CN111718379A

CN111718379A - Platinum substance with structures of L1 and L2 and preparation method and application thereof

Info

Publication number: CN111718379A
Application number: CN201910209775.2A
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: 2020-09-29

Abstract

The invention relates to a platinum substance with structures of L1 and L2, a preparation method and application thereof. Wherein the platinum substance is a platinum compound L1 or L2 or a mixture of the two, which has the following structural formula:

Description

Platinum substance with structures of L1 and L2 and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a platinum substance with structures of formulas L1 and L2, a preparation method thereof and application thereof in medicines, and belongs to the technical field of medicinal chemistry.

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 (a):

lobaplatin has an 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 renal toxicity and less toxicity to bone marrow, and currently marketed lobaplatin for injection is mainly used for treating breast cancer, small cell lung cancer and chronic myelogenous leukemia.

Disclosure of Invention

In order to ensure the safety, effectiveness and controllable quality of the lobaplatin, research on related substances and detection methods of the lobaplatin 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 technical problem to be solved by the invention is as follows: preparation of platinum-based compounds with structures L1 and L2, and establishment of a detection method for the compound as a related substance in lobaplatin and antitumor application of the compound.

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

In one aspect, the present invention provides a platinum species which is a compound of L1 or L2 or a mixture of L1 and L2 having the structural formula:

wherein L1 and L2 can be interchanged.

On the other hand, the invention provides a preparation method of a platinum substance, which is prepared from a compound 6 with the following structural formula:

preferably, in the reaction for preparing the platinum-based substance by the compound 6, a solution of the compound 6 is reacted with the lactic acid compound 7 to obtain a mixture of L1 and L2, and further preferably, the molar ratio of the compound 6 to the lactic acid compound 7 is 1: 1-2; it is further preferable that the pH of the system is adjusted to 6.4 to 6.8 with the lactic acid compound 7, and it is further preferable that the reaction temperature is 25 to 35 ℃, preferably 30 ℃; it is further preferred that the reaction time is from 72 to 96 hours, preferably 87 hours;

wherein, the structural formula of the lactic acid compound 7 is as follows:

preferably, in the above preparation method, the compound 6 is prepared from a compound 5 of the following structural formula:

preferably, in the reaction for producing compound 6 from compound 5, a resin is added for reaction, followed by filtration to obtain a filtrate, the resin is washed to obtain a washing liquid, and the filtrate and the washing liquid are combined to obtain a solution containing compound 6, preferably, the resin is a resin treated with an aqueous sodium hydroxide solution, more preferably, the concentration of the aqueous sodium hydroxide solution is 1 to 2mol/L, still more preferably 1.5 mol/L; it is more preferable to add the resin and react for 0.5 to 2 hours, and it is further preferable to react for 1 hour.

Preferably, in the above preparation method, the compound 5 is prepared from a compound 4.1 of the following structural formula, and more preferably is prepared from a compound 4 of the following structural formula:

wherein X represents halogen element selected from F, Cl, Br or I;

preferably, in the reaction for preparing the compound 5 from the compound 4.1, the compound 4.1 is dispersed into a ketone-water mixed solvent, and then a silver nitrate aqueous solution is added to react away from light to prepare a solution containing the compound 5; it is further preferred that the molar ratio of compound 4.1 to silver nitrate is 1:1-2, further preferred 1: 1.4-2;

preferably, the ketone is selected from acetone, and more preferably, the volume ratio of the acetone-water mixed solvent is acetone: water is 0-9: 1; it is further preferred that the reaction temperature is from 15 to 25 ℃, preferably 20 ℃; the reaction time is 14 to 18 hours, preferably 16 hours.

Preferably, in the above preparation method, the compound 4.1 is prepared from a compound 3 of the following structural formula:

preferably, in the reaction for preparing compound 4.1 from compound 3, a haloplatinate or a haloplatinate, an alkali metal halide, and a hydroxide are reacted to prepare compound 4.1, and more preferably, the molar ratio of compound 3 to the haloplatinate is 1: 0.5-2;

it is further preferred that the haloplatinate is selected from potassium or sodium haloplatinate, it is further preferred that the haloplatinate is selected from potassium or sodium chloroplatinate, it is further preferred that potassium chloroplatinate;

it is further preferred that the alkali metal halide is selected from potassium halide or sodium halide, it is further preferred that the alkali metal halide is selected from potassium iodide or sodium iodide, it is further preferred that potassium iodide;

it is further preferred that the hydroxide is selected from potassium hydroxide or sodium hydroxide, further preferred potassium hydroxide.

Preferably, in the above preparation method, the compound 3 is prepared from the compound 2 with the following structural formula:

preferably, in the reaction for preparing the compound 3 by the compound 2, the compound 2 is reacted with an alcoholic solution of oxalic acid to obtain the compound 3 as a white solid, and more preferably, the molar ratio of the compound 2 to the oxalic acid is 1: 0.5-2; preferably, the reaction system is heated to 65-75 ℃ for reaction, preferably 0.5-2 hours, preferably 70 ℃ for 1 hour; it is further preferred that the separated solid compound 3 is added to a tetrahydrofuran solvent, and the mixture is further mixed at 60 to 70 ℃, preferably 65 ℃, and the purified compound 3 is obtained by solid-liquid separation.

Preferably, in the above preparation method, the compound 2 is prepared from the compound 1 with the following structural formula:

preferably, in the reaction for preparing the compound 2 by the compound 1, borane dimethyl sulfide is added into a tetrahydrofuran solution of the compound 1 for reaction, and further preferably, the molar ratio of the compound 1 to the borane dimethyl sulfide is 1: 5-10;

preferably, the reaction is divided into three stages, wherein the reaction temperature in the first stage is-5-5 ℃, preferably 0 ℃, the reaction temperature in the second stage is 35-45 ℃, preferably 40 ℃, and the reaction temperature in the third stage is 60-70 ℃, preferably 65 ℃; it is further preferred that the reaction time of the first stage is 50 to 70 minutes, preferably 60 minutes, the reaction time of the second stage is 50 to 70 minutes, preferably 60 minutes, and the reaction time of the third stage is 50 to 70 minutes, preferably 60 minutes; it is further preferred to add an alcohol, preferably n-butanol, to the solid obtained in the reaction and to raise the temperature, preferably to 90-110 c, preferably to 100 c.

Preferably, in the above preparation method, the starting material is compound 1:

preferably, the preparation method comprises the following steps:

wherein, in the reaction for preparing the compound 2 by the compound 1, borane dimethylsulfide is added into a tetrahydrofuran solution of the compound 1 for reaction; preferably, the reaction is divided into three stages, the first stage reaction temperature is-5-5 ℃, preferably 0 ℃; the reaction temperature of the second stage is 35-45 ℃, and preferably 40 ℃; the reaction temperature of the third stage is 60-70 ℃, preferably 65 ℃; it is further preferred that the reaction time of the first stage is from 50 to 70 minutes, preferably 60 minutes; the reaction time of the second stage is 50 to 70 minutes, preferably 60 minutes; the reaction time of the third stage is 50 to 70 minutes, preferably 60 minutes; further preferably, n-butanol is added into the solid obtained by the reaction, and the temperature is raised, preferably to 90-110 ℃, preferably to 100 ℃;

and/or, in the reaction for preparing the compound 3 by the compound 2, reacting the compound 2 with an isopropanol solution of oxalic acid to obtain a white solid compound 3; preferably, the reaction system is heated to 65-75 ℃ for reaction, preferably 0.5-2 hours, preferably 70 ℃ for 1 hour; further preferably, the separated solid compound 3 is added into tetrahydrofuran solvent, and is continuously mixed at 60-70 ℃, preferably 65 ℃, and the purified compound 3 is obtained by solid-liquid separation;

and/or, in the reaction for preparing the compound 4 by the compound 3, the compound 3 is reacted with potassium chloroplatinate, potassium iodide and potassium hydroxide to prepare the compound 4, preferably, the compound 3 is mixed with an aqueous solution of potassium hydroxide to obtain a solution F, the compound 3 is mixed with an aqueous solution of potassium chloroplatinate and potassium iodide to obtain a solution E, and the solution F and the solution E are mixed and reacted to obtain the compound 4, preferably, the reaction temperature is 25-35 ℃, preferably 30 ℃, further preferably, the reaction time is 3-5 hours, preferably, the reaction time is 4 hours;

