CN111378006A - Novel double-arm intermediate LND1026-035 for antibody coupling drug and synthetic method thereof - Google Patents
Novel double-arm intermediate LND1026-035 for antibody coupling drug and synthetic method thereof Download PDFInfo
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- CN111378006A CN111378006A CN201811617966.4A CN201811617966A CN111378006A CN 111378006 A CN111378006 A CN 111378006A CN 201811617966 A CN201811617966 A CN 201811617966A CN 111378006 A CN111378006 A CN 111378006A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides a novel double-arm intermediate LND1026-035 for antibody coupling drug and its synthesis method, LND1026-035 can connect two cytotoxic molecules on one antibody connection site, thereby can reduce the administration concentration, or can increase the treatment effect under the same administration concentration. Meanwhile, one antibody carries more cytotoxins, so that the utilization rate of the antibody can be improved, and the production cost of the medicine can be reduced.
Description
Technical Field
The invention relates to the field of organic synthesis, in particular to a molecular structure of a novel double-arm intermediate for antibody coupled drugs and a preparation method thereof.
Background
Antibody Drug Conjugates (ADC) are a novel anti-tumor drug, and the principle is that cytotoxin is connected to an Antibody, and the cytotoxin is transported to a target point through recognition of a specific antigen on the surface of a cancer cell by the Antibody and entering the cancer cell through endocytosis, so that the aim of targeted therapy of malignant tumor is achieved. Compared with the traditional micromolecule antitumor drugs, the ADC has higher specificity and effectiveness due to the fact that the target recognition of the antibody and the high activity of the toxin can be used.
ADCs comprise three distinct components, namely antibodies, linkers and cytotoxins. The antibody realizes targeting, the linker ensures the stability of the ADC in the blood transportation process, and after the ADC reaches an action target, the toxin plays a role in killing cancer cells. Depending on the mechanism of action, the toxins suitable for ADCs are classified into microtubule-like inhibitors (microtubuliinhibitors), DNA damaging agents (DNA damaging agents), RNA polymerase inhibitors (RNA polymerase inhibitors), and the like. Currently, the toxins used in ADCs marketed and in clinical trials are mainly microtubule inhibitors, mainly including compounds designed based on Dolastatin-based (Dolastatin-based) such as MMAE, MMAF and MMAD, and compounds designed based on Maytansine-based (Maytansine-based) such as DM1 and DM 4. In the linker context, the main applications are non-cleavable types, such as Valine-citrulline (Valine-Citriline) and cyclohexyl carboxylic acid (MCC), which remain active after lysosomal hydrolysis and bind to an amino acid residue via a linker region.
In the existing antibody coupling technology, a cytotoxin is usually connected to a connection site of each antibody, the antibody carries the cytotoxin to reach the interior of a tumor cell, and the cytotoxin is released under the action of enzyme, so that the aim of killing the tumor cell is fulfilled.
Disclosure of Invention
The existing antibody coupling technology has limited sites capable of connecting cytotoxin on each antibody, one antibody can carry less than 10 cytotoxins, and in order to kill tumor cells, high antibody drug concentration is generally required.
One of the purposes of the invention is to provide a novel double-arm intermediate LND1026-035 used for antibody coupling drug, which has the structural formula
Another object of the present invention is to provideA novel intermediate compound F of a double-arm intermediate LND1026-035, the structural formula of which is
It is a further object of this invention to provide a method of synthesis of a novel dual arm intermediate LND 1026-035.
The technical scheme for achieving the purpose of the invention is as follows.
A method for synthesizing a double-arm intermediate LND1026-035, the structural formula of LND1026-035 is
The method comprises the following steps:
1) a compound A with a structural formula A and DMAP are dissolved in dichloromethane, wherein the structural formula A is
Stirring for reaction under the action of triphosgene, and adding a compound B with a structural formula B
Stirring for condensation reaction, and separating to obtain compound C with structural formula C
2) Dissolving the compound C in dichloromethane, performing deprotection reaction under the action of diethylamine, and separating to obtain compound D with structural formula D
3) Dissolving a compound D, a compound E with a structural formula E and HATU in dichloromethane, wherein the structural formula E is
Condensation reaction under the action of DIEA, and separationTo obtain a compound F with a structural formula F
4) And dissolving the compound F in dichloromethane, removing a protecting group under the action of diethylamine, and separating to obtain LND 1026-035.
