Discovery of [1,2,4]Triazolo[4,3- a]pyridines as Potent Inhibitors Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

Article abstract of DOI:10.1021/acs.jmedchem.9b00312

Inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction using small-molecule inhibitors is an emerging immunotherapeutic approach. A novel series of [1,2,4]triazolo[4,3-a]pyridines were designed and found to be potent inhibitors of the PD-1/PD-L1 interaction. Among them, compound A22 exhibited the most potent activity, as assessed by homogenous time-resolved fluorescence assay, with an IC₅₀ of 92.3 nM. Furthermore, A22 dose-dependent elevated interferon-γproduction in a co-culture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. We concluded that A22 is a promising lead compound for the development of inhibitors of the PD-1/PD-L1 interaction. In addition, we explored the structure-activity relationships of the newly synthesized [1,2,4]triazolo[4,3-a]pyridines and demonstrated that a ring fusion strategy can be employed for designing analogues of the Bristol-Myers Squibb chemical series. These studies pave the way for future drug design.

Full text of DOI:10.1021/acs.jmedchem.9b00312

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Article
Discovery of [1,2,4]Triazolo[4,3-a]pyridines as Potent Inhibitors Targeting
the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction
Mingze Qin, Qi Cao, Shuaishuai Zheng, Ye Tian, Haotian Zhang,
Jun Xie, Hongbo Xie, Yajing Liu, Yanfang Zhao, and Ping Gong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00312 • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 10, 2019
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Discovery of [1,2,4]Triazolo[4,3-a]pyridines as Potent Inhibitors Targeting the
Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction
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Mingze Qin,*,† Qi Cao, Shuaishuai Zheng, Ye Tian, Haotian Zhang, Jun Xie,
†
†
†
‡
‡
Hongbo Xie, Yajing Liu, Yanfang Zhao, and Ping Gong*, †
§
†
*, †
†Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, 103 Wenhua Road, Shenyang 110016, PR China
‡Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, PR China
§College of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150086,
PR China
ABSTRACT
Inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand
1
(PD-L1) interaction using small-molecule inhibitors is an emerging
immunotherapeutic approach. A novel series of [1,2,4]triazolo[4,3-a]pyridines were
designed and found to be potent inhibitors of the PD-1/PD-L1 interaction. Among
them, compound A22 exhibited the most potent activity, as assessed by homogenous
time-resolved fluorescence (HTRF) assay, with an IC of 92.3 nM. Furthermore, A22
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dose-dependently elevated IFN-γ production in a co-culture model of
Hep3B/OS-8/hPD-L1 and CD3 T cells. We concluded that A22 is a promising lead
compound for the development of inhibitors of the PD-1/PD-L1 interaction. In
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addition, we explored the structure-activity relationships of the newly synthesized
[1,2,4]triazolo[4,3-a]pyridines, and demonstrated that a ring fusion strategy can be
employed for designing analogues of the Bristol-Myers Squibb (BMS) chemical
series. These studies pave the way for future drug design.
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Key words: PD-1/PD-L1 interaction; rational design; structure-activity relationship;
IFN-γ production.
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INTRODUCTION
Immune checkpoint blockade is considered one of the most promising strategies
in the field of immunotherapy.1,2 Programmed cell death-1 (PD-1, also known as
CD279) is a cell surface receptor mainly expressed on different kinds of immune
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system cells, particularly cytotoxic T cells. The interaction of PD-1 with its ligands,
programmed cell death-ligand 1 (PD-L1, CD274) or PD-L2 (CD273) suppresses the
proliferation of T cells and the expression of inflammatory cytokines such as IL-2 and
IFN-γ.5–8 This intrinsic negative feedback system plays an important role in
preventing excessive activation of T cells and in maintaining the homeostasis of
immune system. PD-L1 is expressed by T cells, B cells, dendritic cells, macrophages,
3
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as well as tumor cells. Notably, PD-L1 upregulation is found in almost 30% of solid
and hematologic tumors, including melanoma, non-small cell lung cancer (NSCLC),
renal cancer, and others.6,9–13 Tumor cells utilize the PD-1/PD-L1 regulatory
mechanism to avoid immunologic surveillance, which in turn facilitates their own
proliferation.
Blocking the PD-1/PD-L1 interaction can restore the immune system response
against tumors. To date, five humanized monoclonal antibodies (mAbs) targeting
either PD-1 (nivolumab, pembrolizumab) or PD-L1 (avelumab, atezolizumab,
durvalumab) have gained the U.S. Food and Drug Administration (FDA) acceptance
and exhibited durable antitumor responses in a subset of patients with different
cancers.5,14 However, intrinsic disadvantages of antibody therapy such as high cost,
immune-related side effects, and the lack of oral bioavailability, limit their
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applications. Thus, development of small-molecule inhibitors of the PD-1/PD-L1
pathway is a promising alternative for treatment of tumor patients expressing high
levels of PD-L1.30
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A few series of small-molecule peptides and peptidomimetics targeting the
PD-1/PD-L1 pathway have been reported.15 CA-170 is a first-in-class, orally
bioavailable small-molecule inhibitor of PD-L1, which is currently under evaluation
in a phase-1 clinical trial in patients with advanced solid tumors and lymphomas
(NCT02812875). In addition to PD-L1, it also inhibits v-domain
immunoglobulin-containing suppressor of T cell activation (VISTA) immune
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checkpoint. The chemical structure of CA-170 has not been disclosed, and it is
speculated to be a peptidomimetic.
Recently, scientists from Bristol-Myers Squibb (BMS) disclosed the first class of
non-peptide small-molecule inhibitors of the PD-1/PD-L1 interaction in patent
applications, as exemplified by compounds 1, 2, and 6 (Figure 1).17,18 These
compounds showed excellent inhibition of the PD-1/PD-L1 interaction in the
homogenous time-resolved fluorescence (HTRF) assay. The cocrystal structure of
human PD-L1 complexed with compound 2 was solved, revealing that PD-L1
dimerization was induced upon ligand binding.25 Based on these results, Incyte
investigators also developed PD-1/PD-L1 pathway inhibitors characterized by an
amide fragment, such as compounds 3 and 4.19–21 They showed promising activity at
the biochemical level, further evaluation of these inhibitors in more experimental
models are warranted. Considering the above-described inhibitors, as well as those
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designed by other groups, we established a pharmacophoric model for non-peptide
inhibitors of the PD-1/PD-L1 pathway, as shown in Figure 1.
