Design, Synthesis, and Biological Evaluation of Amidobenzimidazole Derivatives as Stimulator of Interferon Genes (STING) Receptor Agonists

Article abstract of DOI:10.1021/acs.jmedchem.9b01567

Stimulator of interferon genes (STING) is an endoplasmic reticulum-localized adaptor protein (STING receptor) that has been shown to be activated by binding to natural cyclic dinucleotide (CDN) ligands and plays a vital role in innate immune sensing of exogenous or endogenous DNA, which then induces type I interferons and other cytokines. In this paper, we described a series of amidobenzimidazole STING agonists with high potency for the STING receptor and presented the relevant structure-activity relationships (SARs). The relative potencies of compounds 16g, 24b, and 24e were measured by a STING competition binding assay. A more thorough study of the effect on the STING signaling pathway demonstrated that three compounds, 16g, 24b, and 24e, significantly increased the protein levels and mRNA levels of IFN-β, CXCL10, and IL-6, and 24b as a representative compound effectively triggered the phosphorylation of STING, TBK1, and IRF3 in both human peripheral blood mononuclear cells (hPBMCs) and WT THP-1 cells. In addition, compound 24b demonstrated impressive antitumor efficacy in mice with established syngeneic colon tumors by intravenous administration. Furthermore, the pharmacokinetic profile of compound 24b was fully evaluated.

Full text of DOI:10.1021/acs.jmedchem.9b01567

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Article
Design, Synthesis, and Biological Evaluation of Amidobenzimidazole
Derivatives as Stimulator of Interferon Genes (STING) Receptor Agonists
Qiumu Xi, Mingjin Wang, Wenqiang Jia, Minjian Yang, Jinping
Hu, Jing Jin, Xiaoguang Chen, Dali Yin, and Xiaojian Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01567 • Publication Date (Web): 10 Dec 2019
Downloaded from pubs.acs.org on December 10, 2019
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Design, Synthesis, and Biological Evaluation of
Amidobenzimidazole Derivatives as Stimulator of
Interferon Genes (STING) Receptor Agonists
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Qiumu Xi,† Mingjin Wang,†, Ⅱ Wenqiang Jia, Mingjian Yang, Jinping Hu, Jing Jin,
, Ⅱ
‡
†
†
*,†
Xiaoguang Chen, *,† Dali Yin,†, ‡ Xiaojian Wang, *,†,‡
†State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of
Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences,
Beijing 100050, P.R. China. E-mail: wangxiaojian@imm.ac.cn; Fax: +86 10 63165248; Tel: +86
1
0 63165248
‡Department of Medicinal Chemistry, Beijing Key Laboratory of Active Substances Discovery
and Druggability Evaluation, Institute of Materia Medica, Peking Union Medical College and
Chinese Academy of Medical Sciences, Beijing 100050, P.R. China
KEYWORDS: STING receptor agonists, amidobenzimidazole, structure modification, SAR,
antitumor
ABSTRACT: Stimulator of interferon genes (STING) is an endoplasmic reticulum-localized
adaptor protein (STING receptor) that has been shown to be activated by binding to natural
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cyclic dinucleotide (CDN) ligands and plays a vital role in innate immune sensing of exogenous
or endogenous DNA, which then induces type I interferons and other cytokines. In this paper, we
described a series of amidobenzimidazole STING agonists with high potency for the STING
receptor and presented the relevant structure–activity relationships (SARs). The relative
potencies of compounds 16g, 24b and 24e were measured by a STING competition binding
assay. A more thorough study of the effect on the STING signaling pathway demonstrated that
three compounds, 16g, 24b and 24e, significantly increased the protein levels and mRNA levels
of IFN-β, CXCL10 and IL-6, and 24b as a representative compound effectively triggered the
phosphorylation of STING, TBK1 and IRF3 both in human peripheral blood mononuclear cells
(hPBMCs) and WT THP-1 cells. In addition, compound 24b demonstrated impressive antitumor
efficacy in mice with established syngeneic colon tumors by intravenous administration.
Furthermore, the pharmacokinetic profile of compound 24b was fully evaluated.
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INTRODUCTION
Over the past decade, the profound immune-stimulatory capacity of nucleic acids has been
described, and many efforts have been made to identify the receptors and pathways influenced
by nucleic acids.1, 2 Cyclic GMP-AMP synthase (cGAS), a stimulator of interferon genes
(STING) pathway in the cytoplasm of mammalian cells, is referred to a DNA-driven immune
response and is involved in a major DNA-sensing mechanism to detect self or foreign DNA and
trigger the appropriate signaling pathway for host defense.3, 4 Tumor-derived DNA causes cGAS
activation, resulting in the conversion of GTP and ATP into cGAMP, which binds to and
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activates STING. STING activation leads to the recruitment and phosphorylation of the TANK
binding kinase TBK1, the phosphorylation of the interferon regulatory transcription factor IRF3,
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the production of type I interferons and inflammatory cytokines, and the triggering tumor-
+
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specific CD8 T cell to eliminate tumor cells. STING is an important mediator of the innate
immune signaling pathway, especially in defense against pathogen infections, preventing tumors
and maintaining the normal immune functions of the human body.8, 9 Accordingly, the STING
receptor has been actively investigated as a therapeutic target for the treatment of infectious
diseases, cancer immunotherapy and autoimmunity.
