Validation of Phosphodiesterase-10 as a Novel Target for Pulmonary Arterial Hypertension via Highly Selective and Subnanomolar Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b00224

Pulmonary arterial hypertension (PAH) causes pathological increase in pulmonary vascular resistance, leading to right-heart failure and eventual death. Previously, phosphodiesterase-10 (PDE10) was reported to be a promising target for PAH based on the studies with a nonselective PDE inhibitor papaverine, but little progress has been made to confirm the practical application of PDE10 inhibitors. To validate whether PAH is ameliorated by PDE10 inhibition rather than other PDE isoforms, here we report an integrated strategy to discover highly selective PDE10 inhibitors as chemical probes. Structural optimization resulted in a PDE10 inhibitor 2b with subnanomolar affinity and good selectivity of >45 000-fold against other PDEs. The cocrystal structure of the PDE10-2b complex revealed an important H-bond interaction between 2b and Tyr693. Finally, compound 2b significantly decreased the arterial pressure in PAH rats and thus validated the potential of PDE10 as a novel anti-PAH target. These findings suggest that PDE10 inhibition may be a viable treatment option for PAH.

Full text of DOI:10.1021/acs.jmedchem.9b00224

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Article
Validation of Phosphodiesterase-10 as a Novel Target for Pulmonary
Arterial Hypertension via Highly Selective and Subnanomolar Inhibitors
Yi-You Huang, Yanfa Yu, Chen Zhang, Yiping Chen, Qian Zhou, Zhuoming
Li, Sihang Zhou, Zhe Li, Lei Guo, Deyan Wu, Yinuo Wu, and Hai-Bin Luo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00224 • Publication Date (Web): 19 Mar 2019
Downloaded from http://pubs.acs.org on March 19, 2019
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Validation of Phosphodiesterase-10 as a Novel Target for
Pulmonary Arterial Hypertension via Highly Selective and
Subnanomolar Inhibitors
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Yi-You Huang||, Yan-Fa Yu||, Chen Zhang||, Yiping Chen, Qian Zhou, Zhuoming Li, Sihang Zhou,
Zhe Li, Lei Guo, Deyan Wu*, Yinuo Wu*, and Hai-Bin Luo*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
 ABSTRACT
Pulmonary arterial hypertension (PAH) causes pathological increase in pulmonary vascular resistance,
leading to right-heart failure and eventual death. Previously, phosphodiesterase-10 (PDE10) was
reported to be a promising target for PAH based on the studies with a non-selective PDE inhibitor
papaverine, but little progress has been made to confirm the practical application of PDE10 inhibitors.
To validate whether PAH is ameliorated by PDE10 inhibition rather than other PDE isoforms, here we
report an integrated strategy to discover highly selective PDE10 inhibitors as chemical probes.
Structural optimization resulted in a PDE10 inhibitor 2b with subnanomolar affinity and good
selectivity of > 45 000-fold against other PDEs. The cocrystal structure of the PDE10-2b complex
revealed an important H-bond interaction between 2b and Tyr693. Finally, compound 2b significantly
decreased the arterial pressure in PAH rats and thus validated the potential of PDE10 as a novel anti-
PAH target. These findings suggest that PDE10 inhibition may be a viable treatment option for PAH.

INTRODUCTION
Phosphodiesterases (PDEs) are important enzymes that hydrolyze the second messengers cyclic
adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).1 Due to the critical
roles of cAMP and cGMP in physiologic processes, PDEs have attracted wide attention of researchers
as drug targets for treatment of human diseases. Till now, 11 selective PDEs inhibitors have been
approved in clinic, such as the PDE5 inhibitors of sildenafil and tadalafil, which are the treatments for
erectile dysfunction and pulmonary arterial hypertension (PAH).2 In addition to the high expression
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in the lung, PDE10 is expressed at high levels in the medium spiny neurons of the striatum and thought
to modulate the downstream dopaminergic and glutamatergic signaling pathways.3 As a result, several
PDE10 inhibitors have entered clinical Phase I/II trials for the treatment of central nervous system
(CNS) disorders.4-8
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PAH is a debilitating and progressive disease of the pulmonary vasculature and causes right
ventricular failure and eventual death. Although significant advances in understanding of the
pathobiology of PAH have brought the development of several therapeutics against this disease, a clear
unmet medical need exists.9,10 Recently, PDE10 was reported to be a promising target for treatment of
PAH based on studies with a non-selective inhibitor papaverine.11 However, little progresses has been
made to confirm PDE10 as an anti-PAH target. Since the loss of nitric oxide (NO)- and PGI2-driven
cGMP and cAMP signaling is an important hallmark of PAH, agents such as PDE5 inhibitors
sildenafil/tadalafil (elevating cGMP level) and iloprost (elevating cAMP signaling) have been proven
to be one of effective PAH therapeutics.12 In general, PDE10 inhibitors might augment both cAMP
and cGMP signaling13,14 and potentially exert additive effects, suggesting that PDE10 is an appealing
target that potentially opens a new avenue to develop effective anti-PAH therapeutics.
Besides PDE10 and PDE5, several other PDE isoforms including PDE1, PDE2, PDE3 and PDE4,
are also involved in the pathological mechanism of PAH, as indicated by altered expression and
activities in the pulmonary vasculature.15-17 In fact, papaverine as a probe for PAH in a previous
report,11 could inhibit PDE10A, PDE3A, and PDE4A with IC50 values of 40 nM, 287 nM, and 1.6
M,9,5,17 respectively, potentially resulting in a false-positive action of PDE10A in PAH11 due to
contributions from other PDE subtypes (PDE3A and PDE4A). The treatment effect of papaverine on
pulmonary hypertensive hemodynamic parameters in MCT-PAH rats may be related to the multiple
inhibition of PDEs subfamilies such as PDE10, PDE3, and PDE4. The similar phenomenon occurs
with sildenafil, a PDE5 inhibitor, whose therapeutic effect might be linked to some extent to its
inhibition on PDE1C.15 In order to exclude disturbance from other PDE subtypes, highly selective
PDE10A inhibitors are generally valuable to further validate the feasibility of PDE10A as a novel
target of PAH.
Herein, we combined the molecular dynamics (MD)-based design/optimization protocol with an
integrated strategy (cocrystal structure, structural optimization, bioassay, and pharmacodynamic
evaluation), which dramatically accelerated the hit-to-lead process and minimized the need for
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chemical synthesis, to identify a highly selective PDE10 inhibitor 2b (IC50 of 0.44 nM towards PDE10
and selectivity of over 45 000-fold against other PDEs). Compound 2b significantly decreased the
arterial pressure in PAH rats at a dosage of 2.5 mg/kg, thus validated PDE10 as a potential drug target
for PAH.
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 RESULTS AND DISCUSSION
MD-based virtual screening (Figure 1) remarkably accelerated the discovery of PDE10A
inhibitors with novel scaffolds. The commercially available database SPECS (http://www.specs.net),
which consists of approximately 200 000 compounds, was selected for virtual screening and all
compounds were filtered by Lipinski’s Rule of Five19 to improve the pharmacological properties of
screened compounds.
A 3D-pharmacophore model was generated from the X-ray crystal structures of PDE10A in
complex with a variety of ligands (PDB ID: 3QPN, 3QPO, 3QPP, 5DH4, and 5DH5).4,20 In the
established model, pharmacophore features and structural motifs included aromatic rings, hydrophobic
side chains, and H-bond donor and acceptor groups in ligands. Moreover, the volume of the amino
acid residues was added as an exclusion feature. A test set containing 193 active compounds selected
from the Binding Database (http://www.bindingdb.org) and 9187 inactive compounds randomly
generated from the DUD-E database (http://dude.docking.org/), was prepared for the goodness of hit
(GH) test. The GH test score was 0.56, indicating that the model effectively picked out active PDE10A
inhibitors (see Table S1 in the Supporting Information). Furthermore, a PAINS (pan-assay interfering
compound substructures) screen was applied to remove possible false positive compounds using the
online program PAINS-Remover.21 Afterwards, 1091 molecules were retained.
Insert Figure 1 here.
