Novel matrix metalloproteinase inhibitors: Generation of lead compounds by the in silico fragment-based approach

Article abstract of DOI:10.1016/j.bmc.2005.04.051

Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico technique. In order to identify the small fragment interacting with residues in the S1′ pocket of MMP-1 through hydrogen bonds, we performed in silico screening using the LUDI program. As a result, acetyl-l-alanyl-(N-methyl)amide (Ac-l-Ala-NHMe) was selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the l-glutamic acid derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.

Full text of DOI:10.1016/j.bmc.2005.04.051

Bioorganic & Medicinal Chemistry 13 (2005) 4527–4543
Novel matrix metalloproteinase inhibitors: Generation of
lead compounds by the in silico fragment-based approach
Kanji Takahashi, Masahiro Ikura, Hiromu Habashita, Minoru Nishizaki,
Tsuneyuki Sugiura, Shingo Yamamoto, Shingo Nakatani,* 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 8 March 2005; accepted 13 April 2005
Available online 23 May 2005
Abstract—Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico tech-
nique. In order to identify the small fragment interacting with residues in the S10 pocket of MMP-1 through hydrogen bonds,
we performed in silico screening using the LUDI program. As a result, acetyl-L-alanyl-(N-methyl)amide (Ac-L-Ala-NHMe) was
selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the L-glutamic acid
derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic
nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
a zinc-binding moiety attached to a substructure that
interacts with the substrate recognition sites of the en-
zyme. The strongest known class of MMPIs is the
hydroxamates, which have been shown to bind biden-
tately to the catalytic Zn(II) of the enzyme, causing it
to adopt a distorted trigonal bipyramidal geometry.2
The hydroxamate anion forms a short and strong
hydrogen bond with the carboxylate moiety of Glu
219, which is oriented toward the unprimed binding re-
gions, whereas the NH hydroxamate participates in a
hydrogen bond with the carbonyl oxygen of Ala 182.
Thus, several strong interactions occur at the zinc site
without any significant unfavorable contacts.
Matrix metalloproteinases (MMPs), a family of zinc-
containing endopeptidases, have been shown to play
a central role in various physiological and pathophy-
siological events, such as embryonic development,
blastocyte implantation, nerve growth, ovulation, mor-
phogenesis, angiogenesis, tissue resorption, and tissue
remodeling. In addition, the overexpression or dysregu-
lation of MMPs is associated with several disease pro-
cesses, such as tumor metastasis, arthritis,
artherosclerosis, aneurysm, breakdown of the blood–
brain barrier, periodontal disease, skin ulcers, corneal
ulcers, gastric ulcers, and liver fibrosis. In patients with
these diseases, MMPs are considered to be a potential
drug target. In fact, a number of MMP inhibitors
(MMPIs) have been developed, several of which have
shown some efficacy in clinical trials.1
To discover the substructure interacting with the sub-
strate recognition sites of MMPs, NMR-based screening
has proven to be a valuable approach. For this ap-
proach, individual small compounds are mixed with a
15N-labeled protein to evaluate their binding affinity.
By this method, 4-cyanobiphenyl was found to show
weak binding to the S10 pocket in the case of stromelysin
(MMP-3), and then was connected with hydroxamic
acid through an appropriate linker to successfully pro-
duce potent inhibitors of MMP-3. Although this
NMR-based fragment approach is a powerful method
of discovering lead compounds, a significant amount
of isotope-labeled protein is required for screening.3
Inhibition of MMPs is related to the coordination be-
tween the inhibitor molecule (generally as an anion)
and the catalytic metal ion, with or without substitution
of the metal-bound water molecule. MMPIs should have
*
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962
9314; e-mail: s.nakatani@ono.co.jp
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.051

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K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
Table 3. Effect of the C2 substitution of 4-aminobutyric acid analogs
on the activity profiles
In silico virtual screening of small molecules based on
the three-dimensional structure of the target protein is
an attractive way to identify candidates that may inter-
act with a specific site. As structural information about
the target protein is established in more detail, this in sil-
ico approach becomes increasingly reliable. In the case
of MMPs, detailed three-dimensional structural infor-
mation about the binding sites of MMPIs is available
from X-ray crystallography, NMR experiments, and
structure–activity relationship (SAR) data. This situa-
tion prompted us to explore virtual screening with the
de novo program, LUDI,4 in order to identify new sub-
structures that might interact with amino acids around
the S10 pocket of MMPs. Here, we report the results
of our in silico fragment-based approach and the preli-
minary SAR data for the identified lead compounds.
Compound
X
IC50 (lM)
MMP-1 MMP-2 MMP-9 MMP-3
4
H
CH2OH
59%a
6.4
18
40%a
NT
11
NT
NT
54
10
11
12
13
4.0
2.2
12
(+)-CH2OH 88%a
(ꢀ)-CH2OH 69%a
38
1.2
>100
5.7
CH2OBzl
0.53
0.18
a Inhibition percentage at 100 lM.
Compound 3 was prepared as outlined in Scheme 2.
N-Acylation of 25, which was prepared from 24, with
a glutaric anhydride followed by formation of the N-
methylamide by the conventional procedures afforded
26b. Without N-protection in 26a, an exclusive intra-
molecular cyclization took place in the following N-
methylamide formation reaction. Deprotection of
26b with a catalytic hydrogenation resulted in 3.
2. Chemistry
Synthesis of the test compounds listed in Tables 1–4 is
described in Schemes 1–8. Synthesis of compounds
2a,b, and 5 is outlined in Scheme 1. Condensation of
the optically pure glutamic acid derivatives 19a,b with
methylamine afforded 20a,b, respectively. Catalytic
hydrogenation of 20a provided 21a, while acidic depro-
tection of 20b gave 21b. Acidic deprotection of 21a re-
sulted in 2a, while catalytic hydrogenation of 21b
produced 22. The carboxylic acids 2a and 22 were con-
verted to hydroxamic acids (2b and 5) by the condensa-
tion with O-benzylhydroxyamine followed by catalytic
hydrogenation.
Compound 4 was synthesized as outlined in Scheme 3.
N-Acylation of 4-aminobutyric acid with 4-meth-
ylbenzoylchloride in the presence of sodium hydroxide
gave 28, which was converted to the corresponding
hydroxamic acid 4 by the usual method.
Compound 6 was also synthesized from 19b as outlined
in Scheme 4. Condensation of 19b with 4-methyl aniline
afforded 30, catalytic hydrogenation of which gave 31.
Condensation of 31 with O-benzylhydroxyamine pro-
duced 32. Acidic deprotection of 32 resulted in 33, which
was converted to 34 according to the usual N-acetyla-
tion. Deprotection of 34 by catalytic hydrogenation pro-
duced the hydroxamate 6.
Table 1. Activity profiles of newly designed chemical leads (Chart 1)
Compound
IC50 (lM)
MMP-1
MMP-2
MMP-9
MMP-3
2a
2b
3
>100
19
>100
2.4
NTb
38
>100
39%a
>100
NT
>100
59%a
>100
>100
>100
18
>100
40%a
NT
4
Synthesis of 7–8 is described in Scheme 5. N-Acylation
of a protected glutamic acid 35 with 4-methylbenzoyl
chloride afforded 36, deprotection of which by catalytic
hydrogenation provided the carboxylic acid 37. Conden-
sation of 37 with 25 gave 38. Acidic deprotection of 38
5
>100
>100
>100
>100
6
NT
a Inhibition percentage at 100 lM.
b Not tested.
Table 2. Effect of the chemical modification of C1 carboxylic acid moiety of L-glutamic acid analog on the activity profiles
Compound
X
IC50 (lM)
MMP-1
MMP-2
MMP-9
MMP-3
2b
7
CONHMe
COOH
19
2.4
14
38
39%a
>100
41%a
NT
23
NT
NT
8
3.4
9
4.3
>1
>1
84%a
a Inhibition percentage at 100 lM.

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4529
Table 4. Effect of chemical modification of the N-acyl moiety on activity profiles of 4-aminobutyric acid analogs
Compound
R
IC50 (lM)
MMP-1
59%a
MMP-2
18
MMP-9
40%a
MMP-3
4
NT
14
15
16
17
9.6
47%a
6.8
NT
NT
NT
NT
>100
65%a
>100
13
67%a
>100
>100
45%a
2.8
18
>100
3.5
NT
1.0
a Inhibition percentage at 100 lM.
i
Scheme 1. Synthesis of 2a,b, and 5. Reagents: (a) (i) ClCO2 Bu, NMM, THF; (ii) NH2Me; (b) H2, Pd–C, MeOH or 4 N HCl/dioxane; (c) 4-
methylbenzoyl chloride, pyridine or 4-methylbenzoyl chloride, Et3N, CH2Cl2; (d) TFA–H2O or H2, Pd–C, EtOH; (e) HClÆNH2OBzl, EDC, HOBt,
Et3N, DMF; (f) H2, Pd–C, MeOH.
resulted in 39, catalytic hydrogenation of which led to 7.
Condensation of 39 with 4-amino-1-butanol afforded 40,
after which deprotection by the usual method produced
8.
Synthesis of 9 is outlined in Scheme 6. A commercially
available protected glutamic acid 41 was converted to
45 by the following sequence of reactions. Formation
of an activated ester of 41, followed by sodium borohy-

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K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
i
Scheme 2. Synthesis of 3. Reagents: (a) Cbz–Cl, Pr2NEt, CH2Cl2; (b) (i) glutaric anhydride, DMAP, CH3CN; (ii) H2NMe, EDC, HOBt, Pr2NEt,
i
DMF; (c) H2, Pd–C, MeOH.
i
Scheme 3. Synthesis of 4. Reagents: (a) 4-aminobutyric acid, NaOH aq, THF; (b) HClÆNH2OBzl, EDC, HOBt, Pr2NEt, DMF; (c) H2, Pd–C,
MeOH.
Scheme 4. Synthesis of 6. Reagents: (a) 4-methylaniline, EDC, HOBt, Et3N, DMF; (b) H2, Pd–C, EtOAc; (c) HClÆNH2OBzl, EDC, HOBt, Et3N,
DMF; (d) 4 N HCl/dioxane; (e) Ac2O, Pr2NEt; (f) H2, Pd–C, MeOH.
i
dride reduction, gave 42, after which O-alkylation with
ethoxyethylchloride provided 43. Catalytic hydrogena-
tion of 43, followed by N-acylation with 4-meth-
ylbenzoyl chloride, afforded 44, after which alkaline
hydrolysis resulted in 45. Condensation of 45 with O-
(2-methoxypropane-2-yl)hydroxylamine followed by
acidic deprotection produced 9.
Compounds 14–18 were synthesized as described in
Scheme 7. Condensation of carboxylic acids 48a–e with
4-aminobutyric acid ethyl ester afforded 49a–e, alkaline
hydrolysis of which gave 50a–e, respectively. Condensa-
tion of 50a–e with O-(2-methoxypropane-2-yl)hydroxyl-
amine, followed by acidic deprotection, produced 14–18,
respectively.
Synthesis of 10–13 is described in Scheme 7. C2-Alkyl-
ation of a 4-amino-N-benzoyl-butyric acid 28 with ben-
zyloxymethylchloride afforded 46, condensation of
which with O-benzyl hydroxylamine provided 47. Par-
tial hydrogenolysis of 47 gave 13, which was converted
to an alcohol 10 by treatment with boron tribromide.
After optical resolution of 47 using a chiral HPLC col-
oumn, the resulting two enantiomers were deprotected
by catalytic hydrogenation to produce 11 and 12,
respectively.
3. In silico generation of lead compounds
In order to design new MMP inhibitors, we selected the
strategy of connecting two fragments by an appropriate
linker. The first fragment was intended to interact with
the catalytic Zn(II) ion. Based on information about
known MMPIs, we selected formic acid and N-formyl
hydroxylamine as candidate fragments. The second
fragment was expected to form hydrogen bonds around

