New type of metalloproteinase inhibitor: Design and synthesis of new phosphonamide-based hydroxamic acids

Article abstract of DOI:10.1021/jm0103211

A series of phosphonamide-based hydroxamate derivatives were synthesized, and the inhibitory activities were evaluated against various metalloproteinases in order to clarify its selectivity profile. Among the four diastereomeric isomers resulting from the

Full text of DOI:10.1021/jm0103211

J . Med. Chem. 2002, 45, 919-929
919
New Typ e of Meta llop r otein a se In h ibitor : Design a n d Syn th esis of New
P h osp h on a m id e-Ba sed Hyd r oxa m ic Acid s
Masaaki Sawa,* Takao Kiyoi, Kiriko Kurokawa, Hiroshi Kumihara, Minoru Yamamoto, Tomohiro Miyasaka,
Yasuko Ito, Ryoichi Hirayama, Tomomi Inoue,† Yasuyuki Kirii,† Eiji Nishiwaki,† Hiroshi Ohmoto,† Yu Maeda,†
Etsuko Ishibushi,† Yoshimasa Inoue,† Kohichiro Yoshino, and Hirosato Kondo‡
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 11, 2001
A series of phosphonamide-based hydroxamate derivatives were synthesized, and the inhibitory
activities were evaluated against various metalloproteinases in order to clarify its selectivity
profile. Among the four diastereomeric isomers resulting from the chirality at the C-3 and P
atoms, the compound with a (R,R)-configuration both at the C-3 position and the phosphorus
atom was found to be potently active, while the other diastereomeric isomers were almost
inactive. A number of (R,R)-compounds synthesized here exhibited broad spectrum activities
with nanomolar Ki values against MMP-1, -3, -9, and TACE and also showed nanomolar IC50
values against HB-EGF shedding in a cell-based inhibition assay. The modeling study using
X-ray structure of MMP-3 suggested the possible binding mode of the phosphonamide-based
inhibitors.
In tr od u ction
A large number of zinc-dependent metalloproteinases
(MPs) with various functions have been isolated and
characterized recently.Matrixmetalloproteinases (MMPs)
are a family of such zinc-dependent metalloproteinases
that play a significant physiological role in extracellular
matrix remodeling.1 The implication of MMPs in a
number of pathological processes has been reported, and
thus, they are considered to be important therapeutic
targets for the treatment of a wide array of disease
processes such as rheumatoid arthritis, tumor metasta-
sis, multiple sclerosis, and congestive heart failure.2
F igu r e 1. Binding interaction of the phosphinamide inhibitor
The ADAMs (a disintegrin-like and metalloprotein-
in MMP enzyme.
ase-containing protein) are also zinc-dependent metal-
normal and aberrant processes such as wound healing,
blastocyst implantation, SMC hyperplasia, atheroscle-
rosis, and tumor growth.9 Therefore, specific inhibitors
of ADAM enzymes are also considered to be attractive
targets in drug discovery research.
loproteinases that have been implicated in procytokine
conversion processes.3 The importance of such trans-
membrane protein shed by the ADAM enzyme family
has been increasingly recognized. For example, ADAM-
17 was identified as a tumor necrosis factor R (TNF-R)
converting enzyme (TACE) that catalyzes the processing
of membrane-anchored proTNF-R to produce a soluble
form4 that contributes to a variety of inflammatory
diseases.5 Other ADAM enzymes are also thought to be
involved in such important shedding processes. A recent
study indicated that ADAM-9 is involved in the shed-
ding of heparin-binding epidermal growth factor (EGF)-
1ike growth factor (HB-EGF),6 a member of the EGF
family that stimulates cell growth and differentiation,7,8
although the responsible enzyme is not known. HB-EGF
has been implicated as a participant in a variety of
Recently extensive efforts to enhance potency and
selectivity for inhibition of these metalloproteinases
have been reported;2 however, there still remains con-
siderable scope for optimization of increasing enzyme
selectivity in order to reduce side effects. In general,
these studies have focused on the optimization of the
P1′ portions of the inhibitors because X-ray analyses of
the enzyme-inhibitor complex suggested that the S1′
pocket is a selectivity pocket for MMP inhibitors.2,10-12
Recently, Pikul et al. reported that the phosphinamide-
based hydroxamic acid 1 exhibited potent inhibitory
activity against MMPs, and the binding interaction was
proposed on the basis of X-ray data of the inhibitor-
enzyme complex (Figure 1).13 This type of compound has
an additional chiral center at the phosphorus atom
different from those of the corresponding sulfonamide
derivatives, and it has been found that the stereochem-
istry at the phosphorus was very important for activity.
In recent reports, many chiral compounds exhibited
* 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.
† 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/jm0103211 CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/15/2002

920 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
Sch em e 2a
a
(a) R1OH, NaH, THF; (b) SOCl2, catalyst DMF; (c) (3R)-4,
diisopropylethylamine, THF.
F igu r e 2. Strategy for the study of SAR.
Sch em e 3a
Sch em e 1a
a
(a) SOCl2, catalyst DMF; (b) (3R)-4, diisopropylethylamine,
THF or CH2Cl2; (c) H2, Pd-C.
a
(a) (3R)- or (3S)-N-benzyloxy-1,2,3,4-tetrahydroisoquinoline-
3-hydroxamide (4), then R1OH, THF or pyridine; (b) H2, Pd-C.
presence of N-methylmorpholine, the diastereomeric
isomers were produced in an approximately 1:1 ratio,
although the yields for the reactions were decreased.
Deprotection of the benzyl group in 5a with 10% Pd-C
gave the diastereomerically pure hydroxamic acids 6
and 7 that were successfully separated by HPLC
purification. In some cases, these diastereomeric iso-
mers could be purely isolated by recrystallization.
selective profiles in their inhibitions;14-18 however, there
was little information of a structure-activity relation-
ship (SAR) regarding the effects of the chirality on
enzyme selectivity. On the basis of these findings, we
supposed that such a chirality might induce enzyme
selectivity. Thus, we have designed the phosphonamide-
based inhibitor 2 in order to study the SAR in more
detail and to discover a new class of selective MP
inhibitors. In our design, the ester moiety R1 could be
readily converted to various substituents compared to
the corresponding moiety in the phosphinamides 1, and
this modification enables us to identify new interactions
with S2′/S3′ in the enzymes (Figure 2). Moreover, the
modification of R2 and X moieties could provide not only
the SAR information for MPs but structural information
about the responsible enzyme for HB-EGF shedding.
In this paper, we describe the design and syntheses
of novel and potent phosphonamide-based inhibitors for
MP enzymes. New derivatives synthesized here were
assayed for the inhibition of three types of MMPs:
collagenase 1 (MMP-1), stromelysin 1 (MMP-3), and
gelatinase B (MMP-9). In addition, we also tested their
inhibition activitites against the ADAM family, TACE,
and HB-EGF shedding. The SAR for these enzymes was
discussed, and the possible binding mode in MP en-
zymes was also analyzed by computer modeling.
The stepwise synthesis of the phosphonamide was
also developed as shown in Scheme 2 (method B). This
methodology affords 1:1 diastereomeric products in good
yields compared with the one-pot methodology (method
A). Thus, p-methoxyphenylphosphonic dichloride 3 was
treated with the appropriate sodium alkoxide to provide
the diester 8, which was converted to the corresponding
phosphonyl chloride 9 in reflux with thionyl chloride in
the presence of catalytic DMF. The phosphonyl chloride
9 was then coupled with (3R)-1,2,3,4-tetrahydroiso-
quinoline derivative 4 in THF in the presence of
diisopropylethylamine (DIEA) to yield the (3R)-phos-
phonamide 5b as a mixture of two diastereomers (1:1).
A further step was identical to the one described above.
The general procedure for the replacement of R2 by
various groups is shown in Scheme 3 (method C). The
required diethyl phosphonate 10 was prepared accord-
ing to the method reported in the literature.20 Com-
pound 10 was refluxed in thionyl chloride in the
presence of a catalytic amount of DMF to give the
chlorophosphonate 11. Compound 11 was then coupled
with the (3R)-4 in the presence of DIEA to give the (3R)-
phosphonamide 12. Completion of the synthesis was
achieved as described in method A to afford the desired
product 13.
Ch em istr y
The modification of the R1 moiety was performed by
two methods (method A or B). First, the synthesis of
phosphonamide derivatives by a one-pot methodology
is presented in Scheme 1 (method A). Commercially
available p-methoxyphenylphosphonic dichloride 3 was
treated with (3R)- or (3S)-N-benzyloxy-1,2,3,4-tetrahy-
droisoquinole-3-carboxamide 4 (prepared from the cor-
responding carboxylic acid19) in pyridine, and then the
resulting monochloride was allowed to react with the
appropriate alcohol to give the phosphonamide 5a (ca.
