Eutectic medicine ZM851 and application thereof in lung cancer resistance

Abstract

The invention relates to the technical field of biological medicine, and discloses a co-crystal medicine ZM851 and application thereof in lung cancer resistance, a preparation method of the co-crystal medicine ZM851 comprises the following steps: mixing osimertinib, dihydroartemisinin and a mixed solvent, heating, stirring and dissolving, then adding normal hexane, and separating out a yellow solid, namely the co-crystal medicine ZM851. The antitumor activity shown by the eutectic molecule ZM851 in the invention is more than ten times that shown by a non-eutectic mixture of the eutectic molecule ZM851. In addition, in an in-vitro experiment, the ZM851 shows a remarkable inhibition effect on an osimertinib drug-resistant cell strain. The eutectic drug ZM851 has a remarkable inhibition effect on osimertinib drug-resistant cell strains in an in-vitro experiment, which indicates that the eutectic drug ZM851 possibly provides a new treatment scheme for clinical treatment of lung cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cocrystal drug ZM851 and its application in treating lung cancer. Background Art

[0002] Lung cancer is the type of cancer with the highest morbidity and mortality worldwide, of which non-small cell lung cancer (NSCLC) accounts for 80% to 85%, while small cell lung cancer (SCLC) accounts for 10% to 15%. Epidermal growth factor receptor (EGFR) is one of the key targets for the treatment of non-small cell lung cancer. Targeting this target, first-generation to third-generation non-small cell lung cancer treatment drugs have been successfully launched. Osimertinib, as a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), can not only overcome the drug resistance problem caused by methionine (TM790) mutations in the first-generation EGFR-TKI, but also solve the defect of insufficient selectivity of the second-generation EGFR-TKI for wild-type EGFR. However, patients may also develop drug resistance after 9 to 14 months of treatment with osimertinib.

[0003] Therefore, it is particularly important to develop new drugs that can inhibit osimertinib-resistant cell lines. Summary of the invention

[0004] In view of this, the present invention proposes a cocrystal drug ZM851 and its application in anti-lung cancer, aiming to solve the drug resistance problem occurring in existing treatment schemes.

[0005] The present invention provides a compound ZM851, the chemical structure of which is:

[0006]

[0007] The present invention also provides a method for preparing the compound ZM851, comprising:

[0008] Osimertinib, dihydroartemisinin and a mixed solvent are mixed and heated and stirred to dissolve, and then n-hexane is added, and the precipitated yellow solid is the compound ZM851.

[0009] Preferably, the mixed solvent is acetone and water.

[0010] Preferably, the volume ratio of acetone to water is 15:1.

[0011] Preferably, the temperature at which osimertinib, dihydroartemisinin and the mixed solvent are mixed and heated and stirred for dissolution is at least 60°C.

[0012] The present invention also provides an application of the compound ZM851, which includes at least any one of the following:

[0013] A1) Application in the preparation of anti-tumor drugs;

[0014] A2) Use in the preparation of a pharmaceutical composition for preventing or treating drug-resistant tumors;

[0015] A3) Application in the preparation of drug resistance sensitizers for anti-tumor drugs.

[0016] The present invention also provides a co-crystal drug, the active ingredient of the co-crystal drug is the above-mentioned compound ZM851.

[0017] The present invention also provides an application of the above-mentioned co-crystal drug, wherein the application comprises at least any one of the following:

[0018] A1) Application in the preparation of anti-tumor drugs;

[0019] A2) Use in the preparation of a pharmaceutical composition for preventing or treating drug-resistant tumors;

[0020] A3) Application in the preparation of drug resistance sensitizers for anti-tumor drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention successfully synthesized one cocrystal molecule and two non-cocrystal compounds. In particular, the anti-tumor activity exhibited by the cocrystal molecule ZM851 is more than 10 times that of its non-cocrystal mixture. In addition, in in vitro experiments, ZM851 showed a significant inhibitory effect on osimertinib-resistant cell lines.

[0023] The cocrystal drug ZM851 has a significant inhibitory effect on osimertinib-resistant cell lines in in vitro experiments, indicating that it may provide a new treatment option for lung cancer patients in clinical treatment. The application of cocrystal drug ZM851 is expected to improve the treatment effect, prolong the patient's progression-free survival, and may have a positive impact on the patient's overall survival rate.

