The discovery of quinoline derivatives, as NF-κB inducing kinase (NIK) inhibitors with anti-inflammatory effects in vitro, low toxicities against T cell growth

Article abstract of DOI:10.1016/j.bmc.2020.115856

NIK is a critical regulatory protein of the non-classical NF-kB pathway, and its dysregulated activation has been proved to be one of the pathogenic factors in a variety of autoimmune diseases and inflammatory diseases. Nevertheless, its corresponding dev

Full text of DOI:10.1016/j.bmc.2020.115856

Journal Pre-proofs
The Discovery of Quinoline Derivatives, as NF-κB Inducing Kinase (NIK) In-
hibitors with Anti-inflammatory Effects in Vitro, Low Toxicities against T
Cell Growth
Jianing Song, Yuqin Zhu, Weidong Zu, Chunqi Duan, Junyu Xu, Fei Jang,
Xinren Wang, Shuwen Li, Chenhe Liu, Qianqian Gao, Hongmei Li, Yanmin
Zhang, Weifang Tan, Tao Lu, Yadong Chen
PII:
S0968-0896(20)30686-6
https://doi.org/10.1016/j.bmc.2020.115856
BMC 115856
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
11 September 2020
29 October 2020
1 November 2020
Please cite this article as: J. Song, Y. Zhu, W. Zu, C. Duan, J. Xu, F. Jang, X. Wang, S. Li, C. Liu, Q. Gao, H. Li,
Y. Zhang, W. Tan, T. Lu, Y. Chen, The Discovery of Quinoline Derivatives, as NF-κB Inducing Kinase (NIK)
Inhibitors with Anti-inflammatory Effects in Vitro, Low Toxicities against T Cell Growth, Bioorganic &
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115856
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The Discovery of Quinoline Derivatives, as NF-κB
Inducing Kinase (NIK) Inhibitors with Anti-
inflammatory Effects in Vitro, Low Toxicities
against T Cell Growth
Jianing Song ⋚,⊥, Yuqin Zhu†,⊥, Weidong Zu†, Chunqi, Duan†, Junyu Xu†, Fei Jang†, Xinren Wang†, Shuwen
Li†, Chenhe Liu†, Qianqian Gao†,Hongmei Li†, Yanmin Zhang†, Weifang Tan†, 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.
1

ABSTRACT
NIK is a critical regulatory protein of the non-classical NF-kB pathway, and its dysregulated activation has
been proved to be one of the pathogenic factors in a variety of autoimmune diseases and inflammatory
diseases. Nevertheless, its corresponding development of inhibitors faces many obstacles, including the lack of
structure types of known inhibitors, immature activity evaluation methods of compounds in vitro. In this study,
a series of quinoline derivatives were obtained through rational design and chemical synthesis. Among them,
the representative compounds 17c and 24c have excellent inhibitory activities on LPS-induced macrophage
(J774) nitric oxide release and anti-Con A-stimulated primary T cell proliferation. This evaluation method has
good universality and overcomes the obstacles mentioned above, which are faced by the current inhibitor
research to a certain extent. Besides, the compound's toxicity against the growth of T cells under non-stress
conditions was evaluated, for the first time, as an indicator for the investigation to avoid potential safety risks.
Pharmacokinetic properties evaluation of the less toxic compound 24c confirmed its good metabolic behavior
(especially oral properties, F% = 21.7 %), and subsequent development value.
2

INTRODUCTION
The nuclear factor-κB (NF-κB) belongs to a family of inducible transcription factors, which consists of five
structurally related members, including NF-κB1 (p50 / p105), NF-κB2 (p52 / p100), RelA (p65), RelB and c-Rel.1,
2 NF-κB family is a key mediator for regulating innate and adaptive immunity.3, 4 NF-κB regulates the survival
and activation of lymphocytes, which is essential for the normal maintenance of immune response.5 Once NF-
κB dysfunction, it will lead to constitutive activation of the NF-κB signaling pathway. As a result, it mediates the
occurrence and progression of related autoimmune diseases and inflammatory diseases, such as rheumatoid
arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis (PSO), and bronchial
asthma.6, 7
The currently developed inhibitors to block NF-κB signaling indicate that the treatment of autoimmune
and inflammatory diseases is beneficial, but it is challenging to balance therapeutic efficacy and safety.8
Relevant studies showed that comprehensive and in-depth inhibition of NF-κB signaling might lead to serious
side effects, such as liver toxicity during embryonic development, fetal malformations9, and Listeria
monocytogenes infection (patients often have mononuclear complications of cell proliferation) caused by
decreased immunity.10 There is no doubt that a better elucidation of the NF-κB subunit involved in a specific
pathological process will help develop a new generation of anti-autoimmune and inflammatory drugs with high
effects, fewer side effects, and low cost. With in-depth research on the mechanism of NF-κB activation, several
upstream kinases (such as NIK, IKK1, IKK2, and MEKK3) and downstream effector IκB ubiquitin E3 ligase have
been identified, which can selectively regulate NF-κB function. Indeed, they have become promising targets
for drug development.11
NIK (also known as MAP3K14), as a key regulator, participates in the response of NF-κB2 signaling pathway
to TNF superfamily receptors such as CD40, CD27, lymphotoxin β receptor (LTβR), B cell-activating factor
receptor (BAFF- R), tumor necrosis factor receptor superfamily member 12A (TWEAK), etc. Under normal
physiological conditions, TRAF3 connects NIK and TRAF2-cIAPs E3 to form a complex, conducive to the
ubiquitination of NIK protein and its degradation by the proteasome. Unlike the generally low NIK level, when
3

the cytokine CD40L or BAFF binds to its membrane receptor, NIK will accumulate in the cytoplasm, leading to
IKKα phosphorylation, p100 processing, and increased p52 levels. p52 combines with RelB to form a p52 / RelB
heterodimer, which then translates to the nucleus to trigger the expression of target inflammatory factors and
chemokines.12 The non-classical NF-κB pathway regulates a variety of physiological processes, including the
differentiation, maturation, and survival of B cells, and immune behavior of T cells.13-15 Once the NIK is
dysregulated, it can promote the abnormal activation of non-canonical NF-κB, thereby mediating autoimmune
and inflammatory diseases' occurrence and progression. Similar to the classic NF-κB signal, an abnormal NF-
κB2 signal is also an important pathogenic mechanism of RA16, which is related to the high expression of NIK in
patients' synovial endothelial cells.17 Progressive joint injury in patients with RA involves the abnormal
generation and activation of osteoclasts, and this process depends on the NF-κB2 signaling pathway.18 Besides,
the pathogenesis of RA is often accompanied by the prolongation and maturation of B cell survival, which stems
from the abnormal activation of the NF-κB2 pathway by BAFF.19 Excessive BAFF levels in SLE patients up-
regulate the NF-κB2 signaling pathway, thereby promoting the release of auto-antibodies from B cells and
affecting the pathological survival and differentiation of B cells.20 NIK overexpression mediates the
pathogenesis of MS, by over activating autoreactive Th1 and Th17 cells.21-22 Abnormal activations of NIK can
enhance mouse intestine sensitivity to rodent citrobacter and then induce enteritis.23 Similarly, NIK dysfunction
also has a direct or indirect induction effect in the pathogenesis of psoriasis.24-29 Thus, the research of highly
potent and druggable small molecule inhibitors targeting NIK protein has excellent value and prospects.29
Currently, research on NIK inhibitors is scarce, and their structure types are far from abundant (Figure 1).
Aminopyrimidine compound 1, as the first NIK inhibitor, was developed by Amgen.30 Compound 1 has excellent
inhibitory activity against NIK and can notably alleviate the symptoms of toxicity-induced hepatitis and liver
injury in mouse models.31 In 2017, Genentech developed carbamoyl imidazole derivatives represented by
compounds 2 and 3 through high-throughput screening and structural optimization. In particular, 2 performed
better in p52/RelB dimer nuclear transfer inhibition and NF-κB luciferase gene report assay. Besides, they also
developed other series of NIK inhibitors: nicotinamide and pyrazole derivatives represented by 4 (SIM1) and 5,
4

