Drug Discovery against Psoriasis: Identification of a New Potent FMS-like Tyrosine Kinase 3 (FLT3) Inhibitor, 1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-3-(5-(tert-butyl)isoxazol-3-yl)urea, That Showed Potent Activity in a Psoriatic Animal Model

Article abstract of DOI:10.1021/acs.jmedchem.6b00604

Psoriasis is a chronic T-cell-mediated autoimmune disease, and FMS-like tyrosine kinase 3 (FLT3) has been considered as a potential molecular target for the treatment of psoriasis. In this investigation, structural optimization was performed on a lead compound, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (1), which showed a moderate inhibitory activity againt FLT3. A series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized, and structure-activity relationship analysis led to the discovery of a number of potent FLT3 inhibitors. One of the most active compounds, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-fluorophenyl)-3-(5-tert-butylisoxazol-3-yl)urea (18b), was then chosen for in-depth antipsoriasis studies because this compound displayed the highest potency in a preliminary antipsoriasis test. Compound 18b exhibited significant antipsoriatic effects in the K14-VEGF transgenic mouse model of psoriasis, and no recurrence was found 15 days later after the last administration. Detailed mechanisms of action of compound 18b were also investigated. Collectively, compound 18b could be a potential drug candidate for psoriasis treatment.

Full text of DOI:10.1021/acs.jmedchem.6b00604

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Article
Drug Discovery against Psoriasis: Identification of a New Potent
FMS-like Tyrosine Kinase 3 (FLT3) Inhibitor, 1-(4-((1H-pyrazolo[3,4-
d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-3-(5-(tert-butyl)isoxazol-3-
yl)urea, That Showed Potent Activity in a Psoriatic Animal Model
Guo-Bo Li, Shuang Ma, Ling-Ling Yang, Sen Ji, Zhen Fang, Guo Zhang, Li-
Jiao Wang, Jie-Min Zhong, Yu Xiong, Jiang-Hong Wang, Shen-Zhen Huang,
Lin-Li Li, Rong Xiang, Dawen Niu, Ying-Chun Chen, and Sheng-Yong Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00604 • Publication Date (Web): 18 Aug 2016
Downloaded from http://pubs.acs.org on August 18, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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University
Niu, Dawen; State Key Laboratory of Biotherapy and Cancer Center, West
China Hospital, Sichuan University/Collaborative Innovation Center of
Biotherapy
Chen, Ying-Chun; Key Laboratory of Drug Targeting and Drug Delivery
System of Ministry of Education, West China School of Pharmacy, Sichuan
University
Yang, Sheng-Yong; State Key Laboratory of Biotherapy and Cancer Center,
West China Hospital, Sichuan University/Collaborative Innovation Center of
Biotherapy
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Drug Discovery against Psoriasis: Identification of a New Potent FMS-like
Tyrosine Kinase 3 (FLT3) Inhibitor, 1-(4-((1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)
-
3-fluorophenyl)-3-(5-(tert-butyl)isoxazol-3-yl)urea, That Showed Potent Activity
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in a Psoriatic Animal Model
†
,#
†,#
†,‡
†
†
§
GuoꢀBo Li, Shuang Ma, LingꢀLing Yang, Sen Ji, Zhen Fang, Guo Zhang,
†,‡
†
†
§
†
LiꢀJiao Wang, JieꢀMin Zhong, Yu Xiong, JiangꢀHong Wang, ShenꢀZhen Huang,
§
&
†
§
†,*
LinꢀLi Li, Rong Xiang, Dawen Niu, YingꢀChun Chen, and ShengꢀYong Yang
†
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University/Collaborative Innovation Center of Biotherapy, Sichuan 610041,
China
‡
College of Food and Bioengineering, Xihua University, Sichuan 610039, China
§
Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of
Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan,
6
10041, China
&
Department of Clinical Medicine, School of Medicine, Nankai University, Tianjin,
China
Abstract
Psoriasis is a chronic Tꢀcellꢀmediated autoimmune disease, and FMSꢀlike tyrosine
kinase 3 (FLT3) has been considered as a potential molecular target for the treatment
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of psoriasis. In this investigation, structural optimization was performed on a lead
compound,
1ꢀ(4ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyloxy)phenyl)ꢀ3ꢀ(4ꢀchloroꢀ3ꢀ
(trifluoromethyl)phenyl)urea (1), which showed a moderate inhibitory activity againt
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FLT3. A series of pyrazolo[3,4ꢀd]pyrimidine derivatives were synthesized, and
structureꢀactivity relationship analysis led to the discovery of a number of potent
FLT3
inhibitors.
ꢀ(4ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyloxy)ꢀ3ꢀfluorophenyl)ꢀ3ꢀ(5ꢀtertꢀbutylisoxazolꢀ
ꢀyl)urea (18b), was then chosen for inꢀdepth antiꢀpsoriasis studies because this
One
of
the
most
active
compounds,
1
3
compound displayed the highest potency in a preliminary antiꢀpsoriasis test.
Compound 18b exhibited significant antiꢀpsoriatic effects in the K14ꢀVEGF
transgenic mouse model of psoriasis, and no recurrence was found 15 days later after
the last administration. Detailed mechanisms of action of compound 18b were also
investigated. Collectively, compound 18b could be a potential drug candidate for
psoriasis treatment.
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1. Introduction
Psoriasis is a longꢀlasting autoimmune disease characterized by itchy, scaly, and
erythematous skin patches, which has profound psychological effects on patients and
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their family members. Though the pathogenesis of psoriasis has not been fully
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understood, significant evidence indicates that psoriasis is highly related to the
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inappropriate or unregulated activation of autoreactive T cells.
Traditional
approaches for psoriasis treatment through immunosuppression have thus focused on
the direct inhibition of T cells. However, these treatments inevitably cause
considerable toxic side effects, including immunogenicity and immunosuppression of
3
ꢀ5
beneficial immune responses.
Therefore, safe and effective strategies for the
treatment of psoriasis are in great demand at present.
Recent studies have shown that targeted inhibition of antigenꢀpresenting cells
could be a safe and effective approach to treat autoimmune diseases including
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,6ꢀ10
psoriasis.
regulating immune responses.
cells to mediate immune response by ingesting, processing and presenting the
Dendritic cells (DCs) are the main antigenꢀpresenting cells for
11ꢀ14
On the one hand, DCs can effectively activate T
1
5,16
antigens to T cells.
On the other hand, DCꢀTꢀcell interactions drive the
differentiation of T cells towards specific phenotypes. For instance, DCs can stimulate
the T cells to differentiate into Th17 cells, which are thought to play an essential role
in the pathogenesis of autoimmune diseases, through secreting cytokines, including
interleukinꢀ23 (ILꢀ23), transforming growth factor β (TGFꢀβ), and interleukinꢀ6
1
7,18
(ILꢀ6).
