Discovery of 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol- 9(2H)-one as a phosphodiesterase-5 inhibitor and its complex crystal structure

Article abstract of DOI:10.1016/j.bcp.2014.02.013

Phosphodiesterase-5 (PDE5) inhibitors have been approved for the treatment of erectile dysfunction and pulmonary hypertension, but enthusiasm on discovery of PDE5 inhibitors continues for their potential new applications. Reported here is discovery of a series of new PDE5 inhibitors by structure-based design, molecular docking, chemical synthesis, and enzymatic characterization. The best compound, 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57), has an IC₅₀ of 17 nM against the PDE5 catalytic domain and good selectivity over other PDE families. The crystal structure of the PDE5 catalytic domain in complex with 57 was determined at 2 A resolution and showed that 57 occupies the same pocket as other PDE5 inhibitors, but has a different binding pattern in detail. On the basis of the binding pattern of 57, a novel scaffold can be proposed as a candidate of PDE inhibitors.

Full text of DOI:10.1016/j.bcp.2014.02.013

Biochemical Pharmacology 89 (2014) 86–98
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Discovery of 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno
[2,3-c]pyrrol-9(2H)-one as a phosphodiesterase-5 inhibitor and its
complex crystal structure§
Na-Na Shang a,1, Yong-Xian Shao a,b,1, Ying-Hong Cai a,1, Matthew Guan b, Manna Huang b,
*
Wenjun Cui b, Lin He a, Yan-Jun Yu a, Lei Huang a, Zhe Li a, Xian-Zhang Bu a,
,
a,
Hengming Ke a,b, , Hai-Bin Luo
*
*
a School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, PR China
b Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill,
NC 27599-7260, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Phosphodiesterase-5 (PDE5) inhibitors have been approved for the treatment of erectile dysfunction and
pulmonary hypertension, but enthusiasm on discovery of PDE5 inhibitors continues for their potential
new applications. Reported here is discovery of a series of new PDE5 inhibitors by structure-based
design, molecular docking, chemical synthesis, and enzymatic characterization. The best compound, 3-
(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57), has an IC50 of 17 nM
against the PDE5 catalytic domain and good selectivity over other PDE families. The crystal structure
Received 31 December 2013
Accepted 14 February 2014
Available online 22 February 2014
Keywords:
Phosphodiesterase
Guanosine 30,50-cyclic monophosphate
˚
of the PDE5 catalytic domain in complex with 57 was determined at 2 A resolution and showed that 57
occupies the same pocket as other PDE5 inhibitors, but has a different binding pattern in detail. On the
basis of the binding pattern of 57, a novel scaffold can be proposed as a candidate of PDE inhibitors.
ß 2014 Elsevier Inc. All rights reserved.
PDE5 inhibitor
Crystal structure
Structure-based molecular design
1. Introduction
The most successful examples of this drug class are the PDE5
inhibitors that have been approved for the treatment of several
Cyclic nucleotide phosphodiesterases (PDEs) are sole enzymes
hydrolyzing cellular adenosine and guanosine 30,50-cyclic mono-
phosphate (cAMP and cGMP) [1–3]. Cyclic AMP and cGMP are the
second messengers mediating many physiological processes,
including cardiac and smooth muscle contraction, steroid hormone
function, adrenal hyperplasia, inflammation, axon guidance and
regeneration, memory, and circadian regulation [4–8]. For the
critical roles of cAMP and cGMP in cellular processes, PDE
inhibitors have been widely studied as therapeutics for treatment
of human diseases, such as diabetes, Alzheimer’s disease, asthma,
and erectile dysfunction [9–14].
diseases: sildenafil (Viagra), vardenafil (Levetra), tadalafil (Cialis),
avanafil (Stendra), udenafil (Zydena, Korean only), and mirodenafil
(Mvix, Korean only) (Fig. 1) for erectile dysfunction [15–19];
sildenafil (Revatio) and tadalafil (Adcirca) for pulmonary arterial
hypertension [9,20]; and tadalafil for benign prostatic hyperplasia
[21]. In addition, preclinic and clinic trials have shown that PDE5
inhibitors are potent for treatment of many other human diseases,
including lower urinary tract symptoms [22], benign prostatic
hyperplasia [23], cardiovascular diseases [24–27], heart failure and
coronary artery disease [28–30], second Raynaud’s phenomenon
[31], Duchenne muscular dystrophy [32], Peyronie’s disease [33],
§
PDB Accession Codes: The atomic coordinates and structure factors have been deposited into the RCSB Protein Data Bank with accession number 4MD6.
Chemical compounds studied in this article: tert-Butyl-2-(9-oxo-1-phenyl-2,9-dihydrochromeno[2,3-c]pyrrol-3-yl)acetate (ACP37; PubChem CID: 54770534); 3-Benzyl-1-
phenylchromeno[2,3-c]pyrrol-9(2H)-one (ACP42; PubChem CID: 49784789); 3-(4-(tert-Butoxy)benzyl)-1-phenylchromeno[2,3-c]pyrrol-9(2H)-one (ACP43; PubChem CID:
54770536); 3-(9-Oxo-1-phenyl-2,9-dihydrochromeno[2,3-c]pyrrol-3-yl)propanoic acid (ACP52; PubChem CID: 72725677); 3-(4-Hydroxybenzyl)-1-(thiophen-2-yl)chro-
meno[2,3-c]pyrrol-9(2H)-one (ACP57; PubChem CID: 71765666); 3-(4-Hydroxybenzyl)-1-phenylchromeno[2,3-c]pyrrol-9(2H)-one (ACP61; PubChem CID: 71738344); 3-
(4-Hydroxybenzyl)-2-methyl-1-phenylchromeno[2,3-c]pyrrol-9(2H)-one (ACP62; PubChem CID: 71738345).
*
Corresponding authors.
E-mail addresses: phsbxzh@mail.sysu.edu.cn (X.-Z. Bu), hke@med.unc.edu (H. Ke), luohb77@mail.sysu.edu.cn (H.-B. Luo).
1
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bcp.2014.02.013
0006-2952/ß 2014 Elsevier Inc. All rights reserved.

N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
87
Fig. 1. Chemical structures of PDE5 inhibitors.
neurological disorders [34]. Moreover, PDE5 inhibitors have been
(Chromeleon SR9 Build 2673); column, Acclaim1 120 C18
m, 4.6 ꢀ 250 mm; mobile phase, solvent A: water, solvent B:
,
used for anti-platelet therapy [35], wound healing [36], cognition
enhancement [37], and improvement of sperm quality [38].
However, PDE5 inhibitors have been shown to have some adverse
side effects such as vision disturbance [39] and hearing loss [40].
Thus, study of PDE5 inhibitors still attracts great attention of both
academic and industrial researchers, to discover new compounds
that may have potential new applications and also less severe side
effects.
Reported in this paper are the structure-based design, chemical
synthesis, and structural and enzymatic characterization of the
PDE5 inhibitors. The best compound 3-(4-hydroxybenzyl)-1-
(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57) exhibits
strong inhibitory affinity (IC50 = 17 nM) against PDE5 and good
selectivity over other PDE families. The crystal structure of the
PDE5A1 catalytic domain in complex with 57 was solved and the
molecular docking suggested a novel scaffold that may be used as a
molecular core for development of new PDE5 inhibitors.
5 m
CH3CN, flow rate, 1 ml/min; UV wavelength, 254 nm; tempera-
ture, ambient. The linear gradient procedure was used to purify
the compounds: 65%–99% B for 16–26, 33–49, and 60; 40% B + 1/
1000 TFA to 80% B for 50, 52, 54, and 56; 45% to 95% B for 51, 53, 55,
57–59, 61, and 62. Most compounds have purity >95%, as
calculated with the percentage peak area of the analyzed
compounds in the HPLC system. High resolution mass spectra
(HRMS) were recorded on Shimadzu LCMS-IT-TOF.
The syntheses of the new compounds 16–26, 33–36, 38–40, and
48–62 are diagramed in Scheme 1 and described as follows.
Compounds 37, 41–47 were previously reported [41].
2.1.1. 1-(2-Hydroxyphenyl)-3-phenylpropane-1,3-dione (1)
1.36 g 2-Hydroxyacetophenone (10 mmol) and 2.1 g benzoyl
chloride (15 mmol) were dissolved in pyridine (25 mL) with stir at
25 8C. When 2-hydroxyacetophenone was consumed as deter-
mined by using TLC in about 4–5 h, excess of water was added and
the pH of the mixture was adjusted to 5.0 with 10% HCl. The
precipitated product was collected by filtration, giving the ester
intermediate in the form of white powder. The crude product was
dissolved in 50 mL pyridine and 1–1.5eq KOH was added. After the
mixture was heated to 60 8C for 6–8 h, excess of water was added
and the pH of the solution was adjusted to 5.0 with 10% HCl. The
product was precipitated and collected by filtration. The yellow
powder was purified by recrystallization with alcohol to afford
2. Materials and methods
2.1. Reagents and synthesis
All reagents used were commercially available. Amino acids,
DMAP (dimethylaminopyridine) and DCC (dicyclohexylcarbodii-
mide) were bought from GL Biochem (Shanghai) Ltd. Acetophe-
nones, aryl chlorides and all other chemicals were bought from
Alfa Aesar (Tianjin) chemical Ltd. Amino acids were treated
1.99 g product 1, yield 83%; 1HNMR (400 MHz, CDCl3),
d 15.53 (1H),
as follows:
a
-amino groups were protected with Fmoc (9-
12.08 (1H), 7.93 (d, J = 7.5 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 7.55 (t,
J = 7.3 Hz, 1H), 7.47 (dd, J = 16.5, 8.1 Hz, 3H), 7.00 (d, J = 8.4 Hz, 1H),
6.92 (t, J = 7.6 Hz, 1H), 6.83 (1H).
fluorenylmethoxycarbonyl), the side chains of lysine and trypto-
phan were protected with Boc (tert-butyloxy carbonyl), and the
side chains of aspartic acid, glutamic acid and tyrosine were
t
protected as Bu (tert-butyl). Reactions were monitored by thin
2.1.2. 1-(Furan-2-yl)-3-(2-hydroxyphenyl)propane-1,3-dione (2)
1.36 g 2-Hydroxyacetophenone (10 mmol) and 1.95 g furan-2-
carbonyl chloride (15 mmol) were treated by the procedure used
for the synthesis of 1, giving 2.07 g 2 as yellow crystal, yield 90%;
layer chromatography (TLC) on a glass plate coated with silica gel
with fluorescent indicator (GF254). Column chromatography was
performed on silica gel (200–300 mesh). 1H NMR and 13C NMR
spectra were recorded using TMS as an internal standard with a
Burker BioSpin Ultrashield 400 NMR system at 400 MHz and
100 MHz, respectively. The purity of targeted compounds
was determined on a DIONEX Ultimate 3000 HPLC System
1H NMR (400 MHz, CDCl3),
d 15.09 (1H), 12.08 (1H), 7.78
(d, J = 8.1 Hz, 1H), 7.62 (s,1H), 7.47 (dd, J = 8.2, 7.3 Hz, 1H), 7.19
(d, J = 3.5 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.93 (dd, J = 8.0, 7.2 Hz,
1H), 6.79 (s,1H), 6.61–6.59 (m, 1H).

