Identification of N-Phenyl-7 H-pyrrolo[2,3- d]pyrimidin-4-amine Derivatives as Novel, Potent, and Selective NF-κB Inducing Kinase (NIK) Inhibitors for the Treatment of Psoriasis

Article abstract of DOI:10.1021/acs.jmedchem.0c00055

A series of N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives with NF-κB inducing kinase (NIK) inhibitory activity were obtained through structure-based drug design and synthetic chemistry. Among them, 4-(3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-4-morpholinophenyl)-2-(thiazol-2-yl)but-3-yn-2-ol (12f) was identified as a highly potent NIK inhibitor, along with satisfactory selectivity. The pharmacokinetics of 12f and its ability to inhibit interleukin 6 secretion in BEAS-2B cells were better than compound 1 developed by Amgen. Oral administration of different doses of 12f in an imiquimod-induced psoriasis mouse model showed effective alleviation of psoriasis, including invasive erythema, swelling, skin thickening, and scales. The underlying pathological mechanism involved attenuation of proinflammatory cytokine and chemokine gene expression, and the infiltration of macrophages after the treatment of 12f. This work provides a foundation for the development of NIK inhibitors, highlighting the potential of developing NIK inhibitors as a new strategy for the treatment of psoriasis.

Full text of DOI:10.1021/acs.jmedchem.0c00055

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Article
Identification of N-phenyl-7H-pyrrolo[2,3-d] pyrimidin-4-
amine Derivatives as Novel, Potent, and Selective NF-#B
Inducing Kinase (NIK) Inhibitors for the Treatment of Psoriasis
Yuqin Zhu, Yuxiang ma, Weidong Zu, Jianing Song, Hua Wang, You Zhong,
Hongmei Li, Yanmin Zhang, Qianqian Gao, Bo Kong, Junyu Xu, fei jiang,
Xinren Wang, Shuwen Li, Chenhe Liu, Haichun Liu, Tao Lu, and Yadong Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00055 • Publication Date (Web): 01 Jun 2020
Downloaded from pubs.acs.org on June 1, 2020
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Identification of N-phenyl-7H-pyrrolo[2,3-d]
pyrimidin-4-amine Derivatives as Novel, Potent, and
Selective NF-κB Inducing Kinase (NIK) Inhibitors
for the Treatment of Psoriasis
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†
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Yuqin Zhu ,Yuxiang Ma , Weidong Zu , Jianing Song , Hua Wang , You Zhong , Hongmei Li , Yanmin
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Zhang , Qianqian Gao , Bo, Kong , Junyu Xu , Fei Jang , Xinren Wang , Shuwen Li , Chenhe Liu , Haichun
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,†,⋚
,†
Liu , Tao Lu* , Yadong Chen* .
†
School of Sciences, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, PR China.
⋚
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, PR China.
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ABSTRACT
A series of N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives with NF-κB inducing kinase (NIK)
inhibitory activity were obtained through structure-based drug design and synthetic chemistry. Among them,
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-(3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-4-morpholinophenyl)-2-(thiazol-2-yl)but-3-yn-2-ol (12f) was
identified as a highly potent NIK inhibitor, along with satisfactory selectivity. The pharmacokinetics of 12f and
its ability to inhibit interleukin 6 secretion in BEAS-2B cells were better than compound 1 developed by
Amgen. Oral administration of different doses of 12f in an imiquimod-induced psoriasis mouse model showed
effective alleviation of psoriasis, including invasive erythema, swelling, skin thickening, and scales. The
underlying pathological mechanism involved attenuation of pro-inflammatory cytokine and chemokine gene
expression, and the infiltration of macrophages after the treatment of 12f. This work provides a foundation
for the development of NIK inhibitors, highlighting the potential of developing NIK inhibitors as a new strategy
for the treatment of psoriasis.
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INTRODUCTION
As a common chronic inflammatory skin disease, psoriasis affects approximately 2-4% of the worldwide
population. Psoriasis can be divided into psoriasis vulgaris, pustular psoriasis, erythropoietic psoriasis, and
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arthritic psoriasis. Regardless of the type, it is characterized by associated histological and pathological
features, including exaggerated angiogenesis, abnormal growth, differentiation of keratinocytes, and
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prominent immune cell infiltration, such as T cells, neutrophils, and macrophages. Like other autoimmune
and inflammatory diseases, the complex pathogenesis of psoriasis determines the multidirectional and
multipath nature of drug development.
Indeed, biomacromolecules targeting autoimmune and inflammatory processes, particularly belimumab
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-10
(
Benlysta), has contributed significantly in patient care.
Despite substantial advances in the treatment of
autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), there is still
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an urgent need for effective psoriasis treatment strategies. In arthritic psoriasis, monoclonal antibodies
(mAbs) and recombinant proteins targeting cytokines and receptors, such as IL-12, IL-17, IL-23, TNFα, and
TNFR II, have become a research hotspot for major pharmaceutical giants represented by Pfizer Inc. and
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Janssen Biotech Inc.
Small molecule inhibitors yet provide an alternative when biomacromolecules
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targeting specific extracellular cytokines cannot reliably achieve disease remission. Janus kinase (JAK) is a
popular target that has been studied for the treatment of psoriasis. There exists non- or low-selective JAK
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inhibitors tofacitinib , ruxolitinib and baricitinib , but anemia and other hematological toxicities (e.g.,
dose-dependent neutropenia and significant lymphopenia) are often caused by inhibition of the JAK2 subunit
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which prompts researchers to develop more selective JAK inhibitors. Peficitinib as a selective JAK1/3
20-21
inhibitor is in phase II clinical trials for the treatment of psoriasis.
BMS-986165 developed by Bristol-
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Myers Squibb is currently in phase III clinical trials as a potential treatment for psoriasis. Besides, other
types of inhibitors are developed to target autoimmune-disease associated with intracellular proteins such as
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BTK and SYK.
It was believed that patients would benefit from combination therapies where multiple
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pathways or synergies are involved in improving efficacy.
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NF-κB inducing kinase (NIK, also known as MAP3K14) is an essential noncanonical NF-κB signaling
mediator downstream of the corresponding ligands of TNF superfamily receptors, including CD40, CD27,
lymphotoxin β receptor, BAFF-R, receptor activator of NF-κB (RANK), OX40, and Fn14 (TWEAK-R). In the basal
or resting state, TRAF3 links NIK to TRAF2-cIAPs E3 complex, promoting cIAPs-mediated Lys48 ubiquitination
and proteasomal degradation of NIK by the E3 ligase. Once the homeostasis is perturbed, such as the
stimulation of CD40L or BAFF, NIK would accumulate in the cell, leading to phosphorylation of IKKα, p100
processing as well as increased p52 protein levels. p52 then interacts with RelB to form p52/RelB
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heterodimer, which translocates into the nucleus and triggers the expression of inflammaory cytokines and
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chemokines.