respectively. Using compound 1 (B022) as a starting point for the inhibitor design, Zhao's group developed a
series of pyrrolopyrimidin-2-amine compounds.32 They also provided an example of compound 6 (XT2), as a
NIK inhibitor, which can effectively inhibit toxin-induced hepatitis. Our previous work demonstrated the
potential of compound 7 that alleviates the disease phenotype of imiquimod-induced psoriasis model mice. 29
Compound 8 is unique than other inhibitors because its molecular structure does not contain alkynyl linkers
and tertiary alcohol fragments used to match the back pocket’s shape.
NH2
O
NH2
N
NH2
N
O
H2N
O
N
NH
OH
OH
S
N
HO
OH
N
N
Cl
O
N
N
N
O
N
N
O
N
O
O
F
O
2
4 (SIM1)
1 (B022)
3
K = 0.4 0.4 nM
K = 0.23 0.17 nM
K = 0.4 0.3 nM
±
K = 0.47 0.27 nM
±
i
±
±
i
i
i
H
H
H
N
N
N
NH2
N
N
N
N
OH
N
OH
S
N
N
F
OH
S
Cl
CN
HN
N
HN
N
N
N
F
NC
HN
O
OH
6 (XT2)
8
7
5
N
IC50 = 9.1 2.0 nM
IC50 = 1.8 nM
±
IC50 = 9.87 0.27 nM
±
IC50 = 21 nM
Figure 1. Representative NIK inhibitors.
Ignoring that the structural types of known inhibitors are scarce, their development also faces many other
obstacles, including immature cell screening methods, high clearance rates of compounds, and short half-lives
in vivo (compound 1). This situation dramatically limits the verification of NIK protein's biological function and
the exploration of its potential indications. Thus, the development of NIK inhibitors with high druggability and
the enrichment of structural types have become the primary task.33, 34 Inspired by the structure of 3-pyrazole-
indole analog 5, we speculated on the factors that may be unfavorable to its activity and developed a new
design strategy in this work. Through synthetic chemical means, a series of quinoline derivatives with suitable
NIK enzyme inhibitory activities were obtained. The corresponding activity evaluation revolved around their
inhibitory potency against NIK, cell activity, and toxicity, and PK characteristics of the selected compounds.
RESULTS AND DISCUSSION
5

Structure-Based Design Strategy
In 2015, Janssen Pharmaceuticals developed the indole skeleton analog 5, which has a double-digit
nanomolar NIK inhibitory activity (IC50 = 21 nM) and a good anti-proliferation activity (IC50 = 61 nM) against
human leukemia cell line (JJN3, a genetically engineered cell line transfected with NIK plasmid). Given this
series compounds' excellent in vitro activities, Janssen applied for a corresponding Chinese patent. However,
there is no follow-up report of further research. In order to obtain a class of novel quinoline NIK small molecule
inhibitors, a new and reasonable design strategy was proposed based on 5. As described in Figure 2A, the
design strategy includes: a) removing negative spatial trends and the excessively sizeable 4-methyl piperidine
ring. b) the pyrrole ring is grown into pyridine, gives a highly medicinal core: quinoline skeleton. This ring
expansion treatment may benefit the 6-position alkynyl group to point to the back pocket region. c) Introducing
a predominant group that fits well with the back pocket: the thiazolyl fragment. As can be seen from Figure
2B, there are two important amino residues in the back pocket, which are Asp536 and Phe537. Thiazole can
adapt to its volume and may have potential interactions in hydrogen bond and π-π stacking. The application of
above strategy has spawned a representative quinoline derivative 17a.
Subsequently, compound 17a was docked into the NIK crystal structure (RCSB PDB code 4G3E) to explore
the compatibility of 17a with the NIK binding pocket (Figure 2B). The pyrazole fragment occupies the hinge
region and forms corresponding hydrogen bond interactions with Leu474 and Glu472, respectively. With
proper spatial orientation, the alkynyl group smoothly extends into the back pocket area through the narrow
channel formed by the side chains of Met471, Lys431, and Asp536. The thiazole ring locates in a small
hydrophobic pocket behind Met471, where the thiazole ring forms a π-π stacking effect with the benzene ring
of Phe537. The nitrogen atom on the thiazole ring forms a hydrogen bond interaction with Asp536. In the back
pocket, the alkynol hydroxyl group captures Phe537 and Glu442 residues, forming essential hydrogen bonds.
Besides, the lactam structural unit of compound 17a (2-hydroxyquinoline tautomerism) occupies the solvent-
accessible area. The cavity is not fully occupied in this area, making the 2-position of quinoline has great
structural modification potential. The cross-sectional view shows the arrangement and molecular orientation
6

of 17a within the binding site (Figure 2C). The next kinase activity assay confirmed that 17a has a good
inhibitory potency against NIK (IC50 = 38.6 ± 0.25 nM).
Figure 2. (A) Based on the structure of comound 5, the follow-up optimization ideas are proposed. (B)
Representative compound 17a docked into NIK (PDB code 4G3E). (C) Spatial layout of 17a in NIK binding pocket
is presented (PDB code 4G3E).
Chemsitry
As presented in Scheme 1, using 2,4-hydroxyquinoline as the starting material, intermediate 10 was
obtained in a good yield through a nitration reaction in a nitric acid/sulfuric acid reaction system. Subsequently,
the 2-hydroxy group of the quinoline was substituted with bromine to give 11. The bromine atom can be
derived from phosphorus oxybromide or tetrabutylammonium bromide (step b). In step c, acetic acid catalyzed
the hydrolysis of 11 to give 4-bromo-6-nitroquinolin-2-ol (12). Under strict reaction temperature and the
dripping acceleration control of the hydrophilic group, various kinds of chain or cyclic secondary amines
selectively attack the quinoline 2-position to give 13b－13d or 13f with high yields. For the synthesis of 4-(2-
7

((6-nitroquinolin-2-yl)oxy)ethyl)morpholine (13e), Et3N in condition d was replaced by NaH. Under the catalysis
of PdCl2(dppf), 13 was coupled with pyrazole borate, and then, the Boc protecting group was removed by using
trifluoroacetic acid to generate 14. Subsequently, 14 undergoes three-step reaction: Fe/NH4Cl catalyzed nitro
reduction reaction, Sandmeyer reaction, and Sonogashira coupling gave the final product 17a－17f.
Scheme 1. Synthesis of 2-hydrophilic substituted quinoline analogsa
N
OH
O2N
d
e
or
N
R
12
Br
c
O2N
O
13
Br
N
OH
N
OH
N
Br
R
a
b
O
O2N
O2N
B
f
OH
OH
Br
N
9
10
11
BocN N
O2N
N
R
N
R
N
R
g
i
h
S
N
I
H2N
OH
HN N
HN N
17a ~17f
HN N
14
HN N
15
16
17a
17c
17e
17f
: R =
: R = -OH
: R =
: R =
N
N
: R =
O
O
N
N
N
O
17b:
17d
R =
NHCH3
N
aReagents and conditions: (a) HNO3, H2SO4, 0 °C; (b) POBr3, toluene, 110 oC or (n-Bu)4N+Br-, P2O5, toluene; (c)
HOAc, 90 °C, overnight; (d) appropriate amine, Et3N, DCM, 0 °C; (e) NaH, corresponding 4-(2-chloroethyl)
morpholine, DCM, 0 oC － room temperature; (f) PdCl2(dppf), K2CO3, dioxane/H2O, 80 °C; coupling completed,
CF3COOH, DCM, r.t; (g) Fe, NH4Cl, 75% EtOH; (h) NaNO2, KI, p-TsOH, CH3CN/H2O, 0 °C － room temperature; (i)
2-(thiazol-2-yl)but-3-yn-2-ol, PdCl2(PPh3)2, CuI, Et3N/THF = 1:3.
As shown in Scheme 2, the synthesis of different back pocket binding structural units (20) was achieved
through the nucleophilic attack of ketones by terminal alkyne or alkenyl Grignard reagent. Similar to the
8

synthesis of compound 16, 2-hydroxyquinoline was used as the starting material to synthesize intermediate
23. Subsequently, 23 reacted with 20 in the presence of PdCl2(PPh3)2 and CuI to give target compounds 24a－
24f. Using methyl pyrazole borate, the synthesis of compound 28 was similar as that of compound 24a. When
introducing alkenyl groups as linkers (24g), Pd(OAc)2 was required as a catalyst. In Scheme 3, the preparation
of 27a－27b was achieved by two reactions: introduction of amide linker in the presence of Et3N as a base, and
removal of the pyrazole nitrogen “-Boc” protecting group.
Scheme 2. Synthesis of compounds 24a-24ga
O
TMS
n = 1 or 2
18
n
R1
a
b
or
or
MgBr
or
X
R2
N
Y
R2
OH
O
20
X = S or CH2
Y = N or CH2
h
i
or
R1
X
19
X
HO
N
N
N
N N
c
d ~ g
R3
I
24a ~ 24g
O2N
OH
OH
22
23
21
N N
R3
1
2
3
24a
: R = -CH3, R = -CH3, R = H
1
2
24e
: R /R =
1
2
3
24b:
24c:
R = thiophene, R = -CH3, R = H
1
X =
24g
X =
: R = thiazole
R2 = -CH3
1
2
3
R = thiazole, R = -CH3, R = H
1
2
3
24d
: R = pyridine, R = -CH3, R = H
1
2
24f
: R /R =
1
2
3
28
: R = -CH3, R = -CH3, R = -CH3
Reagents and conditions: : (a) n-BuLi, THF, -78 °C ― r .t, and then NaOH (3.0 equiv) in MeOH was dropwised;
o
(b) THF, stirred at 0 °C; (c) HNO3, H2SO4, 0 °C; (d) POBr3, toluene, 110 C or (n-Bu)4N+Br-, P2O5, toluene; (e)
PdCl2(dppf), K2CO3, dioxane/H2O, 80 °C, and then CF3COOH, DCM, r. t; (f) Fe, NH4Cl, 75% EtOH; (g) NaNO2, KI,
p-TsOH, CH3CN/H2O, 0 °C ― room temperature; (h) PdCl2(PPh3)2, CuI, Et3N/THF = 1:3; (i) Pd(OAc)2, PPh3, K2CO3,
toluene, 90 oC, overnight.
Scheme 3. Synthesis of compounds 27a-27ca
9

