The synthesis of furoquinolinedione and isoxazoloquinolinedione derivatives as selective Tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104881

Based on our previous study on the development of the furoquinolinedione and isoxazoloquinolinedione TDP2 inhibitors, the further structure–activity relationship (SAR) was studied in this work. A series of furoquinolinedione and isoxazoloquinolinedione de

Full text of DOI:10.1016/j.bioorg.2021.104881

Bioorganic Chemistry 111 (2021) 104881
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The synthesis of furoquinolinedione and isoxazoloquinolinedione
derivatives as selective Tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors
Hao Yanga, Xiao-Qing Zhua, Wenjie Wangb, Yu Chena, Zhu Hua, Yu Zhanga, De-Xuan Hua,
,c,*
Le-Mao Yua, Keli Agamab, Yves Pommierb, Lin-Kun Ana
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States
c Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, Guangzhou 510006, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Based on our previous study on the development of the furoquinolinedione and isoxazoloquinolinedione TDP2
inhibitors, the further structure–activity relationship (SAR) was studied in this work. A series of furo-
quinolinedione and isoxazoloquinolinedione derivatives were synthesized and tested for enzyme inhibitions.
Enzyme-based assays indicated that isoxazoloquinolinedione derivatives selectively showed high TDP2 inhibi-
tory activity at sub-micromolar range, as well as furoquinolinedione derivatives at low micromolar range. The
most potent 3-(3,4-dimethoxyphenyl)isoxazolo[4,5-g]quinoline-4,9-dione (70) showed TDP2 inhibitory activity
Tyrosyl-DNA phosphodiesterase
Topoisomerase
DNA repair
DNA damage
Furoquinolinedione
Isoxazoloquinolinedione
with IC50 of 0.46 ± 0.15
μ
M. This work will facilitate future efforts for the discovery of isoxazoloquinolinedione
TDP2 selective inhibitors.
1. Introduction
blocking lesions in the absence of tyrosyl-DNA phosphodiesterase 1
(TDP1) [12]. These findings imply that TDP2 is as a rational target for
cancer treatment.
Chemotherapeutics are still one of the most effective approaches for
cancer treatment. However, drug-resistance of cancer cells becomes a
significant impediment to successful chemotherapy and usually leads to
cancer treatment failure. The mechanisms of drug-resistance are
complicated. Among them, the overexpression of DNA repair enzyme in
cancer cells is an important reason [1]. Tyrosyl-DNA phosphodiesterase
2 (TDP2) is a recently discovered DNA repair enzyme that can specif-
ically cleaves 5′-phosphotyrosyl protein-DNA bond, which are mainly
derived from abortive topoisomerase 2 (TOP2)-DNA cleavage complexes
(TOP2cc) [2,3]. TOP2cc can be trapped by various exogenous factors
including TOP2 poisons [4], DNA intercalators [5] and cytosine arabi-
noside [6], resulting in double-stranded DNA damage, and ultimately 5′-
phosphotyrosyl protein-DNA adducts. Such adducts can be firstly
cleaved by TDP2 to give the nicked DNA with clean ends for subsequent
DNA repair. Up-regulated expression of TDP2 results in cells resistant to
TOP2 poison etoposide [2,7]. Conversely, TDP2-deleted or knocked-
down cells and animal model show hypersensitive to etoposide and
elevate levels of TOP2-mediated DNA damage[2,7–10]. TDP2 inhibition
also can lead to cells sensitive to etoposide[11]. Moreover, TDP2 can
catalytically hydrolyze topoisomerase 1 protein-DNA crosslinks and 3′-
In addition, TDP2 has also been identified as a viral protein unlinkase
enzyme involved in the viral translation and RNA synthesis of viruses,
such as picornaviruses and hepatitis B virus [13,14] The genome repli-
cation of these viruses is protein-primed via a tyrosine residue, which
results in 5′-phosphotyrosyl protein-nucleic acid adducts similar to
TOP2cc. Hence, TDP2 is also a potential target for the development of
novel antiviral agents.
Because of the multiple physiological functions of TDP2, the dis-
covery of its inhibitors has attracted attention. To date, several TDP2
inhibitor chemotypes (Fig. 1) have been reported, including deazaflavin
1 [11,15–17], isoquinoline-1,3-dione 2 [18], indenoisoquinoline 3 [19],
diaminoquinoline-2,8-dione 4 [10], benzylidenepyrazolone 5 [20],
disulfides 6 [20], triazolopyridine 7 [21] and quinazolinylaminopyr-
imidinone 8 [22], etc. Among them, the deazaflavin derivatives show
high TDP2 inhibition at submicromolar level (1: IC50 = 0.59 μM), and
are the only reported TDP2 inhibitors showing synergistic effects with
etoposide [11]. However, further study indicated that deazaflavin TDP2
inhibitors remained synergistic with etoposide in TDP2 knockout cells,
implying that they possibly have additional cellular targets beyond
* Corresponding author at: School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
E-mail address: lssalk@mail.sysu.edu.cn (L.-K. An).
https://doi.org/10.1016/j.bioorg.2021.104881
Received 18 January 2021; Received in revised form 17 March 2021; Accepted 28 March 2021
Available online 1 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
TDP2 [17,23]. Although isoquinoline-1,3-dione derivatives show high
TDP2 inhibition (2: IC50 = 1.9
μM), there is no synergistic effect
observed with etoposide. The disulfides 6 are chemically fragile with the
profiles of pan-assay interference structure (PAINS), as well as 4 (redox
cycler) and 5 (Michael acceptor) [24]. Indenoisoquinolines were re-
ported as triple inhibitors of TOP1/TDP1/TDP2 with only moderate
TDP2 inhibition (3: +++) [19]. Triazolopyridines 7 and quinazoliny-
laminopyrimidinones 8 display weak TDP2 inhibition at high micro-
molar concentration range [21]. Therefore, there is a strong need to
discover novel TDP2 selective inhibitors as potential anticancer or
antiviral agents.
In our effort to discover novel TDP2 inhibitors, furoquinolinediones
were found showing good TDP2 inhibition with IC50 values at low
micromolar concentration levels, for example 9 and its isoxazole
analogue 10 with IC50 values of 11 ± 1.0 μM and 1.9 ± 0.28 μM (Fig. 1),
respectively [25]. To obtain the structure–activity relationship (SAR)
and discover novel TDP2 inhibitors with more potency, we report the
synthesis and SAR of furoquinolinedione derivatives and iso-
xazoloquinolinedione derivatives as TDP2 inhibitors in this work.
2. Results and discussion
2.1. Chemistry
To study the effect of the number and position of nitrogen atom in A-
ring on TDP2 inhibitory activity, the furoquinolinedione derivatives
11–15 were synthesized in a one-pot reaction under base condition as
shown in Scheme 1. The reaction of ethyl acetoacetate and 2,3-dichlor-
oacridine-1,4-dione mainly gave the derivative 16 with fused benzene
ring to A-ring (Scheme 1). To study effect of the substituents at A-ring on
TDP2 inhibitory activity, the furoquinolinedione derivatives 19–31 and
33–36 were synthesized as shown in Schemes 2 and 3. According to our
reported method [26], the 8-hydroxyquinoline materials 17a and 17b
were oxidized with sodium chlorate to give the dichloroquinolinedione
intermediates 18a and 18b (Scheme 2). Subsequently, the intermediates
18a (or 18b) reacted with active methylene reagent (AMR), ethyl
Scheme 1. Synthesis of compounds 11–16. Reagents and conditions: (a) Ethyl
acetoacetate, K2CO3, CH3CN, reflux. (b) Ethyl acetoacetate, Cu(OAc)2, K2CO3,
CH3CN, reflux.
Fig. 1. The reported TDP2 inhibitors.
2

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
Scheme 2. Synthesis of compounds 19–31. Reagents and conditions: (a) NaClO3, conc. hydrochloric acid. (b) Ethyl acetoacetate, K2CO3, MeCN, reflux. (c) Secondary
amine, dioxane, rt. (for 23–26, 30 and 31). (d) ArB(OH)2, Ph(PPh3)4, Na2CO3, DME (for 27–28). (e) R1H, NaH, THF (for 29).
Scheme 3. Synthesis of compounds 33–36. Reagents and conditions: (a) Ac2O, MeCN, rt.
acetoacetate to simultaneously give two isomers, N,O-anti isomer 19 (or
21) and N,O-syn isomer 20 (or 22). The target compounds 23–31 were
obtained through nucleophilic substitution or Suzuki reaction of the
chloro-substituted compounds 19 and 20 (Scheme 2) [27,28]. As shown
in Scheme 3, the target compounds 33–36 could be obtained from the
cyclization reaction of 5-bromo-furoquinone material with the hydra-
zines 32a-32d prepared according to the literature [29].
and NBS in the presence of azodiisobutyronitrile (AIBN) to give the
monobromo-substituted derivative 43 and dibromo-substituted deriva-
tive 44 simultaneously. The compounds 45–47 were synthesized via the
nucleophilic substitution of 43 with amine materials. The bromo group
of 43 could be displaced by hydroxy group under 150 OC condition to
give 48, which was subsequently oxidized to give 49.
To study effect of the number and position of nitrogen atom in A-ring
on TDP2 inhibitory activity of the isoxazoloquinolinedione derivatives,
the isoxazoles 50–53 were synthesized as shown in Scheme 6. Under the
catalysis of Mn(OAc)3, the reaction of the 6- and 7-bromoisoquinoline-
5,8-diones with ethyl nitroacetate gave the target products 50 and 51,
respectively [30,31]. According to our reported method [25], the reac-
tion of 6,7-dichloroquinoxaline-5,8-dione with ethyl nitroacetate gave
the target compound 52. Under the catalysis of PhI(OAc)2 [32], the
The furoquinolinedione derivatives 37–49 with various substituent
at 2-position were synthesized as shown in Scheme 4 and 5. Similarly,
the reaction of 6,7-dichloroquinoline-5,8-dione with AMR, such as ethyl
3-oxopentanoate, ethyl 4-methyl-3-oxopentanoate and ethyl 3-oxo-3-
phenylpropanoate, simultaneously gave two furoquinolinedione iso-
mers, N,O-anti isomer and N,O-syn isomer (Scheme 4). As shown in
Scheme 5, bromination could occur between the 2-methyl group of 9
3

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
Scheme 4. Synthesis of compounds 37–42. Reagents and conditions: (a) RCOCH2COOEt, K2CO3, MeCN, reflex.
