New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase a (MMP-2)

Article abstract of DOI:10.1016/j.bmc.2004.01.047

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).

Full text of DOI:10.1016/j.bmc.2004.01.047

Bioorganic & Medicinal Chemistry 12 (2004) 2441–2450
New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as
selective inhibitors of gelatinase A (MMP-2)
Armando Rossello,a,* Elisa Nuti,a Elisabetta Orlandini,a Paolo Carelli,a
Simona Rapposelli,a Marco Macchia,a Filippo Minutolo,a Laura Carbonaro,b
Adriana Albini,c Roberto Benelli,c Giovanni Cercignani,d Gillian Murphye
and Aldo Balsamoa
aDipartimento di Scienze Farmaceutiche, Universitꢀa degli Studi di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
bDipartimento di Chimica e Chimica Industriale, Universitꢀa degli Studi di Pisa, Via Risorgimento, 35, 56126 Pisa, Italy
cIstituto Nazionale per la Ricerca sul Cancro, Via Rosanna Benzi 10, 16132 Genova, Italy
dDipartimento di Fisiologia e Biochimica, Universitꢀa degli Studi di Pisa, Via Santa Maria, 55, 56126 Pisa, Italy
eDepartment of Oncology, University of Cambridge, Hills Road, Cambridge CB2 2XY, UK
Received 12 December 2003; accepted 30 January 2004
Abstract—New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-ana-
logues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix
metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central
skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over
MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug
CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells
over-expressing MMP-2 and MMP-9).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
expression of MMPs.5 Two of them, MMP-2 (gelatinase
A; EC 3.4.24.24) and MMP-9 (gelatinase B; EC
3.4.24.35), seem to play an important role in these
processes, considering that they are involved in meta-
static tumour dispersion and angiogenesis.6–9 Indirect
confirmation of this fact may be found in recent studies
on MMP-2- and MMP-9-deficient mice, which have
been shown to be resistant to cancer growth and
metastasis diffusion, without the development of any
other abnormalities.10;11 All these data, taken together,
indicate that gelatinase A and B may represent an
important target to develop new potential antimetastatic
and antiangiogenic drugs.12;13
During tumour progression, an increased expression of
certain enzymes belonging to the family of matrix
metalloproteinases (MMPs), which are believed to be
involved in metastatic tumour dispersion and angio-
genesis, accompanies the passage from a benign to a
malignant phenotype.1–4 These metallo (zinc) endopep-
tidases are produced and secreted as inactive zymogens
in the extracellular matrix by the tumour cells them-
selves or by surrounding stromal cells stimulated by the
nearby tumour. The pro-enzyme activation occurs by
means of other proteases [i.e., uPa and/or MMPs,
especially of a membrane-associated type, such as MT1-
MMP (MMP14)]. Normally, the homeostasis of MMPs
is maintained by their tissue inhibitors TIMPs, but in
cancer progression, there is often an uncontrolled over-
In the past few years, some potent Ôbroad spectrumÕ
MMP inhibitors (MMPi) have been proposed and tested
against tumours.14 Some of them have entered clinical
trials, but at present none of them is on the market. In
actual fact, some trials with these MMPi were carried
out on tumour forms, which do not express these
enzymes, thus leading to disappointing results.15 As they
were developed as cytostatic agents, they may be used in
Keywords: Gelatinase A inhibitors; MMP-2 selective; Sulfonamide-
based MMP inhibitors.
* Corresponding author. E-mail: aros@farm.unipi.it
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.047

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A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
a prolonged therapy, and therefore their bioavailability
and slow long-term toxicity are very important.16
Compounds such as Marimastat 1, CGS-27023A 2,
Prinomastat (AG-3340) 3 and BMS-275291 4 have
shown a severe musculoskeletal syndrome, with fibro-
proliferative effects in the joint capsule of the knees.17–19
These effects are thought to be linked to an impairment
of normal tissue remodelling governed by the MMP-1
and/or by sheddases such as TNF-a convertase.20 For
these reasons, a lack of activity with respect to MMP-1
is considered to be an important factor in reducing some
of the side effects found for ÔnonselectiveÕ MMPi such as
AG-3340, 3.21 Recently some synthetic MMPi, such as,
for example, BAY 12-9566 (5) and the retrohydrox-
amate-based derivative ABT-518 (6), have shown a high
inhibitory potency versus gelatinases A and B, with
good indices of selectivity.22;23 Also in the class of the
sulfonamido aminoacids, in addition to potent non-
selective gelatinase A and B inhibitors such as CGS
27023A (2) and its related analogue AG-3340 (3), some
more selective MMPi have recently been identi-
fied.22;24–28
9a, which may represent structurally simplified ana-
logues of compounds of type 3, and their oxa-analogues
8a and 10a, in which the methylenic carbon of their
isobutyl group is replaced by an oxygen atom. This
structural modification, already tested in other classes of
enzyme inhibitors,29 inserts into the molecular backbone
of 7a and 9a a new functionality (the oxyaminic oxygen)
able to act as a hydrogen bond acceptor, which may
strengthen the interaction of the new compounds (8a
and 10a) with the active site of the MMPs, thus
improving their biopharmacological properties. The
choice of the p-methoxyphenyl (for 7, 8) or the biphenyl
(for 9, 10) groups was made considering that many of
the known MMPi contain in their molecular structures a
phenyl ring substituted in the para position with more or
less hindered groups, especially of an aromatic or het-
eroaromatic nature.
O
O
O
O
S
S
O
O
OMe
N
HO
N
HO
N
H
X
N
H
X
R
R
7, X = CH2
8, X = O
9, X = CH2
10, X = O
O
O
S
O
O
O
a, R = i-Pr; b, R = Et; c, R = Allyl; d, R = p-BnO-Bn
H
N
O
HO
N
HO
N
H
N
H
N
H
OH
O
N
The use of Ôlengthened chainsÕ on the sulfonamide
group, which is present in many of the sulfonamides
studied, seems to give these known inhibitors selectivity
for MMP-2 and MMP-9, accommodating these linear
chains into the S10 pocket of MMP-2 and MMP-9,
which is described as a long, almost rectilinear canal.30–35
According to our hypothesis, the combination of the
two groups, the oxyaminic oxygen and the biphenyl
group, in consideration of the differences in the S10 and
S20 pockets existing between MMP-2 and MMP-1,
might influence the selectivity for MMP-2 over MMP-1
of type 10 sulfonamides without modifying their inhib-
itory potency significantly. Finally, in order to evaluate
the effects on their inhibitory properties of the replace-
ment of the isopropyl group of 8a and 10a with other
substituents with a different steric hindrance and lipo-
philicity, some of their ethyl (8b), allyl (10c) and
p-benzyloxybenzyl (8d and 10d) analogues were also
synthesized.
