CN117427176A - Drug conjugate and application thereof - Google Patents

Drug conjugate and application thereof Download PDF

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Publication number
CN117427176A
CN117427176A CN202210819270.XA CN202210819270A CN117427176A CN 117427176 A CN117427176 A CN 117427176A CN 202210819270 A CN202210819270 A CN 202210819270A CN 117427176 A CN117427176 A CN 117427176A
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cancer
mmol
compound
drug conjugate
drug
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刘辰
刘源
陈涛
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Yafei Shanghai Biolog Medicine Science & Technology Co ltd
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Yafei Shanghai Biolog Medicine Science & Technology Co ltd
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Abstract

The invention discloses a drug conjugate and application thereof. The drug conjugate is shown as a formula I: R-AA-PABC-D (I) wherein: r is a targeting group, and can also carry out certain modification on the characteristics of the medicine or be used for regulating the water solubility of the medicine and the activation efficiency in the tumor microenvironment; PABC represents a p-aminobenzyl alcohol group; AA represents a group of an amino acid; d represents a drug molecule.

Description

Drug conjugate and application thereof
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a pharmaceutical conjugate with a camptothecin derivative and application thereof.
Background
7-Ethyl-10-hydroxycamptothecin (SN-38) is an active metabolite of Irinotecan (CPT-11), a broad-spectrum antitumor drug, and can be used for treating malignant tumors such as colorectal cancer, liver cancer, lung cancer, breast cancer and the like. SN-38 has extremely poor water solubility and low bioavailability, and limits its wide application in anti-tumor treatment.
Disclosure of Invention
The invention aims to improve the therapeutic efficacy of medicaments such as SN-38 and the like.
In a first aspect of the invention, there is provided a drug conjugate of formula I:
R-AA-PABC-D
(I)
wherein:
r is a targeting group, and can also carry out certain modification on the characteristics of the medicine, or can be used for regulating the water solubility of the medicine and the activation efficiency in the tumor microenvironment;
PABC represents a p-aminobenzyl alcohol group;
AA represents a group of an amino acid;
d represents a drug molecule.
In another embodiment, wherein R isOr R represents that R1 and R2 are formed by click reaction
In another embodiment, R 1 Selected from:
R 2 selected from the group consisting of compounds containing:
wherein n is an integer of 1 to 10.
In another embodiment, wherein R is selected from:
wherein m and n are integers of 1 to 10, respectively.
In another embodiment, AA is selected from the group consisting of lysine, acetyllysine, or lysine-glycine.
In another embodiment, the drug is selected from any one of camptothecins, SN38, irinotecan, an irinotecan derivative, topotecan, 9-aminocamptothecin, 9-nitrocamptothecin, paclitaxel, docetaxel, rapamycin, everolimus, daunorubicin, epirubicin, doxorubicin, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitoxantrone, imiquimod, rassimethide, luoli bine.
In another embodiment, the drug conjugate is selected from the group consisting of:
in a second aspect of the invention there is provided a pharmaceutical composition comprising a pharmaceutical conjugate as provided herein above and a pharmaceutically acceptable excipient.
In a third aspect of the invention there is provided the use of a drug conjugate as provided herein above for the manufacture of a medicament for the treatment of cancer.
In another embodiment, the cancer comprises bladder cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, and leukemia.
Accordingly, the present invention provides a way to increase the bioavailability of SN-38, thereby contributing to its wide application in anti-tumor therapy.
Drawings
Fig. 1 shows the efficacy of drug conjugates S7, S10 and S11 in human fibrosarcoma.
Fig. 2 shows the efficacy of drug conjugates S7, S10 and S11 in a human pancreatic cancer cell model.
Detailed Description
The research of the inventor discovers a conjugate which can be specifically activated by tumor microenvironment and other complex functions, and the structure of the conjugate can be used for part of antitumor drugs, and can play a role in changing the characteristics of targeting, activation, stability, solubility, metabolism, toxicity and drug effect of the connected drugs.
The conjugate has a structure as shown in a general formula I:
R-AA-PABC-D
(Ⅰ)
wherein:
r is a targeting group, and can also carry out certain modification on the characteristics of the medicine or be used for regulating the water solubility of the medicine and the activation efficiency in the tumor microenvironment;
AA represents an amino acid sequence selected from Lys, ac-Lys, lys-Gly;
PABC is p-aminobenzyl alcohol
D represents the following drugs: any one of camptothecins, SN38, irinotecan, topotecan, 9-aminocamptothecin, 9-nitrocamptothecin, paclitaxel, docetaxel, rapamycin, everolimus, daunorubicin, epirubicin, doxorubicin, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitoxantrone, imiquimod, rassimethide, luoli bine.
In one embodiment of the invention, R is
In one embodiment of the present invention, R represents that R1 and R2 are formed by a click reactionWherein,
R 1 selected from any one of the following:
r2 is selected from compounds containing the following groups:
wherein n is an integer of 1 to 10; such as, but not limited to, 1,2,3, 4, 5, 6, 7, 8, 9, 10. In one embodiment of the invention, R is selected from any one of the following groups:
wherein m and n are each an integer of 1 to 10; such as, but not limited to, 1,2,3, 4, 5, 6, 7, 8, 9, 10.
As used herein, "click reaction" refers to the cycloaddition of an alkynyl group to an azido group by Cu (I) catalysis to produce a regioselective 1, 4-disubstituted-1, 2, 3-triazole.
