WO2016065525A1 - Use of oxopiperazinyl amide compounds - Google Patents

Abstract

Disclosed is a use of oxopiperazinyl amide compounds as shown by formula I, and isomers, hydrates or salts thereof in the preparation of drugs for inhibiting PLK1 activity.

Description

New use of oxopiperazine amides Technical field

The present invention relates to the use of an oxopiperazine amide compound, and in particular to a novel use of the compound for inhibiting PLK1 activity.

Background technique

The oxopiperazine amide compounds of formula I are a class of known antibacterial agents. It has a good antibacterial effect on a variety of Gram-negative bacteria, and also has antibacterial effects on many anaerobic bacteria.

Some antibacterial drugs have long been used as anti-tumor drugs. For example, mitomycin, doxorubicin, epirubicin, actinomycin D, bleomycin, pingyangmycin, pilomycin, daunorubicin and the like. CN1911236A also reports that tetracycline antibacterial drugs (such as doxycycline) have anti-tumor, anti-inflammatory and cell survival effects in addition to antibacterial.

However, other antibacterial drugs other than this have not been reported to have anti-tumor effects.

Clinically, it is a common practice to use antibacterial drugs for the treatment of infections caused by cancer. This is because the infiltration of tumors leads to low immunity, and the use of anticancer drugs leads to a decrease in neutrophils, which makes the body more susceptible to bacterial infections, leading to patients with malignant tumors often accompanied by serious bacterial infections (Liang Xinwen, Et al, clinical analysis of nosocomial infections in malignant tumors, Chinese Journal of Hospital Infection, 2002, 12 (4), 259-261). Specific examples are: cefoperazone and sulbactam in the treatment of lung cancer complicated with obstructive pneumonia (Journal of Drug Epidemiology, Vol. 17 No. 4, 2008, 215-217).

However, in the field of medicine, antibacterial drugs and anti-tumor drugs are essentially different. Antibacterial and anticancer are different indications. From the point of view of the object of action, bacteria have cell walls and belong to foreign pathogenic organisms; while tumors have no cell wall and are abnormal proliferation or lesions produced by the body itself, which determines the essential difference between the two drugs in terms of mechanism of action.

The oxopiperazine amide compounds of formula I are also used clinically for anti-infective treatment, including anti-infective treatment in patients with malignant tumors, but it still exerts an antibacterial effect. And it's with the various antibacterial or antineoplastic agents above. The substance is different, and its mechanism of action is to bind to the PBPs protein on the bacterial cell membrane, inhibit the synthesis of bacterial cell walls, and promote bacterial cell apoptosis.

Polo like kinase (PLK) is a serine/threonine kinase, and PLK1 is a subtype of Polo-like kinase. Its structure has an N-terminus and a C-terminus, and its activity is exerted by phosphorylating a substrate. Existing studies have shown that PLK1 is involved in the precise regulation of different stages of cell growth and is a key substance for maintaining the normal operation of the cell cycle. For example, PLK1 can regulate various downstream substrates such as Cdc25, cyclin B, Wee1 and Myt1 during cell G2/M phase transition; PLK1 plays an important role in cell centrosome maturation and isolation, affecting ninein like protein (Nlp) and Kizuna and other centrosome-associated protein activities; PLK1 acts on Mad3, Bub1, PICH and other spindle structure monitoring point related proteins to ensure the normal progression of cell mitosis.

By inhibiting the activity of PLK1, it is possible to control the abnormal proliferation of cells, and it is of great significance in the treatment of cell proliferative diseases such as cancer, neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, inflammatory diseases, and immune-mediated diseases. For example, Fan, Zheng et al. 2005, can significantly prevent the growth and proliferation of tumor cells by significantly inhibiting PLK1. Lane and Nigg 1996 documented that inhibition of PLK1 at the protein level can achieve cell cycle arrest. Liu X, et al. Proc Natl Acad Sci 2003, 100(10): 5789–5794. It is documented that in vitro, the depletion of PLK1 strongly inhibits cell proliferation and reduces cancer cell survival in cancer cells, and knockdown of PLK1 makes cancer Cells are blocked in the G2-M phase, leading to apoptosis in cancer cells.

