Synthesis, pharmacological evaluation and docking studies of new sulindac analogues

Article abstract of DOI:10.1016/j.ejmech.2008.11.012

This paper describes the synthesis, pharmacological evaluation and docking studies of a series of new sulindac analogues. Overall, the designed compounds revealed good, in vivo, antinociceptive activity and satisfactory anti-inflammatory profile. Flexible

Full text of DOI:10.1016/j.ejmech.2008.11.012

European Journal of Medicinal Chemistry 44 (2009) 1959–1971
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, pharmacological evaluation and docking studies of new sulindac
analogues
*
Nelilma C. Romeiro, Ramon D.F. Leite, Lidia M. Lima , Suzana V.S. Cardozo, Ana L.P. de Miranda,
*
Carlos A.M. Fraga, Eliezer J. Barreiro
ˆ
´
Laborato´rio de Avaliaç˜ao e S´ıntese de Substancias Bioativas (LASSBio), Faculdade de Farmacia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, PO Box 68023, RJ 21944-
970, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis, pharmacological evaluation and docking studies of a series of new
sulindac analogues. Overall, the designed compounds revealed good, in vivo, antinociceptive activity and
satisfactory anti-inflammatory profile. Flexible molecular docking with COX-1/COX-2 has shown putative
binding modes of the designed compounds while the theoretical evaluation of cell permeability based on
Lipinski’s rule of five has helped rationalize the biological results.
Received 1 September 2008
Received in revised form
26 November 2008
Accepted 27 November 2008
Available online 6 December 2008
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Molecular docking
Sulindac analogues
NSAIDs
Antinociceptive
Anti-inflammatory drugs
1. Introduction
[8], aspirin (ASA, 2) [9], meclofenamic acid (3) [10] and indo-
methacin (4) [11] (Fig. 1) inhibit both isoforms of COX non-selec-
Prostaglandins act as potent mediators of pain, fever and
inflammation. Prostaglandin synthase (COX) catalyses the
tively or with low selectivity, exerting their anti-inflammatory
activity via inhibition of COX-2, and their deleterious side effects by
inhibition of COX-1 (Fig. 1). In order to provide an effective treat-
ment for inflammatory disorders, the design of selective inhibitors
of COX-2 has been widely described as a new therapeutical
approach, aiming at obtaining new NSAIDs, devoid of the side
effects commonly associated with conventional NSAIDs [12–15].
Examples of selective inhibitors of COX-2 are celecoxib (5) [16],
rofecoxib (6) [17], valdecoxib (7) [18] and etoricoxib (8) [19] (Fig. 1).
Although rofecoxib (6, Fig. 1) has been withdrawn from the market
because of its cardiovascular side effects originated from long-term
treatment, and valdecoxib (7, Fig. 1) has been withdrawn for serious
cutaneous adverse reactions, there still remains a lot of research
work to be done in the design of new safer anti-inflammatory
drugs, selective COX-2 inhibitors [20,21].
Additionally, the action of NSAIDs in other pathologies has been
the subject of many studies. For instance, recent reports have
shown a growing body of evidence that COX-2 expression is
a fundamental step in breast cancer pathogenesis acting through
prostaglandin-dependent and independent mechanisms [22,23].
Epidemiological studies suggest that NSAIDs confer a moderate
degree of benefit against breast cancer [24–26]. Further reports
H
conversion of arachidonic acid to the prostaglandin endoperoxide
PGG2 then PGH2, which serves as the precursor for the formation
of PGs and thromboxanes [1,2]. Two isoforms of COX have been
identified [3–5]. The first, COX-1, is constitutively expressed in most
tissues and is believed to generate prostaglandins for normal
physiological functions. The second isoform, COX-2, is character-
ized by a rapid induction by a variety of stimuli, including mitogens,
hormones, cytokines, and growth factors. Selective inhibition of
COX-2 is desirable in order to avoid gastric side effects generally
associated with inhibition of COX-1, which is responsible for gastric
mucosa protection [3–5].
Non-steroidal anti-inflammatory drugs (NSAIDs) [6,7] exert
their anti-inflammatory, antipyretic and antinociceptive effects by
blocking the production of prostanoids from arachidonic acid
through inhibition of COX activity. Classic NSAIDs like sulindac (1)
* Corresponding authors. Tel./fax: þ55 21 2562 6644.
E-mail addresses: lidia@pharma.ufrj.br, ejbarreiro@ccsdecania.ufrj.br (L.M.
Lima).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.11.012

1960
N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
Fig. 1. NSAIDs, COX-1/COX-2 inhibitors.
have shown the ability of NSAIDs to prevent colorectal cancer,
among other diseases [27–30]. However further work is required to
establish how this enzyme system can be best manipulated for
therapeutic benefit.
containing a methyl sulfoxide group that is converted to the
metabolites sulindac sulfide and sulindac sulfone in vivo (Fig. 1, 9–
10) [38]. It has been reported that sulindac sulfide, and not sulindac
sulfone, blocks prostaglandin synthesis [39]. In addition, sulindac
and its metabolites have been shown to play important roles in the
prevention of colonic carcinogenesis [40–42] and may function as
anti-apoptotic agents [29,43].
Based on the recent renewed interest in sulindac and its
metabolites [40], our group designed and synthesized a new series
of analogues of this classic NSAID, exploring the spatial similarities
with selective NSAIDs of the diarylheterocyclic class, like celecoxib
and rofecoxib (Fig. 1, 5–6). The design concept is disclosed in Fig. 3.
These new sulindac analogues (17a–i) were planned by the
substitution of the principal pharmacophoric group for COX-1
inhibitor activity, represented by the carboxylic acid moiety (A,
Fig. 3), by different neutral amide residues (C, Fig. 3). Otherwise, in
order to accomplish a better COX-1/COX-2 selectivity ratio for these
new series of active compounds (17a–i), the introduction of the
pharmacophoric methyl sulfone unit (D, Fig. 3), characteristic of
several COX-2 selective inhibitors, such as rofecoxib (6), was
planned in replacement for the methyl sulfoxide group (B, Fig. 3)
present in the prototype 1.
One of the existing approaches for the design of such substances
is the molecular modification of existing drugs, e.g., second
generation NSAIDs, which are comprised of modified classic
NSAIDs [31–35]. This approach is based on the introduction of
pharmacophores aiming at improving the selectivity for COX-2.
Examples of this approach have been reported recently and include
aspirin, indomethacin, flurbiprofen, ketoprofen and meclofenamic
acid derivatives with improved selectivity to COX-2 [31–35].
Among these compounds, amide derivatives of indomethacin have
shown great selectivity for COX-2, with a special performance of
aliphatic amides with maximum of eight carbon atoms, secondary
amides and terminally functionalized alkyl chains (Fig. 2) [31–33].
Among the preferred terminal groups in the ester functionalized
chain, the benzyl radical with substituents in position 4 showed
greater selectivity to COX-2. In the meclofenamic acid series, O-
substituted hydroxamate derivatives and alkyl chains functional-
ized with a halogen atom showed greater selectivity among other
modifications [34]. Successful examples of these compounds are
shown in Fig. 2.
The designed compounds (17a–i) were synthesized using
sulindac (1), a known marketed drug, as starting material and have
been submitted to flexible docking studies using the X-ray crys-
tallographic structure of COX-1 and COX-2 [44–48] and FlexE
software [49] in order to build structure–activity relationship
considerations. Finally, Lipinski’s rule of five has been applied with
Sulindac analogues have been explored in our group as new
potential anti-inflammatory agents using safrole, an abundant
natural product available from sassafras oil in Brazil, as starting
material [36,37]. This anti-inflammatory drug functions by non-
selective inhibition of COX-1 and COX-2. Sulindac (1) is a pro-drug,

N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
1961
Fig. 2. Modified NSAIDs with better COX-1/COX-2 selectivity ratio.
Fig. 3. Design concept of sulindac analogues, aimed at a better COX-1/COX-2 selectivity ratio.

