Carbonic anhydrase and matrix metalloproteinase inhibitors. Inhibition of human tumor-associated isozymes IX and cytosolic isozyme I and II with sulfonylated hydroxamates

Article abstract of DOI:10.1016/j.bmc.2007.01.023

A series of sulfonylated hydroxamates were synthesized and evaluated as dual inhibitors of both human carbonic anhydrases (hCAs) and matrix metalloproteinases (MMPs), two metalloenzyme families involved in carcinogenesis and tumor invasion processes. The

Full text of DOI:10.1016/j.bmc.2007.01.023

Bioorganic & Medicinal Chemistry 15 (2007) 2298–2311
Carbonic anhydrase and matrix metalloproteinase inhibitors.
Inhibition of human tumor-associated isozymes IX and cytosolic
isozyme I and II with sulfonylated hydroxamates
Elisa Nuti,a Elisabetta Orlandini,a Susanna Nencetti,a Armando Rossello,a,*
Alessio Innocenti,b Andrea Scozzafavab and Claudiu T. Supuranb,*
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, via Bonanno 6, 56126 Pisa, Italy
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy
b
`
Received 3 July 2006; revised 8 January 2007; accepted 17 January 2007
Available online 19 January 2007
Abstract—A series of sulfonylated hydroxamates were synthesized and evaluated as dual inhibitors of both human carbonic anhy-
drases (hCAs) and matrix metalloproteinases (MMPs), two metalloenzyme families involved in carcinogenesis and tumor invasion
processes. The new derivatives were tested on three CA isozymes, the cytosolic isozymes I and II, and the transmembrane, tumor-
associated isozyme IX, and also on human gelatinases (MMP-2 and MMP-9). Some of the new derivatives proved to be potent and
selective inhibitors of CA II, but only compounds 3b and 6b, devoid of the arylsulfonyl moiety, proved to have a better inhibitory
activity on hCA IX than on hCA I and II, in the micromolar range.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
on cell adhesion and differentiation by its N-terminal
proteoglycan-related region which is absent in other
transmembrane CA isozymes, such as CA XII (which
is present in some tumors3) and CA XIV (which is not
associated with tumors).8 However, only the catalytic
activity of CA IX seems to be involved in oncogenesis,
the role of the proteoglycan-like region being largely
unknown at this moment.
The a-carbonic anhydrases (CAs, EC 4.2.1.1) are a fam-
ily of metalloenzymes involved in the catalysis of an
important physiological reaction: the hydration of CO2
to bicarboþnate and
a
proton ðCO2 þ H2O $
HCO3ꢀ þ H Þ. At least 13 enzymatically active isoforms
have been discovered in higher vertebrates.1 CAs are
involved in pH regulation, secretion of electrolytes, res-
piration,2 and biosynthetic reactions which require CO2/
bicarbonate as substrate such as gluconeogenesis, lipo-
genesis, ureagenesis, and pyrimidine synthesis among
others.3 Other roles for these enzymes were highlighted,
such as calcification and bone resorption.4 The discov-
ery that CA IX, a transmembrane tumor-associated pro-
tein,5 was prevalently expressed in several human cancer
cells and not in their normal counterparts6 suggests a
role for some CA isoforms in oncogenesis.3 Several stud-
ies showed a clearcut relationship between high CA IX
levels in tumors and a poor prognosis.7 CA IX also acts
Tumor cells have a lower extracellular pH (pHe) than
normal cells due to lactic acid produced by glycolysis.9
An acidic pHe contributes to increased tumor progres-
sion by promoting the action of growth factors,10 prote-
ases11 and an increased rate of mutation.12 Recently
CO2 in addition to lactic acid was demonstrated to be
significant source of acidity in tumors,13a pointing out
the implication of CAs (such as CA IX and XII) in
tumor progression.3 The expression of CA IX is both
regulated by the von Hippel-Lindau (VHL) tumor sup-
pressor protein and by hypoxia present in many tumor
types.3,13a Thus, an inactivation of the VHL factor gene
enhances the expression of CA IX,12c whereas hypoxia
induces the expression of CA IX via a direct transcrip-
tional activation of CA9 gene by the hypoxia-inducible
factor-1 (HIF-1).12d Moreover, hypoxia stimulates CA
IX to acidify the pHe (by an yet unknown mechanism
Keywords: Carbonic anhydrase inhibitors; Matrix metalloproteinase
inhibitors; Hydroxamic acid inhibitors.
*
Corresponding authors. Tel.: +39 050 2219562; fax: +39 050 2219605
(A.R.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.); e-mail
addresses: aros@farm.unipi.it; claudiu.supuran@unifi.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.023

E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
2299
of action), proving that the expression levels and the
catalytic activity of CA IX are dependent on the
availability of oxygen within the tumor.13a Many CA
isoforms also participate to metabolons in which various
anion exchangers (AEs) or sodium bicarbonate co-trans-
porters (NBCs) interact physically and functionally with
the enzyme.9–11 Indeed, for allowing the control of their
pH and bicarbonate levels, cells express various anion
and bicarbonate transport proteins that rapidly and
selectively move bicarbonate and/or protons across the
plasma membrane (Fig. 1).13b,c
ogies including arthritis (MMP-1, -3, -13) and metastatic
cancer (MMP-2, -9), and therefore inhibition of these
enzymes has been considered an effective therapeutic
approach.
Inhibition of both CAs as well as MMPs is correlated to
the coordination of the inhibitor molecule to the catalyt-
ic metal ion. Thus, CA and MMP inhibitors must con-
tain a zinc-binding function attached to a scaffold
interacting with other binding regions of the enzymes.17a
The most potent MMPIs reported up to now incorpo-
rate a hydroxamate moiety which is able to bind biden-
tately to the catalytic Zn(II) ion of the enzyme.
Recently, it has been shown that N-hydroxyurea, one
of the simplest hydroxamates known, also binds biden-
CA IX was clearly demonstrated to be involved in the
acidification of the pHe by Svastova et al.13a Teicher
and collaborators showed earlier that acetazolamide
(AAZ) decreased the tumor growth in vivo and
enhanced the action of some chemotherapeutic agents,
such as cisplatin, melphalan, and PtCl4, when used in
combination therapy.14 Several CA IX-selective sulfon-
amide inhibitors were able to reduce the extracellular
acidification of Madin-Darby canine kidney (MDCK)-
CA IX cells in hypoxia but not in normoxia.13a Further-
more, a decrease of the extracellular pH reduces the
cytotoxicity of weakly basic chemotherapeutic drugs
such as paclitaxel, mitoxantrone, and topotecan.15 Tak-
en together, these data suggest the growing interest for
specific CA IX/XII inhibitors in cancer therapy. Such
compounds may prevent the decrease in pHe and may
be used in combinations with other antitumor drugs to
increase the efficacy or the uptake of weakly basic
drugs.9,14 A decrease in pHe is also known to lead to
activation of matrix metalloproteinases (MMPs), zinc
endopeptidases critically involved in extracellular matrix
breakdown and metastasis.13b,16
tately to the Zn(II) ion within the CA active site.17b
A
large number of sulfonylated amino acid hydroxamates
have been developed in the search of potent and selec-
tive inhibitors after the discovery of CGS 27023A
(Fig. 2),18 the first broad-spectrum inhibitor of this class
entered in clinical development.
In a previous paper,19 we reported a series of sulfonylat-
ed hydroxamates structurally related to CGS 27023A
that were also effective as CAIs. The best CA inhibitory
activity was seen with compounds of type A (Fig. 2)
where X was an hydrogen atom, while bulkier X (ben-
zyl, substituted benzyl, etc.) was advantageous for
obtaining an increased activity on MMP. These data
suggested that it should be possible to develop dual
enzyme inhibitors that would inhibit both these metallo-
enzymes, based on the nature of the X and R substitu-
ents in the A formula.
Based on these findings, the aim of this work was to fur-
ther investigate the possibility of inhibiting both CAs
and MMPs, two families of enzymes involved in tumor
formation, growth, and metastasis, by the same class of
In the last few years sulfonylated derivatives have been
reported as MMP inhibitors (MMPIs).16 Up-regulation
of specific MMPs has been associated to various pathol-
Figure 1. Interaction of cytosolic (CA II) and transmembrane (CA IX and XII) isozymes with other proteins involved in pH homeostasis and anion
transport such as (1) the monocarboxylate transporter; (2) the Na+–H+-antiporter; (3) the ATP-dependent Na+–K+-antiporters; (4) the H+– ATP-ase;
(5) acquaporins; (6) membrane-bound CAs (CA IX, XII or XIV); (7) bicarbonate/chloride anion exchangers (AEs).13b The acidic extracellular pH
leads to MMP activation. Blocking of both CA and MMP isoforms involved in such processes (CA II, IX, MMP-2, MMP-9, etc.) may thus lead to
novel approaches to fight cancer.

