Synthesis, biological evaluation, and docking studies of N-substituted acetamidines as selective inhibitors of inducible nitric oxide synthase

Article abstract of DOI:10.1021/jm800846u

New acetamidines structurally related to N-(3-(aminomethyl)benzyl) acetamidine (1, W1400) were designed as inhibitors of inducible nitric oxide synthase (iNOS). Six compounds were found to be selective for iNOS over endothelial nitric oxide synthase (eNOS), and among them, the most active and selective compound was the N-benzylacetamidine 2. A docking study was also performed to shed light on the effects of the structural modifications on the interaction of the designed inhibitors with the NOS.

Full text of DOI:10.1021/jm800846u

J. Med. Chem. 2009, 52, 1481–1485
1481
Synthesis, Biological Evaluation, and Docking Studies of N-Substituted Acetamidines as
Selective Inhibitors of Inducible Nitric Oxide Synthase
Cristina Maccallini,†,| Antonia Patruno,†,| Neva Besˇker,†,| Jamila Isabella Al`ı,† Alessandra Ammazzalorso,†
Barbara De Filippis,† Sara Franceschelli,‡ Letizia Giampietro,† Mirko Pesce,‡ Marcella Reale,§ Maria L. Tricca,† Nazzareno Re,†
Mario Felaco,‡ and Rosa Amoroso*,†
Dipartimento di Scienze del Farmaco, Dipartimento di Scienze del MoVimento Umano, and Dipartimento di Oncologia e Neuroscienze,
UniVersita` “G. d’Annunzio”, Via dei Vestini, 66100 Chieti, Italy
ReceiVed July 10, 2008
New acetamidines structurally related to N-(3-(aminomethyl)benzyl)acetamidine (1, W1400) were designed
as inhibitors of inducible nitric oxide synthase (iNOS). Six compounds were found to be selective for iNOS
over endothelial nitric oxide synthase (eNOS), and among them, the most active and selective compound
was the N-benzylacetamidine 2. A docking study was also performed to shed light on the effects of the
structural modifications on the interaction of the designed inhibitors with the NOS.
Chart 1. 1 and Designed NOS Inhibitors
Introduction
Nitric oxide (NOa) is an important mediator involved in the
regulation of many physiological and pathological processes,
including neurotransmission and smooth muscle relaxation. The
formation of NO is catalyzed by the enzyme nitric oxide
synthase (NOS) via the NADPH- and O2-dependent oxidation
of L-arginine.1 Three distinct isoforms of NOS have been
identified: the constitutive endothelial (eNOS) and neuronal
(nNOS), predominantly expressed in the vascular endothelium
and in the nervous system, respectively, and the inducible
(iNOS), which generates high levels of NO that modulates
inflammation through multiple pathways and plays an important
role in the regulation of immune reactions.2 Overproduction of
NO by iNOS has been implicated in various pathological
processes, including tissue damage and cell apoptosis following
inflammation and ischemia, rheumatoid arthritis, and onset of
colitis.3 Blocking the localized excess production of NO through
inhibition of iNOS has been identified as a potential means of
treating these diseases.4
Different types of NOS inhibitors have been described in the
past few years, and their effects are caused by competition with
the natural substrate of NOS, L-arginine, in the binding site and/
or in the oxidizing center of the enzyme (heme) or by the
interaction with peptide motifs of the enzyme that influence its
dimerization, affinity for cofactors, and interaction with associ-
ated proteins.5 Typical NOS inhibitors described to date include
guanidines, amidines, isothioureas, and thiazines. Moreover,
heterocyclic compounds (such as indazoles, benzoxazoles, and
imidazoles) that incorporate a guanidine-like group into a cyclic
structure have been reported as NOS inhibitors.6
date, 5000- and 2000-fold more potent against purified human
iNOS than eNOS and nNOS, respectively.7 This compound is
a time-, concentration-, and NADPH-dependent inactivator of
iNOS and appears to be the first example of an enzyme inhibitor
that acts without being modified in any way.8
In this paper, we report the synthesis and in vitro evaluation
(activity and selectivity for iNOS vs eNOS) of novel aceta-
midines 2-11 (Chart 1) that are structurally related to the iNOS
selective inhibitor 1. A docking study was also performed to
shed light on the effects of the structural modifications on the
interaction of the designed inhibitors with the NOS.