Scheme 5. Synthesis of compounds 43–49. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux. (b) Secondary amine, DCM, rt (for 45–47). (c) DMSO/H2O, air, 150
℃ (obtained 48 and 49 simultaneously).
reaction of ethyl (Z)-2-(hydroxyimino)-3-oxobutanoate with 2,3-dime-
thylphenol gave the lacking A-ring analogue 53. As shown in Scheme
7, isoxazole derivatives 54–57 were synthesized from a one-pot reaction
of 6,7-dichloroquinoline-5,8-dione with nitroacetate materials. Similar
to the synthesis of 50 and 51, the reaction of 7-bromoquinolinedione
with various nitroacetates 58a-e gave the target isoxazoles 59–63
(Scheme 8). The cyclization between 7-bromoquinolinedione and 64a-g
in the presence of sodium hypochlorite and triethylamine afforded the
target products 65–71 [33].
In our previous investigation [25], N,O-anti furoquinolinedione de-
rivatives showed higher TDP2 inhibitory activity than that of the N,O-
syn isomers. Same SAR was observed in this work. For example, for N,O-
trans/N,O-syn isomers 37 (37 ± 2.0
μ
M)/38 (>111 M), 39 (25 ± 7.7
μ
μ
M)/40 (>111 M), 41 (28 ± 6.4
μ
μ
M)/42 (47 ± 24 μM), the activity of
N,O-trans isomers showed higher inhibitory activity, implying the
important role of nitrogen atom position at A-ring. The TDP2 inhibition
results also indicated that the activity could be decreased by removing
the nitrogen atom from A-ring (11: > 111
at 6-position (61 ± 43
Furthermore, the presence of two N atoms at A-ring could not benefit the
activity (12: 32 ± 3.2 M, 13: > 111 M), even if one of N atoms at 5-
position (12). The fused aromatic ring at A-ring abolished the activity
(16: > 111 M).
According to the TDP2 inhibition (Table 1), the 6-substituent
μ
M), as well as 14 with N atom
Finally, twenty-seven furoquinolinedione derivatives and twenty
isoxazoloquinolinedione derivatives were synthesized and characterized
through HRMS and NMR spectra.
μM) and 15 with N atom at 7-position (>111 μM).
μ
μ
μ
2.2. Enzyme inhibition assays
decreased the activity comparing with 9 (11 ± 1.0 μM), implying that
All target compounds were screened firstly against three enzymes,
recombinant TOP1, TDP1 and TDP2 according to our reported protocol
[34–36]. All compounds did not show obvious inhibition against both
the larger steric bulk of the 6-substituent impaired the activity. For
example, compounds 25–29 with larger 6-substituted group did not
show TDP2 inhibition (>111 μM) except for 24 (44 ± 2.0 μM). Similarly,
TDP1 at the highest concentration tested of 111
μ
M and TOP1 at the
M. Subsequently, the compounds
with TDP2 potency were tested at six or eight three-fold dilution con-
the larger steric bulk of the 8-substituent also impaired the activity (36:
highest concentration tested of 100
μ
28 ± 1.3 μM, 35: 69 ± 3.1 μM, 34: > 111 μM). And, all compounds
(30–33) with 7-substituents did not show inhibitory activity (>111 μM),
centrations from 111 μM to 0.46 μM or 0.051 μM against TDP2 using a
32P-labeled single-stranded oligonucleotide TY19 substrate bearing 5-
phosphotyrosyl group [36]. The TDP2 inhibitory activities were
expressed as IC50 values, defined as the concentration of compound that
inhibits 50% of enzyme activity, and summarized in Tables 1.
implying that the 7-substituents abolished the activity.
Compared with 9, compound 49 (5.2 ± 4.1 μM) with formyl group at
2-position showed increased activity. Other compounds with 2-alkyl
(37, 39), -aromatic (41) and -alkylaminomethyl (45–47) group
4

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
concentration. And, compound 70 showed the highest TDP2 inhibitory
activity with IC50 of 0.46 ± 0.15 μM. This SAR was different from that of
furoquinolinedione analogues, of which the ester function at 3-position
is critical for TDP2 potency [25]. These results implied that the binding
mode of isoxazole derivatives with TDP2 enzyme might be different
from that of furoquinolinediones. The representative gels of TDP2 in-
hibitions are shown in Fig. 2. In TDP2 inhibition assays, deazaflavin 1
was used as a positive control [15]. After being incubated with the in-
hibitors, the band of the enzyme product TP19 decreased in a dose-
dependent manner and almost disappeared at 0.46 μM concentration,
implying the full TDP2 inhibition. Similarly, the tested compounds
showed TDP2 inhibition also in a dose-dependent manner.
2.3. Molecular modeling
To view the molecular interactions of the bioactive iso-
xazoloquinolinedione derivatives with TDP2, we constructed a hypo-
thetical binding model using in-silico docking. The co-crystal structure
of humanized mouse TDP2 (PDB code: 5J3Z) was employed as a tem-
plate because of lack of human TDP2 structure [37]. The reason of using
mTDP2 as a homology modeling template is that the mutated mouse
residue to its human equivalent: H323L, which shows the same inhibi-
tory efficacy on SV-163 as hTDP2 and verifies the reliability of this
template [37]. Moreover, mTDP2 has few key amino acid residue mu-
tations adjacent to the catalytic site, implying it is suitable to be used as
a mimic of hTDP2 [16].
The inhibitors were docked into the binding site. The hypothetical
structure pose of the top-ranked 70 is shown in Fig. 3. elucidated the
binding mode. Fig. 3A shows that 70 binds to the DNA binding channel
adjacent to the site of tyrosyl-DNA phosphoester cleavage, similar to the
direction of TDP2-bound DNA in mTDP2 complex. The polycyclic core
of 70 is arranged along the hydrophobic cavity pocket formed by
Scheme 6. Synthesis of compounds 50–53. Reagents and conditions: (a)
NO2CH2CO2Et, Mn(OAc)3, MeCN. (b) Ethyl acetoacetate, K2CO3, MeCN, reflux.
(c) PhI(OAc)2, MeCN, H2O.
Trp297, Leu305, and Phe315, and forms π-π stacking with Trp297 and
showed moderate activity, implying that the steric bulk is tolerated with
Phe315 residues (Fig. 3B). Two H-bonds (2.7 Å and 2.9 Å) are observed
between the N atom of isoxazole and the guanidine group of Arg266,
implying the importance of the isoxazole ring. Also three H-bonds are
observed between the OCH3 groups and the backbone guanidine NH of
Arg231 (2.7 Å and 2.8 Å) and between the carbonyl group and the amide
group of Asn264 (2.7 Å), implying the importance of the H-bond re-
ceptors. The hypothetical binding mode is in general agreement with the
empirical SAR observed.
respect to inhibitory properties. However, compound 48 (>111 μM)
with 2-hydroxymethyl group did not show the inhibitory activity,
possibly implying that the H-bond donor at 2-position impaired the
activity.
Unlike furoquinolinedione analogues, the isoxazoloquinolinedione
derivatives with N atom at 6- or 7-position still showed good TDP2
inhibitory activity (50: 5.4 ± 0.80 μM and 51: 12 ± 2.4 μM vs. 10: 1.9 ±
0.28 μM). And the deletion of A-ring resulted in the loss of inhibitory
activity (53: > 111 μM), implying the important role of A-ring for the
3. Conclusions
potency. The isoxazole derivatives (54–57, 59–63) with various 3-
alkoxy carbonyl groups showed good inhibitory activity with IC50
Based on our previous study [25], a series of furoquinolinedione and
isoxazoloquinolinedione derivatives were synthesized for the discovery
of selective TDP2 inhibitors and the SAR study. TDP2-based assay
indicated that seven isoxazoloquinolinedione derivatives 65–71 showed
high TDP2 inhibitory activity at sub-micromolar concentration range.
Compound 70 had the highest TDP2 potency with IC50 of 0.46 ± 0.15
values of between 3.1 ± 0.43 and 20 ± 0.18 μM, implying the steric bulk
is tolerated to TDP2 inhibitory potency. Surprisingly, 3-alkyl and 3-aryl
displacement significantly increased TDP2 potency. For example, com-
pounds 65–71 showed high inhibitory activity with IC50 values at sub-
micromolar level ranging 0.46
± 0.15 to 0.99 ± 0.13 μM
Scheme 7. Synthesis of compounds 54–57. Reagents and conditions: (a) NO2CH2CO2R, K2CO3, MeCN.
5

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
Scheme 8. Synthesis of compounds 59–63 and 65–71. Reagents and conditions: (a) Mn(OAc)3, MeCN. (b) NaClO, Et3N, DCM.
isoxazoloquinolinedione scaffold is a productive chemotype for TDP2
inhibitors, and deserves further study as potential anticancer and anti-
viral drugs.
Table 1
The TDP2 inhibitory activity of the synthesized compounds.
Cpd.
IC50
(
μ
M)a
Cpd.
IC50
(μ
M)a
Cpd.
IC50
(μ
M)a
4. Methods and materials
1
0.27 ± 0.07
>111
31
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
>111
>111
>111
51
52
53
54
55
56
57
59
60
61
62
63
65
66
67
68
69
70
71
12 ± 2.4
11
12
13
14
15
16
19
20
21
22
23
24
25
26
27
28
29
30
9.3 ± 2.7
>111
32 ± 3.2
>111
4.1. General experiments
69 ± 3.1
28 ± 1.3
37 ± 2.0
>111
3.1 ± 0.43
3.3 ± 0.65
15 ± 1.3
61 ± 43
>111
All chemical reagents for synthesis were purchased from local com-
mercial suppliers and were used without further purification unless
otherwise indicated. The halo-quinolinedione materials, such as 6,7-
dichloroquinoxaline-5,8-dione, 6,7-dichlorophthalazine-5,8-dione, 6,7-
dichloroisoquinoline-5,8-dione, 2,3-dichloronaphthalene-1,4-dione, 7-
bromoisoquinoline-5,8-dione, 2,3-dichloroacridine-1,4-dione, ethyl 5-
bromo-2-methyl-4,7-dioxo-4,7-dihydrobenzofuran-3-carboxylate, 6,7-
dichloroquinoline-5,8-dione, 6-bromoisoquinoline-5,8-dione were pre-
pared in our laboratory according to the reported methods
[26,29,38–43]. Chemical reaction courses were monitored by silica gel
GF254 thin layer chromatography. Melting points were determined in
open capillary tubes on a MPA100 Optimelt Automated Melting Point
System without being corrected. Nuclear magnetic resonance spectra
were recorded on a Bruker AVANCE III 400 MHz or 500 MHz spec-
trometer using tetramethylsilane as an internal reference. The high-
resolution mass spectra (HRMS) were analyzed on an SHIMADZU
LCMS-IT-TOF mass spectrometer. All compounds tested for biological
activities were analyzed by HPLC and their purities were>95%. The
analysis condition is: detection at 220 nm, 1.0 mL/min flowrate, a linear
gradient of 50%-15% PBS buffer (pH 3) and 50%-85% MeOH in 30 min.
