Tertiary amides with a five-membered heteroaromatic ring as new probes for the translocator protein

Article abstract of DOI:10.1016/j.ejmech.2011.07.025

In this study novel ligands of the translocator protein (TSPO), characterized by a five-membered aromatic heterocycle (i.e. oxazole, isoxazole, oxadiazole), a phenyl ring, and an amide side chain of carboxy or acetic type, were designed using a previously

Full text of DOI:10.1016/j.ejmech.2011.07.025

European Journal of Medicinal Chemistry 46 (2011) 4506e4520
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tertiary amides with a five-membered heteroaromatic ring as new probes
for the translocator protein
, ,
,
,
**
Barbara Cosimelli a 1, Francesca Simorini b 1, Sabrina Taliani b, Concettina La Motta b,
Federico Da Settimo b, Elda Severi a, Giovanni Greco a, Ettore Novellino a, Barbara Costa c,
Eleonora Da Pozzo d, Sara Bendinelli d, Claudia Martini d
*
a Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli "Federico II", Via D. Montesano 49, 80131 Napoli, Italy
b Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
c Dipartimento di Morfologia Umana e Biologia Applicata, Università di Pisa, Via Volta 4, 56126 Pisa, Italy
d Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study novel ligands of the translocator protein (TSPO), characterized by a five-membered aromatic
heterocycle (i.e. oxazole, isoxazole, oxadiazole), a phenyl ring, and an amide side chain of carboxy or
acetic type, were designed using a previously reported pharmacophore/topological model. Most of
compounds showed significant TSPO binding affinity (Ki values in the nanomolar/submicromolar range),
the highest being displayed by oxazolacetamides 6. A number of compounds were tested for their ability
to inhibit the proliferation/viability of human glioblastoma cell line U87MG. The dose-time dependent
cell response to treatment with 6d demonstrated the specificity of the observed effect. The ability of 6d
to induce mitochondrial membrane dissipation (DJm) substantiates the intracellular pro-apoptotic
mechanism activated by ligand binding to TSPO.
Received 7 March 2011
Received in revised form
13 July 2011
Accepted 15 July 2011
Available online 23 July 2011
Keywords:
Translocator protein ligands
Pharmacophore/topological model
Mitochondrial permeability transition
Tertiary amides
Ó 2011 Elsevier Masson SAS. All rights reserved.
Five-membered heterocycles
1. Introduction
well as our previously synthesized 2-phenylindolglyoxylylamide
derivative PIGA [5,6], are able to produce MPT-pore opening that
The 18 kDa translocator protein (TSPO) in the cell is primarily
localized in the outer/inner mitochondrial membrane as a compo-
nent of the megachannel responsible for mitochondrial perme-
ability transition (MPT). As a major component of the mitochondrial
membrane, TSPO mediates various mitochondrial processes
including cholesterol transport and steroid hormone synthesis,
mitochondrial respiration, calcium homeostasis, lipid metabolism,
regulation of immune functions, apoptosis and cell proliferation
[1,2]. It has been observed that cancer cells (eg from breast, ovary,
colon, prostate and brain) express high levels of TSPO and result
sensible to apoptotic cell death induced by binding of ligands to this
protein, thus suggesting TSPO as a potential anti-cancer drug target.
Actually, classic TSPO ligands as the isoquinolinecarboxamide
derivative PK 11195 [3] and the benzodiazepine Ro5-4864 [4], as
lead to dissipation of the mitochondrial membrane potential
DJm), one of the early events of the apoptotic cascade activation
[7,8].
In this view, the development of TSPO ligands characterized by
suitable physical properties may provide both useful tools for
studying MPT-pore functioning and identifying potential thera-
peutic agents against tumours.
Although human, bovine, and murine TSPOs have been isolated,
cloned and sequenced [9], the three-dimensional structure of TSPO
has not yet been determined, as its close association to the
membrane makes the purification and crystallization processes
very difficult to accomplish. Models of the secondary and tertiary
structures of the TSPO have been proposed [10,11], which hypoth-
(
esize five a-helices composed of 21 residues, the N-terminus and
C-terminus being exposed to the mitochondrial and cytoplasmic
spaces, respectively. Furthermore, site-directed mutagenesis
studies demonstrated that the portion of the receptor which
recognizes ligands such as PK 11195 is located on the first cyto-
plasmic loop [11].
* Corresponding author. Fax: þ39 081678630.
** Corresponding author. Fax: þ39 0502219605.
E-mail addresses: barbara.cosimelli@unina.it (B. Cosimelli), simorini@farm.
unipi.it (F. Simorini).
1
These Authors have equally contributed to the research.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.025

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4507
Analysis of the TSPO sequence clearly shows that this protein is
not homologous to any other known protein, apart from a signifi-
cant relationship with CrtK, a member of the protein family
responsible for the carotenoid biosynthesis [12,13]. Unfortunately,
the CrtK three-dimensional structure has not yet been solved and
such
a circumstance makes unfeasible homology building
approaches to construction of TSPO three-dimensional models.
This implies that the rational design of new TSPO ligands, as done in
the present work, must necessarily rely on ligand-based methods.
Some years ago we proposed a pharmacophore/topological
model of TSPO ligands after superimposing the structures of PK
11195 and Ro5-4864, and some pyrrolobenzothiazepine derivatives
(Fig. 1) [14]. The model (see Fig. 2) is made up by a ligand hetero-
atom accepting an H-bond from a receptor site (H1 site), and three
lipophilic pockets: the first one (L1 site) hosting an aromatic ring
and the latter two (L3 and L4 sites) accommodating either aromatic
or aliphatic substituents. Starting from the results obtained at the
date, we designed several novel classes of TSPO ligands [5,15e21],
among which the 2-phenylindolylglyoxylamides, including the
dichlorosubstituded derivative PIGA, recently emerged for their
high affinity and selectivity (Fig. 1).
In this paper, we report the synthesis, the characterization, and
the binding data of new probes for TSPO of general formula I and II
(Fig. 2), where "Het" is a five-membered aromatic heterocycle
(oxazole, isoxazole, oxadiazole), and the amide side chains are
carboxy and acetic type, respectively.
Fig. 2. Design of structures I and II guided by a pharmacophore/topological model of
TSPO ligands.
According to our model, the designed structures should accept
an H-bond from the H1 site via the carbonyl oxygen, orient their
pendant phenyl ring into the L1 pocket and fill the L3 and L4
pockets with the R1 and R2 substituents. The choice of such het-
eroaromatic rings as scaffolds was dictated by their capability of
conferring to structures of type I and II a drug-like character
according to the principles of Lipinski’s rule [22]. In terms of
physical properties, two or three heteroatoms in the aromatic
scaffold contribute to increase the water solubility of these other-
wise extremely hydrophobic structures. The lack of H-bond donor
functions in the selected heteroaromatic rings significantly
simplified the synthetic strategies whose last step is a reaction
between an acyl chloride or a carboxylic acid with a secondary
amine.
a few "asymmetric" (R1 s R2) amides substituted by groups
formerly emerged as significant in the SAR studies of
2-phenylindolglyoxylylamides [15], such as methyl/n-butyl and
ethyl/benzyl.
Compounds type II (Fig. 4) feature a methylene spacer between
the heterocyclic ring and the amide nitrogen in analogy to alpidem
and FGIN-1-27 (Fig. 1). The set of R1 and R2 substituents of the new
acetic derivatives 5 and 6 was limited to identical alkyl chains with
a length comprised in the range C3eC6. Given the “explorative”
character of our work, the X substituents at the para position were
restricted to H, Cl, F, and CH3.
The type I series of compounds was firstly synthesized (Fig. 3).
Substituents R1 and R2 and X were selected by taking into account
the structureeaffinity relationships reported in literature for
various TSPO ligands. With regard to the side chain, we synthe-
sized mostly "symmetric" (R1 ¼ R2 ¼ linear alkyl chains) as well as
Fig. 1. High affinity ligands to TSPO.
Fig. 3. Type I structures.

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B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
Scheme 2. For R1R2NH see Fig. 3.
Compounds 3bee and 4bee,h,k were prepared starting
respectively from commercially available 3-phenylisoxazole-5-
carboxylic acid (13) or 5-phenylisoxazole-3-carboxylic acid (14),
or 5-(4-chlorophenyl)isoxazole-3-carboxylic acid (15) via prelimi-
nary conversion into their correspondent non-isolated acyl-
chlorides and successive reaction with the appropriate amine
(Scheme 3).
The [3 þ 2] cycloaddition of alkenes/alkynes with in situ
generated nitrile oxides is the most convenient method for the
synthesis of five-membered heterocycles as isoxazoles [27e31]. So,
the 3-(4-methylphenyl)isoxazole-5-carboxylic acid (16) was
obtained as depicted in Scheme 4: the 4-methylbenzaldehyde (17)
was converted into the corresponding oxime 18 that, by reaction
with prop-2-yn-1-ol, gave the [3-(4-methylphenyl)isoxazol-5-yl]
methanol (19); subsequent oxidation with K2Cr2O7 gave the desired
16 (Scheme 4), which furnished, analogously, the amide 3s (Scheme
3).
Fig. 4. Type II structures.
2. Chemistry
The synthetic pathway followed to obtain 1,2,4-
oxadiazolecarboxamides 1aeg is outlined in Scheme 1; the key
intermediate was the ethyl 3-phenyl-1,2,4-oxadiazole-5-
carboxylate (9), prepared in accordance with a reported proce-
dure [23]. The ester 9 was directly converted to the desired final
amides 1aeb by heating at reflux with a large excess of diethyl-
amine and dipropylamine, respectively, easily removed by distil-
lation under reduced pressure during the work-up of the reaction.
Derivatives 1ceg, characterized by bulkier side chains, were
synthesized through the oxadiazole-5-acyl chloride 10 [24], with
a stoichiometric equivalent of the secondary amine, in anhydrous
toluene, at room temperature, in the presence of triethylamine.
For the synthesis of oxazolecarboxamides 2aeg (Scheme 2), the
2-phenyloxazole-4-carboxylic acid (11) was prepared following
a described procedure starting from the commercially available
hippuric acid [25,26]. The acid 11 was converted into the corre-
sponding acyl chloride (12) and then reacted with the appropriate
secondary amines in anhydrous toluene, in the presence of trie-
thylamine, to give the oxazolecarboxamides 2aeg.
The synthetic route to obtain compounds characterized by an
oxadiazoleacetamide moiety 5bee,hek (Fig. 4) required the prep-
aration
of
the
corresponding
unsubstituted
and
4-
chlorophenylsubstituted 2-aryloxadiazol-4-yl acetic acids 20 and
21, performed adopting our previously reported procedure [32].
The acids 20 and 21 were condensed with the appropriate
secondary amines in anhydrous tetrahydrofuran solution and in the
presence of carbonyldiimidazole (CDI), to give the target products
5bee,hek (Scheme 5).
The synthesis of oxazoleacetamides 6bee,hes, depicted in
Scheme 5, consisted initially in the preparation of (2-phenyl-1,3-
oxazol-4-yl)acetic acid (22) [32], and of 4-chloro-, 4-fluoro,
4-methyl-phenyl analogues (23) [33], (24) [32], and (25) [33],
respectively, according to previously reported procedures. Then,
the acids 22e25 furnished the final amides 6bee,hes via the
Scheme 1. For R1R2NH see Fig. 3.
Scheme 3. For R1R2NH see Fig. 3.

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4509
Scheme 6. Synthesis of the acetamide 7d.
Scheme 4. Synthesis of the 5-carboxylic acid 16.
3. Biology
reaction with the opportune amines, under the same conditions
used to synthesize oxadiazoleacetamides 5bee,hek.
The binding affinity to TSPO of all the newly synthesized
compounds 1e8 was determined by analyses of their competition
on the [3H]PK 11195 binding in membrane homogenates derived
from kidney, a tissue rich in TSPO. The binding affinity of
compounds to TSPO, expressed as Ki values, are reported in Tables 1
and 2. The subset of amides 6bee was also evaluated for the
binding affinity to the central benzodiazepine receptor (CBR), using
The preparation of N,N-di-n-pentyl-2-(3-phenylisoxazol-5-yl)
acetamide (7d) and N,N-di-n-pentyl-2-(5-phenylisoxazol-3-yl)
acetamide (8d) was not carried out starting from the corresponding
isoxazolylacetic acids, due to the difficulty of their obtainment
through the 1,3-cycloaddition reaction.
In brief, in our hands, the 1,3-cycloaddition reaction between
the in situ generated nitrile oxide and the vinylacetic acid 26 did not
[3H]Ro15-1788 as the radioligand. The compounds, tested at 10
mM
concentration, showed no binding properties in this last assay, with
inhibition percentages <25%, demonstrating their selectivity for
TSPO (data not shown).
give the desired
D
2-isoxazoline. The procedures selected for the
synthesis of 7d and 8d are depicted in Schemes 6 and 7. The amide
27, obtained by the vinylacetic acid 26 via the relevant acylchloride,
was the dipolarophile for the cycloaddition reaction with the
The compounds showing the best TSPO binding affinity, namely
compounds 6d (Ki 32 nM), 6j (Ki 45 nM), 6r (Ki 68 nM), 6n (Ki
11 nM) and 8d (Ki 51 nM), were selected to evaluate their ability to
influence the proliferation/viability of the human glioblastoma cell
line U87MG. As initial screening, U87MG cells were exposed for
24 h at a single concentration of each compound, corresponding to
1000 times the Ki value. On the basis of the results obtained,
compound 6d was in depth investigated to assess whether the
observed effect was dose-and time-dependent, by exposing the
cells to a single incubation time (24 h) and increasing 6d concen-
trations (dose-dependent treatment) and to a single concentration
of 6d for different times (time-dependent treatment).
benzaldoxime (Scheme 6). The
D
2-isoxazoline 28 was oxidized to
the desired isoxazole 7d with potassium permanganate. The
oxidizing agent, although did not give a satisfactory yield, was
however the best one among the attempted (Scheme 6).
The 3-alkylsubstituted-5-arylisoxazoles are typically obtained
by a nitroalkyl derivative and phenylacetylene by using Mukaiya-
ma’s procedure [34], but in our case this procedure or a number of
its modifications were not fully satisfactory. First of all, also in this
case, the carboxylic group is not compatible with the cycloaddition
reaction. We performed the synthesis of amide 30 analogously to
the precedent amide 27 with similar yield (Scheme 7). On the
contrary, the subsequent cycloaddition reaction of 30 with the
phenylacetylene to obtain 8d showed a very low yield; the reported
one is the best result of our attempts in which we changed the
reaction conditions.
Finally, being depolarisation of mitochondrial membrane the
early intracellular event of apoptosis activated by TSPO ligands, the
efficacy of 6d to induce mitochondrial membrane dissipation
(DJm) of U87MG cells was evaluated.
Scheme 5. For R1R2NH see Fig.4.
Scheme 7. Synthesis of the acetamide 8d.

