A synthesis, in silico, in vitro and in vivo study of thieno[2,3-b]pyridine anticancer analogues

Article abstract of DOI:10.1039/c5md00245a

The anticancer activity of the thieno[2,3-b]pyridines was explored by altering the ring size of the cyclo-aliphatic moiety. Five-, six-, seven- and eight-membered derivatives were tested against the NCI60 tumour cell panel. According to this assay the mos

Full text of DOI:10.1039/c5md00245a

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van Rensburg, L. Pilkington, C. Jeon, M. Song, L. Gridle, E. Leung, D. Barker, M. Vuica-Ross, K. Volcho, A.
Zakharenko, O. Lavrik and J. Reynisson, Med. Chem. Commun., 2015, DOI: 10.1039/C5MD00245A.
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A synthesis, in silico, in vitro and in vivo study of
thieno[2,3ꢀb]pyridine anticancer analogues
Homayon J. Arabshahi1, Michelle van Rensburg1, Lisa I. Pilkington1,2, Chae Yeon Jeon1,
Mirae Song1, Ling-Mey Gridel1, Euphemia Leung2, David Barker1, Milena Vuica-Ross3
Konstantin P. Volcho4,5, Alexandra L. Zakharenko6, Olga I. Lavrik5,6, and Jóhannes
Reynisson1*
1School of Chemical Sciences, University of Auckland, New Zealand
2Auckland Cancer Society Research Centre and Department of Molecular Medicine and
Pathology, University of Auckland, New Zealand
3Johns Hopkins University School of Medicine, Baltimore, USA
4N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the
Russian Academy of Sciences, Novosibirsk, Russian Federation
5Novosibirsk State University, Novosibirsk, Russian Federation
6Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, Novosibirsk, Russian Federation
*To whom correspondence should be addressed: School of Chemical Sciences, University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand.
Eꢀmail: j.reynisson@auckland.ac.nz, Tel. 64ꢀ9ꢀ373ꢀ7599 ext. 83746, Fax. 64ꢀ9ꢀ373ꢀ7422
Abstract
The anticancer activity of the thieno[2,3ꢀb]pyridines was explored by altering the ring size of
the cycloꢀaliphatic moiety. Five, six, seven and eight membered derivatives were tested
against the NCI60 tumour cell panel. According to this assay the most active derivative 9a
has a cyclooctane moiety, which suggests that larger aliphatic ring systems are favourable.
For the most sensitive tumour cell line MBꢀMDAꢀ435 derivative 9a has a GI50 = 70 nM and a
LC50 = 925 nM. To explore the biological mechanism of the thieno[2,3ꢀb]pyridines five
derivatives were tested against the TyrosylꢀDNA phosphodiesterase I (TDP1), a
phospholipase D enzyme, using a biochemical assay. The most potent derivative for TDP1
1

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was 9d giving an excellent IC50 at 0.5 ± 0.1 ꢁM. Also, derivative 12 was tested against 97
kinases and no or very limited activity was found excluding this class of bioꢀmolecular
targets. Finally, a mouse xenograft study using derivative 12 was encouraging but the tumour
size/mass reduction was not quite statistically significant.
Introduction
It is now established that a class of thieno[2,3ꢀb]pyridine compounds have a potent anticancer
activity against a variety of tumour cell lines and,1ꢀ3 Their molecular structure is shown in
Fig. 1. The efficacy of the thieno[2,3ꢀb]pyridines was discovered by virtual high throughput
screen (vHTS) against the phospholipase C – γ2 (PLC – γ2) isoform.4 PLC – γ is an
interesting anticancer bioꢀmolecular target regulating a number of cancer cellular functions
including tumorigenesis5, metastasis development6, cancer cell invasion7 and tumour
angiogenesis.8 The administration of thieno[2,3ꢀb]pyridines causes the breast cancer cell line
MDAꢀMBꢀ231 to be severely growth restricted, rounded, G2/M phase population increase in
the cell cycle and slowed proliferation in scratch assays, which is in line with PLC – γ
inhibition making it the putative bioꢀmolecular target.9 Furthermore, However, it is quite
possible that other bioꢀmolecular targets are affected contributing to the overall efficacy of
this class of compounds.
Fig. 1 The basic molecular structure of the anticancer thieno[2,3ꢀb]pyridines (3ꢀaminoꢀ5ꢀoxoꢀNꢀphenylꢀ5,6,7,8ꢀ
tetrahydrothieno[2,3ꢀb]quinolineꢀ2ꢀcarboxamide). Ortho / meta substitution on the phenyl ring give the most
potent anticancer effect.
2

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The substitution pattern of the phenyl moiety of the thieno[2,3ꢀb]pyridines has been
thoroughly explored revealing that ortho / meta substitution on the same side of the phenyl
group gives the best anticancer effect.2, 9 However, other parts of the molecular scaffold have
received less attention such as the cyclohexanone moiety and a classical strategy in medicinal
chemistry is to alter ring sizes in order to optimise the potency of ligands. Thus, by
systematically altering the ring size of the cyclohexane would result in a consistent structural
activity relationship (SAR) series of cyclopentane, cyclohexane, cycloheptane and
cyclooctane derivatives.
The aim of this study was twofold: First, to extend the SAR of the thieno[2,3ꢀb]pyridines by
systematically altering the cyclohexane moiety’s ring size in conjunction with testing against
the National Cancer Institute’s human tumour cell line panel (NCI60).10 Second, to explore
the biological mechanism of action of the thieno[2,3ꢀb]pyridines led by molecular modelling
investigations. Two classes of bioꢀmolecules were considered, i.e., the phospholipase D
enzyme TyrosylꢀDNA phosphodiesterase I (TDP1),11 a promising anticancer target,12, 13 and
kinases, an established class of anticancer targets (see Strebhardt, 2010 and references
therein).14 Finally, the efficacy of the thieno[2,3ꢀb]pyridines was investigated in xenograph
mice models.
Results and Discussion
Synthesis of compounds
Thieno[2,3ꢀb]pyridines were prepared in three steps from the corresponding cyclic ketones
1aꢀc (Scheme 1). Firstly, salts 2aꢀc were formed from the reaction of ketones 1aꢀc with
freshlyꢀprepared sodium methoxide and methyl formate, and were immediately used in the
next reaction by heating with cyanothioacetamide and piperidinium acetate, following this,
acidification with acetic acid provided bicyclic carbonitriles 3aꢀc.
3

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Bromoanilines 5aꢀe were synthesised from the reaction of anilines 4aꢀe with 1 equivalent of
bromoacetyl bromide in the presence of Et3N, providing the desired products 5aꢀe in very
good to excellent yields. These bromoanilines 5aꢀe were coupled with the required bicyclic
carbonitrile 3aꢀc followed by concomitant cyclisation in the basic conditions to give
thieno[2,3ꢀb]pyridines 6a, 7aꢀe, 8a and 8b.
Scheme 1. Reagents and conditions: (i) Na, MeOH, methyl formate, 2aꢀc 52ꢀ67 %; (ii) cyanothioacetamide (1.1
equiv.), piperidinium acetate, water, reflux, 4 h, AcOH, r.t., 12 h, 3aꢀc 30ꢀ59 %; (iii) bromoacetyl bromide (1
equiv.), Et3N (1.1 equiv.), 0 ºC, 1 h, 5aꢀe 70ꢀ100 %; (iv) Na2CO3, EtOH, reflux, 18 h, 6a, 7aꢀe, 8aꢀb 22ꢀ91 %.
