PHENOTHIAZINE FERROPTOSIS INHIBITOR, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

A phenothiazine ferroptosis inhibitor, a preparation method therefor and an application thereof. Specifically provided is a compound represented by formula I, or a salt thereof, or a stereoisomer thereof. The synthesized phenothiazine compound has a good inhibitory effect on ferroptosis and has good drug safety. Moreover, the compound can be used to prepare a ferroptosis inhibitor, and can also be used to prepare a drug for treating diseases related to ferroptosis, such as neurodegenerative diseases, tumors, tissue ischemia reperfusion injury, stroke, cardiovascular diseases, renal failure, and diabetic complications.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. A compound represented by formula IV, a salt thereof, or a stereoisomer thereof: ##STR00058## wherein, ring B is selected from 5- to 6-membered unsaturated heterocyclyl independently substituted by n R.sub.2; n is an integer of 0˜3; each R.sub.2 is independently selected from substituted or unsubstituted C.sub.1˜C.sub.3 alkyl, halogen, amino, —C(O)NR.sub.3R.sub.4, —C(O)R.sub.5 and —C(O)OR.sub.5 ; or two R.sub.2 on the same carbon atom constitute =O; R.sub.3 and R.sub.4 are independently selected from hydrogen, substituted C.sub.1˜C.sub.2 alkyl, and substituted aryl; R.sub.5 is independently selected from hydrogen and C.sub.1˜C.sub.2 alkyl; the substituent of the alkyl is selected from substituted or unsubstituted 5- to 6-membered saturated heterocyclyl, 4- to 6-membered saturated cycloalkyl, —NR.sub.6R.sub.7, aryl, naphthyl, and C.sub.1˜C.sub.3 alkoxy; R.sub.6 and R.sub.7 are independently selected from C.sub.1˜C.sub.3 alkyl; the substituent of the saturated heterocyclyl is selected from C.sub.1˜C.sub.8 alkyl and —C(O)OR.sub.5; the substituent of the aryl is selected from C.sub.1˜C.sub.2 alkyl and nitro; and the heteroatom of the heterocyclyl is selected from N, O and S.

6. A compound represented by formula V, a salt thereof, or a stereoisomer thereof: ##STR00059## wherein, R.sub.1is selected from hydrogen and 4- to 6-membered saturated heterocyclyl; R.sub.2 is null, or R.sub.2 is selected from substituted or unsubstituted C.sub.1˜C.sub.3 alkyl, halogen, substituted or unsubstituted 4- to 6-membered saturated heterocyclyl, —C(O)NR.sub.3R.sub.4, —C(O)R.sub.5 and —C(O)OR.sub.5; R.sub.3 and R.sub.4 are independently selected from hydrogen, substituted or unsubstituted C.sub.1˜C.sub.3 alkyl, 4- to 6-membered saturated heterocyclyl, and substituted 4- to 5-membered unsaturated cycloalkyl; R.sub.5 is selected from hydrogen and C.sub.1˜C.sub.4 alkyl; the substituent of the alkyl is selected from substituted or unsubstituted 4- to 6-membered saturated heterocyclyl, 4- to 6-membered saturated cycloalkyl, —NR.sub.6R.sub.7, aryl, and naphthyl; R.sub.6 and R.sub.7 are independently selected from C.sub.1˜C.sub.2 alkyl; the substituent of the saturated heterocyclyl is selected from C.sub.1˜C.sub.4 alkyl and —C(O)OR.sub.5; the substituent of the unsaturated cycloalkyl is selected from C.sub.1˜C.sub.4 alkyl, nitro, halogen, hydroxyl; and the heteroatom of the heterocyclyl is selected from N, O and S.

7. A compound selected from the following group, a salt thereof, or a stereoisomer thereof: ##STR00060## ##STR00061##

8. A method of inhibiting ferroptosis, comprising administrating the compound, a salt thereof, or a stereoisomer thereof according to claim 1 to a subject in need thereof.

9. A method of treating or preventing a neurodegenerative disease, tumor, tissue ischemia-reperfusion injury, cardiovascular disease, cerebrovascular disease, renal failure or diabetic complication, comprising administrating the compound, a salt thereof, or a stereoisomer thereof according to claim 1 to a subject in need thereof.

10. A pharmaceutical composition comprising the compound, a salt thereof, or a stereoisomer thereof according to claim 1 as an active ingredient, and optionally a pharmaceutically acceptable auxiliary.

11. The method according to claim 9, wherein the cerebrovascular disease is cerebral stroke.

12. The method according to claim 9, wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

13. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is in the form of an oral preparation or an intravenous injection preparation.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0099] FIG. 1 shows the therapeutic effect of the compounds of the present disclosure on ischemic cerebral stroke in SD rats of the rat middle cerebral artery embolism (MCAO) cerebral stroke model, where FIG. A shows the anatomical photos of the rat brain after different treatments, FIG. B shows the effect of different treatments on the volume of cerebral infarction (in FIG. B, * means p<0.05; *** means p<0.001), and FIG. C shows the neurological function score (Longa neurological score with 5 points) after the intravenous injection of 5 mg/kg dose of different treatments.

[0100] The above content of the present disclosure will be further described in detail below through specific implementations in the form of examples. However, it should not be understood that the scope of the above subject matter of the present disclosure is limited to the following examples. All technologies implemented based on the above contents of the present disclosure belong to the scope of the present disclosure.

DETAILED DESCRIPTION

[0101] The raw materials and equipment used in the specific embodiments of the present disclosure are known products, and obtained by purchasing commercially available products.

Example 1 Synthesis of 2-(1-(1-(azetidine-3-amino)-1H pyrazolo[3,4-b]pyridin-5-yl)vinyl)-10H phenothiazine (compound 1)

1. Synthesis of (E)-N′-(1-(10H-phenothiazin-2-yl)ethylene)-4-methylbenzenesulfonylhydrazide (Intermediate I)

[0102] The synthetic route of intermediate I is as follows:

##STR00015##

[0103] 2-Acetylphenothiazine (10.0 g, 41.44 mmol, 1.0 eq) and 4-methylbenzenesulfonylhydrazide (7.72 g, 41.44 mmol, 1.0 eq) were dissolved in 100 mL of MeOH, added with 1 mL of HOAc, and reacted at 60° C. The reaction was monitored by TLC and completed after about 4 h. After cooling to room temperature, a yellow solid appeared. The system was filtered under reduced pressure, washed with MeOH and diethyl ether until the filtrate was colorless, and dried in vacuum to obtain Intermediate I (15 g) with a yield of 88.4%.

2. Synthesis of Intermediate II

[0104] The synthetic route of intermediate II is as follows:

##STR00016##

[0105] Intermediate I (120 mg, 0.293 mmol, 1.2 eq), tert-butyl 3-(5-bromo-1H-pyrazolin[3,4-B]pyridin-1-yl)azelate (86 mg, 0.244 mmol, 1.0 eq), tris(dibenzylidene-BASE acetone)dipalladium(0)Pd.sub.2(dpa).sub.3 (24 mg, 0.03 mmol, 0.1 eq), 2-dicyclohexylphosphorus-2,4,6-triisopropylbiphenyl X-phos (25 mg, 0.03 mmol, 0.2 eq) and anhydrous t-BuOLi (43 mg, 0.537 mmol, 2.2 eq) were dissolved in 10 mL of 1,4-dioxane replaced with argon 3 times, and reacted at 70° C. The reaction was monitored by TLC and completed after about 4h. The system was cooled to room temperature, filtered through celite, and concentrated under reduced pressure. The residue was extracted with saturated aqueous NaHCO.sub.3 solution/DCM. The organic layer was concentrated and separated by column chromatography to obtain Intermediate II (91 mg) with a yield of 55.3%.