All the molecules that passed the pharmacophore model and PAINS screening were docked into
the PDE10A catalytic pocket to predict their binding patterns and the docking results were
preliminarily selected by docking scores. More importantly, most potent and selective PDE10A
inhibitors were reported to interact with the unique PDE10A selectivity pocket, which was first
identified in the cocrystal structure of the clinical candidate PF-2545920 with PDE10A.22 In addition
to the conserved interactions with residues Gln726 and Phe729, PF-2545920 formed an H-bond with
Tyr693 in the selectivity pocket, which was important to maintain potency and selectivity (Figure S1
in supporting information). Based on the evidence, only diverse hits that potentially interacted with
selectivity pocket were pursued in our docking screen, and 47 molecules were retained.
MD simulations were also performed to precisely predict binding patterns and binding free
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energies were estimated via the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA)
and the molecular mechanics/Generalized Born surface area (MM/GBSA) methods.23,24 Finally, only
23 compounds with stable MD simulation trajectories and considerable binding free energies were
purchased and evaluated by bioassay.
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Encouragingly, 13 of the selected 23 compounds exhibited greater than 50% inhibition at 10 μM
(see Table S2 and Figure S2 in the Supporting Information). The hit ratio was up to 57% and eight
novel scaffolds were discovered (Figure S2). Notably, compounds AF-399/41980954 and AF-
399/14387019 showed considerable potency with IC50 values of 39 and 68 nM, respectively (Figure
1). In comparison with the inhibitory profiles of high throughput screening (HTS) reported in previous
studies,22,25,26 our hits had comparable or even better potency towards PDE10A, thus confirming the
efficiency of the MD-based virtual screening.
The cocrystal structure of PDE10A in complex with AF-399/14387019 revealed binding mode
consistent with the predicted one. Among the 13 active compounds identified, compound AF-
399/41980954 exhibited the strongest inhibition, but its imidazo[1,2-a]pyrimidine scaffold and
isomers were already reported in other PDE10 inhibitors.27-30 As we aimed to identify PDE10A
inhibitors with novel scaffolds, AF-399/14387019 was selected for further structural optimization.
The cocrystal structure of PDE10A with hit AF-399/14387019 was determined (PDB ID: 6IJH)
and the electron density in (2Fo–Fc) and (Fo–Fc) unambiguously showed that AF-399/14387019
bound to the catalytic pocket of PDE10A (Figure 2A). The 1,8-naphthalimide group of AF-
399/14387019 was sandwiched by the hydrophobic residues Phe729 on one side and Phe696/Ile692
on the other side, and the carbonyl oxygen formed an H-bond with the amide nitrogen of Gln726. The
H-bond with Gln726 and stacking against Phe729 are two characteristic binding modes conserved in
other PDE inhibitors. Additionally, the ethyl linker of AF-399/14387019 stretched towards the
selectivity pocket, which was partially occupied by the coupled 1, 2, 4-triazole ring. An H-bond
between the nitrogen of triazole ring and phenolic hydroxyl group of Tyr693 was also observed.
Notably, the binding mode observed in the crystal structure superimposed well with the predicted
pattern derived from the MD simulation (Figure 2B), confirming the reliability of the MD-based virtual
screening protocol employed in the present study.
MD simulations greatly facilitated the 1st-round optimization. Currently, research on PDE10A
inhibitors usually begins with HTS to identify novel scaffolds,22,25-27,31-35 and a selective pocket
binding group (SPBG) is then integrated into the scaffold by a linker to promote the interaction with
Tyr693 (see Figure S1 in supporting information).4,34-39 As the selectivity pocket maintains a relatively
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defined shape, only groups with an appropriate volume fit well. However, a universal SPBG for
PDE10A inhibitors has not been determined. As the result, a large number of analogues must be
synthesized to identify a suitable SPBG, which is very time-consuming and costly.
Insert Figure 2 here.
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Based on the cocrystal structure of PDE10A in complex with AF-399/14387019, in silico docking
and MD simulations were used to identify the suitable SPBG for synthesis. As described above, the
1,8-naphthalimide group of AF-399/14387019 functioned as the scaffold to interact with the conserved
Gln726 and the hydrophobic clamp of PDE10A, while the coupled 1, 2, 4-triazole ring partially
occupied the selectivity pocket. In the subsequent modification procedures, different combinations of
linkers and heterocyclic SPBG were designed and subjected to in silico docking and MD simulations
(Figure 3). According to the MM/PBSA binding free energies, the top five compounds (1a and 1c-1f)
were selected and synthesized (Table 1). Quinoline, a common scaffold of current PDE10A inhibitors,
well fits the selectivity pocket and was attached to the 1, 8-naphthalimide core via an ethyl linker to
form compound 1a. Compound 1c was designed with an ethyl linker and 4-phenyl-imidazole scaffold
as the binding group while 1d used 5-methyl-benzimidazole as the binding group. Compounds 1e and
1f contained longer linkers and a benzimidazole scaffold and were designed based on another hit AR-
422/43115142. On the other hand, compound 1b with a pyridine ring was designed as a negative
reference and predicted to be less potent in comparison with AF-399/14387019 (-25.7 vs -28.7
kcal/mol of MM/PBSA binding free energies).
Insert Figure 3 here.
Insert Table 1 here.
Insert Scheme 1 here.
Insert Scheme 2 here.
Insert Scheme 3 here.
As expected, compounds 1a, 1c, and 1d exhibited significant inhibitory potency against PDE10A
with IC50 values ranging from 2.1 to 20 nM, which were better than hit AF-399/14387019. Moreover,
the reference compound 1b exhibited poor potency with an IC50 >1000 nM, showing that groups with
a small volume were not suitable for the selectivity pocket. For compounds 1e and 1f, the low
inhibitory affinities of them against PDE10A were not in accordance with the high predicated binding
free energies. We speculated that configuration of the propyl linker in both compounds are too flexible
to predict binding free energies precisely. Compared to compound 1d, a heteroatom was added to the
benzimidazole motifs in these two compounds, which might affect their inhibitory affinities.
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The 2nd round of optimization resulted in subnanomolar PDE10A inhibitors. Inspired of the
promising results from the 1st round, compound 1d (IC50 of 2.1 nM) was selected as a new starting
compound for the 2nd round design (Figure 3). Binding patterns from stable MD simulations illustrated
that compound 1d deeply occupied the selectivity pocket and formed an H-bond with Tyr693.
However, some space in the selectivity pocket remained unoccupied. Comparing the inhibitory
potencies of compounds 1a and 1b, we found that compounds with larger side chains displayed more
potent binding affinities (Table 1). Thus, large substitution groups such as tetrahydropyran, phenyl and
pyridyl rings were attached to the N-1 position of the 5-methyl-benzoimidazole scaffold. In addition,
the 5-methyl-benzoimidazole scaffold of compound 1d was replaced by 5-fluoro-benzoimidazole or
6-methyl-imidazopyridine.
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Similarly, all designed compounds were subjected to in silico docking and MD simulations that
showed the top five compounds (2b-2f) having binding free energies 5-8 kcal/mol lower than 1d (Table
2). The binding modes from MD simulations predicted that all compounds adopted similar orientations
to that in the PDE10A-1d complex, with some subtle differences. As the phenyl or pyridyl substituted
5-methyl-benzoimidazole in compounds 2b and 2f has a larger volume than 1d, they were expected to
properly occupy the selectivity pocket to enhance potency. However, the space for the tetrahydropyran
group in 2a/2c was relatively narrowed due to the location of residues Met713 and Ile711 near the
selectivity pocket. We speculated that small planar rings may fit better in this space than larger
aliphatic rings (Figure 2). The substituted groups on the benzoimidazole scaffold also affected the
inhibitory activity. The inhibitory potency towards PDE10A will be decreased by perturbation of the
electronic properties of the 1-phenyl-benzimidazole group from 5-methyl (2c) to 5-fluoro (2a),
replacement of the 5-methyl-1-benzoimidazole group (2b) with 6-methyl-imidazopyridine (2d).
Insert Table 2 here.
Insert Scheme 4 here.
