4-Alkyliden-β-lactams conjugated to polyphenols: Synthesis and inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2005.06.041

A series of compounds combining the β-lactam and polyphenol scaffold have been prepared and evaluated for inhibition of human leukocyte elastase and matrix metallo-proteases MMP-2 and MMP-9. The design of these compounds has been based on the 'overlapping

Full text of DOI:10.1016/j.bmc.2005.06.041

Bioorganic & Medicinal Chemistry 13 (2005) 6120–6132
4-Alkyliden-b-lactams conjugated to polyphenols: Synthesis
and inhibitory activity
Gianfranco Cainelli,a Paola Galletti,a Spiridione Garbisa,b,* Daria Giacomini,a,*
Luigi Sartorb and Arianna Quintavallaa
aDepartment of Chemistry ‘‘G. Ciamician’’, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
bDepartment of Experimental Biomedical Sciences, Medical School of Padova, Viale G. Colombo 3, 35121 Padova, Italy
Received 7 April 2005; revised 9 June 2005; accepted 13 June 2005
Available online 3 August 2005
Abstract—A series of compounds combining the b-lactam and polyphenol scaffold have been prepared and evaluated for inhibition
of human leukocyte elastase and matrix metallo-proteases MMP-2 and MMP-9. The design of these compounds has been based on
the Ôoverlapping-typeÕ strategy where two pharmacophores are linked in a single molecule. The most powerful compound against
elastase was an N-galloyl-4-alkyliden b-lactam, [3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-
azetidin-2-ylidene]-acetic acid ethylester, with an IC50 of 0.5 lM; while the most powerful against MMP-2 was a 4-alkyliden
b-lactam arylated on the C-3 hydroxy side chain (3,5-bis-benzyloxy-4-hydroxy-benzoic acid 1-(2-benzyloxycarbonylmethylene-
4-oxo-azetidin-3-yl)-ethyl ester) with an IC50 of 4 lM. Of the total 35 compounds tested, high levels of inhibition of elastase and
of MMPs were separately exerted by distinct molecules.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
b-Lactam compounds really are ÔevergreenÕ bioactive
molecules. Starting from the antimicrobial potency
exerted by naturally occurring bicyclic compounds (pen-
icillins and cephalosporins), today new variants with
monocyclic structure (azetidinones) are showing new
and specific biological activities. In fact, b-lactam relat-
Figure 1.
ed compounds are irreversible inhibitors of a wide range
of serine proteases including elastase,1 b-lactamase,2
human leukocyte elastase (LE), a very potent degrada-
phospholipase A2,3 and bacterial signal peptidase,4 the
tive weapon released by inflammatory cells, and media-
key step in this process being the acylation of the serine
tor of some severe pathologies such as pulmonary
at the enzyme active site by the b-lactam ring.
emphysema. Furthermore, some of these new deriva-
tives were the first b-lactams shown to be active at
micromolar concentrations against two matrix metallo-
proteases instrumental in cancer invasion and metasta-
sis, MMP-2 and MMP-9.6
We recently developed the synthesis of new monocyclic
b-lactam derivatives, 4-alkyliden-azetidinones, charac-
terized by a C@C bond on the C-4 position of the ring
(Fig. 1).5
In animal models, generic matrix protease inhibitors are
able to reduce inflammatory tissue damage, and prevent
tumor dissemination.7 The possibility of acquiring an
extraprotection against excessive proteolytic activities
through designer drugs has led to the development of
a number of synthetic inhibitors; however, when assayed
in clinical trials, these compounds failed to live up to
expectations, mostly because of their systemic toxicity.8
As compared to other b-lactam inhibitors, this novel
structure was responsible for an increased inhibition of
Keywords: b-Lactams; Polyphenols; Inhibitors; Gelatinase; Elastase.
*
Corresponding authors. Tel.: +39 051 209 9528; fax: +39 051 209
9456 (D.G.); fax: +39 049 8276089 (S.G.); e-mail addresses:
garbisa@unipd.it; daria.giacomini@unibo.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.041

G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
6121
Certain plant compounds can exert anti-inflammatory,
anti-tumor growth, anti-angiogenic, anti-invasive, and
anti-metastatic effects without apparent toxicity,9–12
and (ꢀ) epigallocatechin-3-gallate (EGCG), the major
flavonoid contained in green tea, has proved to be a
good inhibitor of MMP-2 and MMP-9,13 macrophage
metallo-elastase MMP-12,14 and leukocyte serine-elas-
tase LE.15
To test whether the introduction of phenolic moieties to
the b-lactam scaffold can increase binding affinity with
specific enzymes, we have designed and synthesized
new molecules with a central 4-alkyliden b-lactam scaf-
fold to which aromatic—in particular, polyphenolic—
branches are anchored, and for some, we have indeed
found improved efficacy in inhibition of LE, MMP-2,
and MMP-9.
Scheme 2. Reagents and conditions: (a) HCl 1 M, CH3CN. (b)
ArCOCl, SnCl4 5%, benzene, reflux.
ditions of the O-acylation of these compounds required
an acidic catalyst, because, as we demonstrated, 4-
alkyliden b-lactams are quite sensitive to basic condi-
tions.16 Thus, the aromatic or phenolic function was
introduced via a tin tetrachloride catalyzed reaction
(Scheme 2).
2. Results and discussion
2.1. Chemistry
Hydrogenolysis of benzyl esters and of O-benzyl phenols
yielded the corresponding carboxylic acids and phenols
14, 18, 23, 24, and 25 (Scheme 3).
Our earlier work demonstrated that 4-alkyliden func-
tionality is essential for switching on inhibitory proper-
ties.6 In the design of these new compounds, we make
use of a central 4-alkyliden b-lactam scaffold which pre-
sents three sites for introduction of molecular diversity:
(1) the C-3 hydroxyethyl side chain, (2) the C-4 carboxy
function, and (3) the b-lactam nitrogen atom.
Starting from carboxylic acid 26, obtained from benzyl
ester 2 via hydrogenolysis, we introduced different aro-
matic moieties by treatment with amines, phenols, or
benzyl alcohols (compounds 27–34, Scheme 4).
Starting from the 4-alkyliden b-lactams 3 and 4, we ob-
tained N-benzoylated derivatives 35–39 by coupling
with the corresponding benzoyl chlorides (Scheme 5).
Compounds 37 and 38 were subsequently treated with
H2 and Pd on C, giving the fully debenzylated deriva-
tives 40 and 41, respectively (Scheme 5).
The Lewis acid-mediated reaction of 4-acetoxy-azetidi-
nones with a-diazo carbonyls represents the key step
in the synthesis of 4-alkylidene-azetidinone precursors.
Thus, compounds 1–4 were prepared starting from com-
mercially available (3R,4R)-4-acetoxy-3-[(1R)-1-(tert-
butyldimethylsilyloxy)-ethyl]-azetidin-2-one and the
corresponding diazoacyl compound (Scheme 1) follow-
ing a procedure already developed in our laboratory.5
All compounds were purified by silica gel chromato-
graphy and new derivatives fully characterized (see
Section 5).
The reaction proceeds smoothly, yielding E and/or Z
isomers of the corresponding 4-alkylidene-b-lactams
with ethyl- or benzyldiazoacetate. Critical to the success
of the reaction was a stoichiometric amount of TiCl4 or
AlCl3, and an excess of the diazo-compound associated
with a requirement for trimethylsilyl protection of the b-
lactam nitrogen atom.
3. Biochemistry
The inhibition exerted on human LE and gelatinases
MMP-2 and MMP-9 by the various molecules was as-
sayed using a chromogenic substrate for elastase, and
gelatin zymography for the gelatinases, as described in
Section 5. The results are shown in the tables as percent-
age of inhibition at 100 lM and IC50 values: Table 1 re-
ports data for 4-alkyliden b-lactams arylated on the
hydroxy side chain, Table 2 for those modified on the
C-4 side chain, and Table 3 for N-benzoyl-4-alkyliden
b-lactams.
The diastereomeric products E and Z were easily sepa-
rated by column chromatography, allowing access to
stereochemically pure compounds.
Treatment of 1, 2, and 3 with HCl in acetonitrile pro-
duced deprotected derivatives 5–7 (Scheme 2). The con-
Scheme 1.

