CN111721842B

CN111721842B - Method for detecting related substances in lobaplatin

Info

Publication number: CN111721842B
Application number: CN201910208846.7A
Authority: CN
Inventors: 窦啟玲; 汪立冬; 常新亮
Original assignee: Hainan Changan International Pharmaceutical Co ltd; Guizhou Yibai Pharmaceutical Co Ltd
Current assignee: Hainan Changan International Pharmaceutical Co ltd; Guizhou Yibai Pharmaceutical Co Ltd
Priority date: 2019-03-19
Filing date: 2019-03-19
Publication date: 2022-07-12
Anticipated expiration: 2039-03-19
Also published as: CN111721842A

Abstract

The invention also provides a method for detecting related substances in lobaplatin, wherein the related substances comprise 6 compounds, and the detection method is an HPLC method or an HPLC-MS method. The chromatographic column of the HPLC method is as follows: the surface of the silica gel is coated with cellulose-tri (3-chloro-4-methyl phenyl carbamate) as a filler. The mobile phase of the HPLC method is n-hexane-ethanol (60-70: 30-40), the flow rate of the HPLC method is 0.8-1.5ml per minute, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is carried out for 30-50 min; preferably, the flow rate is 1.0ml per minute, the detection wavelength is 210nm, the column temperature is 35 ℃ and the isocratic elution time is 40 min. The related substances are applied to cells of lung cancer, liver cancer, small cell lung cancer, breast cancer, blood tumor, leukemia, gastric cancer, ovarian cancer, prostatic cancer and/or renal cancer.

Description

Method for detecting related substances in lobaplatin

Technical Field

The invention relates to the field of medicines, in particular to a method for detecting related substances in lobaplatin, and belongs to the technical field of medicine analysis quality control.

Background

Lobaplatin (Lobaplatin, D19466), also known as Lobaplatin, is a third-generation platinum-based antitumor drug following cisplatin and carboplatin, and its chemical name is: cis- [ trans-1, 2-cyclobutanebis (methylamine) -N, N']- [ (2S) -lactic acid-O1, O2]-platinum (II), formula C₉H₁₈N₂O₃Pt has a molecular weight of 397.34 and a chemical structural formula shown in the following formula (1):

lobaplatin has alkylating action and belongs to an alkylating agent (in a broad sense). Has good antitumor effect, such as inhibiting in vitro AH 135-tumor, B16-melanoma, colon cancer 115, and in vivo mouse P338 leukemia. Lobaplatin is characterized by strong anticancer activity, low toxicity, no accumulative toxicity and nephrotoxicity and less toxicity to bone marrow, and currently marketed lobaplatin is mainly used for treating breast cancer, small cell lung cancer and chronic myelocytic leukemia.

Disclosure of Invention

In order to ensure the safety, effectiveness and controllable quality of the medicine, the research on related substances and detection methods of the related substances is very important. For the drug, due to the existence of three chiral carbons and related substances generated in the preparation process, confirming the structure of the related substances and finding a suitable detection method for controlling the product quality of the drug become technical problems to be solved urgently in the field.

The invention aims to provide a novel detection method which can simultaneously detect a plurality of related substances in lobaplatin.

One skilled in the art will recognize that any substance that affects the purity of a drug is collectively referred to as a related substance (or related substances). Research on related substances is an important part of drug development, and comprises selecting a proper analysis method, accurately distinguishing and determining the content of the related substances, and determining the reasonable limit of the related substances by combining the results of pharmaceutical, toxicological and clinical researches. This study is throughout the entire process of drug development.

Specifically, the present invention is realized by the following technical means.

A method for detecting related substances in lobaplatin, wherein the related substances are selected from the following compounds H1, H2, G1, G2, L1 or L2:

compound H1:

compound H2:

compound G1:

compound G2:

compound L1:

and compound L2:

preferably, in the detection method, the substance concerned is any one of compound H1, compound H2, a mixture of compounds G1 and G2, compound L1, or compound L2.

Preferably, for the detection method, the related substances simultaneously include compound H1, compound H2, a mixture of compounds G1 and G2, compound L1 and compound L2.

Preferably, for the detection method of any one of the preceding claims, wherein said lobaplatin comprises either or both of lobaplatin diastereomer I and lobaplatin diastereomer II.

Preferably, the detection method of any one of the preceding claims, wherein the preparation of compounds H1 and H2 is via intermediates

And/or

Preparing to obtain; the preparation method of the compounds G1 and G2, L1 and L2 is to pass through intermediates

Is prepared.

Preferably, the detection method described in any one of the above, wherein the compounds H1 and H2 are obtained by the following reaction equation (1):

alternatively, the compounds G1 and G2 are obtained by the following reaction scheme (2):

alternatively, the compounds L1 and L2 were obtained by the following reaction equation (3):

among them, still more preferably, in the above reaction equation (1), the trans-diiodide, compound 1, is prepared by the following equation (1-a):

in the above reaction equation (2), the compound 1 (cis oxalate) can be obtained by reacting cis dicyanocyclobutane to obtain diaminomethylcyclobutane and then reacting with oxalic acid as in the reaction equation (3); that is, the compounds G1 and G2 can be prepared by the following reaction formula (1-b):

preferably, the detection method of any one of the preceding claims, wherein the detection method is an HPLC method or an HPLC-MS method.

Preferably, the detection method according to any one of the preceding claims, wherein the HPLC method comprises a column: the surface of the silica gel is coated with cellulose-tri (3-chloro-4-methyl phenyl carbamate) as a filler.

Preferably, the detection method is any one of the detection methods, wherein the mobile phase of the HPLC method is n-hexane-ethanol with a volume ratio of 60-70: 30-40, preferably 63-67: 37-33; more preferably, the mobile phase is n-hexane-ethanol in a volume ratio of 65: 35.

Preferably, the detection method according to any one of the preceding claims, wherein the elution profile of the HPLC method is isocratic elution.

Preferably, for the detection method of any one of the preceding items, wherein the flow rate of the HPLC method is 0.8-1.5 ml/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is 30-50 min; preferably, the flow rate is 1.0ml per minute, the detection wavelength is 210nm, the column temperature is 35 ℃ and the isocratic elution time is 40 min.

Preferably, in the detection method according to any one of the above, the detection method is such that the related substance H1, the related substance L1, and the related substance L2 each do not exceed 0.5 times the peak area of the main component in the control solution, and the total of the related substance G1, the related substance G2, and the related substance H2 does not exceed the peak area of the main component in the control solution, as calculated in terms of the peak area by the main component self-control method without addition of a correction factor.

Preferably, in the detection method according to any one of the preceding claims, if there is a peak of the relevant substance in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of the substances of interest G1, G2 and/or H2 is 2.40-2.70, preferably 2.58, and the relative retention time of the substance of interest H1 is 2.00-2.30, preferably 2.16; the relative retention times of compound L1 are in the range from 1.2 to 1.5, preferably 1.35, and of compound L2 in the range from 3.4 to 3.7, preferably 3.58.

The invention has the following beneficial effects:

the invention confirms the structure of the compound H1, the compound H2, the compound G1, the compound G2, the compound L1 or the compound L2, confirms related substances of the lobaplatin, and can simultaneously detect a plurality of related substances (impurities) in the lobaplatin in order to establish a complete lobaplatin quality detection system. The method has the advantages of high sensitivity, strong specificity, good repeatability and high accuracy.

Drawings

FIG. 1A: the HPLC-MS of the substance H of the present invention;

FIG. 1B: the present invention relates to the MS spectrum in HPLC-MS of substance H;

FIG. 2: of the invention with respect to substance H¹An H-NMR spectrum;

FIG. 3: of the invention with respect to substance H¹³A C-NMR spectrum;

FIG. 4: the invention relates to the Q-NMR spectrum of substance H;

FIG. 5: the invention relates to the UV spectrum of substance H;

FIG. 6: the invention relates to the IR spectrum of substance H;

FIG. 7: the invention relates to the DSC pattern of substance H;

FIG. 8A-1: HPLC chromatogram (wavelength 220nm) for HPLC-MS structure confirmation detection of the substance H1 of the present invention;

FIG. 8A-2: HPLC chromatogram (wavelength 254nm) for HPLC-MS structure confirmation detection of the substance H1 of the present invention;

FIG. 8B: the invention relates to the MS map of substance H1;

FIG. 9A: the invention relates to the SFC profile of substance H1;

FIG. 9B: the molecular three-dimensional structure diagram of the substance H1 of the invention;

FIG. 10A-1: HPLC chromatogram (wavelength 220nm) for HPLC-MS structure confirmation detection of the substance H2 of the present invention;

FIG. 10A-2: HPLC chromatogram (wavelength 254nm) for HPLC-MS structure confirmation detection of the substance H2 of the present invention;

FIG. 10B: the HPLC-MS structure of the related substance H2 confirms the detected MS map;

FIG. 11: the invention relates to the SFC profile of substance H2;

FIG. 12A-1: the HPLC-MS combined structure of the compound G1 of the invention confirms the HPLC spectrum (the wavelength is 215nm) in the detection;

FIG. 12A-2: the HPLC-MS combined structure of the compound G1 of the present invention confirms the HPLC profile (wavelength 210nm) in the detection;

FIG. 12B: the HPLC-MS combined structure of the related substance G1 confirms the MS map in the detection;

FIG. 13: of the invention with respect to substance G1¹An H-NMR spectrum;

FIG. 14: of the invention with respect to the substance G1¹³A C-NMR spectrum;

FIG. 15: the invention relates to the Q-NMR spectrum of substance G1;

FIG. 16: the present invention relates to the UV spectrum of substance G1;

FIG. 17: the invention relates to the IR spectrum of substance G1;

FIG. 18A: the present invention relates to the DSC profile of substance G1;

FIG. 18B: the invention relates to an HPLC spectrogram confirmed by the structure of a substance G1;

FIG. 19A-1: the HPLC-MS combined structure of the compound G2 of the invention confirms the HPLC spectrum (the wavelength is 215nm) in the detection;

FIG. 19A-2: the HPLC-MS combined structure of the compound G2 of the present invention confirms the HPLC profile (wavelength 210nm) in the detection;

FIG. 19B: the MS spectrum in the detection is confirmed by the HPLC-MS combined structure of a related substance G2;

FIG. 20: of the invention with respect to substance G2¹An H-NMR spectrum;

FIG. 21: of the invention with respect to substance G2¹³A C-NMR spectrum;

FIG. 22: the invention relates to the Q-NMR spectrum of substance G2;

FIG. 23: the present invention relates to the UV spectrum of substance G2;

FIG. 24: the invention relates to the IR spectrum of substance G2;

FIG. 25A: the present invention relates to the DSC pattern of substance G2;

FIG. 25B: the invention relates to an HPLC spectrogram for confirming the structure of a substance G2;

FIG. 26: intermediate of the invention for preparing Compounds L1 and L2 Compound 3¹An H-NMR spectrum;

FIG. 27A-1: the HPLC-MS combined structure of the compound L1 confirms the HPLC spectrum (the wavelength is 215nm) in the detection;

FIG. 27A-2: the HPLC-MS combined structure of the compound L1 confirms the HPLC spectrum (the wavelength is 210nm) in the detection;

FIG. 27B: the MS spectrum in the detection is confirmed by the HPLC-MS combined structure of a related substance L1;

FIG. 28: of the invention with respect to the substance L1¹An H-NMR spectrum;

FIG. 29: of the invention with respect to the substance L1¹³A C-NMR spectrum;

FIG. 30: the present invention relates to a Q-NMR spectrum of substance L1;

FIG. 31: the present invention relates to the UV spectrum of substance L1;

FIG. 32: the present invention relates to the IR spectrum of substance L1;

fig. 33A: the present invention relates to the DSC pattern of substance L1;

FIG. 33B: the HPLC spectrogram confirmed by the structure of the related substance L1 of the invention;

FIG. 34A-1: the HPLC-MS combined structure of the compound L2 confirms the HPLC spectrum (the wavelength is 215nm) in the detection;

fig. 34A-2: the HPLC-MS combined structure of the compound L2 confirms the HPLC spectrum (the wavelength is 210nm) in the detection;

FIG. 34B: the present invention relates to the MS spectrum of substance L2;

FIG. 35: of the invention with respect to the substance L2¹An H-NMR spectrum;

FIG. 36: of the invention with respect to the substance L2¹³A C-NMR spectrum;

FIG. 37: the invention relates to the Q-NMR spectrum of substance L2;

FIG. 38: the present invention relates to the UV spectrum of substance L2;

FIG. 39: the present invention relates to the IR spectrum of substance L2;

FIG. 40A: DSC chart of substance L2 according to the present invention;

FIG. 40B: the HPLC spectrogram confirmed by the structure of the related substance L2 of the invention;

FIG. 41: the related substance of the invention is taken as a typical HPLC (high performance liquid chromatography) spectrum in a detection example of the related substance of the lobaplatin;

FIG. 42A-1: dose response plot of substance H against HCCC-9810;

FIG. 42A-2: dose response profile of STSP to HCCC-9810;

FIG. 42B-1: dose response profile of substance H to NCI-H460;

FIG. 42B-2: dose response profile of substance L2 to NCI-H460;

FIG. 42B-3: dose response profiles of STSP to NCI-H460;

FIG. 42C-1: dose response profile of substance H to MDA-MB-453;

FIG. 42C-2: dose response profile of STSP to MDA-MB-453;

FIG. 42D-1: dose response plot of substance H against DU 145;

FIG. 42D-2: dose response plots of STSP versus DU 145;

FIG. 42D-3: dose response plot of substance L1 against SK-OV-3;

FIGS. 42D-4: dose response profiles of STSP to SK-OV-3;

