Design, synthesis and phenotypic evaluation of N-biaryl amides for IL-17A suppression

Article abstract of DOI:10.1039/c6md00427j

A series of N-biaryl amides for Th17 cell phenotypic screening were designed and quickly prepared by microwave-assisted solid-phase organic synthesis. The structure-activity relationship of the three regions of the amides was explored and potent small molecular inhibitors that suppress IL-17A production in mouse Th17 cells were identified. Compound 6h showed excellent suppression of IL-17A with an IC₅₀ of 13 nM, which can be employed as a chemical tool for target identification and Th17 cell-related biological studies.

Full text of DOI:10.1039/c6md00427j

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RESEARCH ARTICLE
Design, synthesis and phenotypic evaluation of N-biaryl amides
for IL-17A suppression†
Received 00th xxx 20xx,
Accepted 00th xxx 20xx
Ruomeng Qiua, Wenli Shib, Ting Caib, Siyu Peib, Ting Tanga, Yafei Huanga, Huan Wanga, Liming
Shaoa, Ju Qiub,∗and Yonghui Wanga,
∗
DOI: 10.1039/x0xx00000x
A series of N-biaryl amides for Th17 cell phenotypic screening was designed and quickly prepared by microwave-assisted
solid-phase organic synthesis. Structure-activity relationship of the three regions of the amides was explored and potent
small molecular inhibitors to suppress IL-17A production in mouse Th17 cells were identified. Compound 6h showed
excellent suppression of IL-17A with an IC50 of 13 nM, which can be employed as a chemical tool for target identification
and Th17 cell-related biology studies.
www.rsc.org/
The interleukin 17 (IL-17) family, a subset of cytokines agonists (e.g.,
1
in Fig. 1).15-18 RORγt activity was measured by
consisting of IL-17A–F, plays important roles in host defence the RORγ FRET peptide recruitment assay.19 Subsequent
against microbial organisms and in the development of biological evaluation of synthetic compounds was carried out
inflammatory diseases.1 The most widely studied cytokine of by the Th17 cell differentiation assay, in which IL-17A
this family, IL-17A, is a pro-inflammatory cytokine which has production was measured for the validation of the compounds
potential effect in various inflammatory conditions such as activity at cellular level.15 During the structure activity
autoimmune diseases, metabolic disorders, and cancer.2-4 relationship (SAR) exploration, we noticed that a set of N-
Current clinical phase III trials of IL-17A inhibitors such biarylbenzamides (e.g.,
2) showed no RORγt activity in the
aslxekizumab (LY2439821), brodalumab (AMG-827), and the FRET assay but had reasonable activity in Th17 cell
recently approved drug secukinumab (AIN457), have shown differentiation assay (e.g., 39% of IL-17A inhibition@0.3μM for
promising results as the proof of concept.5 However, synthetic
2). No activity of 2 in the RORγ FRET assay indicated that such
molecules as ligands for IL-17A/IL-17R have not been found suppression activity of IL-17A was unlikely caused by inverse
yet, indicating that IL-17A/IL-17R is a challenging target for agonism of RORγt. Due to the positive effect of inhibiting IL-
discovery of small molecule inhibitors.
17A production and easy modification/diversification, we
T helper 17 (Th17) cells, a subset of CD4+ T helper cells, are hypothesized that N-biaryl amides have the potential to be
one of the major sources of IL-17A in a variety of human developed into an IL-17A-suppressive small molecular lead
autoimmune inflammatory diseases.6 Th17 cells can be using Th17 cell differentiation assay as cellular phenotypic
differentiated from naïve CD4+ T cells in the presence of TGF-β screening strategy. In this paper, we report design, rapid
and IL-6, which activates downstream signalling factors synthesis and phenotypic evaluation of a series of N-biaryl
including STAT3 and the orphan nuclear receptor RORγt. Other amides as potential suppressors of IL-17A production by Th17
cytokines such as IL-1, IL-21 and IL-23 have also been shown to
be able to promote Th17 cell differentiation in pathological
conditions.7-13 Accordingly, Th17 cell differentiation can be
considered as an exploitable cellular phenotype.
RORγt is a key transcription factor regulating Th17 cell
differentiation and IL-17A production.14 Recently we reported
discovery of a series of novel small molecule RORγt inverse
cells.
Fig. 1. RORγt inverse agonist 1 and IL-17A suppressive hit 2
Based on structure of
3
2, we designed a small library of N-
biaryl amides ( in Fig. 2) with right hand side (RHS) R, middle
Ar1 and left-hand side (LHS) Ar2 as variables. While Ar1 and Ar2
are aromatic moieties, R can be aromatic or non-aromatic
moieties. In order to explore SAR of each component, three
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sets of compounds (4, 5, 6) with one variable at one time were
designed for synthesis and biological evaluation.
to get high conversion and purity of product for the next step.