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4531
i
Scheme 5. Synthesis of 7 and 8. Reagents: (a) 4-methylbenzoyl chloride, Pr2NEt, CHCl3; (b) H2, Pd–C, EtOAc; (c) 25, EDC, DMAP, DMF; (d)
TFA (e) H2, Pd–C, MeOH; (f) 4-amino-1-butanol, EDC, HOBt, Et3N, DMF; (g) H2, Pd–C, MeOH.
Scheme 6. Synthesis of 9. Reagents: (a) HONSu, DCC, THF; (b) NaBH4, THF–H2O; (c) EtOCH2Cl, iPr2NEt, CH2Cl2; (d) H2, Pd–C, MeOH; (e) 4-
methylbenzoyl chloride, Et3N, CH2Cl2; (f) NaOH aq, MeOH; (g) O-(2-methoxypropane-2-yl)hydroxylamine, EDC, HOBt, DMF; (h) HCl, MeOH.
the S10 pocket and to be involved in the van der Waals
interaction with the pocket. LUDI was used as a de
novo program to detect fragments with the possibility
of showing a second interaction with collagenase
(MMP-1).5
is available about the binding sites of MMPIs. Most
MMPIs bind to the same enzyme pocket and occupy
common subregions. Around the S10 pocket, they all
show a common interaction with Pro238 and Leu181.
Each MMPI donates a hydrogen bond to a proline res-
idue (Pro238) and accepts a hydrogen bond from a leu-
cine residue (Leu181). We reasoned that a novel
inhibitor should have the ability to form these two key
hydrogen bonds. It should also contain a lipophilic S10
From the X-ray crystallography and the well-established
SAR data on inhibition of MMPs by numerous inhibi-
tors, detailed three-dimensional structural information

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K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
i
Scheme 7. Synthesis of 10–13. Reagents: (a) LDA, BzlOCH2Cl, HMPA, THF; (b) HClÆNH2OBzl, EDC, HOBt, Pr2NEt, DMF; (c) H2, Pd–C,
MeOH; (d) BBr3, CH2Cl2; (e) HPLC separation then, H2, Pd–C, MeOH.
Scheme 8. Synthesis of 14–18. Reagents: (a) 4-aminobutyric acid ethyl ester hydrochloride, EDC, HOBt, Et3N, DMF; (b) NaOH aq, MeOH; (c) O-
(2-methoxypropane-2-yl)hydroxylamine, EDC, HOBt, DMF; (d) HCl, MeOH.
pocket to satisfy the steric and lipophilic requirements of
the enzyme pocket.
(Fig. 1). As the methyl moiety of Ala was suggested
to be directed towards the zinc site, we decided to con-
nect this methyl group with the first fragment using a
simple linker (methylene group). It was also thought
that the methyl moiety of N-acetyl should be located
in the lipophilic S10 pocket. However, this methyl
group was too small to fill the cavity, so we replaced
it with a 4-methylphenyl group. Based on the above-
mentioned analysis, we designed the glutamic acid
derivatives 2a,b and performed a docking study. As
shown in Figure 2, compound 2b seemed to fit the ac-
tive site of MMP-1.
A search of the standard LUDI library was carried
out to identify candidate second fragments. This
search resulted in a list of 83 small molecules.
Although N-acetyl-L-alanine N-methylamide (Ac-L-
Ala-NHMe) had a relatively low LUDI score, we
tested it because of the ease of synthesis and the orien-
tation of the a-methyl group. A docking study of Ac-
L-Ala-NHMe with MMP-15 suggested the formation
of key hydrogen bonds with Pro238 and Leu181

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4533
Figure 1. Docking study of Ac-L-Ala-NHMe 1 with MMP-1.
Figure 2. Docking study of 2b with MMP-1.
Based on the promising outcome of in silico simulation,
compounds 2a,b were synthesized, and their inhibition
of MMP-1, -2, -9, and -3 was measured in vitro. The car-
boxylic acid 2a did not show any significant inhibitory
activity at 100 lM, but the hydroxamic acid 2b did dem-
onstrated moderate to weak inhibitory activity (Table 1).
loss of inhibitory activity, but the 4-aminobutyric acid
derivative 4 still showed weak inhibition of MMP-1,
MMP-2, and MMP-9. This result indicated that the benz-
amide moiety is more important for interaction with the
target enzyme than the formamide moiety. Conversion
of the 2S-configuration of 2b to a 2R-configuration gave
5, with loss of inhibitory activity against MMP-1,
MMP-2, and MMP-3 at 100 lM. This indicated that the
2S-configuration of 2b plays a role in supporting the inter-
action with the target enzyme, while the 2R-configuration
of 5 seems to disturb interaction with the enzyme. In order
to confirm the importance of hydrogen bonds, a retro-in-
verse analogue of 2b was synthesized and evaluated for
4. Structure–activity relationships
As shown in Table 1, the effect of chemical modification of
the two amide moieties of 2b on the activity profile was
investigated in order to confirm the results of in silico
modeling. The glutaric acid derivative 3 showed complete

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K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
inhibition of MMP-1, -2, and -3. This analogue 6 did not
inhibit the MMPs at 100 lM at all. These results sup-
ported the concept that compound 2b showed its activity
through hydrogen bond interactions at the benzamide
moiety (Chart 1).
A docking study suggested that the benzamide moiety of
2b might be located in the S10 cavity of the enzyme, as
shown in Figure 2. Although all MMPs have a similar
environment around the active site, there are slight dif-
ferences of the environment in the S10 pocket, for exam-
ple, the S10 cavities of MMP-2, -3, and -9 are known to
be deeper and narrower than that of MMP-1.1 To ascer-
tain whether the benzamide moiety of 2b was located
in the S10 cavity, 14–18 were synthesized and their
inhibitory activity was evaluated. Replacement of the
4-methylbenzoyl moiety of 4 with a trans-4-methylcyclo-
hexanoyl moiety gave 14, which showed increased
activity against MMP-1 and reduced activity against
MMP-2. Conversion of the methyl group of 14 to an
n-propyl group afforded 15, which showed a decrease
of activity against MMP-1 and an increase of activity
against MMP-2. Replacement of the 4-methylbenzoyl
moiety of 4 with a benzofuran-2-yl moiety provided
16, with loss of inhibitory activity against MMP-1, -2,
and -3, conversion to the 5-(4-methylphenylethynyl)fu-
ran-2-yl moiety or the 5-(4-chlorophenyl)furan-2-yl moi-
ety resulted in 17 or 18 and both showed a significant
increase of inhibitory activity against MMP-2 and
MMP-3. A similar trend was observed in the case of
substrate-based inhibitors.1a While the selective MMP-
1 inhibitors had short and bulky substituents such as
cyclohexyl or isobutyl groups at the P10 site, the selective
MMP-2 or MMP-3 inhibitors had long substituents like
biphenyl or 4-phenylbutyl groups at that site. These re-
sults strongly suggested that the benzamide moiety was
located in the S10 cavity.
Based on the above information, further optimization
was conducted without chemical modification of the
hydroxamate moiety. The docking study of 2b with
MMP-1 indicated that the N-methylformamide moiety
might not interact with the enzyme and could be lo-
cated in the aqueous environment. Accordingly, its
replacement of this part with a hydrophilic substituent
was investigated. As shown in Table 2, conversion to
the carboxylic acid moiety gave 7, which showed re-
duced MMP-2 inhibitory activity. Replacement of
the methyl group with a 4-hydroxybutyl moiety re-
sulted in 8, with retention of the inhibitory activity
against MMP-1 and MMP-2. In order to reduce the
peptidic nature and maintain hydrophilicity, the N-
methylformamide moiety was replaced with a 1-eth-
oxymethoxymethyl moiety. The resulting compound
9 showed an increase of inhibitory activity against
MMP-1 and MMP-3.
Hydroxamate-containing compounds are known to be
susceptible to hydrolysis to form the corresponding
carboxylic acids in vivo. One promising approach to
prevent such hydrolysis is the introduction of a substi-
tuent near the hydroxamate moiety, so the effect on
the activity profile of incorporation of a hydroxy-
methyl group at the a-position of 4-aminobutyric acid
analog 4 was investigated. Fortunately, introduction
of a hydroxymethyl moiety at the a-position of 4 gave
10, which showed an increase of inhibitory activity
against MMP-1 and MMP-2. (+)-Isomer 11 showed
more potent inhibitory activity against all of the tested
isoforms than (ꢀ)-isomer 12. Introduction of a benzyl-
oxymethyl moiety at the a-position of 4 afforded 13,
which showed a significant increase of inhibitory activ-
ity against all the MMP isoforms tested. As a result,
the carbon next to hydroxamate was also found to
be another site for further chemical modification as
illustrated by 13.
In summary, we succeeded in the generation of structur-
ally new lead compounds that acted as MMPIs using the
in silico fragment approach. As shown in Figure 3, 2b
was designed by replacing the N-acetyl residue of com-
pound 1 with a 4-methylbenzoyl residue, followed by
connection of the methyl moiety of the Ala unit of com-
pound 1 with an N-formylhydroxylamine by a simple
linker, methylene. Compounds 2b, 8, and 11 inhibited
MMP-2 selectively, while 9 and 14 inhibited MMP-1
selectively relative to the other MMP isoforms. Com-
pound 13 inhibited all of the MMP isoforms although
its inhibitory activity against MMP-1 and MMP-2 was
Chart 1. New chemical leads for MMPIs.

