CN119241496A

CN119241496A - Ectonucleotide pyrophosphatase/phosphodiesterase 1 inhibitor and its preparation method and use

Info

Publication number: CN119241496A
Application number: CN202310805811.8A
Authority: CN
Inventors: 张翱; 蒙凌华; 何洁; 马晓雨; 姚雨佳; 陈嫚嫚
Original assignee: Shanghai Institute of Materia Medica of CAS; Shanghai Jiao Tong University
Current assignee: Shanghai Institute of Materia Medica of CAS; Shanghai Jiao Tong University
Priority date: 2023-07-03
Filing date: 2023-07-03
Publication date: 2025-01-03

Abstract

The invention relates to an external nucleotide pyrophosphatase/phosphodiesterase 1 inhibitor, a preparation method and application thereof. Specifically, the compound has a structure shown in a formula I, wherein the definition of each group and substituent is described in the specification. The invention also discloses a preparation method of the compound and application of the compound in preventing and/or treating diseases such as cancers.

Description

External nucleotide pyrophosphatase/phosphodiesterase 1 inhibitor and preparation method and application thereof

Technical Field

The invention relates to the field of medicaments, in particular to an external nucleotide pyrophosphatase/phosphodiesterase 1 inhibitor, a preparation method and application thereof.

Background

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) activates the interferon gene Stimulator (STING) pathway, an important anticancer innate immune pathway. Under microbial infection or pathophysiological conditions including cancer and autoimmune diseases, the cGAS-cGAMP-STING pathway can be activated by cytoplasmic DNA. Cyclic GMP-AMP synthetases (cGAS) belong to the family of nucleotidyl transferases, are important cytoplasmic DNA sensors in the innate immune pathway that are activated upon binding to cytoplasmic dsDNA, catalyzing GMP and AMP to produce the signaling molecule (2 '-5',3 '-5') cyclic GMP-AMP (or 2',3' -cGAMP or cGAMP). 2',3' -cGAMP acts as a second messenger during microbial infection, binding and activating STING on the endoplasmic reticulum membrane, thereby inducing production of type I Interferon (IFN) and inflammatory cytokines, regulating downstream protein expression, inducing cell growth arrest and apoptosis, and producing an anti-infective effect. In addition to its role in microbial infection, STING pathway can also modulate the innate immune recognition of immunogenic tumors by cGAMP and type I interferon, enhancing the anti-tumor immune response, thereby inhibiting the development and progression of cancer. The STING pathway has become a therapeutic target for tumors and autoimmune diseases.

CGAMP is a key stimulator of innate immune response, an endogenous activator of STING, and has anti-tumor immune effects. The outer nucleotide pyrophosphatase/phosphodiesterase 1 (ENPPl) is the primary hydrolase that degrades cGAMP. ENPP1 is a member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family, a type II transmembrane glycoprotein comprising two identical disulfide-bonded subunits. ENPPl proteins have a broad specificity and can cleave a variety of substrates including phosphodiester linkages of nucleotides and nucleotide sugars and pyrophosphate linkages of nucleotides and nucleotide sugars. ENPP1 can hydrolyze nucleoside 5' triphosphates to their corresponding monophosphates, and can also hydrolyze adenosine polyphosphate. cGAMP produced by tumor cells is rapidly hydrolyzed by ENPP1 after being transported outside the cell, and immunosuppressive adenosine molecules are produced, thereby promoting immune escape of the tumor. Inhibition of ENPP1 activity to increase cGAMP levels in the tumor microenvironment is a potential tumor immunotherapy strategy.

Since the demonstration of the function of ENPP1 as a cGAS-STING pathway modulator, a certain study was performed on ENPP1 selective inhibitors, but there are great difficulties in developing ENPP1 inhibitors with drug-like properties, and no such drugs are currently marketed, and only a few molecules are being studied in early clinical and preclinical settings to investigate their role as immunomodulators in tumor immunotherapy. In 2022, the oral ENPP1 small molecule inhibitor RBS2418 entered phase I clinical trial (NCT 05270213), was administered in combination with Keytruda or as monotherapy to treat advanced unresectable, recurrent or metastatic tumors, unfortunately its structure has not been disclosed and its precise structural information is not clear.

QS-1 was a PC-1 inhibitor developed by the company pyroxene (Bioorganic & MEDICINAL CHEMISTRY LETTERS 2009,19,3339-3343), and researchers at the university of stanford reported that this compound exhibited a strong affinity for ENPP1 (K _i =1600 nM) in 2020. Further, the Stenford researchers developed a series of ENPP1 inhibitors containing phosphate groups, representative of which compound 1 exhibited very high affinity (K _i <2 nM) for ENPP1, and in vivo pharmacodynamic studies showed that compound 1 could inhibit the growth of E0771 mouse breast cancer.