88
N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
Scheme 1. Syntheses of 16–26, 33–36, 38–40, 48–62. Only the non-H groups are described as follows. 16: R2 = CH3, R3 = furan-2-yl, R4 = CH2COOtBu; 17: R2 = OCH3,
R
R
R
3 = furan-2-yl, R4 = CH2CH2COOtBu; 18: R2 = CH3, R3 = furan-2-yl, R4 = CH2CH2COOtBu; 19: R2 = CH3, R3 = furan-2-yl, R4 = CH2C6H4(p-OtBu); 20: R3 = furan-2-yl,
4 = CH2C6H4(p-OtBu); 21: R3 = furan-2-yl, R4 = CH2CH2COOtBu; 22: R2 = Br, R3 = furan-2-yl, R4 = CH2CH2COOtBu; 23: R3 = 4-Br-C6H4, R4 = CH2C6H4(p-OtBu); 24:
2 = OCH3, R3 = thiophen-2-yl, R4 = CH2CH2COOtBu; 25: R2 = OCH3, R3 = thiophen-2-yl, R4 = CH2C6H4(p-OtBu); 26: R3 = C6H5, R4 = CH2CH2COOtBu; 33: R3 = naphthalen-2-
yl, R4 = CH2C6H5; 34: R3 = naphthalen-2-yl, R4 = CH2C6H4(p-OtBu); 35: R2 = OCH3, R3 = thiophen-2-yl, R4 = CH2COOtBu; 36: R3 = 4-Br-C6H4, R4 = CH2C6H5; 38: R3 = 4-F-C6H4,
4 = CH2COOtBu; 39: R2 = CH3, R3 = 4-F-C6H4, R4 = CH2COOtBu; 40: R3 = 4-F-C6H4, R4 = N-Boc-1H-indol-3-yl; 48: R2 = OCH3, R3 = thiophen-2-yl, R4 = CH2(CH2)3NHCOOtBu;
R
50: R2 = OCH3, R3 = thiophen-2-yl; 51: R2 = OCH3, R3 = thiophen-2-yl; 52: R3 = C6H5; 53: R2 = CH3, R3 = 4-F-C6H4; 54: R2 = Br, R3 = thiophen-2-yl; 55: R2 = Br, R3 = thiophen-2-
yl; 56: R2 = CH3, R3 = 4-F-C6H4; 57: R3 = thiophen-2-yl; 58: R3 = 4-OCH3-C6H4; 59: R3 = naphthalen-2-yl; 61: R3 = C6H5.
2.1.3. 1-(Furan-2-yl)-3-(2-hydroxy-5-methylphenyl)propane-1,3-
dione (3)
2.1.6. 1-(4-Fluorophenyl)-3-(2-hydroxyphenyl)propane-1,3-dione
(6)
1.5 g 1-(2-Hydroxy-5-methylphenyl)ethanone (10 mmol) and
1.95 g furan-2-carbonyl chloride (15 mmol) were treated by the
procedure used for the synthesis of 1, giving 1.83 g 3 as yellow
1.36 g 2-Hydroxyacetophenone (10 mmol) and 2.37 g 4-fluor-
inebenzoyl chloride (15 mmol) were treated by the procedure used
for the synthesis of 1, giving 2.2 g 6 as yellow crystal, yield 86%; 1H
powder, yield 75%; 1H NMR (400 MHz, CDCl3),
d
15.17 (1H), 11.89
NMR (400 MHz, CDCl3) d 15.60 (1H), 12.02 (1H), 7.98–7.91 (m, 2H),
(1H), 7.64 (dd, J = 1.6, 0.7 Hz, 1H), 7.55 (1H), 7.31–7.28 (m, 1H),
7.20–7.18 (m, 1H), 6.91 (d, J = 8.5 Hz, 1H), 6.78 (1H), 6.62 (dd,
J = 3.5, 1.7 Hz, 1H), 2.35 (3H).
7.76 (d, J = 8.1 Hz, 1H), 7.47 (dd, J = 8.3, 7.2 Hz, 1H), 7.18 (dd,
J = 12.4, 4.9 Hz, 2H), 7.00 (d, J = 8.4 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H),
6.78 (1H).
2.1.4. 1-(5-Bromo-2-hydroxyphenyl)-3-(furan-2-yl)propane-1,3-
dione (4)
2.1.7. 1-(4-Fluorophenyl)-3-(2-hydroxy-5-methylphenyl)propane-
1,3-dione (7)
2.15 g 1-(5-Bromo-2-hydroxyphenyl)ethanone (10 mmol) and
1.95 g furan-2-carbonyl chloride (15 mmol) were treated by the
procedure used for the synthesis of 1, giving 2.84 g 4 as yellow
1.5 g 1-(2-Hydroxy-5-methylphenyl)ethanone (10 mmol) and
2.37 g 4-fluorinebenzoyl chloride (15 mmol) were treated by the
procedure used for the synthesis of 1, giving 2.36 g 7 as yellow
powder, yield 87%; 1H NMR (400 MHz, CDCl3)
d 15.68 (1H), 11.83
powder, yield 92%; 1H NMR (400 MHz, CDCl3)
d
14.98 (1H), 12.02
(1H), 7.85 (s,1H), 7.65 (s,1H), 7.52 (d, J = 8.9 Hz, 1H), 7.21 (d,
J = 3.0 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.70 (s,1H), 6.63–6.60 (m,
1H).
(1H), 8.00–7.94 (m, 2H), 7.53 (s,1H),7.29 (d, J = 8.5 Hz, 1H), 7.18
(dd, J = 12.0, 5.2 Hz, 2H), 6.91 (d, J = 8.5 Hz, 1H),6.77 (s,1H), 2.34
(3H).
2.1.5. 1-(5-Bromo-2-hydroxyphenyl)-3-(thiophen-2-yl)propane-1,3-
dione (5)
2.1.8. 1-(Furan-2-yl)-3-(2-hydroxy-5-methoxyphenyl)propane-1,3-
dione (8)
2.15 g 1-(5-Bromo-2-hydroxyphenyl)ethanone (10 mmol) and
2.19 g thiophene-2-carbonylchloride (15 mmol) were treated by
the procedure used for the synthesis of 1, giving 3.12 g 5 as yellow
1.66 g 1-(2-Hydroxy-5-methoxyphenyl)ethanone (10 mmol)
and 1.95 g furan-2-carbonyl chloride (15 mmol,) were treated by
the procedure used for the synthesis of 1, giving 2.36 g 8 as yellow
powder, yield 91%; 1H NMR (400 MHz, CDCl3)
d 15.19 (s, 1H), 11.64
powder, yield 96%; 1H NMR (400 MHz, CDCl3)
d
15.59 (1H), 11.91
(1H), 7.85–7.83 (m, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 4.9 Hz,
1H), 7.52 (dd, J = 8.9, 2.4 Hz, 1H), 7.21–7.18 (m, 1H), 6.91 (dd,
J = 8.9, 3.0 Hz, 1H), 6.61 (s,1H).
(s, 1H), 7.62 (d, J = 0.9 Hz, 1H), 7.20 (d, J = 3.0 Hz, 1H), 7.19 (dd,
J = 3.5, 0.5 Hz, 1H), 7.10 (dd, J = 9.0, 3.0 Hz, 1H), 6.94 (d, J = 9.0 Hz,
1H), 6.72 (s, 1H), 6.61 (dd, J = 3.5, 1.7 Hz, 1H), 3.84 (s, 3H).

N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
89
2.1.9. 1-(2-Hydroxy-5-methoxyphenyl)-3-(thiophen-2-yl)propane-
2.1.16. tert-Butyl 2-(1-(furan-2-yl)-7-methyl-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)acetate (16)
1,3-dione (9)
1.66 g 1-(2-Hydroxy-5-methoxyphenyl)ethanone (10 mmol)
and 2.19 g thiophene-2-carbonyl chloride (15 mmol) were treated
by the procedure used for the synthesis of 1, the yielded crude
product 9 (2.34 g) was subjected to the following synthesis
without further purification and characterization.
244 mg 3 (1 mmol), 741 mg Fmoc-Asp(OtBu)-OH (1.8 mmol) and
49 mg DMAP (0.4 mmol) were dissolved in pyridine (10 mL), and
then 412 mg DCC (2 mmol) was added. The mixture was stirred at
room temperature about 3 h until 3 disappeared as monitored by
TLC. The reaction temperature was raised to 50 8C for 4–6 h, and a
major yellow spot could be observed by TLC. After the reaction
mixturewasevaporatedundervacuum,15 mLEtOAcwas addedand
side product DCU was filtered. The filtrate was evaporated and
subjected to column chromatography to afford 133 mg 16 as yellow
2.1.10. 1-(2-Hydroxy-5-methylphenyl)-3-(thiophen-2-yl)propane-
1,3-dione (10)
1.5 g 1-(2-Hydroxy-5-methylphenyl)ethanone (10 mmol) and
2.19 g thiophene-2-carbonylchloride (15 mmol) were treated by
the procedure used for the synthesis of 1, giving 1.95 g 10 as yellow
powder, yield 75%; 1H NMR (400 MHz, CDCl3)
d 15.74 (s, 1H), 11.75
(s, 1H), 7.80 (dd, J = 3.8, 1.1 Hz, 1H), 7.61 (dd, J = 5.0, 1.0 Hz, 1H),
7.49 (d, J = 1.3 Hz, 1H), 7.26–7.29 (m, 1H), 7.18 (dd, J = 4.9, 3.9 Hz,
1H), 6.90 (d, J = 8.5 Hz, 1H), 6.68 (s, 1H), 2.34 (s, 3H).
powder, yield 35%; 1H NMR (400 MHz, CDCl3)
d 9.76 (s, 1H), 8.10 (s,
1H), 7.87 (d, J = 3.4 Hz, 1H), 7.45 (s, 1H), 7.39 (dd, J = 8.5, 2.1 Hz, 1H),
7.23 (d, J = 8.5 Hz, 1H), 6.55 (dd, J = 3.4, 1.8 Hz, 1H), 3.82 (2H), 2.43
(3H), 1.53(9H);13CNMR(101 MHz,DMSO)
d173.47, 168.77, 154.26,
146.03, 142.56, 142.07, 134.94, 132.03, 125.67, 121.63, 118.33,
117.18, 112.21, 109.35, 106.98, 106.38, 80.57, 30.36, 27.71 ꢀ 3,
20.23; HRMS calcd. for C22H21NO5[M+H]+: 380.1492, found,
380.1509.
2.1.11. 1-(2-Hydroxy-4-methoxyphenyl)-3-(thiophen-2-yl)propane-
1,3-dione (11)
1.66 g 1-(2-Hydroxy-4-methoxyphenyl)ethanone (10 mmol)
and 2.19 g thiophene-2-carbonylchloride (15 mmol) were treated
by the procedure used for the synthesisof 1, giving 2.5 g 11 as yellow
2.1.17. tert-Butyl 3-(1-(furan-2-yl)-7-methoxy-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)propanote (17)
260 mg 8 (1 mmol), 766 mg Fmoc-Glu(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for the synthesis of 16 to afford 225 mg 17
powder, yield 91%; 1H NMR (400 MHz, CDCl3)
d 15.45 (s, 1H), 12.47
(s, 1H), 7.75 (dd, J = 3.8, 1.1 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.57 (dd,
J = 5.0, 1.1 Hz, 1H), 7.16 (dd, J = 5.0, 3.8 Hz, 1H), 6.57 (s, 1H), 6.48 (dd,
J = 8.9, 2.5 Hz, 1H), 6.45 (d, J = 2.5 Hz, 1H), 3.85 (s, 3H).
as yellow powder, yield 55%, 1H NMR (400 MHz, CDCl3)
d 9.86 (1H),
7.86 (d, J = 3.4 Hz, 1H), 7.75 (d, J = 3.1 Hz, 1H), 7.46–7.44 (m, 1H),
7.29 (d, J = 9.2 Hz, 1H), 7.20 (dd, J = 9.0, 2.9 Hz, 1H), 6.55 (dd, J = 2.9,
1.8 Hz, 1H), 3.91 (3H), 3.10 (2H), 2.68 (2H), 1.50 (9H); 13C NMR
2.1.12. 1-(4-Bromophenyl)-3-(2-hydroxyphenyl)propane-1,3-dione
(12)
(101 MHz, DMSO) d 173.28, 171.21, 154.64, 150.66, 146.19, 142.37,
1.36 g 2-Hydroxyacetophenone (10 mmol) and 3.28 g 4-bro-
mobenzoyl chloride (15 mmol) were treated by the procedure used
for the synthesis of 1, giving 2.39 g 12 as yellow crystal, yield 75%;
141.24, 122.34, 122.26, 118.75, 117.80, 112.18, 112.08, 108.99,
106.89, 106.10, 79.61, 55.46, 34.31, 27.57 ꢀ 3, 19.47; HRMS calcd
for C23H23NO6[M+H]+: 410.1598, found, 410.1610.
1H NMR (400 MHz, CDCl3)
d 15.49 (1H), 12.00 (1H), 7.78 (dd,
J = 13.9, 7.6 Hz, 3H), 7.63 (d, J = 8.0 Hz, 2H), 7.47 (t, J = 7.8 Hz, 1H),
7.01 (d, J = 8.4 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.81 (1H).
2.1.18. tert-Butyl 3-(1-(furan-2-yl)-7-methyl-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)propanoate (18)
244 mg 3 (1 mmol), 766 mg Fmoc-Glu(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
withtheprocedureusedfor16 toafford118 mg18as yellowpowder,
2.1.13. 1-(2-Hydroxyphenyl)-3-(thiophen-2-yl)propane-1,3-dione
(13)
1.36 g 2-Hydroxyacetophenone (10 mmol) and 2.19 g thio-
phene-2-carbonylchloride (15 mmol) were treated by the proce-
dure used for the synthesis of 1, giving 1.62 g 13 as yellow powder,
yield 30%; 1H NMR (400 MHz, CDCl3)
d9.82 (1H), 8.11 (s, 1H), 7.86 (d,
J = 3.4 Hz, 1H), 7.45 (d, J = 1.0 Hz, 1H), 7.40 (dd, J = 8.5, 2.1 Hz, 1H),
7.25 (d, J = 8.5 Hz, 1H), 6.56 (dd, J = 3.4, 1.8 Hz, 1H), 3.10 (2H), 2.68
(2H), 1.50 (9H); 13C NMR (101 MHz, DMSO)
d173.54, 171.20, 154.32,
yield 66%; 1H NMR (400 MHz, CDCl3)
d
15.65(1H), 11.96 (1H), 7.79
(d, J = 3.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 4.9 Hz, 1H),
7.46 (t, J = 7.8 Hz, 1H), 7.17 (t, J = 4.3 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H),
6.92 (t, J = 7.6 Hz, 1H), 6.69 (s,1H).
146.16, 142.39, 141.12, 134.86, 131.84, 125.64, 121.60, 118.02,
117.22, 112.18, 109.10, 106.41, 79.60, 34.31, 27.56 ꢀ 3, 20.23, 19.47;
HRMS calcd. for C23H23NO5[M+H]+:394.1649, found, 394.1644.
2.1.14. 1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)propane-1,3-
dione (14)
2.1.19. 3-(4-(tert-Butoxy)benzyl)-1-(furan-2-yl)-7-
methylchromeno[2,3-c]pyrrol-9(2H)-one (19)
1.36 g 2-Hydroxyacetophenone (10 mmol) and 2.56 g 4-
methoxybenzoyl chloride (15 mmol) were treated by the proce-
dure used for the synthesis of 1, giving 1.67 g 14 as yellow crystal,
244 mg 3 (1 mmol), 827 mg Fmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 106 mg 19 as yellow
yield 62%; 1H NMR (400 MHz, CDCl3)
d
15.76 (1H), 12.13 (1H),7.95
powder, yield 25%; 1H NMR (400 MHz, DMSO)
d 12.57 (1H), 7.95
(dd, J = 31.0, 7.8 Hz, 2H), 7.76 (d, J = 7.9 Hz, 1H), 7.44 (t, J = 7.6 Hz,
1H), 6.98 (d, J = 7.2 Hz, 3H), 6.91 (t, J = 7.4 Hz, 1H), 6.76 (s,1H), 3.88
(3H).
(s,1H), 7.84 (s,1H), 7.74 (d, J = 3.4 Hz, 1H), 7.54–7.49 (m, 1H), 7.36
(d, J = 8.5 Hz, 1H), 7.19 (d, J = 8.3 Hz, 2H), 6.90 (d, J = 8.3 Hz, 2H),
6.68–6.65 (m, 1H), 4.10 (2H), 2.40 (3H), 1.25 (9H); 13C NMR
(101 MHz, DMSO) d 173.59, 154.38, 153.28, 146.13, 142.45, 141.22,
2.1.15. 1-(2-Hydroxyphenyl)-3-(naphthalen-2-yl)propane-1,3-dione
(15)
134.92, 134.14, 131.98, 128.55 ꢀ 2, 125.65, 123.67 ꢀ 2, 121.67,
118.01, 117.32, 112.79, 112.22, 109.20, 106.58, 77.62, 28.47 ꢀ 3,
20.25; HRMS calcd. for C27H25NO4[M+H]+: 428.1856, found,
428.1863.
1.36 g 2-Hydroxyacetophenone (10 mmol) and 2.85 g 2-
naphthoyl chloride (15 mmol) were treated by the procedure
used for the synthesis of 1, giving 2.4 g 15 as brown powder, yield
83%; 1H NMR (400 MHz, CDCl3)
d
15.62 (1H), 12.11 (1H), 8.49 (s,
2.1.20. 3-(4-(tert-Butoxy)benzyl)-1-(furan-2-yl)chromeno[2,3-
c]pyrrol-9(2H)-one (20)
230 mg 2 (1 mmol), 828 mg Fmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
1H),7.96–7.93 (m, 1H), 7.91 (s,2H), 7.87 (d, J = 7.6 Hz, 1H), 7.82 (d,
J = 7.5 Hz, 1H), 7.60–7.52 (m, 2H), 7.46 (t, J = 7.3 Hz, 1H), 7.01 (d,
J = 8.3 Hz, 1H), 6.96–6.91 (m, 2H).

