Benzodioxane-benzamides as new bacterial cell division inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.09.100

A SAR study was performed on 3-substituted 2,6-difluorobenzamides, known inhibitors of the essential bacterial cell division protein FtsZ, through a series of modifications first of 2,6-difluoro-3-nonyloxybenzamide and then of its 3-pyridothiazolylmethoxy analogue PC190723. The study led to the identification of chiral 2,6-difluorobenzamides bearing 1,4-benzodioxane-2-methyl residue at the 3-position as potent antistaphylococcal compounds.

Full text of DOI:10.1016/j.ejmech.2014.09.100

European Journal of Medicinal Chemistry 89 (2015) 252e265
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzodioxaneebenzamides as new bacterial cell division inhibitors
Giuseppe Chiodini a, Marco Pallavicini a, Carlo Zanotto b, Massimiliano Bissa c,
,
Antonia Radaelli c d, Valentina Straniero a, Cristiano Bolchi a, Laura Fumagalli a,
,
, *
Paola Ruggeri a, Carlo De Giuli Morghen b d, Ermanno Valoti a
a
ꢀ
Dipartimento di Scienze Farmaceutiche, Universita di Milano, Via Mangiagalli 25, I-20133 Milano, Italy
Department of Medical Biothechnologies and Translational Medicine, Universita di Milano, Via Vanvitelli 32, I-20129 Milano, Italy
Department of Pharmacological and Biomolecular Sciences, Universita di Milano, Via Balzaretti 9, I-2013 Milano, Italy
CNR Institute of Neurosciences, Cellular and Molecular Pharmacology Section, Universita di Milano, Via Vanvitelli 32, I-20129 Milano, Italy
b
ꢀ
c
ꢀ
d
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A SAR study was performed on 3-substituted 2,6-difluorobenzamides, known inhibitors of the essential
Received 10 August 2014
Received in revised form
9 September 2014
Accepted 15 September 2014
Available online 18 October 2014
bacterial cell division protein FtsZ, through
a series of modifications first of 2,6-difluoro-3-
nonyloxybenzamide and then of its 3-pyridothiazolylmethoxy analogue PC190723. The study led to
the identification of chiral 2,6-difluorobenzamides bearing 1,4-benzodioxane-2-methyl residue at the 3-
position as potent antistaphylococcal compounds.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
FtsZ
Antibacterial activity
Benzodioxane
2,6-Difluorobenzamide
Staphylococcus aureus
1. Introduction
the development of new antibacterial agents, such as taxane I [6],
benzimidazole II [7], guanidinomethyl biaryl III [8], OTBA IV [9],
The increasing number of bacterial infections that are resistant
to current antibiotic therapies urgently requires the development
of new antibacterial agents exhibiting novel mechanisms of action.
The essential bacterial cell division protein FtsZ has been recently
recognized as an appealing target for antibiotics discovery because
it is absent in humans and highly conserved among important
bacterial pathogens [1,2]. When bacteria divide, FtsZ polymerizes in
a GTP-dependent manner at mid-cell to give filaments, which form
the Z-ring. This cytokinetic structure recruits other cell division
proteins and contracts enabling cell separation and division [3].
Accordingly, altering proper FtsZ assembly results in bacterial
growth inhibition and cell death [4]. FtsZ is structurally and func-
and 2,6-difluoro-3-nonyloxybenzamide V [10], to name just a few
(Fig. 1).
In particular, the design of 3-substituted benzamides has
started from the weak antibacterial 3-methoxybenzamide (3-
MBA) [11]. A series of modifications, aimed at improving po-
tency and optimizing drug-like properties while retaining the on-
target inhibition of cell division, has led to the potent derivative
PC190723, a 2,6-difluorinated 3-MBA analogue, bearing a meth-
yleneoxy 2-linked 6-chloro-thiazolo[5,4-b]pyridine at position 3
in lieu of methoxyl (Fig. 2) [12]. Key stages of 3-MBA optimization
have been 2,6-difluorination, elongation of the alkoxy substituent
up to nonyloxy, replacement of this latter with heteroaryloxy or
heteroarylmethoxy systems, and chlorination of pyridine nucleus
of the best selected pyridothiazolylmethoxy derivative to give
PC190723 [13] and its two prodrugs TXY541 and TXY436 [14].
Very recently, further improvements over PC190723 were re-
ported to be achieved for in vitro and in vivo properties by
replacing the thiazolopyridine with a phenyl-substituted oxazole
and by making the pseudo-benzylic carbon of the methyleneoxy
linker secondary and stereogenic through the introduction of
diverse hydroxylated alkyl residues, as best exemplified by com-
pound VI (Fig. 2) [15,16].
tionally homologous to mammalian b-tubulin, a long-known target
for successful anticancer chemotherapeutics, but the low sequence
homology between the two proteins makes it possible to discover
FtsZ-targeting compounds without cytotoxicity to eukaryotic cells
[5]. By different investigation approaches, several FtsZ-specific
compounds have been recently identified as promising leads for
* Corresponding author.
E-mail address: ermanno.valoti@unimi.it (E. Valoti).
http://dx.doi.org/10.1016/j.ejmech.2014.09.100
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
253
Fig. 1. Chemical structures of some FtsZ-specific compounds.
Such an optimization route has indicated that the amide func-
tion is critical for the on-target inhibition of cell division and that
few substitutions are tolerated at benzamide ring, while there is
scope for further improvement of the 3-alkyloxy substituent. Ac-
cording to these findings, we considered a series of novel modifi-
cations of V and of PC190723 (a) at the amide function by
introduction of a small, polar N-substituent (compounds 1, 2 and 4)
or by dimerization through a NeN0-ethylene linker (compound 3)
or by replacement with CN or carbonyl bioisosteres (compounds
5e8) [17], (b) at the alkyl chain by introduction of alcoholic and
aminic functions at the end of the chain (compounds 9e10) or by
replacement with a benzo-condensed heteroalicycle, such as 1,4-
benzodioxane (compounds 11e13), and (c) at both amide and
alkyl chain by hybridizing 2 with 11 (compound 14) (Fig. 3).
In previous researches, we had successfully used the 1,4-
benzodioxane-2-methyl residue, substituted or unsubstituted
at the aromatic ring, to design other bioactive molecules, such
as a1-AR antagonists [18e20], neuronal nicotinic agonists [21,22]
and farnesyl transferase inhibitors [23,24]. Compared to the up to
now described heteroaryl bicycles, such as benzothiazole and
pyridothiazole, which have been modified by introducing sub-
stituents at the six-membered ring, 1,4-benzodioxane chemotype
has the additional option of stereoisomerism. Its C(2) becomes
stereogenic when bound to the rest of the molecule and, in case of
high activity of the racemate, investigation of the single enantio-
mers acquires great interest for the further development of the lead
molecule, as demonstrated by the heterogeneous ligands we have
previously studied [18e22]. Revealingly, the antibacterial activity of
the latest compound VI [16], reported when our present investi-
gation were just completed, has been found to be greatly influenced
by the configuration of its pseudo-benzylic stereocentre, which is
very close to the endocyclic stereocentre of our benzodioxane de-
rivatives 11e14, if the benzamidic portions are superimposed: the R
enantiomer of VI is about 100-fold more potent than the S
enantiomer.
Here, we report the synthesis and the antibacterial activity of
compounds 1e14, a series of analogues of V and, ultimately, of its
congener PC190723, within which we identified compound 13 ꢀ its
S enantiomer in particular ꢀ as a novel potent lead for inhibitors of
bacterial cell division.
Fig. 2. Design of 3-MBA analogues leading to oxazole-benzamide VI.

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G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
Fig. 3. Chemical structures of compounds 1e14.
2. Chemistry
with 6-chloro-2-mesyloxymethyl-1,4-benzodioxane (32) or 7-
chloro-2-mesyloxymethyl-1,4-benzodioxane (36), afforded com-
pounds 12 and 13, respectively (Scheme 3). The intermediate 32
was synthesized from O-benzylated 5-chlorosalicylaldehyde,
which was oxidized to the formyl ester 27 and hydrolysed to the
phenol 28. The phenolic function was alkylated with epichlori-
drine and the benzyl group was removed by hydrogenolysis to give
30. Successive treatment with sodium hydroxide yielded 2-
hydroxymethyl-6-chloro-1,4-benzodioxane (31), which was O-
mesylated. The positional isomer of 31, namely the intermediate
35, was prepared from 5-chlorosalicylaldehyde by O-alkylation
with epichloridrine, oxidation to formyl ester 34, and one-step
conversion into 35 in the presence of sodium hydroxide. Finally,
treatment with mesyl chloride provided 36.
The two enantiomers of 13 were synthesized from the tosyl
esters of (R)- and (S)-solketal [29], readily available by resolution of
racemic glycerol acetonide [30] and successive tosylation, through
identical synthetic steps. The sequence of reactions leading to (S)-
13 is shown in Scheme 4. Nucleophilic displacement of tosylate
from (S)-3-tosyloxy-1,2-propanediol by 2-acetyl-4-chlorophenate
provided the phenyl ether (R)-37, whose carbonyl group was
oxidized to ester with m-chloroperbenzoic acid. The ester (R)-38
was hydrolysed and the resultant phenol (R)-39 was O-benzylated.
After removing the acetonide protecting group, the two hydroxyls
of (S)-41 were mesylated and the phenol debenzylated. Intra-
molecular nucleophilic substitution of the mesylate at the sec-
ondary carbon of the glycerol skeleton yielded (S)-7-chloro-2-
mesyloxymethyl-1,4-benzodioxane (S)-44, which was converted
into (S)-13 by reaction with 2,6-difluoro-3-hydroxybenzamide in
the presence of potassium carbonate.
The synthesis of compounds 1e8 is shown in Scheme 1. o,p-
Difluorophenol was O-alkylated with nonyl bromide and the
resulting phenyl ether 15 was treated with n-butyllithium and car-
bonic anhydride to give 2,6-difluoro-3-nonyloxybenzoic acid (16).
The acid was converted into the corresponding benzoyl chloride 17
and reacted with ammonia, ethanolamine and ethylenediamine to
yield V, 1 and 3 respectively. Reaction of 1 with SOCl2 provided the
N-chloroethylamide 18, which was converted into the azide 19 and
then reduced to the amine 2. The benzodiazepine 4 was prepared
from 20, the methyl ester of 17, by treatment with ethylenediamine.
Dehydration of V gave the nitrile 5, from which the tetrazole 8 was
obtained by reaction with sodium azide in the presence of zinc
chloride. The N-chloroethylamide 18 was cyclised to the oxazoline 6
by triethylamine, while the imidazoline 7 was synthesized from 15
by conversion into the aldehyde 21 and subsequent reaction with
ethylenediamine and trichloroisocyanuric acid.
The benzamides 9 and 10 were synthesized, as racemates, from
2,6-difluoro-3-hydroxybenzamide, which was prepared according
to literature methods [25] with minor modifications (Scheme 2).
The alkylation of the phenolic function with 1-(4-chlorobutyl)-2,3-
isopropylideneglycerol (22), prepared from racemic solketal and 1-
chloro-4-tosyloxybutane, and the successive removal of the ketal
protecting group from 23 gave 9. The amide 10 was obtained by O-
alkylation of 2,6-difluoro-3-hydroxybenzamide with the phthali-
mide of 3-(4-tosyloxybutoxy)-2-hydroxypropylamine (25) and
subsequent phthalimide deprotection. The alkylating agent 25 was
synthesized by nucleophilic opening of the epoxide ring of racemic
4-oxiranylmethoxy-1-butanol [26] by phthalimide and successive
tosylation of the primary OH of 24.
The hybrid 14 was synthesized by O-alkylation of methyl 2,6-
difluoro-3-hydroxybenzoate with 2-mesyloxymethylbenzodioxane,
conversion into benzoyl chloride, reaction with mono-Boc-
ethylenediamine, and removal of Boc protecting group.
The benzodioxane derivative 11 was obtained by O-alkylation
of 2,6-difluoro-3-oxybenzamide with 2-mesyloxymethyl-1,4-
benzodioxane (Scheme 3) [27,28]. The same reaction, performed

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
255
Scheme 1. Reagents: (a) Nonyl bromide, potassium carbonate, acetone. (b) CO2, n-BuLi, THF. (c) Thionyl chloride, toluene. (d) 30% NH3 in water, THF. (e) Trimethyl orthoformate, 98%
H2SO4, methanol. (f) Ethylenediamine, ethanol. (g) EEthanolamine, TEA, DCM. (h) Thionyl chloride. (i) NaN3, NaI, DMF. (j) PPh3, water, THF. (k) Ethylenediamine, DCM. (l) (CF3CO)2O,
Py, dioxane/EtOH. (m) TEA, 1,2-dichloroethane. (n) Ethylenediamine, trichloroisocyanuric acid, methanol. (o) n-BuLi, DMF, THF. (p) NaN3, ZnCl2, water, DMF.
Scheme 2. (a) NaH, THF. (b) K2CO3, NaI, DMF. (c) 10% HCl, methanol. (d) Phthalimide, 2-propanol. (e) Tosyl chloride, pyridine. (f) K2CO3, DMF. (g) Hydrazine hydrate, ethanol.

