Synthesis and biological activities of 11- and 12-substituted benzophenanthridinone derivatives as DNA topoisomerase IB and tyrosyl-DNA phosphodiesterase 1 inhibitors

Abstract

Herein, a series of 11- or 12-substituted benzophenanthridinone derivatives was designed and synthesized for the discovery of dual topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Enzyme-based assays indicated that two compounds 12 and 38 showed high TOP1 inhibitory potency (+++), and four compounds 35, 37, 39 and 43 showed good TDP1 inhibition with IC₅₀ values ranging from 10 to 18 µM. 38 could induce cellular TOP1cc formation, resulting in the highest cytotoxicity against HCT-116 cells (0.25 µM). The most potent TDP1 inhibitor 43 (10 µM) could induce cellular TDP1cc formation and enhance topotecan-induced DNA damage and showed strong synergistic cytotoxicity with topotecan in both MCF-7 and MCF-7/TDP1 cells.

Graphical Abstract

A SAR research of 11- or 12-substituted benzophenanthridinone derivatives led to identify potent dual TOP1 and TDP1 inhibitor 43 that can enhance the ability of topotecan to induce DNA damage, shows synergy in combination with topotecan in both MCF-7 and MCF-7/TDP1 cells.

Introduction

DNA topoisomerase IB (TOP1) is ubiquitous and essential enzymes that solve the topological problems that accompany DNA replication, transcription and genomic stability^[1–4], and a validated target for the development of anticancer agents^[5–8]. TOP1 inhibitors, such as camptothecins and indenoisoquinolines can trap the normally transient TOP1-DNA cleavage complex (TOP1cc) and cause TOP1-mediated DNA damage, resulting in cancer cell death^[8–10]. TOP1-mediated DNA damage can be repaired through several pathways, including homologous recombination (high accuracy and low efficiency), cell cycle checkpoint signaling (often absent in cancer cells) and tyrosyl-DNA phosphodiesterase 1 (TDP1)-dependent pathways^[11–14].

TDP1 is a member of the phospholipase D superfamily^[15]. It can specifically hydrolyze the 3′-phosphotyrosyl bonds of TOP1ccs to generate DNA breaks with 5′-hydroxyl and 3′-phosphate ends for further DNA repair^[16,17]. Following the recruitment and activation of poly(ADP-ribose) polymerase (PARP1), the resulting DNA breaks can be repaired by polynucleotide kinase phosphatase (PNKP), DNA polymerase β and DNA ligase III through the (X-ray repair complementation group 1) XRCC-1-dependent pathway^[18–23]. In this pathway, TDP1-catalyzed hydrolysis of the 3′-phosphotyrosyl bond is a key step for initiating the repair of TOP1-mediated DNA damage, which can lead to cancer cell resistance to TOP1 inhibitors^[24]. Indeed, TDP1-overexpressing cells show resistance to camptothecin (CPT) ^[25,26]. Conversely, cells with TDP1 deficiency or mutated (SCAN1 cells), and TDP1 knockout mice show hypersensitivity to CPT^[27–33]. Recently, several TDP1 inhibitors have been reported to show a synergistic anticancer activity with TOP1 inhibitors in vitro and in vivo^[31,34–48]. Therefore, TDP1 is a rational target for the discovery of anticancer drugs^[49,50].

As TOP1 is a validated anticancer target, TOP1 inhibitors have been well developed and several camptothecin analogues are extensively used in clinical cancer treatment^[5,10,51]. Topotecan (TPT) ^[52,53], Irinotecan^[54], and the recently antibody drug conjugates Trastuzumab deruxtecan and Sacituzumab govitecan have been approved by the Food and Drug Administration (FDA) ^[10,55,56]. Additionally, 10-hydroxycamptothecin is clinically approved in China^[57,58], and Belotecan in Korea^[8]. Despite their outstanding efficacy, camptothecin TOP1 inhibitors suffer from several drawbacks including chemical instability, acute and chronic toxicity including drug-related leukemia, and drug resistance. Subsequently, to counter these limitations, much attention is paid to discovery of non-camptothecin inhibitors^[59]. In addition, small molecular TDP1 inhibitors have been also developed recently, and several TDP1 scaffold have been reported^[60], including steroids^[48,61], methyl-3,4-dephostatins^[62], arylidenethioxothiazolidinones^[63], thioxopyrimidinedione^[64], benzopentathiepines^[65], indenoisoquinolines^[66,67], usnic acid derivatives^[39], aminoadamantanes^[36], 7-hydroxycoumarin derivatives^[35,47], and N,2-diphenylimidazo[1,2-a]amines^[43], benzodipyrans^[68]. Additionally, recently TDP1 co-crystal structures with inhibitors could provide the structural basis for the rational design of TDP1 inhibitors^[43,69].

The physiological function of TDP1 is to specifically hydrolyzes the 3′-phosphotyrosyl bond of TOP1ccs. Based on this point, the dual activities against TOP1 and TDP1 will act on multiple stages of cancer genesis and development, and may play a greater anticancer effect. A few dual TOP1 and TDP1 inhibitors have been identified^{[37,51,67,70]}. The first reported dual TOP1 and TDP1 inhibitors were indenoisoquinoline derivatives such as the representative bisindenoisoquinoline inhibitor 1 is shown in Figure 1^[67]. However, further study indicated that several indenoisoquinoline derivatives also inhibited tyrosyl-DNA phosphodiesterase 2^[71], a DNA repair enzyme hydrolyzing primarily the 5′-phosphotyrosyl bonds of TOP2 and TOP3 cleavage complexes^[72–74], implying that indenoisoquinolines might be promiscuous inhibitors.

Figure 1.

The structures of the reported dual TOP1 and TDP1 inhibitors and the representative oxynitidine derivatives.

In our effort to discover novel dual TOP1 and TDP1 inhibitor chemotypes, two chalcone derivatives 2 and 3 (Figure 1) have been identified from the roots of Isodon ternifolius Kudo as dual inhibitors^[42]. However, they just show moderate inhibitory activities for both TOP1 and TDP1. In addition, oxynitidine (4, Figure 1) derivatives have also been identified as dual inhibitors with the ability to trap cellular TDP1-DNA complexes and with anticancer activity in vitro and in vivo^[37,46,75]. The representative molecule 5 shows a strong TOP1 inhibition (+++), but a weak TDP1 inhibition (12% enzyme inhibitory activity at 100 μM concentration) ^[37]. Structure-activity relationship (SAR) analysis has indicated that the lactam skeleton of oxynitidine derivatives is important for TOP1 inhibition. Molecular modeling indicates a hydrogen bond (2.9 Å) formed between the lactam oxygen atom of 5 and the key residue Arg364 possibly contribute to the TOP1 inhibition^[37]. And, near the C-ring of 5, there is a hydrophilic binding pocket comprising the key residues Asn352 and Glu356 of TOP1, which could accommodate an additional polar side chain for a TOP1 inhibitor. In addition, the molecular modeling between 5 and TDP1 (PDB 6DJD) indicates that the benzophenanthridinone skeleton binds to the DNA binding region of TDP1 (Figure S1, Supporting Information) ^[69]. And, there is also a hydrophilic binding pocket comprising the key residues His263, and Ser399, Ser400, Asn516, Ala521 and Glu538 of TDP1 near the C-ring of 5. This observation inspired us to design the 11- or 12-substituted benzophenanthridinone derivatives for the discovery of dual TOP1 and TDP1 inhibitors. In this work, we report the synthesis of the designed compounds, their TOP1 and TDP1 inhibitory activities and anticancer potential.

Results and Discussion

Chemistry

Because of the potential role of the lactam skeleton for TOP1 inhibition, the designed compounds retain the oxynitidine scaffold (Figure 1, N-methylpyridinone structure). The synthesis of the 12-substituted benzophenanthridinone derivatives is illustrated in Scheme 1. According to Cheng’s synthetic method^[76], the key intermediate 8 was prepared from the reaction of the Schiff base 6 with 7, prepared through Sonogashira coupling reaction from 5-bromobenzo-[d][1,3]dioxole and 3-butyn-1-ol. Following the Swern oxidation of the hydroxyl group of 8^[77], the obtained aldehyde intermediate 9 was further oxidized by NaClO₂ under NaH₂PO₄ condition^[78], and subsequently cyclized to give the benzophenanthridinone 10. After hydrolysis of 10, the reaction of 11 with the corresponding chloride material in the presence of K₂CO₃ and KI gave the 12-substituted benzophenanthridinone derivatives 12-23 (Table 1).