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Agonists of human STING can generally be divided into two groups (natural cyclic
dinucleotides (CDNs) and amidobenzimidazoles) based on differences in their chemical
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structures (Figure 1). CDNs have four known natural structures, cyclic di-AMP, cyclic di-
GMP,11 cyclic di-GMP-AMP 12 and 2’, 3’-cGAMP.13, 14 Indeed, several modified cyclic
dinucleotides that show favorable hSTING agonist activities are currently being developed as
antitumor agents; for example, ADU-S100 has been promoted to phase Ib clinical trials in
patients with diverse, solid, accessible tumors for achievable intratumoral delivery.15, 16 On the
other hand, a series of amidobenzimidazole hSTING agonists were obtained by a high-
throughput screening strategy, and they are active in humans and suitable for systemic
administration. Subsequently, a linking strategy was developed to synthesize two linked
symmetry-related amidobenzimidazole STING agonists with enhanced binding to STING and
improved cellular function. Immediately after that, the interaction mechanisms between STING
and a representative amidobenzimidazole (compound 1) and two linked-amidobenzimidazoles
(compound 2) were revealed.17, 18
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Figure 1. Chemical structures of hSTING agonists. (A) The four known natural CDNs and a
representative modified CDN (ADU-S100) as STING agonists. (B) Amidobenzimidazole (1, 3,
4
) and two linked symmetry-related amidobenzimidazoles (2) as STING agonists.
In an effort to discover potent human STING agonists, we designed and synthesized a series of
amidobenzimidazole derivatives to explore their structure–activity relationships (SARs).
Beginning with a binding model analysis, the interactions between the amidobenzimidazole and
STING (PDB ID: 6DXG) showed that the terminal amide formed a hydrogen bond with Ser241,
and the carboxamide formed a hydrogen bond with Thr263. Based on the hydrogen bond
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between the carboxamide and Thr263, four modification sites (R , R , R and R ) were chosen to
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guide the derivatization of these amidobenzimidazole compounds (Figure 2). At the same time,
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amidobenzimidazole compound 3 and 4 were chosen as positive control. Subsequently, we
performed an SAR study based on an evaluation of their STING-dependent activities. This study
led to the identification of three compounds, 16g, 24b and 24e, that exhibit micromolar STING
agonist activities, and the potency of 24b is superior to the potencies of 16g and 24e. In addition
to the SAR study, the relative potency of compounds 16g, 24b and 24e binding to STING, the
effects of compounds 16g, 24b and 24e on the STING signaling pathway, their capacity for
tumor inhibition and the pharmacokinetic profile of 24b as a representative compound were fully
evaluated and analyzed in detail.
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Figure 2. Four modification sites used to design amidobenzimidazole derivatives.
RESULT AND DISCUSSION
Chemistry. The synthetic pathway utilized to prepare propyl-1H-benzo[d]imidazole
derivatives with substituents at R and R is outlined in Scheme 1. Methyl 4-chloro-3-methoxy-
1
3
5
-nitrobenzoate (5a) and 2-chloro-1-methoxy-3-nitrobenzene (5b) were both commercially
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available. Initially, methyl 4-chloro-3-methoxy-5-nitrobenzoate (5a) was converted to 4-chloro-
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-methoxy-5-nitrobenzamide (6) via amination with an ammonia solution. Then, 5a, 5b and 6
were subjected to substitution reactions with propan-1-amine with potassium carbonate in N-
methyl-2-pyrrolidinone to provide corresponding intermediates 7a~7c.19 The subsequent
reductions of 7a~7c were performed with Pd/C and hydrogen to afford intermediates 8a~8c.
Intermediates 8a~8c were then reacted with cyanogen bromide, and the loss of 1 eq of HBr
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afforded intermediates 9a~9c with a benzimidazole scaffold. Finally, condensation reactions
between benzimidazoles 9 with different aromatic heterocyclic carboxylic acids afforded propyl-
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H-benzo[d]imidazole derivatives 10a~10k.21
Scheme 1. Preparation of propyl-1H-benzo[d]imidazole derivatives with substituents at R and
1
R3
O
NO2
H2N
Cl
b
O
6
a
O
O
NO2
Cl
R1
NO2
R1
NH2
R1
R1
b
c
d
N
N
e
N
N
R3
O
NH2
NH
N
H
N
H
O
O
O
O
O
5b
7a~7c
8a~8c
9a~9c
10a~10k
b
NO2
Cl
O
O
7a~10a
7b~10b
R
1
= H
7c~10c
R
R
1
1
=
NH2
NH2
O
O
5
a
R
1
=
10d~10k
=
O
Reagents and conditions: (a) NH OH, 50 ℃; (b) n-PrNH , K CO , NMP, 120 ℃; (c) H , Pd/C,
4
2
2
3
2
DCM, 25 ℃; (d) BrCN, MeOH, 60 ℃; (e) R COOH, HATU, DIPEA, DMF, 25 ℃.
3
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The synthetic procedure of benzyl (1H-benzo[d]imidazol-1-yl)propyl carbamate derivatives
with substituents at R and R is described in Scheme 2. Compounds 5a, 5b and 6 were subjected
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to substitution reactions with benzyl (3-aminopropyl)carbamate with potassium carbonate in N-
methyl-2-pyrrolidinone to provide corresponding intermediates 11a~11c.19 The subsequent
reductions of 11a~11c were performed with zinc powder and acetic acid to give corresponding
intermediates 12a~12c. Intermediates 12 were then reacted with cyanogen bromide, and the loss
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of 1 eq of HBr afforded intermediates 13 with a benzimidazole scaffold. Finally, condensation
reactions between benzimidazoles 13 with different aliphatic and aromatic heterocyclic
carboxylic acids afforded benzyl (1H-benzo[d]imidazol-1-yl)propyl carbamate derivatives
14a~14n.21
Scheme 2. Preparation of benzyl (1H-benzo[d]imidazol-1-yl)propyl carbamate derivatives with
substituents at R and R
1
3
O
NO2
Cl
H2N
b
O
6
a
O
O
NO2
Cl
R1
NO2
R1
NH2
R1
R1
O
b
c
d
N
N
e
N
N
R3
NH2
NH
cbz
cbz
N
H
N
N
H
N
H
H
O
O
O
O
O
5
b
1
1a~11c
12a~12c
HN
cbz
HN
cbz
b
NO2
Cl
O
O
13a~13c
14a~14n
11a~14a
11b~14b
R
1
=
=
H
11c~14c
14d~14n
R1=
NH2
NH2
O
O
5a
R
1
R1=
O
Reagents and conditions: (a) NH OH, 50 °C; (b) NH (CH ) NHCbz, K CO , NMP, 120 °C; (c)
4
2
2 3
2
3
Zn, CH COOH, DCM, 25 °C; (d) BrCN, MeOH, 60 °C; (e) R COOH, HATU, DIPEA, DMF, 25
3
3
°C.
