Pkn3 kinase inhibitors, pharmaceutical compositions, and methods of making and using pkn3 kinase inhibitors

Abstract

The application discloses a PKN3 kinase inhibitor, a pharmaceutical composition and a preparation method and application of the PKN3 kinase inhibitor. The PKN3 kinase inhibitor RI of the application further prevents the RAF family protein from forming a stable compound with CDC37-HSP90 by inhibiting the activation of CDC37, reduces the stability and activation level of the RAF protein, realizes the down-regulation of the MAPK signal path, and inhibits the proliferation and survival of tumor cells. Meanwhile, the PKN3 kinase inhibitor RI is combined with a MEK inhibitor, the treatment effect is excellent, and the drug resistance problem caused by single-path inhibition can be overcome. The PKN3 kinase inhibitor RI of the application is a novel molecular targeted drug which can target the RAF regulation mechanism and is suitable for BRAF wild-type melanoma, and fills the technical blank of long-term lack of effective treatment drugs and means for the BRAF wild-type melanoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor targeted therapy and molecular target drug development, and particularly relates to a PKN3 kinase (Protein Kinase N3) inhibitor, a pharmaceutical composition, and a preparation method and application of the PKN3 kinase inhibitor. BACKGROUND

[0002] Melanoma is a highly malignant skin tumor with high invasiveness and rapid metastasis, and has limited response to traditional chemotherapy and radiotherapy. At present, the main strategies for treating melanoma in clinic include immune checkpoint inhibitor therapy, BRAF / MEK (B-Raf proto-oncogene, serine / threonine kinase, Mitogen-Activated Protein Kinase Kinase) targeted therapy, and combination therapy, etc. In the aspect of molecular targeted therapy, a variety of small molecule inhibitors have been developed for treating BRAFV600E mutant melanoma, such as Dabrafenib, Vemurafenib and Trametinib, etc. These drugs effectively delay tumor progression and improve patient prognosis by inhibiting BRAF kinase or its downstream MEK kinase. However, about 40% to 50% of melanoma patients are BRAF wild type (WT), and lack BRAFV600E mutation in vivo, so they are not suitable for the existing BRAF targeted therapy. This part of patients has no obvious effect on the above drugs, and still lack effective targeted treatment means, and the clinical treatment demand has not been met for a long time.

[0003] Current research on MAPK (Mitogen-Activated Protein Kinase) pathway mainly focuses on direct kinase targets in the RAS-BRAF-MEK-ERK cascade, but the mechanism of RAF (Rapidly Accelerated Fibrosarcoma) protein maturation, stabilization and activation is still not well understood. Recent research has found that the HSP90-CDC37 (Heat Shock Protein 90-Cell Division Cycle 37) chaperone complex plays a key role in the folding and activation of RAF protein. CDC37 (Cell Division Cycle 37) can enhance its ability to bind and stabilize RAF protein through specific site phosphorylation. However, the upstream kinase that regulates CDC37 activity and its drug accessibility are still unknown.

[0004] In summary, the existing related technologies have the following main problems:

[0005] 1. Existing BRAF inhibitors are ineffective for BRAF wild-type melanoma, and treatment options are limited;

[0006] 2. There is a lack of targeted drugs that act on the RAF maturation process, and no drugs have been developed to target the stabilization mechanism of RAF protein;

[0007] 3. No upstream regulatory factors have been found that can be targeted by drugs, such as protein kinases that regulate CDC37 phosphorylation;

[0008] 4. Some broad-spectrum kinase inhibitors have the problem of non-specificity of target sites and large side effects, which affect their clinical application;

[0009] 5. Direct inhibitors of HSP90 / CDC37 may induce heat shock response and accompanying toxicity, and do not have ideal selectivity and safety;

[0010] 6. There is no marketed drug or published literature reporting PKN3 (Protein Kinase N3) kinase as a target for melanoma treatment. SUMMARY

[0011] In view of the fact that there is no effective molecular targeted therapy for BRAF wild-type melanoma in the prior art, especially in the aspect of RAF protein activation and MAPK signal pathway regulation, the related mechanism has not been fully explored, and there is a lack of upstream regulatory target for drug intervention, the present application provides a PKN3 kinase inhibitor, a pharmaceutical composition, and a preparation method and application of the PKN3 kinase inhibitor. The present application develops a novel molecular targeted drug which can target the RAF regulation mechanism and is suitable for BRAF wild-type melanoma, and can fill the technical blank of long-term lack of effective therapeutic drugs for BRAF wild-type melanoma.

