HPK1 INHIBITOR, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

The present invention relates to a compound of general formula (1) and a preparation method therefor and use of the compound of general formula (1) or isomers, crystalline forms, pharmaceutically acceptable salts, hydrates or solvates thereof as an HPK1 inhibitor. The compound of the present invention can be used for preparing a medicament for treating or preventing related diseases mediated by HPK1.

##STR00001##

Claims

1. A compound with a structure as shown as general formula (1), or isomers, crystalline forms, pharmaceutically acceptable salts, hydrates or solvates thereof: ##STR00203## in formula (1): “*” denotes a chiral center; n is an integer of 0, 1, 2 or 3; X is H, halogen, C.sub.1-3 alkyl, C.sub.1-3 haloalkyl or C.sub.3-6 cycloalkyl; Y is —O—, —NH— or —N(C.sub.1-3 alkyl)-; when each R is linked to a different carbon atom, each R is independently H or C.sub.1-3 alkyl; when two R are simultaneously linked to the same carbon atom, the two R may independently be H or C.sub.1-3 alkyl or form a carbon group (C═O) with a carbon atom linked thereto; R.sup.1 is H, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, heterocycloalkyl, hydroxy-substituted C.sub.1-6 alkyl, halogen-substituted C.sub.1-6 alkyl, C.sub.1-6 alkoxy-substituted C.sub.1-6 alkyl or cyano-substituted C.sub.1-6 alkyl; A is the following group: ##STR00204## wherein “**” denotes a position linking to a group B, X.sub.1 is N, CH, CF, C—Cl, C-Me or C—CN, X.sub.2 is N, CH, CF, C—Cl, C-Me or C—CN, X.sub.3 is N or CH, Y.sub.1 is N or CH, and Y.sub.2 is N, CH, C-Me or C—CN; B is C.sub.6-10 aryl or 5-10 membered heteroaryl, wherein the 5-10 membered heteroaryl consists of at least 1 carbon atom and 1-4 heteroatoms selected from the group consisting of N, O and S; wherein the N or S atom may be oxidized; wherein ring carbon atoms of the 5-10 membered heteroaryl may be optionally substituted with oxygen to form a carbonyl group (C═O); and the C.sub.6-10 aryl and 5-10 membered heteroaryl are optionally substituted with 1-5 substituents independently selected from R.sup.2; R.sup.2 is independently halogen, CN, NO.sub.2, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, cyano-substituted C.sub.1-6 alkyl, hydroxy-substituted C.sub.1-6 alkyl, C.sub.1-3 alkoxy-substituted C.sub.1-6 alkyl, C.sub.1-3 haloalkoxy-substituted C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, 4-7 membered heterocycloalkyl, 5-7 membered heteroaryl, OR.sup.3, SR.sup.3, C(O)R.sup.3, S(O)R.sup.3, S(O).sub.2R.sup.3, C(O)OR.sup.3, OC(O)R.sup.3, NR.sup.4R.sup.5, P(O)R.sup.4R.sup.5, C(O)NR.sup.4R.sup.5, OC(O)NR.sup.4R.sup.5, —C.sub.1-6 alkyl-NR.sup.4R.sup.5, —O—C.sub.1-6 alkyl-NR.sup.4R.sup.5, —C.sub.1-6 alkyl-OR.sup.3, —O—C.sub.1-6 alkyl-R.sup.3, -hydroxy-substituted C.sub.1-6 alkyl-R.sup.3, —NR.sup.6C(O)R.sup.3, —NR.sup.6S(O).sub.2R.sup.3, —NR.sup.6CO—C.sub.1-3 alkyl-R.sup.3 or —NR.sup.6CO—C.sub.1-3 alkyl-NR.sup.4R.sup.5; or two adjacent R.sup.2 substituents on ring B, together with atoms linked thereto, form a fused 4-7 membered heterocycloalkyl or C.sub.3-7 cycloalkyl, wherein the fused 4-7 membered heterocycloalkyl contains at least 1 carbon atom and 1-4 heteroatoms selected from the group consisting of N, O and S; wherein the N or S atom may be oxidized; wherein ring carbon atoms of the fused 4-7 membered heterocycloalkyl may be optionally substituted with oxygen to form a carbonyl group (C═O); and the fused 4-7 membered heterocycloalkyl or the C.sub.3-7 cycloalkyl is optionally substituted with 1-5 substituents independently selected from R.sup.7; each R.sup.3 is independently H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, C.sub.3-6 cycloalkyl, 4-7 membered heterocycloalkyl, C.sub.6-10 aryl, 5-7 membered heteroaryl, cyano-substituted C.sub.1-6 alkyl, hydroxy-substituted C.sub.1-6 alkyl, C.sub.1-3 alkoxy-substituted C.sub.1-6 alkyl or C.sub.1-3 haloalkoxy-substituted C.sub.1-6 alkyl; each of R.sup.4 and R.sup.5 is independently H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, C.sub.3-6 cycloalkyl, cyano-substituted C.sub.1-6 alkyl, hydroxy-substituted C.sub.1-6 alkyl, C.sub.1-3 alkoxy-substituted C.sub.1-6 alkyl or C.sub.1-3 haloalkoxy-substituted C.sub.1-6 alkyl, C.sub.6-10 aryl-substituted C.sub.1-6 alkyl or 5-10 membered heteroaryl-substituted C.sub.1-6 alkyl, or R.sup.4 and R.sup.5, together with an N atom, form 4-12 membered heterocycloalkyl, the 4-12 membered heterocycloalkyl optionally being substituted with 1-5 substituents independently selected from R.sup.7; each R.sup.6 is H or C.sub.1-3 alkyl; each R.sup.7 is OH, CN, NH.sub.2, NHMe, NMe.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl or C.sub.1-6 alkoxy.

2. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), X is H, F, Cl, Me, Et, CF.sub.3, isopropyl or cyclopropyl.

3. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), Y is —O—, —NH—, —N(Me)— or —N(Et)—.

4. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), R is H or Me.

5. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), R.sup.1 is H, Me, Et, ##STR00205##

6. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), A is the following group: ##STR00206## ##STR00207## and wherein “**” denotes a position linking to a group B.

7. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), B is ##STR00208## R.sup.2 is independently H, CH.sub.3, F, Cl, OCH.sub.3, CF.sub.3, CN, CONH.sub.2, ##STR00209## NHSO.sub.2CH.sub.3, NHCOCH.sub.3, PO(CH.sub.3).sub.2, SO.sub.2CH.sub.3, SO.sub.2NH.sub.2, SO.sub.2NHCH.sub.3, CH.sub.2NHCH.sub.3, CH.sub.2NHCH.sub.2CH.sub.3, ##STR00210## CH(CH.sub.3)NHCH.sub.3, CH.sub.2CN, CH.sub.2OCH.sub.3, ##STR00211## NHCOCH.sub.2N(CH.sub.3).sub.2, ##STR00212## OCON(CH.sub.3).sub.2, ##STR00213## ##STR00214## OCH.sub.2CH.sub.2CH.sub.2NH.sub.2, OCH.sub.2CH.sub.2CH.sub.2N(CH.sub.3).sub.2, ##STR00215## m is an integer of 0, 1, 2 or 3.

8. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), B is ##STR00216## ##STR00217## ##STR00218## ##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223## ##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229##

9. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in general formula (1), B is ##STR00230## ##STR00231## ##STR00232## ##STR00233## ##STR00234## ##STR00235##

10. The compound, or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein the compound has one of the following structures: ##STR00236## ##STR00237## ##STR00238## ##STR00239## ##STR00240## ##STR00241## ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## ##STR00254## ##STR00255## ##STR00256## ##STR00257## ##STR00258## ##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263## ##STR00264## ##STR00265## ##STR00266## ##STR00267##

11. A pharmaceutical composition, comprising a pharmaceutically acceptable excipient or carrier, and the compound or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1 as an active ingredient.

