Synthesis and biological evaluation of 5-aminoethyl benzophenanthridone derivatives as DNA topoisomerase IB inhibitors

Abstract

DNA topoisomerase IB (TOP1) regulates DNA topological structure in many cellular metabolic processes and is a validated target for development of antitumor agents. Our previous study revealed that the benzophenanthridone scaffold is a novel chemotype for the discovery of TOP1 inhibitors. In this work, a series of novel 5-aminoethyl substituted benzophenanthridone derivatives have been synthesized and evaluated for TOP1 inhibition and cytotoxicity. Compound 12 exhibits the most potent TOP1 inhibition (+++) and cytotoxicity in human cancer cell lines with GI₅₀ values at nanomolar concentration range. 12 induces the cellular TOP1cc formation and DNA damage, resulting in HCT116 cell apoptosis. The pharmacokinetics, acute toxicity and antitumor efficiency in vivo of 12 were also studied.

1. Introduction

DNA topoisomerase IB (TOP1) is an essential nuclear enzyme that regulates DNA topological structure in many essential cellular metabolic processes including transcription and replication [1–3]. To perform its functions, TOP1 cleaves one strand of the DNA through a transesterification reaction by nucleophilic attack of its catalytic tyrosine (Tyr723 for human TOP1) to the DNA phosphodiester backbone to form the enzyme-DNA covalent cleavage complexes (TOP1cc). TOP1cc are transient intermediates under normal physiological circumstances as they reverse into the intact DNA and release TOP1 [4,5]. TOP1 inhibitor, such as camptothecin (CPT, Fig. 1) can bind at the interface of TOP1cc [6–8], which stabilizes TOP1cc and prevents further religation of the nicked DNA, resulting in DNA damage and triggering cell death [4,5]. Therefore, TOP1 is a validated target for the discovery of anticancer agents [4,6,9,10].

Fig. 1.

Structures of the camptothecins in clinical uses, indenoisoquinolines in clinical trials and the oxynitidine TOP1 inhibitors found in our laboratory.

To date, four well-known camptothecin derivatives (Fig. 1) have been approved for clinical treatment of cancers, including Topotecan and Irinotecan approved by FDA [11–13], 10-hydroxycamptothecin (HCPT, in China) and Belotecan (in South Korea) [4,14–16]. In spite of their effectiveness in solid tumors, camptothecin TOP1 inhibitors suffer from many shortcomings, such as chemical instability under physiological condition, poor solubility, bone marrow dose-limiting toxicity, severe gastrointestinal toxicity for Irinotecan and drug efflux-mediated resistance [9,17]. Therefore, many investigations have focused on the discovery of non-camptothecin TOP1 inhibitors. And three chemotypes of non-camptothecin TOP1 inhibitors, including indolocarbazole, dibenzonaphthyridinone and indenoisoquinoline derivatives, have been developed [9,18,19]. Three indenoisoquinolines LMP400L, MP744 and LMP776 (Fig. 1) are in clinical trials [19].

Natural products are important sources for medicinal chemistry and drug development. To find novel non-camptothecin TOP1 inhibitors, we studied our in-house natural product library and found several chemotypes inhibiting TOP1, including oxynitidine and meroterpenoid derivatives. Further investigation indicated that several meroterpenoid derivatives showed high TOP1 relaxing inhibition, but were not able to trap and stabilize TOP1cc and could be classified as TOP1 catalytic inhibitors [20,21]. On the contrary, oxynitidine (1, Fig. 1) exhibited weak TOP1 cleavage inhibitory activity (+/0) [22]. Structural optimization of 1 gave several TOP1 inhibitors with higher inhibitory activity, including NTD-96 (2, Fig. 1). 2 shows increased TOP1 inhibition (+++) and traps TOP1cc at genome binding sites different from CPT [22]. Further investigation indicated that 2 targets to TOP1 in cells, inducing cellular TOP1cc formation and DNA damage, and exhibits good antitumor activity both in vitro and in vivo [22]. Structure-activity relationship (SAR) analysis indicated that [22]: 1) the carbonyl group at the 6-position (benzophenanthridinone derivatives) is important for TOP1 inhibition. Most of the 6-aminoalkyloxy benzophenanthridine derivatives have no TOP1 inhibition at 100 μM concentration and show weak cytotoxicity. Furthermore, the reduced derivatives (dihydrobenzophenanthridine) show weak both TOP1 inhibition and cytotoxicity; 2) substitution of aminoethyl group at the 5-position may increase both TOP1 inhibition and cytotoxicity. It is noteworthy that bigger substituents at the 5-position, for example aminopropyl group did not significantly increase the potency of the drugs as TOP1 inhibitors. To further study the spatial effect of the terminus of the aminoethyl group at 5-position on the TOP1 inhibition, in this work, a series of novel 5-aminoethyl substituted benzophenanthridinone derivatives was synthesized and biologically evaluated.

2. Results and discussion

2.1. Chemistry

The synthesis of the designed 5-aminoethyl substituted benzophenanthridinone derivatives is outlined in Scheme 1. Similar to our previous publication [22], the hydroxy group of the Schiff base 3 prepared from the reaction of 6-bromoveratraldehyde with 2-aminoethanol was protected using methoxymethyl (MOM) group to give the intermediate 4, which was directly used for the next reaction without further purification. The intermediate 6 was obtained in two steps. First, under nickel-based catalysis [23], the cyclization reaction of 4 with 5, prepared through Sonogashira coupling reaction according to the reported method [24], gave a ternary ammonium salt intermediate. In the second step, the resulting ternary ammonium salt intermediate was oxidized by K₃Fe(CN)₆ to give the intermediate 6. Following the Swern oxidation of the hydroxy group of 6, the cyclization reaction under concentrated hydrochloric acid condition gave the intermediate 8 with simultaneous deprotection of MOM group. The replacement of hydroxy group of 8 with bromine gave the bromide 9 in 91% yield. Following replacement reaction of 9 with NaN₃, the Pd/C catalytic reduction reaction under hydrogen atmosphere gave the target amine 11 in 51% yield for the two steps (from 9). 11 reacted with formaldehyde to form a Schiff base, which could be reduced by zinc powder to give the target product 12 in 59% yield. The acylation reaction of 12 with various acyl chloride in dichloromethane gave the target products 13–19 with N-methyl amide group as the terminus of the side chain at the 5-position. Similarly, the acylation of 11 gave the target products 20–25. Finally, we synthesized fifteen benzophenanthridone derivatives with various termini groups of the aminoethyl side chain at the 5-position, including amino group, methylamino group, amide group, sulfonamide group and phosphoamide group. Their structures and purity were assessed through HRMS, 1D and 2D NMR spectra, and HPLC method.

