Optimization and biological evaluation of nicotinamide derivatives as Aurora kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.016

Aurora kinases are known to be overexpressed in various solid tumors and implicated in oncogenesis and tumor progression. A series of nicotinamide derivatives were synthesized and their biological activities were evaluated, including kinase inhibitory act

Full text of DOI:10.1016/j.bmc.2019.07.016

Accepted Manuscript
Optimization and Biological Evaluation of Nicotinamide Derivatives as Aurora
Kinase Inhibitors
Baohui Qi, Xingwei Xu, Ying Yang, Huan He, Xupeng Yue
PII:
S0968-0896(19)30874-0
https://doi.org/10.1016/j.bmc.2019.07.016
BMC 15000
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 June 2019
2 July 2019
4 July 2019
Please cite this article as: Qi, B., Xu, X., Yang, Y., He, H., Yue, X., Optimization and Biological Evaluation of
Nicotinamide Derivatives as Aurora Kinase Inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.07.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Optimization and Biological Evaluation of Nicotinamide Derivatives as Aurora Kinase
Inhibitors
Baohui Qi*, Xingwei Xu, Ying Yang, Huan He, Xupeng Yue
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, Guangdong Province,
China
Abstract: Aurora kinases are known to be overexpressed in various solid tumors and implicated
in oncogenesis and tumor progression. A series of nicotinamide derivatives were synthesized and
their biological activities were evaluated, including kinase inhibitory activity against Aur A and
Aur B and in vitro antitumor activity against SW620, HT-29, NCI-H1975 and Hela cancer cell
lines. In addition, the study of antiproliferation, cytotoxicity and apoptosis was performed
meanwhile. As the most potent inhibitor of Aur A, 4-((3-bromo-4-fluorophenyl)amino)-6-chloro-
N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)nicotinamide (10l) showed excellent
antitumor activity against SW620 and NCI-H1975 with IC50 values were 0.61 and 1.06 μM,
while the IC50 values of reference compound were 3.37 and 6.67 μM, respectively. Furthermore,
binding mode studies indicated that compound 10l forms better interaction with Aur A.
Keywords: Antitumor; Aurora kinases; Kinase inhibitors; nicotinamide; SAR
1. Introduciton
Protein kinases (PKs) catalyzing phosphorylation of serine, threonine, or tyrosine residues of
predetermined target proteins are deregulated in numerous diseases [1]. As an important subclass
of PKs, Aurora kinase family could phosphorylate serine/threonine amino acid residues and is
involved in the regulation of various stages of mitosis, such as centrosome maturation, mitotic
1

spindle formation, chromosome segregation and cytokinesis during mitosis [2,3].
Aurora kinase family includes three Aurora kinase isoforms designated as Aurora A (Aur A),
Aurora B (Aur B) and Aurora C (Aur C), which have quite different and nonoverlapping
functions in mitotic processes [4-7]. Aur A plays a pivotal role in centrosome maturation, the
mitotic spindle assembly and entry into mitosis [8,9]. Aur B is essential for chromosome
condensation, chromosome orientation on the mitotic spindle and the spindle-assembly
checkpoint, as well as the final stages of cytokinesis [10,11]. Aur C which appears to have
overlapping functions with Aur B during mitotic cell division process is a chromosomal
passenger protein [12].
All three Aurora kinases are known to be overexpressed in various solid tumors and implicated
in oncogenesis and tumor progression. However, recent study suggested that the sub-type
selective Aur A inhibitors might have advantages over pan-selective Aurora kinase inhibitors, by
avoiding Aur B-mediated neutropenia [13]. Nevertheless, Aurora kinases have also become
promising antitumor targets, and a number of structurally diverse small molecular inhibitors have
been reported already, including pyrimidine derivative Tozasertib (VX-680), pyrimidoazepine
derivative Alisertib (MLN8237), 3-aminopyrazole derivative Danusertib (PHA-739358),
phthalazine derivative AMG900 and quinazoline derivative Barasertib (AZD1152) (Fig. 1) [14-
19].
O
N
NH
N
N
N
NH
N
O
HN
N
HN
N
H
O
N
F
N
H
N
N
O
Cl
O
N
S
O
N
N
COOH
Danusertib (PHA-739358)
Tozasertib (VX-680)
Alisertib (MLN8237)
N
O
N
N
H2N
N
HO
N
S
HN
O
N
N
NH
HN
N
N
N
H
O
F
AMG900
Barasertib (AZD1152)
2

Fig. 1 The structures of representative Aurora kinase inhibitors.
Nicotinamide and its derivates, known as a scaffold with multiple biological activities, were
widely used in drug design, such as tissue-nonspecific alkaline phosphatase (TNAP) inhibitors,
acetylcholinesterase inhibitors, psychiatric and neurodegenerative agents, and anticancer agents,
etc [20-24]. Based on the structure activity relationship (SAR) of type Ⅱ c-Met inhibitors [25,
26], nicotinamide fragments were introduced into Cabozantinib in our previous work.
Unfortunately, the c-Met inhibitory activity of designed compounds was not satisfactory (data not
shown). To our delight, their in vitro antitumor activity against various cancer cell lines was more
potent than that of Cabozantinib. Kinase profile study indicated that they were potent Aurora
kinase inhibitors. In this present article, more nicotinamide derivates were synthesized and
evaluated for their antitumor activity. In addition, detailed SAR and antitumor mechanism were
disclosed concurrently (Fig. 2).

 H
H
H
N
N
A
R2
N
O
O
O
HN
O
N
F
O
N
R1
O
O
O
O
R1 = H, F, Cl, Br, etc;
R2 = H, F;
A = 2-chloropyridine, pyridinone,N-methylpyridinone, etc.
Fig. 2 Design of target compound derived from Cabozantinib.
2. Results and Discussion
2.1. Chemistry
As shown in Scheme 1, selective acid-catalyzed displacement of the 4-chlorine atom of ethyl
4,6-dichloronicotinate with different substituted aromatic amines to yield 4-arylamino-6-
chloropyridine esters 1a-k [27]. The N-methylpyridone ester 3 were prepared by quaternization
of the ethyl 6-chloro-4-(phenylamino)nicotinate 1a with dimethylsulfate followed by in situ
treated with a mixture of AcOH, TEA and EtOH [28]. Hydrolysis of obtained ethyl esters under
basic conditions gave the acid 4 which were coupled with amine 8a by HATU and DIPEA in
3

