New strategy for antedrug application: Development of metalloproteinase inhibitors as antipsoriatic drugs

Article abstract of DOI:10.1021/jm010349c

Phosphonamide-based inhibitors were synthesized and evaluated for the inhibitory activities against the shedding of epidermal growth factors, amphiregulin and heparin-binding EGF-like growth factor, that would participate in the development of psoriasis. All compounds exhibited excellent inhibitory activities for these EGF sheddings; however, they also inhibited matrix metalloproteinases (MMPs). To avoid adverse effects reported by the clinical development of MMP inhibitors, the antedrug concept was introduced. Among the phosphonamide inhibitors, the 2,2,2-trifluoroethyl ester 8d and 2,2-difluoroethyl ester 8c showed rapid decomposition in human plasma, which is an essential property for the antedrug. Topical applications of these compounds significantly suppressed TPA-induced epidermal hyperplasia in murin skin, a model of psoriasis. These results suggested that the phosphonamide-based inhibitors have a therapeutic potential for the treatment of psoriasis as an antedrug application.

Full text of DOI:10.1021/jm010349c

930
J . Med. Chem. 2002, 45, 930-936
New Str a tegy for An ted r u g Ap p lica tion : Develop m en t of Meta llop r otein a se
In h ibitor s a s An tip sor ia tic Dr u gs
Masaaki Sawa,* Takako Tsukamoto,† Takao Kiyoi, Kiriko Kurokawa, Fumio Nakajima, Yuichiro Nakada,†
Koichi Yokota,‡ Yoshimasa Inoue,§ Hirosato Kondo,| and Kohichiro Yoshino
Department of Chemistry, R&D Laboratories, Nippon Organon K.K., 1-5-90, Tomobuchi-cho,
Miyakojima-ku, Osaka 534-0016, J apan
Received J uly 27, 2001
Phosphonamide-based inhibitors were synthesized and evaluated for the inhibitory activities
against the shedding of epidermal growth factors, amphiregulin and heparin-binding EGF-
like growth factor, that would participate in the development of psoriasis. All compounds
exhibited excellent inhibitory activities for these EGF sheddings; however, they also inhibited
matrix metalloproteinases (MMPs). To avoid adverse effects reported by the clinical development
of MMP inhibitors, the antedrug concept was introduced. Among the phosphonamide inhibitors,
the 2,2,2-trifluoroethyl ester 8d and 2,2-difluoroethyl ester 8c showed rapid decomposition in
human plasma, which is an essential property for the antedrug. Topical applications of these
compounds significantly suppressed TPA-induced epidermal hyperplasia in murin skin, a model
of psoriasis. These results suggested that the phosphonamide-based inhibitors have a
therapeutic potential for the treatment of psoriasis as an antedrug application.
In tr od u ction
against matrix metalloproteinases (MMPs). However,
it was thought that the inhibition of MMPs may cause
undesirable side effects.
Psoriasis is an inflammatory skin disease and affects
1-3% of the world’s population. The feature of the
disease is epidermal hyperproliferation of epidermal
keratinocytes.1-3 Several systemic and topical therapies
are available, such as methotrexate, retinoids, corticos-
teroid, vitamin D3 analogue, and tazarotene, but the
effects of these agents are not sufficient.2 In addition,
undesirable adverse effects limit the use of these agents
for the therapy. Although the etiology of psoriasis has
not been clearly elucidated, aberrant expression of
growth factors for epidermal keratinocyte, such as
amphiregulin (AR)4 and heparin-binding EGF-like growth
factor (HB-EGF),5 would participate in the development
of psoriatic inflammation.3,6-8 These epidermal growth
factors (EGFs) are synthesized as membrane-anchored
precursors, which are converted to the soluble growth
factors.8 Several studies have suggested that zinc-
dependent metalloproteases (MPs) are involved in the
processing of these membrane-anchored precursors;9-12
however, the responsible enzymes have not been iso-
lated yet. Therefore, inhibitors of such EGF production
in the epidermis are considered to be effective agents
for the treatment of psoriasis.
In fact, it has been reported that adverse effects such
as musculoskeletal syndrome were observed in the
clinical studies of MMP broad inhibitors.14 To overcome
such side effects, the antedrug concept was introduced.
The term “antedrug” was first defined by Lee et al. in
1982 as locally effective drugs that topically showed
activity at the site of application but rapidly metabolized
to inactive compounds upon entry into the systemic
circulation15 and thus resulted in no systemic toxicity.
This antedrug concept has been mostly applied to
antiinflammatory glucocorticoid derivatives to reduce
their side effects.16-19 We expected that the phospho-
namide derivatives 1 would be easily metabolized to
inactive compounds in blood because the P-OR ester
bond in 1 was expected to be easily hydrolyzed by
nonspecific plasma esterases (Scheme 1).20,21 Then, it
was expected that the resulting phosphonic acid 2 would
be spontaneously decomposed to inactive compounds 3
and 4.22
In this paper, we describe the synthesis and evalua-
tion of the phosphonamide-based derivatives as EGF
shedding inhibitors. These compounds were also tested
for the stability in human plasma to evaluate the
potential of the antedrug. In addition, the degradation
mechanism in plasma was investigated by using 31P
NMR. Finally, the selected compounds were subjected
to in vivo assay using 12-O-tetradecanoylphorbol-13-
acetate (TPA)-induced inflammation and hyperplasia in
murine skin, a model of psoriasis.
