Phenotypic Screening-Based Identification of 3,4-Disubstituted Piperidine Derivatives as Macrophage M2 Polarization Modulators: An Opportunity for Treating Multiple Sclerosis

Article abstract of DOI:10.1021/acs.jmedchem.8b01635

Multiple sclerosis (MS) is a disease of the autoimmune-mediated disorder in the central nervous system, for which no effective therapeutic agent is currently available. The regulation of macrophage polarization toward M2 is a general benefit for treating MS. The gene biomarker-based phenotypic screening approach was developed, and 3,4-disubstituted piperidine derivative S-28 was identified as a lead compound modulating macrophage M2 polarization. Further SAR studies resulted in the discovery of the most potent modulator D11 that showed good oral bioavailability and significant in vivo therapeutic effects. Mechanistic studies demonstrated that the M2 polarization macrophages modulated by D11 mainly functioned through inhibiting the proliferation of T-cells and activating the phosphorylation of Stat3 and Akt. Therefore, the gene biomarker-based phenotypic screening was demonstrated as a promising tool for the discovery of novel macrophage M2 polarization modulators. Compound D11 may serve as a promising starting point for the development of therapeutics to treat MS.

Full text of DOI:10.1021/acs.jmedchem.8b01635

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Article
Phenotypic Screening-based Identification of 3,4-Disubstituted
Piperidine Derivatives as Macrophage M2 Polarization
Modulators: An Opportunity for Treating Multiple Sclerosis
qin jie weng, Jinxin Che, Zhikang Zhang, Jiahuan Zheng, Wenhu Zhan, Sendong Lin, Tian
Tian, Jincheng Wang, Renhua Gai, Yongzhou Hu, Bo Yang, Qiaojun He, and Xiaowu Dong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01635 • Publication Date (Web): 11 Mar 2019
Downloaded from http://pubs.acs.org on March 12, 2019
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Phenotypic Screening-based Identification of 3,4-
Disubstituted Piperidine Derivatives as Macrophage
M2 Polarization Modulators: An Opportunity for
Treating Multiple Sclerosis
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Qinjie Weng,b,‡ Jinxin Che,a,‡ Zhikang Zhang,b,‡ Jiahuan Zheng,b Wenhu Zhan,a
Sendong Lin,a Tian Tian,a Jincheng Wang, b Renhua Gai, b Yongzhou Hu,a Bo Yang,b
Qiaojun He,b Xiaowu Dong,a,*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, Hangzhou Institute of Innovative
Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058,
China
b Institute of Pharmacology & Toxicology, Zhejiang Province Key Laboratory of Anti-
Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, 310058, China
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‡
These authors contributed equally to this work
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KEYWORDS: Multiple sclerosis; Phenotypic screening; Gene biomarker; 3,4-
disubstituted piperidine derivative; Macrophage M2 polarization; Modulator
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ABSTRACT: Multiple sclerosis (MS) is a disease of the autoimmune-mediated disorder
in the central nervous system (CNS), for which no effective therapeutic agent is currently
available. The regulation of macrophage polarization towards M2 is a general benefit for
treating MS. The gene biomarker-based phenotypic screening approach was developed
and 3,4-disubstituted piperidine derivative S-28 was identified as a lead compound
modulating macrophage M2 polarization. Further SAR studies resulted in the discovery
of the most potent modulator D11 that showed good oral bioavailability and significant in
vivo therapeutic effects. Mechanistic studies demonstrated that the M2 polarization
macrophages modulated by D11 mainly functioned through inhibiting the proliferation of
T-cells and activating the phosphorylation of Stat3 and Akt. Therefore, the gene
biomarker-based phenotypic screening was demonstrated as a promising tool for the
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discovery of novel macrophage M2 polarization modulators. Compound D11 may serve
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as a promising starting point for the development of therapeutics to treat MS.
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1. Introduction
Multiple sclerosis (MS) represents an immune-mediated chronic and demyelinating
disease of the central nervous system (CNS),1,
featuring perivascular leukocyte
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infiltrates, astrogliosis, axonal damage, and loss of function3, 4 In particular, MS is the
most common cause of neurological disability among young adults, affecting
approximately one in 1,000 individuals in both Europe and North America.5 Clinically,
there are various MS treatment approaches available that have been shown to decrease
the frequency of relapses and delay disease progression. Examples include beta-
interferons, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, natalizumab,
and ocrelizumab.6 However, there is no effective therapeutic agent that cures MS is
currently available. Most of the above-mentioned anti-MS drugs only relief the
development of MS progression. Moreover, the application of developed anti-MS-drugs
is often limited by frequently occurring side effects, including flu-like symptoms and the
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development of other autoimmune disorders by interferon-β or fingolimod.7, 8 Hence, it is
of particular importance to identify more effective compounds with new mechanisms of
action. Therefore, the development of more effective compounds as alternative
approaches for the treatment of MS remains a critical, albeit unmet, scientific goal in MS
research.
