BENZIMIDAZOLE DERIVATIVE, PREPARATION METHOD THEREFOR, AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to and claims the benefit of PCT/CN2023/142436 filed December 27, 2023, which is incorporated herein by reference.
TECHNICAL FIELDThe present application relates to the technical field of benzimidazole derivatives, in particular to a benzimidazole derivative, a preparation method therefor, and a use thereof.
BACKGROUNDBenzimidazole derivatives have a wide range of biological activities, enabling them to be a remarkable class of substances in biotechnological and pharmaceutical researches and applications. Researches have shown that a strong non-covalent interaction exists between benzimidazole compounds and the minor groove of DNA rich in A-T sequence, allowing the benzimidazole compounds to show great potential in fields such as antibacterial, anticancer, antiparasitic, and antiviral applications. However, there are limitations to traditional benzimidazole compounds in their applications in the field of biotechnology due to unfavorable factors such as cytotoxicity, and mutagenicity.
Fluorescence imaging is increasingly becoming an indispensable technique in the field of preclinical biomedical research. This technique has a number of advantages, such as cost-effectiveness, ease of implementation, enhanced sensitivity, and minimal optical damage to biological samples. A large Stokes shift (Δλ > 120 nm ) is a key indicator of a high-performance fluorescent dye, as a narrow Stokes shift often causes spectral crosstalk through excitation-emission overlap, thereby reducing the signal-to-background ratio and hindering high-resolution fluorescence imaging. However, in the study of DNA fluorescent dyes, the Stokes shift of most existing DNA fluorescent dyes is very small.
Accordingly, the endeavor to enhance the Stokes shift in DNA fluorescent dyes has become a primary focus of research, presenting a significant challenge in this field.
SUMMARY OF THE INVENTIONAn object of the present application is to provide a novel benzimidazole derivative, a preparation method therefor, and a use thereof.
In order to achieve the above object, the present application adopts technical solutions as follows:
One aspect of the present application discloses a benzimidazole derivative. The benzimidazole derivative comprises a thiophene-benzimidazole structure, and is a compound represented by structural formula I:
structural formula I
where n is an integer of 0-3, R1 is a C1-6 alkyl group, and R2 is selected from at least one of hydrogen, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, a C1-6 alkylamino group, a C1-4 haloalkyl group, and a substituted or unsubstituted pyrazole ring.
When n is 0, the thiophene ring and the pyrazine ring is connected to the same benzimidazole unit; when n is 1, a dibenzimidazole is comprised, with the thiophene ring being connected to one benzimidazole unit and the pyrazine ring being connected to the other benzimidazole unit; when n is 2, a tribenzimidazaole is comprised; and when n is 3, a tetrabenzimidazole is comprised. By substituted pyrazole ring is meant that the hydrogen at any position of the pyrazole ring is substituted by a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkoxy group, a structure represented by structural formula III, a structure represented by structural formula IV, a structure represented by structural formula V, or a structure represented by structural formula VI.
It is to note that the benzimidazole derivative of the present application has a thiophene ring connected to the benzimidazole ring, thereby having a thiophene-benzimidazole structure. It has been found in the research of the present application that such a modification enables a larger Stokes shift to be exhibited. Moreover, the benzimidazole derivative shows no obvious genotoxicity to cells, which reduces induction of gene mutation, allowing it to be better used for DNA staining.
It is appreciated that a key of the present application lies in using the thiophene-benzimidazole structure to expand the Stokes shift of the benzimidazole derivative. In principle, substitutions can be made at any positions, including R1 and R2, using groups conventionally used in the art, so long as the thiophene-benzimidazole structure of the benzimidazole derivative of the present application is not affected.
In one embodiment of the present application, the substituted or unsubstituted pyrazole ring is formed from a reaction of an azide compound.