[0012] The technical scheme adopted by the present application is as follows:

[0013] I. A PKN3 kinase inhibitor

[0014] The structural formula of the PKN3 kinase inhibitor RI is specifically as follows:

[0015] .

[0016] II. A preparation method of the PKN3 kinase inhibitor as described above

[0017] The preparation method comprises the following steps:

[0018] S1, 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, (4-chloro-3-hydroxyphenyl)boronic acid and a palladium catalyst are added to a solvent containing potassium carbonate in a molar ratio of 1.5-2:2-3:0.1-0.2 to obtain a reaction mixture; the solvent is a mixed solution of 1,4-dioxane and water;

[0019] S2, the reaction mixture is stirred at 80-96 DEG C under a protective atmosphere for 13-20 hours;

[0020] S3, after the reaction is completed, the reaction mixture is diluted with ethyl acetate, and then washed, dried, filtered and concentrated to obtain a crude product;

[0021] S4, the crude product is purified by using a silica gel column chromatography to obtain the PKN3 kinase inhibitor RI;

[0022] In the purification process, a mixed solution of ethyl acetate and n-hexane is used as an eluent, and the volume ratio of ethyl acetate to n-hexane is 1-2:2-3.

[0023] III. Application of the PKN3 kinase inhibitor as described above or the PKN3 kinase inhibitor obtained by the preparation method as described above

[0024] The application is specifically: the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate for screening or preparing a drug for treating tumors.

[0025] Further, the application is: the combination of the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate and the MEK inhibitor for preparing a drug for treating tumors.

[0026] Preferably, the tumor includes adrenal cortex cancer, mesothelioma and uveal melanoma.

[0027] More preferably, the tumor includes BRAF V600E mutant melanoma and BRAF wild-type melanoma.

[0028] Most preferably, the tumor is BRAF wild-type melanoma.

[0029] Preferably, the administration dose of the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate is selected from 10-50 mg / kg per day.

[0030] Four, a drug for treating tumors

[0031] The main active ingredient of the drug is the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate, and the PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI as described above or the PKN3 kinase inhibitor RI obtained by the preparation method as described above.

[0032] Five, a pharmaceutical composition for treating tumors

[0033] The main active ingredient of the drug composition includes the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate, and the MEK inhibitor. The PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI as described above or the PKN3 kinase inhibitor RI obtained by the preparation method as described above.

[0034] The beneficial effects of the present application are as follows:

[0035] 1. The present application proposes a new mechanism that has not been disclosed in the literature or existing drugs: PKN3 kinase is a key upstream regulator of the RAF-MAPK pathway, which promotes RAF maturation and activation by phosphorylating CDC37 (Ser13). This mechanism is different from the classic site of existing BRAF or MEK inhibitors, and has complementary and alternative value.

[0036] 2、The application provides a novel PKN3 kinase small molecule inhibitor RI, which inhibits the activation of CDC37, further prevents the RAF family protein from forming a stable complex with CDC37-HSP90, reduces the stability and activation level of the RAF protein, and thus realizes down-regulation of the MAPK signal pathway and inhibition of the proliferation and survival of tumor cells.

[0037] 3、The PKN3 kinase inhibitor RI provided by the application has a remarkable tumor inhibition effect, is much better than the control drugs such as PP1, dabrafenib, vemurafenib and trametinib, and is suitable for a wide range of indications, especially for wild-type BRAF melanoma and other melanomas that cannot use BRAF V600E targeted inhibitors.

[0038] 4、The PKN3 kinase inhibitor RI provided by the application, when used in combination with a MEK inhibitor, not only has excellent treatment effect, but also can significantly overcome the drug resistance problem caused by single-pathway inhibition.

[0039] 5、The PKN3 kinase inhibitor RI provided by the application not only has the effects of excellent safety, low side effect and good drug property, but also has the industrialization advantages of mature compound synthesis process, easy industrial amplification and suitability for clinical development.

