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• Topoisomerases (Top), an important component of nuclear enzymes, play an essential role in modulating DNA supercoiling during DNA transcription, replication, and chromatin assembly [1]. DNA topoisomerases Top1 and Top2 induce transient DNA single-strand or double-strand breaks by cleaving single or double DNA strands, respectively [1]. In the past decades, important progress has been achieved in the development of Top inhibitors [2-5]. Several Top inhibitors, such as topotecan (TPT, a Top1 inhibitor) and etoposide (Eto, a Top2 inhibitor), have been approved and successfully applied in clinic for cancer therapy [6].

  In the field of DNA targeted drugs, small molecules that stabilize G-quadruplex (G4) DNA are also considered as promising anticancer strategy [7-11]. G4 structures are formed by stacking of two or more G-quartets, i.e., cyclic planar arrangements of four guanines [12-14]. When promoters are folded into G4 structures, transcription is repressed at oncogene level [15]. Since oncogenes are overexpressed only in cancer cells [16], antitumor drugs that simultaneously promoting G-quadruplex formation also at oncogene promoters level could dramatically reinforce the anticancer activity [17]. Thus, simultaneously targeting G4 and Top would be a promising strategy in antitumor drug discovery. Moreover, synergistic antitumor effects between G4 and Top ligands, such as dibenzoquinoxaline (1) [18], indenoisoquinoline (2) [19] and quinolinoquinoxaline (3) [20] have been confirmed (Fig. 1A).

  Figure 1

  

  Figure 1.  Chemical structures of representative G4 and Top1/2 ligands and design rationale of target compounds. (A) Representative G4 ligands, dual Top1/G4 ligands and dual Top2/G4 ligand. The scaffold and side chain are depicted in different colors. (B) Chemical structures of evodiamine, rutaecarpine and highly active evodiamine derivates. (C) Overlay of the three-dimensional structures of evodiamine (carbon in yellow), rutaecarpine (carbon in blue) and reported G4 ligands (1, carbon in gray; 2, carbon in green; and 3, carbon in pink) in their energy minimized states. (D) Design of triple G4 and Top1/2 ligands and chemical structure of compound 15g.

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  G4 ligands generally bear a large planar heteroaromatic core and a side chain with a terminal amine group [20-23]. For example, G4 ligands BRACO-19 (4) [17-19], Pidnarulex (CX-5461, 5) [24] and quindoline (6) (Fig. 1A) [7,25] have been reported to possess potent antitumor potency [26]. Natural products containing planar polycyclic system offer favorable templates to design new G4 ligands by conjugating alkyl amine side chains [20].

  Evodiamine (7a) and rutaecarpine (8a) (Fig. 1B) are two main bioactive indolopyridoquinazoline alkaloids isolated from Evodia rutaecarpa, and have diverse biological activities [27-29]. Several evodiamine derivatives (e.g., compounds 7b, 7c and 7d, Fig. 1B) were identified as Top1/2 dual inhibitors with potent antitumor activities by our group [30-32]. The indolopyridoquinazoline skeleton bearing a large planar heteroaromatic core shares structural similarity with reported G4 ligands (Fig. 1C). Thus, we envisioned that G4 and Top1/2 ligands could be designed by incorporating the alkyl amine-containing moiety into the indolopyridoquinazoline scaffold. As a conceptual validation study, herein the first G4 and Top1/2 triple ligands were designed and assayed (Fig. 1D).

  The synthetic route of evodiamine derivatives were depicted in Scheme 1 and Table S1 (Supporting information). In the presence of sodium hydride (NaH), target compounds 10a-o were obtained by nucleophilic substitution reaction via treating compounds 7 [27] with various commercially available 3-chloro propyl amines (9). Target compounds 11f-o were obtained by demethylation of 10f-o in the presence of BBr₃ at −78 ℃. Similarly, intermediate 12 was prepared from compounds 7 with 1,3-dibromopropane reaction in the presence of potassium hydroxide (KOH). Then, 12 was treated various commercially available 3-chloro propyl amines (9) to afford target compounds 10p-x. Compounds 11s-x were obtained by demethylation of 10s-x in the presence of BBr₃ at −78 ℃.

  Scheme 1

  

  Scheme 1.  Synthesis of evodiamine derivatives. Reagents and conditions: (a) DMF, NaH, 65 ℃, 12 h, yield 43%−78%; (b) DCM, BBr₃, under N₂, −78 ℃, 2 h, yield 24%−85%; (c) DMF, KOH, 1,3-dibromopropane, r.t., 12 h, yield 35%−80%; (d) 10p-q, 10s-t, 10v-w: DMF, K₂CO₃, 65 ℃, 12 h, yield 32%−60%; (e) 10r, 10u, 10x: MeCN, r.t., 72 h, yield 44%−81%.

