3-Amino-thieno[2,3-b]pyridines as microtubule-destabilising agents: Molecular modelling and biological evaluation in the sea urchin embryo and human cancer cells

Article abstract of DOI:10.1016/j.bmc.2016.11.041

A series of 3-amino-thieno[2,3-b]pyridines was prepared and tested in a phenotypic sea urchin embryo assay to identify potent and specific molecules that affect tubulin dynamics. The most active compounds featured a tricyclic core ring system with a fused cycloheptyl or cyclohexyl substituent and unsubstituted or alkyl-substituted phenyl moiety tethered via a carboxamide. Low nano-molar potency was observed in the sea urchin embryos for the most active compounds (1–5) suggestive of a microtubule-destabilising effect. The molecular modelling studies indicated that the tubulin colchicine site is inhibited, which often leads to microtubule-destabilisation in line with the sea urchin embryo results. Finally, the identified hits displayed a robust growth inhibition (GI₅₀of 50–250?nM) of multidrug-resistant melanoma MDA-MB-435 and breast MDA-MB-468 human cancer cell lines. This work demonstrates that for the thieno[2,3-b]pyridines the most effective mechanism of action is microtubule-destabilisation initiated by binding to the colchicine pocket.

Full text of DOI:10.1016/j.bmc.2016.11.041

Accepted Manuscript
3-Amino-thieno[2,3-b]pyridines as microtubule-destabilising agents: Molecu-
lar modelling and biological evaluation in the sea urchin embryo and human
cancer cells
Chatchakorn Eurtivong, Victor Semenov, Marina Semenova, Leonid
Konyushkin, Olga Atamanenko, Jóhannes Reynisson, Alex Kiselyov
PII:
S0968-0896(16)31016-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.041
BMC 13408
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 October 2016
16 November 2016
21 November 2016
Please cite this article as: Eurtivong, C., Semenov, V., Semenova, M., Konyushkin, L., Atamanenko, O., Reynisson,
J., Kiselyov, A., 3-Amino-thieno[2,3-b]pyridines as microtubule-destabilising agents: Molecular modelling and
biological evaluation in the sea urchin embryo and human cancer cells, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.11.041
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3-Amino-thieno[2,3-b]pyridines as microtubule-destabilising agents:
Molecular modelling and biological evaluation in the sea urchin
embryo and human cancer cells
Chatchakorn Eurtivong1, Victor Semenov2, Marina Semenova3,4, Leonid Konyushkin2, Olga
Atamanenko2, Jóhannes Reynisson1*, Alex Kiselyov5
1 School of Chemical Sciences, University of Auckland, New Zealand
2 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian
Federation
3 N. K. Kol’tsov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow,
Russian Federation
4 Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus
5 Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology,
Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation
*To whom correspondence should be addressed: School of Chemical Sciences, University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand.
E-mail: j.reynisson@auckland.ac.nz, Tel. 64-9-373-7599 ext. 83746, Fax. 64-9-373-7422
Abstract
A series of 3-amino-thieno[2,3-b]pyridines was prepared and tested in a phenotypic sea urchin
embryo assay to identify potent and specific molecules that affect tubulin dynamics. The most active
compounds featured a tricyclic core ring system with a fused cycloheptyl or cyclohexyl substituent
and unsubstituted or alkyl-substituted phenyl moiety tethered via a carboxamide. Low nano-molar
potency was observed in the sea urchin embryos for the most active compounds (1–5) suggestive of a
microtubule-destabilising effect. The molecular modelling studies indicated that the tubulin
colchicine site is inhibited, which often leads to microtubule-destabilisation in line with the sea
urchin embryo results. Finally, the identified hits displayed a robust growth inhibition (GI50 of 50–
250 nM) of multidrug-resistant melanoma MDA-MB-435 and breast MDA-MB-468 human cancer
cell lines. This work demonstrates that for the thieno[2,3-b]pyridines the most effective mechanism
of action is microtubule-destabilisation initiated by binding to the colchicine pocket.
1

