Discovery of trisubstituted pyrazolines as a novel scaffold for the development of selective phosphodiesterase 5 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104322

Celecoxib, is a selective cyclooxygenase-2 (COX2) inhibitor with a 1,5-diaryl pyrazole scaffold. Celecoxib has a better safety profile compared to other COX2 inhibitors having side effects of systemic hypertension and thromboembolic complications. This may be partly attributed to an off-target activity involving phosphodiesterase 5 (PDE5) inhibition and the potentiation of NO/cGMP signalling allowing coronary vasodilation and aortic relaxation. Inspired by the structure of celecoxib, we synthesized a chemically diverse series of compounds containing a 1,3,5-trisubstituted pyrazoline scaffold to improve PDE5 inhibitory potency, while eliminating COX2 inhibitory activity. SAR studies for PDE5 inhibition revealed an essential role for a carboxylic acid functionality at the 1-phenyl and the importance of the non-planar pyrazoline core over the planar pyrazole with the 5-phenyl moiety tolerating a range of substituents. These modifications led to new PDE5 inhibitors with approximately 20-fold improved potency to inhibit PDE5 and no COX-2 inhibitory activity compared with celecoxib. PDE isozyme profiling of compound 11 revealed a favorable selectivity profile. These results suggest that trisubstituted pyrazolines provide a promising scaffold for further chemical optimization to identify novel PDE5 inhibitors with potential for less side effects compared with available PDE5 inhibitors used for the treatment of penile erectile dysfunction and pulmonary hypertension.

Full text of DOI:10.1016/j.bioorg.2020.104322

Bioorganic Chemistry 104 (2020) 104322
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of trisubstituted pyrazolines as a novel scaffold for the
development of selective phosphodiesterase 5 inhibitors
,
Mohammad Abdel-Halima *, Heather Tinsleyb, Adam B. Keetonc, Mohammed Weama,
Noha H. Attaa, Mennatallah A. Hammama, Amr Hefnawya, Rolf W. Hartmannd,
,
*
Matthias Engeld, Gary A. Piazzac, Ashraf H. Abadia
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
b Department of Biology, University of Montevallo
c Department of Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36608, USA
d Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Celecoxib, is a selective cyclooxygenase-2 (COX2) inhibitor with a 1,5-diaryl pyrazole scaffold. Celecoxib has a
better safety profile compared to other COX2 inhibitors having side effects of systemic hypertension and
thromboembolic complications. This may be partly attributed to an off-target activity involving phosphodies-
terase 5 (PDE5) inhibition and the potentiation of NO/cGMP signalling allowing coronary vasodilation and aortic
relaxation. Inspired by the structure of celecoxib, we synthesized a chemically diverse series of compounds
containing a 1,3,5-trisubstituted pyrazoline scaffold to improve PDE5 inhibitory potency, while eliminating
COX2 inhibitory activity. SAR studies for PDE5 inhibition revealed an essential role for a carboxylic acid
functionality at the 1-phenyl and the importance of the non-planar pyrazoline core over the planar pyrazole with
the 5-phenyl moiety tolerating a range of substituents. These modifications led to new PDE5 inhibitors with
approximately 20-fold improved potency to inhibit PDE5 and no COX-2 inhibitory activity compared with cel-
ecoxib. PDE isozyme profiling of compound 11 revealed a favorable selectivity profile. These results suggest that
trisubstituted pyrazolines provide a promising scaffold for further chemical optimization to identify novel PDE5
inhibitors with potential for less side effects compared with available PDE5 inhibitors used for the treatment of
penile erectile dysfunction and pulmonary hypertension.
Celecoxib
PDE5
Pyrazoline
cGMP elevation
PDE5 inhibitors
1. Introduction
systemic hypertension and thromboembolic complications, including
myocardial infarction, among patients receiving the COX2 inhibitor
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most
widely prescribed drugs all over the world due to their anti-
inflammatory, antipyretic, and analgesic effects which are exerted
through inhibiting cyclooxygenase (COX), isozymes COX1 and COX2
[1]. Long-term use of conventional NSAIDs, that non-selectively inhibit
COX1 and COX2, causes gastric and renal toxicities that is attributed to
COX1 inhibition and the suppression of physiologically important
prostaglandins. The identification and characterization of an inducible
COX2 isozyme in the 1990s that is localized primarily in inflamed tissues
was the start of a race to develop safer NSAIDs that selectivity target
COX2, collectively known as coxibs. Coxibs have reduced gastric and
renal toxicity compared to classical NSAIDs [2–4], but clinical trials
involving large numbers of subjects revealed an increased risk of
rofecoxib (Vioxx®) which resulted in a number of coxibs being pulled
from the market. This unexpected toxicity was attributed to an imbal-
ance between thromboxane and prostacyclin levels whereby only pros-
tacyclin synthesis is inhibited by coxibs leading to unopposed
vasoconstriction [5]. Among the coxibs, celecoxib (A, Fig. 1) (Cele-
brex®) was an exception as it showed lower cardiovascular risk and
remains on the market [6]. As previously reported, the improved car-
diovascular safety profile of celecoxib may be attributed to a potenti-
ating effect on the NO/cGMP signalling pathway through an off-target
effect involving the inhibition of phosphodiesterase 5 (PDE5) [7]. Thus,
the PDE5 inhibitory activity of celecoxib may compensate for a decrease
of prostacyclin-dependent cAMP generation by concomitantly
increasing cGMP levels, which is supported by evidence that PDE5
* Corresponding authors.
E-mail addresses: mohammad.abdel-halim@guc.edu.eg (M. Abdel-Halim), ashraf.abadi@guc.edu.eg (A.H. Abadi).
https://doi.org/10.1016/j.bioorg.2020.104322
Received 7 July 2020; Received in revised form 25 August 2020; Accepted 24 September 2020
Available online 28 September 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
inhibitors have cardioprotective properties [8]. In addition, several
other NSAIDs, most notably, sulindac, have been reported to inhibit
PDE5 [9], which also has cardioprotective properties [10]. However,
none of the other coxibs inhibit PDE5, which suggests a specific property
of celecoxib that might be optimized chemically to develop novel PDE5
inhibitors useful for the treatment of erectile dysfunction and pulmonary
arterial hypertension but do not have side effects associated with the
non-selective inhibition of other PDE isozymes [7].
out in order to increase the activity toward PDE5 inhibition. Impor-
tantly, the effect of partial saturation of the pyrazole to the Δ2-pyrazo-
line on the PDE5 inhibitory activity and on the general PDE isoform
selectivity was also investigated. This modification was expected to have
a major impact on the inhibitory profile, as it incorporated a tetrahedral
kink in the 3D shape. If successful, it would also disrupt the agglomer-
ation of aromatic ring systems, which can lead to disadvantageous
pharmacokinetic properties [24]. Further modifications involved the
replacement of the electronegative trifluoromethyl group at position 3
of the pyrazole with t-butyl. As an alkyl, the t-butyl is anticipated to
result in reducing COX-2 inhibitory activity [25]. Fig. 2 summarizes the
planned modifications.
Cyclic nucleotide PDEs are a large superfamily of phosphohydrolases
which degrade the 3′-cyclic phosphate bond in adenosine and/or gua-
nosine 3′,5′-cyclic monophosphate (cAMP and/or cGMP) [11]. PDE5 is a
cGMP-specific PDE isozyme that terminates the vasodilatory effect of
nitric oxide (NO) in smooth muscles by degrading cGMP. Selective PDE5
inhibitors increase intracellular cGMP levels that lead to cGMP-
dependent protein kinase (PKG) activation and prolonged vasodilata-
tion [11,12]. Sildenafil (Viagra®) (B) and tadalafil (Cialis®) (C) are
among the most potent PDE5 inhibitors on the market; both drugs are
clinically approved for the treatment of male erectile dysfunction and
pulmonary arterial hypertension (Fig. 1) [13]. Both drugs were found to
have non-selective effects on other PDE isozymes leading to side effects
including visual disturbances, headache, flushing, nasal congestion,
nasopharyngitis, and dyspepsia. For example, sildenafil inhibits PDE6
due to the high similarity in amino acid sequence as well as secondary
structural element of the catalytic domains of PDE5 and PDE6. In
addition, tadalafil was shown to inhibit PDE11 [14].
2.2. Chemistry
Scheme 1 shows the synthesis of the planned pyrazoline derivatives
in two steps. In the first step, a Claisen–Schmidt reaction was carried out
between pinacolone and aryl aldehydes, using 10% aq KOH in methanol
to yield the enone derivatives. In the second step, enones were heated
with the respective aryl hydrazine hydrochloride under inert atmo-
sphere to give the desired pyrazoline derivatives. Oxidation of the pyr-
azoline ring to pyrazole was accomplished via reflux with DDQ in
benzene. Two of the carboxylic acid derivatives were converted to the
respective ethyl esters using acetyl chloride in refluxing ethanol
(Scheme 1).
One of the known tools in lead discovery is to enhance a desired
minor effect of an existing drug and eliminate its major pharmacological
action, an approach known as selective optimization of side activities
(SOSA). This approach has the potential to shorten the time and costs to
reach more drug-like lead compounds [15]. In our continued efforts to
develop novel PDE5 inhibitors [16–20] and based on the reported off-
target activity of celecoxib involving PDE5 inhibition, the present
work aims to modulate the COX2/PDE5 inhibitory activities of celecoxib
to develop a chemically, and potentially biologically distinct class of
selective PDE5 inhibitors with reduced side effects.
2.3. Biological evaluation
All the newly synthesized celecoxib analogues were screened for
their in vitro ability to inhibit human recombinant PDE5, COX1 and
COX2 enzymes at concentrations of 50, 100 and 100 μM, respectively.
Compounds showing>50% inhibition at the screening dose were tested
in a dose–response curve to determine the IC50. Results of PDE5 inhi-
bition are shown in Table 1. All the tested compounds were inactive
against COX1 and COX2 enzymes at a concentration of 100 µM except
compounds 31, 32 and 33, which showed weak inhibition with IC50
values of 80, 85 and 34 µM respectively (see Table S1, Supporting
information).
