Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines

Article abstract of DOI:10.1016/j.bmcl.2016.12.009

3-Amino-2-arylcarboxamide-thieno[2,3-b]pyridines are a known class of antiproliferative compounds with activity against the phospholipase C enzyme. To further investigate the structure activity relationships of these derivatives a series of analogues were prepared modifying key functional groups. It was determined that modification of the 3-amino and 2-aryl carboxamide functionalities resulted in complete elimination of activity, whilst modification at C-5 allowed compounds of greater activity to be prepared.

Full text of DOI:10.1016/j.bmcl.2016.12.009

Accepted Manuscript
Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thieno-
pyridines
Michelle van Rensburg, Euphemia Leung, Natalie A. Haverkate, Chatchakorn
Eurtivong, Lisa I. Pilkington, Jóhannes Reynisson, David Barker
PII:
S0960-894X(16)31276-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.009
BMCL 24498
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 August 2016
14 November 2016
2 December 2016
Please cite this article as: van Rensburg, M., Leung, E., Haverkate, N.A., Eurtivong, C., Pilkington, L.I., Reynisson,
J., Barker, D., Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines, Bioorganic
& Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.009
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Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines
Michelle van Rensburga, Euphemia Leungb, Natalie A. Haverkate,a Chatchakorn Eurtivonga,
Lisa I. Pilkington,a Jóhannes Reynissona and David Barker*a
aSchool of Chemical Sciences, University of Auckland, New Zealand
bAuckland Cancer Society Research Centre and Department of Molecular Medicine and
Pathology, University of Auckland, New Zealand
*To whom correspondence should be addressed: School of Chemical Sciences, University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand.
E-mail: d.barker@auckland.ac.nz, Tel. 64-9-373-7599, Fax. 64-9-373-7422
Keywords: cancer; thieno[2,3-b]pyridine; phospholipase C; tyrosyl-DNA
phosphodiesterase I; antiproliferative
Abstract
3-Amino-2-arylcarboxamide-thieno[2,3-b]pyridines are a known class of antiproliferative
compounds with activity against the phospholipase C enzyme. To further investigate the
structure activity relationships of these derivatives a series of analogues were prepared
modifying key functional groups. It was determined that modification of the 3-amino and 2-
aryl carboxamide functionalities resulted in complete elimination of activity, whilst
modification at C-5 allowed compounds of greater activity to be prepared.
The potent antiproliferative activity of thieno[2,3-b]pyridines has driven substantial synthetic
interest in this area.1–7 Activity of these compounds has been reported to be due to their
ability to inhibit phospholipase C (PLC), an enzyme that plays a key role in cell signalling
pathways involved in cell proliferation and motility.8,9 More recently, reported interactions
with tyrosyl-DNA phosphodiesterase I (TDP1) may also account for the antiproliferative
activity exhibited by this class of compounds.10 Previously, a range of thieno[2,3-b]pyridine
analogues were prepared and tested for their antiproliferative activity against the National
Cancer Institute’s human tumour cell lines (NCI-60).3 These analogues consisted of two
main types, those containing a fused cyclohexyl ring adjacent to the pyridine ring (derivative
1) and those lacking this moiety (derivatives 2 and 3, Figure 1). These two groups contained
further variations, with the former group differing by the incorporation of a ketone or alcohol
functionality at C-5, whilst the latter group included modifications to the 3-amino group.
1

