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WO2015196944A1 - Conjugué de molécule analogue de cytotoxique de gnrh ainsi que son procédé de préparation et son utilisation - Google Patents

Conjugué de molécule analogue de cytotoxique de gnrh ainsi que son procédé de préparation et son utilisation Download PDF

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Publication number
WO2015196944A1
WO2015196944A1 PCT/CN2015/081781 CN2015081781W WO2015196944A1 WO 2015196944 A1 WO2015196944 A1 WO 2015196944A1 CN 2015081781 W CN2015081781 W CN 2015081781W WO 2015196944 A1 WO2015196944 A1 WO 2015196944A1
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cancer
aph
cells
ser
leu
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Chinese (zh)
Inventor
刘克良
周宁
马永涛
王晨宏
冯思良
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Institute of Pharmacology and Toxicology of AMMS
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Institute of Pharmacology and Toxicology of AMMS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides

Definitions

  • the invention belongs to the field of medicine and chemistry, and particularly relates to a GnRH analogue-cytotoxic molecule conjugate, a preparation method thereof and use thereof for preparing a medicament for preventing and/or treating a tumor.
  • Gonadotropin-releasing hormone also known as luteinizing hormone releasing hormone (LHRH)
  • LHRH luteinizing hormone releasing hormone
  • GnRH is a decapeptide hormone secreted by the hypothalamus in a pulsed form (Glp–His–Trp–Ser–Tyr–Gly–Leu–Arg–Pro–Gly) ⁇ NH 2 ), a key molecule that regulates the reproductive system. It is produced by neurons in the hypothalamus and reaches the pituitary through the portal vein. In the pituitary, GnRH activates its receptor GPCR, a seven-fold transmembrane G-protein coupled receptor, which in turn promotes the synthesis and secretion of LH (luteinizing hormone) and FSH (follicle stimulating hormone).
  • GnRH also plays a role in the hypothalamus and other organs outside the pituitary. GnRH receptors are found in uterus, ovaries, fallopian tubes, placenta, breast, prostate, peripheral blood mononuclear cells, colon, rectal and renal tumor cells, as well as in lymphoma and melanoma. Even the receptor for GnRH is found in the digestive system. In addition, the GnRH receptor has a limited distribution in normal tissues, which makes it possible to use the GnRH receptor as a receptor for targeted administration of antitumor drugs and to use GnRH analogs as targeting groups.
  • a GnRH analogue as a targeting group, through a degradable linker such as a disulfide bond, an ester bond, a hydrazone bond, etc., with cytotoxic molecules such as: doxorubicin, daunorubicin, paclitaxel, docetaxel Race, methotrexate, camptothecin, etc. are directly conjugated, enter the tumor cells in a receptor-mediated manner, and then release in the specific microenvironment inside the tumor cells (acidic environment and high expression of specific enzymes) Free cytotoxic molecules are released, ultimately inhibiting tumor growth.
  • cytotoxic molecules such as: doxorubicin, daunorubicin, paclitaxel, docetaxel Race, methotrexate, camptothecin, etc.
  • a first aspect of the invention relates to a compound of the formula (I), a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic,
  • Aaa 1 is L or D type Glp or Nal
  • Aaa 2 is an L or D type His or Cpa
  • Aaa 3 is L or D type Trp or Pal
  • Aaa 5 is the L or D-Tyr or Aph (L-Hor);
  • Aaa 6 is Lys or Aph of L or D type
  • Aaa 8 is L or D type Arg or ILys
  • Aaa 10 is L or D type Gly or Ala;
  • X is selected from the group consisting of H, Ac, -ab, -Xaa 1 -Xaa 2 -ab;
  • Y is selected from the group consisting of H, Cbm, -ab, -Xaa 1 -Xaa 2 -ab;
  • Xaa 1 and Xaa 2 are each independently a hydrophilic amino acid
  • a is linked to the peptide chain via an amide bond (the carboxyl group of a forms an amide bond with the amino group on the peptide chain).
  • the small molecule-modified paclitaxel b forms a disulfide bond by a disulfide bond (eg, a disulfide bond of b2, b4 is exchanged with a sulfhydryl group on a) or a thioether bond (eg, b1,
  • a disulfide bond eg, a disulfide bond of b2, b4 is exchanged with a sulfhydryl group on a
  • a thioether bond eg, b1
  • the maleimide on b3 is nucleophilically substituted with a thiol group on a to form a thioether bond
  • the -Xaa 1 -Xaa 2 for example, a peptide fragment is selected from L or D form, -Arg-Gln, -Gln-Arg , -Arg-Arg, -Gln-Gln, -Arg -Ser, -Arg-Asp.
  • the -Xaa 1 -Xaa 2 is bonded to a through an amide bond.
  • the a and b are linked by a thioether bond or a disulfide bond.
  • -Xaa 1 -Xaa 2 is bonded to the polypeptide chain by forming an amide bond with an ⁇ -amino group of Aaa 1 or a side chain amino group of Aaa 6 .
  • Xaa 1 in -Xaa 1 -Xaa 2 is located at the C-terminus.
  • X is bonded to Aaa 1 by substituting H on the ⁇ -amino group of Aaa 1 .
  • Y is bonded to Aaa 6 by substituting H on the side chain amino group of Aaa 6 .
  • R1-R19 corresponds to a modifying group as shown in Table 1.
  • the above 1-58 number is the compound number, which may represent the compound.
  • the amino acid is an L-form amino acid unless otherwise specified.
  • a second aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound according to any one of the first aspects of the invention, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic, and a pharmaceutically acceptable carrier or Shape agent.