3. Synthesis of compound 1

[0106] The synthetic route of compound 1 is as follows:

##STR00017##

[0107] 91 mg of intermediate II was added with 2 mL of dichloromethane and 3 mL of trifluoroacetic acid, reacted at room temperature for half an hour. The resultant was rotated to dryness, dissolved in DCM, and purified by TLC with PE:EA=1:1 as the developing solvent, and separated to obtain the target product compound 1 (67 mg) with a yield of 91.2%.

[0108] .sup.1H NMR and HRMS data of compound 1 are as follows: [0109] .sup.1H NMR. (400 MHz, DMSO) δ 8.61 (s,1H), 8.55 (d, J=1.8 Hz, 1H), 8.38 (s, 1H), 8.19 (d, J=1.8 Hz, 1H), 6.95 (dt, J=15.5, 7.4 Hz, 3H), 6.79-6.71 (m, 2H), 6.63 (d, J=7.7 Hz, 2H), 6.04-5.97 (m, 1H), 5.60 (s, 1H), 5.51 (s, 1H), 4.53 (dd, J=13.8, 6.9 Hz, 4H), 4.49-4.42 (M, 1H). [0110] HRMS (ESL) calcd for C.sub.23H.sub.19N .sub.5S [M+H].sup.+ 398.1361 found; 398.1363.

Example 2 Synthesis of Compound 2

[0111] ##STR00018##

[0112] Compound 2 was prepared by a synthetic method similar to that of Example 1 with a yield of 36.4%.

[0113] .sup.1H NMR and HRMS data of compound 2 are as follows: [0114] .sup.1H NMR (400 MHz, DMSO) δ 8.54 (t, J=5.4 Hz, 2H), 8.16 (s, 1H), 8.11 (s, 1H), 7.03- 6.88 (m, 3H), 6.86- 6.69 (m, 2H), 6.60 (d, J=9.1 Hz, 2H)5.57 (s, 1H), 5.50 (s, 1H), 4,38 (d, J=7.0 Hz, 2H), 3.82 (d, J=9.1 Hz, 2H), 3.25 (t, J=10.7 Hz, 2H), 2.23 (ddd, J=10.9, 7.6, 3.9 Hz, 1H), 1.38-1.20 (M, 4H). [0115] HRMS mk, (ESI) calcd for C.sub.26H.sub.24N.sub.4OS [M+H].sup.+ 441.1671 found: 441.1673.

Example 3 Synthesis of Compound 3

[0116] ##STR00019##

[0117] Compound 3 was prepared by a synthetic method similar to that of Example 1 with a yield of 45.1%.

[0118] .sup.1H NMR and HRMS data of compound 3 are as follows: [0119] .sup.1H NMR (400 MHz, DMSO) δ 8.58-8.47 (m, 2H), 8.15 (s, 1H), 8.10 (d, J=2.0 Hz, 1H), 6.95 (dt, J=17.5, 5.4 Hz, 3H), 6.81 (dd, J=8.0, 1.7 Hz, 1H), 6.75 (td, J=7.6, 1.1 Hz, 1H), 6.60 (dd, J=10.4, 1.4 Hz, 2H), 5.57 (s,1H), 5.50 (s, 1H), 4.39 (d, J=7.5 Hz, 2H), 1.63-1.60 (m, 2H), 1.51 (td, J=7.9, 2.8 Hz, 2H), 1.37-1.29 (m, 2H), 1.24 (d, J=6.9 Hz, 2H), 1.16 (d, J=6.7 Hz, 1H). [0120] HRMS m/z, (ESI) calcd for C.sub.26H.sub.24N.sub.4S [M+H].sup.+ 425.1722 found: 425.1725.

Example 4 Synthesis of Compound 4

[0121] ##STR00020##

[0122] Compound 4 was prepared by a synthetic method similar to that of Example 1 with a yield of 39.5%.

[0123] .sup.1H NMR and HRMS data of compound 4 are as follows: [0124] .sup.1H NMR (400 MHz, DMSO) δ 8.57-8.49 (m, 2H), 8.16 (s, 1H), 8.11 (d, J=2.1 Hz, 1H), 6.94 (ddd, J=10.4, 8.2, 1.1 Hz, 3H), 6.83-6.72 (m, 2H), 6.60 (dd, J=9.6, 1.4 Hz, 2H), 5.57 (s,1H), 5.50(s, 1H), 4.38 (d. J=7.0 Hz, 2H), 3.91 (d, J=12,4 Hz, 2H), 2.74-2.57 (m, 2H), 2.17 (ddd, J=14.9, 9.5, 3.9 Hz, 1H), 1.54-1.44 (m, 2H),1.39 (d, J=6.8 Hz, 9H), 1.20-1.10 (m, 2H). [0125] HRMS m/z (ESI) calcd for C.sub.31H.sub.33N.sub.5O.sub.2S [M+H].sup.+ 540.2355 found: 540.2357.

Example 5 Synthesis of Compound 5

[0126] ##STR00021##

[0127] Compound 5 was prepared by a synthetic method similar to that of Example 1 with a yield of 52.1%.

[0128] .sup.1H NMR and HRMS data of compound 5 are as follows: [0129] .sup.1H NMR (400 MHz, DMSO) δ 11.33 (s, 1H) 8.55 (s, 1H), 7.86 (s, 1H), 7.47 (s, 1H), 7.38 (d, J=8.5 Hz, 2H), 7.11 (dd, J=19.7, 11.2 Hz, 2H), 6.97 (d, J=7.8 Hz, 1H), 6.91 (d, J=7.7 Hz, 2H), 6.84-6.70 (m, 3H), 6.61 (d, J=5.6 Hz, 2H), 5.33 (s, 1H), 5.32 (s, 1H), 1.23 (s, 6H). [0130] HRMS m/z. (ESI) calcd for C31H24N4O3S [M+H]+ 533.1509 found: 533.1571.

Example 6 Synthesis of Compound 6

[0131] ##STR00022##

[0132] Compound 6 was prepared by a synthetic method similar to that of Example 1 with a yield of 53.5%.

[0133] .sup.1H NMR and HRMS data of compound 6 are as follows: [0134] .sup.1H NMR. (400 MHz, DMSO) δ 11.61 (s, 1H), 8.52 (s, 1H), 8.41 (t, J=5.7 Hz, 1H), 7.53 (s, 1H) 7.40 (t, J=6.0 Hz, 1H) 7.13 (dd, J=8.5, 1.5 Hz, 1H), 7.08 (d, J=1.2 Hz, 1H), 6.99-6.88 (m, 3H), 6.81-6.71. (m, 2H), 6.63-6.56 (m, 2H), 5.36 (s, 1H). 5.35 (s, 1H), 3.63-3.53 (m, 5H), 3.44-3.39 (m, 2H), 2.43 (s, 5H). [0135] HRMS m/z (ESI) calcd for C.sub.29H.sub.28N.sub.4O.sub.2S [M+H].sup.+ 497.1933 found: 497.1935.

Example 7 Synthesis of Compound 7

[0136] ##STR00023##

[0137] Compound 7 was prepared by a synthetic method similar to that of Example 1 with a yield of 54.2%.

