A co-crystal drug ZM889 and its application in resisting lung cancer

Abstract

The application relates to the technical field of biological medicine, and discloses a co-crystal drug ZM889 and application thereof in resisting lung cancer, and a preparation method of the co-crystal drug ZM889, which comprises the following steps: mixing osimertinib, ristocetin and a mixed solvent, heating, stirring and dissolving, and then adding n-hexane; and yellow solid separated out is the co-crystal drug ZM889. The co-crystal molecule ZM889 exhibits enhanced antitumor activity compared with a non-co-crystal counterpart; in an in-vitro experiment, the co-crystal drug ZM889 shows a significant inhibitory effect on an osimertinib-resistant cell strain. The co-crystal drug ZM889 and the preparation method thereof bring a new strategy to the field of lung cancer resistance treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a co-crystal drug ZM889 and its application in anti-lung cancer. BACKGROUND

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

[0003] Therefore, it is particularly crucial to develop new drugs that can inhibit osimertinib-resistant cell lines. SUMMARY

[0004] In view of this, the present application provides a co-crystal drug ZM889 and its application in anti-lung cancer, aiming to overcome the drug resistance problem of existing treatment drugs and provide a new treatment strategy.

[0005] The present application provides a compound ZM889, and the chemical structural formula of the compound ZM889 is:

[0006]

[0007] The present application also provides a preparation method of the above-mentioned compound ZM889, comprising:

[0008] After osimertinib, reserpine and mixed solvent are mixed and heated and stirred to dissolve, n-hexane is added, and the yellow solid separated out 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 for heating and stirring to dissolve osimertinib, reserpine and mixed solvent after mixing is at least 60℃.

[0012] The application also provides an application of the compound ZM889, which at least includes any one of the following:

[0013] A1) the preparation of an anti-tumor drug;

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

[0015] A3) the preparation of a drug resistance sensitizer for an anti-tumor drug.

[0016] The application also provides a co-crystal drug, and the active ingredient of the co-crystal drug is the compound ZM889.

[0017] The application also provides an application of the co-crystal drug, which at least includes any one of the following:

[0018] A1) the preparation of an anti-tumor drug;

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

[0020] A3) the preparation of a drug resistance sensitizer for an anti-tumor drug.

[0021] Compared with the prior art, the application has the beneficial effects that:

[0022] The application successfully prepared one co-crystal molecule and two non-co-crystal compounds. Among these molecules, the co-crystal molecule ZM889 showed much higher anti-tumor activity than the non-co-crystal counterparts. Further in vitro experiments showed that ZM889 had a significant inhibitory effect on osimertinib-resistant cell lines.

[0023] The significant inhibitory effect of the co-crystal drug ZM889 on osimertinib-resistant cell lines in in vitro experiments suggests that it may provide a new treatment option for lung cancer patients in clinical treatment. The use of the co-crystal drug ZM889 is expected to improve the treatment effect, prolong the progression-free survival of patients, and may have a positive impact on the overall survival rate of patients.

[0024] In summary, the co-crystal drug ZM889 and the preparation method thereof of the application introduce a new strategy for the field of anti-lung cancer treatment, and are expected to play a key role in future clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are for purposes of illustration only and are not considered a limitation of the application. Moreover, like reference numerals are used to designate identical components throughout the specification. In the drawings:

[0026] Figure 1 1H NMR spectrum of compound ZM886;

[0027] Figure 2 XRD spectrum of compound ZM886;

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

[0029] Figure 4 Raman spectrum of compound ZM886;

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

[0031] Figure 6 1H NMR spectrum of compound ZM887;

[0032] Figure 7 XRD spectrum of compound ZM887;

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

[0034] Figure 9 Raman spectrum of compound ZM887;

[0035] Figure 10 TG and DSC spectra of compound ZM887;

[0036] Figure 11 1H NMR spectrum of compound ZM889;

[0037] Figure 12 XRD spectrum of compound ZM889;

[0038] Figure 13 Fourier transform infrared spectrum of compound ZM889;

[0039] Figure 14 Raman spectrum of compound ZM889;