90
N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
with the procedure used for 16 to afford 194 mg 20 as yellow
powder, yield 47%; 1H NMR (400 MHz, DMSO)
12.60 (1H), 8.19
2.1.25. 3-(4-(tert-Butoxy)benzyl)-7-methoxy-1-(thiophen-2-
yl)chromeno[2,3-c]pyrrol-9(2H)-one (25)
d
(dd, J = 7.9, 1.6 Hz, 1H), 7.83–7.81 (m, 1H), 7.79–7.76 (m, 1H), 7.71
(dt, J = 6.5, 2.7 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.37–7.31 (m, 1H),
7.21 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 6.69 (dd, J = 3.4,
276 mg 9 (1 mmol), 829 mg Fmoc-Tyr(tBu)-OH(1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 353 mg 25 as yellow
1.8 Hz, 1H), 4.12 (2H), 1.25 (9H); 13C NMR (101 MHz, DMSO)
d
powder, yield 77%; 1H NMR (400 MHz, DMSO)
d 12.35 (1H), 8.02
173.51, 156.18, 153.28, 146.09, 142.51, 141.13, 134.12, 133.98,
128.56 ꢀ 2, 126.21, 123.69 ꢀ 2, 122.87, 122.04, 118.18, 117.51,
112.95, 112.24, 109.31, 106.50, 77.60, 28.45 ꢀ 3; HRMS calcd. for
(d, J = 3.5 Hz, 1H), 7.57 (dd, J = 13.4, 3.9 Hz, 2H), 7.43 (d, J = 9.1 Hz,
1H), 7.31 (dd, J = 9.0, 3.0 Hz, 1H), 7.20–7.13 (m, 3H), 6.91 (d,
J = 8.4 Hz, 2H), 4.11 (1H), 3.85 (3H), 1.25 (9H); 13C NMR (101 MHz,
C
26H23NO4[M+H]+: 414.1700, found, 414.1719.
CDCl3) d 175.34, 155.11, 154.23, 151.58, 142.37, 133.19, 132.81,
128.88 ꢀ 2, 127.84, 126.35, 125.23, 124.52 ꢀ 2, 123.28, 122.69,
122.56, 118.63, 111.71, 107.98, 106.77, 78.51, 55.77, 29.32,
28.82 ꢀ 3; HRMS calcd. for C27H25NO4S[M+H]+: 460.1577, found,
460.1564.
2.1.21. tert-Butyl 3-(1-(furan-2-yl)-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)propanoate (21)
230 mg 2 (1 mmol), 766 mg Fmoc-Glu(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 170 mg 21 as brown
2.1.26. tert-Butyl 3-(9-oxo-1-phenyl-2,9-dihydrochromeno[2,3-
c]pyrrol-3-yl)propanoate (26)
powder, yield 45%; 1H NMR (400 MHz, DMSO)
d 12.36 (1H), 8.15
(dd, J = 7.9, 1.5 Hz, 1H), 7.80 (s, 1H), 7.74–7.67 (m, 2H), 7.46 (d,
J = 8.2 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 6.66 (dd, J = 3.3, 1.8 Hz, 1H),
3.02 (2H), 2.68 (2H), 1.31 (9H); 13C NMR (101 MHz, DMSO)
173.96, 171.69, 156.61, 146.56, 143.00, 141.50, 134.49, 126.68,
123.30, 122.44, 118.67, 117.96, 112.88, 112.71, 109.71, 106.80,
80.16, 34.79, 28.07 ꢀ 3, 19.97; HRMS calcd. for C22H21NO5[M+H]+:
380.1492, found, 380.1506.
240 mg
1
(1 mmol),
766 mg
Fmoc-Glu(OtBu)-OH
(1.8 mmol), 49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol)
were treated with the procedure used for 16 to afford 318 mg
26 as yellow powder, yield 82%; 1H NMR (400 MHz, DMSO)
d
12.16 (1H), 8.11 (dd, J = 29.6, 7.1 Hz, 3H), 7.68 (d, J = 6.7 Hz, 1H),
7.46 (s, 3H), 7.33 (dd, J = 14.5, 7.0 Hz, 2H), 3.04 (2H), 2.70 (2H),
1.32 (9H); 13C NMR (101 MHz, DMSO)
d 174.01, 171.23, 155.91,
d
141.56, 133.84, 130.96, 128.08 ꢀ 2, 127.66 ꢀ 2, 127.56, 127.37,
126.37, 122.61, 121.92, 117.32, 112.58, 107.14, 79.67, 34.34,
27.58 ꢀ 3, 19.56; HRMS calcd. for C24H23NO4[M+H]+: 390.1700,
found, 390.1697.
2.1.22. tert-Butyl 3-(7-bromo-1-(furan-2-yl)-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)propanoate (22)
309 mg 4 (1 mmol), 766 mg Fmoc-Glu(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 342 mg 22 as yellow
27–32 were used as intermediates and not characterized.
powder, yield 75%; 1H NMR (400 MHz, DMSO)
d
12.19 (1H), 8.18 (d,
2.1.27. 3-(4-(tert-Butoxy)benzyl)-1-(4-fluorophenyl)-7-
methylchromeno[2,3-c]pyrrol-9(2H)-one (27)
272 mg 7 (1 mmol), 828 mg Fmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 254 mg 27.
J = 2.5 Hz, 1H), 7.83 (dd, J = 7.6, 3.7 Hz, 2H), 7.71 (d, J = 3.1 Hz, 1H),
7.44 (d, J = 8.9 Hz, 1H), 6.68 (dd, J = 3.3, 1.7 Hz, 1H), 3.00 (2H), 2.89
(2H), 1.33 (9H); 13C NMR (101 MHz, DMSO)
d 171.99, 171.16,
155.08, 145.85, 142.67, 140.80, 136.36, 128.24, 123.57, 120.06,
118.55, 114.72, 112.66, 112.23, 109.48, 105.80, 79.65, 34.19,
27.57 ꢀ 3, 19.41; HRMS calcd. for C22H20NO5Br[M+H]+: 458.0598,
found, 458.0592.
2.1.28. tert-Butyl3-(7-bromo-9-oxo-1-(thiophen-2-yl)-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl) propanoate (28)
325 mg
5
(1 mmol) and 766 mg Fmoc-Glu(OtBu)-OH
2.1.23. 1-(4-Bromophenyl)-3-(4-(tert-butoxy)benzyl)chromeno[2,3-
c]pyrrol-9(2H)-one (23)
(1.8 mmol), 48.8 mg DMAP (0.4 mmol) and 412 mg DCC
(2 mmol) were treated with the procedure used for 16 to afford
142 mg 28.
319 mg 12 (1 mmol), 828 mg Fmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 280 mg 23 as yellow
2.1.29. 7-Bromo-3-(4-(tert-butoxy)benzyl)-1-(thiophen-2-
yl)chromeno[2,3-c]pyrrol-9(2H)-one (29)
325 mg 5 (1 mmol), 828 mg Fmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 142 mg 29.
powder, yield 56%; 1H NMR (400 MHz, DMSO)
d 12.47(1H), 8.13
(dd, J = 32.4, 7.9 Hz, 3H), 7.69 (dd, J = 18.7, 7.7 Hz, 3H), 7.49 (d,
J = 8.5 Hz, 1H), 7.34 (t, J = 7.1 Hz, 1H), 7.21 (d, J = 8.2 Hz, 2H), 6.91
(d, J = 8.2 Hz, 2H), 4.14 (2H), 1.25 (9H); 13C NMR (101 MHz, DMSO)
d
174.10, 155.91, 153.36, 141.91, 133.99, 133.89, 131.02 ꢀ 2,
130.06, 129.49 ꢀ 2, 128.54 ꢀ 2, 126.40, 126.03, 123.72 ꢀ 2, 122.79,
121.90, 120.72, 117.40, 113.68, 107.65, 77.62, 28.52, 28.46 ꢀ 3;
HRMS calcd. for C28H24NO3Br[M+H]+: 502.1012, found, 502.0999.
2.1.30. tert-Butyl 3-(1-(4-fluorophenyl)-7-methyl-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl) propanoate (30)
272 mg 7 (1 mmol), 766 mg Fmoc-Glu(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 193 mg 30.
2.1.24. tert-Butyl 3-(7-methoxy-9-oxo-1-(thiophen-2-yl)-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)propnoate (24)
276 mg 9 (1 mmol), 766 mg Fmoc-Glu(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 297 mg 24 as yellow
2.1.31. 3-(4-(tert-Butoxy)benzyl)-1-(thiophen-2-yl)chromeno[2,3-
c]pyrrol-9(2H)-one (31)
246 mg 13 (1 mmol), 828 mg Fmoc-Tyr(tBu)-OH(1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 280 mg 31.
powder, yield 70%; 1H NMR (400 MHz, DMSO)
d 12.15 (1H), 8.00
(d, J = 3.6 Hz, 1H), 7.57 (dd, J = 8.6, 4.0 Hz, 2H), 7.44 (d, J = 9.1 Hz,
1H), 7.32 (dd, J = 9.1, 3.1 Hz, 1H), 7.17–7.13 (m, 1H), 3.02 (2H),
2.69 (2H), 1.33 (9H); 13C NMR (101 MHz, DMSO)
d
173.57, 171.18,
2.1.32. 3-(4-(tert-Butoxy)benzyl)-1-(4-
154.64, 150.58, 141.23, 133.25, 127.25, 126.05, 125.94, 122.41,
122.17, 121.37, 118.71, 112.07, 107.02, 106.59, 79.71, 55.50,
34.36, 27.61 ꢀ 3, 19.54; HRMS calcd. for C23H23NO5S[M+H]+:
426.1370, found, 426.1378.
methoxyphenyl)chromeno[2,3-c]pyrrol-9(2H)-one (32)
270 mg 14 (1 mmol), 828 mgFmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 100 mg 32.

