Discovery of dehydroabietic acid sulfonamide based derivatives as selective matrix metalloproteinases inactivators that inhibit cell migration and proliferation

Article abstract of DOI:10.1016/j.ejmech.2017.07.020

A series of dehydroabietic acid (DHAA) dipeptide derivatives containing the sulfonamide moiety were designed, synthesized and evaluated for inhibition of MMPs as well as the effects of in vitro cell migration. These compounds exhibited relatively good inhibition activity against MMPs with IC₅₀ values in low micromolar range. A docking study of the most active compound 8k revealed key interactions between 8k and MMP-3 in which the sulfonamide moiety and the dipeptide group were important for improving activity. It is noteworthy that further antitumor activity screening revealed that some compounds exhibited better inhibitory activity than the commercial anticancer drug 5-FU. In particular, compound 8k appeared to be the most potent compound against the HepG2 cell line, at least partly, by inhibition of the activity of MMP-3 and apoptosis induction. The treatment of HepG2 cells with compound 8k resulted in inhibition of in vitro cell migration through wound healing assay and G1 phase of cell cycle arrested. In addition, 8k-induced apoptosis was significantly facilitated in HepG2 cells. Thus, we conclude that DHAA dipeptide derivatives containing the sulfonamide moiety may be the potential MMPs inhibitors with the ability to suppress cells migration.

Full text of DOI:10.1016/j.ejmech.2017.07.020

Accepted Manuscript
Discovery of dehydroabietic acid sulfonamide based derivatives as selective matrix
metalloproteinases inactivators that inhibit cell migration and proliferation
Ri-Zhen Huang, Gui-Bin Liang, Xiao-Chao Huang, Bin Zhang, Mei-Mei Zhou, Zhi-Xin
Liao, Heng-Shan Wang
PII:
S0223-5234(17)30542-1
10.1016/j.ejmech.2017.07.020
EJMECH 9583
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 May 2017
Revised Date: 11 July 2017
Accepted Date: 12 July 2017
Please cite this article as: R.-Z. Huang, G.-B. Liang, X.-C. Huang, B. Zhang, M.-M. Zhou, Z.-X. Liao,
H.-S. Wang, Discovery of dehydroabietic acid sulfonamide based derivatives as selective matrix
metalloproteinases inactivators that inhibit cell migration and proliferation, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.020.
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ACCEPTED MANUSCRIPT
Graphical abstract
Discovery of dehydroabietic acid sulfonamide based derivatives as selective
matrix metalloproteinases inactivators that inhibit cell migration and
proliferation
Ri-Zhen Huang, Gui-Bin Liang, Xiao-Chao Huang, Bin Zhang, Zhi-Xin Liao, and Heng-Shan
Wang
O
C
HN
H
O2
S
N
H
N
N
O
H
O
CH3
CH3
8k
Apoptosis
Cell proliferation inhibition
Cell Migration inhibition
Cell cycle arrest
Selective MMP-3 inhibition

