Structure-based discovery of highly selective phosphodiesterase-9A inhibitors and implications for inhibitor design

Article abstract of DOI:10.1021/jm301189c

A new series of phosphodiesterase-9 (PDE9) inhibitors that contain a scaffold of 6-amino-pyrazolopyrimidinone have been discovered by a combination of structure-based design and computational docking. This procedure significantly saved the load of chemical synthesis and is an effective method for the discovery of inhibitors. The best compound 28 has an IC₅₀ of 21 nM and 3.3 μM, respectively, for PDE9 and PDE5 and about 3 orders of magnitude of selectivity against other PDE families. The crystal structure of the PDE9 catalytic domain in complex with 28 has been determined and shows a hydrogen bond between 28 and Tyr424. This hydrogen bond may account for the 860-fold selectivity of 28 against PDE1B, in comparison with about 30-fold selectivity of BAY73-6691. Thus, our studies suggest that Tyr424, a unique residue of PDE8 and PDE9, is a potential target for improvement of selectivity of PDE9 inhibitors.

Full text of DOI:10.1021/jm301189c

Article
pubs.acs.org/jmc
Structure-Based Discovery of Highly Selective Phosphodiesterase-9A
Inhibitors and Implications for Inhibitor Design
Fei Meng,†,∥ Jing Hou,‡,∥ Yong-Xian Shao,‡,∥ Pei-Ying Wu,§,∥ Manna Huang,† Xinhai Zhu,†
Yonghong Cai,‡ Zhe Li,‡ Jie Xu,‡ Peiqing Liu,‡ Hai-Bin Luo,*,‡ Yiqian Wan,*,† and Hengming Ke*,‡,§
†School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, People's Republic of China
‡School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People's Republic of China
§Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina,
Chapel Hill, North Carolina 27599-7260, United States
S
* Supporting Information
ABSTRACT: A new series of phosphodiesterase-9 (PDE9) inhibitors that contain
a scaffold of 6-amino-pyrazolopyrimidinone have been discovered by a
combination of structure-based design and computational docking. This procedure
significantly saved the load of chemical synthesis and is an effective method for the
discovery of inhibitors. The best compound 28 has an IC50 of 21 nM and 3.3 μM,
respectively, for PDE9 and PDE5 and about 3 orders of magnitude of selectivity
against other PDE families. The crystal structure of the PDE9 catalytic domain in
complex with 28 has been determined and shows a hydrogen bond between 28 and
Tyr424. This hydrogen bond may account for the 860-fold selectivity of 28 against
PDE1B, in comparison with about 30-fold selectivity of BAY73-6691. Thus, our
studies suggest that Tyr424, a unique residue of PDE8 and PDE9, is a potential
target for improvement of selectivity of PDE9 inhibitors.
series of PDE9 inhibitors (Scheme 1) that contain a scaffold of
6-amino-pyrazolopyrimidinone and are capable of directly
INTRODUCTION
■
The second messengers adenosine and guanosine 3′,5′-cyclic
monophosphate (cAMP and cGMP) modulate many physio-
logical processes, such as cardiac and smooth muscle
contraction, steroid hormone function, platelet aggregation,
apoptosis, leukocyte migration, adrenal hyperplasia, inflamma-
tion, axon guidance and regeneration, memory, and circadian
regulation.1−6 Cyclic nucleotide phosphodiesterases (PDEs)
hydrolyze intracellular cAMP and cGMP and thus play key
roles in the cellular processes.7−10 Family-selective PDE
inhibitors have been widely studied as therapeutics for the
treatment of human diseases.11−17 The most successful example
of this drug class is the PDE5 inhibitor sildenafil that has been
marketed as drugs (Viagra and Revatio) for the treatments of
male erectile dysfunction and pulmonary hypertension.11,12
PDE9 is a family of cGMP-specific enzymes and has the
lowest KM among cGMP-specific PDE families.7−10 Selective
PDE9 inhibitors have been studied as potential therapeutics for
the treatment of diabetes and CNS diseases such as Alzheimer's
disease.18−30 The crystal structures of the PDE9A catalytic
domain in complex with the inhibitors20,31−33 and the substrate
cGMP34 have not only provided insight into the catalytic
mechanism but also served as templates to lead to discovery of
PDE9 inhibitors with high affinity.18,20−22,30 However, some of
these PDE9 inhibitors showed only moderate selectivity against
PDE1,18,20 which might cause side effects for the treatment of
CNS diseases since PDE1 is abundant in the brain.30 To
explore improvement of the selectivity, we designed a novel
Scheme 1. Chemical Structure of Our Compounds
forming a hydrogen bond with Tyr424 that is unique to
PDE9 and PDE8. The best compound in this series of the
PDE9 inhibitors showed about 3 orders of magnitude of
selectivity against PDE1. We also reported a combined
procedure of structure-based inhibitor design and computa-
tional docking, which significantly saved the load of chemical
synthesis and is thus an effective method for the discovery of
PDE9 inhibitors.