2
1
N
O
O
O
O
H
S
HS
N
O
N
H
N
H
O
HO
N
O
N
H
O
O
S
N
3
Cl
4
OCF3
O
O
O
O
O
S
HO
N
O
HO
S
O
O
6
5
Starting from the hypothesis that in many classes of
peptide-mimic enzyme inhibitors, even small structural
modifications may significantly influence their potency
and/or selectivity, we planned the synthesis of new
arylsulfonamido compounds, taking as a structural
model CGS 27023A, 2, whose molecular frame is par-
tially included also in the structure of the more active
AG-3340 3 (IC50 ¼ 83 pM on MMP-2).
2. Chemistry
The synthetic route to the N-arylsulfonyl-N-alkoxy-
aminoacetohydroxamic acids 8a,b,d and 10a,c,d is
reported in Scheme 1. para-Substituted arylsulfonyl
chlorides 11 and 12 were coupled with the appropriate
O-alkylhydroxylamines 13a–d in the presence of N-
methylmorpholine to give the corresponding O-alkyl-
sulfonamides 14a,b,d and 15a,c,d. Reaction of 14 and 15
with tert-butylbromoacetate yielded the corresponding
tert-butyl esters 16a,b,d and 17a,c,d. The deprotection of
16 and 17, followed by the reaction with O-(tert-butyl-
In order to carry out a preliminary screening on the
principal MMPs, we synthesized the already known
N-(4-methoxyphenyl)sulfonyl-N-isobutyl-acetohydrox-
amic acid 7a25 and its N-biphenylsulfonamido analogue

A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
2443
R1
O
O
R1
O
H2N
S
a
O
R
O
+
S
HN
O
R
Cl
11, R1 = OMe
12, R1 = Ph
13a-d
14a,b,d; R1 = OMe
15a,c,d; R1 = Ph
b
R1
R1
O
O
O
O
O
S
N
O
S
c-e
HO
N
t-BuO
O
R
N
O
R
H
16a,b,d; R1 = OMe
17a,c,d; R1 = Ph
8a,b,d; R1 = OMe
10a,c,d; R1 = Ph
a, R = i-Pr; b, R = Et; c, R = Allyl; d, R = p-BnO-Bn
Scheme 1. Reagents and conditions: (a) NMM, anhyd THF, rt; (b) BrCH2CO2–But, Cs2CO3, Bu4NHSO4, anhyd DMF, rt, 3 days; (c) TFA, anhyd
CH2Cl2, 0 ꢁC, 5 h; (d) TBDMSONH2, EDCl, anhyd CH2Cl2, 0–25 ꢁC, 24 h; (e) TFA, anhyd CH2Cl2, 0 ꢁC, 5 h.
dimethylsilyl)hydroxylamine in the presence of EDCI
gave the O-silylated hydroxamates 20a,b,d and 21a,c,d,
which were transformed by acid cleavage into the cor-
responding hydroxamic acids 8a,b,d and 10a,c,d. The
carba-analogues of 8a and 10a (7a and 9a) were pre-
pared using a known synthetic method.36
tuted acetohydroxamic acids 7–10 and of the reference
drugs CGS 27023A (2) and Ag-3340 (3).6
For the more interesting compounds and reference
drugs 2, 3, selectivity indices for MMP-2 over the other
MMPs studied are also reported, expressed as ratios of
their inhibitory indices (Table 1). On MMP-2, the two
isobutylsulfonamides 7a and 9a show a good potency,
with an IC50 near to 10 nM; the reference drug 2 shows a
slightly lower inhibitory activity (IC50 ¼ 20 nM) while
Ag-3340 (3) is more active by two orders of magnitude
(IC50 ¼ 0.083 nM). On the same MMP-2, while the oxa-
analogue of 7a, compound 8a, proves to be less active by
3. MMP inhibition
Table 1 shows the inhibitory indices (IC50) towards some
of the principal MMPs of the N-arylsulfonyl-N-substi-
Table 1. Inhibitory activity of 7a, 8a,b,d, 9a, 10a,c,d, 2 and 3 towards some of the principal MMPs and, for the more active compounds, the MMP-2
selectivitya (in parentheses)
IC50 (nM)6
Compound
X
R
MMP-1
MMP-2
6.9
MMP-3
MMP-7
MMP-9
7a
CH2
i-Pr
220
118
2.9
(32)a
4800
(1.3)a
(17)a
7800
(2.2)a
(0.4)a
3350
(0.9)a
2200
(0.2)a
1830
(0.4)a
34
8a
O
i-Pr
3600
9600
2100
13
8b
O
Et
8d
O
p-Bn–O–Bn
i-Pr
9a
CH2
3270
6500
(500)a
4500
(375)a
7800
(236)a
10,000
(244)a
22
(251)a
>50,000
(>4000)a
13,000
(394)a
>50,000
(>4000)a
55
(2.6)a
10a
10c
10d
O
i-Pr
12
>50,000
(>4000)a
200
(17)a
520
O
Allyl
33
(16)a
O
p-Bn–O–Bn
41
640
(16)a
8
2, CGS27023A
3, Ag-3340
20
100
(5)a
54
(2.8)a
(1.1)a
(0.4)a
0.26
(3.1)a
8.2
(99)a
0.083
0.23
(2.8)a
(650)a
a Selectivity for MMP-2 over each of the other MMPs, is expressed as the ratio of the IC50 value for MMPn over the value for MMP-2.

2444
A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
more than two orders of magnitude than 7a, the oxa-
analogue of 9a, compound 10a, shows an inhibitory
activity (IC50 ¼ 12 nM) practically identical to that of 9a
(IC50 ¼ 13 nM). Among the other N-alkyloxy sulfon-
amides, while the p-methoxyphenyl substituted ones
8b,d show an inhibitory activity similar to that of the
corresponding O-i-Pr homologue (8a), the p-biphenyl
substituted ones (10c,d) are slightly less active than their
O-i-Pr homologue 10a. Also at the level of MMP-9, the
isobutyl derivative 7a appears to be the most active of
the sulfonamidic compounds (IC50 ¼ 2.9 nM), while its
oxa-homologue is the least active (IC50 ¼ 3350 nM).
Also the isobutyl derivative 9a shows a good inhibitory
activity (IC50 ¼ 34 nM) while its oxa-homologue 10a
exhibits an inhibitory activity six times lower
(IC50 ¼ 200 nM). On the same enzyme, the p-MeO
substituted compounds 8b,d prove to be slightly more
active than their homologue 8a, whereas the p-Ph-
substituted ones 10c and 10d are slightly less active than
their homologue 10a. On MMP-9, Ag-3340 3 shows a
sub-nanomolar inhibitory activity (IC50 ¼ 0.26 nM). At
the level of MMP-1, among the sulfonamido derivatives
7–10, only the isobutyl one 7a shows an IC50 in the sub-
micromolar range, while the other compounds are
scarcely active, with an IC50 ranging from 3270 nM of 9a
practically devoid of any selectivity; also Ag-3340 3
proves to be devoid of MMP-3/MMP-2 and MMP-9/
MMP-2 selectivity, but proves to be more selective, with
respect to CGS 27023A 2, towards the other MMPs
(MMP-1/MMP-2 and MMP-7/MMP-2 ratios of 99 and
650, respectively).
