Synthesis and biological evaluation of quinoline derivatives as potential anti-prostate cancer agents and Pim-1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.016

In this work, a series of quinoline derivatives were designed and synthesized as antitumor agents. Most quinolines showed potent anti-proliferative activity against human prostatic cancer PC-3 cell line. Among which, 9d, 9f and 9g were the most effective compounds with GI₅₀ values of 2.60, 2.81 and 1.29 μM, respectively. Structure-activity relationship analysis indicated that the secondary amine linked quinoline and pyridine ring played an important role in the anti-proliferative effects. Mechanistic studies revealed that 9g was a potential Pim-1 kinase inhibitor with abilities of cell cycle arrest and apoptosis induction. Considering of the increased activity of Pim-1 in prostate cancer, such compounds have potential to be developed as anti-prostate cancer agents.

Full text of DOI:10.1016/j.bmc.2016.03.016

Accepted Manuscript
Synthesis and biological evaluation of quinoline derivatives as potential anti-
prostate cancer agents and Pim-1 kinase inhibitors
Kun Li, Ying Li, Di Zhou, Yinbo Fan, Hongye Guo, Tianyi Ma, Jiachen Wen,
Dan Liu, Linxiang Zhao
PII:
S0968-0896(16)30165-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.016
BMC 12865
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 January 2016
5 March 2016
7 March 2016
Please cite this article as: Li, K., Li, Y., Zhou, D., Fan, Y., Guo, H., Ma, T., Wen, J., Liu, D., Zhao, L., Synthesis
and biological evaluation of quinoline derivatives as potential anti-prostate cancer agents and Pim-1 kinase
inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.03.016
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.

Synthesis and biological evaluation of quinoline derivatives as potential anti-prostate cancer agents and Pim-1
kinase inhibitors
Kun Lia, Ying Lib, Di Zhoua, Yinbo Fana, Hongye Guoa, Tianyi Maa, Jiachen Wena, Dan Liua* and Linxiang Zhaoa*
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
*Corresponding authors.
Tel./Fax: +86 024 2398 6420.
E-mail address: sammyld@163.com (D. Liu);
linxiang.zhao@vip.sina.com (L. Zhao)

Abstract
In this work, a series of quinoline derivatives were designed and synthesized as antitumor agents. Most quinolines
showed potent anti-proliferative activity against human prostatic cancer PC-3 cell line. Among which, 9d, 9f and 9g
were the most effective compounds with GI50 values of 2.60, 2.81 and 1.29 μM, respectively. Structure-activity
relationship analysis indicated that the secondary amine linked quinoline and pyridine ring played an important role in
the anti-proliferative effects. Mechanistic studies revealed that 9g was a potential Pim-1 kinase inhibitor with abilities of
cell cycle arrest and apoptosis induction. Considering of the increased activity of Pim-1 in prostate cancer, such
compounds have potential to be developed as anti-prostate cancer agents.
Keywords: Quinoline derivatives; Anti-proliferative activity; Pim-1 kinase inhibitor; Prostate cancer

1. Introduction
The quinoline scaffold is known to have diverse biological and pharmacological activities, such as anti-AIDs,1
anti-psychotic,2 anti-malaria,3 and anti-cancer activity.4 Certain compounds with such a pharmacophore have been
developed into marketed drugs, like Saquinavir, Aripiprazole, Chloroquine, Cabozantinib, etc.5 Aided by
structure-guided docking strategy, compound 9d, a novel quinoline derivative was identified as a urokinase receptor
(uPAR) and α5β1-integrin interaction disrupt agent, thus blocking ERK activation and metastasis.6 Herein, a series of
substituted quinoline derivatives were synthesized and their anti-tumor activity was tested against human prostatic
cancer PC-3 cell line. Structure-activity relationship (SAR) and mechanisms of action were illustrated. It turns out to be
that it was inhibition of Pim-1 that led to cell cycle arrest and apoptosis induction, rather than ERK. Biological enzyme
activity assay and molecular docking analysis revealed that these compounds were potential Pim-1 inhibitors.
(Figure 1. should be listed here.)
Figure 1. The structure of 2-(pyridin-2-ylamino)-quinolin-8-ol (9d).
Pim-1 belongs to the proviral insertion site in moloney murine leukemia virus (PIM) proteins family and is a
highly evolutionarily conserved serine/threonine kinase.7 It has been proved that Pim-1 kinase has an extensive range of
biological roles, including cell survival, proliferation, differentiation, and anti-apoptosis.8 High expression level of
Pim-1 is frequently observed in a number of hematologic and solid malignancies, such as prostate cancer.9 Therefore,
it’s reasonable to highlight Pim-1 as a potential therapeutic target to develop anticancer agents.10 Most Pim-1 inhibitors
functioned as ATP competitors or mimics, such as ruthenium containing organometallic complexes, indolocarbazoles
and pyridazines.11 Several compounds have entered into clinical trials, unfortunately, none of them was approved as
anticancer drug.12 Current work represents that quinolines were potential anticancer agents as a new group of Pim-1
inhibitor.

2. Results and discussion
2.1. Chemistry
The synthetic route adopted for preparation of target compounds 3a–c was outlined in Scheme 1. The starting
material quinoline 1 was oxidated by m-chloroperoxybenzoic acid (m-CPBA) in chloroform at ambient temperature, to
give the key intermediate quinoline N-oxide 2. Condensation of 2 with various substituted 2-aminopyridine provided
compounds 3a–c conveniently.13
(Scheme 1. should be listed here.)
Scheme 1. Synthesis of 3a–c. Reagents and conditions: (a) m-CPBA, CHCl3, r.t., 2 h; (b) appropriate pyridines, p-TsCl,
10% KOH aq., CH2Cl2, r.t., 2 h.
The synthesis of compounds 8a–i, and 9d–i was described in Scheme 2. The key intermediates 6a–c could be
readily yielded by oxidation of substituted 8-hydroxyquinoline 4a–c, which were protected by a methyl group using the
methyl iodide under basic conditions previously. Subsequently, the coupling of 6a–c with appropriate pyridines
afforded compounds 7a–i. An efficient demethylation of 7a–c was carried out in good yield with tribromoborane under
nitrogen environment to provide thioethers 8a–c. On the other side, 7d–i were hydrolyzed by concentrated hydrochloric
acid to give 8d–i. Afterwards, deprotection was performed to afford compounds 9d–i.
(Scheme 2. should be listed here.)

Scheme 2. Synthesis of 8a–i and 9d–i. Reagents and conditions: (a) methyl iodide, K2CO3, (CH3)2CO, r.t. to 60°C, 8 h;
(b) m-CPBA, CHCl3, r.t., 2 h; (c) appropriate pyridines, p-TsCl, 10% KOH aq., CH2Cl2, r.t., 2 h; (d) HCl, 100°C; (e)
BBr3, dry CH2Cl2, 0°C, 30 min, r.t., 2 h, reflux, 9 h.
Compounds 13a–c were prepared as depicted in Scheme 3. Briefly, 4a–c were reacted with MsCl in the presence
of Et3N, followed by N-oxidation. Then condensation with 2-hydroxypyridine was performed to give compounds 12a–c,
which were hydrolyzed with NaOH to afford corresponding ethers 13a–c.
(Scheme 3. should be listed here.)
Scheme 3. Synthesis of 13a–c. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, 0°C, 1 h; (b) m-CPBA, CHCl3, r.t., 2
h; (c) appropriate pyridines, p-TsCl, 10% KOH aq., CH2Cl2, r.t., 2 h; (d) 1M NaOH aq., EtOH, (CH3)2CO, reflux, 8 h.
2.2. Biological results and discussion
All target compounds were initially tested for their anti-proliferative activity against human prostatic cancer PC-3
cell line using MTT assay. The mean values of three independent experiments, expressed as 50% cell growth inhibitory
concentration (GI50) values, were presented in Table 1.