2:1 mixture of two diastereomers) in a one-pot reaction.
In this step, if THF was used as the solvent in the
Another series of derivatives shown in Table 3 was
obtained by reaction of the appropriate cyclic amine 14a
or 14b with ethyl p-methoxyphenylchlorophosphonate
15, shown in Scheme 4 (method D). The (R)-cyclic amine
14a or 14b was respectively prepared from natural or
unnatural amino acid derivatives under Pictet-Spen-
gler conditions.21 The conversion of the R3 moiety into
hydroxamic acid was done with general methods.

Phosphonamide-Based Hydroxamic Acids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 921
Sch em e 4a
Ta ble 1. Effects of Stereochemistry on in Vitro Activity for the
Phosphonamide Derivatives
Ki (nM)a
stereochemistry
(C-3, P)
IC50 (nM)a
compd
MMP-1 MMP-3 MMP-9 TACE HB-EGF
18
19
20
21
(R,S)
(R,R)
(S,R)
(S,S)
>850
4.58
759
>650
5.20
>650
>650
>800
5.05
349
>780
7.15
>780
>780
>10 000
230
>10 000
>10 000
a
(a) 4-Methoxyphenylchlorophosphonic acid ethyl ester (15),
>850
>800
diisopropylethylamine, THF; (b) NaOH, then WSC, HOBt, hy-
droxylamine hydrochloride or H2, Pd-C.
a
See Experimental Section for details of experimental assays.
Ta ble 2. In Vitro Profile of R1-Modified Phosphonamide
Derivatives
F igu r e 3. ORTEP view of X-ray structure of 18.
To determine the absolute configuration at the phos-
phorus atom, compound 18 was crystallized and ana-
lyzed by X-ray diffraction.22 As shown in Figure 3, the
configuration at the phosphorus atom of compound 18
was determined to be S. For all diastereomeric pairs in
this series, the configuration of phosphorus was as-
signed by a characteristic signal pattern in 1H NMR.
All compounds synthesized here were found to be
stable under assay conditions. However, decompositions
of the compounds were observed under acidic conditions
(below pH 4) due to the lability of P-N bonds.23
a
See Experimental Section for details of experimental assays.
b
Diastereomeric mixture. nd, not determined.
Resu lts a n d Discu ssion
range, while IC50 values against HB-EGF shedding are
in the micromolar range. This difference in the order of
inhibition activities seems to attribute to the difference
in assay system.27 As shown in Table 1, (3R,R)-
compound 19 exhibited potent inhibitory activity against
all enzymes but the other isomers showed no activity
or moderate activities. The importance of the R config-
uration at the R-carbon of the hydroxamate was well
documented;2,28-30 however, (3S,R)-compound 20 showed
moderate activity for MMP-1 and -9, while both (3R,S)-
compound 18 and (3S,S)-compound 21 showed no activ-
ity. These results suggested that the stereochemistry
of the phosphorus was more important for the activity
than that of R-carbon of the hydroxamate.
Because the (3R,R)-configuration was found to be very
important for the activity, the SAR study for the ester
group R1 was performed with (3R,R)-phosphonamide
derivatives, and the results are shown in Table 2. All
of the compounds in the series exhibited significantly
potent activity against MMPs with almost similar Ki
values. The increase of steric bulk of the R1 group
resulted in a dramatic decrease of the inhibitory activity
In the course of the study of the MMP inhibitor in
our laboratory, we have found that conversion of an
acyclic structure into a bicyclic structure resulted in the
enhancement of the inhibitory activity.24 Matter et al.
also reported that rigid bicyclic inhibiors based on the
1,2,3,4-tetrahydroisoquinoline ring were potent MMPs
inhibitors.25 Therefore, we chose the 1,2,3,4-tetrahy-
droisoquinoline ring as the X moiety in the initial SAR
study.
At first, we synthesized all four diasteromeric isomers
of the ethyl ester derivative as typical phosphonamide
inhibitors (18-21) and tested for the inhibition of MMP-
1, -3, -9, and TACE to study the effect of the stereo-
chemistry on the activities. Recombinant human colla-
genase-1 (MMP-1), stromelysin 1 (MMP-3), gelatinase
B (MMP-9), and TACE were used for these assays. We
also examined their abilities to inhibit HB-EGF release
from fibrosarcoma HT-1080 transfectants expressing
alkaline phosphatase (AP)-tagged HB-EGF stimulated
by 12-O-tetradecanoylphorbol 13-acetate (TPA).26 It can
be seen from the results (Tables 1-4) that Ki values for
MMPs and TACE inhibitions are in the nanomolar

922 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
Ta ble 3. In Vitro Profile of R2-Modified Phosphonamide
for HB-EGF shedding (compounds 23-28). The phene-
tyl derivative 27 and biphenylethyl derivative 28 were
significantly less potent for HB-EGF, and compound 28
showed a 50-fold decrease of inhibitory activity for HB-
EGF shedding compared to compound 22. These bulky
derivatives also showed a modest activity against TACE.
Incorporation of a nitrogen atom into the phenyl group
in compound 27 resulted in a dramatic improvement of
inhibitory activity for HB-EGF (compound 30), although
this replacement led to a further decrease of the
inhibitory activity against TACE. Introduction of a
diethylamino group (compound 29) resulted in a de-
crease of the inhibitory activity for MMP-3 and HB-EGF
shedding and a modest decrease for TACE. However,
no significant changes of the activities against MMP-1
and -9 were observed with these compounds. These
results suggested that MMP-3 and the ADAM enzymes
appear to be sensitive to the modification of the ester
groups in the phosphonamides, while MMP-1 and -9
have little sensitivity to the ester groups. This sensitiv-
ity to MMP-3 was supported by the fact that the
phosphinamide-based inhibitors showed similar inhibi-
tory profiles.13
Derivatives
On the basis of the results of SAR for the ester group,
the R1 group seems to have little interaction with the
S2′/S3′ site of MMPs, especially of MMP-1. Therefore,
it might be useful to introduce additional properties to
the inhibitors, such as water solubility, without any loss
of inhibitory potencies.
The effects of the substituents R2 attached to the
phosphonamide moiety were shown in Table 3. Intro-
duction of a long alkoxyalkyl chain at the para position
of the phenyl ring resulted in a significant decrease of
the inhibitory activity against MMP-1 (compound 40),
while the inhibitory activity for the other enzymes was
maintained or increased compared to that of the parent
compound 19. These results suggested that the substit-
uents R2 would bind to the S1′ pocket of MP enzymes
because this pocket is deep for most MMP enzymes but
it is short for MMP-1.10,30 On the other hand, bulky
substituents at the para position of the phenyl ring
increased the inhibitory activity for MMP-9 (compounds
41-44). It was noteworthy that compound 42 was an
extremely potent inhibitor against MMP-9, and it was
at least 250-fold potent compared with the best phos-
a
See Experimental Section for details of experimental assays.
b
Methyl ester derivatives.
a possibility of a specific interaction of the fluorine atom
with the residues in the S1′ pocket of MMP-1, such as
a hydrogen bond. The m-fluorophenyl and o-fluorophe-
nyl substituents decreased the inhibitory activity against
MMP-1 and HB-EGF shedding (compounds 35 and 36).
These lower activities of compounds 35 and 36 can be
explained not only by the narrowness of the S1′ pocket
but also by the inability of the fluorine atoms to develop
the putative hydrogen bonding.
Replacement of 1,2,3,4-tetrahydroisoquinoline ring
with other heterocycles provided insight into the struc-
tural requirements of the S1/S2 binding site (Table 4).
Many of the compounds were quite potent inhibitors of
all enzymes. â-Carboline derivative 50 was slightly less
potent for HB-EGF shedding. It was noteworthy that
reduction of ring size (proline derivative 52) resulted
in a significant loss in potency for all enzymes. This
observation may imply a limit to the dihedral angle
between hydroxamate and phosphonamide acceptable
to these enzymes.
To better understand the observed SAR, the binding
model of the 19-MMP-3CD complex was constructed
using recently published crystal structures of MMP-3
complexed with a phophinamide derivative.31 As shown
in Figures 4 and 5, the hydroxamate was tightly bound
to the zinc ion and the p-methoxyphenyl moiety was
placed in the S1′ pocket. The oxygen atom of the
phosphonamide was positioned at the hydrogen bond
distance with the main chain of Leu-164 and Ala-165.
