Synthesis and bioactivity of 3,5-dimethylpyrazole derivatives as potential PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.03.031

A series of 3,5-dimethylpyrazole derivatives containing 5-phenyl-2-furan moiety were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. Bioassay results showed that the title compounds exhibited considerable inhibitory activity agains

Full text of DOI:10.1016/j.bmcl.2018.03.031

Accepted Manuscript
Synthesis and bioactivity of 3,5-dimethylpyrazole derivatives as potential PDE4
inhibitors
De-Kun Hu, Dong-Sheng Zhao, Min He, Hong-Wei Jin, Yong-Mei Tang, Lian-
Hui Zhang, Gao-Peng Song, Zi-Ning Cui
PII:
S0960-894X(18)30220-8
https://doi.org/10.1016/j.bmcl.2018.03.031
BMCL 25685
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 November 2017
2 March 2018
12 March 2018
Please cite this article as: Hu, D-K., Zhao, D-S., He, M., Jin, H-W., Tang, Y-M., Zhang, L-H., Song, G-P., Cui, Z-
N., Synthesis and bioactivity of 3,5-dimethylpyrazole derivatives as potential PDE4 inhibitors, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.031
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Synthesis and bioactivity of 3,5-dimethylpyrazole derivatives
as potential PDE4 inhibitors
De-Kun Hua,#, Dong-Sheng Zhaob,#, Min Hea, Hong-Wei Jinc, Yong-Mei Tangd,
Lian-Hui Zhanga, Gao-Peng Songd,*,Zi-Ning Cuia, *
a
State Key Laboratory for Conservation and Utilization of Subtropical
Agro-bioresources, Integrative Microbiology Research Centre, Guangdong Province
Key Laboratory of Microbial Signals and Disease Control, South China Agricultural
University, Guangzhou 510642, China
b Department of pharmacy, Quanzhou Medical College, Quanzhou 362100, China
c State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
d College of Materials and Energy, South China Agricultural University, Guangzhou
510642, China
To whom Correspondence should be addressed. Telephone/Fax: +86-20-85288229.
E-mail: ziningcui@scau.edu.cn (Z. C.), vinsin1021@126.com (G. S.)
#These authors contributed equally to this paper.

Abstract:
A series of 3,5-dimethylpyrazole derivatives containing 5-phenyl-2-furan moiety were
designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. Bioassay
results showed that the title compounds exhibited considerable inhibitory activity
against PDE4B and blockade of LPS-induced TNFα release. Among the designed
compounds, compound If showed the best inhibitory activity against PDE4B with the
IC50 value of 1.7 µM, which also showed good in vivo activity in animal models of
asthma/COPD and sepsis induced by LPS. The primary structure–activity relationship
(SAR) study and docking results suggested that introduction of the substituent groups
to the phenyl ring at the para-position, especially methoxy group, was helpful to
enhance inhibitory activity against PDE4B.
Keywords: 3,5-dimethylpyrazole derivatives; synthesis; PDE4 inhibitor; SAR;
molecular simulation

Cyclic nucleotide phosphodiesterases (PDEs), which include 11 diverse families
according to their structures and properties, are essential in the metabolism of the
secondary signal messengers, cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate(cGMP). 1,2 It is also well known that cAMP, specifically
hydrolyzed by the PDE4, regulates the function of airway smooth muscle,
inflammatory cells, and immune cells. 3,4 When PDE4 is inhibited, the accumulation
of cAMP leads to activating the cascades of specific protein phosphorylation, which
contributes to lots of functional responses such as suppression of TNFα production.
Therefore, PDE4 inhibitors are an important type of anti-inflammatory drugs for the
treatment of asthma and chronic obstructive pulmonary disease (COPD), which have
been a major public health burden worldwide. 5-7
Since the discovery of rolipram and piclamilast (Fig. 1),3,8 the first and second
representative drug of diether derivatives of catechol class, which is an important type
of selective PDE4 inhibitors, 9,10 PDE4 inhibitors have been extensively studied in the
past few years. It was reported the 4-(3,4-dialkoxyphenyl) moiety (Fig. 1) was
essential for PDE4 inhibition where the catechol ether oxygens played an important
7
role in binding to the enzyme. Further study shows that the modification of the
4-(3,4-dialkoxyphenyl) moiety to the 8-methoxyquinoline-5-carboxamides (such as
11
SCH 365351) could enhance the PDE4 inhibitory activity.
What is more,
replacement of the amide portion of SCH 365351 with either a five-membered
heterocyclic oxazole moiety or furan ring is helpful to enhance potent PDE4
inhibitory activity. 11-14