Thus, targeted inhibition of DCs may provide a new and effective
therapeutic strategy for psoriasis treatment by downꢀregulating the Tꢀcellꢀmediated
immune responses. Furthermore, it has been demonstrated that the FMSꢀlike tyrosine
kinase 3 (FLT3) plays a critical role in regulating the generation and maturation of
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DCs. FLT3 is known to express in hematopoietic progenitors,
and stimulation of
FLT3 via its ligand either in vitro or in vivo could drive the expansion and
22ꢀ24
differentiation of hematopoietic progenitors towards to a DC phenotype.
FLT3 is
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also highly expressed in mature DCs, suggesting that signals through this receptor
could be an important aspect of maintaining DC function. In a recent study,
Whartenby et al reported that inhibition of FLT3 signaling could induce apoptosis in
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both mouse and human DCs. Additionally, we for the first time detected a
+
significant accumulation of FLT3 DCs in human psoriatic lesions compared with
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normal skin from same psoriasis patients. All of these studies indicate that FLT3 is
likely a potential molecular target for the treatment of psoriasis through interference
with DCs.
We
previously
disclosed
an
FLT3
inhibitor,
1
ꢀ(4ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyloxy)phenyl)ꢀ3ꢀ(4ꢀchloroꢀ3ꢀ(trifluoromethyl)p
2
8
henyl)urea (1, Figure 1a), which showed an IC value of 39 nM against FLT3. This
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compound indeed exhibited certain antiꢀpsoriatic effect in vascular endothelial growth
factor (K14ꢀVEGF) transgenic homozygous mice; the K14ꢀVEGF transgenic mouse
model is one of the most commonly used psoriasis models, which can develop a
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9ꢀ32
cutaneous inflammatory condition resembling psoriasis.
Nevertheless, the activity
was at a moderate level, which could be mainly due to the moderate potency against
FLT3 of this compound. We shall in this investigation perform a further structural
modification to improve its potency against FLT3. The structureꢀactivity relationship
(
SAR) will also be discussed. The most active compounds will be evaluated for their
antiꢀpsoriatic effects in vivo, and mechanisms of action will be examined as well.
<Figure 1 here>
2
. Results and Discussion
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2
.1 Synthesis of 4-Phenoxy-1H-pyrazolo[3,4-d]pyrimidine Derivatives
All target compounds were prepared by routes outlined in Schemes 1ꢀ5. Scheme
shows the synthetic routes for compounds 7a-c. Firstly, the regioisomeric 3ꢀ and
ꢀaminoisoxazole intermediates 4a-c were synthesized in slightly different ways. The
ꢀaminoisoxazoles 4a and 4c were obtained by initial attack of the hydroxylamine
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nitrogen on the cyano group of the ketonitriles (3a and 3b) at pH 7ꢀ8 at 55 ℃,
followed by ring closure under acidic conditions (pH 3ꢀ4) at 55 ℃. The
5
ꢀaminoisoxazole 4b was acquired by initial attack of the hydroxylamine nitrogen on
the ketone group of 4,4ꢀdimethylꢀ3ꢀoxopentanenitrile (3a) and ring closure at pH 8 at
00 ℃ for 2.5 h. Secondly, reaction of 4ꢀchloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (5)
with 4ꢀaminophenolate through nucleophilic substitution produced intermediate
ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyloxy)aniline (6). Finally, treatment of 3ꢀ or
1
4
5ꢀaminoisoxazoles 4a-c with triphosgene led to corresponding isocyanates using a
33
similar method as that described in our previous work. The resulting isocyanates
then reacted with 6 to give final products 7a-c in 58ꢀ89% yields.
<
Scheme 1 here>
The general synthetic routes for compounds 9a-f, which contain a substituted
pyrazole group in ring B, are depicted in Scheme 2. Esters 2a-c reacted with
acetonitrile to produce ketonitriles 3aꢀc in 68%ꢀ84% yields. Intermediates 8a-f were
then obtained through a ring closure process of 3a-c with alkyl or aryl hydrazines.
The hydrazines used here were commercially available or synthesized, and
cyclopentylhydrazine was synthesized according to the method described by
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Ranatunge et al. The target compounds 9a-f were finally synthesized from 8a-f
using a method similar to that for 7a-c in 58%ꢀ92% yields.
<
Scheme 2 here>
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Scheme 3 presents the synthetic routes for compounds 12a, 12b and 14.
Commercially available 1ꢀchloroꢀ3,3ꢀdimethylbutanꢀ2ꢀone (10a) and 2ꢀbromoꢀ1ꢀ
phenylethanone (10b) reacted with thiocarbamide in EtOH at 90 ℃ to produce
2
ꢀaminothiazoles 11a and 11b, respectively. Treatment of 11a, 11b and
benzo[d]thiazolꢀ2ꢀamine (13), which was purchased from the market, with
triphosgene in the presence of triethylamine afforded the corresponding isocyanates,
which then reacted with 4ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyloxy)aniline (6) to
produce the desired products 12a, 12b and 14 in good yields.
<
Scheme 3 here>
Synthetic routes for compounds 16a-d are displayed in Scheme 4. Compounds
6a and 16b, which contain a methyl or isopropyl substituent at the Nꢀ1 of
1
pyrazolopyrimidine, were prepared from the corresponding analogue 7a in the
presence of sodium hydroxide in N,Nꢀdimethylformamide (DMF) at room
temperature and 60 ℃, respectively. Compounds 16c and 16d were obtained using a
twoꢀstep
procedure
involving
substitution
of
the
(6)
1ꢀNH
of
4ꢀ(1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyloxy)aniline
by
3
ꢀchloroꢀN,Nꢀdimethylpropanꢀ1ꢀamine or 4ꢀ(2ꢀchloroethyl)morpholine in DMF at 100
℃ under basic conditions and subsequent formation of ureas as described above.
<
Scheme 4 here>
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Scheme 5 shows the synthetic routes for compounds 18a-e, 19a-d, and 20.
Reactions of 4ꢀchloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (5) with various 2ꢀ or
3
ꢀsubstituted 4ꢀaminophenols gave intermediates 17a-e in the presence of sodium
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hydroxide. Compounds 18a-e were then prepared by reaction of intermediates 17a-e
with 5ꢀ(tertꢀbutyl)isoxazolꢀ3ꢀamine (4a), respectively, with yields ranging from 51%
to quantitative. Compounds 19a-d and 20 bearing a pyrazoleꢀurea moiety were
synthesized using corresponding pyrazoleisocyanates and intermediate 17b at a
temperature of 120 ℃.
