CN108853110A - A kind of compound A-28 is as histone methyltransferase NSD3 activity inhibitor and its application - Google Patents
A kind of compound A-28 is as histone methyltransferase NSD3 activity inhibitor and its application Download PDFInfo
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- CN108853110A CN108853110A CN201810971369.5A CN201810971369A CN108853110A CN 108853110 A CN108853110 A CN 108853110A CN 201810971369 A CN201810971369 A CN 201810971369A CN 108853110 A CN108853110 A CN 108853110A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a kind of compound A-28s as histone methyltransferase NSD3 activity inhibitor and its medicinal usage.The compound A-28 has chemical structure shown in Formulas I：Chemistry is entitled：6-amino-9-(2-(p-tolyloxy)ethyl)-9H-purine-8-thiol；The medicinal usage, which refers to using at least one of the compound A-28 or its hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer, precursor compound, is used to prepare anti-tumor drug as active constituent.Experiment shows that NSD3 enzymatic activity can be effectively suppressed in compound A-28 of the present invention, and the horizontal IC50 value of zymetology is 0.69 ± 0.06 μm of ol/L；And it can obviously inhibit the growing multiplication of Lines H460.Compound of the present invention has the function of inhibiting tumor cell proliferation, it is expected to be used to prepare anti-tumor drug as active constituent, have prospect in medicine.
Description
Technical field
The invention belongs to pharmaceutical technology fields, are specifically related to a kind of histone methyltransferase NSD3 activity inhibitor
And its application.
Background technique
Histone methylated is one of most important modified mechanism of epigenetic modification.Histone methylated and its regulation person
The generation of a variety of diseases such as the unconventionality expression of histone methyltransferase and hereditary disease, autoimmune disease, aging and cancer
It is closely related, especially it is of great significance in the occurrence and development of tumour.Therefore, histone methyltransferase is considered as potential
Antineoplaston novel targets.
In recent years, it made breakthrough progress by the research and development of the inhibitor of target spot of histone methyltransferase.Currently, group
Protein methyltransferase EZH2 inhibitor EPZ-6438 (Epizyme company) entered I/II clinical trial phase in 2014, was used for
Treat non-Hodgkin lymphoma, advanced malignance and malignant mesothelioma patient；Histone methyltransferase DOT1L inhibitor
EPZ-5676 (Epizyme company) also entered Phase I clinical trial in 2014, for treating acute leukemic patient.In addition, also
There is the micromolecular inhibitor of multiple histone-lysine methyltransferases, such as SETD7 inhibitor PFI-2, G9a (EHMT2) and GLP
(EHMT1) inhibitor UNC0638, A-366, SMYD2 inhibitor LLY-507 etc. is in the preclinical study stage, these researchs pair
It is most important in the research and development of antineoplastic target therapeutic agent, it has broad application prospects.Histone methyltransferase NSD3 (also known as
WHSC1L1, Wolf-Hirschhorn syndrome candidate1-like1) the 36th lysine of histone H 3 can be made
(H3K36) di-methylation (me2) and tri-methylated (me3).The NSD3 assignment of genes gene mapping on the position human chromosome 8p11.23, with
The occurrence and development of Several Kinds of Malignancy have important relationship.Firstly, 8p11-12 chromosome interval where NSD3 is in breast cancer,
It is highly expanded in the cancers such as lung cancer, cancer of pancreas and neoplastic hematologic disorder, disclosing the section and tumor development has strong correlation.
Secondly, multiple studies have shown that NSD3 gene itself plays a significant role in the occurrence and development of malignant tumour.In breast cancer, bladder
NSD3 is found highly to express in the tumours such as cancer, Head and neck squamous cell carcinoma, has the function of promoting tumour formation；Acute myelogenous
T (8 is found in leukaemic；11)(p11.2；P15) NUP98-NSD3 fusion, specific mechanism of action are unclear；
Find that NSD3-NUT fusion, the fusion protein of expression pass through in NUT center line cancer (NUT midline carcinoma)
It plays an important role in terms of tumor cell differentiation retardance and proliferation in conjunction with BRD4 albumen；In acute myelocytic leukemia
(AML) in cell, the NSD3 isomers for containing only first, the end N PWWP structural domain mediates BRD4-CHD8 protein to combine, directly
Connect the proliferation and differentiation for influencing tumour cell.
NSD3 is by 1 SET (Su (var), Enhancer of zeste, and Trithorax) structural domain, 2 PWWP
(Pro-Trp-Trp-Pro motif) structural domain and 4 PHD structural domain (Plant Homeodomain) compositions.Wherein SET is tied
Structure domain is catalytic center, PWWP and PHD structural domain usually participates in many biologies such as the relevant transcriptional control of chromatin and DNA reparation
Process.