N-(3-(Aminomethyl)benzyl)acetamidine (1, W1400) is the
Chemistry
most selective inhibitor of purified human iNOS reported to
Compounds 2-4, 9, and 10 were prepared under mild
conditions by the addition of the appropriate amine to S-2-
naphthylmethyl thioacetimidate hydrobromide9 (see Scheme 1).
Acetamidine 3 was obtained in racemic and chiral forms, while
4 was prepared as its racemate.
* To whom correspondence should be addressed. Phone: +39-0871-
3554686. Fax: +39-0871-3554911. E-mail: ramoroso@unich.it.
†
Dipartimento di Scienze del Farmaco.
These authors contributed equally to this work.
Dipartimento di Scienze del Movimento Umano.
Dipartimento di Oncologia e Neuroscienze.
|
The treatment of BOC-protected m-xylylenediamine10 (12)
with benzyl 4-methylbenzenesulfonate afforded a mixture of
N-benzyl (13) and N-dibenzyl (14) derivatives that were
separated by chromatography. Treatment of 13 and 14 with
CF3COOH followed by NaOH afforded the deprotected inter-
mediates 15 and 16. Reaction of 15 with S-2-naphthylmethyl
‡
§
a Abbreviations: NO, nitric oxide; NOS, nitric oxide synthase; iNOS,
inducible NOS; eNOS, endothelial NOS; NADPH, nicotinamide adenine
dinucleotide phosphate; rmsd, root-mean-square deviation; MCMM, Monte
Carlo multiple minimum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane
sulfonic acid; Ts, tosylate; HPLC, high performance liquid chromatography.
10.1021/jm800846u CCC: $40.75
2009 American Chemical Society
Published on Web 02/09/2009

1482 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Brief Articles
Scheme 1a
a Reagents and conditions: (a) RNH2, absolute EtOH, 0 °C, 1-3 h.
Scheme 2a
Figure 1. Percent of eNOS and iNOS activities compared with those
of untreated samples (C) and 1. iNOS and eNOS activity was detected
in the 11000g supernatant of THP-1 cell homogenates. Cells were
induced to express iNOS with 10 µg/mL of bacterial LPS (Sigma) for
24 h after a 1 h preincubation of the inhibitors (150 µM). The
concentrations of drugs was selected on the basis of the results from
pilot studies using three different concentration of the inhibitor (50,
100, 150 µM) (data not shown).
aqueous solution of NaOH, as outlined in Scheme 4 (see
Supporting Information).
Results and Discussion
To assess the ability of test compounds to inhibit iNOS and
eNOS, a preliminary biological evaluation was performed in
vitro by using a whole-cell assay on THP-1 cells, a human
myelomonocytic leukemia cell line. Figure 1 illustrates the iNOS
and eNOS inhibition in the presence of a 150 µM 2-11; results
are expressed as percentage of NOS activity and compared with
those of 1. Among the amidines 2-4, differing from 1 by the
absence of the 3-aminomethyl group and the addition of a
methyl or an ethyl group on the benzylic carbon connected to
the acetamidine nitrogen (2 and 4, respectively), 2, (S)-3, and
rac-4 showed good iNOS and almost negligible eNOS inhibi-
tion. Amidines 5-8 were synthesized to investigate the effects
of amine substitution at the 3-aminomethyl group. The addition
of one or two benzyl groups to give 5 or 6 did not seem to
influence iNOS activity with respect to 1 but resulted in an
increased eNOS inhibition and thus in a lower selectivity. The
introduction of 4-methylpyridine or tosylate group (Ts) as
N-substituents (7 and 8, respectively) led to inhibition of iNOS
but not of eNOS. Compounds 9 and 10 were designed to develop
a new series of acetamidines in which the aromatic ring of 1
was replaced by a N-substituted piperidine linked to the
acetamidine function in the 4-position. Both molecules showed
good iNOS inhibition, but only 9 seemed to be somewhat
selective. Finally, 11, with a phenyl separated from acetamidinic
group by a sulfone, showed inhibition of iNOS and eNOS. On
the basis of this preliminary study, acetamidines that did not
significantly affect eNOS activity, i.e., 2, (S)-3, rac-4, 7-9, were
selected for determination of IC50 and selectivities (ratio of
eNOS IC50/iNOS IC50), performing an enzymatic activity assay
(Table 1). The IC50 values obtained for 1 are in line with those
found in literature.4 All compounds showed good iNOS IC50
and selectivities ranging from 57 to 1750. Amidines 2, rac-4,
and 9 resulted in more potent iNOS inhibitors than 1, while
(S)-3, 7, and 8 are less potent. These results seem to indicate
that the removal of the aminomethyl group, rather than its
substitution, improves the iNOS inhibition. Concerning the
selectivity, the synthesized compounds can be considered very
promising. With the exception of 7, the above amidines are very
selective for iNOS over eNOS, according to the literature4a
defining as “highly selective” agents showing 50- to 100-fold
a Reagents and conditions: (a) BzOTs, dry CH3CN, N2, reflux, 5 days;
(b) CF3COOH, 0 °C, 1 h; (c) NaOH, room temp; (d) S-2-naphthylmethyl
thioacetimidate hydrobromide or ethylacetamidate hydrochloride, absolute
EtOH, room temp, 7 h.
