Design, synthesis, and cytotoxic activity of 2-amino-1,4-naphthoquinone-benzamide derivatives as apoptosis inducers

Abstract

A new series of 2-amino-1,4-naphthoquinone-benzamides 5a-n was designed based on previously reported potent cytotoxic agents. These compounds were synthesized from the reaction of 1,4-naphthoquinone, 4-aminobenzoic acid, and appropriate amine derivatives in good yields. Cytotoxic activities of the target compounds 5a-n were evaluated against three cancer cell lines MDA-MB-231, SUIT-2, and HT-29 by MTT assay and the obtained in vitro data. All newly synthesized compounds were more potent than positive control cisplatin against MDA-MB-231 cell line and less potent than this control against SUIT-2 cell line. Moreover, most of the synthesized compounds were more potent than cisplatin against HT-29 cell line. Among the synthesized compounds, compound 5e was the most potent cytotoxic agent against all the three evaluated cell lines. Moreover, cell cycle analysis showed that derivatives 5f and 5l dose-dependently increase the percentage of sub-G1 cells. Induction of apoptosis as an important mechanism of anti-cancer drugs was confirmed morphologically in the most potent new compounds 5e, 5f, 5g, and 5l by Hoechst 33,258 staining.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-78468-2.

Introduction

Cancer is the second most common cause of death in the world¹. The most important available methods of treating this disease are chemotherapy and surgery². Chemotherapeutic agents that help to destroy cancer cells by different mechanisms, almost invariably suffer from low efficacy and high toxicity³. Therefore, design of effective and safe new chemotherapeutic agents is a golden target for medicinal chemists^4–6.

Apoptosis induction is a common mechanism of action of many chemotherapeutic agents, which leads to the destruction of malignant cells⁷. Therefore, if the newly designed compounds can induce apoptosis, they can be further evaluated as promising anticancer agents⁸.

1,4-Naphthoquinone is an important anti-cancer pharmacophore that is found in a number of chemotherapeutic agents such as doxorubicin, daunorubicin, and mitoxantrone and natural cytotoxic compounds such as lawsone, lapachol, juglone, and plumbagin (Fig. 1)^9–15.

Fig. 1.

Chemical structures of 1,4-naphthoquinone, anticancer drugs, and cytotoxic agents containing 1,4-naphthoquinone moiety.

In addition, a large number of synthetic 1,4-naphthoquinone derivatives with cytotoxic effects have been reported¹⁶. In Fig. 2, some of these derivatives are showed^17–19. As can be seen in this figure, compounds A-D are derivatives of 2-amino-1,4-naphthoquinone that showed cytotoxic effects via induction of apoptosis. On the other hand, potent cytotoxic compounds D in addition to 2-amino-1,4-naphthoquinone moiety, have benzamide unit in their structures (Fig. 2). Therefore, in the present work, new structures of 2-amino-1,4-naphthoquinone-benzamide scaffold were designed and evaluated as potential apoptosis-inducing agents.

Fig. 2.

2-Amino-1,4-naphthoquinone derivatives A-D with cytotoxic effect and new designed 1,4-naphthoquinone-benzamide derivatives as potent cytotoxic agents.

Results and discussion

Chemistry

As illustrated in Scheme 1, 2-amino-1,4-naphthoquinone-benzamides 5a-n were prepared. 1,4-Naphthoquinone 1 was treated with 4-aminobenzoic acid 2 in DMF at 80 °C for 12 h to produce 4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzoic acid 3. In the next step, target compounds 5a-n were synthesized via a nucleophilic substitution reaction between compound 3 and different amine derivatives 4a-n in the presence of TBTU and NEt₃ in DMF at room temperature for 24 h.

Scheme 1.

Synthetic procedure for preparation of 2-amino-1,4-naphthoquinone-benzamide derivatives 5a-n.

Scheme 2.

SARs diagram of the aliphatic derivatives 5a-d.

Assessment of the antiproliferative effect

The antiproliferative effects of 14 synthesized compounds 5a-n were assessed against breast cancer cell line MDA-MB-231, pancreatic cancer cell line SUIT-2, and colorectal adenocarcinoma cell line HT-29 by MTT assay²⁰. The antiproliferative activity of cisplatin and doxorubicin were also evaluated as positive control agents. Obtained IC₅₀ values (the concentration required to cause 50% cell growth inhibition) and structures of the newly synthesized compounds 5a-n are listed in Table 1.

Table 1.

Antiproliferative activity of synthesized compounds 5a-n against MDA-MB-231, SUIT-2, and HT-29 cell lines.

