Discovery of Evodiamine Derivatives as Highly Selective PDE5 Inhibitors Targeting a Unique Allosteric Pocket

Article abstract of DOI:10.1021/acs.jmedchem.0c00983

Clinical use of phosphodiesterase-5 (PDE5) inhibitors is limited by several side effects due to weak isoform selectivity. Herein, a unique allosteric pocket of PDE5 is identified by molecular modeling and structural biology, which enables the discovery of highly selective PDE5 inhibitors from natural product evodiamine (EVO). The crystal structure of PDE5 with bound EVO derivative (S)-7e revealed that binding of (S)-7e to the novel allosteric pocket induced dramatic conformation changes in the H-loop with a maximum 24 ? movement of their Cα atoms. This movement directly blocks the binding of substrate/inhibitors to the PDE5 active site, which is different from all traditional PDE5 inhibitors such as sildenafil, tadalafil, and vardenafil. These derivatives showed >570-fold selectivity over PDE6C and PDE11A and achieved potent efficacy for the effective treatment of pulmonary hypertension in vivo.

Full text of DOI:10.1021/acs.jmedchem.0c00983

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Article
Discovery of Evodiamine Derivatives as Highly Selective
PDE5 Inhibitors Targeting a Unique Allosteric Pocket
Tianhua Zhang, Zengwei Lai, Suying Yuan, Yi-You Huang,
Guoqiang Dong, Chunquan Sheng, Hengming Ke, and Hai-Bin Luo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00983 • Publication Date (Web): 14 Aug 2020
Downloaded from pubs.acs.org on August 14, 2020
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Discovery of Evodiamine Derivatives as Highly Selective PDE5
Inhibitors Targeting a Unique Allosteric Pocket
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Tianhua Zhanga,b#, Zengwei Lai , Suying Yuan , Yi-You Huang , Guoqiang Dong , Chunquan
a,c#
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c*
b*
a, d,*
Sheng , Hengming Ke , Hai-Bin Luo
a
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, P. R. China
Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The
b
University of North Carolina, Chapel Hill, North Carolina 27599-7260, United States
c
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
d
Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life and
Pharmaceutical Sciences, Hainan University, Haikou 570228, Hainan, China.
Abstract: Clinical use of phosphodiesterase-5 (PDE5) inhibitors is limited by several side effects due
to weak isoform selectivity. Herein, a unique allosteric pocket of PDE5 is identified by molecular
modelling and structural biology, which enables the discovery of highly selective PDE5 inhibitors
from natural product evodiamine (EVO). The crystal structure of PDE5 with bound EVO derivative
(S)-7e revealed that binding of (S)-7e to the novel allosteric pocket induced dramatic conformation
changes of the H-loop with a maximum 24 Å movement of their Cα atoms. This movement directly
blocks the binding of substrate/inhibitors to the PDE5 active site, which is different from all traditional
PDE5 inhibitors such as sildenafil, tadalafil, and vardenafil. These derivatives showed >570 fold
selectivity over PDE6C and PDE11A and achieved potent efficacy for the effective treatment of
pulmonary hypertension in vivo.
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. Introduction
Phosphodiesterases (PDEs) hydrolyze the second messengers of cyclic adenosine and guanosine 3', 5'-
monophosphate (cAMP and cGMP)1-13, which play critical roles in regulation of a vast number of
physiological processes such as cardiovascular diseases and cancers.1,5-6 Human genome contains 21
PDE genes that are categorized into 11 families.1-4 PDE molecules are divided into a variable
regulatory domain and a conserved catalytic domain at C-terminus. For the unique roles of PDEs on
decomposition of cellular cAMP/cGMP, over ten family-selective PDE inhibitors have been approved
as drugs for treatment of several diseases. The most successful example of this drug class are the PDE5
inhibitors sildenafil and tadalafil, which have been used against erectile dysfunction (ED) and
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pulmonary hypertension (PH) . However, the current PDE5 inhibitors often have poor selectivity of
less than 100 fold over PDE6 or PDE11, leading to several side effects, such as include headache,
facial flushing, nasal congestion, dyspepsia, back and muscle pain.1-4,13-14 Recently, a novel PDE5
inhibitor (TPN171) with fewer side effects and an excellent pharmacokinetic profile has being
14
investigated in a phase II clinical trial for the treatment of PH. Therefore, it is urgent to develop new
therapeutic strategies and more effective therapeutics with an improved safety profile to overcome
these intrinsic drawbacks,2,13-14, which would be a welcome contribution to ED and PH treatment.2
To toggle this challenging, discovery of allosteric inhibitors15-20 targeting PDE5 with high
selectivity might be a promising strategy. In our previous screening of inhibition on PDE5 by natural
products and their synthetic derivatives, we found that evodiamine (EVO, Figure 1 and 2)21-22, a
traditional Chinese medicine for the treatment of several diseases such as neurological disorders and
cancers23-24, inhibited PDE5 with an IC of 2.1 µM. Herein, we reported discovery of a new category
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of highly selective PDE5 inhibitors from the natural product EVO and identification of a unique
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allosteric pocket in PDE5 (Figure 1) for the first time by using EVO synthetic derivatives. As a result,
structure-based design leads to identify novel inhibitors targeting the allosteric pocket of PDE5, which
show great selectivity over other PDEs and achieved potent efficacy for the treatment of PH in vivo.
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Figure 1. Rational design of highly selective PDE5 inhibitors by targeting a novel allosteric site. a)
The predicted allosteric pocket II by software Allofinder based on the crystal structure of PDE5A-
sildenafil (PDB ID: 2H42). b) EVO derivative 7b was identified as a highly selective PDE5 inhibitor.
c) Structural biology and hit optimization enables the discovery of leads (S)-7d and (S)-7e for the
effective treatment of PH. The PDB ID for the crystal structure of PDE5A-(S)-7e is 6VBI.
Figure 2. Chemical structures of evodiamine and its synthesized derivatives.
2. Results and discussion
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Allosteric pockets prediction by the modelling approach. First, the crystal structure of PDE5 (PDB
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ID: 2H42) was hunted for the potential allosteric pockets by Allofinder . As a result, two sites (I and
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II) were predicted with the pocket volumes of 1365 (the substrate site) and 468 Å , respectively.
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Second, we virtually screened an in-house natural product library by targeting the predicted site II and
found that EVO derivatives 7b and its (S)-isomer (S)-7b possibly bind to PDE5. Their inhibition on
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PDE5 were assayed following our previously published protocol . The results showed that compounds
EVO, 7b, and (S)-7b inhibited PDE5 with IC values of 2.1 µM, 340 nM, and 110 nM (Table 1),
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respectively. Third, in screening of PDE6 inhibition by the three compounds, we found that EVO, 7b,
and (S)-7b inhibited PDE6C with their IC values > 20 M, which suggested that 7b and (S)-7b
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possibly exhibit higher selectivity of > 64 fold and 190 fold than sildenafil (5-10 folds2,13-14).
Structural optimizations for leads with potent affinity and high isoform selectivity. Encouraged
by the relatively high selectivity of 7b/(S)-7b plus their novel EVO scaffold different from current
PDE5 inhibitors, we performed hit-to-lead structural optimizations on EVO (Scheme 1 and Figure 2)
so as to discover leads with strong binding affinity and high isoform selectivity, following the protocols
described in the early publication.21-24 Derivatives with superior predicted binding affinity using
molecular docking were synthesized. This rational design has resulted in the candidates (Table 1) and
substantially reduced the synthetic load.
