Design and biological evaluation of phenyl imidazole analogs as hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1111/cbdd.13799

The hedgehog (Hh) signaling pathway is involved in diverse aspects of cellular events. Aberrant activation of Hh signaling pathway drives oncogenic transformation for a wide range of cancers, and it is therefore a promising target in cancer therapy. In the principle of association and ring-opening, we designed and synthesized a series of Hh signaling pathway inhibitors with phenyl imidazole scaffold, which were biologically evaluated in Gli-Luc reporter assay. Compound 25 was identified to possess high potency with nanomolar IC₅₀, and moreover, it preserved the inhibition against wild-type and drug-resistant Smo-overexpressing cells. A molecular modeling study of compound 25 expounded its binding mode to Smo receptor, providing a basis for the further structural modification of phenyl imidazole analogs.

Full text of DOI:10.1111/cbdd.13799

DR CHIYU SUN (Orcid ID : 0000-0001-5736-4022)
Article type
: Research Letter
Title Page
Design and biological evaluation of phenyl imidazole analogs as
hedgehog signaling pathway inhibitors
Chiyu Sun,*a Ying Zhang,b Han Wang,a Zhengxu Yin,a Lingqiong Wu,a Yanmiao Huang,a
Wenhu Zhanga, Youbing Wang a and Qibo Hu a
a School of Pharmacy, Shenyang Medical College, Shenyang 110034, China.
b School of Chemical Engineering, Shenyang University of Chemical Technology, Shenyang
110142, China.
Corresponding author: scy_dream@126.com,
Tel.: +86-24-62215689,
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13799
This article is protected by copyright. All rights reserved

Acknowledgments
This work was supported financially by Liaoning Science and Technology Project
Management (grant number 20170520026); the Science and Technology Fund Project of
Shenyang Medical College (grant numbers 20191027).
Conflict of Interest Statement
The authors report no conflict of interest.
Abstract
The hedgehog (Hh) signaling pathway is involved in diverse aspects of cellular events.
Aberrant activation of Hh signaling pathway drives oncogenic transformation for a wide range of
cancers, and it is therefore a promising target in cancer therapy. In the principle of association and
ring-opening, we designed and synthesized a series of Hh signaling pathway inhibitors with
phenyl imidazole scaffold, which were biologically evaluated in Gli-Luc reporter assay.
Compound 25 was identified to possess high potency with nanomolar IC50, moreover, it preserved
the inhibition against wild-type and drug-resistant Smo-overexpressing cells. A molecular
modelling study of compound 25 expounded its binding mode to Smo receptor, providing a basis
for the further structural modification of phenyl imidazole analogs.
Keywords hedgehog signaling, inhibitors, phenyl imidazole, Smo receptor, modelling
1. Introduction
The Hedgehog（Hh）signaling pathway is not only associated with the maintenance and
repair of many human tissues, but also plays an important role in cell growth, survival and
metastasis (Sharpe et al., 2015; Makley et al., 2013; Owens et al., 2017; Galperin et al., 2019). Hh
signaling is silent in normal cells, however, its abnormal activation is associated with basal cell
carcinoma (BCC), medulloblastoma (MB), pancreatic cancer, acute myeloid leukemia (AML),
colon and prostate cancer (DeBerardinis et al., 2014; Berman et al., 2002; Mathew et al., 2014;
This article is protected by copyright. All rights reserved