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B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
Table 1
Compared with compounds of series I, those of type II feature
a methylene spacer between the heterocyclic ring and the amide
nitrogen. Since symmetrical di-n-butyl- and di-n-pentyl-carbox-
amides exhibited the best affinity within the series 1e4, the
synthesis of the new acetic derivatives 5 and 6 was focused on
compounds bearing R1 and R2 with a length comprised in the range
C3eC6.
The replacement of the carboxamide with an acetamide side
chain resulted in a significant general enhancement of affinity
(Table 2), both in oxadiazoles 5bee and in oxazoles 6bee, with
compound 6d (R1 ¼ R2 ¼ n-pentyl) showing Ki ¼ 32 nM.
As several of the most potent TSPO ligands (PK 11195, Ro 5-4864,
alpidem, and the indolylglyoxylamide PIGA - Fig. 1) possess
a chlorine on their pendant phenyl ring, both oxadiazoleacetamides
and oxazoleacetamides were likewise functionalized to give
derivatives 5hek, 6hek with X ¼ Cl. Actually, such a modification
increased affinity only for compounds with short N-alkyl chains
(compare 5h vs. 5b, and 5i vs. 5c), whereas it was slightly disad-
vantageous for compounds with longer N-alkyl chains (compare 5j
vs. 5d, and 5k vs. 5e).
Receptor binding affinity of compounds 1e4 (structures I) for TSPO.
Cmpd
X
R1
R2
Ki (nM)a or % of
inhibition at 10
m
M.
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
3b
3c
3d
3e
3s
4b
4c
4d
4e
4h
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2CH3
CH2CH3
>10,000
>10,000
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
CH2C6H5
772 ꢀ 54
914 ꢀ 87
4193 ꢀ 125
4873 ꢀ 310
>10,000
CH3
CH2CH3
(CH2)3CH3
CH2CH3
>10,000
>10,000
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
CH2C6H5
(CH2)3CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)5CH3
3462 ꢀ 260
613 ꢀ 45
4601 ꢀ 370
5487 ꢀ 455
>10,000
CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)5CH3
>10,000
651 ꢀ 56
379 ꢀ 25
7000 ꢀ 568
2700 ꢀ 158
>10,000
620 ꢀ 47
382 ꢀ 25
5800 ꢀ 568
>10,000
H
H
4eCH3
H
H
H
H
Further attempts to improve affinity were focused on oxazo-
leacetamides 6 by inserting at the para position of the phenyl ring
a small electron-withdrawing or electron-donating group (X ¼ F,
4-Cl
4-Cl
Table 2
4k
>10,000
Receptor binding affinity of compounds 5e8 (structures II) for TSPO.
PK11195
Ro 5-4864
9.3 ꢀ 0.5
23 ꢀ 3.1
a
The concentration of tested compounds that inhibited [3H]PK11195 binding at
rat kidney mitochondrial membranes (IC50) by 50% was determined with six
concentrations of the displacers, each performed in triplicate. Ki values are the
means ꢀ SEM of three determinations.
4. Discussion
Tables 1 and 2 list the binding affinities at the TSPO of the
heteroarylcarboxamides I and heteroarylacetamides II, respec-
tively, expressed as Ki values or percentages of inhibition of [3H]PK
11195 binding.
Within compounds of type I, several amides showed low or
moderate affinity to TSPO (Table 1). The affinity trend was similar
for oxadiazole, oxazole and isoxazole series, with the best results
(micromolar Ki values) being achieved with R1 ¼ R2 ¼ n-butyl,
n-pentyl; in particular, derivatives 1c, 2d, 3d, 4d were the most
active in their respective subsets.
Compounds with one, or both amide alkyl chains shorter than 4
carbon atoms showed to be ineffective (carboxamides 1a, 1b, 1g;
2a, 2b, 2g; 3b; 4b).
N-Ethyl-N-benzyl amides, namely 1f, 2f, did not perform better
than their “symmetric” counterparts of similar molecular weight
(1e and 2e).
The Ki values of 1d, 2d, 3d and 4d, all bearing two n-pentyl
groups on the amide nitrogen and differing only in the heterocyclic
ring, vary from 379 nM to 914 nM. Such a narrow range of potency
suggested that the four different heterocyclic rings were more or
less equivalent without possessing any particular element in
interacting with the TSPO binding cleft.
The introduction of a chlorine or a methyl group at the para
position of the phenyl ring of a few derivatives did not give any
appreciable improvement in affinity (compare 3s vs 3e, 4h vs 4b
and 4i vs 4e). Thus, further attempts in optimizing potency in series
1e4 by the introduction of other substituents (X) were not made.
Although affinity was low throughout the series of the synthe-
sized compounds, the best performing amides could be considered
a starting point for the design of new TSPO ligands, thus the
subsequent step performed was the modification of the amide side
chain.
Cmpd
X
R1
R2
Ki (nM)a
5b
5c
5d
5e
5h
5i
5j
5k
6b
6c
6d
6e
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
7d
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
H
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)4CH3
(CH2)4CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)4CH3
(CH2)4CH3
4340 ꢀ 370
489 ꢀ 36
133 ꢀ 10
1100 ꢀ 100
3400 ꢀ 250
267 ꢀ 18
152 ꢀ 11
3200 ꢀ 235
1698 ꢀ 150
346 ꢀ 25
32 ꢀ 4
H
H
H
151 ꢀ 12
942 ꢀ 85
177 ꢀ 15
45 ꢀ 3
4-Cl
4-Cl
4-Cl
4-Cl
4-F
4-F
4-F
4-F
4eCH3
4eCH3
4eCH3
4eCH3
H
704 ꢀ 56
1630 ꢀ 190
88 ꢀ 3
11 ꢀ 1
157 ꢀ 14
2200 ꢀ 188
487 ꢀ 41
68 ꢀ 7
877 ꢀ 65
257 ꢀ 20
51 ꢀ 5
8d
PK11195
Ro5-4864
H
9.3 ꢀ 0.5
23 ꢀ 3.1
a
The concentration of tested compounds that inhibited [3H]PK11195 binding at
rat kidney mitochondrial membranes (IC50) by 50% was determined with six
concentrations of the displacers, each performed in triplicate. Ki values are the
means ꢀ SEM of three determinations.

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4511
CH3). Affinity was increased by the presence of a p-fluorine (6n,
Ki ¼ 11 nM), whereas it was slightly diminished by a p-methyl.
Taken together, the above results suggest that: i) the pendant
astrocytoma), characterized by high TSPO density and high degree
of resistance to current conventional DNA alkylating chemotherapy,
was selected as experimental model. In the initial screening, the
compounds were examined for their ability to inhibit proliferation/
viability of U87MG cells at the concentration of 1000 times their Ki
value after 24 h incubation time (see Fig. 5). After the cell exposure
with each compound the percentage of proliferating/viable cells
were calculated with respect to control cells (100%) and resulted:
9.3 ꢀ 2.1% for 6d, 72.6 ꢀ 2.1% for 6j, 5.3 ꢀ 0.9% for 6r, 98 ꢀ 1.2% for
6n and 24.8 ꢀ 1.6% for 8d (Fig. 5). The Fig. 5 shows also the
percentage of proliferating/viable cells exposed to the TSPO ligand
PIGA (76.0 ꢀ 1.7%), used as reference compound. Among the tested
compounds, 6d showed the best balance in binding affinity value
and efficacy in inhibiting proliferation/viability of U87MG cells,
thus it was further investigated to exclude aspecific cytotoxic
effects. In particular, experiments were performed to assess
whether the observed effect was dose- and time-dependent.
Concerning the dose-dependent treatment, cells were exposed
to a single incubation time (24 h) and increasing 6d concentrations,
corresponding to 15, 30 and 150 times Ki value. Cell treatments
with 6d concentrations of 30 and 150 times Ki value revealed
a significant reduction of living cells with respect to control
(p < 0.01) (Fig. 6A). Concerning the time-dependent cell treatment,
U87MG cells were exposed to a single concentration of 6d (30 times
of Ki value) for 24, 48 and 72 h. As shown in Fig. 6B, cell treatment
with 6d significantly inhibits the exponential growth of cells after
48 and 72 h incubation time (p < 0.05). In summary, 6d was able to
reduce U87MG cell proliferation/viability in a dose-time dependent
manner, supporting the specificity of the effect (see Fig. 6A, B).
Finally, the ability of 6d to cause the dissipation of mitochon-
drial membrane potential was studied to substantiate the intra-
cellular pro-apoptotic mechanism activated following ligand-TSPO
binding. This effect was investigated by flow cytometric analysis,
with the use of the mitochondrial potentiometric probe JC-1 and
the uncoupling agent carbonylcyanide-m-chlorophenylhydrazone
(CCCP) as a positive control. Representative examples of the cyto-
metric analysis are given in Fig. 7A. The majority of untreated
control cells (99%) showed high fluorescence emission in both
channels and were found in the upper right quadrant of the plot.
The remaining (1%) of the untreated control cells showed low
emission of fluorescence in FL2, therefore plotting in the lower
right quadrant. An increase in the percentage of the cells plotting in
the lower right quadrant, consistent with the DJm dissipation, was
seen in cells exposed to 6d (30 times of Ki value concentration). In
phenyl ring of our ligands might be involved in a
p-stacking
interaction with an electron-rich aromatic moiety of the TSPO
binding cavity; ii) the X substituent face the steric boundaries of the
L1 pocket. Both hypotheses are consistent with the electron-
withdrawing and steric properties of the X substituent beneficial
and, respectively, unfavourable to potency in series 6. Thus, the
p-fluorine, electron-withdrawing and small, is the best one; the
methyl electron-donating and relatively bulky is the worst one; the
hydrogen and the p-chlorine exert weaker effects on affinity
according to their intermediate electronic properties and
dimensions.
Inside every subset of four compounds with amide side chains
ranging from C3 to C6 (5bee, 5hek, 6hek, 6leo, 6pes), affinity
followed the same order, that is di-n-pentyl > di-n-butyl > di-n-
hexyl > di-n-propyl, with the only exception for oxazoleacetamides
unsubstituted on the pendant phenyl 6bee, where the di-n-hexyl
derivative is about two-fold more potent than the di-n-butyl one.
Concerning the isoxazole series, we prepared and evaluated
only the acetamides 7d and 8d owing to a more difficult synthetic
route (see Chemistry). The n-pentyl groups attached to the amide
nitrogen of these two compounds were chosen as those best per-
forming in the series 5 and 6. Indeed, 8d displayed a potency
(Ki ¼ 51 nM) comparable with that of 6d (Ki ¼ 32 nM).
The observation that the most active compounds were always
the di-n-pentyl ones, both in the carboxamide and in the acetamide
derivatives, suggests that these alkyl chains possess the best steric
complementarity with the lipophilic pockets L3 and L4, once the
basic scaffold of the molecule has been anchored to the receptor.
Moreover, the heterocyclic core engages better interactions within
the receptor binding cleft when the link of the side amide function
is longer and more flexible (acetic type).
Differently from the results obtained with the above discussed
carboxamides (Table 1), in which the nature of the heterocyclic core
seemed not to be crucial in the receptor site interaction,
a comparison of the Ki values of the four acetamides differing in the
heterocycle ring and bearing two n-pentyl on the amide nitrogen
(5d, 6d, 7d and 8d) reveals that 6d and 8d (Ki values of 32 nM and
51 nM, respectively) are significantly more potent than 5d and 7d
(Ki values of 133 nM and 257 nM, respectively). These findings
might be attributed to the different H-bonding geometric proper-
ties of nitrogen and oxygen as acceptors in aromatic heterocycles.
Nobeli and co-workers investigated the directionality and relative
strengths of H-bonds between CꢁOH and aromatic rings containing
nitrogen and/or oxygen heteroatoms, using crystal structure data
and theoretical calculations [35]. These authors showed that the H-
bonds in the CꢁOH/O(furan) system are more widely scattered
around the aromatic ring plane compared with the
CꢁOH/N(pyridine) system. More specifically, the interaction
energy is destabilized up to 5.0 kJ/mol upon deviations of the CꢁOH
hydrogen from the pyridine plane by 25e35ꢂ or from the furan
plane by 50ꢂ. In the quoted article, Nobeli’s group concluded that
“substituting oxygen for nitrogen heterocycles will generally have
a marked effect on the binding of a protein ligand” [35].
In light of the above-mentioned studies, given that the TSPO
binding site might contain an H-bond donor group placed out of the
plane of the ligand heterocycle, only the endocyclic oxygen of 6d
and 8d, but not the corresponding nitrogen of 5d and 7d, would
form a significantly attractive H-bond with the receptor protein.
Compounds 6d, 6j, 6r, 6n and 8d showing the best values of
binding affinity to TSPO (nanomolar Ki values), were assayed in vitro
to determine their ability to induce death in cancer cells. The
human U87MG cell line of glioblastoma multiforme (GBM, Grade IV
Fig. 5. Effect of the TSPO ligands on U87MG cell proliferation/viability. U87MG cells
were exposed for 24 h at a single concentration of each compound (6d, 6j, 6n, 6r, 8d)
corresponding to 1000 times the Ki value and used in the MTS assay. The percentage of
proliferating cells exposed to each compound was calculated with respect to untreated
control cells (100%). The figure shows data (means ꢀ SEM) obtained from 3 experi-
ments performed in triplicate.