Dysfunction of the NIK protein drives the development of many autoimmune and
inflammatory diseases. In synovial endothelial cells of RA patients, NIK is highly expressed, along with the
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+
increased BAFF serum levels.
Furthermore, Peli1-deficiency mice exhibited splenic CD19 B cell
hyperproliferation and increased production of its antigen-specific antibodies ( IgM, IgG2a, and IgG3), due to
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accumulation of NIK in the cell, leading to spontaneous SLE.
Related liver disease studies also show that
one of the common pathological features in patients with primary biliary cirrhosis is hepatocyte-specific
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overexpression of NIK, which further promotes liver inflammation and injury.
Similarly, NIK plays a direct
37-42
-/-
or indirect role in the pathogenesis of psoriasis.
In 2015, Etemadi et al. reported that TRAF2 but not
wild-type keratinocytes showed increased levels of NIK and p52, which led to many psoriasis associated-
37
inflammatory gene expression, including Csf1 (M-CSF), IL-23, TNFSF9 (4-1BBL), Cr1l and Cxcl16. In addition
to local tissue cell-associated inflammatory regulation, dysfunctional NIK also mediates the behavior of
immune T cells in autoimmune-like disease. It has recently been established that NIK signaling not only
promotes the differentiation of αβ T and γδ T cells but also acts as the hardwiring in thymic epithelial cells to
38-40
mediate γδ T cell cytokine production.
Furthermore, in TRAF2-ablated DKO mice, constitutive NF-κB2
activity resulting from NIK accumulation triggers aberrant enhancement of T cell activation and premature
differentiation of Th17 effector T cells, but not T-helper 1 (Th1), and eventually develops autoimmune
diseases such as psoriasis. Subsequent research confirmed that the corresponding inflammatory phenotype
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of the DKO mice could be reversed by a haplodeficiency of NIK. Accordingly, development of novel NIK
inhibitors for the treatment of psoriasis has extremely high research value.
Currently, the design and application of NIK inhibitors are scarce, and only a few cases of NIK inhibitors
have been reported. Amgen is the first to design a series of compounds containing alkynol fragments, which
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have good NIK inhibitory potency with a typical representative being compound 1 (K = 0.4 ± 0.3 nM). In 2016,
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Chen’s group confirmed the efficacy of 1 on toxin-induced acute hepatitis and liver injury. After the X-ray
crystal structure of the NIK kinase domain was determined, Janssen Pharmaceuticals and Genentech
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discovered another series of NIK inhibitors with high NIK inhibitory activity and selectivity (compounds 2-4).
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Compounds 2 and 3 can effectively inhibit LTβR-mediated p52/RelB nuclear translocation and the
expression of related target genes. Both are in the preclinical stage, and the initial indication is the same as
SLE.
Figure 1 Representative NIK inhibitors.
Although the efficacy of NIK small molecule inhibitors against SLE and toxin-induced liver inflammation
and injury has been well established (compounds 3 and 1, respectively), there is no reported case of NIK
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small molecule inhibitor as potential therapeutics for psoriasis.
In this study, we report the design and
synthesis of N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as novel NIK inhibitors. A promising
lead molecule 12f was identified from this series after a joint effort of medicinal chemistry synthesis and
bioassay evaluation of kinase selectivity, macrophage nitric oxide secreted inhibition and T cell anti-
proliferation screening. The pharmacokinetics (PK) properties of 12f were also evaluated, and it is more
stable than the starting compound 1 developed by Amgen. The in vivo study in an imiquimod (IMQ)-induced
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psoriasis mouse model demonstrated that compound 12f could significantly improve the disease phenotype,
and it also showed evidence of the underlying pathological characteristics.
RESULTS AND DISCUSSION
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Structure-Based Design Strategy
Indolinoline-based compound 1 (developed by Amgen) is the first NIK inhibitor that has been shown to
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be effective in vivo when administered by injection in acute hepatitis and liver injury models. Analyzing the
binding pattern of ligand 1 to NIK indicated that the interaction surface in the solvent accessible region
showing onset of van der waals clashes. The size of this region may allow us to open the tetrahydropyrrole
ring and grow new substituents at the appropriate site. Moreover, we hypothesized that the pyrimidine ring
grows into a pyrrolopyrimidine fragment that could engage the hinge region with potential H-bond (Figure
2A). Starting from compound 1, a structure-based design including ring-opening of tetrahydropyrrole,
cyclization of the aminopyrimidine ring, and the introduction of hydrophilic groups, was utilized, resulting in a
new molecule 12k with a distinct scaffold (Figure 2B).
Subsequently, compound 12k was docked into the NIK crystal structure (RCSB PDB code 4G3E) to
investigate the compatibility of 12k with the target. As shown in Figure 2C, overlay of compounds 1 and 12k
in the active site looks reasonable. Besides, a similar binding pattern of 12k with NIK protein was surfaced,
compared to that of compound 1. The 4-amino-pyrrolo[2,3-d]pyrimidine unit forms two conserved hydrogen
bonds with Leu474 in the hinge region of NIK. The acetylenic alcohol unit forms two hydrogen bonds with
Glu442 and Phe537 in the alpha-C helix and the DFG region, respectively. The thiazole nitrogen atom captures
Asp536 to form hydrogen-bond interaction. The methyl-piperazine fits into the solvent accessible region near
the G-loop. The next work demonstrated moderate NIK inhibitory activity (IC = 78.3 ± 5.53 nM, Figure 2B).
50
Following this design idea, structural optimization was performed on compound 12k, and a series of
molecules were synthesized and evaluated in our kinase assay.
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Figure 2. (A) The binding mode of ligand 1 with NIK and the assumptions of design strategies. (B) The design
strategy from compound 1 to 12k. The corresponding NIK inhibitory activities were displayed (C) The overlay
of compound 12k to ligand 1 (PDB code 4G3E). Only the hydrogen bonding interaction between 12k and NIK
is presented.
Chemsitry
Synthetic routes to the N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine analogs are depicted in Scheme 1.
Compound 7 was obtained in two steps starting with 4-chloro-7H-pyrrolo[2,3-d]pyrimidine. In the second
step, AgOTf was used as a catalyst and the electron-withdrawing tosyl protecting group allowed the
nucleophilic aromatic reaction to occur readily. After the nitro reduction reaction with Fe/NH Cl, subsequent
4
diazotization reaction afforded 10 in acceptable yield. Removal of the N-tosyl group was carried out to
provide 11 using LiOH or NaOH in EtOH. Target compounds N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine
analogs 12 were obtained via the sonogashira coupling reaction. Conversion of intermediate 9 to target
analogs 14 were then achieved in two steps: the deprotection step, and the condensation reaction of 13 with
acyl chloride or isocyanate in the presence of Et N. Similarly, using Et N as a base, the nucleophilic attack of
3
3
the piperazine nitrogen atom to appropriate acyl chloride gave 15. Then, we constructed the alkynol
fragment on the core skeleton via the coupling reaction and obtained the target compound 16. As shown in
Scheme 2, compounds 21a-f were prepared in good overall yields via a six-steps conversion, similar to the
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reactions shown in Scheme 1. The difference was in the reaction step a: using AgOTf or Pd (dba) as a
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catalyst and 2-fluoro-5-nitroaniline as a starting material to react with the appropriate nitrogen-containing
aromatic heterocyclic compounds.