N
N
N
N
S
O
O
27a
27b
N
N
H2N
a
b
H
H
N N
HN N
N N
Boc
Boc
27
26
25
aReagents and conditions: (a) appropriate acyl chloride, Et3N, DMF, 0 °C; (b) CF3COOH, DCM, room
temperature.
Structure-Activity-Relationship (SAR) Studies
Taking 17a as the starting point of structural modification, the introduction of a larger volume of
methylamino at the 2-position of quinoline gave 17b, and its activity was slightly improved compared to the
former. Replacement of the methylamino group with different flexible hydrophilic substituents greatly affects
the NIK inhibitory activities of compounds (17c and 17d). Among them, the inhibition of 17c towards NIK was
significantly improved (IC50 = 5.2 ± 0.04 nM). However, when the terminal of a flexible hydrophilic group was
substituted with dimethylamino (17d), its activity decreased, which might be due to repulsion between the
terminal methyl group and amino acid residues surrounding protein cavity at this position (Figure 3).
Subsequently, cyclization of the terminal dimethylamino group of 17d to a morpholine ring obtained 17e. It
was found that its inhibitory effect against NIK was almost lost, indicating the reasonableness and reliability of
the above speculation. To further investigate the volume of the cavity in the solvent accessible area, the methyl
piperazine ring was introduced into the quinoline 2-position to generate the compound 17e, which retained
the potency on NIK (compare 17d with 17f).
Table 1. Structure optimization of 2-position substituents of quinoline in solvent accessible region
Solvent
accessible region
N
R
S
N
OH
HN N
10

NIK IC50(nM)a
Compounds
1
R
NIK IC50(nM)a
Compounds
17d
R
% enzyme
activityb
N
/
4.9 ± 0.34
159.6 ± 1.13
N
O
N
17a
17b
17c
-OH
38.6 ± 0.25
26.0 ± 0.18
5.2 ± 0.04
17e
17f
91.97±0.24%
174.8 ± 0.97
O
N
-NHCH3
N
N
O
aKinase inhibition assay was performed at 10 µM ATP concentration, and the values are the mean ± SD from
two independent experiments. bEnzyme activity assay was investigated at 2 µM dosing compound
concentration and 10 µM ATP concentration.
Figure 3. Representative compound 17d docked into NIK (PDB code 4G3E).
Further work mainly investigated the match of different tertiary alcohol fragments to the back pocket and
the types and characteristics of linker structures that can smoothly pass through the narrow channels formed
by the side chains of Met471, Lys431, and Asp536. As shown in Table 2, a variety of tertiary alcohol fragments
with different shapes, sizes, and electrical properties were preferentially investigated. When R1 and R2 were
methyl groups (24a), the activity was greatly reduced. Substitution of methyl group with five-membered, or
11

six-membered aromatic ring afforded 24b －24d. Summarizing the corresponding activity data showed that
five- or six-membered aromatic rings are conducive to the improvement of activities. Specifically, compound
24c more potently inhibits the protein activity of NIK (IC50 = 22.1 ± 2.46 nM), demonstrating the importance of
the hydrogen bonding interaction between the thiazole nitrogen atom and Asp536 (compare 24b with 24c).
The cyclization of R1 and R2, as a six-membered saturated aliphatic ring, reduces the activity of 24e (compare
to 24f) by nearly 10-fold, which may be due to its excessive bulk. Subsequently, the alkynyl group was replaced
with a different linker to investigate the linker characteristics that can smoothly pass through narrow channel
of the back pocket. Using vinyl as a linker (24g, IC50 = 2080 nM), the activity was decreased by nearly 100-fold
(compare 24g with 24c). The more flexible amide bond as a linker is unfavorable for binding between the
corresponding compound and the NIK active pocket (compare 27a or 27b with 24f). Besides, methylation
blocks "-NH-" on the pyrazole structural unit, resulting in the complete loss of the activity of compound 28,
which verifies the importance of hydrogen bonding interaction in hinge region to maintain good ligand NIK
inhibitory activity.
Table 2. Structures and their inhibitory activities against NIK of target compounds
Asp536
pocket
Back
region
N
R2
R1
X
O
H
Phe537
N N
R3
Glu442
Leu474
NIK IC50(nM)a
% enzyme activityb
Compounds
R1
R2
X
R3
1720 ± 32.16
523.8 ± 12.57
22.1 ± 2.46
24a
-CH3
-CH3
H
24b
24c
-CH3
-CH3
H
H
S
N
S
12

590.9 ± 21.47
6050 ± 76.25
24d
24e
-CH3
H
H
N
564.1 ± 16.3
2080 ± 24.83
24f
H
H
N
24g
-CH3
S
O
O
N
72.26 ± 1.26%
27a
H
N
H
S
83.60 ± 1.37%
99.09 ± 0.08%
27b
28
H
N
H
-CH3
-CH3
-CH3
aKinase inhibition assay was performed at 10 µM ATP concentration, and the values are the mean ± SD from
two independent experiments. bEnzyme activity assay was investigated at 2 µM dosing compound
concentration and 10 µM ATP concentration.
Inhibitory Activities of the Compounds on the Release of Nitric Oxide (NO) in J774 Macrophages
and Anti-splenic T cell Proliferation Activities
In the pathogenesis of various autoimmune and inflammatory diseases (such as RA, SLE, and PSO), NIK's
dysregulated activation can regulate the prolonged survival of B cells, excessive proliferation of T cells, and
deepened infiltration of macrophages.20-22, 29 Thus, investigation of the compounds’ inhibitory effects on
lipopolysaccharide (LPS) induced NO release in macrophages, and concanavalin A (Con A) stimulated splenic T
cell proliferation is of great significance for evaluating its cell activity. Six compounds with better NIK inhibitory
potency (17a－17d, 17f, and 24c) were selected for further cell-level activity assay. The results showed that at
1 µM concentration, four compounds inhibited LPS-induced NO release in J774 cells by more than 40%.
Consistent with its high enzyme inhibitory efficacy, the activity of 17c was optimal at 51%, which indicated that
the introduction of appropriate flexible hydrophilic groups might increase the inhibition of NO release of in
J774 cells. In other parallel experiments, these compounds all showed notably anti-T cell proliferation activities
13

at two dose levels (2 µM and 0.4 µM). Interestingly, although there is an apparent distinction between 17c and
24c in inhibitory activities on NIK enzyme and macrophage NO release, their T cell anti-proliferative activities
of both performed better, and the inhibitory efficacy were similar.
Table 3. Anti-inflammatory activity screening of selected compounds at cellular level.
NO Inhibition rate (%)a
Proliferation-Inhibitory rate (%)b
Cpds NIK IC50 (nM)
At 1 μM
At 2 μM
At 0.4 μM
19.04± 0.03
23.31±0.03
18.40±0.68
33.05±0.49
19.93±0.81
19.38±0.12
36.14±0.84
1
43.90± 0.12
31.45±0.34
46.48±0.75
51.44±0.58
34.63±0.28
41.03±0.70
43.77±0.74
59.57± 0.09
27.25±0.44
34.50±0.88
53.13±0.22
22.72±0.34
36.72±0.08
52.46±0.71
4.9± 0.34
38.6 ± 0.25
26.0 ± 0.18
5.2 ± 0.04
17a
17b
17c
17d 159.6 ± 1.13
17f
24c
174.8 ± 0.97
22.1 ± 2.46
aNO inhibition rate % = (model group NO concentration – compound group NO concentration) / (model group
b
NO concentration – control group NO concentration) × 100%. The proliferation of splenic T cells induced by
Con A was tested under the treatment of six selected compounds. Proliferation -Inhibitory rate (%) = (test hole
absorbance – blank hole absorbance) / (control hole absorbance – blank hole absorbance) × 100%. a,bThe values
are the mean ± SD from three independent experiments, n = 3.
Toxicity of Representative Compounds for Normal Growth and Differentiation of Splenic T Cells
As an important indicator for the evaluation of anti-inflammatory small molecule inhibitors, the lower
toxicity of the compound to normal growth and differentiation of T cells (non-Con A stimulation), the higher
its subsequent development potential. It is expected that small molecules can target to suppress the expression
of inflammation-related target genes and the secretion of inflammatory cytokines in immune cells, instead of
directly killing immune cells to eliminate inflammation.35 Taking compound 1 developed by Amgen and
compound 7 discovered in another part of our work as positive drugs, we examined the T cell growth toxicity
of six compounds in this series. As shown in Figure 4, the compounds’ T cell toxicity is different from each
other. Among them, the toxicity of 17c and 17f is similar to that of compound 1, and the inhibition rate is close
to 30%. Besides, the 17d and 24c toxicity performance were comparable to 1, and both were low. 17a did not
14

