Design, synthesis and evaluation of N2,N4-diaminoquinazoline based inhibitors of phosphodiesterase type 5

Article abstract of DOI:10.1016/j.bmcl.2018.11.043

We describe the design, synthesis and evaluation of a series of N²,N⁴-diaminoquinazoline analogs as PDE5 inhibitors. Twenty compounds were prepared and these were assessed in terms of their PDE5 and PDE6 activity, ex-vivo vasodilation response, mammalian cytotoxicity and aqueous solubility. Molecular docking was used to determine the binding mode of the series and this was demonstrated to be consistent with the observed SAR. Compound 15 was the most active PDE5 inhibitor (IC₅₀ = 0.072 ± 0.008 μM) and exhibited 4.6-fold selectivity over PDE6. Ex-vivo assessment of 15 and 22 in a rat pulmonary artery vasodilation model demonstrated EC₅₀s of 1.63 ± 0.72 μM and 2.28 ± 0.74 μM respectively.

Full text of DOI:10.1016/j.bmcl.2018.11.043

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of N2,N4-diaminoquinazoline based
inhibitors of phosphodiesterase type 5
Nattakarn Pobsuka, Tamkeen Urooj Parachab, Nattiya Chaichamnongc,d, Nattapas Salaloya,
Praphasri Suphakune, Supa Hannongbuaa, Kiattawee Choowongkomone, Dumrongsak Pekthongb,
⁎
⁎
Krongkarn Chootipf, Kornkanok Ingkaninand, , M. Paul Gleesong,
a Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand
c Division of Applied Thai Traditional Medicine, Faculty of Public Health, Naresuan University, Phitsanulok 65000, Thailand
d Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand
e Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
f Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand
g Department of Biomedical Engineering, Faculty of Engineering, King Mongkut’s Institute of Technology, Ladkrabang 10520, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords:
We describe the design, synthesis and evaluation of a series of N2,N4-diaminoquinazoline analogs as PDE5 in-
hibitors. Twenty compounds were prepared and these were assessed in terms of their PDE5 and PDE6 activity,
ex-vivo vasodilation response, mammalian cytotoxicity and aqueous solubility. Molecular docking was used to
determine the binding mode of the series and this was demonstrated to be consistent with the observed SAR.
Drug design
Phosphodiesterase type 5
Quinazoline analogs
Solubility
Compound 15 was the most active PDE5 inhibitor (IC50 = 0.072
0.008 µM) and exhibited 4.6-fold selectivity
over PDE6. Ex-vivo assessment of 15 and 22 in a rat pulmonary artery vasodilation model demonstrated EC50s of
1.63
0.72 µM and 2.28
0.74 µM respectively.
Approximately 17.7 million people die each year as a result of
cardiovascular diseases (CVDs) according to the World Health
Organization (WHO).1 CVDs are disorders of the heart and blood vessels
that can result from factors including a poor diet, high blood pressure,
high blood cholesterol, diabetes, etc. There exists a huge healthcare
market which includes erectile dysfunction (ED)2–5 and pulmonary
arterial hypertension (PAH).6 PDE5 is an enzyme that is highly dis-
tributed in smooth muscle tissue located in the heart, lung, corpus ca-
vernosum, liver, brain, and stomach. The enzyme catalyzes the hydro-
lysis of cyclic guanosine monophosphate (cGMP)7 and has emerged as a
key target to treat both CVDs and ED8–11 with drugs including Sildenafil
incorporation of a range of substituents at N2-,N4-position and quina-
zoline ring. Twenty compounds were synthesized according to methods
described in Scheme 1. The compounds were evaluated against PDE5
and PDE6, their vasodilation effect was assessed in an ex-vivo rat pul-
monary artery model and mammalian cytotoxicity and phosphate
buffer solubility were determined.
Substitution of 2,4-dichloroquinazoline with amines was achieved
using triethylamine (TEA) in tetrahydrofuran (THF) giving compounds
1–10 following the general procedures reported elsewhere.21–23 Sub-
stitution at the 2-position of intermediates 1–10 was performed under
acidic condition and/or heat to give compounds 11–30 as solids, with
yields ranging from 15% to 90%.21,23 The purity of compounds were
confirmed as > 95% by HPLC analysis. The 1H-, 13C-NMR and mass
spectra of all compounds were obtained and are in agreement with the
proposed structures (see supporting information).
(Viagra®) and Tadalafil (Cialis®).12–16 Nevertheless,
a number of
common side effects exist including headaches, abnormal vision,
muscle pain and diarrhea.3,4 Given the effectiveness of the target, there
is continuing interest in the discovery of novel PDE5 inhibitors.
In this study we report our efforts to profile the activity of pre-
viously unreported activity of N2,N4-diaminoquinazolines derivatives at
PDE5. We utilized molecular docking to PDE5 active site and scaffold
similarity to known PDE5 inhibitors (Fig. 1) to facilitate the design and
modification of new compounds (Fig. 2).17–20 We investigated the
A radioactive assay adapted from Sonnengburg et al. was used to
determine the inhibitory activity of compounds towards PDE5 and 6
(see supporting information).24 Reported are the concentrations re-
quired to inhibit the enzymes by 50% (IC50) and the percent inhibition
at the highest concentration tested (Table 1).25,26 The cytotoxicity of
⁎ Corresponding authors.
E-mail addresses: kornkanoki@nu.ac.th (K. Ingkaninan), paul.gl@kmitl.ac.th (M.P. Gleeson).
https://doi.org/10.1016/j.bmcl.2018.11.043
Received 10 September 2018; Received in revised form 19 October 2018; Accepted 20 November 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Pobsuk,N.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2018.11.043