Keywords: Microtubule-destabilisation; sea urchin embryo assay; anticancer; thieno[2,3-
b]pyridines; molecular modelling
Introduction
Tubulin is the main protein component of polymeric microtubules that play essential roles in mitotic
spindle formation, intracellular transport, cell shape maintenance and motility.1, 2 During mitosis,
chromosomes are separated along the mitotic spindles before the formation of two daughter cells.1, 2
It is established that deregulated division of cancer cells is dependent on mitosis making
microtubules an attractive target for anticancer chemotherapeutics.1 Drugs that affect microtubules
dynamics are typically characterised as microtubule-destabilising or stabilising agents, depending on
their mode of action.3 The majority of reported microtubule-targeted drugs bind to three distinct
pockets on the tubulin molecule, i.e., the colchicine, Vinca alkaloid or taxol sites.1, 2 Unfortunately,
many anti-tubulin agents exhibit narrow efficacy/safety windows, multiple drug resistance profiles
and are often generally cytotoxic to healthy cells.3, 4 Thus, there is a considerable incentive to
develop novel selective tubulin-binding agents with improved therapeutic profiles.
Recently, a series of 3-amino-thieno[2,3-b]pyridines (AThPs) were identified as effective anti-
proliferative agents against a number of human tumour cell lines.5-9 AThP derivatives (Figure 1, I)
have previously been shown to display high cytotoxicity against a tumorigenic cell line.10 Analysis
of the literature further suggests that biological target specificity for this class of compounds is
dictated by the substitution pattern of the core thieno[2,3-b]pyridine bicyclic system. Namely, related
3-amino-2-keto-7H-thieno[2,3-b]pyridin-6-ones (Figure 1, II) were found to inhibit ubiquitin C-
terminal hydrolase-L1 (UCH-L1).11 Also, the AThP core (Figure 1, III) was introduced into a series
of potent P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and the breast
cancer resistance protein (BCRP1) modulators.12 In a representative example, administration of
AThP IV (Figure 1) to human breast cancer cell line MDA-MB-231 caused growth restriction,
rounding of cell shape, reduced motility and increase in G2/M phase population most likely by
phospholipase C- (PLC-) inhibition.13, 14 Series of related cytotoxic molecules V (Figure 1) were
shown to inhibit in vitro tubulin polymerisation by binding to the colchicine site.15
2

O
O
O
NH2
NH2
NH2
NH2
O
O
O
Cl
O
R
S
S
S
Ar
Ar
HN
S
Ar
N
N
H
N
N
S
IV
II
III
I
X
O
R3
R3
N
NH2
NH2
R
O
O
S
OCH3
S
S
Ar
Ar
HN
N
X = H; CH3
n
n
H3CO
OCH3
n = 0 - 2
V
VI
VII
Figure 1. Biologically active 3-amino-thieno[2,3-b]pyridines I–IV, VI, VII and their analogs 1-benzothiophen-2-
yl(3,4,5-trimethoxyphenyl)methanones V.
In order to glean further insight into the biological mechanism of the AThPs, series VI and VII
(Figure 1) were prepared and tested in a phenotypic sea urchin embryo assay as well as in a panel of
human tumour cell lines. A set of specific developmental alterations exhibited by sea urchin embryo
upon treatment with selective anti-tubulin agents has been validated and used successfully by our
team in the past.16-18 Specifically, two distinct processes during sea urchin embryogenesis are directly
affected by microtubule modulators, cleavage and ciliary swimming after hatching. The compounds
cause cleavage alteration/arrest with distinguishing tuberculate shape of arrested eggs and unique
changes of embryo motility, namely, settlement to the bottom of the culture vessel and rapid
spinning around the animal-vegetal axis. 16, 17 In this study, we used molecular modelling to elucidate
the binding of the VI and VII series to tubulin, their microtubule dynamics effect in the sea urchin
embryos and also the efficacy in the NCI60 human tumour cell line panel.19
Results and Discussion
Two distinct series of AThP VI (1–5) and VII (6–40) with carboxamide and ketone linkers,
respectively, are easily accessed via the 3-component condensation of ketones, aldehydes and
cyanothioacetamide, followed by reaction with 2-halo-acetamides or 2-halo-acetophenones as shown
3

