Highly water-soluble matrix metalloproteinases inhibitors and their effects in a rat adjuvant-induced arthritis model.

Article abstract of DOI:10.1016/S0968-0896(02)00109-8

A new series of succinate-based dual inhibitors against matrix metalloproteinases (MMPs) and tumor necrosis factor alpha converting enzyme (TACE) possessing highly-water solubility was designed, synthesized, and evaluated for enzyme inhibition. Incorporat

Full text of DOI:10.1016/S0968-0896(02)00109-8

Bioorganic & Medicinal Chemistry10 (2002) 2569–2581
Highly Water-Soluble Matrix Metalloproteinases Inhibitors and
Their Effects in a Rat Adjuvant-Induced Arthritis Model
Tetsunori Fujisawa, Katsuhiro Igeta, Shinjiro Odake, Yasuo Morita,
Junko Yasuda and Tadanori Morikawa*
Research Institute, Daiichi Fine Chemical Co., Ltd., 530 Chokeiji, Takaoka, Toyama 933-8511, Japan
Received 17 January2002; accepted 22 March 2002
Abstract—A new series of succinate-based dual inhibitors against matrix metalloproteinases (MMPs) and tumor necrosis factor a
converting enzyme (TACE) possessing highly-water solubility was designed, synthesized, and evaluated for enzyme inhibition.
0
Incorporating of acidic or basic functional groups at the P2 position afforded sufficient water solubilitywithout significant loss of
0
inhibitorypotencies. Compound 18e, which had a guanidino group at the P2 position as the basic functional group, exhibited
broad inhibition against target enzymes for a relatively long period in rat plasma (b t1/2; 2.0 h) after sc administration when com-
pared with compounds possessing acidic functional groups (18a and 18b). Consequently, the representative compound 18e together
with compound 18b, Marimastat and Trocade were evaluated in the rat adjuvant-induced arthritis model, a model of chronic car-
tilage destruction. It is concluded that the newlysynthesized highlywater-soluble compound
18e showed significant activityin
suppressing hindpaw swelling and the bone destruction with a minimal administration period (days 3–7). # 2002 Elsevier Science
Ltd. All rights reserved.
Introduction
sis.10 For example, increased levels of fibroblast
collagenase (MMP-1) and stromelysin-1 (MMP-3) have
been observed in the cartilage and synovium of patients
with osteoarthritis (OA) or rheumatoid arthritis (RA),
Matrix metalloproteinases (MMPs) are zinc endo-
metallopeptidases that are involved in the degradation
and remodeling of connective tissues. This familyof
enzymes shows proteolytic activity towards virtually all
of the constituents of the extracellular matrix. The
members of this family, currently numbering 28, can be
classified into four groups, which are: the collagenases
that cleave triple-helical interstitial collagen; the gelati-
nases that cleave denatured collagen, elastin, and types
IV and V collagen; the stromelysins that mainly cleave
proteoglycans; and the membrane-type MMPs that
have a C-terminal transmembrane domain for anchor-
ing to the cell membrane. The MMPs are involved in
crucial physiological and physiopathological events,
such as wound healing, nerve growth, angiogenesis, and
11
and are correlated with the severityof the disease.
RA, showing chronic and progressive polyarthritis as its
main symptoms, is a generalized inflammatory disease
with multi organ disorder. Destruction of joint cartilage
introduces a serious permanent functional disorder. The
cartilage matrix consists mainlyof collagen (90% of
type II and other types including IX, XI)12 and aggrican
(chondroitin 6-sulfate, chondroitin 4-sulfate, and kera-
tan sulfate are incorporated as core proteins). The
major constituent of type II collagen is degraded by
MMP-1, neutrophil collagenase (MMP-8), and col-
lagenase-3 (MMP-13).13 MMP-3 can also degrade type
II collagen around the N-terminal region which forms
intermolecular cross-linkages. Moreover, MMP-3 also
destroys the NC2 domain of type IX collagen, which
plays an important role in the construction of collagen
networks. On the other hand, cleavage of core protein
at the G1-G2 domain is conducted byvarious MMPs
[MMP-1, 2, 3, matrilysin (MMP-7), 8, gelatinase B
(MMP-9), and 13]. From this evidence, MMP-3 is
pregnancy. In these physiological processes, MMP
1ꢀ5
activityis tightlyregulated.
However, excessive
MMPs syntheses and releases can lead to connective
tissue degradation and destruction, which occurs in
tumor invasion, metastasis,6 corneal ulceration,7 arthri-
tis disease,8 periodontal disease,9 and multiple sclero-
*Corresponding author. Tel.: +81-776-21-3456; fax: +81-766-21-
4439; e-mail: morikawa@daiichi-fcj.co.jp
14
thought to be one of the keyenzymes for RA.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00109-8

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T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
Given the inflammatoryphenomenon found in RA,
tumor necrosis factor a (TNF-a), a critical pro-inflam-
matory cytokine produced by monocytes or macro-
phages, plays an important role by inducing other pro-
inflammatorycytokines such as IL-1 b, IL-6, and IL-8.
Elevated TNF-a concentrations have been demon-
strated in RA disease.15 TNF-a is initiallyexpressed as a
26 kDa cell-associated form which is then proteolyti-
callyprocessed byTNF- a converting enzyme (TACE)
to yield a monomeric soluble form of 17 kDa secreted in
a mature form.16 This secreted protein contains one
intrachain disulfide bridge and exists as a dimer or tri-
mer in circulation.17 The mature TNF-a exerts its mul-
tiple biological effects via interaction with two
structurallyand functionallydistinct high-affinity
receptors: TNFR1 (p55) and TNFR2 (p75).18 Binding
of TNF-a to these receptors results in the activation of
several transduction pathways. From these bindings,
suppression of MMPs activity, especially MMP-1 and 3,
together with TACE activitycould relieve the severityof
RA.
Results and Discussion
Design
From a conformational analysis of MMP-3 complexed
with a succinyl based MMP inhibitor by X-ray crystal-
0
and the P1 and P2 residues have little interaction with
0
lography, the P1 isobutyl group fits into the S1 pocket,
0
MMP-3 and are directed awayfrom the active site into
the solvent.25 According to this observation, introduc-
tion of hydrophilic functional groups, which interact
with the water molecules existing outside the active site,
at P1 and/or P2 residues could increase the water solu-
bilityof succinly based MMPs inhibitors. As the P
residue incorporates a succinate unit with (S)-configur-
ation, it seems to be troublesome to resolve diastereoi-
somers of succinate units after the introduction of
various hydrophilic functional groups on the P1 residue.
0
1
0
The P2 position, which is constructed from an amino
acid, is more convenient both for introducing the func-
tional group and obtaining chiral compounds than is
the P1 position. As the P1 residue and an aromatic ring
0
at the P2 residue are preferable for in vitro activity, we
Our initial objective was to confirm the efficacyof dual
inhibitors of MMPs and TACE in a rat arthritis model
using a systemic administration route. Although oral
administration is preferable, in order to consider the
initial objective of assessing the intrinsic potencyof the
dual inhibitor a subcutaneous (sc) injection route pro-
vides a more direct result byignoring other biological
factors, e.g., intestinal adsorption rate, and hepatic
metabolism. Give the sc injection for systemic adminis-
tration, acquiring a highlywater-soluble propertyis a
prerequisite for inhibitors. Generallypeptide based
drugs are poorlyin water soluble. In several MMPs
inhibitors progressed into clinical trials (Fig. 1), sulfo-
namide type compounds, represented as Prinomastat
419 or CGS-27023A 520 include a heterocyclic ring
which can improve the water solubility. Regarding suc-
cinate-based type compound depicted as Batimastat 121
or Marimastat 2,22,23 efforts to acquire the highlywater-
soluble derivatives have not been conducted (water
solubilitywere <1 and 1.4 mg/mL,24 respectively). In
this study, we have investigated the synthesis of highly
water-soluble dual inhibitors based on the chemical
structure of succinate-based type compound, without
anyloss of inhibitorypotencies.
designed dual inhibitors with the new physical char-
acteristics depicted in Figure 2 as a general formula.
Chemistry
The 2,3-disubstituted succinic acid half esters 11 were
prepared according to the sequence shown in Scheme 1.
Diazotization of the amino group in d-leucine following
intracyclization by carboxylate and simultaneous ring
opening bybromo anions proceeded predominantlyin a
stericallyconserved reaction that was accompanied by
2–3% racemization. The benzyl esterification was con-
ducted bythe removal of water byazeotropic distilla-
tion with catalytic p-toluenesulfonic acid (TsOH) to give
8. Then, the C2–C3 bond formation at succinate was
accomplished bythe reaction of a-bromo carboxylate 8
with activated methylene of malonic acid diester to give
tricarbonate 9.26 After introduction of a cinnamyl
group, consequent hydrogenolytic removal of the two
benzyl groups and decarboxylation at the malonyl moi-
etyof 10 in the presence of tertiaryamine afforded
compound 11.26 The crude 11, which was obtained as a
colorless oil, contained of 3(R),2(S) and 3(S),2(S) forms
Figure 1. Hydroxamic acid based MMP inhibitors.