Scheme 3a
a Reagents and conditions: (a) 4-chloromethylpyridine or TsCl, dry
DMSO, room temp, 24 h; (b) CF3COOH, 0 °C, 1 h; (c) NaOH, room temp;
(d) S-2-naphthylmethyl thioacetimidate hydrobromide or ethylacetamidate
hydrochloride, absolute EtOH, room temp, 24 h.
thioacetimidate hydrobromide or 16 with ethylacetamidate
hydrochloride led to the desired acetamidines 5 and 6 as their
hydrobromide or hydrochloride salts, respectively (Scheme 2).
Condensation of 12 with 4-(chloromethyl)pyridine or p-
toluensulphonyl chloride provided the Boc-protected intermedi-
ates 17 and 18, respectively. Submitting intermediates 17 and
18 to deprotection conditions (CF3COOH followed by NaOH)
afforded amines 19 and 20, which were subsequently condensed
with S-2-naphthylmethyl thioacetimidate hydrobromide or ethy-
lacetamidate hydrochloride, respectively, to provide the desired
7 and 8 (Scheme 3).
Acetamidine 11 was prepared as free base by reaction of
acetamidine hydrochloride with benzenesulfonyl chloride in an

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1483
Table 1. Inhibition of NOS by Selected Acetamidinesa
IC50 (µM)
compd
iNOS
eNOS
selectivity eNOS/iNOS
2
(S)-3
rac-4
7
8
9
1
0.20 ( 0.03
0.45 ( 0.02
0.25 ( 0.02
0.35 ( 0.03
0.5 ( 0.05
0.25 ( 0.03
0.33 ( 0.06
350 ( 70
300 ( 60
80 ( 22
1750
666
320
57
600
360
3300
20 ( 2
300 ( 30
90 ( 6
1100 ( 104
a NOS inhibition at 20 µM arginine. Experiments were independently
performed at least three times.
Figure 3. Top-ranked docked conformation of 4 (a), 7 (b), and 9 (c)
to iNOS.
Figure 2. Schematic drawing of the active site of iNOS. The main
interactions of 1 with residues and cofactors important for binding are
illustrated.
of the benzyl with a piperidyl, 9 and 10, or a benzenesulphonyl
core, 11. The most stable docking conformation of 4 (Figure
3a) shows essentially the same position of the benzylic backbone
as that observed in the X-ray structure of 1, with the phenyl
ring on one of the heme pyrrole rings but differently oriented,
probably because of the lack of the 3-aminomethyl group and
its interaction with the propionate arms of the heme cofactor.
