Design, synthesis and preliminary bioactivity evaluations of 8-hydroxyquinoline derivatives as matrix metalloproteinase (MMP) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111563

Matrix metalloproteinases (MMPs) play important roles in many diseases including cancer. With moderate metal-binding affinity, 8-hydroxyquinoline has gained much interest in current drug design and development. Specially, it has been reported that 8-hydroxyquinoline derivatives serve as MMP-2 inhibitors with micromolar IC₅₀ values. In the current study, a series of 8-hydroxyquinoline derivatives were designed and synthesized as new MMP-2 and MMP-9 inhibitors. The most active compounds 5e and 5h not only displayed good inhibitory activities against MMP-2/9 with IC₅₀ at submicromolar level, but also possessed potent anti-proliferative, anti-invasive and anti-angiogenesis activity in A549 cell line. Western blot also revealed that 5e and 5h down-regulate the expression of MMP-2 and MMP-9 in A549 cell line. Moreover, flow cytometry analysis indicated that compound 5e could promote apoptosis of A549 cells in vitro. Molecular docking analysis also revealed favorable binding modes of 5e in the active sites of MMP-2 and MMP-9.

Full text of DOI:10.1016/j.ejmech.2019.111563

Accepted Manuscript
Design, synthesis and preliminary bioactivity evaluations of 8-hydroxyquinoline
derivatives as matrix metalloproteinase (MMP) inhibitors
Chen Chen, Xinying Yan, Hao Fang, Xuben Hou
PII:
S0223-5234(19)30687-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111563
Article Number: 111563
Reference:
EJMECH 111563
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2019
Revised Date: 23 July 2019
Accepted Date: 23 July 2019
Please cite this article as: C. Chen, X. Yan, H. Fang, X. Hou, Design, synthesis and preliminary
bioactivity evaluations of 8-hydroxyquinoline derivatives as matrix metalloproteinase (MMP) inhibitors,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111563.
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Design, synthesis and preliminary bioactivity evaluations of
8-hydroxyquinoline derivatives as matrix metalloproteinase
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(MMP) inhibitors
Chen Chen a, Xinying Yang a, Hao Fang a, Xuben Hou a, *
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Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
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School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012, Jinan,
Shandong, PR China
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*Address correspondence to this author:
Prof. Xuben Hou, Department of Medicinal Chemistry, School of Pharmaceutical Sciences
Shandong University, 44 West Wenhua Road, Jinan, PR China.
E-mail: hxb@sdu.edu.cn
Tel. 86-531-88382009
Fax. 86-531-88382548
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Abstract：Matrix metalloproteinases (MMPs) play important roles in many diseases including
cancer. With moderate metal-binding affinity, 8-hydroxyquinoline has gained much interest in
current drug design and development. Specially, it has been reported that 8-hydroxyquinoline
derivatives serve as MMP-2 inhibitors with micromolar IC50 values. In the current study, a series
of 8-hydroxyquinoline derivatives were designed and synthesized as new MMP-2 and MMP-9
inhibitors. The most active compounds 5e and 5h not only displayed good inhibitory activities
against MMP-2/9 with IC50 at submicromolar level, but also possessed potent anti-proliferative,
anti-invasive and anti-angiogenesis activity in A549 cell line. Western blot also revealed that 5e
and 5h down-regulate the expression of MMP-2 and MMP-9 in A549 cell line. Moreover, flow
cytometry analysis indicated that compound 5e could promote apoptosis of A549 cells in vitro.
Molecular docking analysis also revealed favorable binding modes of 5e in the active sites of
MMP-2 and MMP-9.
Keywords:
8-hydroxyquinoline
MMP-2/9 inhibitors
Anti-migration/invasion
Anti-angiogenesis
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1. Introduction
Matrix metalloproteases (MMPs) are zinc-dependent endopeptidases that proteolytically
degrade various components of the extracellular matrix (ECM). To date, at least 24 MMPs have
been identified in human, which could be classified into five subfamilies based on their
specificities [1]. Among various MMPs, MMP-2 and MMP-9 (also known as gelatinase A and B)
serve as important gelatinases. The structures of both MMP-2 and MMP-9 consist of an
N-terminal predomain, a prodomain and a catalytic domain with a zinc-binding site (Fig. 1). The
predomain could guide gelatinases to the endoplasmic reticulum and the prodomain could
maintain the latency of the gelatinases. The catalytic domain of MMP-2 and MMP-9 also
comprises three repeatedly collagen-binding type II of fibronectin (Fi) inserts.
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Fig. 1. Structures of MMP-2 and MMP-9.
MMP-2 and MMP-9 play critical roles in tumor proliferation, migration, invasion and
angiogenesis. For example, MMP-2 and MMP-9 could induce the release of
cell-membrane-precursors of growth factors, such as insulin-like growth factors (IGFs) and
epidermal growth factor receptor (EGFR) ligands, which promote tumor proliferation [2].
Moreover, MMP-2 and MMP-9 could regulate proliferation signals by shedding and activating the
transforming growth factor TGF-β [3]. The ECM is a natural barrier that prevents tumor cell
invasion and metastasis [4]. Since MMP-2 and MMP-9 exert proteolytic activities and degrade
physical barriers, developing MMP-2/9 inhibitors should prevent tumor cell invasion and
metastasis. During tumor angiogenesis progression, MMP-2 and MMP-9 play critical roles through
participating in the degradation of the vascular basement membrane and remodeling of the ECM [5].
MMP-2 and MMP-9 are also known as "angiogenic switches" which promote tumor vascularization
[6]. Expression of both MMP-2 and MMP-9 are upregulated in angiogenic lesions. MMP-9 can
release VEGF to render normal islets angiogenic [7]. When the MMP-2 expression is
down-regulated by antisense oligonucleotides, tumor cells lose their angiogenic ability and tumor
growth is inhibited [8]. It had been reported that MMP inhibitors could induce cell apoptosis [9,
10] and down-regulate MMP-2 or MMP-9 expression [9]. For example, previously reported
MMP-2/9 inhibitor could interact synergistically with ligands of the TNF receptor superfamily to
induce apoptosis in a cell-type-specific manner [11]. Therefore, inhibition of MMP-2/9 serves as a
potentially valuable strategy for anti-cancer drug design and development.
Hydroxamic acids derivatives are the most common MMPs inhibitors, however, several
hydroxamate-based MMP inhibitors failed in clinical trial due to low isoform selectivity and
various toxicities, such as joint stiffness, swelling and arthralgia[12]. Currently, development of
non-hydroxamate inhibitors against specific MMPs is of foremost interest. 8-Hydroxyquinoline is
an important scaffold in drug development [13] and its derivatives (e.g. Clioquinol) have already
been used in clinical therapy [14]. Previously, Jacobsen and coworkers screened hundreds of
fragments and found that 8-hydroxyquinoline possessed inhibitory activity against MMPs [15].
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Further structural modification suggested that aromatic substitutions at either the 5- or 7-positions
of 8-hydroxyquinoline could yield MMP-2 inhibitors (Compound A and B in Fig. 2).
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Fig. 2. Reported MMP inhibitors.
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As far as the structure of MMPIs is concerned, it should contain a zinc binding group and a
chemical moiety protruding into the deep and hydrophobic S1’ pocket [16]. For instance, known
MMP inhibitor NNGH (Fig.2, MMP-8, -9, -12, -13, and -20 with Ki values of 9, 2.6, 4.3, 3.1, and
17 nM, respectively) mainly interacts with the S1’ pocket[17]. Docking analysis (Fig. 3)
suggested that 8-hydroxyquinoline chelated the zinc ion and lead compound B is surrounded by
less occupied pockets (e.g. S1’ pocket and S1 pocket). Inspired by these analyses above, we
designed a new series of MMP inhibitors using 8-hydroxyquinoline as the zinc binding group.
Flexible linkers with different lengths were introduced between 8-hydroxyquinoline and aromatic
substitutions of quinoline to improve interactions with these pockets, especially interactions with
S1’ pocket. This paper describes the synthesis and evaluation of these compounds, as well as their
effects on cancer proliferation, migration, angiogenesis and apoptosis.
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Fig. 3. Predicted binding mode of compound B against MMP-2.
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2. Results and discussion
2.1. Chemistry
The synthetic routes of all target compounds are outlined in Schemes 1 and 2. As shown in
Scheme 1, commercially available starting materials 1a-1e were protected with a Boc group and
then coupled with different amines. Amides 3a-3j were converted to intermediates 4a-4j by
deprotection of the amine. Amines 4a-4j were synthesized according to literatures [18, 19]. Then,
compounds 4a-4j were reacted with 5-chloro-8-hydroxyquinoline-7-carbaldehyde or
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8-hydroxyquinoline-5-carbaldehyde via reductive amination to obtain target compounds 5a-5s. As
shown in Scheme 2, compound 7 was synthesized by protecting the OH group of 6. Compounds
9a-9f were afforded via 8a-f reacting with N-methylaniline, 2-methylbenzimidazole and saccharin
according to published literatures [20-22]. Intermediate 7 underwent a coupling reaction with 9a-f
to provide 10a-11i. Deprotection of the TBS group yielded the target compounds 11a-11i.
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Scheme 1. Synthesis of target compounds 5a-s. Reagents and conditions: (a) (Boc)2O, 1,4-dioxane,
1M aq NaOH, rt, 8h; (b) R1-NH2, EDCI, HOBT, DCM, rt, 6h; (c) HCl-AcOEt (d)
5-chloro-8-hydroxyquinoline-7-carbaldehyde or 8-hydroxyquinoline-5-carbaldehyde, MeOH, rt,
4h; NaBH4, rt, 1h.
