of tissues under the
control of tissue inhibitor of metalloproteinases (TIM-
Ps), which bind to and block the activity of MMPs.
As a result, a stoichiometric imbalance between MMPs
and TIMPs could theoretically result in a variety of
pathological states, ranging from tumor metastasis to
rheumatoid arthritis and multiple sclerosis.1
In the preceding paper,3 we reported on the fragment-
based generation of a new chemical lead using the in
silico technique. Search of a standard LUDI library,6
followed by conventional molecular design, resulted in
lead structure 1 that was targeted for further optimiza-
tion. Here we report on the discovery of a series of N-ac-
yl c-aminobutyric hydroxamic acids, 10 and 11 (Fig. 1),
that are effective MMP inhibitors.
At least 20 members of this enzyme family, which share
significant sequence homology, have been reported.1,2
They can be divided into (1) collagenases (MMP-1, -8,
-13, and -18), (2) gelatinases (MMP-2 and -9), (3) strom-
elysins (MMP-3, -10, and -11), and (4) membrane-type
MMPs (MT-MMPs) (MMP-14, -15, -16, and -17).
2. Chemistry
Synthesis of the test compounds listed in Tables 1–4 is
outlined in Schemes 1–4. Synthesis of compounds 1–16
is described in Scheme 1. Condensation of 21b–m and
21p with O-2-methoxypropane hydroxylamine, followed
by acidic deprotection, afforded 2–13 and 16, respective-
ly. Condensation of 21a, 21n, and 21o with O-ben-
zylhydroxyamine, followed by catalytic hydrogenation,
resulted in 1, 14, and 15, respectively.
For more than 30 years, MMPs have been heralded as
promising targets for the treatment of the above-men-
Keyword: Matrix metalloproteinase inhibitor.
*
Corresponding author. Tel.: +81759611151; fax: +81759629314;
e-mail: ikura@ono.co.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.059

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M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
Synthesis of 19 and 20 is described in Schemes 4A
and B, respectively. A key intermediate 36 for the
synthesis of both compounds 19 and 20 was derived
from the malic acid dimethyl ester 32. Chemoselective
reduction of 32 with borane dimethyl sulfide complex
afforded the diol 33.4 Tosylation of 33, followed by
SN2 substitution of the formed tosylate 34 with sodi-
um azide, produced the azide 35. Catalytic hydroge-
nation of 35, followed by N-acylation with an acid
chloride, afforded 36. O-Alkylation of 36 with chloro-
methyl methyl ether in the presence of diisopropyleth-
ylamine resulted in 37. Treatment of 37 with aqueous
sodium hydroxide gave an elimination product 38,
which was converted to the hydroxamic acid 19 by
the condensation with O-2-methoxypropane hydroxyl-
amine. Compound 39 was prepared by O-acylation of
36 with an acetic anhydride in pyridine. Another
elimination product 40 was obtained by treatment
of the acetate 39 with DBU. Alkaline hydrolysis of
40 led to 41, which was converted to a hydroxamic
acid 20 by condensation with O-2-methoxypropane
hydroxylamine.
Figure 1. A new class of inhibitors of matrix metalloproteinases.
Synthesis of 17 is described in Scheme 2. N-Methylation
of 225 with methyl iodide in the presence of sodium hy-
dride gave 23. Alkaline hydrolysis of 23, followed by the
condensation with O-2-methoxypropane hydroxyl-
amine, provided hydroxamic acid 17.
3. Results and discussion
Compound 18 was prepared from 25 as outlined in
Scheme 3. O-Alkylation of 25 with ethyl 5-bromopent-
anoate in the presence of potassium carbonate yielded
26, after which catalytic hydrogenation resulted in 27.
Palladium-catalyzed coupling of trifluoromethane sulfo-
nate 28, which was prepared from 27 using trifluorome-
thanesulfonic anhydride, with the boric acid 29 resulted
in 30. Alkaline hydrolysis of 30 gave 31, which was con-
verted to the hydroxamic acid 18 by the condensation
with O-2-methoxypropane hydroxylamine.
The compounds listed in Tables 1–4 were tested for their
inhibitory activity against MMP-1 (human collagenase),
MMP-2 (human gelatinase A), MMP-9 (human gelatin-
ase B), and MMP-3 (recombinant human stromelysin).
Since the discovery of the N-benzoyl c-aminobutyric
hydroxamic acid 1 as a new chemical lead for an
MMP-2 and MMP-9 inhibitor, further chemical modifi-
cation of 1 to identify superior structure has been
continued.
Table 1. Effect of the para-substituent of N-benzoyl residue on the activity profiles
Compound
R
IC50 (lM)
MMP-1
59%a
MMP-2
18%a
MMP-9
40%a
MMP-3
NTb
1
2
>100%a
>100
NTb
65%a
49%a
0.038
0.13
2.2
NTb
NTb
NTb
NTb
NTb
>100
>100
2.8
3
4
5
6
>100
NTb
16
17
a Inhibition percentage at 100 lM.
b Not tested.

M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
Table 2. Effect of the para-substituent of N-benzoyl residue on the activity profiles
4243
Compound
R
IC50 (lM)
MMP-9
MMP-1
NTb
MMP-2
0.038
MMP-3
2.8
MMP-3/MMP-2
4
7
NTb
NTb
NTb
NTb
NTb
0.052
73
65
21.1
0.058
0.13
3.8
16
8
NTb
59%a
53%a
>100
123
—
9
0.801
0.0029
0.0071
34%a
10
11
1.8
620
117
0.83
a Inhibition percentage at 100 lM.
b Not tested.
Table 3. Effect of the para-alkoxy substituent at N-benzoyl residue on the activity profiles
Compound
R
IC50 (lM)
MMP-1
>100
MMP-2
0.75
MHP-9
NTa
MMP-3
19
MMP-3/MMP-2
25
12
13
NTa
NTa
NTa
>100
0.038
0.032
NTa
2.9
76
500
460
—
14
15
0.011
0.043
NTa
16
13
0.028
16
>100
>100
a Not tested.
As described in Figure 2, optimization of the N-benzoyl
residue was expected to be the simplest way to obtain an
increase of MMP inhibitory activity. Accordingly,
chemical modification was started with optimization of
the para-substituent of the N-benzoyl residue of 1. Sec-
ond, optimization of the c-aminobutyric hydroxamic
acid moiety of 1 was performed to find the best spacer
for connecting the hydroxamic acid and the N-benzoyl
residue.
Optimization of the para-methyl residue of the N-benzo-
yl moiety of 1 was carried out as shown in Table 1.
Chemical modification was focused on identification of
a partial structure that was optimal for the S10 pocket

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M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
Table 4. Effect of the chemical modification of the 4-aminobutyric acid moiety of 10 on the activity profiles
Compound
X
IC50 (lM)
MMP-1
53%a
MMP-2
0.0029
MMP-9
NTb
MMP-3
1.8
10
17
18
19
20
>100
>10
1.4
>0.1
0.81
0.47
NTb
NTb
NTb
NTb
9.9
>100
35
>100
>100
17
a Inhibition percentage at 100 lM.
b Not tested.
Scheme 1. Synthesis of 1–16. Reagents: (i) MeOCMe2ONH2, EDCÆHCl, HOBtÆH2O, Et3N or iPr2NEt, DMF then HCl, MeOH; (ii) BzlONH2ÆHCl,
EDCÆHCl, HOBtÆH2O, Et3N, DMF then H2, Pd–C, MeOH, THF.
