Use of acridine derivative in cancer treatment

Abstract

A use of an acridine derivative in cancer treatment. Specifically, because the described acridine derivative has the ability to stably intercalate into two groups of DNA molecules, by administering an effective amount of the described acridine derivative or a salt thereof to a cancer patient, the DNA molecular structure of cancer cells can be destroyed, thereby effectively achieving the effect of treating cancer or delaying the progression of cancer.

Description

Use of acridine derivatives in anticancer activities Technical Field

[0001] The present invention relates to a second use of a compound, particularly an anti-cancer use of an acridine derivative. Background Art

[0002] Acridine compounds, discovered in 1870, are composed of polycyclic heteroaromatic rings, similar in structure to anthracene, with a nitrogen atom replacing the central CH group of one of the benzene rings. In 1922, scientists discovered that an acridine derivative, trypaflavin, possessed antimicrobial activity and was subsequently used in disinfectants and antimicrobial agents. Subsequent research has shown that acridine compounds and their derivatives possess numerous physiological activities, including anti-inflammatory, antiviral, and antimalarial properties.

[0003] Previous studies have shown that acridine compounds embed between DNA and RNA by forming hydrogen bonds and π-π stacking forces between base pairs, causing DNA cross-linking and breakage during replication, thereby inhibiting telomerase activity. Therefore, acridine compounds are considered to be an embedding compound that can be used for drug resistance. However, single embedding drugs lack selective specificity and frequently have side effects.

[0004] Summary of the Invention

[0005] The main purpose of the present invention is to provide an anti-cancer use of an acridine derivative, wherein the acridine derivative can be embedded in a DNA molecule, thereby changing or destroying the structure of the DNA molecule and affecting the function of the DNA molecule, thereby achieving the effect of treating or improving cancer.

[0006] Therefore, in order to achieve the above-mentioned object, the present invention discloses an acridine derivative or a salt thereof for use in preparing a pharmaceutical composition for treating cancer, and the structural formula of the acridine derivative is shown in Formula (A): XYZ, wherein X and Z are selected from acridine compounds, quinolone, A quinoxaline or 1,8-naphthyridine derivative, wherein at least one of X and Z is an acridine compound; and Y is an alkane chain having 4 to 9 carbon atoms or an alkane chain in which one of the methane groups is replaced by formamide. By administering an effective amount of an acridine derivative or a salt thereof to a cancer patient, the acridine derivative can be incorporated into the DNA molecules of cancer cells through X and / or Z in formula (A), thereby disrupting the DNA molecular structure of the cancer cells and effectively inhibiting the growth of the cancer cells, thereby treating the cancer or delaying its progression.

[0007] To go further, the structural formulas of X, Y, and Z are as follows:

[0008] X is

[0009] Y is

[0010] Z is

[0011] In an embodiment of the present invention, the structural formula of the acridine derivative is any one of the following formulas (I) to (XIV):

[0012] In one embodiment of the present invention, the cancer is colorectal cancer, lung cancer, breast cancer, or cervical cancer.

[0013] In one embodiment of the present invention, the pharmaceutical composition is a double-intercalating drug, which interacts with the DNA double helix in target cells through the X and Z structures in formula (A), thereby changing the DNA molecular structure and inhibiting DNA replication, transcription, and other functions.

[0014] In the next embodiment of the present invention, the compound of formula (A) is used to prepare a dual intercalating drug.

[0015] In another embodiment of the present invention, the structural formula of the acridine derivative is shown in any one of Formulas (I) to (VIII) above, which has two acridine compounds that interact with two groups of DNA molecules respectively, so that the acridine derivative or its salt is embedded in the two groups of DNA molecules.

[0016] In another embodiment of the present invention, the acridine derivative has an asymmetric structure, as shown in Formulas (IX) to (XIV), with an acridine compound at one end and a nitrogen-containing heterocyclic compound at the other. Acridine derivatives such as those shown in any of Formulas (IX) to (XIV) are capable of interacting with DNA molecules, allowing the acridine derivative or a salt thereof to be intercalated into two DNA molecules.

[0017] Another embodiment of the present invention discloses a method for disrupting DNA structure, comprising the following steps:

[0018] Step A: providing an effective amount of an acridine derivative or a salt thereof as represented by the above formula (A).

[0019] Step B: The acridine derivative reacts with at least one DNA molecule, so that the X and Z structures in formula (A) are respectively inserted between the base pairs of the DNA molecule, thereby destroying the structure of the DNA molecule.

[0020] The structural formula of the acridine derivative in step A is any one of formula (I) to formula (XIV) above.

[0021] The DNA molecule in step B contains CG / CG and / or TA / TA bases for interacting with the acridine derivative.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides an anti-cancer use of an acridine derivative. The acridine derivative can be embedded in a DNA molecule, thereby changing or destroying the structure of the DNA molecule and affecting the function of the DNA molecule, thereby achieving the effect of treating or improving cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows the melting point analysis results of the Z3 complex.

[0025] FIG2 shows the melting point analysis results of the Z5 complex.

[0026] Figure 3 shows the thermal melting point analysis results of Z3 compound, Z5 compound and d(CGTATACG)2.

[0027] Figure 4 shows the complete crystal structure and electron density map of the Z3 crystal, where the d(CGTATACG)2 sequence is orange; the Z3 compound is cyan; atoms in the structure are represented by spheres, 42 water molecules are presented as red spheres, and manganese ions are presented as purple spheres.

[0028] FIG5 is a schematic diagram of the Z3 crystal structure.

[0029] FIG6 shows the results of analyzing the π-stacking force formed by the Z3 compound in a DNA molecule (first insertion site), obtained using Discovery Studio software. (A) is the overall π-stacking force; (B) is the distance between the base and the π-stacking force.

