Identification and optimization of novel 6-acylamino-2-aminoquinolines as potent Hsp90 C-terminal inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.07.080

In order to discover novel Hsp90 inhibitors targeting the C-terminal ATP binding pocket, a novobiocin derivative based ROCS model was constructed for virtual screening. Compound 13 was identified as the lead compound and then systematical structure activity relationship (SAR) study was conducted. These efforts led to compound 69, which exhibited potent anti-proliferative activities against MCF7 and SKBr3 breast cancer cell lines. In 4T1 mice breast cancer models, 69 exhibited potent tumor growth inhibition and anti-metastasis effect. Compound 69 as a potent antitumor agent targeting the Hsp90 C-terminal is worthy of further pre-clinical study.

Full text of DOI:10.1016/j.ejmech.2017.07.080

Accepted Manuscript
Identification and optimization of novel 6-acylamino-2-aminoquinolines as potent
Hsp90 C-terminal inhibitors
Fen Jiang, An-ping Guo, Jia-cheng Xu, Hui-Jie Wang, Xiao-fei Mo, Qi-Dong You,
Xiao-Li Xu
PII:
S0223-5234(17)30602-5
10.1016/j.ejmech.2017.07.080
EJMECH 9643
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 June 2017
Revised Date: 28 July 2017
Accepted Date: 31 July 2017
Please cite this article as: F. Jiang, A.-p. Guo, J.-c. Xu, H.-J. Wang, X.-f. Mo, Q.-D. You, X.-L. Xu,
Identification and optimization of novel 6-acylamino-2-aminoquinolines as potent Hsp90 C-terminal
inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.080.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Identification and optimization of novel 6-acylamino-2-aminoquinolines
as potent Hsp90 C-terminal inhibitors
Fen Jianga,b, An-ping Guoa,b, Jia-cheng Xua,b, Hui-Jie Wanga,b, Xiao-fei Moa,b, Qi-Dong Youa,b* and
Xiao-Li Xua,b*
a
State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing
210009, China
Abstract
In order to discover novel Hsp90 inhibitors targeting the C-terminal ATP binding pocket, a novobiocin
derivative based ROCS model was constructed for virtual screening. Compound 13 was identified as the
lead compound and then systematical structure activity relationship (SAR) study was conducted. These
efforts led to compound 69, which exhibited potent anti-proliferative activities against MCF7 and
SKBr3 breast cancer cell lines. In 4T1 mice breast cancer models, 69 exhibited potent tumor growth
inhibition and anti-metastasis effect. Compound 69 as a potent antitumor agent targeting the Hsp90
C-terminal is worthy of further pre-clinical study.
Keywords
Hsp90 C-terminal inhibitor
anti-proliferative activity
anti-metastasis
1. Introduction
As the most important chaperone in mammalian cells, heat shock protein 90 (Hsp90) is critical for the
maturation, conformation stability and function of many other proteins that are called clients.[1] Some
clients are notorious oncogenic proteins, such as Her2, Akt, Raf-1, CDK6. Cancer cells depend on the
chaperone function of Hsp90 to stabilize these mutated and overexpressed oncoproteins. In addition,

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Hsp90 is 2 to 10 folds higher expressed in tumor cells than in normal cells and identified as a novel
tumor bio-marker.[2-4] Inhibiting the chaperone function of Hsp90 can induce degradation of the
oncogenic clients, thereby blocking the cancer cell dependent multiple signaling pathways and leading
to death of the cancer cells.[5] Thus, as promising anti-tumor agents, novel Hsp90 inhibitors with
different mechanisms are developed in the past years.[6]
The N-terminal domain of Hsp90 incorporates an ATP-binding pocket, the chaperone function of
Hsp90 depends on the hydrolysis process of ATP and the subsequent conformation change of the
protein.[7] Many small molecules that are called “Hsp90 N-terminal inhibitors” have been developed to
occupy this pocket to inhibit the chaperone function of Hsp90.[8] In pre-clinical study, these inhibitors
exhibited promising anti-tumor effects. However, in clinical trials, limited efficacy was observed. Most
of the clinical trials of these inhibitors have been terminated due to the unsatisfactory curative effect.
One possible explanation is the potent induction of heat shock response (HSR) of these N-terminal
inhibitors.[9] The HSR leads to increased expression of Hsp90 and the protective proteins Hsp70 and
Hsp27. These induced heat shock proteins play critical roles in the initiation and progression of cancer,
that can neutralize the antitumor effects of the Hsp90 N-terminal inhibitors.[10-13] To overcome the
undesired HSR, Hsp90 inhibitors with novel mechanisms are pursued.
Besides the N-terminal one, another ATP-binding pocket was discovered at the C-terminal domain of
Hsp90.[14-17] Inspired by the discovery process of N-terminal inhibitors, small molecules can be
developed to target this new-discovered C-terminal ATP-binding pocket to inhibit the chaperone
function of Hsp90. As the first discovered Hsp90 C-terminal inhibitor, novobiocin provides
proof-of-concept for Hsp90 C-terminal inhibition. Novobiocin binds to the C-terminal ATP-binding
pocket and manifests weak client degradation inducing effect (～700 µM in SKBr3 cells).[16] To
improve the Hsp90 inhibition activity and simplify the structure, extensive SAR studies for novobiocin
have been conducted.[18-25] As the pioneer of this field, the research group of Blagg identified many
potent novobiocin derivatives, such as compound 2 and 3.[22, 24] These derivatives exhibited potent
anti-proliferative activities against various cancer cell lines. Most importantly, unlike the N-terminal
inhibitors, these C-terminal inhibitors didn’t induce HSR. Thus, C-terminal inhibitors provide new

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chance for Hsp90 as an anti-cancer target, and the development of novel Hsp90 C-terminal inhibitors is
meaningful.
Fig. 1. Representative Hsp90 C-terminal inhibitors.
Up to now, though different types of Hsp90 C-terminal inhibitors have been developed, including
novobiocin and derivatives (1−4), deguelin and derivatives (5−6), dihydropyrimidinones (7−8) and other
natural original compounds (9−11) (Fig. 1), the structure diversity is still insufficient.[26-31] As no
co-crystal structure of Hsp90 C-terminal inhibitor has been reported, structure based rational design of
novel Hsp90 C-terminal inhibitors is limited. As a complement, ligand based drug design may promote
the discovery process. Rapid Overlay of Chemical Structures (ROCS) is a useful ligand based drug
design method.[32] The ROCS model includes both the chemical features and the molecular shape and
orientation of the reference molecule. In our previous study, the ROCS method was successfully applied
in the discovery of Hsp90 N-terminal inhibitors.[33, 34] To discover novel Hsp90 C-terminal inhibitors,
a ROCS model based virtual screening process was conducted. In this study, the novobiocin derivative 2
was selected as the reference molecule to generate the ROCS model. Then the model was used to screen
the Chemdiv database and led to the hit compound 13. Compound 13 manifested potent

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anti-proliferative activities against breast cancer cells and induced significant degradation of the Hsp90
clients. Further structure modification led to compound 69, which possessed improved anti-proliferative
activities and physicochemical properties. In 4T1 mice breast cancer models, 69 exhibited potent
antitumor effects with no obvious toxicity.
2. Results and discussion
2.1. ROCS model based virtual screening
ROCS is a shape-based method to validate the 3D similarity between molecules. Similarity of a
candidate to the reference compound is ranked by combo score ranging from 0 to 2. The closer the
combo score is to 2, the more match is the candidate to the model. In this study, the novobiocin
derivative 2 was selected as the reference compound to generate the ROCS model (Fig. 2). Then the
model was used to screen the Chemdiv database (including 1293896 compounds). As results, 20
compounds (shown in supporting information) with higher combo scores and diverse structures were
selected and purchased form the Chemdiv database.
Fig. 2. Process of the ROCS model based discovery of Hsp90 C-terminal inhibitors. In the ROCS model, the molecular
shape of 2 was depicted in gray shadow and the chemical features were represented with various spherule.
In the subsequent MTT assay to evaluate the anti-proliferative activities of the purchased 20
compounds, the IC50 values of the three compounds that exhibited over 90% growth inhibition rate at 50

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µM against MCF7 cells were determinated (Table 1). All the three compounds exhibited potent
antiproliferative activities against MCF7 and SKBr3 breast cancer cell lines with IC50 values lower than
10 µM. Among which, compound 13 possessed the best activities.
Table 1. Antiproliferative activities of the selected compounds
Compd
Anti-proliferative activities (IC50, µM)a
ID
Structure
MCF7
SKBr3
Z999-0009C
8.41 ± 0.76
8.09 ± 0.35
12
OCH3
CH3
H
N
C095-0286
4.86 ± 0.12
5.38 ± 0.46
3.33 ± 0.30
13
O
N
N
H3C
N
C498-0931
4.99 ± 1.49
14
AT13387b
－
－
0.032 ± 0.006
0.045 ± 0.013
b
aEach data represents mean ± SD of at least three independent experiments. The clinical Hsp90 N-terminal inhibitor
AT13387 was used as the positive control.
As important bio-marker, Hsp90 inhibition induces significant client degradation. To verify whether
the potent anti-proliferative activities of the three compounds were Hsp90 inhibition dependent, Western
blot assay was conducted. As shown in Fig. 3, compound 13 decreased the expression levels of Akt, Erk
and CDK6, indicating that 13 could inhibit the chaperone function of Hsp90. Compound 12 and 14
manifested no influence on the client expression levels, and they exhibited the cytotoxicity based on
other mechanisms. Based on the Western blot results, compound 13 was identified as the lead compound
for further modification.