in Scheme 1.20-23 All the compounds for the biological testing were purchased from Chemical Block
Ltd. (www.chemblock.com), and they were synthesised using the approach shown in Scheme 1.
Hal
O
R4
HN
R5
R3
N
NH2
R2
R1
O
R4
R6
R7
R6
S
HN
R5
R3
R3
R7
1-5
O
O
R2
R1
R1
c
N
a
b
Hal
O
R2
S
N
H
80-98%
S
R4
R3
N
NH2
R2
R1
O
NH2
R6
R5
N
S
R4
6-40
R6
R5
Scheme 1. Reagents and conditions: (a) R3CHO, KOH, MeOH; (b) 2-cyanothioacetamide;
(c) 2-halo-acetamides or 2-halo-acetophenones. R1–R7 are listed in Table 1.
Derivatives 1–5 have a tricyclic AThP ring system linked to a phenyl ring via a carboxamide moiety.
Compounds 6–40 include cycloalkyl (6–19), bicyclic (20–36), quinuclidine (37–39), and piperidine
(40) cores with a carbonyl linker. Specifically, molecules 20–28 contain small substitutions at
position groups R1 – R3 in the AThP ring system, whereas molecules 29–36 have aromatic or
heterocyclic substituents at R1.
1
Prior to biological testing, the structures of the acquired compounds were confirmed by H NMR-,
MS-spectroscopies as well as melting points were determined. This data is given in the SI. AThPs 1–
40 were tested in the phenotypic sea urchin embryo assay and in the NCI60 human tumour cell line
panel and the results are given in Table 1.
4

Table 1. The effects of AThPs on the sea urchin embryos and human cancer cells.
R3
O
NH2
R3
H3CO
H3CO
O
NH2
R2
R1
O
S
O
OCH3
N
R2
R1
O
R4
R6
NH
S
N
N
H
S
HN
R5
N
R4
OH
H3CO
Phenstatin
R7
1-5
6-40
Nocodazole
R6
R5
OCH3
Sea urchin embryo effects, EC
NCI60 screen
(M)a
Compound
#
R1
R2
R3
R4
R5
R6
R7
Mean
Cleavage Cleavage
Embryo
spinning
0.1
0.5
5
Mean GI,
b
GI50, Mc
0.0389d
0.06e
alteration
arrest
0.001
0.05
0.5
0.05
0.1
%
Nocodazole
Phenstatin
-
-
-
-
-
-
-
-
-
-
H
H
H
H
H
Me
Br
H
Me
OMe
F
Cl
Br
-
-
Me
H
Me
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
-
-
0.005
0.01
0.05
0.01
0.01
0.01
>4
–(CH2)3–
–(CH2)4–
–(CH2)4–
–(CH2)5–
–(CH2)5–
–(CH2)3–
–(CH2)3–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
H
H
H
H
CF3
H
H
H
H
H
H
H
H
Me
OMe
OMe
OMe
–OCH2O–
–OCH2O–
H
Me
H
Me
H
H
-
-
-
-
-
-
-
-
-
-
-
-
-
76.4f
69.7f
0.42f
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.2
0.5
0.2
>4
>4
>4
0.5
>4
>5
>4
>4
>4
>5
>5
>4
>4
>5
>4
>4
1.00f
NDg
0.04
>4
85.3f
90.0f
0.69f
3.09f
0.2
>4
NDg
NDg
NDg
1
>4
19.3
16.4
0.02
0.02
0.5
0.2
2 (TE)h
>4
0.2
>4
26.7
7.8
8.1
0.5
>4
0.5
0.05
0.5
>4
H
0.2 (TE)h
4 (TE)h
>4
78.9
60.5
0.28
3.24
OMe
OMe
OMe
>4
NDg
NDg
NDg
NDg
NDg
4-Py
H
CF3
H
>4
0.025
>4
>4
0.5 (TE)h
>4
H
H
-
-
H
H
Br
1
>4
5

