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WO2016070712A1 - 氨基酸衍生物及其应用 - Google Patents

氨基酸衍生物及其应用 Download PDF

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Publication number
WO2016070712A1
WO2016070712A1 PCT/CN2015/092451 CN2015092451W WO2016070712A1 WO 2016070712 A1 WO2016070712 A1 WO 2016070712A1 CN 2015092451 W CN2015092451 W CN 2015092451W WO 2016070712 A1 WO2016070712 A1 WO 2016070712A1
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amino acid
cancer
group
acid derivative
compound
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PCT/CN2015/092451
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English (en)
French (fr)
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臧仕宁
范恒梅
朱伟
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广州德汇行医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/56—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having carbon atoms of carboxamide groups bound to carbon atoms of carboxyl groups, e.g. oxamides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups

Definitions

  • This invention relates to amino acid derivatives and their use.
  • the diagnosis and treatment of tumors is a hot spot in existing medical research.
  • the treatment of tumors mainly includes surgery and medication.
  • Surgical treatment is mainly applied to tumors that have not metastasized.
  • the surgical results vary according to the tumor, and are more restricted, and the damage to normal humans is greater.
  • Chemotherapy is mainly based on chemotherapy, and well-known, chemotherapy drugs have a strong killing effect on normal cells, and side effects are large, and their use is also limited.
  • Another object of the present invention is to provide an application of the above amino acid derivative for the preparation of an antitumor drug.
  • Another object of the present invention is to provide an antitumor drug.
  • X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;
  • R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;
  • Y, Y 1 , Y 2 are independently polypeptides
  • the amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;
  • the amino acid derivative does not include And other compounds which have been disclosed to meet the above formula.
  • R, R' are independently selected from the group consisting of glutamic acid, glutamine, citrulline, ornithine, cystine, aspartic acid, asparagine, threonine, serine ,leucine,isoleucine,glycine,cysteine,methionine,tryptophan,tyrosine,phenylalanine,valine,lysine, Side chain substituents of hydroxylysine, hydroxyproline, valine, lysine, histidine, and simple modifications thereof.
  • Y, Y 1 and Y 2 are independently 2 to 12 peptides.
  • Y, Y 1 and Y 2 are independently 2 to 12 tumor targeting peptides.
  • the pharmaceutically acceptable salt of the amino acid derivative is selected from the group consisting of sodium, potassium, calcium, zinc, lithium, iron, magnesium salt, sulfonate;
  • the pharmaceutically acceptable ester of the amino acid derivative is selected from the group consisting of C2 ⁇ The ester between C4;
  • the pharmaceutically acceptable ether of the amino acid derivative is selected from the group consisting of an ether between C2 and C4.
  • amino acid derivatives in the preparation of antitumor drugs, the structural formula of amino acid derivatives is as shown in Formulas I to VI,
  • X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;
  • R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;
  • Y, Y 1 , Y 2 are independently polypeptides
  • the amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;
  • the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, lymphocytic leukemia. , osteosarcoma, lymphosarcoma.
  • An antitumor drug whose active ingredient is at least one of the amino acid derivatives shown above.
  • the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, Lymphocytic leukemia, osteosarcoma, lymphosarcoma.
  • the synthesis process of amino acid derivatives includes the following synthetic routes:
  • X is selected from the group consisting of an oxalyl group and a simple modification thereof, or a residue obtained by coupling a compound containing an oxalyl group with an amino acid, and a simple modification thereof.
  • the amino acid derivative of the invention can target lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin.
  • Lactate degeneration, citrate synthase, ⁇ -ketoglutarate dehydrogenase complex protein activity in malignant tumor tissues such as cancer, granulocyte leukemia, lymphocytic leukemia, osteosarcoma, lymphosarcoma, etc., specifically interfere with tumor cell production ATP destroys its energy metabolism, induces tumor cell death and apoptosis, enhances the inhibition of cancer cell proliferation, growth, metastasis, etc. It has high-efficiency and low-toxic anti-tumor pharmacodynamic activity, and has high energy consumption and high consumption. All kinds of malignant tumors have better killing or inhibition effects.
  • the amino acid derivative of the present invention has good water solubility and is easy to prepare into an injection preparation.
  • the amino acid derivative of the invention has simple synthesis process, low cost, easy purification and industrial production.
  • Figure 1 is a 1 H NMR spectrum of Compound 2
  • Figure 2 is a 13 C NMR spectrum of Compound 2
  • Figure 3 is a 1 H NMR spectrum of Compound 3
  • Figure 4 is a 13 C NMR spectrum of Compound 3;
  • FIG. 5 is a 1 H NMR spectrum of Compound 4.
  • Figure 6 is a 13 C NMR spectrum of Compound 4.
  • Figure 8 is a 13 C NMR spectrum of Compound 6;
  • Figure 10 is a 13 C NMR spectrum of Compound 7;
  • FIG. 11 is a 1 H NMR spectrum of Compound 8.
  • Figure 12 is a 13 C NMR spectrum of Compound 8.
  • Figures 13 to 18 are in vitro antitumor profiles of different compounds
  • Figure 19 is the effect of different compounds on the resting metabolic energy of animals
  • Figure 20 is the effect of different compounds on ATP content in animal transplanted tumor tissues
  • Figures 21 to 31 show the tumor inhibition rate of different compounds in different tumor models in animals.
  • X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;
  • R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;
  • Y, Y 1 , Y 2 are independently polypeptides
  • the amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;
  • the amino acid derivative does not include And other compounds which have been disclosed to meet the above formula.
  • a simple modification of a group refers to a simple substitution or addition of a group without changing the structure of the main body of the group, such as replacement of one or more of H by a hydroxyl group; or halogenation, Or esterification and etherification with a small molecule of C2 to C4; when the group has a carboxyl group or an amine group, the carboxyl group or the amine group is amidated.
  • the amino acid derivative of the present invention may be either L-form or D-form.
  • the structure of L-form is closer to the structure of natural amino acids, but it is also easily metabolized in vivo, with shorter half-life and shorter onset time; D-amino acid derivatives can also be identified and better combined in vivo. It is more difficult to be metabolized, has a longer half-life, and has a longer onset of action.
  • Different amino acid derivatives can be selected as antitumor active ingredients as needed.
  • the amino acid derivative when the amino acid derivative is Or a pharmaceutically acceptable salt, ester, ether or amide thereof, the amino acid derivative can be formulated into an oral preparation and has excellent antitumor activity.
  • Tumor cell targeting polypeptide (2-10 peptide) has a targeted distribution in tumor tissues, which can be better located in tumor cells and taken up by tumor cells, and is more prone to pharmaceutically active.
  • Y, Y 1 and Y 2 in the above structural formula are independently 2 to 12 peptides, and further, Y, Y 1 and Y 2 are independently 2 to 10 peptides, 2 to 8 peptides, 2 to 7 peptides, 2 to 4 peptides. More preferably, Y, Y 1 and Y 2 are independently 2 to 12 tumor targeting peptides. Further, Y, Y 1 and Y 2 are independently 2 to 10, 2 to 8, 2 to 7 , 2 to 4 tumor targeting peptides, especially those which have been confirmed to be more easily taken up by tumor cells and used for protein synthesis. Peptide.
  • the pharmaceutically acceptable salt of the amino acid derivative is selected from the group consisting of sodium, potassium, calcium, zinc, lithium, iron, magnesium salt, sulfonate;
  • the pharmaceutically acceptable ester of the amino acid derivative is selected from the group consisting of C2 ⁇ The ester between C4;
  • the pharmaceutically acceptable ether of the amino acid derivative is selected from the group consisting of an ether between C2 and C4.