DMF to afford target compound 9b (Scheme 3). The 6-chloro-4-anilinonicotinate 1a was
converted to the desired intermediate 5 by refluxing in AcOH/H2O [29]. The synthesis of target
compounds 9a, 10a-l and 11-13 was carried out in a manner similar to that of compound 9b
(Scheme 2 and Scheme 3).
R1
R1
Cl
N
NH
NH2
NH
COOEt
ii
i
COOH
COOEt
Cl
R1
Cl
N
2a-k
Cl
N
1a-k
NH
NH
ii
COOEt
iii
COOH
NH
COOEt
O
N
O
N
1a, 2a:
1b, 2b:
1c, 2c:
1d, 2d:
1e, 2e:
R
R
R
R
1 = H;
1 = 3-F;
1 = 4-F;
1 = 2-F;
1 = 2-Cl;
1 = 3-Cl;
CH3
CH3
Cl
N
3
(R1 = H)
4
(R1 = H)
1a
R
1f, 2f:
R
NH
NH
ii
iv
1g, 2g:
1h, 2h:
R
R
1 = 4-Cl;
1 = 2-Br;
NH
COOEt
COOH
COOEt
1i, 2i:
1j, 2j:
1k, 2k:
R
R
R
1 = 3-Br, 4-F;
1 = 2-F, 4-Br;
1 = Ph.
O
N
H
5
O
N
Cl
N
H
1a
6
Scheme 1. The synthesis of intermediates 2a-k, 4 and 6. Reagents and condition: i) EtOH, concd HCl
(cat.), 90 °C, 15 h; ii) NaOH, EtOH, 50 °C, 6 h; iii) a. Me2SO4, CHCl3, 0 °C to reflux, b. TEA, AcOH,
EtOH, reflux 4 h; iv) AcOH, H2O, reflux, 48 h.
10a:
10b:
10c:
10d:
10e:
10f:
10g:
10h:
10i:
R
R
R
R
R
R
1 = H, R2 = H;
1 = 3-F, R2 = H;
1 = 4-F, R2 = H;
1 = 2-F, R2 = H;
1 = 2-Cl, R2 = H;
1 = 3-Cl, R2 = H;
N
Cl
R2
O
NH2
R2
O
NO2
ii
H
R2
O
N
Cl
N
O
O
i
O
HN
iii
O
O
O
O
R1
O
O
R1 = 4-Cl, R2 = H;
R1 = 2-Br, R2 = H;
R1 = 3-Br, 4-F, R2 = H;
R1 = 2-F, 4-Br, R2 = H;
R1 = Ph, R2 = H;
N
8a:
8b:
N
7a:
7b:
N
R2 = H;
R2 = F.
R
R
2 = H;
2 = F.
10a-l
10j:
10k:
10l:
R1 = 3-Br, 4-F, R2 = F.
Scheme 2. The synthesis of intermediates10a-l. Reagents and condition: i) 4-nitrophenol or 2-fluoro-4-
nitrophenol, PhCl, DIPEA, reflux, 13 h; ii) Fe, 90% EtOH-H2O, AcOH, reflux, 4 h; iii) 2a-k, HATU,
DIPEA, DMF, rt, 2 h.
4

H
A
R2
O
NH2
R2
O
N
O
HN
R1
i
O
O
O
O
N
N
8a, 8b
9a, 9b, 11-13
H
N
N
O
N
O
9a:
R
1 = H, R2 = H, A =
9b:
R
1 = H, R2 = H, A =
11:
R
1 = 3-Br, 4-F, R2 = F, A =
Br
Cl
N
12:
R1 = 3-Br, 4-F, R2 = F, A =
13:
R
1 = 3-Br, 4-F, R2 = F, A =
N
Scheme 3. The synthesis of intermediates 9a-b and 11-13. Reagents and condition: i) 4,6 or carboxylic
acid;, HATU, DIPEA, DMF, rt, 2 h.
2.2. Biological evaluation and Structure activity relationship
Taking Cabozantinib as reference compound, in vitro kinase assay was performed at a single-
dose concentration of 0.2 μM over Aur A and Aur B by mobility shift assay. In vitro antitumor
activity against SW620, a cancer cell line usually used in the study of developing Aurora kinase
inhibitors, was identified by MTT assay (Table 1). In general, four compounds (10h-j and 10l)
showed high potency over 70% inhibitory rate against Aur A, and most of these compounds were
less inhibitive against Aur B (13.7–57.1% inhibition at 0.2 μM).
Preliminary modification of the ring A was carried on by introducing 2-pyridinone 9a, N-
methylpyridinone 9b and 2-chloropyridine 10a into the target compounds. As can be seen in
Table 1, 10a exhibited more potent kinase inhibitory activity and cellular activity among the three
compounds. Therefore, more 2-chloropyridine derivates 10b-l were synthesized and evaluated for
their biological activity in order to disclose the influence of R1 substitutions on terminal phenyl
ring. The biological results indicated that halo substituted phenyl ring led to better activity,
especially in o- or m-position. In comparison with fluoro and chloro analogues 10b-g, bromo
analogues 10h-j showed a considerable improvement in both kinase inhibitory activity and
cellular activity.
Modification came to the central benzyl ring (R2) in the following work. As shown in table 1,
incorporation of fluorine led to over 2-fold improved antitumor activity and slightly increase in
Aur A inhibitory potency (10l vs 10i).
5

With these preferred groups at R1 (R1 = 3-Br, 4-F) and R2 (R2 = F) in place, additional
structural variations were introduced to the heterocyclic core A. The Aur A potency drastically
declined due to the replacing of 2-chloropyridine 10l with 6-bromoquinoline 11 or 2-chloro-6-
methylpyridine 13, suggesting that incorporation of bulky groups in ring A was unprofitable.
Table 1. Enzyme inhibitory activity and in vitro antitumor activity against human colon cancer
cell SW620.
H
A
R2
O
N
O
HN
R1
O
O
N
Inh@0.2 μM (%)a
SW620
Compd.
9a
A
R1
R2
H
IC50 (μM)b
Aur A
Aur B
H
N
O
H
H
29.9
38.7
18.9 ± 1.95
4.43 ± 0.42
N
O
9b
H
40.9
31.1
N
N
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
10a
10b
10c
10d
10e
10f
H
H
H
H
H
H
H
H
H
48.2
48.6
47.1
51.5
68.3
55.1
51.9
78.2
52.1
49.6
51.6
50.1
57.1
31.9
38.5
56.1
1.92 ± 0.18
5.10 ± 0.46
6.82 ± 0.55
8.10 ± 0.76
3.60 ± 0.31
4.33 ± 0.42
2.38 ± 0.21
6.90 ± 0.62
3-F
4-F
2-F
2-Cl
3-Cl
4-Cl
2-Br
10g
10h
6

