TWI807691B - Salt form and crystal form of 1,2,4-triazine-3,5-dione compounds and preparation method thereof - Google Patents

Abstract

The present invention discloses a salt form and a crystal form of a 1,2,4-triazine-3,5-dione (formula I) compound and a preparation method thereof, and use thereof in the treatment and/or the preparation of pharmaceuticals. The use is in the treatment of diseases associated with thyroid hormone receptors.

Description

Salt form, crystal form and preparation method of a 1,2,4-triazine-3,5-dione compound

The invention relates to a salt form, a crystal form and a preparation method of a 1,2,4-triazine-3,5-dione compound.

Thyroid hormones play a key role in the normal growth and development of the body and in maintaining metabolic balance (Physiological Reviews 2001, 81(3), 1097-1126.). Thyroid hormones are produced by the thyroid gland and are secreted into the circulatory system (hypothalamic/pituitary/thyroid system) in two different forms, T4 and T3, with T4 being the predominant form secreted by the thyroid and T3 being the more physiologically active form. T4 is converted to T3 by tissue-specific deiodinases, which are present in all tissues but mainly in liver and kidney.

The physiological activity of thyroid hormone is mainly mediated by thyroid hormone receptor (THR) (Endocrine Reviews 1993, 14, 348-399.). THR belongs to the superfamily of nuclear receptors that form heterodimers with retinoid receptors that act as ligand-induced transcription factors. THR has a ligand-binding domain, a DNA-binding domain, and an amino-terminal domain, and regulates gene expression through interactions with DNA response elements and with various nuclear co-activators and co-repressors. THR is encoded by the expression α and β of different genes located on human chromosomes 17 and 3. Different protein isoforms are produced by alternative splicing of primary transcripts. Each gene produces two isoforms, namely THRα1, THRα2, THRβ1, and THRβ2. THRβ1 and THRβ2 were differentially expressed from the promoters, and the two isoforms differed only at the amino terminus. THRα1 and THRα2 arise from differential splicing of pre-mRNAs, mainly at the carboxyl terminus. THRα1, THRβ1, and THRβ2 bind thyroid hormones. Studies have shown that thyroid hormone receptor subtypes can differ in their contribution to specific physiological responses. THRβ1 plays an important role in regulating thyroid-stimulating hormone and thyroid hormone in the liver, and THRβ2 plays an important role in regulating thyroid-stimulating hormone. face plays a major role. Studies have shown that two subtypes of THR α and β coexist in the liver, of which THRβ related to lipid metabolism accounts for 70-80%. In the heart, THRα is related to increased heartbeat and cardiac output (Endocrinology 2001, 142, 544; J. Biol. Chem. 1992, 267, 11794.).

Thyroid hormone is metabolized in target organs and mainly excreted in bile. Its physiological role in mammals is mainly manifested in growth and differentiation and life-sustaining functions, such as the control and regulation of heart rate, blood cholesterol and triglyceride concentrations, and systemic metabolic rate and body weight. From the perspective of pathophysiology, tachycardia, arrhythmia, heart failure, fatigue, shortness of breath, skeletal sarcopenia and osteoporosis are observed in hyperthyroidism such as Graves disease (Physiol. Rev. 2001, 81, 1097; J. Steroid. Biochem. Mol. Biol. 2001, 76, 31.).

The therapeutic use of thyroid hormone itself is limited by adverse side effects associated with hyperthyroidism, especially cardiovascular toxicity. A thyroid hormone analog, if it can avoid the adverse effects of hyperthyroidism and hypothyroidism while maintaining the beneficial effects of thyroid hormone, it may be used in the treatment of responsive diseases, such as metabolic diseases including obesity, hyperlipidemia, hypercholesterolemia, diabetes and other diseases such as liver steatosis and nonalcoholic steatohepatitis (NASH), atherosclerosis, cardiovascular disease, hypothyroidism, thyroid cancer, thyroid disease, etc.

Thyroid hormone analogs with different structures from the compounds of the present invention WO2007009913 discloses a pyridazinone derivative, especially Example 8 (compound 31, namely MGL-3196), as a thyroid hormone analog with THRβ selectivity and liver tissue selectivity, which has achieved good results and may be used to treat various diseases. However, MGL-3196 still has problems such as insufficient activity and rapid metabolism in the body.