2. Results and discussion
2.1. Design concept and synthetic strategy
2.3.1. Structure-activity relationships for PDE5 inhibition
Our initial efforts were aimed at abolishing the COX2 inhibitory
activity of celecoxib while keeping the PDE5 inhibition by fine modu-
lation of certain moieties reported to be necessary for COX2 inhibition.
This was envisaged mainly by the bioisosteric replacement of the sul-
fonamide group at the 1-phenyl of celecoxib with a carboxylic acid
functionality [21]. It was previously reported that the COX2 inhibitory
activity and selectivity of celecoxib and related analogues of the diaryl
heterocycle scaffolds are dependent on the presence of the p-sulfon-
amide or p-methylsulfonyl groups. The carboxylic acid group was also
reported to cause total loss of COX2 activity when it was used as a
replacement for the sulfonamide in the related 1,2-diarylcyclopentenes
COX-2 inhibitors [22], and significantly reduced the activity in the
diaryl pyrazole scaffold [23]. In addition, further modifications at the 1-
phenyl but also the 5-phenyl of celecoxib were systematically carried
2.3.1.1. First probe compound and bioisosteric replacement of
sulfonamide. Compound 22 was the first compound synthesized in this
series by adopting the main structural modifications mentioned above to
the structure of celecoxib, where the pyrazole ring is converted to the
Δ2-pyrazoline; the p-sulfonamide is converted to a carboxylic acid
function; and the trifluoromethyl is converted to t-butyl. Compound 22
showed no COX inhibition and a 4-fold improvement in the PDE5
inhibitory activity compared to celecoxib (IC50 8.4 vs. 37 μM, respec-
tively). Moreover, replacing the carboxylic acid function with the more
acidic sulfonic acid moiety in compounds 31 and 32 kept the PDE5
inhibitory activity with only about 2-fold reduction in potency (com-
pound 32 compared to 7). However, these 2 compounds were able to
retain some inhibitory activity against COX2 with IC50 values of 80 and
Fig. 1. The chemical structure of celecoxib (A) sildenafil (B) and tadalafil (C).
2

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
Fig. 2. The modifications adopted to the structure of celecoxib.
Scheme 1. Reagents and conditions: (i) 10%KOH, MeOH, ice cooling then room temperature, overnight (ii) 1.5 equiv Ph-NH-NH2.HCl derivative, DMF, 85 ◦C, 5 h.
(iii) 1.5 equiv DDQ, benzene, reflux, 5 h. (iv) EtOH, acetyl chloride, reflux, 5 h.
85
μ
M respectively (Table S1, Supporting information). The suggested
preventing the phenyl from getting deeply in a certain pocket. Using the
strong electron-withdrawing but less lipophilic nitro group gave com-
pounds of comparable PDE5 inhibitory activity to their halogen conge-
modifications including the use of the carboxylic acid functionality
fulfilled the required activity shift toward PDE5.
ners (compounds 20 and 21 with IC50 values of 3.2 and 5.7
μM
respectively). The use of the more hydrophilic cyano moiety at the para
2.3.1.2. Modifications at position 5 of the pyrazoline. The p-methyl
moiety at the 5-phenyl of compound 22 (that was originally present in
celecoxib) was deleted or replaced by other substituents of different
electronic, lipophilic or steric nature. In addition, the substitution
pattern on ring was varied. In addition, bulkier aromatic systems such as
β-naphthyl and biphenyl as well as heteroaromatic systems were tested
instead of the 5-phenyl.
position led to lower activity than the respective halogens (compound
23, IC50 = 13.8 μM).
In addition, several electron-donating substituents were evaluated
on the 5-phenyl, starting with the methoxy group; indeed, the intro-
duction of the p-methoxy substituent gave a 4-fold increase in activity
compared to the methyl analogue 22 (compound 15, IC50 = 2 μM).
However, moving the methoxy group to the ortho position greatly
Deleting the p-methyl in compound 22 gave a slightly more active
reduced the activity (compound 14, IC50 > 50
μM). This may be
compound as PDE5 inhibitor (compound 1, IC50 = 6.2 μM). Then, the
attributed to the electronic effect of the oxygen atom in terms of the
electronic complementarity with the binding site rather than the steric
effect, as it is shown above that the ortho-bromo derivative is still
methyl group in 22 was replaced with several mono halogen sub-
stitutions including fluoro, chloro and bromo substituents with different
substitution pattern on the ring to give compounds (2–10). All of the
halogen-substituted 5-phenyl analogues showed equivalent or up to 4-
fold higher potency to inhibit PDE5 compared to the methyl deriva-
tive 22, with the best activity displayed by compounds 5 and 6 having
showing significant activity (compound 8, IC50 = 6.9 μM). Interestingly,
combining both the ortho and para methoxy groups in compound 16 led
to intermediate PDE5 inhibitory activity (IC50 = 23.5 M). By contrast,
compound 17 with the 2,5-dimethoxy substituent showed reduced PDE5
inhibitory activity (IC50 > 50 M).
Extending the p-methoxy group in compound 15 to the longer chain
μ
ortho and meta chloro substituents (IC50 values are 1.9 and 2
μM
μ
respectively), while the p-fluoro derivative was the least active in this set
of monohalogentated compounds (compound 4, IC50 = 7.7 μM). Based
ethoxy retained PDE5 inhibitory potency (compound 19, IC50 = 2.9 μM).
on this pronounced increase in potency with the mono-chloro de-
rivatives, the dichloro derivatives (11–13) were synthesized. However,
having two chloro substituents on the phenyl did not yield an additive
effect in improving the potency to inhibit PDE5. Compounds 11 (with
Similar to what was observed with the ortho-methoxy, the ortho-ethoxy
in compound 18 showed a high reduction in PDE5 inhibitory potency
(compound 18, IC50 > 50 μM).
The p-hydroxyl moiety (compound 24, IC50 = 2.1
comparable PDE5 inhibitory activity to the p-methoxy (compound 15,
IC50 = 2 M), with no extra advantage offered by the HBD function.
Furthermore, increasing the bulk of the 5-aryl to biphenyl or
μM) showed
2,4-dichloro substitution, IC50 = 2.7
substitution, IC50 = 5.5
pound 13 (with 2,6- dichloro substitution, IC50 = 28.8
gested some steric clash that may be caused by this di-ortho substitution,
μ
M) and 12 (with 3,4-dichloro
M) were markedly more active than com-
M) which sug-
μ
μ
μ
3

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
Table 1
PDE5 inhibition by the synthesized compounds.
Cpd #
Ar
R
PDE5 % inh at 50
PDE5
Cpd #
Ar
R
PDE5 % inh at 50
PDE5 IC50
μM
IC50
6.2
(μ
M)a
μ
M
(
μ
M)a
1
phenyl
4-
88
20
3-nitrophenyl
4-nitrophenyl
4-methylphenyl
4-cyanophenyl
4-hydroxyphenyl
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
3-COOH
2-COOH
4-SO3H
87.6
3.2
COOH
4-
2
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-bromophenyl
3-bromophenyl
4-bromophenyl
2,4-dichlorophenyl
3,4-dichlorophenyl
2,6-dichlorophenyl
2-methoxyphenyl
4-methoxyphenyl
86.3
94.4
80
3.3
3.7
7.7
1.9
2
21
91
5.7
COOH
4-
3
22
93
8.4
COOH
4-
4
23
75
13.8
2.1
COOH
4-
5
100
95.7
85
24
77.1
100
80
COOH
4-
6
25
[1,1′-biphenyl]-4-
yl
35.6
21.8
ND
COOH
4-
7
3.3
6.9
3.1
7.2
2.7
26
naphthalen-2-yl
COOH
4-
8
100
91.4
88
27
furan-2-yl
23.6
28.1
100
46
COOH
4-
9
28
thiophen-2-yl
4-chlorophenyl
4-chlorophenyl
4-fluorophenyl
4-chlorophenyl
4-chlorophenyl
Phenyl
ND
COOH
4-
10
11
12
13
14
15
16
17
18
19
29
9.9
COOH
4-
97
30
ND
COOH
4-
100
99.1
49.8
95
5.53
28.8
ND
2
31
80
9.3
COOH
4-
32
4-SO3H
85
8.7
COOH
4-
33
4-
57.5
72
48.7
18.9
31.3
ND
COOH
4-
SO2NH2
34
–
COOH
4-
2,4-
85.8
47
23.5
ND
ND
2.9
35
4-fluorophenyl
4-chlorophenyl
3-nitropheyl
–
–
–
–
–
75
dimethoxyphenyl
2,5-
COOH
4-
36
10.5
16
dimethoxyphenyl
2-ethoxyphenyl
COOH
4-
42.5
95.3
37
ND
COOH
4-
4-ethoxyphenyl
Celecoxib
66.5
37
COOH
a
Values are mean of at least two experiments; standard deviation < 12%; ND: not determined, for compounds that showed<50% inhibition at 50 µM, no IC50 was
determined. Tadalafil was used as a positive control (IC50 = 5 nM).
naphthyl (compounds 25 and 26 respectively), led to a remarkable drop
in activity (IC50 values of 35.6 and 21.8 M respectively) with the
bulkier biphenyl showing the lower potency. In comparison to com-
pound 1 with the unsubstituted phenyl (IC50 = 6.2 M), replacing the 5-
phenyl with the isosteric furyl (compound 27) or thienyl (compound 28)
rings was detrimental to the activity. To summarize, the 5-phenyl
seemed tolerant to a wide range of substituents with diverse electronic
and lipophilic nature, including halogens, nitro, methoxy, ethoxy and
μ
μ
Fig. 3. SAR summary.
4

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
Fig. 4. cGMP elevation in live HEK-293 cells stably expressing a cGMP biosensor. Tadalafil (A), Celecoxib (B), compound 11(C) and compound 19 (D) could show a
dose dependent increase in the measured luminescence. All samples were pretreated with sodium nitroprusside to activate the endogenous soluble guanylyl cyclase.
hydroxy substituents, with the substitution pattern more limiting in case
of the alkoxy substituents. The phenyl ring at position 5 was irreplace-
able by other bulkier aryls or heteroaryl rings.