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Macrophages exhibit a dynamic role in host defense and the maintenance of tissue
homoeostasis. This necessitates a delicate balance between the proinflammatory and
immunomodulatory functions to ensure appropriate responses to environmental stimuli.
In general, macrophages can be broadly classified as M1 (classical) and M2 (alternative)
subtypes based on function.9 M1 macrophages are activated by LPS and/or IFN-γ to
elaborate proinflammatory cytokine production and tissue inflammation.10, 11 Conversely,
M2 macrophages can be characterized by high expression of arginase 1 (Arg1), mannose
receptor C-type 1 (Mrc1), resistin-like molecule alpha1 (Fizz1) on cell surface marker
CD206, stimulated by Th2 cytokines IL-4 or IL-13 to promote helminthic immunity,
fibrosis, allergy and immunomodulation.12, 13
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The encephalomyelitis (EAE) model is a standard model for MS.14 Recently,
macrophages have been shown to actively participate in the pathogenesis of EAE
progression.14-16 In particular, during the induction phase of EAE, it was found that the M1
macrophages proportion increases in the spleen, resulting in the draining of lymph nodes
(dLNs) in C57BL/6 mice with early EAE.17 In this state, M1-polarized macrophages were
found to exhibit the ability to induce neuronal destruction,18 with symptoms of ongoing
EAE worsening19. However, M2-polarized macrophages have been demonstrated to
produce pro-repair molecules, including the brain-derived neurotropic factor (BDNF), IL-
10, and ferritin.20-23 After administration of M2-activated macrophages, the development
of an EAE model could be significantly suppressed.24, 25 Importantly, pro-repair molecules
secreted by M2 macrophages favor the restoration of myelin and axons, which indicates
particularly beneficial characteristics for the potential cure of MS.26-28 Therefore, the
inflammatory phenotype of macrophage cells is crucial for the EAE progression, revealing
that the regulatory control of macrophage polarization may be a promising strategy for
the treatment of MS. However, the molecular mechanism of regulating macrophages M2-
polarization is still not very clear. To the best of our knowledge, there is no specific
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effective regulator to treat or control MS using this strategy. Therefore, it would be of
particular interest to explore small molecular compounds that could induce the M2-
polarization of macrophages for MS therapy.
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In recent years, interest in phenotypic screening as a means for small-molecule drug
discovery has continued to increase as an alternative to target-based screening.29, 30
However, there is an ever-growing curiosity to elucidate potential benefits of combining
phenotypic and genomic data, in an effort to advance small-molecule drug discovery.
Accordingly, the distinct biomarkers of two inflammatory phenotypes (M1 and M2) of
macrophage cells described herein render phenotypic screening suitable to find small
molecular regulators for macrophages M2-polarization. In previous work, we have
established an effective evaluation system of macrophage polarization based on gene
biomarkers such as Arg1 and Mcp1.31 As a continued study for identifying novel
macrophage M2 polarization modulators, we wonder whether this evaluation system can
be used for the discovery of novel macrophage M2 polarization modulators for the
treatment of MS. Thus, similarity search and SAR studies involving different structural
moieties of different lead compounds were carried out based on in-house database
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screening. This led to the identification of the most potent (both in vitro and in vivo)
compound D11 (14-folds upregulation of M2 marker Arg1). Further mechanism studies of
compound D11 demonstrated that D11 mainly functioned through inhibiting the
proliferation of T-cells in EAE mouse model, and Stat3 and Akt proteins may be important
nodes for its regulation of macrophage M2 polarization.
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2. Results and Discussion
2.1 Phenotypic screening of a structural diverse compound library
A structurally diverse compound library containing approximately 20,000 compounds
was established in-house for phenotypic screening, assisted by cluster analysis. A total
of 28 compounds with significantly different structural skeletons were selected (for
corresponding compound structures see Figure S1). The murine macrophage cell line
RAW264.7 was treated with different compounds for 24 hours. Then, the relative
expression of macrophage M1 polarization biomarker Mcp1 and M2 polarization
biomarker Arg1 were tested. As shown in Figure 1, several compounds demonstrated a
good M2 polarization-inducing activity such as compound S-2, S-7, S-9, S-11, S-13 and
S-28 (for corresponding data see Table S1). Specifically, treatment with compound S-28
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(also termed as PZ8 in our database) was accompanied by the most remarkable elevation
of the M2 marker Arg1 mRNA expression and the least upregulation of M1 marker Mcp1
mRNA expression among all studied compounds.