6122
G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
Scheme 3. Reagents: (a) H2, Pd on C, THF–MeOH.
Scheme 4.
OH derivatives, only compound 23, characterized by a
galloyl fragment, presented significant potency.
a
Regarding the substituent R on the 4-alkyliden-carboxy
side chain, in general benzyl esters exerted a better inhib-
itory activity, while carboxylic acid had a lower potency.
Four compounds in Table 1 showed some activity
against MMPs. Only two members of this group—
namely 19 and 23—were effective in blocking MMP-2
activity, with 19 showing the lowest IC50 (4 lM), but
23 significant against MMP-9 with an IC50 of 50 lM.
If we compare 19, 21, and 23, it is interesting to note
that the structural motif that switched on the inhibition
against MMPs was the 3,4,5-trihydroxy framework (gal-
loyl moiety) with improved and specific activity of the
3,5-dibenzyloxy-4-hydroxy derivative 19 against MMP-
2. Moreover, when compared to EGCG—which has a
15 lM IC50 against MMP-2—compound 19 represents
an excellent inhibitor with a potency equivalent to
EGCG and the best of the b-lactams.
Scheme 5. Reagents: (a) ArCOCl, K2CO3, acetone. (b) H2, Pd on C,
THF–MeOH.
When used at 100 lM, the majority of the 18 com-
pounds of the first group (Table 1) showed a certain de-
gree of LE inhibition, but for six of them the inhibition
was greater than 75%, with compounds 15 and 22 close
to 100%, and 15 showing the lowest IC50 (1 lM). The
substitution pattern on the C-3 aromatic fragment was
important; in particular a higher number of substituents
afforded higher activity. It should be underlined that the
inhibitory potency against LE increases with hydropho-
bic substituents on the aromatic fragment (compounds
13, 15, and 22), with a significant effect of the para-ben-
zyloxy one (cf. compounds 19, 21, and 15). Among the
Data in Table 2 show that modifications of the C-4 car-
boxy residue had little influence on the anti-LE activity,
even if of the 10 members of this group 27 and 34 are
worthy of mention, with LE inhibition percentages at
100 lM of 74 and 81, respectively.
Structural modifications on C-4 conferred anti-gelatina-
sic potency to some extent, even if the majority of these
derivatives showed weak inhibition of MMP-2 and

G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
6123
Table 1. 4-Alkyliden b-lactams arylated on the C-3 hydroxy side chain: human LE, MMP-2, and MMP-9 inhibition results
Compound
Ar
R
Isomer
Human LE
MMP-2
MMP-9
Inhibition % IC50 (lM)
Inhibition %
at 100 lM
IC50 (lM)
Inhibition %
at 100 lM
IC50 (lM)
at 100 lM
8
Et
Z
11
—
—
—
—
—
9
Et
Et
E
Z
39
29
—
—
—
—
—
—
—
—
—
—
10
11
12
13
14
15
16
17
18
19
20
21
Bn
Et
Et
H
Z
Z
E
Z
Z
Z
Z
Z
Z
Z
Z
50
82
50
11
98
15
77
59
59
79
58
50
25
—
33
—
—
43
—
—
—
81
—
—
—
—
—
—
—
—
—
—
4
—
24
—
—
10
—
—
—
36
—
—
—
—
—
—
—
—
—
—
—
—
—
100
—
Et
Et
Bn
H
1
—
10
60
Et
Bn
Et
25
15
—
—
n.d.a
(continued on next page)

6124
G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
Table 1 (continued)
Compound
Ar
R
Isomer
Human LE
MMP-2
Inhibition %
MMP-9
Inhibition %
Inhibition %
at 100 lM
IC50 (lM)
IC50 (lM)
IC50 (lM)
at 100 lM
at 100 lM
22
23
24
25
Et
Et
Et
H
Z
Z
Z
Z
97
6
—
—
—
—
82
5
20
—
—
61
—
—
70
—
—
70
—
—
50
—
—
37
a n.d., not determined, due to non-constant slope of the inhibition exerted.
MMP-9 activity. Only compounds 29 and 33 had signif-
icant potency against MMP-2, with IC50 of 40 and
70 lM, respectively.
MMPs at O-galloyl or N-galloyl derivatives with free
phenolic OH.
The Ki was studied for one member of each group
against both LE and MMP-2 (the most sensitive of the
two gelatinases). With the exception of compounds 29
and 40 (see below), the Ki was in good agreement with
the calculated IC50, and indicates that the type of inhibi-
tion was non-competitive, as shown by the example,
double-reciprocal plots in Figure 2 and Table 4. The
non-competitive inhibition inferred for these com-
pounds indicates that the inhibitory effects observed
are due to allosteric binding rather than binding to the
active site.19
Acylation of the b-lactam nitrogen atom is crucial for
LE inhibition; in fact, five out of seven members of
the third group (Table 3) exerted an LE inhibition
>75% at 100 lM, with 36 close to 100% and showing
the best IC50 (0.5 lM). This is an important result given
that one of the best inhibitors of LE was EGCG, with an
IC50 of 0.3 lM. Ethyl ester 36 worked better than benzyl
ester 37 with a 100-fold improvement in IC50. Results in
Table 3 confirm the preference for LE inhibitor of a
higher lipophilic character, with the exception of 41.
We have already demonstrated the efficiency of N-acyl-
ation for LE inhibition.6 The electron-withdrawing
character of this N1 side chain should have an influence
on the b-lactam reactivity, that is, the sensitiveness of
the C-2 carbonyl toward nucleophilic attack.17 This fact
has a counterpart in a lower stability of these com-
pounds at pH values higher than 7.8, and in a higher
sensitivity to alcohols.18
In the case of 29 and 40, the chromogenic assay for
MMP-2 gave absorbance values higher in the presence
of compound (potential inhibitor) than in its absence,
preventing full deduction of the Ki (n.d. in Table 4);
comprehension of this unexpected effect requires further
investigation.
The most active compounds of each group were tested
for short-term (16 h) cytotoxicity at 1, 10, and 100 lM
on non-transformed breast epithelial (HBL-100) and
fibrosarcoma (HT-1080) cell lines. As shown in Table
4, the most potent compound against LE (36, IC50
0.5 lM) was totally non-cytotoxic on non-transformed
cells when used at concentration 20· its IC50, whereas
the cell viability of transformed cells was affected 14%
at the same concentration. The second most effective
compound against LE, 15 (IC50 1 lM), was completely
non-cytotoxic on non-transformed cells even when used
at concentration 100· its IC50, whereas it appreciably
affected the viability of transformed cells even at 10·.
Compound 27, the most effective of the second group
against LE, was considerably cytotoxic on both cell
lines at 4· its IC50 (25 lM). The most effective com-
pound against MMP-2 was 19, showing a Ki of
Gelatinases MMP-2 and MMP-9 are poorly inhibited by
compounds in Table 3, with the exception of 40 which,
bearing a galloyl moiety, showed >90% inhibition at
100 lM, and an IC50 of 15 lM against MMP-2, and 70
against MMP-9. The inhibition of MMPs by 4-alkyliden
b-lactams could depend on a specific interaction with the
NH of the azetidinone;6 however, the special characteris-
tics of compound 40 could result from the presence of the
3,4,5-phenolic triad that may replace the loss of NH
interaction in this N-acylated compound.
The results in Tables 1–3 revealed that the potency of
these new 4-alkyliden b-lactams was specifically located
at particular substitutions: N-acylation with lipophilic
benzyloxy-galloyl moiety against LE, whereas for

G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
6125
Table 2. 4-Alkyliden b-lactams modified on the C-4 side chain: human LE, MMP-2, and MMP- 9 inhibition results
Compound
XR
Human LE
MMP-2
Inhibition %
MMP-9
Inhibition % IC50 (lM)
Inhibition %
at 100 lM
IC50 (lM)
IC50 (lM)
at 100 lM
at 100 lM
2
48
—
—
—
—
—
4
32a
74
—
25
—
4
—
—
—
—
—
—
27
28
29
—
—
—
—
46
65
—
40
30
—
—
—
30
31
40
26
—
—
32
13
—
—
18
5
—
—
32
33
19
43
—
—
47
55
—
70
4
—
—
20
34
81
35
—
—
—
—
a Compound 4 has an E geometry of the C=C bond.
1.6 lM, with only 26% in viability at 100 lM on HBL-
100 cells (Table 4).
leader-compound azetidin-2-one L-680,833 shows, in
fact, an IC50 of 9 lM20), compound 19 represents the
best inhibitor of MMP-2, with an IC50 of 4 lM. Of
the total 35 compounds tested, high levels of inhibition
of elastase and MMPs were separately exerted by dis-
tinct molecules, and in absence of cytotoxic effects.
4. Conclusions
In summary, we designed and synthesized a series of
substituted 4-alkyliden b-lactams combining the b-lac-
tam with a polyphenol scaffold, and evaluated these
new compounds for inhibition of human LE and matrix
metallo-proteases, MMP-2 and MMP-9. The presence
of polyphenolic substituents in these b-lactams was
determinant for the corresponding biochemical activity:
the most potent non-competitive inhibitor against LE
(IC50 0.5 lM) was 36, an N-galloyl-4-alkyliden-b-lac-
tam. While the latter results to be thus the best b-lac-
tam-based inhibitor of human LE (the reference
5. Experimental
5.1. General
Commercial reagents were used as received without addi-
tional purification. Anhydrous solvents were obtained
commercially and used without further drying. H and
1
13C NMR values were recorded on a VARIAN Mercury
400, INOVA 300 or GEMINI 200 instrument using a

