Co-crystal medicine ZM889 and application thereof in lung cancer resistance

Abstract

The invention relates to the technical field of biological medicine, and discloses a co-crystal medicine ZM889 and application thereof in lung cancer resistance, a preparation method of the co-crystal medicine ZM889 comprises the following steps: mixing osimertinib, reserpine and a mixed solvent, heating, stirring and dissolving, then adding n-hexane, and separating out a yellow solid, namely the co-crystal medicine ZM889. The antitumor activity of the eutectic molecule ZM889 disclosed by the invention is enhanced compared with that of a non-eutectic counterpart of the eutectic molecule ZM889; in an in-vitro experiment, the eutectic drug ZM889 shows a remarkable inhibition effect on an osimertinib drug-resistant cell strain. According to the eutectic medicine ZM889 and the preparation method thereof, a new strategy is provided for the field of anti-lung cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cocrystal drug ZM889 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 ZM889 and its application in anti-lung cancer, aiming to overcome the drug resistance problem of existing therapeutic drugs and provide a new treatment strategy.

[0005] The present invention provides a compound ZM889, the chemical structure of which is:

[0006]

[0007] The present invention also provides a method for preparing the compound ZM889, comprising:

[0008] Osimertinib, reserpine 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 ZM889.

[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, reserpine 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 ZM889, 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 ZM889.

[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 prepared one cocrystal molecule and two non-cocrystal compounds. Among these molecules, the cocrystal molecule ZM889 showed much higher anti-tumor activity than its non-cocrystal counterparts. Further in vitro experiments showed that ZM889 had a significant inhibitory effect on osimertinib-resistant cell lines.

[0023] The cocrystal drug ZM889 showed significant inhibitory effects on osimertinib-resistant cell lines in in vitro experiments, suggesting that it may provide a new treatment option for lung cancer patients in clinical treatment. The use of cocrystal drug ZM889 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 ZM889 and its preparation method of the present invention introduce a new strategy into 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 is the 1HNMR spectrum of compound ZM886;

[0027] Figure 2 is the XRD spectrum of compound ZM886;

[0028] Figure 3 is the Fourier transform infrared spectrum of compound ZM886;

[0029] Figure 4 is the Raman spectrum of compound ZM886;

[0030] Figure 5 TG spectrum and DSC spectrum of compound ZM886;

[0031] Figure 6 is the 1HNMR spectrum of compound ZM887;

[0032] Figure 7 is the XRD spectrum of compound ZM887;

[0033] Figure 8 is the Fourier transform infrared spectrum of compound ZM887;

[0034] Fig. 9 is the Raman spectrum of compound ZM887;

[0035] Fig.10 TG spectrum and DSC spectrum of compound ZM887;

[0036] Fig.11 is the 1HNMR spectrum of compound ZM889;

[0037] Fig.12 is the XRD spectrum of compound ZM889;

[0038] Fig.13 is the Fourier transform infrared spectrum of compound ZM889;

[0039] Fig.14 is the Raman spectrum of compound ZM889;

[0040] Fig.15 TG spectrum and DSC spectrum of compound ZM889. 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 ZM886

[0044] Weigh osimertinib (200 mg, 0.4 mmol) and omeprazole (138.2 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 white-brown solid precipitates. Filter and wash to obtain 250 mg of a white-brown powder, which is compound ZM886, with the chemical formula:

[0045]

[0046] 2. Compound ZM887

[0047] Weigh osimertinib (200 mg, 0.4 mmol) and piperine (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 light brown solid. Filter and wash to obtain 240 mg of a light brown powder, which is compound ZM887, with the chemical formula:

[0048]

[0049] 3. Preparation of Compound ZM889

[0050] Weigh osimertinib (200 mg, 0.4 mmol) and reserpine (122 mg, 0.2 mmol) in a reaction tube, add 1.5 mL of dichloromethane, heat to 40 °C and stir to dissolve. Add 3 mL of n-hexane, slowly cool to room temperature, stir overnight, and a brown solid precipitates. Filter and wash to obtain 240 mg of brown powder, which is compound ZM889, 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 ZM886, ZM887 and ZM889.