(Fig. 2) and subtype selectivity. As illustrated by the re-
sults of evaluation of 2 and 3, introduction of a larger
para-alkyl residue slightly improved the extent of inhibi-
tion of MMP-2. Replacement of the para-methyl residue
of the N-benzoyl moiety of 1 with a para-chlorophenyl
moiety gave 4 which showed a marked increase of both

M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
4245
Scheme 2. Synthesis of 17. Reagents: (a) Mel, NaH, DMF; (b) NaOH, MeOH; (c) MeOCMe2ONH2, EDCÆHCl, HOBtÆH2O, DMF; (d) HCl, MeOH.
Scheme 3. Synthesis of 18. Reagents: (a) EtO2C(CH2)4 Br, K2CO3, DMF; (b) H2, Pd–C, MeOH; (c) Tl2O, pyridine; (d) Pd(PPh3)2Cl2, Et3N, OMF;
(e) NaOH, THF, MeOH; (f) MeOCMe2ONH2, EDCÆHCl, HOBt H2O, DMF; (g) HCl, MeOH.
MMP-2 inhibition and MMP-3 inhibition. Replacement
of the para-chloro residue of 4 with para-alkyl residues,
such as n-propyl and n-heptyl residues, led to 5 and 6,
respectively, with a decrease of inhibitory activity for
both MMP-2 and MMP-3. These results indicated that
the more electron-rich para-aryl moieties 4–6 were supe-
rior to the para-alkyl residues 1–3 as a para-substituent
R that was optimum for the S10 pocket. In particular, a
para-chlorophenyl residue performed best among the
para-aryl residues tested.
structures. Based on the relatively weaker inhibitory
activity of 8 and 9, their hydrophilic nature seemed
to be unsuited to the S10 pocket.
Because of the equipotency of the para-methoxyphenyl
analog 7 and the para-chlorophenyl analog 4, the
para-butoxyphenyl analog 12 and three para-phenoxy-
phenyl analogs 13–15 were synthesized and tested
(Table 3). Replacement of the para-methoxyphenyl res-
idue of 7 with a para-butoxyphenyl residue led to 12,
with a more than 10-fold decrease of inhibitory activity
for MMP-2, while replacement of the methoxyphenyl
residue with a phenoxyphenyl residue afforded 13 that
retained both potent inhibitory activity and subtype
selectivity (MMP-3/MMP-2 ratio). Again, an electron-
rich phenoxy residue was found to be more favorable
than a larger alkoxy residue. Based on the above find-
ings, another two analogs 14 and 15 were synthesized
and evaluated. Introduction of a methoxy residue at
the para-position of the phenoxy moiety of 13 afforded
14, which retained inhibitory activity for MMP-2 and
showed decreased inhibition of MMP-3. Demethylation
of the methoxy residue of 14 gave 15, which also re-
tained inhibitory activity for MMP-2 and showed de-
creased inhibition of MMP-3. Compounds 14 and 15
also showed potent inhibitory activity against MMP-9,
with 14 being more potent than 15. It was interesting
that the phenol analog 15 showed stronger activity
against MMP-2 than compound 8. This was speculated
to be due to the difference of three-dimensional struc-
tures between the biphenyl moiety of 8 and the para-
phenoxybenzoyl moiety of 15. To confirm the rationale
for attempting para-substitution of N-benzoyl residues, a
meta-nbutoxy isomer 16 was synthesized and evaluated.
Further chemical modification of the para-substituent
R was continued as shown in Table 2. Based on the
marked increase of inhibitory activity shown by the
para-chlorophenyl analog 4, para-methoxyphenyl and
para-hydroxyphenyl analogs 7 and 8 were also synthe-
sized and evaluated. The para-methoxyphenylbenzoyl
analog 7 showed stronger inhibitory activity than the
para-hydroxyphenylbenzoyl analog 8, while the inhibi-
tory activity of 7 for MMP-2 and MMP-3 was close
to that of 4. Among the heteroaryl analogs, such as
9 and 10, that were tested, 10 showed significantly
stronger inhibitory activity and greater selectivity rela-
tive to 4 and 5, although the pyrrole analog 9 showed
significantly weaker activity relative to 4 and 7. Based
on the significant increase of potency and subtype
selectivity of 10, the para-chlorocinnamyl analog
11 was also prepared and evaluated. Compound 11
demonstrated slightly less potent inhibition and lower
selectivity (MMP-3/MMP-2 ratio) relative to 10, that
it also showed potent inhibitory activity against
MMP-9. As a result, conjugated and electron-rich
para-substituents, such as benzofuran-2-yl and para-
chlorocinnamyl, were identified as the optimum partial

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M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
Scheme 4. (A) Synthesis of 19. Reagents: (a) i–BH3ÆSMe2, THF then NaBH4; ii—TsOH Æ H2O, MeOH; (b) TsCl, pyridine; (c) NaN3, DMF; (d) H2,
Pd–C, MeOH then HCl; (e) 4-(1-benzofuran-2-yl)benzoyl chloride, Et3N, CH2Cl2; (f) MOMCl, Pr2NEt, CH2Cl2; (g) NaOH, THF, MeOH; (h)
i
MeOCMe2ONH2, EDCÆHCl, HOBtÆH2O, DMF; (i) HCI, MeOH. (B) Synthesis of 20. Reagents: (a) Ac2O, pyridine; (b) DBU, THF; (c) NaOH,
THF, MeOH; (d) MeOCMe2ONH2, EDCÆHCl, HOBt H2O, DMF; (e) HCI MeOH.
moiety of 10 resulted in 17, with marked reduction of
MMP inhibitory activity. Replacement of the amide
moiety of 10 with an ether oxygen afforded 18, which
showed loss of inhibitory activity for MMP-2. As a re-
sult, formation of the hydrogen bonds of the amide moi-
ety with Pro238 and Leu181, as described in Figure 2,
was strongly suggested to be important for the potent
MMP-2 inhibitory activity of 10. Introduction of a
trans-double bond into the alkylene of the c-aminobu-
tyric hydroxamic acid moiety could be suggested to reg-
Figure 2. Design of N-benzoyl residue.
ulate the length and direction of the two
pharmacophores, that is the hydroxamic acid and the
Compound 16 showed less than 50% inhibition of
MMP-1, MMP-2, and MMP-3 at 100 lM. Thus,
para-substitution was superior to meta-substitution for
increasing the inhibitory activity, as illustrated by the
loss of inhibitory activity with compound 16.
N-benzoyl moiety. Introduction of a conjugated trans-
2,3-double bond into the trimethylene moiety of 10
afforded 19, which showed 277-fold weaker inhibitory
activity for MMP-2. Introduction of a non-conjugated
3,4-trans-double bond into the trimethylene moiety of
10 yielded 20, which also showed a remarkable decrease
of inhibitory activity. Thus, the optimum three-dimen-
sional arrangement of the two pharmacophores (zinc-
binding hydroxamic acid and N-benzoyl residue) was
confirmed to be important for potent inhibitory activity.
Optimization of the spacer that connected the hydroxa-
mic acid and the N-benzoyl residue was carried out
using the best-performing benzofuran-2-yl analog 10,
as shown in Table 4. N-Methylation of the N-benzoyl

M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
4247
The secondary amide moiety of 10 was found to be
essential for potent activity because it controlled the
conformation and/or the hydrogen bond with Pro238
and Leu181, as described in Figure 2.
