Structure-Activity Relationship Study of Amidobenzimidazole Analogues Leading to Potent and Systemically Administrable Stimulator of Interferon Gene (STING) Agonists

Article abstract of DOI:10.1021/acs.jmedchem.0c01900

Activation of the stimulator of interferon gene (STING) has emerged as an exciting immuno-oncology therapeutic strategy; however, the first-generation STING agonists, cyclic dinucleotide (CDN) analogues, have suffered from many disadvantages and failed in

Full text of DOI:10.1021/acs.jmedchem.0c01900

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Article
Structure−Activity Relationship Study of Amidobenzimidazole
Analogues Leading to Potent and Systemically Administrable
Stimulator of Interferon Gene (STING) Agonists
Zilan Song,# Xiyuan Wang,# Yan Zhang,# Wangting Gu, Ancheng Shen, Chunyong Ding, Han Li,
Ruoxuan Xiao, Meiyu Geng, Zuoquan Xie,* and Ao Zhang*
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ABSTRACT: Activation of the stimulator of interferon gene
(STING) has emerged as an exciting immuno-oncology
therapeutic strategy; however, the first-generation STING agonists,
cyclic dinucleotide (CDN) analogues, have suffered from many
disadvantages and failed in clinical trials. Therefore, non-CDN
small-molecule STING agonists are urgently needed. In view of the
unique structure of the high potency of dimeric amidobenzimida-
zole STING agonist 5, a structural elaboration was conducted by
modifying several structural hotspots of this scaffold. Triazole 40
was identified as a new potent STING activator, possessing EC50
values of 0.24 and 39.51 μM for h- and m-STING, respectively.
This compound has a slightly better pharmacokinetic profile and is
>20-fold more aqueously soluble than 5. It activated the STING
signaling dramatically by directly binding and stabilizing all h-STING isoforms and m-STING. In vivo, intermittent administration of
40 was found to have significant antitumor efficacy with good tolerance in two mouse tumor models.
of type I interferon (IFN) as well as many other
proinflammatory cytokines.11−13 These events can eventually
result in promotion of the adaptive immune response by
activation of cytotoxic CD8 T cells. Therefore, pharmaco-
logical activation of STING by agonists has emerged as an
exciting yet challenging strategy of IO therapy for various
diseases such as cancer, virus, and autoimmune diseases.14−16
Many efforts have been focused on STING agonists by
developing chemically more stable cyclic dinucleotide (CDN)
analogues of 2′,3′-cGAMP, resulting in the most upfront
compound 1 (ADU-S100, Figure 1) bearing dithio-mixed
linkages.17 This compound significantly enhanced STING
signaling by inducing much higher levels of IFN-β and showed
remarkably tumor growth suppression or even regression in
several mice models, including BALB/c mice bearing CT26
colon and 4T1 mammary carcinomas after three intermittent
intratumoral (IT) injections.18 This is the first STING agonist
approaching into clinical trials jointly developed by Aduro and
INTRODUCTION
■
The human immune system including innate and adaptive
immunity is precisely controlled by a number of costimulatory
and coinhibitory signaling molecules.1 Aberrations of these
immune signaling pathways can cause many diseases including
cancer. In the past decade, the successful launch of several
monoclonal antibodies (mAbs) targeting the suppressive
immune checkpoint proteins has established a new era of
immuno-oncology (IO) therapy as a vibrant cancer treat-
ment.2,3 However, compared to the success of checkpoint
inhibitors, development of small molecules targeting the
immunostimulatory signaling proteins is still in the infancy
with much unknown and challenge.4−6 The stimulator of
interferon gene (STING) is a stimulatory molecule residing on
the endoplasmic reticulum and plays a pivotal role in innate
immunity by acting as a downstream sensor of both exogenous
and endogenous DNA.7−9 2′,3′-cGAMP (Figure 1), a cyclic
dinucleotide (cyclic GMP-AMP) synthesized by cyclic
guanosine monophosphate (GMP)-adenosine monophosphate
(AMP) synthase (cGAS) upon detecting tumorous or virus
DNA in the cytosol, has been found as the endogenous ligand
of STING.10 Binding of STING with 2′,3′-cGAMP activates
STING by forming dimers or oligomers resulting in down-
stream signaling cascade via recruitment of the serine/
threonine protein kinase (TBK1), phosphorylation of the
interferon regulatory transcription factor IRF3, and production
Received: November 3, 2020
Published: January 20, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c01900
© 2021 American Chemical Society
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Figure 1. Representative STING agonists and our optimized hotspots (in blue color).
Novartis.19,20 Unfortunately, Novartis recently terminated its
clinical trials based on the marginally clinical responses either
on monotherapy or in combination with PD-1 antibody
spartalizumab.20 Many other low-weight small-molecule
STING agonists have also been reported,16 however, most
with distinct species specificity between human and mouse
STING. For example, 5,6-dimethylxanthenone-4-acetic acid
(2, DMXAA)21,22 and 10-carboxymethyl-9-acridanone (3,
CMA)23 are the very early low-weight small-molecule
STING agonists, but they are only active for m-STING,
other than for h-STING.
subsequent secretion of IFN-β with an value of EC50 130
nM. Different from CDNs (e.g., compound 1), dimeric 5 can
be administered intravenously (iv), and intermittent dosing of
5 at 1.5 mg/kg in subcutaneous CT26 tumors results in
significant tumor growth inhibition and improved survival. At
the end of 2019, Wang and co-authors25 reported a procedure
to optimize the amidobenzimidazole monomer and identified a
lead compound 6 with improved biochemical and cellular
potency, but the in vivo antitumor efficacy was modest. Very
recently, Addona and co-workers26 at Merck reported an orally
available STING agonist MSA-2 (Figure 1), which has the
potential to target the acidic tumor microenvironment. In the
meantime, Lairson and co-workers27 at Scripps reported a
small-molecule STING agonist SR-717, which can be
administered intraperitoneally.
app
Recently, Ramanjulu and co-workers24 at GlaxoSmithKline
reported a small-molecule STING agonist 4 (Figure 1) bearing
a key amidobenzimidazole (ABZI) component and showing
reproducible inhibition against 3H-cGAMP binding to STING
with an apparent inhibitory constant (IC50APP) of 14 μM. The
crystal structure of this compound in complex with the h-
STING C-terminal domain indicates that 4 binds in the same
binding pocket of the endogenous ligand cGAMP, with each of
two molecules of 4 bound with one STING subunit without
contacts with each other. Subsequently, these authors used a
linkage strategy to connect two molecules of 4 to form a dimer.
After further modification, compound 5 (diABZI) was
identified to exhibit a synergic effect in human peripheral
blood mononuclear cells by inducing STING activation and
Despite the structural novelty and significant antitumor
efficacy of the dimeric compound 5, limited structure−activity
relationship (SAR) study has been reported. In this regard, we
conducted a structural elaboration on this dimeric scaffold by
modifying several hotspots of compound 5, including the
readily tautomeric N-acyl 1H-imidazol-2(3H)-imine compo-
nent, the polaric terminal amido moiety, and the linkage
(marked in blue in Figure 1). This approach led to
identification of a new lead compound with improved safety
profile, good systemic plasma exposure, and high antitumor
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a
Scheme 1. Synthesis of Compounds 16−19 and 21 and 22
a
Reactions and conditions: (i) Cs2CO3, DMF, and rt; (ii) ethyl 2-(diethoxyphosphoryl)acetate, NaH, DMF, and 0 °C to rt; (iii) LiOH·H2O,
MeOH/H2O (1:1), and rt; (iv) 5% Pd/C, H2, MeOH, and rt; and (v) for 16 and 17: (1) EDCI, HOBt, DIPEA, DMF, and rt and (2) AcOH and
90 °C; for 18: 4 N HCl aqueous solution, MeOH, and 100 °C; and for 19: 4 N HCl aqueous solution, H2O, and 100 °C.
activity. Herein, we describe our synthesis and biological
characterization of these new STING agonists.
gave carboxylic acid derivative 19, whereas using MeOH as the
solvent gave ester derivative 18. Likewise, a similar event
occurred in the preparation of compounds 21 and 22.
RESULTS AND DISCUSSION
The key intermediates 29a−29e were synthesized as
described in Scheme 2. Iodination of commercially available
2-methoxy-6-nitroaniline (23) with I2 afforded 24 in 71%
yield. Diazotization of 24 followed by chlorination led to
intermediate 25 in 88% yield. Cu-catalyzed Sonogashira
coupling of iodide 25 with trimethylsilylacetylene yielded 26
in 64% yield. Subsequent deprotection delivered 27a in 93%
yield. By following a similar literature procedure,24 27a−27d
were treated with tert-butyl (E)-(4-aminobut-2-en-1-yl)-
carbamate affording 28a−28d in 26−52% yields, respectively.
The click reaction of 28a with 1-azido-2-(2-methoxyethoxy)-
ethane yielded 29a in 52% yield. In the meantime, preparation
of intermediate 29b was achieved by treating 28b with 2,2-
dimethoxyethan-1-amine in 56% yield. Treating 28c with
DMF-DMA or 1,1-dimethoxy-N,N-dimethylethan-1-amine
gave corresponding methylene benzamide derivatives, which
were then subjected to cyclization with hydrazine acetate to
afford compounds 29c and 29d in 64 and 49% yields,
■
Chemistry. The synthesis of compounds 16−19 and 21
and 22 bearing at least one flexible chain to replace the
terminal pyrazole-5-carboxamide component is shown in
Scheme 1. The intermediate 9 was obtained in 55% yield by
alkylation of 3-methyl-1H-pyrazole-5-carbaldehyde (7) with
bromoethane (8). The subsequent Horner−Wadsworth−
Emmons reaction of intermediate 9 with triethyl phosphonoa-
cetate delivered intermediate 10 in 44% yield. Hydrolysis of
ester 10 using LiOH·H2O afforded acrylic acid 11 in 49%
yield. Hydrogenation of 10 led to intermediate 12 in 84%
yield, which was then hydrolyzed to provide propanoic acid 13
in 76% yield. Condensation of the intermediate 14, prepared
according to a literature procedure,24 with acids 11, 13, or 2-
oxopentanedioic acid (15) was sluggish, and the corresponding
products 16−19 were obtained in 13−21% overall yields. It is
of note that different solvents affected the formation of
compounds 18 and 19. The reaction with water as the solvent
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a
Scheme 2. Synthesis of Intermediates 29a−29e
a
Reactions and conditions: (i) I2, Ag2SO4, EtOH, and rt; (ii) TsOH·H2O, NaNO2, CuCl, MeCN, H2O, and 0 °C to rt; (iii) ethynyltrimethylsilane,
PdCl2(PPh3)2, CuI, DIPEA, THF, and 0 °C to rt; (iv) K2CO3, MeOH, and rt; (v) tert-butyl (E)-(4-aminobut-2-en-1-yl)carbamate, DIPEA, EtOH,
120 °C, and sealed tube; (vi) for 29a: 1-azido-2-(2-methoxyethoxy)ethane, sodium ascorbate, CuSO4·5H2O, t-BuOH/H2O = 2:1, and rt; for 29b:
2-azidoethan-1-ol, sodium ascorbate, CuSO4·5H2O, t-BuOH/H2O = 2:1, rt; (vii) NaOMe, MeOH, and rt; (viii) 2,2-dimethoxyethan-1-amine,
AcOH, and 50 °C; (ix) for 29d: DMF-DMA and 100 °C; for 29e: 1,1-dimethoxy-N,N-dimethylethan-1-amine, 100 °C; (x) hydrazine acetate,
EtOH, and 50 °C; (xi) 2-aminoethan-1-ol, MeOH, and 70 °C; (xii) SOCl2, DCM, and 0 °C to rt; and (xiii) NaHCO3, H2O, and 0 °C to rt.
respectively.The ammonolysis reaction of ester 28d with 2-
aminoethan-1-ol produced amide 30 in 85% yield, which was
then chlorinated with SOCl2 followed by cyclization under a
basic condition to deliver 29e in 45% overall yield over two
steps.
Dehydration of 32a with POCl3 afforded 32b in 82% yield.
The intermediates 32c and 32d were obtained by following
similar procedures to those for preparation of 29b and 29c in
58 and 72% yields, respectively (Scheme 3). Substitution of
29a−29e with 32a−32d produced 33a−33l in 31−50% yields.
Reduction of the nitro group in 33a−33l followed by
imidazole ring fusion using 1-ethyl-3-methyl-1H-pyrazole-5-
carbonyl isothiocyanate was conducted by following a
literature procedure.24 The dimeric compounds 34−45 were
obtained in 27−42% yields over two steps.
As described in Scheme 4, compound 48 was obtained by a
procedure similar to that for preparation of compounds 34−
45. It has to be mentioned that the same synthetic route was
not suitable for synthesis of compounds 55 and 56. Instead,
the intermediate 50 needs to be prepared first. Reduction of 49
with sodium dithionite followed by treating with 1-ethyl-3-
methyl-1H-pyrazole-5-carbonyl isothiocyanate in the presence
of EDCI and Et3N achieved compound 50. Subsequent
deprotection of 50 followed by substitution with halogenated
compounds 51 and 52 yielded compounds 53 and 54 in 41
and 38% overall yields, respectively. The target products 55
and 56 were prepared in 25 and 31% yields, respectively, by
following literature procedures.24 Dimeric compounds 64−66
with structural modifications on the linker were implemented
as shown in Scheme 5 by following similar procedures as
described above.
Activation of New Compounds on Both h- and m-
STING. To quickly identify potent STING agonists for further
study, we first determined activation folds of new compounds
against h- and m-STING by respectively measuring the
secreted embryonic alkaline phosphatase (SEAP) reporter of
interferon-stimulated gene (ISG) in human THP1-Blue-ISG
cells and the secreted luciferase reporter of ISG in mouse
RAW-Lucia cells, compared to the control. In our initial
experiments, we found that compound 5 (diABZI) is more
sensitive for h-STING and less sensitive for m-STING.
