CN116655638B - Deuterated PRMT5 inhibitors - Google Patents

Deuterated PRMT5 inhibitors Download PDF

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CN116655638B
CN116655638B CN202310523973.2A CN202310523973A CN116655638B CN 116655638 B CN116655638 B CN 116655638B CN 202310523973 A CN202310523973 A CN 202310523973A CN 116655638 B CN116655638 B CN 116655638B
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inhibition
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CN116655638A (en
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付家胜
覃华
朱伟波
孙大庆
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Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Abstract

The present invention provides a deuterated PRMT5 kinase inhibitor, pharmaceutical compositions containing the compounds and methods of treating cell proliferative disorders, such as cancer, using the compounds of the invention.

Description

Deuterated PRMT5 inhibitors
The present application claims priority from the chinese patent office filed at 2022, 5/12, under the name CN202210511776.4, entitled "deuterated PRMT5 inhibitor", the entire contents of which are incorporated herein by reference.
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a quinoline-substituted PRMT5 inhibitor, a pharmaceutical composition containing the compound and a method for treating cell proliferation diseases such as cancers by using the compound.
Background
Protein arginine methyltransferases (protein arginine methyltransferase, PRMTs) play an important role in protein methylation, such as being involved in variable cleavage, post-transcriptional regulation, processing of RNA, cell proliferation, cell differentiation, apoptosis, and tumor formation, among others. Currently, 11 members of this family (PRMTs 1-11) have been identified, and PRMTs can be classified into 3 classes depending on the way they catalyze arginine methylation: form I includes PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8, catalyzed forms of monomethyl (MMA) and asymmetric dimethyl (acma); type II is symmetrical dimethyl (sDMA), including PRMT5 and PRMT9; type III is PRMT7.
PRMT5 was first isolated in a protein complex that interacted with Janus tyrosine kinase 2 (Jak 2), and is therefore also known as Jak binding protein 1 (JBP 1). PRMT5, an epigenetic enzyme, symmetrically methylates arginine residues of histone or non-histone substrates, affects multiple target genes and multiple signaling pathways, and thus performs multiple biological functions. Studies show that PRMT5 is also an oncogene, is highly expressed in various tumors, and the expression level is closely related to the occurrence, development and prognosis of the tumors.
The metabolic pathway in which CYP450 enzymes participate is a very important metabolic pathway in the clearance process of compounds in vivo, inhibition of the activity of this family of enzymes can bring about changes in clearance and pharmacokinetics in the body of drugs, and drug-drug interactions (DDI) mediated by CYP450 enzymes is an important factor in drug efficacy and drug safety considerations (J Pharmacol Exp Ther.2006Jan [ J ].2006,316 (1): 336-48). CYP3A4 is a major metabolizing enzyme of the CYP450 family, and inhibition of DDI by CYP3A4 can lead to serious safety problems (The AAPS Journal volume 24,Article number:12 (2022)). CYP3A4 is the P450 isozymes with the most abundant content in the liver and intestinal walls, and can participate in the metabolism of about 50% of clinical drugs, and drugs (drug) for inhibiting or inducing CYP3A4 can influence the pharmacokinetics of other drugs used in combination when used in combination clinically, so that the exposure of the drugs in plasma is influenced to cause pharmacokinetic DDI. Inhibitory (including reversible, mechanistic inactivation) drug interactions have increased efficacy, but drugs with a narrower therapeutic window are prone to cause clinical adverse reactions, which can be life threatening in severe cases. And the pharmacokinetics DDI caused by induction causes less medication safety problem, but can reduce the curative effect of the medicine. Therefore, predicting DDI likely to be caused by a new drug is important in evaluating candidate drug properties in the development of new drugs (Zhang Qing et al: quantitative prediction of in vivo drug-drug interactions based on in vitro CYP3A4 inhibition and induction data: pharmaceutical journal Acta Pharmaceutica Sinica [ J ] 2010,45 (8): 952-959).
Inhibition of P450 enzymes by drugs is generally classified into reversible inhibition (including competitive inhibition, non-competitive inhibition, and anti-competitive inhibition) and irreversible inhibition. The reversible inhibitor forms a complex with the enzyme (or enzyme-substrate complex) by non-covalent bond, prevents the normal enzymatic reaction between the enzyme and the substrate, and can continue to perform normal enzymatic reaction with the substrate without being affected by the enzyme activity after the inhibitor is removed. In irreversible inhibition, the inhibitory effect of the inhibitor on the enzyme does not disappear immediately after removal of the inhibitor, but exhibits a time-dependent characteristic. This phenomenon is commonly referred to as time-dependent suppression (TDI).
Drugs have a variety of mechanisms for the production of TDI by P450 enzymes, of which the mechanistic inhibition (mechanism based inhibition, MBI) is the most important mechanism of TDI, i.e. the inhibitor can be converted via CYP mediation to electrophilic reactive metabolites (reactive metabolite) which can interact with the enzyme (mainly in a covalently bound form) resulting in a change in the enzyme structure to inactivate it. The production of MBI requires a process of metabolism of the P450 enzyme, so that inhibition of the P450 enzyme by the inhibitor requires a certain time, and synthesis of a new P450 enzyme in vivo requires a certain time (generally 4-7 d), so that inhibition may occur for a certain period of time even if the inhibitor is removed. Compared with reversible inhibition, TDI brings more serious medication safety problems, because many medicines needing combined medication need to be taken for a long time, which tends to cause the inhibited CYP subtype to be inhibited for a long time, and even if medicines producing TDI are stopped, the inhibition effect can still last for a period of time; meanwhile, since the MBI-producing inhibitor is also a substrate of the P450 enzyme, after the enzyme activity is inhibited, the metabolism of the enzyme is also blocked, so that the exposure in the body is increased unevenly; furthermore, covalent modification of the P450 enzyme by reactive metabolites may lead to hapten production, may cause autoimmune reactions, and have serious consequences (Xie Shanshan et al. Time-dependent inhibition studies of cytochrome P450 enzymes and their role in the development of new drugs [ J ]. J. Chinese New drug & clinical J. Chin J New Drugs Clin Rem,2013,32 (6): 419-424). At present, the PRMT5 inhibitor enters clinical medicines including GSK-3326595, JNJ-64619178, PF-06939999 and the like, and more safe PRMT5 kinase inhibitors still need to be developed.
Disclosure of Invention
The invention provides a compound or pharmaceutically acceptable salt shown in a formula (A),
wherein R is 1 Is NH 2 ；
R 2 Is halogen;
L 1 selected from-CH 2 -CH 2 -or-ch=ch-;
R 3 selected from C 1-4 Alkyl or hydroxy;
m is selected from 0, 1, 2 or 3;
R 4 is NH 2 。
Certain embodiments of the present invention, said R 2 Is Br.
Certain embodiments of the present invention, said R 3 Selected from methyl or hydroxy.
The present invention also provides a compound or pharmaceutically acceptable salt represented by formula (A-1):
wherein R is 9 Selected from H or methyl, R 1 、R 2 、R 4 、L 1 As defined by formula (a).
In certain embodiments of the invention, the compound or pharmaceutically acceptable salt thereof is selected from:
the invention also provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The invention also provides application of the compound shown in the formula or pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a medicament for treating cancer.
The compound has good inhibition effect on PRMT5 methylase and human pancreatic cancer cell line MIA PaCa-2, has the potential advantage of higher cardiac safety, has small inhibition effect on CYP3A4 enzyme, has lower possibility of drug interaction (DDI), has lower risk of time-dependent inhibition (TDI), and has good pharmacokinetic property. Compared with the prior art, the compound has higher safety.