AcOEt. The organic layer was washed with brine and
dried over MgSO4. Removal of the volatiles in vacuo
provided a solid, which was washed with Et2O to give
21a (2.00 g, 90%) as a white crystal. TLC Rf = 0.58
(CHCl3/MeOH/AcOH, 18/2/1); 1H NMR (200 MHz,
CDCl3) d 7.73 (d, J = 8.4 Hz, 2H), 7.40 (m, 1H), 7.21
(d, J = 8.4 Hz, 2H), 3.47 (m, 2H), 2.41 (t, J = 7.3 Hz,
2H), 2.38 (s, 3H), 1.94 (m, 2H).
In summary, further optimization of a new chemical
lead 1, which was discovered by the in silico fragment-
based lead generation technique,6 was carried out
successfully. Chemical modification was focused on the
4-N-(4-methyl)benzoyl moiety of 1, and a series of N-
benzoyl c-aminobutyric hydroxamic acids were synthe-
sized and evaluated. 4-(para-Substituted phenyl)benzoyl
analogs 4 and 7 showed stronger inhibitory activity,
while the 4-(para-alkylphenyl)benzoyl analogs 5 and 6
showed weaker inhibition. Introduction of electron-rich
para-substituents, such as benzofuran-2-yl and para-
chlorocinnamyl moieties, into the N-benzoyl moiety
was found to be highly effective to enhance the MMP
inhibitory activity, as illustrated by compounds 10 and
11. As shown by the complete loss of inhibitory activity
in the case of 16, para-substitution of the N-benzoyl
moiety was also required for strong activity. Among
those tested, c-aminobutyric hydroxamic acid was found
to be the best spacer for connecting the two pharmaco-
phores. A secondary amide was a superior linkage com-
pared to the ether or N-methyl amide with respect to the
formation of hydrogen bonds with amino acid residues
Pro238 and Leu181 in the S10 pocket.
4.1.2. N-[4-(Hydroxyamino)-4-oxobutyl]-4-methylbenza-
mide (1). To a stirred solution of 21a (885 mg) in
DMF (5 mL) were added O-benzyl hydroxylamine
hydrochloride (638 mg), EDCÆHCl (844 mg), HOBt
(673 mg), and diisopropylethylamine (1.74 mL). After
being stirred at room temperature for 40 h, the reac-
tion mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and
evaporated. The resulting residue was purified by col-
umn chromatography on silica gel to give a protected
hydroxamate. A heterogeneous mixture of the above-
mentioned residue and 10% palladium on carbon
(504 mg) in MeOH (4 mL) was vigorously stirred un-
der H2 gas for 2 h. Catalyst was removed by filtration
and the filtrate was concentrated to give 1 (293 mg,
65%) as a white powder. TLC Rf = 0.23 (CHCl3/
MeOH, 10/1); MS (APCI, neg. 40 V) m/z 235
(MꢀH)ꢀ; IR (KBr) 1674, 1613, 1608, 1564, 1510,
1458, 1436, 1360, 1332, 1313, 1258, 1233, 1197,
1178, 1101, 1024, 969 cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6) d10.39 (s, 1H), 8.70 (s, 1H), 8.40 (t,
J = 5.2 Hz, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.25 (d,
J = 8.1 Hz, 2H), 3.24 (dt, J = 6.6, 5.2 Hz, 2H), 2.35
(s, 3H), 2.02 (t, J = 7.7 Hz, 2H), 1.74 (m, 2H).
4. Experimental
4.1. General chemical procedures
Analytical samples were homogeneous as confirmed by
thin-layer chromatography (TLC) and yielded spectro-
scopic data consistent with the assigned structures. All
1H NMR spectra were obtained with a Varian Gemi-
ni-200 or MERCURY-300 spectrometer. The chemical
shift values are reported in ppm (d) and coupling con-
stants (J) in Hertz (Hz). Fast atom bombardment
(FAB) and electron ionization (EI) mass spectra were
obtained with a JEOL JMS-DX303HF or JMS-700
spectrometer. Atmospheric pressure chemical ionization
(APCI) mass spectra were determined by a Hitachi M-
1200H spectrometer. Matrix assisted laser desorption
ionization-time-of-flight (MALDI-TOF) mass spectra
were obtained on a PerSeptive Voyager Elite spectrom-
eter. IR spectra were measured using a Perkin-Elmer
FTIR 1760X or JASCO FTIR-430 spectrometer. Col-
umn chromatography was carried out using silica gel
[Merck silica gel 60 (0.063–0.200 mm), Wako Gel
C200, Fuji Silysia FL60D, or Fuji Silysia BW-235].
TLC was also performed on silica gel (Merck TLC plate,
silica gel 60 F254).
4.1.3. Synthesis of esters 21b–p. Compounds 21b–p were
prepared from the corresponding methyl or ethyl esters,
respectively, according to the usual alkaline hydrolysis.
4.1.4. 4-Butyl-N-[4-(hydroxyamino)-4-oxobutyl]benzam-
ide (2). To a stirred solution of 21b (400 mg, 1.52 mmol)
in DMF (3 mL) were added HOBt (470 mg, 2.0 equiv),
EDCÆHCl (590 mg, 2.0 equiv), and O-2-methoxypro-
pane hydroxylamine (320 mg, 2.0 equiv). After being
stirred at room temperature for 150 min, the reaction
mixture was quenched with satd NH4Cl and extracted
with AcOEt. The organic layer was washed with brine,
dried over MgSO4, and evaporated to give a protected
hydroxamate. The pH value of a solution of the protect-
ed hydroxamate in MeOH was adjusted to 3 with 1 N
HCl. After being stirred at room temperature for
20 min, the solution was diluted with AcOEt and treated
with satd NaHCO3. The organic layer was washed with
brine, dried over MgSO4, and evaporated. The resulting
crystalline solid was washed with Et2O to give 2
(211 mg, 50%) as a white powder. TLC Rf = 0.37
(CHCl3/MeOH/AcOH, 9/1/0.5); MS (MALDI, Pos.)
m/z 301 (M+Na)+, 279 (M+H)+; IR (KBr) 3214, 2935,
2849, 1672, 1610, 1564, 1435, 1314, 1176, 1100, 1023,
4.1.1. 4-[(4-Methylbenzoyl)amino]butanoic acid (21a). To
a stirred suspension of 4-aminobutyric acid (1.13 g,
11.0 mmol) in THF (21 mL) was added 1 N NaOH
(21.0 mL, 21.0 mmol) at 0 °C, followed by the addition
of 4-methylbenzoyl chloride (1.55 g, 10.0 mmol). After
being stirred for 3 h at room temperature, the reaction
mixture was acidified with 2 N HCl and extracted with
967, 907, 851, 764, 688, 529, 412 cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6) d10.40 (br s, 1H), 10.20 (s, 1H),
8.41 (t, J = 5.4 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.25
(d, J = 8.0 Hz, 2H), 3.23 (dt, J = 5.4 Hz, 7.0 Hz, 2H),
2.62 (t, J = 8.2 Hz, 2H), 2.02 (t, J = 7.0 Hz, 2H),

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M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
1.83-1.66 (m, 2H), 1.64 ꢁ 1.48 (m, 2H), 1.30 (sextet,
J = 7.2 Hz, 2H), 0.90 (t, J = 7.2 Hz, 3H).
J = 8.5 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.05 (d,
J = 8.8 Hz, 2H), 3.81 (s, 3H), 3.26 (q, J = 6.5 Hz, 2H),
2.03 (t, J = 7.5 Hz, 2H), 1.83–1.69 (m, 2H).
Compounds 3–16 were prepared from 21c–p, respective-
ly, according to the same procedures as described above.
4.1.10.