As shown in Table 1, most of the synthesized compounds exhibited potent anti-proliferative activity with GI50
values less than 30 μM. Generally, the variations of substituents on quinoline ring together with the hetero-atom linked
quinoline and pyridine ring have a notable influence on the anti-proliferative activity. Compounds 9d (GI50 = 2.60 μM),
9f (GI50 = 2.81 μM) and 9g (GI50 = 1.29 μM) with a hydroxyl group at C-8 position of quinoline displayed the most
potent inhibitory effects, while compounds 8d (GI50 = 7.47 μM), 8f (GI50 = 11.85 μM) and 8g (GI50 = 29.22 μM) with a
methoxyl group possessed inferior anti-proliferative activity. However, all of them were superior to compounds 3a and
3c without a substituent at C-8 position of quinoline ring, indicating a fine tuning of functional groups at this position
was beneficial for the activity. Replacement of H by Cl at quinoline C-5 position could lead to a significant decline of
anti-proliferative activity as exemplified by 8d (GI50 = 7.47 μM) and its analog 8g (GI50 = 29.22 μM), which could be
further confirmed by 8f, 9e, 9f vs. 8i, 9h, 9i. Once the C-4’ position of pyridine ring was substituted by CH3 or Cl, the
anti-proliferative effects were reduced or disappeared in most cases, as exemplified by 9g (R1 = H, GI50 = 1.29 μM) vs.
9h (R1 = CH3, GI50 = 10.23 μM), 9i (R1 = Cl, GI50 = 25.54 μM). Exceptionally, compound 8e (R1 = CH3, GI50 = 3.82
μM) showed a slight improvement in activity as compared to 8d (R1 = H, GI50 = 7.47 μM). Furthermore, a sharp
decrease in activity was observed in 8a (X = S, GI50 = 20.87 μM) and 13a (X = O, GI50 > 30 μM) as compared to 9d (X
= NH, GI50 = 2.60 μM), suggesting the -NH- moiety was essential for the inhibition activity. The merit of -NH- moiety
might be attributed to it’s interaction with the target protein as a hydrogen bond donor.
(Table 1. should be listed here.)
Table 1. The anti-proliferative activity of target compounds against PC-3 cell line.
PC-3
GI50 (µM) a
Compd.
R3
R2
R1
X
3a
3b
3c
8a
H
H
H
H
H
H
H
CH3
Cl
NH
NH
NH
S
> 30
26.7 6.90
> 30
H
OH
H
20.87 2.58

8b
8c
OH
OH
Cl
Br
H
H
H
S
10.19 0.04
10.22 0.22
7.47 1.91
3.82 0.52
11.85 0.54
29.22 2.66
> 30
S
8d
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
8e
H
CH3
Cl
H
8f
H
8g
Cl
Cl
Cl
H
8h
CH3
Cl
H
8i
> 30
9d
2.60 0.67
5.84 1.02
2.81 0.03
1.29 0.4
10.23 0.23
25.54 6.19
9e
OH
H
CH3
Cl
H
9f
OH
H
9g
OH
Cl
Cl
Cl
H
9h
OH
CH3
Cl
H
9i
OH
13a
13b
13c
SGI-1776
OH
> 30
> 30
OH
Cl
Br
H
O
OH
H
O
> 30
4.86 0.16
a GI50 value is the concentration that inhibits 50% of cell growth compared with the untreated cells. Each value was
measured at least three times.
The most promising compound 9g was selected to investigate the mechanism of action in PC-3 cells by flow
cytometry. As shown in Figure 2A, after treatment with 9g at the concentration of 8 μM and 12 μM for 24 h separately,
there was a slight increase in the G0/G1 phase (65.2% and 64.5%, respectively) as compared to untreated cells (57.1%).
As to the 16 μM concentration, an increase in the Sub-G1 phase (31.5%) was observed as compared to untreated cells
(1.7%), indicating apoptosis occurred in PC-3 cells. In general, these results indicated that the anti-proliferation of 9g
was mediated by cell cycle arrest as well as apoptosis induction. To further study the apoptosis effects, PC-3 cells were
treated with 9g for a prolonged 48 h at above-mentioned concentrations. There was a significant increase in the number
of cells underwent apoptosis at 48 h than 24 h when treated with 9g. (Figure 2B).
(Figure 2. should be listed here.)

Figure 2. The effects of 9g on PC-3 cell cycle progression. PC-3 cells were treated with 9g at various concentrations (0,
8, 12 and 16 μM) for 24 h (A) or 48 h (B), then harvested, stained with PI, and analyzed by flow cytometry.
Next, the levels of cell cycle and apoptosis related proteins were determined by western blot assay in PC-3 cells
treated with 9g at different concentrations for 24 h. The results (as shown in Figure 3) indicated that 9g could
efficiently decrease the level of c-Myc as well as the phosphorylation level of 4E-BP1 and BAD. Whereas, an increased
level of p21 and p27 was observed in a concentration-dependent manner. Previously it was reported that 9d could
inhibit ERK phosphorylation in T-HEp3 cells after short time treatment.6 Interestingly, we found that 9g could increase
the phosphorylation level of ERK in PC-3 cells. Therefore, it is prone to illustrate that 9g undergone different
mechanism in PC-3 cells comparing with T-HEp3 cells. It has been proved that BAD and 4E-BP1 could be
phosphorylated by Pim-1, thus promoting cell survival and proliferation.14 Moreover, Pim-1 could destabilize p21 and
p27 through phosphorylation at the same time.15 As a whole, compound 9g could lead to a decreased phosphorylation
level of BAD and 4E-BP1, and an increased level of p21 and p27, 9g might be a promising Pim-1 direct inhibition
agent.
(Figure 3. should be listed here.)

Figure 3. The regulation of cell cycle and apoptosis related proteins in PC-3 cells treated with 9g for 24 h.
Further more, compounds 3a, 8a and 9g were selected to examine their Pim-1 kinase inhibition activity at a range
of 10 concentrations and the IC50 values were calculated. Compound 9g was more potent to inhibit Pim-1 activity with
IC50 value of 0.75 μM (Figure 4), while 3a and 8a were less effective (IC50 > 10 μM). SGI-1776 was used as positive
control with IC50 of 0.048 μM in the assay. These profiles in combination with cell growth inhibition data of compounds
3a, 8a and 9g listed in Table 1 indicated that Pim-1 was a potential target of these compounds.
(Figure 4. should be listed here.)
Figure 4. The inhibitory activities of compounds 3a, 8a and 9g on Pim-1 kinase. The ratio between the signals of 620
and 665 nm was used in the calculation of the inhibition rates.
Docking analysis was carried out to further illustrate the biological activity of these compounds against Pim-1
using the Discovery Studio 3.0 software package. Compounds 3a, 8a and 9g with diverse Pim-1 inhibitory activity were

selected for docking evaluation as representative examples and the predicted binding modes were shown in Figure 5.
(Figure 5. should be listed here.)
Figure 5. The binding modes of compound 9g (B), 3a (C), 8a (D) and their superimposition (A) in the active site of
Pim-1 kinase (1YXT in PDB). H-Bonding interactions were presented with green line.
The docking studies revealed that these compounds interacted with Pim-1 in a non-ATP mimetic binding mode
without forming the typical hydrogen bond with the hinge region. Superimposition of 3a, 8a and 9a (Figure 5A)
revealed a similar positioning in the active site of Pim-1 kinase. Analysis of the docked complexes suggested that the
most potent compound 9g (Figure 5B) with a hydroxyl at C-8 position of quinoline ring allowed the bidentate H-bonds
formation with residue Asn172 and residue Asp186 (a crucial interaction for the binding with receptor protein, also
found in many known inhibitors co-crystallized with Pim-1). In addition, the -NH- linked quinoline ring and pyridine
ring as a hydrogen-bond donor formed a third hydrogen bond with the polar residue Asp128. Owing to the absence of
hydroxyl at C-8 position, compound 3a (Figure 5C) interacted with Pim-1 by means of only one hydrogen bond
exhibited inferior Pim-1 inhibition activity. As for Compound 8a (Figure 5D), the -NH- moiety was replaced by -S-,
which led to a tremendous change of dihedral angle between quinoline ring and pyridine ring. In such circumstances,
only one hydrogen bond was presented between the hydroxyl group and residue Asn172, causing a sharp decrease in
Pim-1 inhibition activity. These docking results were in parallel with the observation in enzymatic assay. Apart from