[0030] FIG7 shows the results of analyzing the π-stacking forces formed by the Z3 compound in a DNA molecule (second insertion site), obtained using Discovery Studio software. (A) is the overall π-stacking force; (B) is the distance between the base and the π-stacking force.

[0031] FIG8 is a schematic diagram of the detailed bonding of the Z3 crystal (I).

[0032] FIG9 is a schematic diagram of the detailed bonding of the Z3 crystal (II).

[0033] Figure 10 shows the complete crystal structure and electron density map of the Z5 crystal, where the d(CGTATACG)2 sequence is represented in yellow and blue; the Z5 compound is represented in cyan and pink: atoms in the structure are represented by spheres, 109 water molecules are presented as red spheres, and magnesium ions are presented as green spheres.

[0034] FIG11 is a schematic diagram of the Z5 crystal structure.

[0035] FIG12 shows the results of analyzing the π-stacking forces formed by the Z5 compound in DNA molecules, obtained using Discovery Studio software. (A) is the overall π-stacking force; (B) is the distance between the bases and the π-stacking force.

[0036] FIG13 is a schematic diagram of the detailed bonding of the Z5 crystal (I).

[0037] FIG14 is a schematic diagram of the detailed bonding of the Z5 crystal (II).

[0038] FIG15A shows the cytotoxicity analysis results of Hct116 cells treated with different concentrations of Z3 compound for 48 hours.

[0039] FIG15B shows the cytotoxicity analysis results of HCT116+ch3 cells treated with different concentrations of Z3 compound for 48 hours.

[0040] FIG16A shows the cytotoxicity analysis results of Hct116 cells treated with different concentrations of Z5 compound for 48 hours.

[0041] FIG16B is a graph showing the cytotoxicity of HCT116+ch3 cells treated with different concentrations of Z5 compound for 48 hours. FIG17 is a schematic diagram showing the crystal structure of QA5 crystals.

[0042] FIG18 is a schematic diagram of the crystal structure of QA4 crystal.

[0043] FIG19 is an analysis of the effects of Z3 compound, Z5 compound, QA4 compound and QA5 compound at different concentrations on the cell cycle of HCT cells.

[0044] FIG20 is an analysis of the effects of Z3 compound, Z5 compound, QA4 compound and QA5 compound at different concentrations on the cell cycle of A549 cells.

[0045] FIG21 shows CD spectra of the reactions of NA5, NA6, NA7 and NA8 compounds with A:A mismatched base pair sequences, respectively, wherein A:A in the figure represents A:A mismatched base pair sequence.

[0046] FIG22 shows CD spectra of the reactions of NA5, NA6, NA7 and NA8 compounds with T:T mismatched base pair sequences, respectively, wherein B:B in the figure represents a T:T mismatched base pair sequence.

[0047] FIG23 shows CD spectra of the reactions of NA5, NA6, NA7 and NA8 compounds with C:C mismatched base pair sequences, respectively, wherein C:C represents a C:C mismatched base pair sequence.

[0048] FIG24 shows CD spectra of the reactions of NA5, NA6, NA7 and NA8 compounds with G:G mismatched base pair sequences, respectively, wherein D:D represents a G:G mismatched base pair sequence.

[0049] FIG25 is an analysis of the results of NA5, NA6, NA7 and NA8 compounds inducing apoptosis in A549 cells at different concentrations. DETAILED DESCRIPTION

[0050] The present invention provides an acridine derivative for use in anticancer treatment, wherein the acridine derivative has a structural formula of XYZ (hereinafter referred to as Formula (A)), and at least one of X and Z is an acridine compound. Because the acridine derivative disclosed herein has the ability to stably intercalate into DNA molecules, administering an effective amount of the acridine derivative disclosed herein or a salt thereof to a cancer patient can disrupt the DNA molecular structure of cancer cells, thereby effectively treating cancer or delaying cancer progression.

[0051] Specifically, Y in formula (A) is a chain-like linker unit connected to X and Z at both ends; X and Z are respectively a cyclic chimeric unit for interacting with base pairs of DNA molecules to disrupt DNA structure and function.

[0052] In one embodiment of the present invention, X is an acridine compound, Z is an acridine compound, quinolone Quinoxaline or a derivative of 1,8-naphthyridine, such as 2-amino-7-methyl-1,8-naphthyridine.

[0053] In another embodiment of the present invention, X is an acridine compound, a quinolone wherein Z is an acridine compound or a derivative of 1,8-naphthyridine, such as 2-amino-7-methyl-1,8-naphthyridine; and Z is an acridine compound or a derivative of 1,8-naphthyridine, such as 2-amino-7-methyl-1,8-naphthyridine.

[0054] In one embodiment of the present invention, the structural formulas of X, Y, and Z are respectively as follows:

[0055] X is

[0056] Y has a main chain of 4-9 atoms in length and is chemically bonded to X and Z at both ends. Y is an alkane chain with 4 to 9 carbon atoms or one of the methane groups in the alkane chain is replaced by formamide, such as

[0057] Z is

[0058] In one embodiment of the present invention, the acridine derivative represented by formula (A) has a symmetrical structure, meaning that both X and Z are acridine compounds and are symmetrically disposed at both ends of Y.

[0059] In another embodiment of the present invention, the acridine derivative represented by formula (A) has an asymmetric structure, that is, one of X and Z is an acridine compound and the other is a heterocyclic compound, such as quinone. It is a derivative of 1,8-naphthyridine or 1,8-naphthyridine.

[0060] Specifically, when both X and Z are The compounds of formula (A) disclosed in the present invention are shown in Table 1 below.

