CN111808079A - Indole ASK1 small molecule inhibitor and preparation method and application thereof - Google Patents

Abstract

The invention discloses an indole ASK1 small molecule inhibitor and a preparation method and application thereof, wherein the inhibitor comprises a compound shown as a general formula (I) and a stereoisomer or pharmaceutically acceptable salt thereof. The experimental data prove that the inhibitor is used for non-alcoholic fatHas certain treatment effect on the sexual hepatitis and the ulcerative colitis and good application prospect.

Description

Indole ASK1 small molecule inhibitor and preparation method and application thereof

technical field

The invention mainly relates to a class of compounds and a preparation method and application thereof, in particular to an indole ASK1 small molecule inhibitor and a preparation method and application thereof.

Background technique

Apoptosis signal-regulated kinase 1 (ASK1) belongs to the mitogen-activated protein kinase (MAPK) family. The MAPK signaling pathway is one of the important signal transduction systems in organisms, and is involved in mediating cell proliferation, differentiation, apoptosis, and inflammation. Physiological processes such as reactions. The MAPK family includes three subfamilies of extracellular signal-regulated kinase (ERK), ^P38 MAPK and JNK. The MAPK family consists of three-level kinases. First, mitogen-activated protein kinase kinase kinase (MAP3K) is activated by stimuli inside and outside the cell. The activated MAP3K phosphorylates and activates MAP2K, which in turn activates MAPK. ASK1 belongs to ^MAP3K5 and is upstream of the P38 MAPK and JNK signaling pathways.

ASK1 can be activated in response to a series of stress conditions, such as oxidative stress, endoplasmic reticulum stress, and calcium influx. Activated ASK1 activates the P38 MAPK and JNK pathways by phosphorylating ^MKK3 /6 and MKK4/7. ASK1 plays an important role in regulating cellular physiological processes such as apoptosis, cytokine responses, cell differentiation, and innate immune responses. Studies have shown that ASK1 has a clear correlation with many diseases.

Relevant literature reports show that ASK1 small-molecule inhibitors exhibit therapeutic potential for a variety of diseases. For example, GS-444217 has obvious effects in various animal experimental models of renal fibrosis and inflammation, K811, K812 prolong mouse survival in SOD1 ^G93A transgenic mouse model of ALS (Amyotrophic Lateral Sclerosis), K811 Inhibition of gastric cancer cell growth in vitro and in vivo experiments, GS-4997 has been clinically tested for pulmonary hypertension, diabetic nephropathy, etc. However, the current structure of ASK1 small molecule inhibitors is relatively simple, and further research is needed.

SUMMARY OF THE INVENTION

Purpose of the invention: The purpose of the present invention is to provide an indole ASK1 small molecule inhibitor, which can affect apoptosis signal-regulated kinase 1, thereby showing potential for the treatment of various diseases. Another object of the present invention is to disclose the preparation method of the inhibitor and its application in non-alcoholic steatohepatitis and ulcerative colitis medicines.

Technical solution: The indole ASK1 small molecule inhibitor of the present invention comprises a compound represented by general formula (I) and a stereoisomer or a pharmaceutically acceptable salt thereof:

M ¹ is selected from N or CH;

R ¹ are the same or different, and each is independently selected from a hydrogen atom, an alkoxy group, a halogen, a heterocyclic group, an aryl group, a heteroaryl group, -(CH ₂ ) _n OR ⁴ , -(CH ₂ ) _n NR ⁵ R ⁶ , -(CH ₂ ) _n NR ⁵ C(O)R ⁴ , n is 0, 1, 2, 3, 4 or 5; wherein the heteroaryl group is not substituted or is substituted by an alkyl group, an alkoxy group, substituted with one or more substituents in heterocyclyl;

R ² are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a halogen, an aryl group, and a heteroaryl group;

^R is selected from alkyl, hydroxyalkyl or heterocyclyl;

R ⁴ is selected from alkyl;

R ⁵ and R ⁶ are the same or different, and each is independently selected from a hydrogen atom, a heterocyclic group;

X is selected from N(R ² );

m is 0, 1, 2, 3 or 4;

p is 1 or 2.

For the indole ASK1 small molecule inhibitor, M ¹ is N:

In the indole ASK1 small molecule inhibitor, X is an imino group, and R ³ is an isopropyl group, as shown in the general formula (III):

In the indole ASK1 small molecule inhibitor, R ¹ is selected from hydrogen atom, C _1-6 alkoxy, halogen, 6-membered heterocyclic group, 6-membered aryl, 5-6-membered heteroaryl, substituted 5-6-membered heteroaryl group and -(CH ₂ ) _n NR ⁵ C(O)R ⁴ , preferably hydrogen atom, C _1-3 alkoxy group, halogen, 6-membered heterocyclic group, 6-membered aryl group, 5- 6-membered heteroaryl, substituted 5-membered heteroaryl and -(CH ₂ ) _n NHC(O)R ⁴ , more preferably hydrogen atom, methoxy group, chlorine atom, fluorine atom, 3,6-dihydro-2H -pyran-4-yl, 1,2,3,6-tetrahydropyridin-4-yl, phenyl, pyrazolyl, thienyl, furyl, pyridyl, 1-(1-ethoxyethyl )-1H-pyrazol-4-yl, 1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl, 1-methyl-1H-pyrazol-4-yl, Acetamide group; R ² is selected from hydrogen atom, C _1-6 alkyl group, 5-6 membered heteroaryl group, halogen, preferably hydrogen atom, C _1-3 alkyl group, 5 membered heteroaryl group and halogen, more preferably hydrogen atom, methyl, furyl; R ³ is selected from C _1-6 alkyl, C _1-6 hydroxyalkyl, preferably C _1-3 alkyl, C _1-3 hydroxyalkyl, more preferably 1-hydroxypropane- 2-yl, isopropyl; R ⁴ is C _1-6 alkyl; R ⁵ is a hydrogen atom; R ⁶ is tetrahydro-2H-pyran-4-yl.

The indole ASK1 small molecule inhibitor is selected from the following compounds: N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl) -1H-Indole-2-carboxamide (I-1), 7-chloro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2 -yl)-1H-indole-2-carboxamide (I-2), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2- yl)-6-methoxy-1H-indole-2-carboxamide (I-3), 6-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazole) -3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I-4), 5-fluoro-N-(6-(4-isopropyl-4H-1,2,4) -Triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I-5), N-(6-(4-isopropyl-4H-1,2,4- Triazol-3-yl)pyridin-2-yl)-1-methyl-1H-indole-2-carboxamide (I-6), (R)-N-(6-(4-(4-( 1-Hydroxypropan-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I-7), N-( 3-(4-Isopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1H-indole-2-carboxamide (I-8), 6-fluoro-N-( 3-(4-Isopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1H-indole-2-carboxamide (I-9), N-(6-(4 -Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-methyl-1H-indole-2-carboxamide (I-10), 3-( Furan-3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I-11), 7-acetamido-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole- 2-Carboxamide (I-12), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(1H-pyridine Azol-4-yl)-1H-indole-2-carboxamide (I-13), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine -2-yl)-5-(1H-pyrazol-4-yl)-1H-indole-2-carboxamide (I-14), 6-(1-(1-ethoxyethyl)-1H -Pyrazol-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2- Carboxamide (I-15), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1H-pyrazole- 4-yl)-1H-indole-2-carboxamide ( I-16), N-(3-(5-(4-isopropyl-4H-1,2,4-triazol-3-yl)-1H-indazol-3-yl)phenyl)-4 -(Trifluoromethyl)benzamide (I-17), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)- 6-(1-(Tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-1H-indole-2-carboxamide (I-18), N-(6-( 4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-methyl-1H-pyrazol-4-yl)-1H-indole -2-Carboxamide (I-19), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(thiophene- 3-yl)-1H-indole-2-carboxamide (I-20), 7-(furan-3-yl)-N-(6-(4-isopropyl-4H-1,2,4- Triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I-21), N-(6-(4-isopropyl-4H-1,2,4-triazol) Azol-3-yl)pyridin-2-yl)-7-phenyl-1H-indole-2-carboxamide (I-22), N-(6-(4-isopropyl-4H-1,2) ,4-Triazol-3-yl)pyridin-2-yl)-7-(pyridin-4-yl)-1H-indole-2-carboxamide (I-23), 7-(3,6-di Hydro-2H-pyran-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole -2-Carboxamide (I-24), N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(1, 2,3,6-Tetrahydropyridin-4-yl)-1H-indole-2-carboxamide (I-25), N-(6-(4-isopropyl-4H-1,2,4- Triazol-3-yl)pyridin-2-yl)-6-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-2-carboxamide (1-26).