Me
Me
Me
Me
Me
NH2
NH2
NH2
H
H
H
H
H
CN
CN
CN
H
H
H
H
H
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
Cl
Me
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.5
1
>4
>4
1
4
>4
4
>4
4 (TE)h
>4
>4
>10
>4
>4
>4
>4
>4
>4
18.4
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
NDg
NDg
OMe
–OCH2CH2O–
H
H
OMe
OMe
–OCH2O–
OMe
–OCH2O–
H
H
OMe
OMe
OMe
>4
>4
>4
>4
14.2
2.0
F
Cl
OMe
OMe
NDg
0.5
4.6
43.5
37.4
17.1
>4
2-MeO-C6H4
2-MeO-C6H4
4-Py
NAi
>4
7.94
5.13
>4
4
>4
>4
>4
4
>4
>4
>4
>4
>4
>4
OMe
>4
>4
>4
>4
4-Py
H
H
H
H
NDg
NDg
NDg
2-Thienyl
2-Thienyl
2-Thienyl
OMe
Cl
OMe
OMe
>4
52.6
7.3
6.31
3,4-Methylene-
dioxy-C6H3
N
H
NAi
NDg
H
H
H
H
-
>4
>4
>4
37
N
N
H
H
H
H
OMe
H
OMe
OMe
H
H
H
H
-
-
-
>4
>4
4
>4
>4
>4
>4
>4
>4
11.5
11.7
13.3
38
39
40
N
a The sea urchin embryo assay was conducted as described previously.16 Fertilised eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds. Duplicate
measurements showed no differences in effective threshold concentration (EC) values.
b GI %: single dose inhibition of cell growth at 10 M concentration.
c GI50: concentration required for 50% cell growth inhibition. This is the mean for the 60 cell lines tested in the NCI60 panel.
d NCI ID 238159.
6

e Data from Ref.24
f Data from Ref. 6
g ND: not determined.
h TE: tuberculate eggs typical for microtubule destabilisers.
i NA: not tested, insoluble in 96% ethanol.
7

Biological evaluation
Sea urchin embryo assay results
Analysis of the data presented in Table 1 suggests that many of the AThPs display anti-
proliferative cytotoxic properties. In the in vivo phenotypic sea urchin embryo assay, the
AThPs 1–4 demonstrated the strongest effect, i.e., caused pronounced cleavage alteration in
the 10 – 50 nM range, cleavage arrest was observed at 40 – 500 nM concentrations, and
embryo spinning (0.2 – 5 M), suggesting antimitotic microtubule-destabilising activity
comparable with the activity of phenstatin, a known destabilising agent. In this series, both
cycloheptane and cyclohexane rings were well tolerated, since compounds 2 and 4 exhibited
similar potencies. Introduction of methyl groups at the R6 and R7 positions resulted in
derivative 3 with similar activity, whereas the replacement of the cyclohexane ring in 3 with
cyclopentane (1) reduced antimitotic effect. Interestingly, derivative 5 substituted with the m-
CF3 group at R4 did not affect sea urchin embryo development up to 4 M concentration.
Tricyclic AThPs 6–40 with carbonyl linker displayed weaker antimitotic effects upon linker
replacement (ketone vs. amide) compared to the 1-4 series. For the 6-40 family the
unsubstituted compound 8 showed the strongest microtubule-destabilising effect (embryo
spinning EC = 0.5 M). In addition, compounds 9, 14, 15, and 18 can be considered as
tubulin-targeting antimitotics, since they induced formation of tuberculate eggs typical for
microtubule-destabilising agents.16 The p-substituted methyl (6), methoxy (10), and halogen
(7, 11–13) derivatives were less active, unable to induce cleavage arrest and embryo spinning
up to 4 M concentration. Similar to series 1–5, the replacement of the cyclohexane ring in 9
with cyclopentane yielded less active molecule 6. Introduction of phenyl, 4-pyridine, and CF3
groups at R3 resulted in the loss of effect (15 vs. 16 and 17; 18 vs. 19).
In general, bicyclic AThPs 20–36 as well as quinuclidine (37–39), and piperidine (40)
derivatives exhibited minimal or no antimitotic effect. Only p-methyl substituted compound
22 showed weak microtubule-destabilising activity, inducing the formation of tuberculate
8