To remove possible false hits, an online program PAINS Remover (pan-assay interfering
compound substructures, http://cbligand.org/PAINS) was used to perform the screening before
synthesis (Scheme 1) and all the designed compounds passed this test. Finally, the synthesized EVO
derivatives were screened at concentration 10 M to decide whether they are determined their IC50
values.
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Table 1. Chemical structures and experimental IC of PDE5 ligands.
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6
*
Entry Compound
Structure
IC (µM)
50
1
2
3
4
5
6
7
8
7a
2.1 ± 0.3
1.3 ± 0.2
>50
0
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0
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0
O
N
(S)-7a
(R)-7a
7b
N
H
N
O
N
N
N
H
O
O
O
OMe
OMe
OMe
OH
N
0.34 ± 0.02
0.11 ± 0.01
2.6 ± 0.4
N
N
H
N
(S)-7b
(R)-7b
7c
N
H
N
N
N
H
N
O
N
1.7 ± 0.3
N
N
H
O
O
OH
N
(S)-7c
0.42 ± 0.06
N
H
N
OH
N
9
(R)-7c
>10
N
H
N
O
OMe
OMe
OMe
N
10
11
7d
0.053 ± 0.005
0.042 ± 0.001
>10
N
H
N
MeO
MeO
O
N
(S)-7d
(R)-7d
(S)-7e
N
H
N
O
N
1
1
2
3
N
H
N
MeO
OMe
O
OMe
N
0.035 ± 0.003
N
N
H
*
Sildenafil served as the positive control with an IC of 5.0 nM to validate the assay conditions.
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NH2
NHCHO
b
N
a
R1
R1
R1
N
H
N
N
H
H
3
a R =H
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a R =H
2a R1=H
2b R1=11-OMe
2c R1=9-OMe
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3b R =11-OMe
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b R =11-OMe
1c R =9-OMe
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e
R2
O
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O
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COOH
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R2
NH2
d
c
O
O
N
H
O
O
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4
a R =H
b R =OMe
5a R =H
5b R =OMe
6a R =H
2
2
2
6b R =OMe
2
2
2
O
O
9
R2
10
R1
11
N
OH
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7a R1=H, R2=H
N
f
7b R =H, R =OMe
7b
3
1
2
N
N
7d R =11-OMe, R =OMe
N
1
2
N
H
H
1
2
1
2
7e R1=9-OMe, R2=OMe
7
7c
o
Scheme 1. Reagents and conditions: (a) ethyl formate, reflux; (b) POCl , CH Cl , 0 C-rt; (c)
3
2
2
o
o
triphosgene, THF, reflux; (d) NaH, CH I, DMF, 0 C-rt; (e) CH Cl , rt; (f) BBr , CH Cl , -78 C.
3
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2
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2
2
Structure-Activity Relationships (SARs). As shown in Table 1, the designed EVO derivatives
showed a significant variation on the inhibitory potencies against PDE5A. Our optimization efforts on
compound 7a started by adding the methoxyl or hydroxyl group at the R position of ring E (Table 1
2
and Scheme 1). Compared with compound 7a (IC : 2.1 M), compound 7c with a hydroxyl group at
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the R position gave a similar IC of 1.7 M against PDE5, while compound 7b with a methoxyl
2
50
group at the same position exhibited a more potent IC of 340 nM toward PDE5, indicating that the
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methoxyl group was more suitable to involve interactions with of PDE5 than the hydroxyl group at the
same R position. The methoxyl group at the R position of ring E was kept unchanged, 7d with another
2
2
methoxyl group at the 11/R position of ring A showed higher potency with an IC of 53 nM against
1
50
PDE5, which is 40-fold better than 7a. This affinity improvement appears to be dominantly contributed
by the introduction of two methoxyl groups on 7a.
These (S)-isomers exhibited more potent inhibition against PDE5 than their (R)-isomers. Since
compound 7a has a chiral center, we separated the racemic form and obtained the chiral enantiomers.
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As a result, the IC (1.3 M) of its (S)-isomer (S)-7a was similar to that (2.1 M) of the racemic form
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7a, however, the IC of (R)-7a dropped to > 50 M and made more than 23-fold loss of binding
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affinity relative to 7a, indicating that the (R)-isomer weakened the binding with PDE5 than the (S)-
isomer. Similar trend was observed for compounds 7b, 7c, and 7d, confirming that these (S)-isomers
showed over 20-fold improvement of binding affinity than their (R)-isomers, e.g., (S)-7d and (R)-7d
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(42 nM vs >10 M), (S)-7b and (R)-7b (110 nM vs 2.6 M), (S)-7c and (R)-7c (420 nM vs >10 M).
Based on the putative binding pattern between PDE5 and (S)-7b by molecular docking, the methoxyl
group at the R position possibly formed H-bond interactions with adjacent residues Tyr612 and
2
His617 in the predicted allosteric pocket II (Figure S1), accounting for its higher affinity (IC = 110
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nM) than (S)-7a (1.3 M). In view of the above trend, we only synthesized another isomer (S)-7e by
replacing the methoxyl moiety from the 11/R position of (S)-7d to the 9/R position of ring A, which
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gave a little better IC value of 35 nM than (S)-7d (42 nM).
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Notably, (S)-7d exhibited better pharmacokinetic (PK) profile with an oral bioavailability of 14%
(
Table 2) that (S)-7e (1%), confirming that the methoxyl group at the 11/R position of ring A rather
1
than the 9/R position could improve their metabolic stability. In view of their more potent affinities
1
(42 and 35 nM) against PDE5 and metabolic stability, thus (S)-7d or/and (S)-7e was/were subjected
to subsequent studies.
Table 2. Pharmacokinetic Profile of (S)-7d in SD rats.
t1/2
h)
tmax
(h)
Cmax
AUC(0-t)
AUC(0-∞)
MRT(0-t)
F
(
(ng/mL)
(h·ng /mL) (h·ng /mL) (h)
(%)
i.v. 0.22  0.04 0.083
 1797  378 519  108 520  109 0.14  0.01 -
155  82 3.19  0.39 14  7
p.o. 1.43  0.12 0.83  1.01 54.4  44.5 146  75
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Excellent Selectivity of (S)-7d and (S)-7e Across PDEs. The two compounds (S)-7d and (S)-7e
exhibited at least 570-fold selectivity over other PDEs except PDE10 (Table 3). Especially worth
mentioned is >3500 fold selectivity of (S)-7e over PDE6C, relative to only 5-10 folds of sildenafil,2,13-
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thus implying potential relief of side effects.
It is noted that at concentration 100 M, the two compounds (S)-7d and (S)-7e were inactive
against Top1 and Top2 mediated relaxation of supercoiled DNA,23-24 indicating that they lost the
ability to inhibit Top1 and Top2.
*
Table 3. Isoform Profiling of (S)-7d and (S)-7e (IC , µM) Across PDEs.
