CN117486761A - Aryl-containing chloroethyl nitrosourea compound and preparation method and application thereof - Google Patents

Aryl-containing chloroethyl nitrosourea compound and preparation method and application thereof Download PDF

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CN117486761A
CN117486761A CN202311430690.XA CN202311430690A CN117486761A CN 117486761 A CN117486761 A CN 117486761A CN 202311430690 A CN202311430690 A CN 202311430690A CN 117486761 A CN117486761 A CN 117486761A
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chloroethyl
aryl
preparation
pharmaceutical composition
nitrosourea compound
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习保民
郑多
王霆
刘叔文
阳衡
韩家坤
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Guangzhou Kelang Medical Technology Co ltd
Shenzhen University
Southern Medical University
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Guangzhou Kelang Medical Technology Co ltd
Shenzhen University
Southern Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/66—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to halogen atoms or to nitro or nitroso groups
    • C07C275/68—N-nitroso ureas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C273/00—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C273/18—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas
    • C07C273/1854—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas by reactions not involving the formation of the N-C(O)-N- moiety
    • C07C273/1863—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas by reactions not involving the formation of the N-C(O)-N- moiety from urea
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D319/18—Ethylenedioxybenzenes, not substituted on the hetero ring

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Abstract

The invention belongs to the field of medicines, and provides an aryl-containing chloroethyl nitrosourea compound. The chloroethyl nitrosourea compound containing the aryl has the following structural general formula:wherein R is selected from-CH 3 、‑CH 2 CH 3 Or (b)Ar is selected from benzene, substituted benzene,Or (b)The aryl-containing chloroethyl nitrosourea compound disclosed by the invention has higher capacity of penetrating through brain barrier, fully plays the role of aziridine ions, has excellent anti-glioma cell activity, and simultaneously shows anti-tumor cell activity on various tumors.

Description

Aryl-containing chloroethyl nitrosourea compound and preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to an aryl-containing chloroethyl nitrosourea compound, and a preparation method and application thereof.
Background
Brain gliomas are primary intracranial tumors produced by brain and spinal cord glioblastoma, and world health organization classifies brain gliomas into four classes, i, ii being low-grade gliomas being more benign, iii, iv being high-grade gliomas. Among them, glioblastomas belonging to class iv (glioblastoma multiforme, GBM) are the most common malignant tumors in the central nervous system, which are significantly more aggressive and recurrent than other intracranial tumors, and seriously threaten the life health of patients. The current treatment of GBM is that of,the tumor tissue is firstly removed by maximum surgery, and radiotherapy and chemotherapy are assisted after the surgery [1,2] . Temozolomide (TMZ), a first-line drug for chemotherapy of GBM patients, is an alkylating agent of the imidazotetrazine class, continuing until now since clinical use in 1999.
Nitrosourea drugs can be applied to GBM treatment to a certain extent, for example, lomustine and temozolomide can be combined to improve the survival time of a patient with newly diagnosed glioblastoma [3] . Small clinical trials have shown that the combination combines different degrees of DNA damage or potential additional or even synergistic effects and that no organ toxic response typical of nitrosoureas is observed during the combination. Dimitrios Pletsas [4] The novel compound 3- (2-phenethyl) substituted imidazole tetrazine designed and synthesized by the et al, the biological activity of which comes from an aziridine ion which is an active intermediate formed by in vivo conversion and is mainly used for modifying guanine-N7 sites on DNA, so that the problem of drug resistance of GBM chemotherapeutic drugs can be alleviated to a certain extent [4] . However, the therapeutic effect of 3- (2-phenylethyl) substituted imidazotetrazine on brain gliomas is still not ideal.
Disclosure of Invention
The invention provides chloroethyl nitrosourea compounds containing aryl, which can improve the capacity of a drug penetrating through brain barrier, fully exert the effect of aziridine ions and improve the effect of treating brain glioma.
