CN111825599A - Medical application of novel Neddylation inhibitor - Google Patents

Abstract

The invention relates to the formula I_AThe compounds or the pharmaceutically acceptable salts and the medical application of the compounds or the pharmaceutically acceptable salts and the medical compositions thereof in preventing, delaying or treating diseases in which NEDDylation pathways participate or are mediated, and I_AThe participated combined medication.

Description

Medical application of novel Neddylation inhibitor

Technical Field

The invention relates to discovery of a novel Neddylation inhibitor and medical application thereof, and particularly provides a formula I_AUse of compound or its pharmaceutically acceptable salt and its medicinal composition in preventing and treating diseasesAnd the application in the medical application of delaying or treating diseases in which the NEDDylation pathway participates or mediates, in particular the application in the anti-tumor field. At the same time, a class I is provided_AThe involved multi-drug combination therapy anti-tumor method.

Background

Degradation of proteins is mainly via two pathways: lysosomal degradation pathways and ubiquitin (ubiquitin) -mediated proteasomal degradation pathways. In eukaryotic cells, the ubiquitin-protease system (UPS) selects target protein molecules from intracellular protein substrates under the action of a series of specific enzymes, and specifically modifies these target proteins, a process known as Ubiquitination. Ubiquitination modification plays an important role in the localization, metabolism, function, regulation and degradation of proteins. Meanwhile, it is involved in the regulation of almost all vital activities such as cell cycle, proliferation, apoptosis, differentiation, metastasis, gene expression, transcriptional regulation, signal transmission, injury repair, inflammatory immunity, and the like. In the field of tumor research, numerous studies have also demonstrated that ubiquitination modification plays a crucial role in tumorigenesis, metastasis, and tumor microenvironment formation.

Similar to the process of ubiquitination, ubiquitination is also coupled to substrates by the same enzymatic reaction (E1-E2-E3). Neddylation is one of the most important ubiquitination pathways. Unlike inactivation, Neddylation is not normally directly involved in protein degradation, but rather acts as a protein activation signal to regulate the activity of various transcription factors within the cell. The Neddylation modification process is mainly that a ubiquitin-like molecule NEDD8 (neural progenitor cell-expressed degraded down-regulated protein 8) is subjected to three-step enzymatic cascade reaction and finally combined with a target protein (figure 1). The modification process is that NEDD8 is catalyzed and activated by NEDD8 activating enzyme E1(NEDD8-activating enzyme E1, NAE) under the condition of ATP, the activated NEDD8 is transferred to one of two binding enzymes UBE2M (or UBC12) or UBE2F through sulfhydrylation reaction, and the NEDD8 is transferred to substrate protein from E2 under the catalysis of E3 ligase to complete the modification.

Unlike E3 ligase in ubiquitination systems, which has up to 4 families and over 600 subtypes, the Neddylation E3 ligase has only 10 and contains mostly ubiquitin-related ring (reaction intervening New gene) structural signature domains. Because of the modifications that directly regulate Cullin-RING ubiquitin ligases (CRLs), the Neddylation E3 ligase RBX1 (RING-box proteins 1, also known as ROC1) and RBX2 (also known as ROC2/SAG) are the two most studied classes. The CRL ubiquitin ligase family can specifically regulate about 20% of substrates degradation mediated by the ubiquitin-proteasome system, and the substrates include cell cycle regulatory protein, transcription factor, signal transduction molecule, oncogenic protein, oncostatin, DNA replication regulatory protein and the like. The Cullins protein family (Cullins-1, 2, 3, 4a, 4b, 5, 7, 9, where Cullins-7 and Cullins-9 are less studied) is the backbone protein of CRLs, and Neddylation E3 ligase RBX1 catalyzes the Neddylation modification of Cullins-1, 2, 3, 4a, 4b in the Cullins family by pairing with Neddylation-modified UBE 2M. Similarly, NEDD8E3 ligase RBX2 catalyzes the Neddylation modification of cullin-5 in the cullins family by pairing with Neddylation modified UBE 2F.

In 2009, milnenium Pharmaceuticals, Inc, reported for the first time that adenine nucleotide (AMP) analog MLN4924 was able to inhibit secondary activation of NAE by ATP by competing with ATP for the NAE protein binding pocket, and finally blocking modification of CRLs by Neddylation, through a high-throughput drug screening model. The subsequent drug-protein crystal complex structure also proves that MLN4924 and NEDD8 are covalently bound in NAE adenylylation functional domain (the side chain of methyl sulfamate in MLN4924 structure is bound with cysteine sulfydryl of NEDD 8), and the candidate drug enters the clinical stage III experiment of acute/chronic myelogenous leukemia at present. Depending on the insight of NAE inhibitors, several nucleoside drugs with methyl sulfamate side chains were designed, synthesized and used for screening for Neddylation pathway inhibitory activity. However, the potential metabolic toxicity and easy drug resistance of nucleoside antitumor drugs and almost complete coverage of the core structure by patents may cause that the drugs face huge obstacles in the aspects of patent application and intellectual property protection. Therefore, the development of a non-nucleoside Neddylation antitumor small molecule inhibitor is a necessary trend.