[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 using 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 , used for conventional powder compression tests and liquid pool tests. The experimental data in this article are obtained by KBr tableting method, grinding KBr to 2μm with an agate mortar. Add a certain amount of sample (2-5mg) and KBr (100-120mg), grind and mix until there are no obvious particles. Place it in a mold and then press it 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 ZM886

[0061] like Figure 1 As shown, the nuclear magnetic resonance hydrogen spectrum (1H NMR, 500 MHz, DMSO-d6) of ZM886 showed the following characteristics: 10.21 (singlet, 1H), 9.16 (singlet, 1H), 8.69 (singlet, 1H), 8.34 (doublet, J = 5.3 Hz, 1H), 8.25 (doublet, J = 8.1 Hz, 1H), 7.91 (singlet, 1H), 7.30-7.21 (multiplet, 2H), 7.16 (triplet, J = 7.5 Hz, 1 H), 7.05 (singlet, 1H), 6.49-6.40 (multiplet, 1H), 6.29 (doublet, J = 16.9 Hz, 1H), 5.78 (doublet, J = 9.4 Hz, 1H), 3.92 (singlet, 3H), 3.87 (singlet, 3H), 2.90 (triplet, J = 5.7 Hz, 2H), 2.73 (singlet, 3H), 2.31 (triplet, J = 5.8 Hz, 2H). These data correspond to the H NMR spectrum of osimertinib. In addition, the nuclear magnetic resonance hydrogen spectrum (1H NMR, 500MHz, DMSO-d6) data of omeprazole are: δ13.40 (singlet, 1H), 8.19 (singlet, 1H), 7.10 (singlet, 1H), 6.93 (doublet, J = 8.8Hz, 1H), 4.77 (doublet, J = 13.5Hz, 1H), 4.69 (doublet, J = 13.6Hz, 1H), 3.81 (singlet, 3H), 3.69 (singlet, 3H). The two hydrogen atoms at 7.54 (triplet, J = 8.6Hz, 2H) belong to osimertinib and omeprazole respectively; the 12 hydrogen atoms at 2.26-2.09 (multiplet, 12H), 6 of which come from osimertinib and the other 6 from omeprazole. By calculation, 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 ZM886 is shown below: Figure 2As 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 omeprazole shows characteristic peaks at 2θ angles of 9.24°, 11.1°, 12.32°, 15.76°, 17.14°, 19.3°, 19.72°, 20.4°, and 23.88°. However, the X-ray diffraction pattern of ZM886 does not show new characteristic peaks at these angles, and it is preliminarily judged that no new crystalline phase is generated.

[0063] like Figure 3 As shown in Table 1, the ZM886 FT-IR data revealed that the infrared characteristic peaks of the osimertinib and omeprazole cocrystal molecule were significantly shifted, weakened, and changed compared with the API. Specifically, the amide characteristic peak of the osimertinib API was located at 3428.64 cm -1 (ν N-H )、1576.49cm -1 (ν C＝O )、1533.96cm -1 (δ N-H ), while in the eutectic molecule, these characteristic absorption peaks shift to 3436.11 cm -1 (ν N-H )、1589.33cm -1 (ν C＝O )、1540.08cm -1 (δ N-H ). The characteristic peak of omeprazole is 3460.74 cm -1 (ν N-H )、1628.73cm -1 (δ N-H ), the characteristic absorption peak in the eutectic molecule shifts to the low frequency direction to 3436.11 cm -1 (ν N-H )、1540.08cm -1 (δ N-H ). The reason for this change is that after the formation of hydrogen bonds, the characteristic peaks of the API shift to varying degrees.