The chloroethyl nitrosourea compound containing the aryl has the following structural general formula:
wherein R is selected from-CH 3 、-CH 2 CH 3 Or->Ar is selected from benzene, substituted benzene and->
The invention aims to provide a preparation method of chloroethyl nitrosourea compounds containing aryl.
The preparation method of the chloroethyl nitrosourea compound containing the aryl comprises the following steps:
R 1 reacting-NH-Ar with 2-chloroethanol to generate mono-or di-2-hydroxyethyl aniline, carrying out halogenation reaction on hydroxyl, substituting phthalimido, reacting with hydrazine hydrate to convert into amino, carrying out elimination reaction on the amino and chloroethyl isocyanate to generate chloroethyl urea, and carrying out nitrosation reaction to generate the chloroethyl nitrosourea compound containing aryl, wherein R 1 Selected from-H, -CH 3 or-CH 2 CH 3 Ar is selected from benzene, substituted benzene,
It is an object of the present invention to provide a pharmaceutical composition.
The pharmaceutical composition comprises an aryl group-containing chloroethyl nitrosourea compound and one or more of the aryl group-containing chloroethyl nitrosourea compounds and pharmaceutically acceptable salts thereof.
Further, the pharmaceutical composition is an oral preparation or an injection.
Further, the injection is an intravenous injection preparation, an intraperitoneal injection preparation or a subcutaneous injection preparation.
It is an object of the present invention to provide the use of a pharmaceutical composition as described above for the preparation of a medicament for the treatment of tumors.
Further, the tumor comprises one or more of breast cancer, lung cancer, nasopharyngeal carcinoma, colon cancer liver cancer, cervical cancer and glioma.
The beneficial effects are that:
the aryl-containing chloroethyl nitrosourea compound disclosed by the invention has higher capacity of penetrating through brain barrier, fully plays the role of aziridine ions, has excellent anti-glioma cell activity, and simultaneously shows anti-tumor cell activity on various tumors.
Drawings
FIG. 1 is a graph showing the concentrations of TMZ and HJ03 in the blood or brain 30 minutes after administration of 66mg/Kg provided in Effect example 3;
FIG. 2 shows various blood indexes of mice in different groups after 7 days of oral administration provided in effect example 4;
FIG. 3 is a graph showing the change in body weight of mice during administration provided in effect example 4;
fig. 4 is an evaluation of anti-glioma activity of compound HJ03 provided in effect example 5 in vivo.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings, but are not to be construed as limiting the scope of the invention.
The research designs an aryl-containing chloroethyl nitrosourea compound, the molecular structural general formula of which is shown in the figure, wherein the aryl-containing chloroethyl nitrosourea compound comprises 1 to 2 structural units of beta-chloroethyl nitrosourea, and the beta-chloroethyl group can greatly increase the lipophilicity of the medicament, so that the medicament is delivered to the center through the BBB to a greater extent; introducing electron donating groups such as methoxy and methyl or electron withdrawing groups such as Cl, br, -CN and the like into the 2,3,4 positions of benzene rings of the chloroethyl nitrosourea compound containing aryl, and substituting the benzene rings to become other aromatic rings; the aryl-containing chloroethyl nitrosourea compound can react with DNA to cause apoptosis in vivo to achieve the purpose of resisting tumor.
-CH 3 ,-CH 2 CH 3 ；Ar＝substituted phenyl,/>
Structural general formula of chloroethyl nitrosourea compound containing aryl
The present invention provides example 1, a series of aryl group-containing chloroethyl nitrosoureas were prepared and designated as HJ01-HJ22, wherein Ar and R groups in HJ01-HJ22 are as shown in the following table:
when (when)When the chloroethyl nitrosourea compound containing the aryl is prepared by the following synthetic route:
scheme 1
When R= -CH 3 ,-CH 2 CH 3 When the chloroethyl nitrosourea compound containing the aryl is prepared by the following synthetic route:
R 1 selected from-CH 3 or-CH 2 CH 3。
Scheme 2
Scheme 1 differs from scheme 2 in that the Ar group is present in the initial starting material xx in scheme 2One hydrogen on attached amino group being-CH 3 or-CH 2 CH 3 The aryl group-containing chloroethyl nitrosourea compound produced by the reaction has a structural unit of beta-chloroethyl nitrosourea, and the aryl group-containing chloroethyl nitrosourea compound produced in the synthetic route 1 has two structural units of beta-chloroethyl nitrosourea. In the present invention, xx is any number from 01 to 25.