FIGS. 42D-5: dose response plot of substance G1 against K562;

FIGS. 42D-6: dose response plot of substance G2 against K562;

FIGS. 42D-7: dose response plot of substance L1 against K562;

FIGS. 42D-8: dose response plot of STSP versus K562;

FIGS. 42D-9: dose response plot of substance L2 against K562;

FIGS. 42D-10: dose response plots of STSP versus K562;

FIG. 42E-1: dose response profile of substance G1 to Jurkat Clone E6-1;

FIG. 42E-2: dose response profile of substance L1 to Jurkat Clone E6-1;

FIG. 42E-3: dose response profile of substance L2 to Jurkat Clone E6-1;

FIG. 42E-4: dose response profile of substance G2 to Jurkat Clone E6-1;

FIGS. 42E-5: dose response plot of substance H against Jurkat Clone E6-1;

FIGS. 42E-6: dose response profiles of STSP to Jurkat Clone E6-1;

FIG. 42F-1: dose response profile of substance G2 to AGS;

FIG. 42F-2: dose response profile of substance L1 to AGS;

FIG. 42F-3: dose response plot of substance L2 against AGS;

FIG. 42F-4: dose response profile of substance G1 to AGS;

FIG. 42F-5: dose response profile of substance H to AGS;

FIGS. 42F-6: dose response plots of STSP versus AGS;

FIG. 42G-1: dose response plot of substance H against HL-60;

FIG. 42G-2: dose response plots of STSP versus HL-60;

FIG. 42G-3: dose response profile of substance G2 to HL-60;

FIGS. 42G-4: dose response plot of L1 against HL-60;

FIGS. 42G-5: the dose response plot of substance L2 against HL-60;

FIGS. 42G-6: dose response plot of substance G1 against HL-60;

FIG. 42H-1: dose response profile of substance H to SK-NEP-1;

FIG. 42H-2: dose response profiles of STSP to SK-NEP-1;

FIG. 42H-3: the dose response plot of substance G2 for SK-NEP-1;

FIGS. 42H-4: the dose response plot of substance L1 against SK-NEP-1;

FIGS. 42H-5: the dose response plot of substance L2 against SK-NEP-1;

FIGS. 42H-6: the dose response plot of substance G1 for SK-NEP-1;

FIG. 42I-1: dose response profile of substance H versus 95-D;

FIG. 42I-2: dose response profiles of STSP versus 95-D;

FIG. 42I-3: dose response profile of substance G1 versus 95-D;

FIG. 42I-4: dose response profile of substance G2 versus 95-D;

FIGS. 42I-5: a dose response plot of substance L2 versus 95-D;

FIG. 42J-1: dose response profile of substance H to THP-1;

FIG. 42J-2: dose response plot of STSP versus THP-1;

FIG. 42J-3: dose response profile of substance G2 to THP-1;

FIGS. 42J-4: a dose response plot of substance L1 against THP-1;

FIG. 42J-5: a dose response plot of substance L2 against THP-1;

FIGS. 42J-6: dose response profile of substance G1 to THP-1;

FIG. 42K-1: dose response profile of substance H to OVCAR-3;

FIG. 42K-2: dose response profiles of STSP to OVCAR-3;

FIG. 42K-3: dose response profile of substance G1 to OVCAR-3;

FIG. 42K-4: dose response profile of substance G2 to OVCAR-3;

FIG. 42K-5: the dose response plot of substance L2 against OVCAR-3;

FIG. 43: the detection methodology of the invention verifies the chromatogram of the hollow white solution;

FIG. 44: the chromatogram of the RS solution in the detection methodology verification is adopted;

FIG. 45: the linear plot of diastereomer II in the validation of the detection methodology of the invention;

FIG. 46: the linear graph of diastereomer I in the detection methodology validation of the invention;

FIG. 47: the linear plot of diastereomer H2 in the validation of the detection methodology of the present invention;

FIG. 48: the invention detects the linear graph of related substance H1 in the methodological verification;

FIG. 49: the invention detects the linear graph of the related substance G1 in the methodology validation;

FIG. 50: the invention detects the linear graph of the related substance G2 in the methodological verification;

FIG. 51: the invention detects the linear graph of related substance L1 in the methodological verification;

FIG. 52: the detection methodology of the present invention validates the linear plot of the related substance L2.

Detailed Description

The invention finds and prepares the preparation of new substances related to the quality of lobaplatin and a detection method for controlling the quality of lobaplatin, and particularly provides a detection method for a plurality of related substances of lobaplatin related to the quality of lobaplatin.

In the present invention, any substance affecting the purity of the lobaplatin medicament is collectively referred to as "related substance affecting the quality of lobaplatin" or "related substance affecting the quality", simply referred to as "related substance" (also referred to as "related substance" in some cases herein), for example, a peak of related substance affecting the quality of lobaplatin appearing in an XRD diffraction peak for detecting the quality of lobaplatin, simply referred to as "related substance peak"; the "related substance" in the present invention is sometimes an "impurity" known to those skilled in the art to affect the purity of the drug, however, the "related substance" in the present invention is not limited to the category of "impurity" but also includes substances having a certain anticancer activity even higher than that of lobaplatin, which belong to the category of materials related to lobaplatin with respect to the active molecule "lobaplatin", and the principles of their anticancer activity or other positive effects and functions in developing new drugs have not been fully studied. The research of the related substances in the invention is an important content of drug development, and comprises the steps of selecting a proper analysis method, accurately distinguishing and measuring the content of impurities and determining the reasonable limit of the impurities by integrating the results of pharmaceutical, toxicological and clinical researches, wherein the research is carried out in the whole process of drug development.

In a preferred embodiment of the present invention, the present invention provides a method for detecting the quality of a lobaplatin bulk pharmaceutical drug or formulation, which comprises the step of measuring a related substance affecting the quality of lobaplatin therein, wherein the related substance is any one of the following compounds H1, H2, G1, G2, L1 or L2, or a mixture of any two or more of them:

compound H1:

compound H2:

compound G1:

compound G2:

compound L1:

compound L2:

wherein the detection method is an HPLC method or an HPLC-MS method; the chromatographic column of the HPLC method comprises the following steps: the surface of the silica gel is coated with cellulose-tri (3-chloro-4-methyl phenyl carbamate) as a filling agent; the mobile phase of the HPLC method is n-hexane-ethanol with the volume ratio of 60-70: 30-40, preferably 63-67: 37-33; preferably, the mobile phase is n-hexane-ethanol with a volume ratio of 65:35, and the elution mode is isocratic elution.

Preferably, the flow rate of the HPLC method is 0.8-1.5ml per minute, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is performed for 30-50 min; preferably, the flow rate is 1.0ml per minute, the detection wavelength is 210nm, the column temperature is 35 ℃ and the isocratic elution time is 40 min.

Preferably, wherein the related substance H1, the related substance L1, and the related substance L2 each do not exceed 0.5 times the peak area of the main component in the control solution, as calculated by the peak area of the main component self-control method without addition of a correction factor, and the total of the related substance G1, the related substance G2, and the related substance H2 should not exceed the peak area of the main component in the control solution.

Preferably, the detection method is characterized in that if a peak of a relevant substance exists in the test sample-lobaplatin solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, preferably 2.58, and the relative retention time of related substance H1 is 2.00-2.30, preferably 2.16; the relative retention time of compound L1 was 1.2-1.5, preferably 1.35, and the relative retention time of compound L2 was 3.4-3.7, preferably 3.58.

The relative retention time of the material refers to the retention time relative to lobaplatin, specifically relative to lobaplatin diastereomer II. Specifically, as the lobaplatin compound, 2 isomers, lobaplatin diastereomer I and lobaplatin diastereomer II, which are represented by the following structural formulae, are known:

lobaplatin diastereomer I (RRS for short):

lobaplatin diastereomer II (SSS for short):

the preparation of compounds such as H1, H2, G1 or G2, L1 or L2 which are substances related to the quality of lobaplatin and found by the present invention, the confirmation of the structure of these novel substances and the antitumor activity of these novel substance compounds will be described below by way of examples; the method for detecting related substances in lobaplatin of the present invention will be further described in detail by examples.

EXAMPLE 1 preparation of Compound H

The preparation of substance H and H1 and H2 is shown in the following equations (A), (B) and isolation scheme (C); the compound is prepared according to the method described in example 1 of patent No. CN102093226B, and the compound 1A (trans-diaminomethylcyclobutane oxalate) is obtained after structural identification, and then the diiodide shown in the formula 1, namely the compound 1, is prepared according to the following reaction formula (A) by using the oxalate as a raw material; then, a compound represented by the formula (H) (also referred to as a compound H) is obtained from the compound 1 by the following reaction formula (B); then separating the H compound by SFC (supercritical fluid chromatography) to obtain a compound H1 and a compound H2, as shown in a separation process formula (C). Wherein the reaction conditions of temperature, time, solvent, etc. are merely exemplified to designate the temperatures of some embodiments, the process for producing H of a substance in the present invention is not limited to those shown in the following reaction formulas (A), (B) and (C).

The sources of reagents used in the following preparations were as follows:

the compound was prepared according to the method described in example 1 of patent No. CN102093226B, and the compound 1A (trans-dimethylaminocyclobutane oxalate) was confirmed by structural identification, and then the diiodide compound represented by formula 1, i.e., compound 1, was prepared according to reaction formula (a) using the oxalate as a starting material for preparing compound H according to reaction formula (B). Specifically, the preparation of compound 1 (diiodo compound 1 described in examples 5 and 6 below is also prepared by the following procedure) is as follows:

compound 1A (30.0g,101.9mmol), potassium chloroplatinite (36.0g,86.7mmol), potassium iodide (86.0g,518.1mmol) and potassium hydroxide (24.0g,427.7mmol) were dissolved in 170mL, 180mL,87mL and 120mL of purified water, respectively, to give solutions A, B, C and D.

And ii, heating the liquid B to 30 ℃. Stirring the material A and scattering.

And iii, adding the solution C to the solution B, and stirring for 0.5h to obtain a solution E.

Iv, adding the solution D to the solution A, stirring, clarifying the system, and filtering by using a 0.45-micron filter membrane to obtain a solution F.

V. add F to E and precipitate a yellow solid, continue stirring at 30 ℃ for 2 hours.

Vi. filtration, washing the filter cake with purified water (100mLX6) to a halide-free residue. The filter cake was dried by rotary evaporator to give compound 1(35g) as a yellow powder.

Hereinafter, a specific process for preparing compound H by the reaction formula (B) using compound 1 as a starting material and processes for preparing H1 and H2 by compound H are described as follows:

1) preparation of Compound 2

Compound 1(20g,35.5mmol) was dispersed in purified water (84mL) and acetone (12mL) to give feed A. Silver nitrate (10.91g,64.2mmol) was dissolved in purified water (32mL) and added to feed A and stirred at 30 ℃ for 18 h in the dark. Filtration, the filter cake was washed 6 times with water (20mL x6), and the filtrates were combined to give 250mL of compound 2 solution which was used directly in the next step.

2) Preparation of Compound 3

The resin (80g) was treated three times with 1.5mol/L aqueous sodium hydroxide (120 mL). 250mL of compound 2 solution and treated resin (80g) were placed in a three-necked flask and stirred at 30 ℃ for 1 hour in the dark. Filtration, resin with purified water 100mL washing 6 times (100mL 6), combined washing liquid and filtrate to get compound 3(850 mL) solution directly for the next step.

3) Preparation of Compound H

To the solution of compound 3, a 20 mass% aqueous solution of hydroxypropionic acid was added, the pH was adjusted to 6.4, and the mixture was stirred at 35 ℃ for 36 hours in the dark. Filtering, and concentrating the filtrate under reduced pressure to dryness. The residue was dissolved in acetone (9mL) and purified water (18 mL), recrystallized at 10 ℃ for 120H, filtered, and spun dry to give the relevant substance H (3.5g) as an off-white solid.

4) Preparation of Compounds H1 and H2

The related substance H obtained above was separated by SFC (supercritical fluid chromatography, apparatus model used: Waters 80Q Preparative SFC system) to obtain related substances H1 (Compound H1) and H2 (Compound H2). Wherein the separation conditions are as follows: column DAICEL CHIRALCEL OD (250mm 30mm,10 μm), mobile phase A: [ supercritical CO₂]Mobile phase B, [ methanol, + volume ratio is 0.1% NH₃·H₂O](ii) a Isocratic elution, 0-14min, 75% by volume mobile phase A + 25% by volume mobile phase B). Among them, substances H1 and H2 were specifically separated by SFC supercritical fluid chromatography (H1 was labeled H1 for compounds whose chromatograms came first, i.e., which showed short retention times, and H2 was labeled H2 for substances whose chromatograms come later, i.e., which showed long retention times), after which the following confirmation of structure was carried out, particularly single crystals were carried outAfter the culture, the substance H1 successfully obtains the related detection data such as single crystal diffraction peak; however, the substance H2 did not obtain ideal single crystal diffraction data, and after confirming the structure of the substance H1 from the structure, the structure of the substance H2 was estimated. In the following structure confirmation examples and activity test examples, compounds H1 and H2 both corresponded to compound H1 and compound H2 prepared in this example 1, that is, compound H1 referred to herein was obtained first (i.e., the retention time was short) when the compound was prepared under the above-mentioned liquid chromatography conditions, and compound H2 referred to herein was obtained after (i.e., the retention time was long) when the compound was prepared under the above-mentioned liquid chromatography conditions.

