Design, synthesis, and evaluation of dihydropyranopyrazole derivatives as novel pde2 inhibitors for the treatment of alzheimer’s disease

Article abstract of DOI:10.3390/molecules26103034

Phosphodiesterase 2 (PDE2) has been regarded as a novel target for the treatment of Alz-heimer’s disease (AD). In this study, we obtained (R)‐LZ77 as a hit compound with moderate PDE2 inhibitory activity (IC50 = 261.3 nM) using a high‐throughput virtual screening method based on molecular dynamics. Then, we designed and synthesized 28 dihydropyranopyrazole derivatives as PDE2 inhibitors. Among them, compound (+)‐11h was the most potent PDE2 inhibitor, with an IC50 value of 41.5 nM. The molecular docking of PDE2‐(+)‐11h reveals that the 4‐(trifluoromethyl)ben-zyl)oxyl side chain of the compound enters the H‐pocket and forms strong hydrophobic interactions with L770/L809/F862, which improves inhibitory activity. The above results may provide insight for further structural optimization of highly potent PDE2 inhibitors and may lay the foundation for their use in the treatment of AD.

Full text of DOI:10.3390/molecules26103034

molecules
Article
Design, Synthesis, and Evaluation of Dihydropyranopyrazole
Derivatives as Novel PDE2 Inhibitors for the Treatment of
Alzheimer’s Disease
Yan Zhou 1 , Jinjian Li 1, Han Yuan 1, Rui Su 1, Yue Huang 1, Yiyou Huang 1, Zhe Li 1, Yinuo Wu 1, Haibin Luo 1,2
,
Chen Zhang 1,* and Ling Huang 1,2,
*
1
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China;
zhouy598@163.com (Y.Z.); lijinj15@163.com (J.L.); yuanhan0806@163.com (H.Y.); surui20@163.com (R.S.);
huangy266@mail2.sysu.edu.cn (Y.H.); huangyy287@mail.sysu.edu.cn (Y.H.); lizhe5@mail.sysu.edu.cn (Z.L.);
wyinuo3@mail.sysu.edu.cn (Y.W.); luohb77@mail.sysu.edu.cn (H.L.)
School of Pharmaceutical Sciences, Hainan University, Haikou 510006, China
2
*
Correspondence: zhangch328@mail.sysu.edu.cn (C.Z.); huangl72@mail.sysu.edu.cn (L.H.)
Abstract: Phosphodiesterase 2 (PDE2) has been regarded as a novel target for the treatment of
Alzheimer’s disease (AD). In this study, we obtained ( )-LZ77 as a hit compound with moder-
R
ate PDE2 inhibitory activity (IC50 = 261.3 nM) using a high-throughput virtual screening method
based on molecular dynamics. Then, we designed and synthesized 28 dihydropyranopyrazole
derivatives as PDE2 inhibitors. Among them, compound (
hibitor, with an IC50 value of 41.5 nM. The molecular docking of PDE2-(
+
)-11h was the most potent PDE2 in-
)-11h reveals that the
+
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
4-(trifluoromethyl)benzyl)oxyl side chain of the compound enters the H-pocket and forms strong
hydrophobic interactions with L770/L809/F862, which improves inhibitory activity. The above
results may provide insight for further structural optimization of highly potent PDE2 inhibitors and
may lay the foundation for their use in the treatment of AD.
Citation: Zhou, Y.; Li, J.; Yuan, H.;
Su, R.; Huang, Y.; Huang, Y.; Li, Z.;
Wu, Y.; Luo, H.; Zhang, C.; et al.
Design, Synthesis, and Evaluation of
Dihydropyranopyrazole Derivatives
as Novel PDE2 Inhibitors for the
Treatment of Alzheimer’s Disease.
Molecules 2021, 26, 3034. https://
doi.org/10.3390/molecules26103034
Keywords: dihydropyranopyrazole derivatives; PDE2 inhibitors; Alzheimer’s disease
1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disease with clinical
manifestations that include memory loss; cognitive dysfunction; disorientation; and loss
Academic Editor: Andrea Trabocchi
of language, writing, and logical reasoning skills [1,2]. The pathogenesis of Alzheimer’s
disease is complex and involves environmental and genetic factors, which, to date, have
not been fully clarified. In the past few decades, researchers have proposed several
Received: 23 April 2021
Accepted: 14 May 2021
Published: 19 May 2021
hypotheses, including the cholinergic hypothesis [
3], amyloid cascade hypothesis [4], and
inflammation hypothesis [ ]. Currently, two types of drugs are approved by the Food
5
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
and Drug Administration (FDA) for the treatment of AD: cholinesterase inhibitors (ChEI)
and N-methyl-D-aspartate (NMDA) receptor antagonists, such as donepezil, galantamine,
rivastigmine, and memantine, which can improve memory and cognitive function to a
certain extent but cannot definitively cure this disease [6]. Therefore, developing safe and
effective drugs for the treatment of AD is of great significance.
Phosphodiesterases (PDEs) are a group of enzymes that regulate cell signaling and
synaptic transmission by hydrolyzing the phosphodiester bonds of cGMP and cAMP, which
Copyright:
© 2021 by the authors.
act as second messengers [
play an important role in neuroprotection and the enhancement of neuroplasticity [8–10]
PDE2, which is a dual-substrate enzyme that hydrolyzes both cAMP and cGMP, has re-
ceived extensive attention due to its high levels of expression in the forebrain, especially in
the hippocampus and cortex, and in other parts of the brain [11]. Recent studies clearly
indicate that PDE2 inhibitors can regulate neuronal plasticity, reduce tau phosphorylation
7]. A large number of studies have indicated that PDE inhibitors
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
.
Molecules 2021, 26, 3034. https://doi.org/10.3390/molecules26103034
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Molecules 2021, 26, 3034
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and beta amyloid (A
β) aggregation, promote inflammatory cytokines and apoptosis, in-
crease the levels of Brain-Derived Neurotrophic Factor (BDNF) and cAMP/cGMP, and
improve cognitive impairment [11
target for the treatment of AD.
–14]. Therefore, PDE2 has been regarded as a novel
Although PDE2 was discovered some time ago, research on PDE2 inhibitors only
started in the 2000s. The determination of the crystal structure of PDE2 facilitated the
development of PDE2 inhibitors (Figure 1) [15,16]. EHNA was first discovered as an in-
hibitor of PDE2 [17]. Its application was restricted to its adenosine deaminase inhibitory
activity [11]. Then, studies clearly indicated that BAY 60-7550, the first selective PDE2
inhibitor reported by Bayer in 2002, could reverse functional impairments by decreasing
hippocampal neurodegeneration and by enhancing hippocampal neuronal plasticity. How-
ever, the development of BAY 60-7550 was terminated due to its shortcomings in solubility
and pharmacokinetics [18
,19]. PF-05180999, another PDE2 inhibitor, was developed by
Pfizer and entered the first phase of clinical trials as a treatment for schizophrenia and
migraine, but there have been no further reports [
PDE2 inhibitors is of great significance.
7]. Thus, the development of selective
Figure 1. Chemical structures of several reported PDE2 inhibitors.
In previous research, we developed an efficient virtual screening method based
on molecular dynamics and successfully applied it to discover highly potent PDE10 in-
hibitors [20,21]. In this study, we used this method to obtain (R)-LZ77 as a hit compound
with moderate PDE2 inhibitory activity (IC50 = 261.3 nM) through high-throughput screen-
ing. In order to improve its activity and selectivity, we designed and synthesized a series
of dihydropyranopyrazole derivatives based on the structure of (R)-LZ77, aiming to obtain
compounds with better inhibitory activity against PDE2. Then, we evaluated the inhibitory
activity of target compounds against PDE2 and their selectivity relative to other PDEs.
Furthermore, molecular docking studies and decomposition of binding free energies were
also performed to explore the binding modes of PDE2 and inhibitors.
According to the molecular docking of (
that (1) the hit compound ( )-LZ77 forms stacking interactions with F862 through the
benzene ring in the middle and forms a hydrogen bond with Q859 through the cyano group
on the pyranopyrazole; (2) the oxygen atom on the ( )-LZ77 pyranopyrazole skeleton
R)-LZ77 and PDE2 (Figure 2), we found
R
π–π
R
forms a hydrogen bond with Y655 through crystallization water; and (3) the benzyl side
chain extends outside of the pocket without entering the hydrophobic pocket (H pocket)
formed by L770/H773/T805/L809/F862/I870. Therefore, if a side chain can be introduced
to (
R)-LZ77 and simultaneously occupy the H pocket, it can significantly improve the
inhibitory activity against PDE2 and the selectivity relative to other PDEs.

Molecules 2021, 26, 3034
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Figure 2. Structure and putative binding mode of (
R
)-LZ77 with PDE2 according to molecular
docking studies and the design strategy for PDE2 inhibitors.
In this research, in order to improve activity and selectivity, first, the position and
type of substituents on the benzene ring were changed. Second, the position and length
of the side chains on the benzene ring connected to the pyran ring were changed to bind
to the H pocket in the PDE2 catalytic pocket, thereby increasing the activity. A total
of 28 compounds were designed and synthesized to evaluate their effects on inhibitory
activity against PDE2, aiming to provide insights for the subsequent design of highly
potent and highly selective PDE2 inhibitors.
2. Results
2.1. Chemistry
The synthesis of target compounds 4a
of potassium carbonate, 2-hydroxybenzaldehyde with different substituents reacts with
benzyl chloride for O-alkylation reaction to obtain compounds 3a . Then, compounds
4a are obtained through the ring-closure reaction of aromatic aldehyde, malononitrile,
–c is outlined in Scheme 1. In the presence
–
c
–
c
and pyrazolinone catalyzed by a three-component base, which involves a tandem Michael
addition/Thorpe–Ziegler-type reaction to generate the dihydropyrano[2,3-c] pyrazole
core [22].
Scheme 1. Reagents and conditions: (
lamine, 3-methyl-5-pyrazolone, ethanol, 80 C, 15 min.
a
) K2CO3, acetonitrile, 80 ◦C, 5 h; (
b
) malononitrile triethy-
◦
The synthesis of target compounds 8a–d and 11a–t is illustrated in Scheme 2. In the
presence of HATU and DIPEA, 2-hydroxy-5-methoxybenzaldehyde is condensed with
acids that substitute different chain lengths and different substituents at room temperature