Therefore, we screened the activation potency of new
compounds by treating with human THP1-Blue-ISG cells at
a concentration of 10 μM whereas with mouse RAW-Lucia
cells at higher concentrations of 50 μM.
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a
Scheme 3. Synthesis of Compounds 34−45
a
Reactions and conditions: (i) POCl3 and 100 °C; (ii) NaOMe, MeOH, and rt and then 2,2-dimethoxyethan-1-amine, AcOH, and 50 °C; (iii)
DMF-DMA and 100 °C and then hydrazine acetate, EtOH, and 50 °C; (iv) 4 N HCl in 1,4-dioxane, MeOH, and rt; (v) DIPEA, i-PrOH, 120 °C,
sealed tube, and 2 days; and (vi) (a) Na2S2O4, NH3·H2O, THF/H2O = 1:1, and rt and (b) 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl
isothiocyanate, EDCI, Et3N, DMF, and 0 °C to rt.
As shown in Table 1, we first tested compounds from
modification of the readily tautomeric pyrazole-5-carboxamide
component of compound 5 by changing the amido linkage
with an ethylenyl or ethyl moiety. The resulting compounds 16
and 17 showed no activation against both h- and m-STING
with activation fold less than 1.0 compared to the control,
whereas the reference compound 5 showed approximately 26-
fold and 8-fold activation against h-STING (10 μM) and m-
STING (50 μM), respectively. Interestingly, compound 18
bearing a 3-(methoxycarbonyl)propionyl moiety to replace the
pyrazole-5-carboxamide component of 5 retained significant
activation in the human THP1-Blue-ISG cells with an
activation fold of 6.16; however, nearly no activation was
observed in the mouse RAW-Lucia cell. Compound 19,
bearing terminal carboxylic groups in the tails of the dimer,
retained a similar activation fold against h-STING (9 fold) but
was still inactive against m-STING. Meanwhile, we selectively
replaced one pyrazole-5-carboxamido edge and kept the other
one intact, yielding acid 21 and ester 22, respectively. Both
compounds lost activation against both h- and m-STING.
These results indicate that the two down-side pyrazole-5-
carboxamido edges are not only important to the activation
potency but also highly sensitive to different species.
Next, we modified the two up-side primary amide moieties
of 5 with the aim to reduce the polarity and increase stability.
As shown in Table 2, replacement of the two amido functions
with two cyano groups afforded compound 34, which retained
good activation against h-STING with an activation fold of 7.9
at 10 μM but showed no activation against m-STING at 50
μM. Conversion of the two amido moieties to heterocyclic
triazole and imidazole motifs yielded compounds 35 and 36,
respectively, which showed distinct activation patterns.
Triazole 35 showed significant activation against h-STING
(4.9-fold) and no activation against m-STING. However,
imidazole 36 only showed marginal activation for h-STING
(1.22-fold) and no activation for m-STING. Interestingly,
compound 37 carrying a triazole on one side and a cyano on
the other side also showed strong activation against h-STING
with an activation fold of 11.10, whereas the activation for m-
STING was negligible.
Meanwhile, we also tested compounds with only one of the
terminal amido groups in 5 replaced by other functional
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a
Scheme 4. Synthesis of Compounds 48, 55, and 56
a
Reactions and conditions: (i) DIPEA, i-PrOH, 120 °C, sealed tube, and 2 days; (ii) (a) Na2S2O4, NH3·H2O, THF/H2O = 1:1, and rt; (b) 1-ethyl-
3-methyl-1H-pyrazole-5-carbonyl isothiocyanate, EDCI, Et3N, DMF, and 0 °C to rt; and (c) TBAF, AcOH, THF, and 0 °C to rt.
groups. As shown in Table 3, using a heterocycle to mask the
left-side amido group but retain the H-bonding ability
generated compounds 38−42. All these analogues showed
markedly high activation for h-STING with activation folds
ranging between 17.25 and 19.21, whereas their activation for
m-STING is moderate. Triazole 40 generated greater
activation than imidazole 38 and dihydrooxazole 39 with
activation folds of 19.21, 17.50, and 18.30, respectively, for h-
STING, indicating that the triazole moiety can well mimic
both H-bonding donor and acceptor properties of the amido
motif. Importantly, compared to 38 and 39, triazole 40 also
showed modest activation for m-STING at both 50 μM with
an activation fold of 3.20. Further substitution on the triazole
moiety yielded compounds 41 and 42, all retaining significant
activation for h-STING with activation folds of 18.75 and
17.25, respectively; however, the activation for m-STING was
reduced. As a comparison, we also kept the left-side amido
moiety of compound 5 intact but replaced the right-side one,
leading to compounds 43−45. All three compounds still
retained significant activation on h-STING with activation
folds of 13.38−17.31; however, again, only the triazole 44
showed certain stimulation on m-STING with an activation
fold of 2.17 at 50 μM.
Since triazoles 40 and 44 are able to activate both h- and m-
STING, we further optimized this compound by incorporating
aqueous substituents or replacing the but-2-ene linker with
other variations. As shown in Table 4, introducing a water
soluble hydroxypropyl chain delivered compound 48, which
retained significant activation on h-STING with an activation
fold of 16.66; however, no activation was observed on m-
STING. Fusing 1,4-dioxane to the benzamide component led
to compound 55, which nearly completely lost the activation
effects on both h- and m-STING. Notably, compound 56
obtained by fusing a furan moiety to the benzamide
component exhibited high activation on h-STING with an
activation fold of 17.04; unfortunately, no obvious activation
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a
Scheme 5. Synthesis of Compounds 64−66
a
Reactions and conditions: (i) (a) DMF-DMA and 100 °C and (b) hydrazine acetate, EtOH, and 50 °C; (ii) 4 N HCl in 1,4-dioxane, MeOH, and
rt; (iii) DIPEA, EtOH, sealed tube, and 120 °C; (iv) DIPEA, i-PrOH, 120 °C, sealed tube, and 2 days; and (v) (a) Na2S2O4, NH3·H2O, THF/H2O
= 1:1, and rt and (b) 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate, EDCI, Et3N, DMF, and 0 °C to rt.
on m-STING was observed. Switching the but-2-ene linkage to
butyl or cyclohexyl afforded compounds 64−66; however, all
showed marginal activation on both h- and m-STING.
EC50 Values of Selected Compounds against Both h-
and m-STING. With the preliminary data above, we further
tested the compounds that showed obvious stimulatory effects
on h- and/or m-STING at various concentrations and
calculated their EC50 values on h-STING or m-STING by
measuring the secreted luciferase reporter of interferon-
stimulated gene (ISG) in human THP1-Dual cells and
mouse RAW-Lucia cells, respectively. As shown in Table 5,
compound 5 is extremely potent for h-STING with an EC50
value less than 0.1 nM, whereas the activation potency for m-
STING is modest by showing an EC50 value of 3.0 μM.
Compounds 18, 34, and 37, in which both the two down-side
pyrazole-5-carboxamide components or the two up-side
benzamido moieties were replaced, showed reduced activation
effects with EC50 values of 29.1, >50, and 26.0 μM,
respectively, for h-STING, and there is no activation for m-
STING (EC50 > 50 μM). Imidazole 38, dihydrooxazole 39,
and methyl triazole 41 all showed modest potency on h-
STING with EC50 values of 3.28, 1.54, and 2.55 μM,
respectively; however, they were inactive on the m-STING.
In parallel to the preliminary results, triazole 40 showed a high
activation effect on the h-STING, with an EC50 value of 0.24
μM, only slightly less potent than compound 5. To be glad,
this compound showed somewhat activation on the m-STING,
although the activation potency is rather modest with an EC50
value of 39.51 μM.
Among the right-side amido-replacing analogues, triazole 44
remains as more potent than nitrile 43 and imidazole 45 with
EC50 values of 0.33, 1.02, and 0.79 μM, respectively, but
triazole 44 is less potent than the triazole 40 (0.33 vs 0.24
μM). Compounds 43 to 45 showed negligible potency on m-
STING with EC50 values greater than 50 μM. The analogues
48 and 66 showed much less activation effects at both h- and
m-STING.
From the results above, triazole 40 turns out as the most
potent STING activator with EC50 values of 0.24 and 39.51
μM for h- and m-STING, respectively. This compound is less
potent than compound 5; however, in view of the well-
accepted drug-like properties and safety profile of triazole
compounds in drug discovery, compound 40 was selected for
further profiling, side-by-side with the reference compound 5.
Meanwhile, a docking study was also conducted (Figures S1
and S2, see the Supporting Information). It was found that the
triazole moiety in compound 40 is an excellent substituent for
the amido group of compound 5. This component forms a
hydrogen bond at the top with Ser241 and has a π−π
interaction with Try240. The nitrogen atom in the pyrazole
ring forms a new hydrogen bond with Ser162. This result
partially rationalizes the high potency of the triazole 40.
Pharmacokinetic Properties and Aqueous Solubility
Study of Compound 40. Since the CDN analogues, such as
2′,3′-cGAMP and ADU-S100, are mainly administered by
intratumoral (IT) administration, therefore, we first tested the
pharmacokinetic (PK) properties of triazole compound 40 in
rats. As shown in Table 6, compound 40, in comparison to
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a
Table 1. Activation Folds of New Compounds against m- and h-STING
a
Data were the average values of at least two repeated experiments.
compound 5, shows an overall similar PK profile in rats at 1
mg/kg iv injection, with a slightly higher plasma exposure (678
vs 657 h ng/mL) and slightly lower clearance (23.9 vs 26 mL/
min/kg).
In the meantime, we also tested the aqueous solubility of
compounds 40 and 5 by conversion to their corresponding
hydrochloride salts. As shown in Table 7, compared to the
modest solubility of 5, the HCl salt form of compound 40 is
completely aqueously soluble and its solubility is >20-fold
higher than that of the HCl salt form of 5 (71.33 vs 3.23 mg/
L). In addition, despite the high loading of the N atom, both
compounds showed no obvious hERG inhibition with IC50
values greater than 40 μM, indicating their negligible cardiac
toxicity.
Compound 40 Potently Activated the STING Signal-
ing. The THP1-Dual cell bearing both the ISG reporter and
NF-κB reporter was used to determine the activation of
compound 40 on STING signaling. As shown in Figure 2A,B,
compound 40 dramatically activated the ISG signaling and NF-
κB signaling in a dose-dependent manner. The reference
compound 5 also showed high potency for both signaling
processes; however, a dose-dependent manner is not obvious.
Next, we determined the cytokine secretion of THP1-Dual
cells under treatment with compound 40 or 5. As expected,
both compounds dramatically increased the secretion of IP-10
and IFN-β at tested concentrations (Figure 2C,D). At a high
concentration of 10 μM, compounds 5 and 40 exhibited
similar high potency on IP-10 and IFN-β secretion, while at
lower concentrations (0.1 and 0.01 μM), compound 5 showed
more potency on IP-10 secretion than 40.
Meanwhile, the STING downstream signaling proteins were
assessed as well after treatment with compound 40 or 5 for 4 h.
As shown in Figure 2E, both compounds remarkably increased
the phosphorylation of the TBK1/IRF3 signaling pathway in a
dose-dependent manner, demonstrating the activation of
STING signaling by the two compounds. Furthermore, the
STING-knockout THP1-Dual cells (THP1-Dual-KO-STING)
were used to evaluate the specificity of compounds. Treatment
with this cell showed that knockout of STING completely
blocked the activation on ISG signaling (Figure 2F), indicating
the specificity of both compounds 40 and 5 on the STING
signaling.
Compound 40 Directly Activated Human STING
Isoforms and Mouse STING. To confirm whether
compound 40 could directly target on STING protein, we
used the differential scanning fluorimetry (DSF) assay to test
the binding of this compound on various human STING
isoforms and mouse STING. As shown in Figure 3A−E, the
Tm value of the melt curve was increased after treatment with
compound 40 as compared to the vehicle, similar to that of the
positive control 5. The melt curve shifted to higher
temperature, indicating that compounds 40 and 5 can directly
bind and stabilize human STING protein isoforms,29 including
hSTING-R232, hSTING-H232, hSTING-293Q, and hSTING-
AQ, as well as mouse wild-type STING protein R231.
Combining these findings with the previous results on
THP1-Dual cells bearing STING isoform of HAQ, we
concluded that compound 40 has the capacity of activating
all the five main STING isoforms. In addition, the binding
ability of compounds 40 and 5 to human wild-type STING
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a
Table 2. Activation Folds of Compounds against h- and m-STING
a
Data were the average values of at least two repeated experiments.
(R232) was determined using a commercial competition assay
kit by homogeneous time-resolved fluorescence (HTRF)
technology. Compound 40 has an EC50 value of 6.2 nM,
compatible to the endogenous ligand 2′,3′-cGAMP (5.8 nM),
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a
Table 3. Activation Folds of Compounds against h- and m-STING
a
Data were the average values of at least two repeated experiments.
while the EC50 value of compound 5 was extremely low (<0.1
nM) (Figure S3, see the Supporting Information). To further
determine the activation of this compound on different STING
isoforms, we used 293-Dual-hSTING-R232 cells and 293-
Dual-hSTING-H232 cells to determine its activation on
hSTING-R232 and hSTING-H232, respectively. The results
showed that compound 40 potently activated ISG signaling in
a dose-dependent manner in both two reporter cells (Figure
3F,G), while compound 5 exhibited high activity as well on
hSTING-R232 and hSTING-H232 but without an obvious
dose-dependent manner. In addition, activation of compound
40 on mouse STING protein was also tested using both RAW-
Lucia and RAW-Lucia-KO-STING cells. Interestingly,
although compound 40 is 13-fold less potent than compound
5 against m-STING with EC50 values of 39.51 and 3 μM,
respectively (Table 5), both compounds dramatically activated
the ISG signaling in RAW-Lucia cells. Further, no activity was
observed in RAW-Lucia-KO-STING cells (Figure 3H),
demonstrating compound 40 specifically targeted on mouse
STING signaling.
bearing orthotopic 4T1 tumors were intravenously (iv)
received comparable dose of compound 40 or 5 at 10 mg/
kg at days 1, 4, and 7. Compound 40 was found to dramatically
suppress the tumor growth as compared to the vehicle control
(Figure 4A). Although compound 5 was found more potent
and induced tumor regression, unfortunately, high toxicity was
observed and all the tested mice were dead after the third
injection, indicating that a lower dose should be used for
compound 5 to avoid potential overdose toxicity.24 Compound
40 was well tolerated, although slight weight loss was observed
after the third injection but mice were recovered well afterward
(Figure 4B).
