Optimization of Chromeno[2,3- c]pyrrol-9(2 H)-ones as Highly Potent, Selective, and Orally Bioavailable PDE5 Inhibitors: Structure-Activity Relationship, X-ray Crystal Structure, and Pharmacodynamic Effect on Pulmonary Arterial Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.8b01209

To further explore the structure-activity relationship around the chromeno[2,3-c]pyrrol-9(2H)-one scaffold, 19 derivatives as inhibitors against PDE5 were discovered. The most potent inhibitor 3 has an IC₅₀ of 0.32 nM with remarkable selectivity and druglike profile. Oral administration of 3 (1.25 mg/kg) caused comparable therapeutic effects to sildenafil (10.0 mg/kg) against pulmonary arterial hypertension. Further, different binding patterns from sildenafil were revealed in cocrystal structures, which provide structural templates for discovery of highly potent PDE5 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b01209

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Brief Article
Optimization of Chromeno[2,3-c]pyrrol-9(2H)-ones as Highly
Potent, Selective, and Orally Bioavailable PDE5 Inhibitors:
Structure-Activity Relationship, X-ray Crystal Structure, and
Pharmacodynamic Effect on Pulmonary Arterial Hypertension
Deyan Wu, Yadan Huang, Yiping Chen, Yi-you Huang, Haiju Geng, Tianhua
Zhang, Chen Zhang, Zhe Li, Lei Guo, Jianwen Chen, and Hai-Bin Luo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01209 • Publication Date (Web): 27 Aug 2018
Downloaded from http://pubs.acs.org on August 27, 2018
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Optimization of Chromeno[2,3-c]pyrrol-9(2H)-ones as Highly Po-
tent, Selective, and Orally Bioavailable PDE5 Inhibitors: Structure-
Activity Relationship, X-ray Crystal Structure, and Pharmacodynam-
ic Effect on Pulmonary Arterial Hypertension
Deyan Wu‡, Yadan Huang‡, Yiping Chen‡, Yi-You Huang, Haiju Geng, Tianhua Zhang, Chen
Zhang, Zhe Li, Lei Guo, Jianwen Chen, Hai-Bin Luo*
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School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, P. R. China.
ABSTRACT: To further explore the structure-activity relationship around the chromeno[2,3-c]pyrrol-9(2H)-one scaffold,
nineteen derivatives as inhibitors against PDE5 were discovered. The most potent inhibitor 3 has an IC50 of 0.32 nM with
remarkable selectivity and druglike profile. Oral administration of 3 (1.25 mg/kg) caused comparable therapeutic effects to
sildenafil (10.0 mg/kg) against pulmonary arterial hypertension. Further, different binding patterns from sildenafil were
revealed in co-crystal structures, which provide structural templates for discovery of highly potent PDE5 inhibitors.
INTRODUCTION
11b complex, we further designed and synthesized five
compounds as potent inhibitors. As a result, compound 3
exhibits a highly potent affinity of 0.32 nM with remarka-
ble pharmacodynamic effects and druglike profile (meta-
bolic stability, inhibition of hERG potassium channels,
cytochrome P450 inhibition, plasma protein binding, and
pharmacological safety), indicating that 3 could be used
as a promising treatment or chemical probe against PAH.
Pulmonary arterial hypertension (PAH) is a severely pro-
gressive and debilitating chronic illness that induces high
pressure in the pulmonary arteries, leading to right-heart
failure and eventual death.1,2 Recently, significant advanc-
es in PAH pathology have been achieved for the develop-
ment of several effective therapeutic targets in this dis-
ease and three signal pathways have been validated for its
pathogenesis and progression (endothelin pathway, pros-
tacyclin pathway, and NO/cGMP pathway).3-5 In general,
the inhibition of phosphodiesterase-5 (PDE5) via the
NO/cGMP signal pathway6,7, would lead to pulmonary
vasodilation and smooth muscle cell growth inhibition5,8.
Currently, two famous PDE5 inhibitors such as sildenafil
and tadalafil were clinically used as anti-PAH agents.3,5,9,10
Compared with other anti-PAH drugs, PDE5 inhibitors
have relatively few adverse effects and are safe for patients
who are with mild to moderate renal or hepatic dysfunc-
tion.11,12
Recently, we reported several chromeno[2,3-c]pyrrol-
9(2H)-one analogues as potent inhibitors against PDE5.
