Synthesis and biological evaluation of non-peptide αvβ3/α5β1 integrin dual antagonists containing 5,6-dihydropyridin-2-one scaffolds

Article abstract of DOI:10.1016/j.bmc.2007.07.049

Small constrained non-peptidic molecules consisting of a polyfunctionalized rigid core, carrying appendages corresponding to arginine and aspartic acid side chains, have been recently reported to be promising for drug development. In this work, the 5,6-di

Full text of DOI:10.1016/j.bmc.2007.07.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7380–7390
Synthesis and biological evaluation of non-peptide avb3/a5b1 integrin
dual antagonists containing 5,6-dihydropyridin-2-one scaffolds
Fides Benfatti,a Giuliana Cardillo,a,* Serena Fabbroni,a Patrizia Galzerano,a
Luca Gentilucci,a Riccardo Juris,a Alessandra Tolomelli,a Monica Baiula,b
b
b,
*
`
Antonino Sparta and Santi Spampinato
a
`
Dipartimento di Chimica ‘‘G. Ciamician’’, Universita di Bologna, Via Selmi 2, 40126 Bologna, Italy
b
`
Dipartimento di Farmacologia, Universita di Bologna, Via Irnerio 48, 40126 Bologna, Italy
Received 20 March 2007; revised 16 July 2007; accepted 25 July 2007
Available online 22 August 2007
Abstract—Small constrained non-peptidic molecules consisting of a polyfunctionalized rigid core, carrying appendages correspond-
ing to arginine and aspartic acid side chains, have been recently reported to be promising for drug development. In this work, the
5,6-dihydropyridin-2-one was envisaged as a scaffold to turn into potential integrin ligands, introducing a carboxylic acid and a
basic appendage. The synthesis and the antiadhesion activity of a small library of peptidomimetics capable to recognize avb3
and a5b1 integrins has been herein reported.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ponents like fibronectin, fibrinogen, vitronectin, and
osteopontin through recognition of the Arg-Gly-Asp
(RGD) tripeptide sequence.4 This sequence is also essen-
tial for the binding of a5b1 integrin to fibronectin, which
has been unambiguously recognized as proangiogenic
receptor.5 a5b1 integrin may regulate the function of
integrins avb3 on endothelial cells during their migration
in vitro or angiogenesis in vivo. Activation of a5b1
potentiates avb3-mediated migration on vitronectin,
whereas a5b1 integrin antagonists inhibit avb3-mediated
cell spreading. Therefore, antagonists of both integrins,
block the same pathway of angiogenesis.6 In this paper,
we report the design, synthesis, and blockade of fibro-
nectin-mediated cell adhesion of novel avb3/a5b1 integrin
dual antagonists, whose activity could be synergistically
effective in preventing angiogenesis.
Integrins are a large family of heterodimeric transmem-
brane glycoproteins involved in the attachment of a cell
to the extracellular matrix (ECM) and in signal trans-
duction from the ECM to the cell.1 These adhesion
mechanisms are of fundamental importance in a diverse
range of biological processes, including cell differentia-
tion, apoptosis, embryonic cell migration, maintenance
of tissue integrity, and blood coagulation.2 Alterations
or aberrations in integrin-mediated cell adhesion have
been connected with the pathogenesis of several diseases
such as atherosclerosis, osteoporosis, cancer and a vari-
ety of inflammatory disorders, making integrins an
attractive target for the development of therapeutic
agents.3 The identification of key recognition motifs
within integrin ligands is the starting point for the devel-
opment of antagonists. To date, these motifs have been
identified for only a few subtypes. avb3 integrin has been
deeply investigated as it is involved-tumor proliferation
and metastasis through the formation of new blood ves-
sels. avb3 integrin binds to a wide number of ECM com-
The X-ray analysis7 of the complex between avb3 inte-
grin and c(RGDfV) ligand shows that the ligand inter-
acts mainly through electrostatic interactions. Arg and
Asp form a charged clamp that binds regions with oppo-
site charges in the protein: Asp interacts with a metal
cation in the b subunit and Arg with two Asp in the a
subunit.
Keywords: avb3-Integrin; a5b1-Integrin; Dual antagonist; Dihydro-
pyridinone.
Several efficient classes of ligands, containing the RGD
sequence, have been reported in the literature.8 These
structures share as common features conformational
*
Corresponding authors. Tel.: +39 051 2099570; fax: +39 051
2099456; e-mail addresses: giuliana.cardillo@unibo.it; spampi@
biocfarm.unibo.it
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.049

F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
7381
restraints able to give a proper orientation to the periph-
eral substituents.
Linear and cyclic peptides containing the RGD se-
quence, showing high affinity toward avb3 integrins,
have significant therapeutic potential but serious limita-
tions especially for oral dosing. The need for antagonists
with higher bioavailability and lower molecular weight
has prompted several research groups to develop small
constrained non-peptidic molecules mimicking the
RGD motif, which would be more promising for drug
development.9
Figure 1. Model A, B, C integrin ligands.
Most of the structures proposed so far consist of a poly-
functionalized rigid core, linked to appendages corre-
sponding to arginine and aspartic acid side chains.10
The basicity and the length of the arginine-mimicking
group was found to play a central role. Moreover, the
presence of a carboxylic function, mimicking the aspar-
tic acid residue in the original binding motif, is a funda-
mental feature to create anionic interaction with the
metal cation in the receptor active site.11 Many hetero-
cyclic scaffolds have been employed to maintain the
acidic and the basic ends of the molecule at the appro-
priate distance and with the suitable conformation for
binding interaction.
Ac-Asp-Arg-Leu-Asp-Ser-OH (H3534) was measured as
a positive control.15
2. Results
2.1. Synthesis of model A antagonists
The highly functionalized racemic compound 1a12 was
chosen as precursor in the design of model A avb3 inte-
grin antagonists. The introduction of the basic function
was carried out through nitrogen acylation with Cbz-
aminoalkanoic chloride, followed by hydrolysis of the
ester function and hydrogenation (Scheme 2).
We identified the 5,6-dihydropyridin-2-one as scaffold,12
easily prepared through a short concise synthesis
(Scheme 1).
Compounds 6a and 7a were isolated in 55% overall yield
and were tested for their ability to perturb initial cell
attachment mediated by avb3 and a5b1 integrin. The
results obtained are reported in Table 1. All the com-
pounds did not exhibit a potent inhibition of avb3-
and a5b1-mediated cell adhesion (Table 1).
This heterocycle may be converted into a potential inte-
grin ligand introducing the acidic and the basic append-
ages as reported in Models A, B, and C (Fig. 1).
To evaluate the biological activity of these novel com-
pounds in a cellular environment, we tested their ability
to perturb initial cell attachment mediated by avb3 inte-
grin and a5b1 integrin using cell adhesion assays. The
integrin ligand fibronectin (10 lg/ml) was immobilized
on tissue culture plates. The ability of human melanoma
cell line SK-MEL 24, expressing amb3 integrin,13 and hu-
man erythroleukemic cell line K562, expressing a5b1
integrin,14 to adhere to fibronectin in the presence or ab-
sence of the assayed compounds was examined. The ant-
iadhesion activity of the well-known integrin antagonist
Under these experimental conditions, reference com-
pound H3534 caused a noteworthy inhibition of avb3-
mediated cell adhesion and was less potent toward
a5b1-mediated cell adhesion (Table 1).
Although compounds 6a and 7a were designed with a
considerable difference in the distance between acidic
Scheme 2. Reagents and conditions: i—Cl-CO-(CH2)nNHCbz, pyri-
dine, CH2Cl2, rt; ii—LiOH, THF/MeOH/H2O; iii—H2, Pd/C, MeOH,
rt.
Scheme 1. Synthetic route to 5,6-dihydropyridin-2-one 1.