  • a third aspect of the invention relates to a process for the preparation of a compound according to any one of the first aspects of the invention, a stereoisomer thereof, a solvate thereof or a salt thereof which is not physiologically toxic, comprising the steps of:
  • 6-amino acid modification According to the solid phase synthesis Boc, Fmoc cross-protection strategy, the peptide sequence was synthesized from the C-terminus to the N-terminus to the 6-position amino acid, and the 20-50% piperidine/DMF solution was removed from the 6-position amino acid side chain amino group.
  • the Fmoc protecting group is added with a modifying unit (p-methylbenzyl-protected mercaptopropionic acid), DIC, HOBT, and the ninhydrin is detected after 4 hours of reaction.
  • N-terminal modification including simultaneous modification of N-terminal and 6-position amino acids: synthesis of peptide sequence from C-terminus to N-terminus to 6-position amino acid or N-terminus according to solid phase synthesis Boc and Fmoc cross-protection strategy, 20-50% piperidine
  • the acyl/DMF solution was used to remove the Fmoc protecting group on the amino acid side chain amino acid of the 6-position amino acid, and the modified unit (p-methylbenzyl-protected mercaptopropionic acid), DIC, HOBT was added. After the reaction for 4 hours, the ninhydrin was detected. After the reaction was completed, the reaction was completed.
  • the peptide resin is lysed with anhydrous HF, dissolved in 10-30% aqueous acetic acid solution, and lyophilized to obtain crude peptide, which is purified by medium pressure chromatography. ;
  • a fourth aspect of the invention relates to the use of a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for the manufacture of a medicament for the prevention and/or treatment of a tumor.
  • the tumor comprises, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; chorion Cancer; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; Leukemia; liver cancer; lung cancer (eg small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lip , tongue, mouth and pharynx; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testi
  • a fifth aspect of the invention relates to a method of preventing and/or treating a tumor comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a compound according to any one of the first aspects of the invention, a stereoisomer thereof Or a step of its physiologically toxic salt.
  • the tumor comprises, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; chorion Cancer; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; Leukemia; liver cancer; lung cancer (eg small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lip , tongue, mouth and pharynx; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; test
  • the subject is a mammal, such as a bovine, an equine, a sheep, a porcine, a canine, a feline Animals, rodents, primates; wherein the preferred subject is a human.
  • a sixth aspect of the invention relates to a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for use in the prevention and/or treatment of a tumor.
  • composition according to any one of the sixth aspects of the present invention, or a stereoisomer thereof, or a physiologically toxic salt thereof wherein the tumor includes, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer ; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; Gastric cancer; neoplasms in epithelial cells; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (eg, small cells and non-small cells); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; Myeloma; neuroblastoma; oral cancer (eg, lips, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; reti
  • a seventh aspect of the invention relates to the use of a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for the preparation of a reagent for killing and/or killing tumor cells.
  • the agent is for use in an in vitro method.
  • the agent is for use in an in vivo method.
  • the tumor cell is a tumor cell line, or a tumor cell from a subject.
  • the tumor cells include, but are not limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; bone cancer cells; brain and CNS cancer cells; Cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; esophageal cancer cells; Gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Hodgkin's and non-Hodgkin's lymphoma cells; Tumor cell Tumor cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; pancreatic cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells; Cancer cells of the respiratory system; sar
  • An eighth aspect of the invention relates to a method of killing and/or killing a tumor cell, the method comprising administering to the cell an effective amount of a compound according to any one of the first aspects of the invention, a stereoisomer thereof or no physiological toxicity thereof Salt.
  • the method is carried out in vitro.
  • the method is carried out in vivo.
  • the tumor cell is a tumor cell line, or a tumor cell from a subject.
  • the tumor cells include, but are not limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; bone cancer cells; brain and CNS cancer cells; Cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; esophageal cancer cells; Gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Hodgkin's and non-Hodgkin's lymphoma cells; Tumor cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ovarian cancer cells; pancreatic cancer cells; prostate cancer cells; retinal tumor cells; rhabdomyosarcoma cells Rectal cancer cells;
  • a ninth aspect of the invention relates to a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof for use in killing and/or killing tumor cells.
