From Celecoxib to a Novel Class of Phosphodiesterase 5 Inhibitors: Trisubstituted Pyrazolines as Novel Phosphodiesterase 5 Inhibitors with Extremely High Potency and Phosphodiesterase Isozyme Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.0c01120

A ligand-based approach involving systematic modifications of a trisubstituted pyrazoline scaffold derived from the COX2 inhibitor, celecoxib, was used to develop novel PDE5 inhibitors. Novel pyrazolines were identified with potent PDE5 inhibitory activit

Full text of DOI:10.1021/acs.jmedchem.0c01120

pubs.acs.org/jmc
Article
From Celecoxib to a Novel Class of Phosphodiesterase 5 Inhibitors:
Trisubstituted Pyrazolines as Novel Phosphodiesterase 5 Inhibitors
with Extremely High Potency and Phosphodiesterase Isozyme
Selectivity
Mohammad Abdel-Halim,* Sara Sigler, Nora A. S. Racheed, Amr Hefnawy, Reem K. Fathalla,
Mennatallah A. Hammam, Ahmed Maher, Yulia Maxuitenko, Adam B. Keeton, Rolf W. Hartmann,
Matthias Engel, Gary A. Piazza, and Ashraf H. Abadi*
Cite This: J. Med. Chem. 2021, 64, 4462−4477
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: A ligand-based approach involving systematic
modifications of a trisubstituted pyrazoline scaffold derived from
the COX2 inhibitor, celecoxib, was used to develop novel PDE5
inhibitors. Novel pyrazolines were identified with potent PDE5
inhibitory activity lacking COX2 inhibitory activity. Compound
d12 was the most potent with an IC50 of 1 nM, which was three
times more potent than sildenafil and more selective with a
selectivity index of >10,000-fold against all other PDE isozymes.
Sildenafil inhibited the full-length and catalytic fragment of PDE5,
while compound d12 only inhibited the full-length enzyme,
suggesting a mechanism of enzyme inhibition distinct from
sildenafil. The PDE5 inhibitory activity of compound d12 was
confirmed in cells using a cGMP biosensor assay. Oral
administration of compound d12 achieved plasma levels >1000-fold higher than IC50 values and showed no discernable toxicity
after repeated dosing. These results reveal a novel strategy to inhibit PDE5 with unprecedented potency and isozyme selectivity.
attributed to an off-target mechanism involving the inhibition
of phosphodiesterase 5 (PDE5), which leads to vasodilatation
that may offset the vasoconstriction resulting from inhibition of
prostacyclin biosynthesis.8−11
INTRODUCTION
■
Celecoxib is a non-steroidal anti-inflammatory drug (NSAID)
approved for the treatment of pain and swelling associated with
rheumatoid arthritis or osteoarthritis. Celecoxib (4-[5-(4-
methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-
benzenesulfonamide),1 unlike other NSAIDs that suppress
prostaglandin synthesis by non-selective inhibition of cyclo-
oxygenases types 1 and 2 (COX1 and COX2), belongs to a
distinct subclass of NSAIDs referred to as coxibs that
selectively inhibit the inducible COX2 isozyme. By sparing
COX1 and physiological levels of prostaglandins, coxibs were
developed to reduce the risk of gastric ulceration and other
toxicities associated with the inhibition of constitutively
expressed COX1.2 After FDA approval of several coxibs,
prolonged use was unexpectedly found to have cardiovascular
toxicities, including myocardial infarction and stroke.3,4 These
toxicities are linked to imbalance between prostacyclin and
thromboxane levels, in which the biosynthesis of the former is
suppressed by selective COX2 inhibitors.5−7 Unlike other
COX2 inhibitors, such as rofecoxib that was withdrawn from
the market because of risks associated with cardiovascular
toxicity, celecoxib is still marketed and is considered to be less
cardiotoxic. The reduced toxicity of celecoxib may be partly
PDE5 is a cGMP-specific PDE that plays an important role
in the regulation of cGMP levels in multiple cell types.12−14
Like all PDEs, PDE5 is a dimeric protein, each monomer
consisting of a C-terminal catalytic domain and N-terminal
regulatory domain.14 The regulatory domain of PDE5 contains
tandem GAF domains (GAF-A and GAF-B) and a site for
phosphorylation by cyclic nucleotide-dependent protein
kinases. PDE5 is allosterically regulated by binding of cGMP
to GAF-A domain, leading to an increase in its enzymatic
activity.15,16
Received: June 30, 2020
Published: April 1, 2021
https://doi.org/10.1021/acs.jmedchem.0c01120
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 4462−4477
4462

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
PDE5 is highly abundant in smooth muscle cells and plays
an important role in regulating contractility.16 PDE5 became
an important therapeutic target for the treatment of erectile
dysfunction and pulmonary arterial hypertension, which led to
the development of potent PDE5 inhibitors, such as sildenafil,
vardenafil, and tadalafil.17,18
More recently, several potent PDE5 inhibitors were
reported; however, they show significant cross-reactivity with
PDE6 and PDE11, similar to currently approved PDE5
inhibitors.19−22 Fiorito et al. reported a highly potent
tetrahydrobenzo[b][1,6]naphthyridine derivative with about
500-fold selectivity vs PDE6; however, no selectivity data were
provided regarding the other PDE isoforms.23
All the classical PDE5 inhibitors like sildenafil, vardenafil,
and tadalafil are known to act through binding within the
catalytic domain of PDE5, competing with cGMP.24−26
However, it was reported that the N-terminal 46 amino acids
in GAF-B domain (Glu420 through Gly466) are required for
high potency of vardenafil but not for sildenafil, despite having
a similar mode of binding, as shown in cocrystal structures.27
Here, we show that the previously reported off-target activity
of celecoxib on PDE5 occurs by a unique mechanism of
inhibition that requires the presence of the regulatory domain.
As described below, we employed a ligand-based approach to
uncouple PDE5 and COX2 inhibitory activities using a
pyrazoline scaffold derived from celecoxib. A novel series of
pyrazolines were found with high potency and selectivity to
inhibit PDE5 while lacking COX2 inhibitory activity, providing
distinct advantages over currently available PDE5 inhibitors.
Figure 1. Inhibition of PDE5 full length vs the catalytic domain by
celecoxib and sildenafil.
of the sulfonamide group at the 1-phenyl with a carboxylic acid
functional group. The second modification was the replace-
ment of trifluoromethyl group with t-butyl at position 3 of the
pyrazole. Finally, partial saturation of the pyrazole to the non-
coplanar Δ2-pyrazoline was found to improve PDE5 inhibitory
potency. These modifications led to compound A (Figure 2)
with an IC50 of 8.4 μM against PDE5, giving more than 4-fold
improved potency compared to celecoxib (IC50 = 37 μM). The
subsequent optimization of compound A involved the
replacement of the 4-tolyl moiety by several mono-substituted
aryls (details are recently published);29 this led to compound B
that has a 4-methoxyphenyl group replacing the 4-tolyl (Figure
2). Compound B showed a further 4-fold increase in potency
against PDE5 (IC50 = 2 μM). In this work, compound B was
selected as a template for optimization and to explore SAR, as
discussed in the following sections (Figure 2). The details of
modifications of celecoxib to compounds A and B have been
recently published.29
Chemical Synthesis. The synthesis of the pyrazoline
derivatives was performed in two steps. In the first step, the
required enones were furnished through a Claisen−Schmidt
condensation between aromatic aldehydes and pinacolone or
acetophenone analogues. In the second step, the respective
enones were reacted with 4-hydrazinobenzoic acid hydro-
chloride to yield the desired pyrazolines using the previously
reported regioselective synthesis of 1,3,5-triarylpyrazolines
(Schemes 1 and 2).30 Following a rigidification strategy,
bridging atom(s), particularly, −CH2− (a48), −CH2−CH2−
(a49), or −O−CH2− (a50), were introduced between the
phenyl at position 3 and C-4 of the original 1,3,5-trisubstituted
pyrazoline system (Scheme 2).
RESULTS AND DISCUSSION
■
Synthetic Strategy and Approach. Our rationale to
develop novel PDE5 inhibitors was inspired from previous
studies suggesting that celecoxib inhibits PDE5 and from our
earlier synthetic efforts, which revealed that certain pyrazoline
derivatives yielded PDE5 inhibitors with improved potency.
To this end, we characterized the PDE5 inhibitory activity of
celecoxib using the PDE5 catalytic domain as well as the full-
length enzyme and compared its inhibitory activity with
sildenafil. As anticipated, sildenafil inhibited both the catalytic
domain and the full-length enzyme with IC50 values of 4.9 and
3 nM, respectively.27 Surprisingly, celecoxib was found to only
inhibit the full-length enzyme, with an IC50 of 37 μM, whereas
the activity of the catalytic domain was not affected at
concentrations up to 100 μM. Figure 1 shows the dose−
response curves for sildenafil and celecoxib against both the
PDE5 catalytic domain and the full-length PDE5. These results
suggested that celecoxib inhibits PDE5 by a mechanism that is
different from conventional PDE5 inhibitors, which compete
with cGMP for binding to the catalytic domain. Instead,
celecoxib appeared to exert a mode of action that was strictly
dependent on the regulatory domain. Thus, relying on binding
to a less conserved regulatory site, celecoxib was identified as a
promising lead compound to develop a new class of inhibitors
with better selectivity profile over classical substrate-compet-
itive inhibitors.28 Applying a ligand-based approach starting
from celecoxib, systematic structural modifications were
undertaken to enhance potency and selectivity to inhibit
PDE5.
The chemical optimization included three rounds of
synthesis, each of which was followed by biological evaluation,
in which the first round involved simple variations of
compound B with substitutions at positions 3 and 5, as
shown in Figure 2. A second round of optimization involved
extending the substitution at position 1 from a carboxylic acid
with various amide derivatives. Amides were synthesized by the
reaction of the benzoic acid derivatives with the suitable amine
precursor using EDCI·HCl as a coupling agent in CH2Cl2
Our initial synthetic efforts aimed at enhancing the PDE5
inhibitory activity of celecoxib while abolishing its COX2
inhibitory action by modulating essential features required for
COX2 inhibition.1 This was successfully done by replacement
4463
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. Optimization of the PDE5 inhibitory activity starting from celecoxib.
(Scheme 3). A third round included combining the favorable
structural features obtained from the first two rounds (Figure
2). To confirm the importance of the non-coplanar pyrazoline
system for the PDE5 activity, compounds a4 and d9 were
oxidized to the corresponding pyrazole analogues using DDQ
in refluxing benzene (Schemes 1 and 3).
Biological Activity. All the benzoic acid derivatives and
the amides were evaluated for their ability to inhibit full-length
recombinant PDE5. Each compound was evaluated in two
steps. For the first screening step, the percentage of PDE5
inhibition was determined at a concentration of 10 μM.
Compounds showing greater than 50% inhibition were re-
tested with an extended concentration range to determine
PDE5 inhibitory potency by calculation of IC50 values. The
results for PDE5 inhibition are shown in Tables 1−3.
