Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo

Article abstract of DOI:10.1016/j.bmc.2020.115584

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it's necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.

Full text of DOI:10.1016/j.bmc.2020.115584

Journal Pre-proofs
Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors
targeting the Hedgehog pathway with improved antitumor activity in vivo
Nannan Tian, Huanxian Wu, Huiwu Zhang, Danni Yang, Lin Lv, Zichao
Yang, Tingting Zhang, Dongling Quan, Lei Zhou, Ying Xie, Yimei Xu, Ning
Wei, Jiajie Zhang, Mian Chen, John C. Schmitz, Yuanxin Tian, Shaoyu Wu
PII:
DOI:
Reference:
S0968-0896(20)30414-4
https://doi.org/10.1016/j.bmc.2020.115584
BMC 115584
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
15 April 2020
28 May 2020
1 June 2020
Please cite this article as: Tian, N., Wu, H., Zhang, H., Yang, D., Lv, L., Yang, Z., Zhang, T., Quan, D., Zhou, L.,
Xie, Y., Xu, Y., Wei, N., Zhang, J., Chen, M., Schmitz, J.C., Tian, Y., Wu, S., Discovery of [1,2,4]triazolo[4,3-
a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in
vivo, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115584
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Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors
targeting the Hedgehog pathway with improved antitumor activity in vivo
Nannan Tiana,f,1, Huanxian Wu a,1, Huiwu Zhanga,1, Danni Yanga, Lin Lva, Zichao Yanga,
Tingting Zhanga, Dongling Quana, Lei Zhoua, Ying Xieb, Yimei Xuc, Ning Weid, Jiajie
Zhanga, Mian Chene, John C. Schmitzd,*, Yuanxin Tiana,*, Shaoyu Wua,*
a
Guangdong Provincial Key Laboratory of New Drug Screening, School of
Pharmaceutical Science, Southern Medical University, Guangzhou, 510515, PR China;
b State Key Laboratory for Quality Research of Chinese Medicines, Macau University of
Science and Technology, Taipa, Macau, China;
c The Center for Disease Control and Prevention of Xinjiang Uygur Autonomous Region,
Urumqi Xinjiang, 830002, PR China;
d Cancer Therapeutics Program, UPMC Hillman Cancer Center, University of Pittsburgh,
Pittsburgh, PA, USA;
e
Oxford Transplant Centre, Churchill Hospital, Oxford University Hospitals NHS
Foundation Trust, Old Road, Headington, Oxford, OX3 7LE, United Kingdom;
f
Key Laboratory of Orthopaedics and Traumatology, The First Affiliated Hospital of
Guangzhou University of Chinese Medicine, The First Clinical Medical College,
Guangzhou University of Chinese Medicine, Guangzhou, PR China.
Abbreviations: Hh, Hedgehog; Gli1, glioma-associated oncogene 1; Ptch, patched; Smo,
Smoothened; ATO, Arsenic Trioxide; AML, Acute myeloid leukemia; TNBC, triple-negative breast
cancer ; FDA, Food and Drug Administration; DIPEA, N,N-Diisopropylethylamine; SPR, surface
plasmon resonance; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; Shh,
Sonic Hedgehog; IC50, half-maximal inhibitory concentration; LD50, half-leathal dose; Ac-Tub,
Acetylated α-tubulin; IFD, Induce-fit docking; PK, pharmacokinetics.
*Corresponding authors.
E-mail
addresses:
Shaoyu
Wu
(wushaoyu@smu.edu.cn),
Yuanxin
Tian(tyx523@smu.edu.cn), John C. Schmitz(schmitzjc@upmc.edu).
1 These authors contributed equally to the work.
1

Abstract:
Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer
survival than other breast cancer types. Recent studies have demonstrated that the
abnormal Hedgehog (Hh) pathway is activated in TNBC and that these
treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened
（Smo ） protein is a vital constituent in Hh signaling and an attractive drug target.
Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical
application of Smo inhibitors is limited to adult patients with BCC and AML, with many
side effects. Therefore, it’s necessary to develop novel Smo inhibitor with better profiles.
Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo
inhibitors. Four of these novel compounds showed directly bound to Smo protein with
stronger binding affinity than VIS. The new compounds showed broad anti-proliferative
activity against cancer cell lines in vitro, especially triple-negative breast cancer cells.
Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and
apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and
reduced Smo protein and mRNA expression. Furthermore, the expression of the
downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly
inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models
than VIS with lower toxicity. Hence, these results support further optimization of this
novel scaffold to develop improved Smo antagonists.
Keywords: Hedgehog pathway; Smoothened inhibitor; triple-negative breast cancer
2

1. Introduction
Hedgehog signaling is necessary for the growth and formation of numerous tissues
1
during embryogenesis, which is mostly quiescent in adults . The main constituents of
this pathway include the Hh ligands (Sonic, Desert, Indian), Patched receptor (Ptch),
Smoothened receptor (Smo), and the terminal transcription factors Gli1-3, that regulate
the transcription of Hh target genes 1-3. When the Hh ligand binds to Ptch directly, the
inhibition of Ptch on Smo is reduced, thus activating the Hh signaling pathway4.
Subsequently, activated Smo translocates into the primary cilia, resulting in activation
and nuclear accumulation of Gli, which regulates cell proliferation, survival and
differentiation 4, 5. It had be reported that abnormal activation of Hh signaling is related to
the progression of multiple cancers such as medulloblastoma, basal cell carcinoma
(BCC), acute myeloid leukemia (AML), rhabdomyosarcoma, prostate, lung and breast
cancers 1, 4, 6. Smo, as a G protein-coupled receptor, is a vital constituent in Hh signaling
pathway and an attractive drug target4. Smo gets more attention than other components of
Hh signaling pathway because of its flexibility and feasibility7, 8.
The first Smo inhibitor Vismodegib (GDC-0449, VIS) was approved by the US
Food and Drug Administration (FDA) in 2012 for the treatment of BCC patients 9. After
that ， many other Smo inhibitors have been come out, including the approved drugs
Sonidegib (NVP-LDE225), Glasdegib (PF-04449913) and Patidegib (IPI-926) 10 (Fig. 1).
11, 12. Clinical trials of the Smo inhibitors demonstrated not only hopeful results in BCC
patients, but also provided medical cures for patients cannot be treated with traditional
surgery or radiotherapy13, 14. The abnormal activation of the Hh signaling pathway in the
3

