CN111087389A

CN111087389A - Medicine for treating glioblastoma

Info

Publication number: CN111087389A
Application number: CN201911224491.7A
Authority: CN
Inventors: 张世华; 李洪滨; 杨慧; 刘千子; 蔡栋梁
Original assignee: Jiamusi University
Current assignee: Jiamusi University
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-05-01
Anticipated expiration: 2039-12-03
Also published as: CN111087389B

Abstract

The invention relates to a compound of formula I, pharmaceutically acceptable salts thereof, a preparation method and application thereof, wherein R is₁And R₂Independently selected from H, C_1‑5Alkyl, - (CH)₂)_n‑NR₃R₄，R₃And R₄Independently selected from H or C_1‑5Alkyl, n is 1, 2, 3, 4 or 5. The compound has a good treatment effect on glioblastoma, the concentration content in normal tissue cells after the administration is obviously reduced, the potential risk of side effects is small, and the compound has a great clinical application prospect.

Description

Medicine for treating glioblastoma

Technical Field

The invention relates to the field of pharmacy, in particular to a medicine for treating glioblastoma, a preparation method and application thereof in resisting tumors, especially in treating glioblastoma.

Background

Glioblastoma (GBM; world health organization grade IV glioma) is the most common primary brain tumor in adults. The prognosis of GBM remains poor even with the advances in active surgical resection (active surgical intervention) and post-operative radiotherapy and chemotherapy, which are the most advanced preoperative and intraoperative neuroimaging techniques currently in the field. Median survival (median survival) under the current best post-operative treatment regimen is only 15 months. Therefore, it is imperative to identify new candidate therapeutic agents. One challenge in defining new therapeutic approaches is the heterogeneity of GBM. Intratumoral heterogeneity originates from genetic and non-genetic epigenetic factors. Furthermore, emerging concepts propose a continuously progressing aberrant and malignant differentiation process that inhibits the generation of a tumor cell phenotype from the initiation of stem-like oxygen cells (stem-like cells) of small subsets. These stem cell-like cells are defined by their ability to: (i) self-renewal (self-renewal); (ii) tumor initiation and proliferation in various xenograft paradigms; and (iii) multipotency (multipotency), i.e., its ability to differentiate into astrocytes (astrocytes), oligodendrocytes (oligodendrocytes), and neurons. It is further hypothesized that these cells are resistant to most of the current radiation and chemotherapy treatments, and it is believed that treatments directed against stem cell-like cells may improve the dire record of current conventional treatments. Based on this consideration, in vitro techniques, which are commonly applied in the field of stem cell research, have begun to be adapted for the isolation, expansion and better characterization of tumor cells with stem cell characteristics. Cells proliferate as monolayer cultures under adherent conditions, which can provide significant advantages over "spheroid" cultures.

In recent years, research and search of a targeted marker of a tumor and development of a novel targeted drug become an effective way for treating the tumor, and the patent CN201610177363.1 finds that nestin plays an important role in-vitro proliferation and in-vivo growth of glioblastoma tumor cells, provides a targeted drug Nes0694 for treating glioblastoma, has a good inhibition effect on glioblastoma, and provides a novel targeted drug and a chemotherapy method for treating glioblastoma.

However, the targeted drug is an effective treatment means for tumor, and after the drug is taken, the targeted drug is often accompanied with a lot of side effects, which affect the postoperative recovery of patients and increase the pain of patients. Further animal experiments show that after the drug is administered, Nes0694 in blood enters into the body and then reaches the tissues of the whole body along with blood circulation, and quickly enters into each tissue cell in a passive diffusion mode, so that the Nes0694 not only enters into tumor cells, but also has relatively high concentration in normal tissues, and the drug concentration in the blood is rapidly reduced after 1 hour. Because the antitumor drug is distributed to various tissues of the whole body relatively more, the content of the drug in tumor cells is affected, and more importantly, the damage and side effects on other normal tissues of the body are increased. Therefore, the molecular structure design and improvement of the medicine can find a medicine which can maintain a more lasting and higher concentration in blood than that of the Nes0694 medicine, so that the result is that the medicine has a very good antitumor effect and the damage to normal tissues is reduced.