hydrogen bonding, the pyridine ring of these inhibitors, especially compound 9g, showed hydrophobic and van der
Waals interactions with residues Leu44, Gly45 and Val52 (P-loop) of the receptor protein, which likely provided a
significant contribution to Pim-1 binding and inhibition. By analyzing the events mentioned above, we draw the
conclusion that the hydroxyl group at C-8 position of quinoline ring and the -NH- moiety played a crucial role in
enhancing the Pim-1 inhibition ability.
3. Conclusion
We reported herein the synthesis of a series quinoline derivatives as anti-proliferative agents. The specific
structure-activity relationship of these quinolines was summarized in detail simultaneously. 9g as the most excellent
compound with GI50 value of 1.29 μM was used for further mechanistic studies. The results indicated that the tumor cell
growth inhibitory effects were correlated with the decreased levels of phosphorylated BAD and 4E-BP1 as well as the
increased levels of p21 and p27, which led to the discovery of Pim-1 as a potential target of this quinoline derivative.
Further enzymatic assay revealed that 9g exhibited a good potency in inhibiting Pim-1 (IC50 = 0.75 μM). Molecular
docking analysis showed that the hydroxyl on quinoline ring and the secondary amine linked quinoline and pyridine
ring contributed to the Pim-1 activity inhibition. This study provides a novel class of Pim-1 inhibitors bearing quinoline
scaffold, which deserves further research.
4. Experimental section
4.1. Chemistry
The melting points were determined with an electrically heated X-4 digital visual melting point apparatus and were
1
uncorrected. Mass spectra (MS) were measured with a Finnigan MAT/USA spectrometer (LC-MS). H NMR and 13C
NMR spectrum were recorded on a Bruker ARX-300 or AV-400 instrument with tetramethylsilane (TMS) used as the
internal standard. Chemical shifts were reported in ppm (δ). Infrared (IR) spectra (KBr disks) were recorded on a
Bruker IFS55 instrument. High–resolution mass spectra were obtained on Bruker microTOF–Q in the ESI mode
(HR–ESI–MS). All reactions were performed with commercially available reagents and they were used without further

purification. All reactions were monitored by thin-layer chromatography (TLC) carried on fluorescent precoated plates
GF254 (Qindao Haiyang Chemical, China) and detection of the components was made by short UV light. Column
chromatography was performed with silica gel 60 (200–300 mesh).
4.1.1. General procedure for the synthesis of 3a–c
The m-chloroperoxybenzoic acid (m-CPBA) (9.49 g, 0.055 mol) was added to a solution of quinoline 1 (3.51 g,
0.027 mol) in CHCl3 (100 mL) at 0°C within 30 min and the mixture was stirred for 24 h at room temperature. Then, the
reaction was quenched with saturation NaHCO3 and extracted with CHCl3 (50 mL × 3). The combined organic extracts
were washed with brine (50 mL), dried with Na2SO4, filtered and evaporated. Finally, the resulting residue was purified
by column chromatography on silica gel as indicated to give 2 as a brown solid.
The p-toluenesulfonyl chloride (1.9g, 0.01 mol) and substituted 2-aminopyridines (0.01 mol) were added to a
solution of 2 (1.45g, 0.01 mol) in CH2Cl2 (60 mL) and stirred for 30 min. Then, 10% NaOH (40 mL) was added and
stirred for another 2 h at room temperature. The water layer was separated, and several times extracted with CH2Cl2.
The combined CH2Cl2 solutions were dried with Na2SO4, filtered and evaporated. The resulting residue was then
purified by column chromatography on silica gel as indicated to give 3 as a white solid.
4.1.1.1. N-(pyridin-2-yl)quinolin-2-amine (3a)
Yield: 40.2%. Mp: 105-108°C. ESI-MS: m/z, 222.1 [M+H]+. IR (KBr): 3275, 1585, 1529, 1433, 1299, 1226, 816
1
cm-1. H-NMR (300 MHz, DMSO-d6) δ: 6.95 (t, 1H, J=5.7Hz, H-5’), 7.34 (m, 1H, H-7), 7.60 (m, 2H, H-3, 6), 7.76 (m,
3H, H-4’, 5, 8), 8.13 (d, 1H, H-4), 8.27 (d, 1H, J=5.7Hz, H-6’), 8.50 (d, J=8.4Hz, 1H, H-3’), 10.05 (s, 1H, -NH-). HRMS
(ESI+) m/z calcd for C14H12N3 [M+H]+ 222.1031, found: 222.1023.
4.1.1.2. N-(4-methylpyridin-2-yl)quinolin-2-amine (3b)
Yield: 37.5%. Mp: 90-93°C. ESI-MS: m/z, 236.1 [M+H]+. IR (KBr): 3289, 3062, 1583, 1531, 1429, 1224, 828,
1
753 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 2.35 (s, 3H, CH3-4’), 6.80 (d, 1H, J=5.1Hz, H-5’), 7.32 (m, 1H, H-7), 7.62
(m, 2H, H-3, 6), 7.76 (m, 2H, H-5, 8), 8.12 (m, 2H, H-4, 6’), 8.29 (s, 1H, H-3’), 9.96 (s, 1H, -NH-). 13C-NMR (400 MHz,

CDCl3) δ: 21.67 (-CH3), 113.30 (C-3), 114.05 (C-3’), 115.23 (C-4’), 118.75 (C-10), 123.78 (C-6), 124.54 (C-8), 126.91
(C-5), 127.59 (C-7), 129.84 (C-4), 137.83 (C-6’), 147.38 (C-9), 149.47 (C-4’), 153.06 (C-2’), 154.06 (C-2). HRMS
(ESI+) m/z calcd for C15H14N3 [M+H]+ 236.1188, found: 236.1181.
4.1.1.3. N-(4-chloropyridin-2-yl)quinolin-2-amine (3c)
Yield: 41.3%. Mp: 163-165°C. ESI-MS: m/z, 256.1, 258.1 [M+H]+. IR (KBr): 3255, 3174, 3007, 1580, 1560, 1531,
1
1422, 1281, 1226, 880, 814 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 7.07 (d, 1H, J=5.4Hz, H-5’), 7.38 (t, 1H, J=7.8Hz,
H-7), 7.54 (d, 1H, J=8.9Hz, H-3), 7.64 (d, 1H, J=7.8Hz, H-6), 7.78 (m, 2H, H-5, 8), 8.17 (d, 1H, J=9.0Hz, H-4), 8.26 (d,
1H, J=5.4Hz, H-6’), 8.72 (s, 1H, H-3’), 10.34 (s, 1H, -NH-). HRMS (ESI+) m/z calcd for C14H11ClN3 [M+H]+ 256.0642,
found: 256.0636.
4.1.2. General procedure for the synthesis of compounds 7a–i.
The K2CO3 (2.7g, 0.02 mol) or KOH (1.6g, 0.03 mol) was added to a solution of 5-substituted 8-hydroxy quinoline
4a–c (0.01 mol) in acetone or THF (200 mL) at ambient temperature and the mixture was stirred for 30 min. Then the
methyl iodide (CH3I) (2.8g, 0.02 mol) was added dropwise and stirred overnight. The mixture solution was concentrated
to dryness, added water and extracted with CH2Cl2 (150 mL × 3). Finally, the organic layers were combined, washed
with brine, dried with Na2SO4, filtered and evaporated. 5a–c were obtained as brown oil, which could be used without
further purification.
The m-chloroperoxybenzoic acid (m-CPBA) (9.49g, 0.055 mol) was added to a solution of 5-substituted 8-methoxy
quinoline (0.027 mol) in CHCl3 (100 mL) at 0°C within 30 min and the mixture was stirred for 24 h at room
temperature. Then, the reaction was quenched with saturation NaHCO3 and extracted with CHCl3 (50 mL × 3). The
combined organic extracts were washed with brine (50 mL), dried with Na2SO4, filtered and evaporated. Finally, the
resulting residue was purified by column chromatography on silica gel as indicated to give 6a–c as brown solid .
The p-toluenesulfonyl chloride (1.9g, 0.01 mol) and substituted pyridines (0.01 mol) were added to a solution of
6a–c (0.01 mol) in 60 mL CH2Cl2 and stirred for 30 min. Then, 10% NaOH (40 mL) was added and stirred for another 2