With the low binding free energies, compounds 2b, 2e, and 2f showed improved PDE10 inhibition
compared with compound 1d, and had the IC50 values ranging from 0.44 to 1.0 nM. In particular,
compound 2b having the pyridyl group at the N-1 position of the 5-methyl-benzoimidazole scaffold
displayed inhibitory potency of 0.44 nM, which equates to 4- and 154-fold increases in binding affinity
compared with 1d and AF-399/14387019, respectively. Notably, the inhibitory potency of lead
compound 2b was comparable to the PDE10A clinical candidates TAK-063 and PF-2545920 that
have IC50 values of 0.3 nM and 0.37 nM, respectively.
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Compound 2b shows high selectivity against other PDE isoforms.
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In the two-round optimization, a series of compounds with different substituents on the side chain
of hit AF-399/14387019 were designed and 12 compounds with properly predicted binding free
energies were synthesized. Among them, compound 2b with the pyridyl group at the N-1 position of
the 5-methyl-benzoimidazole scaffold exhibited highly potent inhibition of 0.44 nM towards PDE10A
and > 20 μM against other PDEs (> 45 000-fold selectivity, Table 3), suggesting that it represents a
suitable chemical probe to investigate the role of PDE10 in PAH with minimal interference from other
PDEs. In general, the discovery of drug leads requires several rounds of design/optimization of linkers,
substituent groups, and substantial amounts of synthetic work. In our approach, we took full advantage
of binding free energy predictions to substantially accelerate the hit-to-lead process and minimize
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chemical loads of synthetic work.
Insert Table 3 here.
Insert Table 4 here.
Ligand profiling was performed to screen the binding affinities of compound 2b at the Guangzhou
Institutes of Biomedicine and Health against over twenty different targets in a panel of receptors, ion
channels, and other enzymes,40-46 which would be beneficial to rule out other its non-target activities.
The in vitro binding affinities of compound 2b were shown in Table 4 and most of them (Estrogen
receptor ER, Estrogen receptor ER, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, ABL, Aurora A,
Aurora B, PLK1, EGFR, ZAK, BRD4, COX2, HDAC1, HDAC6, AChE, and BChE) were > 10.0 M
except that against hERG potassium channel. The IC50 of 2b towards hERG potassium channel was
4.7 M, which was over 1000-fold higher than its inhibitory potency of 0.44 nM against PDE10A.
The cocrystal structure validated the predicted binding pattern of compound 2b with PDE10A.
The cocrystal structure of PDE10A with lead 2b was determined (PDB ID: 6IJI). As shown in Figure
4, the 1, 8-naphthalimide group of 2b served as the scaffold to interact with the conserved residue
Gln726 and hydrophobic clamp of PDE10A catalytic pocket. In comparison with hit AF-
399/14387019, 2b involves another H-bond interaction with Try693 in the selectivity pocket, which
contributes to its high inhibitory potency against PDE10A and excellent selectivity over other PDEs.
The binding pattern in the cocrystal structure of PDE10A-2b was superposed well with the predicted
mode, validating that our modeling protocols were feasible and practical for design of PDE inhibitors,
especially those with well-defined or special sub-pockets such as in the cases of PDE2, PDE4, and
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Insert Figure 4 here.
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The pharmacokinetic profile assessment in SD rats after intravenous administration of 2.5 mg/kg
2b was performed, and 2b showed a Cmax of 3500.87 ng/mL at Tmax 0.083 h and AUC (0-t) of 1097.38
h*ng/mL in Table 5, respectively, indicating that it could give a reasonable blood exposure. In addition,
its plasma protein binding (PPB) was 98%. On the basis of its pharmacokinetic profile, compound 2b
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was further subjected to pharmacodynamics studies in vivo.
Insert Table 5 here.
The highly selective PDE10A inhibitor 2b significantly decreased the arterial pressure in PAH
rats. The in vivo therapeutic effects of compound 2b against PAH were measured. PAH was
successfully induced in rats after 3 weeks of monocrotaline (MCT) injections, as shown by increase
of the mean pulmonary artery pressure (mPAP), right ventricle hypertrophy index (RVHI%), and wall
thickness percentage (WT%) of the model group in comparison with the control group (Figure 5A-
5C). As a positive control, PDE5 inhibitor sildenafil at a dose of 10 mg/kg (p. o.) effectively
ameliorated these medical indicators in the rats, further verifying the feasibility of our in vivo rat
models. Under the same conditions, the values of mPAP (Figure 5A), RVHI% (Figure 5B), and WT%
(Figure 5C) from the group treated with 2.5 mg/kg 2b (i.p.) were significantly improved compared
with the model group. Body weight results (Figure 5D) demonstrated the low toxicity of compound
2b. In addition, PDE10 inhibitor 2b could greatly increase the concentration of cAMP (Figure 5E) in
lung and slightly increase the level of cGMP (Figure 5F) compared with the model group,
demonstrating that the in vivo activity of compound 2b was mediated by both cAMP and cGMP and
the former played the major role. PDE5 inhibitor sildenafil remarkably improved the concentration of
cGMP in lung, while the concentration of cAMP remained unchanged, suggested that the in vivo
activity of sildenafil was predominantly mediated by cGMP rather than cAMP. Thus, our results
suggested that lead compound 2b is an effective anti-PAH agent.
PDE isoforms of PDE1, PDE2, PDE3, PDE4 PDE5, and PDE10 are expressed and regulated in
the pulmonary vasculature of patients with PAH.13 High selective inhibitors will be needed to identify
which specific PDE is related to PAH. The cGMP-specific PDE5 is the most abundant isoform
expressed in lung, and two PDE5 inhibitors (sildenafil and tadalafil) were approved as treatments for
human PAH. Since compound 2b shows high inhibitory potency against PDE10A (IC50 of 0.44 nM)
and high selectivity over other PDEs (selectivity index > 45 000-fold), the anti-PAH effect of 2b in
rats would be achieved via selective inhibition on PDE10A rather than other PDEs, validating that
PDE10 is a potential target for treatment of PAH.
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Insert Figure 5 here.
Both PDE10A inhibitors 2b and PF-2545920 prevented HPASMC phenotypic transformation
induced by platelet-derived growth factor. Vascular remodeling plays an important role in the
development and complications of PAH. Vascular smooth muscle cells (VSMC) phenotypic
transformation works as a major initiating factor for vascular remodeling in hypertension.50 VSMC
phenotypic transformation is characterized by an increase in synthetic protein including osteopontin
(OPN) and proliferating cell nuclear antigen (PCNA), as well as a reduction in contractile proteins
such as α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α).51 Thus, with the results
obtained in the pharmacodynamic experiments, both PDE10A inhibitor 2b and PF-2545920 developed
by Pfizer were applied in the platelet-derived growth factor (PDGF)-BB induced HPASMC model to
assess the effects on VSMC phenotypic transformation. The results showed that both compound 2b
and PF-2545920 reduced the level of OPN and PCNA (Figure S3A and S3B), and increased the level
of α-SMA and SM22α (Figure S3C and S3D). Compound PF-2545920 gave a better result on the
regulation of OPN and SM22α, and gave a comparable result on the regulation of PCNA and α-SMA.
These results indicated these two compounds modulate VSMC phenotypic transformation and further
supported that PDE10 could work as a potential anti-PAH target.
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 CONCLUSION
In summary, an effective hit-to-lead strategy was introduced to identify a highly potent and selective
PDE10A inhibitor 2b (IC50 of 0.44 nM towards PDE10A and > 45 000-fold selectivity over other
PDEs), which served as a probe to validate PDE10 as a potential anti-PAH target.
Remarkably, a hit ratio of 57% was achieved and 8 novel scaffolds were discovered by the MD-
based virtual screening, confirming that the screening strategy was efficient and could serve as a
complementary approach to the costly HTS method. By docking and MD simulations, the two-round
optimization of hit AF-399/14387019 were rapidly advanced and resulted in the discovery of
compound 2b. The cocrystal structures of PDE10A in complex with AF-399/14387019 and 2b
revealed that their binding patterns were consistent with the predicted modes, verifying the MD-based
design/optimization strategy used in the present study. Compound 2b significantly decreased PAH in
rats in vivo, and this improvement is probably achieved by selectively inhibiting PDE10A based on
the high PDE selectivity of 2b. These findings are consistent with PDE10A inhibition being a valid
approach for treatment of PAH.