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The preparation of compounds 16g~16i is illustrated in Scheme 3. Compound 6 were
subjected to substitution reactions with tert-butyl (3-aminopropyl)carbamate or tert-butyl (4-
aminobutyl)carbamate with potassium carbonate in N-methyl-2-pyrrolidinone to provide
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corresponding intermediates 16g-1 and 16h-1. The subsequent reductions of 16g-1 and 16h-1
were performed with Pd/C and hydrogen to give corresponding intermediates 16g-2 and 16h-2.
Intermediates 16g-2 and 16h-2 were then reacted with cyanogen bromide, and the loss of 1 eq of
HBr afforded intermediates 16g-3 and 16h-3 with a benzimidazole scaffold.20 Finally,
condensation reactions between benzimidazoles 16g-3 and 16h-3 with different aromatic
heterocyclic carboxylic acids afforded tert-butyl (1H-benzo[d]imidazol-1-yl)propyl carbamate
derivatives 16g and tert-butyl (1H-benzo[d]imidazol-1-yl)butyl carbamate derivatives 16h and
6i.21
1
Scheme 3. Preparation of tert-butyl (1H-benzo[d]imidazol-1-yl)alkyl carbamate derivatives with
substituents at R and R
1
3
O
O
O
O
O
O
NO2
Cl
NO2
R2
NH2
R2
N
N
R3
a
H2N
b
2
H N
c
2
H N
N
N
d
H2N
O
NH2
NH
N
N
H
H
R2
R2
O
O
O
O
O
5b
16g~16i
1
1
6g-1
6h-1
16g-2
16h-2
16g-3
16h-3
Boc
1
6g-1~16g-3
R
2
=
Boc
16g
6h
6i
R
2
=
N
N
H
H
H
N
H
N
1
R
R
2
2
=
=
Boc
Boc
1
6h-1~16h-3
R
2
=
Boc
H
N
1
Reagents and conditions: (a) NH OH, 50 ℃; (b) NH (CH ) NHBoc or NH (CH ) NHBoc,
4
2
2 3
2
2 4
K CO , NMP, 120 ℃; (c) H , Pd/C, DCM, 25 ℃; (d) BrCN, MeOH, 60 ℃; (e) R COOH, HATU,
2
3
2
3
DIPEA, DMF, 25 ℃.
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The preparation of derivatives with different R groups started from compound 4 (Scheme 4).
2
Hydrogenating compound 4 with a mixture of methanol and Pd/C furnished key intermediate 15,
which was then condensed with different aromatic heterocyclic and aliphatic carboxylic acids to
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give corresponding derivatives 16a~16e with different R groups. In addition, subsequent
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hydrolysis of condensation product 16e furnished corresponding oxobutanoic acid 16f.
Scheme 4. Preparation of derivatives at R site
2
O
O
N
N
N
N
H2N
O
NH
O
N
N
O
O
N
N
N
N
2
H N
N
N
NH
O
H2N
b
a
NH
O
HN
O
O
O
HN
R5
H2N
O
4
15
16a~16e
O
1
6a
R5=
16d
16e
R5=
R5=
O
O
O
O
N
N
O
N
N
N
O
N
2
H N
H2N
NH
16b R5=
O
O
NH
N
N
O
O
16c
R5=
N
c
HN
HN
O
O
O
O
OH
O
1
6e
16f
Reagents and Conditions: (a) H , Pd/C, methanol, 50 °C; (b) R COOH, HATU, DIPEA, DMF,
2
5
25 °C; (c) 0.5 mol/L LiOH, MeOH, 45 °C, acidification.
The preparation of target compounds 24a~24g with different substituents at the R site is
4
outlined in Scheme 5. The demethylation of intermediate 6 was performed with boron tribromide
in methylene chloride to give phenol 17. Further elaboration of the side chains attached to phenol
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2
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6
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
1
7 (summarized in step (b)) furnished intermediates 18b and 18c. Compound 19 was converted
to benzamide 20 via amination with an ammonia solution. Then, 18b, 18c and 20 were subjected
to substitution reactions with benzyl (3-aminopropyl)carbamate with potassium carbonate in 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
19
methyl-2-pyrrolidinone to provide corresponding nitrobenzenes 21a, 21b, and 21c. Synthesized
nitrobenzenes 21 were then reduced to corresponding anilines 22 in the presence of zinc powder
and acetic acid. Subsequently, anilines 22a, 22b, and 22c were reacted with cyanogen bromide to
2
0
furnish intermediates 23a, 23b and 23c. Finally, the condensation of benzimidazoles 23 and
different aromatic heterocyclic carboxylic acids afforded derivatives 24a~24g.21
Scheme 5. Preparation of derivatives at R site
4
O
O
O
O
NO2
Cl
NO2
Cl
NO2
Cl
H2N
2
H N
b
2
H N
NO2
a
H2N
O
N
H
N
O
O
OH
O
H
R6
d
R4
6
17
18b~18c
21a~21c
18b R
6
=
O
21a
1b
4
R = H
O
O
N
2
R
4
=
1
8c R
6
=
N
O
O
d
O
O
O
2
1c
R
4
=
NO2
Cl
NO2
Cl
c
O
H2N
1
9
20
O
O
O
O
NH2
N
N
R3
e
2
H N
O
f
H2N
g
H2N
NH2
NH
N
N
N
H
N
H
O
R4
R4
R4
22a~22c
HN
HN
O
O
O
2
2a
4
R =
H
O
O
O
N
2
2b
R
4=
O
22c
R
4
=
O
2
3a~23c
24a~24g
2
3a
3b
R
4
=
H
O
O
N
2
R
4=
O
23c
R
4
=
O
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9
Reagents and conditions: (a) BBr , DCM, 25 °C; (b) R X, K CO , DMF, 70 °C; (c) NH OH,
3
6
2
3
4
5
0 °C; (d) NH (CH ) NHCbz, K CO , NMP, 120 °C; (e) Zn, CH COOH, DCM, 25 °C; (f) BrCN,
2 2 3 2 3 3
MeOH, 60 °C; (g) R COOH, HATU, DIPEA, DMF, 25 °C.