N. Pobsuk et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
O
O
vs 27). Compound 25, containing 3-amino-5-methylpyrazole, was
H
N
O
N
found to be inactive (IC50 = 3.706
0.642 µM) suggesting a methy-
N
N
N
lene linker to the aromatic ring may be critical. The SAR associated
with the R2 position showed greater variability. Introduction of small
polar substituents such as amide and sulfonamide (14–16 & 20–29)
were preferable over larger substituents such as the 4-morpholino and
4-phenylurea exemplars (12, 13, 18 & 19). Compounds lacking sub-
stituents on the phenyl ring (11 & 17) displayed weak to no PDE5 in-
hibitory activity. Addition of a methoxy substituents to the 6- and 7-
position of the quinazoline ring had no dramatic effect on the activity.
However, the use of the 5-chloropyrimidine scaffold led to the abol-
ishment of all PDE5 activity suggesting the bicyclic system is critical for
activity. Additional selectivity data against PDE6 was obtained
(Table 2). Sildenafil was shown to have a 6.5-fold selectivity over PDE6
compared to compound 15 which shows 4.6-fold selectivity and com-
pound 22, which shows 4.0 fold selectivity.
N
H
N
O
O
N
S
N
H
O
O
O
Sildenafil
IC50= 2.0 nM
Tadalafil
IC50 = 12.0 nM
O
N
O
O
O
HN
N
N
Cl
N
O
N
N
-
+
N
N
O Na
NH
O
O
CHEMBL551052
IC50= 2.9 nM
E4021
We undertook computational docking of compounds to PDE5 (PDB
code: 3HC8)19 using GOLD5.1 to help rationalize the observed SAR.
Briefly, cofactors and solvent were initially removed from the crystal
structure, and docking was performed using default settings. A H-bond
between the Gln817 H-bond donor and substrate acceptor was a re-
quirement for a valid solution. Illustrated in Fig. 2 is the docked solu-
tion of compound 15 along with the experimental solutions for Silde-
nafil (PDB code: 1UDT)10 and CHEMBL551052 (PDB code: 3HC8).19
The guanidinyl core of Sildenafil makes two H-bond interactions to the
amide sidechain of Gln817, two pi-stacking interactions with Phe786
and Phe820 and multiple H-bonds to active site water molecules though
its sulfonamide moiety. The quinazoline ring of CHEMBL551052 makes
comparable pi-stacking interactions with Phe786 and Phe820, interacts
with Gln817 with its pyridyl group and forms multiple solvent inter-
actions.
IC50 = 3.9 nM
Fig. 1. Structures of PDE5 inhibitors marketed drugs (Sildenafil and Tadalafil),
CHEMBL55105219 and E4021.17
the compounds against adenocarcinomic human alveolar basal epithe-
lial cells (A549 cells, ATCC CCL-185) were assessed using an MTT based
assay and are reported as an IC50 (Table 2). Compounds 11–30 showed
affinities for PDE5 in range of 0.07 to > 10 µM (Table 1). Compounds
15 and 22 were found to be the most potent of this series showing IC50
values against PDE5 of 0.072 ( 0.008) and 0.089 ( 0.011) µM re-
spectively. Sildenafil, which was used as standard, displayed an IC50 of
0.002 ( 0.0008) µM. At the R1 position, the furfurylamino and 2-
thiophenemethylamino substituents displayed comparable if slightly
weaker activity than the corresponding benzyl amino derivatives
(compounds 22 vs 24 and 28 vs 29). The benzylamino derivatives also
exhibited better PDE5 activity than the corresponding 3-methox-
ybenzylamino compounds by between 2 and 20-fold (15 vs 16 and 26
The quinazoline ring of compound 15 docks in a conformation that
sees the overlap of the quinazoline with the bicyclic ring system of
CHEMBL551052. The former is found to interact with Gln817 via the 2-
position nitrogen H-bond acceptor and make the required π-stacking
O
N
O
c
a
b
NH
2
N
HOH
HOH
S
HOH
HOH
O
HOH
HOH
N
HOH
N
S
O
O
Phe786
HN
HOH
HN
N
HOH
Phe786
Phe786
N
O
N
O
N
N
N
O
HN
NH
N
O
Phe820
Phe820
Phe820
N
O
O
O
O
NH
NH
2
2
NH
2
Gln817
Gln817
Gln817
Fig. 2. Sildenafil (top left, PDB code: 1UDT) and compound 15 (top right) bound to PDE5 enzyme. Shown in the bottom panels are 2D ligand interactions diagrams
showing the interactions between PDE5 and Sildenafil (a), CHEMBL551052 (b) and compound 15 (c).
2