In short, our studies not only show that PDE10 inhibitors are potential therapeutics for treatment
of PAH, but also open a new avenue to effectively discover highly selective PDE inhibitors via the
integrated strategy.
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 EXPERIMENTAL SECTION
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Chemistry. Unless specified, all reagents and starting materials were purchased from commercial
sources and directly 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 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 compounds was determined by reverse-phase high-performance liquid
chromatography (HPLC) 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).
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General procedures for synthesis of compounds 1a-1b. A suspension of 1,8-naphthalic
anhydride 7 (198 mg, 1.0 mmol) and appropriate aliphatic amine (2.0 mmol) in ethanol (10 mL) was
heated under reflux for 3 h. After evaporation of solvent under reduced pressure, the crude product
was purified on a silica gel column with chloroform/methanol (100:1) to give compounds 1a-1b as a
colorless solid.
2-(2-(quinolin-2-yl)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1a). Colorless solid.
Yield: 85%; purity: 96.6%; Mp: 199.7-210.3 oC; 1H NMR (400 MHz, CDCl3) δ 8.61 (dd, J = 7.3, 1.1
Hz, 2H), 8.24 (dd, J = 8.3, 1.0 Hz, 2H), 8.10 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.78 (dt, J
= 8.2, 5.7 Hz, 3H), 7.68 – 7.63 (m, 1H), 7.53 – 7.45 (m, 2H), 4.77 – 4.71 (m, 2H), 3.45 (dd, J = 8.4,
6.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 164.1×2, 159.4, 148.0, 136.2, 133.8×2, 131.6, 131.2×2,
129.2, 129.1, 128.2, 127.4, 126.9×2, 126.9, 125.8, 122.8, 121.6×2, 39.9, 37.1; HRMS (ESI-TOF) m/z
calcd for C23H16N2O2 [M+H]+ 353.1285, found 353.1275.
2-(2-(pyridin-2-yl)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1b). Colorless solid.
Yield: 83%; purity: 99.9%; Mp: 143.7-145.4 oC; 1H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 7.2 Hz,
2H), 8.54 (d, J = 4.2 Hz, 1H), 8.21 (d, J = 8.1 Hz, 2H), 7.75 (t, J = 7.7 Hz, 2H), 7.59 (td, J = 7.7, 1.6
Hz, 1H), 7.26 (d, J = 6.9 Hz, 1H), 7.12 (dd, J = 6.9, 5.3 Hz, 1H), 4.64 – 4.55 (m, 2H), 3.28 – 3.18 (m,
2H); 13C NMR (101 MHz, CDCl3) δ 164.0×2, 159.0, 149.4, 136.3, 133.9×2, 131.6, 131.2×2, 128.2,
126.9×2, 123.3×2, 122.7, 121.5, 40.1, 36.4; HRMS (ESI-TOF) m/z calcd for C19H14N2O2 [M+H]+
303.1128, found 303.1122.
General procedures for synthesis of compounds 1c-1d. To a mixture of appropriate amine (1.0
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mmol) in dichloromethane(5 mL) was added HATU (570 mg, 1.5 mmol), N, N-diisopropylethylamine
(516 mg, 4.0 mmol) and 9 (269 mg, 1.0 mmol), and stirred at room temperature for 12 h. The mixture
was diluted with saturated sodium bicarbonate aqueous solution and extracted with dichloromethane
(300 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo to get a residue. To a solution of this residue in acetic acid glacial (5 mL) was
added ammonium acetate (1.38 g, 18.0 mmol). The resulting mixture was heated at reflux for 7 h.
After being cooled to room temperature, brine was added and the mixture was separated. The aqueous
phase was extracted with ethyl acetate (300 mL), the organic phase was dried over sodium sulfate.
After filtration and concentration, the residue was purified by column chromatography on silica gel
(petroleum/ethyl acetate, 1:1) to yield compounds 1c-1d.52
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2-(2-(4-phenyl-1H-imidazol-2-yl)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1c).
Colorless solid. Yield: 63%; purity: 99.9 %; Mp: 132.5-136.3 oC; 1H NMR (400 MHz, DMSO – d6) δ
8.47 (dd, J = 9.9, 7.8 Hz, 4H), 7.90 – 7.83 (m, 2H), 7.65 – 7.58 (m, 2H), 7.42 (s, 1H), 7.28 (t, J = 7.6
Hz, 2H), 7.14 (t, J = 7.3 Hz, 1H), 4.47 – 4.36 (m, 2H), 3.04 (t, J = 7.5 Hz, 2H); 13C NMR (101 MHz,
DMSO – d6) δ 163.9×2, 146.0, 134.7×2, 131.8, 131.1×3, 128.8×2, 127.9, 127.6×3, 126.3, 124.5×3,
122.7×2, 39.1, 26.8; HRMS (ESI-TOF) m/z calcd for C23H17N3O2 [M+H]+ 368.1394, found 368.1384.
2-(2-(5-methyl-1H-benzo[d]imidazol-2-yl)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1d). Colorless solid. Yield: 54%; purity: 96.7%; Mp: 138.0-142.0 oC; 1H NMR (400 MHz, MeOD) δ
8.52 (dd, J = 7.3, 1.0 Hz, 2H), 8.39 – 8.34 (m, 2H), 7.84 – 7.78 (m, 2H), 7.32 (d, J = 8.2 Hz, 1H), 7.25
(s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 4.63 (t, J = 7.1 Hz, 2H), 3.30 (d, J = 7.1 Hz, 2H), 2.43 (s, 3H); 13C
NMR (101 MHz, DMSO– d6) δ 163.8×2, 152.2, 134.8×2, 134.7, 131.8, 131.1×2, 131.0, 128.0, 127.6×2,
127.5, 123.3×2, 122.6, 114.9, 114.3, 38.7, 27.5, 21.7; HRMS (ESI-TOF) m/z calcd for C22H17N3O2
[M+H]+ 356.1394, found 356.1385.
General procedures for synthesis of compounds 1e-1f. Sodium hydride (48 mg, 1.2 mmol) was
added to a solution of benzimidazole (1.0 mol) in DMF (5 mL) at 0 oC. The mixture stirred at 0 oC for
15 min, and 11 (317 mg, 1.0 mmol) was added at 0 oC. The reaction was stirred at room temperature
for 2 h, quenched with MeOH and then concentrated to dryness. The resulting crude was purified by
silica gel column chromatography (CH2Cl2) to afford compounds 1e-1f as a colorless solid.
2-(3-((1H-benzo[d]imidazol-2-yl)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(1e) . Colorless solid. Yield: 84%;purity: 95.6%; Mp: 197.9-200.5 oC; 1H NMR (400 MHz, CDCl3) δ
8.60 (d, J = 7.3 Hz, 2H), 8.23 (d, J = 8.2 Hz, 2H), 7.76 (td, J = 8.2, 2.0 Hz, 2H), 7.39 (d, J = 7.2 Hz,
1H), 7.15 – 7.01 (m, 3H), 4.28 (dd, J = 8.8, 4.0 Hz, 2H), 4.09 (t, J = 7.0 Hz, 2H), 2.34 – 2.27 (m, 2H);
13C NMR (101 MHz, CDCl3) δ 164.6×2, 147.6, 141.6, 134.4×2, 133.9, 131.6×2, 131.5, 128.2, 127.1×2,
122.3×2, 121.7, 119.9, 116.3, 107.8, 88.3, 80.6, 78.9, 50.5, 41.2, 37.9, 28.3;HRMS (ESI-TOF) m/z
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calcd for C22H18N4O2 [M+H]+ 371.1503, found 371.1500.
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2-(3-((1H-benzo[d]imidazol-2-yl)thio)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione(1f).