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4535
Figure 3. Molecular design of a L-glutamic acid analog 2b.
stronger than that against MMP-9 and MMP-3. These
new L-glutamic acid or 4-aminobutyric acid-based struc-
tures should undergo diverse chemical modifications to
identify MMP isoform-selective inhibitors because of
cost-effectiveness and good functionality. Further opti-
mization of these compounds will be reported in the
near future.
5.1. tert-Butyl N2-[(benzyloxy)carbonyl]-N1-methyl-L-
glutaminate (20a)
To a stirred solution of 5-tert-butyl N-carbobenzoxy-L-
glutamate (19a) (1.00 g, 2.97 mmol) in THF (6 mL) were
added N-methylmorpholine (0.40 mL, 3.6 mmol) and
isobutyl chloroformate (0.50 mL, 3.6 mmol) at ꢀ20 °C.
After stirring at ꢀ20 °C for 15 min, excess amount of
aqueous methylamine solution (40%) was added to the
mixture, and then it was warmed to room temperature.
The reaction mixture was diluted with EtOAc and
washed with 1 N HCl, saturated NaHCO3, and brine.
The organic layer was dried over MgSO4. Removal of
the volatiles in vacuo provided a solid, which was puri-
fied by silica gel chromatography with MeOH–CHCl3
(3:97) to give the title compound 20a in quantitative
yield as a white powder: TLC Rf = 0.73 (CHCl3–MeOH,
9:1); MS (APCI, pos. 20 V) m/z 351 (M+H)+; 1H NMR
(200 MHz, CDCl3): d 7.38–7.31 (m, 5H), 6.24 (br s, 1H),
5.65 (m, 1H), 5.10 (s, 2H), 4.20–4.12 (m, 1H), 2.81 (d,
J = 5.0 Hz, 3H), 2.49–2.24 (m, 2H), 2.17–1.85 (m, 2H),
1.44 (s, 9H).
5. Experimental
Analytical samples were homogeneous as confirmed
by thin-layer chromatography (TLC), and yielded
spectroscopic data consistent with the assigned struc-
1
tures. All H NMR spectra were obtained with a Var-
ian Gemini-200 or MERCURY-300 spectrometer. The
chemical shift values are reported in ppm (d) and
coupling constants (J) in hertz (Hz). Fast atom bom-
bardment (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 deter-
mined by a Hitachi M-1200H spectrometer. Matrix-
assisted laser desorption ionization (MALDI) mass
spectra were obtained on a PerSeptive Biosystems
VoyagerTM Elite spectrometer. IR spectra were mea-
sured using a Perkin–Elmer FTIR 1760X or JASCO
FTIR-430 spectrometer. Column 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 per-
formed on silica gel (Merck TLC plate, silica gel 60
F254). The following abbreviations for solvents and
reagents are used: THF, tetrahydrofuran; EtOAc,
ethylacetate; MeOH, methanol; EtOH, ethanol;
DMF, N,N-dimethylformamide; CH2Cl2, dichloro-
methane; CHCl3, chloroform; EDCÆHCl, 1-[3-(dimeth-
5.2. tert-Butyl N1-methyl-N2-(4-methylbenzoyl)-L-glut-
aminate (21a)
Catalytic hydrogenation of 20a (1.18 g, 3.37 mmol) in
MeOH (12 mL) was conducted at room temperature
for 21 h in the presence of 5% palladium on carbon
(50 mg) at atmospheric pressure. Removal of the cata-
lyst by filtration followed by evaporation of the sol-
vent afforded the amine in 98% yield as a white
amorphous powder: TLC Rf = 0.29 (CHCl3–MeOH,
9:1). To a stirred solution of the above amine
(500 mg, 2.31 mmol) in pyridine (3 mL) was added 4-
methylbenzoyl chloride (0.30 mL, 2.8 mmol) at 0 °C.
After stirring at room temperature overnight, the reac-
tion mixture was diluted with EtOAc. The resulting
solution was washed with 1 N HCl, saturated NaH-
CO3, and brine, and dried over MgSO4. Removal of
the volatiles in vacuo provided an oily residue, which
was purified by silica gel chromatography with
MeOH–CHCl3 (3:97) to give 21a (499 mg, 65% yield)
as a white powder: TLC Rf = 0.46 (CHCl3–MeOH,
9:1); MS (APCI, pos. 20 V) 355 (M+H)+. 1H NMR
ylamino)propyl]-3-ethylcarbodiimide
hydrochloride;
HOBt, 1-hydroxybenzotriazole; HMPA, hexamethyl-
phosphoramide; TFA, trifluoroacetic acid; DMSO,
dimethylsulfoxide; AcOH, acetic acid; Et2O, diethyle-
ther; BuLi, butyllithium; FITC, fluorescein isothiocya-
nate; MOCAc, 7-methoxycoumarin-4-acetyl; Dpa,
N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl; Dnp,
2,4-dinitrophenyl.

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K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
(200 MHz, CDCl3): d 7.74 (d, J = 8.0 Hz, 2H), 7.37 (d,
J = 6.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 6.61–6.55
(m, 1H), 4.59 (q, J = 6.6 Hz, 1H), 2.84 (d,
J = 4.9 Hz, 3H), 2.65–2.08 (m, 4H), 2.40 (s, 3H),
1.43 (s, 9H).
1H), 2.76 (s, 3H), 2.56–2.30 (m, 2H), 2.34 (s, 3H),
2.22–1.84 (m, 2H).
5.6. N5-(Benzyloxy)-N1-methyl-N2-(4-methylbenzoyl)-L-
glutamamide (23a) and N5-hydroxy-N1-methyl-N2-(4-
methylbenzoyl)-L-glutamamide (2b)
5.3. Benzyl N2-(tert-butoxycarbonyl)-N1-methyl-D-gluta-
minate (20b) and benzyl N1-methyl-N2-(4-methylbenzoyl)-
D-glutaminate (21b)
To a stirred solution of 2a (257 mg, 0.924 mmol) in DMF
(1 mL) were added O-benzylhydroxylamine hydrochlo-
ride (177 mg, 1.11 mmol), 1-hydroxybenzotriazole mono-
hydrate (170 mg, 1.11 mmol), triethylamine (0.155 mL,
1.11 mmol), and 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (213 mg, 1.11 mmol) at
0 °C. After stirring at room temperature for 1 h, the reac-
tion mixture was diluted with EtOAc. The resulting sus-
pension was washed with 1 N HCl, saturated NaHCO3,
and brine, and dried over MgSO4. Removal of the vola-
tiles in vacuo provided 23a (205 mg, 58% yield) as a white
powder. Compound 23a: TLC Rf = 0.44 (CHCl3–MeOH,
9:1). Catalytic hydrogenation of 23a (205 mg,
0.699 mmol) in a mixed solvent of MeOH–CHCl3 (2:1,
3 mL) was conducted at room temperature in the presence
of 5% palladium on carbon (22 mg) at atmospheric pres-
sure. Removal of the catalyst by filtration followed by
evaporation of the solvent afforded a solid. The resulting
solid was purified by silica gel chromatography with a gra-
dient from MeOH–CHCl3 (3:97) to MeOH–CHCl3 (1:9)
to give 2b (77 mg, 49% yield) as a white amorphous pow-
der. Compound 2b: TLC Rf = 0.69 (CHCl3–MeOH, 4:1);
MS (FAB, pos.) m/z 294 (M+H)+; IR (KBr) 3267, 1636,
The amide 20b was obtained as a white powder in 80%
yield from 5-benzyl N-(tert-butoxycarbonyl)-D-gluta-
mate (19b) according to the analogous procedures as
described for the preparation of 20a. Compound 20b:
TLC Rf = 0.20 (EtOAc–n-hexane, 1:1). A solution of
20b (3.34 g, 10.0 mmol) in 4 N HCl/dioxane (50 mL)
was stirred at room temperature for 1 h. The reaction
mixture was evaporated to give the amine hydrochlo-
ride quantitatively as a yellow oil. To a stirred solution
of the above amine hydrochloride (10.0 mmol) in
CH2Cl2 (50 mL) were added triethylamine (3.4 mL,
24 mmol) and 4-methylbenzoyl chloride (1.39 mL,
10.5 mmol) at 0 °C. After stirring at room temperature
overnight, the solution was washed with 1 N HCl, sat-
urated NaHCO3, and brine, and dried over MgSO4.
Removal of the volatiles in vacuo provided 21b
(2.39 g, 65% yield) as a white powder: TLC Rf = 0.50
(CHCl3–MeOH, 30:1); MS (EI, pos.) m/z 368 (M+);
1H NMR (200 MHz, CDCl3): d 7.90–7.70 (m, 2H),
7.40–7.10 (m, 8H), 6.60–6.30 (br, 1H), 5.12 (s, 2H),
4.80–4.50 (m, 2H), 2.81 (d, J = 4.8 Hz, 3H), 2.40 (s,
3H), 2.80–2.10 (m, 3H).
1
1538, 1189, 838, 753, 433 cmꢀ1; H NMR (200 MHz,
DMSO-d6): d 10.37 (br s, 1H), 8.69 (m, 1H), 8.43 (d,
J = 7.6 Hz, 1H), 7.85–7.79 (m, 3H), 7.25 (d, J = 8.0 Hz,
2H), 4.40–4.25 (m, 1H), 2.59 (d, J = 4.6 Hz, 3H), 2.06 (s,
5.4. N1-Methyl-N2-(4-methylbenzoyl)-L-a-glutamine (2a)
30
3H), 2.10–1.85 (m, 4H); optical rotation ½aꢁ +7.96° (c
D
A solution of 21a (499 mg, 1.49 mmol) in a mixed sol-
vent of TFA–H2O (3 mL:0.3 mL) was stirred at room
temperature for 1 h. The reaction mixture was evapo-
rated to give 2a (416 mg, 100% yield) as a white powder:
TLC Rf = 0.40 (CHCl3–MeOH, 9:1); MS (FAB, pos.)
m/z 279 (M+H)+; IR (KBr) 3343, 2944, 1647, 1527,
1503, 1454, 1415, 1290, 1210, 946, 897, 834, 761, 653,
0.515, MeOH); HRMS (FAB) calcd for C14H20N3O4:
294.1454. Found: 294.1457.
5.7. N5-(Benzyloxy)-N1-methyl-N2-(4-methylbenzoyl)-D-
glutamamide (23b) and N5-hydroxy-N1-methyl-N2-(4-
methylbenzoyl)-D-glutamamide (5)
1
566, 534, 484 cmꢀ1; H NMR (200 MHz, DMSO-d6): d
The hydroxamate 23b was obtained as a white powder
in 61% yield from 22 according to the analogous proce-
dures as described for the preparation of 23a. Com-
pound 23b: TLC Rf = 0.69 (CHCl3–MeOH, 5:1). The
hydroxamic acid 5 was obtained as a white powder in
94% yield from 23b according to the analogous proce-
dures as described for the preparation of 2b. Compound
2b: TLC Rf = 0.30 (CHCl3–MeOH, 10:1); MS (APCI,
neg. 20 V) m/z 292 (MꢀH)ꢀ; IR (KBr) 3322, 1652,
1615, 1537, 1506, 1449, 1413, 1384, 1302, 1257, 1184,
12.13 (br s, 1H), 8.33 (d, J = 7.8 Hz, 2H), 7.86–7.78
(m, 3H), 7.26 (d, J = 8.1 Hz, 2H), 4.40–4.25 (m, 1H),
2.59 (d, J = 4.8 Hz, 3H), 2.36 (s, 3H), 2.30–2.23 (m,
30
D
2H), 2.05–1.82 (m, 2H); optical rotation ½aꢁ +5.65° (c
0.54, MeOH); HRMS (FAB) calcd for C14H19N2O4:
279.1345. Found: 279.1339.
5.5. N1-Methyl-N2-(4-methylbenzoyl)-D-a-glutamine (22)
Catalytic hydrogenation of 21b (2.38 g, 6.46 mmol) in
EtOH (65 mL) was conducted at room temperature for
1 h in the presence of 10% palladium on carbon
(344 mg) at atmospheric pressure. Removal of the cata-
lyst by filtration followed by evaporation of the solvent
afforded 22 (1.63 g, 91% yield) as a white powder: TLC
Rf = 0.20 (CHCl3–MeOH, 5:1); MS (FAB, pos.) 279
(M+H)+; IR (KBr) 3339, 3260, 1735, 1698, 1651, 1531,
1305, 1416, 1306, 1210, 947, 870, 835, 761, 692,
1075, 1031, 834, 752, 686, 600 cmꢀ1 1H NMR
;
(200 MHz, CD3OD): d 7.72 (d, J = 8.0 Hz, 2H), 7.20
(d, J = 8.0 Hz, 2H), 4.50–4.44 (m, 1H), 2.67 (s, 3H),
2.31 (s, 3H), 2.22–1.85 (m, 4H); optical rotation
31
½aꢁ ꢀ5.76° (c 0.545, MeOH); HRMS (FAB) calcd for
D
C14H20N3O4: 294.1454. Found: 294.1456.
5.8. Benzyl benzyloxycarbamate (25)
534 cmꢀ1
;
7.73–7.60 (m, 2H), 7.22–7.14 (m, 2H), 4.66–4.52 (m,
1H NMR (200 MHz, CDCl3 + CD3OD): d
To a stirred suspension of 24 (16.0 g, 100 mmol) in
CH2Cl2 (100 mL) were added N,N-(diisopropyl)ethyl-