The structural formulas of the above-mentioned specific compounds are shown in the following table:

The described indole ASK1 small molecule inhibitor, the pharmaceutically acceptable salt is the salt formed by the inhibitor and an acid or a base, and the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, Methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid ; The base is an inorganic base containing alkali metal cations, alkaline earth metal cations or ammonium cation salts.

The preparation method of the indole ASK1 small molecule inhibitor comprises the following steps:

(1) methyl 3-aminobenzoate or methyl 6-aminopyridine-2-carboxylate A is first converted into hydrazide compound B;

(2) compound C is obtained through ring-closure reaction, and compound C is reacted with carboxylic acid compound D to obtain compound (I);

The corresponding acid or base solution is added to the solution of compound (I) prepared by the above method, and the solvent is removed under reduced pressure after the salt formation is completed to obtain the pharmaceutically acceptable salt of the ASK1 inhibitor.

A pharmaceutical composition comprising the indole ASK1 small molecule inhibitor according to claim 1 and one or more pharmaceutically acceptable carriers, diluents or excipients.

Application of the indole ASK1 small molecule inhibitor or the pharmaceutical composition in the preparation of ASK1 inhibitor drugs.

Application of the indole ASK1 small molecule inhibitor or the pharmaceutical composition in the preparation of medicines for treating inflammatory diseases and diseases related to ASK1; the inflammatory diseases are nonalcoholic steatohepatitis, ulcerative colon inflammation.

Beneficial effects: Compared with the prior art, the present invention has the following remarkable features: the indole ASK1 small molecule inhibitor exhibits good ASK1 kinase inhibitory activity, which is superior to the AP1-HEK293 cell activity of GS-4996, and the compound I-1 exhibited better stability of human liver microsomes than GS-4997, and compounds I-1 and I-4 exhibited good in vivo pharmacokinetic properties. The experimental data prove that compound I-4 has certain therapeutic effect on both nonalcoholic steatohepatitis and ulcerative colitis.

Description of drawings

Figure 1 shows the effect of I-4 compound on the colon length of DSS-induced colitis mice;

Figure 2 shows the effect of I-4 compound on the histopathology of colon in mice with DSS-induced colitis.

Detailed ways

The structures of the compounds of the present invention were confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) and mass spectrometry (MS). Compound purity was determined by high performance liquid chromatography (HPLC). The measurement of ¹ H-NMR was carried out with Bruker Advance 300 and Bruker Advance 400 nuclear magnetic instruments, and the solvents were deuterated dimethyl sulfoxide (DMSO-d ₆ ) and deuterated chloroform (CDCl ₃ ), and the internal standard was tetramethylsilane ( TMS).

Advion Mass expression CMS mass spectrometer was used for MS measurement, and Agilent 1260 Infinity liquid chromatography system was used for HPLC measurement.

Thin-layer chromatography (TLC) adopts TLC silica gel plate (Yantai Jiangyou Silica Gel Development Co., Ltd.), self-made silica gel TLC plate adopts GF254 silica gel (Qingdao Ocean Chemical Factory), and silica gel column chromatography generally adopts 200-300 mesh silica gel (Qingdao Marine Chemical Plant).

The starting materials in the examples of the present invention are known and commercially available, or can be synthesized using or according to methods known in the art.

Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring, and reactions sensitive to oxygen and moisture are carried out in a dry nitrogen atmosphere and in a dry solvent, and the reaction temperature is in degrees Celsius.

Example 1

Preparation of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I-1)

The preparation of the first step 6-aminopyridinecarboxylhydrazide

Methyl 6-aminopicolinate (5.0 g, 32.9 mmol) was dissolved in MeOH, N ₂ H ₄ ·H ₂ O (3.22 g, 65.8 mmol) was added, the reaction solution was heated to reflux for 3 h; cooled to room temperature, a solid was precipitated, and the After filtration, the filter cake was washed with EA, and the target product (4.5 g, 90%) was obtained after vacuum drying.

ESI-MS m/z: 153.1[M+H] ⁺ .

Preparation of the second step 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine

6-Aminopicolinic acid hydrazide (4.5 g, 29.61 mmol) was dissolved in anhydrous Dioxane, triethyl orthoformate (5.92 mL, 35.53 mmol) was added, and the reaction was carried out at 75° C. for 1 h under the protection of N ₂ . CH ₃ COOH (5.08 mL, 88.83 mmol) and cyclopropylamine (2.05 mL, 29.61 mmol) were added through a syringe, and the reaction was carried out at 110° C. for 4 h; TLC detected the end of the reaction, the reaction solution was diluted with water and adjusted to pH 10, extracted with EA, and dried with EA Spin dry, and the target product (4.81 g, 80%) was obtained by silica gel column chromatography.

ESI-MS m/z: 204.0[M+H] ⁺ .

The third step is the preparation of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide

1H-Indole-2-carboxylic acid (300 mg, 1.86 mmol) was dissolved in 10 mL of DCM, 2 drops of DMF were added, and oxalyl chloride (355 mg, 2.79 mmol) was added under ice bath conditions. After 10 min, the reaction was performed at RT for 2 h, and the DCM was removed by spinning off to obtain the acid chloride. Standby; 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (343 mg, 1.68 mmol) was dissolved in DCM, DIEA (434 mg, 3.36 mmol) was added, Acyl chloride was added under ice bath conditions, and the reaction was performed at RT for 4 h after 10 min; TLC detected the end of the reaction. After removing DCM, diluting with water, extracting with DCM/MeOH 10:1, drying the organic phase and rotating it to dryness, silica gel column chromatography was performed and purified on a large silica gel plate. The desired product (180 mg, 31%).

¹ H NMR(300MHz, DMSO)δ11.89(s,1H),10.67(s,1H),8.90(s,1H),8.23(d,J=8.2Hz,1H),8.04(t,J=8.0 Hz, 1H), 7.86(d, J＝7.5Hz, 1H), 7.71(d, J＝8.0Hz, 1H), 7.58(d, J＝1.4Hz, 1H), 7.50(d, J＝8.2Hz, 1H), 7.26(t, J＝7.2Hz, 1H), 7.10(t, J＝7.4Hz, 1H), 5.68-5.73(m, 1H), 1.48(d, J＝6.7Hz, 6H).

ESI-MS m/z: 347.2[M+H] ⁺ .