[0061] Table 1: Acridine derivatives disclosed in the present invention (I)

[0062] When X is And Z is The compounds of formula (A) disclosed in the present invention are shown in Table 2 below.

[0063] Table 2: Acridine derivatives disclosed in the present invention (II)

[0064] When X is And Z is The compounds of formula (A) disclosed in the present invention are shown in Table 3 below.

[0065] Table 3: Acridine derivatives disclosed in the present invention (III)

[0066] The acridine derivatives of formula (A) disclosed in the embodiments of the present invention interact with sequences containing TA / TA and / or CG / CG bases through X and Z, thereby stably intercalating into DNA molecules containing the sequences, causing structural deformation of the DNA, inhibiting topoisomerase II from binding to DNA, and thereby disrupting cell replication and transcription, thereby affecting DNA molecular function and treating or mitigating cancer.

[0067] The DNA molecule refers to a double helical structure containing a sequence of TA / TA and / or CG / CG bases.

[0068] The DNA molecule comprises CGTTAACG, CGTATACG, TACGCGT, AACGCGAT, ATCGCGTT, ACCGCGCT, AGCGCGGT, or a sequence as shown in SEQ ID No. 1 (TTCGTACGAA) or SEQ ID No. 2 (TCGTATACGA), or a sequence having a similarity of more than 90% to the above sequence.

[0069] Among them, the acridine derivatives represented by any one of Formulas (I) to (XIV) were designed based on acridine by the inventors of this case in collaboration with the laboratory of Professor Hong Yisheng of the Department of Chemistry at Changhua Normal University in Taiwan, China.

[0070] Furthermore, the acridine derivatives represented by any of Formulas (I) to (VIII) are acridine compounds or derivatives thereof at both ends. For example, the difference between the acridine derivative represented by Formula (I) (hereinafter referred to as Z3 compound) and the acridine derivative represented by Formula (III) (hereinafter referred to as Z5 compound) lies only in the length of the linker unit (Y), i.e., the Z3 compound contains two acridine compounds and an alkyl chain with a length of 4 carbon atoms, and the Z5 compound contains two acridine compounds and an alkyl chain with a length of 5 carbon atoms.

[0071] In any of the acridine derivatives represented by Formula (XIII) and Formula (XIV), only one end is an acridine compound. The difference between the acridine derivative represented by Formula (XIII) (hereinafter referred to as QA4 compound) and the acridine derivative represented by Formula (XIV) (hereinafter referred to as QA5 compound) lies only in the length of the alkyl chain, i.e., the alkyl chain in the QA4 compound is 4 carbon atoms in length, while the alkyl chain in the QA5 compound is 5 carbon atoms in length.

[0072] In the disclosed embodiments, the interactions between the acridine derivative and the DNA molecule are primarily π-π stacking forces and base interactions. For example, when the DNA molecule has the sequence d(CGTATACG)2, the complex formed between the Z3 compound and the DNA molecule exhibits 20 π-π stacking forces, with the acridine ring interacting with each base at varying strengths. The complex formed between the Z5 compound and the DNA molecule exhibits 18 π-π stacking forces, with one acridine ring interacting with three bases.

[0073] In addition to the aforementioned primary forces, the acridine derivatives disclosed herein interact with guanine, forming reverse-Hoogsteen-like hydrogen bonding forces. For example, when a DNA molecule has the sequence d(CGTATACG)2, triple helical base pairs form at the terminal bases of the DNA molecule after the reaction between the acridine derivatives and the DNA molecule. This indicates that, after the acridine derivatives disclosed herein are embedded in the DNA molecule, they bring the bases of two adjacent DNA double helices closer together, forming a special hydrogen bonding force, which further enhances the structural stability of the chimeric structure.

[0074] Although the lengths of the alkyl chains or linker units in the acridine derivatives disclosed herein vary, this does not affect the ability of the acridine derivatives to chimerize with DNA molecules. For example, the alkyl chain lengths of compounds Z3 and Z5 differ by one carbon atom, yet both compounds can stably chimerize with DNA molecules.

[0075] Furthermore, after the acridine derivatives of the present invention react with DNA molecules to form crystals, in addition to the aforementioned interactions between the acridine derivatives and the bases of the DNA molecules, the metal ions participate in the interaction with the phosphate groups of the bases, and the water in the structure forms hydrogen bonds with the nitrogen atoms of the acridine derivatives and the oxygen or nitrogen atoms of the bases, all of which can further stabilize the crystal structure.

[0076] The "pharmaceutical composition" disclosed in the present invention refers to a composition containing an effective amount of an active ingredient and / or a pharmaceutically acceptable carrier, wherein the effective amount is 0.001%-100%; and in the present invention, the active ingredient is a compound represented by any one of Formulas (I) to (XIV) above; the pharmaceutically acceptable carrier may be an active agent, an adjuvant, a dispersant, a wetting agent, a coating agent or a suspending agent, etc.

[0077] The "dual intercalating drugs" disclosed herein refer to drugs that carry two intercalating units. During treatment, each intercalating unit interacts with the DNA double helix and inserts into different base pairs. Furthermore, depending on the structure of the compound contained within, dual intercalating drugs can intercalate into a single DNA molecule or two DNA molecules. For example, the acridine derivatives disclosed herein intercalate into two DNA molecules through intermolecular crosslinking. Dual intercalating drugs intercalate between adjacent stacked base pairs in the DNA double helix through covalent or non-covalent interactions with DNA, such as van der Waals forces, π-stacking, hydrogen bonds, and hydrophobic forces. This can distort the DNA backbone, interfere with DNA-protein interactions, destabilize the DNA structure, or cause other structural changes, disrupting DNA replication, transcription, and repair.