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Scheme 2. Synthesis of target compounds 11a-i. Reagents and conditions: (a) TBSCl, imidazole,
DCM; (b) R3-H, K2CO3, DMF, rt or 120 ; (c) oxalyl chloride, DCM, rt, 1h; NaHCO3, DCM, rt,
5h; (d) HCl, MeOH.
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2.2. MMP-2 and MMP-9 inhibitory activity
The inhibitory activities against MMP-2 and MMP-9 of these quinoline derivatives were
determined using Colorimetric Drug Discovery Kit (BML-AK408, BML-AK410, Enzo®Life
Sciences). Known MMP inhibitors (Compound A [15], NNGH [23] and Prinomastat [12]) were
used as positive control. As shown in Table 1, target compounds with aromatic substitutions at the
5-positions of quinoline are ineffective against MMP-2 and MMP-9, with only 5m, 5o, 5s, 11c and
11d exhibiting obvious MMP inhibition (IC50 < 10 ꢀM). In contrast, compounds 5a-5j, which
possess aromatic substitutions at the 7-positions, showed good inhibitory activities against
MMP-2 and MMP-9. In addition, it seems that the various lengths of linkers have different
influence on MMP inhibitory activity. For compounds 5a-5e, 5e with five-methylene linker
displayed the most potent inhibitory activity against MMP-2. Among compounds 5f-5j, compound
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5h with three-methylene linker showed the best inhibitory activity with lowest IC50 values. These
results revealed the importance of a suitable linker length in the inhibition of MMPs.
It should be noted that 5e and 5h are inhibitors of both MMP-2 and MMP-9 with IC50 values
ranging from 0.66 ꢀM to 1.23 ꢀM. On the other hand, compound 11c and 11d displayed
selectivity of MMP-2 over MMP-9 (more than 7-fold). The IC50 values of 11c and 11d against
MMP-2 are 7.46 ꢀM and 6.22 ꢀM, whereas their inhibition ratios for MMP-9 are 35% and 26% at
50 ꢀM (Supplementary material Table S2).
Table. 1. IC50 values of target compounds against MMP-2 and MMP-9.
Cpd.
R1
n
MMP-2
MMP-9
IC50a(ꢀM)
2.1±0.33
3.5±0.01
3.2±0.09
3.6±0.21
0.66±0.01
Cpd.
R3
n
MMP-2
MMP-9
a
a
IC50 (ꢀM)
IC50 (ꢀM) IC50a(ꢀM)
Ph-
Ph-
Ph-
Ph-
Ph-
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3
4
5
1
2
3
4
5
1
2
3
4
5
6
2
3
4
3.2±0.52
3.3±0.01
3.8±0.48
3.0±0.57
0.70±0.02
8.8±0.34
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5
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3
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5
5a
5b
5c
5d
5e
5f
11a
11b
11c
11d
11e
11f
11g
11h
11i
>
>
10
10
>
>
>
>
>
>
>
>
>
>
>
>
10
10
10
10
10
10
10
10
10
10
10
10
7.5±0.60
6.2±0.83
N
N
>
>
>
>
>
>
>
>
10
10
10
10
10
10
10
10
N
N
>
>
10
10
N
N
5g
5h
5i
>
10
N
N
0.81±0.14
7.6±0.76
3.6±0.50
1.3±0.09
3.1±0.05
1.7±0.48
5j
11j
11k
11l
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
5k
5l
>
>
10
10
>
>
10
10
5.7±1.1
10
8.8±0.19
5.1±0.30
10
6.0±0.21
5m
5n
5o
5p
5q
5r
5s
A
>
>
>
>
>
10
10
10
>
>
10
10
>
10
8.8±0.19
8.8±0.21
5.3±0.14
11±0.47
0.0091±0.0021
0.0088±0.0016
NNGH
0.0018±0.00034 0.0033±0.00017
Prinomastat
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a IC50 were expressed as the mean ± standard deviation of three separate determinations
2.3. Molecular docking study
Molecular docking studies were performed to investigate the binding modes of our new
inhibitors in the active sites of MMP-2 and MMP-9. The most potent MMP-2/9 inhibitor
compound 5e displayed favorable interactions with both MMP-2 and MMP-9 (Fig. 4 and Fig. 5).
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In the case of MMP-2 (Fig. 4), the 8-hydroxyquinoline of 5e chelates the zinc ion through the N
atom and the OH group. Importantly, the 6-(methyleneamino)-N-phenylhexanamide of 5e inserted
into the hydrophobic S1’ pocket of MMP-2 enzyme formed by Leu82, Leu83, Tyr142, Ile141,
Pro140, Ala139 and Leu116. In addition, hydrogen bond interactions could be found between
compound 5e and amino acid residues at the enzyme active site (Ala139 and Arg149). Regarding
the docking result of 5e with MMP-9 (Fig. 5), the 8-hydroxyquinoline also chelates with zinc ion.
The side chain of 5e could enter into the hydrophobic S1’ pocket surrounded by amino acid
residues Leu397, Tyr420, Leu188 and Glu402. Hydrogen bonds with Glu402 of MMP-9 were
observed in the predicted 5e-MMP-9 complex. Some literatures reported that the big and
hydrophobic S1’ pocket was more important for MMP inhibition than the small S2’ pocket [16].
Compared with the predicted binding mode of compound B (Fig. 3), the improved MMPs
inhibition activity of 5e may come from its good interactions with the S1’ pockets.
Moreover, we predicted the binding modes of 5b and 5d in MMP-2. As shown in Fig. 6,
compounds 5b and 5d only partially occupy the S1’ pocket due to their shorter linkers. Moreover,
the interactions between 8-hydroxyquinoline and zinc ion in 5b and 5d are weaker than 5e (Fig. 6).
These results would be helpful to understand the better MMP inhibitory activity of 5e than 5b and
5d. Furthermore, we performed molecular docking of 11c with MMP-2 and MMP-9 to investigate
its isoform selectivity. According to the calculated docking scores, the predicted binding affinity of
11c with MMP-2 (docking score=7.32) is higher than MMP-9 (docking score=4.98), possibly due
to the good interactions between 11c and S1’ pocket in MMP-2 (Supplementary material, Figure
S1). This result may explain the reason why 11c was more potent for MMP-2 (IC50 =7.5 ꢀM) than
MMP-9 (IC50 >50ꢀM).
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Fig. 4. Predicted binding mode of compound 5e in MMP-2 enzyme (PDB: 1HOV).
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Fig. 5. Predicted binding mode of compound 5e in MMP-9 enzyme (PDB: 1GKC).
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Fig. 6. (A) Comparison of the predicted binding modes of 5e and 5b in MMP-2. (B) Comparison
of the predicted binding modes of 5e and 5d in MMP-2.
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2.4. In vitro anti-proliferative assay
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We further evaluated the anti-proliferative activities of 5a, 5b, 5d, 5c, 5d, 5e, 5h and 5j, which
possessed good MMP-2/9 inhibitory activities, using MTT assays. All of these target compounds
were screened against different cancer cell lines (HL-60, K562, KG1, A549, PC-3 and MCF-7)
along with human umbilical vein endothelial cells (HUVECs). The IC50 values were summarized
in Table 2. All compounds exhibited inhibitory activities against six cancer cell lines with
comparable or slightly improved anti-proliferative activities when compared to known MMP-2
inhibitor compound A. Interestingly, hematologic tumor cells (K562 and KG1) seem to be more
sensitive to our compounds than solid tumor cells (PC-3 and MCF-7). On the other hand,
hydroxamate-based MMP inhibitors NNGH exerted 29-187 ꢀM anti-proliferation activities for
different cancer cell lines and Prinomastat possessed no cytotoxicity in these cancer cell lines.
Table. 2. Anti-proliferative activities of 5a, 5b, 5c, 5d, 5e, 5h, 5j, A, NNGH and Prinomastat.
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Compound
IC50 （ꢀM）
A549
HL60
K562
KG1
PC-3
MCF-7
13±0.03
13±1.7
HUVEC
2.9±0.03
1.7±0.20
1.9±0.05
2.9±0.04
2.3±0.07
1.8±0.08
4.2±0.69
3.6±0.04
143±9.5
2.3±0.43
1.1±0.07
1.6±0.11
1.6±0.10
2.3±0.09
0.69±0.09
4.7±0.02
4.6±0.12
52±4.4
7.4±0.37
9.7±0.55
17±0.31
13±0.24
11±0.14
6.4±0.69
10±0.95
1.9±0.10
7.7±0.06
8.4±0.18
8.3±0.10
22±0.29
5a
>
5
>
>
50
50
1.3±0.09
1.9±0.04
1.9±0.19
1.8±0.11
4.0±0.57
2.6±0.12
4.6±0.03
29±1.1
5b
9.0±1.0
7.4±0.64
4.6±0.14
7.8±0.63
12±0.61
42±1.6
5c
>
50
5d
>
>
>
>
>
50
50
50
50
50
5e
5h
5j
>
25
>25
51±1.2
96±4.3
18±1.2
A
52±0.02
187±10
NNGH
Prinomastat
>
>
250
500
>
500
>
500
>
500
>
500
>
500
>500
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a IC50 were expressed as the mean ± standard deviation of three separate determinations
2.5. Transwell migration and invasion assay
Since MMP-2 and MMP-9 were associated with increased tumor migration and invasion in lung
cancer[24] and overexpression of MMP-2 and MMP-9 leads to poor prognosis in patients with
NSCLC[25]. We further evaluated the effects of most active inhibitors (5e and 5h) on tumor cell
migration and invasion using A549 cell line (lung cancer).
The migration assay was performed using transwell chambers where fetal bovine serum (FBS)
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acted as a chemoattractant. Briefly, A549 cells were incubated with compounds 5e, 5h and A for
24h. Then cells were seeded into the upper compartment of transwell chambers, whereas 10%
FBS was used as chemoattractant and placed in the lower compartment. After 24h incubation,
cells in the upper side of the filter were removed and cells that migrated to the lower surface were
fixed, stained, captured and counted.