Meanwhile, it has been reported that the combination of Neddylation inhibitor MLN4924 and histone deacetylation reagent can effectively improve the antitumor effect of the drug (Liang Zhou, et al. blood,2016,127,2219). In view of the above, the present invention will also utilize the advantage of Neddylation inhibitors to develop related drug combination experiments.

Disclosure of Invention

The invention utilizes in vitro enzyme activity verification, cell in vivo NEDDyaniton channel blocking experiment and cell activity screening to finally obtain the formula I_AA compound having a blocking pathway for NEDD 8-cullins.

The present invention is directed to providing formula I_AThe compound or the pharmaceutically acceptable salt thereof and the medical application of the medicinal composition thereof, in particular to the application in preventing, delaying or treating diseases involved or mediated by a NEDDylation pathway, especially in the anti-tumor field.

The invention is to broaden I_AProvides a new anti-tumor therapeutic strategy in combination with Histone Deacetylase (HDAC) inhibitors.

To achieve the above object, the present invention provides a compound having the formula I_AA compound of formula (I), or a pharmaceutically acceptable salt thereof:

the synthesis of the above compounds can be obtained from published relevant literature and patents.

It is a further object of the present invention to provide formula I_AThe compound or the pharmaceutically acceptable salt thereof can be applied to the anti-tumor field. Wherein the tumor is lung cancer, liver cancer, gastric cancer, lip cancer, esophageal cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, uterine cancer, gallbladder cancer, laryngeal cancer, cerebroma, squamous carcinoma, hemangioma, prostatic cancer, intestinal cancer, renal cancer, bone cancer, tongue cancer, lymph cancer, pancreatic cancer, bladder cancer, melanoma, leukemia, skin cancer, lipoma, cervical cancer, thyroid cancer and thymus cancer.

The biological activity test result of the invention shows that the provided compound I_AHas the function of inhibiting NEDDylation pathway and simultaneously represents compound I_AHas certain inhibition effect on the growth of tumor cell strains. Therefore, the compound can be used for developing antitumor drugs.

In the present invention, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

"pharmaceutically acceptable acid addition salts" refers to salts with inorganic or organic acids which retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formates, acetates, 2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, caprates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbates, salicylates, 4-aminosalicylates, napadisylates, and the like. These salts can be prepared by methods known in the art.

"pharmaceutically acceptable base addition salts" refers to salts with inorganic or organic bases which maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

In the present invention, "pharmaceutical composition" refers to the compound of the present invention and the field generally accepted for delivery of biologically active compounds to mammals (such as human) medium preparation. The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of active ingredients and exert biological activity.

The term "pharmaceutically acceptable" as used herein refers to a substance (e.g., carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an undesirable biological response or interacting in an undesirable manner with any of the components contained in the composition.

In the present invention, a "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, or emulsifier that is approved by the relevant governmental regulatory agency for human or livestock use.

The terms "preventing", "prevention" and "prevention" as used herein include reducing the likelihood of occurrence or worsening of a disease or condition in a subject.

The term "treatment" and other similar synonyms as used herein include the following meanings:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease or condition, but has not been diagnosed as having the disease or condition;

(ii) inhibiting the disease or disorder, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the state of the disease or condition; or

(iv) Alleviating the symptoms caused by the disease or disorder.

As used herein, the term "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" refers to an amount of at least one agent or compound that is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent after administration. The result may be a reduction and/or alleviation of signs, symptoms, or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound of the present disclosure that is clinically necessary to provide a significant relief from the condition. An effective amount suitable in any individual case can be determined using techniques such as a dose escalation assay.

The terms "administering", "administration", "administering", and the like as used herein refer to a method capable of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, via the duodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Administration techniques useful in The compounds and methods of The present invention are well known to those skilled in The art, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

The terms "drug combination", "administering other treatment", "administering other therapeutic agent" and the like as used herein refer to a drug treatment obtained by mixing or combining more than one active ingredient, including fixed and unfixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one compound of the invention and at least one co-agent to a patient in the form of a single entity or a single dosage form. The term "non-fixed combination" means that at least one compound of the invention and at least one synergistic formulation are administered to a patient simultaneously, in combination, or sequentially at variable intervals as separate entities.