[0078] In the following, to illustrate the technical features and effects of the present invention, several embodiments are given and described in detail with reference to the accompanying drawings.

[0079] The Z3 compound (structure shown in the above formula (I)), the Z5 compound (structure shown in the above formula (III)), the NA5 compound (structure shown in the above formula (IX)), the NA6 compound (structure shown in the above formula (X)), the NA7 compound (structure shown in the above formula (XI)), the NA8 compound (structure shown in the above formula (XII)), the QA5 compound (structure shown in the above formula (XIII)), and the QA4 compound (structure shown in the above formula (XIV)) used in the following examples were designed and obtained by the inventors of this case in collaboration with the laboratory of Professor Hong Yisheng of the Department of Chemistry at Changhua Normal University in Taiwan, China. Among them, the molecular weight of the Z3 compound is 442.57, the molecular weight of the Z5 compound is 456.59, the molecular weight of the QA5 compound is 435.53, and the molecular weight of the QA4 compound is 421.50.

[0080] The cell lines used in the following examples are all cell lines that can be easily obtained by those skilled in the art and therefore do not need to be deposited separately.

[0081] The DNA molecules used in some of the following examples are designed according to the present invention and contain sequences of CG / CG and TA / TA bases, and the DNA molecules have a double-stranded helical structure. The nucleotide composition of the sequences is as follows: CGTTAACG, CGTATACG, TACGCGT, SEQ ID NO.: 1 (TTCGTACGAA), SEQ ID NO.: 2 (TCGTATACGA).

[0082] Example 1: Melting Point Temperature Analysis (I)

[0083] The d(CGTATACG)2 sequence (final concentration 3 μM) was added to various concentrations (0, 3, 6, and 15 μM) of the Z3 or Z5 compound. The reaction was allowed to react in a 4°C incubator for 16-18 hours to form the Z3 or Z5 complex. The absorbance of each complex under varying temperatures was measured using a UV / visible spectrometer, as shown in Figures 1 and 2. The actual melting points of the Z3 and Z5 compounds were then calculated using a thermal melting analysis system, as shown in Figure 3.

[0084] The results in Figures 1 to 3 show that the melting point of the d(CGTATACG)2 sequence alone is 27.68°C. When the ratio of the compound to the d(CGTATACG)2 sequence is 1:2, the melting point of the Z3 complex is 32.8°C and the ΔTm value is 5.12°C. The melting point of the Z5 complex is 31.69°C and the ΔTm value is 4.01°C. When the ratio of the compound to the d(CGTATACG)2 sequence is 1:5, the melting point of the Z3 complex is 40.93°C and the ΔTm value is 13.25°C. The melting point of the Z5 complex is 36.11°C and the ΔTm value is 8.43°C.

[0085] The results in Figures 1 to 3 show that the Z3 compound and Z5 compound disclosed in the present invention can effectively stabilize DNA, and the stability of both compounds increases with increasing concentration. Moreover, regardless of whether the concentration ratio of the d(CGTATACG)2 sequence to the Z3 compound or the Z5 compound is 1:2 or 1:5, the temperature increased by the Z3 compound is significantly higher than that of the Z5 compound, indicating that the Z3 compound requires a higher temperature to unwind DNA, that is, the thermal stability of the Z3 compound is higher than that of the Z5 compound.

[0086] Example 2: Crystal structure analysis of the Z3 complex

[0087] The Z3 complex prepared in Example 1 is placed under suitable crystal growth conditions for cultivation to form a single crystal, which is a Z3 crystal with a diamond shape and light yellow color. In this example, the suitable crystal growth conditions are obtained by vapor diffusion method.

[0088] The crystal structure analysis of Z3 crystal is carried out. The analysis steps are as follows: first, the crystal data is collected by diffractometer, then the crystal data is processed by HKL2000 software, and the crystal resolution is calculated and analyzed. Cell parameters are α, β, and γ are all 90°; completeness is 99.6%. Based on this parameter information, sca, log, and cif files are generated for phase angle analysis.

[0089] Computational analysis of the sca file revealed that each asymmetric unit (ASU) contains a d(CGTATACG)2 sequence (DNA molecule) and a Z3 compound. Molecular replacement was used to obtain a preliminary correct phase, and a Fourier transform was performed to obtain the crystal electron density map and molecular model. The Z3 crystal structure was then refined and adjusted using Phenix and WinCoot CCP4 software, respectively, adjusting base and compound positions and rotation angles. The resulting R-factors were 21.85% and 24.57% respectively. Finally, an electron density map was created using Phenix and rendered using PyMOL, resulting in the overall Z3 crystal structure. This structure contains 328 DNA atoms and 34 Z3 compounds. Water and metal molecules were manually added at appropriate locations based on the electron density map, resulting in a structure containing 42 water molecules and 2 metal molecules, as shown in Figure 4.

[0090] The d(CGTATACG)2 sequence of each asymmetric unit stacks end-to-end with the Z3 compound, embedding the two tandem double helices. The acridine ring of the Z3 compound spans the d(CGTATACG)2 sequences embedded in the two asymmetric units, interacting with the upper and lower asymmetric units. The bases in the d(CGTATACG)2 sequence are numbered from 5' to 3', as shown in Figure 5, where the numbering is divided into C1 to G8 for Chain A, C9 to G16 for Chain B, C17 to G24 for Chain C, C25 to G32 for Chain D, C33 to G40 for Chain E, and C41 to G48 for Chain F.