  • amino acid derivatives in the preparation of antitumor drugs, the structural formula of amino acid derivatives is as shown in Formulas I to VI,
  • X is selected from the group consisting of oxalyl and simple modifications thereof, or a residue after coupling reaction of an oxalyl-containing compound with an amino acid, and a simple modification thereof;
  • R or R' is independently selected from the group consisting of an L-type amino acid constituting a human protein, or a side chain substituent of a D-type amino acid, and a simple modification thereof, or a side chain substituent constituting an intermediate of natural amino acid metabolism in various human bodies;
  • Y, Y 1 , Y 2 are independently polypeptides
  • the amino acid derivative further includes a pharmaceutically acceptable salt, ester or ether thereof;
  • amino acid derivative is And its simple modifications.
  • the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, lymphocytic leukemia. , osteosarcoma, lymphosarcoma.
  • An antitumor drug whose active ingredient is at least one of the amino acid derivatives shown above.
  • the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, esophageal cancer, gastric cancer, gallbladder cancer, ovarian cancer, nasopharyngeal cancer, tongue cancer, skin cancer, breast cancer, prostate cancer, melanoma, skin cancer, granulocyte leukemia, Lymphocytic leukemia, osteosarcoma, lymphosarcoma.
  • the synthesis process of amino acid derivatives includes the following synthetic routes:
  • X is selected from the group consisting of an oxalyl group and a simple modification thereof, or a residue obtained by coupling a compound containing an oxalyl group with an amino acid, and a simple modification thereof.
  • the corresponding solid phase synthesis can be carried out on the basis of the above synthesis steps.
  • the amino acid derivative of the present invention has a simple structure, it can also be directly entrusted to an existing compound synthesis company for synthesis.
  • the metabolism of tumor cells is vigorous, and a large amount of amino acids, small molecules for energy supply, etc. are required during the growth process.
  • the amino acid derivative of the present invention acts on tumor cells, a large amount of targeted accumulation in tumor tissues and tumor cells is observed. Aggregation, which can feedbackly inhibit the activity of citrate synthase and ⁇ -ketoglutarate dehydrogenase complex protein in tumor tissues through the mechanism of elevated substrate levels (the two enzymes are rate-limiting enzymes for the Krebs cycle)
  • it can specifically interfere with the tumor cell mitochondrial complex IV cytochrome c oxidase complex to produce ATP, thereby inducing tumor cell death and apoptosis, and enhancing the inhibition of cancer cell proliferation, growth, metastasis, etc.
  • the metabolic rate is significantly lower than that of tumor cells, and the amino acid derivative of the present invention is less ingested, and the energy metabolism and substance synthesis of the cells are also less affected, and there is no obvious killing effect. Therefore, the amino acid derivative of the present invention
  • the amino acid derivative of the invention has the effect of inhibiting and killing tumors from tumors, and can be applied to the treatment of tumors at various stages, and can also be combined with existing anti-tumor drugs to better anti-tumor. effect.
  • the amino acid derivatives of the present invention can also be used in combination to block the energy metabolism of the tumor as much as possible to achieve a better anti-tumor effect.
  • the white solid insoluble solid was filtered off, and the organic phase was washed once with 5% aqueous NaHCO 3 solution, saturated brine, 10% aqueous KHSO 4 and brine, and the organic phase was collected, dried and concentrated to give a viscous compound 2
  • the yield was 90% and the HPLC purity was 95%.
  • the nuclear magnetic data of Compound 2 is:
  • the nuclear magnetic data of Compound 3 is:
  • the nuclear magnetic data of the compound 4 is: 1 H NMR (400 Hz, CD 3 OD) ⁇ : 4.47-4.54 (m, 1H), 2.39-2.46 (m, 2H), 2.01-2.33 (m, 2H), the hydrogen spectrum is as Figure 5;
  • the white solid insoluble solid was filtered off, and the organic phase was washed once with 5% aqueous NaHCO 3 solution, saturated brine, 10% aqueous KHSO 4 and brine, and the organic phase was collected, dried and concentrated to give viscous compound 6
  • the yield was 88% and the HPLC purity was 96%.
  • the nuclear magnetic data of Compound 6 is:
  • the nuclear magnetic data of Compound 7 is:
  • the nuclear magnetic data of Compound 8 is:
  • L-929 cells were supplemented with 10% fetal bovine serum and penicillin (100 units), streptomycin in a gaseous environment of 5% ⁇ 1% carbon dioxide at 37 ⁇ 1 ° C and in a humidified environment containing tissue culture flasks containing the recommended media. 100 ⁇ g) and other antibiotics (Invitrogen, USA) cell culture medium (Invitrogen, USA) were cultured to achieve the cell density required for the experiment. The monolayer cells were rinsed twice with PBS, the PBS was aspirated, and digested in a 0.25% trypsin/EDTA, 37 ⁇ 1 °C tissue culture flask until the cells were separated and floated.
  • Test drug Confluent single layer percentage Intracellular granule Dissolve level reaction 100 ⁇ M Compound 2 no ⁇ 50% Have no 2 mild 100 ⁇ M compound 3 no ⁇ 50% Have no 2 mild 100 ⁇ M Compound 4 Have ⁇ 20% Have no 1 slight 100 ⁇ M Compound 6 Have 0% Have no 0 no response 100 ⁇ M Compound 7 Have 0% Have no 0 no response 100 ⁇ M Compound 8 Have 0% Have no 0 no response Cisplatin no >70% no no 4 severe
  • Negative control (NS) Have 0% Have no 0 no response
  • Blank control (solvent) Have 0% Have no 0 no response
  • Toxicity of compounds 2, 3, 4, 6, 7, and 8 to L929 cells showed that 5% DMEM dilution at 100 ⁇ M, 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, and 6.25 ⁇ M was applied to L929 cells over 24 hours. No significant cytotoxicity was observed at a maximum concentration of 100 ⁇ M.
  • Drugs 2, 3, 4, 6, 7, 8, control cisplatin human lung cancer H460, H1650, SPC-A-1, A549 cell line, liver cancer HepG2 cell line, breast cancer MCF-7 cell line, colon cancer HT -29 cell line, prostate cancer PC-3, DU145 cell line, cervical cancer Hela cell line, gastric cancer MGC-803 cell line, DMEM medium, 10% FBS calf serum, MTT reagent, DMSO, 96-well plate, enzyme label Instrument and so on.
  • Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) were synthesized from the applicant's laboratory.
  • the number of cells is 4 ⁇ 10 3 /L, and 100 uL per well;
  • RESULTS Compounds 2, 3, 4, 6, 7, and 8 were diluted in 5% DMEM at 0.5, 2.5, 5.0, 10, 25, 50, and 100 ⁇ M for in vitro lung cancer H460, H1650, and SPC-A-1. , A549 cells, liver cancer HepG2 cells, breast cancer MCF-7 cells, colon cancer HT-29 cells, prostate cancer PC-3, DU145 cells, cervical cancer Hela cells, gastric cancer MGC-803 cells, observed after 24 hours, observed compound 2 The 50% inhibition rate (IC 50 ) of the above cells at 3, 4, 6, 7, and 8.
  • Compounds 2, 3, 4, 6, 7, and 8 act on lung cancer H460, H1650, SPC-A-1, A549 cells, liver cancer HepG2 cells, breast cancer MCF-7 cells, colon cancer HT-29 cells, and prostate cancer PC, respectively.
  • -3 DU145 cells, cervical cancer Hela cells, gastric cancer MGC-803 cells, after 24 hours incubation, MTT assay for anti-tumor activity found that compounds 2, 3, 4, 6, 7, 8 have certain anti-tumor effects (respectively See Figures 13 to 18), but the anti-tumor activity of the amino acid derivative in vitro is not high compared to the positive control group.
  • SPF grade KM mice were housed in an independent ventilation system (IVC) facility in an SPF laboratory animal chamber. Group raising, free drinking water, eating, laboratory temperature is 20 ⁇ 25 ° C, humidity is 40 ⁇ 70%. Animal room conditions are always stable to ensure the reliability of the test results.
  • Feed and drinking water The feed is SPF grade granular rat material purchased from Guangdong Medical Laboratory Animal Center. Nutrition and sanitation meet the requirements of SPF laboratory animals. Drinking water is free to ingest.
  • Clinically used route oral administration.