Some drugs which inhibit PLK1 activity have been reported in the prior art, such as: CN103435608A discloses a pyridinopyrimidine PLK1 inhibitor, CN103435594A discloses an aminoquinazoline PLK1 inhibitor, and CN103408546A discloses an amino group. A PLK1 inhibitor of steroids, CN102174035A discloses a PLK1 inhibitor of an aromatic dihydrazide, CN101636399A discloses a PLK1 inhibitor of pyrimidodiazepines, and CN101568539A discloses an imidazolinoneaminopyrimidine PLK1 inhibitors, CN101541800A, disclose a PLK1 inhibitor of pteridine derivatives, but these drugs are in the research and development stage, requiring new synthesis, raw materials are not easy to obtain, quality control of raw materials is not mature, and their structures are different. There is no obvious regularity, which makes it difficult to predict new PLK1 inhibitors by the structure of existing compounds.

By studying the oxopiperazine amide compounds of formula I, a new use is obtained which is different from the prior art.

Summary of the invention

One of the objects of the present invention is to provide a novel use of an oxopiperazine amide compound, and the second object is to find a new option for an anticancer drug in the clinic.

One technical solution of the present invention is: an oxopiperazine amide compound in the preparation of a drug for inhibiting PLK1 activity For use, the oxopiperazine amide compound is selected from the group consisting of a compound of formula I, an isomer, hydrate or salt thereof,

In formula I,

R ₁ is a C ₁ -C ₆ alkyl group;

R _{2 is} selected from a substituted or unsubstituted C ₁ -C ₆ alkyl group, or a substituted or unsubstituted phenyl group;

R ₃ is a -CONH-R ₄ group, wherein R _{4 is} selected from Formula Ia or Formula Ib;

In formula Ia or Ib,

Y is oxygen or sulfur;

R _{5 is} selected from the group consisting of acetyl, 1-methyl-tetrazolyl, or 5-methyl-[1.3.4]thiadiazolyl;

R _{6 is} selected from the group consisting of hydrogen, methoxy or -NH-CHO groups.

Preferably, R ₁ of formula I is ethyl; R ₂ is hydroxyethyl, phenyl or hydroxy substituted phenyl.

Further preferably, the compound of formula I is selected from any of the following compounds of formula II to formula VIII:

The drug contains an oxopiperazine amide compound.

Preferably, the drug further comprises a beta-lactamase inhibitor.

When the drug contains a beta-lactamase inhibitor:

Preferably, the weight ratio of the oxopiperazine amide compound to the β-lactamase inhibitor in the drug is 1:10 to 10:1; more preferably, the weight ratio is 1:1 to 8:1; further preferably The weight ratio is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1.

Preferably, the beta-lactamase inhibitor is clavulanic acid, sulbactam, or an isomer, hydrate or salt of clavulanic acid, or an isomer, hydrate or salt of sulbactam. More preferably, the beta-lactamase inhibitor is potassium clavulanate or sulbactam sodium.

Further preferably, the medicament comprises: a sodium salt of a compound of formula II and sodium sulbactam, sodium of a compound of formula III Salt and sulbactam sodium, the sodium salt of the compound of formula IV and sulbactam sodium, or the sodium salt of the compound of formula V and sulbactam sodium.

Still more preferably, the medicament comprises: a sodium salt of a compound of the formula II in a weight ratio of 1:1, 2:1, 4:1 or 8:1 and sulbactam sodium in a weight ratio of 1:1, 3:1 a sodium salt of a compound of the formula III of 6:1 or 8:1 and a sulbactam sodium, a sodium salt of the compound of the formula IV in a weight ratio of 2:1 or 10:1 and sodium sulbactam, or a weight ratio of 1:1 , 2:1, 5:1 or 10:1 compound sodium salt of formula V and sulbactam sodium.

The drug may contain no excipients or a pharmaceutically acceptable excipient depending on the dosage form.

