New isoxazolidinone and 3,4-dehydro-β-proline derivatives as antibacterial agents and MAO-inhibitors: A complex balance between two activities

Article abstract of DOI:10.1016/j.ejmech.2016.09.007

Among the different classes of antibiotics, oxazolidinone derivatives represent important drugs, since their unique mechanism of action overcomes commonly diffused multidrug-resistant bacteria. Anyway, the structural similarity of these molecules to monoa

Full text of DOI:10.1016/j.ejmech.2016.09.007

Accepted Manuscript
New isoxazolidinone and 3,4-dehydro-β-proline derivatives as antibacterial agents
and MAO-inhibitors: A complex balance between two activities
Lucia Ferrazzano, Angelo Viola, Elena Lonati, Alessandra Bulbarelli, Rosario
Musumeci, Clementina Cocuzza, Marco Lombardo, Alessandra Tolomelli
PII:
S0223-5234(16)30735-8
10.1016/j.ejmech.2016.09.007
EJMECH 8873
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 July 2016
Revised Date: 1 September 2016
Accepted Date: 2 September 2016
Please cite this article as: L. Ferrazzano, A. Viola, E. Lonati, A. Bulbarelli, R. Musumeci, C. Cocuzza,
M. Lombardo, A. Tolomelli, New isoxazolidinone and 3,4-dehydro-β-proline derivatives as antibacterial
agents and MAO-inhibitors: A complex balance between two activities, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.007.
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ACCEPTED MANUSCRIPT
Journal
New isoxazolidinone and 3,4-dehydro-ꢀ-proline derivatives as antibacterial
agents and MAO-inhibitors: a complex balance between two activities
.
Lucia Ferrazzanoa, Angelo Violaa, Elena Lonatib, Alessandra Bulbarellib, Rosario
Musumecib, Clementina Cocuzzab, Marco Lombardoa, Alessandra Tolomellia*.
aDepartment of Chemistry, University of Bologna, Via Selmi 2, 40126, Bologna, Italy
bDepartment of Medicine and Surgery, University of Milano-Bicocca, Via Cadore 48, 20900, Monza, Italy
Among the different classes of antibiotics, oxazolidinone derivatives represent
important drugs, since their unique mechanism of action overcomes commonly diffused
multidrug-resistant bacteria. Anyway, the structural similarity of these molecules to
monoamino oxidase (MAO“ inhibitors, like toloxatone and blefoxatone, induces in many
cases loss of selectivity as a major concern. A small library of compounds based on
isoxazolinone and dehydro-β-proline scaffold was designed with the aim to obtain
antibacterial agents, evaluating at the same time the potential effects of structural
ABSTRACT
features on MAO inhibitory behaviour. The structural modification introduced in the
backbone, starting from Linezolid model, lead to a significant loss in antibiotic activity,
while a promising inhibitory effect could be observed on monoamino oxidases. These
interesting results are also in agreement with docking experiments suggesting a good
binding pose of the synthesized compounds into the pocket of the oxidase enzymes, in
particular
of
MAO-B.
1. Introduction
pharmacokinetic oxazolidinone ring and interacts with fixed
residues of the PTC pocket through van der Waals
interactions. The acetamide NH on the tail participates in
hydrogen bond with the phosphate group on a residue in the
upper part of the pocket. The oxazolidinone fits, together with
the C5-tail, in the direction of the ribosomal tunnel, orienting
the ring-C toward the inter-subunit interface. Ring-B is
sandwiched through ꢄ-staking with two residues and
participates in a T-shaped interaction, while the C2 fluorine
points out of the pocket to contribute to the coordination of a
putative magnesium ion that appears upon Linezolid binding in
this particular configuration. The morpholine in Ring-C, does
not give particular interactions with the binding site but has
great importance in the modulation of molecule
pharmacokinetic.[4-5] Despite all these favourable features,
there are several cases of resistance to Linezolid.[6]
Considering their efficacy and evaluating the chance to
introduce modifications to reduce resistance effects,
oxazolidinones were deeply studied in their structure-activity
relationships (SAR“ in order to identify bioisosters of each part
of the molecule, in particular of Ring-A. These studies revealed
that bioisosteric modifications on Ring-A should maintain i“ an
sp2 centre directly linked to Ring-B, ii“ an oxygen atom properly
located and iii“ a C5-chiral centre stereochemically defined
carrying the acylaminomethyl chain.[7] These requirements
were confirmed by the results in terms of inhibition of the
The increase of phenomena of antibiotic resistance became a
central matter in public health programs, since it represents a
problem not only for people already infected, but also for the
diffusion of resistant bacteria to other people.[1] The problem
grew with the appearance of multidrug-resistant ESKAPE
pathogens
(Enterococcus
spp, Staphylococcus aureus,
Klebsiella spp, Acinetobacter baumannii, Pseudomonas
aeruginosa, Enterobacter spp“, usually causes of chronic
infections like skin infections, meningitis, urogenital tract
diseases and respiratory tract infections. This emergency
justifies the continuous research for new type of antibiotics.
Between the most recent classes of antibiotics, oxazolidinones
represent a great innovation in the attempt of limiting the
diffusion of ꢁsmartlyꢂ modified Gram-positive bacteria. Linezolid
(Zyvox®“ represents a well-known antimicrobial agent able to
fight Vancomicyn-Resistant Enterococci (VRE“, Methicillin-
Resistant Staphylococcus aureus (MRSA“ and Penicillin-
Resistant Streptococcus pneumoniae (PNSSP“.[2-3] Like other
molecules belonging to the family of oxazolidinones,
Linezolidꢃs mechanism of action regards the binding to
peptidyltransferase centre (PTC“, corresponding to 50S
subunit of prokaryotic ribosome. The structure of Linezolid
consists in four chemical moieties that can justify its interaction
with the ribosomal binding site (Figure 1“. Ring-A is the

ACCEPTED MANUSCRIPT
proliferation of resistant bacteria reported for dihydrofuran-2-
one (a“, tetrahydrofuran-2-one (b“, pyrrolidin-2-one (c“, 3-
phenylisoxazoline (d“, 3-phenylisoxazolidone (e“ and 1,2,4-
oxadiazole (f“ derivatives.
Figure 1. Structure of Linezolid and its analogues and structure of blefoxatone and toloxatone.
In vitro Minimum Inhibitory Concentration (MIC“ microbiological
tests, assessing the antibacterial activity, show how the
gradually removal of one or more of the three features reported
above results in decrease of antimicrobial activity (Figure 2“.
[8-9-10-11]
bioactivity: if the ligand is a MAO-inhibitor, the reduction of
FAD goes through an anionic semiquinone intermediate to the
fully reduced form and no reoxidation of FAD is observed; if
the ligand is not an inhibitor, no intermediate is detected and
only the fully reduced form of FAD can be observed.[18]
Linezolid shows
a weak reversible MAO-A and MAO-B
O
O
O
O
O
O
O
N
inhibition.[19] In the literature, a modified C5 side chain (-
CONH2“ has been reported known as theꢁreverse amideꢂ.
The analogues containing this group have been easily
obtained through total synthesis and showed a reduction in
MAO inhibition and myelotoxicity, usually observed for long
period treatment using Linezolid.[20] SAR studies showed that
lipophilic substrates like oxazolidinones with an aromatic ring
near to a nitrogen/oxygen/sulphur atom find a long and deep
hydrophobic cavity. The substrates are aligned between two
tyrosine residues, placing their amine near to N5 of flavin for
catalysis.[21] Therefore, in the development of new
oxazolidinone derivatives is essential to focus on the
enhancement of antibacterial activity and on the overcoming of
potential side activities of this class of molecules. Starting from
all these considerations, we are searching for new analogues
of Linezolid with acceptable antibacterial activity, evaluating at
same time the potential effects of structural modifications on
the inhibitory behaviour on MAO of these molecules (Figure 1“.
For this purpose, we decided to change first of all Ring-A by
the introduction of i“ a racemic isoster of oxazolidinone,
isoxazolidinone 3, or ii“ 3,4-dehydro-ꢀ-proline 12, we recently
reported in literature as rigid five-membered heteroaromatic
core for biological applications.[22] As regard isoxazolidine
ring, we kept two of the three necessary features to obtain a
good bioisoster of oxazolidinone: a sp2 centre linked to Ring-B
and an oxygen atom. We reported herein the synthesis of this
scaffold, obtained through TBAF induced cyclization of
intermediate 2 (Scheme 1“. Two products were designed by
changing the substitution on C1 of the heterocycle,[23] to
evaluate if the presence of substituent in this position can
influence the activity. In the case of 3,4-dehydro-ꢀ-proline
containing molecules, which keep only the sp2 centre as
positive feature for interaction, worse results were expected.
For both classes of compounds, as a consequence of the
N
N
N
O
N
O
O
N
oxazolidinone
(e)
(a)
(b)
(d)
(c)
(f)
lacking of needed features
reduced/none activity
Figure 2. Bioisoster of Ring-A classified on the base of reduction of activity.
The few trials to change Ring-B did not give significant
improvements in terms of activity, while several examples of
changes in Ring-C are reported in literature and regard four
major conceptual frameworks like fusion of ring B and C,
separation of ring-B and C by a spacer, appendage of
stabilizing moieties on Ring-C and new Ring-C. [10-12] Taking
into account that the principal metabolites of Linezolid are
produced during the lactone or lactame pathways, regarding
so the morpholine ring oxidation,[13-14-15] a widely used
substitution is piperazine and its derivatives, as reported for
Eperezolid and its analogues. Usually, Linezolid analogues
have a C5 aminomethyl functionalized tail as amide, urea and
thiourea. With these moieties, the resulting structures are quite
similar to those of monoamino oxidase (MAO“ inhibitors, like
toloxatone and blefoxatone (Figure 1“, that are active against
the two human isoforms of monoamino oxidase (MAO-A and
MAO-B“, responsible for the metabolism of monoamino
neurotrasmitters.[16] In particular, MAO-A inhibitors have been
successfully applied as anti-depressant, while molecule that
are able to modulate MAO-B activity have been used in the
treatment of Parkinsonꢃs disease and they are now receiving
great attention for the protective effect on vascular and
neuronal tissues, thus suggesting their possible use in the
prevention and retard of Alzheimerꢃs disease.[17]
These molecules convert amines into imines through the
reduction of the cystinyl-FAD cofactor of MAO, then reoxidized
by H2O2. The presence of ligands close to FAD influences the

