WO2024125628A1 - Phosphate ester prodrug of malt1 inhibitor - Google Patents

Abstract

The present disclosure provides a phosphate ester prodrug of a Malt1 inhibitor. Specifically, the present disclosure provides a prodrug as represented by formula (I) or a pharmaceutically acceptable salt thereof and a use thereof in preparation of a drug.

Description

A phosphate prodrug of a Malt 1 inhibitor

This application claims the priority of Chinese Patent Application No. 2022116209806 filed on December 16, 2022. This application cites the entire text of the above Chinese Patent Application.

Technical Field

The present invention relates to a compound represented by formula I and a preparation method and use thereof, and belongs to the field of medicine.

Background technique

Mucosa-associated-lymphoid-tissue lymphoma-translocation 1 (MALT1) is an important protein molecule in the upstream of the NF-κB signaling pathway. It forms a complex CBM with B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10) and caspase-recruitment domain (CARD) containing membrane-associated guanylate kinase protein 1 (CARMA1), which transmits the signal of proximal antigen receptor protein to IκB kinase (IKK), thereby activating the NF-κB signaling pathway. Excessive activation of the MALT1-NF-κB signaling pathway is closely related to the occurrence of inflammation and tumors.

Patent application PCT/CN2022/099499 discloses a class of Malt 1 inhibitors. The present disclosure introduces the entire contents of patent application PCT/CN2022/099499 into the present disclosure.

Summary of the invention

In a first aspect, the present disclosure provides a prodrug of a compound of formula I or a pharmaceutically acceptable salt thereof,

In a second aspect, the present disclosure also provides a prodrug of a compound as shown in Formula I, wherein the prodrug can be selected from a phosphate prodrug of the compound of Formula I, a dicarboxylic acid ester prodrug of the compound of Formula I, or an amino acid ester prodrug.

In some embodiments, the prodrug of the compound of formula I as described in the first aspect can be a phosphate prodrug of the compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, the phosphate prodrug of the compound of formula I may be a compound structure shown in formula II-a,

n is an integer selected from 1-5, preferably n is an integer selected from 1-3, and more preferably n is an integer selected from 1-2.

In some embodiments, the prodrug of the compound of formula I as described in the first aspect may be a dicarboxylic acid ester prodrug of the compound of formula I.

In certain embodiments, the dicarboxylic acid ester prodrug of the compound of formula I is a structure shown in formula II-b

m is selected from integers of 1-5, preferably m is selected from integers of 1-3, and more preferably m is selected from integers of 1-2.

In some embodiments, the prodrug of the compound of formula I as described in the first aspect may be an amino acid ester prodrug of the compound of formula I, wherein the amino acid may be a common amino acid structure.

Wherein, the amino acid is preferably a naturally occurring amino acid. More preferably, the amino acid is an α-amino acid. Also preferably, the amino acid is an L-configuration. Preferred amino acids include the twenty essential amino acids. Preferred amino acids are lysine (Lys), leucine (Leu), isoleucine (Ile), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), methionine (Met), alanine (Ala), valine (Val), proline (Pro), histidine (His), tyrosine (Tyr), serine (Ser), norleucine (Nor), arginine (Arg), phenylalanine (Phe), tryptophan (Trp), hydroxyproline (Hyp), homoserine (Hsr), carnitine (Car), ornithine (Ort), canavanine (Cav), asparagine (Asn), glutamine (Gln), carnosine (Can), taurine (Tau), S-methylenecysteine (Djk), gamma-aminobutyric acid (GABA), cysteine (Cys), cystine (Dcy), sarcosine (Sar), threonine (Thr), and the like. More preferred amino acids are the twenty essential amino acids, Lys, Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Thr, Arg, Phe, Trp, Gln, Asn, Cys and Ser.

In certain embodiments, the amino acid ester prodrug of the compound of formula I is a structure shown in formula II-c

R is a pendant group or side chain of an amino acid.

In a third aspect, the present disclosure also provides a compound as shown in Formula III or a pharmaceutically acceptable salt thereof,

In some embodiments, the compound or pharmaceutically acceptable salt thereof as described in the first, second and third aspects, wherein the pharmaceutically acceptable salt can be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

"Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbates, salicylates, 4-aminosalicylates, naphthalene disulfonates, and the like. These salts can be prepared by methods known in the art.