and/or in the reaction for preparing the compound 5 by the compound 4, dispersing the compound 4 into an acetone-water mixed solvent, adding a silver nitrate water solution, and reacting in a dark place to prepare a solution containing the compound 5 for the next reaction; preferably, the volume ratio of the acetone-water mixed solvent is acetone: water 0-9:1 reaction temperature 15-25 deg.c, preferably 20 deg.c; the reaction time is 14 to 18 hours, preferably 16 hours;

and/or, in the reaction for preparing the compound 6 by the compound 5, adding resin for reaction, then filtering to obtain filtrate, washing the resin to obtain washing liquid, combining the filtrate and the washing liquid to obtain a solution containing the compound 6 for further reaction, preferably, wherein the resin is a resin treated by a sodium hydroxide aqueous solution, preferably, the concentration of the sodium hydroxide aqueous solution is 1-2mol/L, and more preferably, 1.5 mol/L; more preferably, the resin is added for reaction for 0.5 to 2 hours, and more preferably for reaction for 1 hour;

and/or, in the reaction for preparing the platinum-based substance by the compound 6, the solution of the compound 6 is reacted with the lactic acid compound 7 to obtain a mixture of L1 and L2, preferably, the pH of the system is adjusted to 6.4 to 6.8 by the lactic acid compound 7, further preferably, the reaction temperature is 25 to 35 ℃, preferably 30 ℃, and the reaction time is 72 to 96 hours, preferably 87 hours.

It is preferable thatThe preparation method comprises the following steps: separating the prepared mixture of the compounds L1 and L2 by liquid chromatography to obtain a compound L1 and a compound L2; preferably, the liquid chromatography is performed as NH₄HCO₃The aqueous solution of (A) is a mobile phase A, and acetonitrile is used as a mobile phase B; the concentration of the mobile phase A is preferably 8-12m mol/L, preferably 10m mol/L; it is further preferred that the volume of mobile phase B is increased from 0 to 20% and the volume of mobile phase A is decreased from 100% to 80% in 0-20min by using gradient elution.

On the other hand, the invention provides a detection method of platinum substances, which is characterized in that the method is an HPLC method or an HPLC-MS method; preferably, the detection conditions of the HPLC method are as follows: coating cellulose-tri (3-chloro-4-methylphenyl carbamate) on the surface of silica gel as a filling agent, and taking n-hexane-ethanol (the volume ratio is 60-70:30-40) as a mobile phase, wherein the flow rate is 0.8-1.5 mL/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is carried out for 30-50 min; preferably, n-hexane-ethanol (volume ratio is 63-67:37-33) is used as a mobile phase, and the column temperature is 33-37 ℃; more preferably, the mobile phase is n-hexane-ethanol (volume ratio of 65:35), the flow rate is 1.0 mL/min, the detection wavelength is 210nm, the column temperature is 35 ℃, and the isocratic elution time is 40 min.

The invention also provides the application of the platinum substance as a relevant substance index in the quality standard of the lobaplatin bulk drug or preparation for control.

In another aspect, the present invention provides a method for detecting the quality of a lobaplatin bulk drug or preparation, which comprises the step of measuring a related substance affecting the quality of lobaplatin, wherein the related substance is the above compound, and the related substance affecting the quality of lobaplatin is detected by an HPLC method or an HPLC-MS method;

preferably, the HPLC method detection conditions are as follows: coating cellulose-tri (3-chloro-4-methylphenyl carbamate) on the surface of silica gel as a filling agent, and taking n-hexane-ethanol (the volume ratio is 60-70:30-40) as a mobile phase, wherein the flow rate is 0.8-1.5 mL/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is carried out for 30-50 min; more preferably, n-hexane-ethanol (volume ratio is 63-67:37-33) is used as a mobile phase, and the column temperature is 33-37 ℃; more preferably, the mobile phase is n-hexane-ethanol (volume ratio of 65:35), the flow rate is 1.0 mL/min, the detection wavelength is 210nm, the column temperature is 35 ℃, and the isocratic elution time is 40 min;

more preferably, if a related substance peak exists in the chromatogram of the test solution, the peak areas of the compound L1 and the compound L2 in the test solution are respectively not more than 0.5 times of the peak area of the main component in the control solution by taking a 1% diluent of the test solution as the control solution and calculating the peak areas according to the main component self-control method without adding a correction factor; wherein, the 1% refers to 1% of the test solution.

It is further preferred that, if a peak of a substance of interest, if present, is located in a chromatogram of a test solution by identifying a chromatographic peak in a typical chromatogram with the substance of interest, the relative retention time of compound L1 is 1.2 to 1.5 and the relative retention time of compound L2 is 3.4 to 3.7.

In another aspect, the invention provides a pharmaceutical composition containing a platinum-based substance, wherein the pharmaceutical composition is a pharmaceutical preparation, and preferably the pharmaceutical composition is a pharmaceutical preparation for injection.

Preferably, the pharmaceutical composition comprises an adjuvant, and preferably, the adjuvant is one or more selected from the group consisting of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, an antioxidant, and a matrix; it is further preferred that the auxiliary material is selected from fillers and/or antioxidants.

On the other hand, the invention provides the application of the platinum substance or the pharmaceutical composition in preparing antitumor drugs.

Preferably, the above use, wherein the tumor is a cell of lung cancer, leukemia, gastric cancer, ovarian cancer and/or renal cancer; preferably, the tumor is a leukemia cell.

Preferably, the application is the application of the compound L1 in preparing anti-THP-1 tumor drugs and the application of the compound L2 in preparing anti-Jurkat Clone E6-1 and/or THP-1 tumor drugs.

To enable formulation in the compositions of the present invention, pharmaceutically acceptable excipients may be added in the preparation of these formulations, such as: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, antioxidants, bases, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crospolyvinylpyrrolidone, low-substituted hydroxypropylcellulose, croscarmellose sodium, etc.; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the adhesive comprises: starch slurry, polyvinylpyrrolidone, hydroxypropylmethylcellulose, and the like; the sweetener comprises: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, and the like; the flavoring agent comprises: sweeteners and various essences; the preservative comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroacetidine acetate, eucalyptus oil, etc.; the antioxidant includes: sodium sulfite, sodium metabisulfite, dibutyl phenol, sodium bisulfite, sodium thiosulfate, tert-butyl p-hydroxyanisole, thiourea, vitamin c, propyl gallate, alpha-tocopherol, ascorbyl palmitate; the matrix comprises: PEG6000, PEG4000, insect wax, etc. Therefore, it is within the scope of the present invention to add any other substance that contributes to the formation of a stable drug effect to the compound of the present invention.

The invention has the following beneficial effects:

the platinum compounds L1 and L2 are synthesized and prepared, and have an anti-tumor effect; the invention also separates two diastereoisomers of L1 and L2, and confirms that L1 and L2 are related substances of lobaplatin, thereby laying a better foundation for establishing a complete lobaplatin quality detection system. The invention provides a method for detecting platinum compounds with structural formulas L1 and L2 as related substances in lobaplatin quality standard, which has the advantages of high sensitivity, strong specificity, good repeatability and high accuracy.