initiation and development of various cancers urges the investigation of the Smo
inhibitors in other cancers.
Despite significant results with Smo-targeted antagonists or other Hh signaling
inhibitors, the clinical application of Smo inhibitors is limited to adult patients with BCC
and AML, with many side effects, including diarrhea, constipation, loss of appetite,
alopecia, muscle cramps, and fatigue. Therefore, more and more attention were paid on
discovering novel inhibitors with better profiles.
Previous findings have shown that overexpression of the Hh pathway predicts poor
outcome for breast cancer patients 15. Triple-negative breast cancer (TNBC)，a subset of
breast cancers, have poorer survival than other breast cancer types. Recent studies have
demonstrated that the Hh pathway is activated in TNBC and that these treatment-resistant
cancers are sensitive to inhibition of the Hh pathway 16-18. Early phase clinical trials have
revealed antitumor activity of the Smo inhibitor Sonidegib in TNBC 19, 20. Thus, the Hh
pathway is a promising target for treatment of TNBC and development of novel Smo
inhibitors with superior therapeutic properties is crucial for improved patient outcomes.
We designed and synthesized Twenty [1,2,4]triazolo[4,3-a]pyridines were as novel
Smo inhibitors. Then screened the bioactivities of the novel compounds.
4

Fig. 1. Chemical structure of Smo inhibitors, our designed compounds, and the
binding conformation of VIS to Smo protein. Yellow dot line represents the H-bonds, and
the number on it is the distance of the H-bone.
2. Results and discussion
2.1. Analysis of Co-crystal complex of Vismodegib with Smo and design strategy
The co-crystal structures of human Smo receptor combined with VIS reported
recently provided target structure and interaction details for structure-based drug
discovery21. According to binding analysis based on the crystal structure, the VIS
molecule can be divided into three parts (Fig. 1): the red part including the chloro-phenyl
ring and amide linker, acts as the core skeleton of Smo protein targeting moiety. It can
occupy the central hydrophobic pocket of the transmembrane domain. The H-bond
formed with the Asp384 and the potential H-bonds with Arg400 can stabilize the
conformation of VIS and play an important role in its binding affinities 21. Based on
chemical structure/binding conformation analysis, we designed novel Smo inhibitors for
the treatment of Hh-dependent malignancies. Considering that triazole derivatives
including pyridine derivatives show a wide range of biological activities (insecticidal,
5

anti-inflammatory, anti-tumor, microbicides and anti-bacterial and relaxing blood vessels
22,
23), we replaced the pyridyl of VIS (blue part; Fig. 1) with
[1,2,4]triazolo[4,3-a]pyridine. As an electron-deficient group, it maintains the pi-pi
interaction with the Ar ring within the target protein. The N atoms from the
[1,2,4]triazolo[4,3-a]pyridine group are expected to form H-bonds with Smo protein
(such as Tyr394) contributing to its antagonistic activity, similar to the role of pyridine in
VIS. In order to further optimize compound activity, the chlorophenyl-methylsulfone
moiety (magenta part; Fig. 1) was replaced with different five- or six-membered aromatic
rings.
2.2. Chemistry
A series of [1,2,4]triazolo[4,3-a]pyridines were synthesized according to the process
outlined in Scheme 1. 3-Chloro-2-hydrazinylpyridine and 2-Chloro-5-nitrobenzoic acid
were activated by addition of phosphorus oxychloride and then reduced by Fe to afford
compound 4. Different carboxylic acid groups were formed into corresponding acyl
chloride compounds under dichlorosulfoxide and then reacted with compound 4 under
dichloromethane and DIPEA to afford compounds TPB1-TPB20. The structures of the
1
target compounds were proved by H NMR, 13C NMR and HRMS (see Supporting
information).
6

Scheme 1. Synthetic route for compounds TPB1-TPB20. Conditions: (a) reflux, 8 h; (b)
80℃, reflux, 4 h; (c) reflux, 3 h; (d) stirred overnight at room temperature.
2.3. Inhibitory activity of compound TPB1-TPB20 on Hedgehog pathway
To determine whether compounds TPB1–TPB20 have significant effect on the
activation of the Hh pathway, their inhibitory effect was assessed using NIH3T3 cells
transfected with 8×Gli-binding site firefly luciferase reporter assay 24. VIS, the Smo
antagonist, was a control to verify the assay (Table 1). The results show that most of our
compounds displayed moderate activity against Hh signaling pathway. Among them,
compounds TPB3, TPB14, TPB15 and TPB17 offered considerable potency in this
assay with IC50 of 0.102 µM, 0.096 µM, 0.090 µM and 0.093 µM, respectively (VIS, IC50
0.100 µM) (Table 1). The result demonstrated that our compounds can block the Hh
7

signaling pathway at nanomolar concentrations. In contrast, the other compounds showed
poor inhibitory activities.
8