40-Hydroxy-N-[4-(hydroxyamino)-4-oxobutyl]-
1,10-biphenyl-4-carboxamide (8). Yield 36%; TLC
Rf = 0.23 (CHCl3/MeOH/AcOH, 9/1/0.5); MS (FAB,
Pos.) m/z 315 (M+H)+; IR (KBr) 3310, 1632, 1543,
1495, 1381, 1300, 1265, 1206, 1186, 1110, 829, 769,
4.1.5.
4-Cyclohexyl-N-[4-(hydroxyamino)-4-oxobu-
tyl]benzamide (3). Yield 56%; TLC Rf = 0.40 (CHCl3/
MeOH/AcOH, 9/1/0.5); MS (FAB, Pos.) m/z 305
(M+H)+; IR (KBr) 3320, 2923, 2849, 1631, 1546, 1503,
1447, 1306, 1185, 1099, 1029, 996, 843, 766, 662,
673, 626, 529 cmꢀ1 1H NMR (200 MHz, DMSO-d6)
;
d10.40 (s,1H), 9.80–9.45 (br s, 1H), 8.53–8.44 (m, 1H),
7.89 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.56
(d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 3.31–3.20
(m, 2H), 2.03 (t, J = 7.4 Hz, 2H), 1.83–1.68 (m, 2H).
1
632 cmꢀ1; H NMR (200 MHz, DMSO-d6) d10.39 (s,
1H), 8.43-8.36 (m, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.34
(d, J = 8.4 Hz, 2H), 3.30–3.13 (m, 2H), 2.63–2.54 (m,
1H), 2.01 (t, J = 7.6 Hz, 2H), 1.86–1.65 (m, 6H), 1.48–
1.24 (m, 6H).
4.1.11. N-[4-(Hydroxyamino)-4-oxobutyl]-4-(1H-pyrrol-
1-yl)benzamide (9). Yield 74%; TLC Rf = 0.31 (CHCl3/
MeOH/AcOH, 9/1/0.5); MS (MALDI, Pos.) m/z 310
(M+Na)+, 288 (M+H)+; IR (KBr) 3322, 3175, 1632,
4.1.6. 40-Chloro-N-[4-(hydroxyamino)-4-oxobutyl]-1,10-
biphenyl-4-carboxamide (4). Yield 87%; TLC Rf = 0.34
(CHCl3/MeOH/AcOH, 9/1/0.5); MS (MALDI, Pos.)
m/z 371 (M+K)+, 355 (M+Na)+, 333 (M+H)+; IR
(KBr) 3308, 3180, 3020, 1632, 1542, 1484, 1454, 1390,
1545, 1519, 1474, 1331, 845, 723 cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6) d 10.40 (s, 1H), 9.00–8.24 (br s,
1H), 8.52 (t, J = 5.6 Hz, 1H), 7.94 (d, J = 8.5 Hz, 2H),
7.68 (d, J = 8.5 Hz, 2H), 7.50–7.44 (m, 1H), 6.34–6.29
(m, 1H), 3.38–3.31 (m, 2H), 2.04 (t, J = 7.5 Hz, 2H),
1.76 (quint, J = 7.5 Hz, 2H).
1302, 1103, 1003, 972, 942, 825, 769, 685, 623 cmꢀ1
;
1H NMR (200 MHz, DMSO-d6) d10.41 (s, 1H), 8.62–
8.52 (m, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.82–7.72 (m,
4H), 7.55 (d, J = 8.4 Hz, 2H), 3.35–3.20 (m, 2H), 2.04
(t, J = 7.5 Hz, 2H), 1.83–1.69 (m, 2H).
4.1.12.
4-(1-Benzofuran-2-yl)-N-[4-(hydroxyamino)-4-
oxobutyl]benzamide (10). Yield 38%; TLC Rf = 0.22
(CHCl3/MeOH/AcOH, 100/10/1); MS (FAB, Pos.) m/z
339 (M+H)+; IR (KBr) 3320, 1633, 1541, 1451, 1306,
1030, 854, 807, 748 cmꢀ1; 1H NMR (200 MHz, DMSO-
d6) d10.39 (br s, 1H), 8.59 (t, J = 5.8 Hz, 1H), 8.01 (d,
J = 9.0 Hz, 2H), 7.96 (d, J = 9.0 Hz, 2H), 7.67 (m, 2H),
7.57 (d, J = 0.5 Hz, 1H), 7.39–7.23 (m, 2H), 3.27 (q,
J = 5.8 Hz, 2H), 2.03 (t, J = 7.6 Hz, 2H), 1.76 (m, 2H).
4.1.7. N-[4-(Hydroxyamino)-4-oxobutyl]-40-propyl-1,10-
biphenyl-4- carboxamide (5). Yield 60%; TLC Rf = 0.40
(CHCl3/MeOH/AcOH, 9/1/0.5); MS (FAB, Pos.) m/z
341 (M+H)+; IR (KBr) 3302, 3180, 2954, 1630, 1544,
1475, 1371, 1305, 1222, 1096, 1044, 1029, 1003, 972,
1
943, 834, 812, 769, 687, 658, 628, 536 cmꢀ1; H NMR
(200 MHz, DMSO-d6) d 10.40 (s, 1H), 8.70 (br s, 1H),
8.57–8.49 (m, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.74 (d,
J = 8.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.31 (d,
J = 8.5 Hz, 2H), 3.31–3.20 (m, 2H), 2.61 (t, J = 7.4 Hz,
2H), 2.04 (t, J = 7.2 Hz, 2H), 1.76 (quint, J = 7.2
Hz, 2H), 1.62 (sextet, J = 7.4 Hz, 2H), 0.92 (t,
J = 7.4 Hz, 3H).
4.1.13. 4-[(E)-2-(4-Chlorophenyl)vinyl]-N-[4-(hydroxya-
mino)-4-oxobutyl]benzamide (11). Yield 78%; TLC
Rf = 0.17 (CHCl3/MeOH/AcOH, 100/10/1); MS (FAB,
Pos.) m/z 359 (M+H)+; IR (KBr) 3266, 1636, 1544,
1503, 1489, 1437, 1323, 1097, 970, 843, 814, 710,
1
563 cmꢀ1; H NMR (200 MHz, DMSO-d6) d 10.39 (br
4.1.8. 40-Heptyl-N-[4-(hydroxyamino)-4-oxobutyl]-1,10-
biphenyl-4-carboxamide (6). Yield 84%; TLC Rf = 0.34
(CHCl3/MeOH/AcOH, 9/1/0.5); MS (FAB, Pos.) m/z
397 (M+H)+, 279; IR (KBr) 3305, 3185, 2921, 1631,
1543, 1494, 1454, 1307, 1096, 835, 815, 770, 627,
s, 1H), 8.50 (t, J = 5.8 Hz, 1H), 7.86 (d, J = 8.4 Hz,
2H), 7.67 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H),
7.44 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 16.2 Hz, 1H),
7.30 (d, J = 16.2 Hz, 1H), 3.25 (q, J = 5.6 Hz, 2H),
2.02 (t, J = 7.6 Hz, 2H), 1.74 (m, 2H).
1
610 cmꢀ1; H NMR (200 MHz, DMSO-d6) d 10.40 (br
s, 1H), 8.57–8.50 (m, 1H), 7.93 (d, J = 8.4 Hz, 2H),
7.74 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.30
(d, J = 8.4 Hz, 2H), 3.32–3.22 (m, 2H), 2.62 (t,
J = 7.7 Hz, 2H), 2.04 (t, J = 7.3 Hz, 2H), 1.76 (quint,
J = 7.3 Hz, 2H), 1.69–1.52 (m, 2H), 1.38–1.17 (m, 8H),
0.86 (t, J = 6.6 Hz, 3H).