N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
91
2.1.33. 3-benzyl-1-(naphthalen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-
one (33)
1H), 7.31 (dd, J = 12.1, 5.7 Hz, 3H), 3.78 (2H), 1.43 (9H); 13C NMR
(101 MHz, DMSO) 173.97, 168.78, 162.92, 160.48, 155.84, 142.42,
d
290 mg 15 (1 mmol), 697 mg Fmoc-Phe-OH (1.8 mmol), 49 mg
DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated with the
procedure used for 16 to afford 148 mg 33 as yellow powder, yield
133.98, 129.82, 129.74, 127.37, 127.34, 126.82, 126.38, 122.79,
121.86, 117.28, 115.18, 114.97, 107.34, 107.05, 80.70, 30.45,
27.70 ꢀ 3; HRMS calcd. for C23H20NO4F [M+H]+: 394.1449, found,
394.1464.
37%; 1H NMR (400 MHz, DMSO)
d 12.56 (1H), 8.66 (s, 1H), 8.25–
8.19 (m, 2H), 8.02–7.92 (m, 3H), 7.73 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H),
7.57–7.49 (m, 3H), 7.38–7.32 (m, 5H), 7.21 (dd, J = 9.4, 4.3 Hz, 1H),
2.1.38. tert-Butyl 2-(1-(4-fluorophenyl)-7-methyl-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)acetate (39)
4.23 (2H); 13C NMR (101 MHz, DMSO)
d 174.10, 155.98, 141.96,
139.48, 133.95, 132.76, 132.24, 128.49 ꢀ 2, 128.10 ꢀ 2, 128.08,
127.51, 127.42, 127.38, 126.46, 126.43, 126.29, 126.22, 126.17,
125.96, 122.79, 122.03, 117.42, 113.40, 107.71, 29.35; HRMS calcd.
for C28H19NO2 [M+H]+: 402.1489, found, 402.1506.
272 mg 7 (1 mmol), 741 mg Fmoc-Asp(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 260 mg 39 as yellow
powder, yield 64%; 1H NMR (400 MHz, DMSO)
d 12.31 (1H), 8.13
(dd, J = 8.4, 5.6 Hz, 2H), 7.93 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.32
(dd, J = 16.3, 8.4 Hz, 3H), 3.77 (2H), 2.38 (3H), 1.43 (9H); 13C NMR
2.1.34. 3-(4-(tert-Butoxy)benzyl)-1-(naphthalen-2-
yl)chromeno[2,3-c]pyrrol-9(2H)-one (34)
(101 MHz, DMSO) d 174.04, 168.80, 162.88, 160.44, 154.06, 142.52,
290 mg 15 (1 mmol), 828 mg Fmoc-Tyr(tBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 203 mg 34 as yellow
134.94, 131.87, 129.79, 129.71, 127.44, 127.41, 126.64, 125.83,
121.50, 117.09, 115.17, 114.96, 107.17, 80.69, 30.46, 27.71 ꢀ 3,
20.28; HRMS calcd. for C24H22NO4F [M+H]+: 408.1606, found,
408.1605.
powder, yield 43%; 1H NMR (400 MHz, DMSO)
d 12.55 (1H), 8.69
(1H), 8.28–8.20 (m, 2H), 8.03–7.92 (m, 3H), 7.71 (d, J = 7.0 Hz, 1H),
7.58–7.47 (m, 3H), 7.40–7.33 (m, 1H), 7.25 (d, J = 7.6 Hz, 2H), 6.93
(d, J = 7.7 Hz, 2H), 4.19 (2H), 1.25 (9H); 13C NMR (101 MHz, DMSO)
2.1.39. tert-Butyl 3-((1-(4-fluorophenyl)-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)methyl)-1H-indole-1-
carboxylate (40)
d
174.10, 155.98, 153.36, 141.94, 134.04, 133.95, 132.76, 132.23,
128.58 ꢀ 2, 128.48, 128.07, 127.51, 127.38, 126.47, 126.43, 126.27,
126.22, 125.94, 123.74 ꢀ 2, 122.78, 122.04, 117.41, 113.55, 107.70,
77.63, 28.60, 28.47 ꢀ 3; HRMS calcd. for C32H2NO3[M+H]+:
474.2064, found, 474.2051.
258 mg 6 (1 mmol), 948 mg Fmoc-Trp(BOC)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 213 mg 40 as yellow
powder, yield 42%; 1H NMR (400 MHz, DMSO)
d 12.36 (1H), 8.16
(dd, J = 7.9, 1.5 Hz, 1H), 8.11 (dd, J = 8.8, 5.5 Hz, 2H), 8.03 (d,
J = 8.1 Hz, 1H), 7.74 (t, J = 6.3 Hz, 2H), 7.56–7.50 (m, 2H), 7.36–7.28
(m, 4H), 7.24 (t, J = 7.2 Hz, 1H), 4.21 (2H), 1.61 (9H);13C NMR
2.1.35. tert-Butyl 2-(7-methoxy-9-oxo-1-(thiophen-2-yl)-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)acetate (35)
276 mg 9 (1 mmol), 741 mg Fmoc-Asp(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 279 mg 35 as yellow
(101 MHz, DMSO) d 174.05, 155.95, 149.03, 141.32, 134.78, 134.03,
129.99 ꢀ 2, 129.91 ꢀ 2, 129.68, 127.37, 126.67, 126.38, 124.44,
123.37, 122.80, 122.57, 121.88, 119.11, 118.24, 117.32, 115.09,
114.87, 114.73, 111.86, 107.14, 83.61, 27.63 ꢀ 3, 19.21; HRMS
calcd. for C31H25N2O4F [M+H]+: 509.1871, found, 509.1859.
powder, yield 68%; 1H NMR (400 MHz, DMSO)
d 12.31 (1H), 8.03 (d,
J = 2.8 Hz, 1H), 7.61–7.56 (m, 2H), 7.43 (d, J = 9.1 Hz, 1H), 7.32 (dd,
J = 9.0, 3.1 Hz, 1H), 7.18–7.14 (m, 1H), 3.85 (3H), 3.77 (2H), 1.44
(9H); 13C NMR (101 MHz, DMSO)
d
173.49, 168.77, 154.72, 150.46,
2.1.40. tert-Butyl (4-(7-methoxy-9-oxo-1-(thiophen-2-yl)-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)butyl) carbamate (48)
276 mg 9 (1 mmol), 843 mg Fmoc-Lys(BOC)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 351 mg 48 as brown
powder, yield 75%; 1H NMR (400 MHz, DMSO)
d 12.14 (1H), 8.00 (s,
142.18, 133.10, 127.38, 126.26, 126.16, 122.52, 122.19, 121.82,
118.69, 106.98, 106.82, 106.52, 80.72, 55.50, 30.42, 27.72 ꢀ 3;
HRMS calcd. for C22H21NO5S[M+H]+: 412.1213, found, 412.1228.
2.1.36. 3-Benzyl-1-(4-bromophenyl)chromeno[2,3-c]pyrrol-9(2H)-
one (36)
1H), 7.59 (s, 1H), 7.55 (d, J = 3.4 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.31
(d, J = 9.0 Hz, 1H), 7.16 (s, 1H), 6.78 (1H), 3.85 (3H), 2.98 (2H), 2.79
319 mg 12 (1 mmol), 697 mg Fmoc-Phe-OH (1.8 mmol), 49 mg
DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated with the
procedure used for 16 to afford 270 mg 36 as yellow powder, yield
(2H), 1.68 (2H), 1.47 (2H), 1.36 (9H); 13C NMR (101 MHz, DMSO)
d
173.65, 155.55, 154.59, 150.66, 141.04, 133.35, 127.21, 125.89,
125.79, 122.37, 122.18, 120.90, 118.75, 113.86, 106.94, 106.64,
77.28, 55.49, 29.13, 28.20 ꢀ 3, 26.39, 23.15; HRMS calcd. for
63%; 1H NMR (400 MHz, DMSO)
d 12.48 (1H), 8.17 (dd, J = 7.9,
1.5 Hz, 1H), 8.09 (d, J = 8.6 Hz, 2H), 7.74–7.69 (m, 1H), 7.67 (d,
J = 8.6 Hz, 2H), 7.48 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.32
(d, J = 4.1 Hz, 4H), 7.21 (dd, J = 8.4, 4.6 Hz, 1H), 4.19 (2H); 13C NMR
C
25H28N2O5S [M+H]+: 469.1792, found, 469.1793.
(101 MHz, DMSO)
d
174.58, 156.40, 142.41, 139.81, 134.51,
2.1.41. 1-([1,10-biphenyl]-4-yl)-3-(4-(tert-
131.52 ꢀ 2, 130.54, 130.00 ꢀ 2, 128.96 ꢀ 2, 128.54 ꢀ 2, 126.89,
126.67, 126.54, 123.30, 122.38, 121.24, 117.91, 114.03, 108.14,
29.78; HRMS calcd. for C24H16NO2Br [M+H]+: 430.0437, found,
430.0431.
The synthesis of compounds 37, 41–47 was previously reported
[41].
butoxy)benzyl)chromeno[2,3-c]pyrrol-9(2H)-one (49)
250 mg 23 (0.5 mmol), 79 mg phenyl boronic acid (0.65 mmol)
and 25 mg dichloro(1,10-bis(diphenylphosphino)ferrocene) palla-
dium(II)-dichloromethane adduct (PdCl2 (dppf), 0.03 mmol) were
dissolved in 2 ml dioxane, and 2 ml 2 M K2CO3 was added. React in
microwave at 150 8C for 20 min, monitored by using TLC. Then
20 ml ethyl acetate was added and washed with water, organic
layer was evaporated and subjected to column chromatography to
afford 205 mg 49 as yellow powder, yield 82%; 1H NMR (400 MHz,
2.1.37. tert-Butyl 2-(1-(4-fluorophenyl)-9-oxo-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)acetate (38)
258 mg 6 (1 mmol), 741 mg Fmoc-Asp(OtBu)-OH (1.8 mmol),
49 mg DMAP (0.4 mmol) and 412 mg DCC (2 mmol) were treated
with the procedure used for 16 to afford 122 mg 38 as yellow
DMSO) d 12.45 (1H), 8.22 (dd, J = 19.5, 8.0 Hz, 3H), 7.82–7.69 (m,
5H), 7.50 (t, J = 7.7 Hz, 3H), 7.43–7.32 (m, 2H), 7.23 (d, J = 8.2 Hz,
2H), 6.92 (d, J = 8.2 Hz, 2H), 4.16 (2H), 1.25 (9H); 13C NMR
powder, yield 31%; 1H NMR (400 MHz, DMSO)
(dd, J = 8.6, 5.8 Hz, 3H), 7.70 (t, J = 7.1 Hz, 1H), 7.45 (d, J = 8.3 Hz,
d
12.34 (1H), 8.14
(101 MHz, DMSO)
d 174.06, 155.93, 153.35, 141.89, 139.52, 139.04,
134.05, 133.91, 130.00, 128.91 ꢀ 2, 128.56 ꢀ 2, 128.09 ꢀ 2, 127.50,