[0138] .sup.1H NMR and HRMS data of compound 7 are as follows: [0139] .sup.1H NMR (400 MHz, DMSO) δ 8.61 (s, 1H) 7.97 (d, J=0.8 Hz, 1H), 7.77 (s, 1H), 7.52 (d, J=0.8 Hz, 1H), 6.97 (ddd, J=19.1, 11.7, 4.0 Hz, 3H), 6.85-6.73 (m, 2H), 6.71 (d, J=1.6 Hz, 1H), 6.66 (d, J=7.9 Hz, 1H), 6.01 (d, J=2.0 Hz, 1H), 5.32 (d, J=2.0 Hz, 1H), 4.32-4.14 (m, 4H), 3.85-3.68 (m, 4H). [0140] HRMS m/z (ESI) calcd for C.sub.24H.sub.21N.sub.5OS [M+H].sup.+ 428.1467 found: 428.1469.

Example 8 Synthesis of Compound 8

[0141] ##STR00024##

[0142] Compound 8 was prepared by a synthetic method similar to that of Example 1 with a yield of 58.4%.

[0143] .sup.1H NMR and HRMS data of compound 8 are as follows: [0144] .sup.1H NMR (400 MHz, DMSO) δ 8.52 (d, J=2.0 Hz 2H), 8.30 (s, 1H), 8.16(d, J=2.1 Hz, 1H), 6.95 (ddd, J=17.8, 8.8, 1.2 Hz, 3H), 6.80 (dd, J=8.0, 1.8 Hz, 1H), 6.75 (td, J=7.6, 1.2 Hz, 1H), 6.62-6.54 (m, 2H), 5.82 (tt, J=8.1, 5.5 Hz, 1H), 5.60 (s, 1H),. 5.49 (s, 1H), 4.39 (dd, J=30.0,21.9 Hz, 4H), 1.43 (s, 9H) [0145] HRMS m/z (ESI) calcd for C.sub.28.sub.27N.sub.5O.sub.2S [M=H].sup.+ 498.1885 found: 498.1887.

Example 9 Synthesis of Compound 9

[0146] ##STR00025##

[0147] Compound 9 was prepared by a synthetic method similar to that of Example 1 with a yield of 52.8%.

[0148] .sup.1H NMR and HRMS data of compound 9 are as follows: [0149] .sup.1H NMR (400 MHz, DMSO) δ 11.48 (s, 1H), 8.53 (s, 1H), 8.10 (d, J=7.8. Hz, 1H), 7.99 (s, 1H), 7.79 (s, 1H), 7.29 (s, 1H) 7.13 (d, J=7.8 Hz, 1H), 7.05-6.85 (m, 3H), 6.85-6.70 (m, 2H) 6.61 (s, 2H), 5.42 (s 1H), 5.38 (s, 1H), 3.58 (s, 4H), 3.39 (d, J=5.2 Hz, 2H), 2.45 (d, J=15.9 Hz, 6H). [0150] HRMS m/z (ESI) calcd for C29H28N4O2S [M+H]+ 497.1933 found: 497.1933.

Example 10 Synthesis of Compound 10

[0151] ##STR00026##

[0152] Compound 10 was prepared by a synthetic method similar to that of Example 1 with a yield of 55.1%.

[0153] .sup.1H NMR and HRMS data of compound 10 are as follows: [0154] .sup.1H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 9.95 (s, 1H), 8.53 (s, 1H), 8.31 (s, 1H), 8.05 (dd, J=20.0, 8.3 Hz, 1H) 7.42-7.34 (m, 1H), 7.24 (d, J=8.2 Hz, 1H), 7.09-6.87 (m, 3H), 6.81 (dd, J=7.9, 1.4 Hz, 1H), 5.75 (t, J=7.4 Hz, 1H), 6.60 (d, J=7.4 Hz, 2H), 5.44 (s, 2H). [0155] HRMS m/z (ESI) calcd for C.sub.23H.sub.16N.sub.2OS [M+H].sup.+ 369.0983 found: 369.0985.

Example 11 Synthesis of compound 11

[0156] ##STR00027##

[0157] Compound 11 was prepared by a synthetic method similar to that of Example 1 with a yield of 46.3%.

[0158] .sup.1H NMR and HRMS data of compound 11 are as follows: [0159] .sup.1H NMR (400 MHz, DMSO) δ 10.64 (s, 1H), 8.56 (s, 1H), 7.23(d, J=1.9 Hz, 1H), 7.10 .(dd, J=8.3, 2.0 Hz, 1H), 6.97 (ddd, J=7.9, 4.2, 1.4Hz, 2H), 6.91 (d, J=7.9 Hz, 2H), 6.78-6.71 (m, 2H), 6.62 (dd, J=7.9, 0.8 Hz, 1H), 6.57 (d, J=1.7 Hz, 1H), 5.37 (s 2H), 3.48 (s, 2H). [0160] HRMS m/z (ESI) calcd for C.sub.22H.sub.16N.sub.2OS [M+H].sub.+ 389.0704 found: 389.0706.

Example 12 Synthesis of Compound 12

[0161] ##STR00028##

[0162] Compound 12 was prepared by a synthetic method similar to that of Example 1 with a yield of 48.6%.

[0163] .sup.1H NMR and HRMS data of compound 12 are as follows: [0164] .sup.1H NMR (400 MHz, DMSO) δ 8.56 (s, 1H), 8.29 (d, J=8.7 Hz, 1H),7.80 (s. 2H), 7.56 (d, J=1.6 Hz, 1H), 7.40 (dd, J=8.6, 1.7 Hz, 1H), 7.04-6.88 (m,3H), 6.86 - 6.70 (m, 2H), 6.61 (dd, J=9.6, 1,2 Hz, 2H), 5.63 (s,1H), 5.57 (s, 1H), 4.36 (q, J=7.1 Hz, 2H), 2.62 (s, 3H), 1.36 (t, J=7.1 Hz, 3H). [0165] HRMS m/z (ESI) calcd for C.sub.27H.sub.23N.sub.3O.sub.2S [M+H].sup.+ 454.1511 found: 454.1513.

Example 13 Synthesis of Compound 13

[0166] ##STR00029##

[0167] Compound 13 was prepared by a synthetic method similar to that of Example 1 with a yield of 49.6%. [0168] .sup.1H NMR and HRMS data of compound 13 are as follows:

[0169] .sup.1H NMR (400 MHz, DMSO) δ 8.56 (s, 1H), 6.97 (td, J=7.8, 1.4Hz, 1H), 6.94 - 6.88 (m, 2H), 6.88 - 6.82 (m, 1H), 6.77 (d, J=1.8 Hz, 2H), 6.75 (d, J=2.0 Hz, 1 H), 6.72 (d, J=1.7 Hz, 1H), 6.63 (dd, J=7.9, 0.9 Hz, 1H), 6.59 (d, J=1.7 Hz, 1H), 5.31 (s, 2H), 4.25 (s, 4H). [0170] HRMS m/z (ESI) calcd for C.sub.22H.sub.17NO.sub.2S [M+H].sub.+ 360.0980 found: 360.0982.

Example 14 Synthesis of Compound 14

[0171] ##STR00030##

[0172] Compound 14 was prepared by a synthetic method similar to that of Example 1with a yield of 52.6%.

[0173] .sup.1H NMR and HRMS data of compound 14 are as follows: [0174] .sup.1H NMR (400 MHZ, DMSO) δ 8.90 (dd, J=4.2, 1.6 Hz, 1H), 8.54 (s, 1H), 8.40 (d, J . 7.7 Hz, 1H), 8.02 (d, J=8.7 Hz, 1H), 7.90 (d, J=1.8 Hz, 1H) 7.72 (dd, J=8.7, 2.0 Hz, 1H) 7.54 (dd, J=8.3, 4.2 Hz, 1H), 7.02-6.88 (m, 3H), 6.83 (dd, J=7.9, 1,8 Hz, 1H), 6.75 (td, J=7.6, 1.1 Hz, 1H), 6.60 (dd, J=4.2, 2.5 Hz, 2H), 5.60 (s, 1H), 5.58 (s, 1H). [0175] HRMS m/z (ESI) calcd for C.sub.23H.sub.16N.sub.2S [M+H].sup.+ 353.1034 found: 353.1036.