However, under the test conditions of the present invention, compound 1 showed a 80-fold decrease in activity of more than 50nM for inhibition IC ₅₀ of ENPP1 in MDA-MB-231 breast cancer cells, although it showed 0.6nM for inhibition IC ₅₀ of ENPP1 enzyme activity. In addition, the compounds have phosphate groups with impermeable cell membranes, the effective dose of the compounds in-vivo pharmacodynamic tests is up to 300mg/kg, and only subcutaneous administration can be adopted, so that the further development of the medicaments as candidate new medicaments is severely limited.

Disclosure of Invention

The invention aims to provide a compound shown in a formula I, a preparation method thereof and application thereof in preventing and/or treating diseases such as cancers.

In a first aspect of the present invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt, racemate, R-isomer, S-isomer thereof,

Wherein,

R ₁、R₂、R₃ and R ₄ are each independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy, cyano, hydroxy;

R ₅ is selected from the group consisting of halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, cyano, hydroxy;

R ₆ is selected from the group consisting of substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-7 membered heterocycloalkyl containing 1,2 or 3 heteroatoms selected from N, O or S, NR ₈R₉, substituted by 1,2 or 3 substituents selected from halogen, hydroxy, cyano, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy;

r ₈、R₉ is each independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, halo C1-C4 alkyl, C2-C4 alkenyl, halo C2-C4 alkenyl, C2-C4 alkynyl, halo C2-C4 alkynyl, C1-C4 alkoxy, cyano, hydroxy;

X is selected from N, CR _a;

R _a is selected from the group consisting of hydrogen, cyano;

R ₇ is hydrogen;

Or R _a and R ₇ together with the C to which they are each attached form a substituted or unsubstituted radical selected from the group consisting of phenyl, a 5-7 membered heteroaryl group containing 1,2 or 3 heteroatoms selected from N, O or S, a 5-7 membered heterocyclyl group containing 1,2 or 3 heteroatoms selected from N, O or S, a C4-C8 monocyclic cycloalkyl, a C4-C8 bridged cycloalkyl, a C4-C8 spirocycloalkyl, said substitution being by 1,2 or 3 substituents selected from halogen, hydroxy, cyano, C1-C3 alkyl, C1-C3 alkoxy, C2-C4 alkenyl, C2-C4 alkynyl, oxo.

In another preferred embodiment, R ₁、R₂、R₃ is hydrogen;

r ₄ is C1-C4 alkoxy.

In another preferred embodiment, R ₄ is selected from the group consisting of methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy.

In another preferred embodiment, R ₅ is selected from the group consisting of halogen, C1-C4 alkyl, cyano, hydroxy.

In another preferred embodiment, the halogen is selected from the group consisting of F, cl, br, I.

In another preferred embodiment, the C1-C4 alkyl is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl.

In another preferred embodiment, R ₅ is cyano.

In another preferred embodiment, R ₆ is selected from the group consisting of substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-6 heterocycloalkyl containing 1, 2, or 3 heteroatoms selected from N, O or S, NR ₈R₉, substituted by 1, 2, or 3 substituents selected from halogen, hydroxy, cyano, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy;

R ₈、R₉ is each independently selected from the group consisting of hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, cyano, hydroxy.

In another preferred embodiment, R _a is selected from the group consisting of hydrogen, cyano;

r ₇ is hydrogen.

In another preferred embodiment, the compound is selected from the group consisting of:

In a second aspect of the invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of a compound of the first aspect of the invention.

In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient selected from the group consisting of immune checkpoint inhibitors, tyrosine kinase inhibitors, PARP inhibitors, steroid drugs.

In a third aspect of the invention there is provided the use of a compound according to the first aspect of the invention for the manufacture of a medicament for the prophylaxis and/or treatment of diseases associated with external nucleotide pyrophosphatase/phosphodiesterase 1.

In another preferred embodiment, the disease associated with outer nucleotide pyrophosphatase/phosphodiesterase 1 is a disease of high expression of outer nucleotide pyrophosphatase/phosphodiesterase 1.

In another preferred embodiment, the agent is an external nucleotide pyrophosphatase/phosphodiesterase 1 inhibitor.

In another preferred embodiment, the outer nucleotide pyrophosphatase/phosphodiesterase 1-related disorder is selected from the group consisting of cancer, autoimmune disease, inflammatory disease, neurodegenerative disease.