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B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
reduce U87MG cell proliferation/viability in a dose-time dependent
manner, and to cause the dissipation of mitochondrial membrane
potential. These data substantiate the specificity of the inhibition of
U87MG cell growth and the intracellular pro-apoptotic mechanism
activated by ligand binding to TSPO protein.
6. Experimental section
6.1. Chemistry
Melting points were determined using a Reichert Köfler hot-
stage apparatus and are uncorrected. Routine nuclear magnetic
resonance spectra were recorded on a Varian Mercuryplus 400
spectrometer operating at 400 MHz or on a Varian Gemini 200
spectrometer operating at 200 MHz. Mass spectra were obtained on
a ThermoQuest Finnigam GCQplus spectrometer using a direct
injection probe and an electron beam energy of 70 eV. Evaporation
was performed in vacuo (rotary evaporator). Analytical TLC was
carried out on Merck 0.2 mm precoated silica gel aluminum sheets
(60 F-254). Silica gel 60 (230e400 mesh) was used for column
flash-chromatography. Elemental analyses were performed by our
Analytical Laboratory and agreed with theoretical values to within
ꢀ0.4%. The acid 13 is commercially available from Bionet Research,
Cornwell. The acids 14 and 15 are from SigmaeAldrich. The
commercially available products were used as received without
additional purification. Anhydrous solvents (Toluene, THF) were
freshly distilled over sodium.
6.2. Synthesis
6.2.1. General procedure for the synthesis of N,N-dialkyl-3-phenyl-
1,2,4-oxadiazole-5-carboxamides 1aeb
3-Phenyl-1,2,4-oxadiazole-5-carboxylate
9
[23] (0.44 g,
Fig. 6. Dose and time-response curve of U87MG cell proliferation/viability following
treatment with compound 6d. The dose-and time-response effect exerted by 6d on
U87MG cell proliferation/viability was estimated using trypan blue exclusion dye
assay. The percentage of living cells after exposure to each treatment was calculated
with respect to untreated control cells, which was assigned a value of 100%. (6A) shows
the doseeresponse curve following cell exposure with increasing concentrations of 6d
(15, 30 and 150 times the Ki value) for 24 h. (6B) shows the time-response curve
following cell exposure to a single concentration of 6d (30 times the Ki value) for 24, 48
and 72h. The figures show data (means ꢀ SEM) obtained from 3 experiments per-
formed in triplicate.
2.0 mmol) was refluxed in a large excess of diethylamine or di-n-
propylamine (40.0 mmol) for 2 h (TLC analysis); the resulting
solution was evaporated to dryness to yield crude 1aeb. Product 1a
was a white solid purified by crystallization; compound 1b was
a colourless oil, purified by column chromatography.
6.2.1.1. N,N-Diethyl-3-phenyl-1,2,4-oxadiazole-5-carboxamide (1a).
Yield: 20%, mp: 45.0e46.0 ꢂC (from petroleum ether). 1H NMR
(200 MHz, DMSO-d6) d: 8.08e8.03 (m, 2H, H-Ar); 7.65e7.55 (m, 3H,
particular, significant changes in DJm were observed after treat-
ment for 48 h (24%; p < 0.05), as shown in Fig. 7B.
H-Ar); 3.57e3.45 (m, 4H, 2ꢃCH2); 1.30e1.14 (m, 6H, 2ꢃCH3). 13C
NMR (50 MHz, DMSO-d6) d: 169.1; 167.2; 154.7; 131.9; 129.3; 127.1;
125.3; 43.0 (39.9); 14.1 (12.3). IR cmꢁ1: 1682; 1651; 1566; 1354;
1293; 1135; 1118; 862; 715; 691; 613. EI-MS m/z (%): 131 (100); 246
(Mþþ1). Anal.: calcd for C13H15N3O2: 63.66%; 6.16%, 17.13%, found: C
64.02%; 6.31%, 17.50%.
5. Conclusion
In conclusion, a number of novel TSPO ligands 1e8, featuring
a five-membered heteroaromatic ring as scaffold, were synthesized
and tested. The structures were designed on the basis of a phar-
macophore/topological model summarizing the key pharmaco-
phoric interactions of well-known TSPO ligands. The highest
affinities were displayed by oxazolacetamides 6, with 6n turning
out the most potent compound (Ki ¼ 11 nM). The structureeaffinity
relationships derived from these novel compounds allowed us to
probe the TSPO binding cleft in the perspective of further molecular
design studies.
A subset of selected compounds (6d, 6j, 6r, 6n and 8d), showing
the best values of TSPO binding affinity, were assayed in vitro to
determine their ability to induce death in glioblastoma multiforme
U87MG cell line. In the initial screening, 6d, 6r and 8d, after 24 h
incubation time, resulted to inhibit proliferation/viability of
U87MG cells at the concentration of 1000 times their Ki value.
Ligand 6d was selected and further investigated to assess the
specificity of the observed effect. Actually, 6d showed to be able to
6.2.1.2. 3-Phenyl-N,N-di-n-propyl-1,2,4-oxadiazole-5-carboxamide (1b).
Yield: 36%. Colourless oil by column chromatography (petroleum
ether:ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR (200 MHz, DMSO-
d6) d: 8.08e8.03 (m, 2H, H-Ar); 7.65e7.56 (m, 3H, H-Ar); 3.48e3.40
(m, 4H, 2ꢃCH2); 1.71e1.58 (m, 4H, 2ꢃCH2); 0.95e0.79 (m, 6H, 2ꢃCH3).
13C NMR (50 MHz, DMSO-d6)
d: 169.3; 167.4; 155.3; 132.0; 129.4;
127.2; 125.4; 49.7 (47.2); 21.6 (20.0); 11.1 (10.7). IR cmꢁ1: 1662; 1443;
1300; 1214; 1139; 1100; 715; 692. EI-MS m/z (%): 131 (100); 274
(Mþþ1). Anal.: calcd for C15H19N3O2: 65.91%; 7.01%, 15.37%, found: C
66.30%; 7.31%, 15.22%.
6.2.2. General procedure for the synthesis of N,N-dialkyl-3-phenyl-
1,2,4-oxadiazole-5-carboxamides 1ceg and N,N-dialkyl-2-phenyl-
1,3-oxazole-4-carboxamides 2aeg
A solution of the appropriate secondary amine (1.0 mmol) in
2 ml of anhydrous toluene was added dropwise to a stirred

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4513
Fig. 7. Flow cytometry analyses of mitochondrial membrane potential dissipation by 6d in U87MG cells. Following U87MG cell treatment with a single concentration of 6d (30
times the Ki value) for various incubation times (12, 24 and 48 h), the cells were stained with JC-1 and analysed by FACS. 7A: Representative examples of dot-blots of the fluo-
rescence pattern of DMSO-treated (control) or 6d-, and CCCP-treated cells (positive control) stained with JC-1. Cells with polarized mitochondria are found in the upper right
quadrant of plots, corresponding in high emission in both FL 1 (x-axis) and FL 2 (y-axis) channels. After treatment, mitochondrial depolarization is evident as a decrease in the
fluorescence emission in the FL 2 and an increase in FL 1 channels, lower right (LR) quadrant. 7B: Histograms show mean values of cell percentages either in the upper right
(polarized mitochondria) and lower right quadrant of DJm analysis plots derived from three independent experiments.
solution, cooled at
0
ꢂC, of 3-phenyl-1,2,4-oxadiazol-5-
DMSO-d6)
d: 169.1; 167.2; 155.0; 131.9; 129.2; 127.0; 125.3; 48.1
carbonylchloride 10 [24] or 2-phenyloxazolcarbonylchloride 11
[25,26] (1.0 mmol) in 10 ml of the same solvent, followed by the
dropwise addition of a solution of triethylamine (0.14 ml, 1.0 mmol)
in 2 ml of anhydrous toluene. The reaction mixture was stirred at
room temperature for 3e20 h (1,2,4-oxadiazolcarboxamides 1ceg)
or 1e2 h (oxazolecarboxamides 2aeg) (TLC analysis). Triethylamine
hydrochloride was removed by filtration and the toluene solution
was washed with water, dried on MgSO4 and evaporated to dryness
to yield crude 1ceg, 2aeg, successively purified by flash-
chromatography or crystallization.
(45.5); 28.3 (28.0); 27.9 (26.3); 21.8 (21.6); 13.8 (13.7). IR cmꢁ1
:
1661; 1446; 1279; 1187; 1135; 774; 719; 692. EI-MS m/z (%): 132
(100); 330 (Mþþ1). Anal.: calcd for C19H27N3O2: 69.27%; 8.26%,
12.76%, found: C 68.89%; 8.04%, 12.38%.
6.2.2.3. N,N-Di-n-hexyl-3-phenyl-1,2,4-oxadiazole-5-carboxamide (1e).
Yield: 39%. Colourless oil by column chromatography (cyclo-
hexane:ethyl acetate ¼ 98:2 v/v as eluant). 1H NMR (200 MHz, DMSO-
d6) d: 8.07e8.02 (m, 2H, H-Ar); 7.65e7.57 (m, 3H, H-Ar); 3.50e3.42
(m, 4H, 2ꢃCH2); 1.73e1.52 (m, 4H, 2ꢃCH2); 1.38e1.11 (m, 12H,
6ꢃCH2); 0.91e0.75 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-d6)
d:
6.2.2.1. N,N-Di-n-butyl-3-phenyl-1,2,4-oxadiazole-5-carboxamide (1c).
Yield: 34%. Colourless oil by column chromatography (petroleum
ether:ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR (200 MHz, DMSO-
169.1; 167.2; 155.0; 131.9; 129.2; 127.0; 125.3; 48.1 (45.6); 30.8 (30.7);
28.2 (26.6); 25.9 (25.4); 22.0 (21.9); 13.8 (13.7). IR cmꢁ1: 1661; 1525;
1351; 1296; 1228; 1180; 1139; 1105; 1030; 712; 688. EI-MS m/z (%):
131 (100); 358 (Mþþ1). Anal.: calcd for C21H31N3O2: 70.55%; 8.74%,
11.75%, found: C 70.59%; 8.70%, 11.76%
d6) d: 8.07e8.02 (m, 2H, H-Ar); 7.64e7.60 (m, 3H, H-Ar); 3.51e3.43
(m, 4H, 2ꢃCH2); 1.70e1.56 (m, 4H, 2ꢃCH2); 1.42e1.15 (m, 4H,
2ꢃCH2); 0.96e0.79 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-d6)
d:
169.1; 167.2; 154.7; 131.9; 129.2; 127.0; 125.3; 47.9 (45.3); 30.4
(28.7); 19.5 (19.1); 13.6 (13.4). IR cmꢁ1: 1659; 1455; 1300; 1204;
1137; 953; 722; 692. EI-MS m/z (%): 131 (100); 302 (Mþþ1). Anal.:
calcd for C17H23N3O2: 67.75%; 7.69%, 13.94%, found: C 68.11%; 7.93%,
13.58%.
6.2.2.4. N-Benzyl-N-ethyl-3-phenyl-1,2,4-oxadiazole-5-carboxamide
(1f). Yield: 58%. Colourless oil by column chromatography (cyclo-
hexane:ethyl acetate ¼ 98:2 v/v as eluant). 1H NMR (200 MHz,
DMSO-d6) d: 8.09e7.97 (m, 2H, H-Ar); 7.63e7.57 (m, 3H, H-Ar);
7.40e7.28 (m, 5H, H-Ar); 4.83 and 4.75 (2s, CH2C6H5); 3.57e3.43
(m, 2H, CH2); 1.26e1.07 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-
6.2.2.2. N,N-Di-n-pentyl-3-phenyl-1,2,4-oxadiazole-5-carboxamide
(1d). Yield: 46%. Colourless oil by column chromatography
(cyclohexane:ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR (200 MHz,
d6) d: 169.1 (168.1); 167.5 (167.4); 155.5 (155.3); 136.4 (136.3); 132.1
(132.0); 129.4 (129.3); 128.6; 127.8 (127.7); 127.6 (127.5); 127.3
(127.2); 125.4; 51.2 (47.9); 43.2 (40.8); 13.90 (11.8). IR cmꢁ1: 3066;
3037; 1663; 1600; 1567; 1354; 1281; 1180; 1126; 962; 720. EI-MS
m/z (%): 132 (100); 308 (Mþþ1). Anal.: calcd for C18H17N3O2:
70.34%; 5.58%, 13.67%, found: C 70.73%; 5.15%, 13.81%.
DMSO-d6) d: 8.07e8.02 (m, 2H, H-Ar); 7.66e7.57 (m, 3H, H-Ar);
3.50e3.41 (m, 4H, 2ꢃCH2); 1.72e1.54 (m, 4H, 2ꢃCH2); 1.32e1.15
(m, 8H, 4ꢃCH2); 0.92e0.78 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz,

