Design, Synthesis, and Pharmacological Evaluation of 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-aryl Propanamides as Novel Smoothened (Smo) Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.6b01247

A series of novel Smo antagonists were developed either by directly incorporating the basic skeleton of the natural product artemisinin or by first breaking artemisinin into structurally simpler and stable intermediates and then reconstructing into diversified heterocyclic derivatives, equipped with a Smo-targeting bullet. 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-arylpropanamide 65 was identified as the most potent, with an IC₅₀ value of 9.53 nM against the Hh signaling pathway. Complementary mechanism studies confirmed that 65 inhibits Hh signaling pathway by targeting Smo and shares the same binding site as that of the tool drug cyclopamine. Meanwhile, 65 has a good plasma exposure and an acceptable oral bioavailability. Dose-dependent antiproliferative effects were observed in ptch+/-;p53-/- medulloblastoma cells, and significant tumor growth inhibitions were achieved for 65 in the ptch+/-;p53-/- medulloblastoma allograft model.

Full text of DOI:10.1021/acs.jmedchem.6b01247

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Article
Design, Synthesis and Pharmacological Evaluation of
-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-aryl
Propanamides as Novel Smoothened (Smo) Antagonists
2
Gang Liu, Jun Yang, Juan Wang, Xiaohua Liu, Wenjing Huang, Jie Li, Wenfu Tan, and Ao Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01247 • Publication Date (Web): 13 Oct 2016
Downloaded from http://pubs.acs.org on October 14, 2016
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Design, Synthesis and Pharmacological Evaluation of
2ꢀ(2,5ꢀDimethylꢀ5,6,7,8ꢀtetrahydroquinolinꢀ8ꢀyl)ꢀNꢀaryl
Propanamides as Novel Smoothened (Smo) Antagonists
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†
,ǁ,§
‡,§
‡,§
†,ǁ
‡
ǁ
Gang Liu, Jun Yang, Juan Wang, Xiaohua Liu, Wenjing Huang , Jie Li,
,‡
*,†,ǁ
Wenfu Tan,* and Ao Zhang
†
CAS Key Laboratory of Receptor Research, and Synthetic Organic & Medicinal
Chemistry Laboratory (SOMCL), Shanghai Institute of Materia Medica (SIMM),
University of Chinese Academy of Sciences, Shanghai 201203, China
‡
Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai
2
01203, China
ǁ
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210,
China
§
These authors contributed equally to this work.
*
To whom correspondence should be addressed. For W.T.: phone: +86ꢀ21ꢀ51980039;
fax: 86ꢀ21ꢀ51980039; Eꢀmail: wftan@fudan.edu.cn.
For
A.Z.:
phone:
+86ꢀ21ꢀ50806035;
fax:
86ꢀ21ꢀ50806035;
Eꢀmail:
aozhang@simm.ac.cn.
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Abstract.
A series of novel Smo antagonists were developed either by directly incorporating
the basic skeleton of the natural product artemisinin or by first breaking artemisinin
into structurally simpler and stable intermediates and then reconstructing into
diversified heterocyclic derivatives, equipped with a Smoꢀtargeting bullet.
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ꢀ(2,5ꢀDimethylꢀ5,6,7,8ꢀtetrahydroquinolinꢀ8ꢀyl)ꢀNꢀarylpropanamide
65
was
identified as the most potent with an IC50 value of 9.53 nM against the Hh signaling
pathway. Complementary mechanism studies confirmed that 65 inhibits Hh signaling
pathway by targeting Smo and shares the same binding site as that of the tool drug
cyclopamine. Meanwhile, 65 has a good plasma exposure and an acceptable oral
bioavailability. Doseꢀdependent antiproliferative effects were observed in
ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells, and significant tumor growth inhibitions were
achieved for 65 in the ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma allograft model.
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INTRODUCTION
The hedgehog (Hh) signaling pathway plays a pivotal role in the embryogenesis
1
and tissue maintenance by controlling cell proliferation and differentiation. In adults,
the Hh pathway is significantly downregulated, but reactivation usually occurs during
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tissue repair. The Hh ligands (Sonic, Desert, and Indian), the transmembrane receptor
Patched (Ptch), the signal transducer Smoothened (Smo), and the transcription factors
3ꢀ5
Gli1ꢀ3 are the major components that regulate the transcription of Hh target genes.
Ptch is a 12ꢀpass transmembrane receptor that negatively regulates the sevenꢀpass
transmembrane receptor Smo. Upon Ptch binding to the Hh ligand, the repression of
Smo by Ptch is disarmed, and Smo is consequently released to initiate a downstream
signaling cascade leading to the activation of transcription factors of the Gli family. It
has been reported that aberrant Hh pathway activation is involved in tumorigenesis of
various cancers, especially basal cell carcinoma (BCC) and medulloblastoma (MB) as
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ꢀ10
the most commonly identified Hhꢀdependent tumors.
