Design, synthesis and biological evaluation of novel benzoxaborole derivatives as potent PDE4 inhibitors for topical treatment of atopic dermatitis

Article abstract of DOI:10.1016/j.ejmech.2021.113171

In this work, a series of structurally novel benzoxaborole derivatives were designed, synthesized and biologically evaluated as PDE4 inhibitors for battling atopic dermatitis (AD). Among them, the majority exhibited superior PDE4B inhibitory activities to that of the lead compound Crisaborole, an approved PDE4 inhibitor. In particular, 72, the most potent PDE4B inhibitor throughout this series, displayed 136-fold improved enzymatic activity (IC₅₀ = 0.42 nM) as compared to Crisaborole (IC₅₀ = 57.20 nM), along with favorable isoform specificity. In the phorbol ester (PMA)-induced mouse ear oedema model, 72 exerted remarkably greater efficacy than Crisaborole at the same dosage (P < 0.05). Moreover, the ointment of 72 exerted dramatically enhanced therapeutic potency than the ointment of Crisaborole (P < 0.05) in the calcipotriol-induced mouse AD model. In addition to the potent in vitro and in vivo activity, 72 displayed favorable safety in the repeated oral dose toxicity study and did not exhibit phototoxicity. With the above attractive biological performance, 72 is worthy of further functional investigation as a novel anti-AD therapeutic agent.

Full text of DOI:10.1016/j.ejmech.2021.113171

European Journal of Medicinal Chemistry 213 (2021) 113171
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of novel benzoxaborole
derivatives as potent PDE4 inhibitors for topical treatment of atopic
dermatitis
Zhaoxing Chu a, , Qinlong Xu , Qihua Zhu a , Xiaodong Ma , Jiajia Mo ,
b
,
c
c
,
d
,
b
d
c
c
c
c
c
c
c
c
Gaofeng Lin , Yan Zhao , Yuanfeng Gu , Lincui Bian , Li Shao , Jing Guo , Wenfeng Ye ,
Jiaming Li , Guangwei He
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 211198, China
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, 21009, China
Hefei Institute of Pharmaceutical Industry Co., Ltd., Hefei, 230088, China
d
c
,
**
a, b, *
, Yungen Xu
a
b
c
d
Department of Medicinal Chemistry, Anhui University of Chinese Medicine, Hefei, 230031, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 November 2020
Received in revised form
In this work, a series of structurally novel benzoxaborole derivatives were designed, synthesized and
biologically evaluated as PDE4 inhibitors for battling atopic dermatitis (AD). Among them, the majority
exhibited superior PDE4B inhibitory activities to that of the lead compound Crisaborole, an approved
PDE4 inhibitor. In particular, 72, the most potent PDE4B inhibitor throughout this series, displayed 136-
fold improved enzymatic activity (IC50 ¼ 0.42 nM) as compared to Crisaborole (IC50 ¼ 57.20 nM), along
with favorable isoform specificity. In the phorbol ester (PMA)-induced mouse ear oedema model, 72
exerted remarkably greater efficacy than Crisaborole at the same dosage (P < 0.05). Moreover, the
ointment of 72 exerted dramatically enhanced therapeutic potency than the ointment of Crisaborole
7
January 2021
Accepted 7 January 2021
Available online 12 January 2021
Keywords:
Benzoxaborole derivatives
Atopic dermatitis
Isoform specificity
In vivo efficacy
Safety
(
P < 0.05) in the calcipotriol-induced mouse AD model. In addition to the potent in vitro and in vivo
activity, 72 displayed favorable safety in the repeated oral dose toxicity study and did not exhibit
phototoxicity. With the above attractive biological performance, 72 is worthy of further functional
investigation as a novel anti-AD therapeutic agent.
©
2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
Massive efforts have been undertaken by the research com-
munity to unravel the exact etiology of AD. Although it remains
ambiguous, numerous studies have implicated the epidermal bar-
rier defects and the dysregulation of the immune system in the
pathogenesis of AD [6,7]. Cyclic adenosine monophosphate
(cAMP)-related signaling cascades play a critical role in regulating
the immune function. Under normal physiological conditions, the
cellular level of cAMP is retained by a dynamic equilibrium be-
tween the biosynthesis and cyclic nucleotide phosphodiesterases
(PDEs)-catalyzed decomposition [8]. The involvement of PDE4, the
most diverse family of PDEs, in a broad spectrum of inflammatory
disorders renders it a promising therapeutic target for these dis-
eases. Upon the inhibition of PDE4, the elevated intracellular cAMP
level triggers specific protein phosphorylation cascades and
consequently induces a variety of functional responses in the in-
flammatory cells, exemplified by the downregulation of tumor
Atopic dermatitis (AD) is the most common chronic inflamma-
tory skin disorder affecting young children. The prevalence of AD is
continuously increasing, with approximately 20% of children and
1
%e3% of adults suffering from it in developed countries [1].
Depending on the duration of the lesions, individuals bearing AD
experience pruritus, erythema, and dermatitic plaques which may
weep, crust or scale [2,3]. Currently, topical corticosteroids are
frequently prescribed for battling AD in clinic. However, the toxicity
resulted from the long-term treatment hindered their extensive
application. Hence, the development of nonsteroidal tropical anti-
inflammatory agents for combating AD is in urgent demand [4,5].
*
Corresponding author. Department of Medicinal Chemistry, China Pharma-
ceutical University, Nanjing, 211198, China.