>111
9.9 ± 2.2
14 ± 2.9
94 ± 29
>111
25 ± 7.7
>111
10 ± 1.48
8.7 ± 0.54
11 ± 3.8
21 ± 5.5
>111
28 ± 6.4
47 ± 24
21 ± 5.5
27 ± 2.5
31 ± 4.2
22 ± 6.4
31 ± 6.4
>111
37 ± 7.0
44 ± 2.0
>111
20 ± 0.18
0.82 ± 0.28
0.94 ± 0.24
0.99 ± 0.13
0.74 ± 0.25
0.96 ± 0.20
0.46 ± 0.15
0.74 ± 0.25
>111
>111
>111
>111
5.2 ± 4.1
5.4 ± 0.80
>111
a
The IC50 was expressed as mean ± SD from at least three independent ex-
periments unless otherwise indicated.
μ
M. For the furoquinolinediones, compound 49 with a formyl substit-
uent at 3-position showed the highest TDP2 potency with IC50 of 5.2 ±
4.1 μM. The SAR for TDP2 inhibition was analyzed. In addition, enzyme-
based assays also showed that the synthesized compounds have no
inhibitory activity against both TDP1 and TOP1 at the highest tested
concentration, 111 μM for TDP1 and 100 μM for TOP1, respectively,
indicating that the furoquinolinedione and isoxazoloquinolinedione
derivatives are selective TDP2 inhibitors, consistent with our previous
results
[25].
In
summary,
this
work
indicates
that
6

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
Fig. 2. Representative gels for the testing of the active compounds against Rec TDP2. Lane 1, DNA alone; lane 2, DNA and recombinant TDP2; lanes 3–26, DNA,
recombinant TDP2 and the active compounds at tested concentration. Tested concentrations were 0.051, 0.15, 0.46, 1.4, 4.1, 12.3, 37 and 111 M. The deazaflavin 1
was tested as the positive control at 0.0019, 0.0056, 0.017, 0.051, 0.15, 0.46, 1.4 and 4.1 M. TY19 and TP19 are the substrate and product of TDP2, respectively.
μ
μ
Fig. 3. The hypothetical binding mode of isoxazole derivative 70. (A) An inhibitor steric fit in hypothetical binding mode of 70 to TDP2 (surface). (B) Molecular
interactions of 70 (green carbon atoms spheres and sticks representation) in complex with TDP2 (cartoon representation, residues in the proximity of the ligand
shown as sticks). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4.2. General procedure for the synthesis of compounds 11–14, 16,
19–22, 37–42, 52 and 54–57.
4.2.1. Ethyl 2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxylate (11)
Luminous yell1ow solid, yield 95%, mp, 159.7–161.5 ℃ (Lit. [45]
149.3–159.9 ◦C). H NMR (400 MHz, CDCl3) δ 8.23–8.16 (m, 2H), 7.75
(dd, J = 8.8, 2.0 Hz, 2H), 4.45 (q, J = 7.1 Hz, 1H), 2.72 (s, 3H), 1.45 (t, J
= 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 178.6, 173.4, 164.4, 162.0,
151.3, 134.1, 133.6, 131.5, 128.2, 127.3, 126.4, 113.7, 61.5, 14.2, 14.1.
HRMS (ESI) m/z: 307.0590 [M + Na]+, calcd for C16H12O5Na 307.0577.
The cyclization reaction between dichloroquinolinedione reagents
(2 mmol) and AMR under K2CO3 condition was conducted according to
our reported method [44]. Briefly, to
a solution of dichlor-
oquinolinedione reagents (2 mmol) and K2CO3 (2.4 mmol) in acetoni-
trile (50 mL), a solution of AMR (6 mmol) in acetonitrile (2 mL) was
added dropwise at room temperature. The yellow reaction solution was
refluxed and stirred for 3 h and cooled to room temperature. The reac-
tion solution was added with H2O (20 mL) and extracted with ethyl
acetate (20 mL × 3). The combined organic layer was dried with
Na2SO4, filtered and concentrated. The residue was purified by column
chromatography on silica gel using ethyl acetate/petroleum ether as
eluent to give the target compounds.
4.2.2. Ethyl 2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]isoquinoline-3-
carboxylate (12)
1
Yellow solid, yield 23%, mp, 143.7–145.4 ℃. H NMR (400 MHz,
CDCl3) δ 9.35 (s, 1H), 9.01 (d, J = 4.9 Hz, 1H), 7.91 (d, J = 4.9 Hz, 1H),
4.38 (q, J = 7.1 Hz, 2H), 2.67 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 178.1, 172.2, 165.5, 161.6, 155.6, 150.7, 149.2,
137.0, 128.5, 126.0, 118.42, 113.9, 61.7, 14.2, 14.1. HRMS (ESI) m/z:
286.0706 [M + H]+, calcd for C15H12NO5 286.0710.
7

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
4.2.3. Ethyl 2-methyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]isoquinoline-3-
4.2.10. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-
carboxylate (37)
carboxylate (13)
1
Yellow solid, yield 20%, mp, 146.2–148.1 ℃. H NMR (400 MHz,
Yellow solid, yield 5%, mp, 144.6–147.9 ℃.1H NMR (400 MHz,
CDCl3) δ 9.05 (d, J = 2.1 Hz, 1H), 8.56–8.51 (m, 1H), 7.71 (dt, J = 7.6,
3.9 Hz, 1H), 4.49–4.40 (m, 2H), 3.18–3.09 (m, 2H), 1.51–1.43 (m, 3H),
1.43–1.35 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 176.6, 172.4, 169.8,
162.0, 154.4, 150.5, 149.1, 134.7, 128.7, 128.4, 127.3, 113.2, 61.8,
21.4, 14.0, 12.0. HRMS (ESI) m/z: 300.0869 [M + H]+, calcd for
C16H14O5N 300.0866.
CDCl3) δ 9.36 (s, 1H), 9.02 (d, J = 3.6 Hz, 1H), 7.91 (d, J = 4.1 Hz, 1H),
4.37 (q, J = 7.1 Hz, 2H), 2.67 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 177.7, 172.7, 165.2, 161.6, 156.0, 150.8, 148.4,
139.1, 128.3, 124.3, 119.5, 113.7, 61.7, 14.2, 14.2. HRMS (ESI) m/z:
286.0705 [M + H]+, calcd for C15H12NO5 286.0710.
4.2.4. Ethyl 7-methyl-5,9-dioxo-5,9-dihydrofuro[2,3-g]quinoxaline-8-
carboxylate (14)
4.2.11. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-
1
Yellow solid, yield 13%, mp, 176.6–178.0 ℃. H NMR (400 MHz,
carboxylate (38)
1
CDCl3) δ 9.06 (dd, J = 4.8, 2.2 Hz, 2H), 4.48 (q, J = 7.2 Hz, 2H), 2.81 (s,
3H), 1.49 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 175.1, 170.3,
166.3, 161.6, 151.2, 148.3, 148.2, 145.4, 143.8, 128.6, 114.0, 61.9,
14.2, 14.0. HRMS (ESI) m/z: 287.0668 [M + H]+, calcd for C14H11N2O5
287.0662.
Yellow solid, yield 21%, mp, 157.7–159.5 ℃. H NMR (400 MHz,
CDCl3) δ 9.04 (dd, J = 4.7, 1.7 Hz, 1H), 8.52 (dd, J = 7.9, 1.7 Hz, 1H),
7.71 (dd, J = 7.9, 4.7 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 3.12 (q, J = 7.6
Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.6 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 177.4, 171.5, 169.7, 161.6, 154.2, 151.6, 147.6, 135.4,
130.6, 128.1, 127.6, 112.9, 61.6, 21.7, 14.1, 12.0. HRMS (ESI) m/z:
300.0869 [M + H]+, calcd for C16H14O5N 300.0866.
4.2.5. Ethyl 2-methyl-4,11-dioxo-4,11-dihydrofuro[2,3-b]acridine-3-
carboxylate (16)
1
Yellow solid, yield 16%, mp, 223.9–225.1 ℃. H NMR (400 MHz,
4.2.12. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-
carboxylate (39)
CDCl3) δ 9.08 (s, 1H), 8.47 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H),
7.99–7.91 (m, 1H), 7.76 (t, J = 7.2 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H),
2.77 (s, 3H), 1.50 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
176.5, 172.1, 165.5, 162.2, 151.6, 149.5, 148.2, 137.2, 133.3, 131.7,
129.9, 129.8, 129.6, 128.3, 125.5, 114.3, 61.8, 14.2, 14.1. HRMS (ESI)
m/z: 336.0856 [M + H]+, calcd for C19H14NO5 336.0866.
Yellow solid, yield 15%, mp, 115.4–115.8℃. 1H NMR (400 MHz,
CDCl3) δ 9.05 (d, J = 4.4 Hz, 1H), 8.55 (d, J = 7.8 Hz, 1H), 7.70 (dd, J =
7. 7, 4.7 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 3.79–3.65 (m, 1H), 1.48 (t, J
= 7.1 Hz, 3H), 1.41 (d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
176.8, 1 72.3, 162.1, 154.3, 150.4, 149.1, 134.7, 128.8, 128.5, 127.3,
112.4, 61.8, 27.8, 20.5, 14.0. HRMS (ESI) m/z: 314.1033 [M + H]+,
calcd for C17H16O5N 314.1023.