Scheme 1.

The synthesis of the target compounds Reagents and conditions: (a) MeOH, NH₂(CH₂)₂OH, rt. (b) MOMCl, NaH, THF, 0 °C. (c) I) N₂, Ni(cod)₂, P(o-Tol)₃, MeCN, 80 °C; ii) CsOH, K₃[Fe(CN)₆], MeOH, H₂O, 80 °C. (d) (COCl)₂, DMSO, TEA, DCM, −60 °C. (e) concd. hydrochloric acid, MeOH, rt. (f) PBr₃, TCM, rt. g) NaN₃, DMSO, rt. (h) Pd/C, H₂, THF, rt. (i) aqueous HCHO, Zn, CH₃CO₂H, H₂O, rt. (j) Acyl chloride, DIPEA, DCM, reflux. (k) Acyl chloride, DIPEA, DCM, rt.

2.2. TOP1 inhibition

The synthesized compounds were tested for TOP1 inhibitory activity through TOP1-mediated cleavage assay using a 3’-[³²P]-labeled double-stranded DNA fragment as substrate along with CPT and LMP744, an indenoisoquinoline TOP1 inhibitor [25], as positive controls [26]. All compounds were tested at four concentrations, 0.1, 1.0, 10 and 100 μM. The TOP1-mediated cleavage activity of the compounds was semiquantitatively graded consistent with prior publications based on the number and intensities of the DNA cleavage bands relative to the TOP1 inhibition of CPT at 1 μM concentration [22,25]: 0, no inhibitory activity; +, between 20% and 50% inhibitory activity; ++, between 50% and 75% inhibitory activity; +++, between 75% and 95% inhibitory activity; ++++, equal inhibitory activity to CPT. The TOP1-mediated cleavage activity of the synthesized compounds is summarized in Table 1. Compared to the parent 1, most of the synthesized compounds showed increased TOP1 inhibitory activity except for three compounds 13, 22 and 24, which showed equal activity to 1. Compound 12 showed the most potent TOP1 inhibition of +++, equal to compound 2 [22]. Five compounds 11, 18, 19, 20 and 23 showed moderate TOP1 inhibitory activity (++). Compared with 2, the bigger termini of the side chain seem to decrease of TOP1 inhibitory activity. Representative TOP1-mediated cleavage assay gel is shown in Fig. 2. Compounds 11, 12, 18–20 and 23 exhibited the ability to induce TOP1-mediated cleavage bands in a dose-dependent manner with cleavage sites similar to LMP744 but not to CPT. For example, the cleavage sites 17, 35 and 79 could be induced by 11, 12, 18–20 and 23 but not by CPT, implying that the synthesized benzophenanthridinone derivatives trap TOP1cc at different DNA sequence from CPT.

Table 1.

TOP1 cleavage inhibitory activity and cytotoxicity of the synthesized compounds.

Compd.	R	TOP1 inhibitiona	GI50 ± SD(μM)b
			HCT116	MCF-7	DU145	A549
CPT	/c	++++	0.009 ± 0.001	0.012 ± 0.002	0.21 ± 0.069	0.099 ± 0.017
1	/	0/+	74 ± 4.4	31 ± 4.6	64 ± 11	39 ± 26
2	NMe2	+++	0.076 ± 0.007	0.34 ± 0.098	0.018 ± 0.002	0.79 ± 0.11
11	NH2	++	0.29 ± 0.019	0.10 ± 0.001	0.014 ± 0.001	0.98 ± 0.014
12	NHMe	+++	0.036 ± 0.003	0.090 ± 0.001	0.002 ± 0.001	0.97 ± 0.001
13		0/+	86 ± 2.5	35 ± 1.7	65 ± 1.2	42 ± 0.98
14		+	7.9 ± 0.15	5.8 ± 0.89	10 ± 1.2	15 ± 1.5
15		+	0.96 ± 0.04	1.5 ± 0.15	15 ± 0.59	7.9 ± 0.23
16		+	2.5 ± 0.18	3.8 ± 0.25	3.1 ± 0.30	5.3 ± 0.41
17		+	40 ± 1.2	64 ± 0.57	48 ± 1.2	35 ± 2.5
18		++	9.2 ± 0.14	10 ± 0.54	5.3 ± 0.21	14 ± 1.4
19		++	5.9 ± 1.2	1.8 ± 0.47	3.6 ± 0.25	1.9 ± 0.034
20		++	5.2 ± 0.13	3.0 ± 0.63	1.3 ± 0.32	4.3 ± 0.21
21		+	30 ± 2.1	21 ± 1.0	45 ± 1.2	35 ± 0.14
22		0/+	34 ± 1.1	38 ± 0.78	79 ± 1.0	61 ± 0.15
23		++	1.1 ± 0.047	0.89 ± 0.12	1.6 ± 0.021	2.9 ± 0.027
24		0/+	51 ± 1.5	48 ± 1.9	39 ± 2.2	41 ± 2.0
25		+	5.3 ± 0.11	2.2 ± 0.27	7.8 ± 0.52	1.3 ± 0.006

^aTOP1 cleavage inhibitory activity of the synthesized compounds was semiquantitatively graded relative to CPT at 1 μM concentration: 0, no inhibition; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity; ++++, equal activity.

^bGI₅₀ values (means ± SD) were calculated from MTT assay, and defined as the concentrations of compounds that resulted in 50% cell growth inhibition. The experiments were repeated at least three times independently.

^c“/” means “inapplicable”.

Fig. 2.

Representative gel of TOP1-mediated DNA cleavage assay. Lane 1, DNA alone; lane 2, DNA and TOP1; lane 3, DNA and TOP1 with CPT (1.0 μM); lane 4, DNA and TOP1 with LMP744 (1.0 μM); lanes 5–28, DNA and TOP1 with the indicated compounds at 0.1, 1.0, 10 and 100 μM concentration, respectively. The arrows at left indicate the cleavage site positions.