*
* Corresponding author.
E-mail addresses: heguangwei@ahipi.com (G. He), xyg@cpu.edu.cn (Y. Xu).
necrosis factor alpha (TNF-a) production [9e11].
https://doi.org/10.1016/j.ejmech.2021.113171
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
The four subtypes of PDE4, termed as PDE4A, B, C and D, are
predominantly expressed in inflammatory and immune cells,
including B cells, T cells and macrophages. In particular, PDE4B and
PDE4D participate in modulating the liberation of pro-
inflammatory mediators and regulating neutrophil function,
thereby attracting considerable attention in both academic area
and pharmaceutical industry as anti-inflammatory targets. So far,
Roflumilast (1), Apremilast (2), Cilomilast (3) and Crisaborole (4), as
PDE4 inhibitors, have been approved for treating inflammation-
related diseases (Fig. 1). While 1e3 are applied to treat asthma
and arthritis upon oral administration, 4 is a nonsteroidal topical
anti-AD agent, which is capable of suppressing the release of in-
PDE4B inhibitor with excellent performance both in vitro and
in vivo. In addition to the remarkable efficacy, 72 featured favorable
safety profile. Owing to these advantages, it has the potential to be
an anti-AD candidate for further development.
2. Results and discussion
2.1. Chemistry
Scheme 1 described the synthetic route to compounds 29e32
[16e21]. 1-(2,4-Dihydroxyphenyl) ethan-1-one 6, as the starting
material, was condensed with triethyl orthoformate to afford 7-
flammatory cytokines, including TNF-a, IL-2, IL-5 and interferon-g
(
IFN- ) [12]. However, approximately 25% patients fail to benefit
g
hydroxy-4H-chromen-4-one
7
[22].
7-Hydroxy-2,2-
from 4 and need rescue therapy with either topical corticosteroids
or topical tacrolimus [13,14]. Additionally, some patients receiving
Crisaborole treatment suffer from local skin irritation.
dimethylchroman-4-one 10 was prepared via condensation of
resorcinol with 3,3-dimethacrylic acid and subsequent intra-
molecular cyclization in the presence of NaOH. Subsequently, 7 and
10e12 were subjected to SNAr reaction with 5-fluoro-2-
For surmounting the aforementioned drawbacks, we have
recently initiated a medicinal chemistry campaign to discover
effective and more specific PDE4 inhibitors. Our insight into the
crystal structure of AN2898 (5, Fig. 1), a Crisaborole derivative,
complexed with PDE4B catalytic site provided a foundation for the
rational design of novel PDE4 inhibitors from 4. AN2898 assumed a
distinct binding mode from other PDE4 inhibitors with the boron
atom coordinated to the water molecule held and activated by zinc
and magnesium ions (Fig. 2A and B). Provided this, the boron
adopted a tetrahedral form and mimicked the phosphate group of
AMP, hence conferring similar interactions with the metal ions [15].
These interactions were necessary for PDE4 binding affinity.
Nonetheless, the substituted phenyl of AN2898 was merely located
at the entrance to the adenine pocket formed by side chains of
residues Phe 446, Ile 410 and Gln 443 (Fig. 2C). Therefore, further
interactions with residues in this pocket may be beneficial to
enhancing the potency and specificity. Based on this consideration,
a structure-based drug design approach was employed in this work
via replacing the substituted phenyl of Crisaborole with structurally
diverse bicyclic fragments for exploiting the adenine pocket (Fig. 3).
Besides, as revealed by the reported SARs and pharmacophore
feature of the benzoxaborole-drived PDE4B inhibitors, electron-
withdrawing groups and H-bond acceptors on the substituents
tethered to the oxygen at C-5 position of the benzoxaborole core
were beneficial to the enzymatic activity. Hence, we prioritized
introduction of bicyclic scaffolds bearing electron-withdrawing
substituents or H-bond acceptors to replace the cyanophenyl.
Upon the strategy stated above, a novel series of Crisaborole ana-
logues were designed, synthesized, and biologically evaluated,
which led to the discovery of 72, a highly potent and selective
2 3
nitrobenzaldehyde in the presence of K CO to provide 13e16.
Reduction of the nitro functionality of 13e16 and the following
Sandmeyer reaction led to the generation of 21e24 as the in-
termediates. Afterwards, they were converted into corresponding
boric acid esters 25e28, which further underwent one-pot reduc-
tion of the aldehyde moiety and the intramolecular cyclization, as
well as the hydrolysis after treatment with 6 N HCl to furnish target
compounds 29e32 [23,24].
Scheme 2 depicted the preparative method for compounds
63e72. SNAr reaction of the imidazopyrimidine derivative 33 or the
quinoline derivatives 34e42 with 2-bromo-5- hydrox-
2 3
ybenzaldehyde in the presence of K CO provided 43 and 44e52,
respectively. They were then transformed into corresponding boric
acid esters, which experienced a similar route to Scheme 1 to afford
63 and 64e72 as the target compounds [25e27]. The structures of
13
1
all the compounds were characterized by C NMR, H NMR and
HRMS.