92
N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
127.09, 126.48 ꢀ 2, 126.27 ꢀ 2, 123.73 ꢀ 2, 122.74, 122.04, 117.38,
113.35, 107.56, 77.62, 28.56, 28.47 ꢀ 3; HRMS calcd. for C34H2NO3
[MꢁH]ꢁ: 498.2075, found, 498.2068.
1H), 7.16 (dd, J = 8.7, 5.0 Hz, 1H), 3.02 (2H), 2.72 (2H); 13C NMR
(101 MHz, DMSO) 173.47, 172.34, 155.02, 140.72, 136.40, 132.85,
128.30, 127.37, 126.44, 126.37, 123.50, 122.03, 120.11, 114.68,
112.97, 106.40, 33.03, 19.33; HRMS calcd. for C18H12NO4S
Br[M+H]+: 417.9743, found, 417.9741.
d
2.1.42. 3-(7-methoxy-9-oxo-1-(thiophen-2-yl)-2,9-
dihydrochromeno[2,3-c]pyrrol-3-yl)propanoic acid (50)
212 mg 24 (0.5 mmol) was dissolved in 20 ml CH2Cl2 and 5 ml
TFA was dropped in. The mixture was stirred at room temperature
until reactant disappeared as monitored by using TLC. Then the
reaction mixture was evaporated in vacuum and subjected to
column chromatography to yield 107 mg 50 as red powder, yield
2.1.47. 7-Bromo-3-(4-hydroxybenzyl)-1-(thiophen-2-
yl)chromeno[2,3-c]pyrrol-9(2H)-one (55)
254 mg 29 (0.5 mmol) was treated with the procedure used for
50 to afford 72 mg 55 as yellow powder, yield 32%; 1H NMR
(400 MHz, DMSO)
d 12.41 (1H), 9.21 (1H), 8.20 (d, J = 2.4 Hz, 1H),
58%; 1H NMR (400 MHz, DMSO)
d
12.23 (1H), 12.16 (1H), 8.00 (s,
8.02 (d, J = 3.2 Hz, 1H), 7.84 (dd, J = 8.8, 2.4 Hz, 1H), 7.58 (d,
J = 4.9 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.19–7.14 (m, 1H), 7.08 (d,
J = 8.3 Hz, 2H), 6.70 (d, J = 8.3 Hz, 2H), 4.04 (2H); 13C NMR
1H), 7.58 (s, 2H), 7.44 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.15
(s, 1H), 3.85 (3H), 3.04 (2H), 2.72 (2H); 13C NMR (101 MHz, DMSO)
d
173.59, 173.49, 154.63, 150.62, 141.15, 133.25, 127.24, 126.02,
(101 MHz, DMSO) d 172.39, 155.73, 155.06, 140.87, 136.41,
125.94, 122.39, 122.17, 121.23, 118.76, 112.37, 106.97, 106.67,
55.50, 33.11, 19.39; HRMS calcd. for C19H15NO5S [M+H]+:
370.0744, found, 370.0759.
132.88, 129.29, 128.94 ꢀ 2, 128.35, 127.37, 126.43, 123.59,
122.08, 120.11, 115.20 ꢀ 2, 114.71, 113.69, 106.51, 28.28; HRMS
calcd. for C22H14NO3SBr[M+H]+: 451.9951, found, 451.9961.
2.1.43. 3-(4-hydroxybenzyl)-7-methoxy-1-(thiophen-2-
2.1.48. 3-(1-(4-Fluorophenyl)-7-methyl-9-oxo-2,9-
yl)chromeno[2,3-c]pyrrol-9(2H)-one (51)
dihydrochromeno[2,3-c]pyrrol-3-yl)propanoic acid (56)
210 mg 30 (0.5 mmol) was treated with the procedure used for
50 to afford 63.8 mg 56 as yellow powder, yield 35%; 1H NMR
The tert-butyl protection of 25 (229 mg, 0.5 mmol) was
removed by dissolving 25 in 20 ml CH2Cl2 and 5 ml TFA with
stir at room temperature until the reactant disappeared as
monitored by TLC. The reaction mixture was evaporated in
vacuum and subjected to column chromatography to afford
91 mg 51 as yellow powder, yield 45%; 1H NMR (400 MHz, MeOD)
(400 MHz, DMSO)
7.53 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.31 (t, J = 8.8 Hz,
2H), 3.06 (2H), 2.74 (2H), 2.40 (3H); 13C NMR (101 MHz, DMSO)
d 12.23 (2H), 8.15–8.09 (m, 2H), 7.93 (s, 1H),
d
174.13, 173.55, 162.78, 160.34, 154.16, 141.45, 134.80, 131.64,
129.80, 129.72, 127.58, 127.55, 126.02, 125.75, 121.46, 117.17,
115.05, 114.84, 112.64, 107.19, 33.10, 20.27, 19.38; HRMS calcd.
for C21H16NO4F[M+H]+: 366.1136, found, 366.1137.
d
7.92 (d, J = 3.7 Hz, 1H), 7.66 (d, J = 3.0 Hz, 1H), 7.41 (d, J = 5.1 Hz,
1H), 7.35 (d, J = 9.1 Hz, 1H), 7.26 (dd, J = 9.1, 3.1 Hz, 1H), 7.10 (d,
J = 8.6 Hz, 3H), 6.72 (d, J = 8.4 Hz, 2H), 4.09 (2H), 3.88 (3H); 13C
NMR (101 MHz, DMSO)
d 173.61, 155.69, 154.67, 150.62, 141.25,
133.26, 129.52, 128.92 ꢀ 2, 127.26, 126.05, 125.97, 122.43, 122.23,
121.26, 118.79, 115.19 ꢀ 2, 113.11, 106.98, 106.73, 55.51, 28.32;
HRMS calcd. for C23H17NO4S [M+H]+: 404.0951, found, 404.0951.
2.1.49. 3-(4-Hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-
c]pyrrol-9(2H)-one (57)
tert-Butyl protection of 31 (215 mg, 0.5 mmol) was removed
with the procedure used for 51 to afford 112 mg of 57 as red
crystal, yield 60%; 1H NMR (400 MHz, DMSO)
d 12.33 (1H), 9.21
(1H), 8.16 (dd, J = 7.9, 1.4 Hz, 1H), 8.04–8.00 (m, 1H), 7.74–7.67 (m,
1H), 7.56 (d, J = 5.0 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.33 (t,
J = 7.5 Hz, 1H), 7.15 (dd, J = 5.0, 3.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H),
6.69 (d, J = 8.5 Hz, 2H), 4.05 (2H); 13C NMR (101 MHz, DMSO)
d
173.80, 156.06, 155.71, 141.03, 133.99, 133.16, 129.48, 128.93 ꢀ 2,
127.30, 126.27, 126.16, 122.79, 121.93, 121.65, 117.45, 115.20 ꢀ 2,
113.40, 106.98, 28.32; HRMS calcd. for C22H15NO3S [MꢁH]ꢁ:
372.0700, found, 372.0703.
2.1.44. 3-(9-Oxo-1-phenyl-2,9-dihydrochromeno[2,3-c]pyrrol-3-
yl)propanoic acid (52)
195 mg 26 (0.5 mmol) was treated with the procedure used for
50 to afford 117 mg 52 as yellow powder, yield 70%; 1H NMR
(400 MHz, DMSO)
(t, J = 7.6 Hz, 1H), 7.48 (dd, J = 14.7, 7.8 Hz, 3H), 7.38–7.30 (m, 2H),
3.07 (2H), 2.76 (2H); 13C NMR (101 MHz, DMSO)
174.03, 173.55,
d
12.18 (1H), 8.12 (dd, J = 30.4, 7.9 Hz, 3H), 7.72
d
155.99, 141.46, 133.84, 130.95, 128.08 ꢀ2, 127.70 ꢀ 2, 127.57,
127.24, 126.35, 122.63, 121.92, 117.39, 112.89, 107.21, 33.12,
19.41; HRMS calcd. for C20H15NO4 [M+H]+: 334.1074, found,
334.1081.
2.1.50. 3-(4-Hydroxybenzyl)-1-(4-methoxyphenyl)chromeno[2,3-
c]pyrrol-9(2H)-one (58)
2.1.45. 1-(4-Fluorophenyl)-3-(4-hydroxybenzyl)-7-
226 mg 32 (0.5 mmol) was treated with the procedure used for
50 to afford 111 mg 58 as brown crystal, yield 56%; 1H NMR
methylchromeno[2,3-c]pyrrol-9(2H)-one (53)
227 mg 27 (0.5 mmol) was treated with the procedure used for
50 to afford 112 mg 53 as yellow powder, yield 56%; 1H NMR
(400 MHz, DMSO) d 12.30 (1H), 9.20 (1H), 8.13 (s, 2H), 7.94 (s, 1H),
7.51 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.30 (t, J = 8.3 Hz,
2H), 7.10 (d, J = 7.6 Hz, 2H), 6.70 (d, J = 7.7 Hz, 2H), 4.04 (2H), 2.40
(3H); 13C NMR (101 MHz, DMSO)
d 174.17, 162.79, 160.34, 155.69,
154.21, 141.53, 134.87, 131.74, 129.86, 129.78, 129.58, 128.96 ꢀ 2,
127.56, 127.54, 126.09, 125.80, 121.54, 117.22, 115.18 ꢀ 2, 115.07,
114.86, 113.50, 107.28, 28.43, 20.29; HRMS calcd. for C25H18NO3F
[M+H]+: 400.1343, found, 400.1345.
(400 MHz, DMSO) d 12.16 (1H), 9.20 (1H), 8.10 (dd, J = 39.1, 6.3 Hz,
3H), 7.69 (t, 1H),7.46 (d, J = 6.9 Hz, 1H), 7.31 (t, 1H),7.06 (dd,
J = 26.7, 6.2 Hz, 4H), 6.69 (d, J = 6.1 Hz, 2H), 4.04 (2H), 3.82 (3H);
13C NMR (101 MHz, DMSO)
d 174.47, 159.42, 156.48, 156.16,
141.73, 134.29, 130.22, 129.66 ꢀ 2, 129.45 ꢀ 2, 128.22, 126.88,
124.07, 123.08, 122.51, 117.83, 115.67 ꢀ 2, 114.04 ꢀ 2, 113.26,
107.19, 55.67, 28.91; HRMS calcd. for C25H19NO4 [MꢁH]ꢁ:
396.1241, found, 396.1257.
2.1.51. 3-(4-Hydroxybenzyl)-1-(naphthalen-2-yl)chromeno[2,3-
c]pyrrol-9(2H)-one (59)
2.1.46. 3-(7-Bromo-9-oxo-1-(thiophen-2-yl)-2,9-
237 mg 34 (0.5 mmol) was treated with the procedure used for
50 to afford 52 mg 59 as brown powder, yield 25%; 1H NMR
dihydrochromeno[2,3-c]pyrrol-3-yl)propanoicacid (54)
237 mg 28 (0.5 mmol) was treated with the procedure used for
50 to afford 135 mg 54 as yellow powder, yield 65%; 1H NMR
(400 MHz, DMSO)
d 12.51 (1H), 9.24 (1H), 8.67 (1H), 8.24 (ddd,
J = 9.9, 8.3, 1.7 Hz, 2H), 8.01–7.92 (m, 3H), 7.72 (t, J = 8.6 Hz, 1H),
7.57–7.48 (m, 3H), 7.35 (dd, J = 11.1, 3.9 Hz, 1H), 7.16 (d, J = 8.5 Hz,
2H), 6.72 (dd, J = 6.6, 4.7 Hz, 2H), 4.12 (2H); 13C NMR (101 MHz,
(400 MHz, DMSO)
d 12.26 (2H), 8.21–8.17 (m, 1H), 8.01 (m,1H),
7.86–7.81 (m, 1H), 7.58 (t, J = 4.5 Hz, 1H), 7.46 (dd, J = 8.8, 5.0 Hz,