Cortes et al., 2019; Vesci et al., 2018; Domenech et al., 2012). Aberrant Hh signaling is mainly
driven by either ligand-dependent or Patched (Ptch) mutation mechanism, and induces Ptch to
release smoothened (Smo) protein, promoting the translocation of its downstream Gli protein into
the nucleus to express Hh target gene (Trinh et al., 2014; Mas et al., 2010; Wahid et al., 2016;
Salaritabar et al., 2019). Undoubtedly, there is an increasing level of interests in modulating the
Hh signaling pathway for cancer treatment.
Smo is the most studied Hh pathway component as a drug target, and its inhibition leads to
down-regulation of those genes associated with cancer growth and progression. Most Hh pathway
inhibitors suppress the function of Smo receptor. To date, Vismodegib (1), Sonidegib (2) and
Glasdegib (3) have been the Smo antagonists approved by the U.S. Food and Drug Administration
(FDA) for the treatment of BCC and AML (Angelaud et al., 2016; Robarge et al., 2009; Lindsley,
2016; Oan et al., 2010; Munchhof et al., 2012; Sheridan, 2019) (Figure 1). Despite the significant
accomplishment in the development of Hh pathway inhibitors, the clinical use of 1 and 2 is
severely restricted by virtue of their several adverse effects including diarrhea, taste disturbance,
hair loss and muscle spasms (Ghirga et al., 2018). Alternatively, acquired resistance to these drugs
has become a major barrier for their continued advancement (Pricl et al., 2015). For instance, Smo
D473H mutation was discovered from metastatic BCC and MB patient with relapse after treatment
with 1 (Yauch et al., 2009; Dijkgraaf et al., 2011). Smo D477G, a comparable murine Smo mutant,
in the mouse MB model was drug-resistant as well (Coni et al., 2013). Albeit Smo mutation has no
effect on Hh signal transduction, it diminished the affinity of ligands to Smo receptor and
disrupted their binding. This finding highlights the continued efforts and interests in research and
development of novel chemotype Hh pathway inhibitors.
Figure 1 should be here.
In the past decade, some benzimidazole analogs as potential Hh inhibitors have been
discovered by the high-throughput screening campaign, for instance, compound 3, HhAntag691
and SANT-2 (Bariwal et al., 2019). HhAntag691 has been early reported by Curis and Genentech
as a potent Hh inhibitor with low nanomolar affinity for SMO (Romer et al., 2004). SANT-2 is a
known Smo antagonist with an IC50 of 98 nM in the Shh light II assay (Chen et al., 2002). Later,
lead optimization of SANT-2 identified TC132 (4) with an IC50 value of 80 nM, slightly more
potent than SANT-2. Büttner et al reported that exchange of benzimidazole core in compound 4
with other heterocyclic rings, such as indole, benzothiazole and benzoxazole, led to decrease in
activity (Büttner et al., 2009). Therefore, benzimidazole is an efficacious moiety for Hh inhibition.
This article is protected by copyright. All rights reserved

In addition, the benzyloxy group was regarded as a bioactive structure normally found in
antineoplastic agent like lapatinib (5) (Petrov et al., 2006). In our effort to probe novel Hh
pathway inhibitors, we introduced benzyloxy moiety into compound 4 to replace the metabolically
labile trimethoxy groups (Figure 2). Encouragingly, this modification led to compound 6 with
enhanced anti-Hh activity as measured in Gli-Luc reporter assay (IC50 of 0.07 μM as comparison
to 0.09 μM for compound 4). In an attempt to further pursue the chemical structural space,
replacement of benzimidazole with phenyl imidazole moiety obtained compound 25 according to
ring-opening strategy. The emergence of a rotatable bond between phenyl and imidazole was able
to reduce its rigidity, which was expected to improve Smo-binding affinity. The recent
determination of Smo-vismodegib crystal structures (PDB code 5L7I) allowed us to examine the
interaction patterns between the ligands and Smo receptor (Byrne et al., 2016). The predicted
Smo-binding affinity of compound 25 was superior to compound 4, because their docking score
were -11.65 kcal/mol and -8.49 kcal/mol, respectively. In this study, a series of phenyl imidazole
derivatives were prepared, and their evaluation on Hh signaling pathway inhibition was reported.
Figure 2 should be here.
2. Results and discussion
The synthetic routes for the target compounds were outlined in Schemes 1-2. Methylparaben
was etherified with substituted benzyl bromide in acetone to afford compounds 6a-l, which was
hydrolyzed to the key intermediates 7a-l in refluxing ethanol for 2 hours. The intermediate 3-(1H-
benzo[d]imidazol-2-yl)-4-chloroaniline was synthesized in accordance to the reported procedure
(Büttner et al., 2009), before its amidation with 7a-l gave the target compounds 12-16. 2-chloro-5-
nitrobenzonitrile reacted with sodium methylate and ammonium chloride in alkaline conditions to
form amidine hydrochloride 9. Then, the intermediate 10 was obtained via condensation of 9 and
2-bromoacetophenone. 10 and stannous chloride refluxed in acidic ethanol solution, and its
reduction product was intermediate 11. Finally, 11 reacted with 7a-l to give the target compound
17-28.
Scheme 1 should be here.
Scheme 2 should be here.
The target compound involoved the following three regions: A, B and a linker. Region A
contained benzimidazole moiety and phenyl imidazole moiety. Region B involved
This article is protected by copyright. All rights reserved

