CN113072556B

CN113072556B - Medicine for treating glioma and preparation method thereof

Info

Publication number: CN113072556B
Application number: CN202110343447.9A
Authority: CN
Inventors: 梁绍栋; 孟祥慧; 张万里; 刘英伟; 初金玉; 庄亚严; 刘魁力; 于雪; 白吉祥; 王书惠
Original assignee: Mudanjiang Medical University
Current assignee: Mudanjiang Medical University
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2022-02-01
Anticipated expiration: 2041-03-30
Also published as: CN113072556A

Abstract

The invention relates to a medicament for treating glioma and a preparation method thereof. In particular, the medicament comprises 3-methoxy-4- [ 4-oxo-7- (tetrahydro-pyran-4-yl) -3, 4-dihydro-imidazo [5,1-f ] [1,2,4] triazin-2-yl ] -pyrrolidine-1-carboxylic acid tert-butyl ester and a pharmaceutically acceptable carrier, and optionally further comprises other active ingredients for the treatment of gliomas. The invention also relates to a preparation method of the medicine and application of the compound in preparing a medicine for treating glioma.

Description

Medicine for treating glioma and preparation method thereof

Technical Field

The present invention relates to the fields of neurosurgery and oncology therapy. More particularly, the present invention relates to a novel compound for the treatment of glioma, a medicament comprising said compound, and a process for the preparation of said medicament.

Background

Gliomas (gliomas) are primary malignant tumors generated by brain and spinal glia cell canceration, are the most common primary tumors in the central nervous system, have the morbidity of about 40-50% of craniocerebral tumors, and have the annual incidence rate of 3-8 persons/10 ten thousand persons. Gliomas can be further classified into subtypes such as astrocytomas, oligoblastoma, ependymomas, mixed gliomas, and the like, according to the degree of similarity between the morphology of tumor cells and normal brain gliomas. These different types of gliomas all have the characteristics of invasive growth and unclear boundaries with surrounding normal tissues, so that the gliomas present the situations of difficult thorough removal of operations, high recurrence rate, high mortality rate, low cure rate and the like. Except for the fact that a small number of patients with low-grade gliomas such as hairy cell astrocytomas (according to the WHO grading system, gliomas are classified into WHO I-IV grades, I and II belong to low-grade gliomas, and III and IV belong to high-grade gliomas) can be cured through operations, most of patients with gliomas are easy to relapse after receiving various treatment measures, particularly patients with glioblastomas (WHO IV grades), the average survival time of the patients is only about 1 year, and the 5-year survival rate of the patients does not exceed 10%, so that the patient becomes one of the most troublesome treatment problems in neurosurgery.

Currently, the basic treatment of glioma is still focused on traditional treatments such as surgery, radiation therapy and chemotherapy. In particular, current treatment of gliomas remains surgery as the primary treatment modality, and the extent of tumor resection is closely related to the prognosis of the patient. However, because glioma itself has the characteristic of invasive growth and the brain function has particularity, it is difficult to achieve biological complete resection in the true sense clinically. Therefore, how to remove the tumor to the maximum extent with the precondition of maximally protecting the brain function is a great problem that the glioma surgery has to face. Radiation therapy is also an important means of treating gliomas. For high-grade glioma, the total dose of common radiotherapy is 54-60 Gy, and the division is performed for 30-33 times. However, for low-grade gliomas, there is always controversy regarding the best opportunity for post-operative radiation therapy and the risk of long-term radioactive neurotoxicity. In the field of chemotherapy, temozolomide is recommended as a standard first-line chemotherapeutic drug for glioblastoma, and is also frequently used for first-line chemotherapy for other gliomas such as astrocytoma and the like. In addition, it is also known that valproic acid, kemplan or interferon alpha/beta may have some sensitizing effect on temozolomide chemotherapy and radiotherapy. Although these treatments have developed rapidly and have made considerable progress in recent years, unfortunately the improvement in clinical therapeutic efficacy has not been satisfactory. Therefore, there is still a need for novel therapeutic approaches, in particular drugs for the treatment of gliomas, to meet the growing clinical demand.