According to the above synthetic route, example 1 provides a stepwise preparation of aryl group containing chloroethyl nitrosoureas:
the preparation method of A01-A25 is the same, and comprises the following steps:
1mmol of the starting material xx, 2.5mmol (201 mg) of 2-chloroethanol and 0.8mL of aqueous sodium hydroxide solution were successively added to a sealed tube and reacted at 50℃for 4 hours, then the temperature was raised to 110℃and the reaction was continued with stirring for 20 hours. After cooling to room temperature, 5mL of water was added and stirred for a while, and extracted with ethyl acetate. The organic layers were combined, extracted again with saturated sodium chloride solution, and the organic layer was dried over anhydrous magnesium sulfate. Concentrated under reduced pressure, and purified by column chromatography on silica gel (100-200 mesh) (ethyl acetate: petroleum ether=1:4) to give Axx.
In the examples provided by the present invention, a02, 03, 05 are commercially available directly. Whether A02, 03, 05 prepared by the synthetic route or A02, 03, 05 purchased directly, C02, 03, 05 can be obtained by the preparation method of C01-C25, and the preparation method is within the protection scope of the invention. A02, 03 and 05 are N, N-bis (2-hydroxyethyl) -m-methylaniline, N-bis (2-hydroxyethyl) -m-chloroaniline and N, N-bis (2-hydroxyethyl) -p-methylaniline respectively.
The preparation method of C01-C25 is the same, and comprises the following steps:
a25 mL round bottom flask was taken, 0.44mmol Axx was dissolved in 5mL acetonitrile solution, the flask was placed in an ice-water bath, phosphorus oxychloride (202.4 mg,1.32 mmol) was added dropwise, and after the addition was completed, the reaction system was heated to reflux for 2 hours at 100 ℃. After heating was stopped, the mixture was cooled to room temperature, acetonitrile was removed by rotary evaporation under reduced pressure, 5mL of cold water was added, followed by stirring for a while, extraction was performed with dichloromethane, and the organic layers were combined and then extracted with saturated NaHCO 3 The solution is extracted again, and the mixture is extracted again,the organic layer was dried over anhydrous magnesium sulfate. Concentration under reduced pressure gives crude Bxx. 0.34mmol Bxx and potassium phthalimide (191 mg,1.03 mmol) are dissolved in 5mL DMF and reacted overnight at 100 ℃. The hot solution of the reaction system was slowly poured into 10mL of cold water (containing 10g of ice and 1g of potassium carbonate), and after standing for 1 hour, a yellow precipitate was obtained by filtration, washed with distilled water, and dried under vacuum to obtain a yellow solid Cxx.
In the examples provided herein, B23-B25 are commercially available directly. Whether B23-B25 prepared by the synthetic route or B23-B25 purchased directly, C22-C25 can be obtained by the preparation method of C01-C25, and the preparation method is within the protection scope of the invention. B23-B25 are respectively N- (2-chloroethyl) -N-methylaniline, N- (2-chloroethyl) -N-ethylaniline and N- (2-chloroethyl) -N, 4-dimethylaniline.
The preparation method of E01-E25 is the same, and comprises the following steps:
0.2mmol of Cxx was dissolved in 5mL of ethanol, hydrazine hydrate (100 mg,2 mmol) was added, and the mixture was refluxed at 90℃for 2 hours, and the reaction was allowed to stand at room temperature. The white precipitate was filtered off, the ethanol in the filtrate was dried by swirling, the flask was placed under an ice bath, 8ml of dichloromethane was added, the white flocculent precipitate was filtered off, and the dichloromethane was evaporated off to give a clear yellow oil Dxx. 0.18mmol of Dxx was dissolved in 3mL of methylene chloride solution, chloroethyl isocyanate was dissolved in 2mL of methylene chloride, and the methylene chloride solution of chloroethyl isocyanate was dropwise added to the methylene chloride solution of Dxx to react overnight at room temperature. The resulting white precipitate was washed with DCM, filtered and dried to give compound Exx.