T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
2571
as the major products, together with 3(R),2(R) and
3(S),2(R) forms as contaminants that were caused by
the reaction mechanism of diazotization together with
bromination of d-leucine and utilizing a synthetic route
for preparation of 11 from 9. To obtain the pure,
desired 3(R),2(S) form, we examined the crystallization
of 11 with 35 kinds of amine including chiral amines.
Several amines gave crystals that were subjected to
recrystallization from an appropriate solvent. It was
found that tert-butylamine gave a sufficient diaster-
eomeric excess (de) and enantiomeric excess (ee). The
relative stereochemistrywas inferred byobservation
of nuclear Overhauser effect (NOE) between the C-2
methine, the C-3 methine and the one C-4 methine at
compound 13, which was prepared as described by
Beckett (Scheme 2).27
3. The succinate unit 11 was coupled with 14a–f using
EDC/HOBt as the coupling reagent. The tert-butyl
esters 15a–f were converted to O-benzylhydroxamate
17a–f bytreatment with acid followed bycoupling with
O-benzylhydroxylamine. To construct an acylimidoy-
liminomethyl group, compounds that had a cyano
group on the aromatic ring 15d were hydrogenated in
AcOH and subsequently reacted with ethyl acylimidate
to give 16a–c. N,N0-Diprotected guanidinomethyl com-
pound 16d was also obtained byreaction of the amino
methyl
intermediate
and
1H-pyrazole-N,N0-
bis(benzyloxycarbonyl)carboxamidine,28 instead of
ethyl acylimidate. Carboxylic acids 16a–d were coupled
to O-benzylhydroxylamine using EDC/HOBt to yield
17g–f. O-sulfonation was carried out on O-benzylhy-
droxamate compound 17a using sulfur trioxide pyridine
complex, which was subsequentlyhydrogenated to give
18a. Other target compounds (18b–j) were prepared by
catalytic hydrogenation of 17b–j.
The preparation of compounds in which R1 represents
hydrophilic functional groups is summarized in Scheme
Inhibition of MMP-1, 3 and TACE
Initially, the inhibitions of MMP-1, 3 and TACE, which
have been assumed to playa crucial role in RA, were
evaluated byin vitro assasy together with the water
solubilityof those compounds and the results were
compared with those for unsubstituted compound
(R1=H) in Table 1. Due to the introduction of hydro-
philic functional groups on the aromatic ring of
phenylalanine, the inhibitory potency for MMP-1 was
decreased, but that for MMP-3 was almost tolerated
against Comparative compound. Furthermore, it is
noteworthythat after installation of a hdyrophilic
Figure 2. General formula of highlywater-soluble inhibitor.
0
functional group at the P2 position, a dramatic increase
in water solubilitywas observed. There was at least a
10-fold increase of water solubilitywhen compared with
unsubstituted compounds. In particularly, acidic func-
tional groups represented bysulfonate ( 18a and 18b)
possessing compounds showed at least 100-fold increa-
ses in water solubility.
Half-life measurements in rat plasma
Before evaluation of these inhibitors in the rat adjuvant-
induced arthritis model, we selected several typical
compounds and measured plasma t1/2 in the b-phase in
rats after sc dosing together with the area under the
plasma concentration time curve (Table 2). The values
in Table 2 indicate the amount of the administered
compound plus its active metabolites, since we esti-
mated the value based on ex vivo MMP-3 activity
Scheme 1. Preparation of succinate units. Reagents: (a) (1) aq
NaNO2/aq.HBr; (2) BnOH/TsOH in c-hexane reflux; (b) benzyl tert-
butylmalonate/tert-BuOK; (c) cinnamyl bromide/NaH; (d) (1) H2/Pd-
C, (2) N-ethylmorpholine in toluene reflux, (3) tert-butylamine.
.
Scheme 2. Determination of the relative stereochemistryof 11c. Reagents: (a) BH3 THF; (b) TFA/CH2Cl2.

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T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
Scheme 3. Syntheses of highly water-soluble MMPs inhibitors 18a–j. Reagents: (a) 4 N HCl in EtOAc; (b) EDC/HOBt; (c) (1) 95% aq TFA, (2)
ethyl acylimidate hydrochloride/Et3N (for 16a–c) or 1H-pyrazole-N,N0-bis(benzyloxycarbonyl)carboxamidine (for 16d); (d) BnONH2/EDC/HOBt;
(e) (1) 95% aq TFA, (2) BnONH2/EDC/HOBt; (f) (1) sulfur trioxide pyridine complex (for 18a), (2) H2/Pd-C (for 18a–j).
Table 1. Inhibition of MMP-1, MMP-3 and TACE byhydrophilic MMPs inhibitors and their solubilityfor water
IC50 (nM)a
Compd
R1
MMP-1
MMP-3
TACE
Solubility
(mg/mL)
Comparative compd.
—H
—OSO3Na
—SO3Na
—NH2
—CH2NH2
0.15
0.4
4
3
5
6
5
7
6
8
5
4
1
8
6
5
15
3
10
10
NDd
120
270
134
210
130
140
120
160
420
130
1>
>100
>100
10b
18a
18b
18c
18d
18e
18f
18g
18h
18i
10b
—NHC(=NH)NH2
—C(=NH)NH2
—CH2NHC(=NH)Me
—CH2NHC(=NH)Et
—CH2NHC(=NH)Ph
—CH2NHC(=NH)NH2
30b
80c
70c
70c
10
4
40c
18j
80c
aConcentration required for 50% inhibition of enzyme activity. Standard deviations are less than 10% of the mean values. Details of the enzyme
assays are described in Experimental.
bAcOH salt.
cHCl salt.
dNot done.

T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
2573
measurements. As predicted, acidic sulfonate sub-
showed the similar efficacyexcept for days 0–3 admin-
stituted compounds (18a and 18b) showed a short half-
life compared with compounds having substitutent of
basic functional groups (18e and 18g). Compared with
Marimastat and Trocade, compound 18e was shown to
have a superior half-life and almost the same AUC
value as Trocade.
istration. It is note worthythat the shorter period
administration (days 3–7) also caused sufficient sup-
pression. But late stage administrations (days 7–20 and
days 14–27) did not improve the swelling. These results
indicate that MMPs and/or TACE act at an earlystage
in this model and playa pivotal role in swelling together
with bone destruction. The effects of indomethacin
(typical non-steroidal anti-inflammatory drug) and
methotrexate (slow-acting anti-rheumatic drug), the
common RA therapeutic medicines in this model are
also shown in Table 4. Indomethacin showed moderate
efficacy(45.7% inhibition of hindpaw swelling), even
for late stage administration (days 14–27). On the con-
trary, at early stage administration, only 10.7% inhibi-
tion of hindpaw swelling was observed. Although
methotrexate exhibited 51.9% inhibition at earlystage
administration (days 0–7), hindpaw swelling was exa-
cerbated with late stage administration (ꢀ20.9%, days
14–27). From the studyof these administration points,
it can be concluded that several different pathogenic
factors including MMPs and TACE participated in the
rat adjuvant-induced arthritis model. Combined
administration of compound 18e and indomethacin
might have an additional or synergistic effect in RA
patients bysuppressing alternative pathogenic factors.
Inhibition of MMP-2, 7, 9, metalloelastase (MMP-12),
13 and membrane-type 1 MMP (MMP-14)
In addition to IC50 determinations against other MMPs
and TACE, 18e was evaluated together with 18b, Mar-
imastat, and Trocade (Table 3). As a result, 18b and 18e
showed similar broad spectrum inhibitoryprofiles against
MMPs and TACE. Marimastat exhibited broad and
strong inhibitorypotencies against MMPs, and a one
order of magnitude decrease in potencyagainst TACE
compared with 18e. On the other hand, Trocade showed
relativelyMMP-7 and 13 selective inhibitorypotencies.
Effect of water-soluble MMP inhibitors on the rat
adjuvant-induced arthritis model
Initially, compound 18e was evaluated in the rat adju-
vant-induced arthritis model. 18e was administrated sc
in three doses (3, 10, 30 mg/kg/day) from day 0 and
continued throughout the 20 days of the experiment
with three doses. The time course of hindpaw volumes are
depicted in Figure 3. Although, in the saline-adminis-
tered control group, the hindpaw volume dramatically
increased from day10 after adjuvant injection, 18e
suppressed this swelling efficientlyand dose-depen-
dently. The X-ray scoring data, which was utilized by
measuring the severityof joint destruction, also
improved depending on doses. Representative radio-
graphs of hindpaws from normal rats and rats treated
with saline or compound 18e (30 mg/kg/day) after
injection of adjuvant are shown in Figure 4. Compared
with the saline-treated control, the hindpaw from a rat
treated with compound 18e shows less bone deminer-
alization, joint space narrowing, and bone erosion at the
distal tibia, talus, calcaneus, and metatarsus. To deter-
mine the administration point during the experiment,
we explored several administration periods (Table 4).
Compared to administration everyday(dasy 0–20),
early stage administrations (days 0–5 and days 0–7)
We also measured the efficacyof other developing
MMPs inhibitors represented as Marimastat and Tro-
cade together with 18b and 18e (Table 5). Under the
same administration conditions for route (sc), dose
(30 mg/kg), and timing (days 3–7), Trocade which is a
MMP-7 and 13 selective inhibitor, showed a lack of
efficacyagainst hindpaw swelling and bone destruction,
suggesting that MMP-7 and 13 are not important at an
earlystage in this model. Probabl,y due to its short
half-life in rat plasma (0.58 h), 18b showed only20.6%
Table 2. Effect of hydrophilic functional group on pharmacokinetic
properties in the rat after subcutaneous administration
R1
t1/2b (h)
.
AUC (ng/mL h)
Compd.
18a
18b
18e
18g
Marimastat
Trocade
ꢀOSO3Na
0.33
0.58
2.00
1.83
1.15
1.03
436
1738
13659
8148
9790
ꢀSO3Na
ꢀNHC(=NH)NH2
ꢀCH2NHC(=NH)Me
—
—
14058
Figure 3. Effect of compound 18e on hindpaw swelling in adjuvant
arthritis rats. Compound 18e were administered subcutanouslyto the
back of rats on day0–20. Indomethacin was given orallydaily. Nor-
mal rats received no treatment. Data represent meanꢁSE with 6 rats
per group. *P<0.05 as compared with the control bytwo-tailed
Dunnett’s multiple comparison. #P<0.05 as compared with the con-
trol bytwo-tailed Student’s t-test.
Pharmacokinetic parameter t1/2
ꢀ represents half-lives for second
phase. The values were estimated based on the ex vivo MMP-3 activity
measurements for plasma concentration. Data shown are mean values
(n=3). Area under the plasma concentration time curves are extra-
polate based on a 0–4 h (AUC(0ꢀ4 h)) value byWinNonlin Standard
(Ver. 1.5) software.

