PKN3 kinase inhibitor, pharmaceutical composition and preparation method and application of PKN3 kinase inhibitor

Abstract

The invention discloses a PKN3 kinase inhibitor, a pharmaceutical composition and a preparation method and application of the PKN3 kinase inhibitor. The PKN3 kinase inhibitor RI further prevents RAF family protein and CDC37-HSP90 from forming a stable compound by inhibiting activation of CDC37, and reduces the stability and activation level of RAF protein, so that down-regulation of an MAPK signal channel is realized, and proliferation and survival of tumor cells are inhibited; meanwhile, the PKN3 kinase inhibitor RI and the MEK inhibitor are combined for use, so that the treatment effect is excellent, and the drug resistance problem caused by single-channel inhibition can be remarkably solved. The PKN3 kinase inhibitor RI is a novel molecular targeting drug which can target an RAF regulation mechanism and is suitable for BRAF wild type melanoma, and fills the technical blank that the BRAF wild type melanoma is lack of effective treatment drugs and means for a long time.

Description

Technical Field

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

[0002] Melanoma is a highly malignant skin tumor characterized by highly aggressive biological behavior and rapid metastasis, and limited response to traditional chemotherapy and radiotherapy. Currently, the main clinical treatment strategies for melanoma include immune checkpoint inhibitors, BRAF / MEK (B-Raf proto-oncogene, serine / threonine kinase, mitogen-activated protein kinase kinase) targeted therapy, and combination therapy. In the field of molecular targeted therapy, several small molecule inhibitors, such as dabrafenib, vemurafenib, and trametinib, have been developed for the treatment of melanoma with the BRAFV600E mutation. These drugs effectively delay tumor progression and improve patient prognosis by inhibiting BRAF kinase or its downstream kinase, MEK. However, approximately 40% to 50% of melanoma patients are BRAF wild-type (WT), lacking the BRAFV600E mutation, and therefore are not suitable for existing BRAF-targeted therapies. These patients have no significant response to the aforementioned drugs, and effective targeted therapies remain a long-standing unmet clinical need.

[0003] Currently, research on the MAPK (Mitogen-Activated Protein Kinase) pathway primarily focuses on direct kinase targets within the RAS-BRAF-MEK-ERK cascade, but a deeper understanding of the maturation, stabilization, and activation mechanisms of the RAF (Rapidly Accelerated Fibrosarcoma) protein remains lacking. Recent studies have revealed 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 proteins. CDC37 (Cell Division Cycle 37) can enhance its ability to bind and stabilize RAF proteins through phosphorylation at specific sites. However, the upstream kinases that regulate CDC37 activity and their drug accessibility remain largely unknown.

[0004] In summary, the existing related technologies have the following main problems: 1. Existing BRAF inhibitors are ineffective against BRAF wild-type melanoma, and treatment options are limited; 2. There is a lack of targeted drugs that act on the RAF maturation process, and there is no drug that targets the stabilization mechanism of RAF protein; 3. No upstream regulatory factors that can be targeted by drugs, such as protein kinases that regulate CDC37 phosphorylation, have been found; 4. Some broad-spectrum kinase inhibitors have problems with target nonspecificity and significant toxic side effects, which affect their clinical application; 5. Direct inhibitors of HSP90 / CDC37 may induce heat shock response and associated toxicity and lack ideal selectivity and safety; 6. Currently, there are no marketed drugs or public literature reporting PKN3 (Protein Kinase N3) kinase as a target for melanoma treatment. Summary of the Invention

[0005] To address the lack of effective molecularly targeted therapies for BRAF wild-type melanoma in the prior art, particularly regarding RAF protein activation and MAPK signaling pathway regulation, where the related mechanisms of action have not been fully explored and upstream regulatory targets amenable to drug intervention are lacking, the present invention provides a PKN3 kinase inhibitor, a pharmaceutical composition, and a preparation method and application of the PKN3 kinase inhibitor. The present invention develops a novel molecularly targeted drug suitable for BRAF wild-type melanoma that targets the RAF regulatory mechanism, potentially filling the long-standing technological gap in the lack of effective therapeutics for BRAF wild-type melanoma.