The preparation method of HJ01-HJ25 is the same, and comprises the following steps:
exx and mixed acid solvent (including acetic acid and acetic anhydride) were weighed into a round bottom flask, the reaction system was maintained at 0-5℃and 15mg of sodium nitrite was added three times over 1 hour, and after the addition was completed, the reaction was continued at this temperature for 2 hours. After the reaction, the reaction system was warmed to 10-15℃and stirred with 5mL of ice water for a while, extracted with dichloromethane (5 mL. Times.3), the organic layers were combined and the organic layer was saturated with NaHCO 3 The solution was washed and dried over anhydrous magnesium sulfate. Silica gel (100-200 mesh) column chromatography separation and purificationEluent: petroleum ether: ethyl acetate=3:1, v/v) to yield HJxx. Wherein, the proportion of E06, acetic acid and acetic anhydride in the preparation process of HJ06 is 0.1mmol: 55.6. Mu.L: 277.8. Mu.L; the ratio of E15, acetic acid and acetic anhydride in the preparation of HJ15 was 0.1mmol:18.4 μl:92.1 μl; the ratio of Exx, acetic acid and acetic anhydride in the rest of HJxx preparation process is 0.1mmol:50 μl:250 mul.
In the synthesis of HJ01-HJ25, the amounts of Exx are shown in table 1 below:
TABLE 1
Aryl group-containing chloroethyl nitrosoureas HJxx and their corresponding intermediates Axx, cxx and Exx are shown in table 2 below.
TABLE 2
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Axx mass, yield, and nmr spectrum are shown in table 3 below:
TABLE 3 Table 3
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Cxx mass, yield, and nmr spectrum are shown in table 4 below:
TABLE 4 Table 4
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Exx mass, yield, and nmr spectrum are shown in table 5 below:
TABLE 5
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HJxx mass, yield, appearance, mass spectrum and nuclear magnetic resonance spectrum are shown in table 6 below:
TABLE 6
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In order to verify the safety and effectiveness of the aryl group-containing chloroethyl nitrosourea compound in treating glioma, the following effect examples are provided.
Effect example 1
Verification of anti-tumor cell Activity
The method comprises the following steps: (1) Human breast cancer MDA-MB-231 and MCF7 cells, human lung cancer A549 and PC9 cells, human nasopharyngeal carcinoma SUNE-1 cells, human colon cancer HCT116 cells, human liver cancer HepG2 cells and human cervical cancer Hela cells are obtained, the cells are cultured to 90% density, the cells are digested by pancreatin, collected, counted, and inoculated into 96-well plates (three blank control wells are arranged) according to 5,000 cells/well. Placing at 37deg.C, 5% CO 2 The incubator was left overnight.
(2) After 24 hours, HJ01, HJ02, HJ03 and TMZ stock solutions (100 mM concentration) were diluted with fresh medium to the corresponding final concentrations of drug-containing medium and added to wells with two thousandths of DMSO as drug control.
(3) After 72 hours, the medium was aspirated, and after adding 100. Mu.L of complete medium and 10. Mu.L of CCK-8 per well for 2 hours, the OD450 value was determined by a microplate reader. Data analysis: cell OD value-blank control hole OD value, cell OD value of deducting background, calculate cell survival rate again. Cell viability = OD value of dosed/OD value of control (non-dosed) cells x 100%.