[0091] As can be seen from the content of Figure 5, the Z3 compound is embedded in the d(CGTATACG)2 sequences of different asymmetric units, and the acridine ring of the Z3 compound is respectively inserted into the adjacent stacked bases at the ends. The d(CGTATACG)2 sequence changes the spatial position of some bases due to binding with the Z3 compound. At the C1, G2, C7 and G8 positions of Chain A and the C9, G10, C15 and G16 positions of Chain B, the acridine ring of the Z3 compound is embedded, making the spatial distance between bases different from that of other bases.

[0092] Furthermore, please refer to Figures 6 and 7, which respectively show the analysis results of the two embedding sites of the Z3 compound in the d(CGTATACG)2 sequence, and further observe the interaction forces between the two embedding sites, as shown in Tables 1 and 2, respectively. Among them, Complex I contains Chain A and Chain B, Complex II contains Chain C and Chain D, and Complex II' contains Chain E and Chain F.

[0093] The results in Table 1 indicate that the acridine ring of compound Z3 exhibits π-π stacking interactions with bases C1 and G2 in Chain A and C15 and G16 in Chain B, with at least 10 stacking interactions. Furthermore, the acridine ring on the other side also exhibits 10 stacking interactions with bases C23 and G24 in Chain C and C25 and G26 in Chain D. Furthermore, two weaker hydrogen bonds were observed between the bases and the Z3 compound, formed between the acridine ring and C15-N4 on Chain B and C23-N4 on Chain C.

[0094] The results in Table 2 indicate that π-π stacking interactions exist between bases C7 and G8 in Chain A and C9 and G10 in Chain B. On the other hand, interactions occur with bases C33 and G34 in Chain E and C47 and G48 in Chain F. Furthermore, weak hydrogen bonds are observed between the C7-N4 and C47-N4 acridine rings in Chain A and Chain F, respectively. The two acridines in compound Z3 are linked by an N-alkyl group, and some hydrophobic interactions are observed between the bases and the carbon atoms of the N-alkyl group at the linker.

[0095] The results in Tables 4 and 5 show that there are a total of 20 stacking forces in the Z3 crystal structure, and the acridine ring and each base also have different strengths of interaction forces, which affect the Z3 compound's embedding into the base and tend to favor one strand.

[0096] Table 4: π-π stacking forces of Z3 compound-d(CGTATACG)2 (first embedding site)

[0097] Table 5: π-π stacking forces of Z3 compound-d(CGTATACG)2 (second embedding site)

[0098] In addition, the DNA molecules in the asymmetric unit structure have hydrogen bonding forces. There are four pairs of C:G base pairs, namely C1:G16, G2:C15, C7:G10, and G8:C9, and each base pair is paired with each other by three hydrogen bonds. There are four pairs of A:T base pairs, namely T3:A14, A4:T13, T5:A12, and A6:T11, and each base pair is paired with each other by two hydrogen bonds.

[0099] Please refer to Figures 8 and 9 again. It can be seen that there is a reversed Hoogsteen hydrogen bond between the two DNA molecules. The G2-N3, N2 of Chain A and the G24-N3, N2 of Chain C form two stable G:G base pairs. The hydrogen bond distances are and G16-N3 of Chain B interacts with N2 and G26-N3 and N2 of Chain D, and the hydrogen bond distances are and These results indicate the presence of a triple helix base pair at the terminal bases. These forces form C:G:G hydrogen bonds with adjacent double helix bases, increasing structural stability. In other words, the Z3 compound disclosed in this invention can stably embed into DNA molecules through its flanking acridine rings.

[0100] Example 3: Crystal structure analysis of the Z5 complex

[0101] The Z5 complex was cultured under suitable crystal growth conditions to obtain multiple single nucleic acid crystals (hereinafter referred to as Z5 crystals). Each Z5 crystal was cubic in appearance, light yellow in color, with a smooth and polished surface and distinct edges and corners.

[0102] Referring to the analysis method described in Example 2, the crystal structure of Z5 crystal was analyzed and the crystal space group was P21; the crystal resolution was The lattice coefficient is Phase angle analysis is performed based on the following parameter information: α and γ are both 90°, and β is 92.666°; the integrity is 96.4%; the average signal-to-noise ratio (Average I / σ) is 45.658; Rmerge is 0.022; and the redundancy parameter is 3.6.

[0103] After molecular replacement, the Z5 crystal obtained a correct initial phase. The structure was then manually refined using WinCoot CCP4 based on the electron density map. Base pair positions, bond lengths, and angles were carefully adjusted and refined. The final structure refinement yielded an R-work value of 21.83% and an R-free value of 22.68%. As shown in Figure 10, the complete crystal structure of the Z5 crystal consists of two DNA molecules, the Z5 compound, and two magnesium ions. The DNA comprises 656 atoms, the Z3 compound comprises 70 atoms, and water molecules were manually added at appropriate locations based on the electron density map. The overall structure contains 39 water molecules.

[0104] As shown in Figure 10, the asymmetric unit in the Z5 crystal is composed of two groups of DNA molecules (d(CGTATACG)2 sequence) and two Z5 compounds. The acridine ring of the Z5 compound will also be embedded across the two different groups of DNA molecules and interact with the upper and lower asymmetric units. Further, as shown in Figure 11, the bases in the d(CGTATACG)2 sequence are numbered from the 5th end to the 3rd end, where the numbers are C1 to G8 of Chain A, C9 to G16 of Chain B, C17 to G24 of Chain C, C25 to G32 of Chain D, C33 to G40 of Chain E, and C41 to G48 of Chain F. As shown in Figure 11, the Z5 compound will be embedded in different DNA molecules, and the acridine ring of the Z5 compound will be inserted into the bases stacked adjacent to the ends.

