4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivatives, their preparation methods and applications

Abstract

The application discloses a 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl) aniline derivative and a preparation method and application thereof, and belongs to the technical field of biological medicines. A structural formula is shown in the following formula I: wherein the R substituent is selected from any one of hydrogen, halogen, a carboxyl group, a cyano group, a nitro group and an alkoxy group. The application takes 4-position substituted o-phenylenediamine and 4-[bis(beta-chloroethyl)amino]benzaldehyde as raw materials, and obtains a target molecule through a condensation reaction. At present, similar nitrogen mustard or benzimidazole drug synthesis needs multiple steps of reaction, and separation and purification is complicated. The application has only one step of synthesis, a simple separation method and low requirement on equipment. In vitro anti-tumor experiments show that the target compounds synthesized by the application all have obvious anti-tumor effects, exhibit strong pharmacological activities in inhibiting tumor cell proliferation, and have wide research and development value in the field of anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to 4-(1H-benzis[d]imidazole-2-yl)-N,N-bis(2-chloroethyl)aniline derivatives, their preparation methods and applications. Background Technology

[0002] Nitrogen mustard is one of the earliest drugs used clinically for cancer treatment. Its pharmacologically active functional group is the alkylated portion; for example, the active group of aromatic nitrogen mustard is N,N-bis(2-chloroethyl)aniline. Currently marketed aromatic nitrogen mustard drugs include chlorambucil. Its mechanism of action involves reacting with DNA, RNA, or enzymes in cells, forming covalent bonds that inactivate these macromolecules, thus inhibiting cell replication and achieving anti-tumor effects. Besides the alkylated portion, the carrier portion of nitrogen mustard drugs primarily affects the drug's physicochemical properties, as well as its pharmacokinetic properties such as absorption and distribution in vivo. By selecting different carriers, it is possible to improve drug selectivity and efficacy while reducing toxicity. Structural modifications of nitrogen mustard compounds are mainly concentrated on the carrier portion; therefore, it is still necessary to develop novel nitrogen mustard drugs and obtain structure-activity relationships between different carriers, providing an important theoretical basis for developing highly effective and low-toxicity nitrogen mustard anti-tumor drugs. Summary of the Invention

[0003] The purpose of this invention is to provide 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivatives, their preparation methods and applications, in order to solve the problems existing in the prior art.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] One of the technical solutions of this invention is a 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative, the structural formula of which is shown in Formula I:

[0006]

[0007] The R substituent is selected from any one of hydrogen, halogen, carboxyl, cyano, nitro and alkoxy.

[0008] The second technical solution of the present invention, the method for preparing the 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative, includes the following reaction:

[0009]

[0010] The process includes the following steps: adding 4-substituted o-phenylenediamine and 4-[bis(β-chloroethyl)amino]benzaldehyde to a solvent, heating the reaction, and then recrystallizing the reaction product to obtain the 4-(1H-benzis[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative.

[0011] The third technical solution of the present invention is the application of the 4-(1H-benzis[d]imidazole-2-yl)-N,N-bis(2-chloroethyl)aniline derivative in the preparation of antitumor drugs, wherein the tumor is cervical cancer or non-small cell lung cancer.

[0012] The fourth technical solution of the present invention is an anti-tumor drug comprising the 4-(1H-benzimidazole-2-yl)-N,N-bis(2-chloroethyl)aniline derivative.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] This invention uses 4-substituted o-phenylenediamine and 4-[bis(β-chloroethyl)amino]benzaldehyde as raw materials to obtain the target molecule through a condensation reaction. Currently, the synthesis of similar nitrogen mustard or benzimidazole drugs requires multiple steps and cumbersome separation and purification. This invention requires only one step in synthesis, with a simple separation method and low equipment requirements. In vitro antitumor experiments show that the target compounds synthesized in this invention all have significant antitumor effects, exhibiting strong pharmacological activity in inhibiting tumor cell proliferation, and have broad research and development value in the field of antitumor drugs. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The figure shows the experimental results of the anti-proliferative activity of product 1a prepared in Example 1 of the present invention against A549 cells and HeLa cells.

[0017] Figure 2 The figure shows the experimental results of the anti-proliferative activity of product 1b prepared in Example 2 of the present invention against A549 cells and HeLa cells.

[0018] Figure 3 The figure shows the experimental results of the anti-proliferative activity of product 1c prepared in Example 3 of the present invention against A549 cells and HeLa cells.

[0019] Figure 4The figure shows the experimental results of the anti-proliferative activity of the product prepared in Example 4 of this invention against A549 cells and HeLa cells after 1 day.