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

In some embodiments, the compound or a pharmaceutically acceptable salt thereof as described in the first aspect, the second aspect, and the third aspect, wherein the pharmaceutically acceptable salt is a sodium salt.

In a fourth aspect, the present disclosure also provides a compound represented by formula IV or a pharmaceutically acceptable salt thereof,

In a fifth aspect, the present disclosure provides an isotope substituted prodrug or a pharmaceutically acceptable salt thereof as described in any one of the first to fourth aspects, preferably, the isotope substitution is deuterium atom substitution.

In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising the prodrug or a pharmaceutically acceptable salt thereof as described in any one of the first to fourth aspects or the isotope substitution as described in the fifth aspect and a pharmaceutically acceptable excipient.

In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or a pharmaceutically acceptable salt thereof.

In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable excipient based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable excipient.

The present disclosure also provides use of a prodrug or a pharmaceutically acceptable salt thereof as described in any one of the first to fourth aspects, or an isotope substitution according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect in the preparation of a medicament for preventing and/or treating a disorder associated with MALT1.

The present disclosure also provides a prodrug or a pharmaceutically acceptable salt thereof as described in any one of the first to fourth aspects, or a prodrug according to the fourth aspect. The isotope substitution according to the fifth aspect or the pharmaceutical composition according to the sixth aspect is used for preventing and/or treating diseases related to MALT1.

The present disclosure also provides use of a prodrug or a pharmaceutically acceptable salt thereof as described in any one of the first to fourth aspects, or the isotope substitution according to the fifth aspect, or the pharmaceutical composition according to the sixth aspect in the preparation of a drug for preventing and/or treating autoimmune diseases, inflammatory diseases, cancer, and tumors.

The present disclosure also provides a prodrug or a pharmaceutically acceptable salt thereof as described in any one of the first to fourth aspects, or an isotope substitution according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect for preventing and/or treating autoimmune diseases, inflammatory diseases, cancer, and tumors.

The autoimmune diseases and inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus or vasculitic diseases, the cancers or tumors, such as primary cancers of the hematopoietic system or solid tumors, including chronic myeloid leukemia, myeloid leukemia, non-Hodgkin's lymphoma and other B-cell lymphomas.

The present disclosure provides a prodrug of a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the compound of formula I has good malt1 inhibitory activity, and when it is prepared in the form of a prodrug, its water solubility and pK effect can be significantly improved.

Terminology explanation:

"Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.

"Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or domestic animals.

As used herein, an "effective amount" or "therapeutically effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

Detailed ways

The present invention is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

Example

The structures of the compounds were determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts (δ) are given in units of 10 ^-6 (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, and the solvent used was deuterated dimethylformamide. The analytes were dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), and the internal standard was tetramethylsilane (TMS).

MS was measured using Shimadzu 2010 Mass Spectrometer or Agilent 6110A MSD mass spectrometer.

High performance liquid chromatography (HPLC) was performed using Shimadzu LC-20A systems, Shimadzu LC-2010HT series, Shimadzu DGU-20A5R, Shimadzu LC-30AD, Shimadzu SIL-30AC or Agilent 1200 LC high pressure liquid chromatograph (Ultimate XB-C18 3.0*150mm column or Xtimate C18 2.1*30mm column).

Chiral HPLC analysis was carried out using Chiralpak IC-3 100×4.6mm I.D., 3um, Chiralpak AD-3 150×4.6mm I.D., 3um, Chiralpak AD-3 50×4.6mm I.D., 3um, Chiralpak AS-3 150×4.6mm I.D., 3um, Chiralpak AS-3 100×4.6mm I.D., 3μm, ChiralCel OD-3 150×4.6mm I.D., 3um, Chiralcel OD-3 100×4.6mm I.D., 3μm, ChiralCel OJ-H 150×4.6mm I.D., 5um, and Chiralcel OJ-3 150×4.6mm I.D., 3um columns.

The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm-0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm.

Column chromatography generally uses Yantai Huanghai silica gel 100-200 mesh, 200-300 mesh or 300-400 mesh silica gel as the carrier.