Interpretation of the terms
The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense.
The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present invention prepared with relatively non-toxic acids or bases. These salts may be prepared during synthesis, isolation, purification of the compound, or the purified compound may be used alone in free form to react with a suitable acid or base. When the compound contains a relatively acidic functional group, reaction with an alkali metal, alkaline earth metal hydroxide or organic amine gives a base addition salt, including cations based on alkali metal and alkaline earth metal. When the compound contains a relatively basic functional group, it is reacted with an organic acid or an inorganic acid to give an acid addition salt.
The compounds of the present invention exist as isomers, such as cis-trans isomers, enantiomers, diastereomers, as well as racemic and other mixtures thereof, all of which are within the scope of the present invention.
The term "enantiomer" refers to stereoisomers that are mirror images of each other.
The term "diastereoisomer" refers to a stereoisomer of a molecule having two or more chiral centers and having a non-mirror image relationship between the molecules.
The term "cis-trans isomer" refers to a configuration in which a double bond or a single bond of a ring-forming carbon atom in a molecule cannot rotate freely.
Unless otherwise indicated, with solid wedge bondsAnd wedge-shaped dotted bond->Representing the absolute configuration of a stereogenic center.
Stereoisomers of the compounds of the invention may be prepared by chiral syntheses or chiral reagents or other conventional techniques. For example, one enantiomer of a compound of the invention may be prepared by asymmetric catalytic techniques or chiral auxiliary derivatization techniques. Or by chiral resolution techniques, a single configuration of the compound is obtained from the mixture. Or directly prepared by chiral starting materials. The separation of the optically pure compounds in the invention is usually accomplished by using preparative chromatography, and chiral chromatographic columns are used to achieve the purpose of separating chiral compounds.
The invention also includes isotopically-labeled compounds comprising isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, e.g. 2 H、 3 H、 13 C、 11 C、 14 C、 15 N、 18 O、 17 O、 31 P、 32 P、 35 S、 18 F and F 36 Cl. Compounds of the present invention containing the above isotopes and/or other isotopes of other atoms are within the scope of this invention. Preferably, the isotope is selected from the group consisting of: 2 H、 3 H、 11 c and C 18 F. More preferably, the radioisotope is 2 H. Specifically, deuterated compounds are intended to be included within the scope of the present invention.
Where the bond of a substituent may be cross-linked to a ring, the substituent may be bonded to any atom on the ring. For example, structural unitsRepresents a substituent R 3 Substitution may occur at any position on ring a.
The term "pharmaceutically acceptable carrier" refers to a medium commonly accepted in the art for delivery of biologically active agents to animals, particularly mammals, and includes adjuvants, excipients or vehicles, such as diluents, preservatives, fillers, flow modifying agents, disintegrants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, and dispersing agents, depending on the mode of administration and the nature of the dosage form. Pharmaceutically acceptable carriers are formulated within the purview of one of ordinary skill in the art according to a number of factors. Including but not limited to: the type and nature of the active agent formulated, the subject to which the composition containing the agent is to be administered, the intended route of administration of the composition, and the therapeutic indication of interest. Pharmaceutically acceptable carriers include both aqueous and nonaqueous media and a variety of solid and semi-solid dosage forms. Such carriers include many different ingredients and additives in addition to the active agent, and additional ingredients included in the formulation for a variety of reasons (e.g., stabilizing the active agent, adhesive, etc.) are well known to those of ordinary skill in the art. The term "excipient" generally refers to the carrier, diluent, and/or medium required to make an effective pharmaceutical composition.
The term "prophylactically or therapeutically effective amount" means that the compound of the invention, or a pharmaceutically acceptable salt thereof, is a sufficient amount of the compound to treat a disorder at a reasonable effect/risk ratio applicable to any medical treatment and/or prophylaxis. It will be appreciated that the total daily amount of the compounds of formula (I) or pharmaceutically acceptable salts and compositions of the present invention will be determined by the physician within the scope of sound medical judgment. For any particular patient, the particular therapeutically effective dose level will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the specific composition employed; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular compound employed; duration of treatment; a medicament for use in combination with or simultaneously with the particular compound employed; and similar factors well known in the medical arts. For example, it is common in the art to start doses of the compound at levels below that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
The term "optionally substituted" means that the species and number of substituents may be any on the basis of being chemically realizable, unless otherwise indicated, e.g., the term "optionally substituted with one or more R 1c Substituted "means that one or more R's may be substituted 1c Substituted or not by R 1c And (3) substitution. When any variable (e.g. R 1c ) Where the composition or structure of a compound occurs more than once, its definition is independent in each case. For example, if a group is substituted with 0-2R 1c Substituted, the radicals may optionally be substituted by up to two R 1c Substituted, and R in each case b There are independent options.
The term "alkyl" is used to denote a straight or branched saturated hydrocarbon group unless otherwise specified. Preferably C 1-6 More preferably C 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, n-hexyl and the like.
The term "halogen" means a fluorine, chlorine, bromine or iodine atom unless otherwise specified.
It is specifically stated that combinations of all substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
In the examples of the present invention, the title compound is named after the compound structure is converted by Chemdraw. If the compound name is inconsistent with the compound structure, the compound name can be determined in an auxiliary way by combining the related information and the reaction route; cannot be confirmed by other methods, and the structural formula of the given compound is subject to. The preparation method of some compounds in the present invention refers to the preparation method of the aforementioned analogous compounds. It will be appreciated by those skilled in the art that the ratio of the reactants, the reaction solvent, the reaction temperature, etc. may be appropriately adjusted depending on the reactants when using or referring to the preparation method to which they are applied.
The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, but is not meant to be limiting in any way. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the invention without departing from the spirit and scope of the invention.
Summary of the laboratory instruments:
the structure of the compounds of the present invention is determined by Nuclear Magnetic Resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS), or ultra-efficient liquid chromatography-mass spectrometry (UPLC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR was performed using Bruker Neo 400M or Bruker Assend 400 nuclear magnetic instruments with deuterated dimethyl sulfoxide (DMSO-d) 6 ) Deuterated methanol (CD) 3 OD) and deuterated chloroform (CDCl) 3 ) Heavy water (D) 2 O), internal standard is Tetramethylsilane (TMS).
LC-MS was performed using Agilent 1260-6125B single quadrupole mass spectrometer for determination of LC-MS, column Welch Biomate column (C18, 2.7 μm, 4.6X150 mm) or waters H-Class SQD2, column Welch Ultimate column (XB-C18, 1.8 μm, 2.1X150 mm) mass spectrometer (ion source electrospray ionization).
Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was performed using a Waters UPLC H-class SQD mass spectrometer (electrospray ionization as the ion source).
HPLC determinations used Waters e2695-2998 or Waters ARC and Agilent 1260 or Agilent Poroshell HPH high performance liquid chromatography.
The thin layer chromatography silica gel plate uses smoke table Jiang You silica gel to develop GF254 silica gel plate of the limited company or GF254 silica gel plate of the new material of the limited company on the opal market, the specification adopted by TLC is 0.15 mm-0.20 mm, the preparation is 20 multiplied by 20cm, and column chromatography is generally used for forming 200-300 mesh silica gel as a carrier.
The starting materials in the examples of the present invention are known and commercially available or may be synthesized using or according to methods known in the art.
All reactions of the invention were carried out under continuous magnetic stirring under dry nitrogen or argon atmosphere, with the solvent being a dry solvent, and the reaction temperature being in degrees celsius or in degrees celsius, without specific description.