[0105] Referring again to Figure 12 and the results in Table 6, we can see that the acridine ring of compound Z5 has at least nine π-π stacking forces with bases C1 and G2 in Chain A and C15 and G16 in Chain B. The acridine ring on the other side also has nine stacking forces with bases C23 and G24 in Chain C and C25 and G26 in Chain D. Therefore, there are a total of 18 stacking forces in the Z5 crystal structure.

[0106] In addition, there are some weaker hydrogen bonds between the bases and the Z5 compound. On Chain B, there is a weaker set of hydrogen bonds between C15 and N4, and on Chain C, there are two weaker sets of hydrogen bonds between C23 and N4, which form the connection points of the Z5 compound with the acridine ring. Hydrophobic interactions are also found between the N-alkyl chain and the bases.

[0107] Table 6: π-π stacking forces of Z5 compound-d(CGTATACG)2

[0108] Furthermore, hydrogen bonds exist between the bases of the DNA molecules in the asymmetric unit structure. There are four C:G base pairs, namely C1:G16, G2:C15, C7:G10, and G8:C9, and each base pair is paired with three hydrogen bonds. There are four A:T base pairs, namely T3:A14, A4:T13, T5:A12, and A6:T11, and each base pair is paired with two hydrogen bonds. In addition, there is a reversed Hoogsteen hydrogen bond force between the two DNA molecules (as shown in Figures 13 and 14). Chain A's G2-N3, N2 and Chain C's G24-N3, N2 form two stable G:G base pairs, with hydrogen bond distances of 1:1. and G16-N3 of Chain B interacts with N2 and G26-N3 and N2 of Chain D, and the hydrogen bond distances are and These results demonstrate the presence of triple-helical bases at the terminal ends of the DNA molecule. These bases form C:G:G hydrogen bonds with adjacent double-helical bases, enhancing structural stability. In other words, the Z5 compound disclosed herein can stably intercalate into DNA molecules through its flanking acridine rings.

[0109] Example 4: Cytotoxicity Analysis (I)

[0110] In this example, two colorectal cancer cell models, HCT116 and HCT116+ch3, were used as test subjects. The HCT116 cell line harbors an MMR gene deletion, while the HCT116+ch3 cell line harbors a normal MMR gene. Specifically, the cells were cultured in a 96-well plate at a concentration of 5,000 cells / well (100 μL) at 37°C for 16-18 hours. After the cells adhered to the bottom, different concentrations of the compound Z3 or the compound Z5 were added, respectively. The concentrations of the compound Z3 were 1, 3, 5, 10, and 15 μM, and the concentrations of the compound Z5 were 0.1, 0.5, 1, 5, and 10 μM. The compounds were allowed to react with cells in a 37°C incubator for 48 hours. The supernatant from each well was removed, and 0.5 mg / mL MTT (100 μL) was diluted with culture medium. The diluted MTT was then added to a 96-well plate and incubated in the dark for 3 hours. After the supernatant was removed, the cells were dissolved in 100 μL / well DMSO and reacted for 5 minutes. The absorbance was measured at a wavelength of 570 nm using an ELISA reader and converted to cell viability. Finally, the IC50 concentration was calculated using GraphPab Prism 8 software. The results are shown in Tables 7 to 10, Figures 15, and 16, respectively.

[0111] From the results in Tables 7 to 10, it can be seen that both Z3 compound and Z5 compound can inhibit the growth of HCT116 or HCT116+ch3 cell lines. Specifically, when the concentration of Z3 compound is 5 μM, the survival rate of HCT116 cell line is 51.15±3.59%, and the survival rate of HCT116+ch3 cell line is 42.51±1.16%; when the concentration of Z5 compound is 1 μM, the survival rate of HCT116 cell line is 45.72±

[0112] The survival rate of the HCT116+ch3 cell line was 3.56%, and the survival rate of the HCT116+ch3 cell line was 29.58±6.88%. The results in Figures 15 and 16 show that the half maximal inhibitory concentrations (IC50) of the Z3 compound and the Z5 compound were as low as 3-5 μM and 0.5-0.7 μM, respectively.

[0113] This indicates that the Z3 and Z5 compounds disclosed herein possess anticancer activity. In other words, administering an effective amount of the Z3 or Z5 compounds disclosed herein to a cancer patient effectively treats the cancer or slows the progression of the cancer. This indicates that the Z3 or Z5 compounds disclosed herein, or their salts, can serve as active ingredients in targeted anticancer drugs.

[0114] Table 7: Cytotoxicity of Z3 compound after 48 hours of culture on HCT116 cells

[0115] Table 8: Cytotoxicity of Z3 compound after 48 hours of culture on HCT116+ch3 cells

[0116] Table 9: Cytotoxicity of Z3 compound after 48 hours of culture on HCT116 cells

[0117] Table 10: Cytotoxicity of Z3 compound after 48 hours of culture on HCT116+ch3 cells

[0118] Melting Point Temperature Analysis (II)

[0119] Referring to the experimental process disclosed in Example 1, the actual melting point values ​​and changes of the QA4 compound or QA5 compound after forming the QA4 complex or QA5 complex with the d(CGTATACG)2 sequence at different concentration ratios were tested, as shown in Tables 11 and 12 below.

[0120] Table 11: Thermal melting point temperature and temperature difference of QA4 complex

[0121] Table 12: Thermal melting point temperature and temperature difference of QA5 complex

[0122] The results in Tables 11 and 12 indicate that the compounds QA4 and QA5 disclosed in the present invention can effectively stabilize DNA molecules and maintain stable stability at different concentrations.

[0123] Example 6: Melting Point Temperature Analysis (III)

[0124] Referring to the experimental procedure disclosed in Example 1, the actual melting point values ​​and changes of the Z3 complex, Z5 complex, QA4 complex, or QA5 complex formed by the Z3 compound, Z5 compound, QA4 compound, or QA5 compound with the d(CGTTAACG)2 sequence at different concentration ratios were tested, as shown in Tables 13 to 16 below.

