Discovery, Synthesis, and Evaluation of Oxynitidine Derivatives as Dual Inhibitors of DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1), and Potential Antitumor Agents

Xiao-Ru Zhang; Hao-Wen Wang; Wen-Lin Tang; Yu Zhang; Hui Yang; De-Xuan Hu; Azhar Ravji; Christophe Marchand; Evgeny Kiselev; Kwabena Ofori-Atta; Keli Agama; Yves Pommier; Lin-Kun An

doi:10.1021/acs.jmedchem.8b00639

. Author manuscript; available in PMC: 2021 Apr 30.

Published in final edited form as: J Med Chem. 2018 Oct 31;61(22):9908–9930. doi: 10.1021/acs.jmedchem.8b00639

Discovery, Synthesis, and Evaluation of Oxynitidine Derivatives as Dual Inhibitors of DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1), and Potential Antitumor Agents

Xiao-Ru Zhang ^†,^#, Hao-Wen Wang ^†,^#, Wen-Lin Tang ^†, Yu Zhang ^†, Hui Yang ^†, De-Xuan Hu ^†, Azhar Ravji ^‡, Christophe Marchand ^‡, Evgeny Kiselev ^‡, Kwabena Ofori-Atta ^‡, Keli Agama ^‡, Yves Pommier ^‡,^*, Lin-Kun An ^†,^*

PMCID: PMC8085681 NIHMSID: NIHMS1684587 PMID: 30336023

Abstract

Tyrosyl–DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)–DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC₅₀ value of 7 μM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future efforts for the discovery of natural product-based TOP1 and TDP1 inhibitors.

Graphical Abstract

graphic file with name nihms-1684587-f0011.jpg

INTRODUCTION

Topoisomerase IB (TOP1) is an essential nuclear enzyme controlling the topology of DNA in many cellular metabolic processes.^1–4 To perform its functions, TOP1 cleaves one strand of DNA by nucleophilic attack of its active site tyrosine on the DNA phosphodiester backbone (Figure 1A), resulting in nicks in the target DNA as part of TOP1–DNA covalent cleavage complexes (TOP1cc, Figure 1B). TOP1cc are transient intermediates under normal physiological circumstances and reverse via 5′-OH attack of the nicked strand on the TOP1–DNA phosphotyrosyl group, thus restoring the intact DNA and releasing TOP1.^4,5 TOP1 inhibitors, such as camptothecin (1, Figure 2S, Supporting Information), bind at the interface of TOP1cc by intercalating between the base pairs at the site of cleavage and forming key hydrogen bonds with TOP1.⁶ This stabilizes TOP1cc and prevents further religation of the broken DNA. When replication and transcription machineries encounter the trapped TOP1cc, DNA damage is generated, triggering cell death.^4,5 To date, four well-known camptothecin TOP1 inhibitors (Figure 2S, Supporting Information) have been approved for clinical treatment of cancers, including irinotecan (2) and topotecan (3), approved by the FDA,^7–9 10-hydroxycamptothecin (4, HCPT, in China), and belotecan (5, in South Korea).^5,10,11 Therefore, TOP1 is a validated target for the discovery of antitumor agents.^5,12–14

Figure 1. — Schematic for TOP1 and TDP1 mediated biochemical reactions. (A) TOP1 binds to DNA and cleaves one strand of a dsDNA by forming a reversible covalent 3′-phosphotyrosyl bond. (B) The resulting TOP1cc may be trapped and stabilized by TOP1 inhibitors (black stick) such as camptothecin and indenoisoquinoline analogues, resulting in replication/transcription-mediated DNA damage and cell death. (C) TOP1cc could be proteolyzed to expose the DNA-3′-phosphotyrosyl bond, which can be cleaved by TDP1 to leave the 3′-phosphate end DNA (D). (E) The DNA breaks may be repaired sequentially by the XRCC-1 dependent base excision repair (BER) pathway.

The DNA damage generated from TOP1cc can be repaired through several pathways, including homologous recombination (BRCA1, BRCA2, CtIP, Mre11, Rad52), cell cycle checkpoint signaling, XPF/ERCC1, Mre11, CtIP, and tyrosyl–DNA phosphodiesterase-dependent pathways, etc.^15,16 Tyrosyl–DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D superfamily.¹⁷ It catalytically hydrolyzes the 3′-phosphotyrosyl bond (Figure 1C) associated with TOP1cc. TDP1 generates DNA breaks with 5′-hydroxyl and 3′-phosphate ends (Figure 1D) for further DNA repair by the XRCC-1-dependent pathway, including polynucleotide kinase phosphatase (PNKP), poly(ADP-ribose) polymerase 1 (PARP1), DNA ligase III (Lig3), and DNA polymerase β (Polβ), finally resulting in resealed DNA (Figure 1E).^18,19 In this pathway, TDP1-catalyzed hydrolysis of the 3′-phosphotyrosyl bond is a key step for initiating the repair of TOP1-mediated DNA damage. In addition, TDP1 catalyzes hydrolysis of 3′-blocking lesions generated by DNA oxidation and alkylation,^20–22 as well as 5′-phosphotyrosyl bond involved in topoisomerase II-mediated DNA damage, implying a broader role of TDP1 in the maintenance of genomic stability.^18,20,23,24 The physiological importance of TDP1 is further emphasized by the discovery of a rare neurodegenerative disease called spinocerebellar ataxia with axonal neuropathy (SCAN1), in which TDP1 bears a H493R mutation in its active site.²⁵

Because TDP1-deficient cells are hypersensitive to TOP1 inhibitors, it has been suggested that overexpression of TDP1 will confer resistance to TOP1-mediated DNA damage, and therefore TDP1 inhibitors would have the ability to sensitize cancer cells to TOP1 inhibitors.¹⁸ Indeed, cells overexpressing TDP1 show resistance to camptothecin.^26,27 Conversely, TDP1 knockout mice, human cells deficient in TDP1 and the SCAN1 mutation all show hypersensitivity to camptothecin,^20,28–33 and some TDP1 inhibitors show synergistic activity to camptothecin derivatives.^34–36 Together, these observations imply that TDP1 is a rational target for the development of novel antitumor agent.^18,37–39

Because of the unique physiological functions of TOP1 and TDP1, the discovery of their inhibitors has attracted attention. As TOP1 inhibitors, camptothecin derivatives have been well investigated, and four derivatives (2–5, Figure 2S, Supporting Information) are clinically available, as well as several tumor-targeted derivatives currently in clinical trials. Despite their effectiveness in solid tumors, camptothecin inhibitors suffer from well-established limitations, including chemical instability under physiological condition, poor solubility, bone marrow dose-limiting toxicity, severe gastrointestinal toxicity,⁴⁰ and drug efflux-mediated resistance.¹² Because of these shortcomings, much attention is directed toward the discovery of noncamptothecin inhibitors. To date, noncamptothecin TOP1 inhibitors have been reported, and three chemotypes, including indolocarbazoles, dibenzonaphthyridinone, and indenoisoquinolines, have been developed. The indenoisoquinolines LMP744 (48, Figure 2S, Supporting Information), LMP400 (Indotecan), and LMP776 (Indimitecan) are in clinical trials.^{12,13,41–43}

Compared to TOP1 inhibitors, the discovery of TDP1 inhibitors was initiated relatively recently, and there is a limited number of chemotypes reported as TDP1 inhibitors, which leaves much room for development.^37,39,44 Among TDP1 inhibitors, sodium orthovanadate (7, Figure 2) was the first reported TDP1 inhibitor mimicking the DNA substrate in the transition state but with inhibition only at millimolar concentration.^45,46 Recently, a few more potent inhibitors with IC₅₀ values at low micromolar or submicromolar concentrations have been reported, such as aminoglycoside neomycin,⁴⁷ methyl-3,4-dephostatin,⁴⁸ arylidenethioxothiazolicinone,⁴⁹ thioxopyrimidinedione (CD00509),⁵⁰ and benzopentathiepine.⁵¹ A few derivatives of the clinically developed TOP1 inhibitors, the indenoisoquinolines have been reported to also inhibit TDP1.^52–56 The indenoisoquinoline 10 (Figure 2) showed consistent TDP1 inhibition with IC₅₀ value of 1.52 μM.⁵² Recently, three chemotypes (Figure 2), 7-hydroxycoumarin derivatives (11), usnic acid enamines (8), and aminoadamantanes (9), were reported to inhibit TDP1 and synergize with camptothecin derivatives.^34–36

Figure 2. — Structures of representative reported TDP1 inhibitors.

RESULTS AND DISCUSSION

Hit Discovery for TOP1 Inhibitor.

Natural products are an important source for medicinal chemistry and drug development.^57,58 To find novel TOP1 inhibitors, we screened our in-house natural product library containing more than 900 natural products using a TOP1-mediated DNA relaxation assay. Several chemotypes were found to inhibit TOP1, including meroterpenoid derivatives and oxynitidine (6, Figure 1S, Supporting Information). The meroterpenoid derivatives showed high TOP1 relaxing inhibition, but did not have the ability to trap and stabilize TOP1cc,⁵⁹ and therefore could be classified as TOP1 catalytic inhibitors.⁶⁰ By contrast, TOP1-mediated DNA cleavage assay indicated that 6 was able to induce the formation of TOP1cc in a dose-dependent manner (Figure 1S, Supporting Information). It is noteworthy that the cleavage sites induced by 6 are different from that induced by camptothecin and similar to the indenoisoquinoline 48 (Figure 2S, Supporting Information). For example, the cleavage sites 17, 35, and 79 can be induced by 6 and 48 but not by camptothecin, implying that this novel TOP1 inhibitor might target the genome at different sites and translate to different cellular effects compared with camptothecin.¹²

It was reported that nitidine can intercalate into double-stranded DNA (dsDNA) and inhibit TOP1.^61–63 Because 6 is the 6-oxo analogue of nitidine, a FRET melting assay using F10T (5′-FAM-dTATAGCTATAHEG-TATAGCTATA-TAMRA-3′) as substrate was conducted to detect the enhanced melting temperature (ΔT_m) and investigate the interaction of 6 with dsDNA. Intercalation of small molecules into dsDNA stabilizes the DNA double-helix structure, resulting in an increased melting temperature by about 5–8 °C.⁶⁴ The FRET melting assay indicated that 6 showed low ΔT_m value (0.4 °C) compared to the intercalator 49 (Figure 2S, Supporting Information) with ΔT_m value of 5.8 °C (Table 1),⁶⁵ implying that 6 seems not be a DNA intercalator.

Table 1.

TOP1 and TDP1 Inhibitions, Enhanced Melting Temperature with dsDNA and Cytotoxicity of the Indicated Compounds

graphic file with name nihms-1684587-t0012.jpg

graphic file with name nihms-1684587-t0013.jpg

Open in a new tab

TOP1 cleavage inhibitory activity of synthesized compounds was semiquantitatively expressed relative to CPT at 1 μM as follows: 0, no inhibition; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity; ++++, equal activity.

TDP1 inhibition was determined by using a fluorescence assay and the percentage inhibition of the compounds at 100 μM concentration was calculated. Every experiment was repeated at least three times independently.

ΔT_m = T_m(DNA + compound) − T_m(DNA). Every experiment was repeated at least twice independently.

GI₅₀ values (means ± SD) were defined as the concentrations of compounds that resulted in 50% cell growth inhibition and obtained from MTT assay. Every experiment was repeated at least three times independently.

“/” mean “ inapplicable”.

“ND” mean ” not determined”.

These results indicate that the oxynitidine scaffold is a novel chemotype for discovery and development of new classes of TOP1 inhibitors.

Chemistry.

To synthesize the benzophenanthridinone derivatives, the synthetic route was designed as shown in Scheme 1 according to the method reported by Korivi et al.⁶⁶ The reaction of 2-bromo-4,5-dimethoxybenzaldehyde with 2-hydroxyethylamine or 3-hydroxypropanylamine gave the imine intermediate 12a or 12b in 100% yield. To avoid oxidation during Swern oxidation at step e (Scheme 1), the hydroxy groups of 12a and 12b were protected with methoxymethyl (MOM) group to give intermediates 13a and 13b, which were directly used without purification to react with 14, prepared through Sonogashira coupling reaction from 5-bromobenzo-[d][1,3]dioxole and 3-butyn-1-ol. Under Ni-based catalysis,⁶⁷ the cyclization reaction of 13a/b with 14 gave the intermediates 15a/b in 45–57% yield for two steps (from 12a/b). The structures of 15a and 15b were confirmed using 1D and 2D NMR spectra and HRMS. Following the Swern oxidation of the hydroxyl group of 15a and 15b,⁶⁸ cyclization reaction under acid conditions gave the alcohol products 17a and 17b with simultaneous deprotection of MOM group.

Following the chlorination of 17a/b with SOCl₂, the target benzophenanthridinone derivatives (19a/b–25a/b) were obtained from the reaction of 18a/b with the corresponding amines.

The dihydrobenzophenanthridine derivatives (26a/b–32a/b) were obtained from the reduction of the corresponding benzophenanthridinone substrates (19a/b–25a/b) with LiAlH₄.

The structures of all the benzophenanthridinone and dihydrobenzophenanthridine derivatives were characterized through IR, NMR and HRMS spectra. The signals of the lactam carbonyl stretching vibration were between 1630 and 1642 cm⁻¹. The structure of dihydrobenzophenanthridine derivative 20b was also confirmed with X-ray single crystal analysis (Figure 3).

Figure 3. — Perspective ORTEP drawing of compound **20b**.

The 6-alkoxy substituted benzophenanthridinone derivatives (39a/b–45a/b) were synthesized as shown in Scheme 2. The isoquinolinone 34 was obtained from the nickel-catalyzed cyclization of 14 and 33, prepared from the reaction of 2-bromo-4,5-dimethoxybenzaldehyde with 4-methoxyphenylmethanamine. Following the Swern oxidation of 34, the key intermediate 36 was prepared from the acid-catalytic cyclization of 35, meanwhile, the p-methoxybenzyl (PMB) group was removed. As shown in Scheme 2, two synthetic pathways were designed for the target products 39a–45a (Scheme 2, steps e and f) and 39b–45b (Scheme 2, steps g and h).

Surprisingly, the reaction of 36 with 1,3-dibromopropane under strong base conditions (NaH) mainly gave the product 38 with 3-bromopropyl group attached to oxygen atom at 6-position in 86% yield (Scheme 2, step g) but not the product with 3-bromopropyl group attached to the nitrogen atom of lactam similar to that reported in many publications.^69–71 The reaction of 38 with amines gave the corresponding benzophenanthridines 39b–45b with 3-alkylamino propoxy substituents at 6-position.

The reaction of 36 with 1,2-dichloroethane gave a mixture of intermediate 46 (Figure 4) and 18a, which are inseparable through silica gel column chromatography. It was proposed that the 2-chloroethyl group might first attach to the oxygen atom at 6-position to give 46. Compound 46 was not stable and would undergo a transformation according to the pathway proposed by Nguyen et al.,⁵² in which 46 undergoes intramolecular cyclization and elimination of chlorine atom to produce an intermediate 47 as shown in Figure 4. Nucleophilic attack of the chloride anion to 47 gave the products 18a or 46, respectively. Therefore, the products 39a–45a with the 2-alkylamino ethoxy group at 6-position were synthesized through the steps e and f of Scheme 2, in which 36 first was chlorinated with POCl₃ to give intermediate 37,⁷² and then reaction of 37 with 2-hydroxy ethylamines under strong base condition gave the target products 39a–45a.

In summary, three groups of oxynitidine derivatives, the benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and their identity and purity assessed through HRMS, 1D and 2D NMR spectra, and HPLC methods.

TOP1 Inhibition.

All synthesized compounds were evaluated for TOP1 inhibition through our TOP1-mediated cleavage assay using a 3′-[³²P]-labeled double-stranded DNA fragment as substrate.⁷³ The compounds were tested at 100, 10, 1, and 0.1 μM concentrations, using camptothecin 1 and indenoisoquinoline 48 as positive controls, and semiquantitatively scored on the basis of visual inspection of the number and intensities of the DNA cleavage bands relative to the TOP1 inhibition of 1 at 1 μM concentration: 0, no inhibitory activity; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity; ++++, equal activity to 1 (Table 1). For the benzophenanthridinone derivatives 19a/b–25a/b, both alkylaminoethyl and alkylaminopropyl substituents attached to the nitrogen atom of lactam increased the TOP1 inhibition, except 23b with 3-morpholinopropyl group attached at 5-position, which showed equal activity to that of parent molecule 6 (+/0, very minor activity less than 10% TOP1 inhibition of 1). Bigger substituents at the 5-position decreased TOP1 inhibitory activity. Most benzophenanthridinone derivatives showed limited TOP1 inhibitory activity of +, except compounds 19a (+++) and 21a (++), with high and moderate TOP1 inhibitory activity, respectively. The compounds 17a and 17b with a hydroxy terminus showed slightly increased TOP1 inhibition of +. Compound 19a was the most potent among the three synthesized chemotypes with TOP1 inhibition of +++. Figure 5A demonstrates that 19a exhibits cleavage sites similar to 48 but not to 1. It was reported that the dibenzonaphthyridinone derivative, ARC-111, a TOP1 inhibitor that was developed in clinical trials,⁴¹ exhibits different cleavage sites from 1 and 48,⁷⁴ implying that, although 19a and ARC-111 are structurally similar, they trap TOP1cc at different DNA sequences.

Figure 5. — (A) Representative gel of TOP1-mediated DNA cleavage assay. Lane 1, DNA alone; lane 2, DNA and TOP1; lane 3, DNA and TOP1 with 1 (1 μM); lane 4, DNA and TOP1 with 48 (1 μM); lanes 5–8, DNA and TOP1 with **19a** at 0.1, 1.0, 10, and 100 μM concentration, respectively. The arrows at left indicate the cleavage site positions. The gel of TOP1-mediated unwinding assay using supercoiled pBR322 DNA (B) or relaxed pBR322 DNA (C) as substrate, respectively. Lane 1, DNA alone; lane 2, DNA and TOP1; lanes 3–5, DNA and TOP1 with the known DNA intercalator, ethidium bromide (EB) at 0.3, 0.6, 1.2 μg/mL, respectively; lanes 6–8, DNA and TOP1 with **19a** at 1, 3, 9 μM, respectively. Rx, relaxed DNA. Sc, supercoiled DNA. (D) Hypothetical binding mode of **19a** in the ternary TOP1–DNA–drug cleavage complex (PDB 1K4T). **19a** was shown as yellow carbon atoms ball and stick representation. All distances were measured from heavy atom to heavy atom.

Most of the dihydrobenzophenanthridine derivatives, such as 26a/b–31a/b and 32a, showed equal or slightly increased TOP1 inhibitory activity (+/0 or + ) compared to the parent 6. Among this series of derivatives, the most potent compound 32b with 3-imidazolylpropyl group at the 5-position showed moderate TOP1cc inhibitory activity (scored + +). Surprisingly, most of benzophenanthridine derivatives did not exhibit TOP1 inhibitory activity except compounds 40a (+) and 43a (+/0), showing weak activity.

Molecular modeling was performed to rationalize the molecular binding mode of the benzophenanthridinone derivatives within the TOP1–DNA complex. A hypothetical binding model was built using in silico docking from the X-ray crystal of the TOP1–DNA–ligand ternary complex (PDB 1K4T).⁷⁵ Compounds were energy minimized and docked into the binding model. As shown in Figure 5D, the benzophenanthridinone scaffold of 19a intercalates in the DNA break made by TOP1 and readily stacks with the +1 and −1 base pairs flanking the DNA cleavage site. The A- and B-ring of 19a stack with the bases of noncleaved strand (C and A), while the C- and D-ring stack with the scissile strand bases (G and T). In addition, the dimethylaminoethyl substituent of 19a extends into the minor groove of the DNA. Also, a hydrogen bond (2.9 Å) was observed between the lactam oxygen atom and Arg364, implying the importance of hydrogen bond acceptor, which is consistent with the cytotoxicity of 19a against the prostate cancer cells DU145-RC0.1, resistant cells with a R364H mutation in TOP1. DU145-RC0.1 cells showed high resistance to 19a (Table 4). The interaction between the two oxygen atoms in the dioxole ring and Asn722 (3.7 Å) or Thr718 (3.6 Å), respectively, might also contribute to the trapping of TOP1cc.

Table 4.

Cytotoxicity of the 19a in Drug-Resistant Isogenic Human Cancer Cell Lines

	GI₅₀ ± SD (μM)^a
compd	parental cell line	resistant subline	resistance ratio^b
	HCT116	HCT116-siTOP1
19a	0.076 ± 0.010	0.45 ± 0.31	5.9
1	0.009 ± 0.001	0.075 ± 0.014	8.3
	DU-145	DU145-RC0.1
19a	0.018 ± 0.002	2.38 ± 0.34	132
1	0.021 ± 0.016	4.73 ± 0.68	225
	MCF-7	MCF-7/ADR
19a	0.34 ± 0.098	0.95 ± 0.35	2.8
DOX	0.15 ± 0.003	11.67 ± 1.94	77.8
	HepG2	HepG2/ADR
19a	0.30 ± 0.050	3.20 ± 0.40	10.7
DOX	0.19 ± 0.048	9.04 ± 0.14	47.6

Open in a new tab

Resistance ratio was calculated by dividing the GI₅₀ of the mutant cell line by the GI₅₀ of the corresponding parental cell line.

TDP1 Inhibition.

Because of the unique function of TDP1 to repair TOP1-mediated DNA damage, and the recently reported inhibition of TDP1 by the indenoisoquinoline TOP1 inhibitors,^54–56 TDP1 inhibition by the synthesized compounds were also screened through a fluorescence assay.⁵⁰ A quenched fluorescent single-stranded oligonucleotide (5′-FAM-AGGATCTAAAAGACTT-BHQ-3′) was used as substrate. The compounds were tested at 100 μM concentration. Twelve of them, 19a, 21b, 22b, 39a/b, 40a/b, 41a/b, 42b, and 44a/b, exhibited TDP1 inhibition with the percentage inhibition ranging from 12% to 98% (Table 1). The most potent TOP1 inhibitor 19a (+++) showed low TDP1 inhibition (12% at 100 μM). None of the dihydrobenzophenanthridine derivatives exhibited TDP1 inhibition up to 100 μM concentration.

Seven compounds 39a/b, 40a/b, 41a/b, and 42b with notable TDP1 inhibition (>50%) were further tested to determine their IC₅₀ values (Table 2), expressed as the concentration of compound that inhibits 50% of TDP1 activity. Compound 41a was the most potent with an IC₅₀ value of 7.0 ± 1.4 μM in a dose-dependent manner (Figure 6A).

