Design, synthesis and activity of BBI608 derivatives targeting on stem cells

Article abstract of DOI:10.1016/j.ejmech.2018.03.054

STAT3 plays a vital role in maintaining the self-renewal of tumor stem cells. BBI608, a small molecule identified by its ability to inhibit gene transcription driven by STAT3 and cancer stemness properties, can inhibit stemness gene expression and kill stemness-high cancer cells isolated from a variety of cancer types. In order to improve the pharmacokinetic properties of BBI608 and the antitumor activity, a series of BBI608 derivatives were designed and synthesized here. Most of these compounds were more potent than BBI608 on HepG2 cells, compound LD-8 had the most potent inhibitory activity among them and was 5.4-fold more potent than BBI608 (IC₅₀ = 11.2 μM), but had considerable activity on normal liver cells L-02. Compounds LD-17 (IC₅₀ = 3.5 μM) and LD-19 (IC₅₀ = 2.9 μM) were found to possess significant inhibitory activities and good selectivity. The results showed that compound LD-19 was worthy to investigate further as a lead compound according to its potent inhibitory activity, ideal ClogP value and better water solubility.

Full text of DOI:10.1016/j.ejmech.2018.03.054

Accepted Manuscript
Design, synthesis and activity of BBI608 derivatives targeting on stem cells
Qifan Zhou, Chen Peng, Fangyu Du, Linbo Zhou, Yajie Shi, Yang Du, Dongdong Liu,
Wenjiao Sun, Meixia Zhang, Guoliang Chen
PII:
S0223-5234(18)30297-6
10.1016/j.ejmech.2018.03.054
EJMECH 10319
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2018
Revised Date: 13 March 2018
Accepted Date: 19 March 2018
Please cite this article as: Q. Zhou, C. Peng, F. Du, L. Zhou, Y. Shi, Y. Du, D. Liu, W. Sun, M. Zhang, G.
Chen, Design, synthesis and activity of BBI608 derivatives targeting on stem cells, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.054.
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ACCEPTED MANUSCRIPT

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Design, Synthesis and Activity of BBI608 Derivatives Targeting
on Stem Cells
Qifan Zhoua, 1, Chen Pengb, 1, Fangyu Dua, Linbo Zhoua, Yajie Shia, Yang Dua, Dongdong Liua,
Wenjiao Suna, Meixia Zhangb, *, Guoliang Chena, *
a
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang 110016, People's Republic of China
b
Institute of Metabolic Disease Research and Drug Development, China Medical University, 77 Puhe Road, Shenyang North New Area,
Shenyang 110122, People's Republic of China
*
Corresponding author.
E-mail addresses: spucgl@163.com (M. Chen). mxzhang@cmu.edu.cn (M. Zhang)
1 Both authors contributed equally to this work
Abstract
STAT3 plays a vital role in maintaining the self-renewal of tumor stem cells.
BBI608, a small molecule identified by its ability to inhibit gene transcription driven
by STAT3 and cancer stemness properties, can inhibit stemness gene expression and
kill stemness-high cancer cells isolated from a variety of cancer types. In order to
improve the pharmacokinetic properties of BBI608 and the antitumor activity, a series
of BBI608 derivatives were designed and synthesized here. Most of these compounds
were more potent than BBI608 on HepG2 cells, compound LD-8 had the most potent
inhibitory activity among them and was 5.4-fold more potent than BBI608 (IC50=11.2
µM), but had considerable activity on normal liver cells L-02. Compounds LD-17
(IC50=3.5 µM) and LD-19 (IC50=2.9 µM) were found to possess significant inhibitory
activities and good selectivity. The results showed that compound LD-19 was worthy
to investigate further as a lead compound according to its potent inhibitory activity,
ideal ClogP value and better water solubility.
Key Words: BBI608 derivatives, STAT3 inhibitors, tumor stem cells, antitumor
activity

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Introduction
Tumor stem cells are kinds of cancer cells that have the ability to self-renewal
and differentiate, and are usually considered to be potential to develop into cancer. If
the tumor stem cells do not be cleared in the course of cancer treatment, cancer cells
are easy to relapse and metastasis, as cancer stemness can differentiate into new types
of cancer [1]. Wnt, Hedgehog (Hh), Notch, BMP, Bmi-1, PI3K/Akt and STAT3 are
the main signal pathways of tumor stem cells which regulate the self-renewal and
differentiation. Increased interest has been devoted toward molecules able to inhibit
STAT3 [2,3].
Signal transducer and activator of transcription 3 (STAT3) is a kind of
cytoplasmic protein that can be activated by different cell signaling factors, including
epidermal growth factor receptor (EGFR), vascular epidermal growth factor
receptor(VEGFR), fibroblast growth factor (FGFR), Src family-related kinase, Abl
family-related kinase, Insulin-like growth factor receptor (IGFRs), hepatocyte growth
factor receptor (HGFRs) and interleukin-6 (IL-6) [4]. Stimulated by extracellular
signals, STAT3 monomer can be activated through phosphorylation of tyrosine
residues. Dimerization is happened between activated STAT3 monomers via the SH2
domain to form homo-dimers and then enter into the nucleus to combine the specific
DNA sequence, and regulate the transcription and expression of downstream target
genes. After STAT3 completes signal transduction, the nuclear protein tyrosine
phosphorylases make the phosphorylated STAT3 dephosphorylate, which returns to
cytoplasm [5]. Recent studies have shown that since STAT3 regulates the self-renewal
and differentiation of tumor stem cells, whose action is considered an important factor
for maintaining the self-renewal of tumor stem cells and turns into a new target for
targeting cancer stem cells [6].
Researches on STAT3 inhibitors mostly stay in the initial stage, development of a
potent inhibitor is imminent. In 2006, Schust et al. reported compound Stattic (Fig. 1)
[7], the first non-peptides STAT3 inhibitor, which stays in preclinical study now, is
capable of blocking STAT3 phosphorylation by inhibiting the function of the SH2

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domain of STAT3. HO-3867 (Fig. 1) reported by Selvendiran et al., which could
selectively inhibit the phosphorylation of STAT3 protein [8], and not affect the
expression of other STAT proteins [9], is currently in preclinical study. Ren et al.
screened a novel STAT3 inhibitor, Niclosamide (Fig. 1) [10], having a similar mode
of action with HO-3867, potently inhibiting STAT3 with IC50 of 0.7 μM. S3I-201
(Fig. 1) [11,12] and BP-1-102 (Fig. 1) [13] are STAT3 inhibitors containing salicylic
acid structure, can block phosphorylation of STAT3 monomer. The latter is now in
preclinical study, the Kd value of binding affinity with STAT3 is 504 nM at
concentration of 4-6.8 µM. Song et al. designed a series of compounds containing
anthraquinones and found that STA-21 (Fig. 1) displayed powerful anti-breast cancer
activity [14]. Napabucasin, known as BBI608 (Fig. 1), a small molecule identified
by its ability to inhibit gene transcription driven by STAT3 and cancer stemness
properties in phase III clinical trials at Sumitomo Dainippon Pharma for the treatment
of metastatic colorectal carcinoma, pancreatic cancer and gastric cancer. BBI608 can
inhibit stemness gene expression and block spherogenesis or kill stemness-high
cancer cells isolated from a variety of cancer types. These data demonstrate that
targeting cancer stemness is a novel approach to develop the next generation of cancer
therapeutics to suppress cancer relapse and metastasis [15].