2.3.2. Effect on intracellular cGMP levels
Both compounds 11 and 19 were selected for further testing against
PDE5 in cells, since both compounds belong to the cluster of the most
potent PDE5 inbhiotrs developed in this series (Table S2, Supporting
information) and they have the most lipophilic 5-aryl system with
electron withdrawing halogen (2,4-dichloro, compound 11) or electron
donating groups (ethoxy, compound 19). Compounds 11 and 19 showed
the highest clogD values compared to the most related potent analogues,
thus raising the probability for cell membrane penetration (Table S2,
Supporting information); this was estimated to overcome the effect of
the highly polar –COOH which may slightly impair cellular perme-
ability. Compounds 11 and 19 along with celecoxib and tadalafil were
tested in luciferase cGMP biosensor assay using HEK293 cells stably
transfected with a genetically encoded GAF-Luc construct containing a
cGMP-specific binding domain from the human PDE5A GAF-A domain
fused with modified firefly luciferase. This cGMP biosensor construct
measures the relative intracellular cGMP concentration in cells by
luminescence. Real-time monitoring of luminescence in the treated cells
showed that compounds 11 and 19 and tadalafil as a positive control
caused a concentration-dependent increase in intracellular cGMP levels
(Fig. 4). In correspondence with their inhibitory effects on recombinant
PDE5 activity, celecoxib showed the least effect on cGMP levels, while
tadalafil was the most potent and effective. Despite its ability to increase
2.3.1.3. Confirmation of the SAR by further probe compounds. As the
main modifications were introduced in combination (replacement of the
sulfonamide with –COOH and change the core ring to pyrazoline), we
decided to verify the impact of each modification on the PDE5 inhibitory
potency.
Replacing the carboxylic acid group in compound 7 with a sulfon-
amide group reduced the PDE5 inhibitory activity by about 15-fold and
partly regained the COX2 inhibition (compound 33, PDE5 IC50 = 48.7
μ
M, COX2 IC50 = 34 μM). Compound 33 confirmed the deleterious effect
of the –COOH group on COX2 inhibition and its essential role in PDE5
inhibition. Besides, the p-COOH in compound 3 was esterified, or shifted
to o- or m- positions to further test the importance of this group. Notably,
shifting the –COOH group to the meta position led to a 3-fold reduction
in the PDE5 inhibitory activity (compound 29), while shifting it to the
ortho position diminished the activity (compound 30); this indicated
that the –COOH mediates a critical interaction in the p-position that was
dramatically influenced by this shift. Esterification of the p-COOH
moiety with ethyl alcohol in compounds 36 and 37 diminished the PDE5
inhibitory activity, which may be due to the increased size of the sub-
stituent, or the loss of the potential polar interactions mediated by the
free carboxylate.
Table 2
Additionally, the importance of the non-coplanar pyrazoline system
for the PDE5 inhibitory activity was confirmed by the oxidation of
compounds 1 and 4 to the corresponding pyrazole analogues using DDQ
to yield compounds 34 and 35, respectively. Restoring the core ring
planarity in the pyrazole system led to a significant reduction in the
PDE5 inhibitory activity by 3–4 times (compounds 34 and 35 compared
to compounds 1 and 4). The loss of the core ring planarity in the pyr-
azoline derivatives with the resulting change in the orientation of the 5-
aryl substituent appeared to be one of the main reasons for enhancing
the PDE5 inhibition.
Selectivity profiles of compound 11 vs all PDE isoforms. Compound 11 was
screened against all PDE isoforms at 50 µM.a
PDE isoform
% inhibition at 50
PDE isoform
% inhibition at 50
µM
µM
PDE1A (cAMP)
PDE1A (cGMP)
PDE2A (cAMP)
PDE2A (cGMP)
PDE3A (cAMP)
PDE3B (cAMP)
2.6
PDE5A (cGMP)
PDE6C (cGMP)
PDE7A (cAMP)
PDE8A (cAMP)
PDE9A (cGMP)
PDE10A
97
22
16.2
9.1
4
5.6
19.9
12.4
6.7
10.7
3.6
(cAMP)
Altogether, our probe compound syntheses corroborated that the
bioisosteric replacement of the sulfonamide and the partial saturation at
the central ring are both essential to the optimization towards selective
PDE5 inhibitors. Fig. 3 summarizes the most important SAR conclusions.
PDE3A (cGMP)
PDE3B (cGMP)
36.2
15.7
1.6
PDE10A
4.5
0.9
5.9
(cGMP)
PDE11A
(cAMP)
PDE4B2
(cAMP)
PDE11A
(cGMP)
aValues are mean of at least two experiments; standard deviation < 15%.
5

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
intracellular cGMP levels, celecoxib did not show a clear concentration-
dependent effect, which can be attributed to its lower potency against
PDE5. Both compound 11 and 19 showed a substantially higher efficacy
in increasing intracellular cGMP levels in HEK239 cells when compared
to celecoxib.
4.1.1.2. (E)-1-(2-Fluorophenyl)-4,4-dimethylpent-1-en-3-one (E2). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and o-flourobenzaldehyde; oil; yield: 1.48 g (72%) [28].
4.1.1.3. (E)-1-(3-Fluorophenyl)-4,4-dimethylpent-1-en-3-one (E3). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and m-fluorobenzaldehyde; yellow solid; yield: 1.4 g (68%);
mp 43–44 ◦C [28].
2.3.3. PDE isoform selectivity
Since compound 11 showed higher cellular potency than compound
19 (Fig. 4), it was selected to be tested for its PDE isozyme selectivity
using all 11 recombinant human PDE isozymes. The compound exhibi-
ted remarkable selectivity for PDE5 with an IC50 of 2.7 µM without
significantly affecting the activity of all other PDE isozymes at concen-
trations up to 50 µM, including PDE6 and PDE11. This suggested that
trisubstituted pyrazolines may provide an attractive scaffold for PDE5
inhibition with a promising PDE isozyme selectivity and the potential for
fewer side effects. The results are summarized in Table 2.
4.1.1.4. (E)-1-(4-Fluorophenyl)-4,4-dimethylpent-1-en-3-one (E4). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and p-fluorobenzaldehyde ; oil; yield: 1.4 g (68%) [29].
4.1.1.5. (E)-1-(2-Chlorophenyl)-4,4-dimethylpent-1-en-3-one (E5). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and o-chlorobenzaldehyde; transparent oil; yield: 1.67 g
(75%) [28].
3. Conclusion
4.1.1.6. (E)-1-(3-Chlorophenyl)-4,4-dimethylpent-1-en-3-one (E6). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and m-chlorobenzaldehyde; yellow oil; yield: 1.6 g (72%)
[28].
Through systematic structural modifications, the off-target effect of
celecoxib to inhibit PDE5 was enhanced while eliminating COX-2
inhibitory activity. The developed trisubstituted pyrazolines represent
previously unrecognized scaffold for the development of novel PDE5
inhibitors with the potential for improved isozyme selectivity and
reduced side effects. The carboxylic acidic group at the para position of
the 1- phenyl together with non-coplanar pyrazoline were shown to be
essential features to boost the PDE5. Extension of the scaffold might be
required to bring the PDE5 inhibitory activity of this novel class to
greater potency while retaining PDE5 isozyme selectivity.
4.1.1.7. (E)-1-(4-Chlorophenyl)-4,4-dimethylpent-1-en-3-one (E7). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and p-chlorobenzaldehyde; white solid; yield: 1.78 g (80%);
mp 85–85.5 ◦C [30].
4.1.1.8. (E)-1-(2-Bromophenyl)-4,4-dimethylpent-1-en-3-one (E8). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and o-bromobenzaldehyde; Yellowish white solid; yield:
2.18 g (82%); mp 49–50 ◦C [28].
4. Experimental section
4.1. Chemistry
4.1.1.9. (E)-1-(3-Bromophenyl)-4,4-dimethylpent-1-en-3-one (E9). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and m-bromobenzaldehyde; Yellow oil; yield: 2.21 g (83%)
[28].
Solvents and reagents were obtained from commercial suppliers and
used as received without further purification. 1H NMR and 13C NMR
spectra were obtained using a Bruker DRX 500 spectrometer, or Varian
Mercury 400 spectrometer. The chemical shifts are referenced to the
residual protonated solvent signals and occasionally TMS was used as a
reference. At least 95% purity in all the tested compounds (Table 1) was
obtained using HPLC coupled with mass spectrometry. Mass spectra
(HPLC-ESI-MS) were obtained using a TSQ quantum (Thermo Electron
Corporation) instrument prepared with a triple quadrupole mass de-
tector (Thermo Finnigan) and an ESI source. All samples were inserted
using an autosampler (Surveyor, Thermo Finnigan) by an injection
4.1.1.10. (E)-1-(4-Bromophenyl)-4,4-dimethylpent-1-en-3-one
(E10).
Synthesized according to the general procedure for enone synthesis
using pinacolone and p-bromobenzaldehyde; Yellowish white solid;
yield: 2.08 g (78%); mp 101 ◦C [31].
4.1.1.11. (E)-1-(2,4-Dichlorophenyl)-4,4-dimethylpent-1-en-3-one
(E11). Synthesized according to the general procedure for enone syn-
thesis using pinacolone and 2,4-dichlorobenzaldehyde; yellow oil; yield:
2.05 g (80%) [32].
volume of 10
μL. The MS 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
5
◦
pressure of 4.0⋅10 Pa, a capillary temperature of 400 C, a capillary
voltage of 35 V and an auxiliary gas pressure of 1.0⋅105 Pa. The sta-
tionary 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). HPLC-
4.1.1.12. (E)-1-(3,4-Dichlorophenyl)-4,4-dimethylpent-1-en-3-one
(E12). Synthesized according to the general procedure for enone syn-
thesis using pinacolone and 3,4-dichlorobenzaldehyde; white solid;
yield: 2.1 g (82%); mp 89 ◦C [33].
Method: flow rate of 400 μL/min. The percentage of B started at an
initial of 5%, was increased up to 100% during 16 min, kept at 100% for
2 min, and flushed back to 5% in 2 min. Melting points were determined
using a Buchi B-540 melting point apparatus and are uncorrected.