The invention aims to solve the technical problems, overcome the toxic side effect generated by the existing Nes0694 medicament, and provide a novel medicament for resisting tumors, particularly glioblastoma, which selectively conveys anti-tumor medicaments to target tissues in a body, reduces the medicament content in normal tissues, improves the curative effect and reduces the toxic and side effect on the normal tissues on the basis of maintaining the anti-tumor activity of the original targeted medicaments.

Disclosure of Invention

One of the purposes of the present invention is to provide a novel anti-tumor compound based on the prior art, wherein the novel anti-tumor compound has a good treatment effect on glioblastoma, small damage to normal tissues in vivo, small potential risk of side effects, and a large clinical application prospect. To achieve this object, the present invention provides a compound having the structure shown in formula I:

and pharmaceutically acceptable salts thereof;

wherein R is₁And R₂Independently selected from H, C_1-5Alkyl, or- (CH)₂)_n-NR₃R₄，R₃And R₄Independently selected from H or C_1-5Alkyl, n is 1, 2, 3, 4 or 5.

Further, said C_1-5The alkyl group is selected from methyl, ethyl, propyl or isopropyl.

Preferably, the compounds of formula I are selected in particular from the following compounds:

further, the pharmaceutically acceptable salt is a salt formed by the compound of the formula I and an inorganic acid or an organic acid, and is preferably a salt formed by the compound of the formula I and hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid or malic acid.

Another object of the present invention is to provide a process for the preparation of the compound of formula I, the process for the preparation of the compound of formula I as claimed in claim 1, comprising the steps of:

step (1): synthesis of 2, 6-bis (bromotriphenylphosphine methyl) pyridine (II): dissolving 2, 6-dibromomethylpyridine in an organic solvent, heating to 50-80 ℃, adding triphenylphosphine, keeping the temperature, stirring and reacting for 6-15 hours, cooling to room temperature, adding petroleum ether, filtering and drying the solid to obtain 2, 6-bis (bromotriphenylphosphine methyl) pyridine (II), wherein the reaction formula is as follows:

step (2): synthesis of compounds of formula I: adding a compound shown in the formula II into an ether or nitrile solvent, cooling to-75 ℃ to-30 ℃, slowly dropwise adding 1.1-2.0 equivalent of n-butyllithium, after completely dropwise adding, continuously stirring for half an hour at the temperature, then adding a solution of a compound shown in the formula III dissolved in the ether or nitrile solvent, continuously reacting for 1-3 hours, heating to room temperature, carrying out quenching reaction, adding ethyl acetate for extraction, concentrating an organic phase, and carrying out rapid column chromatography separation to obtain a compound shown in the formula I, wherein the reaction formula is as follows:

preferably, the organic solvent in step (1) is selected from acetonitrile, dichloromethane, dichloroethane, DMF or DMSO; the molar ratio of the 2, 6-bis (bromotriphenylphosphine methyl) pyridine (II) to the triphenylphosphine is 1: 2-3.

Further, in the step (2), the molar ratio of the compound of formula II to the compound of formula III is 1: 1.05-1.2; preferably 1: 1.05-1.1. Further, preferably, the ethereal solvent is selected from THF and dioxane.

It is a further object of the present invention to provide a composition comprising said compound of formula I and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient.

The specific dosage form of the pharmaceutical composition can be selected according to the actual needs in the prior art, such as tablets, powders, pills, capsules, suppositories, granules, suspensions, oral liquids, injections and other pharmaceutical dosage forms; among them, tablets and capsules for oral administration contain conventional excipients such as: fillers, lubricants, dispersants, diluents, and binders.

The invention further aims to provide application of the compound of the formula I, the pharmaceutically acceptable salt thereof or the composition in preparation of anti-tumor drugs, wherein the tumors are preferably glioblastoma, liver cancer, lung cancer, cervical cancer, leukemia and gastric cancer, and particularly preferably glioblastoma.

Drawings

Fig. 1 a-1 e: profiles of the compound of the invention and the drug Nes0694 in ICR mice.

