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

Abstract

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 derivatives were synthesized and tested for enzyme inhibitions. Enzyme-based assays indicated that isoxazoloquinolinedione derivatives selectively showed high TDP2 inhibitory 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 with IC₅₀ of 0.46 ± 0.15 μM. This work will facilitate future efforts for the discovery of isoxazoloquinolinedione TDP2 selective inhibitors.

1. Introduction

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 specifically 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 arabinoside [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^′-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.

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 replication 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 discovery 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 quinazolinylaminopyrimidinone 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 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 reported as triple inhibitors of TOP1/TDP1/TDP2 with only moderate TDP2 inhibition (3: +++) [19]. Triazolopyridines 7 and quinazolinylaminopyrimidinones 8 display weak TDP2 inhibition at high micromolar concentration range [21]. Therefore, there is a strong need to discover novel TDP2 selective inhibitors as potential anticancer or antiviral agents.

Fig. 1.

The reported TDP2 inhibitors.

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 isoxazoloquinolinedione 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-dichloroacridine-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 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 hydrazines 32a-32d prepared according to the literature [29].

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.

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.

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 isomers, 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 and NBS in the presence of azodiisobutyronitrile (AIBN) to give the monobromo-substituted derivative 43 and dibromo-substituted derivative 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 °C condition to give 48, which was subsequently oxidized to give 49.

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 °C (obtained 48 and 49 simultaneously).

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 reaction of 6,7-dichloroquinoxaline-5,8-dione with ethyl nitroacetate gave the target compound 52. Under the catalysis of PhI(OAc)₂ [32], the reaction of ethyl (Z)-2-(hydroxyimino)-3-oxobutanoate with 2,3-dimethylphenol 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].

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.

Scheme 7.

Synthesis of compounds 54–57. Reagents and conditions: (a) NO₂CH₂CO₂R, K₂CO₃, MeCN.

Scheme 8.

Synthesis of compounds 59–63 and 65–71. Reagents and conditions: (a) Mn(OAc)₃, MeCN. (b) NaClO, Et₃N, DCM.

Finally, twenty-seven furoquinolinedione derivatives and twenty isoxazoloquinolinedione derivatives were synthesized and characterized through HRMS and NMR spectra.

2.2. Enzyme inhibition assays

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 TDP1 at the highest concentration tested of 111 μM and TOP1 at the highest concentration tested of 100 μM. Subsequently, the compounds with TDP2 potency were tested at six or eight three-fold dilution concentrations 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.

Table 1.

The TDP2 inhibitory activity of the synthesized compounds.

Cpd.	IC50 (μM)a	Cpd.	IC50 (μM)a	Cpd.	IC50 (μM)a
1	0.27 ± 0.07	31	>111	51	12 ± 2.4
11	>111	33	>111	52	9.3 ± 2.7
12	32 ± 3.2	34	>111	53	>111
13	>111	35	69 ± 3.1	54	3.1 ± 0.43
14	61 ± 43	36	28 ± 1.3	55	3.3 ± 0.65
15	>111	37	37 ± 2.0	56	15 ± 1.3
16	>111	38	>111	57	9.9 ± 2.2
19	94 ± 29	39	25 ± 7.7	59	14 ± 2.9
20	>111	40	>111	60	10 ± 1.48
21	21 ± 5.5	41	28 ± 6.4	61	8.7 ± 0.54
22	>111	42	47 ± 24	62	11 ± 3.8
23	37 ± 7.0	43	21 ± 5.5	63	20 ± 0.18
24	44 ± 2.0	44	27 ± 2.5	65	0.82 ± 0.28
25	>111	45	31 ± 4.2	66	0.94 ± 0.24
26	>111	46	22 ± 6.4	67	0.99 ± 0.13
27	>111	47	31 ± 6.4	68	0.74 ± 0.25
28	>111	48	>111	69	0.96 ± 0.20
29	>111	49	5.2 ± 4.1	70	0.46 ± 0.15
30	>111	50	5.4 ± 0.80	71	0.74 ± 0.25

^aThe IC₅₀ was expressed as mean ± SD from at least three independent experiments unless otherwise indicated.