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Fig. 3. Western blot analysis of the client levels in MCF7 cells after incubation for 24 h with various concentrations of
12−14. The clinical Hsp90 N-terminal inhibitor AT13387 (1.0 µM) was selected as the positive control. β-Actin was
used as the control for protein loading.
2.2. Binding mode analysis of compound 13.
Though no co-crystal structure was reported, many computational efforts have been made to predict
the binding site of Hsp90 C-terminal inhibitors.[35-38] Based on the reported putative binding site, we
conducted docking study to analyze the binding mode of compound 13. The yeast Hsp90 homologue
(PDB code: 2CG9) was selected as the receptor. Results showed that compound 13 fitly bound into the
predicted C-terminal ATP-binding pocket (Fig. 4). The 4-methylquinoline core (Part A) formed
hydrophobic interactions with residues Ala597(A), Ile592(B) and Pro504(B). The benzamide fragment
(Part B) inserted into a big hydrophobic pocket and was surrounded by the hydrophobic residues
Phe329(B), Leu331(B), Phe332(B), Leu427(B) and Ile505(B). The hydrophobic pocket was occupied
incompletely and more hydrophobic groups could be introduced on the benzamide fragment. The
nitrogen atom of the acylamino formed hydrogen bond with residue Gln596(A). The N-ethyl piperazine
(Part C) targeted into a polar region, with potential for further modification. In addition, a nitrogen atom
of the piperazine group formed hydrogen bond with residue Arg591(B). These results gave us great
guidance for subsequent structure modification.

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Fig. 4. (A) The structure of compound 13. 13 is divided into three parts. (B) Docking analysis of the binding mode
between Hsp90 C-terminal and compound 13 (protein structure from PDB 2CG9). The carbon atoms of compound 13
and the residues of Hsp90 C-terminal are shown in green and yellow, respectively. The hydrogen bonds are indicated
by magenta dashed lines.
2.3. Structure modification of compound 13
Based on the docking analysis of compound 13, the benzamide fragment inserted into a big
hydrophobic pocket. Hydrophobic substituents on the benzene might be favorable. Thus, various
aromatic amides were investigated to elucidate the SARs of this region, leading to target compounds
18−46. The antiproliferative activities of the compounds against MCF7 and SKBr3 breast cancer cell
lines were evaluated. As summarized in Table 2, compound 18 exhibited decreased anti-proliferative
activities, of which the 3-methoxy on the benzene was removed. For mono-substituted benzamide
compounds, compounds with 4-position substituents manifested the most potent antiproliferative
activities, indicting 4-substitution was favorable. Removing the substituents from 4-position to
3-position resulted in decreased antiproliferative activities. As the exception, the 4-methoxy substituted
compound 20 exhibited lower antiproliferative activities than the 3-methoxy homologue 13. During the
MTT assay, 20 was observed to be precipitated, the poor solubility might decrease the antiproliferative
activities. The 2-position substituted compounds exhibited significant decreased anti-proliferative
activities compared with the 3- or 4-position substituted homologues. This might be caused by the
detrimental influence of 2-substitution on the conformation of the amide bond. The disubstituted
benzamide compounds 35−39 manifested retained or improved activities, among which, 38 with

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3,4-dichloro substitution possessed the best anti-proliferative activities. The heteroaromatic compounds
40−43 exhibited poor activities. However, 44 and 45 that incorporating benzene fused heterocycles
possessed potent activities. These results were consistent with the hydrophobic feature of the pocket.
Among all the synthesized derivatives, compound 38 with 3,4-dichloro benzamide fragment exhibited
the best anti-proliferative activities. Thus, it was selected as the favorable fragment for further structure
modification.
Table 2. SAR exploration of the part B.
Anti-proliferative activities (IC50, µM) a
Structure
Compd
Ar =
MCF7
SKBr3
4.86 ± 0.12
3.33 ± 0.30
13
18
19
20
21
22
23
24
25
26
27
28
14.19 ± 1.40
13.20 ± 0.87
7.69 ± 0.76
10.90 ± 0.56
7.80 ± 0.21
6.74 ± 0.09
15.84 ± 1.17
3.06 ± 0.04
32.60 ± 0.92
8.92 ± 0.19
4.03 ± 0.25
18.41 ± 1.35
15.66 ± 0.99
9.00 ± 0.72
29.22 ± 0.58
13.71 ± 0.36
11.40 ± 0.71
23.91 ± 0.61
9.85 ± 1.34
38.07 ± 5.86
24.87 ± 2.01
19.78 ± 0.93

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Cl
29
30
31
32
﹥50
﹥50
3.82 ± 0.06
1.53 ± 0.02
27.52 ± 0.30
17.82 ± 1.75
3.02 ± 0.35
39.16 ± 8.08
11.64 ± 0.23
5.94 ± 0.60
3.65 ± 0.19
4.61 ± 0.24
13.30 ± 2.66
6.31 ± 1.16
2.71 ± 0.17
12.55 ± 1.01
1.98 ± 0.17
2.16 ± 0.11
2.66 ± 0.02
37.51 ± 3.87
13.15 ± 0.81
10.58 ± 0.11
﹥50
33
34
35
36
37
38
39
40
41
42
43
44
5.23 ± 0.13
Cl
1.38 ± 0.30
Cl
6.09 ± 0.11
41.19 ± 4.03
14.72 ± 0.43
26.56 ± 0.55
﹥50
N
3.76 ± 0.30
7.06 ± 0.13
2.95 ± 0.06
45
2.55 ± 0.09
N
H
AT13387b
－
0.032 ± 0.006
0.045 ± 0.013
b
aEach data represents mean ± SD of at least three independent experiments. The clinical Hsp90 N-terminal inhibitor
AT13387 was used as the positive control.
Then, the modification of the Part C was conducted. As shown in Table 3, transformation of the

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N-ethyl piperazine into N-methyl piperazine (57) and N-isopropyl piperazine (58) had slight influence
on the anti-proliferative activities. While other hydrophobic secondary amine substituted compounds
(59−63) exhibited dramatically decreased activities. For primary amine substituted compounds, the
methylamine substituted 64 exhibited retained anti-proliferative activities, while 65 which incorporating
a more hydrophobic ethylamine manifested significant decreased activities. These results indicated that
hydrophobic substituents were unfavorable for this region. Considering the polar feature of the
sub-pocket and the low solubility of 38, introducing solubilizing groups at this position might improve
the anti-proliferative activities and solubility. In addition, based on the reported SARs of novobiocin
derivatives, polar amine substituents were favorable for part C. Thus, compounds 66−71 incorporating
polar and flexible amines were synthesized. As shown in results, these compounds exhibited potent
anti-proliferative activities, especially compound 69, possessed improved activities compared with
compound 38.
Physicochemical property evaluation is very important during drug discovery. Thus, parameters
including solubility, LogD7.4 and permeability of some selected compounds were evaluated. The lead
compound 38 had poor drug-like properties with low solubility (11.3 µg/ml) and moderate permeability
(25.86 × 10−6 cm/s, PH 7.4) (Table 4). Compound 66−71 that incorporating polar and flexible amines
exhibited reduced LogD7.4 and 6−11 folds improved solubility compared with 38. In addition, 69 and 70
exhibited nearly 2 folds improved permeability. These two compounds possessed balanced
physicochemical properties. Considering compound 69 had high anti-proliferative activities and suitable
physicochemical properties, it was chosen for further biological evaluation.
Table 3. SAR exploration of the part C.
Anti-proliferative activities (IC50, µM)a
Structure
Compd
R =
MCF7
SKBr3
1.38 ± 0.30
2.16 ± 0.11
38