Compound	R	IC50 (µM)
		MDA-MB-231	SUIT-2	HT-29
5a		12.1 ± 2.6	27.9 ± 5.3	14.9 ± 2.5
5b		4.3 ± 0.9	16.6 ± 2.6	5.1 ± 1.1
5c		10.0 ± 4.2	21.9 ± 2.8	58.3 ± 9.5
5d		1.7 ± 0.3	7.2 ± 2.4	15.2 ± 3.9
5e		0.4 ± 0.1	0.7 ± 0.1	0.5 ± 0.2
5f		1.8 ± 0.1	1.5 ± 0.4	0.8 ± 0.4
5g		0.9 ± 0.3	2.8 ± 0.7	0.9 ± 0.3
5h		12.4 ± 3.4	32.4 ± 4.0	16.7 ± 0.9
5i		14.2 ± 3.9	12.1 ± 2.0	8.7 ± 0.7
5j		29.4 ± 5.2	23.1 ± 3.1	13.5 ± 2.3
5k		24.2 ± 2.4	27.3 ± 4.9	> 100
5l		0.4 ± 0.1	1.9 ± 0.4	1.1 ± 0.4
5m		12.5 ± 0.9	9.4 ± 2.1	> 100
5n		19.3 ± 1.6	17.5 ± 3.1	> 100
Cisplatin	-	31.5 ± 6.5	0.6 ± 0.11	25.4 ± 1.8
Doxorubicin	-	0.4 ± 0.1	0.1 ± 0.03	0.2 ± 0.02

As can be seen in Table 1, all the synthesized compounds 5a-n were more potent than cisplatin against MDA-MB-231 cell line. The most potent compounds 5e and 5l possessing the IC₅₀ values of 0.4 µM were 78.8-fold more active than the positive control cisplatin. All compounds 5a-n were also effective against SUIT-2 cell line but their antiproliferative activities were less than standard controls. Moreover, our new compounds, with the exception of compounds 5c, 5k, 5m, and 5n, demonstrated cytotoxic activity more than cisplatin against HT-29 cell line. The most potent compound against HT-29 (compound 5e, IC₅₀ = 0.5 µM) was 50.8-times more active than cisplatin. The comparison of IC₅₀ values of new compounds 5a-n against all studied cell lines with doxorubicin revealed that only compounds 5e and 5l against MDA-MB-231 cell line exhibited cytotoxic effects similar to doxorubicin, while other observed cytotoxic effects were weaker than doxorubicin.

Structurally, our newly synthesized compounds 5a-n could be divided into three groups: aliphatic derivatives 5a-d, aromatic derivatives 5e-l, and aliphatic-aromatic derivatives 5m-n.

Among the aliphatic derivatives, the most potent compound against cell lines MDA-MB-231 and SUIT-2 was isobutyl derivative 5d and the most effective compound against HT-29 cells was propyl derivative 5b. The latter derivative was also the second potent compound among the aliphatic derivatives against MDA-MB-231 and SUIT-2 cell lines. Structure-activity relationships (SARs) diagram of compounds 5a-d was depicted in Scheme 2.

SAR survey on aromatic derivatives 5e-l was schematically showed in Scheme 3. As can be seen in Table 1 and Scheme 3, the most potent compound against all studied cell lines in this series and all the new synthesized compounds was un-substituted derivative 5e. 3-Nitrophenyl derivative 5l of this series also demonstrated excellent cytotoxic effect (equal with compound 5e) against MDA-MB-231 cell line. Introduction of substituents such as 4-CH₃ and 2,4-diCH₃ on phenyl ring of compound 5e, in the case of compounds 5f and 5g, led to a slight reduction in the cytotoxicity while introduction of halogen atoms on phenyl ring of compound 5e dramatically decreased cytotoxic effect as observed in 2-chloro derivative 5h, 3-chloro derivative 5i, 2,4-dichloro derivative 5j, and 4-bromo derivative 5k.

Scheme 3.

SARs diagram of the aromatic derivatives 5e-l.

Observed IC₅₀ values also demonstrated that aliphatic-aromatic derivatives 5m-n exhibited high cytotoxic effect against MDA-MB-231, low cytotoxic effect against SUIT-2, and were inactive against HT-29.

The comparison of cytotoxic effects of the new 2-amino-1,4-naphthoquinone-benzamides (compounds 5 ) against breast cancer cell line MDA-MB-231 with their corresponding analogs of template compounds B, which were tested against breast cancer cell line MCF-7 is shown in Fig. 3¹⁸. This comparison demonstrated that phenyl and 4-mthylpheny derivatives of series 5 were more potent than their corresponding analogs of series B. In contrast, 3-chlorophenyl and 4-bromophenyl of series 5 were less potent than their corresponding analogs of series B.