  • Pre-test data After a single oral administration of the compound 8 (20140304) synthesized by the applicant at the maximum dose, that is, the maximum concentration of 1800. mg/kg ⁇ bw, the mortality rate of the mice was 0%.
  • Grouping of animals 140 animals with quarantine and weight requirements were randomly divided into 14 groups according to body weight and sex, 10 in each group, half male and half female. Preparation of test drug: Weighed compound 8 freeze-dried powder injection, dissolved in physiological saline. Route and number of administration: single oral administration. Dosing volume: 10 ml/kg. Fasting time of animals: fasting for 6 to 12 hours before administration and fasting for 4 hours after administration.
  • Observation and examination General observation: Immediately after administration, observe the reaction of the animals, and continue to observe for 4 hours, and then observe once every morning for 14 days, record the performance and characteristics of the animal poisoning, the time of occurrence of toxicity, and the disappearance time. And the time of death of the animal.
  • Body weight measurement before administration (d 1 ), 24 hours after administration (d 2 ), day 4 (d 4 ), day 8 (d 8 ), day 11 (d 11 ), and day 15 (d 15 ) Weigh the animal's body weight.
  • Pathological examination poisoning death or sudden death of animals in time for necropsy, other animals at the end of the observation period after necropsy, when the tissue and organ changes in volume, color, texture, etc., the pathological organization of the changed tissue and organs Learn to check.
  • Anatomical organs include thymus, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), lymph nodes, bladder, prostate, testis ( With epididymis), uterus, ovaries.
  • Observation indicators detailed records of animal reactions, including appearance, behavioral activities, mental state, appetite, urine and color, fur, skin color, breathing, nose, eyes, oral cavity with abnormal secretions, weight changes and death.
  • mice did not die. After the gavage, the mice contracted and did not like the activity. The analysis was caused by the excessive concentration of the drug and the excessive volume of the gavage. It indicates that the toxicity of compound 8 is low, and the maximum tolerated dose of mice is about 1800 mg/kg, which is equivalent to 512 times of the clinically recommended dose.
  • RESULTS The CCM metabolites produced by MEDGRAPHIC of the United States were used to allow the nude mice to rest without stimulation before the test. The measurements were taken after 30 minutes of calm (the measured indicators were O 2 , CO 2 and exhaled breath in inhalation and exhalation, according to The inspiratory and exhaled nitrogen concentrations are constant, and the inspiratory volume is calculated. The oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) are combined, and the total nitrogen output (24) is combined with the 24-hour urine.
  • the ambient humidity during the test is 50-65%, the temperature is 18-24 ° C, the atmospheric pressure is 101 ⁇ 102.4 kPa (758-768 mmHg), and these data are input into the computer for instrument calibration.
  • the energy consumption is calculated according to the formula 3.94 ⁇ VO 2 + 1.11 ⁇ VCO 2 - 2.17 ⁇ N.
  • REE Resting Energy Expenditure, total daily energy consumption: kJ/d
  • Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) were synthesized from the applicant's laboratory. As can be seen from Fig. 19, on the 10th day and the 14th day after administration, the model group and the cisplatin group and the compounds 2, 3, 4, 6, 7, and 8 groups can significantly reduce the energy metabolism of the tumor tissue.
  • the ATP (ATP Assay Kit) was used to detect ATP (adenosine 5'-triphosphate) levels in tumor-bearing nude mice.
  • ATP adenosine 5'-triphosphate
  • the fluorescence is required to be developed by ATP.
  • the production of fluorescence is proportional to the concentration of ATP in a certain concentration range. This makes it possible to detect the ATP concentration in the solution with high sensitivity.
  • the test kit used can detect ATP at concentrations as low as 5 nmol/L.
  • the concentration of ATP in conventional cell or tissue lysates is only 0.1-1 ⁇ mol/L, and the intracellular ATP level of some common cells is about 10 nmol/mg protein.
  • the concentration of ATP in a cell or tissue is detected, the ATP concentration can be determined by lysing the lysate provided by the kit.
  • Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) were synthesized from the applicant's laboratory.
  • the effects of compounds 2, 3, 4, 6, 7, and 8 on ATP content in tumor-bearing nude mice A549 xenografts are shown in Figure 20.
  • the model group and the cisplatin group and the compounds 2, 3, 4, 6, 7, and 8 all significantly reduced the ATP content of the tumor tissue, but compared with the cisplatin group.
  • Compounds 2, 4, and 6 have significant differences in the inhibition of ATP production.
  • mice Healthy Balb-c nude mice, male and female, about 4 weeks old, weighing 18-22g, were purchased from Guangdong Medical Laboratory Animal Center. Nude mice with a tumor diameter of about 0.5-1.0 cm were taken for 2-3 weeks after inoculation for the experiment.
  • mice 70 healthy Balb-c nude mice, male, 4 to 5 weeks old, weighing 18-22 g.
  • the anterior right axilla was subcutaneously and observed after 1-2 weeks of inoculation.
  • the tumor formation rate was 98.6%, and nude mice with tumor growth of about 0.5 ⁇ 0.5 cm were selected for the experiment.
  • Model nude mice bearing a tumor volume of approximately 0.5 ⁇ 0.5 cm were randomly assigned, Compound 2 (20140701), Compound 3 (20140506), Compound 4 (20140305), Compound 6 (20140508), Compound 7 (20140602), Compound 8 (20140304) Synthesis from the inventor's laboratory. Therefore, the tumor-bearing animals were divided into eight groups, a model group, a positive control group, a compound 2 group, a compound 3 group, a compound 4 group, a compound 6 group, a compound 7 group, and a compound 8 group, and 8 animals in each group.
  • Model group equal volume of normal saline; positive control group: 10 ⁇ M/kg (cisplatin solution); compound 2 group: 50 ⁇ M/kg; compound 3 group: 50 ⁇ M/kg; compound 4 group: 50 ⁇ M/kg; compound 6 group: 50 ⁇ M /kg; Compound 7 group: 50 ⁇ M/kg; Compound 8 group: 50 ⁇ M/kg; each group was administered intraperitoneally (ip) according to 0.1 ml/10 g.
  • Dosing daily for 14 consecutive days All were administered by intraperitoneal injection (ip), weighed before daily administration, and the amount of each dose was determined according to the daily body weight. Weigh the next day and measure the tumor volume. On the 15th day, the animals were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor were taken out, weighed separately, and stored in 10% formaldehyde.
  • V 1/2 ⁇ a ⁇ b 2 (a, b respectively indicate the length and width of the tumor)
  • Tumor weight inhibition rate (1 - average tumor weight of the administration group / average tumor weight of the control group) ⁇ 100%
  • Tumor volume inhibition rate (1 - (volume before administration - volume after administration) / (volume before administration - volume after administration)) ⁇ 100%.
  • Compound 3 and Compound 8 were P>0.05 as compared with the positive control drug cisplatin, i.e., the efficacy of Compound 8 and Compound 2 against lung cancer H1650 proliferation was comparable to that of cisplatin.
  • Compounds 2 and 6 were P>0.05 as compared with the positive control drug cisplatin, i.e., the potency of Compounds 2 and 6 against lung cancer H460 proliferation was comparable to that of cisplatin.
  • Compounds 2, 3 and 7 were P>0.05 compared with the positive control drug cisplatin, i.e., the effects of Compounds 2, 3 and 7 against lung cancer A549 proliferation were comparable to those of cisplatin.