Table 2.

TDP1 Inhibition of the Active Compounds

	IC₅₀ (μM)^a
compd	fluorescence assay	gel-based assay
39a	24 ± 0.80	16 ± 0.40
39b	18 ± 1.7	13 ± 4.0
40a	58 ± 20	40 ± 14
40b	15 ± 2.7	27 ± 5.2
41a	7.0 ± 1.4	8.2 ± 1.3
41b	20 ± 1.7	21 ± 1.2
42b	19 ± 4.8	20 ± 5.4

Open in a new tab

IC₅₀ values were defined as the concentration of compound that inhibits 50% of enzyme activity. Every experiment was repeated at least three times independently.

Figure 6. — (A) TDP1 inhibition curves of the active compounds determined using fluorescence assay. Tested concentrations were 0.41, 1.23, 3.70, 11.1, 33.3, and 100 μM. (B) Hypothetical binding mode of **41a** (red carbon atoms ball and stick representation) in the complex with TDP1 (PDB 1RFF). All distances were measured from heavy atom to heavy atom. (C) Representative TDP1 inhibition gels of active compounds. Lane 1, DNA alone; lane 2, DNA and recombinant TDP1; lanes 3–26, DNA, recombinant TDP1 and the active compounds at tested concentration. Tested concentrations were 0.05, 0.15, 0.46, 1.4, 4.1, 12.3, 37, and 111 μM. N14Y and N14P are the substrate and product of TDP1, respectively.

To further establish their TDP1 inhibition, these seven active compounds 39a/b, 40a/b, 41a/b, and 42b, were further tested by a gel-based assay, in which a single-stranded 5′-³²P-labeled oligonucleotide N14Y containing 3′-phosphotyrosyl group was used as substrate.⁷⁶ Their IC₅₀ values are summarized in Table 2. Compound 41a was again found the most potent TDP1 inhibitor (IC₅₀ = 8.2 μM). Representative TDP1 inhibition gels are shown in Figure 6C. The indenoisoquinoline 10 was used as the positive control.⁵² Five compounds, 39a/b, 40b, and 41a/b, showed full inhibition at the highest concentration (111 μM) with progressive dose dependency.

To obtain a molecular view of the drug-induced TDP1 inhibition, a hypothetical binding model was built by using in silico docking. The TDP1 catalytic region containing two lysine residues (Lys265 and Lys495) and two histidine residues (His263 and His493) was constructed as the binding site from the TDP1 X-ray crystal structure (PDB 1RFF).⁷⁷ The inhibitors were docked into the binding site. The hypothetical structure pose of the top-ranked 41a is shown in Figure 6B. The polycyclic core of 41a lies along the DNA binding groove in the proximity of the catalytically important Phe259 residue, indicating a potential π–π stacking stabilizing drug TDP1 complex (Figure 6B), while the pyrrolidinyl ethyl side chain is directed to the catalytic site of TDP1 through a narrow channel (Figure 3S, Supporting Information). Two hypothetical hydrogen bonds form between the nitrogen atom in the pyrrolidinyl group and the catalytic residues His493 (3.3 Å) and Asn283 (3.1 Å), implying the importance of the side chain to TDP1 inhibition. In addition, the 1,3-dioxole ring is close to Glu538, and hydrogen bond forms between the oxygen atom and the amide group of Glu358 with the distance of 3.7 Å, which might also contribute to TDP1 inhibition.

Interaction with DNA.

To evaluate the interaction of the synthesized compounds with DNA, FRET melting assays were performed using F10T as a self-annealing substrate. The ΔT_m values of the compounds are summarized in Table 1. The FRET melting assay indicated that none of the synthesized compounds significantly increase the melting temperature of F10T at 2 μM concentration and that 27b has the highest ΔT_m value of 1.9 °C. Potent TOP1 inhibitor 19a demonstrated a low ΔT_m value of 0.5 °C, implying that the synthesized compounds could not stabilize the DNA double helix and increase the melting temperature by acting as classical DNA intercalators.

The binding of classical DNA intercalators between the base pair of circular DNA also produce TOP1-mediated unwinding effect, thereby limiting TOP1-mediated DNA relaxation.⁷⁸ To further evaluate 19a, TOP1-mediated unwinding assays in the presence of excess TOP1 were performed.⁷⁹ As shown in Figure 5B, the DNA intercalator ethidium bromide (EB) showed a distributed DNA ladder along with the increased tested concentration using supercoiled pBR322 DNA as substrate. On the contrary, 19a produced no TOP1-mediated unwinding effect up to 9 μM concentration. The assay using relaxed pBR322 DNA as substrate was also performed (Figure 5C)⁷⁹ and confirmed that 19a had no unwinding effect.

Thus, we conclude from the FRET and TOP1-mediated unwinding assays, the specificity for TOP1 of 19a and its lack of detectable binding to DNA outside of TOP1cc.

Cytotoxicity Assays.

The cytotoxicity of the compounds was assessed by MTT assay against five human tumor cell lines, including colon cancer (HCT116), leukemia (CCRF-CEM), prostate cancer (DU-145), nonsmall cell lung cancer (A549), and hepatocarcinoma (Huh7) cell lines. The cells were incubated with the compounds for 72 h in a five-dose assay, with concentrations ranging from 0.01 to 100 μM. After drug treatments, MTT solution was added to measure the percentage of cellular growth. The GI₅₀ values, defined as the concentrations of the compounds that result in 50% cell growth inhibition, are plotted and summarized in Table 1.

Compared to the parent 6, most benzophenanthridinone derivatives 17a/b and 19a/b–25a/b showed increased cytotoxicity against the five human cancer cell lines, which is consistent with their TOP1 inhibition. Compound 19a with the highest TOP1 potency (+++) also showed the highest cytotoxicity against these five cancer cell lines, with GI₅₀ values at nanomolar range (0.076 μM for HCT116, 0.029 μM for CCRF-CEM, 0.018 μM for DU145, 0.79 μM for A549, and 0.12 μM for Huh7). Although compounds 20a and 22a with alkylaminoethyl substituents at the 5-position showed weak TOP1 inhibition of +, they showed significant cytotoxicity against HCT116 (0.21 μM for 20a, 0.16 μM for 22a), CCRF-CEM (0.18 μM for 20a, 0.32 μM for 22a), and DU145 (0.054 μM for 20a, 0.96 μM for 22a) cell lines. Compound 21a with TOP1 inhibition of ++ showed cytotoxicity against the CCRF-CEM (0.62 μM) and DU145 (0.19 μM) cell lines with GI₅₀ values at submicromolar range. Compared to the compounds with hydroxyl terminus (17a/b) on the side chain at the 5-position, the compounds with alkylamino termini, including dimethylamino (19a/b), diethylamino (20a/b), pyrrolidinyl (21a/b), and piperidinyl (22a/b) groups, showed increased cytotoxicity against HCT116 cells, while the morpholinyl (23a/b) and 4-methylpiperazinyl (24a/b) termini led to decreased cytotoxicity. Compounds 23a/b and 24b showed low cytotoxicity against A549 and Huh7 cell lines, with GI₅₀ values more than 100 μM. The compounds with shorter side chain at the 5-position showed more cytotoxicity than that with corresponding longer side chain against HCT116 cells, for example, 17a vs 17b and 19–23a vs 19–23b, exceptions are the compounds with 4-methylpiperazinyl (24a/b) and imidazolyl (25a/b) termini.

Among the dihydrobenzophenanthridine derivatives (26a/b–32a/b), 26b with diethylaminopropyl substituent at the 5-position exhibited the highest cytotoxicity against HCT116 (GI₅₀ = 0.27 μM), CCRF-CEM (GI₅₀ = 0.12 μM), and DU145 (GI₅₀ = 0.081 μM) cell lines. The other five dihydrobenzophenanthridine derivatives, 26a, 28b, 29b, and 30a/b, showed high cytotoxicity against CCRF-CEM cells with GI₅₀ values at submicromolar concentration, while two dihydrobenzophenanthridine derivatives 28b and 29b showed high cytotoxicity against HCT116 cells, with GI₅₀ values of 0.66 and 0.64 μM, respectively. 32b showed increased cytotoxicity against these five cancer cell lines compared to parent 6, which was consistent with its TOP1 inhibition of ++. Contrary to the benzophenanthridinones, the dihydrobenzophenanthridines with a shorter side chain at the 5-position showed lower cytotoxicity against HCT116 cells than that corresponding to compounds with a longer side chain (for example 26a–30a vs 26b–30b). Exceptions are the compounds with 4-methylpiperazinyl (31a/b) and imidazolyl (32a/b) termini.

The benzophenanthridines (39a/b–45a/b) with alkoxy substituents at the 6-position generally showed decreased cytotoxicity compared to benzophenanthridinones (19a/b–25a/b) with the corresponding side chain at the 5-position. Although the benzophenanthridines 39a/b and 41a/b did not show TOP1 inhibition at up to 100 μM concentration, they had good TDP1 inhibitory activity, with IC₅₀ values at low micromolar concentration, and showed good cytotoxicity against four cancer cell lines, including CCRF-CEM, DU145, A549, and Huh7 cells, with GI₅₀ values between 1.29 and 8.95 μM, suggesting additional cellular targets.

Compound 19a, being the most potent inhibitor of TOP1 and the most cytotoxic, was selected and submitted to the National Cancer Institute (NCI, USA) for testing against the 60 tumor cell lines representing nine tissue types (NCI-60).^80–82 According to the NCI established procedures, the cells were incubated with 19a for 48 h and stained with sulforhodamine B dye. Cell growth inhibition (GI₅₀ in Table 3) was calculated relative to cells without treatment. High cytotoxicity was observed for 19a with a mean graph midpoint (MGM) for growth inhibition of all cancer cell lines of 0.145 μM, and its GI₅₀ values against HCT116, SR, NCI-H522, and UACC-62 cell lines were at nanomolar range (<100 nM).

Table 3.

Cytotoxicity of 19a against Individual NCI-60 Cell Lines

panel	cell line	GI₅₀ (μM)^a	panel	cell line	GI₅₀ (μM)
	MGM^b	0.145	colon cancer	COLO 205	0.144
leukemia	CCRF-CEM	0.144		HCC-2998	0.63
	K-562	0.156		HCT116	0.0855
	MOLT-4	0.118		HCT-15	0.427
	RPMI-8226	0.14		HT29	0.149
	SR	0.0669		KM12	0.875
non-small cell lung cancer	A549/ATCC	0.244		SW-620	0.345
	EKVX	0.79	renal cancer	786-0	0.166
	HOP-62	0.18		A498	0.347
	HOP-92	0.558		ACHN	0.16
	NCI-H226	0.512		CAKI-1	0.186
	NCI-H23	0.201		RXF 393	0.516
	NCI-H322M	0.428		SN 12C	0.258
	NCI-H460	0.141		TK-10	0.722
	NCI-H522	0.076		UO-31	0.157
CNS cancer	SF-268	0.283	breast cancer	MCF7	0.118
	SF-295	0.177		MDA-MB-231/ATCC	0.826
	SF-539	0.186		HS 578T	1.86
	SNB-19	0.233		BT-549	0.291
	SNB-75	0.239		T-47D	0.111
	U251	0.14		MDA-MB-468	0.14
melanoma	LOX IMVI	0.112	ovarian cancer	IGROV1	0.312
	MALME-3M	0.284		OVCAR-3	0.582
	M14	0.149		OVCAR-4	0.557
	MDA-MB-435	0.395		OVCAR-5	0.595
	SK-MEL-2	0.886		OVCAR-8	0.554
	SK-MEL-28	0.861		NCI/ADR-RES	0.29
	SK-MEL-5	0.181		SK-OV-3	0.24
	UACC-257	0.528	prostate cancer	PC-3	0.317
	UACC-62	0.0966		DU-145	0.215

Open in a new tab

GI₅₀ values were defined as the compound concentrations that resulted in 50% cell growth inhibition. The cells were incubated for 2 days with the tested compounds.

MGM: mean graph midpoint for growth inhibition of all human cancer cell lines.

Because the NCI-60 database contains several thousand chemicals and anticancer drugs which have been tested across the cancer cell line panel⁸² (http://discover.nci.nih.gov/cellminer and http://discover.nci.nih.gov/cellminercdb), we were able to perform a pattern comparison analysis with 19a. The drugs with the highest correlation with 19a were all TOP1 inhibitors including camptothecin and its derivatives as well as the indenoisoquinolines. Figure 7 shows the high correlation between the cytotoxicity of 19a and camptothecin and the clinical indenoisoquinoline LMP400.

Figure 7. — Pattern comparison analysis of **19a** using GI₅₀ data from NCI-60 assays. Individual dots represent a cell line of the NCI-60. Red lines represent regression line. R-square values were 0.58 and 0.40 for camptothecin and the indenoisoquinoline indotecan (LMP400), respectively. P-values were <0.0001 for both comparisons.

The cytotoxicity of 19a was further evaluated against a panel of isogenic camptothecin- and doxorubicin-resistant cell lines using MTT assay (Table 4). The HCT116-siTOP1 subline was developed by transfection of colon cancer parental cells HCT116 with short hairpin RNA vectors expressing siRNA for TOP1.⁸³ Compared to the parental cell HCT116, the HCT116-siTOP1 subline showed 8.3-fold resistant to 1, of which TOP1 is the only known cellular target,^12,84 and about 5.9-fold to 19a, implying that TOP1 is a major cellular target of 19a.

The camptothecin-resistant prostate cancer DU145-RC0.1 cells have a R364H mutation in TOP1 relative to the wild-type parental DU-145.⁸⁵ The TOP1 with R364H mutation is catalytically active but leads to RC0.1 cells being resistant to 1 because the R364 residue is close to the catalytic tyrosine and can stabilize the open form of TOP1cc.^75,86 The DU145-RC0.1 cells were highly resistant to 1 (225-fold) and 19a (132-fold), which is consistent with the molecular modeling (Figure 5D) showing hydrogen bonding between R364 and 19a.

P-Glycoprotein (P-gp) mediated drug efflux is generally responsible for classical multiple drug resistance.⁸⁷ The chemotherapeutic agent doxorubicin (DOX) is a substrate of P-gp, and both breast cancer MCF-7/ADR (77.8-fold) and hepatocellular HepG2/ADR sublines (47.6-fold), which overexpress P-gp, are highly resistant to doxorubicin (77.8- and 47.6-fold, respectively; Table 4).⁸⁸ Compound 19a appeared to be less a P-gp substrate (Table 4) with a resistance ratio of 2.8 (MCF-7/ADR:MCF-7) and 10.7 (HepG/ADR:HepG2), respectively.

Synergistic Effects of 41a and 41b with Camptothecin.

The combined effects of the TDP1 inhibitors 41a and 41b were tested with camptothecin in MCF-7 human breast cancer cells using MTT assay. As shown in Figure 8A, after 96 h incubation at 37 °C, the cytotoxicity of camptothecin against MCF-7 cells increased in the presence of 41a. At 2.5 μM concentration, almost 60% of cells were killed by camptothecin itself and over 90% of cells were killed when being coincubated with 41a at 5 μM concentration, which implies that a supra-additive effect of 41a with camptothecin is consistent with TDP1 inhibition in cells. Similarly, 41b showed a synergistic effect with camptothecin in MCF-7 cells (Figure 8B).

Figure 8. — Synergistic effect of **41a** (A) and **41b** (B) with camptothecin. The MCF-7 cells were incubated with camptothecin and the tested compounds for 96 h. Every experiment was repeated four times.

Induction of Cellular TOP1cc and DNA Damage by 19a.

The immunocomplex of enzyme to DNA (ICE) assay was conducted to evaluate the induction of TOP1cc by 19a in HCT116 cancer cells. Figure 9A shows that 19a induces cellular TOP1cc in a dose-dependent manner, similar to the positive control 1.

To assess the DNA damage in cancer cells induced by 19a, γH2AX foci were assessed by immunofluorescence microscopy in human colon cancer HCT116 cells treated with 19a. After incubation with 19a for 3 h, HCT116 cells were stained with γH2AX antibodies. γH2AX foci were induced by 19a at 1 μM concentration (Figure 9C), consistent with DNA damage due to the trapping of cellular TOP1cc by 19a.

Cancer Cell Apoptosis Induced by 19a.

Flow cytometry assays were also conducted in HCT116 cells to assess the induction of apoptosis by 19a. After being incubated with 19a for 24 h, apoptotic HCT116 cells were detected. Figure 9B shows that 19a could significantly induce HCT116 cells apoptosis in a dose-dependent manner. Approximately 60.9% (25.11% early apoptotic cells and 35.8% late apoptotic cells) of the treated cells were scored as apoptotic following 24 h incubation with 19a at 2 μM.

Pharmacokinetics of 19a.

In vivo pharmacokinetic (PK) study of 19a was conducted in Sprague–Dawley (SD) rats. The SD rats were divided into two groups (n = 3) and treated by intravenous injection (iv) at 1 mg/kg dose and intragastrical administration (ig) at 5 mg/kg dose, respectively. Plasma samples were collected up to 24 h postdosing and the concentration of 19a was measured. The PK parameters are summarized in Table 5. Following iv administration, the AUC_0→24h was 119 ± 10 h·ng/mL and T_1/2 was 0.855 ± 0.011 h. After oral administration, the T_max, C_max, and AUC_0→24h were 1.17 ± 0.76 h, 23.5 ± 16 h·ng/mL, and 89.7 ± 36 h·ng/mL, respectively. The bioavailability (F) was 15.5%.

Table 5.

Pharmacokinetic Parameters of 19a

	mean ± SD
parameters	iv (1 mg/kg)^a	ig (5 mg/kg)^b
T_max (h)		1.17 ± 0.76
C_max (ng/mL)		23.5 ± 16
AUC_0→t (h·ng/mL)	119 ± 10	89.7 ± 36
AUC_0→∞ (h·ng/mL)	121 ± 10	109 ± 33
MRT_INF (h)	1.01 ± 0.023	4.73 ± 3.3
T_1/2 (h)	0.855 ± 0.011	3.08 ± 3.4
F (%)		15.5 ± 5.4

Open in a new tab

iv means intravenous injection.

ig means intragastrical administration.

Acute Toxicity of 19a in Vivo.

To assess the toxicity of 19a, Kunming male mice were randomly divided into six groups (n = 4) and administered with 19a by ip injection at single doses of 300, 240, 192, 154, and 123 mg/kg, respectively. The control group was injected with sterile water. All mice survived after 7 days of administration with 19a in the groups of 192 mg/kg dose (Figure 10A), 154 mg/kg dose (data not shown), and 123 mg/kg dose (data not shown). Four mice died within 5 days in the 300 mg/kg group, and one mouse died within 7 days in the 240 mg/kg dose group (Figure 10A).

Figure 10. — Antitumor activity of **19a** in the HCT116 xenograft model. (A) Percent survival of mice treated with **19a** at doses of 300, 240, and 192 mg/kg. The effects of **19a** on body weight (B), tumor size (C), and tumor weight (D) at the dose of 5 and 10 mg/kg. Statistically significant difference mean tumor weight compared with the control, **: P < 0.01.

Antitumor Efficiency of 19a in Vivo.

Because 19a showed high TOP1 inhibition and cytotoxic activity in vitro, its antitumor efficiency in vivo was assessed in a human colon cancer HCT116 xenograft nude mice model. Male nude mice were randomly divided into three groups (n = 6) and administered with saline or with 19a at 10 or 5 mg/kg dose by ip injection daily. Administration of 19a significantly reduced tumor volume in a dose-dependent manner (Figure 10C). Meanwhile, the mice treated with 19a at 5 mg/kg dose showed no obvious body loss compared to the saline control group. Body weight loss was observed in the 10 mg/kg group (Figure 10B). Tumor weight inhibitions (TWI) by 19a were 59.6% (10 mg/kg, P < 0.01) and 32.7% (5 mg/kg) (Figure 10D). These results demonstrate the antitumor activity of 19a.

CONCLUSIONS

Here we report that the natural product oxynitidine scaffold represents a productive chemotype for TOP1 and TDP1 inhibitors. In this work, three subgroups of oxynitidine derivatives (benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives) were synthesized and evaluated as inhibitors of TOP1 and TDP1 and for cytotoxicity against human cancer cell lines (HCT116, CCRF-CEM, DU-145, A549, and Huh7 cell lines). Enzyme- and cell-based assays show that the benzophenanthridinone derivative 19a with dimethylaminoethyl substituent at the 5-position is potent at trapping TOP1cc (+++) and exhibits antiproliferative activity in human cancer cell lines at nanomolar concentration range GI₅₀ values. Compound 19a also shows consistent antiproliferative activity in the NCI-60 cell lines with MGM value of 0.145 μM. Six benzophenanthridine derivatives, 39a/b, 40b, 41a/b, and 42b, exhibit TDP1 inhibition with IC₅₀ values between 7.0 and 24 μM in biochemical assays. Consistent with TDP1 inhibition, both 41a and 41b show synergy in combination with camptothecin in MCF-7 human breast cancer cells. Cell-based assays demonstrated that 19a induces the formation of cellular TOP1cc and DNA damage in HCT116 cells, promotes apoptosis, and is a poor substrate of P-gp, a drug efflux protein responsible for multidrug resistance. The PK, acute toxicity, and antitumor efficiency of 19a suggest its potential value for further development.

EXPERIMENTAL SECTION

General Experiments.

The major chemical reagents for synthesis were purchased from Alfa Aesar, Sigma-Aldrich Co., or Aladdin Reagent Database Inc. (Shanghai) and were used without further purification unless otherwise indicated. 4-(Benzo[d][1,3]dioxol-5-yl)but-3-yn-1-ol (14) was prepared in our lab according to the Korivi’s method.⁶⁶ Chemical reaction courses were monitored by silica gel GF₂₅₄ thin layer chromatography (TLC). 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 spectrometer using tetramethylsilane as an internal reference. Mass spectra were analyzed on an Agilent 6120 (Quadrupole LC-MS) mass spectrometer. The high-resolution mass spectra 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 was detection at 220 nm, 1.0 mL/min flow rate, and a linear gradient of 50% to 15% PBS buffer (pH 3) and 50% to 85% MeOH in 35 min.

All animals were obtained from Laboratory Animal Center of Sun Yat-sen University. All procedures were approved by the Animal Ethics Committee of Sun Yat-sen University, in accordance with National Institute of Health and Nutrition Guidelines for the Care and Use of Laboratory Animals.