In another preferred embodiment, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, lung cancer.

In another preferred embodiment, the cancer is advanced cancer.

In another preferred embodiment, the cancer is a recurrent and/or metastatic cancer.

In another preferred embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, psoriasis, arthritis.

In another preferred embodiment, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, psoriasis, arthritis.

In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of Parkinson's disease and Alzheimer's disease.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Detailed Description

The present inventors have conducted extensive and intensive studies to obtain a compound having excellent ENPP1 inhibitory activity at a cellular level through structural optimization such as introduction of a substituent at the para position of a piperidine ring nitrogen atom. On this basis, the inventors completed the present invention.

Terminology

In the present invention, unless otherwise indicated, terms used have the ordinary meanings known to those skilled in the art.

In the present invention, the term "halogen" refers to F, cl, br or I.

In the present invention, "C1-C6 alkyl" means a straight-chain or branched alkyl group comprising 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, neopentyl, t-pentyl, or the like. The terms "C1-C4 alkyl", "C1-C3 alkyl" have similar meanings.

In the present invention, the term "C2-C6 alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms containing one double bond, including without limitation ethenyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl and the like. The term "C2-C4 alkenyl" has a similar meaning.

In the present invention, the term "C2-C6 alkynyl" refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms containing one triple bond, and includes, without limitation, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like. The term "C2-C4 alkynyl" has similar meanings.

In the present invention, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The term "C3-C6 cycloalkyl" has similar meaning.

In the present invention, the term "C1-C6 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, including without limitation methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like. Preferably C1-C4 alkoxy, more preferably C1-C3 alkoxy.

In the present invention, the term "heterocyclyl" or "heterocycloalkyl" is a 4-8 membered heterocyclyl containing 1,2 or 3 heteroatoms selected from N, O, S, including, but not limited to, the following groups:

In the present invention, the term "aromatic ring" or "aryl" has the same meaning, preferably "C6-C10 aryl". The term "C6-C10 aryl" refers to an aromatic cyclic group having 6 to 10 carbon atoms, such as phenyl, naphthyl, and the like, which does not contain heteroatoms in the ring.

In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning and refers to a heteroaromatic group containing one to more heteroatoms. For example, "C3-C10 heteroaryl" refers to an aromatic heterocycle containing 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen, and 3 to 10 carbon atoms. Non-limiting examples include furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring. Heteroaryl groups may be optionally substituted or unsubstituted.

In the present invention, the term "halo" refers to substitution with halogen.

In the present invention, the term "deuterated" means substituted with deuterium.

In the present invention, the term "substituted" means that one or more hydrogen atoms on a particular group are replaced with a particular substituent. The specific substituents are those described in the foregoing for each of the examples or are those found in each of the examples. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of the group, which may be the same or different at each position. Those skilled in the art will appreciate that combinations of substituents contemplated by the present invention are those that are stable or chemically achievable. Such as, but not limited to, halogen, hydroxy, carboxy (-COOH), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 3-to 12-membered heterocyclyl, aryl, heteroaryl, C1-C8 aldehyde, C2-C10 acyl, C2-C10 ester, amino, C1-C6 alkoxy, C1-C10 sulfonyl, and the like.

In the present invention, the term 1-6 refers to 1,2, 3, 4, 5 or 6. Other similar terms each independently have similar meanings. The term "plurality" refers to 2-6, such as 2, 3, 4, 5 or 6.

It will be understood that when a group is present in a compound in a plurality of different positions at the same time, the definition of each position is independent of the other and may be the same or different. That is, the term "selected from the group consisting of" and the term "each independently selected from the group consisting of" have the same meaning.

Compounds of formula (I)

The invention obtains a novel piperidine ENPP1 inhibitor containing quaternary carbon, the compound maintains high inhibition activity on ENPP1 molecular level, simultaneously remarkably improves the inhibition activity on cell ENPP1, and most of compound IC ₅₀ values are smaller than 10nM under the same batch test condition as compound 1.

In particular to a compound which is shown in a formula I or pharmaceutically acceptable salt, racemate, R-isomer and S-isomer thereof,

Wherein each group is as defined above.

In another preferred embodiment, any one of R ₁、R₂、R₃、R₄、R₅、R₆、R₇ and X in the compound is independently a group corresponding to the specific compound of the present invention.

As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salt-forming acids include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like, organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and the like, and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, and the like.

