CN104693199A - 2,9-bis-styryl substituted phenanthroline compounds, and preparation method and application thereof - Google Patents

Abstract

The invention provides 2,9-bis-styryl substituted phenanthroline compounds specifically combined with G-tetrastrobila-structure nucleic acid and a preparation method thereof, and application of the 2,9-bis-styryl substituted phenanthroline compounds in tumor resistance. The structural formula is disclosed as Formula I. The compounds disclosed as Formula I can quickly judge whether the sample to be detected is G-tetrastrobila-structure nucleic acid by ultraviolet-visible absorption spectrum or fluorescence spectrum. The drug effect test proves that the compounds disclosed as Formula I in vitro have strong inhibiting actions on multiple tumor cell strains. The 2,9-bis-styryl substituted phenanthroline compounds can be used for preparing anticancer drugs.

Description

2,9-bisstyrene-substituted o-phenanthroline compounds, preparation method and application thereof

technical field

The invention belongs to the field of medicine, and in particular relates to 2,9-bisstyrene substituted o-phenanthroline compounds and a preparation method and application thereof.

Background technique

G-quadruplex (G-quadruplex) is a nucleic acid sequence rich in guanine (G), through the formation of Hoogsteen base pairing between chains or corresponding G bases in the chain, so that four or four pieces of G-rich nucleic acid A special nucleic acid secondary structure formed by aggregation of fragments [S. Burge et al. Nucleic Acids Res, 2006, 34, 5402-5415]. G-rich sequences are commonly found in genomes with important functions, such as telomeres, gene promoter regions, immunoglobulin switch regions, etc. [J.L.Huppert et al.Nucleic Acids Res,2005,33,2908-2916; 2007,35,406- 413; A.K. Todd et al. Nucleic Acids Res, 2005, 33, 2901-2907]. And it is closely related to the formation mechanism of human lifespan, cancer, HIV and other diseases [T.A.Brooks et al.FEBS Journal 2010,277,3459-3469]. At present, G-quadruplex has become an important drug target. for the discovery of new anticancer drugs. Therefore, it is of great significance to develop molecular probes and antitumor drugs that have selective optical responses to the G-quadruplex structure.

At present, people have discovered some compounds that can bind to the G-quadruplex of nucleic acids, such as diquinoline derivatives, diindole derivatives and bisbenzimidazole derivatives, etc. Some of these compounds show the activity of inhibiting tumor cell proliferation . However, most of the compounds have low selectivity to G-quadruplexes, and can bind to single- and double-stranded nucleic acids at the same time [T.Ou et al. ChemMedChem2008, 3, 690-713]. In addition, the optical properties of most compounds do not change significantly after binding to the G-quadruplex, so they cannot be used for the detection of the G-quadruplex structure.

Contents of the invention

One of the objectives of the present invention is to provide a 2,9-bisstyrene-substituted o-phenanthroline compound and a preparation method thereof.

The 2,9-distyrene-substituted o-phenanthroline compound provided by the present invention has a structural formula as shown in formula I:

In the above formula I, _R1 and _R2 are independently selected from any one of the following: H, halogen, C1-C6 alkyl disubstituted amino, C1-C8 alkyl, substituted or unsubstituted heteroatom-containing ring alkyl;

The heteroatom in the substituted or unsubstituted heteroatom-containing cycloalkyl group is selected from at least one of the following: N, O and S; the cycloalkyl group is a 3-6 membered cycloalkyl group;

The substituent in the substituted heteroatom-containing cycloalkyl group is selected from at least one of the following: C1-C6 alkyl, -(CH ₂ ) _n -OH (n=1-8), -(CH ₂ ) _n -Ar (n=1-6, Ar represents an aromatic group).

The 2,9-bisstyrene-substituted o-phenanthroline compounds shown in the above formula I are preferably any of the following:

The 2,9-bisstyrene-substituted o-phenanthroline compound shown in the above formula I is prepared according to the method comprising the following steps:

Condensing the compound shown in formula II with the compound shown in formula III and the compound shown in formula IV to obtain 2,9-bisstyrene-substituted o-phenanthroline compounds shown in formula I;

In the above formula III and formula IV, R ₁ and R ₂ are independently selected from any one of the following: H, halogen, C1-C6 alkyl disubstituted amino, C1-C8 alkyl, substituted or unsubstituted hetero Atom cycloalkyl;

The heteroatom in the substituted or unsubstituted heteroatom-containing cycloalkyl group is selected from at least one of the following: N, O and S; the cycloalkyl group is a 3-6 membered cycloalkyl group;

The substituent in the substituted heteroatom-containing cycloalkyl group is selected from at least one of the following: C1-C6 alkyl, -(CH ₂ ) _n -OH (n=1-8), -(CH ₂ ) _n -Ar (n=1-6, Ar represents an aromatic group).

The molar ratio of the compound represented by the formula II to the compound represented by the formula III and the compound represented by the formula IV is 1:1:1 in turn.

The temperature of the condensation reaction is 90°C-120°C, and the time is 12h-36h.

The condensation reaction is carried out in an organic solvent, and the organic solvent may specifically be toluene.

Another object of the present invention is to provide the application of 2,9-bisstyrene-substituted o-phenanthroline compounds represented by the above formula I as molecular probes in identifying the G-quadruplex structure of nucleic acids.

