Discovery of a 6-(pyridin-3-yl)benzo[d]thiazole template for optimization of hedgehog and PI3K/AKT/mTOR dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.06.049

Abstract Vismodegib is the first FDA approved cancer therapy based on inhibition of aberrant hedgehog signaling. Like most cancer therapies, vismodegib suffered from resistance, even during clinical development. Numerous reports demonstrated that simultan

Full text of DOI:10.1016/j.bmcl.2015.06.049

Accepted Manuscript
Discovery of a 6-(pyridin-3-yl)benzo[d]thiazole Template for Optimization of
Hedgehog and PI3K/AKT/mTOR Dual Inhibitors
Zhaohui Yang, Haikuo Ma, Zhijian Sun, Lusong Luo, Sheng Tian, Jiyue Zheng,
Xiaohu Zhang
PII:
S0960-894X(15)00642-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.049
BMCL 22839
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
6 April 2015
28 May 2015
12 June 2015
Please cite this article as: Yang, Z., Ma, H., Sun, Z., Luo, L., Tian, S., Zheng, J., Zhang, X., Discovery of a 6-
(
pyridin-3-yl)benzo[d]thiazole Template for Optimization of Hedgehog and PI3K/AKT/mTOR Dual Inhibitors,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.049
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Graphical Abstract
Vismodegib is the first FDA approved cancer therapy based on inhibition of aberrant hedgehog
signaling. Like most cancer therapies, vismodegib suffered from resistance, even during clinical
development. Numerous reports demonstrated that simultaneous blockage of hedgehog and
PI3K/AKT/mTOR pathways resulted in significantly superior outcomes compared with single
agent alone in a number of animal disease models. The dual hedgehog and PI3K/AKT/mTOR
inhibition represented a promising approach not only to overcoming the resistance but also to
delaying its onset. Here we report
a
series of compounds based on
a
6-(pyridin-3-yl)benzo[d]thiazole template which have demonstrated significant inhibition of both
hedgehog and PI3K/AKT/mTOR signaling pathways. This new scaffold can serve as a lead for
further optimization.
1

Discovery of a 6-(pyridin-3-yl)benzo[d]thiazole Template for Optimization of Hedgehog and
PI3K/AKT/mTOR Dual Inhibitors
a,1
Zhaohui Yang , Haikuo Ma , Zhijian Sun , Lusong Luo , Sheng Tian , Jiyue Zheng ,
a,1
b
b,*
a
a
a,*
Xiaohu Zhang
a
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and
College of Pharmaceutical Sciences, Soochow University, Su Zhou, Jiangsu 215021, P. R. China
b
BeiGene (Beijing) Co., Ltd., No. 30 Science Park Road, Zhongguancun Life Science Park,
Beijing 102206, P. R. China
1
these authors contributed equally to this paper
*
Corresponding author. (X.H.Z) Tel.: +86 512 65880380. Fax: +86 512 65880380. Email:
xiaohuzhang@suda.edu.cn. (L.S.L) Email: lusong.luo@beigene.com.
2

Abstract: Vismodegib is the first FDA approved cancer therapy based on inhibition of aberrant
hedgehog signaling. Like most cancer therapies, vismodegib suffered from resistance, even during
clinical development. Numerous reports demonstrated that simultaneous blockage of hedgehog
and PI3K/AKT/mTOR pathways resulted in significantly superior outcomes compared with single
agent alone in a number of animal disease models. The dual hedgehog and PI3K/AKT/mTOR
inhibition represented a promising approach not only to overcoming the resistance but also to
delaying its onset. Here we report
a
series of compounds based on
a
6-(pyridin-3-yl)benzo[d]thiazole template which have demonstrated significant inhibition of both
hedgehog and PI3K/AKT/mTOR signaling pathways. This new scaffold can serve as a lead for
further optimization.
Keywords: hedgehog pathway, PI3K/AKT/mTOR pathway, polypharmacology, GPCR, kinase,
cancer therapy
Abbreviations
AKT, protein kinase B; BCC, basal cell carcinoma; CNS, central nervous system; GPCR, G
protein-coupled receptor; HBA, hydrogen bond acceptor; Hh, Hedgehog; MB, modulloblastoma;
mTOR, mammalian target of rapamycin; N-G-L, NIH3T3-GRE-Luc reporter gene assay; PI3K,
phosphoinositide 3-kinase; Ptch, Patched; SAR, structure-activity relationship; Smo, Smoothened;
SMO-BCB, BODIPY-Cyclopamine binding assay; SUFU, suppressor of fused.
3