The ethyl ester group seems to lie along the binding
phinamide-based inhibitor (R1′ ) Me; R2′ ) Ph; R3′
)
Bn; R4′ ) CH2iPr, IC50 ) 20.6 nM for MMP-9).13
Insertion of an alkyl chain between the phenyl ring and
the phosphonamide moiety resulted in a slight decrease
of the inhibitory activity for all enzymes, but the alkenyl
chain dramatically decreased the activity for MMP-1
(compounds 45 and 46). Interestingly, the aniline
derivative (compound 38) was a moderately potent
inhibitor for TACE and HB-EGF shedding, but the
pyridine derivative (compound 37) was a poor inhibitor
for those enzymes. Substitution of the p-methoxy group
in the phenyl ring with a p-methyl group (compound
33) resulted in a decrease of the potency against MMP-
3, -9, TACE, and HB-EGF shedding but did not affect
the activity for MMP-1. The p-fluorophenyl derivative
34 showed strong inhibition against MMP-1 but showed
modest activity for MMP-3 and -9. As a result, the
selectivity for MMP-1 vs MMP-3 and -9 was increased
to 66-fold and 54-fold, respectively. This result implied

Phosphonamide-Based Hydroxamic Acids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 923
Ta ble 4. In Vitro Profile of Phosphonamide Derivatives
Modified with Various Ring
MP enzymes. The tetrahydroisoquinoline ring was
stacked on a S1/S2 site and also appears to produce a
van der Waals interaction. This modeling study strongly
supported the observed results that the R-isomer at the
phosphorus center was only active, and the S-isomer
showed no potency because of the conflict with the
residues (Leu-164 and Ala-165) of MMP-3.
Con clu sion s
The design and synthesis of a new generation of
metalloproteinases inhibitors have been described.
Among the four diastereomeric isomers 18-21, com-
pound 19 having an R-configuration both at the R-car-
bon of hydroxamate and at the phosphorus atom was
found to be a potent inhibitor for all MP enzymes, while
the other diastereomeric isomers were almost inactive.
We have synthesized a number of (R,R)-derivatives
modified in each three portions, R1, R2, and X. Evalu-
ation of these compounds in in vitro assays showed that
most of compounds were potently active against all
enzymes. For the modification of the R1 group, we
showed that compound 28 with a biphenyl group was
surprisingly less potent against HB-EGF shedding
compared to compound 22. Introduction of long alkoxy-
alkyl chains for the R2 group resulted in a significant
decrease of the inhibitory activity for MMP-1 (compound
40), while the activity for the other enzymes was
maintained. In contrast, bulky substituents for the R2
group increased the inhibitory activity for MMP-9
(compounds 41-44). Regarding the SAR for the X
moiety, reduction of the ring size (proline derivative 52)
resulted in a significant decrease of the inhibitory
activity against all enzymes. Subsequent modeling work
suggested that key interactions of enzyme and inhibitor
provided the explanation for the observed stereoselec-
tivity. These results revealed the potential of phosphon-
amide derivatives as a new type of candidate for
metalloproteinase inhibitors.
a
See Experimental Section for details of experimental assays.
b Synthesized from (+)-form of the corresponding cyclic amine 14a ,
although the configuration at C-7 was not established. c Methyl
ester derivatives.
Exp er im en ta l Section
1. Gen er a l Meth od s a n d Ma ter ia ls. All commercially
available starting materials and solvents were reagent grade.
Melting points were uncorrected. 1H NMR spectra were
measured at 250 MHz on a Bruker DPX-250 using CDCl3 or
DMSO-d6 as the solvent. Mass spectra were determined on a
Perceptive Biosystems Voyager-DE RP spectrometer. Elemen-
tal analyses were performed by Sumika Chemical Analysis
Service, Ltd., J apan, and the results are within (0.4% of the
calculated values unless otherwise noted.
F igu r e 4. Docking model of MMP-3-19 complex.
(3R )-2-t er t -B u t o x y c a r b o n y l-1,2,3,4-t e t r a h y d r o is o -
qu in ole-3-ca r boxylic Acid . To a solution of (3R)-1,2,3,4-
tetrahydroisoquinole-3-carboxylic acid hydrochlorde19 (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
F igu r e 5. Expected binding mode of 19 in MMP-3.
groove of substrate backbone and to produce a van der
Waals interaction. The fact that steric bulk ester
derivatives (compounds 23-28) dramatically decreased
the potency for HB-EGF shedding suggested that the
corresponding groove in the responsible enzyme for HB-
EGF shedding might be narrow compared to the other

924 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
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 (4). (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
(19): 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); MALDI-
TOF MS m/z 429 [M + K]+, 413 [M + Na]+, 391 [M + H]+.
Anal. (C19H23N2O5P) C, H, N. Calcd: C, 58.46; H, 5.94; N, 7.18.
Found: C, 58.00; H, 6.14; N, 7.14.
Compounds 20 and 21 were synthesized from (3S)-N-
benzyloxy-1,2,3,4-tetrahydroisoquinole-3-carboxamide utilizing
similar reaction conditions.
(3S)-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
(20): colorless solids; 23% yield; 1H NMR (DMSO-d6) δ 1.16
(t, J ) 7.0 Hz, 3H), 2.90-3.05 (m, 2H), 3.70-3.90 (m, 1H),
3.81 (s, 3H), 3.90-4.05 (m, 1H), 4.16 (dd, J ) 7.6 and 16.0 Hz,
1H), 4.32 (dd, J ) 4.9 and 16.0 Hz, 1H), 4.40-4.50 (m, 1H),
7.00-7.20 (m, 6H), 7.72 (dd, J ) 8.8 and 12.4 Hz, 2H), 8.80
(br s, 1H), 10.60 (br s, 1H); MALDI-TOF MS m/z 429 [M +
K]+, 413 [M + Na]+, 391 [M + H]+. Anal. (C19H23N2O5P) C, H,
N.
(3S)-N-Benzyloxy-1,2,3,4-tetrahydroisoquinole-3-carboxam-
ide was synthesized from (3S)-1,2,3,4-tetrahydroisoquinole-3-
carboxylic acid hydrochlorde19 utilizing smilar reaction con-
ditions.
(3S)-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
1
(21): colorless solids; 25% yield; H NMR (DMSO-d6) δ 1.32
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 A). Syn th esis of (3R)-N-Hyd r oxy-
2-[(R an d S)-eth oxy(4-m eth oxyph en yl)ph osph or yl]-1,2,3,4-
tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (18 a n d 19).
Dia ster eom er s of (3R)-N-Ben zyloxy-2-[(RS)-eth oxy(4-
m e t h o x y p h e n 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 -
qu in olin e-3-ca r boxa m id e (5a ). To a solution of 4-methox-
yphenylphosphonic dichloride (398 mg, 1.77 mmol) in 5 mL of
pyridine was added (3R)-N-benzyloxy-1,2,3,4-tetrahydroiso-
quinole-3-carboxamide 4 (500 mg, 1.77 mmol, prepared from
(3R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid19 and O-
benzylhydroxylamine), 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 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).
(t, J ) 7.0 Hz, 3H), 2.90-3.15 (m, 2H), 3.76 (s, 3H), 3.95-4.20
(m, 3H), 4.40-4.60 (m, 2H), 6.90-7.10 (m, 6H), 7.56 (dd, J )
8.8 and 12.3 Hz, 2H), 8.77 (br s, 1H), 10.56 (br s, 1H); MALDI-
TOF MS m/z 429 [M + K]+, 413 [M + Na]+, 391 [M + H]+.
Anal. (C19H23N2O5P) C, H, N.
Compounds 22-24, 26-30 were also synthesized using a
procedure similar to the procedure for the preparation of 19.
(3R)-N-H yd r oxy-2-[(R)-m et h oxy(4-m et h oxyp h en yl)-
ph osph or yl]-1,2,3,4-tetr ah ydr oisoqu inoline-3-car boxam ide
(22): colorless solids; 12% yield; 1H NMR (DMSO-d6) δ 2.80-
3.05 (m, 2H), 3.48 (d, J ) 11.1 Hz, 3H), 3.80 (s, 3H), 4.14 (dd,
J ) 7.5 and 16.1 Hz, 1H), 4.30 (dd, J ) 4.9 and 16.1 Hz, 1H),
4.35-4.45 (m, 1H), 7.00-7.15 (m, 6H), 7.69 (dd, J ) 8.9 and
12.4 Hz, 2H), 8.79 (br s, 1H), 10.58 (br s, 1H); MALDI-TOF
MS m/z 415 [M + K]+, 399 [M + Na]+, 377 [M + H]+. Anal.
(C18H21N2O5P) C, H, N.