1962
N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
the aim to predict cell permeability behavior of the new sulindac
analogues (17a–i) [50]. The compounds were also evaluated for
anti-inflammatory and antinociceptive activities in vivo.
sulindac, used as positive control, were administered by oral admin-
istration at a screening dose of 300
mol/kg and also evaluated for its
m
ulcerogenic potential [55]. The results are disclosed in Tables 1 and 2.
The results demonstrated that all compounds showed anti-
nociceptive activity in the tested dose (Table 1). The most potent
antinociceptive derivatives were 17b (LASSBio-952) and 17a
(LASSBio-852), bearing an ethyl amide and methyl amide side
chain, respectively, with 59.4% and 43.4% of inhibition of the
induced abdominal constrictions. The results obtained with the
homologous 17c (LASSBio-854, 38.6%) and 17d (LASSBio-869,
34.6%), with amide side-chains of three and four carbon atoms,
respectively, suggest some steric limitation in the putative bio-
receptor (Table 1). This assumption is further corroborated by the
low activity found for derivative 17e (LASSBio-905, 13.9%), which
has a bulky diethyl amide side chain. Aromatic amide side-chains
are also not tolerated due to the low antinociceptive activity
demonstrated by compound 17i (LASSBio-895, 18.9%). As expected,
derivative 20 (LASSBio-906, Scheme 1), synthesized in order to
attribute the importance of the methyl sulfone subunit introduced
in the structural design of this new bioactive series, which bears
a methyl amide side chain and the methyl sulfoxide group, showed
the lowest antinociceptive effect (6.3%) (Table 1), confirming the
pharmacophoric profile of the methyl sulfone group for the anti-
nociceptive activity.
2. Results and discussion
2.1. Synthesis
The synthesis of compounds (17a–i) was performed in three
overall steps, using sulindac (1) as starting material (Scheme 1). In
the synthetic approach compound methyl 2-5-fluoro-2-methyl-
1-[1-(4-methylsulfonylphenyl)-(Z)-methylidene]-1H-3-indenylace
tate (19) was identified as the key precursor. This compound was
obtained in ca. 85% overall yield from sulindac, exploring the
oxidation of methyl sulfoxide group and the esterification of the
carboxylic acid group (Scheme 1) [51].
With an attractive method for access to the key intermediate
(19), we next performed the condensation of this compound with
functionalized alkyl and aryl amines, using aluminum chloride in
dichloromethane boiled at reflux, under nitrogen atmosphere [52],
to afford the corresponding sulfone-amides derivatives 17b–i in
moderate to excellent yields (Scheme 1). The methyl amide deriv-
ative (17a) was obtained in satisfactory yield by aminolysis of ester
(19), using aqueous solution of methylamine 40% [53].
The methyl amide-sufoxide derivative (20) was obtained in two
reaction steps, in 50% overall yield, exploring the esterification of
carboxylic acid group of sulindac (1), followed by aminolysis of the
corresponding ester using aqueous methylamine 40% (Scheme 1).
The application of non-classic bioisosteric strategy [56] resulted in
the design of compound 17f (LASSBio-870) with significant analgesic
activity (35.6%, Table 1). Subsequent bioisosteric replacement of
methylene in the piperidine moiety of compound 17f by oxygen or by
sulfur atom result in the structural design of compounds 17g (LASSBio-
853) and 17h (LASSBio-868), bearing a morpholine and thiomorpho-
line amide subunit, respectively. As depicted in Table 1, derivative 17h
(LASSBio-868) showed 47.8% of inhibition of the induced constrictions,
close to the value observed for the prototype sulindac, 49%. Celecoxib,
a selective COX-2 inhibitor, showed a superior inhibition of the
constrictions by 55.6% (Table 1).
2.2. Biological assays
The antinociceptive and anti-inflammatory activities of the new
sulindac analogues (17a–i, Fig. 3) were evaluated using the acetic acid
induced mice abdominal constrictions test and the carrageenan-
induced rat paw edema test, respectively [54]. All compounds and
Scheme 1. Conditions: (a) OxoneÒ, THF:MeOH:H2O, r.t., 1 h, 90%; (b) MeOH, H2SO4 cat., reflux, 1 h, 95%; (c) 1: AlCl3, CH2Cl2, N2, r.t.; 2: functionalized amines, CH2Cl2, reflux, 24 h,
50–94%; (d) aqueous methylamine 40%, 50 ꢀC, 72 h, 45–50%.

N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
1963
Table 1
in a variety of COX-2 inhibitors [17,19]. Thus, we may hypothesize
that 18 (LASSBio-843, sulindac sulfone) works through the inhi-
bition of COX-1 and COX-2, in a dual fashion, with some selectivity
towards COX-2, in opposition to what has been reported in the
literature in an in vitro assay of cancer cell lines [39]. Nevertheless,
the interference of 18 (LASSBio-843, sulindac sulfone) in other
enzyme pathways related to the inflammatory response cannot be
discarded [57].
Compound 17a (LASSBio-852), bearing a methyl amide subunit,
showed reasonable anti-inflammatory activity, in addition to the
already mentioned good antinociceptive profile, inhibiting edema
formation in 35%. The sulfoxide derivative of 17a, compound 20
(LASSBio-906), showed an anti-inflammatory activity at the same
magnitude of the former, inhibiting edema formation in 28%,
however without showing an antinociceptive effect.
Furthermore, analogues bearing bulky or hydrophobic aromatic
amides in the side-chains, such as 17b (LASSBio-952), 17c (LASSBio-
854), 17d (LASSBio-869), 17e (LASSBio-905), 17f (LASSBio-870), 17g
(LASSBio-853), 17h (LASSBio-868) and 17i (LASSBio-895), did not
present good anti-inflammatory profiles, inhibiting edema formation
very poorly (Table 2). These compounds showed a better anti-
nociceptive profile than anti-inflammatory one. Surprisingly,
compound 17h (LASSBio-868), that bears a thiomorpholine group in its
side chain, and was one of the most active compounds in the anti-
nociceptive test, showed no significant anti-inflammatory activity.
We may hypothesize that this result is due to some sort of steric
blockade exerted by this group on its biomolecular target. Finally,
another curious result was the low anti-inflammatory activity of 19
(LASSBio-842), the methyl ester analogue, which is expected to be
transformed, in vivo, into its carboxylic acid metabolite 18 (LASS-
Bio-843, sulindac sulfone) (Table 2). As has been hypothesized in
the analysis of the antinociceptive results (Table 1), it is possible
that the time window in which the biological experiments have
been performed was not wide enough to allow full biotransfor-
mation of this compound.
Effect of sulindac analogues on the inhibition of abdominal constrictions induced by
acetic acid (0.1 N, i.p.) in mice.
Compound
Constrictions count
% Inhibition
Vehicle control (Arabic gum 5%)
60.6 ꢁ 1.2
30.9 ꢁ 2.1*
26.7 ꢁ 1.7*
34.3 ꢁ 2.8*
24.6 ꢁ 2.4*
37.2 ꢁ 1.8*
39.6 ꢁ 3.8*
52.6 ꢁ 3.8
39.0 ꢁ 2.2*
46.5 ꢁ 3.1*
31.6 ꢁ 2.7*
49.5 ꢁ 3.5
39.8 ꢁ 1.3*
47.7 ꢁ 1.5*
57.2 ꢁ 2.2
–
Sulindac
Celecoxib
17a
17b
17c
17d
17e
17f
17g
17h
17i
18
19
20
49%
56%
43%
59%
39%
35%
14%
36%
22%
48%
19%
34%
21%
6%
All test compounds were administered at a dose of 300
m
mol/kg p.o. N ¼ 8–10
animals. Results are expressed as mean ꢁ SEM.
comparison with vehicle control group.
*p > 0.05 (Student’s t-test).
% of inhibition obtained by
Curiously, the sulindac sulfone acid derivative 18 (LASSBio-843),
described as an inactive metabolite of sulindac (1), showed similar
analgesic activity to compounds 17d (LASSBio-869) and 17f (LASS-
Bio-870) with an inhibition of 34.3%. Finally, compound 19 (LASSBio-
842), an synthetic intermediate, that bears a methyl ester side chain,
was also evaluated and showed low antinociceptive activity, inhib-
iting the abdominal constrictions in only 21.2% (Table 1).
The results for the anti-inflammatory activity of sulindac
analogues (17a–i), administered p.o. at a dose of 300 mmol/kg
(Table 2), indicated that the most active compounds were sulindac
(1), sulindac sulfone (18, LASSBio-843) and 17a (LASSBio-852), that
inhibited edema formation in 67%, 48% and 35%, respectively.
According to what was expected, sulindac exerted a significant
ulcerative behavior in the gastric mucosa in the carrageenan-
induced rat paw edema assay (ulcerogenic potential ¼ 2.0) sup-
porting its non-selective COX mechanism of action. However, 18
(LASSBio-843, sulindac sulfone) and 17a (LASSBio-852) had
a lower or null ulcerogenic potential (0.4) (Table 2), probably
because it bears a pharmacophoric methyl sulfone group, present
2.3. Molecular modeling
2.3.1. Molecular docking with COX-1 and COX-2
In an attempt to further corroborate the design strategy of the
new sulindac analogues (17a–i), aimed at a better COX-1/COX-2
selectivity ratio, and try to establish the structure–activity rela-
tionship of the designed compounds, we performed flexible dock-
ing studies using available X-ray crystal structures of COX-1/COX-2
with bound inhibitors [44–48]. This theoretical study was per-
formed using the software FlexE [49] with protein ensembles of
both enzymes under study [44–48]. The proposed interaction
modes of the ligands into the active site of COX-1 and COX-2 were
determined as the highest scored conformations (best-fit ligand)
among 30 conformational and binding modes generated according
to FlexX scoring function, which corresponds to the structure with
Table 2
Effect of sulindac analogues on the inhibition of carrageenan-induced rat paws
edema (1 mg/paw; i.p.).
Compound
Edema (m
l)a % Inhibition Ulcerogenic potential
Vehicle control (Arabic gum 5%) 569 ꢁ 45
–
–
2
0
0
0
1.4
2
1.0
0
0
4
0
0.4
0
0
Sulindac
Celecoxib
17a
17b
17c
17d
17e
17f
17g
17h
17i
18
19
20
190 ꢁ 28*
370 ꢁ 27*
370 ꢁ 37*
411 ꢁ 54*
414 ꢁ 27*
531 ꢁ 11
415 ꢁ 19*
461 ꢁ 9*
546 ꢁ 57
503 ꢁ 22
497 ꢁ 48
299 ꢁ 28*
484 ꢁ 34
406 ꢁ 32*
67%
35%
35%
27%
20%
7%
27%
19%
4%
11%
12%
48%
15%
28%
the most favorable free energy of binding, i.e.
(Fig. 4). G values shown in this study have been based on PM3
charges [58] due to a better correlation between binding energy
Gbind) and COX inhibition. Fig. 4 demonstrates that FlexE was able
DGbind (kJ/mol)
D
(D
to predict the non-selective COX profile of sulindac sulfide (9) and
the selective COX-2 profile of celecoxib (5) (Fig. 1).
In the analysis of the docked complexes, we have tried to get
some qualitative assessment on the molecular basis of the biolog-
ical activity of closely related compounds in our series, trying to
correlate the biological data to the molecular design, keeping in
mind that the assays have been performed in vivo, increasing the
complexity of the biological response and, thus, of correlation to
theoretical data. Only the best docking scores have been considered
in this analysis (Figs. 5–8).
All test compounds were administered at a dose of 300
animals.
m
mol/kg p.o. N ¼ 8–10
*p > 0.05 (Student’s t-test).
a
Edema was calculated as volume difference between carrageenan- and saline-
treated paws after 3 h of carrageenan injection. Results are expressed as mean -
ꢁ SEM. % of inhibition obtained by comparison with the vehicle control group.

