Design, synthesis and biological evaluation of new Axl kinase inhibitors containing 1,3,4-oxadiazole acetamide moiety as novel linker

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Design, synthesis and biological evaluation of new Axl kinase inhibitors containing

1,3,4-oxadiazole acetamide moiety as novel linker

Congjun Xu, Yufei Han, Sicong Xu, Ruxin Wang, Ming Yue, Yu Tian, Xiaofan Li,

Yanfang Zhao, Ping Gong

PII:

S0223-5234(19)31019-0

DOI:

https://doi.org/10.1016/j.ejmech.2019.111867

EJMECH 111867

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 16 July 2019

Revised Date: 31 October 2019

Accepted Date: 7 November 2019

Please cite this article as: C. Xu, Y. Han, S. Xu, R. Wang, M. Yue, Y. Tian, X. Li, Y. Zhao, P. Gong,

Design, synthesis and biological evaluation of new Axl kinase inhibitors containing 1,3,4-oxadiazole

acetamide moiety as novel linker, European Journal of Medicinal Chemistry (2019), doi: https://

doi.org/10.1016/j.ejmech.2019.111867.

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Design, synthesis and biological evaluation of new Axl kinase inhibitors containing 1,3,4-oxadiazole

acetamide moiety as novel linker

Congjun Xu^a,b*, Yufei Han^a, Sicong Xu^a, Ruxin Wang^a, Ming Yue^a, Yu Tian^a, Xiaofan Li^a, Yanfang Zhao^a,

Ping Gong^a

a

Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of

Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning Province,

PR China

b

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, People’s Republic of

China

Corresponding author. Tel: +86-024-43520216; Fax: +86-024-43520216;

E-mail address: gongpinggp@126.com

Abstract

Using the principle of bioisosteric replacement, we present a structure-based design approach to obtain

new Axl kinase inhibitors with significant activity at the kinase and cellular levels. Through a stepwise

structure-activity relationships exploration, a series of 6,7-disubstituted quinoline derivatives, which

contain 1,3,4-oxadiazol acetamide moiety as novel Linker, were ultimately synthesized with Axl as the

primary target. Most of them exhibited moderate to excellent activity, with IC₅₀values ranging from 0.032

to 1.54 µM against the tested cell lines. Among them, the most promising compound 47e, as an Axl kinase

inhibitor (IC₅₀= 10 nM), shows remarkable cytotoxicity against A549, HT-29, PC-3, MCF-7, H1975 and

MDA-MB-231 cell lines. More importantly, 47e also shows a significant inhibitory effect on EGFR-TKI

resistant NSCLC cell lines H1975/gefitinib. Meanwhile, this study provides a novel type of linker for Axl

kinase inhibitors, namely 1,3,4-oxadiazol acetamide moiety, which is out of the scope of the "5- atoms role

".

Keywords: Axl, EGFR, drug resistance, 1,3,4-oxadiazole, Isosteresis

1. Introduction

Lung cancer remains one of the most common malignant cancers worldwide, with approximately 1.8

million new cases diagnosed annually. Among them, non-small cell lung cancer (NSCLC) accounts for

about 85%. NSCLC targeted therapy has now become critically important, and the epidermal growth factor

receptor (EGFR) tyrosine kinase is a particular popular target, but its drug resistance has become a major

challenge in the targeted treatment of NSCLC. The receptor tyrosine kinase Axl is one of most

phosphorylated RTKs in over 50% of non-small cell lung cancer cells (NSCLCs). Accumulating evidence

suggests that Axl is an effective target for the treatment of non-small cell lung cancer and overcoming the

drug resistance of EGFR-TKI[1-3].

Axl belongs to the TAM family as Mer and Tyro-3[4]. Upon activation by its endogenous ligand

growth arrest-specific 6 (Gas6)[5], Axl exhibits a key role in many fundamental cellular processes,

including survival[6], adhesion, proliferation[7], differentiation[8], migration, invasion and angiogenesis

though activating the downstream signaling pathways. Apart from NSCLC, overexpression of Axl has been

observed in a wide range of tumor types, both liquid and solid, such as prostate carcinoma[9], breast

cancer[10], pancreatic cancer[11], leukemia[12], and in most cases high Axl expression correlates with a

poorer patient prognosis.

Moreover, overexpression and/or overactivation of Axl are considered as an important mechanism of

drug-resistance in NSCLC[13]. It has been reported that treatment of resistant cell lines with Axl inhibitors

restored the sensitivity of the cells to some targeted drugs[14]. Thus, Axl becomes a new promising

molecular target for anticancer drug discovery.

In the past decade, several well-characterized receptor tyrosine kinase inhibitors were discovered as Axl

inhibitors with low IC₅₀values, which were reported as multitarget kinase inhibitors. Cabozantinib[15]

(Cometriq^TM, 1), a Met/VEGFR2 inhibitor, was approved by FDA in 2012 for the treatment of patients with

progressive metastatic medullary thyroid cancer (MTC). While identified as c-MET and VEGFR2 inhibitor,

Cabozatinib inhibits other kinases such as Axl at the low nanomolar level (IC₅₀= 7 nM in cell free

assay)[16]. In addition, Liu, L. has reported that Cabozatinib almost inhibited Axl phosphorylation

completely in lapatinib-resistance breast cancer cells at 100 nM and restored sensitivity to lapatinib in

treated cells, suggesting that activation of Axl might be a mechanism of drug resistance [17]. Also as

quinoline derivatives, Foretinib is an extremely potent VEGFR2 inhibitor (IC₅₀= 0.035 nM, in cell-free

assays) that also inhibits a wide number of kinases, including Met, Kit, Ret, and Axl (IC₅₀= 11 nM, in

cell-free assays) in the low nanomolar range [18]. Other inhibitors such as NPS-1034, LY2801663 and

MGCD265 are also multitarget kinase inhibitors. R428 (BGB324, 3) is recognized as a first selective Axl

inhibitor with an IC₅₀value of 14.0ꢀnM. However, recent reports suggest that R428 also inhibit other

kinases (e.g. RET, VEGFR2 and Flt3) as potently as Axl[19, 20].

Figure 1. The representative small-molecule Axl inhibitors

Although increasing attention has been focused on the development of Axl kinase inhibitors, improving

the selectivity of Axl inhibitors is still an urgent problem to be solved. Ke Ding’ team developed a series of

6,7-dimethoxyquinazolin derivatives exhibiting considerable selectivity[20, 21]. However, there is a lack of

further investigation on the antitumor activity and overcoming drug resistance of these compounds. Thus,

in this paper, we developed a class of new inhibitors primarily targeting Axl, and explored their antitumor

activity and overcoming EGFR-TKI resistance.

2. Results and discussion

2.1 Design strategy of the new compounds

As shown in Figure 1, significant structural similarities are found in Cabozatinib, Forotinib and other

related multitarget kinase inhibitors such as NPS-1034 (4), LY2801663 (5), MGCD265 (6), 7[20]and 8[21].

These inhibitors include three chemical moieties: the hinge-binding heterocycles at the left side; middle

linker highlighted in red; the terminal para-F phenyl ring.

The SAR studies[22] about these compounds suggested that hinge-binding heterocycles and terminal

phenyl ring (usually 4-fluorophenyl) appear to be critical for kinase activity. Hence, at the beginning of this

study, we introduced five-membered azole heterocycles as bioisosteres into the linker to meet our previous

study “5-atoms role”[23] and got four series of compounds (23a-b, 24a-b, 25a-b and 26a-b in Figure 2

Step A). With the goal of targeting Axl, these compounds were evaluated for their in vitro inhibitory

activity against Axl kinase. The results indicated that they did not show excellent Axl kinase inhibitory

activity, but it can be seen that the cycloamino substituted propoxy substituent at position 7 of quinolines

plays a role in the interaction with the active site of Axl (shown in Table 1). In the molecular docking

experiment, it was found that the cyclic amino group was embedded in the hydrophilic pocket of Axl kinase,

which increased the affinity. (shown in Figure S1 in SI).

Figure 2. The design strategy of target compounds. (Step A): Based on the principle of bioisosteric

replacement, the amide bond is replaced by various five-membered azole heterocycles. (Step B):

CAD-based and SAR-based drug design.

Based on the result above, an assumption was drawn that the replacement of amides with

five-membered heterocycles and the removal of methylene to meet the "5-atoms role" would lead to an

increase in the rigidity of the Linker, which would be detrimental for target compounds into the Axl kinase

binding site. From this assumption, one carbon atom was added at the carbonyl alpha position to increase

molecular flexibility and several alkyl groups were introduced to limit conformations (Figure 2 Step B).

As proof of this concept, two compounds were docked to the binding sites of with AXL (PDB code:

5u6b) to gain an insight into the possible binding modes (shown in Figure 3). The simulation showed a

clear difference in the ligand positioning between the two subtypes. Compared with 23b without

“methylene”, compound 42a containing the “methylene” is shown to interact with multiple residues in the

binding site, such as Arg727, Asn728, Lys619, Met674 and Leu593 and the extension length and flexibility

enhances its affinity and selectivity (Figure 3B). On the other hand, compound 42a with the “methylene”

exhibits a "V" conformation that is more suitable for embedding into the Axl protein to improve affinity.

Namely, the quinoline fragment occupies the intermediate active site, and the cyclic amino substituted

propoxy group is embedded in the hydrophilic pocket A as well as (4-fluorophenyl)-1,3,4-oxadiazol moiety

into the active pocket B. (Figure 3B and Figure S1). The experiments are in good agreement with the

results obtained from the Axl % Inhibition experiment.

Figure 3. Docking studies of AXL (PDB code: 5u6b) and two compounds. (A): Compound 23b without

methylene in the binding site of Axl. (B): Docking poses of compound 42a containing the “methylene” in

the binding site of Axl.

As a result, other three series compounds (41a, 42a-42e and 43a-47e in Figure 2B) were designed and

synthesized, in which, a novel Linker has been developed. Afterwards, a three-carbon tether which

contained different cyclic tertiary amines were introduced at the 7-position of quinolines. After a stepwise

structure-activity relationships (SAR) exploration, a series of 6,7-disubstituted-4-benzyloxyquinoline

derivatives, which contain 1,3,4-oxadiazol acetamide moiety were ultimately synthesized as Axl inhibitors.

1,3,4-oxadiazole is commonly utilized pharmacophore has been subjected to extensive study[24-26]

due to their metabolic profile[27] and pharmacokinetic property[28, 29]. In addition, 1,3,4-oxadiazol

acetamide moiety enhances hydrogen bonding with the Axl protein and affects the conformation of the

compounds so that the compounds can sufficiently occupy the active pocket (Figure 3 and Figure S1).

Hence, 1,3,4-oxadiazol acetamide moiety may be a novel type of linker for Axl kinase inhibitors.

2.2 Chemistry

Compounds 23a-26a and 23b-26b were synthesized according to the routes respectively outlined in

scheme 1. The key intermediates 9 and 10a-e, as shown in Figure 4, were synthesized using a convenient

procedure starting from 1-(3,4-dimethoxyphenyl) ethanone or 1-(4-hydroxy-3-methoxyphenyl)ethanone,

which was illustrated in detail in our previous study[23, 30]. Intermediate 11 was obtained from

commercially available substituted benzoate via hydrazinolysis with 80% hydrazine monohydrate.

Subsequently, condensation with ethyl oxalyl monochloride[31] acnd the intramolecular cyclization[32] in

the presence of POCl₃afforded the ethyl 5-aryl-1,3,4-oxadiazole-2-carboxylate 12. 12 was hydrolyzed and

then reacted with oxalyl chloride to give the key intermediate 5-aryl-1,3,4-oxadiazole-2-carbonyl chloride

13, which underwent a condensation reaction with intermediate 6,7-disubstituted-4-phenoxyquinolines 9

and 10c respectively to give access to the desired target compounds 23a-b. In this way, 24a-b, 25a-b and

26a-b were prepared via cyclization, hydrolysis, acyl chlorination and condensation from 14, 17 and 20

respectively. Substituted benzonitrile reacted with hydroxylamine hydrochloride in EtOH to give

intermediate N-hydroxybenzimidamide 17[33]. 14 was obtained from substituted acetophenone via

substitution, ammonolysis[34]. 20 was obtained from substituted acetophenone after reaction with ethyl

oxalate in the presence of t-BuOK in THF[35].

Scheme 2 depicts the preparation of compounds 41a-47e. Intermediate 17, obtained by conventional

synthesis as described in Scheme 1, reacted with malonate monoethyl ester to afford amide intermediate,

which transformed to oxazole acetate 27. 27 was hydrolyzed and acylated with oxalyl chloride to give the

key intermediate 28, which underwent

a

condensation reaction with intermediate

6,7-disubstituted-4-phenoxyquinolines 10 to yield the desired target compound 41a. The target compounds

42a-42e and 43a-47e were similarly prepared from compounds 14 or 11 respectively.

`

Figure 4. The structure of intermediates 9 and 10a-e

Scheme 1. Reagents and conditions: (i) 80% hydrazine monohydrate, EtOH, reflux; (ii) ethyl oxalyl monochloride,

TEA, DCM, 0 ^oC to rt; (iii) POCl₃, 85 ^oC; (iv) 1) LiOH, MeOH/THF, H₂O; 2) oxalyl chloride, DCM; (v) 9 or 10c, TEA,

DCM; (vi) NBS, DCM; (vii) hexamethylenetetramine, 6N HCl; (viii) NH₂OH^.HCl, TEA, EtOH; (ix) diethyl oxalate,

t-BuOK, THF; (x) NH₂OH^.HCl, MeOH, 60 ^oC.

