Discovery and Optimization of Chromone Derivatives as Novel Selective Phosphodiesterase 10 Inhibitors

Article abstract of DOI:10.1021/acschemneuro.0c00024

Phosphodiesterase 10 (PDE10) inhibitors have received much attention as promising therapeutic agents for central nervous system (CNS) disorders such as schizophrenia and Huntington's disease. Recently, a hit compound 1 with a novel chromone scaffold has shown moderate inhibitory activity against PDE10A (IC₅₀ = 500 nM). Hit-to-lead optimization has resulted in compound 3e with an improved inhibitory activity (IC₅₀ = 6.5 nM), remarkable selectivity (>95-fold over other PDEs), and good metabolic stability (RLM t_1/2 = 105 min) by using an integrated strategy (molecular modeling, chemical synthesis, bioassay, and cocrystal structure). The cocrystal structural information provides insights into the binding pattern of 3e in the PDE10A catalytic domain to highlight the key role of the halogen and hydrogen bonds toward Tyr524 and Tyr693, respectively, thereby resulting in high selectivity against other PDEs. These new observations are of benefit for the rational design of the next generation PDE10 inhibitors for CNS disorders.

Full text of DOI:10.1021/acschemneuro.0c00024

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Article
Discovery and Optimization of Chromone Derivatives
as Novel Selective Phosphodiesterase 10 Inhibitors
Yan-Fa Yu, Chen Zhang, Yi-You Huang, Sirui Zhang, Qian Zhou, Xiangmin Li,
Zengwei Lai, Zhe Li, Yuqi Gao, Yinuo Wu, Lei Guo, Deyan Wu, and Hai-Bin Luo
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00024 • Publication Date (Web): 27 Feb 2020
Downloaded from pubs.acs.org on February 28, 2020
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Discovery and Optimization of Chromone Derivatives as
Novel Selective Phosphodiesterase 10 Inhibitors
Yan-Fa Yu||, Chen Zhang||, Yi-You Huang||, Sirui Zhang, Qian Zhou, Xiangmin
Li, Zengwei Lai, Zhe Li, Yuqi Gao, Yinuo Wu, Lei Guo*, Deyan Wu*, and Hai-
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Bin Luo
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006,
China
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ABSTRACT
Phosphodiesterase 10 (PDE10) inhibitors have received much attention as promising
therapeutic agents for central nervous system (CNS) disorders such as schizophrenia
and Huntington’s disease. Recently, a hit compound 1 with a novel chromone scaffold
has showed moderate inhibitory activity against PDE10A (IC50 = 500 nM). Hit-to-lead
optimization has resulted in compound 3e with an improved inhibitory activity (IC50 of
6.5 nM), remarkable selectivity (> 95-fold over other PDEs), and good metabolic
stability (RLM t1/2 = 105 min) by using an integrated strategy (molecular modeling,
chemical synthesis, bioassay, and cocrystal structure). The cocrystal structural
information provides insights into the binding pattern of 3e in the PDE10A catalytic
domain to highlight the key role of the halogen and hydrogen bonds toward Tyr524 and
Tyr693, respectively, thereby resulting in high selectivity against other PDEs. These
new observations are of benefit for the rational design of the next generation PDE10
inhibitors for CNS disorders.
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Keywords: Phosphodiesterase 10A, chromone derivatives, cocrystal structure,
metabolic stability, molecular docking, schizophrenia
INTRODUCTION
Schizophrenia is a common psychiatric illness characterized by basic personality
changes, thinking, emotion and behavior splitting, mental activities, and environmental
disharmony.1 Schizophrenia affects ~ 0.7 % of the world’s population, especially
affecting a large number of young adults with lifelong social and communication
disorders.2 Currently, the pathophysiology of schizophrenia has not yet been fully
elucidated meanwhile schizophrenia can be roughly characterized by positive
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(delusions, hallucinations, thinking and behavior disorder), negative (emotional apathy,
social dysfunction and lack of motivation) and cognitive (impairment of action,
attention, learning and memory) symptoms.3 Although typical and atypical
antipsychotic drugs, including chlorpromazine, olanzapine and risperidone, show
curative effect on positive psychotic symptoms by means of modulating dopamine 2
(D2) and 5-HT receptors, they are less effective in alleviating negative symptoms and
cognitive disorders. Furthermore, most current antipsychotic agents have non-ignorable
adverse effects, including extrapyramidal syndrome (such as acute dystonia),
vegetative system dysfunction (such as xerostomia and postural hypotension), weight
gain and hyperprolactinemia.4-6 Up to now, the medical needs for treatment of
schizophrenia is unmet, especially for a definite curative effect on negative symptoms
and cognitive symptoms as well as treatment resistance. Therefore, discovery and
development of new drugs targeting novel targets for schizophrenia is still an urgent
demand.7
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Phosphodiesterases (PDEs) as a superenzyme family (PDE 1~ 11) are in charge of
hydrolyzing the ubiquitous secondary messengers adenosine 3',5'-cyclic
monophosphate (cAMP) to 5'-AMP and guanosine 3',5'-cyclic monophosphate (cGMP)
to 5'-GMP, respectively.8 Inhibition of PDEs can prolong or enhance the effects of
physiological processes mediated by cAMP and/or cGMP, and has proved to be
therapeutic for various diseases,9-11 such as PDE5 inhibitor sildenafil for erectile
dysfunction and pulmonary arterial hypertension (PAH) 12, 13
It has been reported
.
that abnormalities in cAMP signalling pathways was responsible for the
pathophysiology of symptoms in schizophrenia.14, 15 In addition, cGMP might be
involved in the pathophysiology of cognitive symptoms in schizophrenia, since
it related to short-term changes in excitability in striatal neurons.16
Phosphodiesterase 10 (PDE10), a dual-substrate enzyme hydrolyzing both cAMP
and cGMP, is highly expressed in the medium spiny neurons of the striatum (MSNs) in
central nervous system (CNS) of many mammalian species.17 It is thought to be a
promising drug target for CNS disorders such as schizophrenia and Huntington’s
disease.18-20 PDE10 knockout mice were observed with reduced hyperactivity response
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in the locomotor activity model, which were likely triggered to have schizophrenic-like
behaviors by phencyclidine.21 Phosphodiesterase 10A (PDE10A) inhibitors are
expected to modulate via cAMP- and cGMP-dependent mechanisms both the dopamine
D1-direct and D2-indirect striatal pathways and regulate the phosphorylation status of
a panel of glutamate receptor subunits in the striatum.7 Papaverine, a commercial drug
capable of inhibiting PDE10, was prior reported to exhibit curative effect in rat models
of schizophrenia.21, 22 During the last two decades, an increasing number of patents on
novel PDE10 inhibitors have been applied by pharmaceutical companies, but few
results of clinical trials on the possibility of using PDE10 inhibitors as drug candidates
for schizophrenia or Huntington’s disease have been published (Figure 1).23-28 Despite
the promising safety profile of MP-10 exploited by Pfizer, clinical trials were
terminated at Phase II (NCT01939548) due to dissatisfactory pre-specified criteria for
efficacy (NCT01939548) as well as schizophrenic patients suffering an acute
exacerbation of symptoms (NCT01175135). Recently, a clinical Phase II study of the
effects of PDE10 inhibitor TAK-063 on the primary and secondary endpoints may be
suggestive of antipsychotic activity, but the higher risk of extrapyramidal syndromes
has also been found in contrast with the placebo group.29 Only OMS824 from Omeros
pipeline has received the designation of Orphan Drug for treating Huntington’s disease
as well as the designation of Fast Track drug for treating cognitive impairment observed
in patients suffering from Huntington’s disease, but the results are yet to be published.25
These clinical trial results highlight that PDE10 inhibitors are still urgently expected to
be developed to improve CNS disorder outcomes with reduced adverse effects.
A pre-screening of our internal compound library for novel PDE10 inhibitors
identified compound 1 (Figure 2) with a novel chromone scaffold, which had never
been reported among PDE10 inhibitors. As compound 1 only showed modest inhibitory
activity against PDE10 with an IC50 value of 500 nM, further structural optimizations
with the aid of cocrystal structure and molecular modeling were performed to find
compound 3e with remarkably improved inhibitory activity (IC50 = 6.5 nM) and
metabolic stability (RLM t1/2 of 105 min).
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Figure 1. Structure of PDE10A inhibitors in clinical trials.
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Figure 2. Structure of hit compound 1.