Detailed Description

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art and are intended to be within the scope of the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned in this application are herein incorporated by reference.

Example 1: preparation of Compound I-a

Step (1): synthesis of 2, 6-bis (bromotriphenylphosphine methyl) pyridine (II)

Dissolving (5.0mmol)2, 6-dibromomethylpyridine in acetonitrile, heating to 70 ℃, adding (5.25mmol) triphenylphosphine, keeping the temperature, stirring and reacting for 8 hours, cooling to room temperature, adding petroleum ether, performing suction filtration to obtain a solid product, and performing vacuum drying to obtain 3.78g, 96% of 2, 6-bis (bromotriphenylphosphine methyl) pyridine (II).

Step (2): synthesis of Compound I-a:

adding the compound (0.47g, 0.6mmol) of the formula II prepared in the step (1) into THF, cooling to-75 ℃, slowly dropwise adding 1.15 equivalents of n-butyllithium, after the dropwise addition is completed, continuing to stir at the temperature for half an hour, then adding a solution of the compound (0.31g, 1.23 mmol) of the formula III-a dissolved in THF solvent, continuing to react for 2 hours, raising the temperature to room temperature, adding a buffer solution with pH of about 7 to quench the reaction, adding ethyl acetate to extract, concentrating the organic phase, separating by silica gel column flash chromatography (eluent is a mixed solution of petroleum ether and ethyl acetate which is 6: 1) to obtain the compound of the formula I-a, and concentrating under reduced pressure to obtain 0.33g of the compound of the formula I-a with the yield of 91%.

¹H-NMR(d⁶-DMSO，400MHz):δ7.92～7.52(m，2H)，7.42～ 7.12(m，1H)，4.32(t，2H)，4.16(d，2H)，3.74(d，2H)， 3.62(s，2H)，2.78(s，6H)，2.56(s，6H)，2.35(m，4H)，2.21～2.25(m，12H)，1.78(t，12H)。

ESI MS m/z 602.0[M+Na]⁺。

Example 2: preparation of Compound I-b

Adding the compound (0.47g, 0.6mmol) of the formula II prepared in the step (1) into THF, cooling to-75 ℃, slowly dropwise adding 1.15 equivalents of n-butyllithium, after the dropwise addition is completed, continuing to stir at the temperature for half an hour, then adding a solution of the compound (0.26g, 1.26 mmol) of the formula III-b in dioxane solvent, continuing to react for 3 hours, raising the temperature to room temperature, adding a buffer solution with the pH of about 7 to quench the reaction, adding ethyl acetate to extract, concentrating an organic phase, separating by flash chromatography (the eluent is a mixed solution of petroleum ether and ethyl acetate which is 5: 1) to obtain the compound of the formula I-b, and concentrating under reduced pressure to obtain 0.27g of the compound of the formula I-b with the yield of 87%.

¹H-NMR(d⁶-DMSO，400MHz):δ8.01～7.67(m，2H)，7.47～ 7.16(m，1H)，4.17(t，2H)，4.09(d，2H)，3.71(d，2H)， 3.59(s，2H)，3.15(m，2H)，2.74(s，6H)，2.51(s，6H)，2.35 (m，4H)，2.11～2.18(m，4H)，1.52(t，6H)；

ESI MS m/z 518.0[M+Na]⁺。

Example 3: preparation of Compounds I-c

Adding the compound (0.47g, 0.6mmol) of the formula II prepared in the step (1) into THF, cooling to-75 ℃, slowly dropwise adding 1.15 equivalents of n-butyllithium, after the dropwise addition is completed, continuing to stir at the temperature for half an hour, then adding a solution of the compound (0.22g, 1.23 mmol) of the formula III-c dissolved in THF solvent, continuing to react for 2 hours, raising the temperature to room temperature, adding a buffer solution with pH of about 7 to quench the reaction, adding ethyl acetate to extract, concentrating the organic phase, separating by silica gel column flash chromatography (eluent is a mixed solution of petroleum ether and ethyl acetate 8: 1) to obtain the compound of the formula I-c, and concentrating under reduced pressure to obtain 0.26g of the compound of the formula I-c with the yield of 93%.