Molecules 2021, 26, 3034
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to obtain a series of intermediates (7). Then, the series of compounds 7a
–d
and malononi-
trile, N-methylmorpholine and 3-methyl-5-pyrazolone are ring-closed in ethanol at room
temperature to obtain the target compounds 8a . The synthesis of target compounds
11a is similar to the synthetic method of Scheme 1, except that the substitution position
of the hydroxyl group on the aromatic aldehyde is changed from 3 to 2.
–
d
–
t
Scheme 2. Reagents and conditions: (a) HATU, DIPEA, anhydrous CH2Cl2, room temperature,
overnight; ( ) malononitrile, 3-methyl-5-pyrazolone, N-methylmorpholine, ethanol, room tempera-
b
ture, overnight; (
c
) K2CO3, acetonitrile, 80 ◦C, 5 h; (
d
) malononitrile, triethylamine, pyrazolinone,
◦
ethanol, 80 C, 15 min.
Considering the effect of chiral structure on the biological activity of compounds, the
[α]2D0
chiral isomers ( )-11h 46.7 (c 1.0, THF), tR = 24.847 min, ee value, >99%) and
)-11h
[α]2D0 = +46.5 (c 1.0, THF), tR = 33.976 min, ee value, >99%) were prepared by the
separation of ( -11h with chiral HPLC (HPLC chromatogram of ( )-11h, )-11h and
(+)-11h are included in the Supplementary Materials).
The synthesis of intermediate 9o is shown in Scheme 3. In the presence of tetrahydroa-
−
(
=
−
(
+
(
±
)
±
(
−
luminum lithium, the carboxyl group in 12 is reduced to a hydroxyl group to obtain 13
,
and then, triphosgene is used for chlorination to obtain intermediate 9o.

Molecules 2021, 26, 3034
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◦
Scheme 3. Reagents and conditions: (
a
) LiAlH4, THF, 85 C, 6 h; (
b
) triphosgene, triethylamine,
CH2Cl2, room temperature, 6 h.
The synthesis of target compound 16 is summarized in Scheme 4. In the presence of
DMAP, 11h reacts with di-tert-butyl methyl dicarbonate to produce 14. Then, 14 reacts
with methyl iodide in the presence of potassium carbonate to obtain 15, which is converted
to 16 in the presence of trifluoroacetic acid.
Scheme 4. Reagents and conditions: (
K2CO3, CH3CN, 60 C, 5 h; (c) trifluoroacetic acid, DCM, room temperature, 1 h.
a
) DMAP, (Boc)2O, THF, room temperature, 8 h; (
b) CH3I,
◦
2.2. Inhibitory Activity of PDE2 Inhibitors
In vitro inhibitory potencies against PDE2 of the hit compound and its synthesized
derivatives were determined using EHNA (IC50 = 2460 nM), which was purchased from
SIGMA and used as the reference compound. The inhibitory activities of target compounds
against PDE2 are summarized in Table 1. Most of the target compounds demonstrated
moderate to high inhibitory activity against PDE2. Among them, compound (+)-11h was
the most potent PDE2 inhibitor, with an IC50 value of 41.5 nM. We summarize the structure–
activity relationship as follows: (1) the activity of the compound with a substituent at
the 5-position of the benzene ring connected to the pyran ring (11f, 79.2% inhibition at
0.5
the 5-position substituents, the methoxyl group (11h, IC50 = 75.5 nM) is more active
than methyl (11k, IC50 = 153.5 nM), trifluoromethoxyl (11p, 17.4% inhibition at 0.5 M),
difluoromethoxyl (11q, 6.6% inhibition at 0.5 M), hydroxyl (11m, no inhibition at 0.5 M),
and tert-butyl (11j, no inhibition at 0.5 M). (3) The side-chain substitution at position 2 has
a better impact on inhibitory activity (11f, 79.2% inhibition at 0.5 M) than that at position
3 (4c, 43.4% inhibition at 0.5 M). (4) The trifluoromethyl substituent (11h, IC50 = 75.5 nM)
on the benzene ring on the side chain is more active than chlorine (11g IC50 = 807.5 nM),
trifluoromethoxyl (11i, 48.8% inhibition at 0.5 M), fluorine (11j IC50 = 807.5 nM), and
methoxyl (11e, IC50 = 458.4 nM). (5) When the amino group on the pyran ring (11h
µM) is higher than that at the 4-position (11c, 29.0% inhibition at 0.5 µM). (2) Among
µ
µ
µ
µ
µ
µ
,
µ
,
,

Molecules 2021, 26, 3034
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IC50 = 75.5 nM) is replaced by a dimethylamino group (16, 9.6% inhibition at 0.5
µM), the
activity decreases. (6) When the side chain is connected by an ester bond and the length of
the ester bond is extended, the activity of these compounds is not improved (Figure 3).
After these analyses, the best compound 11h was resolved: (
tivity, while ( )-11h was almost inactive. Notably, the most potent compound (
IC50 = 41.5 nM) was more potent than the hit compound ( )-LZ77 (IC50 = 261.3 nM). Thus,
+
)-11h had better ac-
−
+)-11h
(
R
(+)-11h was identified as a potent PDE2 inhibitor for subsequent study.
Table 1. Inhibitory activities of synthesized compounds against PDE2.
Inhibition Ratio (%)
IC50 (nM) a
Comp.
n
R1
R2
R3
R4
R5
0.5 µM
0.1 µM
(R)-LZ77
4a (LZ77)
4b
1
1
1
1
0
0
2
3
1
1
1
1
1
1
1
1
1
1
1
1
1
OCH3
OCH3
H
H
H
H
H
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
CF3
Cl
OCF3
CF3
Cl
Cl
OCH3
H
Cl
H
OCH3
Cl
F
CF3
CF3
CF3
OCF3
CF3
CF3
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
67.5
57.2
14.1
43.4
13.4
39.1
4.12
6.12
27.0
14.3
29.0
17.7
48.0
79.2
44.4
87.3
87.3
93.6
48.8
0
30.4
18.2
-
-
-
-
-
-
261.3
407.2
±
±
-
-
-
-
-
-
-
-
-
-
±
11.2
37.7
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
4c
8a
8b
8c
8d
11a
11b
11c
11d
11e
11f
11g
-
-
-
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
C(CH3)3
CH3
-
17.5
48.1
15.1
63.3
-
75.9
-
458.4
16.2
115.4 ± 1.3
807.5
±
15.3
11h
75.5 ± 2.0
(−)-11h
(+)-11h
11i
11j
11k
-
41.5 ± 3.4
H
H
H
-
-
-
72.0
38.3
153.5 ± 3.3
11l
1
H
CH3
CF3
NH2
13.4
-
-
11m
11n
11o
11p
11q
11r
11s
11t
1
2
3
1
1
1
1
1
1
H
H
H
H
H
H
H
Cl
H
OH
OCH3
OCH3
OCF3
OCHF2
OCH3
OCH3
OCH3
OCH3
CH3
CH3
CH3
CH3
CH3
H
C6H5
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N(CH3)2
0
20.7
8.2
17.4
6.6
60.5
75.3
10.3
9.6
-
-
-
-
-
-
-
-
-
22.7
46.3
-
-
445.3 ± 3.4
153.7 ± 6.2
-
-
16
-
a
Values are presented as the means ± SD of three experiments.

Molecules 2021, 26, 3034
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Figure 3. Summarized SARs of synthesized compounds.
2.3. Remarkable Selectivity of Compound (+)-11h against PDE2 Compared with Other PDEs
The selectivity profile of compound ( )-11h against PDE2 compared with other PDE
+
families was also evaluated, and the results are listed in Table 2. Its inhibition of PDE4D2
and PDE8A1 was very weak (IC50 > 10,000 nM). Its IC50 value against PDE1C was 289-
fold higher than that against PDE2A. In short, (
compared with several other PDEs.
+)-11h has high selectivity against PDE2
Table 2. Selectivity index of (+)-11h against PDE2 compared with several other PDE Families.
PDEs (Catalytic Domain)
IC50 (nM)
Selectivity Index
PDE2A (580–919)
PDE1C (2–634)
PDE4D2 (86–413)
PDE8A1(480–820)
30.31 ± 3.4
8764 ± 667.5
>10,000
-
289
>1149
>1149
>10,000
2.4. Molecular Docking
Similar to the results of the above activity analyses, the docking results of (
+)-11h
with PDE2 (Figure 4A) show that in compound ( )-11h, the oxygen atom of the 5-position
+
methoxyl group on the benzene ring connected to the pyran ring can form a hydrogen
bond with Y827, which explains its improved activity compared with methyl substi-
tution. However, bulky residues such as Y827 and M847 may cause steric hindrance
at this position, so when the methyl group is replaced by tert-butyl and other larger
groups, the activity of the compound decreases. The side chain at position 2 on the
benzene ring can enter the H pocket and form stronger hydrophobic interactions with
L770/H773/T805/L809/F862/I870, thus enhancing the inhibitory activity. Moreover, the
size of the H pocket is just large enough to accommodate the benzyl side chain with CF3 at
position 4. Either an increase or decrease in the size of the side chain length reduces the
activity of the compound. The volume of Cl and F is smaller than that of CF3 and it does
not completely occupy the H pocket, so the activity is not as high as it is with CF3. OCF3
has an extra oxygen atom, which increases its volume, so its activity also decreases. The
superimposed diagram of the docking results of (R)-LZ77 and (+)-11h (Figure 4B) clearly
shows that the side chain at position 2 on the benzene ring connected to the pyran ring
enters the H pocket but that the side chain at position 3 does not. In addition, after changing
the chirality of the compound, the molecular docking pose (Figure 4C) suggests that the
pyranopyrazole skeleton moves away from the pocket and loses its ability to interact with
F862 and Q859, so the inhibitory activity of ( )-11h against PDE2 weakens or disappears.
−