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4537
amine (34.8 mL, 200 mmol) and benzyl chloroformate
(14.3 mL, 100 mmol) at 0 °C. The reaction mixture
was stirred at 0 °C for 2 h and diluted with EtOAc.
The resulting suspension was washed with 2 N HCl,
and brine, and dried over MgSO4. Removal of the vol-
atiles in vacuo provided an oily solid, which was tritu-
rated with n-hexane to give 25 (21.9 g, 85% yield) as a
white powder: TLC Rf = 0.24 (EtOAc–n-hexane, 1:4);
1H NMR (200 MHz, CDCl3): d 7.35 (m, 10H), 5.18 (s,
2H), 4.87 (s, 2H).
(21 mL, 21 mmol) at 0 °C and 4-methylbenzoyl chloride
27 (1.55 g, 10.0 mmol). After stirring at room tempera-
ture for 3 h, the reaction mixture was acidified with
2 N HCl and extracted with EtOAc. The organic layer
was washed with brine and dried over MgSO4. Removal
of the volatiles in vacuo provided a solid, which was
washed with diethyl ether to give 28 (2.00 g, 90% yield)
as colorless crystals: 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).
5.9. 5-{(Benzyloxy)[(benzyloxy)carbonyl]amino}-5-oxo-
pentanoic acid (26a) and benzyl N-[5-(methylamino)-5-
oxopentanoyl]benzyloxycarbamate (26b)
5.12. N-{4-[(Benzyloxy)amino]-4-oxobutyl}-4-methyl-
benzamide (29)
To a stirred solution of glutaric anhydride (1.26 g,
11.0 mmol) in acetonitrile (10 mL) were added 25
(2.57 g, 10.0 mmol) and 4-(dimethylamino)pyridine
(12 mg, 1.0 mmol) at room temperature. The mixture
was stirred for 40 h at room temperature. Glutaric anhy-
dride (1.26 g, 11.0 mmol) and 4-(dimethylamino)pyri-
dine (50 mg, 4.2 mmol) were added to it. The mixture
was stirred for 3 h at room temperature, and concen-
trated under reduced pressure. The residue was acidified
with 1 N HCl, and extracted with EtOAc. The extract
was washed with brine, dried over MgSO4, and filtrated.
Concentration under reduced pressure gave a white pow-
der, which was triturated with hexane to afford 26a as a
white powder (4.57 g): TLC Rf = 0.67 (CHCl3–MeOH–
AcOH, 18:2:1). To a stirred mixture of 26a (1.11 g,
2.99 mmol), methylamine (40% in MeOH, 0.50 g,
5.98 mmol), N,N-(diisopropyl)ethylamine (1.56 mL,
8.97 mmol), and HOBt (686 mg, 4.48 mmol) in DMF
(5 mL) was added EDCÆHCl (859 mg, 4.48 mmol) at
0 °C. The mixture was stirred for 64 h at an ambient tem-
perature, and diluted with EtOAc. The suspension was
washed with brine, 1 N HCl, brine, satd NaHCO3, and
brine, and dried over MgSO4. Removal of the volatiles
in vacuo provided a white powder, which was triturated
with Et2O to give 26b (68 mg, 7% yield from 25) as a col-
orless silky powder: 1H NMR (CDCl3, 200 MHz): d 7.41
(m, 5H), 7.34 (m, 5H), 5.54 (m, 1H), 5.27 (s, 2H), 4.90 (s,
2H), 2.85 (t, J = 7.0 Hz, 2H), 2.80 (d, J = 5.2 Hz, 3H),
2.24 (t, J = 6.6 Hz, 2H), 2.02 (m, 2H).
The title compound was obtained as a white powder in
76% yield from 28 according to the analogous proce-
dures as described for the preparation of 23a: TLC
1
Rf = 0.52 (CHCl3–MeOH, 10:1); H NMR (200 MHz,
CDCl3): d 9.24 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.37
(m, 5H), 7.23 (d, J = 8.2 Hz, 2H), 6.66 (m, 1H), 4.90
(s, 2H), 3.46 (dt, J = 6.0, 5.8 Hz, 2H), 2.39 (s, 3H),
2.17 (m, 2H), 1.92 (m, 2H).
5.13. N-(4-Hydroxyamino-4-oxobutyl)-4-methylbenz-
amide (4)
The title compound was obtained as colorless crystals in
85% yield from 29 according to the analogous procedures
as described for the preparation of 2b: 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): d
;
10.39 (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 (td,
J = 6.6, 5.2 Hz, 2H), 2.35 (s, 3H), 2.02 (t, J = 7.7 Hz,
2H), 1.74 (m, 2H); HRMS (FAB) calcd for
C12H17N2O3: 237.1239. Found 237.1244.
5.14. Benzyl N2-(tert-butoxycarbonyl)-N1-(4-methylphe-
nyl)-D-glutaminate (30)
To a stirred solution of 19b (6.75 g, 20.0 mmol) in DMF
(20 mL)
5.10. N-Hydroxy-N0-methylpentanediamide (3)
were
added
p-methylaniline
(4.31 g,
30.0 mmol), triethylamine (8.36 mL, 60.0 mmol), 1-
hydroxybenzotriazole monohydrate (3.67 g, 24.0 mmol),
and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (4.60 g, 24.0 mmol) at 0 °C. The reaction
mixture was stirred at room temperature for 16 h. The
solution was diluted with EtOAc, and washed sequen-
tially with 1N HCl, saturated NaHCO3, and brine.
The organic layer was dried over MgSO4. Removal of
the volatiles in vacuo provided brown oil, which was
recrystallized from EtOAc–n-hexane (1:4) to give 30
(8.14 g, 98% yield) as colorless crystals: TLC Rf = 0.54
The title compound was obtained as colorless crystals in
81% yield from 26 according to the analogous proce-
dures as described for the preparation of 2b. TLC
Rf = 0.61 (EtOAc–AcOH–H2O, 3:1:1); MS (APCI, neg.
40 V) m/z 159 (MꢀH)ꢀ; IR (KBr) 3224, 2942, 1646,
1562, 1459, 1413, 1291, 1165, 1092, 1071, 1023, 604,
439 cmꢀ1 1H NMR (200 MHz, DMSO-d6): d 10.30–
;
9.30 (br, 1H), 9.30–8.30 (m, 1H), 7.69 (m, 1H), 2.55
(d, J = 4.8 Hz, 3H), 2.05 (t, J = 7.3Hz, 2H), 1.94 (t,
J = 7.7 Hz, 2H), 1.70 (tt, J = 7.7, 7.3 Hz, 2H); HRMS
(FAB) calcd for C6H13N2O3: 161.0926. Found 161.0926.
1
(EtOAc–n-hexane, 2:3); H NMR (200 MHz, CDCl3):
d 8.30 (br s, 1H), 7.35 (m, 7H), 7.11 (d, J = 8.0 Hz,
2H), 5.38 (d, J = 8.0 Hz, 1H), 5.16 (d, J = 13.0 Hz,
1H), 5.13 (d, J = 13.0 Hz, 1H), 4.28 (m, 1H), 2.62 (m,
1H), 2.50 (m, 1H), 2.31 (s, 3H), 2.22 (m, 1H), 2.02 (m,
1H), 1.44 (s, 9H).
5.11. 4-[(4-Methylbenzoyl)amino]butanoic acid (28)
To a stirred suspension of 4-aminobutyric acid (1.13 g,
11.0 mmol) in THF (21 mL) were added 1 N NaOH