General Procedure for Synthesis of Schiff’s Base 12a and 12b.

The reaction solution of 6-bromoveratraldehyde (9.8 g, 40 mmol) and amine (ethanolamine or 3-aminopropanol, 42 mmol) in methanol (200 mL) was stirred at room temperature for 12 h and then concentrated under reduced pressure. The residue was washed with petroleum ether (2 × 10 mL) to give a white solid, yield 100%. The Schiff’s base intermediates were pure as shown by their ¹H NMR spectra and were used for next synthesis without further purification.

N-(2-Hydroxylethyl)-6-bromoveratraldimine (12a).

¹H NMR (CDCl₃) δ 8.58 (s, 1H), 7.54 (s, 1H), 6.99 (s, 1H), 3.95–3.89 (m, 8H), 3.79 (t, J = 4.7 Hz, 2H).

N-(3-Hydroxylprapyl)-6-bromoveratraldimine (12b).

¹H NMR (CDCl₃) δ 8.53 (s, 1H), 7.45 (s, 1H), 7.00 (s, 1H), 3.91–3.86 (m, 8H), 3.82 (t, J = 6.2 Hz, 2H), 1.96 (quint, J = 6.0 Hz, 2H).

General Procedure for the Synthesis of 13a and 13b.

Under nitrogen gas, the solution of NaH (60%, 6.69 g, 167 mmol) in THF (100 mL) was stirred and cooled to 0 °C for 15 min and then added dropwise to a solution of 12a (or 12b, 33 mmol) in THF (100 mL). The reaction solution was stirred at 0 °C for 1 h, and then chloromethyl methyl ether (15 mL, 198 mmol) was added dropwise and stirred for 1 h. The reaction was quenched by the addition of ethanol (20 mL) at 0 °C. The reaction solution was concentrated under reduced pressure. The residue was dissolved in chloroform (100 mL) and was washed with saturated saline (3 × 50 mL). The organic layer was dried (MgSO₄) overnight and concentrated under reduced pressure to give crude intermediates 13a (or 13b), which was used immediately for next synthesis without further purification.

General Procedure for Synthesis of 15a and 15b.

Under nitrogen gas, the reaction solution of 13a (or 13b, 2.7 mmol), 14 (520 mg, 2.73 mmol), Ni(cod)₂ (40 mg, 0.14 mmol), and P(o-Tol)₃ (80 mg, 0.26 mmol) in freshly distilled MeCN (10 mL) was stirred at 80 °C for 3 h. Then K₃[Fe(CN)₆] (6.6 g, 20 mmol), CsOH solution (50% w/w in water, 3.6 mL, 40 mmol), MeOH (20 mL), and water (20 mL) were added. The reaction mixture was stirred vigorously at 80 °C for 12 h. The reaction mixture was cooled and extracted with chloroform (3 × 50 mL). The combined organic layers were dried (MgSO₄) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the target product.

3-(Benzo[d][1,3]dioxol-5-yl)-4-(2-hydroxyethyl)-6,7-dimethoxy-2-(2-(methoxymethoxy)ethyl)isoquinolin-1(2H)-one (15a).

White solid, yield 45% (from 12a). ¹H NMR (CDCl₃) δ 7.89 (s, 1H), 7.11 (s, 1H), 6.91 (d, J = 7.7 Hz, 1H), 6.79 (s, 1H), 6.77 (d, J = 7.9 Hz, 1H), 6.05 (s, 2H), 4.48 (s, 2H), 4.07–3.95 (m, 8H), 3.77–3.62 (m, 4H), 3.20 (s, 3H), 2.80–2.66 (m, 2H). ¹³C NMR (CDCl₃) δ 161.4, 153.6, 149.1, 148.0, 147.9, 140.1, 131.8, 128.3, 123.7, 119.1, 111.5, 110.5, 108.4, 108.1, 103.9, 101.5, 96.1, 64.4, 62.3, 56.1, 56.1, 55.0, 45.6, 32.1. ESI-MS m/z: 458.1 [M + H]⁺. The structure was further confirmed with 2D NMR spectra.

3-(Benzo[d][1,3]dioxol-5-yl)-4-(2-hydroxyethyl)-6,7-dimethoxy-2-(3-(methoxymethoxy)propyl)isoquinolin-1(2H)-one (15b).

White solid, yield 57% (from 12b). ¹H NMR (CDCl₃) δ 7.90 (s, 1H), 7.11 (s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.79–6.74 (m, 2H), 6.07 (s, 2H), 4.48 (s, 2H), 4.02 (s, 3H), 4.01 (s, 3H), 3.71 (t, J = 6.7 Hz, 2H), 3.44 (t, J = 6.3 Hz, 2H), 3.27 (s, 3H), 2.82–2.67 (m, 2H). ¹³C NMR (CDCl₃) δ 161.3, 153.4, 149.1, 148.0, 139.9, 131.7, 128.4, 123.4, 111.3, 110.1, 108.5, 108.1, 103.7, 101.6, 96.0, 93.0, 91.5, 65.3, 62.4, 56.2, 56.1, 55.1, 44.0, 32.0, 29.2. ESI-MS m/z: 472.2 [M + H]⁺. The structure was further confirmed with 2D NMR spectra.

General Procedure for Synthesis of 16a and 16b.

To a solution of (COCl)₂ (215 μL, 2.5 mmol) in freshly distilled CH₂Cl₂ (5 mL), DMSO (365 μL, 5 mmol) was added dropwise at −60 °C and the reaction solution was stirred for 15 min. A solution of 15a (or 15b, 0.5 mmol) in freshly distilled CH₂Cl₂ (3 mL) was added dropwise and stirred at −60 °C for 30 min, and then Et₃N (1.5 mL, 10 mmol) was added dropwise. The reaction solution was brought to room temperature and stirred for 2 h. The reaction was quenched by the addition of water (1 mL) at 0 °C. The reaction mixture was extracted with CH₂Cl₂ (3 × 20 mL). The organic layer was washed with saturated saline (3 × 30 mL) and dried (MgSO₄) overnight and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the target product.

2-(3-(Benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-2-(2-(methoxymethoxy)ethyl)-1-oxo-1,2-dihydroisoquinolin-4-yl)-acetaldehyde (16a).

White solid, yield 85%. ¹H NMR (CDCl₃) δ 9.57 (t, J = 1.8 Hz, 1H), 7.90 (s, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.81–6.73 (m, 3H), 6.08 (s, 2H), 4.50 (s, 2H), 4.09 (d, J = 6.8 Hz, 2H), 4.03 (s, 3H), 3.97 (s, 3H), 3.79–3.67 (m, 2H), 3.53–3.47 (m, 2H), 3.21 (s, 3H). ¹³C NMR (CDCl₃) δ 199.7, 161.6, 153.8, 149.3, 148.4, 148.2, 141.8, 131.6, 128.0, 123.7, 119.5, 110.2, 108.7, 108.3, 106.4, 103.4, 101.7, 96.2, 64.4, 56.2, 56.1, 55.1, 45.9, 44.6. ESI-MS m/z: 455.2 [M + H]⁺.

2-(3-(Benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-2-(3-(methoxymethoxy)propyl)-1-oxo-1,2-dihydroisoquinolin-4-yl)-acetaldehyde (16b).

White solid, yield 87%. ¹H NMR (CDCl₃) δ 9.58 (s, 1H), 7.91 (d, J = 2.4 Hz, 1H), 6.96–6.90 (m, 1H), 6.80–6.74 (m, 3H), 6.08 (s, 2H), 4.71–4.60 (m, 2H), 4.50 (s, 2H), 4.02 (s, 3H), 3.96 (s, 3H), 3.52–3.44 (m, 4H), 3.29 (s, 3H), 1.88–1.83 (m, 2H). ¹³C NMR (CDCl₃) δ 199.6, 161.4, 153.7, 149.3, 148.4, 148.3, 141.5, 131.4, 128.0, 123.3, 119.7, 109.8, 108.8, 108.4, 106.3, 103.4, 101.7, 96.1, 65.3, 56.2, 56.1, 55.1, 44.5, 44.3, 29.2. ESI-MS m/z: 470.2 [M + H]⁺.

General Procedure for the Synthesis of 17a and 17b.

The reaction solution of 16a (or 16b, 0.5 mmol) and concentrated hydrochloric acid (0.4 mL) in MeOH (10 mL) was added to a 50 mL round-bottomed flask. The flask was sealed with a rubber stopper. The reaction solution was stirred at room temperature overnight, and then the formed precipitate was filtered and washed with saturated sodium bicarbonate and water consecutively. The crude solid was dried and purified by silica gel column chromatography to give the target product.

12-(2-Hydroxyethyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo-[1,2-c]phenanthridin-13(12H)-one (17a).

White solid, yield 97%, mp = 228.2–229.7 °C. ¹H NMR (CDCl₃) δ 8.00 (d, J = 8.8 Hz, 1H), 7.91 (s, 1H), 7.59–7.57 (m, 2H), 7.25 (s, 1H), 7.19 (s, 1H), 6.11 (s, 2H), 5.08 (t, J = 5.6 Hz, 1H), 4.54 (t, J = 4.2 Hz, 2H), 4.37–4.33 (m, 2H), 4.12 (s, 3H), 4.06 (s, 3H). ¹³C NMR (CDCl₃) δ 165.8, 154.0, 149.8, 147.5, 147.5, 135.4, 131.9, 129.4, 123.8, 120.8, 118.7, 118.3, 117.3, 108.5, 105.0, 102.8, 102.0, 101.7, 64.0, 56.8, 56.3, 56.2. HRMS (ESI) m/z: 394.1276 [M + H]⁺, calcd for C₂₂H₂₀NO₆ 394.1285. ESI-MS m/z: 394.1 [M + H]⁺.

12-(3-Hydroxypropyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridin-13(12H)-one (17b).

White solid, yield 96%, mp = 263.4–264.2 °C. ¹H NMR (CDCl₃) δ 7.98 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.60 (s, 1H), 7.59–7.55 (m, 2H), 7.19 (s, 1H), 6.12 (s, 2H), 4.72 (t, J = 6.6 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H), 3.54 (t, J = 6.6 Hz, 2H), 2.16–2.07 (m, 2H). ¹³C NMR (CDCl₃) δ 165.0, 153.7, 149.8, 147.6, 147.4, 135.1, 131.7, 129.0, 123.5, 121.1, 119.4, 118.4, 117.5, 108.9, 104.9, 102.8, 102.3, 101.6, 60.1, 56.2, 56.2, 48.3, 32.8. HRMS (ESI) m/z: 408.1457 [M + H]⁺, calcd for C₂₃H₂₂NO₆ 408.1442. ESI-MS m/z: 408.1 [M + H]⁺.

General Procedure for the Synthesis of 18a and 18b.

To a solution of 17a (or 17b, 0.5 mmol) in freshly distilled chloroform (10 mL), SOCl₂ (0.8 mL) was added dropwise through a syringe. The reaction solution was stirred at room temperature for 1 h and then quenched by the addition of water (5 mL) at 0 °C. The precipitate was filtered and washed with saturated sodium bicarbonate and water consecutively. The residue was purified by silica gel column chromatography to give the target product.

12-(2-Chloroethyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo-[1,2-c]phenanthridin-13(12H)-one (18a).

White solid, yield 94%, mp = 236.5–237.3 °C. ¹H NMR (DMSO) δ 8.83 (d, J = 9.0 Hz, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 6.33 (s, 2H), 5.76 (t, J = 9.0 Hz, 2H), 5.35 (t, J = 9.0 Hz, 2H), 4.20 (s, 3H), 4.04 (s, 3H). ESI-MS m/z: 412.1 (100%), 414.0 (33%) [M + H]⁺.

12-(3-Chloropropyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo-[1,2-c]phenanthridin-13(12H)-one (18b).

White solid, yield 96%, mp = 235.5–236.1 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.59–7.55 (m, 2H), 7.51 (s, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 4.69 (t, J = 6.8 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.39 (t, J = 6.4 Hz, 2H), 2.43–2.33 (m, 2H). HRMS (ESI) m/z: 426.1090 [M + H]⁺, calcd for C₂₃H₂₁NO₅Cl 426.1103. ESI-MS m/z: 426.1 (100%), 428.1 (33%) [M + H]⁺.

General Procedure for the Synthesis of 19a/b–25a/b.

The solution of 18a (or 18b, 0.87 mmol), NEt₃ (870 mg, 8.7 mmol), and amines (in pressure vessel for dimethylamine, 8.7 mmol) in PhMe (20 mL) was stirred and heated under reflux for 3–6 h, and then, cooled to room temperature. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give the target product.

12-(2-(Dimethylamino)ethyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (19a).

White solid, yield 65%, mp = 136.4–138.0 °C. IR (KBr, cm⁻¹), 1632, 1611, 1590. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.66 (s, 1H), 7.57–7.54 (m, 2H), 7.18 (s, 1H), 6.10 (s, 2H), 4.66 (t, J = 7.0 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.78 (t, J = 7.0 Hz, 2H), 2.21 (s, 6H). ¹³C NMR (CDCl₃) δ 164.7, 153.6, 149.7, 147.5, 147.3, 135.5, 131.7, 128.9, 123.3, 121.3, 119.6, 118.3, 117.3, 108.8, 104.8, 102.9, 102.3, 101.5, 57.6, 56.2, 56.1, 50.1, 45.5. HRMS (ESI) m/z: 421.1761 [M + H]⁺, calcd for C₂₄H₂₅N₂O₅ 421.1758.

12-(3-(Dimethylamino)propyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (19b).

White solid, yield 70%, mp = 173.2–174.3 °C. IR (KBr, cm⁻¹), 1612 (sh), 1582.¹H NMR (CDCl₃) δ 7.97 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H),7.58–7.53 (m, 2H), 7.48 (s, 1H), 7.19 (s, 1H), 6.13 (s, 2H), 4.55 (t, J = 7.2 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H), 2.51 (t, J = 7.2 Hz, 2H), 2.34 (s, 6H), 2.18 (quint, J = 7.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 164.7, 153.6, 149.6, 147.5, 147.4, 135.3, 131.7, 129.0, 123.3, 121.0, 119.5, 118.3, 117.4, 108.8, 104.8, 102.9, 102.0, 101.6, 56.2, 56.1, 55.5, 49.7, 43.5, 25.1, HRMS (ESI) m/z: 435.1919 [M + H]⁺, calcd for C₂₅H₂₇N₂O₅ 435.1914.

12-(2-(Diethylamino)ethyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (20a).

White powder, yield 86%, mp = 129.1–129.5 °C. IR (KBr, cm⁻¹), 1640, 1612, 1592. ¹H NMR (CDCl₃) δ 7.96 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.70 (s, 1H), 7.59–7.52 (m, 2H), 7.17 (s, 1H), 6.10 (s, 2H), 4.64 (t, J = 6.4 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.82 (t, J = 6.4 Hz, 2H), 2.39 (q, J = 7.2 Hz, 4H), 0.83 (t, J = 7.2 Hz, 6H). ¹³C NMR (CDCl₃) δ 165.0, 153.7, 149.8, 147.6, 147.5, 135.8, 131.8, 129.2, 123.3, 121.3, 119.9, 118.5, 117.6, 109.0, 104.9, 103.0, 102.8, 101.7, 56.4, 56.3, 51.6, 50.6, 47.5, 12.0. HRMS (ESI) m/z: 449.2078 [M + H]⁺, calcd for C₂₆H₂₉N₂O₅ 449.2071.

12-(3-(Diethylamino)propyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (20b).

White solid, yield 60%, mp = 106.5–108.1 °C. IR (KBr, cm⁻¹), 1637, 1613, 1581. ¹H NMR (CDCl₃) δ 7.99 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.59 (s, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.53 (s, 1H), 7.20 (s, 1H), 6.12 (s, 2H), 4.56 (t, J = 7.0 Hz, 2H), 4.13 (s, 3H), 4.07 (s, 3H), 2.63–2.43 (m, 6H), 2.22–2.10 (m, 2H), 0.98 (t, J = 6.5 Hz, 6H). ¹³C NMR (CDCl₃) δ 164.7, 153.6, 149.7, 147.6, 147.4, 135.4, 131.7, 129.0, 123.4, 121.2, 119.5, 118.4, 117.3, 108.7, 104.9, 102.8, 102.1, 101.6, 56.3, 56.2, 50.1, 49.4, 46.6, 25.3, 10.8. HRMS (ESI) m/z: 463.2233 [M + H]⁺, calcd for C₂₇H₃₁N₂O₅ 463.2227. ESI-MS m/z: 463.2 [M + H]⁺. The structure was also confirmed with and single crystal analysis. Compound 20b was crystallized from dichloromethane as white cubic crystals. Data collections for 20b were performed at 103 K on a Xcalibur Nova diffractometer, using Cu Kα radiation (λ = 1.54184 Å). The determination of crystal class and unit cell parameters was carried out by CrysAlisPro. The raw frame data was processed using CrysAlisPro (Rigaku Oxford Diffraction) to yield the reflection data file. The structure of 20b was solved by use of SHELXTL (Bruker, 2005) program. Refinement was performed on F² anisotropically for all the non-hydrogen atoms by the full-matrix least-squares method. The hydrogen atoms were placed at the calculated positions and were included in the structure calculation without further refinement of the parameters. Molecular formula = C₂₇H₃₂N₂O₄, molecular mass = 449.57, monoclinic, a = 9.41991(12) Å, b = 10.11440(12) Å, c = 23.7285(4) Å, β = 91.6653(13)°, V = 2259.82(5) Å³, T = 103 K, space group P2₁/c (no. 14), Z = 4, μ (Cu Kα) = 0.710 mm⁻¹, 10230 reflections measured, 4283 unique (R_int = 0.0458) which were used in all calculations. The final R (reflections) = 0.0500, wR² (reflections) = 0.1495. Crystallographic data for compound 20b has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1579803. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44(0)1223–336033 or deposit@ccdc.cam.ac.uk].

2,3-Dimethoxy-12-(2-(pyrrolidin-1-yl)ethyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (21a).

White solid, yield 47%, mp = 209.2–210.3 °C. IR (KBr, cm⁻¹), 1639, 1610, 1594. ¹H NMR (CDCl₃) δ 7.93–7.88 (m, 2H), 7.65 (s, 1H), 7.52 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.12 (s, 1H), 6.08 (s, 2H), 4.66 (t, J = 7.2 Hz, 2H), 4.08 (s, 3H), 4.05 (s, 3H), 2.91 (t, J = 7.2 Hz, 2H), 2.52–2.42 (m, 4H), 1.73–1.61 (m, 4H). ¹³C NMR (CDCl₃) δ 164.7, 153.5, 149.6, 147.5, 147.3, 135.4, 131.6, 129.0, 123.2, 121.1, 119.6, 118.2, 117.3, 108.8, 104.7, 102.8, 102.6, 101.5, 56.2, 56.1, 54.3, 54.1, 51.0, 23.6. HRMS (ESI) m/z: 447.1916 [M + H]⁺, calcd for C₂₆H₂₇N₂O₅, 447.1914. ESI-MS m/z: 447.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-(pyrrolidin-1-yl)propyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (21b).

White solid, yield 65%, mp = 205.8–207.4 °C. IR (KBr, cm⁻¹), 1640, 1612, 1596. ¹H NMR (CDCl₃) δ 7.95 (d, J = 8.8 Hz, 1H), 7.88 (s, 1H), 7.56 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.48 (s, 1H), 7.16 (s, 1H), 6.10 (s, 2H), 4.57 (t, J = 7.2 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.59 (s, br 4H), 2.52 (t, J = 7.2 Hz, 2H), 2.19 (quint, J = 7.2 Hz, 2H), 1.77 (s, br 4H). ¹³C NMR (CDCl₃) δ 164.7, 153.6, 149.7, 147.6, 147.4, 135.3, 131.7, 129.0, 123.4, 121.2, 119.5, 118.3, 117.3, 108.8, 104.9, 102.9, 102.1, 101.6, 56.2, 56.2, 53.8, 53.3, 49.9, 27.4, 23.4. HRMS (ESI) m/z: 461.2067 [M + H]⁺, calcd for C₂₇H₂₉N₂O₅ 461.2071.

2,3-Dimethoxy-12-(2-(piperidin-1-yl)ethyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (22a).

White solid, yield 44%, mp = 178.3–180.1 °C. IR (KBr, cm⁻¹), 1642, 1612, 1596. ¹H NMR (CDCl₃) δ 7.98 (d, J = 8.8 Hz, 1H), 7.93 (s, 1H), 7.74 (s, 1H), 7.58 (s, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 4.69 (t, J = 6.6 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H), 2.69 (t, J = 6.6 Hz, 2H), 2.34–2.23 (m, 4H), 1.40–1.29 (m, 6H). ¹³C NMR (CDCl₃) δ 165.0, 153.5, 149.5, 147.5, 147.3, 135.6, 131.6, 129.1, 123.2, 121.2, 119.7, 118.2, 117.5, 108.9, 104.7, 102.8, 102.7, 101.5, 57.2, 56.2, 56.1, 54.6, 49.8, 25.8, 24.2. HRMS (ESI) m/z: 461.2076 [M + H]⁺, calcd for C₂₇H₂₉N₂O₅ 461.2071. ESI-MS m/z: 461.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-(piperidin-1-yl)propyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (22b).

White solid, yield 60%, mp = 110.6–112.3 °C. IR (KBr, cm⁻¹), 1639, 1612, 1592.¹H NMR (CDCl₃) δ 7.98 (d, J = 8.7 Hz, 1H), 7.91 (s, 1H), 7.60–7.55 (m, 2H), 7.48 (s, 1H), 7.19 (s, 1H), 6.10 (s, 2H), 4.55 (d, J = 7.2 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 2.50 (d, J = 7.2 Hz, 2H), 2.34–2.13 (s, 6H), 1.51–1.30 (m, 6H). ¹³C NMR (CDCl₃) δ 164.6, 153.7, 149.7, 147.6, 147.5, 135.1, 131.7, 128.9, 123.5, 121.0, 119.4, 118.3, 117.4, 108.7, 104.9, 102.9, 102.0, 101.6, 56.2, 56.1, 55.7, 53.9, 50.1, 25.4, 25.2, 23.9. HRMS (ESI) m/z: 475.2227 [M + H]⁺, calcd for C₂₈H₃₁N₂O₅ 475.2227.

2,3-Dimethoxy-12-(2-morpholinoethyl)-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridin-13(12H)-one (23a).