Molecules 2021, 26, 3034
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Figure 4.
) Putative binding mode of compound (
studies. ( ) Putative binding mode of compound (
) Decomposition of important residue contributions to the total binding free energies for (R
(
A
) Putative binding mode of compound (+)-11h with PDE2 in molecular docking studies.
(
B
+
)-11h and (
)-11h with PDE2 in molecular docking studies.
)-LZ77
R)-LZ77 with PDE2 in molecular docking
C
−
(D
and (+)-11h with PDE2.
2.5. Decomposition of Binding Free Energies
The decomposition of binding free energies was used to explore the contribution
of each residue in the protein to the binding of the inhibitor. Eight-nanosecond (8 ns)
molecular dynamics simulations were performed for complexes of ((
(( )-11h)–PDE2, and 100 snapshots extracted from trajectories in the last 1 ns were used to
calculate the MM-PBSA binding free energies and to perform energy decomposition. The
predicted binding free energies were 27.30 and 30.02 kcal/mol for (( )-LZ77)–PDE2
and (( )-11h)–PDE2, respectively. In general, residues that possess interaction energies
R)-LZ77)–PDE2 and
+
−
−
R
+
lower than -1 kcal/mol are considered important for the recognition of ligands. The energy
decomposition results further suggest that such residues mainly contribute to the binding
of (
2.22 kcal/mol), Q812 (
I866 ( 1.11 and 1.30 kcal/mol), F830 (
1.05 kcal/mol). Among these residues, F862, L770, and Q859 contribute most to the total
binding free energies. This is in agreement with the binding patterns, in which both ( )-
LZ77 and ( )-11h form remarkable hydrophobic interactions with F862/L770 and direct
R
)-LZ77 and (
+
)-11h with PDE2: F862 (
1.14 and 1.67 kcal/mol), Q859 (
1.45 and 1.22 kcal/mol), and M847 (
−
3.45 and
−
4.42 kcal/mol), L770 (
1.82 and 1.57 kcal/mol),
1.77 and
−
2.58 and
−
−
−
−
−
−
−
−
−
−
−
R
+
hydrogen bonds with Q859 through their dihydropyranopyrazole scaffolds. L809 and F862
had major differences in their energy contributions: their binding free energy contributions
to ((+)-11h)–PDE2 were 1.46 and 0.97 kcal/mol lower than those to ((R)-LZ77)–PDE2. Since
L770, L809, and F862 are involved in the composition of the unique H pocket, the enhanced
interaction with L809 and F862 may account for the improved PDE2 inhibition by (+)-11h
relative to (R)-LZ77 (Figure 4D).
3. Materials and Methods
3.1. Chemistry
Unless otherwise specified, all compounds were purchased from commercial sources
(Bide Chemical Co., Ltd., Xiangtan, Hunan, China; Sigma-Aldrich, St. Louis, MO, USA;

Molecules 2021, 26, 3034
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1
and J&K Chemical Ltd., Beijing, China), and no further purification was required. H-NMR
and 13C-NMR spectra were recorded at room temperature on a Bruker AVANCE 400 or
500 spectrometer (Karlsruhe, Germany) with TMS as an internal reference. Peaks were
labeled as single (s), doublet (d), two doublets (dd), doublet of triplets (dt), triplet (t), or
multiplet (m). A Shimadzu LCMS-IT-TOF high-resolution mass spectrometer (Kyoto, Japan)
was used for molecular weight confirmation. The purity of compounds was confirmed to be
over 95% and was determined by reversed-phase high performance liquid chromatography
(HPLC) with a TC-C18 column (250 mm
×
4.6 mm, 5 µm) using acetonitrile/water (70%,
v/v) as the mobile phase (1.0 mL/min) at 25 ◦C.
3.1.1. General Procedure for Synthesis of Intermediate Compounds 3a–c
Potassium carbonate (1.5 mmol) was added to a solution of aldehyde (1.0 mmol)
with different substituents and benzyl chloride (1.0 mmol) with different substituents or
1-(2-chloroethyl)-4-trifluoromethylbenzene (1.0 mmol) in acetonitrile (15 mL). The mixture
was heated to 80 ◦C for 5 h. After the reaction was complete and the solvent was spin-
dried under reduced pressure, the mixture was diluted with water (20 mL) and extracted
with ethyl acetate (60 mL), and the organic layer was separated and dried over anhydrous
sodium sulfate. After the evaporation of solvent under reduced pressure, the crude product
was purified on a silica gel column to produce compounds 3a–c as a white solid.
3-((4-Chlorobenzyl)oxy)-4-methoxybenzaldehyde (3a). White solid. Yield, 80%. 1H-NMR
(
400 MHz, CDCl3)
δ 9.82 (s, 1H), 7.48 (dd, J = 8.2, 1.9 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.40
(d, J = 8.6 Hz, 2H), 7.37–7.33 (m, 2H), 7.00 (d, J = 8.2 Hz, 1H), 5.15 (s, 2H), 3.97 (s, 3H).
1
3-Methoxy-5-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (3b). White solid. Yield, 84%. H-
NMR (400 MHz, CDCl3) 9.93 (s, 1H), 7.68 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H),
δ
7.12–7.06 (m, 2H), 6.80 (t, J = 2.3 Hz, 1H), 5.19 (s, 2H), 3.88 (s, 3H).
3-((4-Chlorobenzyl)oxy)-5-methoxybenzaldehyde (3c). White solid. Yield, 77%. 1H-NMR
(400 MHz, CDCl3)
δ 9.93 (s, 1H), 7.39 (s, 4H), 7.12–7.03 (m, 2H), 6.79 (t, J = 2.3 Hz, 1H), 5.09
(s, 2H), 3.87 (s, 3H).
3.1.2. General Procedure for Synthesis of Compounds 4a–c
Aldehyde (1.0 mmol), malononitrile (1.0 mmol), and triethylamine (1.0 mmol) were
added to ethanol (10 ml) and reacted at 85 ◦C for 5 min, and then, pyrazolone was added
and reacted at the same temperature for 15 min. After completion, the solid was filtered
out, washed with ethanol, and dried in a vacuum drying oven to obtain target compounds
4a–c.
6-Amino-4-(3-((4-chlorobenzyl)oxy)-4-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-
1
5-carbonitrile (4a). White solid. Yield, 61%. H-NMR (400 MHz, DMSO-d6)
δ
12.03 (s, 1H),
7.42 (s, 4H), 6.92 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.80 (s, 2H), 6.72 (dd, J = 8.3, 2.0 Hz,
1H), 5.06–4.98 (m, 2H), 4.50 (s, 1H), 3.75 (s, 3H), 1.69 (s, 3H). 13C-NMR (126 MHz, DMSO-
d6)
δ 161.23, 155.15, 148.51, 147.58, 137.30, 136.61, 136.04, 132.85, 130.13, 128.81, 121.32,
120.70, 113.94, 112.56, 98.12, 69.51, 57.82, 56.03, 36.25, and 10.13. HRMS (ESI) calculated for
C22H19ClN4O3 [M + H]+: 423.1218, found: 423.1222. Purity: 99.66% (by HPLC).
6-Amino-4-(3-methoxy-5-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (4b). White solid. Yield, 42%. H-NMR (400 MHz, DMSO-d6)
δ
12.08 (s, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 6.86 (s, 2H), 6.50 (t, J = 2.1 Hz,
1H), 6.40 (s, 1H), 6.35 (s, 1H), 5.18 (s, 2H), 4.53 (s, 1H), 3.71 (s, 3H), 1.78 (s, 3H). 13C-NMR
(126 MHz, DMSO-d6)
δ 161.47, 160.91, 159.65, 155.17, 147.54, 142.36, 136.08, 128.74 (q,
J = 31.5 Hz), 128.55, 125.75(q, J = 4.2 Hz), 124.72 (q, J = 272.6 Hz), 121.24, 106.88, 106.70,
99.54, 97.72, 68.75, 57.23, 55.62, 36.79 and 10.21. HRMS (ESI) calculated for C23H19F3N4O3
[M + H]+: 457.1482, found: 457.1501. Purity: 99.86% (by HPLC).
6-Amino-4-(3-((4-chlorobenzyl)oxy)-5-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-
1
5-carbonitrile (4c). White solid. Yield, 52%. H-NMR (400 MHz, DMSO-d6)
δ
12.08 (s, 1H),