12. Use of the compound or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, in the preparation of a medicament for treating, regulating and/or preventing related diseases mediated by HPK1.

13. A method for treating an HPK1-mediated disease, comprising administering to a subject the compound or the isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1.

14. The method according to claim 13, wherein the disease is cancer, and the cancer is a hematological cancer or a solid tumor.

Description

DETAILED DESCRIPTION

Example 1 Synthesis of 5-(2-Fluorophenyl)-3-(3-Methyl-1,2,3,4,4a,5-Hexahydrobenzo[b]Pyrazino[1,2-d][1,4]Oxazin-8-Yl)-1H-Indazole-6-Carbonitrile

[0074] (Compound 1)

##STR00073##

[0075] Step 1: Synthesis of Compound 1-1

[0076] Tert-butyl 3-hydroxymethylpiperazine-l-carboxylate (1.0 g, 4.63 mmol) and 1,2-difluoro-4-nitrobenzene (809 mg, 5.1 mmol) were dissolved in DMF (20 mL), DIPEA (1.8 g, 13.89 mmol) was added, and the reaction solution was heated to 120° C. and reacted overnight; after the completion of the reaction as detected by LC-MS, the reaction solution was added into water (100 mL) and extracted with EA (50 mL×3), the organic phases were combined, washed with water (50 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated, and the resulting residue was purified by column chromatography (PE/EA=10/1 to 5/1) to obtain Compound 1-1 (1.0 g, yield 61%) in the form of a yellow solid, ESI-MS m/z: 356.1 [M+H]+.

[0077] Step 2: Synthesis of Compound 1-2

[0078] Compound 1-1 (3.6 g, 10 mmol) was dissolved in DMF (30 mL), NaH (440 mg, 11 mmol) was added under ice-bath cooling, and the reaction solution was heated to 80° C. and reacted overnight; after the completion of the reaction as detected by LC-MS, the reaction solution was cooled, poured into ice water (100 mL) and extracted with EA (50 mL×2), the organic phases were combined, washed with water (150 mL×2) and saturated brine (50 mL) and concentrated, and the resulting residue was purified by column chromatography (PE/EA=20/1 to 10/1) to obtain Compound 1-2 (2.1 g, yield 62%) in the form of a yellow solid, ESI-MS m/z: 336.1 [M+H]+.

[0079] Step 3: Synthesis of Compound 1-3

[0080] Compound 1-2 (2.0 g, 6.0 mmol) was dissolved in EA (40 mL), HCl/dioxane (4.0 M, 10 mL) was added, and the reaction solution was stirred at room temperature for 3 h; after the completion of the reaction as detected by LC-MS, the reaction solution was directly concentrated to obtain Compound 1-3 (2.0 g, yield 100%) in the form of a yellow solid, ESI-MS m/z: 236.1 [M+H]+. The obtained initial product was directly used in the next step without purification.

[0081] Step 4: Synthesis of Compound 1-4

[0082] The crude product 1-3 obtained from the previous step was dissolved in MeCN (20 mL), aqueous formaldehyde (35%-40%, 0.5 mL) and HOAc (360 mg, 6.0 mmol) were added, and the reaction solution was stirred at room temperature for 1 h, added with NaBH.sub.3CN (0.76 g, 12 mmol) and reacted at room temperature overnight; after the completion of the reaction as detected by LC-MS, the reaction solution was concentrated, and the resulting residue was dissolved in EA (50 mL), washed with aqueous NaHCO.sub.3 (50 mL), concentrated, and purified by column chromatography (DCM/MeOH=100/1 to 20/1) to obtain Compound 1-4 (1.0 g, yield 66%) in the form of a yellow solid, ESI-MS m/z: 250.1 [M+H]+.