The commercial compounds were acquired from ChemBridge (6b, 6c, 7f),15 ChemDiv (7g,
7j),16 Specs (7h),17 Key Organics (7i, 7k, 8c)18 and InterBioScreen (9a, 9b, 9c, 9d, 10, 11,
12).19
Tumour cell lines
Twenty one derivatives of the thieno[2,3ꢀb]pyridine family were tested against the NCI60
human tumour cell lines. The molecular structures are shown in Table 1 along with the
average growth inhibition of the NCI60 panel compared to untreated cells, i.e., 100% growth
4

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is the control of untreated cells thus the lower the percentage number the greater the
inhibition. When the ring size on the leftꢀhand side of the derivatives is considered, a clear
trend for increased inhibition is seen for the larger seven (n=2) and eight (n=3) membered
rings rather than the five (n=0) and six (n=1) membered derivatives. This is reflected in
derivatives 8a, 8b, 9a and 9d being the most active with values of < 15% as compared to
untreated cells at 100%. Ethyl substitution (7hꢀk) on the cyclohexyl ring is not favourable as
compared to the unꢀsubstituted derivatives (7aꢀg). The substitution pattern on the phenyl ring
favours the meta (X2) position with derivatives 8b and 9d being the most active compounds
tested, e.g., 9d (mꢀCF3, 7.0%) vs. 9c (oꢀCF3, 80.2%). This is in line with previous finding for
the anticancer sensitivity of the cyclohexanone thieno[2,3ꢀb]pyridine series.1ꢀ3 Also, when the
six membered derivatives are compared to their cyclohexanone counterparts, similar NCI
mean values are observed, e.g., for the unꢀsubstituted phenyl ring derivatives ~30% inhibition
is observed.1 The cyclohexanone moiety has been linked to general cell cytotoxicity20, 21 and
the similar growth inhibition of the two series leads to the conclusion that the biological
effect of the cyclohexanone thieno[2,3ꢀb]pyridine series is not caused by unspecific cytotoxic
effects.
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NCI Mean
79.5
23.6
101.7
30.3
88.1
99.4
34.5
90.6
39.9
28.2
61.1
36.5
60.3
69.7
14.7
10.0
52.1
12.5
29.0
80.2
7.0
n
0
0
0
1
1
1
1
1
1
1
1
1
1
1
2
2
2
3
3
3
3
R
H
H
X1
H
Me
F
X2
H
Me
H
X3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
6a
6b
6c
7a
7b
7c
7d
7e
7f
H
H
H
H
H
H
H
Cl
F
CF3
H
H
CF3
H
H
H
H
H
H
H
Me
OMe
H
7g
7h
7i
Et*
Et*
Et*
Et*
H
F
Me
H
H
F
Me
H
CF3
H
H
7j
H
7k
8a
8b
8c
9a
9b
9c
9d
H
H
H
H
H
Cl
H
H
H
Cl
CF3
H
H
H
H
CF3
*Tested as the racemates
Table 1. The 3ꢀaminoꢀNꢀphenylthieno[2,3ꢀb]pyridineꢀ2ꢀcarboxamide structural derivatives. The NCI Mean
percentages (%) at 10 ꢁM as compared to untreated cells at 100% growth, i.e., the lower percentage numbers
represent greater growth inhibition.
Twelve derivatives were selected for dose response testing due to their favourable inhibition
at 10 ꢁM and the results are given in Table 2. The Growth Inhibition at 50% (GI50) and
Lethal Concentration at 50% (LC50) were derived from these measurements (for further
explanation see Shoemaker, 2006).10 GI50 is the concentration for 50% of maximal inhibition
of cell proliferation and therefore reflects the cytostatic concentration whereas LD50 is the
concentration causing 50% cell death giving the cytotoxic potential of the compounds. Four
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tumour cell lines were particularly affected, i.e., MDAꢀMBꢀ435 (melanoma), SFꢀ295 (Central
Nervous System, CNS), 786ꢀ0 (renal) and MDAꢀMBꢀ468 (breast). Other cell lines also
showed good response, e.g., SNBꢀ75 (CNS), OVCARꢀ3 (ovarian) and ACHN (renal). The
complete data set for the NCI data is given in the Supplementary Information section.
MDAꢀMBꢀ435
SFꢀ539
GI50
786ꢀ0
GI50
MDAꢀMBꢀ468
GI50
52
LC50
LC50
72400
X/X
X
LC50
14300
X/X
X
GI50
197
LC50
22700
X/X
X
241
367
759/656
4130
420
41700
371
71200
X/X
X
6b
7a
7f
172/207
305
416/491
4740
368
10000
398
332/303
1750
239
150
133
13500
X
X
X
X
X
X
7g
7h
7i
347
X
104
Y
X
X
244
X
110
X
X
X
701
X
471
X
7j
205
X
X
307
1940
94300
11300
465
X
6240
254
394
X
8a
8b
9a
9b
9d
2530
52/88
275
1580
354/858
1750
1600
31000
X/X
X
953/897 247/217 7670/3200
20900
10200/35700 291/334
2650
46600
6650
6120
6070
2250
798
2100
59200
1710
15400
X: no response at 100 ꢁM. Y: no data.
Table 2. The GI50 (50% growth inhibition) and LC50 (50% lethal concentration) in nanomolar (nM) are shown
for four tumour cell lines: MDAꢀMBꢀ435 (melanoma), SFꢀ295 (CNS), 786ꢀ0 (renal) and MDAꢀMBꢀ468
(breast). Derivatives 7a and 9a were tested twice and both values are given.
It can be seen from the GI50 values presented in Table 2 that compounds 6b and 9a are the
most active. It has been previously observed that ortho / meta substitution on the phenyl ring
with small moieties, as seen in derivative 6b, is favourable in suppressing cancer cell growth
for the cyclohexanone thieno[2,3ꢀb]pyridines.2 Considering the LC50 values, derivative 9a is
the most potent with nanoꢀmolar sensitivity for the MDAꢀMBꢀ435 tumour cell line (925 nM).
The MDAꢀMBꢀ435 and MDAꢀMBꢀ468 cell lines appear to the most affected, which is in
accordance with previous observations for the cyclohexanone thieno[2,3ꢀb]pyridines.1ꢀ3 It is
interesting to note that five of the derivatives (see Table 2) are not only cytostatic but also kill
the cells (cytotoxic), which makes this class of compounds even more interesting for
anticancer treatment. Close structural analogues of the compounds presented here, i.e., with a
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cyclohexanone moiety rather than a cyclohexane, are predominately cytostatic rather than
cytotoxic.1, 2
TyrosylꢀDNA phosphodiesterase I (TDP1)
TDP1 is a member of the phospholipase D (PLD) superfamily,11 which is related to the PLC
enzymes, the putative target of the thieno[2,3ꢀb]pyridines.9 Also, there is evidence conserved
regions for the sequence similarity between the PLC and PLD enzyme classes.22, 23 The idea
emerged whether TDP1 could also be a bioꢀmolecular target of the thieno[2,3ꢀb]pyridines
since both are phospholipases. TDP1 is a promising cancer target as it plays an important role
in removal of DNA lesions, generated by DNA topoisomerase I (Top1) inhibitors, such as
13
camptotecin.12,
Recently, it has been reported that TDP1 depletion can sensitize
glioblastomaꢀresistant cancer cells to the alkylating agent temozolomide.24 We (Arabshahi
and Reynisson) were already working on the molecular modelling of TDP1 inhibitors25 and
docking of the thieno[2,3ꢀb]pyridines to TDP1 (see Molecular Modelling section) gave
promising results, which can be attributed to serendipity. This phenomenon is known to have
had a significant impact on drug discovery and development.26 Ligand 12 was chosen for
testing because it is a more potent analogue of its derivate lacking the methyl substitution on
the ortho position (GI50 = 18.5 nM vs. 58 nM for MDAꢀMBꢀ435), which has a wellꢀdefined
biological profile.2, 9 Derivative 12 gave an IC50 of 2.2 ꢁM using a new oligonucleotideꢀbased
fluorescence assay.25 Four more derivatives were consequently tested and the results are
given in Table 3.
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TDP1, IC50
(ꢁM)
n
3
3
0
1
1
R
X1
Cl
X2
H
H/H
H/H
H/H
O
1.0 ± 0.3
0.5 ± 0.1
>15
9b
9d
10
11
12
H
CF3
H
CF3
H
CF3
Cl
1.7 ± 0.2
2.2 ± 0.2
O
Me
Table 3. The inhibition of the TDP1 enzyme by the thieno[2,3ꢀb]pyridine structural derivatives.
From the data in Table 3 it is clear that the thieno[2,3ꢀb]pyridines do inhibit the TDP1
enzyme with derivative 9d being the most active with nanoꢀmolar potency. Derivative 10,
with a five membered ring (n=0), is the least active, which fits with the trend that larger ring
systems are preferable for anticancer activity.