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Scheme 1. Synthesis of N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine analogues
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Reagents and conditions: (a) TsCl, NaH, THF, 0 °C ; (b) AgOTf, Dioxane, 130 °C ; (c) corresponding amine, K CO ,
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DMF, 90 °C or relative alcohol, NaH, DMF, 0 °C ; (d) Fe, NH Cl, 75% EtOH; (e) NaNO , KI, p-TsOH, CH CN/H O,
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°C – r.t; (f) NaOH, 95% EtOH, 40 °C ; (g) PdCl (PPh ) , CuI, Et N/THF = 1:3; (h) appropriate acyl chloride or
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isocyanate, Et N, DMF, 0 °C ; (i) appropriate sulfonyl chloride or acetyl chloride, Et N, DMF, 0 °C .
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Scheme 2. Synthesis of compounds 21a-21f
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Reagents and conditions: (a) when X = Cl, AgOTf, Dioxane, 130 °C or when X = Br, Pd (dba) , K CO , t-BuOH,
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00 °C ; (b) morpholine, K CO , DMF, 90 °C ; (c) Fe, NH Cl, 75% EtOH; (d) NaNO , KI, p-TsOH, CH CN/H O, 0 °C –
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room temperature; (e) PdCl (PPh ) , CuI, THF/ Et N = 3:1, 40 °C .
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Structure-Activity-Relationship (SAR) Studies
The structural modification starting with the 1-methylpiperazine ring in 12k as hydrophilic groups at this
position may help improve potency and modulate physical properties. Removal of 1-methylpiperazinyl is not
tolerated and 12a loses NIK inhibitory activity. The introduction of “-N(CH ) ” to the 2-position of the phenyl
3
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ring (12b) (IC50 = 22.5 ± 3.18 nM) is well tolerated and showed enhanced activity compared to 12k. To
investigate whether substitution of flexible chain-like hydrophilic groups has an impact on the potency,
analogs 12c and 12d were synthesized and we observed that the methyl ether group could retain the activity.
We also examined 5 or 6-membered cyclic hydrophilic groups including: pyrrole (12e), morpholine (12f),
piperazine (12g), functionalized piperazine rings with different substituents (12h-12l). Among these
compounds, it is noteworthy that 12f stood out for its enhanced NIK inhibitory activity (IC : 9.87 ± 0.15 nM)
50
compared to compound 12k. Besides, (S)-3-methyl-piperazine (12i, IC : 69.16 ± 5.19 nM) showed better
50
activity than that of 12h and 12j. The introduction of a larger ethyl group than the methyl group on the “-N-”
atom of the piperazine ring had no effect on the inhibitory activity against NIK (12k-12l). In order to obtain
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compounds with different TPSA values (forecast with ChemBioDraw software), we introduced functional
groups containing different numbers of the oxygen atom on the piperazine nitrogen site. Fortunately, the
corresponding compounds (16a-16c) exhibited good inhibitory potency similar to that of 12f. Bulkier rings
such as 4-(piperidin-4-yl)-morpholine (12m, IC : 15.3 ± 2.25 nM) and 1-methyl-1,4-diazepane (12n, IC : 60.1
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50
0
1
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0
±
5.62 nM) retained the activity, suggesting that the cavity of the solvent accessible area could accommodate
larger substituents. An oxygen atom was introduced to link the phenyl and six-membered cyclic hydrophilic
group, 12o and 12p, but showed decreased inhibitory efficiency against NIK. Taken together, exploration of
the solvent accessible area showed that a hydrophilic group at 2-position of phenyl is necessary for the
activity.
Table 1. Optimization of the hydrophilic substituents in the solvent accessible area
a
a
Compounds
1
R
NIK IC (nM)
Compounds
12j
R
NIK IC (nM)
50
50
/
4.7±0.12
1639±85
199.9±7.03
78.3±5.53
12a
12k
12l
1
2b
22.5±3.18
72.4±3.12
34.5±7.20
17.8±1.49
1
2c
16a
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12d
484±19.85
16b
16c
8.22±0.26
16.2±1.07
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1
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2
2
2
2
2
2
2
2
2
3
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3
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0
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9
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2
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9
0
1
2e
106.7±23.53
1
2f
9.87±0.15
47.9±3.48
12m
12n
15.3±2.25
60.1±5.62
1
2g
12h
209.5±13.44
69.16±5.19
12o
12p
112±8.94
705±9.80
1
2i
a
Kinase inhibition assay was performed at 10 µM ATP concentration, and the values are the mean ± SD from
two independent experiments.
Further investigation focused on the interactions of the alkynol fragment with the back pocket, as
presented in Table 2. Several tertiary alcohol fragments were examined, based on their shapes, sizes and
electrical properties. The inhibitory activity towards NIK of 12q was largely lost. This may be due to the loss of
the afore-mentioned H-bond interactions formed between thiazole and pocket residues. Comparing the
affinity of 12r and 12s with NIK protein, the five-membered ring seems to be more suitable for the volume of
the back pocket than the six-membered ring. Interestingly, replacement of the cyclopentyl with thiophene
substituent resulted in an approximately 40-fold increase in potency of the inhibitors (12t and 12u),
suggesting the importance of electrical properties of aromatic heterocycles. Deduced from our design
strategy shown in Figure 2, the molecular flexibility of the N-phenylpyrimidine-4-amine skeleton was higher
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than the 4-(indolin-1-yl)pyrimidine-2-amine, which may be harmful to the orientation of the alkyne.
Considering this, we hypothesized that a more flexible linker such as an amide bond could perform better
when the corresponding fragment passes through the methioine gatekeeper into the back pocket. However,
the results are negative (compare 12s with 14b or 14c with 12f, respectively). Specifically, compounds 14a
and 14b exhibited the same level of inhibition against NIK, despite the different lengths of respective linkers.
Based on the principle of electronic isosterism, the introduction of an amide bond to replace alkynyl linker of
12f yields 14c, leading to a large decrease in its inhibitory activity against NIK.
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Structures and their inhibitory activities against NIK of target compounds
(
%enzyme
(% enzyme
activity at 1.0
activity at 1.0
Compounds
R
b
Compounds
12u
R
b
μM)
μM)
a
a
IC (nM)
IC (nM)
50
50
1
2q
2714±37
50.4±1.59
(
77.27±0.44%)
nd
1
2r
2s
1270±64
645±14.2
14a
14b
(
76.40±1.05%)
nd
1
12t
2240±77
14c
1404±62
a
Kinase inhibition assay was performed at 10 µM ATP concentration, and the values are the mean ± SD from
two independent experiments.
