Design, synthesis and evaluation of novel tetrahydrothieno[3,2-c]pyridine derivatives as potent smoothened antagonists

Article abstract of DOI:10.1039/c6md00020g

Hedgehog (Hh) signalling plays an important role in embryonic development and adult tissue homeostasis. Since activation of the Hh signalling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer

Full text of DOI:10.1039/c6md00020g

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Design, synthesis and evaluation of novel tetrahydrothieno[3,2-
c]pyridine derivatives as potent smoothened antagonists†
Xiaofeng Chen, Chiyu Sun, Jingzhou Zhang, Huaiwei Ding,* and Hongrui Song,**
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, PR China
†The authors declare no competing interests.
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Hedgehog (Hh) signalling plays an important role in embryonic development and adult tissue homeostasis.
Since activation of the Hh signalling pathway is implicated in several types of human cancers, inhibitors
of this pathway could be promising anticancer agents. The smoothened (Smo) receptor mediates Hh
signalling. In the present study, we synthesised a series of novel tetrahydrothieno[3,2ꢀc]pyridine
derivatives using a scaffold hopping strategy. Compounds with this novel scaffold demonstrated
promising Hh and Smo inhibition, indicating that this novel scaffold can serve as a starting point for
further optimisation.
erties 13. Nevertheless, Infinity Pharmaceuticals reported disappoꢀ
inting results from a phase II study of IPIꢀ926 in patients with
1. Introduction
metastatic chondrosarcoma and subsequently suspended the trials
The hedgehog (Hh) pathway is a key signalling transduction
for myelofibrosis in 2012. To date , two Hh signalling pathway
pathway that regulates many critical cellular processes, including
inhibitors, vismodegib (GDCꢀ0449) and sonidegib (NVPꢀ
cell proliferation, embryological development, tissue growth and
LDE225), have been approved by the United State Food and
stemꢀcell maintenance 1ꢀ3. Hh proteins were originally identified
as a highly conserved protein family in fruit flies. This family
Drug Administration for the treatment of locally advanced BCC
14
.
includes sonic Hh, desert Hh and Indian Hh; all of these act as
Unfortunately, the development of resistance to these compoꢀ
ligands of the 12ꢀpass transmembrane protein Patched (Ptch) . In
unds has become a major hurdle for their continued development.
the absence of the Hh ligand, Ptch inhibits the activity of the
Treatment of an MB patient with GDCꢀ0449 produced initially
membrane receptor , smoothened (Smo), which resembles Gꢀ
positive results, but relapse occurred owing to a point mutation in
protein coupled receptors in its general topology. Upon binding to
Smo (D473H), the molecular target of GDCꢀ0449; this rendered
an Hh ligand, Ptch relieves the suppression of Smo, which moves
the patient insensitive to further GDCꢀ0449 treatment 15. The
into the primary cilia where it promotes the dissociation of the
molecular mechanisms explaining resistance to vismodegib weꢀ
complex of suppressor of fused and gliomaꢀassociated oncogene
re reported and published for the first time in 2014 16. The
homologue (GLi). This allows GLi to translocate to the nucleus,
Smo mutations found in sonidegib resistant tumours, at position
where it regulates the expression of genes involvedꢀin uncontrollꢀ
477 17, differed from those found during vismodegib treatment.
ed cellular proliferation and tumour growth most notably in basal
cell carcinoma (BCC)
This is the only acquired resistance reported so far in humans.
4,5
and medulloblastoma (MB) 6. Thus,
Novel potent Hh signalling pathway inhibitors are still urgently
needed as probes to identify clinical profiles for Hh signaling
targeting strategy.
inhibition of aberrant Hh signalling provides a promising
approach to the development of a novel anticancer therapy.