Figure 1. The designed strategy for the title compounds.
In our previous work, we found two series of pyrazole derivatives exhibited
significant PDE4B inhibitory activity (Fig. 1).15-16 The primary SARs demonstrated
that the five-membered heterocyclic moiety was essential for the bioactivity through
integral hydrogen bonds and a π-π stacking interaction. However, it is interesting that
we can not find the π-π stacking interaction in furan-pyrazole moiety like found in
oxazole-pyrazole moiety. Moreover, the π-π stacking interaction enhanced when the

pyrazole ring was substituted to 3,5-dimethylpyrazole ring. Based on these results, in
order to obtain the novel PDE4B inhibitors and get the further relationship between
the bioactivities and the primary SARs, 3,5-dimethylpyrazole derivatives Ia-Io
containing furan moiety as bioisosteric surrogates of oxazole moiety were further
designed and synthesized (Fig. 1).
The synthetic route of title compounds Ia-Io was shown in Scheme 1. The key
intermediate 2 was synthesized from substituted aniline by Meerwein arylation
reaction according to our previous procedure.17,18 Treatment of 5-substituted
phenyl-2-furan carboxylic acid 2 with thionyl chloride in anhydrous toluene under
reflux provided the 5-phenyl-2-furancarbonyl chloride, then followed by treatment
with 3,5-dimethyl-pyrazole in anhydrous dichloromethane afforded the compounds
Ia-Io (see the Supplementary data for the details).
Scheme 1. The synthetic route of the title compounds Ia-Io. Reagents and conditions:
(a) NaNO2, hydrochloric acid, 0–5 oC, 3 h; (b) furoic acid, CuCl2 (cat.), acetone–H2O,
rt, 5 h; (40–65%, two steps) (c) SOCl2, anhydrous toluene, reflux, 3 h; (d)
3,5-dimethylpyrazole, anhydrous dichloromethane, reflux, 4 h (75–91%, two steps).
R1 = Ia: H, Ib: 4-Cl, Ic: 4-F, Id: 4-NO2, Ie: 4-CH3, If: 4-OCH3, Ig: 4-Br, Ih: 3-Cl, Ii:
3-F, Ij: 3-NO2, Ik: 2-Cl, Il: 2-F, Im: 2-NO2, In: 2,4-di-F, Io: 2,6-di-F.
All the title compounds Ia-Io were evaluated for their inhibition against PDE4B

with rolipram as the reference drug. As a standard, compounds with IC50 values ≤ 20
µM against PDE4B, were tested for their inhibition against PDE4D. The results,
expressed as IC50, are summarized in Table 1. What is more, the inhibition of
blockade of LPS-induced TNF-α release was also listed in Table 1.
Table 1. Impact on enzymatic potency (PDE4) and inhibition of TNF-α release from
human blood mononuclear cells stimulated with lipopolysaccharide a
Compds.
R1
PDE4B
IC50 (µM)
70.3 4.2
3.9 0.5
PDE4D
IC50 (µM)
NTb
TNFα
IC50 (µM)
178.5 7.9
25.1 1.1
24.3 1.5
41.7 2.3
37.1 1.8
10.4 0.8
154.2 6.3
58.4 2.0
70.8 2.6
69.3 3.1
112.3 5.7
98.2 4.1
168.2 6.8
19.2 0.8
88.7 3.2
16.5 1.0
H
Ia
Ib
4-Cl
26.4 1.2
49.3 1.8
87.3 3.4
52.9 2.4
9.6 0.7
NTb
4-F
7.2 0.5
Ic
4-NO2
4-CH3
4-OCH3
4-Br
15.4 1.5
8.4 0.4
Id
Ie
If
1.7 0.3
Ig
51.1 1.9
20.9 1.1
20.2 1.2
35.9 2.8
52.4 2.1
45.3 1.8
62.7 2.9
3.2 0.4
Ih
NTb
3-Cl
Ii
3-F
NTb
Ij
NTb
3-NO2
2-Cl
Ik
NTb
Il
NTb
2-F
Im
In
NTb
2-NO2
2,4-di-F
2,6-di-F
19.6 1.1
88.2 2.8
2.5 0.6
14.5 1.2
1.9 0.4
Io
rolipram
a Results are the average of at least three assays. b NT, not tested.
As shown in Table 1, all the compounds except compounds Ia, Ig, Ik and Im
exhibited moderate to good inhibitory activity against PDE4B with the IC50 range of