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O
HO
O
O
F
N
F
O
O2N
O2N
NH
S
Cl
Cl
N
OH
Niclosamide
O
Stattic
HO-3867
O
O
OH
OH
O
OH
OH
F
F
O
O
S
N
O
F
F
S
N
N
H
O
O
F
S31-201
O
BP-1-102
O
OH
O
O
O
O
O
BBI 608
STA-21
Fig. 1. Chemical structures of some STAT3 inhibitors
Few of BBI608 derivatives are yet reported up to now. Modifications of BBI608
can be classified into three categories. (See Fig. 2) Type I (A~C) with compounds
which mainly transforms the quinone of BBI608 into phenolic hydroxyl and then
change into various types of esters pro-drugs to improve the solubility, that can be
transformed into active molecular, 4,9-dihydroxynaphtho[2,3,b]furan, in vivo [16].
Type II (D~F) are compounds with diverse side chains, sulfoxide or sulfone or
propionyl, replacement of acetyl of BBI608. Type III (G~I) are compounds reported
by R&D enterprises of BBI608, Japan Sumitomo pharmaceutical and Boston
Biopharmaceutical Technology Co. Ltd recently [17,18]. X represents carbon,
nitrogen or oxygen, and Y represents a carbonyl group, an amino group, a sulfone
group or a sulfoxide group, wherein R, R1, R2 and R3 represent different alkyl groups
from C1 to C6.

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Fig. 2. Chemical structures of some BBI608 derivatives
These findings prompted us to investigate more derivatives of BBI608 to
examine their structure activity relationships in the hope of discovering more potent
STAT3 inhibitors as antitumor agents targeting on stem cells. Due to the lack of
H-bond donor (HBD), we modified the side chain of BBI608 by methods of hybrid
and bioisosterism, assisted by computer-aided design and drafting (CADD),
introduced nitrogen atoms to replace the original oxygen atoms of the side chain in
this work. Bisantrene (Zantrene) is a kind of cell growth inhibitor containing
anthracycline (Fig. 3), has the similar antitumor spectrum compared with doxorubicin,
but is less toxic [19]. In this paper, as shown in Fig. 3, we have designed a scaffold
that assembled BBI608 and “polar tail” of bisantren by employing the molecular
hybridization approach to obtain LD-1. Then we synthesized LD-2 and LD-3 by the
same chemical method and replaced the unsaturated imine with different substituted
amino groups, affording compounds from LD-5 to LD-21. It was desirable to change
the lipo-hydro partition coefficient and increase solubility to improve

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pharmacokinetic properties. Consequently, a series of derivatives were synthesized
and evaluated for their antitumor activities. Their structure-activity relationships were
summarized and the mechanism of action that the compounds exerted their STAT3
inhibitory activities was examined by molecular docking studies.
Fig. 3. Schematic diagram depicting the procedure for the design of the target compounds

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2. Results and discussion
2.1. Chemistry
The studied compounds were obtained through a number of diverse approaches,
summarized in Schemes 1−3. The strategy adopted for the synthesis of BBI608 is
outlined in Scheme 1. The 3,4-dibromobutan-2-one (2) was obtained by addition with
starting material α,β-unsaturated butanone in the presence of Br2 [20]. The
intermediate
5
(2-hydroxy-1,4-naphthoquinone)
was
synthesized
from
1,4-naphthoquinone via Thiele-winter reaction, hydrolysis and oxidation [21]. The
subsequent cyclization and oxidation reaction of 2-hydroxy-1,4-naphthoquinone 5
with 3,4-dibromobutan-2-one 2 yielded BBI608.
The general strategy for the synthesis of LD-1~LD-3 is illustrated below
(Scheme 2). 2-Hydrazonoimidazolidine hydroiodide 8 was synthetized from
imidazolidine-2-thione 7 according to the published reference [22]. Respectively,
2-hydrazinoimidazolidine hydroiodide 8, p-toluenesulfonyl hydrazide and
hydroxylamine hydrochloride were reacted with BBI608 via condensation to yield the
target compounds LD-1~LD-3. LD-4 was obtained from BBI608 via reduction and
chlorination (Scheme 3). Different alkyl amines or N-heterocycles were reacted with
LD-4 to product the target compounds LD-5~LD-21 (Scheme 3).
Scheme 1. Reagents and conditions: (a) Br2, DCM, 0℃, 10 min (b) Ac2O, H2SO4, 50℃,30 min (c) 37% HCl (aq),
MeOH, reflux, 1h; 30% H2O2, rt (d) DBU, DMF, 0℃, 2 h (e) DBU, DMF, 50℃, 6 h.

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Scheme 2. Reagents and conditions: (f) CH3I, N2H4.H2O, Et2O, rt, 8h (g) Conc. H2SO4, MeOH, 55℃, 4 h (h)
NH2OH.HCl, Py, MeOH, 50℃, 2 h (i) p-Toluenesulfonyl hydrazide, Py, MeOH, 60℃, 1.5 h.
Scheme 3. Reagents and conditions: (j) NaBH4, DMF, H2O, rt, 30min (k) SOCl2, DCM, reflux, 1 h (l) HNR1R2,
CH3CN, reflux, 2h.

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2.2. In vitro biological activity assay and structure-activity relationships
Table 1 The inhibition potencies of compounds in vitro on HepG2 cells
IC50 (µM)
HepG2 cells
Sw
Cpd.
R
CLogP a
Selectivity c
(mg/mL) b
L-02 cells
8.1
LD-1
LD-2
7.4
6.1
1.40
1.96
0.68
0.30
1.1
1.6
9.7
5.4
LD-3
5.9
2.92
0.025
0.9
LD-4
LD-5
16.6
2.3
63.8
3.1
2.53
1.45
0.055
0.48
3.9
1.4
LD-6
3.6
6.2
2.19
0.27
1.7
LD-7
LD-8
LD-9
2.2
1.9
9.5
1.2
2.2
1.74
1.62
2.88
0.48
0.81
0.24
0.6
1.1
1.5
14.4
LD-10
2.2
3.1
0.92
2.10
1.4
LD-11
LD-12
LD-13
LD-14
LD-15
26.0
2.7
43.2
3.3
3.14
2.23
2.89
1.75
3.15
0.098
1.00
1.7
1.2
1.4
0.8
1.3
5.8
7.9
0.096
0.32
25.6
14.5
21.7
18.7
0.25