4. Cellular studies
The most potent and MMP-2 selective of the new
inhibitors, the biphenylsulfonamide 10a, was tested in
an in vitro model of invasion on matrigel, carried out on
cellular lines of fibrosarcoma HT1080 (tumoural cells
that over-express MMP-2 and MMP-9).37 Figure 1
shows the effects of 10a (Fig. 1a) and of drug 2 (Fig. 1b)
on the invasion process, in comparison with the control
results (Fig. 1c). Normally HT1080 cells plated on ma-
trigel in complete medium, rapidly organize into Ôcord-
like structuresÕ with an Ôinhibitor-insensitiveÕ behaviour,
indicating the lack of toxic effects. From these colonies
some small elongations, representing the invasive cells,
can be observed (red arrows). Both compounds (10a and
2), at the same concentration of 50 nM, show a signifi-
cant reduction in the total number of invasive elonga-
tions observed in controls, thus suggesting similar
anti-invasive properties (see red arrows in Fig. 1a and b
and compare their amount with the control in Fig. 1c).
to 13,000 nM of 10c, or practically inactive, with an IC50
>
5 · 104 nM (10a,d). On the same MMP-1, the reference
drugs 2 and 3 are potent inhibitors, with IC50 values in
the nanomolar range. On MMP-3, all compounds 7–10,
with the only exception of 7a, which shows an IC50 value
of 118 nM, are very poor inhibitors (IC50 values >4 lM),
while the two reference drugs 2 and 3 are very potent
(IC50 in the nanomolar or sub-nanomolar range,
respectively). The compound, which is most active on
MMP-2 (10a) appears to be practically inactive on
MMP-7, while the two reference drugs 2 and 3 show a
good inhibitory activity. An analysis of the selectivity
indices reported in parentheses in Table 1 for the
N-alkoxy-N-sulfonamides 8a, 10a,c,d and the reference
compounds 2 and 3 shows that 10a and 10d are the ones
with the highest selectivity profile (MMP-1/MMP-2
ratio >4000 for both compounds, MMP-3/MMP-2
ratios of 375 and 244 for 10a and 10d, respectively,
MMP-7/MMP-2 ratio >4000 for 10a and MMP-9/
MMP-2 ratios of 17 and 16 for 10a and 10d, respec-
tively). The N-isobutyl-sulfonamidic compounds 7a and
9a (similar in their inhibitory potency towards MMP-2
to 10a, 10c and 10d) show a lower MMP-1/MMP-2
selectivity ratio that is 32 and 251, respectively. On the
MMPs tested, the reference compound 2 appears to be
A quantification of the inhibitory effect in the matrigel
test of 10a and of drug 2 is shown in Figure 2.
5. Conclusions
The replacement of the methylenic carbon atom linked
to the sulfonamidic nitrogen of 7a and 9a with an oxy-
gen atom has a negative effect at the level of all the
MMPs studied. This fact can be seen in their structurally
related sulfonamides 8a and 10a. The only exceptions
are the biphenyl-substituted compounds 9a and 10a,
where the oxygenated analogue 10a maintains the same
inhibitory activity as the corresponding methylenic
substituted 9a at the level of MMP-2. In these type 10
oxy-substituted compounds, also the other two biphenyl
homologues of 10a, the derivatives 10c and 10d, appear
to possess a good ability to inhibit MMP-2. As a con-
Figure 1. In vitro inhibition of HT1080 sarcoma cells growth on matrigel by 10a (a), CGS 27023A, 2 (b) and control (c).

A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
2445
comparison of compounds were recorded on a Mattson
1000 FTIR spectrometer. Nuclear magnetic resonance
(1H NMR) spectra were recorded on a Varian Gemini
200 (200 MHz) in a ca. 2% solution of CDCl3 or DMSO-
d6 for all compounds. Peak positions are given in parts
per million (ppm, d units). The proton magnetic reso-
nance assignments were established on the basis of the
expected chemical shifts and the multiplicity of the sig-
nals. Mass spectra were recorded on a HP-5988 A
spectrometer using a direct injection probe and an
electron beam energy of 70 eV. Reactions were routinely
monitored by thin-layer chromatography (TLC) on
0.25 mm silica gel plates (Merck 60F254) and hydroxamic
acids were visualized with FeCl3 aqueous solution. Flash
chromatography or preparative medium pressure liquid
chromatography (MPLC) were carried out through
glass columns containing 40–63 lm silica gel (Macherey-
Nagel Silica Gel 60) The MPLCs were performed using
a chromatographic apparatus consisting of a Buchi 681
pump, a Knauer differential refractometer detector and
a Philips PM 8220 pen recorder. Solvents and reagents
were obtained from commercial sources in the appro-
priate grade and were used without further purification
unless otherwise indicated. Reactions were run under an
argon atmosphere. Elemental analyses were carried out
by our analytical laboratory and were consistent with
theoretical values to within 0.4%.
Figure 2. Means of invasive elongations/field for 10a and for 2 as
compared to the control. ꢀStudentÕs test.
sequence, for the more lipophilic biphenyl-substituted
molecules of type 10, it may be hypothesized that the
presence of the oxyaminic group may give to these types
of sulfonamide-based inhibitors an appreciable MMP-2
selectivity, in particular with respect to MMP-1. The
MMP-2 selectivity of type 10 compounds seems to be
interesting, even when compared with that of the refer-
ence model compounds CGS 27023A (2) and Ag-3340
(3), which are poorly selective.
6.1.1. General procedure for the preparation of sulfon-
amides (14a,b,d, 15a,c,d).
A solution of the sulfonyl
chloride 9 or 10 (7.0 mmol) in anhydrous THF (18.0 mL)
was added dropwise to a stirred and cooled (0 ꢁC)
solution of the appropriate O-alkyl hydroxylamine
hydrochloride 11a–d (7.0 mmol) and N-methylmorpho-
line (1.5 mL, 14 mmol) in anhydrous THF (18.0 mL).
After 30 min under these conditions, the reaction mix-
ture was allowed to rest at rt for 4 days and was then
diluted with H2O (100 mL) and extracted with EtOAc
(100 mL) giving, after work-up, crude residues, which
were purified by flash chromatography on silica gel to
yield 14a,b,d, 15a,c,d as pure solids.
Finally, the selective and potent N-biphenylsulfonyl-N-
isopropoxy-aminoacetohydroxamic acid 10a proves to
be effective also in an in vitro model of invasion on
matrigel carried out on cellular lines of fibrosarcoma
HT1080 (tumoural cells over-expressing MMP-2).