Aberrant activation of the Hh
pathway can result from abnormal upꢀregulation of the Hh ligands, loss of Ptch, Smo
7,9,11
mutations, and gene amplification/chromosomal translocation of Gli1 or Gli2.
Consequently, inhibition of Hh pathway signaling has become an attractive anticancer
3
,5,7,12,13
chemotherapeutic strategy.
Since the natural steroidal alkaloid cyclopamine was identified as the first Smo
1
4
antagonist to block Hh signaling by directly binding to Smo, a number of
12,13,15,16
Smoꢀtargeting small molecules have been developed in recent years.
1
7ꢀ19
Benzamide 1
(GDCꢀ0449, vismodegib) from Genentech is the firstꢀinꢀclass Smo
antagonist that was approved by the FDA in 2012 for the treatment of locally
20,21
advanced or metastatic BCC. Three years later, sonidegib
(NVPꢀLDE225, 2), a
structurally similar analogue of 1 from Novartis, was also approved by FDA in 2015
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as the second clinically prescribed Smo antagonist. In spite of the significant
achievement in the development of Smoꢀtargeted antagonists or other Hh signaling
pathway inhibitors, the clinical application of 1 and 2 is strictly limited to adult BCC
patients, due to many side effects including diarrhoea, constipation, decreased
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appetite, hair loss, muscle spasms, and tiredness.
Such side effects can be severe
in children, especially in the skeletal system. Meanwhile, development of drug
resistance and patient relapse have also been reported after the clinical use of 1 for
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three months, possibly due to Smo mutations.
Therefore, new Hh signaling
pathway inhibitors, especially new Smo antagonists with structures distinct from
benzamides 1 and 2, are highly needed.
The natural product artemisinin (3) is the active component of the sweet
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wormwood plant Artemisia annua L. Its unique 1,2,4ꢀtrioxane structural feature and
significant antimalarial activity have opened a new paradigm for antimalarial drug
discovery, having resulted in a number of artemisinin analogues (artemalogs) such as
dihydroartemisinin, artemether, arteether and artesunate that have been clinically used
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for decades as the antimalarial treatment.
To fully take advantage of the unusual
endoperoxide and multiple cyclic structure of 3, an increasing number of artemalogs
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have been explored as treatment for many other diseases, especially cancer. Among
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which, benzyl ether 4 and dimer 5 were reported to be among the most potent
showing twoꢀdigit nM potency against mouse lymphocytic leukemia L1210 and
human erythroleukemic Kꢀ562 cell lines, respectively (Figure 1). Unfortunately, such
high cellular antitumor potency failed to translate into tumor growth inhibition in
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vivo. For example, dimer 5, one of the most potent artemalogs in vitro, only shows
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moderate tumor growth inhibition in the HLꢀ60 human leukemia xenografts.
In view of the reported disappointing outcomes in the development of artemalogs
as cytotoxic anticancer agents, we recently proposed to develop molecularly targeted
anticancer therapies derived from 3 by combining the typical skeleton of 3 or its novel
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fragmented intermediates with a molecularly targeted drug bullet.
Among several
approaches we tried, we were delighted to find that incorporation of a Smo
antagonistic structural motif into the structure of 3 led to highly potent Smoꢀtargeting
antagonists. As shown in Figure 2, two strategies were used in our design, including 1)
direct merging the basic skeleton of 3 with 4ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline ꢀ the
17,20
wellꢀestablished Smoꢀtargeting bullet
3 into stable key intermediates and then reconstructing with the Smoꢀtargeting bullet
Figure 2). Compounds from strategy one are direct analogues of 3 (series I and II),
as in 1 and 2; 2) first breaking endoperoxide
(
whereas compounds from strategy two belong to derivatives of 3 lacking the basic
skeleton (endoperoxide and polyoxycycle).
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RESULTS AND DISCUSSION
Chemistry. The synthesis of artemalogs 6 and 7 was shown in Scheme 1. Starting
3
2
33
from commercial substrate 3, acid 8 and ringꢀcontracted acid 10 were prepared via
several steps according to literature procedures. Condensation of 8 with
4
ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline in the presence of 1ꢀ[bis(dimethylamino)
methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate (HATU)
and 1ꢀhydroxyꢀ7ꢀazabenzotriazole (HOAT) delivered the hybrid product 6 in 62%
3
4
yield. Reduction of 6 by Zn/HOAc led to the deoxygenated derivative 9 in 54%
yield. Synthesis of the ringꢀcontracted analog 7 was accomplished by following a
similar reaction condition as that for preparation of 6 in 56% yield.