[0006] The technical solution adopted in the present invention is as follows: 1. A PKN3 kinase inhibitor The structural formula of the PKN3 kinase inhibitor RI is specifically: .

[0007] 2. A method for preparing the PKN3 kinase inhibitor described above The preparation method comprises the following steps: S1. Add 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, (4-chloro-3-hydroxyphenyl)boric acid, and a palladium catalyst 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; S2. Stir the reaction mixture under a protective atmosphere at 80-96° C. for 13-20 hours; S3. After the reaction is completed, the mixture is diluted with ethyl acetate, washed, dried, filtered and concentrated to obtain a crude product; S4. Purifying the crude product using silica gel column chromatography to obtain the PKN3 kinase inhibitor RI; During the purification process, a mixed solution of ethyl acetate and n-hexane was used as the eluent, with the volume ratio of ethyl acetate:n-hexane being 1~2:2~3.

[0008] III. Use of the PKN3 kinase inhibitor as described above or the PKN3 kinase inhibitor obtained by the preparation method as described above The specific application is: the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate is used to screen or prepare drugs for treating tumors.

[0009] Furthermore, the application is: the combination of the PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof and a MEK inhibitor is used to prepare a drug for treating tumors.

[0010] Preferably, the tumor includes adrenocortical carcinoma, mesothelioma and uveal melanoma.

[0011] More preferably, the tumor comprises BRAF V600E BRAF mutant melanoma and BRAF wild-type melanoma.

[0012] Most preferably, the tumor is a BRAF wild-type melanoma.

[0013] Preferably, the dosage of the PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof is selected from 10 to 50 mg / kg per day.

[0014] 4. A drug for treating tumors The main active ingredient of the drug is a PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and the PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI described above or a PKN3 kinase inhibitor RI obtained by the preparation method described above.

[0015] 5. A pharmaceutical composition for treating tumors The main active ingredients of the pharmaceutical composition include a PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a MEK inhibitor. The PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI described above or a PKN3 kinase inhibitor RI prepared by the above-described preparation method.

[0016] The beneficial effects of the present invention are as follows: 1. This invention proposes a novel mechanism that has not been used in published literature or existing drugs: PKN3 kinase is a key upstream regulatory factor in the RAF-MAPK pathway, promoting RAF maturation and activation by phosphorylating CDC37 (Ser13). This mechanism is different from the classic sites of existing BRAF or MEK inhibitors and has complementary and alternative value.

[0017] 2. The present invention proposes a novel small molecule inhibitor of PKN3 kinase, RI. RI inhibits the activation of CDC37, further preventing RAF family proteins from forming a stable complex with CDC37-HSP90, reducing the stability and activation level of RAF protein, thereby achieving downregulation of the MAPK signaling pathway and inhibiting the proliferation and survival of tumor cells.

[0018] 3. The PKN3 kinase inhibitor RI proposed in this invention has a significant anti-tumor effect, which is far superior to control drugs such as PP1, dabrafenib, vemurafenib and trametinib, and has a wide range of indications, especially for wild-type BRAF melanoma and other tumors that cannot be treated with BRAF. V600E Targeted inhibitors for melanoma.

[0019] 4. The combined use of the PKN3 kinase inhibitor RI and the MEK inhibitor proposed in the present invention not only has excellent therapeutic effects, but also can significantly overcome the drug resistance problem caused by single pathway inhibition.

[0020] 5. The PKN3 kinase inhibitor RI proposed in the present invention not only has excellent safety, low side effects, and good drugability, but also has industrial advantages such as mature compound synthesis technology, easy industrial scale-up, and suitability for clinical development.