Another preferred class of salts are salts of the compounds of the invention with bases, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butylamine, ethylenediamine, hydroxyethylamine, dihydroxyethylamine, and triethylamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The examples of the present invention specifically describe the preparation method of the compound of the formula I of the present invention, but these specific methods do not limit the present invention in any way. The compounds of the present invention may also be conveniently prepared by optionally combining the various synthetic methods described in this specification or known in the art, such combinations being readily apparent to those skilled in the art to which the present invention pertains.

Typically, the starting materials and reagents used in the preparation of the compounds of the invention are commercially available unless otherwise indicated.

Pharmaceutical compositions and methods of administration

The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound.

Because the compound of the present invention has excellent antitumor activity, the compound of the present invention and various crystalline forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient are useful for the treatment, prevention and alleviation of diseases associated with tumors.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. By "safe and effective amount" is meant an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 10-1000mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

By "pharmaceutically acceptable carrier" is meant one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The pharmaceutical composition is injection, capsule, tablet, pill, powder or granule.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, such as, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, such as, for example, glycerol, (d) disintegrants, such as, for example, agar-agar, calcium carbonate, potato starch, or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) slow solvents, such as, for example, paraffin, (f) absorption accelerators, such as, for example, quaternary ammonium compounds, (g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, such as, for example, kaolin, and (i) lubricants, such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., antineoplastic agents).

The methods of treatment of the present invention may be administered alone or in combination with other therapeutic means or therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective administration dose, and the daily administration dose is usually 1 to 2000mg, preferably 50 to 1000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Compared with the prior art, the invention has the following main advantages:

(1) The compounds have excellent ENPP1 inhibitory activity at the cellular level;

(2) The compound has better metabolic stability in animals and better anti-tumor effect when being combined with other therapies such as radiotherapy and the like.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1

Synthesis of compound S1:

synthesis of Compounds 1-3:

Tert-butyl 4- (aminomethyl) -4-fluoropiperidine-1-carboxylate (200 mg,0.861 mmol) and (tert-butoxycarbonyl) ((4- (dimethylamino) pyridin-1-bromo-1-yl) sulfonyl) amide (337 mg,1.12 mmol) were dissolved in 6ml dichloromethane and N, N-diisopropylethylamine (450 μl,2.58 mmol) was added and reacted at room temperature until the reaction was complete. The reaction was directly spin dried and then column loaded onto silica gel with DCM CH ₃ oh=100:1 to yield 291mg of product 1-3.

Synthesis of Compounds 1-4:

291mg of Compound 1-3 (0.708 mmol) was dissolved in 13ml of dichloromethane, 2.5ml of trifluoroacetic acid was added with stirring, and the reaction was completed at room temperature for about two hours. The reaction solvent is distilled off under reduced pressure, and the crude product is obtained and is directly put into the next step.

Synthesis of compound S1:

Compound 1-4 (75 mg,0.355 mmol) and 4-chloro-8-methoxyquinoline-3-carbonitrile 1-5 (60 mg,0.274 mmol) were dissolved in 3ml of N, N-dimethylformamide and N, N-diisopropylethylamine (143. Mu.l, 0.823 mmol) was added and heated to 80℃until the reaction was complete. The reaction solution was extracted with water and ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and loaded onto a column with silica gel, DCM: CH ₃ OH=20:1 to give crude product, slurried with ethyl acetate, and suction filtered to give 44mg of compound S1.¹H NMR(400MHz,DMSO-d₆)δ8.61(s,1H),7.52–7.44(m,2H),7.29(dd,J＝7.1,1.9Hz,1H),6.88(t,J＝6.9Hz,1H),6.58(s,2H),4.12–4.04(m,2H),3.93(s,3H),3.44–3.35(m,2H),3.16(dd,J＝10.0,6.1Hz,1H),3.10(d,J＝6.9Hz,1H),2.04–1.85(m,4H).

Example 2

Synthesis of compound S2:

synthesis of Compound 2-3:

The synthesis of compound 2-3 is identical to that of compound 1-3, except that compound 2-1 is substituted for 1-1.

Synthesis of Compounds 2-4:

The synthesis of compound 2-4 is identical to that of compound 1-4, except that compound 2-3 is substituted for 1-3.

Synthesis of compound S2:

the synthesis of compound S2 is the same as that of compound S1, except that 1-4 is replaced with compound 2-4 and 1-5 is replaced with compound 2-5.