According to the present invention, a pyridazinone compound represented by formula I is used as a thyroid hormone inhibitor, that is, 2-(3,5-dichloro-4-((5-isopropyl-6-oxyl-1,6-dihydropyridin-3-yl)oxy)phenyl)-(fluoromethyl)-1,2,4-triazine-3,5( 2H , 4H )-dione, and the salt form, crystal form and preparation method of the compound are further provided. However, the solubility of the free base of the compound of formula I is poor, and the crystallinity of the free base is poor, which brings huge challenges to the druggability. For example, poor crystallinity leads to poor fluidity in the formulation process of the preparation, making it difficult to form the preparation or poor chemical stability, etc., and low solubility leads to a decrease in the bioavailability of the drug. It is well known that the active compound can be improved by crystallization to form a pharmaceutically stable and fluid crystalline compound, or by selecting an appropriate salt form thereof. However, it is not always easy to select an appropriate salt for a specific active substance to exhibit a stable crystalline state, because the properties of the salts formed by different compounds with the same salt-forming agent are very different. Therefore the purpose of the present invention is to provide the crystal form of the compound of formula I and the form of the salt, satisfying the above-mentioned requirements of solubility, handling and stability; it is further surprising to find that the addition salt formed by the compound has better pharmacokinetic properties in mice than the free compound of formula I.

One aspect of the present invention provides the crystal form A of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 12.30±0.20, 14.02±0.20, 24.66±0.20, 25.22±0.20, 28.58±0.20,

In some schemes of the present invention, the X-ray powder diffraction pattern of the above formula (I) compound crystal form A has characteristic diffraction peaks at the following 2θ angles: 12.30±0.20, 14.02±0.20, 17.02±0.20, 20.18±0.20, 23.52±0.20, 24.66±0.20, 25.22±0.20, 25.86±0. 20, 28.30±0.20, 28.58±0.20.

In some schemes of the present invention, the X-ray powder diffraction pattern of the compound crystal form A of the above formula (I) has characteristic diffraction peaks at the following 2θ angles: 12.30±0.20, 14.02±0.20, 17.02±0.20, 18.86±0.20, 20.18±0.20, 22.36±0.20, 23.52±0.20, 23.72±0.20, 24.66±0.20, 25.22±0.20, 25.86±0.20, 28.30±0.20, 28.58±0.20, 29.74±0.20, 32.86±0.20. The 2θ angle expressed in the X-ray powder diffraction pattern in the present invention is due to the existence of errors in equipment detection and other reasons. Usually, ±0.20 is used to represent the deviation, and ±0.10 is further used to represent the deviation. Further, ±0.05 is used to represent the deviation, and ±0.02 is further used to represent the deviation. The above expressions all represent the specific numbers of the 2θ angle of the X-ray powder diffraction pattern to be expressed in the present invention.

In some aspects of the present invention, the crystal form A of the compound of formula (I) has an XRPD pattern substantially the same as that in FIG. 1 .

In some schemes of the present invention, the XRPD spectrum analysis data of the compound crystal form A of the above formula (I) are shown in Table 1:

In some schemes of the present invention, the above formula (I) compound crystal form A has a differential scanning calorimetry curve with a peak of the maximum endothermic peak at 308.85°C; in some schemes of the present invention, the DSC spectrum of the above formula (I) compound crystal form A is shown in Figure 2.

In some schemes of the present invention, for the crystal form A of the compound of formula (I), its thermogravimetric analysis curve has a weight loss of 0.0494% at 120°C and a weight loss of 0.5069% at 250°C. In some schemes of the present invention, the TGA spectrum of the crystal form A of the compound of formula (I) above is shown in FIG. 3 .

In some schemes of the present invention, the preparation method of the above-mentioned crystal form A: Weigh the compound of formula (I) into a reaction flask, add an appropriate amount of glacial acetic acid, and heat to a clear state; then slowly cool down to room temperature, and a large amount of white solids precipitate; place the solid in the reaction flask, add an appropriate amount of absolute ethanol, and heat until a suspension is obtained; slowly cool to room temperature, and the solids are separated to obtain crystal form A of the compound of formula (I).

Another aspect of the present invention also provides a pharmaceutically acceptable salt of the compound of formula (I), the pharmaceutically acceptable salt is selected from the group consisting of sodium salt, potassium salt, L-arginine salt, choline salt, meglumine salt, diethylamine salt and L-lysine salt, preferably sodium salt, choline salt and L-lysine salt, more preferably sodium salt and choline salt.

The present invention further provides the sodium salt of the compound of formula (I) as shown in formula (II), the molar ratio of the compound of formula (I) to the cation or anion of the salt is 1:1,

The present invention provides the sodium salt formula (II) of the compound of formula (I) in one of its crystal forms, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.48±0.20, 19.14±0.20, 22.30±0.20, 23.60±0.20, 26.92±0.20; Crystal form, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: .66±0.20.

In some solutions of the present invention, the X-ray powder diffraction pattern of the sodium salt formula (II) crystal form of the above-mentioned compound of formula (I) is substantially the same XRPD pattern as in FIG. 4 .

In some schemes of the present invention, the crystal form XRPD spectrum analysis data of the sodium salt formula (II) of the compound of the above formula (I) is shown in Table 2:

In some solutions of the present invention, the above-mentioned sodium salt of the compound of formula (I) and the crystalline form of formula (II) have a peak value of a maximum exothermic peak at 326.25° C. in the differential scanning calorimetry curve. In some schemes of the present invention, the DSC spectrum of the sodium salt formula (II) crystal form of the above-mentioned compound of formula (I) is shown in FIG. 5 .