We have already reported that a series of phospho-
namide-based inhibitors (1) exhibited potent inhibition
against HB-EGF shedding (IC50 < 1 µM).13 Moreover,
these inhibitors also have potent inhibitory activities
* To whom correspondence should be addressed. Current address:
Chemistry Research Laboratories, Drug Research Division, Dainippon
Pharmaceutical Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053,
J apan. Phone: +81-6-6337-5902. Fax: +81-6-6338-7656. E-mail:
masaaki-sawa@dainippon-pharm.co.jp.
Ch em istr y
† Department of ADME.
‡ Department of Pharmacology.
The syntheses for the phosphonamide derivatives
were shown in Scheme 2. Commercially available p-
methoxyphenylphosphonic dichloride 5 was treated with
(3R)-1,2,3,4-tetrahydroisoquinoline derivative 6 in py-
§ Department of Molecular Biology.
| Current address: Manufacturing Technology R&D Laboratories,
Shionogi & Co., Ltd., 1-3 Kuise Terajima 2-chome, Amagasaki, Hyogo
660-0813, J apan.
10.1021/jm010349c CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/15/2002

Development of Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 931
Sch em e 1
pounds were observed under acidic conditions (below pH
4) because of the lability of the P-N bond as previously
described.13
Resu lts a n d Discu ssion
At first, we synthesized and evaluated the ethyl ester
derivative as a typical phosphonamide inhibitor. The
previous work in our laboratory has demonstrated that
the stereochemistries at the C-3 and the phosphorus
atom of the phosphonamide inhibitors were very im-
portant for the inhibitory activities against MPs.13
Namely, the compounds having (R,R)-configuration only
showed potent inhibitory activities, as shown in Table
1 (compounds 8a and 9a ). We therefore subjected only
(R,R)-isomers in the series to further investigations.
Sch em e 2a
Initially, we tested the stability of the ethyl ester 8a
in human plasma for 60 min at 37 °C (Table 1). The
result was expressed as a percentage of the test com-
pound remaining. Contrary to our expectation, no
decomposition was observed in plasma after incubation
for 1 h. Then, we designed and synthesized compounds
8b-8e, having various ester groups, to find unstable
compounds in plasma. Generally, a good leaving group
would facilitate not only chemical hydrolysis but also
enzymatic hydrolysis. Thus, fluorine atoms were intro-
duced into the ester group as electron-withdrawing
functions to decrease the stability of the ester bond
(compounds 8b-8e). All compounds synthesized here
were essentially stable in an aqueous solution at pH
4-9 and also under inhibitory assay conditions. As
shown in Table 1, all compounds exhibited potent
inhibitory activities against HB-EGF and AR shedding
(compounds 8b-8e). These compounds also showed
potent inhibition against MMP-1, -3, and -9 with na-
nomolar Ki values. These compounds were then sub-
jected to the stability test in plasma. As shown in Table
1, it was found that the trifluoroethyl ester 8d was
extremely rapidly decomposed in plasma within 1 min.
Interestingly, the difluoroethyl ester 8c showed a
moderate decomposition rate (half-life in plasma, ca. 10
min) compared with 8d . The monofluoroethyl ester 8b
and the trifluoropropyl ester 8e were shown to be stable
in plasma for at least 1 h. The metabolite of compound
8d was identified as compound 3 by LC-MS/MS, and
the metabolite 3 showed no inhibitory activity against
EGF shedding and MMPs as expected.
These results suggested that the trifluoroethoxy group
and the difluoroethoxy group attached to the phospho-
rus atom may act as better electron-withdrawing groups.
As a result, an increase in the positive charge on the
phosphorus atom would facilitate enzymatic hydrolyses
of the compounds. Such a dramatic change of the
hydrolysis rate for a phosphate monoester by an alka-
line phosphatase has been reported previouly.24 The
study suggested that the hydrolysis rates were linearly
dependent on the pKa values of the leaving groups,
which are closely related to the positive charge on the
phosphorus atom. To ascertain our assumption, the
electronic environments at the phosphorus atoms of
these compounds were evaluated by measuring 31P
NMR. As shown in Figure 1, the chemical shifts were
moved downfield by the substitution of fluorine atoms.
The chemical shift of compound 8d was 25.9 ppm, which
was 2.5 ppm lower field than that of compound 8a . The
a
(a) Method A: (1) (3R)-N-benyloxy-1,2,3,4-tetrahydroisoquino-
line-3-hydroxamide (6), then ROH, pyridine. Method B: (1) ROH
(2.1 equiv), NaH, THF; (2) KOH, then SOCl2, catalyst DMF; (3)
6, diisopropylethylamine, THF. (b) H2, Pd-C.
