Azasugar-Based MMP/ADAM Inhibitors as Antipsoriatic Agents

Article abstract of DOI:10.1021/jm0304313

As a part of synthetic studies on MMP (matrix metalloproteinase)/ADAM (a disintegrin and metalloproteinase) inhibitors, we have preliminarily communicated that azasugar-based compound 1a exhibited a potential inhibitory activity on some metalloprotease-ca

Full text of DOI:10.1021/jm0304313

1930
J . Med. Chem. 2004, 47, 1930-1938
Aza su ga r -Ba sed MMP /ADAM In h ibitor s a s An tip sor ia tic Agen ts
Hideki Moriyama,†,‡ Takahiro Tsukida,*†,‡,| Yoshimasa Inoue,‡ Kohichi Yokota,‡ Kohichiro Yoshino,‡
Hirosato Kondo,‡,| Nobuaki Miura,† and Shin-Ichiro Nishimura*,§
J apan Bioindustry Association, Hokkaido Collaboration Center N-21, W-12, Kita-Ku, Sapporo 001-0021, J apan, R&D
Laboratories, Nippon Organon K.K., 1-5-90, Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016, J apan, and Division of
Biological Sciences, Graduate School of Science, Hokkaido University, N-21, W-11, Kita-Ku, Sapporo 001-0021, J apan
Received September 3, 2003
As a part of synthetic studies on MMP (matrix metalloproteinase)/ADAM (a disintegrin and
metalloproteinase) inhibitors, we have preliminarily communicated that azasugar-based
compound 1a exhibited a potential inhibitory activity on some metalloprotease-catalyzed
proteolytic reactions. To find promising candidates for the topical treatment of psoriasis, we
investigated stability in aqueous solution of compound 1a and its derivative 1b and then
optimized the P1′ substuent (2-5). In the present study, we synthesized novel derivatives of
compound 1a and evaluated their inhibitory activity toward MMP-1, -3, and -9, TACE, and
HB-EGF shedding, from a viewpoint of versatility of azasugars as a functional scaffold. As a
result, it was found that compound 1b demonstrated desirable inhibitory activity as an
antipsoriatic agent, and some of the derivatives showed selective inhibitory activity. In addition,
it was found that compound 1b exhibited a significant therapeutic effect on a mouse TPA-
induced epidermal hyperplasia model. Therefore, compound 1b could become a promising
candidate as a practical antipsoriatic agent.
In tr od u ction
mis, namely ADAM12 inhibitors, are expected to be
effective therapeutic agents for the treatment of skin
diseases such as psoriasis caused by the proliferation
of keratinocytes. It has already been reported that
N-{DL-[2-(hydroxylaminocarbonyl)methyl]-4-methylpen-
tanoyl}-L-3-(2′-naphthyl)alanine 2-aminoethylamide
(TAPI), a peptide-based MMP inhibitor, inhibited the
proteolytic cleavage of HB-EGF precursor.12 Moreover,
Tokumaru et al. have reported that peptide-type MMP
inhibitors suppressed the proliferation of keratinocytes
both in in vitro and in vivo models.8 Therefore, MMP
inhibitors would be potential lead compounds for the
development of antipsoriatic agents.13
Psoriasis is a complex inflammatory skin disease and
affects 2% of the adult population. The nature of the
disease is characterized by hyperproliferation of epi-
dermal keratinocytes.1,2 Several systemic and topical
therapies are available such as methotrexate, retinoid,
cyclosporine, glucocorticoid, and analogues of 1,25-
dihydroxyvitamine D3 [1,25(OH)2D3], but the effects of
these agents are not sufficient.2 In addition, safety
concerns prevent widespread application. Although the
etiology of psoriasis has not been clearly elucidated, it
was demonstrated that the characterized proliferation
of keratinocytes is induced by a number of growth
factors, such as epidermal growth factor (EGF), trans-
forming growth factor-R (TGF-R), amphiregulin (AR),3
and heparin-binding EGF-like growth factor (HB-
EGF).4-7 Among these growth factors, recently, it has
been reported that HB-EGF might be the most impor-
tant for the proliferation of keratinocytes.8 These epi-
dermal growth factors were synthesized as membrane-
anchored precursors, which are converted to the soluble
growth factors.9 Several studies have suggested that
some zinc-dependent metalloproteinases are involved in
the processing of these membrane-anchored precur-
sors.8,10 Moreover, it has been recently reported that
ADAM12 might be one of most important enzymes to
shed HB-EGF from its precursor protein, proHB-EGF.11
Therefore, inhibitors of HB-EGF production in epider-
On the other hand, there have been considerable
efforts in the design and synthesis of MMP/ADAM
inhibitors, and a lot of cyclic small molecule inhibitors
have been reported and showed blocking activity against
metalloproteinases.14 Recently, we reported novel met-
alloproteinase inhibitors based on an azasugar scaf-
fold.15 An R-configuration of the hydroxamic acid at the
C-2 position and the introduction of an arylsulfonyl
moiety at the N-1 position would become crucial for
practical design of the potential metalloproteinase
inhibitors (Chart 1).15b The azasugar compounds de-
signed were expected to have improved solubility, due
to the hydroxyl group at 3-, 4-, and 5-positions. In
addition, control of stereochemistry of the azasugar
derivatives would be facile, because they were prepared
from L-threitol, L-gulono-1,4-lactone, L-glucono-1,5-lac-
tone, and D-gulonic γ-lactone, commercial available
starting materials.15
* To whom correspondence should be addressed. T.T.: Tel: +81 6
6401 8199, fax: +81
6 6401 1371; e-mail: takahiro.tsukida@
shionogi.co.jp. S.-I.N.: Tel: +81 11 706 9043, fax: +81 11 706 9042;
e-mail: shin@glyco.sci.hokudai.ac.jp.
In this series, we found that compound 1a bearing
the 2R,3R,4R,5S-configuration exhibited potent inhibi-
tory activity toward MMP-1, -3, and -9 and TACE
(TNF-R converting enzyme, ADAM17).15a Next, our
interest has been focused on the discovery of promising
† J apan Bioindustry Association.
‡ Nippon Organon K.K.
§ Hokkaido University.
| Current address: Manufacturing Technology R&D Laboratories,
Shionogi & Co., Ltd., 1-3 Kuise Terajima 2-chome, Amagasaki, Hyogo
660-0813 J apan.
10.1021/jm0304313 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/13/2004

Azasugar-Based MMP/ ADAM Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1931
Ch a r t 1. Practical Design of Novel Azasugar-Based Metalloproteinase Inhibitor
Ta ble 1. Stability of Azasugar Derivatives 1a ,b in Aqueous
Solution
candidates for the topical treatment of psoriasis. For this
purpose, we investigated stability of compound 1a in
aqueous solution. This property is important for topical
application, because the drug must be in a solution for
a long time. As a result, a stereoisomer 1b was designed,
and then modification of P1′-substuent of 1b was
investigated (Figure 1).