¹H NMR(400MHz,DMSO-d₆)δ8.57(s,1H),7.50–7.41(m,2H),7.27(d,J＝7.3Hz,1H),6.55(s,2H),6.38(t,J＝6.7Hz,1H),4.61(s,1H),4.01(d,J＝12.9Hz,2H),3.92(s,3H),3.45(t,J＝12.3Hz,2H),2.90(d,J＝6.8Hz,2H),1.80(t,J＝12.6Hz,2H),1.56(d,J＝13.3Hz,2H).

Example 3

Synthesis of compound S3:

synthesis of Compound 2-3:

Synthesis of Compounds 2-4:

Synthesis of compound S3:

The synthesis of compound S3 is identical to that of compound S1, except that compounds 2-4 are substituted for compounds 1-4.

¹H NMR(400MHz,DMSO-d₆)δ8.68(d,J＝4.2Hz,1H),7.56(d,J＝4.7Hz,2H),7.35–7.25(m,1H),6.58(s,2H),6.41(d,J＝7.0Hz,1H),4.68(s,1H),3.94(d,J＝3.6Hz,3H),3.66(d,J＝11.8Hz,2H),3.59(s,2H),2.96(d,J＝6.8Hz,2H),1.91(s,2H),1.64(d,J＝12.8Hz,2H).

Example 4

Synthesis of compound S4:

Synthesis of Compound 4-2:

4-chloro-8-methoxyquinoline-3-carbonitrile 1-5 (150 mg,0.686 mmol) and 4-methyl-4- (BOC-aminomethyl) piperidine 4-1 (204 mg,0.892 mmol) were dissolved in 9ml of N-butanol, N-diisopropylethylamine (358 μl,2.06 mmol) was added, and heated to 100℃under nitrogen until the reaction was complete. Water was added to the reaction solution, extraction was performed with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate after washing with saturated saline, and was loaded onto a column with silica gel, DCM: CH ₃ OH=50:1, to give 251mg of product 4-2.

Synthesis of Compound 4-3:

The synthesis of compound 4-3 is identical to that of compound 1-4, except that compound 4-2 is substituted for 1-3.

Synthesis of Compound 4-4:

The synthesis of compound 4-4 is identical to that of compound 1-3, except that compound 4-3 is substituted for 1-1.

Synthesis of compound S4:

compound 4-4 (180 mg, 0.365 mmol) was dissolved in 2ml DCM and a solution of 4M hydrochloric acid in 1, 4-dioxane (1 ml) was added at 0℃and reacted overnight at room temperature. After the reaction was completed, filtration was carried out, the filter cake was dissolved in methanol, the mixture was loaded onto a column with silica gel, and DCM: CH ₃ OH=50:1 gave 18mg of the product S4.¹H NMR(400MHz,Methanol-d₄)δ8.59(s,1H),7.64(d,J＝8.6Hz,1H),7.55(t,J＝8.2Hz,1H),7.29(d,J＝7.8Hz,1H),4.03(s,3H),3.81–3.62(m,4H),3.03(s,2H),1.86(d,J＝13.3Hz,2H),1.63(d,J＝13.5Hz,2H),1.14(s,3H).

Example 5

Synthesis of compound S5:

Synthesis of Compound 5-3:

Paralylsulfonyl chloride 5-2 (397 mg,2.08 mmol) was dissolved in 5.7ml of anhydrous DCM and a solution of 4-cyano-4- (hydroxymethyl) -1-piperidinecarboxylic acid tert-butyl ester 5-1 (500 mg,2.08 mmol), triethylamine (578 mg,4.16 mmol) and 4-dimethylaminopyridine (25 mg,0.208 mmol) in anhydrous DCM (0.7 ml) was added at 0deg.C and reacted overnight at room temperature. After the reaction was completed, water was added, DCM was used for extraction, and the mixture was washed with 3M aqueous hydrochloric acid, saturated sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, and then silica gel was loaded onto the column, PE: ea=5:1 to obtain 311mg of product 5-3.

Synthesis of Compounds 5-4:

Compound 5-3 (311 mg,0.789 mmol) was dissolved in 2.5ml of N, N-dimethylformamide and sodium azide (103 mg,1.58 mmol) was added and heated to 100℃until the reaction was complete. Water was added to the reaction solution, extraction was performed with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate after washing with saturated saline, and a sample was applied to a column with silica gel, and 167mg of product 5-4 was obtained by using PE: EA=8:1.

Synthesis of Compounds 5-5:

Compound 5-4 (167 mg,0.630 mmol) was dissolved in 4ml ethanol, 10% palladium on carbon (68 mg,0.0630 mmol) was added and reacted to completion under a hydrogen atmosphere. After the reaction was completed, celite was filtered and the filtrate was column packed on silica gel with DCM CH ₃ oh=50:1 to give 90mg of product 5-5.