benzyloxyphenyl group. The amide bond was the linker. The inhibitory activity of compound 12-
28 on Hh signaling pathway was evaluated by Gli-Luc reporter kit. Compound 1 was positive
control and compound 4 was lead compound. The results expressed as half maximal inhibitory
concentration (IC50) values and are presented in Table 1. Initially, region A in the tested
compounds was focused. Delightedly, the phenyl imidazole derivatives exhibited more potency as
compared with the benzimidazole counterparts (12 vs 18, 13 vs 19, 14 vs 26, 15 vs 27 and 16 vs
24), indicating that ring-opening structural modification enhanced their Hh inhibition. Further
investigations were performed to study the effect of different substituents on the phenyl ring
(region B) on Hh inhibition. The introduction of fluoro atom (22) or methyl (27) in the para-
position was superior to trifluoromethyl surrogate (18). Ortho-fluoro (24) derivative exhibited
improved potency than ortho-chlorine analogue (19). Besides, meta-fluoro derivative (25, IC50 =
0.01 μM) displayed higher activity as compared to other electron-withdrawing groups such as
meta-trifluoromethyl (23, IC50 = 0.02 μM) and meta-chlorine (28, IC50 = 1.16 μM). Moreover, the
anti-Hh activity of fluoro atom in the meta- position (25) was stronger as compared to that in
ortho- (24, IC50 = 0.04 μM) or para-position (22, IC50 = 0.43 μM). Although double chlorine
substituents (20, 21 and 26) were effective against Hh signaling, they were inferior in potencies to
25. Compared with 4, four of the target compounds (20, 23, 24 and 25) showed higher potency
with IC50 values less than 0.06 μM. Compound 25 was identified as the most potent compound in
this study. More importantly, the potency of compound 25 was 2-fold higher as compared to 1,
suggesting that it was a promising Hh pathway inhibitor.
Table 1 should be here.
The Hh inhibitory activities of this series of compounds probably attributed to their
interaction with Smo, since compound 25 effectively competed with BODIPY-cyclopamine in
CHO-K1 cells overexpressing wild-type (WT) Smo, with IC50 values of 17 nM (Figure 3). Next, it
was hypothesized that 25 might be active against the drug-resistant Smo mutant, therefore, we
over-expressed both WT mouse Smo and its mutant D477G with GFP in the NIH3T3-Gli-Luc
reporter cell line. Consistent with previous reports, mutation of Smo can confer resistance to 1. As
presented in Figure 4, compound 1 suppressed WT Smo-overexpressing cells with an IC50 of 20
nM, however, its inhibition of mutant Smo-overexpressing cells declined sharply. On the contrary,
compound 25 inhibited WT and mutant Smo-overexpressing cells with similar potencies (IC50 =
14 nM for WT, IC50 = 25 nM for mutant). The 2-fold shift in IC50 indicated that the D477G
mutation did not significantly interfere with the binding of 25 to Smo. Next, the cytotoxicity assay
This article is protected by copyright. All rights reserved