ridine, and then the resulting monochloride was allowed
to react with the appropriate alcohol to give the phos-
phonamide 7 (ca. 2:1 diastereomeric mixture) in a one-
pot reaction (method A). Alternatively, p-methoxyphenyl-
phosphonic dichloride 5 was treated with the appropriate
sodium alkoxide to provide the corresponding diester
and then selectively hydrolyzed by potassium hydroxide
to give the corresponding monoester, which was con-
verted to the corresponding phosphonyl chloride during
reflux with thionyl chloride in the presence of catalytic
DMF. The resulting phosphonyl chloride was then
coupled with (3R)-1,2,3,4-tetrahydroisoquinoline deriva-
tive 6 in THF in the presence of diisopropylethylamine
(DIEA) to yield the phosphonamide 7 as a mixture of
the 1:1 diastereomer (method B). Deprotection of the
benzyl group in 7 with 10% Pd-C gave the diastereo-
merically pure hydroxamic acids 8 and 9 that were
successfully separated by HPLC purification. Compound
9a was crystallized and analyzed by X-ray diffraction
analysis,23 and the stereochemistry at the phosphorus
atom of compound 9a was determined to be S configu-
ration.13 The stereochemistries of the other compounds
1
were assigned by a characteristic signal pattern in H
NMR, especially in an aromatic region. All compounds
synthesized here were found to be stable under assay
conditions. However, the decompositions of the com-

932 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
Ta ble 1. In Vitro Potency and Stability of the Phosphonamide Derivativesa
IC50 (µM)
HB-EGF
0.23
>10
Ki (nM)
stability in human plasma
(% compd remaining after 60 min)
compd
R
AR
MMP-1
MMP-3
MMP-9
8a
9a
8b
8c
8d
8e
3
Et
Et
0.35
NT
4.59
>850
10.5
5.20
>650
9.07
5.05
>800
10.5
100
NT
96
CH2CH2F
CH2CHF2
CH2CF3
0.18
0.51
0.73
0.31
0.47
1.47
0.95
0.97
NT
6.00
6.57
2.21
6.67
6.75
3.33
4.97
3.68
4.46
0 (t1/2 ≈ 10 min)
0 (t1/2 < 1min)
CH2CH2CF3
99
NT
>10
>850
>650
>800
a
See Experimental Section for details of experimental assays. NT ) not tested.
Ta ble 2. Effect of Phosphonamide Derivatives on TPA-Induced
Epidermal Hyperplasiaa
epidermal
compd
thickness (µm)
% decrease
TPA + vehicle
TPA + 8a
62.2 ( 1.5
44.4 ( 7.3b
39.7 ( 3.4c
30.5 ( 4.8c
23.2 ( 1.7c
45.6
57.7
81.3
TPA + 8c
TPA + 8d
vehicle only
a
See Experimental Section for details of experimental assays.
Data represent the mean ( standard error of five animals.
b
Significant difference from (TPA + vehicle) treated group. p <
0.05. c p < 0.01 (Student’s t-test).
reason for this difference may be that the fluorine atoms
in the phosphonamide ester moiety would improve the
pharmacokinetic profiles, such as skin permeability, and
this resulted in more potent antiinflammatory effects.
In fact, the trifluorinated compound 8d exhibited the
most potent inhibitory activity for TPA-induced epider-
mal hyperplasia (30.5 µm, 81.3% decrease). Further-
more, compound 8d inhibited TPA-induced hyperplasia
in a dose-dependent manner.27 These data indicated
that compounds 8c and 8d would be stable in the
epidermal and would regulate EGF production to sup-
press phorbol-induced inflammation and epidermal hy-
perplasia. Considering the potent topical antiinflam-
matory activity and its rapid decomposition property in
plasma, it was strongly suggested that compound 8d
would have great potential as the antedrug, and thus,
topical use of compound 8d was the most promising
candidate for the treatment of psoriasis.
F igu r e 1. Chemical shifts of the 31P NMR spectra of the
phosphonamide derivatives.
resonance of compound 8c also moved downfield by 1.9
ppm compared with that of compound 8a . This lowering
of the chemical shifts presumably resulted from an
increase in the positive charge on the phosphorus atom.
However, the extension of the methylene chain moved
the chemical shift of compound 8e to higher field similar
to that of the monofluoroethyl ester 8b. These results
suggested that the degradation of the phosphonamide
inhibitors in plasma would be dependent on the elec-
tronic environments on the phosphorus atom.
From the results of the studies for the enzyme
inhibition and the plasma stability assay, compounds
8c and 8d were found to have great potential as the
antedrug, and thus these compounds were subjected to
further in vivo experiments.
Con clu sion s
In the present paper, we provided a novel strategy
for the application of a metalloproteinase inhibitor as
the antedrug. The phosphonamide-based metallopro-
teinase inhibitors showed potent activities against HB-
EGF and AR shedding, which seem to be involved in
hyperproliferation of epidermal keratinocytes. The tri-
fluoroethyl ester derivative 8d showed extremely rapid
decomposition in human plasma. The difluoroethyl ester
8c also showed moderate decomposition; however, the
ethyl ester 8a and monofluoroethyl ester 8b were stable
in plasma. The results of 31P NMR suggested that an
increase in the positive charge on the phosphorus atom
would strongly affect the decomposition rate. Topical
applications of compounds 8c and 8d significantly
reduced TPA-induced inflammation and epidermal hy-
perplasia. These results suggested that compound 8d
may represent a novel approach to the treatment of
psoriasis.
To evaluate the antipsoriatic activities of compounds,
we investigated the effects of these inhibitors on TPA-
induced hyperplasia in murine skin, which is one of the
psoriatic models.25,26 It is well-known that topical ap-
plication of phorbol esters induces cutaneous inflam-
mation and epidermal hyperproliferation. As shown in
Table 2, TPA induced a significant increase in epidermal
thickness (62.2 µm), compared with acetone treatment
(23.2 µm). Topical application of 8a effectively sup-
pressed TPA-induced inflammation with a 45.6% de-
crease at a dose of 100 µg/head. Compounds 8c and 8d
also significantly inhibited TPA-induced hyperplasia
(57.7% and 81.3% decrease, respectively). These results
suggested that compounds 8c and 8d existed in an
active form at a target site, although these compounds
were promptly metabolized in plasma. It was notewor-
thy that 8c and 8d showed more potent antiinflamma-
tory activities than the parent stable compound 8a . The

Development of Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 933
column chromatography on silica gel, eluting with AcOEt to
give the title compound as a diasteromeric mixture (ca. 2:1,
466 mg, 55%): 1H NMR (CDCl3) δ 1.19 (t, J ) 7.1 Hz, 1H),
1.31 (t, J ) 7.1 Hz, 2H), 2.75-2.95 (m, 1H), 3.30-3.45 (m, 1H),
3.81 (s, 2H), 3.85 (s, 1H), 3.90-4.30 (m, 4H), 4.45-4.55 (m,
0.3 H), 4.75-4.85 (m, 2H), 6.85-6.95 (m, 3H), 7.05-7.20 (m,
3H), 7.25-7.35 (m, 5H), 7.45-7.70 (m, 2H), 9.71 (br s, 0.3H),
10.11 (br s, 0.7H).