4.1.1.13. (E)-1-(2,6-Dichlorophenyl)-4,4-dimethylpent-1-en-3-one
(E13). Synthesized according to the general procedure for enone syn-
thesis using pinacolone and 2,6-dichlorobenzaldehyde; yellow oil; yield:
2.18 g (85%) [34].
4.1.1. General procedure for enone synthesis
The appropriate ketone (10 mmol) was reacted with corresponding
aryl aldehyde (10 mmol) using the same procedure we previously re-
ported in ref [26].
4.1.1.14. (E)-1-(2-Methoxyphenyl)-4,4-dimethylpent-1-en-3-one (E14).
Synthesized according to the general procedure for enone synthesis
using pinacolone and o-anisaldehyde; oil; yield: 1.73 g (79%) [28].
4.1.1.1. (E)-4,4-Dimethyl-1-phenylpent-1-en-3-one
(E1). Synthesized
according to the general procedure for enone synthesis using pinacolone
and benzaldehyde; oil; yield: 1.34 g (71%) [27].
4.1.1.15. (E)-1-(4-Methoxyphenyl)-4,4-dimethylpent-1-en-3-one (E15).
Synthesized according to the general procedure for enone synthesis
6

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
using pinacolone and p-anisaldehyde; oil; yield: 1.64 g (75%) [29].
(79%) [45].
4.1.2. General procedure for pyrazoline synthesis
4.1.1.16. (E)-1-(2,4-Dimethoxyphenyl)-4,4-dimethylpent-1-en-3-one
(E16). Synthesized according to the general procedure for enone syn-
thesis using pinacolone and 2,4-dimethoxybenzaldehyde; yellow oil;
yield: 1.79 g (72%) [35].
A mixture of the enone derivative (2 mmol) and the corresponding
aryl hydrazine hydrochloride (3 mmol) were reacted together using the
same procedure we previously reported in ref [26].
4.1.2.1. 4-(3-tert-Butyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic
acid (1). The title compound was prepared by reaction of 4,4-dimethyl-
1-phenylpent-1-en-3-one (E1) and 4-hydrazinobenzoic acid hydrochlo-
ride according to the general procedure for pyrazoline synthesis. The
product was purified by CC (CH2Cl2/CH3OH 50:1); white powder;
yield: 38%; mp 233–234 ◦C; 1H NMR (500 MHz, DMSO‑d6) δ 12.04 (s,
1H), 7.55 (d, J = 8.9 Hz, 2H), 7.21 (t, J = 7.5 Hz, 2H), 7.18–7.02 (m,
3H), 6.74 (d, J = 8.9 Hz, 2H), 5.24 (dd, J = 11.7, 5.1 Hz, 1H), 3.48 (dd, J
4.1.1.17. (E)-1-(2,5-Dimethoxyphenyl)-4,4-dimethylpent-1-en-3-one
(E17). Synthesized according to the general procedure for enone syn-
thesis using pinacolone and 2,5-dimethoxybenzaldehyde; yellow oil;
yield: 1.71 g (69%) [35].
4.1.1.18. (E)-1-(2-Ethoxyphenyl)-4,4-dimethylpent-1-en-3-one
(E18).
Synthesized according to the general procedure for enone synthesis
using pinacolone and o-ethoxybenzaldehyde; yellow oil; yield: 1.86 g
(80%) [29].
= 17.7, 11.8 Hz, 1H), 2.63 (dd, J = 17.7, 5.1 Hz, 1H), 1.07 (s, 9H); 13
C
NMR (126 MHz, DMSO‑d6) δ 167.23, 161.45, 147.80, 142.21, 130.72,
128.97, 127.37, 125.53, 118.95, 111.29, 62.15, 42.54, 33.52, 27.82; MS
(ESI): m/z = 323.2 (M++H).
4.1.1.19. (E)-1-(4-Ethoxyphenyl)-4,4-dimethylpent-1-en-3-one
(E19).
Synthesized according to the general procedure for enone synthesis
using pinacolone and p-ethoxybenzaldehyde; yellow oil; yield: 1.86 g
(80%) [36].
4.1.2.2. 4-(3-(tert-Butyl)-5-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (2). The title compound was prepared by reaction of 1-
(2-fluorophenyl)-4,4-dimethylpent-1-en-3-one (E2) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); beige powder; yield: 70%; mp 224–226 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.19 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.35–7.18 (m, 2H),
7.08 (dt, J = 26.9, 7.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 5.52 (dd, J =
11.8, 4.8 Hz, 1H), 3.62 (dd, J = 17.7, 12.0 Hz, 1H), 2.80 (dd, J = 17.7,
4.8 Hz, 1H), 1.17 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 167.67,
162.25, 159.86 (d, J = 245.1 Hz), 148.05, 131.32, 130.04 (d, J = 8.2
Hz), 128.76 (d, J = 13.7 Hz), 127.86 (d, J = 4.0 Hz), 125.34 (d, J = 3.3
Hz), 119.67, 116.41 (d, J = 21.0 Hz), 111.63, 56.93, 41.74, 34.03,
28.32; MS (ESI): m/z = 341(M++H).
4.1.1.20. (E)-4,4-Dimethyl-1-(3-nitrophenyl)pent-1-en-3-one (E20). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and 3-nitrobenzaldehyde; yellow solid; yield: 1.4 g (60%);
mp 92–93 ◦C [37].
4.1.1.21. (E)-4,4-Dimethyl-1-(4-nitrophenyl)pent-1-en-3-one (E21). Syn-
thesized according to the generalprocedure for enone synthesis using
pinacolone and p-nitrobenzaldehyde; yellow solid; yield: 1.91 g (82%);
mp 125–126 ◦C [38].
4.1.1.22. (E)-4,4-Dimethyl-1-p-tolylpent-1-en-3-one (E22). Synthesized
according to the general procedure for enone synthesis using pinacolone
and p-tolualdehyde; white solid; yield: 1.6 g (79%); mp 79–80 ◦C [39].
4.1.2.3. 4-(3-(tert-Butyl)-5-(3-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (3). The title compound was prepared by reaction of 1-
(3-fluorophenyl)-4,4-dimethylpent-1-en-3-one (E3) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); beige powder; yield: 53%; mp 218–220 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.18 (s, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.42–7.29 (m, 1H),
7.03 (dt, J = 13.7, 8.0 Hz, 3H), 6.84 (d, J = 8.6 Hz, 2H), 5.39 (dd, J =
11.6, 4.8 Hz, 1H), 3.58 (dd, J = 17.8, 11.8 Hz, 1H), 2.76 (dd, J = 17.8,
4.9 Hz, 1H), 1.17 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 167.68,
162.82 (d, J = 244.5 Hz), 162.06, 148.14, 145.56 (d, J = 6.6 Hz), 131.62
(d, J = 8.1 Hz), 131.27, 122.02 (d, J = 2.5 Hz), 119.76, 114.74 (d, J =
20.8 Hz), 112.93 (d, J = 21.9 Hz), 111.84, 62.12, 42.85, 34.02, 28.29;
MS (ESI): m/z = 341 (M++H).
4.1.1.23. (E)-4-(4,4-Dimethyl-3-oxopent-1-enyl)benzonitrile (E23). Syn-
thesized according to the general procedures of enone synthesis using
3,3-dimethylbutan-2-one and 4-formylbenzonitrile; yellow oil; yield:
1.54 g (72%) [40].
4.1.1.24. (E)-1-(4-Hydroxyphenyl)-4,4-dimethylpent-1-en-3-one (E24).
Synthesized according to the general procedure for enone synthesis
using pinacolone and 4-hydroxybenzaldehyde; yellow oil; yield: 1.84 g
(90%) [41].
4.1.1.25. (E)-1-(Biphenyl-4-yl)-4,4-dimethylpent-1-en-3-one (E25). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and 4-biphenylcarbaldehyde; yellow oil; yield: 2.17 g (82%)
[42].
4.1.2.4. 4-(3-tert-Butyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (4). The title compound was prepared by reaction of 1-(4-
fluorophenyl)-4,4-dimethylpent-1-en-3-one (E4) and 4-hydrazinoben-
zoic acid hydrochloride according to the general procedure for pyrazo-
line synthesis. The product was purified by CC (CH2Cl2/CH3OH 50:1);
white powder; yield: 33%; mp 222–224 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 7.68 (d, J = 9.0 Hz, 2H), 7.24 (tt, J = 5.1, 2.5 Hz, 2H),
7.20–7.11 (m, 2H), 6.86 (d, J = 8.9 Hz, 2H), 5.40 (dd, J = 11.7, 5.0 Hz,
1H), 3.58 (dd, J = 17.7, 11.7 Hz, 1H), 2.75 (dd, J = 17.7, 5.0 Hz, 1H),
1.19 (s, 9H); 13C NMR (126 MHz, DMSO‑d6) δ 167.21, 161.48, 161.29
(d, J = 243.2 Hz), 147.68, 138.30 (d, J = 2.9 Hz), 130.74, 127.61 (d, J =
8.2 Hz), 119.10, 115.75 (d, J = 21.4 Hz), 111.34, 61.41, 42.45, 33.51,
27.80; MS (ESI): m/z = 341.2 (M++H).
4.1.1.26. (E)-4,4-Dimethyl-1-(naphthalene-2-yl)pent-1-en-3-one (E26).
Synthesized according to the general procedure for enone synthesis
using pinacolone and 2-naphthaldehyde; yellow oil; yield: 1.81 g (76%)
[43].
4.1.1.27. (E)-1-(Furan-2-yl)-4,4-dimethylpent-1-en-3-one
(E27). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and furan-2-carbaldehyde; yellow oil; yield: 1.32 g (86%)
[44].