<
Scheme 5 here>
2
.2 SAR of 4-Phenoxy-1H-pyrazolo[3,4-d]pyrimidine Derivatives
2.2.1 Variation of the 3-Position of the Urea Moiety (Ring B)
In our previous study, we found that the substituent of 3ꢀposition of the urea
moiety (ring B, Figure 1b) of compound 1 has a significant influence on the activity
against FLT3. Therefore, in this investigation, various substituted 5ꢀmembered
aromatic heterocyclic rings were used to replace the original ring B. The inhibitory
potencies (IC50) against FLT3 of the synthesized compounds 7a-c, 9a-f, 12a-b, and 14
are shown in Table 1. From Table 1, we can see that compounds 7a, 7b, 9c, and 12a,
containing
regioisomeric
5ꢀ(tertꢀbutyl)isoxazole,
3ꢀ(tertꢀbutyl)isoxazole,
3ꢀ(tertꢀbutyl)ꢀ1Hꢀpyrazoleꢀ1ꢀcarboxylic acid, and 4ꢀ(tertꢀbutyl)ꢀthiazole, respectively,
displayed 3ꢀ or 4ꢀfold more potent activity against FLT3 (IC50: 11nM, 8nM, 10nM,
and 8nM, respectively) than compound 1. In contrast, compounds 9b, 9d, 12b, and 14,
containing a heterocyclic ring with larger substituents at ring B, showed decreased
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activities. Especially, compound 14 with a phenylꢀsubstituted thiazole ring exhibited
significantly reduced ability to inhibit FLT3. We noticed that the most potent
derivatives 7a, 7b, and 12a actually have highly similar substituents at ring B, which
are 5ꢀ(tertꢀbutyl)isoxazole, 3ꢀ(tertꢀbutyl)isoxazole, and 4ꢀ(tertꢀbutyl)ꢀthiazole,
respectively. Considering that 5ꢀ(tertꢀbutyl)isoxazole has often been used as a key
motif in drugs or drug candidates like Quizartinib, implying a good biocompatibility,
we thus chose the 5ꢀ(tertꢀbutyl)isoxazole moiety as an optimal substituent for further
structural optimization.
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<
Table 1 here>
2
.2.2 Substitution of the N-1 Position of Pyrazolo[3,4-d]pyrimidine
To examine the substitution effect of the Nꢀ1 position in the
pyrazolo[3,4ꢀd]pyrimidine moiety, we introduced various substituents at this position
and fixed the 3ꢀposition of the urea moiety as the optimal 5ꢀ(tertꢀbutyl)isoxazole
group. Inhibitory activities of the synthesized compounds 16a-d are presented in
Table 2. Obviously, either large (16c, 16d) or small (16a, 16b) substitution at the Nꢀ1
position negatively affected the inhibitory activities against FLT3. Therefore, the NH
in the pyrazolo[3,4ꢀd]pyrimidine moiety may be an important pharmacophore for
binding with FLT3, and substitution at the Nꢀ1 position of pyrazolo[3,4ꢀd]pyrimidine
is not beneficial for the inhibitory activity improvement.
<
Table 2 here>
2.2.3 Replacement on the 1-Phenyl Group of the Urea Moiety (Ring A)
We further examined the possible influences of substitutions on the 1ꢀphenyl
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group of the urea moiety (Figure 1b, ring A). The ring B moiety was still fixed as the
optimal 5ꢀtertꢀbutylꢀisoxazole group, and no substitution occurred at the Nꢀ1 position
of pyrazolo[3,4ꢀd]pyrimidine. The inhibitory activities of synthesized target
compounds 18a-e, in which different substituents were attached to the 2ꢀ or 3ꢀposition
of ring A, are shown in Table 3. Compounds 18b, 18c, and 18e with the 2ꢀposition of
ring A replaced by fluoride, chlorine or methyl exhibited more potent activities
against FLT3 than unsubstituted 7a. Compared with substitution at the 2ꢀposition of
ring A, substitution at the 3ꢀposition of ring A is less favorable for the inhibitory
activity, particularly substitution by a bulky group (for example, see 18d vs 18e).
From here, a preliminary conclusion might be made that substitution at the 2ꢀposition
of ring A by fluoride or chlorine helps to enhance the bioactivity of target compounds.
To examine this point, we synthesized several other compounds, in which the
2ꢀposition of ring A was substituted with fluoride and ring B was replaced by other
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ꢀmembered aromatic heterocyclic rings, including 3ꢀtertꢀbutylꢀ1ꢀmethylꢀ1Hꢀpyrazole
(19a),
ꢀtertꢀbutylꢀ1ꢀcyclopentylꢀ1Hꢀpyrazꢀole
3ꢀtertꢀbutylꢀ1ꢀ(4ꢀfluorophenyl)ꢀ1Hꢀpyrazole
3ꢀtertꢀbutylꢀ1ꢀphenylꢀ1Hꢀpyrazole
(19b),
(19c),
and
3
(19d),
1
ꢀmethylꢀ3ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (20). Bioactivities of these compounds are
also shown in Table 3. From Table 3, we can see that compounds containing
substituent fluorine at the 2ꢀposition of ring A only exhibited comparable potencies
compared with their counterparts (19a vs 9a, 19b vs 9d, 19c vs 9b, and 20 vs 9e).
These results imply that substitution at the 2ꢀposition of ring A by fluoride or chlorine
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does not mean that it must help to significantly improve the bioactivity of target
compounds.
<
Table 3 here>
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Collectively, the structural optimization and SAR studies led to the discovery of a
number of novel compounds that exhibited a higher potency against FLT3 than
original lead compound 1. We then chose five most active compounds, namely 7b,
1
2a, 18a, 18b, and 18c, to perform a preliminary in vivo antiꢀpsoriasis study with the
K14ꢀVEGF transgenic mouse model. All of these compounds showed good
antiꢀpsoriatic activity in this model, and compound 18b is the most potent one.
Therefore, further inꢀdepth studies of antiꢀpsoriasis and mechanisms of action were
subsequently carried out on compound 18b.
2
.3 In Vitro Effects of Compound 18b on Plasmacytoid DCs and Myeloid DCs
The plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) are two main
subpopulations of immature DCs, both of which are highly aggregated in psoriatic
27
lesions. We first examined the effects of compound 18b on the generation of pDCs
and mDCs in vitro. For this purpose, mature bone marrowꢀderived cells (BMDCs)
were separated and cultured according to a standard protocol as described in materials
and methods section. The FLT3 ligand (FLT3L) was supplemented to BM culture to
+
+
35
induce the generation of FLT3 CD11c DCs as described previously. As shown in
Figure 2a, the numbers of pDCs and mDCs were reduced after compound 18b
treatment. These results indicate that compound 18b could suppress the generation of
pDCs and mDCs from BMDCs.