[0040] In summary, the application constructs a new treatment mechanism based on the PKN3-CDC37-RAF signal axis, provides a novel targeted treatment strategy with a clear mechanism, remarkable effect and specific target for BRAF wild-type melanoma, effectively makes up for the shortcomings of existing treatment drugs, and has important clinical application prospect and industrialization value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The synthesis reaction schematic diagram of the PKN3 kinase inhibitor RI provided by the application is shown in the figure;

[0042] Figure 2 The chemical structural formula of PP1 and RI is shown in the figure; wherein (a) is the chemical structural formula of PP1, and (b) is the chemical structural formula of RI;

[0043] Figure 3 The molecular docking sites of PP1 and RI combined with PKN3 kinase are shown in the figure; wherein (a) is the molecular docking site of PP1 combined with PKN3 kinase, and (b) is the molecular docking site of RI combined with PKN3 kinase;

[0044] Figure 4 The bioavailability comparison schematic diagram of PP1 and RI is shown in the figure;

[0045] Figure 5 The comparison chart of the changes of MAPK signal key proteins in melanoma cells before and after PP1 or RI treatment is shown in the figure;

[0046] Figure 6 Figure 1 is a graph showing melanoma cell colony formation before and after treatment with PP1 or RI;

[0047] Figure 7 Figure 2 is a picture of tumors in a cell-derived xenograft model (CDX);

[0048] Figure 8 Figure 3 is a graph showing tumor growth curves in a cell-derived xenograft model (CDX);

[0049] Figure 9 Figure 4 is a graph showing tumor growth curves in a spontaneous mouse model of melanoma after treatment with PP1 or RI

[0050] Figure 10 Figure 5 is a graph showing mouse survival in a spontaneous mouse model of melanoma after treatment with PP1 or RI;

[0051] Figure 11 Figure 6 is a graph showing tumor growth curves in a patient-derived xenograft model (PDX);

[0052] Figure 12 Figure 7 is a graph showing tumor weight in a patient-derived xenograft model (PDX). DETAILED DESCRIPTION

[0053] The present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0054] The present application provides a PKN3 kinase inhibitor in a first aspect. The PKN3 kinase inhibitor is an ATP-competitive inhibitor, and its mechanism of action is as follows: RI selectively binds to the PKN3 kinase pocket, blocks the phosphorylation of the Ser13 site of the CDC37 molecular chaperone, interferes with the recruitment of the CDC37 to the RAF family proteins (ARAF, BRAF, CRAF) by inhibiting the stabilization of the CDC37 and HSP90 complex, prevents the RAF family proteins from forming stable complexes with CDC37-HSP90, and thus causes the RAF proteins to be unstable, degraded, lose the downstream MEK / ERK activation function, and achieve the purpose of inhibiting the MAPK pathway, inhibiting the proliferation and survival of tumor cells.

[0055] The structural formula of the PKN3 kinase inhibitor RI is specifically as follows:

[0056] The present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0057] The first aspect of the present application provides a PKN3 kinase inhibitor. The PKN3 kinase inhibitor is an ATP competitive inhibitor, and its mechanism of action is as follows: RI selectively binds to the PKN3 kinase pocket, blocks the phosphorylation of the Ser13 site of the CDC37 molecular chaperone, interferes with the recruitment of the CDC37 to the RAF family proteins (ARAF, BRAF, CRAF) by inhibiting the stabilization of the CDC37 and HSP90 complex, prevents the RAF family proteins from forming a stable complex with the CDC37-HSP90, and further causes the RAF proteins to be unstable, degraded, lose the downstream MEK / ERK activation function, and achieve the purpose of inhibiting the MAPK pathway and inhibiting the proliferation and survival of tumor cells.

[0058] The structural formula of the PKN3 kinase inhibitor RI is specifically as follows:

[0059] .

[0060] The second aspect of the present application provides a preparation method of the PKN3 kinase inhibitor RI.

[0061] The preparation method specifically comprises the following steps:

[0062] S1, under a protective atmosphere, 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine, (4-chloro-3-hydroxyphenyl)boronic acid and a palladium catalyst are sequentially added into a solvent containing potassium carbonate to obtain a reaction mixture.

[0063] Preferably, the molar ratio of 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine, (4-chloro-3-hydroxyphenyl)boronic acid and the palladium catalyst is 1.5-2:2-3:0.1-0.2.

[0064] Preferably, the mass-volume ratio of potassium carbonate to the solvent is 1-1.5:18-25.

[0065] Preferably, the solvent is a mixed solution of 1,4-dioxane and water, and the volume ratio of 1,4-dioxane to water is preferably 13-20:4-8.

[0066] Preferably, the palladium catalyst is PdCl2(dppf)·CH2Cl2.

[0067] S2, the reaction mixture is stirred vigorously (600-800 rpm) at 80-96°C for 13-20 hours under a protective atmosphere until the reaction is completed.