Using the 2,9-bisstyrene substituted o-phenanthroline compound shown in the above formula I as a method for molecular probe identification of nucleic acid G-quadruplex structure, comprising the following steps:

(1) Dissolve the nucleic acid sample to be detected and the reference nucleic acid sample in a buffer respectively to obtain a nucleic acid sample solution A to be detected and a reference nucleic acid sample solution B; use an organic solvent to dissolve the 2,9-bis After the styrene-substituted o-phenanthroline compound is dissolved, it is then diluted with the buffer solution to obtain detection solution C;

(2) Mix the nucleic acid sample solution A to be detected with the detection solution C to obtain a mixed solution 1, mix the reference nucleic acid sample solution B with the detection solution C to obtain a mixed solution 2, and then mix the obtained The solution 1 and the mixed solution 2 are incubated respectively to obtain the mixed solution 1 after incubation and the mixed solution 2 after incubation; then the mixed solution 1 after incubation and the mixed solution 2 after incubation are subjected to the following a) or b):

a) The post-incubation mixed solution 1 and the post-incubation mixed solution 2 are respectively subjected to ultraviolet-visible absorption spectrum analysis, and the 2,9-bisstyrene-substituted ortho-phenanthrene represented by formula I in the post-incubation mixed solution 1 is The ultraviolet-visible absorption spectrum of the roline compound is compared with the ultraviolet-visible absorption spectrum of the 2,9-bisstyrene-substituted o-phenanthroline compound shown in the formula I in the mixed solution 2 after the incubation, thereby judging Whether the nucleic acid sample to be detected is a nucleic acid with a G-quadruplex structure;

or

b) Fluorescence spectrum analysis is performed on the mixed solution 1 after incubation and the mixed solution 2 after incubation, and the 2,9-bisstyrene-substituted o-phenanthrolines represented by formula I in the mixed solution 1 after incubation are The fluorescence spectrum of the compound is compared with the fluorescence spectrum of the 2,9-bisstyrene-substituted o-phenanthroline compound shown in the formula I in the mixed solution 2 after incubation, so as to determine whether the nucleic acid sample to be detected is G - A nucleic acid with a quadruplex structure.

In the above method step (1), the nucleic acid sample to be detected is a nucleic acid having a G-quadruplex structure.

The nucleic acid sample to be detected can specifically be: Hum24 (its sequence is shown in sequence 1 in the sequence listing), c-myc (its sequence is shown in sequence 2 in the sequence listing), TBA (its sequence is shown in sequence 3 in the sequence listing shown), 22AG K ⁺ (wherein, the sequence of 22AG is shown in sequence 4 in the sequence listing) or 22AG Na ⁺ (wherein, the sequence of 22AG is shown in sequence 4 in the sequence listing).

The reference nucleic acid sample is a nucleic acid with a non-G-quadruplex structure, and may be a single-stranded nucleic acid or a double-stranded nucleic acid.

The reference nucleic acid sample can specifically be ss-DNA1 (its sequence is shown as sequence 5 in the sequence listing), ss-DNA2 (it is the complementary sequence of ss-DNA1) or dsDNA (ss-DNA1+ss-DNA2).

The buffer is Tris-HCl buffer or phosphate buffer.

The pH value of the buffer solution is 6-8.

For the nucleic acid sample solution A to be detected, the molar concentration of the nucleic acid sample to be detected is 0.25 μM-60 μM.

In the reference nucleic acid sample solution B, the molar concentration of the reference nucleic acid sample is 0.25 μM-60 μM.

The organic solvent can specifically be dimethyl sulfoxide.

In the detection solution C, the molar concentration of the 2,9-bisstyrene-substituted o-phenanthroline compound represented by the formula I is 0.5 μM-20 μM.

In step (2) of the above method, in the mixed solution 1, the molar ratio of the nucleic acid sample to be detected to the 2,9-bisstyrene-substituted o-phenanthroline compound represented by formula I is 0.125-6.

In the mixed solution 2, the molar ratio of the reference nucleic acid sample to the 2,9-bisstyrene-substituted o-phenanthroline compound shown in formula I is 0.125-6.

In the above a), when it is observed that the ultraviolet-visible absorption spectrum of the 2,9-bisstyrene-substituted o-phenanthroline compound shown in the formula I in the mixed solution 1 after the incubation is obvious in the range of 310-550nm increase, and a new absorption peak appears in the range of 470-550nm, the nucleic acid sample to be detected is confirmed to be a nucleic acid with a G-quadruplex structure; otherwise, it is a nucleic acid with a non-G-quadruplex structure.

In b), when it is observed that the fluorescence emission spectrum of the 2,9-bisstyrene-substituted o-phenanthroline compound shown in formula I in the mixed solution 1 after the incubation has a fluorescence peak in the range of 450-630 nm And the fluorescence intensity increases, and is higher than the fluorescence that occurs in the fluorescence emission spectrum of the 2,9-bisstyrene-substituted o-phenanthroline compound shown in the formula I in the mixed solution 2 after incubation in the range of 450-630nm Intensity (generally higher than 2 times the fluorescence intensity of the reference nucleic acid sample mixture), then the nucleic acid sample to be detected is confirmed to be a nucleic acid with a G-quadruplex structure; otherwise, it is a nucleic acid with a non-G-quadruplex structure. nucleic acid.

Another object of the present invention is to provide the use of 2,9-bisstyrene-substituted o-phenanthroline compounds represented by formula I.

The purposes of the 2,9-bisstyrene substituted o-phenanthroline compounds shown in the formula I provided by the present invention is its application in the following aspects:

1) the application in the preparation of eukaryotic tumor cell proliferation inhibitors; 2) the application in the preparation of drugs for preventing and/or treating tumors.

The eukaryote is a mammal; the tumor cell is a cancer cell; the cancer cell is a lung cancer cell, a breast cancer cell or a prostate cancer cell.

The lung adenocarcinoma cells are specifically drug-resistant lung cancer cells A549T.

The breast cancer cells are specifically human breast cancer cells MCF-7.

The lung cancer cells are specifically human lung cancer cells A549.