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G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
Scheme 3. (a) K2CO3, DMF. (b) m-Chloroperbenzoic acid, DCM. (c) NaOH, methanol. (d) Epichloridrine, K2CO3, DMF. (e) H2, 5% Pd/C, ethyl acetate. (f) NaOH, methanol. (g) Mesyl
chloride, TEA, DCM. (h) K2CO3, 2,6-difluoro-3-hydroxybenzamide, DMF. (i) Epichloridrine, K2CO3, DMF. (j) m-Chloroperbenzoic acid, DCM. (k) NaOH, methanol. (l) Mesyl chloride,
TEA, DCM. (m) K2CO3, 2,6-difluoro-3-hydroxybenzamide, DMF.
3. Results and discussion
MIB of 4, 7 and 8 were not determined as preliminary assays
showed high toxicity on Vero cells (TD90 < 0.125 g/mL).
m
The antibiotic activities of V, as a reference, and of 1e14 are
provided in Table 1.
Inhibition of bacterial growth was tested on both a Gram-
positive (Staphylococcus aureus) and a Gram-negative (Escherichia
coli) bacterium. In particular, the minimal inhibiting concentration
In compounds 1e4 the crucial amide function of V is main-
tained, but made secondary by introducing a small polar substit-
uent at the nitrogen atom, which improves water solubility and
potential for additional hydrogen bonding to the FtsZ protein, or by
introducing an ethylene linker to the amide nitrogen of another
identical molecule. The results indicate that such modifications are
not tolerated. However, compound 2 retains a moderate antibac-
terial activity against S. aureus and, interestingly, when tested on
(MIC), i.e. the lowest compound dose (
inhibited, and the minimal bactericidal concentration (MBC), i.e.
the minimal dose ( g/mL) at which cell growth is inhibited after
mg/mL) at which growth is
m
compound removal, were determined. The compounds showing
the highest antibacterial activities were tested for cytotoxicity both
on non-human Vero and human MRC-5 primate cells and the doses
E. coli, it is able to inhibit cell growth with a MIB and MIC of 20 mg/
mL in striking contrast with reference compound V and all the
other compounds of the series, which are devoid of activity against
(mg/mL) reducing the viability of these cells by 50% (TD50) or 90%
E. coli. Unfortunately, its TI is low: TD90, 80 mg/mL, is only four times
(TD90) are reported. The therapeutic index (TI) was also determined
the MBC. Replacement of the amide function with CN or bioisosteric
and defined as the ratio between TD90 and MBC values. MIC and
heterocycles (compounds 5e8) is also highly detrimental to
Scheme 4. (a) K2CO3, 5-chloro-2-hydroxyacetophenone, DMF. (b) m-Chloroperbenzoic acid, DCM. (c) NaOH, methanol. (d) K2CO3, benzyl bromide, DMF. (e) 10% aqueous HCl,
methanol. (f) Mesyl chloride, TEA, DCM. (g) H2, 5% Pd/C, THF. (h) K2CO3, DMF. (i) K2CO3, 2,6-difluoro-3-hydroxybenzamide, DMF.

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
257
Table 1
Antibiotic activities and cytotoxicities of 2,6-difluoro-3-nonyloxybenzamide (V) and of compounds 1e14.
Compd
S. aureus
E. coli
MRC-5
Toxicity
Vero
TD50
90
Toxicity
MIC (
m
g/ml)
MBC (
0.25
mg/ml)
TI
>800
MIC (
m
g/ml)
TD50
100
(
m
g/ml)
TD90
(
mg/ml)
(
mg/ml)
TD90 (mg/ml)
V
1
2
0.1
>100
10
>100
>100
20a
>200
>200
20
4
35
80
60
120
3
4
>100
nd
>100
5
6
7
>100
>100
nd
>100
>100
8
nd
9
>100
>100
5
1
0.5
0.25
1
20
>100
>100
>100
>100
>100
>100
>100
>100
10
11
12
13
(S)-13
(R)-13
14
80
2.5
0.5
0.5
2.5
40
320
>1600
>1600
>320
650
500
600
400
800
>800
>800
>800
550
450
>200
>200
800
>800
>200
>200
MIC, Minimal Inhibitory Concentration; MBC, Minimal Bactericidal Concentration; TD50 and TD90, compound concentration reducing cell viability of 50% and 90%, respectively;
TI, ratio between TD90 of MRC-5 and MBC values; nd, not determined.
a
MBC ¼ 20 g/ml and TI ¼ 4.
m
antibacterial activity thus confirming the critical role of the amide
function.
cells with >800 mg/mL TD90 values, toxic doses significantly higher
than those determined for V. The two enantiomers of 13 were
synthesized and separately tested for biological activity. The results
indicated that the anti-staphylococcal activity primarily resides
On the basis of these findings, which indicated that modifica-
tions at the amide function of V prejudice antibacterial activity,
small polar substituents were introduced at the end of the long
alkyl chain instead of at the amide group still pursuing log P
reduction and interaction potential improvement. The last five
carbons of the nonyl chain were replaced by 2,3-dihydroxypropoxy
or by 3-amino-2-hydroxypropoxy residue. The resultant analogues
9 and 10 proved to be much less active than V. These results are
consistent with previous observation that polar substituents, such
as OH and NH2, at the phenyl ring of the nonyl replacing benzo-
thiazolylmethyl system are deleterious, while hydrophobic sub-
stituents, such as Cl, Br, Ph, EtO, Pr, highly beneficial [13]. A
potential binding site for the alkyloxy substituent of these benza-
mides has been identified as a cleft between helix 7 and the C-
terminal domain of FtsZ and the proposed binding mode shows
preference for hydrophobic substituents [12a,31]. At this point of
our traditional SAR-directed design, we considered the reported
productive replacement of the alkyl residue of V with a series of 5-
and 6-membered heterocycles, unsubstituted or phenyl substituted
or benzo-condensed [13]. This kind of modification suggested us
the replacement of nonyl with 1,4-benzodioxan-2-ylmethyl res-
idue. As previously underlined, such a benzo-condensed hetero-
alicycle gives further optimization chances: introduction of
substituents at the aromatic ring or at the methylene linker,
incorporation of heteroatoms into the benzene portion, opening of
the dioxane cycle to give phenoxyethyl ethers, isosteric re-
placements of dioxane oxygens, and, last but not least, ‘chiral
switch’ of the enantiomers at the stereogenic dioxane C(2).
Therefore, we tested the racemate of compound 11 and found a
with the S isomer, which shows 0.25
on S. aureus, while the corresponding values of the R enantiomer
are 1 and 2.5 g/mL.
mg/mL MIC and 0.5 mg/mL MBC
m
To establish whether the mechanism of the antibacterial activity
of 13 is associated with alteration of FtsZ Z-ring formation, S. aureus
was grown in the presence of MIC, 2 ꢁ MIC and 4 ꢁ MIC 13. As
shown in Fig. 4, optical microscopy revealed a significant increase
in the cell volume of S. aureus in response to exposure with 13. Such
an alteration of bacterial morphology is thought to result from
interfering with proper FtsZ function. Ballooning of the cocci was
observed also in the V positive control. These findings, which are
consistent with those reported for other 3-substituted 2,6-
difluorobenzamides, candidate 13 to a series of modifications
aimed at optimizing its profile as a new antibacterial agent.
4. Conclusion
A series of modifications of 2,6-difluoro-3-nonyloxybenzamide
(V) and then of its analogue PC190723, both interfering with FtsZ
function and thus inhibiting bacterial cell division, were accom-
plished in order to identify new potent bactericidal agents. Com-
pound 13, which bears 7-chloro-1,4-benzodioxanyl in lieu of the
good 5 mg/mL MIC on S. aureus. The successive steps were the hy-
bridization of 11 with 2, the only compound active against E. coli, to
give 14, and the introduction of a hydrophobic susbstituent, such as
Cl, at the benzene ring of benzodioxane. The hybridization was
unproductive: 14 was not more active against S. aureus than 11 and
did not retain the activity against E. coli of 2. In contrast, the 6-Cl
and 7-Cl substituted analogues of 11, namely the compounds 12
and 13, were found significantly more potent than 11: they showed
Fig. 4. Cells of S. aureus were cultured in the absence of any FtsZ-targeting compound
(NT) or in the presence of V or 13 and analysed by phase-contrast microscopy. S. aureus
balloons in response to contact with V and 13.
2.5 and 0.5
mg/mL MBC on S. aureus, respectively. Furthermore,
compound 13 had a very low cytotoxicity on both MRC-5 and Vero