Fig. 3.

Cytotoxic effects of the template compounds B in comparison to their corresponding analogs of new compounds 5 against breast cancer cell lines.

In Figs. 4 and 5, the comparison of the cytotoxic effects of template compounds C and D against A549 cell line with their corresponding analogs among the new compounds 5 are shown¹⁹. Although the cell lines used in the study of series C and D are different from our report, it seems that the majority of the new compounds can produce better cytotoxic effects.

Fig. 4.

Comparison of the cytotoxic effects of the template compounds C and their corresponding analogs among new compounds 5.

Fig. 5.

Comparison of the cytotoxic effects of the template compounds D and their corresponding analogs among new compounds 5.

Based on the results of the MTT assay, four compounds, including 5e, 5f, 5g, and 5l, exhibited the lowest IC₅₀ values, particularly against MDA-MB-231 cells and were selected for further examination regarding their effect on cell cycle alterations and apoptosis in this cell line.

Flow cytometric analysis of cell cycle alterations

RNase/propidium iodide (PI)-based flow cytometry was used to analyze cells in different cell cycle phases²¹. The most potent new cytotoxic agents 5e, 5f, 5g, and 5l based on the MTT results were selected to investigate their possible effect on the distribution of MDA-MB-231 cells in different phases of cell cycle. The pattern that would be expected from continuously dividing cancer cells was displayed by untreated control cells. The results showed that, in general, these derivatives can increase the Sub-G1 phase in a dose-dependent manner compared to the control cells, and this increase was significant at the dose of 10 µM and 30 µM in compound 5f and in the dose of 30 µM in compound 5l. The cells in the sub-G1 phase of cell cycle are apoptotic and this increase clearly indicates that the compounds are capable of apoptosis induction in cancer cells. It should be noted that the compounds were unable to significantly alter the G1, S, or G2 phases. Table 2; Fig. 6 depict the distribution of control cells and treated cells at different phases of the cell cycle.

Table 2.

Percentage of breast cancer cell line MDA-MB-231 in each phase of cell cycle after being treated with the potent new cytotoxic compounds 5e-g and 5l.

Compound	Sub-G1	G1	S	G2/M
5e (10 µM)	0.58 ± 0.06	66.64 ± 4.32	10.17 ± 2.34	22.62 ± 5.34
5e (30 µM)	0.59 ± 0.08	67.76 ± 4.04	8.81 ± 1.34	22.84 ± 3.24
5f (10 µM)	0.97 ± 0.70 *	67.07 ± 2.75	9.79 ± 5.39	22.17 ± 2.75
5f (30 µM)	1.04 ± 0.23 *	67.88 ± 2.47	9.06 ± 1.70	22.02 ± 3.11
5g (10 µM)	0.57 ± 0.23	66.31 ± 1.69	10.88 ± 3.85	22.24 ± 2.45
5g (30 µM)	0.66 ± 0.25	66.45 ± 4.06	10.24 ± 2.68	22.65 ± 4.55
5l (10 µM)	0.90 ± 0.12	66.97 ± 2.10	8.91 ± 0.67	23.23 ± 1.75
5l (30 µM)	1.55 ± 0.32 *	67.04 ± 0.62	8.36 ± 1.25	23.05 ± 1.62
Control	0.42 ± 0.11	67.22 ± 4.27	11.05 ± 3.07	21.32 ± 3.45

*There was a significant difference between the treated and untreated cells (P < 0.05).

Fig. 6.

Flow cytometric analysis of cell cycle distribution of MDA-MB-231 cells treated with synthesized derivatives.

MDA-MB-231 cells were seeded in 12-well plates at a density of 1.25 × 105 cells/ml and treated with compounds 5e, 5f, 5g, and 5l at 10 and 30 µM for 48 h. The cells were then trypsinized, washed with PBS and then fixed in 70% ice-cold ethanol at − 20 °C. Subsequently, the fixed cells were washed again with PBS and stained with propidium iodide (PI, 20 µg/ml) and RNase (200 µg/ml) for 30 min. In the end, 20,000 events were analyzed by a FACSCalibur flow cytometer (BD Biosciences). Distinct phases of cell cycle including sub-G1, G1, S and G2/M phases are shown in each histogram. The percentage of cells in different phases were also calculated and reported in Table 2. The experiments were repeated at least three times. A representative histogram for each treatment is shown.