on Hh-driven cancer cells was performed (Figure 5). DAOY cell lines were a suitable human MB
model with constitutive Hh activation, which was reported to be resistant to 1 in vitro. Although 1
was less active against DAOY cells, compound 25 apparently decreased proliferation and survival
of DAOY cells. Moreover, the cell viabilities of 25 were 51 % and 32 % at concentrations of 1 μM
and 10 μM, respectively.
Figure 3 should be here.
Figure 4 should be here.
Figure 5 should be here.
To further elucidate the binding mode of this series of compounds with Smo receptor, a
detailed molecular docking study was performed. The predicted binding affinity of compound 25
was the most among all, with the docking score of − 11.65 kcal/mol (Table S1). As shown in
Figure 6A, compound 1 (yellow) bound in the pocket closer to the upper opening of Smo, and the
binding orientations of compound 1 and 25 (blue) superimposed well with each other. In the
binding mode (Figure 6B and 6C), these two compounds shared similar hydrogen bond with
Arg400 to amide bond, and Tyr394 shared to nitrogen atom of pyridine or imidazole. In
accordance with pyridine ring on 1, imidazole ring on 25 allowed π-π interaction with Phe391 and
Trp281. These common interactions suggested the importance of imidazole ring and amide bond
in region A and linker. Differently, the fluorine on 25 formed a hydrogen bond with Lys395. The
phenyl rings in region A and B formed π-π interaction with Trp281 and Phe484. Similar results
were observed in the conformations of other compounds (Figure S1). The computational modeling
study explained the preferable Hh inhibition of compound 25 at the molecular level.
Figure 6 should be here.
3. Conclusion
In summary, a series of structural modified phenyl imidazole analogs are designed on the
basis of pharmacological association and ring-opening strategy. The synthesized compounds were
evaluated in Gli-luciferase assay, and compounds 20, 23, 24 and 25 exhibited more potent activity
than the lead compound TC132. In particular, compound 25 showed the highest Hh inhibitory
potency with an IC50 value of 0.01 μM, which was 2-fold higher than the launched drug
vismodegib. Our preliminary investigation indicated that meta-fluoro benzyloxy group was well
This article is protected by copyright. All rights reserved

tolerated for enhancement of activity in the target compounds. Additionally, both wild-type and
mutant Smo were effectively suppressed by 25, and it displayed moderate antiproliferation against
DAOY cells in vitro. Computational simulations offered the molecular basis for rationalizing Hh
inhibition of the phenyl imidazole derivatives. Further studies on the structural optimization of
these derivatives are currently underway in our laboratory.
References
Angelaud, R., Reynolds, M., Venkatramani, C., Savage, S., Trafelet, H., Landmesser, T., Demel,
P., Levis, M., Ruha, O., Rueckert, B., & Jaeggi, H. (2016). Manufacturing development and
genotoxic impurity control strategy of the hedgehog pathway inhibitor vismodegib. Org
Process Res Dev, 20, 1509-1519. doi: 10.1021/acs.oprd.6b00208
Bariwal, J., Kumar V., Dong, Y.X., & Mahato, R. I. (2019). Design of hedgehog pathway
inhibitors for cancer treatment. Med Res Rev, 39, 1137-1204. doi: 10.1002/med.21555
Berman, D.M., Karhadkar, S.S., Hallahan, A.R., Pritchard, J.I., Eberhart, C.G., Watkins, D.N.,
Chen, J.K., Cooper, M.K., Taipale, J., Olson, J.M., & Beachy, P.A. (2002). Medulloblastoma
growth inhibition by hedgehog pathway blockade. Science, 297, 1559-1561. doi:
10.1126/science.1073733
Büttner, A., Seifert, K., Cottin, T., Sarli, V., Tzagkaroulaki, L., Scholz, S., & Giannis, A. (2009).
Synthesis and biological evaluation of SANT-2 and analogues as inhibitors of the hedgehog
signaling pathway. Bioorg Med Chem, 17, 4943-4954. doi: 10.1016/j.bmc.2009.06.008
Byrne, E. F., Sircar, R., Miller, P. S., Hedger, G., Luchetti, G., Nachtergaele, S., Tully, M. D.,
Mydock-McGrane, L., Covey, D. F., Rambo, R. P., Sansom, M. S., Newstead, S., Rohatgi, R.,
& Siebold, C. (2016). Structural basis of smoothened regulation by its extracellular domains.
Nature, 535, 517-522. doi: 10.1038/nature18934
Chen J.K., Taipale J., Young K.E., Maiti T., & Beachy P.A. (2002). Small molecule modulation of
smoothened activity. Proc Natl Acad Sci
U
S
A, 99, 14071-14076. doi:
10.1073/pnas.182542899
This article is protected by copyright. All rights reserved