In one embodiment of the invention, the amino acid in AA is selected from at least one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, epsilon-acetyl lysine, arginine, histidine, and like functional fragments of amino acids.
In some specific embodiments, AA is selected from: lysine, acetyl lysine or lysine-glycine.
In some embodiments, preferably, AA is lysine.
In one embodiment of the present invention, D is selected from any one of SN-38, irinotecan, camptothecin, 10-hydroxycamptothecin.
The structure of the drug conjugate provided by the invention can be selected from any one of the following:
synthesis of conjugates
The conjugates of formula (I) described above may be synthesized using standard synthetic techniques or well known techniques in combination with the methods described herein. In addition, the solvents, temperatures and other reaction conditions mentioned herein may vary.
The starting materials for the synthesis of the conjugates of formula (I) may be obtained synthetically or from commercial sources such as, but not limited to, aldrich Chemical co (Milwaukee, wis.) or Sigma Chemical co (St. Louis, mo.). In one aspect, the conjugates described herein are according to methods well known in the art. In one aspect, the conjugates described herein are prepared according to the methods described in the specific examples below.
The synthesis of the conjugates of formula (I) is outlined in the examples.
By "pharmaceutically acceptable" is meant herein a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and which is relatively non-toxic, e.g., administration of a material to an individual does not cause an undesired biological effect or interact in a deleterious manner with any of the components thereof in which it is contained.
The term "fragment" refers to a specific portion or functional group of a molecule. Chemical fragments are generally considered to be chemical entities contained in or attached to a molecule.
The term "cancer", as used herein, refers to an abnormal growth of cells that is not controllable and is capable of metastasis (transmission) under certain conditions. Cancers of this type include, but are not limited to, solid tumors (e.g., bladder, intestine, brain, chest, uterus, heart, kidney, lung, lymphoid tissue (lymphoma), ovary, pancreas, or other endocrine organ (e.g., thyroid), prostate, skin (melanoma), or hematological tumors (e.g., non-leukemia).
The terms "treat," "course of treatment," or "therapy" as used herein include alleviation, inhibition, or amelioration of symptoms or conditions of a disease; inhibit the occurrence of complications; improving or preventing underlying metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or preventing or treating signs caused by diseases or symptoms.
Therapeutic use
The invention provides application of a drug conjugate of a formula (I), and the conjugate drug has high activation and release efficiency in tumor tissues or aspartic acid activation experiments.
The invention also provides the use of a drug conjugate of formula (I) for the preparation of an anti-tumour agent which can be used in the treatment and immunotherapy of different anti-cancer types including: bladder, brain, breast/breast, cervical, colon-rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testes, and hematological cancers.
Because different parts of the conjugate specifically activated in the tumor microenvironment have great influence on the functions of targeting, activating, stabilizing, toxicity, drug effect and the like of the final drug, the use of the conjugate connector specifically activated in the invention can effectively reduce the toxicity of the connected drug, so that the final drug has new targeting, activating and metabolic characteristics, the effect of treating tumors is increased, new tumor indications and anti-tumor metastasis effects are generated, and brand-new structures and functions are generated.
The invention discovers that the characteristics of the drug conjugate are as follows:
(1) The conjugate can change D into a plurality of medicines with therapeutic or auxiliary therapeutic effects on tumors through group conversion.
(2) The target activation is achieved by activating in tumor microenvironment. The medicine can not activate in normal tissues, is a medicine without cytotoxicity or low toxicity, and can not kill normal cells.
(3) The conjugate is activated by the microenvironment of tumors, so that the conjugate can be activated in various tumors, and the coupled linker is subjected to various screening, so that the solubility is obviously improved, the condition limited by the tumor indication of the coupled drug can be directly changed, and the conjugate is developed into a broad-spectrum or special targeted antitumor drug.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. All percentages, ratios, proportions, or parts are by weight unless otherwise indicated. The units in weight volume percent are well known to those skilled in the art and refer, for example, to the weight of solute in 100 milliliters of solution (grams). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Example 1
Synthesis of Compound S1
Synthesis of Compounds 1-2
Fmoc-Lys (NH) 2 ) -OH (5.52 g,15.0 mmol) was dispersed in dichloromethane (40 mL) and TMS-Cl (3.40 g,31.5 mmol) was added dropwise under nitrogen and the reaction stirred for 3 hours at 50℃in an oil bath. Cooled to room temperature, DIPEA and MMT-Cl were added under ice bath, and stirring was continued at room temperature overnight. The majority of the organic solvent was removed by rotary evaporation, ethyl acetate and water were added to the residual solid, the mixture was separated, extracted, the reaction solution was washed with water (40 ml, ph=4.5) in this order, saturated brine was washed, the separated solution was dried over anhydrous sodium sulfate, the obtained organic phase was evaporated to dryness under reduced pressure to obtain a crude product, and then, petroleum ether/ethyl acetate=10/1 was used for beating to obtain Fmoc-Lys (MMT) -OH pale yellow solid (8.0 g, yield: 83%).
Synthesis of Compounds 1-3
Fmoc-Lys (MMT) -OH (5.5 g,8.59 mmol) was dissolved in dichloromethane (40 mL), cooled to 0deg.C, p-aminobenzyl alcohol (1.27 g,10.31 mmol) and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline EEDQ (3.82 g,15.4 mmol) were added dropwise under nitrogen protection and the reaction stirred for 18 hours. The reaction solution was diluted with dichloromethane (50 mL), washed with water (40 mL), separated, the remaining aqueous phase was extracted with dichloromethane (30 ml×3), the organic phase was dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure to give a crude product, and then subjected to basic silica gel column separation (eluent: petroleum ether/ethyl acetate=1/10 to 1/2) to give Fmoc-Lys (MMT) -PABOH as a pale yellow solid (4.4 g, yield: 69%).