Pd(dppf)Cl2 was selected as the catalyst and other conditions
such as solvent (NMP), base (K3PO4·3H2O) and microwave
reaction temperature (90oC) were chosen based on the
reaction results shown in Table 1. A typical experimental
procedure for preparation of N-biaryl amides was described
using 6h as an example.23
Table 1. Optimization of the Suzuki reaction conditionsa
Fig. 2. General structure3 and SAR Sets 4-6of N-biaryl amides
Solid phase organic synthesis (SPOS) has been a powerful tool
for the quick preparation of compound libraries used for
various types of screenings and hit to lead/lead optimization in
drug discovery.20 Microwave-assisted organic synthesis (MAOS)
is being also widely used in reaction optimization and solution-
phase parallel synthesis mainly due to its ability to shorten
reaction time.21 Microwave-assisted solid phase organic
synthesis (MASPOS), combining advantages of SPOS and MAOS,
not only fulfils green chemistry’s needs (environmental
friendly– energy efficiency and less organic waste), but enables
us to efficiently prepare SAR compounds containing various
functional moieties related to a similar scaffold for hit to lead
Temp.
(oC)
Yieldb
(%)
Entry
1c
2c
3
X
Solvent
DMF
Base
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
4-Br
4-Br
4-Br
4-Br
Na2CO3
110
110
110
110
110
110
110
110
100
90
92
91
90
4
DMA
NMP
Na2CO3
Na2CO3
PEG400:H2O
(1:1)
4
Na2CO3
optimization
A general synthetic route for preparing library
Scheme 1. Bromo aryl amines were loaded into 2,6-
.
3
was shown in
5
NMP
NMP
NMP
NMP
NMP
NMP
NMP
K2CO3
95
100
89
72
89
100
70
7
dimethoxy-4-polystyrenebenzyloxy-benzaldehyde resin (DMHB
resin) though reductive amination.22 The Suzuki reaction of the
6
K3PO4·3H2O
Cs2CO3
resulting resin-bound bromides
under microwave conditions produced resin-bound biaryl
amines . The resin-bound amines reacted with different
carboxylic acids formed the resin-bound N-biaryl amides 10
which upon acidic cleavage of resin yielded the desired N-
biaryl amides . Initially, 21 carboxylic acids, 5 bromo aryl
8 and various boronic acids
7
9
9
8
K3PO4·3H2O
K3PO4·3H2O
K3PO4·3H2O
K3PO4·3H2O
,
9
3
amines and 10 aryl boronic acids were selected for library
synthesis.
10
11c
80
aReaction Conditions: 11 (96.9mg, 0.52mmol), 12 (100mg, 0.13mmol),
Pd(dppf)Cl2 (14.3mg, 0.02mmol), base(0.39mmol), 1mL solvent
bubbled with nitrogen, microwave radiation at appointed temperature.
bYield was based on LC-MS relative peak areas monitoring at 254nm.
cBy-product existed.
A total of 34 compounds (21 from Set
4, 4 from Set 5 and 9
Scheme 1. General synthesis of N-biarylamidesa
from Set ) were screened in mouse primary Th17 cell
6
differentiation assay at concentration of 0.3 μM.24 Percentages
of maximum inhibitions for each set of compounds were
summarized in Tables 2-4.
a Reagents and conditions: (a) DMHB-resin, Na(OAc)3BH, DIPEA, 15%
HOAc in NMP, rt; (b) various aryl boronic acids, Pd(dppf)Cl2,
K3PO4·3H2O, NMP, microwave radiation, 90 oC, 1h; (c) various
carboxylic acids, DIC, DMF:DCE (1:1, v/v), microwave radiation, 60 oC,
1h; (d) 50% TFA in DCE, 30min, rt.
The RHS SAR of the amides was shown in Table 2. Substitution
on ortho-position of the phenyl ring (4b, 2, 4c) slightly
influenced the suppressive effect on IL-17A production with
F >Cl > CF3. Replacing phenyl (4b) with pyridine (4d) lowered
the activity. Interestingly, 2,6-di-substitution on the phenyl
The Suzuki reaction is the key step of the whole synthetic
route and its reaction conditions had to be optimized in order
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ring (4e and 4f) displayed much enhanced inhibitory effect
4n
4o
4p
4q
4r
34
30
35
9
compared to no or single ortho-substitution. Inserting one or
two CH2 between phenyl ring and carbonyl group (4g-4i
decreased potency to inhibit IL-17A production. Changing the
phenyl ring to cycloalkyl (4j 4n 4p) or hetero-cycloalkyl (4k
4m 4o) did not improve the inhibitory activity. Other non-
)
,
,
,
,
aromatic groups (4l, 4q-4t) decreased the inhibitory effect to
IL-17A production.