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<
Figure 2 here>
Because DCs (pDCs and mDCs) may have impact on the differentiation of T cells
4
,36ꢀ38
towards Th1 and Th17 cells through secretion of certain cytokines,
we next
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assessed the influence of compound 18b on secretion of cytokine interferon (IFN)ꢀγ
and ILꢀ23 in the culture system of DCs. Using the same protocol described above,
DCs were induced with FLT3L with or without addition of compound 18b and the
supernatants were detected by Elisa. As shown in Figure 2b, compound 18b could
dramatically decrease IFNꢀγ and ILꢀ23 levels even at 10 nM.
We then further examined the effects of compound 18b on the key downstream
signaling components of FLT3 pathway including signal transducer and activator of
transcription 3 and 5 (STAT3 and STAT5), which are critical transcription factors
39,40
involved in production of many inflammatory cytokines.
The results from western
blot analysis showed that the phosphorylation and activation of STAT3 and STAT5 in
DCs were decreased after compound 18b treatment for 1 h (Figure 2c). Taken together,
these results indicated that compound 18b could decrease the number of DCs and
inhibit cytokine secretion of DCs through the blockade of FLT3 signaling pathway.
2.4 In Vivo Effects of Compound 18b on K14-VEGF Transgenic Mice
The in vivo antiꢀpsoriatic effects of compound 18b were evaluated using the
transgenic K14ꢀVEGF mice. The heavy symptom K14ꢀVEGF mice were treated with
compound 18b or vehicle by smearing over the lesion surface uniformly. As shown in
Figure 3a, after a continuous treatment of 18b for 21 days, an obvious improvement
of psoriatic symptom was observed in the compound 18b treated group compared
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with the vehicle group. There was no recurrence 15 days later after the last
administration. Histology also showed that compound 18b treatment led to a
significant alleviation in the psoriatic disease indexes including epidermal thickness,
focal parakeratosis, lack of granular layer, and abundant infiltrated lymphocytes
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(
Figure 3b). This was further confirmed by the Baker scores (Figure 3c); the Baker
score system is a traditional evaluation method in skin disease. From Figure 3c, we
can see that the pathological scores of the mice treated with compound 18b were
significantly decreased compared with those of the vehicleꢀtreated controls. Thus we
conclude that compound 18b could effectively alleviated psoriasis in K14ꢀVEGF
transgenic mice.
<Figure 3 here>
2
.5 Mechanisms of Action of Compound 18b
The mechanisms of action underlying the in vivo antiꢀpsoriatic effects of
compound 18b were further examined. Firstly, we examined the direct effect of
+
+
compound 18b on the population of FLT3 CD11c DCs (pDCs and mDCs) in
K14ꢀVEGF transgenic mice, because it has been shown that a high expression of
+
+
FLT3 and a significant accumulation of FLT3 CD11c DCs are typical characteristics
27,41
of psoriatic lesions.
After external administration of compound 18b to the
+
+
K14ꢀVEGF mice for 29 days, we tested the number of FLT3 CD11c DCs in
psoriatic lesions by flow cytometric quantifications. As shown in Figure 4a, the
+
+
number of FLT3 CD11c DCs in the skin was significantly decreased (about 69.6%)
in compound 18bꢀtreated mice compared with control group mice. The populations of
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pDCs and mDCs in FLT3 CD11c DCs were further analyzed. We found that the
+
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+
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+
+
numbers of FLT3 CD11c B220 DCs (pDCs) and FLT3 CD11c MHC class II DCs
mDCs) were both greatly reduced in compound 18bꢀtreated mice compared with
those in vehicleꢀtreated mice (Figure 4b).
(
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<Figure 4 here>
Secondly, we examined the effects of compound 18b on the defining histological
features of psoriasis including marked infiltration of T cells, elongated/hyperplastic
blood vessels, and the presence of various proꢀinflammatory cytokines, chemokines,
and growth factors in the vehicleꢀtreated and compound 18bꢀtreated psoriatic lesions.
As shown in Figure 5, compound 18b treatment significantly reduced the expressions
of CD8, CD31, VEGF, CD54, and phosphorylated STAT3 (pSTAT3) compared with
vehicle treatment. The decrease in CD8 (79%) indicates inhibition of the infiltration
+
of CD8 inflammatory lymphocytes. The percent of CD31 and VEGF expression
were reduced about 94% and 90% respectively, implying that compound 18b
suppressed the proliferation and dilation of blood vessels. The reduction of expression
of CD54 was about 87%, which indicates a decrease in endothelial cell activation.
Finally, the reduction in pSTAT3 (64%) expression implies the inhibition of
keratinocyte hyper proliferation. Taken together, these results showed that compound
18b could suppress the infiltration of T cells, the abnormal growth of blood vessels,
and the expression of proꢀinflammatory chemokines and growth factors in psoriatic
lesions.
<Figure 5 here>
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Finally, we examined the impact of compound 18b on cytokines related to
42ꢀ44
Th17/Th1 pathways, because psoriasis is thought to be a mixed Th17/Th1 disease.
+
+
Flow cytometric analysis showed that the number of Th17 (ILꢀ17 CD4 ) cells in the
lymph nodes was considerably lower in the compound 18bꢀtreated group compared
with the vehicleꢀtreated group (Figure 6a). The expressions of IFNꢀγ and ILꢀ17 in
blood were also significantly downꢀregulated by compound 18b, as IFNꢀγ and ILꢀ17
are thought to be produced by Th1 and Th17 inflammatory pathway, respectively.
These results indicated that compound 18b treatment suppressed the Th17/Th1
pathways.
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<Figure 6 here>
3. Concluding Remarks
In this investigation, a series of 4ꢀphenoxyꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine
derivatives were synthesized. SAR studies led to discovery of a number of potent
FLT3 inhibitors. One of the most potent FLT3 inhibitors, compound 18b, showed the
highest antiꢀpsoriatic activity in a preliminary in vivo antiꢀpsoriasis study. This
compound was then carried out further inꢀdepth in vitro and in vivo antiꢀpsoriatic
studies. The in vitro results indicated that compound 18b could reduce the generation
of DCs and inhibit cytokine secretion of DCs through the blockade of FLT3 signaling
pathway. The in vivo studies showed that compound 18b could effectively alleviate
psoriasis in K14ꢀVEGF transgenic mice. The mechanism of action studies revealed
+
+
that compound 18b could suppress the population of FLT3 CD11c DCs (including
pDCs and mDCs), the infiltration of Tꢀcell, and the levels of related proꢀinflammation
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chemokines and growth factors, as well as the expression of cytokines related to
Th17/Th1 pathways. Collectively, compound 18b obtained here could be a potential
drug candidate for the treatment of psoriasis.
