Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid X Receptor (FXR) agonists

Article abstract of DOI:10.1016/j.bmc.2012.04.021

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 μM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7- one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.

Full text of DOI:10.1016/j.bmc.2012.04.021

Bioorganic & Medicinal Chemistry 20 (2012) 3429–3445
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid
X Receptor (FXR) agonists
Maura Marinozzi a, , Andrea Carotti a, Emanuele Sansone a, Antonio Macchiarulo a, Emiliano Rosatelli a,
⇑
Roccaldo Sardella a, Benedetto Natalini a, Giovanni Rizzo b, Luciano Adorini b, Daniela Passeri b,
Francesca De Franco b, Mark Pruzanski c, Roberto Pellicciari a
a Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Via del Liceo, I-06123 Perugia, Italy
b Intercept Pharmaceuticals Italia Srl, Via Togliatti, I-06073 Corciano, Italy
c Intercept Pharmaceuticals, 18 Desbrosses Street, New York, NY 10013, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 February 2012
Revised 2 April 2012
Accepted 7 April 2012
Available online 20 April 2012
A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid
X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds
were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 lM.
1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization.
A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and
evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most
of them exhibited low micromolar range of potency and very high efficacy.
Keywords:
Farnesoid X Receptor (FXR)
Non-steroidal FXR agonist
Virtual screening
Ó 2012 Elsevier Ltd. All rights reserved.
1H-Pyrazole[3,4-e][1,4]thiazepin-7-one
Heterocyclic chemistry
1. Introduction
and glucose and lipids metabolism.7 FXR has also been suggested
to counteract pro-inflammatory and pro-atherogenic responses in
Farnesoid X Receptor (FXR) belongs to a family of nuclear recep-
tors that are ligand-inducible transcription factors. FXR is highly
expressed in the liver and intestine and is activated by bile acids,
such as chenodeoxycholic acid (CDCA) and cholic acid (CA), and
their corresponding conjugates, at physiological concentrations.1–
cardiovascular diseases.8 Moreover, the bile acid-FXR interaction
plays a pivotal role in regulating liver inflammation and regenera-
tion as well as in regulating extent of inflammatory responses, bar-
rier function and prevention of bacterial translocation in the
intestinal tract.
3
Activated FXR binds with high affinity to an inverted repeat-1
All these evidences make FXR a promising potential target for
the treatment of a variety of metabolic disorders, including hyper-
lipidemia, cholelithiasis, cholestasis, and diabetes mellitus.9
Over the past few years, many efforts have been dedicated to
the search of highly potent steroidal- and non-steroidal FXR
modulators ( Fig. 1). Based on structural modification of CDCA,
(IR-1) response element on DNA as a heterodimer with the retinoid
X receptor (RXR) regulating in such a way the expression of various
transport proteins and biosynthetic enzymes that maintain choles-
terol and bile acid homeostasis.4 Indeed, activation of FXR by bile
acids or synthetic agonists results in (a) transcriptional repression
of cholesterol 7
a
-hydroxylase (CYP7A1), the rate-limiting enzyme
our group reported in 2002, 6a-ethylchenodeoxycholic acid
in the bile acid biosynthesis pathway, (b) induction of the small
heterodimer partner (SHP), a transcriptional repressor found in
the liver and intestine, and (c) induction of genes encoding for
some bile acid transport proteins, such as intestinal bile acid-
binding protein (IBABP)5 and bile salt export pump (BSEP).6 Fur-
thermore, bile acids-mediated FXR activation has been recently
recognized as a major underlying pathway for energy homeostasis
(obeticholic acid, INT-747, 1) as a highly potent and orally available
FXR full agonist.10 Positive data from two phase II clinical trials
using INT-747 (1), recently demonstrated the clinical utility of this
compound in the treatment of primary biliary cirrhosis, a chronic
inflammatory cholestatic condition in the liver, and type
diabetes.
2
In addition to steroidal FXR agonist, such as INT-747 (1) and
MFA-1 (2),11 a number of synthetic non-steroidal FXR agonists
have been reported in the literature in last decade.
⇑
Corresponding author. Tel.: +39 075 5855159; fax. +39 075 585 5161.
E-mail address: maura@chimfarm.unipg.it (M. Marinozzi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.021

3430
M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
CO2H
O
OH
HO
OH
HO2C
MFA-1
2
6ECDCA, INT-747
1
O
CO2H
N
Cl
Cl
O
N
CO2CH3
Cl
N
O
GW 4064
fexaramine
3
4
O
N
O
F
F
O
HN
N
H
O
O
O
N
XL 335
6
5
Figure 1. Structures of selected steroidal- and non-steroidal FXR agonists.
GW4064 (3) was the first high affinity non-steroidal FXR ago-
nist to be reported in 2000 by Maloney et al.12 Although this com-
pound suffered from the presence in its structure of a potential
toxicophore such as the trans-stilbene moiety, that is also respon-
sible for UV light instability of the compound, and from a poor oral
bioavailability, 3 was the first chemical tool to demonstrate potent
FXR binding and activation in biochemical and cellular in vitro as-
says as well as pharmacological effects in different rodent models
of metabolic diseases. Also the optimization of initial hits identified
by the screening of natural product-like libraries provided a num-
ber of potent FXR agonists, fexaramine (4), in particular.13 Another
recent example is the identification of bioactive triterpenes from
Ganoderma lucidum.14,15
Since these first reports a large amount of work has been fo-
cused towards the identification of novel non-steroidal FXR ago-
nists whether by the modification of the prototype GW4064 (3)
or by the exploration of structurally different scaffolds. Following
the first approach, the structural modifications were focused on
the replacement of the stilbene side-chain of the prototype,16 as
well as around the 3-position of the isooxazole ring, that repre-
sents the heterocyclic core of the compound.17 Whereas the search
for novel structurally diverse FXR agonists resulted in the identifi-
cation of a novel pyrazoline-3,5-dione-based scaffold hit com-
pound 518 and in a novel series of azepino[4,5-b]indoles leading
up to the clinical candidate XL335 (WAY-362450, FXR-450, 6).19
Starting from a virtual screening study that successfully has led
to identify novel non-steroidal classes of FXR modulators, herein
we report the synthetic efforts aimed to optimize the activity of
the pyrazole[3,4-e][1,4]thiazepin-7-one-based class of compounds.
While instrumental to depict structure–activity relationships with-
in this class of FXR ligands, the results have allowed to disclose
s( )24, s( )37 and s( )38 as novel FXR agonists in low micromolar
range of potency and with very high efficacy.
2. Results and discussion
2.1. Virtual screening
A virtual screening experiment has been carried out using the
crystal structures of FXR in complex with agonists. In particular,
a two-step approach was adopted for this part of the study, using
firstly a ligand-based filtering and then a structure-based screen-
ing. The ligand-based filtering was carried out by developing a
pharmacophoric model of the ligand-target interactions observed
in five selected X-ray structures of FXR (pdb codes: 3BEJ,11
1OSH,13 1OSV,10 3DCT,16 and 3DCU16) as detailed in the method

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3431
section. The particularity of our approach, with respect to others
already published,14,15 resides in the contemporary usage of all
the ligands resumed in a ‘supermolecule’ coming from X-ray com-
plexes and use it as starting point for the features’ mapping. As a
result, the pharmacophore model was composed of 13 interaction
points, including 2 hydrogen bond acceptors, 1 hydrogen bond do-
nor, 7 aromatic points, 1 hydrophobic centre, 2 negative charged
points (Fig. 2A). Excluded volumes were also added using the res-
idues of the aligned binding site as detailed in the method section
(Fig. 2B).
H3CO
A
OCH3
S
N
N
O
N
H
B
Then, three million of commercially available compounds from
the DrugLike (2 million) and the LeadLike (1 million) subsets of the
ZINC 8 database20 were funnelled through the pharmacophoric
model. We decided to use the ZINC database, with respect to other
data sources, to manage a single comprehensive repository cover-
ing nearly 45 compound vendors, furthermore already divided in
‘ready to use’ subsets with LeadLike and DrugLike properties. Thus,
760 compounds (630 from the Druglike dataset and 130 from the
Leadlike dataset) were identified as virtual hits, matching a mini-
mum number of 7 interaction sites out of 13. These compounds
were further submitted to a structure-based screening, consisting
in a cross-docking of the molecules into three FXR structures with
two different programs, namely GLIDE 5.521 in extra precision dock-
ing mode (XP) and Leadfinder22 in docking mode, as detailed in the
method section (pdb codes: 3BEJ,11 1OSV,10 3DCT16). The aim of
using different programs and crystal structures was firstly to over-
come the limits of the single programs in the parametrization of
the interactions and secondly, to better cover the receptor flexibil-
ity on the side-chains of the binding site. From the resulting list of
docking scores, we selected those compounds endowed with scor-
ing values better than those of the co-crystallized FXR ligands used
for the pharmacophore generation. Accordingly, the selected vir-
tual hits were 263 from GLIDE docking and 256 from Leadfinder
docking, with 178 the compounds being present in both scoring
lists. The removal of the ligands with more than two chiral centres
and duplicates led to a final set of 328 virtual hits. To select the
most chemically diverse compounds, a clustering analysis with a
Tanimoto similarity factor23 of 0.8 was performed on the resulting
hits, and 149 representative compounds were selected for purchas-
ing. Among them, 117 molecules were successfully retrieved and
tested by AlphaScreen assay.24 As a result, 47 active hit compounds
were identified, with 34 of them showing activity below a concen-
7
2.2. Hit optimization
Starting from the hit 7, we planned a chemical strategy with the
aim of exploring substitution patterns of rings A and B. Although
the main goal was to optimize both potency and efficacy of 7, a
second objective was the development of a structure–activity
relationship (SAR) scheme for this class of compounds and use this
information to infer a putative binding mode of 7 into the receptor.
Accordingly,
a first series of dihydro-1H-pyrazolo[3,4-e][1,4]
thiazepin-7-one derivatives were synthesized and evaluated for
their agonistic activity at FXR. As shown in Table 1, in this series
we explored the effects of modifications on the aromatic ring A
of 7 by the synthesis of derivatives 8–34 (series I).
In agreement with virtual screening results, the anti-couples
proved to be less active than the corresponding syn-ones demon-
strating that the relative disposition of the substituents is a crucial
parameter for the activity. Accordingly, we will only comment on
the biological results of the syn-couples. As shown in Table 1, many
of the compounds belonging to the first series (15 out of 24)
showed agonistic activity higher than that of the hit 7. In particu-
lar, the initial replacement of the 2,4-dimethoxy substituent with
4-biphenyl group was found to increase the FXR potency ꢀ10-fold,
although a concomitant reduction in efficacy was observed [cf.
s( )8 vs 7].
The introduction of substituents on the distal phenyl group of
s( )8 afforded different results depending from position and sub-
stituent properties. meta- and para-Substitutions by carboxylic
acid and ester [s( )13–16]) as well as by styryl moiety caused a
completely loss of activity. The presence of a methoxy group in
ortho- [s( )9], meta- [s( )10] and para-positions [s( )11] was not
influential for the activity with respect to s( )8. The replacement
of the distal phenyl group of s( )8 by heterocycles, such as 2-
and 3-thiophenes [s( )18 and s( )19, respectively], 2- and 3-fur-
anes [s( )20 and s( )21, respectively] and 2-benzofurane [s( )22]
was more or less neutral in terms of EC50, whereas resulted in a
tration of 20 lM. The analysis of these compounds involved several
criteria, including chemical novelty, synthetic versatility, solubility
and chemical stability. After such analysis, 3,6-dimethyl-1-(2-
methylphenyl)-4-(2,4-dimethoxyphenyl)-4,8-dihydro-1H-pyra-
zole[3,4-e][1,4]thiazepin-7-one (7, EC50 = 10.5 lM, efficacy = 63%)
was selected as a new scaffold for a further hit-to-lead optimiza-
tion on the way to novel FXR modulators.
Figure 2. (A) Pharmacophore hypothesis: 2 hydrogen bond acceptors (red dot with arrows), 1 hydrogen bond donor (cyan dot with arrow), 7 aromatic points (orange circle),
1 hydrophobic centre (green dot), 2 negative charges (red dot). (B) Excluded volumes (yellow dots) generated by the superimposed backbones of FXR.