In one embodiment of the present application, the substituted or unsubstituted pyrazole ring is a structure represented by structural formula II:
structural formula II
where R3 is hydrogen, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkoxy group, a structure represented by structural formula III, a structure represented by structural formula IV, a structure represented by structural formula V, or a structure represented by structural formula VI;
structural formula III
structural formula IV
structural formula V
structural formula VI
It is to note that the case where R2 is a substituted or unsubstituted pyrazole ring is equivalent to further connecting a pyrazole ring on the basis of connecting a thiophene ring. It has been further found in the research of the present application that addition of the pyrazole ring causes the benzimidazole derivative of the present application to not only exhibit no mutagenic activity in a gene mutation test, but also show no obvious influence on cell cycle distribution. This further increases the safety of the benzimidazole derivative of the present application, making it a non-toxic substitute in DNA staining applications. It is appreciated that a key of the present application lies in further connecting a pyrazole ring on the basis of connecting a thiophene ring. Therefore, in addition to the specific structures of R3 shown above, other similars, such as any pyrazole rings formed from various azide compounds, may be suitable for the present application.
Another aspect of the present application discloses a use of the benzimidazole derivative of the present application in the preparation of a DNA fluorescent dye.
Yet another aspect of the present application discloses a DNA fluorescent dye, which comprises the benzimidazole derivative of the present application.
It is to note that a key of the benzimidazole derivative of the present application lie in not inducing cytotoxicity, not generating a mutagenic effect, and having a larger Stokes shift. These features enable the benzimidazole derivative of the present application to be better used in a DNA fluorescent dye, solving problems, such as high cytotoxicity, associated with existing DNA fluorescent dyes, and allowing it to be better applied in human medicine.
Still another aspect of the present application discloses a preparation method for preparing the benzimidazole derivative of the present application, comprising reacting compound 5 with o-phenylenediamine or o-phenylenediamine having at least one benzimidazole to obtain the benzimidazole derivative comprising a thiophene-benzimidazole structure:
compound 5
It is to note that reacting compound 5 directly with o-phenylenediamine will obtain the benzimidazole derivative comprising a monobenzimidazole ring connected to the thiophene ring; reacting compound 5 with o-phenylenediamine having one benzimidazole will obtain the benzimidazole derivative comprising a dibenzimidazole ring and having a thiophene-benzimidazole structure; and by parity of reasoning, reacting compound 5 with o-phenylenediamine having two benzimidazole will obtain the benzimidazole derivative comprising a tribenzimidazole ring and having a thiophene-benzimidazole structure.
In an embodiment of the present application, the benzimidazole comprises a piperazine ring.
It is to note that reacting compound 5 with o-phenylenediamine having at least one benzimidazole, wherein the benzimidazole comprises a piperazine ring, will obtain the benzimidazole derivative of the present application comprising a pyrazine ring and a thiophene ring at the same time.
It is to further note that compound 5, o-phenylenediamine, o-phenylenediamine having one benzimidazole, o-phenylenediamine having two benzimidazole, o-phenylenediamine having three benzimidazole, and those o-phenylenediamine compounds where the benzimidazole comprises a piperazine ring can all be prepared by using existing techniques or directly purchased. A key of the present application lies in that, as found by research, reacting compound 5 with o-phenylenediamine or o-phenylenediamine having at least one benzimidazole can obtain the benzimidazole derivative of the present application which has a larger Stokes shift and is more suitable for use in a DNA dye.
In one embodiment of the present application, the preparation method of the present application further comprises reacting the benzimidazole derivative having a thiophene-benzimidazole structure with an azide compound to obtain a benzimidazole derivative having a pyrazole ring.
Still yet another aspect of the present application discloses a method for modifying a benzimidazole derivative, comprising connecting a thiophene ring to a benzimidazole ring of the benzimidazole derivative, such that the Stokes shift of the benzimidazole derivative is expanded by means of the resulting thiophene-benzimidazole structure.
It is to note that a key of the present application lies in that, as found by research, a benzimidazole derivative having a thiophene-benzimidazole structure has a larger Stokes shift. Therefore, a benzimidazole derivative can be modified by connecting a thiophene ring to a benzimidazole ring, such that the modified benzimidazole derivative has a larger Stokes shift.
In an embodiment of the present application, the method for modifying a benzimidazole derivative further comprises connecting a pyrazine ring to the benzimidazole ring of the benzimidazole derivative, and/or connecting a substituted or unsubstituted pyrazole ring on the thiophene ring. By substituted pyrazole ring is meant that the hydrogen at any position of the pyrazole ring is substituted by a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkoxy group, a structure represented by structural formula III, a structure represented by structural formula IV, a structure represented by structural formula V, or a structure represented by structural formula VI.