Colorless solid .Yield: 75%; purity: 95.7%; Mp: 177.3-178.2 oC; 1H NMR (400 MHz, CDCl3) δ 10.04
(s, 1H), 8.65 (d, J = 7.3 Hz, 2H), 8.26 (d, J = 8.2 Hz, 2H), 7.80 (t, J = 7.7 Hz, 2H), 7.69 – 7.61 (m,
1H), 7.43 (d, J = 5.0 Hz, 1H), 7.25 – 7.19 (m, 2H), 4.54 – 4.46 (m, 2H), 3.36 (t, J = 6.6 Hz, 2H), 2.18
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(dd, J = 14.0, 7.0 Hz, 2H); C NMR (101 MHz, CDCl3) δ 164.4×2, 148.8, 144.1, 134.9, 134.2×2,
131.6, 131.4×2, 128.2, 127.0×2, 122.7, 122.5×2, 122.0, 118.7, 110.2, 39.0, 31.3, 30.0; HRMS (ESI-
TOF) m/z calcd for C22H17N3O2S [M+H]+ 388.1114, found 388.1115.
General procedures for synthesis of compounds 2a-2f. To a mixture of appropriate amine (1.0
mmol) in CH2Cl2 (15 mL) were added HATU (380 mg, 1.5 mmol), N, N-diisopropylethylamine (516
mg, 4.0 mmol) and 9 (322 mg, 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 AcOH (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
compounds 2a-2f as a colorless solid.
2-(2-(5-fluoro-1-(tetrahydro-2H-pyran-4-yl)-1H-benzo[d]imidazol-2-yl)ethyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (2a). Colorless solid. Yield: 72%; purity: 98.9%; Mp: 236.6-
237.9 oC; 1H NMR (400 MHz, CDCl3) δ 8.62 (dd, J = 7.3, 1.0 Hz, 2H), 8.25 (dd, J = 8.3, 0.9 Hz,
2H), 7.78 (dd, J = 8.1, 7.4 Hz, 2H), 7.54 (dd, J = 8.9, 4.5 Hz, 1H), 7.39 (dd, J = 9.3, 2.5 Hz, 1H), 7.04
– 6.96 (m, 1H), 4.84 (ddd, J = 12.2, 7.9, 4.5 Hz, 1H), 4.65 – 4.59 (m, 2H), 4.22 (dd, J = 11.7, 4.4 Hz,
2H), 3.73 (dd, J = 11.9, 10.5 Hz, 2H), 3.41 – 3.35 (m, 2H), 2.66 (dd, J = 12.8, 4.6 Hz, 2H), 1.96 (dd,
J = 12.8, 2.7 Hz, 2H); 13C NMR (101MHz, CDCl3) δ 164.0×2, 160.2, 157.8, 152.7, 134.2×2, 131.7,
131.4×2, 130.0, 128.2, 127.0×2, 122.5×2, 112.0 (d, J = 10.0 Hz), 110.3 (d, J = 25.9 Hz), 105.5 (d, J =
23.8 Hz), 67.5×2, 53.2, 38.5, 31.4×2, 26.95; HRMS (ESI-TOF) m/z calcd for C26H22N3O3F [M+H]+
444.1718, found 444.1706.
2-(2-(5-methyl-1-(pyridin-4-yl)-1H-benzo[d]imidazol-2-yl)ethyl)-1H-benzo[de]isoquinoline
-1,3(2H) -dione (2b). Colorless solid. Yield: 78%; purity: 99.7 %; Mp: 200.6-206.6 C; 1H NMR
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(400 MHz, CDCl3) δ 8.75 (dd, J = 4.5, 1.6 Hz, 2H), 8.54 (dd, J = 7.3, 1.0 Hz, 2H), 8.23 (dd, J = 8.3,
1.0 Hz, 2H), 7.75 (dd, J = 8.1, 7.4 Hz, 2H), 7.53 (s, 1H), 7.44 (dd, J = 4.5, 1.6 Hz, 2H), 7.10 (dd, J =
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2.7, 0.9 Hz, 2H), 4.69 (t, J = 7.4 Hz, 2H), 3.32 (t, J = 7.4 Hz, 2H), 2.49 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ 164.0×2, 151.8×2, 151.1, 148.8, 143.7, 143.3, 134.1×2, 133.3, 132.8, 131.6, 131.3×2,
126.9×2, 124.8, 122.5, 121.4×2, 119.6×2, 109.3, 38.3, 26.4, 21.5; HRMS (ESI-TOF) m/z calcd for
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C27H20N4O2 [M+H]+ 433.1659, found 433.1656.
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2-(2-(5-methyl-1-(tetrahydro-2H-pyran-4-yl)-1H-benzo[d]imidazol-2-yl)ethyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (2c). Colorless solid. Yield: 68%; purity: 95.0%; Mp: 200.3-204.1
oC; 1H NMR (400 MHz, CDCl3) δ 8.64 (d, J = 7.2 Hz, 2H), 8.25 (d, J = 8.1 Hz, 2H), 7.79 (t, J = 7.8
Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.3 Hz, 1H), 4.83 (t, J = 4.3 Hz, 1H), 4.63 (dd, J = 9.1,
6.9 Hz, 2H), 4.22 (dd, J = 11.7, 4.4 Hz, 2H), 3.73 (t, J = 11.2 Hz, 2H), 3.40 – 3.33 (m, 2H), 2.70 (qd,
J = 12.6, 4.7 Hz, 2H), 2.48 (s, 3H), 1.94 (dd, J = 12.8, 2.7 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ
164.0×2, 151.1, 143.7, 134.1×2, 131.7, 131.6, 131.4×2, 131.3, 128.2, 126.9×2, 123.5×2, 122.5, 119.7,
111.2, 67.6×2, 53.0, 38.7, 31.5×2, 27.0, 21.3; HRMS (ESI-TOF) m/z calcd for C27H25N3O3 [M+H]+
440.1969, found 440.1956.
2-(2-(6-methyl-3-(pyridin-4-yl)-3H-imidazo[4,5-b]pyridin-2-yl)ethyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (2d). Colorless solid. Yield: 74%;purity: 98.3%; Mp: 223.3-223.8 oC; 1H
NMR (400 MHz, CDCl3) δ 8.73 (dd, J = 4.6, 1.6 Hz, 2H), 8.54 (dd, J = 7.3, 1.0 Hz, 2H), 8.24 (dd, J =
8.3, 0.9 Hz, 2H), 8.18 (d, J = 1.6 Hz, 1H), 7.81 (d, J = 1.0 Hz, 1H), 7.76 (dd, J = 8.0, 7.5 Hz, 2H), 7.50
(dd, J = 4.6, 1.6 Hz, 2H), 4.70 (t, J = 7.2 Hz, 2H), 3.39 (t, J = 7.3 Hz, 2H), 2.49 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 164.0×2, 152.6, 151.4×2, 146.7, 145.0, 142.1, 135.0, 134.1×2, 131.6, 131.4×2, 128.9,
128.2, 127.4, 127.0×2, 122.4×2, 121.7×2, 38.1, 27.0, 18.6; HRMS (ESI-TOF) m/z calcd for
C28H21N5O2 [M+H]+ 434.1612, found 434.1609.
2-(2-(6-methyl-3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)ethyl)-1H-benzo[de]isoquinoline-
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1,3(2H)-dione (2e). Colorless solid. Yield: 54%; purity: 99.9%; Mp: 230.2-231.1 C; H NMR (400
MHz, CDCl3) δ 8.52 (dd, J = 7.3, 1.0 Hz, 2H), 8.22 (dd, J = 8.3, 0.9 Hz, 2H), 8.16 (d, J = 1.5 Hz, 1H),
7.79 – 7.71 (m, 3H), 7.46 (d, J = 3.9 Hz, 4H), 7.42 – 7.38 (m, 1H), 4.69 (t, J = 7.2 Hz, 2H), 3.30 (t, J
= 7.2 Hz, 2H), 2.47 (s, 3H);13C NMR (101 MHz, CDCl3) δ 163.9×2, 153.6, 147.6, 144.7, 134.8, 134.5,
133.9×2, 131.6, 131.2×2, 129.7×2, 128.8, 128.3, 128.0, 127.6×2, 126.9, 126.8×2, 122.6×2, 38.2, 26.8,
18.6; HRMS (ESI-TOF) m/z calcd for C27H20N4O2 [M+H]+ 433.1659, found 433.1651.