Although the development of small-molecule inhibitors disrupting the
PD-1/PD-L1 interaction is considered an attractive strategy worldwide, its
implementation is still challenging. Pharmacodynamic as well as pharmacokinetic
properties of the aforementioned non-peptide inhibitors should be investigated more
comprehensively to identify a suitable candidate compound. Meanwhile, alternative
inhibitors with a novel scaffold are needed for future drug design.
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Here, we report the discovery of a novel series of [1,2,4]triazolo[4,3-a]pyridines,
which potently inhibited the PD-1/PD-L1 interaction. Using a docking-based ring
fusion strategy, as well as data from a preliminary study of structure-activity
relationships (SARs), we devised [1,2,4]triazolo[4,3-a]pyridine as an attractive
scaffold to develop PD-1/PD-L1 inhibitors. Detailed structural modifications were
conducted accordingly, leading to the identification of compound A22, which
exhibited a high level of inhibitory activity, as assessed by the HTRF assay, with an
IC value of 92.3 nM. In addition, A22 dose-dependently elevated IFN-γ secretion in
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a co-culture model of Hep3B/OS-8/hPD-L1 and CD3 T cells.
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Figure 1. Pharmacophoric model and chemical structures of non-peptide PD-1/PD-L1
pathway inhibitors.
CHEMISTRY
The preparation of compounds A4–6 and A10–31 is described in Scheme 1. The
Suzuki–Miyaura coupling between commercially available reagents 7a and 7b with
phenylboronic
acid
or
2
-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
provided the intermediates 8a–c.19,22 The nucleophilic attack of hydrazine hydrate to
methyl 5-bromo-2-chloronicotinate (9) gave rise to intermediate 10, which then
reacted with trimethyl orthoformate or triethyl orthoformate to yield intermediates
1
1a and 11b. A subsequent hydrolysis reaction of 11a and 11b yielded acids 12a and
2b, respectively. Intermediates 13a–d were prepared by amidation of acids 12a and
1
12b with an appropriate amine selected from 8a–c.19,20 By adopting a similar coupling
methodology between intermediates 13a–d and
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane, intermediates 14a–d were generated,
which in turn were oxidized by sodium periodate to give 15a–d, respectively.19,23 The
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NaBH CN-mediated reductive amination was applied to convert aldehydes 15a–d to
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the desired compounds, A4–6 and A10–31.17,24
Scheme 1. Synthesis of compounds A4–6 and A10–31a
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a
Reagents and conditions: (a) phenylboronic acid, Pd(OAc) , K CO , EtOH/H O
2
2
3
2
(
1/1),
2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane,
PdCl (dppf), K CO , 1,4-dioxane/H O (2/1), 100 °C, 10 h, 62%, or
rt,
overnight,
87%,
or
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2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane,
Pd(PPh ) , Cs CO , toluene/EtOH/H O (10/10/3), 95 °C, 12 h, 59%; (b)
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hydrazine hydrate (80%), 1,4-dioxane, 60 °C, 5 h, 80%; (c) trimethyl orthoformate or
triethyl orthoformate, 100 °C, 10 h, 71–79%; (d) 1N NaOH, MeOH, reflux, 30 min,
8
1–87%; (e) 8a–c, HATU, DIPEA, DMF, rt, overnight, 26–88%; (f)
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane, PdCl (dppf), Cs CO ,
,4-dioxane/H O (20/7), 90 °C, 4 h, 47–62%; (g) OsO , NaIO , 1,4-dioxane/H O
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(
5/1), rt, 3 h, 86–97%; (h) appropriate amine, HOAc, NaBH CN, DMF, rt, 8–24 h,
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6–32%.
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The synthesis of compounds A7–9 is outlined in Scheme 2. The starting material,
2
3
compound 16, was reacted with ethane-1,2-diol to conveniently yield acetal 17. The
conversion of intermediate 17 to 21 was achieved in a four-step synthetic procedure
under conditions similar to those described for the preparation of intermediate 13a
from 9. Compounds A7–9 were obtained by deprotection of 21 in the presence of
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p-toluenesulfonicꢀacid, followed by reductive amination using NaBH CN and
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appropriate amines.
Scheme 2. Synthesis of compounds A7–9a
aReagents and conditions: (a) ethane-1,2-diol, p-toluenesulfonic acid, toluene, reflux,
1
2 h, 67%; (b) hydrazine hydrate (80%), 1,4-dioxane, 60 °C, 7 h, 83%; (c) trimethyl
orthoformate, 100 °C, 12 h, 72%; (d) 1N NaOH, methanol, reflux, 1 h, 76%; (e) 8a,
HATU, DIPEA, DMF, rt, 2 h, 89%; (f) p-toluenesulfonic acid, acetone/H O (3/2), 70
2
°
C, 40 h, 76%; (g) appropriate amine, acetic acid, NaBH CN, DMF, rt, 6–12 h,
3
22–37%.
The synthesis of compounds A1–3 is depicted in Scheme 3. The preparation of
-bromoimidazo[1,2-a]pyridine-8-carboxylic acid (24) was accomplished by
6
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cyclization of 23 with 2-chloroacetaldehyde. A sequential amidation, the
Suzuki–Miyaura coupling, and oxidization reactions were utilized, which readily
converted intermediate 24 to 27. In the last step, compounds A1–3 were prepared
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from 27 under NaBH CN-mediated reductive amination conditions, as described
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above.
Scheme 3. Synthesis of compounds A1–3a
aReagents and conditions: (a) 2-chloroacetaldehyde (40%), EtOH, reflux, 7 h, 82%;
(b)
8a,
HATU,
DIPEA,
DMF,
rt,
3
h,
85%;
(c)
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane,
PdCl (dppf),
K CO ,
2
2
3
1
2
,4-dioxane/H O (4/1), 60 °C, 15 h, 53%; (d) OsO , NaIO , 1,4-dioxane/H O (5/1), rt,
2 4 4 2
h, 92%; (e) appropriate amine, acetic acid, NaBH CN, DMF, rt, 5–10 h, 16–21%.