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B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
6.2.2.5. N-n-Butyl-N-methyl-3-phenyl-1,2,4-oxadiazole-5-carboxamide
(m, 12H, 6ꢃCH2); 0.86e0.79 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz,
(1g). Yield: 63%. Colourless oil by column chromatography
DMSO-d6) d: 160.6; 159.6; 143.0; 137.8; 130.9; 129.0; 126.3; 125.9;
(cyclohexane:ethyl acetate
¼
97:3 v/v as eluant). 1H NMR
47.7 (45.8); 31.0 (30.9); 28.9 (27.1); 26.1 (25.8); 22.0 (21.9); 13.8
(13.7). IR cmꢁ1: 3099; 1628; 1563; 1331; 1105; 712; 689. EI-MS m/z
(%):356 (100, Mþ). Anal.: calcd for C22H32N2O2: 74.12%; 9.05%, 7.86%,
found: C 73.87%; 9.12%, 8.23%.
(200 MHz, DMSO-d6) : 8.08e8.02 (m, 2H, H-Ar); 7.65e7.55 (m,
d
3H, H-Ar); 3.55e3.46 (m, 2H, CH2); 3.19 and 3.07 (2s, 3H, CH3);
1.71e1.52 (m, 2H, CH2); 1.43e1.14 (m, 2H, CH2); 0.97e0.81 (m, 3H,
CH3). 13C NMR (50 MHz, DMSO-d6)
d: 169.1 (169.0); 167.2; 155.0
(154.8); 131.9 (131.8); 129.3 (129.2); 127.1 (127.0); 125.3 (125.2);
49.8 (47.1); 35.9 (33.1); 29.7 (28.1); 19.4 (19.0); 13.6 (13.4). IR
cmꢁ1: 3018; 1665; 1597; 1559; 1538; 1412; 1347; 1303; 1197;
1139; 767; 719; 702. EI-MS m/z (%): 131 (100); 260 (Mþþ1). Anal.:
calcd for C14H17N3O2: 64.85%; 6.61%, 16.20%, found: C 65.47%;
7.11%, 15.84%.
6.2.2.11. N-Benzyl-N-ethyl-2-phenyl-1,3-oxazole-4-carboxamide (2f).
Yield: 27%. mp: 110.0e111.0 ꢂC (from petroleum ether). 1H NMR
(200 MHz, DMSO-d6) d: 8.71 (s, 1H, H-5); 8.00e7.86 (m, 2H, H-Ar);
7.67e7.21 (m, 8H, H-Ar); 5.07 and 4.67 (2s, CH2C6H5); 3.66e3.25
(m, 2H, CH2); 1.22e1.07 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-
d6) d: 161.4 (160.9); 160.0 (159.9); 143.5 (143.3); 138.0 (137.8); 137.5
(137.3); 131.1; 129.2; 129.1; 128.4; 127.7; 127.4 (127.1); 126.1; 50.4
(47.8); 42.5 (40.4); 14.2 (12.2). IR cmꢁ1: 3126; 3092; 1611; 1559;
1098; 951; 740; 709. EI-MS m/z (%): 201 (100); 306 (Mþ). Anal.:
calcd for C19H18N2O2: 74.49%; 5.92%, 9.14%, found: C 74.36%; 6.03%,
9.19%.
6.2.2.6. N,N-Diethyl-2-phenyl-1,3-oxazole-4-carboxamide (2a). Yield:
99%. Colourless oil by column chromatography (petroleum ether:-
ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR (200 MHz, DMSO-d6)
d:
8.61 (s, 1H, H-5); 8.02e7.97 (m, 2H, H-Ar); 7.60e7.53 (m, 3H, H-Ar);
3.70e3.40 (m, 4H, 2ꢃCH2); 1.22e1.04 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6)
d
: 160.5; 159.7; 142.6; 137.5; 130.9; 129.1;
6.2.2.12. N-Butyl-N-methyl-2-phenyl-1,3-oxazole-4-carboxamide (2g).
Yield: 53%. Colourless oil by column chromatography (cyclo-
hexane:ethyl acetate ¼ 90:10 v/v as eluant). 1H NMR (200 MHz,
126.2; 126.0; 42.2; (40.1); 14.6 (12.7). IR cmꢁ1: 3153, 3106, 3065,
1621, 1556, 1334, 1109, 856, 712, 692. EI-MS m/z (%): 72 (100); 244
(Mþ). Anal.: calcd for C14H16N2O2: 70.34%; 5.58%, 13.67%, found: C
70.73%; 5.15%, 13.81%.
DMSO-d6) d: 8.62 (s, 1H, H-5); 8.00e7.98 (m, 2H, H-Ar); 7.88e7.55
(m, 3H, H-Ar); 3.77e3.47 (m, 2H, CH2); 3.27 and 2.97 (2s, 3H, CH3);
1.67e1.51 (m, 2H, CH2); 1.32e1.22 (m, 2H, CH2); 0.95e0.83 (m, 3H,
6.2.2.7. 2-Phenyl-N,N-dipropyl-1,3-oxazole-4-carboxamide (2b). Yield:
CH3). 13C NMR (50 MHz, DMSO-d6)
d: 160.9; 159.7; 142.9 (142.6);
68%. Colourless oil by column chromatography (petroleum ether:-
137.4; 130.9; 129.1; 126.2; 125.9; 49.3 (47.0); 35.9 (33.5); 30.3
(28.5); 19.5 (19.2); 13.6 (13.4). IR cmꢁ1: 3099; 1631; 1556; 1334:
1109; 931; 781; 709; 692. EI-MS m/z (%): 190 (100); 258 (Mþ).
Anal.: calcd for C15H18N2O2: 69.74%; 7.02%, 10.84%, found: C 69.54%;
6.99%, 10.46%
ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR (200 MHz, DMSO-d6)
d:
8.61 (s, 1H, H-5); 8.01e7.97 (m, 2H, H-Ar); 7.59e7.55 (m, 3H, H-Ar);
3.64 (t, 2H, CH2); 3.34 (t, 2H, CH2); 1.67e1.53 (m, 4H, 2ꢃCH2);
0.89e0.83 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-d6)
d: 160.7;
159.7; 143.0; 137.7; 130.9; 129.1; 126.3; 125.9; 49.3 (47.5); 22
(20.4); 11.2 (10.8). IR cmꢁ1: 3167; 1624; 1556; 1330; 1105; 712; 692.
EI-MS m/z (%): 190 (100); 272 (Mþ). Anal.: calcd for C16H20N2O2:
70.56%; 7.40%, 10.29%, found: C 70.21%; 7.49%, 10.31%.
6.2.3. General procedure for the synthesis of N,N-dialkyl-3-
arylisoxazole-5-carboxamides 3bee,s and N,N-dialkyl-5-
arylisoxazole-3-carboxamides 4bee,h,k
Thionyl chloride (1.2 mL) was added at 0 ꢂC to the 3-
phenylisoxazole-5-carboxylic acid 13 or the 5-phenylisoxazole-3-
carboxylic acid 14 or the 5-(4-chlorophenyl)isoxazole-3-
carboxylic acid 15 or the 3-(4-methylphenyl)isoxazole-5-
carboxylic acid 16 (0.3 g, 1.58 mmol). The obtained suspension
was stirred and heated at reflux for 16 h and then cooled at 0 ꢂC. At
this temperature, a new addition of thionyl chloride (1.2 mL) was
followed by another heating at reflux for 2 h. The reaction mixture
was cooled at room temperature and the thionyl chloride excess
was evaporated to dryness in vacuo. Anhydrous THF (2 mL) was
added to the crude product. To the resulting solution, cooled
at ꢁ5 ꢂC, was added dropwise a solution of appropriate amine
(3.16 mmol) in dry THF (2 mL). The reaction mixture was stirred at
room temperature for ca. 90 min (TLC, petroleum ether/ethyl
acetate) and then the solid mass was filtered off and washed with
THF. The filtrate was evaporated to dryness; the residue was treated
with saturated sodium bicarbonate solution (20 mL) and extracted
with dichloromethane (3ꢃ15 mL). The combined organic phases
were dried (Na2SO4) and evaporated to dryness to give the crude
product, purified by flash-chromatography to give the desired
amide.
6.2.2.8. N,N-Dibutyl-2-phenyl-1,3-oxazole-4-carboxamide (2c). Yield:
65%. Colourless oil by column chromatography (petroleum ether:-
ethyl acetate ¼ 99:1 v/v as eluant). 1H NMR (200 MHz, DMSO-d6)
d:
8.60 (s, 1H, H-5); 8.00e7.92 (m, 2H, H-Ar); 7.59e7.54 (m, 3H, H-Ar);
3.66 (t, 2H, CH2); 3.36 (t, 2H, CH2); 1.65e1.51 (m, 4H, 2ꢃCH2);
1.32e1.21 (m, 4H, 2ꢃCH2); 0.94e0.83 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6) d: 160.6; 159.6; 143.0; 137.7; 130.9; 129.1;
126.3; 125.9; 47.4 (45.5); 31.0 (29.3); 19.7 (19.4); 13.7 (13.6). IR
cmꢁ1: 3160; 3065; 1624; 1559; 1334; 1108; 712; 689. EI-MS m/z (%):
190 (100); 300 (Mþ). Anal.: calcd for C18H24N2O2: 71.97%; 8.05%,
9.33%, found: C 72.09%; 8.19%, 9.03%.
6.2.2.9. N,N-Dipentyl-2-phenyl-1,3-oxazole-4-carboxamide (2d). Yield:
89%. Colourless oil by column chromatography (cyclohexane:ethyl
acetate ¼ 98:2 v/v as eluant). 1H NMR (200 MHz, DMSO-d6)
d: 8.61
(s, 1H, H-5); 8.02e7.96 (m, 2H, H-Ar); 7.59e7.55 (m, 3H, H-Ar); 3.67
(t, 2H, CH2); 3.37 (t, 2H, CH2); 1.64e1.56 (m, 4H, 2ꢃCH2); 1.40e1.18
(m, 8H, 4ꢃCH2); 0.91e0.83 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz,
DMSO-d6) d: 160.6; 159.6; 143.0; 137.7; 130.9; 129.1; 126.3; 125.9;
47.7 (45.7); 28.6 (28.5); 28.3 (26.8); 21.8 (21.7); 13.8 (13.7). IR cmꢁ1
:
3106; 1638; 1559; 1331; 1105; 774; 712; 689. EI-MS m/z (%): 190
(100); 328 (Mþ). Anal.: calcd for C20H28N2O2: 73.14%; 8.59%, 8.53%,
found: C 73.13%; 8.99%, 8.20%.
6.2.3.1. 3-Phenyl-N,N-dipropylisoxazole-5-carboxamide (3b). Yield:
60%. Purified by column chromatography (petroleum ether:ethyl
acetate ¼ 4:1 v/v as eluant). mp: 46.5e47.0 ꢂC (from petroleum
6.2.2.10. N,N-Dihexyl-2-phenyl-1,3-oxazole-4-carboxamide (2e). Yield:
ether). 1H NMR (400 MHz, CDCl3)
d: 7.84e7.81 (m, 2H, H-Ar);
55%. Colourless oil by column chromatography (cyclohexane:ethyl
7.49e7.46 (m, 3H, H-Ar); 7.08 (s, 1H, H-4); 3.51e3.44 (m, 4H,
2ꢃCH2); 1.75e1.67 (m, 4H, 2ꢃCH2); 0.97 (t, 3H, CH3); 0.93 (t, 3H,
acetate ¼ 98:2 v/v as eluant). 1H NMR (200 MHz, DMSO-d6)
d: 8.60
(s, 1H, H-5); 8.00e7.94 (m, 2H, H-Ar); 7.58e7.53 (m, 3H, H-Ar);
3.67e3.36 (m, 4H, 2ꢃCH2); 1.70e1.50 (m, 4H, 2ꢃCH2); 1.38e1.15
CH3).13C NMR (100 MHz, CDCl3)
d: 165.7; 162.3; 158.0; 130.4; 129.0;
128.2; 126.85; 106.2; 50.3; 48.5; 22.6; 20.6; 11.4; 11.0. Anal.: calcd