show toxicity, and the T cells grew well in this administration group. Besides, the compound 17c, which has the
best cell activity, is about three times as toxic as the positive compound 7. Integrating the above macrophage,
T cell activity, and growth toxicity data of compounds found that 24c is a highly active and less toxic compound.
Accordingly, 24c can be selected for the subsequent determination of metabolic stability and pharmacokinetic
parameters in SD rats.
Figure 4. Using known NIK inhibitors 1 and 7 as the positive control group, the toxicity of selected six
compounds on T cells' normal growth at a concentration of 10 µM was investigated. 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.
Multi-Species Liver Microsomal Metabolic Stability and In Vivo Pharmacokinetic (PK)
Parameters of Compound 24c
As shown in Table 4, we evaluated the metabolic stability of 24c in human liver microsomes (HLM),
monkey liver microsomes (MLM), dog liver microsomes (DLM), and rat liver microsomes (RLM). 24c has
moderate metabolic stability of human liver microsomes, and its clearance rate was 40.237 mL/min/kg. When
switching to monkey-derived liver microsomes, the compound's metabolic half-life was basically maintained
(T1/2 = 33.185 min), but the clearance rate increased to 60.156 mL/min/kg. Inconsistently, in dog and rat liver
microsomes, the stability of 24c decreased significantly, which corresponds to the clearance data in the liver
15

microsomes of these two species. The data presented in Table 5 show the pharmacokinetic performance of
24c under two administration modes: intravenous (IV) and oral (PO). When test compound 24c (1.0 mg/kg)
was injected into the tail vein, the C0 was as high as 1003 ng/mL, the area under the curve was good (AUC0−∞
=
408 ng h/mL), and the half-life was slightly shorter (T1/2 = 0.382 h ) accompanied by a moderate clearance rate.
Correspondingly, at 0.9 h after oral gavage (i.g.) administration of compound 24c, plasma concentration
reached the maximum. Besides, the area under the curve (AUC0−∞ = 885 ng h/mL) did not reach the ideal, and
the half-life was 1.19 h. It is worth mentioning that the oral bioavailability of 24c (F% = 21.7%) is acceptable,
promoting its potential for follow-up research. Based on its good activities in vitro, low T cell survival toxicity,
and acceptable PK properties, compound 24c may be considered to explore the potentially applicable
symptoms of NIK dysregulation or overexpression and verify its related biological mechanisms.
Table 4. Metabolic stability of compound 24c in liver microsomes of multiple species
T1/2
CLint, in vivo
CLint, microsome
Compound Species
Human
R2 a
(µL/min/mg proteins)c
(min)b
(mL/min/kg)d
0.9999 33.099
0.9922 33.185
0.9983 14.224
0.9917 12.554
42.579
41.775
97.451
110.427
40.237
60.156
Monkey
24c
Dog
140.330
198.768
Rat
aR2 is the correlation coefficient of the linear regression for determination of the kinetic constant. bElimination
half-life (T1/2). cIntrinsic clearance in LMs (CLint, in vitro), dIntrinsic hepatic clearance (CLint, in vivo). CLint, in vivo = CLint,
(mL/min/mg microsomal protein) × Scaling factor (physiological parameter) = CLint, in vitro (mL/min/mg
in vitro
microsomal protein) × Microsomal protein (mg protein/g liver) × Liver weight (g liver/kg body weight).
Table 5. Pharmacokinetic parameters of compound 24c in Sprague Dawley Rata
IV
PO
PK parameters (n=3)b
SD
PK parameters (n=3)a
SD
(1.0 mg/Kg)
(10 mg/Kg)
AUC (0-∞)(ng·h/mL)
408
126 AUC(0-∞)(ng·h/mL)
885
137
16

C0 (ng/mL)
T1/2 (h)
1003
0.382
2.73
113
0.087
0.393
12.2
Cmax (ng/mL)
T1/2 (h)
311
1.19
0.900
21.7
92
0.194
0.173
3.3
Vd (L/kg)
Tmax (h)
F(%)
CL (mL/kg/min)
43.3
aAfter intravenous injection (i.v., 1.0 mg / kg) or gavage (i.g., 10 mg / kg) of compound 24c, the blood
concentration of mice was recorded over time. Corresponding in vivo pharmacokinetic parameters are given.
Injection formulation (prescription, DMSO : PEG200 : Saline = 20 : 20 : 60, v / v / v. concentration, 0.2 mg·mL-
1). Oral formulation (0.5% CMC-Na / 0.2% Tween 80. concentration, 1 mg·mL-1). bPK parameters are the mean
± SD from three independent experimental rats, n = 3.
CONCLUSIONS
Inspired by the structure of 3-pyrazole-indole analog 5, a further design strategy was put forward. The
structural activity modification revolves around the following aspects: a) The introduction of flexible chain-like
and cyclic hydrophilic moieties with different sizes at the 2-position of quinoline to explore the characteristics
of the solvent-accessible region. b) Investigation and verification of the dominant fragments of the back pocket.
c) Replacement of alkynyl linker. Subsequently, compounds 17c and 24c attracted our attention for their
outstanding inhibitory potency against NIK and cell activities. Further, T cell toxicity analysis confirmed that 17c
performed poorly, and the toxicity was significantly higher than that of control groups (compounds 1 and 7).
The cross-species liver microsomal metabolism stability of 24c in vitro and SD rat pharmacokinetics (PK)
properties in vivo are acceptable, especially its oral properties (F% = 21.7%), which prompts it has the potential
for subsequent development. In short, a series of quinoline derivatives, with potent NIK inhibition, developed
in this work have enriched the structural types of inhibitors to a certain extent. Two inflammatory cell models
were constructed: LPS-induced (J774) NO release in macrophages and Con A-stimulated splenic T cell
proliferation. This screening technique has certain universality. Besides, the toxicity study of NIK inhibitors is
included in the evaluation system, which may help to avoid the toxicity problems of inhibitors and reduce the
risk of development.
17

EXPERIMENTAL SECTION
ADP-Glo™ Kinase Inhibition Assay
The kinase assay was carried out by Nanjing Anakang Biotechnology Co., Ltd. The experimental method
followed the report in another part of our work.29 Reagents: human recombination MAP3K14 protein (Carna,
catalog no. 07-102), NIK ADP-Glo assay kit (Promega, catalog no. V9101). The assay was performed in two
steps: (1). Terminate the kinase reaction with an equal volume of ADP-Glo reagent and deplete the excess ATP.
(2). Convert ADP to ATP with kinase detection reagent, and then use luciferase/luciferin reaction to measure
the newly generated ATP. The Preparation of buffer solution: (a) General biochemical reaction buffer: 100 µM
Na3VO4, 2 mM DTT, 0.02 mg/mL BSA, 2 mM MgCl2, Tris pH 7.5, 5% DMSO. (b) 2.5 × NIK (5 nM) biochemical
analysis buffer. (c) 25 µM 2.5 × ATP assay buffer. 1.0 µL DMSO solution (5%) of the compounds were added to
a specific well (96-well plate). Buffer was added to the positive and negative control wells. 2 µL 2.5 × NIK and
4 µL 2.5 × ATP was added to both compounds and the positive control wells. Besides, for negative control wells,
2 µL 2.5 × ATP and 2 µL assay buffer were added. The reaction mixture was incubated for 120 min at room
temperature. After the incubation was completed, 5 µL ADP-Glo reagent was added to each well, and the plate
was incubated for another 40 min. Subsequently, 10 µL of detection reagent was added to each well, and their
luminescence intensity was measured after 40 min of incubation. According to the conversion curve of ATP to
ADP, the luminous intensity is converted into ADP concentration. The IC50 value was calculated by Prism
(GraphPad software) curve fitting.
Griess Reagent Method to Measure the Release of Nitric Oxide from LPS-Induced J774
Macrophages
The macrophages (extracted from mouse abdominal cavity) were inoculated in a 96-well plate (200 uL,
concentration: 2 × 104 cell/mL), and the medium was (5% bovine fetal serum, 1% penicillin-streptomycin, 1%
non-essential amino acids, 2% L-glutamine). The plate was incubated at 37 °C and 5% CO2 for 24 h. The original
medium was removed, and replaced with the medium 1640 (197 μL/well). DMSO solution of the test
18

