Exploring the cycloheptathiophene-3-carboxamide scaffold to disrupt the interactions of the influenza polymerase subunits and obtain potent anti-influenza activity

Article abstract of DOI:10.1016/j.ejmech.2017.06.015

With the aim to identify small molecules able to disrupt PA-PB1 subunits interaction of influenza virus (flu) RNA-dependent RNA polymerase, and based on previous structural and computational information, in this paper we have designed and synthesized a new series of cycloheptathiophene-3-carboxamide (cHTC) derivatives. Their biological evaluation led to highlight important structural insights along with new interesting compounds, such as the 2-hydroxybenzamido derivatives 29, 31, and 32, and the 4-aminophenyl derivative 54, which inhibited viral growth in the low micromolar range (EC₅₀ = 0.18–1.2 μM) at no toxic concentrations (CC₅₀ > 250 μM). This study permitted to obtain among the most potent anti-flu compounds within the PA-PB1 interaction inhibitors, confirming the cHTC scaffold as particularly suitable to achieve innovative anti-flu agents.

Full text of DOI:10.1016/j.ejmech.2017.06.015

Accepted Manuscript
Exploring the cycloheptathiophene-3-carboxamide scaffold to disrupt the interactions
of the influenza polymerase subunits and obtain potent anti-influenza activity
Jenny Desantis, Giulio Nannetti, Serena Massari, Maria Letizia Barreca, Giuseppe
Manfroni, Violetta Cecchetti, Giorgio Palù, Laura Goracci, Arianna Loregian, Oriana
Tabarrini
PII:
S0223-5234(17)30461-0
10.1016/j.ejmech.2017.06.015
EJMECH 9510
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 November 2016
Revised Date: 13 April 2017
Accepted Date: 7 June 2017
Please cite this article as: J. Desantis, G. Nannetti, S. Massari, M. Letizia Barreca, G. Manfroni,
V. Cecchetti, G. Palù, L. Goracci, A. Loregian, O. Tabarrini, Exploring the cycloheptathiophene-3-
carboxamide scaffold to disrupt the interactions of the influenza polymerase subunits and obtain
potent anti-influenza activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.06.015.
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ACCEPTED MANUSCRIPT
Exploring the cycloheptathiophene-3-carboxamide scaffold to
disrupt the interactions of the influenza polymerase subunits and
obtain potent anti-influenza activity
a,1
b,1
a
a
Jenny Desantis , Giulio Nannetti , Serena Massari , Maria Letizia Barreca , Giuseppe Manfroni
a
a
b
c,*
b,2,
a,2,
,
Violetta Cecchetti , Giorgio Palù , Laura Goracci , Arianna Loregian *, Oriana Tabarrini *
a
b
c
Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy
Department of Molecular Medicine, University of Padua, 35121 Padua, Italy
Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy
*Corresponding authors:
Oriana Tabarrini
Department of Pharmaceutical Sciences,
University of Perugia, 06123 Perugia, Italy,
Tel: +39 075-585-5139, Fax: +39 075 585 5115
E-mail: oriana.tabarrini@unipg.it
Arianna Loregian
Department of Molecular Medicine,
University of Padua, 35121 Padua, Italy,
Tel: +39 049 8272363, Fax: +39 049 8272355
E-mail: arianna.loregian@unipd.it
Laura Goracci
Department of Chemistry, Biology and Biotechnology,
University of Perugia, 06123 Perugia, Italy
Tel: +39 075 5855632, Fax, +39 075 45646
E-mail: laura.goracci@unipg.it
1
Co-first authors.
Co-last authors.
2
TITLE RUNNING HEAD: Cycloheptathiophene derivatives with anti-influenza activity.
1

ACCEPTED MANUSCRIPT
ABSTRACT
With the aim to identify small molecules able to disrupt PA-PB1 subunits interaction of influenza
virus (flu) RNA-dependent RNA polymerase, and based on previous structural and computational
information, in this paper we have designed and synthesized a new series of cycloheptathiophene-3-
carboxamide (cHTC) derivatives. Their biological evaluation led to highlight important structural
insights along with new interesting compounds, such as the 2-hydroxybenzamido derivatives 29,
31, and 32, and the 4-aminophenyl derivative 54, which inhibited viral growth in the low
micromolar range (EC = 0.18 – 1.2 µM) at no toxic concentrations (CC > 250 µM).
5
0
50
This study permitted to obtain among the most potent anti-flu compounds within the PA-PB1
interaction inhibitors, confirming the cHTC scaffold as particularly suitable to achieve innovative
anti-flu agents.
Keywords: PA-PB1 interaction, influenza virus polymerase, protein-protein interaction inhibitors,
influenza virus inhibitors.
1. INTRODUCTION
Influenza virus (flu) is a major cause of high morbidity and mortality in humans due to respiratory
infectious diseases. The high mutation rate and genomic re-assortment among different strains lead
flu to elude vaccine-induced immunity and to develop resistance to the current antiviral drugs,
which are limited to neuraminidase and M2 ion channel inhibitors. Therefore, the development of
anti-flu therapeutics exploiting new targets is highly desirable. To this end, the inhibition of one or
more components of the viral RNA-dependent RNA polymerase (RdRp) could have a bright future
[
1,2]. Indeed, RdRp is essential for virus transcription, replication, and evolution. Moreover, its
structure is highly conserved among the different flu A, B, and C strains [3-6]. RdRp is a
heterotrimeric complex composed of polymerase acidic protein (PA), polymerase basic protein 1
(PB1), and polymerase basic protein 2 (PB2) subunits that work together in a tightly associated and
2

ACCEPTED MANUSCRIPT
coupled fashion [7]. Successful examples of RdRp complex inhibitors are favipiravir (T-705,
Avigan®), a PB1 inhibitor that has been approved in Japan to treat pandemic flu [8,9], and VX-787,
a PB2 inhibitor that is currently in phase II clinical development [10].
An alternative and appealing strategy to inhibit the RdRp functions is to interfere with its correct
assembly through protein-protein interaction (PPI) inhibitors. This approach may have the major
advantage to reduce the probability of generating escape mutants [11-15].
During the last four years, several remarkable efforts were focused on the identification of small
molecules as PA-PB1 subunits interaction inhibitors [15]. In this context, our groups gave the major
contribution starting from a structure-based virtual screening, which led to identify some interesting
hit compounds that were successively optimized [16-21]. In particular, the structural evolution of
hit compound 1 (Fig. 1) allowed to improve both the antiviral and PA-PB1 inhibitory activity, as in
the p-chloro derivative 2 [19] (Fig. 1), together with the delineation of useful structure-activity
relationship (SAR) insights.
In this study, a further optimization of the cycloheptathiophene-3-carboxamide (cHTC) scaffold has
been performed leading to obtain among the most potent anti-flu compounds within the PA-PB1
interaction inhibitors. Computational studies were also conducted rationalizing their binding mode
within the PA cavity.
Fig. 1. Structures of cHTC derivatives 1 and 2.
3

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2. CHEMISTRY
The synthesis of the cHTC derivatives 21-24, 29-34, 36, and 37 was accomplished by applying the
two-steps Gewald synthesis, as outlined in Scheme 1. Thus, the 2-cyano-N-(substituted)-acetamides
4
[22], 5, 6 [22], 7-9, and 10 [23], prepared by reacting 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-
oxopropanenitrile 3 [22] with the appropriate amines in toluene, were used in a first Knoevenagel
condensation with cycloheptanone. The successive cyclization step performed in the presence of
sulfur and N,N-diethylamine in EtOH gave synthones 11 [19], 12-16, and 17 [24]. The reaction of
synthones 11-17 with the appropriate acyl chlorides in dry pyridine led to the methoxyl derivatives
1
8 [25], 19, 20, 24-28, 34, and 35. The successive O-demethylation performed with BBr in dry
3
CH Cl provided the hydroxyl target compounds 21-23, 29-33, 36, and 37.
2
2
An alternative strategy was used for the synthesis of the target compounds 47-52 (Scheme 2), all
characterized by the o-hydroxyl phenyl ring at C-2 position, entailing the preparation of the key
intermediate 40 suitable to be widely functionalized at the C-3 position. Synthone 40 was in turn
prepared by reacting ethyl 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylate 38
[
26] with the 2-methoxybenzoyl chloride in dry pyridine, to obtain derivative 39 that was then
hydrolyzed under basic conditions. Coupling reaction of 3-carboxylic acid 40 with the appropriate
alkyl amines in presence of benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) and collidine in dry CH Cl gave the desired 2-methoxybenzamido
2
2
derivatives 41-46. Of note, the coupling reaction carried out in the same conditions but using
anilines for the synthesis of the target compounds shown in Scheme 1, did not furnish the desired
compounds but gave only the cyclized cyclohepta[4,5]thieno[2,3-d][1,3]oxazin-4-one derivatives.
The o-hydroxy target derivatives 47-52 were then obtained by O-demethylation of methoxyl
intermediates 41-46 in the presence of BBr in dry CH Cl .
3
2
2
4

