Co-crystal medicine ZM888 and application thereof in lung cancer resistance

Abstract

The invention relates to the technical field of biological medicine, and discloses a co-crystal medicine ZM888 and application thereof in lung cancer resistance, a preparation method of the co-crystal medicine ZM888 comprises the following steps: mixing osimertinib, naringenin and a mixed solvent, heating, stirring and dissolving, then adding n-hexane, and precipitating a yellow solid, namely the co-crystal medicine ZM888. The eutectic drug ZM888 disclosed by the invention shows a remarkable inhibition effect on an osimertinib drug-resistant cell strain in an in-vitro experiment, which indicates that the eutectic drug ZM888 possibly provides a new treatment scheme for clinical treatment of lung cancer patients. The application of the eutectic drug ZM888 is expected to improve the treatment effect and prolong the progression-free lifetime of patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cocrystal drug ZM888 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 ZM888 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 proposes a compound ZM888, the chemical structural formula of the compound ZM888 is:

[0006]

[0007] The present invention also provides a method for preparing the compound ZM888, comprising:

[0008] Osimertinib, naringenin 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 ZM888.

[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, naringenin 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 ZM888, 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 ZM888.

[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 cocrystal molecules, ZM888 is a cocrystal formed by osimertinib and naringenin, and its chemical structure reflects the combination of these two components. Studies have found that the anti-tumor activity of ZM888 on the drug-resistant cell line PC-9OR is significantly better than that of osimertinib alone. As a cocrystal drug, ZM888 shows the potential to overcome the drug resistance problem of osimertinib, and its significant anti-tumor effect on drug-resistant cell lines has been confirmed in in vitro experiments.

[0023] The cocrystal drug ZM888 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 the cocrystal drug ZM888 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 ZM888 of the present invention and its preparation method have brought 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 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 ZM888;

[0037] Fig.12 is the XRD spectrum of compound ZM888;

[0038] Fig.13 is the Fourier transform infrared spectrum of compound ZM888;

[0039] Fig.14 is the Raman spectrum of compound ZM888;

[0040] Fig.15 TG spectrum and DSC spectrum of compound ZM888. 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 ZM888

[0050] Weigh osimertinib (200 mg, 0.4 mmol) and naringenin (109 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 and stir overnight to precipitate a yellow solid. Filter and wash to obtain 128 mg of a yellow powder, which is compound ZM888, with the chemical formula:

[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 ZM888.

[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]

[0067]

[0068] Table 2

[0069] Attribution <![CDATA[Osimertinib (cm -1 )]]> <![CDATA[ZM886(cm -1 )]]> VN-H 3205.13 3213.95 VxV VCN 1353.63 1353.48 8c-N 1538.3 1418.60 8N-H 1555.4 1553.49 VO=0 1621.07 1618.60 YO-H YN-H 989.06 990.70

[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. ZM888 structural characterization analysis

[0083] like Fig.11As shown, the nuclear magnetic resonance hydrogen spectrum (1H NMR, 500 MHz, DMSO-d6) of ZM888 showed the following characteristics: 10.21 (s, 2H), 9.15 (s, 2H), 8.69 (s, 2H), 8.34 (d, J = 5.2 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 7.90 (s, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 6.2 Hz, 4H), 7.16 (t, J = 7 .6Hz, 2H), 7.05(s, 2H), 6.48-6.41(m, 2H), 6.28(d, J=16.8Hz, 2H), 5.78(d, J=10.2Hz, 2H), 3.93(s, 12H), 2.90(t, J=5.9Hz, 4H), 2.30(d, J=6.0Hz, 4H), 2.22(s, 12H), these data correspond to the hydrogen nuclear magnetic resonance spectrum of osimertinib. The nuclear magnetic resonance hydrogen spectrum of naringenin (1H NMR, 500MHz, DMSO-d6) shows: δ12.17 (s, 1H), 9.60 (s, 1H), 7.32 (d, J = 8.1Hz, 2H), 6.80 (d, J = 8.1Hz, 2H), 5.87 (s, 2H), 5.44 (d, J = 12.7Hz, 1H), 3.23 (s, 1H). At 2.73 (s, 7H), there are 7 hydrogen atoms, 6 of which belong to osimertinib and 1 to naringenin. Through calculation, we can conclude that the molar ratio of API (active pharmaceutical ingredient) to CCF (cocrystal former) in the cocrystal molecule is 2:1.

[0084] The X-ray diffraction (XRD) pattern of ZM888 is shown below: Fig.12 As shown. The XRD spectrum 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 XRD spectrum of naringenin shows characteristic peaks at 2θ angles of 10.8°, 11.48°, 15.8°, 17.22°, 18.1°, 19.88°, 20.4°, 21.18°, 22.28°, 23.76°, 24.34°, 25.34°, 25.78°, 27.68°, and 30.06°. The XRD spectrum of ZM888 shows new characteristic peaks at 2θ angles of 5.9°, 7.32°, 11.1°, 12.86°, 14.24°, 18.96°, and 24.7°, which preliminarily indicates the formation of a new crystalline phase.