Example 15 Synthesis of Compound 15

[0176] ##STR00031##

[0177] Compound 15 was prepared by a synthetic method similar to that of Example 1 with a yield of 51.3%.

[0178] .sup.1H NMR and HRMS data of compound 15 are as follows: [0179] .sup.1H NMR (400 MHz, DMSO) δ 8.56 (s, 1H), 7.39 (dd, J=12.2, 5,0 Hz, 2H), 7.11 (dd, J=8.3, 1.5 Hz, 1H), 6.97 (dd, J=10.9, 4.3 Hz, 1H), 6.92 (dd, J=7.4, 3.7 Hz, 2H), 6.78-6.72 (m, 2H), 6.63 (d, J=7.8 Hz, 1H), 6.56 (d, J=1.7 Hz, 1H), 5.48 (s, 1H), 5.43 (s, 1H). [0180] HRMS m/z (ESI) calcd for C.sub.21H .sub.13F.sub.2NO.sup.2S [M +H].sup.+ 382.0635 found: 382.0637.

Example 16 Synthesis of Compound 16

[0181] ##STR00032##

[0182] Compound 16 was prepared by a synthetic method similar to that of Example 1 with a yield of 49.4%.

[0183] .sup.1H NMR and HRMS data of compound 16 are as follows: [0184] .sup.1H NMR (400 MHz, DMSO) δ 8.55 (s, 1 H) 8.01 (d, J=8.3 Hz, 1H), 7.15 (s. 1H), 7.09 (d, J=8.3 Hz, 1H), 6.97 (td, J=7.7, 1.4 Hz, 1H), 6.93-6.87 (m, 2H), 6.79 -6.70 (m, 2H), 6.63 (dd, J=7.9, 0.9 Hz, 1H), 6.58 (d, J=1.7 Hz, 1H) 5.33 (s, 1H), 5.33 (s, 1H) , 4.09 (dd, J=14.2, 5.7 Hz, 2H), 3.19-3.06 (m, 2H), 2.16 (s, 3H), [0185] HRMS m/z (ESI) calcd for C.sub.24H.sub.20N.sub.2OS [M+H].sup.+ 385.1296 found:. 385.1298.

Example 17 Synthesis of compound 17

[0186] ##STR00033##

[0187] Compound 17 was prepared by a synthetic method similar to that of Example 1 with a yield of 48.5%.

[0188] .sup.1H NMR and HRMS data of compound 17 are as follows: [0189] .sup.IH NMR (400 MHz, DMSO) δ 8.56 (s, 1H), 7.62 (d, J=1.5 Hz, 1H), 7.16 (s, 1H), 6.97 (dd, J=11.0, 4.3 Hz, 1H) 6.90 (dd, J=7.4, 3.9 Hz, 2H), 6.80-6.71 (m, 2H), 6.61 (dd, J=12.1, 4.5 Hz, 2H), 6.53 (s, 1H), 5.24 (s, 1H), 5.19 (s, 1H), 3.49 (t, J=8.4 Hz,2H), 2.96 (t, J=8.3 Hz, 2H). [0190] HRMS m/z (ESI) calcd for C.sub.21H.sub.17N.sub.3S [M+H].sub.+ 344.1143 found: 344.1145.

Example 18 Synthesis of Compound 18

[0191] ##STR00034##

[0192] Compound 18 was prepared by a synthetic method similar to that of Example 1 with a yield of 50.9%.

[0193] .sup.1H NMR and HRMS data of compound 18 are as follows: [0194] .sup.1H NMR (400 MHz, DMSO) δ 8.55 (s, 1H), 8.29 (s, 1H), 7.82 (s, 2H), 7.68 (d, J=8.6 Hz, 1H), 7.52 (dd, J=8.6, 1.5 Hz, 1H), 7.03-6.87 (m, 3H), 6.78 (ddd, J=15.0, 11.0, 4.5 Hz, 2H), 6.61 (d, J=8.5 Hz, 2H), 5.56 (s, 1H), 5.52 (s, 1H), 4.36 (q, J=7.1 Hz, 2H), 2.64(s, 3H) 1.35 (t, J=7.1 Hz, 3H). [0195] HRMS m/z (ESI) calcd for C27H23N3O2S [M+H]+ 454.1511 found: 454.1513.

Example 19 Synthesis of Compound 19

[0196] ##STR00035##

[0197] Compound 19 was prepared by a synthetic method similar to that of Example 1 with a yield of 49.9%.

[0198] .sup.1H NMR and HRMS data of compound 19 are as follows: [0199] .sup.1H NMR (400 MHz, DMSO) δ 8.55 -8.50 (m, 2H), 8.20 (s, 1H), 8.13 (d, .J=2.0 Hz,1H), 7.00 -6.89(m, 3 H), 6.82 (dd, J=8.0, 1.8 Hz, 1H), 6.75 (td, J=7.6, 1.0 Hz, 1H), 6.64-6.55 (m, 2H), 5.71-5.61 (m, 1H), 5.59 (s, 1H), 5.49 (s, 1H), 4.13 (ddd, J=22.8, 12.1, 7.2 Hz, 2H), 3.94 (ddd, J=14.3, 6.2, 3.9 Hz, 2H), 2.49 - 2.38 (m, 2H). [0200] HRMS m/z (ESI) calcd for C24H20N4OS [M+H]+ 413.1358 found: 413.1360.

Example 20 Synthesis of Compound 20

[0201] ##STR00036##

[0202] Compound 20 was prepared by a synthetic method similar to that of Example 1 with a yield of 41.2%.

[0203] .sup.1H NMR and HRMS data of compound 20 are as follows: [0204] .sup.1H NMR (400 MHz, DMSO) δ 8.60-8.41 (m, 2H), 8.17 (s, 1H), 8.11 (d, J=1.9 Hz, 1H), 6.94 (dt, J=18.3, 9.1 Hz, 3H) 6.83 (dd, J=7.9, 1.6 Hz, 1H), 6.75 (t, J=7.5 Hz, 1H), 6.58 (dd, J=7.3, 4.8 Hz,2H), 5.58 (s,1H), 5.48 (s, 1H), 4.80 (ddd, J=15.1, 9.6, 3.9 Hz 1H), 2.93 (d, J=10.8 Hz, 2H), 2.25 (s, 3H), 2.23-1.98 (m, 4H), 1.90 (d, J=10.8 Hz, 2H). [0205] HRMS m/z (ESI) calcd for C.sub.26H.sub.25N.sub.5S [M+H].sup.+ 440.1831 found: 440.1833.

Example 21 Synthesis of Compound 21

[0206] ##STR00037##

[0207] Compound 21 was prepared by a synthetic method similar to that of Example 1 with a yield of 46.5%.

[0208] .sup.1H NMR and HRMS data of compound 21 are as follows: [0209] .sup.1H NMR (400 MHz. DMSO) δ 8.60-8.45 (m, 2H), 8.14 (s, 1H), 8.10 (d, J=2.0 Hz, 1H), 7.01-6.89 (m, 3H), 6.81 (dd, J=8.0, 1.8 Hz,1H), 6.75 (td, J=7.6, 1.1 Hz, 1H), 6.65-6.53 (m, 2H), 5.57 (s, 1H), 5.49 (s, 1H), 4.50 (d, J=7.2 Hz, 2H), 2.89 (dt, J=15.1, 7.5 Hz, 1H), 1.97 (dd, J=8.2, 6.5 Hz, 2H), 1.90-1.78 (m, 4H). [0210] HRMS m/z (ESI) calcd for C.sub.25H.sub.22N.sub.4S [M+H].sub.+ 411.1565 found 411.1567.