[0125] Table 13: Thermal melting point temperature and temperature difference of Z3 complex

[0126] Table 14: Melting point temperature and temperature difference of Z5 complex

[0127] Table 15: Thermal melting point temperature and temperature difference of QA4 complex

[0128] Table 16: Thermal melting point temperature and temperature difference of QA5 complex

[0129] Example 7: Melting Point Temperature Analysis (IV)

[0130] Referring to the experimental process disclosed in Example 1, the actual melting point temperature values ​​and changes of the Z3 compound, Z5 compound, QA4 compound or QA5 compound after forming the Z3 complex, Z5 complex, QA4 complex or QA5 complex with the d(TACGCGTA)2 sequence at different concentration ratios were respectively detected, as shown in Tables 17 to 20 below. Among them, the columns with no data in each table are indicated by "--" because the absorption light value is too high to measure the data.

[0131] Table 17: Thermal melting point temperature and temperature difference of Z3 complex

[0132] Table 18: Melting point temperature and temperature difference of Z5 complex

[0133] Table 19: Thermal melting point temperature and temperature difference of QA4 complex

[0134] Table 20: Thermal melting point temperature and temperature difference of QA5 complex

[0135] Example 8: Melting Point Temperature Analysis (V)

[0136] Referring to the experimental procedure disclosed in Example 1, the actual melting point values ​​and changes of the Z3 complex, Z5 complex, QA4 complex, or QA5 complex formed by the Z3 compound, Z5 complex, QA4 complex, or QA5 complex with the d(TTCGTACGAA)2 sequence at different concentration ratios were tested, as shown in Tables 21 to 24 below. Columns with no data in each table are indicated by "--" because the absorption wavelength was too high to measure the data.

[0137] Table 21: Melting point temperature and temperature difference of Z3 complex

[0138] Table 22: Melting point temperature and temperature difference of Z5 complex

[0139] Table 23: Thermal melting point temperature and temperature difference of QA4 complex

[0140] Table 24: Thermal melting point temperature and temperature difference of QA5 complex

[0141] Example 9: Melting Point Temperature Analysis (VI)

[0142] Referring to the experimental procedure disclosed in Example 1, the actual melting point values ​​and changes of the Z3 complex, Z5 complex, QA4 complex, or QA5 complex formed by the Z3 compound, Z5 compound, QA4 compound, or QA5 compound with the d(TCGTATACGA)2 sequence at different concentration ratios were tested, as shown in Tables 25 to 28 below.

[0143] Table 25: Melting point temperature and temperature difference of Z3 complex

[0144] Table 26: Melting point temperature and temperature difference of Z5 complex

[0145] Table 27: Thermal melting point temperature and temperature difference of QA4 complex

[0146] Table 28: Thermal melting point temperature and temperature difference of QA5 complex

[0147] Based on the results of Examples 6 to 9, it can be seen that the acridine derivatives represented by any of the structural formulas (I) to (XIV) disclosed herein are capable of stably binding to sequences containing CG / CG and TA / TA bases, or DNA molecules composed thereof, and are thermally stable. In other words, the acridine derivatives represented by any of the structural formulas (I) to (XIV) disclosed herein are capable of disrupting or altering the structure of DNA molecules by intercalating acridine within sequences containing CG / CG and TA / TA bases, or DNA molecules composed thereof.

[0148] Example 10: Crystal structure analysis of the QA5 complex

[0149] Referring to the experimental steps and processes of Examples 2 and 3, the QA5 crystal structure was analyzed. The QA5 crystals were prepared using a DNA molecule with the sequence d(CGTTAACG)2 and a QA5 compound at a concentration ratio of 1:2, using the same preparation method as in Example 1. The analysis results are shown in Table 29 and Figure 17 .

[0150] Table 29: Crystal structure analysis results of QA5 crystals

[0151] Referring to Figure 17 , the bases in the d(CGTATACG)2 sequence are numbered from the 5-end to the 3-end, where the numbers are C1 to G8 for Chain A, C9 to G16 for Chain B, C17 to G24 for Chain C, and C25 to G32 for Chain D. The QA5 compound has an acridine ring and a quinoline ring inserted into the adjacent stacked bases at the ends, respectively.

[0152] Example 11: Crystal structure analysis of the QA4 complex

[0153] As described in Example 10, referring to the experimental procedures of Examples 2 and 3, the crystal structure of the QA4 complex was analyzed, wherein the QA4 crystal was prepared by a DNA molecule sequence of d(CGTTAACG)2 and a QA4 compound at a concentration ratio of 1:2.

[0154] Referring to Figure 18 , the bases in the d(CGTATACG)2 sequence are numbered from the 5-end to the 3-end, where the numbers are C1 to G8 for Chain A, C9 to G16 for Chain B, C17 to G24 for Chain C, and C25 to G32 for Chain D. The QA4 compound has an acridine ring and a quinoline ring inserted into the adjacent stacked bases at the ends, respectively.

[0155] The results of Example 10 and Example 11 indicate that the acridine derivatives of Formula (XIII) or (XIV) disclosed herein are indeed stably embedded in DNA molecules via the cyclic units on both sides, causing damage or changes in the DNA molecular structure, thereby achieving the effect of affecting the function of the DNA molecule.