RESULTS
■
Design of a New Series of PDE9 Inhibitors. Because
most current PDE9 inhibitors contain a pharmacophore of
pyrazolopyrimidinone,18,20−23 we chose 6-amino-pyrazolopyr-
imidinone as the scaffold to design a new series of PDE9
inhibitors by using the template structure of PDE9 in complex
Received: August 13, 2012
Published: September 17, 2012
© 2012 American Chemical Society
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Figure 1. Design of a new type of PDE9 inhibitors. (A) Surface presentation of 10r binding to the pocket of PDE9A2. Color codes are white for
carbon, blue for nitrogen, red for oxygen of PDE9A2, and orange for chlorine. Inhibitor 10r is shown in both surface and stick models (light green).
(B) Design of a scaffold that may potentially form a hydrogen bond with Tyr424 and fit tightly to the PDE9 pocket.
a
Scheme 2. Syntheses of Intermediates 7 and 8
a
Reagents and conditions: (a) EtOH, 60 °C. (b) 30% H2O2/25% NH3·H2O = 1:3 (v/v), rt. (c) Urea, 200 °C. (d) PCl5, POCl3, reflux. (e) 2 N (aq)
KOH, 60 °C.
with (R)-BAY73-669132 [10r, 1-(2-chlorophenyl)-6-(3,3,3-
trifluoro-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidine-
4(5H)-one]. As revealed by the crystal structure, 10r forms two
hydrogen bonds with invariant Gln453 and stacks against
Phe456 (Figure 1A), thus accounting for its IC50 of 22 nM
against PDE9.32 The structure also shows that Tyr424 of
PDE9A2 does not form a hydrogen bond with 10r but is
located at another site to the small pocket where the
fluoromethyl group of 10r occupies (Figure 1A). To target
interaction with Tyr424, we manually constituted a linker chain
with various lengths and different substitution groups in a
graphic terminal. The linker contains an alanine, in the hope
that the methyl side chain or the Cβ atom would interact with
Gly452 and Phe441 to prevent the substitution from entering
the neighboring small subpocket (top right in Figure 1B) so as
to steer the chain toward Tyr424. The amide group of the
alanine linker would form a hydrogen bond with Tyr424 for
improvement of selectivity against other PDE families. Finally, a
phenyl group at the end of the substitution is designed to
occupy the partially open hydrophobic pocket that is composed
of Met365, Phe441, and Val460 of PDE9A2 for further
enhancement of the inhibitor affinity (Figure 1B). This idea
was originated on the basis of Bayer's compound BR4872 (Drs.
Andreas Knorr and Frank Wunder, personal communication)
and was then verified by docking various fragments into the
binding pocket of PDE9A2.
With the Surflex-Dock35,36 program, the X-ray crystal
structure of PDE9A in complex with the inhibitor (S)-
BAY73-6691 (10s; PDB code, 3K3H)32 was chosen to validate
our docking. In the docking with the bound inhibitor, the top
30 poses of the docked conformations have the RMSDs in a
range of 0.4−1.9 Å from the true position in the crystal
structure, and eight of them were less than 1.5 Å, thus
indicating that the Surflex-Dock program could produce reliable
docking results. Under identical docking conditions, the
manually designed fragments were docked into the binding
pocket of PDE9A. This manual design and computational
docking led to identification of the basic fragment shown in
Scheme 1 and significantly saved the load of synthesis.
Chemical Synthesis. A series of compounds with various
substituents of R and R′ (Scheme 1) were synthesized by
following the similar protocols in literature.37,38 Thus, the
commercially available arylhydrazines (1) were converted to
the corresponding pyrazoles (3) by reacting with ethoxymethyl-
enemalononitrile (2). Subsequent oxidation yielded carbox-
amide (4) that then underwent intermolecular cyclization by
urea to deliver 1-aryl (akyl)-4,6-dihydroxypyrazolo[3,4-d]-
pyrimidine (5). Chlorination of 5 by phosphorus oxychloride
in the presence of phosphorus pentachloride yielded 1-
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a
Scheme 3. Syntheses of Intermediates 14−21
a
Reagents and conditions: (a) DMF, 1-methyllimidazole, then DECP, rt. (b) MeOH/HCl, 0 °C.
aryl(akyl)-4,6-dichloropyrazolo[3,4-d] pyrimidine (6). Hydrol-
ysis of 6 by KOH produced 1-aryl(alkyl)-6-chloro-4-
hydroxypyrazolo[3,4-d] pyrimidines (7 and 8) (Scheme 2).
The reaction of 2-((tert-butoxycarbonyl)amino)propanoic
acid (11) with aromatic amines (12) yielded compounds 13.
Subsequent deprotection of 13 in HCl (g)-methanol solution
afforded the corresponding compounds 14−21 (Scheme 3).
The desired compounds 26−35 were prepared according to
well-documented methods,37,38 as outlined in Scheme 4.
next section. The R substitutions at the m-, o-, and p-positions
of the phenyl ring are also important for binding of the
inhibitors. A comparison of 28 with 31 and 32 shows that
substitution of the methoxyl at the p-position (28) rather than
at the o- or m-position increases inhibitory potency by 10−15-
fold (Table 1). This result may be reasonably explained by the
crystal structure, in which the m- and o-substitutions might
disturb the van der Waals' interaction with Met365. Among the
p-substitutions tested here, the methoxyl group is most potent
(compare 26−30 and 33). This is perhaps due to the
hydrophilic nature of the nearby binding pocket so that too
much hydrophobicity would not favor the inhibitor binding.