13,14 Among them, compound 1 (Figure 1) exhibits an inhib-
itory affinity (IC50) of 5.6 nM and could be used as a lead
against PAH. To further explore the structure-activity
relationships around the scaffold and enrich their mo-
lecular diversity, compound 2 was identified as a potent
intermediate inhibitor towards PDE5. Subsequently, the
co-crystal structure of PDE5 with bound 11b showed 11b
occupies the active pocket with a different binding mode
from sildenafil. Based on the binding mode of the PDE5-
RESULTS AND DISCUSSION
Chemistry. As shown in Scheme 1-3, we developed an
efficient route to obtain derivatives 2-3, 11a-m, 16, and
17a-c. Herein, we use compound 4 as the starting material
(Scheme 1), and it was treated with diodomethane and
K2CO3 in acetonitril to acquire the intermediate 5, fol-
lowed by treatment with NaOH to afford the acid 6.15,16
The Boc group of compound 6 was deprotected by TFA in
dichloromethane to generate the amino acid 7.17 Then, the
Fmoc group was introduced with FmocCl and aqueous
Na2CO3 in dioxane to yield the N-Fmoc amino acid (8).14,18
Compounds 10a-10n were synthesized by the reaction of
ethyl
thiazole-2-carboxylate
(9)
and
2’-
hydroxyacetophenones in toluene with the base of sodi-
um hydride. Then, compounds 2 and 11a-m were synthe-
sized as previously reported steps.13,14,19 Compounds 3, 16,
and 17a-c were synthesized through the route outlined in
Scheme 3. Additionally, 3-Fluoro-5-bromophenol (12) was
acetylated with acetyl chloride and followed by Friedel-
Crafts acetylation to afford 1-(4-bromo-2-fluoro-6-
hydroxyphenyl)ethenone (14).20
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Figure 1. Structural optimization of compound 1 resulted in novel and highly potent PDE5 inhibitors with improved
pharmacodynamic profile.
Then, the same procedures as Scheme 2 were used to
yield compound 16, which was coupled with amines to
afford 3, and 17a-c via Buchwald-Hartwig cross coupling
reaction, respectively. Experimental and analytical infor-
mation for 2-3, 11a-m, 16, and 17a-c are provided in the
Supporting Information.
PAINS screening of the newly designed nineteen de-
rivatives was carried out via an online program “PAINS
aReagents and conditions: (a) diodomethane, K2CO3, ace-
Remover” (http://www.cbligand.org /PAINS)21 in order to
tonitril, ref. 16h; (b) 1N NaOH, MeOH, rt, 4 h; (c) TFA,
prevent false positive results, and all the derivatives
DCM, rt, 1 h; (d) 9-Fluorenylmethyl chloroformate, diox-
passed this screening.
ane, H2O, 0°C, 1 h, rt, 1 h.
Structure−Activity Relationships (SARs).
Scheme 2. Synthesis of compounds 2 and 11a-m. a
We previously reported several chromeno[2,3-
c]pyrrol-9(2H)-one inhibitors towards PDE5 as anti-PAH
agents, and lead 1 has an IC50 of 5.6 nM.13,14 Herein, to
further explore and enrich the structure-activity relation-
ships around the scaffold, nineteen derivatives were syn-
thesized. Their inhibitory activities against PDE5 are
showed in Table 1 and all the derivatives show considera-
ble inhibitory potencies (less than 10 nM). Specifically,
compounds 2 and 11a-11b have a different substitution at
the 6-position, in which the introduction of OH/F groups
(2 and 11b, IC50 = 1.05 nM and 1.41 nM, respectively) at the
aReagents and conditions: (a) NaH, toluene, 0°C to 60°C, 2
h; (b) 8, DMAP, DCC, pyridine, rt, 3 h, 50°C, 6 h.
6-position resulted in tighter binding than that with
methoxyl group (11a). Comparing compounds 11b, 11d,
and 11j with fluorine substitutions at the 6-position, 5-
Scheme 3. Synthesis of compounds 3 and 17a-c. a
position, and 4-position, respectively, 11b shows the best
IC50 of 1.41 nM, which indicates that fluorine atoms in the
6-position possibly favor the binding mode of chemicals
with PDE5. Comparing compounds 11m, 11e, and 11k with
chlorine substitutions at the 3-position, 4-position, and 5-
position, respectively, 11e shows the worst IC50 of 9.63 nM,
which indicates that halogen atoms in the 5-position pos-
sibly disfavor the binding mode of chemicals with PDE5.
A similar trend is observed for 11f and 11l or 11d and 11j.
Thus, compounds2 and 11b were identified as potent in-
termediate inhibitors for subsequent study.
aReagents and conditions: (a) Ac2O, NaOAc, 110°C, 2 h; (b)
AlCl3, 140°C, 3 h; (c) 9, NaH, toluene, 0°C to 60°C, 2 h; (d)
8, DMAP, DCC, pyridine, rt, 3 h, 50°C, 6 h; (e) RNH2,
Pd(dppf)2Cl2, BINAP, t-BuONa, DMSO, 100°C, 24 h.
Scheme
1.