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Figure 1. Compound S-28 promoted macrophages M2 polarization most effectively; (blue)
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis for expression of
macrophage M2 phenotype gene Arg1 in RAW264.7 cells treated with 28 compounds
(300 nM) for a total 24 hours. IL-4 (20 ng/ml) was used as positive control; (purple) qRT-
PCR analysis for expression of macrophage M1 phenotype gene Mcp1 in RAW264.7 cells
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treated with 28 compounds (300 nM) for 24 hours. LPS (50 ng/ml) was used as positive
control.
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Compound S-28 (Figure 2) was composed of four rings, a, c and d rings represented
aryl rings, and the b ring represented a saturated (3S, 4S)-piperidine ring. In order to
explore the structure-activity relationship of compound S-28 and in an effort to identify
more potent M2 polarization modulators, three strategies were applied: A) A structure
similarity search was performed using a Molport database for the purpose of extending
the structural diversity based on the skeleton of compound S-28; B) It was also found that
two chiral centers were present in the b ring. Therefore, the influences on M2 polarization
modulation activity of the chiral centers was also evaluated; C) In order to explore SAR
at the initial stage, the four different ring systems and two rings in compound S-28 were
retained such as the a-b and c-d ring systems. Substituted phenyl, heterocycles or
saturated chains were selected as substituents of the a-b or c-d ring systems.
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F
A
B
a
d
N
Structure similarity search
N
F
H
c
(S)
N
R2
(S)
b
R1
(R)
O
(R)
Chirality evaluation
N
H
HCl
S-28
N
H
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C
a-b ring system
d
N
N
F
R1
N
H
R1, R2 =
c
N
H
F
R2
O
c-d ring system
F
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H
N
R =
R
N
N
O
S
b
F
O
N
H
Figure 2. Different strategies of structure modification for SAR study
2.2 The synthetic route of compounds B1, C1-C11 and D1-D21
Synthesis of biaryl carboxylic acid 3a-c, 6a-c, and 9a-j. The synthetic route for the
production of the acid fragments is shown in Scheme 1. Different esters (1a-b, 4, 7a-d)
were used as starting materials. The coupling reaction with 1-methyl-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole provided compounds 2a-b, 5 and 8a-d.
After electrophilic reaction with NBS or NCS and hydrolysis, the biaryl carboxylic acids
3a-c, 6a-c and 9a-j were obtained.
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O
O
Br
N
3a-c
N
N
O
OH
b, c
a
N
N
N
R2
R2
N
R2
COOMe
N
R1
3a
3b
3c
R1= Br, R2= Me
R1= H, R2= Me
R1= Br, R2= H
1a
1b
R2= Me
R2= H
2a
2b
R2= Me
R2= H
O
O
Cl
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OH
O
N
N
b, c
a
N
N
N
N
6a
6b
6c
R1= H
R1= Cl
R1= Br
R1
6a-c
COOMe
5
4
O
O
Br
R2
R2
OH
R1
N
O
b, c
R2
a
N
N
COOMe
N
9a-j
8a-d
7a-d
9f
R1= H, R2= 3-Cl
R1= Cl, R2= 2-F
R1= Cl, R2= 3-Cl
R1= H, R2= H
9a
9b
9c
9d
9e
R1= Br, R2= 3-Cl
R1= Cl, R2= 3-Me
R1= Br, R2= 3-Me
R1= Cl, R2= H
9g
9h
9i
7a
8a
8b
8c
8d
R2= 3-Cl
R2= 3-Me
R2= H
R2= 3-Cl
R2= 3-Me
R2= H
7b
7c
7d
9j
R1= Br, R2= H
R2= 2-F
R2= 2-F
R1= H, R2= 2-F
Scheme 1. (a) 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole,
Pd(PPh3)4, K3PO4, DMF; (b) NBS or NCS, dichloromethane; (c) NaOH, H2O/EtOH.