Hedgehog signaling pathway is a pivotal developmental pathway responsible for patterning and
organogenesis in early embryonic development. Hedgehog (Hh) signaling pathway is relatively
dormant in adults, with limited functions in tissue repair and maintenance. In canonical hedgehog
signaling, the secreted proteins Sonic hedgehog, Indian hedgehog and Desert hedgehog bind to the
negative regulator Patched (Ptch), relieving the suppression of Ptch to a GPCR protein
Smoothened (Smo). Activated Smo orchestrates a signaling cascade which involves the
glioma-associated oncogene transcription factors’ (Gli1, Gli2, and Gli3) dissociation from
suppressor of fused (SUFU) and entrance to the nucleus, ultimately leading to specific gene
1
expressions mediated by the Gli family transcription factors. Aberrant hedgehog signaling is
connected to numerous cancers. Mutational activation (ligand independent) of Hh from
inactivation of the negative components (Patched, SUFU) or activation of the positive component
(
smoothened) leads to cancers such as basal cell carcinoma (BCC), modulloblastoma (MB), and
2
rhabdomyosarcoma. Ligand dependent activation was observed in colorectal, gastric, pancreatic,
3
prostate, lung, haemotalogical cancers, and so on. Inhibition of the abnormal Hh activity
4
represents a promising strategy for the treatment of cancers.
4
Smoothened is the most studied Hh pathway component as a drug target. The most advanced
Smo antagonist, GDC-0449 (vismodegib), was approved by the FDA in 2012 for the treatment of
5
basal cell carcinoma which was not suitable for operation. There are numerous Smo antagonists
such as NVP-LDE225, BMS-833923, LY2940680, PF-04449913, NVP-LEQ506 (Figure 1) in
6
advanced developmental stages.
Figure 1. Chemical structures of clinical Hh signaling pathway inhibitors.
Like most cancer therapies, vismodegib suffered from resistance even during clinical
development. A patient with metastatic MB went into remission after initial treatment with
vismodegib; however, the cancer reoccurred shortly after, and the patient succumbed to the
7
disease. Later analysis demonstrated that a mutation in Smo, codon 473 from Asp to His (D473H)
8
led to the diminished binding of vismodegib to the mutated Smo. Similar resistance mutations
had also been observed for NVP-LDE225 in a mouse MB model driven by a Ptch-mutant, albeit
9
the identified mutations were different from the vismodegib induced mutation. Numerous work
aiming to overcome the resistance developed by the D473H mutation had been reported. Allosteric
inhibitors ALLO-1 and ALLO-2 identified by Norvatis scientists demonstrated a distinctive
1
binding mode and can inhibit vismodegib resistant Smo. It appeared that antagonists which bind
0
4