2.2. PDE4B inhibitory activity
All the target compounds were evaluated for their PDE4B
inhibitory activities with Crisaborole as the positive reference. The
results demonstrated that a majority of them exhibited superior
PDE4B inhibitory activities to that of Crisaborole, thereby validating
the rationality of the compound design. In particular, six com-
pounds, that are 64, 65, and 69e72, displayed single-digit nano-
molar or subnanomolar IC50 values. Compound 72 was the most
active PDE4B inhibitor throughout this series with IC50 value of
0.42 nM, which was 136-fold more potent than Crisaborole. From
the enzymatic activity shown in Table 1, some valuable structure-
activity relationships (SARs) can be concluded. In general, quino-
line as the bicyclic substituent was beneficial to PDE4B inhibitory
activity, which can be revealed by the more potent activities of
6
4e67 and 69e72 than those of 29e32. According to the biological
data of 64e66, it became evident that the position of COOMe at the
quinoline moiety did not significantly affect the potency. However,
replacement of the COOMe (64) at the C-5 position of the quinoline
with COOEt (67) and COOH (68) led to a decrease in PDE4B inhib-
itory activity. Particularly, the introduction of polar COOH weak-
ened the activity to submicromolar level. When COOH was replaced
3 3
by non-polar COCH and CF groups, the decrease in activity was
reversed, and both the resultant compounds 69 and 70 exhibited
single-digit nanomolar IC50s. Hence, polar substituents at the
quinoline had a detrimental effect on potency. To our delight, when
CF
3
substituted the C-6 position of the quinoline, 72, the most
Fig. 1. The chemical structures of the four approved PDE4 inhibitors and Crisaborole
derivative AN2895.
potent compound throughout this series, was attained.
2

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
Fig. 2. Co-crystal structure of AN2898 with the active site of PDE4B catalytic domain [15].
Fig. 3. The design rationale of target Crisaborole derivatives.
2.3. Effect on phorbol myristate acetate (PMA)-induced mouse ear
2.4. Isoform specificity
oedema
Compound 72, as the representative of this series, was then
evaluated for its specificity over other PDE isoforms. In general, the
specificity of 72 for PDE4B was more favorable over that of Crisa-
borole, according to experimental results shown in Table 3. In
particular, it exhibited over 10,000-fold selectivity over PDE3A,
PDE3B and PDE10A, and the specificity over these isoforms was
much higher than that of Crisaborole.
Based on the PDE4B inhibitory activity, nine compounds were
chosen for evaluating their anti-inflammatory activities against the
PMA-induced mouse ear oedema [28,29], and the results were
shown in Table 2. Compared with the blank, the model group
showed a dramatically increased swelling degree (P < 0.01), indi-
cating the availability of the model. The results illustrated that the
swelling degree was obviously reduced (P < 0.05, P < 0.01) by a
majority of compounds except 65 and 71 after 1 mg/ear. Among the
effective compounds, compound 72 induced the most remarkable
decrease in swelling degree with the inhibition rate of 65.85%, and
its efficacy surpassed that of Crisaborole at the same dosage. The
distinguished efficacy of 72 was consistent with its high PDE4B
inhibitory activity.
2.5. Effect on calcipotriol-induced AD in mice
By virtue of its attractive performance stated above, 72 was
formulated as ointment and further evaluated for its efficacy
against calcipotriol-induced mouse AD model [30,31]. As revealed
by the data presented in Table 4, treatment with the ointment of 72
3

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
Scheme 1. Reagents and conditions: (i) 70% HClO
4
, Triethyl orthoformate, rt., 10min; (ii) 3,3-dimethacrylic acid, ZnCl
2
, POCl
3
, rt., 2 h; (iii) 5% NaOH, rt., 1 h; (iv) 5-fluoro-2-
ꢀ
CN, ꢁ10 ꢀC, 0.5 h; (vii) B
ꢀ
nitrobenzaldehyde, K
ꢀ
3
.5 h; (viii) NaBH , 6 N HCl, CH OH, 0 C,1 h.
2 3
CO , DMF, rt., 24 h; (v) Fe, NH
4
Cl, EtOH/H
2
O, 80 C, 2.5 h; (vi) CuBr, t-BuONO, CH
3
2
pin
2
, Pd (dppf)Cl
2
, AcOK, 1, 4-dioxane, 90 C,
1
4
ꢀ
ꢀ
Scheme 2. Reagents and conditions: (i) 2-bromo-5-hydroxybenzaldehyde, K
CH OH, rt., 0.5 h.
2
CO
3
, DMF, 100 C, rt., 24 h; (ii) B
2
pin
2
, pd (dppf)Cl
2
, AcOK, 1, 4-dioxane, 100 C, 2 h; (vi) NaBH
4
, HCl,
3
caused obvious decrease in swelling degree at the dose of 30 mg/
ear. Importantly, the ointment with 2% 72 resulted in more
remarkable efficacy than ointment with 2% Crisaborole at the same
dosage, and the inhibition rate was 73.33%.
comparing the 400 mg/kg and 700 mg/kg dose groups with the
normal control group in autopsy at the end of the experiment.
These results indicated that compound 72 exhibited favorable
safety with a no observed adverse effect level (NOAEL) dose over
400 mg/kg.