Synthesis of 3-amino-4-aryl piperidine 14a-d. The synthetic route to obtain a series of
3,4-disubstituted piperidine compounds is shown in Scheme 2. Compounds 10a-c
underwent R or S Jorgensen-Hayashi reagent catalyzed cyclization with compound 10 to
provide the (3S,4S) compound 12a-c and (3R,4R) compound 12d. Treatment of
compound 12a-d with EtSiH, and following in the presence of Fe/NH4Cl provided 3-
amino-4-aryl piperidine.
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R3
R3
R3
R3
NO2
c
a
d
NO2
NO2
NH2
HN
Boc
N
N
N
CHO
Boc
12a-c
Boc
13a-c
Boc
14a-c
10a-c
11
10a
10b
10c
R3 = 3-F
R3 = 4-Cl, 3-CF3
R3 = 3,4-diF
13a
13b
13c
14a
R3 = 3-F
R3 = 3-F
R3 = 4-Cl, 3-CF3
R3 = 3,4-diF
12a
12b
12c
R3 = 3-F
R3 = 4-Cl, 3-CF3
R3 = 3,4-diF
14b
14c
R3 = 4-Cl, 3-CF3
R3 = 3,4-diF
10
11
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F
F
F
b
c
d
NO2
NH2
NO2
N
N
N
Boc
13d
Boc
14d
Boc
12d
Scheme 2. (a) (S)-2-(diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine, dichloromethane; ii.
TFA,
dichloromethane;
(b)
(R)-2-(diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine,
dichloromethane; ii. TFA, dichloromethane; (c) i. EtSiH, TFA; ii. Boc2O, TEA,
dichloromethane; (d) Fe, NH4Cl, EtOH/H2O.
Synthesis of target compounds B1, C1-C11, and D1-D21. The synthetic route for the
production of the target compounds is shown in Scheme 3. Treatment of aryl acids with
aliphatic amines in the presence of EDCI and HOBt provided the condensed
intermediates. Then, deprotection with HCl in ethyl acetate yielded the target compounds
B1, C1-C11, D1-D19. For compounds D20 and D21, L-tartrate was introduced to replace
hydrochloride as a counteranion. In doing so, the target compounds could be obtained as
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white solids as opposed to colorless oils in the case of hydrochloride as a counteranion.
The purities of all synthesized compounds were over 95 %.
9
F
10
11
12
13
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F
F
15c, C3
15f, C6
R =
R =
15a, C1
15d, C4
15b, C2
15e, C5
R =
R =
R =
R =
a, b
H
NH2
R-COOH
+
R1
N
F
O
N
15a-f
O
N
N
HCl
H
Boc
14a
N
O
S
C1-C6
F
O
N
16a, C7
R1= H, R2= 4-F-Ph
16d, C10
16e, C11
R1= Et, R2=Et
R1= H, R2=
F
R1
N
OH
a,b
16b, C8
16c, C9
R1= H, R2= 4-MeO-Ph
R1= H, R2=Et
R1
R2
N
+
N
R2
H
O
N
N
N
F
16a-e, 14d
9e
H
HCl
C7-C11
14d, B1
R1= H, R2=
N
H
HCl
R1
X
R3
R2
X
O
D1
D2
D3
D4
D5
D6
X=C, Y=N, R1 = Br, R2 = Me, R3 = 3-F
X=C, Y=N, R1 = H, R2 = Me, R3 = 3-F
X=C, Y=N, R1 = H, R2 = Me, R3 = 3-CF3-4-Cl
X=C, Y=N, R1 = Br, R2 = H, R3 = 3-F
X=N, Y=C, R1 = H, R2 = H, R3 = 3,4-diF
X=N, Y=C, R1 = Cl, R2 = H, R3 = 3,4-diF
R3
N
R2
N
OH
R1
NH2
a, b
H
Y
N
+
Y
N
N
N
O
N
Boc
14a-c
D7 X=N, Y=C, R1 = Br, R2 = H, R3 = 3,4-diF
HCl
H
3a-c
D1-D7
D8
D9
R1 = Br, R2 = 3-Cl, R3 = 3-F
R1 = Cl, R2 = 3-Me, R3 = 3-F
R1 = Br, R2 = 3-Me, R3 = 3-F
R1 = Cl, R2 = H, R3 = 3-F
R1
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
R3
N
R2
N
b
H
R
1 = H, R2 = 2-F, R3 = 3-F
N
R1 = H, R2 = 3-Cl, R3 = 3-CF3-4-Cl
R1 = Cl, R2 = 3-Me, R3 = 3-CF3-4-Cl
R1 = Cl, R2 = 2-F, R3 = 3-CF3-4-Cl
R1 = Cl, R2 = H, R3 = 3-CF3-4-Cl
R1 = Cl, R2 = 3-Cl, R3 = 3,4-diF
R3
O
R2
O
OH
N
H
R1
N
D8-D19
NH2
HCl
a
+
R
1 = Cl, R2 = H, R3 = 3,4-diF
R1
N
N
R1 = H, R2 = 2-F, R3 = 3,4-diF
R1 = H, R2 = H, R3 = 3,4-diF
R1 = Br, R2 = H, R3 = 3,4-diF
Boc
14a-c
R3
R2
N
9a-j
b, c
N
H
N
HOOC
OH
O
D20, D21
N
H
HO
COOH
Scheme 3. (a) EDCI, HOBt, DIPEA; (b) EA, HCl; (c) L-Tartaric acid.