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As showed in Table 3, the 2-aminopyrimidine unit was introduced into the backbone to replace the
pyrrolopyrimidine fragment of 12k, and compound 21a exhibited a similar excellent inhibitory activity to 12f.
Subsequently, different nitrogen-containing aromatic heterocycle fragments were introduced to investigate
the binding tolerance of NIK the hinge region. Indeed, the ring-opening treatment of the dihydroindole
skeleton increases the flexibility of the molecule, and this is not conducive to the formation of crucial
hydrogen bonds in the hinge region. Interestingly, 7-azaindole (21b) and 1H-pyrazolo[3,4-d]pyrimidine (21e)
structural units (IC : 6.59 ± 0.09 nM, and 8.63 ± 0.31 nM, respectively) can still capture the hinge region
1
1
1
1
1
1
1
1
1
1
2
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2
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5
5
5
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0
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9
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2
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5
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7
8
9
0
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2
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5
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9
0
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2
3
4
5
6
7
8
9
0
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interactions, which confirms the feasibility of cyclization strategy. Based on the prediction that the 5-position
of 7-azaindole could be a potential metabolic site, we introduced fluorine or chlorine atom to the position.
The corresponding compounds were then excluded because of their reduced inhibition towards NIK, which
might be due to clashes with the binding pocket (21c-21d). Reduced potency of compound 21f (IC : 179 ±
50
5
.68 nM) confirms the importance of the “-NH-” unit to maintain the optimal “two-anchor” type hydrogen
bonding interactions at the hinge binding mode.
Table 3. Optimization of the nitrogen heterocyclic aromatic substituents in the hinge area
NIK
NIK
Compounds
Het
Compounds
21d
Het
a
a
IC (nM)
IC (nM)
50
5
0
2
1a
1b
11.4±0.81
6.59±0.09
>10000
2
21e
8.63±0.31
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21c
1378±29
21f
179±5.68
a
Kinase inhibition assay was performed at 10 µM ATP concentration, and the values are the mean ± SD from
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two independent experiments.
Taken together, we investigated the NIK inhibitory activities of a series of N-phenyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine derivatives by exploring hydrophilic groups in the solvent accessible region, scaffolds
engaging the hinge region as well as back pocket binding fragments. Compound 12f showed optimal NIK
inhibitory activity, and thus the binding mode of compound 12f within NIK was elucidated using a docking
model (Figure 3). Similarly, the pyrrolopyrimidine unit forms two conserved hydrogen bonds with Leu474.
What is noticeable is that the generation of a new hydrogen bond between the oxygen atom of the
morpholine ring and Arg410 or Gly411, respectively (Figure 3A and 3B). This interaction further anchors the
spatial conformation of the ligand in the pocket, which likely contributes to the inhibitory activity against NIK
(
compare 12f with 12k).
Figure 3. Binding mode analysis of compound 12f to NIK protein. The solvent accessible region of NIK protein
occupied by hydrophilic fragment of the ligand is particularly highlighted. (A) Compound 12f docked into NIK
(
PDB code 4G3E). (B) Compound 12f docked into NIK (PDB code 6G4Y).
Kinase-Inhibition Profile of Compound 12f
The pyrrolopyrimidine fragment has been widely used as a core moiety to construct hydrogen-bonding
interactions between the small molecule and the hinge region. Thus, a wild-type screening (Reaction Biology
Corp.) was performed to investigate the selectivity of 12f against 268 kinases (Figure 4A and Table S1). To
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better estimate the binding potency against six types of selected kinases, the IC value of compound 12f was
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determined (Figure 4B). Compound 12f displayed around 4-20 fold higher inhibitory activity against NIK (IC :
50
9
.7 ± 1.7 nM) than Aurora B (IC : 43.2 ± 3.4 nM), TRKC (IC : 50.1 ± 4.7 nM), LRRK2 (IC : 94.6 ± 8.4 nM) and
50 50 50
ACK1 (IC : 191.7 ± 5.3 nM). It should be mentioned that 12f also has weak inhibitory activities against JAK1
50
1
1
1
1
1
1
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(
IC : 182.6 ± 6.4 nM) and JAK3 (IC : 892 ± 5.2 nM). Taken together, these data establish 12f as a selective
50 50
and highly potent inhibitor of NIK with satisfactory selectivity.
Figure 4. (A) A wild-type screening (Reaction Biology Corp.) was performed to investigate the selectivity of
12f against 268 kinases in a single-dose duplicate mode at a concentration of 1 μM. The 10 µM ATP
concentration was conducted. Staurosporine served as a positive control. The % Ctrl = [(test compound signal
−
positive control signal)/(negative control signal − positive control signal)] × 100%. (B) Further inhibitory
33
activity of compound 12f against selected kinases using the P -radiolabeled assay. The values are the mean
from two independent experiments.
Nitric Oxide Production-Inhibitory and Anti-Proliferative Activity of Selected Compounds in
Vitro
Previous studies indicate that NIK inhibitors have essential effects on B cell survival, T cell proliferation,
28, 38, 41
and macrophage infiltration in the pathogenesis of autoimmune diseases such as SLE and psoriasis.
Thus, the nitric oxide (NO) production-inhibitory activity of J774 peritoneal macrophages and the anti-
proliferative activity against primary T cells isolated from the spleen were considered for the next screening.
A total of fifteen compounds were selected for cell activity assay. Using compound 1 as a positive control, we
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can see that most of the compounds showed satisfactory activities, and eight compounds (12f, 12g, 12i, 12k,
1
2l, 12n, 12o and 21b) presented higher inhibition rates than 60%. Encouragingly, with the stimulation of
µM LPS, the inhibition rate of compound 12f on macrophage NO production was as high as 86.28±0.09%,
1
and its low toxicity was further proven in Figure S1. Besides, we also determined whether the selected
compounds affect the proliferation of primary T cells, which were isolated from the spleen. Consistently,
compounds 12f, 12i and 21b showed better anti-proliferative activities against splenic T cells than others, at
different concentrations of 2 µM and 0.4 µM. These data confirmed that compound 12f to be a highly potent
anti-inflammatory NIK inhibitor, which was considered for further studies.