2574
T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
Figure 4. Representative radiographs of hindpaw; (A) normal rat, (B) treated with saline after adjuvant injection, (C) treated with compound 18e
(30 mg/kg/day) after adjuvant injection.
Table 3. Inhibitoryof various MMP and TACE bycompounds 18b, 18e, Marimastat and Trocade
Compd.
IC50 (nM)a
MMP-9
MMP-1
MMP-2
MMP-3
MMP-7
MMP-12
MMP-13
MMP-14
TACE
270
130
1807
>100,000
18b
18e
Marimastat
Trocade
4
6
1
2
4
6
NDc
0.7
2 (20)b
9
1
1
9
NDc
0.064
5
0.16
0.07
0.74
1.79
NDc
4
3
21
2 (5)b
23 (7)b
13 (6)b
314
50 (200)b
294 (527)b
545 (59)b
17
aConcentration required for 50% inhibition of enzyme activity. Standard deviations are less than 10% of the mean values. Details of the enzyme
assays are described in Experimental.
bIC50 values in parentheses are given from references.
cNot done.
Table 4. Effects of administration timing on hindpaw swelling and bone destruction in adjuvant arthritis rats
Drugs
Dose
(mg/kg)
Route
Timing
Hindpaw Swelling
inhibition (%)
Bone destruction
inhibition (%)
Compd 18e
30
30
30
30
30
30
sc
sc
sc
sc
sc
sc
Day0–3
Day0–7
Day3–7
Day7–20
Day14–27
Day0–20
27.6
70.3 a
74.4 a
15.3
28.5
86.2a
89.0a
26.6
ꢀ17.4
ꢀ24.8
64.0 a
70.4a
Indomethacin
Methotrexate
1
1
1
po
po
po
Day0–7
Day14–27
Day0–20
10.7
45.7 b
70.3 b
ꢀ2.7
61.7b
86.4b
0.2
0.2
0.2
ip
ip
ip
Day0–7
Day14–27
Day0–20
51.9
ꢀ20.9
68.5 b
58.7
ꢀ19.2
79.8b
sc, subcutaneously; po, per oral; ip, intraperitoneally. The conditions are described in Experimental.
aP<0.05 as compared with the control bytwo-tailed Dunnett’s multiple comparison.
bP<0.05 as compared with the control bytwo-tailed Student’s t-test.
Table 5. Effects of MMP inhibitors on hindpaw swelling and bone destruction of adjuvant arthritis rats
Compd
Dose (mg/kg)
Route
Timing
Hindpaw swelling
% inhibition
Bone destruction
% inhibition
18b
18e
Marimastat
Trocade
30
30
30
30
sc
sc
sc
sc
Day3–7
Day3–7
Day3–7
Day3–7
20.6
53.5
30.3
2.1
46.9
62.0
35.7
ꢀ5.4
Drugs were administered subcutaneouslyto the back of rats once a dayfrom day3 to 7. sc, Subcutaneously. The conditions are described in
Experimental.

T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
2575
inhibition of hindpaw swelling. Marimastat, which has a
broad MMPs inhibitoryprofile and moderate TACE
inhibitorypotency, exhibited 30% inhibition of hind-
paw swelling and improved X-rayscoring. Previously,
examples of succinyl-based MMPs inhibitors have
been reported to produce anti-arthritic activityin the
rat adjuvant-induced arthritis model.29ꢀ31 Those
informations and our result of the major difference
between compound 18e and Trocade suggest that
soluble TNF-a might be more operative than MMPs
in the rat adjuvant-induced model and suppression of
TNF-a release is required for the prevention of bone
destruction.
tions were measured in a JASCO DIP-140 apparatus.
1H NMR was recorded on a JEOL JMN-AL300 spec-
trometer (300 MHz), and chemical shifts are given in
ppm (d) from tetramethylsilane (TMS), which was used
as the internal standard. Mass spectra were obtained on
a JEOL JMS 700 spectrometer byfast atom bombard-
ment (FAB) ionization techniques using glycerol as a
matrix. All organic extracts were dried with anhydrous
MgSO4 prior to solvent removal on a rotaryevaporator
under reduced pressure.
Benzyl 2(R)-bromo-4-methylpentanoate (8). To a solu-
tion of d-leucine (50.0 g, 381 mmol) in 3 N aq HBr
(570 mL), aq sodium nitrite [42.1 g, (610 mmol) was dis-
solved in 100 mL of water] was added at 0–2 ꢂC and
stirred at the same temperature for 3 h. The organic
materials were extracted with EtOAc, washed with
brine, evaporated to give crude 2(R)-bromo-4-methyl-
pentanoic acid (66.9 g, 90%) as a slightlybrown oil. A
mixture of crude 2(R)-bromo-4-methylpentanoic acid
(66.9 g, 343 mmol), benzyl alcohol (42.6 mL, 412 mmol),
p-toluenesulfonic acid monohydrate (6.52 g, 34.3 mmol),
and toluene (700 mL) was heated under reflux with
azeotropic trapping of water for 2 h. The reaction mix-
ture was washed with satd aq NaHCO3 and brine, then
evaporated in vacuo to leave a slightlybrown oil, which
was purified byflash column chromatography(eluent;
n-hexane: EtOAc=50:1) to give 8 (64.6 g, 66%) as a
Conclusion
We have developed new dual inhibitors of MMPs and
TACE that were highlywater-soluble, without sig-
nificant deletion of their activities. The sc injection of
high concentration inhibitors becomes possible through
these compounds. The compounds (18e), which pos-
sesses a basic hydrophilic functional group (guanidino)
0
on the P2 aromatic ring, showed longer duration in rat
plasma level after sc injection than compounds (18a and
18b), which have acidic functional groups at the same
position. The representative compound (18e) was con-
firmed as possessing intrinsic efficacyagainst the rat
adjuvant-induced arthritis model, bydecreasing the
hindpaw swelling and bone destruction. We have found
that the best administration point in this model was in
the earlystage immediatelyfollowing adjuvant injec-
tion; days 3–7 administration gave the shortest period
of administration with sufficient efficacy. Treatment
with 18e and then with indomethacin is expected to
have an additional or synergistic effect in patients with
RA.
ꢂ
1
colorless oil: [a]D25 +31.8 (c=1.0, MeOH). H NMR
(CDCl3) d ppm; 0.92 (3H, d, J=6.5 Hz), 0.94 (3H, d,
J=6.4 Hz), 1.67 (1H, m), 1.90 (2H, m), 4.30 (1H, t,
J=7.0 Hz), 5.20 (2H, s), 7.32 (5H, m). HRFABMS
calcd for C13H18O2Br: 285.0490, found: 285.0516.
Dibenzyl 3(RS)-tert-butyloxycarbonyl-2(R)-iso-butylsuc-
cinate (9). To a solution of benzyl tert-butylmalonate
(24.9 g, 99.6 mmol) in DMF (60 mL), potassium tert-
butoxide (13.4 g, 120 mmol) was added byportions with
stirring at 0 ꢂC. The mixture was stirred for 1 h at room
temperature and re-cooled to 0 ꢂC. A solution of 8
(28.4 g, 99.6 mmol) in DMF (60 mL) was added drop-
wise to the cooled mixture over a period of 1 h. After
stirring for 15 h at 5 ꢂC, AcOEt (2 L) was added to the
reaction mixture, which was then partitioned and
washed successivelywith brine, 1 N aq HCl, sat. aq
NaHCO3, and brine (twice for each). The organic layer
was evaporated in vacuo to leave a slightlyyellow oil,
which was purified byflash column chromatography
(eluent; n-hexane: EtOAc=20:1) to give 9 (40.0 g, 89%)
as a colorless oil: [a]D25 +16.7ꢂ (c=1.0, MeOH). 1H
NMR (CDCl3) d ppm; 0.82 (3H, d, J=6.8 Hz), 0.84
(3H, d, J=6.7 Hz), 1.15–1.82 (12H, m), 3.21 (1H, m),
3.73 (1H, m), 5.14 (4H, m), 7.32 (10H, m). HRFABMS
calcd for C27H35O6: 455.2434, found: 455.2437.
Experimental
Chemistry
All commercial chemicals and solvents are reagent
grade and used without further purification unless
otherwise specified. The following solvents and reagents
have been abbreviated: N,N-dimethylformamide
(DMF), tertahydrofurane (THF), trifluoroacetic acid
(TFA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt).
Reaction were monitored bythin-layer chromatography
on 0.25-mm silica gel plates (Silica gal 60 F254, Merck)
and visualized with UV light, iodine vapors, 5% phos-
phomolybdic acid in 95% ehtanol, or 0.7% ninhydrin in
ethanol. Final compound were typically purified either
flash column chromatographyon silica gel BW-300
(200–400 mesh, Fuji Silysia Chemical Ltd.) or reverse
phase flash column chromatographyon C 18-bonded
silica gel Chromatorex-ODS DM1020T (Fuji Silysia
Chemical Ltd.).
Dibenzyl 3(RS)-tert-butyloxycarbonyl-3-cinnamyl-2(R)-
iso-butylsuccinate (10). To a solution of 9 (9.49 g,
20.9 mmol) in DMF (100 mL) was added 60% sodium
hydride (1.0 g, 25.1 mmol) by portions with stirring at
room temperature. The mixture was stirred for 2 h at
room temperature and cooled to 0 ꢂC. Cinnamyl bro-
mide (5.36 g, 27.2 mmol) was added byportions to the
cooled mixture which was then stirred at 5 ꢂC for 15 h.
Melting points were determined on a Yanagimoto melt-
ing point apparatus without correction. Optical rota-