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7.48–7.41 (m, 4H), 6.86 (s, 2H), 6.47 (t, J = 2.1 Hz, 1H), 6.36 (d, J = 10.9 Hz, 2H), 5.05
(s, 2H), 4.53 (s, 1H), 3.71 (s, 3H), 1.80 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.47,
160.88, 159.78, 155.18, 147.48, 136.49, 136.09, 132.85, 130.06, 128.86, 121.25, 106.87, 106.61,
99.49, 97.73, 68.82, 57.25, 55.61, 36.78 and 10.26. HRMS (ESI) calculated for C22H19ClN4O3
[M + H]+: 423.1225, found: 423.1218. Purity: 99.79% (by HPLC).
3.1.3. General Procedure for Synthesis of Intermediate Compounds 7a–d
The acid (1.0 mmol), HATU (1.0 mmol), and DIPEA (2.0 mmol) were added to anhy-
drous methylene chloride (10 mL) to react for 1 h, and then, aldehyde (1.1 mmol) was added
to react overnight at room temperature. After the reaction was complete, water (30 mL)
was added for extraction, and the organic layer was separated and dried with anhydrous
sodium sulfate. After evaporation of the solvent under reduced pressure, the crude product
was purified on a silica gel column to produce compounds 7a–d as a white solid.
2-Formyl-4-methoxyphenyl-4-(trifluoromethoxy)benzoate (7a). White solid. Yield, 60%. 1H-
NMR (400 MHz, CDCl3)
δ 10.14 (s, 1H), 8.30–8.25 (m, 2H), 7.42 (t, J = 1.6 Hz, 1H), 7.37 (d,
J = 8.2 Hz, 2H), 7.22 (d, J = 1.7 Hz, 2H), 3.89 (s, 3H).
1
2-Formyl-4-methoxyphenyl-4-(trifluoromethyl)benzoate (7b). White solid. Yield, 61%. H-NMR
(400 MHz, CDCl3) 10.15 (s, 1H), 8.37 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H), 7.46–7.43
(m, 1H), 7.27–7.24 (m, 2H), 3.92 (s, 3H).
δ
1
2-Formyl-4-methoxyphenyl-3-(4-chlorophenyl)propanoate (7c). White solid. Yield, 50%. H-
NMR (400 MHz, CDCl3) 9.93 (s, 1H), 7.33 (d, J = 3.1 Hz, 1H), 7.32–7.27 (m, 2H), 7.20 (d,
δ
J = 8.4 Hz, 2H), 7.14 (dd, J = 8.9, 3.2 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 3.85 (s, 3H), 3.07 (t,
J = 7.4 Hz, 2H), 2.95 (t, J = 7.4 Hz, 2H).
2-Formyl-4-methoxyphenyl-4-(4-chlorophenyl)butanoate (7d). White solid. Yield, 47%. 1H-
NMR (400 MHz, CDCl3)
δ 10.05 (s, 1H), 7.35 (d, J = 3.1 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H),
7.15 (dt, J = 6.9, 3.6 Hz, 3H), 7.05 (d, J = 8.9 Hz, 1H), 3.85 (s, 3H), 2.73 (t, J = 7.6 Hz, 2H), 2.65
(t, J = 7.4 Hz, 2H), 2.13–2.02 (m, 2H).
3.1.4. General Procedure for Synthesis of Compounds 8a–d
Aldehyde (1.0 mmol), malononitrile (1.0 mmol), and N-methylmorpholine (1.0 mmol)
were added to ethanol (10 mL) and reacted at room temperature for 5 min, and then,
pyrazolone was added and reacted at the same temperature overnight. After completion,
the solvent was spin-dried under reduced pressure; then, the mixture was diluted with
water (20 mL) and extracted with dichloromethane (60 mL), and the organic layer was
separated and dried over anhydrous sodium sulfate. After evaporation of the solvent
under reduced pressure, the crude product was purified on a silica gel column to yield
compounds 8a–d.
2-(6-Amino-5-cyano-3-methyl-1,4-dihydropyrano[2,3-c]pyrazol-4-yl)-4-methoxyphenyl-4-(trifluoromethoxy)
1
benzoate (8a). White solid. Yield, 47%. H-NMR (400 MHz, DMSO-d6)
δ
11.98 (s, 1H), 8.09
(d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.8 Hz, 1H), 6.93 (dd, J = 8.9, 3.0 Hz,
1H), 6.79 (s, 3H), 4.69 (s, 1H), 3.76 (s, 3H), 1.78 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ
163.69, 161.25, 157.41, 155.13, 152.47, 142.35, 136.54, 136.13, 132.53, 128.20, 124.93, 121.12,
121.01, 120.39 (q, J = 258.3 Hz), 113.15, 96.36, 56.30, 55.89 and 10.09. HRMS (ESI) calculated
for C23H17F3N4O5 [M + H]+: 487.1224, found: 487.1240. Purity: 99.38% (by HPLC).
2-(6-Amino-5-cyano-3-methyl-1,4-dihydropyrano[2,3-c]pyrazol-4-yl)-4-methoxyphenyl-4-(trifluoromethyl)
1
benzoate (8b). White solid. Yield, 47%. H-NMR (400 MHz, DMSO-d6)
δ 11.97 (s, 1H), 8.13
(d, J = 7.3 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 9.0 Hz, 1H), 6.94 (dd, J = 8.8, 2.9 Hz,
1H), 6.87 (s, 1H), 6.80 (s, 2H), 4.69 (s, 1H), 3.77 (s, 3H), 1.78 (s, 3H). 13C-NMR (126 MHz,
DMSO-d6)
δ 163.68, 161.22, 157.41, 155.02, 142.38, 136.19, 133.54 (q, J = 31.9 Hz), 132.92,
130.85, 126.11 (q, J = 2.5 Hz), 125.03, 124.24 (q, J = 273.0 Hz), 121.03, 115.92, 113.18, 96.27,
56.21, 55.90 and 10.09. HRMS (ESI) calculated for C23H17F3N4O4[M + H]+: 471.1275, found:
471.1267. Purity: 96.99% (by HPLC).

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2-(6-Amino-5-cyano-3-methyl-1,4-dihydropyrano[2,3-c]pyrazol-4-yl)-4-methoxyphenyl-3-(4-chlorophenyl)
1
propanoate (8c). White solid. Yield, 31%. H-NMR (400 MHz, DMSO-d6)
δ 12.04 (s, 1H),
7.32 (q, J = 8.5 Hz, 4H), 6.87–6.85 (m, 2H), 6.85 (s, 2H), 6.78 (s, 1H), 4.51 (s, 1H), 3.73 (s, 3H),
2.87–2.64 (m, 4H), 1.73 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ
171.16, 161.33, 157.07,
155.33, 142.33, 139.79, 136.36, 136.27, 131.28, 130.69, 128.75, 124.83, 120.95, 115.71, 112.90,
96.66, 56.40, 55.83, 34.90, 29.83 and 10.04. HRMS (ESI) calculated for C24H21ClN4O4[M +
H]+: 465.1324, found: 465.1328. Purity: 99.55% (by HPLC).
2-(6-Amino-5-cyano-3-methyl-1,4-dihydropyrano[2,3-c]pyrazol-4-yl)-4-methoxyphenyl-4-(4-chlorophenyl)
1
butanoate (8d). White solid. Yield, 33%. H-NMR (400 MHz, DMSO-d6)
δ 12.03 (s, 1H), 7.34
(d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 8.8 Hz, 1H), 6.88–6.83 (m, 3H), 6.78
(s, 1H), 4.62 (s, 1H), 3.73 (s, 3H), 2.62 (t, J = 7.8 Hz, 2H), 2.46–2.30 (m, 2H), 1.87–1.77 (m,
2H), 1.75 (s, 3H).13C-NMR (101 MHz, DMSO-d6)
δ 171.74, 161.37, 157.05, 155.30, 142.37,
140.95, 136.39, 136.22, 130.95, 130.68, 128.72, 124.92, 120.97, 115.64, 112.89, 96.68, 56.31, 55.82,
34.08, 32.92, 26.24 and 10.05. HRMS (ESI) calculated for C25H23ClN4O4 [M + H]+: 479.1481,
found: 479.1472. Purity: 99.09% (by HPLC).
3.1.5. General Procedure for Synthesis of Intermediate Compounds 10a–q and 10t
The synthesis method was the same as that used for 3a–c.
1
4-Methoxy-2-((4-methoxybenzyl)oxy)benzaldehyde (10a). White solid. Yield, 82%. H-NMR
(400 MHz, DMSO)
δ 10.20 (s, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.44 (d, J = 8.6 Hz, 2H), 6.99–6.95
(m, 2H), 6.81 (d, J = 2.2 Hz, 1H), 6.65 (dd, J = 8.7, 1.8 Hz, 1H), 5.20 (s, 2H), 3.86 (s, 3H), 3.76
(s, 3H).
1
2-(Benzyloxy)-4-methoxybenzaldehyde (10b). White solid. Yield, 80%. H-NMR (400 MHz,
CDCl3) δ 10.41 (s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.48–7.37 (m, 5H), 6.59 (dd, J = 8.7, 1.8 Hz,
1H), 6.54 (d, J = 2.2 Hz, 1H), 5.19 (s, 2H), 3.88 (s, 3H).
2-((4-Chlorobenzyl)oxy)-4-methoxybenzaldehyde (10c). White solid. Yield, 83%. 1H-NMR
(
400 MHz, CDCl3)
δ 10.38 (s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.40 (s, 4H), 6.60 (dd, J = 8.7,
1.7 Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H), 5.15 (s, 2H), 3.88 (s, 3H).
1
2-(Benzyloxy)-5-methoxybenzaldehyde (10d). White solid. Yield, 86%. H-NMR (400 MHz,
CDCl3) 10.53 (s, 1H), 7.47–7.34 (m, 6H), 7.14 (dd, J = 9.0, 3.3 Hz, 1H), 7.03 (d, J = 9.1 Hz,
δ
1H), 5.18 (s, 2H), 3.83 (s, 3H).
5-Methoxy-2-((4-methoxybenzyl)oxy)benzaldehyde (10e). White solid. Yield, 79%.1H-NMR
(400 MHz, DMSO)
δ 10.33 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 9.1 Hz, 1H), 7.25 (dd,
J = 9.1, 3.2 Hz, 1H), 7.17 (d, J = 3.1 Hz, 1H), 6.97–6.93 (m, 2H), 5.16 (s, 2H), 3.76 (s, 3H), 3.75
(s, 3H).
2-((4-Chlorobenzyl)oxy)-5-methoxybenzaldehyde (10f). White solid. Yield, 81%. 1H-NMR
(
400 MHz, CDCl3) δ 10.51 (s, 1H), 7.42–7.38 (m, 4H), 7.37 (d, J = 3.2 Hz, 1H), 7.14 (dd,
J = 9.0, 3.2 Hz, 1H), 6.99 (d, J = 9.1 Hz, 1H), 5.14 (s, 2H), 3.83 (s, 3H).
2-((4-Fluorobenzyl)oxy)-5-methoxybenzaldehyde (10g). White solid. Yield, 83%. 1H-NMR
(
400 MHz, CDCl3)
δ 10.50 (s, 1H), 7.45–7.39 (m, 2H), 7.37 (d, J = 3.2 Hz, 1H), 7.14 (dd,
J = 8.3, 2.5 Hz, 1H), 7.12–7.08 (m, 2H), 7.01 (d, J = 9.1 Hz, 1H), 5.13 (s, 2H), 3.83 (s, 3H).
1
5-Methoxy-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10h). White solid. Yield, 82%. H-
NMR (400 MHz, CDCl3) 10.55 (s, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H),
δ
7.39 (d, J = 3.2 Hz, 1H), 7.14 (dd, J = 9.0, 3.2 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 5.24 (s, 2H),
3.84 (s, 3H).
5-Methoxy-2-((4-(trifluoromethoxy)benzyl)oxy)benzaldehyde (10i). White solid. Yield, 76%.
1H-NMR (400 MHz, CDCl3)
δ 10.50 (s, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 3.2 Hz, 1H),
7.25 (d, J = 7.4 Hz, 2H), 7.12 (dd, J = 9.0, 3.3 Hz, 1H), 6.98 (d, J = 9.1 Hz, 1H), 5.15 (s, 2H),
3.81 (s, 3H).