6126
G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
Table 3. N-benzoyl-4-alkyliden b-lactams: human LE, MMP-2, and MMP-9 inhibition results
Compound
Ar
R
Human LE
MMP-2
Inhibition %
MMP-9
Inhibition %
Inhibition %
at 100 lM
IC50 (lM)
IC50 (lM)
IC50 (lM)
at 100 lM
at 100 lM
35
36
37
Et
Et
Bn
94
4.5
15
—
40
—
97
85
0.5
25
21
—
—
—
21
—
—
35
38
39
40
Bn
Et
H
2
78
24
—
8
—
15
92
—
—
15
—
—
52
—
—
70
—
41
H
87
9
—
—
—
—
5 mm probe. All chemical shifts have been quoted relative
to deuterated solvent signals, delta in ppm, J in Hz. FT-
IR: Nicolet 205 measured as films or nujol mull between
5.3. Cytotoxicity test
Human breast epithelial (HBL-100) and fibrosarcoma
(HT-1080) cells were used for cytotoxicity tests. Cells
(104) were seeded onto 96-well plastic plates (Falcon,
BD Bioscience) and incubated at 37 ꢁC in DulbeccoÕs
modified EagleÕs medium supplemented with 10% fetal
calf serum, with or without 1, 10, and 100 lM b-lac-
tams. After 16 h, the number of viable cells was deter-
mined by CellTiter 96ꢂ assay (Promega, Madison WI,
USA) and expressed as ratio of absorbance (A490) of
treated versus control cells, as already reported.1
NaCl plates and reported in cmꢀ1. TLC: Merck 60 F254
;
Column chromatography: Merck silica gel 200–300 mesh,
HPLC–MS: HPLC: Agilent Technologies HP1100, col-
umn ZOBRAX-Eclipse XDB-C8 Agilent Technologies.
The products were eluted with CH3CN/H2O, gradient
from 30 to 80% of CH3CN, 0.5 mL minꢀ1. Some acid
products were eluted with CH3CN/H2O mixture contain-
ing 0.1% of formic acid. MS: Agilent Technologies
MSD1100 single-quadrupole mass spectrometer, full-
scan mode from m/z 50 to m/z 2600, scan time 0.1 s in
positive ion mode, ESI spray voltage 4500 V, nitrogen
5.4. Substrate degradation by LE
gas 35 psgi, drying gas flow 11.5 mL minꢀ1, fragmentor
25
voltage 20 V. The ½aꢁ was determined with a Perkin El-
Human LE was solubilized (250 mU mLꢀ1) in Hepes
buffer (0.1 M Hepes, 0.5 M NaCl, and 10% DMSO,
pH 7.8). All the compounds and elastase substrate were
freshly prepared 20· in DMSO. Dilutions of the com-
pounds were pre-mixed with the enzyme in micrometers
wells, and maintained for 15 min at 4 ꢁC. Then, 5 lL of
the substrate (8 mM concentrated) were added to
100 lL final volume, and the mixture was incubated at
37 ꢁC. At 20-min intervals, the intensity of the color
developed by the digested substrate was measured at
D
mer 343 polarimeter. Melting points were determined
using a Buchi apparatus and are uncorrected.
¨
5.2. Materials for biological assays
Elastase from human leukocytes (SE563) was from Elas-
tin Products Company, Owensville MO, USA; elastase
substrate N-methoxysuccinyl-ala-ala-pro-val p-nitroani-
lide was from Sigma.

G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
6127
Figure 2. Double-reciprocal plots demonstrating non-competitive inhibition of human LE (15, 27, 36) and MMP-2 (19) by increasing concentration
of compound (S substrate), and the calculated Ki. Examples of triplicate experiments, analyzed at 60 min; the points represent the mean values of
three wells, with SD <10%.
Table 4. Inhibition constant (Ki) and short-term (16 h) cytotoxicity of some 4-alkyliden b-lactams on non-transformed epithelial (HBL-100) and
transformed fibrosarcoma (HT-1080) cells, expressed as percent viability
Compound
Ki (lM)
HBL-100
Compound conc. (lM)
10
HT-1080
Compound conc. (lM)
10
1
100
1
100
15
27
36
19
29
40
1.5a
20.1a
0.7a
100
100
100
100
99
100
92
100
27
41
74
2
99
97
95
62
91
86
90
88
95
55
41
1
100
94
1.6b
n.d.
100
100
97
61
14
72
98
73
n.d.
91
23
n.d., not determined.
a Inhibition constant for LE.
b Inhibition constant for MMP-2.
405 nm using a Titertek Mutiskan (Flow Laboratories),
and the control background subtracted (in triplicate
experiments). The reactions developed linearly for as
long as 120 min; data from 60 min were used for deter-
mining all IC50s. Double-reciprocal plot of the results al-
lowed deduction of the type and Ki of inhibition exerted
over LE by compounds 15, 27, and 36.
of medium in 0.1% gelatin-containing 8% polyacryl-
amide, in the presence of SDS. After electrophoresis,
the gels were washed twice for 15 min with 2.5% Triton
X-100, incubated overnight at 37 ꢁC in Tris buffer
(50 mM Tris–HCl, 200 mM NaCl, and 10 mM CaCl2,
pH 7.4). For gelatinase inhibition assays, compounds
were freshly solubilized in DMSO and diluted in Tris
buffer used for developing the zymogram. The gel slab
was then cut into slices corresponding to the lanes and
then put in different tanks containing the stated concen-
trations of inhibitors.
5.5. Zymographic analysis
Aliquots of gelatinase-containing medium conditioned
by human neuroblastoma cells (for MMP-2) or HT-
1080 human fibrosarcoma cells (for MMP-9) were as-
sayed as described.5 Without heating the samples,
zymography was performed by electrophoresing 15 lL
The gels then were stained for 30 min with 30% metha-
nol/10% acetic acid containing 0.5% Coomassie brilliant
blue R-250, and destained in the same solution without

6128
G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
dye. Clear bands on the blue background represent
areas of gelatinolysis. Digestion bands were quantitated
using an image analyzer system with GelDoc 2000 and
Quantity One software (Bio-Rad); the densitometric val-
ues were expressed as percentage of control bands on the
same gel, and the values used for IC50 determination.
until total conversion. After, saturated NaCl solution
was added and the mixture extracted with CH2Cl2 (5·