[0068] S3, after the reaction is completed, the reaction mixture is diluted with ethyl acetate, and the diluted mixture is sequentially washed with water and saturated brine, and then sequentially dried, filtered and concentrated to obtain a crude product.

[0069] In this step, anhydrous sodium sulfate can be used for drying.

[0070] In this step, a rotary evaporator can be used for concentration.

[0071] S4, the crude product is purified by using a silica gel column chromatography to obtain the PKN3 kinase inhibitor RI.

[0072] Preferably, a mixture of ethyl acetate and n-hexane is used as an eluent in the purification process.

[0073] Preferably, the volume ratio of ethyl acetate to n-hexane is 1-2:2-3.

[0074] Optionally, in the above preparation process, the protective atmosphere can be any one or a combination of two or more of nitrogen, helium, neon, argon, krypton and radon.

[0075] The third aspect of the present application provides an application of the PKN3 kinase inhibitor RI.

[0076] The PKN3 kinase inhibitor RI or its pharmaceutically acceptable salt or hydrate can be used for screening or preparing a drug for treating tumors.

[0077] Further, the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate can also be combined with a MEK inhibitor for screening or preparing a drug for treating tumors.

[0078] The PKN3 kinase inhibitor RI is metabolically stable in vivo, has good oral bioavailability, and has a plasma half-life of 4-6 hours.

[0079] Preferably, the tumors include adrenocortical carcinoma, mesothelioma and uveal melanoma.

[0080] More preferably, the tumors include BRAF V600E mutant melanoma and BRAF wild-type melanoma.

[0081] Most preferably, the tumors are BRAF wild-type melanoma.

[0082] Preferably, the administration dose of the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate is selected from 10-50 mg / kg per day.

[0083] Most preferably, the administration dose of the PKN3 kinase inhibitor RI or its pharmaceutically acceptable salt or hydrate is 25 mg / kg per day.

[0084] Specifically, the application principle of the PKN3 kinase inhibitor RI is as follows:

[0085] The PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate can inhibit the MAPK pathway and inhibit the proliferation of tumor cells by selectively binding to the PKN3 kinase pocket, blocking the phosphorylation of the Ser13 site of the CDC37 molecular chaperone by the PKN3 kinase, inhibiting the stabilization of the CDC37 and HSP90 complex, interfering with the recruitment of the RAF family proteins (ARAF, BRAF, CRAF) by CDC37, inducing the instability and degradation of the RAF proteins, and finally losing the downstream MEK / ERK activation function.

[0086] The fourth aspect of the present application provides a medicine for treating tumors. The main active ingredient of the medicine is the PKN3 kinase inhibitor RI or its pharmaceutically acceptable salt or hydrate.

[0087] The fifth aspect of the present application provides a pharmaceutical composition for treating tumors. The main active ingredients of the pharmaceutical composition include the PKN3 kinase inhibitor RI or its pharmaceutically acceptable salt or hydrate and the MEK inhibitor.

[0088] Preferably, the MEK inhibitor is trametinib.

[0089] Optionally, the medicine and the pharmaceutical composition can be prepared into tablets, capsules, injections or sustained-release dosage forms as needed.

[0090] Optionally, the administration route of the medicine and the pharmaceutical composition can be oral, intraperitoneal injection or intravenous injection.

[0091] Further, the medicine and the pharmaceutical composition both further include formulation adjuvants.

[0092] Preferably, the formulation adjuvants include one or more of a combination of lactose, starch, talc, hydroxypropyl methyl cellulose and polyvinyl pyrrolidone and the like pharmaceutically acceptable carriers.

[0093] Preferably, for the above-mentioned medicine and the pharmaceutical composition, the tumors include adrenocortical carcinoma, mesothelioma and uveal melanoma.

[0094] More preferably, for the above-mentioned medicine and the pharmaceutical composition, the tumors include BRAF V600E mutant melanoma and BRAF wild-type melanoma.

[0095] Most preferably, for the above-mentioned medicine and the pharmaceutical composition, the tumors are BRAF wild-type melanoma.