Synthesis of Compounds 5-6:

The synthesis of compound 5-6 is identical to that of compound 1-3, except that compound 5-5 is substituted for 1-1.

Synthesis of Compounds 5-7:

the synthesis of compounds 5-7 was identical to that of compounds 1-4, except that compounds 5-6 were substituted for compounds 1-3.

Synthesis of compound S5:

the synthesis of compound S5 is the same as that of compound S1, except that compounds 5-7 are substituted for 1-4 .¹H NMR(400MHz,DMSO-d₆)δ8.75(d,J＝1.0Hz,1H),7.66–7.49(m,2H),7.33(dd,J＝7.4,1.6Hz,1H),7.23(t,J＝7.1Hz,1H),6.73(s,2H),3.95(s,3H),3.82(d,J＝13.1Hz,2H),3.54(t,J＝12.4Hz,2H),3.23(d,J＝7.2Hz,2H),2.11(d,J＝13.4Hz,2H),2.01–1.91(m,2H).

Example 6

Synthesis of compound S9:

Synthesis of Compound 9-2:

Compound 5-5 (64 mg,0.268 mmol) was dissolved in 1ml anhydrous DCM and triethylamine (75 μl,0.536 mmol) was added and 4-morpholinosulfonyl chloride 9-1 (75 mg,0.402 mmol) was added at 0deg.C and reacted at room temperature until the reaction was complete. The reaction was diluted with DCM, washed with 10% sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and loaded onto a column with silica gel, DCM: CH ₃ oh=100:1 to give 45mg of product 9-2.

Synthesis of Compound 9-3:

the synthesis of compound 9-3 is identical to that of compound 1-4, except that compound 9-2 is substituted for 1-3.

Synthesis of compound S9:

The synthesis of compound S9 is the same as that of compound S1, except that 1-4 is replaced with compound 9-3.

¹H NMR(400MHz,DMSO-d₆)δ8.70(s,1H),7.92(t,J＝6.8Hz,1H),7.58–7.49(m,2H),7.29(dd,J＝7.3,1.6Hz,1H),3.92(s,3H),3.80(d,J＝13.0Hz,2H),3.62(t,J＝4.7Hz,4H),3.49(t,J＝12.5Hz,2H),3.29(s,2H),3.04(t,J＝4.8Hz,4H),2.08(d,J＝13.3Hz,2H),1.96–1.85(m,2H).

Example 7

Synthesis of compound S12:

synthesis of Compound 12-2:

Compound 5-5 (90 mg,0.375 mmol) was dissolved in 1.2ml anhydrous acetonitrile and 1- (fluorosulfonyl) -2, 3-dimethyl-1H-imidazol-3-ium triflate 12-1 (123 mg,0.375 mmol) was added at 0deg.C and reacted at room temperature until the reaction was complete. Water was added to the reaction solution, extraction was performed with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate after washing with saturated saline, and was loaded onto a column with silica gel, DCM: CH ₃ OH=100:1, to give 75mg of product 12-2.

Synthesis of Compound 12-4:

Compound 12-2 (75 mg,0.234 mmol), N-methyl propargylamine 12-3 (21. Mu.l, 0.234 mmol) and DBU (36. Mu.l, 0.234 mmol) were dissolved in 600. Mu.l acetonitrile and reacted at 50℃until the reaction was complete. The reaction was directly spin dried and then column loaded onto silica gel with DCM CH ₃ oh=100:1 to give 48mg of product 12-4.

Synthesis of Compound 12-5:

The synthesis of compound 12-5 is the same as that of compound 1-4, except that compound 12-4 is substituted for 1-3.

Synthesis of compound S12:

The synthesis of compound S12 is the same as that of compound S1, except that compounds 12-5 are substituted for 1-4.

¹H NMR(400MHz,DMSO-d₆)δ8.75(s,1H),7.99(t,J＝6.7Hz,1H),7.63–7.52(m,2H),7.33(d,J＝7.3Hz,1H),3.98(d,J＝2.5Hz,2H),3.95(s,3H),3.83(d,J＝13.2Hz,2H),3.53(t,J＝12.4Hz,2H),3.40(t,J＝2.4Hz,1H),3.26(d,J＝6.8Hz,2H),2.80(s,3H),2.12(d,J＝13.3Hz,2H),1.93(t,J＝12.3Hz,2H).