Scheme 1.

Synthesis of 10-23.

Reagents and conditions: (a) NH₂Me, MeOH, rt. (b) i) N₂, Ni(cod)₂, P(o-Tol)₃, MeCN; ii) CsOH, K₃[Fe(CN)₆], MeOH, H₂O. (c) (COCl)₂, DMSO, TEA, DCM. (d) i) NaClO₂, NaH₂PO₄·2H₂O, 2-methyl-2-butene, acetone; ii) Ac₂O, AcOK. (e) KOH, H₂O, MeOH. (f) R(CH₂)_nCl, KI, K₂CO₃, DMF.

Table 1.

The TOP1 and TDP1 Inhibitory Potency, and Cytotoxicity of 10-23.

					GI50 ± SD (μM)[c]
Cpd	n	R	TOP1 Inhibition[a]	IC50 (μM) for TDP1[b]	HCT-116	MCF-7
4	/d	/	0/+	>111	75 ± 4.4	>100
10	0	Ac	+	>111	>100	>100
11	0	H	0	>111	>100	>100
12	2	NHMe	+++	>111	1.3 ± 0.12	2.2 ± 0.06
13	3		+	>111	12 ± 1.2	33 ± 5.0
14	2	NMe2	+	37–111	4.6 ± 0.41	8.5 ± 0.91
15	3		+	>111	36 ± 1.3	54 ± 4.9
16	2		+	>111	>100	22 ± 7.7
17	3		++	>111	1.8 ± 0.32	6.6 ± 1.3
18	2		+	37–111	7.7 ± 0.56	7.0 ± 1.3
19	3		++	>111	1.5 ± 0.21	0.93 ± 0.07
20	2		++	>111	2.9 ± 0.45	2.7 ± 0.23
21	3		0	>111	16 ± 3.6	64 ± 3.9
22	2	NH(CH2)2NMe2	+	>111	9.1 ± 1.0	9.1 ± 2.4
23	1	COOEt	0	>111	>100	91 ± 5.9

^[a]TOP1 cleavage inhibitory activity of the compounds was semiquantitatively expressed relative to CPT at 1 μM as follows: 0, no inhibition; 0/+, less than 20%; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity.

^[b]IC₅₀ values for TDP1 inhibition, defined as the concentration of compound that inhibits 50% of enzyme activity, were detected through the gel-based assays.

^[c]The cytotoxicity GI₅₀ value was defined as the concentration corresponding to 50% cell growth inhibition and obtained from MTT assay. Every experiment was repeated at least three times independently.

^[d]“/” means “inapplicable”.

The synthesis of 11-substituted benzophenanthridinone derivatives is shown in Scheme 2. Using piperonylacetic acid and dimethyloxybenzaldehyde as starting materials, the key intermediate 30 was prepared according to our reported method^[79]. The selective demethylation of 30 gave the pyridinone intermediate 31^[80]. Following the methylation in the lactam of 31^[81], a three-step reaction of 32, including a cyclization under acetic anhydride and AcOK condition, gave the benzophenanthridinone 33. The 11-substituted benzophenanthridinone derivatives 34-45 (Table 2) were prepared from the reaction of 33 with the corresponding chlorides in the presence of K₂CO₃ and KI.

Scheme 2.

Synthesis of 33-45.

Reagents and conditions: (a) i) ICl, AcOH, rt; ii) MeOH, H₂SO₄. (b) N₂, TMSA, PdCl₂(PPh₃)₂, CuI, NEt₃, THF. (c) K₂CO₃, MeOH. (d) i) AgNO₃, I₂, MeOH; ii) KMnO₄, H₂O/MeCN. (e) i) SOCl₂, reflux; ii) NH₃/H₂O, THF. (f) PdCl₂(PPh₃)₂, NEt₃, MeCN. (g) i) Me₃OBF₄, DCM; ii) CF₃COOAg, DCM. (h) NaBr, TsOH·H₂O, MeOH. (i) MeI, K₂CO₃, DMF. (j) i) K₂CO₃, H₂O, MeOH; ii) Ac₂O, AcOK; iii) K₂CO₃, H₂O, MeOH. (k) R(CH₂)_nCl, KI, K₂CO₃, DMF.

Table 2.

The TOP1 and TDP1 Inhibitory Potency, and Cytotoxicity of 33–45.

					GI50 ± SD (μM)[c]
Cpd	n	R	TOP1 Inhibition[a]	IC50 (μM) for TDP1[b]	HCT-116	MCF-7
4	/d	/	0/+	>111	75 ± 4.4	>100
33	0	H	+	>111	>100	>100
34	2	NMe2	+	37–111	11 ± 0.48	27 ± 1.5
35	3		0	18 ± 6.9	0.55 ± 0.17	1.2 ± 0.10
36	2	NEt2	0	>111	2.1 ± 0.23	1.6 ± 0.17
37	3		+	15 ± 2.4	2.4 ± 0.11	2.0 ± 0.10
38	2		+++	37–111	0.25 ± 0.02	6.4 ± 1.0
39	3		+	15 ± 2.8	1.3 ± 0.21	1.4 ± 0.13
40	2		++	>111	15 ± 1.08	18 ± 0.67
41	3		+	>111	3.3 ± 0.73	7.9 ± 1.0
42	3	OH	+	>111	38 ± 2.31	51 ± 1.6
43	3	NH(CH2)2OH	+	10 ± 3.8	0.29 ± 0.19	2.2 ± 1.2
44	3		0/+	>111	10 ± 1.2	7.0 ± 0.55
45	3		0	>111	>100	85 ± 3.8

^[a]TOP1 cleavage inhibitory activity of the compounds was semiquantitatively expressed relative to CPT at 1 μM as follows: 0, no inhibition; 0/+, less than 20%; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity.

^[b]IC₅₀ values for TDP1 inhibition, defined as the concentration of compound that inhibits 50% of enzyme activity, were detected through the gel-based assays.

^[c]The cytotoxicity GI₅₀ value was defined as the concentration corresponding to 50% cell growth inhibition and obtained from MTT assay. Every experiment was repeated at least three times independently.

^[d]“/” means “inapplicable”.

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

TOP1 Inhibition

All the synthesized benzophenanthridinone derivatives were tested for TOP1 inhibition through the TOP1-mediated DNA cleavage assay using a 3′-[³²P]-labeled double-stranded DNA oligonucleotide as substrate^[82]. The compounds were tested at 100, 10, 1 and 0.1 μM concentrations using CPT and the indenoisoquinoline TOP1 inhibitor LMP744 as positive controls^[83].

The TOP1 inhibitory activity was semiquantitatively expressed relative to CPT at 1 μM: 0, no inhibition; 0/+, less than 20%; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity, and summarized in Tables 1 and 2.

As shown in Table 1, compared to 4 (0/+), the attachment of an aminoalkoxyl group at 12-position could increase the TOP1 inhibition. All the 12-aminoalkoxyl substituted analogues have TOP1 inhibitory potency ranging from + to +++ except 21. Among them, three analogues 17, 19, and 20 showed moderate TOP1 inhibition (++), and 12 (+++) showed the most potency equal to 5. The attachment of a hydroxy (11) or an alkoxy group without nitrogen atom (23) did not improve the TOP1 inhibition. Similarly, the attachment of an aminoalkoxyl group at 11-position also could increase the TOP1 inhibition (Table 2). And, 38 showed strong TOP1 inhibition of +++, as well as one analogue 40 with moderate inhibition of ++ and six analogues 34, 37, 39 and 41-43 with weak inhibition of +.

Representative cleavage gels of 12, 19, 38 and 40 are shown in Figure 2. The cleavage sites induced by the novel TOP1 inhibitors were similar to that induced by indenoisoquinoline control LMP744 but not to CPT, implying the novel benzophenanthridinone TOP1 inhibitors target the same genome sites as oxynitidine and its analogue 5^[37].

Figure 2.

Representative gels of TOP1-mediated DNA cleavage assay. Lane 1, DNA alone; lane 2, DNA and TOP1; lane 3, DNA and TOP1 with CPT (1 μM); lane 4, DNA and TOP1 with LMP744 (1 μM); lanes 5–20, DNA and TOP1 with the tested compounds at 0.1, 1.0, 10, and 100 μM concentration, respectively. The arrows at left indicate the cleavage site positions.