¹H-NMR(d⁶-DMSO，400MHz):δ7.83～7.54(m，2H)，7.42～ 7.21(m，1H)，4.51(s，2H)，3.97(d，2H)，3.61(d，2H)， 2.69(s，6H)，2.42(s，6H)，1.87(s，12H)；

ESI MS m/z 460.0[M+Na]⁺。

Example 4: in vitro assay of Compounds of the invention

1. Experimental Material

1.1 drugs and reagents: newborn calf serum (Hangzhou Sijiqing Co., Ltd.), MEM culture solution (Gibco Co., Ltd.), RPMI1640 culture solution (Gibco Co., Ltd.), pancreatin (Gibco Co., Ltd.), MTT (Zhengzhou Boxing Biotechnology Co., Ltd.); examples 1-3 Compounds I-a, I-b, I-c, Nes0694 (white powder, > 98% purity, prepared according to the method of CN 201610177363.1); temozolomide (Jiangsu Tianshili di Yi pharmaceutical Co., Ltd.).

1.2 test cells:

growing human glioblastoma cells (U87MG), human lung cancer cells (A549), human breast cancer cells (MCF-7) and human cervical cancer cells (Hela) in MEM or RPMI1640 medium containing 10% (volume percent) newborn calf serum, 100U/mL penicillin and 100U/mL streptomycin at 37 ℃ with 5% (volume fraction) CO₂The culture medium was changed once for 2 days, and when the culture flask was filled with the medium, the cells were digested with 2.5g/L trypsin and passed through the passage, and the cells were cultured until the logarithmic phase.

1.3 Instrument: CO 2₂An incubator (MCO-15AC, SANYO), an automatic microplate reader (Mk3, produced by Thermro of America), and an inverted microscope (XDS-200D, limited company of optical instruments of Chuikan, Shanghai).

1.4 statistical methods: statistical processing was performed using SPSS-12.0 and the data were expressed as x. + -. s.

2. Method and results

2.1 preparing a drug stock solution: quantitatively weighing 0.01mmol of compounds I-a, I-b, I-c, Nes0694 (prepared according to the method in the patent CN 201610177363.1) and temozolomide, adding 0.5mL of DMSO, dissolving, diluting with 0.5mL of culture solution, and preparing into 1 × 10⁴Mu mol/L of the drug stock solution for later use.

2.2 MTT method for determining the inhibition effect of the compounds I-a, I-b, I-c and Nes0694 on human glioblastoma cells (U87MG), human lung cancer cells (A549), human breast cancer cells (MCF-7) and human cervical cancer cells (Hela): taking the above cells cultured to logarithmic growth phase, counting the cells by 5X 10⁴One cell/mL (2.5X 10 for K562 cells)⁵one/mL) of the cells were inoculated into a 96-well plate, 100. mu.L of the liquid was added to each well, and the stock solution was decanted after the cells were attached to the wall (K562 cells were removed); then adding culture solution containing 0.3% volume fraction of fetal calf serum, and starving and culturing; after 24 hours, replacing the culture solution, adding the prepared drug stock solution, respectively preparing seven different concentrations of 0.16 mu mol/L, 0.32 mu mol/L, 0.63 mu mol/L, 1.25 mu mol/L, 2.5 mu mol/L, 5 mu mol/L and 10 mu mol/L as measurement groups, simultaneously setting normal cells as control groups (6 multiple holes are arranged in each group), and continuously culturing for 24 hours; then 20. mu.L of MTT was added to each well, all the liquid was removed after 4h, 150. mu.L of DMSO was added to dissolve the solid, and the mixture was allowed to stand at 37 ℃ for 0.5 h; detecting with an automatic microplate reader at a wavelength of 570nm, and referencing at a wavelength of 630 nm; the half Inhibitory Concentrations (IC) of the compounds I-a, I-b, I-c and Nes0694 on the above cells were calculated₅₀). The inhibition rate was (1-OD value of measurement group/OD value of normal control group) × 100%. And calculating IC of each compound against tumor cells using Probit analysis in combination with inhibition rate₅₀Statistical analysis was performed using SPSS 11.0 software.