2576
T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
The solvent was evaporated under reduced pressure,
and EtOAc (500 mL) was added to the residue. The
mixture was washed successivelywith brine, 1 N aq
HCl, sat. aq NaHCO3, and brine (twice for each). The
organic layer was evaporated in vacuo to leave a slightly
brown oil, which was purified byflash column chroma-
tography(eluent; n-hexane: EtOAc=20:1) to give 10
and stirred at 4 ꢂC for 1 h. The reaction mixture was
evaporated in vacuo to leave crude 13 which was pur-
ified byflash column chromatography(eluent; n-hexane:
EtOAc=9:1) to give 13 (208 mg, 74%) as a colorless oil:
1H NMR (CDCl3) d ppm; 0.86 (3H, d, J=6.4 Hz), 0.90
(3H, d, J=6.6 Hz), 1.01–1.25 (2H, m), 1.43–1.91 (5H,
m), 2.47–2.59 (2H, m), 2.62–2.76 (2H, m), 4.01 (1H, dd,
J=3.6, 9.0 Hz), 4.18 (1H, dd, J=5.2, 9.0 Hz), 7.13–7.35
(5H, m). HRFABMS calcd for C17H25O2: 261.1855,
found: 261.1825.
1
(10.8 g, 91%) as a colorless oil: H NMR (CDCl3) d
ppm; 0.75–0.98 (6H, m), 1.04–1.16 (1H, m), 1.20–1.50
(10H, m), 1.84–1.95 (1H, m), 2.78–2.90 (2H, m), 3.13–
3.26 (1H, m), 4.99–5.21 (4H, m), 6.08–6.27 (1H, m),
6.30–6.45 (1H, m), 7.14–7.42 (15H, m). HRFABMS
calcd for C36H43O6: 571.3060, found: 571.3086.
Typical procedure for the preparation of compound 15.
N-[4-tert-Butyloxy-2(R)-isobutyl-3(S)-phenylpropylsucci-
nyl]-L-tyrosine N-methylamide (15a). To a mixture of 11
.
3(S)-tert-Butyloxycarbonyl-6-phenyl-2(R)-isobutylhexa-
noic acid (11). To a solution of 10 (10.0 g, 17.5 mmol)
in methanol (120 mL) was added 5% Pd-C (50% wet
catalyst, 2.5 g), and the mixture was vigorously stirred
under a hydrogen atmosphere at room temperature for
7 h. The catalyst was filtered off and then methanol was
evaporated in vacuo to give dicarboxylic acid inter-
mediate as a colorless oil. The residue was dissolved in
toluene (400 mL) and refluxed in a presence of N-ethyl-
morpholine (2.23 mL, 17.5 mmol) for 2 h. The reaction
mixture was washed successivelywith 1 N aq HCl, and
brine (twice for each), then evaporated in vacuo to leave
a yellow oil. The residue was dissolved in EtOAc
(150 mL) and tert-butylamine (1.84 mL, 17.5 mmol) was
added to foam a tert-butylamine salt as a colorless solid,
which was recrystallized from EtOAc to give 11 (color-
less solid, 2.36 g, 32%) as tert-butylamine salt. Enantio-
meric excess (ee); 97.3%, diastereomeric excess (de);
97.2% [The ee and de were determined bychiral HPLC
[column; Chiralpack AD (Daicel Chemical Industries),
detection; 220 nm, eluent; n-hexane/2-propanol/TFA=
98:2:0.1, column temperature; 30 ꢂC, flow rate; 1 mL/
min]. 1H NMR (CDCl3) d ppm; 0.86 (3H, d, J=6.6 Hz),
0.89 (3H, d, J=6.4 Hz), 1.00–1.13 (1H, m), 1.27 (9H, s),
1.42 (9H, s), 1.46–1.78 (6H, m), 2.40–2.69 (4H, m), 5.37
(3H, brs), 7.08–7.30 (5H, m). HRFABMS calcd for
C21H33O4: 349.2379, found: 349.2371.
(4.21 g, 10 mmol), H-Tyr-NHMe HCl (2.54 g, 11 mmol)
[prepared from Boc-Tyr-NHMe (14a) (3.24 g, 11 mmol)
and 4 N HCl in EtOAc (60 mL) as usual], HOBt (1.49 g,
11 mmol), and DMF (70 mL), EDC (2.11 g, 11 mmol)
was added under stirring at ꢀ15 ꢂC. The mixture was
stirred at ꢀ15 ꢂC for 1 h and further at room tempera-
ture overnight. The reaction mixture was diluted with
EtOAc (210 mL) and washed successivelywith brine, 1
N aq HCl, 10% aq Na2CO3 and brine (twice for each),
then evaporated in vacuo to leave a pale yellow oil. The
residue was crystallized from Et2O to give 15a (3.57 g,
68%) as a colorless solid: [a]D25 +9.0ꢂ (c=0.5, MeOH).
1H NMR (CDCl3) d ppm; 0.78 (3H, d, J=6.4 Hz), 0.81
(3H, d, J=6.4 Hz), 0.82–1.74 (16H, m), 2.11–2.70 (4H,
m), 2.74 (3H, d, J=4.7 Hz), 2.81–2.94 (1H, m), 2.94–
3.10 (1H, m), 4.65 (1H, m), 6.32 (1H, m), 6.59 (1H, m),
6.80–7.42 (9H, m). HRFABMS calcd for C31H45N2O5:
525.3328, found: 525.3350.
N-[4-tert-Butyloxy-2(R)-isobutyl-3(S)-phenylpropylsucci-
nyl]-L-40-sulfophenylalanine N-methylamide sodium salt
1
(15b). Yield; 60%. H NMR (MeOH-d4) d ppm; 0.74
(3H, d, J=6.7 Hz), 0.82 (3H, d, J=6.7 Hz), 1.01–1.76
(16H, m), 2.21–2.84 (7H, m), 2.84–3.21 (2H, m), 4.52–
4.70 (1H, m), 6.97–7.40 (7H, m), 7.69–7.85 (2H, m). FAB-
MS: m/z 611 [M+Na]+, 589 [M+1]+. HRFABMS:
calcd for C31H44N2NaO7S 611.2767, found 611.2796.
tert-Butyl 3(R)-hydroxymethyl-5-methyl-2(S)-phenylpro-
pylhexanoate (12). To a solution of free form of 11
(669 mg, 1.92 mmol) in dryTHF (10 mL), borane-THF
complex (1.0 M solution in THF, 2.2 mL) was added at
ꢀ10 ꢂC and stirred at room temperature for 4 h under a
nitrogen atmosphere. To the reaction mixture, satd aq
NH4Cl (50 mL) was added and organic materials were
extracted with EtOAc followed bywashing with 10% aq
Na2CO3 and brine, and then evaporated in vacuo to
leave crude 12 which was purified byflash column
chromatography(eluent; n-hexane: EtOAc=9:1) to give
12 (440 mg, 69%) as a colorless oil: 1H NMR (CDCl3) d
ppm; 0.86 (3H, d, J=6.4 Hz), 0.89 (3H, d, J=6.4 Hz),
1.01–1.30 (2H, m), 1.34–1.92 (15H, m), 2.38–2.53 (1H,
m), 2.63 (2H, t, J=6.8 Hz), 3.48–3.67 (2H, m), 7.11–
7.34 (5H, m). HRFABMS calcd for C21H35O3:
335.2586, found: 335.2580.
N-[4-tert-Butyloxy-2(R)-isobutyl-3(S)-phenylpropylsucci-
nyl]-L-40-nitrophenylalanine N-methylamide (15c). Yield;
83%. 1H NMR (CDCl3) d ppm; 0.74 (3H, d, J=6.4 Hz),
0.80 (3H, d, J=6.4 Hz), 0.90–1.74 (16H, m), 2.21–2.60
(4H, m), 2.64 (3H, d, J=4.7 Hz), 2.98–3.20 (2H, m),
4.46–4.68 (1H, m), 5.90–6.18 (2H, m), 7.16–7.73 (7H,
m), 8.02–8.23 (2H, m). HRFABMS: calcd for
C31H44N3O6 554.3230, found 554.3246.
N-[4-tert-Butyloxy-2(R)-isobutyl-3(S)-phenylpropylsucci-
nyl]-L-40-cyanophenylalanine
N-methylamide
(15d).
Yield; 87%. 1H NMR (CDCl3) d ppm; 0.81 (3H, d,
J=6.4 Hz), 0.84 (3H, d, J=6.2 Hz), 0.96–1.82 (16H, m),
2.07–2.70 (4H, m), 2.72 (3H, d, J=4.6 Hz), 2.96–3.12
(2H, m), 4.56–4.67 (1H, m), 6.02–6.14 (1H, m), 6.48–
6.57 (1H, m), 6.92–7.75 (9H, m). HRFABMS: calcd for
C32H44N3O4 534.3332, found 534.3322.
3(R)-Isobutyl-2(S)-phenylpropyl-ꢀ-butyrolactone (13). To
a solution of tert-butyl ester 12 (360 mg, 1.08 mmol) in
CH2Cl2 (7.2 mL), TFA (414 mL, 5.40 mmol) was added
N-[4-tert-Butyloxy-2(R)-isobutyl-3(S)-phenylpropylsucci-
nyl]-L-40-[N,N0-bis-(benzyloxycarbonyl)guanidino]phenyl-
alanine N-methylamide (15e). Yield; 70%. 1H NMR