[0096] The specific embodiments of the present application are as follows:

[0097] Embodiment 1

[0098] This embodiment is prepared according to the method as Figure 1The synthetic reaction formula shown, the PKN3 kinase inhibitor RI was prepared by the following process:

[0099] S1, under argon atmosphere, the following raw materials were added into the reaction bottle in turn:

[0100] 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (CAS No. 862728-61-8): 500 mg, 1.852 mmol;

[0101] (4-chloro-3-hydroxyphenyl)boronic acid (CAS No. 915201-06-8): 399.0 mg, 2.315 mmol;

[0102] PdCl2(dppf)·CH2Cl2(CAS No. 95464-05-4): 136.4 mg, 0.167 mmol;

[0103] Potassium carbonate K2CO3: 1.279 g, 9.26 mmol;

[0104] Solvent: 1,4-dioxane (16.7 mL) and water (5.9 mL).

[0105] S2, the reaction system was stirred vigorously at 90°C for 16 hours.

[0106] S3, after the reaction was completed, 200 mL of ethyl acetate was added for extraction, and then the organic phase was washed with 100 mL of water and 50 mL of saturated brine in turn. After drying, filtering and rotary evaporation, the crude product was obtained.

[0107] S4, the crude product was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane, volume ratio 1-2:2-3), to obtain a light yellow solid RI (513.6 mg, yield 87.3%).

[0108] The structure of the PKN3 kinase inhibitor RI obtained in this example is characterized as follows:

[0109] 1 H NMR (600 MHz, CDCl3) δ: 8.36 (s, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.21 (dd, J = 8.2, 1.9 Hz, 1H), 5.43 (s, 2H), 1.82 (s, 9H).

[0110] 13C NMR (150 MHz, CDCl3) δ: 157.48, 154.36, 152.25, 140.80, 134.12, 130.06, 121.32, 120.87, 116.33, 99.61, 60.70, 29.18.

[0111] HRMS (ESI): [M+H]+, C 15 H 17 ClN5O + The theoretical value is 318.1116, and the measured value is 318.1112.

[0112] The PKN3 kinase inhibitor RI prepared in this embodiment was mainly designed based on PP1 (synonym: AGL 1872; EI 275; CAS number 172889-26-8). The chemical structural formulas of PP1 and RI are as follows: Figure 2 As shown in (a) and (b), the molecular docking sites for PP1, RI, and PKN3 kinase are respectively as follows: Figure 3 As shown in (a) and (b).

[0113] Depend on Figure 3 It is evident that by introducing chlorine atoms and hydroxyl structures, the hydrogen bonding and hydrophobic interactions between RI and amino acid residues (such as K588) in the PKN3 kinase pocket are enhanced, while the selectivity and affinity are also significantly improved.

[0114] In addition, by Figure 4 It is evident that the bioavailability of RI was significantly improved compared to PP1 (P = 0.0061).

[0115] Example 2

[0116] This embodiment measures the inhibitory effect of the PKN3 kinase inhibitor RI on PKN3 kinase.

[0117] This embodiment uses ADP-Glo TM The Promega kinase assay kit, using the CDC37 peptide as a substrate, was used to evaluate the inhibitory effect of RI on PKN3 kinase in vitro. The results showed that the IC50 of RI was [missing value]. 50 The half-maximal inhibitory concentration (HMIC) was 0.3407 nM; and it had no significant inhibitory effect on PKN1 and PKN2.

[0118] The embodiment also measures the binding constant KD of RI and PKN3 kinase by Surface Plasmon Resonance (SPR) experiment, and the binding affinity is far better than that of the control drug PP1 (KD≈200 nM).

[0119] The Thermal Shift Assay (TSA) in the embodiment shows that the Tm value of PKN3 kinase is increased by about 6.3°C after binding with RI, that is, the binding of RI enhances the thermal stability of PKN3 kinase, thereby further verifying the direct targeting binding ability of RI.

[0120] Embodiment 3

[0121] The embodiment shows the anti-proliferation activity of RI in melanoma cells.

[0122] In the embodiment, CCK-8 detection, clone formation experiment and Western Blot detection are performed on BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903).

[0123] The CCK-8 detection results show that after RI treatment, the activities of BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903) are significantly decreased, and the IC 50 The IC50 values of MM1, MEWO, A375 and UACC903 are 23.17 nM, 57.39 nM, 35.41 nM and 11.46 nM, respectively.

[0124] The Western Blot results are shown in Figure 5 . Among them, DMSO represents the blank control group, RI represents after RI treatment, and PP1 represents after PP1 treatment. It can be seen that in BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903), CDC37 S13 phosphorylation is inhibited, and the RAF / MEK / ERK phosphorylation level is down-regulated, verifying that it is a non-traditional MAPK kinase direct inhibitor.