RESULTS AND DISCUSSION
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Rational Structural Design Based on the Binding Mode of Compound 1 with
PDE10A
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Molecular docking and molecular dynamics (MD) simulations were performed to
investigate the possible binding pattern between compound 1 and PDE10A. As shown
in Figure 3, the chromone scaffold of 1 formed a hydrogen bond with the conserved
Gln726 and π−π stacking interaction with the hydrophobic clamp contributed by
Phe729 and Ile692/Phe696, which were two characteristic interactions conserved in all
PDE inhibitors.30 Additionally, the ethyl linker stretched towards the unique selectivity
pocket (Q2 pocket), which provided an optimal access for the thiazol-5-ylmetyl
benzimidazole motif of 1 to be inserted into and an available H-bond formed with
Tyr693.
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Figure 3. The predicted binding mode of the hit compound 1 with PDE10A.
As is known, most potent and selective PDE10A inhibitors were reported to interact
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with this unique selectivity Q2 pocket.31, 32 Therefore, different substituents on the
benzimidazole moiety of 1 were firstly designed to obtain more appropriate binding
groups toward Q2 pocket. The chromone scaffold of 1 was retained to keep the
representative interactions with conserved Gln726 and Phe729. Prior to chemical
synthesis, the binding modes of designed compounds with PDE10A were predicted by
molecular docking and dynamics simulations, and the binding free energies were
estimated by MM-GBSA method. In addition, the root mean square deviation (RMSD)
plots and hydrogen/halogen bond analysis for each system during MD simulations are
shown in Figure S1, S2 and S3 in Supporting Information.33, 34 According to the
computational results, potential compounds were selected for synthesis and biological
evaluation. The structure-activity relationship (SAR) results are summarized in Table
1.
Table 1. The inhibitory activity of compounds 1a-1k against PDE10A and their
estimated binding free energies.
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Cp. R1
R2
R3
X
IC50 (nM)a GBTOT (kcal/mol)b
1a
1b
1c
H
H
Cl
H
H
H
CH3
CH3 S=O
CH3
S
>1000
>1000
112 ± 8
-29.07±2.86
-29.49±2.29
-36.01±2.30
C
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1d
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H
H
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OCH3
CF3
C
C
>100
>100
-33.72±2.36
-32.73±2.54
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H
H
H
H
H
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CH3
CH3
CH3
CH3
CH3
Cl
C
C
C
C
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C
355 ± 16
>100
-39.77±2.23
-39.04±2.88
-38.95±4.06
-38.47±3.18
-38.45±2.76
-38.70±3.02
F
F
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>100
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76 ± 6
95 ± 11
1j
N
1k
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a Values are means of three independent experiments.
b Binding free energies are relative values estimated by MM-GBSA method.
The First-round of Optimization Assisted with MD Simulations to Result in 1i with
Inhibitory Potency Increased by 10-Fold
The hit compound 1 could be roughly divided into three major parts, including a
chromone scaffold, an ethyl linker and a substituted benzimidazole moiety (selective
pocket binding group, SPBG). Firstly, while the substitution at the N-1 position of
SPBG was absent, the key ethyl linker replaced simply by a thioethyl or sulfoxide ethyl
group afforded 1a or 1b with a remarkable lost in PDE10A inhibitory activity.
Comparing with 1a or 1b, compound 1c based on the ethyl linker had a simple -Cl
substituent at the 6-position of the chromone scaffold to surprisingly exhibit increasing
inhibition with an IC50 value of ~112 nM, indicating the ethyl linker as a crucial
fragment for inhibitory potency. Remarkable increases of ~7 kcal/mol in the absolute
values of predicted binding free energies (MM-GBSA total energy, GBTOT) from 1a
and 1b to 1c were also observed, further convincing that the ethyl group rather than the
thioethyl or sulfoxide ethyl group was favorable to binding Q2 pocket. However, -
OCH3 or -CF3 substituent replacing the -CH3 group at the 5-position of the
benzimidazole fragment was investigated to obtain 1d or 1e with no enhancement in
potency, which was in consistence with the predicted binding free energy values.
Given that trifluoromethyl and the methyl groups were comparable in size, the
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decreased affinity of 1e might be attributed to the unfavorable electrostatic repulsions
while 1d with a methoxy group could provide steric hindrance toward the narrow space
of Q2 pocket. As the methyl group fixed at the 5-position of benzimidazole, various
substituents at the N-1 position of benzimidazole were designed to tune the inhibitory
potency such as benzyl, fluorobenzyl, phenyl and pyridyl groups. Binding patterns and
affinities for 1f-1k with PDE10 were pre-estimated by molecular docking and MD
simulations. All these compounds were predicted to possess better binding affinities
with an increase in the absolute values of GBTOT from 2.4 to 3.7 kcal/mol, which
suggested that aromatic rings at 5-position might benefit for binding. As depicted in
Table 1, compounds with benzyl (1f) or fluorobenzyl (1g, 1h) group at the N-1 position
showed slightly improved inhibitory activity while compounds with phenyl (1i, 1k) or
pyridyl ring (1j) exhibited a great enhancement of inhibitory potency. It was worth
mentioning that 1i owning 5-methyl-1-pheyl benzimidazole group showed a 10-fold
enhancement in inhibitory activity (IC50 = 52 nM) comparing with 1. However, the 4-
pyridyl ring on SPBG instead of the phenyl ring could give rise to a slight decrease of
inhibitory activity. The SAR study indicated that the phenyl ring was a preferable
substituent at the N-1 position of benzimidazole, which fitted the selective pocket of
PDE10 better.
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The synthetic route of benzimidazole derivatives 1a-1b is outlined in Scheme 1.
3-(hydroxymethyl)-4H-chromen-4-one 3 was synthesized from commercially available
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4-oxo-4H-chromene-3-carbaldehyde 2.
Brominating of 3 with CBr4 gave 4.
Compound 4 was reacted with 5-methyl-1,3-dihydro-2H-benzo[d]imidazole-2-thione
to obtain 1a, which was oxidized by Oxone to afford compound 1b.36,37
The synthetic route of 7a-7i is outlined in Scheme 2. A Buchwald–Hartwig
reaction of 8 with corresponding aryl amine gave the nitro aniline analogues 10a-10e.38
Catalytic hydrogenation of 10a-10e yielded the phenylenediamines 7d-7h. On the other
hand, a reaction of the 11 with an excess of aniline gave the nitro aniline derivative 13.
The compound 13 was treated under catalytic hydrogenation conditions to give 7i.
The synthesis of benzimidazole analogues 1c-1k is shown in Scheme 3. One pot
reaction of 2 or 5 with cyclic malonate in the presence of triethylamine and formic acid
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(1:1) under reflux for 2 h gave carboxylic acid 6a-6b.39 Compound 6a or 6b was
condensed with the phenylenediamine derivative 7a-7i in the presence of HATU and
DIPEA and then cyclized in acetic acid to give 1c-1k.40
Scheme 1. General procedures used to synthesize compounds 1a-1b.
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Reagents and conditions: (a) Al2O3, isopropyl alcohol, 75 °C, 4 h; (b) CBr4, PPh3,
CH2Cl2, rt, 4 h; (c) Et3N, DMF, 80 °C, 12h; (d) Oxone, ethyl acetate, rt, 1h.
Scheme 2. General procedures used to synthesize compounds 7a-7i.
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Reagents and conditions: (a) Pd2(dba)3, PhMe, Cs2CO3, BINAP, 110 oC, 24 h; (b) H2,
Pd(OH)2/C, EtOH, rt, 6 h or SnCl2, ethyl acetate, reflux, 2 h; (c) K2CO3, DMSO, 120
oC, 12h; (d) SnCl2, ethyl acetate, reflux, 2 h.
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Scheme 3. General procedures used to synthesize compounds 1c-1k.
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Reagents and conditions: (a) Cyclic malonate, Et3N, HCO2H, reflux, 2-3 h; (b) i) 7a-7i,
HATU, DIPEA, CH2Cl2, rt, 12 h; ii) Acetic acid, 90 oC,12 h.
Halogen Bonds were Important to Increase Inhibitory Potency Illustrated by
Cocrystal Structures
To validate the binding modes between the chromone inhibitors and PDE10A, the
cocrystal structure of compound 1i with PDE10A was successfully determined (PDB
ID: 6KO0). As shown in Figure 4A, the electron density in (2Fo-Fc) and (Fo-Fc)
unambiguously showed that 1i bound to the catalytic pocket of PDE10A. The chromone
moiety of 1i served as the scaffold to be sandwiched in the hydrophobic clamp between
Phe729 and Phe696/Ile692, while the carbonyl oxygen provided a strong H-bond with
the amide nitrogen of conserved Gln726. Furthermore, the 5-methyl-1-phenyl
benzimidazole group of 1i was fitted well into the Q2 pocket meanwhile an H-bond
between the 3-N of the benzimidazole group and Tyr693 was also observed. Notably,
the binding mode revealed by cocrystal structure was consistent with the predicted
pattern derived from MD simulations (Figure 4B), illustrating that the MD-based
protocol employed in the present study was reliable.
