Utilization of halogen bond in lead optimization: A case study of rational design of potent phosphodiesterase type 5 (PDE5) inhibitors

Article abstract of DOI:10.1021/jm200644r

For proof-of-concept of halogen bonding in drug design, a series of halogenated compounds were designed based on a lead structure as new inhibitors of phosphodiesterase type 5. Bioassay results revealed a good correlation between the measured bioactivity and the calculated halogen bond energy. Our X-ray crystal structures verified the existence of the predicted halogen bonds, demonstrating that the halogen bond is an applicable tool in drug design and should be routinely considered in lead optimization.

Full text of DOI:10.1021/jm200644r

BRIEF ARTICLE
pubs.acs.org/jmc
Utilization of Halogen Bond in Lead Optimization: A Case Study of
Rational Design of Potent Phosphodiesterase Type 5 (PDE5) Inhibitors†
Zhijian Xu,‡,# Zheng Liu,§,# Tong Chen,‡,# TianTian Chen,‡ Zhen Wang,‡ Guanghui Tian,‡ Jing Shi,§
Xuelan Wang,§ Yunxiang Lu,|| Xiuhua Yan,‡ Guan Wang,‡ Hualiang Jiang,‡ Kaixian Chen,‡ Shudong Wang,^
Yechun Xu,*,‡ Jingshan Shen,*,‡ and Weiliang Zhu*,‡
‡State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Shanghai 201203, China
§Topharman Shanghai Co., Ltd., 1088 Chuansha Road, Shanghai 201209, China
Department of Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
^School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
S Supporting Information
b
ABSTRACT: For proof-of-concept of halogen bonding in drug design, a series of halogenated compounds were designed based on
a lead structure as new inhibitors of phosphodiesterase type 5. Bioassay results revealed a good correlation between the measured
bioactivity and the calculated halogen bond energy. Our X-ray crystal structures verified the existence of the predicted halogen
bonds, demonstrating that the halogen bond is an applicable tool in drug design and should be routinely considered in lead
optimization.
’ INTRODUCTION
serine protease factor Xa inhibitors.7 More recently, Baumli et al.
reported halogen bonds formed by DRB that are specific for the
CDK9 kinase hinge region and contribute its high selectivity
for the protein.15 On the basis of an indication of a halogen
bond between a 4-chlorophenyl moiety of a ligand and human
cathepsin L, Hardegger et al. presented a systematic study on
halogen bond in proteinꢀligand complexes.11 Clearly, while the
rational computational application of halogen bonding to drug
design and lead optimization remains largely unexplored, a case
study as proof-of-concept might help to convince and stimulate
researchers to intentionally apply halogen bonding in drug
development.
PDE5, which contains two cGMP-specific phosphodies-
terases, bacterial adenylyl cyclase, FhLA transcriptional regulator
(GAF) domains (GAF A and GAF B) in the N-terminal and a
catalytic domain in the C-terminal, catalyzes the specific hydro-
lysis of cGMP to 50-GMP. It is well-known as the target of
sildenafil, an effective drug for the treatment of male erectile
dysfunction and pulmonary arterial hypertension (Figure S1 in
Supporting Information). Our group has synthesized a novel lead
compound 6-ethyl-2-(5-(4-methylpiperazin-1-ylsulfonyl)-2-
propoxyphenyl)pyrimidin-4(3H)-one (Figure 1a, X = H, 1),
structurally simpler but with weaker inhibitory activity than
sildenafil (Table 1). Molecular docking of 1 to PDE5 (PDB
entry 2H42) suggests that a halogen bond could be introduced
between pyrimidinyl-5C of the ligand (Figure 1a) and the Y612
phenolate oxygen atom of the protein in the process of structural
optimization (Figure 1b,c). We performed a PDB survey that
Halogen bonding, a specific intermolecular interaction be-
tween a halogen atom and an electron-rich partner (O, N, or S),
has been studied extensively using computational chemistry and
has found a role in molecular engineering for material design.1ꢀ5
Recently, it has also received recognition in biological systems as
a distinct class of hydrogen-bond-like interactions, holding vast
potential for applications in biological engineering.6ꢀ12 Database
surveys reveal that halogen bonding is a prevalent interaction
between halogenated ligand and target protein,9ꢀ12 and around
25% of the “top 200 brand name drugs by retail dollar in 2009”
possess halogen atoms in their molecular structures. Therefore,
halogens have a key role in drug development.