White solid, yield 48%, mp = 220.5–221.1 °C. IR (KBr, cm⁻¹), 1639, 1611, 1594. ¹H NMR (CDCl₃) δ 7.93 (d, J = 8.7 Hz, 1H), 7.88 (s, 1H), 7.64 (s, 1H), 7.54 (s, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.14 (s, 1H), 6.09 (s, 2H), 4.66 (t, J = 6.2 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 3.34 (s, br 4H), 2.67 (t, J = 6.2 Hz, 2H), 2.21 (s, br 4H). ¹³C NMR (CDCl₃) δ 165.2, 153.5, 149.6, 147.5, 147.4, 135.6, 131.5, 129.0, 123.2, 121.2, 119.7, 118.3, 117.6, 108.9, 104.8, 102.8, 102.5, 101.6, 66.9, 56.6, 56.2, 56.2, 53.4, 49.3. HRMS (ESI) m/z: 463.1875 [M + H]⁺, calcd for C₂₆H₂₇N₂O₆ 463.1864. ESI-MS m/z: 463.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-morpholinopropyl)-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridin-13(12H)-one (23b).

White solid, yield 60%, mp = 203.1–205.0 °C. IR (KBr, cm⁻¹), 1639, 1610, 1595. ¹H NMR (CDCl₃) δ 7.94 (d, J = 8.5 Hz, 1H), 7.90 (s, 1H), 7.58–7.48 (m, 3H), 7.16 (s, 1H), 6.10 (s, 2H), 4.63 (t, J = 6.2 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 3.42 (s, br 4H), 2.12 (s, br 4H), 2.08–1.96 (m, 4H). ¹³C NMR (CDCl₃) δ 164.6, 153.5, 149.7, 147.5, 147.2, 135.9, 131.6, 128.9, 123.0, 121.1, 119.7, 118.3, 117.2, 108.8, 104.8, 102.8, 102.4, 101.5, 66.9, 56.2, 56.2, 55.3, 53.3, 49.8, 25.3. HRMS (ESI) m/z: 477.2032 [M + H]⁺, calcd for C₂₇H₂₉N₂O₆ 477.2020.

2,3-Dimethoxy-12-(2-(4-methylpiperazin-1-yl)ethyl)-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (24a).

White solid, yield 42%, mp = 207.5–208.3 °C. IR (KBr, cm⁻¹), 1635, 1613, 1590. ¹H NMR (CDCl₃) δ 7.93 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.61 (s, 1H), 7.54 (s, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.15 (s, 1H), 6.10 (s, 2H), 4.67 (t, J = 6.8 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.67 (t, J = 6.8 Hz, 2H), 2.64–2.56 (m, 2H), 2.38–2.32 (m, 4H), 2.23–2.16 (m, 5H). ¹³C NMR (CDCl₃) δ 165.2, 153.5, 149.6, 147.5, 147.4, 135.5, 131.5, 129.0, 123.2, 121.2, 119.8, 118.2, 117.6, 109.0, 104.8, 102.8, 102.4, 101.5, 56.2, 56.2, 55.9, 54.7, 52.3, 49.5, 45.5. HRMS (ESI) m/z: 476.2177 [M + H]⁺, calcd for C₂₇H₃₀N₃O₅ 476.2180. ESI-MS m/z: 476.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-(4-methylpiperazin-1-yl)propyl)-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (24b).

White solid, yield 41%, mp = 170.9–172.8 °C. IR (KBr, cm⁻¹), 1631, 1614, 1590. ¹H NMR (CDCl₃) δ 7.96 (d, J = 8.7 Hz, 1H), 7.91 (s, 1H), 7.56 (s, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.52 (s, 1H), 7.18 (s, 1H), 6.11 (s, 2H), 4.61 (t, J = 6.8 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.33–2.08 (m, 13H), 1.99 (t, J = 6.8 Hz, 2H). ¹³C NMR (CDCl₃) δ 164.7, 153.5, 149.7, 147.5, 147.2, 135.8, 131.6, 128.9, 123.2, 121.4, 119.7, 118.3, 117.3, 108.8, 104.8, 102.8, 102.5, 101.5, 56.3, 56.2, 54.9, 52.5, 49.9, 45.8, 29.7, 25.7. HRMS (ESI) m/z: 490.2334 [M + H]⁺, calcd for C₂₈H₃₂N₃O₅ 490.2336. ESI-MS m/z: 490.2 [M + H]⁺.

12-(2-(1H-Imidazol-1-yl)ethyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (25a).

White powder, yield 88%, mp = 254.2–255.6 °C. IR (KBr, cm⁻¹), 1630, 1609, 1588. ¹H NMR (CDCl₃) δ 7.92–7.86 (m, 2H), 7.55–7.50 (m, 2H), 7.32 (s, 1H), 7.17 (s, 1H), 7.00 (s, 1H), 6.73 (s, 1H), 6.53 (s, 1H), 6.12 (s, 2H), 4.90 (t, J = 6.4 Hz, 2H), 4.37 (t, J = 6.4 Hz, 2H), 4.11 (s, 3H), 4.07 (s, 3H). ¹³C NMR (CDCl₃) δ 164.7, 154.0, 149.9, 147.7, 147.7, 137.2, 134.7, 131.7, 129.6, 129.2, 123.9, 120.6, 119.2, 118.5, 118.5, 117.8, 108.7, 105.3, 103.1, 101.8, 101.5, 56.4, 56.3, 51.9, 44.8. HRMS (ESI) m/z: 444.1550 [M + H]⁺, calcd for C₂₅H₂₂N₃O₅ 444.1554. ESI-MS m/z: 444.1 [M + H]⁺.

12-(3-(1H-Imidazol-1-yl)propyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (25b).

White powder, yield 86%, mp = 200.1–200.9 °C. IR (KBr, cm⁻¹), 1631, 1610, 1591. ¹H NMR (CDCl₃) δ 7.99 (d, J = 8.8 Hz, 1H), 7.90 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.58 (s, 1H), 7.31 (s, 1H), 7.19 (s, 1H), 7.06 (s, 1H), 6.89 (s, 1H), 6.61 (s, 1H), 6.11 (s, 2H), 4.51 (t, J = 6.8 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H), 3.71 (t, J = 6.8 Hz, 2H), 2.35 (quint, J = 6.8 Hz, 2H). ¹³C NMR (CDCl₃) δ 164.7, 153.9, 149.9, 147.8, 147.6, 136.9, 135.2, 131.8, 129.5, 129.0, 123.8, 121.1, 119.5, 118.6, 118.4, 117.5, 108.8, 105.1, 103.0, 101.8, 101.7, 56.4, 56.3, 48.8, 44.3, 30.2. HRMS (ESI) m/z: 458.1703 [M + H]⁺, calcd for C₂₆H₂₄N₃O₅ 458.1710. ESI-MS m/z: 458.2 [M + H]⁺.

General Procedure for Synthesis of 26a/b–32a/b.

Under nitrogen gas, to a solution of oxynitidine analogue (19a/b–25a/b, 0.45 mmol) in dried THF (100 mL), the solution of LiAlH₄ (3.6 mL, 9.0 mmol) in THF was added at 0 °C dropwise, and then the reaction solution was stirred and heated under reflux for 3–6 h. The reaction was quenched by the addition of aqueous NaOH solution (5%, 5 mL) at 0 °C. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give the target product.

2-(2,3-Dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]-phenanthridin-12(13H)-yl)-N,N-dimethylethan-1-amine (26a).

White powder, yield 60%, mp = 109.4–109.6 °C. ¹H NMR (CDCl₃) δ 7.74 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.12 (s, 1H), 6.80 (s, 1H), 6.06 (s, 2H), 4.21 (s, 2H), 4.00 (s, 3H), 3.95 (s, 3H), 2.88 (t, J = 6.4 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 2.24 (s, 6H). ¹³C NMR (CDCl₃) δ 148.8, 148.7, 148.2, 147.6, 142.9, 131.0, 126.6, 125.5, 125.2, 124.9, 123.9, 119.9, 110.2, 106.4, 104.5, 101.2, 100.8, 58.4, 56.3, 56.2, 50.8, 50.0, 46.0. HRMS (ESI) m/z: 407.1960 [M + H]⁺, calcd for C₂₄H₂₇N₂O₄ 407.1965. ESI-MS m/z: 407.2 [M + H]⁺.

3-(2,3-Dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]-phenanthridin-12(13H)-yl)-N,N-dimethylpropan-1-amine (26b).

White powder, yield 58%, mp = 82.4–83.0 °C. ¹H NMR (CDCl₃) δ 7.68 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.78 (s, 1H), 6.05 (s, 2H), 4.16 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 2.75 (t, J = 7.6 Hz, 2H), 2.29 (t, J = 7.6 Hz, 2H), 2.21 (s, 6H), 1.81 (quint, J = 7.6 Hz, 2H). ¹³C NMR (CDCl₃) δ 148.9, 148.7, 148.2, 147.5, 143.0, 131.0, 126.6, 125.6, 125.1, 125.0, 123.9, 120.0, 110.1, 106.5, 104.5, 101.1, 100.8, 57.3, 56.3, 56.2, 50.0, 45.6, 26.8. HRMS (ESI) m/z: 421.2121 [M + H]⁺, calcd for C₂₅H₂₉N₂O₄ 421.2122. ESI-MS m/z: 421.3 [M + H]⁺.

2-(2,3-Dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]-phenanthridin-12(13H)-yl)-N,N-diethylethan-1-amine (27a).

White powder, yield 78%, mp = 68.1–68.2 °C. ¹H NMR (CDCl₃) δ 7.80 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.78 (s, 1H), 6.04 (s, 2H), 4.20 (s, 2H), 3.99 (s, 3H), 3.94 (s, 3H), 2.88 (t, J = 6.4 Hz, 2H), 2.75 (t, J = 7.2 Hz, 2H), 2.50 (q, J = 7.2 Hz, 4H), 1.01 (t, J = 7.2 Hz, 6H). ¹³C NMR (CDCl₃) δ 149.0, 148.8, 148.2, 147.6, 143.1, 131.1, 126.6, 125.7, 125.4, 124.9, 123.7, 119.9, 110.3, 106.8, 104.5, 101.1, 101.0, 56.4, 56.3, 52.0, 50.9, 50.0, 47.4, 11.9. HRMS (ESI) m/z: 435.2281 [M + H]⁺, calcd for C₂₆H₃₁N₂O₄ 435.2278.

3-(2,3-Dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]-phenanthridin-12(13H)-yl)-N,N-diethylpropan-1-amine (27b).

White solid, yield 81%, mp = 119.5–121.5 °C. ¹H NMR (CDCl₃) δ 7.68 (d, J = 8.5 Hz, 1H), 7.65 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.79 (s, 1H), 6.05 (s, 2H), 4.17 (s, 2H), 3.99 (s, 3H), 3.95(s, 3H), 2.74 (t, J = 7.6 Hz, 2H), 2.60–2.50 (m, 4H), 2.50–2.42 (m, 2H), 1.88–1.79 (m, 2H), 1.02 (t, J = 7.0 Hz, 6H). ¹³C NMR (CDCl₃) δ 148.8, 148.6, 148.1, 147.4, 143.0, 130.9, 126.5, 125.5, 125.0, 124.8, 123.7, 119.9, 110.0, 106.4, 104.4, 101.0, 100.7, 56.2, 56.1, 50.7, 50.1, 49.9, 46.8, 26.2, 11.6. HRMS (ESI) m/z: 449.2447 [M + H]⁺, calcd for C₂₇H₃₃N₂O₄ 449.2435. ESI-MS m/z: 449.24 [M + H]⁺.

2,3-Dimethoxy-12-(2-(pyrrolidin-1-yl)ethyl)-12,13-dihydro-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (28a).

White solid, yield 55%, mp = 84.3–85.8 °C. ¹H NMR (CDCl₃) δ 7.70–7.64 (m, 2H), 7.48 (d, J = 8.5 Hz, 1H), 7.29 (s, 1H), 7.10 (s, 1H), 6.81 (s, 1H), 6.04 (s, 2H), 4.21 (s, 2H), 3.98 (s, 3H), 3.95 (s, 3H), 3.12 (d, J = 7.2 Hz, 2H), 2.86 (d, J = 7.2 Hz, 2H), 2.73–2.64 (m, 4H), 1.87–1.81 (m, 4H). ¹³C NMR (CDCl₃) δ 148.9, 148.7, 148.2, 147.5, 141.7, 130.9, 126.4, 125.3, 125.2, 125.1, 124.0, 119.8, 110.1, 106.5, 104.4, 101.1, 100.5, 56.2, 56.1, 54.2, 51.2, 50.3, 23.3. HRMS (ESI) m/z: 433.2120 [M + H]⁺, calcd for C₂₆H₂₉N₂O₄ 433.2122. ESI-MS m/z: 433.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-(pyrrolidin-1-yl)propyl)-12,13-dihydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (28b).

White solid, yield 61%, mp = 135.8–137.5 °C. ¹H NMR (CDCl₃) δ 7.70–7.64 (m, 2H), 7.47 (d, J = 8.5 Hz, 1H), 7.29 (s, 1H), 7.10 (s, 1H), 6.78 (s, 1H), 6.03 (s, 2H), 4.16 (s, 2H), 3.98 (s, 3H), 3.94 (s, 3H), 2.76 (t, J = 7.2 Hz, 2H), 2.54–2.47 (m, 6H), 1.93–1.83 (m, 2H), 1.78 (s, br 4H). ¹³C NMR (CDCl₃) δ 148.8, 148.6, 148.0, 147.4, 143.0, 130.9, 126.5, 125.4, 124.9, 124.9, 123.7, 119.9, 110.0, 106.4, 104.4, 101.0, 100.7, 56.2, 56.1, 54.2, 53.8, 49.8, 49.7, 27.8, 23.4. HRMS (ESI) m/z: 447.2273 [M + H]⁺, calcd for C₂₇H₃₁N₂O₄ 447.2278.

2,3-Dimethoxy-12-(2-(piperidin-1-yl)ethyl)-12,13-dihydro-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (29a).

White powder, yield 67%, mp = 140.4–141.5 °C. ¹H NMR (CDCl₃) δ 7.82 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 7.11 (s, 1H), 6.79 (s, 1H), 6.05 (s, 2H), 4.23 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 2.92 (t, J = 5.8 Hz, 2H), 2.61 (t, J = 6.4 Hz, 2H), 2.47–2.26 (m, 4H), 1.67–1.57 (m, 6H). ¹³C NMR (CDCl₃) δ 148.9, 148.7, 148.2, 147.6, 143.0, 131.0, 126.6, 125.6, 125.4, 124.9, 123.8, 119.9, 110.3, 106.6, 104.4, 101.1, 101.1, 58.3, 56.3, 56.2, 55.3, 50.8, 49.2, 26.0, 24.5. HRMS (ESI) m/z: 447.2275 [M + H]⁺, calcd for C₂₇H₃₁N₂O₄ 447.2278. ESI-MS m/z: 447.3 [M + H]⁺.

2,3-Dimethoxy-12-(3-(piperidin-1-yl)propyl)-12,13-dihydro-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (29b).

White powder, yield 53%, mp = 131.0–131.5 °C. ¹H NMR (CDCl₃) δ 7.68 (d, J = 8.4 Hz, 1H), 7.66 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.78 (s, 1H), 6.05 (s, 2H), 4.16 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 2.73 (t, J = 7.6 Hz, 2H), 2.44–2.34 (m, 4H), 2.32 (t, J = 7.6 Hz, 2H), 1.86 (quint, J = 7.6 Hz, 2H), 1.62–1.55 (m, 6H). ¹³C NMR (CDCl₃) δ 148.9, 148.6, 148.2, 147.5, 143.1, 131.0, 126.6, 125.5, 125.0, 124.9, 123.9, 120.0, 110.1, 106.5, 104.5, 101.1, 100.8, 56.8, 56.3, 56.2, 54.8, 50.0, 49.9, 26.0, 24.5. HRMS (ESI) m/z: 461.2432 [M + H]⁺, calcd for C₂₈H₃₃N₂O₄ 461.2435. ESI-MS m/z: 461.3 [M + H]⁺.

2,3-Dimethoxy-12-(2-morpholinoethyl)-12,13-dihydro-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (30a).

White solid, yield 58%, mp = 119.9–121.5 °C. ¹H NMR (CDCl₃) δ 7.95 (s, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.32 (s, 1H), 7.13 (s, 1H), 6.80 (s, 1H), 6.07 (s, 2H), 4.25 (s, 2H), 4.01 (s, 3H), 3.97 (s, 3H), 3.75 (t, J = 4.6 Hz, 4H), 2.93 (t, J = 6.2 Hz, 2H), 2.62 (t, J = 6.2 Hz, 2H), 2.47 (t, J = 4.2 Hz, 4H). ¹³C NMR (CDCl₃) δ 148.8, 148.7, 148.1, 147.5, 142.7, 130.9, 126.5, 125.6, 125.2, 124.8, 123.7, 119.8, 110.1, 106.5, 104.3, 101.0, 100.9, 67.0, 57.9, 56.2, 56.1, 54.3, 50.7, 48.9. HRMS (ESI) m/z: 449.2073 [M + H]⁺, calcd for C₂₆H₂₉N₂O₅ 449.2071. ESI-MS m/z: 449.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-morpholinopropyl)-12,13-dihydro-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (30b).

White solid, yield 72%, mp = 138.2–1139.9 °C. ¹H NMR (CDCl₃) δ 7.70 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.77 (s, 1H), 6.05 (s, 2H), 4.16 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 3.72 (t, J = 4.4 Hz, 4H), 2.77 (t, J = 7.2 Hz, 2H), 2.44 (s, br 4H), 2.39 (d, J = 7.2 Hz, 2H), 1.83 (quint, J = 7.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 148.8, 148.6, 148.1, 147.5, 142.9, 130.9, 126.5, 125.5, 124.9, 123.8, 119.9, 110.0, 106.4, 104.4, 101.0, 100.8, 67.0, 56.2, 56.1, 53.8, 49.8, 49.4, 25.3. HRMS (ESI) m/z: 463.2212 [M + H]⁺, calcd for C₂₇H₃₁N₂O₅ 463.2227.

2,3-Dimethoxy-12-(2-(4-methylpiperazin-1-yl)ethyl)-12,13-dihydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (31a).

White solid, yield 62%, mp = 165.8–167.0 °C. ¹H NMR (CDCl₃) δ 7.86 (s, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.78 (s, 1H), 6.05 (s, 2H), 4.23 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 2.90 (t, J = 6.4 Hz, 2H), 2.67–2.32 (m, 10H), 2.30 (s, 3H). ¹³C NMR (CDCl₃) δ 148.7, 148.6, 148.1, 147.5, 142.8, 130.9, 126.5, 125.5, 125.2, 124.8, 123.7, 119.8, 110.1, 106.4, 104.3, 101.0, 100.9, 57.4, 56.2, 56.1, 55.1, 53.7, 50.7, 49.2, 46.0. HRMS (ESI) m/z: 462.2398 [M + H]⁺, calcd for C₂₇H₃₂N₃O₄ 462.2387. ESI-MS m/z: 462.2 [M + H]⁺.

2,3-Dimethoxy-12-(3-(4-methylpiperazin-1-yl)propyl)-12,13-dihydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (31b).

White solid, yield 53%, mp = 52.9–54.8 °C. ¹H NMR (CDCl₃) δ 7.71–7.65 (m, 2H), 7.49 (d, J = 8.5 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.78 (s, 1H), 6.05 (s, 2H), 4.16 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 2.75 (t, J = 7.2 Hz, 2H), 2.60–2.35 (m, 10H), 2.29 (s, 3H), 1.84 (quint, J = 7.6 Hz, 2H). ¹³C NMR (CDCl₃) δ 148.8, 148.6, 148.0, 147.4, 142.9, 130.9, 126.5, 125.5, 124.9, 124.9, 123.8, 119.9, 110.0, 106.5, 104.4, 101.0, 100.8, 56.2, 56.1, 55.7, 55.0, 53.1, 49.8, 49.7, 45.9, 25.7. HRMS (ESI) m/z: 476.2525 [M + H]⁺, calcd for C₂₈H₃₄N₃O₄ 476.2544.

12-(2-(1H-Imidazol-1-yl)ethyl)-2,3-dimethoxy-12,13-dihydro-[1,3] dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (32a).

White powder, yield 67%, mp = 199.2–200.4 °C. ¹H NMR (CDCl₃) δ 7.67 (d, J = 8.6 Hz, 1H), 7.56–7.46 (m, 2H), 7.31 (s, 1H), 7.13 (s, 1H), 7.10 (s, 1H), 6.96 (s, 1H), 6.93 (s, 1H), 6.74 (s, 1H), 6.04 (s, 2H), 4.12 (s, 2H), 4.09 (t, J = 6.4 Hz, 2h), 4.00 (s, 3H), 3.96 (s, 3H), 3.11 (t, J = 6.4 Hz, 2H). ¹³C NMR (CDCl₃) δ 149.2, 149.0, 148.5, 147.8, 141.4, 137.5, 131.1, 129.8, 126.3, 125.4, 125.4, 124.8, 124.5, 119.9, 119.3, 110.1, 106.8, 104.5, 101.3, 100.0, 56.4, 56.2, 52.7, 50.8, 45.5. HRMS (ESI) m/z: 430.1759 [M + H]⁺, calcd for C₂₅H₂₄N₃O₄ 430.1761. ESI-MS m/z: 430.2 [M + H]⁺.

12-(3-(1H-Imidazol-1-yl)propyl)-2,3-dimethoxy-12,13-dihydro-[1,3] dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (32b).

White powder, yield 55%, mp = 143.2–144.3 °C. ¹H NMR (CDCl₃) δ 7.68 (d, J = 8.4 Hz, 1H), 7.54–7.49 (m, 2H), 7.38 (s, 1H), 7.31 (s, 1H), 7.12 (s, 1H), 7.02 (s, 1H), 6.83 (s, 1H), 6.75 (s, 1H), 6.06 (s, 2H), 4.14 (s, 2H), 4.00 (s, 3H), 3.95 (s, 3H), 3.92 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.08 (quint, J = 7.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 149.1, 148.8, 148.3, 147.7, 142.0, 137.0, 131.0, 129.6, 126.4, 125.4, 125.2, 125.0, 124.2, 120.0, 118.8, 109.9, 106.6, 104.6, 101.2, 100.3, 56.3, 56.2, 50.8, 49.6, 45.1, 30.0. HRMS (ESI) m/z: 444.1920 [M + H]⁺, calcd for C₂₆H₂₆N₃O₄ 444.1918.