So far, about more than 60 kinds of the histone methyltransferase of discovery rely ammonia including more than 50 kinds of histones
Acid methyltransferase.In the histone-lysine methyltransferase having found, there is 50% methyl transferase activity to pass through reality
Proved recipe method is verified, and most of closely related with tumour generation.However, in histone-lysine methyltransferase inhibitor
In research field, most of research focuses primarily upon the histones such as EZH1 and EZH2, DOT1L, SETD7, EHMT1 and EHMT2 and relies
Propylhomoserin transmethylase has been found the histone methyltransferase closely related with human tumor occurrence and development for other
It studies in contrast less.Histone methyltransferase NSD3 is particularly significant during tumor development, is one potential
Antineoplastic new target.Consequently found that the NSD3 inhibitor of high activity is especially heavy for application of the NSD3 in targeting cancer therapy
Will with it is urgent, for these histone methyltransferase find high activity Non-specific inhibitor for antineoplastic target medicine
Object research and development are of great significance.
Summary of the invention
The present invention provides a kind of histone methyltransferase NSD3 activity inhibitor, the inhibitor be compound of formula I or
Its pharmaceutically acceptable salt
The present invention provides new opplication of the inhibitor in preparation tumor.
The present invention also provides new opplication of the compound of formula I in preparation tumor.
Preferred tumour of the present invention is lung cancer, breast cancer, cancer of pancreas, osteosarcoma, head-neck carcinoma, and the lung cancer is preferred
For non-small cell lung cancer.
Preferred drug of the present invention be its hydrate of compound of formula I, pharmaceutically acceptable salt, tautomer,
Drug made of stereoisomer or precursor compound and one or more pharmaceutically acceptable carriers.The carrier includes medicine
The diluent of field routine, excipient, filler, adhesive, wetting agent, disintegrating agent, sorbefacient, surfactant,
Absorption carrier, lubricant etc..
Injection, tablet, pulvis, granule, pill, capsule, oral solution, paste, creme can be made in drug of the present invention
Etc. diversified forms.The drug of above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.
The drug is to treat tumour by inhibition of histone transmethylase NSD3 activity, can by injection, injection,
Collunarium, eye drip, infiltration, absorption, the method physically or chemically mediated import body such as muscle, intradermal, subcutaneous, vein, mucous membrane group
It knits；Or body is imported after other material mixings or package.
Virtual screening is integrated in present invention use and the method for measuring is found and found from from ChemDiv compound library
Compound of formula I (hereinafter referred to as A8) can inhibit NSD3 enzymatic activity, and the anti-tumor activity of combination cell experimental verification compound,
Although the research also prematurity about inhibitor A8 is to clinical stage is entered, these researchs are for finally developing anti-NSD3 medicine
Object is most important, with important application prospects.
The present invention carries out prior assessment using validity of the computer simulation method to drug molecule, and real using biology
Proved recipe method detects and verifies activity, obtains effective NSD3 enzyme inhibitor A8 (chemical name：6-amino-9-(2-(p-
Tolyloxy) ethyl) -9H-purine-8-thiol), the horizontal IC50 value of zymetology is 0.69 ± 0.06 μm of ol/L；To H460
Lines, which have, inhibits growth, and IC50 value in 5~10 μm of ol/L, has good NSD3 respectively
Enzyme inhibitory effect.
Detailed description of the invention
Fig. 1：IC50 value figure of the A8 on NSD3 protein level.
Fig. 2：A8 inhibits NSD3 enzymatic activity in the cell.
Fig. 3：A8 is to H460 non-small cell lung cancer cell growth inhibition effect.
Specific embodiment
In order to confirm the antitumous effect of the compounds of this invention, the present invention is done below in conjunction with the accompanying drawings and the specific embodiments
Further description out.
1. experimental method
1.1 virtual screenings based on receptor
It uses firstAlbumen preparation module Protein Preparatio n Wizard couple in software package
Crystal structure (the PDB of NSD3：4YZ8) handled.Using Discov ery Studio 2.5 to ChemDiv database
The pretreatment of object, including duplicate removal, removal salt ion and inorganic matter are closed, and carries out construction standard.Using
LigPrep module in 9.0 generates the possibility ionization state and tautomer of compound under the conditions of pH=7.4.