[0125] The clone formation experiment results are shown in Figure 6 . It can be seen that when the RI treatment concentration is 50 nM, the clone number of the four kinds of melanoma cells is decreased by more than 90%, and compared with the PP1 treatment group, the cell proliferation is significantly inhibited.

[0126] This embodiment shows that in BRAF wild-type melanoma cells, RI treatment can significantly reduce the phosphorylation level of CDC37, inhibit the expression of RAF protein, and further inhibit cell proliferation, with no significant difference from BRAF mutant cells, and a wider range of applications.

[0127] Example 4

[0128] This embodiment demonstrates the in vivo tumor inhibition effect of RI in a mouse subcutaneous tumor model.

[0129] In this embodiment, MM1 cells (5x10 6 ) were first inoculated subcutaneously on the back of nude mice, and after successful modeling, the mice were randomly divided into groups (n=6):

[0130] ① Solvent control group (corn oil);

[0131] ② RI treatment group (25 mg / kg);

[0132] Once a day for 14 consecutive days.

[0133] The results are shown in Figure 7 and Figure 8 It can be seen that in the ② RI treatment group, the tumor volume inhibition rate reached 80%, which was significantly higher than that of the ① solvent control group.

[0134] In addition, in the mice of the ② RI treatment group, there was no obvious weight loss, no histotoxicity in HE staining of major organs, and p-ERK / p-MEK in tumor tissue was also significantly down-regulated.

[0135] Example 5

[0136] This embodiment demonstrates the in vivo tumor inhibition effect of RI in a spontaneous mouse model of melanoma.

[0137] This embodiment uses a spontaneous mouse model of melanoma: GEMM model (Tyr-CreERT2 / Braf CA / Pten fl / fl ), and randomly divided into groups:

[0138] ① Solvent control group (corn oil);

[0139] ② RI low-dose group (10 mg / kg);

[0140] ③ RI high-dose group (25 mg / kg);

[0141] ④ PP1 treatment group (25 mg / kg).

[0142] The results are shown in Figure 9 and Figure 10As shown, compared with the ① solvent control group and the ④ PP1 treatment group, the RI treatment group (the ② RI low-dose group and the ③ RI high-dose group) significantly delayed the tumor formation time, reduced the tumor volume, and improved the survival rate. At the same time, during the treatment, no weight loss, major organ lesions or severe toxicity were observed in the RI treatment group (the ② RI low-dose group and the ③ RI high-dose group), indicating that RI has good safety. In addition, the RI treatment group (the ② RI low-dose group and the ③ RI high-dose group) also observed that p-ERK / p-MEK in tumor tissue was significantly down-regulated, and Ki67 positive cells were significantly reduced.

[0143] Example 6

[0144] This example demonstrates the synergistic anti-tumor effect of the combination of RI and the MEK inhibitor Trametinib.

[0145] In this example, a BRAF wild-type melanoma PDX model (patient-derived xenograft model) was first established, and after successful modeling, the mice were randomly divided into groups:

[0146] ① solvent control group;

[0147] ② Dabrafenib group (Dabrafenib, 25 mg / kg);

[0148] ③ RI treatment group 25 mg / kg;

[0149] ④ Trametinib group (Trametinib, 1 mg / kg);

[0150] ⑤ Dabrafenib + Trametinib group;

[0151] ⑥ RI + Trametinib group.

[0152] The treatment cycle was 21 days, and the evaluation indicators included tumor volume, survival time, and signal pathway protein expression, and the results are shown in Figure 11 and Figure 12 .

[0153] The results showed that the tumor volume inhibition rate of the ⑥ RI + Trametinib group was > 90%, which was significantly better than each single-drug group (the ④ Trametinib group was about 60%, and the ③ RI treatment group was about 80% tumor inhibition rate), and the combination regimen showed a significant synergistic effect.

[0154] The current FDA-approved targeted drugs (such as vemurafenib and dabrafenib) are only suitable for BRAF V600E / KAbout 50% of melanoma is BRAF wild type, which has no effective targeted therapy. In the BRAF wild type melanoma PDX mouse model, RI showed more effective tumor inhibition than dabrafenib and trametinib. It is proved that: RI can be used in combination with existing MEK inhibitors (such as trametinib), and the combination regimen is better than single drug, which can synergistically inhibit the activity of MAPK pathway and delay drug resistance; In the background of BRAF wild type, the combination therapy significantly reversed the drug resistance of MEK inhibitor alone, providing theoretical and experimental basis for combination therapy.