Table 1. Chemical structures and activity of compounds (TPB1-TPB20).
Cl
N
N
N
O
Cl
R
N
H
IC50 of Gli-luc
reporter (µM)
IC50 of Gli-luc
reporter (µM)
Compd
TPB1
R
Compd
TPB11
R
O
S
O
0.770  0.070
0.727  0.145
0.102  0.020
2.725 0.334
0.285.5  0.020
1.767  0.307
＞10
4.274  0.222
Cl
F
TPB2
TPB3
TPB4
TPB5
TPB6
TPB7
TPB8
TPB9
TPB12
TPB13
TPB14
TPB15
TPB16
TPB17
TPB18
TPB19
TPB20
8.218  0.457
5.204  0.381
0.096  0.002
0.090  0.007
＞10
Cl
Cl
CF3
CF3
Cl
Cl
Cl
Cl
F
Cl
N
Cl
Cl
O
S
O
Cl
N
CF3
0.093  0.013
＞10
N
Cl
Cl
S
＞10
N
N
F
＞10
＞10
1.079  0.107
N
Cl
N
O
TPB10
VIS
2.979  0.299
0.1000 0.022
O
S
O
aIC50 denote the drug concentration that inhibits 50% of Gli-luc reporter. Values represent the
mean ± S.D. from 3–5 separate experiments.
9

2.4. Antiproliferative activities of the compounds against human cancer cell lines
Aberrant activation of the Hh pathway is associated with the development of
multiple cancers 1, 4, 6. However, clinical application of FDA-approved Smo inhibitors is
currently limited to the treatment of adult BCC and AML. To expand the potential use of
Smo inhibitors for other cancers, we evaluated the anti-proliferative effects of our novel
compounds (TPB1–TPB20) by MTT assay against eight human cancer cell lines breast
(MDA-MB-231, MDA-MB-468, MCF-7, T47D), glioma (U251), non-small cell lung
(A549), and cervical (AN3-CA, Hela). The IC50 values are summarized in Table 2.
Interestingly, most new compounds had anti-proliferative activity against these cell lines
that were significantly better than VIS. While VIS was somewhat active in
MDA-MB-468, AN3-CA and Hela cells with IC50 values of 79.0, 93.0 and 61.7 µM,
respectively, it was >100 µM for the other five cell lines. This is consistent with previous
studies demonstrating that VIS had low activity against breast cancer, even though the
25
Hh pathway was active in these cells as evidenced by strong Gli inhibitor activity
.
TPB15 showed the most extensive anti-proliferative activity in 7 of the 8 cell lines tested.
Of note, we found that TPB15 had no effect on growth of MCF-10A cells, a normal breast
epithelial cell, whereas VIS treatment inhibited their growth. Clonal formation assays
were also performed with TPB15 using the two most sensitive cell lines (MDA-MB-231;
MDA-MB-468) (Fig. S1). We obtained IC50 values of 5.63 ± 1.51 and 3.28 ± 0.74 µM,
respectively, confirming the anti-growth effect of TPB15. To further validate TPB15, the
EdU assay was performed on MDA-MB-468 and MDA-MB-231 cells. The result showed
that the cell-proliferation capacity of these cells treated with TPB15 was significantly
lower compared to the control group (Fig. S2).
10

Table 2. Anti-proliferative effects of compounds (TPB1-TPB20).
IC50 (µM)
Cmpd
MDA-MB MDA-MB
MCF-7
T47D
A549
76.9 ± 7.4 73.5 ± 7.1 67.6 ± 7.9
>100 >100 >100
U251
AN3-CA
Hela
MCF10A
-468
-231
TPB1
TPB2
>100
58.2 ± 2.0
>100
>100
>100
>100
59.9 ± 1.6 92.2 ± 6.9 83.8 ± 2.6
>100
>100
>100
TPB3 54.8 ± 5.5 66.1 ± 9.1 71.9 ± 16.9 30.2 ± 5.3 52.7 ± 1.7 43.1 ± 9.6 68.4 ± 8.7
TPB4
>100
>100
>100
>100
>100
>100
58.4 ± 8.1
78.2 ± 6.0
94.2 ± 3.9
>100
>100
73.6 ± 7.5
>100
>100
TPB5 21.6 ± 0.9
>100 61.3 ± 12.4 >100 84.8 ± 19.0 >100
TPB6
TPB7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
TPB8
>100
74.3 ± 1.5
79.8 ± 6.5
TPB9
92.0 ± 4.4 96.4 ± 11.2 >100
>100 88.9 ± 10.2 >100
>100 79.0 ± 19.4 >100 75.5 ± 15.5
TPB10
TPB11
TPB12
TPB13
>100
>100
>100
>100
>100
>100
44.6 ± 5.6
>100
>100
>100
>100
>100
86.4 ± 7.7
>100
37.2 ± 1.3
75.0 ± 2.4
>100
>100
>100
>100
>100
>100 81.6 ± 19.7 >100
>100 62.8 ± 7.6 70.8 ± 6.8
>100
>100
TPB14 22.3 ± 1.1
>100 41.2 ± 12.5 61.9 ± 16.1
TPB15 28.6 ± 3.2 29.5 ± 3.2 61.5 ± 4.4 42.3 ± 8.2 64.9 ± 37.5 85.2 ± 3.9 44.5 ± 9.8 66.3 ± 14.6 >100
TPB16 34.3 ± 4.8
TPB17 46.0 ± 7.9
>100
>100
>100
>100
>100
>100
>100
48.3 ± 7.0 86.3 ± 20.0
>100 >100
>100 87.5 ± 25.8
>100 >100
93 ± 4.5 61.7 ± 7.6 89.5 ± 12.3
>100
>100 87.6 ± 32.1 46.8 ± 22.3 >100
TPB18 57.6 ± 10.1 >100
>100
>100
>100
>100
>100
>100
>100
>100
72.3 ± 2.7
35.8 ± 8.1
42.7 ± 6.5
>100
>100
>100
>100
>100
TPB19
TPB20
VIS
>100
>100
89.7 ± 5.2
>100
79 ± 3.2
>100
aIC50 denote the drug concentration that inhibits 50% of cell growth. Values represent the mean ± S.D. from 3–5
separate experiments.
2.5. Apoptosis and cycle arrest of cells induced by compound TPB15
To understand its mechanism(s) of action, we studied the impact of TPB15 on cell
cycle distribution. In MDA-MB-468 cells (Fig. 2A, 2C), TPB15 arrested cells in G2/M
whereas it arrested MDA-MB-231 cells in G0/G1-phase (Fig. S3A, S3C) in a
dose-dependent manner. In addition, TPB15 increased apoptosis in the MDA-MB-468
cells (Fig. 2B, 2D) and MDA-MB-231 cells (Fig. S3B, S3D) in
a
11