The chiral preparative column used was DAICEL CHIRALPAK IC (250mm*30mm, 10um) or Phenomenex-Amylose-1 (250mm*30mm, 5um).

The CombiFlash rapid preparation instrument uses Combiflash Rf150 (TELEDYNE ISCO).

The average kinase inhibition rate and _IC50 value were determined using NovoStar microplate reader (BMG, Germany).

The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Clear Blue QL-500 hydrogen generator or a HC2-SS hydrogenator.

The hydrogenation reaction is usually carried out by evacuating the vacuum, filling with hydrogen, and repeating the operation three times.

Microwave reactions were performed using a CEM Discover-S 908860 microwave reactor.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of column chromatography used for purifying the compound and the developing solvent system of thin layer chromatography, the volume ratio of the solvent is adjusted according to the polarity of the compound, generally water and acetonitrile are used as the mobile phase, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

Example 1

N-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(1-oxo-1,2-dihydroisoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide 1

first step

5-Hydrazinoisoquinoline 1b

Dissolve isoquinolin-5-amine (5.1 g, 35.37 mmol) in concentrated hydrochloric acid (50 mL), add an aqueous solution (20 mL) of sodium nitrite (3.66 g, 53.06 mmol) at 0°C, react for 30 minutes, add a concentrated hydrochloric acid solution (20 mL) of stannous chloride (19.95 g, 88.43 mmol) dropwise, react at room temperature for 3 hours, adjust the pH to 12-14 with a 20% aqueous sodium hydroxide solution, extract with ethyl acetate (30 mL*3), concentrate the organic phase under reduced pressure, and purify the residue by silica gel column chromatography eluting with ethyl acetate to obtain the title compound 1b (2.33 g, yield: 40.0%).

MS (ESI): m/z = 159.0 [M+H] ⁺ .

Step 2

1-(Isoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid ethyl ester 1d

5-Hydrazinoisoquinoline (2.33 g, 14.64 mmol) and (Z)-2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutanoic acid ethyl ester 1c (3.52 g, 14.64 mmol) were dissolved in ethanol (40 mL), reacted at 60° C. for 3 hours, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography eluting with ethyl acetate to obtain the title compound 1d (2.56 g, yield: 52.1%).

MS (ESI): m/z = 336.4 [M+H] ⁺ .

third step

5-(4-(Ethoxycarbonyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)isoquinoline 2-oxide 1e

Dissolve 1-(isoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid ethyl ester (2.56 g, 7.64 mmol) in dichloromethane (30 mL), add m-chloroperbenzoic acid (3.95 g, 22.9 mmol) at 0°C, react at room temperature overnight, wash with half-saturated sodium bisulfite solution (20 mL*2) and potassium carbonate solution (20 mL*2) in sequence, dry the organic phase, and concentrate in vacuo to give the title compound 1e (2.50 g, yield: 93.21%).

MS (ESI): m/z = 352.4 [M+H] ⁺ .

the fourth step

1-(1-Chloroisoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid ethyl ester 1f

5-(4-(Ethoxycarbonyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)isoquinoline 2-oxide (2.5 g, 7.12 mmol) was dissolved in chloroform (30 mL), phosphorus oxychloride (1.33 mL, 14.23 mmol) was added at room temperature, and the reaction was carried out at 60°C for 3 hours. The reaction was quenched with water (20 mL) under ice bath, and the organic phase was concentrated in vacuo. The mixture was purified by silica gel column chromatography with petroleum ether and ethyl acetate to give the title compound 1f (1.96 g, yield: 74.49%).

MS (ESI): m/z = 370.43 [M+H] ⁺ .

the fifth step

1-(1-Oxo-1,2-dihydroisoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid 1 g

Ethyl 1-(1-chloroisoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (1 g, 2.71 mmol) was dissolved in concentrated hydrochloric acid (15 mL), reacted at 120°C for 3 hours, and concentrated in vacuo to give 1 g (864 mg, yield: 98.83%) of the title compound.

MS (ESI): m/z = 324.4 [M+H] ⁺ .