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E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
give the corresponding N-alkyloxysulfonamides 8a–h.
Reaction of 8a–h with tert-butylbromoacetate or
tert-butyl 2-bromo-3-methyl butanoate yielded the
corresponding tert-butyl esters 9a–d,f–h and 10d,e.
Deprotection of the tert-butyl esters with TFA followed
by condensation with O-(tert-butyldimethylsilyl)hydrox-
ylamine in the presence of EDCI gave the O-silylated
hydroxamates 13a–d,f–h and 14d,e, which were trans-
formed into the corresponding hydroxamic acids 1a–d,
f–h and 2d,e by acidic cleavage.
Compounds 4c, 5b,c, and 6b were synthesized as out-
lined in Scheme 2. Treatment of O-alkylhydroxylamine
hydrochloride 7b,c with glyoxylic acid afforded the cor-
responding N-(alkyloxy)iminoacetic acids 15b,c. The
addition of alkyllithiums to oximes21 15b,c provided
the N-alkyloxyamino acids 16c, 17b,c, and 18b which,
after condensation withO-(tert-butyldimethylsilyl)hy-
droxylamine in the presence of EDCI followed by acidic
deprotection, gave hydroxamic acids 4c, 5b,c, and 6b,
respectively.
Figure 2.
Compounds 3b,c were prepared from 15b,c as described
in Scheme 3. Reduction of N-(alkyloxy)iminoacetic
acids 15b,c with borane-triethylamine complex in hydro-
alcoholic solution in presence of HCl yielded the corre-
sponding N-alkyloxyaminoacetic acids 22b,c. Treatment
of 22b,c withO-(tert-butyldimethylsilyl)hydroxylamine
in the presence of EDCI followed by acidic deprotection
provided hydroxamic acids 3b.
3. Results and discussion
Compounds 1–6 and standard, clinically used CAIs,
such as acetazolamide AAZ, methazolamide MZA, eth-
oxozolamide EZA, dichlorophenamide DCP, and
indisulam IND (Fig. 4), have been tested for the inhibi-
tion of two cytosolic, ubiquitous isozymes of human ori-
gin, that is, hCA I and hCA II,1,2 as well as the human
tumor-associated isoform hCA IX.
Figure 3.
inhibitors, that is, the hydroxamates. As hydroxamates
(except N-hydroxyurea17b) were never investigated until
now as CA IX inhibitors but only against isoforms CA
I, II, and IV,19 in this paper, we report the preparation
of sulfonylated hydroxamates of type B where R1 is an
alkyl group20 and the arylsulfonyl moiety is a 4-meth-
oxybenzenesulfonyl, like in CGS 27023A, and their
interaction with several physiologically relevant CA
and MMP isoforms, that is, CA I, II, IX, and MMP-2
and MMP-9. Moreover, we synthesized a series of
Compounds 1–6 have also been tested in vitro on
MMP-2 (human gelatinase A) and MMP-9 (human
gelatinase B), using CGS 27023A as reference drug.
The two gelatinases play a significant role in some key
functions of tumor cells, facilitating metastatic tumor
dispersion and angiogenesis, resistance to apoptosis,
and activation of EGF receptors.22 Inhibition data
against these enzymes are shown in Tables 1 and 2.
N-alkyloxy amino acid hydroxamates of type
C
The following SAR may be observed for compounds
1a–d,f–h, 2d,e (type B) presented in Table 1: (i) sulfony-
lated hydroxamates reported here generally act as better
inhibitors against the cytosolic isozymes I and II than
against the tumor-associated isozyme hCA IX, being
particularly active on hCA II, with KI values in the
range of 91–97 nM; (ii) these derivatives are also much
more effective on hCA I and II than the reference drug
CGS 27023A, some of them having KI values in the
same range as the clinically used derivatives; (iii) the
introduction of the R substituent in the a-position to
the hydroxamate moiety does not seem to affect the
(Fig. 3) in order to prove the importance of the sulfon-
amide moiety for the inhibitory activity on both these
classes of enzymes.
2. Chemistry
Synthesis of compounds 1a–d,f–h, 2d,e is described in
Scheme 1. 4-Methoxybenzenesulfonyl chloride was cou-
pled with the appropriate O-alkylhydroxylamine hydro-
chloride 7a–h in the presence of N-methylmorpholine to

E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
2301
t
Scheme 1. Reagents: (i) ClSO2PhOCH3, N-methylmorpholine, THF; (ii) BrCHRCO2 Bu, Cs2CO3, Bu4NHSO4, DMF; (iii) TFA, CH2Cl2;
(iv) TBDSiONH2, EDCI, CH2Cl2; (v) TFA, CH2Cl2.
Scheme 2. Reagents: (i) HCOCO2H, AcONa, MeOH/H2O 1:1; (ii) RLi, THF; (iii) TBDMSiONH2, EDCI, CH2Cl2; (iv) TFA, CH2Cl2.
biological activity (e.g., 1d compared to 2d). (iv) The
most interesting compounds from the point of view of
CA-binding affinity of this series are 1d and 2d, which
show a 100-fold selectivity for hCA II over the other iso-
zymes. They both bear an i-Pr group in the R1 position,
so this substituent seems to be the best among those con-
sidered to achieve this kind of selectivity profile.
the best affinities; (vii) they are all less active on gelatin-
ases than CGS 27023A.
The most potent compound of this series is 2d which has
an i-Pr group in R1 and R substituents. This compound
displays good inhibitory activity against both hCA II
(KI = 94 nM) and MMP-9 (IC50 = 370 nM).
(v) These derivatives display a low micromolar inhibito-
ry activity on gelatinases, being particularly effective on
MMP-9; (vi) the a-substitution leads to an increased
activity on both gelatinases, with 2d and 2e showing
In Table 2 are reported the biological results for com-
pounds 3b,c, 4c, 5b,c, and 6b (type C): (i) N-alkyloxy
amino acid hydroxamates presented here generally act
as less effective inhibitors of hCA I and II than the

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E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
Considering these results, it should be concluded that
the lack of the arylsulfonyl moiety causes a 500- to
1000-fold decrease in hCA I, II inhibitory activity
(e.g., 3b compared to 1b) without affecting activity on
hCA IX, and a 90- to 300-fold reduction of gelatinase
inhibition together with an inversion of selectivity pat-
tern between MMP-2 and MMP-9.
4. Conclusions
In conclusion, the present paper reports the design and
synthesis of a series of sulfonylated hydroxamates as
dual inhibitors of both, hCAs and MMPs, families of
enzymes involved in carcinogenesis and tumor invasion
processes. The new derivatives have been tested on three
CA isozymes, the cytosolic isozymes I and II, and the
transmembrane, tumor-associated isozyme IX, and also
on human gelatinases (MMP-2 and MMP-9).
Scheme 3. Reagents: (i) BH3–TEA, HCl 1 N, EtOH; (ii) TBDM-
SiONH2, EDCI, CH2Cl2; (iii) TFA/H2O.
previous series of derivatives; (ii) the most interesting
compounds are 3b and 6b, which are selective for hCA
IX over hCA I, II, showing also the best activities on
hCA IX over both series of derivatives (KI = 6.7 and
7.1 lM, respectively, on this CA isoform); (iii) like
observed for the previous series, the a-substitution (R)
does not affect the inhibitory activity on hCAs, general-
ly. Compound 6b seems to be the only case in which it is
possible to see an influence of the R substituent. In-fact,
the introduction of a sec-butyl group in R maintains a
similar selectivity for hCA IX over hCA I with respect
to its R un-substituted analogue 3b but is able to
increase about 2.5 times the selectivity for hCA IX over
hCA II. Also the R1 substituent seems to influence hCA
affinity: in fact 4-chloro-benzyl-derivatives clearly appear
to be more active than benzyl analogues on hCA I.
All reported derivatives, similarly to the clinically used
sulfonamides, act as better CA II than CA IX inhibitors
(Table 1), with compounds 1d and 2d being selective
inhibitors of CA II over CA I and IX, in the nanomolar
range. Compound 2d was also the most active on
MMP-2, as the best MMP inhibitory activity was seen for
a-substituted derivatives. Only compounds 3b and 6b,
devoid of the arylsulfonyl moiety, proved to have the best
inhibitory activity on hCA IX than on hCA I and II, in the
micromolar range.
Thus, although these compounds are not as effective as
the clinically used sulfonamides on the single enzymes,
their peculiar selectivity profile and the ability to inhibit
both CAs and MMPs make these derivatives interesting
for more detailed SAR studies.
(iv) Against gelatinases these compounds are less active
than sulfonamide derivatives; (v) they present an inverse
activity profile with respect to sulfonamides, becoming
more effective on MMP-2 than on MMP-9, also if in
the micromolar range; (vi) the a-substituted compounds
have an increased activity on both gelatinases (e.g., 5b
compared to 3b) except when R becomes a too bulky
group, as sec-butyl in 6b. (vii) O-benzyl derivatives
prove to be more active on gelatinases than 4-chloro-
benzyl analogues (e.g., 3b compared to 3c and 5b
compared to 5c), thus the best inhibitor of this series
on gelatinases is 5b (IC50 = 33.7 lM on MMP-2).
5. Experimental
5.1. General synthetic methods and materials
All commercially available starting materials and sol-
vents were of reagent grade. Solutions containing prod-
ucts were dried over anhydrous sodium sulfate
(Na2SO4). Melting points were determined on a Kofler
Figure 4.