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Discovery of dehydroabietic acid sulfonamide based derivatives as selective
matrix metalloproteinases inactivators that inhibit cell migration and
proliferation
Ri-Zhen Huanga, 1, Gui-Bin Liangb, 1, Xiao-Chao Huanga, Bin Zhangb, Mei-Mei Zhoub, Zhi-Xin
Liaoa , and Heng-Shan Wangb
a Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast
University, Nanjing 211189, China
b
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
(Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi
Normal University, Guilin 541004, PR China
* Corresponding author.
*Corresponding author. E-mail addresses: zxliao@seu.edu.cn (Z.-X. Liao), whengshan@163.com
(H. -S. Wang).
1 Co-first author: These authors contributed equally to this work.
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Abstract
A series of dehydroabietic acid (DHAA) dipeptide derivatives containing the sulfonamide
moiety were designed, synthesized and evaluated for inhibition of MMPs as well as the effects of
in vitro cell migration. These compounds exhibited relatively good inhibition activity against
MMPs with IC50 values in low micromolar range. A docking study of the most active compound
8k revealed key interactions between 8k and MMP-3 in which the sulfonamide moiety and the
dipeptide group were important for improving activity. It is noteworthy that further antitumor
activity screening revealed that some compounds exhibited better inhibitory activity than the
commercial anticancer drug 5-FU. In particular, compound 8k appeared to be the most potent
compound against the HepG2 cell line, at least partly, by inhibition of the activity of MMP-3 and
apoptosis induction. The treatment of HepG2 cells with compound 8k resulted in inhibition of in
vitro cell migration through wound healing assay and G1 phase of cell cycle arrested. In addition,
8k-induced apoptosis was significantly facilitated in HepG2 cells. Thus, we conclude that DHAA
dipeptide derivatives containing the sulfonamide moiety may be the potential MMPs inhibitors
with the ability to suppress cells migration.
keywords: Dehydroabietic acid; Dipeptide; Sulfonamide; Matrix metalloproteinases; Migration
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1. Introduction
Cancer metastasis, the essential hallmarks of cancer, is initiated by migration and invasion of
cancer cells [1]. Metastasis has been a major impediment to effective cancer treatment with
conventional chemotherapeutic drugs and has been the leading cause of cancer-associated death in
several cancers including liver, breast and lung cancers. Notably, several proteins play important
role to initiate or repress cancer metastasis such as matrix metalloproteinases (MMPs) [2].
Therefore, targeting pivotal inhibition of metastasis-associated proteins or signaling pathways are
the critical point for efficient cancer treatment.
The MMPs, which play a crucial role in many normal physiological functions, are a group of
structurally related zinc-dependent endopeptidases involved in the degradation of extracellular
matrix (ECM) components and a diverse array of non-ECM proteins [3]. Recent studies revealed
that MMPs frequently up-regulated in cancer cells, facilitated migration and have a significant
effect on the ability of cancer cells to grow in a secondary site [4-6]. It is also well-known that
MMPs participate in several steps of cancer progression, including cancer cell growth, apoptosis,
migration, invasion, and angiogenesis, thereby playing a key role in the development of human
breast, colon, thyroid, lung, and prostate cancers [7, 8]. In particular, stromelysin-1 (MMP-3) has
been the subject of intense research due to its presence in the vicinity of melanomas and metastatic
tumors associated with breast cancer; it also functions as an active precursor to the action of other
endopeptidases [9-11]. Therefore, MMPs could serve as promising targets for the development of
new therapeutics to suppress cancer metastasis and down-regulation the expressions of MMPs
may have benefits in enhancing the efficacy of anticancer drugs. Some MMPs inhibitors have
been employed as sensitizers in the established cancer therapy over recent years. Despite evident
and convincing data, indicating that MMP inhibitors have a high potential in clinical trial for
cancer [12], their use in the clinic has been stalled due to their limited proof of efficacy and their
adverse side effects, which might be related to insufficient target validation [13-15]. In this reason,
gaining selectivity between the metalloproteases without decreasing inhibitory potency is a
primary goal in the development of MMP inhibitors, which would help to improve their efficacy
and avoid undesirable side effects.
Dehydroabietic acid (DHAA) is a naturally occurring diterpenic resin acid and has been found
to exhibit a broad spectrum of biological activities, especially anticancer activities [16-19].
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Moreover, previous research has shown that DHAA derivatives could act at various stages of
tumor development to inhibit tumor initiation and promotion, as well as to induce tumor cell
differentiation and apoptosis [20-22]. These findings suggest that DHAA may be a promising
starting tool for the discovery of new anticancer agents. On the other hand, sulfonamides are
currently an important group of organic compounds and have been reported for potent antitumor
activity against numerous types of cancers [23, 24]. Derivatives containing sulfonamides moiety
have been widely used as MMPs or carbonic anhydrase inhibitors, some of which are already in
clinical trial [25-28] (Fig.1). Also, from the literature survey it was found that aryl sulfonamides
might act as antitumor agents through several mechanisms [29]. Furthermore, peptides, which are
among the most versatile bioactive molecules, have been reported to act as the inhibitors of some
proteins and to exert their action by binding to membrane receptors [30-32]. In the present work,
we designed and synthesized a series of DHAA dipeptide sulfonamides derivatives as selective
MMPs inhibitors which suppress the migration of liver cancer cells. A docking analysis using the
crystal structure of the MMP-3 was performed to clarify the binding mode of the designed
inhibitors. Moreover, growth inhibitory effects of these compounds were evaluated against four
human tumor cell lines. The migration inhibition, apoptosis inducing effects and cell cycle arrest
in HepG2 cells by the representative target compound 8k was also investigated.
2. Results and Discussion
2.1 Chemistry
The general procedures for the synthesis of DHAA dipeptide sulfonamides derivatives are
shown in Scheme 1. As shown in Scheme 1, compound 3 was synthesized by the treatment of
phenylalanine 1 with phthalic anhydride (2) in the presence of acetic acid according to the
literature [33]. Compound 4 was then obtained by the condensation of compound 3 and oxalyl
chloride, followed by treatment with series of aromatic primary amines. Compounds 5 were
synthesized by the treatment of compounds 4 with hydrazine hydrate in the presence of ethanol at
room temperature. Compound 6 was synthesized by the treatment of dehydroabietic acid with
vitriol at room temperature [34]. Compound 6 was treated with oxalyl chloride to offer compound
7. Compounds 8 were finally acquired by the condensation of compound 7 and compounds 5 (1:2)
for 18 h in the presence of triethylamine at room temperature. Compounds 9 were finally acquired
by the condensation of compound 7 and compounds 5 (1:1) for 6 h in the presence of
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triethylamine at room temperature. The structures of target compounds 8 and 9 were then
confirmed by 1H NMR, 13C NMR and high resolution mass spectrometry (HRMS).
2.2 Biological evaluation
2.2.1 In Vitro MMPs Assays
The synthesized DHAA derivatives containing sulfonamide dipeptide moiety were assayed in
vitro against human recombinant MMP-3, MMP-8 and MMP-9 using synthetic fluorogenic
substrates according to a previously reported procedure [35]. The broad spectrum inhibitor
CGS-27023A was taken as positive control. The IC50 values obtained in the performed in vitro
inhibition assays of DHAA derivatives are summarized in Table 1.
As shown in Table 1, the newly synthesized DHAA derivatives are potent MMPs inhibitors,
with IC50 values mostly in micromolar or submicromolar levels. As can be seen from Table 1, the
sulfonic acid group in compounds 9 formed a sulfonamide to give compounds 8, substantially
increased MMPs inhibitory activities, which may be due to sulfonamides derivatives were more
active than sulfonic acid derivatives. Notably, when comparing the inhibitory activities of the
different DHAA derivatives toward the investigated MMPs, most of the compounds were found to
preferentially inhibit MMP-3. It was established that the structure of the lipophilic group as well
as the configuration of the dipeptide scaffold significantly affected MMP inhibitory activity and
selectivity. Among them, the most potent MMP-3 inhibitor, compound 8k bearing a lipophilic
methyl, effectively inhibited MMP-3 in the submicromolar range (IC50 = 0.4 µM), with
selectivities over the other tested MMPs ranging from 6.6 to 8.8. The effects of the configuration
of amino acid showed that the
amino acid group generally enhanced MMP-3 inhibition,
L-
compared with a
amino acid. Thus, compound 8n showed weak inhibition against MMP-3,
D-
MMP-8 and -9 in the micromolar range. In contrast, its diastereomer 8o, possessing an amino
L-
acid scaffold, was strikingly 2.5−33.5-fold more active toward all three tested MMPs with IC50 1.0,
17.7, 20.9 µM against MMP-3, MMP-8, MMP-9, respectively. On the other hand, analysis of the
effects of the terminal substituents at the R1 position of dipeptide moiety showed that a methyl
group generally enhanced MMP-3 inhibition, compared with a bromine or fluorine group
substitution. When the bromine of compound 8e was replaced with a methyl group at the
3-position on the phenyl ring (compound 8k), the inhibitory potency was increased by 90-fold
against MMP3 when compared to 8e. Replacing the 4-position on the phenyl ring with a methyl
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group at R1 to yield compound 8n and 8o, respectively, resulted in a marked increase in potency,
thus demonstrating that the lipophilic properties of the substituents may play a critical role in the
inhibitory activities against MMP-3. However, the introduction of a fluorine group hindered the
inhibitory activity. Moreover, analogs with a chlorine and fluorine fragments at R1 were much less
active or completely inactive. A variation at R1 could cause a substantial difference in inhibitory
potency, presumably due to different affinity to the binding pocket. Moreover, by the comparison
of IC50 values of different position substituent, it also demonstrated that meta- or para-substituent
at R1 will enhance inhibition activities.
2.2.2. Molecular docking
To understand the interactions between the most active compound 8k and the target of interest
(MMP-3), we performed molecular docking calculations on the active site of MMP-3 using
SYBYL-X 2.0 software and the results are summarized in Fig. 2 and Table 2. The interacting
mode of compound 8k (docking score 11.09), with the best docking score and interaction in the
binding site of the MMP-3 receptor, is described and shown in Fig. 2a. Some key residues, such as
LEU164, ALA165, HIS205, TYR223 and PRO221, as well as hydrogen bonds between the
selected compound and the residues are also labeled. As shown in Fig. 2a, compound 8k was
stabilized predominantly by hydrogen-bond formation with the hydrophilic or hydrophobic groups
of TYR155, LEU164, VAL198, TYR220 and PRO221. The sulfonamide group, which is the
primary important moiety, formed a hydrogen-bond with the HIS166 carbonyl oxygen of the
active site. More important, the sulfonamide moiety coordinates the catalytic zinc atom in a
monodentate fashion (Fig. 2a). Furthermore, the carbonyl oxygen of the dipeptide group in the
sulfonamide chain as an acceptor established one hydrogen bond with LEU164. In addition, the
polar hydrogen of the amide moiety formed a hydrogen bond with the backbone carbonyl of
PRO221, which confirmed that this moiety is also crucial for binding. The benzyl group of
compound 8k plunge deeply into S1’pocket, the enzyme’s specificity pocket of MMP-3 in the
same fashion as the binding mode of the ligand. In here, the benzyl ring is engaged in multiple
hydrophobic interactions with VAL198, HIS201, TYR220, PRO221, LEU222 and TYR223 side
chains. The methyl-aniline group is found sandwiched between ASN162 and LEU222, whereas
the DHAA moiety is flanked by lipophilic residues such as HIS205 and PHE210. Clearly, all these
interactions endow 8k with a low micromolar inhibitory activity toward MMP-3. The binding
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mode of 1SLN-ligand in the MMP-3 binding pocket is shown in Fig. 2b. Hydrogen bonds with
LEU164, GLU202, PRO221 and TYR223 of MMP-3 were observed in the docked structure.
2.2.3 Cytotoxicity Measurement
The in vitro antiproliferative activity of the DHAA derivatives was evaluated by MTT assay
against NCI-H46, HepG2, SKOV-3 and MCF-7 tumor cell lines, with doxorubicin (DOX) as the
positive control. The results are shown in Table 3.
As shown in Table 3, most of the test compounds exhibited inhibitory activity against the tested
tumor cell lines, indicating that the introduction of sulfonamide and dipeptide moiety on the
DHAA skeleton markedly increased anti-tumor activity. Moreover, the antiproliferative activities
of the tested compounds correlated well with their ability to inhibit MMP-3, with activities
depending on the substituents at the R1 position, and followed the order -CH3> -H > -OCH3> -Br>
-F. Most of these compounds possessed methyl moiety in meta- or para-positions of the benzene
ring which may have had certain steric electronic properties which enhanced lipophilicity and the
ability to penetrate the cellular membrane leading to an increased antiproliferative effect. In
addition, the activities of the sulfonamide derivatives 8 were better than those of the sulfoacid
derivatives 9. In this regard, compound 8k with a methyl moiety at meta-positions of the benzene
ring was the most potent compound, depending on the cell line tested, with IC50 values of
10.6±0.8, 4.2±1.1, 7.6±0.3 and 8.5±1.1 µM against NCI-H460, HepG2, SKOV-3 and MCF-7
cancer cells, respectively, and thus was more potent and effective than DHAA.
As the selectivity of antitumor agents for cancer cells over non-malignant cells is important to
avoid numerous severe side effects, all compounds were tested using a non-cancerous liver cell
line (HL-7702). As shown in Table 3, the cytotoxicity activities of most compounds against cancer
cells was much higher than that against HL-7702 normal cells, making them good candidates as
anticancer drugs. It was worth noting that all the DHAA derivatives demonstrated lower
cytotoxicity on HL-7702 than the commercial anticancer drug DOX. These results showed that the
targeted compounds had selective and significant effect on the cell lines.
2.2.4. Compound 8k inhibited the migration of HepG2 cell in vitro
Metastasis plays an important role in later period of cancer progression. Thus, the inhibition of
metastasis is vital for efficient cancer treatment. Migration of cells is a key attribute of practically
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each biological process, particularly cancer cells where it is known to connect with the tumor
progression and metastatic cascade [36]. As cell migration is connected with the metastatic
activity of cancer cells, therefore we have investigated the effect of compound 8k on HepG2 cells
using wound healing assay. The migration of HepG2 cells was recorded by microscopic
observations at 0 h, 24 h and 48 h after treatment with 5 µM and 10 µM of compound 8k. Results
from Fig. 3 clearly showed that there was almost complete healing of wound in control after 48 h,
however healing was strongly suppressed in the cells treated with 8k.
2.2.5 Cell cycle analysis
To determine the possible role of cell cycle arrest in 8k-induced growth inhibition, HepG2 cells
were treated with different concentrations of compound 8k. Cell cycle distribution was
investigated by flow cytometric analysis following staining of DNA with propidium iodide (PI).
After treatment with compound 8k at different concentrations for 24 h, it was observed that S
phase cells gradually decreased and G2 phase cells did not change significantly, while G1 phase
cells compared with the control cells gradually increased, respectively (Fig. 4). These results
suggest that target compound 8k mainly arrested HepG2 cells in the G1 phase.
2.2.6 Compound 8k induces apoptosis in HepG2 cells
In order to confirm whether 8k-induced reduction in cell viability was responsible for the
induction of apoptosis, HepG2 cells were co-stained with PI and Annexin-V/FITC, and the
number of apoptotic cells was estimated by flow cytometry (Fig. 5). Four quadrant images were
observed by flow cytometry analysis: the Q1 area represented damaged cells which appeared
during the process of cell collection, the Q2 region showed necrotic cells and later stage apoptotic
cells; early apoptotic cells were located in the Q3 area and the Q4 area showed normal cells. A
dose-dependent increase in the percentage of apoptotic cells was noted after the cells were treated
with compound 8k at the concentrations of 5 µM and 10 µM for 24 h. As shown in Fig. 5, few
(5.07%) apoptotic cells were present in the control panel, in contrast, the percentage rose to 13.30%
at the concentration of 5 µM after treatment with 8k for 24 h. At concentrations of 10 µM, there
was a further increase to 50.30% after treatment with 8k. These results clearly confirmed that
compared with the control, compound 8k effectively induced apoptosis in HepG2 cells in a
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dose-dependent manner.
2.2.7 Apoptosis assay by acridine orange/ethidium bromide (AO/EB) staining
To further characterize the cell apoptosis induced by compound 8k, AO/EB staining was carried
out to evaluate the accompanying changes in morphology. The cytotoxicity of compound 8k was
evaluated in HepG2 cells following treatment with 5 µM and 10 µM for 24 h. HepG2 cells not
treated with 8k were 24 h controls. The results (Fig. 6) showed that at both concentrations, the
morphology of 8k-treated HepG2 cells had changed significantly. The cell nuclei were stained
yellow green or orange, and the morphology showed pycnosis, membrane blebbing and cell
budding characteristic of apoptosis. The nearly complete absence of red-stained cells showed that
8k treatment was associated with low toxicity. The results thus demonstrated that compound 8k
induced apoptosis with low toxicity.
3. Conclusion
A series of DHAA sulfonamide dipeptide derivatives were designed and synthesized as
migration inhibitors which targeted MMPs with good IC50 values in low micromolar range. These
compounds exhibited better inhibitory activities toward MMP-3 than MMP-8 and MMP-9, with
IC50 values range from 0.4-59.7 µM. A docking study of the most active compound 8k revealed
key interactions between 8k and MMP-3 in which the sulfonamide moiety and the dipeptide group
was important for improving activity. It is noteworthy that further antitumor activity screening
revealed that some compounds exhibited better inhibitory activity than the commercial anticancer
drug 5-FU. In particular, compound 8k (IC50 = 4.18 ± 1.08 µM) exhibited the best anticancer
activity against the HepG2 cell line and displayed slightly weaker inhibitory activity than
doxorubicin. The exposure of compound 8k to HepG2 cells resulted in inhibition of in vitro cell
migration through wound healing assay. The apoptosis-inducing activity investigated by flow
cytometry revealed that compound 8k markedly induced HepG2 cells apoptosis. The apoptosis
inducing effect of 8k was further analysed by AO/EB staining. In addition, cell cycle analysis
indicated that compound 8k arrested the HepG2 cell line in G1 phase. Consequently, the rational
design of DHAA sulfonamide dipeptide derivatives offers significant potential for the discovery of
a new class of MMPs inhibitors with the ability to suppress cancer cells migration. The precise
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mechanism of this action requires further investigation.
4. Experimental
4.1 General information
Compound 3 was synthesized according to the literature [33]. Compound 6 was synthesized
according to the literature [34]. All the chemical reagents and solvents used were of analytical
grade. Silica gel (200-300 mesh) used in column chromatography was provided by Tsingtao
1
Marine Chemistry Co. Ltd. H NMR spectra were recorded on a BRUKER AV-400 spectrometer
with TMS as an internal standard in CDCl3. Mass spectra were determined on an FTMS ESI
spectrometer.
4.2. Synthesis: general procedure for compounds 8a-8u
Compound 3 (1 mmol) added to dry CH2Cl2 (15 mL) and stirred at 0 . After that, oxalyl
chloride (1.5 mmol) was dripped into the mixture and stirred at room temperature for 6 h. After
the reaction, the solvent and excess oxalyl chloride was evaporated under reduced pressure.
Aromatic primary amines (1 mmol) and triethylamine (0.5 mmol) were added to the mixture and
stirred at room temperature for 0.5 h. After the reaction, the solvent was evaporated under reduced
pressure, and the crude product was purified by chromatography on silica gel eluted with
petroleum ether/ethylacetate (V:V = 6:1) to offer compound 4. Compound 4 (1 mmol) and
hydrazine hydrate (3 mmol) were added to ethanol (15 mL) and the mixture was stirred at room
temperature for 8 h. After the reaction was completed, the solvent was evaporated under reduced
pressure, and the crude product was purified by chromatography on silica gel eluted with
petroleum ether/ethyl acetate (V: V = 3: 1) to obtain compounds 5. Compounds 6 (1 mmol) added
to dry H2Cl2 (15 mL) was stirred at 0
and oxalyl chloride (2.0 mmol) was dripped into the
mixture and stirred at room temperature for 6 h. After the reaction, the solvent and excess oxalyl
chloride was evaporated under reduced pressure. Compounds 5 (2 mmol) and triethylamine (1
mmol) were added to the mixture and stirred at room temperature for 18 h. After the reaction, the
solvent was evaporated under reduced pressure, and the crude product was purified by
chromatography on silica gel eluted with petroleum ether/ethyl acetate (V: V = 6:1) to offer
1
compounds 8a-8u. The structures were confirmed by H NMR, 13C NMR and HR-MS (see
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Supporting information).
Compound 8a: Yields 80.1%, as a white solid. Mp: 143.1−148.2 °C. [α]2D0 = +61 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 7.81 (s, 1H), 7.72 (s, 1H), 7.29–7.26 (m, 3H), 7.21–7.15
(m, 8H), 7.14–7.10 (m, 2H), 7.08–7.03 (m, 2H), 6.99 (s, 1H), 6.89–6.79 (m, 4H), 6.63 (d, J = 7.3
Hz, 1H), 5.61 (d, J = 7.3 Hz, 1H), 4.95–4.89 (m, 1H), 4.22–4.17 (m, 1H), 3.66–3.60 (m, 1H),
3.25–3.12 (m, 2H), 3.10–3.04 (m, 1H), 2.98–2.93 (m, 1H), 2.73–2.67 (m, 1H), 2.58–2.51 (m, 1H),
2.19 (d, J = 12.4 Hz, 1H), 1.96 (d, J = 12.5 Hz, 1H), 1.73–1.58 (m, 3H), 1.41–1.31 (m, 3H),
1.26–1.20 (m, 6H, 2 × CH3), 1.17 (s, 3H, CH3), 1.00 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3)
δ 179.30, 169.68, 168.55, 159.59 (1J(C,F) = 242.9 Hz), 159.29 (1J(C,F) = 242.3 Hz), 147.47,
145.04, 141.26, 136.39, 135.46, 134.05, 133.67 (4J(C,F) = 2.8 Hz), 132.87 (4J(C,F) = 2.9 Hz),
129.32, 129.17, 128.95, 128.73, 127.42, 127.14, 125.14, 122.05 (3J(C,F) = 7.9 Hz), 121.60 (3J(C,F)
= 7.8 Hz), 115.47 (2J(C,F) = 22.3 Hz), 115.38 (2J(C,F) = 22.3 Hz), 58.38, 55.40, 47.23, 45.00,
39.41, 38.04, 37.58, 37.06, 36.63, 29.71, 29.56, 29.35, 24.79, 24.28, 24.10, 20.46, 18.26, 16.29.
HR-MS (m/z) (ESI): calcd for C50H54F2N4NaO5S [M + Na]+: 883.3675; found: 883.3665.
Compound 8b: Yields 87.5%, as a white solid. Mp: 148.8−152.1 °C. [α]2D0 = +56 (c 0.1, AcOEt) 1H
NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 9.96 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 7.90–7.88 (m,
1H), 7.62 (d, J = 7.6 Hz, 1H), 7.58 (s, 1H), 7.42–7.46 (m, 1H), 7.37 (d, J = 9.1 Hz, 1H), 7.35–7.31
(m, 1H), 7.28–7.17 (m, 11H), 7.05 (s, 1H), 7.01–6.97 (m, 1H), 4.69–4.57 (m, 1H), 4.17–4.11 (m,
1H), 3.81–3.59 (m, 1H), 3.04–2.81 (m, 4H), 2.62–2.55 (m, 1H), 2.46–2.39 (m, 1H), 2.08 (d, J =
11.8 Hz, 1H), 1.86–1.79 (m, 1H), 1.59 (s, 2H), 1.46 (s, 1H), 1.34–1.24 (m, 6H), 1.12–1.08 (m, 4H),
1.03 (s, 3H, CH3), 0.78 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 177.61, 170.82, 168.96,
153.21 (1J(C,F) = 241.5 Hz), 153.09 (1J(C,F) = 241.2 Hz), 146.60, 144.41, 140.02, 137.90, 136.77,
136.14 (4J(C,F) = 2.8 Hz), 135.65, 135.25 (4J(C,F) = 3.0 Hz), 129.30, 129.02, 128.07, 127.97,
127.71, 126.50, 126.30, 124.15, 121.48, 120.51, 120.00 (3J(C,F) = 7.1 Hz) , 119.43 (3J(C,F) = 6.6
Hz), 119.12 (2J(C,F) = 18.2 Hz), 118.72 (2J(C,F) = 18.3 Hz), 116.96 (2J(C,F) = 21.6 Hz), 116.53
(2J(C,F) = 21.8 Hz), 58.11, 55.29, 46.23, 44.04, 38.56, 37.29, 36.73, 36.56, 35.92, 28.66, 28.22,
24.89, 23.67, 23.05, 19.77, 18.00, 16.13. HR-MS (m/z) (ESI): calcd for C50H52Cl2F2N4NaO5S [M
+ Na]+: 951.2895; found: 951.2883.
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Compound 8c: Yields 62.4%, as a white solid. Mp: 147.2−151.7 °C. [α]2D0 = +65 (c 0.1, AcOEt) 1H
NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.99 (s, 1H), 8.13 (d, J = 9.3 Hz, 1H), 7.65–7.54 (m,
3H), 7.35–7.32 (m, 1H), 7.29–7.18 (m, 12H), 7.16–7.10 (m, 1H), 7.04 (s, 1H), 6.93–6.78 (m, 3H),
4.74–4.56 (m, 1H), 4.23–4.14 (m, 1H), 3.71–3.66 (m, 1H), 3.10–2.92 (m, 3H), 2.89–2.84 (m, 1H),
2.58–2.51 (m, 1H), 2.44–2.35 (m, 1H), 2.08 (d, J = 11.8 Hz, 1H), 1.85–1.77 (m, 1H), 1.58 (s, 2H),
1.43 (s, 1H), 1.30–1.25 (m, 7H), 1.11 (d, J = 6.6 Hz, 3H, CH3), 1.03 (s, 3H, CH3), 0.81 (s, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6) δ 177.59, 170.94, 169.15, 162.12 (1J(C,F) = 240.0 Hz),
161.85 (1J(C,F) = 239.6 Hz), 146.66, 144.28, 140.63 (3J(C,F) = 10.9 Hz), 139.90 (3J(C,F) = 9.7
Hz), 137.93, 136.80, 135.78, 130.39 (3J(C,F) = 9.5 Hz), 129.96 (3J(C,F) = 9.5 Hz), 129.31, 129.02,
128.04, 127.95, 127.73, 126.46, 126.29, 123.90, 115.35 (4J(C,F) = 2.2 Hz), 114.88 (4J(C,F) = 2.2
Hz), 109.84 (2J(C,F) = 21.0 Hz), 109.73 (2J(C,F) = 21.0 Hz), 106.54 (2J(C,F) = 26.0 Hz), 105.92
(2J(C,F) = 26.1 Hz), 58.14, 55.28, 46.22, 44.04, 38.76, 37.28, 36.75, 36.56, 35.94, 28.61, 28.22,
24.75, 23.75, 23.16, 19.76, 17.98, 16.12. HR-MS (m/z) (ESI): calcd for C50H54F2N4NaO5S [M +
Na]+: 883.3675; found: 883.3665.
Compound 8d: Yields 51.6%, as a white solid. Mp: 155.4−158.9 °C. [α]2D0 = +58 (c 0.1, AcOEt) 1H
NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.93 (s, 1H), 8.16 (d, J = 9.3 Hz, 1H), 7.94 (s, 1H),
7.63 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.43 (s, 1H), 7.30–7.15 (m, 12H),
7.18–7.13 (m, 2H), 7.06–7.02 (m, 2H), 4.73–4.59 (m, 1H), 4.20–4.14 (m, 1H), 3.73–3.66 (m, 1H),
3.07–2.89 (m, 4H), 2.63–2.57 (m, 1H), 2.44–2.35 (m, 1H), 2.08 (d, J = 11.9 Hz, 1H), 1.82 (d, J =
13.6 Hz, 1H), 1.61 (d, J = 9.1 Hz, 2H), 1.46 (s, 1H), 1.36–1.23 (m, 7H), 1.12 (d, J = 6.6 Hz, 3H,
CH3), 1.03 (s, 3H CH3), 0.78 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 177.62, 170.93,
169.05, 146.59, 144.31, 140.49, 140.02, 139.62, 137.94, 136.80, 135.62, 130.77, 130.28, 129.32,
129.02, 128.05, 127.95, 127.70, 126.48, 126.29, 126.03, 125.89, 124.11, 122.41, 121.54, 121.45,
121.12, 118.41, 117.90, 58.11, 55.34, 46.24, 44.05, 38.65, 37.29, 36.71, 36.57, 35.94, 28.71, 28.23,
24.92, 23.74, 23.06, 19.79, 18.01, 16.15. HR-MS (m/z) (ESI): calcd for C50H54Br2N4NaO5S [M +
Na]+: 1005.2053; found: 1005.2048.
Compound 8e: Yields 52.4%, as a white solid. Mp: 155.6−158.4 °C. [α]2D0 = +28 (c 0.1, AcOEt) 1H
12