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LD-16
LD-17
LD-18
LD-19
LD-20
LD-21
BBI 608
27.2
3.5
30.2
11.6
3.0
3.32
1.84
0.98
1.65
1.94
0.99
1.66
0.48
0.56
4.40
2.30
1.50
8.50
0.26
1.1
3.3
1.0
2.6
0.7
1.2
1.8
3.0
2.9
7.6
7.4
4.9
3.2
3.8
10.2
18.7
a Octanol-water partition coefficient (logP) was calculated according to the Pharma Algorithms’AB/LogP v2.0
algorithm. b Solubility in pure water (Sw) was predicted on the logSw [mmol/L] scale and converted into mg/ml
units. c Selectivity = IC50 values on L-02 cells/HepG2 cells
There are high expressions of STAT3 gene in human hepatocellular carcinoma
HepG2 cells [23]. Therefore, HepG2 cells were selected for evaluating the anticancer
activities of compounds and the IC50 values of these compounds were determined.
The results of pharmacological activity were analyzed, pharmacokinetic properties
were analyzed by calculating their CLogP and Sw, and the structure-activity
relationship analysis was discussed.
The activities of most compounds were better than that of the lead compound
BBI608, which was consistent with the results of molecular docking. As shown in
Table 1, the inhibitory activity of compounds LD-1~LD-3, which were obtained by
condensation of BBI608 with amine or hydrazine, had been improved, compounds
LD-1 (IC50=7.4 µM), LD-2 (IC50=6.1 µM), LD-3 (IC50=5.9 µM)，were about 1.4-fold,
1.7-fold and 1.7-fold more potent than that of the positive control drug BBI608 with
IC50 value of 10.2 µM. When the C16’ position of the side chain was substituted with
chlorine (LD-4, IC50=16.6 µM), the inhibitory activity declined, indicating that the
halogen group was unfavorable for our lead compound to display activity. Meanwhile,
LD-5, LD-7, LD-8 with the IC50 values of 2.3 µM, 2.2 µM, 1.9 µM, respectively, were
more potent than BBI608, but had considerable activity on normal liver cells L-02.

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However, when cyclohexylamino group was introduced to the corresponding position
(LD-16), the inhibitory activity declined with an IC50 value of 27.2 µM. It indicated
that the substitutions of alkanes with small size on the C16’ amino group are
beneficial to the increase of the inhibitory activity. In contrast, when the N atom on
C16’ position was di-substituted, LD-6 (IC50=3.6 µM) and LD-9 (IC50=9.5 µM),
compared to their mono-substituted derivatives in the corresponding position LD-5
(IC50 =2.3 µM) and LD-7 (IC50 =2.2 µM). Nevertheless, when ethyl groups of LD-9
were substituted with hydroxyl groups on the terminal (LD-10), the inhibitory activity
improved remarkably with an IC50 value of 2.2 µM, which was 4.3-fold and 4.6-fold
more potent than compound LD-9 and BBI608, respectively. In addition, when C16’
position was replaced with cyclic amine, the obtained compounds were much superior
to BBI608, in which the tetrahydropyrrole substituted derivative LD-12 (IC50=2.7
µM), morpholine substituted derivative LD-17 (IC50=3.5 µM), piperazine substituted
derivative LD-18 (IC50=3.0 µM), 4-methylpiperazine substituted derivative LD-19
(IC50=2.9 µM), 4-ethylpiperazine substituted derivative LD-20 (IC50=7.4 µM) and
2-(piperazin-1-yl)ethan-1-ol substituted derivative LD-21 (IC50=3.2 µM), which were
1.4~3.8 fold more potent than BBI608. Furthermore, when transforming the
tetrahydropyrrole of LD-12 aromatization into pyrrole to obtain LD-13, the inhibitory
activity slightly decreased compared to LD-12. When R was imidazole (LD-14), the
inhibitory activity was decreased, IC50 value of 25.6 µM. These results showed that
the C16’ position had enough modification space to greatly improve the inhibitory
activity on HepG2 cells. In conclusion, LD-8, LD-17, and LD-19 showed superior
HepG2 cell inhibitory effects over BBI608. LD-17 and LD-19 exhibited selectivity
via cytotoxicity assay on L-02 cells. At concentrations of 1, 3, 10, 30, 100 µM/L,
LD-8, LD-17, LD-19 and BBI608 detected concentration-dependent increase in the
inhibition rate, as shown in Table 2.

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Table 2 Inhibition rate on HepG2 cells of BBI608, LD-8, LD-17 and LD-19
Inhibition rate on HepG2 cells (%)
Concentration (µM/L)
BBI608 LD-8 LD-17 LD-19
1
3
7.4
29.0
67.5
86.5
90.5
97.8
33.1
50.9
55.0
87.6
100.0
32.3
47.7
68.7
96.4
99.7
23.7
29.6
89.7
96.1
10
30
100
Due to the poor water solubility of BBI608, lots of studies on BBI608 derivatives
have been devoted to improve water solubility through chemical modification. By
calculating the lipid-soluble and water-soluble properties of these derivatives, it was
found that changes in the substituents at the C16 position could significantly
ameliorate the pharmacokinetic properties and increase the antitumor activity. When
hydrophobic groups such as p-toluenesulfonyl (LD-3), halogen (LD-4), phenylethyl
(LD-11), and pyrrole (LD-13) were introduced at the C16 position, the predicted Sw
values were obviously reduced. Saturated the pyrrole ring of LD-13 (Sw=0.096
mg/mL) to obtain LD-12, the water solubility was increased (Sw=1.00 mg/mL) and
its lipid solubility was maintained at an appropriate level (ClogP=2.23). However, the
polar groups such as hydroxyethyl (LD-10), piperazine (LD-18), and
hydroxyethylpiperazine (LD-21) were introduced at the C16 position; the inhibitory
activities were significantly increased while the water solubility was greatly improved.
In contrast, the lipid solubility were reduced (ClogP<1). As expected, the compounds
obtained after the introduction of the methyl or ethyl group in the para-position of the
piperazine (LD-19 and LD-20) significantly improved the water solubility (Sw=2.3,
1.5 mg/mL) while maintaining the lipid solubility (ClogP=1.65, 1.94).

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2.3. Molecular interactions and docking simulations
To gain insight into the interactions between the BBI608 derivatives and STAT3,
molecular docking simulations were performed using the BBI608 derivatives with
STAT3 (PDB code: 1BG1 [24]). Activation of STAT3 induces the phosphorylation of
STAT3 at tyrosine residue 705, which leads to dimerization of two STAT3 monomers
through SH2 domains of the proteins [25, 26]. The activated STAT3 dimers then
translocate into the nucleus and activate the transcription of genes that control cell
proliferation, apoptosis, angiogenesis, and other cell functions. Turkson et al.
demonstrated that the SH2 domain was a potential target for STAT3 inhibitor binding
[27]. Inhibitor design for STAT3 is a challenging issue due to the lack of
crystallographic study of interaction between STAT3 protein and small molecules
reported. Nevertheless, the calculated binding of compound STA21 to STAT3 was
reported [28], whose structure is similar to BBI608 (STA21 could form hydrogen
bonds with Arg595, Arg609, Ile634 in the SH2 domain of the STAT3). As a result, the
docking site in this paper was set in the SH2 region. As shown in Fig. 4A, the docking
results revealed that BBI608 could form hydrogen bonds with Arg595, Ser636,
Arg609 in the SH2 domain. It was possible that BBI608 exerted its STAT3 inhibitory
activities in similar manner with STA21.
The side chain of BBI608 was modified in our work. The results of molecular
docking showed that LD-8 not only formed hydrogen bonds with Ser636 and Arg609
in SH2, but also a new hydrogen bond with IIe634 and Lys591, which enhanced the
interaction between ligand and receptor (Figure. 4B). As reported, the introduction of
mono-substituted amino at C16’ position led to an appreciable potency gain than
compounds di-substituted amino at C16’ position (Table 1, compound LD-6 and
LD-9 versus compound LD-5 and LD-7, respectively), which was consistent with that
secondary amines formed hydrogen bonds with Ile634 as hydrogen bond donors,
thus strengthened bond interaction between ligands and receptors. In contrast, due to
the presence of larger volumes of cyclohexane, LD-16 formed an undesirable steric
hindrance with protein that making the hydrogen bonds with Ile634 disappeared as the