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G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
chloropyridothiazolyl residue of PC190723, showed 0.5
mg/mL MIC
5.1.3. 2,6-Difluoro-3-nonyloxybenzoyl chloride (17)
on S. aureus being minimally toxic to mammalian cells. Phase-
contrast microscopy analysis revealed similar alterations of bacte-
rial morphology in response to contact with 13 and V suggesting
that 13 shares the same mechanism of action as the other FtsZ-
targeting 2,6-difluorobenzamides. The peculiar structure of the
benzo-condensed heteroalicycle benzodioxane, compared to the
heteroaryls up to now used to construct FtsZ inhibiting benza-
mides, offers interesting chances of further optimization, such as
the enantiomer switch. And indeed, when applied to 13, the chiral
switch reveals that the S enantiomer is 4e5-fold more potent than
the R one.
Thionyl chloride (1.8 mL) was added dropwise to a solution of 16
(4.6 g, 15.3 mmol) in toluene (25 mL) under nitrogen. After stirring
at room temperature for 30 min and then at reflux for 2 h, the re-
action mixture was concentrated under vacuum to give 4.78 g
(98.0%) 17 as a brownish liquid: 1H NMR (CDCl3)
d 7.06 (td, 1H,
J ¼ 11.5, 5.9 Hz), 6.85 (m, 1H), 4.04 (t, 2H, J ¼ 6.6 Hz), 1.83 (m, 2H),
1.36 (m, 12H), 0.87 (t, 3H, J ¼ 7.0 Hz).
5.1.4. 2,6-Difluoro-3-nonyloxybenzamide (V)
30% Ammonia solution in water (17 mL) was added dropwise to
a solution of 17 in THF (20 mL) at 4 ꢂC. After stirring at room
temperature for 30 min, the reaction mixture was allowed to warm
to room temperature over-night and then concentrated. The res-
idue was poured into water and the resultant suspension was
filtered to isolate a white solid, which was dried at 50 ꢂC for 3 h to
give 3.82 g (83.4%) of V as a white powder: mp 80.3 ꢂC; 1H NMR
5. Experimental protocols
5.1. Chemistry
Melting points were determined by DSC analysis and corre-
spond to the peak maximum. 1H NMR spectra were recorded
operating at 300 MHz and 13C NMR at 75 MHz. Chemical shifts are
reported in ppm relative to residual solvent (CHCl3 or DMSO) as
internal standard. Signal multiplicity is designed according to the
following abbreviations: s ¼ singlet, d ¼ doublet, dd ¼ doublet of
doublets, ddd ¼ doublet of doublet of doublets, t ¼ triplet,
m ¼ multiplet, br s ¼ broad singlet, br t ¼ broad triplet, td ¼ triplet
of doublets. Coupling constants are reported in Hertz (Hz). Optical
rotations were determined by a PerkineElmer 241 Polarimeter at
25 ꢂC. Elemental analyses (C, H, N, Cl) of the new substances are
within 0.40% of theoretical values. Purifications were performed by
(CDCl3)
d
6.99 (td, 1H, J ¼ 11.5, 6.4 Hz), 6.84 (m, 1H), 6.02 (br s, 2H),
3.96 (t, 2H, J ¼ 6.6 Hz), 1.77 (m, 2H), 1.29 (m, 12H), 0.83 (t, 3H,
J ¼ 7.0 Hz). Anal. Calcd for C16H23F2NO2 (299.36).
5.1.5. 2,6-Difluoro-N-(2-hydroxyethyl)-3-nonyloxybenzamide (1)
A solution of V (4.0 g, 12.55 mmol) in DCM (15 mL) was added
dropwise to a solution of ethanolamine (0.93 g, 15.1 mmol) and
triethylamine (2.1 mL, 15.1 mmol) in DCM (20 mL) at 4 ꢂC. The
mixture was stirred at 4 ꢂC for 30 min and then at room temper-
ature for 2 h. Water (20 mL) was added and the aqueous phase was
extracted with DCM (2 ꢁ 20 mL). The combined organic phases
were washed with 10% HCl (50 mL), saturated aqueous NaHCO3
solution (50 mL) and brine (50 mL), dried over Na2SO4, and
concentrated under vacuum to give an oily residue, which was
purified by flash chromatography. Elution with 1/1 cyclohexane/
ethyl acetate afforded 2.29 g (53.1%) of 1 as a white solid: mp
flash chromatography using silica gel (particle size 40e63
mm,
Merck).
5.1.1. 2,4-Difluoro-1-nonyloxybenzene (15)
A solution of 2,4-difluorophenol (10.0 g, 76.9 mmol) in acetone
(40 mL) was added drop-wise to a suspension of K2CO3 (13.82 g,
100.0 mmol) in acetone (30 mL). The mixture was stirred for 15 min
at room temperature. Nonyl bromide (15.2 mL, 79.5 mmol) was
added drop-wise and the reaction mixture was refluxed over-night,
and then concentrated under vacuum. The residue was dissolved in
ethyl acetate (80 mL) and water (80 mL). The aqueous layer was
separated and extracted with ethyl acetate twice. The organic
phases were combined, washed with 1 M NaOH twice, dried over
Na2SO4, and concentrated to give a residue (20.0 g), which was
purified by flash chromatography on silica gel. Elution with cyclo-
hexane yielded 15.0 g (77.0%) of 15 as a colourless oil: 1H NMR
75.5 ꢂC; 1H NMR (CDCl3)
d
6.95 (td, 1H, J ¼ 9.0, 5.1 Hz), 6.83 (t, 1H,
J ¼ 9.0 Hz), 6.47 (br s, 1H), 3.96 (t, 2H, J ¼ 6.6 Hz), 3.83 (m, 2H), 3.63
(m, 2H), 1.78 (m, 2H), 1.50e1.20 (m, 12H), 0.88 (t, 3H, J ¼ 6.9 Hz). 13
C
NMR (75 MHz, CDCl3) 161.5, 153.2 (dd, J ¼ 237.2, 6.3 Hz), 146.3 (dd,
J ¼ 243.9, 8.6 Hz),144.1 (d, J ¼ 3.4 Hz), 116.9 (d, J ¼ 9.4 Hz), 115.1 (dd,
J ¼ 23.4, 17.4 Hz), 111.1 (dd, J ¼ 17.4, 23.0 Hz), 70.7, 61.9, 42.8, 32.1,
29.7, 29.5, 29.5, 29.4, 26.1, 22.9, 14.3. Anal. Calcd for C18H27F2NO3
(343,41).
5.1.6. 2,6-Difluoro-N-(2-chloroethyl)-3-nonyloxybenzamide (18)
Thionyl chloride (2 mL) was added to 1 (0.50 g, 1.45 mmol). The
mixture was stirred at room temperature for 90 min and concen-
trated. The residue was dissolved in ethyl acetate (10 mL) and water
(10 mL). The aqueous phase was separated and extracted with ethyl
acetate twice. The combined organic phases were washed with
brine (20 mL), dried over Na2SO4, and concentrated to give 380 mg
(72.5%) of 18 as a yellow oil which solidified upon standing: 1H
(CDCl3)
12H), 0.89 (t, 3H, J ¼ 7.0 Hz).
d
6.83 (m, 3H), 3.97 (t, 2H, J ¼ 6.8 Hz), 1.81 (m, 2H), 1.36 (m,
5.1.2. 2,6-Difluoro-3-nonyloxybenzoic acid (16)
A solution of 15 (15.0 g, 58.4 mmol) in dry THF (190 mL) was
cooled at ꢀ78 ꢂC. n-BuLi (26.0 mL, 70.2 mmol, 2.7 M in heptanes)
was slowly added. After stirring at ꢀ78 ꢂC for 2 h, carbon dioxide
was bubbled into the reaction mixture, which was maintained
at ꢀ78 ꢂC for 1 h and then allowed to warm to room temperature
over-night. The white precipitated lithium salt was isolated by
filtration and washed with THF (30 mL). A second crop of precipi-
tated lithium salt was obtained by halving the volume of the filtrate
under vacuum and recovered by filtration. The two precipitates
were combined and dissolved in ethyl acetate (80 mL) and 3 N HCl
(80 mL). The aqueous layer was extracted with ethyl acetate twice.
The organic phases were combined, washed with brine (80 mL),
dried over Na2SO4, and concentrated to give 15.30 g (87.2%) of 16 as
NMR (CDCl3)
d
6.98 (td, 1H, J ¼ 8.8, 5.1 Hz), 6.85 (t, 1H, J ¼ 8.8 Hz),
6.37 (br s,1H), 3.99 (t, 2H, J ¼ 6.7 Hz), 3.82 (m, 2H), 3.74 (m, 2H),1.76
(m, 2H), 1.30 (m, 12H), 0.88 (t, 3H, J ¼ 7.0 Hz).
5.1.7. 2,6-Difluoro-N-(2-azidoethyl)-3-nonyloxybenzamide (19)
Sodium azide (1.40 g, 21.5 mmol) and sodium iodide (60 mg,
0.4 mmol) were added to a solution of 18 (1.59 g, 4.4 mmol) in DMF
(12 mL) and water (4 mL). After stirring at room temperature for
15 min and then at 65 ꢂC for 5 h. The solvents were evaporated
under vacuum and ethyl acetate (20 mL) and water (20 mL) were
added to the residue. The aqueous phase was separated and
extracted with ethyl acetate twice. The combined organic phases
were washed with brine, dried over Na2SO4, and concentrated. The
resultant oily residue was purified by flash chromatography.
Elution with 70/30 cyclohexane/ethyl acetate gave 600 mg (37.0%)
a yellow liquid: 1H NMR (CDCl3)
d 8.63 (br s, 1H), 7.08 (td, 1H,
J ¼ 11.5, 6.1 Hz), 6.86 (m, 1H), 4.02 (t, 2H, J ¼ 6.7 Hz), 1.81 (m, 2H),
1.38 (m, 12H), 0.87 (t, 3H, J ¼ 7 Hz).