1964
N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
Fig. 4. Binding energies (DGbind, kJ/mol) of top scoring solutions of the sulindac analogues as resulting from docking experiments.
Visual inspection of the obtained COX-1 complex with sulindac
using flexible docking (Fig. 5a) reveals a binding pose in which the
carboxylate group forms hydrogen bonds at the mouth of the
cyclooxygenase active site, with Arg120 and Tyr355; the oxygen
atom of the sulfoxide group forms a hydrogen bond with Ser530
and hydrophobic residues, such as Val349, Leu359 and Leu531
surround the phenyl–ethynyl group, within van der Waals contact
distance. The same interactions involving the phenyl–ethynyl
group can be seen for sulindac sulfide (Fig. 5b). Additionally, the
methyl-sulfide group is close to the side chain of Met522 (Fig. 5b),
performing hydrophobic interactions.
The obtained COX-2 complex with sulindac (Fig. 5c) reveals
a binding pose in which this compound hydrogen bonds to Arg120
via its carboxylate group. Additional hydrophobic interactions
involve Ala527, Leu531 and Leu534 (Fig. 5c). Furthermore, sulindac
sulfide also interacts with Arg513 and Tyr355 via its carboxylate
group (Fig. 5d) and is surrounded by Ile345, Met522, Val523 and
Leu531, in close hydrophobic contacts. This result is in agreement
with previous docking studies of this compound [59].
Compound 18 (LASSBio-843, sulindac sulfone) complexed with
COX-1 (Fig. 6a) reveals an interesting binding mode in which the
sulfone group, instead of the carboxylate group, forms hydrogen
bonds with Arg120 and Tyr355. The carboxylate group hydrogen
bonds to Ser530, close to the aromatic pocket comprised by Phe381,
Tyr385 and Trp387 (Fig. 6a).
The visual inspection of the complex of 18 (LASSBio-843,
sulindac sulfone) and COX-2 (Fig. 6b) reveals the same binding
orientation that had been observed in COX-1, involving hydrogen
bonding interactions of the oxygen atoms of the sulfone group at
the mouth of the binding channel with Arg513 and His90.
Furthermore, Val349, Phe518 and Leu531 are within van der Waals
contact. Additional hydrogen bonds are observed between the
oxygen atoms of the carboxylate group and the hydroxyl groups of
Tyr385 and Ser530 (Fig. 6b). This result is in agreement to the
observed binding modes for arachidonic acid and diclofenac with
COX-2 in which their carboxylate groups are hydrogen-bonded to
Tyr385 and Ser530, leading to enzyme inactivity [44,60]. These
works demonstrate that the carboxylate group of an acidic non-
steroidal anti-inflammatory drug can bind to a COX enzyme in an
orientation that precludes the formation of a salt bridge with
Arg120. Experiments of mutations suggested that Ser530 is also
important in time-dependent inhibition by nimesulide and pirox-
icam [44].
Structure–activity relationship analysis of 17a (LASSBio-852),
17b (LASSBio-952), 17c (LASSBio-854) and 17d (LASSBio-869), that
possess one, two, three, four carbon atoms in their side-chains,
respectively, indicates that the amide side chain plays an important
role in their interactions with COX-1, since these compounds show
similar antinociceptive profiles (Table 1), although the ethyl amide
side chain of 17b (LASSBio-952) seems to be ideal for a better
interaction with the bioreceptor, since this compound showed
59.4% of inhibition of abdominal constrictions induced by acetic
acid (Table 1). Interestingly, the increasing, and therefore less
favorable,
DGbind values observed in the docking results for COX-2
(Fig. 4) correlate well with the decreasing anti-inflammatory
activity observed in the carrageenan-induced rat paw edema assay
and with the increasing ulcerogenic action observed for these
compounds, supporting the better anti-inflammatory profile
observed for 17a (LASSBio-852) in COX-2 pathway due to the
absence of gastric ulcerogenicity (Table 2).
Fig. 7 discloses the interactions of 17d (LASSBio-869) and 17b
(LASSBio-952) with COX-1 and COX-2. Compound 17d (LASSBio-869)
forms hydrogen bonds with COX-1 via one of the oxygen atoms of the
sulfone group with Arg120 (Fig. 7a). A few hydrophobic contacts have
been observed, involving Ile345, Val349, Leu359 and Leu531.
Compound 17b (LASSBio-952), that was very active in the anti-
nociceptive bioassay, interacts with COX-1 in an inverted mode as
compared to 17d (LASSBio-869), hydrogen bonding with Arg120 and
Tyr355 through its amide group (Fig. 7b). An additional hydrogen
bonding interaction is observed between one of the oxygen atoms of its
sulfone group and Trp387. Finally, hydrophobic contacts are observed
involving Val116, Leu359, Ala527 and Leu531 (Fig. 7b).
The main interactions observed for 17d (LASSBio-869) and COX-
2 are hydrogen bonds involving the amide group, Arg120 and
Tyr355, in an inverted binding mode (Fig. 7c). Further hydrophobic
interactions are observed with Leu531 and Leu534 (Fig. 7c).

N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
1965
Fig. 5. Residues involved in the interactions with sulindac (a) and sulindac sulfide (b), in the binding site of COX-1 and in the binding site of COX-2 (c) and (d), respectively, as
resulting from the top scored docking solutions obtained with FlexE. Hydrogen bonds are shown as green dashed lines. Only polar hydrogens are shown for clarity. (For inter-
pretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
However, it can be seen that the amide alkyl chain is protruding out
of the entrance of the active site, probably due to steric clash with
side-chains of amino acid residues in the binding site. Compound
17b (LASSBio-952) interacts with COX-2 in the virtually same
binding mode as with COX-1, hydrogen bonding with Arg120 and
Tyr355 through the oxygen atom of the amide group (Fig. 7d) and
with Trp387 through one of the oxygen atoms of its sulfone group.
Only one hydrophobic contact has been observed, involving Val116.
This lack of additional hydrophobic contacts may be an explanation
for the low activity of this compound in the inflammation bioassay.
Despite the lack of correlation of experimental biological
activities with Gbind, the putative binding modes with COX-1 of
D
17h (LASSBio-868), a thiomorpholine derivative which was the
most active compound in the antinociceptive assay and 17g
(LASSBio-853), a morpholine derivative, one the less active (Table
1), have been investigated (Fig. 8). Analysis of the complex of 17g
(LASSBio-853) in its best docked position with COX-1 (Fig. 8a)
reveals hydrogen bonds of the former involving the oxygen atom
of the amide carbonyl with Arg120 and one of the oxygen atoms of
the sulfone group with Tyr385. Also, the 2CH3 group in the indene
Fig. 6. Residues involved in the interactions with 18 (LASSBio-843, sulindac sulfone) in the binding site of COX-1 (a) and COX-2 (b) as resulting from the top scored docking solutions
obtained with FlexE. Hydrogen bonds are shown as green dashed lines. Only polar hydrogens are shown for clarity. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)