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Figure 4. (A) Cocrystal structure of PDE10-1i (PDB ID: 6KO0). The 2Fo-Fc electron
density is contoured in dark blue at 1.0 σ. (B) Alignment of the crystal structure (yellow)
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and the predicted structure (off-white) of PDE10-1i.
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The cocrystal structure of PDE10A with 1i implied that further optimization could
be implemented in several sites. Recently, halogen bonding, noncovalent
intermolecular interaction occurring between Lewis bases (O, N, and S) and Lewis
acids (Cl, Br, and I), had been recognized as a prevalent ligand-protein interaction,
which were successfully utilized to improve the potency of PDE5 inhibitors.41 In our
case of PDE10-1i, the C-6 position of the chromone ring seemed to be an appropriate
site for halogenation since it was close to the phenolic hydroxyl group of Tyr524 with
a distance of 3.8 Å. Small halogen substituents at the C-6 position of chromone were
expected to be well-tolerated and to build up a potential interaction with Tyr524 by
means of halogen bond. Thus, halogen substituents (F, Cl, Br and I) were introduced at
the C-6 position of the chromone moiety to afford compounds 2a, 2b, 2c and 2d,
respectively. Prior to synthetic and bioassay work, binding free energies were also
calculated based on the predicted binding poses of 2a, 2b and 2d with PDE10. As
supposed, halogen bonds were observed for each compound binding PDE10 in docking
patterns and MD trajectories. In addition, 0.7~2.5 kcal/mol increase in the absolute
values of GBTOT from 1i to 2a, 2b and 2d indicated that halogenation was applicable.
As a result, distinct improvement of inhibitory potency through these simple
modifications was inspiring. As depicted in Table 2, fluoro- (2a), chloro- (2b) and
bromo- (2c) substituted derivatives exhibited increased inhibitory activity against
PDE10A by about 2-3 times referring to 1i except iodine analogue 2d, indicating that
the relatively big radius of the iodine atom was detrimental to form halogen bond with
Tyr524. Similarly, introducing 6-methoxy group to the chromone afforded 2e with
potency enhanced by about 3-fold, which might be on account of an extra hydrogen
bond associated with Tyr524.
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Table 2. The inhibitory activities of compounds 2a-2e against PDE10A and their
estimated binding free energies.
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Cp.
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2e
R
F
Cl
Br
I
IC50 (nM)a
17 ± 1
GBTOT (kcal/mol)b
-39.55±2.76
-39.16±2.44
-40.94±3.78
ndc
22 ± 2
34 ± 2
54 ± 5
18 ± 2
OCH3
-39.65±3.01
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a Values are means of three independent experiments.
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b Binding free energies are relative values estimated by MM-GBSA method.
c Not determined.
The cocrystal structure of 2b bound in PDE10A (PDB ID: 6KO1) was also
successfully determined. As shown in Figure 5, 2b primarily interacted with Gln726
and Phe729 of PDE10A via a hydrogen bond and π−π stacking, respectively, besides a
hydrogen bond with Tyr693 in the Q2 pocket. These interactions were similar to that
of 1i. Interestingly, an extra halogen bond was formed between chlorine atom of 2b and
Tyr524, which contributed to the enhancement of inhibitory activity against PDE10A.
The synthesis of benzimidazole analogues 2a-2e are shown in Scheme 4. Initially, one
pot reaction of 5, 14a-14d and cyclic malonate in the presence of triethylamine and
formic acid (1:1) under reflux for 2 h gave carboxylic acid 15a-15e. Compound 15a-
15e were condensed with the phenylenediamine derivative 7g in the presence of HATU
and DIPEA and then cyclized in acetic acid to give 2a-2e.
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Figure 5. (A) Cocrystal structure of PDE10-2b (PDB ID: 6KO1). The 2Fo-Fc electron
density is contoured in dark blue at 1.0 σ. (B) Alignment of the crystal structure (yellow)
and the predicted structure of PDE10-2b (off-white).
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Scheme 4. General procedures used to synthesize compounds 2a-2e.
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Reagents and conditions: (a) Cyclic malonate, Et3N, HCO2H, reflux, 2-3 h; (b) i) 7g,
HATU, DIPEA, CH2Cl2, rt, 12 h; ii) Acetic acid, 90 oC, 12 h.
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The Third-round of Optimization Resulted in a Nanomolar and Metabolically
Stable PDE10A Inhibitor
Although the structure-based optimizations from the hit compound 1 to 2b were
effective and inspiring assisted with molecular docking and MD simulations, high in
vitro clearance of 2b was also observed with half-life (t1/2) of 0.58 min in rat liver
microsomes (RLM) test, which was detrimental for further in vivo study. To improve
the metabolic stability and pharmacokinetic properties of 2b, the third-round
optimization was performed in the possible metabolic sites on the chromone scaffold
as well as the ethyl linker.
Molecular docking suggested that the benzimidazole group of 2b could also
stretched to the unique selective pocket by replacing the ethyl linker with vinyl one.
Molecular docking and MD simulations confirmed that introducing the vinyl linker for
newly designed compounds didn’t cause major alternations in binding patterns and in
binding affinities compared with 2b. The predicted binding free energies (GBTOT) for
3a-3f with PDE10 ranged from -38.10 to -40.16 kcal/mol, which well matches 2i with
39.16 kcal/mol. Compound 3a was designed with a vinyl linker and found to be
improved in both metabolic stability (RLM t1/2 of 8.68 min) and inhibitory potency (IC50
of 7.9 nM). The introduction of a nitrogen atom at the 7-position of the benzimidazole
ring was successful to afford 3b with less inhibitory activity than 3a. Besides, the
metabolic stability of compound 3c with chlorine atom at 5-position of the
benzimidazole ring was further improved with RLM t1/2 of 13.0 min. Compound 3d
regarded as a vinyl analogue of 2b, exhibited both good inhibitory activity (IC50 = 7.7
nM) and good metabolic stability with RLM t1/2 of 23.3 min, thereby corroborating the
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C=C bond linker enhancing the metabolic stability meanwhile the chlorine atom at 5-
position of the benzimidazole ring was benefit for preventing the compound from
metabolic degradation. In other words, the ethyl linker and the methyl group at the 5-
position of the benzimidazole ring of 2b seemed to be simultaneously prone to the
metabolic degradation. (Table 3). The cocrystal structure of PDE10A with 2b implied
that a halogen bond was formed between the chromone scaffold and Tyr524, we
speculated that the C-6 position of chromone scaffold was a probable metabolic site. It
was a deliberate attempt to introduce the fluorine atom at C-6 position of the chromone
scaffold to obtain 3e with a slight improvement on PDE10A inhibitory potency (IC50 =
6.5 nM). More importantly, 3e had a dramatic improvement on metabolic stability with
RLM t1/2 of 105 min. Conversion of monochloro-substituted 3c to dichloro-substituted
3f, in spite of significant enhancement in metabolic stability, led to a significant loss of
inhibitory activity on account of the disruption of the H-bond interaction between 3f
and Gln726 as well as extremely poor solubility. Although the clinical candidate TAK-
063 exhibited more advantages of inhibitory activity and metabolic stability, compound
3e also indicated desirable properties as a potential PDE10 inhibitor. Our findings
suggest that C-6 position of the chromone scaffold is a probable metabolic site in this
series of PDE10 inhibitors.
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Table 3. The inhibitory activities of compounds 3a-3f and TAK-063 against PDE10A，
their estimated binding free energies, and rat microsomal stability.
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Cp.
3a
3b
3c
3d
3e
R1
H
H
X
R2
IC50 (nM)a GBTOT (kcal/mol)b RLM t1/2 (min)c
C CH3
N CH3
C
7.9±0.4
22 ± 4.0
11 ± 1.7
7.7 ± 0.7
6.5 ± 0.4
>100
-39.19±2.53
-38.36±2.20
-38.10±2.40
-40.16±2.71
-39.70±2.61
-39.32±2.22
8.68
ndd
13.0
23.3
105
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H
Cl
Cl C CH3
Cl
Cl C Cl
F
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0.30 ± 0.10
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a Values are means of three independent experiments.
b Binding free energies are relative values estimated by MM-GBSA method.
c t1/2, calculated in vitro elimination half-life
dNot determined.