4.1.1.28. (E)-4,4-Dimethyl-1-(thiophen-2-yl)pent-1-en-3-one (E28). Syn-
thesized according to the general procedure for enone synthesis using
pinacolone and thiophene-2-carbaldehyde; yellow oil; yield: 1.53 g
7

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
4.1.2.5. 4-(3-(tert-Butyl)-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (5). The title compound was prepared by reaction of 1-
(2-chlorophenyl)-4,4-dimethylpent-1-en-3-one (E5) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); orange crystals; yield: 47%; mp 229–231 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 7.69 (d, J = 9.0 Hz, 2H), 7.51 (dd, J = 7.8, 1.4 Hz, 1H),
7.36–7.18 (m, 2H), 6.99–6.86 (m, 1H), 6.75 (d, J = 8.9 Hz, 2H), 5.55
(dd, J = 11.9, 5.2 Hz, 1H), 3.69 (dd, J = 17.8, 11.9 Hz, 1H), 2.71 (dd, J
= 17.8, 5.2 Hz, 1H), 1.16 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ
167.66, 162.20, 147.90, 138.87, 131.48, 131.40, 130.48, 129.75,
128.37, 127.20, 119.83, 111.58, 60.20, 41.66, 34.05, 28.32; MS (ESI):
m/z = 357 (M++H).
4.1.2.10. 4-(5-(4-Bromophenyl)-3-tert-butyl-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (10). The title compound was prepared by reaction of 1-
(4-bromophenyl)-4,4-dimethylpent-1-en-3-one (E10) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); yellow crystals; yield: 32%; mp 222–223 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 12.19 (s, 1H), 7.68 (d, J = 9.0 Hz, 2H), 7.61–7.39 (m, 2H),
7.25–7.08 (m, 2H), 6.84 (d, J = 8.9 Hz, 2H), 5.38 (dd, J = 11.7, 5.0 Hz,
1H), 3.59 (dd, J = 17.8, 11.8 Hz, 1H), 2.75 (dd, J = 17.7, 5.0 Hz, 1H),
1.18 (s, 9H); 13C NMR (126 MHz, DMSO‑d6) δ 167.20, 161.52, 147.61,
141.50, 131.88, 130.78, 127.88, 120.41, 119.18, 111.34, 61.48, 42.28,
33.52, 27.80; MS (ESI): m/z = 401 (M++H).
4.1.2.11. 4-(3-(tert-Butyl)-5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyr-
azol-1-yl) benzoic acid (11). The title compound was prepared by reac-
tion of 1-(2,4-dichlorophenyl)-4,4-dimethylpent-1-en-3-one (E11) and
4-hydrazinobenzoic acid hydrochloride according to the general pro-
cedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:1); off- white powder; yield: 40%; mp 213–214 ◦C;
1H NMR (400 MHz, DMSO‑d6) δ 7.77–7.67 (m, 3H), 7.36 (dd, J = 8.4,
2.1 Hz, 1H), 6.95 (t, J = 10.3 Hz, 1H), 6.76 (d, J = 8.9 Hz, 2H), 5.55 (dd,
J = 11.9, 5.1 Hz, 1H), 3.70 (dd, J = 17.8, 11.9 Hz, 1H), 2.75 (dd, J =
18.0, 5.0 Hz, 1H), 1.17 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 167.64,
162.33, 147.75, 138.00, 133.32, 132.56, 131.45, 129.99, 128.63,
128.56, 120.04, 111.64, 59.88, 41.45, 34.06, 28.31; MS (ESI): m/z =
391 (M++H).
4.1.2.6. 4-(3-(tert-Butyl)-5-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (6). The title compound was prepared by reaction of 1-
(3-chlorophenyl)-4,4-dimethylpent-1-en-3-one (E6) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); orange powder; yield: 40%; mp 210–212 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.17 (s, 1H), 7.68 (d, J = 9.0 Hz, 2H), 7.39–7.28 (m, 2H),
7.26 (t, J = 1.7 Hz, 1H), 7.11 (dt, J = 7.5, 1.3 Hz, 1H), 6.84 (d, J = 8.9
Hz, 2H), 5.39 (dd, J = 11.7, 5.1 Hz, 1H), 3.57 (dd, J = 17.8, 11.8 Hz,
1H), 2.76 (dd, J = 17.8, 5.1 Hz, 1H), 1.16 (s, 9H); 13C NMR (101 MHz,
DMSO‑d6) δ 167.68, 162.09, 148.10, 145.10, 133.93, 131.46, 131.30,
127.91, 126.01, 124.64, 119.79, 111.85, 62.03, 42.85, 34.01, 28.28; MS
(ESI): m/z = 357 (M++H).
4.1.2.12. 4-(3-(tert-Butyl)-5-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)benzoic acid (12). The title compound was prepared by reac-
tion of 1-(3,4-dichlorophenyl)-4,4-dimethylpent-1-en-3-one (E12) and
4-hydrazinobenzoic acid hydrochloride according to the general pro-
cedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:1); greenish yellow powder; yield: 90%; mp
216–218 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 7.70 (d, J = 9.0 Hz, 2H),
7.63–7.54 (m, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.12 (dt, J = 8.2, 2.8 Hz,
1H), 6.86 (t, J = 5.7 Hz, 2H), 5.43 (dd, J = 11.7, 5.0 Hz, 1H), 3.58 (dd, J
4.1.2.7. 4-(3-tert-Butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (7). The title compound was prepared by reaction of 1-(4-
chlorophenyl)-4,4-dimethylpent-1-en-3-one (E7) and 4-hydrazinoben-
zoic acid hydrochloride according to the general procedure for pyrazo-
line synthesis. The product was purified by CC (CH2Cl2/CH3OH 50:1);
white crystals; yield: 41%; mp 208–210 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 12.24 (s, 1H); 7.74 (d, J = 8.9 Hz, 2H), 7.45 (d, J = 8.5 Hz,
2H), 7.27 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 5.45 (dd, J =
11.7, 5.0 Hz, 1H), 3.64 (dd, J = 17.8, 11.8 Hz, 1H), 2.80 (dd, J = 17.7,
5.0 Hz, 1H), 1.23 (s, 9H); 13C NMR (126 MHz, DMSO‑d6) δ 167.21,
161.51, 147.62, 141.08, 131.89, 130.77, 128.96, 127.53, 119.19,
111.34, 61.43, 42.33, 33.52, 27.79; MS (ESI): m/z = 357.1 (M++H).
= 17.8, 11.8 Hz, 1H), 2.80 (dd, J = 17.8, 5.1 Hz, 1H), 1.18 (s, 9H); 13
C
NMR (101 MHz, DMSO‑d6) δ 167.66, 162.18, 148.00, 143.62, 131.89,
131.77, 131.34, 130.43, 128.37, 126.30, 119.95, 111.91, 61.50, 42.68,
34.02, 28.26; MS (ESI): m/z = 391 (M++H).
4.1.2.8. 4-(5-(2-Bromophenyl)-3-(tert-butyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (8). The title compound was prepared by reaction of 1-
(2-bromophenyl)-4,4-dimethylpent-1-en-3-one (E8) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); yellow powder; yield: 74%; mp 200–201 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.00 (s, 1H), 7.73–7.65 (m, 2H), 7.43 (d, J = 4.02 Hz, 1H),
7.29 (d, J = 7.53 Hz, 1H), 7.17–7.15 (m, 2H), 6.89–6.82 (m, 2H), 5.39
(dd, J = 11.37, 5.12 Hz, 1H), 3.59 (dd, J = 17.18, 11.79 Hz, 1H), 2.78
(dd, J = 17.58, 4.59 Hz, 1H), 1.18 (s, 9H); MS (ESI): m/z = 401 (M++H).
4.1.2.13. 4-(3-(tert-Butyl)-5-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)benzoic acid (13). The title compound was prepared by reac-
tion of 1-(2,6-dichlorophenyl)-4,4-dimethylpent-1-en-3-one (E13) and
4-hydrazinobenzoic acid hydrochloride according to the general pro-
cedure for pyrazoline synthesis. The product was purified by CC
1
(CH2Cl2/CH3OH 50:1); beige powder; yield: 60%; mp 225–227 ◦C; H
NMR (400 MHz, DMSO‑d6) δ 12.17 (s, 1H), δ 7.64 (d, J = 8.6 Hz, 2H),
7.57 (d, J = 7.8 Hz, 1H), 7.37 (dt, J = 15.9, 7.9 Hz, 2H), 6.69 (d, J = 8.5
Hz, 2H), 5.89 (dd, J = 13.2, 8.2 Hz, 1H), 3.63 (dd, J = 18.0, 13.3 Hz,
1H), 2.94 (dd, J = 18.1, 8.2 Hz, 1H), 1.22 (s, 9H); 13C NMR (101 MHz,
DMSO‑d6) δ 167.59, 161.68, 148.09, 135.38, 134.75, 134.08, 131.27,
130.75, 129.39, 119.63, 111.22, 59.03, 40.64, 34.18, 28.58; MS (ESI):
m/z = 391 (M+ + H).
4.1.2.9. 4-(5-(3-Bromophenyl)-3-(tert-butyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (9). The title compound was prepared by reaction of 1-
(3-bromophenyl)-4,4-dimethylpent-1-en-3-one (E9) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); yellow powder; yield: 43%; mp 215–217 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 11.99 (s, 1H), 7.71 (d, J = 1.26 Hz, 2H), 7.31 (d, J = 1.35
Hz, 1H), 7.25 (d, J = 1.89 Hz, 1H), 7.22 (d, J = 1.81 Hz, 1H), 7.20–7.19
(m, 1H), 6.94 (d, J = 7.35 Hz, 1H), 6.77–6.72 (m, 1H), 5.50 (dd, J =
11.79, 5.07 Hz, 1H), 3.71(dd, J = 17.77, 11.85 Hz, 1H), 2.70 (dd, J =
17.81, 5.13 Hz, 1H), 1.18 (s, 9H); MS (ESI): m/z = 401 (M++H).
4.1.2.14. 4-(3-(tert-Butyl)-5-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-
1-yl)benzoic acid (14). The title compound was prepared by reaction of
1-(2-methoxyphenyl)-4,4-dimethylpent-1-en-3-one (E14) and 4-hydra-
zinobenzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); yellowish white powder; yield: 70%; mp 217–219 C; 1H NMR
◦
(400 MHz, DMSO‑d6) δ 7.66 (d, J = 8.2 Hz, 2H), 7.22 (s, 1H), 7.05 (d, J
= 8.1 Hz, 1H), 6.81 (d, J = 15.7 Hz, 2H), 6.75 (d, J = 8.1 Hz, 2H),
5.50–5.39 (m, 1H), 3.86 (s, 3H), 3.56 (dd, J = 17.4, 12.0 Hz, 1H),
2.71–2.56 (m, 1H), 1.15 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ
8

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
167.73, 162.43, 156.40, 148.18, 131.29, 129.27, 129.14, 126.14,
121.08, 119.22, 111.90, 111.45, 57.44, 56.06, 41.76, 34.02, 28.37; MS
(ESI): m/z = 353 (M++H).