10 mL), dried on Na2SO4, concentrated and the residue
purified, when necessary, by flash chromatography
(cyclohexane/ethylacetate 4:6) or by trituration in pen-
tane/Et2O. Yield 87%.
5.6. Classification of MMP-2 inhibition
The desilylated compound 6 (0.045 g, 0.226 mmol) was
dissolved in anhydrous benzene (4 mL), then 3,4,5-trim-
ethoxybenzoyl chloride (0.104 g, 0.452 mmol) and final-
ly SnCl4 (0.011 mmol, 12 lL of a solution 1 M in
CH2Cl2) were added. The solution was heated to reflux
and stirred for 4 h. The reaction mixture was then
quenched in a solution of NaHCO3 (5%), extracted with
ethylacetate (4· 10 mL), dried on Na2SO4 and concen-
trated. The residue was purified by flash chromatogra-
phy (cyclohexane/ethylacetate 8:2) to give compound 12.
To further characterize the type of inhibition exerted by
b-lactams 19, 29, and 40 (those with the lowest IC50 of
the respective group) on the gelatinase, a BIOTRAKTM
MMP-2-activity assay system (Amersham Pharmacia
Biotech) was used, following the manufacturerÕs instruc-
tions, with slight modifications.21 The microtitre wells—
coated with anti-MMP-2 antibodies—were saturated
with the enzyme contained in 50 lL of medium condi-
tioned by SK-N-BE human neuroblastoma cells; after
overnight incubation at 4 ꢁC and exhaustive washing,
the gelatinase was activated by treatment with
0.25 mM APMA. The wells were then incubated at
37 ꢁC in the presence of increasing amount of a peptide
substrate, with increasing concentrations of compound,
and the intensity of the color developed by the digested
substrate was measured at 405 nm after 4 h. A double-
reciprocal plot of the results allowed the type and con-
stant (Ki) of inhibition to be deduced.
5.6.3. Compound 8. The obtained yield 55%, white solid.
1H NMR (CDCl3, 200 MHz): d 1.29 (t, J = 7.0 Hz, 3H),
1.54 (d, J = 6.6 Hz, 3H), 4.04 (d, J = 6.6 Hz, 1H), 4.19
(q, J = 7.0 Hz, 2H), 5.22 (s, 1H), 5.51 (quintet,
J = 6.6 Hz, 1H), 7.40–7.63 (m, 3H), 8.03 (m, 2H), 8.67
(br s, 1H). 13C NMR (CDCl3, 50.3 MHz): d 14.3, 18.1,
60.3, 61.5, 67.5, 90.9, 128.4, 129.6, 129.7, 133.2, 151.4,
25
D
164.5, 165.2, 166.8. ½aꢁ ꢀ46.2 (c 0.70, CHCl3). IR
(film): 3397, 1819, 1706, 1653 cmꢀ1
. HPLC–MS:
tR = 11.13 min, m/z: 260.1 [Mꢀ43]+, 304.1 [M+H]+,
321.2 [M+H2O]+, 326.1 [M+Na]+, 342.0 [M+K]+,
629.2 [2M+Na]+. mp 125–127 ꢁC.
Compounds 2 and 4 have been prepared according to
the analogous previously reported procedure5 using
benzyldiazoacetate22 as diazocarbonyl compound.
5.6.4. Compound 9. The obtained yield 20%, white solid.
1H NMR (CDCl3, 200 MHz): d 1.33 (t, J = 6.8 Hz, 3H),
1.45 (d, J = 6.6 Hz, 3H), 4.24 (q, J = 6.8 Hz, 2H), 4.48
(d, J = 4.6 Hz, 1H), 5.41 (d, J = 1.0 Hz, 1H), 5.96 (dr
q, J = 4.6 Hz, J = 6.6 Hz, 1H), 7.42–8.14 (m, 6H). 13C
NMR (CDCl3, 50.3 MHz): d 14.4, 15.4, 60.4, 61.6,
5.6.1. Compound 2. The obtained yield 54%, pale yellow
oil. 1H NMR (CDCl3, 300 MHz): d 0.06 (s, 3H), 0.07 (s,
3H), 0.87 (s, 9H), 1.31 (d, J = 6.3 Hz, 3H), 3.67 (d,
J = 5.1 Hz, 1H), 4.24 (dr q, J = 5.1 Hz, J = 6.3 Hz,
1H), 5.16 (d, J = 12.3 Hz, 1H), 5.21 (d, J = 12.3 Hz,
1H), 5.28 (s, 1H), 7.37 (m, 5H), 8.45 (br s, 1H). 13C
NMR (CDCl3, 50.3 MHz): d ꢀ5.1, ꢀ4.3, 17.9, 22.2,
66.8, 93.6, 128.3, 129.7, 133.0, 133.6, 150.6, 164.0,
25
165.5, 167.0. ½aꢁ +24.0 (c 0.70, CHCl3). IR (film):
D
25.6, 64.8, 65.0, 65.8, 90.2, 127.9, 128.1, 128.5, 136.1,
1818, 1712, 1652 cmꢀ1. HPLC–MS: tR = 11.78 min, m/
z: 260.3 [Mꢀ43]+, 304.1 [M+H]+, 321.0 [M+H2O]+,
326.1 [M+Na]+, 607.5 [2M+H]+, 629.2 [2M+Na]+. mp
119–121 ꢁC.
25
153.8, 166.5, 166.9. ½aꢁ ꢀ14.6 (c 2.42, CHCl3). IR
D
(film): 3295, 1818, 1700, 1653, 1264 cmꢀ1. HPLC–MS:
tR = 27.93 min,
m/z:
377.3
[M+2H]+,
399.4
[M+H+Na]+, 414.5 [M+K]+.
1
5.6.5. Compound 10. Yield 36%, white solid. H NMR
5.6.2. Compound 4. The obtained yield 6%, pale yellow
oil. H NMR (CDCl3, 300 MHz): d 0.09 (s, 3H), 0.10
(CDCl3, 200 MHz): d 1.29 (t, J = 7.2 Hz, 3H), 1.52 (d,
J = 6.2 Hz, 3H), 4.02 (d, J = 6.6 Hz, 1H), 4.20 (q,
J = 7.2 Hz, 2H), 5.22 (d, J = 0.8 Hz, 1H), 5.49 (quintet,
J = 6.2 Hz, 1H), 5.98 (br s, 1H), 6.86 (m, 2H), 7.92 (m,
2H), 8.67 (br s, 1H). 13C NMR (CDCl3, 50.3 MHz): d
1
(s, 3H), 0.91 (s, 9H), 1.21 (d, J = 6.3 Hz, 3H), 4.12 (m,
1H), 4.65 (dr q, J = 4.2 Hz, J = 6.3 Hz, 1H), 5.17 (s,
2H), 5.36 (d, J = 1.2 Hz, 1H), 7.37 (m, 5H), 7.53 (br s,
1H). 13C NMR (CDCl3, 75.5 MHz): d ꢀ5.0, ꢀ4.8,
14.3, 18.1, 60.4, 61.6, 67.1, 91.0, 115.3, 122.0, 132.0,
25
D
17.9, 19.4, 25.7, 64.6, 64.7, 65.7, 92.0, 127.9, 128.1,
151.5, 160.2, 164.8, 165.0, 166.9. ½aꢁ ꢀ22.1 (c 0.50,
25
128.5, 136.1, 153.7, 166.1, 167.9. ½aꢁ +92.6 (c 1.63,
CHCl3). IR (film): 3337, 1825, 1706, 1660 cmꢀ1
.
D
CHCl3). IR (film): 3263, 1822, 1715, 1652, 1258 cmꢀ1
.
HPLC–MS: tR = 9.43 min, m/z: 276.0 [Mꢀ43]+, 320.0
[M+H]+, 337.1 [M+H2O]+, 342.0 [M+Na]+, 661.1
[2M+Na]+. mp 161–163 ꢁC.
HPLC–MS: tR = 25.99 min, m/z: 377.3 [M+2H]+, 399.4
[M+H+Na]+, 789.3 [2M+K]+.
Compounds 8–13, 15–17, and 19–22 were obtained by
SnCl4 catalyzed O-acylation of 3-hydroxyethyl chain
following the representative procedure: compound 1
(0.313 g, 1 mmol) was dissolved in CH3CN (6 mL) and
HCl 1 N (1 mL) was added. The reaction was monitored
by TLC and further 1 mL portions of HCl 1 N as added
5.6.6. Compound 11. The obtained yield 78%, pale
yellow oil. 1H NMR (CDCl3, 300 MHz): d 1.54 (d,
J = 6.3 Hz, 3H), 3.87 (s, 6H), 3.91 (s, 3H), 4.04 (d,
J = 6.3 Hz, 1H), 5.18 (s, 2H), 5.31 (s, 1H), 5.49 (quintet,
J = 6.3 Hz, 1H), 7.27 (m, 2H), 7.36 (m, 5H), 8.91 (br s,
1H). 13C NMR (CDCl3, 50.3 MHz): d 18.1, 56.1, 60.8,