[0024] In summary, the cocrystal drug ZM851 and its preparation method of the present invention bring new strategies to the field of anti-lung cancer treatment and are expected to play a key role in future clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0026] Figure 1 Compound ZM8511 HNMR spectrum;

[0027] Figure 2 is the XRD spectrum of compound ZM851;

[0028] Figure 3 is the Fourier transform infrared spectrum of compound ZM851;

[0029] Figure 4 is the Raman spectrum of compound ZM851;

[0030] Figure 5 TG spectrum and DSC spectrum of compound ZM851;

[0031] Figure 6 is the 1HNMR spectrum of compound ZM852;

[0032] Figure 7 is the XRD spectrum of compound ZM852;

[0033] Figure 8 is the Fourier transform infrared spectrum of compound ZM852;

[0034] Fig. 9 is the Raman spectrum of compound ZM852;

[0035] Fig.10 TG spectrum and DSC spectrum of compound ZM852;

[0036] Fig.11 is the 1HNMR spectrum of compound ZM853;

[0037] Fig.12 is the XRD spectrum of compound ZM853;

[0038] Fig.13 is the Fourier transform infrared spectrum of compound ZM853;

[0039] Fig.14 is the Raman spectrum of compound ZM853;

[0040] Fig.15 TG spectrum and DSC spectrum of compound ZM853. DETAILED DESCRIPTION

[0041] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0042] Example 1

[0043] 1. Preparation of Compound ZM851

[0044] Weigh osimertinib (200 mg, 0.4 mmol) and dihydroartemisinin (114 mg, 0.4 mmol) in a reaction tube, add 1.5 mL of a mixed solvent (acetone: water = 15:1), heat to 60 °C and stir to dissolve. Then add 3 mL of n-hexane to precipitate a yellow solid. Filter and wash to obtain 251 mg of a yellow powder, namely compound ZM851, with the chemical formula:

[0045]

[0046] 2. Preparation of Compound ZM852

[0047] Weigh osimertinib (200 mg, 0.4 mmol) and gefitinib (179 mg, 0.4 mmol) in a reaction tube, add 1.5 mL of a mixed solvent (ethanol: water = 15:1), heat to 80°C and stir to dissolve. Then cool to 60°C, add 3 mL of n-hexane, and a light brown solid precipitates. Filter and wash to obtain 361 mg of a light brown powder, namely compound ZM852, with the chemical formula:

[0048]

[0049] 3. Preparation of Compound ZM853

[0050] Weigh osimertinib (200 mg, 0.4 mmol) and ibrutinib (176 mg, 0.4 mmol) in a reaction tube, add 1.5 mL of a mixed solvent (ethanol: water = 15:1), heat to 80 °C and stir to dissolve. Then cool to 60 °C, add 3 mL of n-hexane, and slowly cool to room temperature to precipitate a light brown solid. Filter and wash to obtain 323 mg of a light brown powder, namely compound ZM853, with the chemical structure:

[0051]

[0052] Example 2 Structural characterization and analysis of the compound prepared in Example 1

[0053] The following is the characterization analysis of ZM851, ZM852 and ZM853.

[0054] 1. Process

[0055] 1H Nuclear Magnetic Resonance (1H NMR): The H NMR data were collected at room temperature by a DSX-500 NMR spectrometer from Bruker Biospin GmbH, Switzerland. The solvent was DMSO-d6 and the internal standard was TMS. Powder X-ray diffraction (PXRD): The PXRD data were obtained by a Rigaku Ultra IV diffractometer from Rigaku Co., Ltd., Japan. The parameters of the Rigaku Ultra IV powder Xray Cu Ka radiation diffractometer were set as wavelength 1.5418A, voltage 40KV, current 40mA, sample form was powder, test target material was copper target, scanning range was 5-90° (2θ), scanning speed was 10° / min, and all sample data were obtained at room temperature.

[0056] Fourier transform infrared spectroscopy (FT-IR): FT-IR data were obtained by the Nicolet iN10 test produced by Thermo Fisher Scientific in the United States. The maximum axis number range of conventional tests is 400-4000cm-1, which is used for conventional powder compression tests and liquid pool tests. The experimental data in this article were obtained by the KBr tableting method, and KBr was ground to 2μm using an agate mortar. A certain amount of sample (2-5mg) and KBr (100-120mg) were added, ground and mixed until there were no obvious particles. It was placed in a mold and then pressed into a transparent or translucent shape.

[0057] Raman spectroscopy (Raman, RM): Raman data is obtained by the Thermo Fisher DXR test. Place the sample to be tested as flat as possible on the sample holder to ensure the accuracy of the spectral data. According to the sample properties and experimental requirements, select the appropriate laser wavelength, power and detector parameters to ensure the clarity and stability of the spectral signal. Before the sample test, a background scan must be performed to eliminate the interference of the instrument background signal on the sample signal.