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Scheme 1. Synthesis of Artemisinin Analogues 6, 7 and 9a
H
H
O
O
O
O
ref 43
O
O
a
H
3 (artemisinin)
H
O
O
N
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H
N
ref 44
COOH
O
Cl
8
6
H
O
b
O
O
H
H
O
H
O
O
COOH
O
10
O
O
H
O
H
N
N
O
a
H
H
N
N
O
O
9
7
Cl
Cl
a
Reagents and conditions: (a) 4-chloro-3-(pyridin-2-yl)aniline, HATU, HOAT, DIPEA, CH Cl ,
2
2
rt, 4 h, 56-62%; (b) Zn, HOAc, rt, 12 h, 54%.
Acid degradation of 3 was conducted by treating with concentrated sulfuric acid
3
5
(con. H
2
SO4) in methanol to afford ketoꢀester 11 in 64% yield, which was then
35
converted to the decalenone acid 12 by treating with Ba(OH)
2
in 77% yield (Scheme
). The target compound 13 was obtained by condensation of 12 with
ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline in the presence of 1ꢀethylꢀ3ꢀ(3ꢀdimethyl
2
4
aminopropyl)carbodiimide hydrochloride (EDCI) and 4ꢀdimethylaminopyridine
36
(DMAP) in 34% yield. Hydrogenation of 12 with Pd/C under H
cisꢀdecalone acid 14 in 91% yield, which was then coupled with
ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline, leading to product 15 in 56% yield. Subsequent
2
afforded
4
36
4
reduction of 15 in the presence of NaBH provided decalin amide 18 as a pair of
inseparable diastereoisomers in 76% yield (9:1). Bromination of 15 with bromine in
the presence of acetic acid (HOAc) led to the key intermediate 16 in 94%. Without
o
further isolation, treating 16 under microwave condition at 160 C resulted in the
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isomerized decalenone derivative 17 in 53% yield. Thiazole derivatives 19 and 20
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7
were synthesized by heterocyclization of 16 with thiourea or thioacetamide in EtOH
in 71% and 37% yield, respectively. Meanwhile, treatment of 20 with acetyl chloride
in dichloromethane gave acetamidothiazole 21 in 92% yield.
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As outlined in Scheme 3, compound 22 bearing a totally new pyridineꢀfused
38
bicyclic framework was prepared by treating 11 with NH OAc and Cu(OAc) in
4
2
7
1% yield. Hydrolysis of the intermediate 22 followed by amidation with
4
ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline under the standard condensation conditions (EDCI,
2 2
DMAP, CH Cl ) provided pyridineꢀfused compound 24 in 43% overall yield.
Meanwhile, quinolinyl aldehyde 25 was obtained in 51% yield by treating 22 with
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SeO2, which was further converted to acid 26 through Pinnick oxidation in 79%
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yield. Subsequent condensation of 26 with 4ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline was
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found quite sluggish and compound 27 was obtained in 27% yield. In addition, indole
2
8 was obtained in 73% yield by treating 14 with phenylhydrazine hydrochloride.
NꢀMethylation of 28 with CH I provided 29, which was further hydrolyzed and
3
amidated to afford the indoleꢀfused compound 30 in 25% overall yield (three steps).
Compound 31 was prepared by methyl esterification of 14 with MeI in 91% yield.
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Claisen condensation of 31 with ethyl formate followed by treating with hydrazine
hydrochloride produced 32 in 47% overall yield. The final compound 33 was
prepared from 32 in 30% overall yield in a similar manner as that for 30.
Scheme 4. Synthesis of Compounds 38a,ba
b
68%
a
AcO
c,d
4%
COOMe
N+
O-
Cl
2
2
N
N
6
81%
COOMe
COOMe
34
35
36
O
O
N
e
f,g
36%
COOMe
N
N
N
H
N
9
2%
N
O
3
7a
3
8a
Cl
CF3
O
O
g
N
CF3
+
H N
H
2
N
N
61%
COOH
O
23
37b
38b
a
Reagents and conditions: (a) m-CPBA, CH Cl , 1 h; (b) (Ac) O, reflux, 5 h; (c) LiOH.H O,
2
2
2
2
EtOH-H O (1:1), rt, 2 h; (d) SOCl , CH Cl , 30 min, rt; (e) morpholine, MeCN, K CO , rt, 3
2
2
2
2
2
3
o
h; (f) LiOH.H O, EtOH-H O (1:1), 50 C, 12 h; (g) EDCI, DMAP, CH Cl , 1 h, then 4-chloro-
2
2
2
2
3
-(pyridin-2-yl)aniline, rt, 12 h.