6.1.1.1. N-Isopropoxy-4-methoxybenzenesulfonamide
The title compound was prepared from O-iso-
In conclusion, the results of this study seem to indicate a
possible structural requirement to be taken into con-
sideration in the design of new sulfonamido-based
MMPi useful in the control of tumoural invasion and
angiogenesis. In addition, compounds structurally
related to 10 may be useful tools in the study of the role
of the single MMPs in certain pathological pro-
cesses.11–13;15
(14a).
propylhydroxylamine hydrochloride 13a and 4-meth-
oxybenzenesulfonyl chloride 11 following the general
procedure. Compound 14a was used without further
purification: a white solid (65%); mp 93–95 ꢁC; 1H NMR
(CDCl3): d 1.17 (d, J ¼ 6:2 Hz, 6H), 3.88 (s, 3H), 4.23
(septet, J ¼ 6:2 Hz, 1H), 6.67 (s, 1H), 6.98–7.02 (m, 2H),
7.83–7.87 (m, 2H). Anal. (C10H15NO4S) C, H, N.
6.1.1.2.
N-Ethyloxy-4-methoxybenzenesulfonamide
(14b). The title compound was prepared from O-ethyl-
oxyhydroxylamine hydrochloride 13b and 4-meth-
oxybenzenesulfonyl chloride 11 following the general
procedure. Compound 14b was used without further
purification: a white solid (76%); mp 64–66 ꢁC; 1H NMR
(CDCl3): d 1.18 (t, J ¼ 7:1 Hz, 3H), 3.88 (s, 3H), 4.01 (q,
6. Experimental
6.1. General methods
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. Infrared (IR) spectra for

2446
A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
J ¼ 7:1 Hz, 2H), 6.89 (s, 1H), 6.98–7.02 (m, 2H), 7.83–
6.1.2.1. tert-Butyl{isopropoxy[(4-methoxyphenyl)sulfon-
yl]amino}acetate (16a). The title compound was pre-
7.88 (m, 2H). Anal. (C9H13NO4S) C, H, N.
pared from 14a and tert-butylbromoacetate following
the general procedure. The crude product was purified
by flash chromatography on silica gel using hexane–
ethyl acetate 3/1 v/v to give 16a as an oil (52%):
Rf ¼ 0:44; 1H NMR (CDCl3): d 1.23 (d, J ¼ 6:2 Hz,
6H), 1.46 (s, 9H), 3.58 (s, 2H), 3.88 (s, 3H), 4.52 (septet,
J ¼ 6:2 Hz, 1H), 6.99–7.04 (m, 2H), 7.78–7.82 (m, 2H).
Anal. (C16H25NO6S) C, H, N.
6.1.1.3. N-Benzyloxy-benzyloxy-4-methoxybenzene-
sulfonamide (14d).
The title compound was prepared
from O-benzyloxy-benzyloxyhydroxylamine hydrochlo-
ride 13d and 4-methoxybenzenesulfonyl chloride 11
following the general procedure. The crude product was
purified by flash chromatography on silica gel using
hexane–ethyl acetate 2/1 v/v to give 14d as a pale yellow
solid (46%): Rf ¼ 0:27; 1H NMR (CDCl3): d 3.87 (s,
3H), 4.90 (s, 2H), 5.07 (s, 2H), 6.89 (s, 1H), 6.92–7.01
(m, 4H), 7.27–7.42 (m, 7H), 7.83–7.88 (m, 2H). Anal.
(C21H21NO5S) C, H, N.
6.1.2.2. tert-Butyl{ethyloxy[(4-methoxyphenyl)sulfon-
yl]amino}acetate (16b).
The title compound was pre-
pared from 14b and tert-butylbromoacetate following
the general procedure. The crude product 16b was used
without further purification: a yellow gel (76%); 1H
NMR (CDCl3): d 1.15 (t, J ¼ 7:1 Hz, 3H), 1.45 (s, 9H),
3.58 (s, 2H), 3.88 (s, 3H), 4.31 (q, J ¼ 7:1 Hz, 2H), 7.00–
7.04 (m, 2H), 7.77–7.82 (m, 2H). Anal. (C15H23NO6S) C,
H, N.
6.1.1.4.
(15a). The title compound was prepared from O-iso-
N-Isopropoxy-1,10-biphenyl-4-sulfonamide
propylhydroxylamine hydrochloride 13a and 1,10-
biphenyl-4-sulfonyl chloride 12 following the general
procedure. Compound 15a was used without further
purification: a white solid (83%); mp 156–158 ꢁC; 1H
NMR (CDCl3) d: 1.21 (d, J ¼ 6:2 Hz, 6H), 4.3 (septet,
J ¼ 6:2 Hz, 1H), 6.78 (s, 1H), 7.42–7.63 (m, 5H), 7.72–
7.77 (m, 2H), 7.96–8.01 (m, 2H). Anal. (C15H17NO3S) C,
H, N.
6.1.2.3.
phenyl)sulfonyl]amino}acetate (16d).
tert-Butyl{benzyloxybenzyloxy[(4-methoxy-
The title com-
pound was prepared from 14d and tert-
butylbromoacetate following the general procedure. The
crude product was purified by flash chromatography on
silica gel using hexane–ethyl acetate 7/2 v/v to give 16d
as a yellow solid (32%): Rf ¼ 0:25; 1H NMR (CDCl3): d
1.47 (s, 9H), 3.61 (s, 2H), 3.84 (s, 3H), 5.06 (s, 2H), 5.25
(s, 2H), 6.92–6.98 (m, 4H), 7.29–7.40 (m, 7H), 7.75–7.80
(m, 2H). Anal. (C27H31NO7S) C, H, N.
6.1.1.5. N-Allyloxy-1,10-biphenyl-4-sulfonamide (15c).
The title compound was prepared from O-allylhydr-
oxylamine hydrochloride 13c and 1,10-biphenyl-4-sulfo-
nyl chloride 12 following the general procedure. The
crude reaction mixture was purified by flash chroma-
tography on silica gel using hexane–ethyl acetate 3/1 v/v
to give 15c as a pale yellow solid (37%): Rf ¼ 0:37; mp
6.1.2.4. tert-Butyl[(1,10-biphenyl-4-ylsulfonyl)(isoprop-
oxy)amino]acetate (17a). The title compound was pre-
pared from 15a and tert-butylbromoacetate following
the general procedure. The crude product was purified
by flash chromatography on silica gel using hexane–
ethyl acetate 7/1 v/v to give 17a as a white solid (34%):
1
116–118 ꢁC; H NMR (CDCl3) d: 4.50 (d, J ¼ 7:3 Hz,
2H), 5.25–5.37 (m, 2H), 5.80–6.10 (m, 1H), 6.98 (s, 1H),
7.42–7.64 (m, 5H), 7.73–7.78 (m, 2H), 7.97–8.02 (m,
2H). Anal. (C15H15NO3S) C, H, N.