TDP1 Inhibition

All the synthesized benzophenanthridinone derivatives were tested for TDP1 inhibition through a gel-based assay with a Cy5-labeled single-stranded oligonucleotide Cy5–14Y substrate at eight three-fold dilution concentrations (111, 37, 12.3, 4.1, 1.4, 0.46 and 0.051 μM) ^[43]. The imidazopyridine TDP1 inhibitor XZ664 (Figure S2, Supporting Information) was used as the positive control^[43]. As shown in Figure 3, the representative TDP1 inhibition gels indicate that the quantity of TDP1 product Cy5–14P decrease with the addition of increased concentration of XZ664, implying that TDP1 activity is inhibited. The IC₅₀ values, defined as the concentration of compound that inhibits 50% of TDP1 activity, were calculated and summarized in Tables 1 and 2.

Figure 3.

Representative gels of TDP1 assay. Lane 1, DNA alone; lane 2, DNA and recombinant TDP1; lanes 3–26, DNA, recombinant TDP1 and the compounds at the concentrations increasing from 0.051 to 111 μM. The imidazopyridine XZ664 was tested as the positive control at the concentrations increasing from 0.15 to 333 μM. Cy5–14Y and Cy5–14P are the substrate and product of TDP1, respectively.

As shown in Table 1, introduction of a side chain at the 12-position did not enhance the TDP1 inhibitory potency. Only two analogues 14 and 18 showed weak TDP1 inhibition (37–111 μM) for 12-substituted benzophenanthridinone derivatives. For 11-substituted benzophenanthridinone derivatives (Table 2), four analogues 35, 37, 39, and 43 showed good TDP1 inhibitory potency with IC₅₀ values ranging 10 to 18 μM concentration (Figure 3). It seems that the longer side chain at 11-position is favorable for TDP1 inhibitory potency, for example, 35 vs 34, 37 vs 36, 39 vs 38. The most potent TDP1 inhibitor 43 (IC₅₀ = 10 ± 3.8 μM) also showed weak TOP1 inhibition (+). And 38 showing high TOP1 inhibition (+++), only showed weak TDP1 inhibition (37–111 μM).

Cellular TOP1cc and TDP1cc Formation

In previous work, we found benzophenanthridinone TOP1 and TDP1 inhibitors can induce cellular TOP1cc and TDP1cc formation^[37,46]. To evaluate the ability of novel inhibitors to trap cellular TOP1ccs, the most potent inhibitor 38 was tested by the immunocomplex of enzyme (ICE) to DNA assay in MCF-7 cells. Lick TPT (Figure 4A), 38 trapped cellular TOP1ccs in MCF-7 cells in a dose-dependent manner, implying that TOP1 is targeted by 38 in cells.

Figure 4.

Cellular TOP1ccs induced by 38 (A) and TDP1cc induced by 43 (B) in MCF-7 cells, detected through ICE assays. (A) Lane 1, untreated control; lanes 2 and 3, cells treated with TPT at 25 and 50 μM, respectively; lanes 4–6, cells treated with 38 at 25, 50, and 100 μM, respectively. (B) Lane 1, untreated control; lanes 2–4, cells treated with TPT (10 μM) and 43 at 10 and 100 μM, respectively; lanes 5 and 6, cells co-treated with TPT (10 μM) and 43 (10 and 100 μM). (C) Hypothetical binding mode of 38 in the ternary TOP1-DNA-drug cleavage complex (PDB 1K4T). 38 is shown as yellow carbon atoms ball and stick representation. Key residues are highlighted in green ball and stick representation. All distances are measured from heavy atom to heavy atom.

ICE assays were also conducted to evaluate whether the most potent TDP1 inhibitor 43 induced the cellular TDP1ccs. As shown in Figure 4B, cellular TDP1ccs significantly enhanced after being treated with high concentration of 43 (100 μM) in MCF-7 cells, but not with TPT (10 μM) and low concentration of 43 (10 μM). After co-treated with TPT and 43 (both at 10 μM and 100 μM), the quantity of cellular TDP1cc significantly enhanced.

These results indicate that the novel benzophenanthridinone TOP1 inhibitors can induce cellular TOP1cc formation in MCF-7 cells, as well as induce cellular TDP1cc formation.

Molecular Modeling

To view the molecular binding mode of novel benzophenanthridinone TOP1 inhibitors within the TOP1cc, the most potent inhibitor 38 (+++) was selected for molecular modeling studies. A hypothetical binding model was built by using in silico docking from the X-ray crystal of the TOP1-DNA-ligand ternary complex (PDB 1K4T) according to our reported method^[37,46]. The inhibitor 38 was energy minimized and docked into the binding model. As shown in Figure 4C, the benzophenanthridinone scaffold of 38 intercalates in the DNA break made by TOP1 and readily stacks with the base pairs flanking the DNA cleavage site. Compared to 4^[37], the benzophenanthridinone scaffold of 38 is flipped within the TOP1-DNA covalent complex. The A- and B-ring of 38 stack with the scissile stand bases (G and T), while C- and D-ring stack with the noncleaved strand bases (C and A). The 11-side chain extents to the major groove of DNA. A hydrogen bond (2.3 Å) was observed between the lactam oxygen atom and Arg364, implying the importance of the lactam fragment in TOP1 inhibition. Also, two hydrogen bonds between the pyrrolidine nitrogen atom of the side chain and Asn352 (3.3 Å), and between an oxygen atom in the dioxole ring and Glu356 (2.8 Å) might contribute to the trapping of TOP1cc.

Cytotoxicity Assays

The cytotoxicity of the synthesized compounds was tested by MTT assay against two human cancer cell lines, colon cancer (HCT-116) and breast cancer (MCF-7). The cells were treated in a five-dose assay for 72 hours at concentrations ranging from 0.01 to 100 μM. The GI₅₀ values, expressed as the concentrations corresponding to 50% cell growth inhibition, are summarized in Tables 1 and 2.

For 12-substituted benzophenanthridinone derivatives (Table 1), seven analogues 12, 14, 17-20, and 22 displayed significant cytotoxicity against both HCT-116 and MCF-7 cells with GI₅₀ values at micromolar concentrations. The most potent TOP1 inhibitor 12 (+++) showed the highest cytotoxicity for HCT-116 cells (GI₅₀ = 1.3 ± 0.12 µM), as well as 19 (++) for MCF-7 cells (GI₅₀ = 0.93 ± 0.07 µM). For 11-substituted benzophenanthridinone derivatives (Table 2), eight derivatives 35–39, 41, 43, and 44 displayed good cytotoxicity against both HCT-116 and MCF-7 cells with GI₅₀ values at micromolar concentrations. The dual inhibitor 38 with the highest TOP1 inhibition (+++) and low TDP1 inhibition (37–111 μM) showed the highest cytotoxicity for HCT-116 (GI₅₀ = 0.25 ± 0.02 µM), and good cytotoxicity for MCF-7 (GI₅₀ = 6.4 ± 1.0 µM), as well as 43 with the most TDP1 inhibition (10 ± 3.8 µM) and low TOP1 inhibition (+) showed high cytotoxicity for both HCT-116 (GI₅₀ = 0.29 ± 0.19 µM) and MCF-7 (GI₅₀ = 2.2 ± 1.2 µM). Although 35 had no TOP1 inhibition, it showed the highest cytotoxicity for MCF-7 (GI₅₀ = 1.2 ± 0.10 µM) and high cytotoxicity for HCT-116 (GI₅₀ = 0.55 ± 0.17 µM).

Synergistic Effects of 35 and 43 with TPT

In our previous study, the benzophenanthridinone dual TOP1 and/or TDP1 inhibitors showed synergistic effect with TPT in MCF-7 cells^[46]. To evaluate the potential synergy of the novel inhibitors in the combination with TPT, a dual TOP1 and TDP1 inhibitor 43 and a selective TDP1 inhibitor 35 were selected for the synergistic test in MCF-7 and MCF-7/TDP1 cells (overexpressing TDP1) using MTT assay. As shown in Figure 5A, after being co-incubated for 96 hours, 43 (1 μM) showed synergistic effect with TPT in MCF-7 cells, and its combination index (CI) values fell in the range of 0.18–0.77 (Figure 5B). Although 35 had weaker TDP1 inhibition then 43, 35 showed a stronger synergistic effect in MCF-7 cells, and its CI values fell in the range of 0.02–0.69, and two doses showed strong synergy (CI < 0.3) and one dose showed very strong synergy (CI < 0.1). In MCF-7/TDP1 cells, both 43 and 35 showed more synergistic effect with TPT than that in MCF-7 cells (Figure 5C). All their CI values fell in 0.003–0.51 (Figure 5D), and two doses of 43 and one dose of 35 showed strong synergy, and two doses of both 43 and 35 showed very strong synergy. These results indicated that the novel benzophenanthridinone inhibitors had synergistic effect with TPT in both MCF-7 and MCF-7/TDP1 cells, and the TDP1 inhibitor with higher potency showed the greater synergistic effect in MCF-7/TDP1 cells.