It is to note that it has been further found in the research of the present application that addition of the pyrazole ring causes the benzimidazole derivative of the present application to not only exhibit no mutagenic activity in a gene mutation test, but also show no obvious influence on cell cycle distribution. Therefore, a substituted or unsubstituted pyrazole ring can be connected to the thiophene ring such that the modified benzimidazole derivative can better be used in a DNA fluorescent dye.
By adopting the above technical solutions, the present application has the following beneficial effects:
The benzimidazole derivative of the present application has a larger Stokes shift thanks to the thiophene-benzimidazole structure. Moreover, the benzimidazole derivative of the present application shows no obvious genotoxicity to cells, which reduces induction of gene mutation, allowing it to be better used for DNA staining.
BRIEF DESCRIPTION OF THE DRAWINGSFigs. 1-6 sequentially show the absorption spectra of the benzimidazole derivatives Benthio Green-0 to Benthio Green-5 synthesized in the examples of the present application;
Figs. 7-12 sequentially show the emission spectra of the benzimidazole derivatives Benthio Green-0 to Benthio Green-5 synthesized in the examples of the present application;
Figs. 13-18 sequentially show the results of DNA staining tests of the benzimidazole derivatives Benthio Green-0 to Benthio Green-5 synthesized in the examples of the present application; and
Fig. 19 shows the density functional calculations of the benzimidazole derivatives Benthio Green-0 to Benthio Green-5 synthesized in the examples of the present application.
DETAILED DESCRIPTION OF THE INVENTIONIn an aim to address the problem of small Stokes shift associated with existing benzimidazole derivative-based DNA fluorescent dyes, a benzimidazole derivative having a large Stokes shift (135 nm to 143 nm) , useful as a DNA fluorescent dye, has been successfully developed in the present application by introducing a thiophene-benzimidazole structure. The benzimidazole derivative can effectively decrease the genotoxicity associated with such class of compounds and reduce induction of gene mutation. This technological innovation significantly enhances the optical and photochemical properties of benzimidazole derivatives, and can expand their application scope in DNA fluorescence imaging.
In embodiments of the present application, the novel benzimidazole derivative, prepared and designated as Benthio Green-0 to Benthio Green-5, exhibits high selectivity and affinity to DNA rich in A-T sequence, offering new possibility for DNA fluorescence labeling techniques.
The present application provides a new solution to solving the problem of small Stokes shift associated with existing DNA probes while reducing the risks of mutagenicity, carcinogenesis, and teratogenicity, thus showing the prospect of broad applications in the field of fluorescent dyes and in biomedical research. Overall, the present application has made innovative progress not only in the development of DNA fluorescent dyes having a large Stokes shift, but also in the safety of the DNA fluorescent dyes.
The present application will be further illustrated below by detailed description of embodiments with reference to the accompanying drawings. In the following embodiments, many details are described so that the present application will be better understood. However, those skilled in the art can readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods, depending on different situations. In some cases, some operations related to the present application are not shown or described in this specification, so as to avoid overwhelming the core part of the present application with excessive description. Detailed description of these relevant operations is not necessary for those skilled in the art, who can have a complete knowledge of the relevant operations in light of the disclosure in the description and the general technical knowledge in the art.
Example 1: Synthesis of compound Benthio Green-0
The benzimidazole derivative Benthio Green-0 of the present example can be directly prepared by reacting compound 5 with o-phenylenediamine having one benzimidazole. Compound 5 can be directly obtained from the prior art (Boese et al. J. Am. Chem. Soc. 1994, 116, 11153-11154) . However, for the convenience of use, a novel synthesis method for compound 5 was developed in the present example. Therefore, the complete synthesis route for the benzimidazole derivative Benthio Green-0 of the present example, including the synthesis of compound 5, is as follows:
The specific steps are as follows:
For the above synthesis route, the specific synthesis steps and process condition details are described as follows:
(1) At room temperature, 2, 5-thiophene dicarboxylate (1.72 g, 10 mmol) and anhydrous ethanol (50 mL) were added sequentially to a reaction flask and magnetically stirred. Then, sulfuric acid fuming (5.88 g, 60 mmol) was slowly added dropwise to the solution. After the dropwise addition was complete, the mixed reaction solution was heated to reflux, and reaction was allowed to proceed for 8 hours. The reaction solution was cooled to room temperature, and 100 mL of saturated sodium bicarbonate solution was added dropwise to the reaction solution. Ethanol was removed from the solution by distillation under vacuum. The reaction was extracted using ethyl acetate (100 mL × 3) . The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated, affording a crude product. Isolation and purification was performed by column chromatography, affording compound 2 as a white solid (2.0 g, yield 88%) .