[0064] Furthermore, the ZM886 Raman data ( Figure 4 and Table 2) show that the Raman characteristic peaks of osimertinib and omeprazole cocrystal molecules also showed obvious shift, weakening or even disappearance compared with the Raman characteristic peaks of the API. The amide characteristic peaks of osimertinib API include 3205.13 cm -1 (ν N-H)、1353.63cm -1 (ν C-N )、1538.3cm -1 (δ C-N )、1621.07cm -1 (ν C＝O )、989.06cm -1 (γ N-H ), while the characteristic absorption peak in the eutectic molecule shifts to 3213.95 cm -1 (ν N-H )、1353.48cm -1 (ν C-N )、1418.60cm -1 (δ C-N )、1618.60cm -1 (ν C＝O )、990.70cm -1 (γ N-H ). This phenomenon is also attributed to the formation of hydrogen bonds between APIs, which causes the characteristic peaks to shift to varying degrees.

[0065] Table 1

[0066] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Omeprazole (cm -1 )]]> <![CDATA[ZM886(cm -1 )]]> <![CDATA[v N-H ]]> 3428.64 3460.74 3436.11 <![CDATA[v O-H ]]> - - - <![CDATA[v C＝O ]]> 1576.49 - 1589.33 <![CDATA[δ N-H ]]> 1533.96 1628.73 1540.08 <![CDATA[δ O-H ]]> - - - <![CDATA[γ N-H ]]> 745.78 - -

[0067] Table 2

[0068]

[0069]

[0070] However, the TG-DSC data of ZM886 showed that ( Figure 5 ), there is no obvious endothermic peak in the DSC curve, so the melting point cannot be determined. Based on the above analysis, it can be concluded that ZM886 does not form a eutectic.

[0071] 2. Structural characterization and analysis of ZM887

[0072] The nuclear magnetic resonance hydrogen spectrum of ZM887 (1H NMR, 500 MHz, DMSO-d6) is as follows Figure 6As shown, the spectrum shows the following chemical shifts: δ10.21 (s, 1H), 9.16 (s, 1H), 8.69 (s, 1H), 8.34 (d, J = 5.3 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.05 (s, 1H), 6.48-6.40 (m, 1H), 6.28 (d, J = 16.9 Hz, 1H), 5.78 (d, J = 10.2 Hz, 1H), 3.93 (s, 3H), 3.87 (s, 3H), 2.90 (t, J = 5.8 Hz, 2H), 2.73 (s, 3H), 2.30 (t, J = 5.9 Hz, 2H), 2.22 (s, 6H). These data correspond to the H NMR spectrum of osimertinib. Another set of data: δ7.01-6.88 (m, 4H), 6.68 (d, J = 14.6 Hz, 1H), 6.05 (s, 2H), 3.52 (s, 4H), 1.61 (s, 2H), 1.48 (s, 4H) belongs to the H NMR spectrum of piperine. At the chemical shift of 7.29-7.12 (multiple peaks, 5H), there are 3 hydrogen atoms belonging to osimertinib and 2 belonging to piperine. By calculation, the molar ratio of API (active pharmaceutical ingredient) to CCF (cocrystal former) in the cocrystal molecule is 1:1.

[0073] The X-ray diffraction pattern (XRD) of ZM887 is as follows Figure 7 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 piperine shows characteristic peaks at 2θ angles of 14.12°, 14.76°, 15.94°, 19.56°, 21.32°, 22.32°, 22.54°, 24.18°, 25.8°, and 28.22°. However, the X-ray diffraction pattern of ZM887 does not show new characteristic peaks at these angles, and it is preliminarily judged that no new crystalline phase is generated.