Table 2. RHS SAR of the amides
22
1
IL-17A
Compd
4a
4b
2
R
inh.%@0.3μMa
4s
4t
11
35
43
39
25
22
71
64
33
19
24
26
35
8
aAll results are an average of at least n=2.
Fixing the RHS moiety as 2,6-difluoro-phenyl, SAR of middle
aryl in N-biaryl amides was summarized in Table 3. 1,4-
Substituted phenyl
inhibiting IL-17A production than the 1,3-substituted phenyl
5a). Small substituents such as F (5c) or Cl (5d) on the phenyl
slightly increased their suppressive activity while pyridine ring
5b) replacing phenyl ring dropped the activity dramatically.
(4e) showed much better effect on
(
4c
(
Table 3. Middle aryl SAR of the amides
4d
4e
4f
F
F
O
IL-17A
inh.%@0.3μMa
Compd
4e
Ar1
4g
4h
4i
71
12
29
82
75
5a
*
*
N
5b
4j
5c
4k
4l
5d
aAll results are an average of at least n=2.
4m
16
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Table 4. LHS SAR of the amides
almost no ROR
γ
indicating that 6h is not a ROR
t activity (IC50 > 50 μM) in ROR
t inverse agonist.
γ FRET assay,
γ
IL-17A
Compd
6a
Ar2
inh.%@0.3μMa
45
17
4
6b
6c
(A)
6d
6e
-7
N
N
*
60
48
93
71
97
7
6f
6g
(B)
Fig. 3. (A) Flow cytometry analysis of IL-17A production in Th17 cells after
treatment of DMSO, 0.3 μM compound 2 and 0.3 μM compound 6h; (B)
Dose-dependent study of 6h in Th17 cell differentiation assay. Error bars
represent SEM (n=3). Data are representative of 2 independent experiments.
4e
6h
In summary, we have designed and quickly prepared a series
of N-biaryl amides for phenotypic screening of Th17 cells,
characterized by IL-17A production, using microwave-assisted
solid-phase organic synthesis. Structure-activity relationship of
the three regions of the amides was explored and potent small
molecular inhibitors to suppress IL-17A production in mouse
primary cell were identified. Compound 6h showed excellent
suppression of IL-17A with IC50 of 13nM, which can be used as
a chemical tool for target identification and Th17 cell-related
biology studies.
6i
aAll results are an average of at least n=2.
LHS SAR of the amides was shown in Table 4. Pyridines (6b, 6c)
and pyrazole (6d) dropped much of their effect on suppressing
IL-17A production compared to the phenyl compound (6a).
Substituents on ortho-position of the phenyl (6e, 6f) enhanced
the inhibitory activity on IL-17A production with Me > Cl.
Putting additional methoxy group on 5-position of 2-Me-
phenyl (6g) or 2-Cl-phenyl (4e) boosted their effects of
Acknowledgements
inhibiting IL-17A production greatly. Replacing -OMe group (4e
)
This work was supported by Grant81573276 from National
Science Foundation of China, Grant2015CB943400 and
2014CB943300 from the Ministry of Science and Technology of
China, Grant 14140902400 from the Experimental Animal of
Shanghai Science and Technology Committee, Grant91542102
and 31570887 from National Science Foundation of China, and
China's Youth 1000-Talent Program to J. Q.
with -OCF3 group (6h) further enhanced the inhibitory activity
with
a maximum inhibition of 97%@0.3μM. However,
switching the -OMe to a polar group like –CO2H (6i) dropped
the activity dramatically, indicating that a polar group at this
position may impair the activity.
Since compound 6h showed a strong inhibitory effect on IL-
17A production by Th17 cells compared to the starting hit
2
(Fig. 3A), we performed a dose-dependent study on 6h in
mouse primary Th17 cell differentiation system. As showed in
Fig. 3B, IL-17A inhibition for 6h is dose-dependent with an IC50
of 13 nM. Like other compounds in the series, 6h showed
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250mL
flask
were
added
2,6-dimethoxy-4-
polystyrenebenzyloxy-benzaldehyde (DMHB resin) (10g,
15mmol) and 100mL 1-methyl-2-pyrrolidinone (NMP) and
swelled for 5 min. 4-Bromoaniline (13g, 75mmol), DIPEA
(13mL, 75mmol) and acetic acid (15mL) were then added.