Interestingly few small molecule inhibitors of TDP1 have been reported so far.25, 27ꢀ29
Thieno[2,3ꢀb]pyridines are therefore a new compound class that inhibit this enzyme and
further development could lead to more potent compounds. The full dataset is given in the
Supplementary Information section.
Molecular modelling
The thieno[2,3ꢀb]pyridines were docked into the putative binding pocket of the TDP1 crystal
structure. It is proposed that both histidine amino acid residues (His263 and His493) play a
role in the biological function of TDP130 and the binding pocket was defined there. This
pocket has been previously used in molecular modelling work resulting in identification of
active compounds thus lending credibility to this binding site.27, 29 Reasonable scores were
observed for the four scoring functions used (see Table S1 in the Supplementary
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Information). Hydrogen bonding was predicted with His263 and Asn516, as shown in Fig.
2A for derivative 9d. The lipophilic pocket (right) and a cleft (left) shown in Fig. 2B were
consistently occupied by either the phenyl moiety or the aliphatic ring system of the
thieno[2,3ꢀb]pyridines. The cyclopentane derivatives tended to have the aliphatic ring in the
lipophilic pocket whereas the larger eight membered derivatives have their phenyl moiety in
the lipophilic pocket as shown for derivative 9d in Fig. 2B.
A
B
Fig. 2 The docked configuration of 9d to the binding site of TDP1 using ASP. (A) Hydrogen bonds are depicted
as green lines between ligand 9d and the amino acid residues His263 and Asn516. (B) The phenyl group of 9d
occupies a lipophilic cavity to the right hand side and the cyclooctane the lipophilic cleft. The protein surface is
rendered. Red depicts a positive partial charge on the surface, blue depicts negative partial charge and grey
shows neutral/lipophilic areas.
In general, a plausible binding is predicted to the TDP1 of the thieno[2,3ꢀb]pyridines,
supporting the results given in Table 3.
Chemical Space
The calculated molecular descriptors (MW, log P, HD, HA, PSA and RB) for derivatives
6aꢀc, 7aꢀk, 8aꢀc, 9aꢀd, 10ꢀ12 are all within the boundaries of drug – like chemical space. The
calculated values are given in Table S2 in the Supplementary Information section. All of the
derivatives have two hydrogen bond donors and in most cases three rotatable bonds, which
are within lead – like chemical space (for definition of these regions see Zhu, 2012,
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references therein and in Table S3 in the SI).31 The polar surface area (PSA) for all
derivatives is relatively low but does not quite reach lead-like chemical space.
Kinase screen
When the molecular structure of the thieno[2,3ꢀb]pyridine molecular scaffold is considered
then it contains the binding moiety mimicking the adenine moiety in ATP, i.e., the sulphur
atom can act as a hydrogen bond acceptor and the NꢀH in the amine moiety as the hydrogen
bond donor linked by three covalent bonds. In theory, this could make it a possible kinase
inhibitor. Kinases are often targeted for anticancer therapeutics, e.g., Checkpoint kinase 1
32, 33
(Chk1).
Also, inhibition of AuroraꢀB kinase leads to DNA damage,34 which might
explain the potency of the thieno[2,3ꢀb]pyridines. Molecular modelling investigations against
the docking scaffolds of Chk1 and AuroraꢀB were conducted. Plausible binding
configurations were predicted in the hinge regions forming hydrogen bonds to the amino acid
residues Glu85/Cys87 (Chk1) and Ala213 (Aurora), i.e., blocking the adenine binding motif
of ATP and possibly rendering the kinases inactive. This might explain the potency of the
thieno[2,3ꢀb]pyridines. In order to check whether they inhibit kinases, derivative 12 was
screened against 97 defined kinases using the KINOMEscanTM at 10 ꢁM.35, 36
In general, no hits were found against any of the 97 kinases, where a hit is defined as < 35%
of remaining activity compared to untreated system at 100%. 48 of the targets have no
inhibition (100%), 38 with > 90% residual activity, four with > 80%, three in the 70% range
and two in the 60% range. The DYRK1B kinase had 57% activity and the acronym stands for
dualꢀspecificity tyrosineꢀ(Y)ꢀphosphorylation regulated kinase 1B. Finally, the RIO Kinase 2
(RIOK2) had 43% residual activity. In view of these results it is highly unlikely that the
thieno[2,3ꢀb]pyridines are exerting their anticancer effect via kinase inhibition and highlights
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the need to experimentally test modelling hypotheses. The full dataset is in the
Supplementary Information.
Mouse xenograft model
In order to investigate the antitumor activity of the thieno[2,3ꢀb]pyridines derivative 12 was
again selected for preliminary mouse xenograft studies. The 786ꢀ0 renal cancer cell line was
chosen because 12 has an GI50 of ~100 nM for this cell line according to the NCI60 cell
based assay.2 A hundred fold concentration was desired in vivo compared to the GI50 in vitro
or in this case
∼10 ꢁM. Since no pharmacokinetic information was available a uniform and
immediate distribution in the vascular system without accounting for the drug halfꢀlife or
drug metabolism was assumed. Female athymic nude/beige mice were used for the
subcutaneous xenograft model. An individual mouse weighs ~40 g and their volume was
simply taken as 40 mL and the desired dose of 12 was derived as 0.16 mg for daily
intraperitoneal injection. The poor water solubility of derivative 12 was a significant doseꢀ
limiting factor, i.e., the dose could only be doubled before reaching aqueous solubility limit.
It was sought to determine whether 12 was able to inhibit 786ꢀ0 tumour initiation after a
palpable tumour developed. As a control, the animals were injected with the vehicle (5%
(vol/vol) DMSO). Despite poor solubility, intraperitoneal injection of 0.16 mg of 12 resulted
in a partial inhibition of tumour growth; however the difference did not quite reach a level of
statistical significance as shown in Fig. 3 where the error bars slightly overlap.
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1200
1000
800
600
400
200
0
Control
Drug
0
4
8
12
16
20
24
Days
Fig. 3 The xenograft tumour volume of the renal cancer 786ꢀ0 with compound 12 and a control for 24 days.
The mice were administered in morning. The error bars show the standard deviation.
Fig. 4 The final mass of the xenograft tumours of the renal cancer 786ꢀ0 with compound 12 and a control after
24 days of dosing. The error bars show the standard deviation.
The final mass of the tumours was determined and the results are depicted in Fig. 4. As for
the tumour volumes, their mass is reduced by the dosing of compound 12 but statistical
significance is not reached (pꢀvalue 0.13). The mice and the removed tumours are shown in
the Supplementary Information section.
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The injected mice showed evidence of mild spasm following injection, but their overall
activity was not reduced. At the time of sacrifice, several mice in the treatment group showed
signs of facial hirsutism (female facial hair) and dark circles around eyes. Together, these
symptoms suggest a possible effect on adrenal glands, either by their damage by possible
intraperitoneal precipitation of 12 or by direct inhibition of hormone synthesis. Due to the
poor water solubility of 12, these results can only be viewed as preliminary and interpretation
is therefore limited. Nevertheless, this work has qualitatively demonstrated the anticancer
potential of the thieno[2,3ꢀb]pyridines and that aqueous solubility issues need to be resolved
before consistent dosing of animals are achieved. Strategies to enhanced water solubility are
now being investigated.
Conclusions
A series of thieno[2,3ꢀb]pyridines with different aliphatic ring sizes was tested against the
NCI60 tumour panel. It was found that larger ring systems are favoured with derivative 9a,
containing an eight membered ring, showing the greatest cytotoxicity. This series has a
similar biological activity to their cyclohexanone counterparts,1, 2 except they have a better
LC50 profile. Subꢀmicro molecular inhibition of the TDP1 DNA repair enzyme was measured
for derivative 9d, demonstrating that the thieno[2,3ꢀb]pyridines inhibit this enzyme, which
was supported with molecular modelling studies. In order to eliminate possible biological
targets of thieno[2,3ꢀb]pyridines, derivative 12 was screened against 97 kinases. No or weak
affinity was observed excluding this important enzyme class. A preliminary mouse xenograft
study showed reduction of tumour size and mass upon intraperitoneal dosing with derivative
12 but the results did not reach a level of statistical significance. There are still many
unanswered questions regarding this class of compounds such as SAR on the thieno[2,3ꢀ
b]pyridine scaffold, e.g., altering the heteroatoms, which other bioꢀmolecular targets are
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possibly modulated contributing to the anticancer efficacy and the pharmacokinetic profile.