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4515
for C16H20N2O2: 70.56%; 7.40%, 10.29%, found: C 70.23%; 7.48%,
10.32%.
NCH2); 3.53e3.48 (m, 2H, NCH2); 1.69e1.60 (m, 4H, 2ꢃCH2); 1.40
(sext, 2H, J ¼ 7.4, CH2); 1.29 (sext, 2H, J ¼ 7.4, CH2); 0.98 (t, 3H,
J ¼ 7.4, CH3); 0.90 (t, 3H, J ¼ 7.4, CH3). 13C NMR (100 MHz; CDCl3)
d:
6.2.3.2. N,N-Dibutyl-3-phenylisoxazole-5-carboxamide (3c). Yield:
21%. Purified by column chromatography (petroleum ether:ethyl
acetate ¼ 5:1 v/v as eluant). mp: 56.9e57.4 ꢂC (from petroleum
169.9; 160.7; 159.7; 130.5; 129.1; 126.9; 125.9; 100.7; 48.7; 46.2;
31.3; 29.5; 20.3; 19.8; 13.9; 13.7. ESI-MS: 323.1 (M þ Naþ). Anal.:
calcd for C18H24N2O2: 71.97%; 8.05%, 9.33%, found: C 72.13%; 8.16%,
9.22%.
ether). 1H NMR (400 MHz; CDCl3)
d: 7.84e7.80 (m, 2H, H-Ar);
7.48e7.44 (m, 3H, H-Ar); 7.07 (s, 1H, H-4); 3.54e3.46 (m, 4H,
2ꢃNCH2); 1.68e1.61 (m, 4H, 2ꢃCH2); 1.40e1.32 (m, 4H, 2ꢃCH2);
0.97 (t, 3H, J ¼ 7.4, CH3); 0.93 (t, 3H, J ¼ 7.5, CH3). 13C NMR
6.2.3.8. N,N-Dipentyl-5-phenylisoxazole-3-carboxamide (4d). Yield:
79%. Purified by column chromatography (petroleum ether:ethyl
acetate ¼ 6:1 v/v as eluant). White wax (from petroleum ether). 1H
(100 MHz; CDCl3) d: 165.7; 162.3; 157.9; 130.4; 129.0; 128.2; 126.9;
106.2; 48.5; 46.7; 31.5; 29.4; 20.2; 19.9; 13.9; 13.7. ESI-MS: 323.2
(M þ Naþ). Anal.: calcd for C18H24N2O2: 71.97%; 8.05%, 9.33%, found:
C 72.07%; 8.17%, 9.05%.
NMR (400 MHz, CDCl3) d: 7.81e7.77 (m, 2H, H-Ar); 7.48e7.45 (m,
3H, H-Ar); 6.80 (s, 1H, H-4); 3.61e3.56 (m, 2H, NCH2); 3.51e3.46
(m, 2H, NCH2); 1.69e1.59 (m, 4H, 2ꢃCH2); 1.42e1.19 (m, 8H,
4ꢃCH2); 0.92 (t, 3H, J ¼ 6.8, CH3); 0.87 (t, 3H, J ¼ 7.0, CH3). 13C NMR
6.2.3.3. N,N-Dipentyl-3-phenylisoxazole-5-carboxamide (3d). Yield:
35%. Purified by column chromatography (petroleum ether:ethyl
acetate ¼ 6:1 v/v as eluant). White wax (from petroleum ether). 1H
(100 MHz; CDCl3) d: 169.9; 160.6; 159.7; 130.4; 129.0; 126.9; 125.9;
100.7; 48.9; 46.4; 29.2; 28.8; 28.7; 27.1; 22.4; 22.2; 14.0; 13.9. ESI-
MS: 350.9 (M þ Naþ). Anal.: calcd for C20H28N2O2: 73.14%; 8.59%,
8.53%, found: C 73.20%; 8.76%, 8.41%.
NMR (400 MHz; CDCl3) d: 7.84e7.80 (m, 2H, H-Ar); 7.48e7.46 (m,
3H, H-Ar); 7.07 (s, 1H, H-4); 3.53e3.45 (m, 4H, 2ꢃNCH2); 1.70e1.63
(m, 4H, 2ꢃCH2); 1.40e1.26 (m, 8H, 4ꢃCH2); 0.92 (t, 3H, J ¼ 6.7, CH3);
6.2.3.9. N,N-Dihexyl-5-phenylisoxazole-3-carboxamide (4e). Yield:
0.89 (t, 3H, J ¼ 7.2, CH3). 13C NMR (100 MHz; CDCl3)
d
: 165.7; 162.3;
50%. Colourless oil by column chromatography (petroleum ether:-
157.8; 130.4; 129.0; 128.2; 126.8; 106.2; 48.7; 46.9; 29.1; 29.0; 28.8;
27.0; 22.4; 22.3; 14.0; 13.9. Mass (ESI): 351.2 (M þ Naþ). Anal.: calcd
for C20H28N2O2: 73.14%; 8.59%, 8.53%, found: C 73.15%; 8.97%, 8.25%.
ethyl acetate ¼ 4:1 v/v as eluant). 1H NMR (400 MHz, CDCl3)
d:
7.81e7.79 (m, 2H, H-Ar); 7.49e7.47 (m, 3H, H-Ar); 6.80 (s, 1H, H-4);
3.61e3.57 (m, 2H, CH2N); 3.51e3.47 (m, 2H, CH2N); 1.67e1.63 (m,
4H, 2ꢃCH2); 1.35e1.24 (m,12H, 6ꢃCH2); 0.92e0.84 (m, 6H, 2ꢃCH3).
6.2.3.4. N,N-Dihexyl-3-phenylisoxazole-5-carboxamide (3e). Yield:
13C NMR (100 MHz, CDCl3)
d: 169.9; 160.7; 159.8; 130.5; 129.1;
33%. Colourless oil by column chromatography (petroleum ether:-
127.0; 125.9; 100.7; 49.0; 46.5; 31.6; 31.3; 29.2; 27.4; 26.7; 26.3;
22.6; 22.5; 14.0; 13.9. Anal.: calcd for C22H32N2O2: 74.12%; 9.05%,
7.86%, found: C 73.93%; 9.12%, 8.10%.
ethyl acetate ¼ 9:1 v/v as eluant). 1H NMR (400 MHz, CDCl3)
d:
7.84e7.82 (m, 2H, H-Ar); 7.49e7.46 (m, 3H, H-Ar); 7.08 (s, 1H, H-4);
3.53e3.46 (m, 4H, 2ꢃCH2N); 1.67e1.64 (m, 4H, 2ꢃCH2); 1.38e1.28
(m, 12H, 6ꢃCH2); 0.92e0.86 (m, 6H, 2ꢃCH3). 13C NMR (100 MHz,
6.2.3.10. 5-(4-Chlorophenyl)-N,N-dipropylisoxazole-3-carboxamide
(4h). Yield: 51%. Purified by column chromatography (petroleum
ether:ethyl acetate ¼ 4:1 v/v as eluant). mp: 96.6e97.1 ꢂC (from
CDCl3) d: 165.7; 162.3; 157.8; 130.4; 129.0; 128.2; 126.85; 106.2;
48.7; 46.9; 31.6; 31.4; 29.4; 27.3; 26.7; 26.4; 22. 6; 22.5; 14.0; 13.9.
Anal.: calcd for C22H32N2O2: 74.12%; 9.05%, 7.86%, found: C 73.91%;
9.10%, 8.20%.
petroleum ether). 1H NMR (400 MHz, CDCl3)
d: 7.73 (d, 2H, H-Ar);
7.46 (d, 2H, H-Ar); 6.80 (s, 1H, H-4); 3.57 (dd, 2H, CH2); 3.47 (dd, 2H,
CH2); 1.69 (m, 4H, 2ꢃCH2); 0.97 (t, 3H, CH3); 0.88 (t, 3H, CH3). 13
C
6.2.3.5. N,N-Dihexyl-3-(4-methylphenyl)isoxazole-5-carboxamide
(3s). Yield: 40%. Colourless oil by column chromatography (petro-
leum ether:ethyl acetate ¼ 4:1 v/v as eluant). 1H NMR (400 MHz,
NMR (100 MHz, CDCl3) d: 168.8; 160.5; 159.9; 136.6; 129.4; 127.1;
125.4; 101.1; 50.5; 48.1; 22.4; 20.7; 11.4; 11.0. Anal.: calcd for
C16H19ClN2O2: 62.64%; 6.24%, 9.13%, found: C 62.55%; 6.01%, 9.20%.
CDCl3) d: 7.72 (AA’ part of the system AA’XX’, 2H, H-Ar); 7.28 (XX΄
part of the system AA’XX’, 2H, H-Ar); 7.05 (s, 1H, H-4); 3.52e3.45
(m, 4H, 2ꢃCH2N); 2.41 (s, 3H, CH3-Ar); 1.67e1.65 (m, 4H, 2ꢃCH2);
1.39e1.22 (m, 12H, 6ꢃCH2); 0.927e0.86 (m, 6H, 2ꢃCH3). 13C NMR
6.2.3.11. 5-(4-Chlorophenyl)-N,N-dihexylisoxazole-3-carboxamide
(4k). Yield: 20%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 9:1 v/v as eluant). 1H NMR
(100 MHz, CDCl3)
d
: 165.5; 162.2; 157.9; 140.6; 129.7; 126.7; 125.3;
(400 MHz, CDCl3) d: 7.74e7.72 (AA’ part of the system AA’XX’, 2H,
106.1; 48.7; 46.9; 31.6; 31.4; 29.4; 27.3; 26. 7; 26.3; 22. 6; 22.5; 21.4;
14.0; 13.9. Anal.: calcd for C23H34N2O2: 74.55%; 9.25%, 7.56%, found:
C 74.59%; 9.18%, 7.45%.
H-Ar); 7.47e7.45 (XX’ part of the system AA’XX’, 2H, H-Ar); 6.79 (s,
1H, H-4); 3.61e3.57 (m, 2H, CH2N); 3.50e3.46 (m, 2H, CH2N);
1.68e1.62 (m, 4H, 2ꢃCH2); 1.32e1.24 (m, 12H, 6ꢃCH2); 0.92e0.84
(m, 6H, 2ꢃCH3). 13C NMR (100 MHz, CDCl3)
d: 168.8; 160.4; 159.9;
6.2.3.6. 5-Phenyl-N,N-dipropylisoxazole-3-carboxamide (4b). Yield:
25%. Purified by column chromatography (petroleum ether:ethyl
acetate ¼ 4:1 v/v as eluant). mp: 55.3e55.8 ꢂC (from petroleum
136.6; 129.4; 127.2; 125.4; 101.1; 49.0; 46.6; 31.6; 31.4; 29.2; 27.4;
26.7; 26.3; 22.6; 22.5; 14.0; 13.9.Anal.: calcd for C22H31ClN2O2:
67.59%; 7.99%, 7.17%, found: C 67.44%; 7.84%, 7.03%.
ether). 1H NMR (400 MHz, CDCl3)
d: 7.82e7.79 (m, 2H, H-Ar);
7.49e7.47 (m, 3H, H-Ar); 6.81 (s, 1H, H-4); 3.59e3.56 (m, 2H, CH2);
3.49e3.46 (m, 2H, CH2); 1.72e1.67 (m, 4H, 2ꢃCH2); 0.98 (t, 3H,
6.2.4. General procedure for the synthesis of N,N-dialkyl-2-(3-aryl-
1,2,4-oxadiazol-5-yl)acetamides 5bee,hek and N,N-dialkyl-2-(2-
aryl-1,3-oxazol-4-yl)acetamides 6bee,hes
CH3); 0.88 (t, 3H, CH3). 13C NMR (100 MHz, CDCl3)
d: 170.0; 160.8;
159.8; 130.5; 129.1; 126.9; 125.9; 100.8; 50.6; 48.1; 22.4; 20.7; 11.4;
11.0. Anal.: calcd for C16H20N2O2: 70.56%; 7.40%, 10.29%, found: C
70.62%; 7.48%, 10.14%.
To a stirred solution of 1,2,4-oxadiazoleacetic acid 20, 21 [32] or
oxazoleacetic acid 22e25 [32,33] (1.33 mmol) in 20 ml of freshly
distilled anhydrous THF, cooled at
0
ꢂC and maintained in
a
nitrogen atmosphere, 1,10-carbonyldiimidazole (216 mg,
6.2.3.7. N,N-Dibutyl-5-phenylisoxazole-3-carboxamide (4c). Yield:
30%. Purified by column chromatography (petroleum ether:ethyl
acetate ¼ 5:1 v/v as eluant). mp: 65.9e66.7 ꢂC (from petroleum
1.33 mmol) was added. After 10 min a solution of an equimolar
amount of the appropriate amine in anhydrous THF was added
dropwise. The reaction mixture was allowed to warm to room
temperature and stirred, under nitrogen atmosphere, for 72 h on
the whole. Based on TLC analysis, an excess of reagents (20e40%)
ether). 1H NMR (400 MHz, CDCl3)
d: 7.82e7.75 (m, 2H, H-Ar);
7.49e7.42 (m, 3H, H-Ar); 6.81 (s, 1H, H-4); 3.63e3.58 (m, 2H,