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4
. Experimental Section
Chemistry Methods
Unless otherwise noted, all starting materials, reagents, and solvents were
purchased from commercial vendors and used as supplied without further purification.
Reactions were monitored by thin layer chromatography (TLC) on Merck silica gel 60
1
13
Fꢀ254 thin layer plates. H NMR and C NMR spectra were recorded on a Bruker
AVꢀ400 spectrometer at 400 MHz and 100 MHz, respectively. Chemical shifts (δ) are
expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal
standard. The following abbreviations are used to designate multiplicities: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; dd, double–doublet; br s, broad signal.
Lowꢀresolution and highꢀresolution mass spectral (MS) data were determined on an
Agilent 1100 Series LCꢀMS with UV detection at 254 nm in lowꢀresonance
electrospray ionization (ESI) mode. All the final compounds were purified to >95%
purity, as determined by highꢀperformance liquid chromatography (HPLC). HPLC
analysis was performed on a Waters 2695 HPLC system equipped with a Kromasil
C18 column (4.6 mm × 250 mm, 5 um).
1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(5-tert-butylisoxazol-3
-yl)urea (7a) The chemical sythesis of intermediate 6 builds upon previously
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published method. H NMR (400 MHz, DMSOꢀd ): δ 14.07 (s, 1H), 8.50 (s, 1H),
7
.67 (s, 1H), 6.96 (d, J=8.8 Hz, 2H), 6.64 (d, J=8.8 Hz, 2H), 5.20 (s, 2H) ppm;
+
LCꢀMS m/z: 228.1 [M + H] .
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A solution of 5ꢀ(tertꢀbutyl)isoxazolꢀ3ꢀamine (4a, 0.308 g, 2.2 mmol) dissolved in
THF (20 ml) was slowly dripped into a stirred solution of triphosgene (0.654 g, 2.2
mmol) in THF (5 mL) using a constant pressure dropping funnel at room temperature.
Triethylamine (0.65 mL, 4.6 mmol) was then added slowly to the reaction mixture
after the aniline was completely added. Subsequently, the reaction was reflux for 2
hours. After evaporation of the solvent, the residue was dissolved in acetonitrile (20
ml) and toluene (20 ml), and compound 6 (0.454 g, 2.0 mmol) was added. Next, the
reaction mixture was stirred at 110 ℃ for 16 h, and the solvent was removed in
vacuo. The residue obtained was purified by column chromatography (eluent gradient
CH
2
Cl
2
:MeOH, 60:1) and recrystallized from EtOAc and petroleum ether. Yield:
1
8
9%, 95.9% HPLC purity. H NMR(400 MHz, DMSOꢀd ): δ 14.13 (s, 1H), 9.55 (s,
6
1H), 8.95 (s, 1H), 8.51 (s, 1H), 8.05 (s, 1H), 7.56 (d, J=8.8 Hz, 2H), 7.27 (d, J=8.8
13
Hz, 2H), 6.52 (s, 1H), 1.30 (s, 9H) ppm; C NMR (100 MHz, DMSOꢀd
6
): δ 180.7,
163.6, 158.9, 157.2, 155.4, 151.9, 147.4, 137.3, 132.3, 122.8, 120.2, 101.8, 93.0, 33.0,
+
2
8.8 ppm; HRMS (m/z): calcd for C19
H
20
N
7
O
3
[M + H] 394.1622, found 394.1604;
+
C H N NaO [M + Na] 416.1442, found 416.1419.
19
19
7
3
1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(3-tert-butylisoxazol-5
-yl)urea (7b) The title compound was synthesized in a manner similar to the synthesis
of 7a using compounds 4b and 6 to give compound 7b, which was purified by column
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chromatography (eluent gradient CH
2
Cl
2
:MeOH, 60:1) and recrystallized from EtOAc
1
and petroleum ether. Yield: 80%, 95.9% HPLC purity. H NMR (400 MHz,
6
DMSOꢀd ): δ 14.14 (s, 1H), 9.56 (s, 1H), 8.94 (s, 1H), 8.51 (s, 1H), 8.06 (s, 1H), 7.56
0
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(
d, J=8.8 Hz, 2H), 7.27 (d, J=8.8 Hz, 2H), 6.52 (s, 1H), 1.30 (s, 9H) ppm; C NMR
100 MHz, DMSOꢀd ): δ 180.2, 163.1, 158.3, 156.7, 154.9, 151.4, 146.9, 136.8,
31.8, 122.3, 119.7, 101.3, 92.4, 32.4, 28.3 ppm; HRMS (m/z): calcd for C19
(
6
1
20 7 3
H N O
+
+
[
M + H] 394.1622, found 394.1604; C19
16.1419.
-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(3-tert-butyl-1-cyclope
19 7 3
H N NaO [M + Na] 416.1442, found
4
1
ntyl-1H-pyrazol-5-yl)urea (9b) A mixture of cyclopentanone (0.84 g, 10 mmol) and
tertꢀbutyl hydrazinecarboxylate (1.32 g, 10 mmol) in methanol (50 ml) was stirred at
ambient temperature for 3 h. Subsequently, the solvent was removed in vacuo, and
tertꢀbutyl 2ꢀcyclopentylidenehydrazinecarboxylate (1.92 g) was obtained as a white
solid in high yield.
Tertꢀbutylꢀ2ꢀcyclopentylideneꢀhydrazinecarboxylate (1.92 g, 9.7 mmol) was
dissolved in 50% acetic acid (30 ml). The resulting solution was then stirred at room
temperature for 2.5 h after the portion wise addition of sodium cyanoborohydride
(0.60 g, 9.6 mmol). The reaction mixture was neutralized with NaOH and extracted
with CH Cl , and the organic layer was dried with anhydrous MgSO and evaporated
2
2
4
to give the reduction product as oil (1.7 g). The product was used without further
purification.
Trifluoroacetic acid (10 ml) was added dropwise to a solution of tertꢀbutyl
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2 2
ꢀcyclopentylꢀhydrazinecarboxylate (1.7 g, 8.55 mmol) in CH Cl (10 ml). The
solution was stirred at ambient temperature for 1.5 h. The solvent was then evaporated
to give the trifuoroacetate salt as colorless oil in high yield.
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3
ꢀTertꢀbutylꢀ1ꢀcyclopentylꢀ1Hꢀpyrazolꢀ5ꢀamine (8b) was synthesized from
4
,4ꢀdimethylꢀ3ꢀoxopentanenitrile (3a) and the trifuoroacetate salt of
cyclopentylhydrazine using a procedure similar to that of 8a (Yield:76%).