Synthesis of N-(4-Methoxybenzyl)-6-bromoveratraldimine (33).

According to the “General Procedure for Synthesis of Schiff’s Base 12a and 12b”, compound 33 was prepared using 4-methoxybenzylamine as material. ¹H NMR (CDCl₃) δ 8.64 (t, J = 1.3 Hz, 1H), 7.59 (s, 1H), 7.27–7.24 (m, 2H), 7.00 (s, 1H), 6.92–6.86 (m, 2H), 4.77 (s, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.81 (s, 3H).

Synthesis of 3-(Benzo[d][1,3]dioxol-5-yl)-4-(2-hydroxyethyl)-6,7-dimethoxy-2-(4-methoxybenzyl)isoquinolin-1(2H)-one (34).

According to the “General Procedure for Synthesis of 15a and 15b”, compound 34 was prepared using 33 as material. White solid, yield 62%. ¹H NMR (CDCl₃) δ 7.91 (s, 1H), 7.13 (s, 1H), 6.79–6.62 (m, 5H), 6.51–6.41 (m, 2H), 5.99 (s, 1H), 5.95 (s, 1H), 5.06 (d, J = 14.4 Hz, 1H), 4.94 (d, J = 14.1 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.69 (s, 3H), 3.64 (t, J = 6.8 Hz, 2H), 2.77–2.64 (m, 2H). ¹³C NMR (CDCl₃) δ 161.7, 158.5, 153.6, 149.1, 147.9, 147.6, 140.0, 131.9, 130.0, 128.0, 128.0, 123.8, 119.7, 113.6, 111.9, 110.4, 108.5, 108.1, 104.0, 101.4, 62.2, 56.1, 56.1, 55.2, 48.6, 32.0. The structure was further confirmed with 2D NMR spectra.

Synthesis of 2-(3-(Benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-2-(4-methoxybenzyl)-1-oxo-1,2-dihydroisoquinolin-4-yl)acetaldehyde (35).

According to the “General Procedure for Synthesis of 16a and 16b”, compound 35 was prepared using 34 as material. White solid, yield 82%. ¹H NMR (CDCl₃) δ 9.56 (t, J = 2.0 Hz, 1H), 7.98 (s, 1H), 6.87–6.72 (m, 7H), 6.52 (s, 1H), 6.05 (s, 2H), 5.19 (d, J = 14.6 Hz, 1H), 5.06 (d, J = 14.7 Hz, 1H), 4.05 (s, 3H), 3.99 (s, 3H), 3.76 (s, 3H), 3.50 (d, J = 2.0 Hz, 2H). ¹³C NMR (CDCl₃) δ 199.6, 161.8, 158.6, 153.8, 149.4, 148.3, 148.0, 141.7, 131.6, 129.9, 128.1, 127.7, 123.7, 119.7, 113.7, 110.0, 108.8, 108.5, 106.6, 103.5, 101.6, 56.2, 56.1, 55.2, 48.8, 44.4. ESI-MS m/z: 488.2 [M + H]⁺.

Synthesis of 2,3-Dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]-phenanthridin-13(12H)-one (36).

The reaction solution of 35 (270 mg, 0.5 mmol) and concentrated hydrochloric acid (0.4 mL) in acetic acid (4 mL) was added to a 50 mL round-bottomed flask. The flask was sealed with a rubber stopper. The reaction solution was stirred at room temperature overnight, and then the formed precipitate was filtered and washed with saturated sodium bicarbonate and water consecutively. The crude solid was dried and purified by silica gel column chromatography to give white solid 36, yield 62%. IR (KBr, cm⁻¹), 1643 (sh), 1500. ¹H NMR (DMSO) δ 11.53 (s, 1 H), 8.36 (s, 1 H), 8.33 (d, J = 8.8 Hz, 1 H), 7.92 (s, 1 H), 7.75 (s, 1 H), 7.62 (d, J = 8.8 Hz, 1 H), 7.41 (s, 1 H), 6.18 (s, 2 H), 4.05 (s, 3 H), 3.93 (s, 3 H). ESI-MS m/z: 350.1 [M + H]⁺.

Synthesis of 13-Chloro-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridine (37).

The reaction solution of 36 (350 mg, 1 mmol), POCl₃ (10 mL), and two drops of DMF was stirred at 100 °C overnight. The reaction was quenched by the addition of water (20 mL) at 0 °C and diluted with ethyl acetate (20 mL). The organic layer was washed with saturated sodium bicarbonate (10 mL) and water (10 mL) consecutively and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give white solid 37, yield 65%. ¹H NMR (CDCl₃) δ 8.57 (s, 1H), 8.20 (d, J = 8.8 Hz, 1H), 7.84 (s, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.74 (s, 1H), 7.23 (s, 1H), 6.13 (s, 2H), 4.17 (s, 3H), 4.11 (s, 3H). The ¹H NMR spectrum is similar to that reported.⁷² ESI-MS m/z: 368.1 (100%), 370.0 (35%) [M + H]⁺.

Synthesis of 13-(3-Bromopropoxy)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (38).

The reaction solution of 36 (300 mg, 0.86 mmol) and NaH (60%, 105 mg, 2.7 mmol) in DMF (20 mL) was stirred at room temperature for 30 min. 1,3-Dibromopropane (869 mg, 4.3 mmol) was added and stirred at room temperature for 3 h, and then the reaction solution was added with water (10 mL) at 0 °C and diluted by ethyl acetate (50 mL). The organic layer was washed with saturated saline (3 × 20 mL) and dried over MgSO₄ and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give white solid 38, yield 86%. IR (KBr, cm⁻¹), 1596. ¹H NMR (CDCl₃) δ 8.47 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.73 (s, 1H), 7.62 (t, J = 8.8 Hz, 1H), 7.60 (s, 1H), 7.19 (s, 1H), 6.09 (s, 2H), 4.88 (t, J = 7.0 Hz, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.69 (d, J = 7.0 Hz, 2H), 2.57 (quint, J = 7.0 Hz, 2H). ¹³C NMR (CDCl₃) δ 156.9, 152.7, 149.2, 148.0, 147.8, 138.7, 131.0, 129.9, 128.1, 123.7, 118.2, 117.4, 113.7, 104.2, 104.2, 102.2, 102.0, 101.2, 63.8, 56.1, 56.0, 32.4, 30.4.). ESI-MS m/z: 470.0 (100%), 472.0 (100%) [M + H]⁺.

General Procedure of Synthesis of 39a–45a.

To a solution of ethanolamine derivatives (2 mmol) and NaH (2 mmol) in dried THF (50 mL), 37 (146 mg, 0.4 mmol) was added. The reaction solution was stirred at 70 °C overnight. The reaction was quenched by the addition of water (10 mL) at 0 °C. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give the target compound, respectively.

2,3-Dimethoxy-13-(2-(dimethylamino)ethoxyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (39a).

White powder, yield 62%, mp = 159.8–160.5 °C. IR (KBr, cm⁻¹), 1620, 1593. ¹H NMR (CDCl₃) δ 8.54 (s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.82 (s, 1H), 7.73 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 6.11 (s, 2H), 4.91 (t, J = 5.6 Hz, 2H), 4.14 (s, 3H), 4.07 (s, 3H), 3.00 (t, J = 5.6 Hz, 2H), 2.46 (s, 6H). ¹³C NMR (CDCl₃) δ 157.2, 152.6, 149.2, 148.0, 147.8, 138.8, 131.0, 130.0, 128.2, 123.6, 118.3, 117.4, 113.9, 104.5, 104.3, 102.2, 102.1, 101.2, 64.2, 58.3, 56.1, 56.1, 46.2. HRMS (ESI) m/z: 421.1763 [M + H]⁺, calcd for C₂₄H₂₅N₂O₅ 421.1758.

2,3-Dimethoxy-13-(2-(diethylamino)ethoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (40a).

White solid, yield 50%, mp = 161.2–162.5 °C. IR (KBr, cm⁻¹), 1620, 1591. ¹H NMR (CDCl₃) δ 8.53 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H), 7.68 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.20 (s, 1H), 6.10 (s, 2H), 4.84 (t, J = 6.2 Hz, 2H), 4.12 (s, 3H), 4.05 (s, 3H), 3.11 (t, J = 6.2 Hz, 2H), 2.77 (q, J = 7.1 Hz, 4H), 1.17 (t, J = 7.1 Hz, 6H). ¹³C NMR (CDCl₃) δ 157.3, 152.6, 149.2, 148.0, 147.8, 138.9, 131.0, 129.9, 128.2, 123.6, 118.3, 117.3, 113.9, 104.5, 104.2, 102.2, 102.1, 101.1, 64.6, 56.0, 51.3, 48.2, 12.3. HRMS (ESI) m/z: 449.2079 [M + H]⁺, calcd for C₂₆H₂₉N₂O₅ 449.2071. ESI-MS m/z: 449.2 [M + H]⁺.

2,3-Dimethoxy-13-(2-(pyrrolidin-1-yl)ethoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (41a).

White powder, yield 53%, mp = 175.2–175.8 °C. IR (KBr, cm⁻¹), 1620, 1593. ¹H NMR (CDCl₃) δ 8.54 (s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.81 (s, 1H), 7.72 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.22 (s, 1H), 6.11 (s, 2H), 4.94 (t, J = 6.0 Hz, 2H), 4.14 (s, 3H), 4.07 (s, 3H), 3.16 (t, J = 6.0 Hz, 2H), 2.82–2.74 (m, 4H), 1.89–1.82 (m, 4H). ¹³C NMR (CDCl₃) δ 157.3, 152.7, 149.3, 148.1, 147.9, 138.9, 131.1, 130.0, 128.3, 123.7, 118.4, 117.5, 114.0, 104.6, 104.4, 102.3, 102.2, 101.3, 65.2, 56.2, 56.2, 55.1, 55.0, 23.7. HRMS (ESI) m/z: 447.1909 [M + H]⁺, calcd for C₂₆H₂₇N₂O₅ 447.1914.

2,3-Dimethoxy-13-(2-(piperidin-1-yl)ethoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (42a).

White powder, yield 43%, mp = 189.0–189.6 °C. IR (KBr, cm⁻¹), 1620, 1594. ¹H NMR (CDCl₃) δ 8.55 (s, 1H), 8.17 (d, J = 9.2 Hz, 1H), 7.81 (s, 1H), 7.71 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.22 (s, 1H), 6.11 (s, 2H), 4.92 (t, J = 6.4 Hz, 2H), 4.14 (s, 3H), 4.07 (s, 3H), 3.03 (t, J = 6.4 Hz, 2H), 2.77–2.60 (m, 4H), 1.73–1.69 (m, 2H), 1.66–1.63 (m, 2H), 1.53–1.45 (m, 2H). ¹³C NMR (CDCl₃) δ 157.3, 152.8, 149.3, 148.1, 147.9, 139.0, 131.1, 130.1, 128.3, 123.7, 118.4, 117.5, 114.1, 104.6, 104.4, 102.3, 102.2, 101.3, 64.1, 58.0, 56.2, 55.3, 26.2, 24.4. HRMS (ESI) m/z: 461.2068 [M + H]⁺, calcd for C₂₇H₂₉N₂O₅ 461.2071.

2,3-Dimethoxy-13-(2-morpholinoethoxy)-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridine (43a).

White powder, yield 76%, mp = 198.4–199.6 °C. IR (KBr, cm⁻¹), 1619, 1593. ¹H NMR (CDCl₃) δ 8.53 (s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.81 (s, 1H), 7.69–7.65 (m, 2H), 7.23 (s, 1H), 6.11 (s, 2H), 4.93 (t, J = 5.8 Hz, 2H), 4.14 (s, 3H), 4.07 (s, 3H), 3.82–3.75 (m, 4H), 3.05 (t, J = 5.8 Hz, 2H), 2.77–2.67 (m, 4H). ¹³C NMR (CDCl₃) δ 157.2, 152.8, 149.3, 148.1, 147.9, 138.9, 131.1, 130.1, 128.3, 123.8, 118.4, 117.5, 113.9, 104.4, 104.3, 102.3, 102.1, 101.3, 67.2, 63.8, 57.7, 56.2, 54.3. HRMS (ESI) m/z: 463.1859 [M + H]⁺, calcd for C₂₆H₂₇N₂O₆ 463.1864.

2,3-Dimethoxy-13-(2-(4-methylpiperazin-1-yl)ethoxy)-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (44a).

White powder, yield 74%, mp = 206.5–207.4 °C. IR (KBr, cm⁻¹), 1622, 1596. ¹H NMR (CDCl₃) δ 8.54 (s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.82 (s, 1H), 7.73–7.63 (m, 2H), 7.23 (s, 1H), 6.11 (s, 2H), 4.92 (t, J = 6.4 Hz, 2H), 4.14 (s, 3H), 4.06 (s, 3H), 3.07 (t, J = 6.4 Hz, 2H), 2.92–2.65 (m, 4H), 2.65–2.41 (m, 4H), 2.32 (s, 3H). ¹³C NMR (CDCl₃) δ 157.2, 152.7, 149.3, 148.1, 147.9, 138.9, 131.1, 130.0, 128.3, 123.7, 118.3, 117.5, 113.9, 104.5, 104.3, 102.3, 102.2, 101.3, 64.1, 57.2, 56.1, 55.3, 53.8, 46.2. HRMS (ESI) m/z: 476.2179 [M + H]⁺, calcd for C₂₇H₃₀N₃O₅ 476.2180.

13-(2-(1H-Imidazol-1-yl)ethoxy)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (45a).

White powder, yield 56%, mp = 238.9–239.1 °C. IR (KBr, cm⁻¹), 1621, 1597. ¹H NMR (CDCl₃) δ 8.47 (s, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.67 (s, 1H), 7.58 (s, 1H), 7.24 (s, 1H), 7.12–7.09 (m, 2H), 6.12 (s, 2H), 5.06 (t, J = 5.2 Hz, 2H), 4.59 (t, J = 5.2 Hz, 2H), 4.15 (s, 3H), 4.06 (s, 3H). ¹³C NMR (CDCl₃) δ 156.4, 153.1, 149.6, 148.2, 148.0, 138.5, 137.7, 131.3, 130.1, 129.8, 128.2, 124.2, 119.5, 118.4, 117.9, 113.6, 104.5, 104.1, 102.4, 101.9, 101.4, 64.9, 56.3, 56.2, 46.5. HRMS (ESI) m/z: 444.1557 [M + H]⁺, calcd for C₂₅H₂₂N₃O₅ 444.1554.

General Procedure for Synthesis of 39b–45b.

The solution of 38 (221 mg, 0.47 mmol), K₂CO₃ (163 mg, 1.18 mmol), KI (20 mg, 0.118 mmol), and amines (in pressure vessel for dimethylamine. 0.7 mmol) in DMF (20 mL) was stirred at room temperature overnight, and then the reaction solution was diluted with ethyl acetate (100 mL) and washed with saturated saline (3 × 50 mL). The organic layer was dried (MgSO₄) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give target product, respectively.

2,3-Dimethoxy-13-(3-(dimethylamino)propoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (39b).

White powder, yield 82%, mp = 148.6–150.4 °C. IR (KBr, cm⁻¹), 1618, 1593. ¹H NMR (CDCl₃) δ 8.56 (s, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.72 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 6.11 (s, 2H), 4.82 (t, J = 7.4 Hz, 1H), 4.14 (s, 3H), 4.07 (s, 3H), 2.60 (t, J = 7.4 Hz, 1H), 2.32 (s, 6H), 2.20 (quint, J = 7.4 Hz, 1H). ¹³C NMR (CDCl₃) δ 157.5, 152.7, 149.3, 148.0, 147.8, 139.0, 131.1, 130.0, 128.3, 123.6, 118.4, 117.4, 114.0, 104.5, 104.4, 102.3, 102.2, 101.2, 64.5, 57.0, 56.2, 56.2, 45.7, 27.5. HRMS (ESI) m/z: 435.1921 [M + H]⁺, calcd for C₂₅H₂₇N₂O₅ 435.1914. ESI-MS m/z: 435.3 [M + H]⁺.

2,3-Dimethoxy-13-(3-diethylamino)propoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (40b).

White powder, yield 61%, mp = 146.2–147.8 °C. IR (KBr, cm⁻¹), 1620, 1594. ¹H NMR (DMSO) δ 8.45 (d, J = 9.0 Hz, 1H), 8.35 (s, 1H), 8.04 (s, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.57 (s, 1H), 7.43 (s, 1H), 6.19 (s, 2H), 4.71 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.93 (s, 3H), 2.70 (t, J = 6.8 Hz, 2H), 2.53 (q, J = 7.2 Hz, 4H), 2.05 (quint, J = 6.8 Hz, 2H), 1.01 (t, J = 7.2 Hz, 6H). ¹³C NMR (DMSO) δ 157.3, 153.3, 149.7, 148.2, 148.0, 138.3, 131.0, 130.1, 127.7, 123.9, 119.6, 117.5, 113.4, 104.7, 104.6, 103.6, 101.8, 101.4, 64.5, 56.4, 55.9, 49.1, 47.0, 26.4, 12.2. HRMS (ESI) m/z: 463.2233 [M + H]⁺, calcd for C₂₇H₃₁N₂O₅ 463.2227. ESI-MS m/z: 463.2 [M + H]⁺.

2,3-Dimethoxy-13-(3-(pyrrolidin-1-yl)propoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (41b).

White powder, yield 80%, mp = 162.8–163.9 °C. IR (KBr, cm⁻¹), 1619, 1594. ¹H NMR (CDCl₃) δ 8.48 (s, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.71 (s, 1H), 7.67–7.57 (m, 2H), 7.18 (s, 1H), 6.09 (s, 2H), 4.77 (t, J = 6.8 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.78 (t, J = 6.8 Hz, 2H), 2.62 (s, br, 4H), 2.24 (quint, J = 6.8 Hz, 2H), 1.83 (s, br, 4H). ¹³C NMR (CDCl₃) δ 157.3, 152.5, 149.1, 147.9, 147.7, 138.8, 130.9, 129.9, 128.2, 123.5, 118.2, 117.2, 113.9, 104.3, 104.2, 102.1, 101.1, 64.5, 56.1, 56.0, 54.4, 53.7, 28.8, 23.5. HRMS (ESI) m/z: 461.2082 [M + H]⁺, calcd for C₂₇H₂₉N₂O₅ 461.2071. ESI-MS m/z: 461.2 [M + H]⁺.

2,3-Dimethoxy-13-(3-(piperidin-1-yl)propoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (42b).

White powder, yield, 81%, mp = 153.7–155.3 °C. IR (KBr, cm⁻¹), 1618, 1593. ¹H NMR (CDCl₃) δ 8.51 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H), 7.69–7.60 (m, 2H), 7.21 (s, 1H), 6.10 (s, 2H), 4.78 (t, J = 6.2 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 2.66 (t, J = 7.6 Hz, 2H), 2.51 (s, br, 4H), 2.24 (quint, J = 7.2 Hz, 2H), 1.72–1.58 (m, 4H), 1.55–1.41 (m, 2H). ¹³C NMR (CDCl₃) δ 157.4, 152.6, 149.2, 147.9, 147.7, 138.9, 130.9, 129.9, 128.2, 123.5, 118.3, 117.3, 113.9, 104.3, 104.3, 102.2, 102.1, 101.1, 64.6, 56.6, 56.1, 56.0, 54.7, 26.6, 25.9, 24.4. HRMS (ESI) m/z: 475.2229 [M + H]⁺, calcd for C₂₈H₃₁N₂O₅ 475.2227. ESI-MS m/z: 475.2 [M + H]⁺.

2,3-Dimethoxy-13-(3-morpholinopropoxy)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (43b).

White powder, yield 65%, mp = 171.8–172.9 °C. IR (KBr, cm⁻¹), 1618, 1593. ¹H NMR (CDCl₃) δ 8.53 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 7.82 (s, 1H), 7.69 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 6.11 (s, 2H), 4.83 (t, J = 6.6 Hz, 2H), 4.13 (s, 3H), 4.07 (s, 3H), 3.76 (t, J = 4.8 Hz, 4H), 2.67 (t, J = 7.2Hz, 2H), 2.56–2.53 (m, 4H), 2.22 (quint, J = 7.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 157.3, 152.6, 149.2, 147.9, 147.7, 138.8, 130.9, 129.9, 128.2, 123.5, 118.2, 117.3, 113.9, 104.3, 104.3, 102.2, 102.0, 101.1, 67.0, 64.3, 56.1, 56.1, 56.0, 53.9, 26.3. HRMS (ESI) m/z: 477.2027 [M + H]⁺, calcd for C₂₇H₂₉N₂O₆ 477.2020. ESI-MS m/z: 477.2 [M + H]⁺.

2,3-Dimethoxy-13-(3-(4-methylpiperazin-1-yl)propoxy)-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridine (44b).

White powder, yield 65%, mp = 142.5–143.8 °C. IR (KBr, cm⁻¹), 1619, 1593. ¹H NMR (CDCl₃) δ 8.55 (s, 1H), 8.18 (d, J = 9.0 Hz, 1H), 7.83 (s, 1H), 7.70 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 6.11 (s, 2H), 4.83 (t, J = 6.4 Hz, 2H), 4.14 (s, 3H), 4.07 (s, 3H), 2.70 (t, J = 7.2 Hz, 2H), 2.67–2.41 (m, 8H), 2.32 (s, 3H), 2.22 (quint, J = 7.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 157.3, 152.6, 149.2, 147.9, 147.7, 138.9, 131.0, 129.9, 128.2, 123.5, 118.3, 117.3, 114.0, 104.4, 104.2, 102.2, 102.1, 101.1, 64.5, 56.1, 56.0, 55.8, 55.2, 53.4, 46.0, 26.7. HRMS (ESI) m/z: 490.2339 [M + H]⁺, calcd for C₂₈H₃₂N₃O₅ 490.2336. ESI-MS m/z: 490.23 [M + H]⁺.

13-(3-(1H-Imidazol-1-yl)propoxy)-2,3-dimethoxy-[1,3)dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridine (45b).