[0058] Synchronous thermal analysis TG-DSC: The test data of synchronous thermal analysis uses the HITACHISTA200 instrument of Hitachi, Ltd. of Japan. The TG-DSC test can simultaneously obtain the curves of TG, DSC and DTG. The sample temperature selection range is from room temperature to below 800°C (including 800°C), the specific test temperature is 30-600°C, the test gas atmosphere is N2, and the heating rate is 10°C / min.

[0059] 2. Results

[0060] 1. Structural characterization and analysis of ZM851

[0061] like Figure 1 As shown, the nuclear magnetic resonance hydrogen spectrum of ZM851 cocrystal presents the following data: 1H NMR (500MHz, DMSO-d6) δ10.20 (s, 1H), 9.15 (s, 1H), 8.68 (s, 1H), 8.33 (d, J = 5.3Hz, 1H), 8.24 (d, J = 8.0Hz, 1H), 7.89 (s, 1H), 7.52 (d, J = 8.2Hz, 1H), 7.26-7.22 (m, 2H), 7.15 (t, J = 7.5Hz,1H),7.04(s,1H),6.46-6.40(m,1H),6.29(s,1H),5.77(d,J=10.1Hz,1H),3.92(s,3H),3.86(s,3H),2.89(t,J=5.8Hz,2H),2.72(s,3H),2.21(s,6H), these data correspond to the H NMR spectrum of osimertinib. On the other hand, 1H NMR (500 MHz, DMSO-d6) δ 6.22 (d, J = 4.3 Hz, 1H), 5.43 (s, 1H), 4.97 (t, J = 3.9 Hz, 1H), 2.19-2.13 (m, 1H), 2.01-1.96 (m, 1H), 1.88-1.76 (m, 2H), 1.68-1.62 (m, 1H), 1.59-1.53 ​​(m, 1H), 1.42-1.3 1(m,3H),1.27(d,J=6.8Hz,3H),1.16-1.09(m,1H),0.89(t,J=6.6Hz,3H),0.83(d,J=7.3Hz,3H),0.78(d,J=7.2Hz,1H) are the H NMR spectrum data of dihydroartemisinin, among which two of the three hydrogen atoms at 2.29(t,J=5.8Hz,3H) belong to osimertinib and one belongs to dihydroartemisinin. Through calculation, we concluded that the molar ratio of API (active pharmaceutical ingredient) to CCF (cocrystal former) in the cocrystal molecule is 1:1.

[0062] The X-ray diffraction (XRD) pattern of ZM851 is shown below: Figure 2 As shown. The X-ray diffraction pattern of osimertinib shows characteristic peaks at 2θ angles of 9.16°, 10.34°, 15.88°, 16.6°, 17.58°, 20.44°, 20.82°, 22.74°, 23.14°, 24.1°, 25.98°, 26.54°, and 27.3°. The X-ray diffraction pattern of dihydroartemisinin has characteristic peaks at 2θ angles of 7.72°, 9.28°, and 11.16°. The X-ray diffraction pattern of ZM851 shows new characteristic peaks at 2θ angles of 5.88°, 15.06°, 18.36°, 24.62°, and 25.26°, which preliminarily indicates that a new crystalline phase has been formed.

[0063] like Figure 3 As shown in Table 1, the FT-IR analysis of ZM851 revealed that the infrared characteristic peaks of the osimertinib and dihydroartemisinin cocrystal molecule have changed significantly compared with the API. Specifically, some characteristic peaks of the API have shifted, weakened in intensity, and changed in morphology. For example, the characteristic peak of the amide spectrum of the osimertinib API is located at 745.78 cm -1 (γ N-H ) and 1533.96cm -1 (δ N-H ), and in the eutectic they shift to 733.81 cm -1 (γ N-H ) and 1571.71cm -1 (δ N-H Similarly, the characteristic peak of the -OH band in dihydroartemisinin is at 3380.76 cm -1( ν O-H ) and 661.99cm -1 (δ O-H ), while in the eutectic molecule, these characteristic peaks move to the low frequency direction to 3375.97 cm -1 (ν O-H ) and 594.96cm -1 (δ O-H The reason for this change is that after the cocrystal is formed, -NH in osimertinib forms a strong NH…O hydrogen bond with -OH in the ligand, which leads to a decrease in the vibration frequency of -OH and -NH and a broadening of the spectral band, which in turn causes the characteristic peaks of the API to undergo different degrees of red shift.