4516
B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
was eventually added at the reaction time of 24 and/or 48 h. The
final solution was evaporated to dryness and the residue was dis-
solved in CH2Cl2. The dichloromethane solution was washed with
1N HCl, 10% NaHCO3 and water, dried on MgSO4 and evaporated to
dryness, obtaining crude 5b-e,h-k, 6b-e,h-s, purified by recrystal-
lization or flash-chromatography.
2ꢃCH2); 1.60e1.19 (m, 8H, 4ꢃCH2); 0.95e0.84 (m, 6H, 2ꢃCH3). 13
C
NMR (50 MHz, DMSO-d6) d: 175.9; 166.8; 164.7; 136.2; 129.4;
128.7; 125.0; 47.3 (45.0); 32.2; 30.5 (29.3); 19.5 (19.4); 13.7 (13.6).
IR cmꢁ1: 1658; 1593; 1569; 1112; 900; 726. Anal.: calcd for
C18H24ClN3O2: 61.79%; 6.91%, 12.01%, found: C 61.49%; 6.45%,
11.70%.
6.2.4.1. 2-(3-Phenyl-1,2,4-oxadiazol-5-yl)-N,N-dipropylacetamide (5b).
Yield: 40%, mp: 89.0e90.0 ꢂC (from petroleum ether). 1H NMR
6.2.4.7. 2-[3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl]-N,N-dipentyla-
cetamide (5j). Yield: 47%. mp: 50.0e55.0 ꢂC (from petroleum
(200 MHz, DMSO-d6)
d: 8.03e7.98 (m, 2H, H-Ar); 7.60e7.54 (m, 3H,
ether). 1H NMR (200 MHz, DMSO-d6)
d: 8.03e7.98 (m, 2H, H-Ar);
H-Ar); 4.34 (s, 2H, CH2); 3.35e3.19 (m, 4H, 2ꢃCH2); 1.69e1.40 (m,
7.60e7.54 (m, 3H, H-Ar); 4.32 (s, 2H, CH2); 3.32e3.21 (m, 4H,
4H, 2ꢃCH2); 0.93e0.79 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-
2ꢃCH2); 1.60e1.18 (m, 12H, 6ꢃCH2); 0.91e0.82 (m, 6H, 2ꢃCH3). 13
C
d6)
d: 175.5; 167.6; 165.0; 131.5; 129.3; 126.9; 126.2; 49.1 (46.9);
NMR (50 MHz, DMSO-d6) d: 175.9; 166.8; 164.7; 136.3; 129.5;
32.2; 25.6 (20.4); 11.1 (10.9). IR cmꢁ1: 1637; 1597; 1327; 1231;
1088; 1023; 801; 719. Anal.: calcd for C16H21N3O2: 66.88%; 7.34%,
14.62%, found: C 66.70%; 7.73%, 14.25%.
128.7; 125.0; 47.6 (45.2); 32.2; 28.5 (28.3); 28.0 (26.7); 21.9 (21.8);
13.9 (13.8). IR cmꢁ1: 1651; 1597; 1135; 1115; 907; 787; 715. Anal.:
calcd for C20H28ClN3O2: 63.56%; 7.47%, 11.12%, found: C 63.88%;
7.90%, 11.16%.
6.2.4.2. N,N-Dibutyl-2-(3-phenyl-1,2,4-oxadiazol-5-yl)acetamide (5c).
Yield: 38%. Colourless oil by column chromatography (petroleum
ether:ethyl acetate ¼ 90:10 v/v as eluant). 1H NMR (200 MHz;
6.2.4.8. 2-[3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl]-N,N-dihex-
ylacetamide (5k). Yield: 44%. Colourless oil by column chroma-
tography (petroleum ether:ethyl acetate ¼ 95:5 v/v as eluant). 1H
DMSO-d6) d: 8.04e7.99 (m, 2H, H-Ar); 7.61e7.57 (m, 3H, H-Ar); 4.33
(s, 2H, CH2); 3.36e3.22 (m, 4H, 2ꢃCH2); 1.60e1.19 (m, 8H, 4ꢃCH2);
NMR (200 MHz, DMSO-d6) d: 8.02e7.98 (m, 2H, H-Ar); 7.60e7.57
0.95e0.84 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-d6)
d
: 175.6;
(m, 3H, H-Ar); 4.32 (s, 2H, CH2); 3.32e3.21 (m, 4H, 2ꢃCH2);
167.6; 164.8; 131.5; 129.3; 126.9; 126.2; 47.3 (45.0); 32.2; 30.5
(29.3); 19.6 (19.5); 13.7 (13.6). IR cmꢁ1: 1658; 1593; 1569; 1112;
900; 726. Anal.: calcd for C18H25N3O2: 68.54%; 7.99%, 13.32%, found:
C 68.63%; 7.82%, 12.93%.
1.54e1.18 (m, 16H, 8ꢃCH2); 0.88e0.80 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6) d: 175.9; 166.8; 164.7; 136.3; 129.4; 128.7;
125.0; 47.6 (45.3); 32.2; 31.0 (30.9); 28.3 (27.0); 25.9 (25.8); 22.0
(21.9); 13.8 (13.7). IR cmꢁ1: 1651; 1597; 1107; 1057; 1016; 797; 716;
690. Anal.: calcd for C22H32ClN3O2: 65.09%; 7.95%, 10.35%, found: C
65.30%; 8.27%, 9.97%.
6.2.4.3. N,N-Dipentyl-2-(3-phenyl-1,2,4-oxadiazol-5-yl)acetamide
(5d). Yield: 28%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR
6.2.4.9. 2-(2-Phenyl-1,3-oxazol-4-yl)-N,N-dipropylacetamide (6b).
Yield: 13%. Colourless oil by column chromatography (petroleum
ether:ethyl acetate ¼ 96:4 v/v as eluant). 1HNMR (200 MHz,
(200 MHz, DMSO-d6) d: 8.03e7.98 (m, 2H, H-Ar); 7.60e7.54 (m, 3H,
H-Ar); 4.32 (s, 2H, CH2); 3.32e3.21 (m, 4H, 2ꢃCH2); 1.60e1.18 (m,
12H, 6ꢃCH2); 0.91e0.82 (m, 6H, 2ꢃCH3) 13C NMR (50 MHz, DMSO-
DMSO-d6) d: 8.01 (s, 1H, H-5); 7.97e7.92 (m, 2H, H-Ar); 7.56e7.51
d6)
d
: 175.5; 167.5; 164.6; 131.4; 129.1; 126.8; 126.1; 47.5 (45.2);
(m, 3H, H-Ar); 3.67 (s, 2H, CH2); 3.25e3.17 (m, 4H, 2ꢃCH2);
32.2; 28.5 (28.3); 28.0 (26.7); 21.8; 13.8. IR cmꢁ1: 1651; 1597; 1135;
1115; 907; 787; 715. Anal.: calcd for C20H29N3O2: 69.94%; 8.51%,
12.23%, found: C 69.57%; 8.82%, 11.85%.
1.62e1.42 (m, 4H, 2ꢃCH2); 0.91e0.77 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6) d: 167.9; 159.8; 136.7; 136.6; 130.3; 129.0;
126.9; 125.6; 49.0 (46.8); 31.2; 21.8 (20.4); 11.2 (11.0). IR cmꢁ1
:
1648; 1587; 1344; 1259; 1098; 1023; 801; 712. EI-MS m/z (%): 286
(Mþ, 100). Anal.: calcd for C17H22N2O2: 71.30%; 7.74%, 9.78%, found:
C 70.95%; 8.00%, 9.51%.
6.2.4.4. N,N-Dihexyl-2-(3-phenyl-1,2,4-oxadiazol-5-yl)acetamide (5e).
Yield: 35%. Colourless oil by column chromatography (petroleum
ether:ethyl acetate ¼ 96:4 v/v as eluant). 1H NMR (200 MHz, DMSO-
d6)
d
: 8.02e7.98 (m, 2H, H-Ar); 7.60e7.57 (m, 3H, H-Ar); 4.32 (s, 2H,
6.2.4.10. N,N-Dibutyl-2-(2-phenyl-1,3-oxazol-4-yl)acetamide (6c).
Yield: 18%. Colourless oil by column chromatography (cyclo-
hexane:ethyl acetate ¼ 95:5 v/v as eluant). 1H NMR (200 MHz,
CH2); 3.32e3.21 (m, 4H, 2ꢃCH2); 1.54e1.18 (m, 16H, 8ꢃCH2);
0.88e0.80 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-d6)
d: 175.5;
167.5; 164.6; 131.4; 129.1; 126.8; 126.1; 47.6 (45.2); 40.1; 32.2
(31.0); 28.3 (27.0); 25.9 (25.8); 22.0 (21.9); 13.8. IR cmꢁ1: 1651;
1597; 1107; 1057; 1016; 797; 716; 690. Anal.: calcd for C22H33N3O2:
71.12%; 8.95%, 11.31%, found: C 71.40%; 8.28%, 11.00%.
DMSO-d6) d: 8.00 (s, 1H, H-5); 7.97e7.92 (m, 2H, H-Ar); 7.55e7.49
(m, 3H, H-Ar); 3.66 (s, 2H, CH2); 3.28e3.21 (m, 4H, 2ꢃCH2);
1.57e1.18 (m, 8H, 4ꢃCH2); 0.93e0.83 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6) d: 167.8; 159.8; 136.7; 136.6; 130.3; 129.0;
126.9; 125.6; 47.3 (44.9); 31.2; 30.8 (29.4); 19.6 (19.5); 13.7. IR
cmꢁ1: 1645; 1583; 1094; 1057; 934; 774; 716; 689.EI-MS m/z (%):
314 (Mþ, 100).Anal.: calcd for C19H26N2O2: 72.58%; 8.33%, 8.91%,
found: C 71.12%; 8.71%, 8.83%.
6.2.4.5. 2-[3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl]-N,N-dipropyla-
cetamide (5h). Yield: 37%. mp: 190.0e195.0 ꢂC (from petroleum
ether). 1H NMR (200 MHz, DMSO-d6)
d: 8.03e7.99 (m, 2H, H-Ar);
7.60e7.54 (m, 3H, H-Ar); 4.34 (s, 2H, CH2); 3.35e3.19 (m, 4H,
2ꢃCH2); 1.69e1.40 (m, 4H, 2ꢃCH2); 0.93e0.79 (m, 6H, 2ꢃCH3). 13
C
6.2.4.11. N,N-Dipentyl-2-(2-phenyl-1,3-oxazol-4-yl)acetamide (6d).
Yield: 23%. Colourless oil by column chromatography (petroleum
ether:ethyl acetate ¼ 98:2 v/v as eluant). 1H NMR (200 MHz, DMSO-
NMR (50 MHz, DMSO-d6) d: 175.8; 166.7; 164.7; 136.1; 129.3;
128.6; 124.9; 49.1 (46.8); 32.2; 21.5 (20.3); 11.1 (10.9). IR cmꢁ1
1637; 1597; 1327; 1231; 1088; 1023; 801; 719. Anal.: calcd for
C16H20ClN3O2: 59.72%; 6.26%, 13.06%, found: C 59.42%; 6.53%,
12.90%.
:
d6) d: 8.01 (s, 1H, H-5); 7.97e7.92 (m, 2H, H-Ar); 7.56e7.50 (m, 3H,
H-Ar); 3.66 (s, 2H, CH2); 3.25e3.19 (m, 4H, 2ꢃCH2); 1.53e1.12 (m,
12H, 6ꢃCH2); 0.88e0.80 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-
d6) d: 167.8; 159.8; 136.8; 136.6; 130.3; 128.9; 126.9; 125.6; 47.4
6.2.4.6. N,N-Dibutyl-2-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]
(45.1); 31.2; 28.5 (28.4); 28.3 (26.9); 21.9; 13.8. IR cmꢁ1: 1645;
1587; 1132; 1057; 938; 780; 716; 689. EI-MS m/z (%):194 (100); 342
(Mþ, 64). Anal.: calcd for C21H30N2O2: 73.65%; 8.83%, 8.18%, found: C
73.32%; 9.13%, 8.06%.
acetamide (5i). Yield: 17%. mp: 76.0e82.0 ꢂC (from petroleum
ether). 1H NMR (200 MHz, DMSO-d6)
d
: 8.04e7.99 (m, 2H, H-Ar);
7.67e7.63 (m, 3H, H-Ar); 4.34 (s, 2H, CH2); 3.32e3.18 (m, 4H,