Optionally, the separation method in the step 1) is to dilute the product with dichloromethane, wash the product with water, dry the product with anhydrous sodium sulfate, spin-dry the product, purify the crude product with medium-pressure reversed phase, select 80g of industrial packed C18 reversed phase column, optionally, the volume ratio of purification gradient of the medium-pressure reversed phase purification is 90/10-10/90, and the time is 1 hour.
Alternatively, the separation method in step 2) is adding water and DCM for extraction, washing the organic phase with saturated common salt water, drying with anhydrous sodium sulfate, and concentrating.
Optionally, the separation method in the step 3) is to concentrate the reaction solution, perform medium-pressure reverse-phase purification, select 40g of industrial packed C18 reverse-phase column, and optionally perform medium-pressure reverse-phase purification with a purification gradient volume ratio of water/acetonitrile of 90/10-10/90 for 1 hour.
Optionally, the separation method in the step 4) includes adding water and DCM for extraction, washing the organic phase with saturated common salt water, drying with anhydrous sodium sulfate, concentrating, performing medium-pressure reverse phase purification, selecting 25g of industrial packed C18 reverse phase column, and optionally performing medium-pressure reverse phase purification with a purification gradient volume ratio of water/acetonitrile 90/10-10/90 for 1 hour.
The above-mentioned raw materials, reagents and the like are commercially available unless otherwise specified.
The novel double arm intermediate LND1026-035 allows for the attachment of two cytotoxic molecules to one antibody attachment site, allowing for lower dosing concentrations or increased therapeutic efficacy at the same dosing concentration. Meanwhile, one antibody carries more cytotoxins, so that the utilization rate of the antibody can be improved, and the production cost of the medicine can be reduced. .
Drawings
FIG. 1 is a mass spectrum of Compound C synthesized according to the present invention.
FIG. 2 is a liquid chromatogram of Compound C synthesized according to the present invention.
FIG. 3 is a mass spectrum of Compound D synthesized according to the present invention.
FIG. 4 is a liquid chromatogram of Compound D synthesized according to the present invention.
FIG. 5 is a mass spectrum of Compound F synthesized according to the present invention.
FIG. 6 is a liquid chromatogram of Compound F synthesized according to the present invention.
FIG. 7 is a mass spectrum of the target product LND1026-035 synthesized by the present invention.
FIG. 8 is a liquid chromatogram of the target product LND1026-035 synthesized by the present invention.
Detailed Description
As used herein, the abbreviations commonly used have the conventional meaning in the art, e.g., the abbreviation DMAP for 4-dimethylaminopyridine and the abbreviation DIEA for N, N-diisopropylethylamine.
The technical solution of the present invention will be further described in non-limiting detail with reference to the following embodiments. It should be noted that the following embodiments are only for illustrating the technical concept and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
The invention provides a method for synthesizing a double-arm intermediate LND1026-035, the reaction route of the synthetic method is as follows:
the method for synthesizing the double-arm intermediate LND1026-035 comprises the following steps:
1) a compound A with a structural formula A and DMAP are dissolved in dichloromethane, wherein the structural formula A is
Adding three under the protection of nitrogen at 0 DEG CPhosgene, stirring and reacting for 5 minutes under the action of triphosgene, adding a compound B with a structural formula B, wherein the structural formula B is
The condensation reaction took place with stirring at 0 ℃ for 5 minutes and LCMS showed less than 5% of compound A in the reaction solution as the end of the reaction. Diluting with dichloromethane, washing with water, drying with anhydrous sodium sulfate, spin-drying, performing medium-pressure reverse-phase purification on the crude product, selecting 80g of industrial packed C18 reverse-phase column, filling C18 (octadecylsilane bonded silica gel filler) in the reverse-phase column, performing medium-pressure reverse-phase purification with a purification gradient volume ratio of water/acetonitrile (90/10-10/90) for 1 hour to obtain a compound C with a structural formula C (C is
