Discovery of novel dual c-Met/HDAC inhibitors as a promising strategy for cancer therapy

Article abstract of DOI:10.1016/j.bioorg.2020.103970

Owing to the low efficacy and acquired resistance in clinical trials of c-Met inhibitors, based on the synergistic effects between c-Met and HDAC, novel c-Met and HDAC dual inhibitors were designed and synthesized. We introduced 2-pyrrolidinone to form the 5-atoms linker for c-Met inhibitor and hydroxamic acid as a zinc binding motif for HDAC inhibitor. The highly active dual inhibitor 15f showed excellent and balanced activity against both c-Met (IC₅₀ = 12.50 nM) and HDAC1 (IC₅₀ = 26.97 nM). In those tested tumor cell lines, 15f exhibits efficient antiproliferative activity with greater potency than Vorinostat (SAHA) and Cabozantinib (XL184). However, by comparing with an equimolar mixture of SAHA and Foretinib, we did not observe the compounds showed clearly synergistic antiproliferative effect. Nevertheless, compound 15f was found to induce apoptosis and cause cell cycle arrest in G2/M phase. This proof-of-concept study provides an efficient strategy for discovery of multitarget antitumor drugs.

Full text of DOI:10.1016/j.bioorg.2020.103970

Journal Pre-proofs
Discovery of Novel Dual c-Met/HDAC Inhibitors as a Promising Strategy for
Cancer Therapy
Hao Hu, Fei Chen, Yuhong Dong, Yajing Liu, Ping Gong
PII:
S0045-2068(20)30120-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.103970
YBIOO 103970
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 January 2020
21 May 2020
22 May 2020
Please cite this article as: H. Hu, F. Chen, Y. Dong, Y. Liu, P. Gong, Discovery of Novel Dual c-Met/HDAC
Inhibitors as a Promising Strategy for Cancer Therapy, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.103970
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Discovery of Novel Dual c-Met/HDAC
Inhibitors as a Promising Strategy for Cancer Therapy
Hao Hu, Fei Chen, Yuhong Dong, Yajing Liu *, Ping Gong*1
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103
Wenhua Road, Shenhe District, Shenyang 110016, PR China
Abstract
Owing to the low efficacy and acquired resistance in clinical trials of c-Met inhibitors, based on the synergistic
effects between c-Met and HDAC, novel c-Met and HDAC dual inhibitors were designed and synthesized. We
introduced 2-pyrrolidinone to form the 5-atoms linker for c-Met inhibitor and hydroxamic acid as a zinc binding
motif for HDAC inhibitor. The highly active dual inhibitor 15f showed excellent and balanced activity against both
c-Met (IC50 = 12.50 nM) and HDAC1 (IC50 = 26.97 nM). In those tested tumor cell lines, 15f exhibits efficient
antiproliferative activity with greater potency than Vorinostat (SAHA) and Cabozantinib (XL184). However, by
comparing with an equimolar mixture of SAHA and Foretinib, we did not observe the compounds showed clearly
synergistic antiproliferative effect. Nevertheless, compound 15f was found to induce apoptosis and cause cell cycle
arrest in G2/M phase. This proof-of-concept study provides an efficient strategy for discovery of multitarget
antitumor drugs.
Keywords: c-Met; HDAC; 2-pyrrolidinone; antitumor drug
1. Introduction
Kinases are responsible for transferring phosphate groups from ATP to specific target molecules and have been
the most intensively pursued classes of drug targets, especially in the treatment of cancers [1]. Since the discovery
of protein kinase activity in 1954, the design and synthesis of different kinds of targeted cancer therapy drugs have
advanced dramatically [2]. However, due to the multifaceted and dynamic nature of tumors, the foremost problem
is that the effectiveness of kinase inhibitors is often impaired by acquired drug resistance [3-4]. It is reported that
during the last three decades, the overall survival rates for cancer patients have not significantly improved owing to
the emerging cancer resistance [5]. Cocktail therapy is one of the solutions to this problem, which was employed
by clinicians to treat unresponsive patients [6]. Unfortunately, the approach of drug cocktails also resulted in
1Correspondence: Prof. Ping Gong and Yajing Liu, Key Laboratory of Structure-based Drug Design and Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China.
E-mail: gongpinggp@126.com; lyjpharm@126.com.

complicated doses/schedule, unpredictable pharmacokinetic profile, drug-drug interactions, and reduced patient
compliance [7-8]. From this point, a multitarget drug (single molecule) is an alternative approach to achieve
multitarget therapy as compared to Cocktail therapy. Therefore, molecular hybridization paradigm is prevailing at
present and becoming an interesting and smart way to defeat the multifaceted cancer disease.
c-Met [the receptor for hepatocyte growth factor/scatter factor, (HGF/SF)][9], which is composed of an
extracellular α chain connected with a membrane-spanning β chain through a disulfide bond [10] is amplify or
overexpress in various cancers that caused poor clinical outcomes[11-14] . In our previous study, based on a survey
of launched and clinical c-Met inhibitors, we had designed series of quinoline derivatives and concluded the ‘‘5
atoms regulation’’ between moiety A and B (Fig. 2) [15-16]. However, similar to other RTK inhibitors, new
research indicated that c-Met inhibition alone is usually not sufficient to block tumor progression due to complex
factors, such as network robustness, bypass crosstalk, compensatory activities and acquired resistance in clinical
trials [17].
linker
5 atoms linker
atoms
5
N
H
H
N
N
A moiety
F
N
F
A
F
O
O
moiety
O
F
O
B moiety
O
B moiety
O
O
O
O
N
N
N
N
O
O
AM7
Foretinib
HO
5 atoms linker
5 atoms linker
N
H
H
H
H
H
N
N
F
N
N
N
A moiety
N
A moiety
A moiety
S
O
O
O
O
O
N
O
O
N
F
O
B moiety
O
O
linker
B moiety
B moiety
O
O
atoms
5
N
O
N
Kirin
Carbozatinib
AMG458
Fig. 2. The representative c-Met kinase inhibitors and the corresponding general structure.
It is easy to get discouraged when things are going bad. But we should not lose heart, because god is at work
in our lives, even in the midst of pain and suffering. Fortunately, researchers have found the will of god which
identified the histone deacetylases (HDACs) and told us the connection between HDAC signal and c-Met signal.
HDACs play a crucial role in the regulation of multiple processes of life, from gene expressions transcription, cell
proliferation, differentiation to protein activities [18]. More importantly, HDACs are also found to be overexpressed
in some human cancers, so they are regarded as promising drug targets for several types of cancers therapy [19-20].
HDAC inhibition process affects c-Met and its downstream signaling pathways both directly and indirectly. In

literature researches, we also found HDAC inhibitors inhibit HGF production (the ligand of c-Met) [21]. As far as
HDAC inhibitors are not only single epigenetic target anticancer drugs but also successful patterns as cytotoxic
agents due to their familiar structures. Except for Romidepsin, almost all the HDAC inhibitors are sharing essential
pharmacophoric structural features essential for the activity [22-23]. Therefore, it is so easy to link them to other
anticancer scaffolds that many dual action HDAC inhibitors have been developed. Inspiringly, most of these hybrids
have higher potency than the constituting parents in fighting of the cancer cells [24]. In this context, developments
of dual HDAC inhibitors are proceeding intensively. There are a number of hybrid (chimeric) drugs that are
currently in preclinical and clinical trials (Fig. 1). CUDC-101 is a first-in-class hybrid inhibitor developed by Cai
et al in 2010 which displayed potent in vitro inhibitory activity against HDAC1, EGFR and HER2 with an IC50 of
4.4, 2.4, and 15.7 nM, respectively [25]. CUDC-907 is a phosphatidylinosital 3-kinase (PI3K) and histone
deacetylase (HDAC) inhibitor in phase II clinical trials at Curis for the treatment of relapsed or refractory diffuse
large B-cell lymphoma (DLBCL) harboring MYC mutations [26-28]. Using successful RTK/HDAC hybrid
inhibitor design, Lu et al reported the first potent c-Met and HDAC hybrid inhibitor 2m which inhibited c-Met
kinase and HDAC1 with IC50 values of 0.71 and 38 nM, respectively [29].
Pictilisib like Cap
O
Erlotinib Cap
Linker
ZBG
ZBG
Linker
N
HN
N
N
N
HN OH
H
N
O
O
N
S
N
HO
O
O
N
N
N
CUDC-101
CUDC-907
O
EGFR/HDAC
PI3K/HDAC
c-Met inhibitor Cap
ZBG
NH2
N
Linker
Essential fragment
of kinese inhibitors
O
O
N
N
Linker
ZBG
N
N
N
H
N
Kinases
HDACs
O
O
2m
c-Met/HDAC
Fig. 1. Structures of representative dual RTK/HDAC inhibitors.
In this report, we present a fantastic idea for cancer drug development from a medicinal chemist’s perspective,
which hybrid the pharmacophores of HDACi with a c-Met inhibitor. On the basis of our knowledge and experience
in the development of c-Met inhibitors, we found 2-pyrrolidinone fragment was widely used as a building block in
the design of anticancer agents [30-31]. Furthermore, 2-pyrrolidinone fragment quite fit the “5 atoms regulation’’.
Moreover, in our previous studies, the six-carbon alkyl linker was found to be optimal for the dual inhibitors. In this

work, taking Foretinib as the lead compound, 2-pyrrolidinone was introduced to form the 5-atoms linker in order to
promote c-Met activity. In addition, the hydroxamic acid side chain were introduced into solvent region of Foretinib
being taken as the zinc-binding group (ZBG) of HDAC inhibitors [32] (Fig. 3).
H
N
N
O
O
Kinase inhibitor Core