Drawings

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

FIGS. 1-2: the HPLC-MS combined structure of the compound L1 confirms the HPLC spectrum (the wavelength is 210nm) in the detection;

FIGS. 1 to 3: the HPLC-MS combined structure of the compound L1 confirms the MS map in the detection;

FIG. 2: process for preparation of compound L1 of the invention¹An H-NMR spectrum;

FIG. 3: process for preparation of compound L1 of the invention¹³A C-NMR spectrum;

FIG. 4: a QNMR spectrum of the compound L1 of the present invention;

FIG. 5: the UV spectrum of Compound L1 of the present invention;

FIG. 6: IR spectrum of compound L1 of the present invention;

FIG. 7: a DSC profile of compound L1 of the present invention;

FIG. 8: an HPLC profile of compound L1 of the present invention;

FIG. 9-1: the HPLC-MS combined structure of the compound L2 confirms the HPLC spectrum (the wavelength is 215nm) in the detection;

FIG. 9-2: the HPLC-MS combined structure of the compound L2 confirms the HPLC spectrum (the wavelength is 210nm) in the detection;

FIGS. 9-3: the HPLC-MS combined structure of the compound L2 confirms the MS map in the detection;

FIG. 10: process for preparation of compound L2 of the invention¹An H-NMR spectrum;

FIG. 11: process for preparation of compound L2 of the invention¹³A C-NMR spectrum;

FIG. 12: a QNMR spectrum of the compound L2 of the present invention;

FIG. 13: the UV spectrum of Compound L2 of the present invention;

FIG. 14: IR spectrum of compound L2 of the present invention;

FIG. 15: a DSC profile of compound L2 of the present invention;

FIG. 16: an HPLC profile of compound L2 of the present invention;

FIG. 17: the HPLC typical patterns of the compounds L1 and L2 of the invention as related substances of lobaplatin;

FIG. 18-1: linear plot of peak area and concentration for lobaplatin diastereomer ii;

FIG. 18-2: linear plot of peak area and concentration of lobaplatin diastereomer i;

FIG. 19: linear plots of peak area and concentration for compound L1 of the invention;

FIG. 20: linear plots of peak area and concentration for compound L2 of the invention;

FIGS. 21-1 and 21-2: the inhibitory activity of the compound L1 and a positive control drug (STSP) on ovarian cancer cells SK-OV-3 is shown respectively;

FIGS. 22-1 and 22-2: respectively shows the inhibitory activity of the compound L1 and a positive control drug (STSP) on leukemia cells K-562;

FIGS. 23-1 and 23-2: the inhibitory activity of the compound L1 and a positive control drug (STSP) on leukemia cells Jurkat Clone E6-1 are respectively shown as a graph;

FIGS. 24-1 and 24-2: the inhibition activity of the compound L1 of the present invention and a positive control drug (STSP) against gastric cancer cell AGS are shown in the figure;

FIGS. 25-1 and 25-2: the inhibitory activity of the compound L1 and a positive control drug (STSP) on leukemia cell HL-60 are respectively shown in a graph;

FIGS. 26-1 and 26-2: respectively shows the inhibition activity of the compound L1 and a positive control drug (STSP) on renal carcinoma cells SK-NEP-1;

FIGS. 27-1 and 27-2: the inhibitory activity of the compound L1 and a positive control drug (STSP) on the THP-1 leukemia cells is respectively shown;

FIGS. 28-1 and 28-2: the inhibitory activity of the compound L2 and a positive control drug (STSP) on lung cancer cell NCI-H460 are respectively shown;

FIGS. 29-1 and 29-2: respectively shows the inhibitory activity of the compound L2 and a positive control drug (STSP) on leukemia cells K-562;

FIGS. 30-1 and 30-2: the inhibitory activity of the compound L2 and a positive control drug (STSP) on leukemia cells Jurkat Clone E6-1 are respectively shown as a graph;

FIGS. 31-1 and 31-2: the inhibition activity of the compound L2 of the present invention and a positive control drug (STSP) against gastric cancer cell AGS are shown in the figure;

FIGS. 32-1 and 32-2: the inhibitory activity of the compound L2 and a positive control drug (STSP) on leukemia cell HL-60 are respectively shown in a graph;

FIGS. 33-1 and 33-2: respectively shows the inhibition activity of the compound L2 and a positive control drug (STSP) on renal carcinoma cells SK-NEP-1;

FIGS. 34-1 and 34-2: the inhibitory activity of the compound L2 and a positive control drug (STSP) on lung cancer cells 95-D is shown in the figure respectively;

FIGS. 35-1 and 35-2: the inhibitory activity of the compound L2 and a positive control drug (STSP) on the THP-1 leukemia cells is respectively shown;

FIGS. 36-1 and 36-2: the inhibitory activity of the compound L2 and a positive control drug (STSP) on ovarian cancer cells OVCAR-3 is shown in the figure.

Detailed Description

The invention provides a preparation method and a detection method of a platinum substance (a compound L1 or L2 or a mixture thereof) and an anti-tumor application thereof. The following will describe, as specific examples, the preparation of platinum-based compounds, the confirmation of the structure of compounds, the measurement of antitumor activity of compounds, and the like.

In a preferred embodiment of the present invention, the method for preparing the platinum species L1 or L2 is as follows:

wherein, in the reaction for preparing the compound 2 by the compound 1, borane dimethylsulfide is added into a tetrahydrofuran solution of the compound 1 for reaction; preferably, the reaction is divided into three stages, wherein the reaction temperature in the first stage is-5-5 ℃, preferably 0 ℃, the reaction temperature in the second stage is 35-45 ℃, preferably 40 ℃, and the reaction temperature in the third stage is 60-70 ℃, preferably 65 ℃; it is further preferred that the reaction time of the first stage is 50 to 70 minutes, preferably 60 minutes, the reaction time of the second stage is 50 to 70 minutes, preferably 60 minutes, and the reaction time of the third stage is 50 to 70 minutes, preferably 60 minutes; further preferably, n-butanol is added into the solid obtained by the reaction, and the temperature is raised, preferably to 90-110 ℃, preferably to 100 ℃;

in the reaction for preparing the compound 3 by the compound 2, the compound 2 is reacted with an isopropanol solution of oxalic acid to obtain a white solid compound 3; preferably, the reaction system is heated to 65-75 ℃ for reaction, preferably 0.5-2 hours, preferably 70 ℃ for 1 hour; then separating out the solid, adding the solid into a tetrahydrofuran solvent, continuously mixing at 60-70 ℃, preferably at 65 ℃, and carrying out solid-liquid separation to obtain a purified compound 3;

in the reaction for producing compound 4 from compound 3, an alkali metal halide or an alkali metal halide and a halogenoplatinate salt, and a hydroxide are reacted to produce compound 4; preferably, the alkali metal halide is selected from potassium halide or sodium halide, further preferably, the alkali metal halide is selected from potassium chloride or sodium chloride or potassium iodide or sodium iodide, further preferably potassium iodide; preferably, the halogenoplatinate salt is selected from potassium or sodium halogenoplatinate, and further preferably, the halogenoplatinate salt is selected from potassium or sodium chloroplatinate; preferably, the hydroxide is selected from potassium hydroxide or sodium hydroxide, further preferably potassium hydroxide; it is further preferred that compound 3, potassium chloroplatinite, potassium iodide and potassium hydroxide are reacted to produce compound 4, it is preferred that compound 3 and an aqueous solution of potassium hydroxide are mixed to obtain solution F, an aqueous solution of potassium chloroplatinite and potassium iodide are mixed to obtain solution E, and solution F and solution E are mixed and reacted to obtain compound 4, it is preferred that the reaction temperature is 25 to 35 ℃, it is preferred that 30 ℃, it is further preferred that the reaction time is 3 to 5 hours, it is preferred that the reaction time is 4 hours;

in the reaction for producing compound 4 by using compound 3, if potassium iodide is not added, the product of the reaction of compound 3 with potassium chloroplatinite is compound 4.2:

if potassium iodide is added, the product of the reaction of compound 3 with potassium chloroplatinite and potassium iodide is compound 4:

compared with the compound 4.2, the compound 4 causes fewer side reactions and fewer impurities when used for preparing subsequent compounds;

in the reaction for preparing the compound 5 by the compound 4, dispersing the compound 4 into an acetone-water mixed solvent, adding a silver nitrate water solution, and reacting in a dark place to prepare a solution containing the compound 5 for the next reaction; preferably, the volume ratio of the acetone-water mixed solvent is acetone: water 0-9:1 (pure water at a ratio of 0: 1), at a reaction temperature of 15-25 deg.c, preferably 20 deg.c; the reaction time is 14 to 18 hours, preferably 16 hours;

in the reaction for preparing the compound 6 by the compound 5, resin is added for reaction, then the filtrate is obtained by filtration, the resin is washed to obtain washing liquid, the filtrate and the washing liquid are combined to obtain a solution containing the compound 6 for the next reaction, preferably, the resin is treated by sodium hydroxide aqueous solution, the concentration of the sodium hydroxide aqueous solution is preferably 1-2mol/L, and the concentration of the sodium hydroxide aqueous solution is more preferably 1.5 mol/L; more preferably, the resin is added for reaction for 0.5 to 2 hours, and more preferably for reaction for 1 hour;

in the reaction for producing the platinum-based substance by compound 6, a solution of compound 6 is reacted with lactic acid compound 7 to obtain a mixture of L1 and L2, preferably, lactic acid compound 7 is used to adjust the system pH to 6.4 to 6.8, more preferably, the reaction temperature is 25 to 35 ℃, preferably 30 ℃, and the reaction time is 72 to 96 hours, preferably 87 hours.