Moreover, we also tested the effects of both compounds on
the CT26 colon tumor model by intratumoral (IT) injection at
days 1, 4, and 7, using the widely used m-STING specific
agonist 2 (DMXAA, Figure 1) as a comparison. As shown in
Figure 4C, compounds 40 and 2 both inhibited CT26 tumor
growth significantly. The effect of 2 is more sustained than that
of 40. The IT injection of both compounds was well tolerated
as assessed by body weight (Figure 4D). These results
demonstrated that compound 40 is well tolerated and exerts
potent antitumor efficacy in allograft tumor models of mice.
Antitumor Activity of Compound 40 in Animal
Models. To evaluate whether the observed immune activating
effect of compound 40 in cells can transform to potent
antitumor activity in vivo, mice models bearing orthotopic
transplanted breast tumor (4T1) or subcutaneous transplanted
colon tumor (CT26) were used for study. First, BALB/c mice
CONCLUSIONS
■
In summary, activation of STING by agonists has emerged as
an exciting strategy of IO therapy for cancer; however, the first-
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a
Table 4. Activation Folds of Triazole Analogues against h- and m-STING
a
Data were the average values of at least two repeated experiments.
advantage of the unique dimeric structure of the high potency
STING agonist 5 (diABZI) bearing two amidobenzimidazole
motifs, we conducted a structural elaboration by modifying
several structural hotspots of this dimeric scaffold, including
the readily tautomeric N-acyl 1-ethyl-3-methyl-1H-pyrazole-5-
imino component, the polaric terminal amido moiety, and the
linkage. Several series of analogues were synthesized, and their
SAR was evaluated against both h- and m-STING. All these
compounds were found generally equally sensitive for h-
STING but much less effective for m-STING. Triazole 40 was
found as the most potent STING activator with EC50 values of
0.24 and 39.51 μM for h- and m-STING, respectively. This
compound possesses a slightly better PK profile and is >20-fold
more aqueously soluble than the reference compound 5 both
in their HCl salt form. Activation of the STING signaling by
compound 40 was evidenced in the THP1-Dual cell bearing
both the ISG reporter and NF-kB reporter. Treatment with
this compound was found to dramatically promote secretion of
IP-10 and IFN-β and increase the phosphorylation of STING
downstream TBK1/IRF3 signaling pathway in THP1-Dual
cells, whereas similar activation was not observed in the
STING-knockout cells. Further, compound 40 was found to
Table 5. EC50 Values of Selected Compounds against Both
h- and m-STING
a
compound
EC50, μM (h-STING)
EC50, μM (m-STING)
5 (diABZI)
<0.0001
29.10 4.52
>50
3.00 1.44
>50
>50
>50
>50
>50
39.51 9.85
>50
>50
>50
18
34
37
38
39
40
41
43
44
45
48
66
26.0 8.86
3.28 0.10
1.54 0.46
0.24 0.03
2.55 0.27
1.02 0.22
0.33 0.05
0.79 0.04
2.66 0.13
>50
>50
>50
>50
a
Data were the average values of at least two repeated experiments.
generation STING agonists, (CDN) analogues, have met
multiple limitations. Therefore, low-weight non-CDN small-
molecule STING agonists are urgently needed. To take
a
Table 6. Pharmacokinetic Parameters of Compound 40
iv (1 mg/kg)
compound
T1/2 (h)
AUClast (h ng/mL)
AUCINF_obs (h ng/mL)
CL_obs (mL/min/kg)
MRTINF_obs (h)
Vss_obs (mL/kg)
5
40
1.14
0.697
657
678
666
705
26
23.9
0.633
0.503
965
722
a
Values are the average of three runs. Vehicle: po, DMSO/0.5% HPMC (5/95, v/v); iv, EtOH/PEG300/NaCl (10/40/50, v/v/v); CL, clearance;
Vss, volume of distribution; T1/2, half-life; and AUC, area under the plasma concentration time curve.
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a
Table 7. Aqueous Solubility and hERG Inhibition of Compound 40
standard aqueous solution
saturated aqueous solution
compound
concentration (mg/mL)
area (mAU s)
area (mAU s)
solubility (mg/mL)
hERG (IC50, μM)
HCl salt of 5
HCl salt of 40
2.26
4.81
1.96212 × 104
4.7300 × 104
2.8048 × 104
7.01476 × 105
3.23
71.33
>40
>40
a
Values are the average of three runs.
Figure 2. Compound 40 potently activated the STING signaling pathway. (A) Activation of compounds on the ISG reporter of THP1-Dual cells
after 24 h treatment, fold change of activation was calculated to the vehicle control. (B) Activation of compounds on the NF-κB reporter of THP1-
Dual cells after 24 h treatment, fold change of activation was calculated to the vehicle control. (C) IP-10 secretion of THP1-Dual cells under the
treatment of compounds for 24 h. (D) IFN-β secretion of THP1-Dual cells under the treatment of compounds for 24 h. (E) THP1-Dual cells were
treated with indicated compounds for 4 h and determined by immunoblotting. (F) Activity of compound on the ISG reporter in THP1-Dual and
THP-Dual-KO-STING cells after 24 h treatment, fold change of activation was calculated to the vehicle control.
recorded with a Varian Mercury 300 or 400 MHz NMR spectrometer.
Chemical shifts (δ) were reported in ppm downfield from an internal
TMS standard, and J values were given in Hz. Low- and high-
resolution mass spectra were obtained in the ESI mode from an Elite
mass spectrometer. Purity of final compounds was determined by
analytical HPLC, which was carried out on an Agilent Technologies
1260 series LC system with ultraviolet wavelengths in UV 254. HPLC
analysis conditions are as follows: XDB-C18, 3.5 μm, 4.6 mm × 150
mm, and H2O/MeOH or H2O/MeCN and 0.1% TFA or 0.1% DEA.
All the assayed compounds showed a chemical purity of 95−100%.
Compounds 14 and 20 were prepared according to the literature
procedures.24,28
directly bind and stabilize all isoforms of human STING
proteins, including hSTING-R232, hSTING-H232, hSTING-
293Q, and hSTING-AQ, as well as mouse STING protein
R231. In vivo, intermittent administration of 40 showed
significant antitumor efficacy and good safety both in BALB/c
mice bearing orthotopic 4T1 breast tumor and in CT26 colon
tumor models.
EXPERIMENTAL SECTION
■
Chemical Reagents and General Method. All commercially
available starting materials and solvents are reagent grade and used
without further purification. Column chromatography was performed
using 300−400 mesh or 200−300 mesh silica gel. Analytical TLC was
carried out employing silica gel 60 F254 plates, and spots were
1-Ethyl-3-methyl-1H-pyrazole-5-carbaldehyde (9). To a solution
of compound 7 (550 mg, 5.0 mmol) and Cs2CO3 (2.44 g, 7.5 mmol)
in DMF (10 mL) was added bromoethane (708 mg, 6.5 mmol) under
a N2 atmosphere. The mixture was stirred at room temperature for 2 h
1
visualized by UV (254 or 365 nm). H and 13C NMR spectra were
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Figure 3. Compound 40 directly activated various h-STING isoforms and m-STING. (A−E) Binding of compounds on human STING isoform
R232, human STING isoform H232, human STING isoform 293Q, human STING isoform AQ, and mouse STING R231 (WT), respectively, as
measured by DSF, shown as the mean value and standard error of mean of the fluorescence, n = 3. (F, G) Activation of compound on the ISG
reporter in 293T-Dual-hSTING-R232 and 293T-Dual-hSTING-H232 cells after 24 h treatment, fold change of activation was calculated to the
control. (H) Activation of compound on the ISG reporter in RAW-Lucia and RAW-Lucia-KO-STING cells after 24 h treatment, fold change of
activation was calculated to the control.
and then diluted with water (60 mL). After extraction with EtOAc (3
× 50 mL), the combined organic phase was washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was
further purified by silica gel column chromatography to afford 9 (378
mg, 55%). 1H NMR (400 MHz, CDCl3): δ 9.87 (s, 1H), 6.52 (s, 1H),
4.15 (q, J = 7.3 Hz, 2H), 1.45 (t, J = 7.3 Hz, 3H).
Ethyl (E)-3-(1-Ethyl-3-methyl-1H-pyrazol-5-yl)acrylate (10). To a
solution of compound 9 (348 mg, 2.5 mmol) and ethyl 2-
(diethoxyphosphoryl)acetate (582 mg, 2.6 mmol) in DMF (10 mL)
at 0 °C was added NaH (90 mg, 3.75 mmol). The mixture was stirred
at room temperature overnight and then quenched with saturated
NH4Cl solution. After extraction with EtOAc (3 × 50 mL), the
combined organic phase was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was further purified
by silica gel column chromatography to afford 10 (213 mg, 44%). 1H
NMR (400 MHz, DMSO): δ 7.42 (d, J = 16.0 Hz, 1H), 6.52 (s, 1H),
6.38 (d, J = 16.0 Hz, 1H), 4.16 (q, J = 7.1 Hz, 2H), 4.07 (q, J = 7.2
Hz, 2H), 2.27 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.1 Hz,
3H).
General Procedure for Synthesis of Compounds 11 and 13.
To a solution of compound 10 or 12 (1.0 mmol) in MeOH/H2O (6
mL, 1:1) was added LiOH·H2O (3 mmol). The mixture was stirred at
room temperature for 2 h. The mixture was concentrated in vacuo.
The residue was acidified with 1 N HCl aqueous solution and
extracted with EtOAc. The combined organic phase was washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
crude products 11 and 13 were obtained in 48−76% yields and used
in the next step without further purification.
General Procedure for Synthesis of Compounds 16 and 17.
The solution of compound 14 (0.1 mmol), EDCI (58 mg, 0.3 mmol),
HOBt (40 mg, 0.3 mmol), and DIPEA (38 mg, 0.3 mmol) in DMF (1
mL) was stirred at room temperature for 30 min under a N2
atmosphere. The crude compound 11 or 13 (0.24 mmol) was
added to the reaction mixture and then stirred overnight. The mixture
was concentrated in vacuo followed by addition of acetic acid (0.7
mL). The reaction was heated to 90 °C for 2 h and then diluted with
water (10 mL). After extraction with EtOAc (3 × 10 mL), the
combined organic phase was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was further purified
by silica gel column chromatography (CH2Cl2/NH3 7.0 M solution in
CH3OH from 50:1 to 15:1) to afford desired compounds 16 and 17.
1-((E)-4-(5-Carbamoyl-2-((E)-2-(1-ethyl-3-methyl-1H-pyrazol-5-
yl)vinyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-
2-en-1-yl)-2-((E)-2-(1-ethyl-3-methyl-1H-pyrazol-5-yl)vinyl)-7-me-
thoxy-1H-benzo[d]imidazole-5-carboxamide (16). White solid;
Ethyl 3-(1-Ethyl-3-methyl-1H-pyrazol-5-yl)propanoate (12). To a
solution of compound 10 (213 mg, 1.1 mmol) in MeOH (5 mL) was
added 5% Pd/C (212 mg). After hydrogenation at room temperature
for 8 h, the mixture was filtered and concentrated in vacuo to deliver a
light yellow oil in 84% yield, which was then used in the next step
without further purification.
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Figure 4. In vivo antitumor effect of compound 40. (A) Tumor growth in 4T1 orthotopic BALB/c mouse model, compounds 40 and 5 were
intravenously injected at days 1, 4, and 7 at a dose of 10 mg/kg. (B) Body weight of 4T1 allograft BALB/c mouse model. (C) Tumor growth in
CT26 subcutaneous BALB/c mouse model, compounds 40 and 5 were intratumorally injected at days 1, 4, and 7 at indicated dosages. (D) Body
weight of the CT26 allograft mouse model. One-way ANOVA analysis, *P < 0.05 and **P < 0.01, as compared to the vehicle.
1
yield: 13%. HPLC purity: 97.5%. H NMR (400 MHz, DMSO): δ
7.95 (br, 2H), 7.81 (s, 1H), 7.79 (s, 1H), 7.59 (d, J = 15.3 Hz, 1H),
7.55 (d, J = 14.6 Hz, 1H), 7.26 (br, 2H), 7.24 (s, 1H), 7.17 (s, 1H),
7.09 (d, J = 16.2 Hz, 1H), 7.02 (d, J = 15.6 Hz, 1H), 6.44 (s, 1H),
6.32 (s, 1H), 5.66 (s, 2H), 5.13 (s, 4H), 4.08 (m, 4H), 3.96 (s, 2H),
3.74 (s, 3H), 3.46 (s, 4H), 2.26 (m, 8H), 2.16 (s, 4H), 1.65 (m, 2H),
1.32 (m, 6H). MS (ESI, [M + H]+) m/z, 816.6. HRMS (ESI) calcd
147.11, 146.41, 132.98, 132.93, 129.19, 129.12, 127.47, 127.05,
124.59, 124.47, 121.38, 121.26, 112.03, 111.68, 68.14, 66.02, 56.71,
54.70, 53.14, 51.36, 46.61, 46.50, 29.13, 28.01, 25.25. MS (ESI, [M +
H]+) m/z, 776.4. HRMS (ESI) calcd for C38H46N7O11+, 776.3250;
found, 776.3267.