3
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
The synthetic routes employed to prepare imidazopyridine derivatives 29, 30 and 31 are
shown in Scheme 6. Methyl 6-chloro-5-nitronicotinate (25) was commercially available and was
reacted with N-Boc-1, 3 -diaminopropane with potassium carbonate in N-methyl-2-pyrrolidinone
2
2
to provide nitropyridine 26. The subsequent reduction of 26 with Pd/C and hydrogen yielded
aminopyridine 27. Then, treating aminopyridine 27 with cyanogen bromide afforded
intermediate 28 with the imidazopyridine scaffold. Condensation reactions to obtain derivative
2
1
29 were achieved under standard condensation conditions, and hydrolysis of ester intermediate
9 using aqueous LiOH produced carboxylic acid 30 in good yield. As indicated in step (e) in
2
Scheme 6, carboxylic acid 30 was subjected to ethyl chloroformate and triethylamine in
tetrahydrofuran to form an active ester intermediate, which was then reacted with ammonia/THF
solution to give desired derivative 31.23
Scheme 6. Preparation of derivatives with different substituents on the imidazopyridine ring
O
O
O
O
NO2
Cl
NO2
NH2
a
O
O
b
N
O
O
O
c
NH2
O
N
N
N
H
N
O
N
N
H
N
O
N
N
H
H
25
26
27
28
HN
O
O
N
N
N
N
N
N
O
O
O
O
O
O
N
N
d
N
e
NH
f
NH
NH
HO
H N
O
N
2
N
N
N
N
N
29
30
31
HN
O
HN
O
HN
O
O
O
O
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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
Reagents and conditions: (a) Benzyl (3-aminopropyl)carbamate, K CO , NMP, 120 °C; (b) H ,
2
3
2
Pd/C, DCM, 25 °C; (c) BrCN, MeOH, 60 °C; (d) 1-Ethyl-3-methyl-1H-pyrazole-5-carboxylic
acid, HATU, DIPEA, DMF, 25 °C; (e) 0.5 mol/L LiOH, MeOH, 25 °C, acidification; (f) Ethyl
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
chloroformate, triethylamine, THF, 25 °C, 0.5 mol/L NH /THF.
3
In vitro Structure and Activity Relationship Studies. Here, the hSTING agonist activities
of all the amidobenzimidazole derivatives were screened based on STING-IRF pathway
induction in THP1-Dual cells and THP1-Dual-KO-STING cells. THP1-Dual cells were derived
from the human THP-1 monocyte cell line with the Lucia luciferase gene, and THP1-Dua KO-
STING cells were generated from THP1-Dual cells by stable knockout of the STING gene. Both
THP1-Dual cells and THP1-Dua KO-STING cells allow the study of the interferon regulatory
factor (IRF) pathway by assessing the activity of Lucia luciferase, and the reporter protein is
readily measurable in the cell culture supernatant when using QUANTI-Luc™, a Lucia
luciferase detection reagent. In addition, to exclude the influence of cytotoxic of compounds on
the fluorescence value, MTS was employed to detect cell activity.
The hSTING agonist activities of all the amidobenzimidazole derivatives were compared to
1
8
that of the positive compounds 3, 4 and 2’,3’-cGAMP. First, keeping the propyl group on the
R site constant (based on 3), an ester group and a hydrogen atom were introduced at the R site
2
1
(Table 1). Compared to compound 3, compounds 10a and 10b showed poor potency against
STING. In addition, a variety of aliphatic and aromatic heterocyclic groups with different
physical and chemical properties were introduced at the R site to yield compounds 10c-10k,
3
which were also found to have inferior STING agonist activities. As illustrated by these
compounds, different substituents at the R and R sites were not tolerated when a propyl group
1
3
was attached to the R site. In a second step, the benzyl propylcarbamate side chain at the R site
2
2
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of the imidazole ring was kept constant (based on 4), and a hydrogen atom and an ester group
were introduced at the R site to give compounds 14a and 14b (Table 2). Compound 14a, which
1
showed poor potency, illustrates that the terminal amide group is necessary for STING
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
activation. Compound 14b showed moderate STING agonist activity (STING EC = 20.70 μM)
5
0
in THP1-Dual cells as well as moderate STING agonist activity (STING-KO, EC = 27.60 μM)
5
0
in THP1-Dual-KO-STING cells, revealing an off-target effect of 14b. As before, a variety of
aliphatic and aromatic heterocyclic groups with different physical and chemical properties were
introduced at the R site to yield compounds 14c-14n. Among the selected groups at the R site,
3
3
the derivatives with a pyridyl group (14e, STING EC = 8.28 μM) and a pyridazinyl group (14f,
50
STING EC = 2.20 μM) exhibited micromolar STING agonist activities with the same order of
50
magnitude. The activities of 14e and 14f were lower than the level of compound 4. In contrast,
larger substituents (quinolone derivative 14c, isoquinoline derivative 14d, indole derivative 14j
and benzene derivative 14k), smaller substituents (ethyl pyrazole derivative 14g, methyl
pyrazole derivative 14h, methyl imidazole derivative 14i and methyl pyrrole derivative 14l) and
aliphatic heterocycles (14m and 14n) at the R site led to inactive compounds. An exploration of
3
various groups at R showed that substituents at the R site with the proper volume are
3
3
advantageous for STING activation. To find the rules governing the activity based on changing
the R site, pyridyl and pyridazinyl groups at the R site were chosen for the next investigation.