In a preferred embodiment of the present invention, the present invention provides a method for detecting the quality of a lobaplatin bulk drug or preparation, which comprises the step of determining a related substance affecting the quality of lobaplatin, wherein the related substance is a compound L1 or L2, the determination method adopts an HPLC method, and the detection conditions are as follows: coating cellulose-tri (3-chloro-4-methylphenyl carbamate) on the surface of silica gel as a filling agent, and taking n-hexane-ethanol (the volume ratio is 60-70:30-40) as a mobile phase, wherein the flow rate is 0.8-1.5 mL/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is carried out for 30-50 min; preferably, the mobile phase is n-hexane-ethanol (volume ratio of 65:35), the flow rate is 1.0 mL/min, the detection wavelength is 210nm, the column temperature is 35 ℃, and the isocratic elution time is 40 min;

more preferably, if a related substance peak exists in the chromatogram of the test solution, the peak areas of the compound L1 and the compound L2 in the test solution are respectively not more than 0.5 times of the peak area of the main component in the control solution by taking a 1% diluent of the test solution as the control solution and calculating the peak areas according to the main component self-control method without adding a correction factor;

it is further preferred that, if there is a peak of the substance of interest in the chromatogram of the test solution, the peak of the chromatogram is located in a typical chromatogram for identification of the substance of interest, and that the relative retention time of compound L1 is 1.2 to 1.5 and that of compound L2 is 3.4 to 3.7.

Wherein the relative retention time refers to the retention time relative to lobaplatin, specifically to lobaplatin diastereomer II. Specifically, as the lobaplatin compound, 2 isomers, i.e., lobaplatin diastereomer I and lobaplatin diastereomer II, are known, and their structural formulae are as follows:

in the present invention, any substance affecting the purity of a drug is collectively referred to as "a substance involved in affecting the quality of lobaplatin" or "a substance involved in affecting the quality", and is simply referred to as "a substance involved", for example, a peak of a substance involved in affecting the quality of lobaplatin, which appears in an XRD diffraction peak for detecting the quality of lobaplatin, is simply referred to as "a substance involved 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.

The sources of reagents used in the following examples are given in Table 1 below, and any reagents or instruments not described in this application are routinely determined by those skilled in the art.

TABLE 1 reagents and apparatus used in the examples

Preparation of the Compound of example 1

Examples 1 to 1

The preparation method comprises the following steps:

1) preparation of Compound 2

Compound 1(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 volume ratio: 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 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 (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 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 1h. Filtration and drying of the filter cake gave compound 3(23.0g) as a 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,70mL 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 washed with purified water (50mL 5) until no halide ions remained. 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. Filtration, washing of the filter cake with purified water (30mL x 5), and combining the aqueous phases gave an aqueous solution of compound 5(300mL) which was used directly in the next step.

5) Preparation of Compound 6

The resin (100g, Mitsubishi chemical, model number DIAION SA10AX) 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 (40mL 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. Adjusting the pH value of the system to 6.6 by using compound 7 lactic acid, heating to 30 ℃, generating a small amount of black slag, and reacting for 87 h. Filtering, and freeze-drying the filtrate. The lyophilized product is subjected to preparative high performance liquid chromatography prep: waters 80Q predictive SFC system; column Phenomenex Synergi Max-RP (250 x 50mm x 10 μm); mobile phase A water (10m mol/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.

In the structure confirmation examples and the activity test examples which follow, compounds L1 and L2 both corresponded to compound L1 and compound L2 prepared in this example, that is, compound L1 referred to herein was a compound obtained first (i.e., the retention time was short) when the compound was prepared under the above-described liquid chromatography conditions, and compound L2 referred to herein was a compound obtained later (i.e., the retention time was long) when the compound was prepared under the above-described liquid chromatography conditions.

Examples 1 to 2

The preparation method comprises the following steps:

1) preparation of Compound 2

Compound 1(24.0g,226.1mmol) was dissolved in dry tetrahydrofuran (480mL) and cooled to-5 ℃. 10M BH3.Me2S (157mL,1.57mol) was added dropwise at-5 ℃ with stirring for one hour at incubation. The system was warmed to 45 ℃ and stirred for 1 hour. The temperature was raised to 70 ℃ and stirred for 50 minutes. 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 2(42.8 g) was obtained and used directly in the next step.

2) Preparation of Compound 3

Compound 2(42.8g, crude) was dissolved in isopropanol (400mL) to give solution A. Oxalic acid (11.5g,127.7mmol) was dissolved in isopropanol (115mL) to give solution B. A large amount of white solid was precipitated by dropping B to solution A. The system was warmed to 65 ℃ and stirred for 1 hour. Filtration, addition of the filter cake to THF (110mL), warming to 65 deg.C, stirring for 1h, filtration, and drying of the filter cake afforded Compound 3(22.8g) as a white solid.

3) Preparation of Compound 4

Compound 3(22.8g,77.52mmol), potassium chloroplatinite (29.1g,70.2mmol), potassium iodide (69.6g,419.0mmol) and potassium hydroxide (19.7g,298.6mmol) were dissolved in 88mL,96mL,70mL and 194mL of purified water to give solutions A, B, C and D, respectively. And heating the solution B to 35 ℃. 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 35 ℃ for 3.5 hours. Filtration and the filter cake washed with purified water (50mL 5) until no halide ions remained. The filter cake was dried by rotary evaporator to give compound 4(23.6g,) as a yellow powder.

4) Preparation of Compound 5

Compound 4(23.6g) was dispersed in water (90mL) and acetone (10.5mL) and stirred for ten minutes. Silver nitrate (10.32g,60.75mmol) was dissolved in water (90mL) and added to the system and stirred at 15 ℃ for 18 h, protected from light. Filtration, washing of the filter cake with purified water (30mL x 5), and combining the aqueous phases gave 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 2 hours. Filtration and washing of the resin with purified water (40mL 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. Adjusting the pH value of the system to 6.4 by using compound 7 lactic acid, heating to 30 ℃, generating a small amount of black slag, and reacting for 96 hours. Filtering, and freeze-drying the filtrate. The residue was subjected to preparative high performance liquid chromatography prep.HPLC (model: Waters 80Q preparative SFC system; column: Phenomenex Synergi Max-RP (250X 50 mm. times.10 μm); mobile phase A: water (10 mmol/L NH)₄HCO₃) And B: acetonitrile](ii) a Elution was carried out with a gradient of 0-20min and the volume of mobile phase B was increased from 0 to 20%) twice to obtain compound L1(0.713g) and compound L2(0.839g) as white solids.

Examples 1 to 3

The preparation method comprises the following steps:

1) preparation of Compound 2

Compound 1(24.0g,226.1mmol) was dissolved in dry tetrahydrofuran(480mL), the temperature was reduced to 5 ℃. At 5 ℃, 10M BH is added dropwise₃.Me₂S (157mL,1.57mol), stirred for one hour with heat preservation. The system was warmed to 35 ℃ and stirred for 1 hour. The temperature was raised to 60 ℃ and stirred for 70 minutes. 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 2(41.4 g) was obtained and used directly in the next step.

2) Preparation of Compound 3

Compound 2(41.4g, crude) was dissolved in isopropanol (400mL) to give solution A. Oxalic acid (11.5g,127.7mmol) was dissolved in isopropanol (115mL) to give solution B. A large amount of white solid was precipitated by dropping B to solution A. The system was warmed to 75 ℃ and stirred for 1 hour. Filtration, addition of the filter cake to THF (110mL), warming to 65 deg.C, stirring for 1h, filtration, and drying of the filter cake afforded Compound 3(22.3g) as a white solid.