The prostate cancer cells are specifically human prostate cancer cells PC-3.

The tumor is cancer; the cancer is lung cancer, breast cancer or prostate cancer.

Eukaryotic tumor cell proliferation inhibitors or drugs for preventing and/or treating tumors prepared by using 2,9-bisstyrene-substituted o-phenanthroline compounds represented by formula I as active ingredients also belong to the protection scope of the present invention.

The eukaryotic tumor cell proliferation inhibitors or drugs for preventing and/or treating tumors can be introduced into the body through injection, spraying, nasal drop, eye drop, penetration, absorption, physical or chemical mediated methods such as muscle, intradermal, Subcutaneous, vein, mucosal tissue; or mixed or wrapped with other substances and introduced into the body.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.

The eukaryotic tumor cell proliferation inhibitor or the drug for preventing and/or treating tumor prepared by using the 2,9-bisstyrene-substituted o-phenanthroline compound as the active ingredient can be made into injection, tablet, Powder, granule, capsule, oral liquid, ointment, cream and other forms. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy.

The present invention discusses the interaction of 2,9-bisstyrene-substituted o-phenanthroline compounds with nucleic acid G-quadruplexes by using the changes of ultraviolet-visible absorption spectrum and fluorescence spectrum, and as a comparison with single-stranded nucleic acid, Interaction of double-stranded nucleic acids.

Because the 2,9-bisstyrene-substituted o-phenanthroline compound of the present invention contains a large conjugated aromatic planar structure, it can form π-π stacking with the G-tetrad plane of the nucleic acid G-quadruplex , but there is no large aromatic planar structure in the structure of single-stranded nucleic acid and double-stranded nucleic acid, so it cannot be combined with 2,9-bisstyrene-substituted o-phenanthroline compounds. Through the ultraviolet-visible absorption spectrum and fluorescence spectrum, it can quickly determine whether the structure of the nucleic acid in the solution is a G-chain structure, a single-stranded nucleic acid, or a double-stranded nucleic acid structure.

The 2,9-bisstyrene-substituted o-phenanthroline compounds have a certain flexibility of the conjugation plane, which makes it easier to stack on the G tetrad plane, and then has a strong bond with the G-quadruplex. Affinity, its special crescent-shaped structure makes it weaker to bind to other secondary structures such as double-stranded nucleic acids. Because the molecule has a large conjugation plane, it is easy to form intermolecular aggregates through van der Waals force in the aqueous solution system, resulting in a decrease in the ultraviolet-visible light absorption of 2,9-bisstyrene-substituted o-phenanthroline compounds, and at the same time The nitrogen-containing group interacts with water to produce a non-radiative transition that causes its fluorescence to disappear, so when the 2,9-bisstyrene-substituted o-phenanthroline compound is mixed with the nucleic acid G-quadruplex sample, it will react with the G- The quadruplex interaction disaggregates the aggregate, and thus combines with the G-tetrad plane in the G-quadruplex in the form of monomer through π-π stacking interaction, which increases the degree of conjugation and leads to new absorption band appears. At the same time, the combination with the G-quadruplex shields its interaction with water molecules, resulting in a greatly enhanced fluorescence of the compound.

The present invention simultaneously studies the antitumor effect of 2,9-bisstyrene substituted o-phenanthroline compounds.

G-quadruplex structures widely exist in telomeres and gene promoter regions, especially the promoter regions of some tumor-related genes have multiple sequences that can form G-quadruplexes [S.Balasubramanian et al.NatRevDrugDiscov, 2011 , 10, 261-275]. Many compounds that can bind to the G-quadruplex exhibit the ability to kill tumor cells or inhibit the proliferative activity of tumor cells. However, most of the compounds have low selectivity to G-quadruplexes, and can combine with single- and double-stranded nucleic acids at the same time, so they are highly toxic to normal cells. Therefore, molecules with high selectivity for G-quadruplex will help to reduce their toxic side effects on normal cells. We added 2,9-bisstyrene-substituted o-phenanthroline compounds into the culture medium containing tumor cells, and cultured them together with tumor cells, and found that they did have a higher activity of inhibiting tumor cell proliferation.

Compared with the prior art, the present invention has the following advantages:

1) The 2,9-bisstyrene-substituted o-phenanthroline compounds provided by the present invention are easy to synthesize, very stable, easy to store, have a strong effect of inhibiting the proliferation of tumor cells, and are useful as antitumor drugs potential;

2) The 2,9-bisstyrene-substituted o-phenanthroline compounds provided by the present invention can specifically bind to the G-quadruplex structure, realizing the difference from single-stranded nucleic acid and double-stranded nucleic acid structure, using ultraviolet - Visible absorption spectrum and fluorescence spectrum can distinguish the G-quadruplex structure of nucleic acid, which is simple, fast, low-cost, and can be detected in real time and on the spot.

Description of drawings

Fig. 1A is the change of ultraviolet-visible absorption spectrum after compound E1 interacts with nucleic acid G-quadruplex.

Fig. 1B is a comparison chart of changes in ultraviolet-visible absorption spectra after compound E1 interacts with reference nucleic acid and nucleic acid G-quadruplex (c-myc).

Fig. 2A is a comparison chart of the fluorescence excitation spectrum changes of the compound E1 after the compound E1 interacts with the nucleic acid G-quadruplex and the reference nucleic acid respectively.

Fig. 2B is a graph showing the change of fluorescence emission spectrum after the compound E1 reacts with different concentrations of nucleic acid G-quadruplex 22AG Na ⁺ .

Fig. 2C is the relative fluorescence value of compound E1 after the reference nucleic acid/nucleic acid G-quadruplex and compound E1 were mixed and incubated at different molar ratios.