R
NH2
NH2
3
X
X
X
Y
H
N
H
N
5
2
S
S
N
N
O
O
1
2
N
N
R = (=O) or OH
X = various substitutions
Y = H, Br or Cl
Y
N
O
S
N
3
Figure 1. First group of thieno[2,3-b]pyridine analogues prepared previously.3
Of these analogues, derivatives 1 were found to be most active, with derivatives 2 and 3
showing poor to no activity.3 After the identification of potent activity in derivatives 1,
docking studies of compounds related to 1 were performed and suggested that the 3-amino
and 2-aryl carboxamide moieties of the ligand are involved in hydrogen bonding within the
PLC enzyme site and are therefore important for activity.1 The only investigation of the
importance of the 3-amino group has been undertaken on derivatives 3. However, due to the
poor activity results of the parent compounds containing the 3-amino group (derivatives 2), it
could not be concluded if this group is important for activity.
Our interest was therefore to further develop an understanding of the structure activity
relationship of active thieno[2,3-b]pyridines. We have previously reported thieno[2,3-
b]pyridine 4 which has been the subject of two studies to further determine its biological
activity (Figure 2).1,11 The related, 2-chlorophenyl derivative 5, was found to have similar
activity against tumour cell lines MDA-MB-435, MDA-MB-468, NCI-H522, SF-295 and K-
562.2 Due to promising results, 5 was selected for further testing at the NCI to establish its
activity, which due to its potency led to in vivo toxicity testing. Subsequently, it has been
found to be non-toxic in non-tumoured animals.
O
NH2
H
N
S
N
O
X
Y
4 X = H, Y = Cl
5
X = Cl, Y = H
Figure 2. Active thieno[2,3-b]pyridines 4 and 5.
The encouraging activity seen for thieno[2,3-b]pyridine 5 prompted our interest to develop a
series of derivatives to investigate the structural activity relationship, determining what
moieties are essential for activity. Docking studies of thienopyridine 5 against the binding
2

site of PLC-δ1 show hydrogen bonding interactions between the 2-carboxamide and 3-amino
groups with Asn312, His311, Arg549 and Glu341, as well as hydrogen bonding interactions
between the ketone group at C-5 with Lys438 and Arg549 (Figure 3).
Figure 3. The docked configuration of derivative 5 to the binding site of PLC-δ1 using
ChemPLP. (A) The protein surface is rendered. The phenyl group occupies a lipophilic cavity
to the left hand side. Red depicts positive partial charge, blue depicts negative partial charge
and grey depicts lipophilic/neutral areas. (B) Hydrogen bonds are depicted as green dotted
lines. The amino acids bolded in stick display style contribute to hydrogen bonds with
derivative 5.
To validate the importance of these interactions to biological activity, synthetic focus was
directed to include modifications to the 3-amino, 2-aryl carboxamide, C-5 substituent and the
size of the fused cycloalkyl ring. As such, a group of sixteen derivatives were targeted for
synthesis (Figure 4).
R
R
N
N
NH2
N
H
N
Cl
O
S
S
N
N
O
n
n
Cl
5 R (=O), n = 1
6a R = H, n = 1
8 R (=O), n = 1
9a R = H, n = 1
6b
9b
R = H, n = 2
R = H, n = 2
6c R = H, n = 3
6d R = H, n = 5
9c R = H, n = 3
9d R = H, n = 5
7
10
R = OH, n = 1
R = OH, n = 1
R
R
N
N
NH2
N
H
N
Cl
O
S
S
N
N
O
Cl
11
13
R (=O)
R (=O)
14 R = OH
12 R = OH
Figure 4. Compounds of interest to further investigate SAR of the thieno[2,3-b]pyridine
family.
3

Derivatives 6a-d were first prepared by the reaction of ketone 15a-d with either sodium
methoxide or sodium ethoxide with methyl- or ethyl formate to give the corresponding salts
16a-d (Scheme 1).12,13 Salts 16a-d were then reacted with cyanothioacetamide and
piperidinium acetate, followed by acidification with acetic acid to give carbonitriles 17a-d.12
Carbonitriles 17a-d proceeded in a coupling reaction with 2-chloro bromoacetamide 19, itself
obtained from the reaction of 2-chloroaniline 18 with bromoacetyl bromide and triethylamine
to give the desired thienopyridines 6a-d.
i
ii
CN
S
O
Na
O
O
n
n
N
n
H
15a-d
16a-d
17a-d
H
N
iii
H2N
iv
Br
O
Cl
19
Cl
18
NH2
a n = 1
H
N
b
n = 2
c n = 3
d n = 5
S
N
n
O
Cl
6a-d
Scheme 1. Reagents and conditions: (i) Methyl formate (1 equiv.), Na, MeOH; or Na (1
equiv), ethyl formate (1 equiv), EtOH (cat.), Et2O, r.t., 24 h; (ii) cyanothioacetamide (1.1
equiv.), piperidinium acetate, water, reflux, 4 h, AcOH, r.t., 12 h, 17a 34%, 17b 67%, 17c
62%, 17d 64% (yields over two steps); (iii) bromoacetyl bromide (1 equiv.), Et3N (1.1
equiv.), CH2Cl2, 0 ˚C, 1 h, 19 80%; (iv) Na2CO3 (1.06 equiv.), EtOH, 100 ˚C, 18 h, 6a 78%,
6b 91%, 6c 14%, 6d 52%.
Derivative 5 was prepared in 40% yield by using a similar approach, coupling 2-chloro
bromoaniline 19 with carbonitrile 21, which is derived from diketone 20 (Scheme 2).3
Alcohol derivative 7 was successfully obtained in 89% yield from the reduction of 5 with
NaBH4.
4