concentration-dependent manner. TPB15 induced 39.70% apoptosis in MDA-MB-468
cells at the highest concentration. To confirm these results, we detected apoptotic proteins
by Western blot. With increasing concentrations of TPB15, Bcl-2 was reduced while Bax
was increased in both cell lines (Fig. S4). These results suggest that the increase of
apoptosis contributed to the anti-proliferative effect of TPB15.
Fig. 2. Analysis of cell cycle and apoptosis in MDA-MB-468 cells. (A) Cells were
incubated with TPB15 (1/2 IC50= 14 µM, IC50=28 µM, 2 IC50=56 µM) for 48 h, followed
12

by fixation, PI staining, and flow cytometric analysis. (B) Cells were treated with TPB15
for 48 h followed by Annexin V-FITC/PI staining and flow cytometry. Values (C, D)
represent the mean  S.D. from three independent experiments; (*) P<0.05; (**) P<0.01;
(***) P<0.001 vs control (DMSO).
2.6. The binding capacity of the compounds to the Smo protein
Molecular docking, surface plasmon resonance (SPR) and BODIPY-cyclopamine
viable cell binding assays were used to evaluate the binding capacity of the compounds to
the Smo protein. Considering the flexibility of Smo, the Induce-fit docking (IFD) method
was used to elucidate the binding model of potent compounds. IFD allows the movement
of both side chains from binding site residues and the rotatable bonds from ligand during
the docking process 26. The complex structure of VIS was retrieved from the RCSB
database (PDB entry: 5L7I 27). The key residues Asp384, Arg400, Gln477, Phe484,
Tyr394 were selected for the grid box of the binding site 28.
The docking results demonstrated that the binding modes of compound TPB14,
TPB15, TPB17, and TPB3 were similar to that of VIS. As we expected, they occupied
binding sites comprised by the entrance of the membrane and the transmembrane domain,
especially TM5, TM6 and TM7 (Fig. 3A). Dichloropyridinyl group of TPB15 oriented
towards the extracellular domain and the entrance of TMD pocket. The
N-4-[1,2,4]triazolo[4,3-a] pyridinyl group, instead of pyridine group from VIS, was
deeply buried in the core of transmembrane helixes (TM5, TM6 and TM7). The
arrangement was stabilized by H-bonds and the pi-pi interaction mentioned below. The
core structure amide linker formed two H-bonds: NH from amide linker and the C=O
from the main chain of Asp384; C=O from amide and the NH from the side chain of
13

Glu518 (Fig. 3A). Furthermore, the N-4-[1,2,4]triazolo[4,3-a] pyridinyl group could be
fixed by cation-pi interactions with Arg400 and by pi-pi interactions with Trp281,
stabilizing the binding conformation. The N atoms from triazolo ring and the OH from
side chain of Tyr394 also formed H-bonds, which also benefited the conformation.
Compounds TPB8 and TPB20 have more flexibility because of the smaller
five-membered ring, which led to weaker binding results and anti-proliferative activities.
Compared with TPB15, compound TPB5 had subtle structural differences. When
dichloropyridinyl replaced by dichlorophenyl, the electron-deficiency of the Ar ring led
to a little weaker binding. The same phenomena were also evident between compounds
TPB17 and TPB13. It was worth noting that the difference of 2-position substitute of
phenyl in compounds TPB13 and TPB14: F atom for compound TPB13 and Cl atom for
TPB14. It is possible that the conjugation effect from Cl atom decreased the pi-pi
interaction with Phe484. As the typical electron-withdrawing groups, the methylsulfone
moiety from TPB10 had the steric hindrance at meta-position of phenyl comparing to Cl
from TPB3. This might explain why TPB10 had low activity (2.979 µM) while TPB3
showed potent activity (0.102 µM). The structure-activity relationship is summarized in
Fig. 3B.
14

Fig. 3. The binding conformation of TPB15 compared to VIS. (A) IFD of TPB15
(yellow) and VIS (pink). (B) Structure-activity relationships of our novel compounds.
Blue dot line represents the H-bonds, and the number on it is the distance of the H-bone.
Since compounds TPB3, TPB14, TPB15 and TPB17 showed improved potency,
SPR experiments were used to characterize the interaction of these compounds with Smo
protein compared to VIS. The results showed that the equilibrium dissociation constants
(KD) between compounds TPB3, TPB14 and Smo were 1.21×10-5 M and 1.36×10-5 M,
respectively, while VIS was 9.75×10-8 M (Table 3, Fig. S5). Surprisingly, compounds
TPB15 and TPB17 exhibited the most powerful interactions with Smo (KD values,
1.03×10-8 M and 1.06×10-8 M). These data indicated that TPB15 and TPB17 bind
significantly tighter with Smo protein than VIS.
15