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The binding mode of 3e with PDE10A was predicted by molecular modeling as
illustrated in Figure 6. Ideally, the vinyl linker together with the benzimidazole group
well matched the narrow Q2 pocket and bonded to the chromone scaffold in the
hydrophobic clamp consisting of Phe729 and Phe696/Ile692. Notably, both hydrogen
bond with Gln726/Tyr693 and the halogen bond with Tyr524 were obviously pointed
out to make sense out of good inhibitory potency. Benzimidazole analogues 3a-3f were
prepared according to the synthesis route in Scheme 5. The carboxylic acid 16a-16c
were obtained from a Knoevenagel-Doebner condensation.42 Compound 16a-16c were
condensed with the phenylenediamine derivative 7g or 7i in the presence of HATU and
DIPEA and then cyclized in acetic acid to give 3a-3f.
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Figure 6. The predicted binding mode of compound 3e with PDE10A.
Scheme 5. General procedures used to synthesize compounds 3a-3f.
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Reagents and conditions: (a) pyridine, malonic acid, reflux, 45 min; (b) i) 7g or 7i,
HATU, DIPEA, CH2Cl2, rt, 12 h; ii) Acetic acid, 90 oC, 12 h.
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Remarkable Selectivity of 3e Against other PDE Isoforms.
Compound 3e with the good affinity and desirable metabolic stability was also
evaluated for the selectivity across other PDE isoforms. As shown in Table 4, 3e
revealed ideal PDE10 selectivity more than 100-fold over PDE1B, 2A, 3A, 4D, 7A,
8A, and 9A except PDE5A. However, the IC50 value of 3e against PDE5A was still
determined as ~620 nM, corresponding to 95-fold selectivity. These results demonstrate
that 3e is a potent and highly selective PDE10A inhibitor, which has the potential to be
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exploited for further pharmacological research in the future.
Table 4. Selectivity of compound 3e toward PDEs isoforms.
PDEs
PDE10A2(449-770)
PDE1B(10-487)
IC50 (μM)
0.0065 ± 0.0004
> 10
Selectivity
-
>1524-fold
430-fold
>1524-fold
131-fold
95-fold
PDE2A(580-919)
PDE3A(679-1087)
PDE4D2(86-413)
PDE5A1(535-860)
PDE7A1(130-482)
PDE8A1(480-820)
PDE9A2(181-506)
2.82 ± 0.43
>10
0.86 ± 0.21
0.62 ± 0.03
> 10
>1524-fold
518-fold
317-fold
3.40 ± 0.67
2.08 ± 0.38
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CONCLUSION
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In summary, pre-screening of our internal compound library discovered a novel
chromone-scaffold hit compound 1 with modest PDE10 inhibitory activity (IC50 = 500
nM). Three rounds of structural optimization were performed with the aid of cocrystal
structure, molecular docking and MD simulations. In particular, the satisfying strategy
for the structural optimization was performed to prevent this series of the chromone
compounds from the metabolic degradation via two available approaches. One is the
rigid vinyl linker instead of the rotatable ethyl linker. Another is that halogen atom is
suitable to be introduced on the chromone scaffold and the benzimidazole group,
indicating the possible metabolic site on the base of this molecular skeleton. This
efficient strategy for novel structural discovery resulted in 13 compounds with IC50
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values ranging from 1 to 100 nM and 3 compounds with an IC50 value less than 10 nM.
The lead compound 3e exhibited sufficient inhibitory activity against PDE10A (IC50 =
6.5 nM), remarkable selectivity against other PDEs (> 95- fold) and desirable metabolic
stability (RLM t1/2 of 105 min). Our study presents an excellent example for efficient
structural optimization of novel PDE10 inhibitors for the potential CNS drug discovery.
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METHODS
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Chemistry
Unless specified, all reagents and starting materials were purchased from commercial
sources and used as received. Analytical thin layer chromatography (TLC) was
performed using pre-coated silica gel plate. Visualization was achieved by UV light
(254 nm). Column chromatography was performed using silica gel. 1H NMR and 13C
NMR spectra were recorded on a BrukerBioSpin GmbH spectrometer at 400.1 and
100.6 MHz, respectively. Coupling constants are given in Hz using TMS as an internal
standard and CDCl3 or DMSO - d6 or MeOD as solvents. Chemical shift is given in
ppm (δ). High-resolution mass spectra (HRMS) were obtained on an IT-TOF mass
spectrometer. The purity of the compounds was determined by reverse-phase high-
performance liquid chromatography (HPLC) analysis and confirmed to be more than
95%. HPLC instrument: SHIMADZU LC-20AT (column, Hypersil BDS C18, 5.0 μm,
4.6 mm×150 mm (Elite); detector, SPD-20A UV/vis detector, UV detection at 254 nm;
elution, MeOH in water (60 %, v/v); T = 25 °C; flow rate = 1.0 mL/min).
3-(Hydroxymethyl)-4H-chromen-4-one (3). A suspension of formylchromone (5.6
mmol, 974 mg) and basic Al2O3 (20 g) in 2-propanol (200 mL) was kept with stirring
o
at 75 C for 4 h. The mixture was filtered by celite and the solvent evaporated. The
crude material was purified by silica gel column chromatography (petroleum/ethyl
acetate, 1:1) to afford 3 (808 mg) as a colorless solid. Yield: 82%; 1H NMR (400 MHz,
CDCl3) δ 8.23 (dd, J = 8.0, 1.3 Hz, 1H), 7.96 (s, 1H), 7.72 – 7.67 (m, 1H), 7.47 (d, J =
8.5 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 4.59 (d, J = 5.8 Hz, 2H). The obtained spectra
match those reported.35
3-(Bromomethyl)-4H-chromen-4-one (4). A solution of triphenylphosphine (469
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mg, 1.7 mmol) in dry CH2Cl2 (2 mL) was added dropwise to a stirred solution of carbon
tetrabromide (592 mg, 1.7 mmol) and 3-(hydroxymethyl)-4H-chromen-4-one (246 mg,
1.4 mmol) in dry CH2Cl2 (11 mL) at room temperature under argon atmosphere. The
reaction was stirred for 4 h at room temperature and was concentrated in vacuo to give
the crude product. This was purified by silica gel column chromatography
(petroleum/ethyl acetate, 2:1) to afford 4 (294 mg) as a colorless solid.43 Yield: 88 %;
1H NMR (400 MHz, CDCl3) δ 8.26 (dd, J = 8.0, 1.7 Hz, 1H), 8.13 (s, 1H), 7.69 (ddd, J
= 8.6, 7.2, 1.7 Hz, 1H), 7.48 – 7.42 (m, 2H), 4.41 (s, 2H).
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3-(((5-Methyl-1H-benzo[d]imidazol-2-yl)thio)methyl)-4H-chromen-4-one (1a).
To 5-methyl-1,3-dihydro-2H-benzo[d]imidazole-2-thione (656 mg, 4.0 mmol) in
dimethylformamide (10 mL), triethylamine (836 μL, 6.0 mmol) and 4 (1434 mg, 6.0
mmol) were added. After stirring at 80 °C overnight, water was added to the mixture
followed by extraction with ethyl acetate. After the ethyl acetate phase was dried with
anhydrous sodium sulfate, it was concentrated and the residue was purified by silica gel
column chromatography (petroleum/ethyl acetate, 1:1) to obtain 1a (978 mg) as a
colorless solid. Yield: 76 %; purity: 99.9 %; 1H NMR (400 MHz, DMSO - d6) δ 12.41
(d, J = 6.2 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H), 8.10 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (ddd,
J = 8.6, 7.2, 1.6 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.54 – 7.48 (m, 1H), 7.46 – 7.36 (m,
1H), 7.24 – 7.13 (m, 1H), 6.95 (d, J = 8.2 Hz, 1H), 4.29 (s, 2H), 2.38 (d, J = 2.0 Hz,
3H) ; 13C NMR (101 MHz, DMSO - d6 ) δ 176.1, 156.3, 155.5, 149.6, 134.7, 133.0,
132.1, 131.1, 126.0, 125.4, 123.6, 123.2, 120.8, 118.9, 110.1, 109.6, 27.0, 21.7; HRMS
(ESI-TOF) m/z calcd for C18H14N2O2S [M+H]+ 323.0849, found 323.0843.
3-(((5-Methyl-1H-benzo[d]imidazol-2-yl)thio)methyl)-4H-chromen-4-one (1b).
