Design, Synthesis, and Biological Evaluation of Imidazo[1,2- a]pyridine Derivatives as Novel PI3K/mTOR Dual Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01736

PI3K-Akt-mTOR signaling pathway has been validated as an effective targeting pathway for cancer therapy. However, no PI3K/mTOR dual inhibitor has been approved by the FDA yet. Therefore, it is still essential to discover a candidate with good efficacy and low toxicity. In our design, a series of imidazo[1,2-a]pyridine derivatives had been synthesized and subjected to activity assessment in vitro and in vivo. 15a was proved to be a potent PI3K/mTOR dual inhibitor with excellent kinase selectivity, modest plasma clearance, and acceptable oral bioavailability. Besides, 15a displayed significant inhibition of tumor growth in HCT116 and HT-29 xenografts without obvious effect on body weight.

Full text of DOI:10.1021/acs.jmedchem.9b01736

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Article
Design, Synthesis, and Biological Evaluation of Imidazo[1,2-
a]pyridine Derivatives as Novel PI3K/mTOR Dual Inhibitors
Ya'nan Yu, Yuqiao Han, Fupo Zhang, Zhenmei Gao, Tong Zhu, Suzhen Dong, and Ming-Liang Ma
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01736 • Publication Date (Web): 18 Feb 2020
Downloaded from pubs.acs.org on February 18, 2020
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Design, Synthesis, and Biological Evaluation of Imidazo[1,2-a]pyridine
Derivatives as Novel PI3K/mTOR Dual Inhibitors
Ya’nan Yu,†,# Yuqiao Han,†,# Fupo Zhang, Zhenmei Gao, Tong Zhu, Suzhen Dong,* and
†
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,†
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Mingliang Ma*,†, ‡
†Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai,
200062, PR China.
‡Key Laboratory of Brain Functional Genomics, Ministry of Education, East China Normal
University, Shanghai, 200062, PR China.
ABSTRACT: PI3K-Akt-mTOR signaling pathway has been validated as an effective targeting
pathway for cancer therapy. However, no PI3K/mTOR dual inhibitor has been approved by FDA
yet. Therefore, it is still essential to discover a candidate with good efficacy and low toxicity. In
our design, a series of imidazo[1,2-a]pyridine derivatives had been synthesized and subjected to
activity assessment in vitro and in vivo. 15a was proved to be a potent PI3K/mTOR dual inhibitor
with excellent kinase selectivity, modest plasma clearance and acceptable oral bioavailability.
Besides, 15a displayed significant inhibition of tumor growth in HCT116 and HT-29 xenografts
without obvious effect on body weight.
INTRODUCTION
Phosphoinositide 3-kinase (PI3K) is a family of lipid kinases playing central roles in regulation
of cellular growth, proliferation, differentiation, migration, apoptosis and autophagy. The family
members catalyze the phosphorylation of phosphatidylinositol 4,5-diphosphate (PIP ) to produce
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phosphatidylinositol 3,4,5-triphosphate (PIP ), which transduces extracellular signals into cells to
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induce cellular response as a second messenger. There are three classes of PI3K members that
have been identified, of which class I has been extensively studied. Class I PI3Ks are further
divided into four isoforms: α, β, γ and δ based on their different regulatory subunits. Dysregulation
of PI3Kα, β, γ and δ has been demonstrated to be associated with many cancers by abnormal
phosphorylation of their effector Akt (Protein kinase B, PKB).1-4 Mammalian target of rapamycin
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(mTOR) is an atypical serine/threonine kinase and regulates cell growth and metabolism in
response to extracellular nutrient and energy factors. Over activation of mTOR signaling can be
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caused by gain-of-function mutation of PI3K or loss-of-function of tumor suppressor PTEN
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(Phosphate and tension homology deleted on chromsome ten) in many cancers. Thus, the PI3K-
Akt-mTOR signaling has long been a therapeutic targeting pathway for development of cancer
treatment.
Although tumorigenesis is often related to abnormal activation of PI3K-Akt-mTOR signaling
pathway, activation of the pathway is not vertical. A negative feedback has been found between
mTOR and PI3K. For example, inhibition of mTOR complex 1 (mTORC1) by rapamycin induced
insulin receptor substrate-1 (IRS-1) overexpression and induced Akt activation resulting in cancer
cell survival, proliferation, and growth, thus compromising the anti-tumor effects of the inhibitor.7
On the other hand, mTOR signaling can be also activated in a PI3K-independent manner.8, 9 These
studies suggest that combination therapy of ablating mTOR function and preventing Akt activation
may have improved anti-tumor activity. Until now, more than 40 inhibitors of this signaling
pathway have reached different stages of clinical development for tumor therapies. But only six of
them (the mTOR inhibitors Temsirolimus and Everolimus; the PI3K inhibitors Idelalisib,
Copanlisib, Alpelisib and Duvelisib) were approved by FDA for clinical use.10, 11
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Several class I PI3K/mTOR dual inhibitors have entered different clinical research stages till
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now, such as Gedatolisib, PQR309, Apitolisib, LY3023414, Omipalisib and so on. (Figure 1)
Among these clinical candidates, Omipalisib (PI3Kα Ki = 0.019 nM; PI3Kβ Ki = 0.13 nM; PI3Kγ
Ki = 0.024 nM; PI3Kδ Ki = 0.06 nM; mTORC1 Ki = 0.18 nM; mTORC2 Ki = 0.3 nM), a potent
PI3K/mTOR dual inhibitor, exhibits the best kinase inhibitory activity at picomolar
concentrations.13 It is currently explored in clinical phase I study for treatment of idiopathic
pulmonary fibrosis, solid tumors and lymphoma. However, all these inhibitors have varying
degrees of adverse events14-16 and no PI3K/mTOR dual inhibitor has been successfully approved
by FDA. Therefore, the development of PI3K/mTOR dual inhibitors with good clinical activity,
few side effects and low toxicity is extremely urgent.
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HO
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BGT226
Dactolisib
Apitolisib
PF-04691502
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PQR309
H
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LY3023414
Gedatolisib
Omipalisib
Figure 1. Structures of PI3K/mTOR dual inhibitors under clinical trials.