N
N
N
N
Cl
Cl
Cl
Cl
10i
10j
10k
10l
3-Br, 4-F
2-F, 4-Br
Ph
H
H
H
F
81.3
70.0
59.9
82.9
30.1
31.6
46.3
30.5
1.33 ± 0.12
2.45 ± 0.20
NDc
3-Br, 4-F
0.61 ± 0.046
N
Br
11
12
3-Br, 4-F
3-Br, 4-F
F
F
6.6
13.7
40.7
ND
N
48.4
1.86 ± 0.16
Cl
N
13
3-Br, 4-F
-
F
-
0.6
0.4
17.7
38.3
ND
Cabozantinib
-
3.37 ± 0.31
a)The results are mean values of two independent determinations. b) The values were an average of three
separate determinations and standard deviations were shown. c) Not determined.
In order to further study of in vitro antitumor activity, more potent compounds 10h, 10i, 10j
and 10l were selected and the in vitro antitumor activity against HT-29, NCI-H1975 and Hela
cancer cell lines were evaluated. Generally, all the four compounds exhibited less antitumor
activity against HT-29, NCI-H1975 and Hela compared with that of SW620 (Table 2).
Compound 10l showed acceptable activity against NCI-H1975 cell lines with IC50 of 1.06 μM
which was approximate 6-fold more potent than that of Cabozantinib (IC50 = 6.67 μM).
Table 2. In vitro antitumor activity of selected compounds against HT-29, NCI-H1975 and Hela
cancer cell lines.
IC50 (μM)a
Compd.
HT-29
>20
NCI-H1975
8.00 ± 0.67
3.54 ± 0.29
NDb
Hela
>20
>20
>20
>20
10h
10i
10j
10l
>20
>20
>20
1.06 ± 0.09
7

Cabozantinib
9.6 ± 0.74
6.67 ± 0.58
>20
a) The values were an average of three separate determinations and standard deviations were shown. b) Not
determined.
2.3. Effects on proliferation
IncuCyte live-cell imaging assay was carried on to evaluate whether potent compound 10i and
10l could inhibit proliferation of cancer cell. As shown in Fig. 3 and Fig. 4, similar inhibition in
cancer cell proliferation was induced by compound 10i (confluence 42.6%) and Cabozantinib (confluence
40.9%) at the concentration of 10.0 μg/mL. However, compound 10l showed less antiproliferative activity
at the same concentration. Lower concentrations (3.33, 1.11, 0.37 and 0.12 μg/mL) of compounds 10i, 10l
and Cabozantinib could not induce antiproliferative activity significantly (data not shown).
Fig. 3 The curves of antiproliferative activity against SW620 cancer cells. The cell confluence was
monitored for 84 h using an IncuCyte ZOOM system in an incubator. SW620 cancer cells were incubated
with 0.1% DMSO or exposed to compound 10i, 10l or Cabozantinib at different concentration.
8

Fig. 4 Phase contrast images (white light) of SW620 cancer cells after 84 h of treatment with
Cabozantinib (10.0 μg/mL), compound 10i (10.0 μg/mL) or not (control). The confluence was 86.5% in
control group. Red fluorescent cells were counted as dead cell.
2.4. Effects on cytotoxicity
In order to compare the effects of target compounds and Cabozantinib upon the kinetics of
cytotoxicity against SW620 cell line, compounds 10i and 10l were selected and real-time
IncuCyte live-cell imaging assay was performed. In general, both compounds showed more
excellent cytotoxicity than that of Cabozantinib in a dose- and time-dependent manner (Fig. 5).
As can be seen in Fig. 5 and Fig. 6, significant cytotoxicity was emerged after being treated with
compound 10l (10.0 μg/mL) for 5 hours, and the number of dead cells reached the maximum at
approximate 36 h.
9

Fig. 5 The kinetics of cytotoxicity study in SW620 cancer cells. The number of dead cells was being
monitored for 84 h using an IncuCyte ZOOM system in an incubator. SW620 cancer cells were incubated
with 0.1% DMSO or exposed to compound 10i, 10l or Cabozantinib at different concentration.
Fig. 6 The images (red light) of study on cytotoxicity against SW620 cancer cells after treatment with
compound 10l (10.0 μg/mL), Cabozantinib (10.0 μg/mL) or not (control) for 36 h. Red fluorescent cells
were counted as dead cells.
2.5. Effects on Apoptosis
In comparison with compound 10i and Cabozantinib, the number of apoptotic cells was
significantly increased from 7 h to 24 h after being treated with compound 10l (10.0 μg/mL).
Lower concentration (3.33 μg/mL) resulted in a loss of potency in apoptosis. These results
indicated that target compounds induced apoptosis in a time- and dose-dependent manner in
SW620 cancer cells.
10

Fig. 7 The kinetic study of apoptosis in SW620 cancer cells. Apoptotic cell death was being monitored for
48 h using an IncuCyte ZOOM system in an incubator. SW620 cancer cells were incubated with 0.1%
DMSO or exposed to compound 10i, 10l or Cabozantinib at different concentration.
Fig. 8 The images (green light) of study on apoptosis against SW620 cancer cells after treatment with
compound 10l (10.0 and 3.33 μg/mL), Cabozantinib (10.0 and 3.33 μg/mL) or not (control) for 36 h.
Green fluorescent cells were counted as dead cells induced by apoptosis.
2.6. Docking study
11

In order to determine the binding mode of this series of inhibitors, the most potent compound
10l was selected and molecular docking analysis was performed by Molecular Operating
Environment (MOE, Chemical Computing Group Inc., Canada). The binding results indicated
that compound 10l occupies the ATP-binding site with the activation loop in a DFG-in
conformation (Fig. 9B and 9D). Three H-bonds could be found in the binding mode: one was
established between the quinoline scaffold and Ala213 in the hinge region of the kinase, another
was between the NH (PhNH-) and Asp274, a third was between NH (-NHCO-) and Asp274 (Fig.
9C and 9D). In addition, the binding affinity was strengthened by arene-H interaction between
quinoline and Leu139. These key interactions might contribute to the high in vitro potency of
compound 10l.
Fig. 9 Docked binding modes of compound 10l with Aur A (PDB entry 2X6D). The compound was
shown by blue sticks, the H-bonds were represented by green dotted lines, and the arene-H and interaction
were shown by red dotted lines.
3. Experimental
3.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used
without further purification. 1H NMR and 13C NMR spectra were generated on Bruker ARX-400
12