H
H
H
N
N
F
R2
O
N
R1
O
N
R3
O
O
O
F
O
5 atoms linker
Merge Key Pharmacophores
O
O
O
O
O
N
N
HO
N
N
O
H
Foretinib
Target Compounds
Key HDAC
Zn Binding Group
O
H
N
OH
N
H
O
Vorinostat
Fig. 3. Design strategy for the c-Met/HDAC dual inhibitors.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of target compounds 15a-15t.
The synthesis of c-Met/HDAC dual inhibitors are depicted in Scheme 1. The key intermediates 8a-8b were
treated with intermediates 12a-12s to give condensed amide products 14a-14t, respectively, which were treated with
freshly prepared hydroxylamine methanol solution to give target compounds 15a-15t [33].
R2
H
R2
O
HO
R2
N
R1
O
NH2
R1
O
N
H
N
O
R3
R1
O
N
O
O
R3
O
O
O
N
O
O
O
O
O
O
+
O
b
a
R3
12a-12s
O
N
O
O
N
HO
5
5
N
N
5
15a-15t
14a-14t
H
8a-8b
14a,15a: R1=H,R2=H,R3=2,4-(OCH3)2
14e,15e: R1=H,R2=H, R3=4-OCF3
14i,15i: R1=H,R2=H,R3=3-Cl,4-F
14m,15m: R1=H,R2=H,R3=4-Br
14q,15q: R1=F,R2=H,R3=H
14b,15b: R1=H,R2=H,R3=2-F
14f,15f: R1=H,R2=H,R3= 4-F
14c,15c: R1=H,R2=H,R3=H
14d,15d: R1=H,R2=H,R3=3,4-(F)2
14g,15g: R1=H,R2=H,R3=2-OCH3
14k,15k: R1=H,R2=H,R3=2-OCF3
14h,15h: R1=H,R2=H,R3=4-CH(CH3)2
14l,15l: R1=H,R2=H,R3=4-Cl
14j,15j: R1=H,R2=H,R3=4-CH2CH3
14o,15o: R1=H,R2=H,R3=2-Br
14s,15s: R1=H,R2=CH3,R3=H
14p,15p: R1=H,R2=H,R3=2-CH3
14t,15t: R1=H,R2=CH3,R3=4-Cl
14n,15n: R1=H,R2=H,R3= 3-F
14r,15r: R1=H,R2=CH3,R3=2-F
Scheme 1. Preparation of derivatives of 15a-15t. Reagents and conditions:( a): HATU, Et3N, DCM, 40 ℃, 3-5 h; (b): NH2OH.HCl,
NaOH, anhydrous, CH3OH, rt, 2-6 h;
The key intermediates 8a-8b were synthesized as outlined in Scheme 2. Starting from commercially available
acetovanillone 1 and ethyl 7-bromoheptanoate, intermediate 2 was prepared via a displacement reaction. The
intermediate 2 treated with fuming nitric acid to obtain intermediate 3. Intermediate 3 treated with DMF-DMA, Fe

powder, phosphorus oxychloride through acylation, cyclization and chlorination reaction to afforded intermediate
6. 7a-7b were obtained through nucleophilic substitution with p-nitrophenol or 2-fluoro-4-nitrophenol in
chlorobenzene, which were reduced by H2/Pd to yield the desired intermediates 8a-8b.
O
O
O
O
O
O
O
O
O
a
O
c
b
O
HO
O
NO2
2
1
3
O
Cl
N
O
O
O
O
O
O
O
O
O
O
N
e
d
O
N
H
O
O
NO2
6
5
4
R1
NO2
R1
O
NH2
7a,8a: R1=H
7b,8b: R1=F
O
N
g
f
O
O
O
O
O
O
O
O
N
7a-7b
8a-8b
Scheme 2. Preparation of intermediates 8a-8b. Reagents and conditions: (a) ethyl 7-bromoheptanoate, K2CO3, DMF, 80℃, 3-4 h;(b)
fuming HNO3, anhydrous DCM, -5 ℃, 6-8 h; (c) DMF-DMA, toluene, 110 °C, 7-8 h; (d) Fe powder, AcOH, r.t, 30 min, 80 °C, 1-2 h; (e)
POCl3, 85 ℃, 6-7 h; (f) 2-fluoro-4-nitrophenol/ p-nitrophenol, PhCl, 140 ℃, 20-24 h; (g) H2, Pd/C(10%), CH3OH,40 ℃, 3-5 h.
The intermediates 12a-12s were synthesized as outlined in Scheme 3 [34]. Briefly, cyclopropane-1,1-
dicarboxylic acid reacted with acetone and acetic anhydride to afford intermediate 10, which was reacted with
different substituted anilines to obtain intermediates 12a-12p, respectively. After treating 12b, 12c, 12l with methyl
iodide, intermediates 12q, 12s, 12t were obtained.
HO
R2
O
O
O
R3
COOH
COOH
a
O
/c
b
N
+
NH2
O
O
R3
9
11a-11p
10
12a-12s
11d,12d: R2=H, R3=3,4-(F)2
11a,12a: R2=H, R3=2,4-(OCH3)2
11e,12e: R2=H, R3=4-OCF3
11b,12b, R2=H, R3=2-F
11f,12f: R2=H, R3= 4-F
11c,12c: R2=H, R3=H
11g,12g: R2=H, R3=2-OCH3
11h,12h: R2=H, R3=4-CH(CH3)2
11l,12l: R2=H, R3=4-Cl
11j,12j: R2=H, R3=4-CH2CH3
11k,12k: R2=H, R3=2-OCF3
11i,12i: R2=H, R3=3-Cl,4-F
11n,12n: R2=H, R3= 3-F
11s,12s:R2=CH3, R3=H
11p,12p: R2=H, R3=2-CH3
11o,12o: R2=H, R3=2-Br
11s,12s:R2=CH3, R3=4-Cl
11m,12m: R2=H, R3=4-Br
11q,12q: R2=CH3, R3=2-F
Scheme 3. Preparation of intermediates 12a-12s. Reagents and conditions: (a) i: conc H2SO4, Ac2O, acetone, rt,10 h; (b) DMF, rt, 12
h; (c) THF, NaH, CH3I, -5 ℃, 5 h;
2.2. Biological evaluation
2.2.1. HDAC1 and c-Met enzymatic activity assay
Initially, inhibitory activities for all compounds were preliminarily tested against c-Met and HDAC1 at 100
nM using compounds SAHA, XL-184 and Foretinibas reference drugs. The biological data listed in Table 1 showed
that most of the compounds exhibited excellent inhibitory activity against HDAC1, however, only seven compounds

inhibited c-Met over 50% rate at 100 nM.
H
R2
O
N
R1
O
N
R3
O
O
O
O
HO
N
N
5
H
Table 1. HDAC1 and c-Met inhibitory activities of target compounds 15a-15t.
Compd.
15a
15b
15c
15d
15e
15f
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
R2
R3
2,4-(OCH3)2
2-F
HDAC1 inhibition@100nMa
c-Met inhibition@100nMa
16.0%
H
79.8%
H
H
53.4%
57.0%
H
35.9%
41.2%
H
3,4-(F)2
4-OCF3
4-F
46.1%
40.1%
H
18.9%
18.2%
H
77.8%
93.0%
15g
15h
15i
H
2-OCH3
4-CH(CH3)2
3-Cl,4-F
4-CH2CH3
2-OCF3
4-Cl
78.4%
56.0%
H
21.6%
19.2%
H
33.6%
26.4%
15j
H
34.9%
51.7%
15k
15l
H
61.1%
21.3%
H
58.9%
41.0%
15m
15n
15o
15p
15q
15r
15s
H
4-Br
43.9%
14.4%
H
3-F
77.9%
60.7%
H
2-Br
71.5%
34.5%
H
2-CH3
H
67.6%
74.0%
H
64.0%
82.1%
H
H
CH3
CH3
2-F
51.2%
12.5%
H
53.7%
39.0%

15t
SAHAb
H
CH3
4-Cl
25.4%
88.0%
NT
15.0%
NT
XL-184b
Foretinibb
91.6%
95.5%
NT
a All compounds were assayed at least twice, and the inhibitory values were averaged.
b Used as a positive control.
NT= not tested.
As presented in Table 1, the effects of regiochemical substitution and electronics on the phenyl ring were
explored. The introduction of electron-donating groups, such as methyl and methoxy, at the 2-position on the phenyl
ring improve both HDAC1 and c-Met inhibition activity (15g, 15p vs 15c). Furthermore, by comparing compounds
15c (HDAC1: inhibition rate 35.9 %; c-Met: inhibition rate 41.2%), 15h (HDAC1: inhibition rate 21.6 %; c-Met:
inhibition rate 19.2%) and 15f ( HDAC1: inhibition rate 77.8 %; c-Met: inhibition rate 93.0%), we could found that
electron withdrawing group at the 4-position might contribute to the activity, while the electron donor might
decrease inhibitory activity. Moreover, 4-position substituted compounds demonstrated the superior HDAC
inhibitory activity than 3-position and 2-position substituted compounds (15f vs 15n vs 15b). In addition,
introduction of double-electron-withdrawing groups (double-EWGs) or double-electron-donating groups (double-
EDGs) on the phenyl ring both led to an obvious lost in c-Met activity comparing to the single substituent, such as
compounds (15a vs 15g, 15d vs 15f). Additionally, introducing steric hindrance at the 4-position of the phenyl ring
decreased inhibitory activities (15m vs 15c, 15h vs 15c).
Based on the previous report that halogen-containing substituents on the phenyl ring in the CUDC-101
displayed excellent inhibitory activities [25], we introduced F atom into R1 to test their effects on inhibitory activities
against HDAC1. From the result of 15c and 15q, we concluded that the introduction of fluorine atom was good for
c-Met and HDAC1 inhibition.
Methylation is used to optimize many properties of a drug candidate. For example, methylating next to a
metabolic hot spot might sterically block metabolism, lengthen the half-life. Methylation has a favorable effect on
solubility or selectivity against off-targets. Indeed, methylation can have a favorable effect on binding affinity [35-
36]. On the basis of the foregoing, our further investigations were performed to study the introduction of a methyl
group to the 3 position of 2-pyrrolidinone core on the kinase activity. To our surprised, the compounds which
containing a methyl group had a dramatic loss in activity (15r vs 15b; 15s vs 15c; 15q vs 15l), The reason might be
lay in that the preferred conformation was destructed by the introducing of methyl group.