ACCEPTED MANUSCRIPT
synthetic route and considering the relevance that the
methylmalonamide. However, by adding TBAF in situ, it is
possible to obtain at room temperature directly the product of
cyclization 3-a,b with yield of 63% and 66% respectively,
through the formation of a tetrabutylammonium hydroxylamino
salt that undergoes cyclization via spontaneous intramolecular
transesterification. The asymmetric synthesis of 3,4-dehydro-
ꢀ-pirrole 12 was achieved as reported in Scheme 1 and was
already reported in previous papers.[26-27] To substitute the
ring-B and ring-C, we proceeded as reported in Scheme 2. 3,4-
difluorobenzoic acid was protected as methyl ester using
SOCl2 and methanol obtaining the intermediate 13 in 63%
yield. This scaffold represents the Ring-B of the final
structures. The fluorine in position 4 was used to introduce
morpholine or piperazine via nucleophilic substitution reaction,
with K2HPO4 in DMSO, obtaining intermediate 14 and 16 in
89%-99% yield. The NH moiety of piperazine was then
functionalized with Boc-group in one case, obtaining
intermediate 17 in quantitative yield, to avoid side reaction of
acylaminomethyl chain has in the structure of MAO-inhibitors,
we introduced a reverse N-ethyl amide without control of
chirality on C5 of heterocycle, in order to understand, from a
preliminary study, the potential efficacy as scaffolds of these
cycles. The ring-B was linked to the heteroaromatic core
through peptidic bond, keeping the fluorine atom in the usual
position. Concerning the Ring-C, we used the morpholine, the
piperazine or N-substituted piperazine, to evaluate if different
groups may modulate the antibiotic efficacy.
2. Results and discussion
2.1. Chemistry
To substitute ring-A, two heterocycles have been used:
isoxazolidin-5-one
3
and 3,4-dehydro-ꢀ-pirrole 12. The
synthesis of functionalized isoxazolidin-5-one is reported in
Scheme 1. The first step of the synthesis is the 1,4-addition of
N,O-bis(trimethylsilyl“hydroxylamine on isopropylidene and
cyclohexylidene malonamides. As previously reported,[24]
alkylidene malonamides react as Michael acceptors in catalytic
asymmetric 1,4-addition of nitrogen nucleofiles. So,
intermediates 2-a,b have been obtained through Knövenagel
this centre in subsequent reactions, and with
N-Boc-Glycine in
the other case. The coupling conditions to obtain intermediate
19 involved COMU and DIPEA and gave the desiderated
intermediate in quantitative yield. The final deprotection step
on intermediates 14, 17 and 19 to remove methyl ester, was
successfully performed at room temperature with LiOH.H2O in
a mixture of THF:H2O=2:1, with yields between 67% and 99%.
The isoxazolidin-5-one and 3,4-dehydro-ꢀ-pirrole NH
condensation of the synthesized
N-ethyl-methylmalonamide 1
with isobutyrraldehyde or cyclohexylcarboxyaldehyde in
presence of a catalytic amount of piperidine, giving the product
2a and 2b in 55% and 56% yield respectively and 1/4 E/Z
ratio. The 1,4-addition on both isomers of alkylidene
malonamide was carried out through Lewis acid catalysis:[25]
in dry DCM, a catalytic amount of Sc(OTf“3 was added to 2
derivatives 3-a,b, (S“-12 and (R“-12 were coupled with the
acidic counterpart 15, 18 and 20, in situ converted into the
corresponding chloride using thionyl chloride and pyridine
affording, after removal of Boc protection from intermediates,
the final products 21-a,b, 23a, (S“-24, (R“-24, (S“-26, (R“-26,
and, after lowering the temperature at -40°C, N,O
-
(S
“-28 and ( “-28. The stability of these compounds in DMSO
R
bis(trimethylsilyl“hydroxylamine was added. The adduct could
be isolated in good yield if the temperature is mantained at -
40°C, to avoid byproducts like the corresponding oxime and
has been verified through HPLC method and no by-products
were detected in 24h study.
Scheme 1. Synthetic route for the synthesis of isoxazolidin-5-one ring and 3,4-dehydro-ꢀ-pirrole. Conditions: a“ ethylamine, TEA, DCM, 0°C->rt, overnight; b“ piperidine, aldehyde,
rt, 3 days; c“ Sc(OTf“3, N,O-bis(trimethylsilyl“hydroxylamine, DCM, -40°C, 1.5h; d“TBAF, DCM, 15 min; e“ piperidine, aldehyde, MW 250W/7 min; f“ CeCl3.H2O, NaBH4, THF:MeOH, -
30°C, 45 min, Pseudomonas cepacia lipase, vinylacetate, Et2O, reflux 30 h; g“ LiHMDA, methylchloroformiate, THF, -78°C, 2h; h“ [Ir(COD“Cl]2, P(OPh3“, allylamine, EtOH, 24h; i“
NaHCO3, Fmoc-Cl, dioxane, rt, overnight ; l“ MTBE, Grubbs-II generation catalyst, 50°C, 20h; m“ TFA, 0°C->rt, overnight; n“ ethylamine, HBTU, TEA, DCM, rt, overnight; o“
piperidine, DMF, rt, 10 min.

ACCEPTED MANUSCRIPT
Scheme 2. Synthetic route for the synthesis of Ring-B and Ring-C. Conditions: p“ SOCl2, MeOH, 0°C->rt, overnight; q“ morpholine/piperazine, K2HPO4, dmso, reflux, 8h; r“
LiOH.H2O, THF:H2O, rt, overnight.
Scheme 3. Synthesis of isoxazolidin-5-one and 3,4-dehydro-ꢀ-pirrole derivatives.
MIC (ꢅg/ml“
MIC-range
MIC50
MIC90
E. faecalis
S. aureus
Strains
ATCC
29212
12 S. aureus isolates
ATCC 29213
Compound
21a
>128
>128
>128
>128
4
>128
>128
>128
>128
2
>128
32->128
>128
>128
64
>128
>128
>128
>128
2
21b
(R
“-24
>128
>128
2
(S
“-24
>128
Linezolid
1-4
Table 1. Antimicrobial activities, expressed in MIC values, MIC-range, MIC50 and MIC90 (ꢅg/ml“ of tested compounds against both Gram-positive standard strains and recently
isolated clinical Staphylococcus aureus strains; Linezolid was used as reference antibiotic agent.
from human hepatocytoma, were chosen in this study since
both MAO-A and -B have been found in human liver tissue and
in hepatocytes as well and because of this cell line is easy to
maintain and proliferate rapidly.[28-29]
Cell viability experiments were performed to define the proper
dose for the evaluation of compounds effects on MAO activity
in HepG2, taking into account the previous data obtained with
Linezolid,[30] used as oxazolidinones reference drug.
2.2 Biological evaluation
The antibacterial activity of all the synthesized products 21-a,b,
23a, (S“-24, (R“-24, (S“-26, (R“-26, (S“-28 and (R“-28 was
evaluated on MRSA and MSSA strains, but after the initial
unsatisfactory results, only the activities of 21a, 21b, 24- (R“
and 24-(S“ compounds were further explored.
The antibacterial activities of 21a, 21b, (R“-24 and (S“-24 by
Increasing concentrations (10-100ꢅg/mL“ of 21a, 21b, (
and ( “-24 were administrated to cells for 24 hours. None of
R“-24
means of MIC values are shown in Table 1. Only for 21b a
modest antibacterial activity (MIC=32-64 ꢅg/ml“ against clinical
strains of S. aureus was demonstrated.
S
compounds treatments induces mortality at 10ꢅg/mL, while at
ten-times increasing concentration we observed only a 10%
Therefore, 21a, 21b, (R“-24 and (S“-24 compounds are weak
decrease in cell viability with 21a, 21b, (
24. Since 21a, 21b, ( “-24 and ( “-24 compounds are all
dissolved in DMSO, the effect of ( “-24 on cell viability is
R“-24 and 20% in (S“-
antibiotics with respect to Linezolid (LZD“; nevertheless, they
might be better utilized as MAO inhibitors. For this reason their
potential MAO activity inhibitory power was analyzed along
with their cytotoxicity in an in vitro system, that, is an excellent
model to perform preliminary screening. HepG2 cells, derived
R
S
S
probably due to the cytotoxicity exerted by high percentage of
solvent (2%“ in which it is solubilized (data not shown“.

ACCEPTED MANUSCRIPT
MAO activity was analyzed in HepG2 cell lysates after 24
hours of treatment with 10-100ꢅg/mL of 21a, 21b, ( “-24 and
“-24 compounds, using the Amplex® Red Monoamine
Oxidase assay. The 21a, 21b and ( “-24 compounds showed
significative inhibitory effect of about 25% already at
10ꢅg/mL. Thus, considering the mild cytotoxicity exerted in
HepG2, we increased ten-times the drug concentration
reaching the 45% of inhibitory effect. On the contrary, high
concentration (100ꢅg/mL“ of Linezolid (LZD“ induces a weak
decrease of enzymatic activity (15%“. In agreement to our
data, experimental evidences demonstrated a low inhibitory
effect of LZD on MAO activity in a cell-free assay.[12]
Taken together, cell viability and MAO activity assays showed
that 21a, 21b and (S“-24 molecules might be promising
compounds for the development of MAO inhibitors, showing a
very low cytotoxicity also at the concentration exerting almost
50% of inhibitory effect. Nevertheless, our compounds deserve
future experiments to confirm their potential inhibitory efficacy,
analyzing also their specificity for MAO-A or MAO-B.
R
(
S
S
a
Figure 3. A“ Cell viability of 21a, 21b,
(R“-24, (S“-24 and Linezolid in HepG2 cells. B“ MAO activity of 21a, 21b, (R“-24, (S“-24 and Linezolid in HepG2 cells.
To get some insight into the binding modes of the newly
prepared compounds, we performed a docking study into the
active site of human monoamine oxidase isoforms A (MAO-A“
and B (MAO-B“. The crystal structures of MAO-A[31] and
MAO-B[32] were retrieved from the Protein Data Bank (PDB
codes: 2Z5X[33] and 2V5Z,[34] respectively“ and processed
using Schrödinger Protein Preparation Wizard.[35] A small
library of compounds was built including all the four possible
stereoisomers of isoxazolidinones 21a and 21b ((R,R“-21a,b,
2.3 Structure-activity relationships
In this work we reported how the substitution of oxazolidinone
ring with isoxazolidinone or 3,4-dehydro-ꢀ-proline led to a loss
of antibacterial activity against the bacterial strains previously
reported. By the prediction of log PKow calculated through EPI
SuiteTM for each compound and for the reference compound
Linezolid, we found a rational trend. All the compounds
containing isoxazolidinone ring have an hydrophilic behaviour
while those containing 3,4-dehydro-ꢀ-proline belong to the
family of lipophilic compounds. In the first family, while the
isopropyl group does not affect the hydrophilicity, the
(
R,S“-21a,b, (S,R“-21a,b, (S,S“-21a,b“, the two enantiomers of
the 3,4-dehydro-ꢀ-pirrole derivative 24 ((R“-24 and (S“-24“ and
“-Linezolid as a reference ligand. The library of compounds
(S
was optimized by molecular mechanic and docking was
performed with AutoDock Vina.[36]
The binding free energies calculated for the human MAO-A
model were invariably much lower for all compounds
cyclohexyl group induces an enhancement of log
P meaning a
more lipophilic behaviour. Regarding the second family,
modulation of lipophilicity depends on the Ring C: going from
morpholine to Gly-piperazine through piperazine, the log P
value is mitigated toward more hydrophilic character. As
examined, with respect to the reference ligand (S“-Linezolid
and only compounds (S“-24, (R“-24, (R,R“-21a and (R,R“-21b
displayed negative binding free-energy values (Table 3“.
shown in Table 2, the compound 21b has log
P value quite
Free energy of binding
Compound
near to that of Linezolid, confirming that the changes in terms
of hydrophilicity/lipophilicity affect considerably the interaction
of these compounds with the binding site.
(kcal/mol“
S
-Linezolid
-24
-6.2
-2.0
-1.7
-0.3
-0.2
S
RR-21b
RR-21a
Compound
21a
log PKow
-0.26
1.10
R-24
21b
Table 3. Docking scores (MAO-A“ for compounds 24,
Linezolid.
(R,R“-21a-b, and (S“-
23a*
-0.50
1.72
1.48
0.10
1.26
(S“-24
(S“-26
(S“-28
Linezolid
Both (
C“ toward the MAO-A aromatic cage, formed by FAD, TYR-
407 and TYR-444. On the other side, ( “-24 is forced to
S“-Linezolid and (S“-24 direct the morpholine ring (ring-
S
penetrate deeper into the binding site of MAO-A in order to
accommodate the bulky isopropyl group in the same space
Table 2. Prediction of lop PKow.. (*the log P was calculated for the neutral form of
this compound“
occupied by the oxazolidinone ring (ring-A“ of (
(Figure 4“. A single hydrogen bond interaction was found for
the tail amide NH bond of ( “-Linezolid with VAL-210, as well
as for the central C=O bond of (S“-24 with THR-336.
S“-Linezolid
S
2.4 Modeling studies

