Solid-phase synthesis and insights into structure-activity relationships of safinamide analogues as potent and selective inhibitors of type B monoamine oxidase

Article abstract of DOI:10.1021/jm070725e

Safinamide, (S)-N²-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than safinamide (IC ₅₀ = 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI = 3455 and 1967, respectively, vs 5918). The highest MAO-B inhibitory potency (IC₅₀ = 17 nM) and a good selectivity (SI = 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of safinamide. Structure-affinity relationships and docking simulations pointed out strong negative steric effects of α-aminoamide side chains and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents. The significantly diverse MAO-B affinities of a number of R and S α-aminoamide enantiomers, including the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic binding sites.

Full text of DOI:10.1021/jm070725e

J. Med. Chem. 2007, 50, 4909-4916
4909
Solid-Phase Synthesis and Insights into Structure-Activity Relationships of Safinamide
Analogues as Potent and Selective Inhibitors of Type B Monoamine Oxidase
Francesco Leonetti,§ Carmelida Capaldi,§ Leonardo Pisani,§ Orazio Nicolotti,§ Giovanni Muncipinto,§ Angela Stefanachi,§
Saverio Cellamare,§ Carla Caccia,# and Angelo Carotti*,§
Dipartimento Farmaco-Chimico, UniVersity of Bari, Via Orabona 4, I-70125 Bari, Italy, and Newron Pharmaceuticals,
Via L. Ariosto 21, Bresso, Italy
ReceiVed June 21, 2007
Safinamide, (S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III
clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an
expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine
oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-
chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than safinamide
(IC50 ) 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI ) 3455 and 1967,
respectively, vs 5918). The highest MAO-B inhibitory potency (IC50 ) 17 nM) and a good selectivity (SI
) 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of safinamide. Structure-affinity
relationships and docking simulations pointed out strong negative steric effects of R-aminoamide side chains
and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents.
The significantly diverse MAO-B affinities of a number of R and S R-aminoamide enantiomers, including
the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic
binding sites.
Chart 1. Chemical Structures of Selective MAO-A (A) and
MAO-B (B) Inhibitor Drugs and of Safinamide
(Methanesulfonate) 7
Introduction
Monoamino oxidases (MAOsa) are flavin adenine dinucle-
otide (FAD) containing enzymes localized in the outer mito-
chondrial membrane.1 MAOs are involved in the oxidative
deamination of important endogenous amines, including monoam-
ine neurotransmitters serotonin (5-HT), norepinephrine (NE),
and dopamine (DA), as well as exogenous amines, comprising
the hypertensive dietary amine tyramine.2 Two different isoen-
zymatic forms have been identified, namely, MAO-A and MAO-
B,3 which differ in their amino acid sequences, three-dimen-
sional structures,4,5 and substrate specificity and sensitivity to
inhibitors.6 Selective MAO-B inhibitors (i.e., selegiline and
rasagiline, Chart 1)7 are used, alone or in combination with
L-DOPA, in the symptomatic treatment of Parkinson’s disease,
whereas selective MAO-A inhibitors (i.e., moclobemide, Chart
1) are employed as antidepressants.8-10 The important side
effects associated with the use of selegiline and to a lesser extent
rasagiline, likely due to their irreversible mechanism of inhibi-
tion, and the growing interest in MAO-B inhibitors as potential
anti-Alzheimer’s agents11 called for new studies aimed at the
discovery of novel potent, selective, and, most importantly, truly
reversible MAO-B inhibitors.
modulation, and reduction of glutamate release in the central
nervous system.16 The synthesis of safinamide has been carried
out at Pharmacia & Upjohn several years ago by a conventional
synthetic pathway from (S)-alaninamide.12
As part of our collaboration with Newron Pharmaceuticals
and in continuation of our long-dated research in the field of
MAO inhibition,17-24 we deemed it important to develop an
alternative synthetic protocol more suited for the preparation
of focused libraries of safinamide congeners and aimed at an
in-depth exploration of structure-affinity (SAFIR) and structure-
selectivity (SSR) relationships and a further optimization of the
pharmacokinetic and pharmacological properties of safinamide.
Multiple parallel syntheses, either in the solid phase or in
solution, are currently used by medicinal chemists to prepare
large libraries of compounds addressing specific pharmacologi-
cal targets or to discover and optimize new leads.25,26 Two
different solid-phase-based approaches are actually employed:
the conventional solid-phase organic synthesis (SPOS) on
polymeric support27 and the synthesis driven by polymer-
supported solid reagents.28 For the preparation of safinamide
and related analogues, we chose the conventional solid-phase
Along this line of research, Newron Pharmaceuticals recently
developed safinamide ((S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}-
alaninamide methanesulfonate) (Chart 1), initially reported by
Pharmacia & Upjohn as a potent anticonvulsant,12 which has
just entered phase III clinical trials in Europe as an anti-
Parkinson drug.13-15 Indeed, safinamide acts by means of
multiple mechanisms of action that comprise MAO-B and
dopamine uptake inhibition, sodium and calcium channel
* To whom correspondence should be addressed. Phone: +39 080
5442782. Fax +39 080 5442230. E-mail: carotti@farmchim.uniba.it.
§ University of Bari.
# Newron Pharmaceuticals.
a Abbreviations: MAO, monoamine oxidase; SPOS, solid-phase organic
synthesis; PEA, phenethylamine; PDB, Protein Data Bank.
10.1021/jm070725e CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/07/2007

4910 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Chart 2. Retrosynthetic Analysis of Safinamide 7a
Leonetti et al.
In our investigation the standard protocols normally used in
SPOS to accomplish a reductive alkylation yielded unsatisfactory
results. The use of benzaldehyde protected as the triisopropyl-
silyloxy derivative (TIPS) and Ti(O-iPr)4 as a Lewis acid to
enhance the electrophilic character of the aldehydic carbonyl
resulted in an almost quantitative yield of the aldimine
intermediate. The subsequent reduction reaction was efficiently
performed with a presonicated suspension of NaBH(OAc)3 in
DCM.29 The H NMR analysis of the product obtained after
1
cleavage showed only traces of the starting amino acid, which
disappeared by adding 1 equiv of triethylamine as a proton
sponge during the formation of the aldimine intermediate. In
the above-described experimental conditions the alkylation
reaction reached completion with a very high yield.