(3R)-N-Hyd r oxy-2-[(RS)-n -bu toxy(4-m eth 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 ox-
1
a m id e (23): a mixture of two diastereomers; 60% yield; H
NMR (DMSO-d6) δ 0.79 (t, J ) 7.3 Hz, 1.2H), 0.93 (t, J ) 7.3
Hz, 1.8H), 1.10-1.60 (m, 2.8H), 1.60-1.75 (m, 1.2H), 2.80-
3.20 (m, 2H), 3.76 (s, 1.8H), 3.81 (s, 1.2H), 3.80-4.60 (m, 5H),
6.85-7.20 (m, 6H), 7.55 (dd, J ) 8.8 and 12.3 Hz, 1.2H), 7.72
(dd, J ) 8.8 and 12.4 Hz, 0.8H), 8.76 (s, 0.6H), 8.79 (s, 0.4H),
10.57 (s, 1H); MALDI-TOF MS m/z 457 [M + K]+, 441 [M +
Na]+, 419 [M + H]+. Anal. (C21H27N2O5P) C, H, N.
(3R)-N-Hyd r oxy-2-[(R)-n -h exyloxy(4-m eth 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 ox-
(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 (18 a n d 19). A mixture of compound 5 (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 acid.
19 (80 mg, 21%) was eluted first, and then 18 (127 mg, 34%)
was eluted.
1
a m id e (24): colorless solids; 8% yield; H NMR (DMSO-d6) δ
0.78 (t, J ) 6.6 Hz, 3H), 1.00-1.30 (m, 6H), 1.45-1.55 (m, 2H),
2.85-3.05 (m, 2H), 3.60-3.70 (m, 1H), 3.80 (s, 3H), 3.80-3.90
(m, 1H), 4.08 (dd, J ) 7.9 and 16.1 Hz, 1H), 4.29 (dd, J ) 4.8
and 16.1 Hz, 1H), 4.40-4.50 (m, 1H), 7.03 (dd, J ) 3.0 and
8.7 Hz, 2H), 7.00-7.15 (m, 4H), 7.70 (dd, J ) 8.7 and 12.4 Hz,
2H), 8.77 (br s, 1H), 10.58 (br s, 1H); MALDI-TOF MS m/z
485 [M + K]+, 469 [M + Na]+, 447 [M + H]+. Anal.
(C23H31N2O5P) C, H, N.
(3R)-N-H yd r oxy-2-[(R)-cycloh exylm et h yloxy(4-m et h -
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 boxa m id e (26): colorless solids; 13% yield; 1H NMR
(DMSO-d6) δ 0.75-1.00 (m, 2H), 1.00-1.20 (m, 3H), 1.30-1.70
(m, 6H), 2.80-3.10 (m, 2H), 3.40-3.50 (m, 1H), 3.60-3.75 (m,
1H), 3.81 (s, 3H), 4.09 (dd, J ) 7.8 and 16.2 Hz, 1H), 4.30 (dd,
J ) 5.1 and 16.2 Hz, 1H), 4.40-4.50 (m, 1H), 7.00-7.15 (m,
6H), 7.72 (dd, J ) 8.7 and 12.4 Hz, 2H), 8.79 (s, 1H), 10.58 (s,
1H); MALDI-TOF MS m/z 497 [M + K]+, 481 [M + Na]+, 459
[M + H]+. Anal. (C24H31N2O5P) 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
(18): 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 m/z 429 [M + K]+, 413 [M + Na]+, 391 [M + H]+.
Anal. (C19H23N2O5P) C, H, N.

Phosphonamide-Based Hydroxamic Acids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 925
(3R)-N-Hydroxy-2-[(RS)-phenethyloxy(4-methoxyphenyl)-
phosphor yl]-1,2,3,4-tetr ah ydr oisoqu in olin e-3-car boxam ide
(27): a mixture of two diastereomers; 8% yield; 1H NMR
(DMSO-d6) δ 2.83 (t, J ) 7.2 Hz, 1H), 2.90-3.05 (m, 3H), 3.74
(s, 1.5H), 3.80 (s, 1.5H), 3.80-4.55 (m, 5H), 6.80-7.35 (m,
11H), 7.45 (dd, J ) 8.7 and 12.2 Hz, 1H), 7.63 (dd, J ) 8.7
and 12.5 Hz, 1H), 8.75 (br s, 0.5H), 8.78 (br s, 0.5H), 10.53 (br
s, 0.5H), 10.57 (br s, 0.5H); MALDI-TOF MS m/z 505 [M +
K]+, 489 [M + Na]+, 467 [M + H]+. Anal. (C25H27N2O5P) C, H,
N.
(3R)-N-H yd r oxy-2-[(R)-(2-b ip h en yl-4-yl-et h yloxy)(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 (28): colorless solids; 20% yield; 1H NMR
(DMSO-d6) δ 2.80-3.00 (m, 4H), 3.79 (s, 3H), 3.80-4.20 (m,
3H), 4.20-4.30 (m, 1H), 4.40-4.50 (m, 1H), 6.90-7.10 (m, 3H),
7.00-7.10 (m, 3H), 7.21 (d, J ) 8.3 Hz, 2H), 7.30-7.40 (m,
1H), 7.44 (t, J ) 7.2 Hz, 2H), 7.52 (d, J ) 9.0 Hz, 2H), 7.60-
7.70 (m, 4H), 8.78 (br s, 1H), 10.59 (br s, 1H); MALDI-TOF
MS m/z 581 [M + K]+, 565 [M + Na]+, 543 [M + H]+. Anal.
(C31H31N2O5P) C, H, N.
(3R )-N -H yd r oxy-2-[(R )-(2-d ie t h yla m in oe t h yloxy)(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 (29): colorless solids; 6% yield; 1H NMR
(DMSO-d6) δ 1.14 (t, J ) 7.4 Hz, 3H), 1.16 (t J ) 7.1 Hz, 3H),
2.95-3.00 (m, 2H), 3.05-3.20 (m, 4H), 3.30-3.40 (m, 2H), 3.81
(s, 3H), 4.00-4.10 (m, 1H), 4.20-4.40 (m, 4H), 7.00-7.15 (m,
6H), 7.76 (dd, J ) 8.8 and 12.8 Hz, 2H), 8.84 (br s, 1H), 10.64
(br s, 1H); MALDI TOF MS m/z 500 [M + K]+, 484 [M + Na]+,
462 [M + H]+. Anal. (C23H32N3O5P) C, H, N.
(3R)-N-H yd r oxy-2-{(R)-[2-(p yr id in e-2-yl)et h yloxy](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 (30): pale-yellow solids; 9% yield; 1H
NMR (DMSO-d6) δ 2.90-3.05 (m, 4H), 3.78 (s, 3H), 3.90-4.10
(m, 2H), 4.15-4.25 (m, 2H), 4.40-4.45 (m, 1H), 6.95-7.25 (m,
9H), 7.55-7.70 (m, 3H), 8.35-8.40 (m, 1H), 8.78 (br s, 1H),
10.58 (br s, 1H); MALDI TOF MS m/z 506 [M + K]+, 490 [M
+ Na]+, 468 [M + H]+. Anal. (C24H26N3O5P) C, H, N.
Compound 32 was synthesized from commercially available
phenylphosphonic dichloride and methanol using a procedure
similar to the procedure for the preparation of 19.
tion 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 diester 8 as a yellow oil
(532 mg, 72%): 1H NMR (CDCl3) δ 1.18 (t, J ) 7.0 Hz, 6H),
3.49 (t, J ) 7.0 Hz, 4H), 3.60-3.70 (m, 4H), 3.85 (s, 3H), 4.10-
4.25 (m, 4H), 6.95 (dd, J ) 3.5 and 8.9 Hz, 2H), 7.78 (dd, J )
8.9 and 12.9 Hz, 2H).
Dia ster eom er s of (3R)-N-ben zyloxy-2-[(RS)-(2-eth oxy-
eth yloxy)(4-m eth oxyp h en yl)p h osp h or yl]-1,2,3,4-tetr a h y-
d r oisoqu in olin e-3-ca r boxa m id e (5b). A mixture of (4-
methoxyphenyl)phosphonic acid bis(2-ethoxyethyl) ester 8 (530
mg, 1.59 mmol) and a catalytic amount of DMF (a few drops)
in thionyl chloride (5 mL) was refluxed for 3 h. The reaction
mixture was concentrated in vacuo, and the residual thionyl
chloride was completely removed by azeotropic evaporation
with toluene to afford 469 mg of crude monochloride 9, which
was used for the next reaction without further purification.
