Discovery of selective phosphonamide-based inhibitors of tumor necrosis factor-α converting enzyme (TACE)

Article abstract of DOI:10.1016/S0960-894X(03)00292-0

A novel series of phosphonamide-based inhibitors of tumor necrosis factor-α converting enzyme (TACE) was discovered by structural modification of tetrahydroisoquinoline derivative 1b, which was extremely weak inhibitor of TACE. (S)-Isomer at the phosphorus atom (7b) displayed potent inhibition for TACE, while selectivity sparing MMP-1, -3, and -9.

Full text of DOI:10.1016/S0960-894X(03)00292-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2021–2024
Discovery of Selective Phosphonamide-Based Inhibitors of Tumor
Necrosis Factor-ꢀ Converting Enzyme (TACE)
Masaaki Sawa,a,* Kiriko Kurokawa,a Yoshimasa Inoue,b
Hirosato Kondoa,y and Kohichiro Yoshinoa
aDepartment of Chemistry, R&D Laboratories, Nippon Organon K.K., 1-5-90, Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016, Japan
bDepartment of Molecular Biology, Nippon Organon K.K., 1-5-90, Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016, Japan
Received 3 February 2003;accepted 17 March 2003
Abstract—A novel series of phosphonamide-based inhibitors of tumor necrosis factor-a converting enzyme (TACE) was discovered
by structural modification of tetrahydroisoquinoline derivative 1b, which was extremely weak inhibitor of TACE. (S)-Isomer at the
phosphorus atom (7b) displayed potent inhibition for TACE, while selectivity sparing MMP-1, -3, and -9.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
selective TACE inhibitors are desirable in the long-term
treatment of TNF-a mediated diseases. This paper will
disclose the discovery of a new series of selective TACE
inhibitors using a phosphonamide scaffold.
Tumor necrosis factor-a (TNF-a) is a pro-inflammatory
cytokine, and elevated TNF-a levels are implicated in a
number of pathologies such as rheumatoid arthritis,
multiple sclerosis, non-insulin dependent diabetes melli-
tus (NIDDM), and inflammatory bowel disease.1 TNF-a
exists in two forms, a 26-kDa membrane-bound form
(proTNF-a) and a 17-kDa soluble form processed by spe-
cific proteases. The enzyme responsible for the processing
of the proTNF-a is TNF-a converting enzyme (TACE),
which is a member of the ADAM familiy.2,3 Therefore,
small molecular weight inhibitors of TACE are considered
to be attractive targets in drug discovery research.1
Inhibitor Design
In the previous paper, only the (R)-isomer at the phos-
phorus atom (1a) was found to be a potent inhibitor of
TACE, collagenase-1 (MMP-1), stromelysin-1 (MMP-3)
and gelatinase B (MMP-9), while the (S)-isomer 1b was
almost inactive for those enzymes (Table 1).4a After
subsequent efforts, we have found that the introduction
of fluorine atoms into the ester moiety of the (S)-iso-
mers led to highly potent and selective inhibition against
MMP-1 (2b, Ki: 6.23 nM for MMP-1, selectivities for
MMP-3 and -9: >104- and 66-fold, respectively).6 This
unexpected appearance of the inhibitory activity of the
(S)-isomer could be explained by the switching of the
binding mode in the enzyme, as shown in Figure 2. We
believe that the different binding mode of this type of
compound would make the compound highly selective.
Therefore, we have extended this observation to cover
other types of metalloproteases, and chosen TACE as a
prototypical enzyme since the active site of TACE was
found to be very similar to those of MMPs.1,7 Initially,
we have tested a series of various ester derivatives of 1b,
however none of compounds showed inhibitory activity
against TACE in the nanomolar range. Based on mod-
eling studies using X-ray structure of TACE, it was
anticipated that the ester groups could not be oriented
We recently reported the discovery of a series of
phosphonamide-based metalloproteinases inhibitors
represented by structure 1a (Fig. 1).4 Although com-
pound 1a shows potent inhibitory activity against
TACE (Ki=7.15 nM), it is also potent inhibitor of
the matrix metalloproteinases (MMPs). It has been
reported that side effects were observed in the clinical
studies of broad-spectrum inhibitors of the MMPs,5
*Corresponding author at current address: Chemistry Research
Laboratories, Drug Research Division, Dainippon Pharmaceutical
Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan.
Tel.: +81-6-6337-5904;fax: +81-6-6338-7656;e-mail: masaaki-
sawa@dainippon-pharm.co.jp
yCurrent address: Manufacturing Technology R&D Laboratories,
Shionogi & Co., Ltd., 1-3 Kuise Terajima 2-chome, Amagasaki,
Hyogo 660-0813, Japan.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00292-0