White powder, yield 62%, mp = 217.4–218.5 °C. IR (KBr, cm⁻¹), 1618, 1591. ¹H NMR (CDCl₃) δ 8.47 (s, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.84 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.25 (s, 1H), 7.12 (s, 1H), 7.03 (s, 1H), 6.13 (s, 2H), 4.83 (t, J = 6.0 Hz, 2H), 4.28 (t, J = 6.8 Hz, 2H), 4.16 (s, 3H), 4.11 (s, 3H), 2.53 (quint, J = 6.4 Hz, 2H). ¹³C NMR (CDCl₃) δ 156.8, 152.9, 149.4, 148.1, 147.8, 138.6, 137.2, 131.2, 130.0, 129.8, 128.1, 123.9, 118.9, 118.2, 117.5, 113.6, 104.3, 104.1, 102.4, 101.9, 101.2, 62.6, 56.1, 56.1, 44.3, 30.7. HRMS (ESI) m/z: 458.1712 [M + H]⁺, calcd for C₂₆H₂₄N₃O₅ 458.1710. ESI-MS m/z: 458.2 [M + H]⁺.

TOP1-Mediated Cleavage Assay.

DNA cleavage assays were performed as previously reported.⁷³ A 3′-[³²P]-labeled 117-bp DNA oligonucleotide was prepared as previously described.⁷³ Approximately 2 nM radiolabeled DNA substrate was incubated with recombinant TOP1 in 20 mL 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 ImageQuant software (Molecular Dynamics). Cleavage sites are numbered to reflect actual sites on the 117-bp oligonucleotide.

TOP1-Mediated Unwinding Assay.

The unwinding assay was performed according to the reported method.⁷⁸ Briefly, the reaction mixture (20 μL) contained supercoiled or relaxed pBR322 DNA (0.5 μg), tested compounds, and excess TOP1 (10 units) in relaxing buffer (10 mM Tris, pH 7.5, 0.1 mM EDTA, 5 mM MgCl₂, 50 mM KCl, 1 mM DTT, 15 μg/mL acetylated BSA). The DNA was incubated with the tested compounds at room temperature for 10 min prior to the addition of TOP1. After being incubated for 30 min at 37 °C, the reaction was terminated by the addition of 4 mL of loading buffer. The results were analyzed using 1% agarose gel in TAE buffer at 5 V/cm. The gel was stained with gel red and visualized with a UV transilluminator.

TDP1 Inhibition Assay.

A. Fluorescence Assay.

The TDP1 fluorescence assay was conducted according to the reported method.⁵⁰ Briefly, a linear oligonucleotide labeled with FAM (donorfluorophore, 6-carboxyfluorescein) and BHQ (Black Hole Quencher), 5′-FAM-AGGATCTAAAAGACTT-BHQ-3′ was designed as a linear quenched fluorescent substrate. TDP1 solution (20 μL/well, 0.02 μL of purified TDP1 (100 nM) in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM DTT) was dispensed into wells of a white 384-well plate (NEST). The tested compound solution in DMSO (5 μL) was pinned into assay plates and incubated at room temperature for 30 min. During this time, the plates were read by a Flash multimode reader (Molecular Devices) at Ex₄₈₅/Em₅₁₀ nm to identify false-positive compounds that had autofluorescence. The linear oligonucleotide substrate (25 μL, 35 nM) was dispensed into the wells to start the reaction. The whole plate was immediately read five times using a kinetic read on the Flash multimode reader (Molecular Devices) (Ex₄₈₅/Em₅₁₀ nm). TDP1 percentage inhibition of the tested compounds was calculated by comparing the rate of increase in fluorescence throughout time for the compound-treated wells to that of DMSO control wells.

B. Gel-Based Assay.⁷⁶

A 5′-[³²P]-labeled single-stranded DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y) was incubated at 1 nM with 10 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, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE. Gels were dried and exposed to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon FLA 9500 scanner (GE Healthcare), and densitometry analyses were performed using the ImageQuant software (GE Healthcare)

Melecular Modeling.

The X-ray crystal structures of the ternary TOP1–DNA–ligand complex (PDB 1K4T) was obtained and cleaned and inspected for errors and missing residues, hydrogens were added, and the water molecules and the ligand were deleted. The ternary complex ligand centroid coordinates for docking were defined using the ligand in the complex structure as the center of the binding pocket. Compounds were constructed and optimized using ChemDraw and saved in SDFfile formats and were corrected using MOE software. Hydrogens were added, and the ligands were minimized by the conjugate gradient method using the MMFF94x force field with MMFF94 charges, a distance-dependent dielectric function, and a 0.01 kcal/mol·Å energy gradient convergence criterion. Induced fit was used for docking with the default parameters. The top 30 docking poses per ligand were inspected visually following the docking runs. The highest-ranked poses for these ligands were merged into the crystal structure. Energy minimizations were performed for the highest-ranked poses for these ligands. The AMBER force field was utilized within the MOE software for energy minimization.

The TDP1 crystal structure (PDB 1RFF) was prepared by removing one of the monomers along with all crystallized waters, the polydeoxyribonucleotide, the TOP1-derived peptide residues, and all metal ions. LigX in AMBER10 force field with MOE. Site Finder was used to get the binding pocket containing Lys265, Lys495, and His493. Docking was performed with induced fit protocol in MOE. The top ligand-binding pose (lowest score) was selected and merged with the prepared protein. The AMBER force field was utilized within the MOE software for energy minimization. The calculation was terminated when the gradient reached a value of 0.05 kcal/mol·Å.

FRET Melting Assay.

FRET melting assay was carried out on a real-time PCR apparatus as the following methods. The oligonucleotide labeled with FAM (donorfluorophore, 6-carboxyfluorescein) and TAMRA (acceptor fluorophore, 6-carboxytetramethylrhodamine), F10T (5′-FAM-d(TATAGCTATA-HEG-TATAGCTATA)-TAM-ARA-3′), was purchased from Sangon Biotech. In the experiment, the annealed oligonucleotide (final concentration of 0.4 μM) were incubated with the tested compounds (2 μM) in a total reaction volume of Tris-HCl buffer (10 mM, pH 7.4) containing 60 mM KCl at 37 °C for 0.5 h. Fluorescence melting curves were determined with a Roche LightCycler 2 real-time PCR machine with excitation at 470 nm and detection at 530 nm. Fluorescence readings were taken at an interval of 1 °C over the range 37–99 °C, with a constant temperature being maintained for 30 s prior to each reading to ensure a stable value.

Cell Culture and MTT Assay.

The cells were cultured on RPMI-1640 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 (predissolved in DMSO) at a five-dose assay ranging from 0.01 to 100 μM (0.001–10 μM for camptothecin) concentration. After incubation for 72 hat 37 °C, MTT solution (50 μL, 1 mg/mL) in PBS (PBS without MTT as the blank) was fed to each well of the culture plate (containing 100 mL medium). After 4h incubation, the formazan crystal formed in the well was dissolved with 100 mL of DMSO for optical density reading at 570 nm.⁸⁹ The GI₅₀ value was calculated by nonlinear regression analysis (GraphPad Prism).

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

Immunodetection of Cellular TOP1–DNA Complex.

The ICE assays for cellular TOP1–DNA adduct was performed according to the reported method.⁹⁰ Briefly, mid log phase HCT-116 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, and the solution was incubated overnight at −20 °C. 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, supernatant was used to quantify the DNA concentration. DNA (2 μg) 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) overnight at 4 °C 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).

γH2AX Detection.

γH2AX staining was performed as described.⁷⁴ Briefly, HCT116 cells (2 × 10⁴ cells/mL) were grown in culture medium and treated with compounds for 3 h at 37 °C. After incubation, cells were fixed in 4% paraformaldehyde/PBS for 15 min at 25 °C and washed three times with PBS buffer. Cells were permeabilized with 0.5% Triton X-100 in PBS at 0 °C for 30 min. Dish was blockedwith 5% goat serum/PBS at 37 °C for 3 h. Immunofluorescence assay was performed using standard methods, and the slides were incubated alternately with phospho-γH2AX (Ser139; no. 9718, Cell Signaling Technology) at 37 °C overnight. The coverslips were washed six times with blocking buffer and then incubated with antirabbit alexa 488-conjugated antibody (A21206, Life Technology) and 2.0 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 37 °C for 2 h. The dishes were again washed six times with blocking buffer. Digital images were recorded using an LSM710 microscope (Zeiss, Germany) and analyzed with ZEN software.

Flow Cytometry.

HCT116 cells (3.0 × 10⁵ cells/mL) were grown in culture medium on a 6-well plate treated with compound at the indicated concentration for 24 h. Then the cells were harvested from the medium and washed with cold PBS, resuspended in 1× binding buffer, and then stained with 5.0 μL of FITC annexin V and 10.0 μL of propidium iodide (KeyGen Biotech, China) for 15 min in the dark. The stained cells were analyzed by using flow cytometry (BD, FACSCalibur, USA) within 1 h. The experiments were repeated three times independently.

Pharmacokinetic Study in Rat.

Male SD rats (weighing 220–250 g, n = 3) were treated with compound 19a predissolved in 10% DMSO and 10% Kolliphor HS15 (a nonionic solubilizer) by iv (1 mg/kg) and by ig administration (5 mg/kg), respectively. Blood samples (200 μL) were collected into heparinized tubes via the jugular vein at the following times: 0.083, 0.25, 0.5, 1, 2, 5, 7, and 24 h after dosing. Plasma samples (100 μL) were obtained after centrifugation for 10 min at 3000 rpm and stored at −20 °C until used for analysis. The plasma was detected through LC-MS-MS.

In Vivo Acute Toxicity.

On the basis of the preliminary experiments, the Kuning male mice were randomly divided into six groups (n = 4) and administered by intraperitoneal (ip) injection. The control group was treated with an equivalent volume of sterile water. The testing groups were treated with compound 19a in a single dose 300, 240, 192, 154, and 123 mg/kg, respectively. The mice were kept under observation for 7 days post-treatment in order to check for any behavioral (poisoning symptoms and body weight) and death. All animals were euthanized by cervical dislocation at the end of the experiments.

In Vivo Antitumor Activity.

Athymic nude mice bearing the nu/nu gene were obtained from Laboratory Animal Center of Sun Yat-sen University and maintained in pathogen-free conditions to establish the model of xenografts of HCT116. Male nude mice 4–5 weeks old weighing 12–15 g were used. HCT116 tumor preinduced in the mice by subcutaneously injecting of HCT116 cells (100 μL, 1 × 10⁷ cells) was implanted. When the implanted tumors had reached a volume of about 80 mm³, the mice were randomly divided into three groups (n = 6) and administered by ip injection. The testing groups were treated with 19a in 10 and 5 mg/kg dose once every day, respectively. The negative control group was treated with an equivalent volume of saline. Tumor volumes (V) were monitored by caliper measurement of the length and width and calculated using the formula: V = (larger diameter × smaller diameter)²/2, and growth curves were plotted using average tumor volume within each experimental group at the set time points. At the end of the experiment, all of the animals were euthanized by cervical dislocation. The tumors were removed and weighed. The tumor weight inhibition (TWI) was calculated according to the formula: TWI = (1 − mean tumor weight of the experimental group/mean tumor weight of the control group) × 100%.

Statistical Analysis.

All data are expressed as the mean ± standard deviation. Statistical comparisons were conducted using a one-way analysis of variance (ANOVA) using the Prism statistical software package (GraphPad Software, USA), followed by Tukey’s test.

Supplementary Material

Supplemental figures

NIHMS1684587-supplement-Supplemental_figures.pdf^{(392.3KB, pdf)}

Compounds table

NIHMS1684587-supplement-Compounds_table.csv^{(7.6KB, csv)}

PDB file 4

NIHMS1684587-supplement-PDB_file_4.txt^{(561.9KB, txt)}

PDB file 3

NIHMS1684587-supplement-PDB_file_3.txt^{(913.4KB, txt)}

CIF file

NIHMS1684587-supplement-CIF_file.txt^{(389.8KB, txt)}

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (no. 81373257), Natural Science Foundation of Guangdong Province (no. S2013010015609), and supported by the Intramural Program of the National Cancer Institute (Center for Cancer Research), National Institutes of Health, Bethesda, Maryland, USA (Z01 BC 006150-19).

ABBREVIATIONS USED

TOP1: topoisomerase IB
TOP1cc: TOP1 cleavage complex
TDP1: tyrosyl DNA-phosphodiesterase 1
dsDNA: double-stranded DNA
P-gp: P-glycoprotein
ICE: immunocomplex of enzyme to DNA
FRET: fluorescence resonance energy transfer
MOM: methoxymethyl
PMB: p-methoxybenzyl
NMR: nuclear magnetic resonance
HRMS: high-resolution mass spectra
TLC: thin layer chromatography
HPLC: high performance liquid chromatography

Footnotes

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00639.

TOP1-mediated cleavage assaygel of compound 6 (PDF)

X-ray crystallography file of compound 20b (CIF)

Docking results of 19a with TOP1cc (PDB)

Docking results of 41a with TDP1 (PDB)

Molecular formula strings (CSV)

The authors declare no competing financial interest.