Figure 5.

The synergistic effects and the combination index (CI) of 35 or 43 with TPT in the MCF-7 (A, B) and MCF-7/TDP1 (C, D) cells. The cells were co-incubated with drugs for 96 h.

DNA Damage Induced by Combined Treatment of 43 and TPT

To evaluate the ability of 43 to enhance TOP1 inhibitor-induced DNA damage, an immunofluorescence assay was conducted to test the γH2AX foci formed in MCF-7 cells after being treated by drugs. As shown in Figure 6A, TPT could obviously induce γH2AX foci formation. And 43 alone had a weak ability to induce DNA damage, about one fifth of that of TPT at 1 μM concentration (Figure 6B), possibly due to its weak TOP1 inhibitory potency (+). However, 43 could increase TPT-induced DNA damage. Compared with TPT treated alone, about 1.9-fold and 2.4-fold γH2AX foci formed after co-treatment with TPT and 43 at 1 and 2 μM, respectively. These results indicated that 43 could effectively enhance the ability of TPT to induce DNA damage in MCF-7 cells, possibly due to its TDP1 inhibitory potency.

Figure 6.

(A) Histone γH2AX foci (red) induced by 43 and TPT alone or combined in MCF-7 cells. The cells were incubated with drugs for 5 h at the indicated concentrations. DNA was stained with DAPI (blue). (B) Quantification of the number of γH2AX foci on the figure (A). P < 0.001 (***) and P < 0.0001 (****). Every experiment was repeated at least three times independently. (C) Flow cytometry histograms of apoptosis in MCF-7 cells induced by 43 at 1, 2, and 4 μM, respectively.

Cancer Cell Apoptosis Induced by 43

Although the dual TOP1 and TDP1 inhibitor 43 exhibited only weak TOP1 inhibitory potency (+), it showed high cytotoxicity against both HCT-116 and MCF-7 cells. To assess the induction of apoptosis by 43, flow cytometry assays were conducted in MCF-7 cells. After being incubated with 43 for 24 hours, the apoptotic MCF-7 cells, especially late apoptotic cells significantly increased in a dose-dependent manner (Figure 6C). At 4 µM concentration, 43 induced approximately late apoptosis in 28.58% cells.

Conclusion

In summary, a series of 11- and 12-substituted benzophenanthridinone derivatives was synthesized and evaluated for TOP1 and TDP1 inhibition against purified TOP1 and TDP1 and in cells, and cytotoxicity against two human cancer cell lines, colon carcinoma HCT116 and breast cancer MCF-7. The TOP1-mediated DNA cleavage assays indicate that the aminoalkoxy displacement at both 11- and 12-position is favorable for TOP1 inhibition. Two derivatives 12 and 38 showed strong TOP1 inhibition (+++), and four compounds 17, 19, 20, and 40 showed moderate potency (++). Furthermore, we demonstrate that the novel TOP1 inhibitor 38 induces TOP1ccs in MCF-7 cells, as detected through ICE assays. TDP1-based gel assays indicate that a longer aminoalkoxy side chain at the 11-position is favorable for TDP1 inhibition. Four compounds 35, 37, 39, and 43 showed TDP1 inhibitory potency with IC₅₀ values ranging from 10 to 18 µM. Three of them 37, 39, and 43 also showed weak TOP1 inhibition (+). And the most potent TOP1 inhibitor 38 (+++) also showed weak TDP1 inhibition (37–111 µM). MTT assay indicated that fifteen compounds 12, 14, 17-20, 23, 35–39, 41, 43, and 44 exhibit high cytotoxicity against both HCT-116 and MCF-7 cells with GI₅₀ values at micromolar concentrations. The dual TOP1 and TDP1 inhibitor 38 showed the highest cytotoxicity for HCT-116 (0.25 ± 0.02 µM), and compound 19 showed the highest cytotoxicity for MCF-7 (0.93 ± 0.07 µM). The combination treatment indicated that both the dual TOP1 and TDP1 inhibitor 43 and the selective TDP1 inhibitor 35 showed strong synergistic cytotoxicity in combination with TPT in both MCF-7 and MCF-7/TDP1 cells. The most potent TDP1 inhibitor 43 was found to induce cellular TDP1ccs, and enhanced TPT-induced DNA damage in MCF-7 cells, resulting in apoptosis. Our previous SAR analysis indicated that the 5-aminoalkyl substituent can increase the dual TOP1 and TDP1 inhibition of benzophenanthridinone derivatives^[37,46,75]. Therefore, modifications of benzophenanthridinone derivatives at 5,11-positions fare warranted for the discovery of dual TOP1 and TDP1 inhibitors.

Experimental Section

General experiments

All chemical reagents for synthesis were purchased from local commercial suppliers and were used without further purification unless otherwise indicated. The key intermediates 9 and 30 were prepared in our lab according to the reported method^[76,79]. 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 a SHIMADZU LCMS-IT-TOF mass spectrometer. All compounds tested for biological activities were analyzed by HPLC and their purities were more than 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.

2,3-Dimethoxy-12-methyl-13-oxo-12,13-dihydro-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-6-yl acetate (10)

To a solution of 9 (260 mg, 0.67 mmol) in freshly acetone (10 mL), 2-methyl-2-butene (375 mg, 5.4 mmol), NaClO₂ (182 mg, 2.01 mmol) and NaH₂PO₄·2H₂O (241 mg, 2.01 mmol) were added. The reaction solution was stirred at room temperature for 12 h. After completion of the reaction, the reaction solution was adjusted to pH < 3 with hydrochloride acid (1N). The solution was extracted with ethyl acetate (20 mL x 3). The combined organic layer was concentrated under reduced pressure to give a crude solid, which was used immediately for next synthesis without further purification.

To a solution of the obtained solid (~180 mg, ~0.45 mmol) in acetic anhydride (5 mL), AcOK (89 mg, 0.90 mmol) was added. The suspension was stirred at 140 °C for 3 h. After completion of the reaction, the suspension was cooled to room temperature and added with water (20 mL). Filtration gave a white solid, which was purified by column chromatography on silica gel using methanol/dichloromethane (1/200, v/v) as eluent to give a white solid 10, yield 76%. The NMR spectral data are similar to that reported in the literature^[84]. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.76 (s, 1H), 7.66 (s, 1H), 7.45 (s, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 4.09 (s, 3H), 4.06 (s, 3H), 3.97 (s, 3H), 2.52 (s, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 169.9, 164.3, 153.5, 150.0, 148.2, 147.5, 142.4, 134.1, 128.5, 124.9, 122.3, 119.3, 116.4, 111.0, 108.7, 103.1, 103.0, 101.9, 98.7, 56.4, 56.3, 41.5, 21.2. HRMS (ESI) m/z: 422.1251 [M + H]⁺, calcd for C₂₃H₂₀NO₇ 422.1240.

6-Hydroxy-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (11)

To a solution of 10 (90 mg, 0.21 mmol) in methanol (30 mL), an aqueous KOH solution (1N, 2 mL) was added dropwise. The yellow reaction solution was stirred at 80 °C for 3 h. After completion of the reaction, the reaction solution was cooled to room temperature, and water (10 mL) was added, and then adjusted to pH < 3 by addition of hydrochloric acid (2N). The reaction solution was extracted with ethyl acetate (25 mL x 3). The combined organic layer was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using methanol/dichloromethane (1/200, v/v) as eluent to give a white solid 11, yield 85%, mp = 185.2–187.9 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.17 (s, 1H), 7.74 (s, 1H), 7.71 (s, 1H), 7.60 (s, 1H), 7.54 (s, 1H), 7.51 (s, 1H), 6.18 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.82 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.6, 153.1, 149.3, 148.9, 147.0, 146.8, 128.8, 128.2, 122.9, 121.7, 118.5, 116.3, 108.2, 103.4, 102.6, 101.6, 100.6, 99.1, 55.8, 55.7, 40.8. HRMS (ESI) m/z: 380.1138 [M + H]⁺, calcd for C₂₁H₁₈NO₆ 380.1134.