In some solutions of the present invention, the crystalline form of the sodium salt formula (II) of the compound of the above formula (I) has a thermogravimetric analysis curve with a weight loss of 0.6243% at 120°C. In some solutions of the present invention, the TGA spectrum of the above crystal form is shown in FIG. 6 .

In some schemes of the present invention, the preparation method of the sodium salt formula (II) of the compound of the above formula (I): put the compound of the formula (I) into a reaction bottle, add it to an organic solvent, and stir at room temperature to form a suspension. Then Na alkali salt aqueous solution was slowly added dropwise to the reaction solution at a molar ratio of 1:1-1.5, stirred magnetically at room temperature, and solids were precipitated and collected to obtain the sodium salt formula (II) of the compound of formula (I).

The present invention further provides the L-lysine salt of the compound of formula (I) as shown in formula (III), the molar ratio of the compound of formula (I) to the cation or anion of the salt is 1:1,

The present invention provides the crystal form in one embodiment of the L-lysine salt formula (III) of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.86±0.20, 16.32±0.20, 22.62±0.20, 25.00±0.20, 25.80±0.20; further one embodiment of the L-lysine salt formula (III) of the compound (I) of the present invention The crystal form provided in , its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.71±0.20, 8.86±0.20, 10.98±0.20, 13.82±0.20, 16.32±0.20, 18.92±0.20, 20.72, 22.62±0.20, 25.00±0.20, 25.80 ±0.20.

In some solutions of the present invention, the X-ray powder diffraction pattern of the crystal form of the L-lysine salt formula (III) of the compound of formula (I) above is substantially the same XRPD pattern as in FIG. 7 .

In some schemes of the present invention, the XRPD spectrum analysis data of the crystal form of the L-lysine salt formula (III) of the compound of the above formula (I) is shown in Table 3:

In some schemes of the present invention, the crystal form of the L-lysine salt formula (III) of the above-mentioned compound of formula (I) has a differential scanning calorimetry curve with a peak value of a maximum endothermic peak at 244.18°C, and a peak value of an exothermic peak at 252.63°C. In some solutions of the present invention, the DSC spectrum of the above crystal form is shown in FIG. 8 .

In some schemes of the present invention, the crystal form of the L-lysine salt formula (III) of the compound of formula (I) above has a thermogravimetric analysis curve with a weight loss of 0.5199% at 120°C. In some solutions of the present invention, the TGA spectrum of the above crystal form is shown in FIG. 9 .

In some schemes of the present invention, the preparation method of the L-lysine salt formula (III) of the compound of the above formula (I): put the compound of the formula (I) into a reaction bottle, add it into an organic solvent, and stir at room temperature to form a suspension. Then L-Lysine (L-Lysine) aqueous solution was slowly added dropwise to the reaction solution at a molar ratio of 1:1-1.5, stirred magnetically at room temperature, and solids were precipitated and collected to obtain the L-lysine salt of the compound of formula (I) formula (III).

The present invention further provides the choline salt of the compound of formula (I) as shown in formula (IV), the ratio of the compound of formula (I) to the cation or anion of the salt can be 1:1,

The present invention provides the crystal form in one embodiment of the choline salt formula (IV) of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.46±0.20, 19.02±0.20, 19.60±0.20, 21.68±0.20, 23.64±0.20; Provided crystal form, its X-ray powder diffraction pattern There are characteristic diffraction peaks at the following 2θ angles: 7.78±0.20, 15.46±0.20±0.20, 18.00±0.20, 19.02±0.20, 19.60±0.20, 21.68±0.20, 23.64±0.20, 24.02±0.20, 25.76±0.20, 29.16±0 .20.

In some aspects of the present invention, the X-ray powder diffraction pattern of the crystal form of the choline salt formula (IV) of the compound of the above formula (I) is substantially the same XRPD pattern as in FIG. 10 .

In some schemes of the present invention, the XRPD spectrum analysis data of the crystalline form of the choline salt formula (IV) of the above-mentioned compound of formula (I) are as shown in Table 4:

In some solutions of the present invention, the crystalline form of the choline salt of the compound of formula (I) above (IV) has a differential scanning calorimetry curve with a maximum endothermic peak at 202.26°C. In some solutions of the present invention, the DSC spectra of the above crystal forms are shown in FIG. 11 .

In some solutions of the present invention, the crystalline form of the choline salt formula (IV) of the compound of formula (I) above has a thermogravimetric analysis curve with a weight loss of 0.0128% at 120°C. In some solutions of the present invention, the TGA spectrum of the above crystal form is shown in FIG. 12 .