The title compound was prepared from 8b and 6 using the procedure described
above for compound 7a, which was purified by column chromatography (eluent
gradient CH Cl :MeOH, 55:1) and purified by recrystallization from EtOAc and
2
2
1
petroleum ether. Yield: 92%, 97.4% HPLC purity. H NMR(400 MHz, DMSOꢀd
14.15 (s, 1H), 9.38 (s, 1H), 8.79 (s, 1H), 8.51 (s, 1H), 8.03 (s, 1H), 7.56 (d, J=8.0 Hz,
H), 7.24 (d, J=8.0 Hz, 2H), 6.05 (s, 1H), 4.63ꢀ4.55 (m, 1H), 1.99ꢀ1.92 (m, 4H),
6
): δ
2
1
3
1.84ꢀ1.81 (m, 2H), 1.60ꢀ1.56 (m, 2H), 1.22 (s, 9H) ppm; C NMR (100 MHz,
DMSOꢀd ): δ 162.8, 159.3, 157.0, 155.3, 149.6, 138.5, 133.8, 132.0, 124.8, 116.9,
6
107.2, 101.6, 96.6, 57.5, 32.4, 30.9, 24.4 ppm; HRMS (m/z): calcd for C24
H N O
29 8 2
[M
+
+
+
H] 461.2408, found 461.2410; C24
483.2232.
-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4-tert-butylthiazol-2-
H N NaO [M + Na] 483.2227, found
28 8 2
1
yl)urea (12a) A mixture of 1ꢀchloroꢀ3,3ꢀdimethylbutanꢀ2ꢀone (10a, 2.0 ml, 15.2
mmol) andthiocarbamide (1.16 g, 15.2 mmol) was reacted for 12 h in refluxed alcohol.
Solvent was evaporated to give the crude product 4ꢀ(tertꢀbutyl)thiazolꢀ2ꢀamine (11a);
Yield: 88%.
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3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The title compound was synthesized in a manner similar to the synthesis of 7a
using compounds 11a and 6 to give compound 12a, which was purified by column
chromatography (eluent gradient CH
2
Cl
2
:MeOH, 60:1) and recrystallized from EtOAc
0
1
2
3
4
5
6
7
8
9
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2
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8
9
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8
9
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
and petroleum ether. Yield: 76%, 100% HPLC purity. H NMR (400 MHz,
DMSOꢀd
.57 (d, J=8.8 Hz, 2H), 7.28 (d, J=8.8 Hz, 2H), 6.65 (s, 1H), 1.26 (s, 9H) ppm; C
NMR (100 MHz, DMSOꢀd ): δ 180.7, 164.0, 158.9, 157.3, 155.4, 151.9, 147.4, 137.3,
32.3, 122.8, 120.2, 103.4, 93.0, 33.0, 28.8 ppm; HRMS (m/z): calcd for
6
): δ 14.13 (s, 1H), 10.65 (s, 1H), 8.96 (s, 1H), 8.51 (s, 1H), 8.06 (s, 1H),
13
7
6
1
+
+
C H N O S [M + H] 410.1394, found 410.1383; C H N NaO S [M + Na]
19 20
7
2
19 19
7
2
4
32.1213, found 432.1208.
1-(5-Tert-butylisoxazol-3-yl)-3-(4-(1-(2-morpholinoethyl)-1H-pyrazolo[3,4-d]
pyrimidin-4-yloxy)phenyl)urea (16d) The title compound was synthesized in a
manner similar to the synthesis of 16c using compounds 15b and 4a to give
compound 16d, which was purified by column chromatography (eluent gradient
CH
2
Cl
2
:MeOH, 40:1) and recrystallized from EtOAc and petroleum ether. Yield:
1
7
6%, 97.9% HPLC purity. H NMR (400 MHz, DMSOꢀd
6
): δ 9.57 (s, 1H), 9.04 (s,
1H), 8.54 (s, 1H), 8.10 (s, 1H), 7.56 (d, J=8.8 Hz, 2H), 7.27 (d, J=8.8 Hz, 2H), 6.51
13
(
s, 1H), 4.56 (br s, 2H), 3.46 (s, 4H), 2.80 (s, 2H), 2.43 (s, 4H), 1.30 (s, 9H) ppm; C
NMR (100 MHz, DMSOꢀd ): δ 180.7, 163.7, 158.8, 155.6, 155.3, 151.9, 147.3, 137.4,
6
1
31.8, 122.8, 120.2, 100.2, 93.0, 66.6, 57.5, 53.4, 33.9, 28.8 ppm; HRMS (m/z): calcd
+
+
31 8 4 30 8 4
for C25H N O [M + H] 507.2463, found 507.2462; C25H N NaO [M + Na]
5
29.2282, found 529.2277.
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1
-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-2-fluorophenyl)-3-(5-tert-butylis
oxazol-3-yl)urea (18a) Sodium hydroxide (0.20 g, 5.0 mmol) was added to a solution
of 4ꢀaminoꢀ3ꢀfluorophenol (0.635 g, 5.0 mmol) in H O (20 ml). The mixture was
stirred at room temperature for 30 minutes. Then,
ꢀchloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (5, 0.77 g, 5.0 mmol) in THF (20 ml) was
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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36
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44
45
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53
54
55
56
57
58
59
60
4
slowly added, and the reaction mixture was heated to 60 ℃ for 2 hours. The solvent
was then partially evaporated on a rotary evaporator. The obtained crude mixture was
extracted with ethyl acetate (2 × 120 ml) and water. The combined organic layers
were dried over MgSO and concentrated, and the residue was purified by column
4
2 2
chromatography (eluent gradient CH Cl :MeOH, 80:1) without final recrystallization
to give 17a (0.502 g, 41%) as a brown solid.
The title compound was synthesized in a manner similar to the synthesis of 7a
using compounds 17a and 4a to give compound 18a, which was purified by column
chromatography (eluent gradient petroleum ether:EtOAc, 3:1) and recrystallized from
1
EtOAc and petroleum ether. Yield: 51%, 97.4% HPLC purity. H NMR (400 MHz,
6
DMSOꢀd ): δ 14.17 (s, 1H), 9.86 (s, 1H), 8.87 (s, 1H), 8.52 (s, 1H), 8.19ꢀ8.15 (m,
2H), 7.44 (d, J=10.4 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 6.50 (s, 1H), 1.29 (s, 9H) ppm;
13
6
C NMR (100 MHz, DMSOꢀd ): δ 180.8, 163.2, 158.7, 157.2, 155.3, 153.8, 151.7,
147.2, 132.2, 121.9, 118.6, 110.5, 101.8, 92.8, 32.9, 28.8 ppm; HRMS (m/z): calcd for
+
C
19
H
18FN
7
NaO
3
[M + Na] 434.1347, found 434.1351.