In a round-bottomed flask (50 mL) equipped with a magnetic stirrer, a solution of 1a
(322 mg, 1.0 mmol) in ethyl acetate (5 mL) was prepared. Oxone was added and the
mixture was stirred at room temperature for 24 h. When the starting sulfane had
completely disappeared, the mixture was quenched by adding H2O (10 mL). The
product was extracted with ethyl acetate and the combined extracts were dried with
anhydrous sodium sulfate. It was concentrated and the residue was purified by silica
gel column chromatography (petroleum/ethyl acetate, 1:1) to obtain 1b (196 mg) as a
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colorless solid. Yield: 58 %; purity: 99.4 %; 1H NMR (400 MHz, DMSO - d6 ) δ 13.38
(s, 1H), 8.29 (d, J = 11.9 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.88 – 7.83 (m, 1H), 7.69
(d, J = 8.4 Hz, 1H), 7.60 – 7.49 (m, 2H), 7.42 – 7.31 (m, 1H), 7.13 (dd, J = 18.5, 8.2
Hz, 1H), 4.48 (d, J = 12.7 Hz, 1H), 4.29 (d, J = 12.8 Hz, 1H), 2.44 (s, 3H ); 13C NMR
(101 MHz, DMSO - d6) δ 175.8, 157.5, 156.4, 135.1, 135.0, 126.4, 126.4, 126.2, 125.5,
125.5, 125.4, 123.3, 119.0, 119.0, 113.9, 113.8, 51.1, 21.8; HRMS (ESI-TOF) m/z
calcd for C18H14N2O3S [M+H]+ 339.0798, found 339.0784.
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General Procedures for Synthesis of Compounds 6a-6b. A mixture of 4-oxo-
benzopyran-3-carboxaldehyde 2 or 5 (1.0 mmol), meldrum's acid (1.0 mmol) and
o
triethylamine formic acid (1:1) 5 mL was refluxed at 100 C for 2 h, until the
disappearance of starting material. It was cooled to room temperature and poured into
ice water. The mixture was acidified to pH = 2 with 6 N HCl. The pale yellow solid
was filtered washed with water and purified by column chromatography. The obtained
spectra match those reported. 39
3-(4-Oxo-4H-chromen-3-yl)propanoic acid (6a). Colorless solid. Yield: 93%; 1H
NMR (500 MHz, CDCl3) δ 8.23 (dd, J = 8.0, 1.2 Hz, 1H), 7.91 (s, 1H), 7.69 – 7.64 (m,
1H), 7.46 – 7.39 (m, 2H), 2.80 – 2.74 (m, 4H).
3-(6-Chloro-4-oxo-4H-chromen-3-yl)propanoic acid (6b). Colorless solid. Yield:
90%; 1H NMR (400 MHz, MeOD) δ 8.14 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.72 (dd, J
= 9.0, 2.6 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 2.74 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.2
Hz,2H).
General Procedures for Synthesis of Compounds 7d-7f. A solution of 2-
nitroaniline (30.0 mmol) and SnCl2·2H2O (26.4 g, 109.1 mmol) was heated in ethyl
acetate at reflux for 2h, the mixture was then cautiously quenched with saturated
sodium bicarbonate aqueous solution. The product was extracted with ethyl acetate and
the combined extracts were dried with anhydrous sodium sulfate. It was concentrated
and the residue was purified by silica gel column chromatography to give the product.37
1
N1-benzyl-4-methylbenzene-1,2-diamine (7d). Colorless solid. Yield: 45%; H
NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 13.7, 6.8 Hz, 1H), 7.39 (d, J = 7.2 Hz, 2H),
7.34 (t, J = 7.4 Hz, 2H), 7.29 (dd, J = 9.2, 4.6 Hz, 1H), 6.57 (d, J = 7.0 Hz, 2H), 4.28
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N1-(4-fluorobenzyl)-4-methylbenzene-1,2-diamine (7e). Colorless solid. Yield:
59%; 1H NMR (500 MHz, CDCl3) δ 7.33 (dd, J = 7.8, 5.6 Hz, 2H), 7.01 (t, J = 8.5 Hz,
2H), 6.63 – 6.49 (m, 3H), 4.24 (d, J = 8.6 Hz, 2H), 3.37 (s, 2H), 2.22 (d, J = 9.3 Hz,
3H).
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N1-(3-fluorobenzyl)-4-methylbenzene-1,2-diamine (7f). Colorless solid. Yield:
42%; 1H NMR (400 MHz, CDCl3) δ 7.33 (dd, J = 13.9, 7.8 Hz, 1H), 7.19 (d, J = 7.6
Hz, 1H), 7.14 (d, J = 9.7 Hz, 1H), 6.99 (td, J = 8.4, 2.2 Hz, 1H), 6.62 (d, J = 5.8 Hz,
2H), 6.55 (d, J = 8.4 Hz, 1H), 4.33 (s, 2H), 3.45 (s, 2H), 2.25 (s, 3H).
General Procedures for Synthesis of Compounds 7g-7h. To a mixture of 9d or
9e (1.0 mmol) in EtOH (10 mL) was added palladium hydroxide (238 mg, 1.0 mmol)
and stirred at room temperature for 6 h under hydrogen atmosphere. The mixture was
then filtered through a pad of celite. The filtrate was concentrated in vacuo to give a
yellow solid, which was purified by silica gel chromatography (petroleum/ethyl acetate,
10:1) to give 7g-7h as colorless solid.
3-Amino-5-methyl-2-(pyridin-4-ylamino)benzene-1-ylium
(7g).
Colorless
1
solid.Yield: 89%; H NMR (400 MHz, CDCl3) δ 8.24 (dd, J = 4.8, 1.5 Hz, 2H), 7.01
(d, J = 7.9 Hz, 1H), 6.67 (d, J = 1.1 Hz, 1H), 6.62 (dd, J = 7.9, 1.3 Hz, 1H), 6.53 (dd, J
= 4.8, 1.5 Hz, 2H), 5.59 (s, 1H), 2.38 – 2.27 (m, 3H).
5-Methyl-N2-(pyridin-4-yl)pyridine-2,3-diamine (7h). Colorless solid. Yield: 81%;
1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 5.3 Hz, 2H), 7.71 (s, 1H), 7.02 (d, J = 5.3
Hz, 2H), 6.91 (s, 1H), 6.63 (s, 1H), 2.25 (s, 3H).
4-Chloro-N1-phenylbenzene-1,2-diamine (7i). A solution of 13 (30.0 mmol) and
SnCl2·2H2O (26.4 g, 109.1 mmol) was heated in ethyl acetate at reflux for 2 h, the
mixture was then cautiously quenched with saturated aqueous NaHCO3. The product
was extracted with ethyl acetate and the combined extracts were dried with anhydrous
sodium sulfate. It was concentrated and the residue was purified by silica gel column
chromatography to give the product (1.9 g). Yield: 30%; 1H NMR (500 MHz, CDCl3)
δ 7.21 (t, J = 7.8 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 6.83 (dd, J = 15.6, 8.3 Hz, 1H), 6.78
(d, J = 2.2 Hz, 1H), 6.72 – 6.69 (m, 3H).
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General Procedures for Synthesis of Compounds 10a-10e. To a mixture of
nitrobenzene (1.0 mmol) in toluene (10 mL) were added aniline (3.0 mmol),
tris(dibenzylideneacetone)dipalladium (Pd2(dba)3, 92 mg, 0.1 mmol), BINAP (23 mg,
0.15 mmol) and cesium carbonate (650 mg, 2.0 mmol). The mixture was then stirred at
110 oC for 24 h under argon atmosphere. After cooling to room temperature, the mixture
was filtered through a pad of celite and the filtrate was then concentrated in vacuo to
afford a residue, which was purified by silica gel chromatography (petroleum/ethyl
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acetate, 50 : 1) to give 10a-10e as red oil. The obtained spectra match those reported.
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N-benzyl-4-methyl-2-nitroaniline (10a). Orange oil. Yield: 75%; H NMR (400
MHz, CDCl3) δ 8.35 (s, 1H), 8.03 (s, 1H), 7.43 – 7.30 (m, 5H), 7.23 (dd, J = 8.7, 1.8
Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 4.56 (d, J = 5.7 Hz, 2H), 2.28 (s, 3H).
1
N-(4-fluorobenzyl)-4-methyl-2-nitroaniline (10b). Orange oil. Yield: 68%; H
NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.00 (s, 1H), 7.31 (dd, J = 8.4, 5.4 Hz, 2H),
7.22 (dd, J = 8.7, 1.8 Hz, 1H), 7.04 (t, J = 8.6 Hz, 2H), 6.69 (d, J = 8.7 Hz, 1H), 4.50
(d, J = 5.7 Hz, 2H), 2.26 (s, 3H).
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N-(3-fluorobenzyl)-4-methyl-2-nitroaniline (10c). Orange oil. Yield: 82%; H
NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.99 (s, 1H), 7.31 (dd, J = 13.9, 7.8 Hz, 1H),
7.20 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 9.6 Hz, 1H), 6.97 (t, J =
8.4 Hz, 1H), 6.66 (d, J = 8.7 Hz, 1H), 4.53 (d, J = 5.8 Hz, 2H), 2.25 (s, 3H).
5-Methyl-3-nitro-N-(pyridin-4-yl)pyridin-2-amine (10d). Red oil. Yield: 53%;
1H NMR (400 MHz, CDCl3) δ 10.13 (s, 1H), 8.51 (s, 2H), 8.43 (d, J = 9.0 Hz, 1H),
8.38 (d, J = 8.2 Hz, 1H), 7.69 (s, 2H), 2.38 (d, J = 9.1 Hz, 3H).