1966
N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
Fig. 7. Residues involved in the interactions with 17d (LASSBio-869) (a) and 17b (LASSBio-952) (b) in the binding site of COX-1 and in the binding site of COX-2 (c) and (d),
respectively, as resulting from the top scored docking solutions obtained with FlexE. Hydrogen bonds are shown as green dashed lines. Only polar hydrogens are shown for clarity.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
system is in close van der Waals contact with Leu359 while the
fluorine atom is close to Ile523 and the morpholine ring is in van
der Waals contact with Val116 (Fig. 8a). However, the best docked
position of LASSBio-868 with COX-1 (Fig. 8b) shows an inversed
binding orientation compared to 17g (LASSBio-853). Finally, one of
the oxygen atoms of the sulfone moiety hydrogen bonds to Tyr355
at the mouth of the channel and the oxygen atom of the amide side
chain forms hydrogen bonds with Ser530 (Fig. 8b).
2.3.2. Lipinski’s rule of five
The bioavailability of compounds 17a–i was assessed using
ADME (absorption, distribution, metabolism, elimination) predic-
tion methods. In particular, we calculated the compliance of
compounds to the Lipinski’s rule of five [50]. Briefly, this simple rule
isbased on the observation thatmostorallyadministereddrugs have
a molecularweight (MW) of500 orless, a log P not higher than 5, five
or fewer hydrogen bond donor sites and 10 or fewer hydrogen bond
Fig. 8. Residues involved in the interactions with 17g (LASSBio-853) (a) and 17h (LASSBio-868) (b) in the binding site of COX-1, as resulting from the top scored docking solutions
obtained with FlexE. Hydrogen bonds are shown as green dashed lines. Only polar hydrogens are shown for clarity. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)

N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
1967
Table 3
a Bruker AC 200 spectrometer at 200 MHz. Splitting patterns are as
follows: s, singlet; d, doublet; dd, double doublet; dt, triple doublet;
q, quartet; br, broad; m, multiplet. 13C NMR spectra were obtained
using the same spectrometer described above at 50 MHz, using
deuterated dimethylsulfoxide or deuterated chloroform as internal
standard. Microanalysis data were obtained using a Perkin–Elmer
240 analyser, using a Perkin–Elmer AD-4 balance. The progress of
all reactions was monitored by TLC performed on 2.0 ꢂ 6.0 cm
aluminium sheets precoated with silica gel 60 (HF-254, E. Merck) to
a thickness of 0.25 mm. The developed chromatograms were
viewed under ultraviolet light at 254 nm. E. Merck silica gel (60–
200 mesh) was used for column chromatography.
Compliance of the sulindac analogues to computational parameters of
bioavailability.
Compounds CLOGPa Molecular Number of hydrogen Number of
PSAb
weight
(MW)
bonding acceptors
hydrogen
bonding donors
Sulindac
Sulindac
sulfide
Celecoxib
17a
17b
17c
17d
17e
17f
17g
17h
17i
18
19
20
3.16
5.31
355.405
339.406
3
2
1
1
125.46
105.45
4.37
2.22
2.75
3.28
3.81
3.56
3.28
2.49
3.22
4.46
2.86
3.48
2.27
381.374
385.454
399.481
413.508
427.534
427.534
439.545
441.518
457.584
447.524
371.404
386.439
369.4548
4
3
3
3
3
3
3
4
3
3
4
4
2
1
1
1
1
1
–
–
–
–
1
1
–
1
145.01
105.96
110.20
102.04
94.85
82.62
85.92
4.1. Synthesis
102.15
112.24
96.23
143.40
119.25
85.72
4.1.1. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-
methyl-1-H-inden-3-yl)acetic acid (18)
To a solution of 1 mmol of sulindac (1) in 12 mL of THF, 4 mL of
MeOH and 4 mL of H2O, at 0 ꢀC, was added 5 mmol of potassium
hydrogen persulfate (OxoneÒ). The reaction mixture was stirred for
1 h at room temperature, when the end of reaction was observed by
TLC. The suspension was evaporated to half of its original, followed
by addition of 50 mL of cold water to result in the formation of
a yellow precipitate, which was filtered, washed with water and
dried under reduced pressure to give sulfone derivative (18) in 90%
of yield, mp ¼ 194–196 ꢀC.
a
Calculated octanol/water coefficient [63].
Polar Surface Area, calculated as the surface area of all O/N/S atoms and
b
hydrogens covalently bonded to these atoms.
acceptor sites (N and O atoms). In addition, we calculated the polar
surface area (PSA) since it is another key property that has been
linked to drug bioavailability. Thus, passively absorbed molecules
with a PSA > 140 Å2 are thought to have low oral bioavailability [61].
NMR 1H (200 MHz, DMSO d6)
d: 2.16 (s, ArCH3); 3.28 (s, SO2CH3);
The results disclosed in Table
3 show that all synthesized
3.57 (s, ArCH2CO2H); 6.71 (td, J ¼ 8.4 Hz, 9.5 Hz and 2.5 Hz, H6);
7.02 (dd, J ¼ 9.5 Hz and 2.5 Hz, H4); 7.12 (dd, J ¼ 5.2 Hz and 8.4 Hz,
H7); 7.36 (s, C]CH); 7.78 (d, J ¼ 8.2 Hz, H2’ and H60); 8.00 (d,
J ¼ 8.2 Hz, H30 and H50) ppm.
compounds comply with these rules, except for celecoxib and
sulindac sulfone, that violate only one parameter, PSA. Theoretically,
these compounds should present good passive oral absorption and
differences in their bioactivity cannot be attributed to this property.
Anal. Calcd. for C20H17O4SF: C, 64.50; H, 4.60. Found: C, 64.49; H,
4.59.
3. Conclusions
4.1.2. Methyl 2-((1Z)-1-(4-(methylsulfonyl)benzylidene)-5-fluoro-
2-methyl-1-H-inden-3-yl)acetate (19)
The results described in this work show that all of the sulindac
analogues have significant antinociceptive activity, measured by
the inhibition of abdominal constrictions induced by acetic acid.
The most active compounds were 17b (LASSBio-952), which has an
ethyl amide side chain; 17a (LASSBio-852), which presents a methyl
amide side chain and 17h (LASSBio-868) that presents the thio-
morpholine amide ring in its side chain.
The anti-inflammatory assay, measured by inhibition of carra-
geenan-induced rat paw edema, showed that 18 (LASSBio-843), the
sulfone metabolite of sulindac, was the most active compound,
followed by 17a (LASSBio-852), confirming these compounds as
potential inhibitors of COX enzymes, without significant gastric
ulcerogenic effects, as compared to sulindac.
Molecular modeling studies with COX-1 and COX-2 revealed an
‘‘inversed’’ orientation in the putative binding mode of 18 (LASSBio-
843), which presents special hydrogen bonding interactions with
amino acid residues of COX-2, like Tyr385 and Ser530, as already
reported for other known NSAIDs [44,45,60]. Although reports in
the literature discard the direct COX activity of this compound, the
results reported herein suggest modulation of the prostaglandin
pathway that may either be dependent or independent of COX
activity [57]. Finally, according to Lipinski’s rule of five, the
synthesized compounds should present good oral absorption.
To a carboxylic acid derivative (1 mmol) were added methanol
(20 mL) and catalytic amounts of H2SO4. The mixture was refluxed
for 1 h underawater separator (Dean–Stark apparatus). The reaction
was stopped and the solvent was removed under reduced pressure.
The total product mixture was dissolved in dichloromethane treated
with NaOH 10% and washed with water. The organic layer was dried
(Na2SO4), filtered and evaporated under reduced pressure to give
desired ester derivative in 95% of yield, mp ¼ 169–171 ꢀC.
NMR 1H (200 MHz, CDCl3)
d: 2.20 (s, ArCH3); 3.14 (s, SO2CH3);
3.57 (s, ArCH2CO2H); 3.71 (s, CO2CH3); 6.56 (td, J ¼ 8.4 Hz, 9.5 Hz
and 2.5 Hz, H6); 6.88 (dd, J ¼ 9.5 Hz and 2.5 Hz, H4); 7.10 (dd,
J ¼ 5.1 Hz and 8.4 Hz, H7); 7.12 (s, C]CH); 7.70 (d, J ¼ 8.0 Hz, H20
and H60); 8.00 (d, J ¼ 8.0 Hz, H30 and H50) ppm.
NMR 13C (50 Hz, CDCl3)
d: 10.7 (ArCH3); 31.7 (ArCH2CO2R); 44.7
(SO2CH3); 52.5 (ArCO2CH3); 106.5 (C4); 110.9 (C6); 123.8 (C7); 127.4
(C]CH); 127.6 (C20 and C60); 129.5 (C2); 130.4 (C30 and C50); 132.4
(C10); 138.3 (C3); 140.0 (C40); 142.6 (C7a); 147.0 (C3a); 161.2 (C1);
166.1 (C5); 170.8 (ArCO2R) ppm.
Anal. Calcd. for C21H19O4SF: C, 65.27; H, 4.96. Found: C, 65.29; H,
4.99.
4.1.3. General procedure for the preparation of 2-5-fluoro-2-
methyl-1-[1-(4-methylsulfonylphenyl)-(Z)-methylidene]-1H-3-
indenyl amide derivatives (17b–i)
4. Experimental
Aluminium chloride (10 mmol) was suspended at room
temperature in dry dichloromethane (20 mL) under nitrogen
atmosphere. The amine (10 mmol) was added dropwise, resulting
in a colorless solution. After 10 min, a solution of the ester deriva-
tive (1 mmol) in dry dichloromethane (10 mL) was, carefully,
Melting points were determined with a Quimis 340 apparatus
and are uncorrected. 1H NMR spectra, unless otherwise stated, were
obtained in deuterated dimethylsulfoxide or deuterated chloroform
containing ca. 1% tetramethylsilane as an internal standard using