[0074] The Fourier transform infrared spectrum (FT-IR) of ZM887 is shown in Figure 8 As shown in Table 3. Compared with the API, the infrared characteristic peaks of the cocrystal molecule were significantly shifted, weakened or even disappeared. The amide characteristic peaks of osimertinib API include 3428.64 cm -1 (ν N-H Extension vibration), 1576.49cm -1 (ν C＝O Extension vibration), 1533.96cm-1 (δ N-H bending vibration), while in the eutectic molecule, these characteristic absorption peaks shift to 3438.21 cm -1 (ν N-H Extension vibration), 1671.45cm -1 (ν C＝O Extension vibration), 1637.94cm -1 (δ N-H Bending vibration). The characteristic peak of piperine is 3447.79 cm -1 (ν N-H Extension vibration), 1585.27cm -1 (δ N-H Bending vibration), 1637.94cm -1 (ν C＝O stretching vibration), while in the eutectic molecule, these characteristic absorption peaks shift to 3438.21 cm -1 (ν N-H Extension vibration), 1637.94cm -1 (δ N- H bending vibration), 1671.45cm -1 (ν C＝O Stretching vibration). This phenomenon occurs due to the formation of hydrogen bonds, which causes the characteristic peaks of the API to shift to varying degrees.

[0075] Table 3

[0076] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Piperine (cm -1 )]]> <![CDATA[ZM887(cm -1 )]]> <![CDATA[v N-H ]]> 3428.64 3447.79 3438.21 <![CDATA[v O-H ]]> - - - <![CDATA[v C＝O ]]> 1576.49 1637.94 1671.45 <![CDATA[δ N-H ]]> 1533.96 1585.27 1637.94 <![CDATA[δ O-H ]]> - - - <![CDATA[γ N-H ]]> 745.78 - -

[0077] Table 4

[0078] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[ZM887(cm -1 )]]> <![CDATA[v N-H ]]> 3205.13 - <![CDATA[v O-H ]]> - - <![CDATA[v C-N ]]> 1353.63 1367.44 <![CDATA[δ C-N ]]> 1538.3 1446.51 <![CDATA[δ N-H ]]> 1555.4 1590.7 <![CDATA[v C＝O ]]> 1621.07 1627.91 <![CDATA[γ O-H ]]> - - <![CDATA[γ N-H ]]> 989.06 -

[0079] like Fig. 9 As shown in Table 4, the Raman data of ZM887 revealed that the Raman characteristic peaks of osimertinib and piperine cocrystal molecules were significantly shifted, weakened in intensity, and even disappeared in some peaks compared with the API. Specifically, the amide band of osimertinib API was at 3205.13 cm -1 (ν N-H )、1353.63cm -1 (ν C-N )、1538.3cm -1 (δ C-N )、1555.4cm -1 (δ N-H )、1621.07cm -1( ν C＝O )、989.06cm -1 (γ N-H), while in the eutectic, these characteristic absorption peaks shift to 1367.44 cm -1 (ν C-N )、1446.51cm -1 (δ C-N )、1590.7cm -1 (δ N-H )、1627.91cm -1 (ν C＝O ). The reason for this change is the formation of hydrogen bonds between the APIs, which causes the characteristic peaks to shift to varying degrees.

[0080] In addition, the TG-DSC graph of ZM887 ( Fig.10 ) shows that there is a weight loss phenomenon in the TG graph before 100°C, which corresponds to the endothermic peak before 100°C in the DSC graph, indicating the process of desolvation and dehydration. The second stage of the TG graph shows a weight loss of about 37%, which corresponds to the melting process of osimertinib-piperine at 322.8°C in the DSC graph. The TG graph also shows that the residual amount of osimertinib-piperine is 40.82% at 600°C.

[0081] Based on the above analysis, it can be concluded that ZM887 did not form a eutectic.