[0083] Step 5: Synthesis of Compound 1-5

[0084] Compound 1-4 (1.0 g, 4.0 mmol) was dissolved in MeOH (50 mL), Pd/C (10%, 200 mg) was added, and the reaction solution was purged with hydrogen and reacted at room temperature overnight; after the completion of the reaction as detected by LC-MS, the reaction solution was filtered and concentrated to obtain Compound 1-5 (800 mg, yield 91%) in the form of a pale yellow solid, ESI-MS m/z: 220.1 [M+H].sup.+. The obtained initial product was directly used in the next step without purification.

[0085] Step 6: Synthesis of Compound 1-6

[0086] Compound 1-5 (440 mg, 2 mmol) was dissolved in MeOH (4 mL), aqueous hydrochloric acid (3.0 M, 2 mL) and NaNO.sub.2 (138 mg, 2 mmol) aqueous solution (1 mL) were added under an ice bath, and the reaction solution was stirred for 30 min under the ice bath, added with bis(pinacolato)diboron (1.52 g, 6 mmol) and stirred for 1 h; after the completion of the reaction as detected by LC-MS, the reaction solution was added with aqueous NaHCO.sub.3 to quench the reaction and extracted with DCM (50 mL×2), the organic phase was dried over anhydrous Na.sub.2SO.sub.4 and concentrated, and the resulting residue was purified by column chromatography (DCM/MeOH=50/1 to 20/1) to obtain Compound 1-6 (500 mg, yield 75%) in the form of a pale yellow gum, ESI-MS m/z: 331.1[M+H]+.

[0087] Step 7: Synthesis of Compound 1-8

[0088] Tert-butyl 5-chloro-6-cyano-1H-indazole-1-carboxylate (2.78 g, 10 mmol) and 2-fluorobenzeneboronic acid (1.40 g, 10 mmol) were dissolved in 1,4-dioxane/H.sub.2O (v/v=5/1, 30 mL), K.sub.3PO.sub.4 (4.24 g, 20 mmol) and Pd XPhos G3 (423 mg, 0.05 mmol) were added, and the reaction solution was heated to 90° C. in argon atmosphere for 5 h; after the completion of the reaction as detected by LC-MS, the reaction solution was cooled, diluted with EA (50 mL) and washed with water (50 mL×2), the organic phase was concentrated, and the resulting residue was purified by column chromatography (PE/EA=20/1 to 5/1) to obtain Compound 1-8 (0.83 g, yield 35%) in the form of a pale yellow solid, ESI-MS m/z: 238.1 [M+H]+.

[0089] Step 8: Synthesis of Compound 1-9

[0090] Compound 1-8 (474 mg, 2 mmol) was dissolved in DMF (10 mL), KOH (280 mg, 5 mmol) and 12 (760 mg, 3 mmol) were added, and the reaction solution was stirred at room temperature for 2 h; after the completion of the reaction as detected by LC-MS, the reaction solution was cooled to room temperature, added with 1 N hydrochloric acid to adjust pH to about 7, and then added with EA/H.sub.2O (50 mL/50 mL), followed by liquid separation, the aqueous phase was extracted with EA (50 mL×2), the organic phases were combined and concentrated under reduced pressure, and the resulting residue was purified by column chromatography (PE/EA=10/1 to 2/1) to obtain Compound 1-9 (425 mg, yield 56%) in the form of a pale yellow solid, ESI-MS m/z: 378.2 [M+H]+.

[0091] Step 9: Synthesis of Compound 1-10

[0092] Compound 1-9 (377 mg, 1.0 mmol) was dissolved in DCM (20 mL), TEA (202 mg, 2.0 mmol) was added, (Boc).sub.2O (262 mg, 1.2 mmol) was finally added, and the reaction solution was stirred at room temperature for 3 h; after the completion of the reaction as detected by LC-MS, the reaction solution was added with EA/H.sub.2O (50 mL/50 mL), followed by liquid separation, the organic phase was concentrated, and the resulting residue was purified by column chromatography (PE/EA=20/1 to 10/1) to obtain Compound 1-10 (402 mg, yield 87%) in the form of a pale yellow solid, ESI-MS m/z: 464.3 [M+H]+.