[0064] Table 1

[0065] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Dihydroartemisinin (cm -1 )]]> <![CDATA[ZM851(cm -1 )]]> <![CDATA[v N-H ]]> 3428.64 - - <![CDATA[v O-H ]]> - 3380.76 3375.97 (peak becomes blunt) vC=0 1576.49 - 1676.24 <![CDATA[δ N-H ]]> 1533.96 - 1571.71 <![CDATA[δ O-H ]]> - 661.99 594.96 <![CDATA[γ N-H ]]> 745.78 - 733.81

[0066] like Figure 4As shown in Table 2, the Raman spectrum of ZM851 reveals that the Raman characteristic peaks of the osimertinib and dihydroartemisinin cocrystal molecule have significantly shifted, weakened, or even disappeared compared with the API. Specifically, the amide characteristic peak of the osimertinib API is located at 3205.13 cm -1 (ν N-H )、1621.07cm -1 (ν C＝O )、989.06cm -1 (γ N-H ) and 1555.4cm -1 (δ N-H ). In the eutectic molecule, these characteristic absorption peaks move to the low frequency direction to 3102.13 cm -1 (ν N- H), 1576.94cm -1 (ν C＝O )、976.54cm -1 (γ N-H ) and 1513.97cm -1 (δ N-H ). The reason for this phenomenon is that after the cocrystal is formed, -NH in osimertinib forms a strong NH…O hydrogen bond with -OH in the ligand, which leads to a decrease in the vibration frequency of -OH and -NH, and then causes the characteristic peaks of the API to undergo different degrees of red shift.

[0067] Table 2

[0068] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Dihydroartemisinin (cm -1 )]]> <![CDATA[ZM851(cm -1 )]]> <![CDATA[ν N-H ]]> 3205.13 - 3102.13 <![CDATA[ν O-H ]]> - 3245.76 - <![CDATA[v C-N ]]> 1353.63 - 1400.89 <![CDATA[δ C-N ]]> 1538.3 - 1464.09 <![CDATA[δ N-H ]]> 1555.4 - 1513.97 <![CDATA[v C＝O ]]> 1621.07 - 1576.94 <![CDATA[γ O-H ]]> - 722.22 - <![CDATA[γ N-H ]]> 989.06 - 976.54

[0069] Furthermore, the thermogravimetric-differential scanning calorimetry (TG-DSC) analysis of ZM851 showed Figure 5 As shown, the four-step weight loss behavior of TGA is revealed. The initial weight loss may be due to the release of water molecules. The second weight loss is attributed to the decomposition of dihydroartemisinin. After losing the cocrystal former, the osimertinib molecules in osimertinib-dihydroartemisinin begin to decompose upon further heating. The DSC curve shows that ZM851 has four endothermic peaks located at 63°C, 83°C and 142°C, respectively. The first two peaks correspond to the endothermic process of the cocrystal removing solvent and water, while the peak at 142°C corresponds to the melting peak of osimertinib-dihydroartemisinin. The four weight loss steps in thermogravimetric analysis (TG) indicate that the osimertinib-dihydroartemisinin cocrystal corresponds to the DSC curve. Based on the above analysis, ZM851 is a cocrystal, in which the ratio of API:CCF is 1:1, and the melting point of the cocrystal is 142°C.

[0070] 2. ZM852 structural characterization analysis

[0071] like Figure 6As shown, the ZM852 cocrystal H NMR spectrum showed the following data: 1H NMR (500MHz, DMSO-d6) δ10.20 (s, 1H), 9.15 (s, 1H), 8.6 (s, 1H), 8.33 (d, J = 5.3 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.26-7.21 (m, 2H), 7.18-7.12 (m, 1H) ,7.04(s,1H),6.47-6.40(m,1H),6.30-6.24(m,1H),5.79-5.75(m,1H),3.91(s,3H),3.86(s,3H),2.89(t,J=5.9Hz,2H),2.72(s,3H),2.30(s,2H),2.21(s,6H), these data correspond to the hydrogen nuclear magnetic resonance spectrum of osimertinib. The 1H NMR (500MHz, DMSO-d6) δ9.55 (s, 1H), 8.50 (s, 1H), 8.13-8.10 (m, 1H), 7.82-7.77 (m, 2H), 7.44 (t, J = 9.1Hz, 1H), 7.20 (s, 1H), 4.18 (t, J = 6.3Hz, 2H), 3.94 (s, 3H), 3.58 (t, J = 4.6Hz, 4H), 2.47 (d, J = 7.0Hz, 2H), 2.39 (s, 4H), 1.99 (m, J = 6.6Hz, 2H) correspond to the H NMR spectrum of gefitinib. Through calculation, we concluded that the molar ratio of API (active pharmaceutical ingredient) to CCF (cocrystal former) in the cocrystal molecule is 1:1.