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M. Sawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2021–2024
Scheme 1. Design of new phosphonamide derivatives.
Figure 1. Structures of phosphonamide-based MMP inhibitors.
available d-leucine benzyl ester 4 was coupled with
4-methoxyphenylchlorophosphonic acid ethyl ester4 in
the presence of N-methylmorpholine to give the phos-
phonamide 5 as a mixture of diastereomers (1:1) in 56%
yield. Deprotection of the benzyl group in 5 with Pd/C
(92% yield), followed by coupling with O-benzylhydroxyl-
amine afforded 6 in 38% yield. Deprotection of the benzyl
group in 6 with 10% Pd/C gave the diastereomerically pure
two hydroxamic acids, which were successfully separated
by HPLC purification (yield: 31% for 7a and 24% for 7b).
The stereochemistries of the compounds were assigned by
a characteristic signal pattern in 1H NMR, especially in an
aromatic region as reported previously.4,6
correctly for occupancy of the S10 pokect due to the
restriction of the bicyclic tetrahydroisoquinoline struc-
ture. We therefore considered that acyclic compounds
such as 3 to allow greater flexibility of the ester groups in
the compounds (Scheme 1). In this preliminary report,
we wish to communicate the synthesis and biological
data for a series of novel acyclic phosphonamide-based
inhibitors which exhibit selective inhibition of TACE.
Chemistry
Compounds discussed below were prepared using syn-
thetic routes shown in Schemes 2 and 3. Commercially
Alternatively, N-(tert-butoxycarbonyl)-d-glutamic acid
1-benzyl ester 8 was coupled with phenethylamine using
Table 1. In vitro profile of phosphonamide derivatives
Compd
R1
R2
Ki (nM)
TACE
MMP-1
MMP-3
MMP-9
1a (R)
1b (S)
Tetrahydroisoquinoline ring
Tetrahydroisoquinoline ring
7.15
(41% @1000)a
4.59
(26% @1000)a
5.20
(9% @1000)a
5.05
(18% @1000)a
7a (R)
7b (S)
H
H
H
H
5.06
76.4
27.6
100
196
69.8
165
(10% @1000)a
40.4
(17% @1000)a
7.29
(17% @1000)a
23.7
12a (R)
12b (S)
(27% @1000)a
(47% @1000)a
(24% @1000)a
a% inhibition at the concentration (nM).
Figure 2. Expected binding mode of the phosphonamide inhibitors in MMPs.

M. Sawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2021–2024
2023
Scheme 2. (a) 4-Methoxyphenylchlorophosphonic acid ethyl ester, N-methylmorpholine;(b) H 2, Pd/C;(c) O-benzylhydroxylamine hydrochloride,
WSC, HOBt;(d) H 2, Pd/C.
Scheme 3. (a) Phenethylamine, WSC, HOBt;(b) 4 N HCl in AcOEt;(c) 4-methoxyphenylchlorophosphonic acid ethyl ester, N-methylmorpholine;
(d) H2, Pd/C;(e) O-benzylhydroxylamine hydrochloride, WSC, HOBt;(f) H 2, Pd/C.
WSC to yield 9. Compound 9 was converted into the
hydroxamates 12a and 12b using standard conditions as
described above.
enzymes. (R)-Isomer 12a was also potent inhibitor of
TACE with Ki value of 27.6 nM. It is interesting to
notice that while inhibition of TACE was decreased, the
inhibition of MMPs was increased significantly com-
pared to the leucine derivative 7a. Contrary to our
expectation, the inhibitory activity of compound 12b
was slightly decreased against TACE (Ki=100 nM).
Interestingly, compound 12b was still selective against
TACE, although the significant enhancement of potency
for MMPs was observed in 12a. Modeling study of 7b
with the active site of TACE suggested that the flexible
Results and Discussion
We firstly prepared d-leucine derivatives 7a and 7b in
the anticipation of the hydrophobic interaction of the
isobutyl group with the unprimed subsites (S1/S2) of the
enzyme based on the modeling study.
The d-leucine derivatives 7a and 7b were tested in vitro
for their ability to inhibit TACE, collagenase-1 (MMP-
1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9)
(Table 1).8 (R)-Isomer 7a was potent inhibitor of TACE
(Ki=5.06 nM) and also moderate inhibitor of MMPs
(Ki=196, 69.8 and 165 nM for MMP-1, -3 and -9,
respectively). It is noteworthy that whereas the (S)-iso-
mer of the tetrahydroisoquinoline derivative 1b showed
no inhibition for TACE, the (S)-isomer of the d-leucine
derivative 7b exhibited inhibitory activity against TACE
(Ki=76.4 nM). Moreover this compound was extremely
weak inhibitor against MMP-1, -3, and -9.
We believed that an additional substituent capable of
interacting with S1/S2 site could provide an opportunity
to modify not only potency but also the selectivity.
Thus, d-gulutamate derivatives 12a and 12b were pre-
pared and tested for their ability to inhibit these
Figure 3. Computer model of 7b in TACE.

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M. Sawa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2021–2024
structure of 7b can adopt the switched binding mode as
in Figure 2c (Fig. 3).9
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Yamamoto, M.;Miyasaka, T.;Ito, Y.;Hirayama, R.;Inoue,
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jima, F.;Nakada, Y.;Yokota, K.;Inoue, Y.;Kondo, H.;
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In summary, we have discovered a novel series of phos-
phonamide-based TACE inhibitors. The (S)-form of the
d-leucine derivative 7b showed potent inhibitory activity
against TACE with a highly selective profile. The dif-
ferent binding mode of this type of compound is likely
to enhance selectivity for TACE. This study reveals the
potential of the phosphonamide derivatives as a new
type of MP inhibitor, and provides a novel concept for
the design of selective inhibitors.
5. (a) Nemunaitis, J.;Poole, C.;Primrose, J.;Rosemurgy, A.;
Malfetano, J.;Brown, P.;Berrington, A.;Cornish, A.;Lynch,
K.;Rasmussen, H.;Kerr, D.;Cox, D.;Millar, A. Clin. Cancer
Res. 1998, 4, 1101. (b) Heath, E. I.;Grochow, L. B. Drugs
2000, 59, 1043.
Acknowledgements
Special thanks are addressed to Ms. Etsuko Ishibushi
for expert technical assistance.
6. Sawa, M.;Kondo, H.;Nishimura, S. Bioorg. Med. Chem.
Lett. 2002, 12, 581.
7. Moss, M. L.;White, J. M.;Lambert, M. H.;Andrews, R. C.
Drug Discov. Today 2001, 6, 417.
References and Notes
8. Recombinant human TACE, collagenase-1 (MMP-1), stro-
melysin 1 (MMP-3), and gelatinase B (MMP-9) were used in
our studies (see ref 4 for assay conditions).
1. Newton, R. C.;Decicco, C. P. J. Med. Chem. 1999, 42,
2295 (a review) and references therein.
2. Stone, A. L.;Kroeger, M.;Sang, Q. X. A. J. Protein Chem.
1999, 18, 447.
3. (a) Black, R. A.;Rauch, C. T.;Kozlosky, C. J.;Peschon, J. J.;
Slack, J. L.;Wolfson, M. F.;Castner, B. J.;Stocking, K. L.;
Reddy, P.;Srinivasan, S.;Nelson, N.;Boiani, N.;Schooley, K. A.;
Gerhart, M.;Davis, R.;Fitzner, J. N.;Johnson, R. S.;Paxton,
9. Models of TACE complexed with the phosphonamide
inhibitors were constructed based on the crystal structure of a
TACE/peptide-based inhibitor complex (PDB code, 1BKC).
Docking and energy minimizations were performed with the
TRIPOS force field within the SYBYL program. All compu-
tations were performed on an Silicon Graphics workstation.