(2) At room temperature, compound 2 (2.0 g, 8.8 mmol) and anhydrous ethanol (20 mL) were added to a reaction flask and stirred magnetically. Sodium borohydride (432.8 mg, 11.5 mmol) was added to the above reaction solution in five batches. Then, the reaction solution was heated from room temperature to 80℃, and reaction was allowed to proceed at the constant temperature for 1 hour. After the reaction was completed, the solvent was removed by distillation under vacuum. Isolation and purification was performed by column chromatography, affording compound 3 as a light yellow oily liquid (1.1 g, yield 68%) .
(3) At 0℃, NaH (240 mg, 60wt%, 6 mmol) and anhydrous DMF (5 mL) were added to a reaction flask, and stirred magnetically. Compound 3 (931 mg, 5 mmol) was dissolved in anhydrous DMF (10 mL) . The resulting solution was slowly added dropwise to the above NaH/DMF solution, and stirring was continued for 30 minutes. 3-bromopropyl (892 mg, 7.5 mmol) was dissolved in anhydrous DMF (5 mL) . The resulting solution was added in one batch to the above reaction solution. The reaction mixture was further stirred to carry out reaction at room temperature for 2 hours. After the reaction was completed, a saturated NH4Cl solution (50 mL) was added to quench the reaction. The reaction was extracted with ethyl acetate (100 mL × 3) . The organic phases were combined, dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography, affording compound 4 as a colorless oily liquid (852 mg, yield 76%) .
(4) The above steps were repeated to obtain a sufficient amount of compound 4. At -78℃, compound 4 (900 mg, 22.5 mmol) and dichloromethane (30 mL) were sequentially added to a reaction flask, and stirred magnetically. To the above reaction solution was added dropwise 1.83 mL of DIBAL-H solution (1 M in hexane, 1.83 mmol) , and stirring was continued to carry out reaction for 1 hour. After the reaction was completed, ethyl acetate (3 mL) was added to quench the reaction. A saturated NaCl solution (15 mL) was added, and extraction was performed using DCM (15 mL × 3) . The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated, affording a yellow residue. The obtained yellow residue was redissolved in DCM (10 mL) , and DMP (0.776 g, 1.83 mmol) was added. The reaction mixture was reacted under stirring at room temperature for 2 hours. After the reaction was completed, a saturated NaHCO3 solution (5 mL) and a 15%Na2S2O3 solution (5 mL) were added, and mixing was performed under stirring for 15 minutes. The mixed solution was extracted with DCM (20 mL × 3) . The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. Isolation was performed by column chromatography, affording compound 5 as a yellow oily liquid (0.189 g, 85%) .
(5) The above steps were repeated to obtain a sufficient amount of compound 5. At room temperature, compound 5 (0.9 g, 5 mmol) , 4- (5- (4-methylpiperazin-1-yl) -1H-benzo [d] imidazol-2-yl) benzene-1, 2-diamine (1.61 g, 5 mmol) , and 100 mL of ethanol were added to a reaction flask. The resulting solution was stirred magnetically and heated to reflux, and reaction was allowed to proceed continuously for 6 hours. 4- (5- (4-methylpiperazin-1-yl) -1H-benzo [d] imidazol-2-yl) benzene-1, 2-diamine is compound 6 (For synthesis thereof, reference can be made to the following reference document: Harapanhalli et al. J. Med. Chem. 1996, 39, 4804-4809) . After the reaction was completed, the solvent was removed by distillation under vacuum, affording a brown residue. The residue was redissolved in DCM (50 mL) , and washed with a saturated NaCl solution (50 mL) . The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated, affording a crude product. The crude product was purified by column chromatography, affording Benthio Green-0 as a yellow powder (1.49 g, yield 62%) .