[0155] Example 7

[0156] In this example, BALB / c mice were used to evaluate the toxicity of RI.

[0157] The acute toxicity and subchronic toxicity evaluation results of RI are as follows:

[0158] Acute toxicity: single oral administration of 2000 mg / kg; no death and no abnormal behavior within 14-day observation period; no gross or histological damage to major organs (heart, liver, kidney, lung, brain).

[0159] Subchronic toxicity (28-day repeated administration): the doses were 10, 25 and 50 mg / kg, respectively; slight ALT elevation occurred in the high-dose group, but there was no liver tissue necrosis; the body weight of each group increased normally, and HE staining showed that the tissue structure of major organs (heart, liver, kidney, lung, brain) was complete.

[0160] In addition, compared with common HSP90 inhibitors (such as Geldanamycin), RI does not induce heat shock protein response and has lower off-target effect.

[0161] This example shows that RI has a good safety window, excellent safety, low side effects and other advantages, and has great potential for clinical development.

[0162] Example 8

[0163] In this example, the pharmacokinetic properties of RI (in mice) were analyzed.

[0164] In this example, LC-MS / MS method was used to detect the change of blood drug concentration of RI in mice. The results showed that when the single oral dose was 25 mg / kg, the peak time T max was 0.1±0.04 h, the peak concentration C max was 278±53.25 ng / mL, the half-life t 1 / 2 was 4.49±0.16 h, and the bioavailability was about 5.21%.

[0165] It can be seen that the RI has a large distribution volume, good tissue penetration and good in vivo drug forming property.

[0166] Example 9

[0167] This example performs adaptive verification of RI in other tumor types (solid tumor extension).

[0168] This example uses the following solid tumor cell lines for in vitro inhibition experiments, respectively:

[0169] ACC (Adrenal Cortical Carcinoma): H295R;

[0170] MESO (Mesothelioma): NCI-H28;

[0171] UVM (Uveal Melanoma): OMM.

[0172] The results show that in the three solid tumor cell lines, the IC 50 are 12.3 nM, 17.6 nM, and 9.5 nM, respectively, and are accompanied by down-regulation of RAF protein and inhibition of p-MEK / p-ERK signal.

[0173] This example shows that RI has good cell activity inhibition effect on the above-mentioned cells, the PKN3 kinase-mediated RAF stable pathway exists widely in various tumor types, and RI has potential for multi-indication expansion.

[0174] Example 10

[0175] This example performs specificity verification, and the results show that in the cells of BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903) with knocked-out PKN3 genes or CDC37 genes, RI cannot inhibit the MAPK pathway or tumor growth.

[0176] This further shows that the target specificity of RI depends on the PKN3-CDC37-RAF axis, thereby distinguishing it from traditional BRAF or MEK inhibitors.

[0177] Comparative Example

[0178] To verify the advantages of the PKN3 kinase inhibitor RI of the compound of the present application in target selectivity, inhibition activity and anti-tumor efficiency compared with existing similar compounds, this example compares and analyzes it with the known PKN3 kinase inhibitor PP1 which is structurally related.

[0179] 1. Inhibition activity (IC 50 ):

[0180] The target of PP1 is PKN3 kinase, and the IC of CDC37-Ser13 phosphorylation inhibition is 0.34 nM. 50 is 3.237 nM;

[0181] The target of RI is PKN3 kinase, and the IC of CDC37-Ser13 phosphorylation inhibition is 0.34 nM. 50 is 0.34 nM.

[0182] It can be seen that the inhibition of RI on PKN3 kinase activity is stronger, and the IC 50 is one order of magnitude lower than that of PP1, showing significantly enhanced activity.

[0183] 2. Selectivity comparison:

[0184] By SPR and ADP-Glo kinase screening method, the inhibition of RI and PP1 on PKN family homologous kinases was tested, and the results are shown in Table 1 below:

[0185] Table 1

[0186] Compound PKN1 inhibition PKN2 inhibition PKN3 inhibition PP1 Moderate Weak Effective RI No significant inhibition No significant inhibition Highly selective inhibition

[0187] As can be seen from Table 1, RI significantly improves the selectivity to PKN3 kinase, avoiding non-specific interference on other PKN subtypes.