Synthesis
of
(S)-2-(Fmoc-amino)-3-
(benzo[d][1,3]dioxol-5-yl)propanoic acid. a
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Table 1. Structure−Activity Relationships of Com-
(such as Ala767, Ile768, Gln775, and Ile778). The electron
density maps of (2Fo-Fc) and (Fo-Fc) clearly revealed the
binding mode of PDE5 with 11b, in which the thiazole
ring conjugated with the purine ring. Compared with our
previously reported inhibitor 5R13 (Figure 1), 11b interacts
much tightly with four hydrophobic residues Val782,
Phe787, Ile813, and Met816. Notably, the benzene ring of
11b is outside the binding pattern, which explains why
most of the compounds listed in Table 1 have improved
inhibitory activities towards PDE5, and different substitu-
ents on this ring can be further optimized.
Based on the acquired binding pattern of 11b, we fur-
ther synthesized another series as potent PDE5 inhibitors
based on the 4-position of the benzene ring outside the
binding pattern (Table 2). The results indicated that all
the compounds showed improved inhibitory activities
except 16, which has a bromine atom at the 4-position
and a fluorine atom at the 6-position. It is gratifying to
see that compound 3, with a (2-methoxyethyl)amino sub-
stitution at the 4-position, exhibits the most potent inhi-
bition of IC50 = 0.32 nM. Finally, compound 3 was selected
as the candidate for subsequent study.
pounds 2 and 11a-ma
Compound
R
IC50 (nM)
1.05±0.31
4.38±0.32
1.41±0.30
6.82±0.34
4.03±0.14
9.63±1.09
3.82±0.10
3.04±0.22
1.63±0.24
2.64±0.39
2.38±0.31
2.64±0.27
1.58±0.23
2.09±0.15
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2
6-OH
6-OMe
6-F
11a
11b
11c
11d
11e
11f
5-Cl-6-OMe
5-F
5-Cl
5-Br
11g
11h
11i
3-Br-5-Cl
3,5-diF
4-OMe
4-F
11j
11k
11l
4-Cl
Crystal structure of PDE5 with bound 17c. Given the
promising biological activities of compounds 17a-c and 3,
we tried to acquire their crystal structures with PDE5.
Among them, the co-crystal structure of PDE5 with 17c
was finally obtained (Figure 2B). The structure indicated
that 17c have the same binding pattern as 11b, but 17c
have another substitution at the 4-position and forms
hydrophobic interactions with the residues Val660 and
Met681. As a result, compounds 3 and 17a-c resulted in
tighter binding than compound 2, and exhibit highly po-
tent PDE5 inhibition.
4-Br
11m
3-Cl
aSildenafil, the reference compound (IC50=5.1 nM)
.
Table 2. Structure−Activity Relationships of Com-
pounds 3, 16, and 17a-c.
Figure 2. Co-crystal structures of the PDE5-11b and
PDE5-17c complexes (PDB ID: 5ZZ2 and 6ACB). (A) Sur-
face model of 11b with PDE5 (yellow sticks); (B) Surface
model of 17c with PDE5 (green sticks). The dotted lines
refer to H-bonds.
Compound R1
R2
H
IC50 (nM)
1.05±0.31
2
OH
Crystal structure of PDE5 with bound 11b. To deter-
mine where compound 11b bind，the unliganded PDE5
and its complex were crystallized by the hanging drop
method. In Figure 2A, 11b captured the same pocket as
typical PDE5 inhibitors, except with a different binding
model. The main skeleton of 11b is clamped by three hy-
drophobic residues Val782, Phe786, and Phe820. In addi-
tion, the pyrrol ring (NH) of 11b involves an H-bond of 2.8
Å with the amide oxygen of residue Gln817, and the thia-
zole ring (S or N) of 11b also makes an H-bond with the
amide nitrogen of residue Gln817 (3.3 Å to the nitrogen)
along with hydrophobic interactions with other residues
16
F
Br
2.37±0.17
0.52±0.09
0.79±0.03
0.39±0.05
17a
17b
18
OH
OH
OH
CH3
3
OH
0.32±0.02
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High selectivity of compound 3 against other PDE
Reasonable druglike profile. Since compound 3 exhib-
families. We have measured the selectivity index of com-
pound 3 against other PDE subtypes (Table 3). Compound
3 exhibited at least 1000-fold selectivity over PDE1B,
PDE3A, PDE7A1, PDE8A1, and PDE9A2. Additionally, the
selectivity index values against PDE2A, PDE10A, PDE4D2,
and PDE6C were 331-fold, 144-fold, 134-fold, and 4-fold
higher than those against PDE5A1, respectively. Under
identical assay condition, the reference drug sildenafil or
vardenafil had a similar selectivity index of 5-fold or 3-fold
to 3 against PDE6C. Specially, 3 exhibited a higher selec-
tivity of 122-fold against PDE11A than the reference drug
tadalafil of 20-fold. In short, compound 3 gives high selec-
tivity over other PDEs except PDE6C.