In terms of inhibitory selectivity, compound 28 shows at least
150-fold selectivity against other PDE families (Table 2).
Especially worth mentioning is the 860-fold selectivity against
PDE1B, which is significantly better than those of other PDE9
inhibitors such as 10r (about 30-fold)18 and PF-04447943 (9,
about 170-fold)30 that has been the best PDE9 inhibitor having
great pharmacological potency for the treatment of CNS
diseases.22,28,30 Compound 28 barely inhibits PDE8 (IC50 of
∼100 μM, Table 2), although PDE8 also contains a tyrosine at
the corresponding position of Tyr424 of PDE9A2, implying
that the inhibitory selectivity would be jointly determined by
multiple residues at the active sites of the PDE families.
Binding of 28 to the PDE9 Catalytic Domain. As
expected by the structure-based design and computational
docking, compound 28 binds to the active site of PDE9 in a
similar pattern as other PDE9 inhibitors.20,32 While the overall
orientation of 28 is comparable with that of 10r, the
pyrazolopyrimidinone ring shows about 0.7 Å positional
translation (Figure 2). This movement should not be surprising
because 10r does not fully occupy the binding pocket of PDE9
(Figure 1A) and thus implies a feasibility to design inhibitors
with various substitution that would lock the orientation of
pyrazolopyrimidinone. Our compound 28 is a good example
for design of PDE9 inhibitors, in which 28 not only retains the
affinity with PDE9 but also shows the significantly improved
selectivity against other PDE families (Table 2).
Scheme 4. Syntheses of Targeted Compounds
Enzymatic Properties of PDE9A Inhibitors. Among
compounds 26−35, 28 shows the highest potency with an IC50
of 21 nM against the PDE9A catalytic domain (Table 1). The
chlorine atom of the 2-chlorophenyl group on the R′
substitution is important for the inhibitor binding, as shown
by the over 300-fold affinity loss when the 2-chlorophenyl
group is replaced with the phenyl group (see comparison
between compounds of 28 and 34, Table 1). However, the
same Cl substitution at R′ has less impact when methox-
ylphenyl at R is changed to an ethoxylphenyl group, as shown
by only 11-fold difference in the potency between 29 and 35
(Table 1). Replacement of the methoxylphenyl group at the R
position with an ethoxylphenyl group reduced the inhibitory
potency of the compound containing the o-Cl-phenyl at R′ by
∼35-fold (comparison between 28 and 29). The explanation to
these changes is not clear but might be due to the fact that the
2-Cl group plays a role in maintaining intramolecular
relationship between the Cl-phenyl group at R′ and the oxy-
phenyl group at R to pick up hydrophobic interactions within
the PDE9 pocket, as revealed by the structure described in the
The electron density maps of (2Fo − Fc) and (Fo − Fc)
clearly revealed that the (S)-enantiomer of 28 binds to the
PDE9 pocket (Figure 2C). This is consistent with the use of
(L)-2-aminopropanoic acid as the source material to synthesize
11. The amide oxygen of 28 forms a hydrogen bond with
tyrosyl OH of Tyr424 as originally designed. The O4 and N5
atoms of pyrimidine of 28 are involved in two hydrogen bonds
with the nitrogen and oxygen of side chain amide of the
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Table 1. Chemical Structures and Affinity of Compounds with PDE9A
invariant Gln453 (2.8 and 2.9 Å), which are comparable with
2.7 and 2.9 Å for the same bonds in the structure of PDE9A-
10r.32 The pyrazolopyrimidine ring of 28 forms aromatic π-
stacking against Phe456 of PDE9A (Figure 2). In addition,
compound 28 contacts via van der Waals' interaction with
residues Phe251, His252, Met365, Ile403, Asn405, Leu420, and
Phe441.
An unusual observation of the structural study is that a third
molecule of 28 binds to molecule B in the dimer of the PDE9A
catalytic domain (Figure 2D). This molecule has weaker
electron density than the two molecules that bind to the active
site of PDE9 (Figure 2D), indicating its partial occupancy. Its B
factor of 81.6 Å2 is significantly higher than the average of 50.2
Å2 for the two molecules of 28 bound at the active site (Table
3), consistently indicating its partial occupancy. The third
molecule of 28 forms van der Waals' interactions with Pro440,
Phe441, Thr451, Ala452, Ile454, and Gly455 of molecule B in
the PDE9 dimer and Ala499 and Glu502 of symmetry-related
molecule A. It also contacts 28 that binds to the active site of
molecule B in the PDE9 dimer (Figure 2D). A structural
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studied as the therapeutics for treatment of the CNS
diseases19−23,25,27,28,30 and PDE1 is abundant in brain,7−9
improvement on the selectivity against PDE1 appears to be
very important for lowering risk of side effects.30 The moderate
selectivity of several PDE9 inhibitors against PDE118,20 might
be explained by the comparability between the active sites of
PDE9 and PDE1, as revealed by the structural superposition
(Figure 3A). The sequence alignment shows that the residues
involved in binding of inhibitor 28 are well conserved, such as
Tyr424 of PDE9A2 versus Phe392 of PDE1B, Phe251 ∼
Tyr222, Leu421 ∼ Met389, and Phe441 ∼ Leu409, in addition
to the identical residues of Met365, Ile403, Leu420, Gln453,
and Phe456. Because Tyr424 is unique to PDE9 and PDE8, it
may be speculated that an inhibitor that is capable of forming a
hydrogen bond with the tyrosyl OH of Tyr424 of PDE9 would
increase its selectivity against PDE1 as well as other PDE
families.20,21,30 This idea was illustrated by the design of 9 that
forms a hydrogen bond indirectly with Tyr424 via a bridging
water and shows improved selectivity (170-fold) against
PDE1C.30 In comparison, our inhibitor 28 that directly
interacts with Tyr424 shows about 860-fold selectivity against
PDE1B.