Synthesis of Compounds 1-4
Fmoc-Lys (MMT) -PABOH (3.9 g,5.23 mmol) was dissolved in tetrahydrofuran (35 mL) and stirred at room temperature. 1, 8-diazabicyclo undec-7-ene (DBU) (0.8 g,5.23 mmol) was dissolved in tetrahydrofuran (5 mL), and then added dropwise to the reaction solution, followed by stirring for 30min. TLC monitored complete consumption of the reaction. Spin-drying under reduced pressure, followed by column separation on basic silica gel (eluent: petroleum ether/ethyl acetate=1/1-0/1, then methanol/ethyl acetate from=0/1-1/20) afforded compound H-Lys (MMT) -PABOH as a white solid (2.5 g, yield: 92%).
Synthesis of Compounds 1-5
H-Lys (MMT) -PABC (2.5 g,4.78 mmol) was dissolved in dichloromethane (35 mL), cooled to 0deg.C, monomethyl fumarate (0.62 g,4.78 mmol) and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ) (1.3 g,5.26 mmol) were added dropwise under nitrogen and stirred at room temperature for 18 hours. TLC monitored completion of the original reaction. The reaction mixture was diluted with dichloromethane (30 mL), washed with water (40 mL), separated, the aqueous phase was extracted with dichloromethane (25 mL. Times.3), the organic phases combined and dried over anhydrous sodium sulfate, and spun-dried to give the crude product. The crude product was separated by basic silica gel column (eluent: petroleum ether/ethyl acetate=2/1-0/1, then methanol/dichloromethane=0/1-1/20) to give the compound methyl-fumaic-Lys (MMT) -PABC as pale yellow oily product (2.43 g, yield: 80%).
Synthesis of Compounds 1-6
Into a three-necked flask was charged TBS-SN 38 (0.78 g,1.56 mmol), 4-Dimethylaminopyridine (DMAP) (0.56 g,4.68 mmol) and a solution of triphosgene (BTC) (0.18 g,0.62 mmol) in DCM (5 mL) under nitrogen was slowly added and stirred for 30min. TLC monitored complete reaction of starting material (reaction quenched with methanol). A solution of methyl-fumaic-Lys (MMT) -PABC (1.07 g,1.68 mmol) in DCM (5 mL) was added dropwise to the reaction and stirred at room temperature for 30min. TLC monitored completion of methyl-fumaic-Lys (MMT) -PABC reaction. After dilution of the reaction with DCM (20 mL), saturated NaHCO was added 3 The solution (40 mL) was washed and then separated, the aqueous phase extracted with DCM (30 mL x 2) and the organic phases combined. The organic phase was washed with saturated NaCl solution (40 mL) and then with anhydrous Na 2 SO 4 And (5) drying. And (5) performing reduced pressure spin drying to obtain a crude product. The crude product is subjected to alkaliAfter column separation on silica gel (eluent: petroleum ether/ethyl acetate=5/1-1/2), methyl-full-Lys (MMT) -PABC-SN 38 (TBS) was obtained as a pale yellow solid (1.44 g, yield: 79%).
Synthesis of Compounds 1-7
Compounds 1-6 (500 mg,0.428 mmol) were dissolved in dichloromethane (35 mL), cooled to 0deg.C, acOH (128 mg,2.14 mmol) and tetrabutylammonium fluoride (TBAF) (1.07 mL,1.07mmol,1.0 mmol/mL) were added sequentially under nitrogen and stirred at room temperature for 30min. TLC monitored completion of the original reaction. The reaction mixture was diluted with dichloromethane (30 mL), and the organic phase was washed with water (40 mL), saturated brine, washed, separated, dried over anhydrous sodium sulfate, and spun-dried to give crude compound 1-7 as pale yellow product (400 mg).
Synthesis of Compound S1
Compounds 1-7 (400 mg, trude) were dissolved in dichloromethane (35 mL), cooled to 0deg.C, anisole (410 mg,3.79 mmol) and dichloroacetic acid (DCA) (1.90 mmol,245 mg) were added sequentially under nitrogen and stirred at room temperature for 30min. TLC monitored completion of the original reaction. The reaction mixture was diluted with methylene chloride (30 mL), and the organic phase was washed with water (40 mL), saturated brine, washed, separated, dried over anhydrous sodium sulfate, and spin-dried to give crude product methyl-fumaric-Lys-PABC-SN 38 (S1) as pale yellow product (300 mg, yield: 77%).
When fumaric acid is substituted for trans-4- (maleimidomethyl) cyclohexanecarboxylic acid and the coupled drug molecule is selected to be irinotecan, compound S15 can be obtained.
Example 2
Synthesis of Compound S2
Synthesis of Compound 2-1
H-Lys (MMT) -PABC (2.5 g,4.78 mmol) was dissolved in dichloromethane (35 mL), cooled to 0deg.C and N was added dropwise under nitrogen protection 3 PEG-COOH (0.97 g,4.78 mmol) and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ) (1.3 g,5.26 mmol), stirred at room temperature for 18 hours. TLC monitored completion of the original reaction. The reaction mixture was diluted with dichloromethane (30 mL), washed with water (40 mL), separated, the aqueous phase was extracted with dichloromethane (25 mL. Times.3), the organic phases combined and dried over anhydrous sodium sulfate, and spun-dried to give the crude product. Separating the crude product by an alkaline silica gel column (eluent: petroleum ether/ethyl acetate=2/1-0/1, then methanol/dichloromethane=0/1-1/20) to obtain a compound N 3 -PEG-Lys (MMT) -PABC was the product (3.2 g, yield: 85%) as a pale yellow oil.