Example 2: Synthesis of compound Benthio Green-1
The synthesis route of the benzimidazole derivative Benthio Green-1 in this example is as follows:
At room temperature, compound Benthio Green-0 (96.5 mg, 0.2 mmol) was dissolved in 10 mL of ethanol, and to the solution was added 21 mg of a palladium-carbon catalyst (10%wt) . Hydrogen gas (about 1 atm) was introduced to the system, the temperature was increased to 60℃, and reaction is allowed to proceed under stirring for 6 hours. After the reaction was completed, the solution was filtered through diatomaceous earth, and the filtrate was collected and concentrated, affording a crude product. The crude product was purified by column chromatography, affording Benthio Green-1 as a yellow powder (96.4 mg, yield 99%) .
Example 3: Synthesis of compound Benthio Green-2
The synthesis route of the benzimidazole derivative Benthio Green-2 in this example is as follows:
At room temperature, compound Benthio Green-0 (96.5 mg, 0.2 mmol) , methyl 2-azidoacetate (27.6 mg, 0.24 mmol) , and cuprous iodide (7.6 mg, 0.04 mmol) were dissolved in 5 mL of methanol (MeOH) , and stirred magnetically for 12 hours. After the reaction was completed, the solvent was removed by distillation under vacuum. Purification was performed by column chromatography, affording compound Benthio Green-2 as a yellow powder (113.4 mg, yield 95%) , corresponding to the synthesis route of Benthio Green-2.
Example 4: Synthesis of compound Benthio Green-3
The synthesis route of the benzimidazole derivative Benthio Green-3 in this example is as follows:
At room temperature, Benthio Green-0 (96.5 mg, 0.2 mmol) , 6-azidohexanoic acid (37.6 mg, 0.24 mmol) , and cuprous iodide (7.6 mg, 0.04 mmol) were dissolved in 5 mL of methanol (MeOH) , and stirred magnetically for 12 hours. After the reaction was completed, the solvent was removed by distillation under vacuum. Purification was performed by column chromatography, affording compound Benthio Green-3 as a yellow powder (110.0 mg, yield 86%) .
Example 5: Synthesis of compound Benthio Green-4
The synthesis route of the benzimidazole derivative Benthio Green-4 in this example is as follows:
At room temperature, Benthio Green-0 (96.5 mg, 0.2 mmol) , 6-azidohexylamine (34.1 mg, 0.24 mmol) , and cuprous iodide (7.6 mg, 0.04 mmol) were dissolved in 5 mL of methanol (MeOH) , and stirred magnetically for 12 hours. After the reaction was completed, the solvent was removed by distillation under vacuum. Purification was performed by column chromatography, affording compound Benthio Green-4 as a yellow powder (123.6 mg, yield 99%) .
Example 6: Synthesis of compound Benthio Green-5
The synthesis route of the benzimidazole derivative Benthio Green-5 in this example is as follows:
At room temperature, Benthio Green-0 (96.5 mg, 0.2 mmol) , 6-azidobutylamine (27.3 mg, 0.24 mmol) , and cuprous iodide (7.6 mg, 0.04 mmol) were dissolved in 5 mL of methanol (MeOH) , and stirred magnetically for 12 hours. After the reaction was completed, the solvent was removed by distillation under vacuum. Purification was performed by column chromatography, affording compound Benthio Green-5 as a yellow powder (113.3 mg, yield 99%) .
Example 7: Performance tests
1. Stokes shift test
The absorption wavelength and emission wavelength of Benthio Green-0 to Benthio Green-5 prepared in the above examples were tested, and the results are shown in Table 1 and Figs. 1-12.
Table 1. Absorption wavelength and emission wavelength of benzimidazole derivatives
The results shown in Table 1 and Figs. 1-12 indicate that the six benzimidazole derivatives Benthio Green-0 to Benthio Green-5 prepared in this study had large Stokes shifts ranging from 133 nm to 143 nm, much larger than the Stokes shift of existing Hoechst 33342.
2. Cytotoxicity test
Ames test was used in this study to analyze the cytotoxicity of the prepared benzimidazole derivatives.