2.3 In vitro evaluation of macrophage polarization gene biomarker expression fold change
Ten different structures were selected from the Molport database based on similarity
search results (Table 1). Unfortunately, among these structures, we could not identify any
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other more potent skeletons. The bioactivity test results (Table 1) indicated that most of
the study compounds exhibited low macrophage M2 or M1 polarization induced activity.
Specifically, compounds A2 and A9 displayed about 4-fold upregulation of Arg1
expression, however, the activity was significantly lower than that of compound S-28.
Table 1. Macrophage polarization gene biomarker expression fold change of compound
A1-A10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
N
F
NH
F
O
O
N
N
N
O
N
N
N
N
NH
N
O
H
O
NH
NH
NH
N
O
N
A1
N
A2
N
A3
NH
N
A5
A4
HN
N
HN
F
N
F
HN
N
F
N
O
HN
N
N
O
N
N
HN
HN
NH
O
NH
O
N
N
N
N
O
O
A6
A10
A7
A9
A8
M2 marker Arg1
M1 marker Mcp1
Cpd.
Folda
1
SEM
0
Folda
1
SEM
0
Ctr
A1
A2
2.35
4.85
0.08
1.04
1.14
1.24
0.42
0.57
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9
A3
A4
A5
A6
A7
A8
A9
A10
0.80
0.10
0.97
0.26
0.50
0.33
0.45
2.22
0.36
2.14
1.52
1.40
1.34
1.95
1.86
2.14
1.53
0.66
0.07
0.39
0.05
0.25
0.18
0.33
0.35
1.48
1.16
1.09
2.22
2.22
3.92
1.33
10
11
12
13
14
15
16
17
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21
22
23
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25
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Gene expression was determined by qRT-PCR, the fold change was calculated as
follows: gene expression level of Dosing group/gene expression level of Control group.
A further study of compound S-28 focused on chirality and the exploration of other ring
systems. As shown in Table 2, compound B1 bearing a (3R,4R)-disubstituted-piperidine
ring showed a dramatical loss of M2 polarization induced activity (0.31-fold upregulation
of Arg1), while the expression of M1 marker Mcp1 could still be retained (1.76-fold
upregulation of Mcp1), indicating that the (3S,4S)-disubstituted-piperidine ring was
indeed essential in maintaining compound activity.
When the c-d ring system was replaced by substituted phenyl or heteroaromatic rings
such as compound C1-C6, both exhibited no obvious influences on Mcp1 expression or
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upregulation of Arg1 expression. As for the a-b ring system that was replaced by other
substituents (compound C7-C11), the results were performed in the same manner as the
lack of the c-d ring system. The ablation of only the a ring (compound C11) or the d ring
(compound C1) caused the loss of induced potency. Therefore, we speculated that the a,
b, c and d rings were both essential for keeping the induce activity of compound S-28.
Table 2. Macrophage polarization gene biomarker expression fold change of compound
B1 and C1-C11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
F
N
F
N
N
F
H
H
R1
N
N
R1
N
N
(R)
(R)
R2
O
O
N
N
H
O
HCl
H
HCl
C7-C11
B1
C1-C6
M2 marker Arg1
M1 marker Mcp1
Cpd.
R1
R2
Folda
1
SEM
0
Folda
1
SEM
0
Ctr
B1
-
-
-
-
0.31
0.14
1.76
0.63
C1
C2
C3
-
-
-
1.01
0.91
1.32
0.43
0.24
0.28
1.72
1.64
1.42
0.23
0.22
0.30
F
F
O
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C4
C5
-
-
1.44
1.76
0.36
0.68
1.54
1.89
0.23
0.43
S
O
7
8
9
N
C6
C7
-
1.62
1.63
0.17
1.18
2.05
2.01
0.40
0.33
N
10
11
12
13
14
15
16
17
18
19
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21
22
23
24
25
26
27
28
29
30
31
32
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35
36
37
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H
H
F
C8
3.86
2.97
2.16
0.19
O
C9
H
Et
Et
2.31
1.96
0.78
0.79
1.83
1.97
0.31
0.54
C10
Et
C11
H
2.11
0.79
2.26
0.56
N
H
HCl
a
Gene expression was determined by qRT-PCR, the fold change was calculated as
follows: gene expression level of Dosing group/gene expression level of Control group.
