Crystal form of eutectic compound of carboplatin and 1, 1-cyclobutane dicarboxylic acid and detection method thereof

Abstract

The invention provides a method for testing a crystal structure of a crystal form of a carboplatin and 1, 1-cyclobutane dicarboxylic acid eutectic compound and the crystal structure. According to the invention, crystal powder of the eutectic compound of carboplatin and 1, 1-cyclobutane dicarboxylic acid is prepared, and Micro-ED (microcrystal diffraction) is utilized to measure and obtain the crystal structure of the crystal form of carboplatin and 1, 1-cyclobutane dicarboxylic acid. Through the structure, the combination and positions of hydrogen bonds in two molecules can be determined, preparation of an aqueous solution is facilitated, and good water solubility and stability are explained.

Description

Technical Field

[0001] This invention relates to a platinum-based eutectic complex, specifically to the crystal structure of a carboplatin-1,1-cyclobutane dicarboxylic acid eutectic compound and its detection method. Background Technology

[0002] Platinum-based chemotherapy drugs are among the most effective drugs for the clinical treatment of malignant tumors. Cisplatin, discovered by Barnett Rosenberg in the 1960s, is a first-generation chemotherapy drug with significant efficacy against tumors and has been used clinically for nearly 30 years. However, adverse reactions such as nephrotoxicity, gastrointestinal reactions, and drug resistance have limited its widespread use, thus promoting the research and development of a new generation of platinum-based chemotherapy drugs.

[0003] Second-generation platinum-based chemotherapy drugs such as carboplatin and third-generation oxaloplatin have been developed, but these drugs all have drawbacks to varying degrees, including poor water solubility, instability, low oral efficacy, significant toxic side effects, and the tendency for sensitive tumors to develop resistance. Therefore, the search for stable, highly effective, low-toxicity, and easy-to-use platinum compounds remains a hot research topic.

[0004] Of particular interest is the cocrystal complex formed by carboplatin and 1,1-cyclobutanedicarboxylic acid. Clinical and animal studies have shown that its toxicity is 20% that of carboplatin. However, the structure of this cocrystal complex has not been elucidated, affecting subsequent research on its mechanism of action, efficacy, and other aspects. This invention is based on in-depth research into this issue. Summary of the Invention

[0005] Based on the above problems and needs, this invention utilizes the structural characteristics of the eutectic complex formed by carboplatin and 1,1-cyclobutanedicarboxylic acid, and takes advantage of the essential differences between ordered self-assemblies and physical mixtures. Through specific freeze-drying and detection methods, the crystal form under freezing conditions (100K) was finally obtained, and its structure was determined by microcrystalline electron diffraction.

[0006] Therefore, the present invention provides a crystal structure of carboplatin combined with 1,1-cyclobutanedicarboxylic acid eutectic, which was detected under freezing conditions.

[0007] Specifically, the crystal structure belongs to the monoclinic crystal system. Based on the system's extinction rules and structural analysis results, the space group is determined to be P21 / c (no. 14), and the cell parameters are a = 5.68(2) Å, b = 12.00(5) Å, c = 23.25(19) Å, α = 90°, β = 95.3(5)°, γ = 90°, V = 1578(16) Å. 3The smallest asymmetric unit contains one carboplatin molecule and one 1,1-cyclobutanedicarboxylic acid molecule (A is carboplatin, B is cyclobutanedicarboxylic acid, where molecule A contains metal Pt), and each unit cell contains four smallest asymmetric units.

[0008] Furthermore, the statistical parameters after the structure is refined and converged are R1 = 0.1717, wR2 = 0.3679, and S = 1.1935.

[0009] More specifically, the chemical formula of a single target molecule within the smallest asymmetric unit of the crystal form is C0. 12 H 20 N₂O₈Pt, with a calculated molecular weight of 515.38 Da and a calculated crystal density of 2.169 g / cm³. 3 .

[0010] The present invention further provides a method for detecting the crystal form, which includes electron microscopy observation of powder crystals composed of carboplatin and 1,1-cyclobutanedicarboxylic acid under frozen conditions. The frozen conditions are liquid nitrogen freezing, with the freezing temperature maintained at 100K.

[0011] The powder crystals of carboplatin and 1,1-cyclobutanedicarboxylic acid composite are prepared as follows: 1) Carboplatin with a purity of not less than 99% and 1,1-cyclobutanedicarboxylic acid are mixed in a molar ratio of 1:2 to prepare a supersaturated aqueous solution (the concentration of each component should be as high as possible, but no crystallization should be maintained). This mixing and preparation operation is carried out at 65℃±5℃ and maintained for about 4 hours to complete the self-assembly reaction.