[0072] The X-ray diffraction (XRD) pattern of ZM852 is as follows: Figure 7 As shown, the X-ray diffraction pattern of osimertinib shows characteristic peaks at 2θ angles of 10.34°, 15.88°, 16.6°, 17.58°, 20.44°, 20.82°, 22.74°, 23.14°, 24.1°, 25.98°, 26.54°, and 27.3°. The X-ray diffraction pattern of gefitinib shows characteristic peaks at 2θ angles of 15.8°, 17.62°, 18.6°, 19.3°, 20.6°, 22.44°, 24°, and 26.34°. The X-ray diffraction pattern of ZM852 shows characteristic peaks at 2θ angles of 10.4°, 20.52°, 20.9°, 23.24°, 24.26°, and 26.36°. Since no obvious new characteristic peaks appear in the product, it is impossible to determine whether a new crystalline phase has been formed.

[0073] like Figure 8As shown in Table 3, ZM852 FT-IR analysis revealed that the infrared spectrum characteristic peaks of osimertinib and gefitinib cocrystal molecules were significantly shifted, weakened and changed compared with the API. Specifically, the amide characteristic peak of osimertinib API was located at 1533.96 cm-1 (δ N-H ) and 1576.49cm -1 (ν C＝O stretching vibration), while in the eutectic molecule, these characteristic absorption peaks move to the high frequency region to 1622.59 cm -1 (δ N-H ) and 1669.51cm -1 (ν C＝O ). Similarly, the secondary amine band of gefitinib API appears at 1575.67 cm -1 (δ N-H ) and 1630.41cm -1 (ν C＝O ), and in the eutectic molecule, these characteristic absorption peaks also shifted to a higher frequency, reaching 1622.59 cm -1 (δ N-H ) and 1669.51cm -1 (ν C＝O ). The reason for this change is attributed to the hydrogen bonding effect produced after the formation of the cocrystal, which causes the characteristic peaks of the raw material to shift to varying degrees.

[0074] Table 3

[0075] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Gefitinib (cm -1 )]]> <![CDATA[ZM852(cm -1 )]]> <![CDATA[v N-H ]]> 3428.64 3397.83 3431.72 <![CDATA[v O-H ]]> - - - <![CDATA[ν C＝O ]]> 1576.49 1630.41 1669.51 <![CDATA[δ N-H ]]> 1533.96 1575.67 1622.59 <![CDATA[δ O-H ]]> - - - <![CDATA[γ N-H ]]> - - -

[0076] like Fig. 9 As shown in Table 4, the Raman spectrum of ZM852 reveals that the Raman characteristic peaks of the osimertinib and gefitinib cocrystal molecules have undergone significant shifts, weakening, or even disappearance compared to the raw material drug. Specifically, the characteristic peak of the amide spectrum of the osimertinib raw material drug is located at 1538.3 cm -1 (δ C-N )、1555.4cm -1 (δ N-H )、1621.07cm -1 (ν C＝O ). In the eutectic molecule, these characteristic absorption peaks move to the low frequency direction to 1535.56cm -1 (δ C-N )、1552.67cm -1 (δ N-H )、1612.85cm -1 (ν C＝O ). For gefitinib API, its secondary amine spectrum characteristic peak is located at 1342 cm -1 (ν C-N)、1616.96cm -1 (δ N-H )、784.54cm -1 (γ N-H ), while in the eutectic, these characteristic absorption peaks are red-shifted to 1340.62 cm -1 (ν C-N )、1552.67cm -1 (δ N-H )、774.28cm -1 (γ N-H ). The reason for this phenomenon is that after the cocrystal is formed, -C=O in osimertinib forms a strong NH…O hydrogen bond with one of the -NH in the ligand, which leads to a decrease in the vibration frequency of -C=O and -NH, and then causes the characteristic peaks of the API to undergo different degrees of red shift.

[0077] Table 4

[0078] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Gefitinib (cm -1 )]]> <![CDATA[ZM852(cm -1 )]]> <![CDATA[v N-H ]]> - - - <![CDATA[v O-H ]]> - - - <![CDATA[v C-N ]]> 1353.63 1342 1340.62 <![CDATA[δX C-N ]]> 1538.3 - 1535.56 <![CDATA[δ N-H ]]> 1555.4 1616.96 1552.67 vc=o 1621.07 - 1612.85 <![CDATA[γ N-H ]]> 989.06 784.54 774.28

[0079] like Fig.10 As shown in Figure 1, TG-DSC analysis of ZM852 revealed a two-stage weight loss process. The first step corresponds to the desolvation and dehydration processes, which are shown as exothermic peaks at 86.3°C and 106°C in the DSC graph, respectively. The second step may be related to the decomposition of osimertinib in ZM852, and its exothermic peak appears at 169°C. In addition, the endothermic peak at 351°C marks the melting process of ZM852. Comprehensive analysis shows that ZM852 did not form a eutectic.