The subsequent adjustment of the skeleton was mainly carried out on the substituents
of the four rings. When the pyridine ring was substituted for the b ring, compound D1-D7
showed no improvements in induced activity (Table 3). Based on the above SAR study,
we deemed the skeleton highly conserved, and fine-adjustment of compound S-28 may
lead to the identification of an even more potent compound. Compounds D8-D21 were
synthesized and tested and the corresponding results are shown in Table 3. Compounds
D8, D11, D12, and D15 demonstrated an over 10-fold up-regulation of the M2 marker
Arg1. As expected, most of the compounds showed less potency in elevating the M1
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marker Mcp1, except for compounds D14-D16. Here, R3 was substituted with 3-CF3-4-
Cl. However, compound D11 showed the strongest ability to promote expression of the
M2 marker Arg1 in RAW264.7 and much lower upregulation of M1 marker Mcp1 mRNA
expression (Figure 3A). Furthermore, RAW264.7 cells were treated with LPS,
accompanied by administration of the potent M2 polarization modulators D8, D11, D12,
and D15. It was found that compound D11 could also reverse the up-regulation of M1
marker Mcp1 expression induced by LPS (Figure 3B). Thus, compound D11 was selected
for the following pharmacokinetic and pharmacodynamic studies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Macrophage polarization gene biomarker expression fold change of compound
D1-D21
F
R1
R1
R1
N
F
R3
R3
R2
N
N
R2
N
N
N
N
H
H
H
N
N
N
N
O
O
O
N
N
H
N
HCl
HCl
H
H
D1-D4
D5-D7
D8-D19
(hydrochloride)
D20, D21
(L-tartrate)
M2 marker Arg1
M1 marker Mcp1
Cpd.
Ctr
R1
R2
R3
Folda
SEM
Folda
SEM
-
-
-
1
0
1
0
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D1
D2
Br
H
3-Me
3-Me
3-Me
H
3-F
3-F
0.71
0.95
0.40
0.78
0.85
0.27
0.56
10.12
8.35
6.54
13.97
10.05
5.08
1.82
10.33
2.82
1.72
1.97
3.99
3.42
0.13
0.12
0.26
0.26
0.54
0.15
0.08
0.82
0.91
1.71
3.18
0.92
1.60
0.01
0.35
0.51
0.23
0.24
0.80
0.28
1.63
1.99
1.37
1.14
1.23
1.01
1.32
2.05
2.44
1.89
1.47
1.54
1.49
4.85
3.54
5.08
1.16
1.50
0.39
0.90
0.18
0.42
0.23
0.05
0.05
0.04
0.10
0.74
0.76
0.35
0.12
0.46
0.32
0.32
0.77
0.11
0.09
0.16
0.15
0.24
D3
H
3-CF3-4-Cl
3-F
10
11
12
13
14
15
16
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18
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59
60
D4
Br
H
D5
-
-
D6
Cl
Br
Br
Cl
Br
Cl
H
-
-
D7
-
-
D8
3-Cl
3-Me
3-Me
H
3-F
D9
3-F
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
3-F
3-F
2-F
3-Cl
3-Me
2-F
H
3-F
H
3-CF3-4-Cl
3-CF3-4-Cl
3-CF3-4-Cl
3-CF3-4-Cl
3,4-diF
3,4-diF
3,4-diF
3,4-diF
Cl
Cl
Cl
Cl
Cl
H
3-Cl
H
2-F
H
H
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D21
Br
H
3,4-diF
2.27
0.39
1.77
0.06
6
7
a
Gene expression was determined by qRT-PCR, the fold change was calculated as
8
9
follows: gene expression level of Dosing group/gene expression level of Control group.
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Figure 3. (A) (Blue) qRT-PCR analysis to determine the expression of macrophage M2
phenotype gene Arg1 in RAW264.7 cells treated with compounds (300 nM) for 24 hours.