[0156] Example 12: Cytotoxicity Analysis (II)

[0157] Referring to the experimental content of Example 4, the colorectal cancer cell line HCT116, the colorectal cancer cell line HCT116+ch3, the colon cancer cell line SW620, the lung cancer cell line A549, the breast cancer cell line MDA-MB-231, the breast cancer cell line 4T1, the normal lung cell line BEAS-2b, and the cervical cancer cell line HeLa were used to detect the cytotoxicity of the Z3 compound, the Z5 compound, the QA4 compound, and the QA5 compound against each cell line, and the IC50 of each compound was obtained. The results are shown in Table 30 below.

[0158] Table 30: Cytotoxicity of various acridine derivatives against cancer cells and normal cells

[0159] Example 13: Cancer Cell Cycle Analysis

[0160] The effects of Z3 compound, Z5 compound, QA4 compound and QA5 compound at different concentrations (100 nM, 500 nM) on the cell cycle of colorectal cancer cell HCT116 and lung cancer cell A549 were analyzed. The results are shown in Figures 19 and 20

[0161] The results of Examples 12 and 13 indicate that the compounds Z3, Z5, QA4, and QA5 disclosed herein are cytotoxic to cancer cells, arresting the cell cycle at the S phase and / or G2 / / M phase, and effectively inhibiting cancer cell proliferation and differentiation. This means that administering an effective amount of the acridine derivatives disclosed herein to a cancer patient can effectively treat cancer or delay cancer progression. In other words, the acridine derivatives disclosed herein can serve as active ingredients for cancer-targeted drugs.

[0162] Example 14: Analysis of NA series compounds

[0163] The NA5, NA6, NA7, and NA8 compounds were reacted with an A:A mismatch base pair sequence (5'-AACGCGAT-3'), a T:T mismatch base pair sequence (5'-ATCGCGTT-3'), a C:C mismatch base pair sequence (5'-ACCGCGCT-3'), and a G:G mismatch base pair sequence (5'-AGCGCGGT-3'), respectively. CD spectroscopy was then used to observe whether the NA5, NA6, NA7, and NA8 compounds could recognize the mismatch base pairs and insert base pairs. The results are shown in Figures 21 to 24.

[0164] The results in Figures 21 to 24 demonstrate that the disclosed compounds NA5, NA6, NA7, and NA8 are capable of recognizing and binding to C:C mismatched base pair sequences. In other words, the disclosed acridine derivatives represented by Formula (IX) or Formula (XII) stably embed themselves into DNA molecules via their flanking cyclic units, disrupting or altering the DNA molecular structure and thereby affecting its function.

[0165] Example 15: Cytotoxicity Analysis (III)

[0166] NA5, NA6, NA7 and NA8 compounds were tested on HCT116, A549 and SW620 cell lines, respectively. The results are shown in Table 31.

[0167] Table 31: Analysis results of half-inhibitory concentration of NA series drugs

[0168] Furthermore, A549 cells were treated with different concentrations (0, 10, 20 μM) of NA5, NA6, NA7, and NA8 compounds, and the cell apoptosis was analyzed. The results are shown in FIG25 .

[0169] From the results in Table 31 and FIG. 25 , it can be seen that the NA series compounds represented by formulas (IX) to (XII) disclosed in the present invention have anti-cancer efficacy and have a specific toxic effect on colorectal cancer cell lines.

Claims

1. Use of an acridine derivative or a salt thereof for preparing a pharmaceutical composition for treating cancer, characterized in that: The structural formula of the acridine derivative is shown in formula (A): XYZ, wherein: X and Z are selected from acridine compounds, quinoline compounds, Quinoxaline or a derivative of 1,8-naphthyridine, and at least one of the two is an acridine compound; Y is an alkane chain having 4 to 9 carbon atoms or an alkane chain in which one methane group is replaced by formamide.

2. The use of the acridine derivative or its salt as claimed in claim 1 for preparing a pharmaceutical composition for treating cancer, characterized in that: X is Y is Z is 3. The use of an acridine derivative or a salt thereof for preparing a pharmaceutical composition for treating cancer according to claim 1, characterized in that: The structural formula of the acridine derivative is selected from the group consisting of compounds represented by the following formula (I) to the following formula (XIV):

4. The use of the acridine derivatives or salts thereof as claimed in claim 1 for preparing a pharmaceutical composition for treating cancer, characterized in that: The cancer is colorectal cancer, lung cancer, breast cancer or cervical cancer.

5. Use of an acridine derivative or a salt thereof as claimed in any one of claims 1 to 4 for preparing a pharmaceutical composition for treating cancer, characterized in that: The pharmaceutical composition is a dual-intercalation drug, which is used to intercalate into a DNA molecule containing a sequence of TA / TA or CG / CG bases through the X and the Z in the formula (A).

6. A method for disrupting DNA structure, characterized in that: It includes the following steps: Step A: providing an effective amount of a compound of formula (A) or a salt thereof, wherein formula (A) is XYZ, wherein: X and Z are selected from acridine compounds, quinoline compounds, Quinoxaline or a derivative of 1,8-naphthyridine, and at least one of the two is an acridine compound; Y is an alkane chain having 4 to 9 carbon atoms or an alkane chain in which one methane group is replaced by formamide; Step B: The compound of formula (A) or its salt reacts with at least one DNA molecule, so that the compound of formula (A) is respectively inserted between the base pairs of the DNA molecule through the X and Z structures in formula A, thereby destroying the structure of the DNA molecule.

7. A method for disrupting DNA structure as claimed in claim 6, characterized in that: In the compound of formula (A), The X is The Y is The Z is 8. The method for destroying DNA structure as claimed in claim 6, characterized in that: The structural formula of the compound of formula (A) is selected from the group consisting of compounds represented by the following formula (I) to the following formula (XIV):

9. The method for destroying DNA structure as claimed in claim 6, characterized in that: The DNA molecule contains CG / CG and / or TA / TA bases.

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