% compd remaining after 1 weeka
compd
pH 4
pH 7.4
pH 9
distilled water
1a
1b
98.7
102.1
75.4
102.5
83.6
104.8
96.1
100.0
a
The details of the assay are described in the Experimental
Section.
ing carboxylic acid 1a ′ (Figure 2). If the stereochemistry
of the hydroxyl group at the 5-position would be
inverted, such as in compound 1b, hydrolysis of the
hydroxamic acid would be diminished, compared to 1a .
In fact, compound 1b was tested under the same
conditions. Interestingly, as we predicted, stability of
compound 1b in aqueous solution after 1 week was
dramatically improved as shown in Table 1.16
Next, we focused on the optimization of the inhibitory
activity against MMPs and TACE and shedding of
HB-EGF by synthesizing new analogues of compound
1b. For this purpose, we investigated some modifica-
tions of the arylsulfonyl moiety of compound 1b.
Figure 1 shows a straightforward strategy of a new
type of analogue of sugar-mimetic metalloproteinase
inhibitors such as compound 1b. Scheme 1 shows
synthetic routes of these new compounds.
The azide intermediate 7,17 which was readily pre-
pared from D-mannono-1,4-lactone 6,18 was hydroge-
nated in the presence of 10% Pd-C, and then the
corresponding amine was reacted with substituted
arylsulfonyl chlorides 8i-iv to give compounds 9i-iv
in 75-85% yields (Scheme 1). The terminal isopropyl-
idene groups in compounds 9i-iv were selectively
cleaved by using cerium chloride heptahydrate and a
catalytic amount of oxalic acid in acetonitrile19 to
provide diols 10i-iv in 67-78% yields, respectively. The
primary hydroxyl groups of diols 10i-iv were selectively
mesylated by the treatment with mesyl chloride at -40
°C in dichloromethane to afford mesylates 11i-iv in
moderate yields. The intramolecular cyclization of com-
pounds 11i-iv in the presence of potassium carbonate
gave compounds 12i-iv. After the removal of the benzyl
group of compound 12iv by hydrogenolysis in the
presence of 10% Pd-C, the corresponding phenol de-
rivative was condensed with 1-bromo-2-butyne in the
presence of potassium carbonate to afford compound 12v
in good yield. Compounds 12ii-v were subjected to the
aminolysis by the treatment with 50% hydroxylamine
aqueous solution in the presence of sodium cyanide in
methanol to afford compounds 13ii-v in 35-65% yields,
respectively. Finally, 3,4-O-isopropylidene groups in
13ii-v were cleaved by treating with Muromac, H+-
form, to provide the target compounds 2-5 in 55-81%
F igu r e 1. Modification of azasugar-based metalloproteinase
inhibitor 1a .
In this paper, we report the systematic syntheses of
compound 1a analogues and their inhibitory activity
toward MMP-1, -3, and -9, TACE, and shedding of HB-
EGF. In addition, compound 1b was subjected to in vivo
assay by using TPA-induced hyperplasia in murine skin,
as a model of psoriasis.
Ch em istr y
At first, to verify whether azasugar derivative 1a
would become a promising candidate for the topical
treatment of psoriasis, we investigated stability in
aqueous solution of compound 1a . Compound 1a was
dissolved and tested in solutions of pH 4, pH 7.4, and
pH 9 and distilled water. Unfortunately, compound 1a
was proved to be unstable in neutral solution, pH 7.4
solution after 1 week, because it was hydrolyzed to the
corresponding carboxylic acid 1a ′ (Table 1). One of the
expected mechanisms of hydrolysis for azasugar-based
compound 1a was postulated as follows: the hydroxamic
acid moiety might be hydrolyzed by the equatorial
hydroxyl group at the 5-position of compound 1a to-
gether with H2O in the solution, to produce correspond-

1932 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Moriyama et al.
F igu r e 2. Expected mechanism of hydrolysis for azasugar-based compound 1a .
Sch em e 1a
a
(a) 10% Pd-C/H2, EtOAc; (b) 8i-iv, 4-DMAP, DMF, 75-85% from 7; (c) cerium chloride heptahydrate, oxalic acid, CH3CN, 67-78%;
(d) methanesulfonyl chloride, Et3N, CH2Cl2, 33-86%; (e) K2CO3, DMF, 68-92%; (f) (i) 10% Pd-C/H2, EtOAc, (ii) 1-bromo-2-butyne, K2CO3,
CH3CN, 83%; (g) 50% aq NH2OH, NaCN, MeOH, 35-65%; (h) Muromac, 55-81%, (i) (i) 1 N NaOH, (ii) NH2OBn, WSC, HOBt, DMF,
73%; (j) (i) Muromac, MeOH, (ii) 10% Pd-C/H2, 69%.
yields, respectively. On the other hand, compound 12i
was subjected to hydrolysis by 1 N sodium hydroxide
solution, followed by the condensation with NH2OBn in
the presence of WSC and HOBt, to give compound 14
in 91% yield. After deprotection of an isopropylidene
group of the compound 14 using Muromac, the corre-
sponding triol derivative was hydrogenated in the
presence of 10% Pd-C to afford a target compound 1b.
Biologica l Activity
Inhibitory activity against TACE, shedding of HB-
EGF, and MMPs (MMP-1, MMP-3, MMP-9) of com-
pound 1-5 were summarized in Table 2. Compound 1b
with a 2R,3R,4R,5R-configuration, having improved
stability in aqueous solution after 1 week, exhibited
similar and satisfactory inhibition toward MMPs, TACE,

Azasugar-Based MMP/ ADAM Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1933
F igu r e 3. Docking study of sugar-mimetic compound 1b with MMP-3.
Ta ble 2. Inhibitory Activity of Azasugar Derivatives 1-5
also suggested that the axial hydroxyl group at the
5-position was not directed to the surface of the protein.