The examples of the preparation of the compounds of this application, in part, result in trifluoroacetate salts, and those skilled in the art will appreciate that the preparation of compounds by trifluoroacetate salts, or by compounds, is a relatively conventional means and that the compounds of the trifluoroacetate structures disclosed herein, or the preparation thereof, may be regarded as equivalent to the disclosed free-form compounds structures and the preparation thereof.
Abbreviations used in the examples of the present invention and their corresponding chemical names are as follows:
abbreviations (abbreviations) Chemical name
9-BBN 9-borabicyclo (3, 1) -nonane
THF Tetrahydrofuran (THF)
PdCl 2 (dppf) [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride
Boc Boc-group
TBDPS Tertiary butyl diphenyl silicon base
TFA Trifluoroacetic acid
Et 3 Si Triethylsilyl group
An intermediate: int-1 preparation method
3-bromo-7-iodoquinolin-2-amine
The reaction route is as follows:
the operation steps are as follows:
step A7-nitroquinoline (25 g,143 mmol) and acetic acid solution (50 mL) were added to the reaction flask. The displacement reaction system was nitrogen and then cooled to c. N-bromosuccinimide (50.6 g, 284 mmol) was added dropwise to the reaction system, and the reaction system was stirred at 100℃for 2 hours.
After TLC monitoring showed the disappearance of starting material, the reaction solution was quenched by addition of saturated aqueous ammonium chloride solution. The reaction mixture was extracted with ethyl acetate (500 mL. Times.3), and the organic phases were combined, washed with saturated brine (300 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3-bromo-7-nitroquinoline (24.5 g).
MS(ESI)M/Z:253.0[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ9.18(d,J＝2.0Hz,1H),8.97(s,1H),8.79(t,J＝11.4Hz,1H),8.41(dd,J＝9.0,2.0Hz,1H),8.23(d,J＝9.0Hz,1H).
Step B3-bromo-7-nitroquinoline (24.5 g,97 mmol) and ammonium chloride (20.0 g,390 mmol) were added to a reaction flask, dissolved in a mixed solvent (ethanol/water=1.5/1, volume ratio, 250 mL), zinc powder (65.4 g,488 mmol) was slowly added at room temperature, and the system was replaced with nitrogen atmosphere. After the completion of the dropwise addition, the reaction system was stirred at 80℃for 2 hours.
After TLC monitoring showed the disappearance of starting material, water was slowly added to the reaction solution for quenching. Dichloromethane (50 mL) and methanol (5 mL) were then added, stirred for 30 minutes, and the reaction solution was filtered. The filtrate was concentrated to give an oily crude product, which was purified by silica gel column chromatography to give 3-bromoquinolin-7-amine (6.2 g)
MS(ESI)M/Z:223.0[M+H] + 。
Step C: to the reaction flask was added sulfuric acid (108 mL) followed by a slow addition of a solution of 3-bromoquinolin-7-amine (6.2 g,27.8 mmol) at 86 mL. After stirring at 0deg.C for 10 min, a solution of sodium nitrite (3.8 g,55.6 mmol) was slowly added dropwise (6 mL). Subsequently stirring was continued for 30 minutes, and a solution of sodium iodide (12.5 g,83.4 mmol) was slowly added to the reaction system at 0 ℃. The reaction solution was stirred at 60℃for 2 hours.
After LCMS monitoring showed the disappearance of starting material, the reaction was cooled to room temperature, ethyl acetate (50 ml) was added to the reaction, stirred for 10 minutes, then filtered, the filter cake was repeatedly washed with a mixed solution of dichloromethane and methanol, the filtrates were combined, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 3-bromo-7-iodoquinoline (4 g).
MS(ESI)M/Z:333.8[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ9.16(dd,J＝5.3,2.3Hz,1H),8.96(t,J＝3.6Hz,1H),8.82(dd,J＝7.8,2.2Hz,1H),8.40(dd,J＝9.0,2.3Hz,1H),8.23(d,J＝9.0Hz,1H).
Step D: 3-bromo-7-iodoquinoline (500 mg,1.5 mmol) was dissolved in dichloromethane (10 mL) at room temperature. M-chloroperbenzoic acid (1.03 g,4 mmol) was slowly added at 0deg.C, and the reaction stirred at room temperature for 12 hours.
LCMS monitors the reaction, quench the reaction mixture by adding water (30 mL) at 0deg.C, then slowly adjust pH to 7 by adding saturated aqueous ammonium chloride, and concentrate under reduced pressure. The mixture was extracted with ethyl acetate (15 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL) and then dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 3-bromo-7-iodoquinoline 1-oxide (160 mg).
MS(ESI)M/Z:349.8[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ8.96(d,J＝2.3Hz,1H),8.73(t,J＝13.1Hz,1H),8.44(d,J＝14.5Hz,1H),7.97(dd,J＝8.6,1.5Hz,1H),7.77(d,J＝8.6Hz,1H).
Step E: 3-bromo-7-iodoquinoline 1-oxide (160 mg,0.45 mmol) was dissolved in chloroform (10 mL) at room temperature. Phosphorus oxychloride (10 mL) was then slowly added at 0 c and the reaction was evacuated and replaced with nitrogen multiple times. The reaction solution was stirred for 12 hours at 70℃in an oil bath.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. Dichloromethane (20 mL) was added and then pH was adjusted to 7 with saturated sodium bicarbonate solution, the mixture was extracted with ethyl acetate (10 ml×3), the organic phases were combined, washed with saturated brine (15 mL), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 3-bromo-2-chloro-7-iodoquinoline (90 mg).
MS(ESI)M/Z:367.8[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ8.96(s,1H),8.44(d,J＝18.2Hz,1H),8.03(t,J＝18.1Hz,1H),7.79(t,J＝10.1Hz,1H).
Step F: 3-bromo-2-chloro-7-iodoquinoline (90 mg,0.24 mmol) was dissolved in the mixed solution (ethanol/ammonia=1/1, volume ratio, 8 mL) at room temperature. The reaction solution was then stirred at 100℃for 20 hours under microwaves.
After LCMS monitoring showed the disappearance of starting material, concentrated under reduced pressure, the mixture was extracted with dichloromethane (5 ml×3), the organic phases were combined, the organic phase was washed with saturated brine (10 mL), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 3-bromo-7-iodoquinolin-2-amine (76 mg).
MS(ESI)M/Z:348.8[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ8.39(d,J＝10.2Hz,1H),7.86(s,1H),7.48(dt,J＝19.4,5.0Hz,2H),6.84(brs,2H).
Example 1
(1S, 2R,3S, 5R) -3- (2- (2-amino-3-bromoquinolin-7-yl) ethyl) -5- (4-amino-5, 6-dihydro-7H-pyrrole [2, 3-d)]Pyrimidin-7-yl-5,5,6,6-d 4 ) Cyclopentane-1, 2-diol
The reaction route is as follows:
the operation steps are as follows:
Step A methyl 2- (4, 6-dichloropyrimidin-5-yl) acetate (25 g,113 mmol) was dissolved in N, N-dimethylformamide (300 mL) at room temperature. (3, 4-dimethylphenyl) formamide (20.7 g,124 mmol) and N, N-diisopropylethylamine (17.5 g,135 mmol) were added at room temperature and the reaction stirred at 60℃for 3 hours.
After LCMS monitoring showed the disappearance of starting material, the reaction was quenched by addition of water (50 mL) and concentrated under reduced pressure. The mixture was extracted with ethyl acetate (100 mL. Times.3), and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give methyl 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) acetate (40 g) as a crude product.