[0040] Figure 15 TG and DSC spectra of compound ZM889. DETAILED DESCRIPTION

[0041] Exemplary embodiments of the present disclosure will be described in greater detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be embodied in various forms without being limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. It is noted that the embodiments in the present disclosure and the features in the embodiments can be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0042] Example 1

[0043] I. Preparation of Compound ZM886

[0044] Osimertinib (200 mg, 0.4 mmol) and omeprazole (138.2 mg, 0.4 mmol) were weighed into a reaction tube, 1.5 mL of mixed solvent (ethanol: water = 15: 1) was added, and the solution was stirred and dissolved at 80°C. Then, the temperature was lowered to 60°C, 3 mL of n-hexane was added, and white-brown solid was precipitated. Filtration and washing yielded 250 mg of white-brown powder, which was compound ZM886, with the chemical structure:

[0045]

[0046] II. Compound ZM887

[0047] Osimertinib (200 mg, 0.4 mmol) and piperine (114 mg, 0.4 mmol) were weighed into a reaction tube, 1.5 mL of mixed solvent (acetone: water = 15: 1) was added, and the solution was stirred and dissolved at 60°C. Then, 3 mL of n-hexane was added, and light brown solid was precipitated. Filtration and washing yielded 240 mg of light brown powder, which was compound ZM887, with the chemical structure:

[0048]

[0049] III. Preparation of Compound ZM889

[0050] Osimertinib (200 mg, 0.4 mmol) and reserpine (122 mg, 0.2 mmol) were weighed into a reaction tube, 1.5 mL of dichloromethane was added, and the solution was stirred and dissolved at 40°C. 3 mL of n-hexane was added, and the temperature was slowly lowered to room temperature. The solution was stirred overnight, and brown solid was precipitated. Filtration and washing yielded 240 mg of brown powder, which was compound ZM889, with the chemical structure:

[0051]

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

[0053] The following is the characterization analysis of ZM886, ZM887 and ZM889.

[0054] I. Process

[0055] 1H Nuclear Magnetic Resonance (1H NMR): The 1H NMR data were collected at room temperature by a DSX-500 nuclear magnetic resonance spectrometer of Bruker Biospin Ltd., Switzerland, with DMSO-d6 as solvent and TMS as internal standard. Powder X-ray Diffraction (PXRD): The PXRD data were obtained by a Rigaku Ultima IV diffractometer of Rigaku Corporation, Japan, with the parameters of wavelength 1.5418 A, voltage 40 KV, current 40 mA, sample type powder, target copper, scanning range 5-90° (2q), scanning speed 10° / min, and all samples were tested at room temperature.

[0056] Fourier Transform Infrared Spectroscopy (FT-IR): The FT-IR data were obtained by a Nicolet iN10 produced by Thermo Fisher Scientific, USA, with the range of 400-4000 cm-1 as the maximum axis number for routine test. The experimental data in this paper were obtained by KBr tabletting method, in which KBr was ground to 2 pm by agate mortar. A certain amount of sample (2-5 mg) and KBr (100-120 mg) were added, ground and mixed until no obvious particles were observed. Then it was placed in a mold and tabletted to form a transparent or translucent shape. -1

[0057] Raman Spectroscopy (RM): The Raman data were obtained by a DXR produced by Thermo Fisher Scientific, USA. The sample to be tested was placed as flat as possible on the sample holder to ensure the accuracy of the spectral data. According to the nature of the sample and the experimental requirements, the appropriate laser wavelength, power and detector parameters were selected to ensure the clarity and stability of the spectral signal. Before the sample test, background scanning must be carried out to eliminate the interference of the instrument background signal on the sample signal.

[0058] ​Simultaneous thermal analysis (TG-DSC): The test data for simultaneous thermal analysis were obtained using a Hitachi 200 instrument from Japan. TG-DSC testing can simultaneously obtain TG, DSC, and DTG curves. The sample temperature range is from room temperature to below 800℃ (inclusive). The specific test temperature is 30-600℃, the test gas atmosphere is N2, and the heating rate is 10℃ / min.