The last step of our synthetic scheme was the insertion of
building block C that might have been accomplished through
conventional benzylic nucleophilic substitutions (NSs) or more
efficiently via a Mitsunobu reaction.30 The latter was preferred
because conventional NSs would have required preventive
protection of the secondary amine present on the support-bound
growing molecule. The Mitsunobu reaction was efficiently
carried out using PBu3 and ADDP. Finally, the cleavage from
the resin using the TFA/H2O/TES mixture afforded the target
molecule in very satisfactory overall yield (nearly 60%) without
any detectable trace of the starting and intermediate materials,
a LG: leaving group.
Scheme 1. Solid-Phase Synthesis of Safinamide and Analogues
(Compounds 1-20 in Table 1)a
1
as checked by H NMR and HPLC analyses.
Starting from these very encouraging results we designed a
small exploratory library of safinamide analogues (Table 1) to
develop insightful SAFIRs and SSRs and to challenge the
efficiency and versatility of our synthetic protocol. At this
preliminary stage, the structural variations were limited to
building blocks A and C (R and R1 substituents in Scheme 1).
a Reagents and conditions: (a) HOBt, DICI, DMF, room temp, 3 h; (b)
20% piperidine in DMF, room temp, 30 min; (c) Ti(O-iPr)4, TEA, THF,
4-[(triisopropylsilyl)oxy]benzaldehyde, room temp, overnight; (d) NaB-
H(OCOCH3)3, DMC, room temp, overnight; (e) TBAF, THF, room temp,
1 h; (f) PBu3, ADDP, THF, R1-benzyl alcohol, room temp, overnight; (g)
TFA/H2O/TES, room temp, 1 h.
N-Fmoc-glycine, -(S)-alanine, -(S)-phenylglycine, and -(S)-
serine were chosen as starting amino acids, and benzyl alcohol
and its 3-F, 3-Cl, 4-Cl, and 4-NO2 derivatives were chosen as
structural variants in the Mitsunobu reaction. To our satisfaction,
all 20 library compounds were obtained in overall yields and
purities comparable to those observed in the synthesis of the
lead compound safinamide.
Biological Assay. Rat brain mitochondria were used as the
source for the two MAO isoforms. MAO enzymatic activities
were assessed with a radioenzymatic assay using 14C-serotonin
(5-HT) and 14C-phenylethylamine (PEA) as selective substrates
for MAO-A and MAO-B, respectively, according to a well-
consolidated procedure.16,31 Inhibition data of both MAO
isoforms were given as IC50 (µM) or as the percentage of
inhibition at the indicated concentration for low-active inhibitors.
Selectivity was expressed as selectivity index (SI), which is the
ratio of the IC50 of MAO-A to the IC50 of MAO-B. For
compounds with sufficiently high aqueous solubility and high
MAO-B affinity (i.e., (S)-6 to (S)-8 and (S)-11 to (S)-13), the
IC50 at MAO-A was measured even in the case of low-active
compounds to determine the SI. Chemical structures, MAO
inhibition, and selectivity data of the 20 library members are
reported in Table 1.
approach because it was considered to lead to cleaner reactions,
easier workup, possible automation, and scalability.
Results and Discussion
Chemistry. The straightforward retrosynthetic analysis of
safinamide (Chart 2) revealed three different building blocks:
(S)-alaninamide A, benzaldehyde B and benzyl derivative C
bearing a suitable leaving group (LG). These building blocks
may be easily assembled, and more importantly, the vast
commercial availability and/or simple synthetic accessibility of
many of their analogues may enable the preparation of libraries
endowed with large molecular diversity.
The synthetic protocol developed for the preparation of our
exploratory library is outlined in Scheme 1. The choice of the
initial building block to be anchored to an appropriate polymeric
support was based on the observation that our target molecule
is a primary amide that could be easily formed from the cleavage
of a carboxylic group attached to a Rink amide resin. As the
first step of our synthesis, we therefore loaded the Fmoc-(S)-
alanine on such a resin through a conventional coupling method
with HOBt and DICI in DMF. The most complex step of our
synthetic pathway was the introduction of building block B on
the support-bound amino acid. Various organic reactions could
be taken into account to link building blocks B and A. We
preferred reductive alkylation over nucleophilic substitution with
benzyl halides or other benzyl derivatives carrying suitable LGs,
since the latter might have led to a bis-alkylation of the primary
amine, a highly probable side reaction in SPOS where a large
excess of alkylating reagent is normally employed to drive the
reaction to completion.
Several interesting insights emerged from the analysis of the
SAFIRs and SSRs. All the synthesized compounds bound
MAO-B isoform with higher affinity than MAO-A, leading to
SIs ranging from 2.3 to 5918. Also, for the low-active MAO-A
inhibitors, whose IC50 was not measured, comparison of the
percentages of inhibition of the two isoforms was indicative of
SI values greater than 1. The highest MAO-B selectivities were
shown by the meta-halogen-substituted derivatives (S)-7 and
(S)-8 and by (S)-12 and (S)-13 belonging to the alanine and

Safinamide Analogues as Inibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4911
Table 1. Inhibition Dataa of 2[(4-Benzyloxy)benzylamino]alkanamides
Scheme 2. Synthesis of (S)- and (R)-Enantiomers of
Tetrahydroisoquinoline Derivative 21a
compd
R
R1
MAO-A
MAO-B
SIc
2.7
6.1
109
1.7
1b
2
3
4
H
H
H
H
H
H
8.5
13.6
37
12.4
21.2
101
580
42
114
72
37%
22%
90
100
84.6
100
47%
38%
35%
34
33%
27%
16%
3.15
2.24
0.34
7.10
9.10
0.26
0.098
0.45
0.033
0.80
2.20
37%
0.41
0.14
0.043
0.11
1.10
7.01
73.8
10.0
10.1
34%
4%
3-F
3-Cl
4-Cl
4-NO2
H
3-F
3-F
3-Cl
3-Cl
4-Cl
4-NO2
H
5
2.3
(S)-6b
(S)-7
(R)-7b
(S)-8
(R)-8
(S)-9
(S)-10
(S)-11b
(S)-12
(S)-13
(R)-13
(S)-14
(S)-15
(S)-16
(S)-17
(S)-18
(S)-19
(S)-20
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
C6H5
C6H5
C6H5
C6H5
C6H5
388
5918
93
3455
90
ndd
ndd
220
714
1967
909
ndd
ndd
ndd
3.4
a Reagents and conditions: (a) K2CO3, KI, acetone, reflux, overnight;
(b) DEAD, PS-PPh3, THF, phthalimide, room temp, overnight; (c)
NH2NH2, H2O, absolute EtOH, reflux, 5 h; (d) 40% CH2O, room temp, 2
h; (e) 23% HCl, room temp, 5 h; (f) (S)- or (R)-2-amino-1-methyl-2-
oxoethyl-2-nitrobenzenesulfonate, DIPEA, DCM, room temp, overnight.