To inspect the molecular binding mode of the synthesized TOP1 inhibitors within the TOP1-DNA complex, molecular modeling was performed. A hypothetical binding model was built using in-silico docking from the X-ray crystal of the TOP1-DNA-ligand ternary complex (PDB ID: 1K4T) [7]. Compounds were energy-minimized and docked into the binding model. As shown in Fig. 3A, the benzophenanthridinone scaffold of 12 intercalates in the DNA break made by TOP1 and readily stacks with the +1 and −1 base pairs flanking the DNA cleavage site, similar to that of 2 [22]. The A- and B-ring of 12 stack with the bases of non-cleaved strand (C and A), while the C- and D-ring stack with the scissile strand bases (G and T). In addition, the methylaminoethyl side chain of 12 extends into the minor groove of the DNA and binds to a limited space (Fig. 3B), which might be the reason why the bigger termini of the side chain decrease TOP1 inhibitory activity. Also, a hydrogen bond (2.9 Å) was observed between the lactam oxygen atom and R364 residue (Fig. 3A), implying the importance of hydrogen bond acceptor, which is consistent with the cytotoxicity of 12 against the prostate cancer cells DU145-RC0.1, resistant cells with a R364H mutation of TOP1 [27]. DU145-RC0.1 cells showed high resistance to 12 (Table 3). In addition, a hydrogen bond observed between the oxygen atom in the dioxole ring and Asn722 (3.5 Å) might also contribute to the TOP1cc inhibition.

Fig. 3.

Hypothetical binding mode of 12 in the ternary TOP1-DNA-drug complex (PDB ID: 1K4T). 12 is shown as green carbon atoms ball and stick representation. (A) TOP1 is shown in cartoon and the base pairs are displayed in blue lines. (B) TOP1 is shown in surface and DNA is shown in cartoon. All distances are measured from heavy atom to heavy atom. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 3.

The cytotoxicity of the compound 12 in drug-resistant human cancer cell lines.

Compd.	GI50 ± SD(μM)a		Resisitance Ratiob
	Parental cell line	Resistant subline
	HCT116	HCT116-siTOP1
CPT	0.009 ± 0.001	0.075 ± 0.014	8.3
12	0.036 ± 0.003	0.32 ± 0.035	8.9
	DU145	DU145-RC0.1
CPT	0.021 ± 0.016	4.73 ± 0.68	225
12	0.002 ± 0.001	0.43 ± 0.006	215
	MCF-7	MCF-7/ADR
DOX	0.15 ± 0.003	11.67 ± 1.94	77.8
12	0.10 ± 0.001	1.37 ± 0.55	13.7

^aGI₅₀ values (means ± SD) were calculated from MTT assay and defined as the concentrations of compounds that resulted in 50% cell growth inhibition after 72 h of drug exposure. The experiments were repeated at least three times independently.

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

2.3. Cytotoxicity

The cytotoxicity of the synthesized benzophenanthridone derivatives was evaluated through MTT assay against four human tumor cell lines: colon cancer HCT116, breast cancer MCF-7, prostate cancer DU-145 and non-small cell lung cancer A549 cell lines. The compounds were incubated with cells for 72 h in a five-dose assay ranging from 0.01 to 100 μM concentration. At the end of the incubation, MTT solution was added to test the percentage growth of tumor cells. The GI₅₀ values, defined as the concentrations of the compounds that resulted in 50% cell growth inhibition, are calculated and summarized in Table 1.

With the increased TOP1 inhibitory activity (+, ++ and +++), the novel synthesized benzophenanthridone analogues 11, 12, 14–21, 23 and 25 exhibited increased cytotoxicity against these four tumor cell lines compared with the parent 1 with TOP1 inhibition of +/0, except for 17, which exhibited decreased cytotoxicity against MCF-7 cells.12 with the most potent TOP1 inhibition of +++ showed the highest cytotoxicity against HCT116 (GI₅₀ = 0.036 μM), MCF-7 (GI₅₀ = 0.090 μM), DU145 (GI₅₀ = 0.002 μM) and A549 (GI₅₀ = 0.97 μM) cell lines. Although 12 has the similar structure and equal TOP1 inhibition to 2, it showed higher cytotoxicity against HCT116, MCF-7 and DU145 cells, which might possibly due to its good solubility and cellular permeability. Indeed, 12 has higher bioavailability (20.4%, Table 4) in vivo than 2 (15.5%) [22]. Furthermore, 12 showed highest cytotoxicity against DU145 cells at low nanomolar concentration (0.002 μM). With the bigger steric terminus of the side chain at the 5-position, the acylated analogues 13–25 showed decreased both TOP1 inhibitory activity and cytotoxicity compared 12, which is consistent with molecular modelling analysis.

Table 4.

Pharmacokinetic parameters of 12.

parameters	Mean ± SD (n = 2)
	iv (1 mg/kg)a	ig (5 mg/kg)b
Tmax (h)	/	2 ± 0
Cmax (ng/ml)	/	19 ± 9
AUC0→t (h·ng/ml)	47.2 ± 1.8	48.3 ± 28
AUC0→∞ (h·ng/ml)	49.8 ± 1.5	/
MRTINF (h)	1.46 ± 0.027	/
T1/2 (h)	1.21 ± 0.14	/
F (%)	/	20.4 ± 11.9

^aiv means intravenous injection.

^big means intragastrical administration.

Compounds 11 and 12 were submitted to the National Cancer Institute (NCI, USA) for further study on cytotoxicity against the 60 cancer cell lines representation nine tissue types (NCI-60) [28–30]. According to the NCI established procedures, the cells were incubated with 11 or 12 for 48 h and stained with sulforhodamine B dye. The GI₅₀ values were plotted and summarized in Table 2. The results indicate that 12 has a higher mean graph midpoint (MGM) for growth inhibition of all cancer cell lines of 0.0977 μM than that of 11 (0.525 μM) and 2 (0.145 μM) [22]. 12 shows high cytotoxicity against 28 cancer cell lines at nanomolar range (<100 nM) and the most cytotoxic against leukemia SR with GI₅₀ of 0.0173 μM.

Table 2.

Cytotoxicity of 11 and 12 against individual NCI-60 cell lines.