To a solution of the above product 9 in 5 mL of dry THF was
added (3R)-N-benzyloxy-1,2,3,4-tetrahydroisoquinole-3-car-
boxamide 4 (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 the title compound 5b as a diasteromeric
mixture (1:1, 435 mg, 78%): 1H NMR (CDCl3) δ 1.17 (t, J )
7.0 Hz, 1.5H), 1.18 (t, J ) 7.0 Hz, 1.5H), 2.90-3.65 (m, 6H),
3.80 (s, 1.5H), 3.85 (s, 1.5H), 3.90-4.20 (m, 4H), 4.35-4.45
(m, 0.5 H), 4.55-4.65 (m, 0.5H), 4.75-4.85 (m, 2H), 6.80-7.65
(m, 13H), 9.91 (br s, 0.5H), 10.24 (br s, 0.5H).
(3R)-N-Hyd r oxy-2-[(R)-(2-eth oxyeth yloxy)(4-m eth oxy-
p 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 boxa m id e (31). The title compound was prepared from
5b following the precedure described for compound 19: color-
1
less solids; 31% yield; H NMR (DMSO-d6) δ 1.00 (t, J ) 7.0
Hz, 3H), 2.90-3.20 (m, 2H), 3.30-3.40 (m, 2H), 3.45-3.55 (m,
2H), 3.80 (s, 3H), 3.75-3.90 (m, 1H), 3.90-4.05 (m, 1H), 4.10-
4.35 (m, 2H), 4.45-4.50 (m, 1H), 7.00-7.15 (m, 6H), 7.72 (dd,
J ) 8.8 and 12.5 Hz, 2H), 8.77 (br s, 1H), 10.57 (br s, 1H);
MALDI-TOF MS m/z 473 [M + K]+, 457 [M + Na]+, 435 [M +
H]+. Anal. (C21H27N2O6P‚0.5H2O) C, H, N.
(3R)-N-Hyd r oxy-2-[(R)-m eth oxy(p 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 boxa m id e (32): color-
less solids; 22% yield; 1H NMR (DMSO-d6) δ 2.85-3.05 (m,
2H), 3.51 (d, J ) 11.1 Hz, 3H), 4.18 (dd, J ) 7.5 and 16.2 Hz,
1H), 4.25-4.45 (m, 2H), 7.05-7.15 (m, 4H), 7.45-7.60 (m, 3H),
7.70-7.80 (m, 2H), 8.80 (br s, 1H), 10.61 (br s, 1H); MALDI-
TOF MS m/z 385 [M + K]+, 369 [M + Na]+, 347 [M + H]+.
Anal. (C17H19N2O4P) C, H, N.
Compounds 25 were also synthesized using a procedure
similar to the procedure for the preparation of 31.
(3R)-N-Hydr oxy-2-[(R)-isopr opyloxy(4-m eth oxyph en yl)-
Compound 50 was synthesized from (3R)-N-benzyloxy-
1,3,4,9-tetrahydro-â-carboline-3-carboxamide and methanol
using a procedure similar to the procedure for the preparation
of 19.
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 ox-
a m id e (25): a mixture of two diastereomers; 56% yield; H
1
NMR (DMSO-d6) δ 1.03 (d, J ) 6.1 Hz, 1.5H), 1.27 (d, J ) 6.2
Hz, 1.5H), 1.31 (d, J ) 6.2 Hz, 1.5H), 1.34 (d, J ) 6.2 Hz, 1.5H),
2.90-3.15 (m, 2H), 3.75 (s, 1.5H), 3.81 (s, 1.5H), 3.90-4.20
(m, 1H), 4.32 (dd, J ) 4.4 and 16.2 Hz, 0.5H), 4.40-4.60 (m,
2H), 4.70-4.80 (m, 0.5H), 6.85-7.20 (m, 6H), 7.53 (dd, J )
8.7 and 12.3 Hz, 1H), 7.73 (dd, J ) 8.7 and 12.5 Hz, 1H), 8.77
(s, 0.5H), 8.78 (s, 0.5H), 10.58 (s, 1H); MALDI-TOF MS m/z
443 [M + K]+, 427 [M + Na]+, 405 [M + H]+. Anal.
(C20H25N2O5P) 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 C). Syn th esis of (3R)-N-Hyd r oxy-
2-[(R)-eth oxy(4-p h en oxyp h en yl)p h osp h or yl]-1,2,3,4-tet-
r a h yd r oisoq u in olin e-3-ca r b oxa m id e (42). 4-P h en oxy-
p h en ylp h osp h on ic Acid Dieth yl Ester (10). A mixture of
diethyl phosphite (2.50 g, 18.1 mmol), triethylamine (2.50 mL,
18.1 mmol), 4-bromodiphenyl ether (4.10 g, 16.5 mmol), and
tetrakis(triphenylphosphine)palladium (0.82 g) was stirred at
90 °C under an argon atmosphere overnight, then cooled to
room temperature. Diethyl ether was added to the reaction
mixture, and the precipitate was filtered off. The filtrate was
concentrated in vacuo, and the residue was purified by column
chromatography on silica gel, eluting with a gradient of
n-hexane/AcOEt 3:2 to AcOEt to give the title compound as a
brown oil (5.09 g, 100%): 1H NMR (CDCl3) δ 1.34 (t, J ) 7.0
(3R)-N-Hydr oxy-2-[(R)-m eth oxy(4-m eth oxyph en yl)ph os-
ph or yl]-1,3,4,9-tetr ah ydr o-â-car bolin e-3-car boxam ide (50):
colorless solids; 5% yield; 1H NMR (DMSO-d6) δ 2.80 (dd, J )
5.7 and 15.6 Hz, 1H), 3.02 (d, J ) 15.6 Hz, 1H), 3.55 (d, J )
11.2 Hz, 3H), 3.80 (s, 3H), 4.22 (dd, J ) 8.2 and 15.0 Hz, 1H),
4.43 (d, J ) 15.0 Hz, 1H), 4.57 (dd, J ) 5.7 and 8.2 Hz, 1H),
6.90-7.10 (m, 4H), 7.25 (d, J ) 7.7 Hz, 1H), 7.33 (d, J ) 7.4
Hz, 1H), 7.73 (dd, J ) 8.8 and 12.4 Hz, 2H), 8.70 (br s, 1H),
10.58 (br s, 1H), 10.70 (br s, 1H); MALDI-TOF MS m/z 454
[M + K]+, 438 [M + Na]+, 416 [M + H]+. Anal. (C20H22N3O5P)
C, H, N.
Alter n a tive 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)-(2-eth oxyeth yloxy)(4-m eth oxyph en yl)ph osph or yl]-
1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (31). (4-
Met h oxyp h en yl)p h osp h on ic Acid Bis(2-et h oxyet h yl)
Ester (8). To a solution of 2-ethoxyethanol (440 mg, 4.89
mmol) in dry THF (50 mL) was added NaH (130 mg, 5.4 mmol),
and the mixture was stirred at room temperature under an
argon atmosphere for 20 min. Then, 4-methoxyphenylphos-
phonic dichloride (500 mg, 2.22 mmol) was added to the
mixture, and the stirring was continued overnight. The reac-

926 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
Hz, 6H), 4.00-4.30 (m, 4H), 7.00-7.15 (m, 4H), 7.15-7.30 (m,
1H), 7.35-7.50 (m, 2H), 7.70-7.85 (m, 2H).
(3R)-N-H yd r oxy-2-[(R)-et h oxy(2-flu or op 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
(36): colorless solids; 8% yield; H NMR (DMSO-d6) δ 1.18 (t,
Dia ster eom er s of (3R)-N-Ben zyloxy-2-[(R)-eth oxy(4-
p h en 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 (12). A mixture of 4-phenoxyphenylphos-
phonic acid diethyl ester 10 (5.0 g, 16.3 mmol) and a catalytic
amount of DMF (a few drops) in thionyl chloride (5 mL) was
refluxed for 1.5 h. The reaction mixture was concentrated in
vacuo, and the residual thionyl chloride was completely
removed by azeotropic evaporation with toluene to afford 4.5
g of monochloride 11, which was used for the next reaction
without further purification. To a solution of the above product
11 (180 mg, 0.60 mmol) in 2 mL of dry CH2Cl2 was added (3R)-
N-benzyloxy-1,2,3,4-tetrahydroisoquinole-3-carboxamide 4 (170
mg, 0.60 mmol) and diisopropylethylamine (150 mg, 1.16
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 chroma-
tography on silica gel, eluting with AcOEt to give the title
compound 12 as a diasteromeric mixture (1:1, 140 mg, 47%):
1H NMR (CDCl3) δ 1.10-1.40 (m, 3H), 2.70-3.00 (m, 1H),
3.25-3.40 (m, 1H), 3.70-4.20 (m, 4H), 4.25-4.60 (m, 1H),
4.75-4.90 (m, 2H), 6.80-7.70 (m, 18H), 9.70 (s, 0.5H), 10.07
(s, 0.5H).