Synthesis of Compound 2-2
Into a three-necked flask, TBS-SN 38 (0.78 g,1.56 mmol), 4-Dimethylaminopyridine (DMAP) (0.56 g,4.68 mmol) and DCM (30 mL) were added slowly a solution of triphosgene (BTC) (0.18 g,0.62 mmol) in DCM (5 mL) under nitrogen and stirred for 30min. TLC monitored complete reaction of starting material (reaction quenched with methanol). Will N 3 A solution of (Ac-Lys (MMT) -PABC (1.2 g,1.68 mmol) in DCM (5 mL) was added dropwise to the reaction and stirred at room temperature for 30min. TLC monitoring N 3 -PEG-Lys (MMT) -PABC reacted completely. After dilution of the reaction with DCM (20 mL), saturated NaHCO was added 3 The solution (40 mL) was washed and then separated, the aqueous phase extracted with DCM (30 mL. Times.2) and the organic phases combined. The organic phase was washed with saturated NaCl solution (40 mL) and then with anhydrous Na 2 SO 4 And (5) drying. And (5) performing reduced pressure spin drying to obtain a crude product. Separating the crude product by an alkaline silica gel column (eluent: petroleum ether/ethyl acetate=5/1-1/2)Obtaining N 3 -Ac-Lys (MMT) -PABC-SN 38 (TBS) as a pale yellow solid (1.7 g, yield: 80%).
Synthesis of Compound 2-3
Will N 3 -Ac-Lys (MMT) -PABC-SN 38 (TBS) (500 mg,0.439 mmol) was dissolved in dichloromethane (35 mL), cooled to 0deg.C, acOH (128 mg,2.14 mmol) and tetrabutylammonium fluoride (TBAF) (1.07 mL,1.07mmol,1.0 mmol/mL) were added sequentially under nitrogen and stirred at room temperature for 30min. TLC monitored completion of the original reaction. The reaction mixture was diluted with dichloromethane (30 mL), and the organic phase was washed with water (40 mL), saturated brine, washed, separated, dried over anhydrous sodium sulfate, and spun-dried to give crude compound 2-3 as pale yellow product (400 mg, crude).
Synthesis of Compounds 2-6
Compound 2-5 (350 mg,0.52 mmol) was dissolved in DMSO (8 mL) and H was slowly added dropwise 2 O until just no turbidity was produced, then a solution of Compound 2-3 (295 mg,0.26 mmol) in DMSO (2 mL) was added to the reaction solution, cuBr (67.4 mg,0.47 mmol) was added with stirring and reacted at room temperature for 2 hours, LCMS showed complete reaction of the starting material. The reaction mixture was diluted with DCM (30 mL), washed with saturated aqueous NaCl solution (20 mL), the insoluble material was filtered off, the aqueous phase was extracted three times with DCM (20 mL), and the organic phases were combined with anhydrous Na 2 SO 4 Drying and rotary evaporation of the solvent gave the crude product which was separated by a basic silica gel column (eluent: DCM/meoh=1/0-20/1) to give the product as a yellow solid (220 mg, yield: 46.7%) as compounds 2-6.
Synthesis of Compound S2
Compounds 2-6 (220 mg,0.173 mmol) were dissolved in dichloromethane (6 mL) and DCA (78.4 mg,0.61 mmol) and anisole (132.3 mg,1.22 mmol) were slowly added to the solution and the reaction was stirred at room temperature for 2 hours. TLC monitored reaction was complete. After the solvent was distilled off under reduced pressure, methyl tert-butyl ether (40 mL) was added to the obtained oily product to fix the product. The solidified product was washed with methyl tert-butyl ether (30 ml x 3) and filtered, and the solid product was dried under reduced pressure to give compound S2 as a pale yellow solid (104 mg, yield: 60%).
Example 3
Synthesis of Compound S3
Compound S2 (50 mg,0.05 mmol) was dissolved in DMF (2 mL), acOSu (8 mg,0.06 mmol) and DIPEA (13 mg,0.1 mmol) were slowly added to the solution and the reaction was stirred at room temperature for 2 hours. TLC monitored reaction was complete. After the solvent was distilled off under reduced pressure, methyl tert-butyl ether (40 mL) was added to the obtained oily product to fix the product. The solidified product was washed with methyl tert-butyl ether (30 ml x 3) and filtered, and the solid product was dried under reduced pressure to give compound S3 as a pale yellow solid (40 mg, yield: 80%).
Example 4
Synthesis of Compound S5
Synthesis of Compound 5a
Compound 2-5 (350 mg,0.52 mmol) was dissolved in DMSO (8 mL) and H was slowly added dropwise 2 O until just no turbidity was generated, then a solution of Compound 3-c (293 mg,0.26 mmol) in DMSO (2 mL) was added to the reaction solution, cuBr (67.4 mg,0.47 mmol) was added with stirring, and reacted at room temperature for 2 hoursLCMS showed complete reaction of starting material. The reaction mixture was diluted with DCM (30 mL), washed with saturated aqueous NaCl solution (20 mL), the insoluble material was filtered off, the aqueous phase was extracted three times with DCM (20 mL), and the organic phases were combined with anhydrous Na 2 SO 4 Drying and rotary evaporation removed the solvent to give the crude product, which was separated by basic silica gel column (eluent: DCM/meoh=1/0-20/1) to give the product as a yellow solid (220 mg, yield: 61%) as compound 5a.