Methyl 2-(6-(6,7-dimethoxy-1-oxo-1,2-dihydroisoquinolin-3-yl)benzo[d][1,3]dioxol-5-yl)acetate (31)

To a solution of 30 (100 mg, 0.25 mmol) in MeOH (30 mL), NaBr (1.55g, 15.0 mmol) and p-toluenesulfonic acid monohydrate (571mg, 3.0 mmol) were added. The solution was stirred at 80 °C for 12 h. And then, the reaction solution was cooled to room temperature and added with NaHCO₃ saturated solution (20 mL). The reaction mixture was stirred vigorously for 15 min, and added with ethyl acetate (30 mL). The organic layer was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using methanol/dichloromethane (1/5, V/V) as eluent to give a yellow solid 31, yield 90%. ¹H NMR (400 MHz, CDCl₃) δ 9.31 (s, 1H), 7.78 (s, 1H), 6.89 (s, 2H), 6.80 (s, 1H), 6.37 (s, 1H), 6.03 (s, 2H), 4.01 (s, 3H), 3.99 (s, 3H), 3.75 (s, 2H), 3.57 (s, 3H). ESI-MS m/z: 398.1 [M + H]⁺.

Methyl 2-(6-(6,7-dimethoxy-2-methyl-1-oxo-1,2-dihydroisoquinolin-3-yl)benzo[d][1,3]dioxol-5-yl)acetate (32)

Under nitrogen gas, to a solution of 31 (450mg, 1.13 mmol) in dry DMF in seal tube, iodomethane (321mg, 2.26 mmol) and K₂CO₃ (781mg, 5.65 mmol) were added sequentially. The reaction mixture was stirred at 80 °C for 5 h. After completion of the reaction, water (10 mL) and ethyl acetate (20 mL) were added to the reaction solution. The organic layer was washed with saturated saline (20 mL x 3). The organic layer was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel using ethyl acetate/petroleum ether (1/3, V/V) as eluent to give a yellow solid 32, yield 89%. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (s, 1H), 6.87 (s, 1H), 6.82 (s, 1H), 6.73 (s, 1H), 6.32 (s, 1H), 6.05 (s, 2H), 4.03 (s, 3H), 3.98 (s, 3H), 3.58 (s, 3H), 3.52–3.45 (m, 2H), 3.30 (s, 3H). ESI-MS m/z: 412.1 [M + H]⁺.

5-Hydroxy-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (33)

The solution of 32 (140 mg, 0.36 mmol) and K₂CO₃ (152 mg, 1.08 mmol) in methanol (30 mL) and water (1 mL) was stirred at 80 °C. After completion of the reaction, the solution was cooled to room temperature and added with water (20 mL), and then adjusted with hydrochloride acid (2N) to pH < 3. The reaction solution was extracted with ethyl acetate (25 mL x 3). The combined organic layer was concentrated under reduced pressure to give a white rude solid.

The solution of the obtained white solid (~143 mg, ~0.36 mmol) and AcOK (71 mg, 0.72 mmol) in acetic anhydride (5 mL) was stirred at 140 °C for 3 h. After completion of the reaction, the solution was cooled to room temperature and added with water (20 mL). The suspension was filtrated to give a white solid. The white solid was dissolved in methanol (20 mL) and water (1 mL). K₂CO₃ (276 mg, 2.0 mmol) was added to the solution. The yellow solution was stirred at 80 °C. After completion of the reaction, the reaction solution was cooled to room temperature and added with water (20 mL), and then extracted with ethyl acetate (25 mL x 3). The combined organic layer was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using ethyl acetate/petroleum ether (1/3, v/v) as eluent to give a yellow solid 33, yield 52%, mp = 159.2–161.3 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 9.02 (s, 1H), 7.78 (s, 1H), 7.59 (s, 1H), 7.18 (s, 1H), 7.07 (s, 1H), 6.11 (s, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.80 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4, 152.6, 152.4, 148.9, 148.1, 144.9, 138.1, 131.9, 128.9, 119.0, 114.9, 109.6, 109.2, 108.3, 107.0, 103.0, 102.8, 101.7, 55.9, 55.9, 42.4. HRMS (ESI) m/z: 380.1125 [M + H]⁺, calcd for C₂₁H₁₈NO₆ 380.1129.

General procedure for the synthesis of compounds 12–23 and 34–45.

Under nitrogen gas, to the solution of 11 (for 12-23) or 33 (for 34-45, 1 mmol) in dry DMF (3 mL), the corresponding chloride material (1.5 mmol), K₂CO₃ (3 mmol) and KI (0.1 mmol) were added sequentially. The reaction mixture was stirred at 80 °C for 5 h. After completion of the reaction, H₂O (10 mL) and ethyl acetate (20 mL) were added. The organic layer was washed with saturated NaCl solution (20 mL x 3), and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give the target compounds.

2,3-Dimethoxy-12-methyl-6-(2-(methylamino)ethoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (12)

White solid, yield 68%, mp = 140.2–140.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.80 (s, 1H), 7.97 (s, 1H), 7.84 (s, 1H), 7.79 (s, 1H), 7.75 (s, 1H), 7.67 (s, 1H), 6.22 (s, 2H), 4.60 (t, J= 6.0 Hz, 2H), 4.07 (s, 3H), 3.92 (s, 3H), 3.83 (s, 3H), 3.54 (t, J= 6.0 Hz, 2H), 2.75 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 162.7, 153.3, 149.5, 147.3, 147.2, 130.3, 128.4, 122.9, 121.6, 118.6, 115.9, 108.1, 104.4, 102.7, 101.9, 99.9, 98.9, 64.3, 56.5, 55.7, 47.6, 40.9, 33.0. HRMS (ESI) m/z: 437.1732 [M + H]⁺, calcd for C₂₄H₂₅N₂O₆ 437.1713.

2,3-Dimethoxy-12-methyl-6-(3-(methylamino)propoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (13)

White solid, yield 40%, mp = 144.2–145.7 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.94 (s, 1H), 7.65 (s, 1H), 7.60 (s, 1H), 7.49 (s, 1H), 7.30 (s, 1H), 6.11 (s, 2H), 4.33 (t, J = 6.0 Hz, 2H), 4.13 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 2.69 (t, J = 6.0 Hz, 2H), 2.38 (s, 6H), 2.21–2.17 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.3, 153.5, 150.8, 149.8, 147.4, 147.3, 130.3, 129.1, 123.8, 122.3, 119.3, 116.9, 108.7, 103.0, 102.7, 101.7, 99.7, 97.6, 66.7, 56.6, 56.3, 56.2, 45.1, 41.4, 27.1. HRMS (ESI) m/z: 451.1781 [M + H]⁺, calcd for C₂₅H₂₇N₂O₆ 451.1791.

6-(2-(Dimethylamino)ethoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (14)

White solid, yield 60%, mp = 139.2–139.7 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.94 (s, 1H), 7.67 (s, 1H), 7.60 (s, 1H), 7.48 (s, 1H), 7.33 (s, 1H), 6.11 (s, 2H), 4.39 (t, J = 6.0 Hz, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.96 (s, 3H), 2.96 (t, J = 6.0 Hz, 2H), 2.46 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 153.5, 150.8, 149.8, 147.5, 147.5, 130.6, 129.0, 124.0, 122.4, 119.5, 116.7, 108.9, 102.9, 102.7, 101.7, 100.0, 97.9, 67.4, 58.5, 56.3, 46.3, 41.4. HRMS (ESI) m/z: 451.1913 [M + H]⁺, calcd for C₂₅H₂₇N₂O₆ 451.1864.

6-(3-(Dimethylamino)propoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (15)

White solid, yield 40%, mp = 144.2–145.7 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.94 (s, 1H), 7.65 (s, 1H), 7.60 (s, 1H), 7.49 (s, 1H), 7.30 (s, 1H), 6.11 (s, 2H), 4.33 (t, J = 6.0 Hz, 2H), 4.13 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 2.69 (t, J = 6.0 Hz, 2H), 2.38 (s, 6H), 2.21–2.17 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.3, 153.5, 150.8, 149.8, 147.4, 147.4, 130.3, 129.1, 123.8, 122.3, 119.3, 116.9, 108.7, 103.0, 102.7, 101.7, 99.7, 97.6, 66.7, 56.6, 56.3, 56.2, 45.1, 41.4, 27.1. HRMS (ESI) m/z: 465.2070 [M + H]⁺, calcd for C₂₆H₂₉N₂O₆ 465.2020.

