Synthesis and identification of quinoline derivatives as topoisomerase I inhibitors with potent antipsoriasis activity in an animal model

Article abstract of DOI:10.1016/j.bioorg.2019.03.073

Psoriasis is a chronic inflammatory and immune-mediated skin disease. Although certain agents have shown clinical success in treating psoriasis, development of safe and effective strategies for the treatment of this condition remains important. Research suggests that DNA topoisomerase I (Topo I)inhibitors may have potent psoriasis-ameliorating effects. Here, 25 quinoline derivatives were synthesized and identified as Topo I inhibitors. These compounds inhibited the 12–O–tetradecanoylphorbol-13-acetate-induced mouse ear inflammation. The most potent analogs, 5i and 5l, suppressed the expression of inflammatory cytokines in lipopolysaccharide-stimulated HaCaT cells. Additionally, the lead compounds significantly improved imiquimod-induced psoriasis-like inflammation in mice. Moreover, the expression levels of cytokines and inflammatory mediators, such as interleukin (IL)-17A, IL-22, IL-23, nuclear factor-κB subunit p65, tumor necrosis factor-α, and interferon-γ, were dramatically inhibited in the dorsal skin of 5i- and 5l-treated mice. These findings indicate that the inhibition of Topo I activity may potentially be an effective strategy for psoriasis treatment.

Full text of DOI:10.1016/j.bioorg.2019.03.073

Bioorganic Chemistry 88 (2019) 102899
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and identification of quinoline derivatives as topoisomerase I
inhibitors with potent antipsoriasis activity in an animal model
a,1
a,d
a,b,1
a
a
a,c
Wen-Jin Zhang , Peng-Hui Li
, Min-Cong Zhao , Yao-Hao Gu , Chang-Zhi Dong ,
a,⁎
Hui-Xiong Chen , Zhi-Yun Du
a
b
c
Institute of Natural Medicine & Green Chemistry, School of Chemical Engineering and Light Industry, Guandong University of Technology, Guangzhou 510006, China
School of Life Science, Huizhou University, Huizhou 516001, China
Universite Paris Diderot, Sorbonne Paris Cite, ITODYS, UMR 7086 CNRS, 15 rue J-A de Baif, 75270 Pairs Cedex 13, France
CNRS, UMR8601, Laboratoire de Chimine et Biochimie Pharmacologiques et Toxicologiques, CBNIT, Universite Paris Descartes PRES Sorbonne Paris Cite, UFR
d
Biomedicale, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Psoriasis is a chronic inflammatory and immune-mediated skin disease. Although certain agents have shown
clinical success in treating psoriasis, development of safe and effective strategies for the treatment of this con-
dition remains important. Research suggests that DNA topoisomerase I (Topo I) inhibitors may have potent
psoriasis-ameliorating effects. Here, 25 quinoline derivatives were synthesized and identified as Topo I in-
hibitors. These compounds inhibited the 12–O–tetradecanoylphorbol-13-acetate-induced mouse ear inflamma-
tion. The most potent analogs, 5i and 5l, suppressed the expression of inflammatory cytokines in lipopoly-
saccharide-stimulated HaCaT cells. Additionally, the lead compounds significantly improved imiquimod-induced
psoriasis-like inflammation in mice. Moreover, the expression levels of cytokines and inflammatory mediators,
such as interleukin (IL)-17A, IL-22, IL-23, nuclear factor-κB subunit p65, tumor necrosis factor-α, and interferon-
γ, were dramatically inhibited in the dorsal skin of 5i- and 5l-treated mice. These findings indicate that the
inhibition of Topo I activity may potentially be an effective strategy for psoriasis treatment.
Psoriasis
Topoisomerase I
Quinoline derivatives
Proinflammatory markers
Imiquimod-induced inflammation
1
. Introduction
etanercept, ustekinumab, and ixekizumab) [11–15]. These drugs are
often effective for moderate to severe psoriasis. However, most of them
Psoriasis is a chronic inflammatory and immune-mediated skin
cause considerable toxicity and side effects, and a long duration of
treatment is needed [2,16,17]. Although the drugs usually have strong
effects in early treatment, they cannot be used in long term because of
immune deregulation [18]. More seriously, once the treatment is
stopped, patients may experience more severe symptoms [19]. Hence, it
is urgent to develop safe and effective strategies for the treatment of
psoriasis [1,20].
disease characterized by hyperproliferation and poor differentiation of
keratinocytes [1–3]. Psoriasis has prevalence ranging from 0.2% to
4
.8%, and associated with economic and clinical factors and sig-
nificantly affects the patient’s quality of life [4]. The mechanism of
pathogenesis has been thoroughly investigated for psoriasis in the last
three decades, and significant evidence indicates that the immune re-
sponse, secreted factors, and cellular mediators play important roles in
the development and maintenance of psoriasis [5–7]. Moreover, studies
have suggested that many cytokines and inflammatory mediators, such
as nuclear factor (NF)-κB, interleukin (IL)-22, IL-6, IL-17, IL-23, inter-
feron (IFN)-γ, and tumor necrosis factor (TNF)-α, are involved and in-
teract as a network in the pathogenesis of psoriasis [8–10]. Currently,
the main approaches to the treatment of psoriasis include systemic
antiproliferative or immunosuppressive agents, such as vitamin D
analogs, methotrexate, and acitretin, as well as biological agents (e.g.,
DNA topoisomerase I (Topo I) is a nuclear enzyme essential for re-
solving topological problems that occur during DNA transcription, re-
plication, and chromosome segregation [21]. Topo I plays a critical role
in cell proliferation and is considered as important target for the pre-
vention of rapid proliferation of cancer cells [22]. Several Topo I in-
hibitors have been widely used in the clinic for decades not only as anti-
cancer drugs, but also for their curative effects on psoriasis [23]. For
instance, camptothecin (CPT, Fig. 1) has exhibited a significant curative
efficacy in the treatment of psoriasis [24]. Lin et al. [25] have
⁎
Corresponding author.
E-mail address: zhiyundu@gdut.edu.cn (Z.-Y. Du).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.03.073
Received 18 January 2019; Received in revised form 26 February 2019; Accepted 30 March 2019
Available online 04 April 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
2.2. QCs as potent Topo I inhibitors
Topo I-mediated supercoiled pBR322 DNA plasmid relaxation assay
was first carried out to evaluate the inhibitory activities of QCs against
Topo I [38]. Compound 5a-5o with different substitutions on the
phenyl ring were first synthesized and evaluated. The results, presented
in Fig. 2, showed that the positive control (CPT) strongly inhibited the
Topo I activity. Compound 5a, 5b, 5d, 5e, 5g, 5h, 5n, 5i and 5l dis-
played potent Topo II inhibition activities at 50 μM (Fig. 2A and
Fig. 2B). Among these compounds, 5i and 5l displayed the best activity
at 20 μM (Fig. 2C). It was found that the substituent in the phenyl ring
as well as its position have significant impact on the inhibition of Topo
II activity. Compounds 5l, 5m and 5n, which contain a fluorine on
phenyl ring, respectively, on the p-position (5l) displayed better activ-
ities than on the ortho (5m)- or meta-positions (5n). In addition,
compounds 5j and 5k with two fluorine on phenyl ring have no effect
on Topo I inhibition. Compound 5i with an fluorine on ortho-position
and an bromine on p-position show much more potency to inhibit Topo
I. Compounds 5o-5y were synthesized on the basis of 5i and 5l. How-
ever, all of these derivatives exhibited poorer Topo I inhibitory activ-
ities than those of 5i and 5l (Fig. 2A–C).
Fig. 1. Structures of CPT and quinoline derivatives.
subsequently revealed that CPT and isoCPT inhibited the growth of
cultured normal human adult keratinocytes in vitro by inducing
apoptosis. Their further results have demonstrated that the therapeutic
mechanism of CPT against psoriasis may be associated with its anti-
proliferative activity and the apoptosis of keratinocytes may be medi-
ated through downregulation of telomerase activity [26,27]. Marazzi
et al. [28] have found that chemical inhibition of Topo I suppressed the
expression of proinflammatory cytokines (e.g., IL-6, IL-8, and TNF-α)
and inhibited the endogenous expression of two key pathogen-asso-
ciated molecular pattern-induced genes, namely, those encoding IFN-β
and IFIT1 (IFN-induced protein with tetratricopeptide repeats 1). Taken
together, Topo I is likely involved in psoriasis and represents a potential
molecular target for the treatment of psoriasis.