[0021] In summary, the present invention constructs a new therapeutic mechanism based on the PKN3-CDC37-RAF signaling axis, providing a new targeted treatment strategy for BRAF wild-type melanoma with a clear mechanism, significant efficacy, and target specificity, effectively making up for the shortcomings of existing therapeutic drugs and having important clinical application prospects and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the synthesis reaction of the PKN3 kinase inhibitor RI provided by the present invention; Figure 2 are the chemical structural formulas of PP1 and RI; wherein, (a) is the chemical structural formula of PP1, and (b) is the chemical structural formula of RI; Figure 3 The molecular docking sites for PP1 and RI to bind to PKN3 kinase; wherein, (a) is the molecular docking site for PP1 to bind to PKN3 kinase, and (b) is the molecular docking site for RI to bind to PKN3 kinase; Figure 4 Schematic diagram comparing the bioavailability of PP1 and RI; Figure 5 This is a comparison of the changes in key MAPK signaling proteins in melanoma cells before and after PP1 or RI treatment; Figure 6 Comparison of melanoma cell clone formation before and after PP1 or RI treatment; Figure 7 This is a picture of a tumor in a cell-derived xenograft model (CDX); Figure 8 Comparison of tumor growth curves in cell-derived xenograft models (CDX); Figure 9 Comparison of tumor growth curves in spontaneous melanoma mouse models after PP1 or RI treatment Figure 10 This is a comparison of mouse survival in a spontaneous melanoma mouse model after treatment with PP1 or RI; Figure 11 Comparison of tumor growth curves in patient-derived xenograft (PDX) models; Figure 12 Comparison of tumor weights in patient-derived xenograft (PDX) models. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] The first aspect of the present invention provides a PKN3 kinase inhibitor. This PKN3 kinase inhibitor is an ATP-competitive inhibitor with the following mechanism of action: RI selectively binds to the PKN3 kinase pocket, blocking PKN3 kinase phosphorylation of the CDC37 molecular chaperone at Ser13. This inhibitor, by inhibiting the stabilization of the CDC37 and HSP90 complex, interferes with CDC37's recruitment of RAF family proteins (ARAF, BRAF, and CRAF), preventing RAF family proteins from forming a stable complex with CDC37-HSP90. This in turn leads to the instability and degradation of RAF proteins, resulting in loss of downstream MEK / ERK activation function, thereby inhibiting the MAPK pathway and suppressing tumor cell proliferation and survival.

[0025] The structural formula of PKN3 kinase inhibitor RI is as follows: The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] The first aspect of the present invention provides a PKN3 kinase inhibitor. This PKN3 kinase inhibitor is an ATP-competitive inhibitor with the following mechanism of action: RI selectively binds to the PKN3 kinase pocket, blocking PKN3 kinase phosphorylation of the CDC37 molecular chaperone at Ser13. This inhibitor, by inhibiting the stabilization of the CDC37 and HSP90 complex, interferes with CDC37's recruitment of RAF family proteins (ARAF, BRAF, and CRAF), preventing RAF family proteins from forming a stable complex with CDC37-HSP90. This in turn leads to the instability and degradation of RAF proteins, resulting in loss of downstream MEK / ERK activation function, thereby inhibiting the MAPK pathway and suppressing tumor cell proliferation and survival.

[0027] The structural formula of PKN3 kinase inhibitor RI is as follows: .

[0028] The second aspect of the present invention provides a method for preparing a PKN3 kinase inhibitor RI.

[0029] The preparation method specifically comprises the following steps: S1. Under a protective atmosphere, 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, (4-chloro-3-hydroxyphenyl)boric acid and a palladium catalyst are sequentially added to a solvent containing potassium carbonate to obtain a reaction mixture.

[0030] Preferably, the molar ratio of 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, (4-chloro-3-hydroxyphenyl)boronic acid and palladium catalyst is 1.5-2:2-3:0.1-0.2.

[0031] Preferably, the mass volume ratio of potassium carbonate to solvent is 1-1.5:18-25.

[0032] 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.

[0033] Preferably, the palladium catalyst is PdCl2(dppf)·CH2Cl2.

[0034] S2. Stir the reaction mixture vigorously (600-800 rpm) at 80-96°C under a protective atmosphere for 13-20 hours until the reaction is complete.

[0035] S3. After the reaction is completed, the mixture is diluted with ethyl acetate, washed with water and saturated brine in sequence, and then dried, filtered and concentrated in sequence to obtain a crude product.

[0036] In this step, anhydrous sodium sulfate can be used for drying.

[0037] In this step, a rotary evaporator can be used for concentration.