1
Rf ¼ 0:25; mp 70–72 ꢁC; H NMR (CDCl3): d 1.26 (d,
J ¼ 6:2 Hz, 6H), 1.47 (s, 9H), 3.64 (br s, 2H), 4.58
(septet, J ¼ 6:2 Hz, 1H), 7.42–7.64 (m, 5H), 7.74–7.78
(m, 2H), 7.91–7.95 (m, 2H). Anal. (C21H27NO5S) C, H,
N.
6.1.1.6. N-Benzyloxy-benzyloxy-1,10-biphenyl-4-sulfo-
namide (15d). The title compound was prepared from
O-benzyloxy-benzyloxyhydroxylamine
hydrochloride
13d and 1,10-biphenyl-4-sulfonyl chloride 12 following
the general procedure. Recrystallization of the crude
product from isopropanol provided 15d as a white solid
6.1.2.5.
tert-Butyl[(1,10-biphenyl-4-ylsulfonyl)(allyl-
oxy)amino]acetate (17c). The title compound was pre-
pared from 15c and tert-butylbromoacetate following
the general procedure. The crude product was purified
by flash chromatography on silica gel using hexane–
ethyl acetate 5/1 v/v to give 17c as a white solid (64%):
1
(68%): mp 176–177 ꢁC; H NMR (DMSO-d6) d: 4.83 (s,
2H), 5.09 (s, 2H), 6.97–7.01 (m, 2H), 7.25–7.51 (m,
10H), 7.72–7.76 (m, 2H), 7.91 (m, 4H), 10.47 (s, 1H).
Anal. (C26H23NO4S) C, H, N.
1
Rf ¼ 0:38; mp 95–97 ꢁC; H NMR (CDCl3): d 1.46 (s,
9H), 3.68 (br s, 2H), 4.82 (d, J ¼ 6:2 Hz, 2H), 5.21–5.37
(m, 2H), 5.84–6.10 (m, 1H), 7.43–7.64 (m, 5H), 7.74–
7.78 (m, 2H), 7.91–7.95 (m, 2H). Anal. (C21H25NO5S) C,
H, N.
6.1.2. General procedure for the preparation of tert-butyl
esters (16a,b,d, 17a,c,d). A solution of the appropriate
sulfonamide (13, 14) (1 mmol) in anhydrous DMF
(3.0 mL) was treated with tert-butylbromoacetate
(1.2 mmol), caesium carbonate (1 mmol) and tetrabutyl-
ammonium hydrogensulfate (1 mmol). The reaction
mixture was stirred for 3 days at rt, then diluted with
H2O (20.0 mL) and extracted three times with ethyl
acetate (20.0 mL) giving, after work-up, crude residues,
which were purified by flash chromatography on silica
gel to yield 16a,b,d, 17a,c,d as pure solids.
6.1.2.6. tert-Butyl[(1,10-biphenyl-4-ylsulfonyl)(benzyl-
oxybenzyloxy)amino]acetate (17d). The title compound
was prepared from 15d and tert-butylbromoacetate
following the general procedure. The crude product was
purified by flash chromatography on silica gel using
hexane–ethyl acetate 4/1 v/v to give 17d as a white solid

A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
2447
1
(60%): Rf ¼ 0:37; mp 105–107 ꢁC; H NMR (CDCl3): d
1.48 (s, 9H), 3.69 (br s, 2H), 5.06 (s, 2H), 5.30 (s, 2H),
6.93–6.98 (m, 2H), 7.32–7.59 (m, 12H), 7.68–7.72 (m,
2H), 7.89–7.93 (m, 2H). Anal. (C32H33NO6S) C, H, N.
6.1.3.6. [(Benzyloxybenzyloxy)(1,10-biphenyl-4-ylsulfon-
yl)amino]acetic acid (19d). The title compound was
prepared from 17d following the general procedure. The
crude product was purified by chromatography on silica
gel using hexane–ethyl acetate 2/1 v/v to give 19d as a
1
solid (28%); mp 62–63 ꢁC; H NMR (CDCl3): d 3.83 (s,
6.1.3. General procedure for the preparation of carboxylic
acids (18a,b,d, 19a,c,d).
2H), 5.01 (s, 2H), 5.25 (s, 2H), 6.90–6.94 (m, 2H), 7.29–
7.57 (m, 12H), 7.65–7.70 (m, 2H), 7.87–7.91 (m, 2H).
Anal. (C28H25NO6S) C, H, N.
Trifluoroacetic acid (4.4 mL,
57 mmol) was added dropwise to a stirred and cooled
(0 ꢁC) solution of the appropriate tert-butyl ester 15,16
(1 mmol) in freshly distilled (P2O5) dichloromethane
(3.4 mL). The solution was stirred for 5 h at 0 ꢁC and the
solvent was then removed in vacuo to give pure 18a,b,d,
19a,c,d as solids.
6.1.4. General procedure for the preparation of O-
TBDMS acid hydroxyamides 20a,b,d, 21a,c,d.
1-[3-
(Dimethylamino)propyl]-3-ethyl carbodiimide hydro-
chloride (EDCI) was added portionwise (1 mmol) to a
stirred and cooled (0 ꢁC) solution of the carboxylic acid
18, 19 (1 mmol) and O-(tert-butyldimethylsilyl-hydrox-
ylamine (1 mmol) in freshly distilled (P2O5) dichlorom-
ethane (18 mL). After stirring at rt for 20 h, the mixture
was washed with water (20 mL) and the organic phase
was dried and evaporated in vacuo. The residue was
purified by flash chromatography to yield pure silyl
esters 20a,b,d, 21a,c,d as oils.
6.1.3.1. {Isopropoxy[(4-methoxyphenyl)sulfonyl]amino}-
acetic acid (18a). The title compound was prepared from
16a following the general procedure. The crude product
18a was used without further purification: a yellow oil
1
(99%); H NMR (CDCl3): d 1.23 (d, J ¼ 6:2 Hz, 6H),
3.74 (s, 2H), 3.89 (s, 3H), 4.52 (septet, J ¼ 6:2 Hz, 1H),
7.01–7.05 (m, 2H), 7.80–7.83 (m, 2H). Anal.
(C12H17NO6S) C, H, N.
6.1.4.1.
2-{Isopropoxy[(4-methoxyphenyl)sulfonyl]-
amino}acetic acid (tert-butyldimethylsilyl)hydroxyamide
(20a). The title compound was prepared from 18a fol-
lowing the general procedure. The crude oil 20a (75%)
was used without further purification; 1H NMR
(CDCl3): d 0.20 (s, 6H), 0.97 (s, 9H), 1.22 (d, J ¼ 6:2 Hz,
6H), 3.66 (s, 2H), 3.89 (s, 3H), 4.41 (septet, J ¼ 6:2 Hz,
1H), 7.01–7.05 (m, 2H), 7.78–7.82 (m, 2H), 8.59 (br s,
1H). Anal. (C18H32N2O6SSi) C, H, N.