h at room temperature. The water layer was separated, and several times extracted with CH2Cl2. The combined CH2Cl2
solutions were dried with Na2SO4, filtered and evaporated. The resulting residue was then purified by column
chromatography on silica gel as indicated to give 7a–i as a white solid.
4.1.2.1. 8-methoxyquinoline (5a)
1
Yield: 90.0%. ESI-MS: m/z, 160.0 [M+H]+, 182.0 [M+Na]+. H-NMR (300 MHz, DMSO-d6) δ: 3.96 (s, 3H),
7.17(m, 1H), 7.51 (m, 3H), 8.29 (m, 1H), 8.84 (m, 1H).
4.1.2.2. 8-methoxy-2-(pyridin-2-ylthio)quinoline (7a)
Yield: 37.3%. Mp: 70-72°C. ESI-MS: m/z, 269.0 [M+H]+. IR (KBr): 2928, 1613, 1590, 1559, 1492, 1460, 1422,
1379, 1321, 1303, 1261 cm-1. 1H-NMR (300 MHz, DMSO-d6) δ: 3.95 (s, 3H, -OCH3), 7.22 (dd, 1H, J=0.9Hz, J=7.5Hz,
H-3’), 7.35 (m, 1H, H-5), 7.49 (m, 3H, H-5’, 6, 7), 7.72 (d, 1H, J=8.9Hz, H-3), 7.83 (td, 1H, J=1.5Hz, J=7.5Hz, H-4’),
8.25 (d, 1H, J=9.0Hz, H-4), 8.54 (d, 1H, J=3.9Hz, H-6’).
4.1.2.3. 5-chloro-8-methoxy-2-(pyridin-2-ylthio)quinoline (7b)
Yield: 47.9%. Mp: 89-90°C. ESI-MS: m/z, 303.3, 305.3 [M+H]+. IR (KBr): 3100, 3037, 2940, 2841, 1604, 1589,
1
1573, 1560, 1464, 1492, 1418, 1384, 1316 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 3.96 (s, 3H, -OCH3), 7.21 (d, 1H,
J=8.4Hz, H-7), 7.39 (m, 1H, H-5’), 7.60 (d, 1H, J=9.0Hz, H-3), 7.65 (d, 1H, J=8.4Hz, H-6), 7.79 (d, 1H, J=7.5Hz,
H-3’), 7.86 (m, 1H, H-4’), 8.39 (d, 1H, J=9.0Hz, H-4), 8.57 (d, 1H, J=4.2Hz, H-6’).
4.1.2.4. 5-bromo-8-methoxy-2-(pyridin-2-ylthio)quinoline (7c)
Yield: 47.3%. Mp: 99-101°C. ESI-MS: m/z, 347.2, 349.2 [M+H]+. IR (KBr): 3447, 3099, 3037, 2938, 2839, 1601,
1
1586, 1573, 1559, 1486, 1461, 1451, 1314 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 4.04 (s, 3H, -OCH3), 6.93 (d, 1H,
J=8.4Hz, H-3), 7.23 (m, 1H, H-5’), 7.55 (d, 1H, J=9.0Hz, H-7), 7.69 (m, 3H, H-3’, 4’, 6), 8.35 (d, 1H, J=8.7Hz, H-4),
8.59 (d, 1H, J=4.8Hz, H-6’).
4.1.3. General procedure for the synthesis of compounds 8a–c.
The intermediates 7a–c (0.80 mmol) were dissolved in dry CH2Cl2 (20 mL), and cooled below -40°C. Then the

BBr3 (1 mL, 2 M in dry CH2Cl2) was added dropwise under nitrogen atmosphere and the mixture was stirred for 30 min.
Warmed to room temperature for 2 h, and heated to boiling point for another 2 h. The reaction was quenched with
saturation NaHCO3 solution in an ice-cooled bath, and the PH of the mixture was adjusted to 7.0. Then the water layer
was separated, several times extracted with CH2Cl2. The combined CH2Cl2 solutions were dried with Na2SO4, filtered
and evaporated. The resulting residue was then purified by column chromatography on silica gel as indicated to give
8a–c as white solid.
4.1.3.1. 2-(pyridin-2-ylthio)quinolin-8-ol (8a)
Yield: 88.9%. Mp: 163-165°C. ESI-MS: m/z, 255.0 [M+H]+. IR (KBr): 3358, 1619, 1583, 1560, 1517, 1278 cm-1.
1H-NMR (300 MHz, DMSO-d6) δ: 7.12 (d, 1H, J=6.6Hz, H-7), 7.38 (m, 4H, H-3’, 5’, 5, 6), 7.65 (d, 1H, J=8.8Hz, H-3),
7.82 (td, 1H, J=1.5Hz, J=7.5Hz, H-4’), 8.23 (d, H, J=9.0Hz, H-4), 8.55 (d, H, J=4.2Hz, H-6’), 9.71 (s, 1H, -OH).
13C-NMR (400 MHz, DMSO-d6) δ: 113.63 (C-7), 118.39 (C-3), 123.60 (C-3’, C-5’), 126.99 (C-5), 127.88 (C-10),
128.35 (C-6), 137.93 (C-4), 138.55 (C-9), 139.59 (C-4’), 149.44 (C-6’), 152.58 (C-8), 154.98 (C-2’), 155.12 (C-2).
HRMS (ESI+) m/z calcd for C14H11N2OS [M+H]+ 255.0592, found: 255.0586.
4.1.3.2. 5-chloro-2-(pyridin-2-ylthio)quinolin-8-ol (8b)
Yield: 97.8%. Mp: 180-182°C. ESI-MS: m/z, 289.2, 291.2 [M+H]+. IR (KBr): 3044, 1608, 1576, 1492, 1459, 1258,
1
1233 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 7.11 (d, 1H, J=8.1Hz, H-7), 7.42 (m, 1H, H-5’), 7.55 (d, 1H, J=2.9Hz,
H-7), 7.58 (d, 1H, J=8.0Hz, H-6), 7.74 (d, 1H, J=8.8Hz, H-3), 7.88 (m, 1H, H-4’), 8.38 (d, H, J=9.0Hz, H-4), 8.58 (dd,
1H, J=0.9Hz, J=4.8Hz, H-6’). HRMS (ESI+) m/z calcd for C14H10ClN2OS [M+H]+ 289.0202, found: 289.0915.
4.1.3.3. 5-bromo-2-(pyridin-2-ylthio)quinolin-8-ol (8c)
Yield: 87.5%. Mp: 92-93°C. ESI-MS: m/z, 333.2, 335.2 [M+H]+. IR (KBr): 3353, 1836, 1736, 1644, 1574, 1558,
1
1256 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 7.07 (d, 1H, J=8.0Hz, H-7), 7.39 (m, 1H, H-5’), 7.56 (d, 1H, J=8.1Hz,
H-6), 7.72 (m, 2H, H-3, 3), 7.85 (td, 1H, J=1.5Hz, 7.5Hz, H-4’), 8.31 (d, 1H, J=9.0Hz, H-4)，8.57 (dd, 1H, J=0.9Hz,
4.8Hz, H-6’), 10.09 (s, 1H, -OH). HRMS (ESI+) m/z calcd for C14H10BrN2OS [M+H]+ 332.9697, found: 332.9691.