Here we present a case study to demonstrate how the
introduction of halogen bonds between a ligand and its target
can be applied to the structural optimization of lead compounds.
The optimized lead compounds are phosphodiesterase type 5
(PDE5) inhibitors, being developed as potential drug candidates
for the treatment of male erectile dysfunction and pulmonary
arterial hypertension.13
Halogen bonding cannot be properly described by force field
and scoring function, as halogen atoms are negatively charged as
a whole but positively charged at certain regions of their atomic
surface.5,9 Consequently the interaction has not achieved a significant
role in drug design and lead optimization. The fact that many
drugs contain halogens in their molecular structure is not due to a
rational computational approach but due to medicinal chemists’
experience and intuition. For example, Valadares and co-workers
identified a novel and previously unreported halogen bond in
thyroid hormone.14 Matter et al. demonstrated a successful
supports the existence of O XꢀC halogen bonds between
3 3 3
utilization of CꢀCl/CꢀBr π interaction to enhance ligandꢀ
Received: May 21, 2011
Published: June 29, 2011
3 3 3
protein binding affinity, based on their long-term development of
r
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Journal of Medicinal Chemistry
BRIEF ARTICLE
organic halogens and the phenolate oxygen of tyrosine (Figure S2
in Supporting Information). To further explore this concept, a
hybrid quantum mechanics/molecular mechanics (QM/MM)
method was employed to estimate the potential halogen bond
strength between the halogenated 1 and PDE5, which gave a
supportive prediction (ΔE, Table 1). The compounds were then
synthesized and assayed for inhibitory activity against PDE5, and
a good correlation was observed between the QM/MM derived
binding energies and the experimentally determined bioactivity
data (Table 1). To further validate the predicted halogen bonds,
we determined the crystal structures of the corresponding com-
plexes that clearly show that the halogen atoms at pyrimidinyl-5C
moiety of ligands form halogen bonds with Y612 in PDE5. Thus,
the study demonstrated that halogen bonding is a practicable tool
for rational drug design and lead optimization.
1UDT).16 The H-loop in this structure was disordered, but a
fully active catalytic domain of PDE5 containing a full length
H-loop cocrystallized with sildenafil was recently resolved (PDB
entry 2H42),17 and this was used for docking for lead optimiza-
tion in this study.
Designing New Halogenated Compounds. Figure 1 dis-
plays the docked complex structure of 1 and PDE5, revealing a
geometrical pattern of the O HꢀC with an angle of 141.1ꢀ
3 3 3
and an interaction distance of 3.8 Å between Y612 oxygen atom
and the 5-hydrogen atom of pyrimidinyl moiety of the lead
compound 1 in the binding pocket (Figure 1c). With the concept
of halogen bonding in mind, we realized that if the hydrogen
atom is substituted by halogen atoms (Cl, Br, or I), a halogen
bond might be introduced between the halogenated 1 and PDE5
to improve the bioactivity of the compounds. Hence, four
halogenated derivatives of 1, where X = F, Cl, Br, and I (namely,
2, 3, 4, and 5, respectively), were designed and subsequently
docked to PDE5. The docked complexes show a halogen
bonding interaction pattern between the newly designed halo-
genated ligands and the phenolate oxygen atom of Y612 in the
binding pocket (Figure S3a and Table S1 in Supporting Infor-
mation). A very similar binding mode between the designed ligands
and sildenafil was observed, including two key interactions, i.e.,
hydrogen bonds with Q817 and a πꢀπ stacking interaction with
F820 (Figure S3 in Supporting Information).17
’ RESULTS AND DISCUSSION
PDE5 Structure Used for Docking. Sildenafil has been widely
studied, and several cocrystal structures with PDE5 have been
resolved. In 2003 the structure of an inactive PDE5 in complex with
sildenafil was determined by the multiwavelength anomalous
dispersion method using Se-Met protein crystals (PDB entry
Predicting Halogen Bond Energy. To further confirm the
validity of this concept, a combined QM/MM scheme, viz., our
own N-layered integrated molecular orbital and molecular
mechanics (ONIOM), was employed to optimize the docked
complexes and estimate the binding energy between the 5C-
halogen atom and the phenolate O of Y612. To achieve this, the
compounds and the side chain of Y612 are in the QM layer
(Figure 2aꢀc), while the remainder of the protein, including
Zn2+ and Mg2+, is in the MM layer. Table 1 summarizes the
optimized geometries of the halogen bonding systems and the
calculated halogen bond energies. The optimized interaction
distances and angles lie within the range of halogen bonding
parameters found in biomolecules,9,10 indicating the potential for
halogen bonding between the designed compounds and PDE5
(Figure S4 in Supporting Information). Moving from F to H, Cl,
Br, and I, a systematic increase in absolute value of the predicted
halogen bond energy was observed, indicating that 3, 4, and 5
should show higher potency compared with lead 1 while 2 might
be less active than 1 (Table 1).