Specifically, the structural confirmation and activity detection examples below show the structural formulae of the substances H1 and H2 as follows:

the molecular formulas are all as follows: c₉H₁₈N₂O₃Pt

Example 2: structure confirmation of compounds H, H1 and H2

1. The structure of compound H obtained in example 1 was confirmed:

1)HPLC-MS:

the HPLC-MS conditions used were:

the name and model of the HPLC-MS instrument used were: agilent 1200LC & Agilent 6110 MSD;

HPLC conditions: gradient elution was performed using octadecylsilane bonded silica as a filler (Agilent ZORBAX SB-Aq,2.1 x 50mm, 5 μm), 0.0375 vol% trifluoroacetic acid as mobile phase A, and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the procedure of Table 1 below; the detection wavelengths are 210nm and 215nm (DAD detector), the column temperature is 50 ℃, and the detection spectrum is shown in figure 1A.

TABLE 1

Wherein the MS condition is as follows: and (3) detecting by using a single four-level rod tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is used, a monitoring mode is full scanning, and the scanning range is 100-1500.

The detection result is shown in the following table 2, and the spectrum is shown in the attached figure 1B, and it can be seen that the related substances are platinum-containing organic substances, and because the isotopes with high platinum element abundance have¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.0]⁺The peak is the sample excimer ion peak, and the [ M' + CH ] appears at about 440.1₃CN+H]⁺Peaks are the sample excimer ion peak and [ 2M' + H ] again at 795.3]⁺Is the peak of the excimer ion after the dimerization of the sample, corresponding to the related substance H (C)₉H₁₈N₂O₃Pt) has molecular weight of 397.33, mass spectrum information and related substance H (C)₉H₁₈N₂O₃Pt) the molecular structure is consistent. The mass spectrum information is consistent with the molecular structure of the related substances of the invention.

TABLE 2

m/e	Fragment ion peak	Remarks to note
			398.1	[M’+H]⁺	Peak of excimer ion of sample
440.1	[M’+CH₃CN+H]⁺	Sample addingExcimer peak of acetonitrile
			795.3	[2M’+H]⁺	Peak of excimer ion after sample dimerization

Note: m' is C₉H₁₈N₂O₃Molecular weight of Pt

2)¹H-NMR:

The instrument name: BRUKERBV-400 NMR spectrometer; chemical shifts and assignments of hydrogen spectra (CD3OD — 400MHz) are shown in Table 3 below:

TABLE 3

Chemical shift (ppm)	Multiplicity of properties	Number of protons	Attribution of Hydrogen
				1.21-1.23	d	3	6
1.57-1.65	m	2	1,1’
				1.90-1.98	m	2	1,1’
2.36-2.89	m	6	3,3’,2,2’
				3.99-4.05	m	1	5

The spectrogram is shown in figure 2, and the hydrogen spectrum data of the sample is identical with the molecular structure of the product.

3)¹³C-NMR:

The instrument name: BRUKERBV-400 NMR spectrometer

Chemical shifts and assignments for carbon spectra (CD3OD — 400MHz) are shown in Table 4 below:

TABLE 4

Chemical shift (ppm)	Type of carbon atom	Number of carbon atoms	Attribution of carbon
				21.72-21.76	Secondary carbon(s)	2	1,1’
22.01-22.17	Primary carbon	1	6
				39.51-40.07	Secondary carbon(s)	2	3,3’
50.59-50.89	Tertiary carbon	2	2,2’
				74.42	Secondary carbon	1	5
194.21-194.25	Quaternary carbon	1	7

The spectrum is shown in figure 3, and it can be seen that,¹³the C-NMR chart has 5 saturated secondary carbon peaks, 2 saturated tertiary carbon peaks, 1 saturated primary carbon peak and 1 unsaturated quaternary carbon peak, which are consistent with the molecular structure of the substance H.

4)Q NMR

The determination was performed by bruker avance NEO 400 using CD3OD as a solvent and coumarin (99.74%) as an internal standard, and the results are shown in table 5 below:

TABLE 5

The calculation formula of W% is as follows:

in the formula, W_ISTDMass (mg) of internal standard;

W_Sammass of sample (mg);

A_Sam/A_ISTDis the area ratio of the sample and the internal standard substance;

MW_SAMis the molecular weight of the sample;

MW_ISTDis the molecular weight of the internal standard;

n_ISTDand n_SamIs the number of protons per functional group;

W_ISTD% is the mass percentage of the internal standard substance,

the spectrum is shown in FIG. 4, and it can be seen from the above table that the calibration content is 97.4%.

5) Ultraviolet absorption spectrum (UV):

UV-2600Series ultraviolet visible spectrometer; measuring the temperature at room temperature; the measuring range is 190-400 nm; measuring solvent water; the spectrum is shown in figure 5, and the maximum ultraviolet absorption wavelength is at 190 nm.

6) Infrared spectrum (IR)

An infrared spectrometer: ALPHA-BRUKER; the measurement conditions were as follows: solid KBr pellets were formed. Measurement range: 4000cm^-1～400cm^-1The measurement results and analysis are shown in the following table 6, and the map is shown in the attached figure 6:

TABLE 6

Absorption peak wave number (cm)^-1)	Type of vibration	Group assignment
			3450.62,3206.31,3127.28	ν_NH	Amino N-H stretching vibration
2968.38,2946.15,2866.99	ν_CH	Alkyl C-H stretching vibration
			1638.50,1578.55	ν_C＝O	C ═ O stretching vibration of carbonyl group
1373.23,1346.20,320.56	δ_CH	Alkyl C-H bending vibration
			1110.83	ν_C-O	Stretching vibration of C-O bond
1042.14	ν_C-N	Stretching vibration of C-N bond

7) Differential Scanning Calorimetry (DSC)

Instrument model METTELER DSC 1; the heating rate is 10.0 ℃/min; the temperature range is 40-350 deg.C, and the spectrum is shown in figure 7.

8) Optical Rotation (OR)

Polarimeter Anton paarpmcp 500; the measuring conditions are that C is 0.5mol/L (water),25 ℃; the results are shown in table 7 below:

TABLE 7

The substance H of the present invention was confirmed by the above-mentioned map

The structure is that the air conditioner is provided with a water inlet pipe,

the substance H comprises 2 isomers H1 and H2,

2. structure confirmation of Compound H1

The following structure confirmation was carried out for H1 obtained in example 1:

1)HPLC-MS:

the HPLC-MS conditions used were:

HPLC conditions: all HPLC-MS instruments are SHIMADZU LCMS-2020; using octadecylsilane chemically bonded silica as filler (Kinetex EVO C182.1 × 30mm, 5 μm), using ammonia water with volume ratio of 0.025% as mobile phase A and acetonitrile as mobile phase B, and performing gradient elution according to the following procedure of Table 8; the detection wavelengths were 220nm and 254nm (PAD detector) and the column temperature was 40 ℃.

MS conditions: and (3) detecting by using a single four-level rod tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is used, a monitoring mode is full scanning, and the scanning range is 100-1000-.

TABLE 8

The detection result is illustrated in figure 8A-1, figure 8A-2 and figure 8B, and it can be seen that the related substances are platinum-containing organic substances, and the isotopes with high platinum element abundance have¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.0]⁺The peak is the sample excimer peak, corresponding to the relevant substance H1 (C)₉H₁₈N₂O₃Pt) has a molecular weight of 397.33, mass spectrometric information and compound H1 (C)₉H₁₈N₂O₃Pt) the molecular structure is consistent. The mass spectrum information is consistent with the structure of the molecule H1 of the related substance of the invention.

2) SFC of substance H1

Wherein, the SFC condition is as follows: agilent 1260series analytical SFC, column: DAICEL CHIRALCEL OD-3(50mm 4.6mm,3 μm), mobile phase A: [ supercritical CO ]₂]Mobile phase B, [ methanol, + volume ratio is 0.05% diethylamine]Gradient elution was carried out according to the procedure of Table 8A below at a flow rate of 3.0mL/min, a column temperature of 35 ℃ and a wavelength of 220 nm.

TABLE 8A

The spectrum of compound H1 is shown in fig. 9A, and it can be seen that the peak of compound H1 appears at a retention time of 2.678 min.

3. Single crystal diffraction of Compound H1

1) The conditions for single crystal culture preparation were as follows:

the sample is dissolved in a solution of acetone-water (1: 1) and placed in a semi-sealed container, the solvent is slowly volatilized at room temperature, crystals with proper sizes are formed in 17 days, the transparency of the crystals is checked by a microscope, and then the crystals are subjected to X-ray detection.

2) Single crystal diffraction equipment and data collection mode;

equipment: rigaku Saturn X-ray with graphite monochromatic Mo-K alpha radiation targetLine diffractometer (Rigaku Saturn diffraction grating-monochromatic-modulated Mo K alpha radiation)

)。

Diameter of single tube: phi is 0.50mm

Distance from crystal to CCD detector: d is 45mm

Tube Voltage (Tube Pressure) 50kV

Tube Current (Tube Flow) 16mA

16961 reflection data are collected together in the range of 2.444to 27.864 degrees of theta, the limit indexes are-12 is less than or equal to h less than or equal to 12, -13 is less than or equal to k less than or equal to 13, and-18 is less than or equal to l less than or equal to 18; at 3365unique reflections (R)_int＝0.0682)。

3) The results of single crystal diffraction are summarized below:

the crystal is a colorless prism having dimensions of 0.10X 0.10mm³(ii) a The symmetry of the crystal structure is assigned to the orthogonal space group (P2(1) 2(1)) with the following parameters:

α＝β＝γ＝90°,

Z＝4,Dc＝2.120 Mg/m³,F(000)＝872,μ(Mo Kα)＝9.944mm^–1andT 113 (2). The three-dimensional structure is shown in fig. 9B.

4) The specific data for single crystal diffraction are as follows: wherein the X-ray crystallographic data are summarized in Table 9 below:

TABLE 9

Wherein the sub-coordinates (x 10^4) and isotropic displacement parameters (A ^2x 10^3) (Atomic coordinates (x 10^4) and specific anisotropic displacement parameters (A ^2x 10^ 3)) are shown in Table 10 below:

watch 10

Wherein the Bond length [ A ] and Bond angle [ deg ] (Bond length [ A ] and angles [ deg ]) are shown in Table 11 below:

TABLE 11

Wherein, the Torsion angle [ deg ] (Torsion angles [ deg ]) is shown in the following table 12

TABLE 12

The bond lengths and bond angles of the Hydrogen bonds [ Aad deg. ] (Hydrogen bonds [ Aad deg. ]) ] are shown in Table 13 below.

Watch 13

The symmetric transformations used to generate equivalent atoms:

#1-x+1/2,-y+1,z+1/2 #2x,y+1,z+1 #3x,y+1,z #4-x+1,y+1/2,-z+1/2 #5x-1/2,-y+1/2,-z #6x,y,z-1 #7-x+1/2,-y+1,z-1/2 #8-x,y-1/2,-z+1/2 #9x-1,y-1,z #10x,y-1,z

in summary, the absolute configuration of H1 was determined to be:

3. structure confirmation of Compound H2

The following structure confirmation was carried out for H2 obtained in example 1:

1)HPLC-MS:

HPLC conditions: the HPLC-MS instrument is SHIMADZU LCMS-2020; performing gradient elution by using octadecylsilane bonded silica gel as filler (Kinetex EVO C182.1 × 30mm, 5 μm), ammonia water with volume ratio of 0.025% as mobile phase A, and acetonitrile as mobile phase B according to the following procedure in Table 14; the detection wavelengths were 220nm and 254nm (PAD detector) and the column temperature was 40 ℃.

MS conditions: detecting by a single four-level rod tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is adopted, a monitoring mode is full scanning, and the scanning range is 100-1000-;

TABLE 14

The detection spectra are shown in FIG. 10A-1, FIG. 10A-2 and FIG. 10B, and it can be seen that the related substances are platinum-containing organic substances, and the isotopes with high abundance of platinum element have¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.0]⁺The peak is the sample excimer peak, corresponding to the relevant species H1 (C)₉H₁₈N₂O₃Pt) has a molecular weight of 397.33, mass spectrometric information and compound H2 (C)₉H₁₈N₂O₃Pt) molecular structure phaseAnd (4) sign. The mass spectral information is consistent with the structure of the molecule H2 of the substance of interest (in the case where the absolute configuration of substance H1 is determined as described above, the configuration of substance H2 can be deduced).

2) SFC detection of substance H2

SFC detection conditions: the instrument model names used were: agilent 1260series analytical SFC, column: DAICEL CHIRALCEL OD-3(50mm 4.6mm,3um), mobile phase A: [ supercritical CO ]₂]Mobile phase B, [ methanol, + volume ratio is 0.05% diethylamine]Gradient elution was carried out according to the procedure in Table 14A below at a flow rate of 3.0mL/min, a column temperature of 35 ℃ and a wavelength of 220 nm.

TABLE 14A

The spectrum of compound H2 is shown in fig. 11, and it can be seen from fig. 11 that the peak substance is H2 when t is 2.805 min.

According to the single crystal diffraction result of H1 and the structure of compound H, the structure of compound H2 can be obtained by reverse extrapolation as follows:

example 3: preparation of Compounds G1 and G2

The preparation reaction process is shown as follows:

the sources of reagents used are detailed in Table A below.