G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
6129
61.7, 66.2, 67.3, 90.5, 106.8, 124.5, 128.2, 128.3, 128.6,
25
135.7, 142.5, 152.3, 152.9, 164.7, 164.8, 166.7. ½aꢁ
J = 6.3 Hz, 3H), 3.82 (s, 6H), 4.23 (d, J = 5.1 Hz, 1H),
5.18 (s, 2H), 5.34 (s, 1H), 5.45 (quintet, J = 6.3 Hz,
1H), 7.20–7.40 (m, 7H), 8.10 (br s, 1H), 9.94 (br s,
1H). 13C NMR (acetone-d6, 75.5 MHz): d 18.0, 56.0,
D
+31.1 (c 4.25, CH3OH). IR (film): 3294, 1824, 1720,
1653 cmꢀ1
. HPLC–MS: tR = 12.00 min, m/z: 412.1
[Mꢀ43]+, 456.0 [M+H]+, 473.1 [M+H2O]+, 478.1
62.0, 65.4, 67.2, 89.9, 107.2, 120.3, 128.2, 128.4, 128.7,
25
D
[M+Na]+, 494.0 [M+K]+, 933.3 [2M+Na]+.
137.2, 141.3, 147.8, 153.7, 164.8, 165.9, 166.2. ½aꢁ
ꢀ10.1 (c 1.58, CHCl3). IR (film): 3295, 1820, 1700,
1658, 1611, 1227 cmꢀ1. HPLC–MS: tR = 10.47 min, m/
z: 398.1 [Mꢀ43]+, 442.1 [M+H]+, 459.2 [M+H2O]+,
480.2 [M+K]+, 905.2 [2M+Na]+.
5.6.7. Compound 12. The obtained yield 50%, white
d 1.29 (t,
solid. 1H NMR (CDCl3, 300 MHz):
J = 7.2 Hz, 3H), 1.54 (d, J = 6.3 Hz, 3H), 3.90 (s, 6H),
3.92 (s, 3H), 4.03 (d, J = 6.3 Hz, 1H), 4.18 (q,
J = 7.2 Hz, 2H), 5.24 (d, J = 0.9 Hz, 1H), 5.49 (quintet,
J = 6.3 Hz, 1H), 7.20 (m, 2H), 8.61 (br s, 1H). 13C
NMR (CDCl3, 75.5 MHz): d 14.3, 18.1, 56.2, 60.3,
5.6.12. Compound 19. The obtained yield 14%, pale
yellow oil. 1H NMR (CDCl3, 200 MHz): d 1.17 (t,
J = 7.2 Hz, 3H), 1.42 (d, J = 6.2 Hz, 3H), 3.89 (d,
J = 6.6 Hz, 1H), 4.07 (m, 2H), 5.08 (s, 4H), 5.11 (s,
1H), 5.34 (quintet, J = 6.6 Hz, 1 H), 5.95 (br s, 1 H),
7.20–7.40 (m, 12H), 8.52 (br s 1H). 13C NMR (CDCl3,
50.3 MHz): d 14.3, 18.1, 60.3, 61.6, 67.3, 71.5, 90.9,
60.9, 61.7, 67.4, 91.0, 106.9, 124.6, 142.6, 151.5, 153.0,
25
D
164.6, 164.9, 166.9. ½aꢁ ꢀ16.4 (c 0.60, CHCl3). IR
(film): 3297, 1825, 1700, 1660, 1222 cmꢀ1. HPLC–MS:
tR = 12.78 min,
m/z:
196.2
[Mꢀ197]+,
417.2
[M+H+Na]+. mp 142–144 ꢁC.
108.9, 120.3, 127.8, 128.3, 128.6, 136.1, 140.6, 145.9,
25
151.4, 164.5, 164.7, 166.8. ½aꢁ ꢀ28.3 (c 0.60, CHCl3).
D
5.6.8. Compound 13. The obtained yield 25%, white
solid. 1H NMR (CDCl3, 200 MHz):
1.33 (t,
IR (film): 3510, 3277, 1819, 1706, 1660 cmꢀ1. HPLC–
MS: tR = 16.34 min, m/z: 334.2 [Mꢀ197]+, 460.2
[Mꢀ43ꢀ29]+, 489.4 [M+Hꢀ43]+, 555.2 [M+H+Na]+,
571.3 [M+H+K]+, 1086.3 [2M+H+Na]+.
d
J = 7.0 Hz, 3H), 1.44 (d, J = 6.2 Hz, 3H), 3.91 (s, 3H),
3.93 (s, 6H), 4.23 (q, J = 7.0 Hz, 2H), 4.45 (m, 1H),
5.40 (d, J = 1.2 Hz, 1H), 5.93 (m, 1H), 7.40 (m, 2H),
7.70 (br s, 1H). 13C NMR (CDCl3, 75.5 MHz): d 14.3,
5.6.13. Compound 20. The obtained yield 23%, pale
yellow oil. 1H NMR (CDCl3, 200 MHz): d 1.54 (d,
J = 6.4 Hz, 3H), 4.02 (d, J = 6.2 Hz, 1H), 5.08 (s, 4H),
5.12 (d, J = 12.4 Hz, 1H), 5.19 (d, J = 12.4 Hz, 1H),
5.27 (s, 1H), 5.49 (quintet, J = 6.2 Hz, 1H), 6.84 (m,
1H), 7.28–7.47 (m, 17H), 8.60 (br s, 1H). 13C NMR
(CDCl3, 75.5 MHz): d 18.1, 61.5, 66.2, 67.6, 70.3, 90.6,
15.4, 56.2, 60.4, 60.9, 61.5, 67.0, 93.5, 107.1, 107.4,
25
124.1, 125.1, 150.9, 153.0, 165.2, 165.7. ½aꢁ +22.0 (c
D
0.70, CHCl3). IR (film): 3271, 1820, 1713, 1660,
. HPLC–MS: tR = 11.30 min, m/z: 350.3
1222 cmꢀ1
[Mꢀ43]+, 394.2 [M+H]+, 411.2 [M+H2O]+, 416.3
[M+Na]+, 809.3 [2M+Na]+. mp 144–146 ꢁC.
107.5, 108.5, 127.6, 128.1, 128.2, 128.3, 128.6, 131.5,
25
135.7, 136.3, 152.0, 159.8, 164.4, 164.9, 166.6. ½aꢁ
5.6.9. Compound 15. The obtained yield 51%, white
1.27 (t,
D
solid. 1H NMR (CDCl3, 200 MHz):
d
ꢀ29.8 (c 0.64, CHCl3). IR (film): 3294, 1821, 1699,
J = 7.0 Hz, 3H), 1.52 (d, J = 6.6 Hz, 3H), 4.00 (d,
J = 6.2 Hz, 1H), 4.15 (m, 2H), 5.14 (s, 6H), 5.20 (s,
1H), 5.45 (quintet, J = 6.6 Hz, 1H), 7.20–7.50 (m,
17H), 8.55 (br s, 1H). 13C NMR (CDCl3, 75.5 MHz):
d 14.5, 18.3, 60.6, 61.8, 67.7, 71.5, 75.4, 91.3, 109.5,
1655, 1445 cmꢀ1
. HPLC–MS: tR = 22.05 min, m/z:
578.2 [M+H]+, 595.2 [M+H2O]+, 600.0 [M+Na]+,
616.1 [M+K]+, 1178.4 [2M+H+Na]+.
5.6.14. Compound 21. The obtained yield 20%, pale
yellow oil. 1H NMR (CDCl3, 300 MHz): d 1.25 (t,
J = 7.2 Hz, 3H), 1.52 (d, J = 6.3 Hz, 3H), 3.99 (d,
J = 6.6 Hz, 1H), 4.16 (m, 2H), 5.07 (s, 4H), 5.19 (s,
1H), 5.47 (quintet, J = 6.6 Hz, 1H), 6.83 (m, 1H),
7.26–7.45 (m, 12H), 8.76 (br s, 1H). 13C NMR (CDCl3,
75.5 MHz): d 14.2, 18.1, 60.3, 61.4, 67.7, 70.3, 90.9,
124.9, 127.8, 128.2, 128.3, 128.5, 128.8, 136.8, 137.6,
25
143.1, 151.7, 152.8, 164.8, 165.0, 167.2. ½aꢁ ꢀ24.0 (c
D
1.00, CHCl3). IR (film): 3271, 1825, 1693, 1646,
. HPLC–MS: tR = 28.62 min, m/z: 460.4
1587 cmꢀ1
[Mꢀ161]+, 645.3 [M+H+Na]+, 661.2 [M+H+K]+,
1266.3 [2M+H+Na]+. mp 144–146 ꢁC.
107.4, 108.5, 127.6, 128.1, 128.6, 131.5, 136.3, 151.5,
25
D
5.6.10. Compound 16. The obtained yield 51%, pale
yellow oil. 1H NMR (CDCl3, 200 MHz): d 1.27 (t,
J = 7.2 Hz, 3H), 1.52 (d, J = 6.2 Hz, 3H), 3.92 (s,
6H), 4.02 (d, J = 6.4 Hz, 1H), 4.17 (q, J = 7.2 Hz,
2H), 5.22 (s, 1H), 5.47 (quintet, J = 6.4 Hz, 1H), 6.00
(s, 1H), 7.28 (m, 2H), 8.75 (br s, 1H). 13C NMR
(CDCl3, 75.5 MHz): d 14.2, 18.1, 56.3, 60.3, 61.6,
159.8, 164.6, 164.9, 166.9. ½aꢁ ꢀ32.6 (c 1.67, CHCl3).
IR (film): 3278, 1822, 1700, 1652, 1595, 1445 cmꢀ1
.
HPLC–MS: tR = 18.88 min, m/z: 472.2 [Mꢀ43]+, 516.2
[M+H]+, 533.2 [M+H2O]+, 538.1 [M+Na]+, 1053.3
[2M+Na]+.
5.6.15. Compound 22. The obtained yield 14%, pale
yellow oil. 1H NMR (CDCl3, 200 MHz): d 1.30 (t,
J = 7.0 Hz, 3H), 1.59 (d, J = 6.6 Hz, 3H), 4.10 (d,
J = 6.2 Hz, 1H), 4.21 (q, J = 7.0 Hz, 2H), 5.22 (s, 1H),
5.58 (quintet, J = 6.2 Hz, 1H), 8.35–8.45 (m, 3H), 8.66
(br s, 1H). 13C NMR (CDCl3, 75.5 MHz): d 14.2, 17.9,
60.5, 61.3, 68.8, 91.3, 122.8 (q, J = 273.0 Hz), 127.1
67.2, 90.8, 106.6, 120.5, 139.6, 146.6, 151.7, 164.9,
25
D
165.0, 167.0. ½aꢁ ꢀ29.2 (c 1.33, CHCl3). IR (film):
3295, 1820, 1699, 1653, 1229 cmꢀ1
.
HPLC–MS:
tR = 9.44 min, m/z: 336.2 [Mꢀ43]+, 380.1 [M+H]+,
397.2 [M+H2O]+, 402.2 [M+Na]+, 418.1 [M+K]+,
781.2 [2M+Na]+.
(m), 130.3 (m), 131.4, 132.5 (q, J = 34.2 Hz), 150.8,
25
D
5.6.11. Compound 17. The obtained yield 60%, pale yel-
low oil. 1H NMR (acetone-d6, 300 MHz): d 1.52 (d,
164.3, 166.8, 167.7. ½aꢁ +7.9 (c 0.67, CHCl3). IR (film):
3272, 1825, 1733, 1655, 1281 cmꢀ1
.
HPLC–MS:

6130
G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
tR = 14.79 min, m/z: 395.9 [Mꢀ43]+, 440.1 [M+H]+,
7.34–7.47 (m, 14H). 13C NMR (CDCl3, 50.3 MHz): d
ꢀ5.0, ꢀ4.7, 17.9, 19.9, 25.6, 64.4, 65.3, 66.1, 70.3,
100.3, 108.0, 108.6, 127.5, 128.0, 128.1, 128.2, 128.5,
457.1 [M+H2O]+, 461.9 [M+Na]+, 478.0 [M+K]+.
Compounds 35–39 were obtained by b-lactam N-acyla-
tion using the corresponding acyl-chlorides. A represen-
tative procedure is the following: compound 4 (0.381 g,
1.017 mmol) and 3,4,5-tribenzyloxybenzoyl chloride
(0.513 g, 1.119 mmol) were dissolved in anhydrous ace-
tone (10 mL); K2CO3 (0.141 g, 1.017 mmol) was added
and the reaction mixture stirred at room temperature
for 4 h. Then the K2CO3 was filtered, the solvent re-
moved, and the crude oily residue immediately purified
by flash chromatography (cyclohexane/ethylacetate
95:5) to give compound 37. Compounds 35–39 are elut-
ed in HPLC analysis as single peaks; however in the ESI
ionization they are N-deacylated.
128.6, 133.0, 135.8, 136.3, 149.7, 159.6, 163.8, 165.0,
25
D
166.0. ½aꢁ +54.8 (c 2.77, CHCl3). IR (film): 1843,
1716, 1659, 1595, 1306 cmꢀ1. HPLC: tR = 9.38 min.
5.6.20. Compound 39. The obtaiend yield 37%, pale
yellow oil. 1H NMR (CDCl3, 200 MHz): d 0.12 (s,
6H), 0.88 (s, 9H), 1.30 (d, J = 6.2 Hz, 3H), 1.34 (t,
J = 7.0 Hz, 3H), 4.25 (q, J = 7.0 Hz, 2H), 4.40 (dd,
J = 2.0 Hz, J = 4.4 Hz, 1H), 4.76 (m, 1H), 6.73 (d,
J = 2.0 Hz, 1H), 8.11 (m, 1H), 8.34 (m, 2H). 13C NMR
(CDCl3, 50.3 MHz): d ꢀ5.1, ꢀ4.6, 14.3, 17.9, 20.3,
25.6, 60.6, 65.0, 65.6, 102.1, 122.7 (q, J = 273.2 Hz),
126.8m, 129.9m, 132.0 (q, J = 34.3 Hz), 133.5, 148.0,
25
D
162.5, 164.3, 165.9. ½aꢁ +132.6 (c 0.76, CHCl3). IR
5.6.16. Compound 35. The obtained yield 41%, white
solid. 1H NMR (CDCl3, 300 MHz): d 0.12 (s, 3H),
0.14 (s, 3H), 0.90 (s, 9H), 1.28 (d, J = 6.3 Hz, 3H),
1.32 (t, J = 7.2 Hz, 3H), 3.92 (s, 6H), 3.95 (s, 3H), 4.23
(q, J = 7.2 Hz, 2H), 4.33 (dd, J = 1.8 Hz, J = 4.5 Hz,
1H), 4.79 (dr q, J = 4.5 Hz,J = 6.3 Hz, 1H), 6.65 (d,
J = 1.5 Hz, 1H), 7.28 (m, 2H). 13C NMR (CDCl3,
50.3 MHz): d ꢀ4.9,ꢀ4.6, 14.3, 17.9, 19.8, 25.7, 56.3,
(film): 1845, 1717, 1665, 1280 cmꢀ1
tR = 26.33 min.
.
HPLC:
All the following compounds were obtained by hydro-
genolysis of the corresponding benzylesters and benzyl-
ethers. A representative procedure is the following:
compound 11 (0.444 g, 0.976 mmol) was dissolved in
an anhydrous mixture THF/MeOH (4 mL/4 mL) and
Pd, 10 wt% on activated carbon (0.045 g) was added.
Finally, the reaction mixture was treated with H2
(1 atm) and stirred at room temperature for 4 h. It was
then filtered and concentrated to give compound 14.
60.3, 61.0, 64.2, 65.4, 100.6, 107.6, 116.2, 125.8, 143.0,
25
149.3, 152.8, 164.3, 164.8, 166.2. ½aꢁ +57.3 (c 0.89,
D
CHCl3). IR (film): 1841, 1719, 1683, 1330 cmꢀ1. HPLC:
tR = 27.50 min. mp 120–122 ꢁC.
5.6.17. Compound 36. The obtained yield 44%, pale
yellow oil. 1H NMR (CDCl3, 300 MHz): d 0.15 (s,
3H), 0.16 (s, 3H), 0.93 (s, 9H), 1.24 (d, J = 6.3 Hz,
3H), 1.33 (t, J = 7.2 Hz, 3H), 4.23 (q, J = 7.2 Hz, 2H),
4.31 (dd, J = 1.8 Hz, J = 4.5 Hz, 1H), 4.79 (dr q,
J = 4.5 Hz, J = 6.3 Hz, 1H), 5.17 (s, 6H), 6.63 (d,
J = 1.8 Hz, 1H), 7.26–7.46 (m, 17H). 13C NMR (CDCl3,
75.5 MHz): d ꢀ4.9, ꢀ4.6, 14.3, 17.9, 19.8, 25.7, 60.3,
64.1, 65.4, 71.4, 75.2, 100.5, 109.9, 125.8, 127.4, 127.5,
5.6.21. Compound 14. Quantitative yield, white syrup.
1H NMR (acetone-d6, 200 MHz): d 1.53 (d, J = 6.2 Hz,
3H), 3.79 (s, 3H), 3.86 (s, 6H), 4.22 (d, J = 5.2 Hz,
1H), 5.26 (s, 1H), 5.46 (dr q, J = 6.2 Hz, J = 5.2 Hz,
1H), 7.29 (m, 2H), 9.81 (br s, 1H). 13C NMR (ace-
tone-d6, 75.5 MHz): d 17.9, 55.8, 60.0, 61.7, 67.6, 90.3,
107.0, 125.2, 142.9, 153.2, 153.6, 164.6, 165.8, 167.0.
25
½aꢁ ꢀ20.8 (c 2.31, CHCl3). IR (film): 3281, 1824,
D
1705, 1654 cmꢀ1. HPLC–MS: tR = 8.07 min, m/z: 322.1
[Mꢀ43]+, 366.2 [M+H]+, 383.1 [M+H2O]+, 388.1
[M+Na]+, 404.0 [M+K]+, 753.2 [2M+Na]+, 769.2
[2M+K]+.
128.0, 128.2, 128.4, 128.5, 136.6, 137.3, 143.3, 149.3,
25
D
152.4, 164.1, 164.6, 166.2. ½aꢁ +47.3 (c 4.16, CHCl3).
IR (film): 1841, 1718, 1683, 1328, 1197 cmꢀ1. HPLC:
tR = 9.64 min.
5.6.22. Compound 18. Quantitative yield, white syrup.
1H NMR (acetone-d6, 300 MHz): d 1.52 (d, J = 6.3 Hz,
3H), 3.86 (s, 6H), 4.21 (d, J = 5.1 Hz, 1H), 5.26 (s,
1H), 5.46 (dr q, J = 6.3 Hz,J = 5.1 Hz, 1H), 7.30 (m,
2H), 8.10 (br s, 1H), 9.81 (br s, 1H). 13C NMR (ace-
tone-d6, 75.5 MHz): d 18.6, 56.6, 62.4, 67.9, 90.8,
5.6.18. Compound 37. The obtained yield 30%, pale
yellow oil. 1H NMR (CDCl3, 200 MHz): d 0.16 (s,
3H), 0.17 (s, 3H), 0.96 (s, 9H), 1.26 (d, J = 6.2 Hz,
3H), 4.35 (d, J = 4.8 Hz, 1H), 4.81 (dr q, J = 6.2 Hz,
J = 4.8 Hz, 1H), 5.20 (s, 6H), 5.26 (s, 2H), 6.74 (s,
1H), 7.30–7.49 (m, 22H). 13C NMR (CDCl3,
50.3 MHz): d ꢀ4.9, ꢀ4.7, 17.9, 19.8, 25.7, 64.2, 65.3,
66.1, 71.4, 75.2, 100.2, 109.9, 125.8, 127.4, 127.5,
107.9, 121.0, 141.9, 148.4, 154.0, 165.4, 166.4, 167.7.
25
½aꢁ ꢀ17.5 (c 1.43, CHCl3). IR (film): 3308, 1824,
D
1710, 1654 cmꢀ1. HPLC–MS: tR = 6.57 min, m/z: 308.1
[Mꢀ43]+, 352.1 [M+H]+, 369.2 [M+H2O]+, 374.1
[M+Na]+, 390.0 [M+K]+, 725.2 [2M+Na]+.
128.0, 128.1, 128.2, 128.5, 128.6, 135.9, 136.6, 137.3,
25
D
149.9, 152.4, 164.1, 164.6, 166.1. ½aꢁ +78.2 (c 1.60,
CHCl3). IR (film): 1845, 1718, 1685, 1335, 1127 cmꢀ1
.
HPLC–MS: tR = 20.27 min, m/z: 376.3 [M+Hꢀ423]+,
5.6.23. Compound 23. Quantitative yield, white syrup.
1H NMR (CD3CN, 200 MHz): d 1.23 (t, J = 7.0 Hz,
3H), 1.42 (d, J = 6.4 Hz, 3H), 4.07 (dd, J = 6.3 Hz,
J = 0.8 Hz 1H), 4.14 (q, J = 7.0 Hz, 2H), 5.14 (s, 1H),
5.37 (quintet, J = 6.4 Hz, 1H), 7.02 (m, 2H). 13C NMR
(CD3CN, 75.5 MHz): d 14.6, 18.4, 60.7, 62.3, 67.8,
398.1 [M+Naꢀ423]+.
5.6.19. Compound 38. The obtained yield 39%, pale
1
yellow oil. H NMR (CDCl3, 300 MHz) d 0.15 (s, 3H),
0.16 (s, 3H), 0.94 (s, 9H), 1.29 (d, J = 6.3 Hz, 3H),
4.36 (d, J = 4.2 Hz, 1H), 4.79 (m, 1H), 5.10 (s, 4H),
5.25 (s, 2H), 6.75 (m, 1H), 6.87 (m, 1H), 7.18 (m, 2H),
91.2, 109.9, 121.8, 138.5, 145.7, 153.2, 165.7, 166.5,
25
D
167.2. ½aꢁ ꢀ21.9 (c 0.32, CH3OH). IR (film): 3353,