2,3-Dimethoxy-12-methyl-6-(2-(piperidin-1-yl)ethoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (16)

White solid, yield 41%, mp = 175.2–177.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.66 (s, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 7.33 (s, 1H), 6.11 (s, 2H), 4.45 (t, J = 6.0 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 3.02 (t, J = 6.0 Hz, 2H), 2.72–2.64 (m, 4H), 1.72–1.66 (m, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 164.2, 153.5, 150.7, 149.8, 147.5, 147.4, 130.7, 128.9, 123.9, 122.5, 119.5, 116.8, 108.9, 102.9, 102.8, 101.7, 99.9, 98.1, 67.1, 58.2, 56.5, 56.4, 55.3, 41.4, 25.9, 24.1. HRMS (ESI) m/z: 491.2211 [M + H]⁺, calcd for C₂₈H₃₁N₂O₆ 491.2177.

2,3-Dimethoxy-12-methyl-6-(3-(piperidin-1-yl)propoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (17)

White solid, yield 40%, mp = 153.2–155.1°C. ¹H NMR (400 MHz, CDCl₃) δ 7.95 (s, 1H), 7.67 (s, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 7.29 (s, 1H), 6.11 (s, 2H), 4.31 (t, J = 6.0 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 2.67–2.62 (m, 2H), 2.53–2.43 (m, 4H), 2.23–2.14 (m, 2H), 1.60–1.42 (m, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 164.1, 153.4, 150.9, 149.7, 147.4, 147.4, 130.4, 128.9, 124.0, 122.4, 119.5, 116.7, 108.9, 102.9, 102.7, 101.6, 99.9, 97.7, 67.3, 56.4, 56.3, 54.8, 41.4, 27.0, 26.0, 24.4. HRMS (ESI) m/z: 505.2393 [M + H]⁺, calcd for C₂₉H₃₃N₂O₆ 505.2333.

2,3-Dimethoxy-12-methyl-6-(2-(4-methylpiperazin-1-yl)ethoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (18)

White solid, yield 50%, mp = 152.7–153.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.66 (s, 1H), 7.61 (s, 1H), 7.47 (s, 1H), 7.30 (s, 1H), 6.11 (s, 2H), 4.41 (t, J = 6.0 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 3.03 (t, J = 6.0 Hz, 2H), 2.81–2.66 (m, 4H), 2.60–2.45 (m, 4H), 2.31 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.1, 153.5, 150.7, 149.7, 147.5, 147.5, 130.7, 128.9, 124.0, 122.4, 119.5, 116.7, 108.9, 102.8, 102.7, 101.7, 99.9, 98.0, 67.2, 57.4, 56.3, 55.2, 53.8, 46.1, 41.4. HRMS (ESI) m/z: 506.2297 [M + H]⁺, calcd for C₂₈H₃₂N₃O₆ 506.2286.

2,3-Dimethoxy-12-methyl-6-(3-(4-methylpiperazin-1-yl)propoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (19)

White solid, yield 39%, mp = 144.3–145.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.66 (s, 1H), 7.60 (s, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 6.11 (s, 2H), 4.31 (t, J = 6.0 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 2.69 (t, J = 6.0 Hz, 2H), 2.66–2.54 (m, 4H), 2.55–2.41 (m, 4H), 2.31 (s, 3H), 2.21–2.14 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 164.1, 153.4, 150.8, 149.7, 147.4, 147.4, 130.4, 128.9, 123.9, 122.3, 119.6, 116.6, 108.9, 102.9, 102.7, 101.7, 99.9, 97.7, 67.1, 56.3, 55.5, 55.0, 53.2, 46.0, 41.4, 27.0. HRMS (ESI) m/z: 520.2482 [M + H]⁺, calcd for C₂₉H₃₄N₃O₆ 520.2442.

6-(2-(1H-Imidazol-1-yl)ethoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (20)

White solid, yield 41%, mp = 184.2–185.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.93 (s, 1H), 7.73 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H), 7.39 (s, 1H), 7.18 (s, 1H), 7.16–7.12 (m, 2H), 6.12 (s, 2H), 4.57–4.49 (m, 4H), 4.11 (s, 3H), 4.05 (s, 3H), 3.94 (s, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 164.1, 153.5, 150.0, 149.9, 147.7, 147.7, 137.7, 131.3, 129.9, 128.7, 123.7, 122.5, 119.6, 116.5, 108.9, 102.9, 102.8, 101.9, 99.4, 98.7, 68.7, 56.4, 56.4, 46.8, 41.4. HRMS (ESI) m/z: 474.1696 [M + H]⁺, calcd for C₂₆H₂₄N₃O₆ 474.1660.

6-(3-(1H-Imidazol-1-yl)propoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (21)

White solid, yield 67%, mp = 159.2–160.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.63 (s, 2H), 7.53 (s, 1H), 7.41 (s, 1H), 7.21 (s, 1H), 7.08 (s, 1H), 6.97 (s, 1H), 6.13 (s, 2H), 4.36 (t, J = 6.4 Hz, 2H), 4.20 (t, J = 5.1 Hz, 2H), 4.10 (s, 3H), 4.06 (s, 3H), 3.96 (s, 3H), 2.48–2.39 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 164.1, 153.5, 150.2, 149.8, 147.6, 147.6, 137.5, 130.8, 129.8, 128.8, 123.6, 122.5, 119.6, 119.2, 116.6, 108.9, 102.9, 102.8, 101.8, 99.4, 97.8, 64.5, 56.4, 43.7, 41.5, 30.9. HRMS (ESI) m/z: 488.1866 [M + H]⁺, calcd for C₂₇H₂₆N₃O₆ 488.1816.

6-(2-((2-(Dimethylamino)ethyl)amino)ethoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (22)

White solid, yield 41%, mp = 227.6–229.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.93 (s, 1H), 7.65 (s, 1H), 7.59 (s, 1H), 7.46 (s, 1H), 7.31 (s, 1H), 6.10 (s, 2H), 4.40–4.34 (m, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.94 (s, 3H), 3.25 −3.19 (m, 2H), 2.90–2.84 (m, 2H), 2.54–2.48 (m, 2H), 2.28 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 164.1, 153.5, 150.6, 149.7, 147.4, 130.6, 128.9, 123.9, 122.4, 119.5, 116.6, 108.8, 102.8, 102.8, 101.7, 99.8, 98.1, 68.1, 58.5, 56.3, 48.8, 46.9, 45.3, 41.4. HRMS (ESI) m/z: 494.2342 [M + H]⁺, calcd for C₂₇H₃₂N₃O₆ 494.2286.

Ethyl 2-((2,3-dimethoxy-12-methyl-13-oxo-12,13-dihydro-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-6-yl)oxy)acetate (23)

White solid, yield 52%, mp = 151.2–152.1 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.93 (s, 1H), 7.75 (s, 1H), 7.61 (s, 1H), 7.42 (s, 1H), 7.26 (s, 1H), 6.12 (s, 2H), 4.90 (s, 2H), 4.33 (q, J = 7.0 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.95 (s, 3H), 1.33 (t, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 169.0, 164.1, 153.5, 150.0, 149.9, 147.7, 131.4, 128.7, 124.0, 122.6, 119.6, 116.4, 108.9, 102.7, 101.8, 100.1, 98.9, 66.9, 61.7, 56.4, 56.3, 41.4, 14.4. HRMS (ESI) m/z: 466.1522 [M + H]⁺, calcd for C₂₅H₂₄NO₈ 466.1496.

5-(2-(Dimethylamino)ethoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (34)

Yellow solid, yield 49%, mp = 190.8–191.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.88 (s, 1H), 7.99 (s, 1H), 7.47 (s, 1H), 7.06 (s, 1H), 7.01 (s, 1H), 6.07 (s, 2H), 4.35 (t, J = 6.0 Hz, 2H), 4.10 (s, 3H), 4.07 (s, 3H), 3.92 (s, 3H), 2.97 (t, J = 6.0 Hz, 2H), 2.37 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 164.3, 153.8, 152.5, 148.9, 148.0, 145.3, 138.1, 131.1, 128.6, 119.4, 116.2, 110.5, 109.4, 108.3, 103.6, 103.4, 102.8, 101.3, 66.7, 58.4, 56.1, 45.7, 42.2. HRMS (ESI) m/z: 451.1859 [M + H]⁺, calcd for C₂₅H₂₇N₂O₆ 451.1864.