[0038] S4. Purify the crude product using silica gel column chromatography to obtain PKN3 kinase inhibitor RI.

[0039] Preferably, during the purification process, a mixed solution of ethyl acetate and n-hexane is used as the eluent.

[0040] Preferably, the volume ratio of ethyl acetate:n-hexane is 1-2:2-3.

[0041] Optionally, in the above preparation process, the protective atmosphere can be any one of nitrogen, helium, neon, argon, krypton and radon, or a combination of two or more.

[0042] The third aspect of the present invention provides a use of a PKN3 kinase inhibitor RI.

[0043] The PKN3 kinase inhibitor RI or its pharmaceutically acceptable salt or hydrate can be used to screen or prepare drugs for treating tumors.

[0044] Furthermore, a PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof can be combined with a MEK inhibitor to screen or prepare drugs for treating tumors.

[0045] The PKN3 kinase inhibitor RI is metabolically stable in vivo, has good oral bioavailability, and a plasma half-life of 4 to 6 hours.

[0046] Preferably, the tumors include adrenocortical carcinoma, mesothelioma, and uveal melanoma.

[0047] More preferably, the tumor comprises BRAF V600E BRAF mutant melanoma and BRAF wild-type melanoma.

[0048] Most preferably, the tumor is a BRAF wild-type melanoma.

[0049] Preferably, the dosage of the PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof is selected from 10 to 50 mg / kg per day.

[0050] Most preferably, the dosage of the PKN3 kinase inhibitor RI or a pharmaceutically acceptable salt or hydrate thereof is 25 mg / kg per day.

[0051] Specifically, the application principle of PKN3 kinase inhibitor RI is as follows: PKN3 kinase inhibitors or their pharmaceutically acceptable salts or hydrates selectively bind to the PKN3 kinase pocket, blocking the phosphorylation of the Ser13 site of the CDC37 molecular chaperone by PKN3 kinase, thereby inhibiting the stabilization of the CDC37 and HSP90 complex, interfering with the recruitment of RAF family proteins (ARAF, BRAF, CRAF) by CDC37, inducing RAF protein instability and degradation, and ultimately losing downstream MEK / ERK activation function, thereby achieving the purpose of inhibiting the MAPK pathway and inhibiting tumor cell proliferation.

[0052] A fourth aspect of the present invention provides a drug for treating tumors, wherein the main active ingredient of the drug is a PKN3 kinase inhibitor RI or a pharmaceutically acceptable salt or hydrate thereof.

[0053] The fifth aspect of the present invention provides a pharmaceutical composition for treating tumors, wherein the main active ingredients of the pharmaceutical composition include a PKN3 kinase inhibitor RI or a pharmaceutically acceptable salt or hydrate thereof and a MEK inhibitor.

[0054] Preferably, the MEK inhibitor is trametinib.

[0055] Alternatively, the drugs and pharmaceutical compositions can be prepared into tablets, capsules, injections or sustained-release dosage forms as needed.

[0056] Alternatively, the drug and pharmaceutical composition may be administered orally, intraperitoneally or intravenously.

[0057] Furthermore, both the drug and the pharmaceutical composition also include formulation excipients.

[0058] Preferably, the formulation excipients include one or a combination of pharmaceutically acceptable carriers such as lactose, starch, talc, hydroxypropyl methylcellulose and polyvinyl pyrrolidone.

[0059] Preferably, for the above-mentioned medicaments and pharmaceutical compositions, the tumors include adrenocortical carcinoma, mesothelioma and uveal melanoma.

[0060] More preferably, for the above-mentioned drugs and pharmaceutical compositions, the tumor comprises BRAF V600E BRAF mutant melanoma and BRAF wild-type melanoma.

[0061] Most preferably, for the above-mentioned medicaments and pharmaceutical compositions, the tumor is BRAF wild-type melanoma.