2.3. QCs display potent anti-inflammatory activities
Because psoriasis is a chronic inflammatory skin disease, anti-in-
flammatory activities of QCs were first evaluated in a mouse ear edema
model induced by 12–O–tetradecanoylphorbol-13-acetate (TPA). [39]
Table 1 and Fig. S1 summarize the weights of the ear edema induced by
topical TPA treatment in the absence or presence of QCs. Compared to
the control group, topically treated with acetone, the average punch
weight increased from 7.2 mg to 17.5 mg in the TPA-treated group
The imiquimod (IMQ)-induced psoriasis-like BALB/c mouse model,
whose characteristics resemble most of the features of clinical psoriasis,
has been widely used in studies of psoriasis. Topical application of a 5%
IMQ cream on mouse skin could lead to hyperkeratosis and scaling of
the skin, mimicking the typical symptoms associated with psoriasis
[
29]. Furthermore, the IMQ model resembles human psoriatic in-
(
Table 1). It was found that the ear weights were significantly lower,
flammation in many aspects, one of them being a major involvement of
the IL-23/IL-17A/IL-22 axis. This has been confirmed by the efficacy of
anti-IL23, anti-IL-17A, and anti-IL-22 antibodies in mouse psoriasis-like
skin inflammation models and anti-IL23/IL-17A antibodies in human
psoriatic patients [30–32].
from 11.2% to 83.7%, in the QCs (5 μM)-pretreated groups. Among
these QCs, 5i and 5l significantly inhibited the ear edema formation, by
approximately 69.3% and 83.7%, respectively, which was comparable
to the effect provoked by CPT (5 μM). In addition, a generally good
correlation was observed between the anti-inflammatory and Topo I
inhibitory activities of these QCs (Table 1).
Quinoline is widely used in the drug design and discovery to de-
velop bioactive molecules with various pharmacological activities, such
as antimalarial, antibiotic, antitumor, and anti-inflammatory activities
Hematoxylin and eosin (H&E) staining was subsequently carried out
to study histological changes induced by TPA in the mouse ears [40].
The results of histological assessment are shown in Fig. 3. Histological
sections of the ears subjected to topical application of TPA showed
significant increases in the dermis thickness and the number of in-
flammatory cells compared to those in the acetone-treated mice
[
33–35]. In this study, a series of quinoline derivatives (QCs, Fig. 1)
were synthesized and identified as potent Topo I inhibitors and anti-
inflammatory agents. The most active compounds were evaluated for
their antipsoriatic effects by employing lipopolysaccharide (LPS)-sti-
mulated HaCaT cells and the IMQ-induced psoriasis-like mouse model.
A further study in the mouse model showed that the leading compounds
exhibited strong effects on the expression of key cytokines associated
with psoriasis. The results revealed that QCs functioned as potential
antipsoriasis agents.
(
Fig. 3A and B). Application of CPT, 5i, and 5l dramatically suppressed
the TPA-induced epidermal hyperplasia and reduced the number of
infiltrated inflammatory cells (Fig. 3B).
2
.4. QCs inhibit the levels of nitric oxide, IL-17, IL-1β, IL-6, and TNF-α in
LPS-stimulated HaCaT cells
2
. Results and discussion
Since 5i and 5l exhibited the most potent Topo I inhibition and anti-
inflammatory activities, these two compounds were selected to in-
vestigate whether they could alleviate psoriasis. We first assessed the
effects of QCs on HaCaT cells (an immortal cell line derived from
human keratinocytes), which are often used to test the effects of drug
candidates on psoriasis [41]. To determine the non-toxic concentrations
of 5i and 5l, cell viability was assessed using the Cell Counting Kit-8
2.1. Chemistry
The synthesis of 25 QCs was accomplished as outlined in Scheme 1.
Various anilines (1a–1g) were treated with ethyl acetoacetate in the
presence of polyphosphoric acid to obtain the corresponding QCs
(
2a–2g) [36]. Compounds 2a–2g were treated with appropriate benzyl
(
CCK-8) assay. As shown in Fig. 4A, 5i and 5l did not exhibit significant
bromides using KOH as the base to obtain 3a–3v in 63–76% yields.
cytotoxicity at the concentration as high as 20 μM. Therefore, we used a
concentration of 10 μM for 5i and 5l in the following antipsoriasis ex-
periments with cultured HaCaT cells. The HaCaT cells were pretreated
with 5i and 5l for 4 h, and inflammation was stimulated with LPS.
Nitric oxide (NO) is generated by immune-activated cells in in-
flammation sites and plays a key role in signaling pathways of in-
flammatory cytokines [42]. Hence, inhibiting the production of NO in
Compounds 3a–3v were oxidized by SeO to obtain 4a–4v in 68–85%
2
yields. The target compounds 5a-5y were finally synthesized in 71–88%
yields through the Hornor–Wadsworth–Emmons reaction [37] of the
rhodanine moiety with the prepared aldehydes (4a–4v). The structures
of QCs were characterized and confirmed by 1H NMR and13C NMR.
2

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
Scheme 1. Synthesis of QCs. Reagents and conditions: (i) PPA, 130 °C, 2 h, (ii) KOH, Me
2
O, appropriate benzyl bromide, rt, 8 h, (iii) SeO , dioxane, 65 °C, 2 h, (iv)
2
Rhodanine moiety, NaOAc, acetic acid, 110 °C, 4 h.
the inflammatory response is important. It was found that LPS drama-
tically increased the level of NO compared with that in the control
group. However, treatment with 5i and 5l significantly reduced the NO
concentration, by 70% and 42% compared to that in the LPS group
(
Fig. 4B). In further experiments, we examined whether 5i and 5l could
regulate proinflammatory cytokines such as IL-17A, IL-1β, IL-6, and
TNF-α. These cytokines play major roles in maintaining pathogenesis
and the course of cutaneous inflammation in psoriasis [43]. As shown in
Fig. 4C–F, the mRNA levels of the cytokines significantly increased
upon treatment with LPS. However, 5i and 5l pretreatment significantly
inhibited the expression levels of these cytokines. Taken together, 5i
and 5l exhibited strong inhibitory effects on the NO production and the
expression levels of inflammatory cytokines. These findings indicate
that 5i and 5l may be potent antipsoriasis agents.
2.5. QCs ameliorate IMQ-induced psoriasis-like skin lesions in mice
The findings showing that 5i and 5l could downregulate in-
flammatory cytokines induced in LPS-stimulated HaCaT cells prompted
us to investigate whether these compounds could affect the develop-
ment of psoriasis. The IMQ-induced psoriasis-like mouse model was
employed for this study as previously described [44]. Compounds 5i
and 5l (50 mg/kg) or CPT (5 mg/kg) were orally administered once
daily, followed by application of 62.5 mg of IMQ to the bare skin on the
back of mice for seven consecutive days. Compared with that of the
Fig. 2. The Topo I inhibitory activities of QCs. (I, II, and III), lane D, pBR322
DNA, lane T, pBR322 DNA + Topo I, lane CPT, pBR322 DNA + Topo II + CPT
(
100 μM), other lanes, pBR322 DNA + Topo I + QCs.
3

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
Table 1
Data of inhibition of TPA-induced mice ear edema and Topo I by QCs.
Cpd
Ear weight
Topo I
Cpd
Ear weight
mg
Topo I
Inhibition
c
Inhibition
mga
Inhibition (%)b
Inhibition (%)
d
Ac
7.2 ± 1.6
n.d.
5m
5n
5o
5p
5q
5r
15.4 ± 2.1
14.6 ± 1.5
13.9 ± 1.3
13.7 ± 1.2
15.2 ± 1.4
14.6 ± 1.7
15.8 ± 1.7
13.7 ± 1.2
15.1 ± 1.4
13.6 ± 1.3
14.4 ± 1.6
14.3 ± 1.5
15.8 ± 1.2
10.8 ± 1.3
23.1
27.6
35.3
37.2
22.4
28.6
16.4
36.2
23.1
37.6
29.7
30.6
16.3
64.5
–
Ac/TPA
17.5 ± 1.2
12.6 ± 1.4
13.4 ± 1.1
14.5 ± 1.7
12.2 ± 1.0
14.1 ± 1.5
16.3 ± 1.2
12.5 ± 1.3
13.7 ± 1.3
8.9 ± 1.1
n.d.
++
+
+
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
47.2
39.6
29.4
51.2
33.4
11.2
48.3
36.5
83.7
18.7
23.4
69.3
+
–
+
–
++
+
+
5s
–
–
5t
++
g
h
i
++
+
5u
5v
5w
5x
5y
CPT
–
+
+++
–
–
j
15.6 ± 1.4
15.1 ± 1.3
10.4 ± 1.2
+
k
l
–
–
+++
+++
a
b
Each bar represents the mean ± SE from 3 mice.
The inhibitory rate (%) was calculated by this formule: inhibitory rate (%) = (1−[A(agent group)−B(control group)]/[C(TPA group)−B(control
group)]) × 100%.
c
The relative potent Topo II inhibition of QCs are present as follows: -, no detectable activity at 50 μM, +, weak activity at 50 μM, ++, weak activity at 20 μM, +
+
+, strong activity at 20 μM.
d
Not or non-detected.
mice in the control group, the body weight rapidly decreased in the
IMQ group, whereas this decline was arrested in the 5i-, 5l-, and CPT-
treated groups after 3 days of treatment (Fig. S2). The topical results
observed after 7 days are shown in Fig. 5A. It was found that the mice in
the topical IMQ-treated group did show the development of an apparent
psoriasis phenotype, including an erythema, scales, and thickening of
the skin. However, the mice, pre-treated with CPT, 5i, and 5l, presented
much attenuated psoriasis phenotype as there was a gradual reduction
in the thickness and scales of skin. The effects were also evident when
comparing the reduced grading of Psoriasis Area and Severity Index
potential value for improving psoriasis-like symptoms in the skin.