4.1.14. 4-Butoxy-N-[4-(hydroxyamino)-4-oxobutyl]benz-
amide (12). Yield 71%; TLC Rf = 0.29 (CHCl3/MeOH/
AcOH, 9/1/0.5); MS (MALDI, Pos.) m/z 333 (M+K)+,
317 (M+Na)+, 295 (M+H)+; IR (KBr) 3321, 2956,
1629, 1543, 1507, 1252, 1178, 1067, 1027, 836, 768,
1
644 cmꢀ1; H NMR (200 MHz, DMSO-d6) d 10.39 (s,
1H), 8.70 (br s, 1H), 8.32 (t, J = 5.8 Hz, 1H), 7.80 (d,
J = 9.0 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 4.01 (t,
J = 6.4 Hz, 2H), 3.35–3.15 (m, 2H), 2.01 (t, J = 7.3 Hz,
2H), 1.81–1.64 (m, 4H), 1.44 (sextet, J = 7.4 Hz, 2H),
0.94 (t, J = 7.4 Hz, 3H).
4.1.9.
N-[4-(Hydroxyamino)-4-oxobutyl]-40-methoxy-
1,10-biphenyl-4-carboxamide (7). Yield 70%; TLC
Rf = 0.26 (CHCl3/MeOH, 9/1); MS (MALDI, Pos.) m/
z 351 (M+Na)+, 329 (M+H)+; IR (KBr) 3324, 3186,
3010, 2952, 1629, 1542, 1495, 1289, 1255, 1206, 1182,
1095, 1036, 1012, 824, 769, 715, 637, 625, 601 cmꢀ1
;
4.1.15. N-[4-(Hydroxyamino)-4-oxobutyl]-4-phenoxyben-
zamide (13). Yield 59%; TLC Rf = 0.25 (CHCl3/MeOH/
AcOH/H2O, 100/10/1/1); MS (MALDI, Pos.) m/z 315
1H NMR (200 MHz, DMSO-d6) d 10.40 (s, 1H), 8.57–
8.47 (m, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.71 (d,

M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
4249
(M+H)+; IR (KBr) 3302, 1633, 1544, 1489, 1370, 1303,
1258, 1165, 1100, 1028, 972, 845, 747, 692, 630 cmꢀ1
mixture was quenched with 1 N HCl and extracted with
AcOEt. The organic layer was washed with brine, dried
over MgSO4, and evaporated to give crude product,
which was crystallized from n-hexane–Et2O to give 34
(5.18 g, 48%). TLC Rf = 0.60 (AcOEt/n-hexane, 1/1);
1H NMR (200 MHz, CDCl3) d7.80 (d, J = 8.4 Hz,
2H), 7.36 (d, J = 8.4 Hz, 2H), 4.32–4.18 (m, 1H), 4.05
(d, J = 5.2 Hz, 2H), 3.70 (s, 3H), 3.03 (br s, 1H), 2.57–
2.53 (m, 2H), 2.46 (s, 3H).
;
1H NMR (200 MHz, DMSO-d6) d 10.36 (br s, 1H),
8.69 (br s, 1H), 8.41 (t, J = 5.6 Hz, 1H), 7.85 (dt,
J = 8.8 Hz, 2.8 Hz, 2H), 7.37–7.46 (m, 2H), 7.19 (tt,
J = 7.2 Hz, 1.2 Hz, 1H), 6.97–7.09 (m, 4H), 3.22 (dt,
J = 6.6 Hz, 5.6 Hz, 2H), 1.99 (t, J = 7.6 Hz, 2H), 1.71
(tt, J = 7.6 Hz, 6.6 Hz, 2H).
4.1.16. N-[4-(Hydroxyamino)-4-oxobutyl]-4-(4-methoxy-
phenoxy)benzamide (14). Yield 71%; TLC Rf = 0.40
(CHCl3/MeOH/AcOH/H2O, 50/10/1/1); MS (APCI,
Neg, 40 V) m/z 343 (MꢀH)-; IR (KBr) 3300, 1634,
4.1.21. Methyl (3S)-4-azido-3-hydroxybutanoate (35). To
a stirred solution of 34 (4.66 g, 16.2 mmol) in DMF
(40 mL) was added sodium azide (1.16 g, 17.8 mmol).
After being stirred at 80 °C for 45 min, the reaction mix-
ture was evaporated to remove DMF and extracted with
AcOEt. The organic layer was washed with brine, dried
over Na2SO4, and evaporated to give 35 (1.96 g, 77%).
TLC Rf = 0.68 (AcOEt/n-hexane, 1/1); 1H NMR
(200 MHz, CDCl3) d 4.28–4.14 (m, 1H), 3.74 (s, 3H),
3.40 (dd, J = 3.1 Hz, 9.6 Hz, 1H), 3.33 (dd, J = 4.1 Hz,
9.6 Hz, 1H), 3.14 (d, J = 4.0 Hz, 1H), 2.58–2.55 (m, 2H).
1543, 1508, 1240, 1101, 1035, 837 cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6) d 10.37 and 9.79 (both br s,
1H), 9.02 and 8.70 (both br s, 1H), 8.40 (t,
J = 5.5 Hz, 1H), 7.82 (d, J = 9.1 Hz, 2H), 7.06–6.91
(m, 6H), 3.79 (s, 3H), 3.21 (m, 2H), 1.96 (m, 2H),
1.72 (m, 2H).
4.1.17. N-[4-(Hydroxyamino)-4-oxobutyl]-4-(4-hydroxy-
phenoxy)benzamide (15). Yield 66%; TLC Rf = 0.25
(CHCl3/MeOH/AcOH/H2O, 50/10/1/1); MS (APCI,
Neg, 40 V) m/z 329 (MꢀH)-; IR (KBr) 3297, 1635,
1543, 1497, 1374, 1326, 1236, 1166, 1096, 971, 855,
4.1.22. Methyl (3S)-4-{[4-(1-benzofuran-2-yl)benzo-
yl]amino}-3-(hydroxy)butanoate (36). To a stirred solu-
tion of 35 (1.00 g, 6.36 mmol) in MeOH (50 mL)
was added 10% palladium on carbon under argon
atmosphere. The resulting heterogeneous mixture was
subjected to the catalytic hydrogenation for an hour.
Catalyst was removed by filtration and the filtrate
was concentrated. The resulting amine was treated
with 4 N HCl/AcOEt to obtain the hydrochloride salt.
To a stirred solution of the hydrochloride salt in
CH2Cl2 (20 mL) were added Et3N (2.7 mL,
19.08 mmol) and 4-(1-benzofuran-2-yl)benzoyl chloride
(1.63 g, 6.36 g) under cooling with an ice-water bath.
After being stirred at room temperature for 30 min,
the reaction mixture was quenched with 1 N HCl
and extracted with AcOEt. The organic layer was
washed with brine, dried over Na2SO4, and evaporat-
ed to give 36 (1.89 g, 84%). TLC Rf = 0.31 (AcOEt/n-
hexane, 1/1); 1H NMR (200 MHz, DMSO-d6) d 8.57
(t, J = 6.0 Hz, 1H), 8.00 (s, 4H), 7.71–7.63 (m, 2H),
7.57 (br s, 1H), 7.40–7.24 (m, 2H), 5.13 (d,
J = 5.6 Hz, 1H), 4.18–4.00 (m, 1H), 3.57 (s, 3H),
3.30 (t, J = 6.0 Hz, 2H), 2.55 (dd, J = 4.0 Hz,
15.0 Hz, 1H), 2.31 (dd, J = 4.0 Hz, 15.0 Hz, 1H).
1
838, 777, 633 cmꢀ1; H NMR (200 MHz, DMSO-d6) d
8.37 (t, J = 5.5 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 6.92
(d, J = 9.1 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.80 (d,
J = 9.1 Hz, 2H), 3.21 (m, 2H), 1.99 (m, 2H), 1.71
(m, 2H).