Scheme 2. Reagents and conditions: (i) monoethyl 2-substituted malonate, EDCI, DCM, 0 °C to rt; (ii) POCl₃, 85°C;

(iii) 1) LiOH, MeOH/THF, H₂O; 2) oxalyl chloride, DCM; (iv) 10, TEA, DCM, 0 °C to rt.

2.3 In vitro cytotoxicity and structure-activity relationships

Target compounds 23a-26a and 23b-26b were evaluated for their in vitro inhibitory activity against Axl

kinase by Mobility shift assay with ATP concentration at Km at 1.0 µM. The cytotoxicity of the eight

prepared compounds were evaluated in vitro against three cancer cell lines, including, HT-29 (human colon

cancer), A549 (human lung adenocarcinoma) and PC-3 (human prostate cancer cell).

Table 1.

The antiproliferative activities and Axl inhibition of target compounds 23a-26b.

IC₅₀(µmol/L) ± SD

Axl %

Compd.

R₁

Het

Inhibition at 1 µM

A549

HT-29

PC-3

23a

24a

25a

26a

23b

24b

25b

26b

Me

5.14 ± 0.06

52.29±3.62

>100

-2.0±0.4

21.5±0.6

-4.5±0.5

-2.2±0.9

73.2±1.4

80.9±2.9

36.6±2.7

11.0±0.0

5.59±0.03

10.30±0.02

40.37±0.13

2.73±0.05

0.24±0.04

0.87±0.01

3.34±0.06

21.91±0.56

>100

N

39.74±0.75

>100

N

O

29.44±1.01

3.81±0.08

0.72±0.01

1.04±0.02

2.04±0.03

7.89±0.16

1.89±0.04

6.38±0.07

24.56±1.10

N

O

N

Cabozantinib^b

Foretinib^b

-

3.75±0.01

0.21

4.56± 0.08

0.25 ± 0.01

8.25 ± 0.63

0.81± 0.09

-

93.8±0.85

ND

^aND = not determined;

^bUsed as the positive control;

^cBold values show the IC₅₀values of the target compounds lower than the values of the positive control;

This first screening results are listed in Table 1. None of compounds 23a-26a bearing

6,7-dimethoxyquinolin moiety (contained in the structure of Cabozantinib), exhibits inhibitory activity on

Axl at 1µM. Interestingly, their analogues (23b-26b) bearing the cyclic amino propanoxy group (similar to

Foretinib) exhibited a higher inhibitory activity against Axl. Especially, compound 24b with the oxazole as

bioisostere exhibited the highest inhibitory activity with the IC₅₀values of 0.24 ± 0.04 µM, 1.89 ± 0.04 µM

and 0.72 ± 0.01 µM against A549, HT-29 and PC-3 cell lines respectively and a percent inhibition close to

81% at 1.0 µM, which indicated the cyclic amino propanoxy group seemed to play an important role in the

interaction with the active site of Axl. Compounds 23b and 24b exhibited better Axl kinase inhibitory

activity compared to 25b and 26b, which indicated 1,3,4-oxadiazole and oxazole moiety are more suitable

as isosteres in this study. Interesting, compounds 25b and 26b showed almost no inhibitory activity against

Axl, but exhibited better cytotoxicity against A549 and PC-3 cell lines than Cabozantinib. To explore the

cause of this phenomenon, we tested the inhibition rate of 26b against five other kinases. The result (Table

S1 in SI) indicated that 26b showed good inhibitory activity against FLT3 and KDR at a concentration of

100 nM. Even, 26b had a percent inhibition 87% at 100 nM against c-Met, which suggested 26b may be a

potential c-Met inhibitor.

Meanwhile, the flexibility of the Linker was investigated by evaluating the target compounds 41a-43a.

In this screening whose results were reported in Table 2, further enhanced activity was observed for

compound 42a and 43a with lower IC₅₀values than the values of the positive control. The results showed

that the activity benefits from the presence of the “methylene”.

Table 2.

The anti-tumor activities and Axl inhibition of target compounds 41a-43a.

IC₅₀(µmol/L) ± SD

Axl %

Compd.

Het

Inhibition at 1 µM

A549

HT-29

PC-3

N

1.16 ± 0.04

0.109±0.004

0.238 ± 0.006

68.0±1.8

83.1±2.4

88.1±6.0

41a

42a

43a

0.106±0.002

0.33 ± 0.15

O

N

0.105±0.001

0.082±0.004

0.54±0.09

0.28 ± 0.08

Cabozantinib

Foretinib

-

3.75±0.01

0.21

4.56± 0.08

8.25 ± 0.63

0.81± 0.09

93.8±0.85

0.25 ± 0.011

ND

^aND = not determined;

Based on the preliminary SAR, we decided to focus our screening on some target compounds bearing

cyclic amino propanoxy group at position 7 of quinoline. As shown in Table 3, when the cyclic amino was

morpholinyl group, compound 42b (a percent inhibition only 24% at 100 nM) afforded lower activities in

the anti-Axl activities compared to 42c-42e. The same trend was observed for compounds 43b-43e. In

addition, compounds containing the 1,3,4-oxadiazole moiety (43b-43e) have better activity than

compounds containing an oxazole moiety (42b-42e). The results suggested that the 1,3,4-oxadiazole moiety

was a privileged scafford for anti-Axl activities.

Encouraged by the observations described above, further investigations were performed to study the

cytotoxic activity of different substituents on the position 4 of phenyl ring. As seen from Table 4, among

these compounds (43f-43i), only 43f (containing 4-bromophenyl fragment) exhibited better cytotoxic

activity than Foretinib, but the activity was still weaker than compound 43a, which bear 4-fluorophenyl

moiety. Accordingly, 4-fluorophenyl derivatives were further studied in our work.

Table 3.

The anti-tumor activities and Axl inhibition of target compounds 42b-42e and 43b-43e.

IC₅₀(µmol/L) ± SD

Axl %

Compd.

R₁

Het

Inhibition at 100 nM

A549

HT-29

PC-3

0.596±0.022

13.74±0.75

2.52±0.03

4.17±0.07

1.042±0.021

4.3±0.12

2.41±0.17

24.0±2.7

55.5±3.2

49.0±1.3

37.0±0.28

27.5±3.7

66.0±0.09

61.5±3.5

58.5±1.9

42b

42c

42d

42e

43b

43c

43d

43e

0.254±0.013

0.178±0.005

1.042±0.006

0.103±0.007

0.071±0.014

0.091±0.011

0.813±0.004

0.57±0.06

0.34±0.03

0.53±0.03

0.68±0.07

0.41±0.03

0.21±0.02

0.40±0.01

3.48±0.07

2.37±0.16

0.813±0.041

Cabozantinib

Foretinib

-

3.75±0.01

0.21

4.56± 0.08

8.25 ± 0.63

0.81± 0.09

-

93.0±0.4

0.25 ± 0.011

ND

^aND = not determined;

Our final modification focused on the R₃and R₄group of the methylene at the alpha position of the

carbonyl group. As shown in Table 4, most of them displayed excellent cytotoxicity against four cancer

cells lines (especially in the inhibition of A549 cell line) with potency from single-digit µM to nanomole

range, which were more potent than Foretinib against one or more cell lines. Three compounds (44a, 45a

and 47e) were found to have 1.8-8.4 fold, 1.1-2.9 fold, 1.38-2.0 fold and 2.0-6.5 fold increase in activity

cytotoxic activity than the positive control Foretinib against A549, HT-29, PC-3 and MCF-7 cell lines in

vitro respectively. In order to further summarize the structure-activity relationship, Axl inhibition activity of

those compounds were evaluated. The results showed that most of those compounds exhibit moderate to

excellent anti-Axl activities with Axl % Inhibition at 100 nM > 50%. In addition, there was a positive

correlation between cytotoxicity and Axl kinase inhibitory activity. As for Cyclic amino (R₁), compounds

with morpholinyl groups are much weaker in terms of cell and kinase activity, which is consistent with the

above conclusion.

Table 4.

Cytotoxicity of compounds 43f-43i and 44a-47e against A549, HT-29, PC-3 and MCF-7.

IC₅₀(µM)

PC-3

Axl %

Entry

R₁

R₃

R₄

R

Inhibition

at 100 nM

A549

HT-29

MCF-7

a

H

Br

Cl

H

OMe

F

ND

43f

43h

43g

43i

0.102±0.006

0.63±0.02

0.583±0.01

2.46±0.03

7.81±0.04

7.52±0.04

1.46±0.10

2.54±0.33

1.96±0.01

1.27±0.04

1.00±0.08

0.554±0.03

2.02±0.03

1.54±0.07

0.570±0.016

1.08±0.05

0.648±0.009

2.48±0.11

1.08±0.02

1.03±0.10

0.871±0.002

0.442±0.003

9.33±0.42

1.05±0.07

0.779±0.015

1.03±0.13

1.62±0.07

0.68±0.02

0.36±0.05

4.73±0.03

0.41±0.06

0.53±0.01

1.18±0.02

1.70±0.03

1.08±0.04

1.26±0.04

0.40±0.02

2.53±0.06

0.97±0.03

1.42±0.06

1.47±0.02

1.49±0.02

1.79±0.08

1.90±0.01

1.05±0.07

1.76±0.05

2.18±0.03

12.95±0.13

1.67±0.02

2.34±0.07

0.59±0.04

0.82±0.11

ND

H

0.92±0.06

ND

H

1.56±0.07

ND

44a

44b

44c

Me

Et

0.032±0.001

0.052±0.004

0.075±0.003

0.076±0.002

0.086±0.002

0.119±0.009

0.049±0.007

0.038±0.006

0.078±0.001

0.092±0.099

0.42±0.02

1.00±0

83.2±0.0

47.9±1.2

80.0±0.8

79.3±2.7

69.7±0.3

78.7±0.5

48.9±2.3

76.0±0.2

75.3±1.1

80.1±1.5

69.9±0.0

42.7±0.1

76.3±0.0

70.3±0.2

73.7±1.0

87.9±0.5

42.7±0.7

82.3±0.3

79.3±0.2

93.7±0.2

88.0±0.1

F

1.05±0.04

1.63±0.03

2.70±0.03

1.04±0.02

0.46±0.03

2.69±0.15

2.22±0.32

1.04±0.09

0.41±0.03

0.37±0.08

1.60±0.13

2.48±0.07

ND

F

44d

44e

F

45a

45b

45c

F

Et

F

Et

F

45d

45e

Et

F

Me Me

F

46a

46b

46c

F

0.275±0.0035

0.597±0.0125

0.263±0.03

0.215±0.011

0.120±0.007

1.66±0.03

F

46d

46e

F

0.47±0.09

0.74±0.03

>100

F

47a

47b

47c

F

2.61±0.22

0.31±0.07

1.01±0.011

2.00±0.06

0.143±0.006

0.150±0.008

0.053±0.008

0.27±0.04

F

47d

47e

F

-

Foretinib

^aND = not determined.

To explore the contribution of Axl kinase inhibition to cytotoxicity, we also tested Axl overexpressing

human breast cancer cell line MDA-MB-231[10, 20, 36] and human noncancerous cell line WI-38. The

results indicated 47e had a significant inhibitory effect on the proliferation of MDA-MB-231, which was

much stronger than that on normal cells (Table S2 in SI).

2.4 In vitro enzymatic assays

As shown in Table 5, compounds 44a, 45a and 47e all exhibited excellent Axl enzymatic potency,

suggesting that the inhibition of Axl may be a mechanism for the antitumor effect of these derivatives.

Compounds 45a and 47e showed the potent activity with an IC₅₀value of 0.036 and 0.010 µM against Axl

respectively, which was comparable to that of the positive control, Foretinib (IC₅₀=0.014 µM), indicating

that these compounds deserve further study with regard to its application in the treatment of cancer.

Table 5.

Inhibitory activities of 44a, 45a, 47e and Foretinib against Axl in vitro.

Compd.

IC₅₀on Axl (µM)^a

44a

0.036

45a

0.077

47e

Foretinib

0.010

0.014

^aThe values are an average of two separate determinations.

2.5 Enzymatic selectivity assays

Compounds 44a and 47e were screened against other 7 tyrosine kinases to examine their selectivity on

Axl over other kinases (Table 6). Compared with its high potency against Axl (IC₅₀=0.036 and 0.010 µM),

47e exhibited inferior inhibitory effects against c-Met (IC₅₀= 0.071 µM), VEGFR-2 (IC₅₀= 0.081 µM) and

c-kit (IC₅₀= 0.063µM). Compound 47e showed inhibitory potency against Flt-3, PDGFRβ, Mer and Tyro3

lower (>10 fold) than that of Axl. These data suggested that compound 47e seem to be an Axl kinase

inhibitor with some selectivity. To identify other kinase targets of 47e, profiling studies were conducted to

evaluate its selectivity toward Axl (Figure 4). Except for Axl, 47e also shows considerable activity against

the other three kinases (Aurora-B, Flt4 and Ron, % Inhibition at 200 nM > 80%).

Table 6.