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Results in Fig. 7 showed that addition of 1, 5 and 10 ꢀM of compounds 5e and 5h lead to an
inhibition of A549 migration as compared to known inhibitor compound A. Compound 5e display
the strongest inhibitory effect on A549 cells migration in a dose dependent manner.
The invasion assay is similar with migration assay and the only difference is that transwell
chambers are precoated with matrigel. Results in Fig. 8 showed that compounds 5e and 5h have
significant inhibitory effects on invasion of A549 cells as the concentration increases.
Furthermore, we compared anti-invasion activities of 5e, NNGH and Prinomastat. Although
compound 5e exhibited weaker MMP inhibitory activities than NNGH and Prinomastat (Table
1), compound 5e displayed similar anti-invasive activities to NNGH and better anti-invasive
activities than Prinomastat (Supplementary material, Figure S2). The LogP values of 5e,
Prinomastat and NNGH have been calculated (Supplementary material, Table S3). The results
found 5e to possess higher LogP values than Prinomastat and NNGH, which may lead to better
membrane permeability. This might be the reason that 5e displayed similar or slightly better
anti-invasive activities than reported inhibitors.
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control
5e
150
5h
A
100
&&
&&
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50
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&&
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**
**
0
1µM
5µM
10µM
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Fig. 7. Anti-migration activity of compounds against A549 cells at 1, 5 and 10 ꢀM, imaging (left)
and cell number (right) of migrated cells. All experiments were performed repeated three times.
Means ± SEM are shown (** p < 0.05)
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Fig. 8. Anti-invasion activity of compounds against A549 cells at 1, 5 and 10 ꢀM, imaging (left)
and cell number (right) of migrated cells. All experiments were performed repeated three times.
Means ± SEM are shown (** p < 0.05)
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2.6. HUVECs tuber formation assay
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Since MMP-2 and MMP-9 are deeply involved in angiogenesis progression, we performed
HUVECs tuber formation assay to examine whether compounds 5e and 5h could inhibit HUVECs
tuber formation. According to Table 2, MTT assay indicated that 5e and 5h possess no
cytotoxicity for HUVEC cells (IC50> 50 ꢀM). Then HUVECs tuber formation assay was
performed in vitro. Results in Fig. 9 showed that 5e and 5h significantly suppressed tube
formation of HUVEC when compared with DMSO control. On the other hand, compound A
exhibited moderate effects on tuber formation (Fig. 9).
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Fig. 9. HUVECs tuber formation assay of 5e, 5h and compound A at concentration of 10 ꢀM.
2.7. Apoptotic assay
In order to evaluate whether compounds 5e and 5h could induce apoptosis in cancer cells, an
Annexin-V/PI staining assay was conducted using A549 cells (Fig. 10). Briefly, A549 cells were
treated with 5e and 5h at the concentrations of 0, 1, 5 and 10 ꢀM for 48 h. At the concentrations of
1 ꢀM, compounds 5e and 5h could induce apoptosis with ratio of early apoptosis reaching 16%.
As the concentration of 5h increase, the percentage of early and late stage apoptotic cells
increased to 38%. According to these results, we conclude that 5e and 5h can effectively induce
apoptosis in A549 cells.
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Fig. 10. Effects of 5e, 5h and A on apoptosis of A549 cells at 1 ꢀM, 5 ꢀM and 10 ꢀM after 48 h.
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2.7. Western blot and Gelatine zymography assay
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In order to further investigate the MMPs inhibitory activities of our new inhibitors at the
cellular level, western blot analysis was performed to measure the effects of 5e and 5h on
MMP-2/9 expression using A549 cells. Results in Fig. 11 indicated that 5e and 5h could markedly
decrease expression levels of both MMP-2 and MMP-9 as the compound concentration increase.
Furthermore, gelatin zymography assay was performed to confirm the inhibitory activities of
compound 5e and 5h on MMP-2 and MMP-9. As shown in Fig. 12, compound 5e and 5h
significantly decreased the gelatinolytic activity of MMP-2 and MMP-9 in a dose dependent
manner.
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Fig. 11. Western blot analysis of MMP-2 and MMP-9 expressions in A549 cell after 24 h
treatment with 5e, 5h at 1, 5 and 10 ꢀM.
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Fig. 12. Gelatine zymography analysis of MMP-2 and MMP-9 in A549 cell after 24 h treatment
with 5e, 5h at 1, 5 and 10 ꢀM.
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3. Conclusions
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In summary, a series of 8-hydroxyquinoline derivatives were designed and synthesized as new
MMP-2/9 inhibitors. The most potent inhibitors 5e and 5h displayed anti-proliferation activities
against several cancer cell lines and exhibited anti-migration/invasion activities on A549 cells.
Moreover, HUVECs tuber formation assay was performed to measure the effects of compounds 5e
and 5h on HUVEC angiogenesis. Our results indicated that compounds 5e and 5h possess good
anti-invasion and anti-angiogenesis activity. Meanwhile, flow cytometry analysis showed that
compound 5e can promote A549 cell apoptosis in vitro. Furthermore, western blot analysis
confirmed that both 5e and 5h could induce down-regulation of MMP-2 and MMP-9 expressions.
Also, molecular docking studies not only revealed the favorable binding modes of 5e with
MMP-2/9, but also help us to understand the SAR of our new inhibitors. In the future, compound
5e could serve as lead compound to develop more potent non-hydroxamate MMP-2/9 inhibitors.
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4. Experimental
4.1. Chemistry
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Reagents and solvents used were of commercially available LR grade quality and without
further purification. All reactions were monitored by TLC on 0.25 mm silica gel plates (60
GF-254). UV light, chloride ferric and iodine vapor were used to visualize the spots. Melting
points were recorded by the RY-1G electrothermal melting point apparatus without correction. 1H
and 13C NMR spectra were recorded on a Brucker DRX spectrometer at 300 MHz or 400 MHz, d
1
in parts per million and J in hertz, using TMS as an internal standard. Significant H NMR data
are reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet)
number of protons. High-resolution mass spectra (HRMS) were conducted on an Agilent 6510
Quadrupole Time-of-Flight LC/MS deliver.
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4.2. General synthesis of compounds
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4.2.1. General procedure for the synthesis of 5a-5s
5-Chloro-8-hydroxyquinoline-7-carbaldehyde (0.21g, 1 mmol) and compound 4a-j (1 mmol)
was dissolved in MeOH (10ml) and reacted at RT for 4 h. Then NaBH4 (3 equiv) was added to the
reaction mixture, the solution was next stirred for 2h at RT. The MeOH was evaporated under
reduced pressure and EtOAc was added. The EtOAc layer was washed with a saturated solution of
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NaHCO3 (×3), brine solution (×3) and then dried and evaporated. The crude product was purified
by column chromatography (DCM/MeOH, 30/1-20/1) yielding target compounds 5a-5s.
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2-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-phenylacetamide (5a)
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Yellow solid (Yield 32%), Mp:116-118℃. H NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 8.99 –
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8.91 (m, 1H), 8.47 (dd, J = 8.5, 1.1 Hz, 1H), 7.77 (s, 1H), 7.69 (dd, J = 8.5, 4.1 Hz, 1H), 7.64 (d, J
= 7.8 Hz, 2H), 7.30 (t, J = 7.9 Hz, 2H), 7.05 (t, J = 7.4 Hz, 1H), 3.97 (s, 2H), 3.35 (s, 2H). 13C
NMR (101 MHz, DMSO) δ 170.41, 151.23, 149.30, 139.23, 139.17, 132.83, 129.13, 128.77,
125.30, 123.69, 123.38, 122.99, 119.68, 118.43, 52.39, 47.72. HRMS (ESI) m/z Calcd [M+H]+
342.1004, found: 342.0981.
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3-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-phenylpropanamide (5b)
Yellow solid (Yield 37%), Mp:120-122℃. 1H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 8.85 (dd,
J = 4.1, 1.3 Hz, 1H), 8.51 (dd, J = 8.5, 1.4 Hz, 1H), 7.57 – 7.48 (m, 4H), 7.34 – 7.27 (m, 2H), 7.07
(t, J = 7.4 Hz, 1H), 4.12 (s, 2H), 3.05 (t, 2H), 2.57 (t, 2H). 13C NMR (101 MHz, DMSO) δ 170.62,
151.82, 149.33, 139.68, 139.37, 132.78, 129.07, 128.53, 125.33, 123.48, 122.95, 122.69, 119.60,
118.15, 47.93, 44.98, 36.80. HRMS (ESI) m/z Calcd [M+H]+ 356.1060, found: 356.1145.
4-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-phenylbutanamide (5c)
Yellow solid (Yield 27%), Mp:78-80℃. 1H NMR (400 MHz, CDCl3) δ 8.84 (d, J = 3.2 Hz, 1H),
8.47 (d, J = 8.5 Hz, 1H), 7.49 (dd, J = 8.5, 4.1 Hz, 1H), 7.44 (d, J = 7.9 Hz, 2H), 7.37 (s, 1H), 7.25
– 7.20 (m, 2H), 7.04 (t, 1H), 4.10 (s, 2H), 2.81 (t, J = 6.0 Hz, 2H), 2.51 (t, J = 6.8 Hz, 2H), 2.01 –
1.90 (m, 2H). 13C NMR (101 MHz, DMSO) δ 170.41, 151.23, 149.30, 139.23, 139.17, 132.83,
129.13, 128.77, 125.30, 123.69, 123.38, 122.99, 119.68, 118.43, 52.39, 47.72. HRMS (ESI) m/z
Calcd [M+H]+ 370.1319, found: 370.1299.