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F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
Table 1. avb3- and a5b1-integrin mediated cell adhesion to fibronectin
in the presence of Model-A like ligands
LiOH in MeOH/THF/H2O and BOC deprotection was
performed in neat trifluoroacetic acid. Enantiomerically
pure 6b was obtained from this reaction sequence in
good yield (Scheme 3).
These compounds, carrying a free amino group and the
carboxylic acid function, have been tested in cell-adhe-
sion assays and the results are reported in Table 2.
Compound
IC50 (lM)a
avb3
a5b1
Compound (6S)-4b showed a significant inhibition of
avb3-integrin mediated cell adhesion with submicromo-
lar IC50. Unfortunately, it was completely uneffective to-
ward a5b1 integrin. On the contrary, the (6R)-5b showed
a lower activity in avb3 integrin mediated adhesion as-
says whereas it was a good inhibition of a5b1-mediated
cell adhesion. Since dual antagonists, capable to block
the adhesion function of both integrins, can be consid-
ered more promising as angiogenesis inhibitors, 4b and
5b do not appear to be attractive for any further thera-
peutical development. Finally, guanidino-derivative 6b
resulted inactive toward both integrins. In conclusion,
comparison of the results obtained for 4b and 6b showed
that no advantage could be derived by the introduction
of the guanidino moiety.
6a
7a
>1000
>1000
0.025
>1000
>1000
259
H3534
a Values are means standard error of three experiments.
and basic moieties, they both show a very low affinity to-
ward the two proangiogenic receptors. Thus, we ex-
plored the possibility to enhance integrin antagonist
properties, modifying our synthetic plan according to
Models B and C.
2.2. Synthesis of model B antagonists
As shown in Figure 1, the model B-like antagonists con-
tain the basic function on the C6 side chain of the dihyd-
ropyridinone. This function has been introduced in the
5,6-dihydropyridin-2-one 1b through a nitro-derivative
precursor. The choice of (S)-p-methoxyphenylethyl-
amine as starting building block allowed to obtain
dihydropyridinones (6S)-1b and (6R)-1b in 43/57 diaste-
reomeric ratio (Scheme 1).16 The diastereomers were
easily separated by flash chromatography on silica gel.
Nitrogen acylation with methyl malonyl chloride gave,
respectively, the intermediates 2b and 3b. Hydrogena-
tion, followed by hydrolysis of the ester, allowed opti-
cally active 4b and 5b to be obtained in good yield
(Scheme 3).
2.3. Synthesis of model C antagonists
The model C-like antagonists contain the basic function
on the amide side chain of the dihydropyiridinone. This
function has been introduced in the intermediate 1c, syn-
thesized via ketene/imine cycloaddition between 2-bro-
mo-3-methyl-chrotonyl chloride and the imine of
benzaldehyde and p-nitro-benzylamine, followed by
treatment with allylamine (Scheme 1).
The affinity and selectivity of a ligand for avb3 integrin is
based on the spatial disposition of the C-terminal car-
boxylic acid and the N-terminal basic group. The dis-
tance has been reported to be optimal when it is about
twelve-thirteen bonds;9a therefore, we modified the
spacer length, introducing methyl malonyl chloride
and ethyl fumaryl chloride, to give rise to 2c and 3c in
70% yield.
The guanidinic group was introduced by treatment of
the intermediate ester deriving from 2b reduction, with
N,N0-Bis(t-butoxycarbonyl)-1-H-pyrazole-1-carboxami-
dine in DMF. The guanidino derivative was then trans-
formed into free carboxylic acid by treatment with
Table 2. avb3- and a5b1-integrin mediated cell adhesion to fibronectin
in the presence of Model-B like ligands
Compound
R
IC50 (lM)a
avb3
0.24 0.04
a5b1
4b
5b
H
H
>1000
21
4
0.018 0.006
NH2
NH
6b
>1000
>1000
Scheme 3. Reagents and conditions: i—(a) H2, Pd/C, MeOH, rt; (b)
LiOH, THF/MeOH/H2O, rt: ii—(a) N,N0-Bis(t-butoxycarbonyl)-1-H-
pyrazole-1-carboxamidine, DMF, rt; (b) TFA, rt.
a Values are means standard error of three experiments.

F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
7383
Moreover, the easy a-alkylation of 2c with NaH in THF
and benzyl bromoacetate gave 4c in 81% yield. The
introduction of the amino function and the consecutive
conversion into the corresponding guanidino-derivatives
allowed to obtain the first small library of model C-like
antagonists. Hydrogenation of 2c and 4c, followed by
treatment with LiOH in methanol/water/THF solution,
gave 8c and 9c, having a free amino group and, respec-
tively, one or two free carboxylic acid functions.
lower activity toward both integrins. The same behavior
was observed for compounds 12c and 13c, which main-
tain the optimal 12-bonds distance but possess a second
carboxylic substituent in the acid side chain.
When we turned our attention to the compounds de-
prived of the guanidinium group, we observed for 7c,
and 8c (Table 3, entries 6,7) an enhanced activity to-
ward avb3-integrin mediated cell adhesion. Compound
9c, corresponding to 12c deprived of the guanidine
moiety, did not show any bioactivity toward the same
receptor. Disappointingly, 7c and 9c were uneffective to
block a5b1 integrin-mediated cell adhesion. The most
interesting result was obtained for compound 8c, hav-
ing IC50 of 0.6 lM (Table 3, entry 5) toward avb3 inte-
grin and 0.17 lM toward a5b1-integrin-mediated cell
adhesion.
The guanidino-derivatives 10c, 11c, and 12c were ob-
tained, starting from hydrogenation products 5c, 6c,
and 7c, in excellent yield following the usual procedure.
In a similar way, the mono methyl ester 13c was isolated
in 80% yield starting from methyl ester 7c (Scheme 4).
The small library of model C-like antagonists was then
tested for biological activity. The results are reported
in Table 3. The IC50 was calculated only for compounds
showing major activity.
The encouraging results observed for compound 8c
avb3-and a5b1-integrin mediated cell adhesion assays
suggest that this substrate could be used as a model to
evaluate the influence of the scaffold stereochemistry
on inhibitory effect.
Starting from 10c, which showed a weak inhibitory ef-
fect, the related compounds 11c, 12c, and 13c differ in
the distance between the carboxylic and the guanidino
functions and for the structure of the carboxylic chain.
The modifications, including elongation and presence
of a second carboxylic moiety both as free acid and as
methyl ester, did not afford any advantage (Table 3, en-
tries 2,3,4). In fact, elongated compound 11c, having
thirteen bonds between acidic and basic ends, showed
To clarify the conformation induced by the heterocycle
stereochemistry, we focused our attention on enantio-
merically pure analogs of 8c. The introduction of
(S)-p-nitro-phenylethylamine allowed diastereomeric
compounds 14c and 15c to be obtained, showing the
critical importance of 5,6-dihydropyridin-2-one C6 con-
figuration on substrate-ligand recognition.17
Following the synthetic pathway above reported for
model-C antagonists, enantiomerically pure (10S, 6R)-
14c and (10S, 6S)-15c were obtained in 63:37 d.r., using
(S)-p-nitro-phenylethylamine as precursor of the amino
group. The presence of a methyl group in the nitrogen
side chain allowed the easy separation of the diastereo-
mers and generated a further conformational constrain.
Both diastereomers (10S, 6R)-14c and (10S, 6S)-15c were
capable to block aVb3- and a5b1-mediated cell adhesion
and, being dual inhibitors could be considered as lead
compounds for any further therapeutical and diagnostic
development. Interestingly, a 200-fold gain in potency to
prevent avb3 driven cell adhesion and 600-fold gain in
potency toward a5b1 were observed for (10S, 6S)-15c
in comparison with the corresponding (10S, 6R)-14c dia-
stereomer. On this basis, a strong influence to the spatial
arrangement of the (S)-C6 aromatic substituent on bio-
active conformation could be ascribed.