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
259
of 19 as a white spongy solid: 1H NMR (CDCl3)
d
6.95 (td, 1H, J ¼ 9.1,
J ¼ 6.7 Hz), 3.63 (m, 2H), 3.50 (m, 2H), 1.76 (m, 2H), 1.45e1.20 (m,
12H), 0.87 (t, 3H, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3) 169, (d,
J ¼ 1.4 Hz), 156.5 (d, J ¼ 244.6 Hz), 144.0, (d, J ¼ 2.3 Hz) 137.1 (d,
J ¼ 4.9 Hz), 112.8 (d, J ¼ 10.5 Hz), 110.2 (d, J ¼ 15.1 Hz), 104.0 (d,
J ¼ 24.2 Hz), 69.6, 50.5, 39.9, 32.1, 29.7, 29.6, 29.4, 26.3, 22.9, 14.3.
Anal. Calcd for C19H30FN2O2 (337.45).
5.3 Hz), 6.86 (t,1H, J ¼ 9.1 Hz), 6.23 (br s, 1H), 3.97 (t, 2H, J ¼ 6.8 Hz),
3.80 (m, 4H), 1.81 (m, 2H), 1.35 (m, 12H), 0.89 (t, 3H, J ¼ 7.0 Hz).
5.1.8. 2,6-Difluoro-N-(2-aminoethyl)-3-nonyloxybenzamide (2)
Triphenylphosphine (640 mg, 2.44 mmol) and water (1 mL)
were added to a solution of 19 (600 mg, 1.62 mmol) in THF (10 mL).
After stirring at room temperature for 2 h, the reaction mixture was
evaporated and the residue was dissolved in ethyl acetate (10 mL)
and water (10 mL). The aqueous phase was separated and extracted
with ethyl acetate twice. The combined organic phases were
washed with brine (20 mL), dried over Na2SO4, and concentrated.
The residue was purified by flash chromatography on silica gel.
Elution with 94/6 DCM/MeOH gave 320 mg of 2 (57.8%) as a yellow
5.1.12. 2,6-Difluoro-3-nonyloxybenzonitrile (5)
Trifluoroacetic anhydride (3.35 mL) was added dropwise to a
solution of V (3.80 g, 12.69 mmol) in 1,4-dioxane (26 mL) and
pyridine (6.7 mL) at 4 ꢂC. The mixture was stirred at 4 ꢂC for 15 min
and then at room temperature for 90 min. After cooling at 4 ꢂC,
water (20 mL) and ethyl acetate (20 mL) were added. The aqueous
phase was separated and extracted with ethyl acetate twice. The
combined organic phases were washed with 10% HCl (25 mL),
saturated aqueous NaHCO3 (25 mL), and brine (25 mL), dried over
Na2SO4, and concentrated to give 3.40 g (95.2%) of 5 as a white
oil: 1H NMR (CDCl3)
d
6.92 (td, 1H, J ¼ 8.8, 5.4 Hz), 6.77 (m, 2H), 3.99
(t, 2H, J ¼ 6.8 Hz), 3.68 (m, 2H), 3.03 (t, 2H, J ¼ 5.8 Hz), 2.40 (br s,
2H), 1.81 (m, 2H), 1.35 (m, 12H), 0.89 (t, 3H, J ¼ 7.0 Hz). 13C NMR
(75 MHz, CDCl3) 161.3, 153.2 (dd, J ¼ 243.3, 5.4 Hz), 147.8 (dd,
J ¼ 253.0, 7.4 Hz), 144.1 (d, J ¼ 11.8 Hz), 116.8, 115.3(d, J ¼ 17.2 Hz),
111.0 (d, J ¼ 22.8 Hz), 70.7, 40.8, 32.1, 29.9, 29.7, 29.6, 29.5, 29.4, 26.1,
22.9, 14.3. Anal. Calcd for C18H28F2N2O2 (342,42).
solid: m.p. 28.2 ꢂC; 1H NMR (CDCl3)
d
7.16 (td, 1H, J ¼ 9.1, 5.4 Hz),
6.93 (t, 1H, J ¼ 9.1 Hz), 4.02 (t, 2H, J ¼ 6.5 Hz), 1.80 (m, 2H), 1.4e1.22
(m,12H), 0.88 (t, 3H, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) 156.2 (dd,
J ¼ 252.8, 2.8 Hz), 153.4 (dd, J ¼ 261.4, 4.5 Hz), 144.3 (dd, J ¼ 9.1,
3.5 Hz), 120.5 (dd, J ¼ 8.9, 4.0 Hz) 111.4 (dd, J ¼ 19.4, 3.1 Hz), 109.5,
93.0 (dd, J ¼ 20.6, 16.3), 70.9, 32.1, 29.7, 29.5, 29.4, 29.3, 26.0, 22.9,
14.3. Anal. Calcd for C16H21F2NO (281.34).
5.1.9. N,N0-1,2-ethanediylbis(2,6-difluoro-3-nonyloxy)benzamide
(3)
A solution of 17 (4.0 g, 12.55 mmol) in DCM (15 mL) was added
dropwise to a solution of ethylenediamine (1.29 g, 21.33 mmol) in
DCM (10 mL) at 4 ꢂC. After stirring at 4 ꢂC for 30 min and then at
room temperature overnight, saturated aqueous NaHCO3 (20 mL)
was added. The aqueous phase was separated and extracted with
DCM twice. The combined organic phases were dried over Na2SO4,
and concentrated. The residue was crystallized from isopropanol to
give 0.95 g (12.1%) of 3 as a white solid: mp 125.0 ꢂC; 1H NMR
5.1.13. 5-(2,6-Difluoro-3-nonyloxyphenyl)-1H-tetrazole (8)
Sodium azide (178 mg, 2.75 mmol) and zinc chloride (204 mg,
1.5 mmol) were added to a solution of 5 (350 mg, 1.25 mmol) in
water (1 mL) and DMF (1 mL). The mixture was stirred at room
temperature for 15 min and then at reflux for 5 h. DMF (1 mL) was
added. After cooling at 4 ꢂC, 10% HCl (3 mL) was slowly added. The
precipitate was isolated by filtration, washed with 10% HCl and then
with water twice, dried under vacuum over night yielding 210 mg
(CDCl3)
d
6.94 (td, 2H, J ¼ 8.8, 5.4 Hz), 6.81 (t, 2H, J ¼ 8.8 Hz), 6.65 (br
s, 2H), 3.97 (t, 4H, J ¼ 6.8 Hz), 3.69 (m, 4H), 1.77 (m, 4H), 1.45e1.20
(m, 24H), 0.89 (t, 6H, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3) 161.7,
153.3 (dd, J ¼ 238.5, 5.7 Hz), 150.1 (d, J ¼ 251. 4 Hz), 144.2 (d,
J ¼ 10.4 Hz), 117.1 (d, J ¼ 8.6 Hz), 114.6 (d, J ¼ 20.5 Hz),111.1 (d,
J ¼ 25.4 Hz), 70.8, 40.4, 32.1, 29.7, 29.5, 29.46, 29.41, 26.1, 22.9, 14.3.
Anal. Calcd for C35H50F4N2O4 (638.78).
(52.9%) of 8 as a yellowish solid: 1H NMR (DMSO-d6)
d 7.47 (td, 1H,
J ¼ 8.9, 5.1 Hz), 7.25 (t,1H, J ¼ 8.9 Hz), 4.11 (t, 2H, J ¼ 6.9 Hz),1.78 (m,
2H), 1.42e1.21 (m, 12H), 0.86 (t, 3H, J ¼ 6.9 Hz). 13C NMR (75 MHz,
DMSO-d) 152.1 (dd, J ¼ 244.9, 4.1 Hz), 149.6 (dd, J ¼ 252.8, 6.0 Hz),
148.0, 144.4 (dd, J ¼ 10.2, 3.2 Hz), 118.6 (d, J ¼ 9.2 Hz), 112.2 (dd,
J ¼ 4.0, 4.1 Hz), 104.6 (dd, J ¼ 19.4, 15.1 Hz), 70.3, 31.9, 29.6, 29.4,
29.3, 29.2, 26.0, 22.7, 14.5. Anal. Calcd for C16H22F2N4O (324.37).
5.1.10. Methyl 2,4-difluoro-3-nonyloxybenzoate (20)
Trimethyl orthoformate (0.92 mL, 8.5 mmol) and 98% sulphuric
acid (0.3 mL) were added to a solution of 16 (1.5 g, 5.0 mmol) in
methanol (15 mL). After stirring at room temperature for 30 min
and then at refux for 24 h, the reaction mixture was concentrated
and the resulting residue was taken up into ethyl acetate and
washed with saturated aqueous NaHCO3 (3 ꢁ 20 mL). The organic
phase was dried over Na2SO4, and concentrated to give 1.48 g
5.1.14. 2-(2,6-Difluoro-3-nonyloxyphenyl)-4,5-dihydrooxazole (6)
Triethylamine (0.55 mL, 3.93 mmol) was added to a solution of
18 (308 mg,1.05 mmol) in 1,2-dichloroethane (3.5 mL). The mixture
was stirred at room temperature for 15 min and then at reflux
overnight. DCM (6 mL) and brine (6 mL) were added. The aqueous
layer was separated and extracted with DCM twice. The combined
organic phases were washed with brine, dried over Na2SO4, and
concentrated. The resultant residue was purified by flash chroma-
tography on silica gel. Elution with 70/30 cyclohexane/ethyl acetate
(94.3%) of 20 as a yellow liquid: 1H NMR (CDCl3)
d 7.02 (td, 1H,
J ¼ 8.8, 5.4 Hz), 6.84 (t, 1H, J ¼ 8.8 Hz), 3.97 (m, 5H), 1.77 (m, 2H),
1.34 (m, 12H), 0.88 (t, 3H, J ¼ 7.0 Hz).
gave 240 mg (70.2%) of 6 as a yellow oil: 1H NMR (CDCl3)
d 7.01 (td,
1H, J ¼ 9.0, 5.1 Hz), 6.83 (t, 1H, J ¼ 9.0 Hz), 4.46 (t, 2H, J ¼ 11.2 Hz),
4.15 (t, 2H, J ¼ 6.5 Hz) 3.98 (t, 2H, J ¼ 6.5 Hz), 1.80 (m, 2H), 1.45e1.23
(m, 12H), 0.86 (t, 3H, J ¼ 6.9 Hz). 13C NMR (75 MHz, CDCl3) 157.6,
154.7 (dd, J ¼ 247.0, 4.8 Hz), 151.5 (dd, J ¼ 255.7, 6.2 Hz), 144.2 (dd,
J ¼ 255.7, 3.6 Hz), 144.2 (dd, J ¼ 10.6, 3.6 Hz), 117.8 (d, J ¼ 6.6 Hz),
110.9 (d, J ¼ 22.5 Hz), 108.2 (dd, J ¼ 21.7, 17.4 Hz), 70.9, 67.8, 55.4,
32.1, 29.7, 29.5, 29.4, 26.1, 22.9, 14.3. Anal. Calcd for C18H25F2NO2
(325.39).
5.1.11. 6-Fluoro-7-nonyloxy-1,2,3,4-tetrahydro-5H-1,4-
benzodiazepin-5-one (4)
Ethylenediamine (3.2 mL, 47.7 mmol) was added to a solution of
20 (1.48 g, 4.7 mmol) in ethanol (10 mL). After stirring at room
temperature for 30 min and then at reflux for 6 h, the reaction
mixture was concentrated and the residue was taken up into ethyl
acetate (15 mL) and water (20 mL). The aqueous phase was sepa-
rated and extracted with ethyl acetate twice. The combined organic
phases were dried over Na2SO4, and concentrated. The resultant
residue was purified by flash chromatography on silica gel. Elution
with ethyl acetate gave 470 mg (28.7%) of 4 as an oil which solid-
5.1.15. 2,6-Difluoro-3-nonyloxybenzaldehyde (21)
A solution of 15 (3.96 g, 15.45 mmol) in dry THF (50 mL) was
cooled at ꢀ78 ꢂC. n-BuLi (7.24 mL, 19.55 mmol, 2.7 M in heptanes)
was slowly added. After stirring at ꢀ78 ꢂC for 1 h, DMF (2.4 mL) was
added dropwise and the mixture was stirred at ꢀ78 ꢂC for 1 h. At
ified upon standing: 1H NMR (CDCl3)
J ¼ 8.8, 4.7), 6.40 (t, 1H, J ¼ 8.8), 4.80 (br s, 1H), 3.92 (t, 2H,
d 8.10 (br s, 1H), 6.75 (dd, 1H,