4.2.6. Ethyl-6-chloro-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]
quinoline-3-carboxylate (19)
1
Yellow solid, yield 16%, mp, 167.7–168.5 ℃. H NMR (400 MHz,
4.2.13. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-
carboxylate (40)
CDCl3) δ 8.49 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 4.49–4.43 (q,
2H), 2.77 (s, 3H), 1.48 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
175.0, 171.4, 165.7, 161.8, 157.0, 150.3, 149.2, 137.2, 128.6, 128.4,
127.0, 114.0, 61. 8, 14.1. HRMS (ESI) m/z: 320.0317 [M + H]+, calcd
for C15H11NO5Cl 320.0320.
Yellow solid, yield 25%, mp, 160.3–162.1℃. 1H NMR (400 MHz,
CDCl3) δ 9.05 (d, J = 4.6 Hz, 1H), 8.53 (d, J = 7.9 Hz, 1H), 7.72 (dd, J =
7.9, 4.7 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.76–3.63 (m, 1H), 1.46 (t, J
= 7.2 Hz, 3H), 1.42 (d, J = 7.0 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
177.6, 172.2, 171.4, 161.8, 154.3, 15 1.4, 147.7, 135.3, 130.5, 128.1,
127.6, 112.0, 61.8, 28.0, 20.5, 14.1. HRMS (ESI) m/z: 314.1030 [M +
H]+ , calcd for C17H16O5N 314.1023.
4.2.7. Ethyl-7-chloro-2-methyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]
quinoline-3-carboxylate (20)
1
Yellow solid, yield 17%, mp, 197.5–198.3 ℃. H NMR (400 MHz,
CDCl3) δ 8.49 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H), 4.47 (q, J =
7.1 Hz, 2H), 2.77 (s, 3H), 1.47 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 176.4, 170.1, 165.8, 161.5, 156.9, 151.4, 147.6, 138.1, 129.3,
128.9, 127.99, 113.8, 61.7, 14.4, 14.2. HRMS (ESI) m/z: 320.0313 [M +
H]+, calcd for C15H11NO5Cl 320.0320.
4.2.14. Ethyl 4,9-dioxo-2-phenyl-4,9-dihydrofuro[2,3-g]quinoline-3-
carboxylate (41)
Bright yellow solid, yield 10%, mp, 306.8–308.6 ℃. 1H NMR (400
MHz, CDCl3) δ 9.06 (dd, J = 4.7, 1.7 Hz, 1H), 8.58 (dd, J = 7.9, 1.7 Hz,
1H), 8.00–7.95 (m, 2H), 7.72 (dd, J = 7.9, 4.7 Hz, 1H), 7.51 (dd, J = 5.1,
1.9 Hz, 3H), 4.53 (q, J = 7.1 Hz, 2H), 1.46 (t, J = 7.1, 3H). 13C NMR
(100 MHz, CDCl3) δ 177.1, 178.0, 162.8, 158.9, 154.3, 149.9, 148.9,
134.9, 131.3, 130.1, 129.2, 129.0, 127.6, 127.5, 127.3, 113.5, 62.7,
13.9. HRMS (ESI) m/z: 348.0867 [M + H]+, calcd for C20H14NO5
348.0866.
4.2.8. Ethyl-2,6-dimethyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-
carboxylate (21)
1
Yellow solid, yield 11%, mp, 142.1–142.9 ℃. H NMR (400 MHz,
CDCl3) δ 8.40 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 4.44 (q, J =
7.1 Hz, 2H), 2.79 (s, 3H), 2.74 (s, 3H), 1.47 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 176.9, 172.6, 165.1, 164.9, 162.2, 150.5, 148.7,
134.8, 128.3, 127.2, 126.1, 114.0, 61.7, 25.3, 14.1, 14.0. HRMS (ESI) m/
z: 300.0860 [M + H]+, calcd for C16H14NO5 300.0866.
4.2.15. Ethyl 4,9-dioxo-2-phenyl-4,9-dihydrofuro[3,2-g]quinoline-3-
carboxylate (42)
Bright yellow solid, yield 17%, mp, 210.4–212.6 ℃. 1H NMR (400
MHz, CDCl3) δ 9.08 (dd, J = 4.6, 1.6 Hz, 1H), 8.53 (dd, J = 7.9, 1.6 Hz,
1H), 8.21–7.84 (m, 2H), 7.72 (dd, J = 7.9, 4.7 Hz, 1H), 7.60–7.42 (m,
3H), 4.51 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.1, 3H). 13C NMR (100 MHz,
CDCl3) δ 178.0, 171.1, 162.7, 158.9, 154.5, 150.9, 148.2, 135.1, 131.4,
130.3, 129.3, 129.0, 127.6, 127.6, 127.6, 127.2, 113.0, 62.5, 13.9.
HRMS (ESI) m/z: 348.0865 [M + H]+, calcd for C20H14NO5 348.0866.
4.2.9. Ethyl-2,7-dimethyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-
Carboxylate (22)
1
Yellow solid, yield 16%, mp, 185.8–187.2 ℃. H NMR (400 MHz,
CDCl3) δ 8.40 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 4.45 (q, J =
7.1 Hz, 2H), 2.79 (s, 3H), 2.74 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 177.4, 171.8, 165.1, 164.6, 161.7, 151.6, 147.0,
135.5, 128.6, 127.8, 127.5, 113.6, 61.6, 25.1, 14.2, 14.2. HRMS (ESI) m/
z: 300.0862 [M + H]+, calcd for C16H14NO5 300.0866.
4.2.16. Ethyl-4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoxaline-3-
carboxylate (52)
Yellow solid, yield 5%, mp, 185.7–186.2 ℃. 1H NMR (400 MHz,
CDCl3) δ 9.15 (dd, J = 8.0, 2.1 Hz, 2H), 4.61 (q, J = 7.1 Hz, 2H), 1.51 (t,
J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.0, 169.6, 165.4,
8

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
157.3, 153.6, 149.4, 148.9, 145.3, 143.9, 120.5, 64.0, 13.7. HRMS (ESI)
4.4.1. Ethyl-6-(dimethylamino)-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-
g]quinoline-3-carboxylate (23).
m/z: 296.0278 [M + Na]+, calcd for C12H8N3O5Na 296.0278.
Red solid, yield 55%, mp, 191.7–192.9 ℃. 1H NMR (400 MHz,
CDCl3) δ 8.18 (d, J = 9.0 Hz, 1H), 6.72 (d, J = 9.0 Hz, 1H), 4.43 (q, J =
7.1 Hz, 2H), 3.30 (s, 6H), 2.69 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H).13C NMR
(100 MHz, CDCl3) δ 177.8, 172.7, 163.5, 162.4, 160.6, 151.5, 149.9,
135.5, 126.6, 117.6, 113.6, 108.2, 61.4, 38.2,38.2, 14.3, 14.0. HRMS
(ESI) m/z: 329.1108 [M + H]+, calcd for C17H17N2O5 329.1132.
4.2.17. Tert-butyl 4,9-dioxo-4,9-dihydroisoxazolo[5,4-g]quinoline-3-
carboxylate (54)
Yellow solid, yield 7%, mp, 147.3–148.1 ℃. 1H NMR (500 MHz,
CDCl3) δ 9.14 (dd, J = 4.6, 1.7 Hz, 1H), 8.61 (dd, J = 8.0, 1.7 Hz, 1H),
7.81 (dd, J = 8.0, 4.6 Hz, 1H), 1.69 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
175.3, 170.7, 165.5, 156.6, 155.2, 154.3, 147.6, 135.8, 130.7, 128.6,
119.9, 86.1, 28.0. HRMS (ESI) m/z: 301.0819 [M + H]+, calcd for
C15H13N2O5 301.0891.
4.4.2. Ethyl-2-methyl-4,9-dioxo-6-(piperidin-1-yl)-4,9-dihydrofuro[2,3-g]
quinoline-3-carboxylate (24)
Red solid, yield 58%, mp, 162.3–163.4 ℃. 1H NMR (400 MHz,
CDCl3) δ 8.15 (d, J = 9.0 Hz, 1H), 6.80 (d, J = 9.1 Hz, 1H), 4.43 (q, J =
7.1 Hz, 2H), 3.83 (d, J = 5.0 Hz, 4H), 2.69 (s, 3H), 1.79–1.63 (m, 7H),
1.44 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.8, 172.4,
163.5, 162.5, 160.0, 151.5, 150.1, 135.7, 126.5, 117.6, 113.6, 108.4,
61.4, 46.0, 46.0, 25.8, 25.8, 24.5, 14.3, 14.0. HRMS (ESI) m/z: 369.1415
[M + H]+, calcd for C20H21N2O5 369.1445.
4.2.18. Tert-butyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-
carboxylate (55)
Yellow solid, yield 8%, mp, 157.1–157.9 ℃.1H NMR (500 MHz,
CDCl3) δ 9.14 (ddd, J = 9.5, 4.6, 1.7 Hz, 1H), 8.67 – 8.59 (m, 1H), 7.80
(td, J = 8.1, 4.7 Hz, 1H), 1.69 (s, 9H). 13C NMR (125 MHz, CDCl3) δ
174.5, 171.7, 164.7, 156.6, 155.7, 154.7, 148.9, 135.6, 128.8, 127.9,
120.3, 86.4, 27.9. HRMS (ESI) m/z: 301.0819 [M + H]+, calcd for
C
15H13N2O5 301.0891.
4.4.3. Ethyl-2-methyl-6-(4-methylpiperazin-1-yl)-4,9-dioxo-4,9-
dihydrofuro[2,3-g]quinoline-3-carboxylate (25)
4.2.19. 4-chlorophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[5,4-g]
quinoline-3-carboxylate (56)
Red solid, yield 80%, mp, 152.8–154.5 ℃. 1H NMR (400 MHz,
CDCl3) δ 8.26 (d, J = 9.1 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 4.45 (q, J =
14.2, 7.1 Hz, 2H), 4.12 (s, 4H), 2.90 (s, 4H), 2.72 (s, 3H), 2.62 (s, 3H),
1.47 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.5, 172.3,
164.0, 162.2, 159.7, 151.2, 149.5, 136.3, 127.0, 119.0, 113.6, 109.2,
61.5, 53.9, 53.9, 44.7, 44.7, 43.2, 14.1, 14.0. HRMS (ESI) m/z: 384.1549
[M + H]+, calcd for C20H22N3O5 384.1554.