Panel	Cell line	GI50 (μM)a		Panel	Cell line	GI50 (μM)
		11	12			11	12
	MGMb	0.525	0.0977	Colon Cancer	COLO 205	0.29	0.0605
Leukemia	CCRF-CEM	0.15	0.0201		HCC-2998	1.02	0.156
	K-562	0.161	0.0394		HCT116	0.285	0.0515
	MOLT-4	0.0633	0.0234		HCT-15	0.42	0.116
	RPMI-8226	0.257	0.0763		HT29	0.168	0.0508
	SR	0.0907	0.0173		KM12	1.1	0.238
Non-Small Cell Lung Cancer	A549/ATCC	0.584	0.0948		SW-620	0.243	0.0658
	EKVX	1.36	0.202	Renal Cancer	786–0	0.464	0.0703
	HOP-62	0.399	0.0439		A498	1.23	0.127
	HOP-92	3.56	0.184		ACHN	0.366	0.0856
	NCI-H226	1.34	0.172		CAKI-1	0.295	0.0644
	NCI-H23	0.451	0.0906		RXF 393	0.929	0.16
	NCI-H322M	0.99	0.147		SN 12C	0.739	0.171
	NCI-H460	0.23	0.0212		TK-10	1.35	0.316
	NCI-H522	0.0875	0.0274		UO-31	0.35	0.104
CNS Cancer	SF-268	0.442	0.0903	Breast Cancer	MCF-7	0.164	0.0292
	SF-295	0.279	0.0346		MDA-MB-231/ATCC	1.28	0.362
	SF-539	1.06	0.124		HS 578T	1.96	0.904
	SNB-19	0.398	0.116		BT-549	0.918	0.202
	SNB-75	0.596	0.0885		T-47D	0.368	0.0757
	U251	0.26	0.0394		MDA-MB-468	0.157	0.0599
Melanoma	LOX IMVI	0.246	0.0389	Ovarian Cancer	IGROV1	0.5	0.145
	MALME-3M	0.613	0.174		OVCAR-3	0.826	0.166
	M14	0.324	0.0581		OVCAR-4	0.782	0.154
	MDA-MB-435	0.759	0.118		OVCAR-5	1.2	0.183
	SK-MEL-2	3.01	0.393		OVCAR-8	0.93	0.132
	SK-MEL-28	3.05	0.573		NCI/ADR-RES	1.01	0.165
	SK-MEL-5	0.614	0.0909		SK-OV-3	1.24	0.157
	UACC-257	1.9	0.122	Prostate Cancer	PC-3	0.532	0.112
	UACC-62	0.278	0.0451		DU145	0.527	0.13

^aGI₅₀ values were defined as the compound concentrations that resulted in 50% cell growth inhibition. The cells were incubated for two days with the tested compounds.

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

The cytotoxicity of 12 was further assessed against a panel of isogenic CPT- and doxorubicin-resistant cell lines through MTT assay. The HCT116-siTOP1 subline was established by transfection of colon cancer parental cells HCT116 with short hairpin RNA vectors expressing siRNA for TOP1 [31]. TOP1 is the only known cellular target of CPT [9,32]. HCT116-siTOP1 subline exhibits 8.3-fold resistant to CPT compared to the parental HCT116 cells (Table 3). Similarly, HCT116-siTOP1 subline exhibits 8.9-fold resistant to 12, implying that TOP1 is a major cellular target of 12. The CPT-resistant prostate cancer DU145-RC0.1 cells have a R364H mutation in TOP1 relative to the wild-type parental DU145 cells [27]. The TOP1 with R364H mutation is catalytically active but leads DU145-RC0.1 cells resistance to CPT because the R364 residue is close to the catalytic tyrosine and can stabilize the open form of TOP1cc [7,8]. Compared to the parental DU145 cells, the DU145-RC0.1 cells show 225-fold resistant to CPT and 215-fold resistant to 12 (Table 3), which is consistent with the molecular modeling result (Fig. 3) showing a hydrogen bond between 12 and R364 residue. P-Glycoprotein (P-gp) mediated drug efflux is generally responsible for classical multiple drug resistance [33]. The chemotherapeutic agent doxorubincin (DOX) is a substrate of P-gp. Compared to the parental MCF-7 cells, the breast cancer MCF-7/ ADR cells overexpressing P-gp are highly resistant to DOX (77.8-fold) and less resistant to 12 (13.7-fold, Table 3) [34], implying 12 might not be a substrate of P-gp. These results indicate that 12 acts as TOP1 inhibitor in cancer cells, similar to its analogue 2 [22].

2.4. Induction of cellular TOP1cc and DNA damage

To assess the induction of TOP1cc by 12, the immunocomplex of enzyme to DNA (ICE) assay in HCT116 cells was performed. As shown Fig. 4A, both the positive control CPT and 12 induce the formation of cellular TOP1cc in a dose-dependent manner. And 12 shows higher ability to induce cellular TOP1cc at 25 μM than CPT.

Fig. 4.

(A) Detection of TOP1-DNA covalent cleavage complexes by in vivo complex of enzyme (ICE) assay in human colon cancer HCT116 cells. Left: lane 1, control; lanes 2 and 3, cells treated with CPT at 25 and 50 μM concentration, respectively. Right: lanes 1–3, cells treated with 12 at 25, 50 and 100 μM concentration, respectively. (B) Histone γH2AX foci induced by 12 in HCT116 cells. Cells were treated with CPT or 12 at 1 μM concentration for 3 h. DNA was stained with DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To evaluate the DNA damaging effect of 12 in cancer cells, γH2AX foci were assessed by immunofluorescence microscopy in human prostate cancer HCT116 cells. After incubation with drugs for 3 h, HCT116 cells were stained with γH2AX antibodies. As shown in Fig. 4B, similar to CPT,12 effectively induces γH2AX foci at 1 μM concentration, implying that DNA damage is induced mainly due to the trapping of cellular TOP1cc by 12.

2.5. Cancer cell apoptosis

To evaluate the induction of apoptosis by 12, flow cytometry assays were performed in HCT116 cells. HCT116 cells were incubated with 12 for 24 h and detected for the apoptotic cells. As shown in Fig. 5, 12 could obviously induce HCT116 cells apoptosis in a dose-dependent manner. 27.95% early apoptotic cells were observed after incubation with 12 at 2 μM.

Fig. 5.

Flow cytometry histograms. HCT116 cells were incubated with 12 for 24 h at 0.5 μM, 1 μM and 2 μM concentration, respectively

2.6. Pharmacokinetic parameters

The pharmacokinetic (PK) study in vivo of 12 was performed in Sprague-Dawley (SD) rats. The SD rats were randomly divided into two groups (n = 2) 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 postdosing and the concentration of 12 was measured. The PK parameters were calculated and summarized in Table 4. After iv treatment, the AUC_0→t is 47.2 ± 1.8 h ng/ml and T_1/2 is 1.21 ± 0.14 h. After ig treatment, T_max is 2 ± 0 h and C_max is 19 ± 9 ng/ml. The bioavailability (F, 20.4%) of 12 is higher than that of the analogue 2 (15.5%), which might be the reason why 12 shows higher cytotoxicity in vitro and antitumor efficiency in vivo (Fig. 7) [22].

Fig. 7.

