Synthesis of novel pyrrolyl-indomethacin derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.09.008

In the present work, we report the synthesis of the novel esters of indomethacin (IDMC) and an ester of reduced IDMC. For this purpose, IDMC is covalently bound by using a spacer chain to the pyrrole (Py) in the 3-position. The innovative pyrrole-indomethacin (3-Py-IDMC) derivates show no cytotoxic effects in primary calvarial osteoblasts. The designed IDMC derivates have been studied because they could be injected locally as a component of polymeric micro-particles. In fact, the new 3-Py-IDMC derivatives will assure their further polymerization since the 2- and 5-monomer positions are free.

Full text of DOI:10.1016/j.ejmech.2012.09.008

European Journal of Medicinal Chemistry 57 (2012) 391e397
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of novel pyrrolyl-indomethacin derivatives
,
Judith Serra Moreno a, Dimitrios Agas a c, Maria Giovanna Sabbieti c, Matteo Di Magno a,
,b,
*
Antonella Migliorini a, M. Antonietta Loreto a
a Chemistry Department, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Istituto di Chimica Biomolecolare, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
c School of Biosciences and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work, we report the synthesis of the novel esters of indomethacin (IDMC) and an ester of
reduced IDMC. For this purpose, IDMC is covalently bound by using a spacer chain to the pyrrole (Py) in
the 3-position. The innovative pyrrole-indomethacin (3-Py-IDMC) derivates show no cytotoxic effects in
primary calvarial osteoblasts. The designed IDMC derivates have been studied because they could be
injected locally as a component of polymeric micro-particles. In fact, the new 3-Py-IDMC derivatives will
assure their further polymerization since the 2- and 5-monomer positions are free.
Received 19 December 2011
Received in revised form
3 September 2012
Accepted 5 September 2012
Available online 12 September 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Indomethacin
NSAID
Ester
Pyrrole
Osteoblasts
Cytotoxicity
1. Introduction
particles. Therefore, the designed polymer is expected to decrease
the drug clearance. The polymeric sample could be locally injected
Indomethacin (IDMC) is a non-steroidal anti-inflammatory drug
(NSAID) commonly used for the treatment of pain and inflamma-
tion by inhibiting the cyclooxygenases (COXs) activities (Fig. 1) [1].
The major drawback of IDMC, if orally ingested, is an undesirable
gastric effect. The intra-articular (IA) administration of NSAIDs is
a valid alternative to minimize the systemic bioavailability and
associated side-effects of oral intake. However, the IA therapy for
osteoarthritis still needs micro-/nano-carrier mediated drug
delivery systems to decrease the drug loading, minimizing the
negative side effects, and to increase the drug bioavailability over
time. In fact, the latter aspect is the key point for a successful drug
delivery carrier. The current NSAIDs delivery vehicles still show
a rapid clearance of therapeutic substances from the synovial space
[2]. In the literature different methods to immobilize NSAIDs into
polymers are proposed such as encapsulation technique [3e5]. In
this perspective, rather to encapsulate the drug, we plan to bind
IDMC derivatives namely, esters of IDMC and an ester of reduced
IDMC, to a monomer for further polymerization as nano-/micro-
to the painful zone, overcoming the oral IDMC administration
problems, and could maintain the bioavailability. The chosen
monomer is pyrrole that leads to polypyrrole (PPy) that is a well
known polymer for its electroconducting properties and biomedical
applications [6]. For instance, PPy can be used as a reservoir for
drug delivery following a redox reaction [7,8].
In this work, we report the covalent binding of IDMC or reduced
IDMC to 3-position of pyrrole (3-Py-IDMC also referred as IDMC
derivates) through a hydrolyzable spacer chain by following esters
(5) and (8) and ester of reduced IDMC (13) strategies, respectively.
The modified 3-Py-IDMC monomers were further studied by per-
forming cytotoxic tests in vitro. The designed monomers will be
copolymerized with pyrrole monomer (3-Py-IDMC/Py) in order to
obtain micro-particles that will be used for IDMC or reduced IDMC
delivery in situ. It is expected that the covalent immobilization of
IDMC or reduced IDMC to the 3-Py could assure their controlled
release from the copolymer micro-particles in respect to the direct
IDMC injection.
2. Results and discussion
* Corresponding author. Chemistry Department, Sapienza University of Rome,
P.le A. Moro 5, 00185 Rome, Italy. Tel.: þ39 06 4991 3675; fax: þ39 06 490631.
E-mail address: mariaantonietta.loreto@uniroma1.it (M.A. Loreto).