Step 6

Benzo[c][1,2,5]oxadiazole-5-amine 1i

5-Bromobenzo[c][1,2,5]oxadiazole 1h (250 mg, 1.25 mmol) was dissolved in aqueous ammonia (1.5 mL) and N-methylpyrrolidone (1 mL), and cuprous oxide (36 mg, 0.25 mmol) was added and reacted in a microwave oven at 140°C for 1 hour. After the reaction was completed, 10 ml of ethyl acetate was added, and then washed twice with 10 ml of water and 10 ml of saturated NaCl aqueous solution respectively. The organic phase was concentrated to give the title compound 1i (180 mg, crude product), which was used directly in the next step without further purification.

MS (ESI) m/z: 136.3 [M+H] ⁺ .

Step 7

N-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(1-oxo-1,2-dihydroisoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide 1

Benzo[c][1,2,5]oxadiazole-5-amine 1i (50 mg, 0.37 mmol) was dissolved in pyridine (3 mL), and 1-(1-oxo-1,2-dihydroisoquinolin-5-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid 1 g (119 mg, 0.37 mmol) was added. Phosphorus oxychloride (113 mg, 0.74 mmol) was added at room temperature. The reaction was carried out at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, purified by C18 reverse phase column chromatography, and freeze-dried to give the title compound 1 (8.3 mg, yield: 5%).

MS (ESI): m/z = 441.6 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.65 (br s, 1H), 11.13 (s, 1H), 8.54 (s, 2H), 8.45 (d, J=8.0 Hz, 1H), 8.13 (d, J=9.6 Hz, 1H), 7.95 (d, J=7.2 Hz, 1H), 7.76-7.64 (m, 2H), 7.31 (br s, 1H), 5.67 (d, J=7.6 Hz, 1H).

Example 2

Sodium (5-(4-(benzo[c][1,2,5]oxadiazol-5-ylcarbamoyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)-1-oxoisoquinolin-2(1H)-yl)methyl phosphate

first step

At room temperature, 2a (148.4 g, 0.337 mol) and paraformaldehyde (151.7 g, 5.06 mol) were suspended in 2000 mL of tetrahydrofuran and heated to 65°C for 1 to 2 hours. After the reaction was complete, the paraformaldehyde was removed by filtration. The filtrate was collected, tetrabenzyl pyrophosphate (236 g, 0.438 mol) was added to the filtrate, the replacement gas was argon, the reaction system was cooled to 0-5°C, LiHMDS (506 mL, 0.506 mol) was slowly added dropwise to the reaction system, after the addition was completed, stirring was continued for 30 minutes, the insoluble matter in the reaction system was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with an eluent system (dichloromethane/methanol=50/1) to obtain the title product 2b (194.5 g, yield: 79%).

MS m/z(ESI):731[M+1] ⁺

¹ H NMR (400 MHz, d ₆ -DMSO): δ 11.11 (s, 1H), 8.56 (d, 1H, J=10.8 Hz), 8.51 (d, 1H, J=8.0 Hz), 8.14 (d, 1H, J=9.6 Hz), 8.03 (d, 1H, J=7.6 Hz), 7.78-7.72 (m, 2H), 7.62 (d, 1H, J=7.6 Hz), 7.32 (s, 10H), 5.91 (dd, 2H, J=9.6, 4.8 Hz), 5.81 (d, 1H, J=8.0 Hz), 5.06 (d, 4H, J=8.0 Hz).

Step 2

Add 2b (193 g, 0.264 mol), 10% Pd/C (19.3 g) and 1500 mL tetrahydrofuran to the reaction flask, and replace the gas in the reaction flask with argon. Stir at room temperature until the raw material is completely converted. After the reaction is completed, filter the Pd/C through diatomaceous earth, wash the Pd/C with tetrahydrofuran and keep it moist. Collect the filtrate and concentrate it under reduced pressure. After concentrating to dryness, add 1000 mL of methanol, filter out a small amount of insoluble impurities, and wash with 500 mL of methanol. Dissolve NaOH (18.9 g, 0.472 mol) in 70 mL of purified water, slowly add it dropwise to the methanol solution under ice bath conditions, and after addition, concentrate the methanol solution under reduced pressure to dryness, redissolve it in water, purify it by reverse phase column chromatography (filler: C18, mobile phase: 10% methanol, 90% water, isocratic elution), concentrate the preparation solution, and freeze-dry to obtain the title compound 2 (86 g, yield: 54.7%).