0
1
2
3
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
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Anti-inflammatory activity screening of selected compounds in vitro.
a
b
NO Inhibition rate (%)
Proliferation-Inhibitory rate (%)
Cpds NIK IC (nM)
50
At 1 μM
At 2 μM
At 0.4 μM
19.04±0.03
13.31±0.04
35.96±0.07
20.33±0.14
38.05±0.24
33.03±0.05
24.12±0.24
32.17±0.11
35.54±0.21
19.93±0.15
16.23±0.04
14.02±0.10
16.91±0.14
27.39±0.09
19.01±0.13
14.77±0.21
1
4.7±0.12
72.4±3.12
9.87±0.15
47.9±3.48
69.16±5.19
78.3±5.53
34.5±7.20
15.3±2.25
60.1±5.62
112±8.94
17.8±1.49
8.22±0.26
16.2±1.07
11.4±0.81
6.59±0.09
8.63±0.31
43.90±0.12
47.97±0.13
86.28±0.09
67.99±0.07
62.86±0.24
78.46±0.04
67.36±0.16
57.69±0.20
67.34±0.13
68.29±0.11
29.27±0.07
34.45±0.08
37.19±0.13
58.53±0.16
60.37±0.18
35.85±0.25
59.57±0.09
27.85±0.10
62.29±0.04
39.87±0.06
49.51±0.19
43.70±0.13
33.67±0.19
34.52±0.09
47.73±0.16
42.90±0.18
31.46±0.08
32.52±0.12
32.26±0.20
31.44±0.25
50.23±0.06
24.70±0.14
12c
12f
1
2g
1
2i
2k
2l
2m
2n
12o
6a
16b
6c
1
1
1
1
1
1
2
2
2
1a
1b
1e
a
Using Griess reagents method, NO inhibition rate % = (model group NO concentration – compound group
b
NO concentration) / (model group NO concentration – control group NO concentration) × 100%. Con A-
induced primary T cell proliferation was investigated under the treatment of selected compounds.
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Proliferation -Inhibitory rate (%) = (test hole absorbance – blank hole absorbance) / (control hole absorbance
a,b
–
blank hole absorbance) × 100%. The values are the mean ± SD from three independent experiments, n = 3.
Resolution of Enantiomers and Determination of the Dominant Configuration
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
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8
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4
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9
0
1
2
3
4
5
6
7
8
9
0
Given the outstanding enzyme and cell inhibitory activities of 12f, further work focused on the
resolution of its enantiomers and determination of respective activities. Under the specific chiral separation
conditions, compound 12f was resolved into 22 and 23. As presented in Scheme 3, compound 22 displayed
about 11 fold higher inhibitory activity against NIK (IC : 11.98 ± 3.13 nM) than that of 23 (IC : 135.70 ± 7.96
50
50
nM). Besides, 22 was more effective in inhibiting NO release of LPS-induced J774 macrophages. Inconsistently,
at high and low dosing concentrations, these three compounds had similar inhibitory potency on the
proliferation of T cells stimulated by Con A in vitro. It is also interesting that 12f and its enantiomer 23 were
both less toxic to normal T cell growth than 22 (Figure 5). In summary, compound 12f, as a racemate, can
exhibit similar anti-T cell proliferation activity and lower toxicity compared to that of the dominant
enantiomer 22. Thus, 12f was considered for further studies on the biological mechanism and efficacy
evaluation in vivo.
a
Scheme 3. Compound 12f enantiomeric resolution and determination of the dominant configuration
b
c
NO Inhibition rate (%)
Proliferation-Inhibitory rate (%)
Cpds NIK IC (nM)
50
At 1 μM
86.28±0.09
100.05±0.22
74.35±0.17
At 2 μM
62.29±0.04
61.17±0.18
60.12±0.06
At 0.4 μM
35.96±0.07
37.60±0.10
39.50±0.08
12f
9.87±0.15
11.98±3.13
135.70±7.96
2
2
3
2
a
Enantiomeric resolution of compound 12f was preformed. Mobile phase: Hexane : EtOH = 50 : 50, v / v.
b
Using Griess reagents method, NO inhibition rate % = (model group NO concentration – compound group
b
NO concentration) / (model group NO concentration – control group NO concentration) × 100%. Con A-
induced primary T cell proliferation was investigated under the treatment of selected compounds.
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6
Proliferation - inhibitory rate (%) = (test hole absorbance – blank hole absorbance) / (control hole absorbance
b,c
–
blank hole absorbance) × 100%. The values are the mean ± SD from three independent experiments, n = 3.
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Figure 5. Toxicity comparison between 12f and its enantiomers (22, 23) against normal growth of T cells at 10
µM concentration. Proliferation - inhibitory rate (%) = (test hole absorbance – blank hole absorbance) /
(
control hole absorbance – blank hole absorbance) × 100%. The values are the mean ± SD from three
independent experiments, n = 3.
PK Data of Selected Compounds Across Multiple Species in Vitro and Vivo
A note to mention is that compound 1 was developed by Amgen as a NIK inhibitor, and then Chen’s
36
group disclosed its efficacy on acute hepatitis liver injury in 2016. In addition, as shown in Table 5, our work
revealed its moderate liver microsomal metabolic stability in human species, but poor metabolic-stability for
monkey, dog, and rat liver microsomes (T1/2 ranged from 6.6 to 9.8 min). Conversely, compound 12f
exhibited better metabolic stability in human liver microsomes (HLM), monkey liver microsomes (MLM), dog
liver microsomes (DLM), rat liver microsomes (RLM), consistent with the lower clearance data in vitro (ranged
between 32.4 µL/min/mg and 76.8 µL/min/mg).
Inspired by the favorable in vitro stability properties of compound 12f, we performed in vivo
pharmacokinetic assessment using intravenous (IV) and per os (PO) administration to Sprague Dawley Rat
(shown in Table 6). After IV administration of 12f at a dose of 1.0 mg/kg, maximum concentration (Cmax
)
reached 97.3 µg/L, area under the curve (AUC0−∞) was 419 ng·h /mL, and the half-life was 0.944 h with low
clearance. Besides, compound 12f exhibited compatible pharmacokinetic properties under PO administration
(
Cmax reached 772 ng/mL at 0.833 h post-dosing, AUC0−∞ = 2680 ng/mL·h, T1/2 = 1.39 h, and F% = 64.0%).
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Overall, these data, especially high oral bioavailability, demonstrate better pharmacokinetic properties of 12f
than compound 1 in rat.