E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
2303
Table 1. Inhibition data of sulfonylated hydroxamates 1a–d,f–h, 2d,e against hCA I, II, IX, and MMP-2, 9 compared to standard CA inhibitors and
to CGS 27023A
OCH3
O
O2S
N
HO
R1
N
O
H
R
B: 1a-d, f-h
2d,e
Compound
IC50 (lM)*
R
R1
hCA Ia
hCA IIa
hCA IXb
MMP-2
MMP-9
AAZ
MZA
0.31
0.78
0.025
1.20
0.031
281
0.012
0.014
0.008
0.038
0.015
8.6
0.025
0.027
0.034
0.050
0.024
7.8
EZA
DCP
IND
CGS 27023A
0.025
9.6
0.0048
2.2
1a
1b
1c
1d
1f
H
Et
Bn
8.91
0.88
156
8.7
0.096
4.2
9.0
7.5
H
1.06
1.5
0.87
0.33
3.4
H
4-Cl–Bn
i-Pr
9.3
9.5
H
4.1
0.091
0.097
0.096
0.095
0.094
9.6
4.9
H
n-tetradecyl
Adamantyl
4-Bn-O-Bn
i-Pr
0.89
0.90
5.9
7.4
7.8
30.0
1.98
2.1
33
2.0
1g
1h
2d
2e
H
H
8.7
9.3
1.83
0.370
0.286
i-Pr
i-Pr
9.0
9.7
0.66
0.73
sec-Bu
9.2
CA inhibition values are expressed as KI (lM).
a Human (cloned) isozymes, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme,23 by the CO2 hydration method.24
* Errors in the range of 5–10% of the reported value (from three different assays).
Table 2. Inhibition data of hydroxamates 3–6 against hCA I, II, IX, and MMP-2, 9 compared to standard CA inhibitors and to CGS 27023A
O
H
HO
N
R1
N
O
H
R
C: 3b,c, 4c
5b,c, 6b
Compound
IC50 (lM)*
R
R1
hCA Ia
hCA IIa
hCA IXb
MMP-2
MMP-9
AAZ
MZA
0.31
0.78
0.025
1.20
0.031
281
0.012
0.014
0.008
0.038
0.015
8.6
0.025
0.027
0.034
0.050
0.024
7.8
EZA
DCP
IND
CGS 27023A
0.025
94
0.0048
256
261
3b
3c
4c
5b
5c
6b
H
Bn
490
94
6.7
8.8
H
Me
4-Cl–Bn
4-Cl-Bn
Bn
10.0
9.8
4.8
4.7
137
68
9.2
7.6
141
n-Bu
n-Bu
sec-Bu
520
10.2
435
7.8
6.1
33.7
81.8
151
81.5
125
352
4-Cl–Bn
Bn
6.9
7.1
267
CA inhibition values are expressed as KI (lM).
a Human (cloned) isozymes, by the CO2 hydration method.
b Catalytic domain of human, cloned isozyme,23 by the CO2 hydration method.24
* Errors in the range of 5–10% of the reported value (from three different assays).
hot-stage apparatus and are uncorrected. 1H NMR
spectra were recorded on a Varian Gemini 200
(200 MHz) using CDCl3 or DMSO-d6 as solvent unless
otherwise indicated. The chemical shift values are
reported in ppm (d) and coupling constants (J) in Hertz
(Hz). Mass spectra were recorded on a GC/MS Trace

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E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
GCQ Plus Thermo Quest Finnigan spectrometer using a
direct injection probe and an electron beam energy of
40 eV. Reactions were routinely monitored by thin-layer
chromatography (TLC) on 0.25 mm silica gel plates
(Merck 60 F254) and hydroxamic acids were visualized
with FeCl3 aqueous solution. Flash chromatography
was carried out through glass columns containing silica
gel 60 (Merck 230–400 mesh). Elemental analyses were
carried out by our analytical laboratory and were con-
sistent with theoretical values to within 0.4%.
procedure. The crude product was purified by flash
chromatography on silica gel (n-hexane/EtOAc, 7:2)
1
to give 8e (62% yield) as white solid. Rf = 0.14; H
NMR (CDCl3) d: 0.88 (t, J = 7.5 Hz, 3H), 1.16 (d,
J = 6.2 Hz, 3H), 1.40–1.58 (m, 2H), 3.88 (s, 3H), 4.03
(m, 1H), 6.7 (s, 1H), 6.98–7.02 (m, 2H), 7.83–7.88 (m,
2H).
5.1.1.6. N-n-Tetradecyloxy-4-methoxybenzenesulfona-
mide (8f). The title compound was prepared from
O-n-tetradecylhydroxylamine hydrochloride and 4-meth-
oxybenzenesulfonyl chloride following the general
procedure. Compound 8f was used without further
The clinically used sulfonamide CA inhibitors (CAIs)
acetazolamide AAZ, methazolamide MZA, ethoxozola-
mide EZA, dichlorophenamide DCP, and indisulam
IND, employed as standard inhibitors in the enzyme
assays, are commercially available from Sigma–Aldrich
or have been prepared as previously described.25
1
purification: white solid (92% yield); H NMR (CDCl3)
d: 0.83 (t, J = 6.0 Hz, 3H), 1.25 (m, 24H), 3.88 (s, 3H),
3.96 (t, J = 6.7 Hz, 2H), 6.83 (s, 1H), 6.98–7.02 (m, 2H),
7.83–7.88 (m, 2H).
5.1.1. General procedure for the preparation of N-alkyl-
A
5.1.1.7. N-Adamantyloxy-4-methoxybenzenesulfona-
mide (8g). The title compound was prepared from
O-adamantylhydroxylamine hydrochloride and 4-meth-
oxybenzenesulfonyl chloride following the general
procedure. The crude product was purified by flash
chromatography on silica gel (hexane/EtOAc, 4:1) to
oxysulfonamides (8a–h).
solution of 4-meth-
oxybenzenesulfonyl chloride (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 hydrox-
ylamine hydrochloride 7a–h (7.0 mmol) and N-meth-
ylmorpholine (1.5 mL, 14 mmol) in anhydrous THF
(18.0 mL). After 30 min under these conditions, the reac-
tion mixture was stirred for 3 days at room temperature.
The resulting mixture was diluted with H2O (100 mL) and
extracted with EtOAc (100 mL) giving, after work-up,
crude residues which were purified by flash chromatogra-
phy on silica gel to yield 8a–h as pure solids.
1
give 8g (48% yield) as white solid. Rf = 0.22; H NMR
(CDCl3) d: 1.56 (m, 6H), 1.76 (d, 6H), 2.15 (m, 3H),
3.88 (s, 3H), 6.36 (s, 1H), 6.97–7.01 (m, 2H), 7.82–7.87
(m, 2H).
5.1.1.8. N-Benzyloxy-benzyloxy-4-methoxybenzene-
sulfonamide (8h). The title compound was prepared as
previously described.20
5.1.1.1. N-Ethyloxy-4-methoxybenzenesulfonamide
(8a). The title compound was prepared as previously
described.20
5.1.2. General procedure for the preparation of tert-butyl
esters (9a–d,f–h, 10d,e). A solution of the appropriate
sulfonamide 8a–h (1 mmol) in anhydrous DMF
(3.0 mL) was treated with tert-butylbromoacetate or
tert-butyl 2-bromo-3-methyl butanoate (1.2 mmol),
cesium carbonate (1 mmol), and tetrabutylammonium
hydrogensulfate (1 mmol). The reaction mixture was
stirred for 3 days at room temperature, diluted with
H2O (20.0 mL) and extracted with EtOAc (3·
20.0 mL) giving, after work-up, crude residues which
were purified by flash chromatography on silica gel to
yield 9a–d,f–h, and 10d,e as pure solids.
5.1.1.2. N-Benzyloxy-4-methoxybenzenesulfonamide
(8b). The title compound was prepared from O-ben-
zylhydroxylamine hydrochloride and 4-methoxyben-
zenesulfonyl chloride following the general procedure.
Compound 8b was used without further purification:
white solid (74% yield); mp 105–107 ꢁC; 1H NMR
(CDCl3) d: 3.87 (s, 3H), 4.96 (s, 2H), 6.87 (s, 1H), 6.96–
7.00 (m, 2H), 7.43 (m, 5H), 7.84–7.88 (m, 2H).
5.1.1.3. N-4-Chlorobenzyloxy-4-methoxybenzenesulf-
onamide (8c). The title compound was prepared from
O-(4-chlorobenzyl)hydroxylamine hydrochloride and
4-methoxybenzenesulfonyl chloride following the gener-
al procedure. Compound 8c was used without further
purification: white solid (76% yield); mp 144–146 ꢁC;
1H NMR (CDCl3) d: 3.87 (s, 3H), 4.92 (s, 2H), 6.85 (s,
1H), 6.96–7.01 (m, 2H), 7.24–7.30 (m, 4H), 7.82–7.86
(m, 2H).
5.1.2.1. tert-Butyl {ethyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}acetate (9a). The title compound was prepared
as previously described.20
5.1.2.2. tert-Butyl {benzyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}acetate (9b). The title compound was pre-
pared from 8b and tert-butylbromoacetate following
the general procedure. Compound 9b was used without
1
further purification: yellow gel (92% yield). H NMR
5.1.1.4. N-Isopropoxy-4-methoxybenzenesulfonamide
(8d). The title compound was prepared as previously
described.20
(CDCl3) d: 1.47 (s, 9H), 3.63 (s, 2H), 3.84 (s, 3H), 5.32
(s, 2H), 6.94–6.98 (m, 2H), 7.31–7.36 (m, 5H), 7.76–
7.80 (m, 2H).
5.1.1.5. N-sec-Butyloxy-4-methoxybenzenesulfona-
mide (8e). The title compound was prepared from O-
sec-butylhydroxylamine hydrochloride and 4-meth-
oxybenzenesulfonyl chloride following the general
5.1.2.3. tert-Butyl {4-Chlorobenzyloxy[(4-methoxyphe-
nyl)sulfonyl]amino}acetate (9c). The title compound was
prepared from 8c and tert-butylbromoacetate following
the general procedure. Compound 9c was used without