3-F
3-Cl
3-Cl
4-Cl
4-NO2
H
(S)-phenylglycine derivatives 16-20 compared to the corre-
sponding congeners of the other three series, indicating that the
amino acid side chain R strongly influenced MAO-B affinity.
In fact, while comparable MAO-B inhibitory potencies were
observed for the alanine and serine derivatives, significantly
lower potency and selectivity emerged from the glycine series.
These results suggested that favorable interactions took place
at the R region irrespective of the different hydrophobic,
electronic, and hydrogen-bonding properties of the methyl and
hydroxymethyl groups.
3-F
3-Cl
4-Cl
4-NO2
ndd
ndd
ndd
To further expand the SAFIR and SSR, the (R)-enantiomers
of three very potent and selective inhibitors (i.e., (S)-7, (S)-8,
and (S)-13) were prepared along with the two enantiomers of
the rigid tetrahydroisoquinoline analogue 21 (Scheme 2).
Structures, inhibition, and selectivity data of these additional
MAO inhibitors are reported in Table 1.
The (R)-enantiomers of serinamide derivative 13 and safi-
namide 7 showed both lower MAO-B affinity and selectivity
than the corresponding (S)-enantiomers: IC50 ) 0.11 µM and
SI ) 909 for (R)-13 compared to 0.043 µM and 1967,
respectively, for (S)-13; IC50 ) 0.45 µM and SI ) 93 for (R)-7
compared to 0.098 µM and 5918, respectively, for (S)-7
(safinamide). Unexpectedly, the corresponding enantiomers of
the safinamide tetrahydroisoquinoline rigid analogue 21 gave
opposite inhibition results, the (S)-enantiomer being less active
and selective than the (R)-enantiomer: (S)-21, IC50 ) 0.24 µM
and SI ) 187.5 compared to 0.017 µM and 2941, respectively,
for (R)-21.
Docking Studies. To assist in the interpretation of SAFIRs
and increase our understanding of the main binding interactions
at MAO active sites, docking studies were performed on
glicinamide derivative 2, (S)-inhibitors 6, 8, 12, 17, and 20, and
both the racemic forms of safinamide 7, serinamide derivative
13 and tetrahydroisoquinoline derivative 21 (Table 1). On the
basis of a recent work conducted in our group,23 the GOLD 3.1
program was chosen for docking simulations on the X-ray
structure of rat MAO-A (rMAO-A, PDB code 1O5W) and on
the homology model of rat MAO-B (rMAO-B) developed from
a human MAO-B (hMAO-B) crystallographic structure (1OJC
code in the PDB)4 as already described.23 Four structural water
molecules, located in proximity to the FAD cofactor and labeled
tetrahydroisoquinoline
rigid analogues of 7
MAO-A
MAO-B
SIc
(S)-21
(R)-21
45
50
0.24
0.017
187.5
2941
a Inhibition data are expressed as IC50 (µM) or as % of inhibition at 10
µM, rMAO-B or 100 µM, rMAO-A; the SEM was always less than (10%.
b Described in ref 12. c SI ) selectivity index, that is, IC50(MAO-A)/
IC50(MAO-B). d nd: not determined.
serine series, respectively. Indeed, in all four series, m-
halogenobenzyloxy derivatives showed SIs greater than the
corresponding unsubstituted lead compounds. The highest SIs
were observed for the 3-F- and 3-Cl-benzyloxyalaninamides
(S)-7 and (S)-8 (SI ) 5918 and 3455, respectively) and for the
3-F- and 3-Cl-benzyloxyserinamides (S)-12 and (S)-13 (SI )
714 and 1967, respectively). Noticeably, the introduction of
halogen substituents in the meta position resulted in a significant
increase of MAO-B affinity in all four amino acid series, leading
in the alanine and serine series to the most potent MAO-B
inhibitors of the entire series examined (i.e., the 3-chloroben-
zyloxy derivatives (S)-8 and (S)-13 showing an IC50 of 33 and
43 nM, respectively). Conversely, a strong drop of MAO-B
affinity resulted from the introduction of a chloro and, more
so, of a nitro group at the para position of the benzyloxy
fragment (compare affinity of 4 and 5 vs 1; (S)-9 and (S)-10 vs
(S)-6; (S)-14 and (S)-15 vs (S)-11; (S)-20 and (S)-19 vs (S)-
16). The notably diverse electronic and hydrophobic properties
of these two para substituents may suggest a negative steric
effect as the most likely cause of the observed decrease in
affinity. An even stronger steric effect might be advocated to
explain the dramatic fall of MAO-B affinity observed for the

4912 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Leonetti et al.
Figure 2. Overlaid docking poses of inhibitors 2, (S)-12, (R)-21, and
(S)-21 into rMAO-B, rendered as colored wire models, show a binding
conformation similar to that observed with safinamide (green stick
model).
Figure 1. Docking pose of safinamide (green stick model) into rMAO-
B. Amino acid residues of the binding site and FAD cofactor are
rendered as stick models with carbon atoms in cyan and yellow,
respectively, while the protein backbone is displayed as a cyan ribbon.