Example 2

7-Chloro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I- 2) Preparation

Reference for the preparation of 7-chloro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide Example 1.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 12.06 (s, 1H), 10.85 (s, 1H), 8.90 (s, 1H), 8.26 (dd, J=6.3, 0.3 Hz, 1H), 8.06 ( t, J=8.7, 1H), 7.84 (dd, J=7.6, 0.6Hz, 1H), 7.68 (d, J=6.0Hz, 1H), 7.55 (d, J=2.1Hz, 1H), 7.37 (dd , J=5.7, 0.6Hz, 1H), 7.12 (t, J=7.8Hz, 1H), 5.54-5.65 (m, 1H), 1.48 (d, J=5.1Hz, 6H).

ESI-MS m/z: 381.1.[M+H] ⁺ .

Example 3

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-methoxy-1H-indole-2-carboxamide ( 1-3) Preparation of

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-methoxy-1H-indole-2-carboxamide The preparation refers to Example 1.

¹ H NMR (300MHz, DMSO) δ 11.70 (s, 1H), 10.56 (s, 1H), 8.88 (s, 1H), 8.23 (d, J=8.3Hz, 1H), 8.02 (t, J=7.9 Hz, 1H), 7.84(d, J=7.5Hz, 1H), 7.63-7.47(m, 2H), 6.93(s, 1H), 6.75(d, J=8.7Hz, 1H), 5.66-6.45(m , 1H), 3.80(s, 3H), 1.47(d, J=6.5Hz, 6H).

ESI-MS m/z: 377.2[M+H] ⁺ .

Example 4

6-Fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I- 4) Preparation

Reference for the preparation of 6-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide Example 1.

¹ H NMR (300MHz, DMSO-d ₆ )δ: 11.95(s,1H), 10.72(s,1H), 8.88(s,1H), 8.21(d, J=8.3Hz,1H), 8.03(t, J=7.9Hz, 1H), 7.85(d, J=7.6Hz, 1H), 7.74(m, 1H), 7.59(s, 1H), 7.20(d, J=9.9Hz, 1H), 6.97(t, J=9.3Hz, 1H), 5.65-6.45(m, 1H), 1.47(d, J=6.6Hz, 6H).

ESI-MS m/z: 365.1[M+H] ⁺ .

Example 5

5-Fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I- 5) Preparation

Reference for the preparation of 5-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide Example 1.

ESI-MS m/z: 365.1[M+H] ⁺ .

Example 6

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-1H-indole-2-carboxamide (I -6) Preparation

Preparation of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1-methyl-1H-indole-2-carboxamide Refer to Example 1.

¹ H NMR (400MHz, DMSO-d ₆ )δ: 10.80(s, 1H), 8.88(s, 1H), 8.18(d, J=8.2Hz, 1H), 8.03(t, J=7.9Hz, 1H) ,7.88(d,J=7.5Hz,1H),7.73(d,J=7.9Hz,1H),7.61(d,J=8.4Hz,1H),7.42(s,1H),7.35(t,J= 7.6Hz, 1H), 7.16 (t, J=7.4Hz, 1H), 5.84–5.68 (m, 1H), 4.04 (s, 3H), 1.45 (d, J=6.6Hz, 6H).

ESI-MS m/z: 361.2[M+H] ⁺

Example 7

(R)-N-(6-(4-(4-(1-hydroxypropan-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H- Preparation of indole-2-carboxamide (I-7)

(R)-N-(6-(4-(4-(1-hydroxypropan-2-yl)-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H- The preparation of indole-2-carboxamide refers to Example 1.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 11.89 (s, 1H), 10.65 (s, 1H), 8.80 (s, 1H), 8.21 (dd, J=8.3, 0.6 Hz, 1H), 8.03 ( t,J＝8.0Hz,1H),7.84(dd,J＝7.6,0.3Hz,1H),7.71(d,J＝7.9Hz,1H),7.55(d,J＝1.5Hz,1H),7.49( d, J＝8.1Hz, 1H), 7.26(m, 1H), 7.09(m, 1H), 5.61(m, 1H), 4.99(t, J＝5.4Hz, 1H), 3.66(m, 2H), 1.48(d, J=6.9Hz, 3H).

ESI-MS m/z: 363.1[M+H] ⁺ .

Example 8

Preparation of N-(3-(4-isopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1H-indole-2-carboxamide (I-8)

Refer to Example 1 for the preparation of N-(3-(4-isopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1H-indole-2-carboxamide.

¹ H NMR (300MHz, DMSO-d ₆ )δ: 11.81(s, 1H), 10.43(s, 1H), 8.89(s, 1H), 8.14(s, 1H), 7.98(d, J=8.2Hz, 1H), 7.70 (d, J=8.0Hz, 1H), 7.57 (t, J=7.9Hz, 1H), 7.48 (d, J=7.7Hz, 2H), 7.35 (d, J=7.7Hz, 1H) ,7.24(t,J=7.6Hz,1H),7.08(t,J=7.5Hz,1H),4.43-4.58(m,1H),1.46(d,J=6.7Hz,6H).

ESI-MS m/z: 346.2[M+H] ⁺ .

Example 9

6-Fluoro-N-(3-(4-isopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1H-indole-2-carboxamide (I-9) preparation

Reference Example 1 for the preparation of 6-fluoro-N-(3-(4-isopropyl-4H-1,2,4-triazol-3-yl)phenyl)-1H-indole-2-carboxamide .

¹ H NMR (300MHz, DMSO-d ₆ )δ: 11.90(s, 1H), 10.45(s, 1H), 8.89(s, 1H), 8.13(t, J=1.6Hz, 1H), 7.99-7.93( m, 1H), 7.74 (dd, J=8.8, 5.5Hz, 1H), 7.57 (t, J=7.9Hz, 1H), 7.49 (d, J=1.5Hz, 1H), 7.35 (d, J=7.7 Hz, 1H), 7.19 (dd, J=9.9, 2.2Hz, 1H), 7.01–6.92 (m, 1H), 4.57–4.43 (m, 1H), 1.46 (d, J=6.7Hz, 6H).

ESI-MS m/z: 364.1[M+H] ⁺

Example 10

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-methyl-1H-indole-2-carboxamide (I -10) Preparation

Preparation of N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-methyl-1H-indole-2-carboxamide Refer to Example 1.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 11.64 (s, 1H), 10.22 (s, 1H), 8.89 (s, 1H), 8.25 (dd, J=8.4, 2.4 Hz, 1H), 8.03 ( m,1H),7.84(dd,J=7.5,2.4Hz,1H),7.68(d,J=8.1Hz,1H),7.45(d,J=8.4Hz,1H),7.30(m,1H), 7.07(t,J=7.8Hz,1H),5.55-5.74(m,1H),2.55(s,3H),1.47(d,J=6.7Hz,6H)

ESI-MS m/z: 361.2[M+H] ⁺ .

Example 11

3-(Furan-3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2 - Preparation of carboxamide (I-11)

The first step 3-bromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide preparation

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (200 mg, 0.58 mmol) was used THF was dissolved, NBS was added and the reaction was carried out at RT for 3 h; TLC detected the end of the reaction. After removing THF, diluted with water, extracted with DCM, the organic phase was dried and spin-dried, and the target product (199 mg, 81%) was obtained by silica gel column chromatography.

ESI-MS m/z: 425.1[M+H]+.

The second step N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-methyl-1H-indole-2-carboxylate Preparation of amides

3-Bromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (100 mg, 0.24 mmol), Pd(dppf)Cl ₂ (18 mg, 0.024 mmol), K ₂ CO ₃ (66 mg, 0.48 mmol) and furan-3-boronic acid pinacol ester (70 mg, 0.36 mmol) in dioxane/H Dissolved in ₂ O, reacted at 100 °C for 6 h; TLC detected the end of the reaction, cooled to room temperature, the reaction solution was diluted with water, extracted with EA, the organic phase was dried and spin-dried, and the target product (69 mg, 69%) was obtained by silica gel column chromatography.