The ethyl group shows favorable hydrophobic contacts with the
Pro344 and Val346 residues. Ligands 5-8, differing from 1
for the presence on the 3-aminomethyl group of aromatic
substituents, show the same interaction of the latter group with
the heme propionate arms and the same orientation of the
benzylic core observed for 1, as exemplified by Figure 3b for
7. The presence of the aromatic substituents, Bz, CH2Pyr, or
Ts, does not lead to significant steric clashes because of the
presence of a sufficiently wide pocket or to significant interac-
tions. However, the pyridine nitrogen in 7 is directed toward
the amine group of Asn348, although the N · · ·N distance of
2.30 Å indicates a weak electrostatic interaction rather than a
hydrogen bond. In fact, 7 is the most potent inhibitor among
5-8. In ligands 9-10 the benzyl core of 1 is replaced by a
piperidyl moiety, which assumes a similar position and is
oriented in such a way to find favorable hydrophobic contacts
with the Pro344 and Val346 residues; the piperidyl nitrogen
does not show any interaction (Figure 3c for 9). From the
docking calculations, a Gscore value for each inhibitor was
calculated and compared with the experimental inhibitory
activities, IC50, measured for 2-4 and 5-7. The experimental
pIC50 values were plotted against the calculated docking scores
and show a poor correlation (R2 ) 0.36). As the considered
inhibitors show polar atoms or charged groups that could give
rise to binding conformations significantly higher in energy than
the lowest conformations in their unbound state, we have
corrected the calculated docking scores for the conformational
energies required for the ligands to adopt their binding
conformations. For each ligand the conformational space was
searched by using the Monte Carlo multiple minimum (MCMM)
method in aqueous solution. The conformational energy penalty,
∆Econf, for each ligand was calculated by subtracting the internal
selectivity. One of the considered compound, 2, displayed a
high iNOS to eNOS selectivity (1750), though lower than the
3300-fold selectivity observed with 1.
Molecular Docking. Docking simulations were performed
to predict the binding mode of all the designed inhibitors in the
human iNOS and eNOS active sites and to evaluate their binding
affinities and isoform selectivity.
To evaluate the effectiveness of the Glide program in the
docking of the considered iNOS ligands, we first docked 1 to
its natural iNOS substrate and compared the resulting geometry
with the corresponding crystal structure. The Glide predicted
geometry is shown in Figure 2 (see Supporting Information)
superimposed with the X-ray structure of the 1-iNOS complex.
The root-mean-square deviation (rmsd) of these two conforma-
tions is 0.341 Å, indicating a good agreement with the crystal
structure.7b The schematic drawing in Figure 2 (above) shows
the main features reproduced by the docking procedure that have
been considered responsible for the high potency of this
inhibitor. In particular, we notice (i) the hydrogen bond
interactions between the amidinic group and the carboxylate
oxygen atoms of the catalytic Glu371 and the Trp366 residues,
(ii) the interaction of the 3-aminomethyl group with both
propionate arms of the heme cofactor, (iii) the position of the
benzene ring atop one of the heme pyrrole rings. All the docked
structures of 2-11 to iNOS share a strong interaction between
the common amidine group and the carboxylate moiety of the
Glu371 and Trp366 residues, and 2-8 also show the same
orientation of the benzylic backbone and 5-8 the interaction
of the 3-aminomethyl group with the heme propionate arms.
Figure 3 displays the results of the docking of 4, 7, and 9. A
comparison of the binding geometries of these compounds with
that of 1 allows us to shed light on the main effects of the
considered structural modifications on its interaction with the
iNOS isoform, i.e., the elimination of the aminomethyl group,
2-4, the addition of a bulky benzyl or tosyl substituent on the
nitrogen of the aminomethyl group, 5-8, and the replacement

1484 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Brief Articles
energy of the lowest conformation in aqueous solution, exclud-
ing the solvation energy, from the energy of the protein bound
conformation after constrained structure optimization.
Although based on a reduced set of seven experimental data,
the above multiple linear regressions are promising, and our
docking protocol can be employed not only for a rough
prediction of binding geometries but also as an exploratory guide
for a qualitative estimation of iNOS binding affinities and iNOS/
eNOS selectivities of this class of N-substituted acetamidine
inhibitors.
The higher inhibitory potency toward iNOS of 2-4 compared
with that of the lead 1 indicates that the removal of the
3-aminomethyl group improves the iNOS inhibition, thus
suggesting that the interaction of the latter group with the
propionate arms of the heme cofactors does not contribute
significantly to the binding energy of this class of aminidine
inhibitors. On the other hand, the slightly lower selectivity over
eNOS of 2-4 indicates the importance of this group in
determining the high isoform specificity of 1, as suggested by
the comparison of the X-ray structures with iNOS and eNOS,
which points out the different position of the heme group in
these two isoforms leading to different interactions of its
propionate arms with the 3-aminomethyl group.11
A multiple linear regression was carried out based on eq 1:
pIC50 ) R + ꢀGscore + γ∆Econf
(1)
This two-parameter equation showed a reasonably good cor-
relation (Figure 5 in Supporting Information) with promising
statistical characteristics [n ) 7 samples, correlation coefficient
R2 ) 0.831, standard error σ ) 0.105, F-test statistic F ) 4.45,
confidence level of 90%] and the best coefficients values R )
7.791, ꢀ ) 0.200, and γ ) 0.147.