[0093] Step 10: Synthesis of Compound 1-11

[0094] Compound 1-10 (232 mg, 0.5 mmol) and Compound 1-6 (198 mg, 0.6 mmol) were dissolved in 1,4-dioxane/H.sub.2O (v/v=5/1, 10 mL), K.sub.3PO.sub.4 (212 mg, 1.0 mmol) and Pd(dppf).sub.2C.sub.1 (73 mg, 0.1 mmol) were added, and the reaction solution was heated at 90° C. in argon atmosphere for 5 h; after the completion of the reaction as detected by LC-MS, the reaction solution was cooled, diluted with EA (50 mL) and washed with water (20 mL×1), the organic phase was concentrated, and the resulting residue was purified by column chromatography (DCM/MeOH=50/1 to 20/1) to obtain Compound 1-11 (168 mg, yield 62%) in the form of a pale yellow solid, ESI-MS m/z: 540.2 [M+H]+.

[0095] Step 11: Synthesis of Compound 1

[0096] Compound 1-11 (162 mg, 0.3 mmol) was dissolved in HCl/dioxane (4.0 N, 2 mL), and the reaction solution was reacted at room temperature for 3 h; after the completion of the reaction as detected by LC-MS, the reaction solution was concentrated directly, the resulting residue was dissolved in DCM (50 mL), added with aqueous NaHCO.sub.3 to give a basic pH, followed by layer separation, the organic phase was dried over anhydrous Na.sub.2SO.sub.4 and concentrated, and the resulting residue was purified by column chromatography (DCM/MeOH=50/1 to 20/1) to obtain Compound 1 (96 mg, yield 73%) in the form of a pale yellow solid.

[0097] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 8.36 (s, 1H), 8.21 (s, 1H), 7.78-7.70 (m, 2H), 7.52 (d, J=8.8 Hz, 1H), 7.42-7.32 (m, 2H), 7.23 (dd, J=10.0, 5.3 Hz, 1H), 6.98 (d, J=8.7 Hz, 1H), 4.29-4.16 (m, 1H), 3.91 (t, J=9.7 Hz, 1H), 3.66-3.72 (m, 1H), 3.09 (s, 1H), 2.85 (d, J=10.3 Hz, 1H), 2.79 (d, J=10.9 Hz, 1H), 2.71-2.62 (m, 1H), 2.19 (s, 3H), 2.13-2.00 (m, 1H), 1.72 (t, J=10.7 Hz, 1H), ESI-MS m/z: 440.2 [M+H]+.

[0098] Tert-butyl (S)-3-hydroxymethylpiperazine-l-carboxylate or tert-butyl (R)-3-hydroxymethylpiperazine-1-carboxylate was taken as a starting material, and the synthesis of Compound 1 can obtain two optical isomers 1-a and 1-b of Compound 1:

##STR00074##

Examples 2-129: Synthesis of Compounds 2-129

[0099] The target compounds 2-129 were obtained according to a similar synthesis method as in Example 1 using different starting materials.