5-(3-(Dimethylamino)propoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (35)

Yellow solid, yield 51%, mp = 148.9–150.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.85 (s, 1H), 7.99 (s, 1H), 7.47 (s, 1H), 7.05 (s, 1H), 7.02 (s, 1H), 6.06 (s, 2H), 4.31 (t, J = 6.7 Hz, 2H), 4.06 (s, 3H), 4.05 (s, 3H), 3.91 (s, 3H), 2.59 (t, J = 7.2 Hz, 2H), 2.37–2.15 (m, 8H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 153.8, 152.4, 148.9, 148.0, 145.3, 138.1, 131.3, 128.6, 119.6, 116.1, 110.4, 109.2, 108.4, 103.7, 103.4, 102.8, 101.3, 67.4, 56.4, 56.1, 45.4, 42.2, 29.7, 27.8. HRMS (ESI) m/z: 465.2020 [M + H]⁺, calcd for C₂₆H₂₉N₂O₆ 465.2020.

5-(2-(Diethylamino)ethoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (36)

Yellow solid, yield 67%, mp = 155.8–157.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.73 (s, 1H), 7.91 (s, 1H), 7.40 (s, 1H), 7.00 (s, 1H), 6.96 (s, 1H), 6.00 (s, 2H), 4.33 (t, J = 6.7 Hz, 2H), 4.00 (s, 3H), 3.99 (s, 3H), 3.85 (s, 3H), 3.10 (t, J = 5.8 Hz, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.05 (t, J = 7.1 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 152.4, 148.9, 148.1, 145.4, 138.2, 131.2, 128.5, 119.6, 116.2, 110.4, 109.2, 108.4, 103.8, 103.5, 102.8, 101.4, 56.1, 52.2, 47.7, 42.2, 11.6. HRMS (ESI) m/z: 479.2170 [M + H]⁺, calcd for C₂₈H₃₁N₂O₆ 479.2176.

5-(3-(Diethylamino)propoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (37)

Yellow solid, yield 71%, mp = 237.6–239.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (s, 1H), 7.80 (s, 1H), 7.64 (s, 1H), 7.31 (s, 1H), 7.27 (s, 1H), 4.33 (t, J = 6.6 Hz, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.80 (s, 3H), 2.52–2.50 (m, 6H), 2.36–2.21 (m, 2H), 1.12 (t, J = 7.0 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.1, 153.4, 152.7, 149.0, 148.3, 145.5, 138.1, 131.6, 128.2, 119.2, 115.9, 109.5, 108.5, 104.3, 103.7, 103.2, 101.9, 56.1, 56.0, 47.0, 42.2. HRMS (ESI) m/z: 493.2324 [M + H]⁺, calcd for C₂₈H₃₃N₂O₆ 493.2333.

2,3-Dimethoxy-12-methyl-5-(2-(pyrrolidin-1-yl)ethoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (38)

Yellow solid, yield 66%, mp = 150.1–150.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.87 (s, 1H), 7.97 (s, 1H), 7.44 (s, 1H), 7.04 (s, 1H), 6.99 (s, 1H), 6.06 (s, 2H), 4.38 (t, J = 6.2 Hz, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.90 (s, 3H), 3.14 (t, J = 6.2 Hz, 2H), 2.66 (t, J = 6.1 Hz, 4H), 1.82 (t, J = 6.1 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 153.8, 152.5, 148.8, 148.0, 145.3, 138.1, 131.2, 128.6, 119.4, 116.1, 109.4, 108.3, 103.6, 102.8, 101.3, 67.7, 56.1, 56.1, 55.2, 54.5, 42.2, 23.5. HRMS (ESI) m/z: 477.1994 [M + H]⁺, calcd for C₂₇H₂₉N₂O₆ 477.2020.

2,3-Dimethoxy-12-methyl-5-(3-(pyrrolidin-1-yl)propoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (39)

Yellow solid, yield 78%, mp = 173.8–175.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.85 (s, 1H), 8.00 (s, 1H), 7.48 (s, 1H), 7.07 (s, 1H), 7.03 (s, 1H), 6.08 (s, 2H), 4.35 (t, J = 7.2 Hz, 2H), 4.08 (s, 3H), 4.06 (s, 3H), 3.93 (s, 3H), 2.81 (t, J = 7.2 Hz, 2H), 2.64 (t, J = 6.0 Hz, 4H), 2.32 (p, J = 7.0 Hz, 2H), 1.86 (t, J = 6.0 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 164.3, 153.9, 152.4, 148.8, 148.0, 145.2, 138.1, 131.3, 128.6, 119.5, 116.1, 110.4, 109.2, 108.4, 103.6, 103.4, 102.8, 101.3, 67.6, 56.1, 54.3, 53.2, 42.2, 29.1, 23.5. HRMS (ESI) m/z: 491.2163 [M + H]⁺, calcd for C₂₈H₃₁N₂O₆ 491.2177.

2,3-Dimethoxy-12-methyl-5-(2-morpholinoethoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (40)

Yellow solid, yield 65%, mp = 182.7–183.5 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.85 (s, 1H), 7.98 (s, 1H), 7.47 (s, 1H), 7.06 (s, 1H), 7.01 (s, 1H), 6.07 (s, 2H), 4.39 (t, J = 5.6 Hz, 2H), 4.08 (s, 3H), 4.06 (s, 3H), 3.91 (s, 3H), 3.73 (t, J = 4.7 Hz, 4H), 3.03 (t, J = 5.6 Hz, 2H), 2.59 (t, J = 4.7 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 153.7, 152.5, 148.9, 148.1, 145.4, 138.2, 131.2, 128.5, 119.6, 116.3, 110.5, 109.4, 108.4, 103.8, 103.4, 102.8, 101.4, 66.8, 66.2, 57.8, 56.3, 56.1, 54.0, 42.2. HRMS (ESI) m/z: 493.1962 [M + H]⁺, calcd for C₂₇H₂₉N₂O₇ 493.1969.

2,3-Dimethoxy-12-methyl-5-(3-morpholinopropoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (41)

Yellow solid, yield 86%, mp = 214.4–215.7 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.85 (s, 1H), 7.99 (s, 1H), 7.47 (s, 1H), 7.07 (s, 1H), 7.01 (s, 1H), 6.07 (s, 2H), 4.32 (t, J = 6.5 Hz, 2H), 4.07 (s, 3H), 4.05 (s, 3H), 3.92 (s, 3H), 3.73 (t, J = 4.7 Hz, 4H), 2.64 (t, J = 7.1 Hz, 2H), 2.47 (t, J = 4.7 Hz, 4H), 2.23 (p, J = 6.7 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 164.3, 153.9, 152.4, 148.8, 148.0, 145.3, 139.3, 138.1, 131.3, 119.6, 116.1, 110.4, 109.1, 108.4, 103.6, 103.4, 102.8, 101.3, 67.3, 67.0, 56.1, 56.1, 55.6, 53.8, 42.2, 26.9. HRMS (ESI) m/z: 507.2123 [M + H]⁺, calcd for C₂₈H₃₁N₂O₇ 507.2126.

5-(3-Hydroxypropoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (42)

Yellow solid, yield 64%, mp = 232.1–233.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.82 (s, 1H), 7.98 (s, 1H), 7.46 (s, 1H), 7.05 (s, 1H), 7.02 (s, 1H), 6.07 (s, 2H), 4.41 (t, J = 6.1 Hz, 2H), 4.19–3.97 (m, 8H), 3.91 (s, 3H), 2.38–2.25 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.2, 153.8, 152.6, 149.0, 148.2, 145.4, 138.1, 131.8, 128.3, 119.2, 115.7, 109.4, 108.5, 104.1, 103.7, 103.1, 101.9, 66.5, 58.1, 55.9, 32.8. HRMS (ESI) m/z: 438.1551 [M + H]⁺, calcd for C₂₄H₂₄NO₇ 438.1547.