TABLE A

Specifically, the preparation of compound G by the above reaction scheme is as follows:

1) preparation of Compound 2A

Compound 1A (24.0g,226.1mmol) was dissolved in anhydrous tetrahydrofuran (THF, 480mL) and cooled to 0 ℃. At 0 ℃, dropwise adding 10mol/L borane dimethyl sulfide (BH)₃.Me₂S, the CAS number of which is 13292-87-0, 157mL of 1.57mol) is added, and stirring is carried out for 1 hour under the condition of heat preservation. The system was warmed to 40 ℃ and stirred for 1 hour. The temperature was raised to 65 ℃ and stirred for 1 hour. TLC (petroleum ether/ethyl acetate 2/1) showed complete reaction of starting material. The system was cooled to 0 ℃, quenched with 480mL methanol, and concentrated to dryness. N-butanol (350mL) was added and the mixture was stirred at 100 ℃ for 16 hours. Crude compound 2A (42.0g) was obtained and used directly in the next step.

2) Preparation of Compound 1

Compound 2A (42.0g, crude) was dissolved in isopropanol (i-PrOH, 400mL) to give solution A. Oxalic acid (11.5g,127.7mmol) was dissolved in isopropanol (115mL) to give solution B. The B liquid was dropped to the A liquid, and a large amount of white solid (crude compound 1) was precipitated. The system was warmed to 70 ℃ and stirred for 1 hour. After filtration, the filter cake was added to THF (110mL) at room temperature, warmed to 65 ℃ and stirred for 1 h. Filtration and drying of the filter cake gave compound 1 (23.0g) as a white solid.

3) Preparation of Compound 2

Compound 1(17.0g,57.7mmol), potassium chloroplatinite (21.5g,51.9mmol), potassium iodide (51.4g,309.7mmol) and potassium hydroxide (14.6g,220.8mmol) were dissolved in 65mL,70mL, 50mL and 143mL of purified water, respectively, to give solutions A, B, C and D. And heating the solution B to 30 ℃. Stirring and scattering the material A. And adding the solution C to the solution B, and stirring for 0.5h to obtain solution E. Adding solution D to solution A, stirring, clarifying, and filtering with 0.45 μm filter membrane to obtain solution F. Solution F was added to solution E and a yellow solid precipitated which was stirred at 30 ℃ for 4 hours. Filtration and washing of the filter cake 6 times with purified water, 100mL each time (100mL x6) until no halogen ions remain. The filter cake was dried by rotary evaporator to give compound 2(19g) as a yellow powder.

4) Preparation of Compound 3

Compound 2(19g) was dispersed in water (82mL) and acetone (9.5mL) and stirred for ten minutes. Silver nitrate (10.4g,61.3mmol) was dissolved in water (31mL) and added to the system and stirred at 20 ℃ for 16 h, protected from light. Filtration, washing of the filter cake with purified water (30 mL. times.5), and combining the aqueous phases gave an aqueous solution of compound 3(200mL) which was used directly in the next step.

5) Preparation of Compound 4

The resin (80g) was treated three times with 1.5mol/mL aqueous sodium hydroxide (150 mL). An aqueous solution of compound 3 was heated to 30 ℃. The treated resin was added to the system in one portion and stirred for 1 hour. Filtration was carried out, and the resin was washed with purified water (50 mL. times.6). The aqueous phases were combined to give an aqueous solution of compound 4(500mL) which was used directly in the next step.

6) Preparation of Compounds G1 and G2

An aqueous solution of compound 4(500mL) was placed in the flask. The pH of the system was adjusted to 6.4 (practically, any pH of 6.4 to 6.8) with lactic acid compound 5, and the mixture was heated to 30 ℃ to form a small amount of black residue, followed by stirring for 87 hours. Filtration and lyophilization of the aqueous phase gave a yellow solid. HPLC (instrument model: SHIMADZU LC-20AP, column: Phenomenex Synergi Max-RP (250X 50mM X10 μm)), and mobile phase A: water (10mM NH)₄HCO₃) And B: acetonitrile](ii) a Elution with a gradient of 0-17.8min, mobile phase B increased from 0 to 17.5%) twice to give compound G1(1.45G) and compound G2(1.54G) as white solids. Wherein the first-to-peak compound is labeled G1 and the last-to-peak compound is labeled G2.

In the structure confirmation examples and the activity test examples which follow, compounds G1 and G2 both correspond to compound G1 and compound G2 which were prepared in the examples, that is, compound G1 referred to herein is a compound which is obtained first (i.e., the retention time is short) when the compound is prepared under the above-mentioned liquid chromatography conditions, and compound G2 referred to herein is a compound which is obtained after (i.e., the retention time is long) when the compound is prepared under the above-mentioned liquid chromatography conditions.

Wherein, G1 and G2 are both any one of the following 2 structures, and when G1 is one of the structures, G2 is the other of the 2 structures; the confirmation process of the structure will be specifically explained by the following structure confirmation examples.

Example 4: structure confirmation of compounds G1 and G2

Sequentially sampling and detecting the purified compounds G1 and G2 obtained in the step 6) in the example 3, wherein the detection comprises high performance liquid chromatography-mass spectrometry (HPLC-MS); nuclear magnetic resonance hydrogen spectrum (¹H NMR); nuclear magnetic resonance carbon spectrum (C)¹³C NMR); ultraviolet absorption spectrum (UV); infrared spectroscopy (IR); differential Scanning Calorimetry (DSC); HPLC, Optical Rotation (OR); quantitative nuclear magnetic resonance (Q NMR).

When the compounds G1 and G2 were subjected to single crystal growth, the specific structures of the final two compounds could not be confirmed due to failure of single crystal growth, but two chiral enantiomers could be confirmed, and these measurements confirmed that the related substances were only two compounds, but the specific compounds could not be confirmed finally. The structural confirmation for compounds G1 and G2 are therefore both assigned. The concrete structure is as follows:

comprises the following steps:

or

Molecular formula C₉H₁₈N₂O₃Pt

1. Structure confirmation of Compound G1

1)HPLC-MS:

The instrument name and model are: agilent 1200LC & Agilent 6110MSD

The HPLC-MS conditions used were:

HPLC conditions: gradient elution was performed using octadecylsilane bonded silica as a filler (Agilent ZORBAX SB-Aq,2.1 x 50mm, 5 μm), 0.0375 vol% trifluoroacetic acid as mobile phase A, and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the procedure of Table 15-1 below; the detection wavelengths were 210nm and 215nm (DAD detector), the column temperature was 50 ℃ and the detection results are shown in FIGS. 12A-1 and 12A-2, and the gradient elution procedure is shown in Table 15-1 below.

TABLE 15-1

MS conditions: a single four-level rod tandem mass spectrometer is used for detection, an electrospray ionization (ESI) ion source is used as an ion source, a positive ion scanning mode is used, a monitoring mode is full scanning, and the scanning range is 100-1000.

The test results are shown in the following table 15-2, and it can be seen that the related substance is an organic substance containing platinum, and the isotope with high abundance of platinum element has¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.1]⁺Peaks are the sample excimer peaks, appearing around 438.1, 439.1, 440.2, [ M' + CH₃CN+H]⁺The peak is the sample excimer peak, corresponding to the related substance G1 (C)₉H₁₈N₂O₃Pt) has molecular weight of 397.33, mass spectrum information and related substance G1 (C)₉H₁₈N₂O₃Pt) the molecular structure is consistent. The mass spectrum information conforms to the molecular structure of the related substances of the invention, and is shown in the attached figure 12B in detail.

TABLE 15-2

m/e	Fragment ion peak	Remarks for note
			397.1,398.1,399.1,400.1	[M’+H]⁺	Peak of excimer ion of sample
438.1,439.1,440.2	[M’+CH₃CN+H]⁺	Excimer peak of sample plus acetonitrile

Note: m' is C₉H₁₈N₂O₃Molecular weight of Pt

2)¹H-NMR:

The name of the instrument: BRUKERBV-400 NMR spectrometer;

¹h NMR chemical shifts and assignments for hydrogen spectra (methanol (CD3OD)400MHz) are as follows: related substance G1 (C)₉H₁₈N₂O₃Pt) contains 4 active hydrogens and 14 inactive hydrogens; sample Hydrogen Spectrum data with G1 (C)₉H₁₈N₂O₃Pt) and the associated chemical shifts are shown in table 16, and are detailed in figure 13.

TABLE 16

Chemical shift (ppm)	Multiplicity of properties	Number of protons	Attribution of hydrogen
				1.31-1.37	m	3	6
1.55-1.65	m	2	1,1’
				2.07-2.19	m	2	1,1’
2.70-3.01	m	6	3,3’,2,2’
				4.12-4.18	m	1	5

3)¹³C-NMR:

The instrument name: BRUKERBV-400 NMR spectrometer;

chemical shifts and assignments of carbon spectra (CD3OD,400MHz) are shown in table 17 below:

TABLE 17

Chemical shift (ppm)	Type of carbon atom	Number of carbon atoms	Attribution of carbon
				20.34-20.37	Secondary carbon	2	1,1’
21.75	Primary carbon	1	6
				35.09-35.65	Secondary carbon(s)	2	3,3’
44.47-44.86	Tertiary carbon	2	2,2’
				74.83	Secondary carbon	1	5
194.23	Quaternary carbon	1	7

¹³The C-NMR spectrum has 5 saturated secondary carbon peaks, 2 saturated tertiary carbon peaks, 1 saturated primary carbon peak and 1 unsaturated quaternary carbon peak, as shown in FIG. 14, which is consistent with the molecular structure of related substance G1.

4) Quantitative nuclear magnetic resonance (Q NMR)

The instrument model is as follows: BrukerAVANCE NEO 400; the solvent used was CD3OD and was determined by the internal standard method, which was benzyl benzoate (99.8 mass%), and the results are shown in table 18 below:

watch 18

The calculation formula of W% is as follows:

in the formula, W_ISTDMass (mg) of internal standard;

W_Sammass of sample (mg);

MW_SAMis the molecular weight of the sample;

MW_ISTDis the molecular weight of the internal standard;

n_ISTDand n_SamIs the number of protons per functional group;

W_ISTD% is the mass percentage of the internal standard substance;

the spectrum is shown in figure 15, and from the above table, it can be seen that the calibration content is 94.86 mass%.

5) Ultraviolet absorption spectrum (UV):

UV-2600Series ultraviolet visible spectrometer; measuring the temperature at room temperature; the measuring range is 190-400 nm; measuring solvent water; the map is shown in figure 16.

6) Infrared spectrum (IR)

An infrared spectrometer: ALPHA-BRUKER; the measurement conditions were as follows: solid KBr pellets were formed. Measurement range: 4000cm^-1～400cm^-1The measurement results and analysis are shown in table 19 below: the spectrum is shown in figure 17.

Watch 19

Absorption peak wave number (cm)^-1)	Type of vibration	Group assignment
			3422.64,3253.80,3128.37	ν_NH	Amino N-H stretching vibration
2978.35,2937.71,2873.05	ν_CH	Alkyl C-H stretching vibration
			1637.54,1615.10	ν_C＝O	C ═ O carbonyl group stretching vibration
1363.46,1336.51	δ_CH	Alkyl C-H bending vibration
			1048.04	ν_C-N	Stretching vibration of C-N bond

7) Optical Rotation (OR)

Polarimeter AntonPaarMCP 500; the determination conditions are that C is 0.5mol/L (water),25 ℃;

the results are given in table 20 below:

watch 20

8) Differential Scanning Calorimetry (DSC)

The instrument model is METTELER DSC 1; the heating rate is 10.0 ℃/min; the temperature is 40-350 deg.C, and the graph is shown in figure 18A, wherein the left limit of the first peak is 122.04 deg.C, the peak is 154.49 deg.C, and the right limit is 161.69 deg.C; the second peak has a left limit of 161.69 deg.C, a peak of 177.73 deg.C and a right limit of 240.05 deg.C.

9) High Performance Liquid Chromatography (HPLC)

The instrument model is as follows: SHIMADZU LC-20AB, column model: waters XSelect CSH-C184.6 x 150mm x 3.5 μm;

the operating conditions for the HPLC were: gradient elution was performed using octadecylsilane bonded silica gel as a filler (Waters xselette CSH C18, 4.6 × 150mm, 3.5 μm), water (+0.0375 vol% trifluoroacetic acid) as mobile phase a and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B, following the procedure of table 20A below; the detection wavelength was 235nm (PDA detector) and the column temperature was 40 ℃.

TABLE 20A gradient elution procedure

The pattern is shown in FIG. 18B, from which it can be seen that the peak of compound G1 appears at a retention time of 7.815 min.

2. Structure confirmation of Compound G2

1)HPLC-MS:

The instrument name and model are: agilent 1200LC & Agilent 6110MSD

The HPLC conditions used were:

wherein the HPLC conditions are as follows: gradient elution was performed using octadecylsilane bonded silica as a filler (Agilent ZORBAX SB-Aq,2.1 x 50mm, 5 μm), 0.0375 vol% trifluoroacetic acid as mobile phase A, and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the following procedure; the detection wavelengths were 210nm and 215nm (DAD detector), the column temperature was 50 ℃ and the detection results were shown in FIGS. 19A-1 and 19A-2, and the gradient elution procedure is shown in Table 21-1 below.

TABLE 21-1

Wherein the MS condition is as follows: and (3) detecting by using a single four-level rod tandem mass spectrometer, wherein the ion source is an electrospray ionization (ESI) ion source, a positive ion scanning mode is used, a monitoring mode is full scanning, and the scanning range is 100-1000-.

The related substance is organic substance containing platinum, and the isotope with high abundance of platinum element is¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in the MS of the samples, [ M' + H ] appears at 397.0, 398.1, 399.1, 400.1, 401.1]⁺The peak is the sample excimer ion peak, and the [ M' + CH ] appears at about 440.2₃CN+H]⁺The peak is the sample excimer peak, corresponding to the related substance G2 (C)₉H₁₈N₂O₃Pt) has molecular weight of 397.33, mass spectrum information and related substance G2 (C)₉H₁₈N₂O₃Pt) molecular structures are consistent, the relevant data are shown in a table 21-2, and the specific spectrogram is shown in a figure 19B.