TABLE-US-00001 TABLE 1 Compound Structure of compound [M + H].sup.+   2 [00075] 452.2  3 [00076] 436.2  4 [00077] 458.2  5 [00078] 454.2  6 [00079] 470.2  7 [00080] 458.2  8 [00081] 465.2  9 [00082] 483.2  10 [00083] 574.2  11 [00084] 533.2  12 [00085] 516.2  13 [00086] 518.2  14 [00087] 533.2  15 [00088] 472.2  16 [00089] 488.2  17 [00090] 497.2  18 [00091] 501.2  19 [00092] 551.2  20 [00093] 513.2  21 [00094] 515.2  22 [00095] 517.2  23 [00096] 541.2  24 [00097] 497.2  25 [00098] 502.2  26 [00099] 541.2  27 [00100] 558.2  28 [00101] 584.3  29 [00102] 612.3  30 [00103] 540.2  31 [00104] 592.2  32 [00105] 565.2  33 [00106] 571.3  34 [00107] 545.2  35 [00108] 584.3  36 [00109] 557.2  37 [00110] 495.2  38 [00111] 509.2  39 [00112] 526.2  40 [00113] 517.2  41 [00114] 423.2  42 [00115] 441.2  43 [00116] 437.2  44 [00117] 453.2  45 [00118] 437.2  46 [00119] 480.2  47 [00120] 426.2  48 [00121] 424.2  49 [00122] 424.2  50 [00123] 462.2  51 [00124] 477.2  52 [00125] 477.2  53 [00126] 475.2  54 [00127] 473.2  55 [00128] 462.2  56 [00129] 463.2  57 [00130] 463.2  58 [00131] 463.2  59 [00132] 462.2  60 [00133] 463.2  61 [00134] 463.2  62 [00135] 462.2  63 [00136] 463.2  64 [00137] 446.2  65 [00138] 430.2  66 [00139] 434.2  67 [00140] 434.2  68 [00141] 477.4  69 [00142] 473.3  70 [00143] 413.2  71 [00144] 429.2  72 [00145] 402.2  73 [00146] 464.2  74 [00147] 470.2  75 [00148] 464.2  76 [00149] 524.3  77 [00150] 438.2  78 [00151] 438.2  79 [00152] 447.1  80 [00153] 474.2  81 [00154] 478.2  82 [00155] 471.2  83 [00156] 498.2  84 [00157] 502.2  85 [00158] 446.2  86 [00159] 473.2  87 [00160] 477.2  88 [00161] 445.2  89 [00162] 472.2  90 [00163] 476.2  91 [00164] 469.2  92 [00165] 496.3  93 [00166] 500.2  94 [00167] 446.2  95 [00168] 473.2  96 [00169] 477.2  97 [00170] 470.2  98 [00171] 497.3  99 [00172] 501.2 100 [00173] 445.2 101 [00174] 472.3 102 [00175] 476.2 103 [00176] 523.3 104 [00177] 496.3 105 [00178] 525.3 106 [00179] 522.3 107 [00180] 537.3 108 [00181] 480.3 109 [00182] 496.3 110 [00183] 498.3 111 [00184] 496.3 112 [00185] 524.3 113 [00186] 497.2 114 [00187] 526.3 115 [00188] 524.3 116 [00189] 497.2 117 [00190] 526.3 118 [00191] 525.3 119 [00192] 498.2 120 [00193] 527.3 121 [00194] 496.3 122 [00195] 510.3 123 [00196] 483.2 124 [00197] 511.3 125 [00198] 523.3 126 [00199] 539.3 127 [00200] 580.3 128 [00201] 565.3 129 [00202] 536.3

Example 130: Assay of Inhibitory Effect of Compound Against Enzyme Activity of HPK1

[0100] The inhibitory effect of the compound against the enzyme activity of HPK1 was determined by using a lantha Screen method with PKC as a substrate. The process comprises the following specific steps: the HPK1 protein was incubated with the serially-diluted compound at 28° C. for 10 min followed by addition of fluoroescein-labeled PKC substrate and ATP to react for 90 min. After termination of the reaction, a Terbium-labeled antibody against phosphorylated PKC was added and incubated for 60 min. The energy transfer of Teribium and fluoroscein was monitored by detecting luminescence at 520 nm and 450 nm to quantify PKC as the level of phosphorylation of HPK1. Inhibition % and IC.sub.50 of the compound were calculated compared to the control group.