and Bruker ARX-100 spectrometers (Bruker Bioscience, Billerica, MA, USA). Column
chromatography was carried out on silica gel (200-300 mesh). High Resolution Mass spectra
(HRMS) were taken in ESI mode on Agilent 6520 LC-HRMS (Agilent, Palo Alto, CA, USA).
3.1.1. Ethyl 6-chloro-4-(phenylamino)nicotinate (1a)
To a stirred a solution of ethyl 4,6-dichloronicotinate (2.0 g, 9.1 mmol) in EtOH (30 mL),
aniline (0.91 g, 10.0 mmol) and 2 drops of concd HCl were added. The mixture was refluxed for
16 h, and then the solution was cooled, whereupon the desired product crystallized out of solution.
The product was filtrated and washed with cold EtOH to give desired compound as white solid
(1.2 g, 47.2%). HRMS (ESI) m/z: 277.0703 [M+H]+, calcd. for 277.0744.
Taking different aromatic amines as starting materials, intermediates 1b-k were prepared by
the same procedure as 1a.
3.1.2. 6-Chloro-4-(phenylamino)nicotinic acid (2a)
A solution of NaOH (0.52 g, 12.9 mmol) in water (20 mL) was added to the mixture of 1a (1.2
g, 4.3 mmol) in EtOH (24 mL), and the suspension was stirred for 5 h at 50 ℃. EtOH was
evaporated under reduced pressure, the pH was adjusted to 3 with diluted HCl, and the solid
residue obtained was filtered to afford 2a (0.92, 86.3%). 1HNMR (400 MHz, DMSO-d6) δ 9.46 (s,
1H), 8.15 (s, 1 H), 7.29-7.34 (m, 4H), 7.21-7.25 (m, 1H), 6.91 (s, 1H). HRMS (ESI) m/z:
247.0235 [M-H]-, calcd. for 247.0274.
Intermediates 2b-k, 4 and 6 were prepared by the same procedure as 2a.
3.1.3. Ethyl 1-methyl-6-oxo-4-(phenylamino)-1,6-dihydropyridine-3-carboxylate (3)
To a solution of intermediate 1a (1.0 g, 3.6 mmol) in CHCl3 (25 mL), dimethyl sulfate (2.0 mL,
21.6 mmol) was added dropwise at 0 ℃.The solution was allowed to warm to rt and then heated
at reflux for 18 h. After cooling to rt, a mixture of triethylamine (10.6 mL), acetic acid (7.1 mL),
and EtOH (7.1 mL) was added and the reaction heated at reflux for a further 3 h. Water (60 mL)
was added and the mixture was extracted with ethyl acetate (3×30 mL). The combined organics
were dried (Na2SO4), filtered and the solvent concentrated in vacuo. The crude product was
purifiedn by flash column chromatography on silica gel (eluant: EtOAc: PE = 1:1) gave the title
13

compound 3 as a white solid (0.68 g, 69.8%). 1HNMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.24
(s, 1H), 7.29-7.34 (m, 4H), 7.21-7.25 (m, 1H), 5.76 (s, 1H), 3.44 (s, 3H), 4.29 (q, J = 7.2 Hz, 2H),
1.38 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z: 273.1203 [M+H]+, calcd. for 273.1239.
3.1.4. Ethyl 6-oxo-4-(phenylamino)-1,6-dihydropyridine-3-carboxylate (5)
Intermediate 1a (4.0 g, 14.5 mmol) was dissolved in a mixture of acetic acid (100 mL) and
water (40 mL), and the solution was heated at reflux for 72 h. The mixture was cooled to rt, and
concentrated in vacuo. The residue was purified by flash column chromatography on silica gel
(eluant: CH2Cl2: MeOH = 100:1) gave the title compound 5 as a white solid (1.5, 41.1%).
1HNMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 9.15 (s, 1H), 8.25 (s, 1H), 7.27-7.33 (m, 4H),
7.20-7.25 (m, 1H), 5.75 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H). HRMS (ESI)
m/z: 259.1042 [M+H]+, calcd. for 259.1083.
3.1.5. 6,7-dimethoxy-4-(4-nitrophenoxy)quinoline (7a)
4-Nitrophenol (12.5 g, 89.6 mmol) was added into a suspension of 4-chloro-6,7-
dimethoxyquinoline (10.0 g, 44.8 mmol) in PhCl (80 mL). The resulting mixture was stirred at
reflux for 16 h. The solvent was evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (150 mL) The solution was washed by 10% NaOH aqueous solution (3×30
mL), water (30 mL) and dried (MgSO4), and evaporated to obtain the title compound as a yellow
solid (9.6 g, 65.7%) without further purification. HRMS (ESI) m/z: 327.0949 [M+H]+, calcd. for
327.0981.
3.1.6. 4-((6,7-dimethoxyquinolin-4-yl)oxy)aniline (8a)
A mixture of 7a (9.6 g, 29.4 mmol), Fe (8.2 g, 0.15 mol) and AcOH (0.5 mL) in 90% EtOH
(100 mL) was refluxed with vigorous agitation for 4 h. The hot solution was filtered through
celite and the filter cake was washed with hot EtOH (20 mL). The combined filtrate was
concentrated under reduced pressure to afford a dark brown solid, which was recrystallized from
EtOH to afford 8a as yellow solid (7.2 g, 82.9%). HRMS (ESI) m/z: 297.1205 [M+H]+, calcd. for
297.1239.
3.1.7. General Procedure for the synthesis of target compounds 9a-b, 10a-l and 11-14
14