The indomethacin ester 5 is carried out by a 3-step synthesis
(Scheme 1).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.008

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J. Serra Moreno et al. / European Journal of Medicinal Chemistry 57 (2012) 391e397
Scheme 2. Synthesis of indomethacin ester 8. Reagents and conditions: (a) TIPSCl, n-
BuLi, THF, ꢁ78 ꢀC; (b) ClCH2COCl, AlCl3, CH2Cl2, 0 ꢀC; (c) indomethacin 1, DMAP, Et3N,
CH2Cl2, r.t.
Fig. 1. Chemical structure of indomethacin.
selected conditions proposing them as potential prodrugs [15]. The
products 5 and 8 leave free the positions 2- and 5- of the Py. It is
well known that the substitution of a functional group on the N- or
the 3-position of the pyrrole allows its polymerization if the
substituent is not too bulky [16]. In fact, the compounds 5 and 8 can
assure their further polymerization because they use a spacer chain
that holds indomethacin moiety far from polymerization center.
A different spacer was used for connecting the reduced indo-
methacin (alcohol 12) to 3-position of Py to give the ester derivative
13. The synthesis pathway followed is depicted in Scheme 3.
The first steps concern the synthesis of the 3-acetic acid pyrrole
11 (key intermediate) that involves the acetylation of the N-tosyl-
pyrrole 3 and the conversion of acetylated 9 into the 3-acetic acid
methyl ester 10 by using the thallium-based oxidative trans-
position. The oxidative transposition was achieved by the use of
thallium trinitrate (Tl(NO3)3), trimethyl orthoformate and mont-
morillonite K10 [17]. The following hydrolysis leads to the targeted
key intermediate 11. On the other hand, the IDMC was reduced to
the corresponding alcohol 12 by using the boraneemethyl sulfide
complex (BH3$SMe2) in THF according to the Wey’s method [18].
The reduced indomethacin was produced with high yield (91%). It
was condensed with the 3-acetic acid pyrrole 11 employing 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and N,N-
dimethyl-4-aminopyridine (DMAP) as showed in the Kalgutkar’s
procedure for other different reduced IDMC esters [19]. The
synthesized ester 13 was isolated by chromatography with a yield
of 62% and its structure was confirmed by spectroscopic techniques.
The 1H NMR spectrum showed the signals of the reduced IDMC
ester and the 3-pyrrole acetate. The obtained structure 13 could
allow its polymerization as well as 5 and 8 compounds.
The innovative monomers were tested in order to study their
biocompatibility using primary calvarial osteoblasts as a cell model.
Since we plan to copolymerize them, it is useful to characterize
their cytotoxicity whenever IDMC, reduced IDMC or un-reacted 3-
Py-IDMC monomer could be released from the copolymer. In fact, it
is expected that the delivery of these compounds is due to the
hydrolysis of the ester bond. The hydrolysis of the ester bond is
a well known strategy to release drugs in vitro or in vivo [20e22].
MTS assay, a colorimetric method that determines the number of
viable cells, showed that treatment with the compounds 5, 8, 13
and IDMC did not affect osteoblast viability (Fig. 2A). In particular,
with Hoechst staining and with BrdU assay, performed to specifi-
cally evaluate the cell proliferation, no statistically significant
differences were found between compounds-stimulated and
unstimulated cultures (Fig. 2B1, B2). Moreover, the synthesis of the
anti-apoptotic protein Bcl2 and the pro-apoptotic protein Bax was
evaluated by western blotting, since a small change in the ratio of
above proteins could reflect a cell perturbation. The obtained
results demonstrated that Bcl2/Bax ratio was not affected by the
tested compounds or by IDMC (Fig. 2C).
The first step was the N-tosylation of the pyrrole that was easily
obtained by tosyl chloride in the presence of sodium hydroxide [9].
Afterward the chloroacetyl chloride reacted with the N-protected
pyrrole 3 following the condition for the simple acetylation of the
N-tosylpyrrole, using AlCl3 in dry dichloromethane [10]. The 3-
chloroacetyl N-tosylpyrrole 4 was obtained in 60% yield, the 2-
acylated isomer was also present in 19% yield. The two products
were separated by chromatography and recognized by 1H NMR
spectra [11]. The last step, that is the reaction of the 3-chloroacetyl
pyrrole N-protected with IDMC, was performed successfully by
using the N,N-dimethyl-4-aminopyridine (DMAP) as the coupling
agent and the triethylamine as the base in agreement with reported
procedure for the glicolamide ester of indomethacin [12].
Dichloromethane was used as the solvent. The product 5 was
purified and obtained in 84% yield. The structure was confirmed by
spectroscopic data. The 1H NMR spectrum shows the methylene
protons of eCOOCH2COe group at
d
5.0. The 13C NMR spectrum
confirms the expected number of C atoms and the ester bond at
w170 ppm.
In addition, it was possible to obtain the IDMC ester 8 with the
unprotected pyrrole starting from the N-triisopropylsilyl (N-TIPS)
pyrrole 6 that was prepared according to reported procedure [13],
as outlined in Scheme 2.