Figure 3 is the inhibitory activity of compound E1 on tumor cells.

Detailed ways

The present invention will be described below through specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents and biological materials used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the synthesis of compound E1

4-(4-Methylpiperazine) benzaldehyde (purchased from Bailingwei) (2.04g, 10mmol) and 2,9-dimethyl-2,9-phenanthroline (purchased from Bailingwei) (1.04g, 5mmol) was dissolved in dry toluene (10mL), reacted at a temperature of 120°C for 24 hours, and the solvent in the system was spin-dried to obtain a crude product, which was purified by silica gel column chromatography with dichloromethane as an eluent to obtain 975mg of The solid is compound E1, and the yield is 35%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.34(t,8H),2.85(d,8H),2.87(s,6H). ¹³ CNMR(CDCl ₃ ,400MHz):δ(ppm):156.71 , 151.24, 145.84, 136.25, 133.84, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 54.95, 48.32, 46.13. HRMS (ESI-TOF) calcd for C ₃₈ H ₄₁ N ₆ [M] ⁺ 513.3385

Embodiment 2, the synthesis of compound E2

Basically identical with embodiment 1, difference is to replace 4-(4-methylpiperazine) benzaldehyde with compound 4-(4-(2-hydroxyethyl) piperazine) benzaldehyde (purchased from Bailingwei), Reaction at 120°C for 24 hours, rotary evaporation of toluene to obtain a crude product, which was separated by silica gel column chromatography using ethyl acetate as eluent to obtain compound E2. Yield: 38%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.65(s,2H),3.34(m,20H),2.53(t,4H). ¹³ CNMR(CDCl ₃ ,400MHz):δ(ppm):156.71 , 151.24, 145.84 _{, 136.25 ,} 133.84, 128.45, 127.85, 127.06, 125.50, _120.02 , 115.51, 59.41, _56.32 , 51.33 ^. .

Embodiment 3: the synthesis of compound E3

Basically the same as Example 1, the difference is to replace 4-(4-methylpiperazine) benzaldehyde with compound 4-(4-morpholine) benzaldehyde (purchased from Bailingwei), react at 120 ° C for 24 hours, and rotate Toluene was evaporated to obtain a crude product, which was subjected to silica gel column chromatography with ethyl acetate as eluent to obtain compound E3. Yield: 42%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.65(t,8H),3.18(t,8H). ¹³ C NMR(CDCl ₃ ,400MHz):δ(ppm):156.71,151.24,145.84,136.25 , 133.84, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 66.35, 54.62. HRMS (ESI-TOF) calcd for C ₃₆ H ₃₄ N ₄ O ₂ [M] ⁺ 554.2682, found 554.2679.

Embodiment 4: the synthesis of compound E4

Basically the same as Example 1, the difference is that the compound 4-diethylaminobenzaldehyde (purchased from Bailingwei) is used instead of 4-(4-methylpiperazine) benzaldehyde, reacted at 120 ° C for 24 hours, and the toluene was removed by rotary evaporation The crude product was obtained, and the crude product was subjected to silica gel column chromatography using ethyl acetate as eluent to obtain compound E4. Yield: 56%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.41(q,8H),1.15(t,12H). ¹³ C NMR(CDCl ₃ ,400MHz):δ(ppm):156.71,151.24,145.84,136.25 , 133.84, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 47.32, 12.96. HRMS (ESI-TOF) calcd for C ₃₆ H ₃₈ N ₄ [M] ⁺ 526.3096, found 526.3098.

Embodiment 5, the synthesis of compound E5

Basically the same as Example 1, the difference is that the compound 4-bromobenzaldehyde (purchased from Bailingwei) is used to replace 4-(4-methylpiperazine) benzaldehyde, react at 120 ° C for 24 hours, and rotate to evaporate toluene to obtain crude Product, the crude product was separated by silica gel column chromatography using ethyl acetate as eluent to obtain compound E5. Yield: 30%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.56(d,4H), 7.42(d,2H),7.02(d,2H). ¹³ C NMR(CDCl ₃ ,400MHz):δ(ppm):156.71,151.24,145.84,136.25,133.84,130.92,128.45,127.85,127.06,125.50,122.30 ,120.02.HRMS(ESI-TOF)calcd for C ₂₈ H ₁₈ N ₂ Br ₂ [M] ⁺ 539.9837,found 539.9834.

Embodiment 6, the synthesis of compound E6

Basically the same as in Example 1, the difference is that the compound 4-butylbenzaldehyde (purchased from Bailingwei) is used instead of 4-(4-methylpiperazine)benzaldehyde, reacted at 120°C for 24 hours, and the toluene is removed by rotary evaporation to obtain The crude product was separated by silica gel column chromatography using ethyl acetate as eluent to obtain compound E6. Yield: 62%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),2.62(t,4H),1.59(m,4H),1.31(m,4H),0.91(t,6H). ¹³ C NMR(CDCl ₃ ,400MHz ): δ(ppm): 156.71, 151.24, 145.84, 141.81, 136.25, 133.84, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 36.23, 33.21, 23.14, _13.98 . HRMS (ESI-TO CF)3 ₃₆ N ₂ [M] ⁺ 496.2878, found 496.2881.

Embodiment 7, the synthesis of compound E7

Basically the same as Example 1, the difference is that the compound 4-piperidine benzaldehyde (purchased from Bailingwei) is used instead of 4-(4-methylpiperazine) benzaldehyde, reacted at 120 ° C for 24 hours, and the toluene is removed by rotary evaporation to obtain The crude product was separated by silica gel column chromatography using ethyl acetate as eluent to obtain compound E7. Yield: 48%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.44(t,8H),1.56(m,12H). ¹³ C NMR(CDCl ₃ ,400MHz):δ(ppm):156.71,151.24,148.52,145.84 , 136.25, 133.84, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 54.95, 24.95. HRMS (ESI-TOF) calcd for C ₃₈ H ₃₈ N ₄ [M] ⁺ 550.3096, found 5850.3092.