Synthesis of Compound S5
Compound 5a (234 mg,0.170 mmol) was dissolved in dichloromethane (6 mL) and DCA (78.4 mg,0.61 mmol) and anisole (132.3 mg,1.22 mmol) were slowly added to the solution and the reaction was stirred at room temperature for 2 hours. TLC monitored reaction was complete. After the solvent was distilled off under reduced pressure, methyl tert-butyl ether (40 mL) was added to the obtained oily product to fix the product. The solidified product was washed with methyl tert-butyl ether (30 ml x 3) and filtered, and the solid product was dried under reduced pressure to give compound S5 as a pale yellow solid (100 mg, yield: 55%).
Example 5
Synthesis of Compound S7
Synthesis of Compound 3-2
DBU (361 mg,2.38 mmol) was added to a solution of Compound 1 (2.4 g,4.75 mmol) in tetrahydrofuran (12 mL) and stirred at room temperature for 2 hours. TLC monitoring showed complete reaction (iodine fumigation was required). The reaction mixture was dried by spin-drying and then separated on a silica gel column (eluent: DCM/meoh=1/0 to 20/1) to give compound 3-2 as a pale yellow oily product (1.2 g, yield: 89.2%).
Synthesis of Compound 3-3
DiPEA (455 mg,3.54 mmol) was added to a solution of Compound 2 (500 mg,1.77 mmol) and EMC-OSu (544 mg,1.77 mmol) in dichloromethane (6 mL) and the reaction was stirred at room temperature for 18 hours. TLC monitoring showed the reaction was complete. The reaction was diluted with dichloromethane (20 mL) and saturated NaHCO was added 3 Aqueous (20 mL) was washed, the solution was separated, the remaining aqueous phase was extracted with dichloromethane (15 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to give the crude product, which was then separated on a silica gel column (eluent: DCM/MeOH=1/0-30/1) to give compound 3-3 as a white solid (780 mg, yield: 93%).
Synthesis of Compounds 3-4
HCl (819. Mu.L) was slowly added dropwise to a solution of Compound 3 (780 mg,1.64 mmol) in hexafluoroisopropanol (15 mL) and the reaction was stirred at room temperature for 1 hour. TLC monitored reaction was complete. The reaction mixture was dried under reduced pressure, dissolved in DCM (30 mL) and taken up in Na 2 SO 4 The water was dried and then the solvent was removed under reduced pressure. The product was precipitated by addition of methyl tert-butyl ether (20 mL), the solid product was washed with methyl tert-butyl ether (15 mL. Times.3) after filtration, and compound 3-4 was obtained as a pale yellow product (1 g, 620mg in theory) after filtration, and the crude product was used directly in the next reaction.
Synthesis of Compound 3-5
Compound 3-4 (1 g, converted to 620mg,1.65mmol in theoretical yield) and PEG 5 OSu (805 mg,1.98 mmol) was dissolved in dichloromethane (10 mL) and after the addition of DiPEA (638 mg, 4.95 mmol) the solution was clear and stirred at room temperature for 4 hours. TLC monitored reaction was complete. ReactionThe solution was diluted with dichloromethane (20 mL) and saturated NaHCO was added 3 Aqueous (20 mL) was washed, the solution was separated, the remaining aqueous phase was extracted with dichloromethane (15 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, and evaporated under reduced pressure to give the crude product, which was then separated on a silica gel column (eluent: DCM/MeOH=1/0-30/1) to give the compound 3-5 as a colorless oily product (700 mg, total yield of two-step reaction: 64%).
Synthesis of Compounds 3-6
Compound 3-5 (350 mg,0.52 mmol) was dissolved in DMSO (8 mL) and H was slowly added dropwise 2 O until just no turbidity was produced, then a solution of Compound 2-3 (295 mg,0.26 mmol) in DMSO (2 mL) was added to the reaction solution, cuBr (67.4 mg,0.47 mmol) was added with stirring and reacted at room temperature for 2 hours, LCMS showed complete reaction of the starting material. The reaction mixture was diluted with DCM (30 mL), washed with saturated aqueous NaCl solution (20 mL), the insoluble material was filtered off, the aqueous phase was extracted three times with DCM (20 mL), and the organic phases were combined with anhydrous Na 2 SO 4 Drying and rotary evaporation of the solvent gave the crude product which was separated by a basic silica gel column (eluent: DCM/meoh=1/0-20/1) to give the product as a yellow solid (220 mg, yield: 46.7%) as compound 3-6.
Synthesis of Compound S7
Compounds 3-6 (220 mg,0.173 mmol) were dissolved in dichloromethane (6 mL) and DCA (78.4 mg,0.61 mmol) and anisole (132.3 mg,1.22 mmol) were slowly added to the solution and the reaction was stirred at room temperature for 2 hours. TLC monitored reaction was complete. After the solvent was distilled off under reduced pressure, methyl tert-butyl ether (40 mL) was added to the obtained oily product to fix the product. The solidified product was washed with methyl tert-butyl ether (30 ml x 3) and filtered, and the solid product was dried under reduced pressure to give compound S7 as a pale yellow solid (104 mg, yield: 60%).