TABLE-US-00002 TABLE 2 Inhibitory effect of the compound disclosed herein against enzyme activity of HPK1 Com- Inhibition Com- Inhibition Com- Inhibition pound rate (%) pound rate (%) pound rate (%) 1 +++ 2 +++ 3 ++ 4 ++ 5 ++ 6 +++ 7 + 8 +++ 9 +++ 10 +++ 11 ++ 12 +++ 13 + 14 +++ 15 +++ 16 +++ 17 +++ 18 +++ 19 +++ 20 +++ 21 +++ 22 +++ 23 +++ 24 +++ 25 +++ 26 +++ 27 +++ 28 +++ 29 +++ 30 +++ 31 ++ 32 +++ 33 +++ 34 ++ 35 +++ 36 +++ 37 +++ 38 +++ 39 +++ 40 ++ 41 +++ 42 +++ 43 +++ 44 +++ 45 +++ 46 +++ 47 +++ 48 +++ 49 +++ 50 +++ 51 +++ 52 +++ 53 +++ 54 ++ 55 +++ 56 +++ 57 +++ 58 +++ 59 +++ 60 +++ 61 +++ 62 ++ 63 +++ 64 +++ 65 ++ 66 ++ 67 ++ 68 +++ 69 +++ 70 +++ 71 +++ 72 +++ 73 ++ 74 ++ 75 ++ 76 ++ 77 +++ 78 +++ 79 ++ 80 ++ 81 ++ 82 ++ 83 +++ 84 +++ 85 ++ 86 ++ 87 ++ 88 +++ 89 +++ 90 +++ 91 +++ 92 +++ 93 +++ 94 ++ 95 +++ 96 ++ 97 ++ 98 +++ 99 +++ 100 +++ 101 +++ 102 +++ 103 +++ 104 ++ 105 +++ 106 +++ 107 +++ 108 ++ 109 +++ 110 +++ 111 +++ 112 +++ 113 +++ 114 +++ 115 +++ 116 ++ 117 +++ 118 +++ 119 ++ 120 +++ 121 +++ 122 +++ 123 +++ 124 +++ 125 +++ 126 +++ 127 +++ 128 +++ 129 +++ 1-a +++ 1-b +++ + means that IC.sub.50 is greater than or equal to 100 nM. ++ means that IC.sub.50 is greater than 10 nM but less than 100 nM. +++ means that IC.sub.50 is less than or equal to 10 nM.

[0101] As can be seen from data in Table 2, the compound disclosed herein has an inhibitory effect against the enzyme activity of HPK1 with an IC.sub.50 value less than or equal to 10 nM, and thus has a strong inhibitory effect against enzyme activity of HPK1.

Example 131: Assay of Inhibitory Effect of Compound Against hERG Channel Activity

[0102] The HEK293 cell line stably expressed by the hERG potassium channel was adopted for detection using manual patch clamp. Firstly, the HEK293 cell line stably expressed by the hERG potassium channel was separated and laid on a cover glass before the patch clamp detection test, and the test detection was carried out after culture for 18 h. Then a capillary glass tube was drawn into a recording electrode, and the recording electrode was contacted onto the cells under an inverted microscope to form a whole-cell recording mode. After necessary compensation and recording of the membrane capacitance and series resistance, current recording was performed according to the set current stimulation protocol. After the hERG current stabilized, administration was started and the current detected in compound-free extracellular fluid for each cell served as its own control group, and multiple cells were repeatedly tested independently. The current acted for each drug concentration and the current of the blank control were standardized, then the inhibition rate corresponding to each drug concentration was calculated, and finally the half-inhibition concentration IC.sub.50 value of each compound was calculated.

TABLE-US-00003 TABLE 3 Inhibitory effect of the compound disclosed herein against hERG channel activity (IC.sub.50) Compound hERG channel inhibitory activity (IC.sub.50, μM) 6 17.8 16 23.6 18 11.8 35 31.8 43 17.4 67 51.2 73 41.2 112 35.6

[0103] As can be seen from data in Table 2, the compound disclosed herein has a weak inhibitory effect against hERG channel, with an inhibition activity IC.sub.50 value greater than 10 μM, which indicates that the compound has better safety in the aspect of heart safety.