Specifically, a Mini Ames kit (catalog number 02110110, IPHASE) was used to evaluate the mutagenic potential through Ames test. The test comprised evaluating bacterial strains TA97a, TA98, TA100, TA1535, and WP2uvrA-pKM101 using a pre-incubation method in the presence and absence of metabolic activation. Briefly, the experimental protocol required activation of the cryopreserved bacterial cultures under appropriate conditions for an activation duration of 12 hours. Before the experiment, a metabolic activation mixture (S9 mixture, which was an extract of ground rat liver comprising microsomal enzymes as main active components, and was gifted along with the Mini Ames kit) was thawed on ice. The test compounds were dissolved in dimethyl sulfoxide (DMSO, catalog number D2650, Sigma) . With preliminary toxicity assessment having been conducted, a concentration of 5 μg/μL was used in this study. The experimental process involved sequentially adding 10 μL of a test compound, 20 μL of a bacterial culture, and 10 μL of a 10%S9 mixture or S9 blank solution to a preheated top agarose culture medium. After thorough mixing, the mixture was quickly poured onto a bottom agarose culture medium layer. After the top agarose culture medium layer solidified, the plate was inverted and incubated at 37℃ for 24 hours. The experimental design included a positive control (astandard mutagen) , a solvent control, and an untreated control. All experiments were conducted in triplicate, and the results were analyzed using the Prism GraphPad 9.0 software package.
In order to further elucidate the biosafety of the newly developed DNA dye, Ames test was used in this study to evaluate the mutagenic potential of Benthio Green-4 and Hoechst 33342, using bacterial strains TA97a, TA98, TA100, TA1535, and WP2uvrA-pKM101, regardless of S9 metabolic activation. Known mutagens were used as positive controls, including 2-aminobacteria for TA97a, TA98, and TA100, and 2-aminoanthracene for WP2uvrA pKM101 and TA1535. The test results are shown in Table 2. The solvent control showed negligible mutagenic activity. The positive controls showed a high mutation frequency, demonstrating a strong mutagenic activity. For example, in TA98, the frequency of 2-amino bacteria was 633 ± 85 (without S9) and 1455 ± 272 (with S9) . Compared with the positive control group, Benthio Green-4 showed a limited mutagenic activity. In comparison with the solvent control DMOS, the only significant increase was in TA97a, with the frequency under both S9 (-) and S9 (+) conditions being 26 ± 1. In bacterial strains TA98, TA100, TA1535, and WP2uvrA-pKM101, there showed no significant difference between Benthio Green-4 and the solvent control. These results indicate that Benthio Green-4 had a very weak mutagenic potential in the bacterial Ames tests. Most of the results of Benthio Green-4 were equivalent to those of the solvent control, and were significantly lower than those of established mutagens.
Table 2. Evaluation of the Mutagenic Potential of Benthio Green-4 through Ames Test
3. TK forward mutation assay
TK forward mutation assay was performed on L5178Y mouse lymphoma cells treated with HAT. The cells were suspended at 5 × 105 cells/mL, and were treated with 5 mg/mL of Benthio Green-4. Dimethyl sulfoxide (DMSO, 0.02%v/v) and methyl methanesulfonate (MMS, 0.02 mL) were used as a negative control and a positive control, respectively. After shaking the cells at 70 rpm at 37℃ for 3 hours, the cells were washed, and resuspended in a complete culture medium. On day 0 (PE0) and day 2 (PE2) , cloning efficiency (CE) was determined by inoculating diluted cells at 8 cells/mL in a 96 well plate (0.2 mL/well, 1.6 cells/well) . The plate was incubated for 12 days, then the wells without colony growth were counted. After treatment, the cells were resuspended in RPMI-1640 containing 10%horse serum, and cultured for 2 days. Cell density was measured every day to determine suspension growth (SG) , relative suspension growth (RSG) , and relative total growth (RTG) . On the second day, the cells were suspended at 1 × 104 cells/mL using 1: 1000 diluted trifluorothymidine (TFT) , and were plated in a 96-well plate (2000 cells/well) . After 12 days of culture, mutant colonies were counted after MTT staining. Large colony mutation frequency (L-MF) , small colony mutation frequency, and total mutation frequency (T-MF) were calculated.