Table 2. Affinity of Inhibitor 28 with PDE Families
proteins
IC50 (μM)
PDE9A2 (181−506)
PDE1B (1−516)
0.021 0.005
18.0 1.6
>50
PDE2A (580−941)
PDE3A (679−1087)
PDE4D2 (86−413)
PDE5A1 (535−860)
PDE7A1 (130−482)
PDE8A1 (480−820)
PDE10A2 (448−789)
>50
15.7 2.4
3.3 0.5
>100
∼100
>50
superposition by using comparable residues 190−493 showed
no substantial conformational differences between molecules A
and B of the PDE9 dimer (rmsd of 0.24 Å) but the
intermolecular interactions in the crystal lattice. Thus, the
partial occupancy of the third 28 in molecule B is probably an
artifact resulting from the crystal lattice packing.
DISCUSSION
■
Implication on Inhibitor Selectivity. While several PDE9
inhibitors have been shown to have high affinity with
PDE9,18,20−22 a concern for these inhibitors is the selectivity
against PDE1. Because PDE9 inhibitors have been extensively
On the other hand, because PDE8 also contains a tyrosine at
the corresponding position of Tyr424, the selectivity against
Figure 2. Binding of 28 to PDE9A2. (A) Surface presentation on binding of inhibitor 28 (green sticks) to the active site of PDE9. The chlorine atom
of 28 is an orange color. Dotted lines represent the hydrogen bonds between 28 and PDE9 residues Tyr424 and Gln453. (B) Superposition of
PDE9-28 (green and cyan) over PDE9-10r (yellow and salmon). Thr451 and Ala452 show significant positional differences between PDE9-28 and
PDE9-10r. The pyrazolopyrimidinone ring has about 0.7 Å difference between two structures. (C) Electron density for 28 bound to chain A of the
PDE9 dimer. The mesh (Fo − Fc) map was drawn from the PDE9 structure with omission of 28 and contoured at 2.5σ. (D) Density for two
molecules of 28 bound to chain B. The lower 28 binds to the active site of PDE9.
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In addition, there is another small pocket (top of Figure 3B)
neighboring the main pocket of the active site, which is
composed of Leu421 and the M loop residues Val447 and
Lys449 and gated by Phe441 and Ala452. The structural
superposition shows that significant positional changes on the
residues in the M loop and N terminal to the invariant Gln453
were induced by some but not all PDE9 inhibitors. Inhibitors
with large substitutions of 10r (PDE code, 3E3K), 10s (3K3H),
and Pfizer inhibitor 7 (3JSW)20 pushed away the Cα atoms of
Thr451 and Ala452 by over 1 Å (3−4 times the rmsd, Figures
2B and 3D). In comparison, IBMX (2HD1), cGMP (3DYQ),
and inhibitor 28 have no direct or weak interaction with Ala452
and Phe441 and thus show no substantial positional movement
(Figure 3B,C). The positional differences of these residues
appear to be the consequence of the inhibitor binding because
the M loop is not involved in crystallographic lattice packing.
Thus, the above comparison implies that the gatekeepers of
Phe441 and Ala452 could be pushed slightly apart and a
suitable substitution on the side chain of alanine of 28 might be
able to enter the small neighboring pocket of Leu421, Val447,
and Lys449, thus leading to a new type of PDE9 inhibitors.
Table 3. Statistics on Diffraction Data and Structure
Refinement
PDE9-28
data collection
space group
P41212
104.3, 270.1
2.7
unit cell (a, c, Å)
resolution (Å)
total measurements
unique reflections
completeness (%)
average I/σ
216409
41120
a
99.7 (98.2)
a
17.4 (4.4)
a
Rmerge
0.093 (0.37)
structure refinement
0.212
R factor
b
R free
0.245 (10.0%)
resolution (Å)
reflections
rms deviation for
bond length
bond angle
average B factor (Å2)
protein
15−2.7
38971
0.007 Å
1.3°
c
52.8 (5390)
EXPERIMENTAL SECTION
d
■
inhibitor
50.2 (62) and 81.6 (31)
61.0 (2)
Protein Expression and Purification. The cDNA of the wild-
type PDE9A2 for expression of the catalytic domain (residues 181−
506) was subcloned and purified following the similar protocols
previously reported.31 In brief, the recombinant plasmid was
transferred into Escherichia coli strain BL21 (Codonplus, Stratagene).