Drawings

FIG. 1 is a graph showing the results of a western blotting-based intracellular NEDDyalton inhibition assay (I)_AShows better intracellular NEDDylation pathway inhibition activity).

FIG. 2 is a graph showing the results of an in vivo tumor cell growth inhibition experiment (demonstration I)_ACombined with histone deacetylation inhibitors, can significantly improve in vivo antitumor activity).

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

1. Western blotting-based intracellular NEDDyalton inhibition assay

(1) Experimental materials:

human lung cancer cell A549, 10% bovine serum culture medium, PBS solution, trypsin (sigma), primary antibody (anti-cullin1 rabbitt, abcom, 2000: 1 dilution), secondary antibody (IgG rabbitt), protein lysate and x 4 SDS-loading.

(2) The experimental method comprises the following steps:

(i) recovery and passage of A549 cells: the cell culture containing A549 was removed from the-80 ℃ freezer and centrifuged (1200rmp, 3 minutes), the supernatant removed, suspended with 2ml of 10% bovine serum medium and transferred to a 10cm petri dish and incubated at 37 ℃ for 24 hours. Selecting cells with good growth, sucking culture solution, washing with PBS, digesting with pancreatin, centrifuging (1200rmp, 3 min), removing supernatant, and adding 2ml of 10% bovine serum culture medium for resuspension;

(ii) inoculating cells: counting the cells, evenly distributing the cells into each culture dish according to 30 ten thousand cells per dish, and incubating overnight in an incubator at 37 ℃;

(iii) test compound (final concentration 100. mu.M) was added and incubated for 6 hours;

(iv) after incubation, quantifying protein;

(iv) preparing gel by 10% SDS-PAGE and performing an electrophoresis experiment;

(v) after 1 hour of membrane conversion, sealing with 20% milk for 1 hour, and adding primary antibody for incubation overnight;

(vi) the membrane was washed and incubated for 1 hour with the addition of secondary antibody.

(3) The experimental results are as follows:

I_Ashows better intracellular NEDDylation pathway inhibition activity, as shown in figure 1.

2. In vitro tumor cell proliferation assay

Evaluation of candidate Compound I Using CCK8_AProliferative activity on human tumor cells.

(1) Experimental materials:

human lung cancer cell A549, human lung cancer cell H1299, human lung cancer cell EKVX, human liver cancer cell SK-HepG1, human liver cancer cell Huh7, human liver cancer cell HepG2, human breast cancer cell T-47D, human stomach cancer cell MKN45, human stomach cancer cell MGC803 and human stomach cancer cell PLC. 10% bovine serum medium, PBS solution, trypsin (sigma), × 10CCK8 (sigma).

(2) The experimental method comprises the following steps:

(i) recovery and passage of A549 cells: the cell culture containing A549 was removed from the-80 ℃ freezer and centrifuged (1200rmp, 3 minutes), the supernatant removed, suspended with 2ml of 10% bovine serum medium and transferred to a 10cm petri dish and incubated at 37 ℃ for 24 hours. Selecting cells with good growth, sucking culture solution, washing with PBS, digesting with pancreatin, centrifuging (1200rmp, 3 min), removing supernatant, and adding 2ml of 10% bovine serum culture medium for resuspension;

(ii) inoculating cells: counting the cells, evenly distributing 3000-5000 cells in each hole into each 96-hole plate, and incubating overnight in an incubator at 37 ℃;

(iii) adding test compounds with different concentrations, and incubating for 24 hours;

(iv) sucking supernatant, adding 100 mu L of 10% CCK8 culture solution into each well, and incubating for 1 hour;

(v) the change of absorbance of each well is detected by a microplate reader under 450nm, and IC is calculated₅₀The value is obtained.

(3) The experimental results are as follows:

the result of the CCK8 proliferation test shows that I_AIn vitro, the proliferation of various tumor cells can be obviously inhibited, and the proliferation inhibiting activity is obviously caused by a positive medicament MLN4924 when 48 hours. As shown in table 1 below.

TABLE 1

3. In vivo tumor cell growth inhibition assay

The in vitro proliferation activity test results demonstrate I_ACan obviously inhibit the proliferation of various human-derived tumor cells. In view of this, the present invention also develops_AIn vivo tumor cell A549 growth inhibition experiment.