2) Dissolving the compound C in dichloromethane, performing deprotection reaction under the action of diethylamine, reacting for 4 hours, and determining that the reaction is finished when LCMS shows that less than 3% of the compound C in the reaction solution. Adding water and DCM for extraction, washing the organic phase with saturated salt water, drying with anhydrous sodium sulfate, and concentrating to obtain compound D with structural formula D
3) Dissolving a compound D, a compound E with a structural formula E and HATU in dichloromethane, wherein the structural formula E is
Condensation reaction occurred by DIEA reaction for 16 hours at room temperature, and LCMS showed less than 3% of compound D in the reaction solution as the end of the reaction. Concentrating the reaction solution, performing medium-pressure reversed-phase purification, selecting 40g of industrial packed C18 reversed-phase column, performing medium-pressure reversed-phase purification operation with a purification gradient volume ratio of water/acetonitrile of 90/10-10/90 for 1 hour to obtain a compound F with a structural formula F, wherein the structural formula F is
4) Dissolving the compound F in dichloromethane, removing the protecting group under the action of diethylamine, reacting at room temperature for 4 hours, and determining that the reaction is finished when LCMS shows that less than 3 percent of the compound F in the reaction liquid. Adding water and DCM for extraction, washing an organic phase with saturated common salt, drying with anhydrous sodium sulfate, concentrating, performing medium-pressure reverse phase purification, selecting 25g of industrial packed C18 reverse phase column, wherein the volume ratio of purification gradient of the medium-pressure reverse phase purification operation is 90/10-10/90, and obtaining LND1026-035 within 1 hour.
Example 1
Dissolving compound A (769mg, 1.52mmol) and DMAP (551mg, 4.52mmol) in dichloromethane (10mL), adding triphosgene (225mg, 0.76mmol) at 0 ℃ under the protection of nitrogen, stirring for 5 minutes to obtain a yellow solution, adding compound B (717mg, 1.52mmol), stirring for 5 minutes at 0 ℃, and LCMS to show that less than 5% of compound A in the reaction solution is considered to be the end of the reaction, adding 40mL of dichloromethane to the reaction solution, washing with water (2 × 50mL), drying with anhydrous sodium sulfate, spin-drying, purifying the crude product by medium pressure reverse phase (80 g of industrial packed C18 reverse phase column is selected), purifying gradient water/acetonitrile (90/10-10/90, v/v), and collecting the pure product for 1 hour, and freeze-drying to obtain compound C as a white solid (550mg, yield 36%).
Compound C (550mg, 0.44mmol) was dissolved in dichloromethane (10mL), diethylamine (2mL) was added and the reaction was allowed to react at room temperature for 4 hours, LCMS showed less than 3% of compound C in the reaction solution as the end of the reaction, water (50mL) was added, DCM (2 × 50mL) was extracted, the organic phase was washed with saturated brine, then dried over anhydrous sodium sulfate, and concentrated to give compound D as a yellow solid (380mg, yield 83%).
Compound D (380mg, 0.36mmol), compound E (64mg, 0.18mmol) and HATU (206mg, 0.54mmol) were added to dichloromethane (5mL) followed by DIEA (93mg, 0.72mmol) and reacted at room temperature for 16 h, LCMS showed less than 3% of compound D in the reaction solution as the end of the reaction. After the reaction solution is concentrated, medium-pressure reversed-phase purification (40 g of industrial packed C18 reversed-phase column is selected), and gradient water/acetonitrile (90/10-10/90, v/v) is purified for 1 hour. The pure product was collected and lyophilized to give compound F as a white solid (104mg, 24% yield).
Dissolving compound F (104mg, 0.043mmol) in dichloromethane (2mL), adding diethylamine (0.5mL), reacting at room temperature for 4 hours, LCMS shows that less than 3% of compound F in the reaction solution is regarded as the reaction is finished, adding water (10mL), DCM (2 × 10mL), extracting, washing the organic phase with saturated common salt, drying with anhydrous sodium sulfate, concentrating, and performing medium-pressure reverse-phase purification (25 g of an industrial packed C18 reverse-phase column is selected), purifying gradient water/acetonitrile (90/10-10/90, v/v) for 1 hour, collecting pure product and freeze-drying to obtain a white solid compound LND1026-035(23mg, yield 24%).