Inhibition of tyrosine kinases by compound 44a and 47e.

IC₅₀(µM ^a

)

Compd.

44a

Axl

c-Met

0.062

VEGFR-2

0.040

c-Kit

Flt-3

>0.2

PDGFRβ

Mer

>0.2

Tyro3

0.190

0.036

0.047

>0.2

0.010

0.071

0.081

0.063

0.103

>0.2

47e

^aThe values are an average of two separate determinations.

Figure 4. Compound 47e was profiled against a panel of 30 kinases at 200 nM using Eurofins's

SelectScreen kinase profiling service.

2.6 Study on overcoming drug resistance of EGFR-TKI.

Since Axl inhibitor 47e was obtained after multiple optimizations, the anti-EGFR-TKI resistance activity

was investigated as shown in Table 7. The Axl inhibitor 47e exhibits prominent inhibition against both

H1975 and gefitinib- resistant H1975.

Table 7.

Anti-proliferative activity towards H1975 and H1975/Gefitinib.

IC₅₀(µM)

Entry

H1975

0.099±0.008

6.5±0.14

H1975/Gefitinib

0.107±0.031

78.7±1. 7

47e

Gefitinib

3. Conclusion

In summary, through a stepwise structure-activity relationship analysis, a series of 6,7-disubstituted

quinoline derivatives containing 1,3,4-oxadiazole acetamide moiety were synthesized and evaluated for

their Axl kinase inhibition and cytotoxicity against A549, HT-29, PC-3 and MCF-7 cancer cell lines in vitro.

Most compounds exhibited moderate to excellent activity, with IC₅₀values ranging from 0.032 to 1.54 µM

against A549, HT-29, PC-3 and MCF-7 respectively. Some selected compounds (44a, 45a and 47e) were

evaluated for the activity against Axl kinase, and compounds 44a and 47e were further evaluated for other

seven tyrosine kinases (c-Met, VEGFR-2, c-Kit, Flt-3, PDGFRβ, Mer and Tyro3) to test the enzyme-based

selectivity. The studies of SARs indicated that the presence of a carbonyl alpha-methylene was of

significant importance to the inhibition potency and selectivity. In a sense, this study provides a novel type

of linker for Axl kinase inhibitors, namely 1,3,4-oxadiazole acetamide moiety, which equipped more

suitable length and flexibility to improve activity. Moreover, the introduction of a three-carbon tether

contained cyclic amino (R₁) into the 7-position of quinolines was advantageous to the potency. To our

delight, the most promising compound 47e, as a Axl kinase inhibitor, showed the better cytotoxic activities

than Foretinib with 1.9 fold, 1.57 fold, 1.54 fold and 2.0 fold increases in activity against A549, HT-29,

PC-3 and MCF-7 cell lines in vitro respectively. What's more, compound 47e exhibits prominent inhibition

against both H1975 and gefitinib- resistant NSCLC cell line H1975/ gefitinib.

4. Experimental protocols

4.1 Chemistry

Unless otherwise noted, all materials were obtained from commercial suppliers and used without further

purification. All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi

Labortechnik, Flawil, Switzerland) and were uncorrected. The IR spectra were recorded by means of the

KBr pellet technique on a Bruker FTS 135 spectrometer. ¹H NMR and ¹³C NMR spectra were generated on

a Bruker ARX-300 or ARX-600 spectrometers (Bruker Bioscience, Billerica, MA, USA). Column

chromatography was carried out on silica gel (200-300 mesh). Mass spectra (MS) were taken in ESI mode

on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Elemental analysis was determined on a

Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba, Milan, Italy).

4.2. Preparation of aromatic-substituted heterocyclic carboxylate esters (12, 15, 18 and 21)

4.2.1. The preparation of 4-fluorobenzohydrazide (11)

To a stirred solution of 4-fluoroethylbenzoate (4.5g, 26.7mmol) in EtOH (30mL) was added

.

NH₂NH₂H₂O (6.67g, 133.5mmol) (80% in H₂O) at room temperature and refluxed at 85°C for 10 h. The

reaction was cooled to room temperature, and evaporated in vacuo to afford a crude reside which was

washed with n-hexane (50mL) and then dried to obtain compound. Yield: (4.2g, quantitative yield), white

solid. MS (ESI) m/z (%): 155.4 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 9.85 (s, 1H), 7.86 (dd, 2H),

7.25 (t, 3H), 4.46 (s, 1H), 3.47 (br s, 1H).

4.2.2. The preparation of ethyl 5-(4-fluorophenyl)-1,3,4-oxadiazole-2-carboxylate (12)

Ethyl oxalyl monochloride (0.64g, 5.25mmol) was added drop-wise to

a

solution of

4-fluorobenzohydrazide (11, 0.78g, 5.0mmol) and triethylamie (1.01g, 10.0mmol) in DCM in an ice bath.

Upon completion of the addition, the reaction mixture was removed from the ice bath and placed in room

temperature for 8h and monitored by thin-layer chromatography (TLC). The mixture was washed with 10%

K₂CO₃(150 mLÕ3) followed by brine (150 mLÕ1), and the organic phase was separated, dried, and

evaporated to yield ethyl 2-(2-(4-fluorobenzoyl)hydrazinyl)-2-oxoacetate as yellow solid, Yield: 83%; MS

(ESI)m/z(%): 255.4 [M+Na]⁺.

Ethyl 2-(2-(4-fluorobenzoyl)hydrazinyl)-2-oxoacetate (2.06g) was added to POCl₃150mL and then

o

heated to 85 C for 3h. The reaction mixture was evaporated and then poured into water, basified with

sodium hydroxide to PH = 7, and extracted with dichloromethane (200 mL Õ 3). The combined extracts

were washed with brine (200 mLÕ2), dried over anhydrous Na₂SO₄, and concentrated in vacuum to give

the corresponding ethyl 5-(4-fluorophenyl)-1,3,4-oxadiazole-2-carboxylate (12) as white solid; MS (ESI)

1

m/z(%): 237.4 [M+Na]⁺; H NMR (400 MHz, CDCl₃) δ 7.92 (dd, J=8.6, 5.3 Hz, 2H), 7.13 (t, J= 8.5 Hz,

2H), 4.40 (q, J= 7.1 Hz, 2H), 1.40 (t, J= 7.1 Hz, 3H).

4.2.3. The preparation of 2-amino-1-(4-fluorophenyl)ethanone hydrochloride (14)

To a solution of 1-(4-fluorophenyl)ethan-1-one (15.0mmol, 1.0 equiv) in 8 mL of dichloromethane were

added NBS (2.72g, 15.3mmol, 1.02equiv) and p-toluenesulfonic acid (2.85g, 15.0mmol, 1.0 equiv). The

reaction mixture was stirred for 24 h at 50 °C. After that time, the solvent was evaporated under reduced

pressure. A water solution of saturated NaHCO₃(30 mL) was then added, and the solution was extracted

with dichloromethane (3 × 30 mL). The organic layers were combined and dried over Na₂SO₄. The solvent

was evaporated, and the residue was subjected to column chromatography (silica gel) using

1

hexanes/CH₂Cl₂(from 9:1 to 4:1) as eluent to obtain 2-bromo-1-(4-fluorophenyl)ethan-1-one. H NMR

(600 MHz, CDCl₃) δ 8.05-8.00 (m, 2H), 7.19-7.13 (m, 2H), 4.41 (s, 2H).

A solution of hexamethylenetetramine (1.42g, 10.1mmol) in CHCl₃was added drop-wise over 30 min to

a solution of 2-bromo-4-fluoroacetophenone (2.0g, 9.2mmol) in dry CHCl₃40 mL at 0 °C. The reaction

mixture was allowed to warm up to room temperature and stirred for 16 h. After completion of the reaction

the solid precipitate was collected by filtration and washed with CHCl₃. The solid obtained was suspended

in EtOH 40mL and conc. HCl 4 mL was added. The mixture was heated to 80 °C for 3 h, cooled to room

temperature, and the solid formed was filtered off. The clear filtrate was concentrated.

2-amino-1-(4-fluorophenyl)ethanone hydrochloride (14, 1.5g) as yellow solid. MS (ESI) m/z(%):

154.3[M+H]⁺; ¹H NMR (600 MHz, CDCl₃) δ 8.50 (s, 1H), 8.15 (m, 2 H), 7.44 (m, 2 H), 4.65 (m, 2 H).

4.2.4. The preparation of ethyl 5-(4-fluorophenyl)oxazole-2-carboxylate (15)

Ethyl oxalyl monochloride (0.64g, 5.25mmol) was added drop-wise to

a

solution of

2-Amino-1-(4-fluorophenyl)ethanone hydrochloride (14, 0.78g, 5.0mmol) and triethylamie (1.01g,

10.0mmol) in dichloromethane in an ice bath. Upon completion of the addition, the reaction mixture was

removed from the ice bath and placed in room temperature for 8h and monitored by thin-layer

chromatography (TLC). The mixture was washed with 10% K₂CO₃(150 mLÕ3) followed by brine (150

mLÕ1), and the organic phase was separated, dried, and evaporated to yield Ethyl

2-((2-(4-fluorophenyl)-2-oxoethyl)amino)-2-oxoacetate as white solid;

Ethyl 2-((2-(4-fluorophenyl)-2-oxoethyl)amino)-2-oxoacetate (2.06g) was added to POCl₃15mL and

then heated to 85^oC for 3h. The reaction mixture was evaporated and then poured into water, basified with

sodium hydroxide to PH 7, and extracted with dichloromethane (200 mL Õ 3). The combined extracts were

washed with brine (200 mLÕ2), dried over anhydrous Na₂SO₄, and concentrated in vacuum to give the

ethyl 5-(4-fluorophenyl)-1,3,4-oxadiazole-2-carboxylate (15) as white solid; MS (ESI) m/z(%):

1

236.3[M+H]⁺; H NMR (400 MHz, CDCl₃) δ7.76 (dd, J=8.7,5.2 Hz, 2H), 7.48 (s,1H), 7.17(t, J=8.6 Hz,

2H), 4.50(q, J=7.1 Hz, 2H),1.46(t, J=7.1 Hz, 3H).

4.2.5. The preparation of 4-fluoro-N-hydroxybenzimidamide (17)

To a solution of 4-fluoro benzonitrile (4.84g, 40mmol) in ethanol (50mL), was added hydroxylamine

hydrochloride (6.5g, 100mmol), followed by addition of triethylamine (5.5g, 52mmol). The reaction mass

was refluxed for 8 h. Excess of solvent was removed under vacuum and the reaction mass was diluted with

water, acidified with dilute HCl and filtered to afford the desired product. MS (ESI) m/z (%): 189.1 [M +

1

H]⁺; H NMR (DMSO-d₆): δ 5.95 (s, 2H), 7.42 (t, J = 8.7 Hz, 1H), 7.69 (m, 1H), 7.83 (dd, 1H), 9.80 (s,

1H).

4.2.6. The preparation of ethyl 3-(4-fluorophenyl)-1,2,4-oxadiazole-5-carboxylate (18)

Ethyl oxalyl monochloride (0.64g, 5.25mmol) was added drop-wise to

a

solution of

4-fluoro-N-hydroxybenzimidamide (17, 0.78g, 5.0mmol) and triethylamie (1.01g, 10.0mmol) in

dichloromethane in an ice bath. Upon completion of the addition, the reaction mixture was removed from

the ice bath and placed in room temperature for 8h and monitored by thin-layer chromatography (TLC).

The mixture was washed with 10% K₂CO₃(150 mLÕ3) followed by brine (150 mLÕ1), and the organic

phase was separated, dried, and evaporated to yield Ethyl 2-(2-(4-fluorobenzoyl)hydrazinyl)-2-oxoacetate

as yellow solid. MS (ESI) m/z (%): 255.1 [M + H]⁺.

Ethyl 2-(2-(4-fluorobenzoyl)hydrazinyl)-2-oxoacetate (2.06g) was added to POCl₃15mL and then

o

heated to 95 C for 3h. The reaction mixture was evaporated and then poured into water, basified with

sodium hydroxide to PH = 7, filtered and obtain the ethyl 5-(4-fluorophenyl)-1,3,4-oxadiazole

-2-carboxylate as yellow solid. MS (ESI) m/z (%): 238.1 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d,

J= 8.4 Hz，2H), 7.66 (d, J= 8.6 Hz，2H), 4.37 (q, J= 7.1 Hz, 2H), 1.42 (t, J= 7.2 Hz, 3H).

4.2.7. The preparation of ethyl 4-(4-fluorophenyl)-2,4-dioxobutanoate (20)

In a three-necked flask acetophenone (5g, 41.61mmol) and diethyl oxalate (6.76mL, 50mmol) were

dissolved in anhydrous DMF 65mL at 0°C and t-BuOK (2g, 83.25mmol) was slowly added, the mixture

was stirred at 0°C for 15 min and 30 min more at room temperature before heating at 45 °C for 1h.