Example 8

Synthesis of compound S31:

Synthesis of Compound 31-3:

Compound 31-1 (100 mg, 0.463 mmol) and compound 31-2 (52. Mu.l, 0.466 mmol) were dissolved in 4ml anhydrous 1, 4-dioxane, palladium acetate (7.8 mg,0.0466 mmol), xanphos (26 mg,0.0466 mmol) and cesium carbonate (304 mg,0.932 mmol) were added and heated to 100℃until the reaction was complete under nitrogen. The reaction was filtered through celite, washed with DCM, the organic phase was dried over anhydrous sodium sulfate after washing with saturated brine, and the column was packed on silica gel with DCM CH ₃ oh=100:1 to give 90mg of product 31-3.

Synthesis of Compound 31-4:

Compound 31-3 (90 mg,0.348 mmol) was dissolved in 1.4ml methanol and 400. Mu.l water, sodium hydroxide (28 mg,0.696 mmol) was added and reacted at 60℃until the reaction was complete. The reaction solution was evaporated to dryness, diluted with water, adjusted to pH 6-7 with 1M hydrochloric acid, extracted with 10% MeOH/DCM, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 86mg of product 31-4.

Synthesis of Compound 31-5:

Compound 31-4 (75 mg,0.306 mmol) was dissolved in 1ml phosphorus oxychloride and heated to 100℃under nitrogen until the reaction was complete. The reaction was dried by spinning, diluted with DCM, and neutral alumina loaded onto a column with DCM: CH ₃ OH=200:1 to give 36mg of product 31-5.

Synthesis of compound S31:

The synthesis of compound S31 is the same as that of compound S2, except that compounds 31-5 are substituted for 1-5.

¹H NMR(400MHz,DMSO-d₆)δ8.90(dd,J＝7.5,1.5Hz,1H),8.17(dd,J＝7.5,1.5Hz,1H),7.73(dd,J＝7.5,1.5Hz,1H),7.64(t,J＝7.4Hz,1H),7.55(t,J＝7.5Hz,1H),7.43(t,J＝10.3Hz,1H),7.05(dd,J＝7.5,1.5Hz,1H),5.34(s,2H),4.67(s,1H),3.88(s,3H),3.63(q,J＝7.2Hz,4H),3.08(d,J＝10.4Hz,2H),1.91(d,J＝7.2Hz,4H).

The following target compounds were prepared by reference to the synthetic methods of the above examples:

biological Activity test

Molecular level ENPP1 enzyme activity detection

1. Experimental materials

AMP-Glo ^TM Assay Kit (Promega, V5011), 2'3' -cGAMP sodium salt (Sigma, SML 1229), recombinant human ENPP1 protein (laboratory expression purification), 384 well plates (PERKIN ELMER, 6008289).

2. Experimental method

The ENPP1 enzyme activity reaction system comprises a reaction buffer (50mM Tris,250mM NaCl,0.5mM CaCl2,1mM ZnCl2,pH 7.5), 5 mu M cGAMP,20ng recombinant human ENPP1 protein and test compounds with different concentration gradients. After 3.5h at room temperature, the reaction was incubated at 90℃for 10min to terminate the enzyme reaction. mu.L of the reaction solution and 5. Mu.L of AMP-Glo ^TM Reagent I were added to 384-well plates, and after shaking for 1min, 10. Mu. L AMP Detection Solution (AMP-Glo ^TM REAGENT II: One solution=1:100). After shaking for 1min, the reaction is carried out at room temperature for 1h, and the luminescence reading value is measured by a multifunctional micropore analysis system (PERKINEIMER ENVISION). GraphPad calculated the inhibition of ENPP1 enzyme activity by the compound and IC ₅₀ values.

The compounds of the invention have obvious inhibition activity on ENPP1, and IC ₅₀ for inhibiting the ENPP1 enzyme activity of partial compounds of the invention is shown in table 1.

Table 1 represents the inhibitory Activity of Compounds against EnPP1 enzyme molecular level

Compounds of formula (I)	IC₅₀(nM)
		Compound 1	0.6
S1	3.7
		S2	7.3
S3	13.8
		S4	3.9
S5	7.0
		S6	12.0
S7	10.2

Cell level ENPP1 activity assay

1. Experimental materials

MDA-MB-231 cells (ATCC), L-15 medium (BasalMedia, L620 KJ), p-Nph-5V-TMP (Sigma, T4510), 96 well plates (Corning, 3599).