Before carrying out virtual screening using molecular docking method, it is necessary first to Glide interconnection method used by verifying
Validity, define NSD3 active site, with the matter of ligand molecular S-adenosyl methionine (SAM) in crystal structure
Centered on the heart, setting Square body region, using the SP (Standard of Glide software
Precision) parameter setting after similarly using LigPrep to handle ligand molecular SAM, is docked to NSD3 activity again
In pocket, discovery Glide can preferably reappear the combination conformation in crystal structure.
Using Glide HTVS (High Throughput Virtual Screening) mode to the library ChemDiv chemical combination
Object is docked and is given a mark, and 300,000 forward compounds of giving a mark are selected；It is docked again using Glide SP mode again
And marking, retain 30,000 forward small molecule binding patterns of giving a mark.In the crystal structure of the combination of SAM and NSD3, with activity
Region residue HIS1224, HIS1274 form three crucial interaction of hydrogen bond.30,000 small molecules generated for docking
Binding pattern takes hydrogen bond criteria as screening conditions, selects and HIS1224, HIS1274 form the combination of 2 or more hydrogen bonds
Mode obtains 697 qualified compound molecules.In order to fully consider the structure diversity of compound, use
Canvas module in Schrodinger carries out clustering, selects a collection of compound and carries out measuring.Discoverable type Iization
Object is closed, (hereinafter referred to as A8) has preferable histone methyltransferase inhibitory activity.
Compound of formula I chemical structural formula (compound of formula I hereinafter referred to as A8)
The external Enzyme assay experimental procedure of 1.2NSD3
1) expression and purification of NSD3 albumen：It is expanded from HEK293 cell line cDNA by PCR method and obtains the part NSD3
Gene (1021-1320aa), is subcloned in prokaryotic expression carrier pGEX-4T1, constructs the recombinant plasmid with GST label,
After being sequenced and identifying, conversion inducing expression in Escherichia coli Rossetta (after 0.5mM IPTG induction, is persistently trained at 16 DEG C
Support 20 hours), purified with GST resin, obtains the NSD3 albumen of GST label.
2) the external Enzyme assay step of NSD3:
1. compound and 2.3 μ g NSD3 albumen are mixed, and placed 15 minutes at 30 DEG C；
2. being separately added into histone H 3 K36me1 polypeptide fragment
(ATKAARKSAPATGGV-K (Me1)-KPHRYRPG-GK (Biotin)) (ultimate density is 0.2 μM) and S-
Adenosyl methionine SAM (ultimate density be 1.0 μM), and 50mM Tris-HCl pH8.5,50mM NaCl,
In 5mM MgCl2,1mM DTT and 0.01%Tween reaction solution, 30 DEG C are reacted 1 hour；
3. in conjunction with cisbio HTRF histone methyltransferase detection kit, using multi-function microplate reader in 620nm and
The corresponding fluorescence values of 665nm wavelength detecting calculate NSD3 activity.
1.3 intracellular NSD3 Enzyme assay experiments
1) compound is added in 6 orifice plates repopulating cell after 16 hours, and is split after continuing culture 48 hours with RIPA lysate
It solves cell and harvests protein sample；Protein immunoblotting measuring H3K36me3 and H3 protein expression.
The experiment of 1.4 cellular level tumor suppressions
1) growth of tumour cell inhibiting rate：5000 cells are planted in 96 orifice plates, control DMSO or not is added after 16 hours
With the compound of concentration, continue to cultivate in 37 DEG C, 5% carbon dioxide incubator；Dojindo cell is added after 72 hours
Counting kit-8 reagent, 37 DEG C reaction 1 hour after 450nm measure absorbance, finally measure the tumour cell of the compound
Inhibiting effect
2. experimental result
The activity inhibition that A8 is directed to histone methyltransferase NSD3 on albumen and cellular level is determined, and
A8 is directed to the inhibitory effect of H460 non-small cell lung cancer cell growing multiplication on a cellular level.
A8 has good NSD3 enzyme inhibitory effect, IC in albumen zymetology level in vitro50Value is 0.69 ± 0.06 μM,
See Fig. 1.
0,2.5,5 μM of compound A-28 is respectively adopted and handles H460 and H1299 cell respectively 48 hours, detection compound exists
Into the cell to the inhibiting effect of H3K36me3 modification, histone H 3 is reference protein.The result shows that with pair that compound is not added
It is compared according to group, under 2.5 μM of concentration, compound distinguishes the inhibiting rate of H3K36me3 modification in H460 and H1299 cell strain
It is 37.3%, 38.1%；Under 5 μM of concentration, compound is in H460 and H1299 cell strain to the inhibiting rate of H3K36me3 modification
Respectively 87.7% and 70.0%, illustrate that A8 can effectively inhibit NSD3 enzymatic activity on a cellular level, reduces H3K36me3 and repair
Decorations, are shown in Fig. 2.