Additionally, some compounds showing potent PDE5
inhibition were screened against COX1 and COX2 at 50
μM. All compounds tested were inactive against COX1 and
COX2 at the screening dose (Table S1, Supporting
Information).
Structure−Activity Relationships (SAR) for PDE5
inhibitory activity. First Round of Optimization. To
optimize the PDE5 inhibitory activity of compound B, we
started the modifications at two major sites: the first at position
3 of the pyrazoline and the second at the 5-phenyl.
Modifications at Position 3 of the Pyrazoline. The
modifications at position 3 were started by replacing the tert-
butyl group in compound B by alicyclic groups like
cyclopropyl, 1-methylcyclopropyl, and cyclohexyl (compounds
a1−a3, respectively), which led to a loss of activity. This
finding emphasizes the important role of a bulky and highly
branched alkyl to maintain activity, indicating that the higher
branching of the tert-butyl group is more favored by the
binding pocket. On the other hand, the replacement of the tert-
4464
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Scheme 1
Scheme 2
butyl in B by a phenyl ring slightly improved the potency,
suggesting that additional CH−π interactions were formed
(compound a4; IC50 = 1.2 μM). This is especially evident by
comparing the cyclohexyl analogue, a3, to a4. However, when
other derivatives with aromatic heterocyclic substitutions such
as pyrrole, pyridine, thiophene, and furan were synthesized, the
activity was completely abolished (compounds a5−a8,
respectively). Additionally, the use of bulkier aromatic systems
4465
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Scheme 3
such as 1-napthyl, 4-biphenyl, and 4-phenoxyphenyl at position
3 of the pyrazoline (compounds a9−a11, respectively) led to
loss of activity, which might be due to the higher steric demand
of these aromatic systems that are not tolerated within the
binding pocket. Even the use of more polar methylenediox-
yphenyl (compound a12) did not recover the potency to
inhibit PDE5. Interestingly, compound a16 with the 3,4-
dimethoxyphenyl group that is the open-chain analogue of
compound a12 showed moderate PDE5 inhibitory activity,
indicating a possible steric clash between the methylene bridge
in a12 and the PDE5 binding pocket.
Next, we investigated the effect of adding different types of
substituents at the 3-phenyl ring of compound a4. Initially, a
mono-halogen substitution with chlorine, bromine, and
fluorine at different positions on the phenyl ring in compound
a4 was synthesized to give compounds a17−a22 and a24 and
a25. This resulted in loss of the PDE5 inhibitory activity (IC50
> 10 μM) apart from compound a19 having a para-fluoro
substitution. The loss of PDE5 inhibitory activity might be
either due to the non-tolerated lipophilic and steric nature of
the substituents or because of their electron-withdrawing
property, which decreases the π-electron density on the phenyl
ring and might be unfavorable here. Consequently, compound
a23 with a 2,4-dichloro substitution was found to be inactive,
too. On the other hand, a hydrophilic electron-donating
substituent like a hydroxyl group showed the best activity at
the ortho position in compound a29 with more than a 3-fold
improvement in potency compared to the unsubstituted
phenyl in compound a4, while at the para position, the
hydroxyl group was tolerated with a 2-fold decrease in PDE5
inhibitory potency (compound a31 compared to a4). Moving
the hydroxyl group to the meta position led to loss of PDE5
inhibitory activity (compound a30). The higher activity
displayed by the ortho-substituted compound a29 (IC50
=
0.33 μM) might be explained by an intra-molecular H-bond
with the imine nitrogen, which stabilizes the coplanar,
biologically active conformation, besides its electron-donating
effect. This is supported by the 10-fold lower activity noted for
the 2-methoxy congener a13, which is expected to stabilize a
non-coplanar conformation due to the ortho effect. In
agreement, some of the activity was recovered by positioning
the methoxy group in meta (a14, IC50 = 1.8 μM), whereas the
para-methoxy analogue was inactive (a15). The latter
unfavorable effect was also apparent in the 3,4-dimethoxy-
substituted derivative, strongly suggesting a steric clash of the
para methyl ether, similar to the methylene bridge in the
4466
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 1. Inhibition of Recombinant PDE5 (the Carboxylic Acid Derivatives)
PDE5 IC
PDE5 IC
a 50
a 50
Cpd #
Ar
R1
(μM)
Cpd #
a25
a26
Ar
R1
(μM)
A
B
4-tolyl
t-Bu
8.4
2
4-methoxyphenyl
4-methoxyphenyl
4-bromophenyl
o-tolyl
ND
0.5
4-methoxyphenyl t-Bu
a1
a2
a3
a4
a5
a6
a7
a8
4-methoxyphenyl cyclopropyl
4-methoxyphenyl 1-methylcyclopropyl
4-methoxyphenyl cyclohexyl
4-methoxyphenyl phenyl
4-methoxyphenyl pyrrol-2-yl
4-methoxyphenyl pyridin-2-yl
4-methoxyphenyl thiophen-2-yl
4-methoxyphenyl furan-2-yl
4-methoxyphenyl napthalen-1-yl
4-methoxyphenyl 4-phenoxyphenyl
4-methoxyphenyl [1,1′-biphenyl]-4-yl
4-methoxyphenyl benzo[d][1,3]dioxol-5-yl
4-methoxyphenyl 2-methoxyphenyl
4-methoxyphenyl 3-methoxyphenyl
4-methoxyphenyl 4-methoxyphenyl
4-methoxyphenyl 3,4-dimethoxyphenyl
4-methoxyphenyl 2-fluorophenyl
4-methoxyphenyl 3-fluorophenyl
4-methoxyphenyl 4-fluorophenyl
4-methoxyphenyl 2-chlorophenyl
4-methoxyphenyl 3-chlorophenyl
4-methoxyphenyl 4-chlorophenyl
4-methoxyphenyl 2,4-dichlorophenyl
4-methoxyphenyl 2-bromophenyl
ND
ND
ND
1.2
ND
ND
ND
ND
ND
ND
ND
ND
3.2
a27
a28
a29
a30
a31
a32
a33
a34
a35
a36
a37
a38
a39
a40
a41
a42
a43
a44
a45
a46
a47
b1
sildenafil
celecoxib
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
3-fluoro-4-methoxyphenyl
3-fluoro-4-methoxyphenyl
3-fluoro-4-methoxyphenyl
4-trifluoromethylphenyl
4-trifluoromethoxyphenyl
4-fluoro-3-methoxyphenyl
3-chloro-4-methoxyphenyl
3,5-difluoro-4-methoxyphenyl t-Bu
2,3-difluoro-4-methoxyphenyl t-Bu
2,5-difluoro-4-methoxyphenyl t-Bu
m-tolyl
p-tolyl
1.56
ND
0.33
ND
2.5
ND
ND
ND
1.19
0.13
0.59
ND
10
10.08
0.9
0.09
0.06
10
ND
ND
0.2
ND
0.003
37
2-hydroxyphenyl
3-hydroxyphenyl
4-hydroxyphenyl
2-ethoxyphenyl
4-ethoxyphenyl
2-nitrophenyl
phenyl
o-tolyl
t-Bu
t-Bu
t-Bu
a9
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
a22
a23
a24
1.8
ND
6.9
t-Bu
t-Bu
ND
ND
3.1
ND
ND
ND
ND
ND
3,5-difluorophenyl
t-Bu
t-Bu
t-Bu
phenyl
3-fluoro-4-methylphenyl
3,5-difluoro-4-ethoxyphenyl
4-methoxyphenyl
a
Full-length PDE5 was used. Values are mean of at least two experiments; standard deviation, <10%; ND: not determined for compounds that
showed less than 50% inhibition at 10 μM, no IC50 was determined.
Table 2. Inhibition of Recombinant PDE5 (the Rigidified
Compounds)
due to the increased steric requirements. This was apparent by
comparing the ortho-ethoxy analogue a32 to the ortho-methoxy
congener a13.
Searching for a replacement of the potentially metabolically
problematic ortho-OH group in compound a29, we synthe-
sized the ortho-tolyl derivative, a26. Methyl offered electron-
donating properties, while a pronounced ortho-effect against
the five-membered pyrazoline ring was avoided due to the
smaller size compared to the methoxy. Indeed, the activity of
a26 (IC50 = 0.5 μM) was only slightly reduced compared with
that of a29 and was superior to those of a27 and a28 (Table
1).
Confirming the deleterious effect of the electron-with-
drawing halogen substituents at the ortho position of the 3-
phenyl ring, a replacement of the ortho electron-donating
substituents in the sub-micromolar inhibitors a26 and a29 by a
nitro group abolished the activity (compound a34).
Rigidification of the Structure. As the final stage of
optimization at the 3-phenyl, we wanted to explore whether a
rigidification of compound a26 could increase PDE5 inhibitory
potency by fixing the dihedral angle between the pyrazoline
and the 3-phenyl. Such a strategy seemed straightforward
based on the favorable effect of the ortho-hydroxy function in
a29. However, the activity of the conformationally restricted
analogue (a48) was not enhanced compared to the plain
a
Cpd #
X
PDE5 IC50 (μM)
a48
a49
a50
−CH2−
−CH2CH2−
1.44
ND
1.16
−O−CH2− (O next to Ph)
a
Full-length PDE5 was used. Values are mean of at least two
experiments; standard deviation, <10%; ND: not determined for
compounds that showed less than 50% inhibition at 10 μM, no IC50
was determined.
benzodioxolyl derivative a16 (cf. above). Thus, the exploration
of the phenyl binding subpocket using different substituents
(also cf. below the para-methyl derivative a28) showed that its
ligand-accessible depth corresponded exactly to the diameter
of a phenyl ring. Accordingly, ethoxy substitutions at the ortho
and para positions in compounds a32 and a33, respectively,
further diminished the PDE5 inhibitory activity, which may be
4467
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 3. Inhibition of Recombinant PDE5 (the Amide Derivatives)
a
Cpd #
R1
4-methoxy
4-methoxy
4-methoxy
3-fluoro-4-methoxy
4-methoxy
4-methoxy
4-methoxy
4-methoxy
4-methoxy
3-fluoro-4-methoxy
3-fluoro-4-methoxy
4-fluoro-3-methoxy
3-chlro-4-methoxy
2,3-difluoro-4-methoxy
3,5-difluoro-4-ethoxy
3-fluoro-4-methyl
4-methoxy
R2
R3
n-butyl
ethyl
n-butyl
n-butyl
n-butyl
R4
X
PDE5 IC50 (μM)
c1
c2
c3
c4
c5
c6
d1
d2
d3
d4
d5
d6
d7
d8
d9
d10
d11
d12
d13
F
phenyl
phenyl
t-Bu
0.41
0.6
1.2
t-Bu
0.41
0.28
0.34
0.078
0.029
0.021
0.04
0.02
1.29
0.19
0.004
0.13
ND
o-tolyl
o-tolyl
phenyl
o-tolyl
o-tolyl
o-tolyl
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
2-hydroxyethyl
methyl
N
O
N
N
N
N
N
N
N
N
N
N
N
N
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
t-Bu
t-Bu
t-Bu
t-Bu
0.09
0.001
0.26
ND
3,5-difluoro-4-methoxy
2,5-difluoro-4-methoxy
3,5-difluoro-4-ethoxy
t-Bu
a
Full-length PDE5 was used. Values are mean of at least two experiments; standard deviation, <10%; ND: not determined, for compounds that
showed less than 50% inhibition at 10 μM, no IC50was determined.
phenyl derivative a4 (Table 2). This result suggested that any
entropic advantage due to the restriction of the rotational
freedom was countered by a negative effect, which may have
resulted from a less favorable fitting of the phenyl into the
pocket, since its orientation was shifted by the steric
constraints of the five-membered central ring. Increasing the
central ring system by one carbon (compound a49) led to a
significant drop in PDE5 inhibitory potency, which might be
explained by a steric collision of the two CH2 units of the
central six-membered ring with the binding cleft. It is also
conceivable that the non-polar methylene units did not
complement well within the hydrophilic patch in the binding
site. The latter hypothesis was supported by our observation
that the oxygen-containing isostere of compound a49 was able
to restore the PDE5 inhibitory activity (compound a50),
which might be due to additional polar interactions offered by
the oxygen. However, none of the rigidified analogues showed
a significant improvement in the potency. Hence, the non-
rigidified, sub-micromolar inhibitor a26, with the 3-o-tolyl
group, was selected for further optimization as it will be
discussed below (Figure 2).