ited subnanomolar inhibition against PDE5 and excellent
selectivity over other PDEs, its druglike profile evalua-
tions were performed. Table 4 shows that compound 3
was stable in rat liver microsomes with T1/2 of 15.07 min
and Eh of 75%, respectively, which is significantly better
than the other compounds of 11b(9.52 min and 83%), 17c
(5.26 min and 90%), and reference drug sildenafil (5.37
min and 89%). Other druglike profile evaluations are
shown in Table 5, such as cytochrome P450 inhibition (3
has an IC50 of > 10 μM against CYP1A2, 2B6, 2D6, and 3A4
and 0.135 μM against CYP2C9, respectively), inhibition of
hERG potassium channels (IC50> 30 μM), plasma protein
binding (PPB) (97.74%), and pharmacological safety (3
was well tolerated up to a dose of 1.5 g/kg and with no
acute toxicity). These results indicate that compound 3
could be used as a promising candidate for further devel-
opment.
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Table 3. Selectivity index of 3 across PDE Families.
3
PDE isozyme
IC50 (nM)
0.32 ± 0.02
>32 000
106 ±3
Selectivity index
Notable therapeutic effects against PAH in rats. The
therapeutic results of compound 3 as anti-PAH agents in
vivo are shown in Figure 3. A dramatical reduction of
mean pulmonary artery pressure (mPAP) for the control
group was observed compared with that of the model
group, while the index of right ventricle hypertrophy
(RVHI) of the control group was significantly decreased
than that of the model group, which demonstrated the
rats was successfully induced with PAH after 3 weeks
monocrotaline (MCT) injection. As a result, the average
mPAPs (19.94 mmHg and 20.86 mmHg) of the groups
treated with 3 and sildenafil citrate at oral doses of 1.25
mg/kg and 10.0 mg/kg were significantly decreased com-
pared with the model group (52.55 mmHg, Figure 3A). For
the RVHI values, similar phenomena were also observed.
It was clear that compound 3 at dose 1.25 mg/kg caused
better therapeutic effects on both mPAP and RVHI than
sildenafil citrate at dose 10.0 mg/kg (Figure 3B). Addition-
ally, the wall thickness percentage (WT %) of the external
diameter for the model group was significantly increased
as well. As shown in Figure 3C, compound 3 and sildenafil
citrate showed remarkable reduction to the WT% values
compared with the model group and performed well.
PDE5A1(535-860)
PDE1B(10-487)
-
>10 000
331
PDE2A(580-919)
PDE3A(679-1087)
PDE4D2(86-413)
PDE6C(1-858)
>32 000
43 ± 1
>10000
134
1.2 ± 0.1
>32 000
4a
PDE7A1(130-482)
>10 000
>10 000
>10 000
144
PDE8A1(480-820) >32 000
PDE9A2(181-506) >32 000
PDE10A(449-770) 46 ± 3
PDE11A(588-911)
39.0±0.4
122b
aUnder identical assay condition, the reference drug
sildenafil or vardenafil had a similar selectivity index of 5-
b
fold or 3-fold to 3 against PDE6C; Under identical assay
condition, the reference drug tadalafil had a selectivity
index of 20-fold against PDE11A.
Table 4. Metabolic Stability of Compounds 11b, 17c, and 3 in Rat Liver Microsomes.
T1/2
Clint
Clapp
Clh
Eh
Compound
k
(min)
9.52
5.26
18.33
15.07
5.37
(mL/min/mg)
0.1456
(mL/min/kg)
262.13
(mL/min/kg)
45.46
(%)
83%
90%
71%
75%
89%
11ba
17cb
3a
3b
0.0728
0.1317
0.2634
472.01
49.42
0.0378
0.0460
0.1291
0.0756
0.0920
0.2582
136.09
39.17
164.86
41.35
sildenafil b
462.69
49.32
a the first experiment; b the second experiment.
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Table 5. The DruglikeProfile of Compound 3.
3
4
5
6
Rat liver microsomes
(T1/2)
hERG inhibition
(IC50)
CYP P450 inhibition
(IC50)
Plasma protein
binding
Acute safety
> 1.5 g/kg
> 10 μM（CYP1A2, 2B6, 2D6, and 3A4)
135 nM(CYP2C9)
15.07 min
>30 μM
97.7%
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Figure 3. Significant effects of compound 3 and sildenafil citrate on pulmonary arterial remodeling of rats in vivo. (A)
Dramatical reduction of hypertension pressure by 3 and sildenafil citrate, which was firstly induced by monocrotaline
(MCT). Comparison of the mPAP values between different groups was provided in (A). (B) Effects of 3 and sildenafil cit-
rate on RVHI of PAH model rats. Comparison of the RVHI% values between different groups was provided in (B). (C) Ef-
fects of 3 and sildenafil citrate on the wall thickness percentage (WT%) of small pulmonary arteries of PAH model rats.