MS m/z(ESI):551[M+1] ⁺

¹ H NMR (400 MHz, D ₂ O): δ 8.44 (d, 1H, J=8.0 Hz), 8.24 (s, 1H), 8.04 (s, 1H), 7.84 (d, 1H, J=7.6 Hz), 7.76 (d, 1H, J=9.6 Hz), 7.63 (t, 1H, J=8.0 Hz), 7.56 (d, 1H, J=7.6 Hz), 7.41 (d, 1H, J=9.6 Hz), 6.10 (d, 1H, J=7.6 Hz), 5.59 (dd, 1H, J=17.4, 6.2 Hz).

Test Example 1: MALT1 biochemical protease assay

Using tetrapeptide as substrate (Ac-LRSR-MCA, PEPTIDE INSTITUTE) and full-length MALT1 protein purified from mammalian HEK293T cells (Strep-MALT1(1-824)-Myc/DDK, ORIGENE TP314639), The activity of MALT1 protease was evaluated in an in vitro assay. The tetrapeptide LRSR was coupled to AMC (7-amino-4-methylcoumarin) and provided a quenched fluorescent substrate for the MALT1 protease. Cleavage of AMC from arginine residues resulted in an increase in the fluorescence of coumarin measured at 450nm (excitation 360nm). The composition of the final detection buffer was 5.625nM MALT1 protein, 2.5μM Ac-LRSR-MCA, 20mM HEPES, 10mM KCl, 1.5mM MgCl·6H2O, 1mM 2Na(EDTA·2Na), 0.01% TritonX-100, 1M Trisodium Citrate Dihydrate, and 10mM DTT. The test compound dissolved in 100% DMSO was added to a 384-well plate (Greiner-781086) in an amount of 200nL per well using Echo. The highest concentration of each test compound was 10 μM or 1 μM, 3-fold gradient dilution, and the concentration range of the test was 10 μM to 0.2 nM. The control wells of the detection buffer without enzyme were used as low controls (LC), and the solvent (1% DMSO) wells that reacted with the enzyme but did not add compound treatment were used as high controls (HC). The compound was incubated with the MALT1 enzyme and substrate at room temperature for 15 hours. Subsequently, fluorescence was measured at 360 nm for excitation and 450 nm for emission in Envision. XLfit was used to fit the inhibition curve and calculate _{the IC 50 value} , which is shown in Table 1.

The _IC50 value (Zprime＞0.5) was calculated using the following formula:

LC = median of low control values

Low control: reaction without MALT1 enzyme

HC = median of high control values

High control: vehicle control without compound

Inhibition % = 100 - [(sample-LC)/(HC-LC) × 100]

Curve fitting formula: fit = (A + ((B-A) / (1 + ((C/x)^D))))

A: Min (Bottom), B: Max (Top), C: IC ₅₀ (inflection point), D: slope (Hill value)

Table 1

Experimental results: The IC ₅₀ value of the compound 1 of the present disclosure for inhibiting MALT1 biochemical protease is 34 nM, which has good inhibitory activity.

Test Example 2: Compound Solubility Determination

1. Preparation of buffer and physiological medium

(1) Preparation method of FaSSIF solution:

Preparation method of simulated fasting state intestinal fluid (FaSSIF):

Step 1: Take 0.420g of sodium hydroxide, 4.470g of disodium hydrogen phosphate, and 6.186g of sodium chloride, and dissolve them in 900mL of purified water. Adjust the pH to 6.50 with 1N sodium hydroxide solution or 1N HCl, dilute to 1000mL with purified water at room temperature, and shake well.

Step 2: Take 2.240 g of FaSSIF/FeSSIF/FaSSGF powder (brand: Biorelevant) and add it to 500 mL of the above buffer solution. Stir until the powder is completely dissolved. Add buffer solution to make up the volume to 1000 mL at room temperature.