Influence of the Chirality of C5. Before we proceeded
further in compounds’ optimization, we examined the
influence of the stereochemistry on the PDE5 inhibitory
activity, where chiral separation was made for the sub-
micromolar inhibitor compound a29 obtained from structural
variations at the 3-phenyl. The racemic derivative was
separated into its respective enantiomers by semi-preparative
chiral HPLC. As indicated in Table S2 (Supporting
Information), the difference between the IC50 values of the
separated enantiomers (isomers 1 and 2) was not substantial,
suggesting that both enantiomers contribute almost equally to
the biological activity of the racemates.
In summary, the most optimal substituents at the 3-position
of the pyrazoline regarding PDE5 inhibition were the
unsubstituted phenyl (compound a4) and a phenyl with
small electron-donating group in the ortho position (com-
pounds a26 and a29), or a t-butyl (compound B).
Modification at 5-Phenyl. Compound B was also optimized
at the 5-phenyl residue. Replacement of the para-methoxy
substituent by the strong electron-withdrawing 4-trifluoro-
methyl abolished the activity (compound a38). However, the
use of the less electron-withdrawing trifluoromethoxy (com-
pound a39) partially restored the activity, suggesting the
importance of the availability of methoxy electrons and a
potential role of methoxy oxygen either as HBA or through its
+M mesomeric effect to the 5-phenyl. Adding an adjacent
meta-fluoro group to the methoxy in compound B led to
compound a37 (IC50 = 0.59 μM) with about 3-fold increase in
potency (a37 compared to compound B), which could be
attributed to a direct lipophilic or dipole−dipole interaction
mediated by the introduced fluorine atom. Surprisingly,
inverting the substitution pattern at the 5-phenyl in a37 led
to more than a 17-fold decrease in PDE5 inhibitory potency by
the positional isomer (a40), confirming the optimum position-
ing of both the fluoro and the methoxy substituents in a37.
Replacing the meta-fluoro in a37 by a chloro substituent gave
compound a41, which showed slightly reduced PDE5
inhibitory potency (compared to a37), indicating that the
4468
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 4. Activity and Selectivity of Some Key Compounds on PDE Isoforms
PDE isoform
PDE1A cAMP
PDE1A cGMP
PDE2A cAMP
PDE2A cGMP
PDE3A cAMP
PDE3A cGMP
PDE4B2 (cAMP)
PDE5A cGMP
PDE6C cGMP
PDE7A (cAMP)
PDE8A (cAMP)
PDE9A cGMP
PDE10A cAMP
PDE10A cGMP
PDE11A cAMP
PDE11A cGMP
IC50 ratio PDE6/5
IC50 ratio PDE11/5 (cGMP)
cpd c6 IC50 (μM)
cpd d3 IC50 (μM)
cpd d4 IC50 (μM)
cpd d8 IC50 (μM)
cpd d12 IC50 (μM)
>10
>10
>50
>50
>10
>10
>10
0.34
>10
>10
>10
>10
>100
>100
13.03
13.04
>29
>10
>10
9.54
13.34
>10
>10
>10
0.021
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.040
>10
>10
>10
>10
>10
>10
>10
>10
>250
>250
>10
>10
>10
>10
>10
>10
>10
0.004
>10
>10
>10
>10
>10
>10
>10
>10
>2500
>2500
>10
>10
>10
>10
>10
>10
>10
0.001
>10
>10
>10
>10
>10
>10
>10
>10
>10,000
>10,000
>10
>10
32.3
49.5
12.73
11.38
>476
574
38.35
a
Values are mean of at least two experiments; standard deviation, <15%
higher electronegativity of fluorine is more crucial for the
activity than the higher lipophilicity of chlorine.
occupation within the pocket. This extension was achieved
through the carboxylic acid moiety at the 1-phenyl.
The next step to optimize a37 (with 3-fluoro-4-methoxy)
was to add a second meta-fluoro to give either the 3,5- or the
2,3-difluoro-4-methoxy derivative, compounds a42 and a43,
respectively. The second fluorine markedly enhanced the
potency by 6- and 10-fold, respectively, compared with the
mono-fluorinated analogue, a37 (Table 1). Interestingly, the
2,5-difluoro-4-methoxy isomer (a44), carrying the fluorine
substituents at opposite positions at the benzene ring, showed
a more than 16-fold lower activity (compound a44 compared
to a37). The importance of the 4-methoxy as an essential
element to generate high potency in combination with fluorine
atoms was further confirmed by compounds a45−a47, where
deletion of the 4-methoxy and maintaining the 3,5-difluoro
abolished the activity (compound a45 compared to a42). In
compound a46, the methoxy was replaced by methyl, which
has similar electron-donating ability but lacks the +M
mesomeric effect and the ability to act as HBA. The methyl
group failed to replace the methoxy in compound a37,
confirming that the methoxy at position 4 is essential for high
potency (a46 compared to a37). Finally, replacing the 4-
methoxy in compound a42 with 4-ethoxy (compound a47)
halved the activity, which can be explained by the higher steric
demand of the ethoxy group. Relative to the original hit
compounds B (IC50 = 2 μM), potency optimization through
the 5-phenyl was achieved by maintaining the electron-
donating methoxy group at the para position and adding
difluoro substituents to yield a42 and a43 (IC50 values of 0.09
and 0.06 μM, respectively), demonstrating that multiple
fluorination improved the PDE5 inhibitory potency of the
hit compound, B, up to 33-fold.
This strategy was started by the synthesis of several
carboxamide derivatives of compound a26 (a sub-micromolar
inhibitor obtained through optimization of the 3-position of
pyrazoline). It was encouraging that the n-butylamide analogue
(compound c5) showed a 2-fold increase in potency. To
further verify the effect of side chain amides on potency,
similar open-chain amide derivatives were prepared for other
carboxylic acid analogues like the hit compound B, a4, or a37.
The synthesized ethyl or butyl amide derivatives showed about
2-fold increase in potency (compounds c1−c4). Using a more
polar amide side chain, like the 2-hydroxyethyl in compound
c6, slightly enhanced the potency, indicating that polar side
chain amides could be tolerated by this part of the pocket (c6
compared to a26). A synopsis of the SAR obtained with c1−c4
and c6 suggested that the morpholine heterocycle could be a
logical continuation of this series. Indeed, morpholine in
compound d2 (IC50 = 0.029 μM) led to a major increase in
potency by 12-fold (d2 compared to compound c6). Probably,
the morpholine provided the right balance between lip-
ophilicity and polarity, along with a conformational restriction.
Then, at one of the last stages of optimization, it was
straightforward to replace the morpholine by the dimensionally
and electronically related N-methylpiperazine (compound d3).
This modification kept the high level of potency with a slightly
lower IC50 of 0.021 μM (Figure 2). Thus, converting the
carboxyl at the 1-phenyl into the N-methylpiperazinylcarbonyl
side chain caused a more than 23-fold increase in potency (d3
compared to a26) while also improving the water solubility
and drug-like properties.
Third Round of Optimization: Combining Favorable
Features. To further optimize PDE5 inhibitory potency, we
combined the N-methylpiperazinylcarbonyl side chain at the 1-
phenyl with the fluorinated 4-methoxyphenyl at position 5 of
the pyrazoline while maintaining the t-Bu at position 3. This
was achieved by reacting compounds a37, a42, and a43 with
N-methylpiperazine; thus, the most potent inhibitors among
the series were obtained, namely, compounds d5, d8, and d12,
displaying IC50 values of 0.020, 0.004, and 0.001 μM,
Second Round of Optimization: Extension of the
Structure through the Modification at the 1-Phenyl.
Although a several-fold increase in potency had been achieved,
none of the previous modifications could reach single-digit
nanomolar potency comparable to marketed PDE5 inhibitors
such as sildenafil or tadalafil. To achieve higher potency, it
appeared reasonable to extend the structure to establish
additional interactions within the binding pocket and greater
4469
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 3. PDE5 isozyme selectivity of compound d12. Inhibition of PDE isozymes by compound d12 at 15 μM (15,000-fold higher concentration
than the PDE5 IC50 value).
Figure 4. (A) d12 potently inhibits the full-length PDE5 but not the catalytic fragment. (B) Sildenafil inhibition of the full-length PDE5 and the
catalytic fragment as shown in Figure 1 for comparison.
respectively (Table 3). Remarkably, the most potent inhibitor,
d12, was three times more potent than sildenafil. Thus,
combining the features that increased the potency at the 1- and
the 5-phenyl proved to be a successful strategy toward more
potent PDE5 inhibitors; particularly, the N-methylpiperazinyl-
carbonyl side chain achieved an up to 90-fold improvement
when it was incorporated in the carboxyl precursor compound
(compare, e.g., a42 and d12, possessing IC50 values of 0.09 and
0.001 μM, respectively). In another series of derivatives, the
favorable feature in compound a26, namely, the 3-o-tolyl, was
combined with the 5-(3-fluoro-4-methoxyphenyl) moiety,
which was found to be favorable for compound a37. The
resulting compound a36 (IC50 = 0.13 μM) showed more than
3-fold improvement in potency compared to compound a26.
However, adding the third feature to a36 by its reaction with
N-methylpiperazine to give the amide derivative only led to 3-
fold increase in potency (d4 compared to a36), indicating that
combining the structural features at positions 1, 3, and 5 of the
pyrazoline was not additive. Table S3 (Supporting Informa-
tion) shows the fold increase in potency after synthesizing
amides with N-methylpiperazine from some selected carboxylic
acid derivatives.