In our previous investigation [25], N,O-anti furoquinolinedione derivatives 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 μM)/38 (>111 μM), 39 (25 ± 7.7 μM)/40 (>111 μM), 41 (28 ± 6.4 μM)/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 μM), as well as 14 with N atom at 6-position (61 ± 43 μM) and 15 with N atom at 7-position (>111 μM). Furthermore, the presence of two N atoms at A-ring could not benefit the activity (12: 32 ± 3.2 μM, 13: > 111 μM), even if one of N atoms at 5-position (12). The fused aromatic ring at A-ring abolished the activity (16: > 111 μM).

According to the TDP2 inhibition (Table 1), the 6-substituent decreased the activity comparing with 9 (11 ± 1.0 μM), implying that 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, the larger steric bulk of the 8-substituent also impaired the activity (36: 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), 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 showed moderate activity, implying that the steric bulk is tolerated with 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 potency. The isoxazole derivatives (54–57, 59–63) with various 3-alkoxy carbonyl groups showed good inhibitory activity with IC₅₀ 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, compounds 65–71 showed high inhibitory activity with IC₅₀ values at sub-micromolar level ranging 0.46 ± 0.15 to 0.99 ± 0.13 μM 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 inhibitions are shown in Fig. 2. In TDP2 inhibition assays, deazaflavin 1 was used as a positive control [15]. After being incubated with the inhibitors, 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.

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 μM. 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 μM. TY19 and TP19 are the substrate and product of TDP2, respectively.

2.3. Molecular modeling

To view the molecular interactions of the bioactive isoxazoloquinolinedione derivatives with TDP2, we constructed a hypothetical binding model using in-silico docking. The co-crystal structure of humanized mouse TDP2 (PDB code: 5J3Z) was employed as a template 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 inhibitory efficacy on SV-163 as hTDP2 and verifies the reliability of this template [37]. Moreover, mTDP2 has few key amino acid residue mutations 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 Trp297, Leu305, and Phe315, and forms π-π stacking with Trp297 and 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 receptors. The hypothetical binding mode is in general agreement with the empirical SAR observed.

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).

3. Conclusions

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 μM. For the furoquinolinediones, compound 49 with a formyl substituent 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 isoxazoloquinolinedione scaffold is a productive chemotype for TDP2 inhibitors, and deserves further study as potential anticancer and antiviral drugs.

4. Methods and materials

4.1. General experiments

All chemical reagents for synthesis were purchased from local commercial 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 prepared 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 spectrometer 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.

4.2. General procedure for the synthesis of compounds 11–14, 16, 19–22, 37–42, 52 and 54–57.

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 dichloroquinolinedione reagents (2 mmol) and K₂CO₃ (2.4 mmol) in acetonitrile (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 reaction 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.

4.2.1. Ethyl 2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxylate (11)

Luminous yellow solid, yield 95%, mp, 159.7–161.5 °C (Lit. [45] 149.3–159.9 °C). ¹H 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.

4.2.2. Ethyl 2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]isoquinoline-3-carboxylate (12)

Yellow solid, yield 23%, mp, 143.7–145.4 °C. ¹H 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.

4.2.3. Ethyl 2-methyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]isoquinoline-3-carboxylate (13)

Yellow solid, yield 20%, mp, 146.2–148.1 °C. ¹H NMR (400 MHz, 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)

Yellow solid, yield 13%, mp, 176.6–178.0 °C. ¹H NMR (400 MHz, 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.

4.2.5. Ethyl 2-methyl-4,11-dioxo-4,11-dihydrofuro[2,3-b]acridine-3-carboxylate (16)

Yellow solid, yield 16%, mp, 223.9–225.1 °C. ¹H NMR (400 MHz, 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.

4.2.6. Ethyl-6-chloro-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (19)

Yellow solid, yield 16%, mp, 167.7–168.5 °C. ¹H NMR (400 MHz, 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.

4.2.7. Ethyl-7-chloro-2-methyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-carboxylate (20)

Yellow solid, yield 17%, mp, 197.5–198.3 °C. ¹H 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.

4.2.8. Ethyl-2,6-dimethyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (21)

Yellow solid, yield 11%, mp, 142.1–142.9 °C. ¹H 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.