arrested eggs. Furthermore, AThPs 21, 25, 26, 28, 31, and 35 displayed weak antiproliferative
activity.
The sea urchin embryo tests revealed that AThPs could target not only tubulin/microtubules,
but also produce other tubulin-unrelated effects. Namely, compounds 16, 17, 19, 27, 30, 32,
and 38–40 were unable to alter cleavage, however caused developmental retardation and
alteration at later stages of development. Interestingly, thienyl derivatives 33–35 exhibited
specific embryotoxicity, inducing embryo disintegration and death at early pluteus,
independently of the stage of exposure and treatment duration. These are qualitative
observations and the mechanisms of these effects remain unclear and need further
investigation in order to be established.
Cytotoxicity in human cancer cell lines
In NCI60 screen compounds 1, 2, 4, and 5 at 10 M concentration inhibited cancer cell
growth by 70% (Table 1). Microtubule-destabilising compounds 14 and 15 also
demonstrated pronounced cytotoxicity (GI50 = 0.28 and 3.24 M, respectively), whereas p-
substituted derivatives 7, 9, 11–13 showed weaker cell growth inhibition as previously
reported.5 Bicyclic AThPs 20–36 (ex., 25) exhibited minimal microtubule modulation in vivo
along with barely detectable cytotoxic properties. Independent studies of 8 using in vitro
(bovine) tubulin polymerisation inhibition assay25 yielded an IC50 value of 13.28 M,
however the molecule did not cause cell cycle arrest in 3T3 cells.
In general, sea urchin embryo results for 1, 2, 4 and 5 were in good correlation with the
NCI60 data reported by Arabshahi et al.6, i.e., human MDA-MB-435 melanoma and MDA-
MB-468 breast cancer cell lines were found to be particularly sensitive to the active series
and the pertinent GI50 values are given in Table 2. Increased conformational flexibility by
cycloalkyl ring expansion showed apparent increase in microtubule-destabilising activity and
cell growth inhibition (molecules 1 vs. 3 and 2 vs. 4). However, compound 1 showed the
9

strongest effect against melanoma MDA-MB-435 cell line with GI50 = 52 nM (Table 2) but a
relatively modest activity in the sea urchin embryo assay compared to other analogues (e.g.,
2–4). This effect was more pronounced for compound 5, which displayed the strongest
general cancer growth inhibition with no potency in the sea urchin assay suggesting an
alternative non-tubulin mediated mechanism of action. A similar outcome was observed for
compound 30 (R1 = 2-methoxyphenyl, R2 = 3, 4-dimethoxyphenyl), it demonstrated a robust
cytotoxic effect (GI50 = 5.13 M) with no activity seen in the sea urchin embryo assay. These
data suggest that specific anti-tubulin effect for the AThPs series is dependent on the
substitution patterns for both the core and the phenyl pharmacophore.
Table 2. Effects of AThPs 1, 2, 4 and 5 on MDA-MB-435 human melanoma and MDA-MB-468 breast cancer
cell lines.
Mean GI50, nMb
Compound #
Mean GI, %a
MDA-MB-435c MDA-MB-468c
1
2
4
5
23.6
30.3
14.7
10.0
52
172
205
2530
197
332
254
394
a GI %: single dose inhibition of cell growth at 10 M concentration.
b GI50: concentration required for 50% cell growth inhibition.
c Data from Ref. 6.
Molecular Modelling
To gain further insight into mode of binding, the AThPs derivatives were docked to the three
main binding sites of tubulin, i.e., colchicine, Vinca alkaloid and taxol pockets using the
respective Protein Data Bank (PDB)26, 27 structures (IDs: 4O2B,28 4EB629 and 1JFF30). Four
scoring functions were employed: GoldScore (GS),31 ChemScore (CS),32, 33 ChemPLP34 and
ASP.35 The co-crystallised ligands were removed and re-docked to their respective binding
sites. Excellent root-mean-square deviations (RMSD) only including the heavy atoms for
Colchicine was obtained with average of 0.394 Å for the four scoring functions used,
10