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  A similar strategy was applied for the synthesis of rutaecarpine derivatives 13a-c, 13d-k and 15f-i (Scheme 2). Intermediate m7 was prepared according to the reported procedure [31]. Intermediate m7 was reacted with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) via oxidation reaction to give rutaecarpine (8a). Then, target compounds 13a-c were prepared by nucleophilic substitution reaction via treating intermediates 8a with various commercially available 3-chloropropyl amines (9). Similarly, intermediate 14 was prepared by the treatment of intermediates 8 with 1-bromo-3-chloropropane reaction in the presence of potassium hydroxide (KOH). Compounds 13d-k and 15f-i were obtained by a similar protocol described in Scheme 1.

  Scheme 2

  

  Scheme 2.  Synthesis of rutaecarpine derivatives. Reagents and conditions: (a) DCM, DDQ, r.t., 8 h, yield 45%−55%; (b) 13a, 13b, 13d-k: DMF, Cs₂CO₃, 65 ℃, 12 h, yield 21%−25%; (c) 13c: DMF, NaH, 65 ℃, 12 h, yield 8%; (d) DMF, KOH, 1-bromo-3-chloropropane, r.t., 12 h, yield 19%−22%; (e) 15f-i: DCM, BBr₃, under N₂, −78 ℃, 2 h, yield 44%−98%.

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  Initially, a total of 40 evodiamine analogues were designed and synthesized by incorporating various alkyl amine side chains onto the evodiamine N13 position (Scheme 1, Table S1). We first evaluated the evodiamine derivatives affinities to a typic c-MYCG4 by the fluorescence resonance energy transfer (FRET) quenching and FRET melting assays [18,19]. Since c-MYC is one of the most important oncogenes that is overexpressed in more than 80% of cancer cells and contributes to cell proliferation, angiogenesis, metastasis and apoptosis [8,33]. Moreover, nuclease hypersensitive element (NHE Ⅲ₁) controls 85%−90% of c-MYC transcriptional activity, which folds into a DNA G-quadruplex (G4) under the transcription-associated negative supercoiling and thus silences c-MYC transcription [9,33,34]. The results showed that template molecules 7d and 8a were unable to induce and stabilize c-MYCG4 (Fig. 2A). After introducing the side chain, only compound 11v decreased the relative fluorescence intensity by more than 42% at the concentration of 100 µmol/L (Fig. S1 in Supporting information). Moreover, only three compounds (10g, 10t and 11g) increased the T_m values of c-MYCG4 by more than 10 ℃ at the concentration of 10 µmol/L (Fig. S1). These results suggested that the scaffold of evodiamine was unfavorable to design potent c-MYCG4 ligands. In addition, terminal alkyl amine groups favorable for the activity (e.g., thiazolyl, isopropyl, dimethyl and methyl substituents) were used in the next round of structural modification.

  Figure 2

  

  Figure 2.  c-MYCG4 binding and stabilizing activity and Top1/2 inhibitory activity of rutaecarpine derivatives. (A) FRET quenching assay of DMSO, 100 mmol/L K⁺, rutaecarpine and its analogues in the presence of labeled c-MYCG4. KCl (100 mmol/L) was employed as positive control in FRET-quenching assay, which decreased the relative fluorescence intensity by 42%. (B) Thermal stabilization values (ΔT_m) of c-MYCG4 in the presence of rutaecarpine and its analogues by FRET melting assays. FRET-melting assay was measured in 10 mmol/L K⁺ using dual-3′-FAM- and 5′-TAMRA-labeled c-MYC Pu22 DNA. (C) Top1 inhibitory activity at 50 µmol/L. (D) Top1 inhibitory activity of compounds 15g, 13k, 13h, 13i, 15h, 15i and 13e ranging from 25 µmol/L to 12.5 µmol/L. (E) Top1 inhibitory activity of compounds 15g and 15i ranging from 10 µmol/L to 1 µmol/L. (F) Top2 inhibitory activity at 50 µmol/L.