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possible result of decreasing of inhibitory activity. More importantly, LD-10 formed
additional hydrogen bonds with Ile634 and Arg595 (Fig. 4C). This mode of action
explained why LD-10 was more potent than BBI608 and compound LD-9 due to the
addition of the hydroxyl group on “flexible tail” of compound. Although the amino
groups at the C16’ position of LD-17~LD-21 were di-substituted and could not form
hydrogen bonds with Ile634, new hydrogen bonds with Arg595 may formed (Fig. 4D),
that results of stronger inhibitory activities compared to LD-15.
Fig. 4. Putative binding modes of A: BBI608 in 3D representation B: compound LD-8 in 3D representation C:
compound LD-10 in 3D representation from molecular docking. D: compound LD-19 in 3D representation
Hydrogen bond is shown in green dash lines and small molecules shows as sticks in 3D representation. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
3 Conclusion
Since STAT3 plays a pivotal role in cell proliferation and survival, it is
recognized as one of the significant oncogenic signaling pathways [4,5,13]. BBI608, a

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small molecule identified by its ability to inhibit gene transcription driven by STAT3
and cancer stemness properties, granted Orphan Drug Designation from the FDA in
the treatment of metastatic colorectal carcinoma, pancreatic cancer, gastric cancer, and
gastroesophageal junction (GEJ) cancer, can inhibit stemness gene expression and kill
stemness-high cancer cells isolated from a variety of cancer types [15]. In order to
improve its pharmacokinetic properties, we designed and synthetized a series of
potent compounds by using the molecular hybridization approach and were
subsequently synthesized and evaluated for their inhibitory activities of HepG2 cells
(STAT3 gene is high expressed [23]) and L-02 cells, used for selective research. Most
of these compounds were more potent than BBI608 on HepG2 cells, compound LD-8
had the most potent inhibitory activity among them and was 5.4-fold more potent than
BBI608 (IC50=11.2 µM), but had considerable activity on normal liver cells L-02.
Compounds LD-17 (IC50=3.5 µM) and LD-19 (IC50=2.9 µM) were found to possess
significant inhibitory activities and good selectivity. Considering inhibitory activity
and water solubility, LD-19 is worth for further study. The structure-activity
relationships revealed that the mono-substituted amino, morpholine, piperazine
derivatives substituted at the C16’ position of the side chain improved the inhibitory
activity. Obviously, there is a very large modification space in the side chain to
increase activity and selectivity of derivatives.
4 Experimental section
4.1. Chemistry
All reagents used were commercially available. Reactions were monitored by
thin-layer chromatography (TLC) on glass plates coated with silica gel with a
fluorescent indicator (GF254). Column chromatography was performed on silica gel
1
13
(160-200 mesh). All the H-NMR and C-NMR spectra were recorded on a Bruker
400 MHz and 600 MHz spectrometer in DMSO-d6. Chemical shifts (δ) were
expressed in parts per million using tetramethylsilane as an internal reference.

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ESI-MS data were obtained using an Agilent 1100 instrument. High-resolution mass
spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 in ESI
mode (Agilent, Santa Clara, CA, USA). Melting points were measured on X-4 digital
melting point instrument. The purity was determined by Shimadzu LC-2010AHT high
performance liquid chromatography.
4.1.1. Synthesis of 3, 4-dibromobutanone (2)
Synthesis of the intermediate compound 2 was carried out according to the
previously reported procedure [20]. A solution of bromine (23.03 g, 0.144 mol) in
dichloromethane (20 mL) was added dropwise at -10℃ to a stirred solution of
α,β-unsaturated butanone (10 g, 0.143 mol) in dichloromethane (20 mL). The reaction
mixture was stirred at 0℃ for 20 min. Then saturated sodium thiosulfate was added,
and the organic layer was washed with water, brine and dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. Compound 2 (31.61 g, 96.3% yield)
was obtained as a pale yellowish oil, used further for next step without purification,.
4.1.2. Synthesis of naphthalene-1, 2, 4-triyltriacetate (Thiele-Winter Reaction) (4)
To a 250 mL three-necked flask was added acetic anhydride 193.8 g (1.898 mol)
at room temperature and 18.58 g (0.190 mol) of 98% conc. sulfuric acid was added
slowly at a rate that maintained a reaction temperature below 30℃. The mixture was
brown, then 1, 4-naphthoquinone 50 g (0.316 mol) was added in portions. After the
mixture was stirred at 50℃ for another 30 min, poured into 500 g crushed ice, stirred
to precipitate a grayish brown precipitate, filtered and the filter was washed with
water (100 mL) and dried. 102 g of gray solid was obtained, which was recrystallized
from methanol (450 mL) to give pure product. (72.92 g; 76.25% yield). M.p.135-136℃
(literature [21]: 134℃).
4.1.3. Synthesis of 2-hydroxynaphthalene-1, 4-dione (5)
To a 500 mL three-necked flask was added 72.92 g (0.241 mol)
naphthalene-1,2,4-triyl triacetate (4), 250 mL of methanol, 65 mL conc. hydrochloric
acid, the mixture was stirred at 70℃ for 1 hour. The mixture was slowly poured into
800 mL of 30% H2O2 and stirred vigorously to precipitate a dark green precipitate and
the reaction was monitored by TLC until disappearance of 4. The solid was filtered

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off and washed with water to give a yellow solid which was dissolved in 500 mL of 2%
sodium hydroxide solution. Then the solution was heated to boiling, filtered and the
filtrate was adjusted to pH 1 with 6 N HCl, a yellow solid was precipitated. The solid
was filtered and dried to give a yellow solid. (38.41 g, 91.6% yield). Compound 5 was
used for next step without further purification. M.p. 188-192℃ (literature [21]:
191-192℃). ESI-MS: m/z 175.1 [M+H]+, 197.1 [M+Na]+, 172.8 [M-H]-.
4.1.4. Synthesis of 2-acetylnaphtho[2,3-b]furan-4,9-dione (BBI608)
A solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 21.92 g, 0.144 mol)
dissolved in N,N-dimethylformamide (20 mL) was added dropwise at a rate that
maintained a reaction temperature below 0℃ to a solution containing 2 (31.61 g,
0.138 mol). After adding completed, the mixture continued to react for another 30 min.
Then 5 (20.88 g, 0.12 mol) was added and a solution of DBU (27.14 g, 0.156 mol)
dissolved in N,N-dimethylformamide (10 mL) was added dropwise at a rate that
maintained a reaction temperature between 30 and 50℃. The mixture was stirred for
another 3 h. The reaction was monitored by TLC until disappearance of 5. The
reaction mixture was slowly poured into a mixture of about ice water mixture (1.5 L)
and stirred for 2 h. After cooling the mixture to 0℃, filtered and the solid was washed
respectively with 5% NaHCO3 (20 mL), 2% acetic acid (20 mL) and water (30 mL).
Subsequently mixed with ethanol (100 mL), filtered and dried to give a brown solid
(12.75 g) which was recrystallized from ethyl acetate (1 L) to give a yellow solid.
(7.83 g, 27.19%). M.p. 224-226℃ (literature [29]: 222-224℃). ESI-MS: m/z 241.1
[M+H]+, 263.0 [M+Na]+. ESI-HRMS: m/z [M+H]+ calcd for C14H9O4: 241.0495;
1
found: 241.0506; [M+Na]+ calcd for C14H8O4Na: 263.0315; found: 263.0327. H
NMR (400 MHz, DMSO-d6) δ: 8.12~8.16(m, 2H), 8.03(s, 1H), 7.91~7.93(m, 2H),
2.60(s, 3H).
4.1.5. Synthesis of compounds LD-1~LD-3
4.1.5.1. Synthesis of 2-hydrazonoimidazolidine hydroiodide (8) [22]
To a 250 mL of three-necks flask was added imidazolidine-2-thione (10g, 0.098
mol), 100 mL of anhydrous ethanol. Subsquently stirring at room temperature for 10
min, methyl iodide (7 mL, 0.118 mol) was added. The mixture was stirred at room