ACCEPTED MANUSCRIPT
Figure 6. Best docking pose of (R,R“-21a (orange carbon atoms“ superimposed to
the best docking pose of (R,R“-21b (purple carbon atoms“ in the active site of MAO-
A. Residues of the binding site are represented with green carbon atoms and FAD
as CPK.
Much better and encouraging results were found in the docking
of compounds 21a, 21b and 24 to the human MAO-B model,
as evidenced by the best calculated binding free energies
reported in Table 4. For all compound examined, a second
almost isoenergetic binding pose was invariably found,
differing only in the relative disposition of the fluorinated
aromatic ring (ring-B“. Moreover, for all compounds with the
exception of (R,R“-21b, two distinct binding poses with very
similar energies were located. They differ for the relative
orientation of the ligand in the binding site, one (A“ having the
morpholine ring (ring-C“ and the other (B“ having the amide tail
directed toward the MAO-B aromatic cage, formed by TYR-398
and TYR-435.
Figure 4. Best docking pose of (
S“-Linezolid (cyan carbon atoms“ superimposed to
the best docking pose of ( “-24 (orange carbon atoms“ in the active site of MAO-A.
S
Residues of the binding site are represented with green carbon atoms and FAD as
CPK.
Free energy of binding
A similar binding pose was found also for the enantiomeric
compound (R“-24, which is forced even deeper into the MAO-A
aromatic cage due to the different spatial disposition of the
isopropyl substituent (Figure 5“. Also in this case, a single
hydrogen bond interaction was found for the tail amide NH
Compound
(kcal/mol“
R-24
-8.4 (A“, -8.9 (B“
-7.4 (A“, -7.6 (B“
-7.2 (A“, -6.8 (B“,
-6.5 (A“, -6.7 (B“
-4.5 (A“,
S
-Linezolid
RR-21a
SR-21a
RR-21b
bond of (R“-24 with PHE-208.
S
-24
-4.1 (A“, -3.6 (B“,
Table 4. Docking scores (MAO-B“ for compounds 24,
Linezolid.
(R,R“-21a-b, and (S“-
The morpholine ring (ring-C“ and the aromatic ring (ring-B“ of
the best docking poses of ( “-24 and of ( “-Linezolid are
R
S
almost perfectly superimposed, confined in the bounding
region between the entrance cavity and the substrate cavity
defined by ILE-199. Accordingly, the amide tails are directed
toward the aromatic cage of MAO-B, between TYR-398 and
TYR-435 (Figure 7“. A network of four hydrogen bonds
contributes to stabilize the docking pose of (R“-24, namely
between the tail NH bond and GLN-206, the tail amide C=O
bond and both TYR-435 and CYS-172 and the central amide
C=O bond and TYR-326. Two relevant hydrogen bond
Figure 5. Best docking pose of (
S“-24 (orange carbon atoms“ superimposed to the
best docking pose of ( “-24 (purple carbon atoms“ in the active site of MAO-A.
R
Residues of the binding site are represented with green carbon atoms and FAD as
CPK.
interactions were found for (S“-Linezolid, one between the tail
NH bond and TYR-398 and the other between the
oxazolidinone C=O bond and ILE-198.
Interestingly, isoxazolidinones (R,R“-21a and b displayed two
almost superimposable docking poses, completely different to
the one of the reference ligand (S“-Linezolid (Figure 6“. The N-
ethyl amide tail of the two isoxazolidinones is in this case
directed toward the aromatic cage of MAO-A, whereas the
morpholine ring (ring-C“ is positioned in the direction of the
cavity entrance, in the pocket formed by VAL-93, LEU-97 and
THR-211.
Figure 7. Best docking pose of
(
S“-Linezolid (pose B, cyan carbon atoms“
superimposed to the best docking pose of (
R“-24 (pose B, orange carbon atoms“ in
the active site of MAO-B. Residues of the binding site are represented with green
carbon atoms and FAD as CPK.
As mentioned previously, the best docking pose for the 3,4-
dehydro-ꢀ-pirrole derivative (
respect to its enantiomer ( “-24, directing the morpholine ring
(ring-C“ toward the aromatic cage of MAO-B and the amide tail
S“-24 is oriented differently with
R

ACCEPTED MANUSCRIPT
Figure 10. Superimposed best docking poses of (S,R“-21a (pose B, orange carbon
atoms“ and pose A, purple carbon atoms“ in the active site of MAO-B. Residues of
the binding site are represented with green carbon atoms and FAD as CPK.
in the direction of the entrance cavity (Figure 8“. A single
hydrogen bond interaction was found for (S“-24, involving the
central amide C=O bond and ILE-199.
Finally, the only suitable docking pose for the hindered
cyclohexyl-substituted isoxazolidinone (R,R“-21b is reported in
Figure 11, superimposed to best docking pose of the isopropyl-
substituted analogue (R,R“-21a. Again, as we found for the
docking poses involving MAO-A, the two poses are almost
perfectly superimposed, with the bulky cyclohexyl substituent
confined in the region delimited by LEU-167, ILE-316 and
LEU-164.
Figure 8. Best docking pose of
(
R“-24 (pose B, orange carbon atoms“
superimposed to the best docking pose of (
S“-24 (pose A, purple carbon atoms“ in
the active site of MAO-B. Residues of the binding site are represented with green
carbon atoms and FAD as CPK.
The possible binding poses (A and B“ for isoxazolidinones
(R,R“-21a and (S,R“-21a, differing only in the configuration of
the isopropyl substituted carbon, are reported in Figures 9 and
10, respectively. Three relevant hydrogen bond interactions
are present in both poses of (R,R“-21a. In pose A they involve
the isoxazolidone carbonyl with TYR-326 and ILE-199 and the
tail amide C=O bond with PRO-102, while in pose B the
hydrogen bonds are formed between the tail NH bond and
GLN-206, the tail amide C=O bond and LEU-171 and the
central amide C=O bond and TYR-326.
Figure 11. Best docking pose of (R,R“-21b (orange carbon atoms“ superimposed to
the best docking pose of (R,R“-21a (pose A, purple carbon atoms“ in the active site
of MAO-B. Residues of the binding site are represented with green carbon atoms
and FAD as CPK.
Not surprisingly, possessing almost an identical orientation in
the binding site of MAO-B, the same network of hydrogen
bonds characterizing (R,R“-21a between the isoxazolidinone
carbonyl with TYR-326 and ILE-199 and the tail amide C=O
bond with PRO-102, was found also for (R,R“-21b .
Modeling studies seem to suggest that all the members of the
new family of compounds synthesized are selective against the
binding to MAO-B receptor. In particular the 3,4-dehydro-ꢀ-
pirrole derivative (
an affinity for the binding site of MAO-B similar or even greater
to that of the reference ligand ( “-Linezolid. Moreover, it is
worth to note that also its enantiomer ( “-24 seems to display
R“-24 seems the more promising, displaying
S
S
a good degree of selectivity against MAO-B receptor. Among
the family of isopropyl-substituted isoxazolidinones 21a, only
the ones possessing the
R
configuration at the C4
S,R“-21a, displayed
Figure 9. Superimposed best docking poses of (R,R“-21a (pose A, orange carbon
atoms“ and pose B, purple carbon atoms“ in the active site of MAO-B. Residues of
the binding site are represented with green carbon atoms and FAD as CPK.
stereocenter, namely R,R“-21a and
(
(
favourable binding energies to MAO-B receptor. Interestingly,
the absolute orientation of the substituent in C4 for these two
compounds is the same of the acetamido-methyl substituent
on the oxazolidinone ring of (
more hindered isoxazolidone analogues 21b, only the RR
stereoisomer displayed a moderate activity against MAO-B
isoform. For all the other stereoisomers, no suitable low energy
binding poses were found, due to the steric clashes invariably
present between the bulky cyclohexyl substituent and the
residues of the binding site.
In the case of (S,R“-21a, two hydrogen bond interactions were
found only for pose A, involving the central amide C=O bond
and TYR-326 and the isoxazolidone carbonyl group with PHE-
168.
S“-Linezolid. Finally, among the
-
3. Conclusion
The balance between antibiotic activity and MAO-inhibition is
an
important
issue
to
be
considered
when
developing oxazolidinone based bioactive compounds. In this
work we reported novel compounds, designed to be Linezolid
analogues, where isoxazolidinone or 3,4-dehydro-ꢀ-
proline were introduced as scaffolds in substitution of