1.0-50 µM. Among these derivatives, compound If displayed the strongest inhibition
activity (IC50 = 1.7 µM) as well as good selectivity against PDE4B toward PDE4D
(selective index = 5.6). Compounds Ib, If and In showed the similar inhibition
against PDE4B as the positive drug rolipram, but exhibited over 5.6-fold higher
selectivity rations for PDE4B over PDE4D than rolipram, confirming that
3,5-dimethylpyrazole derivatives containing furan moiety was appropriate for
obtaining the new PDE4B inhibitors with high affinity and selectivity.
Introduction of different substituents at the 2, 3 or 4-position of the benzene ring
resulted in the improvement of inhibitory activity against PDE4B since compound Ia
(R1 = H) showed the poorest activity, maybe due to increasing the affinity to specific
areas in PDE4B enzyme. It was noted that both the substituent positions on the phenyl
ring and the substituent electronegativity seemed to have a significant impact on
potency. In analyzing the inhibitory activity of compounds Ib-Ig, in which the
structural variations were made in the para-position of the phenyl ring. It was
observed that substitution in the phenyl ring was beneficial to enhance inhibition
against PDE4B. The attachment of electron donor groups, such as methoxy (If, IC50 =
1.7 µM) and methyl (Ie, IC50 = 8.4 µM), to the phenyl ring at the para-position led to
the remarkable increased activity when compared to Ia. The same tendency was also
observed when different halogens were attached to the phenyl ring. However, this
increase Ig (R1 = 4-Br, IC50 = 51.1 µM) was not as significant as the other two
halogens, such as Ic (R1 = 4-F, IC50 = 7.2 µM) and Ib (R1 = 4-Cl, IC50 = 3.9 µM),
respectively. Compound Id (R1 =4-NO2, IC50 = 15.4 µM), which have

electron-withdrawing group NO2, also demonstrated increased inhibitory activity
against PDE4B, although with slightly less intensity than the halogens analogues (Ib
and Ic). Thus, all derivatives containing either electron donating groups or electron
withdrawing groups were more active than the unsubstituted analogue Ia, revealing
the importance of this structural modification at the para-position for inhibition
against PDE4B.
We also analyzed the effect of binding both halogens such as Cl as well as F atoms,
and NO2 group in the other two positions of the phenyl ring. It was found that the
meta-substitution generated the more active derivatives such as Ih and Ii than the
ortho-substitution compounds in this study. In general, the activity in the order of
compounds Ib >Ih > Ik or Id >Ij > Im suggests that the attachment of halogens or
NO2 group to the phenyl ring at the para-position is superior to the other kinds of
substitution position. Moreover, di-substitutions were also evaluated in the phenyl
ring by halogen atoms, deriving compounds In (R1 = 2,4-di-F), and Io (R1 = 2,6-di-F).
These compounds were more active than compound Ia. Especially, In as one of the
most potential in the series with an IC50 = 3.2 µM, displayed higher selective index
than the reference drug rolipram.
The ability of all the title compounds I to inhibit the release of HM-TNFα was
consistent well with their relative ability to inhibit PDE4B.The compound with
4-methoxy substituted group (If) was the most potent among all the derivatives
assessed in this study, approximately 1.6 times more active than rolipram.
LPS induced sepsis model for the measurement of TNF-α inhibition (in Swiss

Albino mice) and neutrophilia inhibition for asthma and COPD (in Sprague Dawley
rats) with selected in vitro active compounds If and In were performed. The details
such as oral dosage and number of animals grouped for the experiments were listed in
Table 2. The results showed that compound In demonstrated better inhibitory activity
against TNF-α release (43.1%) and LPS induced neutrophilia inhibition (36.5%) than
the positive control rolipram (41.7% and 32.8%) and compound In (29.6% and
24.2%).
Table 2. LPS induced TNF-α in SA mice and neutrophil influx in BALF of SD rats
Swiss Albino mice (n = 6)
Sprague Dawley rats (n = 6)
Does
(mg/kg, po)
TNF-α
Inhibition
(%)
Does
(mg/kg, po)
LPS induced
neutrophilia
(% inhibition)
36.5 0.5a’
Compds.
R1
4-OCH3
2.4-di-F
10
10
10
43.1 0.3a
29.6 0.4c
41.7 0.3b
10
10
10
If
24.2 0.4c’
In
rolipram
32.8 0.6b’
The letters a-c’ denoted the results of difference significance analysis. Means followed by the
same letter within the same column are not significantly different (p ≥ 0.05, Fisher’s LSD multiple
comparison test).
The binding of title compounds to the PDE4 structure was explored in order to
consider the inhibitory activity. The bioassay results showed that compound If has the
best activity among all the title compounds. Therefore, docking simulation of
compound If at PDE4B (PDB ID: 1XMY) and PDE4D (PDB ID: 1Q9M) was
conducted using Surflex-Dock in Sybyl 8.0 (see the Supplementary data for the
methods),19,20 and the docking contour maps were shown in Figure 2 and Figure 3.