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N-(4-methyl-2-nitrophenyl)pyridin-4-amine (10e). Orange oil. Yield: 55%; H
NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.49 (d, J = 5.8 Hz, 2H), 8.03 (d, J = 1.1 Hz,
1H), 7.53 (d, J = 8.6 Hz, 1H), 7.38 (dd, J = 8.6, 2.0 Hz, 1H), 7.09 (dd, J = 4.8, 1.4 Hz,
2H), 2.45 – 2.35 (m, 3H).
4-Chloro-2-nitro-N-phenylaniline (13). Aniline (930 mg, 10.0 mmol), and
potassium carbonate (2.7 g, 20.0 mmol) were added to a solution of 1,4-dichloro-2-
nitrobenzene (1.9 g, 10.0 mmol) and DMSO. The resulting mixture was stirred at about
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120 C for about 16 h, and then the solvent was removed by evaporation in vacuo.
Following standard extractive workup with dichloromethane (2×100 mL), the crude
residue was purified by silica gel column chromatography (dichloromethane/methanol,
20:1) to give the title product as a yellow solid (868 mg). Yield: 35%; 1H NMR (400
MHz, CDCl3) δ 9.45 (s, 1H), 8.23 – 8.19 (m, 1H), 7.48 – 7.40 (m, 3H), 7.33 – 7.28 (m,
2H), 7.25 (s, 1H), 7.18 – 7.14 (m, 1H).
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General Procedures for Synthesis of Compounds 1c-1k. To a mixture of
appropriate amine (1.0 mmol) in CH2Cl2 (15 mL) were added HATU (570 mg, 1.5
mmol), N, N-diisopropylethylamine (516 mg, 4.0 mmol) and 6a or 6b (1.2 mmol), and
stirred at room temperature for 12 h. The mixture was diluted with saturated sodium
bicarbonate aqueous solution (100 mL) and extracted with dichloromethane (300 mL).
The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered
and concentrated in vacuo. To the residue was added acetic acid (20 mL), and stirred at
90 oC for 12 h. Then the reaction mixture was concentrated in vacuo. The residue was
diluted with saturated sodium bicarbonate aqueous solution and extracted with
dichloromethane. The organic layer was washed with brine, dried over anhydrous
sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (petroeum/ethyl acetate, 10:1) to give compound 1c-1k as
a colorless solid.40
6-Chloro-3-(2-(5-methyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-one
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(1c). Colorless solid. Yield: 45%; purity: 99.7%; H NMR (500 MHz, CDCl3) δ 8.10
(s, 1H), 7.93 (s, 1H), 7.59 – 7.55 (m, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 8.9 Hz,
1H), 7.32 (s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 3.29 (t, J = 7.1 Hz, 2H), 3.03 (t, J = 7.0 Hz,
2H), 2.45 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 177.3, 154.8, 154.1, 153.5, 134.0×2,
132.3, 131.1, 124.9, 124.5, 123.9, 123.0, 120.0×2, 114.5, 114.1, 28.0, 24.6, 21.6;
HRMS (ESI-TOF) m/z calcd for C19H15N2O2Cl [M+H]+ 339.0895, found 339.0895.
3-(2-(5-Methoxy-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-one
(1d).
Colorless solid. Yield: 53%; purity: 99.9%; 1H NMR (500 MHz, CDCl3) δ 8.15 (d, J =
8.0 Hz, 1H), 7.89 (s, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.40 (dd, J = 13.9, 8.5 Hz, 2H), 7.34
(t, J = 7.5 Hz, 1H), 7.01 (s, 1H), 6.83 (d, J = 8.7 Hz, 1H), 3.81 (s, 3H), 3.24 (t, J = 7.2
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Hz, 2H), 3.02 (t, J = 7.2 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.4, 156.5, 156.0,
154.0, 153.7×2, 133.7×2, 125.4×2, 125.1×2, 123.6, 122.9, 118.2, 111.3, 55.8, 28.3, 24.9;
HRMS (ESI-TOF) m/z calcd for C19H16N2O3 [M+H]+ 321.1234, found 321.1212.
3-(2-(5-(Trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-one
(1e). Colorless solid. Yield: 44%; purity: 99.9%; 1H NMR (400 MHz, Acetone - d6) δ
8.14 (d, J = 10.7 Hz, 2H), 7.90 (s, 1H), 7.76 (t, J = 7.8 Hz, 2H), 7.61 (d, J = 7.5 Hz,
1H), 7.50 (d, J = 8.5 Hz, 1H), 7.49 – 7.44 (m, 2H), 3.28 (t, J = 7.3 Hz, 2H), 3.04 (t, J =
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7.3 Hz, 2H); C NMR (101 MHz, DMSO - d6) δ 177.0, 157.6, 156.4, 154.3, 134.4,
127.0, 125.7, 125.5, 124.3, 123.6, 122.9, 122.8, 122.6, 122.3, 121.9, 118.8, 118.5, 27.8,
24.3; HRMS (ESI-TOF) m/z calcd for C19 H13 N2 O2 F3 [M+H]+ 359.1002, found
359.1000.
3-(2-(1-Benzyl-5-methyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-one
1
(1f). Colorless solid. Yield: 64%; purity: 98.9%; H NMR (500 MHz, CDCl3) δ 8.17
(d, J = 7.8 Hz, 1H), 7.88 (s, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.55 (s, 1H), 7.37 (t, J = 8.1
Hz, 2H), 7.14 (dd, J = 15.7, 8.4 Hz, 3H), 7.09 (d, J = 8.3 Hz, 1H), 7.00 (dd, J = 13.2,
7.8 Hz, 3H), 5.36 (s, 2H), 3.18 (t, J = 7.3 Hz, 2H), 3.03 (t, J = 7.3 Hz, 2H), 2.47 (s, 3H);
13C NMR (101 MHz, CDCl3) δ 177.9, 156.5, 154.4, 153.4, 143.0, 136.2, 133.5, 133.5,
131.7, 128.8×2, 127.6, 126.2×2, 125.7, 124.9, 123.9, 123.8, 122.8, 119.1, 118.1, 109.3,
46.9, 26.3, 24.8, 21.6; HRMS (ESI-TOF) m/z calcd for C26H22N2O2 [M+H]+ 395.1754,
found 395.1726.
3-(2-(1-(4-Fluorobenzyl)-5-methyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-
chromen-4-one (1g). Colorless solid. Yield: 48%; purity: 99.7%; 1H NMR (500 MHz,
CDCl3) δ 8.15 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.55 (s, 1H),
7.38 (d, J = 7.9 Hz, 2H), 7.04 (dd, J = 22.5, 8.0 Hz, 2H), 6.97 (d, J = 14.7 Hz, 2H), 6.80
(t, J = 7.9 Hz, 2H), 5.33 (s, 2H), 3.18 (t, J = 7.2 Hz, 2H), 3.02 (t, J = 7.1 Hz, 2H), 2.45
(d, J = 20.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 177.9, 156.4, 154.3, 153.4, 143.0,
133.5×2, 133.3, 132.0, 131.8, 127.9, 127.9, 125.6×2, 125.0, 123.9, 122.7, 119.2, 118.1,
115.8, 115.6, 109.2, 46.2, 26.2, 25.0, 21.6; HRMS (ESI-TOF) m/z calcd for
C26H21N2O2F [M+H]+ 413.1660, found 413.1660.
3-(2-(1-(3-Fluorobenzyl)-5-methyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-
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chromen-4-one (1h). Colorless solid. Yield: 50%; purity: 98.4%; 1H NMR (400 MHz,
CDCl3) δ 8.17 (d, J = 7.9 Hz, 1H), 7.89 (s, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.56 (s, 1H),
7.38 (t, J = 8.1 Hz, 2H), 7.14 – 7.02 (m, 3H), 6.79 (dd, J = 16.6, 8.0 Hz, 2H), 6.69 (d, J
= 9.4 Hz, 1H), 5.37 (s, 2H), 3.18 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.2 Hz, 2H), 2.48 (s,
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3H); C NMR (101 MHz, CDCl3) δ 177.9, 156.5, 154.3, 153.3, 143.1, 133.5, 133.3,
131.9, 130.5, 130.4, 125.7, 125.0, 123.9, 122.7, 121.8, 119.3, 118.2, 114.7, 114.5, 113.4,
113.2, 109.1, 46.4, 26.2, 25.0, 21.6; HRMS (ESI-TOF) m/z calcd for C26H21N2O2F
[M+H]+ 413.1660, found 413.1652.