N. Pobsuk et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
1
1
Cl
N
R
N
R
NH
2
R
R
R
R
(a)
(b)
N
N
N
2
2
R
R
+
Cl
NH
R
Cl
R
N
R = H or -OCH3
11-30
Scheme 1. Reagents and conditions used to synthetic routes: (a) amine, Et3N in THF at room temperature, (b) 1 M HCl, isopropanol, 90 °C, overnight.
Table 1
Molecular weight (M.W.), c log P, inhibitory activity toward PDE5 and PDE6 of compound 11–30. nd indicated not determined.
ID
Core structure
R1
R2
M.W.a
c log Pa
PDE5 IC50 µM (SE)
PDE6 IC50 µM (SE)
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Quinazoline
Benzylamino
H
326.4
411.5
433.5
405.5
405.5
435.5
332.4
466.6
417.5
411.5
375.5
411.5
375.5
395.4
395.4
5.3
5.2
4.4
3.9
3.9
3.8
5.2
6.4
5.1
3.9
4.1
3.9
4.1
3.0
3.3
1.376 (0.169)
0.525 (0.066)
1.260 (0.092)
0.946 (0.035)
0.072 (0.008)
0.350 (0.045)
1.440 (0.170)
0.775 (0.101)
0.514 (0.021)
0.224 (0.038)
0.177 (0.016)
0.089 (0.011)
0.290 (0.098)
0.116 (0.023)
3.706 (0.642)
0.890 (0.100)
Benzylamino
4-Morpholino
4-SO2N(CH3)2
4-SO2NH2
3-SO2NH2
3-SO2NH2
H
0.450 (0.120)
Benzylamino
nd
Benzylamino
nd
Benzylamino
0.332 (0.047)
3-Methoxybenzylamino
2-Thiophenemethylamino
2-Thiophenemethylamino
2-Thiophenemethylamino
2-Thiophenemethylamino
2-Thiophenemethylamino
2-Thiophenemethylamino
2-Thiophenemethylamino
Furfurylamino
nd
nd
4-NHCONHPh
4-morpholino
4-SO2NH2
4-CONH2
3-SO2NH2
3-CONH2
3-SO2NH2
3-SO2NH2
nd
0.610 (0.090)
nd
nd
0.360 (0.120)
nd
0.190 (0.020)
nd
3-Amino-5-methylpyrazolo
26
27
28
29
6,7-Dimethoxyquinazoline
5-Chloropyrimidine
Benzylamino
3-SO2NH2
3-SO2NH2
3-SO2NH2
3-SO2NH2
465.5
495.6
471.6
455.5
3.6
3.5
3.5
2.7
0.218 (0.074)
0.443 (0.124)
0.555 (0.120)
0.284 (0.122)
nd
nd
nd
nd
3-Methoxybenzylamino
2-Thiophenemethylamino
Furfurylamino
30
Benzylamino
H
310.8
4.6
> 10
33.500 (1.940)
Sildenafil
474.6
1.2
0.002 (0.0008)
0.013 (0.001)
Sodium nitroprusside
a
261.9
0.1
nd
nd
M.W. and c log P was calculated using JChem Version 14.9.100.70.
Table 2
Biological activities, selectivity index (SI), vasodilation effects and solubility at pH7.4
.
ID
PDE5
PDE6
SI
A549 IC50 µM (SE)
SI
Vaso
Sol. pH7.4
IC50 µM (SE)
IC50 µM (SE)
PDE6/PDE5
A549/PDE5
EC50 µM (SE)
mg/ml (µM)
11
12
15
19
22
24
1.376 (0.169)
0.525 (0.066)
0.072 (0.008)
0.514 (0.021)
0.089 (0.011)
0.116 (0.023)
0.890 (0.100)
0.450 (0.120)
0.332 (0.047)
0.610 (0.090)
0.360 (0.120)
0.190 (0.020)
0.65
0.86
4.61
1.19
4.04
1.64
12.29 (1.20)
12.10 (3.60)
11.15 (1.22)
11.37 (3.07)
15.04 (4.44)
26.92 (2.16)
9
nd
0.13 (0.41)
0.25 (0.60)
0.56 (1.37)
0.20 (0.48)
0.14 (0.34)
0.15 (0.38)
23
nd
155
22
1.63 (0.72)
nd
169
2.28 (0.74)
232
nd
Sildenafil
0.002 (0.0008)
0.013 (0.001)
6.50
nd
nd
nd
0.14 (0.05)
nd
Nitroprusside
nd
nd
nd
nd
0.019 (0.01)
nd
interactions with Phe820 and Phe786. The sulfonamide substituent
adopts a position similar to that found in Sildenafil. The benzyl/furfuryl
