Design and synthesis of dual inhibitors for matrix metalloproteinase and cathepsin

Article abstract of DOI:10.1016/S0960-894X(01)00755-7

The first example of dual inhibitors for matrix metalloproteinase (MMP) and cathepsin is described. An appropriate alignment of peptide-parts and two different specific functional groups in one molecule led to the discovery of a potent dual inhibitor (3a)

Full text of DOI:10.1016/S0960-894X(01)00755-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 375–378
Design and Synthesis of Dual Inhibitors for Matrix
Metalloproteinase and Cathepsin
Minoru Yamamoto,a Shoji Ikeda,a,* Hirosato Kondoa and Shintaro Inoueb
aChemistry Group R&D Laboratories, Nippon Organon K.K., 1-5-90 Tomobuchi-Cho, Miyakojima-Ku, Osaka, Japan
bBasic Science Laboratory, Kanebo Ltd., 5-3-28 Kotobuki-Cho, Odawara, Kanagawa, Japan
Received 10 September 2001; accepted 9November 2001
Abstract—The first example of dual inhibitors for matrix metalloproteinase (MMP) and cathepsin is described. An appropriate
alignment of peptide-parts and two different specific functional groups in one molecule led to the discovery of a potent dual inhi-
bitor (3a). # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cathepsin would be useful for the treatment of these
diseases.
Proteinases are classified into four types (metalloprotein-
ase, cysteine proteinase, serine proteinase and aspartic
proteinase) by differences in hydrolysis mechanisms of
peptide bonds.1 A large number of proteinase inhibitors
categorized in peptide-type and non-peptide-type inhi-
bitors have been investigated by many scientists. For
the design of peptide-type inhibitors, it is important to
align appropriately peptide-parts and a specific func-
tional group for respective classes.1,2 Peptide-parts
represent a frame which is mimicked from amino acid
sequence of a substrate cleaved by proteinases. On the
other hand, a specific functional group represents a
group which interacts with the active site of an enzyme
to accelerate hydrolysis of a peptide bond. There are
some general functional groups used for the design of
proteinase inhibitors. For example, in the case of
metalloproteinase inhibitors, the introduction of a
functional group chelating metal in an enzyme, such as
hydroxamic acid, carboxylic acid or phosphonic acid
group, is essential to exhibit potent activity.3 Concern-
ing cysteine proteinase inhibitors, a functional group
interacting with thiol group in an enzyme, such as alde-
hyde or ketone, is needed.4,5
Compounds which have inhibitory activity against two
proteinases belonging to the same class are known. For
instance dual inhibitors against both angiotensine con-
verting enzyme (ACE) and neutral endpeptidase (NEP),
which belong to metalloproteinase, have already been
reported.7 However, dual inhibitors for two proteinases
belonging to different classes are not known yet because
it is very difficult to align appropriately peptide-parts
and two specific functional groups for each class.
This paper reports the synthesis and biological activity
of dual inhibitors for two proteinases belonging to dif-
ferent classes, which are matrix metalloproteinase
(MMP-1) belonging to metalloproteinase and cathepsin
L belonging to cysteine proteinase.
Design for Dual Inhibitor
A representative MMP inhibitor8 and cathepsin inhi-
bitor5 are shown in Figure 1.
It has been reported that the activation of MMPs and
cathepsins is associated with cartilage and bone degra-
dation,6 such as osteoporosis and rheumatoid arthritis.
Therefore a potential dual inhibitor for MMP and
MMP inhibitor 1 has hydroxamic acid as a functional
group chelating zinc in an active site of an enzyme. The
P10 and P20 moieties were optimized from amino acid
sequence of the substrate cleaved by MMP, and the
optimized ones are Leu and Phe, respectively.1,2,9 On
the other hand, aldehyde group is generally used as a
functional group for cathepsin inhibitors because the
aldehyde group interacts with thiol group in the active
*Corresponding author. Tel.: +81-6-6921-8978; fax: +81-6-6921-
9080; e-mail: ikeda.shoji@organon.co.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00755-7