3) Preparation of Compound 4

Compound 3(22.3g,75.82mmol), potassium chloroplatinite (29.1g,70.2mmol), potassium iodide (69.6g,419.0mmol) and potassium hydroxide (19.7g,298.6mmol) were dissolved in 88mL,96mL,70mL and 194mL of purified water to give solutions A, B, C and D, respectively. And heating the solution B to 25 ℃. 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 25 ℃ for 5 hours. Filtration and the filter cake washed with purified water (50mL 5) until no halide ions remained. The filter cake was dried by rotary evaporator to give compound 4(23.0g,) as a yellow powder.

4) Preparation of Compound 5

Compound 4(23.0g) was dispersed in water (90mL) and acetone (10.5mL) and stirred for ten minutes. Silver nitrate (10.32g,60.75mmol) was dissolved in water (90mL) and added to the system and stirred at 25 ℃ for 14 h, protected from light. Filtration, washing of the filter cake with purified water (30mL x 5), and combining the aqueous phases gave 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 0.5 hour. Filtration and washing of the resin with purified water (40mL 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. Adjusting the pH value of the system to 6.8 by using compound 7 lactic acid, heating to 30 ℃, generating a small amount of black slag, and reacting for 72 hours. Filtering, and freeze-drying the filtrate. The residue was subjected to preparative high performance liquid chromatography prep.HPLC (model: Waters 80Q preparative SFC system; column: Phenomenex Synergi Max-RP (250X 50 mm. times.10 μm); mobile phase A: water (10 mmol/L NH)₄HCO₃) And B: acetonitrile](ii) a Elution was carried out with a gradient of 0-20min and the volume of mobile phase B was increased from 0 to 20%) twice to obtain compound L1(0.725g) and compound L2(0.852g) as white solids.

Example 2: structure validation

The purified compounds L1 and L2 obtained in step 6) of example 1-1 were sampled sequentially for detection, including high performance liquid chromatography-mass spectrometry (HPLC-MS), hydrogen nuclear magnetic resonance spectroscopy (H: (R)¹H NMR); nuclear magnetic resonance carbon spectrum (¹³C NMR); ultraviolet absorption spectrum (UV); infrared spectroscopy (IR); differential Scanning Calorimetry (DSC); optical Rotation (OR), liquid chromatography (HPLC).

Since the single crystal cultivation failed and there was no single crystal diffraction check data, the absolute configuration of compounds L1 and L2 could not be confirmed, but two chiral enantiomers could be confirmed, and the other check confirmation data except for single crystal diffraction could confirm only the related substances as two compounds, but could not finally confirm the specific compounds. The following structural confirmations for compounds L1 and L2 are therefore both assigned. The concrete structure is as follows:

molecular formula C₉H₁₈N₂O₃Pt

Molecular weight 397.33

1. Structure confirmation of compound L1:

1)HPLC-MS:

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

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 procedure in Table 2; the detection wavelengths were 210nm and 215nm (DAD detector) and the column temperature was 50 ℃. The detection results are shown in FIGS. 1-1 and 1-2.

TABLE 2 gradient elution procedure

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-.

The detection results are shown in Table 3, and the spectra are shown in the attached figures 1-3, and it can be seen that the compound is a platinum-containing organic substance, and the isotope with high platinum element abundance has¹⁹⁴Pt、¹⁹⁵Pt、¹⁹⁶Pt, and thus in MS of the sample, [ M' + H ] appears at 397.1, 398.1, 399.1, 400.2]+ peak is the sample excimer peak, appearing around 438.1, 439.1, 440.2 [ M' + CH₃CN+H]The + peak is the sample excimer peak, corresponding to Compound L1 (C)₉H₁₈N₂O₃Pt) has a molecular weight of 397.33, mass spectral information and Compound L1 (C)₉H₁₈N₂O₃Pt) the molecular structure is consistent.

Table 3 mass spectrometry interpretation information

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

The spectrum is shown in figure 2, and the hydrogen spectrum¹H NMR chemical shifts and assignments for deuterated methanol (CD3OD) _400MHz) are as follows: compound L1 (C)₉H₁₈N₂O₃Pt) contains 4 active hydrogens and 14 inactive hydrogens.

TABLE 4¹Interpretation of H-NMR spectra

Chemical shift (ppm)	Multiplicity of properties	Number of protons	Attribution of hydrogen
				1.31-1.35	m	3	6
1.55-1.64	m	2	1,1’
				2.07-2.19	m	2	1,1’
2.70-3.10	m	6	3,3’,2,2’
				4.12-4.18	m	1	5

As can be seen, the sample hydrogen spectrum data is compared with L1 (C)₉H₁₈N₂O₃Pt) are identical in molecular structure.

3)¹³C-NMR:

The instrument name: BRUKERBV-400 model NMR spectrometer

The spectrum is shown in figure 3, carbon spectrum: (¹³C NMR chemical shifts and assignments of deuterated methanol (CD3OD) _400MHz) were as follows:

TABLE 5¹³Interpretation of C-NMR spectra

Chemical shift (ppm)	Type of carbon atom	Number of carbon atoms	Attribution of carbon
				20.34-20.37	Secondary carbon	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

As can be seen,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 compound L1.

4)QNMR：

The determination was performed by Bruker AVANCE NEO 400 using deuterated methanol (CD3OD) as a solvent and an internal standard method, and the internal standard substance was Coumarin (Coumarin, 99.74%), as shown in fig. 4, the determination results were as follows:

TABLE 6 QNMR test result chart

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,

as can be seen from Table 6 above, the nominal content was 89.8%.

5) Ultraviolet absorption spectrum (UV):

UV-2600 Series 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 map is shown in figure 6. The measurement results and analysis were as follows:

TABLE 7 IR spectrogram interpretation information

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 is shown in figure 7, wherein the first peak has a left limit of 134.46 deg.C, a peak value of 150.29 deg.C, a second peak has a left limit of 154.94 deg.C, a peak value of 175.96 deg.C, and a right limit of 245.06 deg.C.

8) Optical Rotation (OR)

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

the results are as follows:

TABLE 8 OR test results

9) Liquid Chromatography (HPLC)

The instrument model is as follows: SHIMADZU LC-20 AB; 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 according to the procedure of table 9; the detection wavelength was 235nm (PDA detector) and the column temperature was 40 ℃.

TABLE 9 gradient elution procedure

The spectrum is shown in figure 8.

It can be seen from FIG. 8 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 1200 LC & Agilent 6110 MSD

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 procedure in Table 10; the detection wavelengths were 210nm and 215nm (DAD detector) and the column temperature was 50 ℃. The detection results are shown in FIG. 9-1 and FIG. 9-2.

TABLE 10 gradient elution procedure

The detection results are shown in Table 11, and the spectra are shown in figures 9-3, and it can be seen that the compound is a platinum-containing organic substance, and the isotope with high platinum element abundance has¹⁹⁴Pt,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]⁺The peak is the sample excimer peak, corresponding to Compound L2 (C)₉H₁₈N₂O₃Pt) has a molecular weight of 397.33, mass spectral information and Compound L2 (C)₉H₁₈N₂O₃Pt) the molecular structure is consistent.

Table 11 mass spectrometry interpretation information

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: BRUKERBV-400 model NMR spectrometer

The spectrum is shown in figure 10, and the hydrogen spectrum¹H NMR CD3 OD-400 MHz) were assigned as follows: compound L2 (C)₉H₁₈N₂O₃Pt) contains 4 active hydrogens and 14 inactive hydrogens;

TABLE 12¹Interpretation of H-NMR spectra

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

As can be seen, the sample hydrogen spectrum data is related to compound L2 (C)₉H₁₈N₂O₃Pt) are identical in molecular structure.

3)¹³C-NMR:

The instrument name: BRUKERBV-400 model NMR spectrometer

The spectrum is shown in figure 11, carbon spectrum: (¹³C NMR CD3 OD-400 MHz) and the chemical shifts and assignments were as follows:

watch 13¹³Interpretation of C-NMR spectra

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

As can be seen,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 compound L2.

4)QNMR：

The determination was performed by Bruker AVANCE NEO 400 using deuterated methanol (CD3OD) as a solvent and an internal standard method, and the internal standard substance was Coumarin (Coumarin, 99.74%), as shown in fig. 12, the determination results were as follows:

TABLE 14 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);

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 Table 14 above, the nominal content is 93.2%.