¹ H NMR (400MHz, DMSO) δ 12.10(s, 1H), 9.56(s, 1H), 8.88(s, 1H), 8.31(d, J=8.0Hz, 1H), 8.18(s, 1H), 8.04(t,J＝8.0Hz,1H),7.94(t,J＝1.6Hz,1H),7.90(d,J＝7.5Hz,1H),7.58(d,J＝8.1Hz,1H),7.53( d, J=8.3Hz, 1H), 7.32 (t, J=7.3Hz, 1H), 7.14 (t, J=7.5Hz, 1H), 6.86 (d, J=0.9Hz, 1H), 5.32-5.44 ( m,1H),1.43(d,J=6.7Hz,6H).

ESI-MS m/z: 413.2[M+H] ⁺ .

Example 12

7-Acetylamino-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (I -12) Preparation of

The first step (E)-Preparation of ethyl 2-(2-(2-(2-nitrophenyl)hydrazino)propionate

Dissolve o-nitrophenylhydrazine hydrochloride (1 g, 6.53 mmol) in EtOH, add ethyl pyruvate (731 mg, 6.3 mmol), and react at RT for 12 h; the reaction solution was concentrated to obtain a yellow solid product (1.2 g, 73%).

ESI-MS m/z: 252.1[M+H] ⁺ .

Preparation of the second step 7-nitro-1H-indole-2-carboxylic acid ethyl ester

Take (E)-ethyl 2-(2-(2-(2-nitrophenyl)hydrazino)propionate (1.2g, 4.78mmol) and dissolve it with polyphosphoric acid, and react at 80°C for 3h. TLC detects the end of the reaction , cooled to RT, the reaction solution was diluted with water and adjusted to pH 8 with saturated NaOH. The aqueous phase was extracted with EA, washed with water and saturated brine, dried with EA, and chromatographed on silica gel to obtain the target product (800 mg, 71%).

ESI-MS m/z: 235.1[M+H] ⁺ .

The third step is the preparation of ethyl 7-amino-1H-indole-2-carboxylate

Take 7-nitro-1H-indole-2-carboxylic acid ethyl ester (800 mg, 3.42 mmol) and dissolve it with EtOH/H ₂ O (6:1), add Fe (957 mg, 17.0 mmol), NH ₄ Cl (1.10 g, 20.0 mmol), reacted at 80°C for 4 h; TLC detected the end of the reaction, suction filtered while hot, the filtrate was extracted with DCM, dried with DCM and spin-dried, and the target product (400 mg, 57%) was isolated by silica gel column chromatography.

ESI-MS m/z: 205.1[M+H] ⁺ .

The preparation of the fourth step 7-acetamido-1H-indole-2-carboxylic acid ethyl ester

Take 7-amino-1H-indole-2-carboxylic acid ethyl ester (200mg, 0.98mmol) and dissolve it in DCM, add DIEA (253mg, 1.96mmol), add acetyl chloride (115mg, 1.47mmol), RT reaction for 3h; TLC When the reaction was detected, the reaction solution was concentrated and diluted with water, extracted with EA, washed with water and saturated brine, dried with EA, and separated by silica gel column chromatography to obtain the target product (150 mg, 62%).

ESI-MS m/z: 247.1[M+H] ⁺ .

The fifth step is the preparation of 7-acetamido-1H-indole-2-carboxylic acid

Take 7-acetamido-1H-indole-2-carboxylic acid ethyl ester (150mg, 0.61mmol) and dissolve it with THF/H ₂ O 5:1, add NaOH (253mg, 1.96mmol), RT reaction for 3h; TLC detects the end of the reaction , the reaction solution was extracted with EA, the aqueous phase was adjusted to pH 2 with 2M HCl, extracted with EA, dried with EA, and separated by silica gel column chromatography to obtain the target product (100 mg, 75%).

ESI-MS m/z: 219.1[M+H] ⁺ .

The sixth step 7-acetamido-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxyl Preparation of amides

Preparation of 7-acetamido-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide Refer to Example 1.

¹ H NMR (300MHz, DMSO-d ₆ )δ: 11.82(s, 1H), 10.80(s, 1H), 9.94(s, 1H), 8.89(s, 1H), 8.22(d, J=8.2Hz, 1H), 8.05(t, J=7.9Hz, 1H), 7.96(d, J=7.5Hz, 1H), 7.86(d, J=7.4Hz, 1H), 7.62(s, 1H), 7.44(d, J=7.8Hz, 1H), 7.05 (t, J=7.8Hz, 1H), 5.63–5.80 (m, 1H), 2.18 (s, 3H), 1.47 (d, J=6.5Hz, 6H).

ESI-MS m/z: 404.2[M+H] ⁺ .

Example 13

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(1H-pyrazol-4-yl)-1H-indium Preparation of inole-2-carboxamide (I-13)

The first step 7-bromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide preparation

Preparation of 7-acetamido-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide Refer to Example 1 (third step).

ESI-MS m/z: 425.1[M+H] ⁺ .

The second step 7-bromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide preparation

Take 7-bromo-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2-carboxamide (100mg , 0.24 mmol), Pd(dppf)Cl ₂ (18 mg, 0.024 mmol), K ₃ PO ₄ (102 mg, 0.48 mmol), 1-Boc-pyrazole-4-boronic acid pinacol ester (106 mg, 0.36 mmol), Dissolve with Dioxane/H ₂ O 5:1, react at 110 °C for 3 h; TLC detects the end of the reaction, cools to room temperature, dilutes the reaction solution with water, extracts with EA, EA is dried and spin-dried, and the target product (50 mg, 51 mg) is separated by silica gel column chromatography. %).

¹ H NMR (300MHz, DMSO-d ₆ )δ: 13.14(s, 1H), 11.01(d, J=47.6Hz, 2H), 8.89(s, 1H), 8.45-7.97(m, 4H), 7.82( d, J=7.1Hz, 1H), 7.68–7.33 (m, 3H), 7.15 (s, 1H), 5.59 (s, 1H), 1.47 (d, J=5.2Hz, 6H).

ESI-MS m/z: 413.2[M+H] ⁺ .

Example 14

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1H-pyrazol-4-yl)-1H-indium Preparation of inole-2-carboxamide (I-14)

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(1H-pyrazol-4-yl)-1H-indium Refer to Example 13 for the preparation method of inole-2-carboxamide.

¹ H NMR (300MHz, DMSO-d ₆ ) δ: 12.89(s, 1H), 12.06(s, 1H), 10.83(s, 1H), 8.89(s, 1H), 8.22(d, J=8.0Hz, 1H), 8.04(t, J=7.9Hz, 3H), 7.90(s, 1H), 7.85(d, J=7.3Hz, 1H), 7.50(q, J=8.3Hz, 3H), 5.81–5.66( m, 1H), 1.47 (d, J=6.7Hz, 6H).

ESI-MS m/z: 413.2[M+H] ⁺ .

Example 15

6-(1-(1-Ethoxyethyl)-1H-pyrazol-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazole-3- Preparation of pyridin-2-yl)-1H-indole-2-carboxamide (I-15)

6-(1-(1-Ethoxyethyl)-1H-pyrazol-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazole-3- For the preparation of pyridin-2-yl), refer to Example 13.