A solution of acid (0.6 mmol), DIPEA (0.75 mmol), HATU (0.6 mmol) and aniline (0.5 mmol)
was stirred at rt in DMF (2 mL) for 4 h. The solvent was poured into ice water (6 mL), and the
crude product was isolated by filtration. After purification on silica gel (eluant: EtOAc: PE = 1:1),
the desired adducts were obtained.
N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-6-oxo-4-(phenylamino)-1,6-dihydropyridine-3-
1
carboxamide (9a). Yield 60.8%. White solid. HNMR (400 MHz, DMSO-d6) δ 11.49 (br, 1H),
10.37 (s, 1H), 9.64 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.12 (s, 1H), 7.78 (d, J = 12.0 Hz, 2H), 7.52
(s, 1H), 7.40-7.44 (m, 3H), 7.26-7.29 (m, 4H), 7.16-7.19 (m, 1H), 6.46 (d, J = 5.2 Hz, 1H), 5.64
(s, 1H), 3.95 (s, 3H), 3.94 (s, 3H). HRMS (ESI) m/z: 509.1802 [M+H]+, calcd. for 509.1825.
N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-1-methyl-6-oxo-4-(phenylamino)-1,6-
1
dihydropyridine-3-carboxamide (9b). Yield 64.3%. White solid. HNMR (400 MHz, DMSO-d6)
δ 10.38 (s, 1H), 9.45 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.46 (s, 1H), 7.77-7.80 (m, 2H), 7.52 (s,
1H), 7.39-7.44 (m, 3H), 7.26-7.31 (m, 4H), 7.15-7.18 (m, 1H), 6.46 (d, J = 5.2 Hz, 1H), 5.74 (s,
1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.43 (s, 3H). HRMS (ESI) m/z: 523.1971 [M+H]+, calcd. for
523.1981.
6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-(phenylamino)nicotinamide (10a).
1
Yield 57.7%. White solid. HNMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.82 (s, 1H), 8.67 (s,
1H), 8.50 (d, J = 5.2 Hz, 1H), 7.81-7.91 (m, 2H), 7.54 (s, 1H), 7.46-7.49 (m, 2H), 7.42 (s, 1H),
7.31-7.38 (m, 4H), 7.23-7.29 (m, 1H), 6.91 (s, 1H), 6.48 (d, J = 5.2 Hz, 1H), 3.97 (s, 3H), 3.96 (s,
3H). HRMS (ESI) m/z: 527.1432 [M+H]+, calcd. for 527.1486.
6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-((3-
fluorophenyl)amino)nicotinamide (10b). Yield 64.1%. 1HNMR (400 MHz, DMSO-d6) δ 10.69 (s,
1H), 9.81 (s, 1H), 8.65 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 7.84-7.87 (m, 2H), 7.52 (s, 1H), 7.43-
7.49 (m, 1H), 7.40 (s, 1H), 7.29-7.33 (m, 2H) , 7.22-7.26 (m, 1H), 7.19-7.21 (m, 1H), 7.04-7.07
(m, 1H), 7.03 (s, 1H), 6.46 (d, J = 5.2 Hz, 1H), 5.64 (s, 1H), 3.95 (s, 3H), 3.94 (s, 3H). HRMS
(ESI) m/z: 545.1366 [M+H]+, calcd. for 545.1392.
6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-((4-
15

1
fluorophenyl)amino)nicotinamide (10c). Yield 61.9%. White solid. HNMR (400 MHz, DMSO-
d6) δ 10.68 (s, 1H), 9.69 (s, 1H), 8.67 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 7.84-7.87 (m, 2H), 7.51 (s,
1H), 7.44-7.48 (m, 1H), 7.41 (s, 1H), 7.29-7.32 (m, 2H) , 7.22-7.26 (m, 1H), 7.18-7.21 (m, 1H),
7.04-7.08 (m, 1H), 7.03 (s, 1H), 6.46 (d, J = 5.2 Hz, 1H), 5.64 (s, 1H), 3.95 (s, 3H), 3.94 (s, 3H).
HRMS (ESI) m/z: 545.1405 [M+H]+, calcd. for 545.1392.
6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-((2-
fluorophenyl)amino)nicotinamide (10d). Yield 65.6%. 1HNMR (400 MHz, DMSO-d6) δ 10.72 (s,
1H), 9.79 (s, 1H), 8.69 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 7.86-7.89 (m, 2H), 7.52-7.56 (m, 2H),
7.37-7.41 (m, 2H), 7.28-7.36 (m, 4H), 6.68 (m, 1H), 6.49 (d, J = 5.2 Hz, 1H), 3.96 (s, 3H), 3.95
(s, 3H). HRMS (ESI) m/z: 545.1371 [M+H]+, calcd. for 545.1392.
6-chloro-4-((2-chlorophenyl)amino)-N-(4-((6,7-dimethoxyquinolin-4-
1
yl)oxy)phenyl)nicotinamide (10e). Yield 67.3%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.72 (s, 1H), 10.05 (s, 1H), 8.73 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 7.84-7.88 (m, 2H), 7.59-7.64
(m, 2H), 7.52 (s, 1H), 7.29-7.32 (m, 3H), 6.79 (s, 1H), 6.47 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.94
(s, 3H). HRMS (ESI) m/z: 561.1013 [M+H]+, calcd. for 561.1096.
6-chloro-4-((3-chlorophenyl)amino)-N-(4-((6,7-dimethoxyquinolin-4-
1
yl)oxy)phenyl)nicotinamide (10f). Yield 62.6%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.68 (s, 1H), 9.76 (s, 1H), 8.64 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 7.84-7.87 (m, 2H), 7.52 (s, 1H),
7.42-7.47 (m, 2H), 7.40 (s, 1H), 7.24-7.34 (m, 4H), 6.98 (s, 1H), 6.47 (d, J = 5.2 Hz, 1H), 3.95 (s,
3H), 3.94 (s, 3H). HRMS (ESI) m/z: 561.1018 [M+H]+, calcd. for 561.1096.
6-chloro-4-((4-chlorophenyl)amino)-N-(4-((6,7-dimethoxyquinolin-4-
1
yl)oxy)phenyl)nicotinamide (10g). Yield 61.9%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.67 (s, 1H), 9.75 (s, 1H), 8.64 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 7..87 (d, J = 8.8 Hz, 2H), 7.52
(s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.40 (s, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H),
6.92 (s, 1H), 6.45 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H). HRMS (ESI) m/z: 561.1048
[M+H]+, calcd. for 561.1096.
4-((2-bromophenyl)amino)-6-chloro-N-(4-((6,7-dimethoxyquinolin-4-
16