[0059] II. Results

[0060] 1. Structural Characterization and Analysis of ZM886

[0061] like Figure 1 As shown, the 1H NMR spectrum (1H NMR, 500MHz, DMSO-d6) of ZM886 exhibits the following characteristics: 10.21 (single peak, 1H), 9.16 (single peak, 1H), 8.69 (single peak, 1H), 8.34 (doublet, J = 5.3Hz, 1H), 8.25 (doublet, J = 8.1Hz, 1H), 7.91 (single peak, 1H), 7.30-7.21 (multiplet, 2H), 7.16 (triplet, J = 7.5Hz, 1H). 7.05 (single, 1H), 6.49–6.40 (multiple, 1H), 6.29 (doublet, J = 16.9 Hz, 1H), 5.78 (doublet, J = 9.4 Hz, 1H), 3.92 (single, 3H), 3.87 (single, 3H), 2.90 (triplet, J = 5.7 Hz, 2H), 2.73 (single, 3H), 2.31 (triplet, J = 5.8 Hz, 2H). These data correspond to the 1H NMR spectrum of osimertinib. Additionally, the 1H NMR (500MHz, DMSO-d6) data for omeprazole are as follows: δ 13.40 (single, 1H), 8.19 (single, 1H), 7.10 (single, 1H), 6.93 (doublet, J = 8.8Hz, 1H), 4.77 (doublet, J = 13.5Hz, 1H), 4.69 (doublet, J = 13.6Hz, 1H), 3.81 (single, 3H), 3.69 (single, 3H). The two hydrogen atoms at 7.54 (triplet, J = 8.6Hz, 2H) belong to osimertinib and omeprazole, respectively; of the 12 hydrogen atoms in the 2.26–2.09 (multiplex, 12H) range, 6 are from osimertinib and 6 are from omeprazole. Calculations show that the molar ratio of API (active pharmaceutical ingredient) to CCF (cocrystallization agent) in the eutectic 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 did not show any new characteristic peaks at these angles, suggesting that no new crystal phase has formed.

[0063] like Figure 3 As shown in Table 1, ZM886 FT-IR data revealed significant shifts, weakening, and changes in the infrared characteristic peaks of the osimertinib / omeprazole cocrystal compared to the active pharmaceutical ingredient (API). Specifically, the amide characteristic peak of the osimertinib API is located at 3428.64 cm⁻¹. -1 (ν N-H ), 1576.49cm -1 (ν C＝O ), 1533.96cm -1 (δ N-H In eutectic molecules, these characteristic absorption peaks shift to 3436.11 cm⁻¹. -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. -1 (ν N-H ), 1628.73cm -1 (δ N-H The characteristic absorption peak in the eutectic molecule shifts to a lower frequency direction, reaching 3436.11 cm⁻¹. -1 (ν N-H ), 1540.08cm -1 (δ N-H The reason for this change is that after hydrogen bonds are formed, the characteristic peaks of the active pharmaceutical ingredient shift to varying degrees.

[0064] Furthermore, ZM886 Raman data ( Figure 4 As shown in Table 2, the Raman characteristic peaks of the osimertinib and omeprazole cocrystal molecules also exhibited significant shifts, weakening, or even disappearance compared to the Raman characteristic peaks of the active pharmaceutical ingredient (API). The amide characteristic peak of the osimertinib API includes 3205.13 cm⁻¹. -1 (ν N-H), 1353.63 cm -1 (ν C-N ), 1538.3 cm -1 (δ C-N ), 1621.07 cm -1 (ν C＝O ), 989.06 cm -1 (γ N-H ), while the characteristic absorption peaks in the co-crystal molecule shifted to 3213.95 cm -1 (ν N-H ), 1353.48 cm -1 (ν C-N ), 1418.60 cm -1 (δ C-N ), 1618.60 cm -1 (ν C＝O ), 990.70 cm -1 (γ N-H ). This phenomenon is also attributed to the formation of hydrogen bonds between the raw materials, resulting in different degrees of displacement of the characteristic peaks.

[0065] Table 1

[0066] Home Osimertinib (cm -1 )]]> omeprazole (cm -1 )]]> ZM886 (cm -1 )]]> v N-H ]]> 3428.64 3460.74 3436.11 v O-H ]]> - - - v C＝O ]]> 1576.49 - 1589.33 delta N-H ]]> 1533.96 1628.73 1540.08 delta O-H ]] - - - gamma N-H ]] 745.78 - -

[0067] Table 2

[0068]

[0069]

[0070] However, the TG-DSC data of ZM886 showed Figure 5 ), 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 co-crystal.