5) Ultraviolet absorption spectrum (UV):

UV-2600 Series 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 13, 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 map is shown in figure 14. The measurement results and analysis were as follows:

TABLE 15 IR spectrogram interpretation information

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

The graph is shown in figure 15, 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.

8) Optical Rotation (OR)

the results are as follows:

TABLE 16 OR test results

9) Liquid Chromatography (HPLC)

The instrument model is as follows: SHIMADZU LC-20 AB; 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, according to the procedure of table 17; the detection wavelength was 235nm (PDA detector) and the column temperature was 40 ℃.

TABLE 17 gradient elution procedure

The spectrum is shown in FIG. 16.

It can be seen from FIG. 16 that at a retention time of 8.795min, a peak of compound L2 appeared.

Example 3: detection method (control method of compounds L1 and L2 in lobaplatin quality standard)

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

1. Chromatographic conditions and System suitability test

The type of the chromatograph: SHIMADZU LC-20AD was prepared by coating cellulose-tris (3-chloro-4-methylphenyl carbamate) as a filler (Daicel Chiralcel OZ-3, 4.6 × 150mm, 3.0 μm) on the surface of silica gel, and eluting with n-hexane-ethanol (volume ratio 65:35) as a mobile phase at a flow rate of 1.0 mL/min, a detection wavelength of 210nm, a column temperature of 35 ℃ and isocratic elution for 40 min. The system applicability test solution is continuously injected for 6 times, and the relative standard deviation of the peak area of the main peak of lobaplatin is not more than 4.0 percent.

2. Preparation of test solution

About 100mg of lobaplatin to be tested (in the example, a lobaplatin sample prepared by the method disclosed in example 2 of the specification of patent CN 102020679B and confirmed by structure identification is taken, namely lobaplatin trihydrate is added as lobaplatin to be tested in the example, and the places related to the content of the lobaplatin in each example are measured according to lobaplatin anhydride), precisely weighed, placed in a10 mL volumetric flask, added with methanol for ultrasonic dissolution and diluted to a scale, and shaken up to be used as a test solution.

3. Preparation of System suitability test solution/1% control solution

Precisely measuring 1mL of a test solution, placing the test solution in a10 mL 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 a10 mL 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.

4. 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 there is a peak of related substance in the chromatogram of the test solution, the peak of related substance is identified by the chromatogram of typical chromatogram, and L1 and L2 are typical chromatogram of related substance of lobaplatin in the control method of lobaplatin quality standard, as shown in FIG. 17: the peak for lobaplatin diastereomer I appeared at retention time about t-10.062 min, the peak for lobaplatin diastereomer II appeared at retention time about t-8.550 min, the peak for compound L1 appeared at retention time about t-11.570 min, the peak for compound L2 appeared at retention time about t-30.611 min, the relative retention time for compound L1 (relative to lobaplatin diastereomer II) was about 1.35, and the relative retention time for compound L2 (relative to lobaplatin diastereomer II) was about 3.58; the control limits for compound L1, compound L2 in lobaplatin were: the peak areas of compound L1 and compound L2 were not more than 0.5 times the peak area of the main component in the control solution, respectively, as calculated by the peak area of the main component self control method without adding a correction factor.

Example 4: methodological validation of detection methods

In order to confirm the utility and accuracy of the detection method of the present invention, the specificity, sensitivity and quantitation limits, linearity and range, precision, accuracy (recovery), solution stability, durability, etc. of the detection method are explained below:

1. specificity

A blank solution (i.e., methanol solution) and a resolution solution RS (wherein the concentration of L1 is 0.1mgl/L and the concentration of L2 is 0.1mgl/L) were precisely measured and each 20. mu.L was injected into a liquid chromatograph, and the results of the measurement are shown in Table 18, in which the resolution of the main peak of lobaplatin, the compounds L1 and L2 was more than 1.5.

TABLE 18 specificity results

2. Sensitivity of the probe

The lobaplatin control solution, the compound L1 solution and the compound L2 solution were gradually diluted with a signal-to-noise ratio (S/N) of 10 as a limit of quantitation. The limiting concentration for lobaplatin was 0.0203mg/mL, the limiting concentration for Compound L1 was 0.008mg/mL, the limiting concentration for Compound L2 was 0.008mg/mL, and the limiting results are shown in Table 19.

TABLE 19 quantitative Limit results

3. Linearity and range

With the concentration of lobaplatin diastereomer II as abscissa (X) and peak area as ordinate (Y), as shown in FIG. 18-1, the linearity results are as follows: the concentration and peak area of lobaplatin diastereomer II in the range of 3.994mg/mL-6.04mg/mL show good linear relation, the linear relation is Y-8595033.2484X-2155759.5499, and the correlation coefficient R is²0.9934, indicating good linearity.

With the concentration of lobaplatin diastereomer I as abscissa (X) and peak area as ordinate (Y), as shown in FIG. 18-2, the linear results are as follows: the concentration and peak area of lobaplatin diastereomer I in the range of 3.994mg/mL-5.965mg/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.

With the concentration of compound L1 as abscissa (X) and the peak area as ordinate (Y), the linearity results are shown in fig. 19 as follows: the concentration and peak area of the compound L1 in the range of 0.0202mg/mL-0.1008mg/mL have good linear relation, the linear relation is that Y is 7627070.9325X +1812.0250, and the correlation coefficient R is²It was 1.0000, indicating a good linearity.

With the concentration of compound L2 as abscissa (X) and the peak area as ordinate (Y), the linearity results are shown in fig. 20 as follows: the concentration and peak area of the compound L2 in the range of 0.0200mg/mL-0.1002mg/mL show good linear relation, the linear relation is that Y is 7893984.5309X +5247.3750, and the correlation coefficient R²0.9989, indicating a good linearity.

4. Precision degree

Respectively preparing system applicability solution by experimenters A and B, respectively precisely measuring 20 μ L of the system applicability solution, injecting into a liquid chromatograph, recording a spectrum, and continuously injecting for 6 times, wherein the result is shown in table 20-1, the RSD of the lobaplatin diastereomer ratio (I: II) is less than 0.5%, and the precision is good.

TABLE 20-1 results of precision of diastereomer ratio

Sample solutions were prepared by the experimenters a and B, respectively, and then 20 μ L of each sample solution was precisely measured, injected into a liquid chromatograph, and a spectrum was recorded, and sample injection was performed 6 times in succession, with the results shown in table 20-2, where the content of each compound was RSD (n ═ 6) < 5%, RSD (n ═ 12) < 5%, and the precision was good.

TABLE 20-2 precision results for each sample

5. Accuracy (recovery)

The lobaplatin diastereomer and compounds L1 and L2 were each prepared in parallel as 3 parts of recovery solutions at 50% limiting concentration, 3 parts of recovery solutions at 100% limiting concentration, and 3 parts of recovery solutions at 150% limiting concentration, and the accuracy of each was examined. The results show that:

the recovery rate of the lobaplatin diastereomer I is between 99 and 102 percent, and the recovery rate of the lobaplatin diastereomer II is between 98 and 100 percent;

under the limit concentration of 50%, the recovery rate of L1 is between 90% and 100%, and the recovery rate of L2 is between 100% and 108%; under the limit concentration of 100%, the recovery rate of L1 is between 95% and 100%, and the recovery rate of L2 is between 105% and 108%; under the limit concentration of 150%, the recovery rate of L1 is between 95% and 100%, and the recovery rate of L2 is between 105% and 108%.

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

6. Stability of solution

Respectively measuring the test solution and the lobaplatin control solution, injecting samples for 0h, 1.3h, 2.7h, 4.75h, 6h, 15h, 26h, 48h and 81.5h, and inspecting the content change of the compound. As a result, as shown in Table 21, the content of compound L1, L2 was stable within 81.5 h.

TABLE 21 solution stability results

Remarking:

S_EE× 100% (diastereomer ratio in solution/diastereomer ratio in first chromatogram for each time interval);

S_STD× 100% (area of lobaplatin peak in solution/area of lobaplatin peak in first chromatogram per time interval);

S_im-X× 100% (content of each compound in solution per first chromatogram per time interval) x is L1 or L2.