MS(ESI)M/Z:352.2[M+H] + 。
Step B methyl 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) acetate (40 g,113 mmol) was dissolved in toluene (500 mL) at room temperature. Para-toluene sulfonic acid (10.7 g,56.3 mmol) was added at room temperature and the reaction stirred at 120℃for 3 hours.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (100 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 4-chloro-7- (2, 4-dimethoxybenzyl) -5, 7-dihydro-6H-pyrrolo [2,3-d ] pyrimidin-6-one (30.5 g).
MS(ESI)M/Z:320.3[M+H] + 。
Step C4-chloro-7- (2, 4-dimethoxybenzyl) -5, 7-dihydro-6H-pyrrolo [2,3-d ] pyrimidin-6-one (27 g,84.64 mmol) was dissolved in deuterated methanol solution (500 mL) at room temperature. 0.1M aqueous sodium carbonate deuterium solution (300 mL) was added dropwise at room temperature, and the reaction solution was stirred at 80℃for 3 days.
LCMS monitoring showed 60% product followed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (100 mL. Times.3), the aqueous phases (product in aqueous phase) were combined, the pH of the aqueous phase was adjusted to 5-6 with 1M HCl, then extracted with ethyl acetate (100 mL. Times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and finally concentrated under reduced pressure to give the crude 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) acetic acid-2, 2-d 2 Acid (11 g).
MS(ESI)M/Z:340[M+H] + 。
Step D2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) acetic acid-2, 2-D2 acid (13 g,38.34 mmol) was dissolved in methanol (100 mL) in a three-necked flask. Thionyl chloride (18.25 g,153.4 mmol) was added at 0deg.C and the reaction stirred at room temperature for 5 hours.
After LCMS monitoring showed the disappearance of starting material, the pH was adjusted to 8-9 with saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (100 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained is purified by column chromatography on silica gel to give methyl 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) acetate-d 2 (11g)。
MS(ESI)M/Z:354.0[M+H] + 。
Step E methyl 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) acetate-d 2 (5 g,14.16 mmol) was dissolved in tetrahydrofuran (300 mL) in a three-necked flask. Deuterated lithium aluminum hydride powder (594 mg,14.16 mmol) was added in portions at-40℃and the reaction was stirred at-40℃for 0.5 h.
LCMS monitoring showed the disappearance of starting material followed by 10D at-40 ℃ 2 Quenching with sodium sulfate. The mixture was filtered, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained is purified by column chromatography on silica gel to give 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) ethane-1, 2-d 4 -1-alcohol (3.25 g).
MS(ESI)M/Z:328.2[M+H] + 。
Step F2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) ethane-1, 2-d 4-1-ol (3.25 g,9.94 mmol) and 4-dimethylaminopyridine (121 mg,0.0994 mmol) were dissolved in dichloromethane (40 mL) in a three-necked flask. Methanesulfonyl chloride (2.85 mg,24.85 mmol) and triethylamine (4.52 g,944.73 mmol) were added at 0℃and the reaction was stirred at 0℃to room temperature for 3 hours.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. The mixture was extracted with dichloromethane (30 mL. Times.3), the organic phases were combined, the organic phase was washed with saturated brine (50 mL), and Then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel to give 2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) ethyl-1, 2-d 4 Methanesulfonate (3 g).
MS(ESI)M/Z:406.2[M+H] + 。
Step G2- (4-chloro-6- ((2, 4-dimethoxybenzyl) amino) pyrimidin-5-yl) ethyl-1, 2-d 4-methanesulfonate (3G, 7.41 mmol) and potassium carbonate (2.05G, 2.0 mmol) were dissolved in N, N-dimethylformamide (50 mL) at room temperature, and the reaction solution was stirred at 80℃overnight.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (30 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained is purified by silica gel column chromatography to obtain 4-chloro-7- (2, 4-dimethoxy benzyl) -6, 7-dihydro-5H-pyrrole [2,3-d ]]Pyrimidine-5,5,6,6-d 4 (1.7g)。
MS(ESI)M/Z:310.2[M+H] + 。
Step H4-chloro-7- (2, 4-dimethoxybenzyl) -6, 7-dihydro-5H-pyrrolo [2,3-d ] pyrimidine-5,5,6,6-d 4 (1.7 g,5.5 mmol) was dissolved in trifluoroacetic acid (15 mL) at room temperature and the reaction was stirred at 80℃for 5 hours.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. The mixture was adjusted to pH 8-9 with saturated sodium bicarbonate solution, extracted with ethyl acetate (30 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained is purified by silica gel column chromatography to obtain 4-chloro-6, 7-dihydro-5H-pyrrole [2,3-d ] ]Pyrimidine-5,5,6,6-d 4 (670mg)。
MS(ESI)M/Z:160.2[M+H] + 。
Step I, in a tube sealing process, 4-chloro-6, 7-dihydro-5H-pyrrole [2,3-d ]]Pyrimidine-5,5,6,6-d 4 (640 mg,4.2 mmol) and (3 aS,4S,6R,6 aR) -2, 2-dimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ]][1,3]Dioxin-4-ol (1.18 g,6.3 mmol) was dissolved in toluene (3 mL). Cyanomethylenetri-n-butylphosphine (3.04 g,12.6 m)mol) was dissolved in toluene (3 mL), the above liquid was added dropwise under nitrogen atmosphere, and the reaction solution was stirred at 110℃for 18 hours.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (30 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained is purified by column chromatography on silica gel to give 4-chloro-7- ((3 aS,4R,6 aR) -2, 2-dimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ]][1,3]Dioxol-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d]Pyrimidine-5,5,6,6-d 4 (480mg)。
MS(ESI)M/Z:326.0[M+H] + 。
Step J4-chloro-7- ((3 aS,4R,6 aR) -2, 2-dimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ] at room temperature][1,3]Dioxol-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d]Pyrimidine-5,5,6,6-d 4 (580 mg,1.78 mmol) was dissolved in a mixture of ethanol (50 mL) and aqueous ammonia (150 mL), and the reaction mixture was stirred in an autoclave at 160℃for 60 hours.
After LCMS monitoring showed mostly product, concentrate under reduced pressure. The residue obtained is purified by column chromatography on silica gel to give 7- ((3 aS,4R,6 aR) -2, 2-dimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ]][1,3]Dioxol-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d]Pyrimidine-5,5,6,6-d 4 -4-amine) (370 mg).
MS(ESI)M/Z:307.0[M+H] + 。
Step K adding the compound 7- ((3 aS,4R,6 aR) -2, 2-dimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ] to a three-necked flask][1,3]Dioxol-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d]Pyrimidine-5,5,6,6-d 4 A solution of 4-amine) (370 mg,1.2 mmol) in tetrahydrofuran (4 mL) was then replaced with nitrogen. 9-BBN (0.5M in THF,16.8mL,8.4mmol) was slowly added dropwise to the reaction solution at room temperature, and after the completion of the addition, the reaction solution was heated to 55℃and stirred at this temperature for 1 hour. The reaction system was cooled to room temperature by removing from the oil bath, and then a heavy aqueous solution (4 mL) of potassium phosphate (1.78 g,8.4 mmol) was added to the reaction mixture. After the reaction solution was stirred at room temperature for 30 minutes, 3-bromo-7-iodoquinolin-2-amine (420 mg,1.2 mmol) and PdCl were added 2 (dppf) (87 mg,0.12 mmol) in tetrahydrofuran (4 mL). The resulting reaction mixture was heated to 55℃and stirred overnight.