For example, the lyophilized powder for injection may contain no excipients, and the injection may contain sodium chloride or the like, and the tablet may contain fillers, binders, disintegrators, lubricants commonly used in the pharmaceutical field. Wait. The filler may be selected from the group consisting of starch, pregelatinized starch, dextrin, glucose, sucrose, lactose, lactitol, microcrystalline cellulose, mannitol, sorbitol or xylitol; the binder may be selected from the group consisting of carboxy Methylcellulose sodium, hydroxypropylmethylcellulose, ethylcellulose, povidone, starch syrup, sucrose, powdered sugar, syrup, gelatin, polyethylene glycol; the disintegrant may be selected from cross-linking Sodium carboxymethylcellulose, crospovidone, low substituted hydroxypropylcellulose, sodium carboxymethyl starch or starch; the lubricant may be selected from magnesium stearate, talc or micronized silica gel.

Note: The ratios referred to in the present invention are all weight ratios, and refer to the ratio of free or anhydrate, excluding salt ions or water of crystallization. For example, the sodium salt of the compound of formula III + sulbactam sodium (4:1) means that the weight ratio of the compound of formula III to sulbactam is 4:1; and the compound of formula II monohydrate + sulbactam Sodium (2:1) means that the weight ratio of the compound of formula II to sulbactam is 2:1.

The drug for inhibiting PLK1 activity in the present invention may also be referred to as a PLK1 inhibitor.

Preferably, the effect of inhibiting PLK1 activity is to treat a cell proliferative disorder.

Still preferably, the cell proliferative disorder is selected from the group consisting of cancer, an autoimmune disease, or an inflammatory disease.

Further preferably, the cell proliferative disorder is selected from the group consisting of prostate cancer, ovarian cancer, genitourinary tract cancer, laryngeal cancer, leukemia or cystic fibrosis.

The novel use of the oxopiperazine amide compounds provided by the present invention has the following advantages:

1. The present inventors have found for the first time that a prior class of oxopiperazine amide compounds have a function of inhibiting PLK1 activity, and PLK1 is a subtype kinase of Polo like kinase (PLK) having an N-terminus in its structure. At the C-terminus, the activity of regulating cell growth and the like is exerted by phosphorylating the substrate. A PLK1 inhibitor is a substance that selectively binds to PLK1 kinase and blocks the activity of PLK1 kinase.

Studies have found that the oxopiperazine amides of formula I can significantly inhibit PLK1 kinase, but penicillin has no similar effect, which may be due to the structure of 2,3-dioxo-piperazinecarboxamide. Specific binding of the PLK1 kinase results in the inactivation of the kinase. This is in contrast to the traditional indications of the oxopiperazine amides of the formula I and The mechanism of action is essentially different.

2. The inventors have found that oxoperiperazine amide compounds can effectively reduce cell proliferation by inhibiting PLK1 activity, and can be used for treating cell proliferative diseases such as various cancers, autoimmune diseases, and inflammatory diseases. Expanded indications for existing drugs and provided new options for clinical use.

3. The inventors have unexpectedly discovered that some specific oxopiperazine amide compounds having a specific structure and stereo configuration can effectively inhibit PLK1 activity at a concentration of 150 ng/ml, and can control the tumor cell proliferation rate to about 60. % or less than 70%.

The combination of the oxopiperazine amide compound and the β-lactamase inhibitor shows a better effect than the oxopiperazine amide compound alone, and the combination which is particularly preferred in terms of ratio shows more excellent. Effect.

4. The oxopiperazine amide compound of the present invention has the advantages of less toxic side effects and good safety in inhibiting PLK1 activity, particularly anti-tumor.

5. Compared with the existing compounds which inhibit PLK1 activity, the oxopiperazine amide compounds of the present invention are known and used as compounds, the raw materials are readily available, the synthesis process is mature, the impurities are well studied, and the quality control is mature. Therefore, it has the advantages of being more convenient and easy to obtain.

detailed description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The oxopiperazine amide compounds (compounds having the structure of formula I) to which the present invention relates are known compounds which can be obtained by commercial or literature methods. For example, the CAS numbers of some specific oxopiperazine amides are shown in Table 1:

Table 1: Substituents and CAS numbers for some of the compounds of formula I

The test substance, PLK1 kinase, substrate polypeptide, tumor cell strain, and each reagent, material, and instrument in the following Examples 1 or 2 were purchased from commercial sources, such as Sigma, PerkinElmer, American Custom Chemicals, and Shanghai Jingjing. Pure Biotechnology Co., Ltd., Shanghai Institute of Cell Biology, Chinese Academy of Sciences, etc.