260
G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
the same temperature, water (20 mL) was slowly added. The
mixture was allowed to warm to room temperature and diluted
with ethyl acetate (20 mL). The aqueous layer was separated and
extracted with ethyl acetate twice. The organic phases were com-
bined, washed with brine, dried over Na2SO4, and concentrated.
The resultant residue was purified by flash chromatography on
silica gel. Elution with 90/10 cyclohexane/ethyl acetate gave 2.81 g
The resultant residue was purified by flash chromatography on
silica gel. Elution with 1/1 cyclohexane/ethyl acetate gave 470 mg
(60.6%) of 23 as a colourless liquid: 1H NMR (CDCl3)
d 6.98 (td, 1H,
J ¼ 8.8, 5.1 Hz), 6.86 (td,1H, J ¼ 8.8, 2.4 Hz), 6.03 (br s, 1H), 5.90 (br s,
1H), 4.24 (q, 1H, J ¼ 5.1 Hz), 4.05 (m, 2H), 3.71 (dd, 1H, J ¼ 8.2,
6.6 Hz), 3.47 (m, 4H), 1.78 (m, 4H), 1.40 (s, 3H), 1.36 (s, 3H).
(64.0%) of 21 as a yellow liquid: 1H NMR (CDCl3)
d
10.37 (s, 1H), 7.16
5.1.19. 3-(4-(2,3-Dihydroxypropoxy)butoxy)-2,6-
(td, 1H, J ¼ 9.1, 5.6 Hz), 6.91 (t, 1H, J ¼ 9.1 Hz), 4.05 (t, 1H, J ¼ 6.6 Hz),
difluorobenzamide (9)
1.82 (m, 2H), 1.35 (m, 12H), 0.87 (t, 1H, J ¼ 7.0 Hz).
10% HCl (2.5 mL) was added to a solution of 23 (470 mg,
1.30 mmol) in methanol (2.5 mL). The solution was stirred at room
temperature for 30 min and then concentrated. The residue was
taken up into water (5 mL) and ethyl acetate (5 mL). The aqueous
layer was separated and extracted with ethyl acetate (3 ꢁ 5 mL).
The combined organic phases were dried over Na2SO4, and
concentrated to give 300 mg (93.9%) of 9 as a colourless oil: 1H NMR
5.1.16. 2-(2,6-Difluoro-3-nonyloxyphenyl)-4,5-dihydro-1H-
imidazole (7)
Ethylenediamine (0.13 mL, 1.95 mmol) was added to a solution
of 21 (500 mg, 1.77 mmol) in methanol (15 mL). After 15 min, tri-
chloroisocyanuric acid (170 mg, 0.71 mmol) was added. The reac-
tion mixture was stirre at room temperature for 15 min and then at
45 ꢂC for 6 h. After cooling at 4 ꢂC, saturated aqueous NaHCO3
(7 mL) was added. The solution readily became a suspension, which
was filtered. The filtrate was concentrated and the residue was
taken up into ethyl acetate (10 mL) and water (10 mL). The aqueous
layer was separated and extracted with ethyl acetate twice. The
organic phases were combined, washed with saturated aqueous
NaHCO3 (10 mL) and brine (10 mL), dried over Na2SO4, and
concentrated. The resultant residue was purified by flash chroma-
tography on silica gel. Elution with 90/10 DCM/MeOH gave 220 mg
(CDCl3)
d
7.02 (td, 1H, J ¼ 8.8, 5.1 Hz), 6.86 (td, 1H, J ¼ 8.8, 2.3 Hz),
6.43 (br s, 1H), 6.18 (br s, 1H), 4.08 (m, 2H), 3.84 (m, 1H), 3.75e3.45
(m, 6H), 1.90e1.70 (m, 4H). 13C NMR (75 MHz, DMSO-d) 162.1, 152.3
(dd, J 239.4, 6.8 Hz), 148.6 (dd, J 246.8, 8.3 Hz), 143.9 (d, J 3.1 Hz),
117.2 (dd, J 24.7, 20.6 Hz), 116.0 (d, J 8.9 Hz), 111.6 (dd, J 19.1, 3.7 Hz),
73.0, 71.2, 70.8, 69.9, 63.8, 26.3, 26.2. Anal. Calcd for C14H19F2NO5
(319.30).
5.1.20. 2-(2-Hydroxy-3-(4-hydroxybutoxy)propyl)isoindoline-1,3-
dione (24)
(38.3%) of 7 as a yellow oil: 1H NMR (CDCl3)
d
6.96 (td, 1H, J ¼ 9.0,
A solution of racemic 4-oxiranylmethoxy-1-butanol (2.79 g,
19.1 mmol) in isopropanol (40 mL) was added dropwise to a sus-
pension of phthalimide (3.37 g, 22.9 mmol) in isopropanol (40 mL).
After adding pyridine (0.3 mL, 5.6 mmol), the mixture was refluxed
for 72 h. Upon cooling at room temperature, a precipitate was
formed. The suspension was filtered and the filtrate was concen-
trated. The resultant oily residue was purified by flash chroma-
tography on silica gel. Elution with ethyl acetate gave 2.21 g (39.5%)
2.1 Hz), 6.83 (t,1H, J ¼ 5.2 Hz), 5.77 (br s, 1H), 3.99 (t, 2H, J ¼ 6.7 Hz),
3.80 (s, 4H), 1.77 (m, 2H), 1.48e1.21 (m, 12H), 0.88 (t, 3H, J ¼ 7.0 Hz).
13C NMR (75 MHz, DMSO-d) 156.3, 153.5 (dd, J ¼ 242.3, 5.4 Hz),
149.9 (dd, J ¼ 250.2, 7.1 Hz), 144.0 (dd, J ¼ 10.5, 3.2 Hz), 117.0 (d,
J ¼ 8.8 Hz),111.5 (dd, J 22.5, 2.0 Hz),110.9 (dd, J ¼ 16.8, 21.0 Hz), 70.1,
49.8, 32.0, 29.7, 29.4, 29.36, 29.3, 29.1, 26.1, 22.8,14.6. Anal. Calcd for
C
18H26F2N2O (324.41).
of 24 as a white wax: 1H NMR (CDCl3)
d 7.84 (m, 2H), 7.75 (m, 2H),
5.1.17. 1-(4-Chlorobutyl)-2,3-isopropylideneglycerol (22)
4.07 (m, 1H), 3.86 (m, 2H), 3.65 (m, 1H), 3.71 (m, 4H), 2.8 (br s, 1H),
1.88 (br s, 1H), 1.67 (m, 4H).
A solution of racemic solketal (0.6 g, 4.56 mmol) in dry THF
(5 mL) was added dropwise to a suspension of NaH (109 mg,
4.56 mmol) in dry THF (5 mL). The mixture was stirred at room
temperature for 30 min. A solution of 4-chlorobutyl mesylate
(1.09 g, 4.15 mmol) in dry THF (5 mL) was added dropwise. The
mixture was stirred at room temperature for 30 min and then at
reflux for 4 h. After cooling at 4 ꢂC, water (10 mL) was added and the
mixture was extracted with ethyl acetate (3 ꢁ 10 mL). The com-
bined organic phases were dried over Na2SO4, and concentrated
The resulting residue was purified by flash chromatography on
silica gel. Elution with 90/10 cyclohexane/ethyl acetate gave
5.1.21. 2-(2-Tosyloxy-3-(4-hydroxybutoxy)propyl)isoindoline-1,3-
dione (25)
A solution of 24 (2.21 g, 7.53 mmol) in pyridine (5 mL) was
added dropwise to a stirred solution of tosyl chloride (1.58 g,
8.29 mmol) in pyridine (5 mL) at 4 ꢂC. After 30 min, the reaction
mixture was diluted with ethyl acetate (20 mL) and 10% HCl
(20 mL). The aqueous layer was separated and extracted with ethyl
acetate twice. The combined organic phases were washed with
brine (2 ꢁ 20 mL), dried over Na2SO4, and concentrated. The res-
idue was purified by flash chromatography on silica gel. Elution
with 60/40 cyclohexane/ethyl acetate gave 1.50 g (44.5%) of 25 as a
480 mg (52.2%) of 22 as a colourless liquid: 1H NMR (CDCl3)
(q, 1H, J ¼ 5.1 Hz), 4.06 (dd, 1H, J ¼ 8.2, 6.6 Hz), 3.73 (dd, 1H, J ¼ 8.2,
6.6 Hz), 3.51 (m, 5H), 1.85 (m, 2H), 1.62 (m, 2H), 1.41 (s, 3H), 1.37 (s,
3H).
d 4.27
colourless oil: 1H NMR (CDCl3)
d 7.77 (m, 6H), 7.34 (d, 2H,
J ¼ 8.8 Hz), 4.09 (m, 2H), 3.84 (m, 2H), 3.42 (m, 4H), 2.62 (d, 1H,
J ¼ 4.4 Hz), 2.42 (s, 3H), 1.67 (m, 4H).
5.1.18. 3-(4-((2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy)butoxy)-
2,6-difluorobenzamide (23)
5.1.22. 3-(4-(3-(1,3-Dioxoisoindolin-2-yl)-2-hydroxypropoxy)
butoxy)-2,6-difluorobenzamide (26)
Potassium carbonate (448 mg, 3.24 mmol) and sodium iodide
(484 mg, 3.24 mmol) were added to a solution of 2,6-difluoro-3-
hydroxybenzamide (374 mg, 2.16 mmol) in DMF (3 mL). The
mixture was stirred at room temperature for 15 min. A solution of
22 (480 mg, 2.16 mmol) in DMF (2 mL) was added dropwise and the
reaction mixture was stirred at room temperature for 30 min and
then at 70 ꢂC for 8 h. DMF was evaporated under vacuum and the
residue was taken up into ethyl acetate (10 mL) and water (10 mL).
The aqueous layer was separated and extracted with ethyl acetate
twice. The combined organic phases were washed with 1 M NaOH
(15 mL) and brine (15 mL), dried over Na2SO4, and concentrated.
Potassium carbonate (0.7 g, 5.0 mmol) was added to a solution
of 2,6-difluoro-3-hydroxybenzamide (580 mg, 3.35 mmol) in DMF
(7.5 mL). The mixture was stirred at room temperature for 15 min. A
solution of 25 (1.50 g, 3.35 mmol) in DMF (7.5 mL) was added
dropwise. The reaction mixture was stirred at room temperature
for 30 min and then at 60 ꢂC for 5 h. DMF was evaporated under
vacuum and the residue was diluted with ethyl acetate (20 mL) and
water (20 mL). The aqueous layer was separated and extracted with
ethyl acetate (2 ꢁ 20 mL). The combined organic phases were
washed with 1 M NaOH (20 mL) and brine (20 mL), dried over

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
261
Na2SO4, and concentrated. The resultant residue was purified by
flash chromatography on silica gel. Elution with ethyl acetate gave
and concentrated to give 2.02 g (97.1%) of 28 as a liquid: 1H NMR
(CDCl3)
2H).
d
7.39 (m, 5H), 6.95 (d, 1H, J ¼ 2.2 Hz), 6.81 (m, 2H), 5.09 (s,
840 mg (56.1%) of 26 as a yellow oil: 1H NMR (CDCl3)
d 7.85 (m, 2H),
7.70 (m, 2H), 3.99 (td, 1H, J ¼ 9.0, 5.3 Hz), 6.85 (td, 1H, J ¼ 9.0,
2.4 Hz), 6.17 (br s, 1H), 6.0 (br s, 1H), 4.06 (m, 1H), 3.78 (m, 2H), 2.68
(d, 1H, J ¼ 4.5 Hz), 1.81 (m, 4H).
5.1.27. 3-(2-Benzyloxy-5-chlorophenoxy)-1,2-epoxypropane (29)
Potassium carbonate (1.18 g, 8.59 mmol) was added to a solution
of 28 (2.02 g, 8.59 mmol) in DMF (30 mL). After stirring at room
temperature for 30 min, epichloridrine (2.02 mL, 25.78 mmol) was
added dropwise. The reaction mixture was stirred at 40 ꢂC over-
night, concentrated under vacuum, and diluted with ethyl acetate
(40 mL) and water (40 mL). The aqueous layer was separated and
extracted with ethyl acetate twice. The combined organic phases
were washed with 1 M NaOH (40 mL) and brine (40 mL), dried over
Na2SO4, and concentrated. The resultant residue was purified by
flash chromatography on silica gel. Elution with 90/10 cyclohexane/
ethyl acetate gave 1.50 g (57.9%) of 29 as a pale yellow oil: 1H NMR
5.1.23. 3-(4-(3-Amino-2-hydroxypropoxy)butoxy)-2,6-
difluorobenzamide hydrochloride (10)
Hydrazine hydrate (0.11 mL, 2.45 mmol) was added to a solution
of 26 (440 mg, 0.98 mmol) in ethanol (4 mL). The mixture was
stirred at room temperature overnight and then concentrated. The
residue was treated with 0.5 N HCl (6 mL). The resulting suspension
was filtered and the filtrate was concentrated. Residual water was
removed by azeotropic distillation with ethanol to give 286 mg
(92.3%) of 10 as a white wax: 1H NMR (DMSO-d)
d 8.12 (s, 1H), 7.88
(m, 4H), 7.20 (td, 1H, J ¼ 9.1, 5.2 Hz), 7.05 (td, 1H, J ¼ 9.1, 2.1 Hz), 5.49
(br s, 1H), 4.04 (t, 1H, J ¼ 6.2 Hz), 3.82 (m, 1H), 3.37 (m, 4H), 2.88 (m,
1H), 2.66 (m, 1H), 1.59 (m, 4H). 13C NMR (75 MHz, DMSO-d) 162.1,
152.3 (dd, J 239.1, 7.4 Hz), 148.6 (dd, J 247.4, 8.0 Hz), 143.8 (d, J
10.8 Hz), 117.4 (dd, J 20.6, 4.3 Hz), 116.5, (dd, J 64.1, 8.0 Hz), 111.6 (d, J
21.1 Hz), 72.9, 70.8, 69.9, 66.7, 42.7, 26.3, 26.1. Anal. Calcd for
(CDCl3) d 7.34 (m, 5H), 6.94 (m, 1H), 6.85 (m, 2H), 5.11 (s, 2H), 4.28
(dd, 1H, J ¼ 11.7, 3.5 Hz), 4.00 (dd, 1H, J ¼ 11.2, 5.2 Hz), 3.38 (m, 1H),
2.89 (m, 1H), 2.76 (m, 1H).
5.1.28. 3-(5-Chloro-2-hydroxyphenoxy)-1,2-epoxypropane (30)
A solution of 29 (0.75 g, 2.57 mmol) in ethyl acetate (16 mL) was
added with 5% Pd/C (65 mg) and vigorously shaken under hydrogen
at room temperature until hydrogen uptake ceased. The catalyst
was removed by filtration and the filtrate concentrated to give
C
14H21ClF2N2O4 (354.78).
5.1.24. 3-(1,4-Benzodioxan-2-ylmethoxy)-2,6-difluorobenzamide
(11)
450 mg (88.2%) of 30 as a pale yellow oil: 1H NMR (CDCl3)
d 6.88 (m,
Potassium carbonate (800 mg, 5.75 mmol) was added to a so-
lution of 2,6-difluoro-3-hydroxybenzamide (660 mg, 3.83 mmol) in
DMF (4 mL). After stirring at room temperature for 15 min, a so-
lution of 2-mesyloxymethyl-1,4-benzodioxane (930 mg,
3.83 mmol) in DMF (4 mL) was added. The mixture was stirred at
60 ꢂC for 6 h, concentrated, and diluted with ethyl acetate (20 mL)
and water (20 mL). The aqueous layer was separated and extracted
with ethyl acetate (20 mL) twice. The combined organic phases
were washed with 1 M NaOH (30 mL) and brine (30 mL), dried over
Na2SO4, and concentrated. The resultant residue was purified by
flash chromatography on silica gel. Elution with 1/1 cyclohexane/
ethyl acetate gave 450 mg (36.6%) of 11 as a white solid: m.p.
3H), 4.32 (dd,1H, J ¼ 11.3, 2.6 Hz), 4.00 (dd,1H, J ¼ 11.3, 5.8 Hz), 3.38
(ddd, 1H, J ¼ 5.8, 2.9, 1.6 Hz), 2.96 (m, 1H), 2.76 (dd, 1H, J ¼ 4.7,
2.9 Hz).
5.1.29. 6-Chloro-2-hydroxymethyl-1,4-benzodioxane (31)
2.5 M NaOH (1.15 mL) was added dropwise to a solution of 30
(450 mg, 2.24 mmol) in methanol (6 mL). The mixture was stirred
at room temperature overnight, concentrated, and diluted with
water (10 mL) and ethyl acetate (10 mL). The aqueous layer was
separated and extracted with ethyl acetate (2 ꢁ 10 mL). The com-
bined organic phases were washed with brine (20 mL), dried over
Na2SO4, and concentrated to give 420 mg (93.5%) of 31 as a trans-
95.10 ꢂC; 1H NMR (CDCl3)
d
7.07 (td, 1H, J ¼ 9.1, 5.1 Hz), 6.84 (m, 5H),
lucent wax: 1H NMR (CDCl3)
J ¼ 1.4 Hz), 4.25 (m, 1H), 4.10 (dd, 1H, J ¼ 11.1, 7.5 Hz), 3.87 (qd, 1H,
J ¼ 12.1, 4.5 Hz).
d
6.89 (t, 1H, J ¼ 1.4 Hz), 6.82 (d, 1H,
6.29 (br s, 1H), 6.02 (br s, 1H), 4.59 (m, 1H), 4.38 (dd, 1H, J ¼ 11.5,
2.4 Hz), 4.25 (m, 3H). 13C NMR (75 MHz, DMSO-d) 161.9, 152.8 (dd
J ¼ 240.2, 6.5 Hz), 148.6 (dd J ¼ 247.4, 8.2 Hz), 143.5 (d J ¼ 4.2 Hz),
143.4, 143.3, 122.3, 122.1, 117.8, 117.7, 117.2 (dd J ¼ 20.3, 4.5 Hz),
116.5, 111.7, 72.0, 69.0, 65.1. Anal. Calcd for C16H13F2NO4 (321.28).
5.1.30. 6-Chloro-2-mesyloxymethyl-1,4-benzodioxane (32)
Triethylamine (0.37 mL, 2.7 mmol) was added to a solution of 31
(420 mg, 2.09 mmol) in DCM (6 mL). Mesyl chloride (0.21 mL,
2.70 mmol) was added dropwise at 4 ꢂC. The reaction mixture was
stirred at room temperature for 30 min, cooled at 4 ꢂC, and diluted
with water (20 mL) and DCM (10 mL). The organic layer was
separated, washed with 10% HCl (10 mL), saturated aqueous
NaHCO3 (10 mL), and brine (10 mL), dried over Na2SO4, and
concentrated to give 601 mg of 32 (97.8%) as a yellow oil: 1H NMR
5.1.25. 2-Benzyloxy-5-chlorophenyl formate (27)
m-Chloroperbenzoic acid (2.78 g, 12.4 mmol) was added por-
tionwise
to
a
refluxing
solution
of
2-benzyloxy-5-
chlorobenzaldehyde (2.19 g, 8.86 mmol) in DCM (30 mL). The re-
action mixture was refluxed for 2 h, and then cooled at 4 ꢂC. 10%
Na2S2O5 water solution (15 mL) and saturated aqueous NaHCO3
(15 mL) were added. A white precipitate was formed. The suspen-
sion was filtered and the filtrate was extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic phases were washed with
saturated aqueous NaHCO3 (3 ꢁ 30 mL), dried over Na2SO4, and
concentrated to give 2.29 g (98.3%) of 27 as a yellow oil: 1H NMR
(CDCl3)
d 6.91 (m, 1H), 6.84 (m, 2H), 4.46 (m, 3H), 4.31 (dd, 2H,
J ¼ 11.8, 2.5 Hz), 4.13 (dd, 1H, J ¼ 11.8, 6.5 Hz), 3.09 (s, 3H).
5.1.31. 3-(6-Chloro-1,4-benzodioxan-2-yl)methoxy-2,6-
difluorobenzamide (12)
(CDCl3)
J ¼ 8.7 Hz), 5.09 (s, 2H).
d
8.24 (s, 1H), 7.37 (m, 5H), 7.16 (m, 2H), 6.95 (d, 1H,
Potassium carbonate (304 mg, 2.20 mmol) was added to a so-
lution of 2,6-difluoro-3-hydroxybenzamide (380 mg, 2.20 mmol) in
DMF (6 mL). After stirring at room temperature for 15 min, a so-
lution of 32 (615 mg, 2.20 mmol) in DMF (3 mL) was added. The
reaction mixture was stirred at 55 ꢂC overnight, concentrated, and
diluted with water (20 mL) and ethyl acetate (20 mL). The aqueous
layer was separated and extracted with ethyl acetate (2 ꢁ 20 mL).
The combined organic phases were washed 1 M NaOH (2 ꢁ 20 mL),
and brine (20 mL), dried over Na2SO4, and concentrated. The
5.1.26. 2-Benzyloxy-5-chlorophenol (28)
2.5 M NaOH (4.6 mL) was added dropwise to a solution of 27
(2.29 g, 8.71 mmol) in methanol (25 mL). The mixture was stirred at
room temperature for 5 h, concentrated, diluted with NaH2PO4/
Na2HPO4 pH 7 buffer (40 mL), and extracted with ethyl acetate
(3 ꢁ 30 mL). The combined organic phases were dried over Na2SO4