J ) 7.0 Hz, 3H), 2.91 (d, J ) 4.6 Hz, 2H), 3.75-3.90 (m, 1H),
3.95-4.10 (m, 1H), 4.32 (dd, J ) 8.1 and 16.3 Hz, 1H), 4.35-
4.45 (m, 1H), 4.50 (dd, J ) 4.3 and 16.3 Hz, 1H), 7.05-7.20
(m, 4H), 7.20-7.35 (m, 2H), 7.62 (q, J ) 7.0 Hz, 1H), 7.75-
7.95 (m, 1H), 8.78 (br s, 1H), 10.57 (br s, 1H); MALDI-TOF
MS m/z 417 [M + K]+, 401 [M + Na]+, 379 [M + H]+. Anal.
(C18H20FN2O4P) C, H, N.
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(4-p yr id yl)p h osp h or yl]-
1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (37): color-
1
less solids; 13% yield; H NMR (DMSO-d6) δ 1.20 (t, J ) 7.0
Hz, 3H), 2.85-3.20 (m, 2H), 3.75-3.95 (m, 1H), 3.95-4.10 (m,
1H), 4.15-4.35 (m, 2H), 4.40-4.55 (m, 1H), 7.05-7.25 (m, 4H),
7.70 (dd, J ) 5.9 and 12.9 Hz, 2H), 8.70-8.80 (m, 2H), 8.82
(br s, 1H), 10.64 (br s, 1H); MALDI-TOF MS m/z 400 [M +
K]+, 384 [M + Na]+, 362 [M + H]+. Anal. (C17H20N3O4P) C, H,
N.
(3R)-N-H yd r oxy-2-[(R)-et h oxy(4-a m in op h en yl)p h os-
ph or yl]-1,2,3,4-tetr ah ydr oisoqu in olin e-3-car boxam ide tr i-
flu or oa cetic a cid sa lt (38): colorless solids; 12% yield; 1H
NMR (DMSO-d6) δ 1.12 (t, J ) 7.0 Hz, 3H), 2.85-2.95 (m, 2H),
3.60-3.80 (m, 1H), 3.80-4.00 (m, 1H), 4.13 (dd, J ) 7.7 and
16.3 Hz, 1H), 4.25-4.45 (m, 2H), 5.70 (br s, 2H), 6.56 (dd, J )
3.5 and 8.5 Hz, 2H), 7.00-7.15 (m, 4H), 7.37 (dd, J ) 8.5 and
12.5 Hz, 2H), 8.76 (s, 1H), 10.52 (s, 1H); MALDI-TOF MS m/z
414 [M + K]+, 398 [M + Na]+, 376 [M + H]+. Anal.
(C20H23F3N3O6P) C, H, N.
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(4-p h en 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
(42). The title compound was prepared from 12 following the
precedure described for compound 19: yield 14%; 1H NMR
(DMSO-d6) δ 1.15 (t, J ) 6.9 Hz, 3H), 2.85-3.10 (m, 2H), 3.65-
4.00 (m, 2H), 4.10-4.50 (m, 3H), 7.00-7.30 (m, 9H), 7.35-
7.50 (m, 2H), 7.70-7.85 (m, 2H), 8.80 (s, 1H), 10.60 (s, 1H);
MALDI-TOF MS m/z 491 [M + K]+, 475 [M + Na]+, 453 [M +
H]+. Anal. (C24H25N2O5P‚0.66H2O) C, H, N.
Compounds 33-38, 40, 41, 43, 44, 46 were also synthesized
using a procedure similar to the procedure for the preparation
of 42. Compounds 39 and 45 were synthesized from the
corresponding methyl ester derivative instead of the benzyl-
oxyamide derivative using a procedure similar to the procedure
for the preparation of 12. The obtained methyl esters required
base hydrolysis and amidation to obtain the corresponding
hydroxamic acids 39 and 45, using a procedure similar to
procedure for the preparation of compound 48.
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(2-th ien yl)p h osp h or yl]-
1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (39): color-
less solids; 6% yield; 1H NMR (DMSO-d6) δ 1.18 (t, J ) 7.0
Hz, 3H), 2.70-3.20 (m, 2H), 3.75-4.00 (m, 2H), 4.25 (dd, J )
8.0 and 16.3 Hz, 1H), 4.30-4.50 (m, 2H), 7.00-7.30 (m, 5H),
7.65 (dd, J ) 3.4 and 8.1 Hz, 1H), 7.96 (t, J ) 4.8 Hz, 1H),
8.80 (s, 1H), 10.55(s, 1H); MALDI-TOF MS m/z 405 [M + K]+,
389 [M + Na]+, 367 [M + H]+. Anal. (C16H19N2O4PS) C, H, N.
(3R)-N-Hydr oxy-2-{(R)-eth oxy[4-(2-eth oxyeth oxy)ph en -
yl]phosphoryl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
1
(40): colorless solids; 8% yield; H NMR (DMSO-d6) δ 1.12 (t,
J ) 7.0 Hz, 3H), 1.15 (t, J ) 7.1 Hz, 3H), 2.90-3.00 (m, 2H),
3.49 (q, J ) 7.0 Hz, 2H), 3.65-3.85 (m, 3H), 3.85-4.05 (m,
1H), 4.05-4.25 (m, 3H), 4.25-4.50 (m, 2H), 6.95-7.15 (m, 6H),
7.69 (dd, J ) 8.8 and 12.5 Hz, 2H), 8.78 (s, 1H), 10.57 (br s,
1H); MALDI-TOF MS m/z 487 [M + K]+, 471 [M + Na]+, 449
[M + H]+. Anal. (C22H29N2O6P) C, H, N.
(3R)-N-H yd r oxy-2-[(R)-et h oxy(4-m et h ylp 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
(33): colorless solids; 32% yield; 1H NMR (CDCl3) δ 1.22 (t, J
) 7.1 Hz, 3H), 2.41 (s, 3H), 2.99 (dd, J ) 6.5 and 16.0 Hz,
1H), 3.32 (dd, J ) 4.0 and 16.0 Hz, 1H), 3.75-4.25 (m, 4H),
4.55-4.70 (m, 1H), 6.93 (d, J ) 6.8 Hz, 1H), 7.05-7.35 (m,
5H), 7.55 (br s, 1H), 7.63 (d, J ) 13.0 Hz, 1H), 7.68 (d, J )
13.0 Hz, 1H), 10.36 (br s, 1H); MALDI-TOF MS m/z 413 [M +
K]+, 397 [M + Na]+, 375 [M + H]+. Anal. (C19H23N2O4P) C, H,
N. Calcd: C, 60.96; H, 6.19; N, 7.48. Found: C, 60.47; H, 6.48;
N, 7.16.
(3R)-N-Hydr oxy-2-[(R)-eth oxy(4-biph en yl)ph osph or yl]-
1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (41): color-
1
less solids; 16% yield; H NMR (DMSO-d6) δ 1.19 (t, J ) 7.1
Hz, 3H), 2.85-3.15 (m, 2H), 3.70-3.90 (m, 1H), 3.90-4.10 (m,
1H), 4.22 (dd, J ) 7.5 and 15.9 Hz, 1H), 4.37 (dd, J ) 4.3 and
15.9 Hz, 1H), 4.45-4.55 (m, 1H), 7.00-7.20 (m, 4H), 7.30-
7.60 (m, 3H), 7.60-7.95 (m, 6H), 8.81 (s, 1H), 10.62 (s, 1H);
MALDI-TOF MS m/z 475 [M + K]+, 459 [M + Na]+, 437 [M +
H]+. Anal. (C24H25N2O4P) C, H, N.
(3R)-N-H yd r oxy-2-{(R)-et h oxy[4-(p yr id in e-4-yl-oxy)-
p 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 boxa m id e tr iflu or oa cetic a cid sa lt (43): colorless solids;
9% yield; 1H NMR (DMSO-d6) δ 1.10-1.30 (m, 3H), 2.80-3.10
(m, 2H), 3.70-3.90 (m, 1H), 3.90-4.10 (m, 1H), 4.10-4.40 (m,
2H), 4.40-4.55 (m, 1H), 7.00-7.20 (m, 4H), 7.20-7.30 (m, 2H),
7.30-7.40 (m, 2H), 7.85-8.00 (m, 2H), 8.60-8.75 (m, 2H),
10.70 (s, 1H); MALDI-TOF MS m/z 492 [M + K]+, 476 [M +
Na]+, 454 [M + H]+. Anal. (C25H25F3N3O7P) C, H, N.