4.1.4. General procedure for the synthesis of compounds 8d–8i and 9d–i.
A solution of compounds 7d–i (1.70 mmol) in concentrated HCl (5 mL) was heated for 15 h at the boiling point.
The mixture was evaporated to dryness, and dissolved in water. Then the solution was made alkaline by adding K2CO3
and extracted with CH2Cl2. The combined organic extracts were washed with brine, dried with Na2SO4, filtered and
evaporated. The residue was then purified by column chromatography on silica gel as indicated to give compounds 8d–i.
Finally, 8d–i reacted with BBr3 at the same reaction condition as making 8a–c to yield 9d–i.
4.1.4.1. 8-methoxy-N-(pyridin-2-yl)quinolin-2-amine (8d)
Yield: 70.0%. Mp: 220-222°C. ESI-MS: m/z, 252.2 [M+H]+. IR (KBr): 3394, 2850, 1614, 1604, 1564, 1483, 1274,
1107, 833, 567 cm-1. 1H-NMR (300 MHz, DMSO-d6) δ: 3.96 (s, 3H, -OCH3), 6.93 (m, 1H, H-5’), 7.12 (d, 1H, J=7.8Hz,
H-7), 7.26 (t, 1H, J=7.8Hz, H-6), 7.33 (m, 1H, H-5) 7.69 (d, 1H, J=9.0Hz, H-3), 7.74 (m, 1H, H-4’), 8.10 (d, 1H,
J=9.0Hz, H-4), 8.26 (m, 1H, H-6’), 8.53 (d, 1H, J=8.4Hz, H-3’), 10.05 (s, 1H, -NH-). 13C-NMR (400 MHz, DMSO-d6)
δ: 56.18 (-OCH3), 109.52 (C-7), 112.69 (C-3’), 114.67 (C-3), 117.01 (C-5), 119.75 (C-5’), 123.70 (C-6), 125.27 (C-10),
137.56 (C-4), 138.23 (C-9), 138.49 (C-4’), 147.93 (C-6’), 152.80 (C-8), 154.20 (C-2), 152.62 (C-2’). HRMS (ESI+) m/z
calcd for C15H14N3O [M+H]+ 252.1137, found: 252.1128.
4.1.4.2. 8-methoxy-N-(4-methylpyridin-2-yl)quinolin-2-amine (8e)
Yield: 75.2%. Mp: 186-189°C. ESI-MS: m/z, 266.2 [M+H]+. IR (KBr): 3391, 2831, 1656, 1616, 1556, 1427, 1384,
1
1170, 1105, 839, 749 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 2.34 (s, 3H, -CH3), 3.97 (s, 3H, -OCH3), 6.79 (d, 1H,
J=4.8Hz, H-5’), 7.11 (d, 1H, J=7.8Hz, H-7), 7.25 (t, 1H, J=7.8Hz, H-6), 7.33 (d, 1H, J=7.8Hz, H-5), 7.64 (d, 1H,
J=9.0Hz, H-3), 8.08 (d, 1H, J=9.0Hz, H-4), 8.12 (d, 1H, J=4.8Hz, H-6’), 8.51 (s, 1H, H-3’), 9.97 (s, 1H,
-NH-).13C-NMR (400 MHz, CDCl3) δ: 21.44 (-CH3), 56.07 (-OCH3), 108.45 (C-7), 112.63 (C-3’), 113.99 (C-3), 118.34
(C-5), 119.52 (C-5’), 119.58 (C-6), 123.44 (C-10), 125.45 (C-4), 137.73 (C-9), 147.30 (C-6’), 149.22 (C-4’), 152.31
(C-8), 153.90 (C-2), 154.04 (C-2’). HRMS (ESI+) m/z calcd for C16H16N3O [M+H]+ 266.1293, found: 266.1287.
4.1.4.3. 8-methoxy-N-(4-chloropyridin-2-yl)quinolin-2-amine (8f)

Yield: 80.1%. Mp: 100-103°C. ESI-MS: m/z, 286, 288 [M+H]+. IR (KBr): 3451, 2923, 1622, 1583, 1542, 1427,
1
1384, 1100, 988, 796 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 3.92 (s, 3H, -OCH3), 7.01 (d, 1H, J=5.4Hz, H-5’), 7.09
(d, 1H, J=7.8Hz, H-7), 7.24 (m, 1H, J=7.8Hz, H-6), 7.30 (d, 1H, J=3.9Hz, H-5), 7.46 (d, 1H, J=9.0Hz, H-3), 8.08 (d,
1H, J=9.0Hz, H-4), 8.19 (d, 1H, J=5.4Hz, H-6’), 8.99 (s, 1H, H-3’), 10.34 (s, 1H, -NH-). HRMS (ESI+) m/z calcd for
C15H13ClN3O [M+H]+ 286.0747, found: 286.0743.
4.1.4.4. 5-chloro-8-methoxy-N-(pyridin-2-yl)quinolin-2-amine (8g)
Yield: 76.7%. Mp: 179-182°C. ESI-MS: m/z, 286.1, 288.1 [M+H]+. IR (KBr): 3442, 3260, 3051, 1587, 1546, 1436,
1
1402, 1315, 1250, 1099, 810, 782 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 3.97 (s, 3H, -OCH3), 6.97 (m, 1H, H-5’),
7.11 (d, 1H, J=8.7Hz, H-7), 7.40 (d, 1H, J=8.5Hz, H-6), 7.77 (m, 1H, H-4’), 7.84 (d, 1H, J=9.3Hz, H-3), 8.30 (d, 2H,
J=9.0Hz, H-4, 6’), 8.52 (d, 1H, J=8.4Hz, H-3’), 10.27 (s, 1H, -NH-). HRMS (ESI+) m/z calcd for C15H13ClN3O [M+H]+
286.0747, found: 286.0741.
4.1.4.5. 5-chloro-8-methoxy-N-(4-methylpyridin-2-yl)quinolin-2-amine (8h)
Yield: 63.3%. Mp: 175-178°C. ESI-MS: m/z, 300.1, 302.1 [M+H]+. IR (KBr): 3439, 1500, 1428, 1381, 1249, 1098,
798, 782 cm-1. 1H-NMR (300 MHz, DMSO-d6) δ: 2.35 (s, 3H, -CH3), 3.98 (s, 3H, -OCH3), 6.83 (d, 1H, J=5.1Hz, H-5’),
7.11 (d, 1H, J=8.4Hz, H-7), 7.39 (d, 1H, J=8.4Hz, H-6), 7.78 (d, 1H, J=9.0Hz, H-3), 8.14 (d, 1H, J=5.1Hz, H-6’), 8.28
(d, 1H, J=9.0Hz, H-4), 8.51 (s, 1H, H-3’), 10.20 (s, 1H, -NH-). 13C-NMR (300 MHz, DMSO-d6) δ: 21.40 (-CH3), 56.15
(-OCH3), 109.37 (C-7), 113.27 (C-3), 115.48 (C-3’), 118.35(C-5), 121.09 (C-10), 121.84 (C-5’), 123.10 (C-6), 133.56
(C-4), 139.12 (C-9), 147.40 (C-6’), 148.54 (C-4’), 153.03 (C-8), 153.29 (C-2’), 154.16 (C-2). HRMS (ESI+) m/z calcd
for C16H15ClN3O [M+H]+ 300.0904, found: 300.0897.
4.1.4.6. 5-chloro-8-methoxy-N-(4-chloropyridin-2-yl)quinolin-2-amine (8i)
Yield: 66.0%. Mp: 242-245°C. ESI-MS: m/z, 320.0, 322.0, 324.0 [M+H]+. IR (KBr): 3437, 1572, 1532, 1421,
1
1384, 1098, 929, 786 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 3.97 (s, 3H, -OCH3), 7.10 (d, 1H, J=5.4Hz, H-5’), 7.14
(d, 1H, J=8.4Hz, H-7), 7.44 (d, 1H, J=8.4Hz, H-6), 7.67 (d, 1H, J=9.3Hz, H-3), 8.27 (d, 1H, J=5.4Hz, H-6’), 8.33 (d,