[0020] Figure 5 The figure shows the experimental results of the anti-proliferative activity of product 1e prepared in Example 5 of the present invention against A549 cells and HeLa cells.

[0021] Figure 6 The figure shows the experimental results of the antiproliferative activity of product 1f prepared in Example 6 of the present invention against A549 cells and HeLa cells.

[0022] Figure 7 The figure shows the experimental results of the anti-proliferative activity of 1g of the product prepared in Example 7 of this invention against A549 cells and HeLa cells.

[0023] Figure 8 The figure shows the experimental results of the anti-proliferative activity of the product prepared in Example 8 of this invention on A549 cells and HeLa cells after 1 hour. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0030] This invention provides a 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative, the structural formula of which is shown in Formula I:

[0031]

[0032] The R substituent is selected from any one of hydrogen, halogen, carboxyl, cyano, nitro and alkoxy.

[0033] In some specific embodiments, the 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative is:

[0034]

[0035] This invention also provides a method for preparing the 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative, comprising the following reaction:

[0036]

[0037] The process includes the following steps: adding 4-substituted o-phenylenediamine and 4-[bis(β-chloroethyl)amino]benzaldehyde to a solvent, heating the reaction, and then recrystallizing the reaction product to obtain the 4-(1H-benzis[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative.

[0038] In some specific embodiments, the molar ratio of the 4-substituted o-phenylenediamine to 4-[bis(β-chloroethyl)amino]benzaldehyde is 1:1.

[0039] In some specific embodiments, the solvent is selected from at least one of ethanol, propanol, N,N-dimethylformamide (DMF), and N,N-dimethylacetamide.

[0040] In some specific implementations, the heating reaction is carried out under the catalysis of a catalyst, at a reaction temperature of 110°C to 150°C, and for a reaction time of 4 to 10 hours.

[0041] In some specific embodiments, the catalyst is selected from at least one of acetic acid, propionic acid, and p-toluenesulfonic acid.

[0042] In some specific implementations, the solvent for recrystallization is: dichloromethane:methanol = 1:3 (V / V).

[0043] The present invention also provides the application of the 4-(1H-benzo[d]imidazole-2-yl)-N,N-bis(2-chloroethyl)aniline derivative in the preparation of antitumor drugs, wherein the tumor is cervical cancer or non-small cell lung cancer.

[0044] This invention also provides an antitumor drug comprising the 4-(1H-benzis[d]imidazole-2-yl)-N,N-bis(2-chloroethyl)aniline derivative.

[0045] In some specific implementation plans, pharmaceutically acceptable excipients are also included.

[0046] Benzimidazole compounds, due to their unique chemical structures, exhibit unique biological activities and pharmacological effects, playing an important role in antitumor, antiviral, and antibacterial activities. Introducing different functional groups or substituents onto the imidazole ring can regulate their physicochemical properties and physiological activities. This invention synthesizes a 4-(1H-benzimidazole-2-yl)-N,N-bis(2-chloroethyl)aniline derivative by combining two functional groups: 4-substituted benzimidazole and N,N-bis(2-chloroethyl)aniline.

[0047] This invention utilizes the combination of benzimidazole and nitrogen mustard functional groups to synthesize novel derivatives. The synthetic method is simple and easy to perform, and the separation is straightforward. The prepared target molecules all exhibit excellent antitumor activity, demonstrating strong pharmacological activity in inhibiting tumor cell proliferation.

[0048] Example 1

[0049] The structural formula of compound 1a is as follows:

[0050]

[0051] Preparation of compound 1a:

[0052] o-Phenylenediamine (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give compound 1a.

[0053] Characterization data: 1H NMR (600MHz, DMSO-d6) δ: 12.66 (s, 1H, NH), 8.01 (d, 2H, J = 9.01Hz, ArH), 7.54-7.49 (m, 2H, ArH); 7.16-7.12 (m, 2H, ArH); 6.91 (d, 2H, J = 9.01Hz, ArH), 3.85-3.77 (m, 8H, CH2). 13 C NMR(150MHz,DMSO-d6)δ:151.81,147.73,127.97,121.53,118.23,111.82,51.88,41.09.HR-MS(ESI)calcd for C 17 H 18 Cl2N3[M+H] + 334.0878, found334.0881.

[0054] Example 2

[0055] The structural formula of compound 1b is as follows:

[0056]

[0057] Preparation of compound 1b:

[0058] 4-Methoxy-o-phenylenediamine (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give compound 1b.