Considering the similar potent inhibitory activity of 1a
and 1b, it was indicated that the hydroxyl group at the
5-position is not important for desirable interaction with
MMP-3.
against MMPs, TACE, and Shedding of HB-EGF
rMMP-1 rMMP-3 rMMP-9 TACE
shedding of
compd Ki (nM)a Ki (nM)a Ki (nM)a Ki (nM)a HB-EGF: IC50 (µM)a
1a
1b
2
3
4
8.0
5.3
26
162
>850
128
0.51
0.35
2.0
50
2.1
3.3
0.06
0.097
2.0
47
7.4
14
2.3
6.2
15
21
1.7
0.53
0.35
0.34
0.45
21
0.084
0.028
In Vivo Assa y: Effects on Mice TP A-In d u ced
Ep id er m a l Hyp er p la sia . To evaluate the antipsoriatic
activity of compounds synthesized in this study, we
investigated the effects of these inhibitors on TPA(12-
O-tetradecanoylphorbol 13-acetate)-induced hyperplasia
in murine skin, one of the psoriatic model.21 It is well-
known that topical application of phorbol esters induce
cutaneous inflammation and epidermal hyperprolifera-
tion.21 As shown in Figure 4, TPA induced a significant
increase in epidermal thickness, compared with acetone
treatment. Topical application of compound 1b dose-
dependently suppressed TPA-induced inflammation at
a dose of 1-100 µg/site. This result indicates that
compound 1b would regulate EGF production to sup-
press phorbol-induced inflammation and epidermal hy-
perplasia. Considering the potent topical antiinflam-
matory activity, it was suggested that topical use of
compound 1b was a promising candidate for the treat-
ment of psoriasis.
5
a
The details of the assay are described in the Experimental
Section. All values represent the mean of two determinations.
and shedding of HB-EGF, compared to compound 1a
having a 2R,3R,4R,5S-configuration. This result sug-
gests that stereochemistry at the 5-position of azasugar
compounds might not be essential for inhibitory activity
against metalloproteinases. Interestingly, compound 2,
4, and 5, having a methoxy, benzyloxy, and butyn-2-
yloxy moiety, respectively, showed similar or higher
inhibitory activity toward shedding of HB-EGF, com-
pared to compound 1b, suggesting that the S1′ pocket
of the enzyme responsible for the shedding of HB-EGF
is deeper than that of MMP-1.13b In addition, it was also
found that compound 4 appears to become a selective
MMP/ADAM inhibitor, over MMP-1, because the 4-ben-
zyloxybenzenesulfonylamide unit of 4 would not be
accommodated to the shallow S1′ pocket in MMP-1.20
However, in the case of biphenyl derivative 3, inhibitory
activity against metalloproteinases was dramatically
decreased.
In addition, the binding model of 1b with MMP-3
was constructed using the published crystal structure
of MMP-3 with a sulfonamide derivative (PDB code:
1D8F).14b As shown in Figure 3, the hydroxamic acid
moiety was found to be tightly bound with the zinc ion,
and the phenoxybenzene unit was placed in the S1′
pocket. One of the oxygen atoms of the sulfonamide was
positioned at hydrogen bond distance from the main
chain Leu-164 and Ala-165, like that of other sulfona-
mide-based inhibitors.14b As shown in this model, it was
In conclusion, to find promising candidates for the
topical treatment of psoriasis, the aqueous stability of
compound 1a and its derivative 1b and the structural
modification of the P1 -substituent were investigated.
In this study, the compound 1a analogues were system-
atically synthesized and their inhibitory activity toward
MMP-1, -3, and -9, TACE, and shedding of HB-EGF
were discussed. As a result, it was found that compound
1b, having an improved stability in aqueous solution,
exhibited a similar potent in vitro profile, compared to
compound 1a , and its derivative 4 showed a selective
inhibitory profile over MMP-1. In addition, it was also
suggested that compound 1b was effective on the mice

1934 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Moriyama et al.
mL) was hydrogenated for 4 h at 40 °C, under 4 kgf/cm2
pressure. The catalyst was removed by filtration, the filtrate
was evaporated in vacuo, and the residue was dissolved in
DMF (390 mL). To the solution were added 4-DMAP (18.1 g,
148.1 mmol) and 4-phenoxybenzenesulfonyl chloride 8i (34.5
g, 128.4 mmol) in ice bath, and the reaction mixture was
stirred for overnight at room temperature. The mixture was
diluted with ethyl acetate, and it was washed with 1 N HCl,
sat. NaHCO3 aq., H2O and brine, successively. The organic
layer was dried with MgSO4, and solvent was removed under
reduced pressure. The resulting residue was purified by MPLC
(ethyl acetate/n-hexane; 1:3 f 2:3), to obtain 9i (38.3 g) in 77%
1
yield as a colorless amorphous. H NMR (250 MHz, CDCl3) δ:
1.33 (s, 3H), 1.37 (s, 3H), 1.40 (s, 3H), 1.48 (s, 3H), 3.57 (s,
3H), 3.85-4.3 (m, 6H), 5.46 (d, 1H, J ) 10.5 Hz), 6.95-7.1
(m, 4H), 7.15-7.3 (m, 1H), 7.35-7.5 (m, 2H), 7.7-7.85 (m, 2H).
(2R,4′S,4′′R,5R)-(4-Met h oxyb en zen esu lfon yla m in o)-
[2′,2′,2′′,2′′-tetr a m eth yl-[4′,4′′]bi([1,3]d ioxola n yl)-5′-yl]a ce-
tic Acid Meth yl Ester (9ii). (Yield 85%). 1H NMR (500 MHz,
CDCl3) δ: 1.32 (s, 3H), 1.37 (s, 3H), 1.40 (s, 3H), 1.49 (s, 3H),
3.53 (s, 3H), 3.86 (s, 3H), 3.9-4.03 (m, 2H), 4.03-4.13 (m, 2H),
4.13-4.3 (m, 3H), 5.45 (d, 1H, J ) 10.4 Hz), 7.00 (d, 2H, J )
