CN109293663B - KDM5B inhibitor and preparation method thereof - Google Patents

Abstract

The invention provides a KDM5B inhibitor with a chemical name of 4,5,6, 7-tetrahydro-8H-cyclopenta [ d][1,2,4]Triazolo [1,5-a]A pyrimidin-8-one of the formula

Description

KDM5B inhibitor and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a KDM5B inhibitor and a preparation method thereof.

Background

Cancer is a disease seriously threatening human health and life, and about 1410 ten thousand cancer cases and 820 ten thousand death cases are newly added in 2012 in the world according to the global cancer statistical data published by the world health organization, wherein lung cancer is the most common cancer with the highest death rate in the world. The lung cancer seriously threatens human health, and due to the influence of external factors such as population aging, smoking and environmental pollution, the incidence and the death rate of the lung cancer are far higher than those of other cancers. KDM5B is used as an important histone demethylase, is highly expressed in tissues of lung cancer patients, and is closely related to proliferation, invasion and transfer, poor prognosis, multiple drug resistance and the like of lung cancer. Although KDM5B inhibitors have been reported, none of them enter into clinical development stage. Therefore, the development of a novel lung cancer targeted KDM5B small-molecule compound has great innovation and scientific significance.

Histone lysine-specific demethylases can be divided into the FAD-dependent monoamine oxidase LSD1 and the JMJD family of proteins containing the JmjC domain. LSD1 specifically removes H3K4 and H3K9 single and double methylation, and further activates or inhibits gene expression, while JMJD family protein depends on Fe²⁺And a-ketoglutaric acid (2-OG) catalytic demethylation modification, including multiple subfamilies of KDM2A/B, KDM3A/B, KDM4A-E, KDM5A-D (or JARID1A-D), KDM6A-B, JHDM1D, KDM8 and the like, and selectively catalyzing different substrates. The JMJD family protein JmjC domain is highly conserved with 2 histidines (His) and 1 Glutamines (Glu) in 2-OG and O₂Can catalyze histone methyl lysine to generate unstable hydroxylation intermediate without hydrogen donor, and simultaneously generate 1 molecule of CO₂And succinate, further decomposition of the intermediate to demethylationLysine and formaldehyde. In the JMJD family, KDM5 is divided into four subtypes KAM5A-D, which is the most studied subfamily and specifically removes the H3K4 methylation modification, wherein the research reports that KDM5C/D is associated with tumors are less. The demethylase KDM5 is highly expressed in the lung cancer tissues of patients, is far higher than the tissues beside the cancer and normal tissues, is closely related to the invasion and metastasis of the lung cancer, and the reduction of the expression level of KDM5B induced by siRNA can inhibit the growth, invasion and metastasis of tumor cells. KDM5 is over-expressed in various tumor tissues such as breast cancer, prostate cancer, melanoma, gastric cancer and the like, plays a key role in the processes of proliferation, cycle regulation, metastasis and invasion, differentiation and the like, the expression level of the KDM5 is related to the tumor deterioration degree, and the KDM5 expression level is reduced or knocked out to obviously inhibit the metastasis and invasion of the tumor and the like. Based on the important tumor biological functions of KDM5B and the potential of KDM5B in overcoming tumor drug resistance, the KDM 5B-targeted small molecular compound becomes an important direction in the field of tumor-targeted drug development.

KDM5 is used as an important apparent regulation demethylase, is highly expressed in tissues of a lung cancer patient, is closely related to proliferation, invasion and transfer of the lung cancer, poor prognosis, multiple drug resistance and the like, and KDM5 knockout or small molecule inhibitors can effectively remove drug-resistant cells of the lung cancer, and inhibit the processes of proliferation, transfer, invasion, apoptosis, differentiation and the like. Although KDM5A/B inhibitors with different structural skeleton types are reported, no small molecule inhibitor enters into the clinical development stage due to poor selectivity, undefined action mechanism, obvious off-target effect, lack of intracellular activity or poor PD/PK attribute and the like. Therefore, the development of KDM5B small molecular compounds with high activity, high selectivity and good drug property is an important direction in the research field of tumor-targeted drugs.

Disclosure of Invention

In view of the above, the present invention provides a novel KDM5B inhibitor and a preparation method thereof.

The invention provides a KDM5B inhibitor with a chemical name of 4,5,6, 7-tetrahydro-8H-cyclopenta [ d][1,2,4]Triazolo [1,5-a]A pyrimidin-8-one of the formula

The invention also provides a preparation method of the KDM5B inhibitor, which comprises the following steps: the compound M (3-amino-4-hydrogen-1, 2, 4-triazole) and 2-oxocyclopentanecarboxylic acid methyl ester C₄H₇(CO)₂OCH₃And (2) carrying out reaction at the temperature of 115-125 ℃ under the action of an acidic solvent to synthesize the KDM5B inhibitor, wherein the acidic solvent is organic acid such as glacial acetic acid, hydrochloric acid, sulfuric acid, trifluoroacetic acid or trifluoromethanesulfonic acid. The preparation route of the KDM5B inhibitor is shown as follows:

based on the above, the preparation method of the KDM5B inhibitor further comprises the steps of: 3-amino-4-hydrogen-1, 2, 4-triazole and 2-oxocyclopentanecarboxylic acid methyl ester react completely to obtain KDM5B inhibitor mixed liquor; then removing the acid solvent to obtain a KDM5B inhibitor semi-finished product; drying the KDM5B inhibitor semi-finished product at 55-65 ℃ to obtain a KDM5B inhibitor pure product.