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Proteins
(S)-7d
(S)-7e
PDE5A1 (535-860)
PDE1B (10-487)
PDE2A (580-941)
PDE3A (679-1087)
PDE4D2 (86-413)
PDE6C (2-854)
0.042 ± 0.001
0.035 ± 0.003
>50 (~1200 folds)
>50 (~1200 folds)
>50 (~1200 folds)
>50 (~1200 folds)
>150 (>3500 folds)
>50 (~1200 folds)
>50 (~1200 folds)
>50 (~1200 folds)
3.0 ± 0.4 (72 folds)
>30 (>710 folds)
>20 (>571 folds)
>40 (>1142 folds)
>40 (>1142 folds)
~40 (~1142 folds)
>40 (>1142 folds)
>20 (>571 folds)
>40 (>1142 folds)
>40 (>1142 folds)
2.2 ± 0.27 (62 folds)
>20 (>571 folds)
PDE7A1 (130-482)
PDE8A1 (480-820)
PDE9A2 (181-506)
PDE10A (449-770)
PDE11A (588-911)
*
IC values are the mean of at least three independent experiments with standard deviation.
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Binding of (S)-7e to the allosteric pocket of PDE5. Encouraged by the >570 fold selectivity of
S)-7d and (S)-7e over PDE6C and PDE11A, PDE5 with EVO, (S)-7b, (S)-7d, and (S)-7e were
(
crystallized by the hanging drop method (Table S1) in order to determine whether EVO and its
derivatives bind to the predicted allosteric pocket. Only the co-crystal structure of PDE5-(S)-7e was
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obtained, which showed solid conformations for most residues (Figure 3a-3c), except for disorder
around the M-loop (residues 789-809). The H-loop (residues 659-685) had relatively higher B-factors
than overall average of all atoms, but both (Fo-Fc) and (2Fo-Fc) maps showed solid electron density,
suggesting its fixed conformation upon (S)-7e binding. (S)-7e binds to a novel allosteric pocket in
crystal structure, which is consistent with the predicted allosteric pocket II.
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Surprisingly, (S)-7e non-competitively binds to a novel pocket within the catalytic domain of
PDE5 (“the EVO pocket” for short, Figure 3d), which is different from the competitive binding of all
current PDE5 inhibitors. The EVO pocket is located at the back of the active site and isolated from it
by Tyr612, His 613, His617, and Leu781 of helix H14 (Figure 3d & S2A). It is a deep rectangle with
dimension of about 7 x 11 x 15 Å and is filled with a bundle of water molecules in the unliganded
structure (Figure S2B-S2C). The EVO pocket is composed of a hydrophobic wall (residues Phe564,
Ile778, Leu781, and Tyr612), a polar wall (Asp563, Arg616, and Asn620), and a polar bottom (Asp764
and His617) (Figure 3d & S2D). Binding of (S)-7e to the pocket displaces most water molecules in
the unliganded structure, but one hydrogen-bonded with backbone nitrogen of Ala767, carbonyl
oxygen of Asp764, and the methoxy oxygen of (S)-7e (Figure S2C-S2D). The EVO derivative (S)-7e
forms four H-bonds with the side chains of Asp563, Asn614, His617 of PDE5, and the bound water
(Figure 3d, 2e & S2D), which are apparently the driving force for (S)-7e to displace the water
molecules and to bind the novel allosteric site. In addition, numerous van der Waals interactions with
residues Ile778, Leu781, and Phe564 make (S)-7e well fitted to the pocket.
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Figure 3. The predicted allosteric binding pocket validated by the crystal structure of (S)-7e with
PDE5 (PDB ID: 6VBI). (a) Ribbon diagram of PDE5 in complex with (S)-7e (salmon sticks). (b)
Binding of (S)-7e. The purple mesh is the (Fo-Fc) map contoured at 3.0 . (c) Surface representation
of PDE5-(S)-7e. The EVO pocket and the active site are marked respectively by (S)-7e (salmon sticks)
and sildenafil (purple sticks). (d) Binding of (S)-7e to the EVO pocket. (e) Interactions between (S)-
7e and PDE5. (f) Structural superposition of PDE5-sildenafil (PDB ID: 2H42) over PDE5-(S)-7e. The
comparable portion of the structures is shown in white and pale. The H-loops are shown in green for
PDE5-(S)-7e and purple for PDE5-sildenafil. (g) A close view on the conformational changes of the
H-loops upon ligand binding. The H-loop of the unliganded PDE5 (PDB ID: 2H40) is shown in white.
The (S)-7e binding did not significantly change overall conformations of the PDE5 structure, as
shown by the average difference of 2.3 Å for C atoms from the superposition between the unliganded

PDE5 and PDE5-(S)-7e structures. However, the H-loop (Arg659 – His685) underwent dramatic
changes with maximal 24 Å movement that directly blocks inhibitor/substrate binding to the active
site (Figure 3f-3g). In addition, a small loop of Lys608 - Tyr612 at the EVO pocket showed a 3.4 -
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.0 Å shift from the unliganded PDE5 (Figure S2E), but its significance for the PDE5 catalysis is
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unclear. Another surprising feature of the PDE5-(S)-7e structure is the loss of the divalent metals at
the active site, although the metal binding residues (His617, His653, Asp654, Asp764) show only
subtle positional and conformational changes in the PDE5-(S)-7e structure. The loss of the divalent
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metal ions appears to be the consequence of the allosteric conformational changes of the H-loop, in a
similar way to the divalent metal loss in the PDE5-Vardenafil structure.13
Uniqueness of the allosteric site. The sequence alignment suggests that the EVO pocket may
be specific for PDE5. The four residues, which are shown in bigger red letters (Figure 4a), appear to
jointly determine the inhibitory selectivity of the EVO pocket. First, Ile778, which makes hydrophobic
interactions with the EVO derivatives, has a side chain orientation locked by helix H14 (Figure S2A)
and defines integrity of the pocket. Replacement of Ile778 with large tryptophan will destroy the pocket
so as to abolish binding of the EVO derivatives to PDE1, PDE3, PDE4, PDE7, and PDE8, as shown
by a model of PDE1B-(S)-7e (Figure 4b). Second, the H-bond between the side chain of Asp563 and
the nitrogen on ring B of (S)-7e (Figure 3d) is another determinant for binding of the EVO derivatives.
Replacement of Asp563 with other residues would abolish the H-bond and thus weaken the binding of
EVOs. Third, the H-bond between Asp563 and Arg616 apparently plays a “gate-keeping” role to
control diffusion of the EVO compounds into the pocket. This H-bond (Figure 3d) locks the
conformation of Arg616 to make the EVO pocket open. Replacement of Arg616 with Gln565 in
PDE6C and D563 with Val203 in PDE9 would abolish the hydrogen bond and thus block the entry of
the pocket. This argument is supported by our experimental observation that (S)-7d and (S)-7e do not
effectively inhibit PDE6C and PDE9 (Table 3). PDE2 appears to be special for the correspondence of
a short helix of Pro607 - Leu610 of PDE2A3 to the coil of Asp563 - Glu565 of PDE5, which not only
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deforms the EVO pocket, but also blocks binding of the EVO derivatives (Figure 4c). In PDE10A,
correspondence of small side chains of Gly481/Pro482 of PDE10 to D563/F564 of PDE5 would make
the EVO pocket open for binding of the EVO derivatives (Figure 4d). The compatibility of most
elements of PDE10 with PDE5 would predict a potential binding of the EVO derivatives, in agreement
0
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with the experimental IC of 3 µM (Table 1).