In some schemes of the present invention, the choline salt formula (IV) of the above-mentioned formula (I) compound Preparation method: Put the compound of formula (I) into a reaction bottle, add it into an organic solvent, and stir at room temperature to form a suspension. Then choline (Choline) aqueous solution was slowly added dropwise to the reaction solution at a molar ratio of 1:1-1.5, stirred magnetically at room temperature, and solids were precipitated and collected to obtain the choline salt formula (IV) of the compound of formula (I).

In one embodiment, the use of the salt of the compound of formula (I) and its crystal form provided above in the present invention in therapy and or preparation of medicines, the use is to treat diseases related to thyroid hormone receptors.

In one embodiment, the pharmaceutical composition of the salt of the compound of formula (I) and its crystal form provided above in the present invention.

In one embodiment, the present invention provides a method of treatment, the method comprising administering an effective amount of the salt of the compound of formula (I) and its crystal form provided above to a patient in need thereof.

The beneficial effects of the present invention are as follows: It has been surprisingly found that the free base crystal form has better stability and crystal form stability, which is helpful for making corresponding preparation dosage forms of medicines; the addition salt of the compound of formula (I) has better pharmacokinetic properties in mice than the free base, and the best one is choline salt.

Definition and Description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific phrase or term should not be considered indeterminate or unclear if it is not specifically defined, but should be understood according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

The intermediate compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining it with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

The chemical reactions of the specific embodiments of the present invention are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present invention and the required reagents and materials. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes on the basis of existing embodiments.

The present invention will be specifically described below through examples, and these examples do not imply that Invention of any limitation.

All solvents used in the present invention are commercially available and used without further purification.

Compounds are named according to the conventional naming principles in this field or using ChemDraw® software, commercially available compounds adopt the supplier catalog name, and the description of the free base of the compound of formula (I) all means that the compound of formula (I) itself does not form any cation or anion salt.

experimental reagent

Laboratory equipment

Test instrument parameters

X-ray powder diffraction (X-ray powder diffraction, XRPD)

The equipment is Shimadzu XRD-6000, and the sample is scanned according to the following parameters: the radiation source is Cu~Kα target (1.54056Å), the minimum operating voltage and current of the light pipe are 40kV and 30mA, respectively, and the 2-Theta value of the sample scanning range is from 5o to 50o or (2o to 50o). The scanning speed is 5deg/min.

Thermogravimetric analysis (TGA)

Weigh about 5 mg of sample into a crucible, under nitrogen protection, raise the temperature from 30°C to 400°C at a rate of 20°C/min, and keep at 400°C for 1 min.

Differential scanning calorimetry (DSC)

Weigh about 1~5mg powder sample and place it in a closed aluminum crucible, and make a pinhole on the crucible lid. Under nitrogen protection, the temperature was raised from 30°C to 350°C for differential calorimetry scanning, and kept at 350°C for 1 min. The heating rate is 20°C/min.

polarized light microscope (PLM)

The sample is dispersed in the medium (silicone oil), the sample is observed with a 10X eyepiece and a 10X objective lens, and the image is recorded with a camera computer system.

Fig. 1 is the XRPD spectrogram of the Cu-Kα radiation of formula (I) compound crystal form A; Fig. 2 is the DSC spectrogram of formula (I) compound crystal form A; Fig. 3 is the TGA spectrogram of formula (I) compound crystal form A; Fig. 4 is the XRPD spectrogram of the Cu-Kα radiation of the sodium salt formula (II) crystal form of formula (I) compound; Fig. 5 is the DSC spectrogram of the sodium salt formula (II) crystal form of formula (I) compound; Fig. 6 is the sodium salt of formula (I) compound The TGA spectrogram of formula (II) crystal form; Fig. 7 is the XRPD spectrogram of the Cu-K α radiation of the L-lysine salt formula (III) crystal form of formula (I) compound; Fig. 8 is the DSC spectrogram of the L-lysine salt formula (III) crystal form of formula (I) compound; Fig. 9 is the TGA spectrogram of the L-lysine salt formula (III) crystal form of formula (I) compound; XRPD spectrogram; Fig. 11 is the DSC spectrogram of the choline salt formula (IV) crystal form of formula (I) compound; Fig. 12 is the TGA spectrogram of the choline salt formula (IV) crystal form of formula (I) compound; Fig. 13 is the H-NMR result of the L-lysine salt formula (III) of formula (I) compound.

The embodiments of the present invention will be further described in detail below in conjunction with the drawings and descriptions, but the specific implementation manners are not intended to limit the content of the present invention.