[0071] 2. Structure characterization analysis of ZM887

[0072] The nuclear magnetic resonance hydrogen spectrum (1H NMR, 500 MHz, DMSO-d6) of ZM887 is as follows: Figure 6The spectrum shows the following chemical shifts: δ 10.21 (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 nuclear magnetic resonance hydrogen spectrum of osimertinib. Another set of data: δ 7.01-6.88 (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) belong to the nuclear magnetic resonance hydrogen spectrum of piperine. At the chemical shift 7.29-7.12 (multiplet, 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 (co-crystal former) in the co-crystal molecule is 1:1.

[0073] The X-ray diffraction pattern (XRD) of ZM887 is shown in FIG. 1. Figure 7 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°, 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°, 28.22°. The X-ray diffraction pattern of ZM887 does not show new characteristic peaks at these angles, indicating that no new crystal phase is generated.

[0074] The Fourier transform infrared spectrum (FT-IR) of ZM887 is shown in FIG. 2 and Table 3. Figure 8 Compared with the raw material drug, the infrared characteristic peaks of the co-crystal molecule have obvious shift, weakening or even disappearance. The amide characteristic peaks of the osimertinib raw material drug include 3428.64 cm -1 (ν N-H stretching vibration), 1576.49 cm -1 (ν C＝O stretching vibration), 1533.96 cm-1 (δ N-H (bending vibration), while in eutectic molecules, these characteristic absorption peaks shift to 3438.21 cm⁻¹. -1 (ν N-H (Extensional vibration), 1671.45cm -1 (ν C＝O (Extensional vibration), 1637.94cm -1 (δ N-H (Bending vibration). The characteristic peak of piperine is at 3447.79 cm⁻¹. -1 (ν N-H (Extensional vibration), 1585.27cm -1 (δ N-H (bending vibration), 1637.94cm -1 (ν C＝O (Stretching vibrations), while in eutectic molecules, these characteristic absorption peaks shift to 3438.21 cm⁻¹. -1 (ν N-H (Extensional vibration), 1637.94cm -1 (δ N- (H bending vibration), 1671.45cm -1 (ν C＝O (Stretching vibration). This phenomenon occurs because hydrogen bonds are formed, causing the characteristic peaks of the active pharmaceutical ingredient to shift to varying degrees.

[0075] Table 3

[0076] Home Osimertinib (cm -1 )]]> Piperine (cm -1 )]]> ZM887 (cm -1 )]]> v N-H ]]> 3428.64 3447.79 3438.21 v O-H ]]> - - - v C＝O ]]> 1576.49 1637.94 1671.45 delta N-H ]] 1533.96 1585.27 1637.94 delta O-H ]] - - - N-H ]]> ​ 745.78 - -

[0077] Table 4

[0078] Home Osimertinib (cm -1 )]]> ZM887 (cm -1 )]]> v N-H ]]> 3205.13 - v O-H ]]> - - v C-N ]]> 1353.63 1367.44 C-N ]]> ​ 1538.3 1446.51 delta N-H ]]> 1555.4 1590.7 v C＝O ]]> 1621.07 1627.91 gamma O-H ]] - - gamma N-H ]]> 989.06 -

[0079] like Figure 9 As shown in Table 4, the ZM887 Raman data revealed that the Raman characteristic peaks of the osimertinib-piperine cocrystal molecule showed significant shifts, decreased in intensity, and even the disappearance of some peaks compared to the active pharmaceutical ingredient (API). Specifically, the amide band of the osimertinib API was at 3205.13 cm⁻¹. -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) whereas in the co-crystal, these characteristic absorption peaks shifted to 1367.44 cm -1 (ν C-N ), 1446.51 cm -1 (δ C-N ), 1590.7 cm -1 (δ N-H ), 1627.91 cm -1 (ν C＝O ). The reason for this change is that the formation of hydrogen bonds between the raw materials, resulting in different degrees of displacement of characteristic peaks.