The E. coli cells carrying the pET-PDE9A2 plasmids were grown in LB
medium at 37 °C to absorption A600 = 0.6−0.8, and then, 0.1 mM
isopropyl β-D-thiogalactopyranoside was added to induce the
expression. The cells after induction were grown at 15 °C overnight.
Recombinant PDE9A2 proteins were purified by the column
chromatography of Ni-NTA affinity (Qiagen), Q-sepharose ionic
exchange, and gel filtration. A typical batch of purification yielded 20−
60 mg of PDE9A2 from a 1 L cell culture. The PDE9A2 proteins had
purity greater than 90% as shown by SDS-PAGE.
Zn
Mg
39.9 (2)
water
38.9 (18)
a
b
The numbers in parentheses are for the highest resolution shell. The
c
percentage of reflections omitted for the calculation of R free. The
d
number of atoms in the crystallographic asymmetric unit. The
average B factor of 50.2 Å2 is for the two molecules of 28 that bind at
the active site, while 81.6 Å2 is for the third 28.
PDE8 would be a concern. The enzymatic measurement shows
that inhibitor 28 barely inhibits PDE8A (IC50 = ∼100 μM vs 21
nM for PDE9, Table 2). This might be explained by a distant
relationship between the active sites of PDE8 and PDE9. The
structure comparison shows that the selectivity of PDE9
inhibitors against PDE8 may be determined by several factors:
(1) the poorly comparable positions of several active site
residues such as Phe441 of PDE9A2 versus Val768/Phe769 of
PDE8A, (2) the different conformations of the M loops, and
(3) the mutation of amino acids such as Phe785 of PDE8 to
Val460 of PDE9A2.
Hints on Further Improvement of PDE9 Inhibitors.
While our inhibitor 28 shows high affinity to PDE9 and good
selectivity against other PDE families, it does not seem to fully
occupy the active site of PDE9 (Figure 3B), implying room for
further improvement. The surface presentation of the PDE9
structure reveals an empty space near the metal binding pocket
(left of Figure 3B). Although this space could in principle
accommodate modifications of the current PDE9 inhibitors to
improve their affinity and selectivity, occupation of this space
would require a huge molecule. Because larger inhibitors have
poorer capacity of penetration through the blood barrier,
inhibitors that can fully occupy the active site pocket would be
expected to have poor bioavailability and to be unrealistic.
Nevertheless, there is a small space near the Cβ atom of alanine
of 28 (top right of Figure 3B) and an extension from the chiral
carbon of alanine, such as replacement of the methyl group
with an ethyl or propyl group would be expected to fill the
space and thus improve the affinity and selectivity.
The catalytic domains of PDE4D2 (86−413), PDE5A1 (535−860),
PDE7A1 (130−482), PDE8A2 (480−820), and PDE10A2 (448−789)
were purified by using similar protocols previously published.39−43
PDE1B (1−516) was partially purified at UNC, and PDE2A and
PDE3A were prepared by refolding (unpublished data).
Enzymatic Assay. The enzymatic activities of the catalytic
domains of PDE9A and other PDEs were measured by using cGMP
or cAMP as substrates. The assay buffer contains 20−50 mM Tris-HCl
(pH 7.5−8.0), 10 mM MgCl2 or 4 mM MnCl2, 1 mM DTT, and 10−
20 nM 3H-cGMP or 3H-cAMP (20000−30000 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
3
product H-GMP was precipitated out by 0.25 M BaSO4, whereas
unreacted 3H-cGMP remained in the supernatant. The radioactivity in
the supernatant was measured in 2.5 mL of Ultima Gold liquid
scintillation cocktails (PerkinElmer) by a PerkinElmer 2910 liquid
scintillation counter. For the measurement of IC50 of inhibitors, at
least eight concentrations of inhibitors were used in the presence of
3H-cAMP or 3H-cGMP. The enzyme concentration in a range of 10−
100 ng/mL, which hydrolyzed up to 70% of the substrate, was chosen
for each inhibitory assay. Each measurement was repeated at least
three times. The IC50 values were calculated by nonlinear regression.
Molecular Docking. Fragments output from the manual design
were docked into the active site of PDE9A by using the Surflex-Dock
protocol of program Tripos SYBYL.35,36 For Surflex-Dock in SYBYL
7.3.5, an energy-based protocol approach was used to locate the
binding site. The threshold and bloat values were set to 0.5 and 0,
respectively. The search grid extends 6 Å beyond the protein
dimensions. Ring flexibility and soft grid treatment were turned on.
The obtained poses were evaluated by the consensus scores of
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Figure 3. Structural comparison. (A) Superposition of PDE9A (green sticks) over PDE1B (cyan) and PDE8A (orange). Inhibitor 28 of PDE9A is
shown as purple sticks. IBMX of PDE8A is shown as yellow sticks. (B) A surface presentation for comparison on binding of 28 (green sticks) and
cGMP (cyan) to the PDE9A active site. Residues were obtained from the superposition of the PDE9 complex structures. (C) Comparison on
binding of 28 and IBMX (salmon). (D) Comparison on 28 and Pfizer inhibitor 7 (PF7, yellow). Residues Thr451 and Ala452 showed significant
movement in the PDE9-PF7 structure.