Example 1

Preparation of 2-(3,5-dichloro-4-((5-isopropyl-6-oxyl-1,6-dihydropyridin-3-yl)oxy)phenyl)-(fluoromethyl)-1,2,4-triazine-3,5(2H,4H)-dione (Formula I) compound

first step

2-(3,5-Dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid 1b

2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-cyanide 1a (300 mg, 0.69 mmol, see WO2014043706A1 for the synthesis method) was dissolved in 10 mL of glacial acetic acid, and concentrated hydrochloric acid (2 mL), heated to 120°C for 5 hours. After the reaction was completed, the reaction liquid was cooled to room temperature, added 20 mL of water to dilute, stirred, filtered, and dried to obtain 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid 1b (260 mg, light yellow solid), yield: 84%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.71(s, 1H), 12.21(s, 1H), 7.82(s, 2H), 7.45(s, 1H), 3.21(m, 1H), 1.19(d, J = 6.4Hz, 6H). MS m/z (ESI): 453.8 [M+1] ⁺ .

second step

Methyl 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylate 1c

2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid 1b (200mg, 0.44mmol) was dissolved in 20mL of methanol, slowly added phosphine chloride (2mL) under ice bath, and the reaction solution was heated to 100°C React for 5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residual solid was filtered with petroleum ether:ethyl acetate (10:1), and dried to obtain methyl 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylate 1c (200 mg, light yellow solid). Rate: 97%. MS m/z (ESI): 466.2 [M-1] ^- .

third step

2-(3,5-Dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-(hydroxymethyl)-1,2,4-triazine-3,5( 2H , 4H )-dione 1d

Methyl 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylate 1c (200 mg, 0.43 mmol) was dissolved in 20 mL tetrahydrofuran, and sodium borohydride (82 mg, 2.15 mmol) was added at room temperature, Methanol was then slowly added dropwise. After the reaction was completed, add 20 mL of water and adjust the pH to 6 with 3M dilute hydrochloric acid, extract with ethyl acetate (30 mL×3), combine the organic phases, wash with saturated sodium chloride solution (100 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. 1,6-Dihydropyridazin-3-yl)oxy)phenyl)-(hydroxymethyl)-1,2,4-triazine-3,5(2H , 4H )-dione 1d (150 mg, white solid), yield: 79%.

¹ H NMR (400MHz,DMSO-d ₆ )δ 12.49(s,1H),12.25(s,1H),7.86(s,2H),7.45(s,1H),5.33(t, J =6.4Hz,1H),4.40(d, J =6.0Hz,2H),3.21(m,1H),1.19(d, J =6.4Hz,6H). MS m/z (ESI): 438.2 [M-1] ^- .

the fourth step

2-(3,5-Dichloro-4-((5-isopropyl-6-oxyl-1,6-dihydropyridin-3-yl)oxy)phenyl)-(fluoromethyl)-1,2,4-triazine-3,5(2 H ,4 H )-dione (Formula I) compound

Methyl 2-(3,5-dichloro-4-((5-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)oxy)phenyl)-(hydroxymethyl)-1,2,4-triazine-3,5( 2H , 4H )-dione 1d (100mg, 0.23mmol) was dissolved in 20mL of dichloromethane, DAST (5 drops) was added under ice cooling, and the reaction solution was reacted at this temperature for 30 minutes. After the reaction was completed, the reaction solution was added to 20 mL of ice water, extracted with dichloromethane (20 mL×3), combined organic phases, washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by preparative chromatography to obtain 2-(3,5-dichloro-4-((5-isopropyl-6-oxy-1,6-dihydropyridin-3-yl)oxy)phenyl)-(fluoromethyl)-1,2,4-triazine-3 , 5( 2H , 4H )-diketone ( Formula I ) (45 mg), Yield: 45%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.48(s, 1H), 12.21(s, 1H), 7.81(s, 2H), 7.43(s, 1H), 5.29(d, J =48.0Hz, 1H), 3.21(m, 1H), 1.19(d, J =6.4Hz, 6H). ¹⁹ F NMR (400 MHz, DMSO-d ₆ ) δ -219.9 ppm. MS m/z (ESI): 442.2 [M+1] ⁺ .

Example 2

Preparation of Formula (I) Compound Crystal Form A

Weigh 7.0 g of the free base of the compound of formula (I) obtained by the method in Example 1 into a 250 mL flask, add 150 mL of glacial acetic acid, and heat to 120° C. to a clear state. Then the temperature was slowly cooled to room temperature, and the process lasted for about 2 hours, and a large amount of white solids were precipitated. After the solid was separated, it was dried under reduced pressure and vacuum at 50°C for about 2 hours to obtain a white solid. The solid was placed in a 250 mL flask, 50 mL of absolute ethanol was added, heated to 90° C. for 1 h, and a suspension was obtained. Slowly cool to room temperature, separate the solid and dry under reduced pressure at 50°C for about 2 hours to obtain the compound of formula (I) in the crystal form A.

The Cu-Kα radiation XRPD spectrum, DSC spectrum, and TGA spectrum of crystal form A obtained in this example are shown in Figures 1, 2, and 3, respectively.