[0062] The specific embodiments of the present invention are as follows: Example 1

[0063] This embodiment follows Figure 1 The synthetic reaction formula shown in FIG. 1 is used to prepare the PKN3 kinase inhibitor RI by the following process: S1. Under argon atmosphere, add the following raw materials into the reaction flask in sequence: 3-Bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (CAS No. 862728-61-8): 500 mg, 1.852 mmol; (4-Chloro-3-hydroxyphenyl)boronic acid (CAS No. 915201-06-8): 399.0 mg, 2.315 mmol; PdCl2(dppf)·CH2Cl2 (CAS number 95464-05-4): 136.4 mg, 0.167 mmol; Potassium carbonate K2CO3: 1.279 g, 9.26 mmol; Solvents: 1,4-dioxane (16.7 mL) and water (5.9 mL).

[0064] S2. The reaction system was vigorously stirred at 90°C for 16 hours.

[0065] S3. After the reaction is completed, 200 mL of ethyl acetate is added for extraction, and the organic phase is washed with 100 mL of water and 50 mL of saturated brine in sequence. The washed organic phase is dried, filtered and spin-dried to obtain a crude product.

[0066] 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%).

[0067] The structural characterization of the PKN3 kinase inhibitor RI obtained in this example is as follows: 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).

[0068] 13 C 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.

[0069] HRMS (ESI): [M+H]+, C 15 H 17 ClN5O + , theoretical value 318.1116, measured value 318.1112.

[0070] The PKN3 kinase inhibitor RI prepared in this example was designed based on PP1 (synonymous names: AGL 1872; EI 275; CAS number: 172889-26-8). The chemical structures of PP1 and RI are as follows: Figure 2 As shown in (a) and (b), the molecular docking sites of PP1, RI and PKN3 kinase are respectively as shown in Figure 3 As shown in (a) and (b).

[0071] Depend on Figure 3 It can be seen that by introducing chlorine atoms and hydroxyl structures, the hydrogen bonds and hydrophobic interactions between RI and amino acid residues in the PKN3 kinase pocket (such as K588) were enhanced, and the selectivity and affinity were also significantly improved.

[0072] In addition, by Figure 4 It can be seen that compared with PP1, the bioavailability of RI was also significantly improved (P value was 0.0061).

[0073] Example 2 This example measured the inhibitory effect of PKN3 kinase inhibitor RI on PKN3 kinase.

[0074] This embodiment uses ADP-Glo TM Kinase kit (Promega) was used to evaluate the inhibitory ability of RI on PKN3 kinase in vitro using CDC37 peptide as substrate. The results showed that the IC 50 The half maximal inhibitory concentration (Half maximal inhibitory concentration) value was 0.3407 nM; and it had no significant inhibitory effect on PKN1 and PKN2.

[0075] In this example, the binding constant KD of RI to PKN3 kinase was measured by surface plasmon resonance (SPR) and was found to be less than 10 nM. The binding affinity was much better than that of the control drug PP1 (KD≈200 nM).

[0076] This example also shows that the thermal stability shift assay (TSA) shows that after binding to RI, the Tm value of PKN3 kinase increases by about 6.3°C, that is, the binding of RI enhances the thermal stability of PKN3 kinase, thereby further verifying the direct target binding ability of RI.

[0077] Example 3 This example demonstrates the antiproliferative activity of RI in melanoma cells.

[0078] In this example, CCK-8 assay, colony formation assay, and Western Blot assay were performed using BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903).

[0079] The results of CCK-8 assay showed that the activity of BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903) were significantly decreased after RI treatment. 50 The results were as follows: MM1: 23.17 nM, MEWO: 57.39 nM, A375: 35.41 nM, and UACC903: 11.46 nM.

[0080] Western Blot results are as follows Figure 5 As shown in the figure, DMSO represents the blank control group, RI represents RI-treated cells, and PP1 represents PP1-treated cells. It can be seen that in BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903), CDC37 S13 phosphorylation was inhibited and RAF / MEK / ERK phosphorylation levels were downregulated, confirming the direct inhibition of non-traditional MAPK kinases.

[0081] The results of the clone formation experiment are as follows Figure 6 As shown in the figure, when the RI treatment concentration was 50 nM, the clone number of the four melanoma cell lines decreased by more than 90%, and their cell proliferation was significantly inhibited compared with the PP1 treatment group.