Yellow solid, yield 6%, mp, 145.8–146.5 ℃.1H NMR (500 MHz,
CDCl3) δ 9.15 (dd, J = 4.6, 1.7 Hz, 1H), 8.61 (dd, J = 8.0, 1.6 Hz, 1H),
7.83 (dd, J = 7.9, 4.6 Hz, 1H), 7.28 (d, J = 11.9 Hz, 2H), 7.23 (d, J = 8.2
Hz, 2H), 4.69 (t, J = 7.0 Hz, 2H), 3.14 (t, J = 7.0 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 175.1, 170.5, 165.9, 157.5, 155.3, 153.1, 147.5, 135.9,
135.3, 132.8, 130.7, 130.4, 128.8, 120.0, 67.4, 34.2. HRMS (ESI) m/z:
383.0424 [M + H]+, calcd for C19H12N2O5Cl 383.0429.
4.4.4. Ethyl-2-methyl-6-(methyl(prop-2-yn-1-yl)amino)-4,9-dioxo-4,9-
dihydrofuro[2,3-g]quinoline-3-carboxylate (26)
4.2.20. 4-chlorophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]
quinoline-3-carboxylate (57)
Red solid, yield 57%, mp, 165.6–167.2 ℃. 1H NMR (400 MHz,
CDCl3) δ 8.24 (dd, J = 8.9, 1.9 Hz, 1H), 6.81 (d, J = 8.9 Hz, 1H), 4.64 (s,
2H), 4.44 (q, J = 7.1 Hz, 2H), 3.28 (s, 3H), 2.70 (s, 3H), 2.25 (t, J = 2.4
Hz, 1H), 1.45 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.3,
172.6, 163.8, 162.3, 159.9, 151.3, 149.6, 136.0, 126.9, 118.7, 113.6,
108.9, 78.5, 72.3, 61.5, 39.0, 35.7, 14.3, 14.0. HRMS (ESI) m/z:
353.1112 [M + H]+, calcd for C19H17N2O5 353.1132.
Yellow solid, yield 7%, mp, 137.2–137.9 ℃.1H NMR (400 MHz,
CDCl3) δ 9.24–9.13 (m, 1H), 8.63 (dd, J = 7.9, 1.6 Hz, 1H), 7.82 (dd, J =
7.9, 4.6 Hz, 1H), 7.29 (q, J = 3.1, 2.4 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H),
4.70 (t, J = 7.1 Hz, 2H), 3.17 (t, J = 7.1 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 174.3, 171.6, 165.2, 157.5, 155.8, 153.5, 148.8, 135.6, 135.3,
130.4, 128.8, 128.0, 120.4, 67.5, 34.1. HRMS (ESI) m/z: 383.0434 [M +
H]+, calcd for C19H12N2O5Cl 383.0429.
4.4.5. Ethyl-7-(dimethylamino)-2-methyl-4,9-dioxo-4,9-dihydrofuro[3,2-
g]quinoline-3-carboxylate (30)
Red solid, yield 29%, mp, 177.9–179.2 ℃. 1H NMR (400 MHz,
CDCl3) δ 8.18 (d, J = 9.0 Hz, 1H), 6.73 (d, J = 9.0 Hz, 1H), 4.44 (q, J =
7.1 Hz, 2H), 3.29 (s, 6H), 2.70 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 177.5, 172.7, 164.3, 162.2, 160.5, 151.2, 147.7,
136.2, 127.8, 120.2, 113.7, 108.7, 61.5, 38.2, 38.2, 14.3, 14.2. HRMS
(ESI) m/z: 329.1123 [M + H]+, calcd for C17H17N2O5 329.1132.
4.3. The synthesis of product 15.
To a solution of 7-bromoisoquinoline-5,8-dione (0.48 g, 2 mmol) in
acetonitrile (30 mL), K2CO3(0.83 g, 6 mmol), Cu(OAc)2 (0.91 g, 3 mmol)
and ethyl acetoacetate (2.4 mmol) were added. The yellow reaction
solution was stirred and heated under reflux for 6 h and cooled to room
temperature. The solvent was evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel using ethyl
acetate/petroleum ether (1/2, V/V) as eluent to give compound 15.
Yellow solid, yield 18%, mp, 140.0–141.8 ℃. 1H NMR (400 MHz,
CDCl3) δ 9.91 (s, 2H), 4.46 (q, J = 7.1 Hz, 2H), 2.78 (s, 3H), 1.46 (t, J =
7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.5, 171.8, 166.4, 161.2,
150.0, 146.8, 145.9, 128.2, 125.2, 123.7, 113.7, 61.9, 14.4, 14.1. HRMS
(ESI) m/z: 287.0665 [M + H]+, calcd for C14H11N2O5 287.0662.
4.4.6. Ethyl-2-methyl-4,9-dioxo-7-(piperidin-1-yl)-4,9-dihydrofuro[3,2-g]
quinoline-3-carboxylate(31)
Red solid, yield 49%, mp, 198.5–199.3 ℃. 1H NMR (400 MHz,
CDCl3) δ 8.15 (d, J = 9.1 Hz, 1H), 6.83 (d, J = 9.1 Hz, 1H), 4.43 (q, J =
7.1 Hz, 2H), 3.81 (d, J = 5.3 Hz, 4H), 2.70 (s, 3H), 1.68 (dd, J = 11.9,
6.5 Hz, 6H), 1.44 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
177.3, 172.8, 164.2, 162.2, 160.0, 151.2, 147.9, 136.4, 127.8, 120.2,
113.7, 109.0, 61.5, 45.9, 45.9, 25.7, 25.7, 24.5, 14.2, 14.2. HRMS (ESI)
m/z: 369.1449 [M + H]+, calcd for C20H21N2O5 369.1445.
4.4. General procedure for the synthesis of compounds 23–26, 30 and
31.
4.5. The synthesis of compounds 27 and 28.
The reaction solution of compound 19 or 20 (1 mmol) and secondary
amines (10 mmol) in dioxane (10 mL) was stirred at room temperature
for 3 h. The reaction solution was added with ethyl acetate (20 mL), and
washed with H2O (20 mL × 2). The organic layer was dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue was pu-
rified by column chromatography on silica gel using ethyl acetate/pe-
troleum ether (1/10–1/1, V/V) as eluent to give the target compounds.
Under nitrogen, to a solution of compound 19 (1.87 mmol) in a
mixed solvents (dichloroethane/water = 5:1, 18 mL), (4-methox-
yphenyl)boronic acid or furan-2-ylboronic acid (2.81 mmol) was added.
And then, Pd(PPh3)4 (0.037 mmol) and Na2CO3 (3.74 mmol) were
added to the reaction solution. The resulting solution was heated and
stirred at 70 ℃ for 12 h. After the reaction was completed, the reaction
9

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
solution was cooled to room temperature and added with saturated
Na2CO3 (20 mL), and extracted with CH2Cl2 (20 mL × 3). The combined
organic layer was dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by column chromatography
on silica gel using EtOAc/petroleum ether as eluent to give 27 or 28,
respectively.
4.7.2. Ethyl-2-methyl-8-(2-nitrophenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]
quinoline-3-carboxylate (34)
1
Yellow solid, yield 27%, mp, 203.8–206.7 ℃. H NMR (400 MHz,
CDCl3) δ 9.06 (d, J = 4.4 Hz, 1H), 8.33 (d, J = 8.1 Hz, 1H), 7.75 (t, J =
7.3 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 4.4 Hz, 1H), 7.24 (d, J
= 8.1 Hz, 1H), 4.44 (q, J = 7.0 Hz, 2H), 2.69 (s, 3H), 1.48 (t, J = 7.0 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 176.0, 172.9, 165.6, 162.0, 153.6,
150.7, 149.9, 149.1, 146.8, 134.4, 133.9, 129.9, 129.5, 127.9, 127.8,
125.2, 124.8, 113.7, 61.7, 14.1, 14.0. HRMS (ESI) m/z: 407.0882 [M +
H]+, calcd for C21H15N2O7 407.0874.
4.5.1. Ethyl6-(4-methoxyphenyl)-2-methyl-4,9-dioxo-4,9-dihydrofuro
[2,3-g]quinoline-3-carboxylate (27)
1
Yellow solid, yield 48%, mp, 191.6–192.8 ℃. H NMR (400 MHz,
CDCl3) δ 8.42 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 7.94 (d, J =
8.2 Hz, 1H), 6.96 (d, J = 8.3 Hz, 2H), 4.39 (q, J = 7.0 Hz, 2H), 3.82 (s,
3H), 2.67 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H).13C NMR (100 MHz, CDCl3) δ
176.7, 172.5, 164.8, 162.2, 162.0, 161.2, 150.8, 149.2, 135.2, 129.9,
129.4, 129.4, 128.4, 126.1, 122.3, 114.4, 114.4, 114.0, 61.6, 55.5, 14.2,
14.0. HRMS (ESI) m/z: 392.1142 [M + H]+, calcd for C22H18NO6
392.1129.
4.7.3. Diethyl-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3,8-
dicarboxylate (35)
1
Yellow solid, yield 37%, mp, 170.5–171.7 ℃. H NMR (400 MHz,
CDCl3) δ 9.09 (d, J = 4.7 Hz, 1H), 7.57 (d, J = 4.8 Hz, 1H), 4.54 (q, J =
7.1 Hz, 2H), 4.44 (q, J = 7.1 Hz, 2H), 2.75 (s, 3H), 1.46 (m, 6H). 13
C
NMR (100 MHz, CDCl3) δ 175.5, 171.3, 166.8, 165.9, 161.8, 154.4,
150.4, 149.5, 142.2, 128.4, 124.8, 124.5, 114.0, 62.9, 61.8, 14.1, 14.1,
13.9. HRMS (ESI) m/z: 358.0928 [M + H]+, calcd for C18H16NO7
358.0921.
4.5.2. Ethyl-6-(furan-2-yl)-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]
quinoline-3-carboxylate (28)
1
Yellow solid, yield 32%, mp, 223.4–224.7 ℃. H NMR (400 MHz,
CDCl3) δ 8.52 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.66–7.63 (m,
1H), 7.49 (d, J = 3.5 Hz, 1H), 6.63 (dd, J = 3.2, 1.4 Hz, 1H), 4.46 (q, J =
7.1 Hz, 2H), 2.74 (s, 3H), 1.47 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 176.4, 172.2, 165.0, 162.1, 153.1, 152.4, 150.8, 149.5, 145.4,
135.4, 128.3, 126.1, 121.2, 114.0, 113.5, 113.0, 61.7, 14.2, 14.1. HRMS
(ESI) m/z: 352.0799 [M + H]+, calcd for C19H14NO6 352.0816.