Example 6
Synthesis of Compound S13
Synthesis of Compound 2-2a
A solution of Irinotecan (0.78 g,1.56 mmol), 4-Dimethylaminopyridine (DMAP) (0.56 g,4.68 mmol) and DCM (30 mL) in DCM (5 mL) was slowly added under nitrogen and stirred for 30min. TLC monitored complete reaction of starting material (reaction quenched with methanol). Will N 3 A solution of (Ac-Lys (MMT) -PABC (1.2 g,1.68 mmol) in DCM (5 mL) was added dropwise to the reaction and stirred at room temperature for 30min. TLC monitoring N 3 -PEG-Lys (MMT) -PABC reacted completely. After dilution of the reaction with DCM (20 mL), saturated NaHCO was added 3 The solution (40 mL) was washed and then separated, the aqueous phase extracted with DCM (30 ml x 2) and the organic phases combined. The organic phase was washed with saturated NaCl solution (40 mL) and then with anhydrous Na 2 SO 4 And (5) drying. And (5) performing reduced pressure spin drying to obtain a crude product. Separating the crude product by an alkaline silica gel column (eluent: petroleum ether/ethyl acetate=5/1-1/2) to obtain N 3 -Ac-Lys (MMT) -PABC-SN 38 (TBS) as a pale yellow solid (1.7 g, yield: 80%).
Synthesis of Compound 3-6a
Compound 3-5 (350 mg,0.52 mmol) was dissolved in DMSO (8 mL) and H was slowly added dropwise 2 O until just no turbidity was produced, then a solution of Compound 2-3 (295 mg,0.26 mmol) in DMSO (2 mL) was added to the reaction solution, cuBr (67.4 mg,0.47 mmol) was added with stirring and reacted at room temperature for 2 hours, LCMS showed complete reaction of the starting material. The reaction mixture was diluted with DCM (30 mL), washed with saturated aqueous NaCl solution (20 mL), and insoluble was filtered offThe mixture was separated, the aqueous phase was extracted three times with DCM (20 mL), and the organic phases were combined with anhydrous Na 2 SO 4 Drying and rotary evaporation of the solvent gave the crude product which was separated by a basic silica gel column (eluent: DCM/meoh=1/0-20/1) to give the product as a yellow solid (220 mg, yield: 46.7%) as compound 3-6a.
Synthesis of Compound S13
Compound 3-6a (220 mg,0.173 mmol) was dissolved in dichloromethane (6 mL), DCA (78.4 mg,0.61 mmol) and anisole (132.3 mg,1.22 mmol) were slowly added to the solution and the reaction was stirred at room temperature for 2 hours. TLC monitored reaction was complete. After the solvent was distilled off under reduced pressure, methyl tert-butyl ether (40 mL) was added to the obtained oily product to fix the product. The solidified product was washed with methyl tert-butyl ether (30 ml x 3) and filtered, and the solid product was dried under reduced pressure to give compound S13 as a pale yellow solid (104 mg, yield: 60%).
Example 7
Synthesis of Compound S10
/>
Synthesis of Compound 3-a
H-Lys (MMT) -PABC (2.5 g,4.78 mmol) was dissolved in dichloromethane (35 mL), cooled to 0deg.C and N was added dropwise under nitrogen protection 3 PEG-COOH (0.97 g,4.78 mmol) and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ) (1.3 g,5.26 mmol), stirred at room temperature for 18 hours. TLC monitoring of original reverseShould be complete. The reaction mixture was diluted with dichloromethane (30 mL), washed with water (40 mL), separated, the aqueous phase was extracted with dichloromethane (25 mL. Times.3), the organic phases combined and dried over anhydrous sodium sulfate, and spun-dried to give the crude product. Separating the crude product with alkaline silica gel column (eluent: petroleum ether/ethyl acetate=2/1-0/1, then methanol/dichloromethane=0/1-1/20) to obtain compound N 3 -PEG-Lys (MMT) -PABC was the product (3.2 g, yield: 85%) as a pale yellow oil.
Synthesis of Compound 3-b
A solution of TBS-SN 38 (0.78 g,1..566 mmol), 4-Dimethylaminopyridine (DMAP) (0.56 g,4.68 mmol) and triphosgene (BTC) (0.18 g,0.62 mmol) in DCM (5 mL) under nitrogen was slowly added to the flask and stirred for 30min. TLC monitored complete reaction of starting material (reaction quenched with methanol). Will N 3 A solution of (PEG) -Lys (MMT) -PABC (1.2 g,1.68 mmol) in DCM (5 mL) was added dropwise to the reaction and stirred at room temperature for 30min. TLC monitoring N 3 -PEG-Lys (MMT) -PABC reacted completely. After dilution of the reaction with DCM (20 mL), saturated NaHCO was added 3 The solution (40 mL) was washed and then separated, the aqueous phase extracted with DCM (30 mL. Times.2) and the organic phases combined. The organic phase was washed with saturated NaCl solution (40 mL) and then with anhydrous Na 2 SO 4 And (5) drying. And (5) performing reduced pressure spin drying to obtain a crude product. The crude product was subjected to basic silica gel column separation (eluent: petroleum ether/ethyl acetate=5/1-1/2) to give compound 3-b as a pale yellow solid (1.7 g, yield: 80%).