2
3
Table 1. Biological activities of derivatives 10a ~10k with different substituents at the R and R
1
3
sites and of 2’, 3’-cGAMP. a
O
R1
N
N
R3
NH
R2
R4
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6
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
EC （μM）
MTS（μM）
5
0
Compd
R1
R2
R3
R4
STING -
KO
STING-
STING
STING
KO
O
N
N
N
N
N
N
O
O
O
O
3
3.30
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
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
0a
H
>100
>100
>100
O
10b
O
O
O
N
1
0c
0d
0e
0f
10g
H2N
N
O
1
>100
>100
>100
>100
H2N
O
O
O
O
O
O
O
N
N
O
O
O
O
O
O
O
1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
H2N
H2N
H2N
H2N
H2N
H2N
N
1
N
N
N
N
N
N
1
0h
1
0i
N
1
0j
10k
2
’, 3’-
-
-
-
-
38.60
>100
>100
>100
cGAMP
a
EC is the mean of three experimental determinations.
5
0
a
Table 2. SAR of derivatives 14a ~14n with different substituents at the R and R sites.
1
3
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9
O
R1
N
N
R3
NH
R2
R4
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
EC （μM）
MTS（μM）
50
Compd
R1
R2
R3
R4
STING -
STING-
STING
STING
KO
KO
O
H
N
O
O
O
O
O
N
N
4
0.872
>100
20.70
>100
>100
>100
27.60
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
O
O
O
O
H
N
N
N
N
N
O
O
O
14a
H
O
H
N
1
4b
O
O
O
H
N
N
1
4c
H2N
N
H
N
O
O
1
4d
>100
>100
>100
>100
H2N
O
O
O
O
O
O
N
N
H
N
O
O
O
O
O
O
O
O
1
4e
4f
4g
4h
8.28
2.20
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
H2N
H2N
H2N
H2N
O
O
O
N
H
N
1
H
N
N
N
1
>100
>100
>100
H
N
O
O
N
N
N
N
1
O
O
H
N
14i
14j
O
H
N
N
O
O
>100
>100
>100
>100
H2N
O
O
O
H
N
O
O
O
O
1
4k
>100
>100
>100
>100
>100
>100
>100
>100
H2N
H2N
O
O
H
N
N
1
4l
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2
3
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5
6
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
O
O
H
N
N
O
O
O
O
1
4m
4n
>100
>100
>100
>100
>100
>100
>100
>100
H2N
H2N
O
O
H
N
N
1
a
EC is the mean of three experimental determinations.
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
Keeping the 1-ethyl-3-methyl-1H-pyrazole-5-yl group at the R site constant, we then
3
evaluated the effects of a large variety of side chains at the R site (Table 3). Compared to
2
compound 4, amine 15 showed dramatically lower agonist activity for STING. Attaching a
trimethylacetyl group or a benzoyl group to the side chain of the imidazole ring afforded
compounds 16a, which showed lower potency, and 16b, which displayed inferior agonist
activity. As indicated by compound 16b, the rigidity of the phenyl group in the side chain had an
adverse effect on the potency of compounds. Introducing a 3-(pyridin-3-yl) propanoyl group or a
1
,2,3-trimethoxy-5-propylphenyl group at this side chain gave derivatives 16c and 16d,
respectively, which showed lower or no activity against STING. To reduce the volume of the
substituents at the R site, we attached a carboxylic ester fragment and a carboxylic acid
2
fragment to the side chain to give compounds 16e and 16f, resulting in significant losses of
activity. Interestingly, introducing a tert-butyloxycarbonyl aminopropyl group to the R site gave
2
compound 16g, showing an EC of 1.24 μM, which is similar STING agonist activity to that of
50
compound 4. Considering flexibility and lipid solubility, the tert-butyloxycarbonyl aminobutyl
group is similar to the tert-butyloxycarbonyl aminopropyl group; therefore, we prepared
compound 16h furnished the tert-butyloxycarbonyl aminobutyl group at the R site and the
2
pyridyl group at the R site. In addition, keeping the tert-butyloxycarbonyl aminobutyl group at
3
R site constant, a pyridazinyl group was attached to the R site of compound 16i. Compound
2
3
1
6h showed favorable STING agonist activity, as did 16i. As illustrated by compounds 16g, 16h
and 16i, the tert-butyloxycarbonyl group in the side chain had a favorable effect on the potency
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of the compounds. In brief, the compounds compiled in Table 3 illustrated that a tert-
butyloxycarbonyl group in the side chain of the imidazole ring was tolerated by the STING
receptor.
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
Table 3. SAR study via optimization of the R substituent.
2
O
R1
N
N
R3
NH
R2
R4
EC （μM）
MTS（μM）
5
0
Compd
R1
R2
R3
R4
STING-
KO
STING-
STING
STING
KO
O
O
O
O
H2N
N
N
N
N
N
N
O
O
O
O
1
5
>100
27.50
>100
39.50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
H2N
H2N
H2N
H
N
N
N
N
N
16a
16b
O
H
N
O
N
H
N
1
6c
O
O
O
O
O
H
N
O
1
6d
O
>100
>100
>100
>100
H2N
O
O
O
H
N
N
N
N
N
N
O
O
O
1
6e
6f
6g
>100
>100
1.24
3.00
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
H2N
O
O
O
H
N
HO
1
O
O
H
N
O
N
N
O
O
1
H2N
H2N
O
O
16h
O
N
H
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1
1
1
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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
O
O
N
N
O
1
6i
O
N
2.85
>100
>100
>100
H2N
H
a
EC is the mean of three experimental determinations.