Example 1 Inhibition of PLK1 kinase by oxopiperazine amides

1. Test substance: a compound represented by the formula II to formula VIII of the present invention, amoxicillin, cefuroxime, penicillin, sulbactam, clavulanic acid, hydrates and salts thereof, and related compositions and the like.

2. Test method:

1) Preparation of each group of test solution: DMSO was dissolved in a 20 nM aqueous solution of HEPES to form a DMSO/HEPES solution having a concentration of 20%. The analytes were grouped (multiple control groups, separate groups, combined groups), and each group of the test substances was taken and dissolved in 20% DMSO/HEPES to obtain the test solution of each group, and used. The grouping situation and the highest concentration of the analyte in each group of analytes obtained (for the combined group, the concentration of the compound of formula II to formula VIII in the composition; the concentration is calculated as the free or anhydrous substance of the compound , unit μg / ml) see Table 2. Each group of test solution was diluted with a 20% concentration gradient with 20% DMSO/HEPES solution and diluted to 8 concentration gradients.

2) Preparation of PLK1 kinase solution: 0.07653 g of disodium diglyceride phosphate, 0.025 g of Chaps and 0.012 g of L-cysteine were dissolved in 50 ml of a 20 mM aqueous solution of HEPES to obtain a buffer I. PLK1 kinase was diluted with buffer I to obtain 10 nM of PLK1 kinase solution, which was used.

3) Preparation of PLK1 substrate polypeptide solution: 133 μl of an aqueous MgCl ₂ solution (1 M), 1 μl of an aqueous MnCl ₂ solution (1 M), and 9.867 ml of the buffer I in the step 2) were mixed to obtain a buffer solution II. The substrate polypeptide and ATP were diluted together with buffer II to obtain a PLK1 substrate polypeptide solution containing 300 nM substrate polypeptide and 10 μM ATP, and used.

Detection Mixture(购自PerkinElmer公司)，静置反应1小时后置于

酶标仪(PerkinElmer公司)上读数(激发波长340nm，发射波长分别为615nm和665nm)。测定得到的数据使用GraphPad Prism软件进行评价。 4) Reaction: Each group was carried out separately. The method is: 2 μl of the test solution obtained in the step 1), 2 μl of the PLK1 kinase solution obtained in the step 2) are mixed on a 384-well white test plate. 6 μl of the PLK1 substrate polypeptide solution obtained in the step 3) was added, and after standing at room temperature for 30 minutes, 5 μl of 60 mM EDTA was added to terminate the reaction. Add 5μl of 4x

Detection Mixture (purchased from PerkinElmer), placed after 1 hour of standing reaction

The reading was performed on a microplate reader (PerkinElmer) (excitation wavelength 340 nm, emission wavelengths of 615 nm and 665 nm, respectively). The measured data were evaluated using GraphPad Prism software.

3. Judgment method of test results:

The inhibitory activity of PLK1 is expressed as the concentration of the test substance (IC ₅₀ ) when 50% of PLK1 kinase is inhibited, and the smaller the IC ₅₀ value, the stronger the inhibitory effect on PLK1 kinase. IC ₅₀ values (ng/ml) fall into the following five ranges:

A: IC ₅₀ ≤ 25

B: 25 < IC ₅₀ ≤ 50

C:50<IC ₅₀ ≤150

D: 150 < IC ₅₀ ≤ 250

E: IC ₅₀ >250

4. Experimental results: Table 2 lists the IC ₅₀ value levels of each group of analytes (for the combination group, the IC ₅₀ value level of the compounds of formula II to formula VIII in the composition).

Table 2 IC ₅₀ values of each group of test substances inhibiting PLK1 activity

The results of the test in Table 2 indicate that the oxopiperazine amide compounds of the present invention can effectively inhibit PLK1 kinase activity compared with antibiotics such as penicillin, amoxicillin, cefuroxime, sulbactam and the like having no oxopiperazine amide structure. . The combination of the oxopiperazine amide compound and the β-lactamase inhibitor showed a better effect than the oxopiperazine amide compound alone.

Example 2 Inhibition of tumor by oxopiperazine amides

Hereinafter, prostate cancer and ovarian cancer will be exemplified, and the therapeutic effect of the oxopiperazine amide compound of the present invention on cell proliferative diseases, particularly tumors, will be further described.