to the same site as vismodegib may also block the mutant Smo with different, usual larger
6
d,11
substitutions on the core structure (exemplified by compound 1, Figure 2).
This may be due to
the extended interactions between Smo and the antagonists away from the mutant site. Most
1
2
recently, more human mutations have been identified, which may complicate approaches
targeting mutational Smo. Nevertheless, the potential success of blocking the mutant Smo could
be limited once the Gli2 is up-regulated, as demonstrated by both vismodegib and NVP-LDE225
9
,11a
in the Ptch-mutant MB mouse model.
Gli2 up-regulation represents another mechanism of
resistance for Smo antagonists. There are reports of Gli inhibitors, however, the current Gli
1
3
inhibitors could be hampered by safety and developability hurdles. Alternative pathway
activation represents a third mechanism of resistance. For example, treatment with vismodegib
and NVP-LDE225 both led to an activated PI3K/AKT/mTOR signal pathway in a MB mouse
9
,11a
model.
Simultaneous blockage of Hh and PI3K/AKT/mTOR pathways resulted in significant
tumor regression compared with single agent alone in both cases. The dual hedgehog and
PI3K/AKT/mTOR inhibition represented a promising approach not only to overcoming the
resistance but also to delaying its onset.
Despite encouraging results from combination treatment in animal disease models, a combination
1
therapy may face problems from patients’ compliance, drug-drug interaction risks etc.
4
Alternatively, a compound which can block the Hh and the PI3K pathways simultaneously could
offer significant advantages. Polypharmacology was very successful in the CNS drug development
1
and kinase development. However, it is challenging to develop compounds cross different
5
classes of drug targets. In the current case, Smo is a GPCR and PI3K/AKT/mTOR are kinases.
1
In our pursuit of novel Hh inhibitors, we studied numerous templates under the guidance of the
8
1
recently published Smo crystal structure and a pharmocophore model. We have previously
6
17
1
8a
reported two novel templates based on tetrahydroimidazo[1,2-a]pyrazine
and
Compounds from these templates demonstrated potent Smo
inhibition, improved physical-chemical properties, and good pharmacokinetic profiles
exemplified by compound 2, Figure 2). Alternative ring closure by hybridization of compounds 1
and 2 led to a new template based on 6-(pyridin-3-yl)benzo[d]thiazole, exemplified by compound
1 (Figure 2). Compounds from the 6-(pyridin-3-yl)benzo[d]thiazole template also demonstrated
1
8b
tetrahydrothiazolo[5,4-c]pyridine.
(
1
potent inhibition of Smo. Based on the aforementioned rationale, we also surveyed a number of
PI3K/AKT/mTOR inhibitors in the literature in an effort to dial in kinase activity. We found a
series of pan PI3K inhibitors developed by scientists from Amgen which showed great similarity
1
9
to our template. The leading compound, Amgen compound 3, looked exactly like compound 11,
except that an amide linkage was replaced by a sulphonamide (Figure 2). In theory, the
sulphonamide could provide the same key hydrogen bond acceptor (HBA) interaction, as
5
a
demonstrated by the extensive SAR investigation in the development of vismodegib. Therefore
we hypothesized that compound 3 should be an active Hh inhibitor as well. Here we report the
identification of the 6-(pyridin-3-yl)benzo[d]thiazole template as a valuable lead for the
optimization of dual Hh and PI3K inhibitors.
5

Figure 2. Scaffold morphing strategy and molecular similarity of Hh inhibitor 11 and Amgen pan
PI3K inhibitor 3.
The synthesis of compounds 9-20 was outlined in Scheme 1. Commercially available
2-amino-6-bromobenzothiazole (4) was acetylated and followed by Miyaura borylation to give
pinacol boronate 6. Suzuki coupling of 6 and bromopyridine 7 provided the key intermediate 8,
which was acylated with corresponding acyl chloride to afford amides 9-18. Treatment of
intermediate 8 with benzaldehyde under reductive amination conditions using sodium
triacetoxyborohydride afforded compound 19, or under Strecker reaction conditions using
trimethylsilyl cyanide afforded α-aminonitrile 20.
Scheme 1. Reagents and conditions: (a) Ac
bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, DMSO, 90 °C, 97%. (c) K
,4-dioxane/H O, 80 °C. 8 h, 20%. (d) acyl chloride, pyridine, 0 °C to r. t., 12 h, 20 - 50% for 9-18;
TMSCl, NaBH(AcO) , AcOH, r. t., 12 h, 31% for 19; TMSCN, ZnCl in Et O, THF, r. t. to reflux,
2 h, 25% for 20.
2
O, DMAP, CH2Cl2, r. t., 12 h, 79%. (b)
2
2
CO , Pd(PPh
3
3 4
) ,
1
2
3
2
2
1
The synthesis of compounds 25-28 was outlined in Scheme 2. Dibromide derivative 21 was
prepared from 4 through diazotization-bromination in 87% yield. Displacement of the bromine of
intermediate 21 with cyclopropylamine or cyclopropylmethylamine provided compounds 22a and
22b. Our initial attempt to convert 22a-b into corresponding pinacol boronates for the next step
Suzuki coupling with bromide 7 failed, presumably due to the electron-donating nature of the
amino functional group. Alternatively, acylation of 7 with benzoyl chloride or 3-chlorobenzoyl
chloride, followed by Miyaura borylation furnished pinacol boronates 24a and 24b. Suzuki
6