Table 2. Enzymatic assays for HDAC1/c-Met inhibition.
Compd.
15f
HDAC1 IC50 (nM) ± SDa
c-Met IC50 (nM) ± SDa
12.50 ± 0.42
42.24 ± 1.06
17.84 ± 0.25
NT
26.97 ± 0.24
58.26 ± 1.15
76.24 ± 1.05
14.15 ± 0.25
NT
15p
15q
SAHAb
XL-184b
Foretinibb
37.28 ± 1.10
2.11 ± 0.50
NT
a SD: standard deviation.
b Positive control.
NT= not tested.
Compounds 15f, 15p and 15q were select to screened half maximal inhibitory concentration (IC50) against c-Met
and HDAC1 kinases. As shown in Table 2, the tested compounds exhibited excellent c-Met and HDAC1 enzymatic
potency with IC50 values in the nanomolar range. In particular, compound 15f showed IC50 value against HDAC1
was 26.97 nM, which was less effective than SAHA (IC50 = 14.15 nM). However, the potency of 15f against c-Met
(IC50 = 12.50 nM) had been slightly improved than that of XL184 (IC50 = 37.28 nM), which validating our
compound design strategy that incorporation of zinc binding group to the solvent-exposed phenyl group of Foretinib
could obtain HDAC1 and c-Met dual inhibitory activities.
2.2.2. In vitro cytotoxic activities
To further explore the antitumor effect of these c-Met/HDAC inhibitors, all dual inhibitors were tested against
a panel of three solid tumor cell lines selected as representative of hard-to-treat solid tumors while being known to
be sensitive to c-Met and HDAC inhibitors: HCT-116 (human colon cancer), A549 (human lung adenocarcinoma)
and MCF-7 (human breast cancer) by MTT assay.
Table 3. The antiproliferative activities of 15a-15t against HCT-116, MCF-7and A549 cell lines in vitro.
IC50 a (μmol/L) ± SD
Compd.
R1
H
R2
H
R3
HCT-116
MCF-7
A549
>20
15a
2,4-(OCH3)2
11.90 ± 0.26
2.06 ± 0.21

15b
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
2-F
2.68 ± 0.05
3.45 ± 0.22
2.12 ± 0.02
3.37 ± 0.19
0.54 ± 0.05
3.73 ± 0.12
1.68 ± 0.43
3.55 ± 0.06
5.07 ± 0.34
7.18 ± 0.25
0.14 ± 0.02
3.99 ± 0.04
0.47 ± 0.03
4.08 ± 0.25
0.42 ± 0.06
2.10 ± 0.08
9.01 ± 0.52
7.29 ± 0.38
3.52 ± 0.12
0.68 ± 0.13
4.50 ± 0.08
0.63 ± 0.09
0.57 ± 0.03
0.74 ± 0.10
1.93 ± 0.20
0.77 ± 0.04
0.28 ± 0.02
1.86 ± 0.01
0.80 ± 0.01
2.30 ± 1.10
1.44 ± 0.05
0.78 ± 0.44
1.72 ± 0.47
1.39 ± 0.06
0.77 ± 0.05
1.77 ± 0.21
2.02 ± 0.40
1.14 ± 0.09
3.52 ± 0.24
5.51 ± 0.05
1.57 ± 0.05
0.99 ± 0.20
5.58 ± 0.15
0.52 ± 0.08
1.12 ± 0.10
3.99 ± 0.15
4.44 ± 0.12
8.28 ± 1.21
1.08 ± 0.44
5.60 ± 0.33
10.05 ± 0.55
18.92 ± 1.50
6.79 ± 0.10
2.08 ± 0.34
3.86 ± 0.05
4.38 ± 0.20
1.92 ± 0.31
6.76 ± 0.24
1.98 ± 1.00
1.76± 0.29
>20
15c
H
H
15d
H
3,4-(F)2
4-OCF3
4-F
15e
H
15f
H
15g
H
2-OCH3
4-CH(CH3)2
3-Cl,4-F
4-CH2CH3
2-OCF3
4-Cl
15h
H
15i
H
15j
H
15k
H
15l
H
15m
15n
H
4-Br
H
3-F
15o
H
2-Br
15p
H
2-CH3
H
15q
H
15r
H
H
H
CH3
CH3
CH3
2-F
15s
H
9.97 ± 1.02
8.43 ± 0.02
2.29 ± 0.05
7.34 ± 0.20
0.94 ± 0.04
15t
4-Cl
SAHAb
XL-184b
Foretinibb
SAHA+Foretinib
(1:1)
0.64 ± 0.12
0.62 ± 0.11
0.99 ± 0.06
a Inhibition values represent the average of at least three independent experiments.
b Used as a positive control.

Data in Table 3 illustrated that the target compounds exhibited moderate to potent antiproliferation activities
against one or more tested cancer cell lines with potencies in the single digit micromole range, which was mostly
in accordance with the trend in enzymatic activity. However, by comparing with an equimolar mixture of SAHA
and Foretinib, we did not observe the compounds showed clearly synergistic effect. Interestingly, among the tested
solid tumor cell lines, the human breast cancer cell line MCF-7 seemed to be the most sensitive one to our
compounds, indicating that the target compounds might be used in curing human breast cancer. Among them,
compound 15f exhibited the best activity against all of the tested cell lines with IC50 value of 0.54, 0.28 and 1.08
μM against HCT-116, MCF-7 and A549 cell lines, respectively, indicating that 15f deserve further study with regard
to its application in the treatment of cancer.
2.2.3. Cell apoptosis study
To further explore whether our compounds are accompanied by enhanced cancer cell apoptosis, we then
performed a FITC-Annexin V/propidium iodide (PI) staining and flow cytometry assay of 15f in HCT-116 cells.
As shown in Fig. 4, 3.01% apoptotic cells were observed in control group treated with DMSO, whereas tumor cells
treated with 15f at defined concentrations of 0.2, 1.0 and 5.0 µM for 48 h resulted in 6.97%, 7.08% and 13.21%
cells apoptosis, respectively. The results showed that compound 15f increased the cellular apoptosis in a
concentration dependent manner, which might result from concerted inhibition of c-Met and HDAC1 activity.
Control
0.2 μM
0.47%
1.23%
1.03%
1.49%
Q1
Q3
Q2
Q4
Q1
Q3
Q2
Q4
92.00%
95.76%
2.54%
5.48%
1.0 μM
5.0 μM
0.78%
1.75%
5.33%
3.49%
9.72%
2.51%
Q2
Q4
Q1
Q3
Q1
Q2
92.16%
84.28%
Q3 Q4
Fig. 4. Compound 15f induced apoptosis in HCT-116 cells. Cells were treated with various concentrations of 15f for 48 h and then
analyzed the Annexin V-FITC/PI staining test by flow cytometry analysis. Values represent the mean ± S.D, n = 3. The percentage of
cells in each part is indicated.

2.2.4. Cell cycle analysis
To evaluate whether 15f could induce tumor cell cycle arrest, we then performed a flow cytometry assay to
assess the effect of 15f on the distribution of cell cycle. The cell cycle profiles of HCT-116 after treating with 15f
in different concentrations for 48 h, and the results were illustrated in Fig. 5-6. It is clearly disclosed that 15f showed
an obvious increase in the proportion of cells in G2/M phase in a dose-dependent manner. The percentage of cells
in G2/M phase was gradually increased from 10.61% in the untreated cells to 86.68%, 88.36% and 87.75% in cells
treated with 15f at 0.2, 1.0 and 5.0 µM, respectively.
Fig. 5. Effect of compound 15f on the cell cycle distribution of HCT-116 cells. The experiments were performed three times and a
representative experiment is shown.

120
100
80
60
40
20
0
6.25
7.07
6.73
4.91
7.45
4.8
60.16
29.23
G1
S
G2/M
88.36
87.75
86.68
10.61
A
B
C
D
Fig. 6. Quantitative analysis of cell cycle distributions; (A) Non-treated cells as control group (B) treated with 15f at 0.2 μM (C) treated
with 15f at 1.0 μM (D) treated with 15f at 5.0 μM
2.2.5. Binding Mode of the c-Met/HDAC Dual Inhibitor.
Docking simulations were performed for the selected compound 15f to investigate whether it can form favorite
interactions with the two targets. The best-scored pose of compound 15f with c-Met and HDAC1 are presented in
Fig.7. For the binding mode of 15f with HDAC1 (PDB ID:1C3S, Fig.7 A, B) [37], the hydroxamic acid coordinated
to the catalytic Zn2+ of HDAC1 and formed a hydrogen bond with His131. The molecular docking with 15f in c-
Met (PDB ID:3LQ8) was also performed [32], the result was shown in Fig.7 C, D. The carbonyl of the amide
hydrogen bond with the residue of Lys1110. In addition, the carbonyl of pyrrolidinone formed hydrogen bond with
the residues of Asp1222. The N atom of quinoline ring of 15f made hydrogen bond with Met1160 in the hinge
region. Furthermore, the hydroxamic tail formed two hydrogen bonds with the residues of His1162 and Lys1161.