1
yl)oxy)phenyl)nicotinamide (10h). Yield 69.6%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.68 (s, 1H), 9.75 (s, 1H), 8.63 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 7.84-7.87 (m, 2H), 7.52-7.55
(m, 2H), 7.38-7.40 (m, 4H), 7.29-7.32 (m, 2H), 6.96 (s, 1H), 6.45 (d, J = 5.2 Hz, 1H), 3.95 (s,
13
3H), 3.94 (s, 3H). CNMR (100 MHz, DMSO-d6) δ 165.6, 160.4, 153.6, 153.1, 152.5, 150.7,
150.5, 149.8, 149.3, 146.9, 140.6, 136.4, 131.9, 128.3, 126.2, 123.0 (2C), 122.7, 122.4, 121.8
(2C), 115.6, 114.9, 108.3, 107.1, 103.6, 99.6, 56.2 (2C). HRMS (ESI) m/z: 605.0570, 607.0544
[M+H]+, calcd. for 605.0591, 607.0571.
4-((3-bromo-4-fluorophenyl)amino)-6-chloro-N-(4-((6,7-dimethoxyquinolin-4-
1
yl)oxy)phenyl)nicotinamide (10i). Yield 64.8%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.73 (s, 1H), 9.77 (s, 1H), 8.69 (s, 1H), 8.50 (br, 1H), 7.73-7.86 (m, 3H), 7.32-7.50 (m, 6H),
13
6.76 (s, 1H), 6.47 (br, 1H), 3.95 (s, 6H). CNMR (100 MHz, DMSO-d6) δ 165.8, 160.4, 153.8,
153.1, 152.7, 150.8, 150.6, 149.8, 149.3, 146.9, 136.3, 128.8, 128.0, 123.2 (2C), 121.8 (2C),
120.5, 120.4, 115.6, 114.3, 108.3, 107.1, 103.6, 99.5, 56.2 (2C). HRMS (ESI) m/z: 623.0490,
625.0477 [M+H]+, calcd. for 623.0497, 625.0477.
4-((4-bromo-2-fluorophenyl)amino)-6-chloro-N-(4-((6,7-dimethoxyquinolin-4-
1
yl)oxy)phenyl)nicotinamide (10j). Yield 66.0%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.70 (s, 1H), 9.96 (s, 1H), 8.63 (br, 2H), 7.89 (br, 2H), 7.36-7.58 (m, 7H), 6.86 (s, 1H), 6.58 (br,
1H), 3.98 (s, 6H). HRMS (ESI) m/z: 623.0495, 625.0482 [M+H]+, calcd. for 623.0497, 625.0477.
6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-(naphthalen-1-
1
ylamino)nicotinamide (10k). Yield 54.9%. White solid. HNMR (400 MHz, DMSO-d6) δ 10.74
(s, 1H), 10.18 (s, 1H), 8.72 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.04 (m, 1H), 7.89-7.93 (m, 4H),
7.61-7.63 (m, 4H), 7.53 (s, 1H), 7.41 (s, 1H), 7.33 (m, 2H), 6.49 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H),
3.94 (s, 3H). HRMS (ESI) m/z: 577.1658 [M+H]+, calcd. for 577.1643.
4-((3-bromo-4-fluorophenyl)amino)-6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-
1
fluorophenyl)nicotinamide (10l). Yield 61.8%. HNMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H),
9.62 (s, 1H), 8.62 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 7.95-7.99 (m, 1H), 7.69 (m, 1H), 7.61 (m,
1H), 7.43-7.56 (m, 5H), 6.86 (s, 1H), 6.47 (d, J = 5.2 Hz, 1H), 3.97 (s, 6H). HRMS (ESI) m/z:
17

641.0339, 643.0323 [M+H]+, calcd. for 641.0403, 643.0382.
6-bromo-4-((3-bromo-4-fluorophenyl)amino)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-
1
fluorophenyl)quinoline-3-carboxamide (11). Yield 63.1%. White solid. HNMR (400 MHz,
DMSO-d6) δ 10.73 (s, 1H), 9.63 (s, 1H), 8.62 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.27 (s, 1H), 8.17
(m, 1H), 8.11 (m, 1H), 7.59 (m, 1H), 7.41-7.53 (m, 6H), 6.86 (s, 1H), 6.47 (d, J = 5.2 Hz, 1H),
3.95 (s, 3H), 3.94 (s, 3H). HRMS (ESI) m/z: 735.0012, 737.0015. [M+H]+, calcd. for 735.0054,
737.0034.
2-((3-bromo-4-fluorophenyl)amino)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-
1
fluorophenyl)nicotinamide (12). Yield 66.4%. White solid. HNMR (400 MHz, DMSO-d6) δ
10.85 (s, 1H), 10.20 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.41 (m, 1H), 8.29-8.31 (m, 1H), 8.24-8.27
(m, 1H), 8.00-8.04 (m, 1H), 7.66-7.69 (m, 1H), 7.60-7.64 (m, 1H), 7.56 (s, 1H), 7.51 (t, J = 8.8
Hz, 1H), 7.43 (s, 1H), 7.32 (t, J = 8.8 Hz, 1H), 7.00-7.03 (m, 1H), 6.47 (d, J = 5.2 Hz, 1H), 3.97
(s, 6H). HRMS (ESI) m/z: 607.0733, 609.0718 [M+H]+, calcd. for 607.0792, 609.0772.
4-((3-bromo-4-fluorophenyl)amino)-2-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-
fluorophenyl)-6-methylnicotinamide (13). Yield 63.7%. White solid. MS (ESI) m/z: 655.0
1
[M+H]+. HNMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 9.67 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H),
7.93-7.97 (m, 1H), 7.68 (m, 1H), 7.63 (m, 1H), 7.43-7.54 (m, 5H), 6.85 (s, 1H), 6.48 (d, J = 5.2
Hz, 1H), 3.95 (s, 6H), 2.61 (s, 3H). HRMS (ESI) m/z: 655.0521, 657.0502 [M+H]+, calcd. for
655.0559, 657.0539.
3.2. Kinase Assay [30]
All target compounds were tested for their inhibitory rate (@ 0.2 μM) against Aur A and Aur B
by mobility shift assay. Dilute the compounds to 500 mM by DMSO, then transfer 10 mL of
compound to a 96-well plate as the intermediate plate, add 90 μL kinase buffer to each well.
Transfer 5 μL of each well of the intermediate plate to 384-well plates. Kinase base buffer, FAM-
labeled peptide, ATP and enzyme solution were added. Wells containing the substrate, enzyme,
DMSO without compound were used as DMSO control. Wells containing just the substrate
without enzyme were used as low control. Incubate at room temperature for 10 min. Add 10 μL
18