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4517
6.2.4.12. N,N-Dihexyl-2-(2-phenyl-1,3-oxazol-4-yl)acetamide (6e).
NMR (200 MHz, DMSO-d6)
d: 8.02e7.95 (m, 3H, H-5 and H-Ar);
Yield: 20%. Colourless oil by column chromatography (petroleum
7.41e7.32 (m, 2H, H-Ar); 3.67 (s, 2H, CH2); 3.31e3.17 (m, 4H,
ether:ethyl acetate ¼ 93:7 v/v as eluant). 1H NMR (200 MHz, DMSO-
2ꢃCH2); 1.62e1.41 (m, 4H, 2ꢃCH2); 0.92e0.78 (m, 6H, 2ꢃCH3). 13
C
d6)
d: 8.00 (s, 1H, H-5); 7.96e7.91 (m, 2H, H-Ar); 7.56e7.48 (m, 3H,
NMR (50 MHz, DMSO-d6)
d
: 168.1; 163.3 (J ¼ 248.3); 159.2; 136.9;
H-Ar); 3.65 (s, 2H, CH2); 3.37e3.19 (m, 4H, 2ꢃCH2); 1.58e1.10 (m,
136.7; 128.2 (J ¼ 8.7); 123.6 (J ¼ 3.4); 116.3 (J ¼ 22.1); 49.1 (46.8);
31.1; 21.8 (20.4); 11.2 (11.0). IR cmꢁ1: 1637; 1460; 1378; 1239; 1094;
852; 736. Anal.: calcd for C17H21FN2O2: 67.09%; 6.95%, 9.20%, found:
C 66.68%; 6.61%, 9.60%.
16H, 8ꢃCH2); 0.95e0.70 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz, DMSO-
d6) d: 167.8; 159.8; 136.7; 136.6; 130.3; 128.9; 126.9; 125.6; 47.5
(45.1); 31.3 (29.9); 31.0; 28.6 (27.1); 25.5 (25.4); 22.0; 13.38. IR
cmꢁ1: 1652; 1590; 1102; 1057; 935; 778; 716; 689. EI-MS m/z
(%):194 (100); 370 (Mþ, 43). Anal.: calcd for C23H34N2O2: 74.55%;
9.25%, 7.56%, found: C 74.15%; 9.49%, 7.28%.
6.2.4.18. N,N-Dibutyl-2-[2-(4-fluorophenyl)-1,3-oxazol-4-yl]acet-
amide (6m). Yield: 46%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 90:10 v/v as eluant). 1H NMR
6.2.4.13. 2-[2-(4-Chlorophenyl)-1,3-oxazol-4-yl]-N,N-dipropylaceta-
mide (6h). Yield: 45%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 90:10 v/v as eluant). 1H NMR
(200 MHz, DMSO-d6) d: 8.02e7.95 (m, 3H, H-5 and H-Ar); 7.41e7.33
(m, 2H, H-Ar); 3.65 (s, 2H, CH2); 3.37e3.20 (m, 4H, 2ꢃCH2);
1.52e1.21 (m, 8H, 4ꢃCH2); 0.93e0.83 (m, 6H, 2ꢃCH3). 13C NMR
(200 MHz, DMSO-d6)
d: 8.04 (s, 1H, H-5); 7.97e7.93 (AA’ part of the
(50 MHz, DMSO-d6)
d
: 167.8; 163.1 (J ¼ 248.1); 158.7; 136.8; 136.6;
system AA’XX’, 2H, H-Ar); 7.63e7.58 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.68 (s, 2H, CH2); 3.25e3.18 (m, 4H, 2ꢃCH2); 1.63e1.43 (m,
4H, 2ꢃCH2); 0.92e0.78 (m, 6H, 2ꢃCH3).13C NMR (50 MHz, DMSO-d6)
128.1 (J ¼ 9.1); 123.5 (J ¼ 3.7); 116.2 (J ¼ 22.9); 47.2 (44.9); 31.1; 30.5
(29.4); 19.7 (19.6); 13.7. IR cmꢁ1: 1648; 1497; 1368; 1214; 1060;
838; 732. Anal.: calcd for C19H25FN2O2: 68.65%; 7.58%, 8.43%, found:
C 69.04%; 7.72%, 8.05%.
d
: 168.0; 159.0; 137.2; 136.9; 135.1; 129.3; 127.5; 125.7; 49.1 (46.8);
31.1; 21.8 (20.4); 11.2 (11.0). IR cmꢁ1: 1641; 1427; 1257; 1091; 1014;
839; 729. EI-MS m/z (%):139 (100); 320 (Mþ, 81). Anal.: calcd for
C17H21ClN2O2: 63.64%; 6.60%, 8.73%, found: C 64.01%; 6.96%, 8.73%.
6.2.4.19. 2-[2-(4-Fluorophenyl)-1,3-oxazol-4-yl]-N,N-dipentylaceta-
mide (6n). Yield: 48%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 95:5 v/v as eluant). 1H NMR
6.2.4.14. N,N-Dibutyl-2-[2-(4-chlorophenyl)-1,3-oxazol-4-yl]acet-
amide (6i). Yield: 60%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 93:7 v/v as eluant). 1H NMR
(200 MHz, DMSO-d6) d: 8.02e7.95 (m, 3H, H-5 and H-Ar);
7.45e7.32 (m, 2H, H-Ar); 3.64 (s, 2H, CH2); 3.27e3.16 (m, 4H,
2ꢃCH2); 1.60e1.15 (m, 12H, 6ꢃCH2); 0.87e0.80 (m, 6H, 2ꢃCH3). 13
C
(200 MHz, DMSO-d6)
d: 8.05 (s, 1H, H-5); 7.96e7.93 (AA’ part of the
NMR (50 MHz, DMSO-d6)
d
: 167.8; 163.1 (J ¼ 248.2); 159.0; 136.8;
system AA’XX’, 2H, H-Ar); 7.63e7.59 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.70 (s, 2H, CH2); 3.28e3.21 (m, 4H, 2ꢃCH2); 1.53e1.21
(m, 8H, 4ꢃCH2); 0.94e0.84 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz,
136.6; 128.1 (J ¼ 9.2); 123.6 (J ¼ 2.7); 116.1 (J ¼ 22.9); 47.4 (45.1);
31.2; 28.5 (28.3); 28.2 (26.9); 21.4; 13.3. IR cmꢁ1: 1648; 1456; 1371;
1235; 1098; 838; 736. Anal.: calcd for C21H29FN2O2: 69.97%; 8.11%,
7.77%, found: C 69.87%; 8.27%, 7.39%.
DMSO-d6) d: 167.9; 159.0; 137.2; 136.9; 135.1; 129.3; 127.5; 125.8;
47.2 (44.9); 31.1; 30.8 (29.4); 19.6 (19.5); 13.8 (13.5). IR cmꢁ1: 1655;
1607; 1580; 1092; 1016; 835; 736. EI-MS m/z (%): 348 (Mþ, 100).
Anal.: calcd for C19H25ClN2O2: 65.41%; 7.22%, 8.03%, found: C
65.79%; 7.55%, 8.13%.
6.2.4.20. 2-[2-(4-Fluorophenyl)-1,3-oxazol-4-yl]-N,N-dihexylacetamide
(6o). Yield: 68%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 90:10 v/v as eluant). 1H NMR
(200 MHz, DMSO-d6) d: 8.01e7.94 (m, 3H, H-5 and H-Ar); 7.45e7.32
6.2.4.15. 2-[2-(4-Chlorophenyl)-1,3-oxazol-4-yl]-N,N-dipentylaceta-
mide (6j). Yield: 77%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 95:5 v/v as eluant). 1H NMR
(m, 2H, H-Ar); 3.64 (s, 2H, CH2); 3.37e3.19 (m, 4H, 2ꢃCH2);
1.51e1.22 (m, 16H, 8ꢃCH2); 0.86e0.80 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6)
d
: 167.7; 163.1 (J ¼ 248.1); 159.0; 136.7; 136.6;
(200 MHz, DMSO-d6)
d
: 8.05 (s, 1H, H-5); 7.97e7.93 (AA’ part of the
128.0 (J ¼ 9.2); 123.5 (J ¼ 2.7); 116.1 (J ¼ 22.0); 47.5 (45.1); 31.2
(31.1); 31.0; 28.5 (27.1); 26.0 (25.8); 22.0; 13.8. IR cmꢁ1: 1648; 1497;
1371; 1235; 1060; 838; 736. Anal.: calcd for C23H33FN2O2: 71.10%;
8.56%, 7.21%, found: C 71.29%; 8.42%, 7.00%.
system AA’XX’, 2H, H-Ar); 7.62e7.58 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.66 (s, 2H, CH2); 3.28e3.21 (m, 4H, 2ꢃCH2); 1.57e1.22
(m, 12H, 6ꢃCH2); 0.87e0.81 (m, 6H, 2ꢃCH3). 13C NMR (50 MHz,
DMSO-d6) d: 167.7; 158.9; 137.1; 136.8; 135.0; 129.1; 127.3; 125.6;
47.4 (45.1); 31.1; 28.5 (28.3); 28.2 (26.8); 21.9; 13.8. IR cmꢁ1: 1648;
1423; 1266; 1088; 1013; 839; 723. EI-MS m/z (%): 139 (100); 376
(Mþ, 3). Anal.: calcd for C21H29ClN2O2: 66.92%; 7.76%, 7.43%, found:
C 66.58%; 7.39%, 7.21%.
6.2.4.21. 2-[2-(4-Methylphenyl)-1,3-oxazol-4-yl]-N,N-dipropylaceta-
mide (6p). Yield: 58%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 97:3 v/v as eluant). 1H NMR
(200 MHz, DMSO-d6) d: 7.93 (s, 1H, H-5); 7.84e7.80 (AA’ part of the
system AA’XX’, 2H, H-Ar); 7.34e7.30 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.63 (s, 2H, CH2); 3.35e3.16 (m, 4H, 2ꢃCH2); 2.36 (s, 3H,
CH3); 1.62e1.42 (m, 4H, 2ꢃCH2); 0.91e0.77 (m, 6H, 2ꢃCH3). 13C
6.2.4.16. 2-[2-(4-Chlorophenyl)-1,3-oxazol-4-yl]-N,N-dihex-
ylacetamide (6k). Yield: 48%. Colourless oil by column chroma-
1
tography (petroleum ether:ethyl acetate ¼ 97:3 v/v as eluant). H
NMR (50 MHz, DMSO-d6) d: 168.0; 160.0; 140.1; 136.4; 136.3;
NMR (200 MHz, DMSO-d6)
d: 8.04 (s, 1H, H-5); 7.97e7.93 (AA’ part
129.5; 125.6; 124.2; 49.1 (46.8); 31.2; 21.8 (20.9); 20.4; 11.2 (10.9).
IR cmꢁ1: 1648; 1583; 1497; 1371; 1344; 1060; 825; 729. Anal.: calcd
for C18H24N2O2: 71.97%; 8.05%, 9.33%, found: C 71.57%; 8.42%, 8.98%.
of the system AA’XX’, 2H, H-Ar); 7.63e7.58 (XX’ part of the system
AA’XX’, 2H, H-Ar); 3.66 (s, 2H, CH2); 3.27e3.19 (m, 4H, 2ꢃCH2);
1.62e1.06 (m, 16H, 8ꢃCH2); 0.95e0.82 (m, 6H, 2ꢃCH3). 13C NMR
(50 MHz, DMSO-d6)
d: 167.7; 158.9; 137.1; 136.8; 135.0; 129.1;
6.2.4.22. N,N-Dibutyl-2-[2-(4-methylphenyl)-1,3-oxazol-4-yl]acet-
amide (6q). Yield: 60%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 90:10 v/v as eluant). 1H NMR
127.3; 125.6; 47.5 (45.1); 31.2 (31.0); 30.9; 28.5 (27.1); 26.0 (25.8);
22.0; 13.8. IR cmꢁ1: 1638; 1423; 1299; 1095; 1013; 835; 736. EI-MS
m/z (%): 139 (100); 404 (Mþ, 5). Anal.: calcd for C23H33ClN2O2:
68.21%; 8.21%, 6.92%, found: C 68.31%; 8.28%, 6.58%.
(200 MHz, DMSO-d6) d: 7.95 (s, 1H, H-5); 7.84e7.80 (AA’ part of the
system AA’XX’, 2H, H-Ar); 7.35e7.31 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.64 (s, 2H, CH2); 3.37e3.21 (m, 4H, 2ꢃCH2); 2.36 (s, 3H,
CH3); 1.56e1.18 (m, 8H, 4ꢃCH2); 0.93e0.83 (m, 6H, 2ꢃCH3). 13C
6.2.4.17. 2-[2-(4-Fluorophenyl)-1,3-oxazol-4-yl]-N,N-dipropylaceta-
mide (6l). Yield: 59%. mp: 54.0e57.0 ꢂC (from petroleum ether). 1H
NMR (50 MHz, DMSO-d6) d: 167.8; 160.0; 140.1; 136.4; 136.3; 129.5;

4518
B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
125.6; 124.2; 47.2 (44.8); 31.2; 30.8 (29.4); 20.9 (19.5); 13.7. IR
cmꢁ1: 1641; 1586, 1497; 1207; 1098; 1064; 937; 729. Anal.: calcd
for C20H28N2O2: 73.14%; 8.59%, 8.53%, found: C 72.76%; 8.72%, 8.05%.
70.4; 48.4; 46.2; 31.7; 29.1; 28.9; 27.2; 22.4; 22.3; 14.0; 13.9. Anal.:
calcd for C21H30N2O2: C 73.65%; 8.83%, 8.18%, found: C 73.35%;
9.10%, 8.05%.
6.2.4.23. 2-[2-(4-Methylphenyl)-1,3-oxazol-4-yl]-N,N-dipentylaceta-
mide (6r). Yield: 68%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 95:5 v/v as eluant). 1H NMR
6.2.7. 3-(4-Methylphenyl)isoxazole-5-carboxylic acid (16)
Potassium dichromate (5.0 g, 17.0 mmol) was added to a stirred
solution of H2SO4 20% (130 ml). [3-(4-Methylphenyl)isoxazol-5-il]
methanol 19 (2.1 g, 11.2 mmol) was added to the resulting solution
and the mixture was stirred and heated at reflux for 1 h, after
cooling at room temperature, the solid mass was filtered and
washed with water until pH ¼ 7. The crude product was crystallized
by H2O/MeOH to give white needles of acid 16 (1.38 g; 57%); m.p.
(200 MHz, DMSO-d6) d: 7.95 (s, 1H, H-5); 7.84e7.80 (AA’ part of the
system AA’XX’, 2H, H-Ar); 7.34e7.30 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.63 (s, 2H, CH2); 3.37e3.19 (m, 4H, 2ꢃCH2); 2.36 (s, 3H,
CH3); 1.56e1.20 (m, 12H, 6ꢃCH2); 0.89e0.80 (m, 6H, 2ꢃCH3). 13C
NMR (50 MHz, DMSO-d6) d: 167.8; 160.0; 140.1; 136.4; 136.3; 129.5;
125.6; 124.2; 47.4 (45.1); 31.2; 28.5 (28.4); 28.3 (26.9); 21.9 (20.9);
13.8. IR cmꢁ1: 1651; 1587; 1497; 1375; 1180; 1101; 821; 732. Anal.:
calcd for C22H32N2O2: 74.12%; 9.05%, 7.86%, found: C 74.48%; 9.33%,
7.50%.
204.1e204.8 ꢂC. 1H NMR (400 MHz, DMSO-d6)
COOH); 7.84 (pd, 2H, H-Ar); 7.74 (s, 1H, H-4); 7.33 (pd, 2H, H-Ar);
2.36 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
: 162.6; 161.6;
d: 14.4 (bs, 1H,
d
157.7; 140.4; 129.7; 126.6; 124.9; 107.4; 20.9. Anal.: calcd for
C11H9NO3: C 65.02%; 4.46%, 6.89%, found: C 65.21%; 4.22%, 6.78%.
6.2.4.24. N,N-Dihexyl-2-[2-(4-methylphenyl)-1,3-oxazol-4-yl]acet-
amide (6s). Yield: 54%. Colourless oil by column chromatography
(petroleum ether:ethyl acetate ¼ 93:7 v/v as eluant). 1H NMR
6.2.8. 4-Methylbenzaldehyde oxime (18)
Hydroxylamine hydrochloride (3.5 g, 50.0 mmol) was added in
small portions at room temperature to a solution of NaOH (3 g,
75 mmol) and 4-methylbenzaldehyde (17) (6.0 g, 50 mmol) in
water (8.5 mL). After the addition was complete, the mixture was
diluted in 100 mL of water and saturated with CO2 gas. The reaction
mixture was stirred at room temperature for 1 h and then the solid
obtained was filtered, washed with water and anhydrified (94%);
m.p. 74.8e75.5 ꢂC (lit [36]. m.p. 76e78 ꢂC).
(200 MHz, DMSO-d6) d: 7.95 (s, 1H, H-5); 7.84e7.80 (AA’ part of the
system AA’XX’, 2H, H-Ar); 7.34e7.30 (XX’ part of the system AA’XX’,
2H, H-Ar); 3.63 (s, 2H, CH2); 3.37e3.19 (m, 4H, 2ꢃCH2); 2.36 (s, 3H,
CH3); 1.60e1.12 (m, 16H, 8ꢃCH2); 0.90e0.78 (m, 6H, 2ꢃCH3). 13C
NMR (50 MHz, DMSO-d6) d: 167.8; 160.0; 140.1; 136.4; 136.3; 129.5;
125.6; 124.2; 47.5 (45.1); 31.3 (31.0); 31.0; 28.5 (27.1); 26.0 (25.9);
22.0 (20.9); 13.8. IR cmꢁ1: 1651; 1590; 1497; 1183; 1101; 1064; 821;
736. Anal.: calcd for C24H36N2O2: 74.96%; 9.44%, 7.28%, found: C
75.18%; 9.75%, 7.06%.
6.2.9. [3-(4-Methylphenyl)isoxazol-5-yl]methanol (19)
Prop-2-yn-1-ol (0.96 mL, 16.5 mmol) was added dropwise to
a cooled solution (ꢁ5 ꢂC) of triethylamine (0.16 mL, 1.1 mmol) in
CHCl3 (10 mL), maintaining the temperature in the range ꢁ5O0 ꢂC.
At this temperature a solution of 4-methylbenzaldehyde oxime (18)
(1.5 g,11 mmol) in CHCl3 (15 mL) was added in 15 min and then also
35 mL of NaOCl (5% of chloro active solution) in 35 min. The
biphasic mixture was energetically stirred at room temperature for
2 h. Then the organic phase was separated and the aqueous phase
was extracted with CHCl3 (3ꢃ10 mL). The organic phases collected
were treated with water (3ꢃ20 mL) and then anhydrified on
Na2SO4. The solvent was removed to reduced pressure to give pure
compound 19 (86%); m.p. 54.0e55.0 ꢂC. 1H NMR (400 MHz; CDCl3)
6.2.5. N,N-Dipentyl-2-(3-phenylisoxazol-5-yl)acetamide (7d)
Potassium permanganate (0.253 g, 1.6 mmol) was added
portion-wise to compound 28 in 3 mL of H20 and 0.3 mL of
concentrated H2SO4. The mixture was stirred at room temperature
overnight and then was added NaHSO3 (0.308 g, 3.0 mmol). The
aqueous suspension was extracted with ethyl acetate (3ꢃ10 mL).
The organic phases collected were dried (Na2SO4) and evaporated
to dryness to give the crude product, purified by flash-
chromatography (petroleum ether/ethyl acetate 4:1 v/v) to give
the desired amide 7d (8%) as a white wax after treatment with
petroleum ether. 1H NMR (400 MHz; CDCl3)
d: 7.80e7.77 (m, 2H, H-
Ar); 7.45e7.42 (m, 3H, H-Ar); 6.62 (s, 1H, H-4); 3.90 (s, 2H, CH2);
3.37e3.33 (m, 2H, NCH2); 3.32e3.29 (m, 2H, NCH2); 1.64e1.53 (m,
d
: 7.69 (AA’ part of the system AA’XX’, 2H, H-Ar); 7.27 (XX’ part of
the system AA’XX’, 2H, H-Ar); 6.55 (s, 1H, H-4); 4.82 (s, 2H, CH2);
2.41 (s, 3H, CH3). 13C NMR (100 MHz; CDCl3)
: 171.7; 162.5; 140.2;
4H, CH2); 1.40e1.22 (m, 8H, 4ꢃCH2); 0.95e0.86 (m, 6H, 2ꢃCH3). 13
C
d
NMR (100 MHz; CDCl3)
d
: 171.3; 166.4; 162.7; 130.0; 129.0; 128.4;
129.6; 126.8; 126.2; 99.9; 56.7; 21.3. Anal.: calcd for C11H11NO2: C
69.83%; 5.86%, 7.40%, found: C 69.70%; 5.75%, 7.35%.
126.8; 101.3; 48.7; 46.5; 32.2; 29.1; 28.9; 28.8; 27.2; 22.4; 22.4;
14.0; 13.9. Anal.: calcd for C21H30N2O2: C 73.65%; 8.83%, 8.18%,
found: C 73.33%; 9.11%, 8.08%.
6.2.10. N,N-Dipentylbut-3-enamide (27)
Thionyl chloride (1.2 mL) was added at 0 ꢂC to the vinylacetic
acid 26 (1.0 g, 11.6 mmol). The obtained suspension was stirred and
heated at reflux for 30 min and then cooled at ꢁ5 ꢂC by an ice-salt
bath. At this temperature, was added dropwise a solution of
dipentylamine (4.7 mL, 23.2 mmol) in dry THF (5 mL). The reaction
mixture was stirred at room temperature for 2 h (TLC, petroleum
ether/ethyl acetate 4:1 v/v) and then the solid mass was filtered off
and washed with THF. The filtrate was evaporated to dryness; the
residue was treated with saturated sodium bicarbonate solution
(100 mL) and extracted with dichloromethane (5ꢃ50 mL). The
combined organic phases were dried (Na2SO4) and evaporated to
dryness to give the crude product, purified by flash-
chromatography (petroleum ether/ethyl acetate 4:1 v/v) to give
the desired amide 27 (40%) pure by NMR spectra. 1H NMR
(400 MHz; CDCl3): 6.02e5.94 (m, 1H, CH); 5.17e5.07 (m, 2H,
CH2 ¼ ); 3.31e3.25 (m, 2H, NCH2); 3.20e3.15 (m, 2H, NCH2); 3.10 (d,
2H, J ¼ 6.8, CH2CO); 1.60e1.45 (m, 4H, 2ꢃCH2); 1.40e1.21 (m, 8H,
6.2.6. N,N-Dipentyl-2-(5-phenylisoxazol-3-yl)acetamide (8d)
A solution of 3-nitro-N,N-dipentylpropanamide (30) (0.520 g,
2.0 mmol) and triethylamine (2 drops) in toluene anhydrous (3 mL)
was added dropwise to a solution of phenyl isocyanate (0.4 mL,
4 mmol) and phenylacetylene (0.2 mL, 2 mmol) in toluene anhy-
drous (5 mL). After stirring for 1 h, the reaction mixture was
warmed for an additional hour at 80 ꢂC, cooled and checked (TLC,
petroleum ether/ethyl acetate 4:1 v/v). The solid was filtered off,
and the filtrate was evaporated to dryness to give the crude
product, purified by flash-chromatography (petroleum ether/ethyl
acetate 4:1 v/v) to give the desired amide 8d (6%) as a colourless oil.
1H NMR (400 MHz; CDCl3)
d: 7.80e7.72 (m, 2H, H-Ar); 7.46e7.43
(m, 3H, H-Ar); 6.67 (s, 1H, H-4); 3.80 (s, 2H, CH2); 3.35e3.29 (m, 2H,
NCH2); 3.27e3.23 (m, 2H, NCH2); 1.67e1.47 (m, 4H, CH2); 1.40e1.20
(m, 8H, 4ꢃCH2); 0.95e0.86 (m, 6H, 2ꢃCH3). 13C NMR (100 MHz;
CDCl3) d: 169.9; 167.7; 159.7; 130.1; 129.0; 128.9; 125.8; 100.3;