Substituted pyridineꢀfused derivative 38a were synthesized as described in
Scheme 4. Oxidation of methylpyridine 22 with 3ꢀchloroperbenzoic acid (mꢀCPBA)
gave pyridine Nꢀoxide 34 in 81% yield. Subsequent acetylation of 34 in refluxing
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acetic anhydride provided the 2ꢀacetoxymethyl intermediate 35 in 68% yield.
Hydrolysis of 35 followed by chlorination with SOCl
morpholine delivered the corresponding products 37a in 58% overall yield. Amide
8a was obtained in 45% yield through hydrolysis of 37a and subsequent
condensation with 4ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline. Meanwhile, Nꢀbiphenyl product
2
and subsequent amination with
3
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3
8b was obtained similarly by condensation of acid 23 and aniline 37b in 61% yield.
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As shown in Scheme 5, modification or replacement of the pyridine moiety in 24
by a wide variety of heteroaromatic or aliphatic rings was conducted. First, treatment
o
of 39 with Nꢀbromosuccinimide (NBS) at ꢀ20 C followed by Suzuki coupling with
2
ꢀchloroꢀ5ꢀnitrophenyl boronic acid and reduction of the resulting intermediate by Fe
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
powder in ethanol furnished the required aniline 41 in 16% overall yield (three steps).
42
Two 1,2,4ꢀoxadiazoles 42 and 44 were prepared from 2ꢀchloroꢀ5ꢀnitrobenzoic acid
and 2ꢀchloroꢀ5ꢀnitrobenzonitrile respectively by following literature procedures,
which were in turn reduced with Fe powder to yield precursors 43 and 45 in 82% and
8
4% yields, respectively. Syntheses of 1,3,4ꢀoxadiazole 49a and l,3,4ꢀthiadiazole 49b
43
were accomplished over four steps starting from commercial available methyl
ꢀmethylꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylate (46), which was reacted with hydrazine first,
followed by treating with 2ꢀchloroꢀ5ꢀnitrobenzoic acid to yield 47 in 50% overall
1
43
yield. Subsequent cyclization of 47 mediated by POCl
3
, followed by reduction with
Feꢀpowder afforded the 1,3,4ꢀoxadiazole compound 49a in 73% overall yield. The
1
,3,4ꢀthiadiazole 49b was synthesized in 40% overall yield by cyclization of the
36
intermediate 47 in the presence of Lawesson’s reagent in THF and subsequent
reduction with Fe powder in ethanol. The final products 50ꢀ54 were obtained in
4
1ꢀ53% yields by condensation of acid 23 with corresponding anilines under
aforementioned standard conditions.
Substituted benzoimidazole derivatives 62ꢀ69 were synthesized as described in
Scheme 6. Intermediates 55 and 56 were obtained over two steps from
4
4
2
ꢀchloroꢀ5ꢀnitrobenzoic acid following literature procedures. Conversion of
2ꢀchloroꢀ5ꢀnitrobenzoic acid to the corresponding acid chloride and subsequent
reaction with substituted 1,2ꢀdiaminobenzene followed by cyclization of the resulting
45
intermediate in reflux acetic acid provided 58aꢀd in moderate yields. Reduction of
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58a with Fe powder in ethanol resulted in the corresponding aniline 59 in 82% yield.
Treatment of 58aꢀd with MeI provided the corresponding methylated products 60aꢀe
in 65ꢀ81% yields. It should be noted that 60b, 60c and 60d were generated as a pair of
5
ꢀ and 6ꢀisomers in about 1:1 ratio, which were inseparable by silica gel column.
10
11
12
13
14
15
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17
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22
23
24
25
26
27
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29
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32
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Subsequent reduction of 60aꢀe by Feꢀpowder generated the key building blocks
1aꢀd in 82ꢀ93% yields. The final products 62ꢀ69 were obtained in 41ꢀ53% yields by
6
coupling of the appropriate aniline with the key intermediate 23 under the standard
condensation conditions. The isomeric 68 and 69 were successfully separated by
chromatography and corresponding absolute configuration was deduced by
comparison of their nuclear overhauser effect (NOE) correlation analysis (see
Supporting Information)
.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
StructureꢀActivity Relationship (SAR) Study. All new compounds were
evaluated for their ability to inhibit the Hh signaling pathway by dual luciferase
reporter assays using light II cells, which were NIHꢀ3T3 cells stably transfected with
46
a Gliꢀresponsive firefly luciferase reporter and Renillaꢀluciferase expression vector.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As shown in Table 1, we first evaluated artemisinin analogues 6 and 7 based on our
strategy one (series I and II, Figure 2), and found that both compounds showed good
inhibitory potency against the Hh signaling pathway with IC50 values of 241 nM and
1
80 nM, respectively, which were only 3ꢀ to 6ꢀfold less potent than the clinically
approved drug 1 (39 nM). Compared to 6, the ringꢀcontracted derivatives 7 was
slightly more potent. Interestingly, the deoxygenated compound 9 showing slightly
improved potency, compared to the endoperoxide 6 (184 nM vs 241 nM). These
results indicated that neither the polyoxycyclic nor the endoperoxide component is
necessary for these compounds to block the Hh signaling pathway. As mentioned
earlier, artemisinin framework is generally attributed to the poor PK and cytotoxicity
of many reported direct artemisinin analogues, therefore, the rest of our SAR was
directed to simpler derivatives lacking the polyoxycyclic and endoperoxide motifs
through
ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline motif ꢀ the Smoꢀtargeting bullet, based on our
strategy two (series III and IV, Figure 2).