peptide solution to each well. Incubate at 28 ℃ for specified period of time and stop reaction by
25 μL stop buffer. At last collect data on Caliper program (Perkin Elmer, US) and convert
conversion values to inhibition values.
3.3. MTT assay [26]
Taking Cabozantinb as positive control, the in vitro cytotoxic activity of target compounds was
evaluated with SW620, HT-29, NCI-H1975 and Hela cancer cell lines by the colorimetric MTT
assay. Briefly, the cancer cell lines were cultured in DMEM (Hyclone®) or RPMI 1640
(Hyclone®) supplement with 10% fetal bovine serum (FBS) as well as 1% (v/v) penicillin and
streptomycin. Cells were incubated in 96-well plates at a density of 6 × 103 cells/well and
incubated in 5% CO2 at 37 ℃ for 24 h. DMSO content was less than 0.1%. Then, the cells were
exposed to various concentrations of tested compounds and the cell cultures were continued for
another 72 h. Subsequently, 10 μL of fresh MTT (5 mg/mL) was added to each well and
incubated with cell at 37 ℃ for 4 h. The medium was replaced with 100 μL of DMSO to dissolve
the formed formazan crystals, and the absorbency at 492 nm (for absorbance of MTT formazan)
and 630 nm (for the reference wavelength) was measured with the ELISA reader. The IC50 values
were calculated by using GraphPad Prism 5.0.
3.5. IncuCyte live-cell imaging assays [30]
SW620 cancer cells were incubated in 96-well plates at a density of 5 × 103 cells/well and
incubated in a tissue culture incubator at 37 ℃ and 5% CO2 in an IncuCyte (Essen BioScience).
The cells were cultured for 24 h, and different concentration of compound 10i, 10l (10.0, 3.33,
1.11, 0.37 or 0.12 μg/mL) and Cabozantinib (10.0, 3.33, 1.11 or 0.37 μg/mL) were added. Assay
was performed according to the manufacturer's protocol. All samples consisted of three replicates.
Images were captured by 10 times mirror every 1 h over or 84 h monitor proliferation and
cytotoxicity, and 48 h for apoptosis. Red and green fluorescent signals were measured, and red-
fluorescent cells and green-fluorescent cells were counted as dead cells and apoptotic cells,
respectively.
4. Conclusions
19

In summary, a series of nicotinamide derivatives were synthesized, evaluated for their
biological activities, and SAR was illustrated preliminarily. 4-((3-Bromo-4-fluorophenyl)amino)-
6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)nicotinamide
(10l)
was
identified as the most potent inhibitor of Aur A. In addition, compound 10l showed excellent
antitumor activity against SW620 and NCI-H1975 with IC50 values were 0.61 and 1.06 μM,
respectively. As a potent antitumor agent, 10l performed excellent apoptosis, antiproliferation
and cytotoxicity against SW620 cells in a dose- and time-dependent manner. More compounds
were being synthesized by our group and biological evaluation was also ongoing, including
kinase profiles, cell cycle arrest and inhibition of kinase phosphorylation, etc. In addition,
improving water-solubility and kinase selectivity are also main contents in the following work.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (21562053) and
Science and Technology Foundation of Guizhou Province (Qian Ke He J Zi [2014]2181).
References
[1] Noble, M.E.; Endicott, J.A.; Johnson, L.N. Protein kinase inhibitors: insights into drug design
from structure. Science, 2004, 303: 1800−1805.
[2] Gautschi, O.; Heighway, J.; Mack, P.C.; Purnell, P.R.; Lara, P.N., Jr.; Gandara, D.R. Aurora
kinases as anticancer drug targets. Clin. Cancer Res., 2008, 14: 1639−1648.
[3] Carmena, M.; Earnshaw, W.C. The cellular geography of aurora kinases. Nat Rev Mol Cell
Biol., 2003, 4:842–854.
[4] Carmena, M.; Earnshaw, W.C. The cellular geography of aurora kinases. Nature Rev. Mol.
Cell Biol., 2003, 4: 842−854.
[5] Fu, J.; Bian, M.; Jiang, Q.; Zhang, C. Roles of Aurora kinases in mitosis and tumorigenesis.
Mol. Cancer Res., 2007, 5: 1−10.
[6] Nigg, E.A. Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol
20

Cell Biol., 2001, 2: 21–32.
[7] Vader, G.; Lens, S.M. The Aurora kinase family in cell division and cancer. Bioch Biophy
Acta., 2008, 1786:60–72.
[8] Barr, A.R.; Gergely, F. Aurora-A: the maker and breaker of spindle poles. Cell Sci., 2007,
120: 2987–2996.
[9] Saeki, T.; Ouchi, M.; Ouchi, T. Physiological and oncogenic Aurora – a pathway. Int J Biol
Sci., 2009, 5: 758–762.
[10] Vader, G.; Medma, R.H.; Lens, S.M.A. The chromosomal passenger complex: guiding
Aurora-B through mitosis. J Cell Biol., 2006, 173 (6): 833–837.
[11] Carmena, M.; Ruchaud, S.; Earnshaw, W.C. Making the Auroras glow: regulation of Aurora
A and B kinase function by interacting proteins. Curr. Opin. Cell. Biol., 2009, 21: 796−805.
[12] Sasai, K.; Katayama, H.; Stenoien, D.L.; Fujii, S.; Honda, R.; Kimura, M.; Okano, Y.;
Tatsuka, M.; Suzuki, F.; Nigg, E.A.; Earnshaw W.; Brinkley, W.R.; Sen, S. Aurora-C kinase
is a novel chromosomal passenger protein that can complement Aurora-B kinase function in
mitotic cells. Cell Motil Cytoskelet., 2004, 59: 249–263.
[13] Sarvagalla, S.; Coumar, M.S. Structural biology insight for the design of sub-type selective
Aurora kinase inhibitors. Curr. Cancer Drug Targets, 2015, 15(5), 375-393.
[14] Borisa, A.C.; Bhatt, H.G. A comprehensive review on Aurora kinase: Small molecule
inhibitors and clinical trial studies. European Journal of Medicinal Chemistry, 2017, 140: 1-
19.
[15] Gorgun, G.; Calabrese, E.; Hideshima, T.; Ecsedy, J.; Perrone, G.; Mani, M.; Ikeda, H.;
Bianchi, G.; Hu, Y.; Cirstea, D.; Santo, L.; Tai, Y.-T.; Nahar, S.; Zheng, M.; Bandi, M.;
Carrasco, R.D.; Raje, N.; Munshi, N.; Richardson, P.; Anderson, K.C. A novel Aurora-A
kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma.
Blood, 2010, 115: 5202−5213.
[16] Manfredi, M.G.; Ecsedy, J. A.; Chakravarty, A.; Silverman, L.; Zhang, M.; Hoar, K.M.;
Stroud, S.G.; Chen, W.; Shinde, V.; Huck, J.J.; Wysong, D.R.; Janowick, D.A.; Hyer, M.L.;
21