TABLE 7 cell viability of different cancer cell lines under TMZ and its analogues
As shown in Table 7, HJ01, HJ02 and HJ03 showed better inhibitory activities than 300. Mu.M TMZ on lung cancer cells PC9 and A549, colorectal cancer cells HCT116, cervical cancer cells Hela, nasopharyngeal cancer cells SUNE-1 and liver cancer cells HepG2 at a drug concentration of 10. Mu.M. In addition, the inhibition activity of HJ02 and HJ03 on breast cancer cells MCF7 is better than that of TMZ of 300 mu M at the drug concentration of 10 mu M.
Effect example 2
Verification of anti-glioma cell Activity
The method comprises the following steps: (1) day 1: u251 cells (5000 cells/well) were seeded into 96-well plates.
(2) Day 2: the medium was aspirated and replaced with the medicated medium. TMZ and HJ series compounds were diluted to drug-containing medium at 1600- > 0 μm, 100- > 0 μm fold ratios, 10 drug concentration gradients were set, 3 duplicate wells per group, and three blank wells were set per 96-well plate.
(3) Day 5: after 72 hours, 10. Mu.L of CCK-8 was added to each well and the reading of OD450 was determined with a microplate reader over 2 hours.
(4) Data analysis: the OD value of the cells is firstly used for obtaining the OD value of the cells with background deduction from the OD value of the blank control hole, and then the survival rate of the cells is calculated. Cell viability = OD value of dosed/OD value of control (non-dosed) cells x 100%. And IC50 of the drug was calculated using GraphPad, the results are shown in table 8.
TABLE 8 IC50 of HJ Compounds
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According to the data in Table 8, temozolomide (TMZ) has an IC50 of 357.6.+ -. 52.1. Mu.M for glioma U251 cells, most of the compounds of the invention have an IC50 of < 5. Mu.M for glioma U251 cells, and the activity is significantly better than TMZ, e.g., compound HJ03 has an IC50 of 1.89.+ -. 0.01. Mu.M, which is 1/189 of TMZ.
Effect example 3
Verification of Compound HJ 03's ability to penetrate the blood brain Barrier
The method comprises the following steps: (1) TMZ and HJ03 are prepared into solution by DMSO; theophylline was formulated in methanol as an internal standard solution at 1000 ng/mL.
(2) C57BL/6 mice (6-8 weeks, body weight about 20 g) were intraperitoneally injected with a single dose of TMZ (66 mg/kg), or HJ03 (66 mg/kg). Blood was taken from the eyeballs after injection for 0,0.25,0.5,0.75,1,1.5,2,3 hours, mice were sacrificed by cervical dislocation after blood was taken, brains were immediately taken, brain tissues were gently washed with 0.9% sodium chloride solution, and immediately frozen at-20 ℃.
(3) The blood sample was immediately centrifuged at 3,000rpm for 15 minutes, 100. Mu.L of plasma was taken and added with 10. Mu.L of 85% orthophosphoric acid, 200. Mu.L of internal standard solution and 200. Mu.L of 10mM pH=3.5 ammonium acetate buffer, then 200. Mu.L of methanol and 200. Mu.L of ZnSO were added 4 (100 mM). After vortex mixing for 1 minute, the samples were centrifuged at 12000rpm for 15 minutes and 5. Mu.L of supernatant was injected into the LC-MS/MS system.