A
B
D
C
Fig. 7. (A) Proposed 3D binding mode of compound 15f in the active site of HDAC1 (PDB: 1C3S). (B) Proposed 2D binding mode of
compound 15f in the active site of HDAC1 (PDB: 1C3S). (C) Proposed 3D binding mode of compound 15f in the active site of c-Met
(PDB: 3LQ8). (D) Proposed 2D binding mode of compound 15f in the active site of c-Met (PDB: 3LQ8). The figure was generated
using PyMol (http://www.pymol.org).
3. Conclusions
On the basis of the pharmacophore fusion strategy, through designing and structural optimization targeting on
c-Met and HDAC1, we finally discovered a new compound 15f which potently inhibited c-Met and HDAC1 with
IC50 values of 12.50 and 26.97 nM, respectively. In vitro cellular assays, 15f showed potent activities against HCT-
116, MCF-7 and A549 cell lines with IC50 values of 0.54, 0.28 and 1.08 μM, respectively. Antitumor mechanism
studies showed that 15f could promote induction of apoptosis in a dose-dependent manner. Moreover, 15f could
block the G2/M phase even at 0.2 μM. The molecular docking analysis can be used as a valuable probe to clarify
the two enzymes inhibitory activity. All the studies indicated 15f as a novel “hit” compound targeting on c-Met
and HDAC1 deserve for further investigation and development.
4. Experimental
4.1. Chemistry

Unless otherwise specified, all materials were obtained from commercial suppliers and were used without
further purification. Reactions’ time and purity of the products were monitored by TLC on FLUKA silica gel
aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm. Column chromatography was run on silica
gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All melting points were obtained
on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. Mass
spectra (MS) were taken in ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). 1HNMR and 13C
NMR spectra were performed using Bruker spectrometers (BrukerBioscience, respectively, Billerica, MA, USA)
with TMS as an internal standard.
4.1.1. Preparation of ethyl 7-(4-acetyl-2-methoxyphenoxy)heptanoate (2)
To a solution of 4-hydroxy-3-methoxy-acetophenone (20.0 g, 120.5 mmol) in DMF (200 mL) was added
K2CO3 (17.4 g, 126.5 mmol) and ethyl 7-bromoheptanoate (30.0 g, 126.5 mmol). The reaction mixture was then
stirred at 80 °C for 4 h. The mixture was then poured into cold water (500 mL) with vigorously agitating, the
resulting precipitate was filtered-off, washed with water, and dried under vacuum to afford the title compound 2
(31.3 g, 80.1%) as a white solid. MS (ESI) m/z: 323.05 [M+H] +.
4.1.2. Preparation of ethyl 7-(4-acetyl-2-methoxy-5-nitrophenoxy)heptanoate (3)
A stirred solution of 2 (30.0 g, 93.1 mmol) in CH2Cl2 (300 mL) was cooled to 10 °C, fuming HNO3 (43.5 g,
465.5 mmol) was added at an appropriate rate keeping the temperature below -5 °C. The reaction mixture was
allowed to stir at -5 °C for 6 h, then poured into cold water (200 mL), the organic layer was separated and washed
with water (200 mL), then concentrated under reduced pressure to afford crude 3 as a light yellow solid (29.3 g,
85.7%). MS (ESI) m/z: 390.04 [M+Na] +.
4.1.3. Preparation of ethyl (E)-7-(4-(3-(dimethylamino)acryloyl)-2-methoxy-5-nitrophenoxy)heptanoate (4)
28.0 g (76.3 mmol) of 3 was suspended in 280 mL of toluene at room temperature, then DMF-DMA (24.0 g,
228.9 mmol) was added to a solution. The reaction was heated to 110 °C for 4 h. After cooling to rt, the resultant
solid was collected by filtration, washed with toluene (40 mL), then dried under vacuum to yield 4 as a yellow solid
(24.4 g, 75.8%). MS (ESI) m/z: 445.05 [M+Na] +.
4.1.4. Preparation of ethyl 7-((6-methoxy-4-oxo-1,4-dihydroquinolin-7-yl)oxy)heptanoate (5)
Fe powder (15.9 g, 284.2 mmol) was added to a solution of 4 (24.0 g, 56.8 mmol) in acetic acid (100 mL) at
60 °C in batches, then the mixture was stirred at 80 °C for 2 h. The hot solution was filtered through celite and
washed with hot acetic acid. The combined filtrate was cooled to rt, the resultant solid was collected by filtration
and washed with acetic acid (50 mL) to afford 5 as a yellow solid (12.4 g, 62.9 %). MS (ESI) m/z: 348.02 [M+H]

+
.
4.1.5. Preparation of ethyl 7-((4-chloro-6-methoxyquinolin-7-yl)oxy)heptanoate (6)
A mixture consisting of 12.0 g (34.6 mmol) 5 and 96.0 mL (8 v/m) phosphorus oxychloride was refluxed for
10 h whereby a clear solution was formed. Thereafter, the excess unreacted phosphorus oxychloride was evaporated
in vacuo and the residual oil was poured into ice water. The precipitate formed under vigorous stirring conditions,
thereby the solid was collected by vacuum filtration, dried under reduced pressure to afford product 6 as a pale solid
(10.1 g, 80.2%). 1H NMR (600 MHz, DMSO-d6) δ 8.56 (d, J = 4.8 Hz, 1H), 7.48 (d, J = 4.8 Hz, 1H), 7.37 (s, 1H),
7.29 (s, 1H), 4.09 (t, J = 6.5 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.94 (s, 3H), 2.26 (t, J = 7.4 Hz, 2H), 1.76 (t, J = 7.4
Hz, 2H), 1.58 – 1.48 (m, 2H), 1.42 (t, J = 7.8 Hz, 2H), 1.32 (q, J = 8.4 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H).
4.1.6. General procedure for the preparation of intermediates (7a-7b)
4-nitrophenol (4.1 g, 29.6 mmol) or 2-fluoro-4-nitrophenol (4.6 g, 29.6 mmol) was added to a stirred solution
of 6 (9.0 g, 24.7 mmol ) in 60 mL chlorobenzene, then the reaction mixture was refluxed for 24 h whereby a clear
solution was formed. Then evaporated in vacuo, the resulting precipitate was added to 100 mL DCM, the organic
portion was washed with 10% w/w aqueous sodium bicarbonate solution (3 x 50 mL), the organic layer was
evaporated in vacuo and dried under reduced pressure to afford product 7a-7b.
4.1.6.1. ethyl 7-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)heptanoate(7a)
Yellow solid, yield 52.5%, MS (ESI) m/z (%): 469.00 [M+H]+; 1H NMR (600 MHz, DMSO-d6) δ 8.42 (d, J = 5.3
Hz, 1H), 7.50 (s, 1H), 7.34 (s, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.36 (d, J = 5.2 Hz, 1H), 5.18 (s,
2H), 4.12 (t, J = 6.5 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 2.30 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 6.7 Hz, 2H),
1.57 (p, J = 7.4 Hz, 2H), 1.47 (p, J = 7.5 Hz, 2H), 1.37 (q, J = 8.1 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H).
4.1.6.2. ethyl 7-((4-(2-fluoro-4-nitrophenoxy)-6-methoxyquinolin-7-yl)oxy)heptanoate (7b)
Yellow solid, yield 56.8%, MS (ESI) m/z (%): 487.12 [M+H]+.
4.1.7. General procedure for the preparation of intermediates (8a-8b)
10% Pd/C (0.5 g, 10%w/w) was added to a stirred solution of 7a or 7b (5.0 g) in 50 mL methanol under H2,
then the mixture was stirred at 40 °C for 4 h. After completion of reaction, it was allowed to cool to room temperature.
The solution was filtered through celite and washed with methanol (10 mL), then solvent was evaporated in vacuo
and dried under reduced pressure to afford product 8a-8b.
4.1.7.1. ethyl 7-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)heptanoate (8a)
Yellow solid, yield 91.7% ,MS (ESI) m/z (%): 439.19 [M+H]+.
4.1.7.2. ethyl 7-((4-(4-amino-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)heptanoate (8b)