To confirm the protective effects of 5i and 5l on IMQ-induced
psoriasis, H&E staining was carried out on the mouse back skin on day 7
of treatment. Skin samples were fixed and stained with H&E, as shown
in Fig. 5B and C. The histological evaluation of the mice treated with
topical IMQ revealed extensive psoriasis-like lesions, as demonstrated
by hyperkeratosis, the skin barrier destruction, and modest dermal in-
flammation in the epidermal cuticles of the mice. However, these
changes were dramatically attenuated in the groups treated with CPT,
5i, and 5l (Fig. 5C). These results suggest that 5i and 5l may be of
potential value for improving psoriasis-like symptoms in the skin.
(
PASI, Fig. 5C) scores in CPT, 5i, and 5l treatment group with the to-
pical IMQ-treated group. These results suggest that 5i and 5l may be of
Fig. 3. (A) H&E staining for histological changes of TPA-induced mouse model. (B) Epidermal thickness and infiltrated inflammatory cells folds of control group. C,
the control group, TPA, the topical TPA-treated group, CPT, 5i, and 5l, 5 μM compounds + TPA are indicated. Values are expressed as the mean ± SD (n = 3).
ANOVA followed by Bonferroni’s multiple comparison tests was used. (*) p < 0.05 and (**) p < 0.01 versus control group treated with vehicle, (#) p < 0.05 and
(
##) p < 0.01 versus Ac/TPA-treated group.
4

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
Fig. 4. (A) Determination of the no toxic concentration of 5i and 5l against HaCaT cells. (B) 5i and 5l inhibited the production of NO in LPS (1 μg/mL)-stimulated
HaCaT cells. (C–F) 5i and 5l inhibited the mRNA expression levels of inflammatory cytokines in LPS-stimulated HaCaT cells. The mRNA expression level of TNF-α
(
C), IL-6 (D), IL-1β (E) and IL-17A were inhibited by 5i and 5l. C, the control group, LPS, the topical LPS-treated group, CPT, 1 μM + TPA, 5i and 5l, 10 μM + TPA are
indicated. Values are expressed as the mean ± SD (n = 3). ANOVA followed by Bonferroni’s multiple comparison tests was used. (*) p < 0.05 and (**) p < 0.01
versus control group, (#) p < 0.05 and (##) p < 0.01 versus LPS group.
2
.6. Compounds 5i and 5l suppress the expression of the key cytokines
expression levels of these psoriasis-related markers using western blot
analysis. As presented in Fig. 8, topical application of IMQ upregulated
the expression levels of all these tested proteins. Whereas, a pretreat-
ment with CPT, 5i, and 5l significantly downregulated the expression
levels of IL-17A, IL-22 and IL-23, as well as p65, TNF-α, and IFN-γ, but
in less extent. These findings are consistent with the results obtained by
immunohistochemistry staining and the RT–PCR assay. All these results
demonstrated that 5i and 5l displayed potential antipsoriasis activity by
suppressing the expression of key cytokines and inflammatory media-
tors such as p65, IL-17, IL-22, IL-23, TNF-α, and IFN-γ.
implicated in psoriasis
Since compounds 5i and 5l showed potential antipsoriasis activity
in vivo, the underlying mechanism of action of 5i and 5l was further
investigated. First, direct effects of 5i and 5l on IMQ-induced cytokines
were evaluated using immunohistochemistry staining. It has been re-
ported that the development of the IMQ-induced psoriasis-like skin in
mice is mediated via the IL-23/IL-17 axis [29]. IL-23, secreted by
dendritic cells, can induce the proliferation of T helper 17 (Th17) cells,
which can, in turn, activate and secrete IL-17A, IL-22, and IL-23 [45].
Our results indicated that the expression levels of these key cytokines
was significantly higher in the topical IMQ-treated group than that in
the control group (Fig. 6A–C). However, the expression levels of these
cytokines were significantly lower in the CPT-, 5i-, and 5l-treated
groups than those in the topical IMQ-treated group. In addition, IMQ
application increased the transcription of TNF-α and IFN-γ, two key
proinflammatory cytokines involved in the Th17 response [46], while,
CPT, 5i, and 5l treatment dramatically reduced the expression of TNF-α
and IFN-γ (Fig. 6D and E). Thus, oral administration of 5i and 5l sup-
pressed the expression of inflammatory cytokines implicated in psor-
iasis, including IL-17A, IL-22, IL-23, TNF-α, and IFN-γ.
3
. Conclusion
Psoriasis is a skin disorder with a complicated pathophysiology,
which has not been fully elucidated yet. Although certain agents have
shown clinical success, the treatment of psoriasis remains a significant
problem. [55] In this study, we presented a novel strategy to treat
psoriasis with small molecules inhibiting the Topo I activity. A series of
QCs were synthesized and identified as potent Topo I inhibitors. Some
QCs showed potential anti-inflammatory activity against TPA-induced
mouse ear inflammation. The most potent Topo I inhibitors and anti-
inflammatory agents, 5i and 5l, exhibited moderate cytotoxicity against
HaCaT cells. These two compounds significantly suppressed the pro-
duction of NO and the expression levels of inflammatory cytokines such
as IL-17, IL-1β, IL-6, and TNF-α in LPS-stimulated HaCaT cells.
Moreover, we found that oral gavage of 5i and 5l (50 mg/kg), followed
by IMQ application to the dorsal skin once daily for seven consecutive
days, significantly ameliorated psoriasis symptoms, including an er-
ythema, scaling, and epidermal thickness, compared with those in the
vehicle-treated IMQ-induced model mice. These two compounds were
then studied for their action against psoriasis in vivo. The results re-
vealed that 5i and 5l dramatically inhibited the mRNA expression levels
of cytokines and inflammatory mediators, including IL-17A, IL-22, IL-
To further examine the effects of 5i and 5l on the expression of
cytokines associated with psoriasis, quantitative reverse tran-
scription–polymerase chain reaction (RT–PCR) analysis was used to
study the mRNA expression of key cytokines and inflammatory med-
iator in skin lesions after IMQ treatment. The results presented in Fig. 7
demonstrated a marked increase in the mRNA expression of IL-17A, IL-
2
2, IL-23, TNF-α, p65, and IFN-γ in the IMQ-treated group. However,
5
i, 5l, and CPT pretreatment significantly downregulated the mRNA
expression of the genes encoding these inflammatory factors implicated
in psoriasis.
To better assign the mechanism of action of 5i and 5l on anti-
psoriasis, these two compounds were examined for their effects on the
2
3, p65, TNF-α, and IFN-γ, implicated in psoriasis induced by IMQ in
5

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
Fig. 5. (A) Images of psoriatic skin lesions treatment with 5i and 5l. Photos were taken after 7 days of treatment. (B) Histopathological view of psoriatic skin lesions
on treatment with CPT, 5i, and 5l. H&E-stained paraffin sections, 200×. (C) Data on epidermal thickness, inflammatory cell and PASI scales in psoriatic skin lesions.
C, the control group, IMQ, the topical IMQ-treated group, CPT, 5 mg/kg + IMQ, 5i and 5l, 50 mg/kg + IMQ are indicated. Values are expressed as the mean ± SD
(
n = 6). ANOVA followed by Bonferroni’s multiple comparison tests was used. (*) p < 0.05 and (**) p < 0.01 versus control group, (#) p < 0.05 and (##)
p < 0.01 versus IMQ group.
the back skin of BALB/c mice. Collectively, compounds 5i and 5l ob-
tained here could be potential candidates for the treatment of psoriasis.
Therefore, the study provided evidence to support the hypothesis that
chemical inhibition of Topo I is likely to be an effective strategy for the
treatment of psoriasis.
dual pump Shimadzu LC-20AB system equipped with an Ultimate XB-
C18 column and eluted with methanol/water (80%) at a flow rate of
0.8 mL·min−1
.
4.2. General procedure for the synthesis of intermediates 2a-g
4
. Experiment section
To the solution of 1a-g (1.55 mmol) and ethyl acetoacetate (2.02 g,
1
.55 mmol) was added PPA (12.5 g). The reaction mixture was stirred at
4.1. Chemistry
130 °C for 2 h. Reaction completion was monitored by TLC. The reac-
tion mixture was poured into ice water slowly with vigorous stirring
and the pH was then adjusted to 7–8 using NaOH. The precipitated solid
was filtered and dried in vacuum oven get the products. The products
were taken for the next reaction without further purification.
All of the reagents were commercially available and purchased from
Sigma-Aldrich Chemical Co., TCI Chemicals, AlfaAesar, and Aladdin
(
China), which used without further purification. HPLC grade methanol
was order from Sinopharm (China) and silica gel (200–300 mesh,
1
13
Qingdao Haiyang Chemical Co., Ltd). H and C NMR spectra were
recorded using TMS as the internal standard in DMSO-d
6
with a Bruker
4.3. General procedure for the synthesis of intermediates 3a-v
BioSpin GmbH spectrometer at 400 MHz and 101 MHz, respectively.
The chemical shifts are reported in parts per million (ppm) relative to
The solution of 2a–g (10 mmol) in DMF (20 mL) was added the
1
13
residual DMSO-d
6
(δ = 2.50, H, δ = 39.5, C) in the deuterium agent.
appropriate benzyl bromide (30 mmol) and K
2
CO (50 mmol), and the
3
The following abbreviations are used to designate multiplicities: s,
single, d, double, t, triplet, q, quartet, m, multiplet, dd, double-double,
br s, broad signal. Melting point (mp) was determined using an SRS-
OptiMelt automated melting point instrument. High-resolution mass
spectra (HR ESI-MS) were recorded on Shimadzu LCMS-IT-TO. The
purities of QCs were confirmed by analytical HPLC performed with a
reaction mixture was stirred at room temperature for 8 h. It was then
diluted with DCM (80 mL) and H O (80 mL). The organic layer was
separated, and the aqueous layer was extracted with DCM (80 mL × 2).