8.9 Hz), 7.76 (d, 2H, J ) 8.9 Hz).
(2R,4′S,4′′R,5R)-(Bip h en yl-4-su lfon yla m in o)-[2′,2′,2′′,2′′-
t et r a m et h yl-[4′,4′′]b i([1,3]d ioxola n yl)-5′-yl]a cet ic Acid
Meth yl Ester (9iii). (Yield 85%). 1H NMR (500 MHz, CDCl3)
δ: 1.33 (s, 3H), 1.38 (s, 3H), 1.41 (s, 3H), 1.50 (s, 3H), 3.50 (s,
3H), 3.9-4.05 (m, 2H), 4.05-4.15 (m, 1H), 4.19 (dd, 1H, J )
8.6, 6.3 Hz), 4.25-4.35 (m, 2H), 5.53 (d, 1H, J ) 10.5 Hz),
7.37-7.45 (m, 1H), 7.45-7.53 (m, 2H), 7.55-7.63 (m, 2H), 7.71
(d, 2H, J ) 8.4 Hz), 7.89 (d, 2H, J ) 8.4 Hz).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 10i-iv:
(1′′R,2R,4′R,5′S)-[5′-(1′′,2′′-Dih yd r oxyeth yl)-2′,2′-d im eth yl-
[1,3]d ioxola n -4′-yl]-(4-p h en oxyb en zen esu lfon yla m in o)-
a cetic Acid Meth yl Ester (10i). To a solution of compound
9i (25.81 g, 49.54 mmol) in acetonitrile (300 mL) were added
cerium chloride heptahydrate (36.92 g, 99.08 mmol) and oxalic
acid (223 mg, 2.48 mmol), and the mixture was stirred for 70
min at room temperature. Sodium carbonate was added to the
mixture. The reaction mixture was neutralized and filtered
with a Celite pad, and the Celite pad was washed with ethyl
acetate. The filtrate and washings were combined and evapo-
rated in vacuo. The resulting residue was purified by MPLC
(ethyl acetate/n-hexane; 2:3 f 3:1) to obtain 10i (16.0 g) in
67% yield as a syrup, and starting material 9i (7.0 g) was
recovered. 1H NMR (250 MHz, CDCl3) δ: 1.35 (s, 3H), 1.42 (s,
3H), 2.61 (bs, 1H), 3.61 (s, 3H), 3.65-3.95 (m, 3H), 4.05-4.25
(m, 1H), 4.25-4.4 (m, 2H), 5.46 (d, 1H, J ) 8.6 Hz), 6.95-7.1
(m, 4H), 7.15-7.3 (m, 1H), 7.3-7.5 (m, 2H), 7.7-7.85 (m, 2H).
F igu r e 4. Effect of azasugar-based derivative 1b on mice
TPA-induced epidermal hyperplasia. Data represent the mean
( standard error of five animals. Significant difference from
a
TPA treated group. ** p < 0.01 (student’s t-test).
Experimental Section for details.
See
TPA-induced epidermal hyperplasia model. Moreover,
the binding model of 1b with MMP-3 demonstrated that
the stereochemistry of the hydroxyl group at the 5-posi-
tion would not contribute to the desirable interaction
with MMP-3. Therefore, it was strongly concluded that
compound 1b could become a potential candidate as a
practical antipsoriatic agent.
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 Brucker DPX-250 spectrometer or
500 MHz on a Brucker DPX-500 spectrometer using CDCl3 or
DMSO-d6 as the solvent. TPA was purchased from Sigma (St.
Louis, MO). Mass spectra were determined on a PerSeptive
Biosystems Voyager-DE RP spectrometer or a Brucker BI-
FLEX III spectrometer. Optical rotations were measured at
20 °C on a Perkin-Elmer Polarimeter 343. The results of
elemental analysis are within (0.4% of the calculated values
unless otherwise noted.
Mea su r em en t of th e Sta bility of Su ga r -Ba sed In h ibi-
tor s. Standard solutions were prepared as follows: 5 mg of
compounds 14a and 14b was dissolved in MeOH (10 mL),
respectively. Standard solutions (100 µL) were added to the
flask and then dried under nitrogen atmosphere. To the
resulting residue was added pH 4, pH 7.4, or pH 9 solution or
distilled water (5 mL). One milliliter of each solution was
enclosed in ampules. The ampules were incubated in Air Bath
Unit A (Taiyo) at 37 °C for 0 or 168 h. At the time of 0 or 168
h, MeOH (1 mL) was added to the ampules. The mixture was
analyzed by HPLC (L-column ODS 2.1 × 150 mm). Sample
concentration after 0 and 168 h was evaluated by peak area
of HPLC (three times). Percent compound remaining after 168
h was calculated by the following equation:
(1′′R,2R,4′R,5′S)-[5′-(1′′,2′′-Dih ydr oxyeth yl)-2′,2′-dim eth yl-
[1,3]d ioxola n -4′-yl]-(4-m et h oxyb en zen esu lfon yla m in o)-
1
a cetic Acid Meth yl Ester (10ii). (Yield 74%). H NMR (500
MHz, CDCl3) δ: 1.35 (s, 3H), 1.42 (s, 3H), 2.33 (bs, 1H), 2.83
(bs, 1H), 3.56 (s, 3H), 3.75 (dd, 1H, J ) 11.3, 4.2 Hz), 3.78-
3.82 (m, 1H), 3.82-3.9 (m, 4H), 4.17 (t, 1H, J ) 7.4 Hz), 4.25-
4.4 (m, 2H), 5.5 (bs, 1H), 6.98 (d, 2H, J ) 8.9 Hz), 7.77 (d, 2H,
J ) 8.9 Hz).
(1′′R,2R,4′R,5′S)-(Bip h en yl-4-su lfon yla m in o)-[5′-(1′′,2′′-
d ih yd r oxyet h yl)-2′,2′-d im et h yl[1,3]d ioxola n -4′-yl]a ce-
t ic Acid Met h yl E st er (10iii). (Yield 68%). 1H NMR (500
MHz, CDCl3) δ: 1.36 (s, 3H), 1.44 (s, 3H), 2.20 (s, 1H), 2.74
(bs, 1H), 3.53 (s, 3H), 3.7-3.8 (m, 1H), 3.8-3.85 (m, 1H), 3.85
(m, 1H), 3.85-3.95 (m, 1H), 4,18 (dd, 1H, J ) 7.9, 7.1 Hz),
4.33-4.45 (m, 2H), 5.57 (bs, 1H), 7.38-7.45 (m, 1H), 7.45-
7.55 (m, 2H), 7.55-7.63 (m, 2H), 7.71 (d, 2H, J ) 8.5 Hz), 7.90
(d, 2H, J ) 8.5 Hz).
% compound remaining )
average sample concentration after 168 h/
average sample concentration after 0 h × 100
(1′′R,2R,4′R,5′S)-(4-Ben zyloxyben zen esu lfon yla m in o)-
[5′-(1′′,2′′-d ih yd r oxyeth yl)-2′,2-d im eth yl[1,3]d ioxola n -4′-
yl]a cetic Acid Meth yl Ester (10iv). (Yield 67% from 6). H
1
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 9i-iii:
(2R ,4′S ,4′′R ,5R )-(4-P h e n o x y b e n ze n e s u lfo n y la m in o )-
[2′,2′,2′′,2′′-tetr a m eth yl-[4′,4′′]bi([1,3]d ioxola n yl)-5′-yl]a ce-
tic Acid Meth yl Ester (9i). A solution of compound 717 (30.1
g, 95.30 mmol) and 10% Pd-C (4.3 g) in ethyl acetate (300
NMR (500 MHz, CDCl3) δ: 1.37 (s, 3H), 1.48 (s, 3H), 2.17 (s,
1H), 2.69 (d, 1H, J ) 6.7 Hz), 3.55 (s, 3H), 3.7-4.0 (m, 3H),
4.0-4.25 (m, 2H), 4.25-4.45 (m, 2H), 5.18 (s, 2H), 5.45 (d, 1H,
J ) 11.1 Hz), 7.05 (d, 2H, J ) 8.9 Hz), 7.25-7.5 (m, 5H), 7.78
(d, 2H, J ) 8.9 Hz).