Core Ring: Pyrazole vs Pyrazoline. The importance of
the non-coplanar pyrazoline system for PDE5 inhibitory
activity was confirmed at different stages of optimization by
the oxidation of compounds a4 and d9 to the corresponding
pyrazole analogues using DDQ to yield compounds b1 and F,
respectively. None of the pyrazole analogues showed
significant PDE5 inhibition at 10 μM. The abolishment of
the core ring planarity in the pyrazoline derivatives with the
resulting change in the orientation of the 5-aryl substituent
appeared to be an essential requirement for PDE5 inhibitory
activity.
PDE Isoform Selectivity. Compound c6 and the more
potent d3, d4, d8, and d12 were evaluated for their PDE
isozyme selectivity using recombinant human PDE isozymes
PDE1−PDE11, as summarized in Table 4. The moderately
active compound c6, but also the highly potent PDE5
inhibitors, d3, d4, d8, and d12, showed high selectivity for
inhibiting PDE5 with IC50 values in the nanomolar range,
while the IC50 values against all other PDE isozymes were
greater than 10 μM. Compound d12, the most potent PDE5
inhibitor among the present series, showed unprecedented
PDE5 isozyme selectivity. With a selectivity index of >10,000,
compound d12 would be expected to lack the common side
effects associated with the classical PDE5 inhibitors such as
sildenafil and tadalafil that non-selectively inhibit other PDE
isoforms, namely, PDE6 and PDE11, respectively. The
excellent PDE isozyme selectivity profile of compound d12
may be attributed to the unique mode of action that was also
found for celecoxib (see below). To confirm the PDE isozyme
selectivity of compound d12, we screened the compound
against all PDE isoforms at a concentration of 15 μM (15,000
times the PDE5 IC50), as shown in Figure 3. Even at this
extremely high concentration, compound d12 only cross-
reacted with PDE6 (the closest isoform to PDE5), resulting in
approximately 50% inhibition of activity. By comparison,
sildenafil has been reported to inhibit PDE6 with IC50 values
approximately 7-fold higher than IC50 values to inhibit PDE5,
which accounts for side effects such as visual disturbances.31 In
addition, tadalafil inhibits PDE11 with an IC50 value 5-fold
higher than the IC50 to inhibit PDE5,31 although the
significance of this cross-reactivity is not well known. Thus,
to the best of our knowledge, compound d12 represents the
most selective PDE5 inhibitor published to date.
4470
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 5. cGMP elevation by d3, d4, and d8 in live HEK-293 cells transfected to stably express a cGMP biosensor. The biosensor consists of the
PDE5 GAF-A domain fused to a modified firefly luciferase. Intracellular cGMP levels were stimulated with the guanylyl cyclase activator, SNP, at
the time of treatment to assess the amplifying effect of a PDE5 inhibitor. Luminescence is therefore an indicator of intracellular cGMP levels. A
dose-dependent increase in luminescence was seen when cells were treated with (A) d3, (B) d4, or (C) d8. After 90 min of treatment, a dose-
dependent increase was seen. Background luminescence was subtracted, and data were normalized to the DMSO+SNP control. (D) Dose−
response curves after a 90 min treatment and the corresponding EC50 values.
Figure 6. cGMP elevation by d12 and tadalafil in the cGMP-biosensor assay. A dose-dependent increase in cGMP levels by (A) d12 and (C)
tadalafil. After 90 min of treatment, a dose-dependent increase was observed with (B) d12 and (D) tadalafil.
Mechanism of PDE5 Inhibition. To determine if the
optimized compounds show the same type of dependency on
the presence of the regulatory domain as we observed for
celecoxib, compound d12 (IC50 = 0.001 μM against full-length
enzyme), the most potent compound in the presented series,
was tested against the PDE5 catalytic domain along with
sildenafil for comparison. As shown in Figure 4, compound
d12 failed to reach the IC50 against the PDE5 catalytic domain
even at concentrations reaching 1 μM (1000-fold the IC50 on
the full-length enzyme), while sildenafil inhibited activity with
an IC50 of 5 nM. These results clearly indicate that the action
of compound d12 strongly depends on the presence of
4471
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
regulatory domain, even to a much higher extent than reported
previously for vardenafil.27 To further investigate the mode of
action, we studied the enzyme kinetics of d12 along with
sildenafil. In comparison to sildenafil, which is a purely
substrate competitive inhibitor, the apparent Km of d12
increased only slightly with rising inhibitor concentrations,
ruling out a purely competitive mode of inhibition for d12
(Figure S1, Supporting Information). This was in accordance
with the observation that regions outside the catalytic domain
provided a major contribution to binding. However, there was
no clear tendency toward a non- or uncompetitive mode of
inhibition; most likely, d12 has a mixed mode of inhibition,
with a preferred binding to the apo enzyme, while it can also
bind to the substrate-bound form, both enabled by the high
basal affinity to the adjacent regulatory domain. Hence, the
binding site might be located in the interface between the
active site and the GAF-B domain. Alternatively, allosteric
binding to the regulatory domain with allosteric modulation of
the active site would also be in agreement with our
experimental findings. At any rate, targeting of non-active
site regions, which are mostly less conserved, may explain the
unique PDE isozyme selectivity profile of d12.
Table 5. Intrinsic Clearance (Liver Human Microsomes)
compound
half-life (min)
Clint (μL/min/mg)
d12
22
11
>60
>60
320.4
616.9
<115.5
<115.5
terfenadine
imipramine
propranolol
a
The test compound (1 μM) was pre-incubated with pooled human
liver microsomes (final microsomal protein concentration: 0.1 mg/
mL) in phosphate buffer (pH 7.4) for 5 min in a shaking water bath at
37 ° C. The reaction was initiated by adding an NADPH-generating
system and incubated for 0, 15, 30, 45, and 60 min. The reaction is
stopped by transferring the incubation mixture to acetonitrile/
methanol. Samples are then mixed and centrifuged. Supernatants
are used for HPLC−MS/MS analysis. The assay was carried out in
duplicates, SD < 10%.
a
Table 6. Binding to Human Plasma Proteins
compound
% protein bound
d12
98
13
71
99
acebutolol
quinidine
warfarin
Inhibition of PDE5 in Cells. To determine if PDE5
inhibition and the consequent cGMP elevation occur in treated
cells, a cGMP biosensor assay was developed and used to
measure changes in intracellular levels of cGMP in living cells
in response to treatment with compounds d3, d4, d8, and d12
along with tadalafil. The four compounds showed a dose-
dependent increase in cGMP in HEK-293 cells stably
transfected with the cGMP biosensor (Figures 5 and 6).
Thus, the ability of the compounds to inhibit PDE5 and
increase the intracellular cGMP in live cells was demonstrated.
Generally, the compounds showed a similar rank in their
cellular potency compared to the rank of their cell-free
potency, as can be indicated by the calculated EC50 values to
increase intracellular cGMP levels after 90 min of treatment of
cells with the compounds (Figures 5 and 6). For the two most
potent compounds in the present series (d8 and d12),
compound d8 (EC50 = 4.13 μM) showed higher efficacy than
d12 in cells (EC50 = 6.7 μM).
In Vitro and In Vivo Pharmacokinetic Evaluation and
Tolerance Study. We characterized some key pharmacoki-
netic properties. The intrinsic clearance of d12 was measured
using human hepatic liver microsomes along with terfenadine
(rapidly metabolized), imipramine, and propranolol (relatively
stable) as reference compounds. Although compound d12
displayed higher stability than terfenadine, it had a significantly
shorter half-life than imipramine and propranolol (Table 5).
Additionally, the binding to human plasma protein was
measured using an equilibrium dialysis technique to separate
the fraction of a test compound that is unbound from the
protein bound fraction. Acebutolol, quinidine, and warfarin are
tested in the assay as reference compounds, which yield
protein binding values that represent low, medium, and high
bindings to human plasma proteins, respectively. Compound
d12 elicited high plasma protein binding with 98% (Table 6).
Preliminary studies were conducted to determine if d12
reaches systemic circulation after oral administration and if
repeated dosing is tolerated. As shown in Figure 7 and in Table
7, after a single oral administration of a dose of 150 mg/kg,
d12 reached a Cmax of 3995 ng/mL in 2 h (tmax) and showed a
half-life of 2.3 h. This elimination half-life is in a favorable
range for a drug aiming at the treatment of erectile dysfunction.
a
The samples were dialyzed against phosphate buffered saline (pH
7.4) and the bound and unbound compound fractions quantified by
HPLC−MS/MS analysis. The recovery rates were 100% with each
compound. Assay was carried out in duplicates, SD < 10%.
Based on the results of the oral PK study, d12 was reaching the
systemic circulation and achieved a Cmax corresponding to
approx. 8.5 μM.
Next, we evaluated the tolerance of d12 by mice when given
twice daily for 28 days (three different doses were used, Figure
8). Twice daily treatment with d12 by oral gavage was well-
tolerated without deaths at all three doses tested. Except for
intermittent body weight loss observed in all groups
immediately after the start of the treatment (<5% loss from
the pre-dose body weight), mice in all three d12-treated
groups gained weight over the course of the treatment and
showed no visible signs of toxicity (Figure 8). The necropsy
after termination did not reveal any abnormalities; liver, spleen,
and kidney appeared normal upon closer examination.
Altogether, these results indicate that d12 is a promising
candidate for in vivo evaluation.
Conclusions. Through systematic rounds of chemical
modifications of celecoxib and iterative target-directed screen-
ing, we identified a novel series of trisubstituted pyrazolines as
potent and selective PDE5 inhibitors that lack COX2
inhibitory activity. The mechanism of inhibition appears to
involve binding to the interface region between the regulatory/
catalytic domain or to an allosteric site on the regulatory
domain, although further research is necessary to fully define
the binding site on PDE5. The dependency of the inhibition
on the regulatory domain rather than a conserved PDE5
catalytic site may explain the unique selectivity profile of this
novel class of PDE5 inhibitors. Altogether, the synthetic
strategy presented here led to the development of a new
chemotype for PDE5 inhibition with excellent potency and
PDE isozyme selectivity, which could be further evaluated in in
vivo models. Besides the potential treatment of erectile
dysfunction, further indications for d12 will be explored,
such as neuroinflammatory disorders. In a pilot pharmaco-
logical study, we obtained preliminary data showing that d12
4472
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 7. Plasma pharmacokinetics of d12 in C57BL/6N mice after a single oral administration of a 150 mg/kg dose. Mean SD (n = 2−6).
least 95% for all tested compounds. Mass spectra (HPLC−ESI-MS)
were acquired using a TSQ quantum (Thermo Electron Corporation)
instrument with a triple quadrupole mass detector (Thermo
Finnigan) and an ESI source. Samples were introduced using an
autosampler (Surveyor, Thermo Finnigan) with an injection volume
of 10 μL. The mass detection was determined using a source CID of
10 V and carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas
pressure of 4.0 × 105 Pa, a capillary temperature of 400 °C, a capillary
voltage of 35 V, and an auxiliary gas pressure of 1.0 × 105 Pa. The
stationary phase used was an RP C18 NUCLEODUR 100-3 (125 × 3
mm) column (Macherey & Nagel). The solvent system consisted of
water containing 0.1% TFA (A) and 0.1% TFA in acetonitrile (B).