Comparison of the WT % values between different groups was provided in (C). Here, the data is shown as the mean ±
S.E.M (n = 6-9 per group), and (##) refers to p < 0.01 compared to the control group; (∗) refers to p < 0.05 compared to the
model group ,(∗∗) refers to p < 0.01 compared to the model group.
CONCLUSION
(doublet), dd (doublet of doublets), dt (doublet of tri-
plets), t (triplet), td (triplet of doublets), q (quartet), and
m (multiplet), and coupling constants are reported in Hz.
High resolution mass spectra (HRMS) were recorded on a
MAT-95 spectrometer. The purity of tested compounds
were determined by reverse-phase high performance liq-
uid chromatography (HPLC) analysis confirming to be
more than 95%. HPLC instrument: SHIMADZU LC-20AT
(Detector: SPD-20A UV/VIS detector, UV detection at 254
nm; column: Hypersil BDS C18, 5.0 μm, 4.6 × 150 mm
(Elite); Elution, MeOH in water (70%-90%, v/v); T = 25°C;
and flow rate = 1.0 mL/min.
In summary, nineteen newly designed derivatives as PDE5
inhibitors were discovered with IC50 uniformly < 10 nM.
Medicinal optimization resulted in the most potent inhib-
itor 3 with IC50 of 0.32 nM against PDE5 with high selec-
tivity against other PDE subtypes. After druglike proper-
ties evaluations, oral administration of 3 at dose 1.25
mg/kg caused comparable therapeutic effects to sildenafil
citrate at dose 10.0 mg/kg against PAH. The co-crystal
structure of PDE5 with bound 17c reveals a different bind-
ing pattern from sildenafil. In short, this work provides
not only a molecular basis for understanding their
recongnition mechanism, but also the structural tem-
plates for discovery of highly potent PDE5 inhibitors.
General Procedure for Synthesis of Compounds 3. To
a
solution of 16 (500 mg, 1.0 mmol), 2-
EXPERIMENTAL SECTION
methoxyethanamine (225 mg, 3.0 mmol), and 2,2'-
bis(diphenylphosphino)-1,1'-bisnaphthyl (124 mg, 0.2
mmol,) in DMSO (15 mL) was added 1,1'-
General Methods. All starting materials and reagents
were purchased from commercial suppliers (Adamas, Bide,
Energy, Meryer, Sigma-Aldrich, and J&K) and used direct-
ly without further purification. Silica gel plates with fluo-
rescence F254 (0.1-0.2 mm, Huanghai®) were used for
thin-layer chromatography (TLC) analysis, and chemical
HG/T2354-92 silica gel (200-300 mesh, Haiyang®) was
used for column chromatography. Reactions requiring
anhydrous conditions were performed under argon or a
bis(diphenylphosphino)ferrocene-palladium(II)
dichlo-
ride dichloromethane complex (82 mg, 0.1 mmol) and
sodium tert-butoxide (192 mg, 2.0 mmol). The mixture
was then heated to 100°C for 24 h under an argon atmos-
phere. After the solution had cooled to room temperature
it was diluted with EtOAc (150 mL) and washed with
brine (3 × 100 mL). The organic layer was dried over an-
hydrous Na2SO4 and concentrated to give a crude, which
was purified by silica gel column chromatography (petro-
leum ether:EtOAc, 4:1) to obtain the product 3 (182 mg) as
1
calcium chloride tube. H NMR/13C NMR spectra were
recorded on a Bruker AVANCE III 400 instrument with
tetramethylsilane as an internal standard. The following
abbreviations are used: s (singlet), br (broad signal), d
1
a yellow solid. Yield: 37%; purity: 99%; HNMR (500 M,
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Acetone – d6) δ 13.25 (s, 1H), 7.82 (d, J = 3.1 Hz, 1H), 7.59 (d,
ABBREVIATIONS USED
J = 3.1 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.76 (d,
J = 7.9 Hz, 1H), 6.17 (s, 1H), 6.01 (s, 1H), 5.94 (s, 2H), 4.18 (s,
2H), 3.60 (t, J = 5.3 Hz, 2H), 3.43 (dd, J = 10.2, 5.1 Hz, 2H),
3.35 (s, 3H); 13C NMR (101 MHz, DMSO – d6) δ 178.23,
163.32, 159.06, 157.56, 155.90, 147.73, 146.11, 141.75, 141.50,
133.35, 121.56, 121.20, 119.26, 116.83, 109.20, 108.70, 108.10,
101.26, 99.81, 93.76, 89.75, 70.80, 58.52, 42.49, 29.32;
HRMS (ESI) m/z calcd C25H22N3O6S+ [M+H]+ 492.1224,
found 492.1223.