ChemScore, G_Score, D_score, and PMF_Score. Thirty config-
urations of the ligands were output from the docking program for
analysis.
temperature, 50 °C; initial time, 3 min; ramp, 20 °C/min; final
temperature, 250 °C; and final time, 2 min. EI mass spectra were
recorded on the Thermo DSQ mass spectrometer. Thin-layer
chromatography was performed on precoated silica gel F-254 plates
(0.25 mm, E. Merck) and was visualized with UV light. Element
analyses were carried out on an element vario EL series element
analyzer and have errors within 0.3% for CHN elements. Melting
points were determined on a WRS-1B digital melting point apparatus
and were not corrected. All starting materials and reagents were
purchased from commercial suppliers and used directly without further
purification.
Crystallization and Structure Determination. The crystals of
PDE9A2 were grown by using hanging drop vapor diffusion method.
The PDE9A2 (181−506) (10−15 mg/mL) in a buffer of 50 mM
NaCl, 20 mM Tris·HCl, pH 7.5, 1 mM β-mercaptoethanol, and 1 mM
EDTA was mixed with 2 mM IBMX for 2 h before setting-up of
crystallization against the well buffer of 2.0 M Na formate, 0.1 M
HEPES, pH 7.5, and 5% xylitol at 4 °C. The PDE9A-IBMX complex
crystals were formed after 1 day and reached the maximum size in 2
weeks. Crystals of the PDE9A-28 complex were prepared by soaking
PDE9-IBMX cocrystals in the crystallization buffer plus 5 mM 28 at 25
°C for 3 days. The crystals were flash-frozen in liquid nitrogen by using
the well buffer plus saturated xylitol as the cryosolvent. X-ray
diffraction data were collected at 100 K on Beamline BL17U of
Shanghai Synchrotron Radiation Facility, China (Table 3), and
processed by HKL2000.44 The structure of the PDE9A2-28 complex
was solved by the molecular replacement, using the PDE9A2 catalytic
domain as the initial model. The resulting model was rebuilt by
program O45 and refined by CNS.46
Synthesis of Compounds 26−35. To a 50 mL round-bottom
flask were added iso-PrOH (25 mL), compound 7 or 8 (0.4 mmol), 2-
amino-N-arylpropan-amide (compounds 14−21) (0.48 mmol), and
TEA (0.4 mmol, 0.04 g). The mixture was refluxed for 6 h and then
was allowed to cool to room temperature. After the solvent was
removed, the residue was purified by flash column chromatography on
silica gel (eluting with DCM:MeOH = 25:1) to provide the pure target
compounds. The following are the parameters for compounds 26−35.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-phenylpropanamide (26). Yield, 41%; mp, 138−139
1
1
Analysis of Synthesized Compounds. H NMR and 13C NMR
°C. H NMR (300 MHz, CDCl3): δ 10.80 (s, 1H), 8.64 (br s, 1H),
spectra were recorded at room temperature on a Mercury-Plus 300
instrument with TMS as an internal reference. LC/MS was run on a
LCMS-2010A. GC/MS was run on a Finnigan Voyager with an
electron impact (70 eV) mass selective detector and an innowax 30 m
× 0.25 mm × 0.25 μm capillary apolar column. GC-MS method: initial
8.13 (s, 1H), 7.51−7.46 (m, 2H), 7.35−7.24 (m, 6H), 7.06 (d, J = 6.3
Hz, 2H), 4.56−4.49 (m,1H), 1.52 (d, J = 7.3 Hz, 3H). 13C NMR (75
MHz, CDCl3): δ 170.9, 159.4, 155.2, 152.8, 137.1, 136.7, 131.5, 130.3,
130.1, 129.4, 128.7, 127.2, 124.5, 119.9, 100.5, 51.5, 17.5. ESI-MS: m/z
= 407 [M − H]−. IR (KBr, cm−1): 3310, 2980, 1690, 1610, 1548,
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1253. Anal. calcd for C20H17ClN6O2: C, 58.75; H, 4.19; N, 20.56.
Found: C, 58.56; H, 4.31; N, 20.59.
3H), 7.31−6.97 (m, 4H), 4.56−4.46 (m, 1H), 2.45 (s, 3H), 1.53 (d, J
= 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 170.4, 159.4, 155.1,
152.8, 136.8, 135.3, 134.6, 134.1, 131.6, 130.4, 130.2, 129.4, 127.6,
127.4, 120.5, 100.6, 51.5, 17.4, 16.6. ESI-MS: m/z = 453 [M − H]−. IR
(KBr, cm−1): 3300, 2984, 1684, 1610, 1542, 1256, 1040. Anal. calcd
for C21H19ClN6O2S: C, 55.44; H, 4.21; N, 18.47. Found: C, 55.59; H,
4.05; N, 18.41.