2.6. Repeated dose toxicity
2.7. Phototoxicity
We further performed a preliminary repeated oral dose toxicity
study of 72 in order to evaluate its safety profile. The 14-
consecutive-day oral administration of compound 72 did not
cause any systemic toxicity or animal death even at a dose as high
as 700 mg/kg. No significant difference was observed in the liver/
brain index, kidney/brain index, spleen/brain index and white
blood cell count, ALT, AST, blood pressure, and blood glucose, when
In addition to the repeated oral dose toxicity, we also evaluated
the toxicity of the ointment of 72 induced by UVA irradiation via
Lovelland Sander method [32]. As displayed in Table 5, the ery-
thema and oedema occurred in the 8-methoxypsoralen (8-MOP)
group at 24 h post-irradiation, and the phototoxicity was dimin-
ished from 48 h to 72 h post-treatment. Neither erythema nor
4

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
Table 1
PDE4B inhibitory activity of target compounds bearing different various bicyclic.
Cpd.
Bicyclic substituents
/
PDE4B(IC50, nM)
57.20
Cpd.
Bicyclic substituents
PDE4B(IC50, nM)
10.79
Crisaborole
66
2
3
3
3
9
0
1
2
18.90
65.50
21.88
25.49
67
68
69
70
17.29
124.1
3.05
6.42
6
6
3
4
46.20
6.44
71
72
3.57
0.42
65
8.25
Table 2
Effect of selected compounds on PMA-induced oedema in the mouse ear
Table 3
Selectivity of compound 72 and Crisaborole over other nine PDE isoforms enzymes.
(
Mean ± SD, n ¼ 8).
PDEs
IC50(nM)
Group
Dose (mg/ear)
Swelling (mm)
Inhibition (%)
Crisaborole
72
blank
model
Crisaborole
/
/
0.25 ± 0.04
/
/
△
△
PDE1A
PDE1B
PDE1C
PDE2A
PDE3A
PDE3B
PDE4B
PDE5A
PDE7A
PDE10A
5600 (85-fold)a
17,900 (271-fold)
64.7 (154-fold)a
58.0 (138-fold)a
866.0 (2062-fold)
120.0 (286-fold)a
22.55 ± 8.39
12.73 ± 4.75*
9.53 ± 3.32**
15.65 ± 5.50
10.21 ± 4.91**
9.54 ± 3.49**
12.85 ± 6.27*
13.63 ± 3.12*
9.46 ± 3.99**
14.88 ± 3.80*
a
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
43.55
55.62
30.60
54.72
57.69
45.76
39.56
58.05
34.09
65.85
a
a
71,000 (1076-fold)
64
65
66
67
68
69
70
71
72
a
3630 (55-fold)
a
a
64,000 (970-fold)
58,800 (891-fold)
70.0
8130 (19,357-fold)
a
7190 (17,119-fold)a
0.42
a
a
56,100 (850-fold)
819 (1950-fold)
34.7 (83-fold)
a
a
2030 (31-fold)
a
4520 (10,762-fold)a
97,600 (1479-fold)
#
a)
7.70 ± 2.95**
Selectivity fold.
#P < 0.05 statistical significance compared with Crisaborole; *P < 0.05.
**P < 0.01 compared with model.
△
△
P < 0.05 compared with blank.
the epidermis were observed at 72 h in back skin (Fig. 4B). Thus, the
treatment with the ointment of 72 did not induce phototoxicity.
oedema was monitored in the groups treated by the ointment of 72,
even at a high dose, indicating no phototoxicity of 72.
2.8. Molecular modeling
We then conducted the pathological analysis, and the results
illustrated that the epidermis of Crisaborole-treated (Fig. 4C) and
To elucidate its possible binding mode with PDE4B, compound
72 was docked into the active site of PDE4B on the basis of the
available PDE4B/AN-2898 co-crystal structure (PDB code 3O0J)
[33]. As demonstrated in Fig. 5, 72 (blue) assumed a similar binding
mode to that of AN-2898 (yelow) (Fig. 5B). The benzoxaborole was
7
2-treated (Fig. 4DeF) groups were intact. Besides, no infiltration of
inflammatory cells and necrosis were observed in Crisaborole or
2-treated (Fig. 4DeF) groups. By contrast, in the 8-MOP group,
inflammatory cell infiltration in the dermis and necrotic changes in
7
5

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
Table 4
The swelling inhibition of selected compounds against calcipotriol-induced mouse AD model (Mean ± SD, n ¼ 10).
Group
Dose (mg/ear)
Thickness (mm)
left ear
Swelling
Inhibition (%)
right ear
normal
model
e
0.26 ± 0.02
0.26 ± 0.01
0.29 ± 0.03
0.29 ± 0.03
0.26 ± 0.02
0.55 ± 0.06
0.37 ± 0.05
0.32 ± 0.02
e
e
e
0.29 ± 0.05
0.08 ± 0.04*
0.03 ± 0.01**
e
2% Crisaborole
30
30
32.50
73.33
:
:
2% 72
*
*
P < 0.05.
*P < 0.01vs model.
:
P < 0.05.
P < 0.01 vs Crisaborole.
:
:
Table 5
Skin reaction scores at each time point.
therapeutic efficacy and safety will offer benefits for AD patients. In
this study, we discovered a series of structurally novel benzox-
aborole derivatives as PDE4 inhibitors via structure-based drug
design on the basis of Crisaborole, an approved anti-AD agent. A
majority of them exerted PDE4B inhibitory activities superior to
that of Crisaborole. 72, the most active compound throughout this
series, also exhibited attractive PDE4 specificity, which was 136-
fold more potent than Crisaborole. In both PMA-induced mouse
ear oedema model and calcipotriol-induced mouse AD model, 72
led to more remarkable therapeutic efficacy than Crisaborole at the
same dosage. Moreover, as revealed by the repeated oral dose
toxicity study and the phototoxicity investigation, 72 also displayed
favorable safety profile. Attributed to its aforementioned excellent
in vitro and in vivo biological performance, it has been progressed
into further pre-clinical investigation.