[0080] In addition, the TG-DSC graph of ZM887 ( Figure 10 ) shows that the TG graph has a weight loss 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 osimertinib-piperine melting process at 322.8°C in the DSC graph. The TG graph also shows that the residual amount of osimertinib-piperine at 600°C is 40.82%.

[0081] Based on the above analysis, it can be concluded that ZM887 does not form a co-crystal.

[0082] 3. Structure characterization analysis of ZM889

[0083] As Figure 11As shown, the ZM889 nuclear magnetic resonance hydrogen spectrum data are as follows:1H NMR (500 MHz, DMSO-d6) δ 10.23 (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 corresponds to the nuclear magnetic resonance hydrogen spectrum of osimertinib. And1H NMR (500 MHz, DMSO-d6) δ 10.54 (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 is the nuclear magnetic resonance hydrogen spectrum data of reserpine. In the 7.28-7.19 (m, 5H) region, 4 hydrogen atoms belong to osimertinib and 1 to reserpine; at 3.92 (s, 7H), 6 hydrogen atoms belong to osimertinib and 1 to reserpine; in the 3.89-3.85 (m, 12H) region, 6 hydrogen atoms belong to osimertinib and 6 to reserpine; at 2.73 (s, 7H), 6 hydrogen atoms belong to osimertinib and 1 to reserpine; in the 2.41-2.27 (m, 7H) region, 4 hydrogen atoms belong to osimertinib and 3 to reserpine; at 2.22 (s, 13H), 12 hydrogen atoms belong to osimertinib and 1 to reserpine. By calculation, the molar ratio of API (active pharmaceutical ingredient) to CCF (co-crystal former) in the co-crystal molecule is 2:1.

[0084] As Figure 12As shown, the X-ray diffraction pattern of ZM889 showed characteristic peaks of osimertinib at 2 theta 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°, 27.3°. The X-ray diffraction pattern of reserpine also showed characteristic peaks at 2 theta angles of 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°, 25.62°. However, the X-ray diffraction pattern of ZM889 showed new characteristic peaks at 2 theta angles of 5.86°, 11.02°, 14.22°, 22.02°, which preliminarily indicated the formation of a new crystal phase.

[0085] The Fourier transform infrared spectroscopy (FT-IR) data of ZM889 is shown in Table 4 and Table 5. Figure 13 As shown in Table 4 and Table 5, the infrared characteristic peaks of the co-crystal molecule of osimertinib and reserpine were significantly shifted and changed compared with the original drugs. The amide characteristic peaks of osimertinib original drug were at 3428.64 cm -1 (ν N-H ), 1533.96 cm -1 (δ N-H ), 745.78 cm -1 (γ N-H ), while in the co-crystal molecule, these characteristic absorption peaks shifted to 3447.79 cm -1( ν N-H ), 1637.94 cm -1 (δ N-H ), 613.32 cm -1 (γ N-H ). The characteristic peaks of reserpine were at 3462.15 cm -1 (ν N-H ), 1714.55 cm -1 (δ N-H ), 613.32 cm -1 (γ N-H ), while in the co-crystal molecule, these characteristic absorption peaks also shifted to 3447.79 cm -1 (ν N-H ), 1637.94 cm -1 (δ N-H ), 613.32 cm -1 (γ N-H ). This phenomenon was due to the formation of hydrogen bonds between the original drugs, resulting in the broadening of the spectral band, and thus the characteristic peaks of the original drugs were shifted to different degrees.

[0086] Table 5

[0087] Home Osimertinib (cm -1 )]]> reserpiline (cm -1 )]]> ZM889 (cm -1 )]]> v N-H ]]> 3428.64 3462.15 3447.79 v O-H ]]> - - - v C-O ]]> 1576.49 1733.70 - N-H ]]> ​ 1533.96 1714.55 1637.94 O-H ]]> ​ - - - γN-H ]]> ​ 745.78 618.90 613.32