At last, to verify if the substitution of the rigid benzylic
aminic appendage with the more flexible butandiamine
could afford an improvement with respect to ( )-8c,
compound ( )-3d was synthesized, giving further infor-
mation. In fact, the possibility to introduce different
substituents on the rigid core scaffold offers the oppor-
tunity to synthesize other members of this small library
of 5,6-dihydropyridin-2-one ligands. On these bases, we
changed the heterocyclic nitrogen appendage, with the
aim to modulate basicity and lipophilicity. Thus, race-
mic compound 1d was synthesized starting from the
Scheme 4. Reagents and conditions: i—Methyl malonyl chloride or
ethyl fumaryl chloride, TEA, CH2Cl2, 0 °C to rt; ii—NaH, benzyl
bromoacetate, THF, 0 °C to rt; iii—H2, Pd/C, MeOH, rt; iv—LiOH,
THF/MeOH/H2O, rt; v—(a) N,N0-Bis(t-butoxycarbonyl)-1-H-pyra-
zole-1-carboxamidine, DMF, rt; (b) LiOH, THF/MeOH/H2O, rt; (c)
TFA, rt; vi—(a) N,N0-Bis(t-butoxycarbonyl)-1-H-pyrazole-1-carbox-
amidine, DMF, rt; (b) TFA, rt.

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F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
Table 3. avb3- and a5b1- integrin mediated cell adhesion to fibronectin in the presence of Model-B like ligands
Entry Compound
IC50 (lM)
avb3
a5b1
1
120 17
>1000loffset 0mm
2
3
>1000
>1000
>1000
>1000
4
>1000
>1000
5
6
7
0.6 0.1
0.17 0.07
45
7
>1000
>1000
>1000
8
9
15.1 6.0
35.1 12.0
0.071 0.011
0.057 0.017
10
0.038 0.015
0.043 0.05
aValues are means standard error of three experiments.
imine of benzaldehyde and N-benzyloxycarbonyl-but-
andiamine. This derivative, having only eleven bonds
between acidic and basic functionalities, contains the
terminal amino group linked to a more flexible ali-
phatic side chain in place of the lipophilic aniline moi-
ety. By performing the synthetic sequence reported
above, free amino-acid derivative 3d could be isolated
in good yield (Scheme 5).
Compound 3d gave excellent results in avb3- and a5b1-
integrin mediated cell adhesion assays and turned out
to be the best antagonist in the synthesized library (Ta-
ble 3, entry 10). The dual antagonism toward both pro-
angiogenic integrins might be considered for any future
anticancer therapy and tumor targeting, even though
more potent and selective avb3 integrin antagonists have
already been reported.18 Further studies on this

F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
7385
was allowed to slowly warm to rt. The reaction was fol-
lowed by TLC and then quenched with HCl 0.1 M
(10 mL). The layers were separated and the aqueous
layer was extracted twice with dichloromethane
(10 mL). The combined organic layers were dried over
Na2SO4 and concentrated in vacuo to afford the desired
compound, that was purified by flash chromatography
on silica gel (cyclohexane/ethyl acetate 8/2).
3.3. General procedure for hydrogenation of nitro-
derivatives
Scheme 5. Reagents and conditions: i—(a) allylamine, rt, 3 days; (b)
methyl malonyl chloride TEA, CH2Cl2, 0 °C to rt; ii—(a) H2, Pd/C,
MeOH, rt; (b) LiOH, THF/MeOH/H2O, rt.
To a solution of nitroderivative in MeOH, Pd/C (1
equiv) was added in one portion. The reaction mixture
was stirred vigorously at rt in a hydrogen atmosphere
overnight. The solution was filtered to remove catalyst
and evaporated to afford hydrogenated product, which
was used without purification in the following step.
compound are ongoing to evaluate its antagonistic
activity toward other integrins.
3.4. General procedure for the reaction of free amines
with N,N0-Bis(t-butoxycarbonyl)-1-H-pyrazole-1-
carbox-amidine
In conclusion, we have synthesized and tested in cell
adhesion assays small libraries of non-peptide avb3/
a5b1 integrin antagonists, containing the 5,6-dihydro-
pyridin-2-one scaffold. By changing the length, the
structure, and the position of the side chains linked to
the heterocyclic core, we identified model compound
( )-8c that gave excellent results both in avb3- and
a5b1-integrin mediated cell adhesion assays. With the
aim to verify the importance of C6 stereochemistry, we
synthesized and tested 14c and 15c. These last deriva-
tives had excellent affinity toward both receptors, show-
ing a dual inhibitor attitude. Furthermore, comparison
of semirigid aromatic derivative ( )-8c and ( )-3d
clearly indicates that both moieties are well tolerated,
being their bioactivity in the same range.
To a stirred solution of the amino derivative in DMF at
room temperature, N,N0-Bis(t-butoxycarbonyl)-1-H-
pyrazole-1-carboxamidine (1.2 equiv) was added in one
portion. The reaction was followed by TLC and then
quenched with 0.1 M HCl. The mixture was diluted with
ethyl acetate and extracted (three times). The combined
organic layers were dried over Na2SO4 and concentrated
in vacuo. The residue was purified by chromatography
on silica gel (cyclohexane/ethyl acetate 5/5), to afford
the desired compound.
3.5. General procedures for ester hydrolysis
To a stirred solution of ester in a 3.6:1:1 mixture of
THF/MeOH/H2O at room temperature, LiOH (3 equiv)
was added. The reaction was followed by TLC and then
concentrated in vacuo to afford the acid, which was
purified by chromatography on basic ion-exchange
resin.
3. Experimental
3.1. General synthetic methods
All chemicals were purchased from commercial suppli-
ers and used without further purification. Anhydrous
solvents were purchased in sure seal bottles over molec-
ular sieves and used without further drying. Flash chro-
matography was performed on silica gel (230–400
mesh). NMR Spectra were recorded with 200, 300, or
600 MHz spectrometers. Chemical shifts were reported
as d values (ppm) relative to the solvent peak of CDCl3
set at d = 7.27 (1H NMR) or d = 77.0 (13C NMR). Melt-
ing points are uncorrected. MS analyses were performed
on a liquid chromatograph coupled with an electrospray
ionization–mass spectrometer (LC-ESI-MS), using
H2O/CH3CN as solvent at 25 °C (positive scan 100–
500 m/z, fragmentor 70 V, gradient elution program
from 80% water to 70% acetonitrile in 8 min.).
3.6. General procedure of Boc deprotection
The Boc-derivative was dissolved in CF3COOH (9
equiv). The reaction was followed by TLC and then con-
centrated in vacuo. The residue was diluted with toluene
and concentrated in vacuo (three times) to afford the de-
sired pure compound.
Compound 1a: 1H NMR (300 MHz, CDCl3) dH 1.26 (t,
3H, J = 6.9 Hz), 1.74 (s, 3H), 2.33 (dd, 1H, J = 1.8,
17.4 Hz), 2.55 (dd, 1H, J = 6.0, 15.0 Hz), 2.78 (dt, 1H,
J = 7.2, 15.0 Hz), 3.04–3.16 (m, 3H), 3.42–3.51 (m,
2H), 4.04–4.16 (m, 3H), 4.76 (dd, 1H, J = 1.6, 7.5 Hz),
5.07 (dq, 1H, J = 1.5, 11.7 Hz), 5.17 (dq, 1H, J = 1.5,
17.1 Hz), 5.89 (ddt, 1H, J = 11.7, 17.1, 5.7 Hz), 7.16–
7.37 (m, 10H). 13C NMR (75 MHz, CDCl3) dC 14.1,
18.9, 33.4, 37.5, 43.2, 51.1, 59.6, 60.5, 115.6, 120.9,
126.3, 127.4, 128.5, 133.5, 136.7, 141.1, 164.9, 172.1;
LC-ESI-MS rt 11.86 min, m/z 343 (M+1), 365
(M+Na), 707 (2M+Na).