Group
Dose (mg/kg)
Skin reaction score after UVA irradiation
1
h
24 h
48 h
72 h
normal
e
e
5
0
0
0
0
0
0
0
0
0
8-MOP
3.8 ± 0.8
3.7 ± 0.5
2.5 ± 0.5
72
0
0
0
0
0
0
0
0
0
0
0
0
1
2
0
4
Crisaborole
10
engaged in coordination to Mg2þ and Zn ions, H-bond interaction
with His 234, as well as hydrophobic contact with Met 347.
Importantly, the quinoline substituent occupied the entrance of a
2þ
deep hydrophobic pocket and conferred p-p stacking with the ar-
omatic ring of Phe 414 and Phe 285, which may account for the
increase in enzymatic potency after the introduction of quinoline as
the bicyclic substituent.
4. Experimental section
4.1. Chemistry
3
. Conclusions
The reagents and solvents for reaction were commercially
Given the medicinal potential of PDE4 as anti-AD target, the
exploration of PDE4 inhibitors with favorable isoform specificity,
available, and when necessary, pre-treatment was carried out prior
to use. Tetramethylsilane (TMS) was used as the internal standard
Fig. 4. Representative histopathological findings at 72 h after light exposure in each group (hematoxylin and eosin, scale bar: 50
mm). In the 8-MOP group(B), epidermal thicken
(
arrow head NO.1) and dermal inflammatory cell infiltration (arrow head NO.2) were observed in the back skin. There were no infiltration of inflammatory cells and necrosis
observed in normal (unirradiated) albino guinea pig’s skin (A), crisaborole group (C) and 5 mg/kg (D), 10 mg/kg (E), 24 mg/kg (F) of 72 group.
6

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
Fig. 5. The molecular docking result of 72 into PDE4B catalytic site (
p-
p
stacking, H-bond contact, and the interaction with metal ion were shown as pink, green and gray dashed
line, respectively).
for recording H NMR and 13C NMR spectra, and the spectra were
1
4.1.4. 7-((1-Hydroxy-1,3-dihydrobenzo[c] [1,2]oxaborol- 5-yl)oxy)-
4H-chromen-4-one (29)
4.1.4.1. 2-nitro-5-((4-oxo-4H-chromen-7-yl)oxy)benzaldehyde(13).
To a solution of 7-hydroxy-4H-chromen-4-one (7.54 g, 46.5 mmol,
obtained with
Switzerland). MS spectra were collected with a Hewlett-Packard
100 LC/MSD spectrometer (Agilent, Waldbronn, Germany).
a 400 MHz instrument (Bruker, Fallanden,
1
1eq) in DMF (55 mL) were added 5-fluoro-2-nitrobenzaldehyde
(
8.65 g, 51.2 mmol, 1.1eq) and K
2
CO
3
(9.64 g, 69.8 mmol, 1.5eq),
ꢀ
4.1.1. 7-Hydroxy-4H-chromen-4-one (7)
and the reaction mixture was stirred for 1 h at 80 C. The mixture
was cooled to 0 C, poured to 0.5 N HCl solution (165 mL) and
ꢀ
To a solution of 1-(2,4-dihydroxyphenyl)ethan-1-one (12.0 g,
.08 mmol, 1eq) in triethyl orthoformate (30 mL) was added 70%
HClO (8.4 mL, 0.147 mol, 1.83eq) dropwise within 10 min at room
4
temperature. After being stirred for 3 h, the reaction mixture was
treated with anhydrous diethyl ether (216 mL) and the resultant
mixture was stirred for 10 min. The mixture was filtered and the
filter cake was washed with anhydrous diethyl ether (50 mL ꢂ 3).
The filter cake was added to boiling water (360 mL) in portions. The
mixture was stirred till the water cooled to room temperature.
Following filtration, the precipitate was washed with water
0
stirred for 30 min at room temperature. Following filtration, the
precipitate was washed with water (20 mL ꢂ 3) to afford inter-
1
mediate 13 as a brown solid. Yield 96.7%; H NMR (400 MHz,
DMSO‑d
1H), 8.13 (d, J ¼ 8.8 Hz, 1H), 7.58e7.55 (m, 1H), 7.49e7.47 (m, 2H),
7.31e7.28(dd, J ¼ 8.8 Hz, 1H), 6.38 (d, J ¼ 6.0 Hz, 1H).