REFERENCES

(1).Stewart L; Redinbo MR; Qiu X; Hol WGJ; Champoux JJ A model for the mechanism of human topoisomerase I. Science 1998, 279, 1534–1541. [DOI] [PubMed] [Google Scholar]
(2).Champoux JJ DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem 2001, 70, 369–413. [DOI] [PubMed] [Google Scholar]
(3).Wang JC DNA topoisomerases. Annu. Rev. Biochem 1996, 65, 635–692. [DOI] [PubMed] [Google Scholar]
(4).Pommier Y; Sun Y; Huang SN; Nitiss JL Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat. Rev. Mol. Cell Biol 2016, 17, 703–721. [DOI] [PMC free article] [PubMed] [Google Scholar]
(5).Pommier Y; Leo E; Zhang H; Marchand C DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol 2010, 17, 421–433. [DOI] [PMC free article] [PubMed] [Google Scholar]
(6).Pommier Y; Marchand C Interfacial inhibitors: targeting macromolecular complexes. Nat. Rev. Drug Discovery 2012, 11, 25–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
(7).Choi SH; Tsuchida Y; Yang HW Oral versus intraperitoneal administration of irinotecan in the treatment of human neuroblastoma in nude mice. Cancer Lett. 1998, 124, 15–21. [DOI] [PubMed] [Google Scholar]
(8).Brogden RN; Wiseman LR Topotecan a review of its potential in advanced ovarian cancer. Drugs 1998, 56, 709–723. [DOI] [PubMed] [Google Scholar]
(9).Bracher F; Tremmel T From lead to drug utilizing a mannich reaction: The topotecan story. Arch. Pharm 2017, 350, e1600236. [DOI] [PubMed] [Google Scholar]
(10).Zhang R; Li Y; Cai Q; Liu T; Sun H; Chambless B Preclinical pharmacology of the natural product anticancer agent 10-hydroxycamptothecin, an inhibitor of topoisomerase I. Cancer Chemother. Pharmacol 1998, 41, 257–267. [DOI] [PubMed] [Google Scholar]
(11).Shao J; Huang F; Fu J Clinical study on treatment of non-small cell lung cancer with 10-hydroxycamptothecin. Chin. J. Clin. Oncol 2003, 30, 26–28. [Google Scholar]
(12).Pommier Y Topoisomerase I inhibitors: camptothecins and beyond. Nat. Rev. Cancer 2006, 6, 789–802. [DOI] [PubMed] [Google Scholar]
(13).Pommier Y DNA Topoisomerase I Inhibitors: chemistry, biology, and interfacial inhibition. Chem. Rev 2009, 109, 2894–2902. [DOI] [PMC free article] [PubMed] [Google Scholar]
(14).Beretta GL; Perego P; Zunino F Targeting topoisomerase I: molecular mechanisms and cellular determinants of response to topoisomerase I inhibitors. Expert Opin. Ther. Targets 2008, 12, 1243–1256. [DOI] [PubMed] [Google Scholar]
(15).Pommier Y; Barcelo JM; Rao VA; Sordet O; Jobson AG; Thibaut L; Miao ZH; Seiler JA; Zhang H; Marchand C; Agama K; Nitiss JL; Redon C Repair of topoisomerase I-mediated DNA damage. Prog. Nucleic Acid Res. Mol. Biol 2006, 81, 179–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
(16).Maede Y; Shimizu H; Fukushima T; Kogame T; Nakamura T; Miki T; Takeda S; Pommier Y; Murai J Differential and common DNA repair pathways for topoisomerase I- and II-targeted drugs in a genetic DT40 repair cell screen panel. Mol. Cancer Ther 2014, 13, 214–220. [DOI] [PMC free article] [PubMed] [Google Scholar]
(17).Interthal H; Pouliot JJ; Champoux JJ The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily. Proc. Natl. Acad. Sci. U. S. A 2001, 98, 12009–12014. [DOI] [PMC free article] [PubMed] [Google Scholar]
(18).Dexheimer TS; Antony S; Marchand C; Pommier Y Tyrosyl-DNA phosphodiesterase as a target for anticancer therapy. Anti-Cancer Agents Med. Chem 2008, 8, 381–389. [DOI] [PMC free article] [PubMed] [Google Scholar]
(19).Yang SW; Burgin AB; Huizenga JBN; Robertson CA; Yao KC; Nash HA A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases. Proc. Natl. Acad. Sci. U. S. A 1996, 93, 11534–11539. [DOI] [PMC free article] [PubMed] [Google Scholar]
(20).Murai J; Huang SY; Das BB; Dexheimer TS; Takeda S; Pommier Y Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells. J. Biol. Chem 2012, 287, 12848–12857. [DOI] [PMC free article] [PubMed] [Google Scholar]
(21).Interthal H; Chen HJ; Champoux JJ Human Tdp1 cleaves a broad spectrum of substrates including phosphoamide linkages. J. Biol. Chem 2005, 280, 36518–36528. [DOI] [PMC free article] [PubMed] [Google Scholar]
(22).Menon V; Povirk LF End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair. DNA Repair 2016, 43, 57–68. [DOI] [PubMed] [Google Scholar]
(23).Ashour ME; Atteya R; El-Khamisy SF Topoisomerase-mediated chromosomal break repair: an emerging player in many games. Nat. Rev. Cancer 2015, 15, 137–151. [DOI] [PubMed] [Google Scholar]
(24).Nitiss KC; Malik M; He X; White SW; Nitiss JL Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage. Proc. Natl. Acad. Sci. U. S. A 2006, 103, 8953–8958. [DOI] [PMC free article] [PubMed] [Google Scholar]
(25).Takashima H; Boerkoel CF;John J; Saifi GM; Salih MA; Armstrong D; Mao Y; Quiocho FA; Roa BB; Nakagawa M; Stockton DW; Lupski JR Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebellar ataxia with axonal neuropathy. Nat. Genet 2002, 32, 267–272. [DOI] [PubMed] [Google Scholar]
(26).Barthelmes HU; Habermeyer M; Christensen MO; Mielke C; Interthal H; Pouliot JJ; Boege F; Marko D TDP1 overexpression in human cells counteracts DNA damage mediated by topoisomerases I and II. J. Biol. Chem 2004, 279, 55618–55625. [DOI] [PubMed] [Google Scholar]
(27).Nivens MC; Felder T; Galloway AH; Pena MM; Pouliot JJ; Spencer HT Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase. Cancer Chemother. Pharmacol 2004, 53, 107–115. [DOI] [PubMed] [Google Scholar]
(28).Hirano R; Interthal H; Huang C; Nakamura T; Deguchi K; Choi K; Bhattacharjee MB; Arimura K; Umehara F; Izumo S; Northrop JL; Salih MA; Inoue K; Armstrong DL; Champoux JJ; Takashima H; Boerkoel CF Spinocerebellar ataxia with axonal neuropathy: consequence of a Tdp1 recessive neomorphic mutation? EMBO J. 2007, 26, 4732–4743. [DOI] [PMC free article] [PubMed] [Google Scholar]
(29).El-Khamisy SF; Katyal S; Patel P; Ju L; McKinnon PJ; Caldecott KW Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin. DNA Repair 2009, 8, 760–766. [DOI] [PMC free article] [PubMed] [Google Scholar]
(30).Das BB; Antony S; Gupta S; Dexheimer TS; Redon CE; Garfield S; Shiloh Y; Pommier Y Optimal function of the DNA repair enzyme TDP1 requires its phosphorylation by ATM and/or DNA-PK. EMBO J. 2009, 28, 3667–3680. [DOI] [PMC free article] [PubMed] [Google Scholar]
(31).Katyal S; El-Khamisy SF; Russell HR; Li Y; Ju L; Caldecott KW; McKinnon PJ TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo. EMBO J. 2007, 26, 4720–4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
(32).Perego P; Cossa G; Tinelli S; Corna E; Carenini N; Gatti L; De Cesare M; Ciusani E; Zunino F; Luison E; Canevari S; Zaffaroni N; Beretta GL Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell sensitivity to topoisomerase I inhibition. Biochem. Pharmacol 2012, 83, 27–36. [DOI] [PubMed] [Google Scholar]
(33).Alagoz M; Wells OS; El-Khamisy SF TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy. Nucleic Acids Res. 2014, 42, 3089–3103. [DOI] [PMC free article] [PubMed] [Google Scholar]
(34).Zakharenko A; Luzina O; Koval O; Nilov D; Gushchina I; Dyrkheeva N; Svedas V; Salakhutdinov N; Lavrik O Tyrosyl-DNA phosphodiesterase 1 inhibitors: usnic acid enamines enhance the cytotoxic effect of camptothecin. J. Nat. Prod 2016, 79, 2961–2967. [DOI] [PubMed] [Google Scholar]
(35).Khomenko T; Zakharenko A; Odarchenko T; Arabshahi HJ; Sannikova V; Zakharova O; Korchagina D; Reynisson J; Volcho K; Salakhutdinov N; Lavrik O New inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties. Bioorg. Med. Chem 2016, 24, 5573–5581. [DOI] [PubMed] [Google Scholar]
(36).Ponomarev KY; Suslov EV; Zakharenko AL; Zakharova OD; Rogachev AD; Korchagina DV; Zafar A; Reynisson J; Nefedov AA; Volcho KP; Salakhutdinov NF; Lavrik OI Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors. Bioorg. Chem 2018, 76, 392–399. [DOI] [PubMed] [Google Scholar]
(37).Huang S.-y. N.; Pommier Y; Marchand C Tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitors. Expert Opin. Ther. Pat 2011, 21, 1285–1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
(38).Comeaux EQ; van Waardenburg RCAM Tyrosyl-DNA phosphodiesterase I resolves both naturally and chemically induced DNA adducts and its potential as a therapeutic target. Drug Metab. Rev 2014, 46, 494–507. [DOI] [PubMed] [Google Scholar]
(39).Laev SS; Salakhutdinov NF; Lavrik OI Tyrosyl-DNA phosphodiesterase inhibitors: Progress and potential. Bioorg. Med. Chem 2016, 24, 5017–5027. [DOI] [PubMed] [Google Scholar]
(40).Lian Q; Xu J; Yan S; Huang M; Ding H; Sun X; Bi A; Ding J; Sun B; Geng M Chemotherapy-induced intestinal inflammatory responses are mediated by exosome secretion of double-strand DNA via AIM2 inflammasome activation. Cell Res. 2017, 27, 784–800. [DOI] [PMC free article] [PubMed] [Google Scholar]
(41).Li T-K; Houghton PJ; Desai SD; Daroui P; Liu AA; Hars ES; Ruchelman AL; LaVoie EJ; Liu LF Characterization of ARC-111 as a novel topoisomerase I-targeting anticancer drug. Cancer Res. 2003, 63, 8400–8407. [PubMed] [Google Scholar]
(42).Teicher BA Next generation topoisomerase I inhibitors: rationale and biomarker strategies. Biochem. Pharmacol 2008, 75, 1262–1271. [DOI] [PubMed] [Google Scholar]
(43).Burton JH; Mazcko CN; LeBlanc AK; Covey JM; Ji JJ; Kinders RJ; Parchment RE; Khanna C; Paoloni M; Lana SE; Weishaar K; London CA; Kisseberth WC; Krick E; Vail DM; Childress MO; Bryan JN; Barber LG; Ehrhart EJ; Kent MS; Fan TM; Kow KY; Northup N; Wilson-Robles H; Tomaszewski JE; Holleran JL; Muzzio M; Eiseman J; Beumer JH; Doroshow JH; Pommier Y NCI comparative oncology program testing of noncamptothecin indenoisoquinoline topoisomerase I inhibitors in naturally occurring canine lymphoma. Clin. Cancer Res 2018, DOI: 10.1158/1078-0432.CCR-18-1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
(44).Hu Z; Wang HW; An LK Tyrosyl-DNA phosphodiesterases: potential targets for cancer treatment. Acta Pharm. Sin 2016, 51, 215–225. [PubMed] [Google Scholar]
(45).Davies DR; Interthal H; Champoux JJ; Hol WGJ Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures. J. Mol. Biol 2002, 324, 917–932. [DOI] [PubMed] [Google Scholar]
(46).Davies DR; Interthal H; Champoux JJ; Hol WGJ Crystal structure of a transition state mimic for Tdp1 assembled from vanadate, DNA, and a topoisomerase I-derived peptide. Chem. Biol 2003, 10, 139–147. [DOI] [PubMed] [Google Scholar]
(47).Liao Z; Thibaut L; Jobson A; Pommier Y Inhibition of human tyrosyl-DNA phosphodiesterase by aminoglycoside antibiotics and ribosome inhibitors. Mol. Pharmacol 2006, 70, 366–372. [DOI] [PubMed] [Google Scholar]
(48).Marchand C; Lea WA; Jadhav A; Dexheimer TS; Austin CP; Inglese J; Pommier Y; Simeonov A Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay. Mol. Cancer Ther 2009, 8, 240–248. [DOI] [PMC free article] [PubMed] [Google Scholar]
(49).Sirivolu VR; Vernekar SK; Marchand C; Naumova A; Chergui A; Renaud A; Stephen AG; Chen F; Sham YY; Pommier Y; Wang Z 5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I. J. Med. Chem 2012, 55, 8671–8684. [DOI] [PMC free article] [PubMed] [Google Scholar]
(50).Dean RA; Fam HK; An J; Choi K; Shimizu Y; Jones SJ; Boerkoel CF; Interthal H; Pfeifer TA Identification of a putative Tdp1 inhibitor (CD00509) by in vitro and cell-based assays. J. Biomol. Screening 2014, 19, 1372–1382. [DOI] [PubMed] [Google Scholar]
(51).Zakharenko A; Khomenko T; Zhukova S; Koval O; Zakharova O; Anarbaev R; Lebedeva N; Korchagina D; Komarova N; Vasiliev V; Reynisson J; Volcho K; Salakhutdinov N; Lavrik O Synthesis and biological evaluation of novel tyrosyl-DNA phosphodiesterase 1 inhibitors with a benzopentathiepine moiety. Bioorg. Med. Chem 2015, 23, 2044–2052. [DOI] [PubMed] [Google Scholar]
(52).Nguyen TX; Morrell A; Conda-Sheridan M; Marchand C; Agama K; Bermingam A; Stephen AG; Chergui A; Naumova A; Fisher R; O’Keefe BR; Pommier Y; Cushman M Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors. J. Med. Chem 2012, 55, 4457–4478. [DOI] [PMC free article] [PubMed] [Google Scholar]
(53).Conda-Sheridan M; Reddy PV; Morrell A; Cobb BT; Marchand C; Agama K; Chergui A; Renaud A; Stephen AG; Bindu LK; Pommier Y; Cushman M Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase I (Tdp1) and topoisomerase I (Top1). J. Med. Chem 2013, 56, 182–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
(54).Lv PC; Agama K; Marchand C; Pommier Y; Cushman M Design, synthesis, and biological evaluation of O-2-modified indenoisoquinolines as dual topoisomerase I-tyrosyl-DNA phosphodiesterase I inhibitors. J. Med. Chem 2014, 57, 4324–4336. [DOI] [PMC free article] [PubMed] [Google Scholar]
(55).Nguyen TX; Abdelmalak M; Marchand C; Agama K; Pommier Y; Cushman M Synthesis and biological evaluation of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines as potential dual topoisomerase I (Top1)-tyrosyl-DNA phosphodiesterase I (TDP1) inhibitors. J. Med. Chem 2015, 58, 3188–3208. [DOI] [PMC free article] [PubMed] [Google Scholar]
(56).Wang P; Elsayed MSA; Plescia CB; Ravji A; Redon CE; Kiselev E; Marchand C; Zeleznik O; Agama K; Pommier Y; Cushman M Synthesis and biological evaluation of the first triple inhibitors of human topoisomerase 1, tyrosyl-DNA phosphodiesterase 1 (Tdp1), andtyrosyl-DNA phosphodiesterase 2(Tdp2). J. Med. Chem 2017, 60, 3275–3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
(57).Rodrigues T; Reker D; Schneider P; Schneider G Counting on natural products for drug design. Nat. Chem 2016, 8, 531–541. [DOI] [PubMed] [Google Scholar]
(58).Xiao Z; Morris-Natschke SL; Lee KH Strategies for the optimization of natural leads to anticancer drugs or drug candidates. Med. Res. Rev 2016, 36, 32–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
(59).Qin XJ; Yu Q; Yan H; Khan A; Feng MY; Li PP; Hao XJ; An LK; Liu HY Meroterpenoids with antitumor activities from guava (Psidium guajava). J. Agric. Food Chem 2017, 65, 4993–4999. [DOI] [PubMed] [Google Scholar]
(60).Wu N; Wu XW; Agama K; Pommier Y; Du J; Li D; Gu LQ; Huang ZS; An LK A novel DNA topoisomerase I inhibitor with different mechanism from camptothecin induces G2/M phase cell cycle arrest to K562 cells. Biochemistry 2010, 49, 10131–10136. [DOI] [PMC free article] [PubMed] [Google Scholar]
(61).Wang LK; Johnson RK; Hecht SM Inhibition of topoisomerase I function by nitidine and fagaronine. Chem. Res. Toxicol 1993, 6, 813–818. [DOI] [PubMed] [Google Scholar]
(62).Holden JA; Wall ME; Wani MC; Manikumar G Human DNA topoisomerase I: quantitative analysis of the effects of camptothecin analogs and the benzophenanthridine alkaloids nitidine and 6-ethoxydihydronitidine on DNA topoisomerase I-induced DNA strand breakage. Arch. Biochem. Biophys 1999, 370, 66–76. [DOI] [PubMed] [Google Scholar]
(63).Bai LP; Zhao ZZ; Cai Z; Jiang ZH DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine. Bioorg. Med. Chem 2006, 14, 5439–5445. [DOI] [PubMed] [Google Scholar]
(64).Kumar CV; Turner RS; Asuncion EH Groove binding of a styrylcyanine dye to the DNA double helix: the salt effect. J. Photochem. Photobiol, A 1993, 74, 231–238. [Google Scholar]
(65).Tan J-H; Ou T-M; Hou J-Q; Lu Y-J; Huang S-L; Luo H-B; Wu J-Y; Huang Z-S; Wong K-Y; Gu L-Q Isaindigotone derivatives: a new class of highly selective ligands for telomeric G-quadruplex DNA. J. Med. Chem 2009, 52, 2825–2835. [DOI] [PubMed] [Google Scholar]
(66).Korivi RP; Cheng CH Protecting-group-free total synthesis of isoquinoline alkaloids by nickel-catalyzed annulation of o-halobenzaldimine with an alkyne as the key step. Chem. - Eur. J 2010, 16, 282–287. [DOI] [PubMed] [Google Scholar]
(67).Liu CC; Korivi RP; Cheng CH Cobalt-catalyzed regioselective synthesis of indenamine from o-iodobenzaldimine and alkyne: intriguing difference to the nickel-catalyzed reaction. Chem. - Eur. J 2008, 14, 9503–9506. [DOI] [PubMed] [Google Scholar]
(68).Omura K; Sharma AK; Swern D Dimethyl sulfoxide-trifltioroacetic anhydride: a new reagent for oxidation of alcohols to carbonyls. J. Org. Chem 1976, 41, 957–962. [Google Scholar]
(69).Kiselev E; DeGuire S; Morrell A; Agama K; Dexheimer TS; Pommier Y; Cushman M 7-azaindenoisoquinolines as topoisomerase I inhibitors and potential anticancer agents. J. Med. Chem 2011, 54, 6106–6116. [DOI] [PMC free article] [PubMed] [Google Scholar]
(70).Kiselev E; Sooryakumar D; Agama K; Cushman M; Pommier Y Optimization of the lactam side chain of 7-azaindenoisoquinoline topoisomerase I inhibitors and mechanism of action studies in cancer cells. J. Med. Chem 2014, 57, 1289–1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
(71).Elsayed MSA; Su Y; Wang P; Sethi T; Agama K; Ravji A; Redon CE; Kiselev E; Horzmann KA; Freeman JL; Pommier Y; Cushman M Design and synthesis of chlorinated and fluorinated 7-azaindenoisoquinolines as potent cytotoxic anticancer agents that inhibit topoisomerase I. J. Med. Chem 2017, 60, 5364–5376. [DOI] [PMC free article] [PubMed] [Google Scholar]
(72).Janin YL; Bisagni E A formal new access to the benzo[c]phenanthridine alkaloids, synthesis of nitidine and O-methyl fagaronine analogues. Tetrahedron 1993, 49, 10305–10316. [Google Scholar]
(73).Dexheimer TS; Pommier Y DNA cleavage assay for the identification of topoisomerase I inhibitors. Nat. Protoc 2008, 3, 1736–1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
(74).Sooryakumar D; Dexheimer TS; Teicher BA; Pommier Y Molecular and cellular pharmacology of the novel noncamptothecin topoisomerase I inhibitor Genz-644282. Mol. Cancer Ther 2011, 10, 1490–1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
(75).Staker BL; Hjerrild K; Feese MD; Behnke CA; Burgin AB Jr.; Stewart L The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc. Natl. Acad. Sci U. S. A 2002, 99, 15387–15392. [DOI] [PMC free article] [PubMed] [Google Scholar]
(76).Dexheimer TS; Gediya LK; Stephen AG; Weidlich I; Antony S; Marchand C; Interthal H; Nicklaus M; Fisher RJ; Njar VC; Pommier Y 4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesulfonate) (NSC 88915) and related novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors. J. Med. Chem 2009, 52, 7122–7131. [DOI] [PMC free article] [PubMed] [Google Scholar]
(77).Davies DR; Interthal H; Champoux JJ; Hol WGJ Explorations of peptide and oligonucleotide binding sites of Tdp1 using vanadate complexes. J. Med. Chem 2004, 47, 829–837. [DOI] [PubMed] [Google Scholar]
(78).Pommier Y; Covey JM; Kerrigan D; Markovits J; Pham R DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators. Nucleic Acids Res. 1987, 15, 6713–6731. [DOI] [PMC free article] [PubMed] [Google Scholar]
(79).Pommier Y; Covey JM; Kerrigan D; Markovits J; Pham R DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators. Nucleic Acids Res. 1987, 15, 6713–6731. [DOI] [PMC free article] [PubMed] [Google Scholar]
(80).Shoemaker RH The NCI60 human tumour cell line anticancer drug screen. Nat. Rev. Cancer 2006, 6, 813–823. [DOI] [PubMed] [Google Scholar]
(81).Holbeck SL; Collins JM; Doroshow JH Analysis of Food and Drug Administration-approved anticancer agents in the NCI60 panel of human tumor cell lines. Mol. Cancer Ther 2010, 9, 1451–1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
(82).Reinhold WC; Sunshine M; Varma S; Doroshow JH; Pommier Y Using cellMiner 1.6 for systems pharmacology and genomic analysis of the NCI-60. Clin. Cancer Res 2015, 21, 3841–3852. [DOI] [PMC free article] [PubMed] [Google Scholar]
(83).Miao ZH; Player A; Shankavaram U; Wang YH; Zimonjic DB; Lorenzi PL; Liao ZY; Liu H; Shimura T; Zhang HL; Meng LH; Zhang YW; Kawasaki ES; Popescu NC; Aladjem MI; Goldstein DJ; Weinstein JN; Pommier Y Nonclassic functions of human topoisomerase I: genome-wide and pharmacologic analyses. Cancer Res. 2007, 67, 8752–8761. [DOI] [PubMed] [Google Scholar]
(84).Hsiang YH; Hertzberg R; Hecht S; Liu L Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem 1985, 260, 14873–14878. [PubMed] [Google Scholar]
(85).Urasaki Y; Laco GS; Pourquier P; Takebayashi Y; Kohlhagen G; Gioffre C; Zhang H; Chatterjee D; Pantazis P; Pommier Y Characterization of a novel topoisomerase I mutation from a camptothecin resistant human prostate cancer cell line. Cancer Res. 2001, 61, 1964–1969. [PubMed] [Google Scholar]
(86).Staker BL; Feese MD; Cushman M; Pommier Y; Zembower D; Stewart L; Burgin AB Structures of three classes of anticancer agents bound to the human topoisomerase I-DNA covalent complex. J. Med. Chem 2005, 48, 2336–2345. [DOI] [PubMed] [Google Scholar]
(87).Wu CP; Hsieh CH; Wu YS The emergence of drug transporter-mediated multidrug resistance to cancer chemotherapy. Mol. Pharmaceutics 2011, 8, 1996–2011. [DOI] [PubMed] [Google Scholar]
(88).Zhu J; Wang R; Lou L; Li W; Tang G; Bu X; Yin S Jatrophane Diterpenoids as Modulators of P-Glycoprotein-Dependent Multidrug Resistance (MDR): Advances of Structure-Activity Relationships and Discovery of Promising MDR Reversal Agents. J. Med. Chem 2016, 59, 6353–6369. [DOI] [PubMed] [Google Scholar]
(89).Carmichael J; DeGraff WG; Gazdar AF; Minna JD; Mitchell JB Evaluation of a tetrazolium-based semiautomated colorimetrie assay: assessment of chemosensitivity testing. Cancer Res. 1987, 47, 936–942. [PubMed] [Google Scholar]
(90).Yalowich JC; Wu X; Zhang R; Kanagasabai R; Hornbaker M; Hasinoff BB The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIalpha. Biochem. Pharmacol 2012, 84, 52–58. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental figures

NIHMS1684587-supplement-Supplemental_figures.pdf^{(392.3KB, pdf)}

Compounds table

NIHMS1684587-supplement-Compounds_table.csv^{(7.6KB, csv)}

PDB file 4

NIHMS1684587-supplement-PDB_file_4.txt^{(561.9KB, txt)}

PDB file 3

NIHMS1684587-supplement-PDB_file_3.txt^{(913.4KB, txt)}

CIF file

NIHMS1684587-supplement-CIF_file.txt^{(389.8KB, txt)}

[R1] (1).Stewart L; Redinbo MR; Qiu X; Hol WGJ; Champoux JJ A model for the mechanism of human topoisomerase I. Science 1998, 279, 1534–1541. [DOI] [PubMed] [Google Scholar]

[R2] (2).Champoux JJ DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem 2001, 70, 369–413. [DOI] [PubMed] [Google Scholar]

[R3] (3).Wang JC DNA topoisomerases. Annu. Rev. Biochem 1996, 65, 635–692. [DOI] [PubMed] [Google Scholar]

[R4] (4).Pommier Y; Sun Y; Huang SN; Nitiss JL Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat. Rev. Mol. Cell Biol 2016, 17, 703–721. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] (5).Pommier Y; Leo E; Zhang H; Marchand C DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol 2010, 17, 421–433. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] (6).Pommier Y; Marchand C Interfacial inhibitors: targeting macromolecular complexes. Nat. Rev. Drug Discovery 2012, 11, 25–36. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] (7).Choi SH; Tsuchida Y; Yang HW Oral versus intraperitoneal administration of irinotecan in the treatment of human neuroblastoma in nude mice. Cancer Lett. 1998, 124, 15–21. [DOI] [PubMed] [Google Scholar]

[R8] (8).Brogden RN; Wiseman LR Topotecan a review of its potential in advanced ovarian cancer. Drugs 1998, 56, 709–723. [DOI] [PubMed] [Google Scholar]

[R9] (9).Bracher F; Tremmel T From lead to drug utilizing a mannich reaction: The topotecan story. Arch. Pharm 2017, 350, e1600236. [DOI] [PubMed] [Google Scholar]

[R10] (10).Zhang R; Li Y; Cai Q; Liu T; Sun H; Chambless B Preclinical pharmacology of the natural product anticancer agent 10-hydroxycamptothecin, an inhibitor of topoisomerase I. Cancer Chemother. Pharmacol 1998, 41, 257–267. [DOI] [PubMed] [Google Scholar]

[R11] (11).Shao J; Huang F; Fu J Clinical study on treatment of non-small cell lung cancer with 10-hydroxycamptothecin. Chin. J. Clin. Oncol 2003, 30, 26–28. [Google Scholar]

[R12] (12).Pommier Y Topoisomerase I inhibitors: camptothecins and beyond. Nat. Rev. Cancer 2006, 6, 789–802. [DOI] [PubMed] [Google Scholar]

[R13] (13).Pommier Y DNA Topoisomerase I Inhibitors: chemistry, biology, and interfacial inhibition. Chem. Rev 2009, 109, 2894–2902. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] (14).Beretta GL; Perego P; Zunino F Targeting topoisomerase I: molecular mechanisms and cellular determinants of response to topoisomerase I inhibitors. Expert Opin. Ther. Targets 2008, 12, 1243–1256. [DOI] [PubMed] [Google Scholar]

[R15] (15).Pommier Y; Barcelo JM; Rao VA; Sordet O; Jobson AG; Thibaut L; Miao ZH; Seiler JA; Zhang H; Marchand C; Agama K; Nitiss JL; Redon C Repair of topoisomerase I-mediated DNA damage. Prog. Nucleic Acid Res. Mol. Biol 2006, 81, 179–229. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] (16).Maede Y; Shimizu H; Fukushima T; Kogame T; Nakamura T; Miki T; Takeda S; Pommier Y; Murai J Differential and common DNA repair pathways for topoisomerase I- and II-targeted drugs in a genetic DT40 repair cell screen panel. Mol. Cancer Ther 2014, 13, 214–220. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] (17).Interthal H; Pouliot JJ; Champoux JJ The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily. Proc. Natl. Acad. Sci. U. S. A 2001, 98, 12009–12014. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] (18).Dexheimer TS; Antony S; Marchand C; Pommier Y Tyrosyl-DNA phosphodiesterase as a target for anticancer therapy. Anti-Cancer Agents Med. Chem 2008, 8, 381–389. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] (19).Yang SW; Burgin AB; Huizenga JBN; Robertson CA; Yao KC; Nash HA A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases. Proc. Natl. Acad. Sci. U. S. A 1996, 93, 11534–11539. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] (20).Murai J; Huang SY; Das BB; Dexheimer TS; Takeda S; Pommier Y Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells. J. Biol. Chem 2012, 287, 12848–12857. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] (21).Interthal H; Chen HJ; Champoux JJ Human Tdp1 cleaves a broad spectrum of substrates including phosphoamide linkages. J. Biol. Chem 2005, 280, 36518–36528. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] (22).Menon V; Povirk LF End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair. DNA Repair 2016, 43, 57–68. [DOI] [PubMed] [Google Scholar]

[R23] (23).Ashour ME; Atteya R; El-Khamisy SF Topoisomerase-mediated chromosomal break repair: an emerging player in many games. Nat. Rev. Cancer 2015, 15, 137–151. [DOI] [PubMed] [Google Scholar]

[R24] (24).Nitiss KC; Malik M; He X; White SW; Nitiss JL Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage. Proc. Natl. Acad. Sci. U. S. A 2006, 103, 8953–8958. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] (25).Takashima H; Boerkoel CF;John J; Saifi GM; Salih MA; Armstrong D; Mao Y; Quiocho FA; Roa BB; Nakagawa M; Stockton DW; Lupski JR Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebellar ataxia with axonal neuropathy. Nat. Genet 2002, 32, 267–272. [DOI] [PubMed] [Google Scholar]

[R26] (26).Barthelmes HU; Habermeyer M; Christensen MO; Mielke C; Interthal H; Pouliot JJ; Boege F; Marko D TDP1 overexpression in human cells counteracts DNA damage mediated by topoisomerases I and II. J. Biol. Chem 2004, 279, 55618–55625. [DOI] [PubMed] [Google Scholar]

[R27] (27).Nivens MC; Felder T; Galloway AH; Pena MM; Pouliot JJ; Spencer HT Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase. Cancer Chemother. Pharmacol 2004, 53, 107–115. [DOI] [PubMed] [Google Scholar]