Hz, 2H), 3.56 (dd, J = 17.71, 11.68 Hz, 1H), 2.72 (dd, J = 17.75, 5.01
Hz, 1H), 1.29 (t, J = 6.96 Hz, 3H), 1.19 (s, 9H); MS (ESI): m/z = 367
(M++H).
4.1.2.15. 4-(3-tert-Butyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl) benzoic acid (15). The title compound was prepared by reaction of 1-
(4-methoxyphenyl)-4,4-dimethylpent-1-en-3-one (E15) and 4-hydrazi-
nobenzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); white powder; yield: 30%; mp 208–209 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 7.67 (d, J = 9.0 Hz, 2H), 7.15–7.04 (m, 2H), 6.95–6.80
(m,4H), 5.30 (dd, J = 11.7, 5.0 Hz, 1H), 3.70 (s, 3H), 3.55 (dd, J = 17.7,
11.7 Hz, 1H), 2.72 (dd, J = 17.7, 5.0 Hz), 1.19 (s, 9H); 13C NMR (126
MHz, DMSO‑d6) δ 167.27, 161.46, 158.45, 147.83, 134.12, 130.70,
126.79, 118.87, 114.33, 111.34, 61.66, 55.00, 42.56, 33.55, 27.86; MS
(ESI): m/z = 353.2 (M++H).
4.1.2.20. 4-(3-tert-Butyl-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (20). The title compound was prepared by reaction of 4,4-
dimethyl-1-(3-nitrophenyl)pent-1-en-3-one (E20) and 4-hydrazinoben-
zoic acid hydrochloride according to the general procedure for pyrazo-
line synthesis. The product was purified by CC (CH2Cl2/CH3OH 50:1);
yellow powder; yield: 50%; mp 235–237 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 11.98 (s, 1H), 8.10 (d, J = 10.9 Hz, 2H), 7.67 (t, J = 7.7 Hz,
2H), 7.62 (q, J = 8.0 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 5.58 (dd, J =
11.6, 4.7 Hz, 1H), 3.63 (dd, J = 17.8, 11.8 Hz, 2H), 2.82 (dd, J = 17.9,
4.8 Hz, 1H), 1.17 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 167.64,
162.20, 148.60, 148.01, 144.75, 132.74, 131.35, 131.17, 122.95,
121.10, 120.01, 111.95, 61.85, 42.76, 34.04, 28.27; MS (ESI): m/z =
367.6 (M++H).
4.1.2.16. 4-(3-tert-Butyl-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)benzoic acid (16). The title compound was prepared by reac-
tion of 1-(2,4-dimethoxyphenyl)-4,4-dimethylpent-1-en-3-one (E16)
and 4-hydrazinobenzoic acid hydrochloride according to the general
procedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:1); yellowish green powder; yield: 58%; mp
213–215 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 12.11 (s, 1H), 7.66 (d, J =
8.2 Hz, 2H), 6.74 (dd, J = 13.0, 9.0 Hz, 3H), 6.59 (s, 1H), 6.40 (d, J =
8.4 Hz, 1H), 5.37 (dd, J = 11.5, 4.5 Hz, 1H), 3.84 (s, 3H), 3.70 (s, 3H),
3.51 (dd, J = 17.6, 11.8 Hz, 1H), 2.62 (dd, J = 17.7, 4.5 Hz, 1H), 1.15 (s,
9H); 13C NMR (101 MHz, DMSO‑d6) δ 167.76, 162.37, 160.34, 157.42,
148.17, 131.25, 126.96, 121.49, 119.10, 111.44, 105.41, 99.30, 57.17,
56.12, 55.58, 41.80, 34.01, 28.37; MS (ESI): m/z = 383.6 (M++H).
4.1.2.21. 4-(3-tert-Butyl-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (21). The title compound was prepared by reaction of 4,4-
dimethyl-1-(4-nitrophenyl)pent-1-en-3-one (E21) and 4-hydrazinoben-
zoic acid hydrochloride according to the general procedure for pyrazo-
line synthesis. The product was purified by CC (CH2Cl2/CH3OH 50:1);
yellow powder; yield: 35%; mp 245–248 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 12.21 (s, 1H), 8.27–8.12 (m, 2H), 7.68 (d, J = 8.9 Hz, 2H),
7.47 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.9 Hz, 2H), 5.56 (dd, J = 11.9, 5.1
Hz, 1H), 3.65 (dd, J = 17.8, 11.9 Hz, 1H), 2.79 (dd, J = 17.8, 5.2 Hz,
1H), 1.18 (s, 9H); 13C NMR (126 MHz, DMSO‑d6) δ 167.16, 161.59,
149.63, 147.51, 146.84, 130.85, 127.05, 124.27, 119.50, 111.41, 61.53,
42.18, 33.56, 27.78; MS (ESI): m/z = 368.2 (M++H).
4.1.2.17. 4-(3-tert-Butyl-5-(2,5-dimethoxyphenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)benzoic acid (17).. The title compound was prepared by reac-
tion of 1-(2,5-dimethoxyphenyl)-4,4-dimethylpent-1-en-3-one (E17)
and 4-hydrazinobenzoic acid hydrochloride according to the general
procedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:1); faint yellow powder; yield: 55%; mp
262–264 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 7.69 (d, J = 8.9 Hz, 2H),
7.00 (d, J = 8.9 Hz, 1H), 6.84–6.74 (m, 3H), 6.38 (d, J = 2.9 Hz, 1H),
5.44 (dd, J = 11.8, 4.9 Hz, 1H), 3.83 (s, 3H), 3.57 (s, 3H), 3.53 (dd, J =
17.7, 11.9 Hz, 1H), 2.68 (dd, J = 17.7, 4.9 Hz, 1H), 1.18 (s, 9H); MS
(ESI): m/z = 383.6 (M++H).
4.1.2.22. 4-(3-tert-Butyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic
acid (22). The title compound was prepared by reaction of 4,4-
dimethyl-1-p-tolylpent-1-en-3-one (E22) and 4-hydrazinobenzoic acid
hydrochloride according to the general procedure for pyrazoline syn-
thesis. The product was purified by CC (CH2Cl2/CH3OH 50:1); yellow
powder; yield: 35%; mp 194–196 ◦C; 1H NMR (500 MHz, DMSO‑d6) δ
7.67 (d, J = 8.5 Hz), 7.14 (d, J = 7.9 Hz), 7.09 (d, J = 7.9 Hz), 6.86 (d, J
= 8.6 Hz), 5.32 (dd, J = 11.7, 5.0 Hz), 3.58 (dd, J = 17.7, 11.8 Hz), 2.73
(dd, J = 18.0, 4.8 Hz), 2.25 (s), 1.19 (s); 13C NMR (126 MHz, DMSO‑d6) δ
167.24, 161.43, 147.79, 139.21, 136.52, 130.69, 129.51, 125.45,
118.85, 111.28, 61.91, 42.51, 33.51, 27.84, 20.59; MS (ESI): m/z =
337.2 (M++H).
4.1.2.18. 4-(3-(tert-Butyl)-5-(2-ethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (18). The title compound was prepared by reaction of 1-
(2-ethoxyphenyl)-4,4-dimethylpent-1-en-3-one (E18) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
4.1.2.23. 4-(3-tert-Butyl-5-(4-cyanophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (23). The title compound was prepared by reaction of 4-
(4,4-dimethyl-3-oxopent-1-enyl)benzonitrile (E23) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2:CH3OH
1
◦
50:1); greenish yellow powder; yield: 63%; mp 211–212 C; H NMR
(400 MHz, DMSO‑d6) δ 7.67 (d, J = 9.0 Hz, 2H), 7.21 (ddd, J = 8.4, 6.9,
2.2 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.90–6.74 (m, 4H), 5.48 (dd, J =
11.8, 5.3 Hz, 1H), 4.23–4.06 (m, 2H), 3.59 (dd, J = 17.7, 11.9 Hz, 1H),
1
◦
100:2); white solid; yield: 24%; mp 115–117 C; H NMR (500 MHz,
DMSO‑d6) δ 12.23 (s, 1H), 7.84–7.79 (m, 2H), 7.71–7.67 (m, 2H),
7.41–7.36 (m, 2H), 6.86–6.81 (m, 2H), 5.50 (dd, J = 11.8, 5.0 Hz, 1H),
3.62 (dd, J = 17.8, 11.9 Hz, 1H), 2.77 (dd, J = 17.8, 5.1 Hz, 1H), 1.18 (s,
9H); 13C NMR (126 MHz, DMSO‑d6) δ 167.20, 161.61, 147.62, 147.55,
133.04, 130.88, 126.77, 119.46, 118.58, 111.40, 110.32, 61.74, 42.22,
33.58, 27.81; MS (ESI): m/z = 347.9 (M + H)+.
2.68 (dd, J = 17.7, 5.4 Hz, 1H), 1.39 (t, J = 7.0 Hz, 3H), 1.17 (s, 9H); 13
C
NMR (101 MHz, DMSO‑d6) δ 167.74, 162.35, 155.63, 148.22, 131.26,
129.59, 129.10, 126.27, 121.06, 119.22, 112.85, 111.44, 64.01, 57.47,
41.77, 34.02, 28.37, 15.20; MS (ESI): m/z = 367 (M++H).