Figure 1. Structure and the binding mode of 1 (X = H) to PDE5 from the
docking study. (a) Chemical structure of 1. (b) The docked structure of
1ꢀPDE5 complex. PDE5 is shown in cartoon form with Y612 represented
in stick form in the boxed area. 1is also represented in the boxed area in stick
form. (c) For clear visualization, the hydrogen bonding between 5CꢀHin1
and Y612 in PDE5 extracted from (b) is shown.
Our calculations ignore entropy and solvation effects and, of
course, have some limitations. Nevertheless, the conclusions
derived may cast some light on the important role of halogen
bond in rational drug design.
Table 1. Predicted and Determined Parameters of Halogen Bonds and in Vitro Inhibitory Activity of the Compounds against PDE5
d(X O), Å
3 3 3
—(CꢀX O), deg
3 3 3
compd
X
predicted
determined
predicted
determined
ΔE, kcal/mol
IC50,c nM
1
2
3
4
5
H
F
3.24
3.50
3.43
3.33
3.54
NAa
3.85
3.60
3.35b
NAa
129.5
127.7
126.9
132.6
130.0
NAa
ꢀ2.9
ꢀ2.1
ꢀ3.2
ꢀ3.3
ꢀ4.5
51.8 ( 12.1
90.9 ( 22.4
35.9 ( 11.5
13.3 ( 4.5
7.2 ( 2.1
150.7
141.0
149.2b
NAa
Cl
Br
I
a NA: not available. b Data are the mean value of chain A and chain B in the asymmetric unit. c Data are the mean ( standard deviation (SD) of three
independent experiments. Sildenafil was used as the control with IC50 of 3.3 ( 0.9 nM.
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Figure 3. Plot of ꢀlog IC50 versus halogen bond energies for the side
Figure 2. Optimized structures of PDE5 in complex with 2 (a), 3 (b),
and 4 (c) at the ONIOM(B3LYP:AMBER) level, and the difference
electron density maps contoured at 3.0σ for the complexes of PDE5 and
2 (d), 3 (e), and 4 (f) in the crystal structures, respectively.
chain of Y612.
protocol and model predict quite well the binding of the three
inhibitors into PDE5. It was observed that the methylpiperazine
group in 4 adopts a different conformation from the docking
result due to a crystal packing effect. When the methylpiperazine
group is excluded, the rmsd of the inhibitor between the crystal
structure and the docking conformation (or the QM/MM
optimized conformation) of 4 is 1.23 (1.10) Å.