50
Figure 4. Uniqueness of the PDE5 EVO pocket (PDB ID: 6VBI). (a) Sequence alignment of the EVO
pocket of PDE1-11. Residues in red are involved in interaction with the EVO derivatives. Green color
highlights helices. (b) Surface presentation on the PDE1B pocket (PDB ID: 5W6E). Protrusion of
Trp384 into the pocket (marked with (S)-7e, salmon sticks) ruins the EVO pocket of PDE1B. The
numbers in the parentheses denote PDE1 or PDE5. (c) Superposition of PDE2A (1Z1L, violet) over
PDE5A (6VBI, green). PDE2 and PDE5 residues are labeled respectively with magenta and green
sticks. (d) Superposition of PDE10 (6IJI, pink) over PDE5A (6VBI, green).
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Allosteric regulation mechanism on the PDE5 catalysis. To confirm the critical role of Asp563
6
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for the binding of the EVO derivatives to the allosteric pocket, we performed the D563A mutation that
is expected to abolish the hydrogen bond between Asp563 and the EVO compounds (Figure 3d) and
thus to weaken the binding. Indeed, the D563A mutation increased IC of (S)-7d from 42 nM to 2482
0
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nM, showing 59 fold loss of binding affinity (Figure 5a). In comparison, sildenafil, which binds to the
active site and has no interaction with Asp563, retains its binding affinity (IC = 2.5 nM for the wild
5
0
type and 1.2 nM for the D563A mutant, respectively).
Figure 5. Catalysis and kinetics of PDE5. (a) Inhibition on the wild type (WT) PDE5 and the D563A
mutant by sildenafil and (S)-7d. (b) The turnover of cGMP in the presence of 0–200 nM allosteric
modulator (S)-7d.
For the non-competitive nature, the inhibition of the EVO derivatives on PDE5 must be achieved
via an allosteric mode. The assay on the cGMP turnover showed that K lost about 2 folds and kcat
M
steadily dropped in the presence of 25 - 200 nM (S)-7d (Figure 5b, Table 4), implying that (S)-7d
disturbs both binding and turnover of cGMP. The Hill coefficient changed between 0.78 and 0.91 in
the presence of 25 to 200 nM (S)-7d (Table 4), indicating a negative allosteric mode of regulation.
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Table 4. Kinetic properties of the PDE5A1 catalytic domain (535-860) in the presence of allosteric
modulator.
cGMP
(
k /K )
(
Hill coefficient
KMcGMP(µM)
kcatcGMP(sec-1)
cat
-1
M
-1
s µM )
0
1
2
3
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9
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0
PDE5 + 0 nM (S)-7d
PDE5 + 25 nM (S)-7d
PDE5 + 50 nM (S)-7d
PDE5 + 100 nM (S)-7d
PDE5 + 200 nM (S)-7d
4.22 ± 0.45
11.79 ± 1.10
11.84 ± 1.10
13.22 ± 0.61
12.49 ± 1.22
2.53 ± 0.24
1.99 ± 0.04
1.51 ± 0.09
1.48 ± 0.02
1.05 ± 0.07
0.60 ± 0.04
0.17 ± 0.014
0.13 ± 0.005
0.11 ± 0.004
0.084 ± 0.004
1
0.78 ± 0.04
0.85 ± 0.05
0.91 ± 0.03
0.84 ± 0.05
Combined the kinetic and structural data, we believe that the allosteric changes of the H-loop play
a key role in the PDE5 catalysis. This mechanism, conformational and positional changes of a local
segment without domain movement, is different from classic allosteric modulation,15-19 in which
quaternary structural changes or domain rearrangements upon effecter binding lead to conformational
changes at the active site. Therefore, the regulation of the PDE5 catalysis by conformational change
of the H-loop may represent a new pattern of allosteric modulation.
Drug-like evaluations of (S)-7d. In view of better PK property of (S)-7d with an oral
bioavailability of 14% (Table 2) than (S)-7e (1%), only the former was carried out drug-like profile
and in vivo animal studies.
An automated patch clamp electrophysiology measurement showed that (S)-7d inhibited hERG
1
3,26-28
(
human ether-a-go-go related gene) with an IC of 20 μM (Table 5) in CHO-hERG cells,
50
suggesting a weak inhibitory potency on hERG and remarkable cardiac safety. The inhibitory IC50
values of (S)-7d against five human hepatic CYP P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4)
were >10 μM, which indicates that (S)-7d may not exhibit significant pharmacokinetic interactions
with drugs that are metabolized by the five major CYP isoforms. In addition, to test the maximum
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tolerated dose of (S)-7d, three groups of mice (10 each) were given at single oral doses of 0, 1000, and
1500 mg/kg (S)-7d on the first day and no acute toxicity was observed for all the (S)-7d groups on the
4
5
6
7
8
9
1
1
1
1
1
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6
1
4th day.
0
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0
Table 5. Physicochemical Properties of (S)-7d
Content
Value
-
Pharmacokinetic properties (p.o.)
t1/2
1.43 h
14 %
10 μM
F%
Cytochrome P450 inhibition (IC )
5
0
CYP1A2, 2B6, 2C9, 2D6, and 3A4
hERG inhibition (IC )
20 μM
5
0
Acute toxicity
>1.5 g/kg
Physiological significance of the allosteric site validated by in vivo studies. To assess whether
binding of the EVO derivatives to the allosteric pocket is associated with physiological functions of
PDE5, a mouse model on pulmonary hypertension (PH)13, 26-27 was conducted in vivo using (S)-7d.
The oral daily dose of 20 mg/kg (S)-7d significantly decreased the hypertension pressure and the wall
thickness of small pulmonary arteries, which were induced by monocrotaline (MCT), in a comparable
magnitude of sildenafil (Figure 6a-6b). In addition, hematoxylin and eosin-staining photomicrographs
showed that (S)-7d dilated small pulmonary vessels of the rats, as effective as sildenafil (Figure 6c-
6
f).
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Figure 6. Therapeutic effects of (S)-7d and sildenafil citrate on pulmonary arterial remodeling of rats.
a) Reduction of pulmonary pressure by (S)-7d and sildenafil citrate, which was induced by
(
monocrotaline (MCT). The data were shown as mean pulmonary artery pressure (mPAP ± S.E.M, n =
7-9), p<0.01 for comparison between the control (the first column) and the model (the second column).
(b) Effects of (S)-7d and sildenafil citrate on the wall thickness percentile (WT%) of small pulmonary
arteries of the PAH rats, p<0.01. (c-f) Representative HE-staining photomicrographs of small
pulmonary vessels for different groups (magnification × 400). (c) Control. (d) MCT-treated group. (e)
MCT + sildenafil citrate. (f) MCT + (S)-7d.
3
. Conclusions
In summary, our studies identified a novel/unique allosteric pocket in PDE5 using the EVO derivative
S)-7e whereas all traditional PDE5 inhibitors bind to the substrate binding pocket. Notably, this
(
unique allosteric pocket regulates both enzymatic activity and PH function of PDE5. This work
provides not only a molecular basis for understanding allosteric regulation of the PDE5 catalysis, but
also the structural templates for discovery of highly selective PDE5 inhibitors.