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of compounds 21-24, 29-34, 36, and 37. Reagents and conditions: (i) amines,
toluene, reflux; (ii) cycloheptanone, ammonium acetate, glacial acetic acid, benzene, reflux; (iii)
sulfur, N,N-diethylamine, EtOH, 40−50 °C; (iv) acyl chlorides, dry pyridine, rt; (v) BBr , dry
3
CH Cl , rt.
2
2
5

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of compounds 47-52. Reagents and conditions: (i) 2-methoxybenzoyl
chloride, dry pyridine, rt; (ii) LiOH, H O/THF, 50 °C; (iii) amines, BOP, collidine, dry CH Cl , rt;
2
2
2
(
iv) BBr , dry CH Cl , rt.
3 2 2
3. DESIGN OF cHTC ANALOGUES, BIOLOGICAL EVALUATION, AND DISCUSSION
In the first optimization study of hit compound 1, the attention was mainly focused on the C-2
position of the cHTC scaffold: different substituents replaced the o-fluorine atom, one or two
methylene units were inserted between the C-2 phenyl ring and the core, and the phenyl ring was
replaced by bulky aromatic rings or a cyclohexyl moiety. Fewer modifications involved the
cycloheptane ring, whose size was reduced to cyclohexane or cyclopentane, and the C-3 pyridinyl
carboxamide moiety, which was replaced by smaller groups, such as primary amide, ethyl
carboxylate, and carboxylic acid [19]. From this first set of compounds, clear SAR information
emerged: the cycloheptane cannot be replaced by smaller rings and aromatic moieties both at C-2
and at C-3 position are required to impart potent activity.
By analyzing the predicted binding modes of the best PA-PB1 inhibitors reported so far in the PA
cavity from X-ray crystal structure PDB ID 3CM8 [27], it emerged that two hydrophobic
interactions (one is essential for the interaction with W706) seem sufficient to inhibit PA-PB1
6

ACCEPTED MANUSCRIPT
interaction [15]. When additional hydrophobic interactions do not occur, favorable stabilization of
the PA-small molecule interaction seems to take place with the formation of one (with Q408) or
more favorable H-bonds.
By aligning some PA-PB1 inhibitors, we proposed a first pharmacophore model entailing two
hydrophobic moieties separated by a “polar belt” with two H-bond acceptor points and two H-bond
donor points [20]. More recently, we generated a new pharmacophore model by using all the best
PA-PB1 inhibitors reported to date [15]. It preserves the two hydrophobic moieties and one H-bond
acceptor point, while the two H-bond donor points do not seem critical for intermolecular
interactions. Thus, two hydrophobic moieties and a central H-bond acceptor point appear to be the
minimum structural requirement for PA-PB1 inhibition. However, since eight out of the eleven best
PA-PB1 inhibitors studied matched two H-bond interaction points, additional H-bond interactions
should be exploited to achieve a more efficient PA-PB1 inhibition.
Taking into account the indications coming from the previous SAR coupled with the recent
computational suggestions, a new set of derivatives has been designed by inserting a hydroxyl
group to the different positions of the C-2 phenyl ring (compounds 21-23, Table 1).
Computational studies based on the Fingerprints for Ligands and Proteins (FLAP) algorithm [28]
were performed to analyze their binding poses in the PA cavity from the structure PDB ID 3CM8
[
27]. To be consistent with our previous results [15-17,19-21], the FLAP software was used in the
structure-based” mode; this approach allows to generate binding poses of a ligand in a protein
“
cavity based on the similarity between their GRID fields [29]. Thus, although this approach is not a
standard energy-based docking method, it proved to be successful in identifying or optimizing PA-
PB1 inhibitors [16,19-21]. Similarity scores were used to rank the most reliable poses for
compounds 21-23 (Fig. 2B-D); the FLAP binding pose previously obtained for hit compound 1 [19]
was also reported as a reference in Fig. 2A. Compound 1 interacted with three hydrophobic pockets:
the first defined by W706 and F411, the second defined by P710 and L666, and a central
hydrophobic region mainly generated by the aliphatic chain of E623 and the protein backbone.
7

ACCEPTED MANUSCRIPT
According to our model, the insertion of the hydroxyl group induced a significant change of the
binding mode. Indeed, the key hydrophobic interaction with W706 still occurred for all compounds
1
and 21-23, even though the orientation of the cycloheptathiophene core in compounds 21-23
resulted to be inverted with respect to 1. As a consequence, the hydroxyl-substituted phenyl ring
pointed towards a region of the PA cavity defined by F411, C415, and I621. Hydrophobic and H-
bond interactions contributed to obtain a more favorable binding mode for compounds 21-23. Based
on the FLAP similarity scoring, which takes into account a global estimation of hydrophobic,
hydrophilic, H-bond donor, and H-bond acceptor interactions, compounds 21-23 are almost
equivalent (0.65, 0.63, and 065, respectively), with higher values with respect to compound 1
(0.53). A deeper visual inspection of the binding poses in Figure 2 was performed. The o-hydroxyl
derivative 21 displayed the most efficient hydrophobic interactions between the
cycloheptathiophene moiety with W706 and between the hydroxyphenyl group with the
hydrophobic region defined by F411, C415, and I621 (Fig. 2B). The latter hydrophobic region was
recently described as another key area of PA for the interaction with potent inhibitors [30]. In
addition, compound 21 resulted able to form a H-bond between I621 with its hydroxyl group, and a
second one between the C-3 carbonyl group with Q408. On the contrary, the FLAP binding poses
for the meta and para isomers 22 and 23 resulted shifted towards C415, weakening the hydrophobic
interaction with W706 (Fig. 2C-D). This shift could allow 23 to form a new H-bond interaction
between the p-hydroxyl group and the carbonyl group of C415, while the m-hydroxyl group was not
involved in any specific interaction. Additional H-bonds with I621 and Q408 were still possible for
both compounds 22 and 23.
8

ACCEPTED MANUSCRIPT
Fig. 2. FLAP binding poses for compounds 1 (A), 21 (B), 22 (C), and 23 (D). Some of the PA key
residues located in the cavity are highlighted in stick-mode.
The synthesized derivatives were evaluated for the ability to inhibit both the physical interaction
between fluA PA and PB1 subunits (by ELISA), the antiviral activity (by plaque reduction assays
(PRA)) and in parallel for their cytotoxic effect (by MTT assay), as detailed in the experimental
section. The data are reported in Table 1.
In agreement with the similarity scores obtained by the computational study, the ortho hydroxyl
derivative 21 was the most active by efficiently interfering with PA-PB1 interaction (IC = 15 µM)
5
0
and exhibiting potent antiviral activity (EC = 2.6 µM) at no cytotoxic concentrations (CC > 250
5
0
50
µM; selectivity index (SI) > 96). The shifting of the hydroxyl group to meta and para positions
compounds 22 and 23, respectively) decreased both the PA-PB1 interaction inhibitory activity and
mainly the antiviral effect.
(
9

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Table 1. Structure and Biological Activity of the cHTCs Synthesized in This Study.
ELISA
Cytotoxicity
MTT Assay) in
MDCK cells
PA-PB1
Interaction
Assay
PRA
in MDCK cells
(
Compd
1
R1
R2
b
EC50, µM
c
a
CC50, µM
IC , µM
5
0
F
145 ± 12
90 ± 13
18 ± 2
>250
>250
32 ± 9
2
“
“
“
“
“
MeO
d
18
22 ± 2
15 ± 2
86 ± 8
147 ± 39
>250
2
1
2
3
4
9
0
1
2
3
2.6 ± 0.5
2
2
2
2
3
3
3
3
23 ± 6
26 ± 4
78 ± 11
18 ± 1
22 ± 9
19 ± 2
248 ± 1
>250
MeO
82 ± 3
>250
“
1.2 ± 0.3
>250
“
58 ± 16
20 ± 5
30 ± 1
1.2 ± 0.4
0.18 ± 0.07
>100
>250
>250
“
“
“
69 ± 15
114 ± 21
>250
172 ± 39
1
0

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3
3
3
4
4
4
5
5
5
4
6
7
7
8
9
0
1
2
45 ± 14
24 ± 6
>250
HO
“
26 ± 6
1.8 ± 1.0
28 ± 8
HN
“
118 ± 21
33 ± 6
8.2 ± 1.1
>100
>250
>250
“
“
“
“
“
191 ± 21
>200
>100
172 ± 18
>250
>100
156 ± 8
>200
>100
>250
94 ± 6
>100
>250
197 ± 15
>200
145 ± 7
>250
d
53
101 ± 6
0.26 ± 0.13
d
54
“
65 ± 20
>250
RBV
10 ± 2
41 ± 5
>250
>100
Tat-PB11-15
peptide
35 ± 4
a
Activity of the compounds in ELISA PA-PB1 interaction assays. The IC value represents the compound
50
b
concentration that reduces by 50% the interaction between PA and PB1. Activity of the compounds in plaque
reduction assays with the flu A A/PR/8/34 strain. The EC50 value represents the compound concentration that inhibits
c
5
0% of plaque formation. Activity of the compounds in MTT assays. The CC50 value represents the compound
concentration that causes a decrease of cell viability of 50%. All the reported values represent the means ± SD of data
d
derived from at least three independent experiments in duplicate. Reported in reference [25].
1
1