compounds (1 μM concentration, 3 replicate wells/sample), 2 μL DMSO and 2 μL medium were added to the
drug group, model group, and blank group, respectively. The well plate was placed in a 5% CO2, 37 °C incubator
for 2.0 h. A LPS (200 ug/mL) mixture with 1 μL PBS was added to the control and test groups (final concentration
is 1 ug/mL). After culturing for 22 hours, 100 μL of supernatant from each well was transferred to a new 96-
well plate. Subsequently, 100 μL of Griess reagent (1% sulfonamide, 0.1% naphthalenediamine dihydrochloride,
and 2.5% phosphoric acid) was added to each supernatant. Make a standard curve (double-diluted sodium
nitrite solution, initial concentration: 200 μmol/L, serial dilution eleven times), and use a microplate reader to
read. Linear regression assay was used to determine the concentration of NO in the supernatant (under 540
nm).
Anti-Proliferative Activity of Selected Compounds against Splenic T cells Stimulated by Con A
Primary T cells were isolated from spleen tissue (EasySepTM Mouse T Cell Isolation Kit, Stemcell Technologies,
catalog number: 19851). Primary T cells were suspended in RPMI (1% penicillin/streptomycin, 2% glutamine)
medium diluted in 10% PBS. Subsequently, it was seeded in a 96-well culture plate (cell density: 2 × 106 cells/mL
per well). The drug group was added with 2 μM or 0.4 μM NIK inhibitors (3 replicate wells for each sample),
while the blank control group was added with the same amount of DMSO. After the addition, Con A (Sigma, 5
μg/mL) was used to induce T cell proliferation, and then incubated the plate at 37 °C, 5% CO2 for 48 h. According
to the operating manual (CCK8 assay, MedChemExpress) provided by the manufacturer, the compound's anti-
Con A-dependent T cell proliferation activity was determined.
Pharmacokinetic Evaluation of Compound 24c
Metabolic Stability Assay in Liver Microsomes: The multi-species liver microsomal metabolic stability assay
was based on the test method described by 3D BioOptima Co. Ltd. The types of liver microsomes include rat
liver microsomes (RLM), canine liver microsomes (DLM), monkey liver microsomes (CLM), and human liver
microsomes (HLM). To each well labeled (T0, T5, T15, T30, T60, NCF60), test compound or control drug solution
was added. Subsequently, the mixture of the microsome solution (80 μL/well) and the test compound was
incubated at 37°C for 10 min. 10 μL of 100 mM potassium phosphate buffer was added to the designated wells
19

(NCF60), after the addition, incubate for 1.0 h at 37 °C. A mixture of tolbutamide and labetalol (1:1) was used
to stop the reaction at 5 (T5 wells), 15 (T15 wells), 30 (T30 wells), and 60 (T60 wells) minutes after incubation.
The mixture was vortexed for 5 minutes, centrifuged at 4000 rpm at 4 °C for 20 min, and LC-MS analyzed the
remaining compound in the supernatant. The data was analyzed through first-order kinetics to calculate "T1/2
and "CL".
"
Determination of Pharmacokinetic (PK) Parameters in SD Rats: Six 8-week-old male Sprague-Dawley mice
were fasted overnight and used for pharmacokinetic studies under the intravenous or oral administration
routes. The doses of 1.0 and 10 mg/kg were administered via a single intravenous (IV) rapid bolus and
intragastric administration. Compound 24c was suspended in physiological saline containing 0.5%
Carboxymethyl cellulose sodium salt(CMC-Na) and 0.2% Tween 80 to prepare an oral prescription (1.0 mg/mL).
Processing of the collected plasma samples: the samples were divided into 30 µL aliquots, and Acetonitrile (150
µL) containing 5 ng/mL verapamil and 50 ng/mL glibenclamide was added to each aliquot for protein
precipitation. The mixture was vortexed for 10 min and centrifuged at 3700 rpm for 10 min. An equal volume
of water was added to the separated supernatant (70 µL), and the mixture was vortexed for 10 minutes.
Subsequently, 15 µL mixture was injected into the LC/MS system and analyzed using Agilent Technologies 6430.
Molecular Modeling
The X-ray co-crystal structure of the NIK protein binding with corresponding small molecules was retrieved
from the Protein Data Bank36 (PDB codes: 4G3E). Following the pre-processing wizard (Ligand Preparation
module) in Maestro (Schrodinger 2018), the conformational optimization of the target compound is
implemented under standard settings. According to the standard procedure suggested by the protein
preparation (Protein Preparation module), the NIK protein was pre-processed, and the grid file was generated
(using Receptor Grid Generation, version 11.5). In the standard and ultra-precision modes, the binding
conformation of the ligand and NIK protein is obtained (the docking is set according to the Glide standard).
Among them, only the docking results with low energy configuration and good hydrogen bond geometry trends
are considered. Finally, HprSnap 7 is used to edit pictures.
20

General Procedures.
Unless otherwise specified, all reagents were purchased from suppliers. Commercially available
chemically pure (CP, Chemically Pure) or analytical pure (AR, Analytical Reagent) reagent products were used
without further purification. The products were purified by pressurized fast column chromatography, and the
stationary phase is usually 100-200 mesh or 200-300 mesh silica gel produced by Qingdao Ocean Chemical
Factory. The melting points were determined by an X-4 digital-display micro melting-point apparatus (Beijing
Tech Instrument Company, Ltd., Beijing, China). The NMR spectrums were recorded on the Bruker AVANCE AV-
600 spectrometer (400 or 300 MHz for 1H, 100 or 75 MHz for 13C). The chemical shift (δ) and coupling constants
(J) are reported in ppm and Hz, respectively, and the solvent signal is used as a reference. Mass spectrometry
data were obtained on Agilent 1100 LC / MSD mass spectrometer (Agilent, USA) and Q-tofmicro MS (micromass
company). All reactions were monitored by TLC (Merck Kieselgel GF254), and spots were visualized with UV
light or iodine. HPLC conditions: chromatographic column used C18 (1.8 μM, 4.6 mm × 30 mm, Agilent), mobile
phase A was methanol (chromatographic grade), mobile phase B was purified water (wahaha brand); isocratic
elution, methanol: water = 80: 20, the column temperature was 35 °C, the fluorescence detector was 365 and
220 nM. The compound used for biological evaluation (HPLC analysis) shall have a purity of not less than 95%.
The Preparation of 7-nitronaphthalene-1,3-diol (compound 10) was in accordance with
relevant known literature . 37
The Preparation of 2,4-Dibromo-6-nitroquinoline (11)
To a toluene (100 mL) solution of compound 10 (1.05 g, 5.09 mmol), POBr3 (4.38 g, 15.3 mmol) was added
under N2 atmosphere and room temperature. After the addition, the reaction mixture was moved to 110 °C oil
bath and stirred for 2 h. After the reaction was completed, the reaction was naturally cooled to room
temperature. The mixture was extracted with EA (50 mL × 3), combined organic phase, and concentrated under
vacuum. The residue was chromatographed, eluting with a gradient (PE / EA = 30: 1) to afford title compound
11 (1.38 g, yield 82%) as a white solid. ESI-MS m/z [M + H]＋: 332.7. 1H NMR (300 MHz, CDCl3) δ 9.11 (s, 1H),
8.55 (d, J = 9.1 Hz, 1H), 8.19 (d, J = 9.1 Hz, 1H), 8.01 (s, 1H).
21