ACCEPTED MANUSCRIPT
temperature for 30 min, then at 45℃ for another 1 hour. Completion of the reaction
monitored by TLC, the mixture was cooled to room temperature. Hydrazine hydrate
(8 mL, 0.16 mol) was added and stirred for 8 hours (unpleasant smells were produced).
After removal of most of the solvent in vacuo, cooled the mixture and filtered to give
white solid.(15.09 g, 67.42% yield). M.p. 120-123℃. ESI-MS: m/z 101.2 [M+H]+.
4.1.5.2. Synthesis of 2-(1-(2-(4,5-dihydro-1H-imidazol-2-yl)hydrazono)ethyl) naphtha
[2,3-b]furan-4,9-dione (LD-1)
To a 100 mL of single-necked flask was added BBI608 (0.5 g, 0.002 mol),
2-hydrazonoimidazolidine hydroiodide (2.37 g, 0.012 mol), four drops of conc.
sulfuric acid, dissolved in 50 mL of methanol. The mixture was stirred at 55℃ for 4
hours. After cooling to room temperature, the reaction solution was filtered to give a
yellow solid. (0.76 g, 87.86% yield). M.p. >300℃. ESI-MS: m/z 323.0 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C17H15N4O3: 323.1139; found: 323.1166. 1H NMR
(400 MHz, DMSO-d6): δ 12.01 (1H, s), 8.56 (1H, s, br), 8.10-8.17 (2H, m), 7.88-7.94
(2H, m), 7.84 (1H, s), 3.78 (4H,br), 2.37 (3H, s). 13C NMR (150 MHz, DMSO-d6): δ
180.16, 172.57, 158.00, 156.51, 151.70, 143.73, 134.39, 134.13, 132.59, 132.39,
131.22, 126.46, 126.36, 107.69, 42.87, 13.36.
4.1.5.3. Synthesis of 2-(1-(hydroxyimino)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-2)
To a 50 mL of single-necked flask was added BBI608 (1.0 g, 0.004 mol),
hydroxylamine hydrochloride (0.43 g, 0.0062 mol), pyridine (0.66 g, 0.0082 mol)，
dissolved in ethanol. The mixture was stirred at 55℃ for 2 hours. After cooling to
room temperature, the reaction solution was filtered to give a yellow solid. (0.9 g,
91.8% yield). M.p. 218-221℃ (sublimation was observed at 190℃). ESI-MS: m/z
256.0 [M+H]+, 278.0 [M+Na]+, 532.7 [2M+Na]+, 253.8 [M-H]-. ESI-HRMS: m/z
[M+H]+ calcd for C14H10NO4: 256.0604; found: 256.0597; [M-H]- calcd for
C14H8NO4: 254.0453; found: 254.0470. 1H NMR (400 MHz, DMSO-d6): δ 12.05 (1H,
13
s), 8.07-8.12 (2H, m), 7.87-7.90 (2H, m), 7.40 (1H, s), 2.19 (3H, s). C NMR (150
MHz, DMSO-d6): δ 180.02, 172.64, 156.14, 151.48, 145.18, 134.23, 133.98, 132.58,
132.30, 130.83, 126.39, 126.22, 106.42, 16.89.
4.1.5.4
N'-(1-(4,9-dioxo-4,9-dihydronaphtha[2,3-b]furan-2-yl)ethylidene)-4-methyl

ACCEPTED MANUSCRIPT
benzenesulfonyl hydrazide (LD-3)
To a 10 mL of single-necked flask was added BBI608 (0.1 g, 0.4 mmol),
p-toluenesulfonamide (0.24 g, 1 mmol), two drops of pyridine, dissolved in 5 mL
methanol. The mixture was stirred at 60℃ for 1.5 hour. After cooling to room
temperature, the reaction solution was filtered to give a yellow solid. (0.15 g, 93.7%
yield). M.p. 235-236 ℃. ESI-MS: m/z 409.0 [M+H]+, 431.1 [M+Na]+, 839.1
[2M+Na]+. ESI-HRMS: m/z [M+Na]+ calcd for C21H16N2O5SNa: 431.0672; found:
1
431.0714. H NMR (400 MHz, DMSO-d6): δ 11.12 (1H, s), 8.07-8.13 (2H, m),
7.87-7.92 (2H, m), 7.84(2H, d, J = 8.3 Hz), 7.44 (2H, d, J = 8.3 Hz), 7.43 (1H, s),
2.39 (3H, s), 2.23 (3H, s). 13C NMR (150 MHz, DMSO-d6): δ 179.94, 172.72, 156.23,
151.75, 143.61, 142.85, 135.90, 134.28, 134.04, 132.56, 132.33, 130.81, 129.58,
127.36, 126.40, 126.28, 107.76, 20.93, 13.72.
4.1.6. Synthesis of 2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (9) [16]
To a 100 mL of three-necked flask was added BBI608 (5.0 g, 0.02 mol),
dissolved in 40 mL of N,N-dimethylformamide and 2 mL H2O. Then sodium
borohydride (0.8 g, 0.021 mol) was added in portions at the rate that maintained a
reaction temperature blow 30℃ and the mixture was stirred at room temperature for
another 3 hours. The reaction solution was poured into 200 mL of 2% hydrochloric
acid and stirred for 2 hour. It was extracted with EtOAc (3×20 mL), and the organic
layer was washed with water, brine and dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. And then the crude product was recrystallized
from EtOAc (30 mL) to give a yellow solid. (3.1 g, 61.51%). M.p. 146-148℃.
1
ESI-MS: m/z 243.1 [M+H]+, 265.0 [M+Na]+, 241.2 [M-H]-. H-NMR (400 MHz,
CDCl3): δ 8.17-8.23 (1H, m), 8.13-8.16 (1H, m), 7.72-7.78 (2H, m),6.86 (1H, s), 5.06
(1H, q, J = 6.6 Hz), 2.32 (1H, br), 1.66 (3H, d, J = 6.6 Hz).
4.1.7. Synthesis of 2- (1-chloroethyl)naphtha[2,3-b]furan-4,9-dione (LD-4) [30]
To a 25 mL of single-necked flask was added 9 (0.6 g, 0.0025 mol), dissolved in
dichloromethane (15 mL), and then thionyl chloride (1 mL, 0.014 mol) was added in
one portion. The mixture was stirred for 2 hours at room temperature. It was poured