Methyl 4-(5-Carbamoyl-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-
methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-
1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopro-
poxy)-1H-benzo[d]imidazol-2-yl)-4-oxobutanoate (22). White
solid; yield: 20%. HPLC purity: 95.7%. 1H NMR (400 MHz,
CD3OD): δ 7.87 (d, J = 1.9 Hz, 1H), 7.62 (s, 1H), 7.40 (s, 1H), 7.32
(s, 1H), 6.58 (s, 1H), 5.81 (d, J = 16.6 Hz, 1H), 5.71 (d, J = 15.9 Hz,
1H), 5.25 (s, 2H), 4.96 (d, J = 5.2 Hz, 2H), 4.60 (br, 4H), 3.81 (s,
5H), 3.70 (s, 2H), 3.67 (s, 3H), 3.16 (t, J = 6.8 Hz, 2H), 2.85 (t, J =
6.8 Hz, 2H), 2.61 (s, 6H), 2.20 (s, 3H), 1.79 (s, 2H), 1.38−1.32 (m,
3H). MS (ESI, [M + H]+) m/z, 813.4. HRMS (ESI) calcd for
+
for C44H54N11O5 , 816.4304; found, 816.4292.
(E)-1-(4-(5-Carbamoyl-2-(2-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-
ethyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-
en-1-yl)-2-(2-(1-ethyl-3-methyl-1H-pyrazol-5-yl)ethyl)-7-methoxy-
1H-benzo[d]imidazole-5-carboxamide (17). White solid; yield: 16%.
HPLC purity: 95.9%. 1H NMR (400 MHz, CDCl3 + CD3OD): δ 7.78
(s, 2H), 7.12 (s, 1H), 7.08 (s, 1H), 5.79 (s, 1H), 5.73 (s, 1H), 5.58−
5.48 (m, 1H), 5.46−5.36 (m, 1H), 4.88 (d, J = 4.6 Hz, 2H), 4.83 (d, J
= 5.1 Hz, 2H), 4.08−3.96 (m, 4H), 3.92 (s, 4H), 3.67 (m, 2H), 3.52
(s, 3H), 3.08 (s, 4H), 3.04 (d, J = 4.6 Hz, 4H), 2.98 (m, 4H), 2.70
(br, 2H), 2.23 (s, 3H), 2.21 (s, 3H), 1.79 (m, 2H), 1.41−1.33 (m,
6H). 13C NMR (126 MHz, CDCl3): δ 170.53, 155.88, 155.79, 149.42,
149.26, 146.37, 145.10, 142.99, 142.73, 138.67, 128.51, 128.37,
128.08, 127.55, 126.18, 111.63, 111.12, 104.35, 104.17, 103.58, 65.54,
64.47, 55.28, 54.50, 52.16, 46.03, 45.74, 43.45, 29.58, 27.05, 25.97,
15.29, 10.72. MS (ESI, [M + H]+) m/z, 820.6. HRMS (ESI) calcd for
+
C40H49N10O9 , 813.3678; found, 813.3668.
General Procedure for Synthesis of Compounds 19 and 21.
To a solution of compound 14 or 20 (0.068 mmol) and 15 (32 mg,
0.27 mmol) in H2O (1 mL) was added 4 N HCl aqueous solution (1
mL). The mixture was stirred at 100 °C for 1 h. A white solid was
formed and then filtered. The white solid obtained was washed with
EtOH followed by vacuum drying to afford compound 19 or 21.
(E)-4-(5-Carbamoyl-1-(4-(5-carbamoyl-2-(3-carboxypropanoyl)-
7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-
yl)-7-methoxy-1H-benzo[d]imidazol-2-yl)-4-oxobutanoic Acid (19).
+
C44H58N11O5 , 820.4617; found, 820.4602.
General Procedure for Synthesis of Compounds 18 and 22.
To a solution of compound 14 or 20 (0.068 mmol) and 15 (32 mg,
0.27 mmol) in MeOH (1 mL) was added 4 N HCl aqueous solution
(1 mL). The mixture was stirred at 100 °C for 1 h and then
concentrated in vacuo. The residue was further purified by silica gel
column chromatography (CH2Cl2/NH3 7.0 M solution in CH3OH
from 50:1 to 20:1) to afford 18 or 22.
1
White solid; yield: 17%. HPLC purity: 95.3%. H NMR (400 MHz,
DMSO): δ 12.16 (br, 2H), 9.77 (br, 1H), 8.20 (d, J = 3.3 Hz, 2H),
7.98 (d, J = 1.8 Hz, 1H), 7.93 (d, J = 1.9 Hz, 1H), 7.64 (d, J = 1.9 Hz,
1H), 7.61 (d, J = 2.0 Hz, 1H), 7.51 (s, 2H), 5.73 (m, 1H), 5.60 (m,
1H), 5.19−5.07 (m, 2H), 5.00 (dd, J = 4.7, 2.1 Hz, 2H), 4.12 (s, 2H),
4.00 (br, 2H), 3.72 (s, 3H), 3.66 (br, 2H), 3.42 (br, 2H), 3.23 (br,
2H), 3.08 (br, 2H), 3.04 (t, J = 7.0 Hz, 4H), 2.73 (m, 4H), 2.08 (br,
2H). 13C NMR (126 MHz, DMSO): δ 174.31, 174.24, 166.86,
166.78, 159.82, 159.71, 155.34, 155.08, 147.66, 146.59, 133.50,
133.46, 129.68, 129.55, 127.68, 127.17, 125.16, 125.00, 122.10,
121.80, 112.86, 112.16, 67.77, 63.95, 57.31, 54.09, 51.76, 47.46, 47.04,
29.82, 28.59, 28.54, 23.49. MS (ESI, [M + H]+) m/z, 748.3. HRMS
(ESI) calcd for C36H42N7O11+, 748.2937; found, 748.2934.
Methyl (E)-4-(5-Carbamoyl-1-(4-(5-carbamoyl-2-(4-methoxy-4-
oxobutanoyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-
yl)but-2-en-1-yl)-7-methoxy-1H-benzo[d]imidazol-2-yl)-4-oxobuta-
1
noate (18). White solid; yield: 19%. HPLC purity: 95.4%. H NMR
(400 MHz, DMSO): δ 8.19 (s, 1H), 7.89 (s, 2H), 7.60 (s, 1H), 7.58
(s, 1H), 7.49 (s, 1H), 5.67 (m, 2H), 5.09 (s, 2H), 5.02 (s, 2H), 3.98
(s, 2H), 3.72 (s, 3H), 3.60 (s, 6H), 3.52 (s, 4H), 3.07 (m, 4H), 2.79
(m, 4H), 2.23 (br, 6H), 1.69 (br, 2H). 13C NMR (126 MHz,
DMSO): δ 172.76, 172.72, 166.37, 166.29, 158.90, 158.87, 154.65,
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4-(5-Carbamoyl-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-
1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo-
[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-
benzo[d]imidazol-2-yl)-4-oxobutanoic Acid (21). White solid; yield:
(400 MHz, CDCl3): δ 8.47 (d, J = 1.9 Hz, 1H), 8.11 (br, 1H), 7.48
(d, J = 2.0 Hz, 1H), 5.70 (m, 2H), 4.59 (br, 1H), 4.31−4.22 (m, 2H),
3.90 (s, 3H), 3.89 (s, 3H), 3.73 (s, 2H), 1.44 (s, 9H).
tert-Butyl (E)-(4-((2-Methoxy-4-(1-(2-(2-methoxyethoxy)ethyl)-
1H-1,2,3-triazol-4-yl)-6-nitrophenyl)amino)but-2-en-1-yl)-
carbamate (29a). To a solution of 1-azido-2-(2-methoxyethoxy)-
ethane (65 mg, 0.45 mmol) in a mixture of t-BuOH and H2O (2:1, 3
mL) were added sodium ascorbate (30 mg, 0.15 mmol), CuSO4·
5H2O (4 mg, 0.015 mmol), and 28a (108 mg, 0.3 mmol). The
reaction mixture was stirred at rt for 1 h; then, saturated sodium
bicarbonate solution was added, and the mixture was extracted with
EtOAc. The combined organic phase was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was subjected
to column chromatography to afford the title compound in 52% yield.
1H NMR (400 MHz, CD3OD): δ 8.39 (s, 1H), 8.08 (d, J = 1.9 Hz,
1
21%. HPLC purity: 99.5%. H NMR (400 MHz, DMSO): δ 12.79
(br, 1H), 12.12 (br, 1H), 8.15 (s, 1H), 7.98 (s, 1H), 7.91 (d, J = 1.9
Hz, 1H), 7.66 (d, J = 1.4 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H), 7.44 (s,
1H), 7.36 (s, 1H), 7.33 (d, J = 1.4 Hz, 1H), 6.52 (s, 1H), 5.75 (d, J =
3.9 Hz, 2H), 5.11 (s, 2H), 4.87 (s, 2H), 4.54 (q, J = 7.1 Hz, 2H), 3.85
(t, J = 6.2 Hz, 2H), 3.78 (s, 3H), 3.44 (t, J = 4.6 Hz, 4H), 3.04 (t, J =
6.9 Hz, 2H), 2.72 (t, J = 6.9 Hz, 2H), 2.14 (d, J = 6.8 Hz, 6H), 2.11
(s, 3H), 1.64−1.52 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H). MS (ESI, [M +
+
H]+) m/z, 799.2. HRMS (ESI) calcd for C39H47N10O9 , 799.3522;
found, 799.3545.
4-Iodo-2-methoxy-6-nitroaniline (24). To a solution of com-
pound 23 (12.57 g, 74.8 mmol) and Ag2SO4 (23.3 g, 74.8 mmol) in
EtOH (400 mL) was added I2 (19.0 g, 74.8 mmol). The mixture was
stirred at rt for 1 h and then concentrated in vacuo. The residue was
extracted with EtOAc twice, and the combined organic phase was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The resulting brown solid (15.6 g, 71% yield) was used in the next
1H), 7.55 (d, J = 2.0 Hz, 1H), 5.76−5.56 (m, 2H), 4.62 (t, J = 5.0 Hz,
2H), 4.15 (d, J = 4.9 Hz, 2H), 3.96 (s, 3H), 3.92 (t, J = 5.0 Hz, 2H),
3.67−3.61 (m, 2H), 3.60 (s, 2H), 3.56−3.49 (m, 2H), 3.33 (d, J = 2.6
Hz, 2H), 1.40 (s, 9H).
General Procedure for Synthesis of Compounds 29b and
32c. To a solution of benzonitrile 28b or 32b (1 equiv) in MeOH
was added NaOMe (0.5 equiv). The mixture was stirred at rt for 3 h,
and then 2,2-dimethoxyethan-1-amine (1 equiv) and AcOH (2 equiv)
were added. The mixture was stirred at 50 °C for another 2 h, and
then a solution of 6 N HCl solution was added to adjust pH = 1. The
mixture was stirred at 70 °C for 18 h, concentrated in vacuo, and then
diluted with water. A solution of 2 M Na2CO3 aqueous solution was
added to adjust the pH value of the mixture to 10. The mixture was
filtered, and the solid was dried to afford compound 29b or 32c in 56
and 68% yields, respectively.
1
step without further purification. H NMR (400 MHz, CDCl3): δ
8.08 (d, J = 1.8 Hz, 1H), 7.06 (d, J = 1.8 Hz, 1H), 6.47 (br, 2H), 3.91
(s, 3H).
2-Chloro-5-iodo-1-methoxy-3-nitrobenzene (25). To a solution
of compound 24 (6.02 g, 20.5 mmol) in MeOH (120 mL) was added
TsOH·H2O (13.7 g, 72.0 mmol). The mixture was stirred at 0 °C for
10 min followed by adding NaNO2 aqueous solution (2.48 g in 20
mL) slowly. After that, CuCl (5.94 g, 60.0 mmol) was added to the
mixture, stirred at 0 °C for 30 min, and then stirred overnight at room
temperature. The mixture was concentrated in vacuo and extracted
with EtOAc. The combined organic phase was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography to afford compound 25
(5.65 g) in 88% yield. 1H NMR (400 MHz, CDCl3): δ 7.68 (d, J = 1.7
Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 3.96 (s, 3H).
tert-Butyl (E)-(4-((4-(1H-Imidazol-2-yl)-2-methoxy-6-
1
nitrophenyl)amino)but-2-en-1-yl)carbamate (29b). H NMR (400
MHz, DMSO): δ 12.63 (br, 1H), 8.19 (s, 1H), 7.69 (s, 1H), 7.63 (t, J
= 6.3 Hz, 1H), 7.13 (s, 2H), 6.96 (s, 1H), 5.56 (s, 2H), 4.09 (s, 2H),
3.91 (s, 3H), 3.49 (s, 2H), 1.35 (s, 9H).
4-(3-(2-Chloro-5-(1H-imidazol-2-yl)-3-nitrophenoxy)propyl)-
1
morpholine (32c). H NMR (600 MHz, DMSO): δ 12.96 (br, 1H),
((4-Chloro-3-methoxy-5-nitrophenyl)ethynyl)trimethylsilane
(26). To a solution of compound 25 (2.54 g, 8.1 mmol) in THF (60
mL) were added ethynyltrimethylsilane (3.95 g, 40.3 mmol) and CuI
(54 mg, 0.3 mmol), and to DIPEA (4.9 g, 38.2 mmol) was added
PdCl2(PPh3)2 (282 mg, 0.4 mmol). The mixture was stirred at rt
overnight, concentrated in vacuo, and extracted with EtOAc. The
combined organic phase was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by column
8.10 (d, J = 1.8 Hz, 1H), 7.99 (s, 1H), 7.38 (br, 1H), 7.12 (br, 1H),
4.30 (s, 2H), 3.59 (br, 4H), 2.39 (m, 6H), 2.01 (m, 2H).