4538
K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
5.15. N2-(tert-Butoxycarbonyl)-N1-(4-methylphenyl)-D-a-
glutamine (31)
(11.6 mL, 66.7 mmol) and 4-methylbenzoyl chloride
(4.41 mL, 33.4 mmol) at 0 °C. After stirring at room
temperature for 1 h, saturated NaHCO3 was added to
the reaction mixture with vigorous stirring. After stir-
ring for 30 min, the reaction mixture was then diluted
with EtOAc and washed sequentially with brine, 1 N
HCl, brine, saturated NaHCO3, and brine. The organic
layer was dried over MgSO4. Removal of the volatiles in
vacuo provided a solid, which was triturated with n-hex-
ane to give 36 (12.6 g, quantitative yield) as a white pow-
der: TLC Rf = 0.19 (EtOAc–n-hexane, 1:4).
The title compound was obtained quantitatively as a
colorless oil from 30 according to the analogous proce-
dures as described for the preparation of 22: TLC
Rf = 0.57 (CHCl3–MeOH–AcOH, 18:2:1).
5.16. N2-(tert-Butoxycarbonyl)-N5-benzyloxy-N1-(4-
methylphenyl)-D-glutamamide (32)
The title compound was obtained as a white powder in
98% yield from 31 according to the analogous proce-
dures as described for the preparation of 23a: TLC
Rf = 0.41 (CHCl3–MeOH, 10:1).
5.21. 1-tert-Butyl N-(4-methylbenzoyl)-L-glutamate (37)
The title compound was obtained as colorless crystals
from 36 in 76% yield according to the analogous proce-
5.17. N5-Benzyloxy-N1-(4-methylphenyl)-D-glutamamide (33)
1
dures as described for the preparation of 22: H NMR
(200 MHz, CDCl3): d 7.70 (d, J = 8.2 Hz, 2H), 7.22 (d,
J = 8.2 Hz, 2H), 6.97 (d, J = 7.5 Hz, 1H), 4.71 (m,
1H), 2.47 (m, 2H), 2.38 (s, 3H), 2.28 (m, 1H), 2.05 (m,
1H), 1.49 (s, 9H).
A solution of 32 (3.25 g, 7.56 mmol) in 4 N HCl in 1,4-
dioxane (20 mL) was stirred at room temperature for
3 h. The reaction mixture was evaporated to give an oily
residue, which was triturated with Et2O to give 33
(2.74 g, 96% yield) as a white powder: TLC Rf = 0.24
(CHCl3–MeOH–AcOH, 18:2:1).
5.22. tert-Butyl N5-benzyloxy-N5-[(benzyloxy)carbonyl]-
N2-(4-methylbenzoyl)-L-a-glutaminate (38)
5.18. N2-Acetyl-N5-benzyloxy-N1-(4-methylphenyl)-D-
glutamamide (34)
To a stirred solution of 37 (643 mg, 2.00 mmol) in DMF
(10 mL) were added 25 (515 mg, 2.00 mmol), 4-(dimeth-
ylamino)pyridine (24 mg, 0.20 mmol), and 1-[3-(dimeth-
To a stirred solution of 33 (378 mg, 1.00 mmol) in CH2Cl2
(5 mL) were added N,N-(diisopropyl)ethylamine
(0.38 mL, 2.2 mmol) and acetic anhydride (0.094 mL,
1.0 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 16 h. The mixture was concen-
trated under reduced pressure to give a powder, which
was washed with H2O, 1 N HCl, H2O, saturated NaH-
CO3, H2O, and dried under reduced pressure. The ob-
tained powder was dissolved in MeOH. The solution
was evaporated to give a solid, which was triturated with
Et2O to give 34 (320 mg, 83% yield) as a white powder:
TLC Rf = 0.51 (CHCl3–MeOH, 10:1).
ylamino)propyl]-3-ethylcarbodiimide
hydrochloride
(422 mg, 2.20 mmol) at 0 °C. The mixture was stirred
at room temperature for 16 h, and diluted with EtOAc.
The suspension was washed with brine, 1 N HCl, brine,
saturated NaHCO3, brine, dried over MgSO4. Removal
of the volatiles in vacuo provided a colorless oil, which
was purified by silica gel chromatography with EtOAc–
n-hexane (3:7) to give 38 (992 mg, 88% yield) as a color-
1
less syrup: TLC Rf = 0.27 (EtOAc–n-hexane, 3:7); H
NMR (200 MHz, CDCl3): d 7.72 (d, J = 8.0 Hz, 2H),
7.38 (m, 5H), 7.30 (m, 5H), 7.20 (d, J = 8.0 Hz, 2H),
6.89 (d, J = 7.8 Hz, 1H), 5.25 (s, 2H), 4.89 (d,
J = 10.0 Hz, 1H), 4.85 (d, J = 10.0 Hz, 1H), 4.72 (m,
5.19. N2-Acetyl-N5-hydroxy-N1-(4-methylphenyl)-D-
glutamamide (6)
1H), 2.94 (m, 2H), 2.38 (s, 3H), 2.30 (m, 1H), 2.16 (m,
25
D
1H), 1.49 (s, 9H); optical rotation ½aꢁ +6.84° (c 0.655,
CHCl3).
The title compound was obtained as a white powder in
89% yield from 34 according to the analogous procedures
as described for the preparation of 2b: TLC Rf = 0.13
(CHCl3–MeOH, 10:1); MS (APCI, neg. 40 V) m/z, 292
(MꢀH)ꢀ; IR (KBr) 3269, 2900, 1657, 1607, 1538, 1450,
1381, 1287, 1257, 1109, 1086, 1021, 981, 818, 797, 686,
610, 514 cmꢀ1; 1H NMR (200 MHz, DMSO-d6): d 10.41
(s, 1H), 9.94 (s, 1H), 8.70 (s, 1H), 8.16 (d, J = 7.8 Hz,
1H), 7.48 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H),
5.23. N5-Benzyloxy-N5-[(benzyloxy)carbonyl]-N2-(4-
methylbenzoyl)-L-glutamine (39)
A solution of 38 (980 mg, 1.75 mmol) in trifluoroacetic
acid (5 mL) was stirred at room temperature for 3 h.
The reaction mixture was evaporated to give a crude
oil, which was purified by silica gel chromatography
with AcOH–MeOH–CHCl3 (5:10:85) to give a solid,
which was triturated with diisopropylether to give 39
(495 mg, 56% yield) as a white powder: TLC Rf = 0.30
(CHCl3–MeOH–AcOH, 18:2:1).
4.37 (m, 1H), 2.25 (s, 3H), 2.00 (m, 2H), 1.87 (s, 3H),
31
D
1.95–1.70 (m, 2H); optical rotation ½aꢁ +18.2° (c 0.50,
MeOH); HRMS (FAB) calcd for C14H20N3O4:
294.1454. Found: 294.1453.
5.24. N5-Hydroxy-N2-(4-methylbenzoyl)-L-glutamine (7)
5.20. 5-Benzyl 1-tert-butyl N-(4-methylbenzoyl)-L-
glutamate (36)
The title compound was obtained as a white powder in
95% yield from 39 according to the analogous proce-
dures as described for the preparation of 2b: TLC
Rf = 0.43 (EtOAc–AcOH–H2O, 8:1:1); MS (APCI, neg.
To a stirred solution of 35 (10.0 g, 30.3 mmol) in CHCl3
(50 mL) were added N,N-(diisopropyl)ethylamine

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4539
40 V) m/z 279 (MꢀH)ꢀ; IR (KBr) 3240, 3037, 2978,
;
1242, 1191, 1104, 1021, 979, 839, 755, 579 cmꢀ1 1H
cuo provided a solid, which purified by silica gel chro-
matography with n-hexane– EtOAc (2:3) to give 42
(31.4 g, 72% yield) as a white powder: TLC Rf = 0.35
(n-hexane–EtOAc, 2:3); 1H NMR (200 MHz,
CDCl3 + two drops of CD3OD): d 7.45–7.20 (m, 5H),
5.48 (d, J = 9.0 Hz, 1H), 5.09 (s, 2H), 3.80–3.50 (m,
6H), 2.42 (t, J = 7.4 Hz, 2H), 2.00–1.70 (m, 2H).
2925, 1719, 1636, 1543, 1505, 1452, 1409, 1340, 1292,
NMR (200 MHz, DMSO-d6): d 10.47 (m, 1H), 8.49 (d,
J = 7.4 Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.27 (d,
J = 7.8 Hz, 2H), 4.30 (m, 1H), 2.36 (s, 3H), 1.80–2.20
31
(m, 4H); optical rotation ½aꢁ +1.30° (c 0.50, MeOH);
D
HRMS (FAB) calcd for C13H17N2O5: 281.1137. Found:
281.1143.
5.28. Methyl (4S)-4-{[(benzyloxy)carbonyl]amino}-5-
(ethoxymethoxy)pentanoate (43)
5.25. N5-Benzyloxy-N1-(4-hydroxybutyl)-N2-(4-meth-
ylbenzoyl)-L-glutamamide (40)
To a stirred solution of 42 (12.0 g, 42.7 mmol) in
CH2Cl2 (45 mL) were added N,N-(diisopropyl)ethyl-
amine (22 mL) and chloromethyl ethyl ether (7.95 mL,
85.4 mmol) at 0 °C. After stirring at room temperature
for 80 min, the solution was poured into 1 N HCl and
extracted with EtOAc. The organic layer was washed
with saturated NaHCO3, and brine, and dried over
Na2SO4. Removal of the volatiles in vacuo provided
an oily residue, which was purified by silica gel chromat-
ography with EtOAc–n-hexane (1:2) to give 43 (10.4 g,
72% yield) as a colorless oil: TLC Rf = 0.61 (EtOAc–
n-hexane, 1:1); MS (MALDI, pos.) m/z 378 (M+K)+,
362 (M+Na)+, 340 (M+H)+; 1H NMR (200 MHz,
CDCl3): d 7.37–7.28 (m, 5H), 5.10 (s, 2H), 5.05 (d,
J = 9.0 Hz, 1H), 4.65 (s, 2H), 3.90–3.78 (m, 1H), 3.65
(s, 3H), 3.61–3.52 (m, 4H), 2.41 (t, J = 7.6 Hz, 2H),
1.94–1.84 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H).
To a stirred solution of 39 (140 mg, 0.278 mmol) in
DMF (1 mL) were added 4-amino-1-butanol (37 mg,
0.42 mmol),
1-hydroxybenzotriazole
monohydrate
(65 mg, 0.42 mmol), and 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide hydrochloride (80 mg, 0.42 mmol)
at room temperature. The reaction mixture was stirred
at room temperature for 16 h. The solution was then di-
luted with EtOAc. The mixture was washed sequentially
with saturated NH4Cl, saturated NaHCO3, brine, and
dried over MgSO4. Removal of the volatiles in vacuo
provided 40 (69 mg, 57% yield) as a white powder:
TLC Rf = 0.61 (CHCl3–MeOH–AcOH, 18:2:1); MS
(APCI, neg. 20 V) m/z 440 (MꢀH)ꢀ; 1H NMR
(200 MHz, CDCl3): d 10.85 (br s, 1H), 10.37 (br s,
1H), 7.71 (d, J = 8.1 Hz, 2H), 7.40–7.23 (m, 5H), 7.18
(d, J = 8.1 Hz, 2H), 7.00–6.95 (m, 1H), 4.86 (s, 2H),
4.65–4.50 (m, 1H), 3.56–3.04 (m, 5H), 2.55–1.95 (m,
7H), 1.65–1.43 (m, 4H).
5.29. Methyl (4S)-5-ethoxymethoxy-4-[(4-methylbenzoyl)-
amino]pentanoate (44)
5.26. N5-Hydroxy-N1-(4-hydroxybutyl)-N2-(4-meth-
ylbenzoyl)-L-glutamamide (8)
Catalytic hydrogenation of 43 (3.39 g, 10.0 mmol) in
MeOH (30 mL) was conducted at room temperature for
1 h in the presence of 10% palladium on carbon
(600 mg) at atmospheric pressure. Removal of the cata-
lyst by filtration followed by evaporation afforded the
amine. To a stirred solution of the above amine in CH2Cl2
(30 mL) were added triethylamine (1.8 mL, 13 mmol) and
4-methylbenzoyl chloride (1.5 mL, 11 mmol) at 0 °C.
After stirring at room temperature overnight, the reaction
mixture was diluted with EtOAc. The resulting solution
was washed with 1 N HCl, saturated NaHCO3, and brine,
and dried over MgSO4. Removal of the volatiles in vacuo
provided an oily residue, which was purified by silica gel
chromatography with a gradient from EtOAc–n-hexane
(1:3) to EtOAc–n-hexane (2:3) to give 44 (2.47 g, 76%
yield) as a white powder: TLC Rf = 0.30 (EtOAc–n-hex-
ane, 1:1); MS (MALDI, pos.) m/z 324 (M+H)+.
The title compound was obtained as a white powder in
94% yield from 40 according to the analogous proce-
dures as described for the preparation of 2b: TLC
Rf = 0.23 (CHCl3–MeOH–AcOH, 18:2:1); MS (APCI,
neg. 20 V) m/z 350 (MꢀH)ꢀ; IR (KBr) 3277, 2929,
1641, 1543, 1502, 1450, 1377, 1322, 1189, 1054,
753 cmꢀ1 1H NMR (200 MHz, CDCl3): d 7.74 (d,
;
J = 7.5 Hz, 2H), 7.39 (s, 1H), 7.25 (d, J = 7.5 Hz, 2H),
4.60–4.43 (m, 1H), 3.61–3.56 (m, 2H), 3.31–3.15 (m,
2H), 2.40 (s, 3H), 2.30–2.04 (m, 4H), 1.62–1.48 (m,
31
D
4H); optical rotation ½aꢁ +2.67° (c 0.525, MeOH);
HRMS (FAB) calcd for C17H26N3O5: 352.1872. Found:
352.1872.
5.27. Methyl (4S)-4-{[(benzyloxy)carbonyl]amino}-5-
hydroxypentanoate (42)
5.30. (4S)-5-Ethoxymethoxy-4-[(4-methylbenzoyl)ami-
no]pentanoic acid (45)
To a stirred solution of 5-methyl N-carbobenzoxy-L-glu-
tamate 41 (45.7 g, 155 mmol) in THF (250 mL) were
added N-hydroxysuccinimide (20.5 g, 178 mmol) and
N,N-(dicyclohexyl)carbodiimide (36.8 g, 178 mmol) at
0 °C. The reaction mixture was stirred overnight. The
resulting insoluble substance was removed by filtration.
To the filtrate was added NaBH4 (8.80 g, 233 mmol) at
0 °C. After stirring for 2 h at 0 °C, the reaction mixture
was quenched with 2 N HCl and extracted with EtOAc.
The organic layer was washed with 1 N HCl, and brine,
and dried over MgSO4. Removal of the volatiles in va-
To a stirred solution of 44 (751 mg, 2.33 mmol) in
MeOH (5 mL) at room temperature was added 2 N
NaOH (2.3 mL, 4.6 mmol). The reaction mixture was
stirred at room temperature for 2 h. The mixture was
neutralized with 2 N HCl and extracted with EtOAc.
The organic layer was washed with brine, dried over
MgSO4, and evaporated. The resulting residue was trit-
urated with Et2O–n-hexane to give 45 (510 mg, 72%
yield) as a white powder; MS (APCI, neg. 20 V) m/z
308 (MꢀH)ꢀ; 1H NMR (200 MHz, DMSO-d6): d