[0188] 3. Anti-tumor activity (cell proliferation inhibition):

[0189] The anti-proliferation effect of RI and PP1 in BRAF wild-type melanoma cell MM1 was detected by CCK-8, and the results are shown in Table 2 below:

[0190] Table 2

[0191] Compound MM1 cells IC 50 (nM) Inhibition rate (10 nM, 72h) PP1 128.6 30% RI 23.17 80%

[0192] As can be seen from Table 2, the anti-tumor activity of RI is significantly better than that of PP1.

[0193] 4. Signal pathway inhibition effect

[0194] The phosphorylation of MEK / ERK was detected by Western Blot. The results showed that PP1 only partially inhibited p-MEK and p-ERK at high concentration; RI could significantly down-regulate the phosphorylation of CDC37 S13 site at low dose (10 nM), and inhibit the activation of RAF / MEK / ERK pathway.

[0195] 5. Anti-tumor effect in vivo (mouse model)

[0196] In the BRAF wild-type melanoma mouse tumor transplantation model, RI and PP1 were respectively given to observe the change of tumor volume, and the results are shown in Table 3 below:

[0197] Treatment group Dose Tumor inhibition rate (14 days) Control group - 0% PP1 group 25 mg / kg 37% RI group 25 mg / kg 90%

[0198] As shown in Table 3, RI shows a more superior tumor growth inhibition effect in vivo.

[0199] 6. Survival period

[0200] The survival period of mice in the spontaneous melanoma mouse model after PP1 or RI treatment is shown in Table 4. Figure 10

[0201] It can be seen that RI treatment can significantly prolong the survival period of mice.

[0202] The comparative example shows that, compared with the small molecule PP1, RI has high selectivity and stronger inhibition ability on PKN3 kinase, thereby reflecting more significant anti-tumor activity and signal pathway inhibition effect, and finally producing more effective anti-tumor effect.

[0203] In summary, the present application screens and verifies a new small molecule PKN3 kinase inhibitor RI through reasonable structure design, which indirectly interferes with the stability of RAF and the activation of MAPK signal by inhibiting the PKN3-CDC37 signal axis, has a significant effect in the treatment of BRAF wild-type melanoma, and has good transformation and clinical prospects.

[0204] The above specific embodiments are used to explain and illustrate the present application, rather than limit the present application, and any modification and change made to the present application within the spirit and protection scope of the claims of the present application, falls into the protection scope of the present application.​

Claims

1. A PKN3 kinase inhibitor, characterized in that, The structural formula of the PKN3 kinase inhibitor RI is specifically as follows: 。 2. A method for preparing the PKN3 kinase inhibitor as described in claim 1, characterized in that, The method comprises the following steps: S1, 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine, (4-chloro-3-hydroxyphenyl)boronic acid and a palladium catalyst are added into a solvent containing potassium carbonate in a molar ratio of 1.5-2:2-3:0.1-0.2 to obtain a reaction mixture; the solvent is a mixed solution of 1,4-dioxane and water; S2, the reaction mixture is stirred at 80-96 DEG C under a protective atmosphere for 13-20 hours; S3, after the reaction is completed, the reaction mixture is diluted with ethyl acetate, and then washed, dried, filtered and concentrated to obtain a crude product; S4, the crude product is purified by using a silica gel column chromatography to obtain the PKN3 kinase inhibitor RI; During the purification process, a mixed solution of ethyl acetate and n-hexane is used as an eluent, and the volume ratio of ethyl acetate to n-hexane is 1-2:2-3.

3. Use of the PKN3 kinase inhibitor according to claim 1 or of the PKN3 kinase inhibitor obtainable by the process according to claim 2, characterized in that: The PKN3 kinase inhibitor is used for preparing a medicine for treating tumors. The tumor is an adrenocortical carcinoma, a mesothelioma, a uveal melanoma, a BRAF V600E mutant melanoma or a BRAF wild-type melanoma.

4. Use according to claim 3, characterized in that: The combination of the PKN3 kinase inhibitor and the MEK inhibitor is used for preparing a medicine for treating tumors.

5. A medicament for treating a tumor, characterized by: The main active ingredient of the medicine is the PKN3 kinase inhibitor, and the PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI as claimed in claim 1 or the PKN3 kinase inhibitor RI obtained by using the preparation method as claimed in claim 2.

6. A pharmaceutical composition for treating a tumor, characterized by: The main active ingredients of the medicine composition comprise the PKN3 kinase inhibitor and the MEK inhibitor, and the PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI as claimed in claim 1 or the PKN3 kinase inhibitor RI obtained by using the preparation method as claimed in claim 2.

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