IL-4 (20 ng/ml) was used as positive control. (Purple) qRT-PCR analysis for the
determination of macrophage M1 phenotype gene Mcp1 expression in RAW264.7 cells
treated with compounds (300 nM) for 24 hours. LPS (50 ng/ml) was used as positive
control; (B) qRT-PCR analysis for the determination of macrophage M1 phenotype gene
Mcp1 expression. RAW264.7 cells were pre-treated with LPS (50 ng/ml) for 24 hours and
subsequently treated with compounds (300 nM) for 24 hours. Data is shown as mean ±
S.E.M in the graphs.
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2.4 In vivo pharmacokinetic evaluation
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Definitive single-dose pharmacokinetic studies were conducted in rats (Table 4).
Compound D11 demonstrated excellent absolute oral bioavailability in rats with F values
of 50.63%. In addition, the compound showed good clearance and oral absorption (t1/2=
3.3 h, Cmax= 313 ng/mL, AUC0-t= 4669 μg /L·h) characteristics. These data suggested that
compound D11 constitutes a reasonable starting point for further drug development
studies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Pharmacokinetic parameters of D11 in Sprague-Dawley (SD) Rat
Compound D11
Parameters
50 mg/kg
Oral
10 mg/kg
Intravenous
Tmax（h）
3.25±0.5
0.083±0.02
T1/2（h）
7.77±1.52
6.30±1.90
C
max（μg/L）
313.23±87.45
4669.27±1165.37
4743.69±1166.54
98.425±1.32
1001.40±423.30
1844.42±568.57
1869.93±585.16
98.75±1.287
AUC0-t(μg /L·h)
AUC0-∞(μg /L·h)
R_AUC (t/∞)%
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F
50.63%
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2.5 Compound D11 changes macrophage polarization balance towards M2
In D11-treated RAW264.7 cells, the microarray analysis result indicated that D11
activated M2 macrophage marker genes but suppressed M1 marker genes (Figure 4A,
B). Compared to the passage cell line RAW264.7, bone marrow-derived macrophages
(BMDM) present a more ideal in vitro model to understand the mechanisms controlling
polarization of activated macrophages.32 It was further demonstrated that the M2
macrophages marker CD206 expression was elevated (Figure 5A) and M1 macrophages
marker CD86 expression was suppressed dose-dependently after treatment with D11
(Figure 5B) on BMDMs.
Hence, the BMDM cell line was used for further bio-mechanistic studies of compound
D11. Treatment of BMDMs with 300 nM D11 resulted in an increase of mRNA levels of
the M2 markers Arg1, Mrc1, Fizz1 (Figure 4C). Furthermore, compound D11 at a
concentration of 300 nM reversed the elevation of mRNA levels of the M1 markers Mcp1,
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Inos and Cd86 stimulated by LPS (Figure 4D). Meanwhile, the western blot analysis also
showed that the expression level of macrophage M2 polarization marker Arg1 and Ym1
proteins increased while the M1 polarization marker Tnf-α protein was still remained
(Figure S2). Furthermore, treatment of BMDM cells with 300 nM of D11 increased the
expression of the M2 macrophages marker CD206 (Figure 4E) and suppressed the
expression of the M1 macrophages marker CD86 stimulated by LPS (Figure 4F) as
measured by flow cytometry.
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Figure 4. Compound D11 promotes the polarization of M2 macrophages in combination
with inhibiting M1 polarization ex vivo. (A) The numbers of altered genes after the
RAW264.7 cells were treated with D11; (B) Fold change of macrophage polarization gene
markers; (C) BMDM cells were treated with 300 nM of D11 for 24 hours. qRT-PCR was
performed to investigate the macrophages M2 polarization-associated genes Arg1, Mrc1,
and Fizz1 mRNA expression levels. (D) BMDM cells were pre-treated with LPS (50 ng/ml)
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for 24 hours and then treated with 300 nM of D11 for 24 hours. qRT-PCR was performed
to investigate the macrophages M1 polarization-associated gene Mcp1, Inos, Cd86
mRNA expression levels. (E) BMDM cells were treated with 300 nM of D11 for 24 hours.
The percentage of M2 macrophages (CD206+F4/80+ cells) was analyzed by flow-
cytometry. (F) BMDM cells were pre-treated with LPS (50 ng/ml) for 24 hours and then
treated with 300 nM of D11 for 24 hours. The percentage of M1 macrophages
(CD86+F4/80+ cells) was analyzed by flow cytometry. Data is shown as mean ± S.E.M in
the graphs. *P<0.05, ***P<0.01 versus control.