Antitumor efficiency of 12 in HCT116 (A) and MCF-7 (B) xenograft models. The effects of 12 on tumor size (left) and tumor weight (right) at the dose of 5 mg/kg, 10 mg/kg and 20 mg/kg, respectively. Statistically significant difference in mean tumor weight compared with the control, **: P < 0.01, ***: P < 0.001.

2.7. Acute toxicity in vivo

The acute toxicity of 12 was assessed in Kunming male mice. The mice were randomly divided into six groups (n = 6) and treated with 12 by intraperitoneal injection (ip) at single doses of 300, 240, 192, 153.6 and 122.9 mg/kg. The control group was treated with sterile water. As shown in Fig. 6, after 7 days of administration with 12, all mice survived in the group of 122.9 mg/kg dose, and four mice survived in the 153.6 mg/kg dose group, three mice survived in the 192 mg/kg dose group, two mice survived in the 240 mg/kg dose group. All mice died within 5 days in the 300 mg/kg dose group. The median lethal dose (LD₅₀), defined as the dose to kill half of mice after 7 days, is 192 mg/kg.

Fig. 6.

Effect of 12 on mice survival. Mice were treated with 12 at dose 300 mg/kg, 240 mg/kg, 192 mg/kg 153.6 mg/kg and 122.9 mg/kg, respectively.

2.8. Antitumor efficiency in vivo

The in vivo antitumor efficiency of 12 was assessed in both human colon cancer HCT116 and human breast cancer MCF-7 xenograft mude mice models. For both models, the mice were randomly divided into four groups (n = 6) and treated with 12 at 20 mg/kg, 10 mg/kg or 5 mg/kg dose by ip administration daily. The control group was treated with saline. As shown in Fig. 7, administration of 12 significantly reduced the tumor volume in a dose-dependent manner in both HCT116 and MCF-7 xenograft models. 12 is more antitumor efficient in HCT116 xenograft model than MCF-7 xenograft model. The tumor weight inhibitions (TWI) of 12 at 20 μM dose are 76.9% (in HCT116 model) and 71.8% (in MCF-7 model), respectively.

3. Conclusion

In summary, a series of novel 5-aminoethyl substituted benzophenanthridinone derivatives have been synthesized and evaluated for biochemical activity as TOP1 inhibitors and cellular responses against four human cancer cell lines (HCT116, MCF-7, DU145 and A549). The TOP1-mediated cleavage assay indicates that the derivatives with bigger termini of the side chain at the 5-position show decreased TOP1 inhibitory activity. Compound 12 with methylamino ethyl group at the 5-position exhibits the most potent TOP1 inhibition of +++, and cytotoxicity in human cancer cell lines at nanomolar concentration range GI₅₀ values. 12 also shows consistent cytotoxicity in the NCI-60 cell lines with nanomolar MGM value of 0.0977 μM. Cell-based assays indicate that 12 induces the formation of cellular TOP1cc and DNA damage in HCT116 cells, promotes apoptosis, and is not a substrate of P-gp, a drug efflux protein responsible for multidrug resistance. 12 was also evaluated for PK, acute toxicity and antitumor efficiency in vivo. The results indicate that 12 exhibits antitumor efficiency in both HCT116 and MCF-7 xenograft nude mice models, and good bioavailability (20.4%) in rat model, higher than its analogue 2 (15.5%) with a dimethylaminoethyl group at 5-position, which might be the reason why 12 shows more potent antitumor activity both in vitro and in vivo. These results suggest that benzophenanthridinone scaffold is a chemotype for TOP1 inhibitors, and worth further development.

4. Experimental section

4.1. General experiments

All the required chemical reagents used for synthesis were purchased from Sigma-Aldrich, Alfa Aesar or Aladdin Reagent Database Inc (Shanghai) and used without any further purification unless otherwise indicated. Melting points were determined in open capillary tubes on a MPA100 Optimelt Automated Melting Point System without being corrected. Silica gel GF₂₅₄ thin layer chromatography (TLC) was used to monitor the progress of the chemical reaction. 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 an SHIMADZU LCMS-IT-TOF mass spectrometer. All compounds tested for biological activities were analyzed by HPLC and their purities were more than 95%. The analysis condition is: detection at 220 nm, 1.0 ml/min flow rate, a linear gradient of 50%–15% PBS buffer (pH 3) and 50%–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.

4.2. 12-(2-bromoethyl)-2,3-dimethoxy-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-13(12H)-one (9)

To a solution of 8 (0.5 mmol) in freshly distilled chloroform (10 mL), PBr₃ (1 mL) was added slowly with a syringe. The mixture was stirred at room temperature for 1 h. The resulting gray precipitate was filtered and washed with chloroform, saturated sodium bicarbonate and water, respectively. The crude solid was dried and purified by silica gel column chromatography to give the gray solid 9, yield 91%, ¹H NMR (DMSO) δ 8.84 (d, J = 9.1 Hz, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.73 (s, 1H), 7.69 (s, 1H), 6.33 (s, 2H), 5.76 (t, J = 9.0 Hz, 2H), 5.35 (t, J = 9.0 Hz, 2H), 4.21 (s, 3H), 4.04 (s, 3H). ESI-MS m/z: 455.1 (100%), 457.1 (100%) [M + H]⁺.

4.3. 12-(2-aminoethyl)-2,3-dimethoxy-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-13(12H)-one (11)

To a solution of 9 (456 mg, 1 mmol) in DMSO (50 mL), NaN₃ (130 mg, 2 mmol) was added. The mixture was stirred at room temperature for 16 h. And then, the reactive solution was poured into water (100 mL). The formed gray precipitate was filtered and washed with saturated sodium bicarbonate and water, respectively. The crude solid (10) was dried for the next synthesis without further purification.

The crude solid 10 was dissolved in THF (100 mL). Pd/C (42 mg) was added to the solution. The mixture was stirred at room temperature under hydrogen atmosphere for 2 h. The reaction mixture was filtered and washed with THF. The filtrate was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the white solid 11, yield 51% for the two steps, mp = 264.3–267.1 °C. ¹H NMR (CDCl₃) δ 7.98 (d, J = 8.4 Hz,1H), 7.91 (s,1H), 7.59–7.54 (m, 2H), 7.52 (s, 1H), 7.18 (s, 1H), 6.10 (s, 2H), 4.60 (t, J = 6.0 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.13 (t, J = 6.0 Hz, 2H). ¹³C NMR (CDCl₃) δ 164.9, 153.8, 149.9, 147.7, 147.6, 135.6, 131.9, 129.1, 123.6, 121.3, 119.6, 118.5, 117.4, 109.0, 105.0, 103.0, 102.4, 101.7, 56.4, 56.3, 55.1, 41.5. HRMS (ESI) m/z: 393.1439 [M + H]⁺, calcd for C₂₂H₂₁N₂O₅ 393.1445.