Example 22 Synthesis of Compound 22

[0211] ##STR00038##

[0212] Compound 22 was prepared by a synthetic method similar to that of Example 1 with a yield of 47.5%.

[0213] .sup.1H NMR and HRMS data of compound 22 are as follows: [0214] .sub.1H NMR (400 MHz, DMSO) δ 9.17 (s, 1H), 8.64 (s, 1H), 8.52 (d, J=1.4 Hz, 1H), 8.48 (s, 1H), 8.30 (t, J=5.7 Hz, 1H), 6.97 (ddd, J 16.5, 8.6, 3.9 Hz, 3H), 6.83 (dd, J=7.9, 1.7 Hz, 1H), 6.77 (td, J=7.5, 1.1 Hz. 1H), 6.70(d, J=1.7 Hz, 1H), 6.66 (dd, J=7.9, 0.9 Hz, 1H), 6.04 (d, J=1.3 Hz, 1H), 5.51 (d, 1.3 Hz, 1H), 2.51 (d, J=1.6 Hz, 2H),2.42 (t, J=6.5 Hz, 2H), 2.18 (s, 6H). [0215] HRMS m/z (ESI) calcd for C.sub.25H.sub.24N.sub.6OS [M+H].sup.+ 457.1732 found: 457.1734.

Example 23 Synthesis of Compound 23

[0216] ##STR00039##

[0217] Compound 23 was prepared by a synthetic method similar to that of Example 1 with a yield of 50.4%.

[0218] .sup.1H NMR and HRMS data of compound 23 are as follows: [0219] .sup.1H NMR (400 MHz, DMSO) δ 9.15 (s,1H) 8.64 (s, 1H), 8.53 (d, J=1.3 Hz, 1H), 8.49 (s, 1H), 8.44 (d, J=8.3 Hz, 1H), 6.96 (dd, J=17.0, 7.8 Hz, 3H), 6.83 (dd, J=7.9, 1.7 Hz, 1H), 6.77 (td, J=7.6, 1.1 Hz, 1H), 6.70 (d, J=1.7 Hz, 1H), 6.66 (d, J=7.9 Hz, 1 H), 6.04 (d, J=1.2 Hz, 1H), 5.51 (s, 1H), 4.08-3.97 (m, 1H), 3.90-3.82 (m, 2H), 3.43-3.36(m, 2H), 1.70 (dd, J=9.9, 6.6 Hz, 4H). [0220] HRMS m/z (ESI) calcd for C.sub.26H.sub.23N.sub.5O.sub.2S [M+H].sup.+ 470.1572 found: 470.1574.

Example 24 Synthesis of Compound 24

[0221] ##STR00040##

[0222] Compound 24 was prepared by a synthetic method similar to that of Example 1 with a yield of 49.6%.

[0223] .sup.1H NMR and HRMS data of compound 24 are as follows: [0224] .sup.1H NMR (400 MHz, DMSO) δ 9.15 (s, 1H), 8.58 (dd, J=12.7, 6.5 Hz, 2H), 8.53 (d, J=1.1 Hz, 8.48 (s, 1H) 7.06 - 6.90 (m, 3H), 6.83 (dd, J=7.9, 1.6 Hz, 1H), 6.76 (t, J=7.5 Hz, 1H), 6.67 (dd, J 16.2, 4.7 Hz, 2H), 6.04 (s 1H), 5.52 (s, 1H), 3.83 (dd, J=11.3, 2.4 Hz, 2H), 3.30 -313 (m, 4H), 1.93 -1.73 (m, 1H), 1.57 (d, J=12.8 Hz, 2H),1.23 (dd, J=4.8 Hz, 2H). [0225] HRMS m/z (ESI) calcd for C.sub.27H.sub.25N.sub.5O.sub.2S [M+H].sup.+ 484.1729 found: 484.1731.

[0226] Example 25 Synthesis of Compound 25

##STR00041##

[0227] Compound 25 was prepared by a synthetic method similar to that of Example 1 with a yield of 38.7%.

[0228] .sup.1H NMR and HRMS data of compound 25 are as follows: [0229] .sup.1H NMR (400 , DMSO) δ 9.16(s, 1H), 9.01 (d, J=8.4Hz, 1H), 8.64 (s, 1H), 8.57-8.43 (m, 2H), 8.24 (d, J=8.3 Hz, 1H), 7.95 (d, J=7.9 Hz, 1H), 7.83 (d, J=8.2 Hz, 1H), 7.70 (d, J=7.0 Hz, 1H), 7.61 -7.46 (m, 3H), 7.02-6.90 (m, 3H), 6.82 (dd, J=7.9, 1.7 Hz, 1H), 6.76 (td, J=7.6, 1.1 Hz, 1H), 6.70 (d, J=1.7 Hz, 1 H), 6.66, 7.9 Hz, 1H), 6.04 (d, J=1.3 Hz, 1H), 5.98 (dd, J=14.9, 7.2 Hz, 1 H), 5.51 (d, J=1.2 Hz, 1H) 1.67 (d, J=6.9 Hz, 3H). [0230] HRMS m/z (ESI) calcd for C33H25N5OS [M+H]+ 540.1780 found: 540.1782.

Example 26 Synthesis of Compound 26

[0231] ##STR00042##

[0232] Compound 26 was prepared by a synthetic method similar to that of Example 1 with a yield of 57.9%.

[0233] .sup.1H NMR and HRMS data of compound 26 are as follows: [0234] .sup.1H NMR (400 MHz, DMSO) δ 9.17 (S, 1H), 8.85 (d, J=8.5 Hz, 1H), 8.62 (s, 1H) 8.52 (d, J=1.3 Hz, 1H), 8.48 (s, 1H) 7.43 (d, J=7.2 Hz, 2H), 7.32 (t, J=7.5 Hz, 2H), 7.22 (t, J=7.3 Hz, 1H), 6.99 (dd, J=13.0, 4.7 Hz, 2H), 6.93 (dd, J=5.8, 1.8 Hz, 1H), 6.83 (dd, J=7.9, 1.7 Hz, 1H), 6.76 (td, J=7.6, Hz, 1H), 6.69 (d, J=1.7Hz, 1H) 6.65 (dd, J=7.9, 0.9 H.z, 1H), 6.04 (d, J=1.3 Hz, 1H), 5.51 (d, J=1.3 Hz, 1H), 5.23-5.12 (m, 1H), 1.52 (d, J=7.0 Hz, 3H), [0235] HRMS m/z (ESI) calcd for C.sup.29H.sub.23N.sub.5OS [M+H].sub.+ 490.1625 found: 490.1625.

Example 27 Synthesis of Compound 27

[0236] ##STR00043##

[0237] Compound 27 was prepared by a synthetic method similar to that of Example 1 with a yield of 50.7%.