3
RESULTS AND DISCUSSION
Structure-Based Drug Design. The crystal structure of the 2/PD-L1 complex
(PDB: 5J89) has been solved by Holak’s group and represents the binding mode of
inhibitors disclosed by BMS.25,31 Based on the binding model, compound 2 is located
in a deep, hydrophobic cleft formed by the dimeric PD-L1 protein. The biphenyl
moiety of the inhibitor generates significant hydrophobic π-π, π-alkyl, and π-σ
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interactions, with Tyr56, Met115, and Ala121. The methoxypyridine ring, which is
linked to the biphenyl moiety via an ether bond, substantially contributes to the
inhibitor binding mainly through strong π-π stacking with the Tyr56 residue of one
PD-L1 protein. The N-(2-aminoethyl)acetamide tail is oriented toward the solvent and
interacts with Asp122 and Lys124 through hydrogen bonds (Figure 2A). The
exploration of the 2/PD-L1 complex paves the way for further development of
inhibitors of the PD-1/PD-L1 pathway. The dissection of binding mode of compound
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to PD-L1 revealed a large space between the methoxy group and the pyridine
nitrogen, which could be occupied by an appropriate aromatic ring (Figure 2B). Thus,
a ring fusion strategy could be employed accordingly, based on the chemical structure
of compound 2.
Figure 2. (A) Detailed interactions of compound 2 with the dimeric PD-L1 protein.
(B) Close-up view of compound 2 located in the hydrophobic cleft formed by the
dimeric PD-L1 protein. (C) Docking analysis of A4 with the PD-L1 dimer. (D)
Binding overlap of A4 and 2 in the binding site. The crystal structure of the dimeric
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9
PD-L1 protein was taken from the RCSB Protein Data Bank (PDB: 5J89).
In addition to the ether group, the amide has been proven to be another
privileged linker fragment in the design of PD-L1 inhibitors. Several series of
compounds containing an amide linker were disclosed by Incyte in patent
applications.19–21 Considering the probable advantage in terms of drug-like properties,
the amide was selected as the linker group in our design of new compounds.
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
On the basis of these considerations, we envisioned that a new
[1,2,4]triazolo[4,3-a]pyridine or imidazo[1,2-a]pyridine skeleton could be established.
The biphenyl moiety was retained in our initial work and was connected to a newly
designed scaffold through an amide linker. Compounds A1–9 with selected tail
groups were synthesized and evaluated.
Preliminary Biochemical Evaluation of Compounds A1–9. The activities of
compounds A1–9 as inhibitors of the PD-1/PD-L1 interaction were evaluated by
using the well-established HTRF assay. The biochemical data are presented as IC50
values and listed in Table 1.
Interestingly, [1,2,4]triazolo[4,3-a]pyridine compounds A4–6 exhibited
considerable activity against the PD-1/PD-L1 interaction, with IC values of 733.3,
5
0
7
54.7, and 1487 nM, respectively. When replacing the [1,2,4]triazolo[4,3-a]pyridine
skeleton with an imidazo[1,2-a]pyridine moiety, compounds A1–3 were clearly
inactive at concentrations up to 10 µM. Considering that the hydrogen-bond
interactions created by the hydrophilic tail of the inhibitor strongly contributed to
inhibitor binding, we then investigated the role of tail group orientation in inhibitory
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7
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
activity. Shifting the hydrophilic tails of A4–6 from the 6- to the 5-position of
[1,2,4]triazolo[4,3-a]pyridine gave rise to A7–9. However, a sharply decreased
activity was observed for all three compounds, which clearly indicated that a
hydrophilic group was required at the 6-position of the [1,2,4]triazolo[4,3-a]pyridine
skeleton.
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
Preliminary
biochemical
evaluation
indicated
that
the
[1,2,4]triazolo[4,3-a]pyridine could serve as a privileged scaffold in the design of
PD-1/PD-L1 inhibitors. To further investigate the binding mode of this chemical
series and validate our design strategy, docking analysis of compound A4 with
dimeric PD-L1 was performed. As shown in Figure 2C, A4 fitted well into the
hydrophobic cleft formed by dimeric PD-L1. Regarding the biphenyl moiety, key
interactions such as π-π, π-alkyl, and π-σ interactions with Tyr56, Met115, and
Ala121, were all retained, as for compound 2. The aminoethanol group interacted
with Phe19 and Thr20 through hydrogen bonds. In addition to pyridine, the triazole
moiety also formed π-π stacking and π-anion interactions with Tyr56 and Asp122,
which might be beneficial for the binding of the inhibitor to dimeric PD-L1. Figure
2
D shows that compounds A4 and 2 could be well superimposed.
Table 1. Activities of compounds A1–9 in inhibition of PD-1/PD-L1
interaction.
X
N
N
5
H
R1
6
N
O
A1-9
Compd
X
R1
PD-1/PD-L1
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9
a
IC (nM)
5
0
H
N
A1
A2
A3
A4
A5
A6
A7
A8
A9
CH
>10000
>10000
>10000
6
-
-
OH
CH
CH
N
N
6
OH
O
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
H
N
N
6
-
H
H
N
N
733.3 ± 35.2
754.7 ± 27.8
1487 ± 49.3
>10000
6
-
OH
N
6
-
OH
O
H
N
N
N
6
-
H
H
N
N
N
5
-
OH
N
>10000
5-
OH
O
H
N
N
>10000
N
5-
H
aThe data are generated from two independent experiments.
Structural Optimization of [1,2,4]Triazolo[4,3-a]pyridine Precursors. With
the preliminary biochemical results in hand, a detailed structural optimization
campaign was conducted to identify compounds with improved efficacy. At the
beginning of our research, compound 1 could be easily obtained from commercial
sources and thus was used as the positive control.