We are currently working on these topics and the results presented here have provided us
with invaluable information on how to proceed.
Acknowledgement
The work was partly supported by the grant of Russian Science Foundation No 14ꢀ24ꢀ00038.
Methodology
In silico searching: The thieno[2,3ꢀb]pyridine derivatives were acquired from commercial
sources using the eMolecules37 and ZINC38 web based compound libraries. Substructure and
Tanimoto similarity search methods were used to identify plausible inhibitors.39 The
compounds were purchased from ChemBridge,15 ChemDiv,16 Specs,17 Key Organics18 and
InterBioScreen.19 The obtained compounds were submitted to the National Cancer Institute’s
Developmental Therapeutic Program (DTP) where they were screened against a panel of
sixty human tumour cell lines (NCI60, for further information see references10, 40, 41 and a
complete description in the Supplementary Information section).
TDP1 assay: The recombinant TDP1 was purified to homogeneity by the chromatography on
Niꢀchelating resin and phosphocellulose P11 as described11, 42 using plasmid pET 16BꢀTdp1
kindly provided by Dr. K.W. Caldecott (University of Sussex, United Kingdom).
TDP1ꢀbiosensor 5’ꢀ(5,6 FAMꢀAAC GTC AGG GTC TTC CꢀBHQ1)ꢀ3’ was synthesized in
Laboratory of Medicinal Chemistry, Institute of Chemical Biology and Fundamental
Medicine, Novosibirsk, Russia.
The TDP1 activity measurements were carried out as described.25 Briefly, TDP1ꢀbiosensor
with final concentration of 50 nM was incubated in a volume of 200 ꢁL containing TDP1
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buffer (50 mM Tris–HCl pH 8.0, 50 mM NaCl, 7 mM βꢀmercaptoethanol) supplemented with
a purified 1.3 nM TDP1. The reactions were incubated at a constant temperature of 26°C in a
POLARstar OPTIMA fluorimeter, BMG LABTECH, GmbH, to measure fluorescence every
1 min (Ex485/Em520 nm). The data were imported into MARS Data Analysis 2.0 program
(BMG LABTECH) and the slope during the linear phase (here data from 0ꢀ7 min) was
calculated.
Kinase assay: See Fabian et al.35 and DiscoverX website.36 A complete description is given
in the Supplementary information.
Mouse xenograft model: Female athymic nude/beige mice (Harlan Laboratories) were used
for subcutaneous xenograft model. All experiments were approved by the Institutional
Animal Care and Use Committee at Johns Hopkins Hospital. 786ꢀO cell lines were harvested
from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA.
Trypsinisation was stopped with medium containing 10% foetal bovine serum, and the cells
were washed once in serumꢀfree medium and reꢀsuspended in PBS. Suspensions comprising
single cells with >90% viability were used for the injections. To establish RCC tumour
xenografts, 786ꢀO tumour cells were injected s.c. (3 × 106 cells) into the flanks of 6ꢀweekꢀ
old mice that were of 20 g average body weight. Tumours developed in all injected mice and
were visible within a few days of implantation. Drug was solubilized in DMSO and further
diluted in PBS/0.5% Tweenꢀ80. Drug (4.0 mg/kg) or control was administered every other
day intraperitoneally beginning when the tumours had grown to a diameter of 7 mm. Tumour
volumes were measured using callipers twice a week and calculated with the formula V =
1/2(a2b) where a is the short axis and b the long axis of the tumour. Treatment was continued
until tumours in the control group grew to 15 mm in diameter, at which point the mice were
sacrificed and the tumours were excised and weighed.
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Molecular Modelling: The compounds were docked to the crystal structure of TDP1 (PDB
ID: 1MU7, resolution 2.0 Å),43 which was obtained from the Protein Data Bank (PDB).44, 45
The Scigress Ultra version 7.7.0.47 program46 was used to prepare the crystal structures for
docking, i.e., hydrogen atoms were added, the coꢀcrystallised tungsten(VI)ion was removed
from TDP1 as well as crystallographic water molecules. The Scigress software suite was also
used to build the inhibitors and the MM247 force field was used to optimise the structures.
The centre of the binding pocket for TDP1 was defined as the position of the hydrogen atom
of HIS263, which nitrogen formed a coordination bond with the tungsten ion (x = 8.312, y =
12.660, z = 35.452) with 10 Å radius. Fifty docking runs were allowed for each ligand with
default search efficiency (100%). The basic amino acid residues lysine and arginine were
defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be
deprotonated. The GoldScore (GS),48 ChemScore (CS),49, 50 ChemPLP51 and ASP52 scoring
functions were implemented to validate the predicted binding modes and relative energies of
the ligands using the GOLD v5.2 software suite.
The QikProp 3.253 software package was used to calculate the molecular descriptors of the
compounds. The reliability of the prediction power of QikProp is established for the
molecular descriptors used in this study.54
Synthesis of compounds: All reactions were carried out under a nitrogen atmosphere in dry,
freshly distilled solvents unless otherwise noted. All NMR spectra were recorded on either
Bruker Avance DRX 300 MHz or 400 MHz spectrometers at ambient temperatures.
1
Chemical shifts are reported relative to the solvent peak of chloroform (δ 7.26 for H and δ
13
77.0 for 13C), DMSO (δ 2.50 for 1H and δ 39.5 for C) or acetone (δ 2.05 for 1H and δ 29.8
1
for 13C). H NMR data is reported as position (δ), relative integral, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of triplets; dd, doublet of doublets; tt, triplet of
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triplets; m, multiplet; br, broad peak; qd, quartet of doublets), coupling constant (J, Hz), and
the assignment of the atom. 13C NMR data are reported as position (δ) and assignment of the
atom. All NMR assignments were performed using HSQC and HMBC experiments. Highꢀ
resolution mass spectroscopy (HRMS) was carried out by either chemical ionization (CI) or
electrospray ionization (ESI) on a MicroTOFꢀQ mass spectrometer. Unless noted, chemical
reagents were used as purchased.
General procedure A; Synthesis of salt: To a solution of sodium methoxide, which was
prepared by adding sodium (1.25 equiv) to dry methanol (7 equiv) at 0 ºC, was added a
mixture of ketone 1 (1 equiv) and methyl formate (1 equiv) over 30 mins, dropwise. The
reaction mixture was stirred at r.t. overnight before being diluted with ether and filtered to
give the crude product 2 which was used in the next reaction without further purification.
General Procedure B; Synthesis of carbonitrile: A mixture of salt 2 (1 equiv) and
cyanothioacetamide (1.1 equiv) in water with piperidinium acetate solution was heated at
reflux for 4 h before being acidified with acetic acid while hot. The reaction mixture was
allowed to cool to room temperature and stirred for a further 12 h before the residue was
filtered off, washed with ice water and collected to give the crude carbonitrile 3 which was
used in the next reaction without further purification. [Piperidinium acetate solution: 20 %
acetic acid, 45 % water, 35 % piperidine].
General procedure C; Synthesis of bromoanilines: To a solution of aniline 4 (1 equiv) and
triethylamine (1.1 equiv) in CH2Cl2 at 0 ºC was added bromoacetyl bromide (1 equiv)
dropwise over 15 mins and the mixture continued to stir for 1 h at 0 ºC. The mixture was
diluted with CH2Cl2, washed with 2M HCl, water, sat. aq. NaHCO3, brine, and dried
(Na2SO4) before the solvent was removed in vacuo to give the desired amides 5.
General Procedure D; Synthesis of thieno[2,3-b]pyridines carboxamides: A mixture of 2ꢀ
bromoacetamide 5 (1 equiv), carbonitrile 3 (1 equiv) and anhydrous sodium carbonate (1.06
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equiv) in absolute ethanol was stirred at reflux at 100 ºC for 18 h. The mixture was cooled to
room temperature and the solvent removed in vacuo to give the crude product which was
washed with small amounts of ice water before being recrystalised from methanol to give the
desired thieno[2,3-b]pyridines 6,7 or 8.