5
0
Since substitution at the R , R and R sites has been investigated in detail, our attention
1
2
3
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
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9
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1
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7
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1
2
3
4
5
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8
9
0
shifted to exploring substitution at the R site. As shown by the examples in Table 4, keeping the
4
N-carbobenzyloxy aminopropyl group at the R site constant, we evaluated the effects of
2
substituents at the R site. Compared to compound 4, the absence of a methoxy group at the R
4
4
site markedly influenced the STING agonist activity, as exemplified by compound 24a. The
introduction of a 3-methoxypropoxy group at the R site afforded compound 24b with an EC of
4
50
0.287 μM, corresponding to a 4-fold enhancement of the agonist activity for STING relative to
compound 4. Based on compound 24b, introducing a pyridyl group at R gave compound 24c,
3
which was less potent (EC of 1.90 μM). However, attaching a 3-methoxypropoxy group to the
5
0
R site and maintaining the pyridazinyl group at the R site afforded compound 24d, which was
4
3
significantly less potent against STING. In the next step, a 3-morpholinopropoxy group was
introduced to the R site on the benzene ring, generating compound 24e, which showed an EC
4
50
of 1.14 μM, demonstrating its equal potency against STING. Interestingly, when 3-
morpholinopropoxy was introduced at the R site, regardless of is a pyridyl group or pyridazinyl
4
group was introduced at the R site, compounds 24f and 24g showed poor potency against
3
STING. As illustrated by these two compounds, neither a pyridyl group nor a pyridazinyl were
tolerated at the R site when a 3-morpholinopropoxy group was attached at the R site.
3
4
a
Table 4. SAR study via optimization of the R substituent.
4
O
R1
N
N
R3
NH
R2
R4
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EC （μM）
MTS（μM）
5
0
Compd
R1
R2
R3
R4
STING-
KO
STING-
STING
STING
KO
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
H
N
N
N
N
O
O
2
4a
H
>100
0.287
1.90
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H2N
O
O
O
O
O
O
H
N
O
O
N
N
2
4b
H2N
H2N
O
O
H
N
O
O
2
4c
O
O
O
O
H
N
N
N
O
O
2
4d
4e
24f
4g
>100
1.14
H2N
H2N
H2N
H2N
H
N
O
O
N
N
N
O
O
2
N
N
O
O
H
N
O
O
>100
>100
N
O
H
N
N
O
O
N
O
2
a
EC is the mean of three experimental determinations.
5
0
With the last set of compounds compiled in Table 5, we investigated the activities of
substances possessing an imidazopyridine ring instead of the benzimidazole ring. The
imidazopyridine-containing compounds we prepared are shown in Table 5. We prepared ester 29
and acid 30, which showed poor STING agonist activity, while when we attached an amide
group at the R site of the imidazopyridine ring, the STING agonist activity was lost, as
1
exemplified by compound 31. As illustrated by compounds 16g and 31, replacing the
benzimidazole ring with an imidazopyridine ring caused a significant decrease in the STING
agonist activity.
Table 5. STING agonist activity of imidazopyridine derivatives 29 ~ 31. a
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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
O
N
N
R1
N
N
NH
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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2
3
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9
0
HN
O
O
EC （μM）
MTS（μM）
5
0
Compd
R1
STING
>100
STING-KO
>100
STING
STING-KO
>100
O
O
2
9
>100
>100
>100
O
30
>100
>100
>100
>100
>100
>100
HO
O
3
1
H2N
a
EC is the mean of three experimental determinations.
5
0
Binding to STING protein. The relative potencies of three compounds, 16g, 24b and 24e,
with excellent potency for the STING receptor were measured by a STING competition binding
assay (Figure 3). The results showed us that competition with increasing concentrations of
compounds 3, 4, 16g, 24b, 24e and 2’,3’-cGAMP inhibited the binding of full-length STING
with half maximal inhibitory concentrations (IC ) of 1.7370 μM, 0.5821 μM 1.5390 μM, 0.3794
5
0
μM, 0.9365 μM and 0.0055 μM, respectively. The results of the STING competition binding
assay are consistent with those arising from the in vitro THP-1 Dual cell luminescence assay
(16g EC = 1.24 μM, 24b EC = 0.287 μM, 24e EC = 1.14 μM) and IFN-β release in
5
0
50
50
peripheral blood mononuclear cells (PBMCs) assay (16g EC = 1.79 μM, 24b EC = 0.49 μM,
5
0
50
2
4e EC = 1.11 μM, Figure S1).
50
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2
2
2
2
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3
3
4
4
4
4
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4
4
4
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6
0
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0
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9
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. The relative potencies of 2’, 3’-cGAMP, 3, 4, 16g, 24b and 24e measured by a STING
competition binding assay. Mean response ± s.d.
In vitro STING signaling pathway. In general, the cascade optimization afforded a relatively
elaborate SAR study, and provided several compounds with superior STING receptor agonist
effects. The effects of three compounds (16g, 24b and 24e) with excellent potency for the
STING receptor were evaluated further on the STING signaling pathway.
Activation of STING through direct activation by cGAMP produces type I interferons and
proinflammatory cytokines, such as IL6 and CXCL10.24,
25
Many conventional
chemotherapeutics, targeted anticancer agents and immunological adjuvants are only fully
efficient in the presence of an intact type I interferon signaling pathway.26, 27 CXCL10 is strongly
induced by interferons and NF-κB, which are primarily involved in immune response and serve
+
28, 29
as the key chemokines to recruit CD8 T lymphocytes to eliminate tumor cells.