4.7.4. Ethyl-8-ethyl-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-
3-carboxylate (36)
1
Yellow solid, yield 17%, mp, 131.1–133.3 ℃. H NMR (400 MHz,
CDCl3) δ 8.86 (d, J = 4.9 Hz, 1H), 7.47 (d, J = 4.9 Hz, 1H), 4.43 (q, J =
7.1 Hz, 3H), 3.33 (q, J = 7.4 Hz, 2H), 1.47 (t, J = 7.2 Hz, 5H), 1.32 (t, J
= 7.4 Hz, 5H). 13C NMR (100 MHz, CDCl3) δ 176.4, 174.8, 165.2, 162.2,
157.2, 153.1, 151.4, 150.9, 128.9, 126.8, 125.8, 113.4, 61.6, 27.7, 14.2,
14.1, 14.0. HRMS (ESI) m/z: 314.1034 [M + H]+, calcd for C17H16NO5
314.1023.
4.6. The synthesis of compound 29.
To a solution of 3-hydroxypyridine (0.48 g, 5 mmol) in fresh distilled
tetrahydrofuran (20 mL), NaH (0.60 g, 25 mmol) was added slowly at
0 ℃. And then, the mixture solution was heated and stirred at 50 ℃ for
30 min. A solution of 19 (0.80 g, 2.5 mmol) in fresh distilled tetrahy-
drofuran (5 mL) was added dropwise. The reaction solution was stirred
at 50 ℃ for 3 h and cooled to room temperature. The solvent was
evaporated under reduced pressure. The residue was purified by silica
gel column chromatography to give compound 29. Yellow solid, yield
7%, mp, 168.1–168.8 ℃. 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 8.45
(d, J = 8.5 Hz, 2H), 7.71 (dd, J = 8.3, 1.2 Hz, 1H), 7.35 (dd, J = 7.9, 4.7
Hz, 1H), 7.20 (d, J = 4.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.64 (s, 3H),
1.34 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 175.5, 171.8,
165.4, 164.8, 161.8, 150.5, 149.6, 148.4, 146.5, 143.1, 138.4, 129.0,
128.1, 124.6, 124.1, 115.8, 113.9, 61.6, 14.2, 14.1. HRMS (ESI) m/z:
379.0923 [M + H]+, calcd for C20H15N2O6 379.0925.
4.8. The synthesis of compounds 43 and 44
To a solution of 9 (1.7 g, 9.71 mmol) in CCl4 (120 mL), NBS (1.42 g,
8 mmol) and AIBN (79 mg, 0.48 mmol) were added at room tempera-
ture. The yellow reaction solution was refluxed and stirred for 24 h, and
then cooled to room temperature. The reaction solution was filtered and
concentrated under reduced pressure. The residue was purified by silica
gel column chromatography to give two compounds 43 and 44,
simultaneously.
4.8.1. Ethyl 2-(bromomethyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-
3-carboxylate (43)
1
Yellow solid, yield 44%, mp, 191.3–193.0 ℃. H NMR (400 MHz,
CDCl3) δ 9.01 (d, J = 3. 2 Hz, 1H), 8.49 (d, J = 7.1 Hz, 1H), 7.67 (dd, J =
7.7, 4.5 Hz, 1H), 4.77 (s, 2H), 4.45–4.36 (m, 2H), 1.42 (t, J = 5.7 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 175.9, 172.5, 161.4, 160.9, 154.7,
151.4, 149.0, 134.8, 128.4, 128.2, 127. 5, 115.5, 62.3, 19.1, 14.0. HRMS
(ESI) m/z: 363. 9832 (100%), 365.9836 (100%) [M + H]+, calcd for
C15H11O5NBr 363. 9815.
4.7. General procedure for the synthesis of compounds 33–36.
To a solution of 32a-d (2 mmol) in a mixed solvents acetonitrile (10
mL) and acetic anhydride (5 mL), a solution of ethyl 5-bromo-3-ethoxy-
carbonylfuroquinone (0.31 g, 1 mmol) in acetonitrile (2 mL) was added
dropwise at room temperature. The reaction solution was stirred for 12 h
at room temperature. The solvent was evaporated under reduced pres-
sure. The residue was purified by silica gel column chromatography to
give the target compounds.
4.8.2. Ethyl 2-(dibromomethyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]
quinoline-3- carboxylate (44)
1
Yellow solid, yield 15%, mp, 143.5–146.7 ℃. H NMR (400 MHz,
CDCl3) δ 9.11 (dd, J = 4.7, 1.7 Hz, 1H), 8.61 (dd, J = 7.9, 1.7 Hz, 1H),
7.76 (dd, J = 7.9, 4.7 Hz, 1H), 7.35 (s, 1H), 4.51 (q, J = 7.1 Hz, 2H), 1.52
(t, J = 7.2 Hz, 3H). 13C NMR (100 M Hz, CDCl3) δ 175.7, 172.2, 160.6,
160.1, 154.8, 151.8, 148.8, 134.9, 128.4, 12 7.6, 127.3, 111.8, 62.7,
23.1, 14.0. HRMS (ESI) m/z: 441.8919 (50%), 443.8899 (100%),
445.8882 (50%) [M + H]+, calcd for C15H10O5NBr2 441.8920.
4.7.1. Ethyl-2,7-dimethyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-
carboxylate (33)
1
Yellow solid, yield 39%, mp, 169.6–171.2 ℃. H NMR (400 MHz,
CDCl3) δ 8.85 (s, 1H), 8.31 (s, 1H), 4.44 (q, J = 7.1 Hz, 2H), 2.74 (s, 3H),
2.54 (s, 3H), 1.47 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
176.6, 172.8, 165.3, 162.1, 155.0, 150.6, 147.0, 138.3, 134.4, 128.6,
127.9, 114.0, 61.7, 18.8, 14.1, 14.1. HRMS (ESI) m/z: 300.0870 [M +
H]+, calcd for C16H14NO5 300.0866.
4.9. The synthesis of compounds 45–47
To a suspension of compound 43 (0.73 g, 2 mmol) in fresh distilled
dichloromethane (10 mL), secondary amine materials (4 mmol) was
added and stirred at room temperature. The reaction was stopped after
10

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
the disappearance of starting material (typically for between 4 and 8 h).
The reaction solution was added with water (20 mL) and extracted with
dichloromethane (20 mL × 3). The combined organic layer was washed
with brine and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to give the target
compounds.
300.0503.
4.11. General procedure for the cyclization under Mn(OAc)3 catalysis.
To a solution of bromoquinolinedione materials (210 mg, 0.88
mmol) in fresh distilled acetonitrile (10 mL), the nitroacetate materials
(1.05 mmol) and Mn(OAc)3 (354 mg, 2.63 mmol) were added. The
yellow reaction solution was refluxed and stirred for 12 h and cooled to
room temperature. The reaction solution was filtered and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography to give the target compounds.
4.9.1. Ethyl 2-((4-acetylpiperazin-1-yl)methyl)-4,9-dioxo-4,9-dihydrofuro
[2,3-g]qui noline-3-carboxylate (45)
Yellow solid, yield 35%, mp, 167.7–170.2℃. 1H NMR (400 MHz,
CDCl3) δ 9.07 (d, J = 2.6 Hz, 1H), 8.56 (d, J = 7.5 Hz, 1H), 7.75–7.69 (m,
1H), 4.46 (dd, J = 13.6, 6. 7 Hz, 2H), 4.03 (s, 2H), 3.63 (s, 2H), 3.48 (s,
2H), 2.63 (s, 2H), 2.58 (s, 2H), 2.07 (s, 3H), 1.48 (t, J = 6.9 Hz, 3H). 13
C
4.11.1. Ethyl-4,9-dioxo-4,9-dihydroisoxazolo[5,4-g]isoquinoline-3-
carboxylate (50)
NMR (100 MHz, CDCl3) δ 176.5, 172.4, 168.9, 162.5, 161.7, 154.6,
151.3, 149.0, 134.8, 128.5, 128.2, 127.4, 116.6, 62.2, 52.9, 52.7, 52.5,
46.2, 41.3, 21.3, 14.0. HRMS (ESI) m/z: 412.1503 [M + H]+, calcd for
C21H22O6N3 412.1503.
Yellow solid, yield 5%, mp, 104.9–107.1℃. 1H NMR (400 MHz,
CDCl3) δ 9.48 (s, 1H), 9.14 (s, 1H), 8.00 (d, J = 4.8 Hz, 1H), 4.52 (q, J =
7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
176.0, 172.1, 165.0, 157.6, 156.4, 153.6, 149.8, 136.9, 125.4, 120.2,
119.0, 77.4, 77.0, 76.7, 63.7, 62.3, 14.3, 14.0. HRMS (ESI) m/z:
273.0513 [M + H]+, calcd for C13H9N2O5 273.0506.
4.9.2. Ethyl 2-(morpholinomethyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]
quinoline-3-ca rboxylate (46)
Yellow solid, yield 57%, mp, 139.3–141.8℃. 1H NMR (400 MHz,
CDCl3) δ 9.07 (d, J = 4.5 Hz, 1H), 8.56 (d, J = 7.8H z, 1H), 7.72 (dd, J =
7.7, 4.7 Hz, 1H), 4.46 (q, J = 6.8 Hz, 2H), 3.99 (s, 2H), 3.72 (d, J = 3.7
Hz, 4H), 2.63 (d, J = 3.7 Hz, 4H), 1.48 (t, J = 7.1 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 176.5, 172.4, 162.7, 161.7, 154.5, 151.2, 149.0, 134.8,
128.6, 128.3, 127.4, 116.6, 66.8, 62.1, 53.3, 53.2, 14.0. HRMS (ESI) m/
z: 371.1235 [M + H]+, calcd for C19H19O6N2 371.1238.
4.11.2. Ethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]isoquinoline-3-
carboxylate (51)
Yellow solid, yield 7%, mp, 131.9–133.8 ℃. 1H NMR (400 MHz,
CDCl3) δ 9.48 (s, 1H), 9.14 (d, J = 4.7 Hz, 1H), 8.00 (d, J = 4.8 Hz, 1H),
4.52 (q, J = 7.2 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 175.6, 172.0, 165.2, 157.5, 157.2, 153.4, 149.3, 138.8, 124.5,
120.3, 119.7, 63.7, 14.0. HRMS (ESI) m/z: 295.0310 [M + Na]+, calcd
for C13H8N2O5Na 295.0325.