3432
M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
Table 1
spacer between the distal phenyl group and the oxygen linkage
of s( )23 resulted in the inactive derivative s( )30.
FXR functional activity for dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-ones of series
I, 8–34
By choosing the 4-(phenoxy)phenyl moiety as the reference
substitution at C4-position of the pyrazolo[3,4-e][1,4]thiazepine-
7-one core, we moved to explore different substitutions on phenyl
ring B by the synthesis of compounds 35–45 belonging to the series
II (Table 2).
R1
A
S
Firstly, we shifted the methyl group of s( )23 from the orto- to
the meta- and para-positions of ring B [s( )35 and s( )36]. As a re-
sult, both meta- and para-methyl analogues of s( )23 showed a
drop in efficacy, while keeping the EC50 in the same range of po-
tency of the parent compound.
N
N
H
O
N
Accordingly, a number of substituents endowed with different
electronic and steric properties were introduced at the ortho-posi-
tion of ring B. The biological appraisals of these derivatives showed
that alkyl groups (Et, nPr and iPr) contributed positively to the effi-
cacy [cf. s( )37–39 vs s( )23], with the n-propyl analogue being
the most active compound [(s( )38]. Among halogenated deriva-
tives (41–44), the iodine-substituted derivative (44), showing an
efficacy of 130%, appeared particularly promising in consideration
that it was tested as a mixture of four diastereoisomers. Con-
versely, the introduction of methoxy- or trifluoromethyl groups
afforded compounds [cf. s( )45 and 40 vs s( )23] with poor
efficacy.
8-34
Compounda
R1
EC50
(l
M)
Efficacyc (%)
b
7
2,4-Dimethoxy
4-Phenyl
10.5 0.5
0.99 0.1
1.1 0.3
1.8 0.1
1.2 0.2
3.5 0.4
NAd
63
24
50
41
9
7
3
7
s( )8
s( )9
4-(2-Methoxyphenyl)
4-(3-Methoxyphenyl)
4-(4-Methoxyphenyl)
4-(3-Ethoxyphenyl)
4-(3-Carbomethoxyphenyl)
4-(4-Carbomethoxyphenyl)
4-(3-Carboxyphenyl)
4-(4-Carboxyphenyl)
4-(E-Styryl)
s( )10
s( )11
s( )12
s( )13
s( )14
s( )15
s( )16
s( )17
s( )18
s( )19
s( )20
s( )21
s( )22
s( )23
s( )24
s( )25
s( )26
s( )27
s( )28
s( )29
s( )30
s( )31
s( )32
s( )33
s( )34
38 10
13
2
NAd
NAd
NAd
NA
4-(2-Thienyl)
4-(3-Thienyl)
4-(2-Furanyl)
4-(3-Furanyl)
4-(2-Benzofuranyl)
4-Phenoxy
2.6 0.4
1.4 0.5
3.6 0.2
3.5 0.5
1.5 0.5
1.3 0.5
1.7 0.4
1.7 0.1
2.2 0.1
NTe
85
68
32
40
17
118
3
4
4
2
4
4
Table 2
4-(2-Methoxyphenoxy)
4-(3-Methoxyphenoxy)
4-(4-Methoxyphenoxy)
4-(2-Carbomethoxyphenoxy)
4-(3-Carbomethoxyphenoxy)
4-(4-Carbomethoxyphenoxy)
4-(2-Carboxyphenoxy)
4-(3-Carboxyphenoxy)
4-(4-Carboxyphenoxy)
4-Benzyloxy
240 20
120
70 20
FXR functional activity for dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-ones of series
II, 35–45
2
NTe
NTe
NAd
O
NAd
NAd
NAd
3-Methoxy-4-phenoxy
1.9 0.1
130
7
S
a
The symbol s refers to the racemic couples endowed with syn-disposition of the
substituents at C-4 and C-6 positions. The anti-couples resulted in any case less
active than the corresponding syn-couples. Data relative to the anti-couples are not
shown.
N
N
H
O
N
R2
b
Ligand-dependent recruitment of Src-1 peptide assessed by AlphaScreen assay.
B
hFXR-LBD-GST was incubated with increasing concentrations of the indicated
ligand in the presence of biotinylated Src-1 peptide. The AlphaScreen signal
increases when the complex receptor-co-activator is formed.24
35-45
c
Efficacy: % of compound effect versus 10
NA = not active.
NT = not tested because available in poor amount.
lM of CDCA.
d
e
Compound
R2
EC50
(
lM)
Efficacyd (%)a
c
s( )23a
s( )35a
s( )36a
s( )37a
s( )38a
s( )39a
40b
2-CH3
3-CH3
4-CH3
1.3 0.5
2.4 0.5
3.4 1.4
1.1 0.1
1.1 0.3
1.9 0.1
2.0 0.3
3.9 0.1
2.7 0.2
1.8 0.2
1.3 0.3
3.3 0.9
118
22
11
4
2
2
2-CH2CH3
190 10
240 50
174 20
substantial increase in efficacy in the case of 2-thienyl derivative
s( )18. An encouraging result was obtained by the insertion of an
oxygen bridge between the two ring of the biphenyl moiety of
s( )8: the resulting derivative s( )23 indeed, showed to be a full
agonist with a potency ꢀ10-fold when compared to the hit 7 and
2-CH2CH2CH3
2-CH(CH3)2
2-CF3
50
3
41b
2-F
2-Cl
2-Br
2-I
72 10
42b
57
84
5
6
43b
an efficacy of 118% versus 10 lM of CDCA. By maintaining the po-
44b
130 20
37
sitive ether linkage, different substitutions on the distal phenyl
ring were then explored; notably, the introduction of a methoxy
group at ortho-position resulted in about twofold increase in effi-
cacy [cf. s( )24 vs s( )23]. The meta-substituted methoxy deriva-
tive s( )25 was equipotent with s( )23, whereas a slight decrease
in efficacy was observed with the corresponding para-substituted
derivative [cf. s( )26 vs s( )23]. The decoration of the proximal
aromatic ring of s( )23, exemplified by the introduction of a meth-
oxy group in meta-position, was neutral in potency as well as in
efficacy [cf. s( )31 vs s( )23]. The introduction of one-carbon
s( )45
2-OCH3
4
a
The symbol s refers to the racemic couple endowed with syn-disposition of the
substituents at C-4 and C-6 positions. The anti-couples resulted in any case less
active than the corresponding syn-couples. Data relative to the anti-couples are not
shown.
b
Compound tested as mixture of syn- and anti-couples.
Ligand-dependent recruitment of Src-1 peptide assessed by AlphaScreen assay.
c
hFXR-LBD-GST was incubated with increasing concentrations of the indicated
ligand in the presence of biotinylated Src-1 peptide. The AlphaScreen signal
increases when the complex receptor-coactivator is formed.24
d
Efficacy: % of compound effect versus 10 lM of CDCA.

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3433
2.3. Gene expression profile
mic couples. Medium pressure chromatography of the crude reac-
tion mixtures allowed us in most cases, to obtain the less polar
racemic couples a( )8–14, a( )17–29, a( )33–41 and a( )45, en-
dowed with an anti disposition of the substituents (for the assign-
ment, see below), and the more polar ones, s( )8–14, s( )17–29, s
( )33–39, and s( )45 showing a syn disposition of the substituents.
The separation was not possible for the compounds 42–44 which
were therefore tested as a mixture of four diastereoisomers.
The compounds a- and s-( )13, 14, 27, 28 and 29, characterized
by an ester functionality were then submitted to an alkaline hydro-
lysis to afford the corresponding carboxylic acids a- and s( )15, 16,
30–32 (Schemes 2 and 3).
The precursors 1-substituted-3-methyl-5-aminopyrazole 46a–l
were prepared in high yields following a known procedure involv-
ing the reaction of 3-aminocrotononitrile (48) with the appropriate
arylhydrazine 49 (Scheme 4).26 The commercial unavailable aryl-
hydrazines 49d–l were obtained according to a known protocol27
by reduction with stannous chloride of the corresponding diazo-
nium salts, in turn prepared starting from the corresponding
anilines.
In view of their efficacy values, compounds s( )24, s( )37 and
s( )38 were selected to evaluate their capacity to modulate se-
lected FXR target genes with respect to obeticholic acid (INT-747,
1) (Fig. 3).10 The expression of Ostb, Bsep and Cyp7
a1, three well
known FXR target genes, was therefore investigated by quantita-
tive RT-PCR using RNA from HepG2 cells stimulated with these
compounds. The results demonstrated that all compounds induced
Ostb with an activity of 20–40% compared to 1
lM of INT-747(1).
Cyp7 1 was down-regulated by all derivatives tested in dose-
a
dependent manner, with s( )24 resulting the most effective com-
pound. While compounds s( )24 and s( )38 do not induce Bsep,
the compound s( )37 induced Bsep in dose-dependent manner.
2.4. Chemistry
The general synthetic strategy for the preparation of all the
compounds is outlined in Scheme 1 and involved the condensation
reaction between the appropriate 1-substituted-3-methyl-5-ami-
nopyrazole 46 and an aryl aldehyde 47, carried out in refluxing
toluene with concomitant water removal. The intermediate 2,4-
dihydro-3H-pyrazol-5-imines, thus formed, were then treated with
2-mercaptopropanoic acid to afford the title 1H-pyrazolo[3,4-
e][1,4]thiazepin-7-ones 8–14, 17–29, 33–45.25
The 4-aryl-substituted benzaldehydes 47b–m, needed for the
preparation of the final compounds a- and s( )-9–14,-17–22 were
synthesized in high yields, by a Suzuki coupling reaction protocol
starting from 4-bromobenzaldehyde (50) and the appropriate aryl
boronic acid 51 (Scheme 5).
Since two chiral centers are present in the structure of the final
compounds it was expected to obtain them as mixture of two race-
The reaction of 4-fluorobenzaldehyde (52) with the monosub-
stituted phenols 53a–f, pre-treated with potassium carbonate,
12
10
8
1.2
1.0
0.8
0.6
0.4
0.2
0.0
15
10
5
NT
INT-747
s 24
6
4
2
0
0
(μM)
(μM)
(μM)
-
1
1
10
30
-
1
1
10
30
-
1
1
10
30
1.4
10
120
100
80
NT
INT-747
s 37
1.2
1.0
0.8
0.6
0.4
0.2
0.0
8
6
4
2
0
60
15
10
5
0
(μM)
-
1
1
10
30
(μM)
(μM)
-
1
1
10
30
-
1
1
10
30
12
10
8
1.2
1.0
0.8
0.6
0.4
0.2
0.0
15
10
5
NT
INT-747
s 38
6
4
2
0
0
(μM)
(μM)
(μM)
-
1
1
10
30
-
1
1
10
30
-
1
1
10
30
Figure 3. Regulation of Ostb, Bsep and Cyp7
a1 genes by s( )24, s( )37 and s( )38 assessed by quantitative RT-PCR. The results show mean SD of triplicate samples from a
representative experiment of three performed.

3434
M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
R1
R1
CHO
N
S
S
NH2
N
+
a
R2
N
N
R1
N
H
N
H
O
O
N
N
R2
R2
47a-v
R1 4-phenyl
R1 = 4-(2-methoxyphenyl)
R1 = 4-(3-methoxyphenyl)
R1 = 4-(4-methoxyphenyl)
R1 = 4-(3-ethoxyphenyl)
R1 = 4-(3-CO2CH3phenyl)
46a-l
a:
b:
c:
d:
e:
f:
=
a:
b:
c:
d:
e:
f:
=
=
=
=
=
=
R2 2-CH3
R2 3-CH3
R2 4-CH3
R2 2-C2H5
R2 2-nC3H7
R2 2-iC3H7
a( )8-14,17-29,33-45
s( )8-14,17-29,33-45
for R1 and R2, see Tables 1 and 2
g: R1 = 4-(4-CO2CH3phenyl)
h: R1 = 4-(t-styryl)
i: R1 = 4-(2-thienyl)
g: R2 = 2-CF3
h: R2 = 2-F
i: R2 = 2-Cl
j: R2 = 2-Br
k: R2 = 2-I
j: R1 = 4-(3-thienyl)
k: R1 = 4-(2-furanyl)
l: R1 = 4-(3-furanyl)
m: R1 = 4-(2-benzofuranyl)
n: R1 = 4-phenoxy
l: R2 = 2-OCH3
o:
p:
q:
r:
s:
t:
R1 = 4-(2-methoxyphenoxy)
R1 = 4-(3-methoxyphenoxy)
R1 = 4-(4-methoxyphenoxy)
R1 = 4-(2-CO2CH3phenoxy)
R1 = 4-(3-CO2CH3phenoxy)
R1 = 4-(4-CO2CH3phenoxy)
R1 = 4-benzyloxy
u:
v: R1 = 3-methoxy-4-phenoxy
Scheme 1. Synthesis of 1H-pyrazolo[3,4-e][1,4]thiazepin-7-ones, a( )- and s( ) 8–14, -17–29, -33–45. Reagents and conditions: (a) (i) toluene, reflux;
(ii) 2-mercaptopropanoic acid, toluene, reflux; (iii) medium pressure liquid chromatography.
R1
R1
R1
R1
S
S
S
S
a
a
N
N
N
N
H
N
O
N
H
O
N
N
N
H
O
N
H
O
N
N
s( )-13, -14, -27-29
for R1, see table 1
s( )-15, -16, -30-32
a( )-13, -14, -27-29
for R1, see table 1
a( )-15, -16, -30-32
Scheme 2. Synthesis of 1H-pyrazolo[3,4-e][1,4]thiazepin-7-ones, a( )15, 16,
30–32. Reagents and conditions: (a) 0.1 N NaOH, THF, rt.
Scheme 3. Synthesis of 1H-pyrazolo[3,4-e][1,4]thiazepin-7-ones, s( )15, 16,
30–32. Reagents and conditions: (a) 0.1 N NaOH, THF, rt.
afforded, under microwave irradiation, 4-aryloxy-substituted
benzaldehydes 44o–t used in the preparation of the final com-
pounds a- and s( )24–29. (Scheme 6).
hydrogens. These data allowed us to suppose that in s( )23 the se-
ven-member ring adopt such a conformation as the phenoxyphe-
nyl moiety and the C-6 methyl are both oriented in equatorial
position (graphically represented by conformer A in Figure 4).
The absence of cross peak between the C-6 methyl and the aryl
group suggested that the energetically unfavourable conformation
locating both these substituents in pseudo-axial orientation did
not exist (conformer B in Fig. 4). In the case of a( )23, the cross
peak occurring between the hydrogen at C-6 position and the aro-
matic protons of the aryl moiety at C-4 position suggested that the
preferred azepine ring conformation, graphically depicted by
The assignment of the relative configurations to all the synthe-
sized racemic mixtures started from the spectroscopic analysis of
the two racemic couples, a( )23 and s( )23. In the less polar couple
a( )23, strong NOE occurs between H-6 (quartet at 3.20 ppm) and
the aromatic protons of the aryl moiety at C-4 position, whereas
the NOESY spectrum of the more polar diastereoisomer s( )23
shows NOE between H-4 (singlet at 5.63 ppm) and H-6 (quartet
at 3.81 ppm), suggesting a syn disposition between these two