The reaction of 6 with chloroacetyl chloride lead to the new 3-
chloroacetyl pyrrole 7 in the same conditions reported for Py
acylation but by using different acyl chlorides [14]. The compound 7
was obtained as the main product in 43% yield and a small quantity
(16%) of the 2-acylated NeH pyrrole (pyrrole N-unprotected) was
also isolated. The last step was the coupling reaction carried out in
the same conditions of those for the synthesis of compound 5.
These conditions facilitated the deprotection of the monomer
providing the desired product 8. The 1H NMR and 13C NMR analyses
confirmed the absence of the TIPS group and the success of the last
reaction even if with modest yield (40%).
The two obtained esters 5 and 8 are new esters of IDMC. Both
oxoethylesters could allow the chemical or enzymatic hydrolysis of
IDMC as reported for the IDMC 2-oxopropylester derivates at
Scheme 1. Synthesis of indomethacin ester 5. Reagents and conditions: (a) TsCl, NaOH,
DCE, 0 ꢀC; (b) ClCH2COCl, AlCl3, CH2Cl2, r.t.; (c) indomethacin 1, DMAP, Et3N, CH2Cl2, r.t.

J. Serra Moreno et al. / European Journal of Medicinal Chemistry 57 (2012) 391e397
393
Scheme 3. Synthesis of reduced IDMC ester 13. Reagents and conditions: (a) Ac2O, AlCl3, CH2Cl2, r.t.; (b) Tl(NO3)3$3H2O, montmorillonite K10, CH(OCH3)3, CH3OH, r.t.; (c) 1) NaOH,
CH3OH, reflux; 2) HCl, r.t.; (d) BH3$S(CH3)2, THF, r.t.; (e) 2-(1H-pyrrol-3-yl)acetic acid 11, EDCI, DMAP, CH2Cl2, r.t.
Fig. 2. MTS assay (A). COBs were incubated with compounds 5, 8, 13 (10ꢁ6 M) and IDMC (10ꢁ6 M), for 24 h. Graphics represent results of three independent experiments. Hoechst
stain (B1); BrdU assay (B2). Cells were treated with compounds 5, 8, 13 (10ꢁ6 M) and IDMC (10ꢁ6 M), for 24 h. Graphic represents results of three independent experiments. Bcl2/Bax
ratio (C). Cells were treated with compounds 5, 8, 13 (10ꢁ6 M) and IDMC (10ꢁ6 M) for 24 h. Filters were probed with the rabbit anti-Bcl2 or with the rabbit anti-Bax; then,
membranes were stripped and re-probed with mouse anti-a-tubulin to show equal amount of loading. Graphic represents results of three independent experiments.

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It is well known that the cytoskeleton plays a crucial role not
4. Experimental section
only in cell shaping, but also in many cellular functions. In osteo-
blastic cells cytoskeletal modifications are helpful tools for moni-
toring cell healthiness. Indeed, integrity of the osteoblast structure,
is important for their differentiation as well as for the activation of
osteoclast functions through a mechanism involving cell-to-cell
and/or cell-to-matrix contacts [23]. Therefore, the distribution of F-
actin was investigated by using TRITC-phalloidin labeling. The
results evidenced a defined organization of actin cytoskeleton in
osteoblasts treated with compounds 5, 8, 13 and IDMC, overlapping
to that found in untreated cells (Fig. 3A). Moreover, toluidine blue
and hematoxylin/eosin cytochemical staining, performed to
confirm the above results, showed that the fusiform or polygon
shape of the cells observed in untreated osteoblasts was preserved
after administration of the tested compounds (Fig. 3B, C).
Hence, the above multiple experimental approaches strongly
evinced that these novel 3-Py-IDMC monomers are biocompatible
and could be useful for further copolymerization to obtain micro-
particles for IDMC or reduced IDMC delivery. It will be attended
a drug release by the linker hydrolysis (ester bond). These IDMC
derivates could represent a novel bases for the preparation of Py-
IDMC-based polymeric drugs for local application with less side
effects than the existing oral administrated NSAIDs.
4.1. General experimental methods
Solvents and common reagents were purchased from
a commercial source and used without further purification. All
reactions were monitored by thin layer chromatography (TLC)
carried out on Merck F-254 silica glass plates and visualized with
UV light. 1H NMR was recorded in CDCl3 on Varian Gemini 300
(300 MHz). Chemical shifts are expressed in parts per million (
scale) and are referenced to the residual protons of the NMR solvent
(CHCl3:
d
d
7.26); (s) ¼ singlet; (d) ¼ doublet; (t) ¼ triplet;
(q) ¼ quartet; (dd) ¼ double doublet; (ddd) ¼ double double
doublet; (dt) ¼ double triplet; (dq) ¼ double quartet; (bs) broad
singlet; (m) ¼ multiplet. Coupling constants (J) were expressed in
Hz. 13C NMR was recorded in CDCl3 on Varian Gemini 300 (75 MHz).