¼ 1.6 Hz, J
6
):d
10.26 (s, 1H), 8.30 (d, J ¼ 6.0 Hz, 1H), 8.27 (d, J ¼ 8.8 Hz,
1
2
4.1.4.2. 2-Amino-5-((4-oxo-4H-chromen-7-yl)oxy)benzaldehyde
(17). To a solution of 2-nitro-5-((4-oxo-4H-chromen-7-yl)oxy)
benzaldehyde (13.6 g, 43.7 mmol, 1eq) in MeOH(200 mL) and
DCM(200 mL) was added Pd/C (10%, 1.36 g), and the reaction
mixture was stirred for 24 h at room temperature under H2 at-
mosphere (15 psi). The reaction mixture was filtered through Celite,
and the filtrate was concentrated in vacuo. The residue was purified
by column chromatography using EA/PE (1:10e1:3) as the eluent to
(
100 mL ꢂ 3) to afford intermediate 7 as a brown solid. Yield 58.9%;
1
H NMR (400 MHz, DMSO‑d
6
):
H), 7.88 (d, J ¼ 8.8 Hz, 1H), 6.90e6.93(dd, J
H), 6.85 (d, J ¼ 2.0 Hz, 1H), 6.21 (d, J ¼ 6.0 Hz, 1H).
d
10.77 (s, 1H), 8.14 (d, J ¼ 6.0 Hz,
1
1
1
¼ 2.0 Hz, J ¼ 8.8 Hz,
2
1
4
.1.2. 11-(2,4-Dihydroxyphenyl)-3-methylbut-2-en-1-one (9)
give intermediate 8 as a yellow powder. Yield 39.8%; H NMR
To a round bottom flask were added resorcinol (11 g, 0.1 mol,
(400 MHz, DMSO‑d
J ¼ 8.8 Hz, 1H), 7.47 (d, J ¼ 2.8 Hz, 1H), 7.26e7.23 (m, 1H), 7.19 (s,
2H), 7.11e7.08(dd, J
1H), 6.89 (d, J ¼ 9.2 Hz, 1H), 6.30 (d, J ¼ 6.0 Hz, 1H).
6
):
d
9.82 (s, 1H), 8.23 (d, J ¼ 6.0 Hz, 1H), 8.02 (d,
1
0
eq), 3-methylbut-2-enoic acid (10 g, 0.1 mol, 1eq), ZnCl
2
(20.4 g,
.15 mmol, 1.5eq) and POCl (82 mL). After being stirred for 2 h at
3
1
¼ 2.4 Hz, J ¼ 6.4 Hz, 1H), 6.98 (d, J ¼ 2.4 Hz,
2
room temperature, the reaction mixture was cooled to room tem-
perature and poured to ice water (2 L). Following filtration, the
precipitate was washed with water (100 mL ꢂ 3), and then
4.1.4.3. 2-Bromo-5-((4-oxo-4H-chromen-7-yl)oxy)benzaldehyde
(21). To a solution of 2-amino-5-((4-oxo-4H-chromen-7-yl)oxy)
recrystallized with EtOH/H
white solid. Yield 52.0%; H NMR (400 MHz, DMSO‑d
2
O to give the title intermediate as a
1
6
):
d
ppm 13.27
benzaldehyde (4.96 g, 17.7 mmol, 1eq) in anhydrous CH CN(40 mL)
3
(
s, 1 H), 10.59 (s, 1 H), 7.83 (d, J ¼ 4.8 Hz, 1 H), 6.91 (m, 1 H), 6.34 (d,
were added CuBr
2
(5.93 g, 26.6 mmol, 1.5eq) and t-BuONO (2.74 g,
ꢀ
J ¼ 8.8 Hz, 1 H), 6.25e6.26 (m, 1 H), 2.14 (d, J ¼ 1.2 Hz, 1 H), 2.01 (d,
26.6 mmol, 1.5eq) at ꢁ10 C. The reaction mixture was stirred for
J ¼ 1.2 Hz, 1 H).
3 h at room temperature under N
2
atmosphere. The reaction
mixture was filtered through Celite, and the filtrate was concen-
trated in vacuo. The residue was purified by column chromatog-
4
.1.3. 7-Hydroxy-2,2-dimethylchroman-4-one (10)
To a round bottom flask were added 1-(2,4-dihydroxyphenyl)-3-
raphy using DCM/EA/PE (1:1:10) as the eluent to give intermediate
1
9
as
a
white powder. Yield 55.9%;
):
H NMR (400 MHz,
10.19 (s, 1H), 8.28 (d, J ¼ 6.0 Hz, 1H), 8.08 (d, J ¼ 8.8 Hz,
methylbut-2-en-1-one (10 g, 0.052 mol, 1eq) and 5% NaOH solution
30 mL). After stirring the reaction mixture for 1 h at room tem-
perature, the pH of the mixture was adjusted to 3 with HCl solution
3 N), and precipitation occurred. Following filtration, the precipi-
tate was washed with water (100 mL ꢂ 3) to afford the title inter-
DMSO‑d
6
d
(
1H), 7.90 (d, J ¼ 8.4 Hz, 1H), 7.55 (d, J ¼ 2.8 Hz, 1H), 7.50e7.48 (m,
1H), 7.28 (d, J ¼ 2.0 Hz, 1H), 7.21e7.18(dd, J
1H), 6.35 (d, J ¼ 6.0 Hz, 1H).
1
¼ 2.0 Hz, J ¼ 8.8 Hz,
2
(
1
mediate as a white solid. Yield 99%; H NMR (400 MHz, DMSO‑d
ppm 10.48 (s, 1 H), 7.58 (d, J ¼ 4.2 Hz, 1 H), 6.43 (dd, J ¼ 2.0 Hz,
¼ 4.2 Hz, 1 H), 6.26 (d, J ¼ 1.2 Hz, 1 H), 2.66 (s, 2 H), 1.37 (s, 6 H).