1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-fluorophenyl)-3-(5-tert-butylis
oxazol-3-yl)urea (18b) The title compound was synthesized in a manner similar to
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the synthesis of 7a using compounds 17b and 4a to give compound 18b, which was
purified by column chromatography (eluent gradient petroleum ether:EtOAc, 3:1) and
1
recrystallized from EtOAc and petroleum ether. Yield: 89%, 98.6% HPLC purity. H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (400 MHz, DMSOꢀd ): δ 14.24 (s, 1H), 9.65 (s, 1H), 9.14 (s, 1H), 8.53 (s, 1H),
6
8
6
1
9
4
.31 (s, 1H), 7.70 (d, J=10.8 Hz, 1H), 7.42 (t, J=8.4 Hz, 1H), 7.24 (d, J=9.2 Hz, 1H),
13
6
.53 (s, 1H), 1.31 (s, 9H) ppm; C NMR (100 MHz, DMSOꢀd ): δ 180.8, 162.6,
58.7, 157.2, 155.3, 152.8, 151.9, 138.2, 133.9, 132.2, 124.8, 115.3, 107.4, 101.4,
+
3.1, 33.0, 28.8 ppm; HRMS (m/z): calcd for C19
H
19FN
7
O
3
[M + H] 412.1528, found
+
12.1511; C H FN NaO [M + Na] 434.1347, found 434.1338.
19 18
7
3
1
-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-chlorophenyl)-3-(5-tert-butyli
soxazol-3-yl)urea (18c) The title compound was synthesized in a manner similar to
the synthesis of 7a using compounds 17c and 4a to give compound 18c, which was
purified by column chromatography (eluent gradient petroleum ether:EtOAc, 3:1) and
1
recrystallized from EtOAc and petroleum ether. Yield: 96%, 100% HPLC purity. H
NMR(400 MHz, DMSOꢀd
.26 (s, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.46ꢀ7.41 (m, 2H), 6.52 (s, 1H), 1.30 (s, 9H)
ppm; δ 180.8, 162.7, 158.7, 157.3, 155.3, 151.9, 143.0, 138.6, 132.2, 125.6, 125.0,
20.1, 119.1, 101.5, 93.0, 33.0, 28.8, 16.4 ppm; HRMS (m/z): calcd for C19
6
): δ 14.23 (s, 1H), 9.67 (s, 1H), 9.10 (s, 1H), 8.52 (s, 1H),
8
1
7 3
H19ClN O
+
+
[M + H] 428.1232, found 428.1223; C H ClN NaO [M + Na] 450.1052, found
1
9
18
7
3
4
50.1051.
1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-methylphenyl)-3-(3-tert-butyl-
-methyl-1H-pyrazol-5-yl)urea (18e) The title compound was synthesized in a
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manner similar to the synthesis of 7a using compounds 17e and 4a to give 18e, which
was purified by column chromatography (eluent gradient petroleum ether:EtOAc, 2:1)
and recrystallized from EtOAc and petroleum ether. Yield: 57%, 96.8% HPLC purity.
10
11
12
13
14
15
16
17
18
19
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45
46
47
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49
50
51
52
53
54
55
56
57
58
59
60
1
H NMR (400 MHz, DMSOꢀd ): δ 14.14 (s, 1H), 9.54 (s, 1H), 8.86 (s, 1H), 8.49 (s,
6
1
H), 8.00 (s, 1H), 7.48 (s, 1H), 7.36 (d, J=8.8 Hz, 1H), 7.18 (d, J=8.8 Hz, 1H), 6.52
13
(s, 1H), 2.07 (s, 3H), 1.30 (s, 9H) ppm; C NMR (100 MHz, DMSOꢀd
6
): δ 180.7,
1
1
4
63.3, 158.9, 157.3, 155.5, 151.9, 146.1, 137.4, 132.2, 131.1, 123.1, 121.6, 118.0,
+
22 7 2
01.6, 92.9, 33.0, 28.8, 16.4 ppm; HRMS (m/z): calcd for C20H N O [M + H]
+
08.1779, found 408.1760; C H N NaO [M + Na] 430.1598, found 430.1581.
20 21
7
2
1
-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-fluorophenyl)-3-(3-tert-butyl-
1-phenyl-1H-pyrazol-5-yl)urea (19b) The title compound was synthesized in a
manner similar to the synthesis of 7a using compounds 17b and 8d to give compound
19b, which was purified by column chromatography (eluent gradient CH
2 2
Cl :MeOH,
6
0:1) and recrystallized from EtOAc and petroleum ether.Yield: 86%, 98.3% HPLC
1
purity. H NMR (400 MHz, DMSOꢀd
6
): δ 14.22 (s, 1H), 9.31 (s, 1H), 8.53 (s, 1H),
8
.50 (s, 1H), 8.30 (s, 1H), 7.66 (d, J=2.4 Hz, 1H), 7.55 (d, J=4.4 Hz 4H), 7.43ꢀ7.39
1
3
(m, 2H), 7.21 (d, J=2.4 Hz, 1H), 6.40 (s, 1H), 1.29(s, 9H) ppm; C NMR (100 MHz,
DMSOꢀd ): δ 170.3, 162.2, 160.8, 156.7, 154.8, 151.7, 142.1, 138.6, 136.8, 131.7,
6
129.3, 127.4, 126.0, 124.5, 124.2, 119.2, 118.1, 101.0, 96.1, 32.0, 30.1 ppm; LCꢀMS
+
m/z: 487.2 [M + H] .
1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-fluorophenyl)-3-(3-tert-butyl-
1
-cyclopentyl-1H-pyrazol-5-yl)urea (19c) The title compound was synthesized in a
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manner similar to the synthesis of 7a using compounds 17b and 8b to give compound
1
2 2
9c, which was purified by column chromatography (eluent gradient CH Cl :MeOH,
6
5:1) and recrystallized from EtOAc and petroleum ether. Yield: 94%, 98.2% HPLC
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
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8
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4
5
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8
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6
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8
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purity. H NMR (400 MHz, DMSOꢀd ): δ 14.24 (s, 1H), 9.16 (s, 1H), 8.52 (s, 1H),
6
8
.49 (s, 1H), 8.30 (s, 1H), 7.68 (dd, J=2.4 Hz, J=10.8 Hz, 1H), 7.40 (t, J=8.8 Hz, 1H),
.24 (d, J=8.8 Hz, 1H), 6.05 (s, 1H), 4.55ꢀ4.48 (m, 1H), 1.98ꢀ1.89 (m, 4H), 1.88ꢀ1.80
7
1
3
(
m, 2H), 1.62ꢀ1.56 (m, 2H), 1.22 (s, 9H) ppm; C NMR (100 MHz, DMSOꢀd
6
): δ
62.6, 158.9, 157.2, 155.3, 152.8, 152.6, 139.5, 135.3, 133.5, 132.2, 124.7, 114.9,
07.0, 101.4, 95.2, 57.5, 32.4, 30.9, 24.4 ppm; HRMS (m/z): calcd for C H FN O
2
1
1
24 28
8
+
+
[
M + H] 479.2314, found 4179.2314; C24
501.21338.