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3435
CHO
R1
CHO
NHNH2
X
H2N
a
R12
N
a
+
NH2
N
+
CN
R2
OH
F
48
49a-l
52
44o-t
53a-f
46a-l
a
b
o
p
q
: X = 2-OCH3
: X = 3-OCH3
: R1 = 4-(2-methoxyphenoxy)
: R1 = 4-(3-methoxyphenoxy)
: R1 = 4-(4-methoxyphenoxy)
: R1 = 4-(2-CO2CH3phenoxy)
a:
b:
c:
d:
e:
f:
=
R2 2-CH3;
c: X = 4-OCH3
=
R2 3-CH3;
d
: X = 2-CO2C2H5
e: X = 3-CO2CH3
r
=
R2 4-CH3;
s
: R1 = 4-(3-CO2CH3phenoxy)
=
R2 2-C2H5;
f
: X = 4-CO2CH3
t
: R1 = 4-(4-CO2CH3phenoxy)
=
R2 2-nC3H7;
=
R2 2-iC3H7;
Scheme 6. Synthesis of 4-(aryloxy)benzaldehydes, 44o–t. Reagents: (a) K2CO3,
DMF, microwave irradiation.
g:
=
R2 2-CF3;
h: R2 = 2-F;
i: R2 = 2-Cl;
j: R2 = 2-Br;
k: R2 = 2-I;
H
CH3
Ar
l: R2 = 2-OCH3.
H
6
6
H3C
H
Scheme 4. Synthesis of 3-methyl-5 amino1H-pyrazoles, 46a–l. Reagents and
conditions: (a) 12 N HCl, H2O, reflux.
4
4
S
Ar
S
H
conformer A in Figure 5, was that orienting the aryl moiety and
6-CH in pseudo-axial position, whereas it was unfavourable the
conformation locating the same moieties in pseudo-equatorial
disposition (conformer B in Fig. 5).
s( )23
s( )23
conformer B
conformer A
Figure 4. Graphical representation of the conformations of s( )23.
By moving to compare the H-4 and H-6 chemical shifts in a- and
s( )23 we noted remarkable differences: 3.22 ppm in a( )23 ver-
sus 3.81 ppm in s( )23 for H-6, and 5.21 ppm versus 5.63 ppm
for H-4 in a( )23 and s( )23, respectively. The analysis of the 1H
NMR data of all the other final compounds allows to emerge as a
rule for a specific molecule that both H-4 and H-6 are always more
deshielded in the more polar pair, as observed in the more polar
s( )23. According to this evidence the anti relative configuration
was assigned to all the less polar pairs and the syn one to the more
polar couples.
ring A, should be envisaged as large enough to lodge a phenoxy-
phenyl moiety and composed of aromatic and/or hydrophobic res-
idues. At this regard, it is worth noting that the presence of acidic
or ester groups on ring A substituents [s( )13–16, s( )27–32] is
fully detrimental for the activity. Overall, the occupancy of this re-
gion affects both the activity and the efficacy of the compounds.
Conversely, the second region lodging ring B seems to mostly influ-
ence the efficacy of the compounds rather than their activity. These
data suggest that the ring A is the key-player with respect to the
ring B, giving the higher contribution to the driving force that gov-
ern the binding process. Again, SAR of the explored substituents
pinpoints the hydrophobic and/or aromatic character of residues
facing this part of the binding site.
The scheme of Figure 6 was next used as criteria to select a
putative binding mode of the hit compound 7 and the optimized
lead s( )38 that could be compliant with the properties of regions
1 and 2. The top scored poses obtained by docking experiments of
compounds 7 and s( )38 with the FXR structure (pdb code:
1OSV10) are shown in Figure 7. Unfortunately, these poses (as well
as all of the remaining low scored poses) were not in agreement
with the reported SAR scheme, with the major point of disagree-
ment being the presence of Arg328 in the supposed hydrophobic
region 1.
Thus, although the scoring function of docking experiments was
successful in sorting out true FXR binders in the virtual screening
campaign, it did not propose a compelling binding pose for them.
The fallacy to predict true binding modes in docking screens has
been recently debated.28 In that paper, the authors argued that in
the absence of a correct binding pose for hit compounds, a skeptic
might well infer that the discovery is serendipitous.
Although we have inconsistent data between the SAR scheme
and docking results, we deem that the discovery of 7 as FXR ligand
may still not to be considered serendipitous, given the following
consideration. Our virtual screening is not fully dependent on a
structural hypothesis generated from docking experiments, rather
2.5. Binding mode assessment
The analysis of the results reported in Tables 1 and 2 allows to
draw a preliminary scheme of SAR as composed of two regions
harboring rings A and B (Fig. 6). The first region, accommodating
CHO
CHO
OH
OH
a
R1
B
+
Br
R1
51b-m
50
47b-m
b: R1 = 4-(2-methoxyphenyl)
c: R1 = 4-(3-methoxyphenyl)
d: R1 = 4-(4-methoxyphenyl)
e: R1 = 4-(3-ethoxyphenyl)
f: R1 = 4-(3-CO2CH3phenyl)
h: R1 = 4-(E-styryl)
i: R1 = 4-(2-thienyl)
: R1 = 4-(3-thienyl)
k: R1 = 4-(2-furanyl)
j
l
: R1 = 4-(3-furanyl)
m: R1 = 4-(2-benzofuranyl)
g
: R1 = 4-(4-CO2CH3phenyl)
Scheme 5. Synthesis of the 4-(aryl)benzaldeydes, 47b–m. Reagents and conditions:
(a) NaHCO3, H2O, tetrakis(triphenylphosphine)palladium(0), toluene–EtOH 1:1,
reflux.

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M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
Still, the question is why the proposed binding pose does not
H
CH3
agree with the SAR scheme. One explanation could be that the
adopted semi-rigid docking procedure (ligand free and protein ri-
gid) neglects induced-fit aspects that are pivotal for the correct
positioning of the ligand within the binding cleft of the receptor.
At this regard, molecular dynamics29,30 and crystallization stud-
ies10,11,13,16–19 support the high flexibility of FXR ligand binding
domain.
The fact that compound 7 is successfully identified as FXR bin-
der underlines that a correct prediction of the binding pose is not
mandatory for a successful virtual screening, albeit it is important
for an efficient hit to lead and lead to candidate optimization.
Although solving a crystal structure of the complex should be the
method of choice to determine the binding pose, it is not always
feasible or not readily accessible. In such a case, the development
of SAR schemes may allow to get clues on the right binding pose
adopted by hit compounds into the target receptor.
Ar
H
6
6
H3C
H
4
4
S
S
H
Ar
a( )23
conformer A
a( )23
conformer B
Figure 5. Graphical representation of the conformations of a( )23.
Aromatic/Hydrophobic Residues
Activity/Efficacy
1
3. Conclusion
In an effort to identify novel non-steroidal FXR agonists, a
sequential focused virtual screening approach was adopted to
search within DrugLike (2 million of compounds) and the LeadLike
(1 million of compounds) subsets of the ZINC 8 database. By bio-
logically testing only 117 selected compounds, 34 compounds
were successfully identified as FXR ligands, showing activity below
Aromatic/Hydrophobic Residues
Efficacy
2
a concentration of 20
3,6-dimethyl-1-(2-tolyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thia-
zepin-7-one (7, EC50 = 10.5 M, efficacy = 63%) was selected as a
lM. In particular, 4-(2,4-dimethoxyphenyl)-
Figure 6. SAR analysis observations. The phenyl ring A lies in an hydrophobic/
aromatic region (1) affecting the activity and efficacy of the compounds. Ring B is
lodged into a second aromatic/hydrophobic region (2) influencing the efficacy of the
compounds.
l
new scaffold for an initial hit-to-lead optimization focused to ex-
plore substitution patterns of rings A and B of 7. Briefly, the
replacement of the 2,4-dimethoxyphenyl group of 7 by a biphenyl
or 4-phenoxyphenyl moiety increased the potency of the corre-
sponding derivatives, whereas the introduction at the ortho-posi-
tion of the N-phenyl ring of alkyl groups bulkier than the methyl
one influenced positively the efficacy of the resulting derivatives.
The resulting SAR scheme was instrumental to assess a putative
it implements the combination of a pharmacophoric hypothesis
with an energetical hypothesis from the scoring function of dock-
ing experiments. The 39.8% hit rate resulting from such a combina-
tion is high, sustaining the robustness of this strategy as well as the
validity of the employed scoring function.
Figure 7. (A) Ligand interactions of 7 (left) and s( )38 (right). The docking poses in the 1OSV X-ray complex of FXR with the main residues involved in the interaction with the
phenyl moiety are colored in blue and the trigger triad shown in white. The coactivator helix is shown in red ribbon. (B) Pharmacophoric poses of 7 (left) and s( )38 (right),
with snapshot taken with a common view with the docking poses. The regions occupied by ring A and B are the same as observed in A.

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3437
binding mode of the hit compound 7 and s( )38 into the binding
cleft of FXR. Unfortunately, no binding pose was found in agree-
ment with the SAR scheme, prompting for further in-depth studies.
In conclusion, we got the identification of syn-( )-4-[(4-
(2-methoxyphenoxy)phenyl]-3,6-dimethyl-1-(2-methylphenyl)-4,
8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-one [s( )24], syn-( )-
4-(4-phenoxyphenyl)-3,6-dimethyl-1-(2-ethylphenyl)-4,8-dihy-
dro-1H-pyrazole[3,4-e][1,4]thiazepin-7-one [s( )37] and syn-( )-
4-(4-(phenoxyphenyl)-3,6-dimethyl-1-(2-npropylphenyl)-4,8-dihy-
dro-1H-pyrazole[3,4-e][1,4]thiazepin-7-one [s( )38], three deriva-
tives exhibiting low micromolar range of potency (10-fold higher
than the initial hit) and full efficacy.
Additional studies are ongoing with the aim of further optimize
this series of compounds, also devising compounds combining
fruitful substitutions of rings A and B. Likewise, further work will
be directed to gain insights into the putative binding pose of this
series of compounds into the ligand binding cleft of FXR.
(1H, q, J = 7.2 Hz, 6-CH), 5.68 (1H, s, 4-CH), 7.00 (1H, s, NH),
7.33–7.61 (13H, m, aromatics); 13C NMR (CDCl3, 100 MHz) d
13.36, 15.24, 17.23, 39.01, 45.15, 108.43, 127.02, 127.28, 127.46,
128.25, 128.61, 128.76, 130.28, 131.53, 135.34, 135.58, 136.59,
138.25, 140.41, 140.84, 148.15, 172.65; Anal. Calcd for
C27H25N3OS: C, 73.77; H, 5.73; N, 9.56; O, 3.64; S, 7.29. Found: C,
73.98; H, 5.73; N, 9.56; S, 7.30.
4.2.3. anti-( )-4-(20-Methoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )9]
Light petroleum–ethyl acetate (65:35), yield 31%, white solid,
1H NMR (CDCl3, 400 MHz) d 1.25 (3H, d, J = 7.2 Hz, 6-CH3), 2.00
(3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.25 (1H, q, J = 7.2 Hz, 6-CH),
3.80 (3H, s, OCH3), 5.25 (1H, s, 4-CH), 7.0–7.55 (13H, m, aromatics
and NH); 13C NMR (CDCl3, 100 MHz) d 12.86, 16.44, 17.31, 36.37,
42.61, 55.54, 105.05, 111.35, 120.92, 127.52, 127.60, 128.58,
128.84, 129.69, 130.55, 130.83, 131.74, 135.30, 136.25, 137.05,
137.78, 140.32, 147.93, 156.47, 172.24.
4. Experimental section
4.1. General methods
4.2.4. syn-( )-4-(20-Methoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )9]
Melting points were determined by the capillary method on a
Büchi 535 electrothermal apparatus and are uncorrected. 1H and
13C NMR spectra were taken on a Bruker AC 200 or Bruker AC
400 spectrometers as solutions in CDCl3 unless otherwise indi-
cated. The spin multiplicities are indicated by the symbols s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and bs
(broad). Flash chromatography was performed on Merck silica
gel (0.040–0.063 mm). Medium pressure chromatography (MPC)
was performed on Merck LiChroprep Si 60 Lobar columns. All tar-
get compounds possessed acceptable purity as verified by HPLC
(see Supplementary data).
Light petroleum–ethyl acetate (65:35), yield 31%, white solid,
mp 190–193 °C; 1H NMR (CDCl3, 400 MHz)
d 1.50 (3H, d,
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3), 2.10 (3H, s, 3-CH3), 3.75
(4H, m, 6-CH and OCH3), 5.70 (1H, s, 4-CH), 7.0–7.55 (13H, m, aro-
matics and NH); 13C NMR (CDCl3, 100 MHz) d 13.38, 15.22, 17.27,
38.95, 45.18, 55.53, 60.4, 108.76, 111.30, 120.87, 127.30, 127.81,
128.29, 128.78, 129.90, 130.30, 130.82, 131.55, 135.39, 135.59,
136.61, 137.75, 138.23, 148.24, 156.45, 172.79; Anal. Calcd for
C
28H27N3OS: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S, 6.83. Found: C,
71.86; H, 5.82; N, 8.94; S, 6.84.
4.2.5. anti-( )-4-(30-Methoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )10]
4.2. General procedure for the synthesis of 1H-pyrazole[3,4-
e][1,4]thiazepin-7-ones 8–14, 17–29, 33–45
Light petroleum–ethyl acetate (65:35), yield 37%, white solid,
mp 147–149 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
The appropriate 1-substituted-3-methyl-5-aminopyrazole 46
(1.0 mmol) and the aldehyde 47 (1.0 mmol) were heated under re-
flux in toluene (50 mL) for 7–9 hours; during this period produced
water was removed with the aid of a Dean-Stark apparatus. The
reaction mixture was cooled, 2-mercaptopropanoic acid (1.0 mmol)
was added and the mixture heated again at reflux for 12 hours. The
solvent was removed in vacuo and the residue submitted to med-
ium pressure liquid chromatography. Elution by light petroleum–
ethyl acetate mixtures afforded the desired compounds.
J = 7.2 Hz, 6-CH3), 2.00 (3H, s, 2-CH3), 2.20 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 3.82 (3H, s, OCH3), 5.23 (1H, s, 4-CH),
6.97 (1H, s, NH), 7.0–7.68 (12H, m, aromatics); 13C NMR (CDCl3,
100 MHz) d 12.75, 16.34, 17.28, 36.27, 42.49, 55.29, 104.91,
112.74, 112.90, 119.48, 127.30, 127.49, 128.32, 128.54, 129.83,
130.54, 131.72, 135.21, 136.26, 137.01, 140.21, 141.13, 141.76,
147.83, 159.98, 172.24.
4.2.6. syn-( )-4-(30-Methoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )10]
4.2.1. anti-( )-4-Biphenyl-4-yl-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )8]
Light petroleum–ethyl acetate (65:35), yield 35%, white solid, mp
130–135 °C; 1H NMR (CDCl3, 400 MHz) d 1.48 (3H, d, J = 7.2 Hz, 6-
CH3), 1.85 (3H, s, 2-CH3), 2.10 (3H, s, 3-CH3), 3.80 (4H, m, 6-CH and
OCH3), 5.70 (1H, s, 4-CH), 6.90 (1H, m, aromatic), 7.10 (1H, s, NH),
7.12–7.68 (11H, m, aromatics); 13C NMR (CDCl3, 100 MHz) d 13.21,
15.15, 17.21, 38.96, 45.03, 55.24, 108.62, 112.74, 112.84, 119.51,
127.21, 127.50, 128.287, 128.55, 129.75, 130.29, 131.48, 135.36,
135.50, 136.60, 138.36, 140.71, 141.90, 148.07, 159.90, 173.02; Anal.
Calcd for C28H27N3OS: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S, 6.83.
Found: C, 71.88; H, 5.82; N, 8.93; S, 6.84.
Light petroleum–ethyl acetate (80:20), yield 32%, white solid,
mp 193–194 °C; 1H NMR (CDCl3, 400 MHz)
d 1.29 (3H, d,
J = 7.2 Hz, 6-CH3), 1.98 (3H, s, 3-CH3), 2.18 (3H, s, 2-CH3), 3.28
(1H, q, J = 7.2 Hz, 6-CH), 5.22 (1H, s, 4-CH), 7.13 (1H, s, NH),
7.34–7.65 (13H, m, aromatics); 13C NMR (CDCl3, 100 MHz) d
12.80, 16.37, 17.30, 36.25, 42.50, 104.87, 126.97, 127.24, 127.49,
128.36, 128.54, 128.83, 130.52, 131.72, 135.28, 136.24, 136.99,
140.23, 140.30, 140.99, 147.84, 172.16.
4.2.2. syn-( )-4-Biphenyl-4-yl-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )8]
4.2.7. anti-( )-4-(40-Methoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )11]
Light petroleum–ethyl acetate (80:20), yield 33%, white solid,
mp 232–235 °C; 1H NMR (CDCl3, 400 MHz)
d
1.48 (3H, d,
Light petroleum–ethyl acetate (65:35), yield 28%, white solid,
mp 218–220 °C; 1H NMR (CDCl3, 400 MHz) d 1.25 (3H, d, J = 7.2,
J = 7.2 Hz, 6-CH3), 1.92 (3H, s, 3-CH3), 2.13 (3H, s, 2-CH3), 3.85