Four structural water molecules are represented as red balls. Black
dashed lines are drawn among atoms involved in HB interactions.
search of ligand-target interactions within the enzyme binding
site (see Experimental Section).
as WAT23, WAT82, WAT102, and WAT160 according to the
numbering reported for the hMAO-B crystallographic structure,4
were included in the model. The interested reader is referred to
our original paper23 for a more detailed description of the
computational procedures adopted for docking simulations and
water molecule location and elsewhere32 for an in-depth
discussion of principles and methods implemented in GOLD
for energy calculations, conformational search, and clustering.
A preliminary docking investigation aimed at understanding
how the lead compound safinamide interacted at the rMAO-A
and -B active sites revealed a similar binding mode but with a
different docking score (i.e., much higher for rMAO-B), in
conformity with the remarkable MAO-B selectivity observed
for safinamide (SI ) 5918). The availability of a larger number
of IC50 values for MAO-B inhibitors prompted us to study their
binding at the rMAO-B active site in greater detail. Docking
results showed that close binding modes were largely adopted
by safinamide at rMAO-B. In particular, it was observed that
the terminal amide group, located in front of the isoalloxazine
ring of FAD, acted as a molecular harpoon establishing a
network of hydrogen bonds with structural WAT-160 and both
the carbonyl oxygen atoms of L171 and C172 (Figure 1).
The benzyloxy molecular tail was embedded into a hydro-
phobic pocket delimited by F103, P104, W119, I199, V316 (I
in hMAOB), I164 (L in hMAOB), and Y326, with its m-fluoro
substituent pointing toward I164. A hierarchical cluster analysis,
carried out on the entire family of conformers generated during
the free docking run, revealed that all the docking poses could
be collected in one prevalent geometric group within an rms
value of 3.27 Å, the fitness score for the more stable docking
pose being equal to 64.90 kJ/mol. Starting with the results of
the above simulations, a number of additional docking runs were
executed to evaluate the impact of the different R1 and R
substituents on the inhibitory potency. The top docking pose
of safinamide (Figure 1) was taken as a reference to guide the
binding modes of all of the other safinamide analogues. To this
end, some physical constraints were set to improve the local
By use of this approach, the best docking poses for safinamide
derivatives were chosen not only on the basis of score energy
but also on the minimum rms value resulting from their
superposition onto the safinamide top-scored molecular pose.
Considering all the docking poses having the least rms deviation
in the safinamide binding model (Figure 2), the different R side
chains of the 3-F-benzyloxy derivatives yielded the following
score ranking that was consistent with the inhibitory data
reported in Table 1: (S)-7 (R ) CH3, IC50 ) 0.098 µM, score
) 64.99 kJ/mol, rms ) 0.33 Å) > (S)-12 (R ) CH2OH, IC50
) 0.14 µM, score ) 61.01 kJ/mol, rms ) 0.36 Å) > 2 (R ) H,
IC50 ) 3.30 µM, score ) 58.01 kJ/mol, rms ) 1.56 Å) > (S)-
17 (R ) Ph, IC50 ) 10.0 µM, score ) 54.87 kJ/mol, rms )
0.42 Å).
Figure 3 illustrates that the very low affinity of phenylglycine
inhibitor (S)-17 might be derived from unfavorable steric
interactions of its phenyl ring with Y60 and a carbonyl group
of FAD. The large increase of MAO-B affinity arising from
the introduction of a halogen atom, especially chloro, in the
meta position in the benzyloxy group was well accounted for
by the docking results. Taking into account the (S)-alanine
inhibitors, docking analyses awarded m-halogen derivatives with
about 10 kJ/mol, as shown by the following data: (S)-8 (R1 )
3-Cl, IC50 ) 0.033 µM, score ) 65.10 kJ/mol, rms ) 0.80 Å)
g (S)-7 (R1 ) 3-F, IC50 ) 0.098 µM, score ) 64.99 kJ/mol,
rms ) 0.33 Å) . (S)-6 (R1 ) H, IC50 ) 0.26 µM, score )
56.14 kJ/mol, rms ) 0.76 Å), whereas the m-chloro derivatives
showed both higher potencies and docking scores (data not
shown). Similar considerations hold within the series of seri-
namide inhibitors (data not shown).
Irrespective of the type of R substituents, a detrimental effect
on inhibition was observed when a para substituent was
introduced into the benzyloxy group. This unfavorable effect
was more pronounced for the larger sized nitro group compared
to the chloro substituent. These findings were supported by the
docking simulations of the p-nitro derivatives that afforded lower
scores compared to the corresponding unsubstituted lead

Safinamide Analogues as Inibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4913
Figure 3. Docking pose of inhibitor (S)-17 (displayed as a green stick
model) into rMAO-B. Black dashed lines indicate unfavorable steric
interactions among ligand phenyl side chain with FAD carbonyl oxygen
and side chain of Y60.
Figure 5. Overlapped docking pose of the tetrahydroisoquinoline
enantiomers (S)-21 and (R)-21. Energetically different hydrophobic
interactions are engaged by the two methyl groups, represented in
magenta and gray for the (R)- and (S)-enantiomers, respectively, with
the surrounding hydrophobic side chains of Y398, Y60, and Y 435.
quinoline enantiomers 21 compared to the corresponding
safinamide enantiomers. The close binding modes derived from
docking simulations of the two enantiomers of 21 (Figure 5)
were characterized by significantly different score values (i.e.,
59.14 and 52.59 kJ/mol for (R)- and (S)-enantiomers, respec-
tively), which are consistent with the inhibition data. The
different binding energies primarily originated from a more
favorable hydrophobic interaction engaged by the methyl group
of the (R)-21 enantiomer (shown in magenta in Figure 5)
compared to the methyl group of the (S)-21 enantiomer (shown
in gray in Figure 5) with the surrounding hydrophobic side
chains of Y398, Y60, and Y435. The methyl group of safina-
mide (see Figure 1) and that of its (R)-enantiomer were slightly
displaced compared to the corresponding methyl groups of 21,
and as a consequence, they experienced similar contacts with
the tyrosine side chains just mentioned.