Compounds A10–19 with diverse hydrophilic groups were prepared to further
investigate the SAR of this region. In line with the preliminary docking analysis of the
A4/PD-L1 complex, the major contribution of the tail group could derive from
hydrogen-bond interactions with PD-L1. Compound A10, containing an amide group,
showed moderate activity against the PD-1/PD-L1 interaction (IC , 2945 nM),
5
0
whereas compound A11, with an ester group, was almost inactive. Similarly,
comparisons of A16 (IC , 5566 nM) with A17 (IC , >10000 nM) and A18 (IC ,
5
0
50
50
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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
5130 nM) with A19 (IC , >10000 nM) indicated that the presence of a
5
0
hydrogen-bond donor resulted in higher efficacy. Compounds A13 and A15,
containing sulfamide and hydroxycyclohexyl fragments, exhibited promising
inhibitory activity, with IC values of 834 and 968.1 nM, respectively. Compound
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
5
0
A12, bearing a dihydroxypropyl tail group, displayed the most potent activity, with an
IC value of 374.6 nM. Preliminary investigations indicated that the optimization of
5
0
the tail group was straightforward, and properties such as solubility and cellular
activity could be fine-tuned by modifying this region.
In order to explore the SAR of triazole, an additional methyl group was
introduced to the triazole moiety of A4 and A14, resulting in compounds A20 and
A21, respectively. However, both compounds exhibited a remarkably decreased
activity, suggesting that further modification of triazole was inappropriate.
Table 2. Activities of compounds A10–21 in inhibition of PD-1/PD-L1
interaction.
R2
N
N
N
H
N
R3
O
PD-1/PD-L1
Compd
A10
R2
H
H
H
H
R3
a
IC (nM)
5
0
O
H
N
2945 ± 112.5
>10000
NH2
O
O
H
N
A11
OH
A12
374.6 ± 13.9
834 ± 28.6
N
OH
H
N
NH2
A13
S
O
O
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9
N
A14
A15
A16
A17
H
1347 ± 36.1
968.1 ± 37.5
5566 ± 138.2
>10000
N
H
N
H
OH
H
N
OH
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
O
OH
OH
H
N
H
H
O
O
N
A18
5130 ± 95.2
O
OH
N
O
A19
A20
H
>10000
>10000
O
H
Me
Me
N
OH
N
A21
1
>10000
N
79.5 ± 8.6
aThe data are generated from two independent experiments.
Also, we focused on the biphenyl moiety of newly designed
[
1,2,4]triazolo[4,3-a]pyridines. Holak et al. demonstrated that binding of the PD-L1
inhibitor 6 to PD-L1 provoked Tyr56 movement, facilitating the formation of a deep,
enlarged hydrophobic tunnel, mainly induced by the 2,3-dihydro-1,4-benzodioxinyl
2
6
group of compound 6. This study prompted us to synthesize compounds A22–31
and examine whether the introduction of the 2,3-dihydro-1,4-benzodioxinyl group
was beneficial to the activity of [1,2,4]triazolo[4,3-a]pyridine-based compounds.
Biochemical evaluation demonstrated that the incorporation of the
2,3-dihydro-1,4-benzodioxinyl moiety had a diverse impact on the activity of the
different compounds. For example, both 2,3-dihydro-1,4-benzodioxinyl analogs, A22
(
IC , 92.3 nM) and A23 (IC , 238.3 nM), containing an aminoethanol group,
5
0
50
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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
displayed significantly improved activity, as compared to the biphenyl compounds,
A4 (IC , 733.3 nM) and A5 (IC , 754.7 nM), respectively. Similar results could be
5
0
50
observed by comparing compound A25 (IC , 565.8 nM) with A6 (IC , 1487 nM).
5
0
50
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
However, compound A27 (IC , 3694 nM), bearing a sulfamide group, was less active
5
0
than A13 (IC , 834 nM). Interestingly, compound A26 (IC , 987.8 nM), with a
5
0
50
reverse sulfamide group, displayed a 3.74-fold higher activity compared to A27.
Further, modifications were conducted by replacing the methyl in the “core
group” with a cyano group to investigate whether the electronic density of this region
was crucial for high activity. Compounds A24 (IC , 473.8 nM) and A29 (IC , 486.2
5
0
50
nM), both bearing a dihydroxypropyl tail, exhibited comparable activity. Similar
results were also observed when comparing A26 (IC , 987.8 nM) with A31 (IC ,
5
0
50
9
83.4 nM). These findings clearly suggested that the electronic density of the “core
group” was not critical for inhibitor activity. Compounds A28 and A30 displayed high
efficacy, with IC values of 274.6 and 367 nM, respectively.
5
0
In this chemical series, compound A22 was identified as the most promising
inhibitor, demonstrating an IC50 of 92.3 nM, which was comparable to that of
compound 1 (IC , 79.5 nM). It should be noted that 1 was unlikely to be the most
5
0
active inhibitor among several hundreds of compounds reported by BMS.
Table 3. Activities of compounds A22–31 in inhibition of PD-1/PD-L1
interaction.
N
N
O
O
N
R4
H
N
R5
O
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9
PD-1/PD-L1
Compd
A22
A23
A24
A25
A26
A27
A28
A29
A30
R4
R5
a
IC (nM)
50
H
Me
Me
Me
Me
Me
Me
CN
CN
CN
CN
N
92.3 ± 6.2
238.3 ± 11.7
473.8 ± 20.1
565.8 ± 45.6
987.8 ± 65
OH
OH
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
OH
N
OH
O
H
N
N
H
O
H
N
S
N
H
O
H
N
NH2
S
3694 ± 213.2
274.6 ± 11.7
486.2 ± 20.3
367 ± 16.8
O
O
H
N
OH
OH
N
OH
O
H
N
N
H
O
H
N
S
A31
1
983.4 ± 57.2
79.5 ± 8.6
N
H
O
aThe data are generated from two independent experiments.
Docking analysis of A22 with dimeric PD-L1. As reported by Holak and
colleagues, the 2,3-dihydro-1,4-benzodioxinyl group of compound 6 could induce a
conformational change, involving a Tyr56 residue in one of the PD-L1 molecules,
forming an enlarged interaction interface.26 Thus, docking analysis of A22 was
conducted by using the 6/PD-L1 complex (PDB: 5N2F) as a template. Figure 3A
shows the interaction of A22 with PD-L1 amino acids. The
2,3-dihydro-1,4-benzodioxinyl and central phenyl groups of A22 established multiple
key interactions with PD-L1, such as π-alkyl interactions with Met115 and π-σ
interactions with Ala121, which were quite similar to those exhibited by compound 6.