Sodium (E)ꢀ and (Z)ꢀ(2ꢀoxocyclopentylidene)methanolate 2a: The reaction was carried out
following General Procedure A using cyclopentanone 1a (1.92 ml, 21.7 mmol), methyl
formate (1.34 ml, 21.7 mmol) and sodium (0.55 g, 23.9 mmol) in MeOH (4.5 ml, 0.11 mol)
to give the title compound 2a (1.5 g, 52 %) as a white powder which was used in the next
reaction without further purification.
Sodium (E)ꢀ and (Z)ꢀ(2ꢀoxocyclohexylidene)methanolate 2b: The reaction was carried out
following General Procedure A using cyclohexanone 1b (0.2 ml, 1.9 mmol), methyl formate
(1.9 mmol) and sodium (44 mg, 1.9 mmol) in MeOH (0.43 ml, 11 mmol) to give the title
compound 2b (0.16 g, 57 %) as a cream solid which was used in the next reaction without
further purification.
Sodium (E)ꢀ and (Z)ꢀ(2ꢀoxocycloheptylidene)methanolate 2c: The reaction was carried out
following General Procedure A using cycloheptanone 1c (1.44 ml, 12.3 mmol), methyl
formate (0.76 ml, 12.3 mmol) and sodium (0.28 g, 12.3 mmol) in MeOH (2.8 ml, 0.069 mol)
to give the title compound 2c (1.33 g, 67 %) as a white powder which was used in the next
reaction without further purification.
2ꢀThioxoꢀ2,5,6,7ꢀtetrahydroꢀ1Hꢀcyclopenta[b]pyridineꢀ3ꢀcarbonitrile 3a: The reaction
was carried out following General Procedure B using sodium salt 2a (1.51 g, 11.2 mmol) and
cyanothioacetamide (1.23 g, 12.3 mmol) in water (56 ml) with piperidinium acetate solution
(8 ml) followed by acidification with acetic acid (17 ml) to give the title compound 3a (0.76
g, 38 %) as a redꢀbrown solid which was used in the next reaction without further
purification. m.p. 186ꢀ188 °C, (lit. 190 °C).46 δH (400MHz, (CD3)2SO) 2.04ꢀ2.11 (2H, m, Hꢀ
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6), 2.72 (2H, t, J = 7.5 Hz, Hꢀ5), 2.92 (2H, t, J = 7.8 Hz, Hꢀ7), 8.01 (1H, s, Hꢀ4), 14.38 (1H,
br s, NH). δC (100MHz; (CD3)2SO) 22.4 (Cꢀ6), 28.9 (Cꢀ5), 31.1 (Cꢀ7), 113.1 (CN), 117.6 (Cꢀ
3), 127.4 (Cꢀ4a), 141.8 (Cꢀ4), 160.3 (Cꢀ2), 176.0 (Cꢀ7a). The 1H NMR data was in agreement
with the literature values.46
2ꢀThioxoꢀ1,2,5,6,7,8ꢀhexahydroquinolineꢀ3ꢀcarbonitrile 3b: The reaction was carried out
following General Procedure B using sodium salt 2b (0.2 g, 1.4 mmol) and
cyanothioacetamide (0.15 g, 1.5 mmol) in water (7.3 ml) with piperidinium acetate solution
(1.4 ml) followed by acidification with acetic acid (2.2 ml) to give the title compound 3b (77
mg, 30 %) as a tan solid which was used in the next reaction without further purification.
m.p. >230 °C, (lit. 250ꢀ252 °C).46 δH (400MHz, (CD3)2SO) 1.62–1.73 (2 x 2H, m, Hꢀ6 and Hꢀ
7), 2.47–2.51 (2H, m, Hꢀ8), 2.70 (2H, t, J = 6.3 Hz, Hꢀ5), 7.87 (1H, s, Hꢀ4), 13.88 (1H, br s,
NH). δC (100MHz, (CD3)2SO) 20.5 and 21.0 (Cꢀ6 and Cꢀ7), 25.5 (Cꢀ5), 26.7 (Cꢀ8), 113.4
(CN), 117.1 and 121.5 (Cꢀ3 and Cꢀ4a), 145.8 (Cꢀ4), 153.0 (Cꢀ2), 175.4 (Cꢀ8a). The 1H NMR
data was in agreement with the literature values.46
2ꢀThioxoꢀ2,5,6,7,8,9ꢀhexahydroꢀ1Hꢀcyclohepta[b]pyridineꢀ3ꢀcarbonitrile 3c: The reaction
was carried out following General Procedure B using sodium salt 2c (0.76 g, 4.71 mmol) and
cyanothioacetamide (0.47 g, 4.71 mmol) in water (23.6 ml) with piperidinium acetate
solution (4.4 ml) followed by acidification with acetic acid (7.1 ml) to give the title
compound 3c (0.57 mg, 59 %) as a yellowꢀbrown solid which was used in the next reaction
without further purification. m.p. >230 °C, (lit. 247 °C).46 δH (400MHz; (CD3)2SO) 1.50–1.63
(4H, m, Hꢀ8 and Hꢀ6), 1.74–1.80 (2H, m, Hꢀ7), 2.63–2.66 (2H, m, Hꢀ5), 2.94–2.96 (2H, m,
Hꢀ9), 7.96 (1H, s, Hꢀ4), 13.95 (1H, s, NH). δC (100MHz; (CD3)2SO) 24.9 (Cꢀ8), 26.5 (Cꢀ6),
31.1 (Cꢀ7), 31.6 (Cꢀ5), 32.3 (Cꢀ9), 112.8 (CN), 117.3 (Cꢀ3), 127.6 (Cꢀ4a), 145.8 (Cꢀ4), 159.5
(Cꢀ2), 174.9 (Cꢀ9a). The 1H NMR data was in agreement with the literature values.46
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2ꢀBromoꢀNꢀphenylacetamide 5a: The reaction was carried out following General Procedure
C using aniline 4a (1.0 g, 11.0 mmol), bromoacetyl bromide (0.94 ml, 11.0 mmol) and
triethylamine (1.64 ml, 12.0 mmol) in CH2Cl2 (25 ml) to give the title compound 5a (2.29 g,
99 %) as a cream solid. m.p. 133ꢀ135 °C (lit. 129ꢀ131 °C).47 δH (300MHz, CDCl3) 4.02 (2H,
s, Hꢀ2), 7.17 (1H, t, J = 7.5 Hz, Hꢀ4'), 7.36 (2H, t, J = 7.5 Hz, Hꢀ3' and Hꢀ5'), 7.53 (2H, dd, J
= 1.2, 7.5 Hz, Hꢀ2' and Hꢀ6'), 8.19 (1H, br s, NH). δC (75MHz, CDCl3) 29.5 (Cꢀ2), 120.1 (Cꢀ
1
13
2' and Cꢀ6'), 125.2 (Cꢀ4'), 129.1 (Cꢀ3' and Cꢀ5'), 136.9 (Cꢀ1'), 163.4 (C=O). The H and C
NMR data were in agreement with the literature values.47
2ꢀBromoꢀNꢀ(3'ꢀ(trifluoromethyl)phenyl)acetamide 5b: The reaction was carried out
following General Procedure C using 3ꢀ(trifluoromethyl)aniline 4b (0.40 g, 2.48 mmol),
bromoacetyl bromide (0.22 mL, 2.48 mmol) and triethylamine (0.38 mL, 2.73 mmol) in
CH2Cl2 (2 mL) to give the title product 5b (0.70 g, 100 %) as a brown solid. m.p. 78ꢀ81 °C,
(lit. 82 °C).48 δH (400MHz; CDCl3) 4.04 (2H, s, Hꢀ2), 7.41–7.50 (2H, m, Hꢀ4' and Hꢀ5'), 7.75
(1H, d, J = 8.0 Hz, Hꢀ6'), 7.83 (1H, s, Hꢀ2'), 8.28 (1H, s, NH). δC (100MHz; CDCl3) 29.2 (Cꢀ
2), 116.8 (q, 3JF/C 3.7 Hz, Cꢀ2'), 121.8 (q, 3JF/C 3.8 Hz, Cꢀ4'), 123.1 (Cꢀ6'), 123.7 (q, 1JF/C 270.9
Hz, CF3), 129.7 (Cꢀ5'), 131.6 (q, 2JF/C 32.5 Hz, Cꢀ3') 137.4 (Cꢀ1'), 163.7 (C=O). The 1H NMR
data were in agreement with the literature values.49
2ꢀBromoꢀNꢀ(2′ꢀchlorophenyl)acetamide 5c: The reaction was carried out following General
Procedure C using 2ꢀchloro aniline 4c (2 ml, 19 mmol), bromoacetyl bromide (1.66 ml, 19
mmol) and triethylamine (2.9 ml, 21 mmol) in CH2Cl2 (50 ml) to give the title compound 5c
(3.79 g, 80 %) as a light purple solid, m.p. 89ꢀ91 °C. δH (400MHz, CDCl3) 4.07 (2H, s, Hꢀ2),
7.10 (1H, dt, J = 7.6, 1.6 Hz, Hꢀ4′), 7.30 (1H, dt, J = 7.6, 1.6 Hz, Hꢀ5′), 7.40 (1H, dd, J = 8.0,
1.6 Hz, Hꢀ3′), 8.34 (1H, dd, J = 8.0, 1.2 Hz, Hꢀ6′), 8.79 (1H, br s, NH). δC (100MHz, CDCl3)
29.6 (Cꢀ2), 121.2 (Cꢀ6′), 123.5 (Cꢀ2′), 125.4 (Cꢀ4′), 127.7 (Cꢀ5′), 129.2 (Cꢀ3′), 133.9 (Cꢀ1′),
163.4 (C=O). νmax(ATR)/cmꢀ1 3262, 3028, 2963, 1663, 1538, 1445, 1131, 761, 663. m/z
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(ESI+) 270 (79 %)/272 (100 %)/274 (24 %), [MNa+, 100 %]; 227 (7 %); 159 (4 %). HRMS
(ESI+): found 269.9289, C8H779Br35ClNNaO requires 269.9292.