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Masahiko Hayakawa reported a novel PI3Kα inhibitor containing an arylsulfonylhydrazide-
substituted imidazo[1,2-a]pyridine structure exemplified by PIK-75 (in Figure 2), which showed
significant kinase inhibitory activity with an IC of 0.3 nM. In order to develop a new structural
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class of potent PI3K inhibitors, Okseon Kim discovered HS-173 (in Figure 2, PI3Kα IC = 0.8
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nM) using fragment-growing strategy. They found HS-173 has maintained PI3Kα inhibitory
activity. To avoid hydrolysis of the ester group of HS-173 in the phosphate buffer solution, Yan-
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Hua Fan designed 1i by replacing the C-3 position of the imidazo[1,2-a]pyridine core with an
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amide bond. 1i possesses comparable enzymatic inhibitory activity (in Figure 2, PI3Kα IC = 0.5
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nM) and good anti-proliferative effects on HCT116 and SUN638 cells. Taken together, PI3K
inhibitors containing the structure of imidazo[1,2-a]pyridine demonstrate good inhibitory effect
on both kinase and cancer cell proliferation. In addition, this core still has a large number of sites
can be modified, which interested us a lot.
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Br
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N
PIK-75
HS-173
1i
Figure 2. Structures of PI3K inhibitors containing imidazo[1,2-a]pyridine core.
In this communication, we described our work on designing and synthesizing a series of
imidazo[1,2-a]pyridine derivatives as potent PI3K/mTOR dual inhibitors and the discovery of 15a,
a drug candidate for cancer treatment.
RESULTS AND DISCUSSION
OPTIMIZATION STRATEGY
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According to the docking model of 1i, the nitrogen atom on the pyridyl ring forms a hydrogen
bond interaction with Ser854 in the hinge region. On the basis of maintaining the interaction,
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bioisosterism strategy is employed to replace the linker of 1i. In addition, several sulfonamide
methoxypyridine derivative side chains at the C-6 position and linkers with different substituents
at the C-3 position of the core were brought in. Thus, the basic scaffold was constructed by three
parts which target three active pockets: the affinity binding pocket, the hinge region, and the ribose
binding pocket (Figure 3).
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Hinge Region
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Basic Scaffold
Figure 3. The design strategy based on 1i.
Previous researches13, 18-20 indicated that the sulfonamide methoxypyridine structure is an
essential group for maintaining PI3K inhibitory activity. It forms a hydrogen bond network with
the Trp, Asp and Lys residues in the affinity binding pocket. Generally, compounds with halogen-
substituted benzenesulfonamides, for example, 2,4-difluorophenyl, 4-fluorophenyl, have better
kinase-inhibitory activities than these with alkyl-substituted sulfonamides such as methyl and
cyclopropyl. In addition, alkoxy group and the halogen on the C-2 position of the pyridine can
increase kinase inhibitory activity compared to hydrogen or alkyl group. Therefore, we chose a
phenyl or substituted phenyl group as a substituent for the side chain of the sulfonamide
methoxypyridine.
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Yan-Hua Fan used amide bond as a linker to improve bioavailability compared to HS-173 and
proposed that the introduction of a hydrophilic group can effectively increase the kinase inhibitory
activity. Furthermore, computational modelling showed that replacing amide bond with 1,3,4-
oxadiazole or 1,2,3-triazole will increase a π-π interaction, which may lead to an increase in
activity. Therefore, we tried to extend the hydrophilic group by attaching the amide bond to the
triazole as a linker to solve the solubility problem, and 1,3,4-oxadiazole and 1,2,3-triazole were
also introduced to the core as linkers. Hence, three series of compounds were synthesized.
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Chemistry
Compounds 5a-i were synthesized according to Scheme 1. Compound 1 was prepared starting
with a cyclization reaction between 2-amino-5-iodopyridine and 2-chloro-3-oxopropionic acid
18
ethyl ester. Hydrolysis of ester 1 with NaOH resulted in carboxylic acid derivative 2 and the
corresponding intermediate 3 was obtained through amidation. Intermediate 4 was prepared by
Suzuki reaction between 3 and 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide which was synthesized according to the
1
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reported route , followed by a click reaction to produce 5a-i.
Scheme 1. Synthesis of compounds 5a–ia
F
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O
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S
N
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O
NH
O
NH
N
O
O
O
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O
N
O
O
NH
NH
NH
I
I
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a,b
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c
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N
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5a-i
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Reagents and conditions: (a) NaOH, Ethanol, H O, 80 °C, 0.5 h, then HCl (2 mol/L), 78%; (b)
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Prop-2-yn-1-amine, HATU, DIPEA, THF, 0 °C to RT, 12 h, 91%; (c) 2,4-difluoro-N-(2-methoxy-
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-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide,
Pd(PPh ) ,
3 4
K CO , 1,4-dioxane/H O, 110 °C, 1.5 h, 80%; (d) N -R , CuTC, THF, RT, 12 h, 50-87%.
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Compounds 9a-d were synthesized as shown in Scheme 2. Compound 6 was obtained through
1
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a cyclization reaction between 2-amino-5-bromopyridine and chloroacetaldehyde.18 The new
formed intermediate was treated with 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide by Suzuki coupling to furnish compound 7,
then followed by iodination. TMS acetylenyl group was attached at the C3 position using
Sonogashira coupling reaction to give compound 8. And then, triazole analogues 9a-d were
generated by click reaction with various azide derivatives.
Scheme 2. Synthesis of compounds 9a-da
F
F
F
F
F
F
O
O
O
S
N
S
N
S
N
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N
O
NH
O
TMS
O
NH
NH
N
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O
O
O
Br
a
b,c
d,e
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aReagents and conditions: (a) 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide, Pd(dppf)Cl , K CO , Dioxane, H O, 110 °C,
2
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3
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reflux, 1.5 h, 95%; (b) NIS, CH CN, RT, 18 h, 80%; (c) Trimethylsilylacetylene, PdCl (PPh ) ,
3
2
3 2
PPh , CuI, Et N, THF, 45 °C, 14 h, 71%; (d) TBAF, THF, RT, 30 min, 66%; (e) N -R , CuTC,
3
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THF, RT, 12 h, 60-85%.
The synthetic routes of compounds 15a-r, 18a-e, 22 and 26 were outlined in Scheme 3.
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Ester 10 was synthesized via a cyclization reaction between 2-amino-5-bromopyridine and 2-
chloro-3-oxopropionic acid ethyl ester.18 Hydrazinolysis reaction between 10 and hydrazine
hydrate yielded intermediate 11. Hydrazide 11 was treated with several aldehydes to afford acyl
imine derivatives, which was transformed to compounds 12 and 13 via cyclization. Compound 13
was subjected to electrophilic additions to provide intermediates 14a-f. Then, compounds 12 and
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4a-f were engaged in Suzuki cross-couplings with corresponding boronic esters to afford final
compounds 15a-r, respectively.