7. Durability

The method is characterized in that a system applicability solution is taken, parameters in a liquid chromatography system are properly adjusted, the content detection condition of each compound after the system condition changes is inspected, the result is shown in table 22, and after the system condition slightly changes, the U% of each compound is 92% -102%, so that the method is good in durability.

TABLE 22 durability results

Remarking:

U_iso× 100% (peak area ratio of lobaplatin diastereomer II to lobaplatin main peak in solution after changing conditions/peak area ratio of lobaplatin diastereomer II to lobaplatin main peak in solution before changing conditions);

U_im-X(content of each compound in solution after changing conditions/changeThe content of each compound in the solution before changing the conditions) × 100% and x is L1 or L2.

Example 5: in vitro antitumor Activity assay (Activity assay of Compounds L1 and L2 of the present invention)

1. Reagent and consumable

1) In vitro antitumor activity assays cell lines used are shown in table 23 below, from the chinese academy of cells:

TABLE 23 list of cell lines

Species of species	Cell name
		Lung cancer cell	NCI-H460
Leukemia cells	Jurkat Clone E6-1
		Gastric cancer cell	AGS
Leukemia cells	HL-60
		Renal cancer cell	SK-NEP-1
Lung cancer cell	95-D
		Leukemia cells	THP-1
Ovarian cancer cells	OVCAR-3
		Ovarian cancer cells	SK-OV-3
Leukemia cells	K-562

2) McCoy's 5A medium, chinese Procell, cat #: PM150710

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

4)

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

5) 96-well cell culture plates, Corning, cat #: 3610

6) Envision microplate reader, PerkinElmer, USA

7) FBS, Lonsera, cat #: S711-001S

8) Sodium pyruvate, chinese excell, cat #: PB180422

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

10) β -mercaptoethanol, Gibco, cat No.: 21985

11) DMSO, Sigma, usa, cat #: d8418

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

13) 0.25% pancreatin-EDTA, chinese Procell, cat #: PB180228

14) RPMI-1640 medium, chinese excell, cat No.: PM150110

15) IMDM medium, chinese excell, cat #: PM150510

2. Solutions and buffers

1) Cell growth medium

The cell growth media were prepared as shown in Table 24, and stored at 4 ℃ until use.

TABLE 24 cell growth Medium

Cell name	Culture medium
		NCI-H460	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’s 5A+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/mL Insulin+1％P/S
		SK-OV-3	McCoy's 5A+10％FBS+1％P/S
K-562	RPMI-1640+10％FBS+1％P/S

The% values in the table above are in volume percent.

2) Heat inactivated serum

And (3) carrying out water bath on the serum at the temperature of 56 ℃ for 30 minutes.

3) Treatment of compounds

Compounds L1(3.13g) and L2(3.25g) were each dissolved in DMSO to prepare a 30mM solution, and the solution was stored at-20 ℃ until use. The positive control drug was Staurosporine (Staurosporine) (available from MedChemexpress (MCE), under the product name Staurosporine, cat # HY-15141), STSP for short, a natural product originally isolated in 1977 from the bacterium mycete staurosporius.

3. The experimental method comprises the following steps:

1) 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.

2) Cell passage

Cell passage: adherent cells, when grown 80-90% full in petri dish, were trypsinized with 0.25% (i.e., 0.25g pancreatin added to 100ml pbs solution), then resuspended in fresh medium, and passaged at the appropriate ratio for about 1 passage 2-3 d. Suspending the cells, collecting the cell suspension, centrifuging at 800rpm for 5 minutes, removing the supernatant, resuspending with fresh medium, and passaging at an appropriate ratio for about 1 passage 2-3 d.

3) Preparation of compound working solution concentration

A. Compound single concentration assay

On the day of the experiment, compounds were diluted to 1mM mother liquor using DMSO and further diluted to 50 μ M (5X final concentration) working solution with medium, test concentration of compounds was 10 micromolar and incubation time of compounds was 72 hours, according to assay requirements.

B. Compound IC₅₀Testing

On the day of the experiment, compounds were diluted to 1mM mother liquor as the highest concentration using DMSO and subjected to 2-fold, 3-fold or 5-fold gradient dilutions, followed by further dilutions to 5X final concentration of working solution per concentration point with medium, according to assay requirements.

4) Cell inoculation and drug treatment

1 day before the assay, cells were seeded at different densities in 96-well cell plates depending on the growth rate of the cells, 80. mu.L of cell suspension was seeded per well, 37 ℃ C., 5 vol.% CO₂Incubate overnight. The specific plating density of the cells is shown in table 25 below:

TABLE 25 plating Density of cells

Cell name	Density (cells/well)
		NCI-H460	4000
Jurkat Clone E6-1	10000
		AGS	4000
HL-60	8000
		SK-NEP-1	3000
95-D	3000
		THP-1	15000
OVCAR-3	5000
		SK-OV-3	2000
K-562	2000

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.

After the incubation is finished, detection is carried out according to the operation requirement of a CTG kit (purchased from promega, product number G7572, name celliterglo), a corresponding chemiluminescence value is obtained, and the cell activity is calculated.

The calculation method is as follows:

cell viability ═ addition set RLU value/control (solvent) RLU value × 100%.

5) The experimental results are as follows:

the inhibitory activity of compounds L1 and L2 at a single concentration of 10 μ M was as follows:

table 2610 μ M single concentration inhibitory activity of compound L1:

cell name	Cell survival Rate (%) of Compound	Cell viability of control (%)
			SK-OV-3	48.76	1.63
Jurkat Clone E6-1	10.47	0.93
			K-562	45.89	2.71
AGS	43.82	3.11
			HL-60	33.86	2.03
SK-NEP-1	14.38	3.07
			THP-1	5.44	1.30

Table 2710 μ M single concentration inhibitory activity of compound L2:

cell name	Cell survival Rate (%) of Compound	Cell viability of control (%)
			NCI-H460	48.67	1.54
Jurkat Clone E6-1	9.18	0.93
			K-562	37.79	2.71
OVCAR-3	48.84	5.15
			AGS	35.41	3.11
HL-60	15.08	2.03
			SK-NEP-1	11.87	3.07
THP-1	2.30	1.30
			95-D	37.43	2.51

From the data, the compound L1 or L2 has better inhibitory activity to the cancer cells at the concentration of 10 mu M, the inhibition rate of L1 to THP-1 reaches more than 90 percent, and the inhibition rate of L2 to Jurkat Clone E6-1 and THP-1 reaches more than 90 percent, so that the compound has remarkable tumor inhibitory activity.

The dose-response test (inhibitory activity of compounds on cancer cells) was performed on compounds L1, L2 and control drug (STSP), respectively, and the dose-response curves determined are shown in FIGS. 21-1 to 27-2 and FIGS. 28-1 to 36-2, wherein the concentration is plotted on the abscissa in micromolar and the cell viability is plotted on the ordinate. Reading of IC of Compounds L1 and L2 by dose-response Curve₅₀The values are shown in tables 28 and 29 below, respectively:

TABLE 28 IC of Compound L1₅₀The value:

cell name	Compound IC₅₀	Control (STSP)
			Jurkat Clone E6-1	2.4μM	14.67nM/11.63nM/12.12nM/12.84nM
AGS	6.43μM	6.02nM/5.72nM/5.84nM
			HL-60	6.76μM	17.1nM/17.42nM/17.06nM
THP-1	3.4μM	73.02nM/74.45nM/42.58nM
			SK-OV-3	>10μM	8.46nM
K-562	>10μM	129nM/212.2nM
			SK-NEP-1	2.4μM	12.09nM/12.38nM/11.81nM/10.72nM

TABLE 29 IC of Compound L2₅₀The value:

cell name	Compound IC₅₀	Control (STSP)
			NCI-H460	3.19μM	40.35nM/40.41nM
Jurkat Clone E6-1	1.44μM	14.67nM/11.63nM/12.12nM/12.84nM
			AGS	4.55μM	6.02nM/5.72nM/5.84nM
HL-60	5.19μM	17.1nM/17.42nM/17.06nM
			95-D	4.8μM	56.48nM/50.42nM/69.34nM
THP-1	3.21μM	73.02nM/74.45nM/42.58nM
			OVCAR-3	2.11μM	27.19nM/47.29nM/40.25nM
K-562	>10μM	129nM/212.2nM
			SK-NEP-1	1.66μM	12.09nM/12.38nM/11.81nM/10.72nM

As can be seen from the activity data, the compounds L1 and L2 of the invention have certain inhibitory activity on tumor cells. The activity of L2 is stronger than that of L1, and the activity of L2 has IC (Integrated Circuit) on various tumor cells₅₀Under 5 mu M, the anticancer activity is remarkable, and the compound can be further developed into anticancer drugs for clinical application.