ACCEPTED MANUSCRIPT
NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 10.07 (s, 1H), 8.25 (d, J = 8.8 Hz, 1H), 7.93 (s, 1H),
7.79 (s, 1H), 7.70–7.61 (m, 2H), 7.53–7.49 (m, 1H), 7.33–7.24 (m, 6H), 7.22–7.15 (m, 9H), 7.03
(d, J = 14.4 Hz, 1H), 4.73–4.67 (m, 1H), 4.33–4.15 (m, 1H), 3.73–3.66 (m, 1H), 3.09–2.94 (m,
3H), 2.90–2.84 (m, 1H), 2.71–2.58 (m, 2H), 2.18 (d, J = 11.9 Hz, 1H), 1.88 (d, J = 13.3 Hz, 1H),
1.61–1.54 (m, 3H), 1.49–1.37 (m, 1H), 1.31–1.23 (m, 2H), 1.17 (d, J = 6.7 Hz, 3H, CH3), 1.11 (d,
J = 6.7 Hz, 3H, CH3), 1.04 (s, 3H, CH3), 0.97 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ
177.37, 171.00, 169.36, 146.92, 143.81, 140.45, 139.78, 139.75, 137.85, 136.61, 136.50, 130.73,
130.48, 129.18, 129.13, 128.00, 127.95, 127.78, 126.41, 126.26, 126.12, 125.89, 123.34, 121.72,
121.52, 121.48, 121.30, 118.02, 117.98, 58.22, 55.32, 46.20, 46.17, 43.84, 38.90, 36.88, 36.67,
36.65, 35.67, 28.98, 28.07, 24.50, 24.06, 23.70, 19.65, 18.16, 16.21. HR-MS (m/z) (ESI): calcd for
C50H54Br2N4NaO5S [M + Na]+: 1005.2053; found: 1005.2044.
Compound 8f: Yields 88.9%, as a white solid. Mp: 150.7−154.1 °C. [α]2D0 = +65 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.78 (s, 1H), 7.65 (s, 1H), 7.40 (d, J = 7.7 Hz, 2H),
7.30–7.24 (m, 15H), 7.09 (t, J = 7.0 Hz, 4H), 7.04 (s, 1H), 6.60 (d, J = 7.6 Hz, 1H), 5.57 (d, J =
7.3 Hz, 1H), 4.91–4.97 (m, 1H), 4.16–4.13 (m, 1H), 3.72–3.63 (m, 1H), 3.24–3.16 (m, 3H),
3.04–2.99 (m, 1H), 2.70 (t, J = 15.8 Hz, 2H), 2.27 (d, J = 12.2 Hz, 1H), 1.97 (d, J = 11.8 Hz, 1H),
1.90 (s, 1H), 1.65–1.43 (m, 5H), 1.25 (d, J = 6.7 Hz, 6H, 2 × CH3), 1.19 (s, 3H, CH3), 1.10 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3) δ 179.19, 169.71, 168.35, 147.66, 145.03, 141.45, 137.59,
136.90, 136.83, 135.73, 134.07, 129.45, 129.42, 129.07, 129.03, 128.88, 127.53, 127.22, 125.37,
124.94, 124.61, 120.29, 120.17, 58.63, 55.58, 47.30, 44.76, 39.58, 37.98, 37.50, 37.21, 36.91,
29.82, 29.41, 25.11, 24.47, 24.15, 20.36, 18.40, 16.46. HR-MS (m/z) (ESI): calcd for
C50H56N4NaO5S [M + Na]+: 847.3863; found: 847.3857.
Compound 8g: Yields 85.7%, as a white solid. Mp: 151.2−154.6 °C. [α]2D0 = +37 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 7.74 (d, J = 1.8 Hz, 2H), 7.36 (d, J = 7.7 Hz, 2H),
7.28–7.17 (m, 15H), 7.09–7.03 (m, 4H), 6.99 (s, 1H), 6.65 (d, J = 7.7 Hz, 1H), 5.67 (d, J = 7.4 Hz,
1H), 4.99–4.94 (m, 1H), 4.16–4.11 (m, 1H), 3.67–3.58 (m, 1H), 3.22–3.12 (m, 3H), 3.01–2.96 (m,
1H), 2.71–2.53 (m, 2H), 2.21 (d, J = 12.1 Hz, 1H), 2.03 (s, 1H), 1.94 (d, J = 12.4 Hz, 1H),
1.61–1.39 (m, 5H), 1.20 (d, J = 6.7 Hz, 6H, 2 × CH3), 1.15 (s, 3H, CH3), 1.04 (s, 3H, CH3). 13C
13

ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl3) δ 179.19, 169.85, 168.41, 147.60, 144.96, 141.39, 137.59, 136.90,
136.79, 135.70, 134.09, 129.42, 129.40, 128.98, 128.80, 127.46, 127.15, 125.26, 124.89, 124.56,
120.29, 120.17, 58.61, 55.54, 47.25, 44.68, 39.57, 38.05, 37.42, 37.17, 36.86, 29.79, 29.36, 25.10,
24.48, 24.09, 20.32, 18.35, 16.44. HR-MS (m/z) (ESI): calcd for C50H56N4NaO5S [M + Na]+:
847.3863; found: 847.3857.
Compound 8h: Yields 76.3%, as a white solid. Mp: 128.6−133.7 °C. [α]2D0 = +62 (c 0.1, AcOEt)1H
NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 7.74 (s, 1H), 7.65 (s, 1H), 7.29–7.19 (m, 9H), 7.16 (s,
1H), 7.13–7.09 (m, 3H), 7.07 (d, J = 6.8 Hz, 1H), 7.01 (s, 1H), 6.92 (s, 1H), 6.85 (d, J = 8.0 Hz,
1H), 6.67 (d, J = 8.1 Hz, 1H), 6.63–6.61 (m, 3H), 5.56 (d, J = 7.6 Hz, 1H), 4.97–4.88 (m, 1H),
4.28–4.18 (m, 1H), 3.74 (s, 6H, 2 × OCH3), 3.69–3.69 (m, 1H), 3.26–3.17 (m, 2H), 3.15–3.09 (m,
1H), 3.02–2.97 (m, 1H), 2.74–2.68 (m, 1H), 2.65–2.58 (m, 1H), 2.23 (d, J = 13.3 Hz, 1H), 2.00 (d,
J = 12.6 Hz, 1H), 1.93 (s, 1H), 1.68–1.56 (m, 3H), 1.36 (d, J = 11.8 Hz, 2H), 1.28–1.23 (m, 6H, 2
× CH3), 1.19 (s, 3H, CH3), 1.01 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 179.27, 169.43,
168.33, 160.24, 160.14, 147.72, 145.04, 141.59, 138.75, 138.02, 136.60, 135.59, 133.97, 129.76,
129.69, 129.49, 129.37, 129.23, 128.98, 128.88, 127.66, 127.34, 125.42, 112.43, 112.23, 110.70,
110.52, 106.08, 105.75, 58.65, 55.43, 47.37, 45.08, 39.50, 37.80, 37.73, 37.21, 36.91, 29.66, 29.54,
24.84, 24.41, 24.30, 20.69, 18.39, 16.42. HR-MS (m/z) (ESI): calcd for C52H60N4NaO7S [M +
Na]+: 907.4074; found: 907.4067.
Compound 8i: Yields 79.8%, as a white solid. Mp: 129.1−134.2 °C. [α]2D0 = +44 (c 0.1, AcOEt)1H
NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.71 (s, 1H), 7.58 (s, 1H), 7.25–7.16 (m, 9H), 7.10–7.01
(m, 5H), 6.98 (s, 1H), 6.93 (t, J = 2.1 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H),
6.59–6.56 (m, 2H), 6.53 (d, J = 7.6 Hz, 1H), 5.46 (d, J = 7.2 Hz, 1H), 4.90–4.84 (m, 1H),
4.09–4.04 (m, 1H), 3.69 (d, J = 1.0 Hz, 6H, 2 × OCH3), 3.62–3.53 (m, 1H), 3.22–3.05 (m, 3H),
2.97–2.91 (m, 1H), 2.68–2.56 (m, 2H), 2.19 (d, J = 12.3 Hz, 1H), 1.89–1.83 (m, 2H), 1.48–1.41
(m, 3H), 1.30–1.21 (m, 2H), 1.19–1.16 (m, 6H, 2 × CH3), 1.12 (s, 3H, CH3), 1.04 (s, 3H, CH3). 13C
NMR (100 MHz, CDCl3) δ 179.23, 169.74, 168.32, 160.17, 160.09, 147.68, 145.02, 141.47,
138.82, 138.10, 136.84, 135.68, 134.04, 129.70, 129.42, 129.38, 129.07, 128.87, 127.52, 127.19,
125.41, 112.44, 112.34, 110.87, 110.43, 105.93, 105.85, 58.64, 55.64, 55.39, 55.36, 47.31, 44.85,
14