Embodiment 8, the synthesis of compound E8

Basically the same as Example 1, the difference is that the compound 4-(4-benzylpiperazine) benzaldehyde (purchased from Bailingwei) is used instead of 4-(4-methylpiperazine) benzaldehyde, and the reaction is carried out at 120° C. for 24 hours , rotary evaporating toluene to obtain a crude product, which was subjected to silica gel column chromatography using ethyl acetate as eluent to obtain compound E8. Yield: 26%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.33(m,4H),7.26(d,2H),7.23(d,4H),7.02(d,2H),6.85(m,2H),3.66(s,4H),3.34(t,8H),2.85 (d,8H). ¹³ C NMR (CDCl ₃ , 400MHz): δ (ppm): 156.71, 151.24, 145.84, 138.64, 136.25, 133.84, 130.56, 129.21, 128.84, 128.45, 127.85, 127.23, 120.06, 125. , 115.51, 64.85, 54.95, 52.36. HRMS (ESI-TOF) calcd for C ₅₀ H ₄₈ N ₆ [M] ⁺ 732.3940, found 732.3942.

Embodiment 9: the synthesis of compound E9

4-(4-methylpiperazine) benzaldehyde, 4-diethylaminobenzaldehyde and 2,9-dimethyl-2,9-phenanthroline were dissolved in a molar ratio of 1:1:1 In dry toluene, react at 120°C for 24 hours, and rotary evaporate the toluene to obtain the crude product, which is subjected to silica gel column chromatography with ethyl acetate as eluent to obtain compound E9. Yield: 23%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.44(m,8H),2.36(t,4H),2.27(s,3H),1.16(t,6H). ¹³ C NMR(CDCl ₃ ,400MHz ): δ (ppm): 156.71, 151.24, 145.84, 136.25, 133.84, 130.56, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 57.36, 52.84, 48.32, 46.13, 12.93. ₃₇ H ₃₉ N ₅ [M] ⁺ 553.3205, found 553.3208.

Embodiment 10, the synthesis of compound E10

Dissolve (4-(4-methylpiperazine)benzaldehyde, 4-butylbenzaldehyde and 2,9-dimethyl-2,9-phenanthroline in a molar ratio of 1:1:1 In dry toluene, react at 120°C for 24 hours, and rotary evaporate the toluene to obtain the crude product, which is separated by silica gel column chromatography with ethyl acetate as eluent to obtain compound E10. Yield: 28%.

The confirmed data of the compound structure are:

¹ H NMR(CDCl ₃ ,400MHz):δ(ppm):8.04(d,2H),7.95(d,2H),7.83(m,4H),7.64(d,4H),7.42(d,2H), 7.02(d,2H),6.85(m,2H),3.34(t,4H),2.85(t,4H),2.62(t,2H),2.26(s,3H),1.61(m,4H),0.91 (t,3H). ¹³ C NMR (CDCl ₃ , 400MHz): δ (ppm): 156.71, 151.24, 145.84, 136.25, 133.84, 130.64, 128.45, 127.85, 127.06, 125.50, 120.02, 115.51, 57.346, 562.83 , 36.25, 33.36, 23.17, 13.93. HRMS (ESI-TOF) calcd for C ₃₇ H ₃₈ N ₄ [M] ⁺ 538.3096, found 538.3093.

Example 11, Spectroscopy changes after compound E1 binds to nucleic acid

1. Prepare the sample:

DNA samples: DNA samples were purchased from Beijing Sangong Biotechnology Co., Ltd., the DNA except for the 22AG sequence was dissolved in buffer (10mM Tris-HCl, pH=7.4, 20mM KCl, 100mMNaCl), 22AG was dissolved in Na ⁺ buffer solution (10mM Tris-HCl, 100mM NaCl, 0.1mM EDTA, pH=7.4) to form an antiparallel G-quadruplex secondary structure (22AG Na ⁺ ), 22AG was dissolved in K ⁺ buffer solution (10mM Tris-HCl, 20mM KCl , 0.1 mM EDTA, pH=7.4) to form a G-quadruplex (22AG K ⁺ ) of mixed structure, denatured at 95°C for 10 minutes, and then placed in a refrigerator at 4°C for annealing overnight.

DNA samples and sequences tested include:

Reference nucleic acid:

ss-DNA1: CCAGTTCGTAGTAACCC (see sequence 5 in the sequence listing for its sequence)

ss-DNA2 is the complementary sequence of ss-DNA1

dsDNA: ss-DNA1+ss-DNA2

Nucleic acid to be tested:

nucleic acid G-quadruplex

Hum24: TTAGGGTTAGGGTTAGGGTTAGGG (see sequence 1 in the sequence listing for its sequence)

c-myc: TGAGGGTGGGGAGGGTGGGGAA (see sequence 2 in the sequence listing for its sequence)

TBA: GGTTGGTGTGGTTGG (see sequence 3 in the sequence listing for its sequence)

22AG K ⁺ : AGGGTTAGGGTTAGGGTTAGGG (see sequence 4 in the sequence listing for its sequence)

22AG Na ⁺ : AGGGTTAGGGTTAGGGTTAGGG (see sequence 4 in the sequence listing for its sequence)

Compound E1 solution: Compound E1 was first prepared with dimethyl sulfoxide to prepare a 10mM stock solution, and then diluted the stock solution with buffer (10mM Tris-HCl, pH=7.4, 20mMKCl, 100mMNaCl) to a 0.5-20μM solution C for use in For the test, it is particularly pointed out that the buffer solution (10mM Tris ^- HCl, 100mM NaCl, 0.1mM EDTA, pH=7.4) used for 22AG Na + compound E1 is used for 22AG K ⁺ buffer solution (10mM Tris-HCl, 20 mM KCl, 0.1 mM EDTA, pH=7.4) was diluted to 0.5-20 μM solution C for testing.