The simulated geometry of 1 docked to the eNOS substrate
is in good agreement with the crystal structure,11 with a rmsd
of 0.242 Å (excluding the aminomethyl group atoms, affected
by disorder), and shows essentially the same interactions
discussed for the structure of the complex between 1 and iNOS.
The docked structure explicitly shows the interaction of the
3-aminomethyl group with both propionate arms of the heme
cofactor which cannot be unambiguously observed in the X-ray
structure, since there is no clearly defined electron density
corresponding to the 3-aminomethyl group.11 However, this
result supports the assertion in ref 11 that “some residual electron
density indicates that the amino nitrogen might interact with
the propionate group from one pyrrole ring”. The docked
structures of 2-11 to eNOS also share a strong interaction
between the common amidinic group and the carboxylate moiety
of the Glu363 and Trp358 residues and, whenever possible, the
interaction of the 3-aminomethyl group with propionate arms
above interaction. It is worth noting that 7 shows an hydrogen
bond interaction between the pyridine nitrogen and the amine
group of Asn340, with a N · · ·N distance of 2.00 Å, stronger
than the more labile electrostatic interaction observed with
iNOS. This probably is why 7 is by far the least selective among
the considered inhibitors, showing the lowest selectivity (57)
and the highest eNOS IC50 (20 µM).
Conclusions
In this paper, we report the synthesis of novel acetamidines
structurally related to 1 and the in vitro evaluation of their
activities and selectivities toward iNOS. Among the synthesized
compounds, 2, rac-4, and 9 exhibited inhibitory potency higher
than the lead 1, maintaining a high selectivity over eNOS. In
particular the best inhibition potency (0.20 µM) and selectivity
(1750) were achieved with 1-(benzylamino)ethaniminium bro-
mide (2). A docking study was also performed to shed light on
the effects of the structural modifications on the interaction of
the designed inhibitors with the NOS. The proposed docking
protocol is promising and can be employed for a rough
prediction of binding geometries and as an exploratory guide
for a qualitative estimation of iNOS binding affinities and iNOS/
eNOS selectivities of this class of N-substituted acetamidine
inhibitors.
The plot of the calculated docking scores against the
experimental eNOS inhibitory activities shows a reasonable
correlation between the calculated and experimental values (R2
) 0.54) which, however, could be significantly improved (R2
) 0.83) through the inclusion of the conformational energies
through the same multiple linear approach employed for the
docking to iNOS. The statistic for this two-parameter correlation
on eNOS inhibition is slightly better than that for iNOS
[correlation coefficient R2 ) 0.843, standard error σ ) 0.275,
F-test statistic F ) 10.8, confidence level of 98%] although
the correlation is mainly determined by the ∆Econf contribution.
A comparative analysis of the calculated docking scores for
the considered inhibitors of the two NOS isoforms shows better
docking scores for the iNOS isoform, consistent with the
observed selectivity. An analysis of the various energy contribu-
tions to the docking score of each ligand to the eNOS isoform
shows the presence of bad contacts between the methyl on the
amidine group and the Pro336 residue, which could be
responsible for lower scores and therefore for the observed
selectivity. Prompted by this result, we performed a quantitative
analysis of the logarithm of the IC50 ratio of the two isoforms,
the selectivity, i.e., -log(IC50iNOS/IC50eNOS). A reasonably good
correlation was found for the calculated selectivity logarithm,
Experimental Section
General Procedure for the Synthesis of 2-4, 9, and 10. To a
stirred, cooled (0 °C) solution of the appropriate amine (7.1 mmol)
in EtOH (25 mL) four equal portions of S-2-naphthylmethyl
thioacetamidate hydrobromide (7.1 mmol), prepared according to
literature procedure,9 were added. Reactions were allowed to warm
to room temperature to facilitate complete conversion. The mixture
was filtered. The filtrate was concentrated and partitioned between
H2O (15 mL) and Et2O or CH2Cl2 (20 mL). The aqueous layer
was separated, washed with Et2O (2 × 20 mL), and lyophilized to
afford the desired acetamidine hydrobromide.