After shaking at room temperature for 2 hours, sodium
triacetoxyborohydride (19g, 90mmol) was added
gradually and then the mixture continued to shake for 24
hours at room temperature. The loaded resin was washed
with MeOH, DMF, H2O, MeOH and DCM successively and
dried in vacuum overnight to yield DMHB resin-bound 4-
bromo-benzylamine. (2) To mixture of the above DMHB
resin-bound 4-bromo-benzylamine (100mg, 0.13mmol) in
8.
9.
10.
11.
1mL
NMP
were
added
boronic
(2-chloro-5-
(125mg,
(trifluoromethoxy)phenyl)
acid
0.52mmol), K3PO4·3H2O (104mg, 0.39mmol). After
bubbled with nitrogen for 5 min, the mixture was added
Pd(dppf)Cl2 (14.3mg, 0.15mmol) and set up in microwave
reactor at 90 oC, 1h, pre-stirring for 30 seconds. The
resulting resin was washed with THF, THF:H2O (1:1), H2O,
THF:H2O (1:1), THF, DCM successively and dried in
vacuum for 6 hours. An analytical amount of the resin was
cleaved with 15% TFA in DCM for 10 min. The resulting
solution was evaporated and basified by saturated
sodium carbonate solution and extracted with 0.5mL DCM
detected by LC-MS. LC- MS (ESI): 287.0 [M+H]+. (3)To
mixture of the above DMHB resin-bound 2'-chloro-5'-
12.
13.
14.
(trifluoromethoxy)-[1,1'-biphenyl]-4-amine
0.25mmol) in 2mL DMF:DCE (1:1) were added 2,6-
difluorobenzoic acid (389mg, 2.5mmol), N,N'-
(200mg,
15.
methanediylidenebis(propan-2-amine) (DIC) (381μL,
2.5mmol). The mixture was sealed in a tube and set up in
microwave reactor at 60oC, 1h, pre-stirring for 30
seconds. The resulting resin was washed with MeOH,
DMF, H2O, MeOH and DCM successively and dried in
vacuum overnight, and then treated with 50% TFA in DCE
for 30 min. The cleaved solution was concentrated and
basified by saturated sodium carbonate solution and
extracted with DCM. The combined organic phase was
evaporated and isolated by PTLC to get the desired
compound. LC-MS (ESI): 427.0 [M+H]+. 1H NMR (400 MHz,
CDCl3) δ 7.82 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.57 – 7.35
(m, 4H), 7.22 (s, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.01 (t, J = 8.2
Hz, 2H)
Mouse Th17 differentiation assay and measurement of
IC50: CD4+ T cells were purified from mouse splenocytes
using a commercial CD4+ T cell negative selection kit
(Invitrogen). CD4+ T cells were skewed to Th17 cells by
culturing cells in the presence of anti-CD3(0.25ug/ml,
Bioxcel), anti-CD28(1ug/ml, Bioxcel), anti-IFN-γ(2ug/ml,
Bioxcel), anti-IL-4(2ug/ml, Bioxcel), TGF-β(5ng/ml,
16.
17.
18.
19.
W. Zhang, J. Zhang, L. Fang, L. Zhou, S. Wang, Z. Xiang, Y.
Li, B. Wisely, G. Zhang, G. An, Y. Wang, S. Leung and Z.
Zhong, Molecular Pharmacology, 2012, 82, 583-590.
Peprotech) and IL-6(20ng/ml, Peprotech) for
4 days
before analysis. Compounds or DMSO control were added
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to the culture on day
indicated concentrations. Percentage of IL-17A
production from CD4+
cells were analyzed by
0 of Th17 differentiation at
T
intracellular staining followed by flow cytometry. Dose-
response curves were plotted to determine half-maximal
inhibitory concentrations (IC50) for the compounds using
the GraphPad Prism 5 (GraphPad Software, San Diego CA,
USA). All experiments were performed in compliance with
the relevant laws and institutional guidelines, and all
experiments were approved by the Institutional Animal
Care and Use Committee of the Institute of Health
Sciences, Shanghai Institutes for Biological Sciences of
Chinese Academy of Sciences.
6 | Med. Chem. Comm., 2016, 00, 1-3
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Graphical Abstract
A series of N-biaryl amides was designed, quickly prepared by microwave-assisted solid-phase organic synthesis and
phenotypically evaluated via mouse Th17 cell differiation assay.