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Given the potent antiviral profile of compound 21, the o-hydroxyphenyl moiety was maintained at
the C-2 position while the C-3 position was thoroughly explored in a successive set of derivatives
(compounds 29-33 and 47-52, Table 1).
Para halogenated derivatives 29 and 31 showed a good anti-flu activity in the cellular context, even
better than compound 21, with EC values of 1.2 µM; in addition, the absence of cytotoxicity up to
50
250 µM led to SIs > 200. Also the C-3 thiazole ring improved the anti-flu activity; indeed,
compound 32 emerged as the most potent of the series with an EC of 0.18 µM, which, coupled
5
0
with the lack of cytotoxicity up to 250 µM, led to the impressive SI > 1388. However, compound
2 showed a decreased ability to inhibit the PA-PB1 interaction. Conversely, compound 30, having
3
a nude phenyl ring as C-3 substituent, was found to be a weak inhibitor of both PA-PB1 interaction
and flu growth. The introduction of a methylene unit as a spacer between the aromatic ring and the
scaffold (compounds 47 and 48) as well as the replacement of the aromatic ring with aliphatic
substituents (compounds 33 and 49-52) at the C-3 position were in all cases detrimental.
The o-hydroxyl phenyl moiety still conferred anti-PA-PB1 and above all anti-flu activity when
moved to the C-3 position and coupled with the C-2 p-chlorophenyl ring, as in compound 36, even
though it caused cytotoxicity. FLAP-computational studies suggested that compound 36 and its
analogue 2, which showed a comparable anti-PA-PB1 effect, exhibit a similar binding pose in the
PA cavity, with the p-chloro substituent likely oriented into an enlarged hydrophobic pocket
generated by L666 and a rotated P710 (not shown). For what concerns the other C-3 hydroxyphenyl
compound 37, although showing good ability to inhibit viral growth at no toxic concentrations, only
a weak anti-PA-PB1 activity was observed.
Methoxy intermediates 18 [25], 24, and 34 (Table 1) were also assayed to enrich the SAR. Their
ability to interfere with PA-PB1 binding and mainly the antiviral activity dramatically decreased, as
compared with their counterparts 21, 29, and, 36, respectively. Also the replacement of the
hydroxyl with an amino group both in ortho (compound 53 [25]) and para (compound 54 [25])
positions was detrimental for what concerns the PA-PB1 inhibition (Table 1). However, the p-
1
2

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amino derivative 54 exhibited a very potent anti-flu activity with an EC of 0.26 µM and it was
5
0
found to be not cytotoxic in MDCK cells (CC > 250), reaching an SI > 961.
5
0
4. CONCLUSIONS
The continuous need to identify innovative anti-flu compounds led us to explore the subunits
assembly of RdRp as a possible alternative antiviral target. This approach has already proven to be
successful with the identification of effective PA-PB1 interaction inhibitors endowed with the
ability to inhibit flu growth.
In this study, we came back to the cHTC scaffold that was functionalized with a hydroxyl phenyl
moiety with the aim of exploiting additional H-bond interactions with key residues of the PA
cavity, as suggested by recent computational studies [15]. The synthesis of more than 20
compounds led to identify very interesting derivatives. In particular, C-2 o-hydroxyphenyl
derivatives 21, 29, and 31, functionalized at the C-3 position with 2-pyridinyl, p-chlorophenyl, and
p-fluorophenyl groups, respectively, emerged as the best in inhibiting PA-PB1 interaction with IC50
values of 15- 20 µM. This ability well translated into a potent anti-flu activity with EC of 1.2-2.6
5
0
µM. Two additional compounds, C-3 thiazole derivative 32 and C-2 p-aminophenyl compound 54,
although showing a weaker ability to inhibit PA-PB1 interaction, were endowed with a very potent
anti-flu activity (EC = 0.18 and 0.26 µM), reaching SI values >1388 and >961, respectively. For
5
0
these compounds, further studies are in progress to uncover whether an additional mechanism of
action might contribute to their potent anti-influenza activity.
This study confirmed the cHTC core as particularly suitable to obtain innovative and potent anti-flu
agents.
1
3

ACCEPTED MANUSCRIPT
In this study, we came back to the cHTC scaffold that was functionalized with a hydroxylphenyl
moiety with the aim of exploiting additional H-bond interactions with key residues of the PA
cavity, as suggested by recent computational studies [15]. The synthesis of more than 20
compounds added important SAR insights to the cHTC series of derivatives. In particular, the o-
hydroxyphenyl moiety i) emerged as particularly appropriate to impart PA-PB1 inhibitory activity,
ii) should be placed at the C-2 position of the scaffold (its shifting to the C-3 position as in
compound 37 was detrimental), and iii) has to be coupled with an aromatic ring at the C-3 position
(
C-3 aliphatic derivatives were almost inactive).
The most interesting derivatives identified in this study were the C-2 o-hydroxyphenyl derivatives
1, 29, and 31, functionalized at the C-3 position with 2-pyridinyl, p-chlorophenyl, and p-
fluorophenyl groups, respectively, which inhibited PA-PB1 interaction with IC values of 15-20
2
5
0
µM. This ability well translated into a potent anti-flu activity with EC values of 1.2-2.6 µM. Two
5
0
additional compounds, the C-3 thiazole derivative 32 and the C-2 p-aminophenyl compound 54,
although showing a weaker ability to inhibit PA-PB1 interaction, were endowed with a very potent
anti-flu activity (EC = 0.18 and 0.26 µM), reaching SI values >1388 and >961, respectively. For
5
0
these compounds, further studies are in progress to uncover whether an additional mechanism of
action might contribute to their potent anti-influenza activity.
In conclusion, this study confirmed the cHTC core as particularly suitable to obtain innovative and
potent anti-flu agents.
5. EXPERIMENTAL SECTION
5.1. Computational methods
The binding poses in the PA cavity were generated using the FLAP software in the structure-based
mode (Molecular Discovery Ltd., UK; www.moldiscovery.com). The procedure has been
extensively described elsewhere [17]. As in the previous studies, the main cavity of the
crystallographic structure of a large C-terminal fragment of PA (aa 257-716) (pdb code: 3CM8)
1
4

ACCEPTED MANUSCRIPT
[
27] was used as a template. A total of 50 conformers for each ligand were generated to mimic the
compound flexibility, and the most abundant protonation state of each molecule was used, as
predicted by MoKa [31]. The probes used to generate the GRID Molecular Interaction Fields were
H (shape), DRY (hydrophobic interactions), N1 (H-bond donor) and O (H-bond acceptor)
interactions. The global similarity score (Glob-Pro) was used for ranking.
5
5
.2. Chemistry
.2.1. General experimental procedures
Commercially available starting materials, reagents, and solvents were used as supplied. All
reactions were routinely monitored by TLC on silica gel 60F254 (Merck) and visualized by using
UV or iodine. Flash column chromatography was performed on Merck silica gel 60 (mesh 230-
4
00). After extraction, organic solutions were dried over anhydrous Na SO , filtered, and
2 4
concentrated with a Büchi rotary evaporator at reduced pressure. Yields are of purified product and
were not optimized. HRMS spectra were registered on Agilent Technologies 6540 UHD Accurate
Mass Q-TOF LC/MS, HPLC 1290 Infinity. The purity of all the target compounds was determined
to be >95% by LC/MS on Agilent Technologies 6550 iFUNNEL Q-TOF equipped with HPLC
1290 Infinity with DAD detector. HPLC conditions to assess the purity of final compounds were as
follows: column, Phenomenex AERIS Widepore C4, 4.6mm × 100 mm (6.6 µm); flow rate, 0.85
mL/min; acquisition time, 10 min; DAD 190−650 nm; oven temperature, 30 °C; gradient of
1
13
acetonitrile in water containing 0.1% of formic acid (0−100% in 10 min). H NMR and C NMR
spectra were recorded on Bruker Avance DPX-200 and Bruker Avance DRX-400MHz using
residual solvents such as dimethylsulfoxide (δ = 2.48) as an internal standard. Chemical shifts were
recorded in ppm (δ) and the spectral data are consistent with the assigned structures. The spin
multiplicities are indicated by the symbols s (singolet), d (doublet), t (triplet), q (quartet), m
(multiplet), and bs (broad singolet).
1
5