Chemical shifts are expressed in parts per million (
referenced to the residual carbons of the NMR solvent (CHCl3:
d scale) and are
d
¼ 77.0). Infrared spectra (IR) were obtained using a PERKINe
ELMER 1600 (FT-IR); data are presented as the frequency of
absorption (cmꢁ1). HRMS spectra were recorded with Micromass
Q-TOF micro Mass Spectrometer (Waters). GCeMS spectra were
recorded with SHIMAZU GC-2010 equipped with a capillary column
SLB-5ms (30 m ꢂ 0.25 mm ꢂ 0.25
mm) coupled with a detector
SHIMAZU QP 2010S.
3. Conclusions
4.1.1. 3-(Chloroacetyl)-1-tosyl-1H-pyrrole (4)
In summary, in this work we have reported the synthesis of the
innovative IDMC-based compounds namely, IDMC esters and
reduced IDMC ester. From our knowledge, this is the first time that
IDMC is covalently bound by using a spacer chain to the Py in the 3-
position (3-Py-IDMC). In addition, the innovative pyrrole-
indomethacin monomers showed no cytotoxic effects by using
primary calvarial osteoblasts. It could be attended, therefore, no
cytotoxic effects also for the respective polymer. The new 3-Py-
IDMC derivatives assure their further polymerization since the 2-
and 5-monomer position are free. The obtained polymers will be
new interesting materials potentially applicable in pharmacological
field.
Chloroacetyl chloride (1.32 g, 11.8 mmol) was added slowly to
a suspension of anhydrous AlCl3 (2.23 g, 16.7 mmol) in 20 mL of dry
dichloromethane at 25 ꢀC. The reaction mixture was stirred at r.t.
for 10 min, a solution of N-tosylpyrrole 3 (0.99 g, 4.5 mmol) in
2.5 mL of dry dichloromethane was added slowly and the mixture
was stirred for 2 h at r.t and then poured into an iceewater mixture.
The organic phase was separated and combined with dichloro-
methane extracts (4 ꢂ 10 mL) of the aqueous phase. The combined
organic layer was washed with NaOH solution (1 N), dried (Na2SO4
anhydrous) and evaporated in vacuo. The residue was chromato-
graphed on silica gel (dichloromethane/hexane, 7/3) to afford the
Fig. 3. TRITC-phalloidin labeling for F-actin (A). Cells were incubated with compounds 5, 8, 13 (10ꢁ6 M) and IDMC (10ꢁ6 M) for 24 h. Stress fibers are seen running parallel to the
long axis of the cells and in the cytoplasmic projections both in treated and in untreated cells. Toluidine blue (B); hematoxylin/eosin (C). Cells were incubated with compounds 5, 8,
13 (10ꢁ6 M) and IDMC (10ꢁ6 M) for 24 h. Treated cells are morphologically comparable to control cultures.

J. Serra Moreno et al. / European Journal of Medicinal Chemistry 57 (2012) 391e397
395
pure 3-chloroacetyl N-tosylpyrrole 4 as a white solid in 60% yield,
the 2-acylated isomer was also present in 19% yield. The compound
4 was recognized by 1H NMR spectra [11].
(118 mg, 0.4 mmol), DMAP (9.5 mg, 0.1 mmol), Et3N (1 mL) and
dichloromethane (1 mL) according to the procedure described for 5.
After solvent evaporation, the crude product was purified by
column chromatography on silica gel (hexane/ethyl acetate 6/4).
The obtained product was already deprotected (no TIPS was present
in the molecule). Compound 8 was obtained as a yellow solid (yield
74 mg, 0.16 mmol, 40%); Rf ¼ 0.50 (hexane/ethyl acetate, 1/1). 1H
4.1.2. 2-Oxo-2-(1-tosyl-1H-pyrrol-3-yl)ethyl 2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate (5)
3-(Chloroacetyl)-1-tosyl-1H-pyrrole 4 (560 mg, 1.9 mmol) was
added to a solution of indomethacin 1 (673 mg, 1.9 mmol), DMAP
(46 mg, 0.4 mmol) in Et3N (12 mL) and dichloromethane (12 mL).
The reaction mixture was stirred at r.t. under argon atmosphere.