2
The combined organic layers were dried over Mg
2
SO and concentrated
4
in vacuo to provide a crude product, which was purified by column
chromatography to yield the title compounds 3a-v, respectively.
6

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
Fig. 6. BALB/c mice were treated as indicated in Experiment Section. After treatment of 7 days, mice were sacrificed, and the back skin section were strained by
immunohistochemistry to detect related cytokines in control, IMQ, CPT, 5i, and 5l-treated group. (A) IL-17A. (B) IL-22. (C) IL-23. (D) TNF-α. (E) IFN-γ. C, the control
group, IMQ, the topical IMQ-treated group, CPT, 5 mg/kg + IMQ, 5i and 5l, 50 mg/kg + IMQ are indicated. Values are expressed as the mean ± SD (n = 6).
ANOVA followed by Bonferroni’s multiple comparison tests was used. (*) p < 0.05 and (**) p < 0.01 versus control group treated with vehicle, (#) p < 0.05 and
(
##) p < 0.01 versus IMQ group.
1
3
4
.4. General procedure for the synthesis of intermediates 4a-v
(m, 2H), 7.40 (t, J = 7.2 Hz, 1H), 5.43 (s, 2H), 4.74 (s, 2H). C NMR
(
101 MHz, DMSO-d ) δ 200.7, 167.8, 166.9, 162.0, 153.1, 148.5, 136.2,
6
Compounds 3a-v (5 mmol) was dissolved in 20 mL of dioxane and
131.5, 129.7, 129.3, 129.0, 128.8, 128.6, 128.4, 127.8, 122.2, 120.4,
+
then added to 30 mL of dioxane suspension with SeO
2
(7.5 mmol). The
106.0, 70.7, 45.2. HR ESI-MS (M + H) m/z = 437.0626 (Cacld for
mixture was reacted at 80 °C for 1.5 h under N
2
atmosphere, and
C
22
H
16
N
2
O
4
S ) 437.0621, HPLC purity: 95.4%.
2
1
00 mL of ethyl acetate was added for dilution followed by washing
once with water. Solvents were removed in vacuo, and purification by
column chromatography to yield the title compounds 4a-v, respec-
tively.
4.5.2. (E)-2-(5-((4-((3,5-dimethylbenzyl)oxy)quinolin-2-yl)methylene)-4-
oxo-2- thioxothiazolidin-3-yl)acetic acid (5b)
Rhodanine-3-acetic acid and 4b were used as reactants to give 5b.
1
Yellow solid, yield: 76%, mp: 245.3–246.8 °C. H NMR (400 MHz,
4
.5. General procedure for the synthesis of 5a-5y
DMSO-d ) δ 13.41 (br s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.11 (d,
6
J = 8.5 Hz, 1H), 7.95 (s, 1H), 7.87–7.82 (m, 1H), 7.74 (s, 1H), 7.66 (t,
J = 7.5 Hz, 1H), 6.77–6.71 (m, 2H), 6.52 (t, J = 2.1 Hz, 1H), 5.36 (s,
A mixture of the appropriate aldehydes (4a-v, 1.0 mmol), the rho-
1
3
danine moiety (1.1 mmol), and NaOAc (3.0 mmol) in acetic acid
2H), 4.77 (s, 2H), 3.77 (s, 6H). C NMR (101 MHz, DMSO-d ) δ 200.7,
6
(
10 mL) heated to 110 °C for 4 h. Then, it was cooled to room tem-
167.8, 166.8, 161.9, 161.2, 153.0, 148.4, 138.4, 131.5, 129.7, 128.8,
perature and poured into water (50 mL). The product was then filtered
through the suction pump, washed with water/EtOH (1/1, v/v) to re-
move the excess acetic acid and recrystallized from EtOH.
128.5, 127.8, 122.2, 120.3, 106.3, 105.9, 100.3, 70.6, 55.7, 45.0. HR
+
ESI-MS (M + H)
m/z = 497.0878 (Cacld for
C
24
H
20
N
2
O
6 2
S )
497.0881, HPLC purity: 96.3%.
4
.5.1. (E)-2-(5-((4-(benzyloxy)quinolin-2-yl)methylene)-4-oxo-2-
4.5.3. (E)-2-(5-((4-((4-chlorobenzyl)oxy)quinolin-2-yl)methylene)-4-oxo-
2- thioxothiazolidin-3-yl)acetic acid (5c)
thioxothiazolidin- 3-yl) acetic acid (5a)
Rhodanine-3-acetic acid and 4a were used as reactants to give 5a.
Rhodanine-3-acetic acid and 4c were used as reactants to give 5c.
1
1
Yellow solid, yield: 81%, mp: 231.4–232.6 °C. H NMR (400 MHz,
Yellow solid, yield: 79%, mp: 213.6–215.3 °C. H NMR (400 MHz,
DMSO-d
6
) δ 8.16 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.92 (s,
DMSO-d ) δ 13.40 (br s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.12 (d,
6
1
H), 7.82 (t, J = 7.2, 1H), 7.74 (s, 1H), 7.66–7.58 (m, 3H), 7.49–7.43
J = 8.4 Hz, 1H), 7.96 (s, 1H), 7.85 (t, J = 7.5 Hz, 1H), 7.76 (s, 1H),
7

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
Fig. 7. 5i and 5l reduces the expression levels of cytokines associated with psoriasis. The expression levels of IL-17A, IL-22, IL-23, TNF-α, P65, and IFN-γ were
detected by quantitative RT-PCR. C, the control group, IMQ, the topical IMQ-treated group, CPT, 5 mg/kg + IMQ, 5i and 5l, 50 mg/kg + IMQ are indicated. Values
are expressed as the mean ± SD (n = 6). ANOVA followed by Bonferroni’s multiple comparison tests was used. (*) p < 0.05 and (**) p < 0.01 versus control group
treated with vehicle, (#) p < 0.05 and (##) p < 0.01 versus IMQ group.
7
.69–7.63 (m, 3H), 7.57 (d, J = 8.2 Hz, 2H), 5.43 (s, 2H), 4.77 (s, 2H).
4.5.5. (E)-2-(5-((4-((4-nitrobenzyl)oxy)quinolin-2-yl)methylene)-4-oxo-
2- thioxothiazolidin-3-yl)acetic acid (5e)
1
3
C NMR (101 MHz, DMSO-d ) δ 200.7, 167.8, 166.9, 161.8, 153.1,
6
1
1
48.5, 135.7, 132.1, 131.6, 130.6, 129.7, 128.8, 128.6, 127.9, 122.2,
Rhodanine-3-acetic acid and 4e were used as reactants to give 5e.
+
1
22.0, 120.3, 106.0, 69.9, 45.1. HR ESI-MS (M + H) m/z = 471.0232
Yellow solid, yield: 85%, mp: 222.9–224.3 °C. H NMR (400 MHz,
(
Cacld for C22
H
15ClN
2
O
S
4 2
) 471.0226, HPLC purity: 97.2%.
DMSO-d ) δ 13.39 (br s, 1H), 8.31 (d, J = 8.6 Hz, 2H), 8.25 (d,
6
J = 8.2 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.94 (s, 1H), 7.91–7.83 (m,
3H), 7.75 (s, 1H), 7.68 (t, J = 7.6 Hz, 1H), 5.62 (s, 2H), 4.77 (s, 2H).
13
4
.5.4. (E)-2-(5-((4-((4-bromobenzyl)oxy)quinolin-2-yl)methylene)-4-oxo-
- thioxothiazolidin-3-yl)acetic acid (5d)
C NMR (101 MHz, DMSO-d ) δ 200.7, 167.8, 166.8, 161.6, 153.0,
6
2
1
1
48.5, 147.8, 143.9, 131.6, 129.6, 129.0, 128.8, 128.6, 127.9, 124.2,
Rhodanine-3-acetic acid and 4d were used as reactants to give 5d.
+
22.2, 120.2, 106.0, 69.4, 45.0. HR ESI-MS (M + H) m/z = 482.0413
1
Yellow solid, yield: 82%, mp: 239.6–241.4 °C. H NMR (400 MHz,
(
Cacld for C22
H
15
N
3
O
6
S ) 482.0419, HPLC purity: 98.2%.
2
DMSO-d
6
) δ 13.40 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 8.5 Hz,
1
3
H), 7.97 (s, 1H), 7.85 (t, J = 7.4 Hz, 1H), 7.77 (s, 1H), 7.69–7.61 (m,
1
3
H), 7.52 (d, J = 8.3 Hz, 2H), 5.45 (s, 2H), 4.77 (s, 2H). C NMR
4.5.6. (E)-2-(5-((4-((4-methylbenzyl)oxy)quinolin-2-yl)methylene)-4-
oxo-2- thioxothiazolidin-3-yl)acetic acid (5f)
(
101 MHz, DMSO-d
6
) δ 200.7, 167.8, 166.9, 161.9, 153.1, 148.5, 135.3,
1
1
33.5, 131.6, 130.3, 129.7, 129.1, 128.8, 128.6, 127.9, 122.2, 120.3,
Rhodanine-3-acetic acid and 4f were used as reactants to give 5f.