Table 3. The binding strength of TPB3, TPB14, TPB15, TPB17 and VIS to the Smo
receptor
Cmpd
kaa (1/Ms)
kdb (1/s)
Rmaxc (RU)
KAd (1/M)
KDe (M)
TPB3
TPB14
TPB15
TPB17
VIS
101
111
360
260
179
1.23×10-3
1.51×10-3
3.75×10-6
2.75×10-6
1.74×10-5
41.2
49.2
90.5
8.44
4.63
8.21×104
7.35×104
9.71×107
9.43×107
1.03×107
1.21×10-5
1.36×10-5
1.03×10-8
1.06×10-8
9.75×10-8
a ka = association rate constant
b kd = dissociation rate constant
c Rmax = maximum resonance response at equilibrium
d KA = the equilibrium association constant
e KD = the equilibrium dissociation constant
BODIPY-cyclopamine viable cell binding assay was used to determine whether our
29
compounds can bind directly to the Smo protein . Green-fluorescence was observed in
cells after incubation with BODIPY-cyclopamine (Fig. 4B). Incubation with unlabeled
cyclopamine resulted in decreased green fluorescence, indicating that unlabeled
cyclopamine competitively inhibits the binding of BODIPY-cyclopamine. Cells treated
with TPB15 displayed a clear dose-dependent reduction in the green fluorescence
indicating that compound TPB15 binds to Smo protein intracellularly.
16

Fig. 4. (A) Western blot analysis of the Smo protein in the HEK293 cells transfected with
WT-Smo plasmid. (B) BODIPY-cyclopamine viable cell binding assay.
2.7. Compounds inhibited the expression of Hedgehog pathway signaling proteins and
mRNA
To verify the mechanism of action, the time- and dose-dependent effects of TPB3,
TPB14, TPB15 and TPB17 on Smo protein and mRNA expression were characterized
by Western blot and qRT-PCR. The expression of Smo protein and mRNA was markedly
suppressed in a dose- and time-dependent manner (Fig. 5). We also evaluated the effect
of TPB15 on other members of the Hedgehog pathway. As observed with Smo, the
expression of Shh, Gli1 and Ptch2 protein and mRNA was significantly suppressed in a
dose- and time-dependent manner (Fig. S6). Therefore, these data further support that
compound TPB15 effectively inhibits the Hh signaling pathway.
17

18

Fig. 5. Effect of Smo inhibitors on Smo protein and mRNA in MDA-MB-468 cells. Cells
treated with TPB3 (55 µM), TPB15 (28 µM) or TPB17 (50 µM) for 48 h were harvested
and processed for western blot analysis (A) and qRT-PCR (C). Cells treated with
compounds for 24-72 h were processed by western blot analysis (B) and qRT-PCR (D).
GAPDH was used as an internal control. Data represent the mean ± S.D. from 3
experiments. (*) P<0.05; (**) P<0.01; (***) P<0.001 vs control (DMSO).
2.8. Smo localization upon treatment with TPB15
Smo acts on the primary cilium, transmitting Hh signals, while Smo localization to
cilia is a key regulatory step in Hh pathway activation 30. We tested whether our
compounds can inhibit Hh pathway activation through disruption of Smo translocation.
Primary cilia were visualized by immunofluorescence labeled with anti-acetylated
α-tubulin antibodies. NIH3T3 cells were treated with TPB15 or VIS for 24 h, in the
presence or absence of 200 nM Smo agonist SAG. Confocal imaging revealed that
TPB15 inhibited SAG-induced Smo localization to the primary cilia similar to VIS (Fig.
6A). In addition, Smo protein levels were significantly reduced in the cilia of
MDA-MB-468 cells following TPB15 or VIS treatment (Fig. 6B).
Blocking the
translocation of Smo to the primary cilia has been shown to reduce Gli1 expression and
its target genes associated with proliferation, survival and metastasis 31. These results
indicate that Hh signaling can be suppressed by TPB15 through blocking Smo
translocation into the primary cilia.
19

Fig. 6. Effect of TPB15 on Smo localization. Confocal imaging of primary cilia of
NIH3T3 cells (A) and MDA-MB-468 cells (B) treated with VIS or TPB15 (28 µM) for
24 h in the presence or absence of SAG after immunostaining with anti-acetylated
α-tubulin and Smo antibodies. ***, P<0.001, **, P<0.01 vs DMSO; ###, P<0.001 vs SAG.
2.9. Acute toxicity test
The new compounds showed broad anti-proliferative activity against cancer cell
lines in vitro with low toxicity against normal breast epithelial MCF-10A cells. We
20

hypothesized that our compounds have low toxicity in vivo. To verify this hypothesis, two
of our most active compounds were selected for an acute in vivo toxicity test. The LD50 of
TPB3, TPB15 and VIS were determined using Kunming mice by intragastric
administration according to the improved Karber's method 32. TPB3 (LD50 > 5000 mg/kg)
and TPB15 (LD50 = 3875 mg/kg) had higher LD50 and thus lower toxicity compared to
VIS (LD50 = 2008 mg/kg) (Table 4).
Table 4. Acute toxicity of TPB3, TPB15 and VIS
Dose
Mortality
LD50
Mortality（D/T)
Cmpd
TPB3
（mg/kg）
（%）
（mg/kg）
Female
Male
＞5000
5000
3008
3453
3965
4552
5226
741
0/5
1/5
2/5
3/5
3/5
4/5
1/5
1/5
3/5
4/5
4/5
0/5
0/5
1/5
2/5
3/5
3/5
0/5
2/5
2/5
2/5
4/5
0
10
30
50
60
70
10
30
50
60
80
TPB15
3875
1111
1778
2667
4000
VIS
2008
D/T: dead/treated mice. 95% confidence intervals of TPB15 were between 2807 and 4956
mg/kg. 95% confidence intervals of VIS were between 788 and 3623 mg/kg.
2.10.
TPB15 inhibits xenograft tumor growth in vivo
To evaluate the anti-tumor effect of compound TPB15 in vivo, a subcutaneous
MDA-MB-468 xenograft model was used. Tumor volume was recorded every other day
and the body weight was recorded every day. TPB15 and VIS were dosed at 80 mg/kg
21