After TLC monitoring showed the disappearance of starting material, the reaction solution was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel chromatography to give 7- (2- ((3 aR,4S,6R,6 aS) -6- (4-amino-5, 6-dihydro-7H-pyrrole [2, 3-d) ]Pyrimidin-7-yl-5,5,6,6-d 4 ) -2, 2-dimethyltetrahydro-4H-cyclopentane [ d ]][1,3]Dioxy-4-yl) ethyl) -3-bromoquinolin-2-amine (310 mg).
MS(ESI)M/Z:529.5[M+H] + 。
Step L7- (2- ((3 aR,4S,6R,6 aS) -6- (4-amino-5, 6-dihydro-7H-pyrrolo [2, 3-d) at room temperature]Pyrimidin-7-yl-5,5,6,6-d 4 ) -2, 2-dimethyltetrahydro-4H-cyclopentane [ d ]][1,3]Dioxy-4-yl) ethyl) -3-bromoquinolin-2-amine (310 mg,0.58 mmol) was dissolved in methanol (2 mL). Subsequently, 4M hydrochloric acid-methanol solution (8 mL) was added to the above solution. The reaction solution was stirred at room temperature for 2 hours.
After LCMS monitoring showed the disappearance of starting material, the reaction was concentrated under reduced pressure to crude product. The crude product was dissolved in 5mL of methanol and then 7M methanolic ammonia solution was added to adjust the pH to approximately 8-9. Purifying the obtained solution by preparative high performance liquid chromatography to obtain the final product (1S, 2R,3S, 5R) -3- (2- (2-amino-3-bromoquinolin-7-yl) ethyl) -5- (4-amino-5, 6-dihydro-7H-pyrrole [2, 3-d)]Pyrimidin-7-yl-5,5,6,6-d 4 ) Cyclopentane-1, 2-diol (168 mg).
MS(ESI)M/Z:488.8[M+H] +
1 H NMR(400MHz,DMSO-d 6 )δ8.32(s,1H),7.79(s,1H),7.57(d,J＝8.2Hz,1H),7.30(s,1H),7.10(dd,J＝8.2,1.3Hz,1H),6.56(s,2H),5.91(s,2H),4.78(d,J＝5.6Hz,1H),4.40(d,J＝4.7Hz,1H),4.13(dd,J＝18.1,7.9Hz,1H),3.87(dd,J＝13.2,5.7Hz,1H),3.54(dd,J＝10.2,4.8Hz,1H),2.79–2.61(m,2H),1.91–1.79(m,2H),1.79–1.66(m,1H),1.63–1.48(m,1H),1.27–1.14(m,1H).
Example 2
(1S, 2R,3S, 5R) -3- (2- (2-amino-3-bromoquinolin-7-yl) ethyl) -5- (4-amino-5, 6-dihydro-7H-pyrrole [2, 3-d)]Pyrimidin-7-yl-5,5,6,6-d 4 ) -3-methylcyclopentane-1, 2-diol
The reaction route is as follows:
the operation steps are as follows:
Step A4-chloro-6, 7-dihydro-5H-pyrrolo [2,3-d ] pyrimidine-5,5,6,6-d 4 (430 mg,2.7 mmol) and (3 aS,4S,6R,6 aR) -2, 6-trimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ] [1,3] dioxin-4-ol (802 mg,4.05 mmol) were dissolved in toluene (2 mL) in a closed tube. Cyanomethylene tri-n-butylphosphine (1.95 g,8.1 mmol) was dissolved in toluene (1 mL), the above liquid was added dropwise under nitrogen atmosphere, and the reaction solution was stirred at 110℃for 18 hours.
LCMS monitoring showed the disappearance of starting material followed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (30 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained is purified by column chromatography on silica gel to give 4-chloro-7- ((3 aS,4R,6 aR) -2, 6-trimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ]][1,3]Dioxol-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d]Pyrimidine-5,5,6,6-d 4 (170mg)。
MS(ESI)M/Z:340.2[M+H] + 。
And (B) step (B): 4-chloro-7- ((3 aS,4R,6 aR) -2, 6-trimethyl-6-vinyltetrahydro-4H-cyclopentan [ d ] [1,3] dioxan-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d ] pyrimidine-5,5,6,6-d 4 (370 mg,1.09 mmol), tert-butyl carbamate (384.25 g,3.27 mmol) was dissolved in N, N-dimethylformamide (2 mL) at room temperature. The reaction system is vacuumized and replaced with nitrogen for many times. Tri (dibenzylideneacetone) dipalladium (100 mg,0.11 mmol), 2-dicyclohexylphosphorus-2 ',4',6' -triisopropylbiphenyl (52.44 mg,0.11 mmol) and cesium carbonate (708.5 mg,2.18 mmol) were added under nitrogen. The reaction solution was stirred for 3 hours at 100℃under microwaves.
After LCMS monitoring showed the disappearance of starting material, the reaction was quenched by addition of water (30 mL) and concentrated under reduced pressure. The mixture was extracted with ethyl acetate (15 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel to give 7- ((3 as,4r,6 ar) -2, 6-trimethyl-6-vinyltetrahydro-4H-cyclopentan [ d ] [1,3] dioxy-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d ] pyrimidin-5,5,6,6-d 4-amine) (110 mg).
MS(ESI)M/Z:321.2[M+H] + 。
Step C, adding the compound 7- ((3 aS,4R,6 aR) -2, 6-trimethyl-6-vinyltetrahydro-4H-cyclopentane [ d ] to a three-necked flask][1,3]Dioxol-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d]Pyrimidine-5,5,6,6-d 4-amine) (110 mg,0.34 mmol) in tetrahydrofuran (2 mL) and then the system was replaced with nitrogen atmosphere. 9-BBN (0.5M in THF,4.8mL,2.4mmol) was slowly added dropwise to the reaction solution at room temperature, and after the completion of the addition, the reaction solution was heated to 55℃and stirred at this temperature for 1 hour. The reaction system was cooled to room temperature by removing from the oil bath, and then a heavy aqueous solution (2 mL) of potassium phosphate (508 mg,2.4 mmol) was added to the reaction mixture. After the reaction solution was stirred at room temperature for 30 minutes, 3-bromo-7-iodoquinolin-2-amine (119 mg,0.34 mmol) and PdCl were added 2 (dppf) (24.8 mg,0.034 mmol) in tetrahydrofuran (2 mL). The resulting reaction mixture was heated to 55℃and stirred overnight.
After TLC monitoring showed the disappearance of starting material, the reaction solution was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel chromatography to give 7- (2- ((3 aR,4S,6R,6 aS) -6- (4-amino-5, 6-dihydro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl-5,5,6,6-d 4) -2, 6-trimethyltetrahydro-4H-cyclopenta [ d ] [1,3] dioxo-4-yl) ethyl) -3-bromoquinolin-2-amine (150 mg).
MS(ESI)M/Z:543.1[M+H] + 。
Step D7- (2- ((3 aR,4S,6R,6 aS) -6- (4-amino-5, 6-dihydro-7H-pyrrolo [2,3-D ] pyrimidin-7-yl-5,5,6,6-D4) -2, 6-trimethyltetrahydro-4H-cyclopenta [ D ] [1,3] dioxo-4-yl) ethyl) -3-bromoquinolin-2-amine (150 mg,0.27 mmol) is dissolved in methanol (1 mL) at room temperature. Subsequently, 4M hydrochloric acid-methanol solution (3 mL) was added to the above solution. The reaction solution was stirred at room temperature for 2 hours.