ACCEPTED MANUSCRIPT
into water (30 mL), the aqueous layer was extracted with dichloromethane(2×15 mL).
The combined organic layers were washed with water, brine and dried over anhydrous
Na2SO4. After removal of the solvent in vacuo, the crude product was purified by
flash chromatography involving silica gel column chromatography using Petroleum
Ether/EtOAc=15:1 as an eluant to afford LD-4 as a yellow solid. (0.52 g, 80.2% yield).
M.p. 143-145℃. ESI-MS: m/z 261.0 [M+H]+, 283.0 [M+Na]+, 543.0 [2M+Na]+.
ESI-HRMS: m/z [M+H] + calcd for C14H9ClO3: 261.0313, 263.0283; found: 261.0309,
1
263.0283. H NMR (400MHz, CDCl3): δ 8.17-8.24 (2H, m), 7.74-7.79 (2H, m), 6.93
(1H, s), 5.19 (1H, q, J = 6.9 Hz), 1.96 (3H, d, J = 6.9 Hz). 13C NMR (150 Hz,
DMSO-d6): δ 179.93, 172.87, 160.74, 151.80, 134.23, 134.11, 132.57, 132.10, 130.34,
126.43, 126.29, 105.80, 49.45, 22.25.
4.1.8. General procedure for preparation of compounds LD-5~LD-21
To a solution of 2-(1-chloroethyl)naphtha[2,3-b]furan-4,9-dione (LD-4, 0.5 g,
1.92 mmol) and anhydrous potassium carbonate (0.8 g, 5.77 mmol) in acetonitrile (20
mL) was added the corresponding amine (2.5 mmol). The mixture was refluxed for
2-8 hours under argon. After the solvent was removed in a vacuum, water and ethyl
acetate was then added successively to the residue, and the organic layer was washed
with water and then brine and dried over MgSO4, filtered and concentrated under
reduced pressure. The crude product was purified by flash chromatography involving
silica gel column chromatography.
4.1.8.1. 2-(1-(methyl amino) ethyl) naphtha [2,3-b] furan-4,9-dione (LD-5)
Yellow solid, yield 16.32%. M.p. 133-135℃. ESI-MS: m/z 256.1 [M+H]+, 511.2
[2M+H]+. ESI-HRMS: m/z [M+H]+ calcd for C15H14NO3: 256.0968; found: 256.0990;
1
[M+Na]+ calcd for C15H13NO3Na: 278.0788; found: 278.0814. H-NMR (400 MHz,
CDCl3): δ 8.17-8.23 (2H, m), 7.75-7.77 (2H, m), 6.83 (1H, s), 4.00 (1H, q, J = 6.8 Hz),
2.45 (3H, s), 2.19 (1H, s), 1.56 (3H, d, J = 6.8 Hz). 13C NMR (150 MHz, DMSO-d6):
δ 180.38, 172.46, 166.68, 151.13, 134.13, 133.86, 132.52, 132.10, 130.79, 126.30,
126.18, 103.92, 52.49, 33.42, 19.69.
4.1.8.2. 2-(1-(dimethyl amino)ethyl) naphtha[2,3-b]furan-4,9-dione (LD-6)

ACCEPTED MANUSCRIPT
Yellow solid, yield 42.53%. M.p. 105-108℃. ESI-MS: m/z 270.1 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C16H16NO3: 270.1125; found: 270.1148; [M+Na]+
1
calcd for C16H15NO3Na: 292.0944; found: 292.0969. H-NMR (400 MHz, CDCl3): δ
8.21-8.23 (1H, m), 8.17-8.19 (1H, m), 7.72-7.78 (2H, m), 6.80 (1H, s), 3.99 (1H, q, J
= 7.0 Hz), 2.33 (6H, s), 1.53 (3H, d, J = 7.0 Hz). 13C NMR (150 MHz, DMSO-d6): δ
180.41, 172.59, 163.58, 151.37, 134.18, 133.94, 132.59, 132.15, 130.49, 126.35,
126.23, 105.61, 56.42, 40.85, 14.57.
4.1.8.3. 2-(1-(ethyl amino) ethyl) naphtha [2,3-b] furan-4,9-dione (LD-7)
Yellow solid, yield 23.20%. M.p. 152-154℃. ESI-MS: m/z 270.1 [M+H]+, 292.1
[M+Na]+, 539.0 [2M+H]+. ESI-HRMS: m/z [M+H]+ calcd for C16H16NO3: 270.1125;
found: 270.1132; [M+Na]+ calcd for C16H15NO3Na: 292.0944; found: 292.0956.
1H-NMR(600 MHz, D2O) δ: 7.85-7.87 (2H, m), 7.69-7.71 (2H, m), 7.07 (1H, s), 4.75
(1H, q, J = 7.0 Hz), 3.02~3.10 (2H, m), 1.74 (3H, d, J = 7.0 Hz), 1.24 (3H, t, J = 7.3
Hz). 13C NMR (150 MHz, DMSO-d6): δ 179.93, 172.85, 156.72, 152.25, 134.35,
134.24, 132.47, 131.98, 130.20, 126.47, 126.33, 108.68, 54.80, 49.33, 15.97, 10.90.
4.1.8.4. 2-(1-((2-hydroxypropyl)amino)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-8)
Yellow solid, yield 21.62%. M.p.182-184℃. ESI-MS: m/z 300.2 [M+H]+, 322.1
[M+Na]+, 297.8 [M-H]-. ESI-HRMS: m/z [M+Na]+ calcd for C17H17NO4Na: 322.1050;
1
found: 322.1054. H-NMR(600 MHz, D2O): δ 7.96-7.80 (2H, m), 7.74-7.76 (2H, m),
7.13 (1H, s), 4.82 (1H, q, J = 7.0 Hz), 3.96-4.06 (1H, m), 3.07-3.13 (1H, m),
2.85-2.96 (1H, m), 1.76 (3H, d, J = 7.0Hz), 1.14 (3H, d, J = 6.4 Hz). 13C NMR (150
MHz, DMSO-d6): δ 179.98, 172.87, 156.69, 152.25, 134.38, 134.27, 132.49, 132.00,
130.23, 126.49, 126.35, 108.88, 62.35, 21.02, 15.64.
4.1.8.5. 2-(1-(diethyl amino)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-9)
Yellowish solid, yield 17.49%. M.p.72-75℃. ESI-MS: m/z 298.1 [M+H]+, 320.1
[M+Na]+. ESI-HRMS: m/z [M+H]+ calcd for C16H16NO3: 298.1438; found: 298.1436.
1H-NMR (600 MHz, CDCl3): δ 8.20-8.22 (1H, m), 8.16-8.18 (1H, m), 7.72-7.76 (2H,
m), 6.79 (1H, s), 4.23 (1H, q, J = 6.6 Hz), 2.68-2.74 (2H, m), 2.45-2.53 (2H, m), 1.52
13
(3H, d, J = 6.6 Hz), 1.11 (6H, t, J= 6.8 Hz). C NMR (150 MHz, DMSO-d6): δ
180.33, 172.45, 165.06, 151.14, 134.11, 133.85, 132.50, 132.09, 130.58, 126.28,