coupling between 24a-b and aryl bromide 22a-b led to the final compounds 25-28, respectively.
Scheme 2. Reagents and conditions: (a) iso-pentyl nitrite, CuBr
corresponding amine, dioxane, 80 °C, 12 h, 57-67%. (c) ArCOCl, pyridine, 0 °C, 1 h, 88-95%. (d)
bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, 1,4-dioxane, 100 °C, 12 h, 70-90%. (e) Na CO
Pd(dba) , PCy , 1,4-dioxane/H O, 80 °C, 20-30%.
2
, MeCN, 0 °C, 1 h, 87%. (b)
2
2
3
,
2
3
2
The Hh inhibition activity of the synthesized compounds was tested in a cell based
2
0
NIH3T3-GRE-Luc reporter gene assay with 10 nM SAG as the Hh pathway agonist.
This
assay can identify Smo inhibitors and inhibitors downstream of Smo such as Gli. However, this
assay cannot identify inhibitors interacting with targets upstream of Smo, such as Hh protein
2
inhibitors or Hh acyltransferase inhibitors. The structure-activity-relationship (SAR) was
1
summarized in Table 1.
Table 1. Structure Activity Relationship (SAR) of designed compounds
N-G-L
PI3Kα
N-G-L
PI3Kα
a
a
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
Compd Structure
Compd Structure
(
SMO-BCB
(% inhib.
c
(1 µM) )
(SMO-BCB
b
IC50 (nM) )
(% inhib.
c
(1 µM) )
b
IC50 (nM) )
6
4 ± 21
9
9%
18
19
3400 ± 2200
940 ± 130
b
5.6 )
(
10
11
12
4100 ± 1400
4
1 ± 13
980 ± 280
(93%)
490 ± 1.6
(85%)
20
25
100 ± 27
210 ± 18
b
10 )
(
>10000
7

1
1
1
1
3
4
5
6
7
>10000
5000 ± 2800
170 ± 50
26
27
28
3
150 ± 22
150 ± 1.2
190 ± 32
120 ± 3.8
10%
48%
3%
d
810 ± 230
>10000
1.2 ± 0.9
e
1
Vismodegib
16 ± 2.4
a
Inhibition of luminescence signaling in NIH3T3-GRE-Luc reporter gene assay (N-G-L) with 10
nM SAG as the Hh pathway agonist. Data are expressed as geometric mean values of at least two
runs ± the standard error measurement (SEM).
b
Inhibition of BODIPY-cyclopamine fluorescence signaling in the competitive displacement
experiment using U2OS cells over-express human Smo. Data are expressed from a single IC50
determination.
c
Inhibition of luminescence signaling in Kinase-Glo Plus Luminescent Kinase Assay (Promega).
Data are expressed as geometric mean values of two runs ± the standard deviation (SD) or
percentage of inhibition at 1 µM concentration.
d
19
i
Data is expressed as PI3Kα K (nM) which was reported in the literature.
e
Vismodegib was run as standard in each NIH3T3-GRE-Luc reporter gene assay. Data are
expressed as geometric mean values of four runs ± the standard error measurement (SEM).
The simplest benzamide, compound 9, was a potent Hh inhibitor (64 nM). It was within a few
folds less active when compared with vismodegib (16 nM). Chlorine substitution led to different
results: while meta-chloro substitution resulted in improved activity (compound 11, 41 nM), ortho
and para-chloro substitutions were detrimental to activity (compounds 10 and 12). In contrast,
para-fluoro gave the best result (compound 15, 170 nM), while ortho and meta-fluoro substitution
diminished activity (compounds 13 and 14). Para-substituted methyl group was also detrimental to
Hh inhibition. The overall trend indicated that the para position prefer small substitutions while
the meta position can accommodate larger hydrophobic groups. This was consistent with other
1
0
studies. Attempt to replace the phenyl group with a smaller aliphatic cyclopropane led to
diminished activity (compound 17). Introducing flexibility by addition of a methylene or reduction
of the amide bond both led to reduced Hh inhibition activity (compounds 18, 19). However,
bioisosteric replacement of the amide led to sustained activity, as demonstrated by compound 20
8