General Procedure for Synthesis of Compounds 29c, 29d,
and 32d. A solution of benzamide 28c or 32a (1 equiv) in DMF-
DMA was stirred at 100 °C for 1 h, cooled to rt, and then
concentrated in vacuo. The residue was diluted in EtOH, hydrazine
acetate (2 equiv) was added, and the resulting mixture was heated to
50 °C. After 30 min, the mixture was concentrated in vacuo and the
residue was diluted in H2O. After filtration, the obtained solid was
washed with EtOH and dried. Compounds 29c, 29d, and 32d were
obtained in 49−72% yields.
tert-Butyl (E)-(4-((2-Methoxy-6-nitro-4-(1H-1,2,4-triazol-5-yl)-
phenyl)amino)but-2-en-1-yl)carbamate (29c). 1H NMR (400
MHz, DMSO): δ 14.18 (br, 1H), 8.44 (br, 1H), 8.24 (d, J = 1.9
Hz, 1H), 7.72 (s, 1H), 7.65 (d, J = 1.9 Hz, 1H), 6.96 (t, J = 6.0 Hz,
1H), 5.63−5.46 (m, 2H), 4.11 (d, J = 6.0 Hz, 2H), 3.92 (s, 3H), 3.48
(d, J = 5.5 Hz, 2H), 1.35 (s, 9H).
1
chromatography to afford compound 26 (1.47 g) in 64% yield. H
NMR (400 MHz, CDCl3): δ 7.47 (d, J = 1.7 Hz, 1H), 7.14 (d, J = 1.8
Hz, 1H), 3.96 (s, 3H), 0.27 (s, 9H).
2-Chloro-5-ethynyl-1-methoxy-3-nitrobenzene (27a). To a sol-
ution of compound 26 (1.4 g, 4.9 mmol) in MeOH (25 mL) was
added K2CO3 (0.68 g, 4.9 mmol). The mixture was stirred at rt for 30
min, concentrated in vacuo, and extracted with EtOAc. The combined
organic phase was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by column
chromatography to afford 27a (0.96 g) in 93% yield. 1H NMR
(400 MHz, CDCl3): δ 7.49 (s, 1H), 7.18 (s, 1H), 3.97 (s, 3H), 3.23
(s, 1H).
General Procedure for Synthesis of Compounds 28a and
28d. To a solution of chlorides 27a−27d (1 equiv) and tert-butyl
(E)-(4-aminobut-2-en-1-yl)carbamate (2 equiv) in EtOH was added
DIPEA (2 equiv). The mixture was stirred at 120 °C in a sealed tube
for 2 days and then concentrated in vacuo. The residue was further
purified by column chromatography to afford compounds 28a and
28d in 26 and 52% yields, respectively.
tert-Butyl (E)-(4-((2-Methoxy-4-(3-methyl-1H-1,2,4-triazol-5-yl)-
6-nitrophenyl)amino)but-2-en-1-yl)carbamate (29d). 1H NMR
(400 MHz, CD3OD): δ 8.33 (d, J = 1.9 Hz, 1H), 7.67 (d, J = 1.9
Hz, 1H), 5.76−5.53 (m, 2H), 4.21 (s, 2H), 3.98 (s, 3H), 3.62 (d, J =
2.1 Hz, 2H), 2.49 (s, 3H), 1.42 (s, 9H).
4-(3-(2-Chloro-3-nitro-5-(1H-1,2,4-triazol-5-yl)phenoxy)propyl)-
1
morpholine (32d). H NMR (400 MHz, CD3OD): δ 8.53 (s, 1H),
8.05 (d, J = 1.8 Hz, 1H), 7.98 (d, J = 1.9 Hz, 1H), 4.32 (t, J = 6.1 Hz,
2H), 3.70 (t, J = 4.7 Hz, 4H), 2.63 (t, J = 7.4 Hz, 2H), 2.53 (s, 4H),
2.10 (m, 2H).
tert-Butyl (E)-(4-((4-((2-Hydroxyethyl)carbamoyl)-2-methoxy-6-
nitrophenyl)amino)but-2-en-1-yl)carbamate (30). To a solution of
28d (645 mg, 1.63 mmol) in MeOH (10 mL) was added 2-
aminoethan-1-ol (0.6 mL, 6.5 mmol). The mixture was stirred at 70
°C for 14 h and then concentrated in vacuo. The residue was purified
tert-Butyl (E)-(4-((4-Ethynyl-2-methoxy-6-nitrophenyl)amino)-
1
but-2-en-1-yl)carbamate (28a). Yield: 26%. H NMR (400 MHz,
CDCl3): δ 7.91 (d, J = 1.8 Hz, 1H), 7.82 (br, 1H), 6.94 (s, 1H), 5.68
(d, J = 5.3 Hz, 2H), 4.58 (br, 1H), 4.18 (t, J = 5.2 Hz, 2H), 3.84 (s,
3H), 3.72 (s, 2H), 3.01 (s, 1H), 1.44 (s, 9H).
1
by column chromatography to afford compound 30 in 85% yield. H
Methyl (E)-4-((4-((tert-Butoxycarbonyl)amino)but-2-en-1-yl)-
amino)-3-methoxy-5-nitrobenzoate (28d). Yield: 42%. 1H NMR
NMR (400 MHz, CDCl3): δ 8.07 (d, J = 2.0 Hz, 2H), 7.41 (d, J = 2.0
1663
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J. Med. Chem. 2021, 64, 1649−1669

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H), 7.13 (s, 1H), 5.67 (m, 2H), 4.76 (s, 1H), 4.22 (t, J = 5.0 Hz,
2H), 3.84 (s, 3H), 3.82 (d, J = 4.9 Hz, 2H), 3.71 (d, J = 5.4 Hz, 2H),
3.59 (q, J = 5.2 Hz, 2H), 3.49 (s, 1H), 1.42 (s, 9H).
5.51 (m, 2H), 4.13 (d, J = 5.7 Hz, 2H), 4.07 (d, J = 6.0 Hz, 2H), 4.02
(dd, J = 8.2, 4.4 Hz, 2H), 3.86 (s, 3H), 3.55 (m, 4H), 2.36 (m, 2H),
2.31 (s, 4H), 1.93−1.81 (m, 2H).
tert-Butyl (E)-(4-((4-(4,5-Dihydrooxazol-2-yl)-2-methoxy-6-
nitrophenyl)amino)but-2-en-1-yl)carbamate (29e). To a solution
of 30 (170 mg, 0.4 mmol) in DCM (3 mL) was added SOCl2 (66 μL,
0.9 mmol) at 0 °C. The mixture was stirred at 0 °C for 0.5 h and then
concentrated in vacuo. The residue was added ice water, and the
mixture was alkalized by saturated sodium bicarbonate solution to pH
= 9. After concentration in vacuo, the residue was diluted with EtOAc
and water. The organic phase was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was subjected to
column chromatography to afford compound 29e in 45% yield over
(E)-4-((4-((2-Methoxy-4-(3-methyl-1H-1,2,4-triazol-5-yl)-6-
nitrophenyl)amino)but-2-en-1-yl)amino)-3-(3-morpholinopro-
poxy)-5-nitrobenzamide (33h). Crimson solid; yield: 37%. 1H NMR
(400 MHz, CD3OD): δ 6.98 (d, J = 1.8 Hz, 1H), 6.95 (d, J = 1.9 Hz,
1H), 6.92 (d, J = 1.9 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 5.73 (m, 2H),
3.98 (t, J = 6.1 Hz, 2H), 3.80 (s, 3H), 3.66 (t, J = 4.7 Hz, 4H), 3.60−
3.55 (m, 2H), 3.55−3.49 (m, 2H), 2.54−2.46 (m, 2H), 2.45 (s, 2H),
2.44 (s, 3H), 2.01−1.87 (m, 2H).
(E)-4-((4-((2-Methoxy-4-(1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-
triazol-4-yl)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-3-(3-mor-
pholinopropoxy)-5-nitrobenzamide (33i). Crimson solid; yield:
1
two steps. H NMR (400 MHz, CDCl3): δ 8.28 (d, J = 1.9 Hz, 1H),
1
50%. H NMR (400 MHz, CD3OD): δ 8.35 (s, 1H), 7.93 (s, 1H),
8.03 (t, J = 5.5 Hz, 1H), 7.47 (d, J = 1.9 Hz, 1H), 5.69 (m, 2H), 4.58
(br, 1H), 4.43 (t, J = 9.5 Hz, 2H), 4.29−4.18 (m, 2H), 4.04 (t, J = 9.4
Hz, 2H), 3.88 (s, 3H), 3.73 (s, 2H), 1.44 (s, 9H).
7.82 (s, 1H), 7.39 (s, 1H), 5.63 (s, 2H), 4.64 (t, J = 5.1 Hz, 2H), 4.29
(t, J = 6.0 Hz, 2H), 3.99−3.92 (m, 2H), 3.84 (s, 3H), 3.75−3.64 (m,
13H), 3.57−3.49 (m, 2H), 2.64 (m, 2H), 2.53 (m, 4H), 2.13−2.05
(m, 2H).
General Procedure for Synthesis of Compounds 33a−33l
and 47. To a solution of 29a−29e in MeOH (1 mL) was added 4 N
HCl aqueous solution (1 mL). The mixture was stirred at rt for 1 h
and then concentrated in vacuo. The obtained intermediates were
used without further purification. To a solution of the crude
hydrochloride salts of amines (1 equiv) and corresponding aryl
chlorides 32a−32d (1.2 equiv) in i-PrOH was added DIPEA (3
equiv). The mixture was stirred at 120 °C in a sealed tube for 2 days
and then concentrated in vacuo. The residue was purified by column
chromatography to afford the key intermediates 33a−33l or 47 in
31−50% overall yields.
(E)-3-Methoxy-4-((4-((2-(3-morpholinopropoxy)-6-nitro-4-(4H-
1,2,4-triazol-3-yl)phenyl)amino)but-2-en-1-yl)amino)-5-nitroben-
zamide (33k). Crimson solid; yield: 43%. 1H NMR (400 MHz,
CD3OD): δ 8.33 (s, 1H), 8.25 (s, 1H), 8.18 (s, 1H), 7.53 (s, 1H),
7.40 (s, 1H), 5.69 (s, 2H), 4.18−4.10 (m, 4H), 4.07−3.96, (m, 4H),
3.91 (s, 2H), 3.81 (s, 3H), 3.70 (s, 4H), 2.50 (m, 6H), 2.01 (m, 2H).
(E)-3-(3-((tert-Butyldimethylsilyl)oxy)propoxy)-4-((4-((2-me-
thoxy-6-nitro-4-(1H-1,2,4-triazol-5-yl)phenyl)amino)but-2-en-1-yl)-
amino)-5-nitrobenzamide (47). Crimson solid; yield: 39%. 1H NMR
(400 MHz, CD3OD): δ 8.21 (s, 1H), 7.02 (d, J = 1.8 Hz, 1H), 6.99
(d, J = 1.9 Hz, 1H), 6.92 (d, J = 1.9 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H),
5.74 (t, J = 3.1 Hz, 2H), 5.74 (t, J = 3.1 Hz, 2H), 4.05 (t, J = 6.0 Hz,
2H), 3.82 (s, 3H), 3.79 (t, J = 6.1 Hz, 2H), 3.56 (d, J = 3.4 Hz, 2H),
3.55−3.49 (m, 2H), 1.95 (m, J = 6.1 Hz, 2H), 0.86 (s, 9H), 0.01 (s,
6H).
(E)-4-((4-((4-Cyano-2-(3-morpholinopropoxy)-6-nitrophenyl)-
amino)but-2-en-1-yl)amino)-3-methoxy-5-nitrobenzonitrile (33a).
Crimson solid; yield: 38%. 1H NMR (400 MHz, DMSO): δ 7.93 (m,
2H), 7.35 (d, J = 1.8 Hz, 2H), 5.49 (d, J = 3.1 Hz, 2H), 4.07 (m, 2H),
4.04−3.97 (m, 4H), 3.80 (s, 3H), 3.55 (t, J = 4.6 Hz, 4H), 2.35 (m,
6H), 1.86 (m, 2H).
(E)-N1-(2-Methoxy-6-nitro-4-(1H-1,2,4-triazol-5-yl)phenyl)-N4-(2-
(3-morpholinopropoxy)-6-nitro-4-(1H-1,2,4-triazol-5-yl)phenyl)-
Reduction of 33a−33l and subsequent generation of the imidazole
ring using 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate
were conducted by following literature procedures.24 The final
compounds were purified by silica gel column chromatography
(CH2Cl2/NH3 7.0 M solution in CH3OH from 50:1 to 10:1).
N-((E)-5-Cyano-1-((E)-4-((E)-5-cyano-2-((1-ethyl-3-methyl-1H-
pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-
1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-methoxy-1,3-dihydro-
2H-benzo[d]imidazol-2-ylidene)-1-ethyl-3-methyl-1H-pyrazole-5-
1
but-2-ene-1,4-diamine (33b). Crimson solid; yield: 42%. H NMR
(400 MHz, DMSO): δ 8.36 (s, 2H), 8.19 (d, J = 6.4 Hz, 2H), 7.58 (s,
2H), 5.63 (m, 2H), 4.13 (s, 2H), 4.08 (s, 2H), 4.03 (t, J = 6.2 Hz,
2H), 3.83 (s, 3H), 3.59−3.51 (m, 4H), 2.38 (t, J = 7.0 Hz, 2H), 2.32
(s, 4H), 1.94−1.83 (m, 1H).
(E)-N1-(4-(1H-Imidazol-2-yl)-2-(3-morpholinopropoxy)-6-nitro-
phenyl)-N4-(4-(1H-imidazol-2-yl)-2-methoxy-6-nitrophenyl)but-2-
1
1
ene-1,4-diamine (33c). Crimson solid; yield: 48%. H NMR (400
carboxamide (34). White solid; yield: 32%. HPLC purity: 100%. H
MHz, CDCl3): δ 7.91 (m, 2H), 7.59 (t, J = 7.1 Hz, 2H), 7.31 (dd, J =
4.5, 2.0 Hz, 2H), 7.27 (m, 2H), 5.36 (s, 2H), 4.04 (s, 4H), 3.69 (t, J =
4.7 Hz, 4H), 3.63 (t, J = 6.5 Hz, 2H), 3.49 (s, 3H), 2.40 (s, 4H), 2.35
(t, J = 7.3 Hz, 2H), 1.91−1.77 (m, 2H).