ACCEPTED MANUSCRIPT
oxazolidinone ring. Among the small library of synthesized
43.5, 29.1, 21.8, 14.9. LC-ESI-MS: 8.8 min [M+H]+=200,
compounds, only one showed a modest antibiotic effect, thus
demonstrating that the changes in the molecule backbone
were detrimental for antibacterial activity. On the other hand, a
promising inhibitory effect on to monoamino oxidase (MAO“
was observed and confirmed by docking experiments that
suggested a good interaction of all the members of the new
family of compounds on MAO-B receptor. In particular the 3,4-
[M+K]+=238, [2M+Na]+=421 -isomer: Yellow oil; 1H-NMR (400
Z
MHz, CDCl3“ ꢇ 6.88 (bs, 1H“, 6.79 (d, 3J=10.4 Hz, 1H“, 3.76 (s,
3
3H“, 3.40-3.34 (m, 2H“, 3.26 (m, 1H“, 1.18 (t, J=7.2 Hz, 3H“,
1.06 (d, 3J=6.8 Hz, 6H“.13C-NMR (400 MHz, CDCl3“ ꢇ167.7,
163.3, 160.3, 125.0, 51.3, 43.3, 28.9, 21.8, 14.9. LC-ESI-MS:
8.0 min [M+H]+=200, [M+K]+=238, [2M+Na]+=421.
(2b“ E
-isomer: Yellow oil;1H-NMR (400 MHz, CDCl3“ ꢇ 6.91 (d,
dehydro-ꢀ-pirrole derivative (
displaying an affinity for the binding site of MAO-B.
R
“-24 seems the more promising,
3J=10.4 Hz, 1H“, 6.93 (bs, 1H“, 3.74 (s, 3H“, 3.41-3.30 (m, 2H“,
3.75 (m, 1H“, 1.73-1.64 (m, 5H“, 1.37-1.07 (m, 5H“, 1.17 (t,
3J=7.2 Hz, 3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ164.3, 163.7,
155.3, 125.6, 51.3, 38.0, 33.7, 31.3, 25.2, 24.7, 14.0. LC-ESI-
4. Experimental
4.1. Chemistry
MS: 8.9 min, [M+H]+=240, [M+Na]+=262, [2M+Na]+=501.
Z-
isomer: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 6.80 (d,
3J=10.4 Hz, 1H“, 6.93 (bs, 1H“, 3.74 (s, 3H“, 3.41-3.30 (m, 2H“,
3.75 (m, 1H“, 1.73-1.64 (m, 5H“, 1.37-1.07 (m, 5H“, 1.17 (t,
3J=7.2 Hz, 3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ166.7, 164.1,
155.3, 126.8, 51.5, 38.0, 33.7, 31.3, 25.2, 24.7, 14.0. LC-ESI-
MS: 6.6 min [M+H]+=240, [M+Na]+=262, [2M+Na]+=501.
All chemicals were purchased from commercial suppliers and
used without further purification. Anhydrous solvents were
purchased in sure seal bottles over molecular sieves and used
without further drying. Flash chromatography was performed
on silica gel (230ꢆ400 mesh“. NMR Spectra were recorded
with Mercury Plus 400 MHz spectrometers. Chemical shifts
were reported as δ values (ppm“ relative to the solvent peak of
CDCl3 set at ꢇ= 7.27 ppm (1H-NMR“ or ꢇ = 77.0 ppm (13C-
NMR“, CD3OD set at ꢇ = 3.31 ppm (1H-NMR“ or ꢇ = 49.0 ppm
(13C-NMR“, D2O set at ꢇ = 4.79 ppm (1H-NMR“. Coupling
constants are given in Hertz. Optical rotations were measured
in a Perkin Elmer 343 polarimeter using a 1 dm cuvette and
are referenced to the Na-D line value. LCꢆMS analyses were
performed on a HP1100 liquid chromatography coupled with
an electrospray ionization-mass spectrometer (LCꢆESIꢆMS“,
using H2O/CH3CN or H2O/CH3CN acid for 0.2% of CH2O2 as
solvents at 25° C (positive scan 100ꢆ500 m/z, fragmentor 70
V“.
(3a“, (3b“
In
a 2-necked round-bottom flask, equipped to perform
reaction under N2 flow, 2 (1 eq, 3.44 mmol“ was dissolved in
dry DCM (17.2 ml“ togheter with Sc(OTf“2 and stirred at room
temperature for 15 min. The mixture was cooled to -40 °C and
N,O-bis(trimethylsilyl“ hydroxylamine (1.5 eq, 5.16 mmol“ was
added. After stirring for 1.5 h, Tetrabutylammonium fluoride (2
eq, 6.88 mmol“ was added and the mixture stirred at room
temperature for further 15 min. The reaction was quenched
with water and the crude extracted with DCM. Purification was
pefomed by flash chromatography (silica gel, 8:2-
>7:3=Cy:EtOAc“.
Yield of (3a“: 63%. Yield of (3b“: 66%.
(3a“: Waxy solid 1H-NMR (400 MHz, CDCl3“ ꢇ 6.34 (bs, 1H“,
3.94 (dd, 3J=6.4 Hz, 3J=4.4 Hz, 1H“, 3.39-3.26 (m, 2H“, 3.30
4.2. Synthesis
3
3
(d, J=6.4 Hz, 1H“, 1.91 (m, 1H“, 1.16 (t, J=7.2 Hz, 3H“, 0.99
(d, 3J=6.8 Hz, 3H“, 0.97 (d, 3J=6.8 Hz, 3H“. 13C-NMR (400
MHz, CDCl3“ ꢇ165.9, 163.9, 66.5, 51.4, 35.1, 30.8, 18.9 18.0,
14.4. LC-ESI-MS: 2.4 min, [M+H]+=201, [M+Na]+=223,
[M+K]+=239.
(1“
In
a 2-necked round-bottom flask, equipped to perform
reaction under N2, ethylamine (1.2 eq, 22.3 mmol“ and TEA (2
eq, 37.2 mmol“ were added in dry DCM (19 ml“. Methyl
malonyl chloride (1 eq, 18.6 mmol“ was added dropwise at 0°C
and the mixture stirred at room temperature overnight. The
reaction was quenched with water and the product extracted
with DCM. The product was used without further purifications
(Y%>99%“.
(1“: Pale yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 7.02 (bs,
1H“, 3.73 (s, 3H“, 3.44-3.27 (m, 2H“, 3.29 (s, 2H“, 1.14 (dt,
3J=7.2 Hz, 4J=1.6 Hz, 3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ168.2,
166.4, 51.1, 36.5, 33.7, 14.9. LC-ESI-MS: 1.5 min,
[M+H]+=146, [M+Na]+=178, [2M+Na]+=313.
(3b“: Waxy solid 1H-NMR (400 MHz, CDCl3“ ꢇ 6.27 (bs, 1H“,
3.97 (dd, 3J=6.4 Hz, 3J=5.6 Hz, 1H“, 3.38-3.27 (m, 2H“, 3.31
3
(d, J=5.2 Hz, 1H“, 1.84-1.55 (m, 6H“, 1.27-1.02 (m, 5H“, 1.16
3
(t, J=7.2 Hz, 3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ165.5, 163. 5,
66.9, 51.9, 40.1, 34.7, 28.9, 26.5, 25.7, 25.4, 25.3, 13.9. LC-
ESI-MS: 5.8 min, [M+H]+=241, [M+Na]+=263, [M+K]+=279.
(4“
Mono-tertbutyl
acetoacetate
(1
eq,
24.14
mmol“,
isobutyraldheyde (1.7 eq, 41.03 mmol“ and piperidine (0.15 eq,
3.62 mmol“ were mixed and irradiated using microwaves at
250 watt for 7 min. The reaction was quenched with water and
the crude extracted with EtOAc. The product was isolated
(Y%=64%, E/Z=1/4“ by flash chromatography (silica gel,
(2a“, (2b“
Compound 1 (1 eq, 8.97 mmol“, piperidine (0,15 eq, 1.35
mmol“ and aldehyde (1.7 eq, 15.2 mmol“ were stirred at room
temperature for 3 days. The crude was poured in water and
extracted with EtOAc. Purification by flash chromatography
(silica gel, 9:1=Cy:EtOAc“ allowed to isolate the product as a
mixture of two isomers (Z/E=4/1“.
95:5=Cy:EtOAc“.
1
E
-isomer: Pale yellow oil H-NMR (400 MHz, CDCl3“ ꢇ6.58 (d,
3J=10.6 Hz, 1H“, 2.60 (m, 1H“, 2.34 (s, 3H“, 1.55 (s, 9H“, 1.09
3
(d, J=6.6 Hz, 6H“. 13C-NMR (400 MHz, CDCl3“ꢇ197.3, 165.4,
154.2, 132.9, 79.8, 29.4, 28.7, 24.2, 22.0. LC-ESI-MS: 10.1
Yield of (2a“: 55%. Yield of (2b“: 56%.
min, [M+Na]+=235, [2M+Na]+=467.
Z-isomer: Pale yellow oil
(2a“
E
-isomer: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 6.90 (d,
1H-NMR (400 MHz, CDCl3“ ꢇ6.53 (d, 3J=10.6 Hz, 1H“, 2.65
(m, 1H“, 2.30 (s, 3H“, 1.51 (s, 9H“, 1.45 (d, 3J=6.6 Hz, 6H“.
13C-NMR (400 MHz, CDCl3“ꢇ197.3, 165.4, 154.2, 132.9, 79.8,
3J=10.4 Hz, 1H“, 6.88 (bs, 1H“, 3.76 (s, 3H“, 3.40-3.34 (m, 2H“,
3
3
3.26 (m, 1H“, 1.18 (t, J=7.2 Hz, 3H“, 1.06 (d, J=6.8 Hz, 6H“.
13C-NMR (400 MHz, CDCl3“ ꢇ166.5, 162.1, 160.3, 124.5, 51.2 ,

ACCEPTED MANUSCRIPT
29.6, 28.7, 24.4, 21.9. LC-ESI-MS: 9.3 min [M+Na]+=235,
In a 2-necked round bottom flask, equipped to perform reaction
under N2 flow, [Ir(COD“Cl]2 (0.02 eq, 0.03 mmol“ and P(OPh“3
(0.08 eq, 0.13 mmol“ were dissolved in dry EtOH (8.25 ml“ and
the mixture stirred for 20 min. Allylamine (1 eq, 1.65 mmol“
was added and the mixture was stirred for 20 min. Then 6 (1
eq, 1.65 mmol“ and another aliquot of allylamine (4 eq, 6.6
mmol“ were added and the mixture refluxed for 24 h. The
reaction was quenched with water and the crude was extracted
with EtOAc. After removal of the solvent under vacuum, the
crude was used in the next step without further purifications.
(8“
[2M+Na]+=467.
(5“
In
a 2-necked round-bottom flask, equipped to perform
reaction under N2 flow, Z-4 (1 eq, 8.25 mmol“ was dissolved in
a mixture of THF:MeOH=9:1 (82.5 ml“ and CeCl3.H2O (1 eq,
8.25 mmol“ was added. The mixture was stirred at room
temperature for 30 min, then it was cooled at -30 °C and
NaBH4 (1.1 eq, 9.08 mmol“ was added. After 45 min of stirring
at -30 °C, the reaction was quenched with water and the crude
extracted with EtOAc. Purification by flash chromatography
(silica gel, 95:5=Cy:EtOAc“ allowed to isolate the product
(Y%=92%“. Racemic 5 was then dissolved in Et2O (42.5 ml“,
and vinylacetate (5 eq, 37.9 mmol“ and Pseudomonas cepacia
lipase (35 mg/ml“ were added. The mixture was refluxed for 30
h and then filtered to recover the enzyme. The organic phase
was concentrated and the crude was purified by flash
chromatography (silica gel, 95:5=Cy:EtOAc“ to isolate the
1
3
(7“: Sticky solid; H-NMR (400 MHz, CDCl3“ ꢇ6.89 (q, J= 7.2
Hz, 1H“, 5.86 (m, 1H“, 5.15 (dd, 3J=17.2 Hz, 2J=2 Hz, 1H“, 5.03
(dd, 3J=10.4 Hz, 2J=1.6 Hz, 1H“ 3.22 (dd, 3J=14 Hz, 3J=5.2 Hz,
1H“, 3.08 (d, 3J=9.2 Hz, 1H“, 2.96 (dd, 2J=14 Hz, 3J=6.8 Hz,
3
1H“, 1.92 (m, 1H“, 1.75 (d, J=7.2 Hz, 3H“, 1.46 (s, 9H“, 1.07
(d, 3J=6.4 Hz, 3H“, 0.74 (d, 3J=6.4 Hz, 3H“. 13C-NMR (400
MHz, CDCl3“ ꢇ166.7, 138.4, 136.5, 134.3, 115.0, 80.1, 61.4,
49.7, 32.0, 28.1, 21.0, 20.1, 13.9. LC-ESI-MS: 15.0 min,
product (S“-5 and the corresponding acetate of the
R
[M+H]+=254.
S
-enantiomer [α]D= +19.5,
R-enantiomer [α]D= -
enantiomer, that was hydrolysed with K2CO3 (1 eq“ in MeOH
by stirring the mixture for 30 min at room temperature, and
then quenching it with HCl 1M. The product (R“-5 obtained
from extraction with EtOAc was used without further
purifications.
20.6 (c 1 in CHCl3“.
(8“
A NaHCO3 (4 eq, 5.60 mmol“ solution in water (2.5 ml“,
compound 7 (1 eq, 1.40 mmol“ and Fmoc-Cl (1 eq, 1,4 mmol“
were added to dioxane (1,4 ml“ and the mixture was stirred at
room temperature overnight. The reaction was quenched with
water and the crude extracted with EtOAc and purified by flash
chromatography (silica gel, 95:5=Cy:EtOAc“, to isolate the
products are yellow oils.
Yield of (
S“-5: 94%. Yield of (R“-5: 90%.
E
-isomer: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ6.37(d,
3J=10.4 Hz, 1H“, 4.10 (q, 3J=6.8 Hz, 1H“, 3.03 (m, 1H“, 1.51 (s,
9H“, 1.32 (d, J=6.6 Hz, 3H“, 0.99 (d, J=8 Hz, 6H“. 13C-NMR
3
3
(400 MHz, CDCl3“ ꢇ166.8, 147.3, 131.9, 81.0, 58.6, 29.1, 28.3,
22.6, 22.4.
Z
-isomer: Yellow oil; 1H-NMR (400 MHz, CDCl3“
Yield of (S“-8: 90%. Yield of (R“-8: 89%.
ꢇ5.75 (d, 3J=10 Hz, 1H“, 4.36 (q, 3J=6.4 Hz, 1H“, 3.03 (m, 1H“,
1.51 (s, 9H“, 1.32 (d, 3J=6.6 Hz, 3H“, 0.99 (d, 3J=8 Hz, 6H“.
13C-NMR (400 MHz, CDCl3“ ꢇ167.2, 146.7, 130.1, 81.1, 58.8,
28.2, 27.7, 22.6, 22.4. LC-MS (ESI“: 1.6 min, [M+H]+=215,
Isomer a 1H-NMR (400 MHz, CDCl3“ ꢇ 7.73 (d, 3J=7.2 Hz, 2H“,
7.57 (dd, 3J=7.6 Hz, 3J=7.6 Hz, 2H“, 7.36 (dd, 3J=8.4 Hz,
3
3J=8.4 Hz, 2H“, 7.a30-7.24 (m, 2H“, 6.83 (q, J=7.2 Hz, 1H“,
5.57 (m, 1H“, 4.82 (m, 2H“, 4.52 (m, 3H“, 4.20 (m, 1H“, 3.90
(m, 2H“, 2.62 (m, 1H“, 1.87 (d, 3J=7.2 Hz, 3H“, 1.45 (s, 9H“,
0.82 (d, 3J=6.4 Hz, 3H“, 0.76 (d, 3J=6.4 Hz, 3H“. 13C-NMR (400
MHz, CDCl3, ꢇ166.7, 156.8, 143.3, 142.9, 135.1, 134.6, 129.0,
127.5, 126.9, 124.9, 124.4, 118.8, 80.4, 66.8, 59.5, 47.5, 45.5,
28.0, 26.8, 20.0, 19.7, 14.5. Isomer b 1H-NMR (400 MHz,
[M+Na]+=227, [2M+Na]+=451.
S
-enantiomer [α]D= -26,
R-
enantiomer [α]D= +38 (c 1 in CHCl3“.
(6“
In a 2-necked round bottom flask, equipped to perform reaction
under N2 flow, each enantiomer of -5 (1 eq, 1.83 mmol“ was
3
3
3
Z
CDCl3“ ꢇ 7.73 (d, J=7.2 Hz, 2H“, 7.57 (dd, J=7.6 Hz, J=7.6
3
3
dissolved in THF at -78 °C. LiHMDA (1.5 eq, 2.74 mmol“ was
added and the mixture was stirred for 30 min. Methyl
chloroformiate (2 eq, 3.66 mmol“ was added dropwise and the
mixture was stirred for 1.5 h at -78 °C. The reaction was
quenched with water and the crude extracted with EtOAc. It
was purified by flash chromatography (silica gel,
95:5=Cy:EtOAc“ to isolate the product .
Hz, 2H“, 7.36 (dd, J=8.4 Hz, J=8.4 Hz, 2H“, 7.a30-7.24 (m,
2H“, 6.62 (q, J=7.2 Hz, 1H“, 5.57 (m, 1H“, 4.82 (m, 2H“, 4.52
3
(m, 3H“, 4.20 (m, 1H“, 3.90 (m, 2H“, 2.45 (m, 1H“, 1.87 (d,
3
3J=7.2 Hz, 3H“, 1.45 (s, 9H“, 0.82 (d, J=6.4 Hz, 3H“, 0.76 (d,
3J=6.4 Hz, 3H“. 13C-NMR (400 MHz, CDCl3,ꢇ166.7, 156.8,
144.2, 142.7, 135.3, 133.5, 127.3, 126.9, 124.9, 124.4, 119.8,
80.3, 66.8, 66.1, 47.4, 45.8, 28.0, 27.3, 19.8, 19.2, 14.5. LC-
Yield of (
E
S
“-6: 75%. Yield of (
R
“-6: 78%.
ESI-MS: 19.0 min, 21.5 min, [M+H]+=476, [M+H]+=498.
S-
enantiomer [α]D= +6.38, R-enantiomer [α]D= -2.71 (c 1 in
CHCl3“.
-isomer: Transparent oil; 1H-NMR (400 MHz, CDCl3“ ꢇ5.81
(dd, 3J=9.6 Hz, 4J=1.2 Hz, 1H“, 5.44 (q, 3J=6.4 Hz, 1H“, 3.77
(s, 3H“, 3.15 (m, 1H“, 1.51 (s, 9H“, 1.40 (d, 3J=6.8 Hz, 3H“,
0.99 (d, 3J=6.8 Hz, 6H“. 13C-NMR (400 MHz, CDCl3“ ꢇ165.6,
155.0, 146.7, 131.3, 81.2, 74.0, 54.5, 28.1, 26.2, 22.5, 19.8.
(9“
In a 2-necked round bottom flask, equipped to perform reaction
under N2 flow, enantiopure 8 (1 eq, 1.25 mmol“ was dissolved
in MTBE (6.25 ml“ and Grubbs-II generation catalyst (0.03 eq,
0.04 mmol“ was added. The mixture was stirred at 50 °C for 20
h. The reaction was quenched with vinilacetate and diluited
with EtOAc. The organic layer was washed with water and
concentrated under vacuum. The crude was purified by flash
chromatography (silica gel, 98:2=Cy:EtOAc“ to isolate the
product as a single enantiomer.
LC-ESI-MS: 11.8 min [M+H]+=273, [M+Na]+=295.
Z-isomer :
Transparent oil; 1H-NMR (400 MHz, CDCl3“ ꢇ5.76 (d, 3J=9.6
Hz, 1H“, 4.36 (q, 3J=6.4 Hz, 1H“, 3.77 (s, 3H“, 3.00 (m, 1H“,
1.48 (s, 9H“, 1.32 (d, 3J=6.4 Hz, 3H“, 0.99 (d, 3J=6.8 Hz, 6H“.
13C-NMR (400 MHz, CDCl3“ ꢇ165.6, 155.0, 146.7, 131.3, 81.2,
74.0, 54.5, 28.1, 26.2, 22.5, 19.8. LC-ESI-MS: 8.9 min,
[M+H]+=273, [M+Na]+=295.
S
-enantiomer [α]D= -30.0,
R-
enantiomer [α]D= +28.9 (c 1 in CHCl3“.
Yield of (S“-9: 81%. Yield of (R“-9: 79%.
(7“