Coni, S., Infante, P., & Gulino, A. (2013). Control of stem cells and cancer stem cells by hedgehog
signaling: pharmacologic clues from pathway dissection. Biochem Pharmacol, 85, 623-628.
doi: 10.1016/j.bcp.2012.11.001
Cortes, J.E., Gutzmer, R., Kieran, M.W., & Solomon, J.A. (2019). Hedgehog signaling inhibitors
in solid and hematological cancers. Cancer Treat Rev, 76, 41-50. doi:
10.1016/j.ctrv.2019.04.005
DeBerardinis, A.M., Madden, D.J., Banerjee, U., Sail, V., Raccuia, D.S., DeCarlo, D., Lemieux,
S.M., Meares, A., & Hadden, M.K. (2014). Structure-activity relationships for vitamin d3-
based aromatic a-ring analogues as hedgehog pathway inhibitors. J Med Chem, 57, 3724-
3736. doi: 10.1021/jm401812d
Dijkgraaf, G.J.P., Alicke, B., Weinmann, L., Januario, T., West, K., Modrusan, Z., Burdick, D.,
Goldsmith, R., Robarge, K., Sutherlin, D., Scales, S.J., Gould, S.E., Yauch, R.L., &
deSauvage, F.J. (2011). Small molecule inhibition of GDC-0449 refractory smoothened
mutants and downstream mechanisms of drug resistance. Cancer Res, 71, 435-444. doi:
10.1158/0008-5472.CAN-10-2876
Domenech, M., Bjerregaard, R., Bushman, W., & Beebe, D.J. (2012). Hedgehog signaling in
myofibroblasts directly promotes prostate tumor cell growth. Integr Biol, 4, 142-152. doi:
10.1039/c1ib00104c
Galperin, I., Dempwolff, L., Diederich, W.E., & Lauth, M. (2019). Inhibiting hedgehog: an update
on pharmacological compounds and targeting strategies. J Med Chem, 62, 8392-8411. doi:
10.1021/acs.jmedchem.9b00188
Ghirga, F., Mori, M., & Infante, P. (2018). Current trends in hedgehog signaling pathway
inhibition by small molecules. Bio Med Chem Lett, 28, 3131- 3140. doi:
10.1016/j.bmcl.2018.08.033
Lindsley, C.W. (2016). Novel drug approvals in 2015 and thus far in 2016. ACS Chem Neurosci, 7,
1175-1176. doi: 10.1021/acschemneuro.6b00254
Makley, L.N., & Gestwicki, J.E. (2013). Expanding the Number of ‘Druggable’ Targets: Non-
Enzymes and Protein-Protein Interactions. Chem Biol Drug Des, 81, 22-32. doi:
10.1111/cbdd.12066
This article is protected by copyright. All rights reserved