The A8 for detecting various concentration tests the Tumor growth inhibition of non-small cell lung cancer cell H460, A8 concentration gradient point
It is not 0,2.5,5.0,10.0 and 20.0 μM.Increase the result shows that A8 grows cell in H460 Lines
It is obvious to grow inhibitory effect, and inhibiting effect increases with activity and enhanced, it is statistically significant compared with the control group (different dense
The A8 of degree is respectively 21.7%, 41.3%, 48.3% and 54.5%, P to inhibition rate of tumor cell<0. 05), sees Fig. 3.
To sum up, compound A-28 of the present invention is effective histone methyltransferase NSD3 inhibitor, and A8 is in the cell
NSD3 enzymatic activity can obviously be inhibited, H3K36me3 level is reduced, also have significantly to the growing multiplication of Lines
Inhibiting effect.It can be seen that A8 compound of the present invention can obviously inhibit tumour as histone methyltransferase inhibitor
Cell Proliferation has potential antitumor action, it is expected to be used to prepare anti-tumor drug as active constituent, especially be expected to be used for
The drug of anti-lung cancer is prepared, there is prospect in medicine.
Claims (8)
1. a kind of histone methyltransferase NSD3 activity inhibitor, which is characterized in that the inhibitor be compound of formula I or its
Hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer, precursor compound
2. application of the inhibitor according to claim 1 in preparation tumor.
3. compound of formula I according to claim 1 or its hydrate, pharmaceutically acceptable salt, tautomer, solid
The application of isomers, precursor compound in preparation tumor.
4. application according to claim 3, which is characterized in that the tumour is lung cancer, breast cancer, cancer of pancreas, bone and flesh
Tumor, head-neck carcinoma.
5. application according to claim 4, which is characterized in that the lung cancer is non-small cell lung cancer.
6. application according to claim 3, which is characterized in that the drug is compound of formula I, its hydrate, pharmaceutically
Acceptable salt, tautomer, stereoisomer or precursor compound and one or more pharmaceutically acceptable carrier systems
At drug.
7. application according to claim 6, which is characterized in that the drug is by inhibition of histone transmethylase
NSD3 activity treats tumour.
8. application according to claim 6, which is characterized in that the dosage form of the drug be tablet, capsule, granule,
Pill or other regular dosage forms that can be prepared.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021028854A1 (en) * | 2019-08-14 | 2021-02-18 | Novartis Ag | Piperidinyl-methyl-purineamines as nsd2 inhibitors and anti-cancer agents |
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| EP2277595A2 (en) * | 2004-06-24 | 2011-01-26 | Novartis Vaccines and Diagnostics, Inc. | Compounds for immunopotentiation |
| CN105198828A (en) * | 2015-08-26 | 2015-12-30 | 中国药科大学 | Heterocyclic anthracene ketone histone methyltransferase inhibitor and medical application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2277595A2 (en) * | 2004-06-24 | 2011-01-26 | Novartis Vaccines and Diagnostics, Inc. | Compounds for immunopotentiation |
| CN1947717A (en) * | 2005-10-14 | 2007-04-18 | 卓敏 | Method for treating neuronal and non-neuronal pain |
| CN105198828A (en) * | 2015-08-26 | 2015-12-30 | 中国药科大学 | Heterocyclic anthracene ketone histone methyltransferase inhibitor and medical application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021028854A1 (en) * | 2019-08-14 | 2021-02-18 | Novartis Ag | Piperidinyl-methyl-purineamines as nsd2 inhibitors and anti-cancer agents |
| CN114585622A (en) * | 2019-08-14 | 2022-06-03 | 诺华股份有限公司 | Piperidinyl-methyl-purinamines as NSD2 inhibitors and anticancer agents |
| US11420970B1 (en) | 2019-08-14 | 2022-08-23 | Novartis Ag | Piperidinyl-methyl-purineamines as NSD2 inhibitors and anti-cancer agents |
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Effective date of registration: 20210129 Address after: Room 402-403, 4th floor, science and technology transformation building, No.3 Meishan Road, Xuejia Town, Xinbei District, Changzhou City, Jiangsu Province 213001 Patentee after: PMR (Changzhou) Biotechnology Co.,Ltd. Address before: 213001 No. 1801 Wu Cheng Road, Changzhou, Jiangsu Patentee before: JIANGSU University OF TECHNOLOGY |