Although the halogen substitution at the C5 pyrimidinyl moiety
may lead to a change in lipophilic or space filling interactions, the
three X-ray structures demonstrated that the introduction of
large halogens does not change the position of Y612. Thus, the
shorter distances between the negatively charged phenolate
oxygen atom of Y612 and the overall negatively charged Cl/Br
should be logically attributed mainly to the halogen bonding
between the O and Cl/Br, while the longer distance between the
O and F is due to the repulsion between them. In other words,
the attraction between the electronegative O of Y612 and
partially electropositive halogen atoms Cl and Br around their
cap areas along CꢀX axis,10 namely, halogen bond, is the main
cause for their increased inhibitory activity against PDE5. The
small effect of the introduction of halogens on IC50 may be
Good Correlation between Predicted Halogen Bond En-
ergy and Determined IC50. The IC50 values determined experi-
mentally with a scintillation proximity assay approach are shown
in Table 1. It can be seen that the IC50 data of the compounds
show good correlation with the predicted halogen bond strength,
and 2 is the weakest inhibitor because of the totally electro-
negative surface of organic fluorines.5,9 Our previous QM/MM
computations on model protein kinase systems in complex with
halogenated ligands also reproduced this strength order.10 More-
over, we found that the activities of the five inhibitors (expressed
as negative logarithm IC50) correlate strongly with the binding
energies with correlation coefficient R = 0.93 (Figure 3). The
high quality of the correlation implies that the halogen bonding
interactions contribute significantly to the increased activity. The
most active compound 5 exhibits an IC50 of 7.2 nM, which is 13-
and 7-fold more potent than 2 and 1, respectively. Significantly,
5 is comparable in activity to sildenafil (IC50 ≈ 3.3 nM), although
it has a simpler structure.
Halogen Bond Validation by X-ray Crystallography. The
3D structures of the catalytic domain of PDE5 complexed with
the inhibitors 2, 3, and 4 were determined by X-ray diffraction
with resolutions of 2.65, 2.45, and 1.93 Å, respectively. The
complex structures of PDE5 with 2 and 3 were obtained by
soaking the inhibitors into the crystal of apo PDE5 with space
group of P3121 while that of PDE5 and 4 was solved by
cocrystallization (space group P212121) (Table S2 in Supporting
Information). Figure 2dꢀf shows the difference electron density
maps contoured at 3.0σ for the three inhibitors, which clearly
demonstrated that the halogenated inhibitors bind to the cata-
lytic domain of PDE5. Our determined distances in the crystal
structures between the phenolate oxygen atom of Y612 and the
halogen atoms in 2 and 3, viz. 3.60 and 3.35 Å, are in excellent
agreement with the optimized distances by QM/MM approach,
viz. 3.43 and 3.33 Å, respectively, verifying that halogen bonds do
exist between PDE5 and the two newly designed and synthesized
inhibitors. Superimposition of the crystal structures and the docking
conformations (or the QM/MM optimized conformations) of
the inhibitors resulted in rmsd of 1.13 (0.83), 0.85 (1.12), and
2.72 (2.54) Å for 2, 3, and 4, respectively, indicating that docking
attributed to the weak halogen bonds with O X distances
3 3 3
close to the sum of vdW radii (Table 1). Therefore, a more
careful design to form stronger halogen bond in other systems
may lead to more significant improvement in the bioactivity for
lead optimization.
’ CONCLUSION
We have systematically explored the power of halogen bond-
ing in the context of computational rational drug design. A series
of halogenated compounds as potential PDE5 inhibitors were
designed, prepared, and evaluated. The experimentally deter-
mined bioactivities correlated well with the halogen bond energies
predicted at the QM/MM level between the new inhibitors and
the enzyme. The X-ray crystal structures of PDE5 complexed
with the new inhibitors verified the existence of the predicted
halogen bonds, viz., the bonds between the phenolate oxygen
atom of Y612 and Cl/Br in the ligands. In addition, one of the
newly designed compounds, 5, shows an activity comparable to
that of sildenafil. Therefore, this study demonstrated that the
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BRIEF ARTICLE
Scheme 1. Syntheses of Pyrimidin-4(3H)-one 1 and
5-Halopyrimidin-4(3H)-ones 2ꢀ5a
3-oxopentanoate (0.5 g, 3.8 mmol), and potassium carbonate (0.9 g, 6.4
mmol) in DMF (10 mL) was stirred at 100 ꢀC for 4 h. The mixture was
then cooled to room temperature and taken up in water (50 mL) and
DCM (50 mL). The organic layer was separated, and the aqueous layer
was extracted further with DCM (50 mL). The combined organic layers
were dried (Na2SO4) and concentrated. The residue was purified by
flash column chromatography. After crystallization from EtOAc, 1 was
obtained as white crystals (0.81 g, 60%): mp 151ꢀ152 ꢀC. HPLC purity
98.2%; tR= 16.3 min. 1H NMR (DMSO-d6, 300 MHz) δ: 7.89 (d, J =
2.4 Hz, 1H), 7.84 (dd, J1 = 2.4 Hz, J2 = 8.7 Hz, 1H), 7.39 (d, J = 8.7 Hz,
1H), 4.15 (t, J = 6.3 Hz, 2H), 2.89 (br, 4H), 2.45 (q, J = 7.5 Hz, 2H), 2.36
(br, 4H), 2.16 (s, 3H), 1.75 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H), 0.96 (t, J =
7.5 Hz, 3H). HRMS calcd [M + Na]+ for C20H28N4O4NaS 443.1729,
found 443.1736.