+
1
06.1, 69.9, 45.1. HR ESI-MS (M + H) m/z = 514.9778 (Cacld for
Yellow solid, yield: 73%, mp: 198.7–200.4 °C. H NMR (400 MHz,
C
22
H
15BrN
2
O
4
S
2
) 514.9786, HPLC purity: 96.6%.
DMSO-d ) δ 8.12 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.89 (s,
6
Fig. 8. 5i and 5l suppress the expression of cytokines associated with psoriasis. C, the control group, IMQ, the topical IMQ-treated group, CPT, 5 mg/kg + IMQ, 5l
and 5i, 50 mg/kg + IMQ are indicated.
8

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
1
H), 7.80 (t, J = 7.5 Hz, 1H), 7.68 (s, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.48
5.36 (s, 2H), 4.74 (s, 2H). 13C NMR (101 MHz, DMSO-d
) δ 200.7,
6
(
d, J = 7.8 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 5.35 (s, 2H), 4.75 (s, 2H),
167.8, 166.8, 161.6, 152.9, 151.3, 149.9 (d, J = 12.5 Hz), 148.4, 133.9
(d, J = 5.9 Hz), 131.5, 129.6, 128.7, 128.5, 127.8, 125.4 (d,
J = 6.7 Hz), 122.2, 120.2, 118.2 (d, J = 17.2 Hz), 117.6 (d,
1
3
2
1
1
.33 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 200.8, 167.8, 166.8,
6
62.0, 153.0, 148.4, 138.2, 133.2, 131.4, 129.8, 129.6, 128.7, 128.5,
+
28.3, 127.7, 122.2, 120.4, 105.9, 70.7, 45.0, 21.3. HR ESI-MS
J = 17.5 Hz), 105.8, 69.3, 45.0. HR ESI-MS (M + H) m/z = 473.0417
+
(
M + H) m/z = 451.0762 (Cacld for C23
H
18
N
2
O
4
S
2
) 451.0758, HPLC
(Cacld for C22
H
14
F
2
2
N O
4
S ) 473.0425, HPLC purity: 95.3%.
2
purity: 96.4%.
4
.5.12. (E)-2-(5-((4-((4-fluorobenzyl)oxy)quinolin-2-yl)methylene)-4-
4
.5.7. (E)-2-(5-((4-((4-cyanobenzyl)oxy)quinolin-2-yl)methylene)-4-oxo-
oxo-2- thioxothiazolidin-3-yl)acetic acid (5l)
2
-thioxothiazolidin-3-yl)acetic acid (5g)
Rhodanine-3-acetic acid and 4l were used as reactants to give 5l.
1
Rhodanine-3-acetic acid and 4g were used as reactants to give 5g.
Yellow solid, yield: 86%, mp: 231.8–233.9 °C. H NMR (400 MHz,
1
Yellow solid, yield: 88%, mp: 217.6–218.9 °C. H NMR (400 MHz,
DMSO-d ) δ 13.40 (br s, 1H), 8.17 (d, J = 8.3 Hz, 1H), 8.12 (d,
6
DMSO-d
6
) δ 8.20 (d, J = 8.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.93 (d,
J = 8.5 Hz, 1H), 7.98 (s, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.79 (s, 1H),
7.69–7.61 (m, 3H), 7.29 (t, J = 8.8 Hz, 2H), 5.43 (s, 2H), 4.77 (s, 2H).
J = 8.2 Hz, 2H), 7.87 (s, 1H), 7.83 (t, J = 8.2 Hz, 1H), 7.78 (d,
1
3
J = 8.2 Hz, 2H), 7.68 (s, 1H), 7.67–7.61 (m, 1H), 5.52 (s, 2H), 4.76 (s,
C NMR (101 MHz, DMSO-d ) δ 200.7, 167.8, 166.8, 162.6, 161.8,
6
1
3
2
1
1
H). C NMR (101 MHz, DMSO-d
6
) δ 200.7, 167.8, 166.8, 161.6,
153.0, 148.4, 132.5 (d, J = 3.0 Hz), 131.5, 130.8 (d, J = 8.4 Hz),
53.0, 148.4, 141.9, 133.0, 131.6, 129.6, 128.8, 128.7, 128.6, 127.9,
129.7, 128.7, 128.5, 127.8, 122.2, 120.3, 116.0 (d, J = 21.5 Hz), 105.9,
+
+
22.2, 120.2, 119.1, 111.4, 105.9, 69.6, 45.0. HR ESI-MS (M + H) m/
70.0, 45.0. HR ESI-MS (M + H)
m/z = 455.0526 (Cacld for
z = 462.0554 (Cacld for
C
23
H
15
N
3
O
4
S
2
)
462.0559, HPLC purity:
C
22
H15FN
2
O
4
S ) 455.0532, HPLC purity: 97.5%.
2
9
7.8%.
4
.5.13. (E)-2-(5-((4-((3-fluorobenzyl)oxy)quinolin-2-yl)methylene)-4-
4
.5.8. (E)-2-(4-oxo-2-thioxo-5-((4-((4-(trifluoromethyl)benzyl)oxy)
oxo-2- thioxothiazolidin-3-yl)acetic acid (5m)
quinolin-2-yl) methylene)thiazolidin-3-yl)acetic acid (5 h)
Rhodanine-3-acetic acid and 4m were used as reactants to give 5m.
1
Rhodanine-3-acetic acid and 4h were used as reactants to give 5h.
Yellow solid, yield: 86%, mp: 231.8–233.9 °C. H NMR (400 MHz,
1
Yellow solid, yield: 86%, mp: 231.8–233.9 °C. H NMR (400 MHz,
DMSO-d ) δ 8.14 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.83 (s,
6
DMSO-d
6
) δ 8.13 (d, J = 8.2 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H),
1H), 7.79 (t, J = 7.6 Hz, 1H), 7.65–7.58 (m, 2H), 7.53–7.47 (m, 1H),
1
3
13
7
.83–7.75 (m, 6H), 7.62–7.56 (m, 2H), 5.46 (s, 2H), 4.72 (s, 2H).
C
7.45–7.41 (m, 2H), 7.27–7.19 (m, 1H), 5.40 (s, 2H), 4.73 (s, 2H).
C
NMR (101 MHz, DMSO-d ) δ 200.7, 167.8, 166.8, 161.6, 152.8, 148.3,
6
NMR (101 MHz, DMSO-d ) δ 200.7, 167.8, 166.8, 162.7, 161.7, 152.9,
6
1
41.0, 131.4, 129.5, 129.2, 128.7, 128.6, 128.4, 127.7, 125.9 (q,
148.4, 139.0 (d, J = 7.6 Hz), 131.5, 131.2 (d, J = 8.3 Hz), 129.6,
128.7, 128.4, 127.8, 124.2 (d, J = 2.7 Hz), 122.2, 120.2, 115.6 (d,
J = 274.5 Hz), 124.6, 122.1, 120.1, 105.8, 69.6, 45.0. HR ESI-MS
+
(
M + H) m/z = 505.0437 (Cacld for C23
H
15
F
3
N
3
O
4
2
S ) 505.0442,
J = 20.9 Hz), 115.0 (d, J = 22.0 Hz), 105.8, 69.8, 45.0. HR ESI-MS
+
HPLC purity: 98.4%.
(M + H)
m/z = 455.0537 (Cacld for
C
22
H
15FN
2
O
4
S ) 455.0532,
2
HPLC purity: 96.9%.
4
.5.9. (E)-2-(5-((4-((4-bromo-2-fluorobenzyl)oxy)quinolin-2-yl)
methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5i)
4.5.14. (E)-2-(5-((4-((2-fluorobenzyl)oxy)quinolin-2-yl)methylene)-4-
oxo-2- thioxothiazolidin-3-yl)acetic acid (5n)
Rhodanine-3-acetic acid and 4i were used as reactants to give 5i.
1
Yellow solid, yield: 82%, mp: 246.4–248.1 °C. H NMR (400 MHz,
Rhodanine-3-acetic acid and 4n were used as reactants to give 5n.