3-(2-(5-Methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-one
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(1i). Colorless solid. Yield: 42%; purity: 99.8%; H NMR (500 MHz, CDCl3) δ 8.12
(dd, J = 8.0, 1.5 Hz, 1H), 7.82 (s, 1H), 7.64 – 7.60 (m, 1H), 7.58 (s, 1H), 7.44 – 7.33
(m, 5H), 7.24 – 7.19 (m, 2H), 7.01 (t, J = 9.2 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 3.14 (t,
J = 7.1 Hz, 2H), 3.02 (t, J = 6.9 Hz, 2H), 2.49 (s, 3H). 13C NMR (126 MHz, CDCl3) δ
177.5, 156.4, 154.2, 153.1, 142.8, 135.7, 134.5, 133.4, 132.1, 129.8×2, 128.7, 127.1×2,
125.8, 124.9, 124.1, 123.9, 122.9, 118.9, 118.1, 109.6, 26.4, 24.6, 21.6; HRMS (ESI-
TOF) m/z calcd for C25H20N2O2 [M+H]+ 381.1598, found 381.1598 .
3-(2-(5-Methyl-1-(pyridin-4-yl)-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-
one (1j). Colorless solid. Yield: 55%; purity: 97.7%;1H NMR (400 MHz, CDCl3) δ 8.72
(d, J = 5.0 Hz, 2H), 8.13 (d, J = 7.8 Hz, 1H), 7.86 (s, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.59
(s, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.26 (s, 2H), 7.07 (s, 2H), 3.21 (t, J = 7.1 Hz, 2H), 3.05
(t, J = 7.1 Hz, 2H), 2.50 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.6, 156.4, 153.4,
153.2, 151.7×2, 143.5, 143.1, 133.6, 133.2, 133.0, 125.8, 125.1, 124.7, 123.8, 122.6,
121.2×2, 119.4, 118.1, 109.4, 26.5, 24.8, 21.5; HRMS (ESI-TOF) m/z calcd for
C24H19N3O2 [M+H]+ 382.1550, found 382.1540 .
3-(2-(5-Chloro-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-4-one
(1k). Colorless solid. Yield: 44%; purity: 96.9%; 1H NMR (400 MHz, CDCl3) δ 8.14
(dd, J = 8.0, 1.3 Hz, 1H), 7.86 (s, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.67 – 7.63 (m, 1H),
7.49 – 7.45 (m, 3H), 7.43 – 7.37 (m, 2H), 7.24 (dd, J = 6.5, 2.9 Hz, 2H), 7.17 (dd, J =
8.6, 1.8 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 3.15 (t, J = 7.0 Hz, 2H), 3.04 (t, J = 7.1 Hz,
2H); 13C NMR (126 MHz, CDCl3) δ 177.5, 156.4, 155.6, 153.2, 143.4, 135.1, 135.0,
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133.5, 130.0×2, 129.1, 128.0, 127.1×2, 125.7, 124.9, 123.9, 123.1, 122.7, 118.9, 118.1,
110.9, 26.4, 24.4; HRMS (ESI-TOF) m/z calcd for C24H17N2O2Cl [M+H]+ 401.1051,
found 401.1017 .
General Procedures for Synthesis of Compounds 15a-15e. 15a-15e was
synthesized by the general procedure for synthesis of 6a-6b.
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3-(6-Fluoro-4-oxo-4H-chromen-3-yl)propanoic acid (15a). Colorless solid. Yield:
88%; 1H NMR (400 MHz, MeOD) δ 8.16 (d, J = 0.7 Hz, 1H), 7.79 – 7.73 (m, 1H), 7.62
(ddd, J = 9.2, 4.2, 1.5 Hz, 1H), 7.55 (tdd, J = 9.2, 3.1, 1.4 Hz, 1H), 2.75 (t, J = 7.2 Hz,
2H), 2.65 – 2.58 (m, 2H).
3-(6-Chloro-4-oxo-4H-chromen-3-yl)propanoic acid (15b). Colorless solid. Yield:
85%; 1H NMR (400 MHz, MeOD) δ 8.14 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.72 (dd, J
= 9.0, 2.6 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 2.74 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.2
Hz, 2H).
3-(6-Bromo-4-oxo-4H-chromen-3-yl)propanoic acid (15c). Colorless solid. Yield:
92%; 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 2.2 Hz, 1H), 7.90 (s, 1H), 7.77 – 7.70
(m, 1H), 7.34 (dd, J = 8.9, 2.1 Hz, 1H), 2.85 – 2.68 (m, 4H).
3-(6-Iodo-4-oxo-4H-chromen-3-yl)propanoic acid (15d). Colorless solid. Yield:
85%; 1H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 2.2 Hz, 1H), 7.92 (dd, J = 8.6, 2.3 Hz,
2H), 7.22 (d, J = 8.8 Hz, 1H), 2.80 – 2.72 (m, 4H).
3-(6-Methoxy-4-oxo-4H-chromen-3-yl)propanoic acid (15e). Colorless solid.
Yield: 80%; 1H NMR (500 MHz, CDCl3) δ 7.90 (s, 1H), 7.57 (d, J = 3.1 Hz, 1H), 7.39
– 7.36 (m, 1H), 7.25 (dd, J = 9.2, 3.1 Hz, 1H), 3.89 (d, J = 4.5 Hz, 3H), 2.80 – 2.77 (m,
2H), 2.74 (dd, J = 7.9, 4.2 Hz, 2H).
General Procedures for Synthesis of Compounds 2a-2e. 2a-2e was synthesized
by the general procedure for synthesis of 1c-1k.
6-Fluoro-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-
chromen-4-one (2a). Colorless solid. Yield: 60%; purity: 99.7%; 1H NMR (500 MHz,
CDCl3) δ 7.84 (s, 1H), 7.74 (dd, J = 8.3, 2.6 Hz, 1H), 7.57 (s, 1H), 7.44 (d, J = 6.8 Hz,
3H), 7.41 – 7.33 (m, 2H), 7.22 (d, J = 7.4 Hz, 2H), 7.02 (d, J = 8.2 Hz, 1H), 6.97 (d, J
= 8.2 Hz, 1H), 3.13 (t, J = 7.0 Hz, 2H), 3.01 (t, J = 7.0 Hz, 2H), 2.49 (s, 3H); 13C NMR
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(101 MHz, CDCl3) δ 176.9, 154.0, 153.4, 142.8, 135.7, 134.5, 132.2, 129.9×2, 128.7,
127.1×2, 124.1, 122.3, 121.8, 121.6, 120.2, 120.1, 118.9, 110.6, 110.4, 109.7, 26.3,
24.40, 21.6; HRMS (ESI-TOF) m/z calcd for C25H19N2O2F [M+H]+ 399.1503, found
399.1482 .
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6-Chloro-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-
chromen-4-one (2b). Colorless solid. Yield: 45%; purity: 99.9%; 1H NMR (500 MHz,
CDCl3) δ 8.06 (d, J = 2.1 Hz, 1H), 7.83 (s, 1H), 7.58 – 7.53 (m, 2H), 7.43 (d, J = 6.9
Hz, 3H), 7.33 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 6.5 Hz, 2H), 7.01 (d, J = 8.2 Hz, 1H),
6.97 (d, J = 8.2 Hz, 1H), 3.12 (t, J = 7.0 Hz, 2H), 3.01 (t, J = 7.0 Hz, 2H), 2.48 (s, 3H);
13C NMR (101 MHz, CDCl3) δ 176.4, 154.7, 154.0, 153.3, 142.8, 135.7, 134.48, 133.60,
132.12, 130.78, 129.85×2, 128.72, 127.07×2, 125.14, 124.74, 124.10, 123.0, 119.8,
119.0, 109.7, 26.2, 24.4, 21.6; HRMS (ESI-TOF) m/z calcd for C25H19N2O2Cl [M+H]+
415.1208, found 415.1210 .
6-Bromo-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-
chromen-4-one (2c). Colorless solid. Yield: 59%; purity: 99.5%; 1H NMR (500 MHz,
CDCl3) δ 8.22 (d, J = 2.3 Hz, 1H), 7.83 (s, 1H), 7.70 – 7.67 (m, 1H), 7.57 (s, 1H), 7.47
– 7.41 (m, 3H), 7.29 – 7.26 (m, 1H), 7.22 (dd, J = 7.5, 1.6 Hz, 2H), 6.99 (dd, J = 22.8,
8.2 Hz, 2H), 3.12 (t, J = 7.1 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.48 (s, 3H); 13C NMR
(126 MHz, CDCl3) δ 176.2, 155.1, 153.9, 153.3, 142.7, 136.3, 135.7, 134.5, 132.2,
129.9×2, 128.7, 128.4, 127.1×2, 125.1, 124.1, 123.1, 120.1, 118.9, 118.2, 109.7, 26.2,
24.4, 21.6; HRMS (ESI-TOF) m/z calcd for C25H19N2O2Br [M+H]+ 459.0703, found
459.0690 .