O
O
NH2
O
O
CN
S
i
ii
H
N
S
N
H
N
O
Cl
20
21
5
OH
NH2
iii
H
N
S
N
O
Cl
7
Scheme 2. Reagents and conditions: (i) DMFDMA (1 equiv.), DMF, 24 h;
cyanothioacetamide (1 equiv.), NaH (2 equiv.), DMF, 24 h; conc. HCl, 24 h, 21 82%; (ii) 19,
Na2CO3 (1.06 equiv.), EtOH, 100 ˚C, 18 h, 5 40%; (iii) NaBH4 (1 equiv.), MeOH/THF, 2 h, 7
89%.
Derivatives 11 and 12 were prepared starting from 2,4-pentandione 22 (Scheme 3).
O
O
NH2
i
CN
S
ii
O
O
H
N
S
N
H
N
O
Cl
22
23
11
OH
NH2
H
iii
N
S
N
O
Cl
12
Scheme 3. Reagents and conditions: (i) DMFDMA (1 equiv.), dioxane, 24 h; Na (2 equiv.),
MeOH, cyanothioacetamide (1 equiv.), 1 h, r.t., 4 h reflux, 23 97%; (ii) 19, Na2CO3 (2.0
equiv.), EtOH, 100 ˚C, 48 h, 11 98%; (iii) NaBH4 (1 equiv.), MeOH/THF, 2 h, 12 68%.
With the successful preparation of derivatives 5, 6a-d, 7, 11 and 12, modification of the 3-
amino group was achieved through diazotisation using sodium nitrite to give the
corresponding thienotriazinones 8, 9a-d, 10, 13 and 14 (Scheme 4).
5

R
R
N
N
NH2
i
N
H
N
Cl
O
S
S
N
N
O
n
n
Cl
5 R = (=O), n = 1
8 R = (=O), n = 1
6a
6b
9a
9b
R = H, n = 1
R = H, n = 2
R = H, n = 1
R = H, n = 2
6c R = H, n = 3
6d
R = H, n = 5
7
R = OH, n = 1
R
R
N
N
NH2
i
N
H
N
Cl
O
S
S
N
N
O
Cl
11
13
R = (=O)
R = (=O)
14 R = OH
12 R = OH
Scheme 4. Reagents and conditions: (i) NaNO2, AcOH, H2O, 0 ˚C - r.t., 2-7 d, 8 20%, 9a
56%, 9b 31%, 9c 53%, 9d 76%, 10 43%, 13 81%, 14 93%.
Due to the promising preliminary activity of derivative 7 (see Table 1), acetate derivatives
were prepared to investigate further modifications to C-5 (Scheme 5).
6

O
O
NH2
HN
S
O Cl
N
24a
OH
NH2
H
N
i
+
S
N
O
Cl
O
7
O
N
N
Cl
O
S
N
24b
O
OH
O
N
N
N
N
S
i
N
N
Cl
Cl
O
O
S
N
N
10
25
Scheme 5. Reagents and conditions: (i) acetic anhydride (2 equiv.), DMAP, pyridine, 18-72
h, 24a 41%, 24b 56%, 25 91%.
Derivatives 5, 6a, 7, 8, 9a and 10 were accepted for testing against the National Cancer
Institute’s human tumour cell lines (NCI-60), allowing preliminary analysis of some
compounds at at 10 μM concentration (Table 1).14
Table 1. The NCI mean percentage (%) of growth arrest at 10 μM as well as their percentage
(%) of growth arrest against select tumour cell lines, as compared to untreated cells (100%
growth).
Compound
5
6a
7
8
NCI Mean
HCT116
76
MDA-MB-468
MDA-MB-231
48
99
36
96
89
97
25
91
26
86
88
91
70
100
58
94
81
107
30
96
88
102
9a
10
86
7