Reaction was poured into water-acetic acid and product extracted with ethyl acetate. Organic layers were

combined, washed with water and brine, dried over magnesium sulphate, filtered and concentrated to yield

the title compound. MS (ESI) m/z (%): 237.2 [M - H]⁺

4.2.8. The preparation of ethyl 5-(4-fluorophenyl)isoxazole-3-carboxylate (21)

A mixture of 20 (3 g, 11.03 mmol) and NH₂-OH^.HCl (2.28 g, 33.09 mmol) in MeOH (50 mL) was

heated to reflux overnight. The MeOH was then evaporated and the residue was extracted with ethyl acetate,

dried over Na₂SO₄. After filtration and concentration in vacuo, the crude residue was purified by column to

1

afford 21; MS (ESI) m/z (%): 236.1[M + H]⁺; H NMR (400 MHz, CDCl₃) δ7.85-7.78 (m, 2H), 7.20(t,

J=8.6 Hz, 2H), 6.89(s, 1H), 4.01(s, 3H).

4.3. General procedure for preparation of aromatic ring-substituted heterocycliccarbonyl chloride (13, 16,

19 and 22)

The corresponding aromatic ring-substituted heterocycliccarbonyl esters (12, 15, 18 and 21, 10mmol)

was dissolved in THF 10mL and methanol 40mL. A solution of lithium hydroxide (0.6g, 30mmol) in water

3mL was slowly added. The reaction mixture was stirred at room temperature for 5 h and monitored by

thin-layer chromatography (TLC). The reaction was extracted with dichloromethane (40 mL Õ 3) and the

aqueous phase was acidified with 1N HCl and the corresponding aromatic ring-substituted

heterocycliccarboxylic acid was isolated as white solid.

Thoroughly dried aromatic ring-substituted heterocyclic carboxylic acid 3.5mmol, DMF 1 drops,

anhydrous dichloromethane was added in a three-neck flask under a N₂atomosphere. Oxalyl chloride

(4.2mmol) was drop-wise added at 0 ^oC. The reaction mixture was stirred at room temperature for 4h. And

the reaction was then removed in vacuo. The resulting aromatic ring-substituted heterocycliccarbonyl

chloride (13, 16, 19 and 22) was used immediately without further purification.

4.4. General procedure for preparation of target compounds (23a-b, 24a-b, 25a-b and 26a-b)

A mixture of corresponding carbonyl chloride (13, 16, 19 and 22, 1.5eq.) in DCM was added to a

solution of triethylamine (0.2g, 2.0mmol) and 9 or 10c (0.493g, 1.0mmol) in DCM 10 mL. The reaction

mixture was placed at room temperature for 2 h. The solvent was concentrated in vacuum and the residue

was resolved with DCM (100 mL), washed with water (30 mLÕ 3), brine (30 mL), dried over anhydrous

Na₂SO₄, and concentrated in vacuum. The residue was purified by column chromatography to afford the

target compounds (23a-b, 24a-b, 25a-b and 26a-b).

4.4.1. N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole-2-carboxamide

(23a)

Yield: 62%; m.p. 224 - 126^oC; MS (ESI) m/z (%): 487.3 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.05

(s, 1H), 8.52 (d, J = 5.9 Hz, 1H), 8.22 (dd, J = 8.6, 5.2 Hz, 2H), 7.90 (d, J = 8.8 Hz, 2H), 7.86 (s, 1H), 7.61

(s, 1H), 7.32 - 7.25 (m, 4H), 6.64 (d, J = 6.0 Hz, 1H), 4.13 (s, 3H), 4.10 (s, 3H); ¹³C NMR (101 MHz,

DMSO) δ 165.05, 165.03 (d, J = 251.0 Hz), 160.17, 159.03, 153.07, 151.92, 151.10, 149.86, 149.32,

146.96, 135.45, 130.47 (d, J = 9.5 Hz), 123.14, 121.90, 117.37 (d, J = 22.4 Hz), 115.68, 108.34, 103.85,

99.57, 56.21, 56.19. Anal. calcd. for C₂₆H₁₉FN₄O₅(%): C, 64.20; H, 3.94; F, 3.91; N, 11.52; O, 16.44.

Found (%): C, 64.21; H, 3.93; N, 11.51.

4.4.2.

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-5-(4-fluorophen

yl)-1,3,4-oxadiazole-2-carboxamide (23b)

o

1

Yield: 61%; m.p. 122 - 123 C; MS (ESI) m/z (%): 630.1 [M + H]⁺; H NMR (600 MHz, DMSO) δ

11.28 (s, 1H), 8.49 (d, J = 5.1 Hz, 1H), 8.09 (m, 2H), 7.91 (d, J = 12.7 Hz, 1H), 7.61-7.47（m, 6H）, 6.54 (d,

J = 5.0 Hz, 1H), 4.21 (t, J = 6.1 Hz, 2H), 3.98 (s, 3H), 3.14-2.96(m, 2H), 2.78 – 2.58(m, 2H), 2.07(d, J =

17.0 Hz, 4H), 1.68 (d, J = 12.3 Hz, 2H), 1.48 (s, 1H), 1.25(s, 2H), 0.93 (d, J = 6.4 Hz, 3H). Anal. calcd. for

C₃₄H₃₃F₂N₅O₅(%): C, 64.86; H, 5.28; F, 6.03; N, 11.12; O, 12.70. Found (%): C, 64.85; H, 5.24; N, 11.15.

4.4.3. N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-5-(4-fluorophenyl)oxazole-2-carboxamide (24a)

Yield: 54%; m.p. 242 - 243 ^oC; MS (ESI) m/z (%): 486.1 [M + H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ

10.94 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.06 (dd, J = 8.6, 5.4 Hz, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.52 (d, J =

1.8 Hz, 2H), 7.43 (dd, J = 15.9, 7.0 Hz, 3H), 7.31 (d, J = 8.5 Hz, 2H), 6.50 (d, J = 5.3 Hz, 1H), 3.96 (s, 3H),

3.95 (s, 3H).¹³C NMR (101 MHz, DMSO) δ 170.19, 163.92 (d, J = 249.9 Hz), 160.40, 160.26, 157.64,

153.04, 150.67, 149.82, 149.31, 146.95, 135.97, 128.93 (d, J = 8.9 Hz), 123.46, 122.83, 121.86, 117.02 (d,

J = 22.4 Hz), 115.64, 108.34, 103.71, 100.64, 99.56, 56.19. Anal. calcd. for C₂₇H₂₀FN₃O₅(%): C, 66.80; H,

4.15; F, 3.91; N, 8.66; O, 16.48. Found (%): C, 66.76; H, 4.12; N, 8.62.

4.4.4. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-5-(4-fl

uorophenyl)oxazole-2-carboxamide (24b)

o

1

Yield: 57%; m.p. 211 - 213 C; MS (ESI) m/z (%): 629.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

11.17 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.11 - 7.99 (m, 3H), 7.79 (d, J = 8.4 Hz, 1H), 7.57 - 7.38 (m, 6H),

6.48 (d, J = 5.2 Hz, 1H), 4.19 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 2.92 - 2.78 (m, 2H), 2.47 - 2.37 (m, 2H),

2.04 - 1.81 (m, 5H), 1.65 - 1.52 (m, 2H), 1.40 - 1.26 (m, 2H), 0.89 (d, J = 6.4 Hz, 3H); ¹³C NMR (126 MHz,

CDCl₃) δ 164.54, 162.54, 159.88, 154.52 (d, J = 250.2 Hz), 154.01 (d, J = 53.0 Hz), 152.69, 152.31, 149.91,

148.73, 146.94, 137.99 (d, J = 12.5 Hz), 135.55 (d, J = 9.6 Hz), 127.18 (d, J = 8.4 Hz), 124.02, 122.93 (d, J

= 3.4 Hz), 122.44, 116.44 (d, J = 22.3 Hz), 116.09 (d, J = 3.4 Hz), 115.47, 109.49 (d, J = 23.2 Hz), 108.81,

102.28, 99.51, 67.52, 56.15, 55.41, 53.94, 33.99, 30.66, 26.28, 21.79. Anal. calcd. for C₃₅H₃₄F₂N₄O₅(%): C,

66.87; H, 5.45; F, 6.04; N, 8.91; O, 12.72. Found (%): C, 66.84; H, 5.43; N, 8.93.

4.4.5. N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-3-(4-fluorophenyl)-1,2,4-oxadiazole-5-carboxamide

(25a)

Yield: 61%; m.p. 247 - 249^oC; MS (ESI) m/z (%): 487.2 [M + H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.05

(s, 1H), 8.71 (d, J = 5.9 Hz, 1H), 8.17 (dd, J = 8.6, 5.2 Hz, 2H), 7.90 (d, J = 8.8 Hz, 2H), 7.86 (s, 1H), 7.61

(s, 1H), 7.42 - 7.35 (m, 4H), 6.64 (d, J = 6.0 Hz, 1H), 4.11 (s, 3H), 4.09 (s, 3H). Anal. calcd. for

C₂₆H₁₉FN₄O₅(%): C, 64.20; H, 3.94; F, 3.91; N, 11.52; O, 16.44. Found (%): C, 64.13; H, 3.98; N, 11.51.

4.4.6. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-3-(4-fluo

rophenyl)-1,2,4-oxadiazole-5-carboxamide (25b)

o

1

Yield: 61%; m.p. 136 - 137 C; MS (ESI) m/z (%): 630.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

11.64 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.19 (dd, J = 8.7, 5.5 Hz, 2H), 8.05 (dd, J = 12.8, 2.3 Hz, 1H), 7.84

(d, J = 9.3 Hz, 1H), 7.60 - 7.37 (m, 6H), 6.52 (d, J = 5.0 Hz, 1H), 4.24 (t, J = 6.4 Hz, 3H), 3.97 (s, 3H),

2.96 (dd, J = 12.2, 5.1 Hz, 2H), 2.15 (s, 2H), 1.80 - 1.67 (m, 3H), 1.60 - 1.44 (m, 2H), 0.93 (d, J = 6.4 Hz,

3H). Anal. calcd. for C₃₄H₃₃F₂N₅O₅(%): C, 64.86; H, 5.28; F, 6.03; N, 11.12; O, 12.70. Found (%): C, 64.84;

H, 5.23; N, 11.14.

4.4.7. N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-5-(4-fluorophenyl)isoxazole-3-carboxamide (26a)

o

1

Yield: 43%; m.p. 204 – 205 C; MS (ESI) m/z (%): 486.1 [M + H]⁺; H NMR (400 MHz, DMSO) δ

11.03 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.04 - 7.92 (m, 5H), 7.52 (s, 1H), 7.42 (dd, J = 11.3, 6.3 Hz, 3H),

7.32 (d, J = 8.9 Hz, 2H), 6.50 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); ¹³C NMR (101 MHz, DMSO)

δ 163.08 (d, J = 247.6 Hz), 160.24, 154.42, 153.48, 153.02, 152.66, 150.69, 149.81, 149.30, 146.95, 135.88,

127.65 (d, J = 8.6 Hz), 124.18, 123.80, 123.77, 122.93, 121.81, 116.87 (d, J = 22.3 Hz), 115.64, 108.33,

103.72, 99.55, 56.18, 56.15. Anal. calcd. For C₂₇H₂₀FN₃O₅(%): C, 66.80; H, 4.15; F, 3.91; N, 8.66; O,

16.48. Found (%): C, 66.82; H, 4.12; N, 8.63.

4.4.8. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-5-(4-fl

uorophenyl)isoxazole-3-carboxamide (26b)

o

1

Yield: 61%; m.p. 197 – 199 C; MS (ESI) m/z (%): 629.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

11.23 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.06 (dd, J = 11.9, 3.3 Hz, 2H), 7.95 (dd, J = 8.8, 5.3 Hz, 2H), 7.83

(d, J = 8.5 Hz, 1H), 7.59 - 7.48 (m, 2H), 7.45 - 7.37 (m, 3H), 6.49 (d, J = 5.2 Hz, 1H), 4.19 (t, J = 6.3 Hz,

2H), 3.96 (s, 3H), 2.88 (s, 2H), 2.51 - 2.37 (m, J = 1.6 Hz, 11H), 2.10 - 1.79 (m, 4H), 1.59 (d, J = 11.5 Hz,

2H), 1.41 - 1.27 (m, 1H), 1.22 - 1.10 (m, J = 11.7 Hz, 2H), 0.89 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz,

DMSO) δ 167.04, 160.78 (d, J = 225.9 Hz), 153.75 (d, J = 244.9 Hz), 154.12, 153.67, 152.89, 152.21,

150.00, 149.39, 146.77, 137.38 (d, J = 9.9 Hz), 137.00 (d, J = 11.9 Hz), 132.50 (d, J = 9.4 Hz), 127.72 (d, J

= 8.2 Hz), 124.39 (d, J = 27.9 Hz), 123.71, 116.91 (d, J = 22.3 Hz), 116.51 (d, J = 294.0 Hz), 115.94 (d, J =

22.5 Hz), 109.90 (d, J = 22.6 Hz), 109.13, 102.67, 99.56, 66.91, 56.29, 54.49, 53.05, 32.73, 29.57, 25.28,

21.76. Anal. calcd. for C₃₅H₃₄F₂N₄O₅(%): C, 66.87; H, 5.45; F, 6.04; N, 8.91; O, 12.72. Found (%): C,

66.86; H, 5.46; N, 8.95.