4.1.18. 3-Butoxy-N-[4-(hydroxyamino)-4-oxobutyl]benz-
amide (16). Yield 78%; TLC Rf = 0.31 (CHCl3/MeOH/
AcOH, 9/1/0.5); MS (MALDI, Pos.) m/z 317
(M+Na)+, 295 (M+H)+; IR (KBr) 3377, 3292, 2871,
1633, 1542, 1469, 1318, 1239, 1105, 1040, 975, 800,
1
742 cmꢀ1; H NMR (200 MHz, DMSO-d6) d10.39 (s,
1H), 8.45 (t, J = 5.2 Hz, 1H), 7.43–7.29 (m, 3H), 7.19–
7.01 (m, 1H), 4.01 (t, J = 6.3 Hz, 2H), 3.30–3.18 (m,
2H), 2.02 (t, J = 7.5 Hz, 2H), 1.83–1.64 (m, 4H), 1.49
(sextet, J = 7.3 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H).
4.1.19. Methyl (3S)-3,4-dihydroxybutanoate (33). To a
stirred solution of dimethyl (2S)-2-hydroxysuccinate
(32) (92.5 mmol) in THF (170 mL) were added dropwise
borane dimethyl sulfide complex (95.3 mmol) at room
temperature and then sodium borohydride (4.63 mmol)
under cooling with an ice-water bath. After being stirred
for 10 min, the reaction mixture was warmed up to room
temperature and stirred for additional 30 min. To the
stirred reaction mixture were added MeOH (40 mL)
and p-toluenesulfonic acid monohydrate (4.63 mmol)
at room temperature. After being stirred for 30 min,
the reaction mixture was evaporated. The resulting res-
idue was diluted with MeOH–benzene and evaporated.
This work-up was repeated several times to give 33
(100%) as a yellow oil. TLC Rf = 0.25 (AcOEt).
4.1.23. Methyl (3S)-4-{[4-(1-benzofuran-2-yl)benzo-
yl]amino}-3-(methoxymethoxy)butanoate (37). To a stir-
red solution of 36 (201 mg, 0.57 mmol) in CH2Cl2
(1.0 mL) were added diisopropylethylamine (1.0 mL)
and chloromethyl methyl ether (2.83 mmol). After being
stirred at room temperature overnight, the reaction mix-
ture was quenched with 0.5 N HCl and extracted with
AcOEt. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The resulting residue was
purified by column chromatography on silica gel
(AcOEt/n-hexane, 1/1) to give 37 (176 mg, 78%). TLC
1
4.1.20. Methyl (3S)-3-hydroxy-4-{[(4-methylphenyl)sulfo-
nyl]oxy}butanoate (34). To a stirred solution of 33
(37.3 mmol) in pyridine (50 mL) was added p-toluene-
sulfonyl chloride (37.3 mmol) under cooling with an
ice-water bath. After being stirred for 5 h, the reaction
Rf = 0.38 (AcOEt/n-hexane, 1/1); H NMR (200 MHz,
DMSO-d6)
d 8.65 (t, J = 5.4 Hz, 1H), 8.02 (d,
J = 9.2 Hz, 2H), 7.97 (d, J = 9.2 Hz, 2H), 7.71–7.63
(m, 2H), 7.57 (br s, 1H), 7.40–7.24 (m, 2H), 4.65 (d,
J = 7.0 Hz, 1H), 4.59 (d, J = 7.0 Hz, 1H), 4.20–4.08

4250
M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
(m, 1H), 3.58 (s, 3H), 3.52–3.34 (m, 2H), 2.65 (dd,
J = 15.8 Hz, 4.4 Hz, 1H), 2.54–2.43 (m, 1H).
9.6 Hz, 1H), 5.54 (dt, J = 14.4 Hz, 7.1 Hz, 1H), 3.61 (s,
3H), 3.16 (d, J = 7.1 Hz, 2H).
4.1.24. (2E)-4-{[4-(1-Benzofuran-2-yl)benzoyl]amino}but-
2-enoic acid (38). To a stirred solution of 37 (0.35 mmol)
in THF (2.0 mL) was added 1 N NaOH (1.0 mL). After
being stirred at room temperature overnight, the reac-
tion mixture was quenched with 1 N HCl and extracted
with AcOEt. The organic layer was washed with brine,
dried over MgSO4, and evaporated to give 38 (100%).
TLC Rf = 0.21 (CHCl3/MeOH, 10/1).
4.1.28. (3E)-4-{[4-(1-Benzofuran-2-yl)benzoyl]amino}but-
3-enoic acid (41). Compound 41 (52 mg, 69%) was
obtained from 40 according to the same procedures as
described for the preparation of 38 from 37. TLC
Rf = 0.22 (CHCl3/MeOH/AcOH, 100/10/1); 1H NMR
(200 MHz, DMSO-d6) d 10.39 (d, J = 9.8 Hz, 1H), 8.05
(s, 2H), 7.72–7.64 (m, 2H), 7.61 (d, J = 0.6 Hz, 1H),
7.41–7.24 (m, 2H), 6.95 (dd, J = 14.4 Hz, 9.8 Hz, 1H),
5.54 (dt, J = 14.4 Hz, 7.2 Hz, 1H), 3.05 (d, J = 7.2 Hz, 2H).
4.1.25. 4-(1-Benzofuran-2-yl)-N-[(2E)-4-(hydroxyamino)-
4-oxobut-2-enyl]benzamide (19). To a stirred solution of
38 (110 mg, 0.287 mmol) in DMF (2 mL) were added
HOBtÆH2O (66 mg, 0.431 mmol), EDCÆHCl (83 mg,
0.431 mmol), and O-2-methoxypropane hydroxylamine
(107 lL, 1.15 mmol). After being stirred at room tem-
perature for 2 h, the reaction mixture was quenched with
satd NH4Cl and extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and
evaporated. The resulting residue was purified by col-
umn chromatography on a silica gel to give a protected
hydroxamate (TLC Rf = 0.40, CHCl3/MeOH, 10/1). The
pH value of the solution of the protected hydroxamate
in MeOH was adjusted to 3 with 1 N HCl. After being
stirred at room temperature for 30 min, the solution
was evaporated. The resulting crystalline solid was
washed with Et2O to give 19 (15 mg, 23% from 38) as
a white powder. TLC Rf = 0.20 (CHCl3/MeOH/AcOH,
100/10/1); MS (MALDI, Pos.) m/z 337 (M+H)+, 359
(M+Na)+; IR (KBr) 3313, 1635, 1537, 1497, 1450,
4.1.29. 4-(1-Benzofuran-2-yl)-N-[(1E)-4-(hydroxyamino)-
4-oxobut-1-enyl]benzamide (20). Compound 20 (32 mg,
62%) was prepared from 41 as a pale yellow powder
according to the same procedures as described for the
preparation of 19 from 38. TLC Rf = 0.21 (CHCl3/
MeOH/AcOH, 100/10/1); 1H NMR (200 MHz,
DMSO-d6) d 10.45 (br s, 1H), 10.37 (d, J = 9.6 Hz,
1H), 8.75 (s, 1H), 8.04 (s, 4H), 7.72–7.63 (m, 2H), 7.61
(s, 1H), 7.41–7.24 (m, 2H), 6.94 (dd, J = 14.2 Hz,
9.6 Hz, 1H), 5.53 (dt, J = 14.2 Hz, 7.8 Hz, 1H), 2.76
(d, J = 7.8 Hz, 2H).