E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
2305
1
further purification: yellow oil (80% yield); H NMR
(CDCl3) d: 1.46 (s, 9H), 3.61 (s, 2H), 3.85 (s, 3H), 5.28
(s, 2H), 6.95–7.00 (m, 2H), 7.28–7.30 (m, 4H), 7.74–
7.78 (m, 2H).
was removed in vacuo to give 11a–d,f–h, 12d,e as solids
which were purified by crystallization.
5.1.3.1. {Ethyloxy[(4-methoxyphenyl)sulfonyl]amino}-
acetic acid (11a). The title compound was prepared as
previously described.20
5.1.2.4. tert-Butyl {isopropoxy[(4-methoxyphenyl)sul-
fonyl]amino}acetate (9d). The title compound was pre-
pared as previously described.20
5.1.3.2. {Benzyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}acetic acid (11b). The title compound was prepared
from 9b following the general procedure. Compound
11b was used without further purification: yellow solid
5.1.2.5. tert-Butyl {n-tetradecyloxy[(4-methoxyphe-
nyl)sulfonyl]amino}acetate (9f). The title compound
was prepared from 8f and tert-butylbromoacetate fol-
lowing the general procedure. Compound 9f was used
1
(99% yield); H NMR (CDCl3) d: 3.80 (s, 2H), 3.86 (s,
3H), 4.84 (br s, 1H), 5.28 (s, 2H), 6.97–7.01 (m, 2H),
7.31–7.36 (m, 5H), 7.76–7.80 (m, 2H).
1
without further purification: yellow gel (76% yield); H
NMR (CDCl3) d: 0.87 (t, J = 6.6 Hz, 3H), 1.24 (m,
24H), 1.45 (s, 9H), 3.58 (s, 2H), 3.88 (s, 3H), 4.26 (t,
2H), 6.99–7.04 (m, 2H), 7.77–7.83 (m, 2H).
5.1.3.3. {4-Chlorobenzyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}acetic acid (11c). The title compound was pre-
pared from 9c following the general procedure.
Compound 11c was used without further purification:
yellow oil (98% yield); 1H NMR (CDCl3) d: 3.78 (s,
2H); 3.86 (s, 3H); 5.23 (s, 2H); 6.96–7.02 (m, 2H); 7.30
(m, 4H); 7.75–7.81 (m, 2H).
5.1.2.6. tert-Butyl {adamantyloxy[(4-methoxyphe-
nyl)sulfonyl]amino}acetate (9g). The title compound
was prepared from 8g and tert-butylbromoacetate fol-
lowing the general procedure. The crude product was
purified by flash chromatography on silica gel (n-hex-
ane/EtOAc 4:1) to give 9g (49% yield) as a yellow solid.
5.1.3.4. {Isopropoxy[(4-methoxyphenyl)sulfonyl]ami-
no}acetic acid (11d). The title compound was prepared
as previously described.20
1
Rf = 0.32; H NMR (CDCl3) d: 1.49 (s, 9H), 1.62 (m,
6H), 1.90 (d, 6H), 2.18 (m, 3H), 3.75 (s, 2H), 3.88 (s,
3H), 6.98–7.02 (m, 2H), 7.79–7.83 (m, 2H).
5.1.3.5. {n-Tetradecyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}acetic acid (11f). The title compound was
prepared from 9f following the general procedure.
Compound 11f was used without further purification:
yellow solid (92% yield); 1H NMR (CDCl3) d: 0.87 (t,
J = 6.6 Hz, 3H), 1.25 (m, 24H), 3.55 (br s, 1H), 3.75
(s, 2H), 3.89 (s, 3H), 4.24 (t, J = 7.1 Hz, 2H), 7.01–
7.05 (m, 2H), 7.78–7.83 (m, 2H).
5.1.2.7. tert-Butyl {benzyloxybenzyloxy[(4-methoxy-
phenyl)sulfonyl]amino}acetate (9h). The title compound
was prepared as previously described.20
5.1.2.8. tert-Butyl 2-{isopropoxy[(4-methoxyphe-
nyl)sulfonyl]amino}-3-methylbutanoate (10d). The title
compound was prepared from 8d and racemic tert-butyl
2-bromo-3-methyl butanoate following the general
procedure. The crude product was purified by flash
chromatography on silica gel (n-hexane/EtOAc 5:1) to
give 10d (25% yield) as a yellow oil. Rf = 0.27; 1H
NMR (CDCl3) d: 0.88 (d, J = 6.4 Hz, 6H), 1.16 (brs,
9H), 1.22 (d, J = 6.2 Hz, 3H), 1.24 (d, J= 6.2 Hz, 3H),
2.20 (m, 1H), 3.69 (d, J = 10.6 Hz, 1H), 3.87 (s, 3H),
4.41 (septet, J = 6.4 Hz, 1H), 6.95–7.01 (m, 2H), 7.82–
7.87 (m, 2H).
5.1.3.6. {Adamantyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}acetic acid (11g). The title compound was prepared
from 9g following the general procedure. Compound
11g was used without further purification: yellow solid
1
(99% yield); mp 190–192 ꢁC; H NMR (CDCl3) d: 1.63
(m, 6H), 1.88 (d, 6H), 2.20 (m, 3H), 3.80 (d, 2H), 3.89
(s, 3H), 7.01–7.05 (m, 2H), 7.79–7.83 (m, 2H).
5.1.3.7. {Benzyloxybenzyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}acetic acid (11h). The title compound was pre-
pared as previously described.20
5.1.2.9. tert-Butyl 2-{sec-butyloxy[(4-methoxyphe-
nyl)sulfonyl]amino}-3-methylbutanoate (10e). The title
compound was prepared from 8e and racemic tert-butyl
2-bromo-3-methyl butanoate following the general pro-
cedure. The crude product was purified by flash chroma-
tography on silica gel (n-hexane/EtOAc 8:1) to give 10e
(31% yield) as a dense oil. Rf = 0.3; 1H NMR (CDCl3) d:
0.84–0.96 (m, 9H), 1.17–1.26 (m, 12H), 1.35–1.56 (m,
1H), 1.67–1.80 (m, 1H), 2.20 (m, 1H), 3.68 (d,
J = 10.6 Hz, 1H), 3.87 (s, 3H), 4.23 (m, 1H), 6.95–7.01
(m, 2H), 7.82–7.88 (m, 2H).
5.1.3.8. 2-{Isopropoxy[(4-methoxyphenyl)sulfonyl]ami-
no}-3-methylbutanoic acid (12d). The title compound
was prepared from 10d following the general procedure.
Compound 12d was used without further purification:
1
white solid (87% yield); H NMR (CDCl3) d: 0.92 (d,
J = 6.6 Hz, 6H), 1.21–1.30 (m, 6H), 2.15 (m, 1H), 3.69
(d, J = 10.6 Hz, 1H), 3.87 (s, 3H), 4.43 (septet,
J = 6.4 Hz, 1H), 6.94–6.99 (m, 2H), 7.79–7.84 (m, 2H),
9.19 (br s, 1H).
5.1.3. General procedure for the preparation of carboxylic
acids (11a–d,f–h, 12d,e). TFA (4.4 mL, 57 mmol) was
added dropwise to a stirred and cooled (0 ꢁC) solution
of the appropriate tert-butyl ester 9a–d,f–h, 10d,e
(1 mmol) in freshly distilled CH2Cl2 (4.4 mL). The
solution was stirred for 5 h at 0 ꢁC and the solvent
5.1.3.9. 2-{sec-Butyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}-3-methylbutanoic acid (12e). The title compound was
prepared from 10e following the general procedure. Com-
pound 12e was used without further purification: white
1
solid (100% yield); H NMR (CDCl3) d: 0.84–0.95 (m,