Azasugar-Based MMP/ ADAM Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1935
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 11i-iv:
(1′′S,2R,4′R,5′S)-[5′-(1′′-Hyd r oxy-2′′-m eth a n esu lfon yloxy-
et h yl)-2′,2′-d im et h yl[1,3]d ioxola n -4′-yl]-(4-p h en oxyb en -
zen esu lfon yla m in o)a cetic Acid Meth yl Ester (11i). To a
solution of compound 10i (25.0 g, 51.98 mmol) and triethy-
lamine (5.79 g, 57.18 mmol) in dichloromethane (430 mL) was
added methanesulfonyl chloride (6.25 g, 54.57 mmol) in
dichloromethane (20 mL) at -40 °C, and the mixture was
stirred for 1 h at the same temperature. The reaction mixture
was diluted with chloroform, and the organic layer was washed
H2O and dried with MgSO4. The solvent was removed under
reduced pressure, and the resulting residue was purified by
MPLC (ethyl acetate/cyclohexane; 35:65 f 2:3 f 1:1) to obtain
1H), 7.45-7.53 (m, 2H), 7.55-7.63 (m, 2H), 7.71 (d, 2H, J )
7.6 Hz), 7.88 (d, 2H, J ) 7.6 Hz).
(3a R,4R,7R,7a R)-5-(4′-Ben zyloxyb en zen esu lfon yl)-7-
h yd r oxy-2,2-d im eth ylh exa h yd r o[1,3]d ioxolo[4,5-c]-4-ca r -
boxylic Acid Meth yl Ester (12iv). (Yield 72%). 1H NMR (500
MHz, CDCl3) δ: 1.37 (s, 3H), 1.46 (s, 3H), 3.62 (s, 3H), 3.72
(d, 1H, J ) 13.9 Hz), 3.85 (dd, 1H, J ) 9.8, 2.4 Hz), 4.05-4.2
(m, 2H), 4.39 (s, 1H), 5.08 (d, 1H, J ) 6.3 Hz), 5.15 (s, 2H),
7.05 (d, 2H, J ) 8.9 Hz), 7.3-7.5 (m, 5H), 7.77 (d, 2H, J ) 8.9
Hz).
(3a R,4R,7R,7a R)-5-(4′-Bu t-2′-yn yloxyben zen esu lfon yl)-
7-h yd r oxy-2,2-d im et h ylh exa h yd r o[1,3]d ioxolo[4,5-c]-4-
ca r boxylic Acid Meth yl Ester (12v). A solution of compound
12iv (5.8 g, 12.15 mmol) and 10% Pd-C (800 mg) in ethyl
acetate (200 mL), was hydrogenated for 2 h at 40 °C, under
3kgf/cm2 pressure. The catalyst was removed by filtration, the
filtrate was evaporated in vacuo, and the residue was dissolved
with acetonitrile (70 mL). 1-Bromo-2-butyne (2.4 g, 18.23
mmol) and potassium carbonate (2.52 g, 18.23 mmol) were
added to the solution, and the reaction mixture was stirred
for 30 min at 90 °C. The mixture was diluted with ethyl
acetate, it was washed with H2O and brine, the organic layer
was dried with MgSO4, and solvent was removed under
reduced pressure. The resulting residue was purified by MPLC
(ethyl acetate/n-hexane; 1:2 f 1:1) to obtain 39v (4.4 g) in 83%
yield. 1H NMR (500 MHz, CDCl3) δ: 1.38 (s, 1H), 1.46 (s, 3H),
1.8-1.95 (m, 3H), 2.14 (t, 1H, J ) 1.0 Hz), 3.65 (s, 3H), 3.72
(d, 1H, J ) 13.9 Hz), 3.85 (dd, 1H, J ) 9.8, 2.5 Hz), 4.05-4.2
(m, 2H), 4.39 (s, 1H), 4.65-4.75 (m, 2H), 5.08 (d, 1H, J ) 6.4
Hz), 6.95-7.1 (m, 2H), 7.7-7.85 (m, 2H).
1
10i (13.5 g) in 46% yield. H NMR (250 MHz, CDCl3) δ: 1.34
(s, 3H), 1.41 (s, 3H), 2.91 (d, 1H, J ) 6.5 Hz), 3.13 (s, 3H),
3.61 (s, 3H), 3.85-4.0 (m, 1H), 4.05-4.28 (m, 2H), 4.28-4.4
(m, 2H), 4.54 (dd, 1H, J ) 11.1, 2.5 Hz), 5.47 (d, 1H, J ) 9.5
Hz), 6.95-7.1 (m, 4H), 7.15-7.3 (m, 1H), 7.35-7.5 (m, 2H),
7.7-7.85 (m, 2H).
(1′′S,2R,4′R,5′S)-[5′-(1′′-H yd r oxy-2′′-m et h a n esu lfon yl-
oxyeth yl)-2′,2′-d im eth yl[1,3]d i-oxola n -4′-yl]-(4-m eth oxy-
ben zen esu lfon yla m in o)a cetic Acid Meth yl Ester (11ii).
1
(Yield 73%). H NMR (500 MHz, CDCl3) δ: 1.35 (s, 3H), 1.41
(s, 3H), 3.14 (s, 3H), 3.59 (s, 3H), 3.87 (s, 3H), 3.93 (bs, 1H),
4.1-4.18 (m, 2H), 4,21 (d, 1H, J ) 6.6 Hz), 4.25-4.4 (m, 2H),
4.53 (dd, 1H, J ) 11.1, 2.4 Hz), 5.47 (bs, 1H), 6.97 (d, 2H, J )
8.9 Hz), 7.76 (d, 2H, J ) 8.9 Hz).
(1′′S,2R,4′R,5′S)-(Bip h en yl-4-su lfon yla m in o)-[5′-(1′′-h y-
d r oxy-2′′-m et h a n esu lfon yloxyet h yl)-2′,2′-d im et h yl[1,3]-
d ioxola n -4′-yl]a cetic Acid Meth yl Ester (11iii). (Yield
33%). 1H NMR (500 MHz, CDCl3) δ: 1.35 (s, 3H), 1.43 (s, 3H),
2.87 (d, 1H, J ) 7.6 Hz), 3.14 (s, 3H), 3.56 (s, 3H), 3.94 (bs,
1H), 4.20 (t, 1H, J ) 8.1 Hz), 4.25-4.4 (m, 3H), 4.56 (d, 1H, J
) 11.1 Hz), 5.51 (d, 1H, J ) 9.3 Hz), 7.38-7.45 (m, 1H), 7.45-
7.53 (m, 2H), 7.55-7.65 (m, 2H), 7.72 (d, 2H, J ) 7.4 Hz), 7.89
(d, 2H, J ) 7.4 Hz).