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M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
6-CH3), 1.90 (3H, s, 2-CH3), 2.15 (3H, s, 3-CH3), 3.25 (3H, s, OCH3),
3.80 (1H, q, J = 7.2 Hz, 6-CH), 5.25 (1H, s, 4-CH), 6.90 (2H, m, aro-
matics), 7.02 (1H, s, NH), 7.25–7.55 (10H, m, aromatics); 13C
NMR (CDCl3, 1400 MHz) d 12.79, 16.39, 17.30, 29.69, 36.28,
42.52, 55.35, 104.974, 114.29, 126.77, 127.52, 128.02, 128.36,
128.56, 130.57, 131.75, 132.77, 135.26, 136.26, 137.03, 139.94,
140.27, 147.86, 159.32, 172.20.
4.2.12. syn-( )-4-(30-Carbomethoxybiphenyl-4-yl)-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )13]
Light petroleum–ethyl acetate (65:35), yield 31%, white solid,
mp 184–186 °C; 1H NMR (CDCl3, 400 MHz)
d 1.47 (3H, d,
J = 7.2 Hz, 6-CH3), 1.90 (3H, s, 2-CH3), 2.11 (3H, s, 3-CH3), 3.86
(1H, q, J = 7.2 Hz, 6-CH), 3.96 (3H, s, CO2CH3), 5.71 (1H, s, 4-CH),
6.97 (1H, s, NH), 7.30–7.46 (6H, m, aromatics), 7.52 (1H, t,
J = 7.7 Hz, aromatic), 7.60 (2H, d, J = 8.4 Hz, aromatic), 7.77 (1H,
m, aromatic), 8.03 (1H, m, aromatic), 8.27 (1H, m, aromatic); 13C
NMR (CDCl3, 100 MHz) d 13.34, 15.25, 17.25, 39.06, 45.13, 52.21,
108.38, 127.33, 127.54, 128.19, 128.26, 128.53, 128.78, 128.91,
130.36, 130.73, 131.40, 131.59, 135. 42, 135.49, 136.62, 138.86,
139.80, 140.70, 148.10, 166.96, 172.57; Anal. Calcd for
4.2.8. syn-( )-4-(40-Methoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )11]
Light petroleum–ethyl acetate (65:35), yield 26%, white solid,
mp 112–115 °C; 1H NMR (CDCl3, 400 MHz) d 1.48 (3H, d, J = 7.2,
6-CH3), 1.90 (3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.85 (4H, m, 6-CH
and OCH3), 5.70 (1H, s, 4-CH), 6.93 (2H, m, aromatics), 7.02 (1H,
s, NH), 7.25–7.55 (10H, m, aromatics); 13C NMR (CDCl3, 100 MHz)
d 13.31, 15.26, 17.27, 29.70, 39.07, 45.18, 55.34, 108.63, 114.24,
127.04, 127.34, 128.07, 128.33, 128.60, 130.41, 131.59, 132.94,
135.35, 135.532, 136.665, 137.47, 140.51, 148.17, 159.29, 172.79;
Anal. Calcd for C28H27N3OS: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S,
6.83. Found: C, 71.60; H, 5.79; N, 8.93; S, 6.84.
C29H27N3O3S: C, 70.00; H, 5.47; N, 8.44; O, 9.65; S, 6.44. Found:
C, 69.88; H, 5.48; N, 8.45; S, 6.45.
4.2.13. anti-( )-4-(40-Carbomethoxybiphenyl-4-yl)-3,6-
dimethyl-1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )14]
Light petroleum–ethyl acetate (60:40), yield 34%, white solid,
mp 220–223 °C; 1H NMR (CDCl3, 400 MHz)
d 1.29 (3H, d,
4.2.9. anti-( )-4-(30-Ethoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )12]
J = 7.2 Hz, 6-CH3), 1.98 (3H, s, 2-CH3), 2.18 (3H, s, 3-CH3), 3.27
(1H, q, J = 7.1 Hz, 6-CH), 3.97 (3H, s, CO2CH3), 5.22 (1H, s, 4-CH),
7.07 (1H, s, NH), 7.28–7.43 (6H, m, aromatics), 7.66 (2H, d,
J = 8.3 Hz, aromatics), 7.71 (2H, d, J = 8.3 Hz, aromatics), 8.14 (2H,
d, J = 8.3 Hz, aromatics); 13C NMR (CDCl3, 100 MHz), d 12.77,
16.38, 17.30, 36.30, 42.46, 52.16, 104.73, 126.91, 127.47 127.55,
128.53, 129.12, 130.17, 130.64, 131.79, 135.14, 136.32, 137.00,
139.16, 142.02, 144.65, 147.77, 166.91, 172.02.
Light petroleum–ethyl acetate (70:30), yield 32%, white solid,
mp 96–100 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 1.48 (3H, t, J = 7.1 Hz, OCH2CH3), 1.97 (3H, s, 2-
CH3), 2.20 (3H, s, 3-CH3), 3.26 (1H, q, J = 7.2 Hz, 6-CH), 4.17 (2H,
q, J = 7.1 Hz, OCH2CH3), 5.23 (1H, s, 4-CH), 6.81 (m, 1H, aromatic),
7.13–7.60 (m, 12H, aromatics and NH); 13C NMR (CDCl3,
100 MHz)
d 12.76, 14.83, 16.29, 17.27, 36.20, 42.45, 63.41,
4.2.14. syn-( )-4-(40-Carbomethoxybiphenyl-4-yl)-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )14]
104.92, 113.28, 119.29, 127.23, 127.38, 128.26, 128.52, 129.77,
130.40, 131.62, 135.29, 136.21, 136.92, 140.17, 141.13, 141.66,
147.81, 159.29, 172.38.
Light petroleum–ethyl acetate (60:40), yield 32%, white solid, mp
201–204 °C; 1H NMR (CDCl3, 400 MHz) d 1.47 (3H, d, J = 7.1 Hz, 6-
CH3), 1.91 (3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.84 (1H, q, J = 7.1 Hz,
6-CH), 3.95 (3H, s, CO2CH3), 5.67 (1H, s, 4-CH), 6.89 (1H, s, NH),
7.32–7.46 (6H, m, aromatics), 7.61 (2H, d, J = 8.3 Hz, aromatics),
7.66 (2H, d, J = 8.3 Hz, aromatics), 8.11 (2H, d, J = 8.3 Hz, aromatics);
13C NMR (CDCl3, 100 MHz), d 13.30, 15.25, 17.25, 39.11, 45.11, 52.14,
108.38, 126.95, 127.39 127.68, 128.26, 128.79, 129.08, 130.13,
130.49, 131.64, 135.27, 135.51, 136.61, 139.23, 139.70, 144.78,
148.06, 166.91, 172.50; Anal. Calcd for C29H27N3O3S: C, 70.00; H,
5.47;N, 8.44;O, 9.65;S, 6.44. Found:C, 70.01;H, 5.48;N, 8.43;S, 6.45.
4.2.10. syn-( )-4-(30-Ethoxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )12]
Light petroleum–ethyl acetate (70:30), yield 31%, white solid,
mp 103–107 °C; 1H NMR (CDCl3, 400 MHz) d 1.48 (6H, m, 6-CH3
and OCH2CH3), 1.95 (3H, s, 2-CH3), 2.13 (3H, s, 3-CH3), 3.79 (1H,
q, J = 7.2 Hz, 6-CH), 4.17 (2H, q, J = 7.1 Hz, OCH2CH3), 5.62 (1H, s,
4-CH), 6.81 (1H, m, aromatic), 7.13 (1H, s, NH), 7.14–7.60 (11H,
m, aromatics); 13C NMR (CDCl3, 100 MHz) d 13.28, 14.83, 15.16,
17.22, 38.89, 45.02, 63.40, 108.52, 113.29, 119.35, 127.12, 127.44,
128.25, 128.54, 129.71, 130.12, 131.40, 135.39, 135.62, 136.56,
138.41, 140.66, 141.84, 148.05, 159.23, 173.03; Anal. Calcd for
4.2.15. anti-( )-4-{4-[(E)-2-Phenylvinyl]phenyl}-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )17]
C29H29N3OS: C, 72.02; H, 6.04; N, 8.69; O, 6.62; S, 6.63. Found: C,
72.28; H, 6.05; N, 8.68; S, 6.63.
Light petroleum–ethyl acetate (80:20), yield 29%, white solid,
mp 133–136 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
4.2.11. anti-( )-4-(30-Carbomethoxybiphenyl-4-yl)-3,6-
dimethyl-1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )13]
J = 7.2 Hz, 6-CH3), 2.00 (3H, s, 2-CH3), 2.20 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 5.20 (1H, s, 4-CH), 7.05–7.55 (16H, m,
CH@CH, aromatics and NH); 13C NMR (CDCl3, 100 MHz) d 12.73,
16.33, 17.26, 36.17, 42.55, 104.78, 126.49, 126.68, 127.46, 127.79,
128.24, 128.50, 128.67, 129.22, 130.50, 131.69, 135.23, 136.17,
136.64, 137.06, 141.26, 147.79, 172.13.
Light petroleum–ethyl acetate (65:35), yield 35%, white solid,
mp 198–200 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 1.96 (3H, s, 2-CH3), 2.16 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 3.96 (3H, s, CO2CH3), 5.20 (1H, s, 4-CH),
7.01 (1H, s, NH), 7.35–7.47 (6H, m, aromatics), 7.54 (1H, t,
J = 7.7 Hz, aromatic), 7.60 (2H, d, J = 8.4 Hz, aromatics), 7.81 (1H,
m, aromatic), 8.04 (1H, m, aromatic), 8.31 (1H, m, aromatic); 13C
NMR (CDCl3, 100 MHz) d 12.80, 16.38, 17.30, 36.27, 42.48, 52.23,
104.77, 127.35, 127.53, 128.19, 128.51, 128.96, 130.57, 130.77,
131.36, 131.75, 135.26, 136.28, 136.99, 139.30, 140.59, 141.61,
147.80, 166.96, 172.07.
4.2.16. syn-( )-4-{4-[(E)-2-Phenylvinyl]phenyl}-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )17]
Light petroleum–ethyl acetate (80:20); yield 30%, white solid,
mp 203–206 °C; 1H NMR (CDCl3, 400 MHz)
d
1.48 (3H, d,
J = 7.2 Hz, 6-CH3), 1.90 (3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.85