For the HPLC method, the flow rate was 400 μL/min. The
percentage of B started at 5%, increased up to 100% over 16 min, kept
at 100% for 2 min, and then flushed back to 5% in 2 min. Melting
points were determined using a Mettler FP1 melting point apparatus
and are uncorrected.
Table 7. Pharmacokinetic Parameters for Compound d12
after Oral Adminstration
a
b
PK parameters for d12
PO
Ke (1/h)
0.30
t1/2 (h)
tmax (h)
2.33
2
Cmax (ng/ml)
AUC0‑8h (ng/ml·h)
AUC0‑inf_obs (ng/ml·h)
Vdobs (ml)
3995
14057.5
16162.1
625.0
Clobs (ml/h)
185.6
a
Ke: elimination rate constant, Vd: volume of distribution, Cl:
b
clearance. A single oral treatment (150 mg/kg) in ethanol/Maalox
(10:90, v/v).
General Synthetic Methods and Experimental Details for
Key Compounds. General Procedure for the Enone Synthesis. The
appropriate ketone (10 mmol) was reacted with corresponding aryl
aldehyde (10 mmol) using the same procedure that we previously
reported in ref 32.
(E)-1-(3,5-Difluoro-4-methoxyphenyl)-4,4-dimethylpent-1-en-3-
one (E42) was synthesized according to the general procedure for
enone synthesis using pinacolone and 3,5-difluoro-4-methoxybenzal-
dehyde; white solid; yield: 2.44 g (95.9%); mp 74−75 °C;1H NMR
(500 MHz, DMSO-d6) δ 1.15 (s, 9H), 3.97 (t, J = 1.1 Hz, 3H), 7.35−
7.50 (m, 2H), 7.62−7.72 (m, 2H); 13C NMR (126 MHz, DMSO-d6)
δ 25.58, 42.9, 61.71 (t, 4JC‑F = 3.4 Hz), 112.68 (dd, 2JC‑F = 17.5, 4JC‑F
=
5.8 Hz), 122.91, 130.15 (t, 3JC‑F = 9.3 Hz), 136.94 (t, 2JC‑F = 14.2 Hz),
4
1
3
139.41 (t, JC‑F = 2.5 Hz), 154.82 (dd, JC‑F = 246.1, JC‑F = 6.5 Hz),
Figure 8. Tolerance of d12 in mice after chronic oral administration.
Body weight change (expressed as percent change from the starting
body weight) in athymic nude-Foxn1nu mice over the course of
treatment with d12 administered twice (2×) daily for 28 days by oral
gavage. Mean SD (n = 4).
203.28.
(E)-1-(2,3-Difluoro-4-methoxyphenyl)-4,4-dimethylpent-1-en-3-
one (E43) was synthesized according to the general procedure for
enone synthesis using pinacolone and 2,3-difluoro-4-methoxybenzal-
dehyde; white solid; yield: 2.34 g (92%); mp 128−129 °C; 1H NMR
(500 MHz, DMSO-d6) δ 1.16 (s, 9H), 3.93 (s, 3H),7.07−7.12 (m,
1H), 7.40 (d, J = 15.8 Hz, 1H), 7.54 (d, J = 15.8 Hz, 1H), 7.77 (td, J
= 8.8, 2.2 Hz, 1H); 13C NMR (126 MHz, DMSO-d6) δ 25.80, 42.77,
was effective in a neuroinflammation model (to be published
elsewhere).
4
2
56.60, 109.37 (d, JC‑F = 2.6 Hz), 116.15 (d, JC‑1F = 8.9 Hz), 122.39
2
2
EXPERIMENTAL SECTION
(d, JC‑F = 4.9 Hz), 123.92, 132.81, 140.08 (dd, JC‑F = 245.5, JC‑F
=
■
14.4 Hz), 149.42 (dd, 1JC‑F = 251.1, 2JC‑F = 10.5 Hz), 150.01 (dd, 2JC‑F
Chemistry. Solvents and reagents were obtained from commercial
3
1
suppliers and used as received. H NMR and 13C NMR spectra were
= 7.6, JC‑F = 3.5 Hz), 203.10.
General Procedure for the Pyrazoline Synthesis. A mixture of the
enone derivative (2 mmol) and the corresponding aryl hydrazine
hydrochloride (3 mmol) were reacted together using the same
procedure that we previously reported in ref 32.
4-(5-(4-Methoxyphenyl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (a26) was prepared by reaction of (E)-3-(4-
measured using an A Bruker DRX 500 spectrometer, and in very few
cases, a Varian Mercury VX 300 spectrometer was used for recording
the spectra of some enones. The chemical shifts are referenced to the
residual protonated solvent signals, and occasionally, TMS was used
as a reference. The purity of all the tested compounds (Tables 1−3)
was verified by HPLC coupled with mass spectrometry and was at
4473
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2
2
methoxyphenyl)-1-(o-tolyl) prop-2-en-1-one (E26) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/
CH3OH, 100:0.5); yellow solid; yield: 0.25 g (32.4%); mp 209−210
°C; 1H NMR (500 MHz, DMSO-d6) δ 2.70 (s, 3H, −Ph−CH3), 3.19
(dd, J = 17.4, 5.0 Hz, 1H, Ha−C4), 3.70 (s, 3H, −OCH3), 4.01 (dd, J
= 17.4, 12.0 Hz, 1H, Hb−C4), 5.51 (dd, J = 11.9, 5.0 Hz, 1H, H−C5),
6.86−6.92 (m, 2H, ArCH), 6.97−7.03 (m, 2H, ArCH), 7.16−7.22
(m, 2H, ArCH), 7.23−7.37 (m, 3H, ArCH), 7.44−7.47 (m, 1H,
ArCH), 7.71−7.77 (m, 2H, ArCH), 12.27 (s, 1H, −COOH); 13C
NMR (125 MHz, DMSO-d6) δ 23.45 (−PhCH3), 45.09 (C4), 55.00
(−OCH3), 60.92 (C5), 111.93 (ArCH), 114.40 (ArCH), 119.81
(ArCH), 126.03 (ArCH), 126.98 (ArCH), 128.49 (ArCH), 128.70
(ArCH), 130.42 (ArCH), 130.79 (ArCH), 131.50 (ArCH), 133.73
(ArCH), 136.72 (ArCH), 147.04 (ArCH), 150.43 (ArCH), 158.55
(C3N), 167.20 (CO); MS (ESI): m/z = 386.75 (M + H)+.
4-(3-tert-Butyl-5-(3-fluoro-4-methoxyphenyl)-4,5-dihydro-1H-pyra-
zol-1-yl)-benzoic acid (a37) was prepared by reaction of (E)-1-(3-
fluoro-4-methoxyphenyl)-4,4-dimethylpent-1-en-3-one (E37) and 4-
hydrazinobenzoic acid hydrochloride according to the general
procedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2:CH3OH, 100:2); white solid; yield: 0.16 g (22%); 1H NMR
(500 MHz, DMSO-d6) δ 1.15 (s, 9H, −C(CH3)3), 2.72 (dd, J = 17.8,
5.1 Hz, 1H, Ha−C4), 3.52 (dd, J = 17.7, 11.7 Hz, 1H, Hb−C4), 3.76
(s, 3H, −OCH3), 5.32 (dd, J = 11.6, 5.0 Hz, 1H, H−C5), 6.81−6.85
(m, 2H, ArCH), 6.92 (dd, J = 8.5, 1.4 Hz, 1H, ArCH), 6.99 (dd, J =
12.2, 2.1 Hz, 1H, ArCH), 7.08 (t, J = 8.7 Hz, 1H, ArCH), 7.67−7.73
(m, 2H, ArCH), 12.19 (s, 1H, −COOH); 13C NMR (125 MHz,
DMSO-d6) δ 27.84 (−C(CH3)3), 33.56 (−C(CH3)3), 42.41 (C4),
55.91 (−OCH3), 61.27 (C5), 111.42 (ArCH), 113.26 (d, 2JC‑F = 18.6
246.4, JC‑F = 14.2 Hz, ArCH), 147.49 (ArCH), 147.83 (dd, JC‑F
=
7.2, 3JC‑F = 2.0 Hz, ArCH), 147.97 (dd, 1JC‑F = 245.7, 2JC‑F = 10.5 Hz,
ArCH), 161.84 (C3N), 167.22 (CO); MS (ESI): m/z = 389.16
(M + H)+.
General Procedure for Amide Synthesis. The appropriate amine
(2 equiv) was added to a solution of the respective benzoic acid
derivative (1 equiv) and EDCI·HCl (1.5 equiv) in absolute CH2Cl2
(10 mL). The mixture was stirred overnight at room temperature and
monitored by TLC. After completion of the reaction, the solvent was
removed under reduced pressure. The crude product was purified by
column chromatography on a silica gel to give the desired product.
N-(2-Hydroxyethyl)-4-(5-(4-methoxyphenyl)-3-(o-tolyl)-4,5-dihy-
dro-1H-pyrazol-1-yl) benzamide (c6) was prepared by reaction of 4-
(5-(4-methoxyphenyl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl) ben-
zoic acid (a26) and ethanolamine according to the general procedure
for amide synthesis. The product was purified by CC (CH2Cl2/
CH3OH, 100:2); faint yellow solid; yield: 0.039 g (9%); mp 68−70
°C; 1H NMR (500 MHz, DMSO-d6) δ 2.70 (s, 3H, −Ph−CH3), 3.18
(dd, J = 17.3, 4.2 Hz, 1H, Ha−C4), 3.23−3.29 (m, 2H, −CH2−),
3.43−3.48 (m, 2H, −CH2−),3.70 (s, 3H, −OCH3), 3.99 (dd, J =
17.1, 12.5 Hz, 1H, Hb−C4), 4.68 (s, 1H, −OH), 5.49 (dd, J = 11.8,
4.9 Hz, 1H, H−C5), 6.88 (d, J = 8.0 Hz, 2H, ArCH), 6.97 (d, J = 8.2
Hz, 2H, ArCH), 7.19 (d, J = 8.1 Hz, 2H, ArCH), 7.23−7.29 (m, 2H,
ArCH), 7.33 (d, J = 6.3 Hz, 1H, ArCH), 7.41−7.47 (m, 1H, ArCH),
7.67 (d, J = 8.4 Hz, 2H, ArCH), 8.07 (s, 1H, −NH−); 13C NMR
(125 MHz, DMSO-d6) δ 23.41 (−PhCH3), 41.97 (C4), 45.00
(−CH2−), 55.00 (−CH2−), 59.94 (−OCH3), 61.09 (C5), 111.84
(ArCH), 114.33 (ArCH), 123.96 (ArCH), 126.02 (ArCH), 127.07
(ArCH), 128.32 (ArCH), 128.38 (ArCH), 128.56 (ArCH), 130.58
(ArCH), 131.48 (ArCH), 133.87 (ArCH), 136.57 (ArCH), 145.89
(ArCH), 149.66 (ArCH), 158.50 (C3N), 166.08 (CO); MS
(ESI): m/z = 429.88 (M + H)+.