ACCEPTED MANUSCRIPT
39.50, 37.88, 37.47, 37.23, 36.86, 29.82, 29.40, 25.12, 24.50, 24.11, 20.32, 18.42, 16.45. HR-MS
(m/z) (ESI): calcd for C52H60N4NaO7S [M + Na]+: 907.4074; found:907.4068.
Compound 8j: Yields 76.8%, as a white solid. Mp: 135.8−139.2 °C. [α]2D0 = +63 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.24–7.18 (m, 10H), 7.11–7.02
(m, 6H), 6.98–6.95 (m, 2H), 6.78 (t, J = 7.2 Hz, 2H), 6.53 (d, J = 7.4 Hz, 1H), 5.43 (d, J = 7.5 Hz,
1H), 4.98–4.88 (m, 1H), 4.20–4.15 (m, 1H), 3.67–3.61 (m, 1H), 3.24–3.08 (m, 3H), 3.00–2.94 (m,
1H), 2.66–2.42 (m, 2H), 2.15 (d, J = 6.8 Hz, 6H, 2 × CH3), 1.92 (d, J = 11.5 Hz, 1H), 1.66–1.53
(m, 5H), 1.41–1.34 (m, 2H), 1.26–1.22 (m, 6H, 2 × CH3), 1.09 (s, 3H, CH3), 0.93 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3) δ 179.11, 169.56, 168.28, 147.52, 144.89, 141.32, 138.78, 138.73,
137.47, 136.72, 136.50, 135.55, 134.05, 129.41, 129.27, 129.00, 128.73, 128.66, 127.43, 127.11,
125.57, 125.20, 125.15, 120.83, 120.63, 117.27, 117.10, 58.49, 55.30, 47.24, 45.00, 39.53, 37.95,
37.60, 37.08, 36.65, 29.73, 29.60, 29.37, 24.78, 24.23, 21.42, 20.48, 18.25, 16.31. HR-MS (m/z)
(ESI): calcd for C52H61N4O5S [M + H]+: 853.4357; found:853.4363.
Compound 8k: Yields 78.9%, as a white solid. Mp: 136.6−139.8 °C. [α]2D0 = +36 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.74 (s, 1H), 7.66 (s, 1H), 7.24–7.20 (m, 10H), 7.16–7.07
(m, 6H), 6.99 (d, J = 8.5 Hz, 2H), 6.87 (t, J = 6.3 Hz, 2H), 6.63 (s, 1H), 5.68–5.56 (m, 1H),
5.02–4.89 (m, 1H), 4.11 (d, J = 3.9 Hz, 1H), 3.64–3.61 (m, 1H), 3.23–3.13 (m, 3H), 3.01–2.95 (m,
1H), 2.66 (d, J = 6.3 Hz, 2H), 2.25 (d, J = 7.4 Hz, 6H, 2 × CH3), 2.04–1.94 (m, 2H), 1.57–1.42 (m,
6H), 1.20 (d, J = 5.4 Hz, 6H, 2 × CH3), 1.16 (s, 3H, CH3), 1.06 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 179.11, 169.71, 168.24, 147.55, 144.87, 141.30, 138.81, 138.77, 137.43, 136.78, 136.75,
135.63, 134.01, 129.35, 129.33, 128.94, 128.73, 128.71, 127.38, 127.06, 125.63, 125.27, 120.78,
120.69, 117.25, 117.15, 58.52, 55.51, 47.18, 44.64, 39.48, 37.94, 37.40, 37.11, 36.80, 29.73, 29.28,
25.05, 24.50, 23.96, 21.43, 20.26, 18.30, 16.41. HR-MS (m/z) (ESI): calcd for C52H61N4O5S [M +
H]+: 853.4357; found:853.4363.
Compound 8l: Yields 81.4%, as a white solid. Mp: 135.9−140.1°C. [α]2D0 = +67 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.32–8.29 (m, 1H), 8.16–8.14 (m, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.70
(s, 1H), 7.35–7.18 (m, 9H), 7.12–7.09 (m, 2H), 7.07–6.99 (m, 3H), 6.97–6.88 (m, 2H), 6.84–6.82
15

ACCEPTED MANUSCRIPT
(m, 1H), 6.79–6.74 (m, 1H), 6.48 (d, J = 7.3 Hz, 1H), 5.43 (d, J = 7.5 Hz, 1H), 4.88–4.79 (m, 1H),
4.15–4.10 (m, 1H), 3.73 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.66–3.61 (m, 1H), 3.27–3.22 (m,
1H), 3.14 (d, J = 7.1 Hz, 2H), 2.98–2.93 (m, 1H), 2.71 (t, J = 8.3 Hz, 2H), 2.27 (d, J = 12.1 Hz,
1H), 2.04–2.00 (m, 1H), 1.68–1.60 (m, 5H), 1.51 (d, J = 6.3 Hz, 1H), 1.38 (d, J = 15.4 Hz, 2H),
1.28 (d, J = 6.7 Hz, 3H, CH3), 1.23 (d, J = 6.7 Hz, 3H, CH3), 1.20 (s, 3H, CH3), 1.05 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3) δ 178.30, 169.13, 167.96, 148.13, 148.08, 147.58, 145.04, 141.26,
136.56, 135.63, 134.29, 129.49, 129.40, 129.01, 128.82, 128.74, 127.37, 127.16, 127.09, 126.74,
124.96, 124.41, 124.27, 121.11, 120.98, 119.90, 119.84, 110.09, 110.06, 58.98, 55.72, 55.71,
55.42, 47.26, 44.93, 40.19, 38.63, 37.77, 37.20, 37.17, 29.69, 29.58, 24.70, 24.33, 24.30, 20.71,
18.43, 16.51. HR-MS (m/z) (ESI): calcd for C52H60KN4O7S [M + K]+: 923.3814; found: 923.3816.
Compound 8m: Yields 82.4%, as a white solid. Mp: 136.8−140.4 °C. [α]2D0 = +32 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.77 (s, 1H), 7.72 (s, 1H), 7.28–7.22 (m, 11H), 7.13–7.09
(m, 4H), 7.03 (s, 1H), 6.82–6.70 (m, 5H), 5.72 (d, J = 7.5 Hz, 1H), 5.00–4.95 (m, 1H), 4.17–4.12
(m, 1H), 3.76 (s, 6H, 2 × OCH3), 3.76–3.61 (m, 1H), 3.25–3.12 (m, 3H), 3.03–2.98 (m, 1H),
2.69–2.65 (m, 2H), 2.23 (d, J = 13.7 Hz, 2H), 1.98 (d, J = 12.5 Hz, 1H), 1.58–1.46 (m, 5H),
1.24–1.21 (m, 6H, 2 × CH3), 1.18 (s, 3H, CH3), 1.07 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
178.97, 169.54, 168.18, 156.72, 156.50, 147.53, 144.91, 141.32, 136.83, 135.73, 134.04, 130.57,
129.88, 129.39, 129.37, 128.89, 128.71, 127.35, 127.05, 125.22, 122.13, 121.92, 114.07, 114.05,
58.40, 55.47, 55.33, 47.15, 44.58, 39.57, 38.12, 37.35, 37.08, 36.77, 29.76, 29.28, 25.03, 24.38,
24.06, 20.27, 18.30, 16.38. HR-MS (m/z) (ESI): calcd for C52H60KN4O7S [M + K]+: 923.3814;
found: 923.3810.
Compound 8n: Yields 79.5%, as a white solid. Mp: 139.0−144.6 °C. [α]2D0 = +59 (c 0.1, AcOEt)1H
NMR (400 MHz, CDCl3) δ 8.76 (s, 1H), 7.71 (s, 2H), 7.34–7.12 (m, 11H), 7.11–7.06 (m, 2H),
7.03 (d, J = 8.5 Hz, 2H), 6.96 (t, J = 8.6 Hz, 5H), 6.69 (d, J = 7.0 Hz, 1H), 5.80–5.56 (m, 1H),
5.02–4.85 (m, 1H), 4.25–4.11 (m, 1H), 3.74–3.59 (m, 1H), 3.12 (m, 3H), 2.98 (m, 1H), 2.71–2.50
(m, 2H), 2.26 (s, 6H, 2 × CH3), 2.18 (d, J = 11.3 Hz, 1H), 1.96 (d, J = 16 Hz, 1H), 1.67–1.46 (m,
4H), 1.39–1.29 (m, 2H), 1.24–1.20 (m, 6H, 2 × CH3), 1.14 (s, 3H, CH3), 0.97 (s, 3H, CH3). 13C
NMR (100 MHz, CDCl3) δ 179.16, 169.76, 168.40, 147.55, 144.93, 141.31, 136.65, 135.73,
16

ACCEPTED MANUSCRIPT
135.09, 134.44, 134.37, 134.32, 134.02, 129.49, 129.36, 128.95, 128.72, 127.40, 127.09, 125.09,
120.39, 120.17, 58.51, 55.40, 47.26, 45.03, 39.80, 38.18, 37.60, 37.13, 36.67, 29.66, 29.38, 24.84,
24.35, 24.23, 20.95, 20.51, 18.32, 16.35. HR-MS (m/z) (ESI): calcd for C52H61N4O5S [M + H]+:
853.4357; found:853.4360.
Compound 8o: Yields 75.2%, as a white solid. Mp: 139.7−145.3 °C. [α]2D0 = +35 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.79 (s, 1H), 7.40 (s, 1H), 7.36–7.20 (m, 11H), 7.14–7.04
(m, 9H), 6.52 (d, J = 7.4 Hz, 1H), 5.39 (d, J = 7.1 Hz, 1H), 4.86–4.80 (m, 1H), 4.08–4.03 (m, 1H),
3.70–3.59 (m, 1H), 3.29–3.24 (m, 1H), 3.22–3.13 (m, 2H), 3.00–2.94 (m , 1H), 2.78–2.65 (m, 2H),
2.31 (d, J = 2.7 Hz, 6H, 2 × CH3), 2.03–2.00 (m, 1H), 1.73–1.68 (m, 4H), 1.60–1.49 (m, 2H),
1.41–1.32 (m, 1H), 1.25 (m, 6H, 2 × CH3), 1.20 (s, 3H, CH3), 1.14 (s, 3H, CH3). 13C NMR (100
MHz, CDCl3) δ 179.08, 169.30, 168.05, 147.69, 145.03, 141.53, 136.93, 135.75, 134.91, 134.67,
134.32, 134.24, 133.84, 129.57, 129.56, 129.48, 129.43, 129.14, 128.97, 128.85, 127.59, 127.27,
125.56, 120.32, 120.16, 58.51, 55.62, 47.32, 44.75, 39.59, 37.85, 37.56, 37.22, 36.88, 29.86, 29.43,
25.13, 24.52, 24.18, 21.02, 21.00, 20.34, 18.44, 16.44. HR-MS (m/z) (ESI): calcd for
C52H61N4O5S [M + H]+: 853.4357; found:853.4365.
Compound 8p: Yields 69.4%, as a white solid. Mp: 122.4−126.8 °C. [α]2D0 = +68 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.49 (d, J = 11.9 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.09 (s, 1H), 7.77
(d, J = 6.1 Hz, 2H), 7.28 (m, 9H), 7.17–6.94 (m, 9H), 6.46 (d, J = 7.2 Hz, 1H), 5.48–5.30 (m, 1H),
5.03–4.87 (m, 1H), 4.23–4.13 (m, 1H), 3.71–3.57 (m, 1H), 3.29–3.14 (m, 3H), 3.07–2.95 (m, 1H),
2.72 (s, 2H), 2.30 (d, J = 10.8 Hz, 1H), 2.09 (d, J = 9.0 Hz, 1H), 1.64 (d, J = 12.1 Hz, 3H), 1.38 (s,
3H), 1.31–1.22 (m, 6H, 2 × CH3), 1.20 (d, J = 5.9 Hz, 3H, CH3), 1.07 (s, 3H, CH3). 13C NMR (100
MHz, CDCl3) δ 179.12, 169.73, 168.57, 152.68 (1J(C,F) = 243.2 Hz), 152.61 (1J(C,F) = 242.5 Hz),
147.51, 144.88, 141.52, 136.31, 135.15, 133.64, 129.23, 129.20, 129.08, 128.84, 128.70, 127.54,
127.22, 125.87 (2J(C,F) = 10.3 Hz), 125.33 (3J(C,F) = 8.4 Hz), 124.61 (3J(C,F) = 7.5 Hz), 124.86,
124.78, 124.50 (3J(C,F) = 3.5 Hz), 124.42 (3J(C,F) = 3.3 Hz), 122.06, 121.97, 114.97 (2J(C,F) =
19.4 Hz), 114.92 (2J(C,F) = 19.2 Hz), 58.61, 55.08, 47.25, 44.74, 39.29, 37.61, 37.37, 37.04,
36.86, 29.84, 29.45, 24.70, 24.32, 24.12, 20.64, 18.30, 16.33. HR-MS (m/z) (ESI): calcd for
C50H54F2KN4O5S [M + K]+: 899.3414; found: 899.3409.
17