General Procedure for the Synthesis of 5 and 6. To a stirred
solution of tert-butyl 3-(aminomethyl)benzylcarbamate10 12 (7.9
mmol) in CH3CN dry (20 mL) a solution of benzyl 4-methylben-
zenesulfonate in CH3CN dry (20 mL) was added, under N2, over a
period of 45 min. The solution was stirred at 35 °C for 5 days and
then concentrated. Purification of the residue by column chroma-
tography (cyclohexane/EtOAc 5:5) furnished 13 and 14. Each
compound was solubilized in CH2Cl2 (5 mL) and treated with
CF3COOH (2.5 mL) at 0 °C for 2 h. The mixture was concentrated,
and the residue, solubilized in a mixture of H2O and EtOH (1:1, 1.5
mL), was treated with 2 N NaOH. After evaporation of the solvent,
15 or 16 was obtained. Finally, ethylacetimidate hydrochloride (7.3
mmol) in EtOH (5 mL) was added to a solution of 15 or 16 (8.8 mmol)
in EtOH (2 mL) at room temperature. After 7 h the mixture was
concentrated and the residue suspended in H2O (15 mL) and washed
with Et2O (3 × 15 mL) and AcOEt (2 × 15 mL). The aqueous layer
was lyophilized to give the product 5 or 6.
estimated as differences of the docking scores ∆Gscore
Gscore
)
iNOS
- GscoreeNOS, as a function of the experimental selec-
tivity logarithm or ∆pIC50 ) -log(IC50iNOS/IC50eNOS) with a
correlation coefficient R2 ) 0.852 (Figure 6 in Supporting
Information).

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1485
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General Procedure for the Synthesis of 7 and 8. A solution
of 12 (6.5 mmol) in DMSO dry (3 mL) was added to 4-(chlorom-
ethyl)pyridine or p-toluensulphonyl chloride (4.5 mmol) in DMSO
dry (7 mL) at 0 °C under N2 over 40 min. The mixture was heated
at room temperature and stirred for 24 h. The solvent was removed
and the residue was dissolved in H2O (15 mL) and extracted with
AcOEt (3 × 15 mL). The combined organic extracts were
concentrated to give a residue that was purified by silica gel
chromatography (CH2Cl2/MeOH 9:1) or by crystallization (toluene/
petroleum ether) to afford 17 or 18. Each compound was solubilized
in CH2Cl2 (5 mL) and treated with CF3COOH (2.5 mL) at 0 °C for
2 h. The mixture was concentrated, and the residue, solubilized in
a mixture of H2O and EtOH (1:1, 1.5 mL), was treated with 2 N
NaOH. After evaporation of the solvent, 19 or 20 was obtained.
Finally, S-2-naphtylmethyl thioacetamidate hydrobromide or ethy-
lacetamidate hydrochloride (4 mmol), respectively, was added to a
stirred solution of 19 or 20 (4 mmol) in EtOH (30 mL) at 0 °C
under N2. After 72 h at room temperature, the solvent was removed
and the residue was partitioned between H2O (10 mL) and Et2O
(15 mL). The aqueous phase was washed with AcOEt (3 × 10
mL) and lyophilized to give the desired product 7 or 8.
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Synthesis of 11. To a stirred solution of acetamidine hydrochlo-
ride (10 mmol) and benzenesulfonyl chloride (10 mmol) in CH2Cl2
(20 mL), an aqueous solution of NaOH (20 mmol, 50% w/w) was
added dropwise. After 24 h at room temperature, the mixture was
filtered and H2O (10 mL) was added. The aqueous layer was sep-
arated and lyophilized to give a solid residue that was purified on
semipreparative HPLC (H2O/MeOH 95:5, 1.5 mL/min) to give the
amidine 11.
Acknowledgment. The Italian Ministry for the University
and the Research is acknowledged for financial support (Con-
tracts 2006038520 and 2005033023).
Supporting Information Available: General information on
instrumentation; spectral data and elemental analysis results of 2,
(S)-3, rac-4, 7-9; experimental procedures for biology and model-
ing; Scheme 4, Figures 2, 5 and 6. This material is available free
of charge via the Internet at http://pubs.acs.org.
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