(3R)-N-Hyd r oxy-2-[(R a n d S)-eth oxy(4-m eth oxyp h en -
yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r box-
a m id e (8a a n d 9a ). A mixture of compound 7a (466 mg, 0.97
mmol) and 10% Pd-C (40 wt %, prewashed with EtOH) in
EtOH (20 mL) was stirred at room temperature under H2 for
3 h. Pd-C was filtered off, and the filtrate was concentrated.
The residue was purified by HPLC (YMC-ODS, CH3CN/water
30:70) to afford two diastereomerically pure hydroxamic acids.
8a (80 mg, 21%) was eluted first, and then 9a (127 mg, 34%)
was eluted.
Exp er im en ta l Section
Gen er a l Meth od s a n d Ma ter ia ls. All commercially avail-
able starting materials and solvents were reagent grade.
Melting points were uncorrected. 1H NMR spectra were
measured at 250 MHz on a Bruker DPX-250 spectrometer
using CDCl3 or DMSO-d6 as the solvent. 31P NMR spectra were
recorded at 243 MHz on a Bruker DRX-600 spectrometer using
DMSO as the solvent with 85% H3PO4 as an external refer-
ence. TPA was purchased from Sigma (St. Louis, MO). Mass
spectra were determined on a Perceptive Biosystems Voyager-
DETM RP spectrometer. Elemental analysis was conducted
by Sumika Chemical Analysis Service, Ltd., J apan, and the
results are within (0.4% of the calculated values unless
otherwise noted.
Gen er a l P r oced u r e for P r ep a r in g P h osp h on a m id e
Der iva t ives (Met h od A). (3R)-2-ter t-Bu t oxyca r b on yl-
1,2,3,4-tetr a h yd r oisoqu in ole-3-ca r boxylic Acid . To a solu-
tion of (3R)-1,2,3,4-tetrahydroisoquinole-3-carboxylic acid hy-
drochlorde28 (17.1 g, 80 mmol) in 200 mL of 50% aqueous 1,4-
dioxane was added sodium carbonate (17.0 g, 160 mmol) and
(Boc)2O (21.0 g, 96 mmol), and the mixture was stirred at room
temperature overnight. The reaction mixture was concentrated
to a half-volume in vacuo, and the solution was acidified with
1 N HCl (pH 3). The solution was extracted with AcOEt, and
the organic layer was washed successively with 1 N HCl and
brine and dried over MgSO4. The solvent was evaporated to
give the title compound (13.5 g, 61%): 1H NMR (DMSO-d6) δ
1.37 (s, 4.5H), 1.44 (s, 4.5H), 3.00-3.25 (m, 2H), 4.35-4.60
(m, 2H), 4.60-4.70 (m, 0.5H), 4.80-4.90 (m, 0.5H), 7.15-7.25
(m, 4H).
(3R)-N-Ben zyloxy-2-ter t-bu toxycar bon yl-1,2,3,4-tetr ah y-
d r oisoqu in ole-3-ca r boxa m id e. To a solution of (3R)-2-tert-
butoxycarbonyl-1,2,3,4-tetrahydroisoquinole-3-carboxylic acid
(13.5 g, 48.7 mmol) in DMF (200 mL) was added WSC (10.4 g,
54 mmol), HOBt (8.3 g, 54 mmol), O-benzylhydroxylamine
hydrochloride (8.6 g, 54 mmol), and triethylamine (5.5 g, 54
mmol), and the mixture was stirred at room temperature
overnight. The reaction mixture was diluted with AcOEt (200
mL), washed successively with 1 N HCl, saturated NaHCO3,
and brine, and dried over MgSO4. The solvent was evaporated,
and the residue was recrystallized from AcOEt-hexane to give
the title compound (7.6 g, 41%): 1H NMR (CDCl3) δ 1.41 (s,
4.5H), 1.52 (s, 4.5H), 3.15-3.35 (m, 2H), 4.45-4.75 (m, 2H),
4.80-4.85 (m, 0.5H), 5.00-5.20 (m, 2H), 5.15-5.25 (m, 0.5H),
7.05-7.40 (m, 9H).
(3R)-N-Ben zyloxy-1,2,3,4-tetr a h yd r oisoqu in ole-3-ca r -
boxa m id e (6). (3R)-N-benzyloxy-2-tert-butoxycarbonyl-1,2,3,4-
tetrahydroisoquinole-3-carboxamide (7.6 g, 19.9 mmol) was
dissolved in 4 N HCl/AcOEt (50 mL), and the solution was
stirred at room temperature for 2 h. The precipitates were
filtered and washed with Et2O. The solids were dissolved in
water, neutralized with NaHCO3, and extracted with AcOEt.
The organic layer was washed with brine and dried over
MgSO4. Removal of the solvent gave the title compound (4.6
g, %): 1H NMR (DMSO-d6) δ 2.70-2.80 (m, 2H), 3.30-3.35
(m, 1H), 3.75-3.95 (m, 2H), 4.80 (s, 2H), 6.95-7.15 (m, 4H),
7.30-7.45 (m, 5H).