1968
N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
added. The reaction mixture was stirred for 24 h at reflux, when the
end of reaction was observed by TLC. After that, the reaction
mixture was diluted with 50 mL of dichloromethane and extracted
with aqueous HCl 10% (5 ꢂ 25 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered and evaporated under reduced
pressure to furnish the amines derivatives (17b–i) in good to
excellent yields. Compounds 17b–i were purified by recrystalliza-
tion in ethanol or in hydro-alcoholic mixture (1:1).
J ¼ 9.2 Hz and 2.1 Hz, H4); 7.06 (dd, J ¼ 5.1 Hz and 8.0 Hz, H7); 7.17
(s, C]CH); 7.68 (d, J ¼ 8.1 Hz, H20 and H60); 7.99 (d, J ¼ 8.1 Hz, H30
and H50) ppm.
NMR 13C (50 Hz, CDCl3)
(CON(CH2CH3)2); 31.9
d
:
10.7 (ArCH3); 13.2 and 14.4
(ArCH2CONR2); 40.8 and 42.5
(CON(CH2CH3)2); 44.7 (SO2CH3); 106.7 (C4); 110.9 (C6); 123.6 (C7);
126.6 (C]CH); 127.7 (C20 and C60); 129.5 (C2); 130.4 (C30 and C50);
134.2 (C10); 136.6 (C3); 139.9 (C40); 142.7 (C7a); 147.4 (C3a); 161.2
(C1); 166.1 (C5); 168.6 (ArCONR2) ppm.
4.1.3.1. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-N-ethylacetamide (17b). Derivative 17b was obtain-
ed as yellow crystals in 50% yield, mp 182–185 ꢀC.
Anal. Calcd. for C23H24O3NSF: C, 67.42; H, 6.13; N, 3.28. Found: C,
67.41; H, 6.13; N, 3.29.
NMR 1H (200 MHz, CDCl3)
d
: 1.18 (t, J ¼ 7.2 Hz, CONHCH2CH3);
4.1.3.5. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-1-(piperidin-1-yl)ethanone (17f). Derivative 17f was
obtained as yellow crystals in 82% yield, mp 196–198 ꢀC.
2.21 (s, ArCH3); 3.15 (s, SO2CH3); 3.28 (q, J ¼ 7.2 Hz, CONHCH2CH3);
3.51 (s, ArCH2CONHR); 5.58 (br, CONH); 6.60 (td, J ¼ 8.3 Hz, 9.2 Hz
and 2.2 Hz, H6); 6.87 (dd, J ¼ 9.2 Hz and 2.2 Hz, H4); 7.12 (dd,
J ¼ 4.9 Hz and 8.3 Hz, H7); 7.17 (s, C]CH); 7.72 (d, J ¼ 8.1 Hz, H20
and H60); 8.02 (d, J ¼ 8.1 Hz, H30 and H50) ppm.
NMR 1H (200 MHz, CDCl3)
d: 1.52 (m, CON(CH2)2(CH2)3); 2.17 (s,
ArCH3); 3.13 (s, SO2CH3); 3.45 (m, CON(CH2)2(CH2)3); 3.59 (s,
ArCH2CONR2); 6.53 (td, J ¼ 8.1 Hz, 9.2 Hz and 1.9 Hz, H6); 6.90 (dd,
J ¼ 9.2 Hz and 1.9 Hz, H4); 7.08 (dd, J ¼ 5.1 Hz and 8.1 Hz, H7); 7.10
(s, C]CH); 7.69 (d, J ¼ 8.2 Hz, H20 and H60); 7.99 (d, J ¼ 8.1 Hz, H30
and H50) ppm.
Anal. Calcd. for C22H22O3NSF: C, 66.15; H, 5.55; N, 3.51. Found: C,
66.14; H, 5.55; N, 3.49.
4.1.3.2. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-N-propylacetamide (17c). Derivative 17c was
obtained as yellow crystals in 92% yield, mp 182–184 ꢀC.
NMR 13C (50 Hz, CDCl3)
d: 10.7 (ArCH3); 24.6, 25.7 and 26.5
(CON(CH2)2(CH2)3); 31.9 (ArCH2CONR2); 43.3 and 47.2
(CON(CH2)2(CH2)3); 44.7 (SO2CH3); 106.7 (C4); 110.9 (C6); 123.6
(C7); 126.6 (C]CH); 127.7 (C20 and C60); 129.5 (C2); 130.4 (C30 and
C50); 134.1 (C10); 137.0 (C3); 139.9 (C40); 142.7 (C7a); 147.3 (C3a);
161.2 (C1); 166.1 (C5); 167.8 (ArCONR2) ppm.
NMR 1H (200 MHz, CDCl3)
d
: 0.83 (t, J ¼ 7.4 Hz, CONHCH2CH2CH3);
1.46 (m, CONHCH2CH2CH3); 2.21 (s, ArCH3); 3.14 (s, SO2CH3); 3.17 (m,
CONHCH2CH2CH3); 3.51 (s, ArCH2CONHR); 5.80 (br, CONH); 6.60 (td,
J ¼ 8.3 Hz, 9.2 Hz and 2.3 Hz, H6); 6.86 (dd, J ¼ 9.2 Hz and 2.3 Hz, H4);
7.12 (dd, J ¼ 5.1 Hz and 8.3 Hz, H7); 7.16 (s, C]CH); 7.71 (d, J ¼ 8.1 Hz,
H20 and H60); 8.01 (d, J ¼ 8.1 Hz, H30 and H50) ppm.
Anal. Calcd. for C25H26O3NSF: C, 68.32; H, 5.96; N, 3.19. Found: C,
68.31; H, 5.95; N, 3.22.
NMR
13C
(50 Hz,
CDCl3)
d:
10.7
(ArCH3);
11.3
4.1.3.6. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-1-morpholinoethanone (17g). Derivative 17g was
obtained as yellow crystals in 90% yield, mp 198–201 ꢀC.
(CONHCH2CH2CH3); 22.9 (CONHCH2CH2CH3); 33.9 (ArCH2CONHR);
41.58 (CONHCH2CH2CH3); 44.6 (SO2CH3); 106.4 (C4); 111.4 (C6);
123.9 (C7); 127.9 (C]CH, C20 and C60); 129.5 (C2); 130.3 (C30 and
C50); 133.4 (C10); 138.7 (C3); 140.2 (C40); 142.4 (C7a); 146.7 (C3a);
161.2 (C1); 166.2 (C5); 169.1 (ArCONHR) ppm.
NMR 1H (200 MHz, CDCl3)
d: 2.18 (s, ArCH3); 3.14 (s, SO2CH3);
3.54 (m, CON(CH2)2); 3.60 (s, ArCH2CONR2); 3.67 (m, O(CH2)2); 6.62
(td, J ¼ 8.1 Hz, 9.0 Hz and 2.0 Hz, H6); 6.89 (dd, J ¼ 9.0 Hz
and 2.0 Hz, H4); 7.09 (dd, J ¼ 5.2 Hz and 8.1 Hz, H7); 7.11 (s, C]CH);
7.70 (d, J ¼ 8.2 Hz, H20 and H60); 8.01 (d, J ¼ 8.1 Hz, H30 and
H500) ppm.
Anal. Calcd. for C23H24O3NSF: C, 66.81; H, 5.85; N, 3.39. Found: C,
66.80; H, 5.86; N, 3.40.
4.1.3.3. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-N-butylacetamide (17d). Derivative 17d was
obtained as yellow crystals in 91% yield, mp 161–163 ꢀC.
NMR 13C (50 Hz, CDCl3)
d: 10.8 (ArCH3); 31.6 (ArCH2CONR2);
42.6 and 46.5 (CON(CH2)2); 44.7 (SO2CH3); 66.8 (O(CH2)2); 106.5
(C4); 111.1 (C6); 123.8 (C7); 127.0 (C]CH); 127.9 (C200 and C60);
129.5 (C2); 130.4 (C30 and C50); 133.3 (C10); 136.0 (C3); 139.9 (C40);
142.5 (C7a); 147.0 (C3a); 161.2 (C1); 166.1 (C5); 168.4
(ArCONR2) ppm.
NMR 1H (200 MHz, CDCl3)
d:
0.87 (t, J ¼ 7.0 Hz,
CONHCH2CH2CH2CH3); 1.28 (m, CONHCH2CH2CH2CH3); 1.39 (m,
CONHCH2CH2CH2CH3); 2.21 (s, ArCH3); 3.14 (s, SO2CH3); 3.23 (q,
J ¼ 6.6 Hz, CONHCH2CH2CH2CH3); 3.51 (s, ArCH2CONHR); 5.70 (br,
CONH); 6.61 (td, J ¼ 8.6 Hz, 9.0 Hz and 2.3 Hz, H6); 6.86 (dd, J ¼ 9.0 Hz
and 2.3 Hz, H4); 7.11 (dd, J ¼ 5.2 Hz and 8.6 Hz, H7); 7.17 (s, C]CH); 7.71
(d, J ¼ 8.0 Hz, H20 and H60); 8.02 (d, J ¼ 8.0 Hz, H30 and H50) ppm.
Anal. Calcd. for C24H24O4NSF: C, 65.29; H, 5.48; N, 3.17. Found: C,
65.27; H, 5.49; N, 3.19.
4.1.3.7. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-1-thiomorpholinoethanone (17h). Derivative 17h
was obtained as yellow crystals in 71% yield, mp 196–198 ꢀC.
NMR 13C (50 Hz, CDCl3)
d: 10.7 (ArCH3); 13.8 (CONHCH2-
CH2CH2CH3); 20.1 (CONHCH2CH2CH2CH3); 31.7 (CONHCH2CH2CH2-
CH3); 33.9 (ArCH2CONHR); 39.7 (CONHCH2CH2CH2CH3); 44.6
(SO2CH3); 106.4 (C4); 111.4 (C6); 124.0 (C7); 127.7 (C]CH, C20 and
C60); 129.5 (C2); 130.3 (C30 and C50); 133.4 (C10); 138.6 (C3); 140.2
(C40); 142.4 (C7a); 146.7 (C3a); 161.2 (C1); 166.2 (C5); 169.0
(ArCONHR) ppm.
NMR 1H (200 MHz, CDCl3)
d: 2.18 (s, ArCH3); 2.56 (m, S(CH2)2);
3.13 (s, SO2CH3); 3.60 (s, ArCH2CONR2); 3.84 (m, CON(CH2)2);
6.56 (td, J ¼ 8.1 Hz, 9.0 Hz and 2.1 Hz, H6); 6.91 (dd, J ¼ 9.0 Hz
and 2.1 Hz, H4); 7.09 (dd, J ¼ 5.2 Hz and 8.1 Hz, H7); 7.11 (s, C]CH);
7.69 (d, J ¼ 8.1 Hz, H20 and H60); 8.00 (d, J ¼ 8.1 Hz, H30 and
H50) ppm.
Anal. Calcd. for C23H24O3NSF: C, 67.42; H, 6.13; N, 3.28. Found: C,
67.44; H, 6.15; N, 3.29.
NMR 13C (50 Hz, CDCl3)
d: 10.8 (ArCH3); 27.4 (S(CH2)2); 32.3
(ArCH2CONR2); 44.7 (SO2CH3); 44.9 and 48.9 (CON(CH2)2); 106.7
(C4); 111.1 (C6); 123.8 (C7); 127.0 (C]CH); 127.7 (C20 and C60);
129.5 (C2); 130.4 (C30 and C50); 133.3 (C10); 137.5 (C3); 140.1 (C40);
142.5 (C7a); 147.0 (C3a); 161.2 (C1); 166.1 (C5); 168.1
(ArCONR2) ppm.
4.1.3.4. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-N-diethylacetamide (17e). Derivative 17e was
obtained as yellow crystals in 75% yield, mp 68–70 ꢀC.
NMR 1H (200 MHz, CDCl3)
d: 1.18 (m, CON(CH2CH3)2); 2.17 (s,
ArCH3); 3.13 (s, SO2CH3); 3.4 (m, CON(CH2CH3)2); 3.59 (s, ArCH2
CONR2); 6.53 (td, J ¼ 8.0 Hz, 9.2 Hz and 2.1 Hz, H6); 6.90 (dd,
Anal. Calcd. for C24H24O3NS2F: C, 63.00; H, 5.29; N, 3.06. Found:
C, 63.01; H, 5.29; N, 3.09.
-