(
100 nM). Replacement of the acetamide with amines was well tolerated, as compounds 25, 26, 27
and 28 all showed only small reduction of Hh inhibition activity.
In order to confirm that the hedgehog inhibition of these compounds is due to the inhibition of the
Smo, we tested representative compounds 9 and 11 in the BODIPY-cyclopamine displacement
experiment in the U2OS cells over-expressing human Smo. Both compounds demonstrated potent
activities in displacing BODIPY-cyclopamine (5.6 and 10 nM, respectively, Table 1). Further,
compounds 9 and 11 were tested in a counter screening Wnt pathway assay and were confirmed
that their hedgehog inhibition activity was not due to non-specific cytotoxicity (see Supporting
Information).
1
9
The Amgen compound 3 was synthesized and characterized according to the literature. The pan
PI3K inhibition activity was confirmed (data not shown). Given the molecular similarity, we
decided to test compound 3 in the NIH3T3-GRE-Luc reporter gene assay. Compound 3
demonstrated significant Hh inhibition activity (120 nM).
We then tested compounds 9, 11, 15, 20, 26, and 28 for their PI3Kα inhibition activity in a
Kinase-Glo Plus Luminescent Kinase Assay (Promega). While compounds 9, 15, 26, 28 were not
active, compounds 11 and 20 demonstrated decent PI3Kα inhibition at 980 nM and 490 nM,
respectively (Table 1. detailed experimental procedure and data analysis can be found in
Supporting Information). It was reported that the acetamide participated in key interactions in the
1
kinase hinge binding region. It was therefore detrimental for the kinase activity to replace the
9
acetamide with amines, albeit both acetamide and amine functional groups were well tolerated by
1
9
Smo. The Amgen compound 3 was obtained by an intensive PI3K optimization campaign. The
sulfonamide functionality was found to contribute significantly to the PI3K activity. This is
consistent with our modeling results (Supporting Information). While the sulfonamide
functionality was tolerated in Smo, it led to poor physicochemical properties (e.g. extremely poor
solubility), therefore hampered the enthusiasm to further pursue templates associated with this
functional group.
Vismodegib as the first FDA approved cancer therapy based on inhibition of aberrant hedgehog
signaling suffered from resistance. Numerous reports demonstrated that simultaneous blockage of
hedgehog and PI3K/AKT/mTOR pathways resulted in significantly superior outcomes compared
with single agent alone in a number of animal disease models. The dual hedgehog and
PI3K/AKT/mTOR inhibition represented a promising approach not only to overcoming the
resistance but also to delaying its onset. The data presented above demonstrated that compound 3
is a potent pan PI3K and Hh inhibitor. This may help to explain the broad in vivo anti tumor
1
activities demonstrated by compound 3. Further, close analogues 11 and 20 also demonstrated
9
potent Hh inhibition activity and decent PI3Kα inhibition activity. Therefore, the
6-(pyridin-3-yl)benzo[d]thiazole template can serve as a starting point for the development of dual
Hh and PI3K/AKT/mTOR inhibitors.
In summary, during the process of design and synthesis of a dual Hh and PI3K/AKT/mTOR
template, we discovered that compound 3, a potent pan PI3K inhibitor developed by Amgen, also
possessed potent Hh inhibition. This may help explain the broad in vivo anti tumor activity of this
compound. We also showed that compounds 11 and 20 were potent Hh inhibitors as well as decent
PI3Kα inhibitors, thus establishing that the 6-(pyridin-3-yl)benzo[d]thiazole template may serve as
a lead to optimize dual Hh and PI3K inhibitors for cancer treatment.
9

Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No.81273372,
81473090), China Postdoctoral Science Foundation (2013M541729), Natural Science
Foundation of Jiangsu Province (BK20140313), BM2013003, and PAPD (A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions).
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