[0059] Characterization data: 1 H NMR (600MHz, DMSO-d6) δ: 8.02 (d, 2H, J = 8.98, ArH), 7.48 (d, 1H, J = 8.78Hz, ArH), 7.05 (d, 1H, J = 2.01Hz, ArH ), 6.95 (d, 2H, J = 8.98, ArH), 6.88 (1H, dd, J = 8.78, 2.01Hz, ArH), 3.84-3.77 (overlapped, 11H, OCH3, CH2). 13 C NMR(150MHz,DMSO-d6)δ:154.76,151.45,149.39,126.01,111.99,56.91,51.79,41.09.R-MS(ESI)calcd forC 18 H 20 Cl2N3O[M+H]+ 364.0983, found 364.0986.

[0060] Example 3

[0061] The structural formula of compound 1c is as follows:

[0062]

[0063] Preparation of compound 1c:

[0064] 4-Nitro-o-phenylenediamine (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give compound 1c.

[0065] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ:13.32(s,1H,ArH),8.34(s,1H,ArH),8.07-8.01(overlapped,3 H, ArH), 7.65 (d, 1H, J = 8.85Hz, ArH), 6.93 (d, 2H, J = 9.00Hz, ArH), 3.84-3.73 (m, 8H, CH2). 13 C NMR(150MHz,DMSO-d6)δ:148.67,128.62,117.60,116.90,111.95,51.80,41.04.R-MS(ESI)calcd for C 17 H 17 Cl2N4O2[M+H] + 379.0729, found 379.0730.

[0066] Example 4

[0067] The structural formula of compound 1d is as follows:

[0068]

[0069] Preparation of compound 1d:

[0070] 4-Fluorophenylenediamine (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give compound 1d.

[0071] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ:12.70(d,1H,J=7.41,NH),7.98-7.95(m,2H,ArH),7.55-7.39(m,1H,ArH ),7.27(m,1H,ArH),6.99-6.92(m,1H,ArH),6.88(d,2H,J=8.96Hz,ArH),3.82-3.74(m,8H,CH2). 13 C NMR(150MHz,DMSO-d6)δ:153.74,147.91,136.93,131.66,128.00,111.83,111.14,103.64,97.09,51.86,41.07.R-MS(ESI)calcd forC 17 H 17 Cl2FN3[M+H] + 352.0784, found 352.0781.

[0072] Example 5

[0073] The structural formula of compound 1e is as follows:

[0074]

[0075] Preparation of compound 1e:

[0076] 4-Chloro-o-phenylenediamine (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give compound 1e.

[0077] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ:12.62(s,1H,NH),8.00(d,2H,J=8.98Hz,ArH),7.55(s,1H,ArH),7.51(d,1H,J =8.66Hz, ArH), 7.15 (dd, 1H, J = 8.66, 2.02Hz, ArH), 6.92 (d, 2H, J = 8.98Hz, ArH), 3.84-3.77 (m, 8H, CH2). 13C NMR(150MHz,DMSO-d6)δ:153.27,148.04,128.14,125.80,121.67,125.80,121.67,117.64,111.85,51.84,41.07.R-MS(ESI)calcd for C 17 H 17 Cl3N3[M+H] + 368.0488, found 368.0485.

[0078] Example 6

[0079] The structural formula of compound 1f is as follows:

[0080]

[0081] Preparation of compound 1f:

[0082] 4-Bromo-o-phenylenediamine (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give compound 1f.

[0083] Characterization data: 1 H NMR (600MHz, DMSO-d6) δ: 7.93 (d, 2H, J = 9.01Hz, ArH), 7.62 (s, 1H, ArH), 7.40 (d, 1H, J = 8.60Hz, ArH), 7.20 (dd, 1H, J = 8.60, 1.50Hz, ArH), 6.85 (d, 2H, J = 9.01Hz, ArH), 3.78-3.70 (m, 8H, CH2). 13 C NMR(150MHz,DMSO-d6)δ:153.25,148.02,128.17,124.23,117.83,113.56,111.88,51.89,41.12.R-MS(ESI)calcd for C 17 H 17 Cl3N3[M+H] + 368.0488, found 368.0485.

[0084] Example 7

[0085] The structural formula of 1g of compound is as follows:

[0086]

[0087] Preparation of 1g of compound:

[0088] 3,4-Diaminobenzonitrile (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to give 1 g of the compound.

[0089] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ:8.07-8.04(overlapped,3H,ArH),7.69(d,1H,J=8.38Hz,ArH), 7.58(dd,1H,J=8.38,1.50Hz,ArH),6.95(d,2H,J=9.03Hz,ArH),3.86-3.77(m,8H,CH2). 13 C NMR(150MHz,DMSO-d6)δ:154.61,149.48,130.03,127.51,120.09,114.79,110.79,102.08,49.97,41.11.R-MS(ESI)calcd for C 18 H 17 Cl2N4[M+H] + 359.0830, found 359.0828.