2-(2-(5-methyl-1-phenyl-1H-benzo[d]imidazol-2-yl)ethyl)-1H-benzo[de]isoquinoline-1,3
(2H)-dione (2f). Colorless solid. Yield: 88%;purity: 97.7%; Mp: 208.8-211.9 oC; 1H NMR (400 MHz,
CDCl3) δ 8.52 (d, J = 7.2 Hz, 2H), 8.20 (d, J = 8.2 Hz, 2H), 7.73 (t, J = 7.7 Hz, 2H), 7.52 – 7.38 (m,
6H), 7.01 (t, J = 6.3 Hz, 2H), 4.69 (t, J = 7.3 Hz, 2H), 3.25 (t, J = 7.3 Hz, 2H), 2.48 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 163.9×2, 152.0, 143.0, 136.0, 134.7, 133.9×2, 131.9, 131.6, 131.2×2, 129.8×2,
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128.6, 128.2, 127.3×2, 126.8×2, 124.1×2, 122.6, 119.1, 109.5, 38.4, 26.3, 21.5; HRMS (ESI-TOF) m/z
calcd for C28H21N3O2 [M+H]+ 432.1707, found 432.1703.
General procedures for synthesis of compounds 4a-4b. To a solution of 2-(quinolin-2-
yl)ethanol (3a) or 2-(pyridin-2-yl)ethanol(3b) (2.0 mmol) in distilled dichloromethane (10 mL) was
added thionyl chloride (5.2 mmol) under a calcium chloride tube. The solution was stirred at room
temperature for 1 h. After concentration under reduced pressure, the mixture was diluted with ethyl
acetate (350 mL) and washed with brine (20 mL). The organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure to give compounds 4a-4b as yellow powder.53
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2-(2-chloroethyl)quinoline (4a).Yellow powder. Yield: 94%; H NMR (400 MHz, CDCl3) δ
8.98 (d, J = 8.2 Hz, 1H), 8.82 (s, 1H), 8.13 (d, J = 7.4 Hz, 1H), 8.08 – 8.02 (m, 1H), 7.87 (s, 2H), 4.23
(s, 2H), 3.99 (s, 2H).
2-(2-chloroethyl)pyridine (4b). Yellow powder. Yield: 65%;1H NMR (400 MHz, CDCl3) δ 8.52
(s, 1H), 7.59 (t, J = 6.7 Hz, 1H), 7.23 – 7.06 (m, 2H), 3.89 (dt, J = 5.9, 2.7 Hz, 2H), 3.20 (t, J = 6.2 Hz,
2H).
General procedures for synthesis of compounds 5a-5b. To the solution of 2-(2-
chloroethyl)quinoline or 2-(2-chloroethyl)pyridine (5.0 mmol) in DMF (30 mL) was added sodium
azide (975 mg, 15.0 mmol). The mixture was warmed to 70 oC and stirred for 12 h. After the solution
was cooled to room temperature, water was added. The solution was extracted with ethyl acetate (300
mL) and washed with water and brine. The organic solution was dried over anhydrous sodium sulfate,
concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum/ethyl acetate,
10:1) to afford compounds 5a-5b as a colorless solid.54
2-(2-azidoethyl)quinoline (5a). Colorless solid. Yield: 86%; 1H NMR (400 MHz, CDCl3) δ 8.10
(dd, J = 18.1, 8.4 Hz, 2H), 7.83 (t, J = 10.2 Hz, 1H), 7.73 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.57 – 7.50
(m, 1H), 7.35 (t, J = 10.0 Hz, 1H), 3.91 – 3.81 (m, 2H), 3.31 – 3.22 (m, 2H).
2-(2-azidoethyl)pyridine (5b). Colorless solid. Yield: 79%; 1H NMR (400 MHz, CDCl3) δ 8.61
– 8.55 (m, 1H), 7.64 (td, J = 7.7, 1.8 Hz, 1H), 7.25 – 7.14 (m, 2H), 3.77 – 3.70 (m, 2H), 3.08 (q, J =
6.6 Hz, 2H).
General procedures for synthesis of compounds 6a-6b. To a solution of 5a-5b（5.0 mmol）in
1,4-dioxane /ethanol (30 mL, 1:1, v:v) was added Pd/C (100 mg, 10%). After a 4 h agitation at room
temperature with H2, the mixture was filtered and washed with ethanol. The filtrate was evaporated,
recrystallized from ethanol to give compounds 6a-6b as a brown oil.
2-(quinolin-2-yl)ethanamine (6a). Brown oil. Yield: 92%; 1H NMR (400 MHz, CDCl3) δ 8.06
(dd, J = 14.9, 8.5 Hz, 2H), 7.78 (d, J = 8.1 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H),
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7.31 (d, J = 8.4 Hz, 1H), 3.23 (t, J = 6.5 Hz, 2H), 3.13 (t, J = 6.5 Hz, 2H).
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2-(pyridin-2-yl)ethanamine (6b). Colorless solid. Yield: 91%; 1H NMR (400 MHz, CDCl3) δ
8.52 (d, J = 4.2 Hz, 1H), 7.58 (tt, J = 6.6, 3.3 Hz, 1H), 7.19 – 7.08 (m, 2H), 3.07 (d, J = 24.3 Hz, 2H),
2.91 (t, J = 6.7 Hz, 2H).
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Methyl 3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoate (8). To a solution of 1H-
benzo[de]isoquinoline-1,3(2H)-dione 7 (197 mg, 1.0 mmol) in DMF (30 mL) was added cesium
carbonate (553 mg, 4.0 mmol) and methyl 3-bromopropanoate (495 mg, 3.0 mmol ), and the mixture
was refluxed for 12 h at 120 oC. After completion of the reaction, the solid was removed by filtration
and then poured into water and extracted with ethyl acetate (300 mL), the solvent was evaporated
under reduced pressure to get the crude product. This residue was further purified by silical gel column
chromatography (petroleum/ethyl acetate, 10:1) to afford compound 8 (249 mg).55 Yield: 88%; 1H
NMR (400 MHz, CDCl3) δ 8.60 (d, J = 7.3 Hz, 2H), 8.22 (d, J = 8.2 Hz, 2H), 7.76 (t, J = 7.8 Hz, 2H),
4.51 (t, J = 7.5 Hz, 2H), 3.70 (s, 3H), 2.78 (t, J = 7.5 Hz, 2H).
3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoic acid (9). Compound 8 (2.83 g, 10.0
mmol) was treated with 1N aqueous sodium hydroxide (22 mL, 22.0 mmol) in EtOH (40 mL), and
stirred overnight at room temperature. Additional 1N aqueous sodium hydroxide (22 mL, 22.0 mmol)
was added, and the reaction was stirred for 6 h at room temperature. The mixture was acidified with
1N aqueous HCl to pH = 3-4 and extracted with ethyl acetate (300 mL) and washed with water and
brine. The organic solution was dried and concentrated to give compound 9 as a colorless solid (2.09
g).56 Yield: 78%; 1H NMR (400 MHz, DMSO – d6) δ 12.33 (s, 1H), 8.47 (dd, J = 13.5, 7.3 Hz, 4H),
7.87 (t, J = 7.1 Hz, 2H), 4.27 (t, J = 7.4 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H).
2-(3-bromopropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (10). To a solution of 1,8-
naphthalimide 7 (197 mg, 1.0 mmol) in acetonitrile (30 mL), anhydrous potassium carbonate (553 mg,
4.0 mmol) and 1,3-dibromopropane (597 mg, 3.0 mmol) were added and the mixture was refluxed for
12 h. After completion of the reaction, potassium carbonate was removed by filtration and the solvent
was evaporated under reduced pressure to get the crude product. This was further purified by silica gel
column chromatography (petroleum/ethyl acetate, 10:1) to afford compound 10 (285 mg).57,58 Yield:
90%; 1H NMR (400 MHz, CDCl3) δ 8.63 (dd, J = 14.3, 7.3 Hz, 2H), 8.25 (t, J = 11.3 Hz, 2H), 7.79
(dt, J = 15.5, 7.6 Hz, 2H), 4.37 (dt, J = 14.1, 7.1 Hz, 2H), 3.54 (dt, J = 13.8, 6.9 Hz, 2H), 2.42 – 2.31
(m, 2H).