[0012] 2) Filter the reaction system, and let the filtrate stand at room temperature (20±5℃) in the dark for 10 days. Crystals will be observed to form.

[0013] 3) The filtrate was filtered again to obtain the crystalline compound, and the remaining filtrate was recovered. The purity of the crystals was determined by XRPD analysis, and the content was found to be over 99%.

[0014] 4) Take an appropriate amount of the above complex crystallized and ground into powder, vacuum dried, and dehydrated to obtain carboplatin and 1,1-cyclobutanedicarboxylic acid composite powder crystals.

[0015] This invention obtains the crystal structure of carboplatin combined with 1,1-cyclobutanedicarboxylic acid through research and detection, which helps to explain its good water solubility and stability. It is of great significance for the subsequent preparation of drugs such as aqueous solutions, lyophilized powders, capsules, and tablets, as well as for studying the intermolecular hydrogen bonding linkage mode between the two and the enhancement of their anticancer activity, reduction of toxic side effects, and the development of other cocrystallized drugs. Attached Figure Description

[0016] Figure 1 Ellipsoid diagram of the three-dimensional structure of the smallest asymmetric unit.

[0017] Figure 2 Chemical structure diagram of a molecule.

[0018] Figure 3 The Q peaks near O1 and O3 in molecule B.

[0019] Figure 4 Unit cell molecular packing diagram (projected along the a-axis).

[0020] Figure 5 Unit cell molecular packing diagram (projected along the b-axis).

[0021] Figure 6 Unit cell molecular packing diagram (projected along the c-axis). Detailed Implementation

[0022] The present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the embodiments below, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0023] In the following embodiments, unless otherwise specified, the processes involved (such as temperature control, heating, measurement, collection, preparation of test solution, and testing process) can all be handled in accordance with conventional methods in the art, such as using conventional instruments and methods.

[0024] I. Preparation of ZC2377 powder samples

[0025] The ZC2377 powder sample was prepared according to the method described in Chinese Invention Patent Application No. 202010872938.8, and the specific method is as follows:

[0026] 1) Mix carboplatin with a purity of not less than 99% and 1,1-cyclobutanedicarboxylic acid in a molar ratio of 1:2 to prepare a supersaturated aqueous solution (the concentration of each component should be as high as possible, but no crystallization should be maintained). This mixing and preparation operation is carried out at 65℃±5℃ and maintained for about 4 hours to complete the self-assembly reaction.

[0027] 2) Filter the reaction system, and let the filtrate stand at room temperature (20±5℃) in the dark for 10 days. Crystals will be observed to form.

[0028] 3) The filtrate was filtered again to obtain the crystalline compound. At the same time, the remaining filtrate was recovered. The purity of the crystal was detected by XRPD analysis, and the product content was found to be over 99%.

[0029] 4) Take an appropriate amount of the above complex crystallized and ground into powder, vacuum dried, and dehydrated to obtain ZC2377 powder sample powder (crystal powder).

[0030] II. Preparation of Freeze Crystallization

[0031] I) Experimental Methods

[0032] 1. Micro-ED Experiment

[0033] 1) Sample processing

[0034] a. Take a small amount of ZC2377 (batch number 20240622) powder sample onto a clean glass slide and use a polarizing microscope to confirm its crystallization. For example... Figure 1 The polarized light microscope image shows that the sample contains microcrystals, which can be used for MicroED detection.

[0035] b. Take a small amount of ZC2377 (batch number 20240622) powder sample and disperse it evenly on a cryo-electron microscope grid (Holey Carbon EM grid).

[0036] c. Place the prepared sample carrier into the front end of the liquid nitrogen cryogenic sample holder (Model 626, Gatan), and insert the sample holder into the Talos F200C cryo-electron microscope. Before testing, add liquid nitrogen to the Dewar flask at the end of the cryogenic sample holder to gradually stabilize the temperature of the crystals on the sample carrier at around 100 K.

[0037] d. After inspecting the quality of the microcrystals, 21 crystals with high diffraction resolution (such as...) were selected. Figure 2 Microcrystals were used for MicroED data collection. During the test, the microcrystals were irradiated with a parallel electron beam (NanoProbe mode), and a goniometer continuously and uniformly rotated the microcrystals at a speed of 2.0° / second. Simultaneously, a Ceta-D camera collected MicroED data from the microcrystals in continuous exposure mode. The MicroED data collection angle for the microcrystals was 80-110°, and the exposure time for a single diffraction photograph was 0.5 seconds.