ACCEPTED MANUSCRIPT
The synthesis of some compounds reported in this study, i.e., 3 [22], 6 [22], 11 [19], 17 [24], 18
[
25], 38 [26], 53 [25], and 54 [25], has been previously described. Derivatives 12-14, 16, and 40 are
commercially available but their synthesis has been not published before and is herein reported. The
synthesis of compounds 5 [23], 7 [32], 8 [33], and 9 [34] has been previously described but their
preparation is herein reported through a different synthetic strategy.
5.2.2. General procedure for the preparation of 2-cyano-N-(substituted)acetamides (Method A). To
a solution of the appropriate amine (1.0 equiv) in toluene, compound 3 [22] (1.0 equiv) was added.
The mixture was refluxed for 5 min, and then stirred at room temperature to obtain a precipitate,
which was filtered, washed with Et O, and dried.
2
5.2.3. N-(4-Chlorophenyl)-2-cyanoacetamide (5). The tile compound was prepared starting from p-
1
chloroaniline through Method A, in 84% yield as white solid. H NMR (200 MHz, DMSO-d ): δ
6
3
1
5
.80 (s, 2H, CH ), 7.35 (d, J = 8.8 Hz, 2H, aromatic CH), 7.50 (d, J = 8.8 Hz, 2H, aromatic CH),
2
0.25 (bs, 1H, NH).
.2.4. 2-Cyano-N-(4-fluorophenyl)acetamide (7). The tile compound was prepared starting from p-
1
fluoroaniline through Method A, in 87% yield as white solid. H NMR (200 MHz, DMSO-d ): δ
6
3
.75 (s, 2H, CH ), 7.00-7.20 (m, 2H, aromatic CH), 7.40-7.50 (m, 2H, aromatic CH), 10.50 (bs, 1H,
2
NH).
5.2.5. 2-Cyano-N-(thiazol-2-yl)acetamide (8). The title compound was prepared starting from 2-
1
aminothiazole through Method A, in 87% yield as white solid. White solid. H NMR (200 MHz,
DMSO-d ): δ 4.00 (s, 2H, CH ), 7.20 and 7.45 (d, J = 3.5 Hz, each 1H, aromatic CH) 12.50 (bs,
6
,
2
1H, NH).
5.2.6. 2-Cyano-N-cyclohexylacetamide (9) The tile compound was prepared starting from
1
cyclohexylamine through Method A, in 72% yield as white solid. H NMR (200 MHz, DMSO-d ):
6
δ 1.00-1.45 and 1.50-1.80 (m, each 5H, cyclohexyl CH ), 3.45-3.50 (m, 1H, cyclohexyl CH), 3.55
2
(
s, 1H, CH ), 8.05 (d, J = 7.6 Hz, 1H, NH).
2
1
6

ACCEPTED MANUSCRIPT
5.2.7. General procedure for the preparation of 2-amino-N-(substituted)-cycloheptathiophene-3-
carboxamides (Method B). By applying a two-steps Gewald synthesis, a mixture of the appropriate
-cyano-N-(substituted)acetamide (1.0 equiv), cycloheptanone (4.0 equiv), ammonium acetate (1.3
2
equiv), and glacial acetic acid (3.5 equiv) in benzene (10 ml/mmol) was heated at reflux for 16 h in
a Dean-Stark apparatus. After cooling, the mixture was diluted with CHCl and then washed with
3
H O, 10% NaCO solution, and finally H O. The organic layer was concentrated to dryness to
2
3
2
afford the crude Knoevenagel product, which was used directly in the successive step without
further purification. Thus, to the crude Knoevenagel product (1.0 equiv) dissolved in EtOH, sulfur
(4.0 equiv) and N,N-diethylamine (4.0 equiv) were added. The mixture was maintained at 40-50 °C
for 2 h and then concentrated to dryness to yield a residue, which was treated with a mixture of
cyclohexane/Et O and filtered.
2
5.2.8. 2-Amino-N-(4-chlorophenyl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide
(12).The title compound was prepared starting from 5 through Method B, in 61% yield as red-
1
brown solid. H NMR (400 MHz, DMSO-d ): δ 1.40-1.60 (m, 4H, cycloheptane CH ), 1.65-1.80
6
2
(
m, 2H, cycloheptane CH ), 2.40-2.55 and 2.60-2.70 (m, each 2H, cycloheptane CH ), 5.80 (bs, 2H,
2
2
NH ), 7.25 (d, J = 8.8 Hz, 2H, aromatic CH), 7.60 (d, J = 8.8 Hz, 2H, aromatic CH), 9.55 (bs, 1H,
2
NH).
5.2.9. 2-Amino-N-phenyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (13). The
title compound was prepared starting from 6 [22] through Method B, in 64% yield as red-brown
1
solid. H NMR (200 MHz, CDCl ): δ 1.40-1.70 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H,
3
2
cycloheptane CH ), 2.50-2.60 and 2.75-2.85 (m, each 2H, cycloheptane CH ), 5.05 (s, 2H, NH ),
2
2
2
7
.05 (t, J = 7.2 Hz, 1H, aromatic CH), 7.20-7.35 (m, 2H, aromatic CH), 7.45 (d, J = 7.9 Hz, 2H,
aromatic CH), 9.50 (s, 1H, NH).
.2.10. 2-Amino-N-(4-fluorophenyl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide
14). The title compound was prepared starting from 7 through Method B, in 41% yield as red-
5
(
1
7

ACCEPTED MANUSCRIPT
1
brown solid. H NMR (400 MHz, DMSO-d ): δ 1.40-1.60 (m, 4H, cycloheptane CH ), 1.60-1.90
6
2
(
m, 2H, cycloheptane CH ), 2.50-2.70 (m, 4H, cycloheptane CH ), 5.75 (bs, 2H, NH ), 6.90-7.10
2
2
2
(m, 2H, aromatic CH), 7.70-7.85 (m, 2H, aromatic CH), 9.50 (s, 1H, NH).
5.2.11.
2-Amino-N-(thiazol-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide
(15). The title compound was prepared starting from 8 through Method B, in 40% yield as yellow
1
solid. H NMR (200 MHz, CDCl ): δ 1.50-1.70 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H,
3
2
cycloheptane CH ), 2.50-2.60 and 2.75-2.85 (m, each 2H, cycloheptane CH ), 5.15 (s, 2H, NH ),
2
2
2
6.90 and 7.30 (d, J = 3.6 Hz, each 1H, aromatic CH), 10.00 (s, 1H, NH).
5.2.12. 2-Amino-N-cyclohexyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (16).
The title compound was prepared starting from 9 through Method B, in 55% yield as light-brown
1
solid. H NMR (200 MHz, DMSO-d ): δ 1.00-1.25 and 1.40-1.55 (m, each 5H, cyclohexyl CH ),
6
2
1
.60-1.75 (m, 8H, cycloheptane CH ), 2.50-2.60 (m, 2H, cycloheptane CH ), 3.50-3.60 (m, 1H,
2
2
cyclohexyl CH), 5.50 (bs, 2H, NH ), 7.20 (d, J = 7.6 Hz, 1H, NH).
2
5.2.13. General procedure for C-2 amidation (Method C). A solution of the appropriate synthones
(1.0 equiv) in dry pyridine was added of the suitable benzoyl chloride (2.0 equiv). The reaction
mixture was maintained at room temperature until no starting material was detected by TLC. After
cooling, the reaction mixture was poured into ice/water, obtaining a precipitate which was filtered
and purified as described below.
5.2.14. 2-(3-Methoxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (19). The title compound was prepared starting from 11 [19] through Method C using
3
7
-methoxybenzoyl chloride and purified by flash chromatography eluting with cyclohexane:EtOAc
1
:3, in 41% yield as red-brown solid. H NMR (400 MHz, DMSO-d ): δ 1.50-1.60 (m, 4H,
6
cycloheptane CH ), 1.80-1.90 (m, 2H, cycloheptane CH ), 2.70-2.85 (m, 4H, cycloheptane CH ),
2
2
2
3
.75 (s, 3H, OCH ), 7.05-7.15 (m, 2H, aromatic CH and pyridine CH), 7.35-7.45 (m, 3H, aromatic
3
1
8