The Preparation of 4-bromo-6-nitroquinolin-2-ol (12)
In a 100 mL reaction flask, 2,4-dibromo-6-nitroquinoline (1.34 g, 4.05 mmol) was added and then dissolved
in acetic acid (25 mL). The reaction was heated at 90 °C and stirred overnight. The reaction mixture was slowly
poured into an ice-water mixture, and a large amount of white solid precipitated out. The suspension was
filtered, then filter cake was transferred, and vacuum dried to afforded the title compound 12 (1.03 g, 95%) as
a white solid. ESI-MS m/z [M + H]＋: 270.1. 1H NMR (300 MHz, DMSO-d6) δ 12.57 (s, 1H), 8.57 (d, J = 2.5 Hz, 1H),
8.41 (dd, J = 9.1, 2.5 Hz, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.23 (d, J = 1.7 Hz, 1H).
General Procedure A for the Synthesis of Compounds 13b—13f.
Method A-I: To a solution of compound 11 (1.2 g, 3.62 mmol) in DMF, appropriate primary or secondary
amines in DMF (1.0 mL) were added dropwise at 0 °C, under N2 atmosphere. Subsequently, Et3N (1.5 mL, 10.86
mmol) diluted with DMF (1.0 mL) was added in the same way. After the addition was completed, the mixture
was continuously stirred at this temperature for 2 h. The reaction mixture was concentrated under vacuum.
The residue was chromatographed, eluting with a gradient (DCM / MeOH = 40: 1) to afford title compound 13b
－13d, 13f (yield, 64%-88%).
Method A-II: Compound 12 (1.08 g, 4.00 mmol) was dissolved in DCM (60 mL), and stirred vigorously
under N2 atmosphere at 0 °C. NaH (60%, 480 mg) was added in batches within 5 min, and then stirred for 30
min after the addition. 1-(2-bromoethyl)piperidine (1.66 mL, 12 mmol) was added dropwise. The reaction
mixture was naturally warmed to room temperature and stirred overnight. After the reaction was complete,
the mixture was slowly poured into ice water to quench the excess NaH. The mixture was extracted with EA
(60 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under
vacuum. The residue was chromatographed, eluting with a gradient (PE / EA = 20 : 1) to afford title compound
13e (924mg, 60%) as a yellow solid. ESI-MS m/z [M + H]＋: 383.2. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (s, 1H),
8.35 – 8.28 (m, 2H),7.63 (s, 1H), 4.63 (d, J = 7.5 Hz, 2H), 3.61 (s, 4H), 3.23 – 3.18 (m, 2H), 2.77 (s, 4H).
4-bromo-N-methyl-6-nitroquinolin-2-amine (13b). Compound 13b was prepared according to general
procedure A-I on a 3.0 mmol scale. Purification by column chromatography (2.5% MeOH / DCM) afforded the
22

title compound (638.4 mg, 2.26 mmol, 75% yield). [M + H]+: 283.1. 1H NMR (300 MHz, DMSO-d6) δ 8.62 (d, J =
2.6 Hz, 1H), 8.26 (dd, J = 9.2, 2.7 Hz, 1H), 7.99 – 7.85 (m, 1H), 7.61 (d, J = 9.1 Hz, 1H), 7.29 (s, 1H), 2.95 (d, J =
4.7 Hz, 3H).
4-bromo-N-(2-methoxyethyl)-N-methyl-6-nitroquinolin-2-amine (13c) Compound 13c was prepared
according to general procedure A-I on a 4.0 mmol scale. Purification by column chromatography (2.5% MeOH
/ DCM) afforded the title compound (1.2 g, 3.53 mmol, 88% yield). [M + H]+: 341.1. 1H NMR (300 MHz, DMSO-
d6) δ 8.63 (d, J = 2.6 Hz, 1H), 8.25 (dd, J = 9.2, 2.7 Hz, 1H), 7.66 (s, 1H), 7.58 (d, J = 9.2 Hz, 1H), 3.94 – 3.88 (m,
2H), 3.59 (t, J = 5.4 Hz, 2H), 3.27 (s, 3H), 3.22 (s, 3H).
N1-(4-bromo-6-nitroquinolin-2-yl)-N1,N2,N2-trimethylethane-1,2-diamine (13d) Compound 13d was prepared
according to general procedure A-I on a 3.85 mmol scale. Purification by column chromatography (3% MeOH /
DCM) afforded the title compound (868.8 g, 2.46 mmol, 64% yield). [M + H]+: 354.2. 1H NMR (300 MHz, CDCl3)
δ 8.80 (d, J = 3.0 Hz, 1H), 8.22 (dd, J = 9.0, 3.0 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.21 (s, 1H), 3.78 (t, J = 7.5 Hz,
2H), 3.17 (s, 3H), 2.53 (t, J = 7.5 Hz, 2H), 2.28 (s, 6H).
4-bromo-2-(4-methylpiperazin-1-yl)-6-nitroquinoline (13f) Compound 13f was prepared according to general
procedure A-I on a 2.4 mmol scale. Purification by column chromatography (3% MeOH / DCM) afforded the
title compound (648.5 g, 1.84 mmol, 77% yield). [M + H]+: 352.3. 1H NMR (300 MHz, DMSO-d6) δ 8.64 (d, J = 2.6
Hz, 1H), 8.27 (dd, J = 9.2, 2.7 Hz, 1H), 7.84 (s, 1H), 7.60 (d, J = 9.2 Hz, 1H), 3.81 (t, J = 5.0 Hz, 4H), 2.42 (t, J = 5.0
Hz, 4H), 2.23 (s, 3H).
General Procedure B for the Synthesis of Compounds 14a—14f.
Starting materials 13a－13f (2 mmol, 1.0 equiv), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole-1-carboxylate (1.76 g, 6.0 mmol), PdCl2(dppf) (73.2 mg, 5% mmol), X-Phos (95.2 mg, 10% mmol),
K2CO3 (829.2 mmol, 6 mmol) were suspended in 24 mL dioxane / water (6 : 1), and stirred at 80 °C for 4.0 h
under N2 atmosphere. After completion of reaction, The mixture was extracted with EA (60 mL × 3), resulting
organic layers, which were concentrated under vacuum. The crude product was then transferred to a 50 mL
reaction flask and dissolved in 20 mL DCM. CF3COOH (0.37 mL, 5.0 mmol) was added dropwise to the reaction
23

mixture at room temperature, and after the addition, the mixture was stirred overnight. After the “-Boc”
protecting group was completely removed, the reaction mixture was poured into ice water. K2CO3 was used to
neutralize excess CF3COOH. The mixture was extracted with EA (60 mL × 3). The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated under vacuum. The residue was chromatographed,
eluting with a gradient of (DCM / MeOH = 30: 1) to afford title compounds 14a－14f ( yield, 65%-86%) as a
yellow solid.
6-nitro-4-(1H-pyrazol-4-yl)quinolin-2-ol (14a). Compound 14a was prepared according to general procedure B
on a 4.46 mmol scale. Purification by column chromatography (3% MeOH / DCM) afforded the title compound
(740.0 mg, 2.89 mmol, 65% yield). [M + Na]+: 279.1. 1H NMR (300 MHz, DMSO-d6) δ 13.44 (s, 1H), 12.31 (s, 1H),
8.63 (d, J = 2.5 Hz, 1H), 8.38 (dd, J = 9.1, 2.5 Hz, 2H), 7.98 (s, 1H), 7.51 (d, J = 9.1 Hz, 1H), 6.68 (d, J = 1.2 Hz, 1H).
N-methyl-6-nitro-4-(1H-pyrazol-4-yl)quinolin-2-amine (14b). Compound 14b was prepared according to
general procedure B on a 2.0 mmol scale. Purification by column chromatography (4% MeOH / DCM) afforded
the title compound (463.0 mg, 1.72 mmol, 86% yield). [M + H]+: 270.2. 1H NMR (300 MHz, DMSO-d6) δ 13.38
(s, 1H), 8.68 (d, J = 2.7 Hz, 1H), 8.24 (dd, J = 9.0, 2.7 Hz, 2H), 7.90 (s, 1H), 7.74 – 7.71 (m, 1H), 7.63 (d, J = 9.2 Hz,
1H), 6.86 (s, 1H), 2.98 (d, J = 6.0 Hz, 3H).
N-(2-methoxyethyl)-N-methyl-6-nitro-4-(1H-pyrazol-4-yl)quinolin-2-amine (14c). Compound 14c was
prepared according to general procedure B on a 3.55 mmol scale. Purification by column chromatography (4%
MeOH / DCM) afforded the title compound (880.2 mg, 2.69 mmol, 76 % yield). [M + H]+: 328.3. 1H NMR (300
MHz, DMSO-d6) δ 13.40 (s, 1H), 8.81 – 8.65 (m, 1H), 8.34 (d, J = 6.5 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 7.98 (d, J =
6.4 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.16 (t, J = 5.0 Hz, 1H), 3.92 (s, 2H), 3.60 (t, J = 5.2 Hz, 2H), 3.33 – 3.24 (m,
6H).
N1,N1,N2-trimethyl-N2-(6-nitro-4-(1H-pyrazol-4-yl)quinolin-2-yl)ethane-1,2-diamine (14d). Compound 14d
was prepared according to general procedure B on a 2.46 mmol scale. Purification by column chromatography
(9% MeOH / DCM) afforded the title compound (556.1 mg, 1.63 mmol, 66 % yield). [M + H]+: 341.4. 1H NMR
24