(1′′S,2R,4′R,5′S)-(4-Ben zyloxyben zen esu lfon yla m in o)-
[5′-(1′′-h y d r o x y -2′′-m e t h a n e s u lfo n y lo x y e t h y l)-2′,2′-
d im et h yl[1,3]d ioxola n -4′-yl]a cet ic Acid Met h yl E st er
(11iv). (Yield 86%). 1H NMR (500 MHz, CDCl3) δ: 1.37 (s,
3H), 1.44 (s, 3H), 2.88 (d, 1H, J ) 8.0 Hz), 3.16 (s, 3H), 3.59
(s, 3H), 3.94 (s, 1H), 4.05-4.25 (m, 3H), 4.25-4.45 (m, 2H),
4.56 (d, 1H, J ) 11.1 Hz), 5.15 (s, 2H), 5.44 (d, 1H, J ) 9.6
Hz), 7.06 (d, 2H, J ) 8.9 Hz), 7.3-7.5 (m, 5H), 7.78 (d, 2H, J
) 8.9 Hz).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 12i-iv:
( 3 a R ,4 R ,7 R ,7 a R ) -7 -H y d r o x y -2 ,2 -d i m e t h y l -5 -( 4 ′-
p h en oxyben zen esu lfon yl)h exa h yd r o[1,3]d ioxolo[4,5-c]-
4-ca r boxylic Acid Meth yl Ester (12i). To a solution of 11i
(13.5 g, 24.12 mmol) in DMF (320 mL) was added potassium
carbonate (4.00 g, 28.95 mmol), and the reaction mixture was
stirred for 70 min at 45 °C. The reaction mixture was diluted
with ethyl acetate, and it was washed with H2O and brine (×2)
and then dried with MgSO4. The organic layer was evaporated
in vacuo, and the resulting residue was purified by MPLC
(ethyl acetate/cyclohexane; 2:3) to obtain 12i (10.3 g) in 92%
yield. 1H NMR (250 MHz, CDCl3) δ: 1.35 (s, 3H), 1.44 (s, 3H),
2.23 (s, 1H), 3.65 (s, 3H), 3.65-3.75 (m, 1H), 3.82 (dd, 1H, J )
9.8, 2.5 Hz), 4.0-4.2 (m, 2H), 4.3-4.4 (m, 1H), 5.05 (d, 1H, J
) 6.3 Hz), 6.95-7.1 (m, 4H), 7.15-7.25 (m, 1H), 7.35-7.45
(m, 2H), 7.7-7.8 (m, 2H).
(3 a R ,4 R ,7 R ,7 a R )-7 -H y d r o x y -2 ,2 -d i m e t h y l -5 -(4′-
m eth oxyben zen esu lfon yl)h exa h yd r o[1,3]d ioxolo[4,5-c]-
4-ca r b oxylic Acid Met h yl E st er (12ii). (Yield 68%). 1H
NMR (500 MHz, CDCl3) δ: 1.31 (s, 3H), 1.44 (s, 3H), 2.23 (s,
1H), 3.55-3.75 (m, 4H), 3.75-3.9 (m, 4H), 4.0-4.15 (m, 2H),
4.37 (s, 1H), 5.06 (d, 1H, J ) 6.3 Hz), 6.96 (d, 2H, J ) 8.8 Hz),
7.75 (d, 2H, J ) 8.8 Hz).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 13ii-v:
( 3 a R ,4 R ,7 R ,7 a R ) -7 -H y d r o x y -2 ,2 -d i m e t h y l -5 -( 4 ′-
m eth oxyben zen esu lfon yl)h exa h yd r o[1,3]d ioxolo[4,5-c]-
4-ca r boxylic Acid Hyd r oxy Am id e (13ii). Hydroxylamine
(50% aq) (12 mL) was added to a solution of compound 39ii
(2.09 g, 5.21 mmol) and sodium cyanide (255 mg, 5.21 mmol)
in MeOH (60 mL), and the solution was stirred overnight at
room temperature. The reaction mixture was concentrated in
vacuo, and the resulting residue was purified by MPLC
(CHCl3/MeOH: 50:1 f 30:1). The resulting fraction was
1
lyophilized to afford 13ii (1.06 g) in 50% yield. H NMR (500
MHz, DMSO-d6) δ: 1.24 (s, 3), 1.30 (s, 3H), 3.65 (d, 1H, J )
12.9 Hz), 3.7-3.82 (m, 2H), 3.84 (s, 3H), 4.13 (dd, 1H, J ) 9.7,
2.2 Hz), 4.20 (s, 1H), 4.54 (d, 1H, J ) 6.2 Hz), 5.22 (d, 1H, J
) 3.9 Hz), 7.07 (d, 2H, J ) 8.9 Hz), 7.72 (d, 2H, J ) 8.9 Hz),
8.93 (s, 1H), 10.63 (s, 1H); Anal. (C16H22N2O8S‚0.45H2O) C,
H, N.
(3a R,4R,7R,7a R)-5-(Bip h en yl-4′-su lfon yl)-7-H yd r oxy-
2,2-d im et h ylh exa h yd r o[1,3]d ioxolo[4,5-c]-4-ca r b oxylic
Acid Hyd r oxy Am id e (13iii). (Yield 65%). 1H NMR (500
MHz, DMSO-d6) δ: 1.05 (s, 3H), 1.30 (s, 3H), 3.71 (d, 1H, J )
12.4 Hz), 3.75-3.9 (m, 2H), 4.15 (dd, 1H, J ) 9.7, 2.3 Hz), 4.23
(s, 1H), 4.62 (d, 1H, J ) 6.3 Hz), 5.29 (d, 1H, J ) 3.9 Hz),
7.4-7.48 (m, 1H), 7.48-7.55 (m, 2H), 7.76 (d, 2H, J ) 7.4 Hz),
7.84 (s, 4H), 8.98 (d, 1H, J ) 1.5 Hz), 10.78 (d, 1H, J ) 1.2
Hz); Anal. (C21H24N2O7S‚0.80H2O) C, H, N.