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Abstract

本发明公开了氨基酸衍生物及其应用，氨基酸衍生物含有草酰基，该氨基酸衍生物可干扰细胞的能量代谢，诱导细胞凋亡，具有广泛的应用。

Description

氨基酸衍生物及其应用 技术领域
本发明涉及氨基酸衍生物及其应用。
背景技术
20世纪后期近30年以来癌症发病一直呈上升的趋势，据世界卫生组织(WHO)报告，1990年全球癌症新发病例数约807万，比1975年的517万增加了37.4％。而1997年全球的癌症死亡数约620万，并且按目前的趋势预测，至2020年随着世界人口达80亿，将有2000万新发癌症病例，其中死亡人数将达1200万，且其中绝大部分将发生在发展中国家。
肿瘤严重危害人们的健康，给患者和社会带来巨大的经济负担。有效地对肿瘤进行诊断和治疗具有非常实际的意义，肿瘤的诊断和治疗是现有医学研究的热点。
肿瘤的种类繁多，特性不一，对药物的反应也不一，这导致肿瘤的治疗是极为困难的。现阶段，肿瘤的治疗主要有手术法和药物治疗。手术治疗主要应用于适用于未发生转移的肿瘤，手术效果依肿瘤的不同而有所不同，受限较多，对正常人体的损害较大。药物治疗以化疗为主，而周知的，化疗药物对正常细胞也具有较强的杀伤作用，副作用大，其使用也受到限制。
不管是手术治疗还是药物治疗，对于发生转移的恶性肿瘤都是无能为力的。开发出对各种肿瘤，特别是对发生了转移的肿瘤都具有较好杀伤作用的抗肿瘤药物具有非常实际的意义。
发明内容
本发明的一个目的在于提供一类氨基酸衍生物。
本发明的另一个目的在于提供上述氨基酸衍生物在制备抗肿瘤药物中的应用。
本发明的另一个目的在于提供一种抗肿瘤药物。
本发明所采取的技术方案是：
氨基酸衍生物，其结构通式如式Ⅰ～Ⅵ所示，
Figure PCTCN2015092451-appb-000001
式中：
X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物；
R或R'独立选自组成人体蛋白质的L-型氨基酸，或D-型氨基酸的侧链取代基及其简单修饰物，或构成各种人体内天然氨基酸代谢的中间体的侧链取代基；
Y、Y1、Y2独立为多肽；
所述氨基酸衍生物还包括其药用盐、酯或醚；
所述氨基酸衍生物不包括
Figure PCTCN2015092451-appb-000002
及其他已经公开的符合上述通式的化合物。
作为上述氨基酸衍生物的进一步改进，R、R’独立选自谷氨酸、谷氨酰胺、瓜氨酸、鸟氨酸、胱氨酸、天冬氨酸、天冬酰胺、苏氨酸、丝氨酸、亮氨酸、异亮氨酸、甘氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、苯丙氨酸、缬氨酸、赖氨酸、 羟赖氨酸、羟脯氨酸、脯氨酸、赖氨酸、组氨酸的侧链取代基及其简单修饰物。
作为上述氨基酸衍生物的进一步改进，氨基酸衍生物的结构式为
Figure PCTCN2015092451-appb-000003
Figure PCTCN2015092451-appb-000004
及其简单修饰物。
作为上述氨基酸衍生物的进一步改进，Y、Y1、Y2独立为2～12肽。特别的，Y、Y1、Y2独立为2～12的肿瘤靶向肽。
作为上述氨基酸衍生物的进一步改进，氨基酸衍生物的药用盐选自其钠、钾、钙、锌、锂、铁、镁盐、磺酸盐；氨基酸衍生物的药用酯选自其C2～C4之间的酯；氨基酸衍生物的药用醚选自其C2～C4之间的醚。
氨基酸衍生物在制备抗肿瘤药物中的应用，氨基酸衍生物的结构通式如式Ⅰ～Ⅵ所示，
Figure PCTCN2015092451-appb-000005
式中：
X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物；
R或R'独立选自组成人体蛋白质的L-型氨基酸，或D-型氨基酸的侧链取代基及其简单修饰物，或构成各种人体内天然氨基酸代谢的中间体的侧链取代基；
Y、Y1、Y2独立为多肽；
所述氨基酸衍生物还包括其药用盐、酯或醚；
或如上所述的氨基酸衍生物。
肿瘤选自肺癌、肝癌、结肠癌、食管癌、胃癌、胆囊癌、卵巢癌、鼻咽癌、舌癌、皮肤癌、乳腺癌、前列腺癌、黑色素瘤、皮肤癌、粒细胞白血病、淋巴细胞白血病、骨肉瘤、淋巴肉瘤。
一种抗肿瘤药物，其活性成分为如上所示的氨基酸衍生物中的至少一种。
优选的，肿瘤选自肺癌、肝癌、结肠癌、食管癌、胃癌、胆囊癌、卵巢癌、鼻咽癌、舌癌、皮肤癌、乳腺癌、前列腺癌、黑色素瘤、皮肤癌、粒细胞白血病、淋巴细胞白血病、骨肉瘤、淋巴肉瘤。
氨基酸衍生物的合成工艺，包括如下合成路线：
Figure PCTCN2015092451-appb-000006
Figure PCTCN2015092451-appb-000007
在合成的过程中，根据需要对-OH、-NH2或R基团进行保护和脱保护；
式中：X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物。
本发明的有益效果是：
本发明的氨基酸衍生物，可以靶向性抑制肺癌、肝癌、结肠癌、食管癌、胃癌、胆囊癌、卵巢癌、鼻咽癌、舌癌、皮肤癌、乳腺癌、前列腺癌、黑色素瘤、皮肤癌、粒细胞白血病、淋巴细胞白血病、骨肉瘤、淋巴肉瘤等恶性肿瘤组织的乳酸脱氢酶、柠檬酸合成酶、α-酮戊二酸脱氢酶复合体蛋白活性，特异性干扰肿瘤细胞产生ATP，破坏其能量代谢，从而诱导肿瘤细胞死亡和凋亡，增强对癌细胞增殖、生长、转移等的抑制作用，具有高效低毒的抗肿瘤药效学活性，对上述高耗能高消耗等各种恶性肿瘤均具有较好的杀伤或抑制作用。
本发明的氨基酸衍生物，具有较好的水溶性，易于制成注射制剂。
同时部分氨基酸衍生物口服依然具有较好，甚至更好的抗肿瘤活性，是现有抗肿瘤药物所不具备的，大大方便了患者的使用，克服了现有抗肿瘤药物依赖于注射，安全性低等缺陷。
本发明的氨基酸衍生物，合成工艺简单，成本低廉，易于纯化和工业化生产。
附图说明
图1是化合物2的1H NMR谱图；
图2是化合物2的13C NMR谱图；
图3是化合物3的1H NMR谱图；
图4是化合物3的13C NMR谱图；
图5是化合物4的1H NMR谱图；
图6是化合物4的13C NMR谱图；
图7是化合物6的1H NMR谱图；
图8是化合物6的13C NMR谱图；
图9是化合物7的1H NMR谱图；
图10是化合物7的13C NMR谱图；
图11是化合物8的1H NMR谱图；
图12是化合物8的13C NMR谱图；
图13～18分别是不同化合物的体外抗瘤谱；
图19是不同化合物对动物静息代谢能量的影响；
图20是不同化合物对动物移植瘤组织中ATP含量的影响；
图21～31不同化合物对动物体内不同肿瘤模型的抑瘤率。
具体实施方式
氨基酸衍生物，其结构通式如式Ⅰ～Ⅵ所示，
Figure PCTCN2015092451-appb-000008
式中：
X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物；
R或R'独立选自组成人体蛋白质的L-型氨基酸，或D-型氨基酸的侧链取代基及其简单修饰物，或构成各种人体内天然氨基酸代谢的中间体的侧链取代基；
Y、Y1、Y2独立为多肽；
所述氨基酸衍生物还包括其药用盐、酯或醚；
所述氨基酸衍生物不包括
Figure PCTCN2015092451-appb-000009
及其他已经公开的符合上述通式的化合物。