1H, J=9.3Hz, H-4), 9.03 (s, 1H, H-3’), 10.56 (s, 1H, -NH-). HRMS (ESI+) m/z calcd for C15H12Cl2N3O [M+H]+
320.0357, found: 320.0350.
4.1.4.7. 2-(pyridine-2-amino)quinolin-8-ol (9d)
Yield: 75.4%. Mp: 130-134°C. ESI-MS: m/z, 238.2 [M+H]+. IR (KBr): 3451, 3262, 2924, 1590, 1551, 1460, 1439,
1384, 1319, 1142, 776 cm-1. 1H-NMR (300 MHz, DMSO-d6) δ: 6.94 (m, 1H, H-5’), 7.01 (m, H, H-7), 7.24 (m, 2H, H-5,
6), 7.54 (d, 1H, J=9.0Hz, H-3), 7.76 (m, 1H, H-4’), 8.08 (d, 1H, J=9.0Hz, H-4), 8.26 (m, 1H, H-6’), 8.57 (d, J=8.4Hz,
1H, H-3’), 9.00 (s, 1H, -OH), 10.01 (s, 1H, -NH-). HRMS (ESI+) m/z calcd for C14H12N3O [M+H]+ 238.0980, found:
238.0975.
4.1.4.8. 2-(4-methylpridine-2-amino)quinolin-8-ol (9e)
Yield: 66.7%. Mp: 90-92°C. ESI-MS: m/z, 252.1 [M+H]+. IR (KBr): 3358, 2919, 1647, 1618, 1587, 1567, 1525,
1
1507, 1455, 1430, 1358. H-NMR (300 MHz, DMSO-d6) δ: 2.34 (s, 3H, -CH3), 6.80 (d, 1H, J=4.8Hz, H-5’), 7.03 (d,
1H, J=7.2Hz, H-7), 7.20 (m, 2H, H-5, 6), 7.58 (d, 1H, J=9.0Hz, H-3), 8.08 (d, 1H, J=9.0Hz, H-4), 8.14 (d, 1H, J=4.8Hz,
H-6’), 8.34 (s, 1H, H-3’), 9.07 (s, 1H, -OH), 9.93 (s, 1H, -NH-). HRMS (ESI+) m/z calcd for C15H14N3O [M+H]+
252.1137, found: 252.1131.
4.1.4.9. 2-(4-chloropridine-2-amino)quinolin-8-ol (9f)
1
Yield: 73.2%. Mp: 155-157°C. ESI-MS: m/z, 272.2, 274.2 [M+H]+. H-NMR (400 MHz, DMSO-d6) δ: 7.04 (m,
2H, H-5’, 7), 7.26 (m, 2H, H-5, 6), 7.46 (d, 1H, J=9.0Hz, H-3), 8.10 (d, 1H, J=9.0Hz, H-4), 8.24 (d, 1H, J=5.0Hz, H-6’),
8.92 (s, 1H, H-3’), 9.44 (s, 1H, -OH), 10.27 (s, 1H, -NH-). 13C-NMR (400 MHz, DMSO-d6) δ: 112.12 (C-3’), 112.99
(C-7), 114.75 (C-3), 117.06 (C-5’), 118.31 (C-5), 124.46 (C-6), 125.57 (C-10), 137.37 (C-4), 137.92 (C-9), 144.23
(C-4’), 149.39 (C-8), 151.90 (C-6’), 152.20 (C-2), 155.45 (C-2’). HRMS (ESI+) m/z calcd for C14H11ClN3O [M+H]+
272.0591, found: 272.0584.
4.1.4.10. 5-chloro-2-(pyridin-2-ylamino)quinolin-8-ol (9g)
Yield: 69.2%. Mp: 196-198°C. ESI-MS: m/z, 272.2, 274.2 [M+H]+. IR (KBr): 3442, 2922, 2852, 1589, 1549, 1437,

1384, 1318, 1141, 820, 781 cm-1. 1H-NMR (300 MHz, DMSO-d6) δ: 6.97 (m，1H, H-5’)，7.08 (d，1H，J=8.4Hz, H-7)，
7.38 (d，1H，J=8.4Hz, H-6)，7.77 (m，2H, H-3, 4’)，8.28 (m，2H，H-4, H-6’), 8.53 (m, 1H, H-3’), 9.44 (s, 1H, -OH),
10.19 (s, 1H, -NH-).13C-NMR (400 MHz, DMSO-d6) δ: 112.97 (C-3’), 113.18 (C-7), 116.10 (C-3), 117.67 (C-5’),
119.66 (C-5), 122.29 (C-10), 123.96 (C-6), 134.11 (C-4), 138.27 (C-9), 138.58 (C-4’), 147.97 (C-8), 151.28 (C-6’),
152.83 (C-2), 154.02 (C-2’). HRMS (ESI+) m/z calcd for C14H11ClN3O [M+H]+ 272.0591, found: 272.0584.
4.1.4.11. 5-chloro-2-(4-methylpyridin-2-ylamino)quinolin-8-ol (9h)
Yield: 52.1%. Mp: 179-181°C. ESI-MS: m/z, 286, 288 [M+H]+. IR (KBr): 3407, 2923, 1599, 1568, 1429, 1346,
1
1384, 1305, 1115, 809, 783 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 2.33 (s, 3H, -CH3), 6.83 (d, 1H, J=4.8Hz, H-5’),
7.01 (d, 1H, J=8.1Hz, H-7), 7.29 (d, 1H, J=8.1Hz, H-6), 7.73 (d, 1H, J=9.0Hz, H-3), 8.14 (d, 1H, J=4.8Hz, H-6’), 8.26
(d, 1H, J=9.0Hz, H-4), 8.38 (s, 1H, H-3’), 9.47 (brs, 1H, -OH), 10.09 (s, 1H, -NH-).13C-NMR (400 MHz, DMSO-d6) δ:
112.87 (C-3’), 113.46 (C-7), 116.04 (C-3), 118.78 (C-5’), 119.63 (C-5), 122.27 (C-6), 123.85 (C-10), 133.99 (C-4),
138.40 (C-9), 147.58 (C-4’), 148.99 (C-8), 151.35 (C-6’), 152.90 (C-2), 154.16 (C-2’). HRMS (ESI+) m/z calcd for
C14H9ClN3O [M+H]+ 286.0747, found: 286.0739.
4.1.4.12. 5-chloro-2-(4-chloropyridin-2-ylamino)quinolin-8-ol (9i)
Yield: 63.1%. Mp: 171-173°C. ESI-MS: m/z, 304, 306, 308 [M+H]+. IR (KBr): 3431, 2915, 1576, 1538, 1435,
1
1438, 1394, 1299, 1215, 795, 735 cm-1. H-NMR (400 MHz, DMSO-d6) δ: 7.05(m, 2H, H-5’, 7), 7.31(s, 1H, H-6),
7.60(s, 1H, H-3), 8.27(s, 2H, H-4, 6’), 8.95(s, 1H, H-3’), 9.85(s, 1H, -OH), 10.46(s, 1H, -NH-). HRMS (ESI+) m/z calcd
for C15H12Cl2N3O [M+H]+ 306.0201, found: 306.0196.
4.1.5. General procedure for the synthesis of compounds 13a–c.
The Et3N (6.05 mL) was added to a solution of substituted 8-hydroxyquinoline 4a–c (0.035 mol) in CH2Cl2 (100
mL). To this mixture, the methanesulfonyl chloride (0.038 mol, dissolved in 20 mL CH2Cl2) was added dropwise in an
ice-salt bath and stirred for 1 h. The mixture solution was concentrated to dryness, added water and extracted with
CH2Cl2 (50 mL × 3). The combined organic extracts were washed with brine, dried with Na2SO4, filtered and