6.1.3.2. {Ethyloxy[(4-methoxyphenyl)sulfonyl]amino}-
The title compound was prepared
acetic acid (18b).
from 16b following the general procedure. The crude
product 18b was used without further purification: a
yellow oil (99%); 1H NMR (CDCl3): d 1.15 (t,
J ¼ 7:1 Hz, 3H), 3.76 (s, 2H), 3.89 (s, 3H), 4.28 (q,
J ¼ 7:1 Hz, 2H), 7.01–7.06 (m, 2H), 7.79–7.83 (m, 2H).
Anal. (C11H15NO6S) C, H, N.
6.1.4.2. 2-{Ethyloxy[(4-methoxyphenyl)sulfonyl]amino}-
acetic acid (tert-butyldimethylsilyl)hydroxyamide (20b).
The title compound was prepared from 18b following
the general procedure. The crude product was purified
by flash chromatography on silica gel using hexane–
ethyl acetate 1/1 v/v to give 20b as a yellow oil (20%): 1H
NMR (CDCl3): d 0.20 (s, 6H), 0.97 (s, 9H), 1.19 (t,
J ¼ 7:1 Hz, 3H), 3.62 (s, 2H), 3.89 (s, 3H), 4.21 (q,
J ¼ 7:1 Hz, 2H), 6.98–7.01 (m, 2H), 7.77–7.82 (m, 2H).
Anal. (C17H30N2O6SSi) C, H, N.
6.1.3.3. {Benzyloxybenzyloxy[(4-methoxyphenyl)sulfon-
yl]amino}acetic acid (18d).
The title compound was
prepared from 16d following the general procedure. The
crude product 18d was used without further purification:
1
a yellow solid (96%); H NMR (CDCl3): d 3.78 (s, 2H),
3.85 (s, 3H), 5.04 (s, 2H), 5.21 (s, 2H), 5.40 (br s, 1H),
6.92–7.00 (m, 4H), 7.28–7.40 (m, 7H), 7.77–7.81 (m,
2H). Anal. (C23H23NO7S) C, H, N.
6.1.3.4. [(Isopropoxy)(1,10-biphenyl-4-ylsulfonyl)amino]-
acetic acid (19a). The title compound was prepared from
17a following the general procedure. The crude product
19a was used without further purification: a yellow solid
6.1.4.3. 2-{Benzyloxybenzyloxy[(4-methoxyphenyl)sul-
fonyl]amino}acetic acid (tert-butyldimethylsilyl)hydroxy-
amide (20d). The title compound was prepared from 18d
following the general procedure. The crude oil 20d
1
(86%); mp 125–127 ꢁC; H NMR (CDCl3): d 1.26 (d,
1
J ¼ 6:2 Hz, 6H), 3.82 (s, 2H), 4.58 (septet, J ¼ 6:2 Hz,
1H), 7.43–7.64 (m, 5H), 7.76–7.80 (m, 2H), 7.92–7.96
(m, 2H). Anal. (C17H19NO5S) C, H, N.
(67%) was used without further purification; H NMR
(CDCl3): d 0.17 (s, 6H), 0.97 (s, 9H), 3.57 (s, 2H), 3.86
(s, 3H), 5.06 (s, 2H), 5.09 (s, 2H), 6.95–7.00 (m, 4H),
7.31–7.41 (m, 7H), 7.76–7.81 (m, 2 H), 8.16 (br s, 1H).
Anal. (C29H38N2O7SSi) C, H, N.
6.1.3.5. [(Allyloxy)(1,10-biphenyl-4-ylsulfonyl)amino]-
acetic acid (19c).
The title compound was prepared
from 17c following the general procedure. The crude
product 19c was used without further purification: a
pale yellow solid (92%); mp 150–152 ꢁC; 1H NMR
(CDCl3): d 3.86 (s, 2H), 4.81 (d, J ¼ 6:2 Hz, 2H), 5.23–
5.37 (m, 2H), 5.81–6.01 (m, 1H), 7.43–7.63 (m, 5H),
7.76–7.80 (m, 2H), 7.92–7.96 (m, 2H). Anal.
(C17H17NO5S) C, H, N.
6.1.4.4. [(Isopropoxy)(1,10-biphenyl-4-ylsulfonyl)amino]-
acetic acid (tert-butyldimethylsilyl)hydroxyamide (21a).
The title compound was prepared from 19a following
the general procedure. The crude oil 21a (58%) was used
1
without further purification; H NMR (CDCl3): d 0.20
(s, 6H), 0.90 (s, 9H), 1.26 (d, J ¼ 6:2 Hz, 6H), 3.71 (s,
2H), 4.50 (septet, J ¼ 6:2 Hz, 1H), 7.42–7.64 (m, 5H),

2448
A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
7.76–7.80 (m, 2H), 7.92–7.96 (m, 2H). Anal.
(C23H34N2O5SSi) C, H, N.
(DMSO-d6): d 3.62 (s, 2H), 3.87 (s, 3H), 4.93 (s, 2H),
5.10 (s, 2H), 6.96–7.00 (m, 2H), 7.14–7.41 (m, 9H), 7.73–
7.78 (m, 2H), 10.70 (s, 1H). EI-MS m=z: 214 (1.95), 171
(8.14), 107 (8.86), 91 (100), 44 (34.18). Anal.
(C23H24N2O7S) C, H, N.
6.1.4.5. [(Allyloxy)(1,10-biphenyl-4-ylsulfonyl)amino]-
acetic acid (tert-butyldimethylsilyl)hydroxyamide (21c).
The title compound was prepared from 19c following
the general procedure. The crude pale yellow oil 21c
6.1.5.4.
2-[(Isopropoxy)(1,10-biphenyl-4-ylsulfonyl)-
1
(81%) was used without further purification; H NMR
amino]-N-hydroxyacetamide (10a). The title compound
was prepared from 21a following the general procedure.
Recrystallization from diethyl ether/hexane provided
10a as white solid: mp 99–101 ꢁC (25%); 1H NMR
(CDCl3): d 1.25 (d, J ¼ 6:2 Hz, 6H), 3.79 (s, 2H), 4.48
(septet, J ¼ 6:2 Hz, 1H), 7.44–7.64 (m, 5H), 7.76–7.80
(m, 2H), 7.91–7.96 (m, 2H). EI-MS m=z: 365 (2, M+1),
321 (8), 217 (51), 152 (100), 43 (55). Anal.
(C17H20N2O5S) C, H, N.
(CDCl3): d 0.10 (s, 6H), 0.91 (s, 9H), 3.76 (s, 2H), 4.69
(d, J ¼ 6:2 Hz, 2H), 5.33–5.43 (m, 2H), 5.80–6.00 (m,
1H), 7.46–7.64 (m, 5H), 7.76–7.79 (m, 2H), 7.91–7.95
(m, 2H). Anal. (C23H32N2O5SSi) C, H, N.