ACCEPTED MANUSCRIPT
CH), 7.70-7.80 (m, 1H, pyridine CH), 8.10 (d, J = 8.2 Hz, 1H, pyridine CH), 8.25-8.30 (d, 1H,
pyridine CH), 10.30 and 10.90 (s, each 1H, NH).
5.2.15. 2-(4-Methoxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (20). The title compound was prepared starting from 11 [19] through Method C using
4
-methoxybenzoyl chloride and purified by treatment with Et O, in 34% yield, as red-brown solid.
2
1
H NMR (200 MHz, DMSO-d ): δ 1.40-1.60 (m, 4H, cycloheptane CH ), 1.65-1.80 (m, 2H,
6
2
cycloheptane CH ), 2.60-2.80 (m, 4H, cycloheptane CH ), 3.80 (s, 3H, OCH ), 6.95 (d, J = 8.7 Hz,
2
2
3
2H, aromatic CH), 7.05-7.15 (m, 1H, pyridine CH), 7.70-7.85 (m, 3H, aromatic CH and pyridine
CH), 8.15 (d, J = 8.3 Hz, 1H, pyridine CH), 8.25-8.35 (m, 1H, pyridine CH), 10.30 and 10.90 (s,
each 1H, NH).
5.2.16. General procedure for O-demethylathion (Method D). To a solution of the appropriate
methoxy derivative (1.0 equiv) in dry CH Cl , 1M solution of BBr in CH Cl (6.0 equiv) was
2
2
3
2
2
added dropwise maintaining the temperature at 0 °C. The reaction mixture was stirred at room
temperature for 3-6 h and then quenched with MeOH and water. The organic solvent was removed
under vacuum affording a residue, which was filtered and purified as described below.
5.2.17. 2-(2-Hydroxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (21). The title compound was prepared starting from 18 [25] through Method D and
1
purified by crystallization by EtOH/H O, in 48% yield as light-yellow solid. H NMR (400 MHz,
2
DMSO-d ): δ 1.50-1.65 (m, 4H, cycloheptane CH ), 1.70-1.85 (m, 2H, cycloheptane CH ), 2.65-
6
2
2
2
.70 and 2.70-2.80 (m, each 2H, cycloheptane CH ), 6.85-6.95 (m, 2H, aromatic CH), 7.05-7.15 (m,
2
1H, pyridine CH), 7.35 (t, J = 7.7 Hz, 1H, aromatic CH), 7.80 (t, J = 7.1 Hz, 1H, pyridine CH), 7.95
(d, J = 7.7 Hz, 1H, aromatic CH), 8.05 (d, J = 8.1 Hz, 1H, pyridine CH), 8.30 (d, J = 4.6 Hz, 1H,
13
pyridine CH), 10.40 (s, 1H, NH), 11.65 (bs, 1H, OH), 11.95 (s, 1H, NH); C NMR (101 MHz,
DMSO-d ): δ 27.5, 28.1, 28.7, 28.8, 32.1, 115.1, 117.1, 117.5, 120.1, 120.2, 121.8, 130.9, 131.4,
6
1
4
34.2, 135.3, 137.5, 138.4, 148.5, 152.1, 156.7, 162.0, 164.9. HRMS: m/z calcd for C H N O S
2
2
21
3
3
+
08.1383 (M+H) , found 408.1380.
1
9

ACCEPTED MANUSCRIPT
5.2.18. 2-(3-Hydroxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (22). The title compound was prepared starting from 19 through Method D and
purified by flash chromatography eluting with CHCl :MeOH 98:2, in 13% yield as light-yellow
3
1
solid. H NMR (400 MHz, DMSO-d ): δ 1.50-1.60 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H,
6
2
cycloheptane CH ), 2.65-2.80 (m, 4H, cycloheptane CH ), 6.90-7.00 (m, 1H, aromatic CH), 7.00-
2
2
7
.10 (m, 1H, aromatic CH), 7.20-7.30 (m, 3H, aromatic CH and pyridine CH), 7.80 (t, J = 7.0 Hz,
H, pyridine CH), 8.10 (d, J = 8.3 Hz, 1H, pyridine CH), 8.3 (d, J = 4.1 Hz, 1H, pyridine CH), 9.80
1
1
3
(
s, 1H, OH), 10.25 and 10.90 (s, each 1H, NH); C NMR (101 MHz, DMSO-d ): δ 27.5, 28.1, 28.5,
6
2
1
5
8.8, 32.1, 114.7, 115.0, 118.4, 119.5, 120.1, 124.1, 130.1, 132.0, 134.7, 136.2, 136.9, 138.4, 148.4,
+
52.3, 157.9, 164.3, 164.4. HRMS: m/z calcd for C H N O S 408.1383 (M+H) , found 408.1377.
22
21
3
3
.2.19. 2-(4-Hydroxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (23). The title compound was prepared starting from 20 through Method D and
purified by crystallization by EtOH/DMF followed by flash chromatography eluting with
1
CHCl :MeOH 98:2, in 32% yield as light-yellow solid. H NMR (400 MHz, DMSO-d ): δ 1.45-
3
6
1
.65 (m, 4H, cycloheptane CH ), 1.70-1.85 (m, 2H, cycloheptane CH ), 2.60-2.70 and 2.70-2.80 (m,
2
2
each 2H, cycloheptane CH ), 6.75 (d, J = 8.0 Hz, 2H, aromatic CH), 7.10 (t, J = 7.0 Hz, 1H,
2
pyridine CH), 7.70 (d, J = 8.0 Hz, 2H, aromatic CH), 7.80 (t, J = 7.2 Hz, 1H, pyridine CH), 8.15 (d,
J = 7.7 Hz, 1H, pyridine CH), 8.25-8.35 (m, 1H, pyridine CH), 10.20 (s, 1H, OH), 10.30 and 10.90
1
3
(
s, each 1H, NH); C NMR (101 MHz, DMSO-d ): δ 27.5, 28.1, 28.5, 28.8, 32.1, 115.0, 115.6,
6
120.1, 123.5, 123.8, 130.1, 131.7, 136.1, 137.5, 138.4, 148.3, 152.3, 161.5, 163.9, 164.5. HRMS:
+
m/z calcd for C H N O S 408.1383 (M+H) , found 408.1380.
2
2
21
3
3
5.2.20.
N-(4-Chlorophenyl)-2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (24). The title compound was prepared starting from 12
through Method C using 2-methoxybenzoyl chloride and purified by flash chromatography eluting
1
with cyclohexane:EtOAc 6:4, in 21% yield as red-brown solid. H NMR (200 MHz, CDCl ): δ
3
1
.55-1.65 (m, 4H, cycloheptane CH ), 1.65-1.75 (m, 2H, cycloheptane CH ), 2.55-2.65 and 2.65-
2
2
2
0

ACCEPTED MANUSCRIPT
2
.80 (m, each 2H, cycloheptane CH ), 4.00 (s, 3H, OCH ), 6.95 (d, J = 8.3 Hz, 1H, aromatic CH),
2
3
7.10 (t, J = 7.2 Hz, 1H, aromatic CH), 7.35 (d, J = 8.8 Hz, 2H, aromatic CH), 7.40-7.50 (m, 2H,
aromatic CH and NH), 7.60 (d, J = 8.7 Hz, 2H, aromatic CH), 8.25 (dd, J = 8.07 and 1.9 Hz, 1H,
+
aromatic CH), 12.10 (s, 1H, NH). HRMS: m/z calcd for C H ClN O S 455.1197 (M+H) , found
2
4
23
2
3
455.1196.
5.2.21.
2-(2-Methoxybenzamido)-N-phenyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (25). The title compound was prepared starting from 13 through Method C using 2-
methoxybenzoyl chloride and purified by flash chromatography eluting with cyclohexane:EtOAc
1
7
:3, in 32% yield as white solid. H NMR (200 MHz, DMSO-d ): δ 1.45-1.70 (m, 4H, cycloheptane
6
CH ), 1.75-1.85 (m, 2H, cycloheptane CH ), 2.65-2.80 (m, 4H, cycloheptane CH ), 3.80 (s, 3H,
2
2
2
OCH ), 7.00-7.20 (m, 3H, aromatic CH), 7.30 (t, J = 7.6 Hz, 2H, aromatic CH), 7.50 (t, J = 7.1 Hz,
3
1
1
5
H, aromatic CH), 7.70 (d, J = 7.5 Hz, 2H, aromatic CH), 8.00 (d, J = 6.7 Hz, 1H, aromatic CH),
0.10 and 11.60 (s, each 1H, NH).
.2.22.
N-(4-Fluorophenyl)-2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (26). The title compound was prepared starting from 14
through Method C using 2-methoxybenzoyl chloride and purified by treatment with Et O, in 74%
2
1
yield as light-brown solid. H NMR (200 MHz, DMSO-d ): δ 1.60-2.00 (m, 6H, cycloheptane CH ),
6
2
2
7
1
5
.60-2.75 (m, 4H, cycloheptane CH ), 3.75 (s, 3H, OCH ), 7.00-7.25 (m, 4H, aromatic CH), 7.50-
2 3
.60 (m, 1H, aromatic CH), 7.70-7.80 (m, 2H, aromatic CH), 8.00 (d, J = 7.5 Hz, 1H, aromatic CH),
0.20 and 11.75 (s, each 1H, NH).
.2.23. 2-(2-Methoxybenzamido)-N-(thiazol-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (27). The title compound was prepared starting from 15 through Method C using 2-
methoxybenzoyl chloride and purified by flash chromatography eluting with cyclohexane:EtOAc
1
6
:4, in 32% yield as yellow-orange solid. H NMR (400 MHz, CDCl ): δ 1.55-1.65 (m, 4H,
3
cycloheptane CH ), 1.75-1.85 (m, 2H, cycloheptane CH ), 2.70-2.80 and 2.85-2.90 (m, each 2H,
2
2
2
1