2ꢀBromoꢀNꢀ(2′ꢀfluorophenyl)acetamide 5d: The reaction was carried out following General
Procedure C using 2ꢀfluoroaniline 4d (1 ml, 10 mmol), bromoacetyl bromide (0.9 ml, 10
mmol) and triethylamine (1.58 ml, 11 mmol) in CH2Cl2 (25 ml) to give the title compound 5d
(2.3 g, 96 %) as a light brown solid, m.p. 73ꢀ75 °C. δH (300MHz, CDCl3) 4.04 (2H, s, Hꢀ2),
7.07–7.17 (3H, m, Hꢀ3', Hꢀ4' and Hꢀ5'), 8.21–8.26 (1H, m, Hꢀ6'), 8.42 (1H, br s, NH). δC
(75MHz, CDCl3) 29.2 (Cꢀ2), 114.8 and 115.1 (Cꢀ4' and Cꢀ5'), 121.6 (Cꢀ6'), 124.6 (d, 2JF/C 3.8
2
1
1
Hz, Cꢀ3'), 125.3 (d, JF/C 7.7 Hz, Cꢀ1'), 152.7 (d, JF/C 243.0 Hz, Cꢀ2'), 163.5 (C=O). The H
and 13C NMR data were in agreement with the literature values.50
2ꢀBromoꢀNꢀ(2′ꢀ(trifluoromethyl)phenyl)acetamide 5e: The reaction was carried out
following General Procedure C using 2ꢀaminobenzotrifluoride 4e (1.2 g, 7.5 mmol),
bromoacetyl bromide (0.65 ml, 7.5 mmol) and triethylamine (1.14 ml, 8.2 mmol) in CH2Cl2
(25 ml) to give the title compound 5e (1.48 g, 70 %) as a tan solid, m.p. 95ꢀ98 °C.
δH (400MHz, CDCl3) 4.06 (2H, s, Hꢀ2), 7.28 (1H, t, J = 7.6 Hz, Hꢀ4'), 7.58 (1H, t, J = 7.6 Hz,
Hꢀ5'), 7.64 (1H, d, J = 7.6 Hz, Hꢀ3'), 8.16 (1H, d, J = 8.0 Hz, Hꢀ6'), 8.60 (1H, br s, NH). δC
2
1
(100MHz, CDCl3) 29.2 (Cꢀ2), 120.7 (q, JF/C 29.8 Hz, Cꢀ2'), 123.8 (q, JF/C 271.4 Hz, CF3),
124.1 (Cꢀ6'), 125.2 (Cꢀ4'), 126.2 (q,3JF/C 5.1 Hz, Cꢀ3'), 132.9 (Cꢀ5'), 134.4 (Cꢀ1'), 163.8
(C=O). νmax(ATR)/cmꢀ1 3261, 3026, 2963, 1663, 1533, 1454, 1167, 1110, 662. m/z (ESI+)
304(100 %)/306(96 %), [MNa+, 100 %]; 227(21 %); 159 (10 %). HRMS (ESI+): found
303.9545, C9H779BrF3NNaO requires 303.9555. Found 305.9530, C9H781BrF3NNaO requires
305.9535.
3ꢀAminoꢀNꢀphenylꢀ6,7ꢀdihydroꢀ5Hꢀcyclopenta[b]thieno[3,2ꢀe]pyridineꢀ2ꢀcarboxamide
6a: The reaction was carried out following General Procedure D using 2ꢀbromoꢀNꢀ
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phenylacetamide 5a (60 mg, 0.28 mmol), carbonitrile 3a (50 mg, 0.28 mmol) and anhydrous
sodium carbonate (31 mg, 0.30 mmol) in absolute ethanol (2 ml) to give the title compound
6a (40 mg, 55 %) as a black solid. m.p. 199ꢀ201 °C. δH (400MHz; (CD3)2SO) 2.19–2.27 (2H,
m, Hꢀ6), 3.07–3.14 (4H, m , Hꢀ5 and Hꢀ7), 7.13–7.18 (1H, m, Hꢀ4′), 7.38–7.42 (4H, m, NH2
and Hꢀ3′ and Hꢀ5′), 7.77–7.79 (2H, m, Hꢀ2′ and Hꢀ6′), 8.37 (1H, s, Hꢀ4), 9.43 (1H, br s, NH).
δC (100MHz; (CD3)2SO) 23.2 (Cꢀ6), 29.6 (Cꢀ5), 33.6 (Cꢀ7), 95.7 (Cꢀ2), 121.0 (Cꢀ2′ and Cꢀ
6′), 123.3 (Cꢀ4′), 124.2 (Cꢀ3a), 128.4 (Cꢀ3′ and Cꢀ5′), 128.7 (Cꢀ8a), 133.3 (Cꢀ4), 139.0 (Cꢀ1′),
147.0 (Cꢀ3), 157.2 (Cꢀ4a), 164.0 (C=O), 167.8 (Cꢀ7a). νmax(ATR)/cmꢀ1 3410, 3288, 2952,
1684, 1590, 1509, 1487, 1394, 1226, 742. m/z (ESI+) 332 (100 %), [MNa+, 100 %]; 310 (60
%), [MH+, 60 %]; 219 (80 %); 159 (15 %). HRMS (ESI+): found 332.0818, C17H15N3NaOS
requires 332.0828.
3ꢀAminoꢀNꢀphenylꢀ5,6,7,8ꢀtetrahydrothieno[2,3ꢀb]quinolineꢀ2ꢀcarboxamide 7a: The
reaction was carried out following General Procedure D using 2ꢀbromoꢀNꢀphenylacetamide
5a (105 mg, 0.5 mmol), carbonitrile 3b (93 mg, 0.5 mmol) and anhydrous sodium carbonate
(55 mg, 0.52 mmol) in absolute ethanol (2 ml) to give the title compound 7a (136 mg, 86 %)
as a green solid. m.p. >230 °C. δH (300MHz, (CD3)2SO) 1.76–1.89 (2 x 2H, m, Hꢀ6 and Hꢀ7),
2.88 (2H, t, J = 6.3 Hz, Hꢀ5), 2.95 (2H, t, J = 6.2 Hz, Hꢀ8), 7.06 (1H, tt, J = 7.2, 1.2 Hz, Hꢀ
4'), 7.28–7.34 (4H, m, NH2, Hꢀ3' and Hꢀ5'), 7.66–7.70 (2H, m, Hꢀ2' and Hꢀ6'), 8.18 (1H, s, Hꢀ
4), 9.33 (1H, s, NH). δC (75MHz, (CD3)2SO) 22.3 and 22.4 (Cꢀ6 and Cꢀ7), 28.3 (Cꢀ5), 32.5
(Cꢀ8), 95.8 (Cꢀ2), 121.1 (Cꢀ2' and Cꢀ6'), 123.3 (Cꢀ4'), 124.4 (Cꢀ3a), 128.3 (Cꢀ8a), 128.3 (Cꢀ3'
and Cꢀ5'), 130.8 (Cꢀ4), 139.0 (Cꢀ1'), 146.8 (Cꢀ3), 155.9 (Cꢀ9a), 159.1 (Cꢀ4a), 164.1 (C=O).