TABLE 21-2

Note: m' is C₉H₁₈N₂O₃Molecular weight of Pt

2)¹H-NMR:

The instrument name: BRUKERBV-400 NMR spectrometer;

chemical shifts and assignments of hydrogen spectra (CD3OD — 400MHz) are as follows: related substance G2 (C)₉H₁₈N₂O₃Pt) contains 4 active hydrogens and 14 inactive hydrogens; sample Hydrogen Spectrum data with G2 (C)₉H₁₈N₂O₃Pt) are matched, and the data is shown in table 22, and the detailed spectrum is shown in fig. 20.

TABLE 22

Chemical shift (ppm)	Multiplicity of properties	Number of protons	Attribution of hydrogen
				1.28-1.35	d	3	6
1.44-1.65	m	2	1,1’
				2.10-2.22	m	2	1,1’
2.54-3.00	m	6	3,3’,2,2’
				4.10-4.18	m	1	5

3)¹³C-NMR:

The name of the instrument: BRUKERBV-400 NMR spectrometer;

chemical shifts and assignments of carbon spectra (CD3OD — 400MHz) are given in Table 23 below:

TABLE 23

Chemical shift (ppm)	Type of carbon atom	Number of carbon atoms	Attribution of carbon
				20.34	Secondary carbon	2	1,1’
21.82	Primary carbon	1	6
				35.19	Secondary carbon	2	3,3’
44.40-44.94	Tertiary carbon	2	2,2’
				74.85	Secondary carbon	1	5
194.22	Quaternary carbon	1	7

¹³The C-NMR spectrum contains 5 saturated secondary carbon peaks, 2 saturated tertiary carbon peaks, 1 saturated primary carbon peak and 1 unsaturated quaternary carbon peak, which are consistent with the molecular structure of the related substance G2 shown in the attached FIG. 21.

4) Quantitative nuclear magnetic resonance (Q NMR)

The instrument model is as follows: BrukerAVANCE NEO 400; the solvent used was CD3OD and was determined by the internal standard method, which was benzyl benzoate (99.8%), and the results are shown in table 24 below:

the calculation formula of W% is shown in the following table 24:

TABLE 24

In the formula, W_ISTDMass (mg) of internal standard;

W_Sammass of sample (mg);

A_Sam/A_ISTDis the area ratio of the sample and the internal standard;

MW_SAMis the molecular weight of the sample;

MW_ISTDmolecules as internal standardsAn amount;

n_ISTDand n_SamIs the number of protons per functional group;

W_ISTD% is the mass percentage of the internal standard substance;

the map is shown in FIG. 22, from which it can be seen that the nominal content is 95.49%.

5) Ultraviolet absorption spectrum (UV):

UV-2600Series ultraviolet visible spectrometer; measuring the temperature at room temperature; the measuring range is 190-400 nm; measuring solvent water; the spectrum is shown in figure 23, and the maximum ultraviolet absorption wavelength is at 190 nm.

6) Infrared spectrum (IR)

An infrared spectrometer: ALPHA-BRUKER; the measurement conditions were as follows: solid KBr pellets were formed. Measurement range: 4000cm^-1～400cm^-1The measurement results and analysis are shown in table 25 below: the spectrum is shown in figure 24.

TABLE 25

Absorption peak wave number (cm)^-1)	Type of vibration	Group assignment
			3424.38,3217.09,3133.38	ν_NH	Amino N-H stretching vibration
2975.09,2868.01	ν_CH	Alkyl C-H stretching vibration
			1636.72	ν_C＝O	C ═ O stretching vibration of carbonyl group
1350.46	δ_CH	Alkyl C-H bending vibration
			1110.91	ν_C-O	Stretching vibration of C-O bond
1048.04	ν_C-N	Stretching vibration of C-N bond

7) Optical Rotation (OR)

Polarimeter AntonPaarMCP 500; the measuring conditions are that C is 0.5mol/L (water),25 ℃;

the results are given in Table 26 below:

watch 26

8) Differential Scanning Calorimetry (DSC)

Instrument model METTELER DSC 1; the heating rate is 10.0 ℃/min; the temperature range is 40-350 deg.C, and is shown in figure 25A, wherein the left limit of the first peak is 100.35 deg.C, the peak value is 130.32 deg.C, and the right limit is 150.86 deg.C; the left limit of the second peak was 154.96 deg.C, the peak was 182.38 deg.C, and the right limit was 241.85 deg.C.

9) High Performance Liquid Chromatography (HPLC)

The instrument model is as follows: SHIMADZU LC-20AB, column model: waters XSelect CSH-C184.6 mm 150mm 3.5 μm.

The operating conditions for the HPLC were: gradient elution was performed using octadecylsilane bonded silica gel as a filler (Waters xselette CSH C18, 4.6 × 150mm, 3.5 μm), water (+0.0375 vol% trifluoroacetic acid) as mobile phase a and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B, following the procedure of table 26A below; the detection wavelength was 235nm (PDA detector) and the column temperature was 40 ℃.

TABLE 26A gradient elution procedure

The pattern is shown in FIG. 25B, from which it can be seen that the peak of compound G2 appears at a retention time of 8.800 min.

Example 5: preparation of Compounds L1 and L2

The reaction process in the preparation is as follows:

wherein the following examples prepared compounds L1 and L2 using the following sources of starting materials:

the specific preparation steps are described as follows:

1) preparation of Compound 2

Compound 1(24.0g,226.1mmol) was dissolved in anhydrous tetrahydrofuran (480mL) and cooled to 0 ℃. At 0 ℃, dropwise adding 10mol/L borane dimethyl sulfide (BH)₃.Me₂S, the CAS number of which is 13292-87-0, 157mL of 1.57mol) is added, and stirring is carried out for 1 hour under the condition of heat preservation. The system was warmed to 40 ℃ and stirred for 1 hour. The temperature was raised to 65 ℃ and stirred for 1 hour. TLC (petroleum ether/ethyl acetate 2/1 vol) showed the starting material was completely reacted. The system was cooled to 0 ℃, quenched with 480mL methanol, and concentrated to dryness. N-butanol (350mL) was added and the mixture was stirred at 100 ℃ for 16 hours. Crude compound 2(42.0 g) was obtained and used directly in the next step.

2) Preparation of Compound 3

Compound 2(42.0g, crude) was dissolved in isopropanol (400mL) to give solution A. Oxalic acid (11.5g,127.7mmol) was dissolved in isopropanol (i-PrOH, 115mL) to give solution B. The B liquid is dropped into the A liquid, and a large amount of white solid (crude compound 3) is separated out. The system was warmed to 70 ℃ and stirred for 1 hour. After filtration, the filter cake was added to THF (110mL) at room temperature, warmed to 65 ℃ and stirred for 1 h. Filtration and drying of the filter cake afforded Compound 3(23.0g, which¹The HNMR detection spectrum is shown in FIG. 26) is white solid.

3) Preparation of Compound 4

Compound 3(23.0g,78.2mmol), potassium chloroplatinite (29.1g,70.2mmol), potassium iodide (69.6g,419.0mmol) and potassium hydroxide (19.7g,298.6mmol) were dissolved in 88mL,96mL,70 mL and 194mL of purified water to give solutions A, B, C and D, respectively. And heating the solution B to 30 ℃. Stirring and scattering the material A. Adding solution C to solution B, and stirring for 0.5h to obtain solution E. Adding solution D to solution A, stirring, clarifying, and filtering with 0.45 μm filter membrane to obtain solution F. The solution F was added to the solution E, and a yellow solid precipitated, and stirring was continued at 30 ℃ for 4 hours. Filtration and the filter cake was washed 5 times (50mL x 5) with 50mL of purified water to halogen-free ionic residues. The filter cake was dried by rotary evaporator to give compound 4(24.0g) as a yellow powder.

4) Preparation of Compound 5

Compound 4(24.0g) was dispersed in water (90mL) and acetone (10.5mL) and stirred for 10 min. Silver nitrate (10.32g,60.75mmol) was dissolved in water (90mL) and added to the system and stirred at 20 ℃ for 16 h, protected from light. The filter cake was washed 3 times with 30mL of purified water (30mL x 5) and the aqueous phases were combined to give an aqueous solution of compound 5(300mL) which was used directly in the next step.

5) Preparation of Compound 6

The resin (100g) was treated three times with 1.5mol/L aqueous sodium hydroxide (200 mL). An aqueous solution of compound 5(300mL) was heated to 30 ℃. The treated resin was added to the system in one portion and stirred for 1 hour. Filtration and washing of the resin with purified water 4 times (40mLX 4). The aqueous phases were combined to give an aqueous solution of compound 6(460mL) which was used directly in the next step.

6) Preparation of Compounds L1 and L2

An aqueous solution of compound 6(460mL) was placed in the flask. By chemical combinationThe product 7 was adjusted to pH 6.6 with lactic acid, heated to 30 ℃ and reacted for 87 hours with a small amount of black residue. Filtering, and freeze-drying the filtrate. The lyophilized product was subjected to preparative high performance liquid chromatography prep.HPLC (model of chromatography apparatus: waters 80Q preparative SFC system; column: Phenomenex Synergi Max-RP (250X 50mm X10 μm), mobile phase A: water (10 mmol/L NH)₄HCO₃) And B: acetonitrile](ii) a Eluting with a gradient of 0-20min, increasing the volume of mobile phase B from 0 to 20%) twice to obtain compound L1(0.775g) and compound L2(0.882g) as white solids. Wherein the first-to-peak compound is labeled as L1, and the last-to-peak compound is labeled as L2.

In the structure confirmation examples and the activity test examples which follow, compounds L1 and L2 both correspond to compound L1 and compound L2 which were prepared in the present examples, that is, compound L1 referred to herein is a compound which is obtained first (i.e., the retention time is short) when the compound is prepared under the above-mentioned liquid chromatography conditions, and compound L2 referred to herein is a compound which is obtained after (i.e., the retention time is long) when the compound is prepared under the above-mentioned liquid chromatography conditions.

Wherein, the structural formulas of L1 and L2 are both any one of the following 2 structures, and when L1 is one of the structures, L2 is the other one of the 2 structures; the confirmation process of the structure will be specifically explained by the following structure confirmation examples. The concrete structure is as follows:

or

Molecular formula C₉H₁₈N₂O₃Pt

Example 6: structure confirmation of Compounds L1 and L2

The compounds L1 and L2 prepared in example 5 were subjected to structural detection and confirmation as follows. The chemical structure is as follows: the absolute configuration of the impurities L1 and L2 could not be confirmed because of failure of single crystal cultivation and no single crystal diffraction detection data), but two chiral enantiomers could be confirmed, and these detection confirmation data confirmed that the related substances were only two compounds, but could not finally confirm the specific compounds, so the following confirmation of the structures of the compounds L1 and L2 were both designated or labeled as described above.

1. Structure confirmation of Compound L1

The instrument name and model are: agilent 1200LC & Agilent 6110MSD

The HPLC-MS conditions used were as follows:

HPLC conditions: gradient elution was performed using octadecylsilane bonded silica gel as a filler (Agilent ZORBAX SB-Aq,2.1 x 50mm, 5 μm), 0.0375 vol% trifluoroacetic acid as mobile phase A, and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the procedure of Table 27-1 below; the detection wavelengths were 210nm and 215nm (DAD detector) and the column temperature was 50 ℃. The results are shown in FIG. 27A-1 and FIG. 27A-2.

TABLE 27-1 gradient elution procedure

The results are shown in the following table 27-2 and fig. 27B, and it can be seen that the related substance is an organic substance containing platinum due to the higher abundance of platinum element¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.2]⁺Peaks are the sample excimer peaks, appearing around 438.1, 439.1, 440.2, [ M' + CH₃CN+H]⁺The peak is the sample excimer peak, corresponding to the related substance L1 (C)₉H₁₈N₂O₃Pt) has molecular weight of 397.33, mass spectrum information and related substance L1 (C)₉H₁₈N₂O₃Pt) has a consistent molecular structure, as shown in FIG. 27B.

Watch 27

m/e	Fragment ion peak	Remarks for note
			397.1,398.1,399.1,400.2	[M’+H]⁺	Peak of excimer ion of sample
438.1,439.2,440.2	[M’+CH₃CN+H]⁺	Excimer peak of sample plus acetonitrile

Note: m' is C₉H₁₈N₂O₃Molecular weight of Pt

2)¹H-NMR:

The instrument name: BRUKERBV-400 model NMR spectrometer

Hydrogen spectrum (¹H NMR chemical shifts and assignments for deuterated methanol (CD3OD) _400MHz) are as follows: related substance L1 (C)₉H₁₈N₂O₃Pt) contains 4 active hydrogens and 14 inactive hydrogens; sample Hydrogen Spectrum data with L1 (C)₉H₁₈N₂O₃Pt) are identical in molecular structure and are shown in figure 28 in detail.

Watch 28

3)¹³C-NMR:

The instrument name: BRUKERBV-400 NMR spectrometer;

chemical shifts and assignments of carbon spectra (CD3OD — 400MHz) are given in Table 29 below:

watch 29

Chemical shift (ppm)	Type of carbon atom	Number of carbon atoms	Attribution of carbon
				20.34-20.37	Secondary carbon(s)	2	1,1’
21.74	Primary carbon	1	6
				35.08-35.20	Secondary carbon	2	3,3’
44.47-44.86	Tertiary carbon	2	2,2’
				74.83	Secondary carbon	1	5
194.23	Quaternary carbon	1	7

¹³The C-NMR spectrum contains 5 saturated secondary carbon peaks, 2 saturated tertiary carbon peaks, 1 saturated primary carbon peak and 1 unsaturated quaternary carbon peak, which are consistent with the molecular structure of related substance L1 shown in FIG. 29.