4.5.1 The preparation of ethyl 2-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)acetate (27)

Ethyl hydrogen malonate (0.70g, 5.25mmol) was added drop-wise to

a

solution of

4-fluoro-N-hydroxybenzimidamide (17, 0.78g, 5.0mmol), EDCI (1.5eq.) and triethylamine (1.01g,

10.0mmol) in dichloromethane in an ice bath. Upon completion of the addition, the reaction mixture was

removed from the ice bath and placed in room temperature for 8h and monitored by thin-layer

chromatography (TLC). The mixture was washed with 10% K₂CO₃(150 mLÕ3) followed by brine (150

mLÕ1), and the organic phase was separated, dried and evaporated to yield ethyl

3-((4-fluorobenzimidamido)oxy)-3-oxopropanoate as yellow solid. MS (ESI) m/z (%): 269.1 [M + H]⁺.

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The intermidate was added to POCl₃15mL and then heated to 95 C for 3h. The reaction mixture was

evaporated and then poured into water, basified with sodium hydroxide to PH = 7, filtered and obtain the

ethyl 2-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)acetate as yellow solid. MS (ESI) m/z (%): 251.3 [M +

H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.06 (dd, J= 3.2, 3.4 Hz 2H), 7.42 (t, J= 8.4 Hz, 2H), 4.36 (s, 2H), 4.17

(q, J= 7.4 Hz 2H), 1.20 (t, J= 7.2 Hz 3H).

4.5.2 The preparation of 2-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)acetyl chloride (28)

The ethyl 2-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)acetate (27, 12.5g, 50mmol) was dissolved in

THF 200mL added methanol and a solution of LiOH (1.8g, 75mmol) in water 15mL. The reaction mixture

was stirred at room temperature for 5 h and monitored by thin-layer chromatography (TLC). The reaction

was extracted with dichloromethane (70 mL Õ 3) and the aqueous phase was acidified with 1N HCl and

the corresponding aromatic ring-substituted heterocyclic carboxylic acid was isolated as white solid.

2-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)acetic acid (7.7g, 35mmol), DMF (2 drops), anhydrous

dichlromethane were added under a N₂atomosphere. Oxalyl chloride (5.28g, 42mmol) was drop-wise

added at 0 ^oC. The reaction mixture was stirred at room temperature for 4h. The mixture was then removed

in vacuo, and the resulting 5-(4-fluorophenyl)-1,3,4-oxadiazole-2-carbonyl chloride (28) was used

immediately without further purification.

4.5.3 The preparation of N-(3-fluoro-4-((6-methoxy-7- (3-(4-methylpiperidin-1-yl)propoxy)quin-olin-4-yl)

oxy)phenyl)-2-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)acetamide (41a)

Triethylamine (0.2g, 2.0mmol) was added to a solution of 5-(4-fluorophenyl)-1,3,4-oxadiazole-

2-carbonyl chloride 29 and 10 (0.493g, 1.0mmol) in DCM. The reaction mixture was placed at room

temperature for 2 h. The solvent was concentrated in vacuum and the residue was resolved with

dichloromethane (20 mL), washed with water (10 mLÕ 3), brine (30 mL), dried over anhydrous Na₂SO₄,

and concentrated in vacuum. The residue was purified by column chromatography to afford the target

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1

compound. Yield: 51%; m.p. 214 - 216 C; MS (ESI) m/z (%): 644.2 [M + H]⁺; H NMR (600 MHz,

DMSO) δ 11.26 (s, 1H), 8.53 (d, J = 4.1 Hz, 1H), 8.09 (m, 2H), 7.90 (d, J = 12.7 Hz, 1H), 7.59 (s, 1H), 7.48

(m, 5H), 6.54 (d, J = 3.4 Hz, 1H), 4.41 (s, 2H), 4.28 (t, J = 5.2 Hz, 2H), 3.98 (s, 3H), 3.51 (d, J = 11.6 Hz,

2H), 3.21 (s, 3H), 2.94 (dd, J = 21.9, 10.7 Hz, 2H), 2.32 (s, 2H), 1.79 (d, J = 13.4 Hz, 2H), 1.63 (s, 1H),

1.51 (dd, J = 24.2, 11.9 Hz, 2H), 0.93 (d, J = 6.2 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 175.48,

32.7332.7350, 164.52, 164.45 (d, J = 249.3 Hz), 159.93, 153.85 (d, J = 245.7 Hz), 152.12, 150.00, 149.16,

138.13 (d, J = 9.8 Hz), 136.31 (d, J = 11.9 Hz), 130.03 (d, J = 9.0 Hz), 124.68, 123.10, 117.08, 116.86,

115.87 (d, J = 141.7 Hz), 109.04, 108.54 (d, J = 23.0 Hz), 102.66, 99.66, 66.59, 56.35, 54.06, 52.41, 35.48,

31.26, 28.64, 23.72, 21.58; Anal. calcd. for C₃₅H₃₅F₂N₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N, 10.88; O,

12.43. Found (%): C, 65.25; H, 5.35; N, 10.80.

4.6 General preparation of 42a~42e.

Starting from 2-amino-1-(4-fluorophenyl)ethan-1-one (14), the target compounds were prepared by the

method metioned above.

4.6.1

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluoro

phenyl)oxazol-2-yl)acetamide (42a)

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Yield: 55%; m.p. 221 - 223 C; MS (ESI) m/z (%): 643.1 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.77 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.87 (d, J = 13.1 Hz, 1H), 7.76 (dd, J = 8.7, 5.4 Hz, 2H), 7.64 (s,

1H), 7.54 (s, 1H), 7.49 - 7.29 (m, 5H), 6.46 (d, J = 4.9 Hz, 1H), 4.22 (t, J = 6.0 Hz, 2H), 4.07 (s, 2H), 3.95

(s, 3H), 3.24 - 2.96 (m, 4H), 2.19 - 1.99 (m, 2H), 1.76 - 1.61 (m, 2H), 1.45 (s, 2H), 1.34 - 1.15 (m, J = 15.6,

8.2 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H). Anal. calcd. for C₃₆H₃₆F₂N₄O₅(%): C, 67.28; H, 5.65; F, 5.91; N,

8.72; O, 12.45. Found (%): C, 67.13; H, 5.60; N, 8.73.

4.6.2

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)oxaz

ol-2-yl)acetamide (42b)

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Yield: 56%; m.p. 157 - 158 C; MS (ESI) m/z (%): 631.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.77 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.87 (d, J = 12.7 Hz, 1H), 7.76 (dd, J = 8.7, 5.4 Hz, 1H), 7.64 (s,

1H), 7.53 (s, 1H), 7.46 (d, J = 4.2 Hz, 1H), 7.41 (s, 1H), 7.34 (t, J = 8.9 Hz, 1H), 6.45 (d, J = 5.2 Hz, 1H),

4.21 (t, J = 6.1 Hz, 1H), 4.07 (s, 2H), 3.95 (s, 2H), 3.61 (s, 4H), 2.47 (m, 6H), 2.00 (m, 2H); ¹³C NMR (101

MHz, DMSO) δ 165.87, 161.97 (d, J = 452.7 Hz), 161.16, 158.68, 154.57 (d, J = 203.2 Hz), 152.29, 150.71,

150.02, 149.33, 146.76, 137.61, 137.44, 136.30, 136.19, 126.63, 126.54, 124.67, 123.06, 119.38, 116.77,

116.56, 116.45, 114.98, 109.05, 108.58, 108.35, 102.51, 99.98, 99.53, 75.08, 66.99, 66.06, 56.28, 55.05,

53.35, 37.09; Anal. calcd. for C₃₄H₃₂F₂N₄O₆(%): C, 64.75; H, 5.11; F, 6.03; N, 8.88; O, 15.22. Found (%):

C, 64.68; H, 5.10; N, 8.82;.

4.6.3

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluoro

phenyl)oxazol-2-yl)acetamide (42c)

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Yield: 58%; m.p. 168 - 169 C; MS (ESI) m/z (%): 644.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.78 (s, 1H), 8.47 (d,J = 5.2 Hz, 1H), 7.87 (d, J = 12.9 Hz, 1H), 7.76 (dd, J = 8.7, 5.4 Hz, 2H), 7.64 (s,

1H), 7.42 (m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.1 Hz, 2H), 4.07 (s, 2H), 3.95 (s, 3H), 2.70 - 2.52

(m, 8H), 2.34 (s, 3H), 2.04 - 1.95(m,2H). Anal. calcd. for C₃₅H₃₅F₂N₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N,

10.88; O, 12.43.Found(%):C, 65.20; H, 5.45; N, 10.78.

4.6.4 N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-

fluorophenyl)oxazol-2-yl)acetamide (42d)

1

Yield: 53%; m.p. 120 - 121^oC; MS (ESI) m/z (%): 615.2 [M + H]+ ; H NMR (400 MHz, DMSO) δ

10.76 (s, 1H), 8.48 (d,J = 5.2 Hz, 1H), 7.87 (dd, J = 12.6, 0.7 Hz, 1H), 7.76 (dd, J = 8.7, 5.4 Hz, 2H), 7.64

(s, 1H), 7.56 (s, 1H), 7.48 - 7.42 (m, 3H), 7.34 (t, J = 8.9 Hz, 2H), 6.47 (d, J = 4.8 Hz, 1H)，4.26 (t, J = 6.0

Hz, 2H), 4.07 (s, 2H), 3.96 (s, 3H), 3.07 (dd, J = 13.9, 6.8 Hz, 5H), 2.25 - 2.13 (m, 2H), 1.98 - 1.86 (m, 4H);

¹³C NMR (101 MHz, DMSO) δ 165.89, 162.39 (d, J = 246.0 Hz), 159.77, 158.68, 153.88 (d, J = 246.5 Hz),

151.94, 150.71, 149.91, 149.45, 146.68, 138.23 (d, J = 10.1 Hz), 136.23 (d, J = 12.5 Hz), 132.56 (d, J =

10.0 Hz), 126.59 (d, J = 8.5 Hz), 124.65, 123.05, 116.67 (d, J = 22.3 Hz), 116.49, 115.17, 109.28, 108.49 (d,

J = 22.8 Hz), 102.66, 99.63, 66.32, 56.36, 53.89, 52.15, 37.09, 25.66, 23.07; Anal. calcd. For

C₃₄H₃₂F₂N₄O₅(%): C, 66.44; H, 5.25; F, 6.18; N, 9.12; O, 13.01. Found (%): C, 66.34; H, 5.26; N, 9.10.

4.6.5

N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)o

xazol-2-yl)acetamide (42e)

Yield: 52%; m.p. 104 - 106^oC; MS (ESI) m/z (%): 629.7 [M + H]+; H NMR (400 MHz, DMSO) δ

1

10.77 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.87 (d, J = 13.3 Hz, 1H), 7.76 (dd, J = 8.8, 5.4 Hz, 2H), 7.64 (s,

1H), 7.54 (s, 1H), 7.47 (dd, J = 26.5, 22.0 Hz, 3H), 7.34 (t, J = 8.9 Hz, 2H), 6.46 (d, J = 5.0 Hz, 1H), 4.22 (t,

J = 6.1 Hz, 2H), 4.07 (s, 2H), 3.96 (s, 3H), 2.61 (dd, J = 29.0, 16.6 Hz, 6H), 2.11 (s, 2H), 1.62 (s, 4H), 1.46

(s, 2H).Anal. calcd. for C₃₅H₃₄F₂N₄O₅(%): C, 66.87; H, 5.45; F, 6.04; N, 8.91; O, 12.72. Found (%): C,

66.85; H, 5.40; N, 8.88.

4.7. General preparation of 43a~43i and 44a~47e.

Starting from 4-fluorobenzohydrazide (11), the compounds were prepared by the method mentioned

above.

4.7.1 N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-

fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (43a)

1

Yield: 59%; m.p. 123 - 125^oC; MS (ESI) m/z (%): 644.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.89 (s, 1H), 8.47 (d, J = 5.1 Hz, 1H), 8.08 (dd, J = 8.8, 5.3 Hz, 2H), 7.86 (d, J = 12.3 Hz, 1H), 7.56 - 7.37

(m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.29 (s, 2H), 4.20 (t, J = 6.1 Hz, 2H), 3.95 (s, 3H), 3.12 - 2.94 (m, 2H),

2.77 - 2.57 (m, 2H), 2.07 (d, J = 17.0 Hz, 4H), 1.64 (d, J = 12.3 Hz, 2H), 1.49 - 1.32 (m, 1H), 1.23 (s, 2H),

0.90 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 165.03, 164.58 (d, J = 250.4 Hz), 164.26, 162.35,

159.68, 153.90 (d, J = 246.1 Hz), 152.30, 150.02, 149.32, 146.82, 138.01 (d, J = 9.8 Hz), 136.39 (d, J =

12.2 Hz), 129.66 (d, J = 9.1 Hz), 124.70, 120.44 (d, J = 3.1 Hz), 117.36, 117.13, 115.76 (d, J = 158.1 Hz),

109.11, 108.56 (d, J = 23.2 Hz), 102.53, 99.53, 67.06, 56.28, 54.85, 53.47, 34.23, 28.46, 27.14, 26.11,

22.08; Anal. calcd. For C₃₅H₃₅F₂N₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N, 10.88; O, 12.43. Found (%): C,

65.25; H, 5.45; N, 10.83.