Determination of apoptosis by Hoechst staining

The Hoechst 33,258 staining was used to examine the induction of apoptosis by synthetic compounds²². The MDA-MB-231 cells were exposed to the most potent compounds 5e, 5f, 5g, and 5l for a duration of 48 h.

Cells that undergo apoptosis exhibit different morphological features, including chromatin condensation, as well as nuclear shrinkage and fragmentation. Figure 7 displays the images of treated synthesized compounds 5e, 5f, 5g, and 5l and untreated control cells. The results demonstrated that the synthesized derivatives caused modifications in nuclear morphology in comparison to the control cells, indicating their effectiveness in triggering apoptosis. At a dosage of 10 µM, these compounds exhibited obvious signs of apoptosis, such as nuclear condensation and fragmentation. The ability to induce apoptosis is a key characteristic of successful anticancer therapeutic agents. Therefore, these chemicals have the potential to act as anticancer agents due to their apoptotic effects.

Fig. 7.

Apoptosis induction in MDA-MB-231 cells by synthesized compounds. MDA-MB-231 cells were seeded in six-well plates and treated with compounds 5e, 5f, 5g, and 5l at a concentration of 10 µM for 48 h. The cells were then stained with Hoechst 33,258 (2.5 µg/ml) for 30 min after being fixed with 4% paraformaldehyde. The cells were then observed and imaged under a fluorescence microscope. Red arrows indicate apoptotic cells.

Docking study

The most important mechanism reported for the cytotoxic effects of naphthoquinone is topoisomerase II inhibition¹⁸. Therefore, the best new naphthoquinone derivative 5e was placed in the active site of this enzyme and its interactions were compared with the standard drug doxorubicin. The superposed structure of doxorubicin as a standard topoisomerase II inhibitor and compound 5e in the active site of this enzyme is shown in Fig. 8.

Fig. 8.

Doxorubicin (pink) and most cytotoxic compound 5e (cyan) superimposed in the active site pocket of topoisomerase II.

The detailed binding mode of doxorubicin showed that this agent formed interactions with eleven amino acids Glu376, Asn163, His279, Gly164, Phe142, Ile141, Ala167, Asn150, Ser149, Asn91, Asp94 (unfavorable interaction), Gly161, and Arg162 in the topoisomerase II active site (Fig. 9). Figure 9 also shows the 2D and 3D docking poses of compound 5e. As can be seen in this figure, compound 5e interacted with six amino acids of eleven amino acids that doxorubicin interacted with them: Phe142, Ile141, Ala167, Asn150, Ser149, and Arg162. In addition to these six amino acids, the compound 5e also interacted with amino acids Lys168, Asp86, Ile317, Val90, Lys378, and Glu87.

Fig. 9.

The predicted binding modes of doxorubicin (a) and selected compound 5e (b) in the topoisomerase II active site.

Conclusion

In conclusion, we designed and synthesized 2-amino-1,4-naphthoquinone-benzamides 5a-n as new cytotoxic agents. These compounds were derived from various amines. All the synthesized compounds showed excellent cytotoxic activity against breast cancer cell line MDA-MB-231, more potent than standard drug cisplatin. Representatively, aniline derivative 5e and 3-nitroaniline derivative 5l with IC₅₀ values of 0.4 µM were 78.75 times more potent than cisplatin against MDA-MB-231 cells. Compound 5e was also 50.8-times more active than cisplatin against HT-29 cells. Further biological evaluations including flowcytometric analysis and Hoechst 33,258 staining, revealed that our potent new compounds were apoptosis inducer.

Experimental

Synthesis of 4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzoic acid 3. A mixture of 1,4-naphthoquinone 1 (15 mmol), 4-aminobenzoic acid 2 (15 mmol), and CuSO4 in DMF was stirred at 80 °C for 12 h. After completion of reaction (checked by TLC), the mixture was poured in water and observed participate was collected by filtration. This participate without purification was used for next step.