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3439
(1H, q, J = 7.2 Hz, 6-CH), 5.65 (1H, s, 4-CH), 7.10 (1H, s, NH), 7.25–
7.55 (15H, m, CH@CH and aromatics); 13C NMR (CDCl3, 100 MHz) d
13.29, 15.21, 17.21, 38.94, 45.22, 108.38, 126.48, 126.85, 127.19,
127.69, 127.89, 128.24, 128.53, 128.63, 129.11, 130.21, 131.45,
135.33, 135.54, 136.58, 137.06, 138.45, 148.07, 172.80; Anal. Calcd
for C29H27N3OS: C, 74.81; H, 5.84; N, 9.02; O, 3.44; S, 6.89. Found:
C, 74.82; H, 5.84; N, 9.03; S, 6.90.
J = 7.2 Hz, 6-CH3), 1.96 (3H, s, 2-CH3), 2.20 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 5.23 (1H, s, 4-CH), 6.50 (1H, m, aromatic),
6.73 (1H, m, aromatic), 7.25 (1H, s, NH), 7.27–7.52 (7H, m, aromat-
ics), 7.70 (2H, m, aromatics); 13C NMR (CDCl3, 100 MHz) d 12.68,
16.27, 17.25, 36.19, 42.56, 104.80, 105.42, 111.71, 123.95, 127.44,
128.26, 128.52, 130.16, 130,49, 131.68, 135.23, 136.22, 136.96,
140.93, 142.28, 147.81, 153.33, 172.29.
4.2.17. anti-( )-4-[(4-(2-Thienyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )18]
4.2.22. syn-( )-4-[(4-(2-Furanyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )20]
Light petroleum–ethyl acetate (80:20), yield 30%, white solid;
1H NMR (CDCl3, 400 MHz) d 1.25 (3H, d, J = 7.2 Hz, 6-CH3), 1.99
(3H, s, 2-CH3), 2.20 (3H, s, 3-CH3), 3.25 (1H, q, J = 7.2 Hz, 6-CH),
5.21 (1H, s, 4-CH), 7.10 (1H, m, aromatic), 7.20 (1H, s, NH), 7.25–
7.50 (8H, m, aromatics), 7.60 (2H, m, aromatics); 13C NMR (CDCl3,
100 MHz) d 12.72, 16.31, 17.25, 36.21, 42.47, 104.73, 123.28,
125.05, 125.98, 127.45, 128.08, 128.43, 128.50, 130.48, 131.68,
133.66, 135.25, 136.20, 136.94, 141.13, 143.52, 147.78, 172.19.
Light petroleum-ethyl acetate (70:30), yield 22%, white solid,
mp 147–149 °C; 1H NMR (CDCl3, 400 MHz)
d 1.48 (3H, d,
J = 7.2 Hz, 6-CH3), 1.96 (3H, s, 2-CH3), 2.18 (3H, s, 3-CH3), 3.83
(1H, q, J = 7.2 Hz, 6-CH), 5.70 (1H, s, 4-CH), 6.50 (m, 1H, aromatic),
6.73 (1H, m, aromatic), 7.25–7.50 (8H, m, aromatics and NH), 7.62
(2H, m, aromatics); 13C NMR (CDCl3, 100 MHz) d 13.19, 15.17,
17.20, 38.97, 45.20, 105.38, 108.46, 111.67, 124.10, 127.19,
128.27, 128.55, 130.25, 130,54, 131.46, 135.44, 136.61, 138.08,
142.21, 148.07, 153.38, 173.04; Anal. Calcd for C25H23N3O2S: C,
69.91; H, 5.40; N, 9.78; O, 7.45; S, 7.47. Found: C, 69.88; H, 5.42;
N, 9.76; S, 7.45.
4.2.18. syn-( )-4-[(4-(2-Thienyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )18]
Light petroleum–ethyl acetate (80:20), yield 31%, white solid,
mp 224–227 °C; 1H NMR (CDCl3, 400 MHz)
d 1.50 (3H, d,
4.2.23. anti-( )-4-[(4-(3-Furanyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )21]
J = 7.2 Hz, 6-CH3), 1.91 (3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.79
(1H, q, J = 7.2 Hz, 6-CH), 5.60 (1H, s, 4-CH), 7.04 (1H, s, NH),
7.24–7.46 (7H m, aromatics), 7.54 (1H, d, J = 8.1 Hz, aromatic),
7.60 (1H, d, J = 8.1 Hz, aromatic), 7.85 (2H, d, J = 8.2 Hz, aromatics);
13C NMR (CDCl3, 100 MHz) d 13.28, 15.19, 17.20, 38.97, 45.11,
108.354, 123.29, 125.00, 126.19, 127.23, 128.01, 128.23, 128.721,
130.25, 131.49, 134.08, 135.33, 135.52, 136.58, 138.37, 143.63,
148.08, 172.82; Anal. Calcd for C25H23N3OS2: C, 67.39; H, 5.20; N,
9.43; O, 3.59; S, 14.39. Found: C, 67.19; H, 5.19; N, 9.43; S, 14.40.
Light petroleum-ethyl acetate (75:25), yield 23%, white solid,
mp 223–226 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 1.92 (3H, s, 2-CH3), 2.10 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 5.22 (1H, s, 4-CH), 6.75 (1H, s, aromatic),
7.20 (1H, s, NH), 7.25–7.50 (9H, m, aromatics), 7.75 (1H, s, aro-
matic); 13C NMR (CDCl3, 100 MHz) d 12.71, 16.31, 17.26, 36.18,
42.49, 104.85, 108.62, 125.76, 125.98, 127.45, 128.37, 128.52,
130.50, 131.68, 135.21, 136.20, 136.96, 138.65, 140.63, 143.82,
147.80, 172.28.
4.2.19. anti-( )-4-[(4-(3-Thienyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )19]
4.2.24. syn-( )-4-[(4-(3-Furanyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )21]
Light petroleum–ethyl acetate (80:20), yield 35%, white solid, mp
227–229 °C; 1H NMR (CDCl3, 400 MHz) d 1.27 (3H, d, J = 7.2 Hz, 6-
CH3), 1.97 (3H, s, 2-CH3), 2.18 (3H, s, 3-CH3), 3.25 (1H, q, J = 7.2 Hz,
6-CH), 5.18 (1H, s, 4-CH), 7.10 (s, 1H, NH), 7.25–7.50 (9H, m, aromat-
ics), 7.60 (2H, d, J = 8.2 Hz, aromatics); 13C NMR (CDCl3, 100 MHz) d
12.74, 16.33, 17.28, 36.23, 42.51, 104.86, 120.52, 126.08, 126.45,
126.55, 127.49, 128.40, 128.53, 130.53, 131.72, 135.03, 135.23,
136.25, 136.98, 140.77, 141.45, 147.81, 172.19.
Light petroleum-ethyl acetate (75:25), yield 23%, white solid,
mp 161–163 °C; 1H NMR (CDCl3, 400 MHz)
d 1.50 (3H, d,
J = 7.2 Hz, 6-CH3), 1.88 (3H, s, 2-CH3), 2.07 (3H, s, 3-CH3), 3.76
(1H, q, J = 7.2 Hz, 6-CH), 5.63 (1H, s, 4-CH), 6.75 (1H, s, aromatic),
7.25–7.50 (10H, m, aromatics and NH), 7.75 (1H, s, aromatic);
13C NMR (CDCl3, 100 MHz) d 13.25, 15.17, 17.21, 38.94, 45.14,
108.49, 108.68, 125.85, 126.16, 127.18, 128.26, 128.65, 130.21,
131.45, 132.12, 135.37, 135.53, 136.60, 137.86, 138.62, 143.71,
148.07, 173.07; Anal. Calcd for C25H23N3O2S: C, 69.91; H, 5.40;
N, 9.78; O, 7.45; S, 7.47. Found: C, 69.92; H, 5.42; N, 9.77; S,
7.45.
4.2.20. syn-( )-4-[(4-(3-Thienyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )19]
Light petroleum–ethyl acetate (80:20), yield 34%, white solid,
mp 220–225 °C; 1H NMR (CDCl3, 400 MHz)
d 1.48 (3H, d,
J = 7.2 Hz, 6-CH3), 1.91 (3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.84
(1H, q, J = 7.2 Hz, 6-CH), 5.66 (1H, s, 4-CH), 7.00 (1H, s, NH),
7.25–7.48 (9H, m, aromatics), 7.60 (2H, d, J = 8.2 Hz, aromatics);
13C NMR (CDCl3, 100 MHz) d 13.31, 15.24, 17.23, 39.02, 45.19,
108.40, 120.52, 126.16, 126.32, 126.75, 127.28, 128.265, 128.67,
130.30, 131.54, 135.37, 135.51, 136.61, 138.00, 141.60, 148.11,
172.67; Anal. Calcd for C25H23N3OS2: C, 67.39; H, 5.20; N, 9.43;
O, 3.59; S, 14.39. Found: C, 67.28; H, 5.20; N, 9.44; S, 14.40.
4.2.25. anti-( )-4-[(4-(2-Benzofuranyl)phenyl]-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )22]
Light petroleum-ethyl acetate (65:35), yield 26%, white solid,
mp 210–213 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 2.00 (3H, s, 2-CH3), 2.12 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 5.23 (1H, s, 4-CH), 7.08 (1H, s, NH),
7.26–7.46 (9H, m, aromatics), 7.55 (1H, d, J = 8.0 Hz, aromatic),
7.62 (1H, d, J = 7.4 Hz, aromatic), 7.89 (2H, d, J = 8.2 Hz, aromatics);
13C NMR (CDCl3, 100 MHz) d 13.28, 15.20, 17.22, 39.01, 45.23,
101.69, 108.30, 111.14, 120.93, 122.96, 124.40, 125.28, 127.25,
128.26, 128.67, 129.06, 130.16, 130.30, 131.51, 135.37, 135.48,
136.60, 139.51, 148.08, 154.86, 155.26, 172.92.
4.2.21. anti-( )-4-[(4-(2-Furanyl)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )20]
Light petroleum–ethyl acetate (70:30), yield 21%, white solid,
mp 118–121 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,

3440
M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
4.2.26. syn-( )-4-[(4-(2-Benzofuranyl)phenyl]-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )22]
Anal. Calcd for C28H27N3O3S: C, 69.25; H, 5.60; N, 8.65; O, 9.88; S,
6.60. Found: C, 69.19; H, 5.61; N, 8.64; S, 6.61.
Light petroleum-ethyl acetate (65:35), yield 25%, white solid,
4.2.31. anti-( )-4-[4-(3-Methoxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )25]
mp 240–242 °C; 1H NMR (CDCl3, 400 MHz)
d 1.50 (3H, d,
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3), 2.10 (s, 3H, 3-CH3), 3.92
(1H, q, J = 7.2 Hz, 6-CH), 6.72 (1H, s, 4-CH), 7.00 (1H, s, NH),
7.24–7.46 (9H, m, aromatics), 7.55 (1H, d, J = 8.0 Hz, aromatic),
7.60 (1H, d, J = 7.4 Hz, aromatic), 7.85 (2H, d, J = 8.2 Hz, aromatics);
13C NMR (CDCl3, 100 MHz) d 13.28, 15.20, 17.212, 39.01, 45.22,
101.68, 108.29, 111.13, 120.92, 122.95, 124.39, 125.28, 127.25,
128.25, 128.66, 129.06, 130.15, 130.29, 131.51, 135.37, 135.48,
136.60, 139.50, 148.07, 154.85, 155.25, 172.92; Anal. Calcd for
Light petroleum-ethyl acetate (65:35), yield 29%, white solid,
mp 151–153 °C; 1H NMR (CDCl3, 400 MHz)
d 1.49 (3H, d,
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3), 2.15 (3H, s, 3-CH3), 3.78
(4H, m, 6-CH and OCH3), 5.21 (s, 1H, 4-CH), 6.5–7.5 (m, 13H, aro-
matics and NH); 13C NMR (CDCl3, 50 MHz) d 12.77, 16.29, 17.25,
36.18, 42.18, 55.33, 105.01, 105.28, 109.11, 111.24, 118.61,
127.45, 128.52, 129.28, 130.21, 130.50, 131.67, 135.22, 136.12,
136.64, 136.95, 147.73, 156.53, 157.82, 160.93, 172.31.
C
29H25N3O2S: C, 72.63; H, 5.25; N, 8.76; O, 6.67; S, 6.69. Found:
C, 72.64; H, 5.23; N, 8.77; S, 6.68.
4.2.32. syn-( )-4-[4-(3-Methoxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )25]
4.2.27. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )23]
Light petroleum-ethyl acetate (80:20), yield 32%, white solid,
mp 91–94 °C; 1H NMR (CDCl3, 400 MHz) d 1.27 (d, J = 7.2 Hz, 3H,
6-CH3), 1.98 (s, 3H, 2-CH3), 2.21 (s, 3H, 3-CH3), 3.52 (q, J = 7.2 Hz,
1H, 6-CH), 3.81 (s, 3H, OCH3), 5.21 (s, 1H, 4-CH), 6.5–7.5 (m, 13H,
aromatics and NH); 13C NMR (CDCl3, 50 MHz) d 12.77, 16.29,
17.25, 36.18, 42.18, 55.33, 105.01, 105.28, 109.11, 111.24, 118.61,
127.45, 128.52, 129.28, 130.21, 130.50, 131.67, 135.22, 136.12,
136.64, 136.95, 147.73, 156.53, 157.82, 160.93, 172.31; Anal. Calcd
for C28H27N3O3S: C, 69.25; H, 5.60; N, 8.65; O, 9.88; S, 6.60. Found:
C, 69.21; H, 5.59; N, 8.63; S, 6.61.
Light petroleum-ethyl acetate (80:20), yield 36%, white solid,
mp 93–95 °C; 1H NMR (CDCl3, 400 MHz) d 0.96 (3H, d, J = 7.2 Hz,
6-CH3), 1.97 (3H, s, 2-CH3), 2.14 (3H, s, 3-CH3), 3.22 (1H, q,
J = 7.2 Hz, 6-CH), 5.15 (1H, s, 4-CH), 6.95–7.50 (14H, m, aromatics
and NH); 13C NMR (CDCl3, 100 MHz) d 12.72, 16.34, 17.251,
36.23, 42.18, 105.04, 118.39, 119.27, 123.70, 127.51, 128.51,
129.29, 129.82, 130.58, 131.73, 135.10, 136.20, 136.38, 136.96,
147.68, 156.57, 156.86, 172.12.
4.2.28. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )23]
4.2.33. anti-( )-4-[4-(4-Methoxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )26]
Light petroleum-ethyl acetate (80:20), yield 37%, white solid,
mp 152–155 °C; 1H NMR (CDCl3, 400 MHz)
d
1.46 (3H, d,
Light petroleum-ethyl acetate (65:35), yield 32%, white solid,
J = 7.2 Hz, 6-CH3), 1.90 (3H, s, 2-CH3), 2.10 (3H, s, 3-CH3), 3.81
(1H, q, J = 7.2 Hz, 6-CH), 5.63 (1H, s, 4-CH), 6.95–7.41 (14H, m, aro-
matics and NH); 13C NMR (CDCl3, 100 MHz) d 13.34, 15.24, 17.21,
39.00, 44.91, 108.47, 118.76, 119.14, 123.56, 127.29, 128.25,
129.59, 129.78, 130.32, 131.54, 133.72, 135.30, 135.46, 136.60,
148.09, 156.71, 157.08, 172.67; Anal. Calcd for C27H25N3O2S: C,
71.18; H, 5.53; N, 9.22; O, 7.02; S, 7.04. Found: C, 71.38; H, 5.52;
N, 9.20; S, 7.01.
mp 179–181 °C; 1H NMR (CDCl3, 400 MHz)
d 1.26 (3H, d,
J = 7.2 Hz, 6-CH3), 1.90 (3H, s, 2-CH3), 2.16 (3H, s, 3-CH3), 3.24
(1H, q, J = 7.2 Hz, 6-CH), 3.77 (3H, s, OCH3), 5.15 (1H, s, 4-CH),
6.91–7.47 (13H, m, aromatics and NH); 13C NMR (CDCl3,
100 MHz) d 12.71, 16.31, 17.24, 36.16, 42.15, 55.60, 105.10,
114.86, 117.12, 127.44, 128.51, 129.16, 130.50, 131.65, 135.16,
135.59, 136.09, 136.94, 147.71, 149.43, 156.11, 158.03, 172.35.
4.2.34. syn-( )-4-[4-(4-Methoxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )26]
4.2.29. anti-( )-4-[4-(2-Methoxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )24]
Light petroleum-ethyl acetate (65:35), yield 34%, white solid,
mp 97–99 °C; 1H NMR (CDCl3, 400 MHz) d 1.49 (3H, d, J = 7.2 Hz,
6-CH3), 1.83 (3H, s, 2-CH3), 2.16 (3H, s, 3-CH3), 3.78 (4H, m, 6-CH
and OCH3), 5.59 (1H, s, 4-CH), 6.91–7.02 (7H, m, aromatics and
NH), 7.50–7.73 (6H, m, aromatics); 13C NMR (CDCl3, 100 MHz) d
13.29, 15.19, 17.19, 38.94, 44.86, 55.60, 108.56, 114.84, 117.49,
121.00, 127.22, 128.25, 129.45, 130.25, 131.47, 132.88, 135.25,
135.46, 136.59, 148.08, 149.58, 156.03, 158.28, 172.94; Anal. Calcd
for C28H27N3O3S: C, 69.25; H, 5.60; N, 8.65; O, 9.88; S, 6.60. Found:
C, 69.25; H, 5.59; N, 8.64; S, 6.61.
Light petroleum-ethyl acetate (65:35), yield 32%, white solid,
mp 201–201 °C; 1H NMR (CDCl3, 400 MHz)
d 1.26 (3H, d,
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3), 2.20 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 3.81 (3H, s, OCH3), 5.20 (1H, s, 4-CH),
6.90–7.50 (13H, m, aromatics and NH); 13C NMR (CDCl3,
100 MHz) d 12.72, 16.30, 17.24, 36.15, 42.18, 55.85, 105.11,
112.77, 116.94, 121.10, 121.29, 125.15, 127.43, 128.50, 129.08,
130.47, 131.65, 135.20, 135.67, 136.09, 136.94, 144.52, 147.72,
151.42, 157.41, 172.29.
4.2.30. syn-( )-4-[4-(2-Methoxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )24]
4.2.35. anti-( )-4-[4-(Benzyloxy)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )33]
Light petroleum-ethyl acetate (65:35), yield 31%, white solid,
Light petroleum-ethyl acetate (75:25), yield 31%, white solid,
mp 175–177 °C; 1H NMR (CDCl3, 400 MHz)
d
1.45 (3H, d,
mp 158–160 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 1.87 (3H, s, 2-CH3), 2.09 (3H, s, 3-CH3), 3.80
(4H, m, 6-CH and OCH3), 5.61 (1H, s, 4-CH), 6.96–7.36 (13H, m, aro-
matics and NH); 13C NMR (CDCl3, 100 MHz) d 13.28, 15.22, 17.18,
38.96, 44.92, 55.84, 108.565, 112.73, 117.15, 121.07, 121.30,
125.09, 127.24, 128.25, 128.93, 129.37, 130.27, 131.48, 132.84,
135.23, 135.45, 136.59, 144.51, 148.12, 151.41, 157.70, 172.83;
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3), 2.20 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 5.10 (2H, s, OCH2), 5.14 (1H, s, 4-CH),
6.99 (2H, d, J = 8.2 Hz, aromatics), 7.20 (2H, d, J = 8.10 Hz, aromat-
ics), 7.30 (1H, s, NH), 7.36–7.48 (8H, m, aromatics); 13C NMR
(CDCl3, 100 MHz) d 12.69, 16.30, 17.26, 36.13, 42.20, 70.05,
105.23, 114.78, 127.39, 127.52, 128.03, 128.56, 129.08, 130.41,