and 100 mg/kg per day, respectively (intragastric administration). Smo inhibitors did not
affect mouse body weight (Fig. 7A). The reduction in tumor volume of VIS-treated group
(100 mg/kg) reached 55.78% compared to the vehicle control, while TPB15 (80 mg/kg)
reduced the tumor volume by 64.97% (Fig. 7B). Thus，TPB15 has significant inhibitory
effects on tumor growth in vivo without evidence of toxicity.
Fig. 7. Effect of TPB15 on growth of a breast cancer xenograft. Body weight (A) and
tumor volume (B) were expressed as mean ± SD (n=5/group). **, p<0.01 compared to
control; #, p<0.05 compared to Vismodegib group.
3. Discussion
In summary, we have developed a series of novel [1,2,4]triazolo[4,3-a]pyridines as
potent Smo inhibitors that exhibited anti-proliferative activity against a variety of cancer
cell lines, especially TNBC. The compounds TPB14, TPB15 and TPB17 demonstrated
nanomolar inhibition of the Gli-luciferase reporter assay indicating that the design of
8-chloro-[1,2,4]triazolo[4,3-a]pyridines was successful. Molecular docking simulations
indicated that our compounds have a similar binding mode as VIS to Smo protein.
Surprisingly, the SPR assay demonstrated that compounds TPB15 and TPB17 interacted
22

with Smo protein with stronger affinity than VIS. The potency may be related to the
novel scaffold that potentially increases the H-bond interactions with the hinge region of
Smo. Compounds containing a 3-chlorophenyl and 2,6-dichloro-3-pyridyl moiety,
TPB15 and TPB3, respectively, showed extensive anti-proliferative activity in various
cell lines. This indicated that the introduction of chlorine atoms in phenyl or pyridyl
moieties might improve biological activity. Global cancer epidemic statistics shown that
breast cancer accounts for 11.6% of all the cancer incidence, the second leading cause of
cancer death among women after lung cancer, and TNBC accounts for about 15~20% of
breast cancers33. Currently, there is no FDA-approved targeted therapy for TNBC, so
patients have to undergo a systemic treatment strategy based on conventional
chemotherapy and radiotherapy34. Evidence suggests that about 30% of the TNBCs have
paracrine activation in the Hh signaling pathway; SMO inhibitors could represent
therapeutics of interest for a significant number of women who diagnosed with TNBC 20.
Our results provide evidence that four of the novel compounds suppress the growth of
human breast cancer cells in vitro. TPB15 induced cell apoptosis and cycle arrest in
breast cancer cells, and reduced expression of Bcl-2 while increased the Bax.
Mechanisms results showed that TPB15 effectively suppressed protein and mRNA
expression of members of the Hh pathway, which downregulated Hh target gene
expression. Imaging studies demonstrated that TPB15 suppressed Hh signaling through
blocking Smo translocation into the cilia. Consistent with our in vitro data, we also found
TPB15 significantly reduced the tumor volume with low toxicity. Taken together, our
results show that the [1,2,4]triazolo[4,3-a]pyridine scaffold represents a novel platform
for the development of a novel class of Hh pathway inhibitors for the treatment of TNBC.
23

4. Experimental section
4.1. General
Unless otherwise specified, all chemical and biological reagents are purchased from
commercial suppliers and used without further decontamination. All reactions were
monitored using thin-layer chromatography (TLC) on silica gel plates at 254 nm under a
1
13
UV light. H and C NMR spectra were recorded on a BRUKER AVIII 400 MHz and
101 MHz spectrometers. High resolution mass spectra (HR-ESI-MS) were determined on
Orbitrap Fusion Tribrid (Q-OT-qIT) mass spectrometer (Thermo Fisher Scientific,
Bremen, German). The purity of the targeted compound was measured by high
performance liquid chromatography (HPLC), performed on the LC-2010CHT (Shimadzu,
Japan) using Waters XTERRA MS C18 column （ 3.5 μ m 2.1x100mm ， Waters ，
USA）, eluting with a mixture of solvents MeOH (A) and 0.1% formic acid aqueous (B)
from VA:VB = 20:80 to 80:20. Peaks were detected at 254 nm with a flow rate of 0.25
mL/min.
4.2. Synthesis
4.2.1 Synthesis of 4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)aniline
(Compound 4)
As shown in the Scheme 1., phosphorus oxychloride (400 ml) was added dropwise
at room temperature to a mixture of 3-Chloro-2-hydrazinylpyridine (Compound 1, 20.0
g, 0.139 mol) and 2-Chloro-5-nitrobenzoic acid (Compound 2, 30.0 g, 0.149 mol). The
mixture was then vigorously stirred at reflux temperature for 8 h. After cooling to room
temperature, the reaction solution was poured into ice water and filtered to give the
8-chloro-3-(2-chloro-5-nitrophenyl)-[1,2,4]triazolo[4,3-a]pyridine (Compound 3, 37.8 g,
24