Figure 4. Docking pose of inhibitor (S)-20 (displayed as a green stick
model) into rMAO-B. Black dashed lines indicate unfavorable steric
interactions among the ligand nitro group with side chains of P104,
F103, and W119.
Conclusions
Herein, we reported the SPOS of safinamide and of a small
library of its analogues which were evaluated as MAO inhibitors.
The proposed solid-phase synthetic protocol enabled the facile,
expeditious, and high-yield preparation of a series of potent,
reversible, and selective MAO B inhibitors and paved the way
for the synthesis of larger libraries endowed with higher
structural diversity for an in-depth exploration of SAFIRs and
SSRs. Unlike the coumarin series, safinamide analogues pre-
sented excellent pharmacokinetic properties, such as higher
water solubility and bioavailability, that may anticipate, as it
was indeed found for safinamide, a high in vivo activity and
outstanding therapeutic and toxic profiles. In the present study,
R-aminoalkanamide analogues of safinamide were designed to
explore mostly the substituent effects at the meta and para
positions of the benzyloxy group and to detect the main
interactions engaged by the R-aminoamide side chain. Our
findings indicated that MAO-B affinity and selectivity can be
efficiently modulated by appropriate substitutions on the ben-
zyloxy ring. In particular, as already observed in the coumarin
compounds. The docking pose of the lowest active p-nitro
derivative (S)-20 (Figure 4) suggested unfavorable steric interac-
tions of the nitro group with amino acid side chains of F103,
P104, and W119. Similar steric hindrance was not observed
for the equally substituted 7-benzyloxycoumarin derivative,19
and this may suggest that the anchoring benzyloxy group binds
in a slightly different way in the two classes of inhibitors.
The small but significant decrease of MAO-B affinity
observed moving from the (S)- to the (R)-configuration was
investigated by docking the two enantiomers of the m-chloro-
(S)-serine derivative 13 and safinamide 7. In both cases scoring
values consistent with a decreased inhibitory potency were
found: 66.85 kJ/mol versus 60.42 kJ/mol for the (S)- and (R)-
enantiomers of 13, respectively, and 64.99 kJ/mol versus 63.52
kJ/mol for the (S)- and (R)-enantiomers of safinamide.
Finally, our docking analyses permitted proper interpretation
of the unexpectedly inverted affinities of rigid tetrahydroiso-

4914 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Leonetti et al.
measured for all the chiral compounds listed in Table 1 was always
>98%. H NMR spectral data of compounds 1-20 were reported
in the Supporting Information.
series, lipophilic meta substituents significantly increased both
MAO-B affinity and selectivity. Large and rigid R-aminoamide
side chains were not tolerated, most likely for steric reasons.
The highest inhibitory potencies were observed for the 3-chlo-
robenzyloxyalanine and 3-chlorobenzyloxyserine derivatives
(S)-8 and (S)-13, respectively, and by the (R)-3-F-benzylox-
ytetrahydroisoquinoline derivative 21, which displayed the
highest affinity of the entire series of examined inhibitors (IC50
) 17 nM) along with an outstanding selectivity (SI ) 2941).
Docking studies enabled the identification of the physico-
chemical nature and spatial location of the main binding inter-
actions modulating the enzymatic affinity and selectivity of
safinamide analogues. The high MAO-B selectivity of safina-
mide was nicely interpreted by docking simulations which
yielded a much higher score for rMAO-B than rMAO-A. Fur-
thermore, docking poses of all the inhibitors disclosed favorable
interactions of the benzyloxy group, which took place in the
entrance cavity of the MAO-B binding site, whereas R-amino-
amide groups bind in the FAD region establishing HB interac-
tions with structured water molecules and polar groups of the
enzyme backbone. In conclusion, the present study reinforced
and complemented, especially in the less explored FAD region,
the interaction models proposed by our group for selective
MAO-B inhibition19,20,23 and deepened our understanding of the
structural requirements for high MAO-B affinity and selectivity.
The ease of synthetic accessibility and fine druglike properties
of safinamide analogues make them “privileged molecules” for
further structural modifications in the preparation of novel and
improved neuroprotective agents. However, particular care has
to be taken in the design of new hMAO-B inhibitors because
SAFIR and SSR might vary among species.21,24
1
Synthesis. General Procedure for SPOS of Safinamide 7 and
Analogues (1-6 and 8-20). An amount of 125 mg (0.08 mmol)
of Rink amide resin was placed in 20 different reactor vessels
(QUEST 210 apparatus), followed by swelling with 2 mL of DMF
for 0.5 h. After filtration, a 20% solution of piperidine in DMF
(1.5 mL) was added to each reactor vessel, and the mixture was
shaken for 30 min. The resin was then filtered and washed three
times with 2 mL of DMF. To four separate 25 mL round-bottom
flasks were added 2.0 mmol of the selected Fmoc-protected amino
acid (Gly, Ser, Ala, Phg), HOBt (2.0 mmol, 0.30 g), DICI (2.0
mmol, 0.31 mL), and DMF (10.0 mL). After the mixture was stirred
for 3 min at room temperature, each solution was divided into five
2 mL aliquots and 0.50 mL of each of them was added to the 20
reactor vessels followed by shaking for 3 h. A bromophenol test
was employed to monitor the reaction completion. The resin was
filtered and washed three times with 2 mL of DMF. After Fmoc
deprotection with piperidine in DMF and washing, the amino
terminus was alkylated by adding to each vessel p-triisopropylsi-
lyloxybenzaldehyde (0.40 mmol, 0.12 g), Ti(O-iPr)4 (0.55 mmol,
0.15 g), and TEA (0.08 mmol, 11.1 µL). After the mixture was
stirred at room temperature overnight, the resin was filtered and
washed with anhydrous THF (3 × 2 mL) and anhydrous DCM (3
× 2 mL). A presonicated suspension of NaBH(OAc)3 (0.40 mmol,
0.09 g) in DCM (1.5 mL) was added to the resin. The mixture was
stirred on an orbital shaker overnight, then filtered and washed in
sequence with water (3 × 2 mL), 0.75 M Na2CO3 aqueous solution
(3 × 2 mL), water (3 × 2 mL), THF (3 × 2 mL), and MeOH (3
× 2 mL) and dried under N2 flow. THF (1.5 mL) was added to
each vessel, and the slurries were shaken for 30 min, filtered, treated
with 1 M solution of TBAF in THF (1.5 mL) for 1 h, and then
filtered again and washed with THF (3 × 2 mL). Finally, the
phenolic hydroxyl group was alkylated by the Mitsunobu protocol
using five different benzyl alcohol derivatives (see Table 1) as
follows: 1.3 mL of a 0.2 M solution of the appropriate benzyl
alcohol in DMF was added to the appropriate support-bound 2-(p-
hydroxybenzyl)-2-aminoamide (see Scheme 1) in order to have the
final 20-member library (5 benzyl alcohols × 4 aminoamides). To
each reaction vessel were subsequently added PBu3 (0.24 mmol,
0.06 mL) and ADDP (0.24mmol, 0.06 g), and the slurries were
shaken overnight. The mixtures were filtered off, the resins washed
with THF (3 × 2 mL), DMF (3 × 2 mL), THF (3 × 2 mL), MeOH
(3 × 2 mL), and DCM (3 × 2 mL) and then treated for 1 h with
2 mL of a TFA/H2O/TES (95/2.5/2.5) mixture. The mixture was
filtered and the resin washed with the same mixture of cleavage (3
× 2 mL). The filtrate and washing solvent mixtures were combined
and immediately concentrated with evaporation under reduced
pressure. Toluene (2 × 2 mL) was added during the concentration
step in order to remove the remaining TFA. The compounds were
isolated as free bases, generally with a purity of >90%. When
necessary, the crude compound was purified by flash chromatog-
raphy using a mixture of DCM/MeOH as eluent.