6
):
4.1.4.4. 5-((4-oxo-4H-chromen-7-yl)oxy)-2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan -2-yl) benzaldehyde (25). To a solution of 2-
bromo-5-((4-oxo-4H-chromen-7-yl)oxy)benzaldehyde (1.46 g,
d
1
J
2
7

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
4
.23 mmol,1eq) in 1, 4-dioxane (60 mL) were added bis(pinacolato)
temperature, and the pH was adjusted to 2 with HCl solution (3 N).
diboron (2.15 g, 8.46 mmol, 2eq), AcOK (1.25 g, 12.7 mmol, 3eq) and
Following filtration, the precipitate was washed with water
Pd (dppf)Cl
being stirred overnight at 60 C under N
2
(0.16 g, 0.21 mmol, 0.05eq) at room temperature. After
(3 mL ꢂ 3) to afford the title compound as a white solid. Yield
ꢀ
1
2
atmosphere, the reaction
58.6%. H NMR (400 MHz, DMSO‑d
6
):
d
ppm 9.26 (s, 1 H), 9.20 (d,
mixture was filtered through Celite. The filtrate was concentrated
in vacuo, and the residue was purified by column chromatography
using EA/PE (1:10e1:1) as the eluent to give the title intermediate
as a yellow oil. Yield 96.3%.
J ¼ 7.28 Hz, 1 H), 8.48 (s, 1 H), 7.83 (d, J ¼ 8.03 Hz, 1 H), 7.44 (s, 1 H),
7.32 (d, J ¼ 7.78 Hz, 1 H), 6.98 (d, J ¼ 7.53 Hz, 1 H), 5.03 (s, 2 H), 3.67
13
6
(s, 3 H); C NMR (100 MHz, DMSO‑d ) d 163.36, 162.25, 156.29,
154.88, 147.61, 146.65, 140.71, 132.26, 121.07, 114.92, 102.11, 100.85,
7
0.20, 51.33. ESI-HRMS: m/z calcd for C15
H12BN
3
O
5
[MþH]þ
4.1.4.5. 7-((1-Hydroxy-1,3-dihydrobenzo[c] [1,2]oxaborol-5-yl)oxy)-
326.0948, found 326.0949.
4
H-chromen-4-one (29). To a solution of 5-((4-oxo-4H-chromen-7-
Compounds 64e72 were prepared via a similar procedure to
that for 63, and the data for structural characterization of the in-
termediates and target compounds were provided in the support-
ing information.
yl)oxy)-2-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl) benzalde-
hyde (1.60 g, 4.08 mmol, 1eq) in MeOH(4 mL) was added NaBH
4
ꢀ
(
0.23 g, 6.12 mmol, 1.5eq) in portions at 0 C. After being stirred for
3
0
3
3
0 min at room temperature, the reaction mixture was cooled to
C and quenched with water (1 mL). The pH was then adjusted to
with HCl solution (6 N), and the resultant mixture was stirred for
0 min. Following filtration, the precipitate was washed with water
ꢀ
4.2. Biological evaluation
4.2.1. PDE biochemical assay
(
7
2 mL ꢂ 3) and further purified by prep-TLC (PE/DCM ¼ 1:2) to give
The inhibitory activity against PDEs was tested by measuring the
3
3
3
3
-((1-hydroxy-1,3-dihydrobenzo [c] [1,2] oxaborol-5-yl)oxy)-4H-
hydrolysis of [ H]-cAMP or [ H]-cGMP into [ H]-AMP or [ H]-GMP,
respectively, using a phosphodiesterase scintillation proximity
assay (SPA). The protein was diluted to a concentration of 1e2 nM
1
chromen-4-one as a white solid. Yield 16.7%; H NMR (400 MHz,
DMSO‑d ):
ppm 9.19 (s, 1 H), 8.21 (d, J ¼ 6.0 Hz,1 H), 8.01e8.03 (m,
H), 7.78 (d, J ¼ 7.6 Hz, 1 H), 7.17 (br, 1 H), 7.12 (br, 2 H), 7.10 (t,
6
d
1
with an assay buffer (50 mM Tris pH 7.5, 8.3 mM MgCl
EGTA). For PDE1A, PDE1B, PDE1C, the reaction buffer also contained
0.2 mM CaCl and 0.36 mM calmodulin. The compound solution
2
, 1.7 mM
13
J ¼ 2.4 Hz, 1 H), 6.29 (d, J ¼ 6.0 Hz, 1 H), 4.95 (s, 2 H), C NMR
(
100 MHz, DMSO‑d : 176.10, 161.77, 157.78, 157.31, 133.08, 132.66,
6
)
d
2
1
27.78,120.49,119.54,119.39,116.83,113.15,112.79, 61.61, 70.17. ESI-
experienced 3-fold serial dilution for 10 doses, and Crisaborole was
introduced as the reference compound. The reaction assay was
þ
HRMS: m/z calcd for C16
H11BO
5
[MþH] 295.0778, found 295.0780.
Compounds 30e32 were prepared via a similar procedure to
that for 29, and the data for structural characterization of the in-
termediates and target compounds were provided in the support-
ing information.
initiated by successively adding 80
test compound solution, and 10
(0.5 Ci/mL) to a “low binding” plate, and all reactions were carried
m
L of protein solution, 10
m
L of
3
3
m
L of [ H]-cAMP or [ H]-cGMP
m
ꢀ
out in duplicate. The plate was incubated at 30 C for 30 min, and
the reaction was terminated by addition of phosphodiesterase SPA
4
.1.5. Methyl 5-((1-hydroxy-1, 3-dihydrobenzo[c] [1,2]oxaborol-5-
yl)oxy)pyrazolo [1,5-a] pyrimidine-3-carboxylate (63)
.1.5.1. 5-Hydroxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
benzaldehyde (43). To solution of 2-bromo-5-
beads (50 mL, RPNQ0150, PerkinElmer Inc.). All plates were settled
for 20 min before being counted with a MicroBeta 2 (PerkinElmer
Inc.) counter. All experimental data were analyzed by using
GraphPad Prism 5.0 to determine IC50 values.