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7
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
Concerning the [1,2,4]triazolo[4,3-a]pyridine moiety, face-to-face π-π stacking with
Tyr56 and π-anion interactions with Asp122 were formed by both triazole and
pyridine moieties of A22. The aminoethanol tail of A22 was solvent-accessible and
interacted with Ala121 via two hydrogen bonds.
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
Figure 3. (A) Docking analysis of A22 with the PD-L1 dimer. (B) Binding overlap of
A22 and 6 in the binding site. The crystal structure of the dimeric PD-L1 protein was
taken from the RCSB Protein Data Bank (PDB: 5N2F).
Effect of A22 on IFN-γ production. A great number of studies have
demonstrated that activation of the PD-1/PD-L1 pathway leads to numerous
immunosuppressive effects, including inhibition of T cell proliferation and reduced
secretion of inflammatory cytokines, such as IFN-γ.5–8 On the other hand, blockade of
the PD-1/PD-L1 interaction can enhance T cell response.27–29 A well-established T
cell-tumor co-culture assay was performed to evaluate the effects of A22 on immune
regulation. CD3 T cells isolated from peripheral blood mononuclear cells (PBMCs)
were co-cultured with Hep3B cells stably expressing OS-8 (anti-CD3 single chain
variable fragment) and human PD-L1, followed by the addition of A22 and compound
1
at the indicated concentrations. IFN-γ levels were measured 72 h after stimulation.
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5
6
7
8
9
As shown in Figure 4, A22 significantly elevated the production of IFN-γ in a
dose-dependent manner. In the presence of 5 μM A22, IFN-γ secretion was increased
by 68.95%, which was similar to the effect induced by compound 1 (70.97%). Upon
treatment with 20 μM A22, the IFN-γ level was increased by 205.65%.
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
Figure 4. Effects of compounds 1 and A22 on IFN-γ secretion from T cells
co-cultured with tumor cells. Data are shown as mean ± SEM, *p<0.05, **p<0.01,
*** p<0.001.
CONCLUSIONS
The development of small-molecule inhibitors has emerged as an attractive
approach for the modulation of the PD-1/PD-L1 pathway, but is still in its infancy.
The non-peptide inhibitors identified by scientists from BMS can serve as a promising
starting point. These compounds may possess distinct pharmacokinetic and
pharmacodynamic profiles from peptides, peptidomimetics, and mAbs, and deserve
further investigation. Here, in an attempt to identify potent inhibitors with a novel
scaffold, a docking-based ring fusion strategy was employed. A series of
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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
[1,2,4]triazolo[4,3-a]pyridines were synthesized and their ability to inhibit the
PD-1/PD-L1 interaction was evaluated. Among these chemicals, compound A22
exhibited the most promising activity, with an IC value of 92.3 nM. Furthermore,
5
0
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
A22 significantly promoted T cell response. In a T cell-tumor co-culture assay,
treatment with A22 induced IFN-γ release in a dose-dependent manner. We explored
the SARs of the newly designed [1,2,4]triazolo[4,3-a]pyridines and demonstrated that
a fused ring could be devised for the “aryl group” moiety of the established
pharmacophoric model, thus providing a valuable strategy for the rational design of
PD-1/PD-L1 inhibitors.
Notably, although A22 displayed comparable activity to the BMS compound in
the cellular assay, 1 may not have been the best choice among BMS inhibitors.
Compounds with higher affinities for dimeric PD-L1 would probably be more potent
in cells than A22. In addition, compound toxicity should also be taken into account
when evaluating the cellular activity. Compounds that are active in biochemical
assays may be unsuitable for cell-based assays due to toxicity. More experiments
should be devised to further investigate the immunoregulatory activity and toxicity of
A22.
In conclusion, compound A22 could be employed as a promising lead compound
for the further development of blockers of the PD-1/PD-L1 interaction. We hope that
non-peptide inhibitors will enter oncology clinical trials in the near future.
EXPERIMENTAL SECTION
General methods. Reagents and solvents were obtained from commercial
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sources and used without further purification. All the reactions were monitored by
TLC using silica gel GF/UV 254. Flash chromatography was performed using silica
gel (300–400 mesh). Melting points were determined on a Büchi Melting Point B-540
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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13
apparatus (Büchi Labortechnik, Flawil, Switzerland). The H and C NMR spectra
were recorded on a Bruker AV-400 spectrometer, with TMS as an internal standard.
The low resolution of ESI-MS was recorded on an Agilent 1100 LC-MS spectrometer,
and high-resolution mass spectrometry was performed on an Agilent Accurate-Mass
Q-TOF 6530 in ESI mode. The purity of the synthesized compounds was determined
by high-performance liquid chromatography (HPLC), which was performed on an
Agilent 1260 machine with a C18 reverse-phase column (4.6 mm × 250 mm, 5 μm) at
25 °C. Mobile phase A (100% methanol) and mobile phase B (NaH PO and H PO
2
4
3
4
buffer solution, pH = 2.5) were used in a gradient elution program (0 min, phase A:
%, phase B: 95%; 25 min, phase A: 95%, phase B: 5%) with a flow rate of 1.0
5
mL/min. UV Detection was conducted at 220 nm. The purity of the target compounds
was determined to be higher than 95%.
2-Methyl-[1,1'-biphenyl]-3-amine
-bromo-2-methylaniline (10 g, 54.06 mmol), phenylboronic acid (6.93 g, 56.78
mmol), K CO (19.35 g, 140 mmol) in ethanol (70 mL) and water (70 mL) was added
(8a).
To
a
mixture
of
3
2
3
Pd(OAc) (0.73 g, 3.25 mmol). The mixture was stirred at room temperature
2
overnight under N atmosphere. The catalyst was filtered off and the solvent was
2
removed by evaporation. The residue was dissolved in DCM and washed with brine.