major
structural
operations
of
3,
but
containing
the
4
Table 1. Inhibition of compounds to the Hh pathway activity tested by dual luciferase
a
reporter assays in light II cells.
Gliꢀluc reporter,
IC50 (nM)
Gliꢀluc reporter,
IC50 (nM)
Compound
Compound
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2
41 ± 14.1
180.0 ± 10.4
6
7
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
84 ± 39.3
39.2 ± 2.16
9
a
IC50 values are shown as the mean ± SD (nM) from three separate experiments.
3
5
Since 3 is highly sensitive to both acidic and basic conditions, we treated 3
with H SO followed by Ba(OH) , leading to a stable bicyclic acid 12. With this
structurally stable and nonꢀperoxide acid 12 as the key intermediate, compounds 13,
5, 17 and 18 were obtained and evaluated. Unfortunately, only compound 15
2
4
2
1
containing a naphthalenꢀ2(1H)ꢀone motif retained moderate potency with an IC50
value of 290 nM (Table 2). Enones 13 and 17 as well as the alcoholic 18 were inactive
(> 1 ꢁM) in the Gliꢀluc reporter assay. Further functionalization of ketone 15 led to a
small series of heterocyclic derivatives, amongst which thiazole 21 was inactive, but
pyridine 24, 1ꢀphenylꢀpyrazole 33, Nꢀmethylindole 30, as well as quinoline 27
retained moderate potency with IC50 values ranging between 212–476 nM. Since
pyridine 24 showed the highest potency (212 nM) and its synthesis only needs four
steps from the ample commercial source of 3, it served as our lead for further
structure optimization.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Inhibition of compounds to the Hh pathway activity tested by dual
a
luciferase reporter assays in light II cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Gliꢀluc reporter,
IC50 (nM)
Gliꢀluc reporter,
IC50 (nM)
X =
X =
>1000
>1000
13
21
24
33
30
290 ± 80.3
212 ± 110
15
17
18
>1000
327 ± 33.6
>
1000
476 ± 56.3
270 ± 23.4
>1000
2
7
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9
a
IC50 values are shown as the mean (nM) from three separate experiments.
As shown in Table 3, incorporating a morpholinyl moiety to compound 24 lead to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
38a, which showed slightly improved potency with an IC50 value of 155 nM.
Replacement the Smoꢀtargeting bullet ꢀ 4ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline with a
biphenyl bioisostere led to compound 38b, which only showed modest inhibitory
potency. Meanwhile, optimization of the pyridinyl component within the
4
ꢀchloroꢀ3ꢀ(pyridinꢀ2ꢀyl)aniline motif was conducted and a series of heterocyclic
17
bioisosteres were introduced (50ꢀ55 and 62ꢀ64). Quite disappointingly, introducing
a 2,6ꢀdimethylmorpholinyl (50) on the pyridinyl moiety abolished the inhibitory
activity. Significant difference was observed by replacing the pyridinyl with
3
ꢀcyclopropyl 1,2,4ꢀoxadiazole (51) and 5ꢀmethyl 1,2,4ꢀoxadiazole (52), of which
only the former compound retained good potency whereas the latter one was inactive
220 nM vs 1 ꢁM). Moderate potency was observed on compounds 53 and 54
containing the bioisosetric 5ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ1,3,4ꢀoxadiazole and
ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ1,3,4ꢀthiadiazole, respectively. However, oxadiazole 53
(
5
was 2ꢀfold less potent than thiadiazole 54 (588 nM vs 265 nM). Compounds 62 and
63 containing benzo[d]oxazole and benzo[d]thiazole, respectively, retained similar
potency (243 nM vs 282 nM). To our delight, compound 64 containing the
benzo[d]imidazole component showed much improved potency with an IC50 value of
4
6 nM. It was nearly 5ꢀfold more potent than pyridine 24 (212 nM) and was nearly as
potent as reference compound 1 (39 nM).