4.1.2.19. 4-(3-(tert-Butyl)-5-(4-ethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (19). The title compound was prepared by reaction of 1-
(4-ethoxyphenyl)-4,4-dimethylpent-1-en-3-one (E19) and 4-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); beige powder; yield: 50%; mp 159–160 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 7.67–7.64 (m, 2H), 11.82 (s, 1H), 7.11–7.08 (m, 2H),
6.88–6.83 (m, 4H), 5.30 (dd, J = 11.57, 4.97 Hz, 1H), 3.97 (q, J = 6.96
4.1.2.24. 4-(3-tert-Butyl-5-(4-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (24). The title compound was prepared by reaction of 1-
(4-hydroxyphenyl)-4,4-dimethylpent-1-en-3-one (E24) and 4-hydrazi-
nobenzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); white powder; yield: 65%; mp 259–261 ◦C; 1H NMR (500 MHz,
DMSO‑d6) δ 7.66 (d, J = 9.1 Hz, 2H), 7.06–6.93 (m, 2H), 6.92–6.76 (m,
2H), 6.75–6.59 (m, 2H), 5.23 (dd, J = 11.6, 5.1 Hz, 1H), 3.53 (dd, J =
9

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
17.7, 11.7 Hz, 1H), 2.70 (dd, J = 17.7, 5.1 Hz, 1H), 1.18 (s, 9H); 13C
NMR (126 MHz, DMSO) δ 167.28, 161.45, 156.59, 147.88, 132.43,
130.67, 126.74, 118.72, 115.63, 111.30, 61.80, 42.58, 33.53, 27.84; MS
(ESI): m/z = 339 (M++H).
50:1); faint yellow powder; yield: 61%; mp 245–247 ◦C; 1H NMR (500
MHz, DMSO‑d6) δ 7.70–7.67 (m, 2H), 7.41–7.38 (m, 2H), 7.23–7.20 (m,
2H), 6.86–6.83 (m, 2H), 5.40 (dd, J = 11.7, 5.0 Hz, 1H), 3.58 (dd, J =
17.8, 11.8 Hz, 1H), 2.74 (dd, J = 17.8, 5.0 Hz, 1H), 1.18 (s, 9H); 13C
NMR (126 MHz, DMSO‑d6) δ 167.22, 161.52, 147.63, 141.09, 131.91,
130.79, 128.98, 127.54, 119.18, 111.35, 61.45, 42.36, 33.53, 27.80; MS
(ESI): m/z = 356.7 (M++H).
4.1.2.25. 4-(5-([1,1′-Biphenyl]-4-yl)-3-(tert-butyl)-4,5-dihydro-1H-pyr-
azol-1-yl)benzoic acid (25). The title compound was prepared by reac-
tion of 1-([1,1′-biphenyl]-4-yl)-4,4-dimethylpent-1-en-3-one (25) and 4-
hydrazinobenzoic acid hydrochloride according to the general proced-
ure for pyrazoline synthesis. The product was purified by CC (CH2Cl2/
CH3OH 50:1); white powder; yield: 45%; mp 232–234 ◦C; 1H NMR (500
MHz, DMSO‑d6) δ 12.15 (s, 1H), 7.76–7.54 (m, 6H), 7.42 (t, J = 7.3 Hz,
2H), 7.37–7.21 (m, 3H), 6.88 (d, J = 8.4 Hz, 2H), 5.40 (dd, J = 11.6, 4.9
Hz, 1H), 3.60 (dd, J = 17.7, 11.8 Hz, 1H), 2.79 (dd, J = 17.7, 4.7 Hz,
1H), 1.18 (s, J = 9.5 Hz, 9H); 13C NMR (126 MHz, DMSO‑d6) δ 167.71,
162.03, 148.26, 141.84, 140.06, 139.72, 131.26, 129.35, 127.89,
127.76, 127.02, 126.67, 119.48, 111.80, 62.30, 42.96, 34.05, 28.34; MS
(ESI): m/z = 399 (M++H).
4.1.2.30. 2-(3-tert-Butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (30). The title compound was prepared by reaction of 1-
(4-chlorophenyl)-4,4-dimethylpent-1-en-3-one (E7) and 2-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); white powder; yield: 53%; mp 163–165 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.27 (s, 1H), 7.38–7.33 (m, 2H), 7.32–7.27 (m, 2H), 7.24
(dd, J = 7.6, 1.6 Hz, 1H), 7.10 (ddd, J = 8.7, 7.4, 1.6 Hz, 1H), 6.73 (td, J
= 7.5, 0.9 Hz, 1H), 6.54 (d, J = 8.2 Hz, 1H), 5.27 (dd, J = 11.3, 8.3 Hz,
1H), 3.45 (dd, J = 17.3, 11.3 Hz, 1H), 2.71–2.59 (m, 1H), 1.12 (s, 9H);
13C NMR (101 MHz, DMSO‑d6) δ 170.78, 159.25, 142.61, 141.46,
132.24, 130.42, 129.31, 129.19, 128.54, 123.88, 119.14, 114.67, 63.68,
42.65, 33.96, 28.32; MS (ESI): m/z = 356.7 (M++H).
4.1.2.26. 4-(3-(tert-Butyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-
1-yl)benzoic acid (26). The title compound was prepared by reaction of
4,4-dimethyl-1-(naphthalen-2-yl)pent-1-en-3-one (E26) and 4-hydrazi-
nobenzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
50:1); white powder; yield: 41%; mp 221–223 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 7.86 (t, J = 9.1 Hz, 3H), 7.75 (s, 1H), 7.64 (d, J = 8.4 Hz,
2H), 7.48 (dd, J = 9.3, 4.8 Hz, 2H), 7.28 (d, J = 8.5 Hz, 1H), 6.89 (d, J =
8.4 Hz, 2H), 5.50 (dd, J = 11.7, 5.2 Hz, 1H), 3.65 (dd, J = 17.8, 11.9 Hz,
1H), 2.82 (dd, J = 17.8, 5.1 Hz, 1H), 1.19 (s, 9H); 13C NMR (101 MHz,
DMSO‑d6) δ 167.69, 162.09, 148.40, 140.16, 133.38, 132.80, 131.22,
129.50, 128.19, 128.02, 126.88, 126.46, 124.68, 124.03, 119.56,
111.83, 62.92, 42.99, 34.07, 28.33; MS (ESI): m/z = 373 (M++H).
4.1.2.31. 4-(3-tert-Butyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonic acid (31). The title compound was prepared by reaction
of 1-(4-fluorophenyl)-4,4-dimethylpent-1-en-3-one (E4) and 4-hydrazi-
nobenzene sulfonic acid hydrochloride according to the general pro-
cedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:2); yellow crystals; yield: 25%; mp 92–95 ◦C; 1H
NMR (500 MHz, DMSO‑d6) δ 7.65–7.73 (m, 4H), 7.6 (d, J = 8.57 Hz
,2H), 6.75 (d, J = 5.59 Hz, 2H), 5.28 (dd, J = 11.65, 5.76 Hz, 1H), 3.53
(dd, J = 17.49, 11.69 Hz, 1H), 2.69 (dd, J = 17.51, 5.81 Hz, 1H), 1.17 (s,
9H); 13C NMR (126 MHz, DMSO‑d6) δ 161.73, 146.82, 131.70, 128.63,
127.59, 124.06, 115.68, 115.51, 111.19, 67.39, 62.09, 33.43, 27.91; MS
(ESI): m/z = 377.1 (M++H).
4.1.2.27. 4-(3-tert-Butyl-5-(furan-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ben-
zoic acid (27). The title compound was prepared by reaction of 1-(furan-
2-yl)-4,4-dimethylpent-1-en-3-one (E27) and 4-hydrazinobenzoic acid
hydrochloride according to the general procedure for pyrazoline syn-
thesis. The product was purified by CC (CH2Cl2/CH3OH 50:1); faint
brown powder; yield: 57%; mp 190–191 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.23 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.56 (s, 1H), 7.00 (d,
J = 8.1 Hz, 2H), 6.37 (s, 2H), 5.49 (dd, J = 11.6, 4.5 Hz, 1H), 3.44 (dd, J
= 17.5, 11.7 Hz, 1H), 3.01 (dd, J = 17.5, 4.4 Hz, 1H), 1.21 (s, 9H); 13C
NMR (101 MHz, DMSO‑d6) δ 167.72, 162.23, 153.43, 148.43, 143.22,
131.12, 119.69, 112.01, 110.80, 107.92, 56.21, 39.05, 34.11, 28.30; MS
(ESI): m/z = 312.9 (M++H).
4.1.2.32. 4-(3-tert-Butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonic acid (32). The title compound was prepared by re-
action of 1-(4-chlorophenyl)-4,4-dimethylpent-1-en-3-one (E7) and 4-
hydrazinobenzene sulfonic acid hydrochloride according to the gen-
eral procedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:2); white crystals; yield: 27%; mp 92–95 ◦C; 1H
NMR (500 MHz, DMSO‑d6) δ 7.87 (d, J = 9.01 Hz, 2H), 7.44–7.41 (m,
2H), 7.37 (d, J = 8.53 Hz, 2H), 7.34 (d, J = 8.84 Hz, 2H), 5.28 (dd, J =
11.69, 5.80 Hz, 1H), 3.54 (dd, J = 17.53, 11.74 Hz, 1H), 2.69 (dd, J =
17.52, 5.83 Hz, 1H), 1.2 (s, 9H); 13C NMR (126 MHz, DMSO‑d6) δ
159.57, 144.93, 141.40, 137.93, 131.70, 128.83, 127.70, 126.42,
111.17, 62.14, 42.28, 30.29, 27.91; MS (ESI): m/z = 393 (M++H).
4.1.2.28. 4-(3-tert-Butyl-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (28). The title compound was prepared by reaction of 4,4-
dimethyl-1-(thiophen-2-yl)pent-1-en-3-one (E28) and 4-hydrazinoben-
zoic acid hydrochloride according to the general procedure for pyrazo-
line synthesis. The product was purified by CC (CH2Cl2/CH3OH 50:1);
dark yellow powder; yield: 58%; mp 214–216 ◦C; 1H NMR (400 MHz,
DMSO‑d6) δ 12.20 (s, J = 72.6 Hz, 1H), 7.71 (dd, J = 9.2, 2.0 Hz, 2H),
7.41–7.32 (m, 1H), 7.10 (d, J = 2.7 Hz, 1H), 6.98 (t, J = 5.6 Hz, 2H),
6.94 (dd, J = 5.0, 3.5 Hz, 1H), 5.75 (dd, J = 11.2, 4.2 Hz, 1H), 3.55 (dd,
4.1.2.33. 4-(3-tert-Butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (33). The title compound was prepared by reac-
tion of 1-(4-chlorophenyl)-4,4-dimethylpent-1-en-3-one (E7) and 4-
hydrazinobenzene sulfonamide hydrochloride according to the general
procedure for pyrazoline synthesis. The product was purified by CC
(CH2Cl2/CH3OH 50:1); white powder; yield: 55%; mp 180–182 ◦C; 1H
NMR (400 MHz, DMSO‑d6) δ 7.53 (t, J = 5.7 Hz, 2H), 7.46–7.32 (m, 2H),
7.27–7.14 (m, 2H), 6.97 (s, 2H), 6.94–6.78 (m, 2H), 5.41 (dd, J = 11.7,
5.0 Hz, 1H), 3.59 (dd, J = 17.7, 11.7 Hz, 1H), 2.76 (dd, J = 17.8, 5.0 Hz,
1H), 1.18 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 161.96, 147.04,
141.35, 132.80, 132.39, 129.44, 128.06, 127.55, 111.86, 61.91, 42.79,
34.01, 28.28; MS (ESI): m/z = 392.5 (M++H).