After 24 h evaporation of the solvent in vacuo followed by flash
chromatography on silica gel (hexane/ethyl acetate ¼ 1/1) afforded
the pure product 5 as a yellow solid (yield 991 mg, 1.6 mmol, 84%);
Rf ¼ 0.50 (hexane/ethyl acetate, 6/4). 1H NMR (CDCl3, 300 MHz,
NMR (CDCl3, 300 MHz, 25 ꢀC):
d
¼ 2.37 (s, 3H, CH3CN); 3.84 (s, 2H,
CCH2CO); 3.85 (s, 3H, OCH3); 5.10 (s, 2H, OCH2CO); 6.61 (td,
J ¼ 3.0 Hz, J ¼ 1.6 Hz, 1H, CHpy); 6.68 (dd, J ¼ 9.0 Hz, J ¼ 2.5 Hz, 1H,
CHarom); 6.74 (dt, J ¼ 3.0 Hz, J ¼ 1.6 Hz, 1H, CHpy); 6.92 (d, J ¼ 9.0 Hz,
1H, CHarom); 7.04 (d, J ¼ 2.5 Hz, 1H, CHarom); 7.32 (dt, J ¼ 3.0 Hz,
J ¼ 1.6 Hz, 1H, CHpy); 7.44e7.48 (m, 2H, CHarom); 7.63e7.68 (m, 2H,
CHarom); 8.77 (bs, 1H, NH) ppm. 13C NMR (CDCl3, 75 MHz, 25 ꢀC):
25 ꢀC):
d
¼ 2.34 (s, 3H, CH3CN); 2.38 (s, 3H, CH3CCHarom); 3.79 (s,
d
¼ 13.4; 30.0; 55.8; 66.9; 101.4; 108.5; 112.0; 112.4; 114.9; 119.6;
2H, CCH2CO); 3.81 (s, 3H, OCH3); 5.04 (s, 2H, OCH2CO); 6.61 (dd,
J ¼ 3.3 Hz, J ¼ 1.6 Hz, 1H, CHpy); 6.65 (dd, J ¼ 9.0 Hz, J ¼ 2.5 Hz, 1H,
CHarom); 6.88 (d, J ¼ 9.0 Hz, 1H, CHarom); 6.99 (d, J ¼ 2.5 Hz, 1H,
CHarom); 7.11 (dd, J ¼ 3.3 Hz, J ¼ 2.1 Hz, 1H, CHpy); 7.27e7.31 (m, 2H,
CHarom); 7.40e7.44 (m, 2H, CHarom); 7.61e7.65 (m, 2H, CHarom);
7.73e7.77 (m, 3H, CHpy þ CHarom) ppm. 13C NMR (CDCl3, 75 MHz,
122.3; 123.1; 129.1; 130.7; 130.9; 131.2; 133.9; 136.0; 139.3; 156.2;
168.4; 170.3; 187.7 ppm. HRMS: calcd. for [C25H21ClN2O5 þ Na]þ
~
487.1031; found 487.1036. IR (CHCl3):
n
¼ 3466; 1742, 1680 cmꢁ1
.
M.p.: 125e127 ꢀC.
4.1.6. N-Tosyl-3-acetyl pyrrole (9)
25 ꢀC):
d
¼ 13.3; 21.6; 29.7; 55.6; 66.4; 101.1; 111.8; 112.0; 114.8;
Acetic anhydride (0.44 mL, 4.7 mmol) was added dropwise to
a solution of aluminum chloride (896 mg, 6.7 mmol) in 5.22 mL of
dichloromethane. After 10 min of stirring at room temperature, N-
tosylpyrrole 3 (400 mg, 1.81 mmol) in 0.9 mL dichloromethane was
added. The mixture was stirred for 2 h and poured into water and
ice. The aqueous phase was extracted with CH2Cl2, washed with an
aqueous solution of 1 N NaOH and dried over Na2SO4. The product
was washed with methanol and after evaporation gave 99% of N-
tosyl-3-acetyl pyrrole 9 as pure solid product (470 mg, 1.78 mg).
Rf ¼ 0.5 (hexane/ethyl acetate, 6/4). The product was recognized by
1H NMR spectra [24].
121.7; 124.0; 125.1; 127.2; 129.0; 130.3; 130.5; 130.7; 131.1; 133.8;
134.7; 135.9; 139.1; 146.1; 156.0; 168.2; 170.2; 187.1 ppm. HRMS:
calcd. for [C32H27ClN2O7S þ Na]þ 641.1120; found 641.1124. IR
~
(CHCl3):
n
¼ 1746; 1690 cmꢁ1. M.p.: 134e136 ꢀC.