[0038] e. Sixteen sets of relatively good MicroED data were selected. XDS software was used to index diffraction points, determine unit cell parameters, and integrate diffraction intensity. The XSCALE program was then used to merge the data, resulting in the final diffraction data file. The merged diffraction data file contains a total of 81,872 diffraction points and 3,090 independent diffraction points. During crystal structure analysis, the atomic scattering factor of electrons was used for theoretical structure factor calculation, and SHELXT (Intrinsic phasing algorithm) software was used to complete the structure analysis, obtaining the initial structural model. SHELXL (least squares method) was used for structure refinement. Based on the compound's molecular formula, all non-hydrogen atoms were identified and their anisotropy was refined. The positions of all hydrogen atoms were obtained by combining Fourier difference plots with calculations involving hydrogen addition.

[0039] II) Experimental Results

[0040] 1. Crystal structure of solid raw materials

[0041] Micro-ED results indicate that the structure belongs to the monoclinic crystal system. The space group can be determined as P21 / c (no. 14) based on the system extinction behavior and structural analysis. The cell parameters are a = 5.68(2) Å, b = 12.00(5) Å, c = 23.25(19) Å, α = 90°, β = 95.3(5)°, γ = 90°, V = 1578(16) Å. 3 The smallest asymmetric unit contains one of each of two target molecules (molecule A and molecule B, where molecule A contains metal Pt), and each unit cell contains four smallest asymmetric units. The statistical parameters after structure refinement convergence are R1 = 0.1717, wR2 = 0.3679, and S = 1.1935. The final chemical formula of a single target molecule within the smallest asymmetric unit is determined to be C0. 12 H 20 N₂O₈Pt, with a calculated molecular weight of 515.38 Da and a calculated crystal density of 2.169 g / cm³. 3 Its crystallographic parameters are shown in Table 1.

[0042] Table 1. Crystallographic parameters of ZC2377

[0043]

[0044] Figure 1 The ellipsoidal diagram of the smallest asymmetric unit cell in the crystal is shown (atomic labels are the same as in the appendix). The chemical structure of the molecule is as follows. Figure 2 As shown. Figure 3 The diagram shows Q peaks (corresponding to the residual potential positions of hydrogen atoms in the Fourier difference plot) near O1 and O3 atoms in molecule B. O1 is 1.125 Å from its nearest neighbor Q peak, while this Q peak is 1.509 Å from the nearest O6 atom in molecule A. O3 is 1.052 Å from its nearest neighbor Q peak, while this Q peak is 1.557 Å from the nearest O5 atom in molecule A. These results indicate the presence of hydrogen atoms around O1 and O3 in molecule B, meaning that the protons on the carboxyl groups of molecule B have not transferred to molecule A, and that hydrogen bonds exist between the carboxyl groups of molecule B and the carbonyl groups on the carboxylate groups of neighboring molecule A. Therefore, this structure should be a eutectic.

[0045] Figure 4 , Figure 5 and Figure 6The figures show the projections of the molecular unit cell packing diagrams along the a-axis, b-axis, and c-axis, respectively. The results indicate that the two types of molecules are interleaved by hydrogen bonds along the ab-plane, forming a network layer approximately 1 / 2c thick. This network layer is also packed along the c-axis, with adjacent network layers interacting through van der Waals forces. Hydrogen bonds exist between the hydroxyl groups on the two carboxyl groups of molecule B and the carbonyl groups on the carboxylate groups of neighboring molecule A (O1-H1···O6). 5 And O3-H3···O5), where the distance between H1 and the O6 atom in neighboring molecule A is 1.96 Å, and the distance between H3 and O5 is 1.97 Å. The two amine atoms coordinated to Pt in molecule A also form multiple hydrogen bonds with the carboxyl group of neighboring molecule B (N1-H1A···O2). 1 , N2-H2A···O4 4 and N2-H2B···O2 1 The distance between H1A and the O2 in neighboring molecule B is 2.17 Å, the distance between H2A and the O4 in neighboring molecule B is 2.14 Å, and the distance between H2B and the O2 in neighboring molecule B is 2.07 Å. In addition, the two ammonia molecules coordinated with Pt in molecule A also form multiple hydrogen bonds with other neighboring molecules A (N1-H1B···O8). 2 ,N1-H1C···O5 3 and N2-H2C···O6 3 The distance between H1B and the O8 in neighboring molecule A is 2.33 Å, the distance between H1C and the O5 in neighboring molecule A is 2.25 Å, and the distance between H2C and the O6 in neighboring molecule A is 2.23 Å.