[0090] Example 8

[0091] The structural formula of compound 1h is as follows:

[0092]

[0093] Preparation of compound 1h:

[0094] 3,4-Diaminobenzoic acid (1 mmol) and 4-[bis(β-chloroethyl)amino]benzaldehyde (1 mmol) were added to DMF (20 mL), followed by the addition of propionic acid (20 μL). The mixture was heated at 120 °C for 8 hours. The solvent was then removed, the mixture was washed with water, freeze-dried, and the crude product was recrystallized from dichloromethane:methanol = 1:3 (V / V) to obtain the compound.

[0095] Characterization data: 1H NMR (600MHz, DMSO-d6) δ: 12.73 (s, 1H, NH), 8.01 (d, 2H, J = 8.98Hz, ArH), 7.98 (d, 1H, J =8.18Hz, ArH), 7.39 (s, 1H, ArH), 6.89 (d, 2H, J = 8.98Hz, ArH), 3.85-3.76 (m, 8H, CH2). 13 C NMR(150MHz,DMSO-d6)δ:147.55,127.95,118.76,111.78,51.92,41.12.R-MS(ESI)calcd for C 18 H 18 Cl2N3O2[M+H] + 378.0776, found 378.0779.

[0096] Example of effect

[0097] In vitro antiproliferative activity:

[0098] When the tumor cells (A549 and HeLa) are in the logarithmic growth phase, seed approximately 2 × 10⁶ cells / well in a 96-well culture dish. 3 After 24 hours of cell adhesion, add 100 μM of medium containing 10% FBS to each well of the plate containing different concentrations of the drug, and incubate again at 37°C. After 72 hours of cell culture, discard the medium in the 96-well plate, add 100 μL of CCK8 reagent to each well, and continue incubation for 2 hours. Read the OD of each well using a microplate reader. 450 Numerical values ​​were used to calculate changes in cell activity after treatment with different concentrations of drugs.

[0099] The antiproliferative activity of the products 1a-1h prepared in Examples 1-8 against A549 and HeLa cells are shown in the following results. Figures 1-8 As shown in the figure, the ability to inhibit the proliferation of A549 and HeLa cells continuously increased with increasing compound concentration, and compounds 1a–1h all exhibited good antitumor activity. Among them, compounds 1a–1h generally showed slightly stronger antiproliferative activity against HeLa cells than against A549 cells. The antitumor proliferative activities of different compounds also varied slightly, with compound 1g exhibiting the strongest activity at a half-maximal inhibitory concentration (IC50) against A549 cells. 50The half-maximal inhibitory concentration (WMC) for A549 cells was 21.3 μM, with a half-maximal inhibitory concentration (WMC) of 15.8 μM against HeLa cells. The activity of 1d was second best, close to that of 1g, with WMCs of 23.3 μM and 16.1 μM against A549 and HeLa cells, respectively. Next, the activities of 1h, 1b, and 1c were similar. 1a showed relatively weaker activity, but still exhibited significant anti-proliferative activity against tumor cells, with WMCs of 47.2 μM and 36.7 μM against A549 and HeLa cells, respectively.

[0100] The structure-activity relationship above shows that when the imidazole ring is substituted with a halogen at position 5, all exhibit good activity, with fluorine showing slightly greater activity than chlorine and bromine. Furthermore, the activities of fluorine and cyano groups are similar. When there are no substituents on the imidazole ring, the activity is relatively weaker. These results indicate that the synthesized 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative possesses significant antitumor activity.

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A process for the preparation of a 4-(1 H-benzo[d]imidazol-2-yl)-N,N- bis(2-chloroethyl)aniline derivative, characterized in that, The 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative is: A preparation method of the 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative comprises the following reaction: The method comprises the following steps: adding 4-position substituted o-phenylenediamine and 4-[bis(β-chloroethyl)amino]benzaldehyde into a solvent, heating the reaction, and recrystallizing the reaction product to obtain the 4-(1H-benzo[d]imidazol-2-yl)-N,N-bis(2-chloroethyl)aniline derivative; The heating reaction is performed under catalysis of a catalyst, the reaction temperature is 110-150 DEG C, and the reaction time is 4-10 hours; The catalyst is at least one selected from acetic acid, propionic acid and p-toluenesulfonic acid; The recrystallization solvent is dichloromethane:methanol=1:3 (V / V).

2. The production method according to claim 1, characterized by, The molar ratio of the 4-position substituted o-phenylenediamine and the 4-[bis(β-chloroethyl)amino]benzaldehyde is 1:

1.

3. The production method according to claim 1, characterized by, The solvent is at least one selected from ethanol, propanol, N,N-dimethylformamide and N,N-dimethylacetamide.

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