[0082] 3. ZM889 structural characterization analysis

[0083] like Fig.11As shown, the hydrogen nuclear magnetic resonance data of ZM889 are as follows: 1H NMR (500 MHz, DMSO-d6) δ10.23 (s, 2H), 9.16 (s, 2H), 8.69 (s, 2H), 8.34 (d, J = 4.9 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 7.91 (s, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.16 (t, J = 7.4 Hz, 2H), 7.05 (s, 2H), 6.44 (dd, J = 16.9, 10.2 Hz, 2H), 6.28 (d, J = 16.8 Hz, 2H), 5.78 (d, J = 10.6 Hz, 2H), 2.90 (t, J = 6.0 Hz, 4H), which correspond to the hydrogen nuclear magnetic resonance spectrum of osimertinib. The 1H NMR (500 MHz, DMSO-d6) δ10.54 (s, 1H), 7.35 (d, J = 2.9 Hz, 2H), 6.81 (s, 1H), 6.61 (d, J = 7.2 Hz, 1H), 4.93 (s, 1H), 4.35 (s, 1H), 3.80 (d, J = 2.7 Hz, 3H), 3.76 (d, J = 3.0 Hz, 6H), 3.41 (s, 3H), 3.02 (d, J = 8.3 Hz, 2H), 2.84 (d, J = 16.5 Hz, 2H), 1.99-1.89 (m, 2H), 1.76 (d, J = 15.6 Hz, 2H), which are the nuclear magnetic resonance hydrogen spectrum data of reserpine. In the region of 7.28-7.19 (m, 5H), there are 4 hydrogen atoms belonging to osimertinib and 1 to reserpine; at 3.92 (s, 7H), there are 6 hydrogen atoms belonging to osimertinib and 1 to reserpine; at 3.89-3.85 (m, 12H), there are 6 hydrogen atoms belonging to osimertinib and 6 to reserpine; at 2.73 (s, 7H), there are 6 hydrogen atoms belonging to osimertinib and 1 to reserpine; in the region of 2.41-2.27 (m, 7H), there are 4 hydrogen atoms belonging to osimertinib and 3 to reserpine; at 2.22 (s, 13H), there are 12 hydrogen atoms belonging to osimertinib and 1 to reserpine. By calculation, the molar ratio of API (active pharmaceutical ingredient) to CCF (cocrystal former) in the cocrystal molecule is 2:1.

[0084] like Fig.12As shown, in the X-ray diffraction pattern of ZM889, osimertinib shows characteristic peaks at 2θ angles, which are located at 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 reserpine also shows characteristic peaks at 2θ angles, which are located at 6.66°, 7.26°, 11.84°, 12.18°, 15.36°, 16.5°, 17.58°, 18.48°, 19.62°, 20.88°, 21.84°, 23.8°, 24.88°, and 25.62°. The X-ray diffraction pattern of ZM889 shows new characteristic peaks at 5.86°, 11.02°, 14.22°, and 22.02° at an angle of 2θ, preliminarily indicating the formation of a new crystalline phase.

[0085] The Fourier transform infrared spectroscopy (FT-IR) data of ZM889 are shown in Fig.13 As shown in Table 5, the infrared characteristic peaks of the osimertinib and reserpine cocrystal molecule have undergone significant shifts and changes compared with the API. The amide characteristic peaks of the osimertinib API are located at 3428.64 cm -1 (ν N-H )、1533.96cm -1 (δ N-H )、745.78cm -1 (γ N-H ), while in the eutectic molecule, these characteristic absorption peaks shift to 3447.79 cm -1( ν N-H )、1637.94cm -1 (δ N-H )、613.32cm -1 (γ N-H ). The characteristic peak of reserpine is located at 3462.15 cm -1 (ν N-H )、1714.55cm -1 (δ N-H )、613.32cm -1 (γ N-H ), while in the eutectic molecule, these characteristic absorption peaks also shift to 3447.79 cm -1 (ν N-H )、1637.94cm -1 (δ N-H )、613.32cm -1 (γ N-H ). This phenomenon occurs due to the formation of hydrogen bonds between APIs, which leads to the broadening of the spectral band and the displacement of the characteristic peaks of the APIs to varying degrees.