Claims

1. A crystal of carboplatin and 1,1-cyclobutanedicarboxylic acid co-crystal combination, the structure of the crystal form belongs to monoclinic system, the space group is P21 / c (no. 14) determined by systematic extinction law combined with structure analysis results, the cell parameters are a = 5.68 (2) Å, b = 12.00 (5) Å, c = 23.25 (19) Å, α = 90°, β = 95.3 (5) °, γ = 90°, V = 1578 (16) Å 3 The minimum asymmetric unit contains 2 target molecules each 1, wherein A is carboplatin and B is cyclobutane diacid, molecule A contains metal Pt, and each cell contains 4 minimum asymmetric units.

2. The crystal of claim 1, wherein The statistical parameters of the structure refinement convergence are R1 = 0.1717, wR2 = 0.3679, S = 1.1935.

3. A method of detecting a crystal as claimed in claim 1 or 2, characterised in that, The method comprises the following steps: S1: preparing a crystalline powder of carboplatin and 1,1-cyclobutanedicarboxylic acid eutectic compound; S2: determining the crystal structure of the crystal form by using a cryo-EM technique.

4. The method of claim 3, wherein, S1 specifically comprises the following steps: 1) mixing carboplatin and 1,1-cyclobutanedicarboxylic acid with a purity of not less than 99% at a molar ratio of 1:2 to prepare a supersaturated aqueous solution (the concentrations of the components are as high as possible, but crystallization is not allowed to occur), and the mixing and preparation operation is performed at 65°C±5°C for about 4 hours; 2) filtering the reaction system, and placing the filtrate at room temperature (20±5°C) in the dark for 10 days, during which crystallization is observed to occur; 3) filtering the filtrate again to obtain the crystalline compound, and recovering the remaining filtrate, and detecting the purity of the product by XRPD analysis, and the content of the product is measured to be more than 99%; 4) grinding the crystalline compound into a powder, and vacuum drying to remove the crystallization water, thereby obtaining the powder crystal of the carboplatin and 1,1-cyclobutanedicarboxylic acid combination. S2 specifically comprises the following steps: performing an electron microscope experiment on the powder crystal of the carboplatin and 1,1-cyclobutanedicarboxylic acid eutectic combination under a frozen condition. The freezing is liquid nitrogen freezing, and the temperature of the freezing is maintained at 100K. S2 specifically comprises the following steps:

5. The method of claim 3, wherein, S2-1: placing the powder crystal sample on a clean glass slide, and using a polarizing microscope to confirm the crystallization condition of the sample; 6. The method of claim 5, wherein, S2-2: taking the powder crystal sample and uniformly dispersing the sample onto a cryo-EM grid; 7. The method of claim 3, wherein, S2-3: placing the prepared grid at the front end of a liquid nitrogen freezing sample rod, and inserting the sample rod into a Talos F200C cryo-EM; S2-4: before testing, adding liquid nitrogen into a Dewar flask at the tail end of the freezing sample rod to gradually stabilize the temperature of the crystal on the grid at 100K. The testing process specifically comprises the following steps: The microcrystal is irradiated in a parallel electron beam mode, an angle meter drives the microcrystal to rotate at a constant speed of 2.0° / second, and a Ceta-D camera collects MicroED data of the microcrystal in a continuous exposure mode; the collection angle of the MicroED data of the microcrystal is 80-110°, and the exposure time of a single diffraction photograph is 0.5 seconds. The selected Micro-ED data is subjected to diffraction point indexing, cell parameter determination and diffraction intensity integration by using XDS software, and data merging is completed by using an XSCALE program, thereby obtaining a final diffraction data file; 8. The method of claim 6, wherein, In the process of crystal structure analysis, an atomic scattering factor of an electron is used for theoretical structure factor calculation, and a SHELXT (Intrinsic phasing algorithm) software is used to complete structure analysis, thereby obtaining an initial structure model; A SHELXL program (least squares method) is used to complete structure refinement, all non-hydrogen atoms are identified and anisotropically refined in combination with compound molecular formula information, and all hydrogen atom positions are obtained by Fourier difference maps combined with calculation hydrogenation. ​ ​ ​

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