N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
93
DMSO)
d
174.12, 156.00, 155.71, 141.72, 133.94, 132.76, 132.20,
[42] and are briefly described as follows. The recombinant plasmid
of pET15b-PDE5A1 (535–860) was transferred into E. coli. strain
BL21 (CodonPlus, Stratagene). The E. coli cell carrying the pET15-
PDE5A1 plasmid was grown in LB medium at 37 8C to absorption
129.59, 129.02 ꢀ 2, 128.52, 128.06, 127.50, 127.35, 127.18, 126.46,
126.42, 126.24, 126.20, 125.98, 122.75, 122.02, 117.43, 115.22 ꢀ 2,
114.22, 107.69, 28.50; HRMS calcd. for C28H19NO3 [M+H]+:
418.1438, found, 418.1446.
A
600 = 0.6–0.8, and then 0.1 mM isopropyl b-D-thiogalactopyrano-
side was added to induce the expression at 15 8C for 40 h. The
recombinant PDE5A1 protein was purified by column chromatog-
raphy of Ni-NTA affinity (Qiagen), thrombin cleavage, Q-sepharose
ionic exchange, and Sephacryl S200 gel filtration (GE Healthcare). A
typical batch of purification yielded 20 mg PDE5A1 from 2-L cell
culture. The PDE5A1 protein has purity >95%, as shown by SDS-
PAGE.
The catalytic domains of PDE4D2 (86–413), PDE7A1 (130–482),
PDE8A2 (480–820), PDE9A2 (181–506), and PDE10A2 (448–789)
were purified by using similar protocols previously published [43–
47]. PDE1B (1–516) was partially purified (unpublished data).
2.1.52. 3-Benzyl-2-methyl-1-phenylchromeno[2,3-c]pyrrol-9(2H)-
one (60)
176 mg 3-Benzyl-1-phenylchromeno[2,3-c]pyrrol-9(2H)-one
(42, 0.5 mmol) was dissolved in 20 ml CH3CN or acetone, and
207 mg K2CO3 (1.5 mmol) was added. 355 mg CH3I (2.5 mmol) was
dropped into the mixture quickly, refluxed at 50 8C for 3 h until
reactant disappeared, as monitored by using TLC. The reaction
mixture was evaporated in vacuum, and then 20 ml EtOAc was
added. The resulted solution was washed with 50 ml water and
saturated salt solution. The organic layer was dried over anhydrous
sodium sulfate, and the filtrate was evaporated and subjected to
column chromatography to afford 142 mg 60 as light yellow
2.3. Enzymatic assay
powder, yield 78%; 1H NMR (400 MHz, DMSO)
1H), 7.70 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 6.1 Hz,2H), 7.52–7.44 (m,
4H), 7.37–7.31 (m, 3H), 7.31–7.20 (m, 3H), 4.30 (2H), 3.48 (3H); 13
NMR (101 MHz, DMSO) 173.03, 156.07, 141.47, 138.57, 133.80,
131.02 ꢀ 2, 129.70, 129.10, 128.68 ꢀ 2, 128.24, 127.99 ꢀ 2,
127.79 ꢀ 2, 126.34, 126.10, 122.74, 122.07, 117.54, 113.71,
107.23, 32.08, 28.18; HRMS calcd. for C25H19NO2 [M+H]+:
366.1489, found, 366.1503.
d 8.09 (d, J = 7.7 Hz,
The enzymatic activities of the catalytic domains of PDE5A1 and
other PDEs were measured by using cGMP or cAMP as substrates
and sildenafil and other PDE inhibitors as the reference com-
pounds. The assay buffer contains 50 mM Tris–HCl (pH 8.0),
10 mM MgCl2 or 4 mM MnCl2, 1 mM DTT, 10–30 nM 3H-cGMP or
3H-cAMP (20,000–30,000 cpm/assay, GE Healthcare). The reaction
was carried out at room temperature for 15 min and then
terminated by addition of 0.2 M ZnSO4. The reaction product
3H-GMP or 3H-AMP was precipitated by 0.2 N Ba(OH)2, while
unreacted 3H-cGMP or 3H-cAMP was remained in the supernatant.
Radioactivity of the supernatant was measured in 2.5 ml Ultima
Gold liquid scintillation cocktails (Fisher Scientific) by a Perki-
nElmer 2910 liquid scintillation counter. For the measurement of
IC50, at least eight concentrations of inhibitors were used in the
presence of suitable concentrations of 3H-cAMP/3H-cGMP and the
enzymes that hydrolyze up to 70% of the substrates. Each
measurement was repeated three times. The IC50 values were
calculated by nonlinear regression.
C
d
2.1.53. 3-(4-Hydroxybenzyl)-1-phenylchromeno[2,3-c]pyrrol-9(2H)-
one (61)
The tert-butyl protection of 43 (212 mg, 0.5 mmol) was
removed with the procedure used for 51 to afford 152 mg of 61
as yellow powder, yield 83%; 1H NMR (400 MHz, DMSO)
d 12.39
(1H), 9.20 (1H), 8.17 (dd, J = 7.9, 1.6 Hz, 1H), 8.09 (d, J = 7.4 Hz, 2H),
7.71 (ddd, J = 8.6, 7.2, 1.7 Hz, 1H), 7.52–7.41 (m, 3H), 7.34 (dd,
J = 14.6, 7.2 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H),
4.06 (2H); 13C NMR (101 MHz, DMSO)
d 174.05, 155.96, 155.69,
141.52, 133.88, 130.94, 129.61, 128.98 ꢀ 2, 128.07 ꢀ 2, 127.71 ꢀ 2,
127.57, 127.26, 126.40, 122.69, 121.98, 117.40, 115.19 ꢀ 2, 113.74,
107.30, 28.44; HRMS calcd. for C24H17NO3 [M+H]+: 368.1281,
found, 368.1281.
2.4. Crystallization and structure determination
The crystals of PDE5A1 were grown by the hanging drop vapor
diffusion method. The unliganded PDE5A1 (535–860) (6–10 mg/
mL in a storage buffer of 50 mM NaCl, 20 mM Tris–HCl pH 7.5,
2.1.54. 3-(4-Hydroxybenzyl)-2-methyl-1-phenylchromeno[2,3-
c]pyrrol-9(2H)-one (62)
110 mg 61 (0.3 mmol) was dissolved in 10 ml acetone, and
124 mg K2CO3 (0.9 mmol) was added. 213 mg CH3I (1.5 mmol) was
dropped into the mixture quickly and refluxed at 50 8C for 3 h until
reactant disappeared as monitored by using TLC. The reaction
mixture was evaporated in vacuum, and 20 ml EtOAc was added.
The resulted solution was washed with 50 ml water and saturated
salt solution. The organic layer was dried over anhydrous sodium
sulfate, and the filtrate was evaporated and subjected to column
chromatography to afford 39 mg 62 as yellow powder, yield 34%;
1 mM b-mercaptoethanol and 1 mM EDTA) was vapor-diffused
against the well buffer of 0.2 M MgSO4, 0.1 M Na cacodylate (pH
6.5), and 18% PEG3350 at room temperature. The crystal size of the
unliganded PDE5A1 was improved by micro-seeding against the
same crystallization buffer. The PDE5A1-57 complex was prepared
by soaking the unliganded PDE5A1 crystals in the crystallization
buffer plus 5 mM 57 at 24 8C for 3 days. The crystals were flash-
frozen in liquid nitrogen by using the well buffer plus 20% (w/v)
glycerol as the cryosolvent. The X-ray diffraction data were
collected at 100 K on Beamline X29 of Brookhaven National
Laboratory (Table 3) and processed by HKL2000 [48]. The structure
of the PDE5A1-57 complex was solved by the molecular
replacement [49]. The resulted model was rebuilt by program O
[50] and refined by CNS [51].
1H NMR (400 MHz, CDCl3)
d 8.27 (d, J = 7.8 Hz, 1H), 7.60 (t,
J = 7.6 Hz, 1H), 7.52 (d, J = 7.4 Hz, 2H), 7.47 (t, J = 7.1 Hz, 2H), 7.43
(d, J = 6.8 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 7.7 Hz, 1H),
7.10 (d, J = 7.9 Hz, 2H), 6.79 (d, J = 7.9 Hz, 2H), 5.02 (1H), 4.23 (2H),
3.46 (3H); 13C NMR (101 MHz, DMSO)
d 173.05, 156.09, 155.82,
141.24, 133.78, 131.02 ꢀ 2, 129.73, 128.93 ꢀ 2, 128.49, 128.20,
127.78 ꢀ 2, 126.09, 122.70, 122.06, 117.55, 115.44 ꢀ 2, 114.39,
107.17, 32.05, 27.35; HRMS calcd. for C25H19NO3 [MꢁH]ꢁ:
380.1292, found, 380.1296.
2.5. Docking
The crystal structure of the PDE5A catalytic domain in complex
with sildenafil (PDB code 2H42) was used for the virtual docking by
the CDOCKER protocol embedded in Accelrys Discovery Studio
2.5.5 [52]. In order to validate the reliability of the default
parameters of CDOCKER, the bound sildenafil was docked to the
active site of PDE5, yielding the root-mean-squared deviations
2.2. Protein expression and purification
The subcloning and protein expression of human PDE5A1
catalytic domain (residues 535–860) were previously published

94
N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
˚
(RMSDs) of <1.0 A for 23 out of total 50 poses from the bound
conformation of sildenafil in the crystal. Then, fifty conformations
of each designed compound were randomly generated, optimized,
and evaluated on the basis of both docking score and cluster
population.
inhibitors of 16, 35, 37, 38, and 39 with IC50 = 446, 122, 381, 217,
and 492 nM, respectively. Thus, the above data suggest that a
smaller group linked to OtBu have better inhibitory activity, and
also imply that the t-butanyl group may be a steric obstacle for
interaction with PDE5 so as to scarify the inhibition. The best
substitution on R4 is –CH2C6H4(p-OH), and compounds containing
this substitution showed the IC50 values of 456 (59), 356 (53), 221
(58), 137 (62), 135 (55), 61 (51), 18 (61), and 17 nM (57),
respectively.
In summary, four compounds 42, 51, 57, and 61 show the
potent IC50 values of 77, 61, 17, and 18 nM, and may thus be
deserved for further pharmacological studies. Compound 57 was
selected for more complete assay and shows reasonably good
selectivity over the catalytic domains of the other PDE families
(Table 2).
The new scaffolds were designed on the basis of the PDE5-57
crystal structure and docked by program AutoDock 4.2 [53] into
the active sites of PDE4D2 (79–413) [43], PDE5A1 (535–860),
and PDE9A2 (191–506) [47]. The rigid monomeric PDE catalytic
domains were used for docking of all scaffolds. The default
docking parameters were verified by docking of 57 into the
crystal structure and then used for the scaffold docking. A typical
˚
box with 60 ꢀ 60 ꢀ 60 grid points equally spaced at 0.375 A per
grid was generated using AutoGrid [53]. The following param-
eters and Lamarckian genetic algorithm were used for the
docking: random initial orientation and position, population
size (150), maximum number of energy evaluations (25 million),
maximum number of generations (27,000), mutation rate (0.02),
crossover rate (0.8), and 100 docking runs to output 100 docked
conformations.
3.3. Architecture of the PDE5-57 structure
The crystallographic asymmetric unit contains one PDE5
catalytic domain in complex with 57. The structure of the PDE5-
57 complex (Fig. 2) has the overall similar folding as those of other
PDE5 catalytic domains [42,56–62], as shown by the average
3. Results
˚
positional differences of 0.24 and 0.26 A for 286 and 279
comparable C atoms when PDE5-57 was superimposed over
3.1. Structure-based molecular design
a
the structures of the unliganded PDE5 and its complex with
sildenafil [42]. A significant portion of the H-loop (667–676) and
the M-loop (791–807) is disordered in the PDE5-57 complex. This
is similar to the disorder of the H-loop in the structures of the
unliganded PDE5 and its IBMX complex [42,61], but different from
the ordered conformation of the H-loops in the PDE5 complexes
with sildenafil and vardenafil, in which the H-loops showed large
positional migrations and different conformations [42,59].
We previously reported the synthesis of a series of 1-aryl
chromeno[2,3-c]pyrrol-9(2H)-one (ACPs) that may have antican-
cer activity [41]. Since these compounds contain a chromeno-
pyrrol core similar to that of Johnson’s PDE5 inhibitors RWJ387273
and RWJ444772 [54,55], we tested their ability to bind to the active
site of PDE5. The docking of the ACP compounds to PDE5A shows
that some of them occupy the same pocket as sildenafil does, and
also are capable of forming a hydrogen bond with invariant Gln817
and
p
–p
stack against conserved Phe820, suggesting that these
3.4. Binding of inhibitor 57
compounds may be developed to PDE5A inhibitors. Indeed, the
enzymatic assay confirmed the inhibitory activity of the com-
pounds on PDE5. Thus, the triple ACP ring was chosen as the
pharmacophore and various substitutions on R1 to R5 were
designed to optimize the inhibition on PDE5 (Table 1).
Inhibitor 57 binds to the active site of PDE5 as other PDE5
inhibitors do (Fig. 2). The aryl-chromeno-pyrrol ring of 57 is
sandwiched by the hydrophobic residues of Phe820 on one side
and Val782 and Phe786 on another side (Fig. 2B and C). In addition,
the amine on the pyrrol ring of 57 forms a hydrogen bond with the
˚
3.2. Discovery of aryl chromeno-pyrrol analogs as PDE5 inhibitors
amide oxygen of the Gln817 side chain of PDE5 (2.5 A). This
hydrogen bond with Gln817 and the stack against Phe820 are two
characteristics conserved for the binding of many PDE inhibitors
[62]. The tyrosyl group of 57 interacts with hydrophobic residues
of Val782, Ala783, Phe787, Ile813, and Met816, although the
docking suggested that its OH group may form a hydrogen bond
with the carbonyl oxygen of Leu804 that is disordered in the
crystal. The thiophen ring of 57 forms a hydrogen bond with the
The inhibitory activities of obtained aryl chromeno-pyrrol
analogs were evaluated by enzymatic assay (Table 1). In summary,
most compounds have the IC50 values less than 1
mM against
PDE5A1, and small substitutions on R1, R2, and R5 (–CH3, –OCH3, or
–Br) do not dramatically improve the inhibitory activity (com-
pounds 16–19, 22, 24, 25, 35, 39, 45, 47, 48, 51, 53–56, 60, and 62).
The activity of the compounds largely depends on the substitutions
on R3 and R4, as discussed as follows.
˚
amide nitrogen of Gln817 (3.3 A to the sulfur), in addition to the
hydrophobic interactions with residues of Ala767, Ile768, Gln775,
and Ile778.
The electron density maps of (2Fo ꢁ Fc) and (Fo ꢁ Fc) unam-
biguously revealed the conformation of 57, in which the thiophen
For substitution on R3, the small modifications such as the
exchanges between furan, thiophen, fluorobenzene, and benzene
do not significantly change the IC50 values, as shown by the
comparisons of 16 vs. 39, 17 vs. 24, 21 vs. 26, 37 vs. 38, 50 vs. 56,
and 57 vs. 61 (Table 1). However, significant improvement is
observed for the cases, in which (1) furan (19 or 20) is better than
4-Br-C6H4– (23) and 4-C6H5-C6H4– (49), and (2) thiophen (57) is
better than 4-O(CH3)-C6H4– (58) or naphthalene (59). These data
suggest that the compounds with small substitutions on R3 have
better activity than those with large substitutions. This conclusion
is also supported by the comparisons of 36 vs. 33 and 61 vs. 59.
For R4, substitution with –CH2C6H4(p-OtBu) (23, 25, 34, 43, and
49) or –CH2(CH2)3NHCOOtBu (41 and 46–48) produces poor
ring conjugated with the aryl-chromeno-pyrrol plane, but tilted
2
˚
slightly (Fig. 2C). However, the B-factor of 57 (86 A ) is significantly
2
˚
higher than the average B-factor of the protein atoms (48 A ,
Table 3), suggesting its low occupancy. A possible explanation to
the low occupancy of 57 might be the unfavorable match of the
polar chromenol ring with the hydrophobic residues Phe820,
Val782, and Phe786 (Fig. 2B and C). Another possibility might be
the close contact of a carbon atom on the aryl ring of 57 with an
oxygen of the bound sulfate group in a hydrogen bond distance.
The sulfate group is not an integrated part of the protein, but may
be recruited from the crystallization buffer containing 0.2 M
MgSO4. The sulfate replaced a chelating water of the zinc ion and
its occupancy at the active site is supported by its comparable
inhibition with IC50 > 10
tution yields IC50 in a range 0.8–4
Table 1). The –CH2COOtBu substitution results in the moderate
m
M, while the –CH2CH2COOtBu substi-
M (17, 18, 21, 22, 24, and 26,
m