4.4. 2,3-Dimethoxy-12-(2-(methylamino)ethyl)-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenanthridin-13(12H)-one (12)

To a solution of 11 (470 mg, 0.12 mmol) in acetic acid (140 mL) and water (100 mL), aqueous formaldehyde solution (40%, 90 mL) and Zn (156 mg, 2.4 mmol) was added. The mixture was stirred at room temperature overnight. Ammonia water was added to quench the reaction. The solution was exacted with chloroform (3 × 50 mL). The combined organic layer was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the white solid 12, yield 59%, mp = 211.8–212.0 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.8 Hz, 1H), 7.90 (s, 1H), 7.59–7.54 (m, 2H), 7.54 (s, 1H), 7.18 (s, 1H), 6.10 (s, 2H), 4.61 (t, J = 6.4 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 3.11 (t, J = 6.4 Hz, 2H), 2.36 (s, 3H).¹³C NMR (CDCl₃) δ 165.0,153.9,149.9,147.7,147.6,135.6, 131.9,129.2,123.6,121.2,119.6,118.5,117.4,108.9,105.0,103.1,102.4, 101.7, 56.4, 56.3, 52.0, 51.3, 36.2. HRMS (ESI) m/z: 407.1601 [M + H]⁺,calcd for C₂₃H₂₃N₂O₅ 407.1601.

4.5. General procedure for the synthesis of compounds 13–25

To a solution of 12 (or 11, 0.24 mmol) and DIPEA (2.4 mmol) in freshly distilled dichloromethane (30 mL), the solution of acyl chlorides (0.32 mmol) in dichloromethane (5 mL) was added slowly at an ice bath. And then, the reaction solution was stirred and heated under reflux for 1 h (for 13–19, or at room temperature for 20–25). The reaction solution was cooled to room temperature. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give target product.

4.5.1. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo[1,2-c]phenan-thridin-12(13H)-yl)ethyl)-1,1,1-trifluoro-N-methylmethanesulfonamide (13)

White solid, yield 40%, mp = 217.4–217.8 °C. ¹H NMR (CDCl₃) δ 7.99 (d, J = 8.8 Hz, 1H), 7.87 (s, 1H), 7.62–7.56 (m, 2H), 7.41 (s, 1H), 7.20 (s, 1H), 6.13 (s, 2H), 4.81 (t, J = 6.6 Hz, 2H), 4.11 (s, 2H), 4.06 (s, 2H), 3.82 (s, 2H), 2.87 (s, 3H). ¹³C NMR (CDCl₃) δ 164.6, 153.9, 149.8, 147.8, 147.7, 134.6, 131.8, 129.2, 123.9, 120.5, 119.2, 118.4, 117.7, 108.6, 105.2, 102.9, 101.7, 101.6, 56.2, 56.2, 49.0, 48.8, 35.9. HRMS (ESI) m/z: 539.1116 [M + H]⁺, calcd for C₂₄H₂₂N₂O₇F₃S 539.1094.

4.5.2. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanth-ridin-12(13H)-yl)ethyl)-N-methylcyclopropanesulfonamide (14)

White solid, yield 80%, mp > 280 °C. ¹H NMR (CDCl₃) δ 7.96 (d, J = 8.5 Hz, 1H), 7.88 (s, 1H), 7.56 (d, J = 6.0 Hz, 2H), 7.52 (s, 1H), 7.17 (s, 1H), 6.11 (s, 2H), 4.74 (t, J = 6.5 Hz, 2H), 4.10 (s, 2H), 4.05 (s, 2H), 3.74 (t, J = 6.6 Hz, 2H), 2.78 (s, 3H), 2.08 (d, J = 4.5 Hz, 1H), 1.07 (d, J = 3.6 Hz, 2H), 0.83 (d, J = 6.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 164.6, 153.8, 149.7, 147.7, 147.7, 135.1, 131.8, 129.2, 123.7, 120.7, 119.2, 118.3, 117.4,108.6,104.9,102.9,102.1,101.7, 56.2, 56.1, 50.2, 48.5, 35.7, 26.9, 4.5, 4.5. HRMS (ESI) m/z: 511.1536 [M + H]⁺, calcd for C₂₆H₂₇N₂O₇S 511.1533.

4.5.3. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenan-thridin-12(13H)-yl)ethyl)-N-methyldimethylamine-1-sulfonamide (15)

White solid, yield 83%, mp = 234.7–235.9 °C · ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.6 Hz, 1H), 7.88 (s, 1H), 7.60–7.49 (m, 3H), 7.18 (s, 1H), 6.12 (s, 2H), 4.74 (t, J = 6.5 Hz, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.71 (t, J = 6.5 Hz, 2H), 2.68 (s, 3H), 2.59 (s, 6H). ¹³C NMR (CDCl₃) δ 164.6, 153.8,149.7,147.7,147.6,135.2,131.8,129.2,123.6,120.8,119.3,118.3, 117.4,108.6,104.9,102.8,102.1,101.7, 56.3, 56.2, 49.8, 48.9, 37.8, 37.8, 36.1. HRMS (ESI) m/z: 514.1669 [M+ H]⁺, calcd for C₂₅H₂₈N₃O₇S 514.1642.

4.5.4. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phena-nthridin-12(13H)-yl)ethyl)-N-methylpyrrolidine-1-sulfonamide (16)

White solid, yield 81%, mp = 223.4–224.3 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.7 Hz, 1H), 7.89 (s, 1H), 7.56 (d, J = 6.5 Hz, 2H), 7.54 (s, 1H), 7.18 (s,1H), 6.12 (s, 2H), 4.75 (t, J = 6.6 Hz, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.69 (t, J = 6.6 Hz, 2H), 3.03 (t, J = 6.7 Hz, 4H), 2.64 (s, 3H), 1.82–1.75 (m, 4H).¹³C NMR (CDCl₃) δ 164.6,153.8,149.7,147.7,147.6, 135.3,131.8,129.2,123.5,120.9,119.4,118.2,117.4,108.7,104.9,102.9, 102.2, 101.7, 56.3, 56.2, 50.1, 48.8, 47.9, 47.9, 35.9, 25.5, 25.5. HRMS (ESI) m/z: 540.1811 [M + H]⁺, calcd for C₂₇H₃₀N₃O₇ 540.1799.