(E)-4-((4-((2-Methoxy-6-nitro-4-(4H-1,2,4-triazol-3-yl)phenyl)-
amino)but-2-en-1-yl)amino)-3-(3-morpholinopropoxy)-5-nitroben-
zonitrile (33d). Crimson solid; yield: 45%. 1H NMR (400 MHz,
DMSO): δ 8.15 (dd, J = 5.3, 1.9 Hz, 2H), 8.08 (s, 1H), 7.76 (t, J = 6.3
Hz, 1H), 7.64 (d, J = 1.9 Hz, 1H), 7.56 (t, J = 6.3 Hz, 1H), 7.51 (s,
1H), 7.33 (s, 1H), 7.12 (s, 2H), 5.71−5.53 (m, 2H), 4.12 (t, J = 5.0
Hz, 2H), 4.06 (t, J = 5.1 Hz, 2H), 4.01 (t, J = 6.3 Hz, 2H), 3.84 (s,
3H), 3.54 (t, J = 4.6 Hz, 4H), 2.40 (t, J = 7.1 Hz, 2H), 2.34 (s, 4H),
1.88 (t, J = 6.8 Hz, 2H).
(E)-4-((4-((4-(4,5-Dihydrooxazol-2-yl)-2-methoxy-6-nitrophenyl)-
amino)but-2-en-1-yl)amino)-3-(3-morpholinopropoxy)-5-nitroben-
zamide (33f). Crimson solid; yield: 50%. 1H NMR (400 MHz,
CDCl3): δ 8.22 (d, J = 1.9 Hz, 1H), 8.06 (d, J = 1.9 Hz, 1H), 8.00 (s,
2H), 7.49 (s, 1H), 7.35 (s, 1H), 5.69−5.58 (m, 2H), 4.48 (t, J = 9.5
Hz, 2H), 4.23 (dd, J = 11.9, 5.9 Hz, 4H), 4.07 (dd, J = 12.2, 6.4 Hz,
4H), 3.80 (s, 3H), 3.72 (t, J = 4.6 Hz, 4H), 2.48 (dd, J = 14.6, 7.4 Hz,
6H), 2.01 (t, J = 6.7 Hz, 3H).
(E)-4-((4-((2-Methoxy-6-nitro-4-(1H-1,2,4-triazol-5-yl)phenyl)-
amino)but-2-en-1-yl)amino)-3-(3-morpholinopropoxy)-5-nitroben-
zamide (33g). Crimson solid; yield: 39%. 1H NMR (400 MHz,
DMSO): δ 8.42 (s, 1H), 8.21 (d, J = 1.9 Hz, 1H), 8.17 (d, J = 1.9 Hz,
1H), 8.04 (s, 1H), 7.78 (t, J = 6.3 Hz, 1H), 7.68 (t, J = 6.2 Hz, 1H),
7.62 (d, J = 1.9 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.33 (s, 1H), 5.70−
NMR (400 MHz, CDCl3): δ 7.24 (s, 1H), 6.91 (s, 1H), 6.88 (s, 1H),
6.57 (s, 1H), 6.55 (s, 1H), 5.81 (s, 2H), 4.94 (br, 4H), 4.65−4.44 (m,
4H), 3.96 (br, 2H), 3.73 (s, 3H), 3.64 (br, 4H), 2.31 (br, 6H), 2.20
(s, 3H), 2.20 (s, 3H), 1.78 (br, 2H), 1.36 (m, 6H). 13C NMR (126
MHz, CDCl3): δ 169.15, 169.13, 168.92, 168.89, 153.54, 153.37,
146.58, 146.14, 145.51, 139.68, 129.94, 129.81, 128.42, 121.54,
121.39, 118.69, 110.15, 109.79, 109.11, 109.06, 108.90, 106.78, 77.42,
77.16, 76.91, 67.63, 66.75, 56.19, 55.12, 53.59, 46.58, 45.21, 25.90,
16.18, 16.18, 13.25, 13.23. MS (ESI, [M + H]+) m/z, 814.3. HRMS
+
(ESI) calcd for C42H48N13O5 , 814.3896; found, 814.3899.
1-Ethyl-N-((E)-1-((E)-4-((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-
carbonyl)imino)-7-(3-morpholinopropoxy)-5-(1H-1,2,4-triazol-5-
yl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-me-
thoxy-5-(1H-1,2,4-triazol-5-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-
ylidene)-3-methyl-1H-pyrazole-5-carboxamide (35). White solid;
1
yield: 37%. HPLC purity: 100%. H NMR (500 MHz, CD3OD): δ
8.27 (s, 1H), 8.25 (s, 1H), 7.51 (s, 1H), 7.48 (s, 1H), 7.11 (s, 1H),
7.05 (s, 1H), 6.49 (s, 1H), 6.39 (s, 1H), 5.62 (m, 2H), 4.72 (m, 4H),
4.47 (m, 4H), 3.70 (br, 2H), 3.53 (br, 7H), 2.28 (br, 6H), 2.13 (s,
3H), 2.07 (s, 3H), 1.61 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H), 1.23 (t, J =
7.3 Hz, 3H). 13C NMR (126 MHz, CD3OD): δ 168.90, 160.24,
153.52, 153.48, 147.46, 147.40, 147.13, 146.36, 141.70, 141.55,
131.70, 129.60, 129.15, 126.62, 119.85, 110.66, 105.23, 104.70,
103.80, 67.91, 67.31, 56.48, 56.09, 54.39, 47.24, 45.71, 26.79, 16.66,
16.64, 13.14. MS (ESI, [M + H]+) m/z, 898.4. HRMS (ESI) calcd for
+
C44H52N17O5 , 898.4332; found, 898.4335.
1664
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Journal of Medicinal Chemistry
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Article
1-Ethyl-N-((E)-1-((E)-4-((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-
carbonyl)imino)-5-(1H-imidazol-2-yl)-7-(3-morpholinopropoxy)-
2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-5-(1H-imida-
zol-2-yl)-7-methoxy-1,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-
3-methyl-1H-pyrazole-5-carboxamide (36). White solid; yield: 35%.
HPLC purity: 97.4%. 1H NMR (400 MHz, CDCl3): δ 7.28 (br, 2H),
7.07 (br, 4H), 6.99 (d, J = 1.4 Hz, 1H), 6.95 (d, J = 1.3 Hz, 1H), 6.63
(s, 1H), 6.60 (s, 1H), 5.70−5.67 (m, 2H), 4.85 (m, 4H), 4.59 (m,
4H), 3.55 (m, 4H), 3.49 (m, 2H), 3.31 (s, 3H), 2.24 (br, 4H), 2.21 (s,
3H), 2.19 (s, 3H), 2.17 (m, 2H), 1.51 (m, 2H), 1.39 (m, 6H). 13C
NMR (151 MHz, CD3OD): δ 168.85, 168.64, 153.02, 152.90, 146.60,
146.58, 146.43, 146.40, 146.04, 145.38, 140.21, 140.10, 130.08,
129.77, 128.84, 128.72, 126.71, 123.78, 117.93, 117.83, 109.93,
104.00, 103.42, 101.42, 101.28, 66.71, 66.65, 55.44, 55.03, 53.50,
46.53, 44.71, 44.67, 25.94, 16.27, 13.25, 13.23. MS (ESI, [M + H]+)
J = 1.3 Hz, 1H), 7.52 (s, 1H), 7.36 (s, 1H), 7.16 (d, J = 1.4 Hz, 1H),
6.60 (s, 1H), 6.52 (s, 1H), 5.80 (s, 2H), 4.97 (s, 2H), 4.94 (s, 2H),
4.56 (m, 4H), 3.87 (t, J = 6.1 Hz, 2H), 3.70 (s, 3H), 3.56 (t, J = 4.6
Hz, 3H), 2.31 (m, 6H), 2.19 (s, 3H), 2.18 (s, 3H), 1.68 (m, 2H), 1.33
(m, 6H). 13C NMR (126 MHz, CD3OD): δ 169.74, 167.65, 167.45,
159.13, 152.52, 152.14, 146.09, 146.05, 145.84, 144.67, 140.20,
140.13, 130.39, 129.96, 129.27, 128.11, 127.93, 125.47, 120.07,
118.54, 109.33, 105.21, 104.35, 103.42, 102.52, 66.79, 66.18, 55.17,
54.78, 53.16, 45.86, 44.41, 43.66, 25.61, 15.27, 11.81, 11.78. MS (ESI,
+
[M + H]+) m/z, 874.4. HRMS (ESI) calcd for C43H52N15O6 ,
874.4220; found, 874.4222.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(3-methyl-1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]-
imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-2,3-dihy-
dro-1H-benzo[d]imidazole-5-carboxamide (41). White solid; yield:
31%. HPLC purity: 100%. 1H NMR (400 MHz, CD3OD): δ 7.60 (d,
J = 1.3 Hz, 1H), 7.49 (d, J = 1.3 Hz, 1H), 7.27 (d, J = 1.4 Hz, 1H),
7.12 (d, J = 1.5 Hz, 1H), 6.58 (s, 1H), 6.49 (s, 1H), 5.83−5.68 (m,
2H), 4.92 (d, J = 3.7 Hz, 2H), 4.87 (s, 2H), 4.63−4.51 (m, 4H), 3.83
(t, J = 6.1 Hz, 2H), 3.66 (s, 3H), 3.54 (t, J = 4.6 Hz, 4H), 2.48 (s,
3H), 2.27 (m, 6H), 2.18 (s, 3H), 2.17 (s, 3H), 1.65 (m, 2H), 1.34 (t,
J = 7.2 Hz, 3H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz,
CD3OD): δ 171.23, 168.95, 153.68, 147.63, 147.58, 147.39, 146.24,
141.69, 131.93, 130.82, 129.50, 129.43, 119.99, 110.68, 106.76,
104.86, 103.84, 68.28, 67.60, 56.63, 56.25, 54.59, 49.00, 47.28, 45.89,
27.06, 16.66, 13.19, 13.15. MS (ESI, [M + H]+) m/z, 888.4. HRMS
+
m/z, 896.5. HRMS (ESI) calcd for C46H54N15O5 , 896.4427; found,
896.4438.
N-((E)-5-Cyano-1-((E)-4-((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-
carbonyl)imino)-7-methoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-
1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopro-
poxy)-1,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1-ethyl-3-
methyl-1H-pyrazole-5-carboxamide (37). White solid; yield: 29%.
HPLC purity: 95.4%. 1H NMR (400 MHz, CD3OD): δ 8.38 (s, 1H),
7.51 (s, 1H), 7.14 (s, 1H), 7.08 (s, 1H), 6.64 (s, 1H), 6.45 (s, 1H),
6.34 (s, 1H), 5.66−5.47 (m, 2H), 4.77−4.60 (m, 4H), 4.55−4.32 (m,
4H), 3.66 (br, 2H), 3.58 (s, 3H), 3.50 (m, 4H), 2.31−2.15 (m, 6H),
2.10 (s, 6H), 1.55 (m, 2H), 1.30−1.15 (m, 6H). 13C NMR (151
MHz, CD3OD): δ 167.37, 152.58, 152.16, 146.12, 146.01, 145.77,
145.03, 140.09, 139.72, 130.34, 128.14, 127.77, 125.59, 120.98,
118.40, 118.32, 109.46, 109.25, 108.86, 105.90, 103.32, 102.43, 67.15,
66.04, 54.97, 54.81, 53.04, 45.88, 44.49, 44.40, 25.37, 15.28, 15.23,
11.83, 11.77. MS (ESI, [M + H]+) m/z, 856.4. HRMS (ESI) calcd for
+
(ESI) calcd for C44H54N15O6 , 888.4376; found, 888.4395.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-2,3-
dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholino-
propoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide (42).
+
C43H50N15O5 , 856.4114; found, 856.4097.
1
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-5-(1H-
imidazol-2-yl)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-
but-2-en-1-yl)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]-
imidazole-5-carboxamide (38). White solid; yield: 34%. HPLC
White solid; yield: 37%. HPLC purity: 96.8%. H NMR (400 MHz,
CD3OD): δ 8.29 (d, J = 4.9 Hz, 1H), 7.49 (s, 1H), 7.42 (d, J = 4.4
Hz, 1H), 7.07 (d, J = 6.3 Hz, 1H), 7.01 (s, 1H), 6.59 (d, J = 4.7 Hz,
1H), 6.52 (d, J = 4.5 Hz, 1H), 5.72 (s, 2H), 4.90−4.78 (m, 4H), 4.58
(m, 6H), 3.97 (t, J = 5.0 Hz, 2H), 3.79 (s, 2H), 3.67 (m, 2H), 3.64−
3.57 (m, 7H), 3.57−3.53 (m, 2H), 3.34 (s, 3H), 2.48 (br, 6H), 2.22−
2.14 (m, 6H), 1.72 (s, 2H), 1.33 (m, 6H). 13C NMR (151 MHz,
CD3OD): δ 169.36, 167.09, 151.68, 146.52, 145.75, 144.38, 139.91,
130.25, 129.46, 128.99, 127.70, 127.65, 126.40, 121.21, 117.11,
108.91, 108.83, 104.87, 103.74, 102.43, 100.95, 71.06, 69.37, 68.50,
66.36, 65.71, 57.29, 54.82, 54.33, 52.72, 49.71, 45.45, 44.02, 25.18,
14.85, 11.34, 11.32. MS (ESI, [M + H]+) m/z, 976.4. HRMS (ESI)
1
purity: 98.1%. H NMR (400 MHz, CD3OD): δ 7.41 (d, J = 1.1 Hz,
1H), 7.37 (d, J = 1.2 Hz, 1H), 7.10 (s, 2H), 7.03 (s, 1H), 7.01 (s,
1H), 6.48 (s, 1H), 6.40 (s, 1H), 5.63 (m, 2H), 4.77 (s, 2H), 4.70 (s,
2H), 4.54−4.38 (m, 4H), 3.68 (t, J = 5.9 Hz, 2H), 3.56−3.47 (m,
7H), 2.24−2.16 (m, 6H), 2.11 (s, 3H), 2.10 (s, 3H), 1.56 (m, 2H),
1.25 (m, 6H). 13C NMR (126 MHz, CD3OD): δ 171.18, 168.98,
168.81, 153.90, 153.43, 147.53, 147.45, 147.40, 147.30, 146.04,
141.54, 141.49, 131.82, 131.38, 130.63, 129.52, 129.26, 127.63,
121.45, 119.09, 110.70, 110.65, 106.57, 105.70, 103.97, 102.55, 68.15,
67.53, 56.54, 56.13, 54.52, 47.23, 45.77, 26.97, 16.65, 13.19, 13.14.