ACCEPTED MANUSCRIPT
Compound 8q: Yields 73.3%, as a white solid. Mp: 122.3−126.5 °C. [α]2D0 = +29 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.13 (t, J = 7.8 Hz, 1H), 7.96 (t, J = 7.8 Hz, 1H), 7.69 (s,
1H), 7.63–7.60 (m, 1H), 7.27–7.12 (m, 8H), 7.06–6.94 (m, 9H), 6.40–6.31 (m, 1H), 5.38–5.31 (m,
1H), 4.89–4.32 (m, 1H), 4.09–4.04 (m, 1H), 3.56–3.51 (m, 1H), 3.24–3.01 (m, 3H), 2.98–2.92 (m,
1H), 2.70–2.50 (m, 2H), 2.17 (d, J = 12.6 Hz, 1H), 1.87–1.84 (m, 2H), 1.65–1.35 (m, 5H),
1.19–1.09 (m, 9H, 3 × CH3), 1.04 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 179.12, 169.75,
168.42, 152.72 (1J(C,F) = 243.5 Hz), 152.52 (1J(C,F) = 243.6 Hz), 147.50, 144.88, 141.43, 136.55,
135.20, 133.67, 129.23, 129.20, 129.04, 128.88, 128.78, 128.69, 127.54, 127.21, 125.87 (2J(C,F)
= 10.5 Hz), 125.83 (2J(C,F) = 10.6 Hz), 125.33 (3J(C,F) = 7.2 Hz), 125.17 (3J(C,F) = 7.4 Hz),
124.88, 124.81, 124.48 (3J(C,F) = 3.1 Hz), 124.45 (3J(C,F) = 2.9 Hz), 122.04, 121.85, 115.00
(2J(C,F) = 19.0 Hz), 58.63, 55.34, 47.23, 44.42, 39.36, 37.27, 37.07, 37.04, 36.69, 29.74, 29.39,
25.04, 24.21, 24.18, 20.16, 18.30, 16.29. HR-MS (m/z) (ESI): calcd for C50H54F2KN4O5S [M +
K]+: 899.3414; found: 899.3408.
Compound 8r: Yields 83.6%, as a white solid. Mp: 120.0−125.1 °C. [α]2D0 = +60 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 7.71 (s, 1H), 7.61 (s, 1H), 7.27–7.15 (m, 11H), 7.09 (d, J
= 6.4 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.98 (s, 1H), 6.75–6.65 (m, 5H), 5.62 (d, J = 7.7 Hz, 1H),
4.94–4.88 (m, 1H), 4.21–4.16 (m, 1H), 3.73 (s, 6H, 2 × OCH3), 3.73–3.61 (m, 1H), 3.22–3.07 (m,
3H), 3.00–2.94 (m, 1H), 2.71–2.65 (m, 1H), 2.61–2.54 (m, 1H), 2.19 (d, J = 12.5 Hz, 1H), 1.97 (d,
J = 9.1 Hz, 2H), 1.65–1.57 (m, 3H), 1.38–1.33 (m, 2H), 1.25–1.19 (m, 6H, 2 × CH3), 1.16 (s, 3H,
CH3), 1.00 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 179.10, 169.51, 168.29, 156.78, 156.48,
147.55, 144.99, 141.30, 136.65, 135.73, 134.26, 130.75, 129.97, 129.52, 129.36, 128.98, 128.75,
127.43, 127.13, 125.12, 122.14, 121.82, 114.08, 114.04, 58.44, 55.57, 55.54, 55.31, 47.28, 45.03,
39.74, 38.25, 37.61, 37.15, 36.70, 29.67, 29.41, 24.90, 24.30, 24.28, 20.56, 18.35, 16.38. HR-MS
(m/z) (ESI): calcd for C52H60KN4O7S [M + K]+: 923.3814; found:923.3820.
1
Compound 8s: Yields 82.4%, as a white solid. Mp: 120.2−125.4 °C. [α]2D0 = +29 (c 0.1, AcOEt) H
NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.74 (s,
1H), 7.64 (s, 1H), 7.20–7.11 (m, 8H), 7.02–7.00 (m, 2H), 6.96–6.89 (m, 3H), 6.86–6.79 (m, 2H),
18

ACCEPTED MANUSCRIPT
6.74–6.68 (m, 2H), 6.39 (d, J = 7.6 Hz, 1H), 5.48 (d, J = 7.2 Hz, 1H), 4.82–4.77 (m, 1H),
4.05–4.00 (m, 1H), 3.64 (s, 3H, OCH3), 3.56–3.51 (m, 4H), 3.14–3.08 (m, 3H), 3.06–3.03 (m, 1H),
2.93–2.88 (m, 2H), 2.16 (d, J = 12.8 Hz, 1H), 1.86–1.79 (m, 2H), 1.57–1.38 (m, 5H), 1.17–1.12
13
(m, 6H, 2 × CH3), 1.08 (s, 3H, CH3), 0.98 (s, 3H, CH3). C NMR (100 MHz, CDCl3) δ 178.32,
169.21, 167.89, 148.11, 148.01, 147.41, 144.84, 141.17, 136.78, 135.62, 134.15, 129.33, 128.86,
128.76, 128.65, 127.24, 127.08, 127.03, 126.62, 125.10, 124.37, 124.19, 120.97, 120.90, 119.82,
119.78, 110.10, 110.07, 58.92, 55.64, 55.44, 47.16, 44.59, 39.95, 38.16, 37.29, 37.10, 36.78, 29.79,
29.40, 25.04, 24.22, 24.04, 20.23, 18.40, 16.40. HR-MS (m/z) (ESI): calcd for C52H60KN4O7S [M
+ K]+: 923.3814; found: 923.3815.
Compound 8t: Yields 76.7%, as a white solid. Mp: 121.1−124.6 °C. [α]2D0 = +63 (c 0.1, AcOEt)1H
NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.77 (s, 1H), 7.60 (d, J = 7.9 Hz,
1H), 7.48 (s, 1H), 7.37–7.27 (m, 5H), 7.25–7.22 (m, 3H), 7.20–7.02 (m, 10H), 6.39 (d, J = 7.3 Hz,
1H), 5.24 (d, J = 7.1 Hz, 1H), 4.87–4.81 (m, 1H), 4.12–4.07 (m, 1H), 3.60 (m, 1H), 3.33–3.12 (m,
3H), 2.96–2.91 (m, 1H), 2.70 (t, J = 8.6 Hz, 2H), 2.28 (d, J = 12.5 Hz, 1H), 2.07 (s, 3H, CH3),
1.93 (d, J = 10.1 Hz, 4H), 1.49–1.46 (m, 2H), 1.29–1.26 (m, 4H), 1.25–1.22 (m, 6H, 2 × CH3),
1.17 (s, 3H, CH3), 1.13 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 179.19, 169.43, 168.39,
147.73, 145.02, 141.61, 137.01, 135.69, 135.54, 134.93, 133.72, 130.59, 130.55, 129.42, 129.37,
129.30, 129.22, 129.07, 128.82, 128.68, 127.66, 127.32, 126.79, 126.77, 125.67, 125.61, 125.15,
122.77, 122.29, 58.56, 55.65, 47.33, 44.81, 39.13, 37.51, 37.35, 37.19, 36.74, 29.83, 29.54, 25.11,
24.67, 24.05, 20.16, 18.42, 17.77, 17.37, 16.37. HR-MS (m/z) (ESI): calcd for C52H60N4NaO5S [M
+ Na]+: 875.4176; found: 875.4171.
Compound 8u: Yields 74.5%, as a white solid. Mp: 120.9−124.3 °C. [α]2D0 = +31 (c 0.1, AcOEt)1H
NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.60 (d, J = 8.0 Hz,
1H), 7.56 (s, 1H), 7.36–7.24 (m, 9H), 7.20–7.07 (m, 9H), 6.47 (d, J = 7.3 Hz, 1H), 5.36 (d, J = 7.1
Hz, 1H), 5.00–4.80 (m, 1H), 4.17–4.12 (m, 1H), 3.66–3.60 (m, 1H), 3.32–3.16 (m, 3H), 3.01–2.92
(m, 1H), 2.73 (t, J = 8.6 Hz, 2H), 2.30 (d, J = 12.7 Hz, 1H), 2.09 (s, 3H, CH3), 1.95 (s, 3H, CH3),
1.74–1.61 (m, 3H), 1.52–1.45 (m, 2H), 1.37 (s, 2H), 1.27–1.27 (m, 6H, 2 × CH3), 1.19 (s, 3H,
CH3), 1.15 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 179.17, 169.53, 168.45, 147.71, 145.01,
19