[0088] As Figure 14 shown in Table 6, the Raman data of ZM889 revealed that the Raman characteristic peaks of the osimertinib-rubigallol co-crystal molecule had significant shifts and changes compared with the raw material. Specifically, the amide characteristic peak of the osimertinib raw material was located at 3205.13 cm -1 (ν N-H ), 1353.63 cm -1 (ν C-N ), 1538.3 cm -1 (δ C-N ), 1555.4 cm -1 (δ N-H ), 1621.07 cm -1 (ν C＝O ), 989.06 cm -1 (γ N-H ), while in the co-crystal molecule, these characteristic absorption peaks shifted to 1448.84 cm -1 (ν C-N ), 1551.16 cm -1 (δ C-N ), 1593.02 cm -1 (δ N-H ), 1630.23 cm -1 (ν C＝O ), respectively. The reason for this change is that hydrogen bonds are formed between the raw materials, resulting in different degrees of shifts in the characteristic peaks.

[0089] Table 6

[0090] Home Osimertinib (cm -1 )]]> ZM889 (cm -1 )]]> v N-H ]]> 3205.13 - v O-H ]]> - - v C-N ]]> 1353.63 1448.84 C-N ]]> ​ 1538.3 1551.16 N-H ]]> ​ 1555.4 1593.02 v C＝O ]]> 1621.07 1630.23 O-H ]]> ​ - - N-H ]]> ​ 989.06 -

[0091] Further, the TG-DSC graph of ZM889 Figure 15 shows that the TG graph has a weight loss before 100°C, which corresponds to the dehydration process before 100°C in the DSC graph. The DSC graph has a clear endothermic peak at 245°C, indicating that the osimertinib-rubigallol co-crystal reaches a molten state at 245°C, and this process is represented as a 69.42% weight loss in the TG graph.

[0092] In summary of the above analysis, ZM889 is confirmed as a co-crystal.

[0093] 5. Summary

[0094] The present study used pharmaceutical co-crystal 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 the co-crystal of ZM889 with osimertinib. Specifically, the API of the ZM889 co-crystal molecule has a 2: 1 molar ratio with CCF.

[0095] Example 3 in vitro anti-tumor activity study

[0096] 1. CCK-8 experiment principle

[0097] The 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, the chemical name is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt. Under the action of electron carrier 1-methoxy-5-methyl phenylzolium sulfate dimethyl (1-methoxy PMS), the dehydrogenase in the cell reduces WST-8 to a highly water-soluble yellow formazan product. The amount of formazan generated is directly proportional to the number of living cells, which makes the CCK-8 reagent directly applicable 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 was prepared from basal medium, 10% fetal bovine serum (FBS), and 1% 100 U / mL penicillin and 100 μg / mL streptomycin (P / S). The medium used for NCI-H1975 and NCI-H1650 cells has the same formula. The complete culture medium for NCI-HCT116 cells consists of RPMI-1640 medium without Hepes, 10% fetal bovine serum, and 1% double-antibiotic. The cell freezing solution consists of 55% basal medium, 40% FBS, and 5% DMSO, and is stored in liquid nitrogen. Cells were cultured in a 95% air, 37°C, 5% CO2 environment, and were ensured to be free of mycoplasma and chlamydia contamination.

[0100] In vitro cytotoxicity test: cells in the logarithmic growth phase were selected at 7 x 10 3Cells were seeded in 96-well plates at a density of 5000 cells per well and incubated at 37°C, 5% CO2 for 24 hours to maintain normal physiological pH. After the cells adhered, different concentrations of test compounds were added to three groups of wells, with 0.1% DMSO 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 450 nm wavelength using a microplate reader, and the 50% cell growth inhibition concentration (IC50, Half Maximal Inhibitory Concentration) was calculated by GraphPad Prism software. All data were repeated three times.

[0101] Establishment of drug-resistant cell lines: Drug resistance was induced by the drug concentration gradient method to establish an osimertinib-resistant cell line. The PC-9 cell line was used as the parent cell, and its IC50 value was tested, and a concentration close to the value was selected for culture. After determining the optimal starting concentration, the drug concentration was gradually increased to induce drug resistance in the PC-9 cell line. The culture medium was changed regularly and the growth of the cells under the microscope was observed, and when the cells showed stable tolerance, the drug concentration was appropriately increased. By repeatedly changing the culture medium, subculturing and increasing the drug concentration, until the drug resistance index of the drug-resistant cell line to osimertinib exceeds 15, it is considered that the drug resistance construction is successful. The successfully constructed drug-resistant cell line is represented by PC-9OR.