3.2. General procedure for acylation of 5,6-dihydro-4-
methyl-6-phenyl-pyridin-2-ones 1
To a stirred solution of dihydropyridinone in CH2Cl2
(10 mL) at 0 °C, under argon, TEA (1.5 equiv) and the
acyl chloride (1.5 equiv) were added. The temperature

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F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
Compound 2a: 1H NMR (300 MHz, CDCl3) dH 1.24 (t,
3H, J = 7.2 Hz), 1.64 (s, 3H), 2.19–2.37 (m 2H), 2.41–
2.59 (m, 2H), 2.61–2.80 (m, 1H), 2.99–3.22 (m, 2H),
3.41–3.50 (m, 2H), 3.83 (dd, 1H, J = 7.2, 15.6 Hz),
4.05 (m, 1H), 4.11 (q, 2H, J = 6.0 Hz), 4.23 (dd, 1H,
J = 6.6, 15.6 Hz), 4.89 (bd, 1H, J = 6.9 Hz), 4.97–5.14
(m, 4H), 5.70–5.81 (m, 1H), 7.14–7.20 (m, 2H), 7.24–
7.37 (m, 8H). 13C NMR (75 MHz, CDCl3) dC 14.0,
19.4, 20.1, 33.3, 37.2, 43.4, 50.2, 58.5, 60.5, 66.2, 118.0,
126.0, 127.8, 128.3, 128.6, 133.3, 136.7, 139.8, 140.0,
144.6, 156.3, 162.2, 171.9, 172.3; LC-ESI-MS rt
13.72 min, m/z 548 (M+1), 570 (M+Na).
171.5, 174.5. LC-ESI-MS: rt 5.13 min, m/z 388 (M+1),
410 (M+Na).
1
Compound 7a: H NMR (300 MHz, CD3OD) dH 0.84
(t, 3H, J = 7.2 Hz), 1.20–1.70 (m, 8H), 1.74 (s, 3H),
2.10–2.24 (m, 1H), 2.59–3.06 (m, 7H), 3.10–3.20 (m,
1H), 3.30–3.38 (m, 1H), 3.61–3.67 (m, 1H), 4.14–4.21
(m, 1H), 5.03 (bd, 1H, J = 7.2 Hz), 7.24 (d, 2H, J = 6.9
Hz), 7.31–7.44 (m, 3H).13C NMR (75 MHz, CDCl3)
dC 12.0, 20.2, 22.12, 25.56, 27.1, 28.4, 33.9, 34.5, 34.5,
38.4, 40.9, 45.0, 50.7, 59.8, 127.4, 127.8, 129.2, 130.1,
130.3, 131.0, 141.6, 147.7, 164.6, 176.2. LC-ESI-MS: rt
5.66 min, m/z 444 (M+1), 466 (M+Na).
Compound 3a: 1H NMR (300 MHz, CDCl3) dH 1.11 (t,
3H, J = 7.9 Hz), 1.20–1.45 (m, 6H), 1.56 (s, 3H), 1.81-
1.93 (dt, 1H J = 6.9, 15.3 Hz), 2.03 (dt, 1H, J = 7.8,
15.3 Hz), 2.21–2.45 (m, 3H), 2.55–2.67 (m, 1H), 2.90–
3.12 (m, 5H), 3.70 (dd, 1H, J = 7.2, 14.5 Hz), 3.91–
4.02 (m, 3H), 4.13 (dd, 1H, J = 5.7, 14.5 Hz), 4.71–
5.04 (m, 3H), 4.97 (bs, 2H), 5.59–5.77 (m, 1H), 7.02–
7.24 (m, 10H). 13C NMR (75 MHz, CDCl3) dC 14.1,
19.7, 24.3, 26.3, 29.5, 33.2, 33.6, 37.3, 40.8, 43.5, 50.3,
58.7, 60.6, 66.4, 117.8, 118.0, 126.1, 127.9, 128.0,
128.4, 128.7, 128.9, 130.0, 133.7, 140.1, 144.1, 156.4,
162.4, 172.0, 173.4; IR (film) m 3328, 2934, 1723, 1639,
1529, 1399, 1249 cmꢀ1; LC-ESI-MS rt 19.22 min, m/z
590 (M+1), 612 (M+Na).
1
Compound (6S)-1b: H NMR (300 MHz, CDCl3): dH
1.58 (d, 3H, J = 7.1 Hz), 1.642 (d, 3H, J1,3 = 1.5 Hz),
2.08 (d, 1H, J = 17.1 Hz), 3.09 (ddq, 1H, J = 17.1 7.1,
1.5 Hz), 3.47–3.54 (m, 2H), 3.58 (s, 3H), 4.57 (d, 1H,
J = 7.1 Hz), 5.09 (ddd, 1H, J = 10.2, 1.4, 2.9 Hz), 5.21
(ddd, 1H, J = 17.1, 1.8, 3.0 Hz), 5.83–5.96 (m, 1H,),
5.96 (q, 1H, J = 7.1 Hz), 6.45–6.47 (m, 2H), 6.90–6.93
(m, 2H), 7.00–7.03 (m, 2H), 7.80–7.83 (m, 2H).13C
NMR (75 MHz, CDCl3): dC 17.6, 18.8, 37.9, 50.9,
51.6, 53.0, 55.2, 113.1, 113.4, 115.7, 118.2, 122.5,
123.0, 127.0 127.2, 129.7, 129.5, 130.1, 134.4, 136.7,
146.4, 149.5, 159.0, 163.6. LC-ESI-MS: rt 14.3 min,
m/z 422 (M+1), 444 (M+Na), 865 (2M+Na+).
[a]D = ꢀ53.9 (CHCl3 c 0.5).
Compound 4a: 1H NMR (300 MHz, CDCl3) dH 1.68 (s,
3H), 2.24 (m, 1H), 2.39–2.59 (m, 2H), 2.50 (bd, 1H,
J = 18.0 Hz), 2.67–2.77 (m, 1H), 2.97–3.04 (m, 2H),
3.26 (dd, 1H, J = 7.5, 18.0 Hz), 3.37–3.48 (m, 2H),
3.83 (dd, 1H, J = 7.2, 14.4 Hz), 4.26 (dd, 1H, J = 6.6,
14.4 Hz), 4.87 (bd, 1H, J = 7.5 Hz), 4.97–5.19 (m, 4H),
5.75 (m, 1H), 5.86 (t, 1H, J = 5.1 Hz), 7.15–7.40 (m,
10H), 13C NMR (75 MHz, CDCl3) dC 19.5, 33.2, 37.0,
37.2, 43.2, 50.8, 58.0, 60.3, 66. 6, 118.6, 126.1, 127.9,
128.4, 128.8, 129.0, 129.2, 133.2, 136.5, 139.9, 145.3,
157.0, 163.1, 172.4, 172.9; LC-ESI-MS rt 11.29 min,
m/z 520 (M+1), 542 (M+Na).
1
Compound (6R)-1b: H NMR (300 MHz, CDCl3): dH
1.14 (d, 3H, J = 7.2 Hz), 1.62 (s, 3H), 1.98 (d, 1H,
J = 17.1 Hz), 2.82 (dd, 1H, J = 17.1, 7.2 Hz), 3.42–3.58
(m, 2H), 3.8 (s, 3H), 4.38 (d, 1H, J = 7.2 Hz), 5.09 (d,
1H, J = 10.2 Hz), 5.20 (d, 1H, J = 17.1 Hz), 5.82–5.97
(m, 1H), 6.06 (q, 1H, J = 7.2 Hz), 6.86–6.89 (m, 2H),
7.20–7.23 (m, 2H), 7.31–7.34 (m, 2H), 8.11–8.13 (m,
2H). 13C NMR (75 MHz, CDCl3): dC 16.5, 18.6, 37.6,
50.7, 51.0, 52.9, 55.0, 113.4, 113.8, 115.5, 118.3, 123.2,
123.6, 127.2, 127.5, 128.0, 128.1, 132.9, 134.0, 136.7,
147.0, 150.5, 158.9, 164.0. LC-ESI-MS: rt 14.8 min,
m/z 422 (M+H), 444 (M+Na), 865 (2M+Na).