4
a
hydroxybenzaldehyde (2.0 g, 9.95 mmol, 1eq) in 1,4-
dioxane(45 mL) were added bis(pinacolato)diboron (3.16 g,
4.2.2. Assay for evaluating the effect on PMA-induced mouse ear
oedema
12.44 mmol, 1.25eq), AcOK (2.93 g, 29.8 mmol, 3eq) and Pd (dppf)
Cl
2
(364 mg, 0.49 mmol, 0.05eq) at room temperature. After being
The ethanolic extract containing target compounds were topi-
cally administrated (1.0 mg/ear in ethanol) before PMA application,
and the positive reference group was treated with Crisaborole (1
mg/ear in acetone). The right ear of ICR mice (male, 8-week-old)
ꢀ
stirred for 1 h at 100 C under N
2
atmosphere, the reaction mixture
was cooled to room temperature and filtered through on Celite. The
filtrate was concentrated in vacuo and the residue was purified by
column chromatography using EA/PE (1:10) as the eluent to give
the title intermediate as a pale yellow solid. Yield 87.9%.
received PMA (2
mg/ear, in 20
mL acetone) to induce oedema, while
the left ear, as the blank, received acetone (20
mL). After 4 h, the
animals were euthanized by cervical dislocation, and thickness of
both ears were measured by micrometer. Ear oedema was deter-
mined as the difference between the thickness of both ears. The
inhibition percentage was expressed as a reduction in thickness
compared to the model group.
4
.1.5.2. Methyl 5-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl) phenoxy) pyrazolo [1,5-a] pyrimidine-3-
carboxylate (53). To solution of 5-hydroxy-2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl) benzaldehyde (2.15 g,
.67 mmol, 1eq) in MeOH(20 mL) was added NaBH (426 mg,
1.3 mmol, 1.3eq) in portions. The reaction mixture was stirred for
0 min at room temperature. Afterwards, the pH of the mixture was
a
8
1
3
4
4.2.3. Assay for evaluating the efficacy against calcipotriol-induced
AD in mice
adjusted to 2 with HCl solution (3 N), and the resultant mixture was
stirred overnight at room temperature. The mixture was concen-
trated in vacuo, and the precipitate was filtered and washed with
water (3 mL ꢂ 3) to afford intermediate as a white solid. Yield 57.7%.
According to a published protocol, the low-calcemic analogue of
vitamin D3, calcipotriol (purchased from Sigma, 2 nM, 20 mL, dis-
solved in ethanol), was applied topically to the dorsal side of left ear
for establishing AD mouse model [34]. A total of 32 mice were
randomly assigned into four groups: normal group, AD model
group, the group treated by the ointment of Crisaborole, and the
group treated by the ointment of 72. The ointment of Crisaborole
and the ointment of 72 were administered to the dorsal side of left
ear twice a day (30 mg/kg) for 14 days, while normal group received
ethanol (20 mL). At the 17th day, the animals were anaesthetized
with 10% chloral hydrate. A micrometer (Oditest Kroeplin) was used
for measuring ear thickness, and the obvious increase in ear
4
.1.5.3. Methyl 5-((1-hydroxy-1,3-dihydrobenzo[c] [1,2]oxaborol-5-
yl)oxy)pyrazolo [1,5-a] pyrimidine -3-carboxylate (63). To a solu-
tion of benzo [c] [1,2]oxaborole-1,5(3H)-diol (35 mg, 0.24 mmol,
1
eq) in DMF(2 mL) were added methyl 5-chloropyrazolo [1,5-a]
pyrimidine -3-carboxylate (50 mg, 0.24 mmol, 1eq) and Cs CO
154 mg, 0.47 mmol, 2eq), and the reaction mixture was stirred for
2
3
(
3
ꢀ
0 min at 50 C. Afterwards, the mixture was cooled to room
8

Z. Chu, Q. Xu, Q. Zhu et al.
European Journal of Medicinal Chemistry 213 (2021) 113171
thickness, calculated by subtracting the thickness of the left ear
Acknowledgment
(
vehicle) from the thickness of the right ear (calcipotriol-treated),
indicated that the ear was oedematous. Upon comparing the
treated and non-treated animals, the percentage of inhibition of the
inflammatory reaction was determined.
This work was supported by National Major Scientific and
Technological Special Project for “Significant New Drugs Develop-
ment” (2012ZX09401006), the Outstanding Scientific and Techno-
logical Innovation Team Projects of Jiangsu Province, China (2015).
4
4
.3. Toxicity
Appendix A. Supplementary data
.3.1. Repeated dose toxicity
Healthy male and female Sprague-Dawley rats (8-week old)
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113171.
were randomly divided into three groups with each group con-
taining 6 male animals and 6 female ones. 1% CMC-Na (for vehicle
group) or graded doses of compound 72 (400 and 700 mg/kg) were
administered to corresponding group by oral gavage once daily for
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