4.1.3. N-(Triisopopylsilyl)pyrrole (6)
n-Butyllithium in hexane (2.2 mL of a 2.5 M solution, 5.5 mmol)
was added dropwise to argon-dried pyrrole (compound 2) (335 mg,
5.0 mmol) in 10 mL of anhydrous THF at ꢁ78 ꢀC. Triisopropylsilyl
chloride (965 mg, 5.0 mmol) was added after 10 min and the
reaction warmed to room temperature. The solvent was then
removed, water was added, and the resulting residue was extracted
with diethyl ether. The organic phase was then dried over anhy-
drous sodium sulfate and concentrated by rotatory evaporation. N-
(Triisopropylsilyl)pyrrole (6) was isolated as a colorless oil (yield
972 mg, 4.4 mmol, 87%); Rf: 0.48 (petrol ether 40e70 ꢀC). The
product was recognized by 1H NMR spectra [13].
4.1.7. N-Tosyl-3-methylacetate pyrrole (10)
Thallium trinitrate Tl(NO3)3$3H2O (1.6 g, 3.6 mmol), trimethyl
orthoformiate (4 mL) and montmorillonite K10 (3.2 g) were stirred
in 3.3 mL methanol at room temperature for 45 min. The solvent
was removed to obtain a powder completely dried. N-Tosyl-3-
acetyl pyrrole 9 (470 mg, 1.79 mmol) and the catalyst were stir-
red for 12 h in 29 mL of methanol. The white precipitate of tallium
4.1.4. 3-(Chloroacetyl)-1-(triisopropylsilyl)-1H-pyrrole (7)
salt was filtered and washed with a solution of dichlor-
Chloroacetyl chloride (168 mg, 1.50 mmol) was added slowly to
a stirred slurry of aluminum chloride (125 mg, 1.1 mmol) in anhy-
drous dichloromethane (2.0 mL at 0 ꢀC). After 0.25 h, a solution of 6
(223 mg, 1.0 mmol) in dichloromethane (0.5 mL) was added. The
mixture was stirred for 0.5 h at 0 ꢀC and 0.5 h at r.t. and then poured
into an iceewater mixture. The organic phase was separated and
combined with a dichloromethane extract (4 ꢂ 5 mL) of the
aqueous phase. The organic phase was dried (Na2SO4 anhydrous)
and evaporated in vacuo. The residual material was subjected to
flash chromatography on silica gel (hexane/ethyl acetate ¼ 9/1).
Compound 7 was obtained as a colorless oil (yield 128 mg,
0.43 mmol, 43%); Rf ¼ 0.59 (hexane/ethyl acetate, 7/3). 1H NMR
omethane:methanol (99:1). The solution was evaporated and
filtered. The operation was repeated until the majority of the
precipitate disappeared. 10 mL of diethyl ether was added and the
solution was washed with water and a saturated NaCl solution,
dried over Na2SO4 and evaporated. The yellow oil obtained was
purified by silica gel column chromatography with heptane/ethyl
acetate 9:1 to 7.5:2.5 as eluent to give N-tosyl-3-methylacetate
pyrrole 10 (yield 260.8 mg, 50%). The product was recognized by
1H NMR spectra [17].
4.1.8. 3-Acetic acid pyrrole (11)
2.87 mL NaOH (5 N) in 2.30 mL methanol was added to N-tosyl-
3-methylacetatel pyrrole 10 (260.8 mg, 0.89 mmol) and refluxed for
2 h. The methanol was removed and the residue was washed with
diethyl ether and acidified by using a gradient of aqueous HCl in the
range of 5 Ne0.5 N to pH 3, and finally extracted four times with
diethyl ether. The solution was dried over Na2SO4 anhydrous.
After removal of the solvent, 3-acetic acid pyrrole 11 was obtained
as a white solid (yield 105 mg, 0.84 mmol, 94%). The product
was recognized by 1H NMR spectra [16].
(CDCl3, 300 MHz, 25 ꢀC):
d
¼ 1.10 (d, J ¼ 7.5 Hz, 18H, CH(CH3)2); 1.47
(heptuplate, J ¼ 7.5 Hz, 3H, CH(CH3)2); 4.47 (s, 2H, ClCH2CO); 6.71
(dd, J ¼ 2.9 Hz, J ¼ 1.5 Hz, 1H, CHCHN); 6.75 (dd, J ¼ 2.9 Hz,
J ¼ 2.0 Hz, 1H, CHCHN); 7.49 (dd, J ¼ 2.0 Hz, J ¼ 1.5 Hz, 1H,
CCHN) ppm. 13C NMR (CDCl3, 75 MHz, 25 ꢀC):
d
¼ 11.5; 17.6; 46.3;
110.8; 125.8; 130.4; 133.0; 186.7 ppm. GCeMS: m/z 299 [Mþ], 250
(100). IR (CHCl3):
n
¼ 1678 cmꢁ1
:
~
4.1.5. 2-Oxo-2-(1H-pyrrol-3-yl)ethyl 2-(1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl)acetate (8)
4.1.9. Reduced indomethacin (12)
Compound 8 was prepared from indomethacin 1 (139 mg,
A solution of BH3$SMe2 in 2 M THF (0.8 mL, 1.6 mmol) was
added slowly to an ice-cold solution of indomethacin (500 mg,
0.4 mmol), 3-(chloroacetyl)-1-(triisopropylsilyl)-1H-pyrrole
7

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J. Serra Moreno et al. / European Journal of Medicinal Chemistry 57 (2012) 391e397
1.4 mmol) in THF (7.5 mL). The reaction was warmed up slowly from
the ice-bath and stirred at room temperature for 22 h. Excess BH3
was destroyed by the slow addition of MeOH (0.1 mL). The solvent
was removed under reduced pressure. The crude material was
dissolved in CH2Cl2 (10 mL), washed with saturated NaHCO3 solu-
tion, water, 3 N HCl, water, and saturated NaCl solution, dried over
Na2SO4 anhydrous. The yellowish solid 12 was collected and dried
under vacuum (yield 439 mg, 1.3 mmol, 91%). The product was
recognized by 1H NMR spectra [18].