The synthetic methods and characterization data for other target compounds
8 2
H27FN NaO [M + Na] 501.2135, found
1
13
reported in this study are given in Supporting Information. The H NMR, C NMR,
HRMS spectra and HPLC chromatograms of the target compounds are also shown in
Supporting Information Figure S1ꢀS26.
Kinase Inhibitory Assays
The kinase inhibitory assays were performed according to the KinaseProfiler
assay protocols of Eurofins Pharma Discovery Services UK Limited (Eurofins).
K14-VEGF Transgenic Mice
The K14ꢀVEGF transgenic homozygous mice were purchased from Jackson
Laboratories (Bar Harbor, ME, USA). The high expression of VEGF in the skin tissue
could induce inflammatory skin condition with similar characteristics of psoriasis,
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including erythematous and scaly skin, moderate acanthosis, parakeratosis,
hyperplastic inflamed blood vessels, and typical inflammatory cell infiltrate.
In the experiments, 3ꢀ to 4ꢀmonthꢀold K14ꢀVEGF mice with moderate to serious
psoriasis were chosen and reꢀderived to homozygosity. The same batch of K14ꢀVEGF
transgenic homozygous mice were divided into two groups as follows: treatment
group receiving compound 18b and control groups receiving only vehicle. Mice were
smeared with cream contained 0.05% compound 18b or not on psoriatic lesions for 3
weeks. After the final treatment, the mice were housed and observed for another 15
days. Photographs of mouse head and neck were obtained at day 0, 21 and 36. All
animal experiments described in this study were approved by the Institutional Animal
Care and Use Committee of Sichuan University.
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pDC and mDC Cultures
Balb/c mice were kept under SPF conditions. To generate pDCs and mDCs,
45,46
femurs and tibias of mice were removed and purified as described previously.
Then, BM cells were flushed with PBS using a syringe with a 0.45 mm diameter
6
needle. Whole BM cells (1×10 /ml) were cultured in 6ꢀwell plates in RPMIꢀ1640
ꢀ4
medium supplemented with 10% FCS, 10 M 2ꢀME (SigmaꢀAldrich), sodium
pyruvate (Gibco), 100 ꢁg/ml penicillin (SigmaꢀAldrich), 100 ꢁg/ml streptomycin
(SigmaꢀAldrich) and 100 ng/ml FLT3L (Peprotech) as indicated for 9 days.
Elisa
pDCs and mDCs were induced for 9 days as mentioned before. On day 9,
compound 18b with indicated concentrations dissolved in DMSO was added in the
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culture medium. After 12 h, the content of IFNꢀγ and ILꢀ23 in the culture medium was
analysed by Elisa following the manufacture instructions.
K14ꢀVEGF mice were treated with compound 18b (n=3) or vehicle (n=3) for 29
days as mentioned before. At termination, blood samples were collected as described
previously and kept in 4 °C for 12 h. After centrifugation, the content of IFNꢀγ and
ILꢀ17 in plasma was quantified by Elisa according to the manufacturer’s protocol.
Flow Cytometric Analysis
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pDCs and mDCs were induced for 9 days as mentioned before. On day 9,
indicated concentration of compound 18b dissolved in DMSO was added in the
culture medium. After 24 h, cells were incubated with antibodies against CD11c
PerCP, CD45R/B220 FITC or IꢀA/IꢀE (MHC class II) FITC for 30 min and analysed
47
+
+
by flow cytometric as indicated. pDCs were identified as CD11c B220 and mDCs
+
+
as CD11c MHC class II cells.
The cells in the skin tissue sections and lymph nodes were harvested as indicated
before. For the skin section cells detection, threeꢀ or twoꢀcolor staining was performed
using mouse antiꢀFLT3 PE, antiꢀCD11c PerCP, antiꢀB220 FITC, and IꢀA/IꢀE FITC
antibodies. For the lymphocyte detection, cells were first surfaceꢀstained with
antiꢀCD4 PE followed by fixing and permeabilisation. After the permeabilisation, the
FITCꢀlabeled antiꢀmouse ILꢀ17 antibody was added.
All the antibodies were obtained from BectonꢀDickinson. Cells were analysed
using a flow cytometer (BectonꢀDickinson), and data were processed using FlowJo
software.
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Western Blot Analysis
BMDCs were cultured as above. On day 9, compound 18b was added to the
culture medium for 1 h. Cells were harvested and subjected to western blot analysis
with rabbit antiꢀmouse STAT3, STAT5, pSTAT3 and pSTAT5 antibodies as previously
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described. All the antibodies were purchased from Abcam.
Histology
K14ꢀVEGF mice were treated with compound 18b (n=3) or vehicle (n=3) for 29
days as mentioned before. The skin lesion sections were collected and embedded in
paraffin. Then the HE and immunohistochemistry assays were performed as
48
indicated. The positive areas were assessed using the Imagepro Plus software. All
the antibodies were purchased from Cell Signaling Technology.
Associated Content
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI: 00.0000/acs.jmedchem.0000000.
Supplementary methods section giving the synthetic methods and characterization
data for the target compounds that are not included in the main text; Figure S1ꢀS26
1
13
showing H NMR, C NMR, HRMS spectra and HPLC chromatograms of the target
compounds. Molecular formula strings of the target compounds were uploaded as
“
Molecular Formula Strings.csv”.
Corresponding Authors
Phone: +86ꢀ28ꢀ85164063. Eꢀmail: yangsy@scu.edu.cn.
*
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Author Contributions
#
G.B. Li and S. Ma contributed equally to this work.
Notes
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The authors declare no competing financial interest.
Acknowledgements
This work was supported by the 973 Program (2013CB967204), the National Natural
Science Foundation of China (81325021, 81473140, 81573349), and the Program for
Changjiang Scholars and Innovative Research Team in University (IRT13031).
Abbreviations Used
SAR, structureꢀactivity relationship; DCs, dendritic cells; FLT3, FMSꢀlike tyrosine
kinase 3; FLT3L, FLT3 ligand; IL, interleukin; TGF, transforming growth factor; IFN,
interferon; VEGF, vascular endothelial growth factor; pDCs, plasmacytoid DCs;
mDCs, myeloidDCs; BMDCs, bone marrowꢀderived cells; STAT, signal transducer
and activator of transcription; pSTAT, phosphorylated STAT; DMF,
N,Nꢀdimethylformamide; TLC, thin layer chromatography; TMS, tetramethylsilane;
ESI, electrospray ionization.
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