Compound 16 was generated from ester 10 via Suzuki cross-coupling. Hydrazinolysis of
compound 16 resulted in intermediate 17, which was converted to target compounds 18a-e through
condensation and cyclization.
Scheme 3. Synthesis of compounds 15a-r，18a–ea
R1
R1
R3
NH2
NH
N
N
N
N
N
N
O
O
O
O
R2
O
O
Br
Br
Br
Br
e
N
a
b,c
d
N
N
N
N
N
N
N
N
N
N
10
11
13
14a-f
15a-r
R1
N
N
O
f,c
Br
e
N
N
12
R1
R4
R4
R4
N
N
NH2
O
O
R3
O
R3
R3
O
NH
e
g
h,c
N
N
N
N
N
N
N
N
N
16
17
18a-e
aReagents and conditions: (a) Hydrazine hydrate, Ethanol, 100 °C, reflux, 13 h, 86%; (b)
Acrolein, Acetic Acid, CHCl , RT, 23 h; (c) IBD, DCM, RT, 14 h, 14-53% over two steps; (d)
3
Nucleophiles, DBU, DCM, RT, 12 h, 33-92%; (e) Aryl boronic ester, Pd(dppf) Cl , K CO ,
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Dioxane/H O, 110 °C, reflux, 1.5 h, 33-87%; (f) Methyl 3-formylbenzoate, Acetic Acid, CHCl ,
2
3
7
0 °C, reflux, 3 h; (g) Hydrazine hydrate, Ethanol, 50 °C, reflux, 13 h, 67%; (h) Aldehydes, CHCl3,
Acetic Acid, 70 °C, reflux, 12 h.
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
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
Compound 19 was prepared starting with ethyl bromopyruvate and 2-amino-5-bromopyridine,21
then functionalized to afford intermediate 21. Suzuki coupling of intermediate 21 with boronic
ester produced the desired product 22.
Compound 23, which was synthesized from 2-chloro-3-oxopropionic acid ethyl ester and 2-
amino-4-bromopyridine,18 was used as an intermediate in the preparation of compound 24.
Electrophilic addition of the resulting compound 24 generated intermediate 25, which underwent
Suzuki cross-coupling to get final compound 26. (Scheme 4)
Scheme 4. Synthesis of compounds 22 and 26a
F
N
N
F
N
N
O
Br
Br
O
N
Br
O
O
O
N
N
a,b,c
N
d
e
N
N
O
N
N
N
N
N
N
N
N
N
19
20
21
22
N
N
N
N
F
N
O
N
O
O
N
F
O
N
N
O
a,b,c
e
N
N
d
N
Br
N
N
O
N
Br
Br
N
N
N
N
23
24
25
26
aReagents and conditions: (a) Hydrazine hydrate, Ethanol, 100 °C, reflux, 2 h, 44-87%; (b)
Acrolein, Acetic Acid, CHCl , 70 °C, reflux, 3 h; (c) IBD, DCM, RT, 14 h, 27-31% over two steps;
3
(
d) Nucleophiles, DBU, DCM, RT, 12 h, 34-64%; (e) Aryl boronic ester, Pd(dppf) Cl , K CO ,
2 2 2 3
Dioxane/H O, 110 °C, reflux, 1.5 h, 52-85%.
2
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2
2
2
2
3
3
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3
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5
5
5
5
5
5
5
5
5
6
Structure Activity Relationship (SAR)
The Kinase-Glo Plus luminescent assay was used for screening compounds with inhibitory
activities against PI3Kα.
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
Firstly, the linkers were screened as shown in Table 1. 5a-5d incorporated nitrogen-containing
heterocycles using amide linkage triazole as a linker. And these four compounds exhibited
improved inhibitory activities, compared to that of HS-173, 1i and PI103. When the substituent
was changed to dimethylamino, the activity of 5e (PI3Kα inhibition at 1 nM = 71%) decreased
slightly, which identified a preference for bulky groups in R . We further changed the heterocycles
1
to hydrophobic groups. 5f-5i containing substituted benzene rings showed a significant increase in
the kinase inhibition, with all the inhibition rate exceeding 85% at 1 nM. By contrast, the 1,2,3-
triazole analogues 9a-9c exhibited a mild decrease in inhibitory activities, and dimethylamino
substituted 9c showed the weakest inhibition. Extending the substituent of 9b by one carbon atom,
9d (PI3Kα inhibition at 1 nM = 80%) showed an improved potency in inhibitory activity. When
using 1,3,4-oxadiazole as a linker, we found that 15a (PI3Kα Inhibition at 1 nM = 80%) had the
best activity. Then these compounds were subject to cytotoxicity assay in HCT116 cells and found
that 5a, 5e and 5i had poor cellular inhibitory activities, probably because of their polarity with
poor permeability in cells. However, 9a-9d displayed promising inhibitory activities, whose IC s
5
0
were lower than 1 μM. Among these compounds, 15a displayed the strongest inhibitory activity
in HCT116 cells with an IC value of 0.01 μM. It was more than 1000 times than those of 18a,
50
22, 26, 15o, 15p, 15q and 15r, which was consistent with the kinase data. Hence, 15a was selected
as a lead compound for further structural optimization.
Table 1. SAR studies of the linkersa
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9
F
F
O
S
O
NH
O
R5
N
N
N
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
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
PI3Kα
Inhibition
%) at 1 nM
b
IC (nM) IC (μM)
PI3Kα
50
50
ClogPc
Compd.
R5
HCT-116
(
HS-173
－
－
－
27
68
19
＞5
0.31
4.40
0.25
0.34
－
3.46
4.27
1.22
1
i
PI103
O
N
N
5
a
N
N
N
80
75
86
74
71
85
88
0.37
－
15.9
3.54
5.84
＞10
1.59
0.49
2.13
2.37
3.95
3.29
4.51
3.53
5.62
5.16
H
N
O
N
5
b
N
N
H
N
N
O
N
O
N
5
c
N
1.90
－
H
N
N
N
O
N
N
5
d
N
H
N
O
N
N
H
5
e
N
N
－
N
N
O
O
O
5
f
N
H
0.20
0.20
N
N
N
N
HO
N
H
5
g
N
O
O
O
5
h
N
H
91
95
0.17
0.28
2.32
13.4
5.67
4.35
N
N
N
N
O
O
2
H N
5
i
N
H
N
N
N
9
a
N
56
65
－
－
0.50
0.44
2.72
3.99
N
N
N
N
9
b
N
N
N
O
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8
9
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
N
9
c
N
32
－
0.20
2.72
N
N
N
N
9d
80
80
0.35
0.20
0.37
0.01
4.31
1.94
N
O
N
N
O
1
5a
N
N
N
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
O
1
5b
N
47
77
70
55
－
－
－
－
0.08
0.13
0.13
0.14
3.52
2.87
4.08
2.72
N
N
O
15c
N
N
N
O
O
N
1
5d
N
N
O
1
5e
N
N
N
a
b
–
stands for “not detected”. The proliferation-inhibitory effects of compounds in HCT116
cells were measured after 72 h-treatment using CCK-8 assay. The IC s were done by 8-dose
5
0
c
response in triplicate. clogP were predicted by Chemdraw.