[a]D = +75.3 (CHCl3 c 0.7).
Compound 5a: 1H NMR (300 MHz, CDCl3) dH
1.12–1.66 (m, 6H), 1.72 (s, 3H), 1.98–2.21 (m,
2H), 2.51 (bd, 1H, J = 14.8 Hz), 2.60 (m, 1H),
2.75–2.85 (m, 1H), 3.02–3.24 (m, 5H), 3.84 (dd,
1H, J = 7.4, 14.4 Hz), 4.08 (m, 1H), 4.27 (m, 1H),
4.82 (bd, 1H, J = 7.0 Hz), 4.90–5.18 (m, 4H), 5.40
(bs, 1H), 5.70–85.95 (m, 1H), 7.15–7.40 (m, 10H),
8.60 (bs, 1H). 13C NMR (75 MHz, CDCl3) dC
19.6, 24.3, 26.1, 29.4, 32.9, 37.2, 40.9, 43.1, 43.5,
50.4, 51.0, 58.6, 66.5, 118.0, 126.0, 127.8, 127.9,
128.4, 128.7, 129.5, 129.7, 133.5, 140.1, 144.6,
162.5, 173.5, 175.0; LC-ESI-MS rt. 13.4 min, m/z
562 (M+1), 584 (M+Na).
Compound 2b: 1H NMR (300 MHz, CDCl3): dH 1.59 (d,
3H, J = 6.9 Hz), 1.68 (s, 3H), 2.33 (d, 1H, J = 17.8 Hz),
3.19 (dd, 1H, J = 17.8, 7.2 Hz), 3.27–3.30 (m, 2H), 3.56
(s, 3H), 3.69 (s, 3H), 4.05–4.12 (m, 2H), 4.72 (d,1H,
J = 7.2 Hz), 5.07–5.13 (m, 2H), 5.77–5.90 (m, 1H), 5.93
(q, 1H, J = 6.9 Hz), 6.44–6.47 (m, 2H), 6.93–7.02 (m,
4H), 7.80–7.83 (m, 2H).13C NMR (75 MHz, CDCl3):
dC 17.5, 19.4, 37.7, 41.2, 50.7, 51.8, 52.1 (2), 55.0,
113.1, 113.3, 117.8, 122.6, 122.8, 126.9 (2), 129.5,
129.6, 129.7, 129.8, 133.6, 144.2, 146.5, 148.3, 159.1,
160.9, 166.7, 167.8. LC-ESI-MS: rt 11.6 min, m/z 522
(M+H), 544 (M+Na), 1065 (2M+Na). [a]D = ꢀ92.3°
(CHCl3 c 0.8).
1
Compound 6a: H NMR (300 MHz, CD3OD) dH 0.83
(t, 3H, J = 7.2 Hz), 1.20–1.32 (m, 2H), 1.73 (s, 3H),
2.47–2.63 (m, 3H), 2.69–2.75 (m, 2H), 2.83–3.01 (m,
2H), 3.22–3.46 (m, 3H), 3.48–3.60 (m, 1H), 4.32–4.34
(m, 1H), 4.93 (t, 1H, J = 6.0 Hz), 7.14–7.27 (m, 2H),
7.27–7.37 (m, 3H).13C NMR (75 MHz, CDCl3) dC
11.4, 19.4, 20.7, 31.8, 34.9, 35.2, 37.5, 44.1, 49.1, 50.3,
58.0, 125.8, 127.6, 128.6, 137.3, 140.0, 145.6, 162.6,
Compound 3b: 1H NMR (200 MHz, CDCl3): dH 1.15 (d,
3H, J = 7.1 Hz), 1.67 (s, 3H), 2.22 (d, 1H, J = 17.8 Hz),
2.9 (dd,1H, J = 17.8, 6.7 Hz), 3.31–3.32 (m, 2H) 3.68 (s,
3H), 3.80 (s, 3H), 4.01 (dd,1H, J = 15.0, 6.6 Hz) 4.20
(dd, 1H, J = 15.0, 6.4 Hz), 4.48 (d, 1H, J = 6.7 Hz),
5.09–5.18 (m, 2H), 5.75–5.95 (m, 1H), 6.04 (q, 1H,

F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
7387
J = 7.1 Hz), 6.87–6.91 (m, 2H), 7.18–7.22 (m, 2H), 7.35–
7.39 (m, 2H), 8.11–8.16 (m, 2H).13C NMR (50 MHz,
CDCl3): dC 16.5, 19.5, 37.7, 41.3, 50.8, 51.3, 52.1, 52.2,
55.2, 113.8, 114.0, 117.8, 123.6, 123.7, 127.2, 128.1,
130.1, 132.5, 133.6, 144.5, 147.4, 149.3, 159.1, 161.4,
166.7, 167.8. LC-ESI-MS: rt 12.2 min, m/z 522 (M+H),
544 (M+Na). [a]D = +46.4° (CHCl3 c 0.87).
J = 8.4 Hz), 7.35–7.45 (m, 5H), 8.20 (d, 2H,
J = 8.4 Hz); 13C NMR (75 MHz, CDCl3) dC 19.9, 37.3,
41.8, 48.5, 50.9, 52.2, 57.0, 118.6, 123.9, 126.6, 128.3,
128.6, 128.9, 129.1, 133.1, 138.7, 144.8, 146.0, 147.4,
162.4, 166.3, 167.7; LC-ESI-MS rt 11.1 min, m/z 478
(M+1), 500 (M+Na).
1
Compound 3c: H NMR (300 MHz, CDCl3) dH 1.28 (t,
1
Compound 4b: H NMR (300 MHz, CD3OD): dH 0.90
3H, J = 7.0 Hz), 1.73 (s, 3H), 2.57 (dd, 1H, J = 2.6,
18.4 Hz), 3.18 (dd, 1H, J = 7.6, 18.4 Hz), 3.81 (d, 1H,
J = 15.4 Hz), 4.04–4.37 (m, 4H), 4.60 (dd, 1H, J = 2.6,
7.6 Hz), 5.05–5.23 (m, 2H), 5.45 (d, 1H, J = 15.4 Hz),
5.73–5.96 (m, 1H), 6.81 (d, 1H, J = 15.2 Hz), 6.99 (d,
1H, J = 15.0 Hz), 7.15 (d, 2H, J = 8.8 Hz), 7.28–7.40
(m, 5H), 8.12 (d, 2H, J = 8.8 Hz); 13C NMR (75 MHz,
CDCl3) dC 13.9, 19.6, 37.3, 48.6, 50.6, 57.0, 60.9,
118.6, 123.8, 126.1, 128.1, 128.4, 128.5, 128.8, 129.2,
130.9, 132.7, 134.0, 138.9, 144.7, 146.0, 147.2, 162.3,
164.6, 165.6. LC-ESI-MS rt 11.54 min, m/z 504 (M+1),
526 (M+Na).
(t, 3H, J = 7.4 Hz), 1.41-1.45 (m, 2H), 1.67 (d, 3H,
J = 7.1 Hz), 1.92 (s, 3H), 2.46 (bd,1H, J = 17.4 Hz),
3.10–3.28 (m, 3H), 3.38 (s, 2H), 3.69 (s, 3H), 4.80
(bd,1H, J = 6.3 Hz), 5.74 (q,1H, J = 7.1 Hz), 6.48–6.51
(m, 2H), 6.62–6.68 (m, 4H), 7.18–7.21 (m, 2H).13C
NMR (75 MHz, CD3OD): dC 12.1, 18.9, 20.3, 22.1,
39.9, 51.1, 54.7, 56.0, 68.3, 114.6, 123.8, 129.3, 130.4,
131.0, 131.6, 143.3, 147.9, 160.5, 163.0, 169.9, 169.7.
LC-ESI-MS: rt 7.6 min, m/z 480 (M+H), 502
(M+Na).[a]D = ꢀ144.7 (CHCl3 c 0.6).