(4-(5-(4-Methoxyphenyl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1yl)-
phenyl)(Morpholino) methanone (d2) was prepared by reaction of 4-
(5-(4-methoxyphenyl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl) ben-
zoic acid (a26) and morpholine according to the general procedure
for amide synthesis. The product was purified by CC (CH2Cl2/
CH3OH, 100:2); yellow solid; yield: 0.068 g (15%); mp 144−146 °C;
1H NMR (500 MHz, DMSO-d6) δ 2.70 (s, 3H, −Ph−CH3), 3.17 (dd,
J = 17.3, 5.7 Hz, 1H, Ha−C4), 3.46 (s, 4H, −CH2−NCO−CH2−),
3.56 (t, J = 4.6 Hz, 4H, −CH2−O−CH2−), 3.71 (s, 3H, −OCH3),
3.98 (dd, J = 17.3, 12.0 Hz, 1H, Hb−C4), 5.43 (dd, J = 12.0, 5.7 Hz,
1H, H−C5), 6.88−6.92 (m, 2H, ArCH), 6.97−7.01 (m, 2H, ArCH),
7.29−7.23 (m, 2H, ArCH), 7.24−7.30 (m, 4H, ArCH),7.31−7.35 (m,
1H, ArCH), 7.41−7.45 (m, 1H, ArCH); 13C NMR (125 MHz,
DMSO-d6) δ 23.49 (−PhCH3), 40.10 (C4), 45.15 (−CH2−), 55.00
(−CH2−), 61.27 (−OCH3), 66.11 (C5), 111.95 (ArCH), 114.41
(ArCH), 124.53 (ArCH), 126.01 (ArCH), 127.03 (ArCH), 128.26
(ArCH), 128.55 (ArCH), 128.90 (ArCH), 130.56 (ArCH), 131.47
(ArCH), 134.06 (ArCH), 136.57 (ArCH), 145.15 (ArCH), 149.36
(ArCH), 158.53 (C3N), 169.32 (CO); MS (ESI): m/z = 455.94
(M + H)+.
3
Hz, ArCH), 114.31 (d, JC‑F = 1.2 Hz, ArCH), 119.15 (ArCH),
121.71 (d, 4JC‑F = 3.2 Hz, ArCH), 130.78 (ArCH), 134.97 (d, 3JC‑F
5.1 Hz, ArCH), 146.28 (d, JC‑F = 10.4 Hz, ArCH), 147.74 (ArCH),
151.46 (d, 1JC‑F = 244.9 Hz, ArCH), 161.58 (C3 N), 167.27 (C
O); MS (ESI): m/z = 370.89 (M + H)+.
=
2
4-(3-(tert-Butyl)-5-(3,5-difluoro-4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)benzoic acid (a42) was prepared by reaction of (E)-
1-(3,5-difluoro-4-methoxyphenyl)-4,4-dimethylpent-1-en-3-one
(E42) and 4-hydrazinobenzoic acid hydrochloride according to the
general procedure for pyrazoline synthesis. The product was purified
by CC (CH2Cl2/CH3OH, 100:2); white solid; yield: 0.53 g (68.3%);
1
mp 222−223 °C; H NMR (500 MHz, DMSO-d6) δ 1.18 (s, 9H,
−C(CH3)3), 2.80 (dd, J = 17.8, 5.2 Hz, 1H, Ha−C4), 3.56 (dd, J =
17.8, 11.7 Hz, 1H, Hb−C4), 3.88 (s, 3H, −OCH3), 5.35 (dd, J = 11.7,
5.2 Hz, 1H, H−C5), 6.87 (d, J = 9.0 Hz, 2H, ArCH), 6.94 (d, J = 8.9
Hz, 2H, ArCH), 7.71 (d, J = 9.1 Hz, 2H, ArCH), 12.24 (s, 1H,
−COOH); 13C NMR (126 MHz, DMSO-d6) δ 27.79 (−C(CH3)3),
33.53 (−C(CH3)3), 42.20 (C4), 61.16 (−OCH3), 61.68 (t, 4JC‑F = 2.8
2
4
Hz, C5), 109.74 (dd, JC‑F = 17.5, JC‑F = 5.7 Hz, ArCH), 111.46
(ArCH), 111.54 (ArCH), 119.48 (ArCH), 130.87 (ArCH), 134.76 (t,
3
2JC‑F = 14.2 Hz, ArCH), 138.14 (t, JC‑F = 7.3 Hz, ArCH), 147.62
1
3
(ArCH), 155.18 (dd, JC‑F = 247.7, JC‑F = 6.1 Hz, ArCH), 161.67
(C3N), 167.20 (CO); MS (ESI): m/z = 389.16 (M + H)+.
4-(3-(tert-Butyl)-5-(2,3-difluoro-4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)benzoic acid (a43) was prepared by the reaction of
(E)-1-(2,3-difluoro-4-methoxyphenyl)-4,4-dimethylpent-1-en-3-one
(E43) and 4-hydrazinobenzoic acid hydrochloride according to the
general procedure for pyrazoline synthesis. The product was purified
by CC (CH2Cl2/CH3OH, 100:2); faint yellow solid; yield 0.51 g
(65%); mp 204−205 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.18 (s,
9H, −C(CH3)3), 2.86 (dd, J = 17.7, 4.8 Hz, 1H, Ha−C4), 3.60 (dd, J
= 17.7, 11.9 Hz, 1H, Hb−C4), 3.82 (s, 3H, −OCH3), 5.51 (dd, J =
11.9, 4.8 Hz, 1H, H−C5), 6.79−6.83 (m, 1H, ArCH), 6.86 (d, J = 9.0
Hz, 2H, ArCH), 6.94 (q, J = 7.9 Hz, 1H, ArCH), 7.70 (d, J = 9.0 Hz,
2H, ArCH), 12.22 (s, 1H, −COOH); 13C NMR (126 MHz, DMSO-
d6) δ 27.84 (−C(CH3)3), 33.55 (−C(CH3)3), 41.11 (C4), 56.28
(4-(5-(4-Methoxyphenyl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-
yl)phenyl)(4-methylpiperazin-1-yl)methanone (d3). The title com-
pound was prepared by reaction of 4-(5-(4-methoxyphenyl)-3-(o-
tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid (a26) and 1-methyl-
piperazine according to the general procedure for amide synthesis.
The product was purified by CC (CH2Cl2/CH3OH, 100:2); yellow
1
solid; yield: 0.098 g (21%); mp 127−128 °C; H NMR (500 MHz,
DMSO-d6) δ 2.28 (s, 3H, N−CH3), 2.45 (s, 4H, −CH2−NCH3−
CH2−), 2.69 (s, 3H, −ph-CH3), 3.16 (dd, J = 17.4, 5.7 Hz, 1H, Ha−
C4), 3.50−3.55 (m, 4H, −CH2−NCO−CH2−), 3.70 (s, 3H,
−OCH3), 3.98 (dd, J = 17.3, 12.0 Hz, 1H, Hb−C4), 5.42 (dd, J =
11.9, 5.6 Hz, 1H, H−C5), 6.87−6.90 (m, 2H, ArCH), 6.96−7.00 (m,
2H, ArCH), 7.18−7.23 (m, 2H, ArCH), 7.23−7.29 (m, 4H, ArCH),
7.30−7.35 (m, 1H, ArCH), 7.40−7.44 (m, 1H, ArCH); 13C NMR
(125 MHz, DMSO) δ 30.73 (−PhCH3), 35.74 (C4), 45.68
(−NCH3), 54.96 (−CH2−), 63.46 (−OCH3), 66.79 (C5), 111.97
(ArCH), 114.30 (ArCH), 116.19 (ArCH), 117.34 (ArCH), 119.67
3
(−OCH3), 56.42 (C5), 109.12 (d, JC‑F = 1.5 Hz, ArCH), 111.27
3
(ArCH), 119.27 (ArCH), 121.23 (t, JC‑F = 4.0 Hz, ArCH), 121.71
2
1
(d, JC‑F = 11.9 Hz, ArCH), 130.86 (ArCH), 140.19 (dd, JC‑F
=
4474
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(ArCH), 121.70 (ArCH), 124.28 (ArCH), 126.84 (ArCH), 130.88
(ArCH), 131.41 (ArCH), 145.68 (ArCH), 146.36 (ArCH), 155.42
(ArCH), 158.82 (ArCH), 162.26 (C3N), 167.17 (CO); MS
(ESI): m/z = 469.31 (M + H)+.
incubated for an additional 30 min at 30 °C. FP was measured
according to the manufacturer’s specifications using a Biotek Synergy
4 plate reader.
Experimental Design and Data Analysis. PDE activity was
measured, and potency was expressed by an IC50 value (50%
inhibitory concentration). For enzyme assays, the IC50 value was
determined by testing a range of 10 concentrations with at least two
replicates per concentration. Dose−response curves were analyzed
using PrismTM 4 software (GraphPad) to calculate IC50 values using
a four-parameter logistic equation. All in vitro experiments involved
dose−response analysis, which was repeated at least twice to confirm
the reproducibility of IC50 values.
(4-(3-(tert-Butyl)-5-(2,3-difluoro-4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)phenyl)(4-methylpiperazin-1-yl)methanone (d8)
was prepared by reaction of 4-(3-(tert-butyl)-5-(2,3-difluoro-4-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid (a43)
and N-methylpiperazine according to the general procedure for
amide synthesis. The product was purified by CC (CH2Cl2/CH3OH,
1
100:5); faint yellow solid; yield: 0.21 g (44.6%); mp 92−93 °C; H
NMR (500 MHz, DMSO-d6) δ 1.18 (s, 9H, −C(CH3)3), 2.17 (s, 3H,
N−CH3), 2.27 (s, 4H, −CH2−NCH3−CH2−), 2.83 (dd, J = 17.7, 5.6
Hz, 1H, Ha−C4), 3.44 (s, 4H, −CH2−NCO−CH2−), 3.59 (dd, J =
17.7, 11.9 Hz, 1H, Hb−C4), 3.82 (s, 3H, −OCH3), 5.40 (dd, J = 12.0,
5.6 Hz, 1H, H−C5), 6.82 (dd, J = 18.6, 8.1 Hz, 2H, ArCH), 6.85−
6.90 (m, 1H, ArCH), 6.96 (t, J = 7.9 Hz, 1H, ArCH), 7.19 (t, J = 7.6
Hz, 2H, ArCH); 13C NMR (126 MHz, DMSO-d6) δ 27.88
(−C(CH3)3), 33.48 (−C(CH3)3), 41.14 (C4), 45.59 (−NCH3),
Cyclooxygenase Assay. The colorimetric COX (ovine) inhibitor
screening assay kit (Cayman) was used according to the
manufacturer’s specification.