Recent research has focused on novel types of Smo
antagonist 7ꢀ9. The plant steroidal alkaloid, cyclopamine, was first
identified as an inhibitor of Hh signalling because it produced
teratogenic effects in sheep 10. Cyclopamine was later shown to
bind directly to Smo 11. Subsequent research has identified a
number of smallꢀmolecule Smo antagonists, which are progressiꢀ
ng through clinical trials (Fig. 1)
synthetic cyclopamine analogue IPIꢀ926 (Infinity), LYꢀ2940680
(Lilly) and PFꢀ04449913 (Pfizer). The cyclopamine analogue,
IPIꢀ926 , has demonstrated improved potency and drugꢀlike propꢀ
The aim of the present study was to establish a novel skeleton
for Smo antagonists and to explore the diversity of the Hh signalꢀ
ling pathway inhibitor structure, laying a foundation for further
exploration of the structureꢀactivity relationship (SAR) in order to
develop more effective Smo antagonists. Previous studies have
identified Hh pathway inhibitor molecules using highꢀthroughput
screening campaigns 18,19. The publication of the crystal structure
12
; these include the semiꢀ
20
of Smo protein has provided further opportunities to identify
novel Hh signalling pathway antagonists. Fragmentꢀbased design
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DOI: 10.1039/C6MD00020G
and screening is a highly attractive design tool in medicinal
chemistry. Our investigation of a number of templates for novel
Smo receptor antagonists found that the benzimidazole building
block, was present in numerous Smo inhibitors 21,22, including the
Pfizer compound, PFꢀ04449913 . We replaced the pyridine group
with the benzimidazole fragment of PFꢀ04449913 via the isostere
principle and expected that the benzimidazole group would form
extra hydrogen bond(s) at the receptor binding site, thus
increasing Smo antagonist activity. Additionally, nitrogenꢀor
sulphurꢀcontaining heterocycles are regularly used as isosteres for
amide groups in medicinal chemistry. Interestingly, the use of
heterocycles as amide replacements was recently reported to
improve Smo antagonist activity 23ꢀ25, and we speculated that the
conceptual core of piperidine fused to thiophene would produce
a number of new potential Hh signalling pathway inhibitors.
Hence, the novel compounds were designed using the
tetrahydrothieno[3,2ꢀc]pyridine skeleton as a privileged scaffold
and employing a combination of conceptual cyclisation and
bioisosterism strategies, as shown in Fig. 2 . Subsequently, these
molecules, with diverse side chains, were synthesised and their
biological activities were evaluated .
5
10
15
Fig. 1 Structures of smoothened antagonists in advanced development
20
25
Fig. 2. The isostere and scaffold hopping approach used to design a novel series of smoothened antagonists
of excess triethylamine to yield intermediate (3) and removal of
the protecting group of intermediate (3) in the presence of trifluoꢀ
roacetic acid /dichloromethane afforded the key intermediate (4).
The intermediates (10a) and (10b) were prepared as described
previously 22, by treating the 1,2ꢀphenylenediamine (5) with 3ꢀ
bromoꢀbenzaldehyde or 5ꢀbromoꢀ2ꢀchlorobenzaldehyde to afford
(7a) and (7b) separately, followed by methylation with methyl
iodide in the presence of KOH to produce compounds (8a) and
(8b). These were then coupled with intermediate (4) using Buchꢀ
wald coupling under palladium catalysis to afford the condensatiꢀ
on products (9a) and (9b), which were further hydrolysed to
2. Results and Discussion
2.1. Chemistry
The general synthetic methods used to build the designed
compounds are illustrated in Scheme 1. Production of the key
intermediate (4) involved three transformations. Firstly, commerꢀ
cially available butylꢀ1ꢀpiperidinecarꢀboxylate (1) was chloroforꢀ
mylated using VilsmeierꢀHaack reaction to yield intermediate (2);
this was then reacted with ethyl mercaptoacetate in the presence
2
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produce the free acids (10a) and (10b). The condensation of (10aꢀ
resonance (NMR), 13C NMR, and ESIꢀMS.
10b) with various amines afforded the target compounds (11ꢀ53).
The structures of (11ꢀ53) were confirmed by 1H nuclear magnetic
Scheme 1 Reagents and conditions: (a) POCl3, DMF, CH2Cl2, 0 °C to rt; (b) ethyl mercaptoacetate, Et3N, EtOH, rt; (c) TFA, DCM, rt, 1 h; (d) sodium
bisulfite, 3ꢀbrꢀbenzaldehyde or 5ꢀbromoꢀ2ꢀchlorobenzaldehyde, 70 °C, 3 h; (e) potassium hydroxide, DMSO, MeI, 2h ; (f) Pd(OAc)2, BINAP, toluene,
compound 4, 100 °C, 12 h ; (g) LiOH, THF, rt, overnight ; (h) HATU, CHCl3, ethyl acetate, rt, overnight.