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(4-m eth oxyp h en yl)p h os-
p h or yl]-1,2,3,4-t et r a h yd r oisoq u in olin e-3-ca r b oxa m id e
1
(8a ): colorless solids; H NMR (DMSO-d6) δ 1.14 (t, J ) 7.0
Hz, 3H), 2.90-3.05 (m, 2H), 3.65-3.80 (m, 1H), 3.79 (s, 3H),
3.85-4.00 (m, 1H), 4.14 (dd, J ) 7.6 and 16.1 Hz, 1H), 4.30
(dd, J ) 4.5 and 16.1 Hz, 1H), 4.35-4.45 (m, 1H), 7.02 (dd, J
) 3.1 and 8.9 Hz, 2H), 7.00-7.15 (m, 4H), 7.69 (dd, J ) 8.8
and 12.4 Hz, 2H), 8.78 (br s, 1H), 10.57 (br s, 1H); 31P NMR δ
23.4; MALDI-TOF MS 429 [M + K]+, 413 [M + Na]+, 391 [M
+ H]+. Anal. (C19H23N2O5P) C, H, N.
(3R)-N-Hyd r oxy-2-[(S)-eth oxy(4-m eth oxyp h en yl)p h os-
p h or yl]-1,2,3,4-t et r a h yd r oisoq u in olin e-3-ca r b oxa m id e
(9a ): recrystallization from EtOH-CH3CN gave colorless
1
crystals; mp 165-166 °C; H NMR (DMSO-d6) δ 1.31 (t, J )
7.0 Hz, 3H), 2.90-3.10 (m, 2H), 3.75 (s, 3H), 3.90-4.20 (m,
3H), 4.40-4.60 (m, 2H), 6.90-7.10 (m, 6H), 7.54 (dd, J ) 8.8
and 12.3 Hz, 2H), 8.76 (br s, 1H), 10.54 (br s, 1H); MALDI-
TOF MS 429 [M + K]+, 413 [M + Na]+, 391 [M + H]+. Anal.
(C19H23N2O5P) C, H, N.
Compounds 8b, 9b, 8e, and 9e were also synthesized using
a procedure similar to the procedure for the preparation of 8a
and 9a .
(3R)-N-Hydr oxy-2-[(R)-(2-flu or oeth oxy)(4-m eth oxyph en -
y l)p h o s p h o r y l]-1,2,3,4-t e t r a h y d r o is o q u in o le -3-c a r -
1
boxa m id e (8b): colorless solids; 36%; H NMR (DMSO-d6) δ
2.85-3.00 (m, 2H), 3.81 (s, 3H), 3.85-4.35 (m, 4H), 4.40-4.50
(m, 2H), 4.55-4.70 (m, 1H), 7.00-7.15 (m, 6H), 7.73 (dd, J )
8.8 and 12.6 Hz, 2H), 8.78 (br s, 1H), 10.59 (br s, 1H); 31P NMR
δ 24.3; MALDI-TOF MS 447 [M + K]+, 431 [M + Na]+, 409
[M + H]+. Anal. (C19H22FN2O5P) C, H, N.
(3R)-N-Hydr oxy-2-[(S)-(2-flu or oeth oxy)(4-m eth oxyph en -
y l)p h o s p h o r y l]-1,2,3,4-t e t r a h y d r o is o q u in o le -3-c a r -
1
boxa m id e (9b): colorless solids; 12%; H NMR (DMSO-d6) δ
2.90-3.15 (m, 2H), 3.76 (s, 3H), 3.98 (dd, J ) 7.5 and 17.5 Hz,
1H), 4.10-4.50 (m, 3H), 4.50-4.70 (m, 2H), 4.70-4.85 (m, 1H),
6.90-7.00 (m, 3H), 7.00-7.15 (m, 3H), 7.56 (dd, J ) 8.5 and
12.3 Hz, 2H), 8.78 (s, 1H), 10.58 (s, 1H); MALDI-TOF MS 447
[M + K]+, 431 [M + Na]+, 409 [M + H]+. Anal. (C19H22FN2O5P)
C, H, N.
Syn th esis of (3R)-N-Hyd r oxy-2-[(R a n d S)-eth oxy(4-
m eth oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in o-
lin e-3-ca r boxa m id e (8a a n d 9a ). Dia ster eom er s of (3R)-
N -Be n zyloxy-2-[(R S )-e t h oxy(4-m e t h oxyp h e n yl)p h os-
p h or yl]-1,2,3,4-t et r a h yd r oisoq u in olin e-3-ca r b oxa m id e
(7a ). To a solution of 4-methoxyphenylphosphonic dichloride
(398 mg, 1.77 mmol) in 5 mL of pyridine was added (3R)-N-
benzyloxy-1,2,3,4-tetrahydroisoquinole-3-carboxamide 6,13 and
the mixture was stirred at room temperature for 15 min under
an argon atmosphere. Then, ethanol (0.1 mL, 1.77 mmol) was
added to the solution, and the mixture was stirred at room
temperature for 1 h. The reaction mixture was concentrated
in vacuo, and the remaining pyridine was completely removed
by azeotropic evaporation with toluene. The residual oil was
diluted with AcOEt, and the solution was washed successively
with saturated NaHCO3 and brine and dried over MgSO4. The
solvent was evaporated, and the residue was purified by
(3R)-N-Hydr oxy-2-[(R)-(3,3,3-tr iflu or opr opoxy)(4-m eth -
oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in ole-3-
ca r boxa m id e (8e): colorless solids; 35%; 1H NMR (DMSO-
d6) δ 2.50-2.70 (m, 2H), 2.85-3.05 (m, 2H), 3.80 (s, 3H), 3.80-
3.95 (m, 1H), 4.05-4.20 (m, 2H), 4.25-4.35 (m, 1H), 4.35-
4.45 (m, 1H), 7.00-7.15 (m, 6H), 7.70 (dd, J ) 8.7 and 12.5
Hz, 2H), 8.80 (s, 1H), 10.60 (s, 1H); 31P NMR δ 24.3; MALDI-
TOF MS 497 [M + K]+, 481 [M + Na]+, 459 [M + H]+. Anal.