[0238] .sup.1H NMR and HRMS data of compound 27 are as follows: [0239] .sup.1H NMR (400 MHz, DMSO) δ 8.59 H), 8.52 (d, J=1.9 Hz, 1H), 8.17 (s, 1H), 8.11 (d, J=1.9 Hz, 1H) 7.02 -6.88 (m, 3H), 6.82 (dd, J=8.0, 1.5 Hz, 1H), 6.75 (t, J=7.5 Hz, 1H), 6.67 -6.52 (m, 2H), 5.58 (s, 1H), 5.49 (s, 1H), 4.64 (t, J =5.4 Hz, 2H), 3.86 (t, J=5.4 Hz, 2H), 3.23 (s, 3H). [0240] HRMS m/z (ESI) calcd for C.sub.23H.sub.20N.sub.4OS [M+H].sup.+ 401.1358 found: 401.1360.

Example 28 Synthesis of Compound 28

[0241] ##STR00044##

[0242] Compound 28 was prepared by a synthetic method similar to that of Example 1 with a yield of 53.8%.

[0243] .sup.1H NMR and HRMS data of compound 28 are as follows: [0244] .sup.1H NMR (400 MHz, DMSO) δ 8.57 (s, 1H), 8.52 (d, J=1.8 Hz, 1H), 8.15 (s, 1H), 8.12 (d, J=1.7 Hz, 1H), 7.00 -6.89 (m, 3H), 6.80 (d, J=8.0 Hz, 1H), 6.75 (t, J=7.5 Hz, 1H), 6.60 (dd, J=10.3, 4.7 Hz, 2H), 5.49 (s, 1 H), 5.49 (s 1H), 4.09 (s, 3H). [0245] HRMS m/z (ESI) calcd for C.sub.21,.sub.16N.sub.4S [M+H].sup.+ 357.1096 found: 357.1098.

Example 29 Synthesis of Compound 29

[0246] ##STR00045##

[0247] Compound 29 was prepared by a synthetic method similar to that of Example 1 with a yield of 36.9%.

[0248] .sup.1H NMR and HRMS data of compound 29 are as follows: [0249] .sup.1H NMR (400MHz, DMSO), δ 8.52 (d, J=12.1, Hz, 2H), 8.18 (s, 1H), 8.12 (s, 1H), 6.99-6.89 (m, 3H), 6.82 (d, J=7.9 Hz, 1H), 6.75 (t. J=7.5 Hz, 1H), 6.59 (d, J=11.5 Hz, 2H), 5.59 (s, 1H), 5.48 (s, 1H), 5.07 (td, J=11.0, 5.4 Hz, 1H), 4.11 (d, J=11.5 Hz, 2H), 3.04 (s, 2H), 2.11-1.99 (m, 2H), 1.95, (d, J=10.6 Hz, 2H), 1.44 (s, 9H) [0250] HRMS m/z. (ESI) calcd for C30H31N5O2S [M+H]+ 526.2198 found: 526.2200.

Example 30 Synthesis of Compound 30

[0251] ##STR00046##

[0252] Compound 30 was prepared by a synthetic method similar to that of Example 1 with a yield of 39.8%.

[0253] .sup.1H NMR and HRMS data of compound 30 are as follows: [0254] .sup.1H NMR (400 MHz, MeOD) δ 8.50 (s, 1H), 8.17 (s, 1H), 8.11 (s, 1H), 6.94 (t, J=7.6 Hz, 1H), 6.86 (d, J=7.8 Hz, 2H), 6.78-6.68 (m, 2H), 6.61 -6.52 (m, 2H), 5.88 -5.78 (m, 1H), 5.58 (s, 1H), 5.46 (s, 1H), 4.48 (d, J=6.5 Hz, 4H), 1.50 (s, 9H). [0255] HRMS m/z (ESI) calcd for C.sub.28H.sub.27N.sub.5O.sub.2S [M+H].sup.+ 498.11885 found: 498.1887.

Example 31 Synthesis of Compound 31

[0256] ##STR00047##

[0257] Compound 31 was prepared by a synthetic method similar to that of Example 1 with a yield of 53.4%.

[0258] .sup.1H NMR and HRMS data of compound 31 are as follows: [0259] .sup.1H NMR (400 MHz, DMSO) δ 13.71 (s, 1H), 8.58-8.44 (m, 2H), 8.13 (dd, J=16.0, 1.4 Hz, 2H), 7.00 -6.89 (m, 3H), 6.81 (dd, J=8.0, 1.8 Hz, 1H), 6.75 (td, J=7.6, 1.1 Hz, 1H), 6.59 (dd, J=9.0, 4.8 Hz, 2H), 5.56 (s, 1H), 5.49 (s, 1H). [0260] HRMS m/z (ESI) calcd for C.sub.20H.sub.14N.sub.4S [M+H].sup.+ 343.0939 found: 343.0941.

Example 32 Synthesis of Compound 32

[0261] ##STR00048##

[0262] Compound 32 was prepared by a synthetic method similar to that of Example 1 with a yield of 54.3%.

[0263] .sup.1H NMR and HRMS data of compound 32 are as follows: [0264] .sup.1H NMR (400 MHz, DMSO) δ 8.52 (d, J=2.0 Hz 2H), 8.32 (s, 1H), 8.16 (d, J=2.0 Hz, 1H), 7.00 -6.88 (m, 3H), 6.82-6.71 (m,2H), 6.58 (dd, J=11.0, 4.4 Hz, 2H), 6.22-6.13 (m, 1H), 5.59 (s, 1H), 5.49 (s, 1H), 5.10 (t, J=6.4 Hz, 2H), 5.07-5.02 (m, 2H), 3.38 (q, J=7.0 Hz, 2H).

[0265] HRMS m/z (ESI) calcd for C.sub.24H.sub.20N.sub.4OS [M+H].sup.+ 413.1358 found: 413.1360.

Example 33 Synthesis of Compound 33

[0266] ##STR00049##

[0267] Compound 33 was prepared by a synthetic method similar to that of Example 1 with a yield of 53.6%.

[0268] .sup.1H NMR and HRMS data of compound 33 are as follows: [0269] .sup.1H NMR (400 MHz, DMSO) δ 8.57-8.47 (m, 2H), 8.15 (s, 1H), 8.11 (d, J=2.1 Hz, 1H),6.99 - 6.90 (m, 3H), 6.82 (dd, J=8.0, 1.8 Hz, 1H), 6.75 (td, J=7.6, 1.0 Hz, 1H), 6.62 - 6.54 (m, 2H), 5.58 (s, 1H), 4.51 (q, J=7.2 Hz, 2H), 1.45 (t, J=7.2 Hz, 3H). [0270] HRMS m/z (ESI) calcd for C.sub.22H.sub.18N.sub.4S [M+H].sup.+ 371.1252 found: 371.1255.

Example 34 Synthesis of Compound 34

[0271] ##STR00050##

[0272] Compound 34 was prepared by a synthetic method similar to that of Example 1 with a yield of 55.5%.

[0273] .sup.1H NMR and HRMS data of compound 34 are as follows: [0274] .sup.1H NMR (400 MHz, DMSO) δ 8.51 (s, 1H), 7.46 (d, J=1.2, Hz, 1H), 7.42 (d,. J=8.5 Hz, 1.H),. 733 (d, J=31 Hz, HT), 7.10 (dd, J=8.5, 1.6 Hz, 1H), 6.96 (td, J=7.8, 1.4 Hz, 1H), 6.93-6.88 (m, 2H), 6.81-6.71 (m, 2H), 6.61 (dd, J=5.6, 1.4 Hz, 2H), 6.42 (d, J=2.5 Hz, 1H), 5.34(s, 2H), 3.80 (s, 3H).

[0275] HRMS m/z (ESI) calcd for C.sub.22H.sub.17N.sub.3S [M+H].sup.+ 356.1143 found: 356.1145.

Example 35 Synthesis of Compound 35

[0276] ##STR00051##

[0277] Compound 35 was prepared by a synthetic method similar to that of Example 1 with a yield of 45.4%.