The organic layer was dried and evaporated to afford a crude product, which was
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6
purified by silica gel column chromatography (ethyl acetate/petroleum ether, 1/60 to
1
1
/30) to afford compound 8a (8.63 g, 87.2%). White solid. H NMR (600 MHz,
DMSO-d ) δ 7.40 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 7.2 Hz, 2H),
6
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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4
5
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8
9
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1
2
3
4
5
6
7
8
9
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1
2
3
4
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7
8
9
0
6.93 (t, J = 7.7 Hz, 1H), 6.64 (d, J = 7.9 Hz, 1H), 6.40 (d, J = 7.4 Hz, 1H), 4.92 (s,
2
H), 1.92 (s, 3H). ESI-MS m/z: 184.1 [M+H]+.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylaniline (8b). To a mixture of
3
2
-bromo-2-methylaniline
(1.89
g,
10.21
mmol),
-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(4.02 g, 15.32 mmol), K CO (4.23 g, 30.63 mmol) in 1,4-dioxane (20 mL) and water
2
3
(
10 mL) was added PdCl (dppf) (0.74 g, 1.02 mmol). The mixture was stirred at 100
2
°C for 10 h under N atmosphere. The catalyst was filtered off and the solvent was
2
evaporated. The residue was dissolved in DCM and washed with brine. The organic
layer was dried and evaporated to afford a crude product, which was purified by silica
gel column chromatography (ethyl acetate/petroleum ether, 1/30 to 1/10) to afford
1
compound 8b (1.53 g, 62.1%). White solid. H NMR (600 MHz, DMSO-d ) δ 6.89
6
(dd, J = 15.7, 8.0 Hz, 2H), 6.70 (d, J = 12.1 Hz, 2H), 6.62 (t, J = 9.6 Hz, 1H), 6.38 (d,
J = 7.4 Hz, 1H), 4.86 (s, 2H), 4.27 (s, 4H), 1.93 (s, 3H). ESI-MS m/z: 242.1 [M+H]+.
2
-Amino-6-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzonitrile
(8c).
2-Amino-6-bromobenzonitrile (2 g, 10.21
mmol),
2
-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(2.94 g, 11.22 mmol), Cs CO (6.65 g, 20.4 mmol), and Pd(PPh ) (1.18 g, 1.02 mmol)
2
3
3 4
was added to a mixture of toluene (10 mL), EtOH (10 mL) and water (3 mL). The
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solution was stirred at 95 °C for 12 h under N atmosphere. The catalyst was filtered
2
off and the solvent was concentrated. The residue was dissolved in DCM and washed
with brine. The organic layer was dried and evaporated to afford a crude product,
which was purified by silica gel column chromatography (ethyl acetate/petroleum
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
1
ether, 1/20 to 1/10) to afford compound 8c (1.51 g, 58.6%). White solid. H NMR
(600 MHz, DMSO-d ) δ 7.30 (t, J = 7.9 Hz, 1H), 6.98 (s, 1H), 6.95 (d, J = 1.7 Hz,
6
2
H), 6.76 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 7.4 Hz, 1H), 6.05 (s, 2H), 4.29 (s, 4H).
ESI-MS m/z: 253.1 [M+H]+.
Methyl 5-bromo-2-hydrazinylnicotinate (10). To a mixture of intermediate 9
(12 g, 48.21 mmol) in 1,4-dioxane (60 mL) was added hydrazine hydrate (80%, 12 g,
192 mmol). The mixure was stirred at 60 °C for 5 h when TLC indicated the
completion of the reaction. Being cooled to room temperature, the precipitate was
1
filtered off to afford compound 10 (9.4 g, 79.6%). Yellow solid. H NMR (600 MHz,
DMSO-d ) δ 8.59 (s, 1H), 8.43 (d, J = 1.5 Hz, 1H), 8.14 (d, J = 2.2 Hz, 1H), 4.52 (s,
6
2
H), 3.83 (s, 3H). ESI-MS m/z: 246.0 [M+H]+.
Methyl 6-bromo-[1,2,4]triazolo[4,3-a]pyridine-8-carboxylate (11a). At room
temperature, intermediate 10 (5 g, 20.41 mmol) was added to trimethyl orthoformate
30 mL). The mixture was stirred at 100 °C for 10 h. Upon completion of the reaction,
(
the solution was cooled to room temperature. The precipitate was filtered off, washed,
1
and dried to generate compound 11a (4.09 g, 78.6%). Off-white solid. H NMR (600
MHz, DMSO-d ) δ 9.29 (s, 1H), 9.19 (s, 1H), 8.03 (s, 1H), 3.95 (s, 3H). ESI-MS m/z:
6
2
56.0 [M+H]+.
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Compound 11b was prepared from intermediate 10 in a similar manner as
described for compound 11a, with triethyl orthoformate replacing trimethyl
orthoformate.
0
1
2
3
4
5
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8
9
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Methyl 6-bromo-3-methyl-[1,2,4]triazolo[4,3-a]pyridine-8-carboxylate (11b).
1
White solid. Yield, 71%. H NMR (600 MHz, DMSO-d ) δ 9.01 (s, 1H), 7.99 (s, 1H),
6
3.95 (s, 3H), 2.72 (s, 3H). ESI-MS m/z: 270.0 [M+H]+.
6
-Bromo-[1,2,4]triazolo[4,3-a]pyridine-8-carboxylic acid (12a). To a mixture
of intermediate 11a (1 g, 3.92 mmol) in methanol (10 mL) was added 1N NaOH
aqueous solution (10 mL). The reaction mixture was heated to reflux for 30 min.
Upon completion of the reaction, DCM (5 mL) was added, then the solution was
extracted with water. The aqueous phase was acidized with 1N HCl solution to pH
5
–6. The precipitate was collected via filtration and dried to afford compound 12a
1
(
0.77 g, 81.2%). Yellow solid. H NMR (600 MHz, DMSO-d ) δ 9.27 (s, 1H), 9.15 (s,
6
1H), 7.97 (s, 1H). ESI-MS m/z: 242.0 [M+H]+
Compound 12b was prepared from intermediate 11b in a similar manner as
described for compound 12a.
6
-Bromo-3-methyl-[1,2,4]triazolo[4,3-a]pyridine-8-carboxylic acid (12b).
1
Yellow solid. Yield, 87%. H NMR (600 MHz, DMSO-d ) δ 9.26 (s, 1H), 8.27 (s, 1H),
6
2.78 (s, 3H). ESI-MS m/z: 256.0 [M+H]+
6
-Bromo-N-(2-methyl-[1,1'-biphenyl]-3-yl)-[1,2,4]triazolo[4,3-a]pyridine-8-c
arboxamide (13a). To a solution of 8a (1.5 g, 8.19 mmol) and 12a (2.07 g, 8.6 mmol)
in anhydrous DMF (15 mL) was added HATU (4.67 g, 12.29 mmol), followed by
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DIPEA (2.12 g, 16.38 mmol). The mixture was stirred at room temperature overnight.