ACCEPTED MANUSCRIPT
Isomer a: Transparent oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.75 (d,
(28“: Waxy solid; 1H-NMR (400 MHz, CD3OD“ ꢇ7.34 (d, 3J=14
3
3
3
3
3
3J=6.8 Hz, 2H“, 7.62-7.53 (m, 2H“, 7.38 (dd, J=7.6 Hz, J=6.8
Hz, 1H“, 7.31 (d, J=18.4 Hz, 1H“, 7.10 (dd, J=8.4 Hz, J=8.4
Hz, 1H“, 6.34 (m, 1H“, 5.43 (m, 1H“, 5.48 (d, 3J=16.4 Hz, 1H“,
4.16 (d, 3J=16.4 Hz, 2H“, 3.98 (s, 2H“, 3.78 (q, 3J=7.2 Hz, 2H“,
3.65-3.58 (m, 2H“, 3.27- 3.16 (m, 6H“, 2.29 (m, 1H“, 1.13 (t,
3J=7.2 Hz, 3H“, 1.01 (d, 3J=6.8 Hz, 3H“, 0.94 (d, 3J=6.8 Hz,
3H“. 13C-NMR (400 MHz, CD3OD“ ꢇ169.7, 166.8, 163.1, 153.4,
Hz, 2H“, 7.31 (dd, 3J=7.6 Hz, 3J=7.2 Hz, 2H“, 6.64 (m, 1H“,
3
4.87 (m, 1H“, 4.56 (d, J=5.6 Hz, 2H“, 4.55-4.22 (m, 3H“, 2.30
3
3
(m, 1H“, 1.48 (s, 9H“, 0.93 (d, J=6.8 Hz, 3H“, 0.86 (d, J=6.8
Hz, 3H“. 13C-NMR (400 MHz, CDCl3“ꢇ162.5, 155.0, 144.0,
143.8, 136.8, 136.0, 127.6, 124.9, 124.7, 119.9, 81.3, 68.8,
67.0, 54.2, 47.3, 32.5, 28.0, 19.0, 18.2. Isomer b: Transparent
3
141.1, 139.2, 130.5, 129.4, 124.2, 118.3, 117.0 (d, J=22 Hz“,
3
oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.75 (d, J=6.8 Hz, 2H“, 7.62-
67.5, 57.7, 49.3, 38.5, 34.1, 29.2, 19.1, 17.5, 14.3. LC-ESI-MS:
7.53 (m, 2H“, 7.38 (dd, 3J=7.6 Hz, 3J=6.8 Hz, 2H“, 7.31 (dd,
3J=7.6 Hz, 3J=7.2 Hz, 2H“, 6.64 (m, 1H“, 4.87 (m, 1H“, 4.56 (d,
3J=5.6 Hz, 2H“, 4.55-4.22 (m, 3H“, 1.89 (m, 1H“, 1.48 (s, 9H“,
0.73 (d, 3J=6.8 Hz, 3H“, 0.65 (d, 3J=6.8 Hz, 3H“. 13C-NMR (400
MHz, CDCl3“ꢇ162.5, 154.8, 144.0, 143,8, 136.2, 135.6, 127.0,
124.9, 124.6, 119.9, 81.1, 68.4, 66.7, 53.9, 47.3, 32.2, 28.0,
1.6 min, [M+H]+=446, [M+Na]+=478.
S-enantiomer [α]D= -17,
R-enantiomer [α]D= +32 (c 1 in CHCl3“.
(11“
In a 2-necked round bottom flask, equipped to perform reaction
under N2 flow, 10 (1 eq, 1.01 mmol“, TEA (1.5 eq, 1.52 mmol“
and HBTU (1.5 eq, 1.52 mmol“ were dissolved in dry DCM
(13.5 ml“. Ethylamine (2 eq, 2.02 mmol“ was added and the
mixture stirred overnight at room temperature. The reaction
was quenched with water and the crude extracted with DCM.
Purification by flash chromatography (silica gel, 6:4=Cy:EtOAc“
allowed to isolate the product.
17.8, 17.6. LC-ESI-MS: 15.6 min, [M+H]+=434.
[α]D= -17.7, -enantiomer [α]D= +26.5 ( 1 in CHCl3“.
S-enantiomer
R
c
(10“, (23a“, (26“, (28“
Ester 9 or carbamate 22a-25-27 (1 eq, 1.01 mmol“ was
dissolved in DCM (10.1 ml“ and TFA (15 eq, 15.21 mmol“ was
added at 0 °C. The mixture was stirred at room temperature
overnight and then evaporated under vacuum. The product
was used without further purifications.
Yield of (S“-11: 98%. Yield of (R“-11: 96%.
Isomer a: Pale yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 7.75 (d,
3J=7.2 Hz, 2H“, 7.58 (d, 3J=7.2 Hz, 2H“, 7.47 (bs, 1H“, 7.38
(dd, 3J=7.2 Hz, 3J=7.2 Hz, 2H“, 7.30 (dd, 3J=7.2 Hz, 3J=7.2 Hz,
2H“, 6.24 (m, 1H“, 5.67 (m, 1H“, 4.59 (d, 3J=5.2 Hz, 1H“, 4.48-
Yield of (S“-10, (R“-10, 23a, 26, 28: 99%
(10“ Isomer a: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 7.76 (d,
3
3
3
3J=7.6 Hz, 2H“, 7.76-7.56 (m, 2H“, 7.39 (dd, J=7.2 Hz, J=7.2
4.21 (m, 3H“, 4.05 (m, 1H“, 3.34 (q, J=7.2 Hz, 2H“, 2.31 (m,
Hz, 2H“, 7.32 (dd, 3J=6.8 Hz, 3J=6.8 Hz, 2H“, 6.92 (m, 1H“,
4.66 (d, J=5.2 Hz, 1H“, 4.56 (m, 1H“, 4.43 (m, 1H“, 4.33-4.20
(m, 2H“, 4.09 (dd, J=18.8 Hz, J= 4.8 Hz, 1H“, 2.30 (m, 1H“,
0.94 (d, 3J= 6.8 Hz, 3H“, 0.84 (d, 3J= 6.8 Hz, 3H“. 13C-NMR
(400 MHz, CDCl3“ ꢇ168.7, 156.1, 143.3, 141.4, 140.8, 133.2,
127.8, 127.1, 124.7, 120.0, 68.2, 67.8, 54.5, 47.1, 32.1, 18.9,
1H“, 1.16 (t, J=7.2 Hz, 3H“, 0.70 (d, J=6.8 Hz, 3H“, 0.60 (d,
3J=6.8 Hz, 3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ171.0, 154.8,
143.8, 141.3, 138.7, 138.5, 128.8, 127.6, 124.8, 124.5, 68.8,
66.9, 54.2, 47.3, 34.3, 31.8, 20.9, 14.5. LC-ESI-MS: 11.4 min
[M+H]+=405. Isomer b: Transparent oil; 1H-NMR (400 MHz,
CDCl3“ ꢇ 7.75 (d, 3J=7.2 Hz, 2H“, 7.58 (d, 3J=7.2 Hz, 2H“, 7.47
3
3
3
2
3
1
3
3
3
17.7. Isomer b: Yellow oil; H-NMR (400 MHz, CDCl3“ ꢇ 7.76
(bs, 1H“, 7.38 (dd, J=7.2 Hz, J=7.2 Hz, 2H“, 7.30 (dd, J=7.2
(d, 3J=7.6 Hz, 2H“, 7.76-7.56 (m, 2H“, 7.39 (dd, 3J=7.2 Hz,
Hz, 3J=7.2 Hz, 2H“, 6.24 (m, 1H“, 5.67 (m, 1H“, 4.59 (d, 3J=5.2
Hz, 1H“, 4.48-4.21 (m, 3H“, 4.05 (m, 1H“, 3.34 (q, J=7.2 Hz,
3J=7.2 Hz, 2H“, 7.32 (dd, J=6.8 Hz, J=6.8 Hz, 2H“, 6.92 (m,
3
3
3
1H“, 4.66 (d, 3J=5.2 Hz, 1H“, 4.56 (m, 1H“, 4.43 (m, 1H“, 4.33-
2H“, 1.85 (m, 1H“, 1.16 (t, J=7.2 Hz, 3H“, 0.70 (d, J=6.8 Hz,
3H“, 0.60 (d, 3J=6.8 Hz, 3H“. 13C-NMR (400 MHz, CDCl3“
ꢇ171.0, 154.5, 143.8, 141.2, 138.7, 138.5, 128.3, 126.9, 124.6,
119.8, 68.3, 66.5, 53.9, 47.2, 34.3, 32.1, 20.9, 14.0. LC-ESI-
MS: 10.1 min [M+H]+=405.
enantiomer [α]D= +25.81 (c 1 in CHCl3“.
3
3
2
3
4.20 (m, 2H“, 4.09 (dd, J=18.8 Hz, J= 4.8 Hz, 1H“, 1.87 (m,
1H“, 0.70 (d, 3J= 6.8 Hz, 3H“, 0.62 (d, 3J= 6.8 Hz, 3H“. 13C-
NMR (400 MHz, CDCl3“ ꢇ168.7, 156.1, 143.1, 141.3, 140.5,
133.2, 127.8, 127.1, 124.6, 120.0, 68.2, 67.7, 54.2, 47.1, 32.1,
S
-enantiomer [α]D= -22.37,
R-
18.9, 17.7. LC (acid“-ESI-MS: 10.0 min, [M+H]+=378.
S
-
enantiomer [α]D= -22.22,
R
-enantiomer [α]D= +22.46 (
c
1 in
CHCl3“.
(12“
1
(23a“: Sticky solid; H-NMR (400 MHz,
d-Acetone“ ꢇ7.56 (dd,
Compound 11 (1 eq, 1.01 mmol“ was dissolved in a 30%
solution of piperidine in DMF (1.23 ml“ and stirred at room
temperature for 10 min. The reaction was quenched with water
and the crude extracted with EtOAc. The crude was purified by
flash chromatography (silica gel, 7:3=Cy:EtOAc“ to isolate the
product as pale yellow oil
3J=8.4 Hz, 4J=1.6 Hz, 1H“, 7.48 (dd, 3J=13.6 Hz, 4J=2 Hz, 1H“,
3
7.18 (dd, 3J=8.8 Hz, J=8.8 Hz, 1H“, 5.10 (m, 1H“, 3.77 (d,
3J=2 Hz, 1H“, 3.57 (m, 8H“, 3.20 (q, J=7.2 Hz, 2H“, 2.04 (m,
3
3
3
1H“, 1.10 (t, J=7.2 Hz, 3H“, 1.03 (d, J=6.8 Hz, 3H“, 1.01 (d,
3J=6.8 Hz, 3H“. 13C-NMR (400 MHz,
d-Acetone“ ꢇ171.1, 171.6,
165.9, 154.8 (d, J=233 Hz“, 142.7, 128.0 (d, J=8 Hz“, 127.0 (d,
J=3 Hz“, 119.3 (d, J=3 Hz“, 117.9 (d, J=23 Hz“, 68.5, 50.7,
47.7, 44.3, 35.2 (d, J=13 Hz“, 32.5, 18.4, 18.0, 14.6. LC-ESI-
MS: 1.5 min, [M+H]+=407, [2M+H]+=813.
Yield of (S“-12: 52%. Yield of (R“-12: 55%.
1H-NMR (400 MHz, CDCl3“ ꢇ 6.30 (m, 1H“, 4.57 (bs, 1H“, 4.14-
3
4.03 (m, 2H“, 3.34 (q, J=7.2 Hz, 2H“, 2.36 (m, 1H“, 1.17 (t,
3J=7.2 Hz, 3H“, 1.06 (d, 3J=7.2 Hz, 3H“, 0.90 (d, 3J=7.2 Hz,
3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ163.3, 143.1, 138.9, 70.3,
52.6, 34.3, 30.1, 19.4, 15.8. LC-ESI-MS: 1.7 min [M+H]+=183.
(26“: Sticky solid;1H-NMR (400 MHz,
d-Acetone“ ꢇ 7.44 (d,
3J=14.4 Hz, 1H“, 7.36 (d, 3J=13.2 Hz, 1H“, 7.16 (dd, 3J=8.8 Hz,
3J=8.8 Hz, 1H“, 6.37 (m, 1H“, 5.43 (m, 1H“, 4.52-4.47 (m, 2H“,
S
-enantiomer [α]D= -23.3, R-enantiomer [α]D= +15.8 (c 1 in
3
4.03 (q, J=7.2 Hz, 2H“, 3.67-3.53 (m, 6H“, 3.30-3.24 (m, 2H“,
CHCl3“.
3
3
2.29 (m, 1H“, 1.10 (t, J=7.2 Hz, 3H“, 0.98 (d, J=6.8 Hz, 3H“,
0.91 (d, 3J=6.8 Hz, 3H“. 13C-NMR (400 MHz,
d-Acetone“
(13“
ꢇ169.0, 163.4, 159.2, 140.5, 138.6, 131.2, 128.7, 124.6, 117.3,
114.4, 67.8, 57.4, 47.0, 43.8, 38.8, 33.73, 18.8, 17.0, 14.0. LC-
SOCl2 (5 eq, 47.44 mmol“ was added dropwise in MeOH (76
ml“ at 0°C. After stirring for 15 min, 3,4-difluorobenzoic acid
(1,5 g, 9.49 mmol“ was added and the mixture was stirred at
room temperature overnight. The solvent was removed under
ESI-MS: 1.5 min, [M+H]+=390, [2M+H]+=777.
S-enantiomer
[α]D= -38,
R-enantiomer [α]D= +14 (c 1 in CHCl3“.