N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
1969
4.1.3.8. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-methyl-
1-H-inden-3-yl)-N-phenylacetamide (17i). Derivative 17i was
obtained as yellow crystals in 94% yield, mp 220 ꢀC (with
decomposition).
[54]. Albino mice of both sexes (18–23 g) were used. Compounds
were administered orally (300 mol/kg) as a suspension in 5%
Arabic gum in saline (vehicle). Sulindac (300 mol/kg) was used as
m
m
standard drug under the same conditions. Acetic acid solution was
administered i.p. 1 h after administration of the compounds. 10 min
after the i.p. acetic acid injection the number of constrictions per
animal was recorded for 20 min. Control animals received an equal
volume of vehicle. Antinociceptive activity was expressed as % of
inhibition of constrictions when compared with the vehicle control
group. Results are expressed as mean ꢁ SEM of n animals per group.
The data were statistically analyzed by the Student’s t-test for
NMR 1H (200 MHz, DMSO d6)
d: 2.22 (s, ArCH3); 3.28 (s, SO2CH3);
3.68 (s, ArCH2CONR2); 6.71 (td, J ¼ 8.0 Hz, 9.0 Hz and 2.2 Hz, H6); 7.04
(dd, J ¼ 9.0 Hz and 2.2 Hz, H4); 7.09 (dd, J ¼ 5.2 Hz and 8.0 Hz, H7); 7.11
(s, C]CH); 7.29 (m, H300, H400 and H500); 7.59 (m, H200 and H600); 7.78 (d,
J ¼ 8.3 Hz, H20 and H60); 8.01 (d, J ¼ 8.3 Hz, H30 and H50) ppm.
NMR 13C (50 Hz, DMSO d6)
d: 11.0 (ArCH3); 34.1 (ArCH2
-
CONHPh); 44.1 (SO2CH3); 107.2 (C4); 111.1 (C6); 119.8 (C200 and
C600); 123.8 (C7); 124.0 (C400); 127.5 (C]CH); 127.8 (C20 and C60);
129.3 (C300 and C500); 129.5 (C2); 130.6 (C30 and C50); 134.4 (C10);
138.7 (C3); 139.7 (C40); 140.8 (C100); 142.2 (C7a); 147.9 (C3a); 160.9
(C1); 165.7 (C5); 168.4 (ArCONR2) ppm.
*
a significance level of p < 0.05.
4.2.2. Anti-inflammatory activity
The anti-inflammatory activity was determined in vivo by the
carrageenan-induced rat paw edema test [54]. Fasted albino rats of
both sexes (150–200 g) were used. Test compounds and the anti-
inflammatory standard drugs sulindac and celecoxib were admin-
Anal. Calcd. for C26H22O3NSF: C, 69.78; H, 4.95; N, 3.13. Found: C,
69.79; H, 4.96; N, 3.15.
4.1.4. 2-((1Z)-1-(4-(Methylsulfonyl)benzylidene)-5-fluoro-2-
methyl-1-H-inden-3-yl)-N-methylacetamide (17a)
istered orally at a dose of 300 mmol/kg as a suspension in 5% Arabic
gum in saline (vehicle). Control animals received an equal volume
of vehicle. 1 h later, the animals were then injected with either
0.1 mL of 1% carrageenan solution in saline (0.1 mg/paw) or sterile
saline (NaCl 0.9%) into the subplantar surface of one of the hind
paw, respectively. The paw volumes were measured using a glass
plethysmograph coupled to a peristaltic pump, 3 h after the sub-
plantar administration of carrageenan. The edema was calculated
as the volume variation between the injected paw with carra-
geenan and the saline-treated paw. The anti-inflammatory activity
was expressed as % of inhibition of the edema when compared with
vehicle control group. Results are expressed as mean ꢁ SEM of n
animals per group. The data were statistically analyzed by the
Aminolysis was carried out by mixing ester 19 (1 mmol) with
aqueous methylamine 40% (30 mmol). The reaction mixture was
heated for 72 h at 50 ꢀC. Then, it was cooled, added cold water
(50 mL) and the resulting solid was collected and purified by
column chromatography on silica gel (CH2Cl2 and CH2Cl2:MeOH
1%). Derivative 17a was obtained as yellow crystals in 45% yield, mp
206–208 ꢀC.
NMR 1H (200 MHz, DMSO d6)
d: 2.17 (s, ArCH3); 2.58 (s,
ArCONHCH3); 3.28 (s, SO2CH3); 3.40 (s, ArCH2CONHR); 5.80 (br,
CONH); 6.70 (td, J ¼ 8.3 Hz, 9.0 Hz and 2.1 Hz, H6); 6.95 (dd,
J ¼ 9.0 Hz and 2.1 Hz, H4); 7.13 (dd, J ¼ 5.2 Hz and 8.3 Hz, H7); 7.33
(s, C]CH); 7.77 (d, J ¼ 8.0 Hz, H20 and H60); 8.01 (d, J ¼ 8.0 Hz, H30
and H50) ppm.
*
Student’s t-test for a significance level of p < 0.05.
NMR 13C (50 Hz, DMSO d6)
d
: 10.9 (ArCH3); 26.3 (CONHCH3);
4.2.3. Gastric ulcerogenic potential
33.2 (ArCH2CONHCH3); 43.9 (SO2CH3); 106.8 (C4); 110.8 (C6); 123.6
(C7); 128.9 (C]CH); 127.4 (C20 and C60); 129.8 (C2); 130.1 (C30 and
C50); 134.6 (C10); 138.2 (C3); 140.6 (C40); 141.6 (C7a); 147.9 (C3a);
160.7 (C1); 165.6 (C5); 169.6 (ArCONHCH3) ppm.
Anal. Calcd. for C21H20O3NSF: C, 65.44; H, 5.23; N, 3.63. Found: C,
65.45; H, 5.23; N, 3.65.
Ulcerogenic effects were investigated as described earlier [52] in
the same animals that were submitted to the anti-inflammatory
evaluation. Briefly, animals were euthanized at the end of the
experiments and the stomachs excised along its greater curvature
for visualization of gastric lesions with a stereomicroscope.
4.3. Molecular modeling
4.1.5. 2-((1Z)-1-(4-(Methylsulfinyl)benzylidene)-5-fluoro-2-
methyl-1-H-inden-3-yl)-N-methylacetamide (20)
4.3.1. Selection of protein crystal structures
Derivative 20 was obtained in two steps (Fischer esterification
and aminolysis; see Sections 4.1.2 and 4.1.4), as yellow crystals in
50% overall yield, mp 150–152 ꢀC.
Ligand-bound crystallographic structures of COX-1 and COX-2
are available in the Protein Data Bank [63]. In this study, COX-1
crystal structures 1PXX, 3PGH, 1Q4G, 1HT8 and COX-2 crystal
structures 1CX2, 4COX, 6COX were evaluated and selected for
docking [44–48].
NMR 1H (200 MHz, DMSO d6)
d: 2.20 (s, ArCH3); 2.76 (d,
J ¼ 4.8 Hz, ArCONHCH3); 2.80 (s, SOCH3); 3.51 (s, ArCH2CONHR);
5.77 (br, CONH); 6.58 (td, J ¼ 8.1 Hz, 9.0 Hz and 2.1 Hz, H6); 6.84
(dd, J ¼ 9.0 Hz and 2.1 Hz, H4); 7.16 (dd, J ¼ 5.2 Hz and 8.1 Hz, H7);
7.65 (s, C]CH); 7.72 (d, J ¼ 8.0 Hz, H20 and H60); 7.78 (d, J ¼ 8.0 Hz,
H30 and H50) ppm.
4.3.2. Preparation of the ligands
The preparation of the ligands for FLExE was performed as for
FLExX [64] using Sybyl version 7.3 [65]. First, the ligand coordinates
were generated using the program Sketcher, available in Sybyl
version 7.3. Next, the correct atom types (including hybridization
states) and correct bond types were defined, hydrogen atoms were
added, charges were assigned to each atom, and finally the struc-
tures were energy-minimized. All carboxylic acid groups were
modeled in their ionized forms. The energies of ligand structures
were previously minimized using the semiempirical AM1 method
[66] with PC Spartan v.04 software [67]. PM3 point charges were
assigned to the ligands [58].
NMR 13C (50 Hz, DMSO d6)
d: 10.7 (ArCH3); 26.7 (CONHCH3);
33.8 (ArCH2CONHCH3); 44.0 (SOCH3); 106.3 (C4); 111.3 (C6); 123.9
(C7); 128.8 (C]CH); 124.0 (C20 and C60); 129.7 (C2); 130.4 (C30 and
C50); 132.8 (C10); 139.1 (C3); 141.7 (C7a); 145.8 (C40); 146.7 (C3a);
161.2 (C1); 166.1 (C5); 169.9 (ArCONHCH3) ppm.
Anal. Calcd. for C21H20O2NSF: C, 68.27; H, 5.46; N, 3.79. Found: C,
68.26; H, 5.47; N, 3.75.
4.2. Pharmacology
4.2.1. Antinociceptive activity
4.3.3. Preparation of the ensemble protein structures
The analgesic activity was determined in vivo by the acetic acid-
induced (0.6%; 0.1 mL/10 g) abdominal constriction test in mice
Proteins were prepared for the docking studies using the
Biopolymer module of Sybyl 7.3. Amber7 FF99 charges were