BINAP,
(+/-)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl;
Boc, t-butoxycarbonyl; cGMP, cyclic guanosine monophos-
phate; Clapp, apparent clearane; Clh, hepatic clearane; Clint,
intrinsic clearane; CYP, cytochrome P450s; DCC, Dicyclohex-
ylcarbodiimide; DCM, dichloromethane; DIPEA, ethyldiiso-
propylamine; DMAP, 4-Dimethylaminopyridine; Eh, hepatic
extraction ratio; Fmoc-Cl, fluorenylmethoxycarbonyl chlo-
ride; hERG, the human Ether-a-go-go-Related Gene; MCT,
monocrotaline; mPAP, Mean Pulmonary Artery Pressure;
PAH, Pulmonary arterial hypertension; PDE, phos-
phodiesterase; PDE5, phosphodiesterase 5; RVHI, Index of
Right Ventricle Hypertrophy; SAR, structure-activity rela-
tionship; T1/2, half time; TFA, trifluoroacetic acid; TMS, tet-
ramethylsilane.
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Protein expression and purification. The recombinant
pET-PDE5A1 plasmid (catalytic domain, 535-860) was
subcloned and purified by protocols reported previous-
ly.14,22,23 After being transferred into the E. coli strain BL21
(Codonplus, Stratagene) for overexpression and growing
in TB medium at 37°C until OD600 = 3-4, 0.1 mM isopro-
pyl-β-D-thiogalactopyranoside (DTT) was added to in-
duce PDE5A1 protein expression. The induced cells were
collected after growing at 12°C for an additional 40-48 h.
The PDE5A1 protein was purified through a Ni-NTA col-
umn (QIAGEN), Q-column (GE Healthcare) and Super-
dex 100 column (GE Healthcare). A typical batch of cells
yielded over 10 mg of the PDE5A1 protein from 2 L of TB
cell culture with a purity greater than 95% as shown by
SDS−PAGE. The catalytic domains of PDE1B (10−487),
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
with similar protocols as previously reported.13,24-27 PDE6C
was purchased from BPS Bioscience.
ACKNOWLEDGMENT
This work was supported by the Natural Science Foundation
of China (81522041, 21572279, 81602955, 81703341, and
21702238), Science Foundation of Guangdong Province
(2016A030310144), the Fundamental Research Funds for the
Central Universities (Sun Yat-Sen University) (17ykpy03 and
17ykpy20), Medical Scientific Research Foundation of
Guangdong Province (A2016104), and 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 help with molecular
cloning, expression, purification, crystal structure, and bioas-
say of PDEs.
REFERENCES
ASSOCIATED CONTENT
1. Voelkel, N. F.; Quaife, R. A.; Leinwand, L. A.; Barst, R. J.;
McGoon, M. D.; Meldrum, D. R.; Dupuis, J.; Long, C. S.; Rubin, L.
J.; Smart, F. W.; Suzuki, Y. J.; Gladwin, M.; Denholm, E. M.; Gail,
D. B. Right Ventricular Function and Failure: Report of a National
Heart, Lung, and Blood Institute Working Group on Cellular and
Molecular Mechanisms of Right Heart Failure. Circulation 2006,114,
1883−1891.
2. Thenappan, T.; Shah, S. J.; Rich, S.; GombergꢀMaitland, M. A
USAꢀbased Registry for Pulmonary Arterial Hypertension: 1982ꢀ
2006. Eur. Respir. J. 2007,30, 1103−1110.
3. Badesch, D. B.; Abman, S. H.; Simonneau, G.; Rubin, L. J.;
McLaughlin, V. V. Medical therapy for Pulmonary Arterial
Hypertension: Updated ACCP Evidenceꢀbased Clinical Practice
Guidelines. Chest 2007,131, 1917−1928.
4. Montani, D.; Chaumais, M. C.; Savale, L.; Natali, D.; Price, L. C.;
Jais, X.; Humbert, M.; Simonneau, G.; Sitbon, O. Phosphodiesterase
Type 5 Inhibitors in Pulmonary Arterial Hypertension. Adv. Ther.
2009,26, 813−825.
5. Raja, S. G.; Raja, S. M. Treating Pulmonary Arterial Hypertension:
Current Treatments and Future Prospects. Ther. Adv. Chronic Dis.
2011,2, 359−370.
6. Giordano, D.; De Stefano, M. E.; Citro, G.; Modica, A.; Giorgi, M.
Expression of cGMPꢀbinding cGMPꢀspecific Phosphodiesterase
(PDE5) in Mouse Tissues and Cell Lines Using an Antibody Against
the Enzyme Aminoꢀterminal Domain. Biochim. Biophys. Acta, Mol.
Cell Res. 2001,1539, 16−27.