4540
K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
12.01 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.72 (d,
J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 4.57 (s, 2H),
4.17–3.99 (m, 1H), 3.54–3.39 (m, 4H), 2.32 (s, 3H),
2.23 (t, J = 7.4 Hz, 2H), 1.94–1.57 (m, 1H), 1.07 (t,
J = 7.0 Hz, 3H).
5.33. N-(4-Benzyloxyamino-3-benzyloxymethyl-4-oxobu-
tyl)-4-methylbenzamide (47)
The title compound was obtained as a white powder in
31% yield from 46 according to the analogous procedures
as described for the preparation of 23a: TLC Rf = 0.17
(EtOAc–n-hexane, 3:2); MS (MALDI, pos.) m/z 485
(M+K)+, 469 (M+Na)+, 447 (M+H)+; 1H NMR
(200 MHz, CDCl3): d 9.57 (s, 1H), 7.66 (d, J = 8.1 Hz,
2H), 7.50–7.10 (m, 12H), 6.67 (m, 1H), 4.88 (s, 2H), 4.45
(d, J = 12.1 Hz, 1H), 4.40 (d, J = 12.1 Hz, 1H), 3.49 (m,
4H), 2.47 (m, 1H), 2.38 (s, 3H), 1.85 (m, 2H).
5.31. N-[(1S)-1-[(Ethoxymethoxy)methyl]-4-(hydroxy-
amino)-4-oxobutyl]-4-methylbenzamide (9)
To a stirred solution of 45 (500 mg, 1.62 mmol) in DMF
(10 mL) were added 1-hydroxybenzotriazole monohy-
drate (500 mg, 3.24 mmol), 1-[3-(dimethylamino)pro-
pyl]-3-ethylcarbodiimide
hydrochloride
(620 mg,
3.24 mmol) and O-(2-methoxy-2-propyl)hydroxylamine
(340 mg, 3.24 mmol) at room temperature. After stirring
at room temperature for 3 h, the reaction mixture was
diluted with water, and extracted with EtOAc. The or-
ganic layer was washed with brine, dried over MgSO4,
and evaporated. The residue was purified by silica gel
chromatography with a gradient from CHCl3 to
MeOH–CHCl3 (2:98) to give a solid, which was dis-
solved in MeOH (3 mL). The solution was acidified with
1 N HCl to pH 3. After stirring at room temperature for
10 min, the solution was concentrated in vacuo. The res-
idue was purified by silica gel chromatography with a
gradient from CHCl3 to MeOH–CHCl3 (4:96) to give
9 (96 mg, 18% yield) as a light brown amorphous pow-
der: TLC Rf = 0.30 (CHCl3–MeOH, 9:1); MS (APCI,
neg. 40 V) m/z 323 (MꢀH)ꢀ; IR (KBr) 3236, 2976,
2928, 2878, 1636, 1541, 1506, 1454, 1101, 1038, 838,
5.34. N-(3-Benzyloxymethyl-4-hydroxyamino-4-oxobu-
tyl)-4-methylbenzamide (13)
The titlecompoundwas obtained asa white powderin 82%
yield from 47 according to the analogous procedures as de-
scribed for the preparation of 2b: TLC Rf = 0.36 (CHCl3–
MeOH, 10:1); MS (MALDI, pos.) m/z 395 (M+K)+, 379
(M+Na)+, 357 (M+H)+; IR (KBr) 3249, 3063, 3031,
2922, 2868, 1663, 1636, 1549, 1507, 1455, 1365, 1310,
1206, 1190, 1103, 1077, 1029, 932, 838, 751, 699, 636,
1
613 cmꢀ1; H NMR (200 MHz, DMSO-d6): d 10.52 (s,
1H), 8.86 (s, 1H), 8.32 (t, J = 5.5 Hz, 1H), 7.74 (d,
J = 8.4 Hz, 2H), 7.31 (m, 5H), 7.24 (d, J = 8.4 Hz, 2H),
4.46 (d, J = 12.5 Hz, 1H), 4.44 (d, J = 12.5 Hz, 1H), 3.59
(t, J = 8.8 Hz, 1H), 3.41 (dd, J = 8.8, 5.5 Hz, 1H), 3.21
(m, 2H), 2.44 (m, 1H), 2.35 (s, 3H), 1.66 (m, 2H); HRMS
(FAB) calcd for C20H25N2O4: 357.1814. Found: 357.1818.
1
753, 666, 602 cmꢀ1; H NMR (300 MHz, DMSO-d6): d
10.32 (s, 1H), 8.65 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H),
7.73 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.3 Hz, 2H), 4.57
(s, 2H), 4.10–3.98 (m, 1H), 3.52–3.40 (m, 4H), 2.32 (s,
3H), 2.01–1.94 (m, 1H), 1.91–1.78 (m, 1H), 1.73–1.61
5.35. N-(4-Hydroxyamino-3-hydroxymethyl-4-oxobutyl)-
4-methylbenzamide (10)
To a stirred suspension of 13 (100 mg, 0.28 mmol) in
CH2Cl2 (10 mL) was added 0.98 mL of 1.0 M BBr3 in
CH2Cl2 (0.98 mmol) at 0 °C under argon. The suspension
was stirred at an ambient temperature for 2 h, and
quenched with 1 N HCl. The reaction mixture was
washed with EtOAc. The aqueous layer was concentrated
under reduced pressure. The residue was dissolved in H2O
(10 mL). Silica gel (5 g) was added to the solution, which
was concentrated under reduced pressure and dried in va-
cuo. The powder was bedded on silica gel chromato-
graphy. Elution with AcOH–H2O–EtOAc (2:3:16)
provided a crude oil, which was triturated with Et2O to
give 10 (54 mg, 72% yield) as a beige powder: TLC
Rf = 0.39 (EtOAc–AcOH–H2O, 16:3:2); MS (MALDI,
pos.) m/z 289 (M+Na)+; IR (KBr) 3279, 2932, 2879,
1636, 1549, 1507, 1439, 1408, 1385, 1323, 1310, 1191,
(m, 1H), 1.06 (t, J = 7.1 Hz, 3H); optical rotation
30
D
for C16H25N2O5: 325.1763. Found: 325.1761.
½aꢁ ꢀ23.85° (c 0.535, MeOH); HRMS (FAB) calcd
5.32. 2-Benzyloxymethyl-4-[(4-methylbenzoyl)amino]-
butanoic acid (46)
To a stirred solution of N,N-(diisopropyl)ethylamine
(0.925 mL, 6.60 mmol) in a mixed solvent of dry
THF (5 mL) and HMPA (3 mL) was added 4.05 mL
of 1.63 M n-BuLi in hexane (6.60 mmol) at ꢀ78 °C
under argon. The mixture was stirred at ꢀ78 °C for
15 min. A solution of 4-[(4-methylbenzoyl)amino]buta-
noic acid 28 (442 mg, 2.00 mmol) in dry THF (3 mL)
was added to the above described solution at
ꢀ78 °C. The reaction mixture was allowed to warm
up to room temperature for 30 min. The reaction mix-
ture was cooled at ꢀ78 °C. Benzyloxymethyl chloride
(313 mg, 2.00 mmol) was added to the mixture at
ꢀ78 °C. Stirring was continued at ꢀ78 °C for 2 h fol-
lowed by the addition of 1 N HCl. The reaction mix-
ture was extracted with EtOAc, washed with brine,
and dried over MgSO4. Removal of the volatiles in va-
cuo provided 46 (944 mg, quantitative yield) as a yel-
low oil. This compound was used for the next reaction
without further purification: TLC Rf = 0.67 (CHCl3–
MeOH–AcOH, 18:2:1); MS (MALDI, pos.) m/z 364
(M+Na)+, 342 (M+H)+.
1155, 1119, 1022, 927, 865, 753, 661, 636 cmꢀ1 1H
;
NMR (200 MHz, DMSO-d6): d 8.41 (m, 1H), 7.76 (d,
J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 3.52 (m, 1H),
3.37 (m, 1H), 3.20 (m, 2H), 2.35 (s, 3H), 2.23 (m, 1H),
1.64 (m, 2H); HRMS (FAB) calcd for C13H19N2O4:
267.1345. Found: 267.1353.
5.36. (+)-N-(4-Hydroxyamino-3-hydroxymethyl-4-oxobu-
tyl)-4-methylbenzamide (11) and (ꢀ)-N-(4-hydroxyamino-
3-hydroxymethyl-4-oxobutyl)-4-methylbenzamide (12)
Compound 47 was separated into two fractions by HPLC
using chiral column (tR = 13.66 and 14.90 min, DAICEL