absorbance at 490 nm using a 96 well plate reader. The experiment
was run in triplicate.
4.2.3. Hoechst staining
In order to stain cell nuclei, Hoechst staining was performed
using a 1:800 dilution from 2 mmol/l Hoechst dye stock (Sigmae
Aldrich, Milano, Italy) on COBs treated with compounds 5, 8, 13
(10ꢁ6 M) and IDMC (10ꢁ6 M) for 24.
4.2.4. BrdU assay
4.1.10. 2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)ethyl 2-(1H-pyrrol-3-yl)acetate (13)
BrdU assay was performed using ‘‘Cell Proliferation ELISA, BrdU
(colorimetric)’’ (Roche Diagnostics). Cells were treated for 24 h with
compounds 5, 8 and 13 (10ꢁ6 M) and IDMC (10ꢁ6 M). Then cultures
were labeled with BrdU following the manufacturer’s instructions.
Photometric detection was done with an ELISA reader at 370 nm
wavelength. The experiment was run in triplicate.
EDCI (183 mg, 1.0 mmol), DMAP (6 mg, 0.05 mmol), and 12
(168 mg, 0.3 mmol) were added to a solution of 2-(1H-pyrrol-3-yl)
acetic acid 11 (54 mg, 0.4 mmol) in dichloromethane (1.2 mL) and
stirred overnight at r.t.. Upon dilution with water, the aqueous
solution was extracted with dichloromethane (4 ꢂ 4 mL). The
combined organic layer was washed with water (4 mL), dried
(Na2SO4), filtered, and the solvent concentrated in vacuo. The residue
was chromatographed on silica gel (hexane/ethyl acetate ¼ 7/3) to
afford the esters 13 as a yellow oil (yield 120 mg, 0.3 mmol, 62%);
Rf ¼ 0.38 (hexane/ethyl acetate, 6/4). 1H NMR (CDCl3, 300 MHz,
4.2.5. Western blotting
COBs were plated in 6-well culture dishes (Costar Corp. Celbio,
Italy) at the density of 15,000 cells/cm2 and grown for 5e6 days in
DMEM with 10% heat inactivated FCS, penicillin and streptomycin.
Then, cells were treated with compounds 5, 8,13 (10ꢁ6 M) and IDMC
(10ꢁ6 M) for 24. Proteins were extracted with Cell Lysis Buffer
(EuroClone, Milano, Italy) and the concentration was determined by
the BCA protein assay reagent (Pierce, Celbio, Italy). After SDS-
polyacrylamide gel electrophoresis on 12% gels, proteins were
transferred to PVDF membranes (Bio-Rad, Milano, Italy). The next
steps were performed by ECL Advance Western Blotting Detection
Kit (Amersham Biosciences, Europe, GMBH). Briefly, membranes
were blocked with Advance Blocking Agent in PBS-T (PBS containing
0.1% Tween 20) for 1 h at room temperature. Then, membranes were
incubated with a rabbit anti-Bcl-2 antibody (Santa Cruz Biotech-
nology, Inc., Italy) or with a rabbit anti-Bax antibody (Santa Cruz
Biotechnology, Inc., Italy) both diluted 1:200 in blocking solution for
2 h at room temperature. After washing with PBS-T the blots were
incubated with horseradish peroxidase (HRP)-conjugated donkey
anti-rabbit IgG (EuroClone, Milano, Italy) diluted 1:100,000 in
blocking solution for 1 h at room temperature. After further washing
with PBS-T, immunoreactive bands were visualized using luminol
reagents and Hyperfilm-ECL film (Amersham Biosciences, Europe,
GMBH) accordingly tothe manufacturer’s instructions. To normalize
25 ꢀC):
d
¼ 2.32 (s, 3H, CH3CN); 3.00 (t, J ¼ 7.2 Hz, 2H, CCH2CH2O);
3.51 (s, 2H, CCH2CO); 3.84 (s, 3H, OCH3); 4.29 (t, J ¼ 7.2 Hz, 2H,
CCH2CH2O); 6.13e6.15 (m, 1H, CHarom); 6.65e6.68 (m, 2H, CHarom);
6.71e6.73 (m, 1H, CHarom); 6.89 (d, J ¼ 8.6 Hz, 1H, CHarom); 6.97 (d,
J ¼ 2.5 Hz,1H, CHarom); 7.45e7.48 (m, 2H, CHarom); 7.62e7.65 (m, 2H,
CHarom); 8.11 (bs, 1H, NH). 13C NMR (CDCl3, 75 MHz, 25 ꢀC):
d
¼ 13.1;
23.7; 33.0; 55.7; 63.5; 101.3; 109.1; 111.4; 114.9; 115.3; 115.4; 116.5;
118.0; 129.0; 130.9; 131.0; 131.0; 134.1; 135.2; 139.0; 156.0; 168.2;
172.4. HRMS: calcd. for [C25H23ClN2O4 þ Na]þ 473.1239; found
~
473.1245. IR (CHCl3):
n
¼ 3481; 1730, 1674 cmꢁ1. M.p.: 90e92 ꢀC.