Experimental Section
Chemistry. Starting materials, reagents, and solvents of high
analytical grade were purchased from commercial suppliers. The
Rink amide resin, loading level of 0.64 mmol/g, was from
Novabiochem. Chromatographic separations were performed on
silica gel (15-40 mesh, Merck) by the flash methodology unless
otherwise stated. Tetrahydrofuran (THF) and dichloromethane
(DCM) were made anhydrous by reflux on sodium/benzophenone
ketyl and calcium hydride, respectively, and distillation under N2.
Melting points were determined by the open capillary method on
a Stuart-Scientific SMP3 electrothermal apparatus and are uncor-
rected. 1H NMR spectra were recorded in the indicated deuterated
solvents at 300 MHz on a Varian Mercury 300 instrument. Chemical
shifts are expressed in δ (ppm) and coupling constants J in hertz
(Hz). The following abbreviations were used for multiplicities: br
(broad), s (singlet), d (doublet), t (triplet), dd (double doublet), dt
(double triplet), ddd (double doublet of doublet), q (quintet), m
(multiplet); signals due to OH protons were located by deuterium
exchange with D2O. Elemental analyses (C, H, N) were performed
only for the S and R chiral enantiomers of tetrahydroisoquinoline
derivative 21, prepared through conventional solution-phase syn-
thesis, whereas for all the compounds synthesized by SPOS and
for the intermediates 22-26 as well, the purity was checked by 1H
NMR and HPLC. All the tested compounds had a purity of >95%.
HPLC analyses were carried out on a Waters 1585 system equipped
with a model 2487 UV detector, a Waters XTerra C8 column (3
mm × 250 mm), and different MeOH/H2O mixtures as the mobile
phase. Chiral HPLC analyses were run on a CHIRALPAK IA (4.6
mm × 250 mm) column (Daicel Chemical Industries, Japan) or a
CHIROBIOTIC TAG (4.6 mm × 250 mm) column (Astec
Separation Technologies Inc.). Mobile phases used on the CHIRAL-
PAK column were made of 20-30% of ethanol in hexane, while
60-65% of MeOH in water was used as the mobile phase on the
CHIROBIOTIC TAG column (see Table 1 in the Supporting
Information for additional details). The enantiomeric excess
1H NMR spectral data of compounds 1-20 are described in the
Supporting Information.
2-[3-(3-Fluorobenzyloxy)phenyl]ethanol (22). 3-(2-Hydroxy-
ethyl)phenol (7.2 g, 51.9 mmol), 3-fluorobenzyl bromide (8.9 g,
47.2 mmol), K2CO3 (19.6 g, 141.5 mmol), and KI (1.0 g, 6.1 mmol)
in acetone (65 mL) were refluxed under mechanical stirring
overnight. The reaction mixture was filtered to remove inorganic
salts and evaporated under vacuum to dryness. The crude residue
was dissolved in EtOAc, washed with 0.5 N NaOH and brine, dried
over Na2SO4, filtered, and evaporated to dryness. An amount of
11.8 g of the desired compound (100% yield) was obtained as a
yellow oil. This crude material was utilized in the following reaction
1
without further purification. H NMR (CDCl3) δ 2.85 (t, 2H, J )
7.5), 3.85 (t, 2H, J ) 7.5), 5.06 (s, 2H), 6.79-6.91 (m, 3H), 6.96-
7.06 (m, 1H), 7.11-7.28 (m, 3H), 7.29-7.40 (m, 1H).
2-{2-[3-(3-Fluorobenzyloxy)phenyl]ethyl}isoindole-1,3-di-
one (23). 2-[3-(3-Fluorobenzyloxy)phenyl]ethanol (8.8 g, 35.7

Safinamide Analogues as Inibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4915
mmol), phthalimide (7.4 g, 50.0 mmol), triphenylphosphine polymer
bound (7.4 g, 50.0 mmol, loading of 1 mmol/g), and diethyl
azadicarboxylate (DEAD, 8.7 g, 50.0 mmol) were suspended in
THF (320 mL) and stirred at room temperature overnight. Ad-
ditional DEAD (2.7 g, 15.3 mmol) was added under stirring to the
reaction mixture over a period of 5 h. The reaction mixture was
filtered off, and the filtrate was evaporated to dryness. The crude
residue was dissolved in EtOAc, washed with 1 N KOH and brine,
dried over Na2SO4, filtered, and evaporated to dryness. The residue
was triturated with EtOH, filtered, and then crystallized with EtOH
to give 5.8 g of title compound (43% yield) as white crystals of
was purified by a slow column chromatography, using 99:1 DCM/
MeOH as eluent, to yield 0.068 g (23% yield) of a pure white solid.