LC-MS/MS parameters: chromatographic conditions: elution was performed using Waters Acquity UPLC BEH C column (2.1 x 100mm, particle size 1.7 μm) with (a) (0.1% formic acid) water and (B) (0.1% formic acid) acetonitrile as mobile phases, linear gradient elution. The elution procedure was 10% B phase (initial), 10% B phase (1 min), 10-90% B phase (1 min), 90% B phase (2.5 min), 90-10% B phase (0.1 min), 10% B phase (1.4 min) and total time of 6min. The flow rate was set to 0.3mL/min and the injection volume was 5. Mu.L. Mass spectrometry conditions: the ion source is Electrospray (ESI), and the detection mode is positive ion multi-reaction (MRM) detection. Parent ion optimization parameters: needle inside Diameter (syringed Diameter): 4.61mm, needle pump Flow Rate (Flow Rate): 7uL/min, scan mode (Scan Type): q1MS, scan Rate (Scan Rate): 200Da/s, scan range (Start-Stop): 100 to compound Molecular Weight (MW) +30Da, observing whether the expected parent ion appears, controlling the response value below 3 x 6 and recording. Sub-ion optimization parameters: scan mode (Scan Type): product Ion (MS 2), scan Rate (Scan Rate): 200Da/s, scan range (Start-Stop): 50-MW+20, collision Energy (CE): 5, the response value of the most hadronous ion in the sub-ions of the target compound reaches more than 3 times of that of the parent ion by taking 5eV as interval. Selecting 2-3 sub-ions with the strongest response values as candidates, and recording m/z. The mass to charge ratio of the selected ions should differ from that of the parent ions by not less than 20Da. Optimum declustering voltage (Declustering Potential, DP) and CE optimization were obtained: after inputting the parent ion and the ion information, setting Step as 1eV in the CE range of 5-180eV to collect data, and recording the CE voltage when the response value of each MRM channel is maximum. And selecting the DP, fixing the optimal CE value according to the same method, and recording the optimal DP value. Ion pairs for quantitative analysis were respectively: TMZm/z:195.1 →138.1, CE:12.5, dp:20, HJ03m/z:483.1 →276.9, ce:17.94, dp:27, theophylline m/z as internal standard substance: 181.1 →124.1, ce:24.82, dp:36.
(4) The whole brain was taken up in 100. Mu.L of internal standard solution and homogenized with 200. Mu.L of 10mM pH 3.5 ammonium acetate buffer, 400. Mu.L of chromatographic grade methanol. Then 200. Mu.L of ZnSO was added 4 (100 mM) was then centrifuged at 3,000rpm for 10 minutes at 25 ℃. The supernatant was transferred to a 1.5mL Eppendorf tube and centrifuged at 12,000rpm for 15 minutes, and 5. Mu.L of the supernatant was injected into an LC-MS/MS system. LC-MS/MS system parameters are as above.
The results of the experiment are shown in Table 9 and FIG. 1, and show that HJ03 and TMZ reached maximum concentration in blood after 15 minutes of gastric administration at a dose of 66mg/Kg and reached maximum concentration in brain tissue at 30 minutes, HJ03 could penetrate the blood brain barrier. Although HJ03 was present in the blood at 0.73% of TMZ, the brain was present at 14.7% of TMZ, indicating that HJ03 has a greater ability to penetrate the blood brain barrier than TMZ (fig. 1).
Table 9 pharmacokinetic parameters of TMZ and HJ03
FIG. 1 shows the blood brain concentrations of TMZ and HJ03 (shown as H104) 30 minutes after administration of 66mg/Kg (A, B). (C) brain obtained using the data of (A, B): plasma concentration ratio. (using one-factor T test analysis, <0.05 > is statistically different, <0.01 > is significantly statistically different, and <0.001 > is extremely significantly statistically different)
Effect example 4
Verification of the biosafety of Compound HJ03
The method comprises the following steps: (1) grouping: female C57 mice were purchased 25 (body weight between 18g-22 g) and 8 weeks of age. After one week of adaptation in the animal house, all mice were randomly divided into control and experimental groups, which were divided into HJ03 (132 mg/Kg,66mg/Kg,20mg/Kg,2 mg/Kg) groups for a total of 5 groups according to the doses to be administered.
(2) Administration: HJ03 was suspended with 0.5% cmc Na at the corresponding concentration, administered once daily by gavage, and the mice weights were recorded and the control group was given an equivalent amount of 0.5% cmc Na solution. The change in body weight of the mice is shown in fig. 3.
(3) Blood collection: seven days after administration, 200. Mu.L of whole blood was collected with 15% EDTA-dipotassium vacuum anticoagulation blood collection tube, immediately refrigerated (2-8deg.C), then the residual blood was collected with EP tube, left standing for 30min, centrifuged at 2000-3000rpm for 10min, and serum was collected and refrigerated (2-8deg.C).