General synthetic procedure for 2-amino-1,4-naphthoquinone-benzamides 5a-n. A mixture of 4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzoic acid 3 (1 mmol), appropriate amine derivatives 4a-n (1 mmol), TBTU, and NEt₃ in DMF was stirred at room temperature for 24 h. After completion of reaction (checked by TLC), the mixture was poured in cold water and obtained participate was predicated by recrystallization in ethanol to give pure target compounds 5a-n.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-ethylbenzamide (5a). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.33 (s, H amide), 8.45 (t, J = 5.3 Hz, H8), 8.049 (d, J = 7.4 Hz, H5), 7.97–7.89 (m, H7), 7.89–7.81 (m, H6’, H2’), 7.77 (t, J = 7.3 Hz, H6), 7.47 (d, J = 8.4 Hz, H3’, H5’), 6.257 (s, H4), 3.32–3.22 (m, -CH₂-), 1.12 (t, J = 7.2 Hz, -CH₃);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.4, 165.2, 145.3, 140.7, 134.8, 132.7, 132.4, 130.6, 130.3, 128.3, 126.1, 125.2, 122.3, 103.2, 34.0, 14.8. C₁₉H₁₆N₂O₃ (320): calcd. C, 71.24; H, 5.03; N, 8.74; C, 71.27; H, 4.99; N, 8.77.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-propylbenzamide (5b). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.33 (s, H amide), 8.44 (t, J = 5.6 Hz, H8), 8.044 (d, J = 7.2 Hz, H5), 7.91 (dd, J = 13.1, 8.0 Hz, H2’, H6’), 7.79 (dt, J = 14.3, 7.2 Hz, H6, H7), 7.47 (d, J = 8.2 Hz, H3’, H5’), 6.25 (s, H4), 3.22 (q, J = 6.5 Hz, NH-CH₂-), 1.53 (h, J = 7.1 Hz, -CH₂-), 0.88 (t, J = 7.3 Hz, -CH₃);¹³C NMR (76 MHz, DMSO-d₆) δ 182.81, 181.4, 165.4, 145.3, 140.7, 134.8, 132.7, 132.4, 130.7, 130.3, 128.3, 126.1, 125.3, 125.2, 122.4, 103.2, 41.0, 22.4, 11.4. C₂₀H₁₈N₂O₃ (334): calcd. C, 71.84; H, 5.43; N, 8.38; C, 71.88; H, 5.44; N, 8.41.

N-Butyl-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5c). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d6) δ 9.34 (s, H amide), 8.42 (t, J = 5.6 Hz, H8), 8.06 (d, J = 7.3 Hz, H5), 7.95 (d, J = 7.3 Hz, H7), 7.87 (t, J = 6.9 Hz, H2’, H6’), 7.79 (q, J = 7.2 Hz, H6), 7.47 (d, J = 8.5 Hz, H3’, H5’), 6.26 (s, H4), 3.25 (q, J = 6.6 Hz, NH-CH₂-), 1.50 (p, J = 7.1 Hz, NH-CH₂-CH₂-), 1.32 (h, J = 7.2 Hz, -CH₂-CH₃), 0.90 (t, J = 7.3 Hz, -CH3);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.4, 165.3, 145.3, 140.7, 134.8, 132.7, 132.4, 130.7, 130.4, 128.3, 126.1, 125.2, 122.4, 103.2, 31.2, 19.6, 13.7. C₂₁H₂₀N₂O₃ (348): calcd. C, 72.40; H, 5.79; N, 8.04; C, 72.44; H, 5.81; N, 8.00.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-isobutylbenzamide (5d). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.34 (s, H amide), 8.44 (t, J = 5.9 Hz, H8), 8.05 (d, J = 7.4 Hz, H5), 7.91 (dt, J = 17.8, 8.4 Hz, H7, H2’, H6’), 7.79 (q, J = 7.2 Hz, H6), 7.47 (d, J = 8.2 Hz, H3’, H5’), 6.26 (s, H4), 3.08 (t, J = 6.4 Hz, -CH₂-), 1.88 (s, -CH-), 0.90 (s, -CH₃), 0.88 (s, -CH₃);¹³C NMR (76 MHz, DMSO-d₆) δ 182.81, 181.42, 165.56, 145.40, 140.72, 134.89, 132.75, 132.43, 130.80, 130.39, 128.35, 126.17, 125.29, 122.42, 103.24, 46.73, 28.14, 20.24. C₂₁H₂₀N₂O₃ (348): calcd. C, 72.40; H, 5.79; N, 8.04; found C, 72.41; H, 5.81; N, 8.00.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-phenylbenzamide (5e). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 10.23 (s, H amine), 9.42 (s, H amide), 8.07 (d, J = 7.4 Hz, H8), 8.02 (d, J = 8.2 Hz, H6, H7), 7.96 (d, J = 7.5 Hz, H5), 7.85 (dd, J = 14.6, 7.2 Hz, H2’, H6’), 7.78 (d, J = 7.9 Hz, H2”, H6”), 7.56 (d, J = 8.2 Hz, H3”, H5”), 7.35 (t, J = 7.7 Hz, H3’, H5’), 7.09 (t, J = 7.3 Hz, H4”), 6.32 (s, H4);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.4, 164.7, 145.3, 141.3, 139.2, 134.9, 132.8, 132.4, 130.7, 130.4, 128.9, 128.6, 126.2, 125.3, 123.6, 122.3, 120.3, 120.3, 103.5. HRMS (ESI) m/z: 368.0463. C₂₃H₁₆N₂O₃ (368): calcd. C, 74.99; H, 4.38; N, 7.60; C, 75.01; H, 4.41; N, 7.62.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(p-tolyl)benzamide (5f). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.79 (s, H amines), 9.41 (s, H amide), 8.03 (m, H8, H6, H7, H5), 7.88–7.78 (m, H2’, H6’), 7.54 (d, J = 8.1 Hz, H2”, H6”), 7.20 (d, J = 7.7 Hz, H3”, H5”), 7.05 (d, J = 17.4 Hz, H3’, H5’), 6.32 (s, H4), 2.19 (s, -CH₃);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.4, 164.5, 145.3, 141.1, 134.9, 134.8, 133.7, 133.4, 132.7, 132.3, 130.8, 130.4, 128.8, 126.4, 126.1, 125.2, 122.3, 103.5, 103.4, 17.8. C₂₄H₁₈N₂O₃ (382): calcd. C, 75.38; H, 4.74; N, 7.33; C, 75.40; H, 4.70; N, 7.30.