groups at the 4-position of quinazoline bind within the pocket holding
the ethoxy group of Sildenafil and the longer ether chain of
CHEMBL551052. The weaker activity of the methoxy substituted
phenyl ring is consistent with the fact that it is a rather small pocket.
The predicted binding mode is also consistent with the fact that com-
pound 30, which has a 5-chloropyrimidine core, has negligible affinity.
The compound is not as effective at π-stacking with Phe820 and Phe786
in particular.
selectivity of 169-fold for PDE5. An ex-vivo vasodilation model was
then employed to assess the efficacy of 11 and 22 (Table 2).28 Sildenafil
and nitroprusside were used as standards leading to vasodilation re-
sponses (EC50) of 0.14 ( 0.05) and 0.019 ( 0.01) µM, respectively.
Compounds 15 and 22 displayed EC50 values of 1.63 ( 0.72) and 2.28
(
0.74) µM, respectively. Sildenafil is > 4-fold more active while ni-
troprusside is > 30-fold more active indicating additional effort is
needed to identify further analogs with improved affinity towards
PDE5. Finally, we assessed the solubility of a subset of compounds in
phosphate buffer at pH7.4 using an equilibrium solubility protocol
(Table 2).29 The compounds displayed solubilities in the range of
0.10–0.56 mg/ml (0.22–1.37 µM). Compound 15 was identified as
being the most soluble of all compounds.
The general cytotoxicity of the compounds was assessed using the
human alveolar basal epithelial cell line (ATCC CCL-185)27 with IC50
s
range from 10 to 30 µM. Compound 15, which showed the greatest
activity towards PDE5, also possessed the highest cytotoxicity with an
IC50 of 11.1 µM ( 1.22). Nevertheless, this still corresponds to a se-
lectivity of over 155-fold for PDE5. Compound 22 demonstrated a
In conclusion, we report the inhibition of PDE5 by N2,N4-quinazo-
linediamines derivatives – previously unreported PDE5 inhibitors.
Compounds in this class are reported to have anti-malarial activity30
3

N. Pobsuk et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
which could be consistent with this type of activity.31,32 The binding
mode has been determined via molecular docking and is consistent with
the observed SAR. Compounds 15 and 22 exhibited high PDE5 in-
hibitory activities (IC50) of 0.072 and 0.089 µM, respectively and con-
firmed activity in an ex-vivo rat vasodilation model. Selectivity over the
PDE6 isoform was found to be comparable to Sildenafil (4 vs 6-fold).
The compounds also displayed good selectivity in terms of their
mammalian cytotoxicity.
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The authors are grateful for financial support provided by the
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Kasetsart University Research and Development Institute (KURDI) and
the Development and Promotion of Science and Technology Talents
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