WO2013/110768a1 discloses cyclic guanylate monophosphate (cGMP) specific phosphodiesterase type 9 inhibitors in the form of 3H-imidazo [5,1-f ] [1,2,4] triazin-4-one having structure I, pharmaceutical compositions comprising 3H-imidazo [5,1-f ] [1,2,4] triazin-4-one, and methods for preparing these compounds. The patent application discloses that compounds having structure I can be used for the treatment of cognitive impairment, in particular cognitive impairment associated with neurodegenerative diseases such as cortical dementia or subcortical dementia. This patent application also discloses the compound 3-methoxy-4- [ 4-oxo-7- (tetrahydro-pyran-4-yl) -3, 4-dihydro-imidazo [5,1-f ] [1,2,4] triazin-2-yl ] -pyrrolidine-1-carboxylic acid tert-butyl ester (see compound 23, hereinafter referred to as compound a):

however, this patent application does not disclose that compound a is particularly useful for the treatment of which diseases.

Disclosure of Invention

[ OBJECTS OF THE INVENTION ]

It is an object of the present invention to provide a novel compound which can be used for the treatment of gliomas.

It is another object of the present invention to provide a medicament comprising said compound.

It is a further object of the present invention to provide a process for the preparation of said medicament.

It is still another object of the present invention to provide a novel pharmaceutical use of the compound.

[ technical solution of the present invention ]

The inventors of the present application have conducted intensive studies to develop novel compounds for the treatment of glioma. As a result, the inventors of the present application unexpectedly found through experiments that the compound a disclosed in WO2013/110768a1 has excellent activity for treating glioma. The results of the above studies led to the completion of the technical solution of the present invention.

To this end, in a first aspect of the present invention, there is provided a novel compound which can be used for the treatment of glioma, which is compound a or a pharmaceutically acceptable salt thereof:

in a second aspect of the present invention, there is provided a medicament for the treatment of glioma comprising compound a or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein said compound a has the structure:

the present invention is not particularly limited with respect to the kind of the pharmaceutically acceptable carrier, as long as it is compatible with (i.e., advantageous for and does not cause adverse physical and/or chemical interactions with) compound a or a pharmaceutically acceptable salt thereof. Depending on the route of administration and the particular dosage form of the drug, pharmaceutically acceptable carriers will include conventional pharmaceutically acceptable excipients and other adjuvants and the like, including, but not limited to, disintegrants, binders, lubricants, wetting agents, suspending agents, emulsifiers, isotonicity agents, buffers, dispersants, antioxidants, preservatives, sweetening agents, flavoring agents, and the like.

In one embodiment, the compound a or a pharmaceutically acceptable salt thereof is the only active ingredient formulated in the medicament.

In one embodiment, the compound a or a pharmaceutically acceptable salt thereof is formulated in the medicament in combination with other active ingredients for the treatment of glioma.

The present invention is not particularly limited as to the kind of other active ingredients for treating glioma, as long as it is compatible with (i.e., does not interact adversely physically and/or chemically with) compound a or a pharmaceutically acceptable salt thereof. For example, the other active ingredient for treating glioma may include one or more of temozolomide, cyclohexylnitrosourea, hydroxyurea, fotemustine, procarbazine, imatinib, erlotinib, carboplatin, vincristine.

The medicament of the present invention may be administered by any suitable route. For example, the medicaments of the invention may be administered orally, parenterally (intravenously, intramuscularly or subcutaneously), intraspinally and intracisternally in the form of solids (such as tablets, capsules, pills, powders) or liquids (such as solutions, suspensions or emulsions).

In one embodiment, the medicament contains a therapeutically effective amount of compound a or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the therapeutically effective amount is 0.01-1000mg, preferably 0.1-100mg, more preferably 0.5-50 mg.

In a third aspect of the invention, there is provided a process for the preparation of said medicament, which comprises admixing said compound a or a pharmaceutically acceptable salt thereof and optionally other active ingredients for the treatment of glioma with a pharmaceutically acceptable carrier.

In a fourth aspect of the invention, there is provided the use of compound a, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of glioma.

In one embodiment, the glioma is a glioblastoma.

The above embodiments represent exemplary embodiments of the present invention, but the present invention is not limited to the above embodiments.

Detailed Description

The following examples are provided so that those skilled in the art can more clearly understand the features and effects of the present invention. However, these examples are for illustrative purposes only and are not meant to limit the claims of the present invention in any way.

EXAMPLE 1 in vitro inhibitory Effect of Compound A on C6 glioma cells

[ purpose of experiment ]

The present example was carried out to examine rat C6 glioma cells and examine the inhibitory effect of compound a on the in vitro cell proliferation of glioma cells.

[ Experimental methods ]

1. Cell culture

Rat C6 glioma cells (note: C6 rat brain glioma cells are derived from Benda et al from Wistar germ line rats and are consistent with the pathological characteristics of human glioblastoma) were purchased from the Chinese type culture Collection. Culturing C6 cells in DMEM medium at 37 deg.C under 5% CO₂Subculture in a cell culture incubator at saturated humidity, the medium being supplemented with 10% fetal bovine serum, 100kU/L penicillin and 100mg/L streptomycin.