5-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-phenylpentanamide (5d)
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Yellow solid (Yield 34%), Mp:102-105℃. H NMR (400 MHz, CDCl3) δ 8.82 (d, J = 3.1 Hz,
1H), 8.47 (dd, J = 8.5, 1.3 Hz, 1H), 8.06 (s, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.48 (dd, J = 8.5, 4.1
Hz, 1H), 7.35 (s, 1H), 7.29 (t, J = 7.8 Hz, 2H), 7.08 (t, J = 7.3 Hz, 2H), 4.13 (s, 2H), 2.73 (t, J =
6.5 Hz, 2H), 2.34 – 2.26 (m, 2H), 1.88 – 1.79 (m, 2H), 1.69 – 1.61 (m, 2H). 13C NMR (101 MHz,
DMSO) δ 170.41, 151.23, 149.30, 139.23, 139.17, 132.83, 129.13, 128.77, 125.30, 123.69,
123.38, 122.99, 119.68, 118.43, 52.39, 47.72. HRMS (ESI) m/z Calcd [M+H]+ 384.1473, found:
384.1456.
6-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-phenylhexanamide (5e)
Yellow solid (Yield 24%), Mp:116-118℃. 1H NMR (400 MHz, DMSO) δ 9.86 (s, 1H), 8.91 (d,
J = 2.9 Hz, 1H), 8.45 (d, J = 8.5 Hz, 1H), 7.71 – 7.62 (m, 2H), 7.58 (d, J = 7.9 Hz, 2H), 7.27 (t, J
= 7.8 Hz, 2H), 7.02 (d, J = 7.3 Hz, 1H), 3.96 (s, 2H), 2.54 (t, J = 6.9 Hz, 2H), 2.30 (t, J = 7.4 Hz,
2H), 1.64 – 1.55 (m, 2H), 1.53 – 1.45 (m, 2H), 1.38 – 1.32 (m, 2H). 13C NMR (101 MHz, DMSO)
δ 170.41, 151.23, 149.30, 139.23, 139.17, 132.83, 129.13, 128.77, 125.30, 123.69, 123.38, 122.99,
119.68, 118.43, 52.39, 47.72. HRMS (ESI) m/z Calcd [M+H]+ 398.1629, found: 398.1611.
2-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]acet
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amide (5f)
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Yellow solid (Yield 42%), Mp:113-116 . H NMR (400 MHz, DMSO) δ 10.69 (s, 1H), 8.94
(dd, J = 4.1, 1.4 Hz, 1H), 8.48 (dd, J = 8.5, 1.3 Hz, 1H), 7.97 (t, J = 5.8 Hz, 1H), 7.73 – 7.66 (m,
2H), 7.43 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 6.95 (t, J = 10.9, 4.0 Hz, 1H), 6.91 (t, J =
10.8, 3.9 Hz, 1H), 3.86 (s, 2H), 3.25 (dd, J = 14.2, 6.6 Hz, 2H), 3.11 (s, 2H), 2.76 (t, J = 7.5 Hz,
2H), 2.31 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.28, 149.35, 148.66, 138.50, 135.32, 133.24,
131.91, 128.65, 128.41, 125.54, 122.33, 121.10, 121.06, 120.09, 119.28, 117.83, 110.40, 108.53,
77.38, 77.26, 77.06, 76.74, 51.78, 48.61, 39.45, 24.14, 11.65. HRMS (ESI) m/z Calcd [M+H]+
423.1582, found: 423.1547.
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3-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]pro
panamide (5g)
Yellow solid (Yield 22%), Mp:120-122 . 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 8.34 (d,
J = 8.2 Hz, 1H), 7.96 (d, J = 21.2 Hz, 2H), 7.50 (dd, J = 8.3, 4.2 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H),
7.18 (d, J = 7.7 Hz, 2H), 7.03 (dt, J = 14.5, 6.9 Hz, 2H), 6.13 (s, 1H), 3.90 (s, 2H), 3.45 (dd, J =
12.5, 6.3 Hz, 2H), 2.82 (t, J = 6.6 Hz, 2H), 2.55 (t, 2H), 2.21 (s, 3H), 1.69 (t, 2H). 13C NMR (101
MHz, CDCl3) δ 171.28, 149.35, 148.66, 138.50, 135.32, 133.24, 131.91, 128.65, 128.41, 125.54,
122.33, 121.10, 121.06, 120.09, 119.28, 117.83, 110.40, 108.53, 77.38, 77.26, 77.06, 76.74, 51.78,
48.61, 39.45, 24.14, 11.65. HRMS (ESI) m/z Calcd [M+H]+ 437.1638, found: 437.1551.
4-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]but
anamide (5h)
Yellow solid (Yield 39%), Mp:115-117 . 1H NMR (400 MHz, CDCl3) δ 8.87 (dd, J = 4.2, 1.5
Hz, 1H), 8.47 (dd, J = 8.5, 1.6 Hz, 1H), 7.91 (s, 1H), 7.49 (dd, J = 8.6, 4.2 Hz, 1H), 7.46 (d, J =
7.4 Hz, 1H), 7.34 (s, 1H), 7.24 (s, 1H), 7.12 – 7.07 (m, 1H), 7.07 – 7.02 (m, 1H), 4.03 (s, 2H),
3.44 (d, J = 6.2 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.65 (t, J = 6.7 Hz, 2H), 2.34 (s, 3H), 2.19 (t, J =
7.2 Hz, 2H), 1.85 – 1.80 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 171.28, 149.35, 148.66, 138.50,
135.32, 133.24, 131.91, 128.65, 128.41, 125.54, 122.33, 121.10, 121.06, 120.09, 119.28, 117.83,
110.40, 108.53, 77.38, 77.26, 77.06, 76.74, 51.78, 48.61, 39.45, 24.14, 11.65. HRMS (ESI) m/z
Calcd [M+H]+ 451.1895, found: 451.1872.
5-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]pen
tanamide (5i)
Yellow solid (Yield 25%), Mp:104-106 . 1H NMR (400 MHz, CDCl3) δ 8.90 (dd, J = 4.1, 1.5
Hz, 1H), 8.75 (s, 1H), 8.49 (dd, J = 8.5, 1.5 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.33 (s, 1H), 7.25 – 7.21
(m, 1H), 7.11 – 7.02 (m, 2H), 4.07 (s, 2H), 3.52 (dd, J = 12.6, 6.4 Hz, 2H), 2.92 (t, J = 6.6 Hz,
2H), 2.65 (t, J = 6.6 Hz, 2H), 2.37 (s, 3H), 2.07 (dd, J = 13.2, 5.5 Hz, 2H), 1.66 – 1.54 (m, 2H),
1.55 – 1.45 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 171.28, 149.35, 148.66, 138.50, 135.32,
133.24, 131.91, 128.65, 128.41, 125.54, 122.33, 121.10, 121.06, 120.09, 119.28, 117.83, 110.40,
108.53, 77.38, 77.26, 77.06, 76.74, 51.78, 48.61, 39.45, 24.14, 11.65. HRMS (ESI) m/z Calcd
[M+H]+ 465.2051, found: 465.2023.
6-{[(5-chloro-8-hydroxyquinolin-7-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]hex
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Yellow solid (Yield 32%), Mp:98-101℃. H NMR (400 MHz, CDCl3) δ 8.87 (d, J = 2.8 Hz,
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1H), 8.47 (dd, J = 8.5, 1.4 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.35 (s, 1H), 7.29 – 7.24 (m, 2H), 7.19 –
6.99 (m, 3H), 4.09 (s, 2H), 3.54 – 3.45 (m, 2H), 2.89 (t, J = 6.6 Hz, 2H), 2.65 (t, J = 7.0 Hz, 2H),
2.34 (s, 3H), 2.08 – 2.04 (m, 2H), 1.61 – 1.46 (m, 4H), 1.29 – 1.23 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 171.28, 149.35, 148.66, 138.50, 135.32, 133.24, 131.91, 128.65, 128.41, 125.54, 122.33,
121.10, 121.06, 120.09, 119.28, 117.83, 110.40, 108.53, 77.38, 77.26, 77.06, 76.74, 51.78, 48.61,
39.45, 24.14, 11.65. HRMS (ESI) m/z Calcd [M+H]+ 479.2208, found: 479.2194.
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2-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-phenylacetamide (5k)
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Yellow solid (Yield 33%), Mp:115-118 . 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 1H), 8.82 (dd,
J = 4.1, 1.2 Hz, 1H), 8.49 (dd, J = 8.5, 1.2 Hz, 1H), 7.52 (dd, J = 8.5, 4.2 Hz, 1H), 7.45 – 7.37 (m,
3H), 7.29 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.21 (s, 2H), 3.50
(s, 2H). 13C NMR (101 MHz, CDCl3) δ 169.48, 152.21, 147.73, 138.84, 137.44, 132.37, 129.00,
128.51, 126.96, 125.07, 124.17, 122.00, 119.17, 109.16, 52.73, 51.03. HRMS (ESI) m/z Calcd
[M+H]+ 308.1393, found: 308.1378.
3-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-phenylpropanamide (5l)
Yellow solid (Yield 39%), Mp:121-124 .1H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 8.80 (dd,
J = 4.2, 1.4 Hz, 1H), 8.46 (dd, J = 8.5, 1.4 Hz, 1H), 7.44 (d, J = 7.4 Hz, 1H), 7.42 – 7.39 (m, 1H),
7.35 – 7.28 (m, 2H), 7.24 (t, 2H), 7.13 (d, J = 7.7 Hz, 1H), 7.05 (t, J = 7.3 Hz, 1H), 4.20 (s, 2H),
3.12 (t, 2H), 2.56 (t, 2H). 13C NMR (101 MHz, DMSO) δ 171.02, 152.93, 148.10, 139.70, 139.25,
133.91, 129.11, 128.01, 127.84, 127.04, 123.44, 121.82, 119.49, 110.56, 50.55, 45.76, 37.35.