[0278] .sup.1H NMR and HRMS data of compound 35 are as follows: [0279] .sup.1H NMR (400 MHz, DMSO) δ 8.00 (d, J=8.4 Hz, 1H), 7,78 (d, J=5.5 Hz, 2H), 7.47 (dd, J=7.5, 3,6 Hz, 2H), 7.34-7.30 (m, 1H), 6.99-6.90 (m, 3H), 6.82 (dd, J=7.9, 1.8 Hz, 1H), 6.74 (td, J 7.6, 1.1Hz, 1H), 6.62-6.57 (m, 2H), 5.49 (s, 11H) 5.45 (s, 1H). [0280] HRMS m/z (ESI) calcd for C.sub.21H.sub.14N.sub.2S.sub.2[M+H].sup.+ 359.0598 found: 359.0600.

Example 36 Synthesis of Compound 36

[0281] ##STR00052##

[0282] Compound 36 was prepared by a synthetic method similar to that of Example 1 with a yield of 36.7%.

[0283] .sup.1H NMR and HRMS data of compound 36 are as follows: [0284] .sup.1H NMR(400 MHz, DMSO) δ 8.59 (s, 1H), 8.51 (d, J=1.8 Hz, 1H), 8.22(s, 1H), 8.15 (s, 1H), 7.03 - 6.88 (m, 3H), 6.83 - 6.71(m, 2H), 6.67-6.57 (m, 2H), 5.59 (s, 1H), 5.49 (s, 1H), 5.30-5.13 (m, 1H), 3.28-3.15 (m, 4H), 2.38 (ddd, J=15.1, 13.6, 3.6 Hz,2H),2.14(d, J=12.0 Hz, 2H). [0285] HRMS m/z (ESI) calcd for C.sub.25H.sub.23N.sup.5S [M+H].sub.+ 426.1674 found 426.1676.

[0286] The beneficial effects of the present disclosure are demonstrated below through specific test examples.

Test Example 1 for Biological Activity. Study on the Inhibition Rate of Compounds of the Present Disclosure on Ferroptosis

[0287] In this example, in order to study the inhibitor of ferroptosis, a screening model for ferroptosis was constructed in house, and the details are as follows:

[0288] The screening model for ferroptosis mainly used the MTT cell viability assay. First, the fibrosarcoma cell line was cultured in a dish, and the cells in the logarithmic growth phase were seeded in a 96-well plate in specific numbers (3000-10000 cells/well), 100 μL per well, and then cultured in an incubator at 37° C. and 5% CO.sub.2 to allow the cells to adhere. After 24 h, 100 μL of compounds at certain concentrations prepared in the indicated medium and the ferroptosis inducer Erastin (final concentration of 10 μM) were added, and wells in triplicate were set up for each compound to ensure the accuracy of the results. A negative control group, positive control group (5 μM of ferrostain-1), blank control group and solvent control group were also set. After dosing, cells were placed in the incubator and cultured for 72h. On the day of the MTT test, MTT test solution (5 mg/mL MTT solution dissolved in physiological saline, stored at 4° C. in the dark) was pre-prepared, and 20 μL was added to each well. Cells were placed in the incubator and cultured for another 2-4 h. Then, 50 μL of 20% SDS solution (dissolved in MiliiQ water, added with 1% concentrated hydrochloric acid) was added to each well, and cells were placed in the incubator overnight. The next day, the absorbance value at 570 nm was detected with a microplate reader to calculate the inhibition rate of the drug on ferroptosis. Generally, the absorbance value of the control group should be between 0.8-1.2 as a normal value. After obtaining the absorbance value data, the average value of wells in triplicate was calculated, and the inhibition rate was calculated using the following formula:

Inhibition rate (IR) %=[1 -(A.sub.experimental group−A.sub.blank)/(A.sub.solvent−A.sub.blank)]*100%

[0289] The curve of inhibition rate change was fitted using Graph Pad Prism 5 software to obtain EC.sub.50.

[0290] The compounds prepared in the examples were subjected to the EC.sub.50 test (the EC.sub.50 in the test was obtained from the average of three tests with ferrostain-1 as the positive control), and the results were shown in the following table:

TABLE-US-00001 TABLE 1 EC.sub.50 of compounds of the present disclosure Compound EC.sub.50 (μM) Compound EC.sub.50 (μM) 2 0.038 4 0.076 6 0.015 7 0.024 8 0.055 9 0.012 10 0.015 11 0.033 13 0.037 14 0.006 15 0.010 16 0.010 17 0.089 19 0.013 20 0.0002 22 0.014 23 0.067 24 0.056 25 0.055 26 0.076 27 0.073 28 0.058 29 0.029 30 0.092 31 0.011 32 0.050 33 0.067 34 0.042 35 0.074 36 0.016 Ferrostain-1 0.060

[0291] It can be seen from Table 1 that the compounds prepared in the present disclosure had good inhibitory effects on ferroptosis, especially compound 14 and compound 20, which had excellent inhibitory effects on ferroptosis, significantly better than the positive drug ferrostain-1, and thus can be used for the preparation of a ferroptosis inhibitor and a medicament for treating ferroptosis-related diseases such as neurodegenerative disease, tissue ischemia-reperfusion injury, cerebral stroke, cardiovascular disease, renal failure, and diabetic complications.

Test Example 2 for Biological Activity

[0292] A middle cerebral artery occlusion (MCAO) cerebral stroke model (also called cerebral ischemia model) in rat was established by the suture method, to study the therapeutic effects of the compounds of the present disclosure on ischemic cerebral stroke in SD rats after multiple administrations.

[0293] 30 male SD rats were subjected to anesthesia induced with enflurane at 4%-5% volume fraction and maintained with 1%-2% enflurane mixed with 70% N2O and 30% O2. The middle cerebral artery was occluded by the suture method to establish a rat model of ischemia. The ischemic rats were placed at room temperature to keep their body temperature at 37° C. 1.5 hours later, the suture was pulled out for reperfusion. The experiment was carried out with 3 groups (10 random SD rats in each group): model control group (Vehicle), sample group (administered with compound 14 of the present disclosure), and positive drug group (administered with Fer-1). The animals in each group were subjected to middle cerebral artery occlusion with a suture for 1.5 h before the suture was pulled out for reperfusion, and were dissected 24 h after reperfusion. Each group was administered intravenously once within 30 minutes of middle cerebral artery occlusion with a suture and once at 2 hours after reperfusion, for a total of 2 administrations, and the dosage in each group was 5 mg/kg. The cerebral infarction volume in each group was calculated by TTC staining 1 day after dissection. Cerebral infarction volume =cerebral infarction area% * brain volume. The experimental results are shown in FIG. 1.

Test Example 3 for Biological Activity. Activity Test of Compounds of the Present Disclosure in Rat MCAO Cerebral Stroke Model

[0294] 1. Experiment Method

[0295] Through the middle cerebral artery occlusion (MCAO) cerebral stroke model (also known as cerebral ischemia model) in rat, the therapeutic effects of the compounds of the present disclosure at different doses and single administration on ischemic cerebral stroke in SD rats were studied.

[0296] Male SD rats were subjected to anesthesia induced with 2-3.0% isoflurane, and the middle cerebral artery was occluded by the suture method to establish a rat model of ischemia. The ischemic rats were placed at room temperature to keep their body temperature at 37° C. After 90 min, the suture was pulled out for reperfusion.