2306
E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
1
6H), 1.09 (m, 3H), 1.22–1.29 (m, 3H), 1.39–1.59 (dq, 1H),
1.67–1.84 (dq, 1H), 2.12 (m, 1H), 3.81 (d, J = 10.6 Hz,
1H), 3.87 (s, 3H), 4.30 (m, 1H), 6.02 (br s, 1H), 6.95–
6.99 (m, 2H), 7.80–7.84 (m, 2H).
yield); H NMR (CDCl3) d: 0.23 (s, 6H), 0.99 (s, 9H),
1.62 (m, 6H), 1.85 (d, 6H), 2.20 (m, 3H), 3.80 (d, 2H),
3.89 (s, 3H), 7.01–7.05 (m, 2H), 7.78–7.83 (m, 2H),
8.77 (br s, 1H).
5.1.4. General procedure for the preparation of O-TBDMS
acid hydroxyamides (13a–d,f–h, 14d,e). 1-[3-(dimethyla-
mino)propyl]-3-ethyl carbodiimide hydrochloride (EDCI)
was added portionwise (1 mmol) to a stirred and
cooled (0 ꢁC) solution of the carboxylic acid 11a–d,f–h,
12d,e (1 mmol) and O-(tert-butyldimethylsilyl)hydroxyl-
amine (1 mmol) in freshly distilled CH2Cl2 (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 crude residue was purified by flash chroma-
tography on silica gel to yield pure 13a–d,f–h, 14d,e as
oils.
5.1.4.7. 2-{Benzyloxybenzyloxy[(4-methoxyphenyl)sul-
fonyl]amino}acetic acid (tert-butyldimethylsilyl)hydrox-
yamide (13h). The title compound was prepared as
previously described.20
5.1.4.8. N-(tert-Butyldimethylsilyloxy)-2-[N-isoprop-
oxy(4-methoxyphenyl)sulfonamido]-3-methylbutanamide
(14d). The title compound was prepared from 12d fol-
lowing the general procedure. Compound 14d was used
without further purification: yellow solid (87% yield); 1H
NMR (CDCl3) d: 0.10 (s, 6H), 0.89–0.92 (m, 15H), 1.19–
1.29 (m, 6H), 2.15 (m, 1H), 3.88 (s, 3H), 4.32 (septet,
J = 6.4 Hz, 1H), 6.98–7.02 (m, 2H), 7.80–7.85 (m, 2H),
9.19 (br s, 1H).
5.1.4.1. 2-{Ethyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}acetic acid (tert-butyldimethylsilyl)hy-droxyamide
(13a). The title compound was prepared as previously
described.20
5.1.4.9. N-(tert-Butyldimethylsilyloxy)-2-[N-sec-butyl-
oxy(4-methoxyphenyl)sulfonamido]-3-methylbutanamide
(14e). The title compound was prepared from 12e fol-
lowing the general procedure. The crude product was
purified by flash chromatography on silica gel (n-hex-
ane/EtOAc 4:1) to give 14e (57% yield) as a yellow solid;
1H NMR (CDCl3) d: 0.09 (s, 6H) 0.84–0.91 (m, 18H),
1.17–1.24 (m, 3H), 1.34–1.52 (dq, 1H), 1.65–1.83 (dq,
1H), 2.12 (m, 1H), 3.71 (d, J = 10.6 Hz, 1H), 3.87 (s,
3H), 4.16 (m, H), 6.98–7.02 (m, 2H), 7.80–7.85 (m,
2H), 8.15 (br s, 1H).
5.1.4.2. 2-{Benzyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}acetic acid (tert-butyldimethylsilyl)hy-droxyamide
(13b). The title compound was prepared from 11b fol-
lowing the general procedure. The crude product was
purified by flash chromatography on silica gel (n-hex-
ane/EtOAc 1:1) to give 13b (13% yield) as a yellow solid;
1H NMR (CDCl3) d: 0.16 (s, 6H), 0.95 (s, 9H), 3.58 (s,
2H), 3.86 (s, 3H), 4.96 (s, 2H), 6.96–7.01 (m, 2H),
7.34–7.38 (m, 5H), 7.77–7.82 (m, 2H).
5.1.5. General procedure for the preparation of acid
hydroxyamides (1a–d,f–h, 2d,e). TFA (4.4 mL, 57 mmol)
was added dropwise to a stirred solution of 13a–d,f–h,
14d,e (1 mmol) in freshly distilled CH2Cl2 (4.4 mL)
cooled to 0 ꢁC. The solution was stirred for 5 h at
0 ꢁC and the solvent was removed in vacuo to give a
solid. The crude products were recrystallized from
Et2O and n-hexane to yield 1a–d,f–h, 2d,e as pure
solids.
5.1.4.3. 2-{4-Chlorobenzyloxy[(4-methoxyphenyl)sul-
fonyl]amino}acetic acid (tert-butyldimethylsilyl)hydrox-
yamide (13c). The title compound was prepared from
11c following the general procedure. Compound 13c
was used without further purification: yellow oil (81%
yield); 1H NMR (CDCl3) d: 0.096 (s, 6H); 0.91 (s,
9H); 3.60 (s, 2H); 3.87 (s, 3H); 5.29 (s, 2H); 6.96–6.97
(m, 2H); 7.30–7.33 (m, 4H); 7.74–7.79 (m, 2H).
5.1.4.4. 2-{Isopropoxy[(4-methoxyphenyl)sulfonyl]ami-
no}acetic acid (tert-butyldimethylsilyl)hy-droxyamide
(13d). The title compound was prepared as previously
described.20
5.1.5.1. 2-{Ethyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}-N-hydroxyacetamide (1a). The title compound was
prepared as previously described.20
5.1.5.2. 2-{Benzyloxy[(4-methoxyphenyl)sulfonyl]ami-
no}-N-hydroxyacetamide (1b). The title compound was
prepared from 13b following the general procedure.
The crude product was purified by preparative thin-
layer chromatography (CHCl3/MeOH 9:1) to give 1b
as white solid (4% yield); mp 80–81 ꢁC; 1H NMR
(CDCl3) d: 3.62 (s, 2H), 3.87 (s, 3H), 5.14 (s, 2H),
6.97–7.02 (m, 2H), 7.38 (m, 5H), 7.77–7.80 (m, 2H).
EI-MS, m/z: 51 (38.17), 91 (100), 107 (25.01). Anal.
Calcd for C16H18N2O6S: C, 52.45; H, 4.95; N, 7.65.
Found: C, 52.50; H, 5.05, N 7.55.
5.1.4.5. 2-{n-Tetradecyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}acetic acid (tert-butyldimethylsilyl)hy-droxya-
mide (13f). The title compound was prepared from 11f
following the general procedure. The crude product
was purified by flash chromatography on silica gel (n-
hexane/EtOAc 3:1) to give 13f (4% yield) as a yellow sol-
id; 1H NMR (CDCl3) d: 0.20 (s, 6H), 0.87(t, J = 6.6 Hz,
3H), 0.97 (s, 9H), 1.25 (m, 24H), 3.62 (s, 2H), 3.89 (s,
3H), 4.16 (t, J = 7.1 Hz, 2H), 7.01–7.05 (m, 2H), 7.77–
7.81 (m, 2H), 8.5 (br s, 1H).
5.1.4.6.
2-{Adamantyloxy[(4-methoxyphenyl)sulfo-
5.1.5.3. 2-{4-Chlorobenzyloxy[(4-methoxyphenyl)sul-
fonyl]amino}-N-hydroxyacetamide (1c). The title com-
pound was prepared from 13c following the general
procedure. The crude product was purified by flash
chromatography on silica gel (CHCl3/MeOH 9:1);
nyl]amino}acetic acid (tert-butyldimethylsilyl)hy-droxya-
mide (13g). The title compound was prepared from 11g
following the general procedure. Compound 13g was
used without further purification: yellow solid (75%