After LCMS monitoring showed the disappearance of starting material, the reaction was concentrated under reduced pressure to crude product. The crude product was dissolved in 3mL of methanol and then 7M methanolic ammonia solution was added to adjust the pH to approximately 8-9. The resulting solution was purified by preparative high performance liquid chromatography to give the final product (1 s,2r,3s,5 r) -3- (2- (2-amino-3-bromoquinolin-7-yl) ethyl) -5- (4-amino-5, 6-dihydro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl-5,5,6,6-d 4) -3-methylcyclopentane-1, 2-diol (68 mg).
MS(ESI)M/Z:502.8[M+H] + .
1 H NMR(400MHz,DMSO-d 6 )δ8.32(s,1H),7.79(s,1H),7.57(d,J＝8.2Hz,1H),7.30(s,1H),7.10(dd,J＝8.2,1.5Hz,1H),6.55(s,2H),5.90(s,2H),4.80(d,J＝5.7Hz,1H),4.36(d,J＝5.1Hz,1H),4.25(dd,J＝8.0Hz,1H),4.08(dd,J＝7.6Hz,1H),3.55(t,J＝5.5Hz,1H),2.74–2.57(m,2H),1.70–1.48(m,4H),1.04(s,3H).
Example 3
(1S, 2R,3R, 5R) -3- ((E) -2- (2-amino-3-bromoquinolin-7-yl) vinyl) -5- (4-amino-5, 6-dihydro-7H-pyrrole [2, 3-d)]Pyrimidin-7-yl-5,5,6,6-d 4 ) Cyclopentane-1, 2-diol
The reaction route is as follows:
step A: in a tube seal, 7- ((3 aS,4R,6 aR) -2, 2-dimethyl-6-vinyltetrahydro-4H-cyclopentan [ d ] [1,3] dioxan-4-yl) -6, 7-dihydro-5H-pyrrolo [2,3-d ] pyrimidin-5,5,6,6-d 4-amine) (100 mg,0.33 mmol), 3-bromo-7-iodoquinolin-2-amine (114.8 mg,0.33 mmol) and tetraethylammonium chloride (60.15 mg,0.363 mmol) were dissolved in N, N-dimethylformamide (2 mL). The reaction system is vacuumized and replaced with nitrogen for many times. Palladium acetate (11.1 mg,0.05 mmol) and N, N-diisopropylethylamine (85 mg,0.66 mmol) were added under nitrogen. The reaction was stirred at 75℃in an oil bath for 12 hours.
After LCMS monitoring showed the disappearance of starting material, the reaction was quenched by addition of water (10 mL) and concentrated under reduced pressure. The mixture was extracted with ethyl acetate (20 mL. Times.3), and the organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel to give 7- ((E) -2- ((3 aR,4R,6 aS) -6- (4-amino-5, 6-dihydro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl-5,5,6,6-d 4) -2, 2-dimethyltetrahydro-4H-cyclopenta [ d ] [1,3] dioxo-4-yl) vinyl) -3-bromoquinolin-2-amine (85 mg).
MS(ESI)M/Z:527.0[M+H] + 。
And (B) step (B): 7- ((E) -2- ((3 aR,4R,6 aS) -6- (4-amino-5, 6-dihydro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl-5,5,6,6-d 4) -2, 2-dimethyltetrahydro-4H-cyclopentan [ d ] [1,3] dioxo-4-yl) vinyl) -3-bromoquinolin-2-amine (85 mg,0.162 mol) was dissolved in methanol (2 mL) at room temperature. Subsequently, 4M hydrochloric acid-methanol solution (2 mL) was added to the above solution. The reaction solution was stirred at room temperature for 2 hours.
LCMS showed the disappearance of starting material followed by concentration under reduced pressure at low temperature. The crude product was dissolved in 2mL of methanol and then 7M methanolic ammonia solution was added to adjust the pH to approximately 8-9. The resulting residue was purified by preparative high performance liquid chromatography to give the final product (1 s,2r,3r,5 r) -3- ((E) -2- (2-amino-3-bromoquinolin-7-yl) vinyl) -5- (4-amino-5, 6-dihydro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl-5,5,6,6-d 4) cyclopentane-1, 2-diol (26.7 mg).
MS(ESI)M/Z:487.0[M+H] + 。
1 H NMR(400MHz,MeOD)δ8.24(s,1H),7.85(s,1H),7.56(d,J＝8.4Hz,1H),7.48–7.39(m,2H),6.61(d,J＝16.0Hz,1H),6.47(dd,J＝15.8,7.8Hz,1H),4.33–4.27(m,1H),4.13(t,J＝6.4Hz,1H),3.89(t,J＝6.0Hz,1H),2.82–2.71(m,1H),2.16–2.08(m,1H),1.69–1.61(m,1H).
Biological test evaluation
Test example 1: evaluation of the inhibitory Activity of the Compounds of the invention against PRMT5 methylase
The test adopts a TR-FRET method to measure the inhibition activity of the compound on the short peptide substrate of PRMT5 protein methylation H4 histone, and obtains half inhibition concentration IC of the compound on the inhibition of PRMT5 methylation activity 50 。
1. Experimental materials
PRMT5/MEP50 protein was purchased from BPS Bioscience, histone H4 peptide was purchased from GL Biochem, substrate SAM was purchased from NEB, TR-FRET reagent was purchased from Perkinelmer, and buffer composition was purchased from Sigma.
2. Experimental method
1) Compound dilution was 1:3 serial dilutions, 10 concentration points, titration curves determine the titers of each compound.
2) 100nL of compound was dispensed into each well of a white Greiner 384 well plate, and then a mixture of 5. Mu.L PRMT5-MEP 50/peptide was added thereto for 30 minutes at room temperature.
3) S- (5' -adenosyl) -L-methionine chloride (SAM) was added to initiate each reaction, and the plates were sealed and placed in an incubator at 25℃and pre-incubated with the compounds for 90 minutes.
4) After incubation, 10. Mu.L of detection solution was added to each well, and incubation was continued for 60 minutes, and the TR-FRET signal (excitation light 320 or 340nm, emission light wavelength 665 nm) was detected on an Envision microplate reader (Perkinelmer)
5) Data analysis using GraphPad Prism 6 software, calculation of IC for compounds 50 。
The result of PRMT5 methylase inhibition by the compound of the present invention is shown in Table 1, and the activity data is divided into A, B, C, D four intervals, IC 50 Compounds of less than or equal to 10nM are identified by A, 10nM < IC 50 Compounds of less than or equal to 100nM are identified by B, 100nM < IC 50 Compounds less than or equal to 500nM are identified by C, IC 50 > 500nM is identified by D.
TABLE 1 inhibition of PRMT5 Methylase Activity results
Test example 2: evaluation of proliferation inhibition by Compounds of the present invention on human pancreatic cancer cell line MIAPaCa-2
The experiment adopts a chemiluminescence method to measure the intracellular ATP content to detect the proliferation inhibition effect of the compound on the human pancreatic cancer cell strain MIA PaCa-2, and obtains half inhibition concentration IC of the compound on the proliferation inhibition of the human pancreatic cancer cell strain MIA PaCa-2 50 。
1. Experimental materials
DMEM medium, fetal Bovine Serum (FBS), 100 XPen/Strep, glutaMAX-I supply was purchased from GIBCO corporation. Cell Titer-Glo luminescence Cell viability assay reagents were purchased from Promega corporation.