Claims

1. A platinum species, wherein the platinum species is a compound of L1 or L2 or a mixture of L1 and L2 having the formula:

wherein L1 and L2 can be interchanged.

2. The method for preparing a platinum-based material according to claim 1, which comprises preparing a compound 6 having the following structural formula:

preferably, in the reaction for preparing the platinum-based substance by the compound 6, a solution of the compound 6 is reacted with the lactic acid compound 7 to obtain a mixture of L1 and L2, and further preferably, the molar ratio of the compound 6 to the lactic acid compound 7 is 1: 1-2; it is further preferable that the pH of the system is adjusted to 6.4 to 6.8 with the lactic acid compound 7; further preferably, the reaction temperature is 25-35 ℃, preferably 30 ℃; it is further preferred that the reaction time is from 72 to 96 hours, preferably 87 hours;

wherein, the structural formula of the lactic acid compound 7 is as follows:

3. the method of claim 2, wherein the compound 6 is prepared from a compound 5 of the following formula:

preferably, in the reaction for preparing the compound 6 by the compound 5, resin is added for reaction, then, the reaction solution is filtered to obtain filtrate, the resin is washed to obtain washing liquor, and the filtrate and the washing liquor are combined to obtain a solution containing the compound 6; it is further preferred that the resin is a resin treated with an aqueous solution of sodium hydroxide; more preferably, the concentration of the aqueous sodium hydroxide solution is 1 to 2mol/L, and still more preferably 1.5 mol/L; it is more preferable to add the resin and react for 0.5 to 2 hours, and it is further preferable to react for 1 hour.

4. The method of claim 3, wherein the compound 5 is prepared from a compound 4.1 of the following structural formula, preferably from a compound 4 of the following structural formula:

wherein X represents halogen element selected from F, Cl, Br or I;

further preferably, in the reaction for preparing the compound 5 from the compound 4.1, the compound 4.1 is dispersed in a ketone-water mixed solvent, and then a silver nitrate aqueous solution is added to react away from light to prepare a solution containing the compound 5; it is further preferred that the molar ratio of compound 4.1 to silver nitrate is 1:1-2, further preferred 1: 1.4-2;

it is further preferred that the ketone is selected from acetone, and it is further preferred that the volume ratio in the acetone-water mixed solvent is acetone: water is 0-9: 1; it is further preferred that the reaction temperature is from 15 to 25 ℃, preferably 20 ℃; the reaction time is 14 to 18 hours, preferably 16 hours.

5. The preparation method of claim 4, wherein the compound 4.1 is prepared from a compound 3 with the following structural formula:

6. The method of claim 5, wherein the compound 3 is prepared from the compound 2 of the following structural formula:

preferably, in the reaction for preparing the compound 3 by the compound 2, the compound 2 is reacted with an alcoholic solution of oxalic acid to obtain the compound 3 as a white solid, and more preferably, the molar ratio of the compound 2 to the oxalic acid is 1: 0.5-2; further preferably, the reaction system is heated to 65 to 75 ℃ for reaction, further preferably 0.5 to 2 hours, further preferably 70 ℃ for reaction for 1 hour; it is further preferred that the separated solid compound 3 is added to a tetrahydrofuran solvent, and the mixture is further mixed at 60 to 70 ℃ and further preferably at 65 ℃ to obtain the purified compound 3 by solid-liquid separation.

7. The method of claim 6, wherein the compound 2 is prepared from the compound 1 with the following structural formula:

8. The production method according to any one of claims 2 to 7, wherein the starting material is compound 1:

9. the production method according to any one of claims 2 to 8, which comprises the steps of:

and/or, in the reaction for preparing the platinum-based substance by the compound 6, reacting a solution of the compound 6 with a lactic acid compound 7 to obtain a mixture of L1 and L2, preferably, adjusting the pH of the system to 6.4-6.8 by using the lactic acid compound 7, further preferably, the reaction temperature is 25-35 ℃, preferably 30 ℃; it is further preferred that the reaction time is from 72 to 96 hours, preferably 87 hours.

10. The method of claim 9, comprising the steps of: separating the prepared mixture of the compounds L1 and L2 by liquid chromatography to obtain a compound L1 and a compound L2; preferably, the liquid chromatography is performed as NH₄HCO₃The aqueous solution of (A) is a mobile phase A, and acetonitrile is used as a mobile phase B; the concentration of the mobile phase A is preferably 8-12m mol/L, preferably 10m mol/L; it is further preferred that the volume of mobile phase B is increased from 0 to 20% and the volume of mobile phase A is decreased from 100% to 80% in 0-20min by using gradient elution.

11. The method for detecting a platinum-based substance according to claim 1, wherein the method is an HPLC method or an HPLC-MS method; preferably, the detection conditions of the HPLC method are as follows: coating cellulose-tri (3-chloro-4-methylphenyl carbamate) on the surface of silica gel as a filling agent, and taking n-hexane-ethanol with the volume ratio of 60-70:40-30 as a mobile phase, wherein the flow rate is 0.8-1.5 mL/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is carried out for 30-50 min; preferably, n-hexane-ethanol with the volume ratio of 63-67:37-33 is taken as a mobile phase, and the column temperature is 33-37 ℃; more preferably, the mobile phase is n-hexane-ethanol with volume ratio of 65:35, flow rate is 1.0 mL/min, detection wavelength is 210nm, column temperature is 35 deg.C, and isocratic elution time is 40 min.

12. Use of the platinum-based substance of claim 1 as an indicator of a substance of interest in the quality standards for a lobaplatin-based drug substance or formulation.

13. A method for detecting the quality of a lobaplatin bulk drug or preparation, which comprises the step of measuring related substances affecting the quality of lobaplatin, wherein the related substances are the compound in claim 1, and the related substances affecting the quality of lobaplatin are detected by an HPLC method or an HPLC-MS method;

preferably, the HPLC method detection conditions are as follows: coating cellulose-tri (3-chloro-4-methylphenyl carbamate) on the surface of silica gel as a filling agent, and taking n-hexane-ethanol with the volume ratio of 60-70:30-40 as a mobile phase, wherein the flow rate is 0.8-1.5 mL/min, the detection wavelength is 208-212nm, the column temperature is 30-40 ℃, and isocratic elution is carried out for 30-50 min; more preferably, n-hexane-ethanol with the volume ratio of 63-67:37-33 is taken as a mobile phase, and the column temperature is 33-37 ℃; more preferably, the mobile phase is n-hexane-ethanol with a volume ratio of 65:35, the flow rate is 1.0 mL/min, the detection wavelength is 210nm, the column temperature is 35 ℃, and the isocratic elution time is 40 min;

14. A pharmaceutical composition comprising the platinum-based material according to claim 1, wherein the pharmaceutical composition is a pharmaceutical preparation, preferably the pharmaceutical composition is a pharmaceutical preparation for injection.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutical preparation comprises an adjuvant, preferably the adjuvant is selected from one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, an antioxidant, and a matrix; it is further preferred that the auxiliary material is selected from fillers and/or antioxidants.

16. Use of a platinum group according to claim 1 or a pharmaceutical composition according to claim 14 or 15 for the preparation of an anti-tumor medicament.

17. The use according to claim 16, wherein the tumor is a cell of lung cancer, leukemia, gastric cancer, ovarian cancer and/or renal cancer; preferably, the tumor is a leukemia cell.

18. The use according to claim 16, wherein the compound L1 is used for preparing anti-THP-1 tumor medicament, and the compound L2 is used for preparing anti-Jurkat Clone E6-1 and/or THP-1 tumor medicament.