4
. Experimental
4
.1 Chemistry
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Reagents and solvents were purchased from commercial sources and used without purification. The
purity of all final products tested in vitro was determined by HPLC analysis as being >95%. NMR
spectra were recorded at room temperature on a Bruker Avance 600 spectrometer, operating at 600
4
5
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9
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1
13
MHz for H NMR and 125 MHz for C NMR or a Bruker 300 spectrometer, operating at 300 MHz
1
13
1
for H NMR and 75 MHz for C NMR. Data for H NMR are recorded as follows: chemical shift (δ,
ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet or unresolved, br s = broad singlet,
1
3
coupling constant (s) in Hz, integration). Data for C NMR are reported in terms of chemical shift (δ,
ppm). High resolution mass spectrometry (HRMS) was recorded on an Agilent 6538 UHD Accurate-
Mass Q-TOF LC/MS spectrometer. Chemical HG/T2354-92 silica gel (200-300 mesh, Haiyang®) was
used for chromatography, and silica gel plates with fluorescence F254 (0.25 mm, Huanghai®) were
used for thin-layer chromatography (TLC) analysis.
Synthetic routes for the preparation of the target compounds 7 are depicted in Scheme S1.
Compounds 7c, 7d, and 7e were prepared by a condensation reaction between substituted 3,4-dihydro-
β-carbolines 3 and substituted N-methyl isatoic anhydrides 6, following our previously reported
procedures for 7a and 7b.21,24 The substituted 3,4-dihydro-β-carbolines 3 were obtained using the
formylation reaction followed by intramolecular cyclization from the corresponding commercially
available tryptamine derivatives 1. To synthesize the substituted N-methyl isatoic anhydrides 6,
commercially available 2-aminobenzoic acid derivatives 4 were treated with triphosgene to give
substituted isatoic anhydrides 5, which were methylated to afford compounds 6. Subsequent
condensation of these substituted 3,4-dihydro-β-carbolines 3 with a series of substituted N-methyl
isatoic anhydrides 6 resulted in the desired compounds 7 (7a, 7b, 7d, and 7e). Treatment of 7b with
BBr in DCM afforded the corresponding 4-hydroxyl substituted derivative 7c. Chiral separation was
3
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performed to isolate the enantiomers of 7a-7e. The absolute configurations of the optically pure
products were assigned by comparing the sign of the optical rotation with that reported in literature.
2
1,24
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Preparation of the substituted 3,4-dihydro-β-carbolines (3a-c). The stirred suspension of 1 (10
mmol) in ethyl formate (60 mL) was heated under reflux for 6 h. After the reaction mixture was
evaporated under vacuum, the residue was diluted with DCM (10 mL) and added POCl (12 mmol) at
3
o
0 C. The reaction mixture was stirred at rt for 2 h prior to quenching with 2 N aqueous HCl (100 mL)
and extracting with Et O (15 mL). The combined aqueous phase was basified to pH 10 with aqueous
2
ammonia and extracted with DCM (3 x 60 mL). The combined organic phases were washed with brine,
dried over anhydrous Na SO , filtered and concentrated under reduced pressure to afford the
2
4
substituted 3,4-dihydro-β-carbolines 3, which was used directly in the next step without further
purification.
1
7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indole (3b). Brown solid; 42.3% yield; H NMR (600
MHz, DMSO) δ 11.11 (br s, 1H), 8.28 (s, 1H), 7.42 (d, J = 8.7 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.70
1
3
(
dd, J = 8.7, 2.2 Hz, 1H), 3.77 (s, 3H), 3.76-3.69 (m, 2H), 2.77-2.70 (m, 2H). C NMR (151 MHz,
DMSO) δ 157.73, 151.68, 138.12, 128.25, 120.79, 119.65, 114.72, 110.94, 95.03, 55.60, 48.44, 19.32.
+
MS (ESI) calcd for C H N O [M+H] : 201.10, found 201.31.
1
2
13
2
Preparation of the substituted N-methyl isatoic anhydrides (6a-b). The stirred suspension of 2-
aminobenzoic acid derivatives 4 (10 mmol) and triphosgene (3.3 mmol) in THF (15 mL) was heated
o
at 65 C overnight. The precipitate was collected by filtration, washed with THF, and dried to give
substituted isatoic anhydrides 5. To a stirred solution of 5 (10 mmol) in DMF (20 mL) was added NaH
o
(20 mmol) in portions under nitrogen at 0 C. The mixture was stirred at rt for 1 h, then added CH I
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(15 mmol). After stirring at rt for 6 h, the reaction mixture was added water (80 mL). The resulting
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precipitate was collected by filtration, washed with water and EtOH, and dried to give 6.
1
5
-methoxy-1-methyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (6b). White solid; 60% yield; H
0
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2
3
4
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8
9
0
NMR (600 MHz, DMSO) δ 7.73 (t, J = 8.5 Hz, 1H), 6.92 (dd, J = 12.2, 8.5 Hz, 2H), 3.89 (s, 3H), 3.40
13
(
s, 3H). C NMR (151 MHz, DMSO) δ 161.91, 155.06, 148.52, 144.46, 138.20, 107.07, 106.85,
-
1
00.88, 56.88, 32.64. HRMS (ESI) calcd for C H NO [M+H] :208.0610, found 208.0603.
1
0
9
4
Preparation of the evodiamine derivatives (7b, 7d, and 7e). A solution of substituted N-methyl
isatoic anhydrides 6 (1 mmol) and substituted 3,4-dihydro-β-carbolines 3 (1.2 mmol) in DCM (5 mL)
was stirred overnight at rt. The resulting precipitate was collected by filtration, washed with DCM to
give the target compounds 7b, 7d, and 7e.
1
Evodiamine (7a). H NMR (300 MHz, DMSO) δ 11.06 (br s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.47 (d,
J = 7.3 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.13-6.93 (m, 4H), 6.12 (s, 1H), 4.66-4.60 (m, 1H), 3.24-
13
3
1
3
.15 (m, 1H), 3.00-2.71 (m, 5H). C NMR (75 MHz, DMSO) δ 164.70, 149.25, 136.94, 133.91,
31.07, 128.46, 126.42, 122.32, 120.75, 119.74, 119.36, 118.69, 117.95, 112.12, 111.96, 70.21, 41.33,
+
6.91, 19.95. HRMS (ESI) calcd for C H N O [M+H] : 304.1450, found 304.1443. [Daicel
1
9
18
3
CHIRALPAK AD-H (0.46 cm x 15 cm). hexane/EtOH (0.1% Diethylamine) = 60/40; flow rate = 0.5
2
0
mL/min; detection wavelength = 254 nm; t = 3.21 (R-7a), t = 4.05 (S-7a) min]; (R)-7a [α] = -
1
2
D
20
3
84.1 (c 0.1, CHCl ), ee>99%; (S)-7a [α] = 382.9 (c 0.1, CHCl ), ee>99%.
3 D 3
4
-methoxy-14-methyl-7,8,13b,14-tetrahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(13H)-
1
one (7b). Pale solid; 86% yield; H NMR (600 MHz, DMSO) δ 11.21 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H),
7.43-7.34 (m, 2H), 7.16-7.08 (m, 1H), 7.07-6.99 (m, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.67 (d, J = 8.3 Hz,
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H), 5.83 (s, 1H), 4.49-4.45 (m, 1H), 3.78 (s, 3H), 3.26-3.22 (m, 1H), 2.93-2.76 (m, 2H), 2.63 (s, 3H).