T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
2577
(CDCl3) d ppm; 0.77 (3H, d, J=6.7 Hz), 0.80 (3H, d,
J=6.4 Hz), 0.92–1.14 (1H, m), 1.21–1.77 (15H, m),
2.16–2.64 (4H, m), 2.67 (3H, d, J=4.6 Hz), 2.93 (2H, d,
J=5.9 Hz), 4.50–4.66 (1H, m), 5.13 (2H, s), 5.18 (2H, s),
5.90–6.11 (1H, m), 6.41–6.53 (1H, m), 6.89–7.65 (21H,
m). HRFABMS: calcd for C48H60N5O8 834.4442, found
834.4430.
4.38 (2H, s), 4.52–4.60 (1H, m), 6.99–7.58 (9H, m).
HRFABMS: calcd for C31H45N4O4 537.3441, found
537.3445.
N-[4-Hydroxy-2(R)-isobutyl-3(S)-phenylpropylsuccinyl]-
L - 40 - benzimidoyliminomethylphenylalanine N - methyl-
amide hydrochloride (16c). Yield; 53% overall yield
from 15d: [a]D25 ꢀ9.0ꢂ (c=1.0, MeOH). 1H NMR
(MeOH-d4) d ppm; 0.84 (3H, d, J=6.6 Hz), 0.89 (3H, d,
J=6.4 Hz), 0.95–1.80 (7H, m), 2.14–2.81 (7H, m), 2.95
(2H, d, J=5.6 Hz), 4.30 (2H, s), 4.48–4.63 (1H, m),
6.98–7.82 (14H, m). HRFABMS: calcd for C35H45N4O4
585.3441, found 585.3409.
N-[4-tert-Butyloxy-2(R)-isobutyl-3(S)-phenylpropylsucci-
nyl]-L -40 -[N-(benzyloxycarbonyl)amidino]phenylalanine
1
N-methylamide (15f). Yield; 18%. H NMR (CDCl3) d
ppm; 0.79 (3H, d, J=6.2 Hz), 0.83 (3H, d, J=6.4 Hz),
0.97–1.12 (1H, m), 1.26–1.69 (15H, m), 2.06–2.48 (4H,
m), 2.76 (3H, d, J=4.7 Hz), 3.10 (2H, d, J=6.0 Hz),
4.49–4.62 (1H, m), 5.20 (2H, s), 6.21–6.36 (1H, m),
6.47–6.62 (1H, m), 7.13–7.50 (14H, m), 7.71–7.90 (2H,
m). HRFABMS: calcd for C40H53N4O6 685.3965, found
685.3995.
N-[4-Hydroxy-2(R)-isobutyl-3(S)-phenylpropylsuccinyl]-
L -40 - [N,N0 -bis(benzyloxycarbonyl)guanidinomethyl]phe-
nylalanine N-methylamide (16d). To a solution of N-[4-
hydroxy-2(R)-isobutyl-3(S)-phenylpropylsuccinyl]-l-4-
aminomethylphenylalanine N-methylamide hydrochlo-
ride (500 mg, 0.97 mmol) in DMF (8 mL), 1H-pyrazole-
Typical procedure for the preparation of compound 16.
N-[4-Hydroxy-2(R)-isobutyl-3(S)-phenylpropylsuccinyl]-
L-40-acetimidoyliminomethylphenylalanine N-methylamide
hydrochloride (16a). To the compound 15d (1.73 g,
3.24 mmol), ice-cooled 95% aq TFA (16 mL) was added
and stirred at 5 ꢂC for 4 h, then evaporated in vacuo.
The residue was precipitated from Et2O followed byfil-
tration to give a carboxylic acid intermediate, N-[4-
hydroxy-2(R)-isobutyl-3(S)-phenylpropylsuccinyl]-l-40-
cyanophenylalanine N-methylamide (1.22 g, 79%) as a
colorless solid. To a solution of the carboxylic acid
intermediate (1.22 g) in EtOH (25 mL)-concn. HCl
(1 mL), 5% Pd-C (50% wet, 600 mg) was added and the
mixture was vigorouslystirred under hydrogen atmo-
sphere at room temperature for 10 h. The catalyst was
filtered off and evaporated in vacuo to leave a colorless
residue, which was suspended in water (50 mL) and
stirred for 30 min. The insoluble material was collected
byfiltration and dried over P 2O5 under reduced pressure
to give N-[4-hydroxy-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-l-4-aminomethylphenylalanine N-methyla-
mide hydrochloride (1.15 g, 94%) as colorless solid. To
a solution of the hydrochloride salt (1.15 g) in DMF
(20 mL), ethyl acetimidate hydrochloride (547 mg,
4.43 mmol) and Et3N (903 mL, 6.50 mmol) were added
and stirred at 0 ꢂC for 1 h. The reaction mixture was
adjusted to pH 2 byadding 1 N aq HCl, which was
charged on DIAION HP-20 (Mitsubishi Chemicals;
216 mL) and purified (eluent; 10–80% aq MeOH) to
give 16a (1.11 g, 65%) as a colorless amorphous powder
upon freeze-drying: [a]D25 ꢀ16.5ꢂ (c=1.0, MeOH). 1H
NMR (MeOH-d4) d ppm; 0.76 (3H, d, J=6.3 Hz), 0.80
(3H, d, J=6.2 Hz), 0.94–1.16 (1H, m), 1.29–1.79 (6H,
m), 2.17 (3H, s), 2.26–2.77 (7H, m), 2.89–3.10 (2H, m),
4.38 (2H, s), 4.49–4.59 (1H, m), 6.84–7.54 (9H, m).
HRFABMS: calcd for C30H43N4O4 523.3284, found
523.3301.
N,N0-bis(benzyloxycarbonyl)carboxamidine
(470 mg,
1.16 mmol) and Et3N (162 mL, 1.16 mmol) were added
under stirring in an ice bath and stirred at room tem-
perature overnight. The reaction mixture was con-
centrated under reduced pressure followed by
precipitating from Et2O to give 16d as a colorless solid
(650 mg, 86%): mp 141–145 ꢂC, [a]2D5 ꢀ14.0ꢂ (c=0.71,
DMF). 1H NMR (DMSO-d6) d ppm; 0.74 (3H, d,
J=6.7 Hz), 0.78 (3H, d, J=6.7 Hz), 0.91–1.02 (1H, m),
1.31–1.81 (6H, m), 2.14–2.30 (1H, m), 2.30–2.80 (m),
2.85–3.12 (2H, m), 4.32–4.71 (3H, m), 5.03 (2H, s), 5.19
(2H, s), 6.82–7.64 (20H, m), 7.80–7.95 (1H, m), 8.42–
8.70 (2H, m). HRFABMS: calcd for C45H54N5O8
792.3972, found 792.3996.
Typical procedure for the preparation of compound 17.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-tyrosine N-methylamide (17a). To the
compound 15a (8.92 g, 17.0 mmol), ice-cooled 95% aq
TFA (30 mL) was added and stirred at 5 ꢂC for 3 h, then
evaporated in vacuo. The residue was precipitated from
Et2O followed byfiltration to give carboxlyic acid
intermediate (7.33 g, 92%) as colorless solid. To a mix-
ture of this intermediate (4.68 g, 10 mmol), O-benzylhy-
droxylamine hydrochloride (3.20 g, 20 mmol), HOBt
(2.70 g, 20 mmol) and DMF (70 mL), EDC (2.11 g,
11 mmol) was added under stirring at ꢀ15 ꢂC followed
byadding Et N (2.80 mL, 20 mmol). The mixture was
3
stirred at ꢀ15 ꢂC for 1 h and further at room tem-
perature overnight. The reaction mixture was diluted
with EtOAc (210 mL) and washed successivelywith
brine, 1N aq HCl, satd aq NaHCO3 and brine (twice
for each), then evaporated in vacuo to leave a color-
less residue, which was precipitated from Et2O to
give 17a (3.73 g, 65%) as a colorless solid: mp 217–
220 ꢂC, [a]2D5 +5.6ꢂ (c=1.0, DMF). H NMR (MeOH-
1
d4) d ppm; 0.76 (3H, d, J=6.3 Hz), 0.79 (3H, d,
J=6.4 Hz), 0.84–1.03 (1H, m), 1.47–1.74 (6H, m),
2.01–2.12 (1H, m), 2.28–2.70 (3H, m), 2.71 (3H, s),
2.80–2.93 (1H, m), 2.93–3.08 (1H, m), 4.60 (1H, m),
4.84 (2H, s), 6.69–6.97 (4H, m), 7.00–7.52 (10H, m).
HRFABMS calcd for C34H44N3O5: 574.3281, found:
574.3307.
N-[4-Hydroxy-2(R)-isobutyl-3(S)-phenylpropylsuccinyl]-
L-40-propionimidoyliminomethylphenylalanine N-methyl-
amide hydrochloride (16b). Yield; 59% overall yield
from 15d: [a]D25 ꢀ14.1ꢂ (c=0.2, MeOH). 1H NMR
(MeOH-d4) d ppm; 0.89 (6H, d, J=6.6 Hz), 1.02–1.84
(10H, m), 2.26–2.75 (9H, m), 2.95 (2H, d, J=5.6 Hz),