E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
2307
Rf = 0.45. Recrystallization from Et2O gave 1c as white
solid (9% yield); mp 148–150 ꢁC; H NMR (DMSO-d6)
5.1.6. General procedure for the preparation of
N-(alkyloxy)iminoacetic acids (15b,c). Sodium acetate
(9.76 g, 12 mmol) was added to a solution of glyoxylic
acid hydrate (5.47 g, 60 mmol) and the appropriate
O-alkyl hydroxylamine hydrochloride 7b,c (60 mmol)
in 90 mL of H2O/MeOH 1:1 and the reaction mixture
was stirred overnight at room temperature. The MeOH
was evaporated and the residue was extracted with
EtOAc. The organic phase was then extracted with sat-
urated NaHCO3 solution. The aqueous phase was acid-
ified to pH 3 at 0 ꢁC with HCl 10% and extracted with
EtOAc. The organic extracts were dried and evaporated
to give 15b,c as solids.
1
d: 3.45 (s, 2H), 3.85 (s, 3H), 5.00 (s, 2H), 7.15–7.20
(m, 2H), 7.33–7.46 (m, 4H), 7.74–7.79 (m, 2H), 9.10
(br s, 1H), 10.70 (br s, 1H). EI-MS, m/z: 77 (100), 107
(54.53), 125 (60.47), 171 (39.16). Anal. Calcd for
C16H17ClN2O6S: C, 47.94; H, 4.27; N, 6.99. Found: C,
47.99; H, 4.32; N, 6.89.
5.1.5.4. 2-{Isopropoxy[(4-methoxyphenyl)sulfonyl]ami-
no}-N-hydroxyacetamide (1d). The title compound was
prepared as previously described.20
5.1.5.5. 2-{n-Tetradecyloxy[(4-methoxyphenyl)sulfo-
nyl]amino}-N-hydroxyacetamide (1f). The title com-
pound was prepared from 13f following the general
procedure. Compound 1f was used without further puri-
5.1.6.1. N-(Benzyloxy)iminoacetic acid (15b). The title
compound was prepared from 7b following the general
procedure. Compound 15b was used without further
purification: white solid (84% yield); mp 78–80 ꢁC; H
NMR (CDCl3) d: 5.33 (s, 2H), 6.62 (br s, 1H), 7.39 (s,
5H), 7.56 (s, 1H).
1
1
fication: yellow solid (67% yield); H NMR (CDCl3) d:
0.87 (t, J = 6.6 Hz, 3H), 1.25 (m, 24H), 3.71 (s, 2H),
3.89 (s, 3H), 4.12 (t, J = 7.1 Hz, 2H), 7.01–7.05 (m,
2H), 7.78–7.83 (m, 2H). EI-MS, m/z: 43 (100), 107
(19.28), 149 (64.83), 171 (42.86). Anal. Calcd for
C23H40N2O6S: C, 58.45; H, 8.53; N, 5.93. Found: C,
58.60; H, 8.60; N, 5.88.
5.1.6.2. N-(4-Chlorobenzyloxy)iminoacetic acid (15c).
The title compound was prepared from 7c following the
general procedure. Compound 15c was used without
further purification: white solid (89% yield); mp 120–
1
5.1.5.6. 2-{Adamantyloxy[(4-methoxyphenyl)sulfonyl]-
amino}-N-hydroxyacetamide (1g). The title compound
was prepared from 13g following the general procedure.
Recrystallization from Et2O gave 1g as white solid (62%
yield); mp 190–192 ꢁC; 1H NMR (DMSO-d6) d: 1.55 (m,
6H), 1.81 (m, 6H), 2.11 (m, 3H), 3.79 (d, 2H), 3.87 (s,
3H), 7.15–7.20 (m, 2H), 7.76–7.79 (m, 2H), 10.60 (s,
1H). EI-MS m/z: 135 (100), 171 (8.85), 239 (1.75). Anal.
Calcd for C19H26N2O6S: C, 55.59; H, 6.38; N, 6.82.
Found: C, 55.68; H, 6.47; N, 6.77.
122 ꢁC; H NMR (CDCl3) d: 5.27 (s, 2H), 5.63 (br s,
1H), 7.22–7.90 (m, 4H), 7.54 (s, 1H).
5.1.7. General procedure for the preparation of
N-alkyloxyamino acids (16c, 17b,c, 18b). The appropri-
ate N-(alkyloxy)iminoacetic acid 15b,c (8.38 mmol) was
dissolved in dry THF (40 mL) under nitrogen and
cooled to ꢀ40 ꢁC. A solution of methyl, n-butyl or
sec-butyllithium (16.76 mmol) was added and the mix-
ture was stirred at ꢀ40 ꢁC for 1 h. The reaction was
quenched with saturated NH4Cl, acidified with HCl
10% to pH 3, and extracted with EtOAc. The organic
extracts were combined, washed with brine, dried over
Na2SO4, filtered, and evaporated to provide 16c, 17b,c,
18b as solids.
5.1.5.7. 2-{Benzyloxybenzyloxy[(4-methoxyphenyl)sul-
fonyl]amino}-N-hydroxyacetamide (1h). The title com-
pound was prepared as previously described.20
5.1.5.8. N-Hydroxy-2-[N-isopropoxy(4-methoxyphe-
nyl)sulfonamido]-3-methylbutanamide (2d). The title
compound was prepared from 14d following the general
procedure. Recrystallization from Et2O gave 2d as white
5.1.7.1. 2-(4-Chlorobenzyloxyamino)propanoic acid
(16c). The title compound was prepared from 15c and
methyllithium following the general procedure. Com-
pound 16c was used without further purification: white
1
solid (50% yield); mp 175–176 ꢁC; H NMR (CDCl3) d:
1
0.91 (d, J = 6.6 Hz, 6H), 1.26 (d, J = 6.4 Hz, 3H), 1.30
(d, J = 6.4 Hz, 3H), 2.04 (m, 1H), 3.88 (s, 3H), 4.47 (sep-
tet, J = 6.4 Hz, 1H), 6.97–7.03 (m, 2H), 7.78–7.82 (m,
2H), 8.60 (br s, 1H). EI-MS, m/z: 43 (65.60), 155
(100), 171 (91.26), 361 (M + 1, 2.06). Anal. Calcd for
C15H24N2O6S: C, 49.99; H, 6.71; N, 7.77. Found: C,
50.10; H, 6.85; N, 7.65.
solid (38% yield); mp 102–104ꢁC; H NMR (CDCl3) d:
1.26 (d, J = 7.1 Hz, 3H), 3.72 (q, J = 7.1 Hz, 1H), 3.73
(br s, 2H), 4.68 (s, 2H), 7.20–7.40 (m, 4H). EI-MS, m/
z: 45 (39.19), 77 (19.44), 89 (12.63), 125 (100), 127
(30). Anal. Calcd for C10 H12ClNO3: C, 52.30; H,
5.27; N, 6.10. Found: C, 52.32; H, 5.27; N, 6.11.
5.1.7.2. 2-(Benzyloxyamino)hexanoic acid (17b). The
title compound was prepared from 15b and n-butylli-
thium following the general procedure. Recrystalliza-
tion from CHCl3/n-hexane gave 17b as white solid
(25% yield); mp 135–137 ꢁC; 1H NMR (CDCl3) d:
0.78–1.00 (m, 3H), 1.20–1.70 (m, 6H), 3.61 (t,
J = 6.6 Hz, 1H), 4.18 (br s, 2H), 4.72 (s, 2H), 7.34 (s,
5H). EI-MS, m/z: 45 (6.81), 65 (5.84), 86 (1.81), 91
(100), 107 (3.20), 108 (2.41). Anal. Calcd for C13 H19
NO3: C, 65.80; H, 8.07; N, 5.90. Found: C, 65.82; H,
8.15; N 5.87.
5.1.5.9. N-Hydroxy-2-[N-sec-butyloxy(4-methoxyphe-
nyl)sulfonamido]-3-methylbutanamide (2e). The title com-
pound was prepared from 14e following the general
procedure. Recrystallization from Et2O gave 2e as yellow
1
solid (80% yield); H NMR (CDCl3) d: 0.87–0.97 (m,
6H),1.23–1.29 (m, 3H), 1.40–1.66 (m, 3H), 1.68–1.85 (m,
H), 2.00 (m, 1H), 3.68 (d, 1H), 3.88 (s, 3H), 4.28 (m,
1H), 6.97–7.02 (m, 2H), 7.78–7.83 (m, 2H), 8,70 (br s,
1H). Anal. Calcd for C16H26O6N2S: C, 51.32; H, 7.00;
N, 7.48. Found: C, 51.45; H, 7.10; N, 7.35.