Table 3. Inhibition of compounds to the Hh pathway activity tested by dual
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
luciferase reporter assays in light II cells.
Gliꢀluc reporter,
IC50 (nM)
Structure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38a
155 ± 43.2
O
38b
N
CF3
~1000
H
N
O
Gliꢀluc reporter,
IC50 (nM)
Gliꢀluc reporter,
IC50 (nM)
Y =
Y =
>1000
265 ± 48.0
243 ± 39.1
5
4
50
220 ± 20.0
5
5
1
2
62
63
>1000
282 ± 48.1
45.9 ± 7.79
588 ± 62.4
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53
a
The IC50 values are shown as the mean ± SD (nM) from two separate experiments.
Since compound 64 showed much improved potency, we finely tuned this
10
11
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13
14
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
compound by introducing a small substituent on the benzo[d]imidazole moiety (Table
). To our delight, the Nꢀmethyl substituted benzo[d]imidazole 65 displayed a high
4
potency of 9.53 nM that is 4ꢀ to 5ꢀfold more potent than both the nonꢀsubstituted
benzo[d]imidazole 65 and the reference compound 1. Further introduction of a 5ꢀ or
6
ꢀmethoxy (66) on the benzo[d]imidazole network led to reduced potency (335 nM),
however introducing a 5ꢀ or 6ꢀfluoro substituent afforded compound 67 retaining high
potency of 63 nM. Meanwhile, the 5ꢀ or 6ꢀtrifluotomethoxy substituted analogues 68
and 69 also retained good potency with IC50 values of 84 nM and 53 nM, respectively.
Table 4. Inhibition of compounds to the Hh pathway activity tested by dual
a
luciferase reporter assays in light II cells.
Gliꢀluc reporter,
IC (nM)
Gliꢀluc reporter,
Y =
Y =
IC (nM)
50
50
45.9 ± 7.79
63.2 ± 31.1
84.5 ± 21.2
64
6
6
7
8
9.53 ± 1.19
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
5
6
3
35 ± 91.1
52.6 ± 27.3
6
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
IC50 values are shown as the mean (nM) from three separate experiments.
Cytotoxicity and hERG Inhibition of Potent Compounds. Based on the SAR
above, several compounds were identified showing significant inhibitory potency
against the Hh signaling pathway with IC50 values higher than or compatible to the
reference compound 1. To exclude the chemical structure related untargeted cell
toxicity and cardiac toxicity, compounds 6, 24, 38a, and 65 from different structure
series were selected for evaluation of their inhibitory effects against proliferation of
squamous carcinoma KB cells and against hERG channel. As shown in Table 5, all
these compounds showed no significant antiproliferative effects against the KB cell
(
IC50 > 5 ꢁM), indicating that offꢀtarget related toxicity may not be an issue of
concerns for current compounds. However, compound 38a displayed an alarming
hERG blocking effect (3.3 ꢁM) indicating its cardiac toxicity potential, otherwise the
other compounds (6, 24, 65) showed IC50 values greater than 15 ꢁM against the hERG
channel. Since compound 65 showed highest potency against the Hh signaling
pathway and no significant toxicity to the cell growth and hERG channel, it was
selected for further profiling.
Table 5. Cytotoxicity and hERG Inhibition of Potent Compounds
IC50, nM or µM
Compound
Gliꢀluc reporter
squamous carcinoma
hERG
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KB cells
6
241 ± 14.1 nM
6.61 µM
15.0 µM
> 15.0 µM
> 15.0 µM
24
212 ±110 nM
155 ± 43.2 nM
9.53 ± 1.19 nM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
38a
>20 µM
>20 µM
3.31 µM
65
> 15.0 µM
a
IC50 values are shown as the mean (nM) from three separate experiments.
Compound 65 Inhibited the mRNA Expression of Gli Target Gene Gli1. To
exclude that the high potency of compound 65 observed in the dual luciferase reporter
assay was due to inhibition of the luciferase enzyme itself, we evaluated the effect of
6
5 on the mRNA expression of Gli1, a transcriptional target gene of Gli that was
4
7
frequently used as readout of the Hh signaling pathway activity. As shown in Figure
3, the expression of Gli1 mRNA in light II cells was robustly elevated in response to
Shh conditioned medium (Shh CM), whereas treatment with compounds 65 (0.1 µM)
led to significantly suppression on the mRNA expression of Gli1, equal to that of
compound 1. Therefore, these data further confirm that compound 65 is a potent
inhibitor against the Hh signaling pathway.
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2
2
2
2
2
2
3
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3
3
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0
1
2
3
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Compound 65 inhibited the mRNA expression of Gli target gene Gli1. Light II
cells were subjected to various treatments as indicated for 36 h, and then were collected for
RTꢀqPCR analysis of the Gli1 mRNA expression. The results were expressed as the mean ±
SD.