Yellow solid, yield 89.8% ,MS (ESI) m/z (%): 457.22 [M+H]+.
4.1.8. Preparation of 6,6-dimethyl-5,7-dioxaspiro[2.5]octane-4,8-dione (10)
10.0 g (76.9 mmol) of cyclopropane-1,1-dicarboxylic acid (9) was added together with (9.5 ml, 92.3 mmol) of
acetic anhydride and 0.25 ml of sulfuric acid and heated at 30 to 35 °C. Then (7.5 ml, 92.3 mmol) of acetone is
added dropwise, the temperature of the reaction mixture is kept at 30 to 35 °C. After completion of the addition of
acetone, the reaction mixture is stirred for about 8 hours at 30 to 35 °C. The reaction mixture poured into 100 mL
ice-water, a white crystalline was obtained by filtration. The solid was twice washed with 30 ml portions of cold
water, then dried under reduced pressure to afford product 10 as a white solid (10.6 g, 81.5 %). MS (ESI) m/z:
171.23 [M+H]+.
4.1.9. General procedure for preparation intermediates (12a-12p)
To a solution of 6,6-dimethyl-5,7-dioxaspiro[2.5]octane-4,8-dione (1.0 g, 5.9 mmol) in 10 mL of DMF was
added substituted aniline 11a-11p (6.5 mmol). The reaction mixture was stirred at rt overnight. The reaction
mixture was poured into cold water (10 mL). The precipitate that formed was collected by filtration, washed with
water, and dried under vacuum to afford the desired product 12a-12p.
4.1.9.1. Preparation of 1-(2,4-dimethoxyphenyl)-2-oxopyrrolidine-3-carboxylic acid (12a)
Gray solid; yield: 81.2%; MS (ESI) m/z: 266.17 [M+H] +.
4.1.9.2. Preparation of 1-(2-fluorophenyl)-2-oxopyrrolidine-3-carboxylic acid (12b)
White solid; yield: 89.4%; MS (ESI) m/z: 224.17 [M+H] +.
4.1.9.3. Preparation of 2-oxo-1-phenylpyrrolidine-3-carboxylic acid (12c)
White solid; yield: 84.3%; MS (ESI) m/z: 206.16 [M+H] +; 1H NMR (600 MHz, DMSO-d6) δ 12.74 (s, 1H),
7.70 – 7.58 (m, 2H), 7.45 – 7.32 (m, 2H), 7.22 – 7.11 (m, 1H), 3.95 – 3.78 (m, 2H), 3.60 (dd, J = 9.1, 7.8 Hz, 1H),
2.42 – 2.22 (m, 2H).
4.1.9.4. Preparation of 1-(3,4-difluorophenyl)-2-oxopyrrolidine-3-carboxylic acid (12d)
Yellow solid; yield: 74.4%; MS (ESI) m/z: 242.17 [M+H] +; 1H NMR (600 MHz, DMSO-d6) δ 12.89 (s, 1H),
7.85 (ddd, J = 12.1, 6.8, 2.1 Hz, 1H), 7.53 – 7.37 (m, 2H), 3.89 (m, 1H), 3.83 (m, 1H), 3.63 (m, 1H), 2.34 (dd, J =
36.1, 8.2 Hz, 2H).
4.1.9.5. Preparation of 2-oxo-1-(4-(trifluoromethoxy)phenyl)pyrrolidine-3-carboxylic acid (12e)
White solid; yield: 75.0%; MS (ESI) m/z: 290.16 [M+H] +.
4.1.9.6 Preparation of 1-(4-fluorophenyl)-2-oxopyrrolidine-3-carboxylic acid (12f)
White solid; yield: 74.1%; MS (ESI) m/z: 224.17 [M+H] +.

4.1.9.7. Preparation of 1-(4-methoxyphenyl)-2-oxopyrrolidine-3-carboxylic acid (12g)
Yellow solid; total yield: 82.5%; MS (ESI) m/z: 236.20 [M+H] +.
4.1.9.8. Preparation of 1-(4-isopropylphenyl)-2-oxopyrrolidine-3-carboxylic acid (12h)
White solid; yield: 80.1%; MS (ESI) m/z: 248.23 [M+H] +.
4.1.9.9. Preparation of 1-(3-chloro-4-fluorophenyl)-2-oxopyrrolidine-3-carboxylic acid (12i)
White solid; yield: 67.5%; MS (ESI) m/z: 258.17 [M+H] +.
4.1.9.10. Preparation of 1-(4-ethylphenyl)-2-oxopyrrolidine-3-carboxylic acid (12j)
White solid; yield: 83.1%; MS (ESI) m/z: 234.18 [M+H] +.
4.1.9.11. Preparation of 2-oxo-1-(2-(trifluoromethoxy)phenyl)pyrrolidine-3-carboxylic acid (12k)
White solid; yield: 72.3%; MS (ESI) m/z: 290.16 [M+H] +.
4.1.9.12. Preparation of 1-(4-chlorophenyl)-2-oxopyrrolidine-3-carboxylic acid (12l)
White solid; yield: 81.6%; MS (ESI) m/z: 240.16 [M+H] +.
4.1.9.13. Preparation of 1-(4-bromophenyl)-2-oxopyrrolidine-3-carboxylic acid (12m)
White solid; yield: 70.6%; MS (ESI) m/z: 284.05 [M+H] +.
4.1.9.14. Preparation of 1-(3-fluorophenyl)-2-oxopyrrolidine-3-carboxylic acid (12n)
White solid; yield: 79.9%; MS (ESI) m/z: 224.24 [M+H] +.
4.1.9.15. Preparation of 1-(2-bromophenyl)-2-oxopyrrolidine-3-carboxylic acid (12o)
White solid; yield: 69.1%; MS (ESI) m/z: 284.05 [M+H] +.
4.1.9.16. Preparation of 2-oxo-1-(o-tolyl)pyrrolidine-3-carboxylic acid (12p)
White solid; yield: 88.1%; MS (ESI) m/z: 220.21 [M+H] +.
4.1.10. General procedure for preparation intermediates (12q, 12r, 12s)
Appropriate intermediate 12b, 12c, 12l (10.0 mmol) was suspended in 20 mL of dry tetrahydrofuran at -5°C,
NaH(0.72 g, 30.0 mmol) was added in batches. After stirring for 0.5 h at -5 °C, CH3I (2.1 g, 15.0 mmol) was added
at an appropriate rate keeping the temperature below 0-5 °C. After completion of the addition, the reaction mixture
was stirred for about 2 h at rt. The reaction mixture poured into 100 mL ice-water, acidized to pH 2-3 with 6 N
HCl, the precipitate that formed was collected by filtration, washed with water, and dried under vacuum to afford
the desired product.
4.1.10.1. Preparation of 1-(2-fluorophenyl)-3-methyl-2-oxopyrrolidine-3-carboxylic acid (12q)
White solid; yield: 56.2%, MS (ESI) m/z (%): 238.15 [M+H]+;
4.1.10.2. Preparation of 3-methyl-2-oxo-1-phenylpyrrolidine-3-carboxylic acid (12r)

White solid; yield: 62.7%, MS (ESI) m/z (%): 220.14 [M+H]+.
4.1.10.3. Preparation of 1-(4-chlorophenyl)-3-methyl-2-oxopyrrolidine-3-carboxylic acid (12s)
White solid; yield: 74.0%, MS (ESI) m/z (%): 254.14 [M+H]+.
4.1.11. General procedure for preparation of intermediates (14a-14t)
A mixture of 8a-8b (1.0 mmol) and different intermediates 12a-12s (1.3 mmol), HATU (1.3 mmol), TEA (1.3
mmol) in DCM (15 mL) was stirred at reflux for 3-5 h. After being cooled to rt, the mixture was washed successively
with 10% aqueous potassium carbonate solution (20 mL×3) and brine (20 mL×2), the organic phase was dried over
anhydrous sodium sulfate. The solid was removed by filtration, and the filtrate was concentrated to yield the 14a-
14t, respectively, yeilds 60.7-92.2%. Without any purification, the intermediates were used for next procedure.
4.1.12. General procedure for preparation of target compounds (15a-15t)
A solution of sodium hydroxide (4.0 g, 100.0 mmol) in methanol (14 mL) was added to a stirred solution of
hydroxylamine hydrochloride (4.7 g, 67.2 mmol) in methanol (24 mL) at 0 °C. The reaction mixture was stirred at
0 °C for 1 h. The precipitate was removed by filtration and the filtrate was collected to provide fresh hydroxylamine
solution which was stored in a refrigerator before use. The appropriate intermediates 14a-14t (1.0 mmol) was added
to the above freshly prepared hydroxylamine solution (15 mL) at 0 °C, then NaOH (0.04 g,1.0 mmol) was added to
the solution. The reaction mixture was stirred at room temperature for 2-6 h. The reaction mixture was neutralized
with 6 N HCl. The formed precipitate was collected by filtration, washed with water, dried in vacuo and purified by
silica gel chromatography to afford target compounds 15a-15t.
4.1.12.1.
1-(2,4-dimethoxyphenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-
yl)oxy)phenyl)-2-oxopyrrolidine-3-carboxamide (15a)
Gray solid; Yield: 64.1%; M.p.: 115-116 C; MS (ESI) m/z(%): 673.26 [M+H]+; 1H NMR (600 MHz, DMSO-
d6) δ 10.43 (s, 1H), 10.37 (s, 1H), 8.71 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.52 (s, 1H), 7.38
(s, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 8.6 Hz, 1H), 6.67 (d, J = 2.6 Hz, 1H), 6.56 (dd, J = 8.6, 2.6 Hz, 1H),
6.45 (d, J = 5.2 Hz, 1H), 4.13 (m, 4H), 3.94 (s, 3H), 3.79 (d, J = 5.1 Hz, 6H), 3.66 (m, 2H), 2.33 (m, 1H), 1.98 (t, J
= 7.3 Hz, 2H), 1.81 (t, J = 7.4 Hz, 2H), 1.54 (t, J = 7.6 Hz, 2H), 1.49 – 1.42 (m, 2H), 1.35 (dt, J = 13.9, 6.5 Hz, 2H).
13C NMR (101 MHz, DMSO-d6) δ 171.37 , 169.60 , 168.15 , 160.42 , 160.24 , 156.06 , 152.37 , 149.93 , 149.78 ,
149.23 , 146.88 , 136.99 , 129.54 , 121.95 , 121.29 , 120.32 , 115.50 , 108.91 , 105.36 , 103.45 , 99.90 , 99.62 , 68.70
, 56.22 , 55.89 , 49.75 , 49.06 , 48.57 , 32.71 , 28.87 , 28.80 , 25.75 , 25.55 , 22.50 .
4.1.12.2. 1-(2-fluorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
2-oxopyrrolidine-3-carboxamide (15b)