6-Iodo-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-chromen-
4-one (2d). Colorless solid. Yield: 46%; purity: 97.9%; 1H NMR (500 MHz, CDCl3) δ
8.46 – 8.39 (m, 1H), 7.90 – 7.79 (m, 2H), 7.55 (d, J = 17.1 Hz, 1H), 7.43 (s, 3H), 7.29
– 7.24 (m, 1H), 7.21 (d, J = 3.1 Hz, 2H), 7.17 – 7.10 (m, 1H), 7.04 – 6.95 (m, 2H), 3.13
(d, J = 3.6 Hz, 2H), 3.00 (d, J = 3.5 Hz, 2H), 2.48 (d, J = 3.5 Hz, 3H); 13C NMR (126
MHz, CDCl3) δ 176.0, 155.8, 153.9, 153.3, 142.5, 141.9, 135.6, 134.7, 134.4, 132.3,
129.9×2, 128.8, 127.1×2, 125.5, 124.2, 123.2, 120.2, 118.8, 109.7, 88.6, 26.2, 24.5,
21.6; HRMS (ESI-TOF) m/z calcd for C25H19N2O2I [M+H]+ 507.0564, found 507.0542
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6-Methoxy-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-4H-
chromen-4-one (2e). Colorless solid. Yield: 50%; purity: 99.9%; 1H NMR (500 MHz,
CDCl3) δ 7.81 (s, 1H), 7.58 (s, 1H), 7.48 – 7.39 (m, 4H), 7.31 (d, J = 9.1 Hz, 1H), 7.22
(t, J = 8.0 Hz, 3H), 7.01 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 3.87 (s, 3H), 3.13
(t, J = 7.1 Hz, 2H), 3.02 (t, J = 7.1 Hz, 2H), 2.49 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ 177.3, 156.7, 154.2, 152.9, 151.3, 142.9, 135.8, 134.5, 132.1, 129.8×2, 128.6, 127.1×2,
124.4, 124.0, 123.6, 122.1, 119.5, 119.0, 109.6, 104.7, 55.9, 26.5, 24.6, 21.6; HRMS
(ESI-TOF) m/z calcd for C26H22N2O3 [M+H]+ 411.1703, found 411.1713 .
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General Procedures for Synthesis of Compounds 16a-16c. A mixture of 4-oxo-
4H-chromene-3-carbaldehyde (174 mg, 1.0 mmol) and malonic acid (208 mg, 2.0 mmol)
in the presence of pyridine (5 mL) was refluxed for 45 min with vigorous stirring. Upon
completion, the mixture was cooled to room temperature, the pH adjusted to 1.0 with
concentrated HCl, and the reaction was stirred for additional 30 min. The yellow
colored solid was filtered, washed with 1 M HCl (2 × 20 mL), and dried. The compound
was obtained and recrystallized from MeOH. The obtained spectra match those reported.
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(E)-3-(4-oxo-4H-chromen-3-yl)acrylic acid (16a). Colorless solid. Yield: 85%; 1H
NMR (400 MHz, DMSO - d6) δ 8.87 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.85 (t, J = 7.7
Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 15.9 Hz, 1H),
7.12 (d, J = 15.9 Hz, 1H).
(E)-3-(6-fluoro-4-oxo-4H-chromen-3-yl)acrylic acid (16b). Colorless solid. Yield:
92%; 1H NMR (500 MHz, DMSO - d6) δ 12.43 (s, 1H), 8.88 (s, 1H), 7.83 – 7.70 (m,
3H), 7.41 (d, J = 15.9 Hz, 1H), 7.09 (d, J = 15.9 Hz, 1H).
(E)-3-(6-chloro-4-oxo-4H-chromen-3-yl)acrylic acid (16c). Colorless solid. Yield:
1
82%; H NMR (400 MHz, DMSO - d6 ) δ 8.86 (s, 1H), 8.01 (s, 1H), 7.85 (d, J = 9.0
Hz, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.39 (d, J = 16.0 Hz, 1H), 7.08 (d, J = 15.9 Hz, 1H).
General Procedures for Synthesis of Compounds 3a-3f. 3a-3f was synthesized
by the general procedure for synthesis of 1c-1k.
(E)-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)vinyl)-4H-chromen-4-
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one (3a). Colorless solid. Yield: 43%; purity: 98.3%; H NMR (500 MHz, CDCl3) δ
8.22 (d, J = 7.9 Hz, 1H), 8.14 (s, 1H), 8.00 (d, J = 15.6 Hz, 1H), 7.69 – 7.60 (m, 5H),
7.55 (t, J = 7.3 Hz, 1H), 7.47 (t, J = 7.3 Hz, 3H), 7.41 (t, J = 7.6 Hz, 1H), 7.11 (d, J =
8.3 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 2.50 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
176.7, 156.8, 155.5, 151.3, 143.3, 135.6, 134.7, 133.7, 132.9, 129.9×2, 128.9, 127.9,
127.3×2, 126.2, 125.5, 124.6, 124.2, 120.4, 119.0, 118.7, 118.1, 109.9, 21.7; HRMS
(ESI-TOF) m/z calcd for C25H18N2O2 [M+H]+ 379.1441, found 379.1423 .
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(E)-3-(2-(6-methyl-3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)vinyl)-4H-chromen-
4-one (3b). Colorless solid. Yield: 34%; purity: 99.6%; 1H NMR (400 MHz, CDCl3) δ
8.23 (dd, J = 8.0, 1.3 Hz, 1H), 8.18 (s, 1H), 8.14 (s, 1H), 8.05 (d, J = 15.6 Hz, 1H), 7.86
(s, 1H), 7.72 – 7.69 (m, 1H), 7.68 – 7.62 (m, 3H), 7.57 – 7.53 (m, 2H), 7.52 – 7.46 (m,
2H), 7.42 (t, J = 7.5 Hz, 1H), 2.49 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 176.4, 157.0,
155.4, 152.3, 147.4, 145.2, 135.4, 134.2, 133.8, 129.2×2, 129.0, 128.9, 128.8, 127.7×2,
126.6, 126.2, 125.6, 124.2, 120.2, 118.6, 118.1, 18.8; HRMS (ESI-TOF) m/z calcd for
C24H17N3O2 [M+H]+ 380.1394, found 380.1379 .
(E)-3-(2-(5-chloro-1-phenyl-1H-benzo[d]imidazol-2-yl)vinyl)-4H-chromen-4-
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one (3c). Colorless solid. Yield: 38%; purity: 96.9%; H NMR (500 MHz, CDCl3) δ
8.22 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 8.00 (d, J = 15.6 Hz, 1H), 7.78 (s, 1H), 7.70 –
7.63 (m, 4H), 7.59 (d, J = 7.2 Hz, 1H), 7.46 (dd, J = 13.8, 8.2 Hz, 3H), 7.41 (t, J = 7.6
Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H); 13C NMR (126 MHz, CDCl3)
δ 176.4, 157.1, 155.5, 152.7, 143.9, 135.2, 135.0, 133.8, 130.1×2, 129.3, 129.0, 128.7,
127.3×2, 126.2, 125.6, 124.2, 123.5, 120.1, 118.9, 118.2, 118.2, 111.1; HRMS (ESI-
TOF) m/z calcd for C24H15N2O2Cl [M+H]+ 399.0895, found 399.0914 .
(E)-6-chloro-3-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)vinyl)-4H-
chromen-4-one (3d). Colorless solid. Yield: 41%; purity: 94.9%; 1H NMR (400 MHz,
CDCl3) δ 8.21 (d, J = 2.3 Hz, 1H), 8.14 (s, 1H), 7.99 (d, J = 15.7 Hz, 1H), 7.68 –
7.57 (m, 6H), 7.49 – 7.43 (m, 3H), 7.13 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H),
2.52 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 175.5, 156.7, 153.9, 151.1, 143.3, 135.6,
134.7, 133.9, 133.0, 131.6, 130.0×2, 128.9, 127.3×2, 127.2, 125.6, 125.1, 124.8, 120.5,
119.9, 119.2, 119.0, 109.9, 21.7; HRMS (ESI-TOF) m/z calcd for C25 H17 N2 O2 Cl
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