Mas, C., & Altaba, A.R. ( 2010). Small molecule modulation of hh-gli signaling: Current leads,
trials and tribulations. Biochem Pharmacol, 80, 712-723. doi: 10.1016/j.bcp.2010.04.016
Mathew, E., Zhang, Y.Q., Holtz, A.M., Kane, K.T., Song, J.Y., Allen, B.L., & diMagliano, M.P.
(2014). Dosage-dependent regulation of pancreatic cancer growth and angiogenesis by
hedgehog signaling. Cell Rep, 9, 484-494. doi: 10.1016/j.celrep.2014.09.010
Munchhof, M.J., Li, Q.F., Shavnya, A., Borzillo, G.V., Boyden, T.L., Jones, C.S., LaGreca, S.D.,
Martinez-Alsina, L., Patel, N., Pelletier, K., Reiter, L.A., Robbins, M.D., & Tkalcevic, G.T.
(2012). Discovery of PF-04449913, a potent and orally bioavailable inhibitor of smoothened.
ACS Med Chem Lett, 3, 106-111. doi: 10.1021/ml2002423
Owens, A.E., dePaola, I., Hansen, W.A., Liu, Y.W., Khare, S.D., & Fasan, R. (2017). Design and
evolution of a macrocyclic peptide inhibitor of the sonic hedgehog/patched interaction. J Am
Chem Soc, 139, 12559-12568. doi: 10.1021/jacs.7b06087
Robarge, K.D., Brunton, S.A., Castanedo, G.M., Cui, Y., Dina, M.S., Goldsmith, R., Gould, S.E.,
Guichert, O., Gunzner, J.L., Halladay, J., Jia, W., Khojasteh, C., Koehler, M.F.T., Kotkow,
K., La, H., LaLonde, R.L., Lau, K., Lee, L., Marshall, D., Marsters, J.C., Murray, L.J., Qian,
C.G., Rubin, L.L., Salphati, L., Stanley, M.S., Stibbard, J.H.A., Sutherlin, D.P., Ubhayaker,
S., Wang, S.M., Wong, S., & Xie, M.L. (2009). GDC-0449—A potent inhibitor of the
hedgehog pathway. Bioorg Med Chem Lett, 19, 5576-5581. doi: 10.1016/j.bmcl.2009.08.049
Romer, J. T., Kimura, H., Magdalena, S., Sasai, K., Fuller, C., Baines, H., Connelly, M., Stewart,
C. F., Gould, S., Rubin, L. L., & Curran, T. (2004). Suppression of the Shh pathway using a
small molecule inhibitor eliminates medulloblastoma in Ptc1+/- p53-/- mice. Cancer Cell, 6,
229-240. doi: 10.1016/j.ccr.2004.08.019
Salaritabar, A., Berindan-Neagoe, I., Darvish, B., Hadjiakhoondi, F., Manayi, A., Devi, K.P.,
Barreca, D., Orhan, I.E., Suntar, I., Farooqi, A.A., Gulei, D., Nabavi, S.F., Sureda, A., Daglia,
M., Dehpour, A.R., Nabavi, S.M., & Shirooie, S. (2019). Targeting hedgehog signaling
pathway: paving the road for cancer therapy. Pharmacol Res, 141, 466-480. doi:
10.1016/j.phrs.2019.01.014
Sharpe, H.J., Wang, W.R., Hannoush, R.N., & deSauvage, F.J. (2015). Regulation of the
oncoprotein smoothened by small molecules. Nat Chem Biol, 11, 246 -255. doi:
10.1038/nchembio.1776
This article is protected by copyright. All rights reserved