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(4-flu or oyp 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
(34): colorless solids; 4% yield; H NMR (DMSO-d6) δ 1.00-
1.40 (m, 3H), 2.90-3.15 (m, 2H), 3.70-3.90 (m, 1H), 3.90-
4.10 (m, 1H), 4.10-4.40 (m, 2H), 4.40-4.55 (m, 1H), 7.00-
7.20 (m, 4H), 7.25-7.45 (m, 2H), 7.80-7.95 (m, 2H), 8.80 (s,
1H), 10.62 (s, 1H); MALDI-TOF MS m/z 417 [M + K]+, 401
[M + Na]+, 379 [M + H]+. Anal. (C18H20FN2O4P) C, H, N.
(3R)-N-H yd r oxy-2-[(R)-et h oxy(3-flu or op h en yl)p h os-
(3R)-N-Hyd r oxy-2-{(R)-eth oxy[4-(4-a m in op h en yloxy)-
p 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 boxa m id e (44): colorless solids; 18% yield; 1H NMR
(DMSO-d6) δ 1.15 (t, J ) 7.1 Hz, 3H), 2.85-3.05 (m, 2H), 3.65-
3.80 (m,1H), 3.85-4.00 (m, 1H), 4.05-4.20 (m, 1H), 4.25-4.35
(m, 1H), 4.35-4.50 (m,1H), 6.95-7.15 (m, 10H), 7.74 (d, J )
12.5 Hz, 1H), 7.76 (d, J ) 12.5 Hz, 1H), 10.59 (s,1H); MALDI-
TOF MS m/z 506 [M + K]+, 490 [M + Na]+, 468 [M + H]+.
Anal. (C24H26N3O5P) C, H, N.
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
(35): colorless solids; 18% yield; H NMR (DMSO-d6) δ 1.20
(t, J ) 7.0 Hz, 3H), 2.90-3.10 (m, 2H), 3.70-3.90 (m, 1H),
3.90-4.10 (m, 1H), 4.19 (dd, J ) 7.6 and 16.0 Hz, 1H), 4.31
(dd, J ) 4.9 and 16.0 Hz, 1H), 4.45-4.55 (m, 1H), 7.05-7.20
(m, 4H), 7.40-7.50 (m, 1H), 7.50-7.70 (m, 3H), 8.84 (br s, 1H),
10.65 (br s, 1H); MALDI-TOF MS m/z 417 [M + K]+, 401 [M
+ Na]+, 379 [M + H]+. Anal. (C18H20FN2O4P) C, H, N.

Phosphonamide-Based Hydroxamic Acids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 927
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(styr yl)p h osp h or yl]-1,2,
3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (45): colorless
solids; 6% yield; 1H NMR (DMSO-d6) δ 1.15 (t, J ) 7.1 Hz,
3H), 3.00-3.10 (m, 2H), 3.70-3.85 (m, 1H), 3.85-4.05 (m, 1H),
4.23 (dd, J ) 7.1 and 16.2 Hz, 1H), 4.30-4.50 (m, 2H), 6.62 (t,
J ) 17.9 Hz, 1H), 7.05-7.20 (m, 4H), 7.20-7.50 (m, 4H), 7.55-
7.65 (m, 2H), 8.78 (s, 1H), 10.56 (br s, 1H); MALDI-TOF MS
m/z 425 [M + K]+, 409 [M + Na]+, 387 [M + H]+. Anal.
(C20H23N2O4P) C, H, N.
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(2-p h en yleth yl)p h osp h o-
r yl]-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e (46):
colorless solids; 29% yield; 1H NMR (DMSO-d6) δ 1.08 (t, J )
7.0 Hz, 3H), 1.95-2.25 (m, 2H), 2.60-2.90 (m, 2H), 2.95-3.15
(m, 2H), 3.50-3.75 (m, 1H), 3.75-3.95 (m, 1H), 4.21 (dd, J )
6.8 and 16.0 Hz, 1H), 4.30-4.50 (m, 2H), 7.05-7.35 (m, 9H),
8.77 (s, 1H), 10.56 (s, 1H); MALDI-TOF MS m/z 427 [M + K]+,
411 [M + Na]+, 389 [M + H]+. Anal. (C20H25N2O4P) C, H, N.
3H), 7.02 (dd, J ) 3.0 and 8.7 Hz, 2H), 7.68 (dd, J ) 8.7, 12.2
Hz, 2H), 8.79 (br s, 1H), 10.43 (br s, 1H); MALDI-TOF MS
m/z 367 [M + K]+, 351 [M + Na]+, 329 [M + H]+. Anal.
(C14H21N2O5P) C, H, N.
Compounds 16b were synthesized from the corresponding
methyl ester derivatives 14b instead of from benzyloxyamide
derivatives 14a using a procedure similar to the procedure for
the preparation of 16a . The methyl esters 16b required the
following base hydrolysis and amidation to obtain the corre-
sponding hydroxamic acids 49 and 51.
(3R)-N-Hyd r oxy-5-[(R)-eth oxy(4-m eth oxyp h en yl)p h os-
p h or yl]-4,5,6,7-t et r a h yd r ot h ien o[3,2-c]p yr id in e-6-ca r -
boxa m id e (49). To a solution of 5-[ethoxy-(4-methoxyphenyl)-
ph osph in oyl]-4,5,6,7-t et r a h ydr ot h ien o[3,2-c]pyr idin e-6-
carboxylic acid methyl ester 16b (0.60 g, 1.5 mmol) in 6 mL of
DME was added 3 mL of 1 N NaOH, and the solution was
stirred for 3 h. The reaction mixture was then neutralized with
1 N HCl and extracted with EtOAc. The organic layer was
washed with water, dried over Na2SO4, and evaporated to
afford 0.49 g of the corresponding carboxylic acid. To a solution
of the resulting acid (0.49 g, 1.3 mmol) and triethylamine (1.0
g, 9.9 mmol) in DMF (10 mL) was added WSC (0.31 g, 1.6
mmol) and HOBt (0.25 g, 1.6 mmol) at 0 °C, and the mixture
was stirred for 30 min. Then, hydroxylamine hydrochloride
(1.0 g, 14 mmol) was added to the reaction mixture, and the
stirring was continued overnight. The reaction mixture was
diluted with water and extracted with AcOEt. The organic
layer was dried over Na2SO4 and evaporated. The residue was
purified by HPLC (YMC-ODS, CH3CN/water 72:28), and the
fraction eluted first was lyophilized to afford the title com-
pound 49 (53 mg, 10%) as coloress solids: 1H NMR (DMSO-
d6) δ 1.18 (t, J ) 7.0 Hz, 3H), 2.90-3.15 (m, 2H), 3.82 (s, 3H),
3.70-4.45 (m, 4H), 4.62 (dd, J ) 5.6 and 9.7 Hz, 1H), 6.81 (d,
J ) 5.2 Hz, 1H), 7.04 (dd, J ) 3.1 and 8.7 Hz, 2H), 7.27 (d, J
) 5.2 Hz, 1H), 7.72 (dd, J ) 8.7 and 12.5 Hz, 2H), 8.80 (s,
1H), 10.58 (s, 1H); MALDI-TOF MS m/z 435 [M + K]+, 419
[M + Na]+, 397 [M + H]+. Anal. (C17H21N2O5PS) 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 D). Syn th esis of (3R)-N-Hyd r oxy-
2-[(R)-et h oxy(4-m et h oxyp h en yl)p h osp h or yl]-7-a m in o-
1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxa m id e
(47).
Diaster eom er s of (3R)-N-Ben zyloxy-2-[(R)-eth oxy(4-m eth -
oxyp h e n yl)p h osp h or yl]-7-n it r o-1,2,3,4-t e t r a h yd r oiso-
qu in olin e-3-ca r boxa m id e (16a ). To a solution of (4-meth-
oxyphenyl)chlorophosphonic acid ethyl ester 15 (470 mg, 2.00
mmol, prepared from (4-methoxyphenyl)phosphonic acid di-
ethyl ester) in 7 mL of dry CH2Cl2 was added (3R)-N-
benzyloxy-7-nitro-1,2,3,4-tetrahydroisoquinole-3-carboxam-
ide 14a (628 mg, 1.92 mmol) and diisopropylethylamine (1
mL), and the mixture was stirred at room temperature under
an argon atmosphere for 14 h. The reaction mixture was
diluted with CHCl3. 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 CHCl3/
MeOH 50:1 to give the title compound 16a as a diasteromeric
mixture (1:1, 820 mg, 81%): 1H NMR (CDCl3) δ 1.23 (t, J )
7.0 Hz, 1.5H), 1.34 (t, J ) 7.0 Hz, 1.5H), 2.75-3.05 (m, 1H),
3.35-3.55 (m, 1H), 3.80-3.90 (m, 4H), 3.90-4.20 (m, 3H),
4.20-4.40 (m, 2H), 4.55-4.65 (m, 0.5H), 4.80-4.95 (m, 2H),
6.85-7.05 (m, 2H), 7.36 (br, 5H), 7.45-7.90 (m, 4H), 7.95-
8.10 (m, 1H), 9.99 (br, 0.5H), 10.31 (br, 0.5H).