4.5.5. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenan-thridin-12(13H)-yl)ethyl)-N-methylmorpholine-4-sulfonamide (17)

White solid, yield 85%, mp = 205.7–206.2 °C. ¹H NMR (CDCl₃) δ 7.96 (d, J = 8.7 Hz, 1H), 7.88 (s, 1H), 7.56 (t, J = 4.3 Hz, 2H), 7.27 (s, 1H), 7.17 (s,1H), 6.11 (s, 2H), 4.75 (t, J = 6.5 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.69 (t, J =6.5 Hz, 2H), 3.55 (t, J = 4.0 Hz, 4H), 2.91(t, J = 4.0 Hz, 3H), 2.64 (s, 3H). ¹³C NMR (CDCl₃) δ 164.5, 153.8, 149.8, 147.7, 147.7, 135.1, 131.7, 129.1, 123.6, 120.8, 119.3, 118.3, 117.5, 108.6, 104.9, 102.8, 102.1, 101.7, 66.2, 66.2, 56.5, 56.2, 49.9, 48.9, 45.9, 45.9, 36.3. HRMS (ESI) m/z: 556.1778 [M + H]⁺, calcd for C₂₇H₃₀N₃O₈S 556.1748.

4.5.6. Dimethyl(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5] benzo[1,2-c]phe-nanthridin-12(13H)-yl)ethyl)(methyl) phosphoramidate (18)

White solid, yield 30%, mp = 220.0–221.7 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.6 Hz, 1H), 7.90 (s, 1H), 7.57 (s, 1H), 7.54 (d, J = 5.9 Hz, 2H), 7.18 (s,1H), 6.11 (s, 2H), 4.73 (t, J = 6.5 Hz, 2H), 4.10 (s, 3H), 4.06 (s, 3H), 3.55–3.47 (m, 2H), 3.44 (s, 3H), 3.42 (s, 3H), 2.49 (d, J = 9.8 Hz, 3H). ¹³C NMR (CDCl₃) δ 164.5, 153.6, 149.7, 147.6, 147.5, 135.3,131.7,129.1,123.4,120.9,119.4,118.3,117.4,108.8,104.8,102.8, 102.3, 101.6, 56.3, 56.1, 52.7, 52.7, 50.3, 47.4, 34.3. HRMS (ESI) m/z: 515.1584 [M + H]⁺, calcd for C₂₅H₂₈N₂O₈P 515.1578.

4.5.7. 2,3-Dimethoxy-12-(2-(methyl(2-oxido-1,3,2-dioxaphospholan-2-yl)amino)ethyl)-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-13(12H)-one (19)

White solid, yield 40%, mp = 221.3–222.4 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.8 Hz, 1H), 7.86 (s, 1H), 7.56 (dd, J = 11.7, 6.9 Hz, 2H), 7.51 (s, 1H), 7.18 (s, 1H), 6.12 (s, 2H), 4.74–4.66 (m, 2H), 4.32–4.25 (m, 2H), 4.24–4.19 (m, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.53–3.41 (m, 2H), 2.65 (d, J = 10.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 164.6, 153.6, 149.6, 147.6, 147.5, 135.0, 131.7, 129.2, 123.5, 120.7, 119.3, 118.3, 117.5, 108.5, 104.9,102.9,102.0,101.7, 65.7, 65.6, 56.3, 56.1, 49.1, 47.0, 34.5. HRMS (ESI) m/z: 513.1446 [M + H]⁺, calcd for C₂₅H₃₆N₂O₈P 513.1421.

4.5.8. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-12(13H)-yl)ethyl)cyclopropanesulfonamide (20)

White solid, yield 60%, mp = 245.4–246.2 °C. ¹H NMR CDCl₃) δ 7.97 (d, J = 8.7 Hz, 1H), 7.86 (s, 1H), 7.57 (d, J = 9.7 Hz, 2H), 7.30 (s, 1H), 7.18 (s, 1H), 6.12 (s, 2H), 5.52 (t, J = 6.1 Hz, 1H), 4.62 (t, J = 5.5 Hz, 2H), 4.11 (s, 3H), 4.05 (s, 3H), 3.78 (dd, J = 11.4, 5.7 Hz, 2H), 2.26 (td, J = 8.0, 4.1 Hz, 1H), 1.14–1.02 (m, 2H), 0.92–0.81 (m, 2H). ¹³C NMR (CDCl₃) δ 165.0, 153.9, 149.8, 147.7, 147.6, 135.1, 131.8, 129.2, 123.7, 120.7, 118.9, 118.4, 117.3, 108.6, 105.1, 102.9, 101.7, 101.7, 56.3, 56.2, 52.6, 43.5, 30.2, 5.4, 5.4. HRMS (ESI) m/z: 497.1394 [M + H]⁺, calcd for C₂₅H₂₅N₂O₇S 497.1377.

4.5.9. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-12(13H)-yl)ethyl)dimethylamine-1-sulfonamide (21)

White solid, yield 65%, mp > 280 °C. ¹H NMR (CDCl₃) δ 7.98 (d, J = 8.7 Hz, 1H), 7.86 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.29 (s, 1H), 7.19 (s, 1H), 6.11 (s, 2H), 5.58 (t, J = 5.9 Hz, 1H), 4.59 (t, J = 5.4 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.70 (t, J = 5.4 Hz, 2H), 2.70 (s, 6H).¹³C NMR (CDCl₃) δ 165.1, 153.9, 149.8, 147.7, 147.6, 135.1, 131.9, 129.2, 123.8, 120.7, 118.9, 118.4, 117.3, 108.6, 105.1, 102.9, 101.7, 101.7, 56.3, 56.2, 52.4, 44.2, 37.9. 37.9. HRMS (ESI) m/z: 500.1531 [M + H]⁺, calcd for C₂₄H₂₆N₃O₇S 500.1486.

4.5.10. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phenanthridin-12(13H)-yl)ethyl)pyrrolidine-1-sulfonamide (22)

White solid, yield 85%, mp = 249.7–250.8 °C. ¹H NMR (CDCl₃) δ 7.98 (d, J = 8.7 Hz, 1H), 7.87 (s, 1H), 7.58 (d, J = 7.2 Hz, 2H), 7.31 (s, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 5.41 (t, J = 5.9 Hz, 1H), 4.62 (t, J = 5.4 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.70–3.67 (m, 2H), 3.19 (t, J = 6.6 Hz, 4H), 1.82 (t, J = 6.6 Hz, 4H). ¹³C NMR (CDCl₃) δ 165.0, 153.9,149.8,147.6,147.6,135.2,131.9,129.2,123.7,120.8,119.1,118.4, 117.4,108.7,105.1,102.9,101.8,101.7, 56.3, 56.2, 52.3, 48.0, 48.0, 43.9, 25.5, 25.5.HRMS (ESI) m/z:526.1640 [M + H]⁺,calcd for C₂₆H₂₈N₃O₇S 526.1642.