2. Experimental method
1) Human pancreatic cancer cell line MIA PaCa-2 was counted using a cytometer, and cells were seeded into 96-well plates at a density of 800 cells per well, 150. Mu.L per well. Placing in incubator (37 ℃,5% CO) 2 ) Incubate overnight.
2) Day 0: the test compound was added to the cells of the plates in a gradient of 750nL (9 concentrations, 1:3 ratio dilution) using D300e (TECAN) with a final DMSO concentration of 0.5% and the plates were incubated in a cell incubator for 6 days (37 ℃,5% co 2). Blank control was added to 750nL of DMSO per well.
3) Day 6: mu.L of Cell Titer-Glo reagent was added to each well, and the mixture was shaken at 500rpm for 2 minutes, and incubated at room temperature for 10 minutes under dark conditions to stabilize the luminescence signal.
4) The luminescence signal was detected by an Envision enzyme-labeled instrument (PerkinElmer).
5) Data analysis using GraphPad Prism 6 software, calculation of IC for compounds 50 。
The proliferation inhibition effect results of the compound of the invention on human pancreatic cancer cell line MIA PaCa-2 are shown in Table 2, and the activity data is divided into A, B, C, D sections, and IC 50 Compounds of less than or equal to 10nM are identified by A, 10nM < IC 50 Compounds of less than or equal to 100nM are identified by B, 100nM < IC 50 Compounds less than or equal to 500nM are identified by C, IC 50 > 500nM is identified by D.
TABLE 2 inhibition of human pancreatic cancer cell line MIA PaCa-2
Conclusion: as can be seen from tables 1 and 2, the compounds of the present invention have a better inhibitory effect on PRMT5 methylase and human pancreatic cancer cell line MIA PaCa-2.
Test example 3: in vivo pharmacokinetic assay of the compounds of the invention in CD-1 mice, SD rats and beagle dogs
The pharmacokinetic behavior of the compounds of the present invention in plasma in mice, rats and dogs after intravenous push and oral administration was studied using CD-1 mice, SD rats and beagle dogs as test animals.
1. Test protocol
1.1 test drug:
the compounds of examples 1-3 of the present invention.
1.2 test animals
CD-1 mice total 9, 3/group, male, vendor Vital River.
SD rats total 9, 3/group, male, vendor Vital River.
The beagle dogs are 6, 3/group, male, and the supplier is Marshall.
1.3 administration:
CD-1 mice dosing: 3 mice are administered in each experimental group, the IV dose is 1mg/kg, and the administration volume is 1mL/kg; PO administration doses were 5mg/kg and 60mg/kg, and the administration volume was 10mL/kg. The vehicle was 5% dmso+10% solutol+85% saline.
SD rat dosing: 3 rats in each experimental group, the IV administration dose is 1mg/kg, and the administration volume is 1mL/kg; PO was administered at a dose of 5mg/kg g and a volume of 10mL/kg. The vehicle was 5% dmso+10% solutol+85% Saline.
Beagle dosing: 3 beagle dogs in each experimental group, the IV administration dose is 1mg/kg, and the administration volume is 1mL/kg; PO was administered at a dose of 3mg/kg and a volume of 5mL/kg. The vehicle was 5% dmso+10% solutol+85% Saline.
1.4 experiment apparatus
The centrifuge was purchased from Eppendorf corporation and the pipettor was purchased from Eppendorf corporation.
1.5 sample collection:
after administration, blood was collected intravenously at 0.0833 (IV), 0.25, 0.5, 1, 2, 4, 8 and 24 hours, placed in EDTA-K2 test tubes, and centrifuged at 4℃and 4600rmp for 5min to separate plasma, which was stored at-80 ℃.
1.6 sample treatment:
1) mu.L of plasma sample was precipitated by adding 200. Mu.L of acetonitrile, and after mixing, centrifuged for 15 minutes.
2) The supernatant after the treatment was diluted 3-fold with water and analyzed for the concentration of the test compound by LC/MS.
1.7 liquid phase analysis
Liquid phase: waters ACQuity UPLC class I plus
Mass spectrometry: waters APITQ-XS
Chromatographic column: YMC-Triart phenyl 3 μm (50.2.1 mm)
Mobile phase: a:95% water (0.1% formic acid); b: acetonitrile (containing 0.1% formic acid)
Flow rate: 0.6mL/min
Elution gradient:
Time(min) A(％) B(％)
0.01 100 0
0.50 100 0
2.20 0 100
2.70 0 100
2.71 100 0
3.00 100 0
2. experimental results and analysis
Pharmacokinetic parameters were calculated using WinNonlin, and the pharmacokinetic parameters for intravenous and oral administration of the drug in mice, rats and dogs are shown in the following table:
TABLE 3 pharmacokinetic parameters of the compounds of EXAMPLE 1
Table 4 pharmacokinetic parameters of example 2 compounds
Table 5 pharmacokinetic parameters of example 3 compound
Conclusion: the compounds of the present invention have good pharmacokinetic properties.
Test example 4: effect of the Compounds of the invention on hERG Potassium ion channel stably expressed in Chinese hamster ovary cells
Human ventricle rapid activation delay rectifier potassium current (I) Kr ) Is mediated mainly by cardiac hERG potassium ion channels. Pair I Kr Is the most common mechanism of action of ventricular muscle in prolonged duration due to non-heart-disease drugs. Prolongation of the action potential time course will lead to prolongation of QT interval on clinical electrocardiograms, which is associated with dangerous ventricular arrhythmias and torsades de pointes.
This test uses manual patch clamp to evaluate the effect of the compounds of the invention on the stable expression of hERG (human ether-a-go-go related gene) potassium channel (I Kr ) The concentration-response relationship of the current, and thus the determination of whether the compounds of the invention have inhibition of hERG potassium ion channel.
1. Experimental materials
Extracellular fluid (ECS): naCl,145mM; KCl,4mM; caCl (CaCl) 2 ，2mM；MgCl 2 1mM; HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 10mM; glucose, 10mM, was dissolved in pure water and the pH was adjusted to 7.3-7.4. Chinese hamster ovary cells (CHO-hERG) were derived from Sophion biosciences.
2. Experimental method
1) The mother liquor preparation of the compound of example 1, vehicle control, positive control (JNJ-64619178) was diluted with ECS cocktail to working solutions at concentrations of 0.3 μm, 1 μm, 3 μm, 10 μm and 30 μm; the ECS mixture is a mixture of ECS and DMSO, wherein the ECS ratio is 0.1% (v/v); the blank is DMSO. Two cells were assayed in duplicate for each concentration.
2) CHO-hERG cells in exponential growth phase were collected and resuspended in ECS for use.
3) hERG current was recorded under whole cell patch clamp technique, and recording temperature was room temperature. The patch clamp amplifier output signal is filtered by digital to analog conversion and 2.9KHz low pass. Data recording was collected using the PatchMaster Pro software.
4) The hERG current values were transcribed into Excel tables and IC of compounds to hERG was calculated 50 。
The inhibitory effects of the compounds of the invention and JNJ-64619178 on hERG potassium channel are shown in Table 6.
TABLE 6 inhibition of hERG Current by Compounds of the invention and JNJ-64619178
Remarks: JNJ-64619178 is compound 80 of WO2017032840A1, prepared by the method referred to the patent.
Results: as can be seen from Table 6, half Inhibitory Concentration (IC) of the compound of example 1 on hERG 50 ) IC with a value of 19.50. Mu.M as control compound JNJ-64619178 50 About 2 times the value, indicating the potential advantage of the compound of example 1 for greater cardiac safety.