ACCEPTED MANUSCRIPT
141.57, 137.00, 135.65, 135.58, 134.93, 133.80, 130.58, 130.53, 129.43, 129.36, 129.16, 129.01,
128.89, 128.80, 127.60, 127.27, 126.73, 125.61, 125.21, 122.85, 122.46, 58.56, 55.61, 47.31,
44.79, 39.12, 37.49, 37.42, 37.18, 36.75, 29.84, 29.50, 25.09, 24.66, 24.01, 20.17, 18.41, 17.75,
17.36, 16.36. HR-MS (m/z) (ESI): calcd for C52H60N4NaO5S [M + Na]+: 875.4176; found:
875.4171.
4.3. Synthesis: general procedure for compounds 9a-9i
Compounds 5 (1 mmol) and triethylamine (0.5 mmol) were added to the mixture and stirred at
room temperature for 2 h. After the reaction, the solvent was evaporated under reduced pressure,
and the crude product was purified by chromatography on silica gel eluted with petroleum
ether/ethyl acetate (V : V = 6:1) to offer compounds 9a-9i. The structures were confirmed by 1H
NMR, 13C NMR and HR-MS (see Supporting information).
Compound 9a: Yields 61.8%, as a white solid. Mp: 164.8−169.7 °C. [α]2D0 = +43 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 7.87 (s, 1H), 7.33 (d, J = 10.6 Hz, 1H), 7.28–7.25 (m,
3H), 7.20–7.16 (m, 2H), 7.13 (s, 1H), 7.03–6.97 (m, 2H), 6.70 (t, J = 7.6 Hz, 1H), 6.62 (d, J = 7.0
Hz, 1H), 4.98–4.93 (m, 1H), 4.00–3.94 (m, 1H), 3.26–3.21 (m, 1H), 3.14–3.09 (m, 1H), 2.89–2.83
(m, 1H), 2.74–2.64 (m, 1H), 2.35 (d, J = 12.8 Hz, 1H), 2.11 (d, J = 12.4 Hz, 1H), 1.79–1.75 (m,
5H), 1.57–1.43 (m, 2H), 1.32 (t, J = 6.5 Hz, 6H, 2 × CH3), 1.27 (s, 3H, CH3), 1.21 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3) δ 179.47, 169.84, 162.90 (1J(C,F) = 243.2 Hz), 148.13, 145.69,
143.99, 140.05, 139.29 (3J(C,F) = 10.8 Hz), 136.32, 129.85 (3J(C,F) = 9.2 Hz), 129.57, 129.24,
128.90, 127.35, 124.76, 115.13 (4J(C,F) = 2.5 Hz), 111.03 (2J(C,F) = 21.3 Hz), 107.36 (2J(C,F) =
26.0 Hz), 55.63, 47.39, 44.87, 38.01, 37.63, 37.38, 36.92, 29.73, 28.67, 25.01, 23.89, 23.85, 20.51,
18.38, 16.40. HR-MS (m/z) (ESI): calcd for C35H40FN2O5S [M − H]−: 619.2647; found: 619.2657.
Compound 9b: Yields 68.9%, as a white solid. Mp: 121.8−126.4 °C. [α]2D0 = +12 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.32 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.87 (s, 1H), 7.36–7.23 (m, 5H),
7.18 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.96 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.51 (d, J
= 7.5 Hz, 1H), 4.93–4.88 (m, 1H), 4.05–3.91 (m, 1H), 3.77 (s, 3H, -OCH3), 3.27–3.15 (m, 2H),
2.87–2.72 (m, 2H), 2.34 (d, J = 12.8 Hz, 1H), 2.09 (d, J = 12.6 Hz, 1H), 1.91–1.70 (m, 4H),
20

ACCEPTED MANUSCRIPT
1.63–1.49 (m, 3H), 1.32 (t, J = 7.2 Hz, 6H, 2 × CH3), 1.25 (s, 3H, CH3), 1.21 (s, 3H, CH3). 13C
NMR (100 MHz, CDCl3) δ 178.23, 169.17, 148.34, 148.21, 145.57, 144.31, 139.92, 136.84,
129.53, 129.43, 128.86, 127.14, 124.78, 124.29, 121.06, 119.93, 110.17, 55.72, 55.55, 47.23,
44.53, 38.37, 37.69, 37.39, 36.99, 29.92, 28.64, 24.96, 23.91, 23.83, 20.14, 18.45, 16.46. HR-MS
(m/z) (ESI): calcd for C36H43N2O6S [M − H]−: 631.2847; found: 631.2855.
Compound 9c: Yields 63.7%, as a white solid. Mp: 160.6−166.8 °C. [α]2D0 = +40 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.87 (s, 1H,), 7.87 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.29–7.24 (m,
3H), 7.21 (d, J = 7.6 Hz, 2H), 7.17–7.09 (m, 3H), 7.02 (t, J = 8.0 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H),
5.03–4.98 (m, 1H), 4.01–3.95 (m, 1H), 3.25–3.13 (m, 2H), 2.85–2.79 (m, 1H), 2.72–2.61 (m, 1H),
2.34 (d, J = 12.8 Hz, 1H), 2.12 (d, J = 10.7 Hz, 1H), 1.91–1.67 (m, 5H), 1.47–1.43 (m, 2H), 1.32
(d, J = 6.7 Hz, 6H, 2 × CH3), 1.27 (s, 3H, CH3), 1.21 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
179.03, 169.67, 148.21, 145.61, 144.07, 140.02, 137.63, 136.48, 129.55, 129.41, 128.89, 128.81,
127.26, 124.76, 124.50, 120.09, 55.35, 47.37, 44.79, 38.35, 37.64, 37.38, 36.91, 29.75, 28.66,
25.00, 23.89, 23.88, 20.50, 18.39, 16.41. HR-MS (m/z) (ESI): calcd for C35H41N2O5S [M − H]−:
601.2741; found: 601.2752.
Compound 9d: Yields 67.9%, as a white solid. Mp: 121.3−125.6 °C. [α]2D0 = +38 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.33 (d, J = 7.9 Hz, 1H), 8.07 (s, 1H), 7.88 (s, 1H), 7.29–7.25 (m, 5H),
7.17 (s, 1H), 7.08 (t, J = 7.7 Hz, 1H), 6.97 (t, J = 7.7 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.56 (d, J
= 7.2 Hz, 1H), 4.90–4.85 (m, 1H), 4.01–3.95 (m, 1H), 3.77 (s, 3H), 3.18 (d, J = 7.0 Hz, 2H),
2.94–2.72 (m, 2H), 2.35 (d, J = 12.4 Hz, 1H), 2.10 (d, J = 12.4 Hz, 1H), 1.88–1.68 (m, 5H),
1.50–1.45 (m, 2H), 1.34–1.31 (m, 6H, 2 × CH3), 1.29 (s, 3H, CH3), 1.23 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3) δ 178.15, 169.16, 148.38, 148.14, 145.60, 144.29, 139.95, 136.56, 129.50,
129.40, 128.82, 127.16, 127.10, 124.78, 124.31, 121.11, 119.91, 110.14, 55.73, 55.44, 47.20,
44.67, 38.62, 37.70, 37.44, 37.13, 29.80, 28.65, 24.96, 23.92, 23.83, 20.54, 18.47, 16.53. HR-MS
(m/z) (ESI): calcd for C36H43N2O6S [M − H]−: 631.2847; found: 631.2855.
Compound 9e: Yields 66.1%, as a white solid. Mp: 178.5−183.6 °C. [α]2D0 = +9 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 8.18 (t, J = 7.7 Hz, 1H), 7.86 (s, 1H), 7.33–7.21 (m, 5H),
21