HRMS (ESI) m/z Calcd [M+H]+ 322.1550, found: 322.1522.
4-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-phenylbutanamide(5m)
Yellow solid (Yield 34%), Mp:140-143 . 1H NMR (400 MHz, CDCl3) δ 8.75 (dd, J = 4.2, 1.5
Hz, 1H), 8.47 (dd, J = 8.5, 1.5 Hz, 1H), 8.16 (s, 1H), 7.43 – 7.40 (m, 1H), 7.39 (d, J = 2.8 Hz, 1H),
7.34 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 7.7 Hz, 1H), 7.05 (d, J = 7.3 Hz,
1H), 4.14 (s, 2H), 2.85 (t, J = 6.4 Hz, 2H), 2.46 (t, J = 6.9 Hz, 2H), 1.98 – 1.88 (m, 2H). 13C NMR
(101 MHz, CDCl3) δ 171.61, 151.66, 147.50, 138.70, 138.64, 133.19, 128.65, 127.73, 127.24, 126.54,
123.50, 121.61, 119.65, 108.97, 77.58, 77.46, 77.26, 76.94, 50.90, 49.06, 40.47, 40.26, 40.05, 39.85,
39.64, 39.42, 35.35, 25.62. HRMS (ESI) m/z Calcd [M+H]+ 336.1706, found: 336.1687.
5-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-phenylpentanamide (5n)
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Yellow solid (Yield 31%), Mp:122-124 . H NMR (400 MHz, CDCl3) δ 8.77 (d, J = 3.1 Hz,
1H), 8.55 – 8.47 (m, 1H), 7.48 – 7.36 (m, 4H), 7.29 (d, J = 7.6 Hz, 2H), 7.09 (d, J = 7.6 Hz, 2H),
4.11 (s, 2H), 2.76 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 7.2 Hz, 2H), 1.83 – 1.76 (m, 2H), 1.64 – 1.56 (m,
2H). 13C NMR (101 MHz, DMSO) δ 171.72, 152.78, 148.07, 139.84, 139.23, 133.84, 129.09,
127.84, 127.79, 127.32, 123.36, 121.81, 119.49, 110.51, 50.80, 49.30, 36.82, 29.57, 23.53. HRMS
(ESI) m/z Calcd [M+H]+ 350.1863, found: 350.1863.
6-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-phenylhexanamide (5o)
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Yellow solid (Yield 28%), Mp:125-128 . H NMR (400 MHz, CDCl3) δ 8.80 – 8.74 (m, 1H),
8.52 (d, 1H), 7.49 (d, J = 9.1 Hz, 2H), 7.47 – 7.44 (m, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.31 (t, J =
7.8 Hz, 2H), 7.08 (d, J = 7.7 Hz, 2H), 4.11 (s, 2H), 2.72 (t, J = 7.1 Hz, 2H), 2.34 (t, J = 7.4 Hz,
2H), 1.76 – 1.71 (m, 2H), 1.59 – 1.55 (m, 2H), 1.45 – 1.39 (m, 2H). 13C NMR (101 MHz, DMSO)
δ 171.72, 152.81, 148.05, 139.85, 139.24, 133.83, 129.08, 127.84, 127.78, 127.32, 123.35, 121.78,
119.49, 110.52, 50.82, 49.47, 36.91, 29.73, 27.06, 25.59. HRMS (ESI) m/z Calcd [M+H]+
364.2019, found: 364.2005.
7-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-phenylheptanamide (5p)
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Yellow solid (Yield 31%), Mp:96-98℃.1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 3.0 Hz, 1H),
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8.52 (d, J = 8.5 Hz, 1H), 7.54 – 7.46 (m, 3H), 7.40 (d, J = 7.7 Hz, 1H), 7.31 (t, J = 7.7 Hz, 2H),
7.17 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 6.8 Hz, 2H), 4.12 (s, 2H), 2.70 (t, J = 7.1 Hz, 2H), 2.32 (t, J =
7.5 Hz, 2H), 1.74 (d, J = 26.9 Hz, 2H), 1.51 (d, J = 26.9 Hz, 2H), 1.42 – 1.30 (m, 4H). 13C NMR
(101 MHz, DMSO) δ 171.72, 152.79, 148.06, 139.84, 139.22, 133.84, 129.09, 127.84, 127.80,
127.32, 123.35, 121.79, 119.47, 110.51, 50.83, 49.54, 36.87, 29.84, 29.13, 27.16, 25.63. HRMS
(ESI) m/z Calcd [M+H]+ 378.2176, found: 378.2162.
3-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]propanamid
e(5q)
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Yellow solid (Yield 36%), Mp:92-94 . H NMR (400 MHz, CDCl3) δ 8.74 (dd, J = 4.2, 1.4
Hz, 1H), 8.19 (dd, J = 8.5, 1.4 Hz, 1H), 7.61 (s, 1H), 7.43 – 7.38 (m, 1H), 7.32 – 7.29 (m, 1H),
7.18 (d, J = 7.7 Hz, 1H), 7.14 – 7.10 (m, 1H), 7.04 (ddd, J = 6.5, 3.7, 1.9 Hz, 3H), 3.88 (s, 2H),
3.41 (dd, J = 12.8, 6.5 Hz, 2H), 2.87 – 2.78 (m, 4H), 2.35 – 2.29 (m, 2H), 2.27 (d, J = 6.3 Hz, 3H).
13C NMR (101 MHz, DMSO) δ 171.74, 153.33, 152.93, 148.11, 139.25, 135.65, 133.83, 132.51,
128.78, 127.83, 126.90, 121.83, 120.36, 118.54, 117.76, 110.82, 110.55, 108.09, 55.87, 50.47,
45.82, 36.20, 24.70, 11.67. HRMS (ESI) m/z Calcd [M+H]+ 403.2128, found: 403.2090.
4-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]butanamide
(5r)
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Yellow solid (Yield 27%), Mp:95-98 . H NMR (400 MHz, CDCl3) δ 8.76 (dd, J = 4.2, 1.4
Hz, 1H), 8.44 (dd, J = 8.5, 1.5 Hz, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.44 (d, J = 4.1 Hz, 1H), 7.42 (d,
J = 4.2 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.23 (d, 1H), 7.12 (d, 1H), 7.08 (d, J = 3.2 Hz, 1H), 7.07
– 7.05 (m, 1H), 4.00 (s, 2H), 3.45 (dd, J = 12.8, 6.6 Hz, 2H), 2.86 (t, J = 6.7 Hz, 2H), 2.65 (t, J =
6.7 Hz, 2H), 2.31 (s, 3H), 2.17 (t, J = 7.2 Hz, 2H), 1.78 (t, 2H). 13C NMR (101 MHz, DMSO) δ
172.66, 153.16, 152.92, 148.22, 139.13, 135.63, 133.71, 132.54, 128.77, 128.24, 127.79, 125.35,
121.95, 120.39, 118.58, 117.75, 110.84, 110.58, 108.07, 50.06, 48.83, 33.87, 25.56, 24.62, 11.61.
HRMS (ESI) m/z Calcd [M+H]+ 417.2285, found: 417.2270.
5-{[(8-hydroxyquinolin-5-yl)methyl]amino}-N-[2-(2-methyl-1H-indol-3-yl)ethyl]pentanamide
(5s)
Yellow solid (Yield 33%), Mp:134-136 . 1H NMR (400 MHz, CDCl3) δ 8.77 (dd, J = 4.2, 1.5
Hz, 1H), 8.50 (dd, J = 8.5, 1.5 Hz, 1H), 7.87 (s, 1H), 7.49 – 7.44 (m, 2H), 7.39 (d, J = 7.7 Hz, 1H),
7.13 – 7.02 (m, 4H), 4.08 (s, 2H), 3.50 – 3.44 (m, 2H), 2.88 (t, J = 8.7, 4.6 Hz, 2H), 2.66 (t, J =
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6.9 Hz, 2H), 2.34 (s, 3H), 2.06 (t, J = 11.8, 4.3 Hz, 2H), 1.65 – 1.55 (m, 2H), 1.53 – 1.45 (m, 2H).
13C NMR (101 MHz, CDCl3) δ 172.85, 151.78, 147.57, 138.63, 135.32, 134.15, 133.20, 131.99,
129.57, 128.62, 127.64, 127.24, 125.54, 121.14, 119.33, 117.75, 110.39, 108.80, 50.86, 49.19,
39.90, 36.41, 29.34, 25.77, 24.15, 11.60. HRMS (ESI) m/z Calcd [M+H]+ 431.2441, found:
431.2403.
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7
4.2.2. General procedure for the synthesis of 10a-10l
8
9
Compound 9a-l (1.2 mmol, 1.2 equiv) was dissolved in 20 mL DCM and one drop DMF was
added. Then oxalyl chloride (0.2 mL, 2.4 mmol, 2.4 equiv) was added dropwise and the reaction
was stirred at RT for 1h. After the mixture was evaropated, the residue was dissolved in DCM and
again concentrated in vacuo to offer quantitatively the crude chloride. A solution of chloride in
DCM (10 mL) was suspended with NEt3 (0.3g, 3 equiv) and compound 7 (0.27g, 1mmol) was
added dropwise with stirring. After the addition, the reaction was stirred for 4 h. Then the mixture
was washed with 1M citric acid(×3), saturate solution of NaHCO3(×3) and brine solution(×3). The
solution was concentrated in vacuo, then purified via chlomatography on silica (PE/EA =3:1) to
get 10a-l as a white solid.