4.1.30. Ethyl 4-[[4-(1-Benzofuran-2-yl)benzoyl](meth-
yl)amino]butanoate (23). To a stirred solution of com-
pound 226 (100 mg, 0.29 mmol) and iodomethane
(355 lL, 5.7 mmol) in DMF (3 mL) was added 60%
NaH (13 mg, 0.32 mmol) at 0 °C. After being stirred for
an hour at room temperature, the reaction mixture was
quenched with satd NH4Cl and extracted with AcOEt.
The organic layer was washed with H2O, brine, dried over
MgSO4, and evaporated to give 23 as a crude product.
;
1306, 854, 808, 749 cmꢀ1 1H NMR (200 MHz,
DMSO-d6) d 10.60 (br s, 1H), 8.89 (t, J = 5.8 Hz, 1H),
8.02 (s, 4H), 7.71–7.63 (m, 2H), 7.59 (br s, 1H), 7.40–
7.24 (m, 2H), 6.69 (dt, J = 15.4, 4.8 Hz, 1H), 5.86 (d,
J = 15.4 Hz, 1H), 4.08–4.02 (m, 2H).
4.1.31.
4-[[4-(1-Benzofuran-2-yl)benzoyl](methyl)ami-
no]butanoic acid (24). To a stirred solution of 23
(113 mg, 0.31 mmol) in MeOH (10 mL) was added 2 N
NaOH (3.1 mL). After being stirred for 30 min at room
temperature, the reaction mixture was neutralized with
2 N HCl and extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4, and evapo-
rated. The resulting crystalline solid was washed with
Et2O to give 24 (83 mg, 87% from 22) as a white powder.
TLC Rf = 0.51 (CHCl3/MeOH, 9/1); 1H NMR
(200 MHz, CD3OD) d 7.98 (d, J = 8.4 Hz, 2H), 7.66–
7.59 (m, 1H), 7.57–7.46 (m, 3H), 7.37–7.19 (m, 3H),
3.62 and 3.40 (t and t, J = 7.5 Hz and J = 7.5 Hz, 2H),
3.10 and 3.03 (s and s, 3H), 2.45 and 2.20 (t and t,
J = 7.5 Hz and J = 7.5 Hz, 2H), 2.10–1.75 (m, 2H).
4.1.26. (3S)-3-(acetyloxy)-4-{[4-(1-benzofuran-2-yl)ben-
zoyl]amino}butanoate (39). To a stirred solution of 36
(0.85 mmol) in pyridine (5.0 mL) was added acetic anhy-
dride (0.6 mL). After being stirred at room temperature
for 2 h, the reaction mixture was quenched with 1 N
HCl and extracted with AcOEt. The organic layer was
washed with brine, dried over MgSO4, and evaporated
to give 39 (325 mg, 97%). TLC Rf = 0.52 (AcOEt/n-hex-
ane, 2/1); 1H NMR (200 MHz, CDCl3) d 7.93 (d,
J = 8.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.63–7.52
(m, 2H), 7.37–7.21 (m, 2H), 7.13 (d, J = 0.6 Hz, 1H),
5.50–5.38 (m, 1H), 3.78–3.69 (m, 2H), 3.71 (s, 3H),
2.74 (d, J = 4.2 Hz, 2H), 2.10 (s, 3H).
4.1.32.
4-(1-Benzofuran-2-yl)-N-[4-(hydroxyamino)-4-
4.1.27. Methyl (3E)-4-{[4-(1-benzofuran-2-yl)benzo-
yl]amino}but-3-enoate (40). To a stirred solution of 39
(0.38 mmol) in THF (1.0 mL) was added DBU
(0.872 mmol). After being stirred at 50 °C for 3 h, the
reaction mixture was quenched with 1 N HCl and
extracted with AcOEt. The organic layer was washed
with brine, dried over MgSO4, and evaporated to give
40 (96 mg, 76%). TLC Rf = 0.70 (AcOEt/n-hexane, 2/
1); 1H NMR (200 MHz, DMSO-d6) d 10.41 (d,
J = 9.6 Hz, 1H), 8.05 (s, 4H), 7.71–7.63 (m, 2H), 7.61
(br s, 1H), 7.41–7.24 (m, 2H), 6.98 (dd, J = 14.4 Hz,
oxobutyl]-N-methylbenzamide (17). Compound 17
(65 mg, 85%) was prepared from 24 as a white powder
according to the same procedures as described for the
preparation of 19 from 38. TLC Rf = 0.31 (CHCl3/
MeOH, 9/1); MS (APCI, Neg, 40 V) m/z 351 (MꢀH)ꢀ;
IR (KBr) 3198, 1659, 1607, 1510, 1489, 1450, 1411,
1324, 1260, 1170, 1075, 1032, 919, 850, 808, 748 cmꢀ1
;
1H NMR (200 MHz, CD3OD) d 7.99 (d, J = 8.4 Hz,
2H), 7.66–7.59 (m, 1H), 7.58–7.45 (m, 3H), 7.37–7.20
(m, 3H), 3.70–3.54, and 3.42–3.30 (m, 2H), 3.16–2.95
(m, 3H), 2.30–1.80 (m, 4H).

M. Ikura et al. / Bioorg. Med. Chem. 14 (2006) 4241–4252
4251
4.1.33. Ethyl 5-[4-(benzyloxy)phenoxy]pentanoate (26).
To a stirred solution of 25 (2.0 g, 10.0 mmol) in DMF
(20 mL) were added ethyl 5-bromopentanoate (2.38 g,
15.0 mmol) and K2CO3 (3.45 g, 25.0 mmol). After being
stirred at 80 °C for 3 h, the reaction mixture was neutral-
ized with 1 N HCl and extracted with AcOEt. The organic
layer was washed with brine, dried over Na2SO4, and
evaporated. The resulting residue was triturated with
n-hexane to give 26 (2.88 g, 88%) as a beige powder.
TLC Rf = 0.50 (n-hexane/AcOEt, 4/1); 1H NMR
(200 MHz, CDCl3) d 7.48–7.26 (m, 5H), 6.90 (d,
J = 9.4 Hz, 2H), 6.81 (d, J = 9.4 Hz, 2H), 5.01 (s, 2H),
5.02 (s, 2H), 4.13 (q, J = 7.2 Hz, 2H), 3.97–3.86 (m, 2H),
2.45–2.30 (m, 2H), 1.90–1.70 (m, 4H), 1.26 (t,
J = 7.2 Hz, 3H).
CDCl3 + CD3OD) d 7.79 (d, J = 8.8 Hz, 2H), 7.60–
7.46 (m, 2H), 7.43–7.14 (m, 2H), 6.97 (d, J = 8.8 Hz,
2H), 6.90 (d, J = 1.2 Hz, 1H), 4.10–4.00 (m, 2H), 2.48–
2.34 (m, 2H), 1.96–1.76 (m, 4H).