Upon completion of the reaction, the mixture was poured into water. The precipitate
was collected via filtration, and was triturated with diethyl ether to give compound
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
1
13a (2.91 g, 87.5%). Off-yellow solid. H NMR (600 MHz, DMSO-d ) δ 11.37 (s,
6
1
H), 9.41 (s, 1H), 9.22 (s, 1H), 8.19 (s,1H), 8.15 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.5
Hz, 2H), 7.38 (td, J = 19.1, 7.6 Hz, 4H), 7.11 (d, J = 7.6 Hz, 1H), 2.34 (s, 3H).
ESI-MS m/z: 407.0 [M+H]+.
Compounds 13b–d were prepared in a similar manner as described for
compound 13a.
6
-Bromo-3-methyl-N-(2-methyl-[1,1'-biphenyl]-3-yl)-[1,2,4]triazolo[4,3-a]py
1
ridine-8-carboxamide (13b). Off-yellow solid. 86% yield. H NMR (600 MHz,
DMSO-d ) δ 11.41 (s, 1H), 9.10 (d, J = 1.6 Hz, 1H), 8.19–8.13 (m, 2H), 7.48 (t, J =
6
7
.5 Hz, 2H), 7.42–7.33 (m, 4H), 7.10 (d, J = 7.6 Hz, 1H), 2.76 (s, 3H), 2.33 (s, 3H).
ESI-MS m/z: 421.0 [M+H]+.
6
-Bromo-N-(3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-[1,2,4]
1
triazolo[4,3-a]pyridine-8-carboxamide (13c). White solid. Yield, 79%. H NMR
(
600 MHz, DMSO-d ) δ 11.34 (s, 1H), 9.41 (s, 1H), 9.22 (d, J = 1.7 Hz, 1H), 8.19 (d,
6
J = 1.7 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 7.6 Hz,
1H), 6.94 (d, J = 8.2 Hz, 1H), 6.85–6.78 (m, 2H), 4.29 (s, 4H), 2.35 (s, 3H). ESI-MS
m/z: 465.0 [M+H]+.
6-Bromo-N-(2-cyano-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-[1,2,4]t
1
riazolo[4,3-a]pyridine-8-carboxamide (13d). White solid. Yield, 26%. H NMR
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2
2
2
2
2
2
2
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3
3
3
3
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3
3
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3
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4
4
4
4
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5
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5
5
6
(600 MHz, DMSO-d ) δ 11.87 (s, 1H), 9.42 (s, 1H), 9.25 (s, 1H), 8.21 (d, J = 8.1 Hz,
6
2
H), 7.80 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 1.7 Hz, 1H), 7.09
dd, J = 8.2, 1.9 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 4.32 (s, 4H). ESI-MS m/z: 476.0
[M+H]+.
N-(2-Methyl-[1,1'-biphenyl]-3-yl)-6-vinyl-[1,2,4]triazolo[4,3-a]pyridine-8-car
boxamide (14a). To mixture of 13a (2.3 g, 5.66 mmol),
,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (0.96 g, 6.23 mmol) in 1,4-dioxane
20 mL) and water (7 mL) was added PdCl (dppf) (0.42 g, 0.57 mmol), followed by
(
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
4
(
2
cesium carbonate (3.32 g, 10.2 mmol). The mixture was stirred at 90 °C for 4 h under
N atmosphere. The solution was diluted with water and extracted with ethyl acetate.
2
The organic extract was washed with brine, dried, and evaporated to give a crude
product, which was purified by silica gel column chromatography (ethyl
acetate/petroleum ether, 1/100 to 1/20) to afford compound 14a (1.03 g, 51.2%).
1
White solid. H NMR (600 MHz, DMSO-d ) δ 11.43 (s, 1H), 9.47 (s, 1H), 8.90 (s,
6
1
H), 8.46 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 7.4
Hz, 1H), 7.39–7.33 (m, 3H), 7.10 (d, J = 7.5 Hz, 1H), 6.89 (dd, J = 17.6, 11.1 Hz, 1H),
.06 (d, J = 17.6 Hz, 1H), 5.52 (d, J = 11.0 Hz, 1H), 2.36 (s, 3H). ESI-MS m/z: 355.2
6
M+H]+.
[
Compounds 14b–d were prepared from 13b–d, respectively, in a similar manner
as described for compound 14a.
3-Methyl-N-(2-methyl-[1,1'-biphenyl]-3-yl)-6-vinyl-[1,2,4]triazolo[4,3-a]pyri
1
dine-8-carboxamide (14b). White solid. Yield, 47%. H NMR (600 MHz, DMSO-d )
6
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δ 11.46 (s, 1H), 8.74 (s, 1H), 8.42 (d, J = 1.3 Hz, 1H), 8.20 (d, J = 7.9 Hz, 1H), 7.48 (t,
J = 7.5 Hz, 2H), 7.42–7.32 (m, 4H), 7.09 (d, J = 7.5 Hz, 1H), 6.89 (dd, J = 17.6, 11.1
Hz, 1H), 6.08 (d, J = 17.6 Hz, 1H), 5.51 (d, J = 11.1 Hz, 1H), 2.76 (s, 3H), 2.34 (s,
3H). ESI-MS m/z: 369.2 [M+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
N-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-6-vinyl-[1,2,4]tr
1
iazolo[4,3-a]pyridine-8-carboxamide (14c). White solid. Yield, 62%. H NMR (600
MHz, DMSO-d ) δ 11.40 (s, 1H), 9.47 (s, 1H), 8.89 (s, 1H), 8.45 (s, 1H), 8.14 (d, J =
6
7
.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 7.1 Hz, 1H), 6.97–6.77 (m, 4H),
6.06 (d, J = 17.6 Hz, 1H), 5.52 (d, J = 11.0 Hz, 1H), 4.29 (s, 4H), 2.36 (s, 3H).
ESI-MS m/z: 413.2 [M+H]+.