Claims (10)
1. A double-arm intermediate LND1026-035 for antibody coupling drug, the structural formula is
2. An intermediate compound F for synthesizing a double-arm intermediate LND1026-035, the LND1026-035 has the structural formula
Wherein the intermediate compound F has a structural formula F
3. A method for synthesizing a double-arm intermediate LND1026-035, the structural formula of LND1026-035 is
Characterized in that the method comprises the following steps:
1) a compound A with a structural formula A and DMAP are dissolved in dichloromethane, wherein the structural formula A is
Under the action of triphosgeneStirring for reaction, and adding a compound B with a structural formula B
Stirring for condensation reaction, and separating to obtain compound C with structural formula C
2) Dissolving the compound C in dichloromethane, performing deprotection reaction under the action of diethylamine, and separating to obtain compound D with structural formula D
3) Dissolving a compound D, a compound E with a structural formula E and HATU in dichloromethane, wherein the structural formula E is
Condensation reaction is carried out under the action of DIEA, and a compound F with a structural formula F is obtained through separation operation
4) And dissolving the compound F in dichloromethane, removing a protecting group under the action of diethylamine, and separating to obtain LND 1026-035.
4. The process of claim 3, wherein the separation method in step 1) is that after dichloromethane is diluted, the dichloromethane is washed with water, dried by anhydrous sodium sulfate, dried by spinning, and the crude product is purified by medium pressure reverse phase, and 80g of industrial packed C18 reverse phase column is selected.
5. The process of claim 4, wherein the medium pressure reversed phase purification is performed at a purification gradient volume ratio of water/acetonitrile of 90/10-10/90 for 1 hour.
6. The process of claim 3, wherein the separation in step 2) is performed by adding water, extracting with DCM, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, and concentrating.
7. The method as claimed in claim 3, wherein the separation method in step 3) is to concentrate the reaction solution and purify the reaction solution by medium pressure reverse phase, and 40g of industrial packed C18 reverse phase column is selected.
8. The process of claim 7, wherein the medium pressure reversed phase purification is performed at a purification gradient volume ratio of water/acetonitrile 90/10-10/90 for 1 hour.
9. The process of claim 3, wherein the separation in step 4) is carried out by adding water, DCM for extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, concentrating, medium pressure reverse phase purification, and using 25g of industrial packed C18 reverse phase column.
10. The process of claim 9, wherein the medium pressure reversed phase purification is performed at a purification gradient volume ratio of water/acetonitrile 90/10-10/90 for 1 hour.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874287A (en) * | 2022-05-20 | 2022-08-09 | 联宁（苏州）生物制药有限公司 | Synthetic method of antibody coupled drug-linker LND1042 |
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| TW201827085A (en) * | 2016-12-14 | 2018-08-01 | 美商西雅圖遺傳學公司 | Multi-drug antibody drug conjugates |
| CN108404138A (en) * | 2018-03-09 | 2018-08-17 | 中国药科大学 | A kind of conjugate and its application of targeting CD24 monoclonal antibodies and two alkoxide of diethylamine azo |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0622084A1 (en) * | 1993-04-28 | 1994-11-02 | Eli Lilly And Company | Antibody-drug conjugates |
| CN105669559A (en) * | 2016-01-12 | 2016-06-15 | 南京林业大学 | Pinanyl pyrazole compound and its synthesis method and use |
| CN107915770A (en) * | 2016-10-11 | 2018-04-17 | 联宁（苏州）生物制药有限公司 | A kind of antibody drug conjugates intermediate and preparation method thereof |
| TW201827085A (en) * | 2016-12-14 | 2018-08-01 | 美商西雅圖遺傳學公司 | Multi-drug antibody drug conjugates |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874287A (en) * | 2022-05-20 | 2022-08-09 | 联宁（苏州）生物制药有限公司 | Synthetic method of antibody coupled drug-linker LND1042 |
| CN114874287B (en) * | 2022-05-20 | 2024-04-02 | 联宁（苏州）生物制药有限公司 | Synthesis method of antibody coupling drug-linker LND1042 |
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Application publication date: 20200707 |