(2) Preparation of different pH buffer solutions:

pH 1.0 hydrochloric acid solution: Take 0.9 mL of concentrated hydrochloric acid, add it into 100 mL of ultrapure water, shake well, and you will get a pH 1.0 hydrochloric acid solution.

pH 3.0-8.0 phosphate buffer: weigh 14.2732 g of disodium hydrogen phosphate, add water to 500 mL to obtain 200 mM disodium hydrogen phosphate aqueous solution, shake well to dissolve as solution A, weigh 4.2145 g of citric acid monohydrate, add water to 200 mL, shake well as solution B; take different volumes of solution A and solution B and mix them to obtain phosphate buffers of pH 3.0, 4.0, 5.0, 6.0, 6.8, 7.4 and 8.0.

2. Determination of equilibrium solubility

The equilibrium solubility of Compound 1 and Compound 2 was determined using the standard shake flask method.

3 Experimental results

Conclusion: In different pH and simulated biological media, the water solubility of compound 2 is significantly improved compared with compound 1.

Test Example 3: Pharmacokinetic Evaluation of Compounds 1 and 2

SD rats were used as test animals, and the drug concentration of compound 1 in the plasma at different times after rats were gavaged with compound 1 and compound 2 was determined by LC/MS/MS. The pharmacokinetic behavior of the disclosed compounds in rats was studied, and their pharmacokinetic characteristics were evaluated.

1 Experimental plan

1.1 Preparation of test samples

Accurately weigh appropriate amounts of compound 1 and compound 2, add a suitable volume of 0.5% MC (methylcellulose), vortex and ultrasonicate to fully mix, and prepare the required dosage formulation for oral administration by gavage.

1.2 Experimental animals

SD rats, SPF grade, transferred from the experimental institution animal reserve bank (999M-017), Experimental Animal Management Department of Shanghai Institute of Family Planning Sciences, 4 females.

1.3 Administration

Weigh before administration, and calculate the dosage based on body weight. Administer by oral gavage.

1.4 Blood collection time

0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h after administration.

1.5 Sample collection and disposal

Blood was collected from the jugular vein, about 200uL of each sample was collected, anticoagulated with EDTA-K2, placed on ice after collection, and centrifuged within 1 hour to separate plasma (centrifugation conditions: centrifugal force 6800g, 6 minutes, 2-8°C). The collected plasma samples were stored in a -70°C refrigerator before analysis, and the remaining plasma samples after analysis continued to be stored in a -70°C refrigerator with a shelf life of one month.

2 Bioanalysis and data processing

The concentration of compound 1 in plasma samples was detected, and the metabolites with known ion pair mass-to-charge ratio information were semi-quantitatively analyzed. The accuracy of quality control samples was evaluated while analyzing the samples, and it was required that more than 66% of the quality control samples had an accuracy between 80-120%.

When plotting the plasma drug concentration-time curve, BLQ is recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration is calculated as 0; the BLQ before Cmax (including "No peak") is calculated as 0; and the BLQ after Cmax (including "No peak") is not included in the calculation.

WinNonlin was used to calculate pharmacokinetic parameters such as AUC(0-t), T1/2, Cmax, Tmax and MRT based on the blood drug concentration data at different time points.

3 Experimental results

The pharmacokinetic parameters of the disclosed compounds are as follows:


Conclusion: The pharmacokinetic properties of compound 2 (phosphate prodrug of compound 1) disclosed herein are compared with those of compound 1.
AUC _last/D increased by 3.8 times (10360.06 vs 2147.60), with obvious pharmacokinetic advantages.

Claims (9)

1. A prodrug of a compound represented by formula I or a pharmaceutically acceptable salt thereof,
   
2. The prodrug or a pharmaceutically acceptable salt thereof according to claim 1, wherein the prodrug is a compound represented by formula II-a,
   

   n is selected from integers of 1-5, preferably n is selected from integers of 1-3.
3. The prodrug or a pharmaceutically acceptable salt thereof according to claim 1, wherein the prodrug is a compound represented by formula III,
   
4. The prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt is a sodium salt.
5. The prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the prodrug is
   
6. The isotope substitution of the prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, preferably, the isotope substitution is deuterium atom substitution.
7. A pharmaceutical composition comprising the prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the isotope substitution according to claim 6 and a pharmaceutically acceptable excipient.
8. Use of the prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, the isotope substitution according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and/or treating a disorder associated with MALT1.
9. Use of the prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, the isotope substitution according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and/or treating autoimmune diseases, inflammatory diseases, cancers, and tumors.