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5-(Tert-butyl)-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10j). White solid. Yield, 87%.
1H-NMR (400 MHz, CDCl3)
δ
10.59 (s, 1H), 7.92 (d, J = 2.6 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H),
7.62–7.56 (m, 3H), 6.97 (d, J = 8.7 Hz, 1H), 5.27 (s, 2H), 1.34 (s, 9H).
1
5-Methyl-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10k). White solid. Yield, 89%. H-
NMR (400 MHz, CDCl3) 10.56 (s, 1H), 7.69 (d, J = 7.8 Hz, 3H), 7.58 (d, J = 8.1 Hz, 2H),
7.36 (dd, J = 8.5, 2.1 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 5.25 (s, 2H), 2.35 (s, 3H).
δ
1
2,5-Bis((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10l). White solid. Yield, 79%. H-NMR
(400 MHz, CDCl3) 10.54 (s, 1H), 7.72–7.64 (m, 4H), 7.60–7.55 (m, 4H), 7.46 (d, J = 3.2 Hz,
δ
1H), 7.21 (dd, J = 9.0, 3.3 Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 5.24 (s, 2H), 5.14 (s, 2H).
5-Hydroxy-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10m). White solid. Yield, 56%.
1H-NMR (400 MHz, CDCl3)
δ 10.51 (s, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H),
7.37 (d, J = 3.2 Hz, 1H), 7.11 (dd, J = 8.9, 3.2 Hz, 1H), 6.96 (d, J = 8.9 Hz, 1H), 5.23 (s, 2H).
1
5-Methoxy-2-(4-(trifluoromethyl)phenethoxy)benzaldehyde (10n). White solid. Yield, 69%. H-
NMR (400 MHz, CDCl3) δ 10.40 (s, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.34
(d, J = 3.3 Hz, 1H), 7.13 (dd, J = 9.0, 3.3 Hz, 1H), 6.93 (d, J = 9.1 Hz, 1H), 4.30 (t, J = 6.5 Hz
,
2H), 3.82 (s, 3H), 3.22 (t, J = 6.5 Hz, 2H).
5-Methoxy-2-(3-(4-(trifluoromethyl)phenyl)propoxy)benzaldehyde (10o). White solid. Yield, 78%.
1H-NMR (400 MHz, CDCl3)
10.51 (s, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 3.3 Hz,
δ
1H), 7.34 (d, J = 8.0 Hz, 2H), 7.13 (dd, J = 9.0, 3.3 Hz, 1H), 6.92 (d, J = 9.1 Hz, 1H), 4.07 (t,
J = 6.1 Hz, 2H), 3.83 (s, 3H), 2.92 (t, J = 7.6 Hz, 2H), 2.25–2.14 (m, 2H).
5-(Trifluoromethoxy)-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10p). White solid. Yield,
1
87%. H-NMR (400 MHz, CDCl3)
δ 10.54 (s, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.71 (d, J = 8.2 Hz,
2H), 7.59 (d, J = 8.0 Hz, 2H), 7.42 (dd, J = 9.1, 2.6 Hz, 1H), 7.06 (d, J = 9.1 Hz, 1H), 5.29
(s, 2H).
5-(Difluoromethoxy)-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10q). Diethyl bromo diflu-
oromethylphosphonate (2 mmol) was added in one portion to a solution of 14 m (1 mmol)
and KOH (20 mmol) in CH3CN–H2O (10 mL, 1:1) at 0 ◦C, and the reaction mixture was
allowed to warm to room tempeature. After 20 min, the reaction mixture was diluted with
ethyl acetate (10 mL), and the organic phase was separated. Then, the water phase was
washed with another 10 mL of ethyl acetate, and the combined organic fractions were dried
over anhydrous Na2SO4. Evaporation of the solvent yielded a crude product that was
1
purified on a silica gel column to obtain compound 10q. White solid. Yield, 82%. H-NMR
(400 MHz, CDCl3) δ 10.36 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.49 (d,
J = 1.3 Hz, 1H), 7.27–7.23 (m, 2H), 6.62 (t, J = 72.9 Hz, 1H), 5.18 (s, 2H). 19F-NMR (376 MHz,
CDCl3) δ −62.65 (s), −81.41 (d, J = 72.9 Hz).
4-Chloro-5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde (10t). The synthesis method
1
was the same as that used for 3a
–c
. White solid. Yield, 67%. H-NMR (400 MHz, CDCl3)
δ
10.48 (s, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.44 (s, 1H), 7.14 (s, 1H), 5.22
(s, 2H), 3.94 (s, 3H).
3.1.6. General Procedure for Synthesis of Intermediate Compounds 11a–t
The synthesis method was the same as that used for 4a–c.
6-Amino-4-(4-methoxy-2-((4-methoxybenzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-
1
carbonitrile (11a). White solid. Yield, 41%. H-NMR (500 MHz, DMSO-d6)
δ
11.98 (s, 1H),
7.33 (d, J = 7.3 Hz, 2H), 6.93 (d, J = 7.6 Hz, 2H), 6.89 (d, J = 8.0 Hz, 1H), 6.72 (s, 2H), 6.63 (s,
1H), 6.48 (d, J = 8.3 Hz, 1H), 5.03 (q, J = 11.8 Hz, 2H), 4.89 (s, 1H), 3.75 (s, 3H), 3.72 (s, 3H),
1.74 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.59, 159.45, 159.33, 156.75, 155.56, 135.49,
130.07, 129.66, 129.41, 124.97, 121.47, 114.26, 105.98, 100.07, 98.41, 69.75, 57.35, 55.55, 55.51
and 10.05. HRMS (ESI) calculated for C23H22N4O4 [M + H]+: 419.1714, found: 419.1708.
Purity: 99.77% (by HPLC).

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6-Amino-4-(2-(benzyloxy)-4-methoxyphenyl)-3-methyl-1,4-dihydropyrano [2,3-c] pyrazole-5-carbonitrile
1
(
11b). White solid. Yield, 36%. H-NMR (500 MHz, DMSO-d6)
δ
11.99 (s, 1H), 7.44–7.35
(m, 4H), 7.31 (t, J = 6.8 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.74 (s, 2H), 6.62 (s, 1H), 6.49 (d,
J = 8.4 Hz, 1H), 5.12 (q, J = 12.6 Hz, 2H), 4.94 (s, 1H), 3.72 (s, 3H), 1.75 (s, 3H). 13C-NMR
(126 MHz, DMSO-d6) δ 161.59, 159.48, 156.68, 155.57, 137.60, 135.51, 130.15, 128.89, 128.15,
127.83, 124.97, 121.46, 106.07, 100.04, 98.40, 69.93, 57.36, 55.56 and 10.05. HRMS (ESI)
calculated for C22H20N4O3 [M + H]+: 389.1608, found: 389.1598. Purity: 99.01% (by HPLC).
6-Amino-4-(2-((4-chlorobenzyl)oxy)-4-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-
1
5-carbonitrile (11c). white solid. Yield, 52%. H-NMR (400 MHz, DMSO-d6)
δ 11.97 (s, 1H),
7.44 (m, J = 8.2 Hz, 4H), 6.93 (d, J = 8.3 Hz, 1H), 6.72 (s, 2H), 6.62 (s, 1H), 6.50 (d, J = 8.1 Hz,
1H), 5.11 (q, J = 12.6 Hz, 2H), 4.90 (s, 1H), 3.73 (s, 3H), 1.75 (s, 3H). 13C-NMR (126 MHz,
DMSO-d6)
δ 161.54, 159.52, 156.53, 155.57, 136.60, 135.49, 132.68, 130.27, 129.68, 128.84,
124.83, 121.47, 106.04, 100.12, 98.29, 69.02, 57.28, 55.57 and 10.05. HRMS (ESI) calculated for
C22H19ClN4O3 [M + H]+: 423.1218, found: 423.1219. Purity: 99.91% (by HPLC).
6-Amino-4-(2-(benzyloxy)-5-methoxyphenyl)-3-methyl-1,4-dihydropyrano [2,3-c] pyrazole-5-carbonitrile
1
(
11d). White solid. Yield, 44%. H-NMR (500 MHz, DMSO-d6)
δ
12.03 (s, 1H), 7.43–7.34 (m,
4H), 7.31 (t, J = 6.9 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.81 (s, 2H), 6.76 (d, J = 8.9 Hz, 1H), 6.53
(s, 1H), 5.05 (q, J = 12.3 Hz, 2H), 4.99 (s, 1H), 3.64 (s, 3H), 1.77 (s, 3H). 13C-NMR (126 MHz,
DMSO-d6)
δ 161.82, 155.52, 153.96, 150.05, 137.86, 135.62, 134.07, 128.85, 128.09, 127.84,
121.37, 115.61, 114.39, 112.34, 98.00, 70.69, 56.81, 55.69 and 10.04. HRMS (ESI) calculated for
C22H20N4O3 [M + H]+: 389.1608, found: 389.1601. Purity: 99.93% (by HPLC).
6-Amino-4-(5-methoxy-2-((4-methoxybenzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-
1
carbonitrile (11e). White solid. Yield, 48%. H-NMR (400 MHz, DMSO-d6)
δ
12.01 (s, 1H),
7.33 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.79 (s, 2H), 6.76
(dd, J = 8.9, 3.1 Hz, 1H), 6.52 (d, J = 2.9 Hz, 1H), 4.97 (m, 3H), 3.76 (s, 3H), 3.65 (s, 3H),
1.76 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.81, 159.30, 155.51, 153.89, 150.12, 135.61,
134.09, 129.69, 126.66, 121.37, 115.55, 114.53, 114.23, 112.32, 98.01, 70.54, 56.82, 55.68, 55.52
and 10.04. HRMS (ESI) calculated for C23H22N4O4 [M + H]+: 419.1714, found: 419.1723.
Purity: 99.35% (by HPLC).
6-Amino-4-(2-((4-chlorobenzyl)oxy)-5-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-
5-carbonitrile (11f). White solid. Yield, 48%. 1H-NMR (400 MHz, DMSO-d6)
δ 12.01 (s,
1H), 7.47–7.37 (m, 4H), 7.00 (d, J = 9.0 Hz, 1H), 6.80 (s, 2H), 6.77 (dd, J = 9.0, 3.1 Hz, 1H),
6.56 (d, J = 2.6 Hz, 1H), 5.04 (q, J = 12.4 Hz, 2H), 4.96 (s, 1H), 3.65 (s, 3H), 1.77 (s, 3H).
13C-NMR (126 MHz, DMSO-d6)
δ 161.78, 155.53, 153.97, 149.89, 136.86, 135.63, 133.95,
132.63, 129.69, 128.82, 121.39, 115.76, 114.42, 112.36, 97.91, 69.75, 56.76, 55.70 and 10.04.
HRMS (ESI) calculated for C22H19ClN4O3 [M + H]+: 423.1218, found: 423.1229. Purity:
99.91% (by HPLC).
6-Amino-4-(2-((4-fluorobenzyl)oxy)-5-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-
1
5-carbonitrile (11g). White solid. Yield, 54%. H-NMR (400 MHz, DMSO-d6)
δ 12.02 (s, 1H),
7.47–7.40 (m, 2H), 7.19 (t, J = 8.8 Hz, 2H), 7.02 (d, J = 8.9 Hz, 1H), 6.80–6.75 (m, 3H), 6.55 (d,
J = 2.8 Hz, 1H), 5.02 (q, J = 12.0 Hz, 2H), 4.96 (s, 1H), 3.65 (s, 3H), 1.76 (s, 3H). 13C-NMR
(101 MHz, DMSO-d6) δ 162.10 (d, J = 244.4 Hz), 161.78, 155.53, 153.96, 149.97, 135.62, 134.02,
134.00, 130.13, 130.05, 121.40, 115.70, 115.63 (d, J = 21.4 Hz), 114.46, 112.34, 97.93, 69.94,
56.78, 55.68 and 10.04. HRMS (ESI) calculated for C22H19FN4O3 [M + H]+: 407.1514, found:
407.1521. Purity: 99.85% (by HPLC).
6-Amino-4-(5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11h). White solid. Yield, 50%. H-NMR (400 MHz, DMSO-d6)
δ
12.02 (s, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 9.0 Hz, 1H), 6.81 (s,
2H), 6.78 (dd, J = 8.9, 3.1 Hz, 1H), 6.59 (s, 1H), 5.16 (q, J = 13.2 Hz, 2H), 4.99 (s, 1H), 3.66 (s,
3H), 1.77 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ
161.78, 155.55, 154.03, 149.78, 142.74,
135.65, 133.91, 128.57 (q, J = 39.9 Hz), 128.18, 125.70 (q, J = 3.8 Hz), 124.75 (q, J = 253.7 Hz),