Table 5. Pharmacokinetics of compounds 1 and 12f in liver microsomes of multiple species
T1
/2
CLint, in vivo
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
4
4
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4
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4
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4
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5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
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2
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CLint, microsome
(µL/min/mg proteins)
2
a
Compound Species
R
c
b
d
(
min)
(mL/min/kg)
Human 0.9956 33.940
Monkey 0.9996 6.659
40.844
208.147
192.014
141.205
32.422
76.797
43.312
45.016
38.598
299.732
276.501
254.169
30.639
1
Dog
Rat
0.9982 7.219
0.9687 9.816
Human 0.9060 42.882
Monkey 0.9809 18.048
110.588
62.369
12f
Dog
Rat
0.9765 32.385
0.9215 30.790
81.028
a
2
R is the correlation coefficient of the linear regression for determination of the kinetic constant.
b
c
d
Elimination half-life (T ). Intrinsic clearance in LMs (CL
), Intrinsic hepatic clearance (CLint, in vivo). CLint,
1/2
int, in vitro
=
CLint, in vitro (mL/min/mg microsomal protein) × Scaling factor (physiological parameter) = CLint, in vitro
in vivo
(mL/min/mg microsomal protein) × Microsomal protein (mg protein/g liver) × Liver weight (g liver/kg body
weight).
a
Table 6. The PK parameter of compound 12f in Sprague Dawley Rat
IV
PO
b
a
PK parameters (n=3)
SD
PK parameters (n=3)
SD
(1.0 mg/Kg)
(10 mg/Kg)
AUC (0-∞)(ng·h/mL)
419
377
54.8 AUC(0-∞)(ng·h/mL)
2680
772
111
60
C (ng/mL)
0
56.1
0.112
0.393
Cmax (ng/mL)
T1/2 (h)
T1/2 (h)
0.944
2.73
1.39
0.833
0.0987
0.289
V (L/kg)
d
Tmax (h)
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CL (mL/kg/min)
Individual plasma concentration-time data of 12f, after an i.v./p.o. dose of 1/10 mg kg⁻¹ in Sprague Dawley
Rat, respectively. Corresponding in vivo pharmacokinetic parameters are given. Injection (i.v.) formulation
40.3
4.93
F(%)
64.0
2.7
a
-
1
(
prescription, DMSO : PEG200 : Saline = 20 : 20 : 60, v / v / v. concentration, 0.2 mg·mL ). Oral formulation
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b
(0.5% CMC-Na / 0.2% Tween 80. concentration, 1 mg·mL ). PK parameters are the mean ± SD from three
independent experimental rats, n = 3.
Compound 12f Blocks LPS-Induced Secretion of IL-6 in BEAS-2B Cells and Expression of
Inflammation-Related Genes in J774 Macrophages
The most noticeable features in the pathogenesis of psoriasis are excessive secretion of various cell
44
chemokines and pro-inflammatory factors in epidermal and dermal cells. Thus, we further figured out the
effects of compounds 12f and 1 on the secretion of interleukin-6 (IL-6) in BEAS-2B cells. As seen in Figure 6A,
within a specific concentration range, both compounds exhibited a dose-dependent inhibition of IL-6
secretion against inflammatory model cells. Compound 12f has a relatively higher inhibitory activity than
compound 1, and it inhibits IL-6 secretion at concentrations ranging from 14 nM to 10 μM. Next, we
determined the effectiveness of 12f in regulating inflammation-related gene expression in J774 macrophage
(Figure 6B). The results showed that the mRNA levels of inflammatory cytokines and chemokines (including
TNFα, IL-6, IL-23, IL-1β, and CCL3, respectively) were increased in response to the stimulation of LPS, but
notably decreased by administrating 12f. Moreover, the expression of nitric oxide synthase 2 (NOS2) is
45-46
generally up-regulated in the occurrence of various inflammatory diseases.
Interestingly, NOS2 levels in
J774 macrophages showed a markedly negative regulatory response to the administration of 12f.
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Figure 6. (A) The culture supernatant IL-6 levels in LPS-induced BEAS-2B cells were analyzed and quantified
by Elisa. (B) Quantitative PCR analysis of the mRNA levels of exocrine inflammatory factors (TNFα, IL-6, IL-23,
IL-1β), chemokine (CCL3), and cytokine (NOS2) in LPS-stimulated J774 cells. P values are determined by two-
#
##
###
tailed Student’s t-test, between the normal and model group: P<0.05, P<0.005, P<0.0005, between
model and 12f treatment groups: n.s. (not significant), *P<0.05, **P<0.005, ***P<0.0005. Data are
represented as mean ± SD from two (A) and three (B) biological replicates.
Treatment of 12f Attenuates Psoriasis-like Phenotype in Imiquimod Model Mice
After obtaining promising in vitro activities and in vivo PK results, we further explored the in vivo
pharmacological efficacy of compound 12f on the IMQ-induced psoriasis model using C57 mice. In order to
establish a psoriasis-like dermatitis model, 5% imiquimod cream was evenly applied to the back skin of C57
mice (for seven consecutive days, q.d.). Moreover, 12f was administered orally on the third day and
continued until the end of the experiment (12f-I: 60 mg/kg, 12f-II: 120 mg/kg, i.g./q.d.). The model group of
mice exhibited a typical psoriasis-like dermatitis phenotype, including invasive erythema, roughness, swelling,
and scales. In contrast, the symptoms of the administration group (12f-I, 12f-II) were significantly attenuated
(
Figure 7A). Hematoxylin and eosin staining revealed the occurrence of epidermal acanthosis, as shown in
Figure 7B, and the model group had significant morphological and pathological changes in the back skin
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compared to the normal and drug-treated groups, such as increased skin thickness and severe scales.
Statistical analysis of the back skin thickness of six mice in different experimental groups showed that the skin
thickness of the administration group changed little under the induction of IMQ, confirming a dose-
dependent enhancement of 12f treatment efficacy (Figure 7C). The spleen index of the 12f administration
group was slightly lower than that of the model group, reflecting that IMQ's activation of systemic immunity
was suppressed to a certain extent (Figure 7D). Consistently, Elisa detection of TNFα and IL-17A in serum
revealed that 12f could inhibit mouse secretion of both cytokines in a dose-dependent manner (Figure 7E).
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Figure 7. (A) Representative images of dorsal back from wild-type C57 mice untreated or treated with IMQ or
1
2f for 7 or 4 days. (B) Hematoxylin and eosin (H&E) staining of skin sections from the dorsal back (normal
group: IMQ and 12f were untreated, model group: only IMQ treatment, 12f-I / 12f-II: at 60 mg/kg or 120
mg/kg doses of 12f treatment, along with IMQ induction). Scale bar, 50 μm. (C) Epidermal thickness was
measured in six randomly chosen fields. (D) The weight of the mouse spleen was counted, and each point
represents the value of the spleen weight of a mouse. (E) TNF-α and IL-17A in the mouse serum were
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analyzed and quantified by Elisa. Data are shown as the mean ± SEM based on three independent
experiments.