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  Then, the rutaecarpine scaffold was investigated and 15 N13-substituted derivatives were designed and assayed (Scheme 2, Table S2 in Supporting information). To our delight, 14 out of 15 target compounds exhibited potent c-MYCG4 binding and stabilizing activities at the concentration of 100 µmol/L and 10 µmol/L, respectively (relative decrease of fluorescence intensity: 56.7% to 96.6%; ∆T_m range: 13.3 ℃ to 32.0 ℃ (Figs. 2A and B, Table S3 in Supporting information). Nine compounds (13f, 13g, 13h, 13i, 13j, 13k, 15g, 15h and 15i) decreased the relative fluorescence intensity by more than 70% and increased the T_m value of c-MYCG4 by more than 20 ℃ (Table 1). Moreover, introducing hydroxyl or methoxyl group on the rutaecarpine scaffold led to enhanced c-MYCG4 binding and stabilizing activities (15h > 13j > 13f > 13d, 13g > 15g > 13k > 15i > 13e, Table 1). Notably, N13-substituents also played an important role in c-MYCG4 binding and stabilization (Fig. 2B,Table 1). For example, compounds with N13-(isopropylamino)propyl side chain exhibited better c-MYCG4 stabilizing activity than those with N13-(thiazolylamino)propyl group (13e > 13d, 13g > 13f, 13i > 13h, 13k > 13j, 15g > 15f, 15i > 15h).

  Table 1

  

  Table 1.  The c-MYCG4 binding and stabilizing activities of N13-alkyl amine substituted rutaecarpine by fluorescence FRET quenching and FRET melting assays.^a

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  The compounds with more than 70% relative fluorescence intensity decrease were further selected to evaluate the Top inhibitory activities using the DNA cleavage assay [32]. As shown in Figs. 2C and D, 7 out of 13 compounds (13e, 13h, 13i, 13k, 15g, 15h and 15i) were active against Top1 at the concentration of 50 µmol/L and 25 µmol/L, respectively. The DNA bands treated with compounds 13e and 13h were broadened, which indicated that they had weak Top1 inhibitory activities. However, at a lower concentration of 1 µmol/L, two compounds (15g and 15i) were still active (Fig. 2E). Moreover, 7 out of 13 compounds were active against Top2 at the concentration of 50 µmol/L (Fig. 2F). More specifically, compounds with N13-(isopropylamino)propyl or N13-(thiazolylamino)propyl side chain exhibited potent Top activities, which were consistent with their c-MYCG4 binding and stabilizing activities. Compounds 13i, 15g, 15h and 15i were validated as novel c-MYCG4/Top1/Top2 triple inhibitors.

  On the basis of the c-MYCG4 and Top inhibitory activities, compound 15g (Fig. 1D) was finally selected for further mechanism studies. Circular dichroism (CD) titration assay revealed that the signature of a parallel c-MYCG4 was maintained (Fig. 3A). Fluorescence-based binding assay indicated that compound 15g showed strong c-MYCG4 binding activity with binding affinity K_d value of 156 nmol/L (Fig. 3B). ¹H nuclear magnetic resonance (¹H NMR) titration experiments were conducted to examine the binding interactions of compound 15g with c-MYC. Upon the addition of compound 15g, obvious changes of the tetrad-guanine imino proton signals were observed, confirming the binding interactions (Fig. 3C). Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were further performed using MCF-7 breast cancer cell line. MCF-7 cells were treated with compound 15g at six concentrations (0, 1, 2.5, 5, 10 and 15 µmol/L) for 24 h. As shown in Figs. 3D and E, compound 15g significantly reduced the levels of c-MYC protein and mRNA at the concentration of 2.5 µmol/L and 1 µmol/L, respectively. In contrast, rutaecarpine itself had no effect on the mRNA level and slightly decreased the c-MYC protein level at a high concentration of 10 µmol/L (Figs. 3D and E). These results further demonstrated the c-MYCG4 binding and stabilizing activity and c-MYC downregulation effect of compound 15g.

  Figure 3

  

  Figure 3.  c-MYC inhibitory activities of compound 15g. (A) The signature of c-MYCG4 in the presence of compound 15g by the CD titration assay. (B) Binding affinity of compound 15g with c-MYCG4 using fluorescence-based binding assay. (C) Imino proton regions of the 1D ¹H NMR spectra of c-MYC Pu22 either alone or with compound 15g. (D) Effects of compound 15g and rutaecarpine on the transcription of c-MYC oncogene in MCF-7 cells by Western blotting. (E) Effects of compound 15g and rutaecarpine on the c-MYC expression in MCF-7 cells by RT-PCR. ns = not significant, ^***P < 0.001, ^****P < 0.0001 vs. control group, determined with Student's t-test. At least two independent experiments were done for each condition.