(4) And (3) detection: the collected blood samples were sent to the zhuhai Bai Tong Co., ltd. And five indexes of white blood cell count (WBC), red blood cell count (RBC), neutrophil (Neu), lymphocyte (Lym) and Platelet (PLT) in blood were measured, and the results are shown in FIG. 2.
Fig. 2 results: the HJ03 oral administration of 2, 20, 66mg/Kg dose did not show some statistically significant blood index drop compared to the control group, indicating that continuous one week of HJ03 oral administration of 66mg/Kg did not cause bone marrow suppression in mice. While the dose of 132mg/Kg does not reduce the occurrence of erythrocytes and neutrophils in mice, but significantly reduces the number of leukocytes, lymphocytes and platelets, which has a certain blood toxicity to mice.
Fig. 2 shows blood indices of different groups of mice 7 days after oral administration (statistically significant differences with P <0.05, statistically significant differences with P <0.01, and extremely significant statistical differences with P <0.001, using one-factor T-test analysis).
Figure 3 shows that mice at doses of 66mg/Kg and 132mg/Kg had reduced body weight after 7 days of continuous oral administration of HJ03 compared to the control group, while the other mice administered had insignificant body weight changes.
Effect example 5
Verification of in vivo anti-tumor Activity of Compound HJ03
The method comprises the following steps: (1) cell culture: CT2A-luc cells were incubated with DMEM medium containing 10% FBS,1% diabody at 37℃and 5% CO 2 Culturing under the condition, digesting with 0.25% pancreatin when the cells grow to 90% density, collecting cell liquid, centrifuging, and preparing into cell suspension with PBS solution.
(2) Purchasing animals: c57BL/6 female mice with 6-8 weeks of age were purchased, weighed 17-23g, and placed in animal houses for one week. Mice were purchased from zhuhai Bai Tong Biotechnology Co. All experiments were performed according to the protocols of the ethical committee of animal care and use, of the university of Shenzhen, medical department.
(3) Cell injection: firstly, the mice are subjected to gas anesthesia by isoflurane, hair on the tops of the mice is removed by a dermatome after anesthesia, and then the mice are fixed on an operation table of a brain locator. A3-4 mm incision was made at the midline of the mouse brain, and the coronal and sagittal sutures were found to determine the bregma. The front fontanel mark is used as the origin of coordinates, and the front and back heights of the head of the mouse are adjusted by utilizing the coordinates of the back fontanel, so that the front and back of the head of the mouse are kept horizontal; then, two points are marked by moving 2mm on the left and right sides of the original point, and the left and right heights of the head of the mouse are corrected by the coordinates of the two points, so that the two sides of the head of the mouse are kept horizontal. Suspension in 3. Mu.L PBS 5X 10 4 CT2A-luc cells were injected 1.5mm to the right of the origin, 1mm below, and 3.2mm deep at 0.5 ul/min. After the end of the injection, the injection is completed,waiting for 5min, weighing the mice, and then placing the mice in a thermostatic bath to keep warm until the mice wake up.
(4) Tumor fluorescence imaging monitoring: after the mice were injected with CT2A-luc cells for 7 days, a luciferase substrate stock solution was prepared with DPBS at a concentration of 15mg/mL. The mice were intraperitoneally injected with a stock solution of luciferase substrate at a dose of 10. Mu.L/g, and were placed in an imaging bin for imaging after waiting for 10 min. And determining whether the glioma in-situ model is successfully constructed according to the shooting result.
(5) Grouping and administration: after fluorescence imaging, all mice successfully developed into tumors were randomly divided into control groups,
TMZ (66 mg/Kg) group and HJ03 (20 mg/Kg,2 mg/Kg) group were 4 groups in total. TMZ and HJ03 were suspended in 0.5% sodium carboxymethylcellulose, and administered orally for five days each week, continuously for four weeks, and an equal amount of sodium carboxymethylcellulose solution was used as a control group. The body weight of the mice was recorded daily and the growth of the mice was observed. Tumor growth was detected by an IVIS Spectrum imaging system and photographed to draw a tumor growth curve based on the biological spontaneous light intensity of the mouse head.