1
DMSO-d
6
) δ 8.11 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 8.6 Hz, 1H), 7.93 (s,
Yellow solid, yield: 86%, mp: 231.8–233.9 °C. H NMR (400 MHz,
1
H), 7.85–7.80 (m, 1H), 7.77 (s, 1H), 7.72–7.65 (m, 2H), 7.62 (t,
DMSO-d ) δ 13.45 (br s, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.05 (d,
6
1
3
J = 7.6 Hz, 1H), 7.54–7.50 (m, 1H), 5.44 (s, 2H), 4.76 (s, 2H). C NMR
101 MHz, DMSO-d ) δ 200.7, 167.9, 166.8, 161.6, 160.7, 153.0, 148.4,
J = 8.4 Hz, 1H), 7.90 (s, 1H), 7.82–7.78 (m, 1H), 7.77–7.71 (m, 2H),
(
6
7.63–7.57 (m, 1H), 7.52–7.46 (m, 1H), 7.35–7.28 (m, 2H), 5.45 (s, 2H),
1
3
1
32.4 (d, J = 4.4 Hz), 131.6, 129.7, 128.8, 128.6, 128.4 (d,
4.74 (s, 2H). C NMR (101 MHz, DMSO-d ) δ 200.7, 167.9, 166.8,
6
J = 3.5 Hz), 127.9, 122.8 (d, J = 14.5 Hz), 122.7 (d, J = 9.7 Hz),
161.7, 160.9, 153.0, 148.4, 131.5, 131.3 (d, J = 8.3 Hz), 131.0 (d,
+
1
22.1, 120.1, 119.5, 105.9, 64.6, 45.0. HR ESI-MS (M + H) m/
J = 3.8 Hz), 129.7, 128.7, 128.5, 127.8, 125.2 (d, J = 3.5 Hz), 123.2
z = 532.9652 (Cacld for C22
H
14BrFN
2
O
4
S
2
) 532.9661, HPLC purity:
(d, J = 14.3 Hz), 122.1, 120.2, 116.0 (d, J = 20.7 Hz), 105.8, 65.1,
+
9
7.3%.
45.0. HR ESI-MS (M + H)
m/z = 455.4928 (Cacld for
C
22
H
15FN
2
O
4
S ) 455.0532, HPLC purity: 96.2%.
2
4
.5.10. (E)-2-(5-((4-((3,5-difluorobenzyl)oxy)quinolin-2-yl)methylene)-
-oxo-2- thioxothiazolidin-3-yl)acetic acid (5j)
4
4.5.15. (Z)-2-(5-((6-fluoro-4-((4-fluorobenzyl)oxy)quinolin-2-yl)
methylene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (5o)
Rhodanine-3-acetic acid and 4j were used as reactants to give 5j.
1
Yellow solid, yield: 71%, mp: 266.8–268.5 °C. H NMR (400 MHz,
Rhodanine-3-acetic acid and 4o were used as reactants to give 5o.
1
DMSO-d
6
) δ 8.11–8.03 (m, 2H), 7.91 (s, 1H), 7.83–7.77 (m, 2H), 7.73
Yellow solid, yield: 71%, mp: 262.5–263.9 °C. H NMR (400 MHz,
(
s, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.40–7.34 (m, 1H), 7.22–7.16 (m, 1H),
DMSO-d ) δ 13.46 (s, 1H), 8.23– 7.15 (m, 1H), 7.96 (s, 1H), 7.82–7.72
6
1
3
5
1
1
1
.42 (s, 2H), 4.75 (s, 2H). C NMR (101 MHz, DMSO-d
6
) δ 200.7,
(m, 3H), 7.69–7.65 (m, 2H), 7.31–7.25 (m, 2H), 5.42 (s, 2H), 4.77 (s,
13
67.9, 166.8, 163.6, 161.6, 153.0, 148.4, 132.6 (d, J = 10.1 Hz), 131.5,
2H). C NMR (101 MHz, DMSO-d ) δ 200.5, 167.8, 166.8, 163.8,
6
29.7, 128.7, 128.5, 127.8, 122.7, 122.1, 120.1, 119.7 (d, J = 3.6 Hz),
162.6, 161.3, 159.6, 152.6, 145.6, 132.3 (d, J = 3.0 Hz), 131.9 (d,
J = 9.2 Hz), 130.8 (d, J = 8.4 Hz), 129.5, 128.5, 121.5 (d,
12.3 (d, J = 3.8 Hz), 105.9, 104.7 (d, J = 25.7 Hz), 64.7, 45.0. HR
+
ESI-MS (M + H)
m/z = 473.0419 (Cacld for
C
22
H
14
2
F N
2
O
4
S
2
)
J = 25.9 Hz), 121.1 (d, J = 9.7 Hz), 106.4, 106.1 (d, J = 23.6 Hz),
+
4
73.0425, HPLC purity: 98.1%.
70.1, 45.0. HR ESI-MS (M + H)
m/z = 473.0419 (Cacld for
C
22
H
14
F
2
2
N O
4
S ) 473.0425, HPLC purity: 97.1%.
2
4
.5.11. (E)-2-(5-((4-((3,4-difluorobenzyl)oxy)quinolin-2-yl)methylene)-
-oxo-2- thioxothiazolidin-3-yl)acetic acid (5k)
4
4.5.16. (Z)-2-(5-((6-chloro-4-((4-fluorobenzyl)oxy)quinolin-2-yl)
methylene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (5p)
Rhodanine-3-acetic acid and 4k were used as reactants to give 5k.
1
Yellow solid, yield: 75%, mp: 263.7–265.2 °C. H NMR (400 MHz,
Rhodanine-3-acetic acid and 4p were used as reactants to give 5p.
1
DMSO-d
6
) δ 8.13 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.84 (s,
Yellow solid, yield: 78%, mp: 253.1–254.6 °C. H NMR (400 MHz,
1
H), 7.79 (t, J = 7.5 Hz, 1H), 7.70–7.58 (m, 3H), 7.54–7.43 (m, 2H),
DMSO-d ) δ 13.44 (br s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 8.05 (d,
6
9

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
1
3
J = 2.3 Hz, 1H), 7.92 (s, 1H), 7.86–7.75 (m, 2H), 7.70–7.64 (m, 2H),
7.58–7.48 (m, 1H), 5.45 (s, 2H), 4.77 (s, 2H). C NMR (101 MHz,
1
3
7
.33–7.25 (m, 2H), 5.40 (s, 2H), 4.77 (s, 2H). C NMR (101 MHz,
DMSO-d ) δ 200.3, 167.8, 166.8, 162.7, 160.3, 153.5, 146.8, 134.0,
6
DMSO-d
6
) δ 200.4, 167.8, 166.8, 162.6, 160.9, 153.4, 146.7, 132.3,
132.4 (d, J = 8.3 Hz), 132.2, 132.1, 129.2, 129.0, 128.4 (d,
1
1
32.2 (d, J = 3.0 Hz), 131.9, 130.9 (d, J = 8.4 Hz), 130.8, 129.3,
J = 21.4 Hz), 122.8, 122.6, 120.9 (d, J = 5.8 Hz), 119.6, 119.4, 106.7,
+
28.9, 121.0, 120.9, 116.0 (d, J = 21.5 Hz), 106.6, 70.2, 45.0. HR ESI-
64.9, 45.05. HR ESI-MS (M + H)
m/z = 566.9259 (Cacld for
+
MS (M + H) m/z = 489.0138 (Cacld for C22
H
14ClFN
2
O
4
S
2
) 489.0142,
C
22
H
13BrClFN
2
O
4
S ) 566.9251, HPLC purity: 98.3%.
2
HPLC purity: 97.1%.
4
.5.22. (Z)-2-(5-((4-((4-bromo-2-fluorobenzyl)oxy)-6-(trifluoromethoxy)
4
.5.17. (Z)-2-(5-((4-((4-fluorobenzyl)oxy)-6-methylquinolin-2-yl)
quinolin-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5v)
methylene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (5q)
Rhodanine-3-acetic acid and 4v were used as reactants to give 5v.
1
Rhodanine-3-acetic acid and 4q were used as reactants to give 5q.
Yellow solid, yield: 84%, mp: 229.3–230.8 °C. H NMR (400 MHz,
1
Yellow solid, yield: 88%, mp: 236.7–238.2 °C. H NMR (400 MHz,
DMSO-d ) δ 13.44 (br s, 1H), 8.26 (d, J = 9.2 Hz, 1H), 7.99 (s, 1H),
6
DMSO-d
6
) δ 13.44 (br s, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.93 (s, 1H),
7.95 (d, J = 1.6 Hz, 1H), 7.91 (s, 1H), 7.85–7.79 (m, 1H), 7.75–7.65
13
7
.91 (s, 1H), 7.72 (s, 1H), 7.71–7.61 (m, 3H), 7.35–7.23 (m, 2H), 5.40
(m, 2H), 7.59–7.47 (m, 1H), 5.49 (s, 2H), 4.78 (s, 2H). C NMR
1
3
(
s, 2H), 4.77 (s, 2H), 2.51 (s, 3H). C NMR (101 MHz, DMSO-d
6
) δ
(101 MHz, DMSO-d ) δ 200.3, 167.8, 166.8, 162.0, 161.4, 159.5, 154.0,
6
2
00.8, 167.9, 166.8, 162.6, 161.2, 152.0, 147.0, 137.8, 133.6, 132.5 (d,
146.7, 132.5 (d, J = 8.4 Hz), 131.7, 130.9 (d, J = 8.1 Hz), 129.2, 123.4
J = 3.1 Hz), 130.9 (d, J = 8.4 Hz), 129.9, 128.6, 127.9, 120.8, 120.2,
(q, J = 274.7 Hz), 121.8, 120.6, 119.8, 119.2, 116.0 (d, J = 21.4 Hz),
+
+
1
16.0 (d, J = 21.5 Hz), 105.9, 69.9, 45.0, 21.9. HR ESI-MS (M + H)
112.9, 106.7, 65.0, 45.1. HR ESI-MS (M + H) m/z = 616.9446 (Cacld
m/z = 469.0671 (Cacld for C23
H
17FN
2
O
4
S
2
) 469.0668, HPLC purity:
for C22
H
13BrF
4
N
2
O
5
S ) 616.9438, HPLC purity: 98.4%.