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3.00 ± 0.60
1.71 ± 0.20
6.32 ± 0.67
6.68 ± 0.93
57
58
33.34 ± 9.15
﹥50
﹥50
59
60
61
62
63
41.20 ± 1.02
43.44 ± 5.9
42.49 ± 5.33
15.11 ± 0.18
26.30 ± 7.3
31.10 ± 0.42
11.95 ± 0.81
2.52 ± 0.08
48.61 ± 2.16
2.55 ± 0.43
1.42 ± 0.15
1.28 ± 0.30
6.35 ± 0.29
28.20 ± 2.31
6.67 ± 0.31
3.71 ± 0.23
9.53 ± 0.05
64
65
66
67
68
69
70
1.19 ± 0.02
5.23 ± 1.05
1.80 ± 0.23
9.16 ± 1.75
1.70 ± 0.09
2.69 ± 0.14
71
AT13387b
－
0.032 ± 0.006
0.045 ± 0.013
b
aEach data represents mean ± SD of at least three independent experiments. The clinical Hsp90 N-terminal inhibitor
AT13387 was used as the positive control.
Table 4. Physicochemical properties of the selected derivatives.
Physicochemical properties a
Compd
Log D
Solubility
Pe, pH 7.4b

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PH = 7.4
3.67
(µg/mL)
11.3
(10−6 cm/s)
25.86 ± 1.52
38
1.47
1.66
1.72
2.72
2.64
1.73
121.7
95.2
84.5
70.8
76.2
105.8
3.13 ± 0.77
28.11 ± 3.07
20.80 ± 4.09
47.28 ± 5.99
49.79 ± 4.78
7.71 ± 1.17
66
67
68
69
70
71
aThe distribution coefficient (Log D7.4) was obtained using PULSETM (Pion Inc., MA, USA) and intrinsic aqueous
solubility were obtained using a Gemini Profiler instrument (Pion Inc., MA, USA). Permeability was determined using
the double-sink parallel artificial membrane permeability assay (PAMPA) on a PAMPA Explorer instrument (Pion Inc.,
MA, USA). bValues shown are the mean ± SD (n = 3).
2.4. Clients degradation effects of 69
To verify the potent anti-proliferative activities of compound 69 was Hsp90 inhibition dependent,
western blot assay was performed to determine the client expression levels in MCF7 cells. As shown in
Fig. 5, after treating with various concentrations of 69 for 24 h, the expression levels of Hsp90 clients
(Akt, Erk, CDK6) were dose dependently decreased. In addition, the expression levels of Hsp90 and
Hsp70 were not affected, indicating no HSR was induced. These results confirmed the Hsp90 C-terminal
inhibition activity of compound 69.
Fig. 5. Western blot analysis of the client levels in MCF7 cells after incubation for 24 h with various concentrations of
69. The clinical Hsp90 N-terminal inhibitor AT13387 (1.0 µM) was selected as the positive control. β-Actin was used

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as the control for protein loading.
2.5. Apoptosis induction and antimigratory activity of 69
To evaluate the apoptosis induction effect of 69, Annexin V/PI double staining assay was performed.
The results were shown in Fig. 6A. It was observed that 69 treatment induced obvious apoptosis in
MCF7 cells. After 5 µM or 10 µM 69 treatment, the total apoptotic cells (early and late apoptotic cells)
were increased to 18.01 % and 51.81 %, respectively. These results indicated that the potent
anti-proliferative activities of 69 were apoptosis induction dependent.
Cancer cell migration is a pivotal step in the metastatic process of malignant tumor and discovery of
antimigratory agents is an effective anti-cancer strategy.[39] It has been reported that novobiocin-based
Hsp90 C-terminal inhibitors could disrupt Hsp90α/Aha1 interaction and block the migration of
PC3-MM2 cells.[40] Wound healing assay was conducted to evaluate the antimigratory activity of 69.
As shown in Fig. 6B, 69 treatment efficiently blocked the migration of MCF7 cells in a dose dependent
manner compared with the control.
Fig. 6. (A) Apoptosis induction of 69 in MCF7 cells. Cells were treated with various concentrations of 69 for 24 h, the
samples were labeled with Annexin V-FITC and PI double staining and analyzed by flow cytometry. (B) Antimigratory
activity of compound 69 in MCF7 cells. Wounds were created using 200 µL sterile pipette tip. The cells were treated

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with various concentrations of 69 for 24 h, then imaged. Scale bar, 100 µm.
2.6. In-vivo anticancer effects of 69
The in-vivo anticancer effects of 69 was evaluated in 4T1 induced BALB/c mice breast cancer
models. The growth and metastatic characteristics of this model can mimic the advanced breast cancer
of human. [41] The tumor-bearing mice were intraperitoneally treated with 69 for 2 weeks (10 and 30
mg/kg). As shown in Fig. 7A and 7B, treatment with 69 exhibited potent tumor growth inhibition. The
TGI values of the 10 and 30 mg/kg doses were 26% and 47%, respectively. The in-vivo anti-metastasis
effect was also evaluated (Fig. 7D and 7E). Significant lung metastasis was observed in mice of the
control group, the lung tissue was seriously necrosis. 69 treatment significantly decreased lung
metastasis compared with the control group. In addition, 69 treatment was tolerated, as evidenced no
obvious body weight loss was observed (Fig. 7C).
Fig. 7. In-vivo anticancer effects of 69 in 4T1 induced BALB/c mice breast cancer models. Mice were treated (ip) with

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vehicle control, 69 (10 mg/kg), 69 (30 mg/kg) every day for 2 weeks once the mean tumor volume reached 50 mm3. (A)
Tumor volume changes during treatment. Tumor diameters were measured every two days. Bars represent SEM. ** p <
0.01, Student’s t-test (n = 8). (B) Tumor weight after the final treatment. (C) Body weight changes during the treatment.
(D) Photos of the harvested lung tissues to evaluate the anti-metastasis effect of 69. (E) H&E staining showed that 69
decreased metastatic colons in lung tissues. Scale bar, 50 µm.
2.7. Chemistry
Compounds 18−45 were synthesized according to the route described in Scheme 1.
2-choloroquinolines was refluxed in 1-ethylpiperazine to obtain the intermediate 16. Nitrification in cold
fuming nitric acid and subsequent reduction of the nitro group afforded 17. Then condensed with various
aromatic carboxylic acids to give the desired products.
Schme1. Synthetic route of compounds 18−45a
a Reagents and conditions: (a) 1-Ethylpiperazine, reflux, 4 h; (b) Nitric acid fuming, -10 °C to 0 °C, 4 h; (c) 10 % Pd/C,
H2, tetrahydrofuran (THF), room temperature (rt), 24 h; (d) Aromatic carboxylic acids, EDCI, HOBt, Et3N, DCM, rt, 24
h.
Compounds 57−71 were synthesized using the route described in Scheme 2. 46 was nitrified in cold
fuming nitric acid to afford intermediate 47. Transforming the hydroxyl into chlorine in phosphorus
oxychloride gave 48. The chlorine in 48 was substituted with different secondary amines. Then the nitro
groups were hydrogenated and the obtained amine intermediates were condensed with
3,4-dichlorobenzoyl chloride to afford the target compounds 57−63. For 64−71, primary amines were
introduced in 48 and the NH was BOC-protected to give intermediates 49−56. After nitro reduction and
condensation with 3,4-dichlorobenzoyl chloride, the BOC group was removed to obtain the target
compounds 64−71.
Schme2. Synthetic route of compounds 57−71a

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a Reagents and conditions: (a) Concentrated nitric acid, concentrated sulfuric acid, rt, 2 h; (b) Phosphorus oxychloride,
65 °C, 2 h; (c) Amines, EtOH, 55 , 24 h; (d) Boc2O, Et3N, DCM, rt, 12 h; (e) 10 % Pd/C, H2, THF, rt, 24 h; (f)
3,4-dichlorobenzoyl chloride, Et3N, DCM, rt, 24 h; (g) TFA, DCM, rt, 12h.
3. Conclusion
In this study, ROCS model based virtual screening was successfully conducted to identify compound
13 as a novel Hsp90 C-terminal inhibitor. Further systematical SAR study and physicochemical property
modification led to compound 69, which exhibited potent anti-proliferative activities against two breast
cancer cell lines (MCF7 and SKBr3) and balanced physicochemical properties. In vitro, 69 induced
obvious client degradation and apoptosis in MCF7 cells. In addition, 69 could inhibit the migration of
MCF7 cells. In a highly metastatic 4T1 induced breast xenograft model, 69 significantly reduced tumor
growth and lung metastases. 69, as a potent Hsp90 C-terminal inhibitor, can serve as a novel anti-cancer
agent for further study.
4. Experimental protocols
4.1. Chemistry
All solvents and reagents were obtained commercially. Solvents were dried according to standard
procedures, air and moisture sensitive reactions were performed under nitrogen. Reactions were