N-(2,4-Dimethylphenyl)-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5g). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.79 (s, H amine), 9.41 (s, H amide), 8.09–8.00 (m, H5, H8, H6), 7.95 (s, H7), 7.83 (d, J = 20.7 Hz, H2’, H6’), 7.54 (d, J = 7.1 Hz, H3”, H5”), 7.20 (d, J = 7.1 Hz, H6”), 7.05 (d, J = 15.0 Hz, H3’, H5’), 6.32 (s, H4), 2.28 (s, -CH₃), 2.19 (s, -CH₃);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.4, 164.5, 145.3, 141.2, 135.0, 134.8, 133.8, 133.4, 132.7, 132.4, 130.8, 130.4, 128.8, 126.5, 126.1, 125.2, 122.0, 103.4, 20.5, 17.8. C₂₅H₂₀N₂O₃₃ (396): calcd. C, 75.74; H, 5.09; N, 7.07; C, 75.72; H, 5.06; N, 7.06.

N-(2-Chlorophenyl)-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5h). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.39 (s, H amide), 8.012 (d, J = 25.6 Hz, H8, H6, H7, H5, H6”), 7.74 (d, J = 40.3 Hz, H2’, H6’, H4”, H5”), 7.52 (s, H3’, H6’), 7.35 (s, H3”), 6.344 (s, H4);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.2, 166.9, 144.9, 143.8, 142.4, 134.8, 132.7, 132.2, 130.5, 130.3, 127.3, 126.7, 126.1, 125.2, 122.0, 103.9. C₂₃H₁₅ClN₂O₃ (402): calcd. C, 68.58; H, 3.75; N, 6.95; C, 68.55; H, 3.75; N, 6.99.

N-(3-Chlorophenyl)-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5i). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.39 (s, H amide), 8.04 (d, J = 7.3 Hz, H8, H6”), 8.002–7.91 (m, H6, H7, H5), 7.88–7.81 (m, H5”), 7.80–7.74 (m, H2’, H6’), 7.52 (m, H3’, H5’, H2”), 7.40 (d, J = 7.0 Hz, H4”), 6.34 (s, H4);¹³C NMR (76 MHz, DMSO-d₆) δ 182.9, 181.3, 144.9, 142.5, 134.8, 132.8, 132.3, 130.7, 130.3, 127.3, 126.1, 125.3, 124.3, 122.1, 104.0. C₂₃H₁₅ClN₂O₃ (402): calcd. C, 68.58; H, 3.75; N, 6.95; C, 68.60; H, 3.79; N, 6.93.

N-(2,4-Dichlorophenyl)-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5j). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.38 (s, H1), 8.00 (d, J = 20.6 Hz, H8, H6, H7, H5, H2’, H6’), 7.89–7.64 (m, H3’, H5’), 7.51 (s, H5”, H6”), 7.38 (s, H3”), 6.34 (s, H4);¹³C NMR (76 MHz, DMSO-d₆) δ 182.9, 181.2, 166.7, 145.1, 142.5, 134.8, 132.7, 132.2, 130.5, 130.3, 129.0, 127.1, 126.1, 125.2, 124.3, 122.1, 120.7, 119.1, 109.6, 104.0. C₂₃H₁₄C_l2N₂O₃ (436): calcd. C, 63.18; H, 3.23; N, 6.41; C, 63.21; H, 3.25; N, 6.38.