A
C
E
B
D
F
Figure 2. Model of PDE4 and docking of compounds If (A, B). The entire PDE4B
structure (N-terminal domain, a catalytic domain and C-terminal domain) bound to If
(C, D). The catalytic domain bound to If (E, F). The catalytic domain bound to If
overlaid with rolipram (orange).
The docking orientation revealed that the five-membered 3,5-dimethylpyrazole
moiety as the pivotal pharmacophore formed integral hydrogen bonds with the
conserved glutamine residue (Gln443) (Fig. 2) and the heterocyclic ring was evidently
positioned between the phenylalanine (Phe446) and isoleucine (Ile410) (Fig. 2C),
which formed the cavity accommodating the hydrophobic moiety of compounds If. In
compound If, the oxygen atom of carbonyl group with Gln443 (1.90 Å, Fig. 2C and
2D), which played the same role as the oxygen atoms of methoxy group and

cyclopentyloxy group in rolipram, respectively (Fig. 2E and 2F). Compared to our
previous work15, we found obvious π-π stacking interaction between the
furan-3,5-dimethylpyrazole moiety of compound If and the benzene ring (3.45 Å, Fig.
2C and 2D) in the phenylalanine (Phe446). The para-methoxy group formed
coordinate bond with the Zn2+ (4.02 Å, If, Fig. 2C) and Mg2+ (3.00 Å, If, Fig. 2C and
2D) cations and the remainder of the molecule was displayed to extend into the
catalytic domain in close to both the Zn2+ and Mg2+ cations (Fig. 2C and 2D). The
formation of hydrogen bonds, π-π stacking interaction and the hydrophobic
interactions in the ligand–receptor complex was important for the binding affinity,
which could block the access of cAMP to the catalytic domain and avoid the
hydrolysis of cAMP, which formed the foundation for inhibition of PDE4.
We also superposed the structures of PDE4B (Fig. 3, green) and PDE4D (Fig. 3,
purple) with compound If. The metal cations in PDE4B (Fig. 3A) were Mg2+ and Zn2+,
but which were two Zn2+ cations in PDE4D (Fig. 3B). Meanwhile there was a lack of
a hydrophobic pocket in PDE4D compared with PDE4B. All the results could be the
reasons to the selectivity of the title compounds interacted with the different subtypes
of the enzyme.
In summary, the design and synthesis of 3,5-dimethylpyrazole derivatives
containing 5-phenyl-2-furan moiety as novel PDE4B inhibitors were reported. Their
bioactivity against phosphodiesterase type 4 and TNF-α were evaluated. Compound If
showed the best inhibitory activity against PDE4B and blockade of LPS-induced
TNF-α release among all the title compounds. The primary structure-activity

relationship study and docking results suggested the effect of all the title compounds
on inhibition against PDE4B related to both the substituent types and the substituent
positions on the phenyl ring. All derivatives containing either electron donating
groups or electron withdrawing groups were more active than the unsubstituted
analogue. Introduction of substituent group to the phenyl ring at the para-position,
especially methoxy group, was helpful to enhance inhibitory activity against PDE4B,
which was consistent well with the observed docking simulation.
A
B
C
Figure 3. Docking study of compound If with PDE4B (green, A),
PDE4D (purple, B), the overlapping of PDE4B and PDE4D (C)
Acknowledgements
We acknowledge the financial supports from the National Key Project for Basic

Research (973 Program, 2015CB150600), the National Key Research and
Development Program of China (2017YFD0200504), the Special Fund for the
Research and Construction of Public Service Ability (2016A020217014), the Pearl
River S&T Nova Program of Guangzhou (201506010029).
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Graphical Abstract
Synthesis and bioactivity of 3,5-dimethyl pyrazole
derivatives as potential PDE4 inhibitors
De-Kun Hua,#, Dong-Sheng Zhaob,#, Min Hea, Hong-Wei Jinc, Yong-Mei Tangd,
Lian-Hui Zhanga, Gao-Peng Songd,*,Zi-Ning Cuia, *