¹ H NMR (400MHz, DMSO) δ12.01(s, 1H), 10.74(s, 1H), 8.90(s, 1H), 8.39(s, 1H), 8.23(d, J=8.0Hz, 1H), 8.04(t, J=8.0Hz, 1H), 7.94(s, 1H), 7.85(d, J=7.5Hz, 1H), 7.70(d, J=8.4Hz, 1H), 7.64(s, 1H), 7.54(s, 1H), 7.39(dd, J=8.3, 1.4Hz, 1H), 5.73(dt, J=13.3, 6.6Hz, 1H), 5.58(q, J=5.9Hz, 1H), 3.52–3.44 (m, 1H), 3.29–3.23 (m, 1H), 1.66 (d, J=6.0Hz, 3H), 1.48 (d, J=6.7Hz, 6H), 1.06 (t, J=7.0Hz, 3H) .

ESI-MS m/z: 485.2[M+H] ⁺ .

Example 16

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1H-pyrazol-4-yl)-1H-indium Preparation of inole-2-carboxamide (I-16)

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1H-pyrazol-4-yl)-1H-indium Preparation of inole-2-carboxamide The preparation of the preparation refers to Example 13.

¹ H NMR (300MHz, DMSO-d ₆ )δ: 12.92(s, 1H), 11.88(s, 1H), 10.66(s, 1H), 8.89(s, 1H), 8.23(d, J=8.2Hz, 1H), 8.04(t, J=8.0Hz, 2H), 7.85(d, J=7.6Hz, 1H), 7.68(d, J=8.3Hz, 1H), 7.62(s, 1H), 7.54(s, 1H), 7.38(d, J=8.3Hz, 1H), 5.62-5.73(m, 1H), 1.47(d, J=6.6Hz, 6H).

ESI-MS m/z: 413.2[M+H] ⁺ .

Example 17

N-(3-(5-(4-Isopropyl-4H-1,2,4-triazol-3-yl)-1H-indazol-3-yl)phenyl)-4-(trifluoromethane Preparation of yl)benzamide (I-17)

N-(3-(5-(4-Isopropyl-4H-1,2,4-triazol-3-yl)-1H-indazol-3-yl)phenyl)-4-(trifluoromethane Preparation of phenyl) benzamide The preparation of the preparation refers to Example 13.

¹ H NMR (300MHz, DMSO-d ₆ )δ: 12.07(s, 1H), 10.74(s, 1H), 8.93(s, 1H), 8.89(s, 1H), 8.58(d, J=1.8Hz, 1H), 8.24(d, J＝8.1Hz, 1H), 8.13(d, J＝8.0Hz, 1H), 8.05(t, J＝7.9Hz, 1H), 7.86(d, J＝1.8Hz, 1H) , 7.84(d, J=2.7Hz, 1H), 7.76(s, 1H), 7.63(d, J=1.8Hz, 1H), 7.54(m, 1H), 7.46(d, J=8.3Hz, 1.2Hz , 1H), 5.77–5.66 (m, 1H), 1.48 (d, J=6.7Hz, 6H).

ESI-MS m/z: 424.2[M+H] ⁺ .

Example 18

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1-(tetrahydro-2H-pyran-4- (yl)-1H-pyrazol-4-yl)-1H-indole-2-carboxamide (I-18)

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-(1-(tetrahydro-2H-pyran-4- yl)-1H-pyrazol-4-yl)-1H-indole-2-carboxamide was prepared with reference to Example 13.

¹ H NMR (400MHz, DMSO) δ 11.87(s, 1H), 10.64(s, 1H), 8.90(s, 1H), 8.27(s, 1H), 8.23(d, J=8.1Hz, 1H), 8.04(t, J=8.0Hz, 1H), 7.89(s, 1H), 7.85(d, J=7.6Hz, 1H), 7.69(d, J=8.4Hz, 1H), 7.60(s, 1H), 7.55 (d, J=1.4Hz, 1H), 7.36 (dd, J=8.4, 1.3Hz, 1H), 5.65-5.75 (m, 1H), 4.44 (t, J=7.6Hz, 1H), 3.99 (dd , J=8.3, 2.9Hz, 2H), 3.49 (td, J=11.6, 5.6Hz, 2H), 2.02 (dt, J=12.7, 6.2Hz, 4H), 1.47 (d, J=6.7Hz, 6H) .

ESI-MS m/z: 497.2[M+H] ⁺ .

Example 19

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-methyl-1H-pyrazol-4-yl )-1H-indole-2-carboxamide (I-19) preparation

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-methyl-1H-pyrazol-4-yl )-1H-indole-2-carboxamide was prepared by referring to Example 13.

¹ H NMR (400MHz, DMSO-d ₆ )δ: 12.00(s, 1H), 10.78(d, J=7.1Hz, 1H), 8.89(s, 1H), 8.27(s, 1H), 8.22(d, J=8.3Hz, 1H), 8.10–8.00 (m, 2H), 7.96 (s, 1H), 7.80 (d, J=7.6Hz, 1H), 7.39–7.33 (m, 1H), 7.29–7.20 (m ,2H),5.46-5.65(m,1H),3.95(s,3H),1.46(d,J=6.7Hz,6H).

ESI-MS m/z: 427.2[M+H] ⁺ .

Example 20

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(thiophen-3-yl)-1H-indole-2 - Preparation of Carboxamide (I-20)

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(thiophen-3-yl)-1H-indole-2 - The preparation of the carboxamide refers to Example 13.

¹ H NMR (300MHz, DMSO-d ₆ )δ: 11.13(s, 1H), 10.90(s, 1H), 8.89(s, 1H), 8.26(d, J=7.8Hz, 1H), 8.05(t, J=8.0Hz, 1H), 7.98 (dd, J=2.8, 1.3Hz, 1H), 7.82 (d, J=7.0Hz, 1H), 7.76 (dd, J=5.0, 2.9Hz, 1H), 7.70 ( d, J=7.8Hz, 1H), 7.59–7.52 (m, 2H), 7.41 (dd, J=7.2, 0.9Hz, 1H), 7.24–7.15 (m, 1H), 5.65–5.49 (m, 1H) , 1.46(d,J=6.7Hz,6H).

ESI-MS m/z: 429.1[M+H] ⁺ .

Example 21

7-(Furan-3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2 - Preparation of carboxamide (I-21)

7-(Furan-3-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-1H-indole-2 - The preparation of the carboxamide refers to Example 13.

¹ H NMR (400MHz, DMSO) δ 11.02 (s, 1H), 10.90 (s, 1H), 8.89 (s, 1H), 8.41 (s, 1H), 8.27 (d, J=8.3Hz, 1H), 8.05(t, J=8.0Hz, 1H), 7.87(s, 1H), 7.82(d, J=7.6Hz, 1H), 7.68(d, J=7.9Hz, 1H), 7.53(d, J=1.5 Hz, 1H), 7.43(d, J=7.1Hz, 1H), 7.18(t, J=7.6Hz, 1H), 7.04(s, 1H), 5.59(m, 5.52-5.65, 1H), 1.46(d , J=6.7Hz,7H).

ESI-MS m/z: 413.2[M+H] ⁺ .

Example 22

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-phenyl-1H-indole-2-carboxamide (I -22) Preparation of

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(thiophen-3-yl)-1H-indole-2 - The preparation of the carboxamide refers to Example 13.

¹ H NMR (400MHz, DMSO) δ 11.28(s, 1H), 10.87(s, 1H), 8.89(s, 1H), 8.25(d, J=8.3Hz, 1H), 8.04(t, J=8.0 Hz, 1H), 7.83–7.78 (m, 1H), 7.72 (t, J=7.8Hz, 3H), 7.59–7.52 (m, 3H), 7.47 (t, J=7.4Hz, 1H), 7.29 (dd , J=7.2, 1.0Hz, 1H), 7.22 (t, J=7.5Hz, 1H), 5.47-5.57 (m, 1H), 1.46 (d, J=6.7Hz, 6H).