group at that position reduced compound activity. Nꢀphenyl
piperazine (25), with IC50 of 1.32 ꢁM, displayed similar activity
as that of compound (11). The subsequent introduction of a
halogen atom to the phenyl ring produced 3ꢀchlorineꢀphenyl
piperazine (26) and 4ꢀfluorineꢀphenyl piperazine (27), which
displayed moderate activities (IC50 = 0.38 ꢁM and 0.32 ꢁM ,
respectively). Interestingly, changing the position of the fluorine
atom in compound (28) resulted in a detrimental effect on its
biological activity with respect to that of compound (27), whereas;
3,4ꢀdichlorophenyl piperazine (29) showed improved activity
(IC50 = 0.27 ꢁM). Other substituents reduced potency, such as the
methoxy group in the para or ortho position in compounds (30)
and (31) , with IC50 values of 1.26 ꢁM and 2.35 ꢁM, respectively.
These data appeared to indicate that the introduction of a more
electronꢀwithdrawing group on phenyl improved the biological
activity of these compounds. However, a decrease of Hh
inhibition (IC50 = 1.76 ꢁM) was observed in the presence of a
trifluoromethylꢀphenyl group (32). Replacement of the phenyl
group by a pyridinyl group (34) resulted in moderate activity
(IC50 = 0.34 ꢁM). Addition of a second nitrogen to the heteroaryl
group (35) improved the activity further, with IC50 of 0.15 ꢁM in
this assay.
2.2. Biological evaluation
The Hh signalling pathway inhibitory activities of tetrahydrꢀ
othieno[3,2ꢀc]pyridine derivatives were initially assessed using a
luciferase reporter in NIH3T3 cells stably transfected with a Gliꢀ
reporter construct (Gliꢀluciferase reporter cell lines) 26ꢀ28. This
assay can effectively identify Smo antagonists and Hh inhibitors.
These in vitro 50% inhibitory concentration (IC50) values are
illustrated in Table 1.
Our initial focus was on compounds bearing various basic
substitutions at the carbonyl group. The clearest trend emerging
from the resulting SAR was that some cyclic aliphatic amines
pyrrole (15), methylpiperidine (16), morpholine (17), 4ꢀhydroxy
piperidine (18) and piperazine (19) showed higher activity than
that shown by acyclic aliphatic amines (11ꢀ14). This finding
indicated that the cyclic amine, piperazine (19), showed the most
potent activity.
We then explored the 4ꢀposition displacement of piperazine,
in order to identify some more potent inhibitors. Nꢀethyl
piperazine (20), showed moderate activity (IC50 = 0.35 ꢁM)
compared to that shown by Nꢀmethyl piperazine (19). However,
4ꢀ(methylsulfonyl)piperazine (21) showed promising potency
(IC50 = 0.22 ꢁM). Surprisingly, conversion to the corresponding
amide group (22) by the insertion of a carbonyl group between
the methyl and 4ꢀposition of the piperazine group resulted in a
loss of activity (IC50 = 0.37 ꢁM). Compound 24 also displayed
diminished activity (IC50 = 0.49 ꢁM). In a similar manner, the
Simple anilines were also systematically explored
(compounds 36ꢀ45). As illustrated in Table 1, the majority of
these aniline analogues either retained or improved the Hh
pathway inhibitory potency in these cellꢀbased assays. The
introduction of methyl on aniline reduced activity and lengthened
the hydrocarbon chain , as demonstrated by compound (37);
this was 2ꢀfold less active (IC50 = 1.63 ꢁM) and the propyl
derivative (38) displayed an even more marked drop in potency
larger Boc derivative (23) showed 12ꢀfold lower activity (IC50
2.08 ꢁM) than that shown by compound (19) , perhaps owing to
steric hindrance by Boc. These data demonstrated that a carbonyl
=
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(IC50 = 3.95 ꢁM). Addition of an orthoꢀfluorine (39), a paraꢀ
bromine (41) and orthoꢀmethoxy (42) had no significant impact
Table 1 SAR of designed compounds
on activity (IC50 = 0.36 ꢁM, 0.45 ꢁM and 0.66 ꢁM, respectively).