1
Compound 5b: H NMR (300 MHz, CD3OD): dH 0.94
(t, 3H, J = 7.4 Hz), 1.28 (d, 3H, J = 7.1 Hz), 1.42–1.53
(m, 2H), 1.72 (s, 3H), 2.31 (bd, 1H, J = 17.2 Hz), 2.95
(dd, 1H, J = 17.2, 6.6 Hz), 3.11–3.21 (m, 1H), 3.35 (s,
2H), 3.84 (s, 3H), 4.46 (bd,1H, J = 6.6 Hz), 5.96 (q,
1H, J = 7.1 Hz), 6.71–6.74 (m, 2H), 6.95–6.99 (m, 4H),
7.31–7.34 (m, 2H). 13C NMR (75 MHz, CD3OD): dC
13.6, 17.2, 20.6, 23.1, 38.6, 50.7, 53.4, 57.1, 66.9, 113.8,
123.6, 129.6, 130.2, 131.0, 131.3, 143.0, 148.2, 149.1,
160.6, 164.8, 168.9, 170.1. LC-ESI-MS: 2.4 min, 480
(M+H), 502 (M+Na).[a]D = +23.5 (CHCl3 c 0.2).
Compound 4c: 1H NMR (300 MHz, CDCl3) dH 1.88 (s,
3H), 2.58 (dd, 1H, J = 17.0, 1.4 Hz), 3.30 (dd, 1H,
J = 17.0, 6.2 Hz), 3.60 (m, 1H), 3.68 (s, 3H), 3.72 (d, 1H,
J = 15.6 Hz), 3.90–4.05 (m, 3H,), 4.23 (dd, 1H, J = 14.6,
6.4 Hz), 4.56 (dd, 1H, J = 1.4, 6.2 Hz), 5.49 (d, 1H,
J = 15.6 Hz), 5.05–5.22 (m, 2H), 5.78–5.98 (m, 2H),
7.12–7.38 (m, 12H), 8.13 (d, 2H, J = 8.2); 13C NMR
(75 MHz, CDCl3) dC: 20.3, 37.9, 41.0, 47.4, 50.3, 54.1,
54.3, 58.0, 59.6, 118.8, 121.0, 123.6, 126.9, 127.2, 127.6,
128.0, 128.4, 128.9, 129.1, 131.3, 132.8, 138.0, 142.1,
145.9, 146.1, 162.3, 165.9, 168.1, 169.2. LC-ESI-MS rt
12.9 min, m/z 626 (M+1), 648 (M+Na).
1
Compound 6b: H NMR (300 MHz, CD3OD): dH 0.90
(t, 3H, J = 7.0 Hz), 1.29–1.43 (m, 1H), 1.69 (d, 3H,
J = 6.9 Hz), 1.73 (s, 3H), 2.52 (d, 1H, J = 17.4 Hz),
3.10–3.20 (m, 3H), 3.38 (m, 2H), 3.78 (s, 3H), 5.10
(bd, 1H), 5.88 (q, 1H, J = 7 Hz), 6.60–6.63 (m, 2H),
6.97–7.00 (m, 4H), 7.19–7.22 (m, 2H). 13C NMR
(75 MHz, CD3OD): dC 12.0, 18.5, 20.1, 22.3, 27.9,
31.1, 39.4, 53.8, 54.2, 56.0, 114.6, 125.7, 129.2 (2),
131.4 (2), 131.8, 135.2, 141.4, 145.9, 147.9, 153.1,
160.8, 162.6, 163.9, 180.1. LC-ESI-MS: rt 3.6 min, m/z
522 (M+H), 544 (M+Na). [a]D = ꢀ64.9° (CHCl3 c 0.26).
Compound 5c: Mp 59 °C. 1H NMR (300 MHz, CDCl3)
dH 0.88 (t, 3H, J = 7.5 Hz), 1.35–1.66 (m, 2H); 1.82 (s,
3H); 2.44 (bd, 1H, J = 17.0 Hz), 3.05 (dd, 1H, J = 7.6,
17.0 Hz), 3.30 (d, 1H, J = 15.0 Hz), 3.37 (d, 1H,
J = 15.0 Hz), 3.46–3.62 (m, 2H,), 3.53 (d, 1H,
J = 15.0 Hz), 3.73 (s, 3H), 4.59 (d, 1H, J = 7.6 Hz),
5.53 (d, 1H, J = 15.0 Hz), 6.67 (d, 1H, J = 8.4 Hz);
7.04 (d, 2H, J = 8.4 Hz), 7.16 (d, 2H, J = 8.7 Hz),
7.33–7.39 (m, 3H); 13C NMR (75 MHz, CDCl3) dC:
11.3, 19.6, 20.8, 37.3, 41.5, 48.0, 49.8, 52.1, 55.1, 115.1,
125.9, 126.9, 127.8, 128.9, 129.5, 130.0, 139.5, 144.5,
146.0, 162.1, 166. 7, 168.2. LC-ESI-MS rt 9.9 min, m/z
450 (M+1), 472 (2M+Na).
1
Compound 1c: Mp 75 °C; H NMR (200 MHz, CDCl3)
dH 1.78 (s, 3H), 2.39 (dd, 1H, J = 3.4, 17.4 Hz), 2.99 (dd,
1H, J = 7.5, 17.4 Hz), 3.51 (dd, 2H, J = 1.5, 4.4 Hz),
3.80 (d, 1H, J = 15.8 Hz), 4.47 (dd, 1H, J = 3.4,
7.5 Hz), 5.11 (dd, 1H, J = 1.6, 10.2 Hz), 5.22 (dd, 1H,
J = 1.6, 17.1 Hz), 5.44 (d, 1H, J = 15.8 Hz), 5.80–6.01
(m, 1H), 7.13 (d, 2H, J = 8.4 Hz),7.21–7.39 (m, 5H),
8.14 (d, 2H, J = 8.4 Hz); 13C NMR (75 MHz, CDCl3)
dC 18.9, 37.3, 48.4, 51.0, 58.1, 115.7, 121.9, 123.7,
126.6, 127.8, 128.3, 128.6, 136.5, 139.9, 145.3, 147.1,
165.1. LC-ESI-MS rt 12.9 min, m/z 378 (M+1), 400
(M+Na).
1
Compound 6c: H NMR (300 MHz, CDCl3): d: 0.85 (t,
3H, J = 7.4 Hz); 1.27 (t, 3H, J = 7.3 Hz), 1.35–1.52 (m,
2H); 1.77 (s, 3H); 2.32–3.21 (m, 6H), 3.42–3.47 (m,
1H); 3.48 (d, 1H, J = 14.2 Hz); 3.62–3.76 (m, 1H); 4.14
(q, 2H, J = 7.2 Hz), 4.58 (bd, 1H, J = 8.0 Hz Hz); 5.53
(d, 1H, J = 14.2 Hz); 6.62–6.69 (m, 2H); 7.03 (d, 2H,
J = 8.2 Hz); 7.13–7.22 (m, 3H); 7.34–7.39 (m, 2H). 13C
NMR (50 MHz, CDCl3) major conformer d: 11.3,
14.6, 19.3, 20.3, 27.1, 29.3, 37.6, 48.4, 49.8, 55.6, 60.4,
115.1, 125.8, 126.3, 127.6, 128.6, 129.3, 129.8, 139.5,
143.8, 145.9, 160.1, 171.9, 173.2. LC-ESI-MS rt
11.77 min, m/z: 478 (M+1), 501 (M+Na), 977 (2M+Na).