262
G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
resulting residue was purified by flash chromatography on silica
5.1.36. 3-(7-Chloro-1,4-benzodioxan-2-yl)methoxy-2,6-
gel. Elution with 70/30 toluene/ethyl acetate gave 485 mg (61.8%) of
difluorobenzamide (13)
12 as a white solid: mp 135.5 ꢂC; 1H NMR (DMSO-d)
d
8.17 (br s, 1H),
Potassium carbonate (218 mg, 1.58 mmol) was added to a solu-
tion of 2,6-difluoro-3-hydroxybenzamide (273 mg, 1.58 mmol) in
DMF (3 mL). After stirring at room temperature for 15 min, a solution
of 13 (400 mg, 1.44 mmol) in DMF (3 mL) was added. The reaction
mixture was stirred at 55 ꢂC overnight, concentrated, and diluted
with water (10 mL) and ethyl acetate (10 mL). The aqueous layer was
separated and extracted with ethyl acetate (2 ꢁ 20 mL). The com-
bined organic phases were washed 1 M NaOH (10 mL) and brine
(10 mL), dried over Na2SO4, and concentrated. The resulting residue
was purified by chromatography on silica gel. Elution with 75/25
cyclohexane/ethyl acetate gave 320 mg (62.5%) of 13 as a white
7.82 (br s, 1H), 7.27 (td, 1H, J ¼ 9.4, 5.2 Hz), 7.05 (t, 1H, J ¼ 9.4 Hz),
6.98e6.85 (m, 3H), 4.60 (m, 1H), 4.43 (dd, 1H, J ¼ 2.5, 11.6 Hz), 4.30
(m, 2H), 4.14 (dd, 1H. . J ¼ 7.2, 11.6 Hz). 13C NMR (75 MHz, DMSO-d)
161.9, 152.9 (dd, J ¼ 240.2, 6.5), 148.6 (dd, J ¼ 255.6, 7.9 Hz), 144.2,
143.4 (dd, J ¼ 11.1, 3.2 Hz), 142.4, 122.3, 122.1, 1119.2, 117.6 (d,
J ¼ 9.1 Hz), 117.4 (d, J ¼ 4.6 Hz), 116.7 (d, J ¼ 6.5 Hz), 111.7 (d,
J ¼ 20.1 Hz), 72.1, 68.9, 65.2. Anal. Calcd for C16H12ClF2NO4 (355.72).
5.1.32. 3-(2-Formyl-4-chlorophenoxy)-1,2-epoxypropane (33)
A mixture of potassium carbonate (1.32 g, 9.58 mmol) and 5-
chlorosalicylaldehyde (1.5 g, 9.58 mmol) in DMF (30 mL) was stir-
red at room temperature for 30 min. Epichloridrine (3.8 mL,
47.9 mmol) was added dropwise and stirring was continued over-
night. The reaction mixture was concentrated under vacuum,
diluted with water (30 mL) and ethyl acetate (30 mL). The organic
phase was separated, washed with 1 M NaOH (2 ꢁ 30 mL) and brine
(30 mL), dried over Na2SO4, and concentrated. The resulting residue
was purified by flash chromatography on silica gel. Elution with 80/
20 cyclohexane/ethyl acetate gave 0.90 g (44.1%) of 33 as a yellow
solid: mp 121 ꢂC; 1H NMR (DMSO-d)
d 8.15 (br s, 1H), 7.82 (br s, 1H),
7.27 (m, 1H), 7.04 (m, 1H), 6.93e6.85 (m, 3H), 4.62 (m, 1H), 4.42 (m,
1H), 4.30 (m, 2H), 4.12 (m, 1H). 13C NMR (75 MHz, DMSO-d) 161.9,
154.5,152.9 (dd, J ¼ 240.2, 6.8 Hz),148.7 (dd, J ¼ 247.6, 8.6 Hz),144.0,
143.4 (dd, J ¼ 10.9, 3.2 Hz), 142.6, 125.5, 121.9, 119.2, 117.5 (d,
J ¼ 17.9 Hz), 117.2 (d, J ¼ 20.5 Hz), 116.7 (d, J ¼ 22.6 Hz), 111.7 (d,
J ¼ 23.9 Hz), 72.2, 68.8 65.0. Anal. Calcd for C16H12ClF2NO4 (355.72).
5.1.37. (R)-3-(2-acetyl-4-chloro)phenoxy-1,2-propanediol
acetonide [(R)-37]
liquid: 1H NMR (CDCl3)
d
10.45 (s, 1H), 7.79 (d, 1H, J ¼ 2.8 Hz), 7.48
Potassium carbonate (4.57 g, 33.09 mmol) and 5-chloro-2-
hydroxyacetophenone (5.64 g, 33.09 mmol) were added to a solu-
tion of (S)-3-tosyloxy-1,2-propanediol acetonide (8.24 g,
28.78 mmol) in DMF (60 mL). After stirring at 90 ꢂC overnight, the
reaction mixture was concentrated under vacuum, diluted with
ethyl acetate, and filtered. The filtrate was concentrated and the
resulting oily residue was purified by chromatography on silica gel.
(dd, 1H, J ¼ 8.9, 2.8 Hz), 6.96 (d, 1H, J ¼ 8.9 Hz), 4.41 (dd, 1H, J ¼ 11.2,
2.7 Hz), 4.03 (dd, 1H, J ¼ 11.2, 5.9 Hz), 3.40 (m, 1H), 2.95 (m, 1H),
2.79 (dd, 1H, J ¼ 4.8, 2.7 Hz).
5.1.33. 3-(2-Formyloxy-4-chlorophenoxy)-1,2-epoxypropane (34)
m-Chloroperbenzoic acid (1.33 g, 5.93 mmol) was added por-
tionwise to a refluxing solution of 33 (0.9 g, 4.23 mmol) in DCM
(12 mL). The reaction mixture was refluxed for 1 h. After cooling at
Elution with 80/20 cyclohexane/ethyl acetate gave 8.19 g (98.2%) of
25
(R)-37 as a yellow oil: [
d
a]
¼ ꢀ15.8 (c 1, CHCl3); 1H NMR (CDCl3)
D
7.70 (d, 1H, J ¼ 2.7 Hz), 7.39 (dd, 1H, J ¼ 8.8, 2.7 Hz), 6.90 (d, 1H,
4
ꢂC, 10% Na2S2O5 water solution (10 mL) and saturated aqueous
J ¼ 8.8 Hz), 4.51 (m, 1H), 4.12 (m, 3H), 3.91 (dd, 1H, J ¼ 8.5, 5.6 Hz),
NaHCO3 (10 mL) were added. The suspension was filtered and the
filtrate was extracted with ethyl acetate (3 ꢁ 10 mL). The combined
organic phases were washed with saturated aqueous NaHCO3
(3 ꢁ 30 mL), dried over Na2SO4, and concentrated to give 0.95 g
2.64 (s, 3H), 1.45 (s, 3H), 1.39 (s, 3H).
5.1.38. (R)-3-(2-acetoxy-4-chloro)phenoxy-1,2-propanediol
acetonide [(R)-38]
(97.9%) of 34 as a yellow oil: 1H NMR (CDCl3)
d
7.20 (dd, 1H, J ¼ 8.8,
m-Chloroperbenzoic acid (12.6 g, 55.63 mmol) was added por-
tionwise to a solution of (R)-37 (6.6 g, 23.18 mmol) in dichloro-
methane (60 mL). After refluxing for 5 h, the reaction mixture was
cooled at 4 ꢂC and filtered. The filtrate was concentrated, diluted with
ethyl acetate, washed with saturated aqueous NaHCO3 (4 ꢁ 60 mL),
2.6 Hz), 7.13 (d, 1H, J ¼ 2.6 Hz), 6.96 (d, 1H, J ¼ 8.8 Hz), 4.26 (dd, 1H,
J ¼ 11.3, 3.0 Hz), 3.97 (dd, 1H, J ¼ 11.3, 5.7 Hz), 3.31 (m, 1H), 2.89 (dd,
1H, J ¼ 4.8, 4.2 Hz), 2.79 (dd, 1H, J ¼ 4.8, 2.6 Hz).
dried over Na2SO4, and concentrated to give 6.90 g (99.0%) of (R)-38
5.1.34. 7-Chloro-2-hydroxymethyl-1,4-benzodioxane (35)
25
as a yellow oil: [
a
]
¼ ꢀ13.5 (c 1.6, CHCl3); 1H NMR (CDCl3)
d 7.16 (dd,
D
1 M NaOH (6 mL) was added dropwise to a solution of 34 in
methanol (10 mL). The mixture was stirred at 60 ꢂC for 1 h,
concentrated, and diluted with ethyl acetate (10 mL) and water
(10 mL). The aqueous layer was separated and extracted with ethyl
acetate (3 ꢁ 10 mL). The combined organic phases were washed
with brine (30 mL), dried over Na2SO4, and concentrated to give
1H, J ¼ 8.7, 2.8 Hz), 7.07 (d,1H, J ¼ 2.8 Hz), 6.91 (d,1H, J ¼ 8.7 Hz), 4.41
(m, 1H), 4.03 (m, 4H), 2.34 (s, 3H), 1.45 (s, 3H), 1.39 (s, 3H).
5.1.39. (R)-3-(4-chloro-2-hydroxy)phenoxy-1,2-propanediol
acetonide [(R)-39]
580 mg (65.9%) of 35 as a yellow oil: 1H NMR (CDCl3)
d 6.90 (m, 1H),
6.79 (m, 2H), 4.26 (m, 2H), 4.09 (m,1H), 3.86 (m, 2H), 2.04 (br s,1H).
1 M NaOH (16.5 mL) and then water (15 mL) were added to a
solution of (R)-38 (6.90 g, 22 94 mmol) in methanol. After stirring at
room temperature for 1 h, the reaction mixture was concentrated,
diluted with NaH2PO4/Na2HPO4 pH 7 buffer (20 mL), water (20 mL)
and ethyl acetate (30 mL). The aqueous phase was separated and
extracted with ethyl acetate (3 ꢁ 30 mL). The combined organic
phases were dried over Na22S5O4, concentrated to give 4.42 g (74.5%)
5.1.35. 7-Chloro-2-mesyloxymethyl-1,4-benzodioxane (36)
Triethylamine (0.28 mL, 1.99 mmol) was added to a solution of
35 (300 mg, 1.49 mmol) in DCM (5 mL). Mesyl chloride (0.15 mL,
1.94 mmol) was added dropwise at 4 ꢂC. The reaction mixture was
stirred at room temperature for 30 min, cooled at 4 ꢂC, and diluted
with water (10 mL) and DCM (10 mL). The organic layer was
separated, washed with 10% HCl (10 mL), saturated aqueous
NaHCO3 (10 mL), and brine (10 mL), dried over Na2SO4, and
concentrated to give 400 mg of 32 (96.1%) as a translucent wax: 1H
of (R)-39 as a yellow oil: [
a
]
¼ þ16.3 (c 1, CHCl3); 1H NMR (CDCl3)
D
d
6.94 (m, 1H), 6.78 (m, 2H), 6.49 (br s, 1H), 4.46 (m, 1H), 4.12 (m,
2H), 4.00 (m, 1H), 3.90 (m, 1H), 1.47 (s, 3H), 1.40 (s, 3H).
5.1.40. (R)-3-(2-benzyloxy-4-chloro)phenoxy-1,2-propanediol
acetonide [(R)-40]
NMR (CDCl3)
d
6.91 (m, 1H), 6.84 (m, 2H), 4.47 (m, 2H), 4.31 (dd, 2H,
Potassium carbonate (2.54 g, 18.37 mmol) was added to a so-
lution of (R)-39 (4.32 g, 16.7 mmol) in DMF (40 mL). After 15 min,
J ¼ 11.7, 2.3 Hz), 4.13 (dd, 1H, J ¼ 11.7, 6.2 Hz), 3.10 (s, 3H).