(300 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.74 (d, J = 2.6 Hz, 1H), 8.26 – 8.22 (m, 3H), 7.61 (d, J = 9.2 Hz, 1H), 7.13
(s, 1H), 3.86 (t, J = 6.5 Hz, 2H), 3.36 (s, 3H), 2.56 (t, J = 6.7 Hz, 2H), 2.25 (s, 6H).
4-(2-((6-nitro-4-(1H-pyrazol-4-yl)quinolin-2-yl)oxy)ethyl)morpholine (14e). Compound 14e was prepared
according to general procedure B on a 2.85 mmol scale. Purification by column chromatography (4% MeOH /
1
DCM) afforded the title compound (860.4 mg, 2.32 mmol, 82 % yield). [M + H]+: 370.3. H NMR (400 MHz,
DMSO-d6) δ 13.37 (s, 1H), 8.63 (s, 2H), 8.31 (s, 1H), 7.95 – 7.87 (m, 2H), 7.43 (s, 1H), 4.40 (d, J = 7.7 Hz, 2H),
3.58 (s, 4H), 3.10 – 3.08 (m, 1H), 2.58 (s, 4H), 1.20 (t, J = 7.4 Hz, 1H).
2-(4-methylpiperazin-1-yl)-6-nitro-4-(1H-pyrazol-4-yl)quinoline (14f). Compound 14f was prepared according
to general procedure B on a 2.0 mmol scale. Purification by column chromatography (4% MeOH / DCM)
afforded the title compound (493.8 mg, 1.46 mmol, 73 % yield). [M + H]+: 339.3. 1H NMR (300 MHz, DMSO-d6)
δ 13.39 (s, 1H), 8.75 (d, J = 2.6 Hz, 1H), 8.36 (s, 1H), 8.26 (dd, J = 9.2, 2.7 Hz, 1H), 8.01 (s, 1H), 7.65 (d, J = 9.2 Hz,
1H), 7.34 (s, 1H), 3.88 – 3.84 (m, 4H), 2.49 – 2.45(m, 4H), 2.26 (s, 3H).
General Procedure C for the Synthesis of Compounds 15a—15f.
To a solution of 6-nitro-4-(1H-pyrazol-4-yl) quinoline derivatives 14a－14f (1.4 mmol) in ethanol (9.6 mL)
and water (3.2 mL), Fe (783 mg, 14 mmol) and NH4Cl (748.8 mg, 14 mmol). After the reaction was refluxed at
90°C for 1.0 h, the reaction mixture was filtered through celite and the filter cake was washed with MeOH. The
filtrate was concentrated under vacuum, the resulting residue was extracted with EA (60 mL × 3), and the
organic phases were combined, dried over Na2SO4, and concentrated in vacuo to afford corresponding crude
4-(1H-pyrazol-4-yl)quinolin-6-amine derivatives 15a－15f, which were used without further purification.
General Procedure D for the Synthesis of Compounds 16a—16f.
To the CH3CN (3.0 mL) solution of compounds 15a－15f (1.0 mmol) was added p-toluenesulfonic acid
(516.6 mg, 3.0 mmol) to obtain a suspension. After cooling to 0 oC, an aqueous solution (0.73 mL) of sodium
nitrite (138.0 mg, 2.0 mmol) and KI (414.9 mg, 2.5 mmol) was slowly added dropwise to the reaction system
(during dropping process, the reaction temperature was controlled below 5°C). After the addition, the reaction
mixture was naturally warmed to room temperature and stirred for 2.0 hours. The reaction solution was
25

quenched with an aqueous solution of Na2S2O3 (4.0 mL). The mixture was extracted with CH2Cl2 (50 mL × 3).
The combined organic layer was washed with brine, dried over Na2SO4 and concentrated under vacuum. The
residue was chromatographed, eluting with a gradient of (DCM / MeOH = 30: 1) to afford title compounds 14a
－14f ( yield, 46%-82%) as a yellow solid.
6-iodo-4-(1H-pyrazol-4-yl)quinolin-2-ol (16a). Compound 16a was prepared according to general procedure D
on a 1.0 mmol scale. Purification by column chromatography (4% MeOH / DCM) afforded the title compound
(209.0 mg, 0.62 mmol, 62 % yield). [M + H]+: 338.2. 1H NMR (300 MHz, DMSO-d6) δ 13.33 (s, 1H), 11.84 (s, 1H),
8.02 (d, J = 1.9 Hz, 3H), 7.83 (dd, J = 8.6, 1.9 Hz, 1H), 7.19 (d, J = 8.6 Hz, 1H), 6.51 (s, 1H).
6-iodo-N-methyl-4-(1H-pyrazol-4-yl)quinolin-2-amine (16b). Compound 16b was prepared according to
general procedure D on a 1.0 mmol scale. Purification by column chromatography (4% MeOH / DCM) afforded
the title compound (198.5 mg, 0.57 mmol, 57 % yield). [M + Na]+: 373.1. 1H NMR (300 MHz, DMSO-d6) δ 13.26
(s, 1H), 8.17 (s, 1H), 8.06 (d, J = 2.1 Hz, 1H), 7.79 (s, 1H), 7.72 (dd, J = 8.7, 2.1 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H),
7.13 (s, 1H), 6.71 (s, 1H), 2.91 (d, J = 4.6 Hz, 3H).
6-iodo-N-(2-methoxyethyl)-N-methyl-4-(1H-pyrazol-4-yl)quinolin-2-amine (16c). Compound 16c was
prepared according to general procedure D on a 1.0 mmol scale. Purification by column chromatography (4%
MeOH / DCM) afforded the title compound (332.7 mg, 0.82 mmol, 82 % yield). [M + H]+: 409.3. 1H NMR (300
MHz, CDCl3) δ 8.07 (d, J = 2.0 Hz, 1H), 7.81 (s, 2H), 7.67 (dd, J = 8.8, 2.0 Hz, 1H), 7.44 – 7.41 (m, 1H), 6.78 (s,
1H), 3.83 (t, J = 6.0 Hz, 2H), 3.60 (t, J = 6.0 Hz, 2H), 3.30 (s, 3H), 3.19 (s, 3H).
N1-(6-iodo-4-(1H-pyrazol-4-yl)quinolin-2-yl)-N1,N2,N2-trimethylethane-1,2-diamine (16d). Compound 16d
was prepared according to general procedure D on a 1.0 mmol scale. Purification by column chromatography
(8% MeOH / DCM) afforded the title compound (267.6 mg, 0.64 mmol, 64 % yield). [M + H]+: 422.1. 1H NMR
(300 MHz, DMSO-d6) δ 13.31 (s, 1H), 8.19– 8.12(m, 3H), 7.82 – 7.78 (m, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.05 (s,
1H), 4.06 (d, J = 6.2 Hz, 2H), 3.38 (d, J = 6.1 Hz, 2H), 3.18 (s, 3H), 2.92 (s, 6H).
4-(2-((6-iodo-4-(1H-pyrazol-4-yl)quinolin-2-yl)oxy)ethyl)morpholine (16e). Compound 16e was prepared
according to general procedure D on a 1.0 mmol scale. Purification by column chromatography (7% MeOH /
26

1
DCM) afforded the title compound (274.8 mg, 0.61 mmol, 61 % yield). [M + H]+: 451.2. H NMR (300 MHz,
DMSO-d6) δ 13.36 (s, 1H), 8.26 (s, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.96 – 7.84 (m, 2H), 7.45 (d, J = 9.0 Hz, 1H), 6.62
(s, 1H), 4.38 (t, J = 7.1 Hz, 2H), 3.56 (t, J = 4.6 Hz, 4H), , 3.37 (s, 4H) , 2.56 (d, J = 7.1 Hz, 2H).
6-iodo-2-(4-methylpiperazin-1-yl)-4-(1H-pyrazol-4-yl)quinoline (16f). Compound 16f was prepared according
to general procedure D on a 1.0 mmol scale. Purification by column chromatography (8% MeOH / DCM)
afforded the title compound (193.7 mg, 0.46 mmol, 46 % yield). [M + H]+: 420.2. 1H NMR (300 MHz, DMSO-d6)
δ 13.30 (s, 1H), 8.16 (d, J = 3.0 Hz, 3H), 7.77 (dd, J = 9.0, 3.0 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.18 (s, 1H), 3.72
(t, J = 4.5 Hz, 4H), 2.43 (t, J = 4.5 Hz, 4H), 2.23 (s, 3H).
General Procedure E for the Synthesis of Compounds 17a—17f.
To a suspension containing 16e—16f (1.0 equiv), PdCl2 (PPh3)2 (5% mmol), CuI (10% mmol) in THF / Et3N
= 3: 1 (total concentration: 0.2 M), the corresponding terminal alkynes in THF (1 mL) was added dropwise. In
some cases, anhydrous DMF (2 mL) was added to the reaction system to keep the starting materials completely
dissolved. After the reaction was completed, the mixture was evaporated in vacuo. The residue was
chromatographed, eluting with a gradient of (DCM / MeOH = 15: 1) to afford title compounds 17a －17f
( yield,31%-54%) as a yellow solid.
6-(3-hydroxy-3-(thiazol-2-yl)but-1-yn-1-yl)-4-(1H-pyrazol-4-yl)quinolin-2-ol (17a). Compound 17a was
prepared according to general procedure E on a 0.4 mmol scale. Purification by column chromatography (4%
MeOH / DCM) afforded the title compound (52.1 mg, 0.14 mmol, 36% yield). White solid; mp 285-287 oC. HPLC
analysis: retention time, 2.978 min; peak area, 98.46%. 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 11.93 (s,
1H), 8.96 (s, 1H), 8.24 (s, 1H), 7.87 (s, 1H), 7.76 – 7.66 (m, 2H), 7.56 (dd, J = 8.5, 1.9 Hz, 1H), 7.36 (d, J = 8.5 Hz,
1H), 7.02 (s, 1H), 6.52 (s, 1H), 1.85 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 176.43, 161.80, 143.19, 143.01,
139.81, 133.62, 129.41, 121.06, 120.90, 118.74, 116.80, 116.34, 115.47, 92.19, 83.14, 68.34, 31.97. ESI-MS m/z:
363.2 [M + H]＋.
4-(2-(methylamino)-4-(1H-pyrazol-4-yl)quinolin-6-yl)-2-(thiazol-2-yl)but-3-yn-2-ol (17b). Compound 17b was
prepared according to general procedure E on a 0.4 mmol scale. Purification by column chromatography (5%
27