2. Experimental methods

Washing C6 cells in logarithmic growth phase with phosphate buffer solution, digesting the cells with 0.25% trypsin, discarding the digestion solution, counting, and adjusting cell density to 5 × 10⁴Cells/ml medium were seeded in 96-well plates to a volume of 200 microliters per well, and the 96-well plates were returned to the cell incubator overnight for adherence.

After 24h incubation, the 96-well plates were removed, the medium was discarded, 200. mu.l of medium containing compound A at final concentrations of 0, 10, 20, 40 and 80. mu. mol/L, respectively (each in triplicate) was added, and then returned to the incubator for further incubation for 72 h. After the cell culture was completed, the 96-well plate was taken out, 50. mu.L of a pre-cooled 30W/V% trichloroacetic acid (TCA) solution was added to each well to fix the cells (to make the final concentration of TCA 10%), and the plate was left at 4 ℃ for 1 hour in a refrigerator. The wells were washed with deionized water (× 3), air dried, 100 μ L of 0.4% SRB solution (prepared with 1% acetic acid) was added to each well, after standing for 30min for staining, the staining solution was discarded, and washed 5 times with 1% acetic acid to remove unbound dye, air dried, dissolved with 100 μ L Tris base solution (10mmol/L, pH 10.5), shaken on a microplate shaker for 20min, and the absorbance of each well was measured at 540nm wavelength with a multifunctional microplate reader. The inhibition of cell proliferation by the drug was calculated as follows:

inhibition rate (%) [1- (OD of administration group)₅₄₀value/OD of control group₅₄₀Value)]×100％。

[ Experimental results ]

The results of this experiment are shown in table 1 below.

TABLE 1 inhibition ratio (%) of Compound A on rat C6 glioma cell proliferation

Dosage (μmol/L)	Inhibition ratio (%)
		0	0
10	19.7±4.6
		20	41.3±5.2
40	72.7±8.1
		80	86.5±6.9

Note: the data processing is carried out by SPSS16.0 statistical software, and the data are expressed by means of 'mean value plus or minus standard deviation'.

The above results show that compound a at different concentrations produces a significant cell proliferation inhibition effect after treating C6 cells for 72h, and shows a significant dose dependence: with the increase of the concentration of the compound A, the inhibitory effect of the compound A on the C6 cells is gradually enhanced, and the number of the survival cells is gradually reduced.

EXAMPLE 2 in vivo inhibitory Effect of Compound A on C6 glioma cells

[ purpose of experiment ]

The purpose of this example is to further examine the inhibitory effect of compound a on C6 glioma cells in vivo, on the basis of preliminary demonstration of the inhibitory effect of compound a on C6 glioma cells in vitro in example 1.

[ Experimental animals ]

The experimental animals used in this example were healthy male SD rats (purchased from Tokyo Wintolite laboratory animal technologies, Inc.) weighing between 200 + -20 g. Before the experiment, the animals were placed in a laboratory animal room (room temperature was around 22 ℃, relative humidity was maintained at 50-55%, light and dark conditions were switched every 12 hours) and acclimated for 2 days. Animals can freely ingest clean food and drinking water.

[ Experimental procedures ]

1. Cell culture

Culturing C6 cells in DMEM medium at 37 deg.C under 5% CO₂Subculture in a cell culture incubator at saturated humidity, the medium being supplemented with 10% fetal bovine serum, 100kU/L penicillin and 100mg/L streptomycin.

Before the experiment is started, C6 cells in logarithmic phase are washed by phosphate buffer solution, then 0.25% trypsin is used for digesting the cells, then digestive juice is discarded, the cells are blown and washed by the phosphate buffer solution for 1-2 times to form cell suspension, counting is carried out, the cell concentration is adjusted to be 1 multiplied by 10¹⁰The cell suspension/L was used for seeding.

2. Establishment of rat brain glioma model

After an SD rat is anesthetized by injecting 1% pentobarbital sodium (40mg/kg) through the tail vein, hairs on the top of the head of the rat are removed, the scalp is transversely cut at a position about 1.5cm behind the connecting line of the eye lacerations on the two sides of the top of the head, and the skull mark of the forechimneys is exposed. Drilling a bone hole 1mm in front of the middle point of the chimney by using a self-made dental drill and 3mm beside the sagittal suture, extracting 10 mu L C6 cell suspension by using a micro injector, vertically fixing on a test bed, inserting a needle 6mm under the dura mater through the original bone hole, retreating by 1mm, completely injecting the cell suspension within 10min at the speed of 2 mu L/min, remaining the needle 5min after the injection is finished, slowly retreating the needle, sealing the bone hole by using bone wax, and suturing the scalp incision by using No. 1 silk thread.