4.7.2 N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)

-1,3,4-oxadiazol-2-yl)acetamide (43b)

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Yield: 62%; m.p. 181- 183 C; MS (ESI) m/z (%): 632.8 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.86 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.11 - 8.05 (m, J = 8.8, 5.4 Hz, 1H), 7.85 (d, J = 13.7 Hz, 1H), 7.55

- 7.38 (m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.28 (s, 2H), 4.21 (t, J = 6.1 Hz, 2H), 3.95 (s, 3H), 3.61 (s, 4H),

2.42 (m, 6H), 2.00 (m, 2H); ¹³C NMR (101 MHz, DMSO) δ 165.03, 164.58 (d, J = 248.9 Hz), 164.28,

162.34, 159.69, 153.89 (d, J = 246.2 Hz), 152.32, 150.03, 149.32, 146.78, 137.98 (d, J = 9.9 Hz), 136.40 (d,

J = 11.8 Hz), 129.67 (d, J = 9.4 Hz), 124.70, 120.42 (d, J = 2.9 Hz), 117.35, 117.13, 115.76 (d, J = 160.1

Hz), 109.05, 108.59 (d, J = 23.6 Hz), 102.53, 99.52, 67.04, 66.15, 56.27, 55.11, 53.46, 34.21, 25.64; Anal.

calcd. For C₃₃H₃₁F₂N₅O₆(%): C, 62.75; H, 4.95; F, 6.02; N, 11.09; O, 15.20. Found (%): C, 62.73; H, 4.97;

N, 11.05.

4.7.3 N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-

fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (43c)

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Yield: 53%; m.p. 122 - 124 C; MS (ESI) m/z (%): 645.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.85 (s, 1H), 8.47 (d, J = 5.0 Hz, 1H), 8.08 (m, 2H), 7.92 - 7.77 (m, 1H), 7.56 - 7.34 (m, 6H), 6.45 (d, J =

5.1 Hz, 1H), 4.28 (s, 2H), 4.19 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 3.38 (dd, J = 14.0, 7.0 Hz, 3H), 2.40 (m, 8

H), 2.20 (s, 3H), 2.02 - 1.92 (m, 2H); ¹³C NMR (101 MHz, DMSO) δ 165.03, 164.58 (d, J = 250.3 Hz),

164.28, 162.34, 159.69, 154.00 (d, J = 224.8 Hz), 152.40, 150.04, 149.31, 146.80, 137.83 (d, J = 16.3 Hz),

136.41 (d, J = 12.3 Hz), 129.67 (d, J = 9.1 Hz), 124.71, 120.15 (d, J = 50.2 Hz), 117.36, 117.13, 115.74 (d,

J = 163.6 Hz), 112.16, 108.97, 99.98, 99.50, 67.15, 56.27, 54.70, 54.60, 52.51, 34.21, 26.34, 18.99; Anal.

calcd. For C₃₄H₃₄F₂N₆O₅(%): C, 63.35; H, 5.32; F, 5.89; N, 13.04; O, 12.41. Found (%): C, 63.33; H, 5.30;

N, 13.02.

4.7.4 N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-

fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (43d)

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Yield: 51%; m.p. 131 - 133 C; MS (ESI) m/z (%): 616.7 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.86 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.08 (dd, J = 8.7, 5.4 Hz, 2H), 7.86 (d, J = 13.7 Hz, 1H), 7.56 - 7.36

(m, 6H), 6.45 (d, J = 4.9 Hz, 1H), 4.28 (s, 2H), 4.22 (t, J = 6.2 Hz, 2H), 3.95 (s, 3H), 2.71 (d, J = 33.6 Hz,

6H), 2.08 - 1.98 (m, 2H), 1.76 (s, 4H); ¹³C NMR (101 MHz, DMSO) δ 165.05, 164.59 (d, J = 250.6 Hz),

164.28, 162.35, 159.72, 152.39 (d, J = 58.4 Hz), 149.95, 149.40, 146.74, 138.37 (d, J = 62.7 Hz), 136.74 (d,

J = 59.2 Hz), 129.67 (d, J = 8.6 Hz), 124.72, 120.43, 117.36, 117.14, 115.84 (d, J = 150.5 Hz), 108.95 (d, J

= 48.2 Hz), 102.46, 99.57, 66.59, 56.33, 53.93, 52.29, 34.21, 26.51, 23.24; Anal. calcd. for C₃₃H₃₁F₂N₅O₅

(%): C, 64.38; H, 5.08; F, 6.17; N, 11.38; O, 12.99. Found (%): C, 64.35; H, 5.04; N, 11.35.

4.7.5 N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-

fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (43e)

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Yield: 57%; m.p. 177 - 179 C; MS (ESI) m/z (%): 630.3 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.87 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.08 (dd, J = 8.5, 5.4 Hz, 2H), 7.86 (d, J = 13.9 Hz, 1H), 7.56 - 7.36

(m, 6H), 6.45 (d, J = 5.2 Hz, 1H), 4.29 (s, 2H), 4.21 (t, J = 6.2 Hz, 3H), 3.95 (s, 2H), 2.80 - 2.53 (m, 6H),

2.04 (s, 2H), 1.58 (s, 4H), 1.43 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.44 (d, J = 80.2 Hz), 164.28,

163.34, 162.35, 159.70, 153.66 (d, J = 291.7 Hz), 149.99, 149.36, 146.76, 138.00 (d, J = 7.4 Hz), 137.46 (d,

J = 11.1 Hz), 129.67 (d, J = 9.2 Hz), 124.72, 120.43, 117.36, 117.13, 115.78 (d, J = 154.6 Hz), 109.13,

108.59 (d, J = 23.1 Hz), 102.56, 99.55, 66.95, 56.29, 54.95, 53.77, 34.22, 25.37, 24.71, 20.11 . Anal. calcd.

For C₃₄H₃₃F₂N₅O₅(%): C, 64.86; H, 5.28; F, 6.03; N, 11.12; O, 12.70. Found (%): C, 64.83; H, 5.25; N,

11.14.

4.7.6 2-(5-(4-bromophenyl)-1,3,4-oxadiazol-2-yl)-N-(4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)

propoxy)quinolin-4-yl)oxy)phenyl)acetamide (43f)

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Yield: 65%; m.p. 131 - 132 C; MS (ESI) m/z (%): 705.7 [M + H]⁺; H NMR (600 MHz, DMSO) δ

11.09 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.88 (dd, J = 12.9, 2.0 Hz, 1H), 7.70 (d, J

= 8.6 Hz, 2H), 7.56 (s, 1H), 7.52 - 7.43 (m, 3H), 4.32 (s, 2H), 4.27 (t, J = 5.9 Hz, 2H), 3.96 (s, 3H), 3.51 (d,

J = 11.1 Hz, 2H), 3.22 (s, 2H), 2.92 (dd, J = 36.2, 14.3 Hz, 2H), 2.30 (s, 2H), 1.80 (d, J = 13.5 Hz, 2H),

1.62 (s, 1H), 1.48 (dd, J = 24.4, 11.7 Hz, 2H), 0.93 (d, J = 6.2 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ

165.03, 162.56, 162.00 (d, J = 452.5 Hz), 153.87 (d, J = 245.9 Hz), 151.98, 149.91, 149.37, 146.66, 138.12

(d, J = 10.0 Hz), 137.23, 136.30 (d, J = 12.2 Hz), 130.17, 128.76, 124.69, 122.61, 116.56, 115.14, 109.30,

108.54 (d, J = 23.1 Hz), 102.61, 99.61, 66.52, 56.32, 54.09, 52.46, 34.21, 31.33, 28.62, 23.78, 21.58; Anal.

calcd. For C₃₅H₃₅BrFN₅O₅(%): C, 59.66; H, 5.01; Br, 11.34; F, 2.70; N, 9.94; O, 11.35. Found (%): C,

59.63; H, 5.02; N, 9.95.

4.7.7 2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-

1-yl)propoxy)quinolin-4-yl)oxy)phenyl)acetamide (43g)

Yield: 64%; m.p. 96 - 98 ^oC; MS (ESI) m/z (%): 661.0 [M + H]⁺; ¹H NMR (600 MHz, DMSO) δ 10.93

(s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.86 (dd, J = 3.0 Hz, 1H), 7.70 (d, J = 8.6 Hz,

2H), 7.54 (s, 1H), 7.49 - 7.40 (m, 3H), 6.46 (d, J = 5.1 Hz, 1H), 4.30 (s, 2H), 4.23 (t, J = 6.0 Hz, 2H), 3.96

(s, 3H), 3.21 (s, 2H), 3.11 - 2.98 (m, 2H), 2.97 - 2.76 (m, 2H), 2.14 (s, 2H), 1.71 (d, J = 11.0 Hz, 2H), 1.50

(s, 1H), 1.33 (d, J = 31.8 Hz, 2H), 0.92 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 165.00, 162.54,

161.97 (d, J = 458.3 Hz), 153.89 (d, J = 246.5 Hz), 152.15, 149.97, 149.35, 146.78, 138.05 (d, J = 9.8 Hz),

137.23, 136.36 (d, J = 12.2 Hz), 130.15, 128.74, 124.68, 122.61, 116.56, 115.04, 109.20, 108.56 (d, J =

22.7 Hz), 102.55, 99.56, 66.83, 56.28, 54.39, 52.90, 46.04, 34.23, 32.49, 29.41, 25.10, 21.69, 9.21; Anal.

calcd. For C₃₅H₃₅ClFN₅O₅(%):C, 63.68; H, 5.34; Cl, 5.37; F, 2.88; N, 10.61; O, 12.12. Found (%): C, 63.65;

H, 5.36; N, 10.56.

4.7.8 N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5

-phenyl-1,3,4-oxadiazol-2-yl)acetamide (43h)

o

1

Yield: 54%; m.p. 210 - 212 C; MS (ESI) m/z (%): 626.9 [M + H]⁺; H NMR (600 MHz, DMSO) δ

11.33 (s, 1H), 8.63 (s, 1H), 8.02 (d, J = 6.9 Hz, 2H), 7.94 (dd, J = 12.9, 1.7 Hz, 1H), 7.68 - 7.57 (m, 6H),

6.68 (s, 1H), 4.34 (s, 2H), 4.30 (t, J = 5.9 Hz, 2H), 4.00 (s, 3H), 3.48 (d, J = 11.4 Hz, 2H), 3.25 - 3.19 (m,

2H), 2.96 - 2.87 (m, 2H), 2.38 - 2.31 (m, 2H), 1.87 - 1.77 (m, 4H), 1.75 - 1.68 (m, 1H), 0.92 (d, J = 6.4 Hz,

3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.20, 164.99, 162.37, 154.15 (d, J = 146.5 Hz), 152.43, 150.68,

146.91, 138.69 (d, J = 9.1 Hz), 135.74 (d, J = 13.1 Hz), 132.50, 129.97, 126.94, 124.59, 123.76, 116.66,

115.33, 108.67 (d, J = 1.0 Hz), 108.44, 102.96, 100.09, 66.95, 56.63, 53.86, 52.52, 34.19, 23.52, 22.83,

21.85; Anal. calcd. For C₃₅H₃₆FN₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N, 10.88; O, 12.43. Found (%): C,

65.32; H, 5.45; N, 10.85.

4.7.9 N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5

-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide (43i)

o

1

Yield: 65%; m.p. 119 - 121 C; MS (ESI) m/z (%): 656.8 [M + H]⁺; H NMR (600 MHz, DMSO) δ

10.35 (s, 1H), 8.48 (d, J = 5.1 Hz, 1H), 7.87 (d, J = 13.9 Hz, 2H), 7.94 (dd, J = 12.9, 1.7 Hz, 1H), 7.55-7.48

(m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.32 (s, 2H), 4.25 (t, J = 5.9 Hz, 2H), 3.95 (s, 3H), 3.45 (d, J = 11.1 Hz,

2H), 3.16 (s, 2H), 2.88 (m, 2H), 2.23 (s, 2H), 1.78 (d, J = 11.5 Hz, 2H), 1.58 (s, 1H), 1.39 (dd, J = 21.4,

14.7 Hz, 2H), 0.93 (d, J = 5.0 Hz, 3H); Anal. calcd. For C₃₆H₃₈FN₅O₆(%): C, 67.19; H, 5.80; F, 3.04; N,

11.19; O, 12.78. Found (%): C, 67.14; H, 5.81; N, 11.15.

4.7.10

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluoro

phenyl)-1,3,4-oxadiazol-2-yl)propanamide (44a)

Yield: 64%; m.p. 123 - 125 ^oC; MS (ESI) m/z (%): 658.2 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ 10.86 (s,

1H), 8.47 (d, J = 5.1 Hz, 1H), 8.08 (dd, J = 8.6, 5.4 Hz, 2H), 7.88 (d, J = 11.9 Hz, 1H), 7.56 - 7.36 (m, 6H),

6.45 (d, J = 5.1 Hz, 1H), 4.38 (q, J = 7.1 Hz, 1H), 4.19 (t, J = 6.1 Hz, 2H), 3.95 (s, 3H), 2.95 (d, J = 9.5 Hz,

2H), 2.57 (s, 2H), 2.16 - 1.94 (m, 4H), 1.71 (d, J = 7.1 Hz, 3H), 1.61 (d, J = 12.1 Hz, 2H), 1.36 (s, 1H),

1.21 - 1.09 (m, 2H), 0.89 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 168.64, 165.87, 163.35,

161.53 (d, J = 365.8 Hz), 153.90 (d, J = 245.8 Hz), 152.36, 150.04, 149.30, 146.82, 138.05 (d, J = 10.0 Hz),

136.44 (d, J = 12.1 Hz),129.72 (d, J = 9.0 Hz), 124.70, 120.41, 117.23 (d, J = 22.5 Hz), 116.71, 114.94,

109.02, 108.84, 108.61, 102.50, 99.50, 67.17, 56.26, 54.96, 53.67, 33.91, 30.46, 26.23, 22.12, 15.36. Anal.

calcd. for C₃₆H₃₇F₂N₅O₅(%): C, 65.74; H, 5.67; F, 5.78; N, 10.65; O, 12.16. Found (%): C, 65.70; H, 5.63;

N, 10.63.