ACCEPTED MANUSCRIPT
126.16, 105.14, 52.25, 43.39, 14.79, 13.80.
4.1.8.6. 2-(1-(bis(2-hydroxyethyl)amino)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-10)
Yellowish solid, yield 14.24%. M.p.114-116℃. ESI-MS: m/z 330.1 [M+H]+,
352.1 [M+Na]+, 327.9 [M-H]-. ESI-HRMS: m/z [M+Na]+ calcd for C18H19NO5Na:
1
352.1155; found: 352.1168. H NMR (600 MHz, CDCl3): δ 8.05-8.17 (2H, m),
7.67-7.78 (2H, m), 6.82 (1H, s), 4.25 (1H, q, J = 6.8 Hz), 3.60-3.72 (4H, m), 3.15 (2H,
br), 2.65-2.83 (4H, m), 1.51 (3H, d, J = 6.8 Hz). 13C NMR (150 MHz, DMSO-d6): δ
180.97, 173.07, 165.52, 151.81, 134.73, 134.47, 133.12, 132.70, 131.19, 126.90,
126.77, 105.92, 60.69, 54.61, 53.88, 15.49.
4.1.8.7. 2-(1-(phenethylamino)ethyl) naphtha[2,3-b]furan-4,9-dione (LD-11)
Yellowish solid, yield 16.42%. M.p.193-195℃. ESI-MS: m/z 346.2 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C22H20NO3: 346.1438; found: 346.1446; [M+Na]+
1
calcd for C22H19NO3Na: 368.1257; found: 368.1283. H-NMR(600 MHz, D2O): δ
7.96-8.01 (2H, m), 7.74~7.78 (2H, m), 7.21-7.29 (2H, m), 7.16-7.22 (3H, m), 7.08
(1H, s), 4.75 (1H, q, J = 7.0Hz), 3.21-3.35 (2H, m), 2.90-3.01 (2H, m), 1.71 (3H, d, J
= 7.0 Hz). 13C NMR (150 MHz, DMSO-d6): δ 179.90, 172.84, 156.37, 152.32, 136.81,
134.28, 134.18, 132.44, 131.95, 130.13, 128.49, 128.43, 126.63, 126.41, 126.27,
108.83, 49.73, 45.52, 31.44, 15.85.
4.1.8.8. 2-(1-(pyrrolidin-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-12)
Yellowish solid, yield 14.70%. M.p.73-75℃. ESI-MS: m/z 296.2 [M+H]+, 318.2
[M+Na]+. ESI-HRMS: m/z [M+H]+ calcd for C18H18NO3: 296.1281; found: 296.1313;
1
[M+Na]+ calcd for C18H17NO3Na: 318.1101; found: 318.1135. H NMR (600 MHz,
CDCl3): δ 8.19-8.22 (1H, m), 8.15-8.17 (1H, m), 7.73-7.75 (2H, m), 6.82 (1H, s), 3.90
(1H, q, J = 6.8 Hz), 2.61-2.75 (4H, m), 1.78-1.84 (4H, m), 1.58 (3H, d, J = 6.8 Hz).
13C NMR (150 MHz, DMSO-d6): δ 180.29, 172.51, 164.51, 151.14, 134.11, 133.86,
132.50, 132.08, 130.53, 126.28, 126.15, 104.93, 54.89, 49.96, 22.81, 17.79.
4.1.8.9. 2-(1-(1H-pyrrol-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-13)
Yellow solid, yield 7.83%. M.p.138-140 ℃. ESI-MS: m/z 289.9 [M-H]-.
1
ESI-HRMS: m/z [M+Na]+ calcd for C18H13NO3Na: 314.0788; found: 314.0809. H
NMR (600 MHz, CDCl3): δ 8.25-8.28 (1H, m), 8.22-8.24 (1H, m), 8.17-8.21 (1H, m),

ACCEPTED MANUSCRIPT
8.13-8.16 (1H, m), 7.79-7.81 (2H, m), 7.69-7.76 (2H, m), 4.36 (1H, q, J = 7.2 Hz),
1.76 (3H, d, J = 7.2 Hz). 13C NMR (150 MHz, DMSO-d6): δ 180.29, 172.43, 166.33,
150.91, 134.42, 134.14, 132.81, 132.40, 130.96, 126.58, 126.29, 117.13, 104.67,
54.80, 21.97.
4.1.8.10. 2-(1-(1H-imidazol-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-14)
Yellow solid, yield 14.37%. M.p.145-147℃. ESI-MS: m/z 293.2 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C17H13N2O3: 293.0921; found: 293.0957. 1H-NMR
(600 MHz, CDCl3): δ 8.17-8.27(2H, m), 8.08-8.16 (2H, m), 7.73-7.76 (2H, m),
7.70-7.73 (1H, m), 6.64 (1H, s), 4.30 (1H, q, J = 6.9 Hz), 1.36 (3H, d, J = 6.9 Hz). 13C
NMR (150 MHz, DMSO-d6): δ 180.27, 172.33, 166.84, 165.18, 151.13, 134.15,
133.89, 132.47, 132.02, 131.56, 131.41, 130.77, 128.54, 126.31, 126.14, 104.92,
64.90, 14.08.
4.1.8.11. 2-(1-(cyclohexylamino)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-15)
Yellowish solid, yield 42.09%. M.p.129-131. ESI-MS: m/z 310.1 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C19H20NO3: 310.1438; found: 310.1440; [M+Na]+
1
calcd for C19H19NO3Na: 332.1257; found: 332.1262. H-NMR (400 MHz, CDCl3): δ
8.21-8.23 (1H, m), 8.17-8.19 (1H, m), 7.72-7.78 (2H, m), 6.80 (1H, s), 4.04 (1H, q, J
= 6.8Hz)), 2.63 (2H, br), 2.50 (2H, br), 1.58-1.75 (6H, m), 1.42 (3H, d, J = 6.8 Hz).
13C NMR (150 MHz, DMSO-d6): δ 180.40, 172.56, 163.75, 151.27, 134.14, 133.89,
132.56, 132.14, 130.51, 126.31, 126.19, 105.53, 56.81, 40.70, 25.78, 24.06, 14.61.
4.1.8.12. 2-(1-(cyclohexylamino)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-16)
Yellow solid, yield 12.90%. M.p.143-145℃. ESI-MS: m/z 324.2 [M+H]+, 346.2
[M+Na]+. ESI-HRMS: m/z [M+H]+ calcd for C20H22NO3: 324.1594; found: 324.1610;
[M+Na]+ calcd for C20H21NO3Na: 346.1414; found: 346.1431.1H NMR (600 MHz,
CDCl3): δ 8.20-8.21 (1H, m), 8.16-8.17 (1H, m), 7.73-7.75 (2H, m), 6.81 (1H, s),
4.21(1H, q, J = 6.7 Hz), 2.44 (1H, br), 1.96-1.98 (1H, m), 1.70-1.78 (3H, m),
1.58-1.60 (1H, m),1.53 (3H, d, J = 6.7 Hz), 1.10-1.26 (6H, m). 13C NMR (150 MHz,
DMSO-d6): δ 179.98, 172.85, 156.88, 152.27, 134.36, 134.25, 132.50, 132.01, 130.24,
126.48, 126.33, 108.60, 54.17, 46.60, 28.98, 27.88, 24.57, 23.74, 23.71, 16.58, 14.57.
4.1.8.13. 2-(1-morpholinoethyl) naphtha [2,3-b] furan-4,9-dione (LD-17)