νmax(ATR)/cmꢀ1 3448, 3333, 3180, 2943, 1607, 1590, 1522, 1495, 1318, 1166, 745. m/z
(ESI+) 346 (37 %), [MNa+, 37 %]; 324 (100 %), [MH+, 100 %]; HRMS (ESI+): found
324.1161, C18H18N3OS requires 324.1165.
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3ꢀAminoꢀNꢀ(3′ꢀ(trifluoromethyl)phenyl)ꢀ5,6,7,8ꢀtetrahydrothieno[2,3ꢀb]quinolineꢀ2ꢀ
carboxamide 7b: The reaction was carried out following General Procedure D using 2ꢀ
bromoꢀNꢀ(3′ꢀ(trifluoromethyl)phenyl)acetamide 5b (138 mg, 0.5 mmol), carbonitrile 3b (93
mg, 0.5 mmol) and anhydrous sodium carbonate (55 mg, 0.52 mmol) in absolute ethanol (2
ml) to give the title compound 7b (43 mg, 22 %) as a yellow solid. m.p. 193ꢀ194 °C.
δH (300MHz, (CD3)2CO) 1.79–1.95 (2 x 2H, m, Hꢀ6 and Hꢀ7), 2.87–2.98 (2 x 2H, m, Hꢀ5 and
Hꢀ8), 7.09 (2H, br s, NH2), 7.40 (1H, dt, J = 7.7, 0.7 Hz, Hꢀ4'), 7.54 (1H, t, J = 7.9 Hz, Hꢀ5'),
7.99 (1H, dt, J = 8.8, 0.6 Hz, Hꢀ2'), 8.04 (1H, s, Hꢀ4), 8.27 (1H, s, Hꢀ6'), 8.87 (1H, br s, NH).
δC (75MHz, (CD3)2CO) 23.5 and 23.6 (Cꢀ6 and Cꢀ7), 29.5 (Cꢀ5), 33.7 (Cꢀ8), 97.3 (Cꢀ2),
3
5
117.8 (q, JF/C 4.0 Hz, Cꢀ2'), 120.5 (q, 3JF/C 3.9 Hz, Cꢀ4'), 124.7 (q, JF/C 1.0 Hz, Cꢀ6'), 125.3
(q, 1JF/C 270.0 Hz, CF3), 125.4 (Cꢀ3a), 129.6 (Cꢀ4a), 130.3 (Cꢀ5'), 131.1 (Cꢀ4), 131.2 (q, 2JF/C
31.6 Hz, Cꢀ3'), 141.0 (Cꢀ1'), 148.5 (Cꢀ3), 157.4 (Cꢀ9a), 160.6 (Cꢀ8a), 165.4 (C=O).
νmax(ATR)/cmꢀ1 3441, 3326, 2940, 1591, 1541, 1482, 1439, 1331, 1250, 1165 , 662. m/z
(ESI+) 392 (100 %), [MH+, 100 %]; 381 (22 %); 227 (22 %); 159 (8 %). HRMS (ESI+): found
392.1033, C19H17F3N3OS requires 392.1039.
3ꢀAminoꢀNꢀ(2′ꢀchlorophenyl)ꢀ5,6,7,8ꢀtetrahydrothieno[2,3ꢀb]quinolineꢀ2ꢀcarboxamide
7c: The reaction was carried out following General Procedure D using 2ꢀbromoꢀNꢀ(2′ꢀ
chlorophenyl)acetamide 5c (261 mg, 1.1 mmol), carbonitrile 3b (200 mg, 1.1 mmol) and
anhydrous sodium carbonate (0.12 g, 1.1 mmol) in absolute ethanol (4 ml) to give the title
compound 7c (292 mg, 78 %) as a brown solid. m.p. 228ꢀ229 °C. δH (400MHz, (CD3)2SO)
1.77–1.91 (2 x 2H, m, Hꢀ6 and Hꢀ7), 2.88 (2H, t, J = 6.2 Hz, Hꢀ5), 2.95 (2H, t, J = 6.2 Hz, Hꢀ
8), 7.22–7.27 (3H, m, Hꢀ4' and NH2), 7.36 (1H, dt, J = 1.2, 7.8 Hz, Hꢀ5'), 7.53 (1H, dd, J =
1.2, 8.0 Hz, Hꢀ3'), 7.66 (1H, dd, J = 1.1, 7.9 Hz, Hꢀ6'), 8.18 (1H, s, Hꢀ4), 9.05 (1H, s, NH). δC
(100MHz, (CD3)2SO) 22.3 and 22.4 (Cꢀ6 and Cꢀ7), 28.3 (Cꢀ5), 32.5 (Cꢀ8), 95.7 (Cꢀ2), 124.5
(Cꢀ3a), 126.7 (Cꢀ4'), 127.4 and 127.5 (Cꢀ5' and Cꢀ6'), 128.4 (Cꢀ2'), 128.7 (Cꢀ4a), 129.4 (Cꢀ
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3'), 130.9 (Cꢀ4), 135.2 (Cꢀ1'), 146.8 (Cꢀ3), 155.9 (Cꢀ9a), 159.2 (Cꢀ8a), 163.8 (C=O).
max(ATR)/cmꢀ1 3404, 3283, 2928, 1645, 1585, 1504, 1432, 1303, 1187, 746, 691. m/z (ESI+)
ν
358 (100 %)/360 (38 %), [MH+, 100 %]; HRMS (ESI+): found 358.0778, C18H1735ClN3OS
requires 358.0775. Found 360.0759, C18H1737ClN3OS requires 360.0750.
3ꢀAminoꢀNꢀ(2′ꢀfluorophenyl)ꢀ5,6,7,8ꢀtetrahydrothieno[2,3ꢀb]quinolineꢀ2ꢀcarboxamide
7d: The reaction was carried out following General Procedure D using 2ꢀbromoꢀNꢀ(2′ꢀ
fluorophenyl)acetamide 5d (114 mg, 0.5 mmol), carbonitrile 3b (93 mg, 0.5 mmol) and
anhydrous sodium carbonate (55 mg, 0.52 mmol) in absolute ethanol (2 ml) to give the title
compound 7d (85 mg, 51 %) as a green solid. m.p. >230 °C. δH (400MHz, (CD3)2SO) 1.80–
1.91 (2 x 2H, m, Hꢀ6 and Hꢀ7), 2.88 (2H, t, J = 6.4 Hz, Hꢀ5), 2.95 (2H, t, J = 6.4 Hz, Hꢀ8),
7.17–7.28 (5H, m, Hꢀ3', Hꢀ4', Hꢀ5' and NH2), 7.51 (1H, t, J = 7.8 Hz, Hꢀ6'), 8.18 (1H, s, Hꢀ4),
9.22 (1H, s, NH). δC (100MHz, (CD3)2SO) 22.3 and 22.4 (Cꢀ6 and Cꢀ7), 28.3 (Cꢀ5), 32.5 (Cꢀ
8), 95.5 (Cꢀ2), 115.6 (d, 2JF/C 19.9 Hz, Cꢀ3'), 124.1 (d, 4JF/C 3.1 Hz, Cꢀ5'), 124.4 (Cꢀ3a), 125.8
2
3
(d, JF/C 12.3 Hz, Cꢀ1'), 126.7 (d, JF/C 7.7 Hz, Cꢀ4'), 127.5 (Cꢀ6'), 128.3 (Cꢀ4a), 130.9 (Cꢀ4),
1
146.8 (Cꢀ3), 156.2 (d, JF/C 245.4 Hz, Cꢀ2'), 156.0 (Cꢀ9a), 159.1 (Cꢀ8a), 163.9 (C=O).