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  To investigate the binding modes of compound 15g with Top1 (PDB ID: 1T8I) [35], Top2α (PDB ID: 5GWK) [36] and c-MYCG4 (PDB ID: 2L7V) [37], molecular docking study was performed. As depicted in Fig. 4A, the lactam carbonyl group in the backbone of compound 15g formed a hydrogen bond with Met428 of Top1. Electrostatic interactions between the amino-containing side chain and DNA base DT10 and TGP11 were observed. As for Top2α, Ser763 was interacted with the scaffold of compound 15g through a hydrogen bond, and the side chain formed another hydrogen bond with Arg487 (Fig. 4B). As shown in Fig. 4C, compound 15g could strongly stabilize c-MYCG4 with two of compound 15g binding to the 5′- and 3′-terminal sites of the c-MYCG4. Specifically, the indole and benzopyrimidine moiety in the skeleton of compound 15g in the 3′-site formed π-π stacking interactions with DG13 and DG18, respectively. In addition, the NH in the side chain formed a hydrogen bond with DT23. Similarly, the scaffold of compound 15g formed π-π stacking interactions with the 5′-external tetrads, including DG7, DG11 and DG16. Additionally, a hydrogen bond between the side chain and DG5 was observed.

  Figure 4

  

  Figure 4.  The binding modes of compound 15g. Proposed molecular interaction models of compound 15g with Top1 (A), Top2 (B) and c-MYCG4 (C).

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  Using a competition fluorescence displacement assay [19], we further determined the binding selectivity of compound 15g for c-MYCG4 as compared to a parallel K-Ras promoter G4, a hybrid telomeric G4, and double-stranded (ds) DNA (Fig. 5A). The competition fluorescence displacement assay allows for a straightforward assessment of selective binding toward c-MYCG4 vs the competitors, namely c-MYCG4s (parallel), telomeric G4 (hybrid), dsDNA and K-Ras G4 (parallel). As shown in Fig. 5A, compound 15g showed preferable binding selectivity for parallel and hybrid G4s (c-MYCG4, K-Ras G4 and telomeric G4) over dsDNA, and showed much more selectivity against c-MYCG4s. This result suggested that compound 15g is a selective c-MYCG4 ligand to some degree but its c-MYCG4 selectivity still need to be improved.

  Figure 5

  

  Figure 5.  G4 binding selectivity (A) and in vitro antitumor potency (B) of compound 15g. At least two independent experiments were done for each condition.

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  The in vitro antitumor activity of compound 15g against four human cancer cell lines (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, HCT116 colon cancer cells and HeLa cervical cancer cells) were tested using the CCK8 assay. Camptothecin (CPT) was used as the positive control (Table S1). Compound 15g showed potent antitumor activity with an IC₅₀ value in the range of 1.7-3.3 µmol/L (Fig. 5B). The aqueous solubility was further evaluated under physiological conditions. Compound 15g exhibited moderate aqueous solubility with 41.6 µg/mL at pH 7.4.

  Moreover, the effects of compound 15g on the induction of apoptosis and cell-cycle in MCF-7 cells were evaluated by flow cytometry. As shown in Fig. S3A (Supporting information), compound 15g induced the apoptosis of MCF-7 cells in a concentration-dependent manner. After the exposure of compound 15g to various concentrations (1, 5 and 15 µmol/L) for 24 h, the percentages of apoptotic cells were 25.48%, 32.82%, and 73.01%, respectively. These results demonstrated that compound 15g could effectively inhibit cancer cell growth through apoptosis induction. After the treatment with 1, 5, and 15 µmol/L compound 15g for 24 h, the percentage of cells in the G2 fraction increased from 21.97% to 61.41% with a concomitant decrease in the proportion of cells in other phases of the cell cycle, suggesting the G2 cell-cycle arrest in MCF-7 cells (Fig. S3B in Supporting information).

  In summary, this work reported novel triple G4 and Top1/2 ligands inspired by the indolopyridoquinazoline scaffold of natural products evodiamine and rutaecarpine. A focused library of 55 indolopyridoquinazoline analogues were rationally designed and synthesized. Notably, compound 15g was proven to be a potent antitumor lead compound by triple targeting c-MYCG4 and Top1/2. Among the reported rutaecarpine derivatives, compound 15g represents a highly potent compound in terms of in vitro antitumor activity. It may be used a valuable chemical tool to investigate the biological functions of c-MYCG4 and Top1/2. This proof-of-concept study validated the feasibility of using the planar scaffolds of natural products as templates to design new c-MYCG4 ligands. However, the antitumor potency and c-MYC G-quadruplex selectivity of compound 15g still need to be further improved. Taken together, compound 15g is a lead compound for the discovery of novel antitumor agents and further structural optimizations are in progress.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (Nos. 22077138 to S. Wu, 81725020 to C. Sheng, and 81872742 to G. Dong), the National Key Research and Development Program of China (No. 2020YFA0509200 to C. Sheng) and Shanghai Rising-Star Program (No. 22QA1411300 to S. Wu).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.07.014.