(3a R,4R,7R,7a R)-5-(4′-Bu t-2′-yn yloxyben zen esu lfon yl)-
7-h yd r oxy-2,2-d im et h ylh exa h yd r o[1,3]d ioxolo[4,5-c]-4-
ca r boxylic Acid Hyd r oxy Am id e (13v). (Yield 35%). 1H
NMR (500 MHz, DMSO-d6) δ: 1.25 (s, 3H), 1.30 (s, 3H), 1.75-
1.9 (m, 3H), 3.67 (d, 1H, J ) 13.3 Hz), 3.7-3.85 (m, 2H), 4.14
(dd, 1H, J ) 9.7, 2.0 Hz), 4.54 (d, 1H, J ) 6.2 Hz), 4.75-4.9
(m, 2H), 5.26 (bs, 1H), 7.10 (d, 2H, J ) 8.9 Hz), 7.74 (d, 2H,
J ) 8.9 Hz), 8.96 (s, 1H), 10.75 (s, H); Anal.(C19H24N2O8S)
C, H, N.
(3 a R ,4 R ,7 R ,7 a R )-7 -H y d r o x y -2 ,2 -d i m e t h y l -5 -(4′-
p h en oxyben zen esu lfon yl)h exa h yd r o[1,3]d ioxolo[4,5-c]-
4-ca r boxylic Acid Ben zyloxya m id e (14). To a solution of
compound 12i (10.2 g, 22.0 mmol) in MeOH-1,4-dioxane (30-
150 mL) was added aq 1 N NaOH (55 mL), and reaction
mixture was stirred for 70 min at room temperature. The
reaction mixture was neutralized with 5% citric acid and was
extracted with ethyl acetate. The organic layer was washed
with H2O and brine and dried with MgSO4. The solvent was
(3a R,4R,7R,7a R)-5-(Bip h en yl-4′-su lfon yl)-7-h yd r oxy-
2,2-d im et h ylh exa h yd r o[1,3]d ioxolo[4,5-c]-4-ca r b oxylic
1
Acid Meth yl Ester (12iii). (Yield 87%). H NMR (500 MHz,
CDCl3) δ: 1.35 (s, 3H), 1.44 (s, 3H), 2.26 (s, 1H), 3.62 (s, 3H),
3.74 (d, 1H, J ) 13.8 Hz), 3.84 (d, 1H, J ) 9.7 Hz), 4.05-4.2
(m, 2H), 4.39 (s, 1H), 5.10 (d, 1H, J ) 6.2 Hz), 7.38-7.45 (m,

1936 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Moriyama et al.
concentrated in vacuo. To a solution of the resulting residue
were added WSC (5.48 g, 28.6 mmol), HOBt (3.86 g, 28.6 mmol)
in DMF (200 mL), a solution of O-benzylhydroxylamine
hydrochloride (4.56 g, 28.6 mmol), and DIEA (3.70 g, 28.6
mmol) in DMF (50 mL), and the reaction mixture was stirred
for 2.5 h at room temperature. Then additional amounts of
WSC (299 mg), HOBt (211 mg), O-benzylhydroxylamine
hydrochloride (248 mg), and DIEA (202 mg) were added to the
reaction mixture. The mixture was diluted with ethyl acetate.
The organic layer was washed with aq 0.5 N HCl, sat. aq
NaHCO3, H2O, and brine, successively, and then dried with
MgSO4. The solvent was removed under reduced pressure. The
resulting residue was purified by MPLC (ethyl acetate/
cyclohexane; 1:1) to obtain 14 (8.90 g) in 73% yield as a
69.8, 127.4, 127.5, 128.2, 128.9, 129.6, 138.8, 139.0, 144.2,
165.3; MALDI-TOF MS: 471[M + Na]+, 487[M + K]+ ; Anal.
(C18H20N2O7S‚0.75H2O) C, H, N.
(2R,3R,4R,5R)-1-(4′-Ben zyloxyben zen esu lfon yl)-3,4,5-
tr ih yd r oxyp ip er id in e-2-ca r boxylic Acid Hyd r oxy Am id e
1
(4). (Yield 34% from 12iv). H NMR (500 MHz, DMSO-d6) δ:
3.55-3.67 (m, 2H), 3.70 (d, 1H, J ) 12.7 Hz), 3.78 (s, 1H),
3.8-3.9 (m, 1H), 4.09 (d, 1H, J ) 6.6 Hz), 4.55-4.65 (m, 2H),
5.0-5.25 (m, 3H), 7.12 (d, 2H, J ) 8.8 Hz), 7.3-7.38 (m, 1H),
7.38-7.45 (m, 2H), 7.47 (d, 2H, J ) 7.4 Hz), 7.73 (d, 2H, J )
8.8 Hz), 8.75 (s, 1H), 10.55 (s, 1H); 13C NMR (125 MHz, DMSO-
d6) δ: 48.4, 57.0, 66.5, 68.0, 69.8, 70.1, 115.1, 128.4, 128.6,
129.0, 131.9, 136.8, 161.7, 165.3; MALDI-TOF MS: 461[M +
Na]+, 477[M + K]+ ; Anal.(C19H22N2O8S‚1.30H2O) C, H, N.
1
colorless powder. H NMR (250 MHz, CDCl3) δ: 1.28 (s, 3H),
(2R,3R,4R,5R)-1-(4′-Bu t -2′-yn yloxyb en zen esu lfon yl)-
3,4,5-tr ih yd r oxyp ip er id in e-2-ca r boxylic Acid Hyd r oxy
Am id e (5). (Yield 55%). 1H NMR (500 MHz, DMSO-d6) δ:
1.8-1.9 (m, 3H), 3.55-3.68 (m, 2H), 3.72 (d, 1H, J ) 12.9 Hz),
3.75-3.9 (m, 2H), 4.10 (d, 1H, J ) 6.7 Hz), 4.62 (bs, 2H), 4.75-
4.9 (m, 2H), 5.14 (bs, 1H), 7.06 (d, 2H, J ) 8.9 Hz), 7.74 (d,
2H, J ) 8.9 Hz), 8.81 (s, 1H), 10.57 (s, 1H); MALDI-TOF MS:
423[M + Na]+, 439[M + K]+; Anal. (C16H20N2O8S‚0.90H2O) 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 vector of HB-EGF fused with human
placental alkaline phosphatase (AP) fusion protein that was
constructed as described previously was a generous gift from
Dr. Higashiyama (School of Medicine, Osaka University,
Osaka, J apan). The vector was transfected into HTI080 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 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.
1.42 (s, 3H), 2.39 (s, 1H), 3.39 (d, 1H, J ) 14.5 Hz), 3.61 (d,
1H, J ) 9.1 Hz), 4.05-4.25 (m, 1H), 4.28 (s, 1H), 4.83 (d, 1H,
J ) 11.2 Hz), 4.92 (d, 1H, J ) 11.2 Hz), 5.07 (d, 1H, J ) 5.4
Hz), 6.95-7.1 (m, 4H), 7.15-7.25 (m, 1H), 7.3-7.5 (m, 7H),
7.8-7.9 (m, 2H), 8.94 (s, 1H); Anal. (C25H26N2O8S‚0.70H2O)
C, H, N.