7. Rawson, D. J.; Ballard, S.; Barber, C.; Barker, L.; Beaumont, K.;
Bunnage, M.; Cole, S.; Corless, M.; Denton, S.; Ellis, D.; Floc'h, M.;
Foster, L.; Gosset, J.; Holmwood, F.; Lane, C.; Leahy, D.; Mathias, J.;
Maw, G.; Million, W.; Poinsard, C.; Price, J.; Russel, R.; Street, S.;
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
General Procedures for Bioassays, Crystallization trials,
Druglike Profile of Compound 3, General Procedures for the
Synthesis of Chromeno[2,3-c]pyrrol-9(2H)-ones, and 1H
NMR, 13C NMR, High-resolution mass spectra (HRMS) data
for tested compounds.
Molecular formula strings and some data (CSV).
Accession Codes
The atomic coordinates and structure factors have been
deposited into the RCSB Protein Data Bank with accession
number 5ZZ2 and 6ACB.
AUTHOR INFORMATION
Corresponding Author
*H.-B. L.: phone, +86-20-39943031; fax, +86-20-39943000; e-
mail, luohb77@mail.sysu.edu.cn.
ORCID
Hai-Bin Luo: 0000-0002-2163-0509
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
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Page 7 of 11
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Journal of Medicinal Chemistry
Watson, L. The Discovery of UKꢀ369003, A Novel PDE5 Inhibitor
18. Jørgensen, M. R.; Olsen, C. A.; Mellor, I. R.; Usherwood, P. N.
2
3
4
5
6
7
with the Potential for Oral Bioavailability and Doseꢀproportional
Pharmacokinetics. Bioorg. Med. Chem. 2012,20, 498−509.
8. Raja, S. G.; Danton, M. D.; MacArthur, K. J.; Pollock, J. C.
Treatment of Pulmonary Arterial Hypertension with Sildenafil: From
Pathophysiology to Clinical Evidence. J. Cardiothorac. Vasc. Anesth.
2006,20, 722−735.
R.; Witt, M.; Franzyk, H.; Jaroszewski, J. W. The Effects of
Conformational Constraints and Steric Bulk in the Amino Acid
Moiety of Philanthotoxins on AMPAR Antagonism. J. Med. Chem.
2005,48, 56−70.
19. Yu, Y.; Hu, Y.; Shao, W.; Huang, J.; Zuo, Y.; Huo, Y.; An, L.;
Du, J.; Bu, X. Synthesis of MultiꢀFunctionalized Chromeno[2,3ꢀ
c]pyrrolꢀ9(2H)ꢀones: Investigation and Application of Bakerꢀ
Venkataraman Rearrangement Involved Reactions Catalyzed by 4ꢀ
(Dimethylamino)pyridine. Eur. J. Org. Chem. 2011,2011,
4551−4563.
20. Kónya, K.; Langer, P.; Jordán, S.; Pajtás, D.; Patonay, T.
Synthesis of 6,7ꢀDibromoflavone and Its Regioselective
Diversification via Suzuki–Miyaura Reactions. Synthesis 2017,49,
1983−1992.
21. Baell, J. B.; Holloway, G. A. New Substructure Filters for
Removal of Pan Assay Interference Compounds (PAINS) from
Screening Libraries and for Their Exclusion in Bioassays. J. Med.
Chem. 2010,53, 2719−2740.
9. Humpl, T.; Reyes, J. T.; Holtby, H.; Stephens, D.; Adatia, I.
Beneficial Effect of Oral Sildenafil Therapy on Childhood Pulmonary
Arterial Hypertension. Circulation 2005,111, 3274−3280.
10. Galie, N.; Brundage, B. H.; Ghofrani, H. A.; Oudiz, R. J.;
Simonneau, G.; Safdar, Z.; Shapiro, S.; White, R. J.; Chan, M.;
Beardsworth, A.; Frumkin, L.; Barst, R. J. Tadalafil Therapy for
Pulmonary Arterial Hypertension. Circulation 2009,119, 2894−2903.
11. Archer, S. L.; Michelakis, E. D., Phosphodiesterase Type 5
Inhibitors for Pulmonary Arterial Hypertension.N. Engl. J.
Med.2009,36, 1864−1871.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
32
33
34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
12. Yanagisawa, R.; Kataoka, M.; Taguchi, H.; Kawakami, T.;
Tamura, Y.; Fukuda, K.; Yoshino, H.; Satoh, T. Impact of FirstꢀLine
Sildenafil Monotreatment for Pulmonary Arterial Hypertension. Circ.
J. 2012,76, 1245−1252.
13. Shang, N. N.; Shao, Y. X.; Cai, Y. H.; Guan, M.; Huang, M.; Cui,
W.; He, L.; Yu, Y. J.; Huang, L.; Li, Z.; Bu, X. Z.; Ke, H.; Luo, H.ꢀB.