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monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (GF254) and visualized
1
13
under UV light. Melting points were determined with a Melt-Temp II apparatus. The H NMR and C
NMR spectra were measured on a Bruker AV-300 instrument using deuterated solvents with
tetramethylsilane (TMS) as internal standard. Because of the low solubility of 33, 34 and 41 in
d6-DMSO and other deuterated solvents, they were prepared into hydrochloride and dissolved in D2O to
1
obtain the H NMR. ESI-mass and high resolution mass spectra (HRMS) were recorded on a Water
Q-Tof micro mass spectrometer. The purity (≥ 95%) of the compounds was verified by the HPLC study
performed on Agilent C18 (4.6 mm × 150 mm, 3.5µm) column using a mixture of solvent
methanol/water (1‰ Et3N) at a flow rate of 0.5 mL/min and monitoring by UV absorption at 254 nm.
4.1.1. 2-(4-ethylpiperazin-1-yl)-4-methylquinoline (16)
2-chloro-4-methylquinoline (15) (2.0 g, 11.2 mmol) was dissolved into N-ethylpiperazine (5.7 ml, 45
mmol) and the mixture was refluxed for 4 h. After cooling to room temperature (rt), 20 ml 3 mol/L HCl
solution was added. The aqueous phase was washed twice with DCM, then 4 mol/L NaOH solution was
added to adjust PH to 8. The mixture was extracted with EtOAc (20 mL) for three times. The combined
organic layer was dried over anhydrous Na2SO4, and then concentrated in vacuo to afford 16 as a brown
oil (2.2 g, 76.5%). 1H NMR (300 MHz, CDCl3) δ 7.98–7.90 (m, 2H), 7.79–7.65 (m, 1H), 7.50–7.40 (m,
1H), 7.04 (s, 1H), 3.97 (t, J = 5.0 Hz, 4H), 2.78–2.74 (m, 7H), 2.68 (q, J = 7.2 Hz, 2H), 1.35 (t, J = 7.2
Hz, 3H).
4.1.2 2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-amine (17)
The intermediate 16 (2.0 g, 7.8 mmo) was dropped into 10 ml cold (-10 ) and stirred fuming
HNO3. The reaction was stirred at -10
for 2 h, then stirred at rt for another 2 h. The mixture was
poured into an ice/water mixture (100 ml). Adjusting the PH value to 12 with 4 mol/L NaOH solution,
and the precipitate was filtered off, washed with water and dried to give a yellow solid. The solid was
dissolved in 15 ml THF, 300 mg 10% Pd/C was added. The mixture was stirred under hydrogen for 24 h.
After confirming the progress of the reaction by a thin-layer chromatography, the mixture was filtered
over a pad of Celite to remove Pd/C. The filtrate was concentrated to obtain 17 as a brown oil (1.2 g,
56.9%). 1H NMR (300 MHz, CDCl3) δ 8.71 (s, 1H), 8.29 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 9.2 Hz, 1H),

ACCEPTED MANUSCRIPT
6.91 (s, 1H), 3.88 (t, J = 5.2 Hz, 4H), 2.65 (s, 3H), 2.59 (t, J = 5.4 Hz, 4H), 2.51 (q, J = 8.0 Hz, 2H),
1.16 (t, J = 7.2 Hz, 3H).
General procedures for the synthesis of compounds 18–46
Aromatic carboxylic acid (1.11 mmol), EDCI (213 mg, 1.11 mmol), HOBt (150 mg,1.11 mmol) were
added into 10 ml DCM. The mixture was stirred at rt for 1 h. Then Et3N (0.31 ml, 2.22 mmol) and
intermediate 17 (200 mg, 0.74 mmol) were added. After stirring at rt for 24 h, the reaction was washed
with 2 mol/L NaOH solution (20 ml × 3) and saturated NH4Cl solution (20 ml × 1), dried over
anhydrous Na2SO4 and then concentrated. The residue was purified by normal phase column
chromatography to afford the desired compound.
4.1.3. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (18). White solid (68 mg,
1
24.5%). mp 187.4 −189.3 ℃. H NMR (300 MHz, Methanol-d4) δ 8.22 (s, 1H), 7.90–7.88 (m, 2H),
7.72–7.69 (d, J = 9.2 Hz, 1H), 7.62–7.56 (m, 1H), 7.50–7.40 (m, 3H), 6.97 (s, 1H), 3.66 (s, 4H), 2.54 (s,
7H), 2.35–2.24 (q, J = 7.3 Hz, 2H), 1.10–1.06 (t, J = 6.4 Hz, 3H). HRMS (ESI): calcd for C23H26N4O [M
+ H]+ 375.2181, found 375.2186. Purity: 98.18% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.4. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-2-methoxybenzamide (19). light yellow
solid (114 mg, 38.1%). mp 123.6 ℃−125.5 ℃. 1H NMR (300 MHz, d6-DMSO) δ 10.21 (s, 1H), 8.37 (d, J
= 2.3 Hz, 1H), 7.78–7.75 (dd, J = 9.0, 3.0 Hz, 1H), 7.68–7.66 (dd, J = 9.0, 3.0 Hz, 1H), 7.54–7.48 (m,
2H), 7.20 (d, J = 8.3 Hz, 1H), 7.13 (s, 1H), 7.10–7.05 (m, 1H), 3.84 (s, 3H), 3.72 (m, 4H), 2.63 (s, 4H),
2.56–2.54 (m, 2H), 2.49 (s, 3H), 1.04 (t, J = 7.2 Hz, 3H). HRMS (ESI): calcd for C24H29N4O2 [M +
H]+405.2285, found 405.2279. Purity: 98.99% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.5. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-4-methoxybenzamide (20). light yellow
1
solid (178 mg, 59.6%). mp 220.7 ℃−222.4 ℃. H NMR (300 MHz, DMSO-d6) δ 10.24 (s, 1H),
8.34–8.32 (d, J = 2.2 Hz, 1H), 8.03–7.94 (m, 2H), 7.92 (dd, J = 9.1, 2.4 Hz, 1H), 7.57–7.54 (d, J = 9.0
Hz, 1H), 7.15 (s, 1H), 7.07–7.05 (m, 2H), 3.72 (s, 3H), 3.72 (s, 4H), 3.32 (s, 2H), 2.68 (s, 4H), 2.50 (s,
13
3H), 1.13–1.08 (t, J = 7.2 Hz, 3H). C NMR (75 MHz, DMSO-d6) δ 164.78, 161.84, 156.12, 146.06,
144.47, 143.95, 133.94, 129.60, 126.97, 126.60, 123.84, 122.94, 113.55, 110.45, 55.42, 51.52, 51.32,
43.73, 18.65, 10.89. HRMS (ESI): calcd for C24H29N4O2 [M + H]+ 405.2285, found 405.2293. Purity:

ACCEPTED MANUSCRIPT
98.06% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.6. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-2-methylbenzamide (21). white solid (30
mg, 13.9%). mp 206.5 −208.4 . 1H NMR (300 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.36 (d, J = 1.7 Hz,
1H), 7.82–7.79 (dd, J = 8.9, 2.0 Hz, 1H), 7.54–7.47 (m, 2H), 7.42–7.37 (m, 1H), 7.30–7.28 (m, 2H),
7.13 (s, 1H), 3.64–3.60 (m, 4H), 2.53–2.46 (m, 7H), 2.41 (s, 3H), 2.38–2.26 (m, 2H), 1.04 (t, J = 7.1 Hz,
3H). HRMS (ESI): calcd for C24H29N4O [M + H]+ 389.2336, found 389.2331. Purity: 98.99% by HPLC
(MeOH/H2O = 80:20, 1‰ Et3N).
4.1.7. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-3-methylbenzamide (22). white solid (119
mg, 41.5%). mp 186.5 −188.1 . 1H NMR (300 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.31 (d, J = 2.1 Hz,
1H), 7.93–7.89 (dd, J = 9.0, 2.1 Hz, 1H), 7.81–7.77 (m, 2H), 7.56–7.53 (d, J = 9.0 Hz, 1H), 7.45–7.41
(m, 2H), 7.13 (s, 1H), 3.64–3.60 (m, 4H), 2.54 (s, 3H), 2.50–2.45 (m, 4H), 2.41 (s, 3H), 2.38–2.27 (q, J
= 7.1 Hz, 2H), 1.04 (t, J = 7.1 Hz, 3H). HRMS (ESI): calcd for C24H29N4O [M + H]+389.2336, found
389.2341. Purity: 98.74% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.8. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-4-methylbenzamide (23). white solid (112
mg, 39.0%). mp 218.5 −219.6 . 1H NMR (300 MHz, Methanol-d4) δ 8.24 (d, J = 2.1 Hz, 1H), 7.78 (d,
J = 8.2 Hz, 2H), 7.74–7.70 (dd, J = 10.7, 2.3 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 2H),
7.00 (s, 1H), 3.78 (s, 4H), 2.87 (s, 4H), 2.78–2.71 (d, J = 7.4 Hz, 2H), 2.56 (s, 3H), 2.35 (s, 3H), 1.18 (t,
J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.20, 156.28, 144.38, 144.09, 141.45, 133.69,
132.13, 128.88, 127.63, 126.67, 123.73, 122.89, 113.56, 110.46, 51.88, 51.49, 44.12, 20.99, 18.64, 11.33.
HRMS (ESI): calcd for C24H29N4O [M + H]+ 389.2336, found 389.2341. Purity: 99.52% by HPLC
(MeOH/H2O = 80:20, 1‰ Et3N).
4.1.9. 2-ethyl-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (24). white solid (66
mg, 22.1%). 247.2 −248.3 . 1H NMR (300 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.42 (s, 1H), 7.89 (d, J
= 8.7 Hz, 1H), 7.65 (s, 1H), 7.48–7.41 (m, 2H), 7.37–7.10 (m, 3H), 4.62–4.60 (m, 2H), 3.59–3.52 (m,
4H), 3.15–3.05 (m, 4H), 2.77 (q, J = 7.4 Hz, 2H), 2.58 (s, 3H), 1.32–1.27 (t, J = 7.2 Hz, 3H), 1.21–1.16
(t, J = 7.5 Hz, 3H). HRMS (ESI): calcd for C25H31N4O [M + H]+403.2492, found 403.2487. Purity:
99.54% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).