White solid; Yield: 46.4%; M.p.: 138-139 ºC; MS (ESI) m/z(%): 631.14 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.50 (s, 1H), 10.35 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 11.9
Hz, 1H), 7.57 – 7.42 (m, 3H), 7.38 (s, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.08 – 6.95 (m, 1H), 6.45 (d, J = 5.2 Hz, 1H),
4.14 (t, J = 6.4 Hz, 2H), 3.94 (s, 5H), 3.83 (t, J = 8.6 Hz, 1H), 2.48 – 2.26 (m, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.81 (t,
J = 7.3 Hz, 2H), 1.53 (q, J = 7.7 Hz, 2H), 1.46 (t, J = 7.7 Hz, 2H), 1.35 (q, J = 7.7 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 171.49 , 169.60 , 168.05 , 160.41 , 152.39 , 149.94 , 149.25 , 146.92 , 141.21 , 136.84 , 130.95 , 130.86
, 121.96 , 121.46 , 115.63 , 115.29 , 111.49 ,111.28 , 108.95 , 107.09 , 106.83 , 103.52 , 99.65 , 68.72 , 56.22 , 51.74
, 47.32 , 32.73 , 28.84 , 28.81 , 25.73 , 25.55 , 21.77 .
4.1.12.3.
N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-2-oxo-1-
phenylpyrrolidine-3-carboxamide (15c)
Light yellow solid; Yield: 67.3%; M.p.: 132-134 ºC; MS (ESI) m/z(%): 613.15 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.85 – 7.73 (m, 2H), 7.69 (d, J =
8.1 Hz, 2H), 7.51 (s, 1H), 7.40 (dd, J = 14.5, 6.5 Hz, 3H), 7.30 – 7.22 (m, 2H), 7.18 (t, J = 7.4 Hz, 1H), 6.45 (d, J =
5.2 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H), 4.02 – 3.85 (m, 5H), 3.80 (t, J = 8.6 Hz, 1H), 2.48 – 2.30 (m, 2H), 1.97 (t, J =
7.3 Hz, 2H), 1.81 (t, J = 7.4 Hz, 2H), 1.58 – 1.50 (m, 2H), 1.46 (t, J = 7.2 Hz, 2H), 1.35 (d, J = 7.2 Hz, 2H).
4.1.12.4.
1-(3,4-difluorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-
yl)oxy)phenyl)-2-oxopyrrolidine-3-carboxamide (15d)
1
Yellow solid; Yield: 39.0%; M.p.: 114-116 C; MS (ESI) m/z (%): 649.19 [M+H]+. H NMR (400 MHz,
DMSO-d6) δ 10.50 (s, 1H), 10.36 (s, 1H), 8.69 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.90 (ddd, J = 14.2, 7.5, 2.3 Hz,
1H), 7.80 (d, J = 8.6 Hz, 2H), 7.56 – 7.43 (m, 3H), 7.38 (s, 1H), 7.26 (d, J = 8.5 Hz, 2H), 6.45 (d, J = 5.3 Hz, 1H),
4.13 (q, J = 5.6, 4.7 Hz, 2H), 4.03 – 3.87 (m, 5H), 3.82 (t, J = 8.5 Hz, 1H), 2.43 (dd, J = 15.7, 6.8 Hz, 2H), 1.98 (t,
J = 7.3 Hz, 2H), 1.81 (t, J = 7.3 Hz, 2H), 1.53 (q, J = 7.6 Hz, 2H), 1.50 – 1.42 (m, 2H), 1.35 (q, J = 7.7 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 171.38 , 169.58 , 168.04 , 160.41 , 152.42 , 149.94 , 149.29 , 146.92, 136.80 , 136.59
, 136.49 , 121.94 , 121.51 , 117.97 , 117.79 , 116.47 , 115.54 , 109.53 , 109.28 , 108.92 , 103.56 , 99.63 , 68.71 ,
56.20 , 51.54 , 47.43 , 32.71 , 28.84 , 28.82 , 25.76 , 25.55 , 21.70 .
4.1.12.5.
N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-2-oxo-1-(4-
(trifluoromethoxy)phenyl)pyrrolidine-3-carboxamide (15e)
White solid; Yield: 70.1%; M.p.: 171-172 ºC; MS (ESI) m/z(%): 697.07 [M+H]+;1H NMR (400 MHz, DMSO-
d6) δ 10.51 (s, 1H), 10.36 (s, 1H), 8.69 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.81 (dd, J = 8.7, 5.9 Hz, 4H), 7.52 (s, 1H),
7.48 – 7.33 (m, 3H), 7.26 (d, J = 8.2 Hz, 2H), 6.46 (d, J = 5.1 Hz, 1H), 4.14 (t, J = 6.4 Hz, 2H), 3.94 (m, 5H), 3.83

(m, 1H), 2.44 (d, J = 25.6 Hz, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.81 (t, J = 7.3 Hz, 2H), 1.55 (t, J = 7.4 Hz, 2H), 1.50 –
1.41 (m, 2H), 1.35 (d, J = 7.1 Hz, 2H).
4.1.12.6. 1-(4-fluorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
2-oxopyrrolidine-3-carboxamide (15f)
White solid; Yield: 62.2%; M.p.: 114-115 ºC; MS (ESI) m/z(%):631.21 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.48 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.74 – 7.65 (m,
2H), 7.51 (s, 1H), 7.38 (s, 1H), 7.25 (dt, J = 8.8, 4.5 Hz, 4H), 6.45 (d, J = 5.3 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H), 3.93
(d, J = 8.9 Hz, 5H), 3.79 (t, J = 8.6 Hz, 1H), 2.48 – 2.30 (m, 2H), 1.97 (t, J = 7.3 Hz, 2H), 1.81 (t, J = 7.3 Hz, 2H),
1.53 (q, J = 7.6 Hz, 2H), 1.46 (t, J = 7.6 Hz, 2H), 1.35 (q, J = 8.0 Hz, 2H).
4.1.12.7.
N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-1-(2-
methoxyphenyl)-2-oxopyrrolidine-3-carboxamide (15g)
1
Yellow solid; Yield: 64.1%; M.p.: 105-106 C; MS (ESI) m/z (%): 643.24 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 10.43 (s, 1H), 10.36 (s, 1H), 8.69 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.52 (s,
1H), 7.38 (s, 1H), 7.37 – 7.29 (m, 1H), 7.25 (dd, J = 8.3, 6.6 Hz, 3H), 7.13 (d, J = 8.2 Hz, 1H), 7.06 – 6.92 (m, 1H),
6.45 (d, J = 5.2 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 3.81 (s, 3H), 3.76 – 3.65 (m, 3H), 2.60 – 2.52 (m,
1H), 2.36 (d, J = 8.0 Hz, 1H), 1.98 (t, J = 7.3 Hz, 2H), 1.81 (t, J = 7.2 Hz, 2H), 1.59 – 1.51 (m, 2H), 1.47 (dq, J =
15.0, 8.4, 7.6 Hz, 2H), 1.35 (q, J = 7.7 Hz, 2H).
4.1.12.8.
N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-1-(4-
isopropylphenyl)-2-oxopyrrolidine-3-carboxamide (15h)
White solid; Yield: 55.5%; M.p.: 148-150 ºC; MS (ESI) m/z(%): 655.27 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.47 (s, 1H), 10.35 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 4.0 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.66 – 7.56 (m,
2H), 7.52 (d, J = 2.6 Hz, 1H), 7.38 (s, 1H), 7.26 (p, J = 3.4 Hz, 4H), 6.46 (dd, J = 5.1, 2.6 Hz, 1H), 4.13 (d, J = 7.0
Hz, 2H), 3.93 (dd, J = 12.3, 5.2 Hz, 5H), 3.78 (m, 1H), 2.88 (m, 1H), 2.42 (m, 2H), 1.97 (d, J = 7.8 Hz, 2H), 1.88 –
1.70 (m, 2H), 1.50 (dt, J = 31.5, 7.6 Hz, 4H), 1.35 (m, 2H), 1.23 – 1.13 (m, 6H).
4.1.12.9.
1-(3-chloro-4-fluorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-
yl)oxy)phenyl)-2-oxopyrrolidine-3-carboxamide (15i)
Light yellow solid; Yield: 61.5%; M.p.: 148-149 ºC; MS (ESI) m/z(%):665.13 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) δ 10.50 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 7.99 (dd, J = 6.9, 2.7 Hz, 1H), 7.79
(d, J = 8.5 Hz, 2H), 7.67 (d, J = 9.0 Hz, 1H), 7.49 (dd, J = 16.4, 6.8 Hz, 2H), 7.38 (s, 1H), 7.26 (d, J = 8.5 Hz, 2H),
6.45 (d, J = 5.3 Hz, 1H), 4.14 (t, J = 6.7 Hz, 2H), 3.93 (d, J = 10.4 Hz, 5H), 3.81 (m, 1H), 2.43 (d, J = 17.0 Hz, 2H),