ACCEPTED MANUSCRIPT
Yellow solid, yield 22.29%. M.p.137-139℃. ESI-MS: m/z 312.1 [M+H]+.
ESI-HRMS: m/z [M+Na]+ calcd for C18H17NO4Na: 334.1050; found: 334.1062.
1H-NMR (400 MHz, CDCl3): δ 8.21-8.24 (1H, m), 8.16-8.21 (1H, m), 7.74-7.79 (2H,
m), 6.90 (1H, s), 4.09 (1H, q, J = 6.4 Hz), 3.82 (4H, s), 2.63-2.79 (4H, m), 1.62 (3H, d,
J = 6.4 Hz). 13C NMR (150 MHz, DMSO-d6): δ 180.94, 173.21, 163.68, 152.05,
134.74, 134.50, 133.15, 132.73, 131.04, 126.91, 126.91, 126.79, 106.48, 57.07, 49.74,
15.28.
4.1.8.14. 2-(1-(piperazin-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-18)
Yellow solid, yield 29.35%. M.p.180-182℃. ESI-MS: m/z 311.3 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C18H19N2O3: 311.1390; found: 311.1390. [M+Na]+
1
calcd for C18H18N2O3Na: 333.1210; found: 333.1215. H-NMR (600 MHz, D2O): δ
7.87-7.94 (2H, m), 7.72-7.75 (2H, m), 7.14 (1H, s), 4.77 (1H, q, J = 7.0 Hz),
3.47-3.52 (4H, m), 3.37-3.34 (4H, m), 1.76 (3H, d, J = 7.0 Hz). 13C NMR (150 MHz,
DMSO-d6): δ 179.53, 172.26, 163.14, 151.65, 133.93, 133.80, 131.94, 131.45, 129.73,
126.02, 125.87, 104.09, 56.11, 44.58, 40.96, 13.33.
4.1.8.15. 2-(1-(4-methylpiperazin-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-19)
Yellow solid, yield 33.71%. M.p.154-155℃. ESI-MS: m/z 325.2 [M+H]+, 347.2
[M+Na]+. ESI-HRMS: m/z [M+H]+ calcd for C19H21N2O3: 325.1547; found: 325.1553.
1
[M+Na]+ calcd for C19H20N2O3Na: 347.1366; found: 347.1373. H-NMR (600 MHz,
CDCl3): δ 8.21-8.22 (1H, m), 8.17-8.18 (1H, m), 7.73-7.77 (2H, m), 6.77 (1H, s),
3.96(1H, q, J = 7.0 Hz), 2.55-2.79 (8H, m), 2.33 (3H, s), 1.51(3H, d, J = 7.0 Hz). 13C
NMR (150 MHz, DMSO-d6): δ 180.30, 172.52, 163.40, 151.28, 134.08, 133.84,
132.49, 132.07, 130.40, 126.25, 126.13, 105.63, 56.06, 54.76, 45.52, 14.78.
4.1.8.16. 2-(1-(4-ethylpiperazin-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione (LD-20)
Yellowish solid, yield 19.52%. M.p.80-82℃. ESI-MS: m/z 339.3 [M+H]+.
ESI-HRMS: m/z [M+H]+ calcd for C20H23N2O3: 339.1703; found: 339.1716. [M+Na]+
calcd for C20H22N2O3Na: 361.1523; found: 361.1537. 1H-NMR (600 M Hz, CDCl3): δ
8.21-8.23 (1H, m), 8.17-8.19 (1H, m), 7.72-7.78 (2H, m), 6.77 (1H, s), 3.98 (1H, q, J
= 7.0 Hz), 2.51-2.82(10H, m), 1.51 (3H, d, J = 7.0 Hz), 1.15 (3H, m). 13C NMR (150
MHz, DMSO-d6): δ 180.38, 172.60, 163.42, 151.37, 134.15, 133.90, 132.57, 132.15,

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130.48, 126.32, 126.20, 105.72, 56.13, 52.51, 51.44, 14.92, 11.92.
4.1.8.17. 2-(1-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)naphtha[2,3-b]furan-4,9-dione
(LD-21)
Yellowish solid, yield 14.68%. M.p.108-111℃. ESI-MS: m/z 355.2 [M+H]+, 377.2
[M+Na]+. ESI-HRMS: m/z [M+H]+ calcd for C20H23N2O4: 355.1652; found: 355.1673.
1
[M+Na]+ calcd for C20H22N2O4Na: 377.1472; found: 377.1500. H-NMR(400 MHz,
CDCl3): δ 8.20-8.24 (1H, m), 8.16-8.19 (1H, m), 7.74-7.76 (2H, m), 6.77 (1H, s),
3.98(1H, q, J = 7.0 Hz), 3.73 (2H, t, J = 5.1), 3.59 (1H, m), 2.76-2.82 (4H, m),
2.72-2.76 (2H, m), 2.64-2.72 (4H, m), 1.51 (3H, d, J = 7.0 Hz). 13C NMR (150 MHz,
DMSO-d6): δ 180.38, 172.60, 163.40, 151.36, 134.15, 133.91, 132.57, 132.15, 130.48,
126.33, 126.21, 105.75, 60.06, 58.32, 56.13, 53.31, 14.93.
4.2. In vitro inhibitory activity on HepG2 cells and L-02 cells assay
Human normal liver L-02 cells and the hepatocellular carcinoma HepG2 cells
were purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese
Academy of Sciences. They were cultured in high glucose Dulbecco's Modified Eagle
Medium (gibco, NY, USA) supplemented with 10% fetal bovine serum (TBD science,
Tianjin, China), and penicillin/streptomycin (Sigma, St. Louis, MO, USA) at 37 °C
under 5% CO2 until they were 70-80% confluent. The adherent cells were digested
with 0.25% trypsin (containing EDTA 1 mmol/L) and resuspended in culture medium
and the cell concentration was 6×104 cells/mL.
Cell viability was analyzed by using Cell Counting Kit-8 (CCK-8) (Beyotime,
Shanghai, China), followed by manufacturer’s instructions. The Cells were plated in
96-well plate at a density of 4 × 103 cells/well and grown for 24 h. Following the
addition of different concentrations of compounds LD-1~LD-21 or BBI608, the cells
were further cultured for 48 h, with 0.5% DMSO as the solvent control group. Then
10 µL CCK-8 regent was added to each well and the culture plate was settled at 37 °C
incubation chamber for 2 h. The optical density (OD) at a wavelength of 450 nm was
detected by using a microplate reader. Cell viability was expressed as percentage of

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untreated controls. Each treatment was performed in triplicate. The half inhibitory
concentration IC50 values were calculated by Prism 6 (GraphPad Software).
4.3 Molecular docking
The molecular docking study was performed by AutoDock 4.2.6 [31]. The three
dimensional structure of STAT3 was retrieved from the Protein Data Bank (PDB code:
1BG1 [24]). The binding site was located on the SH2 region. The 3D structures of
BBI608, compounds LD-8, LD-10 and LD-19 were built by using Discovery Studio
4.5 [32]. Both the protein and the ligands were prepared by adding polar hydrogen
atoms and partial charges with the assistance of AutoDockTools 1.5.6. Grid points
were set to 40 at the x, y, z axes. The conformational search was carried out using the
Lamarckian Genetic Algorithm. All other parameters were set to default unless stated
otherwise.
Conflicts of interest
The authors declare no other conflicts of interest.
Acknowledgments
This work was supported by the project 201602707 (Natural Science Foundation
of Liaoning Province).
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