Initially, hydrophilic groups in R , such as substituted benzene rings, thiophene and alkyl groups
1
were introduced while keeping R and R unchanged. It was found that 15a, 15f and 15h had
3
4
remarkable inhibitory activities. When the pyrrolidine ring was introduced, the kinase inhibitory
activity of 15b distinctly decreased. Replacing the pyrrolidine of 15b with six-membered
piperidine afforded compound 15d and resulted in enhanced inhibitory activity. Replacing the
nitrogen heterocycle by a dimethylamino group, the inhibition rate of 15e had a slight decline.
When the substituent was a methoxy group, the inhibition rate of 15f was 79%, which was
comparable to that of 15a. It was found that the inhibitory activity was much better than 15a when
the meta position of the phenyl ring was a hydrophilic carboxylic acid function (15h), and the
activity decreased significantly by 1.5 times when the carboxylic acid was esterified (15g).
Replacement of substituents by the hydrophobic trifluoromethyl group and the methyl group
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resulted in a distinct loss in the inhibitory activity by 2.7 and 1.7 times compared with the carboxyl
group substitution. Altering the phenyl group to thiophene (18c) resulted in the improved
inhibitory potency of PI3Kα and the inhibition rate was 89%. If the substituent was a hydrophilic
ethyl group (18d) or a methyl group (18e), the inhibitory activities were significantly enhanced to
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
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
8
5% and 83%, which was better than 15a. These results suggested that the introduction of a
hydrophilic group such as 1-methyl-4-ethylpiperazine, meta-benzoic acid, ethyl and methyl as R1
substituent could result in high PI3Kα kinase inhibition.
Table 2. SAR studies of 1,3,4-oxadiazole analoguesa
R1
R4
N
N
O
R3
N
N
N
PI3Kα
Inhibition
%) at 1 nM
IC50
(nM)
PI3Kα
b
IC (μM)
5
0
ClogPc
Compd.
R1
R3
R4
HCT-116
(
HS-173
－
－
－
－
－
－
－
－
－
27
68
19
＞5
0.31
4.40
0.25
0.34
－
3.46
4.27
1.22
1
i
PI103
5a
F
F
F
F
O
O
S
N
N
N
N
N
N
1
CH O
80
47
77
70
0.20
－
0.01
0.08
0.13
0.13
1.94
3.52
2.87
4.08
3
H
F
F
F
F
O
O
S
N
H
1
5b
CH O
3
O
O
S
O
N
H
1
5c
CH O
－
3
O
O
S
N
H
15d
CH O
－
3
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9
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
F
O
O
N
O
S
N
H
1
5e
CH O
55
79
58
－
0.17
－
0.14
0.26
2.38
2.72
2.65
5.05
3
F
F
F
F
F
O
O
O
S
S
N
H
1
5f
CH O
3
O
O
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
O
O
N
H
1
5g
CH O
3
HO
F
O
O
O
O
S
S
S
N
H
1
5h
8a
CH O
89
34
54
72
85
83
71
0.19
－
5.95
＞10
0.11
0.28
0.34
0.08
0.03
4.92
5.97
5.57
4.97
3.78
3.25
2.51
3
F
F
F
O
CF3
N
H
1
CH O
3
F
F
O
N
H
1
8b
CH O
－
3
F
F
F
O
O
O
O
S
S
S
S
N
H
1
8c
CH O
－
3
F
F
F
O
N
H
1
8d
8e
CH2CH3
CH O
0.24
0.23
0.22
3
O
N
H
1
CH3
CH O
3
F
O
O
O
N
N
N
N
S
S
N
H
1
5i
CH O
3
Cl
O
N
H
1
5j
5k
5l
5m
15n
5o
5p
15q
CH O
34
53
78
66
－
－
0.07
0.04
0.49
0.47
2.59
1.77
1.50
1.55
3
CF3
O
O
O
S
S
N
N
N
N
N
N
N
H
1
CH O
3
F
O
N
H
1
CH O
0.30
－
3
NO2
O
O
S
N
H
1
CH O
3
O
O
N
N
N
N
S
CH O
N
25
0
－
－
2.22
＞10
＞10
-0.08
0.17
1.34
3
H
1
CH O
3
NH2
F
O
O
N
N
N
N
S
S
N
H
1
Cl
H
81
0.23
F
F
O
O
N
H
29
－
＞10
1.15
F
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5
6
7
8
9
O
O
N
N
S
N
1
5r
H
H
7
－
＞10
0.72
2
2
6
－
－
－
－
－
－
5
0
－
－
＞10
＞10
1.94
1.94
2
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
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
b
–
stands for “not detected”. The proliferation-inhibitory effects of compounds in HCT116
cells were measured after 72 h-treatment using CCK-8 assay. The IC s were done by 8-dose
50
c
response in triplicate. clogP were predicted by Chemdraw.
To further explore the SAR, we investigated the effects of R , R substituents on the imidazo[1,2-
3
4
a]pyridine scaffold. 15i-15o were synthesized by changing R . When replacing a fluorine on the
4
benzene ring with chlorine, the inhibition rate of 15i was slightly decreased to 71%. When the
substituent on the phenyl ring was removed to para-trifluoromethyl or para-fluorine, the activity
was greatly reduced. 15n, which replaced 2,4-difluorophenyl with a methyl group, showed a 3.2-
fold decrease in potency. When there was only an amino group at R , 15o totally lost its inhibitory
4
effect on PI3Kα kinase. Then the effect of R group was examined. Similarly to the methoxy group,
3
1
5p, whose R group is chlorine-substituted, maintained the activity with an IC of 0.23 nM.
3 50
However, the removal of Cl gave the unsubstituted compound 15q, which had a dramatic loss in
potency. In addition, the compound 15r inhibited the kinase by only 7% after removing the
methoxy group and the substituents of the benzene ring, further indicating that the methoxy and
2
,4-difluorophenyl were essential for inhibitory activity in these compounds.