ACCEPTED MANUSCRIPT
vacuum e the product was used without further purifications
(Y%=63%“ as a waxy solid.
(17“
In
a 2-necked round-bottom flask, equipped to perform
1H-NMR (400 MHz, CDCl3“ ꢇ 7.80 (m, 2H“, 7.21 (dd, 3J=8.4
Hz, 3J=18 Hz, 1H“, 3.91 (s, 3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ
164.5, 153.1 (dd, 2JCF=13 Hz, 1JCF=266 Hz“, 149.6 (dd, 2JCF=13
reaction under N2 flow, Boc2O (1 eq, 1.53 mmol“ was dissolved
in dry DCM (8.4 ml“ and 16 (1.1 eq, 1.68 mmol“ was added.
TEA (1.5 eq, 2.30 mmol“ was added dropwise at 0°C. The
mixture was stirred at room temperature overnight, then
poured into HCl 1M and extracted with DCM. The organic layer
was washed with NaHCO3 sat. sol. and brine. The yellow oil
(Y%>99%“ was used without further purifications.
1
4
3
Hz, JCF=247 Hz“, 126.9 (dd, JCF=13 Hz, JCF=5 Hz“, 126.1
4
3
2
(dd, JCF=3 Hz, JCF=7 Hz“, 118.3 (d, JCF=18 Hz“, 116.8 (d,
2JCF=18 Hz“, 51.7. LC-ESI-MS: 8.3 min, [M+H]+=173.
(14“, (16“
1H-NMR (400 MHz, CDCl3“ ꢇ 7.70 (dd, 3J= 29.6 Hz, 3J=1.6 Hz,
1H“, 7.68 (dd, 3J= 34.8 Hz, 3J=1.6 Hz, 1H“, 6.88 (dd, 3J=8.4
Hz, 3J=6.8 Hz, 1H“, 3.86 (s, 3H“, 3.57 (t, 3J= 4.8 Hz, 4H“, 3.12
(t, 3J= 4.8 Hz, 4H“, 1.50 (s, 9H“. 13C-NMR (400 MHz, CDCl3“
13 (1,45 mmol“, morpholine or piperazine (2 eq, 2.90 mmol“
and K2HPO4 (4 eq, 5.80 mmol“ were dissolvend in DMSO (4.4
ml“ and the mixture was refluxed for 8 h. The solution was then
poured into water and washed with EtOAc. The crude was
purified by flash chromatography (silica gel, 8:2=Cy:EtOAc“ to
isolate the product.
1
2
ꢇ165.3, 154.0, 153.8 (d, JCF=246 Hz“, 143.4 (d, JCF=8 Hz“,
4
3
3
126.0 (d, JCF=3 Hz“, 123.0 (d, JCF=8 Hz“, 117.5 (d, JCF=3
2
Hz“, 116.8 (d, JCF=22 Hz“, 79.3, 51.4, 49.3, 27.0. LC-ESI-MS:
11.1 min, [M+H]+=339.
Yield of 14: 89%. Yield of 16: 99%.
(14“: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 7.74 (dd, 3J=
31.6 Hz, 4J=1.6 Hz, 1H“, 7.72 (dd, 3J= 36.8 Hz, 4J=1.6 Hz, 1H“,
(19“
3
3
3
6.94 (dd, J=8.3 Hz, J=8.3 Hz, 1H“, 3.90 (s, 3H“, 3.88 (t, J=
4.8 Hz, 4H“, 3.21 (t, 3J=4.8 Hz, 4H“. 13C-NMR (400 MHz,
CDCl3“ ꢇ 165.7, 154.0 (d, JCF=244 Hz“, 143.0 (d, JCF=8 Hz“,
In
a
2-necked round-bottom flask, equipped to perform
-(Boc“-Gly-COOH (1 eq, 1,26 mmol“
reaction under N2 flow,
N
1
2
was dissolved in DMF (10.5 ml“, COMU (1 eq, 1.26 mmol“ and
DIPEA (3 eq, 3.78 mmol“ were added at 0°C. After stirring for
10 min, 18 (1 eq, 1.26 mmol“ was added and mixture stirred at
room temperature overnight. The reaction was quenched with
HCl 1M and extracted with EtOAc. The organic layer was
washed with NaHCO3 saturated solution and with brine. The
product (Y%>99%“ was used without further purifications as a
waxy solid.
4
3
3
126.2 (d, JCF=3 Hz“, 123.2 (d, JCF=8 Hz“, 117.1 (d, JCF=3
2
Hz“, 117.1 (d, JCF=22 Hz“, 66.4, 51.7, 49.9. LC-ESI-MS: 7.7
min [M+H]+=240.
(16“: Yellow oil; 1H-NMR (400 MHz,
d
-DMSO“ ꢇ 7.69 (dd,
3J=31.6 Hz, J=1.2 Hz, 1H“, 7.66 (dd, 3J=37.2 Hz, J=1.2 Hz,
1H“, 6.88 (dd, 3J=8.4 Hz, 1H“, 3.86 (s, 3H“, 3.15 (t, 3J=4.4 Hz,
2H“, 3.02 (t, J=4.4 Hz, 2H“, 2.58 (m, 4H“. 13C-NMR (400 MHz,
4
4
1
2
d-DMSO“ ꢇ 163.6, 152.2 (d, JCF=241 Hz“, 142.8 (d, JCF=7
1H-NMR (400 MHz, CDCl3“ ꢇ 7.72 (dd, 3J= 27.2 Hz, 3J=1.6 Hz,
4
3
3
3
3
Hz“, 124.7 (d, JCF=2 Hz“, 120.8 (d, JCF=8 Hz“, 116.0 (d,
1H“, 7.70 (dd, J= 32 Hz, J=1.6 Hz, 1H“, 6.88 (dd, J=8.4 Hz,
3JCF=4 Hz“, 115.2 (d, JCF=4 Hz“, 50.0, 44.2, 39.1. LC (acid“-
3J=8.4 Hz, 1H“, 3.99 (s, 2H“, 3.87 (s, 3H“, 3.79 (t, J= 5.2 Hz,
2
3
ESI-MS: 1.3 min, [M+H]+=239.
2H“, 3.55 (t, J= 5.2 Hz, 2H“, 3.23-3.12 (m, 4H“, 1.44 (s, 9H“.
13C-NMR (400 MHz, CDCl3“ ꢇ166.7, 165.3, 162.1, 153.7 (d,
1JCF=244 Hz“, 142.8 (d, 2JCF=9 Hz“, 125.9 (d, 4JCF=3 Hz“, 123.4
(d, JCF=7 Hz“, 117.5 (d, JCF=3 Hz“, 116.8 (d, JCF=22 Hz“,
78.9, 66.0, 51.5, 46.7, 43.8, 27.7 . LC (acid“-ESI-MS: 8.2 min,
[M+H]+=396.
3
(15“, (18“, (20“
3
3
2
Esters 14 or 17 or 19 (1.29 mmol“ was dissolved in a mixture
1:2=H2O:THF (64.5 ml“, then LiOH.H2O (2 eq, 2.58 mmol“ was
added. The mixture was stirred overnight, acidified and the
product extracted with EtOAc .
Yield of 15: 99%. Yield of 18, 20: 99%.
(21-a,b“, (22a“, (24“, (25“, (27“
(15“: White solid, m.p. 199-201 °C; 1H-NMR (400 MHz,
d-
In a 2-necked round bottom flask, equipped to perform reaction
under N2 flow, acid 15 or 18 or 20 (1.5 eq, 0.22 mmol“ was
dissolved in dry DCM (35 ml“ and thionyl chloride (1.5 eq, 0.22
mmol“ was added dropwise at 0 °C. The mixture was stirred at
room temperature for 1 h and pyridine (5 eq, 0.75 mmol“ was
added. The mixture was stirred for 30 min and cyclic amine 3a
or 3b or (S“-12 or (R“-12 (1 eq, 0.15 mmol“ was added. The
reaction was stirred for 3 h and then quenched with water and
extracted with DCM. The crude was purified by flash
chromatography (silica gel, 1:1=Cy:EtOAc“ to isolate the
product.
DMSO“ ꢇ 7.62 (dd, 3J=46 Hz, 4J=1.6 Hz, 1H“, 7.60 (dd,
3J=51.6 Hz, 4J=1.6 Hz, 1H“, 7.06 (dd, 3J=8.4 Hz, 3J=8.4 Hz,
3
3
1H“, 3.72 (t, J=4.4 Hz, 2H“, 3.11 (t, J=4.4 Hz, 2H“, 2.47 (m,
4H“. 13C-NMR (400 MHz,
-DMSO“ 166.2, 153.5 (d,
1JCF=243 Hz“, 143.3 (d, 2JCF=8 Hz“, 126.4 (d, 4JCF=3 Hz“, 123.7
d
ꢇ
3
3
2
(d, JCF=7 Hz“, 118.1 (d, JCF=3 Hz“, 116.6 (d, JCF=22 Hz“,
66.9, 49.7. LC (acid“-ESI-MS: 3.0 min,[M+H]+=226.
(18“: White solid, m.p. 218-220 °C; 1H-NMR (400 MHz,
CDCl3“ ꢇ 7.69 (dd, 3J=29 Hz, 4J=1.6 Hz, 1H“, 7.66 (dd, J=34.2
4
3
3
Hz, J=1.6 Hz, 1H“, 6.88 (dd, J=6.8 Hz, J=6.8 Hz, 1H“, 3.15
(t, J=4.4 Hz, 4H“, 3.02 (t, J=4.4 Hz, 4H“, 1.45 (s, 9H“. 13C-NMR
(400 MHz, CDCl3“ ꢇ 170.4, 155.3, 152.9, 144.4, 127.8, 122.8,
117.9, 117.7, 80.2, 49.7, 49.6, 24,4. LC (acid“-ESI-MS: 6.3 min
[M+H]+=325.
Yield of 21a: 90%. Yield of 21b: 90%. Yield of 22a: 87%. Yield
of 24: 88%. Yield of 25: 93%. Yield of 27: 90%.
(21a“: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ 7.55 (dd, J=
3
4
3
4
22.4, J= 1.6 Hz, 1H“, 7.52 (dd, J= 27.6 Hz, J= 1.6 Hz, 1H“,
(20“: Pale yellow solid, m.p. 223-225 °C; 1H-NMR (400 MHz,
CDCl3“ ꢇ 7.78 (d, 3J=29.6 Hz, 2H“, 7.76 (d, 3J=34.4 Hz, 1H“,
6.91 (dd, 3J=8.4 Hz, 3J=8.4 Hz, 1H“, 5.56 (bs, 1H“, 4.02 (s,
2H“, 3.82-3.80 (m, 2H“, 3.68 (t, 3J= 4.8 Hz, 2H“, 3.24 (t, 3J=4.8
Hz, 2H“, 3.21-3.19 (m, 2H“, 1.45 (s, 9H“. 13C-NMR (400 MHz,
CDCl3“ ꢇ 169.5, 167.5, 155.6, 153.0, 144.1, 126.9, 123.6,
117.9, 117.8, 79.8, 66.6, 47.2, 38.4, 28.3. LC (acid“-ESI-MS:
7.9 min [M+H]+=382.
6.98 (dd, J= 8.4 Hz, J=8.4 Hz, 1H“, 6.13 (bs, 1H“, 5.27 (dd,
3
3
3J=28 Hz, J=6 Hz, 1H“, 3.88 (t, J= 4.4 Hz, 4H“, 3.40 (d, J=
28 Hz, 1H“, 3.33-3.16 (m, 6H“, 2.14 (m, 1H“, 1.13 (t, 3J=7.2 Hz,
3H“, 1.03 (d, 3J=6.8 Hz, 6H“. 13C-NMR (400 MHz, CDCl3“
ꢇ171.4, 171.1, 162.4, 153.9 (d, 1J=245 Hz“, 143.3 (d, 3J=8 Hz“,
3
3
3
3
2
2
126.4 (d, J=4 Hz“, 124.7 (d, J=13 Hz“, 117.5 (d, J=24 Hz“,
117.3 (d, 3J=4 Hz“, 66.6, 66.0, 50.1, 50.0, 49.6, 35.2, 31.5,
17.9, 17.8, 14.3. LC-ESI-MS: 8.5 min, [M+H]+=408,
[M+Na]+=430.