Independently, we assessed all nineteen derivatives, 5-14, 24a, 24b and 25, against three
human tumour cell lines, HCT116, MDA-MB-468 and MDA-MB-231 at 1 μM. Cell
proliferation was measured using a thymidine incorporation assay (Table 2).15
a
Table 2. Antiproliferative activity of compounds 5-25. Values represent relative growth (%)
versus control. All compounds were tested at 1 μM against the specified cell lines.
Compound
5
6a
6b
6c
6d
7
HCT116a
99
MDA-MB-468a
MDA-MB-231a
94
95
81
100
78
14
91
81
90
85
82
80
104
96
96
118
7
104
119
101
56
104
30
97
94
105
92
91
94
94
71
91
11
97
99
99
90
97
96
87
97
98
100
3
92
8
9a
9b
9c
9d
10
11
12
13
14
24a
24b
25
91
100
108
103
100
10
64
93
82
95
98
The two most active compounds, 7 and 24a, were further tested to establish their IC50 values
(Table 3).
Table 3. IC50 values calculated for compounds 7 and 24a.
IC50 (nM)
Compound
HCT116
MDA-MB-468
MDA-MB-231
K562
7
24a
616
184
3
7
546 22
675 21
431 68
192 18
181
4
201
1
It was determined that a pyridine-fused cycloalkyl ring is required for activity as derivatives
11-14 were all inactive. Of the analogues containing a fused ring without further substituents
at C-5, only the eight-membered analogue, 6c, was mildly active with all others showing no
activity at 1 μM. It was determined that acetate (24a) and then alcohol (7) substitution on a
cyclohexane ring resulted in more active compounds, over ketone groups (5) or non-
substitution (6a-d) (Table 2), with 24a and 7 exhibiting IC50 values in the nano-molar range
(Table 3). In all cases, modification of the 3-amino and 2-aryl carboxamide moieties (8-10,
13, 14, 24b and 25) resulted in inactive compounds, with direct comparisons able to be made
8

between compound 7 (11-30% relative growth across the three tumour cell lines) and 10 (80-
96%), as well as between compounds 24a (3-10%), 24b (64-93%) and 25 (93-98%) which
confirms that these groups are essential for bioactivity. These results are in line with
molecular modelling which shows hydrogen bonding between thienopyridine ligands and
amino (Glu341 and Lys438) within the PLC active site. The incorporation of hydrogen
bonding groups at C-5 had mixed results with similar activities seen between derivatives 5
and 6, while derivative 7 had increased activity compared to 5. Interestingly, the
incorporation of an acetate functionality in derivative 24a, which modelling suggests should
have reduced hydrogen bonding, had similar activity results to that of derivative 7.
Twenty two thieno[2,3-b]pyridine derivatives were successfully prepared to allow
investigations into the SAR of this class of compounds. Synthetic focus was directed to aid
in the determination of the importance of the 5-keto functionality on the ring fused to the
pyridine ring, as well as the importance of the 3-amino and 2-carboxamide moieties on the
activity of the compound. Cytotoxicity results showed that modifications are tolerated at C-5
but not at C-2/C-3, which resulted in the loss of activity in all cases. Additionally the size or
absence of the fused cycloalkyl ring effects the activity of the compounds suggesting a
defined binding pocket with specific important interactions. Interestingly, acetate derivative
24a was found to be more active than derivative 5, a result which is not consistent with the
predicted binding pattern from docking studies. It is possible that the addition of an acetate
group in derivative 24a may increase cell permeability. This may be a beneficial discovery
as concurrent studies have shown that thienopyridines have inherently poor aqueous
solubility.13
Acknowledgements
This work was supported by the Auckland Medical Research Fund, Auckland Cancer
Society, the Auckland Cancer Society Research Centre, and the Faculty Research
Development Fund from the University of Auckland.
Supplementary data
Supplementary data (experimental details for the synthesis of compounds) associated with
this article is available.
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Graphical Abstract
Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines
Michelle van Rensburga, Euphemia Leungb, Natalie A. Haverkate,a Chatchakorn Eurtivonga,
Lisa I. Pilkington,a Jóhannes Reynissona and David Barker*a
aSchool of Chemical Sciences, University of Auckland, New Zealand
bAuckland Cancer Society Research Centre and Department of Molecular Medicine and
Pathology, University of Auckland, New Zealand
11