在本发明中，基团的简单修饰物指在不改变基团主体结构的情况下，对基团进行简单的取代、加成，如使用羟基替换其中的一个或多个H；或卤代、或与C2～C4的小分子酯化、醚化；当基团具有羧基或胺基时，羧基或胺基酰胺化。
本发明的氨基酸衍生物，可以是L型的，也可以是D型的。其中L型的结构与天然氨基酸的结构更为接近，但是在体内其同样容易被代谢，半衰期较短，起效时间较短；D型氨基酸衍生物也可以被识别并较好的结合，在体内更难以被代谢，半衰期较长，起效时间更长。可以根据需要选择不同的氨基酸衍生物作为抗肿瘤活性成分。
作为上述氨基酸衍生物的进一步改进，氨基酸衍生物的结构式为
Figure PCTCN2015092451-appb-000010
Figure PCTCN2015092451-appb-000011
及其简 单修饰物。特别的，当氨基酸衍生物为
Figure PCTCN2015092451-appb-000012
或其药用盐、酯、醚或酰胺时，氨基酸衍生物可以制成口服制剂，并具有很好抗肿瘤活性。
肿瘤细胞靶向多肽(2～10肽)具有靶向性分布于肿瘤组织，可以更好的定位于肿瘤细胞并被肿瘤细胞所摄取，更易于发生药学活性
优选的，上述结构通式中的Y、Y1、Y2独立为2～12肽，进一步的，Y、Y1、Y2独立为2～10肽、2～8肽、2～7肽、2～4肽。更佳的，Y、Y1、Y2独立为2～12的肿瘤靶向肽。进一步的，Y、Y1、Y2独立为2～10、2～8、2～7、2～4的肿瘤靶向肽，特别是那些已经被证实更易于被肿瘤细胞摄取并应用于蛋白合成的多肽。
作为上述氨基酸衍生物的进一步改进，氨基酸衍生物的药用盐选自其钠、钾、钙、锌、锂、铁、镁盐、磺酸盐；氨基酸衍生物的药用酯选自其C2～C4之间的酯；氨基酸衍生物的药用醚选自其C2～C4之间的醚。
氨基酸衍生物在制备抗肿瘤药物中的应用，氨基酸衍生物的结构通式如式Ⅰ～Ⅵ所示，
Figure PCTCN2015092451-appb-000013
式中：
X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物；
R或R'独立选自组成人体蛋白质的L-型氨基酸，或D-型氨基酸的侧链取代基及其简单修饰物，或构成各种人体内天然氨基酸代谢的中间体的侧链取代基；
Y、Y1、Y2独立为多肽；
所述氨基酸衍生物还包括其药用盐、酯或醚；
或如上所述的氨基酸衍生物。
特别的，氨基酸衍生物为
Figure PCTCN2015092451-appb-000014
Figure PCTCN2015092451-appb-000015
Figure PCTCN2015092451-appb-000016
及其简单修饰物。
肿瘤选自肺癌、肝癌、结肠癌、食管癌、胃癌、胆囊癌、卵巢癌、鼻咽癌、舌癌、皮肤癌、乳腺癌、前列腺癌、黑色素瘤、皮肤癌、粒细胞白血病、淋巴细胞白血病、骨肉瘤、淋巴肉瘤。
一种抗肿瘤药物，其活性成分为如上所示的氨基酸衍生物中的至少一种。
优选的，肿瘤选自肺癌、肝癌、结肠癌、食管癌、胃癌、胆囊癌、卵巢癌、鼻咽癌、舌癌、皮肤癌、乳腺癌、前列腺癌、黑色素瘤、皮肤癌、粒细胞白血病、淋巴细胞白血病、骨肉瘤、淋巴肉瘤。
氨基酸衍生物的合成工艺，包括如下合成路线：
Figure PCTCN2015092451-appb-000017
在合成的过程中，根据需要对-OH、-NH2或R基团进行保护和脱保护；
式中：X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物。
对于存在多肽结构的氨基酸衍生物，可以在上述合成步骤的基础之上，再进行相应的固相合成即可。
由于本发明的氨基酸衍生物结构简单，也可以直接委托现有的化合物合成公司合成得到。
肿瘤细胞的代谢旺盛，在其生长过程中需要摄取大量的氨基酸、供能小分子等，当本发明的氨基酸衍生物作用于肿瘤细胞时，会在肿瘤组织和肿瘤细胞中大量靶向性累积和聚集，可以通过底物水平升高机制反馈性抑制肿瘤组织的柠檬酸合成酶、α-酮戊二酸脱氢酶复合体蛋白活性(这两种酶为三羧酸循环进行的限速酶)，同时，也可以特异性干扰肿瘤细胞线粒体复合体IV细胞色素C氧化酶复合体产生ATP，从而诱导肿瘤细胞死亡和凋亡，增强对癌细胞增殖、生长、转移等的抑制作用；而正常细胞代谢速率明显低于肿瘤细胞，对本发明的氨基酸衍生物摄取量较少，细胞的能量代谢、物质合成受到的影响同样较小，并不会有明显的杀伤作用。因此，本发明的氨基酸衍生物具有高效低毒的抗肿瘤药效学活性。
本发明的氨基酸衍生物对肿瘤的抑制、杀伤作用不受肿瘤是否转移的影响，可以应用于各阶段肿瘤的治疗，同时也可以与现有的抗肿瘤药物联用，起到更好的抗肿瘤效果。同样的，本发明的氨基酸衍生物之间也可以联用，尽可能多的阻断肿瘤的能量代谢，以起到更好的抗肿瘤效果。
下面结合具体的合成实例，进一步示例性说明本发明的氨基酸衍生物的合成工艺。
氨基酸衍生物的合成
草酸酰谷氨酸(化合物4)合成路线
整体合成路线如下所示，
Figure PCTCN2015092451-appb-000018
具体的合成工艺如下：
化合物2的合成工艺技术路线
称取1.0eq的化合物1溶于适量CH2Cl2中，搅拌条件下，加入2.2eq的Et3N于上述反应液中。在冰浴、搅拌条件下将1.1eq的EtOOC-COCl逐滴加入至上述反应液中，滴加完成后撤去冰浴，室温搅拌，并取样进行HPLC监测，直至原料峰消失后停止反应。滤去白色不溶固体，有机相分别用5％NaHCO3水溶液、饱和食盐水、10％KHSO4水溶液和饱和食盐水各洗涤1次，收集有机相，旋干浓缩并干燥得粘稠状化合物2，收率90％，HPLC纯度95％。
化合物2的核磁数据为：
1H NMR(400Hz,CD3OD)δ：4.28-4.38(m,2H)，2.31-2.35(t,2H)，1.93-2.21(m,2H)，1.44-1.47(d,18H)，1.32-1.37(t,3H)，其氢谱如附图1所示；
13C NMR(400Hz,CD3OD)δ：173.60，171.38，161.07,159.27，83.41，81.92，63.93，54.22，32.50，28.33，28.21，27.30，14.25，其碳谱如附图2所示；
MS(EI)m/z(100％):358.1(M-，100)，理论值359.3；
化合物3的合成工艺技术路线
将化合物2溶于甲醇中，并加入适量1mol/L NaOH水溶液调节其pH 8-9，室温水解，HPLC监测反应。反应结束后，用盐酸调溶液pH 2-3，旋蒸浓缩除去甲醇，用乙酸乙酯萃取两次，合并萃取有机相后，用食盐水洗涤一次，旋干浓缩并干燥得化合物3，收率85％，HPLC纯度96％。
化合物3的核磁数据为：
1H NMR(400Hz,CD3OD)δ：2.31-2.34(t,2H)，1.93-2.21(m,2H)，1.44-1.47(d,18H)，其氢谱如附图3所示；
13C NMR(400Hz,CD3OD)δ：173.62，171.45，162.20,160.07，83.40，81.93，54.21，32.50，28.33，28.21，27.37，其碳谱如附图4所示；
MS(EI)m/z(100％):330.2(M-，100)，理论值331.3；
化合物4的合成工艺技术路线
将化合物3溶于3倍体积的TFA，HPLC监测反应。反应结束后，旋蒸浓缩除去TFA。加适量水溶解，用CH2Cl2洗涤一次，水相经冷冻干燥得白色粉状化合物4，收率90％，纯度96.8％。