(C20H22F3N2O5P) C, H, N.
(3R)-N-Hyd r oxy-2-[(S)-(3,3,3-tr iflu or opr opoxy)(4-m eth -
oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in ole-3-
ca r boxa m id e (9e): colorless solids; 25%; 1H NMR (DMSO-
d6) δ 2.70-2.85 (m, 2H), 2.95-3.15 (m, 2H), 3.75 (s, 3H), 3.92
(dd, J ) 7.5 and 16.1 Hz, 1H), 4.20-4.50 (m, 3H), 4.55-4.60
(m, 1H), 6.90-7.00 (m, 3H), 7.00-7.15 (m, 3H), 7.53 (dd, J )
8.8 and 12.5 Hz, 2H), 8.78 (s, 1H), 10.59 (br s, 1H); MALDI-

934 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
1
TOF MS 497 [M + K]+, 481 [M + Na]+, 459 [M + H]+. Anal.
3-ca r boxa m id e (9d ): colorless solids; H NMR (DMSO-d6) δ
3.00-3.20 (m, 2H), 3.77 (s, 3H), 3.85-3.95 (m, 1H), 4.30-4.40
(m, 1H), 4.55-4.90 (m, 3H), 6.95-7.05 (m, 3H), 7.05-7.15 (m,
3H), 7.56 (dd, J ) 8.8 and 12.6 Hz, 2H), 8.83 (br s, 1H), 10.66
(br s, 1H); MALDI-TOF MS 483 [M + K]+, 467 [M + Na]+,
445 [M + H]+. Anal. (C19H20F3N2O5P) C, H, N.
(C20H22F3N2O5P) C, H, N.
Gen er a l P r oced u r e for P r ep a r in g P h osp h on a m id e
Der iva tives (Meth od B). Syn th esis of (3R)-N-Hyd r oxy-
2-[(R a n d S)-(2,2,2-t r iflu or oet h oxy)(4-m et h oxyp h en yl)-
p h osp h or yl]-1,2,3,4-t et r a h yd r oisoq u in olin e-3-ca r b oxa -
m id e (8d a n d 9d ). (4-Meth oxyp h en yl)p h osp h on ic Acid
Bis(2,2,2-tr iflu or oeth yl) Ester . To a solution of 2,2,2-trif-
luoroethanol (466 mg, 4.66 mmol) in dry THF (50 mL) was
added NaH (123 mg, 5.13 mmol), and the mixture was stirred
at room temperature under an argon atmosphere for 30 min.
Then, 4-methoxyphenylphosphonic dichloride (500 mg, 2.22
mmol) was added to the mixture, and the stirring was
continued overnight. The reaction mixture was quenched with
1 N HCl (50 mL) and extracted with AcOEt. The organic layer
was washed with saturated NaHCO3 and brine and dried over
MgSO4. The solvent was evaporated to give the title compound
as a colorless oil (650 mg, 83%): 1H NMR (CDCl3) δ 3.87 (s,
3H), 4.25-4.50 (m, 4H), 7.01 (dd, J ) 3.9 and 8.9 Hz, 2H),
7.76 (dd, J ) 8.9 and 13.6 Hz, 2H).
(4-Met h oxyp h en yl)p h osp h on ic Acid Mon o(2,2,2-t r i-
flu or oeth yl) Ester . To a solution of (4-methoxyphenyl)-
phosphonic acid bis(2,2,2-trifluoroethyl) ester (650 mg, 1.84
mmol) in 1,4-dioxane (7 mL) was added 1.8 N KOH solution
(3 mL), and the mixture was refluxed for 2 h. The reaction
mixture was acidified with 1 N HCl and extracted with AcOEt.
The organic layer was washed with brine and dried over
MgSO4. The solvent was evaporated to give the title compound
as a colorless oil (477 mg, 96%): 1H NMR (CDCl3) δ 3.86 (s,
3H), 4.15-4.30 (m, 2H), 6.97 (dd, J ) 3.7 and 8.9 Hz, 2H),
7.73 (dd, J ) 8.9 and 13.5 Hz, 2H).