4.2.9. Ethyl-2,7-dimethyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-Carboxylate (22)

Yellow solid, yield 16%, mp, 185.8–187.2 °C. ¹H 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.

4.2.10. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (37)

Yellow solid, yield 5%, mp, 144.6–147.9 °C.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.

4.2.11. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-carboxylate (38)

Yellow solid, yield 21%, mp, 157.7–159.5 °C. ¹H 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.

4.2.12. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (39)

Yellow solid, yield 15%, mp, 115.4–115.8°C. 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.

4.2.13. Ethyl 2-isopropyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinoline-3-carboxylate (40)

Yellow solid, yield 25%, mp, 160.3–162.1°C. 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.

4.2.14. 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 °C. 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.

4.2.15. 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 °C. 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.

4.2.16. Ethyl-4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoxaline-3-carboxylate (52)

Yellow solid, yield 5%, mp, 185.7–186.2 °C. 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, 157.3, 153.6, 149.4, 148.9, 145.3, 143.9, 120.5, 64.0, 13.7. HRMS (ESI) m/z: 296.0278 [M + Na]⁺, calcd for C₁₂H₈N₃O₅Na 296.0278.

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 °C. 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.

4.2.18. Tert-butyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-carboxylate (55)

Yellow solid, yield 8%, mp, 157.1–157.9 °C.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 C15H13N2O5 301.0891.

4.2.19. 4-chlorophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[5,4-g]quinoline-3-carboxylate (56)

Yellow solid, yield 6%, mp, 145.8–146.5 °C.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.

4.2.20. 4-chlorophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-carboxylate (57)

Yellow solid, yield 7%, mp, 137.2–137.9 °C.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.

4.3. 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 °C. 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. General procedure for the synthesis of compounds 23–26, 30 and 31.

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 purified by column chromatography on silica gel using ethyl acetate/petroleum ether (1/10–1/1, V/V) as eluent to give the target compounds.

4.4.1. Ethyl-6-(dimethylamino)-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (23).

Red solid, yield 55%, mp, 191.7–192.9 °C. 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.

4.4.2. 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 °C. 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.

4.4.3. Ethyl-2-methyl-6-(4-methylpiperazin-1-yl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (25)

Red solid, yield 80%, mp, 152.8–154.5 °C. 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.

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)

Red solid, yield 57%, mp, 165.6–167.2 °C. 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.

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 °C. 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.

4.4.6. 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 °C. 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.

4.5. The synthesis of compounds 27 and 28.

Under nitrogen, to a solution of compound 19 (1.87 mmol) in a mixed solvents (dichloroethane/water = 5:1, 18 mL), (4-methoxyphenyl)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 °C for 12 h. After the reaction was completed, the reaction 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.

4.5.1. Ethyl6-(4-methoxyphenyl)-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (27)

Yellow solid, yield 48%, mp, 191.6–192.8 °C. ¹H 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.

4.5.2. Ethyl-6-(furan-2-yl)-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (28)

Yellow solid, yield 32%, mp, 223.4–224.7 °C. ¹H 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.

4.6. 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 °C. And then, the mixture solution was heated and stirred at 50 °C for 30 min. A solution of 19 (0.80 g, 2.5 mmol) in fresh distilled tetrahydrofuran (5 mL) was added dropwise. The reaction solution was stirred at 50 °C 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 °C. 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.

4.7. 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 pressure. The residue was purified by silica gel column chromatography to give the target compounds.

4.7.1. Ethyl-2,7-dimethyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (33)

Yellow solid, yield 39%, mp, 169.6–171.2 °C. ¹H 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.

4.7.2. Ethyl-2-methyl-8-(2-nitrophenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (34)

Yellow solid, yield 27%, mp, 203.8–206.7 °C. ¹H 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.

4.7.3. Diethyl-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3,8-dicarboxylate (35)

Yellow solid, yield 37%, mp, 170.5–171.7 °C. ¹H 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). ¹³C 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.

4.7.4. Ethyl-8-ethyl-2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (36)

Yellow solid, yield 17%, mp, 131.1–133.3 °C. ¹H 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.

4.8. 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 temperature. 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)

Yellow solid, yield 44%, mp, 191.3–193.0 °C. ¹H 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.