[0082] This example shows that in BRAF wild-type melanoma cells, RI treatment can significantly reduce the phosphorylation level of CDC37, inhibit RAF protein expression, and thus inhibit cell proliferation, with no significant difference compared with BRAF mutant cells, and has a wider range of applicability.

[0083] Example 4 This example demonstrates the in vivo tumor-suppressing effect of RI in a mouse subcutaneous tumor model.

[0084] In this example, MM1 cells (5×10 6 ) were inoculated subcutaneously on the back of nude mice, and the mice were randomly divided into groups after successful model establishment (n=6): ①Solvent control group (corn oil); ②RI treatment group (25 mg / kg); The drug was injected intraperitoneally once a day for 14 consecutive days.

[0085] The results are as follows Figure 7 and Figure 8 It can be seen that in the ②RI treatment group, the tumor volume inhibition rate reached 80%, which was significantly higher than that of the ①vehicle control group.

[0086] In addition, there was no obvious weight loss in the mice in the ②RI treatment group, HE staining of major organs showed no tissue toxicity, and p-ERK / p-MEK in tumor tissues was also significantly downregulated.

[0087] Example 5 This example demonstrates the in vivo tumor-suppressing effect of RI in a spontaneous melanoma mouse model.

[0088] This example uses a spontaneous melanoma mouse model: the GEMM model (Tyr-CreERT2 / Braf CA / Pten fl / fl ), and randomly grouped: ①Solvent control group (corn oil); ②RI low-dose group (10 mg / kg); ③RI high-dose group (25 mg / kg); ④PP1-treated group (25 mg / kg).

[0089] The results are as follows Figure 9 and Figure 10 As shown, compared with the vehicle control group (①) and the PP1-treated group (④), the RI-treated groups (② RI low-dose group and ③ RI high-dose group) significantly delayed tumor formation, reduced tumor volume, and improved survival. Furthermore, no weight loss, major organ damage, or severe toxicity were observed in the RI-treated groups (② RI low-dose group and ③ RI high-dose group) during treatment, demonstrating a favorable safety profile. Furthermore, significant downregulation of p-ERK / p-MEK and a significant decrease in Ki67-positive cells were observed in tumor tissues in the RI-treated groups (② RI low-dose group and ③ RI high-dose group).

[0090] Example 6 This example demonstrates the synergistic antitumor effect of RI in combination with the MEK inhibitor Trametinib.

[0091] In this example, a PDX model (patient-derived xenograft model) of BRAF wild-type melanoma was first established. After successful modeling, the patients were randomly divided into the following groups: ① Vehicle control group; ② Dabrafenib group (Dabrafenib, 25 mg / kg); ③RI treatment group 25 mg / kg; ④ Trametinib group (Trametinib, 1 mg / kg); ⑤Dabrafenib + trametinib group; ⑥RI+trametinib group.

[0092] The treatment cycle was 21 days, and the evaluation indicators included tumor volume, survival time, and signaling pathway protein expression. The results were as follows: Figure 11 and Figure 12 shown.

[0093] The results showed that the tumor volume inhibition rate of the ⑥RI+trametinib group was >90%, which was significantly better than that of each single-drug group (the tumor inhibition rate of the ④trametinib group was about 60%, and the tumor inhibition rate of the ③RI treatment group was about 80%). The combination regimen showed a significant synergistic effect.

[0094] Currently FDA-approved targeted drugs (such as vemurafenib and dabrafenib) are only effective for BRAF V600E / KBRAF mutation patients, while approximately 50% of melanomas are BRAF wild-type, have essentially no effective targeted treatment options. RI demonstrated a more effective tumor suppressive effect than dabrafenib and trametinib in a BRAF wild-type melanoma PDX mouse model. This demonstrates that RI can be combined with existing MEK inhibitors (such as trametinib), and that the combined approach is superior to single-agent therapy, synergistically inhibiting MAPK pathway activity and delaying the development of drug resistance. In the BRAF wild-type setting, combined therapy significantly reversed the drug resistance observed with MEK inhibitors alone, providing theoretical and experimental evidence for combined therapy.

[0095] Example 7 In this example, BALB / c mice were used to evaluate the toxicity of RI.