Sheridan, M.H. (2019). Glasdegib: first global approval. Drugs, 79, 207-213. doi:
10.1007/s40265-018-1047-7
Trinh, T.N., McLaughlin, E.A., Gordon, C.P., & McCluskey, A. (2014). Hedgehog signalling
pathway inhibitors as cancer suppressing agents. Med Chem Commun, 5, 117-133. doi:
10.1039/c3md00334e
Pan, S.F., Wu, X., Jiang, J.Q., Gao, W.Q., Wan, Y.Q., Cheng, D., Han, D., Liu, J., Englund, N.P.,
Wang, Y., Peukert, S., Miller-Moslin, K., Yuan, J., Guo, R.B., Matsumoto, M., Vattay, A.,
Jiang, Y., Tsao, J., Sun, F.X., Pferdekamper, A.C., Dodd, S., Tuntland, T., Maniara, W.,
Kelleher, J.F., Yao, Y.M., Warmuth, M., Williams, J., & Dorsch, M. (2010). Discovery of
NVP-LDE225, a potent and selective smoothened antagonist. ACS Med Chem Lett, 1,130-
134. doi: 10.1021/ml1000307
Petrov, K.G., Zhang, Y.M., Carter, M., Cockerill, G.S., Dickerson, S., Gauthier, C.A., Guo, Y.,
Mook, R.A., Rusnak, D.W., Walker, A.L., Wood, E.R., & Lackey, K.E. (2006). Optimization
and SAR for dual ErbB-1/ErbB-2 tyrosine kinase inhibition in the 6-furanylquinazoline series.
Bioorg Med Chem Lett, 16, 4686-4691. doi: 10.1016/j.bmcl.2006.05.090
Pricl, S., Cortelazzi, B., DalCol, V., Marson, D., Laurini, E., Fermeglia, M., Licitra, L., Pilotti, S.,
Bossi, P., & Perrone, F. (2015). Smoothened (SMO) receptor mutations dictate resistance to
vismodegib
in
basal
cell
carcinoma.
Mol
Oncol,
9,
389-397.
doi:
10.1016/j.molonc.2014.09.003
Vesci, L., Milazzo, F.M., Stasi, M.A., Pace, S., Manera, F., Tallarico, C., Cini, E., Petricci, E.,
Manetti, F., DeSantis, R., & Giannini, G. (2018). Hedgehog pathway inhibitors of the
acylthiourea and acylguanidine class show antitumor activity on colon cancer in vitro and in
vivo. Eur J Med Chem, 157, 368-379. doi: 10.1016/j.ejmech.2018.07.053
Wahid, M., Jawed, A., Mandal, R.K., Dar, S.A., Khan, S., Akhter, N., & Haque, S. (2016).
Vismodegib, itraconazole and sonidegib as hedgehog pathway inhibitors and their relative
competencies in the treatment of basal cell carcinomas. ꢀCrit Rev Oncol Hematol, 98, 235-
241. doi: 10.1016/j.critrevonc.2015.11.006
Yauch, R.L., Dijkgraaf, G.J., Alicke, B., Januario, T., Ahn, C.P., Holcomb, T., Pujara, K., Stinson,
J., Callahan, C.A., Tang, T., Bazan, J.F., Kan, Z., Seshagiri, S., Hann, C.L., Gould, S.E., Low,
J.A., Rudin, C.M., & deSauvage, F.J. (2009). Smoothened mutation confers resistance to a
This article is protected by copyright. All rights reserved

hedgehog pathway inhibitor in medulloblastoma. Science, 326, 572-574. doi:
10.1126/science.1179386
This article is protected by copyright. All rights reserved

Figure legends
Figure 1 Representative structures of Smo inhibitors.
Figure 2 Design strategy of novel phenyl imidazole derivatives.
Scheme 1 (a) substituted benzyl bromide, K2CO3, acetone, 80 °C, 5 h; (b)NaOH, aqueous alcohol,
75 °C, reflux, 2 h; (c) (i) (COCl)2, pyridine cat.; (ii) 3-(1H-benzo[d]imidazol-2-yl)-4-chloroaniline,
Et3N, CH2Cl2, rt, 12 h.
Scheme 2 (a) (i) CH3ONa, CH3OH, -15 °C, 2h; NH4Cl, 40 °C, 3h, (ii) 2-bromoacetophenone,
NaHCO3, THF, reflux, 40 °C, 20h; (c) SnCl2•2H2O, ethanol, HCl, 80 °C, reflux, 8 h; (d) HATU,
DIPEA, CH2Cl2, 7a-l, rt, overnight.
Table 1 Hh signaling pathway inhibition of designed compounds
a
Results expressed as the mean ± standard deviation of three separate IC50 determinations. For
each determination, concentration-inhibition curves were acquired in triplicate and then averaged
to afford a single IC50 curve with a 95% confidence interval.
b Used as a lead compound.
c Used as a positive control.
Figure 3 In vitro inhibition of Smo for compound 25. The values are an average of triplicate
separate determinations.
This article is protected by copyright. All rights reserved