  • the invention is used in an in vitro method.
  • the invention is used in an in vivo method.
  • the tumor cell is a tumor cell line, or a tumor cell from a subject.
  • the tumor cells include, but are not limited to, skin basal cancer cells, biliary cancer cells; bladder cancer cells; Bone cancer cells; brain and CNS cancer cells; breast cancer cells; cervical cancer cells; choriocarcinoma cells; colon and rectal cancer cells; connective tissue cancer cells; cancer cells of the digestive system; cancer cells of the endometrium; esophageal cancer cells ; eye cancer cells; cancer cells in the head and neck; gastric cancer cells; renal cancer cells; laryngeal cancer cells; leukemia cells; liver cancer cells; lung cancer cells (eg, small cells and non-small cells); lymphoma cells including Hodgkin And non-Hodgkin's lymphoma cells; melanoma cells; myeloma cells; neuroblastoma cells; oral cancer cells (eg, lips, tongue, mouth and pharyngeal cancer cells); ova
  • the invention also relates to a kit for killing and/or killing tumor cells, the kit comprising a compound according to any one of the first aspects of the invention, a stereoisomer thereof or a non-physiologically toxic salt thereof, And, optionally, instructions for use are also included.
  • the tumor includes, but is not limited to, cutaneous basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer ; cancer of the digestive system; cancer of the endometrium; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; neoplasms in epithelial cells; kidney cancer; laryngeal cancer; leukemia; liver cancer; And non-small cells; lymphomas include Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (eg, lips, tongue, mouth and pharynx); ovarian cancer Pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; testicular cancer; thyroid
  • the compound of the first aspect of the present invention maintains a good GnRH receptor binding activity and has strong tumor cell growth inhibitory activity; and has certain tumor cell selectivity and stability. A substantial increase is beneficial to reduce the side effects of drugs caused by premature release of anti-cancer factors.
  • the compounds of the present invention retain the GnRH receptor agonistic and antagonistic activities of their ligands, respectively, and may play a synergistic therapeutic role for sex hormone-dependent tumors (e.g., prostate cancer, endometrial cancer, breast cancer, etc.).
  • Figure 7 is a graph of acceptor saturation of conjugate 20
  • a side chain amino group means an amino group other than the alpha amino group of the amino acid
  • Ninhydrin refers to ninhydrin indicator reagent:
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient an effective amount of at least one of the formula (I) and/or its stereoisomers or its non-physiologically toxic salts, as well as conventional pharmaceutical excipients or adjuvants.
  • compositions as used herein includes any or all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and sustained release agents, and similar physiologically compatible formulations, Suitable for intravenous, intramuscular, subcutaneous, or other modes of administration, such as oral administration. Depending on the mode of administration, the active compound may be coated to protect the compound from the effects of acid or other natural conditions.
  • physiologically toxic salt refers to a salt or a combination thereof which retains the expected physiological activity of the parent compound without causing any unexpected toxic side effects.
  • the cation according to the salt may be an inorganic salt such as a potassium salt, a lithium salt, a zinc salt, a copper salt, a cerium salt, a cerium salt or a calcium salt, and may also be an organic salt such as a trialkylammonium salt.
  • the formula (I) of the present invention and its stereoisomers, solvates, non-physiological salts or pharmaceutical compositions containing the same may be administered in any manner known per se, such as oral, intramuscular, subcutaneous, etc., for example, for administration of a dosage form, for example Tablets, capsules, buccal tablets, chewable tablets, tinctures, suspensions, transdermal agents, microcapsules, implants, syrups, and the like. It may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems. In order to form a unit dosage form into tablets, various biodegradable or biocompatible carriers well known in the art can be widely used.
  • carrier examples are, for example, saline-based and various buffered aqueous solutions, ethanol or other polyols, liposomes, polylactic acid, vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and the like.
  • the dose of the compound of the formula (I), a stereoisomer thereof, a solvate thereof or a non-physiologically acceptable salt thereof or a pharmaceutical composition containing the same according to the present invention depends on many factors such as the disease to be prevented or treated. Nature and severity, the sex, age, weight, sensitivity and individual response of the patient or animal, the particular compound employed, the route of administration, the number of doses, and the desired therapeutic effect.
  • the above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.
  • the single maximum dose will generally not exceed 30 mg/kg body weight, such as 0.001-30 mg/Kg, preferably 0.01-5 mg/Kg, with a preferred dosage range of 0.5-2 mg/Kg body weight. However, in some cases, it is also possible to use a single dose of 30 mg/kg or more or 0.001 mg/kg or less.