Compound 65 Had no Inhibitory Effect on Transcriptional Factor Activity
Provoked by Tumor Necrosis Factorꢀα (TNFꢀα) and Prostaglandin E2 (PGE2),
respectively. To exclude the possibility that compound 65 may nonꢀspecifically
inhibit the Hh pathway activity, we further tested the effect of compound 65 on two
other unrelated transcriptional factors activity, such as NFꢀκB in response to TNFꢀα,
and Tꢀcell factor (TCF)/lymphoid enhancer factor (LEF) activity stimulated by
4
8
PGE2. As shown in Figure 4, compound 65 had no effects on either the TCF/LEF
activity in response to PGE2 (Figure 4A), or the NFꢀκB activity provoked by TNFꢀα
(Figure 4B); however, significant inhibitions were observed from their respective
inhibitors, (E)ꢀ3ꢀtosylacrylonitrile (BAY11ꢀ7082, NFꢀκB) and
(Nꢀ[2ꢀ((pꢀbromocinnamyl)amino)ethyl]ꢀ5ꢀisoquinolinesulfonamide dihydrochloride
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salt (H89
,
TCF/LEF), which served as positive controls (Figure 4A,B). Hence, these
data suggest that compound 65 possessed selectivity on suppressing the Hh pathway
activity.
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22
23
24
25
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Compound 65 had no effect on inhibiting transcriptional factors activity
provoked by TNFꢀα and PGE2, respectively. A. The HEK293 cells after various
treatments as indicated for 24 h were collected for dual luciferase reporter analysis of the
NFꢀκB activity. B. The LS174T cells after various treatments as indicated for 6 h were
collected for dual luciferase reporter analysis of TCF/LEF activity. The results were
expressed as the mean ± SD.
Compound 65 Inhibited the Hh Signaling Pathway by Targeting Smo. To test
whether the inhibitory effect of 65 on the Hh signaling pathway was due to targeting
49,50
Smo, a central regulator of the Hh signaling pathway,
we then conducted several
control experiments as shown in Figure 5. First, we artificially expressed the Smo
wild type plasmid to activate the Gliꢀluciferase activity in light II cells. Compound 65
was found to doseꢀdependently abolish the Gliꢀluciferase activity provoked by
artificially forced expression of Smo wild type plasmid, similar to compound 1
(Figure 5A), suggesting that 65 inhibited the Hh signaling pathway activity by
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5
6
targeting Smo or molecules downstream of Smo. SmoW539L and SmoD473H were
two Smo mutants which may cause resistance to the clinically approved Smo
7,23,24
antagonists (e.g. 1 and 2).
When compared to the obvious inhibitory effect on the
Gliꢀluciferase activity stimulated by Smo wild type plasmids (Figure 5A),
compound 65 had minimal inhibitory effect on the Gliꢀluciferase activity stimulated
by ectopic expression of either SmoW539L or SmoD473H (Figure 5B, 5C),
indicating that compound 65 was insensitive to SmoW539L and SmoD473H.
Collectively, the capacity of 65 to inhibit the Gliꢀluciferase activity provoked by
ectopic expression of Smo but not by ectopic expression of SmoW539L and Smo
D473H suggested that compound 65 functioned by binding to Smo as an
0
1
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9
0
1
2
3
4
5
6
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9
0
1
2
3
4
5
6
7
8
9
0
5
1,52
antagonist.
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Figure 5. Distinct responses of compound 65 to the Gliꢀluciferase activity stimulated by
Smo (wild type), SmoW539L, and SmoD473H plasmids, respectively. A, compound 65
doseꢀdependently inhibited the Gli luciferase activity initiated by ectopic expression of wild
type Smo plasmid. B and C, compound 65 exhibited minimal inhibitory effect on Gli
luciferase activity provoked by ectopic expression of SmoW539L or SmoD473H mutants.
Light II cells transfected with Smo, SmoW539L or SmoD473H plasmids were exposed to
compound 1 or 65 for 36 h, and then were harvested for dual luciferase reporter assays. The
results were expressed as the mean ± SD.
Compound 65 Shares the Same Binding Site on Smo with Cyclopamine. Having
characterized Smo as the cellular target of compound 65, we further mapped its
binding site on Smo with complementary methods. Given the fact that the vast
majority of currently reported Smo antagonists or agonists were identified to function
by binding the common site in the transmembrane domain of Smo as that of the tool
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drug cyclopamine
—the first reported archetypical Smo antagonist, we set out to
determine whether compound 65 binds Smo in a similar mode. As shown in Figure 6,
the Smo biding site of 65 was tested by using BODIPYꢀcyclopamine, a fluorescent
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derivative of cyclopamine. It was found that compound 65 decreased the binding of
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BODIPYꢀcyclopamine to Smo, analogous to the action of cyclopamine (Figure 6A).