Preparation of nucleic acid solution: Dissolve nucleic acid to be tested in Tris-HCl (pH=7.4) buffer to obtain solution A with a concentration of 0.25 μM to 60 μM; dilute reference nucleic acid with Tris-HCl (pH=7.4) buffer , to obtain a solution B with a concentration of 0.25 μM to 60 μM.

2. Absorption spectrum:

Mix solution A and solution C, solution B and solution C fully respectively, and then incubate the two obtained mixed solutions for 10 minutes respectively, and perform ultraviolet-visible absorption spectrum analysis on the two reaction solutions after the above incubation.

2.1) Take nucleic acid G-quadruplex Hum24, c-myc, TBA, 22AG K ⁺ , 22AG Na ⁺ as an example

Fig. 1A is the change of ultraviolet-visible absorption spectrum after compound E1 interacts with nucleic acid G-quadruplex.

As shown in Figure 1A, add 200 μL of 60 μM nucleic acid G-quadruplex solution to 200 μL of compound E1 solution with a concentration of 10 μM, respectively, and use a UV spectrophotometer to conduct UV-visible absorption of the incubated mixture. Spectral analysis, through observation, it was found that the overall enhancement of the ultraviolet-visible absorption spectrum was caused by the change of compound E1 from the aggregate to the monomer. In addition, a new broad absorption band appeared in the range of 470-550nm in the mixed solution of compound E1 and nucleic acid G-quadruplex.

2.2) Taking reference nucleic acids ss-DNA1, ss-DNA2, and dsDNA as examples

Fig. 1B is a comparison chart of changes in UV-Vis absorption spectrum after compound E1 interacts with reference nucleic acid and nucleic acid G-quadruplex (c-myc).

As shown in Figure 1B, in the UV-Vis absorption spectrum, when 200 μL of 60 μM reference nucleic acid ss-DNA1, ss-DNA2 or dsDNA was mixed and incubated with 200 μL of 10 μM compound E1 solution, the absorption spectrum of compound E1 was overall enhanced, which was It was caused by the change from aggregate to monomer, but the addition of ss-DNA1, ss-DNA2 or dsDNA did not make compound E1 appear a broad absorption band in the range of 470nm to 550nm, indicating that compound E1 only interacted with G-quadruplex combined with high selectivity. Therefore, compare whether there is a broad absorption band in the absorption spectrum of compound E1 in the range of 470nm to 550nm, and whether the absorption peak is significantly enhanced and higher than that of the reference nucleic acid mixture. If so, it can be judged that the nucleic acid to be tested is a G-quadruplex structure. Nucleic acid; otherwise, it is a nucleic acid with a non-G-quadruplex structure.

3. Fluorescence spectroscopy

Compound E1 solution: compound E1 was first formulated with dimethyl sulfoxide to prepare a 10 mM stock solution, and then diluted the stock solution with Tris-HCl (pH=7.4) buffer solution to a solution C with a concentration of 0.5-20 μM for testing.

Preparation of nucleic acid solution: Dissolve nucleic acid to be tested in Tris-HCl (pH=7.4) buffer to obtain solution A with a concentration of 0.1 μM to 30 μM; dilute reference nucleic acid with Tris-HCl (pH=7.4) buffer , to obtain a solution B with a concentration of 0.1 μM to 30 μM.

The solution A and the solution C, and the solution B and the solution C were fully mixed respectively, and then the two obtained mixtures were incubated for 30 minutes respectively, and the fluorescence excitation spectrum and the emission spectrum were analyzed for the two reaction solutions after the above incubation.

3.1) Take nucleic acid G-quadruplex Hum24, c-myc, TBA, 22AG K ⁺ , 22AG Na ⁺ , reference nucleic acid ss-DNA1, ss-DNA2, dsDNA as an example

Add 100 μL of compound E1 solution with a concentration of 4 μM to 100 μL of a nucleic acid G-quadruplex solution with a concentration of 20 μM; add 100 μL of a solution of compound E1 with a concentration of 4 μM into 100 μL of a reference nucleic acid solution with a concentration of 20 μM, and pass A fluorescence spectrometer is used to analyze the fluorescence spectrum of the incubated mixture.

Fig. 2A is a comparison chart of the fluorescence excitation spectrum changes of the compound E1 after the compound E1 interacts with the nucleic acid G-quadruplex and the reference nucleic acid respectively.

As shown in Figure 2A, compound E1 itself has almost no fluorescence in the buffer. After the compound E1 interacts with the reference nucleic acid (ss-DNA1, ss-DNA2, dsDNA), there is still no fluorescence or a small change in fluorescence, but it is different from the nucleic acid G After the action of -quadruplex (Hum24, c-myc, TBA, 22AG K ⁺ , 22AG Na ⁺ ), the fluorescence was significantly enhanced.

Mix 100 μL of compound E1 solution at a concentration of 4 μM with 100 μL of nucleic acid G-quadruplex 22AG at concentrations of 1.0 μM, 2.0 μM, 4.0 μM, 6.0 μM, 8.0 μM, 12.0 μM, 16.0 μM, 20.0 μM, 24.0 μM After the Na ⁺ action, the incubated mixtures were subjected to fluorescence spectroscopic analysis.