4.1.38. 5-[4-(1-Benzofuran-2-yl)phenoxy]-N-hydroxypen-
tanamide (18). Compound 18 (84%) was prepared from
31 as a white powder according to the same procedures
as described for the preparation of 2 from 21b. TLC
Rf = 0.43 (CHCl3/MeOH, 9/1); MS (APCI, neg. 40 V)
m/z 324 (MꢀH)-; IR (KBr) 3212, 2939, 1638, 1505,
1453, 1296, 1254, 1178, 1113, 1034, 975, 920, 885,
834, 799, 751, 526, 442 cmꢀ1 1H NMR (200 MHz,
;
DMSO-d6) d 10.40 (s, 1H), 8.90–8.50 (br s, 1H), 7.85
(d, J = 8.8 Hz, 2H), 7.66–7.54 (m, 2H), 7.34–7.18 (m,
3H), 7.06 (d, J = 8.8 Hz, 2H), 4.04 (t, J = 6.0 Hz, 2H),
2.04 (t, J = 6.6 Hz, 2H), 1.84–1.56 (m, 4H).
4.1.34. Ethyl 5-(4-hydroxyphenoxy)pentanoate (27). To a
stirred solution of 26 (1.50 g, 4.53 mmol) in MeOH
(20 mL) was added 10% palladium on carbon under ar-
gon atmosphere. The resulting heterogeneous mixture
was vigorously stirred under H2 gas for 3 h. Catalyst
was removed by filtration and the filtrate was concen-
trated to give 27 (1.11 g, 100%) as a purple oil. TLC
5. Enzyme assays: MMP-1, MMP-2, MMP-9, and
MMP-3 assays using synthetic substrate
5.1. MMP-1 assay
1
Rf = 0.43 (n-hexane/AcOEt, 2/1); H NMR (200 MHz,
CDCl3) d 6.85–6.70 (m, 4H), 4.77 (br s, 1H), 4.14 (q,
J = 7.2 Hz, 2H), 3.96–3.86 (m, 2H), 2.44–2.32 (m, 2H),
1.86–1.74 (m, 4H), 1.26 (t, J = 7.2 Hz, 3H).
Commercially available assay kits (Yagai, Yamagata
City, Japan) were used. The solutions provided in the kits
were used unless otherwise stated. A 98 lL portion of en-
zyme solution (0.5 U/mL) and 2 lL of inhibitor solution
(DMSO) were incubated with 100 lL of 0.5 mg/mL
FITC-labeled type I collagen solution at 37 °C for 3 h.
After incubation with 300 L of quenching solution on
ice for 30 min, the reaction mixture was centrifuged at
2000g for 15 min. Supernatant was used for measurement
of fluorescence by RF5300-PC. Excitation and emission
wavelengths were 495 and 520 nm, respectively.
4.1.35. Ethyl 5-(4-{[(trifluoromethyl)sulfonyl]oxy}phen-
oxy)pentanoate (28). To a stirred solution of 27
(4.53 mmol) in pyridine (10 mL) was added trif-
luoromethanesulfonyl anhydride (0.76 ml, 4.85 mmol)
under cooling with an ice bath. After being stirred at room
temperature for 20 min, the reaction mixture was extract-
ed with AcOEt. The organic layer was washed with brine,
dried over Na2SO4, and evaporated to give 28 (1.68 g,
98%) as a yellow oil. 1H NMR (300 MHz, CDCl3) d7.18
(d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.13 (q,
J = 7.2 Hz, 2H), 4.00–3.93 (m, 2H), 2.44–2.34 (m, 2H),
1.90–1.76 (m, 4H), 1.26 (t, J = 7.2 Hz, 3H).
5.2. MMP-2 and MMP-9 assays
A mixture of 130 lL MOCAc-Pro-Leu-Gly-Leu-Dpa-
Ala-Arg-NH2 (final concentration: 15 lM) and 20 lL
of inhibitor solution was incubated at 37 °C for 5 min.
Fifty microliters of enzyme solution was added, and
the reaction was performed at 37 °C for 10 min. Its fluo-
rescence was measured by fmax. Excitation and emission
wavelengths were 320 and 390 nm, respectively.
4.1.36. Ethyl 5-[4-(1-benzofuran-2-yl)phenoxy]pentanoate
(30). To a stirred solution of 28 (1.67 g, 4.05 mmol) in
DMF (10 ml) were added benzo[b]furan-2-boronic acid
(29, 1.09 g, 6.75 mmol), Et3N (5 mL), and Pd(PPh3)2Cl2
(316 mg, 0.45 mmol). After being stirred at 50 °C for
4 h, the reaction mixture was extracted with AcOEt.
The organic layer was washed with brine, dried over
Na2SO4, and evaporated. The resulting residue was
purified by chromatography on silica gel and triturated
with n-hexane to give 30 (388 mg, 26%) as a beige pow-
der. TLC Rf = 0.42 (n-hexane/AcOEt, 4/1); 1H NMR
(200 MHz, CDCl3) d 7.79 (d, J = 8.8 Hz, 2H), 7.60–
7.44 (m, 2H), 7.30–7.14 (m, 2H), 6.96 (d, J = 8.8 Hz,
2H), 6.88 (d, J = 0.8 Hz, 1H), 4.14 (q, J = 7.2 Hz, 2H),
4.07–3.97 (m, 2H), 2.50–2.30 (m, 2H), 1.90–1.75 (m,
4H), 1.26 (t, J = 7.2 Hz, 3H).
5.3. MMP-3 assay
A mixture of 190 lL MOCAc-Arg-Pro-Lys-Pro-Val-
Glu-Nva-Trp- Arg-Lys(Dnp)-NH2 (final concentration:
15 lM) and 10 lL of inhibitor solution was incubated
at 37 °C for 5 min. Fifty microliters of enzyme solution
was added and the reaction was performed at 37 °C for
10 min. Its fluorescence was measured by fmax. Excita-
tion and emission wavelengths were 320 and 390 nm,
respectively.
4.1.37. 5-[4-(1-Benzofuran-2-yl)phenoxy]pentanoic acid
(31). Compound 31 (94%) was prepared from 30 as a
pale yellow powder according to the same procedures
as described for the preparation of 38 from 37. TLC
Rf = 0.50 (CHCl3/MeOH, 9/1); 1H NMR (200 MHz,
References and notes
1. (a) Supuran, C. T.; Scozzafava, A. In Proteinase and
peptidase inhibition: Recent potential targets for drug devel-
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London, New York, 2002; pp 35–61; (b) Woessner, J. F.;
Nagase, H. In Matrix Metalloproteinases and TIMPs;
Oxford University Press, 2000; pp 1–223.
5. Compound 22 was prepared by condensation of ethyl 4-
aminobutanoate with 1-benzofuran-2-yl benzoic acid
according to the same procedures as described for the
preparation of 19 from 38: TLC Rf = 0.38 (AcOEt/n-
hexane, 1/1); 1H NMR (200 MHz, DMSO-d6) d 8.57 (t,
J = 5.4 Hz, 1H), 8.01 (d, J = 9.2 Hz, 2H), 7.96 (d,
J = 9.2 Hz, 2H), 7.71–7.62 (m, 2H), 7.57 (br s, 1H), 7.39–
7.23 (m, 2H), 4.04 (q, J = 7.0 Hz, 2H), 3.37–3.25 (m, 2H),
2.36 (t, J = 7.4 Hz, 2H), 1.86–1.72 (m, 2H).
2. Spurlino, J. C.; Smallwood, A. M.; Carlton, D. D.; Banks,
T. M.; Vavra, K. J.; Johnson, J. S.; Cook, E. R.; Falvo, J.;
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