5-Fluoro-6-ethyl-2-[2-propoxy-5-(4-methyl-1-piperazinyl-
sulfonyl)phenyl]pyrimidin-4(3H)-one (2). 2 was prepared in 72%
yield from 8 and methyl 2-fluoro-3-oxopentanoate following a similar
procedure to that described for 1: mp 163ꢀ164 ꢀC. HPLC purity 98.3%;
tR= 10.2 min. 1H NMR (CDCl3, 300 MHz) δ: 8.80 (d, J = 2.1 Hz, 1H),
7.86 (dd, J1 = 2.1 Hz, J2 = 8.7 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 4.26 (t,
J = 6.6 Hz, 2H), 3.09 (br, 4H), 2.74 (q, J = 7.5 Hz, 2H), 2.51 (br, 4H),
2.28 (s, 3H), 2.03 (m, 2H), 1.28 (t, J = 7.5 Hz, 3H), 1.15 (t, J = 7.5 Hz,
3H). HRMS calcd [M + Na]+ for C20H27N4O4NaSF 461.1635, found
461.1630.
a Reagents and conditions: (a) (i) HSO3Cl, 0 ꢀC; (ii) N-methylpiper-
azine, Et3N, DCM, 0 ꢀC; (b) LiHMDS, THF, room temp; (c) K2CO3,
DMF, 100 ꢀC, methyl 3-oxopentanoate or methyl 2-fluoro-3-oxopen-
tanoate; (d) for 3, Cl2 (g), Et3N, DCM, 0 ꢀC; for 4, Br2, Et3N, DCM,
0 ꢀC; for 5, I2, AgNO3, MeOH, room temp.
5-Chloro-6-ethyl-2-[2-propoxy-5-(4-methyl-1-piperazinyl-
sulfonyl)phenyl]pyrimidin-4(3H)-one (3). Chlorine gas was
bubbled into an ice-cold solution of 1 (100 mg, 0.24 mmol) and pyridine
(40 μL, 0.5 mmol) in DCM (10 mL) for 1 min. The resulting mixture
was washed with 1 N Na2S2O3 (aq) (5 mL) and water (5 mL). The organic
layer was dried (Na2SO4) and concentrated in vacuo. After crystal-
lization from EtOAc, 3 was obtained as white crystals (93 mg, 85%): mp
188ꢀ189 ꢀC. HPLC purity 98.3%; tR= 10.1 min. 1H NMR (CDCl3, 300
MHz) δ: 11.25 (1H, br), 8.85 (d, J = 2.1 Hz, 1H), 7.87 (dd, J1 = 2.1 Hz,
J2 = 8.7 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 4.27 (t, J = 6.6 Hz, 2H), 3.08
(br, 4H), 2.85 (q, J = 7.5 Hz, 2H), 2.49 (br, 4H), 2.27 (s, 3H), 2.03
(m, 2H), 1.29 (t, J = 7.5 Hz, 3H), 1.15 (t, J = 7.5 Hz, 3H). HRMS calcd
[M + Na]+ for C20H27N4O4NaSCl 477.1339, found 477.1339.