Mp 153-154 °C. 1H NMR (CDCl3) δ 1.35 (d, 3H, J ) 7.0), 2.67-
2.97 (m, 4H), 3.28 (q, 1H, J ) 7.0), 3.64 (d, 1H, J ) 14.2), 3.77
(d, 1H, J ) 14.2), 5.05 (s, 2H), 5.36 (br, 1H), 6.74 (d, 1H, J )
2.5), 6.79 (dd, 1H, J ) 8.2, 2.5), 6.97 (d, 1H, J ) 8.2), 7.00-7.06
(m, 1H), 7.06-7.24 (m, 3H), 7.29-7.42 (m, 1H).
(S)-2-[6-(3-Fluorobenzyloxy)-3,4-dihydro-1H-isoquinolin-2-yl]-
propionamide (21). The title compound was obtained using the
same procedure described for the synthesis of (R)-2-[6-(3-fluo-
robenzyloxy)-3,4-dihydro-1H-isoquinolin-2-yl]propionamide, start-
ing from 6-(3-fluorobenzyloxy)-1,2,3,4-tetrahydroisoquinoline (0.24
g, 0.95 mmol) and (R)-2-amino-1-methyl-2-oxoethyl-2-nitroben-
zenesulfonate (0.52 g, 1.9 mmol). After column chromatography
purification using 99:1 DCM/MeOH as eluent, 0.075 g (24% yield)
of the title compound was obtained as a pure white solid. Mp 153-
154 °C. 1H NMR (CDCl3) δ 1.35 (d, 3H, J ) 7.0), 2.67-2.97 (m,
4H), 3.28 (q, 1H, J ) 7.0), 3.64 (d, 1H, J ) 14.2), 3.77 (d, 1H, J
) 14.2), 5.05 (s, 2H), 5.36 (br, 1H), 6.74 (d, 1H, J ) 2.5), 6.79
(dd, 1H, J ) 8.5, 2.5), 6.97 (d, 1H, J ) 8.5), 6.99-7.06 (m, 1H),
7.06-7.24 (m, 3H), 7.30-7.40 (m, 1H).
Biological Assay. In Vitro Enzyme Activity Assay. The enzyme
activities were assessed with a radioenzymatic assay using the
selective substrates 14C-serotonin (5-HT) and 14C-phenylethylamine
(PEA) for MAO-A and MAO-B, respectively.
The mitochondrial pellet (500 µg protein) was resuspended in
200 µL of 0.1 M phosphate buffer, pH 7.40, and was added to 50
µL of the solution of the inhibitor (transformed to the methane-
sulfonate salt upon addition of a stoichiometric amount of 0.01 M
methanesulfonic acid to the aqueous solution of the free base) or
of buffer and incubated for 30 min at 37 °C (preincubation). Then
the substrate in 50 µL of buffer (5 µM 14C-5-HT or 0.5 µM 14C-
PEA) was added and the assay mixture was incubated at 37 °C for
30 min (5-HT) or for 10 min (PEA).
The reaction was stopped by adding 0.2 mL of HCl or perchloric
acid for 5-HT or PEA, respectively. After centrifugation, the acidic
radioactive metabolites were extracted with 3 mL of diethyl ether
(for 5-HT) or toluene (for PEA) and the radioactivity of the organic
phase was measured by liquid scintillation spectrometry at 90%
efficiency.
The enzymatic activity was expressed as nanomoles of substrate
transformed per milligram of protein per minute (nmol mg-1 min-1).
The drug inhibition curves were obtained from five to eight
different concentrations (10-10-10-5 M), each in duplicate, and
the IC50 was determined using nonlinear regression analysis
(GraphPad best-fitting computer program). For very low active
inhibitors, the percent of enzyme inhibition was determined in
duplicate at the concentrations indicated in Table.1.
1
sufficient purity for the subsequent reaction. H NMR (CDCl3) δ
2.93-3.03 (m, 2H), 3.90-3.99 (m, 2H), 5.02 (s, 2H), 6.82 (ddd,
1H, J ) 8.3, 2.8, 1.1), 6.86-6.93 (m, 2H), 6.96-7.05 (m, 1H),
7.10-7.25 (m, 3H), 7.30-7.39 (m, 1H), 7.67-7.75 (m, 2H), 7.81-
7.88 (m, 2H).
2-[3-(3-Fluorobenzyloxy)phenyl]ethylamine (24). 2-{2-[3-(3-
Fluorobenzyloxy)phenyl]ethyl}isoindole-1,3-dione (2.0 g, 5.3 mmol)
and hydrazine hydrate (0.58 g, 11.7 mmol) were dissolved in
absolute EtOH (45 mL) under mechanical stirring and refluxed for
5 h. The formed white precipitate was filtered off, and the ethanolic
filtrate was evaporated to dryness, dissolved in DCM, and washed
with water. The organic layer was dried over Na2SO4, filtered, and
evaporated to dryness to yield 1.1 g (82% yield) of the title
compound as a yellow oil. This crude material was utilized in the
1
following reaction without further purification. H NMR (CDCl3)
δ 2.73 (t, 2H, J ) 7.6), 2.97 (t, 2H, J ) 7.6), 5.06 (s, 2H), 6.74-
6.87 (m, 3H), 7.01 (m, 1H), 7.12-7.25 (m, 3H), 7.29-7.41 (m,
1H).