4.9.3. Ethyl 4,9-dioxo-2-(thiomorpholinomethyl)-4,9-dihydrofuro[2,3-g]
quinoline3-carboxylate (47)
Yellow solid, yield 57%, mp, 145.7–147.1℃. 1H NMR (400 MHz,
CDCl3) δ 9.06 (dd, J = 4.7, 1.7 Hz, 1H), 8.55 (dd, J = 7.9, 1.7 Hz, 1H),
7.71 (dd, J = 7.9, 4.7 Hz, 1H), 4.46 (q, J = 7.2 Hz, 2H), 4.01 (s, 2H),
2.90–2.85 (m, 4H), 2.70–2.66 (m, 4H), 1.47 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 176.5, 172.4, 163.0, 161. 8, 154.5, 151.2, 149.1,
134.8, 128.6, 128.3, 127.4, 116.5, 62.1, 54.6, 53.8, 28.0, 14.0. HRMS
(ESI) m/z: 387.1016 [M + H]+, calcd for C19H19O5N2S 387.1009.
4.11.3. 2,2,2-Trifluoroethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]
quinoline-3-carboxylate (59)
Light yellow solid, yield 6%, mp, 165.4–166.1 ℃. 1H NMR (400
MHz, CDCl3) δ 9.17 (dd, J = 4.6, 1.7 Hz, 1H), 8.64 (dd, J = 7.9, 1.7 Hz,
1H), 7.85 (dd, J = 8.0, 4.6 Hz, 1H), 4.88 (q, J = 8.1 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 174.8, 170.3, 166.2, 156.2, 155.4, 152.0, 147.4,
136.0, 130.6, 128.8, 123.7, 120.9, 120.1, 62.0. HRMS (ESI) m/z:
327.0246 [M + H]+ calcd for C13H6F3N2O5 327.0229.
4.10. The synthesis of compounds 48 and 49
4.11.4. 2-Cyanoethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-
Compound 43 (2 mmol) was dissolved in dimethyl sulfoxide (5 mL)
and stirred at 150 ℃ under air. The reaction was continued until the
disappearance of 43. And then, water (20 mL) was added. The resulting
solution was extracted with ethyl acetate (20 mL × 3). The combined
organic layer was washed with brine and evaporated under reduced
pressure. The resulting residue was purified by silica gel column chro-
matography to give the compounds 48 and 49, simultaneously.
carboxylate (60)
1
Yellow solid, yield 20%, mp, 158.9–159.7 ℃. H NMR (500 MHz,
CDCl3) δ 9.09 (d, J = 4.6 Hz, 1H), 8.63–8.50 (m, 1H), 7.77 (dd, J = 7.9,
4.6 Hz, 1H), 4.67 (t, J = 6.4 Hz, 2H), 2.91 (t, J = 6.4 Hz, 2H). 13C NMR
(125 MHz, CDCl3) δ 175.0, 170.4, 166.0, 157.1, 155.4, 152.5, 147.5,
136.0, 130.6, 128.7, 120.0, 116.0, 61.2, 17.9. HRMS (ESI) m/z:
298.0460 [M + H]+ calcd for C14H8N3O5 298.0458.
4.10.1. Ethyl-2-(hydroxymethyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]
4.11.5. 3-Fluorophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]
quinoline-3-carbo-xylate (48)
quinoline-3-carboxylate (61)
1
1
Yellow solid, yield 21%, mp, 164.6–166.6 ℃. H NMR (400 MHz,
Yellow solid, yield 23%, mp, 148.6–149.7 ℃. H NMR (400 MHz,
CDCl3) δ 9.08 (dd, J = 4.6, 1.2 Hz, 1H), 8.55 (dd, J = 7.9, 1.2 Hz, 1H),
7.73 (dd, J = 7.8, 4.7 Hz, 1H), 4.96 (s,2H), 4.48 (q, J = 7.1 Hz, 2H), 3.69
(s, 1H), 1.51 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 176.2,
172.6, 166.7, 162.9, 154.6, 150.9, 149.1, 134.8, 128.3, 128.1, 127.5,
115.2, 62.6, 57.2, 14.0. HRMS (ESI) m/z: 302.0656 [M + H]+, calcd for
C15H12NO6 302.0659.
CDCl3) δ 9.10 (dd, J = 4.7, 1.7 Hz, 1H), 8.52 (dd, J = 7.9, 1.7 Hz, 1H),
7.95 (dd, J = 7.9, 4.6 Hz, 1H), 7.35 (td, J = 8.0, 6.2 Hz, 1H), 7.26–7.16
(m, 2H), 7.06 (td, J = 8.7, 2.6 Hz, 1H), 4.71 (t, J = 6.7 Hz, 2H), 3.13 (t, J
= 6.7 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 181.2, 175.8, 172.3, 172.3,
168.6, 166.2, 163.0, 159.5, 157.8, 153.47, 145.6, 145.5, 140.2, 140.2,
135.5, 133.7, 130.3, 123.7, 121.0, 118.6, 72.2, 38.8. HRMS (ESI) m/z:
367.0724 [M + H]+ calcd for C19H12N2O5F 367.0725.
4.10.2. Ethyl-2-formyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-
carboxylate (49)
4.11.6. 4-Nitrophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-
Yellow solid, yield 6%, mp, 176.8–179.1 ℃. 1H NMR (400 MHz,
CDCl3) δ 10.23 (s, 1H), 9.13 (d, J = 4.1 Hz, 1H), 8.60 (d, J = 7.9 Hz, 1H),
7.77 (dd, J = 7.8, 4.7 Hz, 1H), 4.57 (q, J = 7.1 Hz, 2H), 1.51 (t, J = 7.1
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 178.4, 175.8, 172.8, 160.0,
155.1, 153.3, 152.4, 148.9, 135.2, 129.0, 127.8, 127.7, 123.8, 63.3,
14.0. HRMS (ESI) m/z: 300.0454 [M + H]+, calcd for C15H10NO6
3-carboxylate (62)
1
Yellow solid, yield 31%, mp, 213.1–213.8 ℃. H NMR (400 MHz,
DMSO‑d6) δ 9.10 (d, J = 4.6 Hz, 1H), 8.50 (d, J = 7.9 Hz, 1H), 8.18 (d, J
= 8.2 Hz, 2H), 7.95 (dd, J = 8.1, 4.7 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H),
4.77 (t, J = 6.5 Hz, 2H), 3.27 (t, J = 6.5 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 176.4, 171.0, 167.6, 158.2, 154.8, 148.7, 146.9, 146.3,
11

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
135.4, 130.8, 128.9, 123.9, 118.9, 67.0, 34.2. HRMS (ESI) m/z:
394.0671 [M + H]+ calcd for C19H12N3O7 394.0670.
7.98–7.86 (m, 3H), 7.38–7.28 (m, 2H), 5.70 (s, 2H), 3.19 (s, 3H).13C
NMR (100 MHz, DMSO‑d6) δ 178.4, 166.9, 161.8, 157.7, 154.8, 149.2,
135.2, 134.1, 130.6, 129.8, 128.8, 119.6, 115.8, 61.0, 44.3. HRMS (ESI)
m/z: 385.0486 [M + H]+ calcd for C18H13N2O6S 385.0489.
4.11.7. 2-(((3 s,5s,7s)-Adamantan-1-yl)oxy)ethyl 4,9-dioxo-4,9-
dihydroisoxazolo[4,5-g]quinoline-3-carboxylate (63)
1
Yellow solid, yield 33%, mp, 137.3–138.2 ℃. H NMR (500 MHz,
4.13.4. 4-((4,9-Dioxo-4,9-dihydroisoxazolo[4,5-g]quinolin-3-yl)
methoxy)benzonitrile (68)
CDCl3) δ 9.15 (dd, J = 4.6, 1.7 Hz, 1H), 8.62 (dd, J = 8.0, 1.7 Hz, 1H),
7.83 (dd, J = 8.0, 4.6 Hz, 1H), 4.68–4.58 (m, 2H), 3.83 (t, J = 5.2 Hz,
2H), 2.21–2.11 (m, 3H), 1.77 (d, J = 2.9 Hz, 6H), 1.66–1.58 (m, 6H). 13C
NMR (125 MHz, CDCl3) δ 175.0, 170.6, 165.8, 157.7, 155.2, 153.3,
147.5, 135.9, 130.7, 128.7, 120.1, 72.9, 67.3, 57.6, 41.4, 36.4, 30.5.
HRMS (ESI) m/z: 445.1375 [M + Na]+ calcd for C23H22N2O6 445.1370.
Light yellow solid, yield 21%, mp, 178.6–178.9 ℃. 1H NMR (400
MHz, CDCl3) δ 9.18 (dd, J = 4.6, 1.7 Hz, 1H), 8.61 (dd, J = 7.9, 1.7 Hz,
1H), 7.84 (dd, J = 8.0, 4.7 Hz, 1H), 7.72–7.64 (m, 2H), 7.20–7.11 (m,
2H), 5.59 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 177.3, 170.6, 165.7,
160.9, 157.0, 155.3, 148.1, 135.5, 134.2, 130.3, 128.5, 120.2, 118.7,
115.6, 105.6, 60.4. HRMS (ESI) m/z: 332.0665 [M + H]+ calcd for
C18H10N3O4 332.0666.
4.12. The synthesis of 53.
4.13.5. 4-((4,9-Dioxo-4,9-dihydroisoxazolo[4,5-g]quinolin-3-yl)
methoxy)-2-fluorobenzonitrile (69)
Compound 53 was prepared according to the method [32]. Briefly, to
a solution of 2,3-dimethylphenol (122 mg, 1 mmol) in a mixed solvent
(acetonitrile/water = 2:1, 6 mL), (diacetoxyiodo)benzene (966 mg, 3
mmol) was added and stirred at room temperature for 5 h. And then,
ethyl (Z)-2-(hydroxyimino)-3-oxobutanoate (191 mg, 1.2 mmol) was
added. The reaction solution was stirred for 12 h and added with water
(10 mL) and extracted with ethyl acetate (20 mL × 3). The combined
organic layer was washed with brine and evaporated in vacuo. The
residue was purified by silica gel column chromatography to give the
compound 53. Light yellow solid, yield 5%, mp < 70 ℃. 1H NMR (400
MHz, CDCl3) δ 4.54 (q, J = 7.2 Hz, 2H), 2.16 (s, 6H), 1.47 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 178.4, 174.5, 164.5, 157.9, 152.7,
144.1, 140.4, 118.0, 63.3, 14.0, 13.0, 12.1. HRMS (ESI) m/z: 250.0703
[M + H]+, calcd for C12H12NO5 250.0710.