2
9
5.5%.
4
.5.23. (E)-5-((4-((4-bromo-2-fluorobenzyl)oxy)quinolin-2-yl)
4
.5.18. (Z)-2-(5-((4-((4-fluorobenzyl)oxy)-6-nitroquinolin-2-yl)
methylene)-2- thioxothiazolidin-4-one (5w)
methylene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (5r)
Rhodanine and 4i were used as reactants to give 5w. Yellow solid,
1
Rhodanine-3-acetic acid and 4r were used as reactants to give 5r.
yield: 87%, mp: 217.2–219.1 °C. H NMR (400 MHz, DMSO-d
6
) δ 13.72
1
Yellow solid, yield: 85%, mp: 271.4–272.7 °C. H NMR (400 MHz,
(s, 1H), 8.11–8.03 (m, 2H), 7.80 (t, J = 7.2 Hz, 1H), 7.75–7.64 (m, 4H),
13
DMSO-d
6
) δ 13.48 (br s, 1H), 8.83 (d, J = 2.5 Hz, 1H), 8.51–8.41 (m,
7.60 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 5.43 (s, 2H). C NMR
1
2
H), 8.23 (d, J = 9.3 Hz, 1H), 7.96 (s, 1H), 7.92 (s, 1H), 7.75–7.65 (m,
(101 MHz, DMSO-d ) δ 203.1, 169.7, 162.0, 161.4, 159.5, 153.2, 148.4,
6
1
3
H), 7.35–7.27 (m, 2H), 5.46 (s, 2H), 4.78 (s, 2H). C NMR (101 MHz,
) δ 200.1, 167.8, 166.7, 163.1, 162.7, 156.5, 150.2, 145.2,
132.5 (d, J = 4.5 Hz), 132.1, 131.5, 128.7, 128.4 (d, J = 3.5 Hz),
DMSO-d
6
127.7, 127.6, 123.0, 122.8 (d, J = 12.0 Hz), 120.0, 119.5, 105.8, 64.6.
+
1
1
7
31.8 (d, J = 3.0 Hz), 131.2 (d, J = 8.4 Hz), 130.7, 130.5, 128.6,
HR ESI-MS (M + H) m/z = 474.9519 (Cacld for C20
H12BrFN
2
O
2 2
S )
24.7, 119.2, 119.1 (d, J = 20.9 Hz), 116.1 (d, J = 21.5 Hz), 107.2,
474.9524, HPLC purity: 96.6%.
+
0.7, 45.1. HR ESI-MS (M + H)
m/z = 500.0329 (Cacld for
C
22
H
14FN
3
O
6
S
2
) 500.0336, HPLC purity: 96.9%.
4.5.24. (E)-5-((4-((4-bromo-2-fluorobenzyl)oxy)quinolin-2-yl)
methylene)-3- ethyl-2-thioxothiazolidin-4-one (5x)
4
.5.19. (Z)-2-(5-((4-((4-fluorobenzyl)oxy)-6-methoxyquinolin-2-yl)
Rhodanine-3-ethyl and 4i were used as reactants to give 5x. Yellow
1
methylene)-4- oxo-2-thioxothiazolidin-3-yl)acetic acid (5s)
solid, yield: 84%, mp: 238.1–239.9 °C. H NMR (400 MHz, DMSO-d
6
) δ
Rhodanine-3-acetic acid and 4s were used as reactants to give 5s.
8.13–8.07 (m, 2H), 7.87 (s, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.77 (s, 1H),
1
Yellow solid, yield: 80%, mp: 246.6–248.2 °C. H NMR (400 MHz,
7.75–7.65 (m, 2H), 7.62 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H),
1
3
DMSO-d
6
) δ 13.40 (s, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.92 (s, 1H), 7.72
5.45 (s, 2H), 4.08 (q, J = 6.9 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). C NMR
(
s, 1H), 7.69–7.63 (m, 2H), 7.52–7.44 (m, 1H), 7.40 (d, J = 2.7 Hz, 1H),
(101 MHz, DMSO-d ) δ 167.2, 161.6, 160.0, 153.4, 148.6, 132.5 (d,
6
1
3
7
.31–7.25 (m, 2H), 5.43 (s, 2H), 4.76 (s, 2H), 3.91 (s, 3H). C NMR
J = 4.4 Hz), 131.5, 129.4, 129.0, 128.8, 128.4, 127.8, 125.6, 123.0 (d,
(
101 MHz, DMSO-d
6
) δ 200.7, 167.9, 166.9, 162.5, 160.5, 158.7, 150.4,
J = 8.3 Hz), 122.9 (d, J = 8.5 Hz), 120.2, 119.8, 119.5 (d,
+
1
1
44.4, 132.6, 132.5, 130.7 (d, J = 8.3 Hz), 130.1, 127.1, 123.6, 121.4,
J = 24.4 Hz), 105.9, 64.8, 39.4, 12.6. HR ESI-MS (M + H) m/
16.0 (d, J = 21.5 Hz), 106.2, 100.3, 69.8, 56.1, 45.0. HR ESI-MS
z = 501.9828 (Cacld for C22
H
16BrFN
2
O
2
2
S ) 501.9834, HPLC purity:
+
(
M + H)
m/z = 485.0658 (Cacld for
C
23
H
17FN
2
O
5
S
2
)
485.0662,
97.8%.
HPLC purity: 98.4%.
4
.5.25. (E)-3-(5-((4-((4-bromo-2-fluorobenzyl)oxy)quinolin-2-yl)
4
.5.20. (Z)-2-(5-((4-((4-fluorobenzyl)oxy)-6-(trifluoromethoxy)quinolin-
-yl) methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5t)
methylene)-4- oxo-2-thioxothiazolidin-3-yl)propanoic acid (5y)
2
Rhodanine-3-propanoic acid and 4i were used as reactants to give
1
Rhodanine-3-acetic acid and 4t were used as reactants to give 5t.
5y. Yellow solid, yield: 83%, mp: 256.7–257.9 °C. H NMR (400 MHz,
1
Yellow solid, yield: 75%, mp: 234.5–235.8 °C. H NMR (400 MHz,
DMSO-d ) δ 12.54 (s, 1H), 8.13–8.05 (m, 2H), 7.87 (s, 1H), 7.82 (t,
6
DMSO-d
6
) δ 13.45 (br s, 1H), 8.25 (d, J = 9.2 Hz, 1H), 7.98 (s, 1H),
J = 7.6 Hz, 1H), 7.77 (s, 1H), 7.73–7.67 (m, 2H), 7.62 (t, J = 7.6 Hz,
7
.97 (d, J = 1.5 Hz, 1H), 7.87 (s, 1H), 7.87–7.75 (m, 1H), 7.71–7.63
1H), 7.53 (d, J = 9.6 Hz, 1H), 5.44 (s, 2H), 4.27 (t, J = 8.2 Hz, 2H),
1
3
13
(
m, 2H), 7.31–7.27 (m, 2H), 5.44 (s, 2H), 4.77 (s, 2H). C NMR
101 MHz, DMSO-d ) δ 200.3, 167.8, 166.8, 162.0, 161.4, 159.5, 154.0,
2.65 (t, J = 7.1 Hz, 2H). C NMR (101 MHz, DMSO-d
6
) δ 200.7, 172.3,
(
6
167.2, 162.0, 161.5, 159.5, 153.2, 148.4, 132.4 (d, J = 4.5 Hz), 131.6,
1
1
1
47.0, 146.7, 132.5 (d, J = 8.1 Hz), 131.7 (d, J = 8.5 Hz), 129.2,
129.2, 128.8 (d, J = 4.9 Hz), 128.4 (d, J = 3.5 Hz), 127.8, 123.1,
+
28.4, 125.3 (q, J = 271.5 Hz), 122.6, 120.4, 119.7 (d, J = 20.8 Hz),
122.1, 120.0, 119.6, 119.4, 105.8, 64.6, 31.5. HR ESI-MS (M + H) m/
+
12.9, 106.7, 65.0, 45.1. HR ESI-MS (M + H) m/z = 539.0372 (Cacld
z = 546.9733 (Cacld for C23
H
16BrFN
2
O
4
2
S ) 546.9741, HPLC purity:
for C23
H
17
F N
4 2
O
5
S
2
) 539.0368, HPLC purity: 96.2%.
98.1%.
4
.5.21. (Z)-2-(5-((4-((4-bromo-2-fluorobenzyl)oxy)-6-chloroquinolin-2-
4.6. Topo I-Mediated pBR322 DNA relaxation assay
yl) methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5u)
Rhodanine-3-acetic acid and 4u were used as reactants to give 5u.
The effects of QCs on DNA relaxation catalyzed by Topo I (TaKaRa,
Kyoto, Japan) was evaluated by measuring the relaxation of pBR322
DNA (TaKaRa, Kyoto, Japan) by employing CPT as a standard. The
assay was performed according to the manufacturer’s instructions with
1
Yellow solid, yield: 77%, mp: 236.7–238.4 °C. H NMR (400 MHz,
DMSO-d ) δ 13.44 (s, 1H), 8.12 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 2.3 Hz,
6
1
H), 7.94 (s, 1H), 7.85 (s, 1H), 7.89–7.76 (m, 1H), 7.73–7.67 (m, 2H),
10

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
modification. The reaction mixture was incubated at 37 °C for 30 min.