N-(4-Bromophenyl)-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5k). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 10.34 (s, H amine), 9.41 (s, H amide), 8.06 (d, J = 7.2 Hz, H8), 8.02 (s, H6, H7), 7.95 (d, J = 7.3 Hz, H5), 7–87 (d, J = 7.1 Hz, H2’, H6’), 7.76 (d, J = 8.8 Hz, H2”, H6”), 7.54 (t, J = 8.7 Hz, H3”, H5”, H3’, H5’), 6.32 (s, H4);¹³C NMR (76 MHz, DMSO-d₆) δ 182.9, 181.3, 164.8, 145.2, 141.5, 138.6, 134.9, 132.8, 132.3, 131.4, 130.4, 128.9, 126.2, 125.3, 122.3, 122.1, 115.2, 103.6. C₂₃H₁₅BrN₂O₃ (446): calcd. C, 61.76; H, 3.38; N, 6.26; C, 61.75; H, 3.42; N, 6.25.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-nitrophenyl)benzamide (5l). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.40 (s, H amide), 8.05 (d, J = 7.1 Hz, H4”, H5”, H6”), 7.956 (d, J = 6.2 Hz, H8, H5, H6, H7), 7.87 (d, J = 7.0 Hz, H2”), 7.80 (d, J = 7.4 Hz, H2’, H6’), 7.52 (d, J = 7.7 Hz, H3’, H5’), 6.35 (s, H4);¹³C NMR (76 MHz, DMSO-d₆) δ 182.9, 181.3, 165.2, 148.9, 145.0, 142.5, 140.4, 134.8, 132.8, 132.3, 130.4, 129.0, 126.2, 125.3, 122.2, 114.2, 104.0. HRMS (ESI) m/z: 413.0698. C₂₃H₁₅N₃O₅ (413): calcd. C, 66.83; H, 3.66; N, 10.17; C, 66.87; H, 3.70; N, 10.15.

N-Benzyl-4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzamide (5m). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.36 (s, H amine), 9.05 (s, H amide), 8.06 (d, J = 7.6 Hz, H8), 7.96 (dd, J = 8.6, 2.6 Hz, H6, H7, H5), 7.89–7.77 (m, H2’, H6’), 7.54–7.47 (m, H3’, H5’), 7.32 (s, Ph), 6.28 (s, H4), 4.49 (s, -CH₂-);¹³C NMR (76 MHz, DMSO-d₆) δ 182.8, 181.4, 165.5, 145.3, 141.0, 139.7, 134.9, 132.7, 132.4, 130.4, 130.2, 128.4, 128.2, 127.2, 126.7, 126.1, 125.3, 122.4, 103.3, 42.6. C₂₄H₁₈N₂O₃ (382): calcd. C, 75.38; H, 4.74; N, 7.33; C, 75.40; H, 4.76; N, 7.37.

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-phenethylbenzamide (5n). Yellow solid, Yield: 77%, m.p. 171–173°C;¹H NMR (301 MHz, DMSO-d₆) δ 9.33 (s, H amine), 8.57 (t, J = 5.6 Hz, H amide), 8.04 (d, J = 7.3 Hz, H8), 7.94 (d, J = 7.4 Hz, H5), 7.87 (d, J = 8.3 Hz, H6, H7), 7.82–7.73 (m, H2’, H6’), 7.47 (d, J = 8.3 Hz, H3’, H5’), 7.34–7.15 (m, Ph), 6.25 (s, H4), 2.90 (t, J = 7.3 Hz, NH-CH₂-), 2.84 (t, J = 7.4 Hz, -CH₂-Ph);¹³C NMR (76 MHz, DMSO) δ 182.9, 181.4, 165.6, 145.4, 140.9, 139.6, 134.9, 132.8, 132.4, 130.6, 130.4, 128.7, 128.4, 128.4, 126.2, 126.1, 125.3, 122.5, 103.3, 41.0, 35.2. C₂₅H₂₀N₂O₃ (396): calcd. C, 75.74; H, 5.09; N, 7.07; O, 12.11; C, 75.75; H, 5.10; N, 7.03.

Biological assays. RPMI 1640, DMEM, fetal bovine serum (FBS), trypsin, and phosphate-buffered saline (PBS) were purchased from Biowest (Nuaillé, France). 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigmaaldrich.

Three cell lines including MDA-MB-231 (triple negative human breast adenocarcinoma), SUIT-2 (human pancreatic cancer) and HT-29 (human colorectal adenocarcinoma) were used in this study. MDA-MB-231 and HT-29 cells were obtained from Iranian Biological Resource Center, Tehran, Iran. SUIT-2 cell lines was purchased The Japanese Collection of Research Bioresources (JCRB).