[0086] Table 5

[0087] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[Reserpine (cm -1 )]]> <![CDATA[ZM889(cm -1 )]]> <![CDATA[v N-H ]]> 3428.64 3462.15 3447.79 <![CDATA[v O-H ]]> - - - <![CDATA[v C-O ]]> 1576.49 1733.70 - <![CDATA[δ N-H ]]> 1533.96 1714.55 1637.94 <![CDATA[δ O-H ]]> - - - <![CDATA[ γN-H ]]> 745.78 618.90 613.32

[0088] like Fig.14 As shown in Table 6, the Raman data of ZM889 revealed that the Raman characteristic peaks of the osimertinib and reserpine cocrystal molecule were significantly shifted and changed compared with the API. Specifically, the amide characteristic peak of the osimertinib API was located at 3205.13 cm -1 (ν N-H )、1353.63cm -1 (ν C-N )、1538.3cm -1 (δ C-N )、1555.4cm -1 (δ N-H )、1621.07cm -1 (ν C＝O )、989.06cm -1 (γ N-H ), while in the eutectic molecule, these characteristic absorption peaks shift to 1448.84 cm -1 (ν C-N )、1551.16cm -1 (δ C-N )、1593.02cm -1 (δ N-H )、1630.23cm -1 (ν C＝O ). The reason for this change is the formation of hydrogen bonds between the APIs, which causes the characteristic peaks to shift to varying degrees.

[0089] Table 6

[0090] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[ZM889(cm -1 )]]> <![CDATA[v N-H ]]> 3205.13 - <![CDATA[v O-H ]]> - - <![CDATA[v C-N ]]> 1353.63 1448.84 <![CDATA[δ C-N ]]> 1538.3 1551.16 <![CDATA[δ N-H ]]> 1555.4 1593.02 <![CDATA[v C＝O ]]> 1621.07 1630.23 <![CDATA[γ O-H ]]> - - <![CDATA[γ N-H ]]> 989.06 -

[0091] Furthermore, the TG-DSC graph of ZM889 ( Fig.15 ) shows that there is a weight loss phenomenon in the TG graph before 100°C, which corresponds to the dehydration process before 100°C in the DSC graph. An obvious endothermic peak appears in the DSC graph at 245°C, indicating that the osimertinib-reserpine cocrystal reaches a melting state at 245°C, and this process is manifested in the TG graph as a weight loss of 69.42%.

[0092] Based on the above analysis, ZM889 was confirmed to be a cocrystal.

[0093] 5. Summary

[0094] In this study, three compounds were successfully designed and synthesized using drug cocrystal technology. Through a series of characterization techniques, including 1H NMR, XRD, FT-IR, Raman, and TG-DSC analysis, we confirmed that ZM889 formed a cocrystal with osimertinib. Specifically, the API to CCF molar ratio of the ZM889 cocrystal molecule was 2:1.

[0095] Example 3 In vitro antitumor activity study

[0096] 1. Principle of CCK-8 experiment

[0097] 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.

[0098] 2. Experimental methods and steps

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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).

[0103] Table 10

[0104]

[0105] Table 11

[0106]

[0107] Table 12

[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, osimertinib had significantly reduced sensitivity to the resistant cell line PC-9OR, with IC50 values ​​of 0.0283±0.0075μM and 0.7232±0.2166μM, respectively, and a resistance index of 25.6, which was statistically significant (P<0.01). Compared with ZM889, the mixture was about 2.2 times less active against the resistant cell line PC-9OR. These results indicate that the osimertinib cocrystal molecule is more active than its mixture against the resistant strain PC-9OR, preliminarily revealing 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 the obtained 1 cocrystal molecule and 2 non-cocrystal compounds. The results showed that the cocrystal molecule ZM889 showed certain superiority against intrinsic drug-resistant cell lines. 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 ZM889, characterized in that: The chemical structural formula of the compound ZM889 is:

2. A method for preparing the compound ZM889 according to claim 1, characterized in that: include: Osimertinib, reserpine 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 ZM889.

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 ZM889 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 ZM889 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.

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