6-(3-Fluorobenzyloxy)-1,2,3,4-tetrahydroisoquinoline (25). 2-[3-
(3-Fluorobenzyloxy)phenyl]ethylamine (1.5 g, 6.0 mmol), formal-
dehyde 40% solution in water (0.54 mL, 7.8 mmol), and water (0.54
mL) were stirred at room temperature for 2 h. The reaction mixture
was evaporated to dryness, and an amount of 1.76 mL of 23%
aqueous HCl was added. The mixture was stirred at room
temperature for 5 h. After dilution with some water, 0.5 N aqueous
NaOH was added with cooling up to pH 10. The mixture was
extracted with EtOAc, washed with H2O, dried over Na2SO4,
filtered, and evaporated to dryness to yield 1.4 g (93% yield) of
the title compound as a yellow oil. This crude material was utilized
1
in the subsequent reaction without further purification. H NMR
(CDCl3) δ 2.77 (t, 2H, J ) 6.0), 3.11 (t, 2H, J ) 6.0), 3.95 (s,
2H), 5.03 (s, 2H), 6.69 (d, 1H, J ) 2.7), 6.76 (dd, 1H, J ) 8.4,
2.7), 6.92 (d, 1H, J ) 8.4), 6.95-7.06 (m, 1H), 7.09-7.23 (m,
2H), 7.28-7.41 (m, 1H).
(S)- or (R)-2-Amino-1-methyl-2-oxoethyl-2-nitrobenzene-
sulfonate. The (S)- or (R)-2-hydroxypropionamide (3.4 g, 38.0
mmol), triethylamine (7.7 g, 76.0 mmol), and N,N-dimethylami-
nopyridine (1.2 g, 9.5 mmol) were dissolved in 110 mL of DCM
and cooled to 0 °C. o-Nitrobenzenesulfonyl chloride (16.8 g, 76.0
mmol) was added portionwise, and the mixture was stirred at room
temperature overnight. The mixture was washed with water and a
10% aqueous solution of citric acid, dried over Na2SO4, filtered,
and evaporated to dryness to yield 6.7 g of a brown oil. This crude
material was purified by flash column chromatography, using 95:5
DCM/MeOH as eluent, to yield the title (R)- or (S)-enantiomer as
a pale-yellow solid in a 20-25% yield and an enantiomeric excess
Computational Methods. Computational analyses were con-
ducted on a 16-node Linux cluster employing an openMosix
architecture composed of AMD Athlon XP 2400+ and Intel Xeon
2600 cpus. Molecules and models were displayed and manipulated
on a Silicon Graphics O2+ machine.
Docking Simulations. GOLD, a genetic algorithm-based soft-
ware, was used for our docking study selecting the GOLDScore as
a fitness function. GOLDScore is made up of four components that
account for protein-ligand binding energy: protein-ligand hy-
drogen bond energy (external H-bond), protein-ligand van der
Waals energy (external vdw), ligand internal van der Waals energy
(internal vdw), and ligand torsional strain energy (internal torsion).
Empirical parameters used in the fitness function (hydrogen bond
energies, atom radii and polarizabilities, torsion potentials, hydrogen
bond directionalities, and so forth) are taken from the GOLD
parameter file. The fitness score is taken as the negative of the
sum of the energy terms so that larger fitness scores indicated a
better binding. The fitness function has been optimized for the
prediction of ligand binding positions rather than the prediction of
binding affinities, although some correlation with the latter can be
also found. The protein input file may be the entire protein structure
or a part of it comprising only the residues that are in the region of
the ligand binding site. In the present study, GOLD was allowed
1
of >98%. H NMR (DMSO-d6) δ 1.46 (d, J ) 6.9, 3H), 3.28-
3.61 (br, 2H), 4.45 (q, J ) 6.9, 1H), 7.80-7.86 (m, 2H), 7.88-
7.94 (m, 1H), 8.11-8.14 (m, 1H).
(R)-2-[6-(3-Fluorobenzyloxy)-3,4-dihydro-1H-isoquinolin-2-
yl]propionamide (21). 6-(3-Fluorobenzyloxy)-1,2,3,4-tetrahydroiso-
quinoline (0.23 g, 0.91 mmol) dissolved in DCM (1 mL) was added
dropwise at 0 °C to a solution of (S)-2-amino-1-methyl-2-oxoethyl-
2-nitrobenzenesulfonate (0.5 g, 1.8 mmol) and DIPEA (0.24 g, 1.8
mmol) in DCM (15 mL). The reaction mixture was stirred at room
temperature overnight and extracted with a 5% aqueous solution
of citric acid. The aqueous layer was washed with Et2O, made basic
with 28% NH4OH, and extracted with DCM. The organic layer
was dried over Na2SO4, filtered, and evaporated to dryness to yield
0.14 g of title compound as a white crude solid. This crude material

4916 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Leonetti et al.
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(20) Bru¨hlmann, C.; Ooms, F.; Carrupt, P.-A.; Testa, B.; Catto, M.;
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(21) Novaroli, L.; Reist, M.; Favre, E.; Carotti, A.; Catto, M.; Carrupt,
P.-A. Human Recombinant Monoamine Oxidase B as Reliable and
Efficient Enzyme Source for Inhibitor Screening. Bioorg. Med. Chem.
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(22) Carotti, A.; Altomare, C.; Catto, M.; Gnerre, C.; Summo, L.; De
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(23) Catto, M.; Nicolotti, O.; Leonetti, F.; Carotti, A.; Favia, A.; Soto-
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(24) Novaroli, L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.; Leonetti,
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to calculate interaction energies within a sphere of a 12 Å radius
centered on the phenolic oxygen atom of Y435 of rMAO-B and in
the corresponding Y444 of rMAO-A. When it was explicitly
specified, considering safinamide as a molecular reference, physical
distance constraints were defined between the fluorine atom with
the carbonyl oxygen of I164, the nitrogen of the amide group with
the carbonyl oxygen atoms of C172 and L171, and, finally, the
oxygen of the amide group with WAT160, the spring constant being
set to 30 kJ mol-1 Å-1 and the deviation tolerance to (0.3 Å.
Acknowledgment. The authors gratefully thank MIUR,
Rome, Italy (PRIN Project 2006), and Newron Pharmaceuticals
(Bresso, Italy) for financial support and Dr. Fabiola Miscioscia
for her skillful assistance in the modeling study.
Supporting Information Available: 1H NMR data of com-
pounds 1-20, elemental analyses (C, H, N) of the target compound
21, and table of chromatographic data for the assessment of
enantiomeric excess. This material is available free of charge via
the Internet at http://pubs.acs.org.
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