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4.1.10. 4-ethyl-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (25). pink solid (79
1
mg, 26.6%). mp 181.7 −183.3 . H NMR (300 MHz, DMSO-d6) δ 10.26 (s, 1H), 8.33 (s, 1H),
7.94–7.88 (m, 3H), 7.55 (d, J = 9.0 Hz, 1H), 7.37 (d, J =8.1 Hz, 2H), 7.13 (s, 1H), 3.65 (s, 4H),
2.72–2.65 (q, J = 7.4 Hz, 2H), 2.50 (s, 7H), 2.39 (q, J = 6.8 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H), 1.05 (t, J =
7.1 Hz, 3H). HRMS (ESI): calcd for C25H31N4O [M + H]+403.2492, found 403.2489. Purity: 98.02% by
HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.11. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-2-fluorobenzamide (26). light yellow
solid (226 mg, 77.9%). mp 172.7 −174.1 . 1H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.32 (s,
1H), 7.85–7.79 (m, 1H), 7.70–7.67 (m, 1H), 7.57–7.54 (m, 2H), 7.39–7.27 (m, 2H), 7.15 (s, 1H), 3.67 (s,
4H), 2.53–2.50 (m, 7H), 2.44–2.39 (q, J = 7.0 Hz, 2H), 1.06 (t, J = 7.0 Hz, 3H). HRMS (ESI): calcd for
C23H26FN4O [M + H]+ 393.2085, found 393.2079. Purity: 95.87% by HPLC (MeOH/H2O = 80:20, 1‰
Et3N).
4.1.12. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-3-fluorobenzamide (27). light yellow
1
solid (70 mg, 24.1%). mp 172.9 −174.6 . H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.32 (s,
1H), 7.92–7.79 (m, 3H), 7.42–7.37 (m, 2H), 7.34–7.30 (m, 1H), 7.17 (s, 1H), 3.71 (s, 4H)2.67 (s, 4H),
2.58–2.55 (s, 5H), 1.09 (t, J = 6.5 Hz, 3H). HRMS (ESI): calcd for C23H26FN4O [M + H]+ 393.2085,
found 393.2091. Purity: 97.77 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.13. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-4-fluorobenzamide (28). light yellow
solid (143 mg, 49.2%). mp 204.5
−205.8 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.43 (s,
1H), 7.84–7.81 (d, J = 9.0 Hz, 1H), 7.68–7.65 (d, J = 7.5 Hz, 1H), 7.61–7.58 (d, J = 9.4 Hz, 1H),
7.54–7.51 (d, J = 7.7 Hz, 1H), 7.24–7.19 (m, 2H), 7.11–7.06 (m, 1H), 3.92 (s, 3H), 3.16 (s, 4H), 2.58–
2.54 (m, 6H), 1.25 (t, J = 6.8 Hz, 3H). HRMS (ESI): calcd for C23H26FN4O [M + H]+ 393.2085, found
393.2082. Purity: 99.03% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.14. 2-chloro-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (29). white solid
1
(112 mg, 37.0%). mp 209.0 ℃
−210.4 ℃. H NMR (300 MHz, DMSO-d ) δ 10.59 (s, 1H), 8.34 (s, 1H),
6
7.78–7.75 (dd, J = 8.9, 2.3 Hz, 1H), 7.62–7.44 (m, 5H), 7.15 (s, 1H), 3.64 (s, 4H), 2.53 (s, 3H), 2.49 (s,
4H), 2.40–2.33 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H). HRMS (ESI): calcd for C23H26ClN4O [M + H]+

ACCEPTED MANUSCRIPT
409.1790, found 409.1791. Purity: 96.82% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.15. 3-chloro-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (30). white solid
(140 mg, 46.3%). mp 166.1 −167.8 ℃. 1H NMR (300 MHz, Methanol-d4) δ 8.24 (d, J = 2.0 Hz, 1H),
7.91 (d, J = 1.7 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.74–7.70 (dd, J = 9.0, 2.4 Hz, 1H), 7.60–7.53 (m,
1H), 7.50–7.46 (m, 1H), 7.44–7.41 (m, 1H), 7.00 (s, 1H), 3.75 (t, J = 4.9 Hz, 4H), 2.79 (t, J = 5.0 Hz,
4H), 2.72–2.65 (q, J = 5.7 Hz, 2H), 2.63 (s, 3H), 1.16 (t, J = 7.3 Hz, 3H). HRMS (ESI): calcd for
C23H26ClN4O [M + H]+ 409.1790, found 409.1791. Purity: 97.78% by HPLC (MeOH/H2O = 80:20, 1‰
Et3N).
4.1.16. 4-chloro-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (31). white solid
1
(130 mg, 43.0%). mp 117.3 ℃
−119.0 ℃. H NMR (300 MHz, Methanol-d ) δ 8.25 (d, J = 2.4 Hz, 1H),
4
7.90–7.88 (m, 2H), 7.75–7.71 (dd, J = 9.1, 2.4 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.48 (d, J = 8.7 Hz,
2H), 7.03 (s, 1H), 3.81 (s, 4H), 3.00 (s, 4H), 2.90–2.84 (m, 2H), 2.56 (s, 3H), 1.23 (t, J = 7.3 Hz, 3H).
13C NMR (75 MHz, DMSO-d6) δ 164.21, 156.45, 144.28, 144.24, 136.29, 133.66, 133.24, 129.54,
128.43, 126.68, 123.60, 122.77, 113.69, 110.47, 52.19, 51.62, 44.43, 18.62, 11.78. HRMS (ESI): calcd
for C23H26ClN4O [M + H]+ 409.1790, found 409.1788. Purity: 99.30% by HPLC (MeOH/H2O = 80:20,
1‰ Et3N).
4.1.17. 2-bromo-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (32). white solid (93
mg, 27.8%). mp 197.5 ℃−198.8 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.44 (s, 1H), 7.91 (s,
1H), 7.75 (d, J = 7.6 Hz, 1H), 7.57–7.44 (m, 3H), 7.27–7.10 (m, 2H), 3.57–3.52 (m, 4H), 3.15 (s, 4H),
2.85–2.77 (m, 2H), 2.61 (s, 3H), 1.29–1.25 (m, 3H). HRMS (ESI): calcd for C23H26BrN4O [M + H]+
453.1285, found 453.1291. Purity: 93.14% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.18. 3-bromo-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (33). white solid
(205 mg, 61.2%). mp 287.5 ℃−289.2 ℃. 1H NMR (300 MHz, D2O) δ 8.12 (s, 1H), 7.86 (d, J = 9.0 Hz,
1H), 7.71–7.62 (dd, J = 19.3, 9.1 Hz, 2H), 7.49 (d, J = 7.4 Hz, 1H), 7.34–7.26 (m, 2H), 7.21 (s, 1H) ,
4.62 (s, 4H), 4.00 (s, 4H), 3.59–3.52 (q, J = 7.3 Hz, 2H), 2.62 (s, 3H), 1.59 (t, J = 7.3 Hz, 3H). HRMS
(ESI): calcd for C23H26BrN4O [M + H]+ 453.1285, found 453.1281. Purity: 98.64% by HPLC
(MeOH/H2O = 80:20, 1‰ Et3N).