  • the solid phase synthesis carrier MBHA resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; DIC, HOBT and Boc-protected or Fmoc-protected natural amino acids are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Kaitai New Technology Co., Ltd., TFA, DIEA is a product of ACROS, and Boc- or Fmoc-protected non-natural amino acids are provided by our laboratory, except for instructions.
  • MBHA resin (0.54mmol) was used as the solid phase carrier, and DIC/HOBt was used as the condensing agent.
  • the amino acid sequence of the compound all amino acids and modifying units were sequentially connected according to the Boc and Fmoc cross-protection strategy, and the above peptide resin was placed in HF.
  • 1.0 mL of anisole and 0.5 mL of ethanedithiol were added. After the installation, the system of the HF cutter was evacuated, and the reactor was cooled with liquid nitrogen, and transferred to about 10 mL of liquid HF at 0. °C reaction for 1h.
  • the HF was pumped off with an oil pump, the reactor was removed, solidified by adding anhydrous diethyl ether, and the suspension was transferred to a sand core funnel. It was washed three times with a small amount of cooled anhydrous diethyl ether, and then washed with a 10% aqueous solution of acetic acid until the resin no longer adhered to each other, and the washing liquid was collected, and lyophilized to obtain a white flocculent solid.
  • the conjugates were synthesized in this way: 1, 2, 3, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, 21, 22, 23, 26, 27, 28 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 43, 44, 46, 47, 48, 50, 51, 52, 55, 56, 57.
  • the equimolar amount of the peptide sequence was weighed out with the paclitaxel derivative b2 or b4 in DMSO, and the reaction was stirred at room temperature. After the reaction was completed, the pure product was purified by medium pressure preparative chromatography.
  • the conjugates were synthesized in this way: 4, 8, 12, 16, 20, 24, 25, 29, 33, 37, 41, 45, 49, 53, 54, 58.
  • Example 2 The mass spectrometric characterization data of Example 2 and Example 3 is shown in Table 2.
  • Tumor cell growth inhibitory activity assay method cells in logarithmic growth phase (human breast cancer cell MCF-7 and human colorectal cancer cell line HT-29) were digested, inoculated into a 96-well plate, and the medium was diluted to a cell suspension density. 3 ⁇ 10 4 cells/mL, inoculated at 100 uL per well, and incubated in a cell culture incubator for 24 h.
  • Paclitaxel and conjugate were weighed separately and dissolved in DMSO to prepare 10 -3 mol/L mother liquor.
  • the medium was diluted to 2000 nmol/L, 400 nmol/L, 80 nmol/L, 16 nmol/L, 3.2 nmol/ L, a medium solution of a concentration of 0.64 nmol/L.
  • CHO-K1/G ⁇ 15 cells stably expressing GnRHr were cultured by intracellular calcium ion fluorescence detection technique, and cultured in a 37° C./5% CO 2 incubator.
  • the collected cell suspension was inoculated into a 384-well plate at a density of 20,000 cells per well, and then placed in a 37 ° C / 5% CO 2 incubator for at least 18 h, adding dye, each well 20 ul, then, 10 ul of the configured compound solution was added to the cell plate, and then the cell plate was incubated in a 37 ° C / 5% CO 2 incubator for 1 hour, and finally equilibrated at room temperature for 15 min. Then, by FLIPR detection, for each detection well, the average fluorescence intensity value of 1-20 s is used as a baseline, and the maximum fluorescence intensity value of 21 to 120 s minus the baseline value is the relative fluorescence intensity value.
  • % agonism ⁇ 1 - ( ⁇ RFU compound - ⁇ RFU background ) / ( ⁇ RFU reference agonist - ⁇ RFU background ) ⁇ ⁇ 100
  • % inhibition rate ⁇ 1 - ( ⁇ RFU compound - ⁇ RFU background ) / ( ⁇ RFU reference antagonist - ⁇ RFU background ) ⁇ ⁇ 100
  • the EC 50 or IC 50 was calculated by S curve fitting, and the measurement results are shown in Table 3.
  • IC 50 9.53 ⁇ 0.63 nmol / L;
  • Tumor cell growth inhibitory activity results showed that the conjugate showed good tumor cell growth inhibitory activity in both cells (human breast cancer cells (MCF-7) and human colorectal cancer cells (HT-29)). Individual compounds are even comparable to paclitaxel.
  • the receptor binding activity of the conjugate showed that the conjugates maintained a higher binding activity to the GnRH receptor, indicating that the conjugate has the potential to mediate into the tumor cells via the GnRH receptor.
  • Plasma stability determination method 10 ⁇ L of DMSO solution (12.5 mg/mL) to be tested, added to human plasma 2 mL, vortexed evenly, incubate in a 37 ° C constant temperature oscillator, sample 200 ⁇ L at different time points, and terminate the reaction with three volumes of acetonitrile; High-speed centrifugation (12000 r / min) for 10 min, the supernatant was taken for HPLC analysis, and the prototype retention was calculated from the peak area.
  • the cell selectivity of the conjugate was investigated indirectly by cell receptor saturation assay.
  • the experimental method GnRH receptor-positive MCF-7 cells were used as subjects, and they were divided into two groups, one of which was added with different concentrations of Degarelix.
  • the medium solution was pretreated, and the other group was added to the blank medium, and after incubating for 1 hour in a 37 ° C 5% CO 2 incubator, the medium was aspirated, and then the drug solution of paclitaxel or conjugate was added to examine it in two The difference in growth inhibitory activity of the group of cells.