ACCEPTED MANUSCRIPT
cycloheptane CH ), 4.15 (s, 3H, OCH ), 6.90 (d, J = 3.5 Hz, 1H, aromatic CH), 6.95-7.00 (m, 2H,
2
3
aromatic CH), 7.15 (t, J = 7.5 Hz, 1H, aromatic CH), 7.45 (dt, J = 1.7 and 8.6 Hz, 1H, aromatic
CH), 8.25 (dd, J = 1.7 and 7.8 Hz, 1H, aromatic CH), 11.40 and 12.40 (s, each 1H, NH).
5.2.24. N-Cyclohexyl-2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (28). The title compound was prepared starting from 16 through Method C using 2-
methoxybenzoyl chloride and purified by treatment with Et O, in 89% yield as light-brown solid.
2
1
H NMR (200 MHz, DMSO-d ): δ 1.00-1.30 (m, 5H, cyclohexyl CH ), 1.50-1.90 (m, 13H,
6
2
cyclohexyl CH and cycloheptane CH ), 2.50-2.60 (m, 2H, cycloheptane CH ), 3.50-3.60 (m, 1H,
2
2
2
cyclohexyl CH), 4.00 (s, 3H, OCH ), 7.10 (t, J = 7.5 Hz, 1H, aromatic CH), 7.20 (d, J = 8.5 Hz, 1H,
3
NH), 7.55 (t, J = 7.5 Hz, 1H, aromatic CH), 7.75 (d, J = 8.1 Hz, 1H, aromatic CH), 8.00 (d, J = 7.7
Hz, 1H, aromatic CH), 11.75 (s, 1H, NH).
5.2.25.
N-(4-Chlorophenyl)-2-(2-hydroxybenzamido)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (29). The title compound was prepared starting from 24
1
through Method D and purified by crystallization by EtOH, in 55% yield as light-yellow solid. H
NMR (200 MHz, DMSO-d ): δ 1.40-1.60 (m, 4H, cycloheptane CH ), 1.60-1.80 (m, 2H,
6
2
cycloheptane CH ), 2.60-2.70 (m, 4H, cycloheptane CH ), 6.80-6.90 (m, 2H, aromatic CH), 7.30-
2
2
7.40 (m, 3H, aromatic CH), 7.65 (d, J = 8.8 Hz, 2H, aromatic CH), 7.90 (d, J = 8.0 Hz, 1H,
1
3
aromatic CH), 10.20 (s, 1H, NH), 11.80 and 11.90 (s, each 1H, OH and NH); C NMR (101 MHz,
DMSO-d ): δ 27.6, 28.1, 28.8, 32.1, 117.2, 117.5, 120.1, 121.9, 127.7, 129.0, 131.2, 131.3, 134.2,
6
+
1
35.2, 136.5, 138.2, 156.6, 162.0, 164.2. HRMS: m/z calcd for C H ClN O S 441.1040 (M+H) ,
2
3
21
2
3
found 441.1034.
5.2.26.
2-(2-Hydroxybenzamido)-N-phenyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (30). The title compound was prepared starting from 25 through Method D and
1
purified by crystallization by EtOH, in 30% yield as light-yellow solid. H NMR (400 MHz,
DMSO-d ): δ 1.50-1.70 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H, cycloheptane CH ), 2.65-
6
2
2
2
2

ACCEPTED MANUSCRIPT
2
.80 (m, 4H, cycloheptane CH ), 6.80-6.90 (m, 2H, aromatic CH), 7.05 (t, J = 7.3 Hz, 1H, aromatic
2
CH), 7.20-7.40 (m, 3H, aromatic CH), 7.60 (d, J = 7.7 Hz, 2H, aromatic CH), 7.95 (d, J = 7.1 Hz,
1
3
1
H, aromatic CH), 10.00 (s, 1H, NH), 11.60 (s, 1H, OH), 11.80 (s, 1H, NH); C NMR (101 MHz,
DMSO-d ): δ 27.6, 28.1, 28.8, 28.9, 32.2, 117.1, 117.5, 120.1, 120.5, 122.6, 124.1, 129.1, 131.1,
6
1
4
5
31.4, 134.2, 135.2, 136.4, 139.2, 156.7, 161.9, 164.2. HRMS: m/z calcd for C H N O S
23 22 2 3
+
07.1430 (M+H) , found 407.1427.
.2.27. N-(4-Fluorophenyl)-2-(2-hydroxybenzamido)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (31). The title compound was prepared starting from 26
through Method D and purified by flash chromatography eluting with cyclohexane:EtOAc 8:2, in
1
1
6% yield as light-yellow solid. H NMR (400 MHz, DMSO-d ): δ 1.50-1.65 (m, 4H, cycloheptane
6
CH ), 1.70-1.85 (m, 2H, cycloheptane CH ), 2.65-2.80 (m, 4H, cycloheptane CH ), 6.85-6.95 (m,
2
2
2
2
H, aromatic CH), 7.15 (t, J = 8.8 Hz, 2H, aromatic CH), 7.30 (t, J = 7.7 Hz, 1H, aromatic CH),
.60-7.70 (m, 2H, aromatic CH), 7.90 (d, J = 8.0 Hz, 1H, aromatic CH), 10.20 (s, 1H, NH), 11.70
7
1
3
and 11.90 (s, each 1H, OH and NH); C NMR (101 MHz, DMSO-d ): δ 27.6, 28.1, 28.8, 32.2,
6
1
1
15.7 (d, JC−F = 22 Hz), 117.1, 117.5, 120.1, 122.2 (d, JC−F = 8 Hz), 122.4, 131.1, 131.4, 134.2,
35.2, 135.6, 136.4, 157.1(d, JC−F = 87 Hz), 159.9, 161.9, 164.1. HRMS: m/z calcd for
+
C H FN O S 425.1336 (M+H) , found 425.1335.
23
21
2
3
5.2.28. 2-(2-Hydroxybenzamido)-N-(thiazol-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (32). The title compound was prepared starting from 27 through Method D and
purified by flash chromatography eluting with CH Cl :Acetone 98:2, in 35% yield as light-yellow
2
2
1
solid. H NMR (400 MHz, DMSO-d ): δ 1.50-1.70 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H,
6
2
cycloheptane CH ), 2.65-2.85 (m, 4H, cycloheptane CH ), 6.80-7.05 (m, 2H, aromatic CH), 7.20-
2
2
7.30 (m, 1H, aromatic CH), 7.35 (dt, J = 1.3 and 8.0 Hz, 1H, aromatic CH), 7.50 (d, J = 2.9 Hz, 1H,
aromatic CH), 7.90 (d, J = 7.0 Hz, 1H, aromatic CH), 11.50 (bs, 1H, NH), 11.75 (bs, 1H, OH),
13
1
2.25 (s, 1H, NH); C NMR (101 MHz, DMSO-d ): δ 26.7, 27.6, 28.1, 28.6, 28.7, 32.2, 117.2,
6
2
3

ACCEPTED MANUSCRIPT
117.5, 120.1, 120.4, 131.2, 131.3, 134.3, 135.6, 137.9, 156.8, 162.2. HRMS: m/z calcd for
+
C H N O S 414.0868 (M+H) , found 414.0941.
20
19
3
3 2
5.2.29. N-Cyclohexyl-2-(2-hydroxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (33). The title compound was prepared starting from 28 through Method D and
purified by flash chromatography eluting with cyclohexane:EtOAc 7:3, in 16% yield as light-brown
1
solid. H NMR (400 MHz, DMSO-d ): δ 1.20-1.30 and 1.45-1.60 (m, each 5H, cyclohexyl CH ),
6
2
1
.60-1.85 (m, 6H, cycloheptane CH ), 2.60-2.70 (m, 4H, cycloheptane CH ), 3.50-3.60 (m, 1H,
2
2
cyclohexyl CH), 6.80-6.90 (m, 2H, aromatic CH and NH), 7.35 (t, J = 7.6 Hz, 1H, aromatic CH),
7
1
4
.80 (d, J = 7.6 Hz, 1H, aromatic CH), 7.90 (d, J = 7.6 Hz, 1H, aromatic CH), 11.75 and 11.90 (s,
1
3
H, OH and NH); C NMR (101 MHz, DMSO-d ): δ 25.1, 25.6, 26.7, 17.6, 28.2, 28.7, 32.2, 32.6,
6
8.6, 117.1, 117.7, 120.0, 122.9, 130.7, 131.3, 134.1, 135.1, 135.5, 156.7, 161.7, 164.5. HRMS: m/z
+
calcd for C H N O S 413.1900 (M+H) , found 413.1899.
2
3
28
2
3
5.2.30.
2-(4-Chlorobenzamido)-N-(2-methoxyphenyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (34). The title compound was prepared starting from 17
[
24] through Method C using 4-chlorobenzoyl chloride and purified by crystallization by EtOH, in
1
2
9% yield as light-yellow solid. H NMR (200 MHz, CDCl ): δ 1.50-1.80 (m, 4H, cycloheptane
3
CH ), 1.80-2.00 (m, 2H, cycloheptane CH ), 2.70-2.80 and 2.90-3.00 (m, each 2H, cycloheptane
2
2
CH ), 3.90 (s, 3H, OCH ), 6.80 (dd, J = 1.8 and 7.3 Hz, 1H, aromatic CH), 7.05 (dt, J = 2.0 and 7.5
2
3
Hz, 2H, aromatic CH), 7.45 (d, J = 8.7 Hz, 2H, aromatic CH), 7.90 (d, J = 8.7 Hz, 2H, aromatic
CH), 8.10 (s, 1H, NH), 8.45 (dd, J = 1.8 and 7.4 Hz, 1H, aromatic CH), 12.00 (s, 1H, NH). HRMS:
+
m/z calcd for C H ClN O S 455.1197 (M+H) , found 455.1198.
2
4
23
2
3
5.2.31.
N-(3-((2-Methoxyphenyl)carbamoyl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-2-
yl)picolinamide (35). The title compound was prepared starting from 17 [24] through Method C
using picolinoyl chloride and purified by flash chromatography eluting with CHCl :MeOH 98:2, in
3
1
3
4% yield as light-yellow solid. H NMR (400 MHz, DMSO-d ): δ 1.55-1.70 (m, 4H, cycloheptane
6
2
4