Light yellow solid, yield 21%, mp, 197.1–198.2 ℃. 1H NMR (400
MHz, CDCl3) δ 9.18 (dd, J = 4.8, 1.7 Hz, 1H), 8.61 (dd, J = 8.0, 1.7 Hz,
1H), 7.84 (dd, J = 8.0, 4.6 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 6.94 (ddd, J
= 17.1, 9.5, 2.5 Hz, 2H), 5.59 (s, 2H). 13C NMR (100 MHz, CDCl3) δ
177.3, 170.5, 165.8, 156.6, 155.3, 148.1, 135.5, 134.6, 130.3, 128.5,
120.2, 113.9, 111.7, 103.8, 103.5, 100.0, 60.7. HRMS (ESI) m/z:
350.0573 [M + H]+ calcd for C18H9N3O4F 350.0572.
4.13.6. 3-(3,4-Dimethoxyphenyl)isoxazolo[4,5-g]quinoline-4,9-dione
(70)
Red solid, yield 18%, mp, 234.5–235.6 ℃. 1H NMR (400 MHz,
CDCl3) δ 9.17–9.15 (m, 1H), 8.68–8.65 (m, 1H), 7.98–7.95(m, 1H),
7.91–7.80 (m, 2H), 7.06 (d, J = 8.5 Hz, 1H), 4.03 (d, J = 16.2 Hz, 6H).
13C NMR (100 MHz, CDCl3) δ 177.4, 171.5, 166.5, 160.6, 155.0, 151.8,
149.0, 147.6, 135.9, 131.0, 128.5, 123.0, 118.3, 112.0, 110.9, 56.0.
HRMS (ESI) m/z: 337.0818 [M + H]+ calcd for C18H13N2O5 337.0819.
4.13. General procedure for the synthesis of compounds 65–71.
The compounds 65–71 were prepared according to the literature
method [33]. Briefly, to a solution of 7-bromoquinoline-5,8-dione (60
mg, 0.25 mmol) in dry dichloromethane (5 mL), a solution of NaClO in
water (14%, 0.25 mL), active oxime reagents (0.5 mmol) and Et3N (5
mg, 0.05 mmol) were sequentially added at 0 ℃. And then, the reaction
solution was stirred at room temperature for 12 h. After completion of
the reaction, the solvent was evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel using
EtOAc/petroleum ether as eluent to give the target compounds.
4.13.7. 4-(4,9-Dioxo-4,9-dihydroisoxazolo[4,5-g]quinolin-3-yl)-3-
fluorobenzonitrile (71)
1
Yellow solid, yield 25%, mp, 215.1–215.7 ℃. H NMR (400 MHz,
CDCl3) δ 9.17 (dd, J = 4.7, 1.8 Hz, 1H), 8.59 (dd, J = 7.9, 1.8 Hz, 1H),
7.89 (dd, J = 7.9, 6.7 Hz, 1H), 7.84 (dd, J = 8.0, 4.6 Hz, 1H), 7.69 (dd, J
= 7.9, 1.6 Hz, 1H), 7.63 (dd, J = 9.0, 1.5 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 176.5, 170.8, 165.8, 161.3, 158.8, 155.2, 154.8, 147.8, 135.6,
132.3, 130.6, 128.6, 128.4, 120.3, 120.2, 120.0, 116.7. HRMS (ESI) m/z:
320.0456 [M + H]+ calcd for C17H7N3O3F 320.0466.
4.13.1. 3-(Phenoxymethyl) isoxazolo[4,5-g]quinoline-4,9-dione (65)
1
Yellow solid, yield 27%, mp, 142.9–144.2 ℃. H NMR (500 MHz,
4.14. Recombinant TDP2 assay
CDCl3) δ 9.14 (dd, J = 4.7, 1.7 Hz, 1H), 8.59 (dd, J = 7.9, 1.7 Hz, 1H),
7.80 (dd, J = 8.0, 4.6 Hz, 1H), 7.39–7.30 (m, 2H), 7.04 (dd, J = 17.1, 7.9
Hz, 3H), 5.52 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 177.3, 170.8, 165.5,
158.0, 157.8, 155.1, 148.1, 135.5, 130.4, 129.7, 128.4, 122.1, 120.4,
115.0, 60.4. HRMS (ESI) m/z: 307.0712 [M + H]+ calcd for C17H11N2O4,
307.0713.
TDP2 reactions were carried out according to the previously reported
protocol [36]. The 18-mer single-stranded oligonucleotide DNA sub-
strate (TY18, α32P-cordycepin-3′-labeled) was incubated at 1 nM with
25 pM recombinant human TDP2 in the absence or presence of the tested
compounds for 15 min at room temperature in reaction buffer (50 mM
Tris-HCl, pH 7.5, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT,
4.13.2. 3-((2-Chlorophenoxy)methyl)isoxazolo[4,5-g]quinoline-4,9-dione
40 μg/mL BSA, and 0.01% Tween 20). Reactions were terminated by the
(66)
1
addition of 1 vol of gel loading buffer [99.5% (v/v) formamide, 5 mM
EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol
blue]. Samples were subjected to a 16% denaturing PAGE with multiple
loadings at 12-min intervals. Gels were dried and exposed to a Phos-
phorImager screen (GE Healthcare). Gel images were scanned using a
Typhoon FLA 9500 (GE Healthcare), and densitometry analyses were
performed using the ImageQuant software (GE Healthcare).
Yellow solid, yield 34%, mp: 154.7–155.9 ℃. H NMR (500 MHz,
CDCl3) δ 9.13 (dd, J = 4.6, 1.7 Hz, 1H), 8.59 (dd, J = 7.9, 1.7 Hz, 1H),
7.81 (dd, J = 7.9, 4.6 Hz, 1H), 7.37 (dd, J = 7.8, 1.6 Hz, 1H), 7.25 (dd, J
= 7.8, 1.6 Hz, 1H), 7.16 (dd, J = 8.3, 1.3 Hz, 1H), 6.99 (td, J = 7.8, 1.5
Hz, 1H), 5.56 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 177.2, 170.8, 165.6,
157.5, 155.1, 153.5, 148.1, 135.5, 130.7, 130.4, 128.4, 127.9, 123.90,
115.1, 100.1, 61.6. HRMS (ESI) m/z: 341.0324 [M + H]+ calcd for
C
17H10N2O6 341.0324.
4.15. Molecular modeling
4.13.3. 3-((4-(Methylsulfonyl)phenoxy)methyl)isoxazolo[4,5-g]quinoline-
4,9-dione (67)
The crystal structure of humanized mouse TDP2 (PDB: 5J3Z) was
obtained and tailored by removing one of the polymer, magnesium ions,
glycerin and water molecules. The optimized structure of TDP2 was used
Grey solid, yield 28%, mp, 213.4–214.5 ℃.1H NMR (400 MHz,
DMSO‑d6) δ 9.10 (dd, J = 4.7, 1.7 Hz, 1H), 8.52 (dd, J = 7.9, 1.7 Hz, 1H),
12

H. Yang et al.
Bioorganic Chemistry 111 (2021) 104881
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for molecular modeling. Site Finder was used to get the binding pocket
containing Glu152, Ser229, Asp262 and Leu313 residues. Compounds
were constructed using ChemDraw and saved in SDF file formats, which
were optimized using Discovery Studio software. For compound 70, it
was docked 10 times, starting each time from different orientations and
the default automatic genetic algorithm parameter settings were used.
All torsion angles of compound were allowed to rotate freely and the
results were scored and ranked by using MOE. The selected top ligand-
binding pose was merged into the crystal structure. The AMBER force
field of the MOE was utilized for energy minimization. The calculation
was terminated when the gradient reached a value of 0.05 kcal/mol⋅Å.
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Deazaflavin inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2) specific for the
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[17] E. Kiselev, A. Ravji, J. Kankanala, J.S. Xie, Z.Q. Wang, Y. Pommier, Novel
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Declaration of Competing Interest
[19] P. Wang, M.S.A. Elsayed, C.B. Plescia, A. Ravji, C.E. Redon, E. Kiselev,
C. Marchand, O. Zeleznik, K. Agama, Y. Pommier, M. Cushman, Synthesis and
biological evaluation of the first triple inhibitors of human topoisomerase 1,
tyrosyl-DNA phosphodiesterase 1 (Tdp1), and tyrosyl-DNA phosphodiesterase 2
(Tdp2), J. Med. Chem. 60 (8) (2017) 3275–3288.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[20] B.R. Kossmann, M. Abdelmalak, S. Lopez, G. Tender, C. Yan, Y. Pommier,
C. Marchand, I. Ivanov, Discovery of selective inhibitors of tyrosyl-DNA
phosphodiesterase 2 by targeting the enzyme DNA-binding cleft, Bioorg. Med.
Chem. Lett. 26 (14) (2016) 3232–3236.
Acknowledgement
This work was supported by the Guangdong Basic and Applied Basic
Research Foundation (No. 2019A1515011317), Guangzhou Basic and
Applied Basic Research Project (No. 202002030312) and Guangdong
Provincial Key Laboratory of Construction Foundation (No.
2017B030314030), and supported by the Intramural Program of the
National Cancer Institute (Center for Cancer Research), National In-
stitutes of Health, Bethesda, Maryland, USA (Z01 BCE 006150e19).
[21] C.J.A. Ribeiro, J. Kankanala, J. Xie, J. Williams, H. Aihara, Z. Wang,
Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors, Bioorg. Med.
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[22] C.J.A. Ribeiro, J. Kankanala, K.e. Shi, K. Kurahashi, E. Kiselev, A. Ravji,
Y. Pommier, H. Aihara, Z. Wang, New fluorescence-based high-throughput
screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2
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[23] E. Komulainen, L. Pennicott, D. Le Grand, K.W. Caldecott, Deazaflavin inhibitors of
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