Molecules 2021, 26, 3034
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121.39, 115.87, 114.34, 112.39, 97.88, 69.62, 56.74, 55.70 and 10.04. HRMS (ESI) calculated for
C23H19F3N4O3 [M + H]+: 457.1482, found: 457.1496. Purity: 99.96% (by HPLC).
6-Amino-4-(5-methoxy-2-((4-(trifluoromethoxy)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11i). White solid. Yield, 47%. H-NMR (400 MHz, DMSO-d6)
δ
12.02 (s, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 8.9 Hz, 1H), 6.80
(s, 2H), 6.78 (dd, J = 9.0, 3.2 Hz, 1H), 6.56 (d, J = 2.2 Hz, 1H), 5.09 (q, J = 12.6 Hz, 2H), 4.98
(s, 1H), 3.65 (s, 3H), 1.76 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.77, 155.55, 154.01,
149.85, 148.17, 137.35, 135.64, 133.98, 129.66, 121.42, 121.39, 120.56 (q, J = 256.6 Hz), 115.75,
114.39, 112.37, 97.90, 69.63, 56.78, 55.69 and 10.02. HRMS (ESI) calculated for C23H19F3N4O4
[M + H]+: 473.1431, found: 473.1444. Purity: 99.82% (by HPLC).
6-Amino-4-(5-(tert-butyl)-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11j). White solid. Yield, 36%. H-NMR (400 MHz, DMSO-d6)
δ
11.99 (s, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.19 (dd, J = 8.5, 2.4 Hz, 1H),
7.11 (s, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.77 (s, 2H), 5.18 (q, J = 13.4 Hz, 2H), 4.97 (s, 1H),
1.74 (s, 3H), 1.21 (s, 9H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.78, 155.69, 153.66, 143.39,
142.73, 135.55, 131.42, 128.54 (q, J = 31.5 Hz), 128.07, 126.54, 125.69 (q, J = 3.8 Hz), 124.92,
124.35 (q, J = 252.4 Hz), 121.51, 112.54, 98.01, 68.87, 56.90, 34.18, 31.71 and 10.02. HRMS
(ESI) calculated for C26H25F3N4O2 [M + H]+: 483.2002, found: 483.1984. Purity: 99.84%
(by HPLC).
6-Amino-3-methyl-4-(5-methyl-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11k). Yellow solid. Yield, 40%. H-NMR (400 MHz, DMSO-d6)
δ
12.01 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 7.8 Hz, 2H), 7.02–6.93 (m, 2H), 6.84 (s,
1H), 6.79 (s, 2H), 5.20 (q, J = 13.3 Hz, 2H), 5.01 (s, 1H), 2.19 (s, 3H), 1.76 (s, 3H). 13C-NMR
(101 MHz, DMSO-d6) δ 161.72, 155.58, 153.50, 142.67, 135.61, 132.36, 130.29, 130.05, 128.81,
128.58 (q, J = 31.6 Hz), 128.16, 125.72 (q, J = 3.7 Hz), 124.24 (q, J = 249.1 Hz), 121.47, 113.05,
98.12, 69.08, 56.99, 20.67 and 10.04. HRMS (ESI) calculated for C23H19F3N4O2 [M + H]+:
441.1533, found: 441.1538. Purity: 99.80% (by HPLC).
6-Amino-4-(2,5-bis((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-
5-carbonitrile (11l). White solid. Yield, 31%. 1H-NMR (400 MHz, DMSO-d6)
δ 12.03 (s,
1H), 7.76–7.71 (m, 4H), 7.63 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.02 (d, J = 8.9 Hz,
1H), 6.87 (dd, J = 9.0, 3.0 Hz, 1H), 6.82 (s, 2H), 6.70 (s, 1H), 5.23–5.13 (m, 2H), 5.11 (s, 2H),
4.99 (s, 1H), 1.71 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.78, 155.57, 152.74, 150.10,
142.67, 142.51, 135.64, 128.70 (q, J = 32.0 Hz), 128.59 (q, J = 30.7 Hz), 128.55, 128.20, 125.70 (q,
J = 3.8 Hz), 124.74 (q, J = 272.2 Hz), 124.71 (q, J = 272.6 Hz), 121.40, 116.82, 114.76, 114.33,
113.66, 97.78, 69.58, 69.05, 56.70 and 9.97. HRMS (ESI) calculated for C30H22F6N4O3 [M +
H]+: 601.1669, found: 601.1690. Purity: 99.77% (by HPLC).
6-Amino-4-(5-hydroxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11m). Yellow solid. Yield, 43%. H-NMR (400 MHz, DMSO-d6)
δ
12.03 (s, 1H), 8.92 (s, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 7.7 Hz, 2H), 6.90 (d, J = 8.8 Hz
1H), 6.81 (s, 2H), 6.56 (dd, J = 8.8, 2.9 Hz, 1H), 6.45 (d, J = 2.7 Hz, 1H), 5.18–5.08 (m, 2H), 4.99
(s, 1H), 1.78 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
161.70, 155.50, 152.15, 148.48, 142.94,
,
δ
135.66, 133.94, 128.56 (q, J = 38.2 Hz), 128.20, 125.73 (q, J = 3.4 Hz), 124.76 (q, J = 248.2 Hz),
121.43, 116.07, 114.73, 114.66, 98.21, 69.95, 57.06 and 10.02. HRMS (ESI) calculated for
C22H17F3N4O3 [M + H]+: 443.1326, found: 443.1319. Purity: 99.40% (by HPLC).
6-Amino-4-(5-methoxy-2-(4-(trifluoromethyl)phenethoxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11n). White solid. Yield, 37%. H-NMR (400 MHz, DMSO-d6)
δ
11.96 (s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 6.95 (d, J = 8.9 Hz, 1H), 6.81 (s,
2H), 6.75 (dd, J = 8.9, 3.1 Hz, 1H), 6.54 (s, 1H), 4.80 (s, 1H), 4.18–4.06 (m, 2H), 3.64 (s, 3H),
3.09 (t, J = 6.4 Hz, 2H), 1.63 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.86, 155.41, 153.91,
150.28, 144.23, 135.55, 134.09, 130.31, 127.50 (q, J = 31.9 Hz), 125.54 (q, J = 3.8 Hz), 124.90 (q,
J = 272.2 Hz), 121.35, 115.58, 114.40, 112.36, 98.06, 69.65, 56.76, 55.68, 35.41 and 9.69. HRMS