ESI-MS m/z: 423.2[M+H] ⁺ .

Example 23

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(pyridin-4-yl)-1H-indole-2 - Preparation of carboxamide (I-23)

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(pyridin-4-yl)-1H-indole-2 - The preparation of the carboxamide refers to Example 13.

¹ H NMR (300MHz, DMSO) δ 11.59(s, 1H), 10.84(s, 1H), 8.90(s, 1H), 8.74(s, 2H), 8.25(d, J=8.3Hz, 1H), 8.05(t,J＝8.0Hz,1H),7.83(dd,J＝7.6,2.4Hz,2H),7.75(s,2H),7.60(d,J＝1.8Hz,1H),7.39(d,J =6.3Hz,1H),7.32-7.20(m,1H),5.52-5.62(m,1H),1.45(t,J=8.1Hz,6H).

ESI-MS m/z: 424.2[M+H] ⁺ .

Example 24

7-(3,6-Dihydro-2H-pyran-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2 -yl)-1H-indole-2-carboxamide (I-24) preparation

7-(3,6-Dihydro-2H-pyran-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2 The preparation of -yl)-1H-indole-2-carboxamide refers to Example 13.

¹ H NMR (400MHz, DMSO) δ 11.31(s, 1H), 10.79(s, 1H), 8.89(s, 1H), 8.25(t, J=11.4Hz, 1H), 8.05(t, J=7.1 Hz, 1H), 7.83 (d, J=6.9Hz, 1H), 7.56 (dt, J=26.5, 8.9Hz, 3H), 7.22–7.01 (m, 1H), 6.17 (s, 1H), 5.81–5.47 (m, 1H), 4.27 (d, J=31.8Hz, 2H), 3.97 (d, J=37.7Hz, 2H), 2.13 (d, J=35.7Hz, 2H), 1.44 (t, J=18.2Hz) ,6H).

ESI-MS m/z: 429.2[M+H] ⁺ .

Example 25

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(1,2,3,6-tetrahydropyridine-4 -yl)-1H-indole-2-carboxamide (I-25) preparation

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-7-(1,2,3,6-tetrahydropyridine-4 The preparation of -yl)-1H-indole-2-carboxamide refers to Example 13.

¹ H NMR(300MHz, DMSO)δ11.38(s,1H),10.86-10.63(m,1H),8.88(d,J=26.9Hz,1H),8.26(t,J=12.3Hz,1H), 8.13–7.95 (m, 1H), 7.86 (d, J=7.5Hz, 1H), 7.63 (t, J=9.9Hz, 1H), 7.50 (d, J=19.8Hz, 1H), 7.22–7.04 (m , 3H), 6.08(s, 1H), 5.68(dd, J=13.0, 6.5Hz, 1H), 4.10(s, 3H), 3.66(s, 3H), 1.51(s, 6H).

ESI-MS m/z: 428.2[M+H] ⁺ .

Example 26

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-((tetrahydro-2H-pyran-4-yl) Preparation of amino)-1H-indole-2-carboxamide (I-26)

Preparation of the first step 6-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-2-carboxylic acid ethyl ester

Take ethyl 6-amino-1H-indole-2-carboxylate (500 mg, 2.45 mmol) and dissolve it in DCM, add DIEA (632 mL, 4.9 mmol) and tetrahydropyranone (370 mg, 3.70 mmol), and stir at RT for 2 h , Sodium borohydride acetate (2.6 g, 12.3 mmol) was added, and the reaction was carried out at RT for 2 h. TLC detected the end of the reaction, spun off DCM, the residue was diluted with water, extracted with EA, dried with EA and spin-dried, and the target product (442 mg, 60%) was obtained by silica gel column chromatography.

ESI-MS m/z: 289.1[M+H] ⁺ .

Preparation of the second step 6-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-2-carboxylic acid

Take ethyl 6-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-2-carboxylate (442 mg, 1.47 mmol), dissolve with MeOH/H ₂ O 5:1, add NaOH (118 mg, 2.94 mmol), RT for 4 h. TLC detected the end of the reaction. The reaction solution was extracted with EA after adding a small amount of water. The pH of the aqueous phase was adjusted to 2, the aqueous phase was extracted with EA, and the EA was dried and spin-dried to obtain the target product (300 mg, 78%).

ESI-MS m/z: 261.1[M+H] ⁺ .

The third step N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-((tetrahydro-2H-pyran-4 Preparation of -yl)amino)-1H-indole-2-carboxamide

N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-6-((tetrahydro-2H-pyran-4-yl) The preparation of amino)-1H-indole-2-carboxamide refers to Example 1.

¹ H NMR (400MHz, DMSO) δ 11.85(s, 1H), 8.89(s, 1H), 8.29(d, J=8.1Hz, 1H), 8.07(q, J=8.2Hz, 1H), 7.91( dd, J=7.7, 0.8Hz, 1H), 7.85–7.78 (m, 2H), 7.65 (t, J=8.9Hz, 1H), 7.51 (t, J=7.9Hz, 1H), 5.55-5.65 (m ,J,1H),4.87(s,1H),4.09–3.96(m,2H),3.57(t,J=11.1Hz,2H),3.17(d,J=5.3Hz,1H),2.71(dd, J=15.4, 7.8Hz, 2H), 1.69 (d, J=9.8Hz, 2H), 1.48 (t, J=7.0Hz, 6H).

ESI-MS m/z: 446.2[M+H] ⁺ .

Biological activity test

1. ASK1 protein kinase activity test

In this experiment, the homogeneous time-resolved fluorescence (HTRF) method was used to test the inhibitory activity of the compounds on ASK1 kinase, and it was compared with the positive compound GS-4997.

(1) Add the diluted compounds to the 384-well plate, and add the DMSO diluted in AssayBuffer to the positive control wells and negative control wells respectively.

(2) STK-Substrate 3-biotin/ATP and ASK1 (MAP3K5) were added to compound and positive control wells, and STK-Substrate 3-biotin/ATP and Assay Buffer were added to negative control wells.

(3) Add STK S3 Antibody-Eu after 2.0h incubation at room temperature.

(4) Incubate at room temperature for 1.0 h, measure the fluorescence signal value of each plate well with a microplate reader, calculate the inhibition rate according to the fluorescence signal value, and calculate the IC ₅₀ value by fitting the curve with Prism (GraphPad Software).

The ASK1 protein kinase activity of some example compounds in the present invention is shown in Table 1

Table 1 ASK1 protein kinase activity of some example compounds in the present invention

Compound number ASK1 IC₅₀ Compound number ASK1 IC₅₀ I-1 42nM I-14 31.49nM GS-4997 14nM I-16 37.22nM I-4 32.65nM I-19 18.46nM I-13 38.86nM I-23 23.3nM

The compounds of the above examples have strong ASK1 kinase inhibitory activity. As potent ASK1 small molecule inhibitors, these compounds have great potential clinical application value for the treatment of ASK1-related diseases.

2. AP1-HEK293 cell inhibitory activity test

This method uses AP1 Reporter-HEK293 recombinant cells to evaluate the effects of compounds on the ASK1 downstream signaling pathway at the cellular level.

(1) HEK293-AP1 cells were seeded into a 96-well plate and 100uL Thaw Medium 1 was added.

(2) Incubate the cells in a CO ₂ incubator overnight.