Finally, 22 and 26 were synthesized by changing the binding sites of the side chains of 15a. The
kinase activity data showed that when the substituent at the C-3 position was transferred to the C-2
position, the inhibition rate of 22 was only 5%. In addition, movement of the substituent from C-
6 to C-7 (26) resulted in completely loss of inhibitory activity on the kinase. Therefore, it was
indicated that the C-3 and C-6 positions were active sites of such compounds.
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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
Therefore, 15a which has the best cellular inhibitory activity was selected to measure the
inhibition of PI3K subtypes and mTOR kinase. The data showed that it had good effects on all
PI3K subtypes and mTOR kinase. The effects were comparable or better than the clinic drug
Copanlisib, which had been demonstrated to be a pan-PI3K/mTOR dual-target inhibitor and
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
2
2
greatly inhibit the proliferation of many cancer cells . 15a was also screened at a 1 μM to detect
its kinase selectivity using DiscoverX’s kinomescan technology. Among 97 kinases tested, 15a
only exhibited good affinity for PIK3CA, PIK3CG and PIK3C2B, suggesting excellent kinase
selectivity (Figure 4, Table S1, S2).
Table 3. Activities of 15a against Class I PI3Ks and mTOR.
IC (nM)
5
0
Compd.
PI3Kα
0.11
PI3Kβ
4.57
PI3Kγ
1.68
PI3Kδ
0.18
mTOR
45a
Copanlisib
1
5a
0.20
0.58
1.20
0.50
21
a
22
The IC of Copanlisib against mTOR was cited from reference .
5
0
PI3K/Akt/mTOR signaling pathway is one of the most frequently mutant pathways occurring in
2
3
many cancers. Copanlisib has been shown to have more potent inhibitory activity in PIK3CA
mutant and/or HER2 overexpression cells lines compared with HER2-negative and wild-type
2
2
PIK3CA cancer cell lines. Therefore, some cell lines with PIK3CA mutation (MCF-7, HT-29,
HCT116), Her2 overexpression (LOVO) and PTEN loss (PC3) were chosen to detect the inhibitory
effects of 15a on cell proliferation using the CCK-8 assay. It was shown that 15a performed well
on all these cell lines with submicromolar IC s. Besides, cytotoxicity of 15a was also investigated
5
0
in normal cells to detect its therapeutic window in cells. The results showed that 15a displayed
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much lower cytotoxicity in HUVEC cells with an IC of 15.53 µM, which is several hundreds or
5
0
thousands times higher than that of the cancer cells detected (Table 4). These suggested that 15a
could inhibit cancer cell proliferation at concentration without affecting normal cells. Hence, 15a
was chosen for further antitumor researches.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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Table 4. Anti-proliferative activities of various cell lines. a
IC (µM)
5
0
Compd.
HCT116
0.01
HT-29
0.052
0.95
MCF-7
0.04
PC-3
0.071
ND
LOVO
0.0052
0.053
HUVEC
15.53
15a
Copanlisib
0.12
0.01
35.42
a
The proliferation-inhibitory effects of compounds in indicated cells were measured after 72 h-
treatment using CCK-8 assay. The IC s were done by 8-dose response in triplicate. ND: not
5
0
detected.
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Figure 4. Kinase-selectivity profiling of compound 15a at a concentration of 1 μM
DiscoveRX). A. TREEspot interaction maps for compound 15a against 97 kinases. B. Only PI3K
(
isoforms were hits (mTOR is not included in the screening.). Data for binding interaction are
reported as % control, where low figures stand for stronger hits. S-score is a quantitative indicator
of compound selectivity.
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Then the intracellular PI3K/mTOR pathway inhibitory activities of 15a were evaluated by
Western blot analysis. 15a was observed to inhibit PI3K/Akt/mTOR pathway inside the cancer
cells, as suggested by the dose-dependent decrease of phosphorylation of Akt and its downstream
target P70S6K in HCT116 and HT-29 cell lines (Figure 5). There is a little difference of Akt vs
p70S6K response between HCT116 and HT-29 cells. 15a has more effects on Akt phosphorylation
than p70S6K phosphorylation in HCT116 cells while the situation is contrary in HT-29 cells. The
phenomena might be caused by different cell context of the two cells. They have different gene
mutations. For example, HCT116 cells have KRAS mutation, while HT-29 cells have wild type
KRAS. This kind of difference might have some effects on the sensitivity of the cells to the same
compound. Of course, the accurate mechanism needs further investigation. Meanwhile, Akt
protein level was observed to decrease at 1 µM. The reason might be that many cells underwent
apoptosis and autophagy at that concentration, which led to degradation of proteins including Akt.
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Figure 5. Effects of 15a on pAKT, AKT, p-P70S6K and P70S6K in HCT116 and HT-29 cells.
HCT116 cells and HT-29 cells were treated at the indicated concentrations of 15a for 24 h. The
expression levels of AKT, p70S6K and their phosphorylated forms were analyzed by Western
blotting. GAPDH was used as a loading control.
Docking Modeling
Docking analysis were carried out to examine the binding modes of 15a in the PI3Kα isoform
and mTOR using the AutoDock software (Figure 6). The docking results showed that compound
1
5a and PI3Kα formed four conserved hydrogen bonds, one of which formed between the nitrogen
atom on the imidazo[1,2-a]pyridine ring in the hinge region and Val851. More importantly, the
other two were formed in the affinity pocket. The nitrogen atom on pyridine ring of the side chain,
Asp810, Tyr836 and water formed a hydrogen bond. And the methoxy oxygen atom on the
pyridine ring interacted with Lys802 by another hydrogen bond. Finally, the nitrogen atom on the
N-methylpiperazine ring of compound 15a formed a conserved hydrogen bond with Asn853 in the
Ribose binding pocket region. In addition to the hydrogen bond interaction, the linker 1,2,3-
oxadiazole of the compound 15a also formed a π-π interaction with Trp780. The analysis of 15a
docked into the mTOR model indicated that hydrogen bonds between the nitrogen atom on the
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core and Val2240, nitrogen atom on the N-methylpiperazine ring and Thr2245 were formed and it
remained the π-π interaction of the oxadiazole ring. The proposed binding modes supported the
observed inhibitory activity of 15a to PI3Kα and mTOR kinases.
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Figure 6. Proposed binding modes between compound 15a and the PI3Kα (left, PDB code:
4
JPS) and mTOR (right, PDB code: 4JT6) models.