The results are shown in Table 1.

Table 1: : the inventionMedian Inhibitory Concentration (IC) of compounds on tumor cells₅₀)

Note: compared with the positive control group^*P＜0.05，^**P＜0.01

The results show that the compounds I-a-I-c have cytotoxicity to U87MG cells, Hela cells and MCF-7 cells, have weaker cytotoxicity to A549 cells, especially have stronger cytotoxicity to human glioblastoma cells (U87MG), have advantages compared with Nes0694 and temozolomide, and basically maintain stronger inhibitory activity of the targeted drugs.

Example 5: experiment of the Compound of the present invention for inhibiting tumor-bearing rats

Fisher344 rats (200 × 220g in weight), male and female halves, were taken and two days after feeding, 100ul of U87MG tumor cell suspension (total amount about 1 × 10) was injected on the right dorsal hind limb side of the rats⁷One); the day after inoculation of tumor cells U87MG was randomly divided into 6 groups, 10 each, compound I-a (5mg/kg), I-b (5mg/kg), I-c (5mg/kg), Nes0694 (5mg/kg), temozolomide (5mg/kg) and model (same amount of saline). The injection is administered once daily via tail vein for 2 weeks.

And dynamically observing the anti-tumor effect of the test object by adopting an evaluation method for measuring the tumor diameter. The experimental results are shown in table 2.

The tumor diameter is measured 2-3 times per week, and the rat body weight is also required to be weighed at the same time of each measurement.

The formula for tumor volume is: v is 1/2a × b × c, where a, b, and c represent the length, width, and height of the tumor, respectively. The tumor inhibition rate was calculated according to the following formula:

tumor inhibition (%) - (control tumor volume-treatment tumor volume)/control tumor volume × 100%.

Table 2: determination results of tumor inhibition experiment of compound

The test result shows that: compared with a normal saline control group, the compound group of the invention shows that the tumor volume is obviously reduced under the condition of low dose (5mg/kg) administration, the tumor inhibition rate reaches more than 50 percent, and the compound group is obviously superior to a Nes0694 group and a temozolomide group with the same dose.

Example 6: distribution of inventive and comparative Compounds Nes0694 in mice

To assess the in vivo profile of the compounds of the invention versus the control compound in mice, the test was performed.

The experimental method comprises the following steps: ICR mice were randomly divided into 16 groups, 8 of which (5-6) were injected with Nes0694 via their tail vein at a dose of 5mg/kg body weight. In addition, 8 groups of animals (5 to 6 animals per group) were injected with the compound I-a of the present invention via the tail vein, and the dose was 5mg/kg body weight in terms of the amount of Nes0694 contained therein. In addition, 8 groups of animals (1-2 per group) were fed physiological saline via tail vein as a blank control, and plasma and tissue samples were used to generate a standard curve. ICR mice were injected 0.25, 0.5, 1, 2, 4, 8, 24 and 48 hours after drug administration, blood was immediately centrifuged, and plasma was retained and frozen at-70 ℃ for convenient sample concentration. Then, the animals are sacrificed and the liver, heart, lung and kidney are weighed, blood stain on the tissues is washed away by sterile physiological saline, and then the tissues are cut into small pieces, homogenized by a homogenizer and extracted by solid phase. The samples were labeled with a label and stored frozen at-70 ℃ until detection.

Nes0694 and I-a content in animal plasma and tissue samples were measured by HPLC, and the results are shown in FIG. 1.