yield: 88%) as a gray solid. 1H-NMR (400 MHz, d6-DMSO) δ = 8.64 (s, 1H), 8.53 (d, J =
7.5 Hz, 1H), 8.29 (d, J = 6.6 Hz, 1H), 8.09 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 7.0 Hz, 1H),
7.08 (t, J = 6.9 Hz, 1H). ESI-MS for C12H6Cl2N4O2 ([M+H]+) Calcd:308.99; Found:
309.10. To a suspension of Compound 3 (44.0 g, 0.142 mol) in 250 ml saturated aq.
NH4C1 and 250 mL of EtOH was added Fe powder (31.8 g, 0.569 mol). The reaction
mixture was stirred at 80°C for 4 hours, cooled down to room temperature and then
filtered through Celite. The filter cake was washed with EtOH. The solution was
concentrated in vacuo, and the residue was dissolved in DCM, then washed with
saturated NaHCO3. The organic phase was dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by silica gel column
(DCM/MeOH=20/1)
to
afford
4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)aniline (Compound 4, 14.2 g,
1
yield: 35.9%) as a white solid. H-NMR (400 MHz, d6-DMSO) δ = 8.03 (d, J = 6.9 Hz,
1H), 7.67 (d, J = 7.1 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.02 (t, J = 7.1 Hz, 1H), 6.86 –
6.81 (m, 1H), 5.65 (s, 1H), ESI-MS for C12H8Cl2N4 ([M+H]+) Calcd: 279.01; Found:
279.10.
4.2.2 synthesis of N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3a]pyridin3yl)phenyl)
benzamide (TPB1)
Thionyl chloride (0.356 g; 3 mmol) was added to a mixture of benzoic acid (0.122 g, 1
mmol) in toluene and the mixture was heated at reflux for 3 hours. The reaction was
cooled to room temperature and the solvent was removed under reduced pressure. The
resultant acid chloride was dropwised to a mixture of Compound 4 (0.279 g, 1 mmol)
and DIPEA (0.258g, 2 mmol) in dichloromethane (15ml) at room temperature. The
25

solution was stirred for overnight, then concentrated in vacuo. The residue was purified
by silica gel column (DCM/MeOH=20/1) to afford compound TPB1 (0.382 g, yield: 75%)
1
as a white solid. purity: 99%; mp: 242.3-244.1°C; H-NMR (d6-DMSO) :δ = 10.65 (s,
1H), 8.21 (d, J = 2.5 Hz, 1H), 8.17 (dd, J = 7.0, 0.8 Hz, 1H), 8.13 (dd, J = 8.9, 2.7 Hz,
1H), 8.02 – 7.94 (m, 2H), 7.76 (dd, J = 8.9, 1.6 Hz, 1H), 7.70 (dt, J = 7.2, 0.9 Hz, 1H),
13
7.65 – 7.59 (m, 1H), 7.55 (td, J = 7.1, 1.3 Hz, 2H), 7.06 (t, J = 7.1 Hz, 1H); C-NMR
(d6-DMSO): δ = 166.36 (s), 147.97 (s), 146.19 (s), 139.24 (s), 134.84 (s), 132.41 (s),
130.91 (s), 128.97 (s), 128.21 (s), 127.92 (s), 127.55 (s), 125.60 (s), 124.52 (s), 124.40 (s),
124.13 (s), 120.65 (s), 114.88 (s); HR-ESI-MS for C19H13ON4Cl2 ([M+H]+) Calcd:
383.0461; Found: 383.0460.
4.2.3
Synthesis
of
2-chloro-N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)phenyl)-benzamide
(TPB2)
Thionyl chloride (0.356 g; 3 mmol) was added to a mixture of 2-chlorobenzoic acid
(0.156 g, 1 mmol) in toluene and the mixture was heated at reflux for 3 hours. The
reaction was cooled to room temperature and the solvent was removed under reduced
pressure. The resultant acid chloride was dropwised to a mixture of Compound 4 (0.279
g, 1 mmol) and DIPEA (0.258g, 2 mmol) in dichloromethane (15ml) at room
temperature. The solution was stirred for overnight, then concentrated in vacuo. The
residue was purified by silica gel column (DCM/MeOH=20/1) to afford TPB2 (0.281 g,
1
yield: 68%); purity: 98%; mp: 247.3-248.5°C; H-NMR (d6-DMSO): δ = 10.95 (s, 1H),
8.24 – 8.07 (m, 2H), 8.01 (dd, J = 8.9, 2.6 Hz, 1H), 7.74 (dd, J = 25.8, 8.0 Hz, 2H), 7.67 –
13
7.57 (m, 2H), 7.56 – 7.40 (m, 2H), 7.06 (t, J = 7.1 Hz, 1H); C-NMR (d6-DMSO): δ =
26

165.78 (s), 148.00 (s), 146.17 (s), 138.87 (s), 136.87 (s), 131.92 (s), 131.11 (s), 130.38
(s), 130.23 (s), 129.45 (s), 127.94 (s), 127.84 (s), 125.87 (s), 124.19 (s), 123.87 (s),
123.76 (s), 120.63 (s), 114.87 (s), 99.98 (s); HR-ESI-MS for C19H10ON4Cl3 ([M-H]+)
Calcd: 414.9920; Found: 414.9929.
4.2.4 synthesis of 3-chloro-N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)
phenyl)benzamide (TPB3)
Thionyl chloride (0.356 g; 3 mmol) was added to a mixture of 3-chlorobenzoic acid
(0.156 g, 1 mmol) in toluene and the mixture was heated at reflux for 3 hours. The
reaction was cooled to room temperature and the solvent was removed under reduced
pressure. The resultant acid chloride was dropwised to a mixture of Compound 4 (0.279
g, 1 mmol) and DIPEA (0.258g, 2 mmol) in dichloromethane (15ml) at room
temperature. The solution was stirred for overnight, then concentrated in vacuo. The
residue was purified by silica gel column (DCM/MeOH=20/1) to afford TPB3 (0.342 g,
1
yield: 83%); purity: 98%; mp: 282.1-283.1°C; H-NMR (d6-DMSO): δ = 10.73 (s, 1H),
8.21 – 8.14 (m, 2H), 8.11 (dd, J = 8.9, 2.6 Hz, 1H), 8.04 (t, J = 1.9 Hz, 1H), 7.94 (dt, J =
7.9, 1.3 Hz, 1H), 7.78 (d, J = 8.9 Hz, 1H), 7.74 – 7.67 (m, 2H), 7.60 (t, J = 7.9 Hz, 1H),
7.06 (t, J = 7.1 Hz, 1H); 13C-NMR (d6-DMSO): δ = 13C NMR (101 MHz, DMSO) δ
164.86 (s), 147.99 (s), 146.14 (s), 140.14 (s), 138.93 (s), 136.78 (s), 133.76 (s), 132.24
(s), 131.02 (s), 130.98 (s), 127.93 (s), 127.86 (s), 127.07 (s), 125.66 (s), 124.65 (s),
124.49 (s), 124.15 (s), 120.65 (s), 114.90 (s); HR-ESI-MS for C19H10ON4Cl3 ([M-H]+)
Calcd: 414.9920; Found: 414.9931.
4.2.5 Synthesis of 4-chloro-N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)
phenyl)benzamide (TPB4)
27