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3441
131.62, 134.19, 135.26, 136.10, 136.67, 136.95, 147.76, 158.09,
(1H, q, J = 7.2 Hz, 6-CH), 5.63 (1H, s, 4-CH), 6.96 (2H, d ,
172.47.
J = 8.6 Hz, aromatics), 7.03 (2H, d, J = 8.6 Hz, aromatics), 7.13 (1H,
t, J = 7.4 Hz, aromatic), 7.21–7.41 (9H, m, aromatics and NH); 13C
4.2.36. syn-( )-4-[4-(Benzyloxy)phenyl]-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )33]
NMR (CDCl3, 100 MHz) d 13.30, 15.12, 21.45, 39.07, 44.90,
110.58, 119.50, 119.82, 122.84, 124.28, 126.71, 130.31, 130.41,
130.56, 134.81, 135.02, 137.98, 141.01, 149.02, 157.96, 173.81;
Anal. Calcd for C27H25N3O2S: C, 71.18; H, 5.53; N, 9.22; O, 7.02; S,
7.04. Found: C, 71.17; H, 5.54; N, 9.21; S, 7.04.
Light petroleum-ethyl acetate (75:25), yield 32%, white solid,
mp 199–203 °C; 1H NMR (CDCl3, 400 MHz)
d 1.46 (3H, d,
J = 7.2 Hz, 6-CH3), 1.81 (3H, s, 2-CH3), 2.14 (3H, s, 3-CH3), 3.76
(1H, q, J = 7.2 Hz, 6-CH), 5.10 (2H, s, OCH2), 5.62 (1H, s, 4-CH),
6.96 (2H, J = 8.2 Hz, aromatics), 7.20 (1H, s, NH), 7.26–7.5 (10H,
m, aromatics); 13C NMR (CDCl3, 100 MHz) d 13.30, 15.20, 17.20,
38.91, 44.91, 70.00, 108.61, 114.98, 127.17, 127.46, 127.98,
128.25, 128.54, 129.35, 130.16, 131.43, 135.21, 135.59, 136.58,
136.70, 148.11, 158.39, 172.92; Anal. Calcd for C28H27N3O2S: C,
71.61; H, 5.80; N, 8.95; O, 6.81; S, 6.83. Found: C, 71.60; H, 5.79;
N, 8.93; S, 6.81.
4.2.41. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(4-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )36]
Light petroleum-ethyl acetate (80:20), yield 29%, white solid,
mp 197–199 °C; 1H NMR (CDCl3, 400 MHz)
d 1.26 (3H, d,
J = 7.2 Hz, 6-CH3), 1.99 (3H, s, 3-CH3), 2.48 (s, 4-CH3), 3.25 (1H, q,
J = 7.2 Hz, 6-CH), 5.20 (1H, s, 4-CH), 7.00 (2H, d, J = 8.6 Hz, aromat-
ics), 7.06 (2H, d, J = 7.8 Hz, aromatics), 7.15 (1H, t, J = 7.4 Hz, aro-
matic), 7.24–7.40 (9H, m, aromatics and NH); 13C NMR (CDCl3,
100 MHz) d 12.73, 16.22, 21.14, 36.18, 42.29, 105.68, 118.40,
119.14, 123.60, 125.75, 129.32, 129.78, 130.56, 134.31, 135.30,
136.50, 139.27, 147.77, 156.63, 156.74, 172.04.
4.2.37. anti-( )-4-(3-Methoxy-4-phenoxyphenyl)-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )34]
Light petroleum-ethyl acetate (80:20), yield 32%, white solid,
4.2.42. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(4-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )36]
mp 179–181 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3), 2.11 (3H, s, 3-CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 3.80 (3H, s, OCH3), 5.20 (1H, s, 4-CH),
6.70 (1H, d, J = 8.3 Hz, aromatic), 6.80 (1H, d, J = 8.3 Hz, aromatic),
6.90 (2H, d, J = 8.6 Hz, aromatics), 7.05 (1H, s, NH), 7.10 (1H, d,
J = 8.3 Hz, aromatic), 7.30–7.46 (7H, m, aromatics); 13C NMR
(CDCl3, 100 MHz) d 12.63, 16.21, 17.05, 36.25, 42.45, 55.86,
104.85, 112.39, 117.48, 119.61, 120.23, 122.76, 127.28, 128.34,
129.44, 130.27, 131.48, 135.22, 136.05, 136.69, 138.18, 144.73,
147.59, 151.13, 157.20, 171.98.
Light petroleum-ethyl acetate (80:20), yield 31%, white solid,
mp 97–99 °C; 1H NMR (CDCl3, 400 MHz)
d 1.47 (3H, d,
J = 7.2 Hz, 6-CH3), 1.99 (3H, s, 3-CH3), 2.48 (3H, s, 4-CH3), 3.75
(1H, q, J = 7.2 Hz, 6-CH), 5.60 (1H, s, 4-CH), 6.95 (2H, d,
J = 8.6 Hz, aromatics), 7.02 (2H, d, J = 7.8 Hz, aromatics), 7.13
(1H, t, J = 7.4 Hz, aromatic), 7.27–7.37 (8H, m, aromatics), 7.50
(1H, s, NH); 13C NMR (CDCl3, 100 MHz) d 13.18, 14.92, 21.10,
38.74, 44.55, 109.84, 117.53, 118.73, 119.05, 120.36, 123.48,
125.06, 129.60, 129.74, 130.23, 131.89, 134.07, 134.24, 134.75,
138.63, 147.91, 156.75, 156.93, 172.92; Anal. Calcd for
4.2.38. syn-( )-4-(3-Methoxy-4-phenoxyphenyl)-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )34]
C27H25N3O2S: C, 71.18; H, 5.53; N, 9.22; O, 7.02; S, 7.04. Found:
C, 71.18; H, 5.54; N, 9.21; S, 7.03.
Light petroleum-ethyl acetate (80:20), yield 34%, white solid,
mp 106–109 °C; 1H NMR (CDCl3, 400 MHz)
d 1.48 (3H, d,
4.2.43. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
ethylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )37]
J = 7.2 Hz, 6-CH3), 1.97 (3H, s, 2-CH3), 2.11 (3H, s, 3-CH3), 3.75
(4H, m, 6-CH and OCH3), 5.70 (1H, s, 4-CH), 6.80 (1H, s, NH),
6.80–7.10 (6H, m, aromatics and NH), 7.25–7.46 (7H, m, aromat-
ics); 13C NMR (CDCl3, 100 MHz) d 13.34, 15.21, 17.15, 38.96,
45.24, 56.03, 108.52, 112.59, 117.50, 120.58, 120.85, 122.79,
127.24, 128.27, 129.57, 130.25, 131.49, 135.41, 135.62, 135.78,
136.58, 144.98, 148.12, 151.39, 157.50, 172.87; Anal. Calcd for
Light petroleum-ethyl acetate (80:20), yield 22%, white solid,
mp 156–158 °C; 1H NMR (CDCl3, 400 MHz)
d 1.16 (3H, t,
J = 7.6 Hz, CH2CH3), 1.28 (3H, d, J = 7.2 Hz, 6-CH3), 2.00 (3H, s, 3-
CH3), 2.50 (2H, q, J = 7.6 Hz, 2H, CH2CH3), 3.25 (1H, q, J = 7.2 Hz,
6-CH), 5.24 (1H, s, 4-CH), 6.95 (2H, d, J = 8.6 Hz, aromatics), 7.02
(2H, d, J = 7.8 Hz, aromatics), 7.13 (1H, t, J = 7.4 Hz, aromatic),
7.25–7.51 (9H, m, aromatics and NH); 13C NMR (CDCl3, 100 MHz)
d 12.73, 14.23, 16.31, 23.95, 36.24, 42.20, 104.98, 118.39, 119.24,
13.67, 127.38, 128.62, 129.28, 129.81, 130.00, 130.73, 134.68,
136.22, 136.45, 147.57, 156.59, 156.82, 172.30.
C
28H27N3O3S: C, 69.25; H, 5.60; N, 8.65; O, 9.88; S, 6.60. Found:
C, 69.26; H, 5.62; N, 8.64; S, 6.59.
4.2.39. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(3-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )35]
Light petroleum-ethyl acetate (75:25), yield 37%, white solid,
mp 212–215 °C; 1H NMR (CDCl3, 400 MHz)
d 1.26 (3H, d,
4.2.44. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
ethylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )37]
J = 7.2 Hz, 6-CH3), 2.00 (3H, s, 3-CH3), 2.50 (s, 3H, 3-CH3), 3.26
(1H, q, J = 7.2 Hz, 6-CH), 5.15 (1H, s, 4-CH), 7.00–7.50 (14H, m, aro-
matics and NH); 13C NMR (CDCl3, 100 MHz) d 12.85, 16.33, 21.46,
36.44, 42.56, 106.52, 119.17, 119.91, 123.47, 124.40, 127.31,
130.15, 130.62, 136.13, 137.37, 137.74, 141.21, 148.81, 157.62,
157.75, 173.20.
Light petroleum-ethyl acetate (80:20), yield 20%, white solid,
mp 159–163 °C; 1H NMR (CDCl3, 400 MHz)
d 1.12 (3H, t,
J = 7.6 Hz, CH2CH3), 1.46 (3H, d, J = 7.2 Hz, 6-CH3), 1.90 (3H, s,
3-CH3), 2.41 (2H, q, J = 7.6 Hz, CH2CH3), 3.81 (1H, q, J = 7.2 Hz, 6-
CH), 5.63 (1H, s, 4-CH), 6.95–7.50 (13H, m, aromatics), 7.75 (1H,
s, NH); 13C NMR (CDCl3, 100 MHz) d 10.06, 11.09, 11.99, 20.71,
35.78, 41.65, 105,24, 114.02, 115.53, 115.91, 120.34, 124.02,
125.15, 126.35, 126.55, 126.70, 126.80, 127.41, 130.43, 132.23,
139.34, 144.70, 153.45, 153.85, 169.48; Anal. Calcd for
4.2.40. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(3-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )35]
Light petroleum-ethyl acetate (75:25), yield 36%, white solid,
mp 207–210 °C; 1H NMR (CDCl3, 400 MHz)
d
1.48 (3H, d,
C28H27N3O2S: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S, 6.83. Found:
J = 7.2 Hz, 6-CH3), 1.97 (3H, s, 3-CH3), 2.46 (s, 3H, 3-CH3), 3.80
C, 71.62; H, 5.81; N, 8.94; S, 6.83.