MeOH / DCM) afforded the title compound (61.2 mg, 0.16 mmol, 41% yield). White solid; mp 218-221 oC. HPLC
analysis: retention time, 3.544 min; peak area, 99.69%. 1H NMR (600 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.17 (s,
1H), 7.79 (dd, J = 20.5, 2.6 Hz, 3H), 7.67 (d, J = 3.3 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.45 (dd, J = 8.6, 1.9 Hz, 1H),
7.20 (q, J = 3.9 Hz, 1H), 6.99 (s, 1H), 6.73 (s, 1H), 2.92 (d, J = 4.7 Hz, 3H), 1.87 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 176.64, 158.32, 158.25, 149.12, 143.00, 139.49, 131.95, 128.91, 126.88, 121.85, 120.86, 117.30,
117.26, 114.47, 113.03, 91.94, 84.17, 68.38, 32.03, 28.05. ESI-MS m/z: 376.3 [M + H]＋.
4-(2-((2-methoxyethyl)(methyl)amino)-4-(1H-pyrazol-4-yl)quinolin-6-yl)-2-(thiazol-2-yl)but-3-yn-2-ol (17c).
Compound 17c was prepared according to general procedure E on a 0.4 mmol scale. Purification by column
chromatography (5% MeOH / DCM) afforded the title compound (54.2 mg, 0.12 mmol, 31% yield). White solid;
mp 138-140 oC. HPLC analysis: retention time, 3.807 min; peak area, 98.25%. 1H NMR (400 MHz, DMSO-d6) δ
13.28 (s, 1H), 8.08 – 7.88 (m, 3H), 7.78 (d, J = 3.2 Hz, 1H), 7.68 (d, J = 3.2 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.02 (d, J
= 7.2 Hz, 2H), 3.86 (t, J = 4.0 Hz, 2H), 3.57 (t, J = 6.0Hz, 2H), 3.27 (s, 3H), 3.19 (s, 3H), 1.86 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 176.60, 157.24, 143.00, 140.50, 132.09, 128.89, 127.20, 121.01, 120.86, 117.74, 114.68,
109.69, 92.08, 84.16, 79.64, 70.44, 68.38, 58.67, 49.23, 37.22, 32.02. ESI-MS m/z: 434.4 [M + H]＋.
4-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-(1H-pyrazol-4-yl)quinolin-6-yl)-2-(thiazol-2-yl)but-3-yn-2-
ol (17d). Compound 17d was prepared according to general procedure E on a 0.2 mmol scale. Purification by
column chromatography (8% MeOH / DCM) afforded the title compound (43.8 mg, 0.1 mmol, 49% yield). White
solid; mp 177-179 oC. HPLC analysis: retention time, 4.228 min; peak area, 96.72%. 1H NMR (300 MHz, DMSO-
d6) δ 11.78 (s, 1H), 8.66 (s, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.76 (d, J = 3.3 Hz, 1H), 7.67 (d, J = 3.2 Hz, 1H), 7.25 –
7.22 (m, 1H), 7.11 (s, 2H), 6.47 (dd, J = 3.5, 1.7 Hz, 1H), 3.16 (d, 4H), 2.59 (d, J = 5.7 Hz, 4H), 2.26 (s, 3H), 1.86
(s, 3H), 1.83 – 1.77 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 176.63, 154.20, 151.54, 151.33, 147.72, 142.97,
132.19, 127.83, 127.58, 122.94, 120.85, 120.74, 115.19, 103.82, 98.42, 91.70, 83.80, 68.35, 58.64, 56.99, 54.00,
53.12, 47.00, 32.04, 28.40. ESI-MS m/z: 447.6 [M + H]＋.
4-(2-(2-morpholinoethoxy)-4-(1H-pyrazol-4-yl)quinolin-6-yl)-2-(thiazol-2-yl)but-3-yn-2-ol (17e). Compound
17e was prepared according to general procedure E on a 0.25 mmol scale. Purification by column
28

chromatography (8% MeOH / DCM) afforded the title compound (46.4 mg, 0.1 mmol, 39% yield). White solid;
mp 199-201 oC. HPLC analysis: retention time, 3.508 min; peak area, 96.97%. 1H NMR (300 MHz, DMSO-d6) δ
13.36 (s, 1H), 8.08 – 7.83 (m, 3H), 7.78 (d, J = 3.0 Hz, 1H), 7.68 – 7.63 (m, 3H), 7.07 (s, 1H), 6.63 (s, 1H), 4.39 (t,
J = 6.0 Hz, 2H), 3.56 (t, J = 4.5 Hz, 4H), 3.37 (s, 4H), 2.57 (d, J = 7.0 Hz, 2H), 1.87 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 160.88, 142.26, 139.37, 133.84, 129.87, 120.39, 120.13, 116.52, 116.20, 116.11, 96.33, 80.13,
64.10, 55.71, 53.90, 46.01, 32.05, 9.02. ESI-MS m/z: 476.5 [M + H]＋.
4-(2-(4-methylpiperazin-1-yl)-4-(1H-pyrazol-4-yl)quinolin-6-yl)-2-(thiazol-2-yl)but-3-yn-2-ol (17f). Compound
17f was prepared according to general procedure E on a 0.2 mmol scale. Purification by column
chromatography (8% MeOH / DCM) afforded the title compound (48.1 mg, 0.11 mmol, 54% yield). White solid;
mp 138-140 oC. HPLC analysis: retention time, 4.129 min; peak area, 98.51%. 1H NMR (300 MHz, DMSO-d6) δ
13.27 (s, 1H), 8.23 (s, 1H), 7.88 (d, J = 3.0 Hz, 2H), 7.76 (d, J = 3.0 Hz, 1H), 7.67 (d, J = 3.0 Hz, 1H), 7.55 (d, J = 9.0
Hz, 1H), 7.50 – 7.47 (m, 1H), 7.19 (s, 1H), 6.99 (s, 1H), 3.73 (s, 4H), 2.42 (s, 4H), 2.23 (s, 3H), 1.87 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 176.58, 157.67, 148.37, 143.01, 140.85, 132.12, 128.87, 127.50, 121.49, 120.89,
117.63, 115.33, 110.02, 92.29, 84.07, 68.38, 55.00, 46.25, 44.73, 32.01. ESI-MS m/z: 445.7 [M + H]＋ .The
preparation of compound 20 was in accordance with relevant known literature38, 39
.
6-nitroquinolin-4-ol (22). Compound 22 was prepared according to the synthetic method of compound 10 on
a 60 mmol scale. Purification by column chromatography (10% EA / PE) afforded the title compound (5.13 g,
27 mmol, 45% yield). [M + H]+: 191.0. 1H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 8.84 (d, J = 2.6 Hz, 1H), 8.42
(dd, J = 9.2, 2.7 Hz, 1H), 8.05 (d, J = 4.7 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 6.20 (d, J = 4.7 Hz, 1H).
6-iodo-4-(1H-pyrazol-4-yl)quinoline (23). Compound 23 was prepared according to the synthetic method of
compound 16 on a 4.0 mmol scale. Purification by column chromatography (6% MeOH / DCM) afforded the
title compound (899.1 mg, 2.8 mmol, 70% yield). [M + H]+: 322.1. 1H NMR (300 MHz, DMSO-d6) δ 13.40 (s, 1H),
8.89 (d, J = 4.5Hz, 1H), 8.52 (t, J = 1.5 Hz, 1H), 8.36 (s, 1H), 8.11 – 7.91 (m, 2H), 7.84 (dd, J = 8.8, 1.1 Hz, 1H),
7.55 (d, J = 4.5, Hz, 1H).
29