Dia ster eom er s of (3R)-N-Ben zyloxy-2-[(RS)-(2,2,2-tr i-
flu or oeth oxy)(4-m eth oxyp h en yl)p h osp h or yl]-1,2,3,4-tet-
r a h yd r oisoqu in olin e-3-ca r boxa m id e (7d ). A mixture of (4-
methoxyphenyl)phosphonic acid mono(2,2,2-trifluoroethyl) ester
(477 mg, 1.77 mmol) and a catalytic amount of DMF (a few
drops) in thionyl chloride (5 mL) was refluxed for 2 h. The
reaction mixture was concentrated in vacuo, and the residual
thionyl chloride was completely removed by azeotropic evapo-
ration with toluene to afford 580 mg of crude monochloride,
which was used for the next reaction without further purifica-
tion. To a solution of the above product in 5 mL of dry THF
was added (3R)-N-benzyloxy-1,2,3,4-tetrahydroisoquinole-3-
carboxamide13 (300 mg, 1.06 mmol) and diisopropylethylamine
(363 µL, 2.1 mmol), and the mixture was stirred at room
temperature under an argon atmosphere overnight. The
reaction mixture was concentrated, and the residual oil was
diluted with AcOEt. The solution was washed successively
with water, saturated NaHCO3, and brine and dried over
MgSO4. The solvent was evaporated, and the residue was
purified by column chromatography on silica gel, eluting with
AcOEt to give 255 mg of crude product as a diasteromeric
mixture (1:1). This crude product was used for the next
reaction without further purification.
(3R)-N-Hyd r oxy-2-[(R a n d S)-(2,2,2-tr iflu or oeth oxy)(4-
m eth oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in o-
lin e-3-ca r boxa m id e (8d a n d 9d ). A mixture of compound
7d (255 mg) and 10% Pd-C (200 mg, prewashed with EtOH)
in EtOH (10 mL) was stirred at room temperature under H2
for 3 h. Pd-C was filtered off, and the filtrate was concen-
trated. The residue was purified by HPLC (YMC-Pack ODS,
CH3CN/water 40:60) to afford two diastereomerically pure
hydroxamic acids. The (R)-isomer 8d (25 mg, 12%) was eluted
first, and then the (S)-isomer 9d (28 mg, 13%) was eluted.
Compounds 8c and 9c were also synthesized using a
procedure similar to the procedure for the preparation of 8d
and 9d .
(3R)-N-H yd r oxy-2-[(R)-(2,2-d iflu or oet h oxy)(4-m et h -
oxyphen yl)phosph or yl]-1,2,3,4-tetr ahydr oisoquin ole-3-car -
1
boxa m id e (8c): colorless solids; 30%; H NMR (DMSO-d6) δ
2.99-3.16 (m, 2H), 3.90 (s, 3H), 3.95-4.15 (m, 1H), 4.20-4.45
(m, 3H), 4.45-4.55 (m, 1H), 6.27 (tt, J ) 3.2 and 54.3 Hz, 1H),
7.10-7.25 (m, 6H), 7.82 (dd, J ) 8.7 and 12.6 Hz, 2H), 8.89 (s,
1H), 10.70 (s, 1H); 31P NMR δ 25.3; MALDI-TOF MS 465 [M
+ K]+, 449 [M + Na]+, 427 [M + H]+. Anal. (C19H21F2N2O5P)
C, H, N.
(3R)-N-H yd r oxy-2-[(S)-(2,2-d iflu or oe t h oxy)(4-m e t h -
oxyphen yl)phosph or yl]-1,2,3,4-tetr ahydr oisoquin ole-3-car -
1
boxa m id e (9c): colorless solids; 38%; H NMR (DMSO-d6) δ
3.05-3.25 (m, 2H), 3.85 (s, 3H), 4.02 (dd, J ) 7.1 and 16.3 Hz,
1H), 4.30-4.55 (m, 3H), 4.60-4.70 (m, 1H), 6.45 (tt, J ) 3.4
and 54.6 Hz, 1H), 7.00-7.10 (m, 3H), 7.15-7.20 (m, 3H), 7.65
(dd, J ) 8.8 and 12.5 Hz, 2H), 8.90 (s, 1H), 10.72 (s, 1H);
MALDI-TOF MS 465 [M + K]+, 449 [M + Na]+, 427 [M + H]+.
Anal. (C19H21F2N2O5P) C, H, N.
Sh ed d in g Assa y for EGF Recep tor Liga n d-AP F u sion
P r otein . Expression plasmid of HB-EGF and human placental
alkaline phosphatase (AP) fusion protein that were constructed
as described previously29 were a generous gift from Dr.
Higashiyama (School of Medicine, Osaka University, Osaka,
J apan). The plasmid was transfected into HT1080 cells
(American Type Culture Collection, Rockville, MD) by lipo-
fection using a lipofectamine system (Gibco/BRL, Gaithers-
burg, MD) according to the manufacturer’s directions. Stable
transfectants were selected by growth in G418. Stable trans-
fectants of AR were established in the same way as those of
HB-EGF.
Stable transfectants expressing EGF receptor ligand-AP
fusion protein in MEM (containing 10% FCS) as a culture
medium were seeded in 96-well plates at a density of 2 × 105
cells/well and incubated for 24 h. The cells were washed with
PBS and preincubated with test compounds in MEM (contain-
ing 1% DMSO) for 30 min. TPA (60 nM) was added to
stimulate inducible processing, and the plate was incubated
for 60 min. A 0.1 mL aliquot of the supernatant was trans-
ferred to 96-well plates and heated for 10 min at 65 °C in order
to inactivate endogenous alkaline phosphatases. A 0.1 mL of
substrate solution (1 M diethanolamine, 0.01% MgCl2, 1 mg/
mL p-nitrophenyl phosphate, pH 9.8) was added to each well,
and the plates were incubated for 1-2 h. AP activity was then
determined by the measurement of absorbance at 405 nm with
a microplate reader. The IC50 value was determined with
different inhibitor concentrations by using GraphPad Prism,
version 3.0 (GraphPad Software, Inc.).