evaporated to afford 10a–c as white solid. Then the key intermediates were oxidized and coupled with
2-hydroxypyridine using the method described above, to give compounds 12a–c.
The 12a–c (0.001 mol) was dissolved in a mixture solution of ethanol and acetone (20 mL, v/v = 1:1). To this
solution, NaOH (8 mL, 1 M) was added and refluxed for 8 h. Then the PH of reaction liquid was adjusted to 5~6 by
HCl. The solution was extracted with CH2Cl2 (50 mL × 3), washed with brine, dried with Na2SO4, filtered and
evaporated to afford 13a–c as white solid.
4.1.5.1. 2-(pyridin-2-yloxy)quinolin-8-yl-methanesulfonate (12a)
Yield: 41.5%. Mp: 174-176°C. ESI-MS: m/z, 316.9 [M+H]+. IR (KBr): 3445, 3024, 2997, 2921, 1668, 1602, 1588,
1
1536, 1501, 1473, 1427, 1358, 1318 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 3.53 (s, 3H, -OSO2CH3), 6.47 (td, 1H,
J=0.9Hz, 6.9Hz, H-5’), 6.60 (d, 1H, J=6.2Hz, H-7), 7.60 (m, 1H, H-4’), 7.75 (d, 1H, J=8.5Hz, H-3), 7.84 (m, 1H, H-6),
8.00 (m, 2H, H-5, H-6’), 8.15 (dd, 1H, J=1.2Hz, 5.1 Hz, H-3’), 8.63 (d, 1H, J=8.7Hz, H-4).
4.1.5.2. 5-chloro-2-(pyridin-2-yloxy)quinolin-8-yl-methanesulfonate (12b)
Yield: 42.1%. Mp: 211-213°C. ESI-MS: m/z, 351.2, 353.2 [M+H]+. IR (KBr): 3448, 3029, 2931, 1667, 1599, 1542,
1
1493, 1470, 1390, 1364, 1274 cm-1. H-NMR (300 MHz, CDCl3) δ: 3.35 (s, 3H, -OSO2CH3), 6.40 (t, 1H, J=6.9Hz,
H-5’), 6.71 (d, 1H, J=9.0Hz, H-7), 7.47 (m, 1H, H-4’), 7.69 (m, 2H, H-3, 3’), 8.12 (d, 1H, J=6.4Hz, H-6’), 8.29 (d, 1H,
J=9.0Hz, H-6), 8.71 (d, 1H, J=9.0Hz, H-4).
4.1.5.3. 5-bromo-2-(pyridin-2-yloxy)quinolin-8-yl-methanesulfonate (12c)
Yield: 34.5%. Mp: 232-234°C. ESI-MS: m/z, 395.2, 397.2 [M+H]+. IR (KBr): 3447, 3016, 2932, 1665, 1595, 1541,
1
1491, 1469, 1389, 1363, 1348, 1321, 1275 cm-1. H-NMR (300 MHz, CDCl3) δ: 3.35 (s, 3H, -OSO2CH3), 6.37 (t, 1H,
J=6.6Hz, H-5’), 6.70 (d, 1H, J=8.5Hz, H-7), 7.41 (t, 1H, J=8.1Hz, H-4’), 7.66 (m, 2H, H-3, 3’), 8.11 (d, 1H, J=7.0Hz,
H-6’), 8.28 (d, 1H, J=8.5Hz, H-6), 8.70 (d, 1H, J=9.0Hz, H-4).
4.1.5.4. 2-(pyridin-2-yloxy)quinolin-8-ol (13a)
Yield: 93.6%. Mp: 226-228°C. ESI-MS: m/z, 239.0 [M+H]+. IR (KBr): 3226, 1662, 1595, 1575, 1538, 1502, 1275,

1
1231 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 6.43 (t, 1H, J=6.6Hz, H-5’), 6.55 (d, 1H, J=9.0Hz, H-7), 7.16 (dd, 1H,
J=2.0Hz, 6.9Hz, H-4’), 7.56 (m, 3H, H-3’, 5, 6), 7.87 (d, 1H, J=8.7Hz, H-3), 8.17 (dd, 1H, J=1.8Hz, 4.2Hz, H-6’), 8.43
(d, 1H, J=8.7Hz, H-4), 9.90 (s, 1H, -OH). 13C-NMR (400 MHz, DMSO-d6) δ: 106.33 (C-3’), 113.07 (C-7), 118.12 (C-3),
120.35 (C-5), 121.35 (C-10), 128.64 (C-5’), 128.73 (C-6), 137.57 (C-4), 138.19 (C-9), 138.26 (C-4’), 141.71 (C-6’),
150.59 (C-8), 153.68 (C-2’), 161.95 (C-2). HRMS (ESI+) m/z calcd for C14H11N2O2 [M+H]+ 239.0821, found:
239.0816.
4.1.5.5. 5-chloro-2-(pyridin-2-yloxy)quinolin-8-ol (13b)
Yield: 92.9%. Mp: 238-239°C. ESI-MS: m/z, 273.2, 275.2 [M+H]+. IR (KBr): 3125, 1667, 1594, 1571, 1538, 1504,
1288, 1265 cm-1. 1H-NMR (300 MHz, CDCl3) δ: 6.35 (t, 1H, J=6.9Hz, H-5’), 6.71 (d, 1H, J=9.3Hz, H-7), 7.14 (d, 1H,
J=8.4Hz, H-3’), 7.45 (m, 1H, H-4’), 7.54 (d, 1H, J=9.1Hz, H-6), 7.91 (d, 1H, J=9.0Hz, H-3), 8.10 (dd, 1H, J=1.5Hz,
J=6.9Hz, H-6’), 8.56 (d, H, J=9.0Hz, H-4), 10.28 (s, 1H, -OH). 13C-NMR (400 MHz, DMSO-d6) δ: 106.54 (C-3’),
113.34 (C-7), 119.12 (C-3), 121.40 (C-5), 121.59 (C-10), 125.87 (C-5’), 128.57 (C-6), 134.82 (C-4), 137.98 (C-9),
138.18 (C-4’), 141.89 (C-6’), 151.15 (C-8), 153.29 (C-2’), 161.92 (C-2). HRMS (ESI+) m/z calcd for C14H10ClN2O2
[M+H]+ 273.0431, found: 273.0428.
4.1.5.6. 5-bromo-2-(pyridin-2-yloxy)quinolin-8-ol (13c)
Yield: 91.1%. Mp: 233-235°C. ESI-MS: m/z, 316.8, 318.8 [M+H]+. IR (KBr): 3079, 1665, 1593, 1537, 1501, 1287,
1
1264 cm-1. H-NMR (300 MHz, DMSO-d6) δ: 6.45 (t, 1H, J=6.9Hz, H-5’), 6.58 (d, 1H, J=7.9Hz, H-7), 7.16 (d, 1H,
J=8.4Hz, H-3’), 7.59 (m, 1H, H-4’), 7.66 (d, 1H, J=8.0Hz, H-6), 8.07 (d, 1H, J=8.5Hz, H-3), 8.21 (dd, 1H, J=1.5Hz,
J=6.9Hz, H-6’), 8.58 (d, H, J=8.5Hz, H-4), 10.25 (s, 1H, -OH). HRMS (ESI+) m/z calcd for C14H10BrN2O2 [M+H]+
316.9926, found: 316.9917.
4.2. Biological assays
4.2.1. Anti-proliferative assays
Human prostatic cancer PC-3 cells were cultured in RPMI1640 with 10% (v/v) heat-inactivated fetal bovine serum.

The anti-proliferative activities of target compounds were evaluated by the methyl thiazolyl tetrazolium bromide (MTT)
assay as reported before.[16] Cells (2×103 cells/well) were incubated in a humidified atmosphere with 5% CO2 at 37°C
for 24 h, then various concentrations of each compound mixed in 100 μL medium were added to each well. After
incubated for another 96 h, MTT solution (50 μL of 2 mg/mL) was added per well and incubated for an additional 4 h.
The medium was removed by aspiration and the cells were dissolved in 200 μL DMSO. The absorbance at 570 nm was
measured in the 96-well plate reader. Growth inhibition was calculated and expressed as the ratio of the cell number in
the treated group to that of the untreated group. The GI50 values were calculated according to inhibition ratios. All
experiments were performed three times independently, and the results were reported presented as mean.
4.2.2. Flow cytometry
Human prostatic cancer PC-3 cells seeded in six-well plates were incubated with different concentrations of 9g for
24 and 48 h. After incubation, both floating and adherent cells were collected and washed once with phosphate-buffered
saline (PBS). Then cells were resuspended in ice-cold 70% ethanol, and stored at -20°C overnight. Followed by once
washed with PBS, the cells were treated with 1mg/mL RNase (purchased from Sigma Chemical Co.) at 37°C for 30
minutes. Propidium iodide (PI, purchased from Sigma Chemical Co.) was then add to a final concentration of 50
μg/mL and the DNA content was quantitated by flow cytometry with an excitation wavelength of 488 nm and an
emission wavelength of 625 nm. Data were analyzed using CELLQuest software.
4.2.3. Western blot analysis
The human prostatic cancer PC-3 cells were incubated in presence of the test compound 9g at different
concentrations for 24 h, harvested, and rinsed with ice-cold PBS. Total protein extracts (40 μg) were prepared with
RIPA lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 0.5% sodium
deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 μM leupeptin, and 2 μg/mL aprotinin (pH 8.0)].
Samples were separated on an 10% or 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The
membranes were stained with 0.2% Ponceau S red to assure equal protein loading and transfer. After blocking with 5%

nonfat milk, the membranes were incubated with a specific antibody of Parp, ERK, p-ERK, BAD, p-BAD, 4E-BP1,
p-4E-BP1, p21, p27, c-Myc and β-actin overnight at 4°C. Then incubated with secondary antibodies at 37 °C for 1 h.
Finally, detection was performed with an improved ECL reagent.
4.2.4. Pim-1 inhibition assay
The Ser/Thr KinEase assay kit (CisBio) was used to measure the inhibitory activity of Pim-1 kinase according to
the protocol. The HTRF kinase assay was conducted after optimizing the ATP, enzyme, and substrate concentrations.
Different concentrations of target compounds were incubated in an 8 µL reaction mixture (1 µM substrate S3, 36.7 µM
ATP, 5 mM MgCl2, 1 mM DTT and 1× KinEASE enzymatic buffer). The enzymatic reaction was started by adding 2 μL
Pim-1 (0.333 ng/μL), incubated at room temperature for 50 min, and terminated by adding 10 μL EDTA-containing
detection reagents, which were prepared according to kit instruction. HTRF signal was obtained by reading the plate in
Infinite® F500 microplate reader (Tecan, Switzerland) at 620 and 665 nm simultaneously. The ratio between the signals
of 620 and 665 nm was used in the calculation of the inhibition rate. SGI-1776 and Staurosporine were used as positive
controls with IC50 values of 48 and 22 nM, respectively.

Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grant No. 81273360).

References and notes
[1] Benard, C.; Zouhiri, F.; Normand-Bayle, M.; Danet, M.; Desmaele, D.; Leh, H.; Mouscadet, J. F.; Mbemba, G.;
Thomas, C. M.; Bonnenfant, S.; Le Bret, M.; d'Angelo, J. Bioorg. Med. Chem. Lett. 2004, 14, 2473-2476.
[2] Zajdel, P.; Partyka, A.; Marciniec, K.; Bojarski, A. J.; Pawlowski, M.; Wesolowska, A. Future Med. Chem. 2014, 6,
57-75.
[3] Kumar, A.; Paliwal, D.; Saini, D.; Thakur, A.; Aggarwal, S.; Kaushik, D. Eur. J. Med. Chem. 2014, 85, 147-178.
[4] Solomon, V. R.; Lee, H. Curr. Med. Chem. 2011, 18, 1488-1508.
[5] (a) De Clercq, E. J. Clin. Virol. 2004, 30, 115-133; (b) Burris, K. D. J. Pharmacol. Exp. Ther. 2002, 302, 381-389;
(c) Thome, R.; Lopes, S. C.; Costa, F. T.; Verinaud, L. Immunol. Lett. 2013, 153, 50-57; (d) Elisei, R.;
Schlumberger, M. J.; Muller, S. P.; Schoffski, P.; Brose, M. S.; Shah, M. H.; Licitra, L.; Jarzab, B.; Medvedev, V.;
Kreissl, M. C.; Niederle, B.; Cohen, E. E.; Wirth, L. J.; Ali, H.; Hessel, C.; Yaron, Y.; Ball, D.; Nelkin, B.; Sherman,
S. I. J. Clin. Oncol. 2013, 31, 3639-3646.
[6] Chaurasia, P.; Mezei, M.; Zhou, M. M.; Ossowski, L. PLoS One. 2009, 4, e4617.
[7] (a) Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.; Peng, C.; Kronkaitis, A.; Li, J.; White, A.;
Mische, S.; Farmer, B. J. Biol. Chem. 2005, 280, 6130-6137; (b) Bachmann, M.; Möröy, T. Int. J. Biochem. Cell
Biol. 2005, 37, 726-730.
[8] Wang, Z.; Bhattacharya, N.; Weaver, M.; Petersen, K.; Meyer, M.; Gapter, L.; Magnuson, N. S. J. Vet. Sci. 2001, 2,
167-179.
[9] (a) Amson, R.; Sigaux, F.; Przedborski, S.; Flandrin, G.; Givol, D.; Telerman, A. Proc. Natl. Acad. Sci. U. S. A.
1989, 86, 8857-8861; (b) Ellwood-Yen, K.; Graeber, T. G.; Wongvipat, J.; Iruela-Arispe, M. L.; Zhang, J. F.;
Matusik, R.; Thomas, G. V.; Sawyers, C. L. Cancer Cell. 2003, 4, 223-238; (c) Dhanasekaran, S. M.; Barrette, T. R.;
Ghosh, D.; Shah, R.; Varambally, S.; Kurachi, K.; Pienta, K. J.; Rubin, M. A.; Chinnaiyan, A. M. Nature. 2001,
412, 822-826; (d) Chen, W. W.; Chan, D. C.; Donald, C.; Lilly, M. B.; Kraft, A. S. Mol. Cancer Res. 2005, 3,
443-451.
[10] Casuscelli, F.; Ardini, E.; Avanzi, N.; Casale, E.; Cervi, G.; D'Anello, M.; Donati, D.; Faiardi, D.; Ferguson, R. D.;
Fogliatto, G.; Galvani, A.; Marsiglio, A.; Mirizzi, D. G.; Montemartini, M.; Orrenius, C.; Papeo, G.; Piutti, C.;
Salom, B.; Felder, E. R. Bioorg. Med. Chem. 2013, 21, 7364-7380.
[11] Merkel, A. L.; Meggers, E.; Ocker, M. Expert Opin. Inv. Drug. 2012, 21, 425-436.
[12] Keane, N. A.; Reidy, M.; Natoni, A.; Raab, M. S.; O'Dwyer, M. Blood Cancer J. 2015, 5, e325.
[13] Solekhova, M. A.; Kurbatov, Y. V. Russ. J. Org. Chem. 2005, 41, 128-130.
[14] Drygin, D.; Haddach, M.; Pierre, F.; Ryckman, D. M. J. Med. Chem. 2012, 55, 8199-8208.
[15] (a) Nawijn, M. C.; Alendar, A.; Berns, A. Nat. Rev. Cancer. 2011, 11, 23-34; (b) Brault, L.; Gasser, C.; Bracher, F.;
Huber, K.; Knapp, S.; Schwaller, J. Haematologica. 2010, 95, 1004-1015.
[16] Fan, Y. B.; Li, K.; Huang, M.; Cao, Y.; Li, Y.; Jin, S. Y.; Liu, W. B.; Wen, J. C.; Liu, D. Zhao, L. X. Bioorg.
Med. Chem. Lett. 2016, 26, 1224-1228.

Legends:
Figure 1. The structure of 2-(pyridin-2-ylamino)-quinolin-8-ol (9d).
Figure 2. The effects of 9g on PC-3 cell cycle progression. PC-3 cells were treated with 9g at various concentrations (0,
8, 12 and 16 μM) for 24 h (A) or 48 h (B), then harvested, stained with PI, and analyzed by flow cytometry.
Figure 3. The regulation of cell cycle and apoptosis related proteins in PC-3 cells treated with 9g for 24 h.
Figure 4. The inhibitory activities of compounds 3a, 8a and 9g on Pim-1 kinase. The ratio between the signals of 620
and 665 nm was used in the calculation of the inhibition rates.
Figure 5. The binding modes of compound 9g (B), 3a (C), 8a (D) and their superimposition (A) in the active site of
Pim-1 kinase (1YXT in PDB). H-Bonding interactions were presented with green line.
Scheme 1. Synthesis of 3a–c. Reagents and conditions: (a) m-CPBA, CHCl3, r.t., 2 h; (b) appropriate pyridines, p-TsCl,
10% KOH aq., CH2Cl2, r.t., 2 h.
Scheme 2. Synthesis of 8a–i and 9d–i. Reagents and conditions: (a) methyl iodide, K2CO3, (CH3)2CO, r.t. to 60°C, 8 h;
(b) m-CPBA, CHCl3, r.t., 2 h; (c) appropriate pyridines, p-TsCl, 10% KOH aq., CH2Cl2, r.t., 2 h; (d) HCl, 100°C; (e)
BBr3, dry CH2Cl2, 0°C, 30 min, r.t., 2 h, reflux, 9 h.
Scheme 3. Synthesis of 13a–c. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, 0°C, 1 h; (b) m-CPBA, CHCl3, r.t., 2
h; (c) appropriate pyridines, p-TsCl, 10% KOH aq., CH2Cl2, r.t., 2 h; (d) 1M NaOH aq., EtOH, (CH3)2CO, reflux, 8 h.

Graphical abstract