Then, it was terminated by the additional of dye solution (1% SDS,
and 60% peanut oil) were administered by oral gavage with a stomach-
filling needle prior to application of 62.5 mg of IMQ cream, whereas the
control and model groups received only 200 μL of vehicle. All groups’
mice were sacrificed after 7 day, and the skins were collected for next
experiments.
0
.02% bromophenol blue and 25% glycerol). The mixtures were ap-
plied to 1% agarose gel and subjected to electrophoresis for 1 h at 90 V,
in TAE buffer (40 mM Tris-acetate, 2 mM EDTA). Gels were stained for
−
1
3
0 min in an aqueous solution of Ged Red (0.5 μg·mL ). DNA bands
were visualized by transillumination with UV light and then photo-
graphed with an Alpha Innotech digital imaging system.
4.12. Histopathology and immunohistochemistry analyses
For H&E, the samples from different treatment groups were fixed in
10% formalin and embedded in paraffin for histological examinations.
Sections of the ear or skin samples were cut and mounted on polylysin-
coated slides. Each section was deparaffinized in xylene, rehydrated
through a series of graded alcohols, and subjected to stain with he-
matoxylin and eosin. The thickness of the epidermis was measured
using a Nikon light microscope (Japan) equipped with an ocular mi-
crometer by the magnification (200×) in 15 fields per section. The
number of dermal infiltrating inflammatory cells was determined by
counting the stained cells at five different areas.
4
.7. Animals
Male BALB/c mice (6–7 weeks old) were supplied from the
Experimental Animal Centre of Guang Dong Province (approval docu-
ments: SCXK/20130002). The use of mice was reviewed and approved
by the Ethics Committee for Animal Experimentation of the Guangdong
University of Technology (Guangzhou, China) and was in accordance
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals (the 7th edition, USA).
For immunohistochemistry staining, paraformaldehyde-fixed and
paraffin-embedded ear or skin specimens were deparaffinized, anto-
claved, heat-processed for antigen retrieval in citrate saline buffer, and
incubated with purified rabbit anti-mouse COX-2, P65, IL-17A, IL-22,
IL-23, TNF-α, or IFN-γ (abcam, UK) at 1:50 dilution overnight at 4 °C.
All procedures were performed according to the manufacturers’
guidelines.
4.8. Measurement of ear edema
Mice were randomly divided into difference groups. Control group,
both ears of BALB/c mice were topically treated with 15 μL of acetone
(
vehicle) acetone 30 min prior to application of another acetone. Model
group, both ears of BALB/c mice were topically treated with 15 μL of
acetone 30 min prior to application of 0.08 nmol of TPA in 15 μL of
acetone. Experiment groups, both ears of BALB/c mice were topically
treated with CPT (2 μM) or QCs (4 μM) in 15 μL of acetone 30 min prior
to application of 0.08 nmol of TPA in 15 μL of acetone. All mice were
sacrificed 8 h after TPA (acetone for control group) treatment. Ear
punches (5 mm in diameter) were taken immediately, weighed, frozen,
and stored at −80 °C for next experiment.
4.13. Real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from collected cells or back skin on BALB/
c by using trizol reagent. The synthesis of cDNA on 2 μg RNA was using
the Revert Aid First Strand cDNA Synthesis Kit, and the expression of
relative genes were measured with ABI Stepone plus Fast Real-Time
PCR system by using a Fast Start Universal SYBR Green Master (Rox)
(Roche Diagnostics, China). Primer sequences designed to detect the
gene expression in mice were as follows: IL-17A, forward primer, 5′-
TCA TGT GGT GGT CCA GCT TTC-3′, reverse primer, 5′-GAA GGC CCT
CAG ACT ACC TCA A-3′, IL-22, forward primer, 5′-TTT CCT GAC CAA
ACT CAG CA-3′, reverse primer, 5′-CTG GAT GTT CTG GTC GTC AC-3′,
TNF-α, forward primer, 5′-TAT GGC CCA GAC CCT CAC A-3′, reverse
primer, 5′-GGA GTA GAC AAG GTA CAA CCC ATC-3′, P65, forward
primer, 5′-GTT TCG GGT AGG CAC AGC AAT-3′, reverse primer, 5′-TCG
AGT CTC CAT GCA GCT ACG-3′, IL-23, forward primer, 5′-CTC ACC
GTG ACG TTT AGG GA-3′, reverse primer, 5′-ACT AGA ACT CAG GCT
GGG CAT C-3′, IFN-γ, forward primer, 5′-GTT GCT GAT GGC CTG ATT
GTC-3′, reverse primer, 5′-CGG CAC AGT CAT TGA AAG CCT A-3′,
GAPDH, forward primer, 5′-TGT GTC CGT CGT GGA TCT GA-3′, reverse
primer, 5′-TTG CTG TTG AAG TCG CAG GAG-3′. The GAPDH gene was
used as a reference to normalize the data that was quantitatively ana-
4.9. Cell viability assay
Cell viability was assessed using a Cell Counting Kit-8 assay (CCK-8
from Dojindo, Kumamoto, Japan) according to the manufacturer’s in-
4
structions. Briefly, cells were seeded in 96-well plates (1 × 10 cell per
well) were stimulated with various concentration of 5i and 5l in the
−
1
presence or absence of LPS (1 μg·mL ). CCK-8 was added to each well
of the 96-well plates and incubated for 4 h, and the absorbance at
4
50 nm was measured using a microplate reader (Bio-Tek).
4.10. Measure of NO concentration
5
Briefly, the HaCaT cells (3 × 10 cell per well) were plate in 48-well
plates over night. The cells were pretreated with CPT, 5i and 5l for 4 h
−1
and inflammation was stimulate with the addition of LPS (1 μg. mL
)
−ΔΔCq
for 20 h. Cell culture supernatants were collected and reacted with the
Griess reagent in a 1:1 ratio, and the absorbance at 570 nm was mea-
sured using a microplate reader (Bio-Tek).
lyzed by using the 2
method.
Primers used for detecting the corresponding gene expression in
HaCaT cell line: IL-6, forward primer, 5′-AGA GTA GTG AG GAA CAA
GCC-3′, reverse primer, 5′-TAC ATT TGC CGA AGA GCC CT-3′, TNF-α,
forward primer, 5′-TCC TTC AGA CAC CCT CAA CC-3′, reverse primer,
4.11. Development of IMQ-induced psoriasis-like mice model
5
′-AGG CCC CAG TTT GAA TTC TT-3′, IL-1β, forward, 5′-TGG AGA AGC
One day before IMQ or vaseline treatment, mice were anesthetized
TGT GGC AGC TAC CT-3′, reverse, 5′-GAA CGT CAC ACA CCA GCA
GGT T-3′, IL-17A, forward, 5′-ACT ACA ACC GAT CCA CCT CAC-3′,
reverse, 5′-ACT TTG CCT CCC AGA TCA CAG-3′. β-actin, forward
primer, 5′-AGG GAA ATC GTG CGT GAC AT-3′, reverse primer, 5′-TCC
TGC TTG CTG ATC CAC AT-3′, Cycle parameters were as follows: 95 °C
for 10 min, 40 cycles of 95 °C for 15 sec, and 60 °C for 60 sec.
by intraperitoneal injection of 50 mg/kg of sodium pentobarbital, their
fur was shaved locally with an electric clipper. Mice received a daily
topical dose of 62.5 mg of IMQ cream on the shaved dorsal skin for 7
consecutive days and the equivalent amount of petroleum jelly was
applied to the control group. To examine the efficacy of QCs, mice were
randomly divided into five groups (n = 6 per group): a vehicle-treated
petroleum jelly-induced control group (abbreviated C), a vehicle
treated IMQ-induced model group (IMQ), a CPT (5 mg/kg)-treated
IMQ-induced group (CPT), and a 5i and 5l (50 mg/kg)-treated IMQ-
induced group (5i and 5l). The appropriate agents formulated in 200 μL
of vehicle (consisting of 12% ethanol, 23% normal saline, 5% tween 80,
4.14. Western blot analysis
Back skins collected from BALB/c were kept in −80 °C and in-
cubated in RIPA buffer on ice-bath for 30 min. The skin extractions
were centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was
11

W.-J. Zhang, et al.
Bioorganic Chemistry 88 (2019) 102899
collected and protein concentration was measured by BCA kit for pro-
tein quantification. Equivalent protein (40 μg) from each sample was
load to SDS-PAGE and transferred to PVDF membrane. The PVDF
membranes were blocked with 5% (w/v) BSA in TBST and incubated
with anti-p65, anti-TNF-α, anti-IL-17A, anti-IL-22, anti-IL-23, and anti-
IFN-γ and β-actin (Beyotime Biotechnology Co. Beijing China) over
night at 4 °C, subsequently washed using TBST, probed with specific
secondary antibodies coupled to HRP. An enhanced chemiluminescence
method (ECL) was used to detect the immunoreactive protein by ECL
reagents and the signal was recorded by FuJi medical X-ray film.
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Conflict of interest
(
FLT3) inhibitor, 1–(4-((1H–Pyrazolo[3,4–d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-
The authors declare no competing financial interest.
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This work was supported by the National Science Foundation of
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