2-((4-Hydroxy-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-
amino)-N-(4-methoxyphenyl)propanamide (34). Yield, 41%; mp,
283−285 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.67(s, 1H),
10.17(br s, 1H), 8.06 (d, J = 6.3 Hz, 3H), 7.54 (d, J = 9.0 Hz, 2H),
7.36−7.23 (m, 3H), 7.11 (d, J = 6.3 Hz, 1H), 6.89 (d, J = 9.0 Hz, 2H),
4.55−4.46 (m, 1H), 3.71 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ
170.2, 157.0, 155.0, 153.6, 152.8, 138.7, 135.9, 131.7, 128.7, 125.6,
120.5, 120.3, 113.7, 101.2, 55.0, 50.7, 18.4. ESI-MS: m/z = 403 [M −
H]−. IR (KBr, cm−1): 3280, 2960, 1690, 1504, 1407, 1268, 945. Anal.
calcd for C21H20N6O3: C, 62.37; H, 4.98; N, 20.78. Found: C, 62.56;
H, 4.81; N, 20.59.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(p-tolyl)propan-amide (27). Yield, 45%; mp, 141−143
1
°C. H NMR (300 MHz, CDCl3): δ 10.70 (s, 1H), 8.33 (br s, 1H),
8.11 (s, 1H), 7.50−7.26 (m, 4H), 7.08−6.96 (m, 5H), 4.54−4.45 (m,
1H), 2.29 (s, 3H), 1.49 (d, J = 6.9 Hz, 3H). 13C NMR (75 MHz,
CDCl3): δ 170.6, 159.4, 155.2, 152.9, 136.8, 135.3, 134.5, 134.3, 131.6,
130.4, 130.1, 129.4, 129.3, 127.3, 120.1, 113.9, 100.6, 51.4, 20.9, 17.4.
ESI-MS: m/z = 421 [M − H]−. IR (KBr, cm−1): 3310, 2980, 1690,
1610, 1548, 1246. Anal. calcd for C21H19ClN6O2: C, 59.65; H, 4.53; N,
19.87. Found: C, 59.86; H, 4.35; N, 19.69.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(4-methoxyphenyl)propanamide (28). Yield, 45%;
1
mp, 140−141 °C. H NMR (300 MHz, CDCl3): δ 10.57 (s, 1H),
8.14 (s, 1H), 8.13 (br s, 1H), 7.52−7.29 (m, 4H), 7.11−6.77 (m, 5H),
4.55−4.46 (m, 1H), 3.78 (s, 3H), 1.52 (d, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ 170.6, 158.4, 156.6, 155.2, 152.9, 136.7, 135.3,
131.6, 130.4, 130.1, 130.0, 129.5, 127.3, 121.9, 113.9, 100.6, 55.5, 51.3,
17.5. ESI-MS: m/z = 437 [M − H]−. IR (KBr, cm−1): 3310, 2980,
1680, 1548, 1240. Anal. calcd for C21H19ClN6O3: C, 57.47; H, 4.36; N,
19.15. Found: C, 57.56; H, 4.21; N, 18.95.
N-(4-Ethoxyphenyl)-2-((4-hydroxy-1-phenyl-1H-pyrazolo[3,4-d]-
pyrimidin-6-yl)amino)propanamide (35). Yield, 37%; mp, 277−279
1
°C. H NMR (300 MHz, DMSO-d6): δ 10.67 (s, 1H), 10.16 (br s,
1H), 8.06 (d, J = 6.3 Hz, 3H), 7.53 (d, J = 7.8 Hz, 2H), 7.35−7.31 (m,
3H), 7.25 (d, J = 6.9 Hz, 1H), 6.87 (d, J = 7.8 Hz, 2H), 4.53−4.48 (m,
1H), 3.97 (d, J = 6.9 Hz, 2H), 1.48 (d, J = 5.7 Hz, 3H), 1.48 (d, J = 6.3
Hz, 3H). 13C NMR (75 MHz, DMSO-d6): δ 170.1, 157.0, 154.3,
153.6, 152.8, 138.7, 135.8, 131.6, 128.7, 125.6, 120.4, 120.3, 114.2,
101.2, 62.9, 50.6, 18.4, 14.6. ESI-MS: m/z = 417 [M − H]−. IR (KBr,
cm−1): 3416, 2984, 1690, 1500, 1399, 1240. Anal. calcd for
C22H22N6O3: C, 63.15; H, 5.30; N, 20.08. Found: C, 63.06; H, 5.36;
N, 19.78.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(4-ethoxyphenyl)propanamide (29). Yield, 50%; mp,
169−171 °C. 1H NMR (300 MHz, CDCl3): δ 8.37 (br s, 1H), 8.07 (s,
1H), 7.47−7.43 (m, 2H), 7.36−7.06 (m, 4H), 6.95 (d, J = 6.6 Hz,
1H), 6.76 (d, J = 9.0 Hz, 2H), 4.49−4.46 (m,1H), 3.99−3.93 (q, J =
6.9 Hz, 2H), 1.43 (d, J = 7.5 Hz, 3H), 1.39 (d, J = 7.2 Hz, 3H). 13C
NMR (75 MHz, CDCl3): δ 170.7, 159.3, 155.9, 152.2, 136.7, 135.3,
131.6, 130.3, 130.1, 129.9, 127.3, 121.8, 114.5, 100.6, 63.7, 51.3, 17.4,
14.8. ESI-MS: m/z = 451 [M − H]−. IR (KBr, cm−1): 3310, 2980,
1690, 1610, 1552, 1240, 1050. Anal. calcd for C22H21ClN6O3: C,
58.34; H, 4.67; N, 18.56. Found: C, 58.23; H, 4.70; N, 18.63.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(4-isopropoxyphenyl)propanamide (30). Yield, 47%;
ASSOCIATED CONTENT
* Supporting Information
■
S
Sequence alignment of the active site residues of 21 PDE genes.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
mp, 120−122 °C. H NMR (300 MHz, CDCl3): δ 8.39 (br s, 1H),
8.08 (s, 1H), 7.47−7.23 (m, 4H), 7.09−6.97 (m, 3H), 6.76−6.73 (d, J
= 8.7 Hz, 2H), 4.50−4.42 (m,2H), 1.46 (d, J = 6.9 Hz, 3H), 1.31 (d, J
= 6.0 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 170.6, 159.4, 155.2,
154.7, 152.9, 136.7, 135.2, 131.5, 130.3, 130.1, 129.9, 121.8, 116.1,
100.5, 51.3, 22.0, 17.5. ESI-MS: m/z = 465 [M − H]−. IR (KBr,
cm−1): 3300, 2980, 1690, 1610, 1548, 1230. Anal. calcd for
C23H23ClN6O3: C, 59.16; H, 4.97; N, 18.00. Found: C, 58.89; H,
4.93; N, 17.83.