ACCEPTED MANUSCRIPT
7.15 (s, 1H), 7.12–6.99 (m, 3H), 6.43 (d, J = 7.1 Hz, 1H), 4.96 (d, J = 7.1 Hz, 1H), 4.01–3.95 (m,
1H), 3.30–3.25 (m, 1H), 3.19–3.14 (m, 1H), 2.88–2.70 (m, 2H), 2.32–2.28 (m, 1H), 2.10 (d, J =
12.1 Hz, 1H), 1.84–1.69 (m, 5H), 1.46–1.41 (m, 2H), 1.33 (d, J = 6.7 Hz, 6H, 2 × CH3), 1.24 (s,
3H, CH3), 1.20 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 178.99, 169.78, 152.85 (1J(C,F) =
243.4 Hz), 148.26, 145.58, 144.25, 139.97, 136.38, 129.52, 129.30, 128.93, 127.33, 125.92
(2J(C,F) = 10.6 Hz), 124.97 (3J(C,F) = 7.5 Hz), 124.78, 124.56 (3J(C,F) = 3.6 Hz), 122.27, 115.05
(2J(C,F) = 19.0 Hz), 55.19, 47.31, 44.61, 37.64, 37.60, 37.38, 36.98, 29.73, 28.66, 25.02, 23.91,
23.87, 20.59, 18.41, 16.45. HR-MS (m/z) (ESI): calcd for C35H40FN2O5S [M − H]−: 619.2647;
found: 619.2649.
Compound 9f: Yields 51.4%, as a white solid. Mp: 180.3−187.8 °C. [α]2D0 = +7 (c 0.1, AcOEt) 1H
NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 7.90–7.75 (m, 2H), 7.69–7.61 (m, 2H), 7.43–7.36 (m,
1H), 7.34–7.28 (m, 2H), 7.27–7.19 (m, 3H), 7.16–7.09 (m, 1H), 6.89 (s, 1H), 4.88–4.79 (m, 1H),
4.03–3.98 (m, 1H), 3.22–3.18 (m, 1H), 3.09–3.03 (m, 1H), 2.61–2.55 (m, 1H), 2.44–2.31 (m, 1H),
2.17 (d, J = 11.9 Hz, 1H), 1.90–1.86 (m, 1H), 1.68–1.51 (m, 4H), 1.46–1.24 (m, 3H), 1.14–1.08
(m, 12H, 4 × CH3). 13C NMR (100 MHz, DMSO-d6) δ 178.53, 171.14, 146.04, 143.70, 142.57,
138.55, 136.40, 136.23, 133.14, 129.74, 129.53, 128.63, 128.47, 126.96, 126.76, 126.73, 125.48,
122.72, 116.48, 55.50, 46.97, 44.85, 38.02, 37.00, 36.99, 36.71, 28.99, 28.05, 25.14, 24.68, 24.40,
20.73, 18.71, 16.78. HR-MS (m/z) (ESI): calcd for C35H40BrN2O5S [M − H]−: 679.1846; found:
679.1857.
Compound 9g: Yields 52.7%, as a white solid. Mp: 181.1−187.2 °C. [α]2D0 = +38 (c 0.1, AcOEt) 1H
NMR (400 MHz, DMSO-d6) δ 14.50 (s, 1H), 9.41 (s, 1H), 7.93–7.75 (m, 1H), 7.68–7.62 (m, 1H),
7.63 (d, J = 3.4 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.37–7.34 (m, 1H), 7.32–7.18 (m, 3H),
7.16–7.10 (m, 1H), 6.87 (d, J = 4.1 Hz, 1H), 4.90–4.72 (m, 1H), 4.05–3.98 (m, 1H), 3.22–3.17 (m,
1H), 3.10–3.01 (m, 1H), 2.63–2.53 (m, 1H), 2.44–2.32 (m, 1H), 2.17 (d, J = 12.0 Hz, 1H),
2.02–1.86 (m, 1H), 1.59–1.50 (m, 2H), 1.41–1.28 (m, 3H), 1.13–1.08 (m, 12H). 13C NMR (100
MHz, DMSO-d6) δ 178.56, 171.17, 145.98, 143.69, 142.88, 138.57, 136.41, 136.04, 133.17,
129.76, 129.54, 128.66, 128.49, 126.98, 126.75, 125.52, 122.72, 116.50, 55.50, 46.98, 44.87,
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38.04, 37.00, 36.71, 36.42, 28.98, 28.02, 25.15, 24.71, 24.42, 20.74, 18.72, 16.79. HR-MS (m/z)
(ESI): calcd for C35H40BrN2O5S [M − H]−: 679.1846; found: 679.1858.
Compound 9h: Yields 69.9%, as a white solid. Mp: 178.0−182.9 °C. [α]2D0 = +43 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 8.19 (t, J = 7.9 Hz, 1H), 7.84 (s, 1H), 7.37–7.31 (m, 2H),
7.29 (d, J = 5.0 Hz, 3H), 7.15 (s, 1H), 7.13–7.09 (m, 1H), 7.06–7.02 (m, 2H), 6.33 (d, J = 7.2 Hz,
1H), 4.90–4.84 (m, 1H), 3.98–3.91 (m, 1H), 3.30–3.25 (m, 1H), 3.18–3.12 (m, 1H), 2.79–2.75 (m,
2H), 2.31 (d, J = 13.0 Hz, 1H), 2.05 (d, J = 10.6 Hz, 1H), 1.76–1.64 (m, 5H), 1.56–1.44 (m, 2H),
1.30 (t, J = 7.2 Hz, 6H, 2 × CH3), 1.19 (s, 3H, CH3), 1.17 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ 178.94, 169.48, 152.65 (1J(C,F) = 243.3 Hz), 148.14, 145.49, 144.08, 139.81, 136.52,
129.41, 129.22, 128.94, 127.26, 125.83 (2J(C,F) = 10.3 Hz), 124.82 (3J(C,F) = 7.4 Hz), 124.69,
124.47 (3J(C,F) = 3.7 Hz), 121.92, 114.97 (2J(C,F) = 19.0 Hz), 55.40, 47.19, 44.35, 37.49, 37.31,
37.25, 36.76, 29.77, 28.55, 24.85, 23.82, 23.75, 19.99, 18.27, 16.31. HR-MS (m/z) (ESI): calcd for
C35H40FN2O5S [M − H]−: 619.2647; found: 619.2653.
Compound 9i: Yields 65.9%, as a white solid. Mp: 124.5−128.3 °C. [α]2D0 = +46 (c 0.1, AcOEt) 1H
NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 7.86 (s, 1H), 7.29–7.24 (m, 7H), 7.13 (s, 1H), 6.83 (d, J =
7.5 Hz, 1H), 6.75 (d, J = 9.0 Hz, 2H), 5.15–5.02 (m, 1H), 3.96 (m, 1H), 3.77 (s, 3H), 3.24 (m, 1H),
3.15 (m, 1H), 2.64–2.80 (m, 2H), 2.32 (d, J = 11.9 Hz, 1H), 2.10–2.00 (m, 1H), 1.68–1.39 (m, 7H),
1.31 (m, 6H, 2 × CH3), 1.23 (s, 3H, CH3), 1.17 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
178.91, 169.73, 156.51, 148.19, 145.46, 144.08, 139.82, 136.93, 130.63, 129.46, 129.36, 128.62,
126.99, 124.65, 121.88, 114.01, 55.45, 55.42, 47.10, 44.47, 38.30, 37.57, 37.28, 36.75, 29.82,
28.55, 24.92, 23.82, 23.76, 20.05, 18.28, 16.37. HR-MS (m/z) (ESI): calcd for C36H43N2O6S [M −
H]−: 631.2847; found: 631.2850.
4.4 Inhibition of Matrix Metalloproteinase Activity assay
MMPs inhibition assays were carried out in 50 mM Tris/HCl buffer, pH 6.8, 10 mM CaCl2 at
25 °C as described previously [37]. Assays were performed with a fluorogenic substrate
McaPro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (13 µM) and human MMPs (nanomolar range
concentration) from R&D Systems, except for human MMP-3, MMP-8, and MMP-9 described
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above. Substrate and enzyme concentrations were kept well below 10% substrate utilization to
ensure evaluation of initial rates. Briefly, MMP-3, -8, or -9 and test complexes at varying
concentrations were pre-incubated at 37 °C for 30 min. Continuous assays were performed by
recording the increase in fluorescence induced by the cleavage of fluorogenic substrates. Black,
flat-bottomed, 96-well nonbinding surface plates (Corning-Costar, Schiphol-RijK, Netherlands)
were used for this test. Fluorescence changes were monitored using a Fluoroskan Ascent
microplate reader (Infinite M1000 Pro, Tecan US, Morrisville, NC) equipped with excitation and
emission wavelengths of 325 and 393 nm, respectively. IC50 values of inhibitors were obtained
with iterative fitting package (GraphPad Prism software).
4.5 Molecular docking
All the docking studies were carried out using Sybyl-X 2.0 on a windows workstation. The
crystal structure of the MMPs proteins were retrieved from the RCSB Protein Data Bank (MMP-3:
1SLN) [38]. The synthetic analogues 8 and 9 were selected for the docking studies. The 3D
structures of these selected compounds were first built using Sybyl-X 2.0 sketch followed by
energy minimization using the MMFF94 force field and Gasteiger-Marsili charges. We employed
Powell’s method for optimizing the geometry with a distance dependent dielectric constant and a
termination energy gradient of 0.005 kcal/mol. All the selected compounds were automatically
docked into the binding pocket of MMPs by an empirical scoring function and a patented search
engine in the Surflex docking program. Before the docking process, the natural ligand was
extracted; the water molecules were removed from the crystal structure. Subsequently, the protein
was prepared by using the Biopolymer module implemented in Sybyl. The polar hydrogen atoms
were added. The automated docking manner was applied in the present work. Other parameters
were established by default to estimate the binding affinity characterized by the Surflex-Dock
scores in the software. Surflex-Dock total scores, which were expressed in -log10 (Kd) units to
represent binding affinities, were applied to estimate the ligand-receptor interactions of newly
designed molecules. A higher score represents stronger binding affinity. The optimal binding pose
of the docked compounds was selected based on the Surflex scores and visual inspection of the
docked complexes.
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4.6 Cytotoxicity assay
The cell lines NCI-H460, HepG2, SKOV-3, MCF-7 and HL-7702 were obtained from the
Shanghai Cell Bank in the Chinese Academy of Sciences. NCI-H460, HepG2, SKOV-3, MCF-7
and HL-7702 cell lines were grown on 96-well microtitre plates at a cell density of 10 × 105
cells/well in DMEM medium with 10% FBS. DMEM and FBS were obtained from Gibco-Thermo
(BRL Co. Ltd., USA). The plates were incubated at 37°C in a humidified atmosphere of 5%
CO2/95% air for overnight. Therewith, the cells were exposed to different concentrations of target
compounds, 5-FU and DOX, and incubated for another 48 h. The cells were stained with 10 µl of
MTT at incubator for about 4 h. The medium was thrown away and replaced by 100 mL DMSO.
The O. D. Value was read at 570/630 nm enzyme labeling instrument.
4.7 In vitro cell migration assay/wound healing assay
HepG2 cells (5×105 cells/well) were cultured in 6 well plates as confluent monolayers for 24 h.
Then artificial scratch on the monolayers were created with 200 mL sterile pipette tip and washed
twice with PBS to remove non-adherent cells. The media containing 5 µM and 10 µM of
compound 8k or without compound 8k were added to each well. The migration of cells across the
scratched area were photographed by using phase contrast microscope (Nikon) at 0 h
(immediately), 24 h and 48 h time interval after treatment in three or more randomly selected
fields.
4.8 Cell cycle analysis
The HepG2 cells line were treated with different concentrations of compound 8k. After 24 h of
incubation, cells were washed twice with ice-cold PBS, fixed and permeabilized with ice-cold 70%
ethanol at -20°C overnight. The cells were treated with 100 µg /ml RNase A at 37°C for 30 min
after washed with ice-cold PBS, and finally stained with 1 mg/ml propidium iodide (PI) (BD,
Pharmingen) in the dark at 4°C for 30 min. Analysis was performed with the system software
(Cell Quest; BD Biosciences).
4.9 Apoptosis analysis
HepG2 cells were seeded at the density of 2× 106 cells/mL of the DMEM medium with 10%
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FBS on 6-well plates to the final volume of 2 mL. The plates were incubated for overnight and
then treated with different concentrations compound 8k for 24 h. Briefly, after treatment with
compound 8k for 24 h, cells were collected and washed with PBS twice, and then resuspend cells
in 1×Binding Buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration
of 1× 106 cells /ml. The cells were subjected to 5 µL of FITC Annexin V and 5 µL propidium
iodide (PI) staining using annexin-V FITC apoptosis kit (BD, Pharmingen) followed the 100 µL of
the solution was transfer to a 5 mL culture tube and incubate for 30 min at RT (25°C) in the dark.
The apoptosis ratio was quantified by system software (Cell Quest; BD Biosciences).
4.10 AO/EB Staining
The AO/EB molecular probes were also used to detect apoptotic cells. HepG2 cells (1×106 cells)
were seeded in six-well tissue culture plates. Following incubation, the medium was removed and
replaced with fresh medium plus 10% fetal bovine serum and treated with 5 µM and 10 µM of
compound 8k for 24 h. After the treatment period, briefly, the cells were harvested, suspended in
PBS, stained with 2 µL of AO/EB stain (100 mg/mL) at room temperature for 20 minutes.
Fluorescence was read on a Nikon ECLIPSETE2000-S fluorescence microscope (OLYMPUS Co.,
Japan).
4.11 Statistics
The data were processed by the Student' st -test with the significance level P≤0.05 using SPSS.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 81260472
21362002 and 21431001), Special Research Found for the Doctoral Program of Higher Education
(NO. 20134504110002), State Key Laboratory for Chemistry and Molecular Engineering of
Medicinal Resources, Ministry of Science and Technology of China (CMEMR2016-B06).
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