(3) Slowly pour the medium out of the well plate, dilute the compound to be tested with Assay medium and add it to the cell well, add the Assay medium dilution of the same concentration of DMSO to the cell well as a control, and add the same concentration of DMSO Dilute Assay medium into cell-free wells to remove background fluorescent signal.

(4) Incubation for 1h.

(5) Add the dilution of Assay medium of PMA to the cell wells, add the dilution of Assay medium of 0.1% DMSO to the cell wells as a control, and add the dilution of Assay medium of 0.1% DMSO to the cell-free wells to remove background fluorescence signal.

(6) Perform luciferase detection with the ONE-Step ^TM luciferase detection system according to the operating specification: add ONE-Step ^TM luciferase reagent to the well, shake at room temperature for 15 minutes, and use a luminometer to measure the fluorescence signal.

(7) Data analysis: subtract the background fluorescence value. The inhibition rate of each compound was calculated, and the _IC50 value was fitted according to the inhibition rate.

Table 2

Table 2 AP1-HEK293 cell activity of some example compounds in the present invention

Some example compounds showed better cell activity than the positive drug GS-4997, which preliminarily indicated that the compounds had good physicochemical properties and druggability.

3. Human liver microsome stability

This method preliminarily evaluates the in vitro metabolic stability of the compounds through the stability test of human liver microsomes

Experimental steps:

(1) Buffer A: 1.0L 0.1M potassium dihydrogen phosphate buffer containing 1.0mM EDTA

Buffer B: 1.0L 0.1M Dipotassium Phosphate Buffer with 1.0mM EDTA

Buffer C: 0.1 M potassium phosphate buffer, 1.0 mM EDTA, pH 7.4, 700 mL of buffer B was titrated with buffer A while monitoring with a pH meter.

(2) Reference compound (Ketanserin) and test compound spiking solution:

500 μM spiking solution: Add 10 μL of 10 mM DMSO stock solution to 190 μL of ACN.

Add 1.5 μM spiking solution (0.75 mg/mL) to microsomes: Add 1.5 μL of 500 μM spiking solution and 18.75 μL of 20 mg/mL liver microsomes to 479.75 μL of buffer C on ice.

(3) Prepare NADPH stock solution (6 mM) by dissolving NADPH in buffer C.

(4) Dispense 30 μL of 1.5 μM spiking solution containing 0.75 mg/mL microsomal solution onto assay plates on ice designated for different time points (0, 5, 15, 30, 45 minutes).

(5) For 0 minutes, 135 μL of ACN containing IS was added to the wells of the 0 minute plate, followed by 15 μL of NADPH stock solution (6 mM).

(6) Pre-incubate all other plates at 37°C for 5 minutes.

(7) Add 15 [mu]L of NADPH stock solution (6 mM) to the plate to start the reaction and time it.

(8) At 5 minutes, 15 minutes, 30 minutes and 45 minutes, 135 μL of ACN containing IS was added to the wells of the corresponding plates to stop the reaction.

(9) After quenching, the plate was shaken on a shaker (IKA, MTS 2/4) for 10 minutes (600 rpm) and then centrifuged at 5594 g for 15 minutes (Thermo Multifuge x 3R).

(10) 50 μL of supernatant was transferred from each well to a 96-well sample plate containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for LC/MS analysis.

(11) Fit a curve according to the peak area of the compound at different time points, and calculate the half-life and clearance rate.

Table 3 Liver microsome stability data of some example compounds in the present invention

Compound number T_1/2(minute) Cl_int(mL/min/kg) GS-4997 212 8.21 I-1 821.4 2.12

Compound I-1 showed better metabolic stability of human liver microsomes in vitro than the positive drug GS-4997.

4. PK in rats

Experimental operation

(1) 6 male SD Rat, 198-225g (JH Laboratory Animal Co.LTD), three in the intravenous injection group and three in the oral administration group. The intravenous group was fed with food and water normally; the oral group was fasted overnight and started feeding 4 hours after administration. (2) Intravenous injection group 1mg/kg dorsal foot intravenous injection, oral administration 10mg/kg oral gavage group. (3) At the indicated time points, the animals were manually restrained, and approximately 150 μL of blood samples were collected through the tail vein for continuous infiltration into EDTA-K2 tubes. Blood samples were first kept in wet ice and centrifuged within 15 minutes after sampling to obtain plasma (2000 g, 4°C, 5 minutes). Intravenous group 0.083, 0.25, 0.5, 1, 2, 4, 8, 24h, 8 time points, oral group 0.25, 0.5, 1, 2, 4, 8 and 24h, 7 time points. (4) Blood samples were stored at about -70°C until used for analysis. (5) According to the blood drug concentration at each time point, CL, V _ss , AUC _last , AUC _INF , T _1/2 , MRT _INF were calculated in the oral group; T _max , C _max , AUC _last , AUC _INF , T _1/2 , F.

Table 4 Rat in vivo PK data of compound I-1

NA: Not available

Table 5 Rat in vivo PK data of compound I-4

Compounds I-1 and I-4 show good in vivo pharmacokinetic properties, have the conditions to carry out in vivo efficacy experiments, and have excellent druggability.

5. Study on the pharmacodynamic model of CDAA-induced NASH in mice

Objective: To establish an in vivo pharmacodynamic evaluation model of NASH, and to study the pharmacodynamics of the tested drugs in a non-alcoholic steatohepatitis (NASH) model.

Experimental materials: CDAA feed (Parker Bio: Research Diets: A06071302), 10% neutral formaldehyde fixative (Formalin, Chengdu Lilai Biotechnology Co., Ltd.), methylcellulose (MC, Shanghai McLean Biotechnology Co., Ltd.) ), Tween 80 (Shanghai McLean Biological Co., Ltd.), C57BL/6J mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), animal weight scale (Shanghai Huachao Electric Co., Ltd.), microscope (MIC01964, Zeiss Optics instrument), pipette (Eppendorf)

Experimental operation:

(1) C57BL/6J mice were reared in the SPF cell room for at least one week during the acclimation period.

(2) According to body weight and food intake, they were randomly divided into normal control group (Control), model group (Vehicle), and Example 5 compound 50mpk group. Except for the normal control group, which was fed with normal feed, the rest of the animals were fed with CDAA feed. The CDAA modeling time was 12 weeks, and the administration was from the 5th week to the 12th week. The route of administration was gavage, and the frequency of administration was once a day. Observation by the cage was carried out every day, food intake and body weight were measured once a week before administration; food intake and body weight were measured twice a week after administration, once every 3-4 days.

(3) Fasting for more than 6 hours before dissection, using 3% pentobarbital sodium anesthesia, collecting blood from the heart, centrifuging the supernatant to send samples to detect blood biochemical indicators: ALP, TC, HDL, LDL.

(4) The experimental data of animals in each group were described by means ± standard error (X ± SEM). One-way analysis of variance (One-Way ANOVA) was used for comparison between multiple groups with normality and homogeneity of variance, and LSD test was used for comparison between groups; P<0.05 was considered statistically significant. All statistical analyses were performed using Graphpad Prism 8.0 software.

Experimental results:

Table 6. Effects of I-4 compounds in NASH mouse model

Liver weight (g) ALP(U/L) HDL (mmol/L) LDL/TC Control group 1.097 116.8 2.553 0.06895 Vehicle group 1.534 196.9 1.276 0.1302 Example 4 groups 1.382 158.0 1.404 0.1225

Compared with Vehicle, the I-4 compound down-regulated liver weight, down-regulated ALP, up-regulated HDL, and down-regulated LDL/TC in NASH mice, showing a certain therapeutic potential for NASH.