After injection of Nes0694 and I-a at a dose of 5mg/kg body weight into mice, animal plasma samples taken from different time periods (within 48 hours) were assayed for Nes0694 concentration by HPLC, and the results are shown in FIG. 1 a. As can be seen, the mean blood concentration of Nes0694 after 0.25 hour (15 minutes) into the blood was about 12.67. + -. 0.13. mu.g/mL, and then the blood concentration rapidly decreased within 2 hours. After 0.25 hour of the I-a entering the body, the blood concentration of the drug is about 9.78 +/-0.11 mu g/mL, the decrease is slow, and the blood concentration of the drug in the animal blood of the I-a experimental group exceeds that of the Nes0694 experimental group at each time point of 0.5 hour. These data show that the drug Nes0694 enters the body and reaches the tissues of the whole body along with the blood circulation, and enters the cells of each tissue quickly, so that the drug concentration in the blood is reduced rapidly. After the compound I-a enters the body, the concentration of the compound I-a in blood is reduced slowly so as to maintain the drug concentration which is longer lasting and higher than that of the drug Nes0694, and the result is very favorable for the drug to play the anti-tumor effect. FIGS. 1b, 1c, 1d and 1e show the distribution of the drug in the liver, lung, kidney and heart tissues of animals after administration of Nes0694 or I-a, respectively. As can be seen from fig. 1b, since the liver is the major organ for metabolism of this class of drugs, after intravenous infusion of Nes0694, the concentration of Nes0694 in the liver is higher than in lung, kidney and heart tissues. Unlike Nes0694, the drug concentration in the livers of the animals in the I-a experimental group was lower than that in the Nes0694 experimental group at each time point (0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours). Similar drug distribution profiles were also found in lung and kidney tissues of animals (fig. 1c and fig. 1 d). In addition, the drug concentration in the heart reached a peak around 0.5 hours (4.57 ± 0.12 μ g/g tissue) for the group of Nes0694, followed by a gradual decline (fig. 1 e). The concentration of the drug detected in the heart tissue of the group of I-a animals was lower than that of the group administered with Nes0694, and the peak concentration at 0.5 hour of administration was approximately 30% lower than that of the group administered with Nes0694 on average. The experimental results show that the Nes0694 drug is easier to diffuse in vivo, so that the relatively average systemic tissue is more widely distributed, the expected damage to normal tissues is larger, and the side effect of the drug is more obvious. In contrast, the compounds of the present invention, I-a, have relatively greatly reduced distribution of the drug in normal tissues, thereby reducing the toxic side effects of the drug. Particularly effectively reduces the distribution of the medicine in heart tissues and improves the irreversible damage of the medicine to myocardial cells.

Claims

1. A compound of formula I:

and pharmaceutically acceptable salts thereof;

wherein R is₁And R₂Independently selected from H, C_1-5Alkyl, - (CH)₂)_n-NR₃R₄，R₃And R₄Independently selected from H or C_1-5Alkyl, n is 1, 2, 3, 4 or 5.

2. Compounds of formula I according to claim 1, characterized in that: said C_1-5The alkyl is selected from methyl, ethyl, propyl or isopropyl; n is preferably 1, 2 or 3, particularly preferably 1 or 2.

3. A compound of formula I according to any one of claims 1-3, wherein the compound of formula I is in particular selected from the following compounds:

4. a compound of formula I according to any one of claims 1-3, characterized in that: the pharmaceutically acceptable salt is formed by the compound shown in the formula I and inorganic acid or organic acid, and is preferably formed by the compound shown in the formula I and hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, phosphoric acid, hydrobromic acid, maleic acid, fumaric acid or malic acid.

5. A process for the preparation of a compound of formula I according to claim 1, comprising the steps of:

6. a process for the preparation of a compound of formula I according to claim 5, characterized in that: the organic solvent in the step (1) is selected from acetonitrile, dichloromethane, dichloroethane, DMF or DMSO; the molar ratio of the 2, 6-bis (bromotriphenylphosphine methyl) pyridine (II) to triphenylphosphine is 1: 2-3.

7. The method of claim 5, wherein: the molar ratio of the compound of the formula II to the compound of the formula III in the step (2) is 1: 2.05-2.2; preferably 1: 2.05-2.1.

8. The production method according to claim 5 or 7, characterized in that: the ether solvent in the step (2) is selected from THF and dioxane.

9. A composition comprising a compound of formula I as described in any one of claims 1-4, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient.

10. Use of a compound of formula I according to any one of claims 1 to 4 and pharmaceutically acceptable salts thereof or a composition according to claim 9 for the preparation of a medicament for the treatment of tumors, preferably glioblastoma, liver cancer, lung cancer, cervical cancer, blood cancer, gastric cancer, particularly preferably glioblastoma.