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
263
benzyl bromide (2.0 mL, 16.7 mmol) was added and the mixture
was stirred at 60 ꢂC for 3 h and then concentrated. Water (40 mL)
and ethyl acetate (40 mL) were added. The aqueous layer was
separated and extracted with ethyl acetate (2 ꢁ 40 mL). The com-
analysis on a Kromasil 5-AmyCoat column 250 mm ꢁ 4.6 mm i.d.;
hexane/EtOH 8/2; 2 mL/min; 276 nm); 1H NMR (CDCl3)
7.07 (td,
1H, J ¼ 9.1, 5.1 Hz), 6.89 (m, 2H), 6.82 (m, 2H), 6.10 (br s,1H), 6.01 (br
s, 1H), 4.56 (m, 1H), 4.38 (m, 1H), 4.23 (m, 3H); 13C NMR identical to
that of 13.
d
bined organic phases were dried over Na2SO4 and concentrated to
25
give 5.47 g (93.9%) of (R)-40 as a wax: [
a]
¼ ꢀ14.8 (c 1, CHCl3); 1H
D
NMR (CDCl3)
d
7.37 (m, 5H), 6.93 (m, 1H), 6.88 (m, 2H), 5.07 (s, 2H),
5.1.46. (S)-3-(2-acetyl-4-chloro)phenoxy-1,2-propanediol
4.46 (m, 1H), 4.10 (m, 2H), 3.94 (m, 2H), 1.41 (s, 3H), 1.38 (s, 3H).
acetonide [(S)-37]
Prepared in 80.0% yield from (R)-3-tosyloxy-1,2-propanediol
¼ þ16.3 (c 1, CHCl3); 1H
25
5.1.41. (S)-3-(2-benzyloxy-4-chloro)phenoxy-1,2-propanediol [(S)-
41]
acetonide as described for (R)-37: [
NMR identical to that of (R)-37.
a]
D
10% HCl (27 mL) was added dropwise to a solution of (R)-40
(5.32 g,15.25 mmol) in methanol (55 mL). The reaction mixture was
stirred at 60 ꢂC for 30 min and then cooled at room temperature. A
white precipitate was formed, isolated by filtration and crystallized
5.1.47. (S)-3-(2-acetoxy-4-chloro)phenoxy-1,2-propanediol
acetonide [(S)-38]
Prepared in 77.8% yield from (S)-37 as described for (R)-38:
25
from 2/1 methanol/water to give 2.87 g (60.9%) of (R)-41 as a white
[a
]
¼ þ14.0 (c 1, CHCl3); 1H NMR identical to that of (R)-38.
D
25
solid: mp 119.1 ꢂC; [
d
a]
¼ þ11.35 (c 1, CHCl3); 1H NMR (CDCl3)
D
7.38 (m, 5H), 6.90 (m, 3H), 5.07 (s, 2H), 4.11 (m, 1H), 4.03 (m, 2H),
5.1.48. (S)-3-(4-chloro-2-hydroxy)phenoxy-1,2-propanediol
3.73 (m, 2H), 2.97 (br s, 1H), 2.19 (br s, 1H).
acetonide [(S)-39]
Prepared in 79.0% yield from (S)-38 as described for (R)-39:
25
5.1.42. (R)-3-(2-benzyloxy-4-chloro)phenoxy-1,2-
dimesyloxypropane [(R)-42]
[a
]
¼ ꢀ16.1 (c 1, CHCl3); 1H NMR identical to that of (R)-39.
D
Mesyl chloride (1.74 mL, 22.43 mmol) was added dropwise to a
solution of (S)-41 (2.77 g, 8.97 mmol) and triethylamine (3.24 mL,
23.32 mmol) in dichloromethane (30 mL) at 4 ꢂC. The mixture was
stirred at room temperature for 2 h. After cooling at 4 ꢂC, water
(30 mL) was added. The separated organic layer was washed with
10% HCl (30 mL), saturated aqueous NaHCO3 (30 mL), and brine
(30 mL), dried over Na2SO4, and concentrated. The resultant residue
5.1.49. (S)-3-(2-benzyloxy-4-chloro)phenoxy-1,2-propanediol
acetonide [(S)-40]
Prepared in 78.3% yield from (S)-39 as described for (R)-40:
25
[a
]
¼ þ15.2 (c 1, CHCl3); 1H NMR identical to that of (R)-40.
D
5.1.50. (R)-3-(2-benzyloxy-4-chloro)phenoxy-1,2-propanediol [(R)-
41]
(3.55 g) was crystallized twice from methanol to give 3.29 g (78.9%)
Prepared in 83.3% yield from (S)-40 as described for (S)-41:
25
25
of (R)-42 as a white solid: mp 108.8 ꢂC; [
a
]
¼ þ23.3 (c 1, CHCl3);
[a]
¼ ꢀ10.9 (c 1, CHCl3); mp and 1H NMR identical to those of (S)-41.
D
D
1H NMR (CDCl3)
d
7.39 (m, 5H), 6.96 (d, 1H, J ¼ 2.3 Hz), 6.92 (dd, 1H,
J ¼ 8.5, 2.3 Hz), 6.82 (d, 1H, J ¼ 8.5 Hz), 5.10 (m, 1H), 4.99 (m, 2H),
5.1.51. (S)-3-(2-benzyloxy-4-chloro)phenoxy-1,2-
dimesyloxypropane [(S)-42]
4.51 (m, 2H), 4.20 (m, 2H), 3.05 (s, 3H), 2.83 (s, 3H).
Prepared in 71.3% yield from (R)-41 as described for (R)-42:
25
5.1.43. (R)-3-(4-chloro-2-hydroxy)phenoxy-1,2-dimesyloxypropane
[(R)-43]
[a]
¼ ꢀ23.6 (c 1, CHCl3); mp and 1H NMR identical tothose of (R)-42.
D
A solution of (R)-42 (2.00 g, 4.30 mmol) in THF (20 mL) was
added with 5% Pd/C (200 mg) and vigorously shaken under
hydrogen at room temperature until hydrogen uptake ceased. The
5.1.52. (S)-3-(4-chloro-2-hydroxy)phenoxy-1,2-dimesyloxypropane
[(S)-43]
Prepared in 98.6% yield from (S)-42 as described for (R)-43:
25
catalyst was removed by filtration and the filtrate concentrated to
[a
]
¼ ꢀ6.55 (c 1, CHCl3); 1H NMR identical to that of (R)-43.
D
25
give 1.61 g (98.9%) of (R)-43 as a transparent wax: [
a
]
D
¼ þ5.35 (c 1,
CHCl3); 1H NMR (CDCl3)
d
6.94 (d, 1H, J ¼ 2.3 Hz), 6.80 (dd, 1H,
5.1.53. (R)-7-chloro-2-mesyloxymethyl-1,4-benzodioxane [(R)-44]
J ¼ 8.6, 2.3 Hz), 6.74 (d, 1H, J ¼ 8.6 Hz), 6.37 (br s, 1H), 5.28 (m, 1H),
4.51 (m, 2H), 4.28 (dd, 1H, J ¼ 10.5, 3.6 Hz), 4.16 (dd, 1H, J ¼ 10.5,
7.7 Hz), 3.16 (s, 3H), 3.11 (s, 3H).
Prepared in 97.9% yield from (S)-43 as described for (S)-
25
44:[
a]
¼ ꢀ6.75 (c 1, CHCl3); 1H NMR identical to that of (R)-40.
D
5.1.54. (R)-3-(7-chloro-1,4-benzodioxan-2-yl)methoxy-2,6-
5.1.44. (S)-7-chloro-2-mesyloxymethyl-1,4-benzodioxane [(S)-44]
Potassium carbonate (0.62 g, 4.49 mmol) was added to a solu-
tion of (R)-43 (1.53 g, 4.08 mmol) in DMF (20 mL). The reaction
mixture was stirred at 60 ꢂC for 1 h, and then concentrated. The
residue was taken up into water (20 mL) and ethyl acetate (15 mL).
The aqueous phase was separated and extracted with ethyl acetate
(2 ꢁ 15 mL). The combined organic phases were washed with 1 M
difluorobenzamide [(R)-13]
Prepared in 42.3% yield from (R)-44 as described for (S)-13: mp
25
141.1 ꢂC; [
a]
¼ ꢀ12.5 (c 1, ethyl acetate); tR z 17.5 min (deter-
D
mined by HPLC analysis on
a Kromasil 5-AmyCoat column
250 mm ꢁ 4.6 mm i.d.; hexane/EtOH 8/2; 2 mL/min; 276 nm); NMR
spectra identical to those of (S)-13.
NaOH and twice with brine, dried over Na2SO4 and concentrated to
5.1.55. Methyl 3-(1,4-benzodioxan-2-ylmethoxy)-2,6-
difluorobenzoate (45)
25
give 1.04 g (91.5%) of (S)-44 as a wax: [
NMR (CDCl3)
a]
¼ þ8.8 (c 1, CHCl3); 1H
D
d
6.91 (m, 1H), 6.82 (m, 2H), 4.47 (m, 3H), 4.29 (dd, 1H,
Potassium carbonate (1.23 g, 8.93 mmol) was added to a solu-
tion of methyl 2,6-difluoro-3-hydroxybenzoate (1.68 g, 8.93 mmol)
in DMF (10 mL). After stirring for 15 min, a solution of 2-
mesyloxymethyl-1,4-benzodioxane (2.18 g, 8.93 mmol) in DMF
(10 mL) was added. The reaction mixture was stirred at 65 ꢂC
overnight, concentrated under vacuum, and diluted with ethyl ac-
etate (40 mL) and water (75 mL). The aqueous layer was separated
and extracted with ethyl acetate (2 ꢁ 40 mL). The combined organic
phases were washed with 1 M NaOH (60 mL) and brine (60 mL),
J ¼ 11.7, 2.3 Hz), 4.11 (dd, 1H, J ¼ 11.7, 6.2 Hz), 3.08 (s, 3H).
5.1.45. (S)-3-(7-chloro-1,4-benzodioxan-2-yl)methoxy-2,6-
difluorobenzamide [(S)-13]
Prepared in 42.3% yield from (S)-44 as described for the race-
mate 13. The purification (S)-13 was accomplished by crystalliza-
tion from chloroform instead of chromatography: mp 143.2 ꢂC;
25
[
a]
¼ þ10.7 (c 1, ethyl acetate); tR z 16 min (determined by HPLC
D