[0080] 3. Structural characterization and analysis of ZM853

[0081] like Fig.11As shown in Figure 1, the ZM853 cocrystal H NMR spectrum shows the H spectrum data of osimertinib and ibrutinib. NMR (500MHz, DMSO-d6) data: δ10.21 (s, 1H), 9.16 (s, 1H), 8.68 (s, 1H), 8.33 (d, J = 5.3 Hz, 1H), 7.90 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.26-7.22 (m, 2H), 7.04 (s, 1H), 6.46-6.40 (m, 1H), 6.28 (d, J = 15.8 Hz, 1H), 5.79-5.75 (m, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 2.89 (t, J = 5.8 Hz, 2H), 2.72 (s, 3H), 2.29 (t, J = 5.9 Hz, 3H), 2.21 (s, 6H). Ibrutinib 1H NMR (500 MHz, DMSO-d6) data: δ7.66 (t, J = 6.8 Hz, 2H), 7.43 (t, J = 7.8 Hz, 2H), 6.89-6.83 (m, 1H), 6.75-6.68 (m, 1H), 6.17-6.03 (m, 1H), 5.71 (d, J = 10.5 Hz, 1H), 4.70 (d, J = 23.5 Hz, 1H), 4.56 (d, J = 12.6 Hz, 1H), 4.19 (d, J = 12.2 Hz, 1H), 4.07 (d, J = 13.7 Hz, 1H), 3.20 (q, J = 14.0, 13.2 Hz, 1H), 2.13 (s, 1H), 1.92 (s, 1H), 1.59 (s, 1H). In the region of 8.28-8.21 (m, 2H), two hydrogen atoms belong to osimertinib and ibrutinib, respectively. In the region of 7.21-7.10 (m, 7H), one of the seven hydrogen atoms comes from osimertinib and the remaining six come from ibrutinib. By calculation, the molar ratio of API to CCF in the cocrystal molecule is 1:1.

[0082] like Fig.12As shown, in the X-ray diffraction pattern of ZM853, the characteristic peaks of osimertinib at 2θ angle appear at 10.34°, 15.88°, 16.6°, 17.58°, 20.44°, 20.82°, 22.74°, 23.14°, 24.1°, 25.98°, 26.54°, and 27.3°. The characteristic peaks of the X-ray diffraction pattern of ibrutinib at 2θ angle appear at 10.58°, 16.02°, 18.22°, 18.9°, 19.72°, 20.46°, 21.22°, 21.64°, 22.32°, 23.44°, and 26.44°. The characteristic peaks of the X-ray diffraction spectrum of ZM853 at 2θ angle appear at 18.56°, 19.86°, 20.6°, 20.98°, 21.36°, 26.1°, and 27.48°. No obvious new characteristic peaks appear in the product, so it is impossible to determine whether a new crystal phase is formed.

[0083] The Fourier transform infrared spectroscopy (FT-IR) data of ZM853 are shown in Figure 2. Fig.13 As shown in Table 5, the infrared characteristic peaks of the osimertinib and ibrutinib cocrystal molecules have undergone significant shifts, weakening and changes compared with the API. For example, the characteristic peak of the amide spectrum of the osimertinib API is located at 745.78 cm -1 (γ N-H ) and 1576.49cm -1 (ν C＝O ), while in the eutectic molecule, these characteristic absorption peaks move to the low frequency direction to 611.72 cm -1 (γ N-H ) and 1636.34cm -1 (ν C＝O ). The characteristic peak of Ibrutinib is 618.10 cm -1 (γ N-H ) and 1620.38cm -1 (δ N-H ), while in the eutectic, these characteristic peaks shift to the low frequency direction to 611.72 cm -1 (γ N-H ) and 1614.00cm -1 (δ N-H ). The cause of this phenomenon is that the formation of hydrogen bonds leads to a decrease in vibration frequency, which causes the characteristic peaks of the API to undergo red shifts to varying degrees.

[0084] Table 5

[0085] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Ibrutinib (cm -1 )]]> <![CDATA[ZM853(cm -1 )]]> <![CDATA[v N-H ]]> 3428.64 - - <![CDATA[v O-H ]]> - - - <![CDATA[ν C＝O ]]> 1576.49 1639.53 1636.34 <![CDATA[δ N-H ]]> 1533.96 1620.38 1614.00 <![CDATA[δ O-H ]]> - - - <![CDATA[γ N-H ]]> 745.78 618.10 611.72