13C NMR (151 MHz, DMSO) δ 161.90, 160.50, 151.92, 136.74, 133.12, 129.05, 125.70, 121.91,
1
18.84, 118.37, 111.76, 111.60, 111.40, 110.80, 105.49, 67.64, 55.80, 35.71, 19.74. HRMS (ESI) calcd
+
for C H N O [M+H] : 334.1556, found 334.1549. [Daicel CHIRALPAK AD-H (0.46 cm x 15 cm).
2
0
20
3
2
0
1
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hexane/EtOH (0.1% Diethylamine) = 60/40; flow rate = 0.5 mL/min; detection wavelength = 254 nm;
20
20
t = 3.14 (R-7b), t = 4.98 (S-7b) min]; (R)-7b [α] = -521.9 (c 0.15, CHCl ), ee>99%; (S)-7b [α]
1
2
D
3
D
=
544.2 (c 0.15, CHCl ), ee>99%.
3
4-hydroxy-14-methyl-7,8,13b,14-tetrahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(13H)-one
7c). To a stirred solution of 7b (0.1 g, 0.3 mmol) in DCM (10 mL), a solution of BBr (0.06 mL, 0.6
(
3
mmol) in DCM (5 mL) was added over a period of 2-3 min at -78 °C. The reaction mixture was stirred
under nitrogen atmosphere for 5 h, and then 5 mL of methanol was added. The reaction mixture was
treated with saturated sodium bicarbonate (50 mL) and extracted with DCM (3×50 mL). The combined
organic layers were dried over NaSO and filtered. The solvent was removed under reduced pressure,
4
and the residue was purified by silica gel column chromatography (DCM/MeOH = 100:1) to give
1
compound 7c as a white solid (0.05 g, 50% yield). H NMR (300 MHz, DMSO) δ 12.51 (s, 1H), 11.01
(br s, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.30 (t, J = 8.2 Hz, 1H), 7.11 (t, J = 7.5
Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.39 (d, J = 8.1 Hz, 1H), 6.29 (d, J = 8.2 Hz, 1H), 6.24 (s, 1H), 4.61
1
3
(dd, J = 13.1, 5.2 Hz, 1H), 3.30-3.12 (m, 1H), 3.09-2.89 (m, 4H), 2.81-2.74 (m, 1H). C NMR (75
MHz, DMSO) δ 169.72, 161.82, 149.20, 136.84, 136.14, 131.24, 126.43, 122.44, 119.48, 118.69,
1
12.18, 111.61, 108.04, 106.34, 102.48, 70.81, 41.58, 37.21, 19.77. HRMS (ESI) calcd for C H N O
19 16 3 2
-
[M-H] :318.1243, found 318.1250. [Daicel CHIRALPAK AD-H (0.46 cm x 15 cm). hexane/EtOH (0.1%
Diethylamine) = 60/40; flow rate = 0.5 mL/min; detection wavelength = 254 nm; t = 5.20 (R-7c), t
1
2
=
6.12 (S-7c) min]; (R)-7c, [α]D20 = -326.7 (c 0.2, acetone), ee>99%; (S)-7c, [α]D20 = 359.4 (c 0.2,
acetone), ee=98.5%.
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,11-dimethoxy-14-methyl-7,8,13b,14-tetrahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(13H)-
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5
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8
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one (7d). Pale solid; 79% yield, H NMR (600 MHz, DMSO) δ 11.03 (s, 1H), 7.37 (t, J = 8.6 Hz, 2H),
6
3
.84 (d, J = 2.1 Hz, 1H), 6.71 (d, J = 8.2 Hz, 1H), 6.68-6.62 (m, 2H), 5.74 (s, 1H), 4.44-4.41 (m, 1H),
0
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8
9
0
13
.75 (d, J = 6.9 Hz, 6H), 3.21-3.16 (m, 1H), 2.82-2.73 (m, 2H), 2.58 (s, 3H). C NMR (75 MHz,
DMSO) δ 162.38, 160.89, 156.53, 152.42, 138.03, 133.58, 127.89, 120.48, 119.53, 112.28, 111.89,
11.25, 109.52, 105.81, 95.08, 67.97, 56.20, 55.61, 36.00, 20.27. HRMS (ESI) calcd for C H N O
1
2
1
22
3
3
+
[M+H] :364.1661, found 364.1657. [Daicel CHIRALPAK AD-H (0.46 cm x 15 cm). hexane/EtOH
(
0.1% Diethylamine) = 60/40; flow rate = 0.5 mL/min; detection wavelength = 254 nm; t = 2.13 (R-
1
20
20
7
d), t = 3.88 (S-7d) min]; (R)-7d, [α] = -614.5 (c 0.15, CHCl ), ee>99%; (S)-7d, [α] = 680.3 (c
2 D 3 D
0
.15, CHCl ), ee>99%.
3
4
5
,9-dimethoxy-14-methyl-7,8,13b,14-tetrahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-
1
(13H)-one (7e). Pale solid; 80% yield, H NMR (600 MHz, DMSO) δ 11.22 (br s, 1H), 7.39 (t, J =
8.2 Hz, 1H), 7.02 (t, J = 7.9 Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.67 (d, J =
8
3
.2 Hz, 1H), 6.49 (d, J = 7.7 Hz, 1H), 5.77 (s, 1H), 4.43-4.39 (m, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.26-
1
3
.16 (m, 1H), 3.09 (d, J = 15.7 Hz, 1H), 3.01-2.87 (m, 1H), 2.57 (s, 3H). C NMR (151 MHz, DMSO)
δ 162.29, 160.92, 154.59, 152.52, 138.55, 133.53, 127.55, 123.27, 116.17, 112.14, 111.91, 111.51,
+
106.01, 105.50, 99.72, 67.81, 56.27, 55.51, 35.99, 22.32. HRMS (ESI) calcd for C H N O [M-H] :
2
1
20
3
3
3
62.1505, found 362.1510. [Daicel CHIRALPAK AD-H (0.46 cm x 15 cm). hexane/EtOH (0.1%
Diethylamine) = 60/40; flow rate = 0.5 mL/min; detection wavelength = 254 nm; t = 1.13 (R-7e), t
1
2
= 1.80 (S-7e) min]; (S)-7e, [α]D20 = 592.26 (c 0.15, CHCl ), ee=97.3%.
3
4
.2. Protein expression and purification of the catalytic domains of PDEs
The cDNA of the catalytic domain of human PDE5A1 was generated by site-directed mutagenesis on
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the gene of bovine PDE5A, as previously described. The coding regions for amino acids 535-860 of
PDE5A1 were amplified by PCR and subcloned into the expression vector pET15b. The resultant
plasmid pET-PDE5A1 was transferred into E. coli strain BL21 (Codonplus) for overexpression. The
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o
E. coli cell carrying pET-PDE5A1 was grown in LB medium at 37 C to absorption A600 = 0.7 and
o
then 0.1 mM isopropyl -D-thiogalactopyranoside was added for further growth at 15 C overnight.
Recombinant PDE5A1 was passed through the Ni-NTA affinity column (Qiagen), subjected to
thrombin cleavage to remove the His-tag, and further purified by the columns of Q-Sepharose and
Sephacryl S300 (Amersham Biosciences). A typical batch of purification yielded over 10 mg PDE5A1
with a purity >95% from a 2-liter cell culture. The similar protocols were adopted for the protein
expression and purification of other PDEs.13,26-28 PDE6C was purchased from BPS Bioscience.