[0102] IC50 testing is used to evaluate the toxicity and tolerance of drugs to cells, where a lower IC50 value means stronger drug inhibition effect. In this paper, we evaluate the sensitivity of cells to osimertinib by calculating the resistance index (RI). The formula for calculating the resistance index (RI) is: the IC50 value of the drug-resistant cells divided by the IC50 value of the parent cells. According to the RI value, the drug resistance is classified as follows: 1-5 is low drug resistance; 5-15 is moderate drug resistance; more than 15 is high drug resistance. We tested the activity of five cell lines, including PC-9, PC-9OR (osimertinib acquired drug-resistant cell line), H1975 (osimertinib highly sensitive cell line), H1650 (osimertinib inherently resistant cell line), and HCT116 (human colon cancer cell line).

[0103] Table 10

[0104]

[0105] Table 11

[0106]

[0107] Table 12

[0108]

[0109] In vitro anti-tumor activity studies were conducted on one newly synthesized co-crystal molecule and two non-co-crystal compounds. The results showed that compared with PC-9, the sensitivity of osimertinib to drug-resistant cell line PC-9OR was significantly reduced, and the IC50 values were 0.0283±0.0075 μM and 0.7232±0.2166 μM, respectively, and the drug resistance index was 25.6, and the difference was statistically significant (P<0.01). Compared with ZM889, the activity of the mixture on drug-resistant cell line PC-9OR was about 2.2 times lower. These research results show that the activity of the osimertinib co-crystal molecule on the drug-resistant strain PC-9OR is better than that of its mixture, which preliminarily reveals the potential advantages of co-crystal molecules in anti-tumor activity.

[0110] 3. Summary

[0111] The obtained one co-crystal molecule and two non-co-crystal compounds were evaluated for in vitro anti-non-small cell lung cancer activity. The research results show that the co-crystal molecule ZM889 exhibits certain superiority to the inherent drug-resistant cell line. This preliminarily proves that through the drug co-crystal technology, a new anti-tumor drug for solving the problem of osimertinib drug resistance can be developed.

[0112] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that: the specific embodiments of the present application can still be modified or replaced by the equivalent, without departing from the spirit and scope of the present application. Any modification or equivalent replacement should be covered within the protection scope of the claims of the present application.

Claims

1. A compound ZM889, characterized in that, The chemical structural formula of the compound ZM889 is as follows: ； The compound ZM889 is in eutectic form, and its X-ray diffraction pattern shows characteristic peaks at 5.86°, 11.02°, 14.22°, and 22.02° at a 2θ angle.

2. A method for preparing compound ZM889 according to claim 1, characterized in that, include: Osimertinib, reserpine, and a mixed solvent were mixed, heated, stirred, and dissolved. Then, n-hexane was added, and the precipitated yellow solid was 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, reserpine, and the mixed solvent are heated and stirred to dissolve them is at least 60°C.

6. An application of the compound ZM889 according to claim 1, characterized in that, The application is as follows: Application in the preparation of anti-lung cancer drugs.

7. The application of compound ZM889 according to claim 6, characterized in that, The application is as follows: Use in the preparation of pharmaceutical compositions for the prevention or treatment of drug-resistant lung cancer.

8. The application of compound ZM889 according to claim 6, characterized in that, The application is as follows: Application in the preparation of drug resistance sensitizers for anti-lung cancer drugs.

9. A eutectic drug, characterized in that, The active ingredient of the eutectic drug is compound ZM889 as described in claim 1.

10. The application of the eutectic drug according to claim 9, characterized in that, The application is as follows: Application in the preparation of anti-lung cancer drugs.

11. The application of the eutectic drug according to claim 10, characterized in that, The application is as follows: Use in the preparation of pharmaceutical compositions for the prevention or treatment of drug-resistant lung cancer.

12. The application of the eutectic drug according to claim 10, characterized in that, The application is as follows: Application in the preparation of drug resistance sensitizers for anti-lung cancer drugs.

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