4)Q NMR

It was measured using bruker avance NEO 400 using CD3OD as a solvent and an internal standard method, the internal standard substance was Coumarin (Coumarin, 99.74%), as shown in fig. 30, and the measurement results are shown in table 30 below:

TABLE 30 QNMR test results

The calculation formula of W% is as follows:

in the formula, W_ISTDMass (mg) of internal standard;

W_Sammass of sample (mg);

A_Sam/A_ISTDis the area ratio of the sample and the internal standard;

MW_SAMis the molecular weight of the sample;

MW_ISTDis the molecular weight of the internal standard;

n_ISTDand n_SamIs the number of protons per functional group;

W_ISTD% is the mass percentage of the internal standard substance,

as can be seen from the above table, the nominal content thereof was 89.8 mass%.

5) Ultraviolet absorption spectrum (UV):

UV-2600Series ultraviolet visible spectrometer; measuring the temperature at room temperature; the measuring range is 190-400 nm; measuring solvent water; in detail, as shown in FIG. 31, the maximum ultraviolet absorption wavelength is at 190 nm.

6) Infrared spectrum (IR)

An infrared spectrometer: ALPHA-BRUKER; the measurement conditions were as follows: solid KBr pellets were formed. Measurement range: 4000cm^-1～400cm^-1The measurement results and analysis are shown in table 31 below: the spectrum is shown in figure 32.

Watch 31

Absorption peak wave number (cm)^-1)	Type of vibration	Group assignment
			3420.26,3253.53,3128.09	ν_NH	Amino N-H stretching vibration
2978.35,2937.71,2873.24	ν_CH	Alkyl C-H stretching vibration
			1633.45	ν_C＝O	C ═ O stretching vibration of carbonyl group
1363.15,1336.41	δ_CH	Alkyl C-H bending vibration
			1047.80	ν_C-N	Stretching vibration of C-N bond

7) Optical Rotation (OR)

A polarimeter, AntonPaarMCP 500; the measurement conditions were 0.5mol/L (water) C, 25 ℃;

the results are shown in the following Table 32-1:

TABLE 32-1

Weight (mg)	Volume (mL)	C(g/100mL)	Optical rotation	SpecificRotation (specific rotation)
					24.95	5	0.499	+0.0381°	+7.635°

8) Differential Scanning Calorimetry (DSC)

Instrument model METTELER DSC 1; the heating rate is 10.0 ℃/min; the temperature range is 40-350 deg.C, and the graph is shown in figure 33A, wherein the left limit of the first peak is 134.46 deg.C, the peak value is 150.29 deg.C, the left limit of the second peak is 154.94 deg.C, the peak value is 175.96 deg.C, and the right limit is 245.06 deg.C.

9) Liquid Chromatography (HPLC)

The operating conditions for the HPLC were: the instrument model is as follows: SHIMADZU LC-20AB, gradient eluted using octadecylsilane bonded silica gel as a filler (Waters xsELEct CSH C18, 4.6 x 150mm, 3.5 μm), water (+0.0375 vol% trifluoroacetic acid) as mobile phase a and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the procedure of table 32-2 below; the detection wavelength was 235nm (PDA detector) and the column temperature was 40 ℃.

TABLE 32-2 gradient elution procedure

Time (minutes)	Mobile phase a (% by volume)	Mobile phase B (% by volume)	Flow rate (mL/min)
				0.01	95	5	1.0
5.00	82	18	1.0
				10.00	80	20	1.0
20.00	10	90	1.0
				20.01	95	5	1.0
28.00	95	5	1.0

The spectrum is shown in FIG. 33B.

It can be seen from FIG. 33B that at a retention time of 7.816min, a peak of compound L1 appeared.

2. Structure confirmation of Compound L2

1)HPLC-MS:

The instrument name and model are: agilent 1200LC & Agilent 6110MSD

The HPLC-MS conditions used were as follows:

HPLC conditions: gradient elution was performed using octadecylsilane bonded silica as a filler (Agilent ZORBAX SB-Aq,2.1 x 50mm, 5 μm), 0.0375 vol% trifluoroacetic acid as mobile phase A, and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the following procedure; the detection wavelengths were 210nm and 215nm (DAD detector) and the column temperature was 50 ℃. The results are shown in FIG. 34A-1 and FIG. 34A-2.

TABLE 33-1 gradient elution procedure

Time (minutes)	Mobile phase A (% by volume)	Mobile phase B (% by volume)	Flow rate (mL/min)
				0.00	10	90	1.2
1.50	10	90	1.2

MS detection results are shown in Table 33-2, and the related substances are platinum-containing organic substances, and the isotopes with high platinum element abundance have¹⁹⁴Pt,¹⁹⁵Pt,¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.0]⁺Peaks are the sample excimer peaks, appearing around 438.1, 439.1, 440.1, [ M' + CH₃CN+H]⁺Peaks are sample excimer peaks, corresponding to correlationsSubstance L2 (C)₉H₁₈N₂O₃Pt) has molecular weight of 397.33, mass spectrum information and related substance L2 (C)₉H₁₈N₂O₃Pt) have consistent molecular structures, as shown in FIG. 34B.

TABLE 33-2

m/e	Fragment ion peak	Remarks for note
			397.1,398.1,399.1,400.0	[M’+H]⁺	Peak of excimer ion of sample
438.1,439.1,440.1	[M’+CH₃CN+H]⁺	Excimer peak of sample plus acetonitrile

Note: m' is C₉H₁₈N₂O₃Molecular weight of Pt

2)¹H-NMR:

The instrument name: model BRUKERBV-400 nmr apparatus,

chemical shifts and assignments of hydrogen spectra (CD3OD — 400MHz) are as follows: related substance L2 (C)₉H₁₈N₂O₃Pt) contains 4 active hydrogens and 14 inactive hydrogens; the sample hydrogen spectra data are shown in Table 34 below, together with related species L2 (C)₉H₁₈N₂O₃Pt) are identical in molecular structure and are shown in figure 35 in detail.

Watch 34

Chemical shift (ppm)	Multiplicity of properties	Number of protons	Attribution of Hydrogen
				1.28-1.30	m	3	6
1.52-1.65	m	2	1,1’
				2.07-2.18	m	2	1,1’
2.61-2.98	m	6	3,3’,2,2’
				4.07-4.16	m	1	5

3)¹³C-NMR:

The instrument name: BRUKERBV-400 model NMR spectrometer

Chemical shifts and assignments of carbon spectra (CD3OD — 400MHz) are shown in Table 35 below:

watch 35

Chemical shift (ppm)	Type of carbon atom	Number of carbon atoms	Attribution of carbon
				20.21-20.48	Secondary carbon	2	1,1’
21.83	Primary carbon	1	6
				35.20	Secondary carbon	2	3,3’
44.40-45.05	Tertiary carbon	2	2,2’
				74.85	Secondary carbon	1	5
194.21	Quaternary carbon	1	7

¹³The C-NMR spectrum has 5 saturated secondary carbon peaks, 2 saturated tertiary carbon peaks, 1 saturated primary carbon peak and 1 unsaturated quaternary carbon peak, as shown in FIG. 36, which is consistent with the molecular structure of related substance L2.

4)QNMR：

It was determined using bruker avance NEO 400 using CD3OD as the solvent and an internal standard method, the internal standard being Coumarin (Coumarin, 99.74%), as shown in fig. 37, and the results are shown in table 36 below:

watch 36

The calculation formula of W% is as follows:

in the formula, W_ISTDMass of internal standard (mg);

W_Sammass of sample (mg);

MW_SAMis the molecular weight of the sample;

MW_ISTDis the molecular weight of the internal standard;

n_ISTDand n_SamIs the number of protons per functional group;

W_ISTD% is the mass percentage of the internal standard substance,

as can be seen from the above table, the nominal content is 93.2%.

5) Ultraviolet absorption spectrum (UV):

UV-2600Series ultraviolet visible spectrometer; measuring the temperature at room temperature; the measuring range is 190-400 nm; measuring solvent water; the spectrum is shown in figure 38, and the maximum ultraviolet absorption wavelength is at 190 nm.

6) Infrared spectrum (IR)

An infrared spectrometer: ALPHA-BRUKER; the measurement conditions were as follows: solid KBr pellets were formed. Measurement range: 4000cm^-1～400cm^-1The measurement results and analysis are shown in table 37 below: the spectrum is shown in figure 39.

Watch 37

Absorption peak wave number (cm)^-1)	Type of vibration	Group assignment
			3419.33,3216.73,3133.27	ν_NH	Amino N-H stretching vibration
2974.96,2936.82,2868.12	ν_CH	Alkyl C-H stretching vibration
			1637.12	ν_C＝O	C ═ O stretching vibration of carbonyl group
1350.04,1302.20	δ_CH	Alkyl C-H bending vibration
			1110.91	ν_C-O	Stretching vibration of C-O bond
1046.93	ν_C-N	Stretching vibration of C-N bond

7) Optical Rotation (OR)

the results are shown in the following Table 38-1:

TABLE 38-1

8) Differential Scanning Calorimetry (DSC)

Instrument model METTELER DSC 1; the heating rate is 10.0 ℃/min; the temperature range is 40-350 deg.C, and the graph is shown in figure 40A, wherein the left limit of the first peak is 96.08 deg.C, the peak is 124.04 deg.C, the left limit of the second peak is 154.33 deg.C, the peak is 179.13 deg.C, the left limit of the third peak is 22.80 deg.C, the peak is 232.55 deg.C, and the right limit is 280.40 deg.C.

9) Liquid Chromatography (HPLC)

The operating conditions for HPLC were instrument type: SHIMADZU LC-20AB, gradient elution performed using octadecylsilane bonded silica gel as a filler (Waters xselette CSH C18, 4.6 × 150mm, 3.5 μm), water (+0.0375 vol% trifluoroacetic acid) as mobile phase a and acetonitrile (+0.01875 vol% trifluoroacetic acid) as mobile phase B according to the procedure of table 38-2 below; the detection wavelength was 235nm (PDA detector) and the column temperature was 40 ℃.

TABLE 38-2 gradient elution procedure

The spectrum is shown in FIG. 40B.

From FIG. 40B, it can be seen that at a retention time of 8.795min, a peak of Compound L2 appears.

Example 7: method for detecting related substances in lobaplatin

Measuring according to high performance liquid chromatography (China pharmacopoeia 2015 edition four-part general rules 0512)

Chromatographic conditions and System suitability test

A chromatography instrument model; SHIMADZU LC-20 AD; the silica gel surface was coated with cellulose-tris (3-chloro-4-methylphenyl carbamate) as a filler (Daicel Chiralcel OZ-3, 4.6 x 150mm, 3.0 μm), n-hexane-ethanol (volume ratio 65:35) was used as a mobile phase, the flow rate was 1.0ml per minute, the detection wavelength was 210nm, the column temperature was 35 ℃, and isocratic elution was carried out for 40 min. The system applicability test solution is continuously fed for 6 times, the relative standard deviation of the peak area of the lobaplatin diastereoisomer I is not more than 4.0 percent, and the relative standard deviation of the peak area of the lobaplatin diastereoisomer II is not more than 4.0 percent.

Preparation of test solution

About 100mg of a lobaplatin sample (prepared by the method disclosed in example 2 of the specification of patent CN 102020679B and confirmed by structure identification in this example, lobaplatin trihydrate is added as lobaplatin to be detected in this example, and the lobaplatin content in each example is measured as lobaplatin anhydride) is precisely weighed, placed in a 10ml volumetric flask, added with methanol for ultrasonic dissolution and diluted to a scale, and shaken up to be used as a test solution.

Preparation of System suitability test solution/1% control solution

Precisely measuring 1ml of a test solution, placing the test solution in a 10ml volumetric flask, adding methanol to dilute the test solution to a scale, and shaking the test solution uniformly to serve as a reference stock solution; precisely measuring 1ml of the control stock solution, placing the control stock solution in a 10ml volumetric flask, adding methanol to dilute the solution to a scale, shaking the solution uniformly to serve as a system applicability solution and a 1% control solution.

Assay method

And (4) respectively taking 20 mu l of the system applicability solution and the sample solution, injecting the solutions into a liquid chromatograph, and recording the chromatogram for 40 minutes. If a related substance peak exists in the chromatogram of the test solution, the chromatogram peak in the typical chromatogram is identified by the related substance for positioning; the specific test results are shown in fig. 41. As can be seen, the peak for lobaplatin diastereomer II occurs at t ═ 8.550 min; the peak of lobaplatin diastereomer I appears when t is 10.062; when t is 11.570min, a peak of related substance L1 appears; when t is 18.436min, a peak of related substance H1 appears; when t is 22.043min, peaks of related substances H2, G1 and G2 appear; when t is 30.611min, a peak of related substance L2 appears; the relative retention times of substance G1, substance G2, substance H2 (relative to lobaplatin diastereomer II) were about 2.58, substance H1 (relative to lobaplatin diastereomer II) was about 2.16, substance L1 (relative to lobaplatin) was about 1.35, and substance L2 (relative to lobaplatin non-diastereomer II) was about 3.58; the peak area of each of the related substance H1, the related substance L1 and the related substance L2 should not exceed 0.5% (i.e., should not exceed half of the peak area of the main component in the control solution) as calculated by the main component self-control method without adding a correction factor, and the total of the related substance G1, the related substance G2 and the related substance H2 should not exceed 1.0% (i.e., should not exceed the peak area of the main component in the control solution).