1. Processing of the object to be tested:

Test substance: a compound represented by the formula II to formula VIII of the present invention, amoxicillin, paclitaxel, sulbactam, salts and hydrates thereof, and related compositions and the like.

Solution preparation: The test substances were grouped (multiple control group, single group, combined group), and the test substances in each group were taken separately. First, 1.23 ml of castor oil was added, shaken and dissolved, and then 1.23 ml of absolute ethanol was added. Shake well and dissolve to prepare the mother liquor of each group. The grouping condition and the concentration of the analyte in the obtained group of test substances in the obtained group (for the combined group, the concentration of the compound represented by the formula II to the formula VIII in the composition; the concentration is based on the free or anhydrous substance of the compound, The unit is mg/ml), and the specific grouping and administration are shown in Tables 3 and 4.

0.4 ml of the mother liquor was taken at each administration, and diluted 6-fold with 0.9% NS to obtain a dilution of the analyte.

2. Establishment of a tumor model:

Experimental animals: Female BALB/cA nude mice, aged 35-40 days, weighing 18-22 g. Prostate cancer models and ovarian cancer models were established, respectively.

Prostate cancer model: A human prostate cancer PC-3 cell strain was inoculated subcutaneously in nude mice. The amount of cells inoculated was 3 × 10 ⁶ , and the cells were inoculated to form transplanted tumors and then used in nude mice for 3 generations.

Ovarian cancer model: A human ovarian cancer HO-8910 cell strain was inoculated subcutaneously into nude mice. The amount of cells inoculated was 3 × 10 ⁶ , and the cells were inoculated to form transplanted tumors and then used in nude mice for 3 generations.

Specific method: The tumor tissue in the vigorous growth period was cut into 1.5 mm ³ and inoculated subcutaneously in the right axilla of nude mice under aseptic conditions. The nude mice xenografts were measured with vernier calipers to measure the diameter of the transplanted tumors. After the tumors were grown to 100-300 mm ³ , the animals were randomly divided into groups of 8 animals. Except for the blank group, the situation of each group is consistent with the grouping of the objects to be tested. See Tables 3 and 4 for details.

The prostate cancer model animals were independently operated with the ovarian cancer model animals.

3. Administration:

Intravenous administration, castor oil was administered to the blank group, and each group and each treatment group were given a dilution of the test substance. The drug was administered once a week for a total of 3 weeks. The dose to be administered was adjusted in accordance with the body weight of each animal based on 20 ml of the administration substance (20 ml/kg) per kg of animal body weight. See Tables 3 and 4 for specific administration.

4. Data collection and processing:

The number of animals was counted, the body weight of the animals was weighed, the tumor diameter was measured, and the tumor volume was calculated. The relative tumor growth rate (RT) was used as an evaluation index for antitumor activity, and the smaller the RT value, the stronger the antitumor activity. At the same time, the animal survival rate (RN) and animal body weight change rate (RW) before and after the test were used as indicators for evaluating safety. The high RN value and the difference between the RW value and the value of the blank group indicate that the safety is good. The specific calculation formula is:

RT = (T _{treatment group} / T _{blank group} ) × 100%. among them,

T=V _t /V ₀ , V=1/2×a×b ² ,

RN=(N _t /N ₀ )×100%,

RW = (W _t / W ₀ ) × 100%.

The meaning of each symbol in the calculation formula is as follows:

a - tumor length, b - tumor width, V - tumor volume, T - relative tumor volume,

V ₀ - the tumor volume before treatment (when initially administered 0 days),

V _t - the tumor volume after treatment (after 21 days),

T _{treatment group} - the relative tumor volume of the treatment group,

T _{blank group} - the relative tumor volume of the blank group,

N _t - the number of animals after treatment (after 21 days),

N ₀ - the number of animals before treatment (when initially grouped for 0 days),

W _t - the weight of the animal after treatment (after 21 days),

W ₀ - the body weight of the animal before treatment (at the time of initial group administration 0 days).