MS (ESI, [M + H]+) m/z, 873.4. HRMS (ESI) calcd for
+
calcd for C48H62N15O8 , 976.4900; found, 976.4874.
(E)-1-((E)-4-((E)-5-Cyano-2-((1-ethyl-3-methyl-1H-pyrazole-5-
carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo-
[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-
5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-
5-carboxamide (43). White solid; yield: 42%. HPLC purity: 98.0%.
1H NMR (400 MHz, CDCl3 + CD3OD): δ 7.35 (s, 1H), 7.19 (s, 1H),
+
C44H53N14O6 , 873.4267; found, 873.4267.
(E)-1-((E)-4-((E)-5-(4,5-Dihydrooxazol-2-yl)-2-((1-ethyl-3-methyl-
1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo-
[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-
5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-
benzo[d]imidazole-5-carboxamide (39). White solid; yield: 42%.
7.17 (d, J = 1.4 Hz, 1H), 6.80 (s, 1H), 6.49 (s, 1H), 6.44 (s, 1H), 5.72
(m, 2H), 4.86 (t, J = 4.5 Hz, 4H), 4.46 (q, J = 7.2 Hz, 4H), 3.84 (t, J =
6.5 Hz, 2H), 3.65 (s, 3H), 3.52 (br, 4H), 2.20 (br, 6H), 2.09 (s, 6H),
1.72−1.55 (m, 2H), 1.25 (m, 6H). 13C NMR (151 MHz, CD3OD): δ
169.43, 168.63, 168.39, 153.17, 152.96, 146.46, 146.36, 145.71,
145.33, 139.83, 129.90, 129.34, 128.60, 127.96, 121.27, 120.41,
118.63, 109.94, 109.70, 109.47, 109.03, 106.35, 105.50, 103.97, 67.45,
66.54, 55.65, 55.04, 53.32, 46.27, 45.05, 44.87, 25.64, 15.96, 15.93,
12.88, 12.82. MS (ESI, [M + H]+) m/z, 832.4. HRMS (ESI) calcd for
1
HPLC purity: 100%. H NMR (400 MHz, CD3OD): δ 7.47 (s, 1H),
7.46 (s, 1H), 7.07 (s, 2H), 6.56 (s, 1H), 6.50 (s, 1H), 5.78−5.64 (m,
2H), 4.84 (m, 4H), 4.60−4.45 (m, 6H), 4.01 (t, J = 9.5 Hz, 2H), 3.79
(t, J = 6.1 Hz, 2H), 3.57 (s, 3H), 3.55 (m, 4H), 2.32−2.23 (m, 6H),
2.18 (s, 3H), 2.16 (s, 3H), 1.71−1.62 (m, 2H), 1.36−1.28 (m, 6H).
13C NMR (126 MHz, CD3OD): δ 171.17, 169.02, 166.54, 154.00,
147.66, 146.93, 146.21, 141.57, 131.57, 130.84, 129.57, 129.48,
124.32, 121.68, 110.77, 106.79, 106.40, 69.30, 68.26, 67.60, 56.66,
56.24, 55.17, 54.61, 47.31, 45.96, 27.07, 16.66, 13.18. MS (ESI, [M +
+
C42H50N13O6 , 832.4002; found, 832.4003.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-mor-
pholinopropoxy)-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo-
[d]imidazol-1-yl)but-2-en-1-yl)-7-methoxy-2,3-dihydro-1H-benzo-
[d]imidazole-5-carboxamide (44). White solid; yield: 36%. HPLC
purity: 99.6%. 1H NMR (400 MHz, CD3OD): δ 8.39 (s, 1H), 7.63 (d,
J = 1.1 Hz, 1H), 7.55 (d, J = 1.2 Hz, 1H), 7.27 (s, 1H), 7.20 (d, J =
1.3 Hz, 1H), 6.60 (s, 1H), 6.49 (s, 1H), 5.79 (qt, J = 15.5, 4.9 Hz,
2H), 4.93 (br, 4H), 4.60 (q, J = 7.1 Hz, 2H), 4.52 (q, J = 7.1 Hz, 2H),
+
H]+) m/z, 876.3. HRMS (ESI) calcd for C44H54N13O7 , 876.4264;
found, 876.4262.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo-
[d]imidazole-5-carboxamide (40). White solid; yield: 39%. HPLC
purity: 99.7%. 1H NMR (400 MHz, CD3OD): δ 8.41 (s, 1H), 7.69 (d,
1665
https://dx.doi.org/10.1021/acs.jmedchem.0c01900
J. Med. Chem. 2021, 64, 1649−1669

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3.86 (t, J = 6.1 Hz, 2H), 3.68 (s, 3H), 3.60 (t, J = 4.6 Hz, 4H), 2.43−
2.26 (m, 6H), 2.21 (s, 3H), 2.13 (s, 3H), 1.69 (m, 2H), 1.36 (t, J =
7.1 Hz, 4H), 1.30 (t, J = 7.1 Hz, 4H). 13C NMR (126 MHz,
CD3OD): δ 171.20, 169.06, 154.04, 147.65, 147.58, 147.01, 146.79,
141.50, 131.56, 130.92, 129.97, 129.12, 121.79, 110.70, 106.26,
105.92, 105.58, 68.22, 67.65, 56.66, 56.38, 54.63, 49.00, 47.29, 45.94,
27.07, 16.67, 16.62, 13.17, 13.14. MS (ESI, [M + H]+) m/z, 874.4.
168.38, 166.78, 151.68, 145.81, 145.50, 145.14, 145.05, 139.95,
138.79, 130.69, 128.61, 126.82, 126.20, 123.18, 122.62, 118.26,
116.37, 109.22, 108.03, 103.52, 103.16, 55.73, 45.66, 45.61, 44.64,
16.14, 16.11, 13.13, 13.11. MS (ESI, [M + H]+) m/z, 771.4. HRMS
+
(ESI) calcd for C38H39N14O5 , 771.3222; found, 771.3214.
Conversion of 57 and 27c to target compounds 64−66 were
conducted by following a similar procedure as described above.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-(4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)butyl)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]-
imidazole-5-carboxamide (64). White solid. HPLC purity: 100%. 1H
NMR (500 MHz, DMSO): δ 12.77 (s, 2H), 7.96 (s, 1H), 7.78 (s,
1H), 7.57 (s, 1H), 7.47 (s, 1H), 7.30 (s, 2H), 6.58 (s, 1H), 6.54 (s,
1H), 4.56 (m, 4H), 4.36 (s, 4H), 4.09 (t, J = 6.2 Hz,2H), 3.88 (s,
3H), 3.41 (t, J = 4.6 Hz, 4H), 2.30 (t, J = 7.1 Hz, 2H), 2.18 (br, 4H),
2.09 (s, 3H), 2.08 (s, 3H), 1.86 (br, 4H), 1.83−1.73 (m, 2H), 1.30
(m, 6H). 13C NMR (126 MHz, DMSO): δ 167.68, 166.84, 152.24,
145.38, 144.98, 144.08, 140.01, 130.00, 119.65, 109.16, 105.45,
105.21, 103.32, 66.88, 66.02, 55.90, 54.85, 53.20, 45.57, 43.32, 26.67,
25.78, 16.15, 13.11. MS (ESI, [M + H]+) m/z, 876.5. HRMS (ESI)
+
HRMS (ESI) calcd for C43H52N15O6 , 874.4220; found, 874.4227.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-5-(1H-
imidazol-2-yl)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]-
imidazol-1-yl)but-2-en-1-yl)-7-methoxy-2,3-dihydro-1H-benzo[d]-
imidazole-5-carboxamide (45). White solid; yield: 31%. HPLC
1
purity: 95.2%. H NMR (400 MHz, CD3OD): δ 7.51 (d, J = 1.4 Hz,
1H), 7.40 (d, J = 1.3 Hz, 1H), 7.16 (d, J = 1.5 Hz, 1H), 7.13 (s, 2H),
7.09 (d, J = 1.4 Hz, 1H), 6.56 (s, 1H), 6.46 (s, 1H), 5.83−5.68 (m,
2H), 4.89 (br, 4H), 4.57 (q, J = 7.1 Hz, 2H), 4.48 (q, J = 7.1 Hz, 2H),
3.80 (t, J = 6.1 Hz, 2H), 3.62 (s, 3H), 3.60−3.53 (m, 4H), 2.35−2.21
(m, 6H), 2.18 (s, 3H), 2.10 (s, 3H), 1.65 (m, 2H), 1.33 (t, J = 7.1 Hz,
3H), 1.27 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CD3OD): δ
171.22, 169.01, 153.98, 147.69, 147.60, 147.55, 146.95, 146.81,
141.48, 131.49, 130.88, 129.93, 129.14, 127.75, 121.72, 110.72,
110.66, 106.21, 105.84, 104.77, 68.16, 67.61, 56.67, 56.32, 54.61,
49.51, 49.34, 49.17, 49.00, 48.83, 48.66, 48.49, 48.49, 47.29, 47.25,
45.91, 45.87, 27.07, 16.67, 16.63, 13.17, 13.13. MS (ESI, [M + H]+)
+
calcd for C43H54N15O6 , 876.4376; found, 876.4376.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-
((1s,4s)-4-((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-
7-methoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]-
imidazol-1-yl)cyclohexyl)-7-(3-morpholinopropoxy)-2,3-dihydro-
1H-benzo[d]imidazole-5-carboxamide (65). White solid. HPLC
purity: 99.6% (H2O/MeOH and 0.1% DEA); 99.4% (H2O/MeCN
+
m/z, 873.4. HRMS (ESI) calcd for C44H53N14O6 , 873.4267; found,
873.4264.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)but-2-en-1-yl)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]-
imidazole-5-carboxamide (48). White solid; yield: 38%. HPLC
1
and 0.1% DEA). H NMR (400 MHz, DMSO): δ 14.16 (s, 1H),
13.00 (s, 2H), 8.63 (s, 1H), 8.06 (s, 1H), 7.93 (s, 1H), 7.72 (s, 1H),
7.60 (s, 1H), 7.44 (s, 1H), 7.41 (s, 1H), 6.70 (s, 1H), 6.66 (s, 1H),
5.61 (br, 2H), 5.51 (br, 1H), 4.63 (s, 4H), 4.34 (s, 2H), 4.02 (s, 3H),
3.44 (s, 4H), 2.92 (br, 4H), 2.45 (s, 2H), 2.30 (s, 4H), 2.19−1.88 (m,
12H), 1.38 (t, J = 7.2 Hz, 6H). MS (ESI, [M + H]+) m/z, 902.4.
1
purity: 99.5%. H NMR (600 MHz, DMSO): δ 12.81 (br, 2H), 8.47
(s, 2H), 7.96 (s, 1H), 7.81 (s, 1H), 7.63 (s, 1H), 7.44 (d, J = 1.2 Hz,
1H), 7.31 (s, 1H), 6.50 (d, J = 3.2 Hz, 2H), 5.94−5.75 (m, 2H), 4.92
(d, J = 4.1 Hz, 2H), 4.90 (d, J = 4.3 Hz, 2H), 4.57−4.46 (m, 4H),
4.06 (t, J = 6.4 Hz, 2H), 3.75 (s, 3H), 3.43 (t, J = 6.0 Hz, 2H), 2.09 (s,
3H), 2.07 (s, 3H), 1.71 (p, 2H), 1.25 (m, 6H). 13C NMR (151 MHz,
DMSO): δ 167.50, 167.45, 166.77, 151.98, 151.62, 145.50, 145.01,
144.28, 139.90, 130.62, 129.95, 128.18, 127.96, 119.59, 109.20,
105.43, 105.21, 103.48, 103.12, 65.90, 57.09, 55.79, 45.56, 44.59,
31.81, 16.12, 13.10, 13.08. MS (ESI, [M + H]+) m/z, 805.4. HRMS
+
HRMS (ESI) calcd for C45H56N15O6 , 902.4533; found, 902.4517.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-
((1r,4r)-4-((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-
7-methoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]-
imidazol-1-yl)cyclohexyl)-7-(3-morpholinopropoxy)-2,3-dihydro-
1H-benzo[d]imidazole-5-carboxamide (66). White solid. HPLC
purity: 95.5% (H2O/MeOH and 0.1% DEA); 95.0% (H2O/MeCN
and 0.1% DEA). 1H NMR (400 MHz, CDCl3 + CD3OD): δ 8.19 (s,
1H), 7.64 (s, 1H), 7.59 (s, 1H), 7.47 (s, 2H), 6.74 (s, 1H), 6.68 (s,
1H), 5.36 (s, 2H), 4.65 (m, 4H), 4.50−4.09 (m, 5H), 3.64 (s, 4H),
3.08 (br, 1H), 2.65 (br, 2H), 2.40 (br, 4H), 2.26 (s, 6H), 2.20−1.90
(m, 8H), 1.52−1.40 (m, 6H). MS (ESI, [M + H]+) m/z, 902.4.