B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
4519
4ꢃCH2); 0.93e0.85 (m, 6H, 2ꢃCH3). 13C NMR (100 MHz; CDCl3)
d
:
concentration range (0.1 nMe10
m
M) in 50 mM TriseHCl, pH 7.4, as
170.3; 132.2; 117.3; 48.0; 45.8; 38.6; 29.2; 29.0; 28.7; 27.4; 22.4;
22.3; 14.0; 13.9. Anal.: calcd for C14H27NO: C 74.61%; 12.08%, 6.21%,
found: C 74.20%; 11.60%, 5.98%.
previously described [15]. For the active compounds, the IC50 values
were determined and Ki values were derived in accordance with
the equation of Cheng and Prusoff [37].
6.2.11. N,N-Dipentyl-2-(3-phenyl-4,5-dihydroisoxazol-5-yl)
acetamide (28)
6.3.3. [3H]Ro15-1788 Binding to rat cerebral cortex membranes
Rat cerebral cortex membranes were prepared as previously
described [38]. After differential centrifugation, the crude
membrane fraction obtained was subjected to washing procedures
to remove endogenous GABA [39]. The washed membranes were
N,N-Dipentylbut-3-enamide (27) (4.1 g, 18.0 mmol) was added
dropwise to a cooled solution (ꢁ5 ꢂC) of triethylamine (0.16 mL,
1.1 mmol) in CHCl3 (10 mL), maintaining the temperature in the
range ꢁ5O0 ꢂC. At this temperature a solution of benzaldehyde
oxime (1.5 g, 12 mmol) in CHCl3 (15 mL) was added in 15 min and
then also 35 mL of NaOCl (5% of chloro active solution) in 35 min.
The biphasic mixture was energetically stirred at room temperature
for 3 h. Then the organic phase is separated and the aqueous phase
was extracted with CHCl3 (3ꢃ10 mL). The organic phases collected
were treated with water (3ꢃ20 mL) and then anhydrified on
Na2SO4. The solvent was removed to reduced pressure to give
compound 28 that was purified by flash-chromatography (petro-
leum ether/ethyl acetate 4:1 v/v) to give the desired amide 28
(66%). An analytical sample was obtained by crystallization from
incubated with 0.4 nM [3H]Ro15-1788 for 90 min at 0 ꢂC in 500
mL
of 50 mM Tris-citrate buffer, pH 7.4, as previously described [40].
6.3.4. Cell culture and treatments
The human glioblastoma multiforme (GBM) cell line U87MG
was obtained from the National Institute for Cancer Research (ICLC)
of Genoa. The U87MG cells (1ꢃ106) were seeded onto T-75 flasks
and cultured in RPMI medium supplemented with 10% foetal
bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin,
100 mg/mL streptomycin, and 1% non-essential amino acids, at
37 ꢂC in humidified atmosphere composed of 5% CO2 and 95% O2.
For U87MG cell treatments, the cells were seeded at appropriate
densities depending on the experimental test in complete medium
at 37 ꢂC and 5% CO2. After 24 h the medium was replaced either
with complete culture medium containing DMSO (untreated
control cells) or with complete medium supplemented with the
compounds for the time period indicated. The compounds were
dissolved in DMSO (<1% v/v of medium).
hexane; m.p. 60.0e61.0 ꢂC. 1H NMR (400 MHz; CDCl3)
d: 7.68e7.65
(m, 2H, H-Ar); 7.40e7.36 (m, 3H, H-Ar); 5.20e5.07 (m, 1H, H-5);
3.69e3.60 (m, 1H, 4eCH2); 3.37e3.09 (m, 5H, 2ꢃNCH2 and 4eCH2);
2.99e2.90 (m, 1H, CH2); 2.66e2.57 (m, 1H, CH2); 1.60e1.45 (m, 4H,
2ꢃCH2); 1.38e1.21 (m, 8H, 4ꢃCH2); 0.92e0.86 (m, 6H, 2ꢃCH3). 13
C
NMR (100 MHz; CDCl3) d: 169.0; 157.1; 130.0; 129.6; 128.7; 126.7;
78.5; 48.0; 45.8; 40.9; 38.5; 29.2; 29.0; 28.6; 27.4; 22.4; 22.3; 14.0;
13.9. Anal.: calcd for C21H32N2O2: C 73.22%; 9.36%, 8.13%, found: C
73.53%; 9.04, 8.28%.
6.3.5. Cell proliferation/viability analysis
Cell proliferation/viability was estimated by the colorimetric
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium) conversion assay (Cell Titer 96Ò
Aqueous One Solution Cell Proliferation Assay, Promega) and by
trypan blue (TB) exclusion assay, as previously reported [41]. Con-
cerning the dose-dependent treatment cells were exposed to
a single incubation time (24 h) and increasing 6d concentrations,
corresponding to 15, 30 and 150 times the Ki value. Concerning the
6.2.12. 3-Nitro-N,N-dipentylpropanamide (30)
Thionyl chloride (1.2 mL) was added at 0 ꢂC to the 3-nitropro-
pionic acid 26 (1.38 g, 11.6 mmol). The obtained suspension was
stirred and heated at reflux for 30 min and then cooled at ꢁ5 ꢂC by
an ice-salt bath. At this temperature, was added dropwise a solu-
tion of dipentylamine (4.7 mL, 23.2 mmol) in dry THF (5 mL). The
reaction mixture was stirred at room temperature for 2 h (TLC,
petroleum ether/ethyl acetate 4:1 v/v) and then the solid mass was
filtered off and washed with THF. The filtrate was evaporated to
dryness; the residue was treated with saturated sodium bicar-
bonate solution (100 mL) and extracted with dichloromethane
(5ꢃ40 mL). The combined organic phases were dried (Na2SO4) and
evaporated to dryness to give the crude product, purified by flash-
chromatography (petroleum ether/ethyl acetate 4:1 v/v) to give the
desired amide 30 (45%) pure by NMR spectra. 1H NMR (400 MHz;
time-dependent treatments cells were exposed to
a single
concentration of compound 6d, 30 times the Ki value, for 24 h, 48 h
and 72 h. Each drug concentration was tested in triplicate, and the
experiments were repeated at least three times.
6.3.6. Data analyses
Data were analyzed by use of the GraphPad Prism software
(GraphPad Software, version 5.0; San Diego, CA). Statistical analyses
were performed by one-way ANOVA (with post hoc Bonferroni test).
CDCl3)
d
: 4.72 (t, 2H, J ¼ 6.1, CH2NO2); 3.33e3.27 (m, 2H, NCH2);
3.27e3.21 (m, 2H, NCH2); 2.97 (t, 2H, J ¼ 6.1, CH2CO); 1.65e1.56 (m,
2H, CH2); 1.55e1.46 (m, 2H, CH2); 1.40e1.23 (m, 8H, 4ꢃCH2);
6.3.7. Mitochondrial membrane potential (DJm) Cytofluorimetric
analysis
0.95e0.89 (m, 6H, 2ꢃCH3). 13C NMR (100 MHz; CDCl3)
d: 167.5;
70.45; 47.6; 46.2; 29.9; 29.1; 29.0; 28.6; 27.3; 22.4; 22.3; 14.0; 13.9.
Anal.: calcd for C13H26N2O3: C 60.44%; 10.14%, 10.84%, found: C
60.05%; 10.15%, 10.20%.
Changes in DJm were analized by use of the fluorescent dye JC-
1, as previously described [6,42]. J-aggregate and J-monomer were
recorded with a FACScalibur flow cytometer (Becton Dickinson,
USA) in fluorescence channel 2 (FL2) and in fluorescence channel 1
(FL1), respectively. The cells were selected electronically and
chosen on the basis of morphological characteristics observed on
the forward vs side scatter dot plot. In brief, the cells were seeded in
24-well plates and after treatments with DMSO (control sample) or
with tested compound for 12, 24 and 48 h, cells were collected by
centrifugation. Pellets were suspended in JC-1 solution and incu-
bated for 30 min at room temperature in the dark. After incubation
the pellets were suspended in PBS 1X and their fluorescence was
analyzed by FACS. As a positive control, cells were incubated in the
presence of the uncoupling agent CCCP (carbonylcyanide m-chlor-
ophenylhydrazone) in each experiment.
6.3. Biological methods
6.3.1. Materials
[3H]PK 11195 (S.A. 85.5 Ci/mmol) and [3H]Ro15-1788 (S.A.
83.4 Ci/mmol) were purchased from PerkineElmer Life Sciences. PK
11195 and Ro5-4864 were obtained from SigmaeAldrich. All
reagents were obtained from commercial suppliers.
6.3.2. [3H]PK11195 binding to rat kidney mitochondrial membranes
For binding studies, crude mitochondrial membranes were
incubated with 0.6 nM [3H]PK 11195 in the presence of a compound

4520
B. Cosimelli et al. / European Journal of Medicinal Chemistry 46 (2011) 4506e4520
[19] G. Campiani, I. Fiorini, M.P. De Filippis, A. Garofalo, V. Nacci, S.M. Ciani,
Acknowledgements
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This work was supported by the Tuscan Cancer Institute (Istituto
Toscano Tumori, ITT, Grant 2007).
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