2308
E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
5.1.7.3. 2-(4-Chlorobenzyloxyamino)hexanoic acid (17c).
The crude product was purified by flash chromatography
on silica gel (n-hexane/EtOAc 4:1) to give 21b (60% yield)
as oil; H NMR (CDCl3) d: 0.16 (s, 3H), 0.17 (s, 3H),
0.75–1.10 (m, 12H), 1.05–1.30 (m, 4H), 1.55–1.75 (m,
2H), 3.00–3.30 (m, 1H), 4.65 (s, 2H), 7.20–7.40 (m, 5H).
The title compound was prepared from 15c and n-butylli-
thium following the general procedure. Recrystallization
from CHCl3/n-hexane gave 17c as white solid (27% yield);
mp 141–143 ꢁC; 1H NMR (CDCl3) d: 0.70–0.95 (m, 3H),
1.20–1.40 (m, 4H), 1.42–1.65 (m, 2H), 3.30 (br s, 2H),
3.59 (m, 1H), 4.67 (s, 2H), 7.25–7.40 (m, 4H). EI-MS, m/
z: 45 (16.54), 57 (3.27), 72 (2.51), 77 (17.21), 86 (3.43),125
(100). Anal. Calcd for C13H18Cl NO3: C, 57.46; H, 6.68;
N, 5.15. Found: C, 57.48; H, 6.73; N, 5.15.
1
5.1.9. General procedure for the preparation of acid
hydroxyamides (4c, 5b,c, 6b). TFA (4.4 mL, 57 mmol)
was added dropwise to a stirred solution of 19c, 20b,c,
21b (1 mmol) in freshly distilled CH2Cl2 (4.4 mL) cooled
to 0 ꢁC. The solution was stirred for 5 h at 0 ꢁC and the
solvent was removed in vacuo to give a solid. The crude
products were recrystallized from Et2O and n-hexane to
yield 4c, 5b,c, 6b as pure solids.
5.1.7.4. 2-(Benzyloxyamino)-3-methylpentanoic acid
(18b). The title compound was prepared from 15b and
sec-butyllithium following the general procedure. Com-
pound 18b was used without further purification: oil (25%
yield); 1H NMR (CDCl3) d: 0.81–1.70 (m, 9H), 3.42–3.60
(m, 1H), 4.69 (s, 2H), 5.71 (br s, 2H), 7.20–7.42 (m, 5H).
5.1.9.1. 2-(4-Chlorobenzyloxyamino)-N-hydroxypropan-
amide (4c). The title compound was prepared from 19c
following the general procedure. Recrystallization from
Et2O/n-hexane gave 4c as white solid (53% yield); mp
137–138 ꢁC; 1H NMR (DMSO-d6) d: 1.03 (d,
J = 6.8 Hz, 3H), 3.40–3.60 (m, 1H), 4.64 (s, 2H), 7.30–
7.45 (m, 4H). Anal. Calcd for C10H13ClN2O3: C, 49.09;
H, 5.36; N, 11.45. Found: C, 50.02; H, 5.39; N, 11.40.
5.1.8. General procedure for the preparation of
O-TBDMS acid hydroxyamides (19c, 20b,c, 21b). 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
16c, 17b,c, 18b (1 mmol) and O-(tert-butyldimethylsi-
lyl)hydroxylamine (1 mmol) in freshly distilled CH2Cl2
(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 crude residue was
purified by flash chromatography on silica gel to yield
pure 19c, 20b,c, 21b as oils.
5.1.9.2. 2-(Benzyloxyamino)-N-hydroxyhexanamide
(5b). The title compound was prepared from 20b follow-
ing the general procedure. Recrystallization from Et2O/
n-hexane gave 5b as white solid (60% yield); mp 101–
103ꢁC; 1H NMR (DMSO-d6) d: 0.85–0.95 (m, 3H),
1.00–1.22 (m, 6H), 3.20–3.40 (m, 1H), 4.59 (s, 2H),
7.31 (s, 5H), 10.58 (br s, 1H). MS, m/z: 44 (5.71), 60
(3.64), 65 (10.6), 77 (14.91), 89 (2.61), 91 (100). Anal.
Calcd for C13H20N2O3: C, 61.88; H, 7.99; N, 11.10.
Found: C, 61.92; H, 8.06; N, 11.01.
5.1.8.1.
2-(4-Chlorobenzyloxyamino)-N-(tert-butyl-
dimethylsilyloxy)propanamide (19c). The title compound
was prepared from 16c following the general procedure.
The crude product was purified by flash chromatogra-
phy on silica gel (n-hexane/EtOAc 4:1) to give 19c
5.1.9.3. 2-(4-Chlorobenzyloxyamino)-N-hydroxyhex-
anamide (5c). The title compound was prepared from
20c following the general procedure. Recrystallization
from Et2O/n-hexane gave 5c as white solid (60% yield);
1
(55% yield) as a yellow oil; H NMR (CDCl3) d: 0.16
(s, 6H), 0.95 (s, 9H), 1.20 (d, J = 7.0 Hz, 3H), 3.50 (m,
1H), 4.64 (s, 2H), 7.20–7.40 (m, 4H), 8.37 (br s, 1H).
1
mp 125–126ꢁC; H NMR (DMSO-d6) d: 0.80–1.40 (m,
5.1.8.2. 2-(Benzyloxyamino)-N-(tert-butyldimethylsi-
lyloxy)hexanamide (20b). The title compound was pre-
pared from 17b following the general procedure. The
crude product was purified by flash chromatography
on silica gel (n-hexane/EtOAc 4:1) to give 20b (63%
9H), 3.29 (m, 1H), 4.61 (s, 2H), 6.62 (br s, 2H), 7.15–
7.60 (m, 4H), 10.62 (br s, 1H). MS, m/z: 44 (11.54), 58
(37.43), 77 (9.82), 89 (11.39), 125 (100), 127 (33.11).
Anal. Calcd for C13H19ClN2O3: C, 54.45; H, 6.68; N,
9.77. Found: C, 54.50; H, 6.71; N, 9.70.
1
yield) as oil; H NMR (CDCl3) d: 0.16 (s, 3H), 0.17 (s,
3H), 0.78–0.92 (m, 3H), 0.95 (s, 9H), 1.20–1.38 (m,
4H), 1.40–1.52 (m, 2H), 3.35 (m, 1H), 4.65 (s, 2H),
7.30–7.42 (m, 5H), 8.25 (br s, 1H).
5.1.9.4. 2-(Benzyloxyamino)-N-hydroxy-3-methylpen-
tanamide (6b). The title compound was prepared from
21b following the general procedure. Recrystallization
from Et2O/n-hexane gave 6b as white solid (47% yield);
1
5.1.8.3.
2-(4-Chlorobenzyloxyamino)-N-(tert-butyl-
mp 145–146 ꢁC; H NMR (DMSO-d6) d: 0.60–0.85 (m,
dimethylsilyloxy)hexanamide (20c). The title compound
was prepared from 17c following the general procedure.
The crude product was purified by flash chromatogra-
phy on silica gel (n-hexane/EtOAc 4:1) to give 20c
6H), 0.85–1.55 (m, 5H), 3.05–3.20 (m, 1H), 4.56 (s,
2H), 7.31 (s, 5H), 10.53 (br s, 1H). MS, m/z: 44 (2.35),
65 (5.46), 77 (9.41), 91 (100). Anal. Calcd for
C13H20N2O3: C, 61.88; H, 7.99; N, 11.10. Found: C,
61.90; H, 8.02; N, 11.06.
1
(87% yield) as oil; H NMR (CDCl3) d: 0.17 (s, 6H),
0.79–0.90 (m, 3H), 0.91 (s, 9H), 1.20–1.40 (m, 4H),
1.42–1.70 (m, 2H), 3.35 (m, 1H), 4.63 (s, 2H), 7.20–
7.40 (m, 4H), 8.17 (br s, 1H).
5.1.10. General procedure for the preparation of N-
alkyloxyaminoacetic acids (22b,c). A cooled (0 ꢁC) solu-
tion of the appropriate N-(alkyloxy)iminoacetic acids
15b,c (11.17 mmol) and borane–triethylamine complex
(7.78 mL, 52.65 mmol) in EtOH (21 mL) was treated
dropwise under stirring with HCl 10% (37.5 mL).
5.1.8.4. 2-(Benzyloxyamino)-N-(tert-butyldimethylsilyl-
oxy)-3-methylpentanamide (21b). The title compound
was prepared from 18b following the general procedure.