Synthesis of Compound 3-c
Will N 3 PEG-Lys (MMT) -PABC-SN 38 (TBS) (500 mg,0.403 mmol) was dissolved in dichloromethane (35 mL), cooled to 0deg.C, acOH (128 mg,2.14 mmol) and tetrabutylammonium fluoride (TBAF) (1.07 mL,1.07mmol,1.0 mmol/mL) were added sequentially under nitrogen and stirred at room temperature for 30min. TLC monitoring of the originalThe reaction was complete. The reaction mixture was diluted with dichloromethane (30 mL), and the organic phase was washed with water (40 mL), saturated brine, washed, separated, dried over anhydrous sodium sulfate, and spun-dried to give crude compound 3-c as a pale yellow product (400 mg, crude).
Synthesis of Compounds 3-6
Compound 3-c (350 mg,0.52 mmol) was dissolved in DMSO (8 mL) and H was slowly added dropwise 2 O until just no turbidity is generated, then N 3 A solution of-PEG-Lys (MMT) -PABC-SN 38 (295 mg,0.26 mmol) in DMSO (2 mL) was added to the reaction solution, cuBr (67.4 mg,0.47 mmol) was added with stirring and reacted at room temperature for 2 hours, LCMS showed complete reaction of the starting material. The reaction mixture was diluted with DCM (30 mL), washed with saturated aqueous NaCl solution (20 mL), the insoluble material was filtered off, the aqueous phase was extracted three times with DCM (20 mL), and the organic phases were combined with anhydrous Na 2 SO 4 Drying and rotary evaporation removed the solvent to give the crude product, which was separated by basic silica gel column (eluent: DCM/meoh=1/0-20/1) to give the product as a yellow solid (220 mg, yield: 46.7%) as compound 3-6.
Synthesis of Compound S10
Compounds 3-6 (220 mg,0.12 mmol) were dissolved in dichloromethane (6 mL) and DCA (78.4 mg,0.61 mmol) and anisole (132.3 mg,1.22 mmol) were slowly added to the solution and the reaction was stirred at room temperature for 2 hours. TLC monitored reaction was complete. After the solvent was distilled off under reduced pressure, methyl tert-butyl ether (40 mL) was added to the obtained oily product to solidify the product. The solidified product was washed with methyl tert-butyl ether (30 ml x 3) and filtered, and the solid product was dried under reduced pressure to give compound S7 as a pale yellow solid (128 mg, yield: 70%).
N of the compound PEGn-OSu is any value from 1 to 20, and is the compound S10 when n is 5, and is the compound S11 when n is 8.
LC-MS identification of synthesized compounds
Instrument: agilent 1290-LC-MS
Chromatographic column: waters CORTECS C18, 50X 4.6mm,2.7 μm
Mobile phase: a:0.1% TFA/H2O; b: acetonitrile
Flow rate: 1.0mL/min
Column temperature: 30 ℃,
the results of the identification are shown in Table 1 below
TABLE 1
Example 8
Solubility comparison of the Compounds S2, S3, S5, S7, S10, S11 prepared according to the invention with the control Compound SN38 and irinotecan
The freeze-dried products S2, S3, S7 and S11 prepared by the invention are packaged in a sterile room and are re-dissolved by water for injection. Can be completely dissolved as shown in table 2.
TABLE 2
Compounds of formula (I) Water for injection
SN38 (active metabolite) Is insoluble in water
Irinotecan Is dissolved in water
S2 Dissolving, 2mg/ml
S3 Dissolving, 0.5mg/ml
S5 Dissolving, 5mg/ml
S7 Dissolving, 5mg/ml
S10 Dissolving, 20mg/ml
S11 Dissolving, 25mg/ml
As can be seen from Table 2, the solubility of the modified compounds is greatly improved relative to the active metabolite SN38, and the drugs can be directly dissolved to high concentration by using water for injection or physiological saline without using an organic solvent which is required for dissolving the control compound. The change of dissolution characteristics has a great influence on both the metabolism of the drug distribution and the mode of action of the drug.
Example 9
Solution stability comparison of drug conjugates prepared according to the examples of the present invention with control Compounds
The compounds S5, S7, S10 and S11 are respectively and accurately weighed, 5.0mg of each sample is respectively packaged in a sterile room, 0.5ml of sterilized injection water is added to prepare 10mg/ml of mother solution, and meanwhile, the control compound irinotecan is weighed and dissolved in the injection water. 30ul of the mother solution was taken and 570ul of buffer solution having pH of 5.5 was added to prepare 0.5mg/ml of sample solution. After the sample was clarified, it was placed in a 25deg.C/37deg.C water bath, and after 0 h, samples were taken and tested by HPLC (Agilent 1260, column: eclipse Plus C18,4.6 x 250mm,5 μm, A:0.1% TFA+H2O, B: CH3CN, flow rate: 1.0ml/min, column temperature: 30deg.C, test wavelength: 214 nm) for 0 h, respectively, to obtain solution stability data for the different compounds. The results are shown in Table 3.
Table 3: solution stability data
Compounds of formula (I) 8hr
Irinotecan 95.1％
S5 95.9％
S7 98.3％
S10 98.2％
S11 99.8％
From the data in table 3 above, the modified compound (conjugate) showed a slight increase in S5 relative to irinotecan at 25 ℃ and ph=5.5, whereas S7, S10 and S11 were more pronounced.