1.97 (t, J = 7.3 Hz, 2H), 1.81 (m, 2H), 1.61 – 1.51 (m, 2H), 1.46 (m, 2H), 1.38 – 1.31 (m, 2H).
4.1.12.10. 1-(4-ethylphenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
2-oxopyrrolidine-3-carboxamide (15j)
White solid; Yield: 52.4%; M.p.: 134-135 ºC; MS (ESI) m/z(%): 641.25 [M+H]-; 1H NMR (600 MHz, DMSO-
d6) δ 10.49 (s, 1H), 10.36 (s, 1H), 8.69 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.62 – 7.55 (m,
2H), 7.52 (s, 1H), 7.38 (s, 1H), 7.25 (dd, J = 12.1, 8.5 Hz, 4H), 6.45 (d, J = 5.2 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H),
3.99 – 3.84 (m, 5H), 3.78 (m, 1H), 2.59 (q, J = 7.6 Hz, 2H), 2.49 – 2.34 (m, 2H), 1.98 (t, J = 7.4 Hz, 2H), 1.81 (t, J
= 7.4 Hz, 2H), 1.53 (q, J = 7.5 Hz, 2H), 1.46 (q, J = 7.7 Hz, 2H), 1.35 (q, J = 8.1 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H).
4.1.12.11.
N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-2-oxo-1-(2-
(trifluoromethoxy)phenyl)pyrrolidine-3-carboxamide (15k)
Yellow solid; Yield: 47.3%; M.p.: 111-112 C; MS (ESI) m/z (%): 697.20 [M+H]+;1H NMR (400 MHz,
DMSO-d6) δ 10.45 (s, 1H), 10.35 (s, 1H), 8.68 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.85 – 7.72 (m, 2H), 7.60 – 7.43
(m, 5H), 7.38 (s, 1H), 7.31 – 7.19 (m, 2H), 6.45 (d, J = 5.2 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 3.87 –
3.77 (m, 2H), 3.73 (t, J = 8.5 Hz, 1H), 2.57 (ddd, J = 10.7, 7.6, 3.9 Hz, 1H), 2.48 – 2.34 (m, 1H), 1.97 (t, J = 7.3
Hz, 2H), 1.81 (p, J = 6.7 Hz, 2H), 1.54 (p, J = 7.5 Hz, 2H), 1.45 (q, J = 7.4 Hz, 2H), 1.35 (q, J = 7.7, 7.0 Hz, 2H).
4.1.12.12. 1-(4-chlorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
2-oxopyrrolidine-3-carboxamide (15l)
White solid; Yield: 73.5%; M.p.: 134-136 ºC; MS (ESI) m/z(%): 647.08 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.49 (s, 1H), 10.35 (s, 1H), 8.67 (d, J = 1.8 Hz, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.79 (d, J = 9.0 Hz, 2H), 7.73
(d, J = 9.0 Hz, 2H), 7.51 (s, 1H), 7.47 (d, J = 8.9 Hz, 2H), 7.38 (s, 1H), 7.26 (d, J = 9.0 Hz, 2H), 6.45 (d, J = 5.3
Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H), 4.00 – 3.85 (m, 5H), 3.80 (t, J = 8.6 Hz, 1H), 2.44 (d, J = 8.2 Hz, 2H), 1.97 (t, J
= 7.3 Hz, 2H), 1.81 (t, J = 7.4 Hz, 2H), 1.58 – 1.50 (m, 2H), 1.45 (m, 2H), 1.34 (m, 2H).
4.1.12.13. 1-(4-bromophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
2-oxopyrrolidine-3-carboxamide (15m)
White solid; Yield: 66.5%; M.p.: 188-189 ºC; MS (ESI) m/z(%): 691.05 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.51 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.6
Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.51 (s, 1H), 7.38 (s, 1H), 7.26 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 5.2 Hz, 1H), 4.14
(t, J = 6.5 Hz, 2H), 3.94 (m, 5H), 3.81 (m, 1H), 2.47 – 2.24 (m, 2H), 1.97 (t, J = 7.3 Hz, 2H), 1.81 (t, J = 7.3 Hz,
2H), 1.50 (dt, J = 31.1, 7.5 Hz, 4H), 1.35 (m, 2H).
4.1.12.14. 1-(3-fluorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-

2-oxopyrrolidine-3-carboxamide (15n)
1
Yellow solid; Yield:72.5%; M.p.: 175-177 ºC; MS (ESI) m/z(%): 631.08 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 8.45 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 10.9 Hz,
2H), 7.35 (d, J = 14.9 Hz, 3H), 7.26 (d, J = 8.3 Hz, 3H), 6.45 (d, J = 5.3 Hz, 1H), 4.13 (m, 2H), 3.93 (s, 3H), 3.85
(d, J = 8.6 Hz, 2H), 3.75 (m, 1H), 2.43 (m, 1H), 1.97 (m, 2H), 1.80 (m, 2H), 1.53 (m, 2H), 1.46 (m, 2H), 1.34 (m,
2H).
4.1.12.15. 1-(2-bromophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
2-oxopyrrolidine-3-carboxamide (15o)
White solid; Yield: 84.2%; M.p.: 113-114 ºC; MS (ESI) m/z(%): 691.12 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.47 (s, 1H), 10.36 (s, 1H), 8.68 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.87 – 7.68 (m, 3H), 7.56 – 7.46 (m, 2H),
7.43 (dd, J = 7.9, 1.8 Hz, 1H), 7.41 – 7.29 (m, 2H), 7.29 – 7.17 (m, 2H), 6.45 (d, J = 5.2 Hz, 1H), 4.13 (t, J = 6.5
Hz, 2H), 3.94 (s, 3H), 3.83 – 3.67 (m, 3H), 2.61 (dd, J = 12.7, 8.2 Hz, 1H), 2.47 – 2.36 (m, 1H), 1.97 (t, J = 7.3 Hz,
2H), 1.81 (t, J = 7.3 Hz, 2H), 1.59 – 1.50 (m, 2H), 1.50 – 1.41 (m, 2H), 1.39 – 1.27 (m, 2H).
4.1.12.16.
N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-2-oxo-1-(m-
tolyl)pyrrolidine-3-carboxamide (15p)
White solid; Yield: 83.1%; M.p.: 147-149 ºC; MS (ESI) m/z(%): 627.17 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 10.46 (s, 1H), 10.34 (s, 1H), 8.70 – 8.60 (m, 1H), 8.46 (d, J = 5.3 Hz, 1H), 7.79 (d, J = 9.0 Hz, 2H), 7.56 –
7.42 (m, 3H), 7.38 (s, 1H), 7.27 (dd, J = 14.4, 8.4 Hz, 3H), 7.00 (d, J = 7.5 Hz, 1H), 6.45 (d, J = 5.2 Hz, 1H), 4.14
(t, J = 6.7 Hz, 2H), 4.01 – 3.85 (m, 5H), 3.78 (t, J = 8.6 Hz, 1H), 2.48 – 2.35 (m, 2H), 2.33 (s, 3H), 1.97 (t, J = 7.3
Hz, 2H), 1.81 (p, J = 6.4 Hz, 2H), 1.50 (dp, J = 30.3, 7.4 Hz, 4H), 1.40 – 1.28 (m, 2H).
4.1.12.17. N-(3-fluoro-4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-2-oxo-1-
phenylpyrrolidine-3-carboxamide (15q)
Yellow green solid; Yield: 80.2%; M.p.: 160-162 ºC; MS (ESI) m/z(%): 631.21 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) δ 10.73 (s, 1H), 10.37 (s, 1H), 8.69 (s, 1H), 8.50 (d, J = 5.4 Hz, 1H), 7.95 (dd, J = 12.9, 2.3 Hz, 1H),
7.69 (d, J = 8.1 Hz, 2H), 7.59 – 7.32 (m, 6H), 7.19 (t, J = 7.4 Hz, 1H), 6.52 (d, J = 5.3 Hz, 1H), 4.15 (t, J = 6.5 Hz,
2H), 4.04 – 3.86 (m, 5H), 3.82 (t, J = 8.6 Hz, 1H), 2.49 – 2.33 (m, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.81 (p, J = 6.9 Hz,
2H), 1.54 (p, J = 7.6 Hz, 2H), 1.46 (m, 2H), 1.35 (q, J = 7.9 Hz, 2H).
4.1.12.18. 1-(2-fluorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
3-methyl-2-oxopyrrolidine-3-carboxamide (15r)
White solid; Yield: 81.3%; M.p.: 104-106 ºC; MS (ESI) m/z(%): 645.10 [M+H]+;1H NMR (600 MHz, DMSO-