Fig. 2B is a graph showing the change of fluorescence emission spectrum after the compound E1 reacts with different concentrations of nucleic acid G-quadruplex 22AG Na ⁺ .

As shown in Figure 2B, the fluorescence emission spectrum of the compound E1 in the nucleic acid sample mixture to be tested has a fluorescence peak in the range of 450-630nm, and the fluorescence intensity shows that the concentration of the nucleic acid G-quadruplex 22AG Na ⁺ concentration (0.5μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 6.0 μM, 8.0 μM, 10.0 μM, 12.0 μM) increased. The fluorescence spectrum changes of nucleic acid G-quadruplex Hum24, c-myc, TBA, 22AG K ⁺ are similar to those of 22AG Na ⁺ .

Fig. 2C is the relative fluorescence value of compound E1 after the reference nucleic acid/nucleic acid G-quadruplex and compound E1 were mixed and incubated at different molar ratios.

As shown in Figure 2C, when the molar ratio of reference nucleic acid ss-DNA1, ss-DNA2 or dsDNA to compound E1 was 5:1, the fluorescence intensity of compound E1 did not change significantly.

With the increase of the molar ratio of the nucleic acid G-quadruplex Hum24, c-myc, TBA, 22AG K ⁺ , 22AG Na ⁺ to the compound E1, the fluorescence intensity of the compound E1 gradually increased. When the nucleic acid G-quadruplex When the molar ratio of Hum24, c-myc, TBA, 22AG K ⁺ , 22AG Na ⁺ to compound E1 is 5:1, the fluorescence intensity of compound E1 reaches the maximum, which indicates that compound E1 only binds to the G-quadruplex, It can be used to identify nucleic acid G-quadruplex structures with high selectivity.

Therefore, whether the fluorescence emission intensity of compound E1 in the range of 450-630nm increases significantly, and is higher than 2 times of the fluorescence intensity of compound E1 in the reference nucleic acid mixture, then it can be judged that the nucleic acid to be tested is G-4 A nucleic acid with a chain structure; otherwise, a nucleic acid with a non-G-quadruplex structure.

Example 12. Antiproliferation activity of compounds E1-E10 on tumor cells

1. Experimental steps:

1.1) Seeding plate of cells: 5000 cells (cell lines are MCF-7, A549, A549T, PC3) were inoculated in each well of each 96-well plate, and the volume of culture medium containing cells was 100 microliters per well.

1.2) Addition of the test substance: After the 96-well plate inoculated with cells was placed in a 37°C incubator for 24 hours, the compound E1 was divided into 9 concentration gradients (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM , 80 μM, 90 μM and 100 μM), then add 100 microliters of each concentration of compound E1 to three parallel wells in a 96-well plate, and add no or equal volume of culture medium to the zero well (without cells) .

1.3) Detection of cell viability. After 72 hours, discard the old culture medium, add 110 μL of CCK-8-containing solution (Dojindo Molecular Technologies, Inc) (100 μL of culture medium + 10 μL of CCK-8-containing solution), and incubate at 37°C for 30- After 60 min, the absorbance at 450 nm was detected with a microplate reader.

2. Inhibitory activity of compound E1 on tumor cells

Figure 3 is the inhibitory activity of compound E1 on tumor cells.

As shown in Figure 3, with the increase of the concentration of compound E1, the survival rate of the cells decreased. The concentration of compound E1 at which the cell growth was inhibited by 50% was calculated respectively, expressed as _IC50 value, and the results are shown in Table 1.

Table 1 Compounds E1-E10 inhibit the growth of different tumor cell lines (IC ₅₀ /μM)

cell line Compound E1 Compound E2 Compound E3 Compound E4 Compound E5 MCF-7 2.90 8.91 7.61 2.56 1.89 A549 0.85 4.36 5.41 0.36 3.20 A549T 2.57 5.27 3.68 1.89 2.84 PC3 1.44 6.38 4.26 1.56 3.36 cell line Compound E6 Compound E7 Compound E8 Compound E9 Compound E10 MCF-7 10.61 6.86 2.48 3.47 3.16 A549 8.93 10.13 8.26 2.94 1.23 A549T 11.35 5.64 6.27 4.69 2.78 PC3 9.62 3.89 10.29 5.79 3.66

The results show that the 2,9-bisstyrene-substituted o-phenanthroline compounds of the present invention have strong inhibitory effects on these four tumor cell lines in vitro. Therefore, the 2,9-bisstyrene-substituted o-phenanthroline compounds of the present invention can be used to prepare anticancer drugs.

Claims (10)