3
3
Enzymatic properties. The enzymatic activities were assayed using H-cAMP or H-cGMP as
substrates, as previously reported.13,26-28 Briefly, the wild type proteins were incubated with a reaction
3
3
mixture of 20 mM Tris.HCl, pH 7.5, 10 mM MgCl , 0.5 mM DTT, H-cAMP or H-cGMP (20-40k
2
cpm/assay) at room temperature for 15 min. The reaction was terminated by addition of 0.2 M ZnSO4.
3
3
The reaction product H-AMP or H-GMP was precipitated out by addition of 0.2 N Ba(OH) while
2
3
3
unreacted H-cAMP or H-cGMP stayed in the supernatant. Radioactivity in the supernatant was
measured by a liquid scintillation counter. The inhibition of the reaction was measured at nine
concentrations of inhibitors, using suitable concentrations of the substrates and enzymes. The
inhibition of PDE5 by the lead compounds is shown in Table 1. PDE5 inhibitor sildenafil was used as
a positive control to validate the assay conditions.
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Protein crystallization and structure determination. The unliganded PDE5A1 (10-15 mg/ml) was
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crystallized by vapor diffusion at room temperature against well buffer of 0.2 M MgSO , 0.1 M
4
Tris.base (pH 8.5), 12% PEG 3350, and 2% ethanol. The unliganded PDE5 crystals had the space
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group of P3 21 with unit cell dimensions of a = b = ~ 74 and c = ~ 132 Å (Table S2). The complex of
1
o
PDE5A1-(S)-7e was prepared by mixing 2 mM (S)-7e with 10 mg/mL PDE5A1 at 4 C overnight and
co-crystallized by vapor diffusion against a well buffer of 8% PEG3350, 0.1 M sodium acetate 4.6, 0.2
M ammonium acetate, at 25°C. The PDE5-(S)-7e crystal had the space group of P2 2 2 with unit cell
1
1 1
dimensions of a =~66, b = ~ 86 and c = ~ 124 Å.
The diffraction data were collected on an in-house Oxford Diffraction Xcalibur Nova
2
9
diffractometer, and processed by program HKL2000 (Table S2). The structures were solved by
30
molecular replacement, using the wild type PDE5A as the initial model. The atomic model was
3
1
32
rebuilt by program COOT and refined by program RAFMAC5 .
4
.3 Pharmacokinetic measurement. The in vivo pharmacokinetic properties of (S)-7d and (S)-7e
were analyzed by the Medicilon Company, Shanghai, China. Six male SD rats with a body weight of
30-260 g were purchased from Shanghai SIPPR-BK LAB Animal Ltd., Shanghai, China, and used
2
for the pharmacokinetic analysis. (S)-7d and (S)-7e were dissolved/suspended respectively in 5%
DMSO/10% Solutol/85% water and 5% DMSO/95% (20% HP-β-CD）for intravenous administration
(IV) and oral administration (PO). The formulated compounds at final dosages of 2.5 and 5 mg/kg rat
were administered for IV and PO purposes, respectively. The blood samples were taken at various time
points in a 24 h period. The concentration of the compounds in blood was analyzed by LC-MS/MS
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(
Shimadzu liquid chromatographic system and API4000 mass spectrometer, Applied Biosystems,
Ontario, Canada).
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.4 hERG inhibition. The assays were performed by the Medicilon Company, Shanghai, China.
hERG inhibition was performed using an automated patch clamp electrophysiology measurement in
CHO-hERG cells18, 26-28
.
4.5 Acute Toxicity of (S)-7d. All animal care and experimental protocols were in accordance with
Guide for the Care and Use of Laboratory Animals” (National Institutes of Health Publication, revised
996, No.86-23, Bethesda, MD) and were approved by the Institutional Ethical Committee for Animal
“
1
Research of Sun Yat-sen University. The acute toxicity was tested following the protocols that were
described in our previous study13, 26-28. Thirty KM mice (22 days, 18-20 g) were purchased from the
Laboratory Animal Center of Sun Yat-Sen University (Guangzhou, China) and used to evaluate the
acute toxicity of (S)-7d. Mice were randomly divided into three groups, each of which was given a
single oral dose of 0, 1000, or 1500 mg (S)-7d per kg mice on the first day of the experiment. Mice
were maintained on a 12 h light/dark cycle (light from 7:00 to 19:00) at room temperature and 60−70%
humidity. Sterile food and water were provided according the institutional guidelines. Prior to each
experiment, mice were fasted overnight and allowed free access to water. (S)-7d was dissolved in a
solution of 5% DMSO/10% Solutol/85% water and orally administered. Mice were observed for any
abnormal behavior and mortality and weighed four hours after (S)-7d was administered and then every
2
4 h for 14 days. Animals were sacrificed on the 14th day, and tissue samples of the heart, liver, and
kidney were macroscopically examined for possible damage.
4
.6 Pharmacodynamics effects of (S)-7d in vivo.
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Forty-eight Wister rats (8 weeks, 180−220 g), purchased from the Laboratory Animal Center of
Southern Medical University (Guangzhou, China), were used to evaluate the pharmacodynamics
effects of (S)-7d on PAH. The rats were randomly divided into four groups: control, MCT-treated
model, MCT + (S)-7d (20.0 mg/kg), and positive control (MCT + sildenafil citrate, 10 mg/kg). Rats
were maintained on a 12 h light/dark cycle (light from 7: 00 to 19: 00) at 24 ± 1 °C and 60−70%
relative humidity. Sterile food and water were given according to the institutional guidelines. Prior to
each experiment, the rats were fasted overnight and allowed free access to water. The rats were
administrated with MCT 60 mg/kg except for the control group and then orally fed with drug vehicle
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(control and model groups), compound (S)-7d (20.0 mg/kg), and sildenafil citrate (10.0 mg/kg) for 3
weeks, respectively. Compound (S)-7d and sildenafil citrate were dissolved in 15% Solutol/85% water
solution and orally administrated 0.4 mL per 100 g weight. The method of right cardiac catheter was
applied to measure the pulmonary artery pressure and the mean pulmonary artery pressure (mPAP)
was used for statistics. Marginal left lower pulmonary lobes were harvested after the rats were killed
and immersed in 4% buffered paraformaldehyde at room temperature overnight, and then embedded
in paraffin wax. Paraffin-embedded tissue was sliced at thickness of 5 μm and mounted on glass slides.
33
Tissue sections were stained with hematoxylin and eosin (HE) as previously described . Pulmonary
vascular remodelling was assessed by measuring the percentage medial wall thickness of vessels (50–
2
00 μm diameter). Vessel external diameter and medial wall thickness (MT) were measured using a
light microscope by an observer who was blinded to the treatment of the rats.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website.
The putative binding pattern of (S)-7b with PDE5 by docking, X-ray data of PDE5 in complex
with (S)-7e, Binding of (S)-7e to PDE5, Characterization of target compounds, and HPLC spectra data
for tested compounds (pdf).
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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 codes of 6VBI. Authors will release the atomic coordinates and experimental data upon
article publication.
AUTHOR INFORMATION
Corresponding Authors
*
shengcq@smmu.edu.cn, hengming_ke@med.unc.edu, and luohb77@mail.sysu.edu.cn
T.Z. and Z.L. contributed equally to the work.