[0096] The results of RI's acute toxicity and subchronic toxicity assessment are as follows: Acute toxicity: Single oral administration of 2000 mg / kg; no deaths or behavioral abnormalities during the 14-day observation period; no macroscopic or histological damage to major organs (heart, liver, kidney, lung, brain).

[0097] Subchronic toxicity (28-day repeated administration): The doses were 10, 25, and 50 mg / kg, respectively; the high-dose group showed a slight increase in ALT, but 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, and brain) was intact.

[0098] In addition, compared with common HSP90 inhibitors (such as geldanamycin), RI does not induce heat shock protein response and has lower off-target effects.

[0099] This example shows that RI has a good safety window, excellent safety, low side effects and other advantages, and shows great potential for clinical development.

[0100] Example 8 This example analyzes the pharmacokinetic properties of RI (in vivo in mice).

[0101] This example uses LC-MS / MS to detect the changes in the blood concentration of RI in mice. The results show that after a single oral dose of 25 mg / kg, the peak time T max 0.1±0.04 h, peak concentration C max was 278±53.25 ng / mL, and the half-life t 1 / 2 The duration of treatment was 4.49±0.16 h, and the bioavailability was approximately 5.21%.

[0102] It can be seen that RI has a large distribution volume, good tissue penetration, and good in vivo drugability.

[0103] Example 9 This example verifies the adaptability of RI in other tumor types (solid tumor expansion).

[0104] In this example, the following solid tumor cell lines were used for in vitro inhibition experiments: ACC (Adrenal Cortical Carcinoma): H295R; MESO (mesothelioma): NCI-H28; UVM (Uveal Melanoma): OMM.

[0105] The results showed that in three solid tumor cell lines, IC 50 The results are as follows: 12.3 nM, 17.6 nM, and 9.5 nM, respectively; and all are accompanied by downregulation of RAF protein and inhibition of p-MEK / p-ERK signaling.

[0106] This example shows that RI exhibits a good inhibitory effect on the cell activity of the above cells. The PKN3 kinase-mediated RAF stabilization pathway is widely present in various tumor types, and RI has the potential to expand into multiple indications.

[0107] Example 10 This example performed specific validation, and the results showed that RI failed to inhibit the MAPK pathway or tumor growth in BRAF wild-type melanoma cells (MM1, MEWO) and BRAF mutant cells (A375, UACC903) with PKN3 or CDC37 gene knockout.

[0108] This further demonstrates that the target specificity of RI depends on the PKN3-CDC37-RAF axis, distinguishing it from traditional BRAF or MEK inhibitors.

[0109] Comparative Example To verify the advantages of the PKN3 kinase inhibitor RI of the present invention over existing similar compounds in terms of target selectivity, inhibitory activity and anti-tumor efficacy, this example compared it with the structurally related known PKN3 kinase inhibitor PP1.

[0110] 1. Inhibitory activity (IC 50 ): PP1 targets PKN3 kinase, and phosphorylation of CDC37-Ser13 inhibits IC 50 is 3.237 nM; RI targets PKN3 kinase, and phosphorylation of CDC37-Ser13 inhibits IC 50 is 0.34 nM.

[0111] It can be seen that RI has a stronger inhibitory effect on PKN3 kinase activity, and IC 50 It is one order of magnitude lower than PP1 and shows significantly enhanced activity.

[0112] 2. Selective comparison: The inhibition of RI and PP1 on PKN family homologous kinases was tested by SPR and ADP-Glo ​​kinase screening. The results are shown in Table 1 below: Table 1 Compound PKN1 inhibition PKN2 inhibition PKN3 inhibition PP1 Moderate weak efficient RI No obvious inhibition No obvious inhibition Highly selective inhibition As shown in Table 1, RI significantly improved the selectivity for PKN3 kinase and avoided nonspecific interference with other PKN isoforms.

[0113] 3. Anti-tumor activity (cell proliferation inhibition): The anti-proliferative effects of RI and PP1 in BRAF wild-type melanoma cells MM1 were detected using CCK-8 and are shown in Table 2 below: Table 2 Compound <![CDATA[IC of MM1 cells 50 (nM)]]> Inhibition rate (10 nM, 72h) PP1 128.6 30% RI 23.17 80% As shown in Table 2, the anti-tumor activity of RI was significantly better than that of PP1.