Molecules 2021, 26, 3034
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(ESI) calculated for C24H21F3N4O3 [M + H]+: 471.1639, found: 471.1631. Purity: 98.11%
(by HPLC).
6-Amino-4-(5-methoxy-2-(3-(4-(trifluoromethyl)phenyl)propoxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (11o). White solid. Yield, 48%. 1H-NMR (400 MHz, DMSO-d6)
δ
11.97 (s, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 6.87 (d, J = 8.9 Hz, 1H),
6.77 (s, 2H), 6.76–6.73 (m, 1H), 6.63 (s, 1H), 4.81 (s, 1H), 3.95–3.73 (m, 2H), 3.66 (s, 3H),
2.77 (t, J = 7.5 Hz, 2H), 2.02–1.92 (m, 2H), 1.80 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ
161.82, 155.58, 153.51, 150.74, 147.17, 135.50, 133.40, 129.73, 127.06 (q, J = 34.4 Hz), 125.55 (q,
J = 3.8 Hz), 123.86 (q, J = 260.4 Hz), 121.48, 115.95, 113.67, 112.30, 98.01, 67.93, 56.50, 55.69,
31.97, 30.71, 19.01 and 10.01. HRMS (ESI) calculated for C25H23F3N4O3 [M + H]+: 485.1495,
found: 485.1788. Purity: 98.23% (by HPLC).
6-Amino-3-methyl-4-(5-(trifluoromethoxy)-2-((4-(trifluoromethyl) benzyl) oxy) phenyl)-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11p
)
.
White solid. Yield, 55%. H-NMR (400 MHz, DMSO-d6)
δ
12.08 (s, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 7.7 Hz, 2H), 7.23 (dd, J = 8.9, 2.2 Hz, 1H),
7.17 (d, J = 9.0 Hz, 1H), 7.05 (s, 1H), 6.89 (s, 2H), 5.26 (q, J = 13.2 Hz, 2H), 5.04 (s, 1H),
1.76 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.91, 155.60, 154.64, 142.41, 142.00, 135.71,
134.25, 128.74 (q, J = 35.7 Hz), 128.24, 125.78 (q, J = 3.4 Hz), 123.08 (q, J = 235.6 Hz), 121.60
(q, J = 265.0 Hz), 121.24, 121.18, 114.36, 97.15, 69.39, 55.95 and 9.96. HRMS (ESI) calculated
for C23H16F6N4O3 [M + H]+: 511.1199, found: 511.1196. Purity: 99.87% (by HPLC).
6-Amino-4-(5-(difluoromethoxy)-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11q
)
.
Yellow solid. Yield, 47%. H-NMR (400 MHz, DMSO-d6)
δ
12.09 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.14 (d, J = 8.8 Hz, 1H),
6.98 (dd, J = 9.0, 3.1 Hz, 1H), 6.88 (s, 2H), 6.75 (s, 1H), 5.16 (s, 2H), 4.87 (s, 1H), 1.73 (s,
3H). 13C-NMR (126 MHz, DMSO-d6)
161.80, 155.84, 155.44, 142.88, 142.09, 137.02, 135.76,
δ
128.80 (q, J = 31.9 Hz), 128.63, 125.75 (q, J = 3.78 Hz), 124.70 (q, J = 273.0 Hz), 120.99, 120.66,
117.30 (t, J = 25.8 Hz), 116.60, 114.37, 97.15, 69.05, 56.21 and 9.87. HRMS (ESI) calculated for
C23H17F5N4O3 [M + H]+: 493.1294, found: 493.1279. Purity: 95.59% (by HPLC).
6-Amino-4-(5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,4-dihydropyrano[2,3-c]pyrazole-
1
5-carbonitrile (11r). White solid. Yield, 57%. H-NMR (400 MHz, DMSO-d6)
δ 12.30 (s, 1H),
7.76 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 7.29 (s, 1H), 7.02 (d, J = 8.9 Hz, 1H), 6.94
(s, 2H), 6.78 (dd, J = 8.9, 3.1 Hz, 1H), 6.64 (d, J = 3.1 Hz, 1H), 5.27–5.17 (m, 2H), 5.07 (s,
1H), 3.67 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 162.39, 155.21, 153.95, 149.70, 142.76,
134.77, 128.62 (q, J = 32.8 Hz), 128.21, 126.58, 125.76 (q, J = 3.8 Hz), 124.75 (q, J = 272.6 Hz),
121.32, 115.01, 114.04, 112.06, 100.61, 69.52, 55.74, 55.16 and 31.13. HRMS (ESI) calculated
for C22H17F3N4O3 [M + H]+: 443.1326, found: 443.1323. Purity: 99.17% (by HPLC).
6-Amino-4-(5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-phenyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11s). Yellow solid. Yield, 42%. H-NMR (400 MHz, DMSO-d6)
δ
12.81 (s, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H), 7.45–7.41 (m, 2H), 7.30–7.21
(m, 3H), 6.89 (s, 1H), 6.87 (s, 2H), 6.66 (dd, J = 8.9, 3.1 Hz, 1H), 6.53 (s, 1H), 5.32 (s, 1H),
5.19–5.09 (m, 2H), 3.58 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ 161.06, 156.89, 153.77,
149.66, 142.88, 137.87, 134.06, 129.20, 128.97, 128.72, 128.54 (q, J = 31.9 Hz), 127.94, 126.39,
125.68 (q, J = 3.4 Hz), 124.74 (q, J = 261.7 Hz), 121.16, 115.99, 113.98, 112.51, 97.79, 69.55,
57.68, and 55.65. HRMS (ESI) calculated for C28H21F3N4O3 [M + H]+: 519.1639, found:
519.1627. Purity: 99.66% (by HPLC).
6-Amino-4-(4-chloro-5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
1
c]pyrazole-5-carbonitrile (11t
)
.
White solid. Yield, 44%. H-NMR (500 MHz, DMSO-d6)
δ
12.05 (s, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 6.5 Hz, 2H), 7.23 (s, 1H), 6.87 (s, 3H),
5.30–5.09 (m, 2H), 5.00 (s, 1H), 3.73 (s, 3H), 1.79 (s, 3H). 13C-NMR (126 MHz, DMSO-d6)
δ161.81, 155.56, 149.91, 149.41, 142.20, 135.76, 132.29, 128.67 (q, J = 28.1 Hz), 128.24, 125.72
(q, J = 3.8 Hz), 124.73 (q, J = 272.6 Hz), 121.30, 120.18, 115.64, 114.21, 97.26, 69.79, 56.95,
56.25, and 10.08. HRMS (ESI) calculated for C23H18ClF3N4O3 [M + H]+: 491.1092, found:
491.1089. Purity: 98.50% (by HPLC).

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3-(4-(Trifluoromethyl)phenyl)propan-1-ol (13). Lithium aluminum (6.9 mmol) was slowly
added to a solution of 12 (4.6 mmol) in tetrahydrofuran at 0 ◦C. Then, the mixture was
refluxed at 85 ◦C for 6 h. After the addition was complete, water/10% sodium hydrox-
ide/water = 1:1:3 (mL:mL:mL) was added. Then, the solution was filtered and the filtrate
was collected. After evaporation of the solvent under reduced pressure, the crude product
was purified on a silica gel column to obtain compound 13. White solid. Yield, 56%.
1H-NMR (400 MHz, CDCl3)
δ 7.56 (d, J = 8.1 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 3.71 (dd,
J = 10.3, 5.9 Hz, 2H), 2.83–2.75 (m, 2H), 1.97–1.87 (m, 2H).
1-(3-Chloropropyl)-4-(trifluoromethyl)benzene (9o). Triethylamine (2.5 mmol) was added to
a solution of 13 (1.0 mmol) in anhydrous dichloromethane (10 mL) at 0 ◦C, followed by
◦
triphosgene (0.5 mmol) in one portion. The mixture was stirred at 0 C for 5 min and
then allowed to warm to room temperature. After the reaction was complete, the reaction
mixture was poured into an aqueous solution containing saturated sodium bicarbonate.
After separation of the layers, the aqueous layer was extracted again with dichloromethane
(2
concentrated in vacuo, and the crude product was purified on a silica gel column to obtain
compound 9o. White solid. Yield, 71%. 1H-NMR (400 MHz, CDCl3)
7.58 (d, J = 8.0
×
30 mL). Then, the organic layers were combined, dried with MgSO4, filtered, and
δ
Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 3.55 (t, J = 6.4 Hz, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.17–2.06
(m, 2H).
Tert-butyl-6-amino-5-cyano-4-(5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methylpyrano[2,3-
c]pyrazole-1(4H)-carboxylate (14). (Boc)2O (1.2 mmol) was add◦ed to a solution of 11 h
(1 mmol) and DMAP (0.2 mmol) in tetrahydrofuran (15 mL) at 0 C, and then, the mixture
was stirred at room temperature for 8 h. After completion, the mixture was extracted
with water (30 mL) and the organic layer was separated. After evaporation of the organic
layer under reduced pressure, the crude product was purified on a silica gel column to
obtain compound 14. White solid. Yield, 60%. 1H-NMR (400 MHz, DMSO-d6)
δ 7.68
(d, J = 7.9 Hz, 2H), 7.46 (d, J = 6.7 Hz, 2H), 7.07 (s, 2H), 6.97 (d, J = 8.8 Hz, 1H), 6.78 (d,
J = 8.7 Hz, 2H), 5.19–5.09 (m, 2H), 4.89 (s, 1H), 3.68 (s, 3H), 2.08 (s, 3H), 1.52 (s, 9H). 13C-
NMR (101 MHz, DMSO-d6) δ 161.03, 156.82, 153.78, 149.63, 148.24, 142.52, 140.27, 132.29,
128.73, 128.38(q, J = 33.3 Hz), 128.14, 125.59 (q, J = 3.7 Hz), 124.70 (q, J = 245.8 Hz), 120.72,
116.47, 114.32, 112.78, 105.48, 85.13, 69.23, 56.45, 55.74, 27.87, 27.79 and 13.19. LC/MS (ESI):
557.20 [M + H]+.
Tert-butyl-5-cyano-6-(dimethylamino)-4-(5-methoxy-2-((4-(trifluoromethyl)benzyl) oxy)phenyl)-
3-methylpyrano[2,3-c]pyrazole-1(4H)-carboxylate (15). CH3I (2 mmol) was added to a solution
of K2CO3 (2.1 mmol) and 14 (1 mmol) in acetonitrile (20 mL), and the mixture was stirred
◦
at 60 C for 5 h. After the reaction was complete, the solvent was spin-dried under reduced
pressure. The mixture was diluted with water (20 mL) and extracted with ethyl acetate
(60 mL), and then, the organic layer was separated. After evaporation of the solvent
under reduced pressure, the crude product was purified on a silica gel column to obtain
1
compound 15. White solid. Yield, 53%. H-NMR (400 MHz, DMSO-d6)
δ
7.75 (d, J = 7.9 Hz
,
2H), 7.63 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 1.9 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 9.1
,
2.2 Hz, 1H), 5.19 (s, 2H), 4.93 (s, 1H), 3.84 (s, 3H), 3.73 (s, 3H), 2.20 (s, 3H), 1.87 (s, 3H),
1.52 (s, 9H). 13C-NMR (126 MHz, DMSO-d6)
δ
162.03, 153.33, 149.52, 148.14, 144.10, 142.20,
128.85 (q, J = 31.9 Hz), 128.66, 125.95, 125.70 (q, J = 3.8 Hz), 124.69 (q, J = 291.9 Hz), 117.01,
116.99, 115.75, 114.14, 113.98, 106.59, 84.88, 69.82, 56.23, 55.75, 35.83, 27.90, 24.80 and 13.24.
LC/MS (ESI): 585.19 [M + H] +.
6-(Dimethylamino)-4-(5-methoxy-2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3-methyl-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (16). Compound 15 (1 mmol) was added to the solution of triflu-
oroacetic acid/water = 1:3 (mL:mL), and the mixture was stirred at room temperature
for 1 h. After the reaction was complete, the mixture was diluted with saturated sodium
carbonate, and then, the organic layer was separated and the solvent was spin-dried un-
der reduced pressure. The crude product was purified on a silica gel column to obtain
1
compound 16. White solid. Yield, 77%. H-NMR (400 MHz, DMSO-d6)
δ 11.74 (s, 1H),