2578
T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpropyl-
succinyl]-L-40-sulfophenylalanine N-methylamide, monoso-
dium salt (17b). Yield; 46%. 1H NMR (MeOH-d4) d
ppm; 0.76–0.98 (6H, m), 1.01–1.19 (1H, m), 1.48–1.86
(6H, m), 2.08–2.20 (1H, m), 2.24–2.67 (3H, m), 2.71
(3H, s), 2.96–3.12 (2H, m), 4.48–4.66 (1H, m), 5.04 (2H,
s), 7.06–7.98 (14H, m). FAB-MS: m/z 660 [M+Na]+,
638 [M+1]+. HRFABMS: calcd for C34H43N3NaO7S
660.2719, found 660.2731.
0.89 (3H, d, J=6.6 Hz), 1.04–1.75 (10H, m), 2.20–2.81
(9H, m), 2.84–2.99 (2H, m), 4.40 (2H, s), 4.56–4.75 (1H,
m), 5.02 (2H, s), 6.92–7.65 (14H, m). HRFABMS: calcd
for C38H52N5O4 642.4019, found 642.3997.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl] - L - 40 - benzimidoyliminomethylphenylalanine
N-methylamide hydrochloride (17i). Yield; 92%. 1H
NMR (MeOH-d4) d ppm; 0.84 (3H, d, J=6.4 Hz), 0.86
(3H, d, J=6.7 Hz), 0.96–1.80 (7H, m), 2.01–2.14 (1H,
m), 2.42–2.66 (3H, m), 2.68 (3H, s), 2.83–2.98 (2H, m),
4.40 (2H, s), 4.43–4.54 (1H, m), 5.01 (2H, s), 6.97–8.00
(19H, m). HRFABMS: calcd for C42H52N5O4 690.4019,
found 690.4028.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-nitrophenylalanine N-methylamide (17c).
1
Yield; 63%. H NMR (MeOH-d4) d ppm; 0.78 (6H, d,
J=6.5 Hz), 0.92–1.12 (1H, m), 1.38–1.72 (6H, m), 2.01–
2.14 (1H, m), 2.50–2.78 (6H, m), 3.01–3.22 (2H, m),
4.65–4.78 (1H, m), 5.08 (2H, s), 7.26–7.70 (12H, m),
7.92–8.13 (2H, m). HRFABMS: calcd for C34H43N4O6
603.3183, found 603.3191.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-[N,N0-bis(benzyloxycarbonyl)guanidino-
methyl]phenylalanine N-methylamide (17j). Yield; 76%.
1H NMR (DMSO-d6) d ppm; 0.59–0.79 (6H, m), 0.90–
1.12 (1H, m), 1.54–1.81 (6H, m), 1.85–2.03 (1H, m),
2.40–2.76 (m), 2.81–3.03 (2H, m), 4.25–4.63 (3H, m),
4.76 (2H, s), 5.02 (2H, s), 5.19 (2H, s), 6.81–7.56 (26H,
m), 7.81–8.63 (3H, m). HRFABMS: calcd for
C52H61N6O8 897.4551, found 897.4564.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpropyl-
succinyl]-L-40-cyanophenylalanine N-methylamide (17d).
1
Yield; 77%. H NMR (MeOH-d4) d ppm; 0.79 (3H, d,
J=6.4 Hz), 0.81 (3H, d, J=6.4 Hz), 1.01–1.22 (1H, m),
1.36–1.80 (6H, m), 2.08–2.18 (1H, m), 2.20–2.64 (6H,
m), 2.71 (3H, s), 3.05–3.22 (2H, m), 4.67–4.85 (m), 7.20–
7.81 (14H, m). HRFABMS: calcd for C35H43N4O4
583.3284, found 583.3277.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-O-sulfo-L-tyrosine N-methylamide sodium
salt (18a). To a suspension of 17a (1.96 g, 3.30 mmol)
in DMF (20 mL), sulfur trioxide pyridine complex
(1.60 g, 9.90 mmol) was added and stirred at room tem-
perature for 1.5 h. The reaction mixture was diluted
with 1N aq NaHCO3 (80 mL) then charged on reverse-
phase column chromatographyand purified (eluent; 0–
50% aq MeOH) to give N-[4-(N-benzyloxyamino)-2(R)-
isobutyl-3(S)-phenylpropylsuccinyl]-O-sulfo-l-tyrosine-
N-methylamide sodium salt (1.85 g, 83%) as a colorless
powder. The obtained compound (1.60 g, 2.37 mmol)
was dissolved in MeOH (30 mL) and hydrogenated in
the presence of 5% Pd-C (50% wet, 200 mg) at room
temperature for 1 h. After the catalyst was filtered off,
MeOH was removed byevaporation. The residue was
dissolved in water (20 mL) and lyophilized to give 18a
(1.28 g, 92%) as a colorless amorphous powder: [a]2D5
ꢀ41ꢂ (c=1.0, MeOH). 1H NMR (MeOH-d4) d ppm;
0.74 (3H, d, J=6.4 Hz), 0.77 (3H, d, J=6.4 Hz), 0.98–
1.12 (1H, m), 1.34–1.78 (6H, m), 2.00–2.14 (1H, m),
2.38–2.72 (3H, m), 2.74 (3H, s), 2.84–2.93 (1H, m),
2.93–3.14 (1H, m), 4.44–4.56 (1H, m), 7.14–7.32 (9H,
m). FABMS: m/z 586 [M+Na]+, 564 [M+1]+. Anal.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-[N,N0-bis(benzyloxycarbonyl)guanidino]-
phenylalanine N-methylamide (17e). Yield; 76%. 1H
NMR (DMSO-d6) d ppm; 0.63–0.89 (6H, m), 1.01–1.20
(1H, m), 1.42–1.78 (6H, m), 2.10–2.24 (1H, m), 2.30–
2.58 (m), 2.64 (3H, s), 2.91–3.16 (2H, m), 4.40–4.56 (1H,
m), 4.81 (2H, s), 5.12 (2H, s), 5.18 (2H, s), 6.96–7.88
(24H, m). HRFABMS: calcd for C51H59N6O8 883.4394,
found 883.4420.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40 -[N-(benzyloxycarbonyl)amidino]phenyl-
alanine N-methylamide (17f). Yield; 38%. 1H NMR
(CDCl3) d ppm; 0.84 (3H, d, J=6.4 Hz), 0.86 (3H, d,
J=6.4 Hz), 1.10–1.21 (1H, m), 1.24–1.75 (6H, m), 2.01–
2.14 (1H, m), 2.26–2.55 (3H, m), 2.73 (3H, d,
J=4.7 Hz), 3.01–3.20 (2H, m), 4.56–4.72 (1H, m), 4.86
(2H, s), 5.02 (2H, s), 5.73–5.96 (2H, m), 6.21–6.37 (1H,
m), 6.48–6.65 (1H, m), 6.95–7.80 (20H, m).
HRFABMS: calcd for C43H52N5O6 734.3918, found
734.3924.
.
(C27H36N3NaO8S 0.4H2O) C, H, N.
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl] - L - 40 - acetimidoyliminomethylphenylalanine
N-methylamide hydrochloride (17g). Yield; 76%. 1H
NMR (MeOH-d4) d ppm; 0.78–0.96 (6H, m), 0.96–1.13
(1H, m), 1.54–1.80 (5H, m), 1.89 (3H, s), 2.03–2.18 (4H,
m), 2.54–2.83 (6H, m), 2.98–3.14 (2H, m), 4.36 (2H, s),
4.42–4.61 (1H, m), 6.93–7.69 (14H, m). HRFABMS:
calcd for C37H50N5O4 628.3863, found 628.3821.
Preparation of compound 18b–j. Removal of O-benzyl
group for compounds 17b–j were performed bythe cat-
alytic hydrogenation method utilizing 5% Pd-C as that
described for the preparation of 18a.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-sulfophenylalanine N-methylamide so-
dium salt (18b). Yield; 43%. [a]D25 ꢀ3.73ꢂ (c=1.0,
MeOH). 1H NMR (MeOH-d4) d ppm; 0.86 (3H, d,
J=6.4 Hz), 0.90 (3H, d, J=6.4 Hz), 0.98–1.08 (1H, m),
1.12–1.48 (6H, m), 1.93–2.07 (1H, m), 2.23–2.51 (3H, m),
2.70 (3H, s), 3.04–3.22 (2H, m), 4.68 (1H, dd, J=5.3,
N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl] - L - 40 - propionimidoyliminomethylphenylala-
nine N-methylamide hydrochloride (17h). Yield; 76%.
1H NMR (MeOH-d4) d ppm; 0.87 (3H, d, J=6.4 Hz),