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N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-3-[methyl(phenyl)amino]propanamide(10a)
White solid (Yield 57%), Mp:149-151 .1H NMR (400 MHz, CDCl3) δ 8.77 (dd, J = 4.0, 1.4
Hz, 1H), 8.20 (s, 1H), 7.87 (dd, J = 8.5, 1.4 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.27 (d, J = 8.6 Hz,
2H), 7.18 (dd, J = 8.5, 4.0 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.80 (dd, J = 16.1, 7.9 Hz, 3H), 3.68
(t, J = 6.4 Hz, 2H), 2.93 (s, 3H), 2.66 – 2.58 (m, 2H), 1.05 (s, 9H), 0.24 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-4-[methyl(phenyl)amino]butanamide (10b)
1
White solid (Yield 66%), Mp:144-146 . H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 2.9 Hz,
1H), 7.95 (d, J = 7.7 Hz, 1H), 7.53 (s, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.30 – 7.18 (m, 4H), 7.08 (d,
J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 2H), 6.69 (t, J = 7.3 Hz, 1H), 3.39 (t, J = 7.0 Hz, 2H), 2.90 (s,
3H), 2.44 (t, J = 7.1 Hz, 2H), 2.00 (dd, J = 14.1, 7.0 Hz, 2H), 1.06 (s, 9H), 0.25 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-5-[methyl(phenyl)amino]pentanamide(10c)
Colourless oil (Yield 69%). 1H NMR (400 MHz, CDCl3) δ 8.85 (d, J = 3.0 Hz, 1H), 8.07 – 7.97
(m, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.36 (dd, J = 8.4, 3.9 Hz, 1H), 7.22 (d, J = 8.1 Hz, 2H), 7.13 (d,
J = 8.2 Hz, 1H), 6.76 – 6.65 (m, 3H), 3.40 (s, 2H), 2.95 (s, 3H), 2.49 (t, J = 7.2 Hz, 2H), 1.88 –
1.79 (m, 2H), 1.77 – 1.70 (m, 2H), 1.06 (s, 9H), 0.26 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-6-[methyl(phenyl)amino]hexanamide (10d)
Colourless oil oil (Yield 61%). 1H NMR (400 MHz, CDCl3) δ 8.84 (t, J = 15.7 Hz, 1H), 8.00 (d,
J = 8.5 Hz, 1H), 7.41 (dd, J = 7.6, 2.6 Hz, 1H), 7.34 – 7.24 (m, 1H), 7.20 (d, J = 7.6 Hz, 2H), 7.07
(d, J = 8.2 Hz, 1H), 6.67 (dd, J = 11.3, 7.9 Hz, 3H), 3.30 (dd, J = 8.9, 5.5 Hz, 2H), 2.89 (d, J = 4.9
Hz, 3H), 2.36 (t, J = 7.7 Hz, 2H), 1.74 (s, 2H), 1.59 (d, J = 7.5 Hz, 2H), 1.43 – 1.35 (m, 2H), 1.06
(s, 9H), 0.25 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-3-(2-methyl-1H-benzo[d]imidazol-1-yl)pro
panamide (10e)
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Colourless oil (Yield 41%). 1H NMR (400 MHz, CDCl3). H NMR (400 MHz, DMSO) δ 9.84
(s, 1H), 8.83 (dd, J = 4.0, 1.3 Hz, 1H), 7.84 (dd, J = 8.6, 1.3 Hz, 1H), 7.55 (dd, J = 10.0, 3.8 Hz,
2H), 7.38 (dd, J = 8.6, 4.1 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.20 – 7.14 (m, 2H), 7.13 (d, J = 8.2
Hz, 1H), 4.54 (s, 2H), 2.97 (s, 2H), 2.59 (s, 3H), 1.01 (s, 9H), 0.20 (s, 6H).
4
5
6
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8
9
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-4-(2-methyl-1H-benzo[d]imidazol-1-yl)buta
namide (10f)
White solid (Yield 49%), Mp:133-135 . 1H NMR (400 MHz, DMSO) δ 9.79 (s, 1H), 8.81 (dd,
J = 4.0, 1.4 Hz, 1H), 8.10 (dd, J = 8.6, 1.4 Hz, 1H), 7.40– 7.30 (m, 4H), 7.10 – 7.00 (m, 3H), 4.15
(t, J = 7.0 Hz, 2H), 2.46 (s, 3H), 2.35 – 2.30 (m, 2H), 1.72 – 1.69 (m, 2H), 0.93 (s, 9H), 0.18 (s,
6H).
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N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-5-(2-methyl-1H-benzo[d]imidazol-1-yl)pent
anamide (10g)
White solid (Yield 52%), Mp:142-144 . 1H NMR (400 MHz, DMSO) δ 9.74 (s, 1H), 8.79 (dd,
J = 4.0, 1.4 Hz, 1H), 8.18 (dd, J = 8.6, 1.4 Hz, 1H), 7.46 – 7.35 (m, 4H), 7.10 – 7.00 (m, 3H), 4.15
(t, J = 7.0 Hz, 2H), 2.46 (s, 3H), 2.42 – 2.39 (m, 2H), 1.77 – 1.68 (m, 2H), 1.65 – 1.54 (m, 2H),
0.93 (s, 9H), 0.13 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-6-(2-methyl-1H-benzo[d]imidazol-1-yl)hexa
namide (10h)
White solid (Yield 53%), Mp:132-134 . 1H NMR (400 MHz, DMSO) δ 9.76 (s, 1H), 8.87 (dd,
J = 4.0, 1.3 Hz, 1H), 8.27 (dd, J = 8.6, 1.4 Hz, 1H), 7.56 – 7.42 (m, 4H), 7.18 – 7.10 (m, 3H), 4.20
(t, J = 7.1 Hz, 2H), 2.54 (s, 3H), 2.41 (t, J = 7.1 Hz, 2H), 1.81 – 1.73 (m, 2H), 1.71 – 1.64 (m, 2H),
1.42 – 1.35 (m, 2H), 1.02 (s, 9H), 0.21 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-3-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3
H)-yl}propanamide (10i)
White solid (Yield 63%), Mp:102-104 .1H NMR (400 MHz, CDCl3) δ 8.83 (dd, J = 4.0, 1.3
Hz, 1H), 8.09 (dd, J = 11.2, 4.1 Hz, 2H), 7.99 – 7.80 (m, 3H), 7.56 (d, J = 8.2 Hz, 1H), 7.47 (s,
1H), 7.33 (dd, J = 8.5, 4.1 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 4.31 (t, J = 7.2 Hz, 2H), 3.08 (t, J =
7.2 Hz, 2H), 1.05 (s, 9H), 0.25 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-4-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3
H)-yl}butanamide (10j)
White solid (Yield 59%), Mp:98-100 .1H NMR (400 MHz, DMSO) δ 9.87 (s, 1H), 9.34 (d, J =
1.8 Hz, 1H), 8.91 (dd, J = 4.0, 1.5 Hz, 1H), 8.76 (dd, J = 8.4, 2.0 Hz, 1H), 8.42 – 8.35 (m, 2H),
7.58 (dd, J = 8.6, 4.1 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 3.93 (t, J = 7.0
Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.21 – 2.10 (m, 2H), 1.06 (s, 9H), 0.26 (s, 6H).
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-5-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3
H)-yl}pentanamide (10k)
White solid (Yield 74%), Mp:120-122 . 1H NMR (400 MHz, DMSO) δ 9.83 (s, 1H), 8.88 (dd,
J = 4.0, 1.5 Hz, 1H), 8.33 (d, J = 8.1 Hz, 2H), 8.13 (d, J = 7.3 Hz, 1H), 8.11 – 7.97 (m, 2H), 7.54
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(dd, J = 8.5, 4.0 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 3.80 (t, J = 7.0 Hz,
2H), 1.93 – 1.82 (m, 2H), 1.80 – 1.69 (m, 2H), 1.04 (s, 9H), 0.23 (s, 6H).
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5
6
N-{8-[(tert-butyldimethylsilyl)oxy]quinolin-5-yl}-6-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3
H)-yl}hexanamide (10l)
1
White solid (Yield 63%), Mp:118-120 . H NMR (400 MHz, CDCl3) δ 8.82 (dd, J = 3.9, 1.2
7
8
9
Hz, 1H), 8.09 (dd, J = 8.5, 1.2 Hz, 1H), 7.96 (d, J = 6.8 Hz, 1H), 7.86 – 7.74 (m, 3H), 7.61 (s, 1H),
7.46 (d, J = 8.2 Hz, 1H), 7.34 (dd, J = 8.5, 4.1 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 3.81 (t, J = 7.2
Hz, 2H), 2.46 (t, J = 7.4 Hz, 2H), 1.96 – 1.89 (m, 2H), 1.87 – 1.78 (m, 2H), 1.59 – 1.50 (m, 2H),
1.06 (s, 9H), 0.25 (s, 6H).
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4.2.3. General procedure for the synthesis of 11a-11l
The solution of 10a (0.22g) in MeOH(4ml) was acidified by 2M hydrochloric acid solution to
pH 2. The mixture was stirred at room temperature for 4h. Saturated aqueous NaHCO3 solution
was added and a yellow solid was acquired. The mixture was filtered to get the white solid as the
crude product. The crude product was purified by recrystallization (EtOAc) to give the desired
product as white solid.