3. Animal grouping and administration

50 molded rats were randomly divided into 5 experimental groups (10 rats/group). The name and dosing schedule for each experimental group started 7 days after inoculation of the C6 cell suspension:

(1) model group (animals were administered by gavage with 0.5% CMC-Na aqueous solution at a dose of 10mL 0.5% CMC-Na aqueous solution/1 kg body weight 1 time a day for 15 days);

(2) temozolomide group (temozolomide administered to animals by gavage at a dose of 20mg temozolomide (dissolved in 10mL of 0.5% CMC-Na aqueous solution)/1 kg body weight 1 time a day for 15 days);

(3) compound a low dose group (animals administered compound a by gavage at a dose of 10mg compound a (dissolved in 10ml 0.5% CMC-Na aqueous solution)/1 kg body weight 1 time daily for 15 days);

(4) compound a middle dose group (compound a administered to animals by gavage at a dose of 20mg compound a (dissolved in 10ml of 0.5% CMC-Na aqueous solution)/1 kg body weight 1 time a day for 15 days); and

(5) compound a high dose group (animals were dosed by gavage with compound a at a dose of 40mg compound a (dissolved in 10ml 0.5% CMC-Na aqueous solution) per 1kg body weight, 1 time daily for 15 days).

4. Determination of results

On day 16 after surgery, the experimental animals were sacrificed, the brains were removed by decapitation, the whole brains were fixed in 3% formaldehyde for 1 week, coronal incisions were made along the needle insertion points, the length and length of the tumor were measured, and the tumor volume and tumor inhibition rate were calculated according to the following formula:

tumor volume (V) ═ pi ab²And/6 (a is the major diameter, b is the minor diameter, measured with a vernier caliper).

Tumor inhibition (%) - (average volume of model group tumor-average volume of experimental group tumor)/average volume of model group tumor ] × 100% (tumor inhibition > 30% means that the tumor is sensitive to the drug).

5. Statistical method

The statistical method adopts SPSS16.0 statistical software to process data, the data is expressed by mean value plus or minus standard deviation, and the comparison among groups adopts one-factor variance analysis.

[ Experimental results ]

The results of this experiment are shown in table 2 below.

TABLE 2 summary of tumor volume and tumor suppression Rate data for rats in each experimental group

Note: the results are expressed as mean ± standard deviation, p <0.05 compared to the model group.

3. Discussion of the related Art

The experimental data show that the compound A has obvious inhibition effect on the growth of brain glioma in a tumor-bearing rat body, and is consistent with the results of in vitro experiments. In particular, compound a treatment significantly reduced the tumor volume of C6 glioma in rats and exhibited a dose-dependence: with increasing compound a dose, tumor volume in rats appeared to be in a diminishing state with a corresponding marked increase in tumor suppression rate. In addition, the experimental data also show that the compound A has slightly better inhibiting effect on the growth of in-vivo brain glioma than the positive control medicament temozolomide. In view of the above findings, it can be seen that compound a has a promising prospect for development as an active ingredient of a medicament for treating glioma.

Claims

1. A medicament for treating glioma comprising compound a or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein said compound a has the structure:

2. the medicament of claim 1, wherein the Compound A or a pharmaceutically acceptable salt thereof is the only active ingredient formulated in the medicament.

3. The medicament according to claim 1, wherein the compound a or a pharmaceutically acceptable salt thereof is formulated in the medicament in combination with other active ingredients for the treatment of gliomas.

4. The medicament according to claim 3, wherein the other active ingredient for the treatment of glioma is selected from one or more of temozolomide, cyclohexylnitrosourea, hydroxyurea, fotemustine, procarbazine, imatinib, erlotinib, carboplatin, vincristine.

5. The medicament according to any one of claims 1 to 4, wherein the medicament contains 0.01 to 1000mg of Compound A or a pharmaceutically acceptable salt thereof.

6. A process for the preparation of a medicament according to any one of claims 1 to 5, which comprises admixing the compound a or a pharmaceutically acceptable salt thereof and optionally other active ingredients for the treatment of glioma with a pharmaceutically acceptable carrier.

7. Application of compound A or pharmaceutically acceptable salt thereof in preparation of medicine for treating glioma

8. The use of claim 7, wherein the glioma is a glioblastoma.