ACCEPTED MANUSCRIPT
4.1.19. 4-bromo-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (34). white solid
(119 mg, 35.6%). mp＞300 . 1H NMR (300 MHz, D2O) δ 7.75 (s, 1H), 7.55 (d, J = 9.1 Hz, 1H), 7.33
(d, J = 9.1 Hz, 1H), 6.98–6.88 (m, 5H), 3.59 (s, 4H), 3.26–3.19 (m, 4H), 2.28 (s, 3H), 1.92 (s, 2H), 1.26
(t, J = 7.3 Hz, 3H). HRMS (ESI): calcd for C23H26BrN4O [M + H]+ 453.1285, found 453.1287. Purity:
98.50% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.20. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-3,4-dimethoxybenzamide (35). white
solid (60 mg, 18.7%). mp 212.4 −213.7 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.29 (d, J
= 1.8 Hz, 1H), 7.90–7.86 (dd, J = 9.1, 1.8 Hz, 1H), 7.66 (dd, J = 8.4, 2.1 Hz, 1H), 7.58–7.54 (m, 2H),
7.13–7.08 (m, 2H), 3.85–3.84 (m, 6H), 3.64 (s, 4H), 2.54 (s, 3H), 2.50 (s, 4H), 2.40–2.33 (q, J = 7.1 Hz,
2H), 1.04 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 163.37, 156.17, 145.22, 144.25, 134.19,
132.38, 131.24, 127.32, 124.06, 123.60, 116.56, 113.29, 111.02, 106.23, 99.05, 98.40, 56.67, 56.02,
51.34, 51.03, 43.07, 19.10, 10.13. HRMS (ESI): calcd for C25H31N4O3 [M + H]+435.2391, found
435.2390. Purity: 96.60% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.21.N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzo[d][1,3]dioxole-5-carboxamide (36).
1
light yellow solid (99 mg, 32.0%). mp 211.3 ℃
−212.6 ℃. H NMR (300 MHz, DMSO-d ) δ 10.19 (s,
6
1H), 8.33 (d, J = 2.2 Hz, 1H), 8.32–7.90 (dd, J = 9.1, 2.2 Hz, 1H), 7.64–7.57 (m, 3H), 7.17 (s, 1H), 7.09
(d, J = 8.2 Hz, 1H), 6.16 (s, 2H), 3.70 (s, 4H), 2.61–2.51 (m, 9H), 1.10 (t, J = 7.2 Hz, 3H). HRMS (ESI):
calcd for C24H27N4O3 [M + H]+419.2078, found 419.2082. Purity: 97.96% by HPLC (MeOH/H2O =
80:20, 1‰ Et3N).
4.1.22. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-3,4-difluorobenzamide (37). white solid
1
(175 mg, 57.7%). mp 170.3 ℃
−171.6 ℃. H NMR (300 MHz, DMSO-d ) δ 10.43 (s, 1H), 8.29 (d, J =
6
2.2 Hz, 1H), 8.10– 8.04 (m, 1H), 7.90–7.86 (m, 2H), 7.65–7.55 (m, 2H), 7.15 (s, 1H), 3.68–3.65 (m, 4H),
2.54–2.52 (m, 7H), 2.46–2.40 (q, J = 7.1 Hz, 2H), 1.07 (q, J = 7.0 Hz, 3H). HRMS (ESI): calcd for
C23H25F2N4O [M + H]+411.1983, found 411.1983. Purity: 96.82% by HPLC (MeOH/H2O = 80:20, 1‰
Et3N).
4.1.23. 3,4-dichloro-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (38). white solid
1
(202 mg, 61.7%). mp 224.8 ℃−226.2 ℃. H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.29 (d, J =

ACCEPTED MANUSCRIPT
2.3 Hz, 1H), 8.11–8.04 (m, 1H), 7.90–7.87 (m, 2H), 7.65–7.54 (m, 2H), 7.16 (s, 1H), 3.66 (s, 4H), 2.54
(s, 4H), 2.50 (s, 3H), 2.45–2.38 (q, J = 7.3 Hz, 2H),1.05 (t, J =7.2 Hz, 3H). 13C NMR (75 MHz,
DMSO-d6) δ 162.39, 156.55, 144.41, 144.26, 136.29, 138.11, 134.27, 132.82, 126.72, 126.41, 123.45,
122.70, 113.83, 110.51, 51.31, 51.67, 44.54, 18.61, 11.95. HRMS (ESI): calcd for C23H25Cl2N4O [M +
H]+443.1403, found 443.1403. Purity: 96.82% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.24. 3,5-dichloro-N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (39). white solid
(173 mg, 52.9%). mp 284.1 −285.4 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.27 (dd, J =
6.5, 2.2 Hz, 2H), 7.98 (dd, J = 8.5, 2.1 Hz, 1H), 7.90 (dd, J = 9.0, 3.0 Hz, 1H), 7.83–7.80 (m, 1H), 7.55
(d, J = 9.0 Hz, 1H), 7.13 (s, 1H), 3.64 (s, 4H), 2.54 (s, 3H), 2.43 (s, 4H), 2.40–2.32 (q, J = 7.14 Hz, 2H),
1.04 (t, J = 7.1 Hz, 3H). HRMS (ESI): calcd for C23H25Cl2N4O [M + H]+443.1392, found 443.1392.
Purity: 97.79% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.25. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)furan-2-carboxamide (40). white solid
(197 mg, 63.7%). mp 184.0 ℃−186.4 ℃. mp 184.0 ℃−185.4 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.31
(s, 1H), 8.29 (d, J = 2.2 Hz, 1H), 7.97–7.91 (m, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.37 (m, 2H), 7.17 (m,
1H), 6.74–6.64 (m 1H), 3.70–3.67 (m, 4H), 2.60–2.56(m, 7H), 2.52–2.45 (m, 2H), 1.09 (t, J = 7.2 Hz,
3H). HRMS (ESI): calcd for C24H27N4O3 [M + H]+419.2078, found 419.2082. Purity: 97.25% by HPLC
(MeOH/H2O = 80:20, 1‰ Et3N).
4.1.26. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-1H-pyrrole-2-carboxamide (41). white
solid (247 mg, 91.8%). mp 209.0 ℃−210.4 ℃. 1H NMR (300 MHz, D2O) δ 7.27 (s, 1H), 7.03–6.84 (m,
2H), 6.46 (s, 1H), 6.31 (s, 1H), 5.88 (s, 1H), 5.64 (s, 1H), 3.89–3.76 (m, 2H), 3.38–3.25 (m, 2H),
3.05–2.85 (m, 2H), 2.76–2.67 (m, 2H), 2.66–2.65 (m, 2H), 1.92 (s, 3H), 0.91–0.89 (m, 3H). HRMS
(ESI): calcd for C21H26N5O [M + H]+364.2132, found 364.2128. Purity: 95.80% by HPLC (MeOH/H2O
= 80:20, 1‰ Et3N).
4.1.27. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)nicotinamide (42). light yellow solid (104
mg, 37.4%). mp 152.3 ℃
−153.6 ℃. 1H NMR (300 MHz, DMSO-d ) δ 10.55 (s, 1H), 9.15 (d, J = 1.7 Hz,
6
1H), 8.77–8.59 (dd, J = 4.8, 1.7 Hz, 1H), 8.34–8.32 (m, 2H), 7.89 (dd, J = 11.0, 2.2 Hz, 1H), 7.60–7.56
(m, 2H), 7.15 (s, 1H), 3.66 (s, 4H), 2.55 (s, 4H), 2.52(s, 3H), 2.40 (q, J = 6.9 Hz, 2H), 1.05 (t, J = 7.1 Hz,