N-(8-hydroxyquinolin-5-yl)-3-[methyl(phenyl)amino]propanamide (11a)
White solid (Yield 61%), Mp:165-168℃. 1H NMR (400 MHz, DMSO) δ 9.82 (s, 1H), 8.85 (dd,
J = 4.1, 1.5 Hz, 1H), 8.24 (dd, J = 8.6, 1.5 Hz, 1H), 7.52 (dd, J = 8.6, 4.1 Hz, 1H), 7.40 (d, J = 8.2
Hz, 1H), 7.21 (dd, J = 8.7, 7.3 Hz, 2H), 7.05 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 8.0 Hz, 2H), 6.65 (t,
J = 7.2 Hz, 1H), 3.72 (t, J = 7.0 Hz, 2H), 2.94 (s, 3H), 2.66 (t, J = 7.0 Hz, 2H). 13C NMR (101
MHz, DMSO) δ 171.22, 151.82, 149.31, 148.50, 138.74, 132.64, 129.53, 124.92, 124.82, 124.59,
121.86, 116.45, 112.82, 110.86, 49.14, 38.29, 33.62. HRMS (ESI) m/z Calcd [M+H]+ 322.1550,
found: 322.1541.
N-(8-hydroxyquinolin-5-yl)-4-[methyl(phenyl)amino]butanamide (11b)
1
White solid (Yield 65%), Mp:177-180℃. H NMR (400 MHz, CDCl3) δ 8.76 (d, J = 3.4 Hz,
1H), 8.03 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 7.9 Hz, 2H), 7.30 (s, 1H), 7.23 (d, J = 7.7 Hz, 2H), 7.11
(d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.2 Hz, 2H), 6.72 (t, J = 7.2 Hz, 1H), 3.46 (t, J = 6.9 Hz, 2H),
2.95 (s, 3H), 2.53 (t, J = 7.0 Hz, 2H), 2.16 – 2.05 (m, 2H). 13C NMR (101 MHz, DMSO) δ 172.27,
151.79, 149.54, 148.51, 138.76, 132.57, 129.48, 124.99, 124.92, 124.67, 121.91, 115.98, 112.37,
110.90, 51.84, 38.33, 33.26, 22.56. HRMS (ESI) m/z Calcd [M+H]+ 336.1706, found: 336.1698.
N-(8-hydroxyquinolin-5-yl)-5-[methyl(phenyl)amino]pentanamide (11c)
1
White solid (Yield 51%), Mp:123-126℃. H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 3.6 Hz,
1H), 8.06 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 7.7 Hz, 2H), 7.17 – 7.10 (m,
2H), 6.73 (d, J = 8.3 Hz, 2H), 6.69 (t, J = 7.3 Hz, 1H), 3.40 (t, J = 7.0 Hz, 2H), 2.95 (s, 3H), 2.50
(t, J = 7.2 Hz, 2H), 1.89 – 1.79 (m, 2H), 1.77 – 1.68 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
172.46, 151.17, 149.28, 147.88, 138.08, 131.48, 129.27, 125.22, 124.55, 123.23, 121.84, 116.20,
112.28, 109.25, 52.44, 38.48, 36.63, 26.53, 23.45. HRMS (ESI) m/z Calcd [M+H]+ 350.1863,
found: 350.1858.
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N-(8-hydroxyquinolin-5-yl)-6-[methyl(phenyl)amino]hexanamide (11d)
1
White solid (Yield 55%), Mp:132-135℃. H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 3.4 Hz,
3
4
5
6
7
8
1H), 8.10 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 7.1 Hz, 2H), 7.25 – 7.20 (m, 2H), 7.13 (d, J = 7.5 Hz,
1H), 6.71 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 7.3 Hz, 1H), 3.35 (t, J = 7.3 Hz, 2H), 2.93 (s, 3H), 2.48
(t, J = 7.3 Hz, 2H), 1.91 – 1.79 (m, 2H), 1.71 – 1.63 (m, 2H), 1.51 – 1.44 (m, 2H). 13C NMR (101
MHz, DMSO) δ 172.48, 151.73, 149.51, 148.49, 138.75, 132.50, 129.47, 124.95, 124.62, 121.89,
115.83, 112.32, 110.89, 52.14, 38.37, 36.13, 26.76, 26.27, 25.74. HRMS (ESI) m/z Calcd [M+H]+
364.2019, found: 364.1997.
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N-(8-hydroxyquinolin-5-yl)-3-{2-methyl-1H-benzo[d]imidazol-1-yl}propanamide (11e)
White solid (Yield 57%), Mp:234-236℃.1H NMR (400 MHz, DMSO) δ 9.77 (s, 1H), 8.77 (d, J
= 3.9 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.59 – 7.53 (m, 2H), 7.37 (dd, J = 8.5, 4.1 Hz, 1H), 7.21
(dd, J = 8.5, 4.8 Hz, 1H), 7.19 – 7.12 (m, 2H), 6.94 (d, J = 8.2 Hz, 1H), 4.54 (t, J = 6.6 Hz, 2H),
2.94 (t, J = 6.6 Hz, 2H), 2.58 (s, 3H), 2.09 (s, 1H). 13C NMR (101 MHz, DMSO) δ 170.05, 152.36,
152.28, 148.43, 142.93, 138.79, 135.39, 132.31, 124.90, 124.72, 124.15, 121.88, 121.77, 121.68,
118.64, 110.85, 110.66, 36.04, 14.01. HRMS (ESI) m/z Calcd [M+H]+ 347.1502, found:
347.1504.
N-(8-hydroxyquinolin-5-yl)-4-{2-methyl-1H-benzo[d]imidazol-1-yl}butanamide (11f)
White solid (Yield 69%), Mp:217-220℃. 1H NMR (400 MHz, CDCl3) δ 9.71 (s, 1H), 8.77 (d, J =
3.2 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.52 – 7.41 (m, 3H), 7.35 (d, J = 8.2 Hz, 1H), 7.15 – 7.03 (m,
2H), 6.98 (d, J = 8.2 Hz, 1H), 4.20 (t, J = 7.2 Hz, 2H), 2.50 (s, 3H), 2.43 (d, J = 4.7 Hz, 2H), 2.09 –
1.98 (m, 2H). 13C NMR (101 MHz, DMSO) δ 171.73, 152.13, 151.87, 148.51, 142.86, 138.77,
135.65, 132.59, 124.97, 124.77, 124.66, 121.90, 121.88, 121.59, 118.67, 110.90, 110.29, 43.04,
32.59, 25.45, 13.95. HRMS (ESI) m/z Calcd [M+H]+ 361.1659, found: 361.1638.
N-(8-hydroxyquinolin-5-yl)-5-{2-methyl-1H-benzo[d]imidazol-1-yl}pentanamide (11g)
White solid (Yield 45%), Mp:197-200℃. 1H NMR (400 MHz, DMSO) δ 9.66 (s, 1H), 8.75 (dd,
J = 4.0, 1.2 Hz, 1H), 8.13 (dd, J = 8.5, 1.3 Hz, 1H), 7.46 – 7.39 (m, 3H), 7.30 (d, J = 8.2 Hz, 1H),
7.05 (td, J = 6.9, 1.0 Hz, 2H), 6.94 (d, J = 8.2 Hz, 1H), 4.13 (t, J = 7.1 Hz, 2H), 2.45 (s, 3H), 2.39
– 2.34 (m, 2H), 1.77 – 1.66 (m, 2H), 1.62 – 1.54 (m, 2H). 13C NMR (101 MHz, DMSO) δ 172.22,
152.03, 151.79, 148.51, 142.80, 138.75, 135.63, 132.45, 124.93, 124.83, 124.65, 121.89, 121.82,
121.54, 118.66, 110.89, 110.28, 43.26, 35.64, 29.45, 23.10, 13.99. HRMS (ESI) m/z Calcd
[M+H]+ 375.1815, found: 375.1827
N-(8-hydroxyquinolin-5-yl)-6-{2-methyl-1H-benzo[d]imidazol-1-yl}hexanamide (11h)
1
White solid (Yield 35%), Mp:125-128℃. H NMR (400 MHz, DMSO) δ 9.76 (s, 1H), 9.70 (s,
1H), 8.86 (dd, J = 4.1, 1.4 Hz, 1H), 8.23 (dd, J = 8.6, 1.5 Hz, 1H), 7.56 (dd, J = 8.6, 4.1 Hz, 1H),
7.51 (t, J = 6.6 Hz, 2H), 7.37 (d, J = 8.2 Hz, 1H), 7.19 – 7.10 (m, 2H), 7.03 (d, J = 8.2 Hz, 1H),
4.21 (t, J = 7.2 Hz, 2H), 2.55 (s, 3H), 2.40 (t, J = 7.3 Hz, 2H), 1.83 – 1.73 (m, 2H), 1.72 – 1.65 (m,
2H), 1.43 – 1.35 (m, 2H). 13C NMR (101 MHz, DMSO) δ 172.37, 152.03, 151.74, 148.48, 142.76,
138.72, 135.60, 132.46, 124.93, 124.88, 124.61, 121.89, 121.80, 121.51, 118.62, 110.87, 110.28,
43.40, 35.95, 29.58, 26.43, 25.44, 13.99. HRMS (ESI) m/z Calcd [M+H]+ 389.1972, found:
389.1943.