All cell lines were grown in monolayer cultures and were kept at 37 °C in a humidified incubator containing 5% CO₂. MDA-MB-231 were cultured in DMEM high glucose containing 10% FBS and L-glutamine 2 mM. SUIT-2 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, while HT-29 cells were cultured in DMEM high glucose containing 15% FBS and L-glutamine 4 mM.

Assessment of antiproliferative effect using MTT assay. The antiproliferative activities of the synthesized compounds as well as the reference compounds were evaluated by MTT assay²⁰. The test determines cell viability by assessing the conversion of yellow MTT to purple formazan through the activity of cellular mitochondrial dehydrogenase enzymes. MDA-MB-231, SUIT-2, and HT-29 cells were seeded separately at a density of 3–5 × 10⁵ cells/well in 96-well plates and incubated overnight at 37 °C in 5% CO₂ incubator. Afterwards, the cells were treated with 3–4 concentrations of synthesized compounds 5a-n for 72 h. After the incubation period, supernatants were removed from the wells and 80 µl of MTT solution (0.5 mg/mL in PBS) was added to all wells. After four hours of incubation, formazan crystals formed and were dissolved in 200 µl of DMSO. Ultimately, a microplate reader (BioTek, model Synergy HTX) was used to measure absorbance at 570 nm with background adjustment at 650 nm. The IC₅₀ values of all compounds were calculated using CurveExpert version 1.34 for Windows. At least 3–4 replications of each experiment were conducted.

Flow cytometric analysis of cell cycle. In order to investigate the distribution of cells in various stages of the cell cycle after treatment with the most potent new synthetic compounds 5e, 5f, 5g, and 5l, we conducted flow cytometric analysis using the RNase/propidium iodide test²¹. MDA-MB-231 cells were seeded in a 12-well plate at a density of 1.25 × 10⁵ cells/ml. After incubating at 37 °C for 24 h, the cells were exposed to compounds 5e, 5f, 5g, and 5l at concentrations of 10 and 30 µM for 48 h. The contents of each well were collected after the trypsinization of cells. Afterwards, the cells were washed with PBS and then fixed in 70% ice-cold ethanol and stored at − 20 °C at least for 24 h. Subsequently, the ethanol-fixed cells were washed twice with PBS and suspend in DNA staining solution. This solution comprised 20 µg/ml of propidium iodide (PI) and 200 µg/ml of RNase. The staining process took place at room temperature in the absence of light for a duration of 30 min. Ultimately, 20,000 events were analyzed using FACSCalibur flow cytometer (BD Biosciences), and CellQuest software (BD, USA) was used to determine the proportion of cells in sub-G1, G0/G1, S, and G2/M phases. At least three replications of each experiment were conducted.

Assessment of apoptosis using Hoechst Staining. Apoptosis can be identified using Hoechst 33,258, a DNA binding stain, which is based on the morphological characteristics of DNA fragmentation and nuclear condensation²². MDA-MB-231 cells were seeded in a six-well plate at a density of 1 × 10⁵ cells/ml and incubated overnight at 37 °C. The cells were treated with compounds 5e, 5f, 5g, and 5l at a concentration of 10 µM for 48 h. Afterwards, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed twice with PBS, prior to being stained with Hoechst 33,258 (2.5 µg/ml) at room temperature in the absence of light for 30 min. The stained cells were washed twice with PBS, and the cells were observed and imaged under a fluorescence microscope (Nikon model DS-Ri2).

Molecular docking. Three dimensional (3D) structure of doxorubicin and compound 5e were sketched by MarvinSketch 5.10.4, 2012 software. The 3D X-ray crystal structure of human topoisomerase II protein (PDB ID: 1ZXM) was downloaded from protein data bank (http://www.rcsb.org). Preparation of protein and ligands and molecular docking study were performed with Auto Dock Tools 1.5.6. The grid box with the size of 50 × 50 × 50 Å was determined and the center of grid box was attained at x: 41.996, y: 2.482, and z: 33.507 and exhaustiveness was set to be 50 (other docking parameters were set as default). The obtained results were visualized with Discovery Studio Client 2019 software.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

M.H.S. and N.D. contributed in the synthesis and characterization of compounds, B.H., M.M.T. and B.L. performed biological assays. M.M-K. and M.R.G. performed in silico assays and M.M. and O.F. conceived the idea and designed the experiments. All authors reviewed the manuscript.

Funding

Not applicable.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.