376
M. Yamamoto et al. / Bioorg. Med. Chem. Lett. 12 (2002) 375–378
site. The modification of peptide parts of this inhibitor
was mimicked from the sequence of the left-hand parts
at the cleaved site of the substrate.
position, respectively. We can see that there is a close
similarity between P20 and P30 substituents in MMP
inhibitor 1 and P2 and P1 substituents in cathepsin
inhibitor 2, even though these proteinases belong to
different classes. Therefore we focused on these peptide-
parts and designed dual inhibitor 3a for MMP and
cathepsin by introducing respective specific functional
groups, hydroxamic acid group for MMP and aldehyde
group for cathepsin, into these peptide-parts.
MMP inhibitor 1 contains benzyl group at P20 position
and methyl group at P30 position. Then cathepsin inhi-
bitor 2 also has these groups at P2 position and P1
In addition, we investigated the profile of inhibitory
activity against each enzyme by the modification of
peptide-parts.
Chemistry
Compound 3 was synthesized as shown in Scheme 1.
N,O-Dimethylhydroxylamine 4 was condensed with the
corresponding amino acid protected by benzyloxy-car-
bonyl group (Cbz) to give compound 5. The deprotec-
tion of benzyloxycarbonyl group followed by coupling
with Cbz-amino acid provided compound 6. After
deprotection of benzyloxycarbonyl group of 6, the
resulting amine was condensed with carboxylic acid 910
in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDC) to give benzyloxy-
amide 7. The reduction of 7 with lithium aluminum
hydride provided aldehyde 8, and then the benzyl group
of 8 was deprotected to give compound 3.
Biological Data
The biological data11 of compound 3a are shown in Table
1. As expected, compound 3a exhibited potent inhibi-
tory activity against both MMP-1 and cathepsin L.
Compound 10 having acetal in place of aldehyde group
showed potent inhibition against MMP-1 as well as 3a,
but no inhibition for cathepsin L. This result indicates
Figure 1.
Scheme 1. (a) Cbz-amino acid, EDC, HOBt (5a,d 69%, 5b 96%, 5c 72%); (b) (i) H2/Pd–C; (ii) Cbz-amino acid, EDC,HOBt (6a 71%, 6b 43%, 6c
49%, 6d 84%); (c) (i) H2/Pd–C; (ii) 9, EDC (7a 56%, 7b 78%, 7c 40%, 7d 38%); (d) LiAlH4; (e) H2/Pd–C (3a 39%, 3b 14%, 3c 30%, 3d 27% from
7).

M. Yamamoto et al. / Bioorg. Med. Chem. Lett. 12 (2002) 375–378
377
that acetal did not interact with thiol group in an active
site of cysteine proteinase.
cathepsin L as well as compound 3a. Leucine at the P2
site is also favorable for cathepsin L inhibition. This
result is supported by the S2 pocket of cathepsin L
being considered as a hydrophobic and moderately sized
cavity.13 These results indicate that the appropriate
combination of peptide-parts would be also important
for the design of dual inhibitors of MMP-1 and cathep-
sin L.
Next, we investigated an inhibitory effect on peptide-
parts. As shown in Table 2, the conversion of peptide-
parts (at P20–P30 position for MMP-1, at P1–P2 position
for cathepsin L) gave interesting results. Compound 3b
exhibited potent inhibition against cathepsin L as well
as compound 3a, but less potency against MMP-1. A
small substituent seems to be favorable at P30 position
(R2 substituent) for MMP-1. Actually, it has been
reported that the introduction of Leu at this position
decreased MMP-1 inhibition.12 On the other hand,
compound 3c exhibited potent inhibition against MMP-
1 as well as compound 3a, but less potency against
cathepsin L. The existence of a substituent at P1 posi-
tion (R2 substituent) seems to be essential for the inhi-
bition of cathepsin L. In fact, Yasuma reported that
hydrophobic and bulky groups at P1 position were
favorable for cathepsin L inhibition.13 Compound 3d
showed potent inhibition against both MMP-1 and
The inhibitory activity of these dual inhibitors to other
proteinases (e.g., serine proteinase and aspartic protein-
ase) is not examined, but these inhibitors are probably
considered not to show inhibitory activity to serine
proteinase and aspartic proteinase because the basic
structure of these dual inhibitors is based on the struc-
ture of a selective matrix metalloproteinase inhibitor.
In conclusion, we succeeded in the design and synthesis
of dual inhibitors for MMP and cathepsin. This paper
exhibits the first example of dual inhibitors for two
proteinases respectively belonging to different classes.
We are in the process of investigating more potent dual
inhibitors. This design could be useful for other new dual
inhibitors. For example, a dual inhibitor, such as tumor
necrosis factor-alpha converting enzyme (TACE)14
belonging to metalloproteinase and interleukin-1 con-
verting enzyme (ICE)15 belonging to cysteine proteinase,
might be designed by a similar method.
Table 1. Inhibitory activity of compound 3a against MMP-1 and
cathepsin L
Acknowledgements
A
IC50 (nM)a
Cathepsin L
The authors are grateful to Dr. Yoshimasa Inoue and
Ms. Estuko Ishibushi, Biochemistry Group R&D
Laboratories, Nippon Organon K.K., for the biological
assay.
MMP-1
25
3a
10
15
29
>1000
References and Notes
1
2
3
>1000
>1000
3
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IC50 (nM)a
R1
R2
MMP-1
Cathepsin L
3a
3b
3c
3d
–CH3
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12
15
7
–H
4300
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–CH3
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aSee References and Notes for enzyme assay details.

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The assay for inhibitory activity of cathepsin L: Inhibitory
activities of cathepsin L, purchased from Cosmo Bio Co.
Ltd., were measured using Cbz-Phe-Arg-AMC (Cosmo Bio
Co. Ltd.) as a substrate. Cathepsin L (human kidney) and a
compound were incubated in 400 mM CH3COOH/NaOH
(pH 5.5) containing 4 mM EDTA 2Na at 30 ꢀC for 1 min. A
substrate was added, then incubated at 30 ꢀC for 10 min. The
incubation was terminated by the addition of 100 mM
CH3COOH/NaOH (pH 4.3) containing 100 mM mono-
chloroacetic acid. The fluorescence intensity (Ex 375 nm, Em
445 nm) was measured. The IC50 values were the average of
at least two determinations with a standard deviation of less
than ꢁ30%.