[0086] like Fig.14As shown in Table 6, the Raman data of ZM853 revealed that the Raman characteristic peaks of the osimertinib and ibrutinib cocrystal molecule were significantly shifted, weakened, and changed compared with the API. Specifically, the amide characteristic peak in the osimertinib API is located at 1538.3 cm -1 (δ C-N ) and 1555.4cm -1 (δ N-H ), while in the eutectic molecule, these characteristic absorption peaks move to the high frequency direction to 1554.13 cm -1 (δ C-N ) and 1576.01cm -1 (δ N-H In addition, the C=O absorption peak in osimertinib is located at 1621.07 cm -1 (ν C＝O ), while in the eutectic, the characteristic peak of C=O is 1612.85 cm -1 (ν C＝O The characteristic peak of primary amine of Ibrutinib API is at 1608 cm -1 (δ N-H ), while in the eutectic, this characteristic absorption peak red-shifts to 1576 cm -1 (δ N-H The reason for this change is that after the cocrystal is formed, the -C=O in osimertinib forms a stronger NH…O bond with one of the -NH in the ligand.

[0087] Table 6

[0088]

[0089]

[0090] Further analysis of the TG-DSC data of ZM853 (see Fig.15 ), three endothermic peaks can be observed on the DSC graph. Among them, the peak corresponding to 104°C represents the dehydration process of the eutectic molecule, while the peak at 293°C corresponds to the melting peak of the eutectic molecule. From the TG graph, it can be seen that between 293°C and 483°C, the weight loss is 36.56%. The weight loss in this stage corresponds to the melting process of ZM853 reaching the melting point. The weight loss before 104°C in the TG graph is attributed to the process of the eutectic molecule removing water molecules.

[0091] Based on the above analysis, it can be concluded that ZM853 did not form a eutectic.

[0092] 5. Summary

[0093] The present invention uses drug cocrystal technology to successfully design and synthesize three compounds. Through a series of characterization techniques, including 1H NMR, XRD, FT-IR, Raman and TG-DSC analysis, we confirmed one molecule that forms a cocrystal with osimertinib, namely ZM851. Specifically, the molar ratio of active pharmaceutical ingredient (API) to cocrystal former (CCF) in the ZM851 cocrystal molecule is 1:1.

[0094] Example 3 In vitro antitumor activity study

[0095] 1. Principle of CCK-8 experiment

[0096] Cell Counting Kit-8 (CCK-8) reagent is a simple and accurate method for cell proliferation and toxicity analysis. Its basic principle is based on the WST-8 contained in the reagent, whose chemical name is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2H-tetrazolyl monosodium salt. Under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS), the dehydrogenase in the cell reduces WST-8 to a highly water-soluble yellow formazan product (Formazan Dye). The amount of formazan generated is proportional to the number of living cells. This feature allows the CCK-8 reagent to be directly applied to cell proliferation and toxicity analysis.

[0097] 2. Experimental methods and steps

[0098] Cell source: The cell lines used in this experiment were purchased from the Chinese Academy of Sciences. PC-9, NCI-H1975, and NCI-H1650 cells were cultured in complete medium prepared with RPMI-1640. The RPMI-1640 complete medium used for PC-9 cells is composed of basal medium, 10% fetal bovine serum (FBS), and 1% 100U / mL penicillin and 100μg / mL streptomycin (P / S). The medium formula used for NCI-H1975 and NCI-H1650 cells is the same. The complete medium for NCI-HCT116 cells consists of RPMI-1640 medium without Hepes, 10% fetal bovine serum, and 1% double antibody. The cell cryopreservation solution consists of 55% basal medium, 40% FBS, and 5% DMSO and is stored in liquid nitrogen. The cells were cultured in an environment of 95% air, 37°C, and 5% CO2, and were ensured to be free of mycoplasma and chlamydia contamination.

[0099] In vitro cytotoxicity test: cells in the logarithmic growth phase were selected and cultured at 7×10 3The cells were inoculated in a 96-well plate at a density of 100 μL and incubated for 24 hours at 37°C and 5% CO2 to maintain normal physiological pH. After cell attachment, different concentrations of test compounds were added to the three groups of wells, and 0.1% DMSO was used as a control. After 72 hours, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 0.5 hours. The absorbance was measured at a wavelength of 450 nm using an ELISA reader, and the 50% cell growth inhibition concentration (IC50, Half Maximal Inhibitory Concentration) was calculated using GraphPad Prism software. All data were repeated three times.

[0100] Establishment of drug-resistant cell lines: Induce drug resistance by increasing drug concentration to establish osimertinib-resistant cell lines. PC-9 cell lines were used as parental cells, their IC50 values ​​were tested, and concentrations close to this value were selected for culture. After determining the optimal starting concentration, the drug concentration was gradually increased to induce drug resistance in PC-9 cell lines. The culture medium was replaced regularly and the growth of cells was observed under a microscope. After the cells showed stable tolerance, the drug concentration was appropriately increased. By repeatedly replacing the culture medium, passage, and increasing the drug concentration, the resistance index of the drug-resistant cell line to osimertinib exceeded 15, and the resistance was considered to be successfully established. The successfully constructed drug-resistant cell line is represented by PC-9OR.