4.5.11. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’5’:4,5]benzo [1,2-c]phenanthridin-12(13H)-yl)ethyl)morpholine-4-sulfonamide (23)

White solid, yield 70%, mp = 221.6–222.3 °C. ¹H NMR (CDCl₃) δ 7.98 (d, J = 8.5 Hz,1H), 7.85 (s,1H), 7.58 (d, J = 11.4 Hz, 2H), 7.27 (s, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 5.66 (s, 1H), 4.61 (t, J = 4.8 Hz 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.68 (t, J = 4.8 Hz, 2H), 3.64 (s, 4H), 3.06 (s, 4H). ¹³C NMR (CDCl₃) δ 165.2,154.0,149.9,147.7,147.7,135.0,131.9,129.2, 123.8, 120.7, 118.9, 118.4, 117.4, 108.6, 105.1, 102.9, 101.7, 101.6, 66.1, 66.1, 56.3, 56.2, 52.3, 46.1, 44.4, 44.4. HRMS (ESI) m/z: 542.1623 [M + H]⁺, calcd for C₂₆H₂₈N₃O₈S 542.1592.

4.5.12. Dimethyl(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5] benzo[1,2-c]phena-nthridin-12(13H)-yl)ethyl)phosphoramidate (24)

White solid, yield 62%, mp = 197.2–197.8 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.7 Hz, 1H), 7.88 (s, 1H), 7.56 (d, J = 7.5 Hz, 2H), 7.37 (s, 1H), 7.19 (s,1H), 6.11 (s, 2H), 4.60 (t, J = 5.8 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.55 (s, 3H), 3.52 (s, 3H), 3.50–3.46 (m, 2H). ¹³C NMR (CDCl₃) δ 164.8, 153.8, 149.8, 147.6, 147.5, 135.4, 131.7, 129.1, 123.6, 120.9, 119.3, 118.4, 117.3, 108.7, 104.9, 102.9, 101.9, 101.6, 56.3, 56.2, 53.6, 52.9, 52.9, 41.1. HRMS (ESI) m/z: 501.1423 [M + H]⁺, calcd for C₂₄H₂₆N₂O₈P 501.1421.

4.5.13. N-(2-(2,3-dimethoxy-13-oxo-[1,3]dioxolo[4’,5’:4,5]benzo [1,2-c]phena-nthridin-12(13H)-yl)ethyl)cyclopropanecarboxamide (25)

White solid, yield 80%, mp = 237.6–238.1 °C. ¹H NMR (CDCl₃) δ 7.97 (d, J = 8.7 Hz, 1H), 7.89 (s, 1H), 7.57 (d, J = 9.7 Hz, 2H), 7.42 (s, 1H), 7.17 (s,1H), 6.73 (s,1H), 6.10 (s, 2H), 4.60 (t, J = 4.9 Hz, 2H), 4.11 (s, 3H), 4.07 (s, 3H), 3.88 (d, J = 5.0 Hz, 2H), 0.87 (d, J = 6.7 Hz, 1H), 0.81 (s, 2H), 0.63 (d, J = 4.7 Hz, 2H). ¹³C NMR (CDCl₃) δ 173.5, 165.2, 153.8, 149.7, 147.7, 147.6, 135.4, 131.8, 129.3, 123.7, 120.9, 119.1, 118.2, 117.1, 108.5, 104.9, 102.9, 102.1, 101.7, 56.3, 56.2, 52.2, 40.3, 14.7, 7.0, 7.0. HRMS (ESI) m/z: 461.1732 [M + H]⁺, calcd for C₂₆H₂₅N₂O₆ 461.1707.

4.6. TOP1-mediated cleavage assay

DNA cleavage assays were performed using a 3’-[³²P]-labeled 117-bp DNA oligonucleotide as substrate according to the previously reported method [26]. 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.

4.7. Molecular modelling

The molecular modelling was conducted as previously reported method [22]. Briefly, the X-ray crystal structures of the ternary TOP1-DNA-ligand complex (PDB ID: 1K4T) was obtained and cleaned, 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. The compounds constructed using ChemDraw were saved in SDFfile formats and corrected using MOE software. 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 forcefield was utilized within the MOE software for energy minimization.

4.8. 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 drug treatment experiments, the cancer cells were treated with the compounds (predissolved in DMSO) at a five-dose assay ranging from 0.01 to 100 μM concentration. After incubation for 72 h at 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 4 h incubation, the formazan crystal formed in the well was dissolved with 100 mL of DMSO for optical density reading at 570 nm [35]. The GI₅₀ value was calculated by nonlinear regression analysis (GraphPad Prism).

4.9. Immunodetection of cellular TOP1-DNA complex

The ICE assays for cellular TOP1-DNA adduct was performed according to the reported method [36]. Briefly, mid-log phase HCT116 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 (12,000 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) were dissolved in NaH₂PO₄ buffer (30 μL, 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 HRPconjugated secondary antibodies (Cell Signaling Technology, 1:3000) at room temperature for 1 h. Reactive dots were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

4.10. γH2AX detection

γH2AX staining was performed as described [37]. 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 blocked with 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 (Cell Signaling Technology) at 37 °C overnight. The cover slips were washed six times with blocking buffer and then incubated with anti-rabbit 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.

4.11. Pharmacokinetic study in vivo

The PK study was conducted according to our previous method [22]. Briefly, male SD rats (weighing 220–250 g, n = 2) were treated with compound 12 pre-dissolved in10% DMSO and 10% Kolliphor^® HS15 (a non-ionic 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 and 4 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.

4.12. In vivo acute toxicity

Based on the preliminary experiments, the Kuning male mice were randomly divided into six groups (n = 6) 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 12 in a single dose 300, 240, 192, 153.6 and 122.9 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.

4.13. 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 and MCF-7. Male nude mice 4–5 weeks old weighing 12–15 g were used. Tumor pre-induced in the mice by subcutaneously injecting of cancer 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 four groups (n = 6) and administered by intraperitoneal injection. The testing groups were treated with 12 in 20 mg/kg, 10 mg/kg 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, 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%.

4.14. Statisticl 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.