General procedures for synthesis of compounds 12a-12b. To a solution of nitrobenzene 11a-
11b (1.0 mmol) in dioxne (5.0 mL) was added appropriate aniline (1.1 mmol). The solution was then
refluxed for 4 hours and allowed to cool to room temperature and was diluted with ethyl acetate (75
mL). The organic layer was washed with water (75 mL), brine (75 mL), and dried over anhydrous
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sodium sulfate. After concentration in vacuo the residue was purified by silica gel column
chromatography (petroleum/ethyl acetate, 10:1) to get products 12a-12b as orange oil.
N-(4-fluoro-2-nitrophenyl)tetrahydro-2H-pyran-4-amine (12a). Orange oil. Yield: 52%; 1H
NMR (400 MHz, CDCl3) δ 7.99 (d, J = 5.9 Hz, 1H), 7.92 (dd, J = 9.2, 3.0 Hz, 1H), 7.29 – 7.23 (m,
1H), 6.88 (dd, J = 9.5, 4.5 Hz, 1H), 4.04 (dt, J = 12.0, 3.9 Hz, 2H), 3.78 – 3.68 (m, 1H), 3.64 – 3.54
(m, 2H), 2.13 – 2.04 (m, 2H), 1.75 – 1.65 (m, 2H).
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N-(4-methyl-2-nitrophenyl)pyridin-4-amine (12b). 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).
General procedures for synthesis of compounds 12c-12f. To a mixture of nitrobenzene 11c-
11d (1.0 mmol) in toluene (10 mL) were added appropriate 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 110oC 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 acetate, 50:1) to give compounds 12c-12f as red oil.59
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N-(4-methyl-2-nitrophenyl)tetrahydro-2H-pyran-4-amine (12c). Red oil. Yield: 65%; H
NMR (400 MHz, CDCl3) δ 7.99 (s, 2H), 7.26 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 4.02 (dt, J
= 11.8, 3.7 Hz, 2H), 3.77 – 3.67 (m, 1H), 3.58 (dd, J = 16.4, 6.0 Hz, 2H), 2.26 (s, 3H), 2.06 (d, J =
13.0 Hz, 2H), 1.69 (dd, J = 10.1, 3.9 Hz, 1H), 1.66 – 1.61 (m, 1H).
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5-methyl-3-nitro-N-phenylpyridin-2-amine (12d). Red oil. Yield: 63%; H NMR (400 MHz,
CDCl3) δ 10.01 (s, 1H), 8.34 (s, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.16 (t, J = 7.4
Hz, 1H), 2.33 (s, 3H).
5-methyl-3-nitro-N-(pyridin-4-yl)pyridin-2-amine (12e). 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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4-methyl-2-nitro-N-phenylaniline (12f). Red oil.Yield: 60%; H NMR (400 MHz, CDCl3) δ
9.36 (s, 1H), 8.00 (s, 1H), 7.39 (t, J = 7.8 Hz, 2H), 7.25 (d, J = 7.6 Hz, 2H), 7.23 – 7.15 (m, 3H), 2.29
(s, 3H).
General procedures for synthesis of compounds 13a-13f. To a mixture of 12a-12f (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 compounds 13a-13f as colorless solid.
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5-Fluoro-N2-(tetrahydro-2H-pyran-4-yl)pyridine-2,3-diamine (13a). Colorless solid.Yield:
83%; 1H NMR (400 MHz, CDCl3) δ 6.65 (dd, J = 8.5, 5.5 Hz, 1H), 6.51 – 6.41 (m, 2H), 4.02 (dt, J =
11.9, 3.7 Hz, 2H), 3.50 (td, J = 11.6, 2.3 Hz, 2H), 3.34 (tt, J = 10.2, 4.0 Hz, 1H), 2.03 – 1.96 (m, 2H),
1.57 – 1.46 (m, 2H).
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4-Methyl-N1-(pyridin-4-yl)benzene-1,2-diamine (13b). Colorless solid.Yield: 89%; 1H 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).
4-methyl-N1-(tetrahydro-2H-pyran-4-yl)benzene-1,2-diamine (13c). Colorless solid. Yield:
50%; 1H NMR (400 MHz, CDCl3) δ 6.66 – 6.59 (m, 3H), 4.02 (dt, J = 11.9, 3.8 Hz, 2H), 3.52 (td, J =
11.5, 2.3 Hz, 2H), 3.42 (ddd, J = 10.2, 6.1, 4.2 Hz, 1H), 2.25 (s, 3H), 2.07 – 2.01 (m, 2H), 1.59 – 1.48
(m, 2H).
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5-methyl-N2-(pyridin-4-yl)pyridine-2,3-diamine (13d). 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).
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5-Methyl-N2-phenylpyridine-2,3-diamine (13e). Colorless solid. Yield: 78%; H NMR (400
MHz, CDCl3) δ 7.69 (s, 1H), 7.28 (dd, J = 9.0, 6.8 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H), 6.93 (t, J = 7.3
Hz, 1H), 6.88 (s, 1H), 6.03 (s, 1H), 3.46 (s, 2H), 2.34 – 2.17 (m, 3H).
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4-methyl-N1-phenylbenzene-1,2-diamine (13f). Colorless solid. Yield: 55%; H NMR (400
MHz, CDCl3) δ 7.24 – 7.19 (m, 2H), 7.02 (d, J = 7.9 Hz, 1H), 6.85 – 6.79 (m, 1H), 6.74 – 6.70 (m,
2H), 6.66 (d, J = 1.0 Hz, 1H), 6.60 (dd, J = 7.9, 1.3 Hz, 1H), 5.11 (s, 1H), 3.76 (s, 2H), 2.32 (s, 3H).
Protein expression and purification. The pET15b-PDE10A plasmid for expression of the
recombinant catalytic domain (residues 446-789) was subcloned and purified using previously
reported protocols.60 Briefly, the plasmid was transferred to E. coli strain BL21 (Codonplus,
Stratagene). Then, the E. coli cells carrying the recombinant plasmid were grown in 2xYT medium
(containing 100 μg/mL ampicillin and 30 μg/mL chloramphenicol) at 37 °C until an OD600 = 0.6-0.8
was achieved. Then, 1 mM isopropyl-β-D-thiogalactopyranoside was added to induce the expression
of the PDE10A protein and the culture was incubated at 20 °C for 24 h. A nickel nitriloacetic acid (Ni-
NTA) column (Qiagen) was used to purify PDE10A proteins. The concentration of the PDE10A
fractions was estimated by monitoring the absorbance at 280 nm (calculated using the ProtParam
software). A typical batch of purification yielded 100–200 mg of PDE10A protein from a 1.0 L cell
culture. The catalytic domains of PDE2A (580-919), PDE3A (679-1087), PDE4D (86-413), PDE7A
(130-482), PDE9A (181-506), PDE8A (480-820), PDE10A (449-770), and PDE11A (588-911) were
purified using similar protocols, as previously reported.60-64 PDE1C (2-634) and PDE6C (1−858) were
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purchased from BPS Bioscience.
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Enzymatic assays. The enzymatic activity of the catalytic domain of PDE10A2 was measured with
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3H-cGMP as the substrate in a buffer composed of 50 mM Tris, pH 7.5, 4 mM MgCl2 and 1 mM
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dithiothreitol; 20,000 - 30,000 cpm H-cGMP was used in each assay. Different concentrations of
solubilized inhibitors in DMSO were added to the PDE10A enzyme. The enzymatic reaction was
performed at room temperature for 15 min and then terminated by the addition of 0.2 M ZnSO4. The
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reaction product was precipitated by the addition of 0.2 N Ba(OH)2 and the unreacted H-cGMP
remained in the supernatant. The radioactivity in the supernatant was measured in 2.5 ml of Ultima
Gold liquid scintillation cocktails (PerkinElmer) with a PerkinElmer 2910 liquid scintillation counter.
At least eight concentrations of inhibitors were used to calculate the IC50 values of each inhibitor. Each
measurement was repeated at least three times, and IC50 values were calculated using a nonlinear
regression equation. The mean values of the measurements were considered the final IC50 values and
reported with SD of the three or four measurements. In this assay, papaverine was used as the reference
compound and determined to have an IC50 of 0.1 μM, which is comparable to the value ranging from
0.02 to 0.2 μM in the Binding Database (http://www.bindingdb.org).