Typical patterns marked by related substances G1, G2, H1, H2, L1 and L2 are shown in figure 41.

Example 8: detection method

Chromatographic conditions and System suitability test

Chromatography instrument model; SHIMADZU LC-20AD

Coating cellulose-tris (3-chloro-4-methylphenyl carbamate) on the surface of silica gel as a filler (Daicel Chiralcel OZ-3, 4.6mm, 150mm, 3.0um), using n-hexane-ethanol (volume ratio 60:40) as a mobile phase, with a flow rate of 0.8ml per minute, a detection wavelength of 210nm, a column temperature of 30 ℃, and isocratic elution for 50 min. The system applicability test solution is continuously fed for 6 times, the relative standard deviation of the peak area of the lobaplatin diastereoisomer I is not more than 4.0 percent, and the relative standard deviation of the peak area of the lobaplatin diastereoisomer II is not more than 4.0 percent.

Preparation of test solution

Taking a lobaplatin sample (prepared by the method disclosed in example 2 of the specification of patent CN 102020679B in the present example and confirmed by structure identification, namely, the lobaplatin trihydrate is added as lobaplatin to be detected in the present example, and the content of lobaplatin is calculated as lobaplatin anhydride) is about 100mg, precisely weighing, placing the sample in a 10ml volumetric flask, adding methanol for ultrasonic dissolution, diluting to a scale, and shaking up to obtain a test solution.

Preparation of System suitability test solution/1% control solution

Precisely measuring 1ml of a test solution, placing the test solution in a 10ml volumetric flask, adding methanol to dilute the test solution to a scale, and shaking the test solution uniformly to serve as a reference stock solution; precisely measuring 1ml of the control stock solution, placing the control stock solution in a 10ml volumetric flask, adding methanol to dilute the control stock solution to a scale, and shaking the test solution uniformly to serve as a system applicability solution and a 1% control solution.

Assay method

And (4) respectively taking 20 mu l of the system applicability solution and the sample solution, injecting the solutions into a liquid chromatograph, and recording the chromatogram for 40 minutes. If a related substance peak exists in the chromatogram of the test solution, the related substance is used for identifying the chromatographic peak in the typical chromatogram for positioning: the relative retention times (relative to lobaplatin diastereomer II) of related substance G1, related substance G2, and related substance H2 were about 2.48, the relative retention time (relative to lobaplatin diastereomer II) of related substance H1 was about 2.13, the relative retention time (relative to lobaplatin diastereomer II) of related substance L1 was about 1.38, and the relative retention time (relative to lobaplatin diastereomer II) of related substance L2 was about 3.56; the peak area of the related substance H1, the related substance L1 and the related substance L2 is not more than 0.5% respectively, and the total amount of the related substance G1, the related substance G2 and the related substance H2 is not more than 1.0% calculated by a main component self-control method without adding a correction factor.

Typical patterns identified by related substances G1, G2, H1, H2, L1, L2 are the same as in example 7.

Example 9: method for detecting related substances in lobaplatin

Chromatographic condition and system applicability test

Chromatography instrument model; SHIMADZU LC-20AD

The silica gel surface was coated with cellulose-tris (3-chloro-4-methylphenyl carbamate) as a filler (Daicel Chiralcel OZ-3, 4.6 x 150mm, 3.0um), n-hexane-ethanol (70:30) was used as a mobile phase at a flow rate of 1.5 ml/min, a detection wavelength of 210nm, a column temperature of 40 ℃ and isocratic elution for 40 min. The system applicability test solution is continuously fed for 6 times, the relative standard deviation of the peak area of the lobaplatin diastereoisomer I is not more than 4.0 percent, and the relative standard deviation of the peak area of the lobaplatin diastereoisomer II is not more than 4.0 percent.

Preparation of test solution

Taking a lobaplatin sample (in the embodiment, the lobaplatin sample which is prepared by the method disclosed in the embodiment 2 of the patent CN 102020679B specification and is obtained by structure identification confirmation, namely the lobaplatin trihydrate is added as lobaplatin to be detected in the embodiment, and the content of the lobaplatin is calculated by anhydrous substances) about 100mg, precisely weighing, placing the sample into a 10ml volumetric flask, adding methanol for ultrasonic dissolution, diluting to a scale, shaking uniformly, and taking the sample as a test solution.

Preparation of System suitability test solution/1% control solution

Assay method

And (4) respectively taking 20 mu l of the system applicability solution and the sample solution, injecting the solutions into a liquid chromatograph, and recording the chromatogram for 40 minutes. If a related substance peak exists in the chromatogram of the test solution, the related substance is used for identifying the chromatographic peak in the typical chromatogram for positioning: the relative retention times (relative to lobaplatin diastereomer II) of related substance G1, related substance G2, and related substance H2 were about 2.62, the relative retention time (relative to lobaplatin diastereomer II) of related substance H1 was about 2.19, the relative retention time (relative to lobaplatin diastereomer II) of related substance L1 was about 1.36, and the relative retention time (relative to lobaplatin diastereomer II) of related substance L2 was about 3.52; the peak area of the related substance H1, the related substance L1 and the related substance L2 is not more than 0.5% respectively, and the total amount of the related substance G1, the related substance G2 and the related substance H2 is not more than 1.0% calculated by a main component self-control method without adding a correction factor.

Example 10: in vitro antitumor Activity assay (Activity assay of related substances of the present invention)

The following describes the activity detection experiments carried out on the substance H mixtures, G1, G2, L1 and L2 prepared in the preceding examples.

Reagent and consumable

1. Cell line

Cell line names are shown in Table 39 below.

Watch 39

DMEM medium, chinese excell, cat No.: PM150210

MEM medium, chinese excell, cat # 3: PM150411

McCoy's 5A medium, china Procell, cat #: PM150710

Ham's F-12 medium, chinese Procell, cat #: PM150810

6.

Luminescent Cell visual Assay, Promega, usa, cat #: g7572

7.96 well cell culture plates, American Corning, cat #: 3610

Envision, PerkinElmer, USA

FBS, loncera, cat No.: S711-001S

10. Sodium pyruvate, china Procell, cat #: PB180422

Insulin, Shanghai source culture in China, cargo number: s454

12.β -mercaptoethanol, Gibco, cat No.: 21985

DMSO, Sigma, usa, cat #: d8418

Penicilin & Streptomyces (P/S), China Procell, Cat #: PB180120

15.0.25% pancreatin-EDTA, Chinese Procell, cat #: PB180228

RPMI-1640 medium, chinese Procell, cat No.: PM150110

IMDM medium, chinese excell, cat no: PM150510

Second, solution and buffer solution

1. Cell growth medium

After the preparation is finished, storing the mixture at 4 ℃ for later use; the medium is shown in Table 40 below.

Watch 40

Cell name	Culture medium
		HCCC-9810	RPMI-1640+10％FBS+1％P/S
NCI-H460	RPMI-1640+10％FBS+1％P/S
		MDA-MB-453	DMEM+10％FBS+1％P/S
DU 145	MEM+10％FBS+1％P/S
		SK-OV-3	McCoy's5A+10％FBS+1％P/S
K562	RPMI-1640+10％FBS+1％P/S
		Jurkat Clone E6-1	RPMI-1640+10％FBS+1％P/S
AGS	F-12+10％FBS+1％P/S
		HL-60	IMDM+20％FBS+1％P/S
SK-NEP-1	McCoy’s5A+15％FBS+1％P/S
		95-D	RPMI-1640+10％FBS+1％P/S
THP-1	RPMI-1640+10％FBS+0.05mMβ-mercaptoethanol+1％P/S
		OVCAR-3	RPMI-1640+20％FBS+0.01mg/mlInsulin+1％P/S

Note: the% values in the table above are in volume percent.

Heat-inactivated serum of Heat-inactivated FBS

The serum is put into a water bath for 30 minutes at the temperature of 56 ℃.

3. Compound treatment:

a1 mM solution of compound 3.13g dissolved in DMSO was stored at-20 ℃ until use. The positive control drug is Staurosporine (Staurosporine), STSP for short, which is a natural product isolated from the bacterium mycete staurosporius in 1977, and is commercially available from MedChemexpress (MCE), product name Staurosporine, and product number HY-15141.

II, an experimental method:

reviving cells

The cells to be revived are quickly taken out from the liquid nitrogen tank, melted in a water bath at 37 ℃ and quickly added into the preheated culture medium. Centrifuging for 5min at 1000 rpm, taking out the tube, discarding supernatant, adding fresh preheated culture medium into the tube, resuspending cells, adding cell suspension into culture dish, and centrifuging at 37 deg.C and 5 vol% CO₂And (5) culturing.

Cell passage

Cell passage: adherent cells, digesting the cells with 0.25% pancreatin (prepared by adding 025g pancreatin into 100mlpbs solution) when the cells grow to be 80-90% full in a culture dish, then resuspending the cells with a new culture medium, passaging the cells according to a proper proportion, and passaging for 1 time about 2-3 days. Suspending cells, collecting cell suspension, centrifuging at 800rpm for 5 minutes, removing supernatant, resuspending with fresh culture medium, and passaging according to a proper proportion for 1 time of about 2-3 d.

Cell inoculation and drug treatment

Preparation of compound working solution concentration

Compound single concentration assay

According to the assay requirements, on the day of the experiment, compounds were diluted to 1mM mother liquor using DMSO and further diluted to 50uM (5X final concentration) working solution with medium, final compound concentrations were as shown below, compounds were tested at 10 micromolar concentrations and incubation time of compounds was 72 hours.

Compound IC₅₀Testing

According to the detection requirements, on the day of the experiment, the related substance compound to be tested for activity is diluted to 1mM mother liquor by using DMSO as the highest concentration, and is subjected to gradient dilution by 2 times, 3 times or 5 times, and each concentration point is further diluted to working solution with 5X final concentration by using culture medium.

Cell inoculation and drug treatment

1.1 day before detection, cells were seeded at different densities in 96-well cell plates according to the cell growth rate, 80. mu.L of cell suspension was seeded per well, 37 ℃ C., 5% CO₂Incubate overnight. The specific plating densities of the cells are shown in table 41 below:

table 41

2. According to the experimental requirements, 20 mul of compound working solution is added into each hole, the temperature is 37 ℃, and the CO content is 5 vol%₂Incubate for 72 hours.

3. After the incubation was completed, detection was performed according to the operation requirements of CTG kit (purchased from Promega, Cat. No. G7572, name celltiter-glo) to obtain the corresponding chemiluminescence value, and the cell activity was calculated.

4. Computing

Cell viability ═ addition drug set RLU value/control set (solvent) RLU value × 100%

The experimental results are as follows:

raw absorbance data and% Cell Viability

The dose-response curves measured are shown in FIG. 42A-1 to FIG. 42K-2.

IC of the Compound₅₀The values are shown in table 42 below:

watch 42

As can be seen from the activity data, the inhibitory activity of the related substances reaches the nm level on human ovarian cancer cell strains Jurkat Clone E6-1 and SK-NEP-1, and has certain inhibitory activity on other tumor cells, and the general activity is below 5 mu M.

The inhibitory activity of a single concentration of 10. mu.M compound is shown in Table 43 below.

Watch 43

The data show that the related substance of the invention has better inhibitory activity to the cancer cells under the concentration of 10 mu M, particularly has the inhibitory rate of more than 90 percent to Jurkat Clone E6-1, HL-60, THP-1 and SK-NEP-1, has obvious tumor inhibitory activity, and can be further developed into anticancer drugs for clinical application.

Example 11: methodological validation of detection methods

In order to confirm the utility and accuracy of the detection method of the present invention, the specificity, linearity and range, detection limit and quantification limit, calibration factor, accuracy (recovery rate), precision, solution stability, durability, etc. of the detection method of lobaplatin of the present invention in the previous examples are described below:

1. specificity

A blank solution (i.e., a methanol solution) and a resolution solution RS (each compound concentration is 0.1mg/mL) were precisely measured at 20uL each, and the solution was injected into a liquid chromatograph, and as a result, the separation degree of the main lobaplatin peak and the peak of the related substance was more than 1.5, as shown in Table 1. For the correlated species peaks with poor separation between H2 and G1 and G2, the combination control was performed, and the specificity results are shown in Table 44 below, and the detailed maps are shown in FIGS. 38 and 39.

Watch 44

2. Sensitivity of the probe

Taking a lobaplatin reference substance solution, a related substance H solution, a related substance G1 solution, a related substance G2 solution, a related substance L1 solution and a related substance L2 solution, gradually diluting, and taking a signal-to-noise ratio (S/N)10 as a limit of quantification. The quantitative limit concentration of lobaplatin is 0.0203mg/mL, the quantitative limit concentration of related substance H1 is 0.0197mg/mL, the quantitative limit concentration of related substance H2 is 0.0197mg/mL, the quantitative limit concentration of related substance G1 is 0.0200mg/mL, the quantitative limit concentration of related substance G2 is 0.0200mg/mL, the quantitative limit concentration of related substance L1 is 0.008mg/mL, the quantitative limit concentration of related substance L2 is 0.008mg/mL, and the quantitative limit results are shown in the following table 45.

TABLE 45

3. Linearity

With the concentration of lobaplatin diastereomer II as abscissa (X) and peak area as ordinate (Y), the linear results are as follows: the concentration and the peak area of the lobaplatin diastereomer II in the range of 3.994 mg/mL-6.04 mg/mL have good linear relation, the linear relation is that Y is 8595033.2484X-2155759.5499, and the correlation coefficient R is²0.9934, indicating good linearity, see figures 40-47 for details.