化合物4的核磁数据为：1H NMR(400Hz,CD3OD)δ：4.47-4.54(m,1H)，2.39-2.46(m,2H)，2.01-2.33(m,2H)，其氢谱如附图5所示；
13C NMR(400Hz,CD3OD)δ：176.31，173.86，162.22,160.09，53.45，30.98，27.37，其碳谱如附图6所示；
MS(EI)m/z(100％):113(M--106，100)，理论值219；
草酸酰谷氨酰胺(化合物8)合成路线
整体合成路线如下所示，
Figure PCTCN2015092451-appb-000019
具体的合成工艺如下：
化合物6的合成工艺技术路线
称取1.0eq的化合物5溶于适量CH2Cl2中，搅拌条件下，加入2.2eq的Et3N于上述反应液中。在冰浴、搅拌条件下将1.1eq的EtOOC-COCl逐滴加入至上述反应液中，滴加完成后撤去冰浴，室温搅拌，并取样进行HPLC监测，直至原料峰消失后停止反应。滤去白色不溶固体，有机相分别用5％NaHCO3水溶液、饱和食盐水、10％KHSO4水溶液和饱和食盐水各洗涤1次，收集有机相，旋干浓缩并干燥得粘稠状化合物6，收率88％，HPLC纯度96％。
化合物6的核磁数据为：
1H NMR(400Hz,CD3OD)δ：4.31-4.34(m,1H)，3.57-3.62(m,2H)，2.29-2.33(t,2H)，1.99-2.24(m,2H)，1.50(s,9H)，1.15-1.19(t,3H)其氢谱如附图7所示；
13C NMR(400Hz,CD3OD)δ：177.45，171.38，160.12,159.08，83.47，58.32，54.66，32.41，28.18，27.72，18.37，其碳谱如附图8所示；
MS(EI)m/z(100％):301.2(M-，82)113.1(100)，理论值302.2；
化合物7的合成工艺技术路线
将化合物6溶于甲醇中，并加入适量1mol/L NaOH水溶液调节其pH 8-9，室温水解，HPLC监测反应。反应结束后，用盐酸调溶液pH 2-3，旋蒸浓缩除去甲醇，过滤收集白色不溶物，滤饼用冷水洗涤两次，收集滤饼并干燥得化合物7，收率85％，HPLC纯度95％
化合物7的核磁数据为：
1H NMR(400Hz,CD3OD)δ：4.31-4.35(m,1H)，2.27-2.33(t,2H)，1.97-2.24(m,2H)，1.47(s,9H)，其氢谱如附图9所示；
13C NMR(400Hz,CD3OD)δ：177.47，171.47，162.35,160.32，83.49，54.57，32.38，28.19，27.83，其碳谱如附图10所示；
MS(EI)m/z(100％):273.2(M-，100)，理论值274.2；
化合物8的合成工艺技术路线
将化合物7溶于3倍体积的TFA，HPLC监测反应。反应结束后，旋蒸浓缩除去TFA。加适量水溶解，用CH2Cl2洗涤一次，水相经冷冻干燥得白色粉状化合物8，收率90％，纯度90％。
化合物8的核磁数据为：
1H NMR(400Hz,CD3OD)δ：4.44-4.71(s,1H)，2.31-2.35(m,2H)，2.05-2.28(m,2H)，其氢谱如附图11所示；
13C NMR(400Hz,CD3OD)δ：177.58，173.79，162.27，160.13，53.71，32.50，28.01，其碳谱如附图12所示；
MS(EI)m/z(100％):113.1(M--105，100)，理论值218.2。
下面使用上述方案合成得到的化合物2、3、4、6、7、8进行实验，以证实其功能。
安全性实验
1、氨基酸衍生物对L929细胞毒性实验
细胞培养与细胞毒性评估
L-929细胞在37±1℃的5％±1％的二氧化碳的气态环境中和包含推荐介质的组织培养瓶的湿润环境中，添加10％胎牛血清和青霉素(100units)、链霉素(100μg)等抗生素(美国Invitrogen公司)的细胞培养基(美国Invitrogen公司)中培养，以达到实验要求的细胞密度。用PBS漂洗单层细胞两次，将PBS吸走，在0.25％胰蛋白酶/EDTA，37±1℃的组织培养瓶中消化，直到细胞分离和浮动。
通过我们设计合成的化合物2、3、4、6、7、8对L929细胞的体外细胞毒作用，并按照ISO 10993-part 5的细胞株的形态分析执行。
表1化合物毒性的定性形态分级
Figure PCTCN2015092451-appb-000020
测试项目，观察化合物2、3、4、6、7和8对L929细胞的体外细胞毒作用。各种化合物均采用5％的DMEM进行稀释，配制浓度至100μM，并分别稀释至50μM,25μM，12.5μM,6.25μM的5％的DMEM稀释液，分别加入5％CO2培养的L-929老鼠成纤细胞的融合单层细胞中。另外独立的三份测试液分别为阳性对照(顺铂，阳性对照)，阴性对照(生理盐水)和溶媒对照(5％DMEM)。所有的测试液都在温度为37±1℃的5％±1％CO2中培养24小时。单层细胞在测试中显阳性，试剂对照试液在经过培育后，在显微镜下被检测24±1小时去判定细胞形态的任何变化。
结果
在此研究的条件下，化合物2、3、4、6、7、8在100μM的浓度下，24±1小时的观察，发现对L-929细胞毒性较低或者没有明显的细胞毒性。
经过24小时后温育后，形态观察和生物反应，结果见表2。
表2受试药化合物毒性的定性形态分级
受试药 合流单层 百分比 细胞内颗粒 溶解 级别 反应
100μM化合物2 无 <50％ 有 无 2 温和
100μM化合物3 无 <50％ 有 无 2 温和
100μM化合物4 有 <20％ 有 无 1 轻微
100μM化合物6 有 0％ 有 无 0 没反应
100μM化合物7 有 0％ 有 无 0 没反应
100μM化合物8 有 0％ 有 无 0 没反应
顺铂 无 >70％ 无 无 4 剧烈
阴性对照(NS) 有 0％ 有 无 0 没反应
空白对照(溶媒) 有 0％ 有 无 0 没反应
。
结论
化合物2、3、4、6、7、8对L929细胞的毒性结果表明，在100μM、50μM,25μM，12.5μM,6.25μM的5％的DMEM稀释液作用于L929细胞，经过24小时观察，在最大浓度100μM时发现没有明显的细胞毒性。
2、氨基酸衍生物的体外癌细胞的杀伤实验(IC50)
实验材料及仪器
药物2、3、4、6、7、8、对照品顺铂，人肺癌H460、H1650、SPC-A-1、A549细胞株、肝癌HepG2细胞株、乳腺癌MCF-7细胞株、结肠癌HT-29细胞株、前列腺癌PC-3、DU145细胞株、宫颈癌Hela细胞株、胃癌MGC-803细胞株、DMEM培养基、10％FBS小牛血清、MTT试剂、DMSO、96孔板、酶标仪等。化合物2(20140701)、化合物3(20140506)、化合物4(20140305)、化合物6(20140508)、化合物7(20140602)、化合物8(20140304)来自申请人实验室的合成。
实验方法及步骤
1)收集对数期细胞；
2)调细胞数为4×103个/L，每孔加100uL；
3)培养12小时；
4)添加受试药和阳性对照药浓度梯度分别为0.5、2.5、5.0、10、25、50、100μM浓度的药物及0.5、2.5、5.0、10、25、50、100μM浓度的阳性对照药物顺铂；另外设溶媒对照组
5)培养24后，每孔加MTT(5mg/ml)溶液20μl；