Pharmacokinetic Analysis
15a was further progressed into in vivo pharmacokinetic studies at an intravenous dose of 1
mg/kg and at an oral dose of 5 mg/kg in rats. Moderate plasma clearance was found after
intravenous administration with CL < 1000 mL h−1 kg−1 (Figure 7A, Table 5). Good oral
plasma exposures (AUC0−∞>3000 h·ng/mL) and acceptable oral bioavailability (26.8 %) were
achieved in this species (Figure 7A, Table 5). To investigate the distribution of 15a in tumors,
plasma and tumor concentration at 24 h and 48 h after a single p.o. administration of 15a at 20
mg/kg in nude mice bearing HCT116 human colorectal cancer xenografts had been measured.
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The results showed that 373 ng/mL (about 0.6 µM) 15a was reserved in tumor at 48 h after
administration (Figure 7B).
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Figure 7. Pharmacokinetic/pharmacodynamic evaluations of 15a in SD rats and HCT116 human
colorectal cancer xenografts in nude mice. (A) Mean plasma concentration versus time curves
after single intravenous and oral dosing of 15a to male SD rats (n=3). (B) Plasma and tumor
concentrations of 15a formulated in vehicle containing 5% DMSO, 45% PEG400 following a
single p.o. administration of 20 mg/kg (2 animals/group) in HCT116 xenografts in nude mice.
Table 5. Pharmacokinetic Data for Compound 15a in Rats.
I.V. (1 mg/kg)a
P.O. (5 mg/kg)b
T1/2
CL
Vss
AUC0−∞
T1/2
Cmax
(ng/mL)
AUC0−∞
F%
−
1
−1
(h) (mL h kg ) (mL/kg) (h·ng/mL) (h)
(h·ng/mL)
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339.6
690
2958
7.94
553
3965
26.8
a
b
I.V. formulation: 5% DMSO and 5% Solutol in saline. P.O. formulation: 0.5% CMC. 3 rats
for i.v. and 3 rats for p.o..
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Anti-Tumor Assay In Vivo
Based on the promising biochemical, cellular and pharmacokinetic properties, 15a was subject
to anti-tumor evaluation in vivo. HCT116 and HT-29 xenograft model of BALB/c nude mice were
established and treated with vehicle or 15a by p.o. administration daily. Copanlisib (i.p. injection)
was used as a positive control. As shown in Figure 8, both Copanlisib and 15a significantly
decreased HCT116 tumor growth with tumor growth inhibitions (TGIs) of 57.89 % and 65.77 %,
respectively. Meanwhile, Copanlisib and 15a had no obvious effects on body weight. To further
confirm the effects of 15a on PI3K in vivo, immunohistochemical analysis and HE staining of the
HCT116 tumors collected at the end of the animal studies were performed. As shown in Figure
8
E, the staining of Ki-67, a cell proliferation maker, decreased obviously in tumor slices of 15a-
and Copanlisib-treated mice. Meanwhile, down-regulation of p-AKT levels (indicated by the
brown staining) were also observed. Besides, more massive patchy necrosis in tumors was found
with even eosin-stained proteins in 15a- and Copanlisib-treated groups. Similarly, 15a (20 mg/kg,
p.o.) and Copanlisib (15 mg/kg, i.p.) also greatly suppressed HT-29 tumor growth compared with
control, with TGIs of 68.26% and 60.67%, respectively (Figure 9A, C, D). Besides, 15a hadn’t
caused visible loss of body weight (Figure 9B). Although the in vivo anti-tumor effects are
promising and no obvious decrease of body weight can be observed, liver injury was observed in
our animal studies (Figure S1). The type of liver toxicity, effects on liver enzymes levels and the
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mechanism of inducing toxicity are still under investigation. Therefore, structure modification
should be done to find some new compounds with reserved activity and reduced toxicity.
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Figure 8. 15a inhibited tumor growth of HCT116 xenograft in female BALB/c nude mice. (A)
Tumor volume changes following the treatment of 15a at a dose of 15 mg/kg (p.o., daily) and
Copanlisib (i.p., every second day) at a dose of 15 mg/kg beginning on the 5th day after
inoculation. (B) Average body weights for 15a, Copanlisib and vehicle-treated mice groups. (C)
Tumors removed from vehicle, Copanlisib and 15a-treated mice groups. (D) Tumor weight of
vehicle, Copanlisib and 15a-treated mice groups. (E) Immunohistochemical staining of Ki67 and
p-AKT(S473) and HE staining in tumor tissues from 15a-treated mice and vehicle groups.
Results are expressed as the mean ± SEM (n = 6 for each group), and analyzed by one-way
ANOVA analysis, ****P < 0.0001 vs vehicle.
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Figure 9. 15a inhibited tumor growth of HT-29 xenograft in female BALB/c nude mice. (A)
Tumor volume changes following the treatment of 15a at doses of 10 mg/kg and 20 mg/kg (p.o.,
daily) and Copanlisib (i.p., every second day) at a dose of 15 mg/kg beginning on the 8th day
after inoculation. (B) Average body weights for 15a, Copanlisib and vehicle-treated mice groups.
(
C) Tumors removed from vehicle, Copanlisib and 15a-treated mice groups. (D) Tumor weight
of vehicle, Copanlisib and 15a-treated mice groups. Results are expressed as the mean ± SEM (n
7 for each group), and analyzed by one-way ANOVA analysis, ****P < 0.0001 vs vehicle,
**P<0.001 vs vehicle.
CONCLUSION
=
*
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In conclusion, a new series of imidazo[1,2-a]pyridine derivatives as potent PI3K/mTOR dual
inhibitors was discovered and developed, which led to the discovery of 15a. The tested results
provided a lead compound 15a, which showed a significant inhibitory effect on both PI3K and
mTOR kinases. Furthermore, 15a displayed excellent kinase selectivity, good PK properties and
satisfactory anti-tumor activity in vitro and in vivo, which might be a potential drug candidate for
cancer research. However, 15a also induced obvious liver injury. Hence, our future efforts will be
paid on finding well-tolerated 15a derivatives with good activities.
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EXPERIMENTAL SECTION
Chemistry. All solvents and reagents were used from commercial sources without further
purification. Flash column chromatography was performed using silica gel from Qingdao Haiyang.
1
13
H NMR and C NMR data were recorded on a Bruker Ascend-400 spectrometer using solvent
residual as an internal standard. Chemical shifts are performed in ppm (δ), coupling constants (J)
are in hertz (Hz). ESI-MS spectra were obtained on Bruker micrOTOF-Ⅱ spectrometer. The
purities of final compounds (>95%) were determined by HPLC using Thermo Scientific UltiMate
3
000 HPLC or Waters e2695 (254 nm detection) with a 1.0 mL/min flow rate using a 5−95%
gradient of acetonitrile/water with 0.1% HCOOH over 30 min.