1970
N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
[19] D. Riendeau, M.D. Percival, C. Brideau, S. Charleson, D. Dube´, D. Ethier,
J.P. Falgueyret, R.W. Friesen, R. Gordon, G. Greig, J. Pharmacol. Exp. Ther. 296
(2001) 558–566.
[20] J.M. Dogne, C.T. Supuran, D. Pratico, J. Med. Chem. 48 (2005) 2251–2257.
[21] D.H. Solomon, Arthritis Rheum. 52 (2005) 1968–1978.
[22] B. Arun, P. Goss, Semin. Oncol. 2 (2004) 22–29.
attributed to the protein atoms [68]. The first step in the generation
of suitable protein structures for the ensemble superimposition is
the deletion of extra chains. In this work, chains A were kept while
the others were deleted. Next, Biopolymer protein analysis tool was
used, in a stepwise process of analysis and correction of geometry
parameters. For each structure, the description of an ensemble
contains the definition of the protein atoms, the resolution of
ambiguities in the PDB file, the location of hydrogen atoms at
hetero atoms, and the definition of the active site atoms. The
assignment of hydrogen positions has been made on the basis of
default rules. The side-chains of lysine, arginine and the carbox-
ylate groups of aspartic and glutamic acids have been modeled in
their ionized states. Water molecules contained in the PDB file have
been removed. Finally, the active site of the ensemble has been
defined as the collection of residues within 10.0 Å of the bound
inhibitor and comprised the union of all ligands of the ensemble. All
atoms located less than 10.0 Å from any ligand atom were consid-
ered. 1PXX was used as a reference structure for the united protein
preparation in COX-1 studies, while 1CX2 was used as a reference in
COX-2 studies.
[23] L.R. Howe, Breast Cancer Res. 9 (2007) 210.
[24] R.E. Harris, J. Beebe-Donk, G.A. Alshafie, BMC Cancer 6 (2006) 27–31.
[25] R.E. Harris, J. Beebe-Donk, H. Doss, D. Burr Doss, Oncol. Rep. 13 (2005)
559–583.
[26] D.W. McFadden, D.R. Riggs, B.J. Jackson, C. Cunningham, Int. J. Oncol. 29 (2006)
1019–1023.
[27] M.M. Taketo, J. Natl. Cancer Inst. 90 (1998) 1529–1536.
[28] M.M. Taketo, J. Natl. Cancer Inst. 90 (1998) 1609–1620.
[29] N. Babbar, N.A. Ignatenko, R.A. Casero Jr., E.W. Gerner, J. Biol. Chem. 278 (2003)
47762–47775.
[30] G.A. Piazza, D.S. Alberts, L.J. Hixson, N.S. Paranka, H. Li, T. Finn, C. Bogert,
J.M. Guillen, K. Brendel, P.H. Gross, G. Sperl, J. Ritchie, R.W. Burt, L. Ellsworth,
D.J. Ahnen, R. Pamukcu, Cancer Res. 57 (1997) 2909–2915.
[31] A.S. Kalgutkar, A.B. Marnett, B.C. Crews, R.P. Remmel, L.J. Marnett, J. Med.
Chem. 43 (2000) 2860–2870.
[32] A.S. Kalgutkar, B.C. Crews, S. Saleh, D. Prudhomme, L.J. Marnett, Bioorg. Med.
Chem. 13 (2005) 6810–6822.
[33] S. Khanna, M. Madan, A. Vangoori, R. Banerjee, R. Thaimattam, S.K. Jafar Sadik
Basha, M. Ramesh, S.R. Casturi, M. Pal, Bioorg. Med. Chem. 14 (2006)
4820–4833.
[34] A.S. Kalgutkar, B.C. Crews, S.W. Rowlinson, A.B. Marnett, K.R. Kozak,
R.P. Remmel, L.J. Marnett, Proc. Natl. Acad. Sci. USA 97 (2000) 925–930.
[35] A.S. Kalgutkar, S.W. Rowlinson, B.C. Crews, L.J. Marnett, Bioorg. Med. Chem.
Lett. 12 (2002) 521–524.
[36] E.J. Barreiro, M.E. Lima, Braz. J. Med. Biol. Res. 22 (1989) 1415–1419.
[37] E. Gourzoulidou, M. Carpintero, P. Baumhof, A. Giannis, H. Waldmann,
ChemBioChem 6 (2005) 527–531.
4.3.4. Lipinski’s rule of five
CLOGP, the log of the octanol/water partition coefficient, was
calculated with the CLOGP software [62]. PSA and molecular weight
calculations have been performed with the MOLPROP facility,
available in the QSAR module of Sybyl 7.3 [65].
[38] D.E. Duggan, K.F. Hooke, S.S. Hwang, Drug Metab. Dispos.
241–246.
8 (1980)
[39] J.T. Lim, G.A. Piazza, E.K. Han, T.M. Delohery, H. Li, T.S. Finn, R. Buttyan,
H. Yamamoto, G.J. Sperl, K. Brendel, P.H. Gross, R. Pamukcu, I.B. Weinstein,
Biochem. Pharmacol. 58 (1999) 1097–1107.
Acknowledgements
[40] J.B. Nixon, H. Kamitanib, S.J. Baeka, T.E. Eling, Prostaglandins Leukot. Essent.
Fatty Acids 68 (2003) 323–330.
The authors wish to thank IM-INOFAR (#420.015/2005-1, Br),
PRONEX (Br), CNPq (Br), FAPERJ (Br), FUJB (Br) for financial support
and fellowships (to ALPM, EJB, CAMF and NCR).
[41] Y. Niitsu, T. Takayama, K. Miyanishi, A. Nobuoka, T. Hayashi, T. Kukitsu,
K. Takanashi, H. Ishiwatari, T. Abe, T. Kogawa, M. Takahashi, T. Matsunaga,
J. Kato, Cancer Chemother. Pharmacol. 54 (2004) S40–S43.
[42] K. Gwyn, F.A. Sinicrope, Am. J. Gastroenterol. 97 (2002) 13–21.
[43] B. Jung, V. Barbier, H. Brickner, J. Welsh, A. Fotedar, M. McClelland, Cancer Lett.
219 (2005) 15–25.
References
[44] S.W. Rowlinson, J.R. Kiefer, J.J. Prusakiewicz, J.L. Pawlitz, K.R. Kozak,
A.S. Kalgutkar, W.C. Stallings, R.G. Kurumbail, L.J. Marnett, J. Biol. Chem. 278
(2003) 45763–45769.
[45] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak,
D. Gildehaus, J.M. Miyashiro, T.D. Penning, K. Seibert, P.C. Isakson,
W.C. Stallings, Nature 384 (1996) 644–648.
[1] J.R. Vane, Nature 231 (1971) 232–235.
[2] P.M. Brooks, R.O. Day, New Engl. J. Med. 324 (1991) 1716–1725.
[3] J.Y. Fu, J.L. Masferrer, K. Seibert, A. Raz, P.J. Needleman, Biol. Chem. 265 (1990)
16737–16740.
[4] W. Xie, J.G. Chipman, D.L. Robertson, R.L. Erikson, D.L. Simmons, Proc. Natl.
Acad. Sci. 88 (1991) 2692–2696.
[46] K. Gupta, B.S. Selinsky, C.J. Kaub, A.K. Katz, P.J. Loll, J. Mol. Biol. 335 (2004)
503–518.
[5] J.A. Mitchell, P. Akarasereenont, G. Thiemermann, R.J. Flower, J.R. Vane, Proc.
Natl. Acad. Sci. 90 (1993) 11693–11697.
[6] J.R. Vane, R.M. Botting, Inflamm. Res. 44 (1995) 1–10.
[47] B.S. Selinsky, K. Gupta, C.T. Sharkey, P.J. Loll, Biochemistry 40 (2001)
5172–5180.
[48] P.J. Loll, D. Picot, O. Ekabo, R.M. Garavito, Biochemistry 35 (1996)
7330–7340.
[7] D.L. Simmons, R.M. Botting, T. Hla, Pharmacol. Rev. 56 (2004) 387–437.
[8] D.E. Duggan, K.F. Hooke, E.A. Risley, T.Y. Shen, C.G. Arman, Pharmacol. Exp.
Ther. 201 (1977) 8–13.
[49] H. Clau
ben, C. Buning, M. Rarey, T. Lengauer, J. Mol. Biol. 308 (2001)
[9] W.E. Sneader, Br. Med. J. 321 (2000) 1591–1594.
377–395.
[10] E.A. Meade, W.L. Smith, D.L. DeWitt, J. Biol. Chem. 268 (1993) 6610–6614.
[11] T.Y. Shen, R.L. Ellis, T.B. Windholz, A.R. Matzuk, A. Rosegay, S. Lucas, B.E. Witzel,
C.H. Stammer, A.N. Wilson, F.W. Holly, J.B. Willett, L.H. Sarett, W.J. Holtz,
C.A. Winter, E.A. Risley, G.W. Nuss, J. Am. Chem. Soc. 85 (1963) 488–489.
[12] A. Zarghi, P.N.P. Rao, E.E. Knaus, Bioorg. Med. Chem. 15 (2007) 1056–1061.
[13] M. Biava, G.C. Porretta, G. Poce, S. Supino, S. Forli, M. Rovini, A. Cappelli,
F. Manetti, M. Botta, L. Sautebin, A. Rossi, C. Pergola, C. Ghelardini, E. Vivoli,
F. Makovec, P. Anzellotti, P. Patrignani, M. Anzini, J. Med. Chem. 50 (2007)
5403–5411.
[50] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46
(2001) 3–25.
[51] B.M. Trost, D.P. Curran, Tetrahedron Lett. 22 (1981) 1287–1290.
[52] T. Barf, J. Vallgarda, R. Emond, C. Ha¨ggstro¨m, E. Mosialou, K. Axelsson,
¨
R. Olsson, L. Engblom, N. Edling, Y. Ro¨nquist-Nii, B. Ohman, P. Alberts,
L. Abrahmse´n, J. Med. Chem. 45 (2002) 3813–3815.
[53] N.D. Karis, W.A. Loughlin, I.D. Jenkins, Tetrahedron 63 (2007)
12303–12309.
[54] I.G. Ribeiro, K.C.M. Silva, S.C. Parrini, A.L.P. Miranda, C.A.M. Fraga, E.J. Barreiro,
Eur. J. Med. Chem. 33 (1998) 225–235.
[55] C.C. Chan, S. Boyce, C. Brideau, A.W. Fordhutchinson, R. Gordon, D. Guay,
R.G. Hill, C.S. Li, J. Mancini, M. Penneton, J. Pharmacol. Exp. Ther. 274 (1995)
1531–1537.
[56] L.M. Lima, E.J. Barreiro, Curr. Med. Chem. 12 (2005) 23–49.
[57] V. Quidville, N. Segond, S. Lausson, M. Frenkian, R. Cohen, A. Jullienne, Pros-
taglandins Other Lipid Mediat. 81 (2006) 14–30.
[58] J.J.P. Stewart, J. Comput. Chem. 10 (1989) 209–220.
[59] R. Pouplana, J.J. Lozano, C. Pe´rez, J. Ruiz, J. Comput. Aided Mol. Des. 16 (2002)
683–709.
[14] W.C. Black, C. Brideau, C.-C. Chan, S. Charleson, N. Chauret, D. Claveau,
D. Ethier, R. Gordon, G. Greig, J. Guay, G. Hughes, P. Jolicoeur, Y. Leblanc,
D. Nicoll-Griffith, N. Ouimet, D. Riendeau, D. Visco, Z. Wang, L. Xu, P. Prasit, J.
Med. Chem. 42 (1999) 1274–1281.
[15] F. Julemont, X. de Leval, C. Michaux, J.-F. Renard, J.-Y. Winum, J.-L. Montero,
J. Damas, J.-M. Dogne, B. Pirotte, J. Med. Chem. 47 (2004) 6749–6759.
[16] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter,
M.J. Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier,
S.S. Yu, G.D. Anderson, E.G. Burton, J.N. Cogburn, S.A. Gregory, C.M. Koboldt,
W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, J. Med.
Chem. 40 (1997) 1347–1365.
[60] J.R. Kiefer, J.L. Pawlitz, K.T. Moreland, R.A. Stegeman, W.F. Hood, J.K. Gierse,
A.M. Stevens, D.C. Goodwin, S.W. Rowlinson, L.J. Marnett, W.C. Stallings,
R.G. Kurumbail, Nature 405 (2000) 97–101.
[61] D.E. Clark, S.D. Pickett, DDT 5 (2000) 49–57.
[62] J. Chow, P. Jurs, J. Chem. Inf. Comput. Sci. 19 (1979) 172–178.
[17] X. De Leval, F. Julemont, V. Benoit, M. Frederich, B. Pirotte, J.M. Dogne, Mini
Rev. Med. Chem. 4 (2004) 597–601.
[18.] J.J. Talley, D.L. Brown, J.S. Carter, M.J. Graneto, C.M. Koboldt, J.L. Masferrer,
W.E. Perkins, R.S. Rogers, A.F. Shaffer, Y.Y. Zhang, B.S. Zweifel, K. Seibert, J.
Med. Chem. 43 (2000) 775–777.