4.8.2. Ethyl 2-(dibromomethyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (44)

Yellow solid, yield 15%, mp, 143.5–146.7 °C. ¹H 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.

4.9. 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 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.

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°C. 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). ¹³C 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.

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°C. 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.

4.9.3. Ethyl 4,9-dioxo-2-(thiomorpholinomethyl)-4,9-dihydrofuro[2,3-g]quinoline3-carboxylate (47)

Yellow solid, yield 57%, mp, 145.7–147.1°C. 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.

4.10. The synthesis of compounds 48 and 49

Compound 43 (2 mmol) was dissolved in dimethyl sulfoxide (5 mL) and stirred at 150 °C 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 chromatography to give the compounds 48 and 49, simultaneously.

4.10.1. Ethyl-2-(hydroxymethyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carbo-xylate (48)

Yellow solid, yield 21%, mp, 164.6–166.6 °C. ¹H 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.

4.10.2. Ethyl-2-formyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinoline-3-carboxylate (49)

Yellow solid, yield 6%, mp, 176.8–179.1 °C. 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 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.

4.11.1. Ethyl-4,9-dioxo-4,9-dihydroisoxazolo[5,4-g]isoquinoline-3-carboxylate (50)

Yellow solid, yield 5%, mp, 104.9–107.1°C. 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.

4.11.2. Ethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]isoquinoline-3-carboxylate (51)

Yellow solid, yield 7%, mp, 131.9–133.8 °C. 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.

4.11.3. 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 °C. 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.

4.11.4. 2-Cyanoethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-carboxylate (60)

Yellow solid, yield 20%, mp, 158.9–159.7 °C. ¹H 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.

4.11.5. 3-Fluorophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-carboxylate (61)

Yellow solid, yield 23%, mp, 148.6–149.7 °C. ¹H NMR (400 MHz, 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.

4.11.6. 4-Nitrophenethyl 4,9-dioxo-4,9-dihydroisoxazolo[4,5-g]quinoline-3-carboxylate (62)

Yellow solid, yield 31%, mp, 213.1–213.8 °C. ¹H 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, 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.

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)

Yellow solid, yield 33%, mp, 137.3–138.2 °C. ¹H NMR (500 MHz, 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.

4.12. The synthesis of 53.

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 °C. 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.

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 °C. 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.

4.13.1. 3-(Phenoxymethyl) isoxazolo[4,5-g]quinoline-4,9-dione (65)

Yellow solid, yield 27%, mp, 142.9–144.2 °C. ¹H NMR (500 MHz, 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.

4.13.2. 3-((2-Chlorophenoxy)methyl)isoxazolo[4,5-g]quinoline-4,9-dione (66)

Yellow solid, yield 34%, mp: 154.7–155.9 °C. ¹H 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 C17H10N2O6 341.0324.

4.13.3. 3-((4-(Methylsulfonyl)phenoxy)methyl)isoxazolo[4,5-g]quinoline-4,9-dione (67)

Grey solid, yield 28%, mp, 213.4–214.5 °C.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), 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.

4.13.4. 4-((4,9-Dioxo-4,9-dihydroisoxazolo[4,5-g]quinolin-3-yl)methoxy)benzonitrile (68)

Light yellow solid, yield 21%, mp, 178.6–178.9 °C. 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.

4.13.5. 4-((4,9-Dioxo-4,9-dihydroisoxazolo[4,5-g]quinolin-3-yl)methoxy)-2-fluorobenzonitrile (69)

Light yellow solid, yield 21%, mp, 197.1–198.2 °C. 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 °C. 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.

4.13.7. 4-(4,9-Dioxo-4,9-dihydroisoxazolo[4,5-g]quinolin-3-yl)-3-fluorobenzonitrile (71)

Yellow solid, yield 25%, mp, 215.1–215.7 °C. ¹H 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.

4.14. Recombinant TDP2 assay

TDP2 reactions were carried out according to the previously reported protocol [36]. The 18-mer single-stranded oligonucleotide DNA substrate (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, 40 μg/mL BSA, and 0.01% Tween 20). Reactions were terminated by the 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 PhosphorImager 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).

4.15. Molecular modeling

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 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.