Example 3

The preparation of the sodium salt formula (II) of formula (I) compound

Weigh 502.95 mg of the compound of formula (I) obtained by the method in Example 1 into a 40 mL vial, add 8 mL of isopropanol, and stir at room temperature to form a suspension. Then NaOH aqueous solution (2N aqueous solution) was slowly added dropwise to the reaction solution at a molar ratio of 1:1.05 (600 μL), and it was roughly dissolved and partially adhered to the wall, and a large amount of precipitation occurred in about 0.5 h. Finally, the sample was magnetically stirred at room temperature overnight (about 20 h), and the solid was collected by centrifugation (12000 rpm, centrifuged for 5 min), and vacuum-dried under reduced pressure at 50 ° C for about 20 h overnight to obtain formula (I). The sodium salt of the compound is formula (II).

The Cu-Kα radiation XRPD spectrum, DSC spectrum, and TGA spectrum of the sodium salt formula (II) of the compound of formula (I) in this embodiment are shown in Figures 4, 5, and 6, respectively.

Example 4

The preparation of the L-lysine salt formula (III) of formula (I) compound

Weigh 509.65 mg of the compound of formula (I) obtained by the method in Example 1 into a 40 mL vial, add 10 mL of isopropanol, and stir at room temperature to form a suspension. Then L-Lysine (L-Lysine) aqueous solution (2N in water) was slowly added dropwise to the reaction solution at a molar ratio of 1:1.05 (607 μL) to form a milky form. Finally, the sample was magnetically stirred at room temperature overnight (about 20 h), and the solid was collected by centrifugation (12000 rpm, 5 min); dried under reduced pressure at 50 ° C for about 20 h overnight to obtain the L-lysine salt formula (III) of the compound of formula (I).

The XRPD spectrum, DSC spectrum, and TGA spectrum of the Cu-Kα radiated XRPD spectrum, DSC spectrum, and TGA spectrum of the L-lysine salt formula (III) obtained from the compound of formula (I) in this embodiment are shown in Figures 7, 8, and 9, respectively.

Example 5

The preparation of the choline salt formula (IV) of formula (I) compound

Weigh 501.02 mg of the compound of formula (I) obtained by the method in Example 1 into a 40 mL vial, add 10 mL of isopropanol, and stir at room temperature to form a suspension. Then choline (Choline) aqueous solution (48% in water) was slowly added dropwise to the reaction solution at a molar ratio of 1:1.05 (302 μL), clarified, and finally the sample was magnetically stirred at room temperature overnight for precipitation (about 20 h), and the solid was collected by centrifugation (12000 rpm, centrifuged for 5 min); dried under reduced pressure and vacuum at 50 ° C for about 20 h overnight to obtain the crystal form of the compound of formula (IV).

The Cu-Kα radiation XRPD spectrum, DSC spectrum, and TGA spectrum of the choline salt of the compound of formula (I) obtained in this embodiment are shown in Figures 10, 11, and 12, respectively.

Example 6

The preparation of the magnesium salt of formula (I) compound

Weigh 50.0 mg of the compound of formula (I) obtained by the method in Example 1 into a 10 mL vial, add 2 mL of acetone, and stir at room temperature to form a suspension. Then the magnesium chloride aqueous solution was slowly added dropwise to the reaction solution at a molar ratio of 1:1.05 to obtain a suspension. The suspension was magnetically stirred overnight (about 20 h) at room temperature, and the solid was collected by centrifugation (12000 rpm, centrifuged for 5 min); dried under reduced pressure at 50° C. for about 20 h overnight to obtain a solid. According to XRPD spectrum analysis, no corresponding ideal salt was formed.

Example 7

The preparation of the calcium salt of formula (I) compound

Weigh 50.0 mg of the compound of formula (I) obtained by the method in Example 1 into a 10 mL vial, add 2 mL of acetone, and stir at room temperature to form a suspension. Then calcium chloride aqueous solution was slowly added dropwise to the reaction solution at a molar ratio of 1:1.05 to obtain a suspension. The suspension was magnetically stirred overnight (about 20 h) at room temperature, and the solid was collected by centrifugation (12000 rpm, centrifuged for 5 min); dried under reduced pressure at 50° C. for about 20 h overnight to obtain a solid. According to XRPD spectrum analysis, no corresponding ideal salt was formed.

Example 8

According to the same similar method as in Example 2-7, the preparation of L-arginine, meglumine, and diethylamine salts was completed by using the corresponding cation or anion salts.

Example 9 THRβ binding experiment

Experimental Methods: In vitro analysis of compound agonism on THRβ was performed using a time-resolved fluorescence resonance energy transfer co-initiator peptide recruitment assay. This experiment uses Eu-anti-GST antibody, biotin-SRC2-2 co-initiator peptide, streptavidin-d2, RXRα and GST-tagged THRβ-LBD. Eu-anti-GST antibody indirectly labels THRβ-LBD by binding to the GST tag. Streptavidin-d2 indirectly labels the SRC2-2 co-initiator peptide by binding to the biotin tag. In the presence of RXRα, THRβ-LBD can form heterodimer THRβ-LBD/RXRα with it. Agonists bind to THRβ-LBD/RXRα and cause a conformational change in THRβ-LBD, thereby increasing the recruitment capacity of the heterodimer to the SRC2-2 co-initiator peptide. At the same time, the distance between the resulting d2-labeled SRC2-2 co-initiator peptide and the Eu-anti-GST antibody decreased, increasing the THR-FRET signal. According to different concentrations of chemical The effect of the compound on the activity of THRβ can be used to evaluate the agonistic ability of the compound.