6)孵育4h，终止培养，吸弃上清液；
7)加DMSO100ul/孔，振荡10min；
8)置酶标仪测定，λ＝490nm；
9)记录结果，绘制图表。
按下列公式计算药物对细胞的生长抑制率及IC50：
IR％＝(1-实验组OD值/对照组OD值)×100％
结果：化合物2、3、4、6、7、8在0.5、2.5、5.0、10、25、50、100μM的5％的DMEM稀释液，分别作用于体外肺癌H460、H1650、SPC-A-1、A549细胞、肝癌HepG2细胞、乳腺癌MCF-7细胞、结肠癌HT-29细胞、前列腺癌PC-3、DU145细胞、宫颈癌Hela细胞、胃癌MGC-803细胞，经过24小时观察，观察化合物2、3、4、6、7、8对上述细胞的50％抑制率(IC50)。
化合物2、3、4、6、7、8分别作用于肺癌H460、H1650、SPC-A-1、A549细胞、肝癌HepG2细胞、乳腺癌MCF-7细胞、结肠癌HT-29细胞、前列腺癌PC-3、DU145细胞、宫颈癌Hela细胞、胃癌MGC-803细胞，经过24小时孵育后采用MTT方法检测抗肿瘤活性发现，化合物2、3、4、6、7、8有一定抗肿瘤作用(分别见图13～18)，但是与阳性对照组顺铂相比，氨基酸衍生物的体外其抗肿瘤细胞活性不高。
3、小鼠急性毒性实验
SPF级KM种小鼠，动物饲养在SPF级实验动物室中的独立通风系统(IVC)设施。群养，自由饮水、进食，实验室温度为20～25℃，湿度为40～70％。动物室条件始终保持稳定，以保证试验结果的可靠性。饲料和饮水：饲料为SPF级用颗粒鼠料，购自广东省医学实验动物中心，营养和卫生符合SPF级实验动物要求。饮水自由摄取。
临床拟用途径：口服给药。
预试资料：申请人自行合成的化合物8(20140304)以最大剂量，即1800.mg/kg·bw最大浓度最大体积单次口服给药后，小鼠的死亡率为0％。
动物分组：取检疫合格、体重符合要求的动物140只，按体重和性别随机分为14组，每组10只，雌雄各半。受试药物配制：称取化合物8冻干粉针剂，生理盐水溶解配制。给药途径和次数：单次口服给药。给药容积：10ml/kg。动物空腹时间：给药前禁食6～12小时，给药后禁食4小时。
观察与检查：一般观察：给药后立即观察动物的反应情况，并持续观察4小时,以后每天上午各观察1次，连续观察14天，记录动物中毒表现和特点，毒性反应出现时间、消失时间和动物死亡时间。
体重测定：分别在给药前(d1)、给药后24小时(d2)、第4天(d4)、第8天(d8)、第11天(d11)和第15天(d15)称量动物体重。
病理学检查：中毒死亡或濒死动物及时进行剖检，其他动物在观察期结束后进行剖检，当发现组织器官出现体积、颜色、质地等改变时，则对有改变的组织器官进行病理组织学检查。剖检的组织器官包括胸腺、心、肺、肝脏、脾、胰腺、肾、肾上腺、胃、肠(十二指肠、空肠、回肠、盲肠、结肠、直肠)、淋巴结、膀胱、前列腺、睾丸(连附睾)、子宫、卵巢。
观察指标：详细记录动物反应情况，包括外观、行为活动、精神状态、食欲、大小便及其颜色、皮毛、肤色、呼吸、鼻、眼、口腔有无异常分泌物，体重变化以及死亡等情况。
实验结果：14天内，小鼠无死亡，灌胃后小鼠蜷缩，不喜活动，分析是由于药物浓度过高，灌胃体积过大引起。表明化合物8毒性较低，小鼠最大耐受剂量约1800mg/kg，相当于人临床拟用剂量的512倍。
氨基酸衍生物对细胞能量代谢的影响
1.氨基酸衍生物的抗肿瘤能量代谢作用
方法：采用美国MEDGRAPHIC公司生产的CCM代谢车，测试前让裸鼠静息无刺激，平静30分钟后接受测量(测量的指标是吸气和呼气中的O2、CO2以及呼出气量，根据吸气和呼气中氮气浓度不变的原则，计算出吸气量。由此得出氧气消耗量(VO2)与二氧化碳产生量(VCO2)，结合24小时尿总氮排出量(N)。测试时的环境湿度为50-65％，温度为18-24℃，大气压为101～102.4kPa(758-768mmHg)，并将这些数据输入计算机，用于仪器校正。
再根据公式3.94×VO2+1.11×VCO2-2.17×N，计算出能量消耗。呼吸商按照RQ＝VCO2/VO2计算，非蛋白质呼吸商按照NRQ＝(VCO2-4.754×N)＝(VO2-5.293×N)计算。求出氨基酸衍生物对裸鼠荷瘤A549动物模型静息能量代谢REE(Resting Energy Expenditure，每天能量消耗总量：kJ/d)的影响，结果见图19：
化合物2(20140701)、化合物3(20140506)、化合物4(20140305)、化合物6(20140508)、化合物7(20140602)、化合物8(20140304)来自申请人实验室的合成。从图19可以看出，在给药后第10天和第14天，模型组与顺铂组和化合物2、3、4、6、7、8组都可以显著降低肿瘤组织的能量代谢。
2.ATP生成作用
利用ATP检测试剂盒(ATP Assay Kit)检测荷瘤裸鼠肿瘤组织内的ATP(adenosine 5'-triphosphate)水平。根据萤火虫荧光素酶(firefly luciferase)化荧光素产生荧光时需要ATP提供能量研制而成。当萤火虫荧光素酶和荧光素都过量时，在一定的浓度范围内荧光的产生和ATP的浓度成正比。这样就可以高灵敏地检测溶液中的ATP浓度。所用检测试剂盒可以检测浓度低达5nmol/L的ATP。而常规的细胞或组织裂解液中ATP的浓度仅为0.1-1μmol/L，一些常见细胞的细胞内ATP水平约为10nmol/mg蛋白。检测细胞或组织内ATP浓度时，用本试剂盒提供的裂解液裂解后即可以测定ATP浓度。
化合物2(20140701)、化合物3(20140506)、化合物4(20140305)、化合物6(20140508)、化合物7(20140602)、化合物8(20140304)来自申请人实验室的合成。化合物2、3、4、6、7、8对荷瘤裸鼠A549移植瘤组织中ATP含量的影响，见图20。
从图20看出，在给药后第14天，模型组与顺铂组和化合物2、3、4、6、7、8组都可以显著降低肿瘤组织的ATP含量，但是与顺铂组相比，化合物2、4和6抑制ATP生成的效果具有显著性差异。
氨基酸衍生物的抗肿瘤药效学实验
实验动物：
健康Balb-c裸鼠，雄雌各半，4周龄左右，体重18-22g，购自广东省医学实验动物中心。接种2-3周后取瘤体直径约0.5-1.0cm的裸鼠用于实验。
细胞系：
人恶性肿瘤细胞株H460、H1650、SPC-A-1、A549、HepG2、MCF-7、HT-29、PC-3、DU145、Hela、MGC-803。
SPCA荷瘤裸鼠模型的建立：
健康Balb-c裸鼠70只，雄性，4～5周龄，体重18-22g。人非小细胞肺癌细胞株H460、H1650、SPC-A-1、A549、 HepG2、MCF-7、HT-29、PC-3、DU145、Hela、MGC-803，调整细胞密度约为1×107个/ml，于裸鼠前右腋下皮下接种0.2ml/只，2-3周后待肿瘤长至1.0×1.0cm左右，处死，取肿瘤组织均匀剪碎，套管接种至受试裸鼠前右腋下皮下，接种1-2周后观察，成瘤率达98.6％，挑选肿瘤成长约0.5×0.5cm的裸鼠用于实验。
氨基酸衍生物对H460细胞株荷瘤裸鼠的抑瘤作用
动物分组：