5-Bromo-6-ethyl-2-[2-propoxy-5-(4-methyl-1-piperazinyl-
sulfonyl)phenyl]pyrimidin-4(3H)-one (4). Bromine (40 mg, 0.27
mmol) was added to an ice-cold solution of 1 (100 mg, 0.24 mmol) and
pyridine (22 μL, 0.27 mmol) in DCM (10 mL). The mixture was stirred
for 15 min at 0 ꢀC and washed with 1 N Na2S2O3 (5 mL) and water
(5 mL). The organic layer was dried (Na2SO4) and concentrated. After
crystallization from EtOAc, 4 was obtained as white crystals (107 mg,
90%): mp 182ꢀ183 ꢀC. HPLC purity 98.9%; tR= 10.0 min. 1H NMR
(CDCl3, 300 MHz) δ: 11.12 (1H, br), 8.86 (d, J = 2.1 Hz, 1H), 7.88
(dd, J1 = 2.1 Hz, J2 = 8.7 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 4.27 (t, J = 6.6
Hz, 2H), 3.09 (br, 4H), 2.88 (q, J = 7.5 Hz, 2H), 2.50 (br, 4H), 2.28 (s,
3H), 2.03 (m, 2H), 1.29 (t, J = 7.5 Hz, 3H), 1.15 (t, J = 7.5 Hz, 3H).
HRMS calcd [M + Na]+ for C20H27N4O4NaSBr 521.0834, found
521.0834.
5-Iodo-6-ethyl-2-[2-propoxy-5-(4-methyl-1-piperazinyl-
sulfonyl)phenyl]pyrimidin-4(3H)-one (5). Iodine (254 mg, 1 mmol)
was added to a solution of 1 (420 mg, 1 mmol) and silver nitrate (170 mg,
1 mmol) in MeOH (10 mL) at 0 ꢀC. The mixture was stirred at room
temperature for 30 min. After filtration, the filtrate was poured into water
(25 mL) and extracted with DCM (20 mL). The organic layer was washed
with 1 N Na2S2O3 (5 mL) and water (5 mL), dried, and concentrated. After
crystallization from EtOAc, 5 was obtained as white crystals (410 mg, 75%):
mp 193ꢀ194 ꢀC. HPLC purity 97.5%; tR = 12.2 min. 1H NMR (CDCl3,
300 MHz) δ: 11.12 (1H, br), 8.88 (d, J = 2.1 Hz, 1H), 7.88 (dd, J1 = 2.1 Hz,
J2 = 8.7 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 4.27 (t, J = 6.6 Hz, 2H), 3.10 (br,
4H), 2.92(q,J= 7.5 Hz, 2H), 2.52 (br, 4H), 2.30 (s, 3H), 2.03 (m, 2H), 1.28
halogen bond is a practical and effective tool in drug design and
lead optimization.
’ EXPERIMENTAL SECTION
Molecular Docking. The compounds were docked to PDE5 (PDB
entry 2H42) using Glide in its XP mode in a standard procedure. The
water molecules were removed, while the zinc and magnesium ions were
retained with charge +2, respectively. The compounds were sketched by
Maestro and processed by LigPrep under its default parameters. The
docked conformation of the compound with the lowest energy was
selected for further study.
QM/MM Calculation Procedure. In brief, the systems studied
were divided into two layers: the small molecule and the side chain of
Y612 were placed in the QM layer, while the rest of the protein was in the
MM layer. For the QM layer, the B3LYP/6-31G(d) method was applied
for 1ꢀ4, but the B3LYP/lanl2dz was used for 5.10 The MM layer of the
system was modeled with the AMBER force field. Single-point energy
computations were performed using MP2/6-311+G(d) for 1ꢀ4 and
MP2/6-311+G(d) plus SDD for 5. The binding energies between the
inhibitors and the residue Y612 were computed via the protocol as
described previously.10
Chemistry. 1 and 2 were prepared from the starting material
o-propoxybenzonitrile following the procedure in Scheme 1. Halo-
genation of 1 resulted in the corresponding 5-halopyrimidinone deriva-
tives 3ꢀ5.
All melting points were determined on a Buchi apparatus and are
uncorrected. 1H NMR spectra were determined using a Mercury 300 MHz,
FT NMR spectrometer. HRMS were performed on a Finnigan MAT95
mass spectrometer, and ESIMS was carried out on a Finnigan LCQdeca
mass spectrometer. Reaction solvents were purchased and used without
further purification. HPLC conditions were as follows: column, YMC-Pack
CN 5 μM, 4.6 mm ꢁ 250 mm; solvent system, (A) MeCN; (B) 0.02 M
KH2PO4 (pH 6.0); step gradient, time 0, 30% A; time 20 min, 70% A;
stop time, 25 min; flow rate 1.0 mL/min; UV detection, 220 nm;
injection volume, 5 μL; temperature, 30 ꢀC. The purity of all target
compounds was >95% as confirmed by HPLC.