ACCEPTED MANUSCRIPT
3H). HRMS (ESI): calcd for C22H26N5O [M + H]+376.2132, found 376.2132. Purity: 98.37% by HPLC
(MeOH/H2O = 80:20, 1‰ Et3N).
4.1.28. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)isonicotinamide (43). light yellow solid
(142 mg, 51.1%). mp 217.4
−219.0 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.80–8.79 (m,
2H), 8.35 (d, J = 2.3 Hz, 1H), 7.93–7.91 (m, 3H), 7.59–7.56 (d, J = 9.0 Hz, 1H), 7.18 (s, 1H), 3.83 (s,
4H), 2.62 (s, 4H), 2.62–2.55 (m, 5H), 1.09 (t, J = 7.0 Hz, 3H). HRMS (ESI): calcd for C22H26N5O [M +
H]+376.2132, found 376.2132. Purity: 95.77% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.29. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)quinoline-2-carboxamide (44). brown
solid (126 mg, 40.0%). mp 194.0 ℃
−194.3 ℃. 1H NMR (300 MHz, DMSO-d ) δ 10.87 (s, 1H), 8.65 (d, J
6
= 8.6 Hz, 1H), 8.49 (d, J = 2.2 Hz, 1H), 8.29–8.26 (dd, J = 8.5, 1.9 Hz, 2H), 8.14–8.10 (m, 2H),
7.96–7.90 (m, 1H), 7.79–7.76 (m, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.17 (s, 1H), 3.66 (s, 4H), 2.59 (s, 3H),
2.50–2.48 (m, 4H), 2.39 (d, J = 6.7 Hz, 2H), 1.05 (t, J = 7.1 Hz, 3H). HRMS (ESI): calcd for C26H28N5O
[M + H]+426.2288, found 426.2291. Purity: 99.21% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.30. N-(2-(4-ethylpiperazin-1-yl)-4-methylquinolin-6-yl)-1H-indole-2-carboxamide (45). brown
1
solid (74 mg, 24.2%). mp 124.0 ℃−125.8 ℃. H NMR (300 MHz, DMSO-d6) δ 11.76 (s, 1H), 10.36 (s,
1H), 8.31 (s, 1H), 7.98 (d, J = 7.5 Hz, 1H), 7.83–7.35 (m, 3H), 7.31–7.05 (m, 3H), 3.65 (s, 4H), 2.56 (s,
4H), 2.52 (s, 3H), 2.41–2.40 (m 2H), 1.05 (s, 3H). HRMS (ESI): calcd for C25H28N5O [M +
H]+414.2288, found 414.2288. Purity: 95.33% by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.31. 4-methyl-6-nitroquinolin-2-ol (47)
4-methylquinolin-2-ol (46) (3.0 g, 18.9 mmol) was dissolved in 10 ml concentrated sulfuric acid, then
mixed acid (1.4 ml 65% HNO3 + 1.4 ml concentrated sulfuric acid) was dropped in ice bath. The
mixture was stirred at rt for 2 h. After confirming the reaction was finished, the mixture was slowly
dropped into 100 ml ice water and vigorously stirred. The precipitate was filtered off, washed with water
and dried to give 47 as a yellow solid (3.6 g, 92.0%). 1H NMR (300 MHz, DMSO-d6) δ 12.16 (brs, 1H),
8.50 (s, 1H), 8.33 (dd, J = 9.0, 2.5 Hz, 1H), 7.44 (d, J = 9.3 Hz, 1H), 6.56 (s, 1H), 2.50 (s, 3H).
4.1.32. 2-chloro-4-methyl-6-nitroquinoline (48)
47 (2.2 g, 10.8 mmol) was dissolved in 10 ml phosphorus oxychloride, the reaction was stirred at

ACCEPTED MANUSCRIPT
65 for 6 h. After completely reacted, the mixture was slowly poured into 100ml stirred ice water. The
white precipitate was filtered off, washed with water and dried to give 48 as a white solid (2.4 g, 99.0%).
1H NMR (300 MHz, CDCl3) δ 8.94 (d, J = 1.9 Hz, 1H), 8.50 (dd, J = 9.1, 2.3 Hz, 1H), 8.15(d, J = 9.1
Hz, 1H), 7.42 (s, 1H), 2.81 (s, 3H).
General procedures for the synthesis of compounds 57–71
48 (200 mg, 0.90 mmol) and corresponding amines (5.4 mmol) were stirred at 55 in 20 ml 95%
EtOH for 24 h. Then the mixture was poured into 50 ml water, the precipitate was filtered off and dried
to give a yellow solid.
For the secondary amine substituted compounds (57–63), the yellow solid was dissolved in 15 ml
THF and 50 mg 10% Pd/C was added. The reaction was stirred under hydrogen at rt for 24 h. After
confirming the reaction was complete, the mixture was filtered over a pad of Celite to remove Pd/C. The
filtrate was concentrated to obtain the amine intermediate as a brown oil, which was directly used in the
next step. The intermediate was dissolved in 5 ml anhydrous DCM and Et3N (2.0 eq) was added. Then a
solution of 3,4-dichlorobenzoyl chloride (1.1 eq) in dry DCM was dropped in ice bath. After confirming
the reaction was finished, the mixture was washed with 2 mol/L NaOH solution (20 ml × 3) and brine
(20 ml × 1) successively, dried (Na2SO4) and concentrated. The crude product was purified by
chromatography.
For the primary amine substituted compounds (64–71), protective Boc-group was introduced on the
NH. The yellow solid was dissolved in 15 ml DCM, then Boc2O (2 eq) and Et3N (1 eq) was added, and
the mixture was stirred at room temperature for 12 h. The mixture was poured into water and extracted
three times with DCM. The extract was dried (Na2SO4) and concentrated to give the Boc-protected
intermediates 49−56. Then the nitro group was hydrogenated into amino and condensed with
3,4-dichlorobenzoyl chloride according to the procedures like secondary amine substituted compounds.
After that, the obtained intermediates were dissolved into 15 ml DCM, TFA (10 eq) was added and the
mixture was stirred at room temperature for 12 h. Then the mixture was concentrated, and the residue
was partitioned into aqueous Na2CO3 solution and EtOAc. The organic phase was dried (Na2SO4) and
concentrated. The crude product was purified by chromatography.

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4.1.33. 3,4-dichloro-N-(4-methyl-2-(4-methylpiperazin-1-yl)quinolin-6-yl)benzamide (57). white
1
solid (210 mg, 54.5%). mp 227.5 −229.2 ℃. H NMR (300 MHz, DMSO-d6) δ 10.51 (s, 1H),
8.29–8.25 (m, 2H), 7.97 (dd, J = 8.3, 1.9 Hz, 1H), 7.88 (dd, J = 9.0, 2.3 Hz, 1H), 7.83 (d, J = 8.3 Hz,
1H), 7.55 (d, J = 9.0 Hz, 1H), 7.15 (s, 1H), 3.64 (t, J = 4.7 Hz, 4H), 2.54 (s, 3H), 2.41 (t, J = 4.6 Hz ,
4H), 2.22 (s, 3H). HRMS (ESI): calcd for C22H22Cl2N4O [M + H]+429.1243, found 429.1237. Purity:
98.46 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.34. 3,4-dichloro-N-(2-(4-isopropylpiperazin-1-yl)-4-methylquinolin-6-yl)benzamide (58). white
1
solid (170 mg, 41.4%). mp 228.4 ℃–230.5 ℃. H NMR (300 MHz, DMSO-d6) δ 10.55 (s, 1H),
8.31–8.28 (m, 2H), 8.00 (dd, J = 8.4, 2.0 Hz, 1H), 7.91 (dd, J = 9.0, 2.3 Hz, 1H), 7.85 (d, J = 8.4 Hz,
1H), 7.57 (d, J = 9.0 Hz, 1H), 7.16 (s, 1H), 3.65 (t, J = 4.6 Hz, 4H), 2.70 (p, J = 6.6 Hz, 1H), 2.53–2.51
13
(m, 7H), 1.01 (d, J = 6.5 Hz, 6H). C NMR (75 MHz, DMSO-d6) δ 163.38, 157.03, 144.93, 144.69,
135.76, 134.73, 133.43, 131.77, 131.18, 130.02, 128.44, 127.18, 124.03, 123.20, 114.34, 110.93, 54.23,
48.56, 45.47, 19.10, 18.65. HRMS (ESI): calcd for C24H26Cl2N4O [M + H]+457.1556, found 457.1551.
Purity: 96.14 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.35. 3,4-dichloro-N-(2-(dimethylamino)-4-methylquinolin-6-yl)benzamide (59). white solid (125
mg, 37.2%). mp 207.8 ℃–209.5 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.30 (d, J = 1.7 Hz,
2H), 8.04–8.00 (m, 1H), 7.94–7.91 (m, 1H), 7.90–7.84 (m, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.02 (s, 1H),
3.16 (s, 6H), 2.57 (s, 3H). HRMS (ESI): calcd for C19H17Cl2N3O [M + H]+374.0821, found 374.0815.
Purity: 95.75 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.36. 3,4-dichloro-N-(2-(diethylamino)-4-methylquinolin-6-yl)benzamide (60). white solid (184 mg,
51.0%). mp 232.4 ℃–233.7 ℃. 1H NMR (300 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.26–8.23 (m, 2H), 7.97
(dd, J = 8.4, 2.0 Hz, 1H), 7.87–7.81 (m, 2H), 7.49 (d, J = 9.0 Hz, 1H), 6.90 (s, 1H), 3.63–3.56 (q, J = 7.0
Hz, 4H), 2.53 (s, 3H), 1.15 (t, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C21H22Cl2N3O [M + H]+402.1134,
found 402.1137. Purity: 99.03 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.37. 3,4-dichloro-N-(4-methyl-2-(pyrrolidin-1-yl)quinolin-6-yl)benzamide (61). white solid (212
1
mg, 59.0%). mp 231.9 ℃–233.6 ℃. H NMR (300 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.25 (s, 2H), 7.97
(dd, J = 8.3, 1.8 Hz, 1H), 7.87–7.81 (m, 2H), 7.52 (d, J = 9.0 Hz, 1H), 6.77 (s, 1H), 3.51 (t, J = 6.6 Hz,