(1) Experimental materials:

human lung cancer cell A549, 10% bovine serum culture medium, PBS solution, trypsin (sigma), and female bal/bc mice with 4-5 weeks old;

(2) the experimental method comprises the following steps:

(i) recovery and passage of A549 cells: the cell culture containing A549 was removed from the-80 ℃ freezer and centrifuged (1200rmp, 3 minutes), the supernatant removed, suspended with 2ml of 10% bovine serum medium and transferred to a 10cm petri dish and incubated at 37 ℃ for 24 hours. Selecting cells with good growth, sucking culture solution, washing with PBS, digesting with pancreatin, centrifuging (1200rmp, 3 min), removing supernatant, and adding 2ml of 10% bovine serum culture medium for resuspension;

(ii) inoculating cells: counting the cells, evenly distributing the cells into each culture dish according to 30 ten thousand cells per dish, and incubating overnight in an incubator at 37 ℃;

(iii) centrifuging the suspension cells (centrifuging at 2000rpm for 5min) for collection, washing the cells with PBS twice (centrifuging at 2000rpm for 5min) for collection at 2-3 × 10^{^7}Adding 100 mu L PBS to suspend the cells, mixing uniformly, and storing at room temperature;

(iv) transplanting the 100 mu L of PBS containing the tumor cells into the subcutaneous tissues of the mice, and observing for one week;

(v) after determining the success of tumor inhibition, I is given separately at different doses_A(15mg/kg,30mg/kg),I_AAnd a histone deacetylation inhibitor entinostat, and a blank group, an MLN4924 positive drug group and an HDAC inhibitor group are arranged.

(3) The experimental results are as follows:

I_Aunder the administration dosage of 30mg/kg, the tumor inhibition effect is obviously better than that of a blank group; at the same time, I_AThe tumor volume and size at 30mg/kg dose was not significantly different from 60mg/kg MLN4924, indicating I_AHas good anti-tumor effect in vivo and has small toxic and side effects. Meanwhile, histone deacetylation inhibitors entinostat and I are introduced into the invention_AThe combination administration group of (1) investigates whether the in vivo growth of the tumor can be more effectively inhibited by simultaneously inhibiting NEDDylation targets and histone deacetylation targets. As can be seen from the experimental results, the use of either 10mg/kg entinostat group alone or 15mg/kg I_AThe group showed good in vivo antitumor inhibitory activity, but still was inferior to 30mg/kg I_AGroup and 60mg/kg MLN 492. However, 10mg/kg entinostat and 15mg/kg I_AThe group showed the best in vivo antitumor activity, not only stronger than the group administered alone, but also apparently due to the positive drug group MLN 4924. Demonstration I_ACombined with histone deacetylation inhibitor, the composition can obviously improve the in-vivo antitumor activity. As shown in fig. 2.

4. Conclusion of the experiment

In the present invention, the inventors report that small molecule compound I has Neddylation inhibitory activity_A. In vivo and in vitro experiments prove that_ANot only hasThe strong in vivo and in vitro Neddylation inhibition activity and the anti-tumor experiments show that I_ANot only can obviously inhibit various tumor cells, but also shows better in-vivo anti-tumor activity. By combination with histone deacetylation inhibitors, I_AShows better anti-tumor activity, and no data report about the drug combination mode exists at present. Therefore, the compound provided by the invention can be used for preventing and treating related diseases caused by a Neddylation pathway, particularly tumor-related diseases, and can be used for multi-drug combination.

Claims (8)

1. Formula I_AThe compound or the pharmaceutically acceptable salt and the composition thereof can be used for preparing medicines for preventing, delaying or treating diseases involved or mediated by a NEDDylation pathway.

2. Use according to claim 1, wherein the composition comprises formula I_AThe compound shown in the specification, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. The use according to claim 1, wherein the disease involved in or mediated by the NEDDylation pathway is a tumor or a condition of abnormally high expression of the NEDDylation pathway.

4. Use according to claim 1, characterized in that the use is of the formula I_AThe compound is involved in a multi-drug combination therapy anti-tumor method.

5. The use according to claim 3, wherein the tumor is lung cancer, liver cancer, stomach cancer, lip cancer, esophageal cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, uterine cancer, gallbladder cancer, laryngeal cancer, brain tumor, squamous cancer, hemangioma, prostate cancer, intestinal cancer, kidney cancer, bone cancer, tongue cancer, lymph cancer, pancreatic cancer, bladder cancer, melanoma, leukemia, skin cancer, lipoma, cervical cancer, thyroid cancer or thymus cancer.

6. Use according to claim 3, characterized in that the formula I_AThe compounds shown have the effect of blocking the NEDDylation pathway.

7. The use of claim 4, wherein the multi-drug combination therapy is for anti-tumor treatment of formula I_AThe compound and histone deacetylase are used as main bodies.

8. Use according to claim 5, characterized in that the formula I_AThe compound has obvious effect on proliferation of various tumor cell strains and can accelerate tumor cell apoptosis.

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