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Abstract

La présente invention concerne un conjugué de molécule analogue de cytotoxique de la GnRH tel que représenté par la formule suivante (I) ainsi qu'un de ses procédés de préparation et une de ses utilisations dans la préparation de médicaments de traitement de tumeurs. La molécule cytotoxique est un dérivé du taxol ou un dérivé du docétaxel. Le conjugué peut maintenir l'activité de liaison aux récepteurs de la GnRH et présente une stabilité de plasma relativement bonne ainsi qu'une activité d'inhibition de croissance sur des cellules tumorales. (X)Aaa1Aaa2-Aaa3-Ser-Aaa5-Aaa6(Y)-Leu-Aaa8-Pro-Aaa10-NH2
PCT/CN2015/081781 2014-06-26 2015-06-18 Conjugué de molécule analogue de cytotoxique de gnrh ainsi que son procédé de préparation et son utilisation Ceased WO2015196944A1 (fr)

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CN201410293274.4 2014-06-26
CN201410293274.4A CN105198966B (zh) 2014-06-26 2014-06-26 GnRH类似物-细胞毒分子缀合物、其制备方法及用途

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EP4051309A4 (fr) * 2019-10-31 2023-11-08 Worcester Polytechnic Institute Conjugués lhrh-paclitaxel et leurs procédés d'utilisation
WO2025001852A1 (fr) * 2023-06-25 2025-01-02 南京安吉生物科技有限公司 Conjugué polypeptidique, composition pharmaceutique contenant un conjugué polypeptidique et utilisation associée

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CN108187063B (zh) * 2018-01-09 2020-09-08 沈阳药科大学 白蛋白结合型抗肿瘤药-马来酰亚胺分子前药
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WO2025001852A1 (fr) * 2023-06-25 2025-01-02 南京安吉生物科技有限公司 Conjugué polypeptidique, composition pharmaceutique contenant un conjugué polypeptidique et utilisation associée

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