ACCEPTED MANUSCRIPT
4H), 2.53 (s, 3H),1.87 (t, J = 6.7 Hz, 4H). HRMS (ESI): calcd for C21H19Cl2N3O [M + H]+400.0978,
found 400.0969. Purity: 97.24 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.38. 3,4-dichloro-N-(4-methyl-2-(piperidin-1-yl)quinolin-6-yl)benzamide (62). white solid (203
mg, 54.6%). mp 186.5 –187.2 . 1H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.25 (d, J = 1.9 Hz,
2H), 7.97 (dd, J = 8.3, 2.0 Hz, 1H), 7.90–7.82 (m, 2H), 7.52 (d, J = 9.0 Hz, 1H), 7.13 (s, 1H), 3.67 (t, J =
5.1 Hz, 4H), 2.53 (s, 3H), 1.64–1.56 (m, 6H). HRMS (ESI): calcd for C22H22Cl2N3O [M + H]+414.1134,
found 414.1137. Purity: 95.65 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.39. 3,4-dichloro-N-(4-methyl-2-morpholinoquinolin-6-yl)benzamide (63). white solid (232 mg,
62.1%). mp 115.2 –116.5 . 1H NMR (300 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.30 (d, J = 2.0 Hz, 1H),
8.25 (d, J = 1.8 Hz, 1H), 7.97 (dd, J = 8.4, 1.9 Hz 1H), 7.90 (dd, J = 8.9, 2.0 Hz 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.58 (d, J = 9.0 Hz, 1H), 7.15 (s, 1H), 3.72 (t, J = 4.4 Hz, 4H), 3.61 (t, J = 4.4 Hz, 4H), 2.55 (s, 3H).
13C NMR (75 MHz, DMSO-d6) δ 162.95, 156.68, 144.44, 144.26, 135.24, 134.27, 133.18, 131.29,
130.71, 129.53, 127.95, 126.84, 123.62, 122.97, 113.85, 110.38, 66.10, 45.11, 18.62. HRMS (ESI):
calcd for C21H19Cl2N3O2 [M + H]+416.0927, found 416.0939. Purity: 98.62 % by HPLC (MeOH/H2O =
80:20, 1‰ Et3N).
4.1.40. 3,4-dichloro-N-(4-methyl-2-(methylamino)quinolin-6-yl)benzamide (64). white solid (78 mg,
24.1%). mp 184.5 –185.2 . 1H NMR (300 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.26 (d, J =1.9 Hz, 1H),
8.20 (d, J = 2.1 Hz, 1H), 7.97 (dd, J = 8.4, 1.9 Hz, 1H), 7.85–7.82 (m,2H), 7.50 (d, J = 9.0 Hz, 1H), 6.85
(q, J = 4.9 Hz, 1H), 6.60 (s, 1H), 2.86 (d, J = 4.7 Hz, 3H), 2.45 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
δ 162.98, 156.56, 144.32, 135.24, 134.26, 132.91, 131.28, 130.74, 129.50, 127.95, 126.70, 123.57,
122.72, 118.77, 113.88, 110.53, 54.52, 18.58. HRMS (ESI): calcd for C18H16Cl2N3O [M + H]+360.0665,
found 360.0673. Purity: 96.77 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N ).
4.1.41. 3,4-dichloro-N-(2-(ethylamino)-4-methylquinolin-6-yl)benzamide (65). white solid (67 mg,
20.0%). mp 139.0 –140.7 . 1H NMR (300 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.25 (d, J = 1.4 Hz, 1H),
8.19 (d, J = 1.5 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.84–7.81 (m, 2H), 7.48 (d, J = 8.9 Hz, 1H), 6.81 (t, J
= 4.8 Hz, 1H), 6.60 (s, 1H), 3.42–3.36 (m, 2H), 2.45 (s, 3H), 1.17 (t, J = 7.3 Hz, 3H). HRMS (ESI):
calcd for C19H18Cl2N3O [M + H]+374.0821, found 374.0826. Purity: 97.16 % by HPLC (MeOH/H2O =

ACCEPTED MANUSCRIPT
80:20, 1‰ Et3N).
4.1.42. 3,4-dichloro-N-(2-((2-(dimethylamino)ethyl)amino)-4-methylquinolin-6-yl)benzamide (66).
1
white solid (237 mg, 63.2%). mp 195.5 –196.7 . H NMR (300 MHz, DMSO-d6) δ 10.55 (s, 1H),
8.27 (d, J = 1.8 Hz, 1H), 8.20 (d, J = 2.1 Hz, 1H), 8.00–7.97 (m, 1H), 7.86–7.81 (m, 2H), 7.51–7.46 (m,
1H), 6.71 (t, J = 4.7 Hz, 1H), 6.66 (s, 1H), 3.45 (q, J = 6.5 Hz, 2H), 2.46–2.42 (m, 5H), 2.19 (s, 6H). 13C
NMR (75 MHz, DMSO-d6) δ 162.84, 156.33, 145.07, 142.71, 135.37, 134.15, 132.21, 130.66, 129.57,
129.02, 127.99, 126.07, 123.17, 122.82, 114.33, 113.22, 58.32, 45.29, 38.34, 18.25. HRMS (ESI): calcd
for C21H22Cl2N4O [M + H]+417.1243, found 417.1246. Purity: 99.73% by HPLC (MeOH/H2O = 80:20,
1‰ Et3N).
4.1.43. 3,4-dichloro-N-(2-((2-(diethylamino)ethyl)amino)-4-methylquinolin-6-yl)benzamide (67).
white solid (242 mg, 60.5%). mp 205.5 –206.2 . 1H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 8.15 (s,
1H), 7.99 (s, 1H), 7.72–7.65 (m, 2H), 7.58–7.52 (m, 2H), 6.53 (s, 1H), 5.33 (s, 1H), 3.52 (q, J = 5.4 Hz,
2H), 2.70 (t, J = 5.9 Hz, 2H), 2.60 (q, J = 7.0 Hz, 4H), 2.52 (s, 3H), 1.04 (t, J = 7.1 Hz, 6H). HRMS
(ESI): calcd for C23H26Cl2N4O [M + H]+445.1556, found 445.1552. Purity: 96.54 % by HPLC
(MeOH/H2O = 80:20, 1‰ Et3N).
4.1.44. 3,4-dichloro-N-(4-methyl-2-((2-(pyrrolidin-1-yl)ethyl)amino)quinolin-6-yl)benzamide (68).
white solid (64 mg, 16.1%). mp 193.0 –194.1 . 1H NMR (300 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.25
(d, J = 1.5 Hz, 1H), 8.19 (d, J = 1.8 Hz, 1H), 7.97 (dd, J = 8.5, 2.0 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H),
7.47 (d, J = 9.2 Hz, 1H), 6.77 (t, J = 4.4 Hz, 1H), 6.65 (s, 1H), 3.47 (q, J = 5.6 Hz, 2H), 2.61 (t, J = 6.7
Hz, 2H), 2.44 (s, 3H), 1.69 (s, 4H), 1.22 (s, 4H). HRMS (ESI): calcd for C17H19ClN3O3 [M + H]+443.14,
found 443.1405. Purity: 98.26 % by HPLC (MeOH/H2O = 80:20, 1‰ Et3N).
4.1.45. 3,4-dichloro-N-(4-methyl-2-((2-(piperidin-1-yl)ethyl)amino)quinolin-6-yl)benzamide (69).
1
white solid (180 mg, 43.8%). mp 209.6 –210.3 . H NMR (300 MHz, DMSO-d6) δ 10.46 (s, 1H),
8.26 (s, 1H), 8.20 (s, 1H), 7.97 (d, J = 7.5 Hz, 2H), 7.84 (d, J = 7.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 1H),
13
6.66 (s, 2H), 3.47–3.46 (m, 2H), 2.48–2.45 (m, 5H), 2.39 (s, 4H), 1.50 (s, 4H), 1.33 (s, 2H). C NMR
(75 MHz, DMSO-d6) δ 163.04, 155.55, 143.92, 142.50, 139.42, 138.63, 132.18, 130.48, 129.88, 129.61,
127.96, 125.58, 125.49, 123.07, 114.72, 112.44, 57.88, 54.18, 45.65, 25.54, 24.13, 18.34. HRMS (ESI):