(3R)-N-Hyd r oxy-2-[(R)-eth oxy(4-m eth oxyp h en yl)p h os-
p h or yl]-7-a m in o-1,2,3,4-t et r a h yd r oisoq u in olin e-3-ca r -
boxa m id e (47). The title compound was prepared from 16a
following the precedure described for compound 19: yield 16%;
1H NMR (DMSO-d6) δ 1.16 (qn, J ) 6.5 Hz, 3H), 2.75-3.10
(m, 2H), 3.70-3.90 (m, 3H), 3.90-4.10 (m, 2H), 4.10-4.35 (m,
2H), 4.35-4.50 (m, 1H), 6.45-6.75 (m, 1H), 6.79 (s, 1H), 6.85
(d, J ) 8.3 Hz, 1H), 6.90-7.10 (m, 2H), 7.50-7.75 (m, 2H),
8.70 (br, 1H), 10.59 (br, 1H); MALDI-TOF MS m/z 444 [M +
K]+, 428 [M + Na]+, 406 [M + H]+. Anal. (C19H24N3O5P) C, H,
N.
Compounds 48 and 52 were also synthesized using a
procedure similar to the procedure for the preparation of 47.
The corresponding amines 14a were prepared from the cor-
responding amino acids utilizing the Pictet-Spengler reaction
condition.21
N-Hyd r oxy-6-[(R)-eth oxy(4-m eth oxyp h en yl)p h osp h o-
r yl]-5,6,7,8-tetr a h yd r op yr id o[3,4-b]p yr a zin e-7-ca r boxa -
m id e (48). The title compound was prepared from (+)-N-
ben zyloxy-5,6,7,8-t et r a h yd r opyr id o[3,4-b]p yr a zin e-7-
carboxamide hydrochloride24 following the precedure described
for compound 19: yield 16%; colorless solids; 20% yield; 1H
NMR (DMSO-d6) δ 1.20 (t, J ) 7.0 Hz, 3H), 2.90-3.25 (m, 2H),
3.84 (s, 3H), 3.75-4.15 (m, 2H), 4.25-4.50 (m, 2H), 4.50-4.65
(m, 1H), 7.00-7.10 (m, 2H), 7.60-7.70 (m, 2H), 8.40-8.45 (m,
2H), 8.90 (br s, 1H), 10.84 (br s, 1H); MALDI-TOF MS m/z
431 [M + K]+, 415 [M + Na]+, 393 [M + H]+. Anal.
(C17H21N4O5P) C, H, N.
(2R)-N-Hydr oxy-4-ben zyloxycar bon yl-1-[eth oxy(4-m eth -
oxyp h en yl)p h osp h or yl]p ip er a zin e-2-ca r boxa m id e (51):
1
colorless solids; 8% yield; H NMR (DMSO-d6) δ 1.25 (t, J )
7.0 Hz, 3H), 2.85 (br, 1H), 3.00-3.15 (m, 2H), 3.30-3.50 (m,
1H), 3.79 (s, 3H), 3.86 (d, J ) 11.8 Hz, 1H), 3.97 (q, J ) 10.0
Hz, 2H), 4.05-4.15 (m, 1H), 4.18 (d, J ) 13.6 Hz, 1H), 5.03
(br, 2H), 7.01 (dd, J ) 3.1 and 8.8 Hz, 2H), 7.20-7.40 (m, 5H),
7.69 (dd, J ) 8.8 and 12.4 Hz, 2H), 8.86 (br, 1H), 10.57 (br,
1H); MALDI-TOF MS m/z 516 [M + K]+, 500 [M + Na]+, 478
[M + H]+. Anal. (C22H28N3O7P) C, H, N.
2. Meta llop r otein a se In h ibition Assa y. 2.1. MMP In -
h ibition Assa y. 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.
The 5′-end of each PCR primer was added to a sequence for
the appropriate restriction enzyme site. Amplified DNA frag-
ments 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 incubation with 1 mM p-aminophenylmer-
curic acetate for 1 h at 37 °C. Test compounds were dissolved
in DMSO and diluted with reaction buffer (50 mM Tris, 150
mM NaCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.5). A total of 25
µL of compound solution was mixed with 25 µL of diluted
enzyme solution in a well of a 96-well half-area black micro-
plate (COSTAR) and was incubated for 10 min at 37 °C. The
reaction was started by adding 50 µL of fluorescence-quenching
peptide substrate solution to the well, and incubated for 2
(MMP-1 and MMP-3) or 3 (MMP-9) h at 37 °C. A total of 5 µM
32
(2R)-N-Hyd r oxy-1-[(R)-eth oxy(4-m eth oxyp h en yl)p h os-
p h or yl]p yr r olid in e-2-ca r boxa m id e (52): colorless solids;
7% yield; 1H NMR (DMSO-d6) δ 1.27 (t, J ) 7.1 Hz, 3H), 1.60-
2.00 (m, 4H), 3.00-3.20 (m, 2H), 3.80 (s, 3H), 3.85-4.15 (m,
of MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 (Pep-
tide Institute, Inc.) was used as a substrate for MMP-1 and
MMP-9, and 5 µM of MOCAc-Arg-Pro-Lys-Pro-Val-Glu-Nva-
33
Trp-Arg-Lys(Dnp)-NH2 (Peptide Institute, Inc.) was used as

928 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Sawa et al.
a substrate for MMP-3. After incubation, fluorescence intensi-
ties (Ex/Em ) 320/405 nm) of the wells were measured by a
fluorescence microplate reader (Polarstar; BMG LabTechnolo-
gies, Germany). Ki values were calculated from the percent
inhibition and the Km value of each MMP to the substrate by
using GraphPad Prism.
HB-EGF fused with human placental alkaline phos-
phatase. Special thanks are addressed to Ms. E. Ishi-
bushi for expert technical assistance.
Refer en ces
2.2 TACE In h ibition Assa y. The DNA fragment coding
signal region, prodomain and catalytic domain of TACE, was
amplified by polymerase chain reaction from pBluescriptII-
TACE clone (cloned a full-length cDNA for TACE from human
acute monocytic leukemia cell line, THP-1) as the template.
The 5′-end of each PCR primer was added to a sequence for
the appropriate restriction enzyme site, and the FLAG tag
sequence was also added to the reverse primer. The amplified
DNA fragment was cloned into pFastBac-1 transfer vector (Life
Technologies, Rockville, MD). pFastBac-1/TACE was trans-
formed into DH10Bac cells (Life Technologies, Rockville, MD),
and the recombinant bacmid was isolated, purified, and then
used to generate baculovirus particles in Sf9 insect cells
(Pharmingen, San Diego, CA). Logarithmically growing Sf9
cells were infected with TACE baculovirus at a MOI1. Con-
ditioned media were harvested at 96 h after infection. The
recombinant TACE (rTACE) was purified from the medium
with an anti-FLAG M2 affinity gel column (Sigma, St. Louis,
MO). Purified rTACE had an approximately 90% purity.
The rTACE (final 50 ng/mL) was mixed with the compound
solution and incubated at 37 °C for 10 min in a reaction buffer
(20 mM Tris-HCl (pH 7.5) containing 0.05% Brij-35). The
reaction was initiated by addition of 5 µM (final concentration)
of fluorescence-quenching peptide substrate (Mca-Pro-Leu-Ala-
Glu-Ala-Val-Dap(Dnp)-Arg-Ser-Ser-Ser-Arg-NH2; Bachem AG,
Switerland),34 which contained the cleavage site of proTNF-
R, and the increase of fluorescence intensity (Ex/Em ) 320/
405 nm) was monitored. Ki values were calculated from the
percent inhibition and the Km value of rTACE to the substrate
by using GraphPad Prism, version 3.0 (GraphPad Software,
Inc., San Diego, CA). The Km value of purified rTACE was
about 17 µM, and this value was very similar to that of the
previous report.35
2.3. HB-EGF Sh ed d in g Assa y.25 The expression vector of
HB-EGF fused with human placental alkaline phosphatase
(AP) that was constructed as described previously25 was
obtained from Dr. Higashiyama (School of Medicine, Osaka
University, Osaka, J apan). Fibrosarcoma HT-1080 transfec-
tants expressing AP-tagged HB-EGF in MEM (containing 10%
FCS) as the culture medium was 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 (containing 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
transferred 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 2 h. The AP activity
was then determined by the measurement of absorbance at
405 nm with a microplate reader. The IC50 value was deter-
mined with different inhibitor concentrations by using Graph-
Pad Prism, version 3.0 (GraphPad Software, Inc.).
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