νmax(ATR)/cmꢀ1 3411, 3275, 2938, 1645, 1606, 1507, 1447, 1319, 1244, 1186. m/z (ESI+) 342
(100 %), [MH+, 100 %]; HRMS (ESI+): found 342.1081, C18H17FN3OS requires 342.1071.
3ꢀAminoꢀNꢀ(2′ꢀ(trifluoromethyl)phenyl)ꢀ5,6,7,8ꢀtetrahydrothieno[2,3ꢀb]quinolineꢀ2ꢀ
carboxamide 7e: The reaction was carried out following General Procedure D using 2ꢀ
bromoꢀNꢀ(2′ꢀ(trifluoromethyl)phenyl)acetamide 5e (138 mg, 0.5 mmol), carbonitrile 3b (93
mg, 0.5 mmol) and anhydrous sodium carbonate (55 mg, 0.52 mmol) in absolute ethanol (2
ml) to give the title compound 7e (124 mg, 65 %) as a brown solid. m.p. 188ꢀ190 °C.
δH (400MHz, (CD3)2SO) 1.78–1.91 (2 x 2H, m, Hꢀ6 and Hꢀ7), 2.88 (2H, t, J = 6.4 Hz, Hꢀ5),
2.95 (2H, t, J = 6.4 Hz, Hꢀ8), 7.21 (2H, s, NH2), 7.49 (1H, t, J = 7.6 Hz, Hꢀ4'), 7.58 (1H, d, J
= 7.6 Hz, Hꢀ6'), 7.71 (1H, t, J = 8.0 Hz, Hꢀ5'), 7.76 (1H, d, J = 8.0 Hz, Hꢀ3'), 8.18 (1H, s, Hꢀ
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4), 9.12 (1H, s, NH). δC (100MHz, (CD3)2SO) 22.2 and 22.4 (Cꢀ6 and Cꢀ7), 28.3 (Cꢀ5), 32.5
2
(Cꢀ8), 95.4 (Cꢀ2), 123.7 (q, 1JF/C 271.9 Hz, CF3), 124.4 (Cꢀ3a), 125.7 (q, JF/C 28.9 Hz, Cꢀ2'),
3
126.4 (q, JF/C 4.8 Hz, Cꢀ3'), 126.8 (Cꢀ4'), 128.3 (Cꢀ4a), 130.6 (Cꢀ6'), 130.9 (Cꢀ4), 132.9 (Cꢀ
5'), 135.9 (Cꢀ1'), 146.8 (Cꢀ3), 155.9 (Cꢀ9a), 159.1 (Cꢀ8a), 164.6 (C=O). νmax(ATR)/cmꢀ1
3435, 3305, 2931, 1653, 1588, 1525, 1453, 1290, 1167, 1106, 659. m/z (ESI+) 392 (100 %),
[MH+, 100 %]; HRMS (ESI+): found 392.1042, C19H17F3N3OS requires 392.1039.
3ꢀAminoꢀNꢀphenylꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀcyclohepta[b]thieno[3,2ꢀe]pyridineꢀ2ꢀ
carboxamide 8a: The reaction was carried out following General Procedure D using 2ꢀ
bromoꢀNꢀphenylacetamide 5a (70 mg, 0.34 mmol), carbonitrile 3c (70 mg, 0.34 mmol) and
anhydrous sodium carbonate (38 mg, 0.36 mmol) in absolute ethanol (1.8 ml) to give the title
compound 8a (110 mg, 91 %) as a brown solid. m.p. > 230 °C. δH (400MHz; (CD3)2SO)
1.65–1.70 (2 x 2H, m, Hꢀ8 and Hꢀ6), 1.84–1.90 (2H, m, Hꢀ7), 2.88–2.91 (2H, m, Hꢀ5), 3.08–
3.11 (2H, m, Hꢀ9), 7.08 (1H, tt, J = 7.3, 1.0 Hz, Hꢀ4′), 7.29 (2H, s, NH2), 7.34 (2H, td, J = 8.0,
2.0 Hz, Hꢀ3′ and Hꢀ5′), 7.71 (2H, dd, J = 8.5, 1.0 Hz, Hꢀ2′ and Hꢀ6′), 8.21 (1H, s, Hꢀ4), 9.36
(1H, s, NH). δC (100MHz; (CD3)2SO) 26.2 (Cꢀ8), 27.9 (Cꢀ6), 31.6 (Cꢀ7), 34.5 (Cꢀ5), 38.8 (Cꢀ
9), 96.1 (Cꢀ2), 121.1 (Cꢀ2′ and Cꢀ6′), 123.3 (Cꢀ4′), 124.3 (Cꢀ3a), 128.4 (Cꢀ3′ and Cꢀ5′), 130.4
(Cꢀ4), 134.1 (Cꢀ4a), 139.0 (Cꢀ1′), 147.0 (Cꢀ3), 155.5 (Cꢀ10a), 164.0 (C=O), 164.8 (Cꢀ9a).
νmax(ATR)/cmꢀ1 3382 (NH amine), 3287 (NH amide), 1647 (C=O amide), 1596 (NH amide),
1507 (C=C aromatic), 1447 (CH alkane), 1319 (CꢀN aromatic). m/z (ESI+): 360 (36 %),
[MNa+, 36 %]; 338 (100 %), [MH+, 100 %]. HRMS (ESI+): found MH+ 338.1321,
C19H20N3OS requires 338.1322.
3ꢀAminoꢀNꢀ(3ꢀ(trifluoromethyl)phenyl)ꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀcyclohepta[b]thieno[3,2ꢀ
e
]pyridineꢀ2ꢀcarboxamide 8b: The reaction was carried out following General Procedure D
using 2ꢀbromoꢀNꢀ(3′ꢀ(trifluoromethyl)phenyl)acetamide 5b (60 mg, 0.20 mmol), carbonitrile
3c (40 mg, 0.20 mmol) and anhydrous sodium carbonate (20 mg, 0.22 mmol) in absolute
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ethanol (1.3 ml) to give the title compound 8b (70 mg, 90 %) as a tan solid. m.p. 213ꢀ215 °C.
δH (400MHz; (CD3)2SO) 1.65–1.70 (4H, m, Hꢀ8 and Hꢀ6), 1.84–1.90 (2H, m, Hꢀ7), 2.88–2.90
(2H, m, Hꢀ5), 3.09–3.11 (2H, m, Hꢀ9), 7.40–7.43 (3H, m, NH2 and Hꢀ4′), 7.57 (1H, t, J = 8.0
Hz, Hꢀ5′), 8.01 (1H, d, J = 8.0 Hz, Hꢀ6′), 8.24 (2H, br s, Hꢀ2′ and Hꢀ4), 9.68 (1H, br s, NH).
δC (100MHz; (CD3)2SO) 26.2 (Cꢀ8), 27.9 (Cꢀ6), 31.6 (Cꢀ7), 34.5 (Cꢀ5), 38.8 (Cꢀ9), 95.3
(Cꢀ2), 116.8 (Cꢀ2′), 119.4 (Cꢀ4′), 124.2 (Cꢀ6′), 124.2 (Cꢀ3a), 129.3 (Cꢀ5′), 129.6 (CF3), 130.6
(Cꢀ4), 134.2 (Cꢀ4a), 140.0 (Cꢀ1′), 147.8 (Cꢀ3), 155.6 (Cꢀ10a), 164.3 (C=O), 165.1 (Cꢀ9a).
ν
max(ATR)/cmꢀ1 3396 (NH amine), 3309 (NH amide), 1649 (C=O amide), 1597 (NH amide),
1541 (C=C aromatic), 1439 (CH alkane), 1165 (CꢀN aliphatic). m/z (ESI+): 428 (20 %),
[MNa+, 20 %]; 406 (100 %) [MH+, 100 %]. HRMS (ESI+): found MH+ 406.1190,
C20H19F3N3OS requires 406.1195.
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