[R28] (28).Hirano R; Interthal H; Huang C; Nakamura T; Deguchi K; Choi K; Bhattacharjee MB; Arimura K; Umehara F; Izumo S; Northrop JL; Salih MA; Inoue K; Armstrong DL; Champoux JJ; Takashima H; Boerkoel CF Spinocerebellar ataxia with axonal neuropathy: consequence of a Tdp1 recessive neomorphic mutation? EMBO J. 2007, 26, 4732–4743. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] (29).El-Khamisy SF; Katyal S; Patel P; Ju L; McKinnon PJ; Caldecott KW Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin. DNA Repair 2009, 8, 760–766. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] (30).Das BB; Antony S; Gupta S; Dexheimer TS; Redon CE; Garfield S; Shiloh Y; Pommier Y Optimal function of the DNA repair enzyme TDP1 requires its phosphorylation by ATM and/or DNA-PK. EMBO J. 2009, 28, 3667–3680. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] (31).Katyal S; El-Khamisy SF; Russell HR; Li Y; Ju L; Caldecott KW; McKinnon PJ TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo. EMBO J. 2007, 26, 4720–4731. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] (32).Perego P; Cossa G; Tinelli S; Corna E; Carenini N; Gatti L; De Cesare M; Ciusani E; Zunino F; Luison E; Canevari S; Zaffaroni N; Beretta GL Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell sensitivity to topoisomerase I inhibition. Biochem. Pharmacol 2012, 83, 27–36. [DOI] [PubMed] [Google Scholar]

[R33] (33).Alagoz M; Wells OS; El-Khamisy SF TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy. Nucleic Acids Res. 2014, 42, 3089–3103. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] (34).Zakharenko A; Luzina O; Koval O; Nilov D; Gushchina I; Dyrkheeva N; Svedas V; Salakhutdinov N; Lavrik O Tyrosyl-DNA phosphodiesterase 1 inhibitors: usnic acid enamines enhance the cytotoxic effect of camptothecin. J. Nat. Prod 2016, 79, 2961–2967. [DOI] [PubMed] [Google Scholar]

[R35] (35).Khomenko T; Zakharenko A; Odarchenko T; Arabshahi HJ; Sannikova V; Zakharova O; Korchagina D; Reynisson J; Volcho K; Salakhutdinov N; Lavrik O New inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties. Bioorg. Med. Chem 2016, 24, 5573–5581. [DOI] [PubMed] [Google Scholar]

[R36] (36).Ponomarev KY; Suslov EV; Zakharenko AL; Zakharova OD; Rogachev AD; Korchagina DV; Zafar A; Reynisson J; Nefedov AA; Volcho KP; Salakhutdinov NF; Lavrik OI Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors. Bioorg. Chem 2018, 76, 392–399. [DOI] [PubMed] [Google Scholar]

[R37] (37).Huang S.-y. N.; Pommier Y; Marchand C Tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitors. Expert Opin. Ther. Pat 2011, 21, 1285–1292. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] (38).Comeaux EQ; van Waardenburg RCAM Tyrosyl-DNA phosphodiesterase I resolves both naturally and chemically induced DNA adducts and its potential as a therapeutic target. Drug Metab. Rev 2014, 46, 494–507. [DOI] [PubMed] [Google Scholar]

[R39] (39).Laev SS; Salakhutdinov NF; Lavrik OI Tyrosyl-DNA phosphodiesterase inhibitors: Progress and potential. Bioorg. Med. Chem 2016, 24, 5017–5027. [DOI] [PubMed] [Google Scholar]

[R40] (40).Lian Q; Xu J; Yan S; Huang M; Ding H; Sun X; Bi A; Ding J; Sun B; Geng M Chemotherapy-induced intestinal inflammatory responses are mediated by exosome secretion of double-strand DNA via AIM2 inflammasome activation. Cell Res. 2017, 27, 784–800. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] (41).Li T-K; Houghton PJ; Desai SD; Daroui P; Liu AA; Hars ES; Ruchelman AL; LaVoie EJ; Liu LF Characterization of ARC-111 as a novel topoisomerase I-targeting anticancer drug. Cancer Res. 2003, 63, 8400–8407. [PubMed] [Google Scholar]

[R42] (42).Teicher BA Next generation topoisomerase I inhibitors: rationale and biomarker strategies. Biochem. Pharmacol 2008, 75, 1262–1271. [DOI] [PubMed] [Google Scholar]

[R43] (43).Burton JH; Mazcko CN; LeBlanc AK; Covey JM; Ji JJ; Kinders RJ; Parchment RE; Khanna C; Paoloni M; Lana SE; Weishaar K; London CA; Kisseberth WC; Krick E; Vail DM; Childress MO; Bryan JN; Barber LG; Ehrhart EJ; Kent MS; Fan TM; Kow KY; Northup N; Wilson-Robles H; Tomaszewski JE; Holleran JL; Muzzio M; Eiseman J; Beumer JH; Doroshow JH; Pommier Y NCI comparative oncology program testing of noncamptothecin indenoisoquinoline topoisomerase I inhibitors in naturally occurring canine lymphoma. Clin. Cancer Res 2018, DOI: 10.1158/1078-0432.CCR-18-1498. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R44] (44).Hu Z; Wang HW; An LK Tyrosyl-DNA phosphodiesterases: potential targets for cancer treatment. Acta Pharm. Sin 2016, 51, 215–225. [PubMed] [Google Scholar]

[R45] (45).Davies DR; Interthal H; Champoux JJ; Hol WGJ Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures. J. Mol. Biol 2002, 324, 917–932. [DOI] [PubMed] [Google Scholar]

[R46] (46).Davies DR; Interthal H; Champoux JJ; Hol WGJ Crystal structure of a transition state mimic for Tdp1 assembled from vanadate, DNA, and a topoisomerase I-derived peptide. Chem. Biol 2003, 10, 139–147. [DOI] [PubMed] [Google Scholar]

[R47] (47).Liao Z; Thibaut L; Jobson A; Pommier Y Inhibition of human tyrosyl-DNA phosphodiesterase by aminoglycoside antibiotics and ribosome inhibitors. Mol. Pharmacol 2006, 70, 366–372. [DOI] [PubMed] [Google Scholar]

[R48] (48).Marchand C; Lea WA; Jadhav A; Dexheimer TS; Austin CP; Inglese J; Pommier Y; Simeonov A Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay. Mol. Cancer Ther 2009, 8, 240–248. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] (49).Sirivolu VR; Vernekar SK; Marchand C; Naumova A; Chergui A; Renaud A; Stephen AG; Chen F; Sham YY; Pommier Y; Wang Z 5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I. J. Med. Chem 2012, 55, 8671–8684. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R50] (50).Dean RA; Fam HK; An J; Choi K; Shimizu Y; Jones SJ; Boerkoel CF; Interthal H; Pfeifer TA Identification of a putative Tdp1 inhibitor (CD00509) by in vitro and cell-based assays. J. Biomol. Screening 2014, 19, 1372–1382. [DOI] [PubMed] [Google Scholar]

[R51] (51).Zakharenko A; Khomenko T; Zhukova S; Koval O; Zakharova O; Anarbaev R; Lebedeva N; Korchagina D; Komarova N; Vasiliev V; Reynisson J; Volcho K; Salakhutdinov N; Lavrik O Synthesis and biological evaluation of novel tyrosyl-DNA phosphodiesterase 1 inhibitors with a benzopentathiepine moiety. Bioorg. Med. Chem 2015, 23, 2044–2052. [DOI] [PubMed] [Google Scholar]

[R52] (52).Nguyen TX; Morrell A; Conda-Sheridan M; Marchand C; Agama K; Bermingam A; Stephen AG; Chergui A; Naumova A; Fisher R; O’Keefe BR; Pommier Y; Cushman M Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors. J. Med. Chem 2012, 55, 4457–4478. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R53] (53).Conda-Sheridan M; Reddy PV; Morrell A; Cobb BT; Marchand C; Agama K; Chergui A; Renaud A; Stephen AG; Bindu LK; Pommier Y; Cushman M Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase I (Tdp1) and topoisomerase I (Top1). J. Med. Chem 2013, 56, 182–200. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R54] (54).Lv PC; Agama K; Marchand C; Pommier Y; Cushman M Design, synthesis, and biological evaluation of O-2-modified indenoisoquinolines as dual topoisomerase I-tyrosyl-DNA phosphodiesterase I inhibitors. J. Med. Chem 2014, 57, 4324–4336. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R55] (55).Nguyen TX; Abdelmalak M; Marchand C; Agama K; Pommier Y; Cushman M Synthesis and biological evaluation of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines as potential dual topoisomerase I (Top1)-tyrosyl-DNA phosphodiesterase I (TDP1) inhibitors. J. Med. Chem 2015, 58, 3188–3208. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R56] (56).Wang P; Elsayed MSA; Plescia CB; Ravji A; Redon CE; Kiselev E; Marchand C; Zeleznik O; Agama K; Pommier Y; Cushman M Synthesis and biological evaluation of the first triple inhibitors of human topoisomerase 1, tyrosyl-DNA phosphodiesterase 1 (Tdp1), andtyrosyl-DNA phosphodiesterase 2(Tdp2). J. Med. Chem 2017, 60, 3275–3288. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R57] (57).Rodrigues T; Reker D; Schneider P; Schneider G Counting on natural products for drug design. Nat. Chem 2016, 8, 531–541. [DOI] [PubMed] [Google Scholar]

[R58] (58).Xiao Z; Morris-Natschke SL; Lee KH Strategies for the optimization of natural leads to anticancer drugs or drug candidates. Med. Res. Rev 2016, 36, 32–91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R59] (59).Qin XJ; Yu Q; Yan H; Khan A; Feng MY; Li PP; Hao XJ; An LK; Liu HY Meroterpenoids with antitumor activities from guava (Psidium guajava). J. Agric. Food Chem 2017, 65, 4993–4999. [DOI] [PubMed] [Google Scholar]

[R60] (60).Wu N; Wu XW; Agama K; Pommier Y; Du J; Li D; Gu LQ; Huang ZS; An LK A novel DNA topoisomerase I inhibitor with different mechanism from camptothecin induces G2/M phase cell cycle arrest to K562 cells. Biochemistry 2010, 49, 10131–10136. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R61] (61).Wang LK; Johnson RK; Hecht SM Inhibition of topoisomerase I function by nitidine and fagaronine. Chem. Res. Toxicol 1993, 6, 813–818. [DOI] [PubMed] [Google Scholar]

[R62] (62).Holden JA; Wall ME; Wani MC; Manikumar G Human DNA topoisomerase I: quantitative analysis of the effects of camptothecin analogs and the benzophenanthridine alkaloids nitidine and 6-ethoxydihydronitidine on DNA topoisomerase I-induced DNA strand breakage. Arch. Biochem. Biophys 1999, 370, 66–76. [DOI] [PubMed] [Google Scholar]

[R63] (63).Bai LP; Zhao ZZ; Cai Z; Jiang ZH DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine. Bioorg. Med. Chem 2006, 14, 5439–5445. [DOI] [PubMed] [Google Scholar]

[R64] (64).Kumar CV; Turner RS; Asuncion EH Groove binding of a styrylcyanine dye to the DNA double helix: the salt effect. J. Photochem. Photobiol, A 1993, 74, 231–238. [Google Scholar]

[R65] (65).Tan J-H; Ou T-M; Hou J-Q; Lu Y-J; Huang S-L; Luo H-B; Wu J-Y; Huang Z-S; Wong K-Y; Gu L-Q Isaindigotone derivatives: a new class of highly selective ligands for telomeric G-quadruplex DNA. J. Med. Chem 2009, 52, 2825–2835. [DOI] [PubMed] [Google Scholar]

[R66] (66).Korivi RP; Cheng CH Protecting-group-free total synthesis of isoquinoline alkaloids by nickel-catalyzed annulation of o-halobenzaldimine with an alkyne as the key step. Chem. - Eur. J 2010, 16, 282–287. [DOI] [PubMed] [Google Scholar]

[R67] (67).Liu CC; Korivi RP; Cheng CH Cobalt-catalyzed regioselective synthesis of indenamine from o-iodobenzaldimine and alkyne: intriguing difference to the nickel-catalyzed reaction. Chem. - Eur. J 2008, 14, 9503–9506. [DOI] [PubMed] [Google Scholar]

[R68] (68).Omura K; Sharma AK; Swern D Dimethyl sulfoxide-trifltioroacetic anhydride: a new reagent for oxidation of alcohols to carbonyls. J. Org. Chem 1976, 41, 957–962. [Google Scholar]

[R69] (69).Kiselev E; DeGuire S; Morrell A; Agama K; Dexheimer TS; Pommier Y; Cushman M 7-azaindenoisoquinolines as topoisomerase I inhibitors and potential anticancer agents. J. Med. Chem 2011, 54, 6106–6116. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R70] (70).Kiselev E; Sooryakumar D; Agama K; Cushman M; Pommier Y Optimization of the lactam side chain of 7-azaindenoisoquinoline topoisomerase I inhibitors and mechanism of action studies in cancer cells. J. Med. Chem 2014, 57, 1289–1298. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R71] (71).Elsayed MSA; Su Y; Wang P; Sethi T; Agama K; Ravji A; Redon CE; Kiselev E; Horzmann KA; Freeman JL; Pommier Y; Cushman M Design and synthesis of chlorinated and fluorinated 7-azaindenoisoquinolines as potent cytotoxic anticancer agents that inhibit topoisomerase I. J. Med. Chem 2017, 60, 5364–5376. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R72] (72).Janin YL; Bisagni E A formal new access to the benzo[c]phenanthridine alkaloids, synthesis of nitidine and O-methyl fagaronine analogues. Tetrahedron 1993, 49, 10305–10316. [Google Scholar]

[R73] (73).Dexheimer TS; Pommier Y DNA cleavage assay for the identification of topoisomerase I inhibitors. Nat. Protoc 2008, 3, 1736–1750. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R74] (74).Sooryakumar D; Dexheimer TS; Teicher BA; Pommier Y Molecular and cellular pharmacology of the novel noncamptothecin topoisomerase I inhibitor Genz-644282. Mol. Cancer Ther 2011, 10, 1490–1499. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R75] (75).Staker BL; Hjerrild K; Feese MD; Behnke CA; Burgin AB Jr.; Stewart L The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc. Natl. Acad. Sci U. S. A 2002, 99, 15387–15392. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R76] (76).Dexheimer TS; Gediya LK; Stephen AG; Weidlich I; Antony S; Marchand C; Interthal H; Nicklaus M; Fisher RJ; Njar VC; Pommier Y 4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesulfonate) (NSC 88915) and related novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors. J. Med. Chem 2009, 52, 7122–7131. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R77] (77).Davies DR; Interthal H; Champoux JJ; Hol WGJ Explorations of peptide and oligonucleotide binding sites of Tdp1 using vanadate complexes. J. Med. Chem 2004, 47, 829–837. [DOI] [PubMed] [Google Scholar]

[R78] (78).Pommier Y; Covey JM; Kerrigan D; Markovits J; Pham R DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators. Nucleic Acids Res. 1987, 15, 6713–6731. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R79] (79).Pommier Y; Covey JM; Kerrigan D; Markovits J; Pham R DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators. Nucleic Acids Res. 1987, 15, 6713–6731. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R80] (80).Shoemaker RH The NCI60 human tumour cell line anticancer drug screen. Nat. Rev. Cancer 2006, 6, 813–823. [DOI] [PubMed] [Google Scholar]

[R81] (81).Holbeck SL; Collins JM; Doroshow JH Analysis of Food and Drug Administration-approved anticancer agents in the NCI60 panel of human tumor cell lines. Mol. Cancer Ther 2010, 9, 1451–1460. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R82] (82).Reinhold WC; Sunshine M; Varma S; Doroshow JH; Pommier Y Using cellMiner 1.6 for systems pharmacology and genomic analysis of the NCI-60. Clin. Cancer Res 2015, 21, 3841–3852. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R83] (83).Miao ZH; Player A; Shankavaram U; Wang YH; Zimonjic DB; Lorenzi PL; Liao ZY; Liu H; Shimura T; Zhang HL; Meng LH; Zhang YW; Kawasaki ES; Popescu NC; Aladjem MI; Goldstein DJ; Weinstein JN; Pommier Y Nonclassic functions of human topoisomerase I: genome-wide and pharmacologic analyses. Cancer Res. 2007, 67, 8752–8761. [DOI] [PubMed] [Google Scholar]

[R84] (84).Hsiang YH; Hertzberg R; Hecht S; Liu L Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem 1985, 260, 14873–14878. [PubMed] [Google Scholar]

[R85] (85).Urasaki Y; Laco GS; Pourquier P; Takebayashi Y; Kohlhagen G; Gioffre C; Zhang H; Chatterjee D; Pantazis P; Pommier Y Characterization of a novel topoisomerase I mutation from a camptothecin resistant human prostate cancer cell line. Cancer Res. 2001, 61, 1964–1969. [PubMed] [Google Scholar]

[R86] (86).Staker BL; Feese MD; Cushman M; Pommier Y; Zembower D; Stewart L; Burgin AB Structures of three classes of anticancer agents bound to the human topoisomerase I-DNA covalent complex. J. Med. Chem 2005, 48, 2336–2345. [DOI] [PubMed] [Google Scholar]

[R87] (87).Wu CP; Hsieh CH; Wu YS The emergence of drug transporter-mediated multidrug resistance to cancer chemotherapy. Mol. Pharmaceutics 2011, 8, 1996–2011. [DOI] [PubMed] [Google Scholar]

[R88] (88).Zhu J; Wang R; Lou L; Li W; Tang G; Bu X; Yin S Jatrophane Diterpenoids as Modulators of P-Glycoprotein-Dependent Multidrug Resistance (MDR): Advances of Structure-Activity Relationships and Discovery of Promising MDR Reversal Agents. J. Med. Chem 2016, 59, 6353–6369. [DOI] [PubMed] [Google Scholar]

[R89] (89).Carmichael J; DeGraff WG; Gazdar AF; Minna JD; Mitchell JB Evaluation of a tetrazolium-based semiautomated colorimetrie assay: assessment of chemosensitivity testing. Cancer Res. 1987, 47, 936–942. [PubMed] [Google Scholar]

[R90] (90).Yalowich JC; Wu X; Zhang R; Kanagasabai R; Hornbaker M; Hasinoff BB The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIalpha. Biochem. Pharmacol 2012, 84, 52–58. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Discovery, Synthesis, and Evaluation of Oxynitidine Derivatives as Dual Inhibitors of DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1), and Potential Antitumor Agents

Xiao-Ru Zhang

Hao-Wen Wang

Wen-Lin Tang

Yu Zhang

Hui Yang

De-Xuan Hu

Azhar Ravji

Christophe Marchand

Evgeny Kiselev

Kwabena Ofori-Atta

Keli Agama

Yves Pommier

Lin-Kun An

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

Figure 2.

RESULTS AND DISCUSSION

Hit Discovery for TOP1 Inhibitor.

Table 1.

Chemistry.

Scheme 1. Synthesis of Compounds 19a/b–25a/b and 26a/b–32a/ba.

Figure 3.

Scheme 2. Synthesis of Compounds 39a/b–45a/ba.

Figure 4.

TOP1 Inhibition.

Figure 5.

Table 4.

TDP1 Inhibition.

Table 2.

Figure 6.

Interaction with DNA.

Cytotoxicity Assays.

Table 3.

Figure 7.

Synergistic Effects of 41a and 41b with Camptothecin.

Figure 8.

Induction of Cellular TOP1cc and DNA Damage by 19a.

Figure 9.

Cancer Cell Apoptosis Induced by 19a.

Pharmacokinetics of 19a.

Table 5.

Acute Toxicity of 19a in Vivo.

Figure 10.

Antitumor Efficiency of 19a in Vivo.

CONCLUSIONS

EXPERIMENTAL SECTION

General Experiments.

General Procedure for Synthesis of Schiff’s Base 12a and 12b.

N-(2-Hydroxylethyl)-6-bromoveratraldimine (12a).

N-(3-Hydroxylprapyl)-6-bromoveratraldimine (12b).

General Procedure for the Synthesis of 13a and 13b.

General Procedure for Synthesis of 15a and 15b.

3-(Benzo[d][1,3]dioxol-5-yl)-4-(2-hydroxyethyl)-6,7-dimethoxy-2-(2-(methoxymethoxy)ethyl)isoquinolin-1(2H)-one (15a).

3-(Benzo[d][1,3]dioxol-5-yl)-4-(2-hydroxyethyl)-6,7-dimethoxy-2-(3-(methoxymethoxy)propyl)isoquinolin-1(2H)-one (15b).

General Procedure for Synthesis of 16a and 16b.

2-(3-(Benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-2-(2-(methoxymethoxy)ethyl)-1-oxo-1,2-dihydroisoquinolin-4-yl)-acetaldehyde (16a).

2-(3-(Benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-2-(3-(methoxymethoxy)propyl)-1-oxo-1,2-dihydroisoquinolin-4-yl)-acetaldehyde (16b).

General Procedure for the Synthesis of 17a and 17b.

12-(2-Hydroxyethyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo-[1,2-c]phenanthridin-13(12H)-one (17a).

12-(3-Hydroxypropyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridin-13(12H)-one (17b).

General Procedure for the Synthesis of 18a and 18b.

12-(2-Chloroethyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo-[1,2-c]phenanthridin-13(12H)-one (18a).

12-(3-Chloropropyl)-2,3-dimethoxy-[1,3]dioxolo[4′,5′:4,5]benzo-[1,2-c]phenanthridin-13(12H)-one (18b).

General Procedure for the Synthesis of 19a/b–25a/b.

12-(2-(Dimethylamino)ethyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (19a).

12-(3-(Dimethylamino)propyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (19b).

12-(2-(Diethylamino)ethyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (20a).

12-(3-(Diethylamino)propyl)-2,3-dimethoxy-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (20b).

2,3-Dimethoxy-12-(2-(pyrrolidin-1-yl)ethyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (21a).

2,3-Dimethoxy-12-(3-(pyrrolidin-1-yl)propyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (21b).

2,3-Dimethoxy-12-(2-(piperidin-1-yl)ethyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (22a).

2,3-Dimethoxy-12-(3-(piperidin-1-yl)propyl)-[1,3]dioxolo-[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (22b).

2,3-Dimethoxy-12-(2-morpholinoethyl)-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridin-13(12H)-one (23a).

2,3-Dimethoxy-12-(3-morpholinopropyl)-[1,3]dioxolo[4′,5′:4,5]-benzo[1,2-c]phenanthridin-13(12H)-one (23b).

2,3-Dimethoxy-12-(2-(4-methylpiperazin-1-yl)ethyl)-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (24a).

2,3-Dimethoxy-12-(3-(4-methylpiperazin-1-yl)propyl)-[1,3]-dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (24b).

Scheme 1. Synthesis of Compounds 19a/b–25a/b and 26a/b–32a/b^a.

Scheme 2. Synthesis of Compounds 39a/b–45a/b^a.

B. Gel-Based Assay.⁷⁶