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4541
CHIRALPAK AD, 20% isopropanol in n-hexane,
0.80 mL/min, 256 nm). The fraction with retention time
of 13.66 min was concentrated under reduced pressure
to give an oily residue. The residue was treated with 5%
palladium on carbon under hydrogen atmosphere by
the analogous procedures described for the preparation
of 2b to yield 11 as a brown powder. Compound 11:
TLC Rf = 0.17 (CHCl3–MeOH, 4:1); MS (FAB, pos.)
m/z 289 (M+Na)+, 267 (M+H)+; IR (KBr) 3289, 2925,
1640, 1544, 1505, 1309, 1190, 1120, 1035, 925, 837,
(CHCl3–MeOH–AcOH, 90:10:1); MS (MALDI, pos.)
m/z 322 (M+K)+, 306 (M+Na)+, 284 (M+H)+; 1H
NMR (200 MHz, CDCl3): d 5.78–5.59 (br, 1H), 4.13
(q, J = 7.1 Hz, 2H), 3.28 (q, J = 6.5 Hz, 2H), 2.35 (t,
J = 7.1 Hz, 2H), 2.08–1.75 (m, 7H), 1.54–1.10 (m,
10H), 1.01–0.84 (m, 5H).
5.39. Ethyl 4-[(1-benzofuran-2-ylcarbonyl)amino]butano-
ate (49c)
753 cmꢀ1
;
J = 8.2 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 3.79–3.52 (m,
1H NMR (200 MHz, CD3OD): d 7.70 (d,
The title compound was obtained as a white solid in 69%
yield from 1-benzofuran-2-carboxylic acid 48c according
to the analogous procedures as described for the prepa-
ration of 49a: TLC Rf = 0.20 (n-hexane–EtOAc, 2:1); 1H
NMR (200 MHz, CDCl3): d 7.69–7.65 (m, 1H), 7.54–
7.27 (m, 4H), 6.85 (m, 1H), 4.14 (q, J = 7.4 Hz, 2H),
3.55 (q, J = 6.2 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.07–
1.96 (m, 2H), 1.25 (t, J = 7.4 Hz, 3H).
2H), 3.50–3.25 (m, 2H), 2.38 (s, 3H), 2.38–2.25 (m, 1H),
27
D
1.80 (m, 2H); optical rotation ½aꢁ +19.2° (c 0.14,
MeOH); HRMS (FAB) calcd for C13H19N2O4:
267.1345. Found: 267.1339. The fraction with retention
time of 14.90 min was concentrated under reduced pres-
sure to give an oily residue. The residue was treated with
5% palladium on carbon under hydrogen atmosphere
by the analogous procedures described for the prepara-
tion of 2b to yield 12 as a brown powder. Compound
12: TLC Rf = 0.17 (CHCl3–MeOH, 4:1); MS (FAB,
pos.) m/z 289 (M+Na)+, 267 (M+H)+; IR (KBr) 3289,
2925, 1640, 1544, 1505, 1309, 1190, 1120, 1035, 925,
837, 753 cmꢀ1; 1H NMR (200 MHz, CD3OD): d 7.70 (d,
J = 8.2 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 3.79–3.52 (m,
5.40. Ethyl 4-({5-[(4-methylphenyl)ethynyl]-2-furoyl}-
amino)butanoate (49d)
The title compound was obtained as a light yellow pow-
der in 61% yield from 5-[(4-methylphenyl)ethynyl]-2-
furoic acid 48d according to the analogous procedures
as described for the preparation of 49a: TLC Rf = 0.31
(CHCl3–MeOH–AcOH, 18:2:1); MS (MALDI, pos.)
m/z 378 (M+K)+, 362 (M+Na)+, 340 (M+H)+; 1H NMR
(200 MHz, CDCl3): d 7.44 (d, J = 8.0 Hz, 2H), 7.19 (d,
J = 8.0 Hz, 2H), 7.12 (d, J = 3.7 Hz, 2H), 6.68 (d,
J = 3.7 Hz, 1H), 6.64–6.53 (br, 1H), 4.14 (q, J = 7.1 Hz,
2H), 3.49 (q, J = 6.6 Hz, 2H), 2.46–2.36 (m, 5H), 1.96
(quintet, J = 6.6 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
2H), 3.50–3.25 (m, 2H), 2.38 (s, 3H), 2.38–2.25 (m, 1H),
29
D
1.80 (m, 2H); optical rotation ½aꢁ ꢀ15.2° (c 0.165,
MeOH); HRMS (FAB) calcd for C13H19N2O4:
267.1345. Found: 267.1346.
5.37. Ethyl 4-{[(trans-4-methylcyclohexyl)carbonyl]ami-
no}butanoate (49a)
To a stirred solution of trans-4-methylcyclohexanecarb-
oxylic acid 48a (1.30 g, 9.14 mmol) in DMF (45 mL)
were added ethyl 4-aminobutanoate hydrochloride
(1.99 g, 11.9 mmol), 1-hydroxybenzotriazole monohy-
drate (1.68 g, 11.0 mmol), 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide hydrochloride (2.10 g, 11.0 mmol),
and triethylamine (1.5 mL, 11 mmol) at room tempera-
ture. The reaction mixture was stirred at room tempera-
ture overnight. The solution was then diluted with
EtOAc and washed sequentially with saturated NH4Cl,
saturated NaHCO3, and brine. The organic layer was
dried over MgSO4. Removal of the volatiles in vacuo
provided a solid, which was purified by silica gel chro-
matography with EtOAc–AcOH–CHCl3 (10:3:87) to
give 49a (1.44 g, 62% yield) as a white powder: TLC
Rf = 0.88 (CHCl3–MeOH–AcOH, 90:10:1); MS (MAL-
DI, pos.) m/z 294 (M+K)+, 278 (M+Na)+, 256
5.41. Ethyl 4-{[5-(4-chlorophenyl)-2-furoyl]amino}but-
anoate (49e)
The title compound was obtained as an off-white pow-
der in 60% yield from 5-(4-chlorophenyl)-2-furoic acid
48e according to the analogous procedures as described
for the preparation of 49a: TLC Rf = 0.80 (CHCl3–
MeOH–AcOH, 18:2:1); MS (APCI, pos. 20 V) m/z 336
(M+H)+; 1H NMR (200 MHz, CDCl3): d 7.69 (d,
J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.17 (d,
J = 3.6 Hz, 1H), 6.88–6.78 (m, 1H), 6.72 (d,
J = 3.6.Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.52 (q,
J = 6.6 Hz, 2H), 2.46 (t, J = 6.6 Hz, 2H), 1.99 (quintet,
J = 6.6 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H).
5.42. 4-{[(trans-4-Methylcyclohexyl)carbonyl]ami-
no}butanoic acid (50a)
1
(M+H)+; H NMR (200 MHz, CDCl3): d 5.70 (br s,
1H), 4.14 (q, J = 7.2 Hz, 2H), 3.29 (q, J = 7.2 Hz, 2H),
2.35 (t, J = 7.1 Hz, 2H), 2.07–1.71 (m, 7H), 1.56–1.22
(m, 7H), 1.05–0.84 (m, 4H).
To a stirred solution of 49a (1.33 g, 5.22 mmol) in MeOH
(8 mL) was added 1 N NaOH (6 mL, 6 mmol) at room
temperature. The reaction mixture was stirred at room
temperature for 8 h. The mixture was acidified with
1 N HCl and extracted with EtOAc. The organic layer
was washed with brine and dried over MgSO4. Removal
of volatiles in vacuo provided 50a (1.06 g, 90% yield) as a
white powder: TLC Rf = 0.55 (CHCl3–MeOH–AcOH,
18:2:1); MS (MALDI, pos.) m/z 250 (M+Na)+, 228
5.38. Ethyl 4-{[(trans-4-propylcyclohexyl)carbonyl]ami-
no}butanoate (49b)
The title compound was obtained as a white powder in
69% yield from trans-4-propylcyclohexanecarboxylic
acid 48b according to the analogous procedures as de-
scribed for the preparation of 49a: TLC Rf = 0.20
1
(M+H)+; H NMR (200 MHz, DMSO-d6): d 12.01 (br
s, 1H), 7.67 (t, J = 6.0 Hz, 1H), 3.02 (q, J = 6.0 Hz,