5-(3-((2-Hydroxyethyl)amino)propoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-13(12H)-one (43)

Yellow solid, yield 59%, mp = 124.3– 125.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.82 (s, 1H), 7.97 (s, 1H), 7.44 (s, 1H), 7.03 (s, 1H), 6.97 (s, 1H), 6.06 (s, 2H), 4.32 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 4.04 (s, 3H), 3.90 (s, 3H), 3.67 (t, J = 4.0 Hz, 2H), 2.95 (t, J = 6.8 Hz, 2H), 2.81 (t, J = 5.1 Hz, 2H), 2.22 (q, J = 6.6 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 153.8, 152.4, 148.8, 148.0, 145.2, 138.0, 131.2, 128.6, 119.5, 116.1, 110.3, 109.1, 108.4, 103.5, 103.4, 102.8, 101.3, 67.3, 61.1, 56.1, 56.0, 51.3, 46.5, 42.2, 30.3, 29.7. HRMS (ESI) m/z: 481.1952 [M + H]⁺, calcd for C₂₆H₂₉N₂O₇ 481.1969.

5-(3-(1H-imidazol-1-yl)propoxy)-2,3-dimethoxy-12-methyl-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (44)

Yellow solid, yield 62%, mp = 202.3–204.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.73 (s, 1H), 8.00 (s, 1H), 7.46 (s, 2H), 7.02 (s, 2H), 6.90 (s, 2H), 6.07 (s, 2H), 4.29 (t, J = 6.6 Hz, 2H), 4.19 (t, J = 6.0 Hz, 2H), 4.07 (s, 3H), 4.01 (s, 3H), 3.92 (s, 3H), 2.50 (p, J = 6.3 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.1, 153.2, 152.4, 149.0, 148.2, 145.5, 138.3, 131.1, 128.3, 119.7, 116.4, 110.2, 108.8, 108.7, 103.9, 103.5, 102.8, 101.4, 65.3, 56.1, 56.0, 42.2, 30.9. HRMS (ESI) m/z: 488.1813 [M + H]⁺, calcd for C₂₇H₂₆N₃O₆ 488.1816.

2,3-Dimethoxy-12-methyl-5-(3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)propoxy)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (45)

Yellow solid, yield 65%, mp = 176.1–177.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.80 (s, 1H), 7.98 (s, 1H), 7.47 (s, 1H), 7.36 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 6.3 Hz, 2H), 7.02 (d, J = 13.2 Hz, 2H), 6.07 (s, 2H), 4.44 (t, J = 6.2 Hz, 2H), 4.37 (t, J = 6.3 Hz, 2H), 4.05 (s, 6H), 3.91 (s, 3H), 3.61 (s, 4H), 3.15–3.13 (m, 4H), 2.43 (p, J = 6.3 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 155.1, 153.7, 152.4, 151.2, 148.9, 148.1, 145.4, 138.2, 131.2, 129.7, 128.5, 119.6, 119.4, 116.7 (q, J_C-F = 3.6 Hz), 116.2, 112.9 (q, J_C-F = 3.8 Hz), 110.3, 108.9, 108.5, 103.6, 103.4, 102.8, 101.4, 66.2, 62.7, 56.1, 56.0, 48.8, 42.2, 29.7, 29.5. HRMS (ESI) m/z: 650.2472 [M + H]⁺, calcd for C₃₅H₃₄F₃N₃O₆ 650.2472.

TOP1-Mediated Cleavage Assay.

DNA cleavage assays were performed according to the previously reported method^[82]. A 3′-[³²P]-labeled 117-bp DNA oligonucleotide substrate (2 nM) was incubated with recombinant TOP1 in 10 µL of reaction buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, and 15 mg/mL BSA) at 25 °C for 20 min in the presence of various concentrations of test compounds. The reactions were terminated by adding SDS (0.5% final concentration), followed by the addition of two volumes of loading dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue). Aliquots of each reaction mixture were subjected to 20% denaturing PAGE. Gels were dried and visualized by using a phosphoimager and Image Quant software (Molecular Dynamics). Cleavage sites are numbered to reflect actual sites on the 117-bp oligonucleotide.

TDP1 Inhibition Assay

TDP1 inhibition assays were conducted according to the reported method^[43]. Briefly, a 5′-Cy5-labeled single-stranded DNA oligonucleotide substrate containing a 3′-phosphotyrosine (Cy5–14Y, 5´-GATCTAAAAGACTTpY-3´) was incubated at 1 nM with 40 pM recombinant human TDP1 in the absence or presence of inhibitor for 15 min at room temperature in buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween-20. Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA] and analyzed through a 20% denaturing PAGE. Gel images were scanned using a Typhoon FLA 9500 scanner (GE Healthcare), and densitometry analyses were performed using the ImageQuant software (GE Healthcare).

Cell Culture and MTT Assay

HCT116 cells were cultured on RPMI1640 medium, MCF-7 and MCF-7/TDP1 (overexpression TDP1 in MCF-7 cells) cells were cultured on DMEM medium at 37 °C in a humidified atmosphere with 5% CO₂. All cells to be tested in the following assays had a passage number of 3–6. For the cytotoxicity measurements, the cancer cells were treated with the compounds (pre-dissolved in DMSO) at a five-dose assay ranging from 0.01 to 100 μM (0.001–10 μM for topotecan) concentration. After incubation for 72 h at 37 °C, MTT solution (20 μL, 2.5 mg/mL) in PBS (PBS without MTT as the blank) was fed to each well of the culture plate (containing 100 mL medium). After 4 h incubation, the formazan crystal formed in the well was dissolved with 100 µL of DMSO for optical density reading at 570 nm. The GI₅₀ value was calculated by nonlinear regression analysis (GraphPad Prism).

For the drug combination experiments, human breast cancer MCF-7 or MCF-7/TDP1 cells were incubated with topotecan and the tested compounds for 96 h at 37 °C, and then measured by MTT assay.

Immunodetection of Cellular TOP1cc and TDP1cc

The ICE assays for cellular enzyme-DNA adduct was performed according to the reported method^[46]. Briefly, mid log phase MCF-7 cells were incubated with drugs at the indicated concentration for 1 h, and then the cells were lysed with DNAzol reagent (1 mL) at 25 °C for 30 min. Ethanol (0.5 mL, 100%) was subsequently added and mixed with the lysate. The solution was incubated at −20 °C overnight. The genomic DNA was collected by centrifugation (12000 rpm) at 25 °C for 10 min and washed with 75% ethanol. The precipitated DNA was dissolved in NaOH (8 mM, 0.2 mL). The pH value was adjusted to 7.2 by adding HEPES (1 M). After centrifugation, DNA (2 μg) in supernatant was dissolved in 30 μL of NaH₂PO₄ buffer (25 mM, pH 6.5) and then loaded onto nitrocellulose membranes. Membranes were incubated with rabbit monoclonal to human TOP1 (Abcam, 1:1000) or TDP1 (Abcam, 1:1000) at 4 °C overnight, and then incubated with the appropriate HRP conjugated secondary antibodies (Cell Signaling Technology, 1:3000) at room temperature for 1 h. Reactive dots were detected using Immobilon Western Chemiluminescent HRP substrate (Millipore).

Immunofluorescence Assay

Immunofluorescence staining and confocal microscopy were performed according to the reported method^[46].Briefly, after being incubated with the tested compounds for 5 h at 37 °C, MCF-7 cells were fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.5% Triton X-100/PBS at 37 °C for 30 min, and finally blocked with 5% goat serum/PBS at 37 °C for 3 h. The protocol was followed with the γH2AX antibody (no.9718, Cell Signaling Technology) at 4 °C overnight, and the digital images were recorded using an FV3000 microscope (Olympus, Japan) and analyzed with FV31S-SW software.

Flow Cytometry

MCF-7 cells (2×10⁵) were planted in culture dish and treated with 43 at the indicated concentration for 24 h. The apoptosis detection was applied using the FITC Annexin V/PI apoptosis Detection Kit (KeyGEN). The collected cells were resuspended in 500 μL binding buffer and added with 5 μL of FITC Annexin V and PI dyes. Slowly blending the samples and incubating at 25 °C for 15 min away from light. Finally, the fluorescence-positive cells were quantified by flow cytometry (EPICS XL).

Molecular modeling

The TOP1 crystal structure (PDB 1K4T) was obtained and tailored by removing the ligand and water molecules^[46], while inspected for errors and missing residues, hydrogens were added, the optimized structure of TOP1 was used for molecular modeling. The ligand centroid coordination of the ternary complex as the center of the binding pocket for molecular modeling. Compounds were constructed using ChemDraw and saved in SDF file formats, which were optimized using Discovery Studio software. Compound 38 was docked 30 times, starting each time from different orientations and the default automatic genetic algorithm parameter settings were used. All torsion angles of compounds 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.