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However, itraconazole, another Smo atagonist with binding site dictinct from that of
cyclopamine, had no effect on the binding of BODIPYꢀcyclopamine to Smo (Figure
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A). These results were further recapitulated by FACS analysis (Figure 6B), thus
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suggesting that compound 65 acted on the common binding site similar to that of
cyclopamine. To further confirm this argument, we tested the influence of 65 on the
stimulation of the Hh pathway activity by a specific Smo agonistic tool drug
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nzo[b]thiopheneꢀ2ꢀcarboxamide (SAG), whose binding site on Smo overlaps with that
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of cyclopamine. As shown in Figure 6C, the SAGꢀinduced Gli luciferase activity
was counteracted by increasing concentrations of 65, accompanied with enhancement
of SAG EC50 values (Figure 6D), thus suggesting that compound 65 acted as a
competitive inhibitor of SAG. In conclusion, these data clearly suggested that
compound 65 is a Smo antagonist and shares a same binding mode as that of
cyclopamine.
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Figure 6. Compound 65 shares the same binding site on Smo with cyclopamine. A.
BODIPYꢀcyclopamine competition analysis tested by fluorescent microscope. Photographs
were representatives from three distinct experiments with identical results; B. Cells after
various treatments as indicated were subjected to BODIPYꢀcyclopamine binding analysis by
FACS. CꢀD: Compound 65 is a competitive inhibitor of Smo agonist SAG. Light II cells were
exposed to SAG with or without increasing concentrations of compound 65 for 36 h, and
Gliꢀluciferase activity of light II cells were measured by dual luciferase reporter assays. C.
Compound 65 counteracted the Gliꢀluciferase activity provoked by SAG; D. SAG EC50
values obtained by increasing concentrations of compound 65.
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Figure 7. Compound 65 suppressed proliferation of ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells.
A. Compound 65 inhibited the proliferation of ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells. The
results were expressed as the mean ± SD. B. Compound 65 inhibited the expression of Gli1 at
mRNA level in ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells. The results were expressed as the mean
±
SD. C. Compound 65 treatment caused apoptosis of ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells.
Compound 65 Inhibited the Proliferation of Medulloblastoma Cells Isolated
from ptch+/ꢀ;p53ꢀ/ꢀ Mice. Medulloblastoma spontaneously aroused in ptch+/ꢀ;p53ꢀ/ꢀ
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mice has been widely used to evaluate the anticancer effect of Smo antagonists, as the
growth of medulloblastoma from these mice is addicted to the constitutive Hh activity
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caused by loss of one allele of ptch.
In this regard, medulloblastoma cells from
nude mice allografted with a primary medulloblastoma from a ptch+/ꢀ;p53ꢀ/ꢀmouse
were isolated for testing the antiproliferative effect of compound 65. As revealed by
MTS analysis in Figure 7A, compound 65 doseꢀdependently inhibited the
proliferation of ptch+/ꢀ;p53ꢀ/ꢀmedulloblastoma cells in a fashion similar to that of
compound 1 (Figure 7A). Considering that Hh signaling pathway may transcriptionally
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upꢀregulate the antiꢀapoptotic gene Bclꢀ2 to prevent the cellular apoptosis, we then
tested whether the inhibitory effect of compound 65 on the proliferation of
ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells was caused by inducing apoptosis. Using
annexin Vꢀfluorescein isothiocyanate (FITC) and propidiumiodide (PI) double
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staining meathod, we observed that exposure of compound 65 for 24 h obviously
caused apoptosis of ptch+/ꢀ;p53ꢀ/ꢀ medulloblastoma cells, with the apoptotic rate of
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6.3% compared to 0.9% of the control cohort. Meanwhile, no cytotoxic effect of
compound 65 was observed, as revealed by no cells were stained by PI only (Figure
C).
Pharmacokinetic (PK) Study of Compound 65. Since the most potent compound
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65 displayed favorable profiles in vitro as a novel Smo antagonist, we further
investigated its pharmacokinetic properties in SD rats. After intravenous (i.v.)
injection of 1 mg/kg of the new inhibitor 65, low systemic plasma clearance (CL =
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.31 mL/min/kg) and a good volume of distribution (Vss = 2229 mL/kg) were
obtained. The mean residence time (MRT) was 3.97 h. The oral exposure of
compound 65 at the dose of 3.0 mg/kg was 1836 h•ng/mL, resulting in an acceptable
estimated bioavailability of 27.4% (Table 7). The overall PK parameters of compound
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