  
  
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• 

  Figure 1  Chemical structures of representative G4 and Top1/2 ligands and design rationale of target compounds. (A) Representative G4 ligands, dual Top1/G4 ligands and dual Top2/G4 ligand. The scaffold and side chain are depicted in different colors. (B) Chemical structures of evodiamine, rutaecarpine and highly active evodiamine derivates. (C) Overlay of the three-dimensional structures of evodiamine (carbon in yellow), rutaecarpine (carbon in blue) and reported G4 ligands (1, carbon in gray; 2, carbon in green; and 3, carbon in pink) in their energy minimized states. (D) Design of triple G4 and Top1/2 ligands and chemical structure of compound 15g.

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  Scheme 1  Synthesis of evodiamine derivatives. Reagents and conditions: (a) DMF, NaH, 65 ℃, 12 h, yield 43%−78%; (b) DCM, BBr₃, under N₂, −78 ℃, 2 h, yield 24%−85%; (c) DMF, KOH, 1,3-dibromopropane, r.t., 12 h, yield 35%−80%; (d) 10p-q, 10s-t, 10v-w: DMF, K₂CO₃, 65 ℃, 12 h, yield 32%−60%; (e) 10r, 10u, 10x: MeCN, r.t., 72 h, yield 44%−81%.

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  Scheme 2  Synthesis of rutaecarpine derivatives. Reagents and conditions: (a) DCM, DDQ, r.t., 8 h, yield 45%−55%; (b) 13a, 13b, 13d-k: DMF, Cs₂CO₃, 65 ℃, 12 h, yield 21%−25%; (c) 13c: DMF, NaH, 65 ℃, 12 h, yield 8%; (d) DMF, KOH, 1-bromo-3-chloropropane, r.t., 12 h, yield 19%−22%; (e) 15f-i: DCM, BBr₃, under N₂, −78 ℃, 2 h, yield 44%−98%.

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  Figure 2  c-MYCG4 binding and stabilizing activity and Top1/2 inhibitory activity of rutaecarpine derivatives. (A) FRET quenching assay of DMSO, 100 mmol/L K⁺, rutaecarpine and its analogues in the presence of labeled c-MYCG4. KCl (100 mmol/L) was employed as positive control in FRET-quenching assay, which decreased the relative fluorescence intensity by 42%. (B) Thermal stabilization values (ΔT_m) of c-MYCG4 in the presence of rutaecarpine and its analogues by FRET melting assays. FRET-melting assay was measured in 10 mmol/L K⁺ using dual-3′-FAM- and 5′-TAMRA-labeled c-MYC Pu22 DNA. (C) Top1 inhibitory activity at 50 µmol/L. (D) Top1 inhibitory activity of compounds 15g, 13k, 13h, 13i, 15h, 15i and 13e ranging from 25 µmol/L to 12.5 µmol/L. (E) Top1 inhibitory activity of compounds 15g and 15i ranging from 10 µmol/L to 1 µmol/L. (F) Top2 inhibitory activity at 50 µmol/L.

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  Figure 3  c-MYC inhibitory activities of compound 15g. (A) The signature of c-MYCG4 in the presence of compound 15g by the CD titration assay. (B) Binding affinity of compound 15g with c-MYCG4 using fluorescence-based binding assay. (C) Imino proton regions of the 1D ¹H NMR spectra of c-MYC Pu22 either alone or with compound 15g. (D) Effects of compound 15g and rutaecarpine on the transcription of c-MYC oncogene in MCF-7 cells by Western blotting. (E) Effects of compound 15g and rutaecarpine on the c-MYC expression in MCF-7 cells by RT-PCR. ns = not significant, ^***P < 0.001, ^****P < 0.0001 vs. control group, determined with Student's t-test. At least two independent experiments were done for each condition.

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  Figure 4  The binding modes of compound 15g. Proposed molecular interaction models of compound 15g with Top1 (A), Top2 (B) and c-MYCG4 (C).

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  Figure 5  G4 binding selectivity (A) and in vitro antitumor potency (B) of compound 15g. At least two independent experiments were done for each condition.

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  Table 1.  The c-MYCG4 binding and stabilizing activities of N13-alkyl amine substituted rutaecarpine by fluorescence FRET quenching and FRET melting assays.^a

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