G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
6131
1813, 1695, 1378, 1236 cmꢀ1. HPLC–MS: tR = 6.88 min,
m/z: 308.1 [Mꢀ43]+, 352.1 [M+H]+, 369.2 [M+H2O]+,
374.0 [M+Na]+, 390.0 [M+K]+, 725.2 [2M+Na]+.
5.6.28. Compound 27. The obtained yield 44%, pale yel-
low oil. 1H NMR (CDCl3, 300 MHz) d 0.13 (s, 3H), 0.14
(s, 3H), 0.93 (s, 9H), 1.40 (d, J = 6.3 Hz, 3H), 3.78 (d,
J = 4.8 Hz, 1H), 4.32 (m, 1H), 5.48 (s, 1H), 7.14–7.46
(m, 5H), 8.53 (br s, 1H). 13C NMR (CDCl3,
50.3 MHz): dꢀ5.1, ꢀ4.2, 17.9, 22.3, 25.6, 65.0, 65.1,
5.6.24. Compound 24. Quantitative yield, pale yellow oil.
1H NMR (acetone-d6, 200 MHz): d 1.22 (t, J = 6.8 Hz,
3H), 1.48 (d, J = 6.6 Hz, 3H), 4.13 (q, J = 6.8 Hz, 2H),
4.21 (d, J = 7.0 Hz, 1H), 5.18 (s, 1H), 5.43 (m, 1H),
6.60 (m, 1H), 7.00 (m, 2H), 8.66 (br s, 2H), 9.80 (br s,
1H). 13C NMR (acetone-d6, 75.5 MHz): d 14.6, 18.1,
89.6, 121.7, 125.8, 129.4, 150.5, 155.4, 165.6, 166.1.
25
½aꢁ ꢀ36.3 (c 1.63, CHCl3). IR (film): 3356, 1821,
D
1799, 1714, 1653 cmꢀ1. HPLC–MS: tR = 20.41 min, m/
z: 318.2 [Mꢀ43]+, 362.1 [M+H]+, 384.2 [M+Na]+.
60.2, 62.1, 68.3, 90.7, 108.2, 108.6, 132.8, 153.3, 159.4,
25
D
165.6, 166.0, 166.7. ½aꢁ ꢀ13.0 (c 1.60, acetone). IR
5.6.29. Compound 28. The obtained yield 68%, white
solid. H NMR (acetone-d6, 200 MHz): d 0.08 (s, 3H),
(film): 3351, 1815, 1695, 1603, 1234, 1160 cmꢀ1
.
1
HPLC–MS: tR = 8.66 min, m/z: 292.3 [Mꢀ43]+, 336.2
[M+H]+, 353.1 [M+H2O]+, 358.3 [M+Na]+, 375.2
[M+H+K]+, 693.2 [2M+Na]+.
0.09 (s, 3H), 0.87 (s, 9H), 1.32 (d, J = 6.2 Hz, 3H),
3.84 (d, J = 4.4 Hz, 1H), 4.30 (dr q, J = 4.4 Hz,
J = 6.4 Hz, 1H), 4.97 (d, J = 12.4 Hz, 1H), 5.05 (d,
J = 12.4 Hz, 1H), 5.21 (s, 1H), 6.30 (m, 1H), 6.36 (m,
2H), 8.28 (br s, 2H), 9.68 (br s, 1H). 13C NMR (THF-
d8, 50.3 MHz): d ꢀ5.0, ꢀ4.3, 18.5, 22.4, 26.0, 65.4,
5.6.25. Compound 25. Quantitative yield, white syrup.
1H NMR (acetone-d6, 200 MHz): d 1.49 (d, J = 6.2 Hz,
3H), 4.21 (d, J = 6.2 Hz, 1H), 5.20 (s, 1H), 5.44 (quintet,
J = 6.2 Hz, 1H), 6.60 (m, 1H), 6.95–7.04 (m, 2H), 8.70
(br s, 2H), 9.76 (br s, 1H). 13C NMR (acetone-d6,
65.6, 66.3, 89.8, 102.7, 106.6, 139.6, 155.3, 159.7,
25
166.7, 167.3. ½aꢁ ꢀ10.1 (c 2.90, CH3OH). IR (film):
D
3311,
1810,
1696,
1654
cmꢀ1
.
HPLC–MS:
75.5 MHz): d 18.1, 62.0, 68.3, 90.7, 108.1, 108.6, 132.8,
tR = 11.93 min, m/z: 408.2 [M+H]+, 430.2 [M+Na]+,
446.0 [M+K]+. mp 79–81 ꢁC.
25
153.4, 159.4, 165.6, 165.9, 167.5. ½aꢁ ꢀ2.6 (c 0.98, ace-
D
tone). HPLC–MS: tR = 5.18 min, m/z: 264.4 [Mꢀ43]+,
308.1 [M+H]+, 325.4 [M+H2O]+, 330.0 [M+Na]+,
346.3 [M+K]+, 637.3 [2M+Na]+.
5.6.30. Compound 29. The obtained yield 20%, white
1
syrup. H NMR (acetone-d6, 300 MHz): d 0.10 (s, 3H),
0.12 (s, 3H), 0.90 (s, 9H), 1.37 (d, J = 6.3 Hz, 3H),
3.90 (d, J = 4.8 Hz, 1H), 4.35 (m, 1H), 5.32 (s, 1H),
6.12 (m, 2H), 6.24 (m, 1H), 8.46 (br s, 2H), 9.81 (br s,
1H). 13C NMR (acetone-d6, 75.5 MHz): d ꢀ4.9, ꢀ4.1,
5.6.26. Compound 40. Quantitative yield, white syrup.
1H NMR (CD3CN, 200 MHz) d ꢀ0.01 (s, 6H), 0.77 (s,
9H), 1.13 (d, J = 6.2 Hz, 3H), 4.18 (dd, J = 1.8 Hz,
J = 6.0 Hz, 1H), 4.66 (m, 1H), 6.23 (d, J = 1.8 Hz,
1H), 6.85 (m, 2H), 7.05 (br s, 2H), 7.17 (br s, 1H). 13C
NMR (acetone-d6, 75.5 MHz): d ꢀ4.8, ꢀ4.7, 18.2,
18.5, 22.5, 26.1, 65.7, 65.9, 89.6, 100.7, 101.7, 153.4,
25
156.7, 159.7, 165.0, 167.4. ½aꢁ ꢀ3.9 (c 0.77, CHCl3).
D
IR (film): 3325, 1806, 1700, 1653, 1625 cmꢀ1. HPLC–
MS: tR = 12.05 min, m/z: 394.2 [M+H]+, 416.1
[M+Na]+, 432.0 [M+K]+, 809.3 [2M+Na]+.
19.6, 26.1, 65.1, 66.0, 99.5, 110.7, 122.9, 141.1, 145.9,
25
151.7, 164.8, 165.5, 167.3. ½aꢁ +9.1 (c 0.34, acetone).
D
HPLC–MS: tR = 12.11 min, m/z: 394.1 [Mꢀ43]+, 438.2
[M+H]+, 460.1 [M+Na]+, 476.1 [M+K]+, 897.3
[2M+Na]+.
5.6.31. Compound 30. The obtained yield 50%, pale
yellow oil. 1H NMR (CDCl3, 300 MHz): d 0.07 (s,
3H), 0.08 (s, 3H), 0.86 (s, 9H), 1.33 (d, J = 6.3 Hz,
3H), 3.70 (d, J = 5.1 Hz, 1H), 4.35 (dr q, J = 5.1 Hz,
J = 6.3 Hz, 1H), 5.28 (s, 2H), 5.33 (s, 1H), 7.82 (m,
2H), 7.85 (m, 1H), 8.49 (br s, 1H). 13C NMR (CDCl3,
50.3 MHz): d ꢀ5.1, ꢀ4.3, 17.9, 22.3, 25.6, 64.0, 65.0,
5.6.27. Compound 41. Quantitative yield, white syrup.
1H NMR (acetone-d6, 200 MHz) d 0.14 (s, 3H), 0.15
(s, 3H), 0.91 (s, 9H), 1.30 (d, J = 6.2 Hz, 3H), 4.43 (d,
J = 3.6 Hz, 1H), 4.88 (m, 1H), 6.50 (s, 1H), 6.63 (m,
1H), 6.88 (m, 2H), 8.78 (br s, 3H). 13C NMR (ace-
tone-d6, 75.5 MHz): d ꢀ6.0, ꢀ5.8, 17.2, 18.6, 24.9,
65.1, 89.3, 122.0m, 123.2 (q, J = 272.5 Hz), 127.8 (m),
25
D
132.0 (q, J = 33.6 Hz), 138.9, 155.1, 166.3, 166.6. ½aꢁ
64.2, 64.9, 99.2, 107.4, 107.5, 133.3, 149.9, 158.1,
ꢀ21.2 (c 1.18, CHCl3). IR (film): 3297, 1820, 1699,
25
163.5, 165.0, 166.5. ½aꢁ +14.5 (c 2.82, acetone).
1653 cmꢀ1
. HPLC–MS: tR = 25.41 min, m/z: 512.2
D
HPLC–MS: tR = 12.99 min, m/z: 378.2 [Mꢀ43]+, 422.3
[M+H]+, 444.2 [M+Na]+, 460.2 [M+K]+, 865.4
[2M+Na]+.
[M+H]+, 529.3 [M+H2O]+, 534.0 [M+Na]+.
5.6.32. Compound 31. The obtained yield 62%, pale
yellow oil. 1H NMR (CDCl3, 300 MHz): d 0.10 (s,
3H), 0.11 (s, 3H), 0.91 (s, 9H), 1.34 (d, J = 6.3 Hz,
3H), 3.66 (d, J = 5.4 Hz, 1H), 4.24 (quintet,
J = 6.0 Hz, 1H), 4.51 (dd, J = 6.0 Hz, J = 15.0 Hz,
1H), 4.57 (dd, J = 6.0 Hz, J = 15.0 Hz, 1H), 5.13 (s,
1H), 5.64 (t, J = 6.0 Hz, 1H), 7.30–7.41 (m, 5H), 8.96
(br s, 1H). 13C NMR (CDCl3, 50.3 MHz): d ꢀ4.9,
Compounds 27–34 were obtained starting from 26 by
coupling with the corresponding amine, phenol or alco-
hol. A representative procedure is the following: com-
pound 26 (0.065 g, 0.228 mmol) was dissolved in
anhydrous CH2Cl2 (4 mL) and the solution brought to
0 ꢁC. Then DCC (0.047 g, 0.228 mmol) and benzylamine
(37 lL, 0.342 mmol) were added at 0 ꢁC. The reaction
mixture was stirred at room temperature for 2 h, then fil-
tered and concentrated. The residue was purified by
flash chromatography (cyclohexane/ethylacetate 8:2) to
give compound 31.
ꢀ4.3, 17.9, 22.5, 25.7, 43.3, 64.8, 65.4, 92.2, 127.5,
25
127.6, 128.7, 138.3, 150.3, 166.6, 167.0. ½aꢁ +6.0 (c
D
1.66, CHCl3). IR (film): 3346, 1804, 1682, 1627 mꢀ1
.
HPLC–MS: tR = 14.69 min, m/z: 375.2 [M+H]+, 397.2
[M+Na]+, 771.5 [2M+Na]+.