N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
95
Table 1
Structures of ACPs and their IC50 values (nM) for PDE5A1.
Cpd.
R1
R2
R3
R4
R5
IC50 ꢂ SDa
16
17
18
19
20
21
22
23
24
25
26
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
H
CH3
OCH3
CH3
CH3
H
Furan-2-yl
Furan-2-yl
Furan-2-yl
Furan-2-yl
Furan-2-yl
Furan-2-yl
Furan-2-yl
4-Br-C6H4
Thiophen-2-yl
Thiophen-2-yl
C6H5
CH2COOtBu
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
CH3
446 ꢂ 6
H
CH2CH2COOtBu
CH2CH2COOtBu
CH2C6H4(p-OtBu)
CH2C6H4(p-OtBu)
CH2CH2COOtBu
CH2CH2COOtBu
CH2C6H4(p-OtBu)
CH2CH2COOtBu
CH2C6H4(p-OtBu)
CH2CH2COOtBu
CH2C6H5
4379 ꢂ 450
1667 ꢂ 80
240 ꢂ 44
H
H
H
1997 ꢂ 315
2071 ꢂ 213
823 ꢂ 246
H
H
H
Br
H
H
>10,000 (26%)
1913 ꢂ 60
ꢃ10,000
H
OCH3
OCH3
H
H
H
2142 ꢂ 290
>10,000 (28%)
>10,000 (3%)
122 ꢂ 27
H
H
Naphthalen-2-yl
Naphthalen-2-yl
Thiophen-2-yl
4-Br-C6H4
C6H5
H
H
CH2C6H4(p-OtBu)
CH2COOtBu
H
OCH3
H
H
CH2C6H5
319 ꢂ 78
H
H
CH2COOtBu
381 ꢂ 61
H
H
4-F-C6H4
CH2COOtBu
217 ꢂ 25
H
CH3
H
4-F-C6H4
CH2COOtBu
492 ꢂ 35
H
4-F-C6H4
N-Boc-1H-indol-3-yl
CH2(CH2)3NHCOOtBu
CH2C6H5
CH2C6H4(p-OtBu)
CH2CH(CH3)2
>10,000 (21%)
ꢃ10,000
H
H
C6H5
H
H
C6H5
77 ꢂ 11
H
H
C6H5
>10,000 (40%)
836 ꢂ 17
OCH3
H
H
Thiophen-2-yl
Furan-2-yl
Thiophen-2-yl
Thiophen-2-yl
Thiophen-2-yl
4-C6H5-C6H4
Thiophen-2-yl
Thiophen-2-yl
C6H5
CH3
H
CH2CH(CH3)2
576 ꢂ 7
OCH3
H
CH2(CH2)3NHCOOtBu
CH2(CH2)3NHCOOtBu
CH2(CH2)3NHCOOtBu
CH2C6H4(p-OtBu)
CH2CH2COOH
CH2C6H4(p-OH)
CH2CH2COOH
CH2C6H4(p-OH)
CH2CH2COOH
CH2C6H4(p-OH)
CH2CH2COOH
CH2C6H4(p-OH)
CH2C6H4(p-OH)
CH2C6H4(p-OH)
CH2C6H5
ꢃ10,000
CH3
OCH3
H
>10,000 (41%)
2463 ꢂ 426
>10,000 (6%)
4166 ꢂ 76
61 ꢂ 3
H
H
H
OCH3
OCH3
H
H
H
ꢃ10,000
H
CH3
Br
4-F-C6H4
356 ꢂ 34
H
Thiophen-2-yl
Thiophen-2-yl
4-F-C6H4
2818 ꢂ 370
135 ꢂ 17
H
Br
H
CH3
H
4626 ꢂ 725
17 ꢂ 1
H
Thiophen-2-yl
4-OCH3-C6H4
Naphthalen-2-yl
C6H5
H
H
221 ꢂ 3
H
H
456 ꢂ 24
H
H
902 ꢂ 31
H
H
C6H5
CH2C6H4(p-OH)
CH2C6H4(p-OH)
18 ꢂ 4
H
H
C6H5
137 ꢂ 16
a
The percentiles in parentheses indicate the inhibitory rate at 10,000 nM of the inhibitors.
2
2
˚
˚
B-factor (35 A ) with the overall B-factor for protein atoms (48 A )
from the structural refinement (Table 3). The last possibility might
be due to the fact that 57 does not fit well to the pocket of the
unliganded PDE5 by the soaking experiments since at least 6
conformations of the H-loop at the active site of PDE5 have been
identified [42,59–62].
the thiophen ring of 57 in PDE5 would predict that a large
substitution at R3 results in poor inhibitors, and this indeed
happens to compounds 33 and 34 (IC50 > 10
mM, Table 1). The fit of
the tyrosyl ring of 57 to the hydrophobic environment in the
crystal structure might explain that the compounds containing a
polar ester group at R4 (16–18, 21, 22, 24, 26, 37–39, 41, 46–48, 50,
52, 54, and 56) would not be highly potent inhibitors due to its
polarity or steric hindrance.
The structural information is ready for explanation of the
activity data. The attachment of a methyl group to R5 of 57 would
abolish the hydrogen bond between the nitrogen of pyrrol of 57
and Gln817 (Fig. 2) and thus scarify the potency of the inhibitors 60
and 62 (Table 1). The contact of thiophen with the hydrophobic
residues of Ala767, Ile768, Ile778, and Phe820, may explain the
observation that the benzyl replacement produced an inhibitor
with a similar IC50 (18 nM for 61, Table 1). The dead end location of
3.5. Similarities and differences of 57 binding from those of other
PDE5 inhibitors
Superposition of PDE5-57 over the other PDE5-inhibitor
structures [42,56–62] reveals an overall similar binding between
Table 2
Enzymatic activity of 57 (IC50, mM) against catalytic domains of PDEs.
PDEs
1B2 (10–487)
4D2 (86–413)
5A1 (535–860)
7A1 (130–482)
8A1 (480–820)
9A2 (181–506)
10A2 (447–789)
57
2.00 ꢂ 0.27
1.45 ꢂ 0.11
0.017 ꢂ 0.001
0.99 ꢂ 0.06
2.76 ꢂ 0.73
11.68 ꢂ 0.16
0.75 ꢂ 0.19

96
N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
Fig. 2. Structure of the PDE5-57 complex. (A) Ribbon presentation of the PDE5 catalytic domain (cyan ribbons) in complex with 57 (yellow stick). The red and green ribbons
show the conformations of the H- and M-loops of the unliganded PDE5 and its complex with sildenafil, respectively. The comparable portions of the three structures are
represented by the cyan ribbons. (B) Surface model for the 57 (yellow sticks) binding. The dotted lines represent hydrogen bonds. (C) Interaction of 57 with PDE5. The blue
mesh is the electron density map of (Fo ꢁ Fc) that was calculated from the structure with omission of 57 and contoured at 2.5
s. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of the article.)
57 and other PDE5 inhibitors, but different interactions in detail.
When 57 is compared with sildenafil and tadalafil, the binding of
57 to PDE5 shows a pattern closer to that of tadalafil than sildenafil
(Fig. 3). The interactions of the triple ACP ring of 57 with conserved
Phe820 and invariant Gln817 are closely simulated by the stack of
pyrazolo-pyrimidine ring of sildenafil against Phe820 and two
hydrogen bonds with Gln817 in the PDE5-sildenafil structure [42].
However, the ethoxy group of sildenafil makes much less
contribution to the hydrophobic interactions with a small pocket
made up of Phe787, Ile813, and Met816, than the tyrosyl group of
57 in the PDE5-57 structure, although they occupy the same
location (Fig. 3). Besides, sulfonyl-4-methylpiperazine of sildenafil
and pyrrol of 57 occupy different locations and interact with
different residues.
made up of Val782, Phe786, and Phe820, and form a hydrogen
bond with invariant Gln817. The tyrosyl ring of 57 and benzodioxol
ring of tadalafil occupy the same hydrophobic pocket consisted of
Phe787, Ile813, and Met816, although their detailed interactions
are not exactly comparable (Fig. 3). However, Gln817 forms only
one hydrogen bond with the amine on the indole ring of tadalafil, in
comparison to two hydrogen bonds with the pyrrol amine and
thiophen sulfur of 57.
4. Discussion
4.1. Implication on a new scaffold for development of PDE inhibitors
In comparison, the chromeno-pyrrol and thiophen rings of 57
are well superimposed over the four conjugated ring of pyrazino-
pyrido-indole of tadalafil (Fig. 3). These rings in the structures of
PDE5-tadalafil and PDE5-57 commonly fit to the hydrophobic slot
While the enzymatic and structural studies have shown that 57
is a PDE5 selective inhibitor, its potency may be further improved.
For example, replacement of the triple ACP ring of 57 with a small
ring system may avoid of the unfavorable interactions of the
hydrophobic aryl group with the hydrophilic solvent molecules at
the metal binding pocket. Another possible modification is
replacement of the polar chromeno group of 57 with a hydrophobic
ring in order to favorably fit to the hydrophobic slot of PDE5, which
is composed of Phe820, Val782, Phe786, and Phe787.
On the other hand, since 57 has a different chemical skeleton
from other PDE5 inhibitors (Fig. 1), a fragment of our inhibitors
might be taken as a scaffold for development of PDE inhibitors. An
example of the scaffolds may be methoxyl-phenyl-pyrrol-thio-
phen (5R3, Fig. 1). The docking of 5R3 into the catalytic domains of
cGMP-specific PDE5A1 and PDE9A2, and cAMP-specific PDE4D2
shows that 5R3 binds to the PDEs with the predicted Ki values at
micromolar level. The best poses from the dockings showed the
similar interactions of 5R3 with PDE5 and PDE9, such as stacking
against the conserved phenylalanine and hydrogen-bonding with
the invariant glutamine (Fig. 4). Thus, 5R3 could be used as a
scaffold that has the basic binding affinity with PDE5 and PDE9
while the selectivity may be achieved by targeting at non-
conserved residues in the PDE5 and PDE9 pockets. On the other
hand, 5R3 binding to cAMP-specific PDE4 is completely different
from those of cGMP-specific PDE5 and PDE9 (Fig. 4C). While the
stack against the phenylalanine (Phe372 in PDE4D2) is still
conserved, the pyrrol amine of 5R3 forms a hydrogen bond with
Asn321, instead of the invariant glutamine (Gln369 in PDE4D2). In
short, on the basis of the different binding patterns, 5R3 might be
redesigned to lead to inhibitors for various PDE families.
Table 3
Statistics on diffraction data and structure refinement.
Data collection
PDE5-57
Space group
P3121
˚
Unit cell (a, c, A)
74.1, 132.3
50–2.0 (2.07–2.0)
666,527
˚
Resolution (A)
Total measurements
Unique reflections
Completeness (%)
29,180
100.0 (100.0)
14.0 (5.2)
0.065 (0.688)
Average I/
s
Rmerge
Structure refinement
R-factor/R-free
Reflections
0.217/0.248
28,434/2830
15–2.0
˚
Resolution (A)
RMS deviations for
Bond length
˚
0.0052 A
Bond angle
1.18
2
˚
Average B-factor (A ) (atoms)
Protein 47.5 (2420)
Inhibitor
85.6 (23)
38.3 (1)
41.8 (1)
35.0 (5)
44.0 (75)
Zn
Mg
SO4
Water
Ramachandran plot
Most favored
Allowed
91.0%
7.9%
1.1%
General allowed