The potential mutagenic activity of Benthio Green-4 in eukaryotic cells was evaluated using the L5178Y TK+/-mouse lymphoma gene mutation assay, so as to assess the potential mutagenicity of Benthio Green-4 at a maximum soluble dose of 5 mg/mL. Since Hoechst 33342 exhibited a high toxicity at a much lower concentration than that of Benthio Green-4, it was not used as a reference for the TK gene mutation assay. The cells were treated with Benthio Green-4, the positive control CP, or the solvent control DMSO in the presence or absence of the metabolic activation mixture S9 for 3 hours, and then the TK mutation frequency was determined. In the presence of the metabolic activation mixture S9 (+) , Benthio Green-4 treatment resulted in a TK mutation frequency of 51.7 mutations per 106 live cells, which was even lower than the frequency of 95.96 mutations per 106 live cells for the solvent control, as shown in Table 3. In contrast, the mutation frequency induced by the positive control CP was 17 times higher than that of the solvent control, with 1968.4 mutations per 106 live cells. In the absence of the metabolic activation mixture S9 (-) , Benthio Green-4 again did not increase the TK mutation frequency in comparison with the solvent control, with mutation frequencies of 81 and 68 per 106 live cells, respectively. However, the positive control MMS resulted in an increased mutation frequency of 822 mutants per 106 live cells. In summary, these results indicate that under the experimental conditions studied, Benthio Green-4 did not induce mutagenicity in the L5178Y TK gene mutation experiments with or without metabolic activation.
Table 3. Evaluation of Mutagenic Potential of Benthio Green-4 through TK Mutation Assay
The above results show that the benzimidazole derivatives in this study not only had a larger Stokes shift, but also exhibited smaller genotoxicity, suggesting that the benzimidazole derivatives could greatly reduce the induction of gene mutation, and therefore could be better used for DNA staining.
4. DNA staining experiment
In this study, the DNA marker DL8000 was used to test the DNA staining ability of the six benzimidazole derivatives Benthio Green-0 to Benthio Green-5 prepared. Specifically, 2-fold gradient dilution was performed on the DNA marker DL8000 at an initial concentration of 505 ng/μL to obtain diluted DL8000 at concentrations of 252.5 ng/μ L, 126.25 ng/μL, and 63.12 ng/μL, respectively. A 1%agarose gel containing an xGel Bright Blue-S4 nucleic acid stain was prepared in a 1× TAE buffer. 1 μ L of DL8000, 1 μ L of Benthio Green-0 (or any of the other five benzimidazole derivatives prepared in this study) , and 5 μL of a loading buffer were mixed. The mixed solution was loaded into the holes in the gel, and was run on the gel at 130 V for 30 minutes. Using an X-WB imager, the gel was exposed in the xGel Bright Blue mode for 10 seconds for imaging. The results are as shown in Figs. 13-18, which are the electropherograms showing staining of the six benzimidazole derivatives Benthio Green-0 to Benthio Green-5 sequentially. In the figures, the lanes from left to right are the results of staining of 505 ng, 252.5 ng, 126.25 ng, and 63.13 ng DL8000, respectively.
The results in Figs. 13-18 show that the six benzimidazole derivatives Benthio Green-0 to Benthio Green-5 all had a DNA staining effect on different amounts of DL8000. In conventional agarose gel electrophoresis experiments, an original DNA marker is typically used at 1 μ L to 2 μL. That is, the amount of the DNA marker used is typically 500 ng to 1000 ng. In this study, a good staining effect was obtained with the gradiently diluted DNA marker. Thus it can be seen that the six benzimidazole derivatives in this study had a good DNA staining effect and could be well used for DNA staining. Therefore, the six benzimidazole derivatives in this study could fully meet the needs of DNA staining.
Analysis in view of the above experimental results would suggest that in addition to the six benzimidazole derivatives Benthio Green-0 to Benthio Green-5 synthesized in this study, other benzimidazole derivatives having similar structures would also have the same or equivalent properties. For example, the compound represented by structural formula I is as follows:
structural formula I
where n is an integer of 0-3, R1 is a C1-6 alkyl group, and R2 is selected from at least one of hydrogen, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, a C1-6 alkylamino group, a C1-4 haloalkyl group, and a substituted or unsubstituted pyrazole ring.