Test example 5: inhibition of human liver microsomal CYP450 enzymes by the compounds of the invention
The effect of compounds on CYP enzyme activity is related to the time of action, some compounds have reduced inhibition of enzyme activity over time, while some compounds exhibit time-dependent irreversible inhibition of enzyme activity (time-dependent inhibition, TDI), which affects the interactions between compounds, the in vivo metabolism of compounds, and safety. In the experiment, an inhibition model of the compound on human liver microsomal CYP450 subunit CYP3A4 is established, and the compound is testedThe concentration of the compound of the present invention was measured by setting 7 concentrations, and the time-dependent half-maximal inhibitory concentration (IC 50 ) To show the inhibition of the CYP enzyme by the compounds of the invention, to evaluate whether the compounds of the invention are at risk of TDI (time-dependent inhibition, i.e., time-dependent inhibition).
1. Test protocol
1.1 test drug:
some of the compounds of the invention.
1.2 test materials:
human liver microparticles were purchased from Corning at a concentration of 20mg/mL; potassium phosphate buffer, 100mM; magnesium chloride, 300mM; NADP (nicotinamide adenine dinucleotide phosphate), 65mM; G6P (glucose 6-phosphate), 330mM; g6PDH (glucose 6-phosphate dehydrogenase), 250U/mL; stop solution, acetonitrile (containing internal standard 100ng/mL toluene sulfobutylurea).
1.3 positive controls, test compounds and substrates:
the positive control, test compounds and substrate are shown in table 7.
TABLE 7 positive control, test compound and substrate
1.4 reaction System:
liver microsomal protein concentration, 0.1mg/mL; potassium phosphate buffer, 100mM; positive controls, substrates, test compounds and substrates are described in section 1.3; NADP,1mM; G6P,5.53mM; g6PDH,1.2U/mL; magnesium chloride, 3.3mM.
1.5 reaction procedure:
the reaction system is added with positive control, the test compound is pre-incubated for 10min at 37 ℃, then NADPH (reduced nicotinamide adenine dinucleotide phosphate) cofactor is added for starting the reaction, the reaction is incubated for 30min at 37 ℃, then substrate mixing is added for continuous incubation for 10min at 37 ℃,250 mu M cold stop solution mixing containing internal standard is added at the end of incubation for stopping the reaction, centrifugation is carried out at 4000rpm for 20min, supernatant is separated, and LC-MS/MS analysis is carried out after dilution.
By comparing the half-inhibition concentration (I.C) of the test substance on enzyme time dependence 50 ) The compounds of the invention were evaluated for the risk of TDI (time-dependent inhibition, i.e., immediate dependency inhibition).
2. Experimental results
TABLE 8 inhibitory Activity of the Compounds of the invention and JNJ-64619178 against CYP3A4 enzymes
Results: as can be seen from Table 8, the IC of the compound of example 1 against CYP3A4 enzyme 50 Values higher than JNJ-64619178 indicate that the compounds of the invention have a lower risk of significant time-dependent inhibition (time-dependent inhibition, TDI).
Test example 6: inhibition effect of the compound of the invention on in vitro human liver microsome CYP450 enzyme is examined and evaluated for DDI risk
The metabolic pathway in which CYP450 enzymes participate is a very important metabolic pathway in the in vivo clearance process of compounds, and inhibition of the activity of this family of enzymes can bring about changes in drug clearance and pharmacokinetics, drug-drug interactions (DDI) mediated by CYP450 enzymes are important factors in drug efficacy and drug safety considerations. In this experiment, an inhibition model of the compound on human liver microsomal CYP450 subunit CYP3A4 was established, 7 concentrations were set for the test compound, and half Inhibition Concentration (IC) of the compound of the invention on the CYP enzyme was measured 50 ) To show the inhibition of CYP enzymes by the compounds of the invention, and further evaluate DDI risks of the compounds.
1. Test protocol
1.1 test drug:
some of the compounds of the invention.
1.2 test materials:
human liver microparticles were purchased from Corning at a concentration of 0.127mg/mL; potassium phosphate buffer, 100mM; magnesium chloride, 33mM; NADPH (nicotinamide adenine dinucleotide phosphate), 10mM; stop solution, acetonitrile (containing internal standard 100ng/mL toluene sulfobutylurea).
1.3 positive controls, test compounds and substrates are detailed in Table 9.
TABLE 9 positive control, test compounds and substrates
1.4 reaction System:
liver microsomal protein concentration, 0.127mg/mL; potassium phosphate buffer, 100mM; positive controls, substrates, test compounds and substrates are shown in section 1.3, table 8; NADPH,10mM; magnesium chloride, 33mM.
1.5 reaction procedure:
adding a substrate, a positive control and a compound to be tested into a reaction system, pre-incubating for 10min at 37 ℃, then adding NADPH (reduced nicotinamide adenine dinucleotide phosphate) cofactor to start a reaction, incubating at 37 ℃, wherein 3A4 (meldonium) is incubated for 3min, adding 400 mu M cold stop solution containing an internal standard at the end point of incubation, mixing to stop the reaction, centrifuging at 4000rpm for 20min, separating supernatant, and carrying out LC-MS/MS analysis after dilution.
By comparing half inhibition concentration of test substance to enzyme (I C) 50 ) The compounds of the present invention were evaluated for the risk of DDI (drug-drug interaction), i.e. drug interactions.
2. Experimental results
TABLE 10 inhibitory Activity of the inventive Compounds and JNJ-64619178 against CYP3A4 enzymes
Results: as can be seen from Table 10, the IC of the compound of example 1 against CYP3A4 enzyme 50 The values were significantly higher than JNJ-64619178, indicating lower risk of DDI for the compound of example 1.

Claims (5)

1. A compound represented by the formula (A) or a pharmaceutically acceptable salt thereof,
wherein R is 1 Is NH 2 ；
R 2 Is halogen;
L 1 selected from-CH 2 -CH 2 -or-ch=ch-;
R 9 selected from H or methyl;
R 4 is NH 2 。
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 2 Is Br.
3. A compound, or a pharmaceutically acceptable salt thereof, selected from:
4. a pharmaceutical composition comprising a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
5. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 4, in the manufacture of a medicament for the treatment of cancer.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107922413A (en) * 2015-08-26 2018-04-17 詹森药业有限公司 As the cyclosubstituted nucleoside analog of PRMT5 inhibitor, novel 66 two cyclophanes
CN111527099A (en) * 2017-08-09 2020-08-11 普莱鲁德疗法有限公司 Selective inhibitors of protein arginine methyltransferase 5(PRMT5)
WO2020205867A1 (en) * 2019-04-02 2020-10-08 Aligos Therapeutics, Inc. Compounds targeting prmt5
WO2021079302A1 (en) * 2019-10-22 2021-04-29 Lupin Limited Pharmaceutical combination of prmt5 inhibitors
CN112805006A (en) * 2018-08-07 2021-05-14 默沙东公司 PRMT5 inhibitor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107922413A (en) * 2015-08-26 2018-04-17 詹森药业有限公司 As the cyclosubstituted nucleoside analog of PRMT5 inhibitor, novel 66 two cyclophanes
CN111527099A (en) * 2017-08-09 2020-08-11 普莱鲁德疗法有限公司 Selective inhibitors of protein arginine methyltransferase 5(PRMT5)
CN112805006A (en) * 2018-08-07 2021-05-14 默沙东公司 PRMT5 inhibitor
WO2020205867A1 (en) * 2019-04-02 2020-10-08 Aligos Therapeutics, Inc. Compounds targeting prmt5
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