J = 17.6, 11.2 Hz, 1H), 2.90 (dd, J = 17.6, 4.3 Hz, 1H), 1.21 (s, 9H); 13
C
NMR (101 MHz, DMSO‑d6) δ 167.70, 162.21, 148.33, 145.47, 131.10,
127.18, 125.74, 125.60, 119.92, 112.30, 58.65, 42.99, 34.09, 28.26; MS
(ESI): m/z = 328.8 (M++H).
4.1.2.29. 3-(3-tert-Butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoic acid (29). The title compound was prepared by reaction of 1-
(4-chlorophenyl)-4,4-dimethylpent-1-en-3-one (E7) and 3-hydrazino-
benzoic acid hydrochloride according to the general procedure for
pyrazoline synthesis. The product was purified by CC (CH2Cl2/CH3OH
4.1.3. General procedure for pyrazoline oxidation into pyrazole
A mixture of the pyrazoline derivative (1 mmol) and dichlor-
odicyanoquinone (1.5 mmol) in 10 mL of benzene was heated to reflux
for 5 h. The mixture was cooled to room temperature and filtered
10

M. Abdel-Halim et al.
Bioorganic Chemistry 104 (2020) 104322
through a plug of silica gel wetted with diethyl ether. The filtrate was
20 µL arachidonic acid was added and the reaction allowed to proceed at
25 ◦C for an additional 5 min. Activity was determined by absorbance of
the substrate at a wavelength of 590 nm.
concentrated in vacuo and the residue was purified by CC.
4.1.3.1 4-(3-tert-Butyl-5-phenyl-1H-pyrazol-1-yl)benzoic acid (34). The
title compound was prepared by the oxidation of 4-(3-tert-butyl-5-
phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid (1) according to
the general procedure for pyrazole synthesis. The product was purified
by CC (CH2Cl2/CH3OH 50:1); white powder; yield: 60%; mp
192–194 ◦C; 1H NMR (500 MHz, DMSO‑d6) δ 7.92–7.84 (m, 2H)
7.35–7.23 (m, 7H), 6.6 (s, 1H), 1.24 (s, 9H); MS (ESI): m/z = 321
(M++H).
4.2.2. PDE assay
PDE activity was measured using the IMAP fluorescence polarization
assay (Molecular Devices Inc.) according to the manufacturer’s specifi-
cations. Activity of recombinant PDE enzymes (BPS Biosciences) was
measured through selective binding of the hydrolyzed form (product) of
fluorophore labeled cyclic nucleotide substrate to immobilized metal
coordination complexes. Binding resulted in increased fluorescence
polarization (FP). Tetramethylrhodamine (TAMRA)-cGMP (ex530/
em590 nm) and FAM-cAMP (ex485/em530 nm) substrates, each at final
concentration of 50 nmol/L were incubated with PDE enzymes at con-
centrations empirically determined to yield the EC80 activity (10 µL, 2X
concentrate) in assay buffer (10 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.1
mg/mL bovine serum albumin). Inhibitors were incubated with PDE
enzymes (5 µL, 4X concentrate in assay buffer) for 60 min at 30 ◦C,
followed by incubation with indicated substrate (5 µL, 4X concentrate in
assay buffer) for 30 min at 30 ◦C. Binding reagent (60 µL) was added and
incubated for 10 min at 30 ◦C. FP was measured at the indicated exci-
tation and emission wavelengths using a Synergy4 (Biotek) microplate
reader. IC50 values were determined using GraphPad Prism non-linear
dose–response software package.
4.1.3.2 4-(3- tert-Butyl ꢀ 5-(4-fluorophenyl)-1H-pyrazol-1-yl) benzoic acid
(35). The title compound was prepared by the oxidation of 4-(3-tert-
butyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid
(4) according to the general procedure for pyrazole synthesis. The
product was purified by CC (CH2Cl2/CH3OH 50:1); white powder; yield:
66%; mp180-182 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 7.93–7.90 (m,
2H), 7.33–7.18 (m, 6H), 6.6 (s, 1H), 1.21 (s, 9H); MS (ESI): m/z = 339
(M++H).
4.1.4. General procedure for synthesis of ethyl ester derivatives
A mixture of absolute ethanol (10 mL) and acetyl chloride (0.25 mL)
was stirred on cold for five minutes, then the corresponding benzoic acid
derivative (1 mmol) was added and the mixture was refluxed for 5 h and
the residue was purified using CC.
4.2.3. Cyclic GMP biosensor assay
A luciferase cell-based assay to measure changes in intracellular
cGMP levels was established by stable transfection of HEK293 cells with
a genetically encoded GAF-Luc construct (GloSensor cGMP-40F; Prom-
ega) containing a cGMP-specific binding site from the human PDE5A
GAF-A domain fused with modified firefly luciferase. Following trans-
fection with Lipofectamine LTX (Invitrogen), stable transfectants were
selected with G418 antibiotic. Resulting HEK293-GloSensor cGMP-40F
cells were plated at 6 × 104 cells per well in 100 µL per well of 96-well
assay plates (Corning) and allowed to attach overnight in Dulbecco’s
Modification of Eagle’s Medium (DMEM; Corning) + 10% fetal bovine
4.1.4.1. Ethyl 4-(3-tert-butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-
1-yl)benzoate (36). The title compound was prepared by the esterifica-
tion of 4-(3-tert-butyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-
1-yl)benzoic acid (7) according to the general procedure for ester
synthesis. The product was purified by CC (CH2Cl2/hexane 4:1); white
powder; yield: 50%; mp 144–147 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ
7.68 (d, J = 9.0 Hz, 2H), 7.45–7.32 (m, 2H), 7.25–7.12 (m, 2H), 6.84 (d,
J = 8.9 Hz, 2H), 5.40 (dd, J = 11.7, 4.8 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H),
3.58 (dd, J = 17.8, 11.8 Hz, 1H), 2.74 (dd, J = 17.8, 4.9 Hz, 1H), 1.23 (t,
J = 7.1 Hz, 3H), 1.16 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 166.06,
162.35, 148.20, 141.43, 132.39, 131.07, 129.44, 128.02, 118.71,
111.89, 61.81, 60.21, 42.80, 34.04, 28.27, 14.75; MS (ESI): m/z = 384.6
(M++H).
◦
serum (FBS; Atlanta Biologicals) at 37 C and 5% CO2. Cultures were
pre-incubated at room temperature in CO2 independent media (Gibco)
supplemented with 10% FBS and 5 mmol/L luciferin substrate (Prom-
ega). For measurement of intracellular cGMP levels, endogenous soluble
guanylyl cyclase was activated by addition of 50 µM sodium nitroprus-
side (SNP; Sigma-Aldrich) in combination with indicated concentrations
of test compounds to initiate the experiment. The firefly luciferase
domain was activated by the resulting increase of intracellular cGMP
binding to the GAF-A domain, and produced bioluminescence values in
live cells were measured each minute over a period of 1 h in a Biotek
Synergy4 plate reader.
4.1.4.2. Ethyl 4-(3-tert-butyl-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzoate (37). The title compound was prepared by the esterification
of 4-(3-tert-butyl-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzoic acid (20) according to the general procedure for ester syn-
thesis. The product was purified by CC (CH2Cl2/hexane 4:1); orange
powder; yield: 52%; mp 134–136 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ
8.17–8.04 (m, 2H), 7.70 (d, J = 9.0 Hz, 2H), 7.64 (t, J = 7.6 Hz, 1H),
7.60 (dt, J = 7.7, 1.5 Hz, 1H), 6.96–6.83 (m, 2H), 5.62 (dd, J = 11.7, 4.9
Hz, 1H), 4.25–4.10 (m, 2H), 3.64 (dd, J = 17.9, 11.8 Hz, 1H), 2.84 (dd, J
= 17.9, 4.9 Hz, 1H), 1.24 (t, J = 7.1 Hz, 3H), 1.19 (s, 9H); 13C NMR (101
MHz, DMSO‑d6) δ 166.01, 162.52, 148.58, 148.12, 144.62, 132.73,
131.16, 122.96, 121.12, 119.07, 112.01, 61.75, 60.25, 42.74, 34.05,
28.26, 14.73 ; MS (ESI): m/z = 395.7 (M++H).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
4.2. Biological testing
Financial Support: Research reported in this publication was sup-
ported in part by the National Cancer Institute of the National Institutes
of Health under awards R01CA155638, R01CA197147, and
R01CA131378. Mohammad Abdel-Halim and Mohammad Weam are
grateful to the German Academic Exchange Service (DAAD) for funding
their research visits to Saarland University, Germany.
4.2.1. COX assay
COX-1 and COX-2 activities were measured using purified ovine
COX-1 and COX-2 with colorimetric assay kits obtained from Cayman
Chemical Co. (Ann Arbor, Michigan) according to the manufacturers
protocol. Briefly, 10 µL ovine COX-1 and COX-2 and Hemin were diluted
in 150 µL assay buffer 0.1 M Tris-HCl, pH 8). Inhibitors or vehicle were
added to the reaction in 10 µL of reaction buffer, followed by incubation
at 25 ◦C for 5 min. After the addition of 20 µL the colorimetric substrate,
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
11

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org/10.1016/j.bioorg.2020.104322.
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