ACCEPTED MANUSCRIPT
(21b“: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.54 (dd,
3J=23.6 Hz, 4J=2 Hz, 1H“, 7.51 (dd, 3J=28.8 Hz, 4J=2 Hz, 1H“,
6.90 (dd, 3J=8.4 Hz, 3J=8.4 Hz, 1H“, 6.12 (bs, 1H“, 5.25 (dd,
3J=6 Hz, 3J=28 Hz, 1H“, 3.86 (t, 3J=4.8 Hz, 4H“, 3.44 (d, 3J=28
Hz, 1H“, 3.36-3.13 (m, 6H“, 1.80 (m, 1H“, 1.32-1.05 (m, 5H“,
1.12 (t, 3J=7.2 Hz, 3H“, 0.89-0.82 (m, 5H“. 13C-NMR (400 MHz,
CDCl3“ ꢇ171.6, 171.1, 162.8, 153,6 (d, 1J=245 Hz“, 143.2 (d,
2J=8 Hz“, 126.4 (d, 3J=3 Hz“, 124.9, 117.6 (d, 2J=23 Hz“, 117.3
(d, 3J=4 Hz“, 66.7, 65.7, 50.1, 49.9, 41.2, 35.2, 28.5, 28.3,
25.9, 25.6, 14.2. LC-ESI-MS: 10.3 min, [M+H]+=448,
[M+K]+=486, [2M+Na]+=917.
A
total
of
12
well-characterized
for
their
antibiotic-susceptibility phenotype S. aureus recently isolated
strains, both MRSA and MSSA, were used for the
determination of the in vitro antibacterial activity of the studied
compounds. Staphylococcus aureus ATCC 29213 and E.
faecalis ATCC 29212 reference standard strains were used as
control.
4.3.2 Determination of MICs
The in vitro antibacterial activity of the new synthetized
compounds (21a, 21b,
(S“-24, (R“-24“ was studied by
(22a“: Dark yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.54 (d,
3J=20.4 Hz, 1H“, 7.51 (d, 3J=25.2 Hz, 1H“, 6.90 (dd, 3J=8.4 Hz,
3J=8.4 Hz, 1H“, 6.15 (bs, 1H“, 5.26 (dd, 3J=28 Hz, 3J=6 Hz,
1H“, 3.58 (m, 4H“, 3.34-3.25 (m, 2H“, 3.16-3.09 (m, 4H“, 2.12
determining their minimum inhibitory concentrations (MICs“ by
means of the broth microdilution method according to the
Clinical and Laboratory Standards Institute (CLSI“
guidelines.[37] Linezolid (Sigma Aldrich, Italy“ was used as
reference antibiotic compound for MIC determinations.
Briefly, serial 2-fold dilutions of each compound were made
using the Mueller-Hinton broth in microtitre plates with 96
wells. Dimethyl sulfoxide (DMSO“ was used as solvent for all
the synthetized compounds. An equal volume of the bacterial
inoculum
(1x106 CFU/mL“ was added to each well on the microtitre plate
containing 0.05 mL of the serial antibiotic dilutions. The
microtitre
plate was then incubated at 37 C for 18-24 h after which each
well
3
3
(m, 1H“, 1.46 (s, 9H“, 1.12 (t, J=7.2 Hz, 3H“, 1.02 (d, J=6.8
Hz, 6H“. 13C-NMR (400 MHz, CDCl3“ ꢇ171.6, 171.0, 162.1,
154.6, 154.0 (d, 1J=144 Hz“, 143.4 (d, 3J=8 Hz“, 126.4 (d, 3J=3
Hz“, 124.9 (d, 3J=8 Hz“, 117.8 (d, 3J=3 Hz“, 117.6 (d, 2J=24
3
Hz“, 80.0, 65.9, 49.7 (d, J=4 Hz“, 49.6, 35.3, 31.6, 28.3, 18.0,
17.8, 14.4. LC-ESI-MS: 11.0 min, [M+H]+=507, [M+Na]+=529,
[M+K]+=545.
(24“: Yellow oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.46 (bs, 1H“,
7.36 (d, 3J=24.8 Hz, 1H“, 7.34 (d, 3J=30 Hz, 1H“, 7.05 (dd,
3J=8.4 Hz, 3J=8.4 Hz, 1H“, 6.37 (m, 1H“, 5.44 (m, 1H“, 4.49 (d,
3
3
3J=15.6 Hz, 1H“, 4.17 (d, J=16.4 Hz, 1H“, 3.78 (t, J=4.4 Hz,
4H“, 3.26 (m, 2H“, 3.16-3.07 (m, 4H“, 2.28 (m, 1H“, 1.10 (t,
3J=7.2 Hz, 3H“, 0.97 (d, 3J=6.8 Hz, 3H“, 0.90 (d, 3J=6.8 Hz,
3H“. 13C-NMR (400 MHz, CDCl3“ ꢇ169.8, 163.3, 154.5 (d,
was analyzed for the presence of bacterial growth. The MIC
was
defined as the lowest concentration of antimicrobial agent able
to
cause inhibition of bacterial growth as shown by the lack of
visible turbidity of the culture medium.
1J=247 Hz“, 141.9 (d, J=8 Hz“, 139.1, 135.1, 129.4, 124.6 (d,
2J=60 Hz“, 117.8, 116.0 (d, J=24 Hz“, 67.6, 66.7, 57.4, 50.3,
3
2
34.3, 32.7, 19.3, 17.6, 14.6.
[M+H]+=390, [2M+Na]+=412.
-enantiomer [α]D= -58,
enantiomer [α]D= +41 ( 1 in CHCl3“.
LC-ESI-MS: 11.5 min,
S
R-
c
4.4 Cell cultures conditions
(25“: Transparent oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.28 (dd,
3J=13.6 Hz, 4J=1.6 Hz, 1H“, 7.25 (dd, 3J=18 Hz, 3J=1.6 Hz,
1H“, 6.90 (dd, 3J=8.4 Hz, 3J=8.4 Hz, 1H“, 6.33 (m, 1H“, 5.78
(bs, 1H“, 5.47 (m, 1H“, 4.36 (d, J=14.4 Hz, 1H“, 4.16 (d,
3J=14.8 Hz, 1H“, 3.58 (t, 3J=5.2 Hz, 4H“, 3.38-3-31 (m, 2H“,
3.13-3.05 (m, 4H“, 2.31 (m, 1H“, 1.49 (s, 9H“, 1.16 (t. 3J=7.2
Hz, 3H“, 1.02 (d, 3J=7.2 Hz, 3H“, 0.94 (d, J=7.2 Hz, 3H“. 13C-
NMR (400 MHz, CDCl3“ ꢇ169.7, 163.3, 155.8, 154.6, 142.0,
139.1, 129.7, 129.4, 124.3, 118.3, 116.0, 79.9, 67.6, 57.4,
The effects of synthetized compounds (21a, 21b, (
R“-24 and
(S
“-24“ and Linezolid on cells viability and MAO activity were
studied in vitro on HepG2 cells, (human hepatocellular
carcinoma“. Cells were grown in Dulbeccoꢃs modified eagles
medium (DMEM“ supplemented with 10% heat inactivated
foetal bovine serum (FBS“, 2mM L-glutamine, 100 units/ml
penicillin and 100µg/ml streptomycin and maintained on
collagen coated 75-cm2 flask at 37°C in a 5% CO2 atmosphere.
All reagents for cell culture were from Euroclone, Pero, Italy.
50.0, 34.3, 29.2, 28.4, 17.6, 14.6. LC-ESI-MS: 9.3 min,
+
[M+H]+=489, [M+Na]+=511, [M+K
]
=527, [2M+H]+=976,
+
[2M+Na
]
=999.
S
-enantiomer [α]D= -47,
R-enantiomer [α]D=
4.5 Cell viability assay
+40 (
c
1 in CHCl3“.
(27“: Transparent oil; 1H-NMR (400 MHz, CDCl3“ ꢇ7.30-7.27
(m, 2H“, 6.89 (dd, 3J=8.4 Hz, 3J=8.4 Hz, 1H“, 6.33 (m, 1H“,
Cells were seeded at 45,000 cell/cm2 onto collagen coated 24-
well plates, and after two days treated with increasing
concentrations (10-100ꢅg/mL“ of 21a, 21b, (R“-24 and (S“-24
3
3
5.73 (bs, 1H“, 5.47 (m, 1H“, 4.36 (dd, J=16.4 Hz, J=3.6 Hz,
1H“, 4.15 (d, 3J=15.6 Hz, 1H“, 3.99 (d, 3J=4.4 Hz, 2H“, 3.80-
3.78 (m, 2H“, 3.57-3.54 (m, 2H“, 3.39-3-32 (m, 2H“, 3.14-3.07
(m, 4H“, 2.33 (m, 1H“, 1.44 (s, 9H“, 1.16 (t, 3J=7.2 Hz, 3H“,
compounds. After 24 h treatment, cell viability was evaluated
by means of PrestoBlue® Cell Viability Reagent, a cell
permeable resazurin-based solution that modified by the
reducing power of living cells turns red in colour. Briefly,
PrestoBlue® was administrated to cells according to the
manufacturer's protocol and incubated for 1 h at 37°C. The
absorbance was measured with FLUOstar Omega (BMG
Labtech“ multidetection microplate reader at wavelength of 570
nm and at reference wavelength of 600nm. The same assay
was performed in cells treated with 100ꢅg/mL of Linezolid. All
data are expressed as mean ± SEM of three separate
experiments performed in triplicate. The differences were
calculated by means of Studentꢃs t test. A p value <0.05 was
considered to be statistically significant.
3
3
1.02 (d, J=7.2 Hz, 3H“, 0.94 (d, J=7.2 Hz, 3H“.13C-NMR (400
MHz, CDCl3“ ꢇ169.6, 166.9, 163.3, 155.7, 153.4, 141.3, 139.1
130.3, 129.3, 124.3, 118.4, 116.0 (d, 3J=22 Hz“, 79.7, 67.6,
66.6, 57.4, 49.7, 44.3, 34.3, 29.6, 28.3, 19.3, 17.8, 14.6. LC-
ESI-MS: 7.27 min, [M+H]+=546, [2M+Na]+=569.
[α]D= -30, -enantiomer [α]D= +31 ( 1 in CHCl3“.
S-enantiomer
R
c
4.3 Antibacterial activity assay
4.3.1 Bacterial strains