T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
2579
9.2 Hz), 7.14–7.86 (9H, m). FABMS: m/z 570 [M+Na]+,
ppm; 0.82 (3H, d, J=6.4 Hz), 0.87 (3H, d, J=6.6 Hz),
0.95–1.21 (4H, m), 1.24–1.58 (6H, m), 1.88 (3H, s),
1.96–2.07 (1H, m), 2.24–2.76 (8H, m), 2.89 (1H, dd,
J=10.8, 14.0 Hz), 3.10 (1H, dd, J=4.6, 14.0 Hz), 4.38
(2H, s), 4.71 (1H, dd, J=4.6, 10.8 Hz), 6.91–7.55 (9H,
m). FABMS: m/z 552 [M+1]+. Anal. (C33H49N5O6) C,
H, N.
+
.
548 [M+1] . Anal. (C27H36N3NaO7S 0.3H2O) C, H, N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpropyl-
succinyl]-L-40-aminophenylalanine N-methylamide monoa-
1
cetate (18c). Yield; 82%. H NMR (MeOH-d4) d ppm;
0.83 (3H, d, J=6.3 Hz), 0.85 (3H, d, J=6.4 Hz), 0.96–
1.10 (1H, m), 1.14–1.47 (6H, m), 1.90–2.06 (4H, m),
2.22–2.54 (3H, m), 2.71 (3H, s), 2.83 (1H, dd, J=10.2,
14.0 Hz), 3.12 (1H, dd, J=4.4, 14.0 Hz), 4.51 (1H, dd,
J=4.4, 10.2 Hz), 7.15–7.32 (7H, m), 7.34–7.42 (2H, m).
FABMS: m/z 483 [M+1]+. Anal. (C29H42N4O6) C, H,
N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl] - L - 40 - benzimidoyliminomethylphenylalanine
N-methylamide monoacetate (18i). Yield; 79%. [a]2D5
ꢀ4.3ꢂ (c=1.1, MeOH). H NMR (MeOH-d4) d ppm;
1
0.83 (3H, d, J=6.4 Hz), 0.86 (3H, d, J=6.6 Hz), 0.94–
1.07 (1H, m), 1.22–1.59 (6H, m), 1.93 (3H, s), 2.00–2.14
(1H, m), 2.37–2.62 (3H, m), 2.70 (3H, s), 2.90 (1H, dd,
J=10.7, 14.0 Hz), 3.12 (1H, dd, J=5.3, 14.0 Hz), 4.41
(2H, s), 4.70 (1H, dd, J=5.3, 10.7 Hz), 6.92–7.74 (14H,
m). FABMS: m/z 600 [M+1]+. Anal. (C37H49N5O6) C,
H, N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-aminomethylphenylalanine N-methyla-
mide monoacetate (18d). Yield; 80%. 1H NMR
(MeOH-d4) d ppm; 0.82 (3H, d, J=6.2 Hz), 0.86 (3H, d,
J=6.4 Hz), 0.96–1.09 (1H, m), 1.13–1.50 (6H, m), 1.90–
2.08 (4H, m), 2.20–2.56 (3H, m), 2.69 (3H, s), 2.81 (1H,
dd, J=9.6, 13.9 Hz), 3.10 (1H, dd, J=4.4, 13.9 Hz), 3.99
(2H, s), 4.52 (1H, dd, J=4.4, 9.6 Hz), 7.12–7.30 (7H,
m), 7.33–7.45 (2H, m). FABMS: m/z 497 [M+1]+.
Anal. (C30H44N4O6) C, H, N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-guanidinomethylphenylalanine N-methyl-
amide monoacetate (18j). Yield; 90%. [a]D25 ꢀ9.3ꢂ
(c=0.97, DMF). 1H NMR (MeOH-d4) d ppm; 0.81
(3H, d, J=6.4 Hz), 0.87 (3H, d, J=6.6 Hz), 0.98–1.12
(1H, m), 1.21–1.55 (6H, m), 1.89 (3H, s), 1.96–2.14 (1H,
m), 2.24–2.59 (3H, m), 2.69 (3H, s), 2.90 (1H, dd,
J=9.8, 13.8 Hz), 3.11 (1H, dd, J=4.7, 13.8 Hz), 4.36
(2H, s), 4.68 (1H, dd, J=4.7, 9.8 Hz), 6.97–7.59 (9H,
m). FABMS: m/z 539 [M+1]+. Anal. (C31H46N6O6) C,
H, N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-guanidinophenylalanine N-methylamide
monoacetate (18e). Yield; 93%. [a]D25 ꢀ10.2ꢂ (c=1.0,
MeOH). 1H NMR (MeOH-d4) d ppm; 0.83 (3H, d,
J=6.7 Hz), 0.88 (3H, d, J=6.4 Hz), 0.92–1.19 (1H, m),
1.22–1.58 (6H, m), 1.89 (3H, s), 1.93–2.08 (1H, m), 2.24–
2.56 (3H, m), 2.71 (3H, s), 2.83 (1H, dd, J=10.1, 14.0 Hz),
3.12 (1H, dd, J=4.8, 14.0 Hz), 4.68 (1H, dd, J=4.8,
10.1Hz), 7.13–7.30 (7H, m), 7.32–7.44 (2H, m). FABMS:
m/z 525 [M+1]+. Anal. (C30H44N6O6) C, H, N.
MMP inhibition assay
Inhibitoryactivityof a synthetic compound to MMP-1
and MMP-3 was determined bythe method of Nagai et
al.32 using a fluorescent isothiocyanate (FITC)-labeled
type I collagen (from bovine achilles tendon, Sigma)
and that of Twining33 using a FITC-labeled casein
(Sigma, from bovine milk) as substrate, respectively.
ProMMP-1 and proMMP-3 were purified from the
media of human skin fibroblast NB1RGB cells (RIKEN
Cell Bank, RCB0222) stimulated with IL-1a (Genzyme
Corp. MPLS, MN),34 and their precursor enzymes were
activated with 1 mM 4-aminophenylmercuric acetate
(APMA, Nacalai Tesque, Inc., Kyoto, Japan) at 37 ꢂC
for 2 h.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl]-L-40-amidinophenylalanine N-methylamide
monoacetate (18f). Yield; 96%. [a]D25 ꢀ7.04ꢂ (c=1.0,
MeOH). 1H NMR (MeOH-d4) d ppm; 0.82 (3H, d,
J=6.5 Hz), 0.86 (3H, d, J=6.4 Hz), 0.92–1.10 (1H, m),
1.33–1.70 (6H, m), 1.91 (3H, s), 1.97–2.11 (1H, m),
2.38–2.59 (3H, m), 2.68 (3H, s), 2.88 (1H, dd, J=11.0,
14.2 Hz), 3.13 (1H, dd, J=4.5, 14.2 Hz), 4.74 (1H, dd,
J=4.5, 11.0 Hz), 6.96–7.85 (9H, m). FABMS: m/z 510
[M+1]+. Anal. (C30H43N5O6) C, H, N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl] - L - 40 - acetimidoyliminomethylphenylalanine
N-methylamide monoacetate (18g). Yield; 91%. [a]2D5
MMP-7, MMP-13 and MT1-MMP inhibitoryactivities
were measured bymodifying the method of Yamamoto et
al.35 using a fluorogenic substrate, (7-methoxycoumarin-
4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitropheny]-l-2,3-
diaminopropionyl)-Ala-Arg-NH2 (Peptide Institute,
Inc., Osaka, Japan).36 Recombinant human MMP-7
and MMP-13 were purchased from Oriental Yeast Co.,
Ltd (Shiga, Japan) and Genzyme Corp. (MPLS, MN),
respectively. Recombinant human MT1-MMP lacking
the transmembrane domain (Ala536-Val582) was expres-
sed and purified from Escherichia coli.34 MMP-13, but not
MMP-7 and MT1-MMP, was activated byincubation
with 1 mM APMA for 2 h at 37 ꢂC. One unit of MMP-
7,-13 and MT1-MMP was defined as the amount of the
enzyme required to degrade 1 pmol of the substrate in
ꢀ8.9ꢂ (c=1.0, MeOH). H NMR (MeOH-d4) d ppm;
1
0.81 (3H, d, J=6.4 Hz), 0.85 (3H, d, J=6.4 Hz), 0.94–
1.13 (1H, m), 1.16–1.54 (6H, m), 1.90 (3H, s), 1.97–2.11
(1H, m), 2.24–2.50 (6H, m), 2.71 (3H, s), 2.90 (1H, dd,
J=11.0, 13.9 Hz), 3.12 (1H, dd, J=4.6, 13.9 Hz), 4.36
(2H, s), 4.77 (1H, dd, J=4.6, 11.0 Hz), 6.89–7.62 (9H,
m). FABMS: m/z 538 [M+1]+. Anal. (C32H47N5O6) C,
H, N.
N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenylpro-
pylsuccinyl] - L - 40 - propionimidoyliminomethylphenylala-
nine N-methylamide monoacetate (18h). Yield; 90%.
[a]D25 ꢀ9.7ꢂ (c=1.0, MeOH). 1H NMR (MeOH-d4) d