[0114] 4. Signaling pathway inhibition effect Western blot analysis of MEK / ERK phosphorylation showed that PP1 only partially inhibited p-MEK and p-ERK at high concentrations, while RI significantly downregulated CDC37 S13 phosphorylation at a low dose (10 nM), inhibiting RAF / MEK / ERK pathway activation.

[0115] 5. In vivo anti-tumor effect (mouse model) In a BRAF wild-type melanoma mouse xenograft model, RI and PP1 were administered, respectively, and the changes in tumor volume were observed. The results are shown in Table 3 below: Treatment group dose Tumor inhibition rate (14 days) control group - 0% PP1 group 25 mg / kg 37% RI group 25 mg / kg 90% As shown in Table 3, RI exhibited a superior tumor growth inhibitory effect in vivo.

[0116] 6. Survival Mouse survival in a spontaneous melanoma mouse model after PP1 or RI treatment Figure 10 shown.

[0117] It can be seen that RI treatment can significantly prolong the survival of mice.

[0118] This comparative example shows that compared with the small molecule PP1, RI has high selectivity and stronger inhibitory ability against PKN3 kinase, thereby demonstrating more significant anti-tumor activity and signal pathway inhibition effect, and ultimately producing a more effective anti-tumor effect.

[0119] In summary, the present invention screened and validated a novel small molecule PKN3 kinase inhibitor RI through rational structural design. RI indirectly intervenes in RAF stability and MAPK signaling activation by inhibiting the PKN3-CDC37 signaling axis, has significant effects in the treatment of BRAF wild-type melanoma, and has good translational and clinical prospects.

[0120] The above specific embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A PKN3 kinase inhibitor, characterized in that The structural formula of the PKN3 kinase inhibitor RI is specifically: 。 2. A method for preparing the PKN3 kinase inhibitor according to claim 1, characterized in that: The following steps are involved: S1. Add 3-bromo-1-(tert-butyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, (4-chloro-3-hydroxyphenyl)boric acid, and a palladium catalyst 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; S2. Stir the reaction mixture under a protective atmosphere at 80-96° C. for 13-20 hours; S3. After the reaction is completed, the mixture is diluted with ethyl acetate, washed, dried, filtered and concentrated to obtain a crude product; S4. Purifying the crude product using silica gel column chromatography to obtain the PKN3 kinase inhibitor RI; During the purification process, a mixed solution of ethyl acetate and n-hexane was used as the eluent, with the volume ratio of ethyl acetate:n-hexane being 1~2:2~3.

3. A use of the PKN3 kinase inhibitor according to claim 1 or the PKN3 kinase inhibitor obtained by the preparation method according to claim 2, characterized in that: The PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate is used for screening or preparing drugs for treating tumors.

4. The use according to claim 3, characterized in that: The combination of the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate and the MEK inhibitor is used to prepare a drug for treating tumors.

5. The use according to claim 3 or 4, characterized in that: Such tumors include adrenocortical carcinoma, mesothelioma, and uveal melanoma.

6. The use according to claim 3 or 4, characterized in that: The tumor includes BRAF V600E BRAF mutant melanoma and BRAF wild-type melanoma.

7. The use according to claim 3 or 4, characterized in that: The tumor is a BRAF wild-type melanoma.

8. The use according to claim 3 or 4, characterized in that: The dosage of the PKN3 kinase inhibitor or its pharmaceutically acceptable salt or hydrate is selected from 10 to 50 mg / kg per day.

9. A drug for treating tumors, characterized in that: The main active ingredient of the drug is a PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and the PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI according to claim 1 or the PKN3 kinase inhibitor RI obtained by the preparation method according to claim 2.

10. A pharmaceutical composition for treating tumors, characterized in that: The main active ingredients of the pharmaceutical composition include a PKN3 kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof and a MEK inhibitor. The PKN3 kinase inhibitor is the PKN3 kinase inhibitor RI according to claim 1 or the PKN3 kinase inhibitor RI obtained by the preparation method according to claim 2.

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