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3-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl}-N-(8-hydroxyquinolin-5-yl)propanamide
(11i)
4
5
6
7
8
9
White solid (Yield 38%), Mp:210-213℃.1H NMR (400 MHz, DMSO) δ 9.93 (s, 1H), 8.85 (dd,
J = 4.0, 1.4 Hz, 1H), 8.39 – 8.31 (m, 2H), 8.15 (d, J = 7.6 Hz, 1H), 8.11 – 8.05 (m, 1H), 8.02 (t, J
= 7.1 Hz, 1H), 7.54 (dd, J = 8.6, 4.1 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H),
4.10 (t, J = 7.3 Hz, 2H), 2.97 (t, J = 7.3 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 169.32, 158.93,
152.08, 148.47, 138.78, 137.34, 136.31, 135.77, 132.68, 126.84, 125.58, 124.96, 124.72, 124.43,
122.04, 121.85, 110.86, 35.63, 34.76. HRMS (ESI) m/z Calcd [M+H]+ 398.1702, found: 398.1711
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4-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl}-N-(8-hydroxyquinolin-5-yl)butanamide
(11j)
White solid (Yield 40%), Mp:221-223℃. 1H NMR (400 MHz, DMSO) δ 9.77 (d, 2H), 9.30 (s,
1H), 8.86 (d, J = 4.0 Hz, 1H), 8.73 (d, 1H), 8.41 – 8.26 (m, 2H), 7.58 (dd, J = 8.6, 4.1 Hz, 1H),
7.43 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 3.89 (t, 2H), 2.59 (t, J = 7.3 Hz, 2H), 2.18 –
2.06 (m, 2H). 13C NMR (101 MHz, DMSO) δ 171.53, 157.61, 152.27, 151.81, 148.51, 138.74,
138.13, 132.65, 131.61, 130.75, 127.27, 124.99, 124.85, 124.63, 121.90, 118.48, 110.88, 32.77,
24.32. HRMS (ESI) m/z Calcd [M+H]+ 412.0961, found: 412.0950.
5-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl}-N-(8-hydroxyquinolin-5-yl)pentanamide
(11k)
1
White solid (Yield 46%), Mp:152-154℃. H NMR (400 MHz, DMSO) δ 9.76 (d, J = 2.7 Hz,
2H), 8.86 (dd, J = 4.1, 1.5 Hz, 1H), 8.37 – 8.25 (m, 2H), 8.13 (d, J = 7.5 Hz, 1H), 8.10 – 7.97 (m,
2H), 7.57 (dd, J = 8.6, 4.1 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 3.79 (t, J =
7.1 Hz, 2H), 2.48 (t, J = 7.0 Hz, 2H), 1.90 – 1.80 (m, 2H), 1.81 – 1.67 (m, 2H). 13C NMR (101
MHz, DMSO) δ 172.17, 159.09, 151.76, 148.50, 138.74, 137.26, 136.24, 135.73, 132.58, 126.82,
125.54, 124.98, 124.90, 124.65, 121.99, 121.91, 110.87, 39.03, 35.48, 28.08, 23.03. HRMS (ESI)
m/z Calcd [M+H]+ 426.1118, found: 426.1105.
6-{1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl}-N-(8-hydroxyquinolin-5-yl)hexanamide
(11l)
1
White solid (Yield 44%), Mp:143-146℃. H NMR (400 MHz, CDCl3) δ 8.76 (d, J = 3.5 Hz,
1H), 8.11 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 6.7 Hz, 1H), 7.86 – 7.74 (m, 3H), 7.49 – 7.41 (m, 2H),
7.38 (s, 1H), 7.08 (d, J = 8.1 Hz, 1H), 3.85 (t, J = 7.0 Hz, 2H), 2.50 (t, J = 7.3 Hz, 2H), 1.99 – 1.92
(m, 2H), 1.93 – 1.85 (m, 2H), 1.62 – 1.55 (m, 2H). 13C NMR (101 MHz, DMSO) δ 172.39, 159.09,
151.72, 148.47, 138.72, 137.26, 136.25, 135.73, 132.56, 126.78, 125.53, 124.99, 124.94, 124.66,
121.98, 121.89, 110.85, 39.17, 35.91, 28.20, 26.34, 25.27. HRMS (ESI) m/z Calcd [M+H]+
440.1274, found: 412.1259.
4.3. Colorimetric Screening against MMP-2 and MMP-9.
MMP-2 and MMP-9 inhibitory activities of all target compounds were evaluated by MMP-2
and MMP-9 Colorimetric Drug Discovery Kit (BML-AK408, BML-AK410, Enzo®Life Sciences).
All assays were carried out in clear Costar 96-well, half-area, flat-bottom assay plates. Each well
contained a total volume of 90ꢀL including 50ꢀL assay buffer, 20ꢀL human recombinant MMP-2
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or MMP-9 and 20ꢀL test inhibitors. The enzymes were not added to the blanks and the test
inhibitors were not added to the controls. After a 30 min incubation period at 37 °C, the reaction
was initiated by the addition of 10 ꢀL of chromogenic MMP substrate. Absorbance was monitored
at 412 nm using a microtiterplate reader, and measurements were recorded every minute for 20
min.
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4.4. Molecular docking
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Surflex-dock program in Sybyl-X 2.1.1 was used with default values for all docking studies.
Ligands were optimized using concord method and assigned with Gasteiger-Hückel charges. The
structure of MMP-2 and MMP-9 was downloaded from the Protein Data Bank server with PDB
code: 1HOV and 1GKC. The top-scored pose of selected compounds were selected as the
representative structures.
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4.5. MTT assay
All cell lines were cultured in RPMI1640 medium (10% FBS) at 37°C in a 5% CO2 humidified
incubator. Briefly, Cancer cells were plated at 4000-5000 cells per well (100ꢀL/well) in 96-well
plates for 8 h and then treated with different concentrations of compounds (100ꢀL/well) for 48 h.
0.5% MTT solution (10ꢀL/well) was added and incubation for 4 h. DMSO (150ꢀL/well) was
added to extract formazan formed from MTT. Absorbance was determined using a microtiter-plate
reader at 570 nm. The IC50 values were calculated according to the inhibition ratios.
4.6. Cell migration and invasion assay
A549 cell was plated at 200,000 cells per well (1000ꢀL/well) in 12-well plates for 8 h and then
treated with compound 5e or 5h (1000ꢀL/well) for 24 h. Then cells were harvested and
resuspended as single cells in 1000ꢀL serum-free RPMI1640.
For the migration experiments, we took 30,000 cells in 200ꢀL serum-free RPMI1640. Cells
were added to the upper chamber and the lower chamber was filled with 600ꢀL RPMI1640
supplemented with 10% FBS. Cells were allowed to migrate for 24 h at 37°C in a 5% CO2
humidified incubator. The experiment was terminated by discarding the medium and fixing the
cells with MeOH/Acetic acid (3:1) for 20 min. Non-invading cells on the upper side of the insert
were removed by a cotton-tipped applicator. Staining of the cells on the bottom of the membrane
was performed with crystal violet (0.1%) for 20 min at room temperature and washed with PBS.
Then lower surface was captures and counted. Cells were either counted manually or with the help
of the Image J software.
For the invasion experiments, the chamber membrane was covered with 50µL matrigel (1:10
diluted) for 2 hours at 37°C and the rest of the protocol was identical to that described in the
migration assay.
4.7. HUVECs tuber formation assay
HUVEC were plated at 200,000 cells per well (1000ꢀL/well) in 12-well plates for 8h and then
treated with compound 5e or 5h (1000ꢀL/well) for 24h using serum-free RPMI1640. Then cells
were harvested and resuspended as single cells in 1000ꢀL serum-free RPMI1640.
The wells of 96-well plates were covered with 100µL matrigel (no diluting) for 2 hours at 37°C.
Then cells were carefully layered on the top of the polymerized matrigel. After 6h incubation on
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matrigel, the dimensional organization of the cells was examined under an inverted microscope.
The number of meshes where measured as a direct parameter for HUVEC angiogenesis efficiency,
using the Angiogenesis tool and Image J software.
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4.8. Apoptosis Assay.
A549 cells (1 × 105 cells/mL) were seeded in 12-well plates and treated with compounds 5e, 5h
and A of 1, 5 and 10 ꢀM for 48h. The cells were then harvested and washed with cold PBS. After
the centrifugation and removal of the supernatants, cells were resuspended in 200 ꢀL of binding
buffer, which was then added to 5ꢀL of annexin V-FITC and 10ꢀL of PI. Then the cells were
incubated at room temperature for another 15min in the dark. The stained cells were analyzed by a
flow cytometer.
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4.9. Western blot
A549 cells were treated with 5e, 5h and A for 24h. The assay protocol has been described
previously [26]. Cell lysates were prepared using SDS-PAGE loading bu er and subjected to
SDS-PAGE, they were then transferred to PVDF membranes using the Trans-Blot Turbo Transfer
system (Bio-Rad). After blocking with 10% dry milk in TBST, membranes were probed with
rabbit anti-MMP-2, rabbit anti-MMP-9 or rabbit anti-GAPDH antibody, then were incubated with
secondary antibodies. Images were acquired using Amersham Imager 680.
Zymography in 10% polyacrylamide gel containing 0.1% gelatin was performed according to
the earlier method[27]. Gelatin was used to detect MMP-2 and MMP-9 activity in the culture
medium. Cell supernatants were electrophoresed on an 8% SDS-PAGE containing 2% gelatin. The
gels were washed with 0.25% triton three times. Then the gels were cut and incubated in the
zymography buffer (0.15 M NaCl, 5 mM CaCl2, 0.05% NaN3 and 50 mM Tris-HCl buffer, pH 7.5)
at 37°C for 48 h. After incubation, the gels were stained with 0.25% Coomassie Brilliant Blue
R250, and detained with acetic acid, methanol and water. MMP activity was represented by a
white band of gelatin digestion.
Acknowledgments
This work was supported by National Natural Science Foundation of China (81874288 and
21672127), Key Research and Development Project of Shandong Province (Grant No.
2017CXGC1401), the Fundamental Research Funds of Shandong University (Grant No.
2019GN045) and The Joint Research Funds for Shandong University and Karolinska Institute
(SDU-KI-2019-06).
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Highlights
1. Thirty-one 8-hydroxyquinoline derivatives were designed and synthesized.
2. Compounds 5e and 5h show good MMP-2/9 inhibitory activity.
3. Compounds 5e and 5h exert good anti-proliferative, anti-invasive and anti-angiogenetic
activity.
4. Compounds 5e and 5h down-regulate expression of MMP-2 and MMP-9.