Cellular cGMP Elevation Assay. Human kidney embryonic cells
(HEK-293 cells, obtained from ATCC) were transfected with the
cGMP biosensor (Promega) using Lipofectamine LTX (Invitrogen)
to create a HEK-293 cell line, which stably expresses the cGMP
biosensor. G418 was used to select positive clones, and clones were
screened for luminescence. HEK-293 cells that stably express a cGMP
biosensor were seeded at a density of 60,000 cells per well on tissue
culture-treated microtiter 96-well plates. Cells were incubated
overnight at 37 °C in DMEM media containing 10% FBS. The next
day, media were removed and 100 μL of CO2-independent media
with 10% FBS and 5 mM Luciferin (Promega) was added to each
well. Cells were incubated at room temperature in the dark for 1 h.
Cells were then treated with 50 μM SNP and compound or vehicle
control. Luminescence was monitored for 120 min using a Synergy
H4 Hybrid plate reader (Bio Tek).
Protein Binding. The protein matrix (human) was spiked with the
test compound at 10 μM with a final DMSO concentration of 1%.
The dialysate compartment is loaded with phosphate buffered saline
(PBS, pH 7.4), and the sample side is loaded with equal volume of the
spiked protein matrix. The dialysis plate is then sealed and incubated
at 37 °C for 4 h. After the incubation, samples are taken from each
compartment, diluted with the phosphate buffer followed by addition
of acetonitrile and centrifugation. The supernatants are then used for
HPLC−MS/MS analysis. A control sample is prepared from the
spiked protein matrix in the same manner as the assay samples
(without dialysis). This control sample serves as the basis for the
recovery determination. The recovery determination serves as an
indicator of reliability of the calculated protein binding value. Low
recovery indicates that the test compound is lost during the course of
the assay. This is most likely due to non-specific binding or
degradation of the test compound.
54.54 (−CH2−), 56.43 (C5), 56.72 (−OCH3), 109.18 (ArCH),
3
111.23 (d, 3JC‑F = 8.3 Hz, ArCH), 111.4 (ArCH), 121.30 (dd, JC‑F
=
6.5, 4JC‑F = 2.9 Hz, ArCH), 122.14 (d, 2JC‑F = 11.3 Hz, ArCH), 124.50
(ArCH), 128.78 (ArCH), 140.18 (dd, JC‑F = 246.5, JC‑F = 14.6 Hz,
ArCH), 145.75 (ArCH), 147.77 (dd, JC‑F = 7.8, JC‑F = 2.6 Hz,
1
2
2
3
1
2
ArCH), 147.95 (dd, JC‑F = 244.0, JC‑F = 12.2 Hz, ArCH), 160.68
(C3N), 169.25 (CO); MS (ESI): m/z = 471.25 (M + H)+.
(4-(3-(tert-Butyl)-5-(3,5-difluoro-4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)phenyl)(4-methylpiperazin-1-yl)methanone (d12)
was prepared by reaction of 4-(3-(tert-butyl)-5-(3,5-difluoro-4-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid (a42)
and N-methylpiperazine according to the general procedure for
amide synthesis. The product was purified by CC (CH2Cl2/CH3OH,
1
100:5); faint yellow solid; yield: 0.25 g (53.13%); mp 85−86 °C; H
NMR (500 MHz, DMSO-d6) δ 1.17 (s, 9H, −C(CH3)3), 2.17 (s, 3H,
N−CH3), 2.26 (s, 4H, −CH2−NCH3−CH2−), 2.76 (dd, J = 17.7, 6.3
Hz, 1H, Ha−C4), 3.44 (s, 4H, −CH2−NCO−CH2−), 3.55 (dd, J =
11.1, 6.6 Hz, 1H, Hb−C4), 3.88 (s, 3H, −OCH3), 5.23 (dd, J = 11.7,
6.3 Hz, 1H, H−C5), 6.85 (d, J = 8.8 Hz, 2H, ArCH), 6.99 (d, J = 8.9
Hz, 2H, ArCH), 7.21 (d, J = 8.9 Hz, 2H, ArCH); 13C NMR (126
MHz, DMSO-d6) δ 27.83 (−C(CH3)3), 33.46 (−C(CH3)3), 42.31
4
(C4), 45.59 (−NCH3), 54.54 (−CH2−), 61.69 (t, JC‑F = 2.8 Hz,
2
4
C5), 61.85 (−OCH3), 109.80 (dd, JC‑F = 17.7, JC‑F = 5.5 Hz,
ArCH), 111.67 (ArCH), 111.76 (ArCH), 124.77 (ArCH), 128.76
(ArCH), 134.72 (t, 2JC‑F = 14.2 Hz, ArCH), 138.59 (t, 3JC‑F = 7.3 Hz,
1
3
ArCH), 145.95 (ArCH), 155.20 (dd, JC‑F = 247.5, JC‑F = 6.0 Hz,
ArCH), 160.57 (C3N), 169.21 (CO); MS (ESI): m/z = 471.25
(M + H)+.
In Vivo Pharmacokinetic Study. Female, 11−12 week old C57BL/
6N mice (weight of 19−21 g, Charles River Laboratory, USA) were
used. The animals were housed in a temperature-controlled room
(20−24 °C) and maintained in a 12 h light/12 h dark cycle. Food and
water were available ad libitum. All experimental procedures were
approved by IACUC of University of South Alabama, USA (protocol
number 933458, approved on August 5, 2016) and conducted in
accordance with the regulations of the local animal welfare authorities
(the Animal Welfare Act and PHS Policy on Humane Care and Use
of Laboratory Animals). d12 was formulated in ethanol/Maalox
(10:90, v/v) at a concentration of 10 mg/mL and administed to six
mice by oral gavage at a dose of 150 mg/kg. Blood samples were
taken at 1, 2, or 4 h from two mice per time point (as a survival
bleeding) and from all six mice at 8 h (terminal bleeding). The blood
was collected into K2EDTA tubes, stored on ice, and subsequently
centrifuged at 5000g for 5 min at room temperature, and plasma was
separated and kept at below −20 °C until being assayed by liquid
chromatography mass spectrometry (LC−MS/MS). d12 plasma
concentrations were quantified using a calibration curve based on
calibration standards prepared in drug-free plasma. Results were
analyzed using PKSolver 2.0.33
General Procedure for Pyrazoline Oxidation to Pyrazole. A
mixture of the pyrazoline derivative (1 mmol) and DDQ (1.5 mmol)
in 10 mL of benzene was heated to reflux for 5 h. The mixture was
cooled to room temperature and filtered through a plug of silica gel
wetted with diethyl ether. The filtrate was concentrated in vacuo, and
the residue was purified by CC.
Phosphodiesterase Assay. Recombinant GST-tagged phospho-
diesterase enzymes purified from baculovirus infected sf9 cells were
purchased from BPS Bioscience (PDE1A full length, PDE2A full
length, PDE3A [669-end], PDE3B [592-end], PDE4B2 full length,
PDE5A1 full length, PDE6C full length, PDE7A [122-end], PDE8A1
full length, PDE9A2 full length, PDE10A2 full length, and PDE11A4
full length). Recombinant 6His tagged-PDE5 catalytic domain was
purified from Escherechia coli using a Talon IMAC column (construct
kindly provided by Dr. Hengming Ke at The University of North
Carolina at Chapel Hill). Enzyme (5 μg/mL) was added to the wells
of black 96-well non-binding plates. Immediately, the protein was
treated with compound or vehicle control and 50 nM TAMRA-cGMP
or FAM-cAMP as indicated (Molecular Devices) was added to each
assay well. The plates were incubated for 1.5 h at 30 °C. After
incubation, IMAP FP phosphodiesterase evaluation assay (Molecular
Devices) binding reagent was added to each well and the plates were
Tolerance Study. Female, 8−9 week old athymic nude-Foxn1nu
mice (weight of 18−24 g, Harlan Laboratories, USA) were used. The
animals were housed in a temperature-controlled room (20−24 °C)
4475
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
and maintained in a 12 h light/12 h dark cycle. Food and water were
available ad libitum. All experimental procedures were approved by
IACUC of University of South Alabama, USA (protocol number
933458, approved on August 5, 2016) and conducted in accordance
with the regulations of the local animal welfare authorities (the
Animal Welfare Act and PHS Policy on Humane Care and Use of
Laboratory Animals). Mice were treated with d12 by oral gavage at
doses of 50, 100, or 150 mg/kg twice daily (daily dose of 100, 200,
and 300 mg/kg, respectively) or with d12 vehicle for 28 days. Four
mice were included in each group. d12 was formulated in ethanol/
water/Maalox (7:3:90, v/v/v). Mice were monitored daily for signs of
toxicity and were weighed daily during the first week of treatment and
twice a week for the duration of the study. At the end of the study, all
mice were euthanized and necropsy was conducted.
Mennatallah A. Hammam − Department of Pharmaceutical
Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt
Ahmed Maher − Biochemistry Department, Faculty of
Pharmacy, October University for Modern Sciences and Arts
(MSA), Giza, Egypt
Yulia Maxuitenko − Departments of Oncologic Sciences and
Pharmacology, Mitchell Cancer Institute, University of South
Alabama, Mobile, Alabama 36604, United States
Adam B. Keeton − Departments of Oncologic Sciences and
Pharmacology, Mitchell Cancer Institute, University of South
Alabama, Mobile, Alabama 36604, United States
Rolf W. Hartmann − Pharmaceutical and Medicinal
Chemistry, Saarland University, D-66123 Saarbru
Germany; orcid.org/0000-0002-5871-5231
Matthias Engel − Pharmaceutical and Medicinal Chemistry,
Saarland University, D-66123 Saarbrucken, Germany;
orcid.org/0000-0001-5065-8634
̈
cken,
ASSOCIATED CONTENT
■
sı
* Supporting Information
̈
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01120.
Gary A. Piazza − Departments of Oncologic Sciences and
Pharmacology, Mitchell Cancer Institute, University of South
Alabama, Mobile, Alabama 36604, United States
Molecular formula strings and PDE5 inhibition data;
experimental procedures and analytical data of com-
pounds a1−a25, a27−a36, a38−a41, a44−a50, b1, c1−
c5, d1, d4−d7, d9−d11, d13, F, E1−E41, and E44−
E50; (Figure S1) percent increase in Km values with
rising concentrations of the inhibitor; (Table S1) activity
of some selected compounds against COX1 and COX2;
(Table S2) PDE5 inhibitory activity of compound a29
vs its separated enantiomers; (Table S3) fold increase in
PDE5 inhibitory potency on extending the carboxylic
acids into respective amides with N-methylpiperazine;
HPLC chromatograms for some selected compounds
(PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01120
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research reported in this publication was supported in part by
the National Cancer Institute of the National Institutes of
Health under awards R01CA155638, R01CA197147, and
R01CA131378. We thank Prof. Greg Gorman, Director of
Pharmaceutical Sciences Research Institute, McWhorter
School of Pharmacy Samford University (Birmingham,
Alabama, USA) for analyzing the plasma level of compound
d12.