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Figure 5. D11 promotes macrophages M2 polarization and inhibits M1 polarization dose-
dependently. (A) BMDM cells were treated with indicated concentration of D11 for 24
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hours. The percentage of M2 macrophages (CD206+F4/80+ cells) was analyzed by flow-
cytometry. (B) BMDM cells were pre-treated with LPS (50 ng/ml) for 24 hours and then
treated with indicated concentration of D11 for 24 hours. The percentage of M1
macrophages (CD86+F4/80+ cells) was analyzed by flow cytometry. Data is shown as
mean ± S.E.M in the graphs. *P<0.05, ***P<0.01 versus control group. #P<0.05 versus
LPS group. n.s. P>0.05 versus control or LPS group.
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2.6 Therapeutic effect of compound D11 in EAE model
Since the Myelin Oligodendrocyte Glycoprotein (MOG) has emerged as one of the most
important target antigens in MS,14 the in vivo therapeutic effect of compound D11 was
evaluated in a MOG35-55 induced mouse EAE model. Further safety evaluation of D11 on
healthy mice indicated that no other obvious side effects were observed except for
immunosuppressive activity (which is beneficial in EAE and MS) at 400 mg/kg (Figure S3,
Table S2, and S3). To avoid the potential side effects caused by excessive
immunosuppression and maintain the exposure of D11 being above 300 nM, the doses
of 100 mg/kg and 200 mg/kg were chosen as the therapeutic doses in animal EAE study.
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The therapeutic administration of the drug (D11, 100 mg/kg and 200 mg/kg, ig, qd) was
performed after day 8, right after the appearance of clinical symptoms of EAE (Figure
6A). Dexamethasone (DXM), which is the first choice for the treatment of acute relapses
of multiple sclerosis,33 was also observed therapeutic effect in the EAE model when
treating with dexamethasone as reported,34 so it was chosen as a positive control. The
data indicated that when administered at 200 mg/kg, compound D11 clearly reduced
disease progression, as observed by a significantly lower average clinical score in EAE
mice administrated with 200 mg/kg of compound D11 compared to vehicle-treated EAE
mice (Figure 6B). Accordingly, H&E and LFB histological analysis of the affected spinal
cord indicted that treatment with 200 mg/kg of D11 attenuated demyelination and
inflammation in EAE mice (Figure 6C and D). To study the effect of treatment with
compound D11 on the pro-inflammatory cells Th1, and Th17 infiltration into the CNS, we
isolated mononuclear cells (MNCs) from the CNS (spinal cords and brains) of EAE mice.
Then, we performed flow-cytometry analysis and detected a remarkable percentage
reduction of both Th1 and Th17 in CD4+ T-cells of D11-treated EAE mice (Figure 6E and
F).
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Figure 6. Compound D11 ameliorated clinical severity and limited CNS autoimmunity of
EAE mice. (A) Schematic representation of EAE model. (B) In vivo effect of compound
D11 on EAE mice. EAE mice were treated with D11 (100 mg/kg, 200mg/kg, i.g.),
dexamethasone (10 mg/kg, i.g.) or vehicle (saline, i.g.). Mean clinical scores are shown
(n=5). (C,D) H&E and Luxol fast blue (LFB) staining of spinal cords from vehicle- and
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D11-treated EAE mice on day 14 after induction of EAE. The shown arrows indicate
infiltration of inflammatory cells and demyelination in the spinal cord. Scale bars: 100 μm
(upper), 50 μm (bottom). (E, F) Flow cytometry analysis of Th1 and Th17 (IFN-γ+ and IL-
17+) on the range of CD4+ cells isolated from the CNS of vehicle-treated and 200 mg/kg
D11-treated EAE mice (n=4 mice per group), detected between day 13 to day 19 after
induction of EAE. Data is shown as mean ± S.E.M in the graphs. *P<0.05, **P<0.01,
***P<0.01 versus vehicle control.
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2.7 Compound D11 distorts macrophages polarization towards M2 in vivo
To evaluate the in vivo effect of compound D11 on modulating the polarization of
macrophages, flow cytometry was used to assess the polarization of macrophages in
EAE mice between day 13 to day 19 after induction of EAE. As shown in Figure 7A, the
proportion of M2 macrophages (F4/80+CD206+) in total macrophage populations (F4/80+)
from both peripheral immune organs (spleen and draining lymph nodes) and the CNS
(spinal cord and brain) was found to be significantly elevated in 200 mg/kg D11-treated
mice compared to vehicle-treated EAE mice. Conversely, the percentage of M1
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