Interleukin
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
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4
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5
5
5
5
5
5
5
5
6
IL-6 is often viewed as a proinflammatory cytokine, and it plays a key role in inflammation-
3
0
associated cancer and metabolic disorders. Hence, it is reasonable to detect the above three
tumor-related cytokines to explore the effect of compounds on the STING signaling pathway. To
determine the functional consequences of binding, an enzyme-linked immunosorbent assay
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
(ELISA) was performed to examine the protein levels of IFN-β, the downstream target gene
CXCL10 and IL-6 in peripheral blood mononuclear cells (PBMCs). First, PBMCs were
incubated for 4 h and treated with test compounds 16g, 24b and 24e at four different
concentrations (0 μM, 0.1 μM, 1 μM and 10 μM). The secretions of IFN-β, CXCL10 and IL-6
were measured by ELISA, and then the OD450 values were read. The total secreted cytokine
concentrations (pg/mL) were calculated according to the standard curve, and histograms were
made using GraphPad Prism 8. Similar to 2’, 3’-cGAMP, the three test compounds, 16g, 24b and
2
4e, induced the secretion of IFN-β (Figure 4A). In addition, 10 μM of 16g, 24b and 24e
induced significantly higher levels of IFN-β compared with 139 μM of 2’, 3’-cGAMP. The
performances of these three compounds at doses of 0.1 μM and 1 μM are consistent, and a large
increase in the secretion of IFNβ was observed between doses of 0.1 μM and 1 μM. In terms of
the secretion level of IFN-β, compound 24b was superior to 16g and 24e at the same
concentrations. As indicated in Figure 4B, treatment with 16g, 24b and 24e at 1 μM induced
higher levels of CXCL-10 compared with treatment with 16g, 24b and 24e at 10 μM. Moreover,
1
.39 μM 2’, 3’-cGAMP induced secretion of CXCL10 far below the level seen with 1 μM of
16g, 24b and 24e. Therein, the most dramatic difference between 1 μM and 10 μM of 24b was
observed. In addition, the secretions of IL-6 due to the four compounds were similar to the
secretions of IFN-β. The secretion of IL-6 caused by a dose of 1 μM was dramatically greater
than that caused by a dose of 0.1 μM (Figure 4C). At the same concentration, compound 24e
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was superior to 16g and 24b in the secretion of IL-6. As shown in the results above, 10 μM 16g,
2
4b and 24e induced significantly higher levels of IL-6 compared with 139 μM of 2’, 3’-
cGAMP.
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
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8
9
0
1
2
3
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8
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0
1
2
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9
0
1
2
3
4
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6
7
8
9
0
Next, real-time quantitative PCR analysis (RT- qPCR) was employed to examine mRNA
levels of IFN-β, CXCL10 and IL-6 in PBMCs treated with compounds 16g, 24b and 24e at 10
μM. The results of qPCR (Figure 5A-C) are consistent with those from the ELISA, similar to
what was seen with 69.6 μM 2’,3’-cGAMP, 10 μM 16g, 24b and 24e introduced higher protein
levels and mRNA levels of IFN-β, CXCL10 and IL-6 compared with the blank group. In
addition, compound 24e introduced higher mRNA levels of IFN-β, CXCL10 and IL-6 than 16g
and 24b. In summary, it was demonstrated that compounds 16g, 24b and 24e could induce the
STING signaling pathway, as reflected by the increased protein levels and mRNA levels of IFN-
β, CXCL10 and IL-6.
Studies have shown that STING phosphorylation at Ser366 is required for IRF3 binding and
activation in the DNA-sensing pathway, and STING activation leads to recruitment and
7
phosphorylation of TBK1, phosphorylation of IRF3. To assess the effects of 24b on the STING-
TBK1-IRF3 signaling pathway, an immunoblotting analysis of PBMCs was carried out to
characterize the phosphorylation at Ser366 of STING, the phosphorylation of TBK1 and the
phosphorylation of IRF3 (Figure 5D). The results of the analyses illustrated that compound 24b
markedly induced the phosphorylation of STING, TBK1 and IRF3 and activated the STING-
TBK1-IRF3 signaling pathway.
On the other hand, the increased relative cytokine response and higher protein expression
levels of STING signaling pathway caused by compounds 16g, 24b and 24e were also observed
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4
4
4
4
4
4
4
4
4
4
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5
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5
5
6
in WT THP1 cells. WT THP-1 cell were treated with 2’, 3’-cGAMP (1, 3, 10, 30, 100 μM) and
increasing amounts of compounds 16g, 24b and 24e (0.1, 0.3, 1, 3 and 10 μM) respectively for
24 h, and then the secretion levels of IFNβ, CXCL10 and IL-6 and were assessed by ELISA. As
shown in Figure S2, compound 24b exhibited stronger ability of inducing secretion of IFN-β,
CXCL10 and IL-6 compared with compound 16g and 24e. Subsequently, WT THP-1 cell were
treated with 100 μM 2’3’-cGAMP and 10 μM of compound 16g, 24b and 24e for 4h. Then the
mRNA expression levels of IFN-β, IL-6 and CXCL10 were assessed by qPCR and normalized to
GAPDH expression. The results showed that 10 μM of compound 16g, 24b and 24e introduced
higher mRNA levels of IFN-β, CXCL10 and IL-6 compared with the blank group (Figure S3).
In order to evaluate whether compound 24b affect STING signaling pathway in WT THP-1 cells,
an immunoblotting analysis was performed with compound 24b. 50 μM 2’3’-cGAMP and 10
μM compound 24b were treated with WT THP-1 cells for 30min or 2h (Figure S4), and the
results of the analyses illustrated that compound 24b markedly induced the phosphorylation of
STING, TBK1 and IRF3 and activated the STING-TBK1-IRF3 signaling pathway in WT THP-1
cell.
0
1
2
3
4
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7
8
9
0
1
2
3
4
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7
8
9
0
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9
0
1
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1
2
2
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2
2
2
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0
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9
0
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0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Compounds 16g, 24b and 24e triggered the activation of the STING pathway. (A~C)
PBMCs were treated with 2’,3’-cGAMP (0, 1.39, 13.9, and 139 μM) and increasing amounts of
compounds 16g, 24b and 24e (0, 0.01, 0.1, 1 or 10 μM) for 4 h. The levels of IFN-β (A),
CXCL10 (B) and IL-6 (C) were assessed by an enzyme-linked immunosorbent assay (ELISA).
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Figure 5. Treatment with compound 16g, 24b and 24e at 10 μM triggered the activation of the
STING pathway. The mRNA expression levels of IFN-β (A), CXCL10 (B) and IL-6 (C) were
assessed by real-time quantitative PCR (qPCR) and normalized to GAPDH expression. (D)
Whole PBMC lysates were prepared after treatment with 2’,3’-cGAMP and compound 24b, and
the levels of total STING, phospho-STING, total IRF3, phospho-IRF3, and GAPDH were
assessed by western blotting.