Paclitaxel also gave good results with an average of 1.625 Å but only GS reproduced the
experimentally derived configuration of Vinblastine adequately at 1.307 Å (see
supplementary Table S1), with the GS algorithm giving the most consistent results in general.
The results of the binding scores (see Table S2 in the SI) against the three possible binding
pockets indicate that the Colchicine site is favoured when the GS scores are considered, it has
+60 whereas the Vinblastine and Paclitaxel pockets only have +50 arbitrary units. When the
scores for the Colchicine and Paclitaxel sites are compared ChemPLP gives considerably
better perditions for the former but ASP and CS give similar results. ChemPLP has been
reported to be the best performing scoring function available in the GOLD package.36 This is
supported by experimental results, AThPs do not have the same mode of action as Paclitaxel,
i.e., no mitotic microtubule bundles were seen for the MDA-MB-231 breast cancer cells upon
administration of an AThPs analogue as expected for a compound sharing a mode of action
with Paclitaxel.14 Thus, the docking results indicate that the Colchicine site is favoured as
suggested by Romangoli et al.15 Occupation of this site often causes destabilistation of
microtubules, which is in line with the sea urchin results. Admittedly, it would had been
better to have better predictions for the Vinblastine binding pocket.
Modelling studies for compounds 1-5 for the Colchicine pocket predicted hydrogen bonding
with the amino acid residues Val181 and Thr179 and hydrophobic contacts between the
phenyl ring and lipophilic regions inside the subunit cavity where side chains of
hydrophobic amino acid residues of Ala316, Leu255 Val181 form a cavity. The cycloakyl
moiety is predicted to be oriented towards a large hydrophobic area within the interface
facilitating lipophilic contacts with the amino acid residues Leu248, Tyr224, Gly143, and
Ala180 as shown in Fig. 2B and 2D. Weak anti-tubulin activities of 6–46 tricyclic, bicyclic
and quinuclidinyl AThPs can be explained by limited interaction with the
11

tubulininterface. This hypothesis is supported by the observation that an increase in
cycloalkyl ring size correlates with enhanced tubulin-destabilising effects of the AThPs.
B
Ala316
Leu255
A
Leu248
Val181
Ala180
Thr179
Gly143
Tyr224
D
C
Leu255
Ala316
Leu248
Val181
Ala180
Thr179
Gly143
Tyr224
Figure 2. Binding poses of compounds 1 (A and B) and 4 (C and D). Colchicine (blue) is overlain with the
ligands (A and C). The grey and black polypeptide ribbons belong to the - and -chains, respectively. Green
dotted lines represent hydrogen bonds. The binding site of the protein is rendered where grey is neutral and
green and pink represent hydrogen bond acceptor and donor groups (B and D).
The calculated molecular descriptors (MW, log P, HD, HA, PSA and RB) for derivatives 1-
40 are all within the boundaries of drug – like chemical space with two minor exceptions,
derivative 16 has a log P value of 5.3 and the MW of derivative 36 is 502.5 g mol-1. Indeed,
12

many of the derived values lie within lead-like chemical space (for definition of these regions
see ref.37, references therein). The calculated values are given in Table S3 in the
Supplementary Information section.
Conclusion
In this work it is shown that tubulin destabilisation is one of the main biological mechanism
of action for the AThPs based on the sea urchin embryo data. Derivatives 1–4 have a low
nano-molar activity but the rest of the compounds are less active. An amide linker between
the thienopyridine core and the phenyl group is crucial for the activity, which can be
rationalised with enhanced lipophilic contact at the interface in tubulin compared to the
analogues containing a ketone linker according to molecular modelling to the colchicine site.
The potency in the sea urchin assay is reflected for the NCI60 human tumour panel with
some notable exceptions (e.g., derivative 5) that suggested that this class of compounds has a
number of modes of action, which can be modulated with, e.g., different substitution pattern
on the phenyl ring. It is clear that more work is required in order to identify all of the
biological targets of the AThPs but the results presented here substantially advances our
insight into their mechanism of action.
Methodology
Analysis of AThPs derivatives
1H NMR spectra were recorded on a Bruker DRX-500 (500.13 MHz) instrument. Chemical
shifts were stated in parts per million (ppm) and referenced to the appropriate NMR solvent
peak(s). Low resolution mass spectra (m/z) were recorded on a Finnigan MAT/INCOS 50
mass spectrometer at 70 eV using direct probe injection. Melting points were measured on a
Boetius melting point apparatus and were uncorrected.
13