LeRoy, P.J.; Gershman, R.E.; Silva, M.D.; Germanos, M.S.; Bolen, J.B.; Claiborne, C.F.;
Sells, T.B. Characterization of alisertib (MLN8237), an investigational smallmolecule
inhibitor of Aurora A kinase using novel in vivo pharmacodynamic assays. Clin. Cancer
Res., 2011, 17: 7614−7624.
[17] Fancelli, D.; Moll, J.; Varasi, M.; Bravo, R.; Artico, R.; Berta, D.; Bindi, S.; Cameron, A.;
Candiani, I.; Cappella, P.; Carpinelli, P.; Croci, W.; Forte, B.; Giorgini, M.L.; Klapwijk, J.;
Marsiglio, A.; Pesenti, E.; Rocchetti, M.; Roletto, F.; Severino, D.; Soncini, C.; Storici, P.;
Tonani, R.; Zugnoni, P.; Vianello, P. 1,4,5,6-Tetrahydropyrrolo[3,4-c]pyrazoles:
identification of a potent Aurora kinase inhibitor with a favorable antitumor kinase
inhibition profile. J. Med. Chem., 2006, 49: 7247−7251.
[18] Borges, K.S.; Andrade, A.F.; Silveira, V.S.; Marco Antonio, D.S.; Vasconcelos, E. J.R.;
Antonini, S.R.R.; Tone, L.G.; Scrideli, C.A. The aurora kinase inhibitor AMG 900 increases
apoptosis and induces chemosensitivity to anticancer drugs in the NCI-H295 adrenocortical
carcinoma cell line. Anti-Cancer Drugs, 2017, 28: 634–644.
[19] Linardopoulos, S.; Blagg, J. Aurora Kinase Inhibition: A New Light in the Sky?. J. Med.
Chem., 2015, 58: 5186−5188.
[20] Pinkerton, A.B.; Sergienko, E.; Bravo, Y.; Dahl, R.; Ma, C.; Sun, Q.; Jackson, M.R.;
Cosford,
N.D.P.;
Millán,
J.L.
Discovery
of
5-((5-chloro-2-
methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable
tissue-nonspecific alkaline phosphatase (TNAP) inhibitor. Bioorganic & Medicinal
Chemistry Letters, 2018, 28: 31–34.
[21] Gao, X.; Liu, L.; Liu, H.; Tang, J.; Kang, L.; Wu, H.; Cui, P.; Yan, J. Structure–activity
relationship investigation of benzamide and picolinamide derivatives containing
dimethylamine side chain as acetylcholinesterase inhibitors. Journal of Enzyme Inhibition
and Medicinal Chemistry, 2018, 33 (1): 110–114.
[22] Felts, A.S.; Rodriguez, A.L.; Blobaum, A.L.; Morrison, R.D.; Bates, B.S.; Gray, A.T.; Rook,
J.M.; Tantawy, M.N.; Byers, F.W.; Chang, S.; Venable, D.F.; Luscombe, V.B.; Tamagnan,
22

G.D.; Niswender C.M.; Daniels, J.S.; Jones, C.K.; Conn, P.J.; Lindsley, C.W.; Emmitte,
K.A. Discovery of N‑(5-Fluoropyridin-2-yl)-6-methyl-4-(pyrimidin-5-yloxy)picolinamide
(VU0424238): A Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor
Subtype 5 Selected for Clinical Evaluation. J. Med. Chem. 2017, 60, 5072−5085.
[23] El-Damasy, A.K.; Seo, S.H.; Cho, N.; Kang, S.B.; Pae, A.N.; Kim, F.; Keum, G. Design,
synthesis, in-vitro antiproliferative activity and kinase profile of new picolinamide based 2-
amido and ureido quinoline derivatives. European Journal of Medicinal Chemistry. 2015,
101: 754-768.
[24] Zeidan, M.A.; Mostafa, A.S.; Gomaa, R.M.; Abou-zeid, L.A.; El-Mesery, M.; El-Sayed,
M.A.; Selim, K.B. Design, synthesis and docking study of novel picolinamide derivatives as
anticancer agents and VEGFR-2 inhibitors. European Journal of Medicinal Chemistry, 2019,
168: 315-329.
[25] Qi, B.; Mi, B.; Zhai, X.; Xu, Z.; Zhang, X.; Tian, Z.; Gong, P. Discovery and optimization of
novel 4-phenoxy-6,7-disubstituted quinolines possessing semicarbazones as c-Met kinase
inhibitors. Bioorganic & Medicinal Chemistry, 2013, 21: 5246–5260.
[26] Qi, B.; Yang, Y.; He, H.; Yue, X.; Zhou, Y.; Zhou, X.; Chen, Y.; Liu, M.; Zhang, A.; Wei, F.
Identification of novel N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas showing potent
multi-tyrosine kinase inhibitory activities. European Journal of Medicinal Chemistry, 2018,
146: 368-380.
[27] Spicer, J.A.; Rewcastle, G.W.; Kaufman, M.D.; Black, S.L.; Plummer, M.S.; Denny, W.A.;
Quin, J.; Shahripour, A.B.; Barrett, S.D.; Whitehead, C.E.; Milbank, J.B.J.; Ohren, J.F.;
Gowan, R.C.; Omer, C.; Camp, H.S.; Esmaeil, N.; Moore, K.; Sebolt-Leopold, J.S.;
Pryzbranowski, S.; Merriman, R.L.; Ortwine, D.F.; Warmus, J.S.; Flamme, C.M.; Pavlovsky,
A.G.; Tecle, H. 4-Anilino-5-carboxamido-2-pyridone Derivatives as Noncompetitive
Inhibitors of Mitogen-Activated Protein Kinase Kinase, J. Med. Chem. 2007, 50, 5090-5102.
[28] Zhu, M.; Qui, Z.; Hiel, G. P.; Sieberth, S. M. Photocycloaddition of four-carbon-tethered
pyridones. Intramolecular hydrogen bonding and facilitated amide hydrolysis by a proximal
23

secondary alcohol. J. Org. Chem. 2002, 67, 3487-3493.
[29] Mekheimer, R. A. Fused quinoline heterocycles I. First example of the 2,4-diazido-
quinoline-3-carbonitrile and 1-aryl-1,5-dihydro-1,2,3,4,5,6- hexaazaacephenanthrylene ring
systems. J. Chem. Soc., Perkin Trans. 1 1999, 2183-2188.
[30] Qi, B.;Yang, Y.; Gong G.; He, H.; Yue, X.; Xu, X.; Hu Y.; Li, J.; Chen, T.; Wan, X.; Zhang,
A.; Zhou, G. Discovery of N1-(4-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinolin-
4-yl)oxy)-3,5-difluorophenyl)-N3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)urea as a
multi-tyrosine kinase inhibitor for drug-sensitive and drug-resistant cancers treatment.
European Journal of Medicinal Chemistry, 2019, 163:10-27.
24

Graphical Abstract
25