6-Ethyl-2-[2-propoxy-5-(4-methyl-1-piperazinylsulfonyl)-
phenyl]pyrimidin-4(3H)-one (1). A mixture of 5-(4-methylpiper-
azin-1-ylsulfonyl)-2-propoxybenzimidamide 8 (1.2 g, 3.2 mmol), methyl
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Journal of Medicinal Chemistry
BRIEF ARTICLE
(t, J = 7.5 Hz, 3H), 1.16 (t, J = 7.5 Hz, 3H). HRMS calcd [M + Na]+ for
C20H27N4O4NaSI 569.0695, found 569.0704.
Bioassay. The synthesized compounds 1ꢀ5 were evaluated for their
inhibitory activities against PDE5 isolated from rabbit platelet using
[3H]cGMP SPA kit detailed in the Supporting Information.
(5) Politzer, P.; Lane, P.; Concha, M. C.; Ma, Y.; Murray, J. S.
An overview of halogen bonding. J. Mol. Model. 2007, 13, 305–311.
(6) Voth, A. R.; Khuu, P.; Oishi, K.; Ho, P. S. Halogen bonds as
orthogonal molecular interactions to hydrogen bonds. Nat. Chem. 2009,
1, 74–79.
(7) Matter, H.; Nazare, M.; Gussregen, S.; Will, D. W.; Schreuder,
H.; Bauer, A.; Urmann, M.; Ritter, K.; Wagner, M.; Wehner, V. Evidence
’ ASSOCIATED CONTENT
for CꢀCl/CꢀBr pi interactions as an important contribution to
3 3 3
proteinꢀligand binding affinity. Angew. Chem., Int. Ed. 2009, 48, 2911–
S
Supporting Information. Figures S1ꢀS4, Tables S1 and
b
2916.
S2, additional information on analytical and bioassay data
for 1ꢀ5, 7, and 8, protein purification and crystallization, and
structure determination and refinement. This material is available
free of charge via the Internet at http://pubs.acs.org.
(8) Voth, A. R.; Hays, F. A.; Ho, P. S. Directing macromolecular
conformation through halogen bonds. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 6188–6193.
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in biological molecules. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16789–
16794.
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Zhu, W. Halogen bonding—a novel interaction for rational drug design?
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Accession Codes
†The atomic coordinates 3SHY, 3SHZ, and 3SIE have been
deposited in the Protein Data Bank.
(11) Hardegger, L. A.; Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert,
R.; Stihle, M.; Gsell, B.; Thoma, R.; Diez, J.; Benz, J.; Plancher, J. M.;
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’ AUTHOR INFORMATION
Corresponding Author
*For Y.X.: phone, +86 21 50801267; e-mail, ycxu@mail.shcnc.ac.
cn. For J.S.: phone, +86 21 20231962; e-mail, jsshen@mail.
shcnc.ac.cn. For W.Z.: phone, +86 21 50805020; fax, +86 21
50807088; e-mail, wlzhu@mail.shcnc.ac.cn.
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Author Contributions
#These authors contributed equally to this study.
’ ACKNOWLEDGMENT
This work was supported by NNSF (Grant 20721003), CAS
(Grants KSCX2-YW-R-168 and KSCX2-YW-R-208), the “100
Talents Project” of CAS (to Y.X.), Shanghai S&T Foundation
(Grant 09540703900), National Science & Technology Major
Project (Grants 2009ZX09301-001 and 2009ZX09102-056), Inter-
national Cooperation Project of MOST (Grant 2010DFB73280),
the State Key Laboratory of Drug Research (Grant
SIMM1106KF-05), and Natural Science Foundation of Shanghai
(Grant 11ZR1408700). The authors thank Dr. Nick K. Terrett at
Ensemble Therapeutics for his review of the manuscript.
’ ABBREVIATIONS USED
GAF, cGMP-specific phosphodiesterase, bacterial adenylyl cyclase,
FhLA transcriptional regulator; ONIOM, our own N-layered
integrated molecular orbital and molecular mechanics; PDB,
Protein Data Bank; PDE5, phosphodiesterase type 5; QM/MM,
quantum mechanics/molecular mechanics
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