4.7.11

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-1,3,

4-oxadiazol-2-yl)propanamide (44b)

o

1

Yield: 63%; m.p. 122 - 124 C; MS (ESI) m/z (%): 646.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.91 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.08 (dd, J = 8.8, 5.3 Hz, 2H), 7.88 (dd, J = 12.9, 1.8 Hz, 1H), 7.57

- 7.35 (m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.40 (t, J = 7.0 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.64

- 3.59 (m, 3H), 2.55 (m, 2H), 2.46 (s, 4H), 2.07 - 1.94 (m, 2H), 1.71 (d, J = 7.1 Hz, 3H); ¹³C NMR (101

MHz, DMSO) δ 168.68, 165.87, 163.36, 161.96 (d, J = 445.4 Hz), 153.89 (d, J = 245.9 H) 152.38, 150.03,

149.31, 146.75, 138.02 (d, J = 10.1 Hz), 136.44 (d, J = 12.8 Hz), 129.72 (d, J = 9.0 Hz), 124.70, 120.40,

117.32, 117.21 (d, J = 22.5 Hz), 114.93, 108.93, 108.63, 102.50, 99.48, 67.11, 66.41, 56.26, 55.20, 53.64,

46.16, 25.87, 15.35, 9.06. Anal. calcd. for C₃₄H₃₃F₂N₅O₆(%): C, 63.25; H, 5.15; F, 5.88; N, 10.85; O, 14.87.

Found (%): C, 63.23; H, 5.14; N, 10.84.

4.7.12 N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-

(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)propanamide (44c)

o

1

Yield: 68%; m.p. 120 - 123 C; MS (ESI) m/z (%): 669.0 [M + H]⁺; H NMR (400 MHz, DMSO) δ

11.00 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.11 - 8.05 (m, 2H), 7.89 (dd, J = 12.9, 2.0 Hz, 1H), 7.55 - 7.38 (m,

6H), 6.45 (d, J = 5.1 Hz, 1H), 4.41 (q, J = 7.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 3.04 - 2.96 (m,

3H), 2.57 - 2.51 (m, 8H), 2.28 (s, 3H), 2.02 - 1.94 (m, 2H), 1.70 (d, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz,

DMSO) δ 168.67, 165.89, 163.36, 161.67 (d, J = 443.3 Hz), 153.89 (d, J = 245.9 H), 152.38, 150.04,

149.31, 146.83, 138.11 (d, J = 10.3 Hz), 136.40 (d, J = 12.3 Hz), 129.73 (d, J = 9.0 Hz), 124.70, 120.45,

117.23 (d, J = 22.5 Hz, 3H), 116.71, 114.92, 109.00, 108.82, 108.59, 102.49, 99.50, 67.10, 56.27, 54.52,

52.23, 46.00, 45.19, 26.31, 15.38, 9.26; Anal.calcd. for C₃₆H₃₇F₂N₅O₅(%): C, 63.82; H, 5.51; F, 5.77; N,

12.76; O, 12.14. Found (%): C, 63.84; H, 5.46; N, 12.75.

4.7.13

N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-

1,3,4-oxadiazol-2-yl)propanamide (44d)

o

1

Yield: 54%; m.p. 144 - 146 C; MS (ESI) m/z (%): 630.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.99 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.12 - 8.05 (m, 2H), 7.90 (dd, J = 13.0, 1.7 Hz, 1H), 7.58 - 7.39 (m,

6H), 6.46 (d, J = 5.1 Hz, 1H), 4.41 (q, J = 7.0 Hz, 1H), 4.24 (t, J = 6.1 Hz, 2H), 3.96 (s, 3H), 2.99 (d, J =

6.9 Hz, 7H), 2.22 - 2.05 (m, 2H), 1.85 (s, 4H), 1.70 (d, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ

168.68, 165.89, 163.35, 161.92 (d, J = 441.7 Hz),153.89 (d, J = 246.1 Hz), 152.15, 149.96, 149.36, 146.78,

138.13 (d, J = 9.5 Hz), 136.39 (d, J = 11.8 Hz), 129.72 (d, J = 8.8 Hz), 124.70, 120.45, 117.23 (d, J = 22.4

Hz), 116.67, 115.05, 109.16, 108.81, 108.58, 102.56, 99.54, 66.70, 56.31, 53.84, 52.26, 46.01, 26.82, 23.33,

15.39; Anal. calcd. for C₃₄H₃₃F₂N₅O₅(%): C, 65.06; H, 5.46; F, 3.03; N, 11.16; O, 15.29. Found (%): C,

65.02; H, 5.43; N, 11.12.

4.7.14

N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-

1,3,4-oxadiazol-2-yl)propanamide (44e)

o

Yield: 52%; m.p. 119 - 120 C; MS (ESI) m/z (%): 644.2 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.91 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.15 - 8.03 (m, 2H), 7.88 (dd, J = 12.7, 1.2 Hz, 1H), 7.57 - 7.32 (m,

6H), 6.45 (d, J = 5.1 Hz, 1H), 4.38 (q, J = 7.0 Hz, 1H), 4.20 (t, J = 6.2 Hz, 2H), 3.95 (s, 3H), 2.90 (dd, J =

16.4, 8.4 Hz, 2H), 2.59 (d, J = 4.1 Hz, 4H), 2.09 - 1.97 (m, 2H), 1.70 (d, J = 7.1 Hz, 3H), 1.61 - 1.51 (m,

4H), 1.42 (d, J = 3.8 Hz, 2H); ¹³C NMR (101 MHz, DMSO) δ 168.64, 165.88, 163.37, 161.93 (d, J = 444.1

Hz, 1H), 153.90 (d, J = 246.7 Hz, 1H), 152.32, 150.03, 149.33, 146.82, 138.07 (d, J = 9.6 Hz, 0H), 136.43

(d, J = 13.3 Hz, 0H), 129.73 (d, J = 9.0 Hz, 2H), 124.71, 120.42, 117.24 (d, J = 22.3 Hz, 2H), 116.72,

114.96, 109.08, 108.83, 108.61, 102.52, 99.52, 67.09, 56.28, 55.20, 54.11, 46.10, 25.85, 25.32, 23.84, 15.37.

Anal. calcd. for C₃₅H₃₅F₂N₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N, 10.88; O, 12.43. Found (%): C, 65.25; H,

5.44; N, 10.84.

4.7.15

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluoro

phenyl)-1,3,4-oxadiazol-2-yl)butanamide (45a)

Yield: 64%; m.p. 127 - 129^oC; MS (ESI) m/z (%): 672.4 [M + H]⁺; H NMR (600 MHz, DMSO) δ

1

10.90 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.08 (dd, J = 8.8, 5.3 Hz, 2H), 7.89 (dd, J = 12.9, 2.0 Hz, 1H), 7.54

(s, 1H), 7.52 - 7.44 (m, 4H), 7.42 (s, 1H), 6.47 (d, J = 5.1 Hz, 1H), 4.22 (t, J = 6.8 Hz, 3H), 3.96 (s, 3H),

3.39 (dd, J = 14.0, 7.0 Hz, 2H), 3.13 (s, 2H), 2.79 (s, 2H), 2.26 - 2.15 (m, 2H), 2.10 (s, 2H), 1.69 (d, J =

11.4 Hz, 2H), 1.47 (s, 1H), 1.25 (d, J = 13.8 Hz, 2H), 1.05 (t, J = 7.4 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H); ¹³

C

NMR (101 MHz, DMSO) δ 167.67, 165.03, 164.61 (d, J = 250.1 Hz), 164.14, 153.90 (d, J = 245.0 Hz),

152.19, 149.98, 149.36, 146.78, 137.95 (d, J = 9.1 Hz), 136.48 (d, J = 12.5 Hz), 129.74 (d, J = 9.2 Hz),

124.70, 120.43, 117.24 (d, J = 22.5 Hz); 6.75, 115.01, 109.16, 108.87, 108.65, 102.60, 99.54, 66.89, 56.29,

54.55, 53.11, 46.41, 32.89, 29.61, 25.25, 23.47, 21.80, 12.10. Anal. calcd. for C₃₇H₃₉F₂N₅O₅(%): C, 66.16;

H, 5.85; F, 5.66; N, 10.43; O, 11.91. Found (%): C, 66.12; H, 5.83; N, 10.42.

4.7.16

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-1,3,

4-oxadiazol-2-yl)butanamide (45b)

Yield: 53%; m.p. 128 - 129^oC; MS (ESI) m/z (%): 660.2 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.88 (s, 1H), 8.46 (d, J = 5.0 Hz, 1H), 8.07 (m, 2H), 7.89 (d, J = 13.7 Hz, 1H), 7.57 - 7.40 (m, 6H), 6.46 (d,

J = 5.0 Hz, 1H), 4.21 (m, 4H), 3.95 (s, 3H), 3.64 (s, 4H), 3.09 (m, 2H), 2.19 (m, 3H), 2.03 (s, 2H), 1.04 (t, J

= 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 167.65, 165.02, 164.61 (d, J = 249.9 Hz), 164.14, 153.91 (d,

J = 245.5 Hz), 152.33, 150.03, 149.32, 146.82, 137.93 (d, J = 10.2 Hz), 136.51 (d, J = 12.1 Hz), 129.74 (d,

J = 9.2 Hz), 124.70, 120.41, 117.24 (d, J = 22.3 Hz), 116.74, 114.96, 109.06, 108.89, 108.66, 102.57, 99.50,

67.04, 56.28, 55.13, 53.52, 46.42, 46.15, 23.47, 12.10, 9.08; Anal. calcd. For C₃₅H₃₅F₂N₅O₆(%): C, 63.72;

H, 5.35; F, 5.76; N, 10.62; O, 14.55. Found (%): C, 63.73; H, 5.33; N, 10.61.

4.7.17

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluoro

phenyl)-1,3,4-oxadiazol-2-yl)butanamide (45c)

Yield: 54%; m.p.154 - 155^oC; MS (ESI) m/z (%): 673.7 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.94 (s, 1H), 8.45 (t, J = 4.9 Hz, 1H), 8.07 (dd, J = 8.8, 5.3 Hz, 2H), 7.89 (dd, J = 12.9, 1.7 Hz, 1H), 7.54 -

7.43 (m, 1H), 7.39 (s, 1H), 6.45 (d, J = 5.2 Hz, 1H), 4.21 - 4.15 (m, J = 10.2, 6.2 Hz, 3H), 3.95 (s, 3H), 2.51

- 2.31 (m, 10H), 2.28 - 2.13 (m, 7H), 1.04 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 167.67,

165.03, 164.60 (d, J = 249.6 Hz), 164.13, 153.90 (d, J = 245.7 Hz), 152.40, 150.05, 149.31, 146.84, 137.95

(d, J = 10.1 Hz), 136.50 (d, J = 12.2 Hz), 129.74 (d, J = 9.3 Hz), 124.70, 120.44, 117.24 (d, J = 22.6 Hz);

116.73, 114.92, 109.00, 108.88, 108.65, 102.01, 99.50, 67.15, 56.27, 54.81, 54.65, 52.64, 46.40, 46.08,

45.62, 26.40, 23.48, 12.09; Anal. calcd. For C₃₆H₃₈F₂N₆O₅(%): C, 64.27; H, 5.69; F, 5.65; N, 12.49; O,

11.89. Found (%): C, 64.23; H, 5.70; N, 12.45.

4.7.18

N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-

1,3,4-oxadiazol-2-yl)butanamide (45d)

Yield: 55%; m.p. 133 - 134^oC; MS (ESI) m/z (%): 643.7 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.91 (s, 1H), 8.46 (t, J = 4.8 Hz, 1H), 8.07 (dd, J = 8.8, 5.4 Hz, 2H), 7.88 (dd, J = 14.2, 12.2 Hz, 1H), 7.56

- 7.36 (m, 6H), 6.46 (d, J = 5.1 Hz, 1H), 4.27 - 4.19 (m, 3H), 3.96 (s, 3H), 2.89 (d, J = 23.0 Hz, 6H), 2.25 -

2.14 (m, 2H), 2.14 - 2.04 (m, 2H), 1.82 (s, 4H), 1.04 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ

167.67, 165.03, 164.61 (d, J = 251.0 Hz), 164.14, 153.90 (d, J = 247.6 Hz), 152.21, 150.00, 149.37, 146.81,

137.95 (d, J = 8.9 Hz), 136.50 (d, J = 11.6 Hz), 129.74 (d, J = 9.7 Hz), 124.71, 120.45, 117.24 (d, J = 22.3

Hz, 1H), 116.77, 115.02, 109.14, 108.90, 108.69, 102.07, 99.60 (d, J = 12.5 Hz, 1H), 66.80, 56.31, 53.96,

52.41, 46.42, 27.17, 23.47, 23.37, 12.10; Anal. calcd. For C₃₅H₃₅F₂N₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N,

10.88; O, 12.43. Found (%): C, 65.30; H, 5.45; N, 10.86.