(6) Survival analysis: after the end of the dosing, the survival of the mice was continued to be recorded until the mice died or severe brain damage occurred. The number of days of survival of each dead mouse was noted and a survival curve was drawn.
Results: the HJ03 (20 mg/kg) group significantly inhibited tumor growth in vivo compared to the control group, prolonged survival of mice (p=0.0008), and both the HJ03 (20 mg/kg) group significantly prolonged survival of mice compared to the TMZ group (p=0.0172) and the low dose HJ03 (2 mg/kg) (p=0.009) (fig. 4a, c). Over time, mice in both the HJ03 (2 mg/kg) and HJ03 (20 mg/kg) groups had lower body weight loss than the control group and TMZ group (FIG. 4B).
(A) Tumor volume and growth were measured by bioluminescence imaging in CT2A-luc cell in situ model. (B) statistical graphs of mice body weight during dosing. (C) Based on Kaplan-Meier survival curve, the efficacy of HJ03 treatment was evaluated, and statistical analysis was performed using Log-rank test and P value was calculated.
Reference to the literature
[1]Stupp Roger MD,Mason Warren P.MD,van den Bent Martin J.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[J].Cancer/Radiothérapie,2005,9(3):196-197.
[2]Stupp R,Taillibert S,Kanner A,et al.Effect of Tumor-Treating Fields Plus Maintenance Temozolomide vs Maintenance Temozolomide Alone on Survival in Patients With Glioblastoma[J].JAMA,2017,318(23):2306.
[3]Herrlinger U,Tzaridis T,Mack F,et al.Lomustine-temozolomide combination therapy versus standard temozolomide therapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter(CeTeG/NOA-09):a randomised,open-label,pHase 3trial[J].The Lancet,2019,393(10172):678-688.
[4]Pletsas D,Garelnabi EA,Li L,et al.Synthesis and quantitative structure-activity relationship of imidazotetrazine prodrugs with activity independent of O6-methylguanine-DNA-methyltransferase,DNA mismatch repair,and p53[J].J Med Chem,2013,56(17):7120-7132.

Claims (7)

1. An aryl-containing chloroethyl nitrosourea compound is characterized by having the following structural general formula:
wherein R is selected from-CH 3 、-CH 2 CH 3 Or->Ar is selected from benzene, substituted benzene,
2. The preparation method of the chloroethyl nitrosourea compound containing the aryl is characterized by comprising the following steps:
R 1 NH-Ar and 2-Reacting chloroethanol to generate mono-or di-2-hydroxyethyl aniline, carrying out halogenation reaction, substitution of phthalimido, reaction with hydrazine hydrate to convert into amino, eliminating reaction between the amino and chloroethyl isocyanate to generate chloroethyl urea, and nitrosation reaction to generate the chloroethyl nitrosourea compound containing aryl, wherein R 1 Selected from-H, -CH 3 or-CH 2 CH 3 Ar is selected from benzene, substituted benzene,
3. A pharmaceutical composition comprising one or more of the aryl group-containing chloroethyl nitrosoureas and pharmaceutically acceptable salts of the aryl group-containing chloroethyl nitrosoureas of claim 1.
4. A pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is an oral formulation or an injectable formulation.
5. The pharmaceutical composition of claim 4, wherein the injection is an intravenous injection, an intraperitoneal injection, or a subcutaneous injection.
6. Use of a pharmaceutical composition according to claim 3 for the preparation of a medicament for the treatment of an anti-tumor.
7. The use of the pharmaceutical composition according to claim 6 for the preparation of a medicament for the treatment of an anti-tumor, wherein the tumor comprises one or more of breast cancer, lung cancer, nasopharyngeal carcinoma, colon cancer liver cancer, cervical cancer and glioma.
CN202311430690.XA 2023-10-31 2023-10-31 Aryl-containing chloroethyl nitrosourea compound and preparation method and application thereof Pending CN117486761A (en)

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