(2R,3R,4R,5R)-3,4,5-Tr ih yd r oxy-1-(4′-p h en oxyben zen e-
su lfon yl)p ip er id in e-2-Hyd r oxy Am id e (1b). To a solution
of compound 14 (8.68 g, 15.65 mmol) in MeOH (25 mL) was
added Muromac (19.0 g), and the mixture was stirred for
overnight at room temperature. The insoluble material was
removed by filtration, and the filtrate was concentrated in in
vacuo. The resulting mixture was purified by MPLC (CHCl3/
MeOH; 1:0 f 30:1 f 20:1 f 10:1), to obtain the corresponding
triol (7.35 g) in 91% yield as a colorless powder. 1H NMR (250
MHz, DMSO-d6) δ: 3.65-3.90 (m, 5H), 4.15 (d, 1H, J ) 6.1
Hz), 4.52 (d, 1H, J ) 10.5 Hz), 4.59 (d, 1H, J ) 10.5 Hz), 4.65-
4.75 (m, 2H), 5.26 (d, 1H, J ) 4.3 Hz), 6.95-7.1 (m, 4H), 7.15-
7.25 (m, 1H), 7.3-7.45 (m, 7H), 7.78 (d, 2H, J ) 8.8 Hz), 11.2
(s, 1H).
To a solution of the triol (6.0 g, 11.66 mmol) in MeOH (180
mL) was added 10% Pd-C (1.3 g), and the reaction mixture
was stirred for 2.5 h at 45 °C under a hydrogen atmosphere.
Then, the catalyst was removed by the filtration, and the
filtrate was concentrated in in vacuo. The resulting mixture
was purified by MPLC (CHCl3/MeOH; 20:1 f 10:1 f 5:1) to
obtain 1b (3.76 g) in 76% yield as a colorless powder. mp
1
103.5-112 °C (dec) ; H NMR (250 MHz, DMSO-d6) δ: 3.5-
3.95 (m, 5H), 4.13 (d, 1H, J ) 6.5 Hz), 4.55-4.7 (m, 2H), 5.16
(d, 1H, J ) 4.3 Hz), 7.04 (d, 2H, J ) 8.8 Hz), 7.14 (d, 2H, J )
7.6 Hz), 7.25 (t, 1H, J ) 7.3 Hz), 7.4-7.55 (m, 2H), 7.78 (d,
2H, J ) 8.8 Hz), 8.76 (s, 1H), 10.56 (s, 1H).; MALDI-TOF MS:
425 [M + H]+, 447 [M + Na]+, 463 [M + K]+; [R]D 36° (c ) 0.1,
MeOH); Anal. (C18H20N2O8S‚0.90H2O) C, H, N.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 2-5:
(2R,3R,4R,5R)-3,4,5-Tr ih yd r oxy-1-(4′-m et h oxyb en zen e-
su lfon yl)p ip er id in e-2-ca r boxylic Acid Hyd r oxy Am id e
(2). To a solution of compound 13ii (777 mg, 1.93 mmol) in
MeOH (40 mL) was added Muromac (6.0 g), and the mixture
was stirred overnight at room temperature. The insoluble
material was removed by filtration, and the filtrate was
concentrated in vacuo. The resulting mixture was purified by
MPLC (CHCl3/MeOH; 30:1 f 7:1), and then the fraction was
lyophilized to afford 2 (567 mg) in 81% yield. 1H NMR (500
MHz, DMSO-d6) δ: 3.55-3.73 (m, 3H), 3.73-3.90 (m, 5H), 4.10
(d, 1H, J ) 6.6 Hz), 4.5-4.65 (m, 2H), 5.12 (bs, 1H), 7.03 (d,
2H, J ) 8.9 Hz), 7.72 (d, 2H, J ) 8.9 Hz), 8.84 (s, 1H), 10.55
(s, 1H); 13C NMR (125 MHz, DMSO-d6) δ: 48.4, 56.0, 57.0,
66.6, 68.0, 69.8, 114.4, 129.7, 131.7, 162.6, 165.3; MALDI-TOF
MS: 385[M + Na]+, 401[M + K]+ ; Anal. (C13H18N2O8S)
C, H, N.
(2R,3R,4R,5R)-1-(Biph en yl-4′-su lfon yl)-3,4,5-tr ih ydr oxy-
p ip er id in e-2-ca r boxylic Acid Hyd r oxy Am id e (3). (Yield
80%). 1H NMR (500 MHz, DMSO-d6) δ: 3.6-3.9 (m, 5H), 4.18
(d, 1H, J ) 6.7 Hz), 4.64 (d, 1H, J ) 5.9 Hz), 4.67 (d, 1H, J )
3.0 Hz), 5.19 (d, 1H, J ) 4.5 Hz), 7.4-7.48 (m, 1H), 7.48-7.55
(m, 2H), 7.75 (d, 2H, J ) 7.7 Hz), 7.83 (d, 2H, J ) 8.5 Hz),
7.87 (d, 2H, J ) 8.5 Hz), 8.79 (d, 1H, J ) 1.9 Hz), 10.62 (s,
1H); 13C NMR (125 MHz, DMSO-d6) δ: 48.6, 57.1, 66.6, 68.0,
MMP In h ibition Assa y.22 The assays were performed
using the fluorogenic substrate MCA-Pro-Leu-Gly-Leu-DPA-
Ala- Arg-NH2 (Peptide Institute, Inc.) at a final concentration
of 5 µM (MCA-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

Azasugar-Based MMP/ ADAM Inhibitors
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substrate solution to the 96-well plate, and incubation was
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TACE In h ibition Assa y.23 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 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), and recombinant bacmid was isolated, purified,
and then used to generate baculovirus particle in Sf9 insect
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Sf9 cells were infected with TACE baculovirus at a MOI1.
Conditioned 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 50ng/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-DPA-Arg- Ser-Ser-Ser-Arg-NH2; Bachem AG,
Switzerland), which contained the cleavage site of TNF-a, and
the increase of fluorescence intensity (Ex/Em ) 320/405 nm)
was monitored. Ki values were calculated from the substrate
by using GraphPad Prism, version 3.0 (GraphPad Software,
Inc., San Diego, CA).
In h ibition Assa y for TP A-In d u ced Ep id er m a l Hyp er -
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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 with 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. The present work was supported
by a grant for ‘Research and Development on Glyco-
cluster Controlling Biomolecules’ from the New Energy
and Industrial Technology Development Organization
(NEDO).
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