264
G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
dried over Na2SO4, and concentrated. The resulting residue was
medium (DMEM) supplemented with 10% heat-inactivated
calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin in
an incubator at 5% CO2 atmosphere and 37 ꢂC. The Gram-positive
S. aureus and the Gram-negative E. coli bacterial cells
were grown in LuriaeBertani Broth (LB) medium at 37 ꢂC at
300 rpm.
crystallized from ethanol to give 1.40 g (46.7%) of 37 as a white
solid: mp 86.0 ꢂC; 1H NMR (CDCl3)
d
7.09 (td, 1H, J ¼ 8.8, 5.2 Hz),
6.88 (m, 5H), 4.57 (m, 1H), 4.40 (dd, 1H, J ¼ 11.5, 2.4 Hz), 4.25 (m,
3H), 3.96 (s, 3H).
5.1.56. 3-(1,4-Benzodioxan-2-ylmethoxy)-2,6-difluorobenzoic acid
(46)
5.2.2. Antibacterial activity
2.5 M NaOH (2 mL) and water (7.5 mL) were added to a solution
of 37 (1.40 g, 4.16 mmol) in THF (13 mL). The mixture was stirred at
50 ꢂC for 4 h, concentrated, diluted with water (10 mL) and 10% HCl
(3 mL), and extracted with ethyl acetate (3 ꢁ 20 mL). The combined
organic phases were washed with brine (30 mL), dried over Na2SO4,
and concentrated to give 1.31 g (97.8%) of 38 as a white wax: 1H
The antibacterial activity of the different compounds was
tested on a S. aureus available in the laboratory, and on the DH5
a
strain of E. coli. All of the compounds were dissolved at the final
concentration of 20 mg/ml either in dimethyl sulphoxide (DMSO)
(V, 1, 3e8, 11e14) or in bidistilled water (2, 9, 10) F7c, F10eF13,
F17eF19) and then serially diluted. After incubation at 37 ꢂC for
16 h in aerobic culture tubes, cell concentration was determined
by optical density measurement at 600 nm (OD600) in a Smart-
Spec™ 3000 spectrophotometer (Bio-Rad, Oceanside, CA, USA).
Fresh cell cultures were diluted to 103 cells/ml in a final volume
of 2 mL. Each bacterial sample was grown with different com-
NMR (CDCl3)
d
7.15 (td, 1H, J ¼ 9.0, 5.0 Hz), 6.89 (m, 5H), 4.58 (ddd,
1H, J ¼ 11.3, 6.2, 2.4 Hz), 4.40 (dd, 1H, J ¼ 11.3, 2.4 Hz).
5.1.57. 3-(1,4-Benzodioxan-2-ylmethoxy)-2,6-difluorobenzoyl
chloride (47)
Compound 38 (980 mg, 3.04 mmol) was added portionwise to
thionyl chloride (12 mL). The mixture was stirred at 50 ꢂC for 3 h,
and then concentrated to give 1.01 (97.4%) of crude 39 as a red oil:
pound concentrations, that ranged from 1 to 100
first screening. The compounds active at 1 g/ml (13 and V) were
then evaluated at lower concentrations up to 0.01 g/ml. After
mg/ml for the
m
m
1H NMR (CDCl3)
1H), 4.40 (dd, 1H, J ¼ 11.5, 2.4 Hz), 4.25 (m, 3H).
d
7.20 (td, 1H, J ¼ 9.1, 5.1 Hz), 6.88 (m, 5H), 4.59 (m,
over night incubation at 37 ꢂC, an aliquot of each sample was
harvested under sterile conditions and the OD600 measured to
determine the minimal inhibiting concentration (MIC), i.e. the
lowest compound dose at which growth is inhibited. To also
determine the minimal bactericidal concentration (MBC), i.e. the
minimal dose at which cell growth is inhibited in the absence of
the compound, the remaining cells of each sample were washed
three times with sterile LB to remove the compound, centrifuged
for 10 min at 900 ꢁ g at 4 ꢂC, and the pellet resuspended in LB.
After incubation over night a 37 ꢂC, the absence of growth was
confirmed by OD measurements. LB-diluted compounds were
used as negative controls and incubated for 2 days to verify
sterility.
5.1.58. N-(2-t-butoxycarbonylaminoethyl)-3-(1,4-benzodioxan-2-
ylmethoxy)-2,6-difluorobenzamide (48)
A solution of mono-Boc-ethylenediamine (0.6 g) in pyridine
(7.5 mL) was added to a solution of 39 (1.01 g, 3.04 mmol) at ꢀ5 ꢂC.
The mixture was stirred at the same temperature for 15 min and at
room temperature for 1 h, poured into 10% HCl (90 mL), and
washed extracted with ethyl acetate (3 ꢁ 40 mL). the combined
organic phases were with saturated aqueous NaHCO3 (50 mL) and
brine (50 mL), dried over Na2SO4, and concentrated to give 1.30 g
(92.2%) of 40 as a yellow oil: 1H NMR (CDCl3)
d
7.02 (td, 1H, J ¼ 9.1,
5.1 Hz), 6.88 (m, 5H), 6.70 (br s, 1H), 4.92 (br s, 1H), 4.56 (ddd, 1H,
J ¼ 11.5, 6.3, 2.4 Hz), 4.39 (dd, 1H, J ¼ 11.5, 2.4 Hz), 4.21 (m, 3H), 3.57
(q, 2H, J ¼ 5.4 Hz), 3.39 (m, 2H), 1.44 (s, 9H).
5.2.3. Optical microscopy
Cells were grown over night in the presence of three increasing
concentrations of the most promising compound for each bacte-
rium, starting from the previously determined MIC. S. aureus was
5.1.59. N-(2-aminoethyl)-3-(1,4-benzodioxan-2-ylmethoxy)-2,6-
difluorobenzamide hydrochloride (14)
thus grown in the presence of 13, which was used at 0.5, 1.0, 2.0
ml, whereas E. Coli was grown with 2, and tested at 10, 20, 40 g/ml.
S. aureus was also grown with V at 0.25, 0.5, 1.0 g/ml, as a positive
mg/
10% HCl (1.34 mL) was added dropwise to a solution of 40
(750 mg, 1.61 mmol) in isopropanol (15 mL). The mixture was
refluxed overnight, concentrated, diluted with water (15 mL),
washed with ethyl acetate (15 mL), and, after adjusting pH to 8 by
adding saturated aqueous NaHCO3, extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic extracts were dried over Na2SO4
and concentrated to give an oily residue, which was dissolved into
isopropanol. After addition of 10% HCl (0.57 mL), the alcoholic so-
lution was concentrated to give 575 mg (89.2%) of 14 as a light
m
m
control. Samples were analysed by phase contrast under a Zeiss
Axioskop microscope.
5.2.4. Thiazolyl blue tetrazolium bromide assay
The compounds showing the highest antibacterial activity (13
and V on S. aureus and 2 on E. Coli) were then serially diluted in
DMEM to be tested on Vero and MRC-5 cells by the thiazolyl blue
tetrazolium bromide (MTT) assay (Sigma, St Louis, MO, USA). Cells
were plated at 104 cell/well in a 96-well plate and, after over night
brown solid: 1H NMR (DMSO-d)
d 8.94 (br s,1H), 8.02 (br s, 3H), 7.34
(td,1H, J ¼ 9.4, 5.3 Hz), 7.11 (td,1H, J ¼ 9.4,1.9 Hz), 6.86 (m, 4H), 4.57
(m, 1H), 4.42 (dd, 1H, J ¼ 11.5, 2.4 Hz), 4.33 (m, 2H), 4.12 (dd, 1H,
J ¼ 11.5, 7.2 Hz), 3.57 (q, 2H, J ¼ 6.6 Hz), 2.91 (m, 2H). 13C NMR
(75 MHz, DMSO-d) 160.4, 152.9 (dd, J ¼ 241.1, 6.3), 148.6 (dd,
J ¼ 248.0, 7.7), 143.5, 143.4 (d, J ¼ 3.2 Hz), 143.3, 112.3, 122.1, 117.8,
117.7, 117.2 (d, J ¼ 10.8 Hz), 116.6 (dd, J ¼ 24.2, 19.7), 111.9 (dd,
incubation, 100
tions ranged from 1 to 200
for 13. After 24-h incubation, the compound was removed and cells
were overlaid with 1 mg/ml MTT in 100 l serum-free DMEM for
ml of each compound were added. The concentra-
m
g/ml for 2 and V, and up to 800 g/ml
m
m
J
C
¼
22.8, 3.7), 72.0, 69.0, 65.1, 38.6, 37.5. Anal. Calcd for
18H19ClF2N2O4 (400.81).
3 h at 37 ꢂC. The MTT solution was then removed, substituted with
DMSO for 10 min, and the absorbance was measured at 570 nm. The
percentage of cytotoxicity was calculated by the formula
100 ꢀ (sample OD/untreated cells OD) ꢁ 100. The compound
concentration reducing cell viability by 50% or 90% has been
defined as the toxic dose, TD50 or TD90. The therapeutic index (TI)
was also determined and defined as the ratio between TD90 and
MBC values.
5.2. Biology
5.2.1. Cells
Green monkey kidney cells (Vero) and normal human lung
fibroblasts (MRC-5) were grown in Dulbecco's modified Eagle's

G. Chiodini et al. / European Journal of Medicinal Chemistry 89 (2015) 252e265
265
efflux pumps confers the FtsZ-directed prodrug TXY436 with activity
against Gram-negative bacteria, Biochem. Pharmacol. 89 (2014) 321e328.
[15] N.R. Stokes, N. Baker, J.M. Bennett, J. Berry, I. Collins, L.G. Czaplewski, A. Logan,
R. Macdonald, R. MacLeod, H. Peasley, J.P. Mitchell, N. Nayal, A. Yadav,
A. Srivastava, D.J. Haydon, An improved small-molecule inhibitor of FtsZ with
superior in vitro potency, drug-like properties, and in vivo efficacy, Anti-
microb. Agents Chemother. 57 (2013) 317e325.
[16] N.R. Stokes, N. Baker, J.M. Bennett, P.K. Chauhan, I. Collins, D.T. Davies,
M. Gavade, D. Kumar, P. Lancett, R. Macdonald, L. MacLeod, A. Manhajan,
J.P. Mitchell, N. Nayal, Y.N. Nayal, G.R.W. Pitt, M. Singh, A. Yadav, A. Srivastava,
L.G. Czaplewski, D.J. Haydon, Design, synthesis and structure-activity re-
lationships of substituted oxazole-benzamide antibacterial inhibitors of FtsZ,
Bioorg. Med. Chem. Lett. 24 (2014) 353e359.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.100.
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