ACCEPTED MANUSCRIPT
CH ), 1.75-1.85 (m, 2H, cycloheptane CH ), 2.70-2.80 and 2.80-2.90 (m, each 2H, cycloheptane
2
2
CH ), 3.80 (s, 3H, OCH ), 6.95 (t, J = 7.3 Hz, 1H, aromatic CH), 7.05 (d, J = 7.4 Hz, 1H, aromatic
2
3
CH), 7.10 (t, J = 7.1 Hz, 1H, aromatic CH), 7.55-7.65 (m, 1H, pyridine CH), 7.90 (d, J = 7.7 Hz,
H, pyridine CH), 8.00-8.20 (m, 2H, pyridine and aromatic CH), 8.70 (d, J = 4.7 Hz, 1H, pyridine
CH), 8.80 (s, 1H, NH), 12.20 (s, 1H, NH).
.2.32. 2-(4-Chlorobenzamido)-N-(2-hydroxyphenyl)-5,6,7,8-tetrahydro-4H-
1
5
cyclohepta[b]thiophene-3-carboxamide (36). The title compound was prepared starting from 34
1
through Method D and purified by crystallization by EtOH, in 64% yield as light-yellow solid. H
NMR (400 MHz, DMSO-d ): δ 1.45-1.70 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H,
6
2
cycloheptane CH ), 2.60-2.70 and 2.70-2.80 (m, each 2H, cycloheptane CH ), 6.70-7.00 (m, 3H,
2
2
aromatic CH), 7.60 (d, J = 7.6 Hz, 2H, aromatic CH), 7.80-7.90 (m, 3H, aromatic CH), 9.00 (s, 1H,
1
3
NH), 9.95 and 11.30 (s, each 1H, OH and NH); C NMR (101 MHz, DMSO-d ): δ 27.4, 28.1, 28.4,
6
2
1
5
8.7, 32.1, 115.8, 119.4, 123.1, 124.0, 125.6, 126.3, 129.3, 129.8, 131.9, 132.8, 135.9, 136.5, 137.5,
+
48.6, 163.1, 163.7. HRMS: m/z calcd for C H ClN O S 441.1040 (M+H) , found 441.1037.
2
3
21
2
3
.2.33.
N-(3-((2-Hydroxyphenyl)carbamoyl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-2-
yl)picolinamide (37). The title compound was prepared starting from 35 through Method D and
1
purified by flash chromatography eluting with CHCl , in 10% yield as light-yellow solid. H NMR
3
(
400 MHz, DMSO-d ): δ 1.55-1.65 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H, cycloheptane
6
2
CH ), 2.70-2.80 and 2.85-3.95 (m, each 2H, cycloheptane CH ), 6.80 (t, J = 7.6 Hz, 1H, aromatic
2
2
CH), 6.90 (d, J = 7.8 Hz, 1H, aromatic CH), 7.00 (t, J = 7.5 Hz, 1H, aromatic CH), 7.60 (m, 1H,
pyridine CH), 7.90 (d, J = 7.2Hz, 1H, pyridine CH), 8.05 (t, J = 7.5 Hz, 1H, aromatic CH), 8.15 (d,
J = 7.7 Hz, 1H, pyridine CH), 8.75 (d, J = 4.6 Hz, 1H, pyridine CH), 8.80 (s, 1H, NH), 9.95 (s, 1H,
1
3
OH), 12.15 (s, 1H, NH); C NMR (101 MHz, DMSO-d ): δ 27.3, 27.9, 28.6, 28.7, 31.9, 115.8,
6
1
1
19.5, 121.4, 122.8, 122.9, 125.6, 126.2, 127.9, 132.0, 134.8, 138.3, 138.8, 148.3, 148.5, 149.5,
+
61.1, 163.9. HRMS: m/z calcd for C H N O S 408.1383 (M+H) , found 408.1381.
2
2
21
3
3
2
5

ACCEPTED MANUSCRIPT
5.2.34.
Ethyl
2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxylate (39). The title compound was prepared starting from ethyl 2-amino-5,6,7,8-tetrahydro-
4H-cyclohepta[b]thiophene-3-carboxylate 38 [26] by Method C (2h) using 2-methoxybenzoyl
1
chloride and purified by treatment with cyclohexane, in 100% yield as light-yellow solid. H NMR
(
200 MHz, CDCl ): δ 1.35 (t, J = 7.1 Hz, 3H, CH CH ), 1.50-1.65 (m, 4H, cycloheptane CH ),
3 2 3 2
1
4
.75-1.85 (m, 2H, cycloheptane CH ), 2.65-2.75 and 2.95-3.05 (m, each 2H, cycloheptane CH ),
2 2
.10 (s, 3H, OCH ), 4.35 (q, J = 7.1 Hz, 2H, CH CH ), 6.90-7.20 (m, 2H, aromatic CH), 7.40-7.50
3
2
3
(
m, 1H, aromatic CH), 8.25 (dd, J = 1.8 and 7.8 Hz, 1H, aromatic CH), 13.00 (s, 1H, NH).
.2.35. 2-(2-Methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid
40). A suspension of 39 (3.75 mmol, 1.40 g) and LiOH monohydrate (14.99 mmol, 0.63 g) in a
5
(
mixture H O:THF 1:1 (60 ml) was maintained at 50 °C overnight. After cooling, the reaction
2
mixture was acidified (pH = 6) with 2N HCl yielding a precipitate, which was filtered, washed with
1
water, and purified by treatment with Et O to give 40 as light-yellow solid (0.6 g, 46%). H-NMR
2
(
200 MHz, DMSO-d ): δ 1.45-1.60 (m, 4H, cycloheptane CH ), 1.70-1.80 (m, 2H, cycloheptane
6
2
CH ), 2.60-2.70 and 2.95-3.05 (m, each 2H, cycloheptane CH ), 4.00 (s, 3H, OCH ), 7.10 (t, J = 7.7
2
2
3
Hz, 1H, aromatic CH), 7.20 (d, J = 8.3 Hz, 1H, aromatic CH), 7.55 (dt, J = 1.7 and 8.5 Hz, 1H,
aromatic CH), 8.00 (dd, J = 1.7 and 7.7 Hz, 1H, aromatic CH), 13.00 (s, 1H, NH).
5.2.36. General procedure for C-3 amidation (Method E). To a solution of 40 (1.0 equiv) in dry
CH Cl was added the appropriate alkyl amine (3 equiv) followed by BOP (1.5 equiv) and finally
2
2
collidine (4.0 equiv). The mixture was stirred at room temperature overnight. The mixture was
diluted with EtOAc and successively washed with: water (twice), 0.5 M HCl, saturated NaHCO3
solution, water, and finally brine. The organic layer was dried over Na SO , filtered and then
2
4
evaporated to dryness providing a residue that was purified as described below.
5.2.37.
2-(2-Methoxybenzamido)-N-(pyridin-2-ylmethyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (41). The title compound was prepared through Method E
using (pyridin-2-ylmethyl)amine and purified by flash chromatography eluting with
2
6