Thionyl chloride (0.356 g; 3 mmol) was added to a mixture of 2-chlorobenzoic acid
(0.156 g, 1 mmol) in toluene and the mixture was heated at reflux for 3 hours. The
reaction was cooled to room temperature and the solvent was removed under reduced
pressure. The resultant acid chloride was dropwised to a mixture of Compound 4 (0.279
g, 1 mmol) and DIPEA (0.258g, 2 mmol) in dichloromethane (15ml) at room temperature.
The solution was stirred for overnight, then concentrated in vacuo. The residue was
purified by silica gel column (DCM/MeOH=20/1) to afford TPB4 (0.301 g, yield: 73%);
purity: 99%; mp: >290°C; 1H-NMR (d6-DMSO): δ = 10.70 (s, 1H), 8.20 – 8.14 (m, 2H),
8.11 (dd, J = 8.9, 2.6 Hz, 1H), 8.04 – 7.97 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.71 (dd, J =
7.2, 0.8 Hz, 1H), 7.68 – 7.60 (m, 2H), 7.06 (t, J = 7.1 Hz, 1H); 13C-NMR (d6-DMSO): δ
= 166.36 (s), 147.97 (s), 146.19 (s), 139.24 (s), 134.84 (s), 132.41 (s), 130.91 (s), 128.97
(s), 128.21 (s), 127.92 (s), 127.55 (s), 125.60 (s), 124.52 (s), 124.40 (s), 124.13 (s),
120.65 (s), 114.88 (s); HR-ESI-MS for C19H10ON4Cl3 ([M-H]+) Calcd: 414.9920; Found:
414.9930.
4.2.6 Synthesis of 2,4-dichloro-N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridine
-3-yl)phenyl)benzamide (TPB5)
Thionyl chloride (0.356 g; 3 mmol) was added to a mixture of 2,4-dichlorobenzoic
acid (0.191 g, 1 mmol) in toluene and the mixture was heated at reflux for 3 hours. The
reaction was cooled to room temperature and the solvent was removed under reduced
pressure. The resultant acid chloride was dropwised to a mixture of Compound 4 (0.279
g, 1 mmol) and DIPEA (0.258g, 2 mmol) in dichloromethane (15ml) at room temperature.
The solution was stirred for overnight, then concentrated in vacuo. The residue was
purified by silica gel column (DCM/MeOH=20/1) to afford TPB5 (0.281 g, yield: 63%);
28

1
purity: 98%; mp: 212.3-213.9°C; H-NMR (d6-DMSO): δ = 10.98 (s, 1H), 8.16 (d, J =
6.9 Hz, 1H), 8.11 (d, J = 2.6 Hz, 1H), 8.05 – 7.95 (m, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.77
(d, J = 8.8 Hz, 1H), 7.70 (dd, J = 7.7, 5.9 Hz, 2H), 7.64 – 7.56 (m, 1H), 7.06 (t, J = 7.1
Hz, 1H); 13C-NMR (d6-DMSO): δ = 164.86 (s), 148.00 (s), 146.13 (s), 138.68 (s), 135.69
(s), 135.65 (s), 131.71 (s), 131.15 (s), 130.87 (s), 129.80 (s), 128.05 (s), 127.99 (s),
127.95 (s), 125.91 (s), 124.19 (s), 123.93 (s), 123.80 (s), 120.63 (s), 114.87 (s);
HR-ESI-MS for C19H9ON4Cl4 ([M-H]+) Calcd: 448.9530; Found: 448.9547.
4.2.7 Synthesis of N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)phenyl)-4-
(methylsulfonyl)benzamide (TPB6)
Thionyl chloride (0.356 g;
3
mmol) was added to
a
mixture of
4-(methylsulfonyl)benzoic acid (0.200 g, 1 mmol) in toluene and the mixture was heated
at reflux for 3 hours. The resultant acid chloride was dropwised to a mixture of
Compound 4 (0.279 g, 1 mmol) and DIPEA (0.258g, 2 mmol) in dichloromethane (15ml)
at room temperature. The solution was stirred for overnight, then concentrated in vacuo.
The residue was purified by silica gel column (DCM/MeOH=20/1) to afford TPB6
(0.409 g, yield: 89%); purity: 97%; mp: 267.6-268.9°C; 1H-NMR (d6-DMSO): δ = 10.88
(s, 1H), 8.20 (dd, J = 8.7, 2.1 Hz, 3H), 8.17 (dd, J = 6.9, 0.8 Hz, 1H), 8.12 (dd, J = 8.7,
2.7 Hz, 3H), 7.79 (d, J = 8.8 Hz, 1H), 7.71 (dd, J = 7.2, 0.8 Hz, 1H), 7.06 (t, J = 7.1 Hz,
13
1H), 3.31 (s, 3H); C-NMR (d6-DMSO): δ = 165.13 (s), 148.00 (s), 146.12 (s), 143.92
(s), 139.32 (s), 138.84 (s), 131.03 (s), 129.24 (s), 128.03 (s), 127.95 (s), 127.67 (s),
125.72 (s), 124.69 (s), 124.53 (s), 124.15 (s), 120.65 (s), 114.90 (s); HR-ESI-MS for
C20H15O3N4Cl2S ([M+H]+) Calcd: 461.0242; Found: 461.0234.
4.2.8 Synthesis of N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)phenyl)
29