E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
2309
After stirring for 18 h at room temperature, the resulting
mixture was washed with CHCl3 and then treated at
0 ꢁC with NaOH 1 N until a white precipitate was
formed (pH ꢁ4). The aqueous phase was extracted with
CH2Cl2 and the organic extracts, dried and evaporated,
provided 22b, c as solids.
5.1.12.1. 2-(Benzyloxyamino)-N-hydroxyacetamide (3b).
The title compound was prepared from 23b following the
general procedure. Recrystallization from Et2O gave 3b as
white solid (45% yield); mp 117–119ꢁC; 1H NMR
(DMSO-d6) d: 3.28 (s, 2H), 4.60 (s, 2H), 7.32 (m, 5H),
10.51 (s, 1H). MS, m/z: 44 (10.38), 65 (7.83), 77 (16.21),
91 (100). Anal. Calcd for C9H12N2O3: C, 55.09; H, 6.16;
N, 14.28. Found: C, 55.19; H, 6.18; N, 14.19.
5.1.10.1. 2-(Benzyloxyamino)acetic acid (22b). The
title compound was prepared from 15b following the
general procedure. Compound 22b was used without
further purification: white solid (42% yield); mp 114–
5.1.12.2. 2-(4-Chlorobenzyloxyamino)-N-hydroxyace-
tamide (3c). The title compound was prepared from 23c
following the general procedure. Recrystallization from
CHCl3/n-hexane gave 3c as white solid (95% yield); mp
1
116ꢁC; H NMR (CDCl3) d: 3.57 (s, 2H), 4.66 (s, 2H),
4.89 (br s, 2H), 7.28 (s, 5H). MS, m/z: 45 (9.46), 60
(1.05), 89 (1.26), 91 (100), 105 (0.78), 108 (0.83). Anal.
Calcd for C9H11NO3: C, 59.66; H, 6.12; N, 7.73. Found:
C, 59.69; H, 6.19; N, 7.70.
1
67–69ꢁC; H NMR (DMSO-d6) d: 3.37 (s, 2H), 4.66 (s,
2H), 6.30 (br s, 2H), 7.25–7.50 (m, 4H), 10.13 (s, 1H).
MS, m/z: 45 (100), 51 (78.5), 69 (53.7), 77 (85.13), 125
(56.38), 155 (2.65). Anal. Calcd for C9H11ClN2O3: C,
46.87; H, 4.81; N, 12.15. Found: C, 46.95; H, 4.89; N,
12.05.
5.1.10.2. 2-(4-Chlorobenzyloxyamino)acetic acid (22c).
The title compound was prepared from 15c following
the general procedure. Compound 22c was used without
further purification: white solid (27% yield); mp 107–
5.2. CA inhibition assay
1
109ꢁC; H NMR (CDCl3) d: 2.48 (br s, 2H), 3.64 (s,
2H), 4.69 (s, 2H), 7.26–7.36 (m, 4H). MS, m/z: 45
(22.20), 60 (4.65), 89 (10.22), 125 (100), 126 (7.63), 127
(31.13), 142 (6.77). Anal. Calcd for C9H10ClNO3: C,
50.13; H, 4.67; N, 6.50. Found: C, 50.25; H, 4.77; N,
6.48.
Recombinant human CA isoforms I, II, and IX have
been prepared as reported earlier by our group,23,26
and their activity assayed by a stopped flow CO2 hydra-
tion assay.24 An Applied Photophysics (Oxford, UK)
stopped-flow instrument has been used for assaying
the CA-catalysed CO2 hydration activity. Phenol red
(at a concentration of 0.2 mM) has been used as indica-
tor, working at the absorbance maximum of 557 nm,
with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4
(for maintaining constant the ionic strength), following
the CA-catalyzed CO2 hydration reaction for a period
of 10–100 s. The CO2 concentrations ranged from 1.7
to 17 mM for the determination of the kinetic parame-
ters and inhibition constants. For each inhibitor at least
six traces of the initial 5–10% of the reaction have been
used for determining the initial velocity. The uncata-
lyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions
of inhibitor (1 mM) were prepared in distilled-deionized
water with 10–20% (v/v) DMSO (which is not inhibitory
at these concentrations) and dilutions up to 0.1 nM were
done thereafter with distilled-deionized water. Inhibitor
and enzyme solutions were preincubated together for
15 min at room temperature prior to assay, in order to
allow for the formation of the E–I complex. The inhibi-
tion constants were obtained by non-linear least-squares
methods using PRISM 3, from Lineweaver–Burk plots,
as reported earlier, and represent the mean from at least
three different determinations.23,26
5.1.11. General procedure for the preparation of
O-TBDMS acid hydroxyamides (23b,c). 1-[3-(Dimeth-
ylamino)propyl]-3-ethyl carbodiimide hydrochloride
(EDCI) was added portionwise (1 mmol) to a stirred
and cooled (0 ꢁC) solution of the carboxylic acid 22b,c
(1 mmol) and O-(tert-butyldimethylsilyl)hydroxylamine
(1 mmol) in freshly distilled CH2Cl2 (18 mL). After stir-
ring at rt for 20 h, the mixture was washed with water
(20 mL) and the organic phase was dried and evaporat-
ed in vacuo to provide 23b,c as oils.
5.1.11.1. 2-(Benzyloxyamino)-N-(tert-butyldimethylsi-
lyloxy)acetamide (23b). The title compound was pre-
pared from 22b following the general procedure.
Compound 23b was used without further purification:
1
oil (73% yield); H NMR (CDCl3) d: 0.12 (s, 3H), 0.17
(s, 3H),0.95 (s, 9H), 3.53 (br s, 2H), 4.71 (s, 2H), 7.25–
7.45 (m, 5H), 8.42 (br s, 1H).
5.1.11.2. 2-(4-Chlorobenzyloxyamino)-N-(tert-butyl-
dimethylsilyloxy)acetamide (23c). The title compound
was prepared from 22c following the general procedure.
Compound 23c was used without further purification:
1
oil (80% yield); H NMR (CDCl3) d: 0.12 (s, 3H), 0.17
(s, 3H), 0.95 (s, 9H), 3.52 (br s, 2H), 4.66 (s, 2H),
7.42–7.80 (m, 4H), 8.33 (br s, 1H).
5.3. MMP inhibition assays27
Human recombinant progelatinase A (pro-MMP-2) and
B (pro-MMP-9) from transfected mouse myeloma cells
were supplied by Prof. Gillian Murphy (Department of
Oncology, University of Cambridge, UK). Proenzymes
were activated immediately prior to use with p-amin-
ophenylmercuric acetate (APMA 2 mM for 1 h at
37 ꢁC for MMP-2 and 1 mM for 1 h at 37 ꢁC for
MMP-9). For assay measurements, the inhibitor stock
solutions (DMSO, 100 mM) were further diluted, at sev-
5.1.12. General procedure for the preparation of acid
hydroxyamides (3b, c). TFA (4.4 mL, 57 mmol) was add-
ed dropwise to a stirred solution of 23b,c (1 mmol) in
freshly distilled CH2Cl2 (4.4 mL) cooled to 0 ꢁC. The
solution was stirred for 5 h at 0 ꢁC and the solvent
was removed in vacuo to give a solid. The crude prod-
ucts were recrystallized from Et2O and n-hexane to yield
3b,c as pure solids.

2310
E. Nuti et al. / Bioorg. Med. Chem. 15 (2007) 2298–2311
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