N-(2-Cyano-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-6-vinyl-[1,2,4]tri
1
azolo[4,3-a]pyridine-8-carboxamide (14d). White solid. Yield, 59%. H NMR (600
MHz, DMSO-d ) δ 11.94 (s, 1H), 9.48 (s, 1H), 8.94 (s, 1H), 8.48 (s, 1H), 8.24 (d, J =
6
8.2 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.15 (d, J = 2.2 Hz,
1
H), 7.11 (dd, J = 8.3, 2.2 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.08 (d, J = 17.6 Hz, 1H),
5.53 (d, J = 11.0 Hz, 1H), 5.33 (t, J = 4.8 Hz, 1H), 4.33 (s, 4H). ESI-MS m/z: 424.1
M+H]+.
-Formyl-N-(2-methyl-[1,1'-biphenyl]-3-yl)-[1,2,4]triazolo[4,3-a]pyridine-8-c
arboxamide (15a). Intermediate 14a (0.65 g, 1.84 mmol) was dissolved in
,4-dioxane (25 mL) and water (5 mL). To this mixture was added aqueous solution
[
6
1
of osmium tetraoxide (2% w/w in water, 3.5 mL, 0.276 mmol). After stirred for 10
min, sodium periodate (1.57 g, 7.36 mmol) was added. The mixture was stirred for
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6
further 3 h at room temperature. The precipitate was collected via filtration, and dried
1
to give compound 15a (0.63 g, 96.8%). Yellow solid. H NMR (600 MHz, DMSO-d )
6
δ 11.33 (s, 1H), 10.11 (s, 1H), 9.70 (s, 1H), 9.58 (s, 1H), 8.51 (s, 1H), 8.17 (d, J = 7.8
Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 7.4 Hz, 1H), 7.39–7.37 (m, 2H), 7.35 (d,
J = 7.9 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 2.35 (s, 3H). ESI-MS m/z: 357.1 [M+H]+.
Compounds 15b–d were prepared from 14b–d, respectively, in a similar manner
as described for compound 15a.
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-Formyl-3-methyl-N-(2-methyl-[1,1'-biphenyl]-3-yl)-[1,2,4]triazolo[4,3-a]py
ridine-8-carboxamide (15b). Yellow solid. Yield, 90%. ESI-MS m/z: 371.1 [M+H]+.
N-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-6-formyl-[1,2,4]
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triazolo[4,3-a]pyridine-8-carboxamide (15c). White solid. Yield, 92%. H NMR
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600 MHz, DMSO-d ) δ 11.30 (s, 1H), 10.11 (s, 1H), 9.69 (s, 1H), 9.58 (s, 1H), 8.50
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(s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H),
6.94 (d, J = 8.2 Hz, 1H), 6.88–6.77 (m, 2H), 4.29 (s, 4H), 2.36 (s, 3H). ESI-MS m/z:
15.1 [M+H]+.
N-(2-Cyano-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-6-formyl-[1,2,4]t
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riazolo[4,3-a]pyridine-8-carboxamide (15d). White solid. Yield, 86%. H NMR
(
600 MHz, DMSO-d ) δ 11.83 (s, 1H), 10.12 (s, 1H), 9.71 (s, 1H), 9.62 (s, 1H), 8.52
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(d, J = 0.7 Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 7.5
Hz, 1H), 7.16 (d, J = 1.9 Hz, 1H), 7.11 (dd, J = 8.3, 2.0 Hz, 1H), 7.04 (d, J = 8.3 Hz,
1H), 4.33 (s, 4H). ESI-MS m/z: 426.1 [M+H]+.
Ethyl 2-chloro-6-(1,3-dioxolan-2-yl)nicotinate (17). The solution of
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Page 29 of 57
Journal of Medicinal Chemistry
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intermediate 16 (2.5 g, 11.74 mmol), ethane-1,2-diol (1.46 g, 23.47 mmol), and
p-toluenesulfonic acid (0.48 g, 2.82 mmol) in toluene (20 mL) was stirred under
reflux for 12 h. The precipitate was collected via filtration, and dried to give
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compound 17 (2.03 g, 67.3%). Yellow solid. H NMR (600 MHz, DMSO-d ) δ 8.31
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(d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H), 5.78 (s, 1H), 4.36 (q, J = 7.1 Hz, 2H),
4.11–3.98 (m, 4H), 1.33 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 258.1 [M+H]+.
Compound 21 was prepared from intermediate 17 through a four-step synthetic
procedure which was similar to the conversion of 9 to 13a.
5-(1,3-Dioxolan-2-yl)-N-(2-methyl-[1,1'-biphenyl]-3-yl)-[1,2,4]triazolo[4,3-a]
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pyridine-8-carboxamide (21). Pale-yellow solid. Yield for four steps, 40%. H NMR
(600 MHz, DMSO-d ) δ 11.52 (s, 1H), 9.51 (s, 1H), 8.30 (d, J = 7.1 Hz, 1H), 8.19 (d,
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J = 7.9 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.43–7.33 (m, 5H), 7.10 (d, J = 7.4 Hz, 1H),
.39 (s, 1H), 4.20–4.10 (m, 4H), 2.36 (s, 3H). ESI-MS m/z: 401.2 [M+H]+.
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5-Formyl-N-(2-methyl-[1,1'-biphenyl]-3-yl)-[1,2,4]triazolo[4,3-a]pyridine-8-c
arboxamide (22). To a solution of intermediate 21 (1 g, 2.5 mmol) in acetone (9 mL)
and water (6 mL) was added p-toluenesulfonic acid (1.29 g, 7.5 mmol). The mixture
was stirred at 70 °C for 40 h. Upon completion of the reaction, the precipitate was
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filtered off, and dried to give compound 22 (0.68 g, 76.3%). Yellow solid. H NMR
(600 MHz, DMSO-d ) δ 11.62 (s, 1H), 10.14 (s, 1H), 10.10 (s, 1H), 8.49 (d, J = 7.1
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Hz, 1H), 8.23–8.12 (m, 2H), 7.48 (t, J = 7.2 Hz, 2H), 7.45–7.32 (m, 4H), 7.12 (d, J =
7.6 Hz, 1H), 2.37 (s, 3H). ESI-MS m/z: 357.1 [M+H]+.
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-Bromoimidazo[1,2-a]pyridine-8-carboxylic acid (24). To a mixture of
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