[0101] IC50 test is used to evaluate the toxicity and tolerance of drugs to cells, where lower IC50 values ​​mean stronger drug inhibitory effects. In this article, we evaluated the sensitivity of cells to osimertinib by calculating the resistance index (RI). The resistance index (RI) is calculated as follows: the IC50 value of resistant cells divided by the IC50 value of parental cells. According to the RI value, drug resistance is graded as follows: 1-5 is low resistance; 5-15 is moderate resistance; and more than 15 is high resistance. We tested the activity of five cell lines, including PC-9, PC-9OR (osimertinib acquired resistance cell line), H1975 (osimertinib highly sensitive cell line), H1650 (osimertinib intrinsic resistance cell line) and HCT116 (human colon cancer cell line).

[0102] Table 7

[0103]

[0104]

[0105] Table 8

[0106]

[0107] Table 9

[0108]

[0109] We studied the in vitro antitumor activity of a newly synthesized cocrystal molecule and two non-cocrystal compounds. The results showed that compared with PC-9, the sensitivity of osimertinib to the drug-resistant cell line PC-9OR was significantly reduced, with IC50 values ​​of 0.0283±0.0075μM and 0.7232±0.2166μM, respectively, and the drug resistance index reached 25.6, which was statistically significant (P<0.01). Further comparison showed that the antitumor activity of compound ZM851 was significantly higher than that of osimertinib, with an IC50 value of 0.1237±0.0359μM, and there was also a significant statistical difference between the two (P<0.01). . In the H1975 cell line, the IC50 of osimertinib was 3.4837±1.4693μM, while the cocrystal molecule ZM851 showed better antitumor activity. For the intrinsically resistant H1650 cell line, the IC50 of osimertinib was 4.8187±0.1301μM, and the cocrystal molecule ZM851 also showed better antitumor activity. In the HCT116 human colon cancer cell line, the IC50 of osimertinib was 0.4954±0.1443μM, while the antitumor activity of the cocrystal molecule ZM851 was improved. In the HCT116 cell line, the antitumor activity of ZM851 was 1.2 times that of osimertinib. Compared with ZM851, the mixture was about 26.1 times less active against the resistant cell line PC-9OR. These research results show that the osimertinib cocrystal molecule is more active against the resistant strain PC-9OR than its mixture, which preliminarily reveals the potential advantages of cocrystal molecules in antitumor activity.

[0110] 3. Summary

[0111] The present invention evaluated the in vitro anti-non-small cell lung cancer activity of one cocrystal molecule and two non-cocrystal compounds obtained. The results showed that the activity of the cocrystal molecule ZM851 on the PC-9OR acquired drug-resistant cell line was enhanced by about 5.85 times compared with osimertinib. For the acquired drug-resistant cell line PC-9OR, the anti-tumor activity of the cocrystal molecule ZM851 was more than 10 times higher than that of its non-cocrystal mixture. This preliminarily proves that new anti-tumor drugs can be developed to solve the problem of osimertinib resistance through drug cocrystal technology.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A compound ZM851, characterized in that The chemical structural formula of the compound ZM851 is:

2. A method for preparing the compound ZM851 according to claim 1, characterized in that: include: Osimertinib, dihydroartemisinin and a mixed solvent are mixed and heated and stirred to dissolve, and then n-hexane is added, and the precipitated yellow solid is the compound ZM851.

3. The preparation method according to claim 2, characterized in that: The mixed solvent is acetone and water.

4. The preparation method according to claim 3, characterized in that: The volume ratio of acetone to water is 15:

1.

5. The preparation method according to claim 2, characterized in that: The temperature at which osimertinib, L-proline and the mixed solvent are mixed and heated and stirred for dissolution is at least 60°C.

6. A use of the compound ZM851 according to claim 1, characterized in that: The application includes at least one of the following: A1) Application in the preparation of anti-tumor drugs; A2) Use in the preparation of a pharmaceutical composition for preventing or treating drug-resistant tumors; A3) Application in the preparation of drug resistance sensitizers for anti-tumor drugs.

7. A co-crystal drug, characterized in that: The active ingredient of the co-crystal drug is the compound ZM851 described in claim 1.

8. Use of the co-crystal drug according to claim 7, characterized in that: The application includes at least one of the following: A1) Application in the preparation of anti-tumor drugs; A2) Use in the preparation of a pharmaceutical composition for preventing or treating drug-resistant tumors; A3) Application in the preparation of drug resistance sensitizers for anti-tumor drugs.

Images