N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
97
Fig. 3. Comparison on binding of the PDE5 inhibitors. (A) Superposition of the PDE5-57 complex (cyan ribbons and yellow sticks) over the PDE5-sildenafil structure (green
ribbons and salmon sticks). (B) Comparison on the binding of 57 (yellow sticks) and tadalafil (green). Since the PDE5-tadalafil structure (PDB code 1XOZ) contains a
replacement of the H-loop with the PDE4 fragment, no comparison on the protein structures is shown. (C) Surface model on the binding of inhibitors of 57 (yellow), sildenafil
(salmon), and tadalafil (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 4. Docking of a new scaffold 5R3 to cGMP-specific PDE5A1 (A) and PDE9A2 (B), and cAMP-specific PDE4D2 (C). Dotted lines represent hydrogen bonds. It is visible that the
scaffold 5R3 binds to the PDEs in different orientations and has different patterns of hydrogen bonding in the structures of PDE4, PDE5, and PDE9, but the stacking against the
conserved phenylalanine is retained. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
[7] Piper M, van Horck F, Holt C. The role of cyclic nucleotides in axon guidance.
Acknowledgments
Adv Exp Med Biol 2007;621:134–43.
[8] Zaccolo M, Movsesian MA. cAMP and cGMP signaling crosstalk: role of
phosphodiesterases and implications for cardiac pathophysiology. Circ Res
2007;100:1569–78.
[9] Galie N, Ghofrani HA, Torbicki A, Barst RJ, RubinLJ, Badesch D, etal. Sildenafil citrate
We thank Dr. Howard Robinson at Brookhaven National
Laboratory for help with collection of the diffraction data.
This work is partially supported by US NIH GM59791 (H. Ke),
Natural Science Foundation of China (30973619, 81172931,
21103234, and 81373258), Guangdong Natural Science Founda-
tion (S2011030003190), Guangdong Science Foundation
(2012A080201007), Guangdong Provincial Key Laboratory of
Construction Foundation (2011A060901014), and Fundamental
Research Funds for the Central Universities (11ykzd05 and
09ykpy66).
therapy for pulmonary arterial hypertension. N Engl J Med 2005;353:2148–57.
[10] Blokland A, Schreiber R, Prickaerts J. Improving memory: a role for phospho-
diesterases. Curr Pharm Des 2006;12:2511–23.
[11] Menniti FS, Faraci WS, Schmidt CJ. Phosphodiesterases in the CNS: targets for
drug development. Nat Rev Drug Discov 2006;5:660–70.
[12] Miller CL, Oikawa M, Cai Y, Wojtovich AP, Nagel DJ, Xu X, et al. Role of Ca2+
/
calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating
cardiomyocyte hypertrophy. Circ Res 2009;105:956–64.
[13] Hutson PH, Finger EN, Magliaro BC, Smith SM, Converso A, Sanderson PE, et al.
The selective phosphodiesterase 9 (PDE9) inhibitor PF-04447943 (6-[(3S,4S)-
4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-py-
ran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidin-4-one) enhances synap-
tic plasticity and cognitive function in rodents. Neuropharmacology
2011;61:665–76.
References
[14] Reid DJ, Pham NT. Roflumilast: a novel treatment for chronic obstructive
pulmonary disease. Ann Pharmacother 2012;46:521–9.
[1] Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regula-
tion to clinical use. Pharmacol Rev 2006;58:488–520.
[2] Omori K, Kotera J. Overview of PDEs and their regulation. Circ Res
2007;100:309–27.
[3] Conti M, Beavo JA. Biochemistry and physiology of cyclic nucleotide phos-
phodiesterases: essential components in cyclic nucleotide signaling. Annu Rev
Biochem 2007;76:481–511.
[4] De Felice FG, Wasilewska-Sampaio AP, Barbosa AC, Gomes FC, Klein WL, Ferreira
ST, et al. Cyclic AMP enhancers and Abeta oligomerization blockers as potential
therapeutic agents in Alzheimer’s disease. Curr Alzheimer Res 2007;4:263–71.
[5] Jarnaess E, Taske´n K. Spatiotemporal control of cAMP signalling processes by
anchored signalling complexes. Biochem Soc Trans 2007;35:931–7.
[6] Jin XL, O‘Neill C. cAMP-responsive element-binding protein expression and
regulation in the mouse preimplantation embryo. Reproduction 2007;134:
667–75.
[15] Rotella DP. Phosphodiesterase
5 inhibitors: current status and potential
applications. Nat Rev Drug Discov 2002;1:674–82.
[16] Keating GM, Scott LJ. Vardenafil: a review of its use in erectile dysfunction.
Drugs 2003;63:2673–703.
[17] Kotera J, Mochida H, Inoue H, Noto T, Fujishige K, Sasaki T, et al. Avanafil: a
potent and highly selective phosphodiesterase-5 inhibitor for erectile dys-
function. J Urol 2012;188:668–74.
[18] Oh TY, Kang KK, Ahn BO, Yoo M, Kim WB. Erectogenic effect of the selective
phosphodiesterase type
2000;23:471–6.
5 inhibitor. DA-8159. Arch Pharm Res (NY)
[19] Jung JY, Kim SK, Kim BS, Lee SH, Park YS, Kim SJ, et al. The penile erection
efficacy of a new phosphodiesterase type 5 inhibitor, mirodenafil (SK3530), in
rabbits with acute spinal cord injury. J Vet Med Sci 2008;70:1199–204.

98
N.-N. Shang et al. / Biochemical Pharmacology 89 (2014) 86–98
[20] Udeoji DU, Schwarz ER. Tadalafil as monotherapy and in combination regi-
mens for the treatment of pulmonary arterial hypertension. Ther Adv Respir
Dis 2013;7:39–49.
[42] Wang H, Liu Y, Huai Q, Cai JW, Zoraghi R, Francis SH, et al. Multiple con-
formations of phosphodiesterase-5: implications for enzyme function and
drug development. J Biol Chem 2006;281:21469–79.
[21] Andersson KE, de Groat WC, McVary KT, Lue TF, Maggi M, Roehrborn CG, et al.
Tadalafil for the treatment of lower urinary tract symptoms secondary to
benign prostatic hyperplasia: pathophysiology and mechanism(s) of action.
Neurourol Urodyn 2011;30:292–301.
[22] Mouli S, McVary KT. PDE5 inhibitors for LUTS. Prostate Cancer Prostatic Dis
2009;12:316–24.
[43] Huai Q, Wang H, Sun Y, Kim HY, Liu Y, Ke H. Three dimensional structures of
PDE4D in complex with rolipram and implication on inhibitor selectivity.
Structure 2003;11:865–73.
[44] 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 2007;280:30949–55.
[23] Miller MS. Role of phosphodiesterase type 5 inhibitors for lower urinary tract
symptoms. Ann Pharmacother 2013;47:278–83.
[24] Kass DA, Champion HC, Beavo JA. Phosphodiesterase type 5: expanding roles
in cardiovascular regulation. Circ Res 2007;101:1084–95.
[45] Wang H, Liu Y, Hou J, Zheng M, Robinson H, Ke H. Structural insight into
substrate specificity of phospodiesterase 10. Proc Natl Acad Sci USA
2007;104:5782–7.
[46] Wang H, Yan Z, Yang S, Cai J, Robinson H, Ke H. Kinetic and structural studies of
phosphodiesterase-8A and implication on the inhibitor selectivity. Biochem-
istry 2008;47:12760–68.
[47] Meng F, Hou J, Shao YX, Wu PY, Huang M, Zhu X, et al. Structure-based
discovery of highly selective phosphodiesterase-9A inhibitors and implica-
tions for inhibitor design. J Med Chem 2012;55:8549–58.
[48] Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol 1997;276:307–26.
[49] Navaza J, Saludjian P. AMoRe: an automated molecular replacement program
package. Methods Enzymol 1997;276:581–94.
[50] Jones TA, Zou J-Y, Cowan SW, Kjeldgaard M. Improved methods for building
protein models in electron density maps and the location of errors in these
models. Acta Cryst 1991;A47:110–9.
[51] Bru¨nger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW,
et al. Crystallography & NMR system: a new software suite for macromolecular
structure determination. Acta Cryst 1998;D54:905–21.
[52] Wu G, Robertson DH, Brooks CL, Vieth M. Detailed analysis of grid-based
molecular docking: a case study of CDOCKER—a CHARMm-based MD docking
algorithm. J Comput Chem 2003;24:1549–62.
[25] Kukreja RC, Salloum FN, Das A. Cyclic guanosine monophosphate signaling and
phosphodiesterase-5 inhibitors in cardioprotection.
2012;59:1921–7.
J Am Coll Cardiol
[26] Schwartz BG, Levine LA, Comstock G, Stecher VJ, Kloner RA. Cardiac uses of
phosphodiesterase-5 inhibitors. J Am Coll Cardiol 2012;59:9–15.
[27] Schwartz BG, Jackson G, Stecher VJ, Campoli-Richards DM, Kloner RA. Phos-
phodiesterase type 5 inhibitors improve endothelial function and may benefit
cardiovascular conditions. Am J Med 2013;126:192–9.
[28] Guazzi M, Samaja M, Arena R, Vicenzi M, Guazzi MD. Long-term use of
sildenafil in the therapeutic management of heart failure. J Am Coll Cardiol
2007;50:2136–44.
[29] Cvelich RG, Roberts SC, Brown JN. Phosphodiesterase type 5 inhibitors as
adjunctive therapy in the management of systolic heart failure. Ann Pharmac-
other 2011;45:1551–8.
[30] Kanwar M, Agarwal R, Barnes M, Coons J, Raina A, Sokos G, et al. Role of
phosphodiesterase-5 inhibitors in heart failure: emerging data and concepts.
Curr Heart Fail Rep 2013;10:26–35.
[31] Roustit M, Blaise S, Allanore Y, Carpentier PH, Caglayan E, Cracowski JL.
Phosphodiesterase-5 inhibitors for the treatment of secondary Raynaud’s
phenomenon: systematic review and meta-analysis of randomised trials.
Ann Rheum Dis 2013;72:1696–9.
[53] Huey R, Morris GM, Olson AJ, Goodsell DS. Asemiempirical free energy
force field with charge-based desolvation. J Comput Chem 2007;28:1145–
52.
[32] Percival JM, Adamo CM, Beavo JA, Froehner SC. Evaluation of the therapeutic
utility of phosphodiesterase 5A inhibition in the mdx mouse model of duch-
enne muscular dystrophy. Handb Exp Pharmacol 2011;204:323–44.
[33] Gonzalez-Cadavid NF, Rajfer J. Treatment of Peyronie’s disease with PDE5
inhibitors: an antifibrotic strategy. Nat Rev Urol 2010;7:215–21.
[34] Puzzo D, Sapienza S, Arancio O, Palmeri A. Role of phosphodiesterase 5 in
synaptic plasticity and memory. Neuropsychiatr Dis Treat 2008;4:371–87.
[35] Rondina MT, Weyrich AS. Targeting phosphodiesterases in anti-platelet ther-
apy. Handb Exp Pharmacol 2012;210:225–38.
[54] Jiang W, Guan J, Macielag MJ, Zhang S, Qiu Y, Kraft P, et al. Pyrroloquinolone
PDE5 inhibitors with improved pharmaceutical profiles for clinical studies on
erectile dysfunction. J Med Chem 2005;48:2126–33.
[55] Lanter JC, Sui Z, Macielag MJ, Fiordeliso JJ, Jiang W, Qiu Y, et al. Structure–
activity relationships of N-acyl pyrroloquinolone PDE-5 inhibitors. J Med
Chem 2004;47:656–62.
[56] Sung BJ, Hwang KY, Jeon YH, Lee JI, Heo YS, Kim JH, et al. Structure of the
catalytic domain of human phosphodiesterase 5 with bound drug molecules.
Nature 2003;425:98–102.
[36] Farsaie S, Khalili H, Karimzadeh I, Dashti-Khavidaki S. An old drug for a new
application: potential benefits of sildenafil in wound healing. J Pharm Pharm
Sci 2012;15:483–98.
[57] Zhang KY, Card GL, Suzuki Y, Artis DR, Fong D, Gillette S, et al. A glutamine
switch mechanism for nucleotide selectivity by phosphodiesterases. Mol Cell
2004;15:279–86.
[37] Schmidt CJ. Phosphodiesterase inhibitors as potential cognition enhancing
agents. Curr Top Med Chem 2010;10:222–30.
[38] Dimitriadis F, Giannakis D, Pardalidis N, Zikopoulos K, Paraskevaidis E, Gio-
titsas N, et al. Effects of phosphodiesterase-5 inhibitors on sperm parameters
and fertilizing capacity. Asian J Androl 2008;10:115–33.
[39] Azzouni F, Abu samra K. Are phosphodiesterase type 5 inhibitors associated
with vision-threatening adverse events? A critical analysis and review of the
literature. J Sex Med 2011;8:2894–903.
[40] Khan AS, Sheikh Z, Khan S, Dwivedi R, Benjamin E. Viagra deafness—sensori-
neural hearing loss and phosphodiesterase-5 inhibitors. Laryngoscope
2011;121:1049–54.
[58] Card GL, England BP, Suzuki Y, Fong D, Powell B, Lee B, et al. Structural basis for
the activity of drugs that inhibit phosphodiesterases. Structure 2004;12:
2233–47.
[59] Wang H, Ye M, Robinson H, Francis SH, Ke H. Conformational variations of both
PDE5 and inhibitors provide the structural basis for the physiological effects of
vardenafil and sildenafil. Mol Pharmacol 2008;73:104–10.
[60] Chen G, Wang H, Robinson H, Cai J, Wan Y, Ke H. An insight into the
pharmacophores of phosphodiesterase-5 inhibitors from synthetic and crystal
structural studies. Biochem Pharmacol 2008;75:1717–28.
[61] Huai Q, Liu Y, Francis SH, Corbin JD, Ke H. Crystal structures of phospho-
diesterases 4 and 5 in complex with inhibitor IBMX suggest a conformation
determinant of inhibitor selectivity. J Biol Chem 2004;279:13095–101.
[62] Ke H, Wang H. Crystal structures of phosphodiesterases and implications on
substrate specificity and inhibitor selectivity. Curr Top Med Chem 2007;
7:391–403.
[41] Yu Y, Hu Y, Shao W, Huang J, Zuo Y, Huo Y, et al. Synthesis of multi-
functionalized chromeno[2,3-c]pyrrol-9(2H)-ones: investigation and applica-
tion of Baker–Venkataraman rearrangement involved reactions catalyzed by
4-(dimethylamino)pyridine. Eur J Org Chem 2011;24:4551–63.