挑选肿瘤体积约为0.5×0.5cm的模型荷瘤裸鼠进行随机分组，化合物2(20140701)、化合物3(20140506)、化合物4(20140305)、化合物6(20140508)、化合物7(20140602)、化合物8(20140304)来自发明人实验室的合成。因此，将荷瘤动物分为八组，模型组、阳性对照组、化合物2组、化合物3组、化合物4组、化合物6组、化合物7组、化合物8组，每组8只动物。
剂量设置：
模型组：等体积生理盐水；阳性对照组：10μM/kg(顺铂溶液)；化合物2组：50μM/kg；化合物3组：50μM/kg；化合物4组：50μM/kg；化合物6组：50μM/kg；化合物7组：50μM/kg；化合物8组：50μM/kg；每组均按照0.1ml/10g来腹腔注射(ip)给药。
给药方法：
连续14天每天给药。均采用腹腔注射(ip)给药方式，每日给药前称重，根据每日体重确定每次给药量。隔日称重，量算肿瘤体积。第15天将动物处死，取出心、肝、脾、肺、肾、肿瘤，分别称重后，于10％甲醛中保存。
检测指标：
(1)肿瘤体积(tumor volume,TV)的计算公式为：
V＝1/2×a×b2(a、b分别表示肿瘤的长和宽)
(a,b单位为mm，肿瘤体积V单位为mm3)
(2)肿瘤重量抑制率＝(1-给药组平均瘤重/对照组平均瘤重)×100％
(3)肿瘤体积抑制率＝(1-(给药组给药前体积-给药后体积)/(对照组给药前体积-给药后体积))×100％。
氨基酸衍生物药效学实验结果
1.氨基酸衍生物氨基酸草酰胺对裸鼠体内肺癌H1650模型的抑瘤率如图21所示。
从图21看出，化合物3和化合物8与阳性对照药顺铂相比，P>0.05，即化合物8和化合物2抗肺癌H1650增殖的药效与顺铂相当。
2.氨基酸衍生物氨基酸草酰胺对裸鼠体内肺癌H460模型的抑瘤率如图22所示。
从图22看出，化合物2和6与阳性对照药顺铂相比，P>0.05，即化合物2和6抗肺癌H460增殖的药效与顺铂相当。
3.氨基酸衍生物氨基酸草酰胺对裸鼠体内肺癌SPC-A-1模型的抑瘤率如图23所示。
从图23看出，化合物2、3、4和7与阳性对照药顺铂相比，P>0.05，即化合物2、3、4和7抗肺癌SPC-A-1增殖的药效与顺铂相当。
4.氨基酸衍生物氨基酸草酰胺对裸鼠体内肺癌A549模型的抑瘤率如图24所示。
从图24看出，化合物2、3和7与阳性对照药顺铂相比，P>0.05，即化合物2、3和7抗肺癌A549增殖的药效与顺铂相当。
5.氨基酸衍生物氨基酸草酰胺对裸鼠体内肝癌HepG2模型的抑瘤率如图25所示。
从图25看出，化合物4和6与阳性对照药顺铂相比，化合物4和6抗肝癌HepG2增殖的药效与顺铂相当，但是，化合物2、3和7抗肝癌的药效高于顺铂(P<0.05)。
6.氨基酸衍生物氨基酸草酰胺对裸鼠体内乳腺癌MCF-7模型的抑瘤率如图26所示。
从图26看出，化合物2、3和7与阳性对照药顺铂相比，抗乳腺癌MCF-7增殖的药效与顺铂相当。
7.氨基酸衍生物氨基酸草酰胺对裸鼠体内结肠癌HT-29模型的抑瘤率如图27所示。
从图27看出，化合物2、3、4和7与阳性对照药顺铂相比，抗乳腺癌MCF-7增殖的药效与顺铂相当。
8.氨基酸衍生物氨基酸草酰胺对裸鼠体内前列腺癌PC-3模型的抑瘤率如图28所示。
从图28看出，化合物2、3和7与阳性对照药顺铂相比，其抗前列腺癌PC-3增殖的药效与顺铂相当。
9.氨基酸衍生物氨基酸草酰胺对裸鼠体内前列腺癌DU145模型的抑瘤率如图29所示。
从图29看出，化合物2、3、4和7与阳性对照药顺铂相比，抗前列腺癌DU145增殖的药效与顺铂相当。
10.氨基酸衍生物氨基酸草酰胺对裸鼠体内宫颈癌Hela模型的抑瘤率如图30所示。
从图30看出，化合物2、3和4与阳性对照药顺铂组相比，抗Hela增殖的药效与顺铂相当,但是，化合物7与顺铂组相比，抗Hela增殖的药效高于顺铂。
11.氨基酸衍生物氨基酸草酰胺对裸鼠体内胃癌MGC-803模型的抑瘤率如图31所示。
从图31看出，化合物2、3、7和8与阳性对照药顺铂组相比，抗胃癌MGC-803增殖的药效与顺铂相当。

Claims (10)

  1. 氨基酸衍生物，其结构通式如式Ⅰ～Ⅵ所示，
    Figure PCTCN2015092451-appb-100001
    式中：
    X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物；
    R或R'独立选自组成人体蛋白质的L-型氨基酸，或D-型氨基酸的侧链取代基及其简单修饰物，或构成各种人体内天然氨基酸代谢的中间体的侧链取代基；
    Y、Y1、Y2独立为多肽；
    所述氨基酸衍生物还包括其药用盐、酯或醚；
    所述氨基酸衍生物不包括
    Figure PCTCN2015092451-appb-100002
  2. 根据权利要求1所述的氨基酸衍生物，其特征在于：R、R’独立选自谷氨酸、谷氨酰胺、瓜氨酸、鸟氨酸、胱氨酸、天冬氨酸、天冬酰胺、苏氨酸、丝氨酸、亮氨酸、异亮氨酸、甘氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、苯丙氨酸、缬氨酸、赖氨酸、羟赖氨酸、羟脯氨酸、脯氨酸、赖氨酸、组氨酸的侧链取代基及其简单修饰物。
  3. 根据权利要求1所述的氨基酸衍生物，其特征在于：氨基酸衍生物的结构式为
    Figure PCTCN2015092451-appb-100003
    及其简单修饰物。
  4. 根据权利要求1～3所述的氨基酸衍生物，其特征在于：Y、Y1、Y2独立为2～12肽。
  5. 根据权利要求4所述的氨基酸衍生物，其特征在于：Y、Y1、Y2独立为2～12的肿瘤靶向肽。
  6. 根据权利要求1～5所述的氨基酸衍生物，其特征在于：氨基酸衍生物的药用盐选自其钠、钾、钙、锌、锂、铁、镁盐、磺酸盐；氨基酸衍生物的药用酯选自其C2～C4之间的酯；氨基酸衍生物的药用醚选自其C2～C4之间的醚。
  7. 氨基酸衍生物在制备抗肿瘤药物中的应用，所述氨基酸衍生物的结构通式如式Ⅰ～Ⅵ所示，
    Figure PCTCN2015092451-appb-100004
    式中：
    X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物；
    R或R'独立选自组成人体蛋白质的L-型氨基酸，或D-型氨基酸的侧链取代基及其简单修饰物，或构成各种人体内天然氨基酸代谢的中间体的侧链取代基；
    Y、Y1、Y2独立为多肽；
    所述氨基酸衍生物还包括其药用盐、酯或醚；
    或如权利要求2～6任意一项所示的氨基酸衍生物。
  8. 根据权利要求7所述的应用，其特征在于：肿瘤选自肺癌、肝癌、结肠癌、食管癌、胃癌、胆囊癌、卵巢癌、鼻咽癌、舌癌、皮肤癌、乳腺癌、前列腺癌、黑色素瘤、皮肤癌、粒细胞白血病、淋巴细胞白血病、骨肉瘤、淋巴肉瘤。
  9. 一种抗肿瘤药物，其活性成分为权利要求1～6任意一项所示的氨基酸衍生物中的至少一种。
  10. 氨基酸衍生物的合成工艺，包括如下合成路线：
    Figure PCTCN2015092451-appb-100005
    Figure PCTCN2015092451-appb-100006
    在合成的过程中，根据需要对-OH、-NH2或R基团进行保护和脱保护；
    式中：X选自草酰基及其简单修饰物，或含有草酰基的化合物与氨基酸偶联反应后的残基及其简单修饰物。
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