1. Compound shown in formula I: In the above formula I, _R1 and _R2 are independently selected from any one of the following: H, halogen, C1-C6 alkyl disubstituted amino, C1-C8 alkyl, substituted or unsubstituted heteroatom-containing ring alkyl; The heteroatom in the substituted or unsubstituted heteroatom-containing cycloalkyl group is selected from at least one of the following: N, O and S; the cycloalkyl group is a 3-6 membered cycloalkyl group; The substituent in the substituted heteroatom-containing cycloalkyl group is selected from at least one of the following: C1-C6 alkyl, -(CH ₂ ) _n -OH where n=1-8, -(CH ₂ ) _n -Ar wherein n=1-6, Ar represents an aromatic group. 2. The compound according to claim 1, characterized in that: the compound is any one of the following: 3. A method for preparing the compound according to claim 1 or 2, comprising the steps of: carrying out a condensation reaction of the compound shown in formula II with the compound shown in formula III and the compound shown in formula IV to obtain the compound shown in formula I; In the above formula III and formula IV, R ₁ and R ₂ are independently selected from any one of the following: H, halogen, C1-C6 alkyl disubstituted amino, C1-C8 alkyl, substituted or unsubstituted hetero Atom cycloalkyl; The heteroatom in the substituted or unsubstituted heteroatom-containing cycloalkyl group is selected from at least one of the following: N, O and S; the cycloalkyl group is a 3-6 membered cycloalkyl group; The substituent in the substituted heteroatom-containing cycloalkyl group is selected from at least one of the following: C1-C6 alkyl, -(CH ₂ ) _n -OH where n=1-8, -(CH ₂ ) _n -Ar wherein n=1-6, Ar represents an aromatic group. 4. The method according to claim 3, characterized in that: in the method, the mol ratio of the compound shown in the formula II to the compound shown in the formula III and the compound shown in the formula IV is 1 successively: 1:1; The temperature of the condensation reaction is 90°C-120°C, and the time is 12h-36h. 5. The compound of claim 1 or 2 is used as a molecular probe in identifying the G-quadruplex structure of nucleic acid. 6. A recognition method of nucleic acid G-quadruplex structure, characterized in that: the recognition method of said nucleic acid G-quadruplex structure adopts the compound described in claim 1 or 2 as the nucleic acid G-quadruplex structure identify the probe; The recognition method of described nucleic acid G-quadruplex structure, comprises the steps: (1) Dissolving the nucleic acid sample to be detected and the reference nucleic acid sample in the buffer respectively to obtain the nucleic acid sample solution A to be detected and the reference nucleic acid sample solution B; after dissolving the compound shown in the formula I with an organic solvent, use The buffer solution is diluted to obtain detection solution C; (2) Mix the nucleic acid sample solution A to be detected with the detection solution C to obtain a mixed solution 1, mix the reference nucleic acid sample solution B with the detection solution C to obtain a mixed solution 2, and then mix the obtained mixed solution 1 and The solution 2 is incubated respectively to obtain the mixed solution 1 after incubation and the mixed solution 2 after incubation; then the mixed solution 1 after incubation and the mixed solution 2 after incubation are subjected to the following a) or b): a) Perform UV-Visible absorption spectrum analysis on the mixed solution 1 after incubation and Mixed solution 2 after incubation, and compare the UV-visible absorption spectrum of the compound shown in Formula I in the mixed solution 1 after incubation with the incubation The ultraviolet-visible absorption spectrum of the compound shown in formula I in post-mixing solution 2 is compared, thereby judges whether described nucleic acid sample to be detected is the nucleic acid of G-quadruplex structure; or b) Analyze the fluorescence spectrum of the mixed solution 1 after incubation and the mixed solution 2 after incubation, and compare the fluorescence spectrum of the compound shown in formula I in the mixed solution 1 after incubation with that of the compound shown in the mixed solution 2 after incubation. The fluorescence spectra of the compounds shown in formula I are compared to determine whether the nucleic acid sample to be detected is a nucleic acid with a G-quadruplex structure. 7. method according to claim 6, is characterized in that: in described method step (1), described nucleic acid sample to be detected is the nucleic acid of G-quadruplex structure; The reference nucleic acid sample is a nucleic acid with a non-G-quadruplex structure; The buffer is Tris-HCl buffer or phosphate buffer; The pH value of the buffer is 6-8; For the nucleic acid sample solution A to be detected, the molar concentration of the nucleic acid sample to be detected is 0.25 μM-60 μM; In the reference nucleic acid sample solution B, the molar concentration of the reference nucleic acid sample is 0.25 μM-60 μM; In the detection solution C, the molar concentration of the compound represented by formula I is 0.5 μM-20 μM. 8. The method according to claim 6 or 7, characterized in that: in the method step (2), in the mixed solution 1, the molar ratio of the nucleic acid sample to be detected to the compound shown in formula I is 0.125 -6; In the mixed solution 2, the molar ratio of the reference nucleic acid sample to the compound shown in formula I is 0.125-6; In said a), when it is observed that the ultraviolet-visible absorption spectrum of the compound represented by formula I in the mixed solution 1 after the incubation is significantly enhanced in the range of 310-550 nm, and a new absorption peak appears in the range of 470-550 nm , the nucleic acid sample to be detected is confirmed to be a nucleic acid with a G-quadruplex structure; otherwise, it is a nucleic acid with a non-G-quadruplex structure; In b), when it is observed that the fluorescence emission spectrum of the compound represented by formula I in the mixed solution 1 after incubation has a fluorescence peak in the range of 450-630 nm and the fluorescence intensity increases, which is higher than that of the mixed solution 2 after incubation. If the fluorescence intensity of the fluorescence emission spectrum of the compound shown in formula I appears within the range of 450-630nm, the nucleic acid sample to be detected is confirmed to be a nucleic acid with a G-quadruplex structure; otherwise, it is a nucleic acid with a non-G-quadruplex structure. nucleic acid. 9. The application of the compound described in claim 1 or 2 in the following aspects: 1) Application in the preparation of eukaryotic tumor cell proliferation inhibitors; 2) Application in the preparation of drugs for the prevention and/or treatment of tumors; The eukaryote is a mammal; the tumor cell is a cancer cell; the cancer cell is a lung cancer cell, a breast cancer cell or a prostate cancer cell; The lung adenocarcinoma cells are drug-resistant lung cancer cells A549T; The breast cancer cells are human breast cancer cells MCF-7; The lung cancer cells are human lung cancer cells A549; The prostate cancer cells are human prostate cancer cells PC-3; said tumor is carcinoma; The cancer is lung cancer, breast cancer or prostate cancer. 10. A product whose active ingredient is the compound according to claim 1 or 2, wherein the product is: 1) an inhibitor of eukaryotic tumor cell proliferation; 2) a drug for preventing and/or treating tumors.