Compound 12f Suppressed Epidermal and Dermal Cell Proliferation and Macrophage
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Infiltration in Mouse Back Skin Tissues through Regulating NF-κB2 Signaling Pathway
Characteristics of psoriasis lesions include excessive cell proliferation in epidermal and dermal tissues
and infiltration of immune cells such as macrophages. After orally administering compound 12f to C57 mice
+
for four days, we quantified the population of Ki67 cells in psoriatic lesions by immunohistochemistry to
reveal the extent of epidermal and dermal cell proliferation in skin tissue. As shown in Figure 8A, the
+
population of Ki67 cells in the back skin of the mice was obviously reduced when treated with a low dose of
12f (12f-I, 60 mg/kg), compared to the model group mice. Moreover, by comparing the experimental results
of the high and low dose groups, we obtained preliminary dose-effect relationship of 12f on the inhibition of
cell proliferation in psoriatic skin tissue: normalized to the model group, the 12f-I (60 mg/kg) and the 12f-II
(
(
120 mg/kg) group Ki67 expression percentage decreased by approximately 55% and 83%, respectively
Figure 8B). Subsequently, we investigated the effect of compound 12f on the infiltration of macrophages in
the pathogenesis of psoriasis model mice (Figure 8C). The results of F4/80 immunofluorescence images
showed that the recruitment of macrophages from the back skin tissue of the model group was notably up-
regulated following the induction of IMQ. In contrast, treatment with high or low doses of 12f (12f-I and 12f-
II) notably alleviated this symptom.
Consistent with the above data, compound 12f at different doses exhibited dose-dependent inhibition
of the NF-κB2 pathway by targeting the NIK protein. Furthermore, from Figure 9A and 9B, we found that the
levels of p52, downstream of NIK, in the model group were obviously higher than that in the administration
group, and it decreasing with increased dosing of the 12f administration. We also found reduced expression
of genes associated with the NF-κB2 signaling pathway, such as TNFα, IL-6, IL-10, and IL-12, which in turn
validated the critical role of NIK in the development of psoriasis (Figure 9C). Related pathological regulation
responding to 12f treatment in psoriasis mice was demonstrated (Figure 9D). Together, these results suggest
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that 12f treatment can relieve epidermal and dermal cell proliferation and macrophage infiltration in mouse
back skin tissues by targeting NIK to regulate the activation of NF-κB2 signaling pathway.
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Figure 8. (A) Representative skin sections from the back of the mice, which were stained with Ki67 antibody
(
normal group: IMQ and 12f were untreated, model group: only IMQ treatment, 12f-I / 12f-II: at 60 mg/kg or
1
20 mg/kg doses of 12f treatment, respectively, along with IMQ induction). Scale bar, 50 μm. (B) The
+
percentage of Ki67 cells in the total cells was analyzed by Image J software. (C) F4/80 immunofluorescence
staining revealed the recruitment of macrophages in the back skin tissue of C57 mice. P values are
#
##
###
determined by two-tailed Student’s t-test, compared with normal group: P<0.05, P<0.005, P<0.0005, and
compared with model group: *P<0.05, **P<0.005, ***P<0.0005. Data are represented as mean ± SD.
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Figure 9. (A) After oral administration of compound 12f (12f-I: 60 mg/kg, 12f-II: 120 mg/kg), the levels of NF-
κB2 signaling pathway-related proteins (p100 and p52) were monitored. Quantitative analysis was
normalized to GAPDH. (B) The relative optical densities of specific proteins were recorded. (C) Quantitative
analysis of mRNA levels in the skin of the back of mice (TNFα, IL-6, IL-10, IL-12) to investigate the effect of
high and low doses of 12f treatment on the expression of immune regulation-related genes induced by IMQ
in mice. (D) Pathological signal response to the treatment of 12f as the NIK inhibitor was depicted. P values
#
are determined by two-tailed Student’s t-test, between normal and model group: P<0.05,
#
#
###
P<0.005, P<0.0005, and between model and 12f treatment groups: n.s. (not significant), *P<0.05,
**P<0.005, ***P<0.0005. Data are presented as mean ± SD of three independent experiments.
CONCLUSIONS
In this study, we reported the design and synthesis of N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine
derivatives as novel NIK inhibitors. Through structure-activity relationship studies, compound 12f was
identified as a highly potent NIK inhibitor with high selectivity over 268 wild-type kinases. Besides, compound
1
2f exhibited excellent NO production-inhibitory and anti-proliferative activities against macrophages (J744)
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and splenic T cells, respectively. Compound 12f performed better than a known NIK inhibitor compound 1
developed by Amgen in PK properties and blocking ability towards the secretion of IL-6 induced by LPS in
BEAS-2B cells. Encouragingly, down-regulation of inflammation-related cytokines (including: TNFα, IL-6, IL-10,
IL-12, IL-23, IL-1β, and NOS2) and chemokine (CCL3) gene expression in vitro or in vivo, reduced secretion of
pro-inflammatory factors (TNFα, IL-17A) in serum, the relieved psoriasis-like phenotype, and the results of
hematoxylin and eosin staining, Ki67 immunohistochemistry and F4/80 immunofluorescence staining
indicated that treatment of IMQ-induced mice with compound 12f attenuated the pathological features of
psoriasis. Altogether, this work provided a new idea for the design and development of NIK inhibitors, and
suggests the possibility of using NIK inhibitors as a new treatment of psoriasis for the first time.
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EXPERMENTAL SECTION
Kinase Inhibition Assay
ADP-Glo™ kinase assay: ADP-Glo™ kinase assay was performed by Nanjing Anacon Biotechnology Co., Ltd,
which is one of our partners. Reagents: human recombination MAP3K14 protein (Carna, catalogue no. 07-
1
02), NIK ADP-Glo™ assay kit (Promega, catalogue no. V9101). The assay contains two steps: 1. Add an equal
volume of ADP-Glo reagent to terminate the kinase reaction and deplete the remaining ATP after the kinase
reaction. 2. Add the kinase detection reagents to simultaneously convert ADP to ATP and allow the newly
synthesized ATP to be measured using a luciferase/luciferin reaction. Reaction procedure: 1. Preparation of
the following buffer solution: (a) General reaction buffer: 2 mM DTT, 2 mM MgCl , 100 µM Na VO , 0.02
2
3
4
mg/mL BSA, Tris pH 7.5, 5% DMSO. (b) 2.5 X NIK (5 nM) assay buffer. (c) 2.5 X ATP（25 µM）assay buffers. 2.
For 96-well plate, add 1.0 µL of compound in DMSO (final concentration of DMSO: 5%) to the wells of the
reaction plate. Positive and negative control wells used buffer only. 3. In the compound and positive control
wells, 2 µL 2.5 X NIK and 4 µL 2.5X ATP was delivered. Besides, 2 µL 2.5 X ATP and 2µL assay buffers were
utilized for negative control wells. The reaction mixture was incubated for 120 min at room temperature. 4.
After addition and incubation of 5 µL ADP-Glo™ reagent for 40 min, deliver the 10 µL of detection reagent
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into the wells and incubate for an additional 40 min. 5. Luminescence measurement, using a luminometer.
Luminescence can be correlated to ADP concentrations by using an ATP-to-ADP conversion curve. IC values
50
and curve fits were obtained by Prism (GraphPad Software).
Wild-type kinase screening: To determinate the selectivity of 12f over different kinases, a wild-type
1
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screening was performed by Reaction Biology Corp. (Malvern PA), using Hot-SpotSM kinase assay as
47
described previously. The average % inhibition over 268 kinases were reported (Supplementary Table S1).