1
Compound 2c: H NMR (300 MHz, CDCl3) dH 1.81 (s,
3H), 2.55 (dd, 1H, J = 2.0, 11.9 Hz), 3.18 (dd, 1H,
J = 7.0, 11.9 Hz), 3.33 (s, 2H), 3.72 (s, 3H), 3.83 (d,
1H, J = 15.5 Hz), 4.04–4.26 (m, 2H), 4.59 (dd, 1H,
J = 2.0, 7.0 Hz), 5.06–5.38 (m, 2H), 5.52 (d, 1H,
J = 15.5 Hz), 5.80–6.00 (m, 1H), 7.18 (d, 2H,
1
Compound 7c: H NMR (300 MHz, CD3OD) dH 0.86
(t, 3H, J = 7.2 Hz), 1.21-1.30 (m, 2H); 1.88 (s, 3H);

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F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
2.46 (bd, 1H, J = 16.8 Hz), 3.02 (dd, 1H, J = 7.0,
16.8 Hz), 3.41 (m, 1H), 3.56–4.02 (m, 4H), 3.74 (s,
3H), 3.78 (d, 1H, J = 15.3 Hz), 4.66 (d, 1H,
J = 7.0 Hz), 5.51 (d, 1H, J = 15.3 Hz), 6.95–7.10 (m,
2H); 7.08–7.41 (m, 5H), 7.50–7.62 (m, 2H), 7.33–7.39
(3H, m); 13C NMR (75 MHz, CDCl3) dC: 11.8, 18.9,
22.4, 34.2, 36.9, 43.4, 50.6, 53.1, 124.1, 126.5, 127.3,
127.9, 128.2, 128.8, 131.2, 133.4, 136.1, 139.4, 158.1,
162.1, 166.7, 172.1. LC-ESI-MS rt 8.4 min, m/z 508
(M+1), 530 (M+Na).
(50 MHz, CD3OD) d: 13.2, 20.1, 23.5, 37.5, 40.0, 40.6,
48.3, 58.9, 80.0, 124.6, 128.1, 128.7, 128.8, 129.7,
130.0, 131.5, 134.3, 140.8, 148.6, 151.8, 157.4, 163.1,
175.9, 176.4. LC-ESI-MS rt 3.1 min, m/z: 536 (M+1),
558 (M+Na).
Compound 13c: 1H NMR (200 MHz, CDCl3): d: 0.87 (t,
3H, J = 7.2 Hz); 1.43–1.55 (m, 2H); 1.83 (s, 3H); 2.50
(bd,1H, J = 17.4 Hz); 2.99 (dd, 1H, J = 6.9, 17.4 Hz);
3.16 (m, 2H); 3.21–3.45 (m, 2H); 3.50 (d, 1H,
J = 15.3 Hz); 3.93 (dd, 1H, J = 10.5, 4.2 Hz); 4.56 (bd,
1H, J = 6.9 Hz); 5.60 (d, 1H, J = 15.3 Hz); 7.16–7.58
(m, 9H). 13C NMR (50 MHz, CDCl3) d: 11.8, 18.9,
21.5, 37.2, 39.8, 41.0, 47.1, 53.8, 58.8, 78.9, 122.8,
128.0, 128.6, 128.7, 129.7, 130.7, 131.1, 134.2, 142.7,
149.9, 152.8, 156.1, 163.2, 169.6, 176.4. LC-ESI-MS rt
7.2 min, m/z: 550 (M+1), 572 (M+Na).
Compound 8c: 1H NMR (300 MHz, CD3OD): d: 0.88 (t,
3H, J = 7.5 Hz); 1.30–1.44 (m, 2H); 1.79 (s, 3H); 2.51
(bd,1H, J = 17.4 Hz); 3.16 (dd, 1H, J = 6.6, 17.4 Hz);
3.22 (m, 1H); 3.55 (m, 1H); 3.64 (d, 1H, J = 15.0 Hz);
4.71 (bd, 1H, J = 6.6 Hz); 5.34 (d, 1H, J = 15.0 Hz);
6.68–6.72 (m, 2H), 7.19–7.21 (m, 2H), 7.30–7.41 (m,
3H). 13C NMR (50 MHz, CD3OD) d: 11.3, 18.7, 20.8,
37.3, 40.0, 48.2, 49.8, 58.1, 64.1, 115.1, 125.9, 126.9,
127.8, 128.9, 129.5, 130.0, 134.2, 139.5, 144.5, 150.9,
161.9, 168. 3, 174.4, LC-ESI-MS rt 3.55 min, m/z: 436
(M+1), 458 (M+Na).
1
Compound 14c: H NMR (300 MHz, CDCl3): dH 0.91
(t, 3H, J = 7.2 Hz), 1.21 (d, 3H, J = 7.1 Hz), 1.38–1.45
(m, 2H), 1.62 (s,3H), 2.26 (bd,1H, J = 17.4 Hz), 2.85
(dd, 1H, J = 17.4, 6.8 Hz), 3.01 (m, 1H), 3.12 (d, 1H,
J = 19.1 Hz), 3.44 (d, 1H, J = 19.1 Hz), 3.85 (m, 1H),
4.46 (d, 1H, J = 6.8 Hz), 6.00 (q, 1H, J = 7.1 Hz),
6.68–6.72 (m, 2H), 7.07–7.15 (m, 4H), 7.31–7.33 (m,
3H). 13C NMR (50 MHz, CDCl3): dC 16.4, 18.1, 19.5,
20.6, 22.2, 38.5, 50.0, 51.6, 52.5, 125.9, 127.7, 128.3,
128.4, 128.5, 138.4, 141.9, 143.7, 146.2, 149.5, 160.8,
161.9, 167.2. LC-ESI-MS: rt 2.3 min m/z 450 (M+H),
472 (M+Na), 921 (2M+Na). [a]D = +14.6° (CH3OH c
1.1).
1
Compound 9c: H NMR (300 MHz, D2O): d: 0.64 (t,
3H, J = 6.9 Hz); 1.09–1.22 (m, 2H); 1.59 (s, 3H); 2.36
(dd,1H, J = 16.2, 10.2 Hz); 2.53–2.68 (m, 3H) 3.03–
3.11 (m, 2H), 3.29 (m, 2H); 3.54 (d, 1H, J = 15.3 Hz);
4.52 (m, 1H), 5.00 (d, 1H, J = 15.3 Hz); 6.75–6.78 (m,
2H), 6.79–6.97 (m, 5H), 7.11–7.19 (m, 2H). 13C NMR
(75 MHz, D2O) d: 12.0, 19.9, 22.6, 30.9, 39.0, 43.1,
44.2, 51.9, 124.5, 126.8, 127.1, 127.8, 128.2, 128.5,
130.0, 132.1, 138.1, 139.4, 157.1, 163.0, 175.1, 177.1.
LC-ESI-MS rt 3.20 min, m/z: 494 (M+1), 516 (M+Na).
Compound 15c: 1H NMR (300 MHz, CD3OD): dH 0.85
(t, 3H, J = 7.4 Hz), 1.26–1.41 (m, 2H), 1.63 (s, 3H), 1.67
(d, 3H, J = 7.8 Hz), 2.45–2.55 (m, 1H), 3.05–3.21 (m,
2H), 3.34–3.36 (s, 2H), 3.55–3.65 (m, 1H), 4.70 (m,
1H), 5.71–5.79 (q, 1H, J = 7.8 Hz), 6.42–6.44 (m, 2H),
6.90–7.12 (m, 5H), 7.36–7.41 (m, 2H).13C NMR
(75 MHz, CD3OD): dC 12.0, 18.9, 20.3, 22.1, 29.6,
39.7, 52.7, 54.2, 54.7, 116.6, 127.4, 128.0, 129.6, 130.0,
130.6, 131.6, 142.0, 147.6, 148.1, 163.7, 164.6, 170.6.
LC-ESI-MS: rt 7.47 min m/z 450 (M+H), 472
(M+Na). [a]D = ꢀ120.0° (CH3OH c 0.2).