Crystallization and structure refinement. The crystals of PDE10A2 were grown using the hanging
drop method similar to those previously reported.60-64 Briefly, the unliganded PDE10A2 enzyme (10
mg/mL in a buffer composed of 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA and 1 mM β-
mercaptoethanol) was vapor-diffused against the well buffer of 0.1 M Hepes (pH 7.5), 0.2 M MgCl2,
18% PEG3350 and 50 mM 2-mercaptoethanol. The complex of PDE10A2 with AF-399/14387019 or
compound 2b was prepared by soaking the unliganded crystals in 20 mM AF-399/14387019 or
compound 2b in a buffer of 0.1 M Hepes (pH 7.5), 0.1 M MgCl2, 16% PEG 8000 and 60 mM 2-ME
at 4 °C for 24 h. The crystallization buffer containing 20% ethylene glycol was used as the cryosolvent.
Diffraction data were collected at 100 K on an in-house Oxford Diffraction Xcalibur Nova
diffractometer. The data were processed using the program CrysAlis Pro and the structures were solved
and refined using CCP4.65 The coordinates and structure factors have been deposited in the Protein
Data Bank with PDB ID of 6IJH and 6IJI. Data collection and refinement statistics for all structures
were shown in supporting information.
Pharmacokinetics analysis of 2b. The pharmacokinetic properties of 2b were analyzed by the
Medicilon Company, Shanghai, China. Three male SD rats with a body weight of 230-260 g were
purchased from Shanghai SIPPR-BK LAB Animal Ltd., Shanghai, China, and used for the
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pharmacokinetic analysis of 2b. It was dissolved/suspended in 5% DMSO, 10% Solutol, and 85%
water for intravenous administration (i.v.). A final dosage of 2.5 mg/kg rat of the formulated compound
was administered for i.v. purpose, and the blood samples were taken at various time points during a
24 h period. The concentration of compounds in the blood was analyzed by LC-MS/MS (Shimadzu
liquid chromatographic system and API4000 mass spectrometer, Applied Biosystems, Ontario,
Canada).
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Pharmacodynamics effects of compound 2b against PAH in Wistar rats. All animal care
procedures and experimental protocols61,62 were performed in accordance with the “Guide for the Care
and Use of Laboratory Animals” (National Institutes of Health Publication, revised 1996, No. 86-23,
Bethesda, MD) and approved by the Institutional Ethical Committee for Animal Research of Sun Yat-
sen University. Forty Wistar rats (6-7 weeks, 180-220 g) were purchased from the Laboratory Animal
Center of Southern Medical University (Guangzhou, China) and used to evaluate the anti-PAH effects
of compound 2b on PAH. The rats were randomly divided into four groups as follows: control, model,
compound 2b (2.5 mg/kg), and positive control (sildenafil citrate, 10 mg/kg). The rats were maintained
on a 12 h light/dark cycle (light from 7:00 to 19:00) at 24 ± 1 °C and 60−70% relative humidity. Sterile
food and water were provided according to the institutional guidelines. Prior to each experiment, the
rats were fasted overnight and allowed free access to water. All rats were administered MCT 60 mg/kg,
except for the control group. Then, the rats were treated daily with the drug vehicle (control and model
groups), 2b (2.5 mg/kg, i.p.), and sildenafil citrate (10 mg/kg, p.o.) for 3 weeks. 2b and sildenafil citrate
were dissolved in 5% DMSO/10% Solutol/85% water solution and administered 0.4 mL per 100 g
weight. Rats were anesthetized with pentobarbital sodium (60 mg/kg, i.p.). Mean pulmonary artery
pressure (mPAP) was measured by right heart catheterization. Following pressure measurement, the
heart was divided and the atria dissected. The weight of right ventricle (RV) and left ventricle plus
ventricular septum (LV+S) was measured, and RV/(LV+S) ratio was taken as right ventricular
hypertrophy index (RVHI). Then a part of lung tissues were taken and fixed with 4% paraformaldehyde.
Routine HE staining was performed to observe the pathological changes and calculate the percentage
(WT %) of the medial wall thickness of pulmonary arterioles. The concentrations of cAMP and cGMP
were measured with Abbkine and Cayman ELISA kits according to the manufacturers' instructions,
respectively.
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VSMC culture. Human pulmonary artery smooth muscle cells (HPASMCs) were obtained from BeNa
Culture Collection (Beijing, China). HPASMCs were cultured in Dulbecco’s modified Eagle’s
medium (high glucose) (HDMEM) with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA),
penicillin (100 IU/ml) and streptomycin (100 mg/ml) at 37 °C in a 5% CO2 humidified incubator. Cells
in the four to eight passages were used, and cells at 80–90% confluence were arrested by incubating
in serum-deprived DMEM for 12 h before intervention. PDGF-BB (20 µM) was treated for 24 h.
PDE10A inhibitor 2b and PF-2545920 were added 2 h after the addition of PDGF-BB and were
incubated for 22 h.
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Western blot analysis. Cells were homogenized in lysis buffer, and the supernatant was extracted for
the measurement of total protein with a protein assay kit (BCA; Pierce, Santa Cruz, CA, USA). Equal
amounts of total protein were separated in SDS-PAGE, and transferred to PVDF membranes in
Trisglycine methanol buffer. The bands were visualized using the enhanced chemiluminescent. The
primary antibodies against OPN, PCNA, SM22α and α-tubulin were purchased from Proteintech
(Wuhan, China). Antibodies against -SMA was obtained from Abcam (Cambridge, MA, USA).
■ ASSOCIATED CONTENT
Supporting Information
Detailed protocols for molecular dynamics-based virtual screening, Diffraction data and structure
refinement statistics of crystallography, Extended data series for tested compounds appear in the
Supporting Information. This material is available free of charge via the Internet.
Molecular formula strings (CSV)
Accession Codes
The atomic coordinates and structure factors have been deposited into the RCSB Protein Data Bank
with accession numbers of 6IJH and 6IJI. Authors will release the atomic coordinates and experimental
data upon article publication.
■ AUTHOR INFORMATION
Corresponding Authors
* E-mail: wudeyan3@mail.sysu.edu.cn (W.D. Wu), wyinuo3@mail.sysu.edu.cn (W.Y. Wu), and
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luohb77@mail.sysu.edu.cn (H.-B. Luo); Fax: +86-20-3994 3000
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ORCID
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Hai-Bin Luo: 0000-0002-2163-0509
Deyan Wu: 0000-0001-5855-8662
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Yinuo Wu: 0000-0003-3071-5333
Yi-you Huang: 0000-0002-0712-1310
Author Contributions
|| These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
BINAP, (±)-2,2’-bis(diphenylphosphino)-1,1’-binaphthyl; cAMP, cyclic adenosine monophosphate;
cGMP, cyclic guanosine monophosphate; CNS, central nervous system; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; DIPEA, N,N-diisopropylethylamine; HATU, 2-(7-
Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; IC50, half maximal
inhibitory concentration; MD, molecular dynamics; MCT, monocrotaline; mPAP, mean pulmonary
artery pressure; PAH, pulmonary arterial hypertension; PDE, phosphodiesterase; PDE10,
phosphodiesterase-10; RVHI, right ventricle hypertrophy index; SPBG, selective pocket binding group;
TLC, thin layer chromatography; TMS, tetramethylsilane; WT, wall thickness.
■ ACKNOWLEDGEMENTS
We cordially thank anonymous reviewers' valuable comments and kind proofreading of the manuscript.
This work was supported by Natural Science Foundation of China (21572279, 21877134, 81872727,
81602955, and 81703341), Science Foundation of Guangdong Province (2016A030310144), Pearl
River S&T Nova program of Guangzhou (201806010190),The Fundamental Research Funds for the
Central Universities (Sun Yat-Sen University) (No. 17ykjc03 and 17ykpy20), Guangdong Province
Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2016). We cordially
thank Prof. H. Ke from Department of Biochemistry and Biophysics at the University of North
Carolina, Chapel Hill, for his proofreading and his help with molecular cloning, expression,
purification, crystal structure, and bioassay of PDEs. We cordially thank Dr. Zhengchao Tu from
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Guangzhou Institutes of Biomedicine and Health and Zhiping Zeng from Xiamen University for their
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help to screen the binding affinities of compound 2b against twenty different targets.
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