6. DSS-induced acute colitis model in mice

Experimental methods: (1) ICR mice, weighing 18-22 g, 6-8 weeks old, and adaptively reared for 5 days, were randomly divided into 4 groups: Control, DSS, GS-4997, and Example 4, with 6 mice in each group. (2) Acute colitis was induced by 3% DSS, DSS, GS-4997, and Example 4 were given an aqueous solution containing 3% DSS and allowed to drink freely. The dose of GS-4997 was 25 mg/kg, the dose of Example 5 was 25 mg/kg, and the control group (Control) was given normal drinking water for 7 consecutive days, and was replaced every two days. During the modeling period, drugs were given by gavage, and the control group and DSS group were given the corresponding solvent CMC-Na once a day for 7 consecutive days. (3) After 7 days, the mice were sacrificed by dislocating their cervical vertebrae, and colon tissue was taken to measure the length of the colon. 3 mice in each group were fixed in formalin solution for HE staining, and the remaining tissues were stored at -80°C.

Experimental results: Figure 1 shows the effect of I-4 compound on the colon length of DSS-induced colitis mice.

Compared with the mice in the control group, the colon length of the mice in the DSS group was significantly shortened ( ^### P<0.001). Compared with the DSS group, the colon length of the mice in the I-4 group increased significantly (*P<0.05), while the colon length of the GS-4997 group increased to a certain extent, and there was no significant difference. Figure 2 shows the effect of compound I-4 on colon histopathology in DSS-induced colitis mice.

The colon tissue of the mice in the Control group was intact, and there was no inflammatory cell infiltration. The colon tissue of DSS mice was severely damaged, and a large number of inflammatory cells were infiltrated. However, the colon tissue of the mice in the I-4 compound administration group was relatively intact, and the inflammation was significantly reduced; the tissue damage of the mice in the GS-4997 group was alleviated to a certain extent.

The two experimental results of compound I-4 in DSS-induced colitis mouse model showed that compound I-4 showed a certain therapeutic effect on ulcerative colitis, and was superior to the positive drug GS-4997.

Claims (10)

1. An indole ASK1 small molecule inhibitor, which is characterized by comprising a compound shown as a general formula (I) and a stereoisomer or pharmaceutically acceptable salt thereof:

M¹is selected from N or CH;

R¹is the same or different and is independently selected from hydrogen atom, alkoxy, halogen, heterocyclic radical, aryl, heteroaryl, - (CH)₂)_nOR⁴、-(CH₂)_nNR⁵R⁶、-(CH₂)_nNR⁵C(O)R⁴N is 0, 1,2,3, 4 or 5; wherein the heteroaryl is unsubstituted or substituted by one or more substituents selected from alkyl, alkoxy, and heterocyclyl;

R²the same or different, and each is independently selected from hydrogen atom, alkyl, halogen, aryl, heteroaryl;

R³selected from alkyl, hydroxyalkyl or heterocyclyl;

R⁴selected from alkyl groups;

R⁵and R⁶The same or different, and each is independently selected from hydrogen atom, heterocyclic radical;

x is selected from N (R)²)；

m is 0, 1,2,3 or 4;

p is 1 or 2.

2. The indole ASK1 small-molecule inhibitor according to claim 1, wherein M is¹Is N:

3. the indole ASK1 small-molecule inhibitor according to claim 2, wherein X is imino and R is R³Is isopropyl, and is specifically shown as a general formula (III):

4. the indole ASK1 small molecule inhibitor according to any one of claims 1-2, wherein R is¹Selected from the group consisting of a hydrogen atom, a methoxy group, a chlorine atom, a fluorine atom, a 3, 6-dihydro-2H-pyran-4-yl group, a 1,2,3, 6-tetrahydropyridin-4-yl group, a phenyl group, a pyrazolyl group, a thienyl group, a furyl group, a pyridyl group, a 1- (1-ethoxyethyl) -1H-pyrazol-4-yl group, a 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl group, a 1-methyl-1H-pyrazol-4-yl group, and an acetamido group; r²Selected from hydrogen atom, methyl, furyl; r³Selected from 1-hydroxypropan-2-yl, isopropyl; r⁴Is C_1-6An alkyl group; r⁵Is a hydrogen atom; r⁶Is tetrahydro-2H-pyran-4-yl.

5. The indole ASK1 small molecule inhibitor according to claim 1, selected from the group consisting of: n- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-1), 7-chloro-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-2), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxy-1H-indole-2-carboxamide (I-3), 6-fluoro-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-4), 5-fluoro-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-5), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1-methyl-1H-indole-2-carboxamide (I-6), (R) -N- (6- (4- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-7), N- (3- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) phenyl) -1H-indole-2-carboxamide (I-8), 6-fluoro-N- (3- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) phenyl) -1H-indole-2-carboxamide (I-9), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3-methyl-1H-indole-2-carboxamide (I-10), 3- (furan-3-yl) -N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-11), 7-acetamido-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-12), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7- (1H-pyrazol-4-yl) -1H-indole-2-carboxamide (I-13), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -5- (1H-pyrazol-4-yl) -1H-indole-2-carboxamide (I-14), 6- (1- (1-ethoxyethyl) -1H-pyrazol-4-yl) -N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-15), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6- (1H-pyrazol-4-yl) -1H-indole-2-carboxamide (I-16), N- (3- (5- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) -1H-indazol-3-yl) phenyl) -4- (trifluoromethyl) benzamide (I-17), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6- (1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) -1H-indole-2-carboxamide (I-18), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -4- (1-methyl-1H-pyrazol-4-yl) -1H-indole-2-carboxamide (I-19), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7- (thiophen-3-yl) -1H-indole-2-carboxamide (I-20), 7- (furan-3-yl) -N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-21), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7-phenyl-1H-indole-2-carboxamide (I-22), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7- (pyridin-4-yl) -1H-indole-2-carboxamide (I-23), 7- (3, 6-dihydro-2H-pyran-4-yl) -N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1H-indole-2-carboxamide (I-24), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7- (1,2,3, 6-tetrahydropyridin-4-yl) -1H-indole-2-carboxamide (I-25), N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6- ((tetrahydro-2H-pyran-4-yl) amino) -1H-indole-2-carboxamide (I-26).

6. The indole ASK1 small molecule inhibitor according to claim 1, wherein the pharmaceutically acceptable salt is a salt of the inhibitor with an acid or a base, the acid being hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid; the base is an inorganic base containing a basic metal cation, an alkaline earth metal cation, or an ammonium cation salt.

7. The preparation method of the indole ASK1 small-molecule inhibitor as claimed in claim 1, comprising the following steps:

(1) methyl 3-aminobenzoate or methyl 6-aminopyridine-2-carboxylate A is firstly converted into a hydrazide compound B;

(2) a compound C is obtained through ring closing reaction, and the compound C reacts with a carboxylic acid compound D to obtain a compound (I);

and (3) adding a corresponding acid or alkali solution into the solution of the compound (I) prepared by the method, and removing the solvent under reduced pressure after salt formation is completed to obtain the pharmaceutically acceptable salt of the ASK1 inhibitor.

8. A pharmaceutical composition comprising the indole ASK1 small molecule inhibitor of claim 1 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

9. The use of the indole ASK1 small molecule inhibitor of claim 1 or the pharmaceutical composition of claim 8 for the preparation of an ASK1 inhibitor medicament.

10. Use of the indole ASK1 small molecule inhibitor of claim 1 or the pharmaceutical composition of claim 8 for the preparation of a medicament for the treatment of inflammatory diseases and ASK 1-related diseases; the inflammatory diseases are non-alcoholic steatohepatitis and ulcerative colitis.

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