6.1.4.6. [(Benzyloxybenzyloxy)(1,10-biphenyl-4-ylsulfon-
yl)amino]acetic acid (tert-butyldimethylsilyl)hydroxy-
amide (21d). The title compound was prepared from
19d following the general procedure. The crude product
was purified by flash chromatography on silica gel using
hexane–ethyl acetate 5/2 v/v to give 21d as a yellow oil
6.1.5.5. 2-[(Allyloxy)(1,10-biphenyl-4-ylsulfonyl)amino]-
1
(9%): Rf ¼ 0:35; H NMR (CDCl3): d 0.18 (s, 6H), 0.96
N-hydroxyacetamide (10c).
The title compound was
(s, 9H), 3.63 (s, 2H), 5.06 (s, 2H), 5.14 (s, 2H), 6.97–7.01
(m, 2H), 7.32–7.53 (m, 10H), 7.56–7.61 (m, 2H), 7.71–
7.75 (m, 2H), 7.89–7.94 (m, 2H), 8.11 (s, 1H). Anal.
(C34H40N2O6SSi) C, H, N.
prepared from 21c following the general procedure. The
crude product was purified by flash chromatography on
silica gel using CHCl3–MeOH 9/1 v/v as the eluent.
Recrystallization from diethyl ether/hexane gave 10c as
a white solid (57%): mp 133–135 ꢁC; 1H NMR (DMSO-
d6): d 3.49 (s, 2H), 4.56 (d, J ¼ 6:2 Hz, 2H), 5.20–5.32
(m, 2H), 5.81–5.91 (m, 1H), 7.50–7.57 (m, 3H), 7.76–
7.80 (m, 2H), 7.89–8.03 (m, 4H), 9.04 (s, 1H), 10.69 (s,
1H). EI-MS m=z: 363 (1, M+1), 322 (7), 217 (46), 152
(100), 41 (62). Anal. (C17H18N2O5S) C, H, N.
6.1.5. General procedure for the preparation of acid
hydroxyamides (8a,b,d, 10a,c,d).
Trifluoroacetic acid
(4.4 mL, 57 mmol) was added dropwise to a stirred and
cooled solution (0 ꢁC) of 20a,b,d, or 21a,c,d (1 mmol) in
freshly distilled (P2O5) dichloromethane (3.4 mL). The
solution was stirred under these reaction conditions for
5 h and the solvent was removed in vacuo to give 8a,b,d,
10a,c,d as solids.
6.1.5.6.
sulfonyl)amino]-N-hydroxyacetamide (10d).
2-[(Benzyloxybenzyloxy)(1,10-biphenyl-4-yl-
The title
compound was prepared from 21d following the general
procedure. Recrystallization from diethyl ether/hexane
1
6.1.5.1.
2-{Isopropoxy[(4-methoxyphenyl)sulfonyl]-
gave 10d as yellow solid (94%): mp 75–77 ꢁC; H NMR
amino}-N-hydroxyacetamide (8a). The title compound
was prepared from 20a following the general procedure.
Recrystallization from CHCl3/hexane gave 8a as a white
solid (67%): mp 62–64 ꢁC; H NMR (DMSO-d6): d 1.12
(CDCl3): d 3.67 (br s, 2H), 5.04 (s, 2H), 5.11 (s, 2H),
6.95–6.98 (m, 2H), 7.33–7.57 (m, 12H), 7.67–7.71 (m,
2H), 7.87–7.91 (m, 2H). Anal. (C28H26N2O6S) C, H, N.
1
(d, J ¼ 6:2 Hz, 6H), 3.74 (s, 2H), 3.87 (s, 3H), 4.29
(septet, J ¼ 6:2 Hz, 1H), 7.17–7.22 (m, 2H), 7.76–7.81
(m, 2H), 10.69 (s, 1H). EI-MS m=z: 319 (1, M+1), 276
(2), 171 (36), 107 (22), 45 (100). Anal. (C12H18N2O6S) C,
H, N.
6.1.5.7. N-Isobutyl-1,10-biphenyl-4-sulfonamide (22).
Isobutylamine 23 (4.7 mL, 47.4 mmol) was dissolved in
CHCl3 (36 mL), and the solution was cooled to 0 ꢁC.
1,10-Biphenyl-4-sulfonyl chloride 11 (4.0 g, 15.8 mmol)
was added to this solution. The reaction mixture was
stirred at rt for 1 h and then refluxed for 1 h. After being
cooled back to rt, the reaction mixture was washed three
times with 4 N hydrochloric acid, twice with water and
once with brine, and then it was dried (Na2SO4), and the
solvent was evaporated to give 22 as a solid (4.0 g, 87%):
6.1.5.2. 2-{Ethyloxy[(4-methoxyphenyl)sulfonyl]amino}-
N-hydroxyacetamide (8b). The title compound was pre-
pared from 20b following the general procedure. The
crude product was purified by preparative thin-layer
chromatography using CHCl3–MeOH 9/1 as the eluent
1
1
to give 8b as a yellow solid (3%): H NMR (CDCl3): d
mp 88–90 ꢁC; H NMR (CDCl3): d 0.89 (d, J ¼ 6:8 Hz,
1.16 (t, J ¼ 6:9 Hz, 3H), 3.66 (s, 2H), 3.88 (s, 3H), 4.18
(q, J ¼ 6:9 Hz, 2H), 7.00–7.05 (m, 2H), 7.77–7.82 (m,
2H). EI-MS m=z: 171 (84.19), 107 (67.69), 77 (100), 44
(88.67). Anal. (C11H16N2O6S) C, H, N.
6H), 1.74 (m, 1H), 2.81 (dd, J ¼ 6:6 and 6.8 Hz, 2H),
4.47 (t, 1H), 7.41–7.53 (m, 3H), 7.58–7.64 (m, 2H), 7.69–
7.75 (m, 2 H), 7.89–7.94 (m, 2H). Anal. (C16H19NO2S)
C, H, N.
6.1.5.3.