SMILES (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
Mohammad Abdel-Halim − Department of Pharmaceutical
Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt; orcid.org/0000-
0003-1326-4219; Phone: +201000006222;
ABBREVIATIONS USED
■
cAMP, cyclic adenosine monophosphate; CC, column
chromatography; COX, cyclooxygenase; cGMP, cyclic guano-
sine monophosphate; DDQ, 2,3-Dichloro-5,6-dicyano-1,4-
benzoquinone; EDCI·HCl, N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride; PK, pharmacokinetics;
HEK-293, human kidney embryonic cells; IACUC, Institu-
tional Animal Care and Use Committee
Email: mohammad.abdel-halim@guc.edu.eg
Ashraf H. Abadi − Department of Pharmaceutical Chemistry,
Faculty of Pharmacy and Biotechnology, German University
in Cairo, Cairo 11835, Egypt; orcid.org/0000-0002-
7433-261X; Phone: +202 27589990-8;
Email: ashraf.abadi@guc.edu.eg; Fax: +202-27581041
Authors
REFERENCES
■
Sara Sigler − Departments of Oncologic Sciences and
Pharmacology, Mitchell Cancer Institute, University of South
Alabama, Mobile, Alabama 36604, United States
Nora A. S. Racheed − Department of Pharmaceutical
Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt
Amr Hefnawy − Department of Pharmaceutical Chemistry,
Faculty of Pharmacy and Biotechnology, German University
in Cairo, Cairo 11835, Egypt
Reem K. Fathalla − Department of Pharmaceutical Chemistry,
Faculty of Pharmacy and Biotechnology, German University
in Cairo, Cairo 11835, Egypt; Pharmaceutical and Medicinal
Chemistry, Saarland University, D-66123 Saarbru
Germany
(1) Li, J. J.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Collins,
J. T.; Garland, D. J.; Gregory, S. A.; Huang, H.-C.; Isakson, P. C. 1,2-
Diarylcyclopentenes as selective cyclooxygenase-2 inhibitors and
orally active anti-inflammatory agents. J. Med. Chem. 1995, 38,
4570−4578.
(2) DeWitt, D. L. COX-2-selective inhibitors: the new super aspirins.
Mol. Pharmacol. 1999, 55, 625−631.
(3) FitzGerald, G. A. Coxibs and cardiovascular disease. N. Engl. J.
Med. 2004, 351, 1709−1711.
(4) Krum, H.; Liew, D.; Aw, J.; Haas, S. Cardiovascular effects of
selective cyclooxygenase-2 inhibitors. Expert Rev. Cardiovasc. Ther.
2004, 2, 265−270.
(5) Zarraga, I. G. E.; Schwarz, E. R. Coxibs and heart disease: what
we have learned and what else we need to know. J. Am. Coll. Cardiol.
2007, 49, 1−14.
̈
cken,
4476
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(6) Ju
̈
ni, P.; Nartey, L.; Reichenbach, S.; Sterchi, R.; Dieppe, P. A.;
solubility for the treatment of alzheimer’s disease. J. Med. Chem.
2017, 60, 8858−8875.
(24) Sung, B.-J.; Hwang, K. Y.; Jeon, Y. H.; Lee, J. I.; Heo, Y.-S.;
Kim, J. H.; Moon, J.; Yoon, J. M.; Hyun, Y.-L.; Kim, E.; Eum, S. J.;
Park, S.-Y.; Lee, J.-O.; Lee, T. G.; Ro, S.; Cho, J. M. Structure of the
catalytic domain of human phosphodiesterase 5 with bound drug
molecules. Nature 2003, 425, 98−102.
(25) Turko, I. V.; Ballard, S. A.; Francis, S. H.; Corbin, J. D.
Inhibition of cyclic GMP-binding cyclic GMP-specific phosphodies-
terase (Type 5) by sildenafil and related compounds. Mol. Pharmacol.
1999, 56, 124−130.
(26) Blount, M. A.; Beasley, A.; Zoraghi, R.; Sekhar, K. R.; Bessay, E.
P.; Francis, S. H.; Corbin, J. D. Binding of tritiated sildenafil, tadalafil,
or vardenafil to the phosphodiesterase-5 catalytic site displays
potency, specificity, heterogeneity, and cGMP stimulation. Mol.
Pharmacol. 2004, 66, 144−152.
(27) Blount, M. A.; Zoraghi, R.; Ke, H.; Bessay, E. P.; Corbin, J. D.;
Francis, S. H. A 46-amino acid segment in phosphodiesterase-5 GAF-
B domain provides for high vardenafil potency over sildenafil and
tadalafil and is involved in phosphodiesterase-5 dimerization. Mol.
Pharmacol. 2006, 70, 1822−1831.
(28) Wenthur, C. J.; Gentry, P. R.; Mathews, T. P.; Lindsley, C. W.
Drugs for allosteric sites on receptors. Annu. Rev. Pharmacol. Toxicol.
2014, 54, 165−184.
(29) Abdel-Halim, M.; Tinsley, H.; Keeton, A. B.; Weam, M.; Atta,
N. H.; Hammam, M. A.; Hefnawy, A.; Hartmann, R. W.; Engel, M.;
Piazza, G. A.; Abadi, A. H. Discovery of trisubstituted pyrazolines as a
novel scaffold for the development of selective phosphodiesterase 5
inhibitors. Bioorg. Chem. 2020, 104, 104322.
(30) Huang, Y. R.; Katzenellenbogen, J. A. Regioselective synthesis
of 1,3,5-Triaryl-4-alkylpyrazoles: Novel ligands for the estrogen
receptor. Org. Lett. 2000, 2, 2833−2836.
(31) Bischoff, E. Potency, selectivity, and consequences of
nonselectivity of PDE inhibition. Int. J. Impotence Res. 2004, 16,
S11−S14.
(32) Abdel-Halim, M.; Keeton, A. B.; Gurpinar, E.; Gary, B. D.;
Vogel, S. M.; Engel, M.; Piazza, G. A.; Boeckler, F. M.; Hartmann, R.
W.; Abadi, A. H. Trisubstituted and tetrasubstituted pyrazolines as a
novel class of cell-growth inhibitors in tumor cells with wild type p53.
Bioorg. Med. Chem. 2013, 21, 7343−7356.
(33) Zhang, Y.; Huo, M.; Zhou, J.; Xie, S. PKSolver: An add-in
program for pharmacokinetic and pharmacodynamic data analysis in
Microsoft Excel. Comput. Methods Programs Biomed. 2010, 99, 306−
314.
Egger, M. Risk of cardiovascular events and rofecoxib: cumulative
meta-analysis. Lancet 2004, 364, 2021−2029.
(7) Dogné, J.-M.; Supuran, C. T.; Pratico, D. Adverse cardiovascular
effects of the coxibs. J. Med. Chem. 2005, 48, 2251−2257.
(8) Klein, T.; Eltze, M.; Grebe, T.; Hatzelmann, A.; Kömhoff, M.
Celecoxib dilates guinea-pig coronaries and rat aortic rings and
amplifies NO/cGMP signaling by PDE5 inhibition. Cardiovasc. Res.
2007, 75, 390−397.
(9) Kimmel, S. E.; Berlin, J. A.; Reilly, M.; Jaskowiak, J.; Kishel, L.;
Chittams, J.; Strom, B. L. Patients exposed to rofecoxib and celecoxib
have different odds of nonfatal myocardial infarction. Ann. Intern.
Med. 2005, 142, 157−164.
(10) Graham, D. J.; Campen, D.; Hui, R.; Spence, M.; Cheetham,
C.; Levy, G.; Shoor, S.; Ray, W. A. Risk of acute myocardial infarction
and sudden cardiac death in patients treated with cyclo-oxygenase 2
selective and non-selective non-steroidal anti-inflammatory drugs:
nested case-control study. Lancet 2005, 365, 475−481.
(11) Solomon, D. H.; Avorn, J.; Sturmer, T.; Glynn, R. J.; Mogun,
̈
H.; Schneeweiss, S. Cardiovascular outcomes in new users of coxibs
and nonsteroidal antiinflammatory drugs: High-risk subgroups and
time course of risk. Arthritis Rheum. 2006, 54, 1378−1389.
(12) Mehats, C.; Andersen, C. B.; Filopanti, M.; Jin, S. L. C.; Conti,
M. Cyclic nucleotide phosphodiesterases and their role in endocrine
cell signaling. Trends Endocrinol. Metab. 2002, 13, 29−35.
(13) Conti, M.; Beavo, J. Biochemistry and physiology of cyclic
nucleotide phosphodiesterases: essential components in cyclic
nucleotide signaling. Annu. Rev. Biochem. 2007, 76, 481−511.
(14) Francis, S. H.; Turko, I. V.; Corbin, J. D. Cyclic nucleotide
phosphodiesterases: relating structure and function. Prog. Nucleic Acid
Res. Mol. Biol. 2001, 65, 1−52.
(15) 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, 469−478.
(16) Rybalkin, S. D.; Yan, C.; Bornfeldt, K. E.; Beavo, J. A. Cyclic
GMP phosphodiesterases and regulation of smooth muscle function.
Circ. Res. 2003, 93, 280−291.
(17) Huang, S. A.; Lie, J. D. Phosphodiesterase-5 (PDE5) inhibitors
in the management of erectile dysfunction. P T 2013, 38, 407−419.
(18) Humbert, M.; Ghofrani, H.-A. The molecular targets of
approved treatments for pulmonary arterial hypertension. Thorax
2016, 71, 73−83.
(19) Wu, D.; Huang, Y.; Chen, Y.; Huang, Y.-Y.; Geng, H.; Zhang,
T.; Zhang, C.; Li, Z.; Guo, L.; Chen, J.; Luo, H.-B. Optimization of
chromeno[2,3-c]pyrrol-9(2H)-ones as highly potent, selective, and
orally bioavailable PDE5 inhibitors: Structure−activity relationship,
X-ray crystal structure, and pharmacodynamic effect on pulmonary
arterial hypertension. J. Med. Chem. 2018, 61, 8468−8473.
(20) 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)-pyrimidi-
nones as phosphodiesterase type 5 inhibitors leading to TPN171, a
clinical candidate for the treatment of pulmonary arterial hyper-
tension. J. Med. Chem. 2019, 62, 4979−4990.
(21) Wu, D.; Zhang, T.; Chen, Y.; Huang, Y.; Geng, H.; Yu, Y.;
Zhang, C.; Lai, Z.; Wu, Y.; Guo, X.; Chen, J.; Luo, H.-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.
(22) Zheng, H.; Wu, Y.; Sun, B.; Cheng, C.; Qiao, Y.; Jiang, Y.;
Zhao, S.; Xie, Z.; Tan, J.; Lou, H. Discovery of furyl/thienyl β-
carboline derivatives as potent and selective PDE5 inhibitors with
excellent vasorelaxant effect. Eur. J. Med. Chem. 2018, 158, 767−780.
(23) Fiorito, J.; Vendome, J.; Saeed, F.; Staniszewski, A.; Zhang, H.;
Yan, S.; Deng, S.-X.; Arancio, O.; Landry, D. W. Identification of a
novel 1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine analogue as a
potent phosphodiesterase 5 inhibitor with improved aqueous
4477
https://doi.org/10.1021/acs.jmedchem.0c01120
J. Med. Chem. 2021, 64, 4462−4477