Procedure A for Synthesizing 6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-
3
-yl)-N-((1-(2-(4- methylpiperazin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1, 2-
α]pyridine- 3-carboxamide (5a). To a solution of 2-amino-5-iodopyridine (508 mg, 2.31 mmol)
in 95% EtOH (10 mL), 2-chloro-3-oxopropionic acid ethyl ester (878 mg, 5.27 mmol) was added,
followed by concentrated sulfuric acid (453 mg, 4.62 mmol) added dropwise. The solution was
stirred at room temperature until becoming white. Then, it was stirred at 100 °C for 24 h under
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reflux. After that, the white solid was removed by filtration, and the mixture was concentrated and
diluted with dichloromethane (20 mL), and washed twice with H O (30 mL). The organic phase
2
was extracted and dried (Na SO ). The reaction mixture gave a brown solid after concentration.
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Dichloromethane (5 mL) and petroleum ether (10 mL) were used to recrystallize to give a white
1
solid 1 (461 mg, 63%). H NMR (400 MHz, CDCl ) δ 9.57 (s, 1H), 8.21 (s, 1H), 7.58 (d, J = 9.4
3
1
3
Hz, 1H), 7.51 (d, J = 9.4 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.2 Hz, 3H). C NMR (101
MHz, CDCl ) δ 160.34, 146.89, 141.22, 135.46, 132.56, 118.74, 115.69 (2C), 60.72, 14.42.
3
A solution of 1 (461 mg, 1.46 mmol), NaOH (584 mg, 14.6 mmol) in EtOH (10 mL) and H O
2
(
10 mL) was stirred at 100 °C for 30 min. The reaction mixture was concentrated and H O (15
2
mL) was added. Adjust the solution to pH 5-6 with 1M hydrochloric acid. White solid was
precipitated, which was filtered and dried to give a white solid (393 mg, 78%). Then the solid
(393 mg, 1.14 mmol) was suspended in THF (15 mL). The mixture was stirred at 0 °C for 10 min
after adding DIPEA (293 mg, 2.27 mmol). Then the mixture was stirred for another 20 min after
adding HATU (474 mg, 1.25 mmol). After adding propargylamine (75 mg, 1.36 mmol), the
mixture was naturally warmed to room temperature and stirred for 13 hours. Petroleum ether (15
mL) was added to the reaction mixture to give a solid, which was filtered to give a white solid.
The solid was dissolved in H O (15 mL) and stirred on a stirrer for 15 min, filtered, and dried to
2
1
give white powder 3 (335 mg, 91%). H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 9.05 (t, J =
5
.7 Hz, 1H, NH), 8.34 (s, 1H), 7.68 (d, J = 9.1 Hz, 1H), 7.59 (d, J = 9.3 Hz, 1H), 4.13 (d, J = 3.1
1
3
Hz, 1H), 3.21 (s, 1H). C NMR (101 MHz, DMSO-d6) δ 159.51, 145.43, 137.01, 134.39, 131.90,
1
18.50, 117.40, 81.01, 78.51, 73.15, 27.70.
To an 50 mL reaction bottle, 3 (335 mg, 1.03 mmol), 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide (483 mg, 1.13 mmol),
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potassium carbonate (426 mg, 3.09 mmol), Pd (PPh ) (60 mg, 0.05 mmol) in 1, 4-dioxane (15
3
4
mL) and H O (3 mL) was added. The mixture was stirred at 110 °C for two hours under a nitrogen
2
atmosphere. The mixture was filtered by using the diatomaceous earth, then concentrated in
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vacuum. The crude product was purified by silica gel column chromatography (DCM: MeOH =
1
5
7
0:1) to give 4 (298 mg, 58%). H NMR (400 MHz, CDCl ) δ 9.59 (s, 1H), 8.28 (s, 1H), 8.09 –
3
.98 (m, 2H), 7.92 (s, 1H), 7.75 (d, J = 9.3 Hz, 1H), 7.55 (t, J = 5.6 Hz, 1H, NH), 7.50 (d, J = 9.3
Hz, 1H), 7.11 (t, J = 8.4 Hz, 1H), 6.95 (t, J = 10.4 Hz, 1H), 5.31 (s, 1H), 4.29 (d, J = 3.0 Hz, 2H),
1
3
3
.91 (s, 3H). C NMR (101 MHz, CDCl ) δ 166.22 (dd, J = 258.7, 11.6 Hz), 160.34, 159.81 (dd,
3
J = 259.2, 12.8 Hz), 154.44, 146.73, 140.17, 136.81, 132.99 (d, J = 10.6 Hz), 127.05, 126.74,
126.33, 125.27, 124.54, 123.12 (dd, J = 13.6, 3.7 Hz), 120.70, 118.30, 117.49, 112.35 (dd, J =
2
2.0, 3.5 Hz), 105.76 (t, J = 25.5 Hz), 79.85, 71.53, 54.12, 28.88.
Compound 4 (298 mg, 0.60 mmol) was dissolved in THF (15 mL), 1-(2-azidoethyl)-4-
methylpiperazine (112 mg, 0.66 mmol), CuTC (11 mg, 0.06 mmol) were added and stirred under
nitrogen atmosphere for 12 hours. The reaction mixture was concentrated and purified using silica
gel column chromatography (DCM: MeOH = 30:1) to result in the white product 5a (320 mg,
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0%), m.p. =110 –112 °C. H NMR (400 MHz, DMSO-d ) δ 9.69 (s, 1H, NH), 9.15 (s, 1H), 8.42
6
(s, 1H), 8.17 (s, 1H), 8.04 (s, 1H), 7.94 – 7.77 (m, 3H), 7.76 – 7.66 (m, 1H), 7.57 – 7.40 (m, 1H),
7
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.28 – 7.11 (m, 1H), 4.58 (d, J = 5.7 Hz, 2H), 4.45 (t, J = 6.4 Hz, 2H), 3.72 (s, 3H), 2.73 (t, J =
1
3
.4 Hz, 2H), 2.48 – 2.32 (m, 8H), 2.15 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 164.44 (dd, J
= 252.3, 11.0 Hz), 160.09, 159.16 (dd, J = 256.6, 13.6 Hz), 157.29, 145.94, 144.78, 138.81,
1
37.38, 131.82 (d, J = 10.6 Hz), 129.86, 126.44, 126.30, 125.99, 124.05, 123.96, 123.72, 123.46,
18.26, 117.40, 111.55 (dd, J = 21.9, 3.9 Hz), 105.56 (t, J = 26.0 Hz), 56.64, 53.97 (2C), 53.23,
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Journal of Medicinal Chemistry
Page 28 of 73
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+
5
1.41 (2C), 46.62, 44.50, 34.07. HRMS (ESI) m/z calcd. for C H N O F S (M+H) 667.2375,
30 33 10 4 2
found: 667.2335. Purity: 98.94%.