Figure 4 The inhibition of drug-resistant Smo mutant for compound 25. The inhibition of Gli-Luc
reporter activity by vismodegib (A) and compound 25 (B) in NIH3T3-Gli-Luc cells
overexpressing wild-type Smo or Smo D477G. Error bars represent standard deviation of three
parallel groups (n=3).
Figure 5 Cell viability of DAOY cells treated with the indicated doses of vismodegib and
compound 25 for 48 h. Data represent the average of three independent experiments.
Figure 6 (A) Overlay of compound 25 (blue) and original ligand vismodegib (yellow) in binding
pocket of Smo. (B) Docking conformation of vismodegib (yellow) at the binding site. (C) Docking
conformation of compound 25 (blue) at the binding site. Hydrogen bonds are represented by the
dashed green lines. The dashed pink lines represent π-π interaction. Surrounding amino acid are
shown in grey stick format and labelled.
This article is protected by copyright. All rights reserved

Figure 1
Figure 2
This article is protected by copyright. All rights reserved

Scheme 1
Scheme 2
This article is protected by copyright. All rights reserved

Talbe 1
Compd.
R
Gli-luc reporter Compd.
R
Gli-luc reporter
IC50a (μM)
0.43 ± 0.05
0.02 ± 0.01
0.04 ± 0.02
0.01 ± 0.01
0.09 ± 0.02
0.12 ± 0.07
1.16 ± 0.05
0.09 ± 0.01
0.02 ± 0.01
IC50a (μM)
12
13
14
15
16
17
18
19
20
21
4-CF3
2-Cl
1.86 ± 0.17
1.30 ± 0.06
22
23
24
25
26
27
28
4b
4-F
3-CF3
2-F
2,4-Cl2 0.98 ± 0.08
4-Me
2-F
0.75 ± 0.12
0.17 ± 0.05
1.37 ± 0.11
0.62 ± 0.06
0.41 ± 0.03
3-F
2,4-Cl2
4-Me
3-Cl
-
-
4-CF3
2-Cl
2,3-Cl2 0.06 ± 0.02
3,4-Cl2 0.29 ± 0.08
1c
-
This article is protected by copyright. All rights reserved

Figure 3
Figure 4
This article is protected by copyright. All rights reserved

Figure 5
This article is protected by copyright. All rights reserved

Figure 6
This article is protected by copyright. All rights reserved

Table 1 Hh signaling pathway inhibition of designed compounds
Compd.
R
Gli-luc reporter Compd.
R
Gli-luc reporter
IC50a (μM)
0.43 ± 0.05
0.02 ± 0.01
0.04 ± 0.02
0.01 ± 0.01
0.09 ± 0.02
0.12 ± 0.07
1.16 ± 0.05
0.09 ± 0.01
0.02 ± 0.01
IC50a (μM)
12
13
14
15
16
17
18
19
20
4-CF3
2-Cl
1.86 ± 0.17
1.30 ± 0.06
22
23
24
25
26
27
28
4b
4-F
3-CF3
2-F
2,4-Cl2 0.98 ± 0.08
4-Me
2-F
0.75 ± 0.12
0.17 ± 0.05
1.37 ± 0.11
0.62 ± 0.06
0.41 ± 0.03
3-F
2,4-Cl2
4-Me
3-Cl
-
-
4-CF3
2-Cl
2,3-Cl2 0.06 ± 0.02
3,4-Cl2 0.29 ± 0.08
1c
-
21
a
Results expressed as the mean ± standard deviation of three separate IC50 determinations.
For each determination, concentration-inhibition curves were acquired in triplicate and then
averaged to afford a single IC50 curve with a 95% confidence interval.
b Used as a lead compound.
c Used as a positive control.
This article is protected by copyright. All rights reserved

cbdd_13799_f1.tif
This article is protected by copyright. All rights reserved

cbdd_13799_f2.tif
This article is protected by copyright. All rights reserved

cbdd_13799_f3.tif
This article is protected by copyright. All rights reserved

cbdd_13799_f4.tif
This article is protected by copyright. All rights reserved

cbdd_13799_f5.tif
This article is protected by copyright. All rights reserved

cbdd_13799_f6.tif
This article is protected by copyright. All rights reserved