ACCEPTED MANUSCRIPT
[4] D.N. Wilson, F. Schluenzen, J.M. Harms, A.L. Starosta,
4.6 MAO activity assay
S.R. Connell, P. Fucini, The oxazolidinone antibiotics perturb
the ribosomal peptidyl-transferase center and effect tRNA
positioning, PNAS, 2008, 105 (36“, 13339-13344
Cells were seeded at 45,000 cell/cm2 onto collagen coated 24-
well plates, and after two days treated with 21a, 21b, (
R“-24
[5] S.J. Brickner, M.R. Barbachyn, D.K. Hutchinson, P.R.
and ( “-24 compounds with increasing concentrations (10-
S
Manninen, Linezolid (ZYVOX“, the First Member of a
100ꢅg/mL“. After 24 h treatment cells were harvested and
lysed with a non-denaturizing buffer (10mM Tris pH 7.5,
100mM NaCl, 1mM EDTA, 0.01% Triton X-100“. Then cells
were completely disrupted by sonication with an Ultrasonic
Sonicator bath 2510 (Branson“. MAO inhibition was evaluated
by means of Amplex® Red Monoamine oxidase Assay kit, a
one-step colorimetric method based on the detection of H2O2
in a horseradish peroxidase coupled reaction using 10-acetyl-
Completely New Class of Antibacterial Agents for Treatment of
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3,7-dihydroxyphenoxazine (amplex red reagent“
a
highly
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sensitive stable probe for H2O2. Briefly, according to
manufacturerꢃs protocol, samples were incubated with Amplex
Red working solutions for 30 minutes at room temperature.
The absorbance was measured with a multilabel Victor3
spectrophotometer (Perkin Elmer“ at wavelength of 570 nm.
The same assay was performed in cells treated with 100ꢅg/mL
of Linezolid. All data are expressed as mean ± SEM of three
separate experiments performed in triplicate. The differences
were calculated by means of Studentꢃs t test. A p value <0.05
was considered to be statistically significant.
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4.7 Docking studies
Docking studies on human MAO-A were done on the high
resolution 2Z5X structure bound with the reversible inhibitor
harmine,[33] while the high resolution 2V5Z structure bound
with safinamide was used for human MAO-B studies.[34] In
both cases, the pdb files were prepared for docking using the
Schrödinger Protein Preparation Wizard.[35] Crystallization
supplementary ligands and water molecules were removed
from structure, hydrogens were added and missing side-chains
and loops were filled in. In the case of 2V5Z heterodimer, only
chain A was used. Finally, the protein was refined with an
energy-restrained minimization, using OPLS-2005 force field.
The optimized pdb files were subjected to docking with
AutoDock Vina [36] and PyMOL,[38] using the PyMOL
Autodock plugin.[39] The centroid of the co-crystallized ligands
was used to set up the docking grid cube of size 22.5 ꢀ. In all
cases, the validity of the obtained receptor was tested re-
docking the original co-crystallized ligand, obtaining an RMS of
0.313 for harmine and MAO-A and an RMS of 1.716 for
safinamide and MAO-B. Figures were produced using PyMOL.
[10] A.Renslo, G.W. Luehr,
M.F. Gordeev, Recent
developments in the identification of novel oxazolidinone
antibacterial agents, Bioorg. Med. Chem., 2006, 14, 4227-
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[11] A. Palumbo Piccionello, R. Musumeci, C. Cocuzza, C.G.
Fortuna, A. Guarcello, P. Pierro, A. Pace, Synthesis and
preliminary antibacterial evaluation of Linezolid-like 1,2,4-
oxadiazole derivatives, Eur. J. Med. Chem., 2012, 50, 441-
448.
[12] O.A. Phillips, R. D'Silva, T.O. Bahta, L.H. Sharaf, E.E.
Udo, L. Benov, D.E. Walters, Synthesis and biological
evaluation of novel 5-(hydroxamic acid“ methyl oxazolidinone
derivatives, Eur. J. Med. Chem, 2015, 106, 120-131.
[13] J.G. Slatter, D.J. Stalker, K.L. Feenstra, I.R. Welshman,
J.B. Bruss, J.P. Sams, M.G. Johnson, P.E. Sanders, M.J.
Hauer, P.E. Fagemess, R.P. Styd, G.W. Peng, E.M. Shobe,
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subjects, Drug. Metab. Dispos., 2001, 29, 1136-1145.
[14] M.A. Wynalda, M.J. Hauer, L.C. Wienkers, Oxidation of
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Pharmacokinetics of linezolid in subjects with renal
dysfunctions, Antimicro. Agents Chemoth. 2003, 47, 2775-
2780
Acknowledgements
This study has been carried out with the fundamental
contribution of ꢁFondazione del Monte di Bologna e Ravennaꢂ,
MIUR and University of Bologna. Mr. Andrea Garelli is
gratefully acknowledged for the LC-ESI-MS analysis.
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Graphical Abstract

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New isoxazolidinone and 3,4-dehydro-ꢀ-proline derivatives as antibacterial
agents and MAO-inhibitors: a complex balance between two activities
.
Lucia Ferrazzanoa, Angelo Violaa, Elena Lonatib, Alessandra Bulbarellib, Rosario
Musumecib, Clementina Cocuzzab, Marco Lombardoa, Alessandra Tolomellia*
Highlights
•
•
•
•
The synthesis of novel Linezolid-like derivatives has been carried out
Isoxazolidinone and 3,4-dehydro-ꢀ-proline rings were used as scaffolds
Antibacterial activity was modest but a good affinity for MAO-A and B was observed
Docking experiments suggest a particular affinity for MAO-B