The detailed procedure is as follows.

Prepare a 100X reference compound or compound in DMSO and make a 1:3 equal dilution.

A 100X serial dilution of the reference compound or compound diluted 4X with 1X Reaction Buffer was added to the assay plate.

Prepare a mixed solution of 4X THRβ-LBD, 4X RXRα with 1X reaction buffer, and add it to the assay plate.

Prepare a mixed solution of 2X biotin-SRC2-2, 2X Eu-anti-GST, 2X streptavidin-d2 with 1X reaction buffer, and add it to the assay plate.

Centrifuge at 1000rpm for 1min and incubate at room temperature for 4 hours in the dark.

Read the 665nm and 615nm fluorescence signal values on the EnVision 2104 plate reader, and calculate the Ratio _665nm/615nm .

Experimental results: see Table 5

Example 10 THRα binding experiment

Experimental method: The in vitro analysis of the agonistic effect of the compound on THRα adopts the similar method of the nine kinds of THRβ binding experiments in Example 9, the difference is that THRα is used instead of THRβ.

Experimental results: see Table 6

Example 11

In vitro liver microsome stability assay

experimental method:

(1) Solution preparation

The test substance and the positive control substance verapamil were respectively dissolved in DMSO to 10 mM as a stock solution, and the above 10 mM stock solution was diluted with 70% acetonitrile aqueous solution to a concentration of 0.25 mM.

Prepare the NADPH regeneration system finally containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-PDH and 3.3mM magnesium chloride respectively.

The stop solution was an acetonitrile solution containing tolbutamide and propranolol (both internal standards).

The phosphate buffer was 100 mM K ₃ PO ₄ (pH=7.4) buffer containing 3.3 mM MgCl ₂ .

Liver microsomal incubation system in 100mM phosphate buffer, containing 0.2mg/mL liver microsomal protein and 1μM test substance/positive control

(2) Incubation process

Take 80 μL of the mixed solution from the incubation system, add 400 μL of stop solution to precipitate protein, vortex and add 20 μL of NADPH regeneration system as the 0 min sample point.

Add 130 μL of NADPH regeneration system to the remaining 520 μL of protein drug mixture, mix well, and start incubation. The final incubation system was 650 μL, containing 0.2 mg/mL liver microsomal protein, 1 μM test substance/positive control, 1.3 mM NADP, 3.3 mM G-6-P, 0.6 U/mL G-6-PDH.

The mixed system was slowly shaken and incubated in a water bath at 37 °C. At 5, 10, 30, and 60 min, respectively, 100 μL of the incubation solution was transferred to a new 96-well plate with 400 μL of stop solution in each well, and the protein was precipitated (centrifuged at 4000 × g, 4 °C for 15 minutes).

Take 100 μL of the supernatant, dilute it with water at a ratio of 1:2, and analyze the sample by liquid chromatography-mass spectrometry (LC-MS/MS).

Experimental results: see Table 7.

Example 12

Pharmacokinetic evaluation:

Taking mice as test animals, the drug concentration in blood plasma at different times after the mice were intragastrically administered the compound of formula (I)-formula (IV) was tested. Study the pharmacokinetic behavior of the compound of the present invention in mice, and evaluate its drug metabolism characteristics. Each group of embodiments selects 9 healthy adult male ICR mice with similar body weight, and the oral administration dose is 15.0mg/kg (or equivalently 15.0mg/kg), single administration, and the whole blood is collected before administration and administration 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h respectively. A LC/MS/MS method was established to determine the concentration of unchanged drug in plasma samples. The main pharmacokinetic parameters were calculated using the WinNonlin 6.3 NCA model, see Table 8 below for details.

experimental method:

Experimental drugs: the compound of formula (I) obtained in the above-mentioned embodiment, the sodium salt formula (II) of the compound of formula (I), the L-lysine salt formula (III) of the compound of formula (I) and the choline salt formula (IV) of the compound of formula (I).

Experimental animals: 9 healthy adult male ICR mice, divided into 3 groups on average, 3 mice in each group, Shanghai Sipro-Bikay Experimental Animal Co., Ltd., animal certificate number: SCXK (Shanghai) 2018-0006 2018 0006 010467.

Drug configuration: Accurately weigh a certain amount of formula (I) - formula (IV) compound respectively, and place in 15ml sample Add 5uL of Tween 80 to the tube to make it wet, then add 4mL of 2% hydroxypropyl cellulose (Klucel LF) solution, sonicate and vortex to obtain a uniform suspension. Prepare fresh just before use.