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
MMP s. DNA fragments coding the catalytic domain of human
MMP-1 and human MMP-9 and a DNA fragment coding from
the prodomain to the catalytic domain of human MMP-3 were
amplified by polymerase chain reaction (PCR) from cDNA of
HT1080 cells stimulated with 0.01 µM of TPA. A sequence for
the appropriate restriction enzyme site was added to the 5′-
end of each PCR primer. Amplified DNA fragments were
cloned into a cloning vector and then introduced into a
commercially available expression vector containing a His-6
tag sequence at the end of the N-terminus. Recombinant
proteins were expressed in E. coli cells and purified by Ni-
NTA resin (QIAGEN INC.) and refolded. Recombinant MMP-3
was activated by incubating with 1 mM p-aminophenylmer-
curic acetate for 1 h at 37 °C.
(3R)-N-Hyd r oxy-2-[(R)-(2,2,2-tr iflu or oeth oxy)(4-m eth -
oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in olin e-
1
3-ca r boxa m id e (8d ): colorless solids; H NMR (DMSO-d6) δ
2.90-3.10 (m, 2H), 3.82 (s, 3H), 4.10-4.20 (m, 1H), 4.25-4.35
(m, 2H), 4.35-4.45 (m, 1H), 4.50-4.65 (m, 1H), 7.00-7.15 (m,
6H), 7.75 (dd, J ) 8.8 and 12.8 Hz, 2H), 8.80 (br s, 1H), 10.63
(br s, 1H); 31P NMR δ 25.9; MALDI-TOF MS 483 [M + K]+,
467 [M + Na]+, 445 [M + H]+. Anal. (C19H20F3N2O5P) C, H, N.
MMP In h ibition Assa y. Recombinant MMP-1, -3, and -9
were used at final concentrations of 5, 6.5, and 24 nM,
respectively. The assays were performed using the fluorogenic
(3R)-N-Hyd r oxy-2-[(S)-(2,2,2-tr iflu or oeth oxy)(4-m eth -
oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h yd r oisoqu in olin e-

Development of Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 935
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substrate MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2
(Peptide Institute, Inc.) at a final concentration of 5 µM
(MOCAc-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-
NH2 [Peptide Institute, Inc.] was used for MMP-3). Typical
assays were performed as follows. In a well of a 96-well half-
area black microplate (COSTAR), an enzyme solution (25 µL)
was incubated with 25 µL of a test compound solution (10 mM
DMSO stock solution was diluted to the appropriate concen-
tration with the assay buffer) in 50 mM Tris, 150 mM NaCl,
10 mM CaCl2, 0.05% Brij-35, pH 7.5, for 10 min at 37 °C. Then
the reaction was initiated by adding 50 µL of the substrate
solution to the 96-well plate, and incubation was continued
for 2 h at 37 °C (3 h for MMP-9). The increase in fluorescence
due to cleavage of the substrate was measured with
a
fluorescence microplate reader (excitation/emission ) 320/405
nm, Polarstar; BMG LabTechnologies, Germany). Ki values
were calculated by nonlinear regression analysis using the
percent inhibition and Km values of the substrates for each
MMP with the program GraphPad Prism (GraphPad Software,
Inc., San Diego, CA).
Sta bility Assa y in Hu m a n P la sm a . A stock solution of
test compound (final concentration of 10 µM) was added to
plasma (500 µL) obtained from human (healthy volunteers)
or mice (male ddY, 6 weeks old), and the assay mixture was
incubated for 1 h at 37 °C. A 100 µL aliquot of the assay
mixture was then mixed with 500 µL of ice-cold acetonitrile
and centrifuged at 3000 rpm for 10 min. The solvent of the
supernatant was replaced with an eluent, and the stability of
the compound was then determined by HPLC on a column of
L-column (4.6 mm × 150 mm, Chemicals Evaluation Research
Institute, J apan) with water-acetonitrile eluent. The remain-
ing percent was calculated from the average of two measure-
ments.
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Ectodomain Shedding of Epidermal Growth Factor Receptor
Ligands Is Required for Keratinocyte Migration in Cutaneous
Wound Healing. J . Cell. Biol. 2000, 151, 209-219.
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In h ibition Assa y for TP A-In d u ced Ep id er m a l Hyp er -
p la sia Mod el. Backs of mice were shaved by an electric clipper
and treated with depilatory cream (Eva cream, Tokyo Tanabe,
Tokyo). Three days later, the mice that displayed no evidence
of hair regrowth were used for experiments. A total of 20 µL
of TPA (1 nM) or vehicle (acetone) was applied to the skin
surface of the backs in an area of ca. 1 cm2 using a micropipet.
A minute after TPA application or 1 h prior to TPA application,
a test compound or vehicle (acetone) was topically adminis-
trated to the same area where it was TPA-treated (day 0). The
treatment of the test compound or vehicle was repeated on
days 1 and 2. On day 3, the mice were sacrificed, and skin
tissues treated with TPA-test compound were removed. The
tissue specimen was made and stained by hematoxylin-eosin.
The epidermal thickness was measured as the distance from
the bottom of the stratum corneum to the bottom of the basal
layer using an ocular microscope with a magnification of 400×.
The measurement was performed at 10 different fields within
a width of 6 mm, and the mean was calculated.
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Ack n ow led gm en t. We are greatly acknowledge Dr.
Higashiyama (School of Medicine, Osaka University,
Osaka, J apan) for the gift of the expression vector of
HB-EGF fused with human placental alkaline phos-
phatase. Special thanks are addressed to Ms. Etsuko
Ishibushi and Ms. Mariko Hatakeyama for expert
technical assistance.
(21) Reichenberg, A.; Wiesner, J .; Weidemeyer, C.; Dreiseidler, E.;
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