4.7.19

N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-

1,3,4-oxadiazol-2-yl) butanamide (45e)

Yield: 53%; m.p. 132 - 134^oC; MS (ESI) m/z (%): 658.9 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.89 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 8.07 (dd, J = 8.4, 5.4 Hz, 2H), 7.89 (d, J = 12.3 Hz, 1H), 7.55 (s,

1H), 7.51 - 7.40 (m, 5H), 6.47 (d, J = 5.1 Hz, 1H), 4.27 - 4.18 (m, 3H), 3.96 (s, 3H), 3.14 - 2.77 (m, 6H),

2.26 - 2.09 (m, 4H), 1.68 (s, 4H), 1.51 (s, 2H), 1.04 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ

167.65, 165.02, 164.61 (d, J = 250.4 Hz), 164.14, 153.16 (d, J = 247.6 Hz), 152.10, 149.96, 149.39, 146.77,

137.93 (d, J = 8.9 Hz), 136.51 (d, J = 11.6 Hz), 129.74 (d, J = 9.7 Hz), 124.71, 120.44, 117.25 (d, J = 22.3

Hz, 1H), 116.76, 115.07, 109.26, 108.89, 108.67, 102.59, 99.56, 66.73, 56.31, 53.33, 46.42, 46.16, 24.17,

23.46, 12.10, 9.41, 9.21; Anal. calcd. For C₃₆H₃₇F₂N₅O₅(%): C, 65.74; H, 5.67; F, 5.78; N, 10.65; O, 12.16.

Found (%): C, 65.72; H, 5.65; N, 10.60.

4.7.20N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-

(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-2-methylpropanamide (46a)

Yield: 70%; m.p. 126 - 128^oC; MS (ESI) m/z (%): 672.2 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.02 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.10 (dd, J = 8.8, 5.4 Hz, 2H), 7.88 (dd, J = 13.1, 2.2 Hz, 1H), 7.62

- 7.34 (m,6H), 6.44 (d, J = 5.1 Hz, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.95 (s, 4H), 3.38 (dd, J = 16.5, 9.5 Hz,

2H), 2.89 (d, J = 11.0 Hz, 2H), 2.47 (s, 2H), 2.05 - 1.89 (m, 4H), 1.92(s, 6H), 1.60 (t, J = 12.5 Hz, 2H), 1.41

- 1.27 (m, 1H), 0.89 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 171.19, 169.01, 164.60 (d, J =

249.8 Hz), 164.08, 159.71, 153.73 (d, J = 245.3 Hz), 152.36, 150.04, 149.31, 146.83, 138.06 (d, J = 9.4 Hz),

136.54 (d, J = 13.2 Hz), 129.76 (d, J = 9.2 Hz), 124.37, 120.54, 117.63, 117.21 (d, J = 22.7 Hz), 114.95,

109.63 (d, J = 23.1 Hz), 109.02, 102.54, 99.50, 67.17, 56.27, 54.97, 53.70, 44.97, 33.97, 30.46, 26.25,

24.25, 22.14; Anal. calcd. For C₃₇H₃₉F₂N₅O₅(%): C, 66.16; H, 5.85; F, 5.66; N, 10.43; O, 11.91. Found (%):

C, 66.12; H, 5.84; N, 10.41.

4.7.21

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-1,3,

4-oxadiazol-2-yl)-2-methylpropanamide (46b)

Yield: 64%; m.p. 134 - 135^oC; MS (ESI) m/z (%): 660.2 [M + H]⁺;¹H NMR (400 MHz, DMSO) δ

10.11 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.10 (dd, J = 8.5, 5.4 Hz, 2H), 7.90 (dd, J = 13.1, 1.8 Hz, 1H), 7.64

- 7.36 (m, 6H), 6.43 (t, J = 9.7 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 3.64 - 3.53 (m, 4H), 2.95 (q,

J = 7.1 Hz, 2H), 2.47 (t, J = 7.0 Hz, 2H), 2.39 (s, 4H), 2.03 - 1.92 (m, 2H), 1.81 (s, 6H); Anal. calcd. For

C₃₅H₃₅F₂N₅O₆(%): C, 63.72; H, 5.35; F, 5.76; N, 10.62; O, 14.55. Found (%): C, 63.70; H, 5.34; N, 10.63.

4.7.22

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluoro

phenyl)-1,3,4-oxadiazol-2-yl)-2-methylpropanamide (46c)

Yield: 55%; m.p. 134 - 135^oC; MS (ESI) m/z (%): 673.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

1

10.07 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 8.10 (dd, J = 8.4, 5.4 Hz, 2H), 7.89 (d, J = 11.5 Hz, 1H), 7.63 - 7.33

(m, 6H), 6.44 (d, J = 5.0 Hz, 1H), 4.18 (t, J = 5.9 Hz, 2H), 3.95 (s, 3H), 3.62 (s, 4H), 2.49 (d, J = 7.6 Hz,

6H), 2.24 (s, 3H), 2.04 - 1.92 (m, 2H), 1.81 (s, 6H). Anal. calcd. for C₃₆H₃₈F₂N₆O₅(%): C, 64.27; H, 5.69; F,

5.65; N, 12.49; O, 11.89. Found (%): C, 64.23; H, 5.64; N, 12.44.

4.7.23

N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-

1,3,4-oxadiazol-2-yl)-2-methylpropanamide (46d)

Yield: 65%; m.p. 145 - 146^oC; MS (ESI) m/z (%): 644.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

1

10.05 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.10 (dd, J = 8.6, 5.4 Hz, 2H), 7.89 (dd, J = 13.1, 2.0 Hz, 1H), 7.61

- 7.35 (m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.21 (t, J = 6.2 Hz, 2H), 3.95 (s, 3H), 2.69 (t, J = 6.9 Hz, 2H), 2.59

(s, 4H), 2.09 - 1.96 (m, 2H), 1.81 (s, 6H), 1.73 (s, 4H); ¹³C NMR (101 MHz, DMSO) δ 171.23, 169.02,

164.59 (d, J = 250.5 Hz), 164.08, 159.73, 153.73 (d, J = 245.6 Hz), 152.33, 150.00, 149.30, 146.78, 138.05

(d, J = 10.0 Hz), 136.52 (d, J = 12.3 Hz), 129.75 (d, J = 9.1 Hz), 124.36, 120.53 (d, J = 3.0 Hz), 117.63,

117.19 (d, J = 22.4 Hz),114.94, 109.64 (d, J = 23.1 Hz), 108.95, 102.52, 99.47, 67.03, 56.26, 54.01, 52.56,

44.99, 27.91, 24.22, 23.47; Anal. calcd. For C₃₅H₃₅F₂N₅O₅(%): C, 65.31; H, 5.48; F, 5.90; N, 10.88; O,

12.43. Found (%): C, 65.33; H, 5.44; N, 10.83.

4.7.24

N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-2-(5-(4-fluorophenyl)-

1,3,4-oxadiazol-2-yl)-2-methylpropanamide (46e)

Yield: 53%; m.p. 126 - 127^oC; MS (ESI) m/z (%): 658.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

1

10.02 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.10 (dd, J = 8.8, 5.4 Hz, 2H), 7.88 (dd, J = 13.1, 2.2 Hz, 1H), 7.62

- 7.34 (m, 6H), 6.44 (d, J = 5.1 Hz, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.95 (s, 2H), 3.38 (dd, J = 16.5, 9.5 Hz,

2H), 2.89 (d, J = 11.0 Hz, 2H), 2.47 (s, 2H), 2.05 - 1.89 (m, 4H), 1.60 (t, J = 12.5 Hz, 2H), 1.41 - 1.27 (m,

1H), 0.89 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 171.22, 169.03, 164.59 (d, J = 250.3 Hz),

164.07, 159.71, 153.73 (d, J = 245.6 Hz), 152.37, 150.03, 149.27, 146.84, 138.09 (d, J = 9.8 Hz, 2H),

136.51 (d, J = 12.3 Hz), 129.74 (d, J = 9.1 Hz), 124.34, 120.57, 117.61, 117.18 (d, J = 22.4 Hz), 114.94,

109.62 (d, J = 23.0 Hz); 109.01, 102.51, 99.48, 67.19, 56.24, 55.38, 54.34, 46.04, 45.01, 26.22, 25.69,

24.23; Anal. calcd. For C₃₆H₃₇F₂N₅O₅(%): C, 65.74; H, 5.67; F, 5.78; N, 10.65; O, 12.16. Found (%): C,

65.73; H, 5.65; N, 10.63.

4.7.25 N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-1-

(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)cyclopropane-1-carboxamide (47a)

1

Yield: 64%; m.p. 146 - 147^oC; MS (ESI) m/z (%): 670.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.38 (s, 1H), 8.48 (s, 1H), 8.10 (dd, J = 8.7, 5.4 Hz, 2H), 7.86 (dd, J = 13.0, 1.9 Hz, 1H), 7.58 - 7.34 (m,

6H), 6.46 (d, J = 4.9 Hz, 1H), 4.19 (t, J = 6.2 Hz, 2H), 3.95 (s, 3H), 2.93 (d, J = 10.8 Hz, 2H), 2.53 (d, J =

10.1 Hz, 2H), 2.10 - 1.92 (m, 4H), 1.85 - 1.67 (m, 4H), 1.60 (d, J = 11.7 Hz, 2H), 1.35 (s, 1H), 1.29 - 1.11

(m, J = 24.2, 12.1 Hz, 4H), 0.88 (d, J = 6.4 Hz, 3H); ¹³C NMR (101 MHz, DMSO) δ 166.89, 165.69,

164.54 (d, J = 250.4 Hz), 164.00, 159.67, 153.78 (d, J = 245.2 Hz), 152.37, 150.03, 149.28, 146.88,

δ137.93 (d, J = 9.9 Hz), 136.57 (d, J = 12.2 Hz), 129.76 (d, J = 9.0 Hz), 124.42, 120.69, 117.51, 117.14 (d,

J = 22.5 Hz), 114.96, 109.59 (d, J = 22.7 Hz), 109.00, 102.53, 99.48, 67.19, 56.24, 55.02, 53.74, 34.07,

30.58, 26.36, 23.15, 22.17, 17.32; Anal. calcd. for C₃₇H₃₇F₂N₅O₅(%): C, 65.94; H, 5.38; F, 5.79; N, 10.68;

O, 12.20. Found (%): C, 65.93; H, 5.34; N, 10.64.

4.7.26

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-1-(5-(4-fluorophenyl)-1,3,

4-oxadiazol-2-yl)cyclopropane-1-carboxamide (47b)

Yield: 64%; m.p. 194 - 196^oC; MS (ESI) m/z (%): 658.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

1

10.36 (s, 1H), 8.47 (d, J = 5.1 Hz, 1H), 8.10 (dd, J = 8.6, 5.4 Hz, 2H), 7.86 (d, J = 11.0 Hz, 1H), 7.58 - 7.36

(m, 6H), 6.45 (d, J = 5.1 Hz, 1H), 4.20 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 3.59 (t, J = 4.4 Hz, 4H), 2.51 - 2.44

(m, 2H), 2.40 (s, 4H), 2.04 - 1.94 (m, 2H), 1.81 - 1.67 (m, 4H); ¹³C NMR (101 MHz, DMSO) δ 166.89,

165.69, 164.67 (d, J = 226.9 Hz), 164.01, 159.67, 153.78 (d, J = 245.7 Hz), 152.42, 150.06, 149.31, 146.86,

137.93 (d, J = 9.2 Hz), 136.60 (d, J = 11.2 Hz), 129.78 (d, J = 9.2 Hz), 124.44, 120.73 (d, J = 2.9 Hz),

117.56, 117.16 (d, J = 22.4 Hz), 114.94, 109.60 (d, J = 23.3 Hz), 109.03, 102.54, 99.49, 66.68, 56.26, 55.28,

53.84, 46.09, 26.15, 23.16, 17.28; Anal. calcd. for C₃₅H₃₃F₂N₅O₆(%): C, 63.92; H, 5.06; F, 5.78; N, 10.65;

O, 14.60. Found (%): C, 63.91; H, 5.04; N, 10.64.

4.7.27

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-1-(5-(4-fluoro

phenyl)-1,3,4-oxadiazol-2-yl)cyclopropane-1-carboxamide (47c)

1

Yield: 74%; m.p. 130 - 131^oC; MS (ESI) m/z (%): 671.2 [M + H]⁺; H NMR (400 MHz, DMSO) δ

10.39 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.10 (dd, J = 8.8, 5.3 Hz, 2H), 7.87 (dd, J = 13.0, 2.2 Hz, 1H), 7.55

- 7.38 (m, 6H), 6.46 (d, J = 5.1 Hz, 1H), 4.21 (t, J = 6.2 Hz, 2H), 3.95 (s, 3H), 3.37 (dd, J = 12.9, 5.6 Hz,

Article Doi

Design, synthesis and biological evaluation of new Axl kinase inhibitors containing 1,3,4-oxadiazole acetamide moiety as novel linker

DOI: 10.1016/j.ejmech.2019.111867

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Article abstract of DOI:10.1016/j.ejmech.2019.111867

Full text of DOI:10.1016/j.ejmech.2019.111867

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