Phenotypic sea urchin embryo assay
Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the
Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank. Gametes were
obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater
and fertilised by adding drops of diluted sperm. Embryos were cultured at room temperature
under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The
embryos were observed with a Biolam light microscope (LOMO, St. Petersburg, Russia). For
treatment with the test compounds, 5 mL aliquots of embryo suspension were transferred to
six-well plates and incubated as a monolayer at a concentration up to 2000 embryos/mL.
Stock solutions of compounds were prepared in DMSO at 10 mM concentration followed by
a 10-fold dilution with 96% EtOH. This procedure enhanced the solubility of the test
compounds in the salt-containing medium (seawater), as evidenced by microscopic
examination of the samples. The maximal tolerated concentrations of DMSO and EtOH in the
in vivo assay were determined to be 0.05% and 1%, respectively. Higher concentrations of
either DMSO (0.1%) or EtOH (>1%) caused nonspecific alteration and retardation of the
sea urchin embryo development independent of the treatment stage. Nocodazole (Sigma-
Aldrich) and phenstatin (synthesised as reported previously38) served as reference
compounds. The antiproliferative activity was assessed by exposing fertilised eggs (8–20 min
after fertilisation, 45–55 min before the first mitotic cycle completion) to 2-fold decreasing
concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5–
5.5 h after fertilisation. The effects were estimated quantitatively as an effective threshold
concentration, resulting in cleavage alteration and embryo death before hatching or full
mitotic arrest. At these concentrations all tested microtubule destabilisers caused 100%
cleavage alteration and embryo death before hatching, whereas at 2-fold lower concentrations
the compounds failed to produce any effect. For microtubule-destabilising activity, the
14

compounds were tested on free-swimming blastulae just after hatching (8–10 h after
fertilisation), which originated from the same embryo culture. Embryo spinning was observed
after 15 min to 20 h of treatment, depending on the structure and concentration of the
compound. Both spinning and lack of forward movement were interpreted to be the result of
the microtubule-destabilising activity of a molecule. Video illustrations are available at
http://www.chemblock.com. Experiments with the sea urchin embryos fulfil the requirements
of biological ethics. The artificial spawning did not result in animal death, embryos develop
outside the female organism, and both post-spawned adult sea urchins and the excess of intact
embryos are returned to the sea, their natural habitat.
NCI60 assay
Compounds 7, 9, 11-15, 21, 24, 25, 27-29, 31, 35, 37-40 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 first at 10 μM and for active ligands dose
response curves were generated (NCI60, for further information see ref.19, 39-40 and references
therein). Furthermore, the full description of the protocol is given in the Supplementary
Information.
Molecular modelling
The compounds were docked to the crystal structures of tubulin with the respective PDB IDs:
4O2B (resolution 2.3 Å)28, 4EB6 (resolution 3.47 Å)29 and 1JFF (resolution 3.5 Å).30 The
Scigress v2.641 was used to prepare the crystal structure for docking, i.e. hydrogen atoms
were added, the co-crystallised ligands were removed as well as crystallographic water
molecules. The Scigress software suite was also used to build the chemical structures and
were optimised using the MM242 force field. The centre of the binding were defined in crystal
15

structures: 4O2B as C3 on Colchicine (x = 13.222, y = 8.371, z = -23.331), 4EB6 as C58 on
Vinblastine (x = 13.391, y = 90.610, z = 103.739) and 1JFF as O11 on Paclitaxel (x = 1.403,
y = -16.979, z = 16.391) with 10 Å radius. Fifty docking runs were allowed for each ligand
with default search efficiency (100%). The basic amino acids lysine and arginine were
defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be
deprotonated. The GoldScore (GS),31 ChemScore (CS),32, 33 ChemPLP34 and ASP35 scoring
functions were implemented to validate the predicted binding modes and relative energies of
the ligands using the GOLD v5.4.0 software suite.
The QikProp 3.243 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.44
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Graphical abstract
The thieno[2,3-b]pyridines are shown to induce microtubule-destabilisation explaining their
efficacy against cancer cells.
19