Administration: intragastric administration to ICR mice after fasting overnight, the administration dose is 15.0 mg/kg (or equivalently 15.0 mg/kg).

Operation: Mice were intragastrically administered the compound of formula (I)-formula (IV). Whole blood was collected before administration and 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h after administration, anticoagulated with heparin sodium, and centrifuged at 4°C for 5 minutes to separate the plasma, stored at -80°C for testing, and fed 4 hours after administration.

Determination of the content of the compound to be tested in mouse plasma after intragastric administration of different concentrations of drugs: Take 12.5uL of mouse plasma to be tested at each time after the compound of formula (I)-formula (IV) is administered, mix them in pairs, add 100uL of internal standard solution, 125uL of methanol, vortex mix for 2min, centrifuge at 13000rpm at 4°C for 10min, and take the supernatant for LC-MS/MS analysis.

The results of pharmacokinetic parameters are shown in Table 8.

Conclusion: The sodium salt formula (II), L-lysine salt formula (III) and choline salt formula (IV) compound of the present invention (formula I) have good pharmacokinetic absorption and have obvious pharmacokinetic advantages. Compared with the compound of formula (I), under the same dosage and formulation, some compounds of the present invention unexpectedly show higher Cmax value and exposure, and longer half-life. All the above PK results show that the compounds provided by the present invention have good PK properties and can be used as therapeutic drugs for metabolic related diseases.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies thereof, the present invention also intends to include these modifications and variations.

Claims (18)

A crystal form A of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 12.30±0.20, 14.02±0.20, 24.66±0.20, 25.22±0.20, 28.58±0.20,

The crystal form A as described in Claim 1, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 12.30±0.20, 14.02±0.20, 17.02±0.20, 20.18±0.20, 23.52±0.20, 24.66±0.20, 25.22±0.20, 25.86±0.20, 28. 30±0.20, 28.58±0.20. The crystal form A as described in claim 2 has an XRPD spectrum substantially the same as that in FIG. 1 . The crystal form A as described in Claim 1, wherein its DSC spectrum is shown in FIG. 2 . A pharmaceutically acceptable salt of the compound of formula (I), characterized in that the pharmaceutically acceptable salt is selected from the group consisting of sodium salt, potassium salt, choline salt and L-lysine salt,

The salt as described in claim item 5, wherein the sodium salt of the compound of formula (I) is shown in formula (II), and the molar ratio of the compound of formula (I) to the cation or anion of the salt is 1:1,

The salt as described in Claim 6, wherein the X-ray powder diffraction pattern of the sodium salt of the compound of formula (II) has characteristic diffraction peaks at the following 2θ angles: 10.48±0.20, 19.14±0.20, 22.30±0.20, 23.60±0.20, 26.92±0.20. The salt as described in claim item 7, wherein the X-ray powder diffraction pattern of the sodium salt of the compound of formula (I) (II) has an XRPD pattern substantially the same as that in FIG. 4 . The salt according to claim 6, characterized in that the DSC spectrum of the sodium salt formula (II) of the compound of formula (I) is as shown in Figure 5. The salt as described in claim item 5, wherein the L-lysine salt of the compound of formula (I) is shown in formula (III), and the molar ratio of the compound of formula (I) to the cation or anion of the salt is 1:1,

The salt as described in claim item 10, wherein the X-ray powder diffraction pattern of the L-lysine salt of the compound of formula (I) (III) has characteristic diffraction peaks at the following 2θ angles: 8.86±0.20, 16.32±0.20, 22.62±0.20, 25.00±0.20, 25.80±0.20. The salt according to claim 11, wherein the X-ray powder diffraction pattern of the L-lysine salt of the compound of formula (I) formula (III) has an XRPD pattern substantially the same as that in FIG. 7 . The salt according to claim item 10, wherein the DSC spectrum of the L-lysine salt formula (III) of the compound of formula (I) is shown in FIG. 8 . The salt as described in claim item 5, wherein the choline salt of the compound of formula (I) is shown in formula (IV), and the ratio of the compound of formula (I) to the cation or anion of the salt can be 1:1,

The salt as claimed in item 14, wherein the present invention provides the choline salt of the compound of formula (I) The X-ray powder diffraction pattern of formula (IV) has characteristic diffraction peaks at the following 2θ angles: 15.46±0.20, 19.02±0.20, 19.60±0.20, 21.68±0.20, 23.64±0.20. The salt according to claim 15, wherein the X-ray powder diffraction pattern of the choline salt of the compound of formula (I) (IV) has an XRPD pattern substantially the same as that in Figure 10. The salt as described in claim item 14, wherein the DSC spectrum of the choline salt formula (IV) of the compound of formula (I) is shown in Figure 11. Use of a crystal form A according to any one of claims 1 to 4 or a salt according to any one of claims 5 to 17 in the preparation of a medicament for treating diseases related to thyroid hormone receptors.

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