3442
M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
4.2.45. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
npropylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-
7-one [a( )38]
(1H, s, 4-CH, A), 5.60 (1H, s, 4-CH, B), 7.00–7.90 (14H, m, aromatics
A and B); 13C NMR (CDCl3, 100 MHz) d 12.78, 13.11, 14.89, 16.10,
38.55, 42.09, 44.31, 105.79, 109.50, 117.48, 118.41, 118.70,
119.12, 119.26, 120.36, 121.33, 123.51, 123.66, 124.00, 124.88,
127.70, 127.96, 129.10, 129.498, 129.73, 130.07, 130.62, 130.87,
131.06, 131.23, 131.88, 133.18, 133.44, 133.71, 134.29, 134.52,
136.22, 136.46, 137.01, 148.39, 148.57, 156.53, 156.66, 156.84,
157.02, 172.58, 173.37.
Light petroleum-ethyl acetate (80:20), yield 18%, white solid,
mp 72–73 °C; 1H NMR (CDCl3, 400 MHz) d 0.98 (3H, t, J = 7.3 Hz,
CH2CH2CH3), 1.26 (3H, d, J = 7.2 Hz, 6-CH3), 1.26 (2H, m,
CH2CH2CH3), 2.00 (3H, s, 3-CH3), 2.40 (2H, m, CH2CH2CH3), 3.25
(1H, q, J = 7.2 Hz, 6-CH), 5.24 (1H, s, 4-CH), 6.87–7.50 (13H, m, aro-
matics and NH); 13C NMR (CDCl3, 100 MHz) d 12.72, 14.14, 16.36,
23.29, 33.18, 36.25, 42.15, 104.94, 118.43, 119.19, 123.66, 127.51,
128.65, 129.28, 129.82, 130.59, 130.84, 134.85, 136.26, 136.47,
147.45, 156.62, 156.79, 172.00.
4.2.50. anti+syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
fluorophenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one (41)
Light petroleum-ethyl acetate (75:25), yield 48%, white solid;
1H NMR (CDCl3, 400 MHz) d 1.26 (3H, d, J = 7.2 Hz, 6-CH3, A),
1.40 (3H, d, J = 7.2, 6-CH3, B), 1.95 (3H, s, 3-CH3, A), 1.99 (3H, s,
3-CH3, B), 3.25 (1H, q, J = 7.2 Hz, 6-CH, A), 3.77 (1H, q, J = 7.2 Hz,
6-CH, B), 5.15 (1H, s, 4-CH, A), 5.60 (1H, s, 4-CH, B), 6.90–7.60
(28H, m, aromatics A and B); 13C NMR (CDCl3, 100 MHz) d 12.89,
13.12, 14.77, 16.01, 36.09, 38.49, 42.16, 44.15, 106.09, 110.23,
116.75, 116.95, 117.23, 118.43, 118.72, 119.06, 119.16, 123.49,
123.62, 124.87, 125.23, 129.09, 129.22, 129.53, 129.73, 130.01,
130.84, 130.92, 131.42, 133.94, 135.80, 136.44, 148.97, 149.20,
155.14, 156.69, 156. 79, 156.95, 157.63, 172.66, 173.48.
4.2.46. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
npropylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-
7-one [s( )38]
Light petroleum-ethyl acetate (80:20), yield 20%, white solid,
mp 95–99 °C; 1H NMR (CDCl3, 400 MHz) d 0.85 (3H, t, J = 7.3 Hz,
CH2CH2CH3), 1.48 (5H, m, CH2CH2CH3 and -6-CH3), 1.97 (3H, s, 3-
CH3), 2.35 (2H, m, CH2CH2CH3), 3.75 (1H, q, J = 7.2 Hz, 6-CH),
5.63 (1H, s, 4-CH), 6.95–7.05 (5H, m, aromatics), 7.20–7.50 (9H,
m, aromatics and NH); 13C NMR (CDCl3, 100 MHz) d 13.29, 14.10,
15.18, 23.38, 33.08, 38.92, 44.78, 108.49, 118.78, 119.07, 123.52,
127.25, 128.39, 129.56, 129.77, 130.32, 130.65, 133.95, 135.14,
135.38, 141.17, 147.85, 156.75, 156.99, 172.62; IR:
t 2962.13,
4.2.51. anti+syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
chlorophenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one (42)
1680.66, 1588.57, 1488.78, 1375.96, 1240.00, 1165.76, 1608.85,
908.79, 869.74 cmꢁ1; Anal. Calcd for C29H29N3O2S: C, 72.02; H,
6.04; N, 8.69; O, 6.62; S, 6.63. Found: C, 72.03; H, 6.03; N, 8.67;
S, 6.62.
Light petroleum-ethyl acetate (70:30), yield 56%, white solid;
1H NMR (CDCl3, 400 MHz) d 1.27 (3H, d, J = 7.2 Hz, 6-CH3, A),
1.35 (3H, d, J = 7.2 Hz, 6-CH3, A), 1.95 (6H, m, 3-CH3, A+B), 3.18
(1H, q, J = 7.2 Hz, 6-CH, A), 3.75 (1H, q, J = 7.2 Hz, 6-CH, B), 5.20
(1H, s, 4-CH, A), 5.60 (1H, s, 4-CH, B), 6.80–7.60 (28H, m, aromatics
and NH, A+B); 13C NMR (CDCl3, 50 MHz) d 13.22, 14.94, 16.25,
35.99, 38.64, 42.12, 44.34, 105.76, 109.64, 118.43, 118.75, 119.13,
119.26, 123.54, 123.69, 128.19, 129.33, 129.24, 129.57, 129.79,
130.28, 130.58, 130.83, 131,28, 131.58, 132.35, 133.83, 134.69,
135.90, 136.51, 148.71, 148.99, 157.01, 172.60, 172.82.
4.2.47. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
ipropylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )39]
Light petroleum-ethyl acetate (80:20), yield 18%, white solid,
mp 160–161 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (6H, d,
J = 6.9 Hz, CH(CH3)2), 1.27 (3H, d, J = 7.2 Hz, 6-CH3), 1.99 (3H, s,
3-CH3), 2.60 (1H, m, CH(CH3)2), 3.25 (1H, q, J = 7.2 Hz, 6-CH), 5.25
(1H, s, 4-CH), 6.95 (2H, d, J = 8.6 Hz, aromatics), 7.02 (2H, d,
J = 7.8 Hz, aromatics), 7.13 (1H, t, J = 7.4 Hz, aromatic), 7.20–7.51
(9H, m, aromatics and NH); 13C NMR (CDCl3, 100 MHz) d 12.78,
16.31, 23.59, 28.30, 36.15, 42.20, 104.90, 118.38, 119.25, 123.66,
127.43, 128.61, 128.28, 129.80, 130.92, 133.96, 136.44, 147.43,
156.60, 156.81, 171.97.
4.2.52. anti+syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
bromophenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one (43)
Light petroleum-ethyl acetate (65:35), yield 58%, pale yellow
solid; 1H NMR (CDCl3, 400 MHz) d 1.23 (3H, d, J = 7.2 Hz, 6-CH3,
A), 1.29 (3H, d, J = 7.2 Hz, 6-CH3, B), 1.95 (6H, m, 3-CH3, A+B),
3.25 (1H, q, J = 7.2 Hz, 6-CH, A), 3.82 (1H, q, J = 7.2 Hz, 6-CH, B),
5.15 (1H, s, 4-CH, A), 5.60 (1H, s, 4-CH, B), 6.95–7.75 (28H, m, aro-
matics and NH, A+B); 13C NMR (CDCl3, 50 MHz) d 13.17, 14.90,
16.25, 35.89, 38.74, 42.10, 44.34, 104.76, 108.64, 117.43, 118.75,
119.23, 119.26, 123.54, 123.69, 128.19, 129.33, 129.24, 129.57,
129.79, 130.28, 130.68, 130.83, 131,28, 131.58, 132.35, 133.78,
134.69, 135.90, 136.51, 148.59, 148.99, 157.01, 172.50, 172.80.
4.2.48. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
ipropylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )39]
Light petroleum-ethyl acetate (80:20), yield 18%, white solid, mp
105–107 °C; 1H NMR (CDCl3, 400 MHz) d 1.18 (6H, d, J = 6.9 Hz,
CH(CH3)2), 1.43 (3H, d, J = 7.2 Hz, 6-CH3), 1.92 (3H, s, 3-CH3), 2.55
(1H, m, CH(CH3)2), 3.75 (1H, q, J = 7.2 Hz, 6-CH), 5.61 (1H, s, 4-CH),
6.95 (2H, d, J = 8.6 Hz, aromatics), 7.02 (2H, d, J = 7.8 Hz, aromatics),
7.13 (1H, t, J = 7.4 Hz, aromatic), 7.20–7.51 (9H, m, aromatics and
NH); 13C NMR (CDCl3, 100 MHz) d 13.29, 15.14, 23.79, 28.07,
38.88, 44.76, 108.47, 118.74, 119.11, 123.52, 126.99, 127.18,
128.39, 129.57, 129.76, 130.68, 133.95, 135.61, 147.77, 156.74,
157.01, 172.60; Anal. Calcd for C29H29N3O2S: C, 72.02; H, 6.04; N,
8.69; O, 6.62; S, 6.63. Found: C, 72.04; H, 6.05; N, 8.70; S, 6.62.
4.2.53. anti+syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
iodophenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-
one (44)
Light petroleum-ethyl acetate (75:25), yield 61%, pale yellow
solid; 1H NMR (CDCl3, 400 MHz) d 1.29 (3H, d, J = 7.2 Hz, 6-CH3,
A), 1.47 (3H, d, J = 7.2 Hz, 6-CH3, B), 1.99 (6H, s, 3-CH3, A+B), 3.25
(1H, q, J = 7.2 Hz, 6-CH, A), 3.82 (1H, m, 6-CH, B), 5.20 (1H, s, 4-
CH, A), 5.63 (1H, s, 4-CH, B), 6.80–7.55 (27H, m, aromatics and
NH, A+B), 7.95 (1H, m, aromatic); 13C NMR (CDCl3, 50 MHz) d
14.22, 14.94, 16.26, 36.05, 38.65, 42.12, 44.34, 106.76, 110.66,
118.43, 118.75, 119.13, 120.27, 123.54, 123.69, 128.19, 129.33,
129.24, 129.72, 129.79, 130.18, 130.77, 130.83, 131,28, 131.58,
132.35, 133.93, 134.69, 135.90, 136.51, 148.71, 148.99, 157.07,
172.54, 172.78.
4.2.49. anti+syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
trifluoromethylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one (40)
Light petroleum-ethyl acetate (67:33), yield 54%, white solid;
1H NMR (CDCl3, 400 MHz) d 1.25 (3H, d, J = 7.2 Hz, 6-CH3, A),
1.35 (3H, d, J = 7.2, 6-CH3, B), 1.95 (3H, s, 3-CH3, A and B), 3.25
(1H, q, J = 7.2 Hz, 6-CH, A), 3.75 (1H, q, J = 7.2 Hz, 6-CH, B), 5.22

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3443
4.2.54. anti-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
methoxyphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-
7-one [a( )45]
5.21; N, 8.69; O, 9.93; S, 6.63. Found: C, 69.53; H, 5.20; N, 8.68;
S, 6.62.
Light petroleum-ethyl acetate (67:33), yield 25%, white solid,
4.3.3. anti-( )-4-(40-Carboxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )16]
mp 179–181 °C; 1H NMR (CDCl3, 400 MHz)
d 1.25 (3H, d,
J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 3-CH3), 3.26 (1H, q, J = 7.2 Hz, 6-
CH), 3.91 (3H, s, OCH3), 5.20 (1H, s, 4-CH), 6.95–7.50 (13H, m, aro-
matics), 7.52 (1H, s, NH); 13C NMR (CDCl3, 100 MHz) d 13.02, 16.03,
36.12, 42.32, 56.25, 105.78, 112.47, 118.47, 119.13, 121.71, 123.59,
125.91, 129.26, 129.57, 129.79, 131.02, 136.81, 148.39, 153.76,
156.70, 172.72.
Yield 95%; white solid, mp 255–258 °C; 1H NMR (CDCl3+CD3OD,
400 MHz) d 1.05 (3H, d, J = 7.2 Hz, 6-CH3), 1.75 (3H, s, 2-CH3), 1.98
(3H, s, 3-CH3), 3.03 (1H, q, J = 7.2 Hz, 6-CH), 5.02 (1H, s, 4-CH),
7.14–7.30 (7H, m, aromatics and NH), 7.46 (2H, d, J = 8.3 Hz, aro-
matics), 7.52 (2H, d, J = 8.3 Hz, aromatics), 7.96 (2H, d, J = 8.3 Hz,
aromatics); 13C NMR (CDCl3+CD3OD, 100 MHz) d 12.78, 16.32,
17.30, 36.30, 42.47, 104.83, 126.96, 127.51, 128.42, 128.53,
130.53, 130.73, 131.68, 135.23, 136.30, 136.99, 139.05, 142.23,
145.33, 147.83, 170.93, 173.53.
4.2.55. syn-( )-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
methoxyphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-
7-one [s( )45]
Light petroleum-ethyl acetate (67:33), yield 26%, white solid,
mp 195–197 °C; 1H NMR (CDCl3, 400 MHz)
d 1.42 (3H, d,
4.3.4. syn-( )-4-(40-Carboxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )16]
J = 7.2 Hz, 6-CH3), 2.01 (3H, s, 3-CH3), 3.81 (1H, q, J = 7.2 Hz, 6-
CH), 3.91 (3H, s, OCH3), 5.60 (1H, s, 4-CH), 6.91–7.20 (7H, m, aro-
matics), 7.52–7.65 (6H, m, aromatics), 7.54 (1H, s, NH); 13C NMR
(CDCl3, 100 MHz) d 13.07, 14.80, 38.48, 44.03, 56.49, 109.77,
112.47, 118.787, 119.03, 121.88, 123.45, 126.55, 128.79, 129.51,
129.75, 130.43, 134.43, 136.10, 148.61, 152.68, 156.84, 172.32;
Anal. Calcd for C27H25N3O3S: C, 68.77; H, 5.34; N, 8.91; O, 10.18;
S, 6.80. Found: C, 68.75; H, 5.34; N, 8.90; S, 6.79.
Yield 96%; white solid, mp 168–171 °C; 1H NMR (CDCl3+CD3OD,
400 MHz) d 1.40 (3H, d, J = 7.1 Hz, 6-CH3), 1.88 (3H, s, 2-CH3), 2.05
(3H, s, 3-CH3), 3.82 (1H, q, J = 7.2 Hz, 6-CH), 5.63 (s, 1H, 4-CH),
7.27–7.40 (7H, m, aromatics and NH), 7.55 (2H, d, J = 8.3 Hz, aro-
matics), 7.60 (2H, d, J = 8.3 Hz, aromatics), 8.06 (2H, d, J = 8.3 Hz,
aromatic); 13C NMR (CDCl3+CD3OD, 100 MHz) d 12.91, 14.88,
17.03, 38.78, 44.67, 108.74, 126.78, 127.10, 127.55, 128.16,
128.60, 129.15, 130.29, 131.34, 135.35, 135.59, 136.49, 139.24,
139.58, 144.73, 148.03, 168.57, 173.20; Anal. Calcd for
4.3. General procedure for the synthesis of 1H-pyrazolo[3,4-
e][1,4]thiazepin-7-ones 15, 16, 30–32
0.1 N aqueous sodium hydroxide (50 mL) was added to a solution
of the appropriate 1H-pyrazolo[3,4-e][1,4]thiazepin-7-one methyl
ester in THF (15 mL). The resulting suspension was stirred at
room temperature for 7–8 hours. THF was removed in vacuo and
the resulting aqueous phase was acidified to pH 5 and extracted with
ethyl acetate (2 ꢂ 30 mL). The combined organic phases were
washed with brine (20 mL), dried and the solvent removed in vacuo.
The amorphous white solid thus obtained was dried at 90 °C for 12
hours.
C
28H25N3O3S: C, 69.54; H, 5.21; N, 8.69; O, 9.93; S, 6.63. Found:
C, 69.55; H, 5.22; N, 8.68; S, 6.62.
4.3.5. anti-( )-4-[4-(2-Carboxyphenoxy)phenyl]-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )30]
Yield 97%; white solid, mp 135–137 °C; 1H NMR (CDCl3,
400 MHz) d 1.31 (3H, d, J = 7.2 Hz, 6-CH3), 1.97 (3H, s, 2-CH3),
2.15 (3H, s, 3-CH3), 3.21 (1H, q, J = 7.2 Hz, 6-CH), 5.20 (1H, s, 4-
CH), 6.90–7.55 (13H, m, aromatics, COOH and NH), 8.21 (1H, m,
aromatic); 13C NMR (CDCl3, 100 MHz) d 12.80, 16.35, 17.28,
36.27, 42.11, 104.68, 118.49, 119.69, 124.06, 127.53, 128.50,
129.72, 130.63, 131.75, 133.47, 134.80, 136.21, 147.65, 154.52,
156.64, 166.13, 172.05;
4.3.1. anti-( )-4-(30-Carboxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [a( )15]
Yield 95%; white solid, mp 224–227 °C; 1H NMR (CDCl3,
400 MHz) d 1.26 (3H, d, J = 7.2 Hz, 6-CH3), 1.96 (3H, s, 2-CH3),
2.13 (3H, s, 3-CH3), 3.26 (1H, q, J = 7.2 Hz, 6-CH), 5.25 (1H, s, 4-
CH), 7.25–7.50 (7H, m, aromatics and NH), 7.53 (1H, t, J = 7.7 Hz,
aromatic), 7.60 (2H, d, J = 8.4 Hz, aromatics), 7.75 (1H, m, aro-
matic), 8.08 (1H, m, aromatic), 8.31 (1H, m, aromatic); 13C NMR
(CDCl3, 100 MHz) d 12.54, 16.19, 17.16, 36.16, 42.27, 104.76,
127.22, 127.39, 128.36, 128.57, 128.91, 129.80, 130.52, 131.61,
131.93, 134.80, 136.26, 139.05, 140.51, 141.42, 147.60, 170.52,
172.18.
4.3.6. syn-( )-4-[4-(2-Carboxyphenoxy)phenyl]-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )30]
Yield 95%; white solid, mp 127–130 °C; 1H NMR (CDCl3,
200 MHz) d 1.35 (3H, d, J = 7.2 Hz, 6-CH3), 1.80 (3H, s, 2-CH3),
2.01 (3H, s, 3-CH3), 3.75 (1H, q, J = 7.2 Hz, 6-CH), 5.56 (1H, s, 4-
CH), 6.80–7.50 (11H, m, aromatics), 7.70 (1H, s, NH), 7.95 (1H, m,
aromatic), 8.9 (1H, bs, COOH); 13C NMR (CDCl3, 50 MHz) d 13.22,
15.10, 17.21, 38.88, 44.62, 108.59, 118.30, 119.04, 119.41, 121.06,
123.72, 127.13, 128.27, 129.47, 129.82, 130.21, 131.40, 133.00,
134.51, 135.12, 135.32, 155.70, 156.65, 167.82, 173.55; Anal. Calcd
for C28H25N3O4S: C, 67.32; H, 5.04; N, 8.41; O, 12.81; S, 6.42.
Found: C, 67.33; H, 5.03; N, 8.40; S, 6.41.
4.3.2. syn-( )-4-(30-Carboxybiphenyl-4-yl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-
one [s( )15]
Yield 96%; white solid, mp 245–248 °C; 1H NMR (CDCl3,
400 MHz) d 1.50 (3H, d, J = 7.2 Hz, 6-CH3), 1.96 (3H, s, 2-CH3),
2.13 (3H, s, 3-CH3), 3.80 (1H, q, J = 7.2 Hz, 6-CH), 5.75 (1H, s, 4-
CH), 7.25–7.50 (6H, m, aromatics), 7.52 (1H, t, J = 7.7 Hz, aromatic),
7.60 (2H, d, J = 8.4 Hz, aromatics), 7.70 (1H, s, NH), 7.75 (1H, m, aro-
matic), 8.02 (1H, m, aromatic), 8.27 (1H, m, aromatic); 13C NMR
(CDCl3, 100 MHz) d 13.03, 14.95, 17.11, 38.84, 44.77, 108.63,
127.06, 127.38, 128.17, 128.51, 128.61, 128.82, 129.85, 130.18,
131.32, 131.90, 135.20, 135.38, 136.48, 138.77, 139.46, 140.57,
147.85, 171.00, 173.48; Anal. Calcd for C28H25N3O3S: C, 69.54; H,
4.3.7. anti-( )-4-[4-(3-Carboxyphenoxy)phenyl]-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )31]
Yield 94%; white solid, mp 165–168 °C; 1H NMR (CDCl3+CD3OD,
400 MHz) d 1.24 (3H, d, J = 7.2 Hz, 6-CH3), 1.90 (3H, s, 2-CH3), 2.10
(3H, s, 3-CH3), 3.18 (1H, q, J = 7.2 Hz, 6-CH), 5.15 (1H, s, 4-CH), 7.00
(2H, m, aromatics), 7.20–7.50 (9H, m, aromatics), 8.65 (1H, s, NH),