N.C. Romeiro et al. / European Journal of Medicinal Chemistry 44 (2009) 1959–1971
1971
[63] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res. 28 (2000) 235–242.
[64] M. Rarey, B. Kramer, T. Lengauer, G.A. Klebe, J. Mol. Biol. 261 (1996)
470–489.
[65] SYBYL 7.3 ed., Tripos Inc., 1699 South Hanley Rd., St. Louis, Missouri, 63144,
USA; http://www.tripos.com.
[67] (a) Spartan´ 04; Wavefunction Inc., 18401 Von Karman Avenue, Suite 370.
Irvine, California 92612, USA.
(b) PC SPARTAN Pro User‘s Guide, Wavefunction Inc., California.
[68] D.A. Case, D.A. Pearlman, J.W. Caldwell, T.E. Cheatham III, J. Wang, W.S. Ross,
C.L. Simmerling, T.A. Darden, K.M. Merz, R.V. Stanton, A.I. Cheung, J.J. Vincent,
M. Crowley, V. Tsui, H. Gohike, R.J. Radmer, Y. Duan, J. Pitera, I. Massova,
G.L. Seibel, U.C. Singh, P.K. Weiner, P.A. Kollman, Amber 7, University of Cal-
ifornia, San Francisco, 2002.
[66] M.J.S. Dewar, E.G. Zoebisch, E.F. Healy, J.J.P. Stewart, J. Am. Chem. Soc. 107
(1985) 3902–3909.