Compounds 5b−5i were prepared by a procedure similar to that described for the synthesis of
5a.
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-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-N-((1-(2- (pyrrolidin-1-
yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-3- carboxamide (5b). M.p.
1
=
207 –210 °C. H NMR (400 MHz, CDCl ) δ 9.59 (s, 1H, NH), 8.24 (s, 1H), 8.18 (t, J = 5.9 Hz,
3
1
H), 8.07 (d, J = 2.3 Hz, 1H), 8.00 (q, J = 7.8 Hz, 1H), 7.92 (s, 1H), 7.84 (s, 1H), 7.69 (d, J = 9.3
Hz, 1H), 7.43 (d, J = 9.2 Hz, 1H), 7.05 (t, J = 8.5 Hz, 1H), 6.96 (t, J = 8.9 Hz, 1H), 4.76 (d, J =
.6 Hz, 2H), 4.51 (t, J = 6.7 Hz, 2H), 3.91 (s, 3H), 2.99 (t, J = 6.7 Hz, 2H), 2.56 (s, 4H), 1.75 (s,
5
1
3
4H). C NMR (101 MHz, CDCl ) δ 166.16 (dd, J = 258.6, 11.7 Hz), 160.71, 159.82 (dd, J =
3
2
1
59.3, 12.9 Hz), 154.51, 146.69, 144.82, 140.29, 137.11, 132.89 (d, J = 10.8 Hz), 127.02, 126.71,
26.60, 125.23, 124.20, 123.36, 123.20, 120.70, 118.58, 117.59, 112.19 (dd, J = 20.2, 3.7 Hz),
105.72 (t, J = 25.4 Hz), 55.45, 54.06 (3C), 49.41, 34.52, 23.53 (2C). HRMS (ESI) m/z calcd. for
+
C H N O F S (M+H) 638.2110, found: 638.2067. Purity: 99.82%.
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4 2
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-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-N-((1-(2-
morpholinoethyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-3-
carboxamide
1
(5c). M.p. =200 –205 °C. H NMR (400 MHz, CDCl ) δ 9.60 (s, 1H, NH), 8.40 (s, 1H), 8.28 (s,
3
1
H), 8.07 (s, 1H), 8.03 – 7.95 (m, 1H), 7.91 (d, J = 15.9 Hz, 2H), 7.69 (d, J = 9.3 Hz, 1H), 7.45
(
d, J = 9.3 Hz, 1H), 7.03 (t, J = 8.5 Hz, 1H), 6.96 (t, J = 9.4 Hz, 1H), 4.77 (d, J = 5.8 Hz, 2H),
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4
.49 (t, J = 6.7 Hz, 2H), 3.88 (s, 3H), 3.66 (s, 4H), 2.84 (t, J = 6.6 Hz, 2H), 2.50 (s, 4H). C
NMR (101 MHz, CDCl ) δ 166.11 (dd, J = 258.5, 11.5 Hz), 160.79, 159.82 (dd, J = 259.1, 12.9
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Hz), 154.68, 146.65, 144.98, 140.27, 137.15, 132.83 (d, J = 10.7 Hz), 126.98, 126.95, 126.62,
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25.16, 124.21, 123.47, 123.34, 120.78, 118.57, 117.57, 112.11 (dd, J = 22.1, 3.8 Hz), 105.71 (t,
J = 25.4 Hz), 66.79 (2C), 57.81, 53.99, 53.44 (2C), 47.49, 34.47. HRMS (ESI) m/z calcd. for
1
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0
+
C H N O SF (M+H) 654.2059, found: 654.2061. Purity: 98.96%.
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5
2
6
-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-N-((1-(2- (piperidin-1-
yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-3- carboxamide (5d). M.p.
1
=120 –124 °C. H NMR (400 MHz, CDCl ) δ 9.60 (s, 1H, NH), 8.30 (d, J = 15.5 Hz, 2H), 8.06
3
(s, 1H), 8.00 (q, J = 8.5 Hz, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.43 (d, J =
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.1 Hz, 1H), 7.05 (t, J = 9.2 Hz, 1H), 6.96 (t, J = 9.2 Hz, 1H), 4.76 (d, J = 5.7 Hz, 2H), 4.48 (t, J
= 6.5 Hz, 2H), 3.90 (s, 3H), 2.79 (t, J = 6.6 Hz, 2H), 2.52 – 2.35 (m, 4H), 1.60 – 1.46 (m, 4H),
13
1
1
1
.44 – 1.32 (m, 2H). C NMR (101 MHz, CDCl ) δ 166.16 (dd, J = 258.5, 11.5 Hz), 160.70,
3
59.82 (dd, J = 258.9, 13.1 Hz), 154.49, 144.81, 140.28, 137.14, 132.90 (d, J = 10.5 Hz), 126.96,
26.66, 126.61, 125.20, 124.21, 123.46, 123.28 (dd, J = 13.8, 3.8 Hz), 120.65, 118.57, 117.60,
112.19 (dd, J = 22.0, 3.2 Hz), 105.72 (t, J = 25.5 Hz), 58.14, 54.48, 54.04 (2C), 47.88, 34.51,
+
2
6
5.84 (2C), 24.04. HRMS (ESI) m/z calcd. for C H N O F S (M+H) 652.2266, found:
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52.2269. Purity: 99.22%.
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-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-N-((1-(2-
(dimethylamino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-3-
1
carboxamide (5e). M.p. =88 –89 °C. H NMR (400 MHz, CDCl ) δ 9.59 (s, 1H, NH), 8.35 (s,
3
1
H), 8.27 (s, 1H), 8.06 (s, 1H), 8.00 (q, J = 7.8 Hz, 1H), 7.92 (s, 1H), 7.88 (s, 1H), 7.68 (d, J =
9.3 Hz, 1H), 7.43 (d, J = 9.1 Hz, 1H), 7.05 (t, J = 8.4 Hz, 1H), 6.96 (t, J = 9.3 Hz, 1H), 4.77 (d,
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J = 5.6 Hz, 2H), 4.48 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 2.81 (t, J = 6.4 Hz, 2H), 2.28 (s, 6H). C
NMR (101 MHz, CDCl ) δ 164.67 (dd, J = 253.8, 11.0 Hz), 160.75, 159.19 (dd, J = 254.3, 12.4
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