Example 10
Drug efficacy study of different drug conjugates S7, S10 and S11 in human fibrosarcoma at the same dosage in the invention
1. Animals: nude mice, 6-8 weeks old, were all females (Shanghai Schlenk laboratory animal Co., ltd.).
2. Generating tumor models
1) Corresponding cells were purchased from American type culture Collection (American type culture collection, ATCC) and identified according to the instructions provided by ATCC, and the cells were cultured using modified eagle's medium (DMEM medium for short) containing 10% fetal bovine serum Dalberg (R) at 37℃in 5% carbon dioxide. Cells were used within 15 passages once every 3 days.
2) Tumor production, 5X 10 6 Subcutaneous injection of corresponding cells to the back of nude mice until tumor grows to 100mm 3 The left and right time are randomly grouped, and treatment is started, so that the day of treatment is the first day.
3) Treatment process
The dose of 1/3MTD (20 mg/Kg) was used, once a week for 3 weeks.
4) Grouping and result measurements are shown in Table 4
Table 4: effect of corresponding compound and control on tumor inhibition
5) Results and discussion: the treatment effects of S7, S10 and S11 are greatly improved compared with the original medicines, and the comprehensive comparison of S11 can almost achieve the effect of curing tumors.
EXAMPLE 11 study of the efficacy of different drug conjugates S7, S10 and S11 at equivalent doses in the present invention in human pancreatic cancer cell models
1. Animals: nude mice, 6-8 weeks old, were all females (Shanghai Schlenk laboratory animal Co., ltd.).
2. Generating tumor models
1) Corresponding cells were purchased from American type culture Collection (American type culture collection, ATCC) and identified according to the instructions provided by ATCC, and the cells were cultured using modified eagle's medium (DMEM medium for short) containing 10% fetal bovine serum Dalberg (R) at 37℃in 5% carbon dioxide. Cells were used within 15 passages once every 3 days.
2) Tumor production, 5X 10 6 Subcutaneous injection of corresponding cells to the back of nude mice until tumor grows to 100mm 3 The left and right time are randomly grouped, and treatment is started, so that the day of treatment is the first day.
3) Treatment process
The dose of 1/3MTD (20 mg/Kg) was used, once a week for 3 weeks.
4) Grouping and result measurements are shown in Table 5
Table 5: effect of corresponding compound and control on tumor inhibition
5) Results and discussion: the comparison shows that S7, S10 and S11 are better than irinotecan in treatment effect, so that the drug conjugate in the experimental group can effectively release active drugs, and the treatment effect of the drug S11 is better than that of other experimental groups by combining water solubility and stability data, so that tumors can be cured.
Example 12
S11 drug efficacy study in multiple tumor models
The purpose of the experiment is as follows: the broad spectrum of the antitumor drugs of S11 is known from the multi-tumor model of mice.
Therapeutic drug: s11, diluting the solution to corresponding concentration by using normal saline in the experiment.
Method and results:
1. animals: nude mice, 6-8 weeks old, were all females. (Shanghai Laike laboratory animal Co., ltd.)
2. Generating tumor models
1) Corresponding cells were purchased from American type culture Collection (American type culture collection, ATCC) and identified according to the instructions provided by ATCC, and the cells were cultured using modified eagle medium (DMEM medium for short) containing 10% fetal bovine serum at 37℃and 5% CO 2. Cells were used within 15 passages once every 3 days.
2) Tumor production, 5 x 106 corresponding cells were subcutaneously injected into the backs of nude mice, and when tumors were up to about 100mm3, the tumors were randomly grouped and treatment was started, with the day of treatment starting being the first day.
3) During the treatment, the solution S11 is injected into the tail vein, and the control group is normal saline and is administrated once a week for three weeks.
Table 6: therapeutic effect of drug S11 in multiple tumor model
The result shows that the drug S11 has good drug effect in various tumor models, and the drug can be used as a broad-spectrum tumor therapeutic drug.
The embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims (10)

1. A drug conjugate of formula I:
R-AA-PABC-D
(I)
wherein:
r is a targeting group, and can also carry out certain modification on the characteristics of the medicine, or can be used for regulating the water solubility of the medicine and the activation efficiency in the tumor microenvironment;
PABC represents a p-aminobenzyl alcohol group;
AA represents a group of an amino acid;
d represents a drug molecule.
2. The drug conjugate of claim 1, wherein R isOr R represents +.R 1 and R2 formed by click reaction>
3. The drug conjugate of claim 2, wherein R 1 Selected from:
R 2 selected from the group consisting of compounds containing:
wherein n is an integer of 1 to 10.
4. The drug conjugate of claim 1, wherein R is selected from the group consisting of:
wherein m and n are integers of 1 to 10, respectively.
5. The drug conjugate of claim 1, wherein AA is selected from the group consisting of lysine, acetyllysine, or lysine-glycine.
6. The drug conjugate of claim 1, wherein the drug is selected from any one of camptothecins, SN38, irinotecan, derivatives of irinotecan, topotecan, 9-aminocamptothecin, 9-nitrocamptothecin, paclitaxel, docetaxel, rapamycin, everolimus, daunorubicin, epirubicin, doxorubicin, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitoxantrone, imiquimod, rassimol, luoli bine.
7. A drug conjugate selected from the group consisting of:
8. a pharmaceutical composition comprising the pharmaceutical conjugate of any one of claims 1-7 and a pharmaceutically acceptable excipient.
9. Use of a drug conjugate according to any one of claims 1-7 for the preparation of a medicament for the treatment of cancer.
10. The use of claim 9, wherein the cancer comprises bladder cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and leukemia.
CN202210819270.XA 2022-07-12 2022-07-12 Drug conjugate and application thereof Pending CN117427176A (en)

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