+
(ESI) calcd for C39H45N14O6 , 805.3641; found, 805.3636.
Compounds 55 and 56 were prepared by following similar
procedures as described above.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)but-2-en-1-yl)-2,3,7,8-tetrahydro-1H-[1,4]dioxino[2′,3′:3,4]-
benzo[1,2-d]imidazole-5-carboxamide (55). White solid. HPLC
purity: 99.2%. 1H NMR (400 MHz, DMSO): δ 12.82 (br, 1H), 12.66
(s, 1H), 8.60 (br, 1H), 7.82 (s, 1H), 7.54 (s, 1H), 7.52 (s, 1H), 7.46
(s, 1H), 6.53 (s, 1H), 6.51 (s, 1H), 5.85 (s, 2H), 4.92 (s, 2H), 4.85 (s,
2H), 4.59−4.42 (m, 4H), 4.29 (s, 2H), 4.19 (s, 2H), 3.80 (s, 3H),
2.10 (s, 3H), 2.07 (s, 3H), 1.34−1.23 (m, 6H). 13C NMR (126 MHz,
DMSO): δ 166.78, 165.47, 152.18, 151.69, 145.52, 145.01, 139.82,
138.26, 130.69, 129.54, 128.25, 127.94, 123.39, 119.69, 118.52,
109.20, 106.04, 103.52, 103.15, 64.22, 63.82, 55.79, 45.54, 44.68,
44.42, 16.11, 16.07, 13.06, 13.04. MS (ESI, [M − H]−) m/z, 787.3.
+
HRMS (ESI) calcd for C45H56N15O6 , 902.4533; found, 902.4532.
General Procedure for Synthesis of HCl Salt of 5 and 40. To
a solution of compound 5 or 40 (0.1 mmol) in MeOH (10 mL) was
added 4 N HCl in 1,4-dioxane (1 mL). The mixture was stirred at
room temperature for 4 h and concentrated in vacuo. After repeating
the above operation twice, a white-like solid was formed, washed with
EtOH, and dried under an infrared-ray oven. HCl salt of 5 or 40 was
obtained. For 40·3HCl·2.5H2O (C43H51N15O6·3HCl·2.5H2O), calcd:
C, 50.22; H, 5.78; N, 20.43; found: C, 50.30; H, 5.83; N, 20.23; For
5·3H2O·H2O (C42H56Cl3N13O8·3HCl·H2O), calcd: C, 51.61; H, 5.78;
N, 18.63; found: C, 51.63; H, 5.98; N, 18.57.
Reagents. DMXAA (cat. code: MB3940) was purchased from
Meilunbio (Liaoning, China), and ADU-S100 (cat. code: tlrl-
nacda2r), QUANTI-Luc solution (cat. code: rep-qlc2), and
QUANTI-Blue solution (cat. code: rep-qlc2) were purchased from
InvivoGen (San Diego, USA). Antibodies against phospho-TBK1
(Ser172) (cat. code: 5483S), TBK1 (cat. code: 3504S), phospho-
IRF3 (Ser396) (cat. code:4947S), IRF3 (cat. code: 4302S), and
GAPDH (cat. code: 5174S) were purchased from Cell Signaling
Technology (Beverly, MA). Cytosolic fragments (139−379) of
human STING protein (R232, H232, 293Q, and AQ) and mouse
STING (R231) were purchased from Novoprotein (Shanghai,
China).
−
HRMS (ESI) calcd for C38H39N14O6 , 787.3182; found, 787.3165.
(E)-2-((1-Ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-((E)-4-
((E)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-me-
thoxy-5-(1H-1,2,4-triazol-5-yl)-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)but-2-en-1-yl)-2,3-dihydro-1H-benzofuro[6,7-d]imidazole-5-car-
1
boxamide (56). White solid. HPLC purity: 100%. H NMR (600
MHz, CDCl3 + CD3OD): δ 8.14 (br, 1H), 7.59 (s, 1H), 7.49 (br,
1H), 7.29 (d, J = 2.1 Hz, 1H), 7.17 (d, J = 2.1 Hz, 1H), 7.15 (s, 1H),
6.62 (s, 1H), 6.54 (s, 1H), 6.01 (m, 1H), 5.87 (m, 1H), 5.01 (d, J =
6.3 Hz, 2H), 4.94 (d, J = 6.0 Hz, 2H), 4.59 (m, 2H), 4.54 (q, J = 7.1
Hz, 2H), 3.50 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H), 1.39 (t, J = 7.2 Hz,
3H), 1.36 (t, J = 7.1 Hz, 3H). 13C NMR (151 MHz, DMSO): δ
Cell lines THP1-Blue-ISG cells (cat. code: thp-isg), THP-1-Dual
cells (cat.code:thpd-nfis), THP1-Dual-KO-STING cells (cat. code:
1666
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Article
thpd-kostg), 293T-Dual hSTING-H232 cells (cat. code: 293d-h232),
293T-Dual hSTING-R232 cells (cat. code: 293d-r232), RAW-Lucia
(cat. code: rawl-isg), and RAW-Lucia-KO-STING (cat. code: rawl-
kostg) were purchased from InvivoGen (San Diego, USA). Zeocin
(cat. code: ant-zn-05), Normocin (cat. code: ant-nr-1), and blastcidin
(cat. code: ant-bl-1) were purchased from InvivoGen (San Diego,
USA). All the cells were cultured according to the manufacture's
protocol and incubated at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air.
ISG-Reporter Assay. THP1-Blue-ISG cells or other reporter cells
as indicated (1 × 105 cells/well) were suspended in 180 μL of
medium and added to 96-well plates, and then indicated
concentrations of compound (20 μL) were added to the cells for
24 h. Subsequently, 20 μL of supernatant and 50 μL of QUANTI-Luc
detection reagent (or 180 μL of QUNTI-Blue SEAP detection reagent
for THP1-Blue-ISG) were added into a 96-well black plate (cat. code:
3601; Corning). The luminescence and absorbance at 650 nm were
measured by SpectraMAX Paradigm and SpectraMAX Plus 384
(Molecular Devices, Sunnyvale, CA), respectively.
(10 mg/kg) on days 1, 4, and 7. The CT26 tumor model (n = 6) was
treated with 40 (500 μg/IT) or DMXAA (250 μg/IT) on days 1, 4,
and 7. Tumor volume and body weight were measured twice per
week. Tumor volume (TV) was calculated by the formula: V = (a ×
b2)/2 (a, width; b, length).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01900.
■
sı
Docking mode of compound 40 with h-STING, 1H and
13C spectra of all new compounds, preparation and
elemental analysis of HCl salt of 5 and 40, and aqueous
solubility of HCl salts of 5 and 40 (PDF)
Molecular formula strings and some data (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
NF-kB Reporter Assay. THP1-Dual cells (1 × 105 cells/well)
were suspended in 180 μL of medium and added to 96-well plates,
and indicated concentrations of compound (20 μL) were then added
to the cells for 24 h. Subsequently, 20 μL of supernatant and 180 μL
of QUANTI-Blue detection reagent were incubated at 37 °C in a
humidified atmosphere of 5% CO2 and 95% air. After 1 h, the
absorbance was measured at 650 nm by a SpectraMAX Plus 384
(Molecular Devices, Sunnyvale, CA).
Enzyme-Linked Immunosorbent Assay. THP1-Dual cells were
seeded in 96-well plates at a density of 105 cells/well and treated with
indicated concentrations of 5 or 40 for 24 h. Cell supernatants were
used to test the level of cytokines. Human IP-10 and IFN-β levels in
the supernatant were determined using a human IP-10 ELISA kit (cat.
code: 550926; BD) and human interferon-beta (IFN-β) kit (cat.
code: AL265C; PerkinElmer, MA, USA) according to the protocol.
The cytokine concentration (pg/mL) was calculated according to the
standard curve, and histograms were made by GraphPad Prism 8. The
test was carried out in triplicates.
Zuoquan Xie − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; University of
Chinese Academy of Sciences, Beijing 100049, China;
Phone: +86-21-50806072; Email: zqxie@simm.ac.cn
Ao Zhang − State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica (SIMM), Chinese Academy of
Sciences, Shanghai 201203, China; Shanghai Key Laboratory
for Molecular Engineering of Chiral Drugs, School of
Pharmacy, Shanghai Jiao Tong University, Shanghai
200240, China; College of Pharmacy, Nanjing University of
Chinese Medicine, Nanjing 210023, China; University of
Chinese Academy of Sciences, Beijing 100049, China; State
Key Laboratory of Esophageal Cancer Prevention and
Treatment, Ministry of Education of China, Zhengzhou
University, Zhengzhou 450001, China; orcid.org/0000-
0001-7205-9202; Phone: +86-21-34204020;
Western Blotting. THP1-Dual cells (8 × 105 cells/well) were
seeded into 6-well plate and treated with compound 5 or 40 (3.3, 10,
and 30 μM) for 4 h. Cell lysates were harvested using 1× SDS-PAGE
sample loading buffer (cat. code: P0015; Beyotime, China) and boiled
at 100 °C for 30 min. Cell lysates were run on 10% SDS-PAGE gels
and transferred onto NC membranes. The membranes were probed
with anti-TBK1, phospho-TBK1 (Ser172), anti-IRF3, anti-phospho-
IRF3 (Ser396), and anti-GAPDH. Horseradish peroxidase-conjugated
secondary antibodies were used as secondary antibodies. Finally, the
membrane was detected by enhanced chemiluminescence (ECL Plus;
cat. code: 17050622; Bio-Rad).
Differential Scanning Fluorimetry Assay. Differential scanning
fluorimetry (DSF) was used to evaluate the binding of compound to
STING protein. SYPRO Orange dye (10×) (cat.code: S6651; Life
Technologies), STING protein (5 μM), and compound (50 μM)
were mixed in the 40 μL of reaction buffer, which contained 10 mM
HEPES (pH 7.5) and 150 mM NaCl. Then, the reaction mixture was
transferred to a 96-well plate and measured by a CFX96 real-time
system (Bio-Rad, CA, USA). Briefly, the fluorescence intensity was
monitored as the run program, and the initial temperature was at 30
°C followed by a temperature gradient in which the samples are
heated at a scan rate of 0.5 °C/min until a final temperature of 80 °C.
Tm was calculated as the temperature of median fluorescence from the
initial increase to the highest fluorescence intensity.
Email: ao6919zhang@sjtu.edu.cn
Authors
Zilan Song − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; Shanghai
Key Laboratory for Molecular Engineering of Chiral Drugs,
School of Pharmacy, Shanghai Jiao Tong University,
Shanghai 200240, China; College of Pharmacy, Nanjing
University of Chinese Medicine, Nanjing 210023, China
Xiyuan Wang − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; University of
Chinese Academy of Sciences, Beijing 100049, China
Yan Zhang − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; University of
Chinese Academy of Sciences, Beijing 100049, China
Wangting Gu − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; Shanghai
Key Laboratory for Molecular Engineering of Chiral Drugs,
School of Pharmacy, Shanghai Jiao Tong University,
Shanghai 200240, China
Animal Studies. Animal procedures were approved by the
Institutional Animal Care and Use Committee of the Shanghai
Institute of Materia Medica. BALB/c female mice (6 weeks) were
purchased from LingChang Biotechnology (Shanghai, China). 4T1
cells (5 × 105) were inoculated into the breast fat pad of each mice.
CT26 cells (5 × 105) were injected subcutaneously into the right
armpit of each mice. When the tumors grew to approximately 50−100
mm3, the mice were treated with the indicated compound. The 4T1
tumor model (n = 5) was treated with compound 40 (10 mg/kg) or 5
Ancheng Shen − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; Shanghai
Key Laboratory for Molecular Engineering of Chiral Drugs,
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Han Li − State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica (SIMM), Chinese Academy of
Sciences, Shanghai 201203, China; College of Pharmacy,
Nanjing University of Chinese Medicine, Nanjing 210023,
China
Ruoxuan Xiao − Shanghai Key Laboratory for Molecular
Engineering of Chiral Drugs, School of Pharmacy, Shanghai
Jiao Tong University, Shanghai 200240, China
Meiyu Geng − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China; University of
Chinese Academy of Sciences, Beijing 100049, China
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This work was supported by grants from Chinese NSF (grants
81703327 and 81773565) and “Personalized Medicines-
Molecular Signature-based Drug Discovery and Development”,
Strategic Priority Research Program of the Chinese Academy
of Sciences (grant nos. XDA12020366 and XDA12020226).
Supporting grants from the National Science & Technology
Major Project “Key New Drug Creation and Manufacturing
Program” (grant nos. 2018ZX09711002-11-021 and
2018ZX09711002-017) as well as a start-up grant to the
Research Laboratory of Medicinal Chemical Biology &
Frontiers on Drug Discovery (AF1700037 and
WF220217002) from Shanghai Jiao Tong University and
Shanghai Pujiang Program (18PJD052) are also appreciated.
ABBREVIATIONS USED
■
STING, stimulator of interferon gene; CDN, cyclic dinucleo-
tide; mAbs, monoclonal antibodies; IO, immuno-oncology;
GMP, guanosine monophosphate; AMP, adenosine mono-
phosphate; cGAS, cyclic GMP-AMP synthase; IFN, interferon;
IT, intratumoral; DMXAA, 5,6-dimethylxanthenone-4-acetic
acid; CMA, 10-carboxymethyl-9-acridanone; ABZI, amidoben-
zimidazole; SAR, structure−activity relationship; PK, pharma-
cokinetic; SEAP, secreted embryonic alkaline phosphatase;
ISG, interferon-stimulated gene; DSF, differential scanning
fluorimetry; ECL, enhanced chemiluminescence; Vss, volume
of distribution; T1/2, half-life; Cmax, maximum concentration;
AUC0−∞, area under the plasma concentration time curve
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