Compound 10c: 1H NMR (200 MHz, CDCl3): d: 0.87 (t,
3H, J = 7.2 Hz); 1.21-1.25 (m, 2H); 1.79 (s, 3H); 2.69
(bd,1H, J = 17.2 Hz); 3.11–3.31 (m, 2H); 3.23–3.46 (m,
1H); 3.52 (s, 2H); 3.59 (d, 1H, J = 15.8 Hz); 4.23 (bd,
1H, J = 5.4 Hz); 5.40 (d, 1H, J = 15.8 Hz); 7.21–7.47
(m, 9H). 13C NMR (50 MHz, CDCl3) d: 12.6, 20.9,
22.6, 39.0, 42.9, 48.6, 59.1, 87.0, 125.3, 128.0, 128.6,
128.7, 129.7, 130.7, 131.1, 131.4, 134.4, 140.5, 141.6,
149.9, 154.1, 168.1, 170.4. LC-ESI-MS rt 10.05 min,
m/z: 478 (M+1), 501 (M+Na).
Compound 1d: Isolated as a yellow oil. 1H NMR
(300 MHz, CDCl3): dH 1.54–1.57 (m, 2H); 1.58–1.60
(m, 2H); 1.95 (s, 3H); 2.55 (dd, 1H, J = 17.4, 2.4 Hz),
2.76 (m, 1H), 3.08 (dd, 1H, J = 17.4, 7.2 Hz), 3.18–
3.22 (m, 2H), 4.01 (m, 1H), 4.63 (dd, 1H, J = 2.4, 7.2),
4.98 (bs, 1H), 5.11 (s, 2H), 7.14–7.18 (m, 3H), 7.32–
7.39 (m, 8H). 13C NMR (75 MHz, CDCl3) dC 24.1,
25.1, 26.9, 39.1, 40.3, 46.4, 57.6, 66.2, 118.7, 126.0,
126.1, 127.7, 127.8, 128.1, 128.2, 128.6, 139.6, 144.7,
156.3, 160.3. LC-ESI-MS rt 10.0 min, m/z: 471–473
(M+1), 493–495 (M+Na).
Compound 11c: 1H NMR (300 MHz, CDCl3): d: 0.81 (t,
3H, J = 6.9 Hz); 1.20–1.40 (m, 2H); 1.81 (s, 3H); 2.31
(bd,1H, J = 16.8 Hz); 2.42–2.77 (m, 4H); 3.08 (dd, 1H,
J = 6.5, 16.8 Hz); 3.22 (m, 1H); 3.70 (m, 1H); 3.86 (d,
1H, J = 15.0 Hz); 4.78 (bd, 1H, J = 6.5 Hz); 5.41 (d,
1H, J = 15.0 Hz); 7.20–7.50 (m, 9H); 9.81 (bs, 1H). 13C
NMR (75 MHz, CDCl3) d: 10.7, 18.9, 20.8, 28.4, 28.9,
37.1, 49.5, 50.7, 57.2, 125.6, 126.2, 126.7, 127.8, 128.7,
129.3, 134.2, 136.1, 139.2, 147.2, 151.2, 156.9, 163.3,
173.4. LC-ESI-MS rt 11.84 min, m/z: 492 (M+1), 514
(M+Na).
Compound 2d: Isolated as a yellow oil. 1H NMR
(300 MHz, CDCl3): dH 1.51-1.60 (m, 4H); 1.79 (s, 3H);
2.51 (d, 1H, J = 17.2 Hz); 3.05 (d, 1H, J = 17.2 Hz);
3.16 (m, 2H); 3.19 (m, 2H); 3.27 (m, 2H); 3.69 (s, 3H);
4.00 (m, 2H); 4.66 (bd, 1H, J = 7.27 Hz); 5.11 (m, 4H);
5.81 (m, 1H); 7.14–7.19 (m, 2H); 7.29–7.37 (m, 8H).
13C NMR (75 MHz, CDCl3) dC 19.8, 25.1, 26.9, 29.9,
1
Compound 12c: H NMR (200 MHz, CD3OD): d: 0.87
(t, 3H, J = 7.6 Hz); 1.29–1.45 (m, 2H); 1.88 (s, 3H);
2.68 (bd, J = 16.8 Hz); 2.91 (dd, 1H, J = 12.4,
16.8 Hz), 3.20–2.80 (m, 1H), 3.51–3.98 (m, 4H); 3.82
(d, 1H, J = 15.0 Hz); 4.74 (d, 1H, J = 12.4 Hz), 5.40
(d, 1H, J = 15.0 Hz), 7.19–7.40 (m, 9H). 13C NMR

F. Benfatti et al. / Bioorg. Med. Chem. 15 (2007) 7380–7390
7389
37.5, 40.1, 40.3, 45.1, 50.6, 51.1, 56.7, 67.3, 118.9, 125.8,
125.9, 129.2, 129.3, 130.1, 130.7, 131.5, 133.0, 137.2,
139.9, 145.5, 156.2, 162.2, 167.3. LC-ESI-MS rt
9.2 min, m/z: 548 (M+1), 571 (M+Na).
nitrophenyl N-acetyl-b-D-glucosaminide dissolved at
7.5 mM in 0.09 M citrate buffer solution, pH 5, and
mixed with an equal volume of 0.5% Triton X-100 in
water) was added. This product is a chromogenic sub-
strate for b-N-acetylglucosaminidase that is transformed
in 4-nitrophenol whose absorbance is measured at
405 nm. As previously described,20 there is a linear cor-
relation between absorbance and enzymatic activity. It
is, therefore, possible to identify the number of adherent
cells in treated wells, interpolating the absorbance values
of the unknowns in a calibration curve.
1
Compound 3d: H NMR (300 MHz, CD3OD): dH 0.85
(t, 3H, J = 7.2 Hz); 1.54–1.63 (m, 2H); 1.70–1.75 (m,
2H); 1.80 (m, 3H), 2.65–2.70 (m, 1H); 2.70–2.78 (m,
1H), 2.85–3.02 (m, 2H), 3.10–3.15 (m, 1H), 3.17 (m,
1H); 3.40 (s, 1H), 3.69 (m, 1H); 4.09 (m, 1H), 5.0 (m,
1H), 7.21–7.30 (m, 2H); 7.31–7.45 (m, 3H). 13C NMR
(75 MHz, CDCl3) dC 12.0, 20.8, 26.3, 26.9, 38.4, 41.3,
47.1, 47.5, 49.3, 49.7, 51.0, 59.0, 127.6, 129.1, 130.3,
130.6, 141.3, 141.8, 148.7, 164.4, 169.8, 172.2, 173.9,
175.5. LC-ESI-MS rt 2.9 min, m/z: 402 (M+1), 424
(M+Na).
The reaction was blocked by adding 100 lL of a stop-
ping solution (50 mM glycine, 5 mM EDTA, pH 10.4)
and the plate was read in a Victor2 Multilabel Counter
(Perkin-Elmer, Waltham, Massachusetts, USA).
3.7. Materials for bioassays
Experiments were carried out in quadruplicate. Data
analysis and IC50 values were calculated using Graph-
Pad Prism 3.0 (GraphPad Software Incorporated, San
Diego, CA, USA).
Trypsin/EDTA, non-essential amino acids, minimum
essential medium (MEM), RPMI-1640 with L-gluta-
mine, antibiotic, and antimycotic solution, and glycine
were purchased from Invitrogen (Carlsbad, CA, USA).
Fetal bovine serum (FBS) and phosphate buffered saline
(PBS) were from Cambrex (Walkersville, MD, USA).
Citrate buffer solution, EDTA, DMSO, Triton-X-100,
4-nitrophenyl N-acetyl-b-D-glucosaminide, phorbol 12-
myristate 13-acetate (PMA), pyruvic acid, fibronectin
from human plasma were obtained from Sigma–Aldrich
SRL (Milan, Italy). SK-MEL-24 (human malignant
melanoma) and K-562 (human erythroleukemia) cell
lines were obtained from American Tissue Culture Col-
lection (ATCC, Rockville, MD, USA).
Acknowledgments
We thank MIUR (PRIN 2006), CNR-ISOF and Uni-
versity of Bologna (Funds for selected topics) for finan-
cial support. Mr. Andrea Garelli is gratefully
acknowledged for the LC-ESI-MS analysis
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1
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