2. Experimental method

MDA-MB-231 cells were seeded in 96-well plates. After 24h of incubation, the medium was replaced with reaction buffer (50mM Tris,250mM NaCl,pH 9.5). The substrate p-Nph-5V-TMP was added to a concentration of 1mM while the test compound was added in different concentration gradients and reacted in an air incubator at 37 ℃ for 1h. The OD was read by a microplate reader (Spectra Max 190) at 405 nm. GraphPad calculated the inhibition of ENPP1 activity by the compound and IC ₅₀ values.

The results of the measurement show that the compound of the invention has good inhibition activity on ENPP1 at the cellular level, wherein part of the compounds have better activity than the control compound 1, and the results are shown in Table 2.

Table 2 represents the inhibitory activity of the compounds on ENPP1 at the cellular level

Compounds of formula (I)	IC₅₀(nM)
		Compound 1	50.10±0.97
S1	1.23±0.62
		S2	1.53±0.28
S3	1.84±0.30
		S4	1.07±0.55
S5	0.97±0.16
		S6	2.88±0.13
S7	4.41±0.02

Conclusion based on the sulfonylurea ENPP1 inhibitor QS-1 and the phosphoric acid inhibitor compound 1, the invention introduces substituent groups at the para position of the structural center piperidine ring nitrogen atom, and further preferably carries out structural modification on sulfonylurea groups and aromatic heterocyclic tail chains, so as to design and synthesize a series of ENPP1 inhibitors with quaternary carbon centers. The introduction of the quaternary carbon center limits the rotation of the sulfonylurea group, and remarkably improves the inhibition activity of the compound on ENPP1 at the cellular level. Representative compound S5 inhibited cell ENPP1 with IC ₅₀ <1nM, a fifty-fold improvement over positive control compound 1.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A compound, characterized in that the compound is a compound represented by formula I, or a pharmaceutically acceptable salt, racemate, R-isomer, or S-isomer thereof,

in,

R ₁ , R ₂ , R ₃ and R ₄ are each independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy, cyano, hydroxyl;

R ₅ is selected from the group consisting of halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, cyano, hydroxyl;

R ₆ is selected from the following group: substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-7 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, NR ₈ R ₉ ; the substitution refers to substitution by 1, 2 or 3 substituents selected from the following group: halogen, hydroxyl, cyano, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy;

R ₈ and R ₉ are each independently selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, halogenated C1-C4 alkyl, C2-C4 alkenyl, halogenated C2-C4 alkenyl, C2-C4 alkynyl, halogenated C2-C4 alkynyl, C1-C4 alkoxy, cyano, and hydroxyl;

X is selected from the group consisting of N, CR _a ;

_Ra is selected from the group consisting of hydrogen, cyano;

_R7 is hydrogen;

Or _Ra and _R7 together with the C to which they are connected form a substituted or unsubstituted group selected from the following group: phenyl, 5-7 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N, O or S, 5-7 membered heterocyclyl containing 1, 2 or 3 heteroatoms selected from N, O or S, C4-C8 monocyclic cycloalkyl, C4-C8 bridged ring cycloalkyl, C4-C8 spirocyclic cycloalkyl; the substitution refers to substitution by 1, 2 or 3 substituents selected from the following group: halogen, hydroxyl, cyano, C1-C3 alkyl, C1-C3 alkoxy, C2-C4 alkenyl, C2-C4 alkynyl, oxo.

2. The compound according to claim 1, wherein R ₁ , R ₂ , and R ₃ are hydrogen;

_R4 is a C1-C4 alkoxy group.

3. The compound according to claim 1, characterized in that R ₅ is selected from the group consisting of halogen, C1-C4 alkyl, cyano, and hydroxyl.

4. The compound according to claim 1, characterized in that R ₆ is selected from the group consisting of substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-6 heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from N, O or S, NR ₈ R ₉ ; the substitution refers to substitution by 1, 2 or 3 substituents selected from the group consisting of halogen, hydroxyl, cyano, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy;

R ₈ and R ₉ are each independently selected from the group consisting of hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy, cyano, and hydroxy.

5. The compound according to claim 1, characterized in that _Ra is selected from the group consisting of hydrogen, cyano;

_R7 is hydrogen.

6. The compound according to claim 1, characterized in that the compound is selected from the group consisting of:

7. A pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compound according to claim 1.

8. Use of the compound according to claim 1, characterized in that it is used for preparing a drug for preventing and/or treating diseases related to ectonucleotide pyrophosphatase/phosphodiesterase 1.

9. The use according to claim 8, characterized in that the ectonucleotide pyrophosphatase/phosphodiesterase 1 related disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, and neurodegenerative diseases.

10. The use according to claim 9, characterized in that the cancer is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, and lung cancer.