In vivo PK/PD study of compound 24b. To explore the effect of compound 24b on tumor in
a mouse model and its tumor inhibition ability, we tested compound 24b at two doses, 0.15
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mg/kg and 1.5 mg/kg, and the test compound was injected intravenously on days 1, 3, 5 and 8 in
3
17
mice with approximately 100-mm subcutaneous CT-26 tumors. To our delight, treatment with
0.15 mg/kg compound 24b resulted in significant tumor growth inhibition as measured by tumor
volume (0.15 mg/kg vs. vehicle: *P < 0.05, Figure 6A). However, treatment with 1.5 mg/kg
compound 24b had a modest inhibitory effect on tumor volume. As indicated in Figure 6B,
treatment with 0.15 mg/kg compound 24b resulted in an obvious decrease in tumor weight (0.15
mg/kg vs. vehicle: *P < 0.05), but 1.5 mg/kg compound 24b did not. As we known, the
activation of STING will lead to the recruitment and activation of dendritic cells which
1
1
1
1
1
1
1
1
1
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4
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5
5
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5
5
5
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5
6
0
1
2
3
4
5
6
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8
9
0
1
2
3
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5
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9
0
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9
0
1
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9
0
1
2
3
4
5
6
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9
0
+
eventually activates CD8 T cell in the tumor microenvironment and exerted the anticancer
4
+
effect. In this experiment, we have tested the proportion of CD8 T cells in tumor tissue. As
+
shown in Figure S5, 24b with 0.15 mg/kg could increase more CD8 T cell in tumor than
+
control group. Although 1.5 mg/kg 24b also increase the proportion of CD8 T cell, it was lower
than 0.15 mg/kg group, which may partly explain the different therapeutic effect on the two
doses. In brief, intravenous injection of 0.15 mg/kg compound 24b had a significant inhibitory
effect on tumor growth. Next, to elucidate the metabolic features of compound 24b in vivo, we
determined the mean plasma concentration-time profiles of intravenously delivered 0.15 mg/kg
compound 24b in BALB/c mice (Table 6). Compound 24b exhibited systemic exposure with a
half-life of 0.16 h, an area under the curve of 18.04 h*ng/mL, a mean residence time of 0.08 h, a
time to peak of 0.03 h, a concentration peak of 138 ng/mL, a volume of distribution of 1.18 L/kg
and a clearance rate of 8.6 L/h/kg. The metabolic process of compound 24b were simulated with
Compound Discover 2.1 and found that there were many metabolic sites in the structure of
compound 24b (see supporting information, Figure S6), which might cause short half-life and
easy metabolism. These data support the conclusion that activation of the STING signaling
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pathway is related to the robust antitumor effect of compound 24b, which causes significant
tumor inhibition.
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Figure 6. Efficacy of intravenous injection of 0.15 mg/kg (purple) and 1.5 mg/kg (orange) of
compound 24b or vehicle (blue) in BALB/c mice bearing a single subcutaneous CT-26
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colorectal tumor (~100 mm ). (A) Mice were treated with two intravenous doses of compound
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4b or vehicle on days 1, 3, 5 and 8, and the tumor volumes were measured. The data represent
the mean tumor volume ± s.e.m. (n = 10 per group). The treatment group (0.15 mg/kg) showed a
statistically significant reduction in tumor volume compared to the control group. (B) The weight
of the tumors extracted from tumor-bearing mice at day 8 after initiation of treatment with two
intravenous doses of compound 24b or vehicle. Error bars represent s.d. *p < 0.05
Table 6. Pharmacokinetics of 0.15 mg/kg compound 24b following intravenous bolus in
BALB/c mice. a
t1/2
h
AUC0-t
MRT0-t
h
Tmax
h
Cmax
Vz
l/kg
CL
l/h/kg
compd
4b
h*ng/mL
ng/mL
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0.16
18.04
0.08
0.03
138
1.18
8.6
a
Data are expressed as the mean ± SD of the measured concentration of compound 24b.
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Page 29 of 79
Journal of Medicinal Chemistry
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CONCLUSION
We have reported the design, synthesis, and biological screening of a series of STING-activating
amidobenzimidazole derivatives. By considering multiple pharmacological parameters, cascade
and targeted structural modifications led to an elaborate SAR study of amidobenzimidazole
STING receptor agonists. Briefly, changes in the substituents at the R and R sites were not
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tolerated when a propyl group was attached at the R site, the absence of carboxamide at the R
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site markedly influenced the STING agonist activity, and substituents with proper flexibility and
lipid solubility at the R site and substituents with proper volume at the R site enhanced the
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STING agonist activity. Subsequently, the relative potencies of compounds 16g, 24b and 24e,
which showed excellent potencies for the STING receptor, were subjected to a STING
competition binding assay, and the half maximal inhibitory concentration of 24b was smaller
than those of 16g and 24e. Among the three compounds, in vitro THP-1-Dual cell luminescence
assay, the binding assay and IFNβ release in hPBMCs assay showed that compound 24b is more
potent than 16g and 24e. Next, we employed an ELISA and real-time quantitative PCR (qPCR)
to examine the protein levels and mRNA levels of IFN-β, and the downstream target genes
CXCL10 and IL-6 in PBMCs treated with compounds 16g, 24b and 24e at four different
concentrations (0 μM, 0.1 μM, 1 μM and 10 μM). The results showed that treatment with 10 μM
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6g, 24b and 24e introduced higher levels of IFN-β, CXCL10 and IL-6 relative to what was seen
with 139 μM 2’, 3’-cGAMP. In addition, treatment with 10 μM 16g, 24b and 24e significantly
increased the mRNA levels of IFN-β, CXCL10 and IL-6. At the same time, an immunoblotting
analysis of PBMCs was performed to characterize the STING-TBK1-IRF3 pathway upon
treatment with compound 24b. The results of this analysis demonstrated that treatment with 10
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