d6) δ 10.36 (s, 1H), 9.75 (s, 1H), 8.73 – 8.64 (m, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.73 (dt, J
= 11.9, 2.3 Hz, 1H), 7.51 (m, 2H), 7.47 (dd, J = 8.3, 6.8 Hz, 1H), 7.38 (s, 1H), 7.24 (d, J = 8.7 Hz, 2H), 7.03 (td, J
= 8.3, 2.5 Hz, 1H), 6.44 (d, J = 5.2 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 3.90 (ddd, J = 14.5, 8.2, 5.9 Hz,
2H), 2.78 – 2.70 (m, 1H), 2.12 – 2.04 (m, 1H), 1.98 (t, J = 7.4 Hz, 2H), 1.81 (t, J = 7.5 Hz, 2H), 1.59 (s, 3H), 1.53
(q, J = 7.5 Hz, 2H), 1.50 – 1.41 (m, 2H), 1.35 (q, J = 8.3 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 174.54 , 170.36
, 169.61 , 160.39 , 152.37 , 150.09 , 149.91 , 149.24 , 146.88 , 141.28 ,136.56 , 130.92 , 130.86 ,122.49 , 121.74 ,
115.73 , 115.52 , 111.51 , 108.90 , 107.20 , 107.03 , 99.60 , 68.66 , 56.19 , 55.37 , 54.37 , 45.75 , 32.70 , 30.02 ,
28.81 , 25.76 , 25.55 , 21.53 .
4.1.12.19. N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-3-methyl-2-oxo-1-
phenylpyrrolidine-3-carboxamide (15s)
Yellow solid; Yield: 80.6%; M.p.:133-134 ºC; MS (ESI) m/z(%): 627.17 [M+H]+;1H NMR (600 MHz, DMSO-
d6) δ 10.38 (s, 1H), 9.77 (s, 1H), 8.72 (s, 1H), 8.45 (d, J = 5.1 Hz, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 7.8 Hz,
2H), 7.51 (s, 1H), 7.42 (t, J = 7.6 Hz, 2H), 7.38 (s, 1H), 7.24 (d, J = 8.2 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 6.44 (d, J
= 4.9 Hz, 1H), 4.13 (t, J = 6.4 Hz, 2H), 4.04 – 3.74 (m, 5H), 2.75 (dd, J = 13.9, 7.5 Hz, 1H), 2.08 (dd, J = 12.6, 6.1
Hz, 1H), 1.98 (t, J = 7.3 Hz, 2H), 1.81 (t, J = 7.3 Hz, 2H), 1.59 (s, 3H), 1.54 (t, J = 7.4 Hz, 2H), 1.46 (m, 2H), 1.35
(m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 174.18 , 170.50 , 169.58 , 160.38 , 152.39 , 150.09 , 149.93 , 149.23 ,
146.92 ,139.72 , 136.56 , 129.17 , 125.00 , 122.36 , 121.71 , 120.30 , 115.50 , 108.97 , 103.53 , 99.65 , 68.69 , 56.19
, 54.03 , 45.68 , 32.71 , 30.02 , 28.84 , 28.81 , 25.77 , 25.54 , 21.79 .
4.1.12.20. 1-(4-chlorophenyl)-N-(4-((7-((7-(hydroxyamino)-7-oxoheptyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-
3-methyl-2-oxopyrrolidine-3-carboxamide (15t)
White solid; Yield: 82.3%; M.p.: 139-141 ºC; MS (ESI) m/z(%): 661.03 [M+H]+; 1H NMR (600 MHz, DMSO-
d6) δ 10.36 (s, 1H), 9.75 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 5.3 Hz, 1H), 7.78 (dd, J = 16.9, 8.5 Hz, 4H), 7.51 (s, 1H),
7.48 (d, J = 8.6 Hz, 2H), 7.38 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.44 (d, J = 5.2 Hz, 1H), 4.14 (t, J = 6.5 Hz, 2H),
3.94 (s, 3H), 3.92 – 3.80 (m, 2H), 2.79 – 2.67 (m, 1H), 2.08 (t, J = 5.1 Hz, 1H), 1.97 (t, J = 7.3 Hz, 2H), 1.80 (t, J =
7.4 Hz, 2H), 1.58 (s, 3H), 1.53 (m, 2H), 1.45 (q, J = 7.7 Hz, 2H), 1.34 (td, J = 8.3, 4.2 Hz, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 174.35 , 170.44 , 169.63 , 160.40 , 152.41 , 150.11 , 149.90 , 149.22 , 146.88 , 138.63 , 136.63 , 129.15
, 128.72 , 122.43 , 121.79 , 115.54 , 108.90 , 103.55 , 99.59 , 68.69 , 56.19 , 54.15 , 45.68 , 32.71 , 30.06 , 28.85 ,
28.82 , 25.77 , 25.56 , 21.59 .
4.2. Pharmacology
4.2.1. In vitro antiproliferative assays

The antiproliferative activities of compounds 15a-15t were evaluated against HCT-116, MCF-7 and A549 cell
lines by the standard MTT assay in vitro, with SAHA, Cabozantinib and Foretinib as positive controls. The cancer
cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS).
Approximate 4 × 103 cells, suspended in MEM medium, were plated into each well of a 96-well plate and incubated
in 5% CO2 at 37 C for 24 h. The tested compounds at the indicated final concentrations were added to the culture
medium and incubated for 72 h. Fresh MTT was added to each well at the terminal concentration of 5 μg/mL, and
incubated with cells at 37 C for 4 h. The formazan crystals in each well were dissolved in 100 μL DMSO, and the
absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured
with an ELISA reader. All of the compounds were tested three times in each of the cell lines. The results, expressed
as IC50 (inhibitory concentration 50%), were the averages of three determinations and calculated relative to the
vehicle (DMSO) control by the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
4.2.2 In vitro enzymatic assays
4.2.2.1 In vitro HDAC1 enzymatic assays
The following materials were purchased : HDAC1 (BPS, Cat. No. 50051), 384-well plate (Perkin Elmer, Cat.
No. 6007279). The compounds were dissolved into 10 mM stock in 100% DMSO.
4.2.2.1.1 Experimental Methods
Prepare 1x assay buffer (modified Tris Buffer), then transfer compounds to assay plate by Echo in 100%
DMSO. The final fraction of DMSO is 1%. Then we need to prepare enzyme solution in 1x assay buffer. Adding
trypsin and Ac-peptide substrate in 1x assay buffer to make the substrate solution. Transfer 15 μL of enzyme solution
to assay plate or for low control transfer 15 μL of 1x assay buffer. Reactions were incubated for 15 min at room
temperature and added 10 μL of substrate solution to each well to start reaction. Read the plate on Synergy MX
with excitation at 355 nm and emission at 460 nm after incubating for another 60 min at room temperature.
4.2.2.1.2 Curve fitting
Fit the data in Excel to obtain inhibition values using equation (1)
Equation (1): Inh %=( Max-Signal)/ (Max-Min)*100
Fit the data in XL-Fit to obtain IC50 values using equation (2)
Equation (2): Y=Bottom + (Top-Bottom)/(1+(IC50/X)*HillSlope)
Y is %inhibition and X is compound concentration.
4.2.2.2 In vitro c-Met enzymatic assays
The target compounds were tested for their activity against c-Met Tyrosine kinases through the mobility shift

assay [38-39]. All kinase assays were performed in 96-well plates in a 50 µL reaction volume. The kinase buffer
contains 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer contains 100
mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent 3 and 50 mM EDTA. Dilute the compounds to 500
µM by 100% DMSO, then transfer 10 µL of compound to a new 96-well plate as the intermediate plate, add 90 µL
kinase buffer to each well. Transfer 5 µL of each well of the intermediate plate to 384-well plates. The following
amounts of enzyme and substrate were used per well: kinase base buffer, FAM-labeled peptide, ATP and enzyme
solution. Wells containing the substrate, enzyme, DMSO without compound were used as DMSO control. Wells
containing just the substrate without enzyme were used as low control. Incubate at room temperature for 10 min.
Add 10 µL peptide solution to each well. Incubate at 28 C for specified period of time and stop reaction by 25 µL
stop buffer. At last collect data on Caliper program and convert conversion values to inhibition values. Percent
inhibition = (max − conversion)/(max − min)×100. ‘max’ stands for DMSO control; ‘min’ stands for low control.
4.2.3. Flow cytometry
The HCT-116 cells were seeded in 6-well plates at a seeding density of 105 cells per mL. Twelve hours later,
various concentrations of compound 15f were added. Cells were treated with compound 15f for 48 h. Then cells
were transferred to EP tubes and washed three times with PBS buffer. Then the procedures according to the
operating instructions of the kit were followed. Ultimately, cell apoptosis was analyzed using Annexin-V and
propidium iodide (PI) double staining by flow cytometry. Early apoptotic cells were defined as Annexin-V
positive/PI negative, late apoptotic cells as Annexin-V/PI-double positive and necrotic cells as Annexin-V
positive/PI positive.
4.2.4. Cell cycle distribution analysis
The effects of compounds on cell cycle progression were determined using a standard propidium iodide (PI)
staining procedure followed by flow cytometry analysis. Briefly, HCT-116 cells were seeded in six-well plates
(5×104/well) and then treated with different concentrations of 15f for 48 h. The cells were collected and washed
twice with ice cold PBS, then fixed in ice-cold 70% (v/v) ethanol overnight at 4 C. The cells were washed again
by PBS, and then the cell DNA was stained with 400 μL PI (Beyotime) for 10 min. Data acquisition and analysis
were performed using a flow cytometer.
4.2.5. Molecular docking study
The crystal structure of c-Met (PDB entry code: 3LQ8) in complex with XL880 and HDACs (PDB entry code:
1C3S) in complex with SAHA were used for molecular modeling. The protein structures were prepared using the
protein preparation wizard in Maestro with standard settings. Grids were generated using glide, version 4.5.208,

following the standard procedure recommended by Schrödinger software package version 2014. The conformational
ensembles were docked flexibly using Glide with standard settings in both standard and extra precision mode. Only
poses with low energy conformations and good hydrogen bond geometries were considered. The pictures
elucidating the protein-ligand interactions were produced by Pymol (version 1.7.2.1).
Notes
The authors declare no competing financial interest.
Acknowledgements
This work was supported by grant (No. 81573295) from the National Natural Science Foundation of
China and LiaoNing Revitalization Talents Program (XLYC1808037).
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


A series of novelc-Met/HDAC Dual Inhibitors were designed, synthesized.
15f showed better cytotoxicity against tested cells and comparative enzymatic inhibitory than positive control.
Cell apoptosis study revealed 15f is very effective in the induction of apoptosis in a dose-dependent manner in
HCT-116 cells.


Cell cycle analysis showed that 15f significantly caused G2/M-phase arrest in HCT-116 cells.
Docking mode indicated 15f could form critical bonding interactions with c-Met and HDAC1.