ACCEPTED MANUSCRIPT
1
cyclohexane:EtOAc 5:5, in 15% yield as white solid. H NMR (400 MHz, DMSO-d ): δ 1.50-1.70
6
(
m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H, cycloheptane CH ), 2.60-2.70 and 2.75-2.90 (m,
2
2
each 2H, cycloheptane CH ), 3.75 (s, 3H, CH ), 4.60 (d, J = 5.7 Hz, 2H, CH ), 7.00-7.25 (m, 3H,
2
3
2
aromatic CH), 7.35 (d, J = 7.8 Hz, 1H, aromatic CH), 7.55 (t, J = 7.1 Hz, 1H, aromatic CH), 7.75 (t,
J = 7.6 Hz, 1H, aromatic CH), 8.05 (dd, J = 1.5 and 7.7 Hz, 1H, aromatic CH), 8.35 (d, J = 3.9 Hz,
1H, NH), 8.45-8.55 (m, 1H, aromatic CH), 12.00 (s, 1H, NH).
5.2.38. N-Benzyl-2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (42). The title compound was prepared through Method E using benzylamine and
1
purified by treatment with Et O, in 93% yield as white solid. H NMR (200 MHz, DMSO-d ): δ
2
6
1
.45-1.65 (m, 4H, cycloheptane CH ), 1.70-1.85 (m, 2H, cycloheptane CH ), 2.60-2.75 (m, 4H,
2
2
cycloheptane CH ), 3.80 (s, 3H, CH ), 4.45 (d, J = 5.7 Hz, 2H, CH ), 7.00-7.30 (m, 7H, aromatic
2
3
2
CH and NH), 7.45-7.60 (m, 1H, aromatic CH), 8.00 (dd, J = 1.7 and 7.8 Hz, 1H, aromatic CH), 8.40
t, J = 5.7 Hz, 1H, aromatic CH), 12.00 (s, 1H, NH).
.2.39. N-Isopropyl-2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
(
5
carboxamide (43). The title compound was prepared through Method E using isopropylamine and
1
purified by treatment with Et O, in 54% yield as light-yellow solid. H NMR (200 MHz, DMSO-
2
d ): δ 1.10 and 1.15 (s, each 3H, CH ), 1.45-1.65 (m, 4H, cycloheptane CH ), 1.70-1.80 (m, 2H,
6
3
2
cycloheptane CH ), 2.60-2.75 (m, 4H, cycloheptane CH ), 4.00 (s, 3H, OCH ), 4.00-4.10 (m, 1H,
2
2
3
CH), 7.10 (t, J = 7.5 Hz, 1H, aromatic CH), 7.20 (d, J = 8.3 Hz, 1H, aromatic CH), 7.45-7.60 (m,
1
1
5
H, aromatic CH), 7.75 (d, J = 7.9 Hz, 1H, NH), 8.00 (dd, J = 1.7 and 7.8 Hz, 1H, aromatic CH),
1.90 (s, 1H, NH).
.2.40. N-(tert-Butyl)-2-(2-methoxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (44). The title compound was prepared through Method E using tert-butylamine and
1
purified by treatment with Et O, in 40% yield as light-yellow solid. H NMR (200 MHz, DMSO-
2
d ): δ 1.30 (s, 9H, CH ), 1.50-1.70 (m, 4H, cycloheptane CH ), 1.70-1.85 (m, 2H, cycloheptane
6
3
2
CH ), 2.60-2.75 (m, 4H, cycloheptane CH ), 4.05 (s, 3H, OCH ), 7.10 (t, J = 7.5 Hz, 1H, aromatic
2
2
3
2
7

ACCEPTED MANUSCRIPT
CH), 7.20 (d, J = 8.3 Hz, 1H, aromatic CH), 7.35 (s, 1H, NH), 7.50-7.60 (m, 1H, aromatic CH),
8.00 (dd, J = 1.6 and 7.7 Hz, 1H, aromatic CH), 11.90 (s, 1H, NH).
5.2.41. 2-Methoxy-N-(3-(4-methylpiperazine-1-carbonyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophen-2-yl)benzamide (45). The title compound was prepared through Method E
using 1-methylpiperazine and purified by flash chromatography eluting with CHCl :MeOH 97:3, in
3
1
2
1
2
6% yield as white solid. H NMR (400 MHz, CDCl ): δ 1.50-1.70 (m, 4H, cycloheptane CH ),
3
2
.70-1.90 (m, 2H, cycloheptane CH ), 2.25 (s, 3H, CH ), 2.30-2.40 (m, 2H, cycloheptane CH ),
2
3
2
.45-2.65 (m, 4H, piperazine and cycloheptane CH ), 2.75-2.85 and 3.25-3.35 (m, each 2H,
2
piperazine CH ), 3.55-3.75 (m, 2H, piperazine CH ), 4.00 (s, 3H, OCH ), 6.90 (d, J = 8.3 Hz, 1H,
2
2
3
aromatic CH), 7.10 (t, J = 7.4 Hz, 1H, aromatic CH), 7.45 (t, J = 7.3 Hz, 1H, aromatic CH), 8.25 (d,
J = 7.4 Hz, 1H, aromatic CH).
5.2.42.
2-Methoxy-N-(3-(4-(pyridin-2-yl)piperazine-1-carbonyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophen-2-yl)benzamide (46). The title compound was prepared through Method E
using 1-(pyridin-2-yl)piperazine and purified by flash chromatography eluting with
1
cyclohexane:EtOAc 4:6, in 67% yield as brown oil. H NMR (400 MHz, CDCl ): δ 1.50-1.70 (m,
3
4
2
3
=
H, cycloheptane CH ), 1.75-1.85 (m, 2H, cycloheptane CH ), 2.50-2.70 and 2.80-2.90 (m, each
2
2
H, cycloheptane CH ), 3.25-3.60 (m, 2H, piperazine CH ), 3.65-3.80 (m, 2H, piperazine CH ),
2
2
2
.90-4.05 (m, 4H, piperazine CH ), 4.10 (s, 3H, OCH ), 6.60-6.70 (m, 2H, pyridine CH), 6.90 (d, J
2
3
8.1 Hz, 1H, aromatic CH), 7.10 (t, J = 8.0 Hz, 1H, aromatic CH), 7.40-7.50 (m, 2H, aromatic and
pyridine CH), 8.10-8.20 (m, 1H, pyridine CH), 8.25 (dd, J = 1.8 and 7.8 Hz, 1H, aromatic CH),
11.40 (s, 1H, NH).
5.2.43.
2-(2-Hydroxybenzamido)-N-(pyridin-2-ylmethyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (47). The title compound was prepared starting from 41
1
through Method D and purified by crystallization by EtOH, in 53% yield as light-yellow solid. H
NMR (400 MHz, DMSO-d ,): δ 1.50-1.70 (m, 4H, cycloheptane CH ), 1.75-1.85 (m, 2H,
6
2
cycloheptane CH ), 2.60-2.75 and 2.75-2.90 (m, each 2H, cycloheptane CH ), 4.65 (d, J = 5.3 Hz,
2
2
2
8

ACCEPTED MANUSCRIPT
2
2
8
H, CH ), 6.85-7.00 (m, 2H, aromatic CH), 7.35 (t, J = 7.4 Hz, 1H, aromatic CH), 7.45-7.55 (m,
2
H, pyridine CH), 7.90 (d, J = 7.7 Hz, 1H, aromatic CH), 8.05 (t, J = 7.0 Hz, 1H, pyridine CH),
1
3
.55-8.65 (m, 2H, pyridine CH and NH), 11.75 (s, 1H, OH), 12.10 (s, 1H, NH); C NMR (101
MHz, DMSO-d ): δ 27.6, 28.1, 28.5, 28.6, 32.0, 43.4, 117.0, 117.5, 120.2, 121.3, 122.9, 123.9,
6
131.3, 131.4, 134.3, 135.1, 136.6, 141.0, 146.3, 156.4, 156.8, 162.0, 165.8. HRMS: m/z calcd for
+
C H N O S 422.1539 (M+H) , found 422.1539.
23
23
3
3
5.2.44.
N-Benzyl-2-(2-hydroxybenzamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (48). The title compound was prepared starting from 42 through Method D and
purified by flash chromatography eluting with cyclohexane:EtOAc 8:2, in 9% yield as white solid.
1
H NMR (DMSO-d 400 MHz): δ 1.45-1.70 (m, 4H, cycloheptane CH ), 1.75-2.00 (m, 2H,
6,
2
cycloheptane CH ), 2.60-2.90 (m, 4H, cycloheptane CH ), 4.50 (d, J = 5.8 Hz, 2H, CH ), 6.80-7.10
2
2
2
(m, 2H, aromatic CH), 7.20-7.50 (m, 6H, aromatic CH), 8.00 (d, J = 7.8 Hz, 1H, aromatic CH),
1
3
8
.50-8.60 (m, 1H, NH), 11.75 and 12.00 (s, each 1H, OH and NH); C NMR (101 MHz, DMSO-
d6): δ 27.6, 28.1, 28.6, 28.7, 32.1, 42.8, 117.0, 117.5, 120.2, 122.2, 127.2, 127.6, 128.7, 131.2,
1
4
5
31.3, 134.2, 135.1, 135.7, 139.6, 156.4, 161.8, 165.4. HRMS: m/z calcd for C H N O S
24 24 2 3
+
21.1587 (M+H) , found 421.1586.
.2.45. 2-(2-Hydroxybenzamido)-N-isopropyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide (49). The title compound was prepared starting from 43 through Method D and
1
purified by flash chromatography eluting with CHCl , in 18% yield as white solid. H NMR (400
3
MHz, DMSO-d ): δ 1.10 (d, J = 6.5 Hz, 6H, CH ), 1.50-1.60 (m, 4H, cycloheptane CH ), 1.70-1.80
6
,
3
2
(
m, 2H, cycloheptane CH ), 2.60-2.75 (m, 4H, cycloheptane CH ), 3.95-4.05 (m, 1H, CH), 6.90-
2
2
7.00 (m, 2H, aromatic CH), 7.30 (dt, J = 1.6 and 7.2 Hz, 1H, aromatic CH), 7.80 (d, J = 7.7 Hz, 1H,
1
3
NH), 7.90 (dd, J = 1.7 and 7.8 Hz, 1H, aromatic CH), 11.75 and 11.0 (s, each 1H, OH and NH); C
NMR (101 MHz, DMSO-d ): δ 22.6, 27.6, 28.2, 28.6, 28.7, 32.1, 41.4, 117.1, 117.7, 120.0, 123.0,
6
1
30.7, 131.3, 134.0, 135.0, 135.2, 156.7, 161.8, 164.6. HRMS: m/z calcd for C H N O S
2
0
24
2
3
+
3
73.1587 (M+H) , found 373.1587.
2
9