Molecules 2021, 26, 3034
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7.75 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 3.0 Hz, 1H), 7.05 (d, J = 9.1 Hz
,
1H), 6.88 (dd, J = 9.0, 3.0 Hz, 1H), 5.17 (s, 2H), 4.83 (s, 1H), 3.77 (s, 3H), 3.71 (s, 3H), 1.95 (s,
3H), 1.81 (s, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 161.57, 153.34, 149.44, 142.44, 139.50,
128.72 (q, J = 34.4 Hz), 128.29, 127.35, 125.72 (q, J = 4.2 Hz), 124.72 (q, J = 252.4 Hz), 117.46,
117.27, 115.68, 114.04, 113.68, 97.81, 69.78, 55.91, 55.72, 36.51, 24.78 and 10.53. HRMS
(ESI) calculated for C25H23F3N4O3[M + H]+: 485.1795, found: 485.1783. Purity: 96.81%
(by HPLC).
3.2. Chiral Resolution of Compound 11h
The chiral resolution of compound 11h was performed using Gilson instruments, and
the chromatographic column was a CHIRALPAK®IF column (250 mm
The compound was filtered with a microporous membrane with an injection volume
×
4.6 mm, 5 µm).
of 5 µL, and the mobile phase was Hex/EtOH = 80: 20. The eluent was ultrasonically
degassed for 30 min before use. Flow rate: 1 mL/min; Cycle time: 40 min. (
−
)-11h
:
[α]2D0 = −46.7 (c 1.0, THF), tR = 24.847 min, ee value, >99%; (
+)-11h:
[α]2D0 = +46.5 (c 1.0,
THF), tR = 33.976 min, ee value, >99%.
3.3. In Vitro Assay for PDE2 Inhibitors
3.3.1. Protein Expression and Purification
Purification and expression of the recombinant pET15b-PDE2A plasmid (catalytic
domain, 580–919) was performed following our previously reported method. The plasmid
pET15b-PDE2A (catalytic domain, 580–919) was transferred to thawed competent E. coli
BL21 cells (CodonPlus, Agilent Technologies, Santa Clara, CA, USA), which were grown in
2XYT medium with 100 µg/mL ampicillin and 20 µ
g/mL chloramphenicol at 37 ◦C until
OD600 = 0.6–0.8. Then, 0.1 mM isopropylthiogalactoside was added for induction, and
protein expression was induced by continuing to grow the cells at 16 ◦C for 24 h. After
◦
that, bacteria were collected by centrifugation and frozen overnight at
−
20 C. The bacteria
containing the recombinant plasmid of the target protein were subjected to cell disruption
and passed through a Ni-NTA agarose affinity column, Q-Sepharose ion-exchange column
and other steps. The purity can be 95% or more, and a typical purification batch produces
10–20 mg of PDE2A protein from 1.0 L of cell culture.
3.3.2. Enzymatic Assay
Using the appropriate concentration of 3H-cGMP as the substrate, different compound
solutions were added to the test group, DMSO was added to the negative control and
blank control groups, and EHNA was added to the positive control group, followed by
protein solution (the proteins were all diluted with Assay Buffer); Assay Buffer was added
to the blank group. The enzymatic reaction was performed at room temperature (25 ◦C)
for 15 min, and then, 0.2 M ZnSO4 was added to inactivate PDE2 and to stop the reaction.
Then, 0.2 N Ba(OH)2 solution was added to precipitate the reaction product, and unreacted
3H-cGMP remained in the supernatant. After mixing the system, it was centrifuged. The
radioactivity of the supernatant was measured with a liquid scintillation counter, and the
inhibitory activity of the compound against PDE2 was calculated. The above test was
repeated three times (the IC50 test generally used 9 concentrations, and the single-point
test was directly diluted with DMSO to the required concentration).
3.4. Molecular Docking and Dynamic Simulation
3.4.1. Molecular Docking
The Accelrys Discovery Studio 2.5.5 software (Accelrys, San Diego, CA, USA) was
used for molecular docking research through the CDOCKER method, and the CHARMM
force field and Momany–Rone partial charge method were used to add hydrogen atoms
and charges to the crystal structure of PDE2A (PDB ID: 4HTX, resolution: 1.90 Å). All
ionizable amino acid residues in the system were set to their protonated state at neutral
pH, and the charge of zinc and magnesium ions were +2. (R)-LZ77 was used to define the

Molecules 2021, 26, 3034
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active site of PDE2A and to test the docking. The radius of the input ball was set to 10 Å
from the center of the binding site, and the default values were used for the rest of the
docking parameters. Fifty random images were generated and optimized for each ligand.
The output pose of the docking was evaluated by docking scoring and visual inspection.
3.4.2. Molecular Dynamics (MD) Simulations
MD simulations were conducted using Amber16 (University of California, San Fran-
cisco, CA, USA) for a more precise prediction of binding patterns between each ligand and
PDE2. The partial atomic charges of each ligand were calculated using the Hartree–Fock
method at the 6–31G* level in Gaussian 03 (Gaussian, Inc., Wallingford, CT, USA). Then,
Antechamber (Amber16) was applied to fit the restricted electrostatic potential (RESP)
and to assign the general amber force field (GAFF) parameters. For the PDE2 protein, the
Amber14SB force field was assigned. For the force field parameters of Zn2+ and Mg2+, the
“nonbond model” method was applied for the assignment. The protonation states of amino
acid residues were set according to pH = 7.0. Each simulation system was solvated with
an 8 Å TIP3P water box in the form of a truncated octahedron. Na+ ions were added for
neutralization of the whole system.
Each system was investigated through MD simulations. After energy minimization,
the system was gradually heated in the NPT ensemble from 10 to 300 K over 25 ps. Then,
eight-nanosecond MD simulations were conducted in the NPT ensemble with a constant
pressure and temperature of 1 atm and 300 K, respectively. Periodic boundary conditions
were utilized. An 8 Å cutoff for long-range electrostatic interactions with the partial mesh
Ewald (PME) method was simultaneously employed. The SHAKE algorithm was adopted
to restrict bonds involving hydrogen atoms, and thus, the time step was set to 2 fs. An
Intel(R) Xeon(R) Bronze 3106 CPU and a GeForce RTX 2080Ti GPU were employed to
accelerate the process of MD simulations for each system.
3.4.3. Binding Free Energy Calculations
MM-PBSA binding free energy calculations were performed by extracting 100 snap-
shots of trajectories in the last 1 ns with default parameters assigned. According to the
MM-PBSA method, the binding free energies (∆Gbind) were calculated using Equation (1),
in which Gcomplex, Grec and Glig represent the free energies of the complex, receptor, and
ligand, respectively. Each free energy value is calculated as the sum of the MM energy
E
MM, the solvation free energy Gsolv, and the entropy contribution S, respectively, leading
to Equation (2). EMM represents the gas-phase interaction energy. It is decomposed
∆
into EMM, complex, EMM, rec, and EMM, lig. The solvation free energy represents the sum of
the electrostatic solvation free energy and nonpolar solvation free energy. The entropy
contribution for each system was omitted, since this computational process is extremely
time-consuming for large protein–ligand systems.
∆Gbind = Gcomplex − Grec − Glig
∆Gbind = ∆EMM + ∆Gsolv − T∆S
(1)
(2)
4. Conclusions
In summary, a series of dihydropyranopyrazole derivatives were designed, synthe-
sized, and evaluated as PDE2 inhibitors with the assistance of molecular docking. The
optimal compound is (+)-11h, in which the side chain on the benzene ring connected to
the pyran ring is shifted from the 3-position to the 2-position, the chlorine on the side
chain is replaced by a trifluoromethoxy group, and the methoxy group is shifted from the
4-position to the 5-position. (
an inhibition rate of 93.6% at 0.5
high selectivity against PDE2 compared with several other PDEs. Furthermore, both the
molecular docking of PDE2-( )-11h and energy decomposition reveal that the side chain at
position 2 of the compound enters the H-pocket and forms strong hydrophobic interactions
+)-11h exhibits potent inhibitory activity against PDE2, with
µ
M and an IC50 value of 41.5 nM. In addition, ( )-11h has
+
+

Molecules 2021, 26, 3034
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with L770/L809/F862, which may contribute to its improved inhibitory activity. The above
results may provide insight for further structural optimization of highly potent PDE2
inhibitors and lay the foundation for the use of PDE2 inhibitors in the treatment of AD.
1
Supplementary Materials: The spectra of H-NMR and 13C-NMR for the target compounds are
available online.
Author Contributions: Conceptualization, investigation, project administration, Y.Z., Y.H. (Yue Huang)
J.L., R.S., H.Y., Y.H. (Yiyou Huang), Z.L., Y.W., H.L., C.Z. and L.H.; writing—original draft, Y.Z.,
C.Z. and L.H.; writing—review and editing, H.L. and L.H. All authors have read and agreed to the
published version of the manuscript.
,
Funding: We cordially thank the anonymous reviewers’ valuable comments and kind proofread-
ing of the manuscript. This research was funded by the National Natural Science Foundation of
China (21772241 and 82003652), Guangzhou Pearl River New Star Fund Science and Technology
Planning Project (201806010190). We cordially thank H. Ke from the Department of Biochemistry and
Biophysics at the University of North Carolina, Chapel Hill, for his assistance with the molecular
cloning, expression, purification, determination of the crystal structures and bioassay of PDEs.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available within the article and
Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Sample of the compounds are not available from authors.
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