Discovery of 3ꢀ(4ꢀHydroxybenzyl)ꢀ1ꢀ(thiophenꢀ2ꢀyl)chromeno[2,3ꢀ
22. Li, Z.; Cai, Y. H.; Cheng, Y. K.; Lu, X.; Shao, Y. X.; Li, X.; Liu,
M.; Liu, P.; Luo, H.ꢀB. Identification of Novel Phosphodiesteraseꢀ4D
Inhibitors Prescreened by Molecular Dynamicsꢀaugmented Modeling
and Validated by Bioassay. J. Chem. Inf. Model. 2013,53, 972−981.
23. Lin, T. T.; Huang, Y. Y.; Tang, G. H.; Cheng, Z. B.; Liu, X.; Luo,
H. B.; Yin, S. Prenylated Coumarins: Natural Phosphodiesteraseꢀ4
Inhibitors from Toddalia Asiatica. J. Nat. Prod. 2014,77, 955−962.
24. Park, S. J.; Ahmad, F.; Philp, A.; Baar, K.; Williams, T.; Luo, H.;
Ke, H.; Rehmann, H.; Taussig, R.; Brown, A. L.; Kim, M. K.;
Beaven, M. A.; Burgin, A. B.; Manganiello, V.; Chung, J. H.
Resveratrol Ameliorates Agingꢀrelated Metabolic Phenotypes by
Inhibiting cAMP Phosphodiesterases. Cell 2012,148, 421−433.
25. Shao, Y. X.; Huang, M.; Cui, W.; Feng, L. J.; Wu, Y.; Cai, Y.; Li,
Z.; Zhu, X.; Liu, P.; Wan, Y.; Ke, H.; Luo, H.ꢀB. Discovery of a
Phosphodiesterase 9A Inhibitor as a Potential Hypoglycemic Agent.
J. Med. Chem. 2014,57, 10304−10313.
c]pyrrolꢀ9(2H)ꢀone as
a Phosphodiesteraseꢀ5 Inhibitor and Its
Complex Crystal Structure. Biochem. Pharmacol. 2014,89, 86−98.
14. Wu, D.; Zhang, T.; Chen, Y.; Huang, Y.; Geng, H.; Yu, Y.;
Zhang, C.; Lai, Z.; Wu, Y.; Guo, X.; Chen, J.; Luo, H.ꢀB. Discovery
and Optimization of Chromeno[2,3ꢀc]pyrrolꢀ9(2H)ꢀones as Novel
Selective and Orally Bioavailable Phosphodiesterase 5 Inhibitors for
the Treatment of Pulmonary Arterial Hypertension. J. Med. Chem.
2017,60, 6622−6637.
15. Chang, J.; Chen, R.; Guo, R.; Dong, C.; Zhao, K. Synthesis,
Separation, and Theoretical Studies of Chiral Biphenyl Lignans (α‐
and β‐DDB). Helv. Chim. Acta 2003,86, 2239−2246.
26. Wang, H.; Yan, Z.; Yang, S.; Cai, J.; Robinson, H.; Ke, H. Kinetic
and Structural Studies of Phosphodiesteraseꢀ8A and Implication on
the Inhibitor Selectivity. Biochemistry 2008,47, 12760−12768.
27. Wang, H.; Liu, Y.; Chen, Y.; Robinson, H.; Ke, H. Multiple
Elements Jointly Determine Inhibitor Selectivity of Cyclic Nucleotide
Phosphodiesterases 4 and 7. J. Biol. Chem. 2005,280, 30949−30955.
16. Tran Van, C.; Nennstiel, D.; Scherkenbeck, J. Macrocyclic
Analogues of the Diuretic Insect Neuropeptide Helicokinin I Show
Strong Receptorꢀbinding. Bioorg. Med. Chem. 2015,23, 3278−3286.
17. Muller, J.; Feifel, S. C.; Schmiederer, T.; Zocher, R.; Sussmuth,
R. D. In Vitro Synthesis of New Cyclodepsipeptides of the PF1022ꢀ
type: Probing the AlphaꢀDꢀhydroxy Acid Tolerance of PF1022
Synthetase. Chembiochem 2009,10, 323−328.
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Figure 1. Optimization of chromeno[2,3-c]pyrrol-9(2H)-ones as novel PDE5 inhibitors with improved
inhibitory activity and pharmacodynamic profile.
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Figure 2. Co-crystal structure of the PDE5-11b and PDE5-17c complexes (PDB ID: 5ZZ2 and 6ACB). (A)
Surface model for 11b (yellow sticks) binding. The dotted lines represent hydrogen bonds. (B) Surface
model for 17c (green sticks) binding. The dotted lines represent hydrogen bonds.
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Figure 3. Pharmacodynamic effect of compound 3 on rats with PAH. (A) Reduction of pulmonary artery
pressure by 3 and sildenafil citrate, which was induced by monocrotaline (MCT). The data is shown as the
mean ± S.E.M (n = 6-9/group). Comparison of mPAP between groups: (##) p < 0.01 compared to the
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