[0297] The experiment was carried out with 6 groups: model control group, two administration groups of example compound 14 (2.5 mg/kg in the low-dose group and 5 mg/kg in the high-dose group), and two administration groups of the comparative compound 1 (2.5 mg/kg in the low-dose group and 5 mg/kg in the high-dose group) and positive drug administration group (butylphthalide with administration dose of 5 mg/kg). Each group was assigned to 18 model animals, and within 10 min of reperfusion, compound 14, comparative compound 1 and positive drug were administered via tail vein injection. After 24 h of reperfusion, the animals were scored by NSS to evaluate neurological function, and then the animals were dissected to remove the brain for TTC staining. By determining the size of cerebral infarction and calculating the inhibition rate, the therapeutic effects of the test compounds on ischemic cerebral stroke in rats were evaluated comprehensively. Wherein, the comparative compound 1 is the compound 38 of

[0298] Example 2 in Chinese Patent Application Publication CN108484527A shown in the following formula.

##STR00053##

[0299] The percentage of infarct tissue weight in the whole brain was used as the infarct size (%), and the inhibition rate (%) of each drug treatment group was calculated based on the infarct size. The inhibition rate was calculated with the following formula:

[00001]$\mathrm{Inhibition}\mathrm{rate}\left(\%\right)=\frac{\begin{array}{c}\mathrm{Infarct}\mathrm{size}\mathrm{of}\mathrm{model}\mathrm{group}-\mathrm{Infarct}\\ \mathrm{size}\mathrm{of}\mathrm{treatment}\mathrm{group}\end{array}}{\mathrm{Infarct}\mathrm{size}\mathrm{of}\mathrm{model}\mathrm{group}}\times 100\%$

[0300] 2. Test Results:

[0301] (1) Size of Cerebral Infarction and Inhibition Rate of Cerebral Infarction:

[0302] The high and low dose administration groups of compound 14 and the high and low dose administration groups of control compound 1 can reduce the range of necrosis of brain tissue lesions, as shown in Table 2 below:

TABLE-US-00002 TABLE 2 Size of cerebral infarction and inhibition rate of cerebral infarction in experimental animals Inhibition rate Infarct area Whole brain Infarct size of cerebral Test compound Dosage weight (g) weight (g) (%) infarction (%) Model control — 0.17 ± 0.05 1.23 ± 0.07 14.08 ± 3.67 — Compound 14 2.5 mg/kg 0.15 ± 0.07 1.23 ± 0.08 12.11 ± 5.69 14.0 5 mg/kg 0.12 ± 0.05  1.2 ± 0.08  9.95 ± 4.69 29.3 Comparative 2.5 mg/kg 0.14 ± 0.07 1.24 ± 0.07  11.4 ± 5.64 19.0 Compound 1 5 mg/kg 0.11 ± 0.05  1.2 ± 0.12  9.35 ± 3.54* 33.6 Positive drug 5 mg/kg 0.18 ± 0.09 1.23 ± 0.08 13.93 ± 6.97 1.1 Note: “—” means no data for this item, *means P < 0.05 compared with model control animals.

Test Example 4 for Biological Activity. Effect of Compounds of the Present Disclosure on Human hERG Ion Channel Expressed in HEK293 Cells at Steady State Determined by Patch Clamp

[0303] The hERG ion channel steady-state expressing HEK293 cells were transferred to a perfusion tank and perfused with extracellular fluid at room temperature. Each cell was used as a control. All test compounds were dissolved by shaking with dimethyl sulfoxide and prepared as serial concentrations of 0.3 μM, 1 μM, 3 μM, 10 μM and 30 μM. The test compounds were perfused using a perfusion system utilizing their own gravity. At least two cells were tested for each concentration. After the current was stable (or 5 min), the current magnitude changes before and after the compound was used were compared to calculate the blocking effect of the compound. Test electrodes were pulled with PC-10. Whole-cell patch clamp was used to record, with noise filtered at one-fifth of the sampling frequency. Cells were clamped at −80 mV, depolarized to +60 mV for 850 ms with a square wave lasting 4 s, and then repolarized to −50 mV for 1275 ms to elicit hERG tail currents. This procedure was repeated every 15 seconds. hERG tail currents were pure hERG currents. After the current was stabilized, the administration was performed by continuous extracellular perfusion from low concentration to high concentration. Starting from a low concentration, perfusion was continued until the drug effect was stable, and then perfusion of the next concentration was performed.

[0304] Stimulus delivery and signal acquisition were performed by the Patch Master software.

[0305] The signal was amplified with a patch clamp amplifier with a filtering of 10 KHz. Fit Master, EXCEL, Graph Pad Prism and SPSS 21.0 were used for further data analysis and curve fitting. In data processing, when judging the blocking effect on hERG the peak value of the tail current and its baseline were corrected. The effect of each compound at different concentrations was expressed by the inhibition rate of the tail current.

Inhibition rate %=100×(peak value of tail current before administration-peak value of tail current after administration)/peak value of tail current before administration

[0306] SD<15 of the inhibition rate of all cells at each concentration was taken as the acceptable standard.

[0307] IC.sub.50 values were fitted by the Hill equation:

[00002]$y=\left[\frac{\max -\min }{1+{\left(\frac{\left[\mathrm{drug}\right]}{{\mathrm{IC}}_{50}}\right)}^{n_H}}\right]+\min$

TABLE-US-00003 Test Results Compound Structural formula hERG IC.sub.50 (μM) Terfenadine [00054] 0.043 Comparative Compound 1 [00055] 0.34 Comparative Compound 2 [00056] 0.71 Compound 14 of the present disclosure [00057] >30
Note: The comparative compound 1 is compound 38 of CN108484527A, and the comparative compound 2 is compound 59 of CN111574474A. Terfenadine has been withdrawn from the FDA because of cardiotoxic side effects such as delayed cardiac repolarization due to its severe influence on myocardial ion channels.

[0308] Cardiovascular and cerebrovascular diseases are chronic diseases, which usually require lifelong management and long-term medication. Therefore, in addition to the exact curative effect, the safety of the therapeutic drug must reach a very high level to effectively ensure the safety of patients' long-term medication.

[0309] The main causes of drug-induced cardiotoxicity include blocking the rapid delayed rectifier current (IKr) of the heart, resulting in prolongation of the QT interval in the cardiac action potential time course, and then inducing torsade de pointes (TdP), which can cause sudden death in severe cases.

[0310] In the field of drug development, compounds with significantly lower hERG IC.sub.50 values imply a higher potential risk of cardiotoxicity, and compounds with sufficiently high hERG IC.sub.50 values are considered adequate cardiac safety.

[0311] The above results show that the compounds of the present disclosure exemplified by compound 14 have no significant effect on the human hERG ion channel expressed in HEK293 cells in a steady state, and have good cardiac safety. The cardiac safety data characterized by the hERG inhibitory concentration IC50 value shows that, compared with other structures, the compounds of the present disclosure exemplified by compound 14 have significantly improved cardiac safety by, for example, about 100 times compared with the cardiac safety (IC.sub.50 value for hERG assay) of comparative compound 1, or about 50 times compared with the cardiac safety of compound 59 of CN111574474A.

[0312] In conclusion, the present disclosure has synthesized a new phenothiazine compound, which has a good inhibitory effect on ferroptosis and can be used for the preparation of ferroptosis inhibitor and also a medicament for treating cardiovascular and cerebrovascular diseases such as cerebral stroke, and ferroptosis-related diseases such as neurodegenerative disease, tumor, tissue ischemia-reperfusion injury, renal failure, and diabetic complications. Meanwhile, the compounds of the present disclosure exemplified by compound 14 have very good druggability, such as better cardiac safety, solubility, and no hemolysis reaction, and can be prepared as injections with broad market prospects.