Griess Reagents Method for Detecting Nitric Oxide Production in J744 Peritoneal
Macrophages
Analysis of macrophage nitric oxide production was carried out according to the methods described in the
48-49
4
previous literature.
Peritoneal macrophages (J774) were seeded in a 96-well plate (2*10 cells/mL, 200 uL
per well), with a medium (5% bovine fetal serum, 1% penicillin-streptomycin, 1% nonessential amino acid,
and 2% L-glutamine). After 24 h incubation in 5% CO at 37°C, the medium was discarded. Add the
2
supplemented medium 1640 197 μL/well. Subsequently, add 2 μL DMSO with or without test compound (1
μM concentration, 3 replicate wells per sample) to the test drug group or LPS model control group. For the
blank group, 2 μL of the medium was spiked into corresponding wells. After 2 h incubation in 5% CO2
incubator at 37°C, 1 μL of PBS-configured LPS (200 ug/mL) was added to the LPS control group and each test
group to a final concentration of 1 ug/mL and cultured for additional 22 h. Then, 100 μL supernatant from
each well was transferred to a new 96-well plate. The supernatant was finally treated with 100 μL Griess
reagents (1% sulfa, 0.1% naphthalenediamine dihydrochloride, and 2.5% phosphoric acid). Finally, using
linear regression analysis of the standard curve (serial double dilutions of sodium nitrite from 200 μmol/L to
the eleventh dilution), obtained NO concentrations in the supernatants Ab-sorbance was determined at 540
nm by using the mi-croplate reader.
Anti-Proliferation Assay of Con A-Activated Spleen T Cell
The primary T-cells were isolated from the splenocyte samples using EasySepTM mouse T cell isolation kit
(
Stemcell Technologies, catalogue no. 19851). T cells were suspended in RPMI with 10% PBS (1%
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penicillin/streptomycin, 2% glutamine) and seeded in 96-well culture plates at a density of 2*10 /mL cells per
well. Con A (Sigma) at 5 μg/mL was used to stimulate the T cell proliferation in the presence or absence of
NIK inhibitors (2µM and 0.4µM concentration, 3 replicate wells per sample). Then, incubate the plates for 48
h at 37 °C 5% CO . Compound’s ability to inhibit Con A dependent primary T cell proliferation were measured
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by using CCK-8 assay (MedChemExpress) according to the manufacturer’s instructions. The toxicity analysis
for normal T cells growth was also performed, corresponding data are shown in supplementary figure S1.
The Measurement of IL-6 Production in LPS-Induced Human BEAS-2B Cells
Human BEAS-2B Cells line was purchased from the American Type Culture Collection (ATCC, catalogue
no. CM-1075). Methods: BEAS-2B cells were resuscitated and cultured (Complete growth medium: 89%
DMEM, 10% FBS, 1% penicillin-streptomycin). Then, seed the logarithmic growth phase of BEAS-2B cells into
96-well plates at 5000 cells/well, and incubated under 5% CO for 24 h at 37 °C. After the cells were attached,
2
the test group, the positive control group (dexamethasone), the model group, and the blank control group
were set before the treatment of compounds 12f and 1 (10 µM-0.014 µM, 7 concentrations, 3 replicate-
wells). After 8 hours of administration, add 0.1 mL of LPS ( 20 μg/mL) to each well to stimulate BEAS-2B cells
for 24 hours. The IL-6 levels in the supernatant of the culture were determined by using the human IL-6 Elisa
kit (MultiSciences, catalogue no. 70-EK1062).
Pharmacokinetic Evaluation of Compound 12f
Metabolic stability in liver microsomes: The metabolic stability of compounds 12f and 1 on liver microsomes
of different species was determined according to the procedures described by 3D BioOptima Co. Ltd.,
including: RLM (rat liver microsomes), DLM (dog liver microsomes), CLM (monkeys liver microsomes) and
HLM (human liver microsomes). To each well (T0, T5, T15, T30, T60, NCF60), add the solution of compound
1
2f, 1 or the control working solution. Subsequently, add 80 μL/well to every plate by apricot, incubate the
mixture of microsome solution and compound 12f or 1 at 37°C for 10 min. Add 10 μL 100 mM potassium
phosphate buffer/well to NCF60, incubate at 37°C for 1h. After pre-warming, add 10 μL/well to every plate by
apricot to start reaction. Reactions were terminated at 5, 15, 30 and 60 min of incubation with a chilled
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Journal of Medicinal Chemistry
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mixture of Tolbutamide and Labetalol (1:1). The mixture was vortexed for 5 min, centrifuged at 4000 rpm for
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0 min at 4°C, and the supernatants were analyzed by LC−MS/MS. The data was analysis by the first order
-1
kinetics to calculate t1/2 and Cl. Drug elimination rate constant k (min ), elimination half-life T (min), and in
1/2
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vitro intrinsic clearance CLint, in vitro (µL·min ·mg protein) were calculated according to the following
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equations: k = - slope, T1/2 = 0.693/k, CLint, in vitro = k/Cprotein, where Cprotein (mg·mL ) is the microsomal
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protein concentration in the incubation.
Pharmacokinetic (PK) study: To determine oral bioavailability, 3 SD rats (Beijing Charles River Laboratories)
per group were treated with 12f by a single intravenous (IV) bolus or oral gavage administration at the doses
of 1 and 10 mg/kg, respectively. Injection formulation of compound 12f was prepared in solution (DMSO:
PEG200: Saline = 20: 20: 60, v/v/v). Suspension of 12f in normal saline containing 0.5% CMC-Na and 0.2%
Tween 80 to obtain the oral formulation and using LC-MS/MS-based methods to quantify drug concentration
in plasma. Pharmacokinetic parameters were estimated using Phoenix WinNonlin (version 6.3) from mean
plasma concentration-time profiles. The area under the curve (AUC) was calculated from time versus
concentration data using the linear trapezoidal rule. The oral bioavailability is calculated as the ratio of AUC
for 12f from PO and IV dosage. The calculation is normalized by relative doses. For 12f plasma samples: an
-1
aliquot of 30 µL of the sample was added with 150 µL ACN which contains verapamil, 5 ng·mL , and
-1
glibenclamide, 50 ng·mL for protein precipitation, the mixture was vortexed for 10 min and centrifuged at
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700 rpm for 10 min. Then 70 µL of supernatant was added with 70 µL water and vortexed for 10 min. An
aliquot of 15 µL of the mixture was injected into the system and subjected to LC-MS/MS analysis using an
Agilent Technologies 6430 Triple Quad LC/MS system. Chromatogram signals were integrated and calibrated
using Agilent MassHunter Workstation Software B.06.00. Pharmacokinetic parameters were derived by non-
compartmental analysis from individual 12f plasma concentration versus time profiles (Phoenix WinNonlin,
version 6.3).
Animal Models
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