2-{Benzyloxybenzyloxy[(4-methoxyphen-
yl)sulfonyl]amino}-N-hydroxyacetamide (8d). The title
compound was prepared from 20d following the general
procedure. Recrystallization from CHCl3/hexane gave
8d as a white solid (62%): mp 140–142 ꢁC; H NMR
6.1.5.8. Ethyl[isobutyl-1,10-biphenyl-4-sulfonylamino]-
acetate (24). Sodium hydride (0.27 g of a 60% oil dis-
persion, 6.9 mmol) was suspended in THF (17.6 mL). A
solution of sulfonamide 22 (2.0 g, 6.9 mmol) also in THF
1

A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
2449
(70.7 mL) was added, and the reaction mixture was
stirred for 30 min at rt. Then ethyl bromoacetate
(1.15 mL, 10.35 mmol) was added, and the reaction
mixture was stirred overnight at rt. The reaction was
quenched with a small amount of water, and all the
solvent was removed. The crude mixture was partitioned
between ethyl acetate and water, the aqueous phase was
extracted several times with ethyl acetate, the combined
organic layers were dried (Na2SO4) and the solvent was
evaporated. The crude product recrystallized from hex-
ane provided 24 as a solid (1.24 g, 48%): mp 70–72 ꢁC;
1H NMR (CDCl3): d 0.91 (d, J ¼ 6:6 Hz, 6H), 1.16 (t,
J ¼ 7:1 Hz, 3H), 1.86 (m, 1H), 3.09 (d, J ¼ 7:5 Hz, 2H),
3.99–4.10 (m, 4H), 7.40–7.54 (m, 3H), 7.57–7.63 (m,
2H), 7.66–7.73 (m, 2H), 7.84–7.91 (m, 2H). Anal.
(C20H25NO4S) C, H, N.
HCl 50 mM, pH ¼ 7.5, NaCl 150 mM, CaCl2 10 mM,
Brij 35 0.05% and DMSO 1%. The activated enzyme
(final concentration 155 pM for MMP-2, 2 nM for
MMP-3, 178 pM for MMP-7, 728 pM for MMP-1 and
216 pM for MMP-9) and inhibitor solutions were
incubated in the assay buffer for 4 h. The assay tem-
perature was 25 ꢁC. After the addition of 100 lM
solution of the fluorogenic substrate (Mca-Pro-Leu-
Gly-Leu-Dpa-Ala-Arg-NH2) in DMSO (final concen-
tration 1 lM), the hydrolysis was monitored by con-
tinuously recording the increase in fluorescence (kex
328 nm, kem 393 nm) using a Perkin–Elmer spectroflu-
orimeter LS 50B. Percent inhibitions were calculated
from control reactions without the inhibitor. IC50 was
determined using the formula: Vi=Vo ¼ 1=ð1 þ ½Iꢁ=IC50Þ,
where Vi is the initial velocity of substrate cleavage in
the presence of the inhibitor at concentration ½Iꢁ and Vo
is the initial velocity in the absence of the inhibitor.
Results were analyzed using GraFit.39
6.1.5.9. 2-[Isobutyl-1,10-biphenyl-4-sulfonyl-amino]-N-
hydroxyacetamide (9a).
Ester 24 (1.23 g, 3.28 mmol)
was dissolved in methanol (50 mL). Hydroxylamine
hydrochloride (0.46 g, 6.56 mmol) was added to this
solution, followed by the addition of sodium methoxide,
freshly prepared from sodium (0.226 g, 9.8 mmol) dis-
solved in methanol (5.82 mL). The reaction mixture was
stirred overnight at rt. The reaction was worked up by
partitioning between dilute hydrochloric acid (pH ¼ 3)
and ethyl acetate. The aqueous phase was extracted well
with ethyl acetate, the combined organic layers were
dried and the solvent was evaporated. The product was
purified by flash chromatography on silica gel using
hexane–ethyl acetate 1/1 v/v and then was triturated
with hexane/diethyl ether to give 9a (0.47 g, 40%) as a
6.3. Growth on matrigel
Matrigel, a mixture of basement membrane components
extracted from the EHS tumour,37 was thawed at 4 ꢁC in
an ice-water bath and diluted in water to a final con-
centration of 14 mg/mL. A 0.3 mL volume of this solu-
tion was gently pipetted into a 13-mm-diameter tissue
culture well (24-well plate). After one hour of incubation
at 37 ꢁC, the matrigel was completely polymerized, and
5 · 104 cells in 1 mL of complete medium were carefully
seeded on top of the matrigel layer and treated with
MMP inhibitors. Controls were treated with the carrier
(DMSO) alone. The seeded plates were incubated at
37 ꢁC in a 5% CO2 humidified atmosphere, and were
photographed after 24 h.
1
solid; mp 150–152 ꢁC; H NMR (DMSO-d6): 0.77 (d,
J ¼ 6:4 Hz, 6H), 1.84 (m, 1H), 3.05 (d, J ¼ 7:3 Hz, 2H),
3.68 (s, 2H), 7.41–7.53 (m, 3H), 7.70–7.73 (m, 2H), 7.77–
7.81 (m, 2H), 7.89–7.93 (m, 2H). Anal. (C18H22N2O4S)
C, H, N.
HT1080 sarcoma cells, plated on matrigel in complete
medium, rapidly organize into a network of cord-like
structures with an Ôinhibitor-insensitiveÕ behaviour,
indicating the lack of toxic effects. During this phase,
cells migrate on the surface of matrigel and prolifer-
ate, but do not invade the matrix. After 24 h, it is
possible to note single or small groups of cells pro-
truding from the established ÔcordsÕ; these structures,
indicated in Figure 1 by red arrows, represent the
starting of HT1080 invasive behaviour. Incubation
with 10a or 2 (50 nM) affected the formation of
invading structures. Both molecules strongly reduced
the invasion of matrigel, with a very low number of
HT1080-derived invasive structures compared with
untreated controls.
6.2. Matrix metalloproteinase activation and fluorimetric
assay (MMP-1, -2, -3, -7 and -9).6;38
Type I collagenase (pro-MMP-1, native enzyme derived
from cell culture of human rheumatoid synovial
fibroblasts),
stromelysin
(pro-MMP-3,
human
recombinant) and matrilysin (pro-MMP-7, human
recombinant) was purchased from Calbiochem.
Recombinant human progelatinases A (pro-MMP-2)
and B (pro-MMP9) secreted by transfected mouse
myeloma cells were prepared in our laboratory.
Proenzymes were activated immediately prior to use
with p-aminophenylmercuric acetate (APMA 2 mM for
1 h at 37 ꢁC for pro-MMP-1, APMA 2 mM for 1 h at
25 ꢁC for pro-MMP-2 and pro-MMP-7; APMA 1 mM
for 1 h at 37 ꢁC for pro-MMP-9) and with trypsin
13 lg/mL for 15 min at 37 ꢁC followed by soybean
trypsin inhibitor (SBTI) 50 lg/mL for pro-MMP-3. For
assay measurements, the stock solutions (100 mM) of
the inhibitors in DMSO were further diluted, at seven
different concentrations (1–1500 nM) for each MMP, as
required in the fluorimetric assay buffer (FAB): Tris–
Acknowledgements
The authors would like to thank Mr. Christian Van-
zetto, for his studies on cell cultures carried out at
the Istituto Nazionale per la Ricerca sul Cancro,
Genova.

2450
A. Rossello et al. / Bioorg. Med. Chem. 12 (2004) 2441–2450
Stacey, J. R.; Steinman, D. H.; Albert, D. H.; Bouska, J.
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