4542
K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
2H), 2.19 (t, J = 7.5 Hz, 2H), 2.06–1.91 (m, 1H), 1.74–
1.52 (m, 6H), 1.46–1.18 (m, 4H), 0.98–0.76 (m, 4H).
and O-(2-methoxy-2-propyl)hydroxylamine (555 mg,
5.29 mmol) at room temperature. After stirring at room
temperature for 3 h, the reaction mixture was diluted with
water and extracted with EtOAc. The organic layer was
washed with saturated NH4Cl, saturated NaHCO3, and
brine. The organic layer was dried over MgSO4. Removal
of the volatiles in vacuo provided an oily residue, which
was dissolved in MeOH (3 mL). The resulting solution
was acidified with 1 N HCl to pH 3. After stirring at room
temperature for 30 min, the solution was concentrated in
vacuo. The obtained residue was triturated with Et2O to
give 14 (217 mg, 34% yield) as a white powder: TLC
Rf = 0.29 (CHCl3–MeOH–AcOH, 18:2:1); MS (MALDI,
pos.) m/z 281 (M+K)+, 265 (M+Na)+; IR (KBr) 3306,
2928, 1616, 1546, 1440, 1265, 1222, 1188, 1048, 1000,
5.43. 4-{[(trans-4-Propylcyclohexyl)carbonyl]ami-
no}butanoic acid (50b)
The title compound was obtained as a white powder in
100% yield from 49b according to the analogous proce-
dures as described for the preparation of 50a: TLC
Rf = 0.65 (CHCl3–MeOH–AcOH, 18:2:1); MS (MAL-
DI, pos.) m/z 294 (M+K)+, 278 (M+Na)+, 256
1
(M+H)+; H NMR (200 MHz, DMSO-d6): d 12.00 (s,
1H), 7.74–7.61 (m, 1H), 3.02 (q, J = 6.5 Hz, 2H), 2.19
(t, J = 7.2 Hz, 2H), 2.11–1.92 (m, 1H), 1.78–1.53 (m,
6H), 1.43–1.08 (m, 7H), 0.95–0.89 (m, 5H).
1
949, 720, 661, 580, 544, 441 cmꢀ1; H NMR (200 MHz,
5.44. 4-[(1-Benzofuran-2-ylcarbonyl)amino]butanoic acid
(50c)
DMSO-d6): d 10.37 (s, 1H), 10.20 (s, 1H), 7.69 (t,
J = 5.3 Hz, 1H), 3.07–2.92 (m, 2H), 2.31–1.88 (m, 3H),
1.74–1.52 (m, 6H), 1.46–1.18 (m, 3H), 0.98–0.76 (m,
2H), 0.85 (d, J = 6.6 Hz, 3H); HRMS (FAB) calcd for
C12H23N2O3: 243.1709. Found: 243.1708.
The title compound was obtained as a white powder in
65% yield from 49c according to the analogous proce-
dures as described for the preparation of 50a: TLC
1
Rf = 0.32 (CHCl3–MeOH–AcOH, 100:10:1); H NMR
5.48. trans-N-(4-Hydroxyamino-4-oxobuty)-4-propyl-
cyclohexanecarboxamide (15)
(200 MHz, DMSO-d6): d 8.73 (t, J = 5.4 Hz, 1H),
7.78–7.73 (m, 1H), 7.66–7.61 (m, 1H), 7.51 (d,
J = 0.8 Hz, 1H), 7.49–7.41 (m, 1H), 7.36–7.28 (m, 1H),
3.28 (q, J = 6.6 Hz, 2H), 1.76 (t, J = 7.4 Hz, 2H), 1.83–
1.68 (m, 2H).
The title compound was obtained as a white solid in 91%
yield from 50b according to the analogous procedures as
described for the preparation of 14: TLC Rf = 0.34
(CHCl3–MeOH–AcOH, 18:2:1); MS (MALDI, pos.)
m/z 309 (M+K)+, 293 (M+Na)+, 271 (M+H)+; IR
(KBr) 3283, 2927, 2852, 1651, 1561, 1444, 1392, 1266,
5.45. 4-({5-[(4-Methylphenyl)ethynyl]-2-furoyl}amino)-
butanoic acid (50d)
1208, 1048, 997, 948, 934, 717, 580, 534 cmꢀ1 1H
;
The title compound was obtained as a white solid in
100% yield from 49d according to the analogous proce-
dures as described for the preparation of 50a: TLC
Rf = 0.54 (CHCl3–MeOH–AcOH, 18:2:1); MS (MAL-
DI, pos.) m/z 350 (M+K)+, 334 (M+Na)+, 312
NMR (200 MHz, DMSO-d6): d 10.35 (s, 1H), 7.67 (t,
J = 5.3 Hz, 1H), 2.99 (q, J = 6.5 Hz, 2H), 2.39–1.88
(m, 3H), 1.78–1.61 (m, 6H), 1.45–1.07 (m, 7H), 0.95–
0.76 (m, 5H); HRMS (FAB) calcd for C14H27N2O3:
271.2022. Found: 271.2026.
1
(M+H)+; H NMR (200 MHz, DMSO-d6): d 12.07 (br
s, 1H), 8.51 (t, J = 6.0 Hz, 1H), 7.44 (d, J = 8.1 Hz,
2H), 7.25 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 3.7 Hz, 1H),
6.94 (d, J = 3.7 Hz, 1H), 3.26–3.23 (m, 2H), 2.33 (s,
3H), 2.23 (d, J = 7.5 Hz, 2H), 1.79–1.63 (m, 2H).
5.49. N-(4-Hydroxyamino-4-oxobutyl)-1-benzofuran-2-
carboxamide (16)
The title compound was obtained as a white powder in
47% yield from 50c according to the analogous proce-
dures as described for the preparation of 14: TLC
Rf = 0.16 (CHCl3–MeOH–AcOH, 100:10:1); MS
(FAB, pos.) m/z 263 (M+H)+; IR (KBr) 3306, 3218,
1644, 1603, 1547, 1448, 1419, 1305, 1260, 1183, 969,
5.46. 4-{[5-(4-Chlorophenyl)-2-furoyl]amino}butanoic
acid (50e)
The title compound was obtained as an off-white solid in
100% yield from 49e according to the analogous proce-
dures as described for the preparation of 50a: TLC
Rf = 0.20 (CHCl3–MeOH, 9:1); MS (MALDI, pos.)
1
826, 745, 722 cmꢀ1; H NMR (200 MHz, DMSO-d6): d
10.38 (br s, 1H), 9.30–8.10 (br, 1H), 8.75 (t, J = 6.2Hz,
1H), 7.76 (m, 1H), 7.64 (m, 1H), 7.51 (d, J = 0.6 Hz,
1H), 7.45 (td, J = 7.0, 1.6 Hz, 1H), 7.32 (td, J = 7.6,
1.0 Hz, 1H), 3.25 (q, J = 6.2 Hz, 2H), 2.01 (t,
J = 7.0 Hz, 2H), 1.74 (m, 2H); HRMS (FAB) calcd for
C13H15N2O4: 263.1032. Found: 263.1033.
1
m/z 330 (M+Na)+, 308 (M+H)+; H NMR (200 MHz,
DMSO-d6): d 12.07 (s, 1H), 8.62–8.51 (m, 1H), 7.94 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.17–7.11
(m, 2H), 3.33–3.22 (m, 2H), 2.29 (t, J = 7.2 Hz, 2H),
1.77 (quintet, J = 7.2 Hz, 2H).
5.50. N-(4-Hydroxyamino-4-oxobutyl)-5-[(4-methylphen-
yl)ethynyl]-2-furamide (17)
5.47. trans-N-(4-Hydroxyamino-4-oxobutyl)-4-methyl-
cyclohexanecarboxamide (14)
The title compound was obtained as a yellow powder in
48% yield from 50d according to the analogous proce-
dures as described for the preparation of 14: TLC
Rf = 0.32 (CHCl3–MeOH–AcOH, 18:2:1); MS (MAL-
DI, pos.) m/z 349 (M+Na)+, 327 (M+H)+; IR (KBr)
To a stirred solution of 50a (600 mg, 2.64 mmol) in DMF
(3 mL) were added 1-hydroxybenzotriazole monohydrate
(810 mg, 5.29 mmol), 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (1.01 g, 5.29 mmol),

K. Takahashi et al. / Bioorg. Med. Chem. 13 (2005) 4527–4543
4543
3306, 2210, 1650, 1549, 1439, 1369, 1321, 1225, 1187,
1038, 968, 811, 759, 594, 523 cmꢀ1 1H NMR
6.2. MMP-2 and MMP-9 assay6
;
(200 MHz, DMSO-d6): d 10.40 (br s, 1H), 10.22 (s,
1H), 8.56 (t, J = 5.7 Hz, 1H), 7.47 (d, J = 8.3 Hz, 2H),
7.28 (d, J = 8.3 Hz, 2H), 7.16 (d, J = 3.6 Hz, 1H), 6.96
(d, J = 3.6 Hz, 1H), 3.28–3.14 (m, 2H), 2.36 (s, 3H),
2.00 (t, J = 7.5 Hz, 2H), 1.83–1.64 (m, 2H); HRMS
(FAB) calcd for C18H19N2O4: 327.1345. Found:
327.1347.
A mixture of 130 lL of MOCAc-Pro-Leu-Gly-Leu-
Dpa-Ala-Arg-NH2 (final concentration: 15 lM) and
20 lL of inhibitor solution were incubated at 37 °C for
5 min. 50 lL of enzyme solution was added, and the
reaction was performed at 37 °C for 10 min. Its fluores-
cence was measured by fmax. Excitation and emission
wavelengths were 320 and 390 nm, respectively.
5.51. 5-(4-Chlorophenyl)-N-(4-hydroxyamino-4-oxobu-
tyl)-2-furamide (18)
6.3. MMP-3 assay7
A mixture of 190 lL of MOCAc-Arg-Pro-Lys-Pro-Val-
Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 (final concentration:
15 lM) and 10 lL of inhibitor solution were incubated
at 37 °C for 5 min. 50 lL of enzyme solution was added
and the reaction was performed at 37 °C for 10 min. Its
fluorescence was measured by fmax. Excitation and
emission wavelengths were 320 and 390 nm,
respectively.
The title compound was obtained as a white powder in
62% yield from 50e according to the analogous proce-
dures as described for the preparation of 14: TLC
Rf = 0.38 (CHCl3–MeOH–AcOH, 18:2:1); MS (FAB,
pos.) m/z 323 (M+H)+; IR (KBr) 3205, 3034, 2874,
1739, 1624, 1573, 1546, 1512, 1476, 1303, 1273, 1215,
1187, 1170, 1093, 1043, 1019, 831, 799, 734, 663,
1
599 cmꢀ1; H NMR (200 MHz, DMSO-d6): d 10.40 (s,
1H), 8.64–8.51 (m, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.54
(d, J = 8.4 Hz, 2H), 7.16–7.11 (m, 2H), 3.31–3.18 (m,
2H), 2.08–1.95 (m, 2H), 1.76 (quintet, J = 7.0 Hz, 2H);
HRMS (FAB) calcd for C15H16ClN2O4: 323.0799.
Found: 323.0797.
References and notes
1. (a) Supuran, C. T.; Scozzafava, A. In Proteinase and
Peptidase Inhibition: Recent Potential Targets for Drug
Development; Smith, H. J., Simons, C., Eds.; Taylor and
Francis: London, New York, 2002, p 35; (b) Woessner, J.
F.; Nagase, H. In Matrix Metalloproteinases and TIMPs;
Oxford University Press, 2000, p 1.
2. Spurlino, J. C.; Smallwood, A. M.; Carlton, D. D.; Banks,
T. M.; Vavra, K. J.; Johnson, J. S.; Cook, E. R.; Falvo, J.;
Wahl, R. C.; Pulvino, T. A.; Wendoloski, J. J.; Smith, D. L.
Proteins: Struct. Funct. Genet. 1994, 19, 98.
6. Biology. Enzyme assays: MMP-1, MMP-2, MMP-9,
and MMP-3 assays using synthetic substrate
6.1. MMP-1 assay
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 enzyme solution (0.5 unit/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 cen-
trifuged 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.
3. Hajduk, P. J.; Gomtsyan, A.; Didomenico, S.; Cowart, M.;
Bayburt, E. K.; Solomon, L.; Severin, J.; Smith, R.; Walter,
K.; Holzman, T. F.; Stewart, A.; McGaraughty, S.; Jarvis,
M. F.; Kowaluk, E. A.; Fesik, S. W. J. Med. Chem. 2000,
43, 4781.
4. (a) Bo¨hm, H.-J. J. Comput. Aided Mol. Des. 1992, 6, 69; (b)
Bo¨hm, H.-J. J. Comput. Aided Mol. Des. 1992, 6, 593.
5. For this study, we used the crystal structure with the
following code number: PDB 1HFC.
6. Knight, C. G.; Willenbrock, F.; Murphy, G. A. FEBS Lett.
1992, 296, 263.
7. Nagase, H.; Fields, C. G.; Fields, G. B. J. Biol. Chem. 1994,
269, 20952.