5. Experimental results:

5.1 The test results of prostate cancer model animals are shown in Table 3.

Table 3 Effect of each group of test substances on prostate cancer model animals

5.2 The test results of ovarian cancer model animals are shown in Table 4.

Table 4 Effect of each group of test substances on ovarian cancer model animals

The test results in Tables 3 and 4 indicate that:

The oxopiperazine amide compound of the present invention is effective for inhibiting tumor cell proliferation (RT value less than 60% or 70%) as compared with an antibiotic having no oxopiperazine amide structure such as amoxicillin. The combination of the oxopiperazine amide compound and the β-lactamase inhibitor showed a better effect than the oxopiperazine amide compound alone.

In addition, the oxopiperazine amide compound of the present invention shows good safety (except for RN value higher than paclitaxel, reaching 100% in addition to the effect); the RW values of each individual group and the combined group are similar to those of the blank group, indicating Has little effect on body weight).

Although the present invention has been described in detail above with reference to the preferred embodiments, embodiments, and the invention, the invention may be modified or modified by those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the invention belong to the present invention.

Industrial applicability

The present invention discloses a second use of an oxopiperazine amide compound, that is, for inhibiting PLK1 activity, and further for treating cell proliferative diseases, especially for treating prostate cancer, ovarian cancer, genitourinary tract cancer, laryngeal cancer, leukemia or sac Sexual fibrosis has a definite effect. Expanded indications for existing drugs and provided new options for clinical use.

Claims (14)

The use of an oxopiperazine amide compound for the preparation of a medicament for inhibiting PLK1 activity, the oxopiperazine amide compound being selected from the group consisting of a compound of formula I, an isomer, hydrate or salt thereof;

In formula I, R ₁ is a C ₁ -C ₆ alkyl group; R _{2 is} selected from a substituted or unsubstituted C ₁ -C ₆ alkyl group, or a substituted or unsubstituted phenyl group; R ₃ is a -CONH-R ₄ group, wherein R _{4 is} selected from the group of formula Ia or formula Ib;

In Formula Ia or Formula Ib, Y is oxygen or sulfur; R _{5 is} selected from the group consisting of acetyl, 1-methyl-tetrazolyl, or 5-methyl-[1.3.4]thiadiazolyl; R _{6 is} selected from the group consisting of hydrogen, methoxy or -NH-CHO groups. The use according to claim 1, wherein R ₁ in the formula I is an ethyl group; and R ₂ is a hydroxyethyl group, a phenyl group or a hydroxy group-substituted phenyl group. The use according to claim 1, characterized in that the compound of the formula I is selected from any one of the compounds of the formulae II to VIII:

The use according to claim 1, wherein the drug contains the oxopiperazine amide compound according to claim 1. The use according to claim 4, wherein the drug further comprises a beta-lactamase inhibitor. The use according to claim 5, wherein the drug contains an oxopiperazine amide compound and a β-lactamase inhibitor, and the weight ratio of the two is 1:10 to 10:1. The use according to claim 6, wherein the weight ratio of the oxopiperazine amide compound to the β-lactamase inhibitor is from 1:1 to 8:1. The use according to claim 7, wherein the weight ratio of the oxopiperazine amide compound to the β-lactamase inhibitor is 1:1, 2:1, 3:1, 4:1, 5 :1,6:1, 7:1 or 8:1. The use according to claim 5, wherein the beta-lactamase inhibitor is clavulanic acid, sulbactam, or an isomer, hydrate or salt of clavulanic acid, or sulbactam Isomer, hydrate or salt. The use according to claim 9, wherein the β-lactamase inhibitor is potassium clavulanate or sulbactam sodium. The use according to claim 10, wherein said medicament comprises: a sodium salt of a compound of formula II and sulbactam sodium, a sodium salt of a compound of formula III and sodium sulbactam, a sodium salt of a compound of formula IV and Sulbactam sodium, or the sodium salt of the compound of formula V and sulbactam sodium. The use according to any one of claims 1 to 11, wherein the inhibition of PLK1 activity is treatment of a cell proliferative disorder. The use according to claim 12, wherein the cell proliferative disorder is cancer, an autoimmune disease or an inflammatory disease. The use according to claim 13, wherein the cell proliferative disorder is prostate cancer, ovarian cancer, genitourinary tract cancer, laryngeal cancer, leukemia or cystic fibrosis.