Accession Codes
The atomic coordinates and structure factors have been
deposited into the RCSB Protein Data Bank with accession
number 4GH6.
AUTHOR INFORMATION
Corresponding Author
■
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(3-methoxyphenyl)propanamide (31). Yield, 42%;
mp, 230−232 °C (lit; mp, °C). 1H NMR (300 MHz, CDCl3): δ
8.40(br s, 1H), 8.10 (s, 1H), 7.48−7.45 (m, 2H), 7.36−7.07 (m, 4H),
7.01 (d, J = 6.3 Hz, 1H), 6.62 (d, J = 8.1 Hz, 2H), 4.53−4.45 (m, 1H),
3.74 (s, 3H), 1.50 (d, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ
170.7, 159.9, 159.5, 155.2, 152.8, 138.3, 136.7, 135.2, 131.6, 130.4,
130.1, 129.4, 127.3, 112.1, 109.9, 106.1, 100.5, 55.3, 51.6, 51.4, 17.5.
ESI-MS: m/z = 437 [M − H]−. IR (KBr, cm−1): 3310, 2968, 1683,
1610, 1547, 1290, 1060. Anal. calcd for C21H19ClN6O3: C, 57.47; H,
4.36; N, 19.15. Found: C, 57.39; H, 4.37; N, 19.25.
*Tel: 86-20-39943031. E-mail: luohb77@mail.sysu.edu.cn (H.-
B.L.). Tel: 86-20-84113610. E-mail: ceswyq@mail.sysu.edu.cn
(Y.W.). Tel: 1-919-966-2244. E-mail: hke@med.unc.edu
(H.K.).
Author Contributions
∥These authors contributed equally.
Notes
The authors declare no competing financial interest.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(2-methoxyphenyl)propanamide (32). Yield, 36%; P,
115−117 °C. 1H NMR (300 MHz, CDCl3): δ 8.18 (s, 1H), 8.14 (br s,
1H), 7.42−7.27 (m, 3H), 7.16−7.04 (m, 3H), 6.98−6.84 (m, 3H),
4.61−4.52 (m, 1H), 3.76 (s, 3H), 1.57 (d, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ 170.8, 159.6, 155.5, 152.6, 148.1, 136.7, 135.4,
131.7, 130.2, 129.7, 129.3, 127.0, 126.8, 124.3, 121.0, 120.2, 109.9,
100.6, 55.7, 51.7, 18.34. ESI-MS: m/z = 437 [M − H]−. IR (KBr,
cm−1): 3310, 2990, 1690, 1610, 1539, 1288, 1020. Anal. calcd for
C21H19ClN6O3: C, 57.47; H, 4.36; N, 19.15. Found: C, 57.61; H, 4.31;
N, 19.01.
ACKNOWLEDGMENTS
■
This work is partially supported by US NIH GM59791 (H.K.),
National High Technology Research and Development
Program of China (863 Program, No. 2006AA09Z446),
Natural Science Foundation of China (20872182, 20802095,
and 21103234), Natural Science Foundation of Guangdong
Province (S2011030003190), Natural Science Foundation of
Department of Education in Guangdong Province
(CXZD1006), Natural Science Foundation of Guangzhou
City (2010Y1-C531), and Fundamental Research Funds for
the Central Universities (11ykzd05). The diffraction data were
collected on beamline BL17U of Shanghai Synchrotron
Radiation Facility.
2-((1-(2-Chlorophenyl)-4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)-N-(4-(methylthio)phenyl)propanamide (33). Yield,
1
44%; mp, 161−163 °C. H NMR (300 MHz, CDCl3): δ 10.70 (s,
1H), 8.33 (br s, 1H), 8.13 (s, 1H), 7.52−7.47 (m, 2H), 7.39−7.32 (m,
8556
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ABBREVIATIONS USED
■
PDE, phosphodiesterase; cAMP, adenosine 3′,5′-cyclic mono-
phosphate; cGMP, guanosine 3′,5′-cyclic monophosphate
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