4.2. Experimental animals
Harlan SpragueeDawley ICR (CD-1) male mice (Harlan, Italy)
were used. Mice were sacrificed by CO2 narcosis and cervical
dislocation in accordance with the recommendation of the Italian
Ethical Committee and under the supervision of authorized
investigators.
4.2.1. Primary calvarial osteoblasts (COBs)
the bands, filters were stripped and re-probed with a mouse anti-
tubulin. Bands density were quantified densitometrically.
a-
COBs were obtained from newborn mice by sequential digestion
with 0.1% collagenase (Roche Diagnostic, Milano, Italy) [25]. Cells
were pooled and cultured to confluence in 100-mm dishes and
grown in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen
S.R.L. Milano, Italy) containing 10% heat-inactivated fetal calf serum
(FCS; Invitrogen S.R.L. Milano, Italy), penicillin (100 U/ml), and
4.2.6. Actin labeling
After incubation with compounds 5, 8, 13 (10ꢁ6 M) and IDMC
(10ꢁ6 M) for 24, COBs were rinsed with 0.1 M phosphate-buffered
saline solution (PBS), pH 7.4, and fixed with 4% paraformaldehyde
(PFA) diluted in PBS for 25 min at room temperature. Cells were
permeabilized with 0.3% Triton X-100 for 20 min on ice. Then, COBs
were incubated with 4 ꢂ 10ꢁ6 mol/L phalloidin tetramethylrhod-
amine isothiocyanate (TRITC) conjugate (Sigma Aldrich, Milano
Italy) for 20 min at room temperature. After rinsing in PBS, cover-
slips were mounted on slides with PBS/glycerol (1/1) [28]. Slides
were imaged using a Leica DM 2500 fluorescent microscopy.
streptomycin (50
mg/ml) in a humidified atmosphere of 5% CO2 at
37 ꢀC [26].
4.2.2. Assessment of viable COBs (MTS assays)
The metabolic activity of viable COBs was determined by the
MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium assay as previously described
[27]. Briefly, COBs were plated at the density of 5000 cells/well in
96 culture dishes (Costar Corp., Celbio, Italy) and grown DMEM,
supplemented with 10% heat inactivated FCS, penicillin and strep-
tomycin to approximately 80% confluence. Then, cells were treated
with compounds 5, 8, 13 (10ꢁ6 M) and IDMC (10ꢁ6 M) for 24.
4.2.7. Toluidine blueehematoxylin/eosin
COBs were treated with compounds 5, 8, 13 (10ꢁ6 M) and IDMC
(10ꢁ6 M) for 24. Then, cultures were rinsed with 0.1 M PBS, pH 7.4,
and fixed with 4% PFA diluted in PBS for 25 min at room temper-
ature. After further washing in PBS, cells were stained with tolui-
dine blue (SigmaeAldrich, Milano, Italy) or with hematoxylin/eosin
(SigmaeAldrich, Milano, Italy); then, coverslips were mounted on
slides with mounting medium and were monitored by a Leica DM
2500 optical microscopy.
Subsequently, cells were incubated with 20 ml/well of CellTiter 96
AQueous One Solution Reagent (Promega Italia srl, Milano, Italy) for
2 h in a humidified, 5% CO2, atmosphere. The quantity of formazan
product is directly proportional to the number of living cells in
culture. The colored formazan was measured by reading the

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We thank Sapienza University of Rome (Ateneo Project 2010 “nuovi
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