When n is 1, R1 is -CH3, and R2 is -CH2-CH2-CH3, the compound is Benthio Green-1 synthesized in this study. It is appreciated that replacing the -CH3 of R1 with a similar C1-6 alkyl group on the basis of Benthio Green-1 will not affect and alter the key functional group (s) of Benthio Green-1; therefore, advantages such as a large Stokes shift and a low cytotoxicity also exist. Similarly, replacing the -CH2-CH2-CH3 of R2 of Benthio Green-1 with hydrogen, deuterium, or a similar C1-6 alkyl group, C1-6 alkenyl group, C1-6 alkynyl group, C1-6 alkoxy group, C1-6 alkylamino group, or C1-4 haloalkyl group will not affect and alter the key functional group (s) and properties of Benthio Green-1. In addition, it can be seen from the above analysis that a key of Benthio Green-1 lies in the thiophene-benzimidazole structure and the piperazine ring. On this basis, reducing or increasing the number of the benzimidazole ring, i.e., n=0, or n=2 or 3, will result in properties that are the same as or equivalent to those of Benthio Green-1. When R2 is a substituted or unsubstituted pyrazole ring, the substituted or unsubstituted pyrazole ring is the structure represented by structural formula II:
structural formula II
where R3 is hydrogen, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkoxy group, a structure represented by structural formula III, a structure represented by structural formula IV, a structure represented by structural formula V, or a structure represented by structural formula VI;
structural formula III
structural formula IV
structural formula V
structural formula VI
When R3 is the structure represented by structural formula III, , the compound is Benthio Green-2 in this study; when R3 is the structure represented by structural formula IV, the compound is Benthio Green-3 in this study; when R3 is the structure represented by structural formula V, the compound is Benthio Green-4 in this study; and when R3 is the structure represented by structural formula VI, the compound is Benthio Green-5 in this study. It is appreciated that R3 being hydrogen, i.e., in the case of an unsubstituted pyrazole ring, or R3 being a C1-6 alkyl group, a C1-6 alkenyl group, or a C1-6 alkoxy group will also not affect the key functional group (s) and properties of Benthio Green-2 to Benthio Green-5. Therefore, when R3 is hydrogen, a C1-6 alkyl group, a C1-6 alkenyl group, or a C1-6 alkoxy group, advantages such as a large Stokes shift and a low cytotoxicity also exist.
A person skilled in the art will appreciate that in addition to the above-mentioned groups for replacement for R1, R2, and R3, a simple stable isotope, such as deuterium, will also serve as a replacement that can be reasonably expected.
Example 8: Analysis of the structure and properties of the benzimidazole derivatives
In order to evaluate the effect of a thiophene structure in improving the molecular properties of a DNA fluorescent dye, density functional theory calcullation was used to perform analysis in this study. For the analysis, Hoechst 33342, a known DNA fluorescent dye molecule having a similar molecular structure, was selected as a benchmark for comparison. The results are as shown in Fig. 19.
The results of density functional theory calculation indicate that the addition of a thiophene group would significantly regulate the frontier molecular orbital energy level of a probe molecule. As shown in Fig. 19, in detailed computational analysis, we found that the lowest unoccupied molecular orbital (LUMO) energy level of Hoechst 33342 containing a benzene ring structure was -1.88 eV, while the highest occupied molecular orbital (HOMO) energy level thereof was -4.72 eV. The HOMO was mainly contributed by the benzimidazole moiety and the pyrazine moiety, while the LUMO was mainly contributed by the benzene ring moiety. Further, density functional calculation was conducted on the Benthio Green-1 molecule that we developed, and it was showed that the HOMO energy level contributed by benzimidazole and pyrazine did not change much, while the LUMO significantly decreased to -2.20 eV due to the addition of the thiophene group. This result means that the energy gap of Benthio Green-1 was reduced to 2.55 eV, in significant contrast to the energy gap of 2.84 eV observed with Hoechst 33342. This change led to a significant increase in the Stokes shift of Benthio Green-1. Similar patterns were also observed with the other molecules Benthio Green-2 to Benthio Green-5.
In summary, study has shown that introduction of a thiophene group in the structure of a benzimidazole derivative helps to enhance Stokes shift. It is appreciated that it has been demonstrated in this study that introduction of a thiophene-benzimidazole structure to a benzimidazole derivative can expand the Stokes shift of the benzimidazole derivatives. Therefore, this approach can be used to expand the Stokes shift of a benzimidazole derivative.
The foregoing is further detailed explanation of the present application in conjunction with the specific embodiments, and it cannot be assumed that the specific implementation of the present application is limited to the explanation. For a person of ordinary skill in the art to which the present application pertains, several simple derivations and substitutions may be made without departing from the concept of the present application.