2580
T. Fujisawa et al. / Bioorg. Med. Chem. 10 (2002) 2569–2581
1 min at 37 ꢂC. A mixture of enzyme solution (one unit/
40 mL) and inhibitor solution (10 mL) was incubated
with 50 mL of substrate solution in 50 mM Tris–HCl
(pH 7.5) buffer containing 0.15 M NaCl, 10 mM CaCl2,
0.05% Brij-35 and 0.02% NaN3. Each substrate con-
centration in MMP-7, MMP-13 and MT1-MMP assay
were adjusted to 10, 3 and 5 mM, respectively. Enzyme
reaction was performed at 37 ꢂC, for 0.5 h in MMP-7
and MT1-MMP assayand for 2 h in MMP-13 assay,
and quenched with 100 mL of 3% aqueous acetic acid.
The emergence of fluorescence intensity(ex 325 nm, em
405 nm) was measured using Biolumin 960 (Molecular
Dynamics Pth. Ltd., Key East, VIC).
pound in plasma was separated bycentrifugation
through a 30,000-mol wt cut-off filter (model YMT-30;
Amicon Corp., Beverly, MA) for 3 min at 1,500 g.39 The
plasma filtrate was seriallydiluted and tested for inhi-
bition of rabbit MMP-3 using a fluorogenic substrate, (7
-methoxycoumarin-4-yl)acetyl-Arg-Pro-Lys-Pro-Val-Glu-
Nva-Trp-Arg-Ne-(2,4-dinitrophenyl)-Lys-NH2 (Peptide
Institute, Inc., Osaka, Japan).40 The filtrate dilution
necessaryfor 50% inhibition of rabbit MMP-3 activity
was multiplied bythe known IC concentration to cal-
50
culate initial plasma drug concentrations (The IC50
values of compound 18b, 18e, Marimastat and Trocade
against rabbit MMP-3 were 8, 23, 34 and 353 nM,
respectively). Control experiments showed no effect of
filtrate alone on the MMP-3 assayand <10% binding
of the synthetic compounds to plasma filtrate or the fil-
ter apparatus. It should be emphasized that this method
does not directlyquantifythe amount of parent com-
pound.
MMP-2 and MMP-9 inhibitoryactivities bythe synthetic
compounds were measured using gelatin zymography.37
Briefly, the media of HT-1080 cells containing MMP-2
and MMP-9 were applied to nonreduced SDS-PAGE
using a 7.5% gel containing 0.1% gelatin. After electro-
phoresis at 4 ꢂC, the gels were soaked in 2.5% Triton
X-100 solution with gentlyshaking for 1 h at room tem-
perature. In the presence or absence of a test compound,
the gels were incubated in reaction buffer (50 mM Tris–
HCl, 5 mM CaCl2, 1 mM ZnCl2, 0.05% NaN3, pH 7.5)
for 18 h at 37 ꢂC and were stained with 0.1% Coomassie
Brilliant Blue. A gelatinolytic activity was visualized as
transparent bands on the blue background and was
quantified byNIH image software (Ver. 1.6).
Adjuvant arthritis
Male Lewis rats (Charles River Laboratories, Japan)
weighing about 240 g were used. Adjuvant arthritis was
induced in the rats byan intradermal injection of 0.6 mg
M. tuberculosis (H37RA, Difco Laboratories, Detroit,
MI) in 0.05 mL liquid paraffin into the base of the tail.40
Hindpaw swelling was measured plethymographically
bywater displacement using a volume meter (TK-101;
Muromachi Kikai Co., Ltd., Tokyo, Japan) after adju-
vant injection. Area under the hindpaw swelling time
curve (AUC) was calculated bythe linear trapezoidal
method. At the end of the experiment (on day20 after
adjuvant injection), hindpaws were fixed in 10% buf-
fered formalin and those radiographs were taken in Fuji
Biomedix Co., Ltd. (Yamanashi, Japan). The scoring of
TNFꢁ bioassay
An inhibitoryeffect of a test compound on lipopoly-
saccharide (LPS, from E. coli. 0111:B4, DIFCO,
Detroit, MI)-induced TNFa release was measured in
human acute monocytic leukemia THP-1 cells (ATCC
No. TIB202). In the presence of 1 mg/mL LPS and in the
presence or absence of a seriallydiluted test compound,
THP-1 cells were cultured in 24-well flat-bottomed trays
(Falcon 3047, Becton Dickinson, Sanjose, CA) at 2ꢃ106
cells/well (in 1-mL volume) in a humidified atmosphere
at 37 ꢂC with 5% CO2. After 6 h incubation, TNFa
levels in the supernatants were measured using mouse
fibrosarcoma WEHI-164 cells (ATCC No. CRL1751)
lytic assay.38 WEHI-164 cells were cultured in 96 well
flat-bottomed trays (Falcon 3075, Becton Dickinson,
Sanjose, CA) at 20,000 cells/well (in 0.2-mL volume) in
the presence of 1 mg/mL of actionomycin d-mannitol
(Sigma Chemical Co., St Louis, MO) and a serially
diluted test sample in a humidified atmosphere at 37 ꢂC
with 5% CO2. Recombinant human TNFa (specific
activity1 ꢃ107 U/mg, Genzyme Corp., MPLS, MN) was
used as an internal standard. After 20 h incubation,
20 mL WST-1 (3.3 mg/mL, DOJINDO, Kumamoto,
Japan) was added to the wells and incubated for 4 h,
and then optical densityof culture assessed at 450 nm.
bone degradation in the radiographs was referred to the
41
method byKashima.
The hindpaw joint was divided
to four area of the distal tibia, talus, calcaneus and
metatarsus bones. Each area were graded bythe para-
meter of bone demineralization, bone erosion and joint
space narrowing in a blind fashion from 0 to 4 (0=no
change, 1=slight, 2=mild, 3=moderate, 4=marked
lesion).
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