Therefore, the chemical name of the KDM5B inhibitor provided by the invention is 4,5,6, 7-tetrahydro-8H-cyclopenta [ d ] [1,2,4] triazolo [1,5-a ] pyrimidin-8-one, the structural formula of the inhibitor has a group structural unit of pyrimido 1,2, 4-triazole, a series of compounds targeting KDM5B and having anti-tumor activity can be prepared based on the pyrimido 1,2, 4-triazole basic skeleton, and a new way is opened for searching for a novel anti-tumor drug targeting KDM5B as a target. In addition, the synthesis method of the KDM5B inhibitor provided by the invention is feasible, the yield is high, and the yield reaches more than 82%.

Drawings

Fig. 1 is a graph of the inhibition of KDM5B enzymatic activity by the inhibitors provided by the present invention.

Fig. 2 is a graph of the inhibition of KDM5B enzymatic activity by the inhibitors provided by the present invention.

Detailed Description

The technical solution of the present invention is further described in detail by the following embodiments.

The embodiment of the invention provides a KDM5B inhibitor with the chemical name of 4,5,6, 7-tetrahydro-8H-cyclopenta [ d][1,2,4]Triazolo [1,5-a]A pyrimidin-8-one of the formula

The preparation route of the compound is as follows:

1g (about 11.89mmoL) of 3-amino-4-hydro-1, 2, 4-triazole was added to the reaction flask, 20mL of glacial acetic acid was added, then 2.22mL (about 17.84mmoL) of methyl 2-oxocyclopentanecarboxylate was slowly added dropwise and heated to reflux at 120 ℃ and the reaction was monitored by TLC. After the reaction is finished, the reaction system is put into ice water, a large amount of white solid is separated out, and the white solid is directly obtained by suction filtration. And cleaning the white solid with acetone, and drying in an oven at 60 ℃ for 6h to obtain the pure KDM5B inhibitor. The yield is 82.70 percent by weight calculation, and the melting point is 211.2-216.6 ℃ as a white solid;¹H NMR(400MHz,DMSO-d₆)δ13.39(s,1H),8.16(s,1H),2.91(t,J＝7.7Hz,2H),2.68(t,J＝7.3Hz,2H),2.17-1.99(m,2H)；¹³C NMR(100MHz,DMSO-d₆)δ155.83,154.93,152.11,151.43,110.37,31.78,27.21,22.20；HRMS(ESI):m/z calcd for C₈H₉N₄o (M + H) +, 177.07764; found,177.07671, it was thus confirmed that the KDM5B inhibitors provided by the examples of the present invention have the structure shown above.

Determination of inhibitory Activity

The reaction system of the inhibition activity experimental method is 10 mu L, the reaction system comprises detection buffer solution, histone H3 polypeptide substrate, demethylase KDM5B and a compound KDM5B inhibitor to be detected, the reaction is carried out in a 384-pore plate, and the reaction is carried out for 60min at room temperature. Pre-incubating 3 mu L of LKDM5B inhibitor and 4 mu L of demethylase KDM5B at room temperature for 30min, then adding 3 mu L of histone H3 polypeptide substrate, 5 mu L of receptor microbead coated with rabbit antibody and 5 mu L of histone lysine methylation primary antibody; the DMSO content in the reaction system is 1%; incubate slowly with shaking for 30min at room temperature. Finally 10. mu.L of streptavidin-coupled donor beads (Perkin, 1:125 assay buffer diluted 1: 1) were added. Samples were measured in AlphaScreen for 30min (ensspire Alpha2390Multilabel Reader, PerkinElmer).

1.1 Experimental method feasibility verification

KDM5B inhibitor KDOAM-25 reported in literature is selected as a control, and the activity of the inhibitor KDOAM-25 is determined to be 36nM by the inhibitory activity experimental method under the conditions that the enzyme dosage is 100ng and the concentration of H3 polypeptide is 10 mu M, and is consistent with the activity data reported in the literature, thereby verifying the feasibility of the method. Among them, KDM5B Inhibitor KDOAM-25 is reported under the literature name "Power and Selective KDM5Inhibitor stores Cellular Demethylation of H3K4me3at transformation Start Sites and promotion of MM1S Myeloma Cells", by A.Tumber et al, in Cell Chem Biol, 2017,24(3): 371-.

1.2 Activity experiments of KDM5B inhibitors provided in the examples of the invention

The KDM5B inhibitor 4,5,6, 7-tetrahydro-8H-cyclopenta [ d ] [1,2,4] triazolo [1,5-a ] pyrimidin-8-one provided by the embodiment of the invention is hereinafter referred to as a compound I.

The sample is a pure product of the synthesized KDM5B inhibitor compound I, and the sample stock solution: weighing 1-2mg of sample in 1.5mL of EP tube, preparing 10mM solution with DMSO, storing at 4 ℃, and diluting with DMSO to the required concentration during the experiment. The inhibitory activity of compound I on KDM5B was determined using the above inhibitory activity assay, and the assay data are shown in table 1.

TABLE 1 results of inhibitory Activity of Compound I on enzymatic Activity of KDM5B

The inhibition curves of compound I against KDM5B enzyme activity were plotted against the data in table 1, as shown in fig. 1 and 2. As can be seen from fig. 1: half inhibitory concentration IC of Compound I in replicate experiment 1₅₀369.7nM, half-inhibitory concentration IC of Compound I in replicate experiment 2₅₀236.3nM, average IC of both₅₀303.0nM, mean square deviation 66.7; furthermore, the inhibition curves of the compound I in the repeated experiments 1 and 2 are consistent, so that the compound I provided by the embodiment of the invention has an inhibition effect on KDM5B enzyme and can be used as a KDM5B inhibitor.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.

Claims (1)

1. The application of a compound in preparing KDM5B inhibitor is characterized in that the structural formula of the compound is

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