Diꢀsubstituted analogues (40), (43) and (44) demonstrated deceꢀ
Compound
11
R
H
R1
Gliꢀluc Reporter IC50 a (ꢁM)
1.41 ± 0.02
Compound
33
R
H
R1
Gliꢀluc Reporter IC50 a (ꢁM)
>15
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.56 ± 0.13
1.72 ± 0.01
1.34 ± 0.03
0.26 ± 0.05
0.33 ± 0.11
0.21 ± 0.01
0.28 ± 0.07
0.17 ± 0.06
0.35 ± 0.12
0.22 ± 0.07
0.37 ± 0.09
2.08 ± 0.02
0.49 ± 0.01
1.32 ± 0.02
0.38 ± 0.05
0.32 ± 0.04
0.76 ± 0.16
0.27 ± 0. 01
1.26 ± 0.05
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
H
0.34 ± 0.06
0.15 ± 0.05
0.85 ± 0.08
1.63 ± 0.11
3.95 ± 0.18
0.36 ± 0.02
0.27 ± 0.06
0.45 ± 0.03
0.66 ± 0.11
0.31 ± 0.15
0.35 ± 0.02
0.20 ± 0.03
0.22 ± 0.01
0.06 ± 0.02
0.19 ± 0.03
0.13 ± 0.01
0.24 ± 0.12
0.09 ± 0.01
0.16 ± 0.02
NH
H
H
N
H
H
NH
H
F
F
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
31
H
2.35 ± 0.15
53
54
Cl
0.14 ± 0.03
0.025
32
H
1.76 ± 0.17
Vismodegibb
a The values are an average of triplicate separate determinations.
bUsed as a positive control.
Table 2 In vitro inhibition of Smo for tetrahydrothieno[3,2ꢀc]pyridine derivatives
a
a
compound
15
SMOꢀBCB IC50 (ꢁM)
0.352
compound
47
SMOꢀBCB IC50 (ꢁM)
0.018
4
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17
18
19
21
29
35
45
46
0.294
0.431
0.326
0.373
0.456
0.237
0.261
0.315
48
49
50
51
52
53
0.192
0.135
0.217
0.092
0.213
0.056
Vismodegibb
0.0051
aInhibition of BODIPYꢀcyclopamine fluorescence signalling in the competitive displacement experiment using HEK293 cells overꢀexpressing human Smo.
Data are expressed from a single IC50 determination.
bUsed as a positive control.
nt potency (IC50 = 0.27ꢁM , 0.31 ꢁM and 0.35 ꢁM , respectively).
An attempt to further improve activity using heteroaryl incorporꢀ
ated into aniline (45) produced good activity (IC50 = 0.20 ꢁM).
Next, the influence of a central aromatic ring substitution in
the core region was explored (compounds 46ꢀ53). We prepared a
set of compounds with a chlorine atom on the phenyl. This
introduction afford good potency in the GliꢀLuc reporter assay,
with IC50 values of 0.06 ꢁM, 0.09 ꢁM and 0.14 ꢁM for
compounds (47), (51) and (53). The activity thus approached that
of vismodegib. These data showed that the presence of a chlorine
atom in the central core region was important for inhibition of Hh
signalling .
Synthetic protocols for all compounds, analytical data, and
procedures and methods for in vitro assays are available in the
Supporting Information.
Acknowledgments
The work was supported by Program for Innovative Research
Team of the Ministry of Education of the People’s Republic of
China and Program for Liaoning Innovative Research Team in
University.
References
To further verify whether the Hh inhibitory activities of the
target compounds in Table 1 derived from their inhibition to Smo
receptor, the more potent sixteen compounds were evaluated in a
fluorescent competitive displacement assay (Table 2). In this
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good Hh inhibition by acting as Smo antagonists. SAR studies of
other parts of this novel scaffold are onꢀgoing in our laboratory
and these results will be reported in due course .
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Footnotes
* Corresponding author. E-mail: dinghuaiwei627@163.com (H. W.
Ding);
** Corresponding author. Fax./ Tel: +86 24 23986443;
hongruisonghrs@126.com (H. R. Song)
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
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MedChemComm
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DOI: 10.1039/C6MD00020G
Graphical abstract
Compounds with this novel scaffold demonstrated promising Hh and Smo inhibition, indicating that this novel
scaffold can serve as a starting point for further optimization.