The linear results are given below, with the concentration of the lobaplatin diastereomer i as abscissa (X) and the peak area as ordinate (Y): the concentration and peak area of the lobaplatin diastereomer I in the range of 3.994 mg/mL-5.965 mg/mL show good linear relation, the linear relation is that Y is 8027255.9361X-2805049.4891, and the correlation coefficient R is²0.9977, indicating good linearity.

Taking the concentration of the related substance H2 as the abscissa (X) and the peak area as the ordinate (Y), the linearity results are as follows: the concentration and peak area of the related substance H2 in the range of 0.0236 mg/mL-0.1180 mg/mL have good linear relation, the linear relation is that Y is 8003691.9295X +104.8500, and the correlation coefficient R is²0.9997, indicating a good linearity.

Taking the concentration of the related substance H1 as the abscissa (X) and the peak area as the ordinate (Y), the linearity results are as follows: the concentration and peak area of the related substance H1 in the range of 0.0256 mg/mL-0.1278 mg/mL have good linear relation, wherein the linear relation is that Y is 8282678.6134X +1774.0500, and the correlation coefficient R is²0.9999, indicating a good linearity.

With the concentration of the related substance G1 as abscissa (X) and the peak area as ordinate (Y), the linearity results are as follows: the concentration and peak area of the related substance G1 in the range of 0.0501 mg/mL-0.2505 mg/mL have good linear relation, the linear relation is Y7400098.8024X-13089.7750, and the correlation coefficient R is²It was 1.0000, indicating a good linearity.

With the concentration of the related substance G2 as abscissa (X) and the peak area as ordinate (Y), the linearity results are as follows: the concentration and peak area of the related substance G2 in the range of 0.0500 mg/mL-0.2500 mg/mLHas good linear relation, the linear relation is that Y is 6093148.0000X +8425.1000, and the correlation coefficient R is²0.9998, indicating a good linearity.

With the concentration of the related substance L1 as abscissa (X) and the peak area as ordinate (Y), the linearity results are as follows: the concentration and peak area of the related substance L1 in the range of 0.0202 mg/mL-0.1008 mg/mL have good linear relation, the linear relation is Y7627070.9325X +1812.0250, and the correlation coefficient R²It was 1.0000, indicating a good linearity.

With the concentration of the related substance L2 as abscissa (X) and the peak area as ordinate (Y), the linearity results are as follows: the concentration and peak area of the related substance L2 in the range of 0.0200 mg/mL-0.1002 mg/mL have good linear relation, wherein the linear relation is that Y is 7893984.5309X +5247.3750, and the correlation coefficient R is²0.9989, indicating a good linearity.

4. Precision degree

Respectively preparing system applicability solution by experimenters A and B, respectively precisely measuring the system applicability solution by 20uL, injecting into a liquid chromatograph, recording a spectrum, and continuously injecting for 6 times, wherein the result is shown in the following table 46, the RSD of the lobaplatin diastereomer ratio (I: II) is less than 0.5 percent, and the precision is good.

TABLE 46

Sample solutions are prepared by an experimenter A and an experimenter B respectively, then 20uL of each sample solution is precisely measured and injected into a liquid chromatograph, a spectrum is recorded, and continuous sample injection is carried out for 6 times, so that the results are shown in the following table 47, the content of each related substance is RSD (n-6) < 5%, RSD (n-12) < 5%, and the precision is good.

Watch 47

5. Accuracy of

Lobaplatin diastereomer and each related substance were prepared in parallel with 3 parts of recovery solutions at 50% limiting concentration, 3 parts of limiting concentration and 3 parts of recovery solutions at 150% limiting concentration, respectively, and the accuracy of each was examined. The results are shown in Table 48.

The recovery rate of the lobaplatin diastereomer I is 99-102%, and the recovery rate of the lobaplatin diastereomer II is 98-100%;

under the limit concentration of 50%, the recovery rate of related substance H1 is between 100% and 105%, the recovery rate of related substance H2 is between 100% and 105%, the recovery rate of related substance G1 is between 105% and 110%, the recovery rate of related substance G2 is between 95% and 105%, the recovery rate of related substance L1 is between 90% and 100%, and the recovery rate of related substance L2 is between 100% and 108%;

under the limit concentration of 100%, the recovery rate of related substance H1 is between 100% and 105%, the recovery rate of related substance H2 is between 95% and 110%, the recovery rate of related substance G1 is between 98% and 102%, the recovery rate of related substance G2 is between 98% and 102%, the recovery rate of related substance L1 is between 95% and 100%, and the recovery rate of related substance L2 is between 105% and 108%;

under the limit concentration of 150%, the recovery rate of related substance H1 is between 100% and 105%, the recovery rate of related substance H2 is between 100% and 105%, the recovery rate of related substance G1 is between 100% and 102%, the recovery rate of related substance G2 is between 100% and 105%, the recovery rate of related substance L1 is between 95% and 100%, and the recovery rate of related substance L2 is between 105% and 108%;

the accuracy of the method was thus demonstrated to be good.

Watch 48

Remarking: s_EEX (ratio of diastereomers in solution/ratio of diastereomers in first chromatogram in each time interval) × 100%;

S_STDx (lobaplatin peak area in solution/lobaplatin peak area in first chromatogram) x 100%;

S_im-Xx 100% (content of each relevant substance in solution/first chromatogram) per time interval;

6. durability

Taking a system applicability solution, properly adjusting parameters in a liquid chromatography system, and investigating the content detection condition of each related substance after the system condition changes, wherein the result is shown in the following table 49, and after the system condition slightly changes, the U% of each related substance is between 92% and 102%, which indicates that the method has good durability.

Watch 49

Remarking: u shape_iso X 100 percent (peak area ratio of lobaplatin diastereomer II to lobaplatin main peak in the solution after changing the conditions/peak area ratio of lobaplatin diastereomer II to lobaplatin main peak in the solution before changing the conditions);

U_im-Xx 100% (content of each related substance in the solution after changing the conditions/content of each related substance in the solution before changing the conditions).

Claims

1. A method for detecting related substances in lobaplatin is characterized in that the related substances are any one of or a mixture of any two or more of the following compounds H1, H2, G1, G2, L1 and L2:

compound H1:

compound H2:

、

compound G1:

compound G2:

、

compound L1:

compound L2:

；

wherein the lobaplatin comprises either one or both of lobaplatin diastereomer I and lobaplatin diastereomer II:

lobaplatin diastereomer I:

lobaplatin diastereomer II:

；

the detection method is an HPLC method, and a chromatographic column of the HPLC method comprises the following steps: the surface of the silica gel is coated with cellulose-tri (3-chloro-4-methylphenyl carbamate) as a filling agent, and a mobile phase of the HPLC method is n-hexane-ethanol with a volume ratio of 60-70: 30-40.

2. The detection method according to claim 1, wherein the substance of interest is any one of a mixture of compounds G1 and G2, compound H1, compound H2, compound L1, or compound L2.

3. The method according to claim 1, wherein the related substances include compound H1, compound H2, compound G1, compound G2, compound L1 and compound L2.

4. The assay of claim 1 wherein said compounds H1 and H2 pass through an intermediate

And/or

Preparing to obtain; the compounds G1 and G2, L1 and L2 are via intermediates

Or

And (4) preparing.

5. The assay of claim 2 wherein said compounds H1 and H2 pass through an intermediate

And/or

Preparing to obtain; the compounds G1 and G2, L1 and L2 are via intermediates

Or

Is prepared.

6. The assay of claim 3 wherein compounds H1 and H2 are via an intermediate

And/or

The preparation is carried out; the compounds G1 and G2, L1 and L2 are via intermediates

Or

And (4) preparing.

7. The detection method according to any one of claims 1 to 6, wherein the compounds H1 and H2 are separated to obtain H1 and H2 by obtaining a mixture H according to the following reaction equation (1):

（1）

（2）

（3）。

8. the detection method according to any one of claims 1 to 6, wherein the detection method is an HPLC-MS method.

9. The detection method according to claim 7, wherein the detection method is an HPLC-MS method.

10. The detection method according to any one of claims 1 to 6, wherein the mobile phase of the HPLC method is n-hexane-ethanol in a volume ratio of 63-67: 37-33.

11. The detection method according to claim 10, wherein the mobile phase of the HPLC method is n-hexane-ethanol in a volume ratio of 65: 35.

12. The detection method according to claim 7, wherein the mobile phase of the HPLC method is n-hexane-ethanol in a volume ratio of 63-67: 37-33.

13. The detection method according to claim 8, wherein the mobile phase of the HPLC method is n-hexane-ethanol in a volume ratio of 63-67: 37-33.

14. The detection method according to claim 9, wherein the mobile phase of the HPLC method is n-hexane-ethanol in a volume ratio of 63-67: 37-33.

15. The detection method according to any one of claims 1 to 6, wherein the flow rate of the HPLC method is 0.8 to 1.5ml per minute, the detection wavelength is 208 and 212nm, the column temperature is 30 to 40 ℃, and isocratic elution is 30 to 50 min.

16. The detection method according to claim 15, wherein the flow rate of the HPLC method is 1.0ml per minute, the detection wavelength is 210nm, the column temperature is 35 ℃, and the isocratic elution time is 40 min.

17. The detection method as claimed in claim 7, wherein the flow rate of the HPLC method is 0.8-1.5 ml/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is 30-50 min.

18. The detection method as claimed in claim 8, wherein the flow rate of the HPLC method is 0.8-1.5 ml/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is 30-50 min.

19. The detection method as claimed in claim 10, wherein the flow rate of the HPLC method is 0.8-1.5 ml/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is 30-50 min.

20. The detecting method as claimed in any one of claims 1 to 6, wherein the peak area of each of the substance of interest H1, the substance of interest L1 and the substance of interest L2 is not more than 0.5 times the peak area of the main component in the control solution and the total of the substance of interest G1, the substance of interest G2 and the substance of interest H2 is not more than the peak area of the main component in the control solution, as calculated by the main component self-control method without addition of a correction factor.

21. The detecting method according to claim 7, wherein the peak area of each of the related substance H1, the related substance L1 and the related substance L2 is not more than 0.5 times as large as the peak area of the main component in the control solution, and the total of the related substance G1, the related substance G2 and the related substance H2 is not more than the peak area of the main component in the control solution, as calculated by the main component self-control method without adding a correction factor.

22. The detecting method according to claim 8, wherein the peak area of each of the related substance H1, the related substance L1 and the related substance L2 is not more than 0.5 times as large as the peak area of the main component in the control solution, and the total of the related substance G1, the related substance G2 and the related substance H2 is not more than the peak area of the main component in the control solution, as calculated by the main component self-control method without adding a correction factor.

23. The detecting method according to claim 10, wherein the peak area of each of the related substance H1, the related substance L1 and the related substance L2 is not more than 0.5 times as large as the peak area of the main component in the control solution, and the total of the related substance G1, the related substance G2 and the related substance H2 is not more than the peak area of the main component in the control solution, as calculated by a main component self-control method without adding a correction factor.

24. The detecting method according to claim 15, wherein the peak area of each of the related substance H1, the related substance L1 and the related substance L2 is not more than 0.5 times as large as the peak area of the main component in the control solution, and the total of the related substance G1, the related substance G2 and the related substance H2 is not more than the peak area of the main component in the control solution, as calculated by a main component self-control method without adding a correction factor.

25. The detection method according to any one of claims 1 to 6, wherein, if a peak of a substance of interest is present in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the substance of interest: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, and the relative retention time of related substance H1 is 2.00-2.30; the relative retention time of compound L1 was 1.2-1.5; compound L2 had a relative retention time of 3.4-3.7, said lobaplatin including at least lobaplatin diastereomer II, said relative retention time being the retention time relative to lobaplatin diastereomer II.

26. The detection method according to claim 7, wherein if a peak of a relevant substance exists in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, and the relative retention time of related substance H1 is 2.00-2.30; the relative retention time of compound L1 was 1.2-1.5; compound L2 had a relative retention time of 3.4 to 3.7, the lobaplatin included at least lobaplatin diastereomer II, the relative retention time being the retention time relative to lobaplatin diastereomer II.

27. The detection method according to claim 8, wherein if a peak of a relevant substance exists in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, and the relative retention time of related substance H1 is 2.00-2.30; the relative retention time of compound L1 was 1.2-1.5; compound L2 had a relative retention time of 3.4 to 3.7, the lobaplatin included at least lobaplatin diastereomer II, the relative retention time being the retention time relative to lobaplatin diastereomer II.

28. The detection method according to claim 10, wherein if a peak of a relevant substance exists in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, and the relative retention time of related substance H1 is 2.00-2.30; the relative retention time of compound L1 was 1.2-1.5; compound L2 had a relative retention time of 3.4-3.7, said lobaplatin including at least lobaplatin diastereomer II, said relative retention time being the retention time relative to lobaplatin diastereomer II.

29. The detection method according to claim 15, wherein if a peak of a relevant substance exists in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, and the relative retention time of related substance H1 is 2.00-2.30; the relative retention time of compound L1 was 1.2-1.5; compound L2 had a relative retention time of 3.4-3.7, said lobaplatin including at least lobaplatin diastereomer II, said relative retention time being the retention time relative to lobaplatin diastereomer II.

30. The detection method according to claim 20, wherein if a peak of a relevant substance exists in the test solution, the peak is located by identifying a chromatographic peak in a typical chromatogram with the relevant substance: the relative retention time of related substances G1, G2 and H2 is 2.40-2.70, and the relative retention time of related substance H1 is 2.00-2.30; the relative retention time of compound L1 was 1.2-1.5; compound L2 had a relative retention time of 3.4-3.7, said lobaplatin including at least lobaplatin diastereomer II, said relative retention time being the retention time relative to lobaplatin diastereomer II.