6132
G. Cainelli et al. / Bioorg. Med. Chem. 13 (2005) 6120–6132
Burgey, C. S.; Doherty, J. B.; Dorn, C. P.; Finke, P. E.;
Hagmann, W. K.; Hale, J. J.; Maccoss, M.; Westler, W.
M.; Knight, W. B. Biochemistry 1995, 34, 14331.
2. Strynadka, N. C. J.; Adachi, H.; Jensen, S. E.; Johns, K.;
Sielecki, A.; Betzel, C.; Sutoh, K.; James, M. N. G. Nature
1992, 359, 700.
3. Tew, D. G.; Boyd, H. F.; Ashman, S.; Theobald, C.;
Leach, C. A. Biochemistry 1998, 37, 10087.
4. Paetzel, M.; Dalbey, R. E.; Strynadka, N. C. J. Nature
1998, 396, 186.
5.6.33. Compound 32. The obtained yield 31%, white syr-
up. 1H NMR (acetone-d6, 200 MHz): d 0.08 (s, 3H), 0.09
(s, 3H), 0.87 (s, 9H), 1.29 (d, J = 6.2 Hz, 3H), 3.71 (d,
J = 5.0 Hz, 1H), 4.20 (dd, J = 5.8 Hz, J = 14.2 Hz,
1H), 4.24 (m, 1H), 4.32 (dd, J = 5.8 Hz, J = 14.2 Hz,
1H), 5.31 (s, 1H), 6.59–6.79 (m, 3H), 7.29 (t,
J = 5.8 Hz, 1H), 7.81 (br s, 2H), 9.47 (br s, 1H). 13C
NMR (acetone-d6, 50.3 MHz): d ꢀ4.8, ꢀ4.2, 18.5,
22.5, 26.1, 42.8, 65.2, 66.2, 93.4, 115.7, 115.8, 119.9,
25
132.3, 144.9, 145.8, 149.8, 166.8, 167.9. ½aꢁ ꢀ18.8 (c
5. Cainelli, G.; Giacomini, D.; Galletti, P.; Quintavalla, A.
Eur. J. Org. Chem. 2003, 9, 1765.
D
0.16, acetone). IR (film): 3392, 1804, 1683 cmꢀ1
.
HPLC–MS: tR = 11.34 min, m/z: 407.1 [M+H]+, 429.2
6. Cainelli, G.; Galletti, P.; Garbisa, S.; Giacomini, D.; Sartor,
L.; Quintavalla, A. Biorg. Med. Chem. 2003, 11, 5391.
7. Eccles, S. A.; Box, G. M.; Court, W. J.; Bone, E. A.;
Thomas, W.; Brown, P. D. Cancer Res. 1996, 56, 2815.
8. Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science
2002, 295, 2387.
[M+Na]+, 835.4 [2M+Na]+.
5.6.34. Compound 33. The obtained yield 56%, pale yel-
1
low oil. H NMR (CDCl3, 200 MHz): d 0.07 (s, 3H),
0.08 (s, 3H), 0.87 (s, 9H), 1.31 (d, J = 6.2 Hz, 3H),
3.66 (d, J = 5.4 Hz, 1H), 4.22 (quintet, J = 6.2 Hz, 1H),
4.88 (s, 3H), 7.39 (m, 5H), 7.92 (br s, 1H), 8.81 (br s,
1H). 13C NMR (CDCl3, 50.3 MHz): d ꢀ4.9, ꢀ4.2,
9. Wang, Z. Y.; Huang, M. T.; Ho, C. T.; Chang, R.; Ma,
W.; Ferraro, T.; Reuhl, K. R.; Yang, C. S.; Conney, A. H.
Cancer Res. 1992, 52, 6657.
10. Cao, Y.; Cao, R. Nature 1999, 398, 381.
11. Garbisa, S.; Biggin, S.; Cavallarin, N.; Sartor, L.; Benelli,
R.; Albini, A. Nat. Med. 1999, 5, 1216.
12. Taniguchi, S.; Fujiki, H.; Kobayashi, H.; Go, H.; Miyado,
K.; Sadano, H.; Shimokawa, R. Cancer Lett. 1992, 65, 51.
13. Garbisa, S.; Sartor, L.; Biggin, S.; Salvato, B.; Benelli, R.;
Albini, A. Cancer 2001, 91, 822.
17.9, 22.4, 25.7, 65.2, 65.3, 79.1, 87.8, 128.7, 128.9,
25
129.2, 134.9, 152.8, 166.7, 166.8. ½aꢁ +19.4 (c 3.27,
D
CHCl3). IR (film): 3226, 1813, 1684 cmꢀ1. HPLC–MS:
tR = 12.49 min, m/z: 391.1 [M+H]+, 413.1 [M+Na]+,
430.1 [M+H+K]+, 803.3 [2M+Na]+.
14. Demeule, M.; Brossard, M.; Page, M.; Gingras, D.;
Beliveau, R. Biochim. Biophys. Acta 2000, 1478, 51.
15. Sartor, L.; Pezzato, E.; Garbisa, S. J. Leuk. Biol. 2002, 71,
73.
16. Cainelli, G.; Giacomini, D.; Gazzano, M.; Galletti, P.;
Quintavalla, A. Tetrahedron Lett. 2003, 44, 6269.
17. Gerard, S.; Galleni, M.; Dive, G.; Marchand-Brynaert, J.
Bioorg. Med. Chem. 2004, 12, 129; Beauve, C.; Bouchet,
M.; Touillaux, R.; Fastrez, J.; Marchand-Brynaert, J.
Tetrahedron 1999, 55, 13301.
18. Cainelli, Giacomini, Galletti unpublished results.
19. cf. for instance the X-ray structures of complexes between
b-lactams and porcine pancreas elastase: Taylor, P.;
Anderson, V.; Dowden, J.; Flitsch, S. L.; Turner, N. J.;
Loughrans, K.; Walkinshaw, M. D. J. Biol. Chem. 1999,
35, 24901; Navia, M. A.; Springer, J. P.; Lin, T.-Y.;
Williams, H. R.; Firestone, R. A.; Pisano, J. M.; Doherty,
J. B.; Finke, P. E.; Hoogsteen, K. Nature 1987, 327, 79.
20. (a) Durette, P. L.; Chabin, R. M.; Fletcher, D. S.; Green,
B. G.; Hanlon, W. A.; Humes, J. L.; Knight, W. B.;
Lanza, T. J., Jr.; Mumford, R. A.; Pacholok, S.; MacCoss,
M. Bioorg. Med. Chem. Lett. 1995, 5, 271; (b) Doherty, J.
B.; Shah, S. K.; Finke, P. E.; Dorn, C. P., Jr.; Hagmann,
W. K.; Hale, J. J.; Kissinger, A. L.; Thompson, K. R.;
Brause, K.; Chandler, G. O.; Knight, W. B.; Maycock, A.
L.; Ashe, B. M.; Weston, H.; Gale, P.; Mumford, R. A.;
Andersen, O. F.; Williams, H. R.; Nolan, T. E.; Franken-
field, D. L.; Underwood, D.; Vyas, K. P.; Kari, P. H.;
Dahlgren, M. E.; Mao, J.; Fletcher, D. S.; Dellea, P. S.;
Hand, K. M.; Osinga, D. G.; Peterson, L. B.; Williams, D.
T.; Metzger, J. M.; Bonney, R. J.; Humes, J. L.; Pacholok,
S. P.; Hanlon, W. A.; Opas, E.; Stolk, J.; Davies, P. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 8727.
5.6.35. Compound 34. The obtained yield 98%, pale yellow
oil. 1H NMR (CDCl3, 300 MHz): d 0.07 (s, 3H), 0.08 (s,
3H), 0.87 (s, 9H), 1.32 (d, J = 6.3 Hz, 3H), 3.66 (d,
J = 5.7 Hz, 1H), 4.23 (quintet, J = 5.7 Hz, 1H), 4.63 (d,
J = 6.6 Hz, 2H), 5.16 (s, 1H), 5.79 (t, J = 6.6 Hz, 1H),
7.75 (m, 2H), 7.79 (m, 1H), 8.84 (br s, 1H). 13C NMR
(CDCl3, 50.3 MHz): d ꢀ5.0, ꢀ4.3, 17.9, 22.5, 25.6, 42.3,
64.9, 65.4, 91.5, 121.4m, 123.2 (q, J = 271.2 Hz),
127.5m, 131.9 (q, J = 33.4 Hz), 141.3, 151.5, 166.7,
25
D
166.8. ½aꢁ ꢀ2.9 (c 3.67, CHCl3). IR (film): 3330, 3226,
1800, 1683, 1635 cmꢀ1. HPLC–MS: tR = 19.34 min, m/z:
511.1 [M+H]+, 533.2 [M+Na]+.
Acknowledgments
This research was supported by MURST (60% and
PRIN), University of Bologna (funds for selected top-
ics), University of Padova, and the Associazione Italiana
per la Ricerca sul Cancro. We are grateful to Dr. S. Big-
gin for revision of the English manuscript and to Mrs.
Maurizia Soverini for experimental assistance.
Supplementary data
1H NMR and 13C NMR for all new compounds (2, 4, 8–
25, 27–41), whose synthesis is reported in the experimen-
tal part. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmc.2005.06.041.
`
21. Dona, M.; DellÕAica, I.; Pezzato, E.; Sartor, L.; Calabrese,
F.; Della Barbera, M.; Donella-Deana, A.; Appendino,
G.; Borsarini, A.; Caniato, R.; Garbisa, S. Cancer Res.
2004, 64, 6225.
References and notes
22. Angle, S. R.; Belanger, D. S. J. Org. Chem. 2004, 69, 4361,
and references cited therein; Danheiser, R. L.; Miller, R.
F.; Brisbois, R. G.; Park, S. Z. J. Org. Chem. 1990, 55,
1959.
1. Green, B. G.; Chabin, R.; Mills, S.; Underwood, D. J.;
Shah, S. K.; Kuo, D.; Gale, P.; Maycock, A. L.; Liesch, J.;