3444
M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
7.80 (1H, m, aromatic); 13C NMR (CDCl3, 100 MHz) d 12.37, 15.95,
16.98, 36.05, 41.97, 105.13, 118.56, 120.00, 123.55, 124.89, 127.28,
128.33, 129.25, 129.68, 130.46, 131.48, 132.23, 134.96, 136.16,
136.77, 147.76, 156.23, 156.60, 167.90, 172.62.
To verify the ability of the hypothesis to identify correctly all
the agonists used for the pharmacophore generation, multiple test
runs were performed using different conditions. The best perfor-
mance was achieved setting the MinSites variable of the Phase Find
Matches screening (minimum number of sites to be matched by the
screened compounds to be selected as virtual hits) to 7 and leaving
all the others at default. The MinSites variable of the Phase Find
Matches screening was set to 7 while all the other variables were
leaved at default.
The structure based procedure consisted in a cross docking of all
the pharmacophore virtual hits in three complexes, selected to
cover different binding site conformations. This procedure led to
a final ranking made by six scores for each screened compound
(3 X-rays per two docking programs). The lowest score value ob-
tained by the five known agonists used in the pharmacophore gen-
eration each x-ray docking run have been used as a cut-off to select
only the screened ligands with higher ranking. The grid genera-
tions on each complex and all the docking runs were performed
leaving all the variables at default values. The Tanimoto clustering
was performed using the CACTVS toolkit31 and the SUBSET32
routine.
4.3.8. syn-( )-4-[(4-(3-Carboxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methyphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )31]
Yield 94%; white solid, mp 173–175 °C; 1H NMR (CDCl3,
200 MHz) d 1.35 (3H, d, J = 7.2 Hz, 6-CH3), 1.80 (3H, s, 20-CH3),
2.02 (3H, s, 3-CH3), 3.42 (1H, q, J = 7.2 Hz, 6-CH), 5.56 (1H, s, 4-
CH), 6.98 (2H, m, aromatics), 7.20–7.50 (9H, m, aromatics), 7.60
(1H, m, NH), 7.70 (1H, m, aromatic), 8.23 (1H, bs, COOH); 13C
NMR (CDCl3, 50 MHz)
d 13.19, 15.09, 17.22, 38.86, 44.65,
108.82, 118.66, 118.99, 120.15, 124.02, 125.04, 127.02, 128.34,
129.78, 131.29, 131.59, 134.45, 135.36, 136.64, 148.03, 156.46,
156.83, 169.83, 174.26; Anal. Calcd for C28H25N3O4S: C, 67.32;
H, 5.04; N, 8.41; O, 12.81; S, 6.42. Found: C, 67.31; H, 5.02; N,
8.41; S, 6.41.
4.3.9. anti-( )-4-[4-(4-Carboxyphenoxy)phenyl]-3,6-dimethyl-1-
(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [a( )32]
4.5. Biology
Yield 95%; white solid, mp 220–222 °C; 1H NMR (CDCl3,
400 MHz) d 1.26 (3H, d, J = 7.2 Hz, 6-CH3), 1.95 (3H, s, 2-CH3),
2.13 (3H, s, 3-CH3), 3.25 (1H, q, J = 7.2 Hz, 6-CH), 5.25 (1H, s, 4-
CH), 7.05 (2H, d, J = 8.7 Hz, aromatics), 7.09 (2H, d , J = 8.6 Hz, aro-
matics), 7.31–7.42 (7H, m, aromatics and NH), 8.08 (2H, d,
J = 8.7 Hz, aromatics); 13C NMR (CDCl3, 100 MHz) d 12.78, 16.28,
17.25, 36.28, 42.18, 104.91, 117.55, 119.86, 124.08, 127.41,
128.27, 128.50, 129.55, 130.48, 131.64, 132.36, 135.22, 136.19,
138.16, 147.69, 154.95, 161.80, 170.50, 172.40.
4.5.1. FXR AlphaScreen assay
FXR activity was assessed in a recruitment coactivator assay by
using AlphaScreen technology. Anti-GST-coated Acceptor beads
were used to capture the GST-fusion FXR-LBD, whereas the biotin-
ylated-SRC-1 peptide was captured by the streptavidin Donor
beads. Upon illumination at 680 nm chemical energy is transferred
from Donor to Acceptor beads across the complex streptavidin-Do-
nor/Src-1-Biotin/GSTFXR-LBD/Anti-GST-Acceptor and a signal is
produced. The assay was performed in white, low-volume, 384-
4.3.10. syn-( )-4-[4-(4-Carboxyphenoxy)phenyl]-3,6-dimethyl-
1-(2-methylphenyl)-4,8-dihydro-1H-pyrazole[3,4-
e][1,4]thiazepin-7-one [s( )32]
well Optiplates (Perkin Elmer) using a final volume of 25
3containing final concentrations of 10 nM of purified GST-tagged
FXR-LBD protein, 30 nM biotinylated Src-1 peptide, 20 g/ml
anti-GST-coated Acceptor beads and 10 g/ml of streptavidin Do-
nor bead (Perkin Elmer). The assay buffer contained 50 mM Tris
(pH 7.4), 50 mM KCl, 0.1% BSA, and 1 mM DTT. The stimulation
ll
l
Yield 97%, mp 168–171 °C; 1H NMR (CDCl3, 400 MHz) d 1.51
(3H, d, J = 7.2 Hz, 6-CH3), 1.82 (3H, s, 2-CH3), 2.21 (3H, s, 3-CH3),
3.81 (1H, q, J = 7.2 Hz, 6-CH), 5.71 (1H, s, 4-CH), 7.02 (4H, m, aro-
matics), 7.30–7.40 (7H, m, aromatics), 7.96 (1H, s, NH), 8.02 (2H,
d, J = 8.8 Hz, aromatics); 13C NMR (CDCl3, 100 MHz) d 13.21
15.05, 17.221, 38.90, 44.64, 108.70, 117.44, 120.16, 124.06,
127.10, 128.29, 129.85, 130.20, 131.37, 132.28, 135.40, 135.50,
136.58, 147.97, 155.19, 161.83, 170.43, 173.81; Anal. Calcd for
l
times with 1 ll of tested compound (solubilized in 100% DMSO)
were fixed to 30 min. at room temperature. The concentration of
DMSO in each well was maintained at a final concentration of
4%. After the addition of the detection mix (acceptor and Donor
beads), the plates were incubated in the dark for 4 h at room tem-
perature and then were read in Envision microplate analyzer (Per-
kin Elmer). Dose response curves were done in triplicate and Z0
factor was used to validate the assays. Non linear regression
curves, without constraints, were performed by using four param-
eter equation and GraphPad Prism Software (GraphPad Inc.), to ob-
tain the EC50 values.
C28H25N3O4S: C, 67.32; H, 5.04; N, 8.41; O, 12.81; S, 6.42. Found:
C, 67.31; H, 5.03; N, 8.40; S, 6.43.
4.4. Molecular modelling
All the PDBs used in this work were previously submitted to the
Protein Preparation Wizard protocol of the Schrodinger Suite.21 The
DrugLike (2 million) and the LeadLike (1 million) compounds of the
ZINC 8 database20 were prepared for the calculations removing all
the duplicates and generating all the tautomers, isomers and ioni-
zation states through the Ligprep procedure of the Schrodinger
Suite. A better computational time performance was achieved
transforming the resulting dataset in a 3D Phase database through
a standard procedure that adds a set of conformers to each mole-
cule. After the backbone superposition of the five selected com-
plexes, the ligands were extracted to identify their chemical
features and create the pharmacophoric hypothesis, using the
Manage hypothesis tool of the Phase package (present in the Schro-
dinger Suite). The excluded volumes were added using the residues
of the superimposed protein choosing all the side-chains providing
a larger volume in the binding site with the aim to create a less
restrictive pharmacophore.
4.5.2. FXR target gene expression
The mRNA expression level of three FXR target genes (Ostb, Bsep
and Cyp7a1) was measured by Real-Time Polymerase Chain
Reaction (Q-RTPCR). Total RNA was isolated (Aurum Total RNA
Mini Kit BioRad) from HepG2 cells stimulated with increasing
concentration of tested compound for 18 h. The RNA was random
reverse-transcribed with Iscript cDNA Synthesis kit (BioRad) in
20
ume reaction of Real-Time PCR containing 0.3
and 10 l of 2X SYBR Green PCR Master MIX (Bio-Rad). All reac-
l
l reaction volume. Ten ng template was used in 20
ll final vol-
lM of each primer
l
tions were performed in triplicate and the thermal cycling condi-
tions were: 3 min at 95 °C, followed by 45 cycles of 95 °C for
10 s, and 60 °C for 30 s in iCycler iQ5 instrument (Biorad, Hercules,
CA). The mean value of the replicates for each sample was calcu-
lated and expressed as cycle threshold (CT: cycle number at which

M. Marinozzi et al. / Bioorg. Med. Chem. 20 (2012) 3429–3445
3445
13. Nicolaou, K. C.; Evans, R. M.; Roecker, A. J.; Hughes, R.; Downes, M.; Pfefferkorn,
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15. Grienke, U.; Mihály-Bison, J.; Schuster, D.; Afonyushkin, T.; Binder, M.; Guan, S.
H.; Cheng, C. R.; Wolber, G.; Stuppner, H.; Guo, D. A.; Bochkov, V. N.; Rollinger,
J. M. Bioorg. Med. Chem. 2011, 19, 6779.
each PCR reaction reaches a predetermined fluorescence threshold,
set within the linear range of all reactions). The amount of gene
expression was then calculated as the difference (DCT) between
the CT value of the sample for the target gene and the mean CT va-
lue of that sample for the endogenous control. Relative expression
was calculated as the difference (DDCT) between the
DCT values of
16. (a) Akwabi-Ameyaw, A.; Bass, J. Y.; Caldwell, R. D.; Caravella, J. A.; Chen, L.;
Creech, K. L.; Deaton, D. N.; Jones, S. A.; Kaldor, I.; Liu, Y.; Madauss, K. P.; Marr,
H. B.; McFadyen, R. B.; Miller, A. B.; Navas, F., III; Parks, D. J.; Spearing, P. K.;
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(b) Akwabi-Ameyaw, A.; Bass, J. Y.; Caldwell, R. D.; Caravella, J. A.; Chen, L.;
Creech, K. L.; Deaton, D. N.; Madauss, K. P.; Marr, H. B.; McFadyen, R. B.; Miller,
A. B.; Navas, F., III; Parks, D. J.; Spearing, P. K.; Todd, D.; Williams, S. P.; Wisely,
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Bleicher, K.; Grether, U.; Martin, R. E.; Plancher, J.-M.; Kuhn, B.; Rudolph, M.;
Chen, L. Bioorg. Med. Chem. Lett. 2009, 19, 2595; (b) Bass, J. Y.; Caldwell, R. D.;
Caravella, J. A.; Chen, L.; Creech, K. L.; Deaton, D. N.; Madauss, K. P.; Marr, H. B.;
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the test sample and of the control sample (WT) for each target
gene. The relative quantization value was expressed and shown
as 2-DDCT. All PCR primers were intron spanning designed using
the software Beacon Designer on published sequence data from
the NCBI database.
Supplementary data
Supplementary data (general procedures for the synthesis of
pyrazoles 46a–l, 4-aryl-substituted benzaldehydes 47b–m and 4-
aryloxy-substituted benzaldehydes 47o–t; Characterization data
for compounds 46a–l, 47e, 47i, 47k, 47l, 47p and 47s. Chromato-
graphic estimation of the sample purity of 1H-pyrazolo[3,4-
e][1,4]thiazepin-7-ones 8–14, 17–29, 33–45) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.04.021. These data include MOL files and InChi-
Keys of the most important compounds described in this article.
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2008, 18, 5497.
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Crawley, M. L.; Commons, T.; Green, D. M.; Xu, W.; Hum, W.-T.; Eta, J. E.;
Feingold, I.; Patel, V.; Evans, M. J.; Lai, K.; Borges-Marcucci, L.; Mahaney, P. E.;
Wrobel, J. E. J. Med. Chem. 2010, 53, 1774.
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