#,
ORCID
Hai-Bin Luo: 0000-0002-2163-0509
Chunquan Sheng: orcid.org/0000-0001-9489-804X
ACKNOWLEDGMENTS
This work was supported by the Natural Science Foundation of China (21877134, 81725020,
8
1525020, and 81703341), U.S. NIH Grant GM59791, Science Foundation of Guangzhou City
(201904020023), and Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar
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Funded Scheme (2016). We sincerely thank the anonymous reviewers for their constructive comments
on this paper.
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ABBREVIATION
PDE5, phosphodiesterase-5; EVO, evodiamine; PDEs, phosphodiesterases; cAMP, cyclic adenosine
3
', 5'-monophosphate; cGMP, cyclic guanosine 3', 5'-monophosphate; PH, pulmonary hypertension;
IC , half-inhibitory concentration; PAINS, pan-assay interfering compound substructures; THF,
5
0
tetrahydrofuran; DMF, N,N-Dimethylformamide; PK, pharmacokinetics; p.o., oral administration;
hERG, the human Ether-à-go-go-Related Gene; CYP, cytochrome P450; AUC, under the curves; Cmax
,
peak concentration; T , half time; Tmax, peak time; MRT, mean residence time; F, bioavailability; i.v.,
1/2
intravenous administration; WT, the wild type; MCT, monocrotaline; mPAP, the mean pulmonary
artery pressure; WT%, the wall thickness percentile; HE, hematoxylin and eosin; RLM, rat liver
microsomes; SAR, structure-activity relationships; HPLC, high performance liquid chromatography;
HRMS, high resolution mass spectrum; LC-MS, liquid chromatograph-mass spectrometer; TLC, thin-
layer chromatography; DCM, dichloromethane; DMSO, dimethyl sulfoxide; PCR, polymerase chain
reaction; DTT, dithiothreitol; PEG, polyethylene glycol.
REFERENCES
(
1) Bender, A. T.; Beavo, J. A. Cyclic Nucleotide Phosphodiesterases: Molecular Regulation to
Clinical Use. Pharmacol. Rev. 2006, 58, 488-520.
2) Pissarnitski, D. Phosphodiesterase 5 (PDE 5) Inhibitors for the Treatment of Male Erectile
(
Disorder: Attaining Selectivity Versus PDE6. Med. Res. Rev. 2006, 26, 369-395.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 34
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
3) Maurice, D. H.; Ke, H.; Ahmad, F.; Wang, Y.; Chung, J.; Manganiello, V. C. Advances in
Targeting Cyclic Nucleotide Phosphodiesterases. Nat. Rev. Drug Discovery 2014, 13, 290-314.
4) Baillie, G. S.; Tejeda, G. S.; Kelly, M. P. Therapeutic Targeting of 3',5'-Cyclic Nucleotide
Phosphodiesterases: Inhibition and Beyond. Nat Rev Drug Discov 2019, 18 (10), 770-796.
5) Kokkonen, K.; Kass, D. A. Nanodomain Regulation of Cardiac Cyclic Nucleotide Signaling By
(
0
1
2
3
4
5
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7
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9
0
1
2
3
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5
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2
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4
5
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0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Phosphodiesterases. Annu. Rev. Pharmacol. Toxicol. 2017, 57, 455-479.
(6) Rodrã-Guez, C. I.; Setaluri, V. Cyclic AMP (cAMP) Signaling in Melanocytes and Melanoma.
Arch. Biochem. Biophys. 2014, 563, 22-27.
(
7) Rotella, D. P. Phosphodiesterase 5 Inhibitors: Current Status And Potential Applications. Nat. Rev.
Drug Discov. 2002, 1, 674-682.
8) Galiè, N.; Ghofrani, H. A.; Torbicki, A.; Barst, R. J.; Rubin, L. J.; Badesch, D.; Fleming, T.; Parpia,
(
T.; Burgess, G.; Branzi, A. Sildenafil Citrate Therapy For Pulmonary Arterial Hypertension. New Engl.
J. Med. 2005, 353, 2148-2157.
(
9) Rybalkin, S. D.; Rybalkina, I. G.; Shimizu‐Albergine, M.; Tang, X. B.; Beavo, J. A. PDE5 is
Converted to an Activated State Upon cGMP Binding to the GAF A Domain. EMBO J. 2003, 22, 469-
78.
4
(10)Zoraghi, R.; Bessay, E. P.; Corbin, J. D.; Francis, S. H. Structural and Functional Features in
Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and
Regulation. J. Biol. Chem. 2005, 280, 12051-12063.
(11)Wang, H.; Robinson, H.; Ke, H. Conformation Changes, N-terminal Involvement, and cGMP
Signal Relay in the Phosphodiesterase-5 GAF Domain. J. Biol. Chem. 2010, 285, 38149-38156.
ACS Paragon Plus Environment

Page 29 of 34
Journal of Medicinal Chemistry
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(12)Wang, H.; Liu, Y.; Huai, Q.; Cai, J.; Zoraghi, R.; Francis, S. H.; Corbin, J. D.; Robinson, H.; Xin,
4
5
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5
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Z.; Lin, G. Multiple Conformations of Phosphodiesterase-5: Implications for Enzyme Function and
Drug Development. J. Biol. Chem. 2006, 281, 21469-21479.
0
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(13)(a) Wu, D.W.; Zhang, T.H.; Chen, Y.; Huang, Y.; Geng, H.; Yu, Y.; Zhang, C., Lai, Z.W.; Wu,
Y.; Guo, X.; Chen, J. W.; Luo, L.-B. Discovery and Optimization of Chromeno[2,3-c]pyrrol-9(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. (b) Wang, H.; Ye, M.;
Robinson, H.; Francis, S. H.; Ke, H. Conformational Variations of Both phosphodiesterase-5 and
Inhibitors Provide the Structural Basis for the Physiological Effects of Vardenafil and Sildenafil. Mol.
Pharmacol. 2008, 73, 104-110.
(14) Wang, Z.; Jiang, X.; Zhang, X.; Tian, G.; Yang, R.; Wu, J.; Zou, X.; Liu, Z.; Yang, X.; Wu, C.;
Shi, J.; Li, J.; Suo, J.; Wang, Y.; Zhang, R.; Xu, Z.; Gong, X.; He, Y.; Zhu, W.; Aisa, H. A.; Jiang, H.;
Xu, Y.; Shen, J. Pharmacokinetics-Driven Optimization of 4(3H)-Pyrimidinones as Phosphodiesterase
Type 5 Inhibitors Leading to TPN171, a Clinical Candidate for the Treatment of Pulmonary Arterial
Hypertension. J. Med. Chem. 2019, 62, 4979-4990.
(
15) Wei, J.; Leit, S.; Kuai, J.; Therrien, E.; Rafi, S.; Harwood, H.J.; DeLaBarre, B.; Tong, L. An
Allosteric Mechanism for Potent Inhibition of Human ATP-citrate Lyase. Nature 2019, 568, 566–570.
16) Jaeger, K.; Bruenle, S.; Weinert, T.; Guba, W.; Muehle, J.; Miyazaki, T.; Weber, M.; Furrer,
(
A.; Haenggi, N.; Tetaz, T.; Huang, C.Y.; Mattle, D.; Vonach, J.M.; Gast, A, Kuglstatter, A, Rudolph,
M.G.; Nogly, P.; Benz, J.; Dawson, R.J.P.; Standfuss, J. Structural Basis for Allosteric Ligand
Recognition in the Human CC Chemokine Receptor 7. Cell 2019, 178, 1222-1230.
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