[0085] Table 5

[0086]

[0087] like Fig.13 As shown in Table 6, the ZM888 FT-IR data revealed that the infrared characteristic peaks of the osimertinib and naringenin cocrystal molecules were significantly shifted, weakened, or even disappeared compared with the API. Specifically, the amide characteristic peak of the osimertinib API is located at 3428.64 cm -1 (ν N-H ) and 1533.96cm -1 (δ N-H ), while in the eutectic molecule, these characteristic absorption peaks shift to 3452.58 cm -1 (ν N-H ) and 1513.45cm -1 (δ N-H ). The characteristic peak of naringenin is originally 3519.61cm -1 (ν O-H ) and 718.65cm -1 (δ O-H ), the corresponding characteristic absorption peak in the eutectic molecule shifts to 3452.58 cm -1 (ν O-H The reason for this change is that after the cocrystal is formed, -NH in osimertinib forms a stronger NH…O bond with -OH in the ligand, which causes the vibration frequency of -OH and -NH to change, the spectral band to become wider, and thus the characteristic peaks of the API to shift to varying degrees.

[0088] Further observation of the Raman data of ZM888 ( Fig.14 and Table 6), the Raman characteristic peaks of osimertinib and naringenin cocrystal molecules also show obvious displacement, weakening and changes compared with 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 )、1555.4cm -1 (δ N-H )、1621.07cm -1 (ν C＝O )、989.06cm -1 (γ N-H ), while the characteristic absorption peak of the eutectic molecule shifted to 3206.98 cm -1 (ν N-H )、1448.84cm -1 (ν C-N )、1551.16cm -1(δ C-N )、1593.02cm -1 (δ N-H )、1630.23cm -1 (ν C＝O )、988.37cm -1 (γ N-H ). This phenomenon occurs due to the formation of hydrogen bonds between the APIs, which causes the characteristic peaks to shift to varying degrees.

[0089] Table 6

[0090]

[0091]

[0092] TG-DSC diagram of ZM888 ( Fig.15 ) shows that there is a weight loss phenomenon before 100°C, which corresponds to the endothermic peak before 100°C in the DSC graph, indicating the process of desolvation and dehydration. The DSC graph has an obvious endothermic peak at 358°C, which indicates that the osimertinib-naringenin cocrystal reaches the melting point at 358°C. The corresponding TG graph shows that the weight loss in the second step is 53.84%, indicating that most of the osimertinib-piperine melts after reaching the melting point.

[0093] In summary, ZM888 appears to be in the form of a co-crystal.

[0094] 5. Summary

[0095] 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 one molecule, ZM888, formed a cocrystal with osimertinib. Specifically, the molar ratio of API to CCF of the ZM888 cocrystal molecule was 2:1.

[0096] Example 3 In vitro antitumor activity study

[0097] 1. Principle of CCK-8 experiment

[0098] 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.

[0099] 2. Experimental methods and steps

[0100] 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.

[0101] In vitro cytotoxicity test: cells in the logarithmic growth phase were selected and cultured at 7×10 3 The 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.

[0102] 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.

[0103] The IC50 test is used to evaluate the toxicity and tolerance of drugs to cells, where a lower IC50 value means a stronger drug inhibitory effect. In this article, we evaluated the sensitivity of cells to osimertinib by calculating the resistance index (RI)

[83] . 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, the resistance is graded as follows: 1-5 is low resistance; 5-15 is moderate resistance; and more than 15 is high resistance

[84] . 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).

[0104] Table 7

[0105]

[0106] Table 8

[0107]

[0108]

[0109] Table 9

[0110]

[0111] 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 resistance index reached 25.6, which was statistically significant (P<0.01). Further comparison showed that ZM888 also had significantly better antitumor activity than osimertinib against the drug-resistant cell line PC-9OR, with an IC50 value of 0.5191±0.7631μM. Compared with ZM888, the mixture was about 10.2 times less active against the drug-resistant cell line PC-9OR. These research results show that the activity of osimertinib cocrystal molecules against the drug-resistant strain PC-9OR is better than that of its mixture, which preliminarily reveals the potential advantages of cocrystal molecules in antitumor activity.

[0112] 3. Summary

[0113] 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 ZM888 on the PC-9OR acquired drug-resistant cell line was enhanced by about 1.4 times compared with osimertinib, and it also showed certain superiority on the intrinsic drug-resistant cell line. For the acquired drug-resistant cell line PC-9OR, the anti-tumor activity of the cocrystal molecule ZM888 was more than 10 times higher than that of its non-cocrystal mixture. This preliminarily proves that through drug cocrystal technology, a new anti-tumor drug can be developed to solve the problem of osimertinib resistance.

[0114] 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 ZM888, characterized in that The chemical structural formula of the compound ZM888 is:

2. A method for preparing the compound ZM888 according to claim 1, characterized in that: include: Osimertinib, naringenin 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 ZM888.

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 ZM888 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 ZM888 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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