WO2020038278A1 - 新型喜树碱衍生物及其制备方法和应用 - Google Patents

新型喜树碱衍生物及其制备方法和应用 Download PDF

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WO2020038278A1
WO2020038278A1 PCT/CN2019/100802 CN2019100802W WO2020038278A1 WO 2020038278 A1 WO2020038278 A1 WO 2020038278A1 CN 2019100802 W CN2019100802 W CN 2019100802W WO 2020038278 A1 WO2020038278 A1 WO 2020038278A1
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integer
compound
camptothecin derivative
cancer
formula
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French (fr)
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谢永美
杨细飞
赵宇
罗波
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深圳市塔革特生物医药科技有限公司
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Priority to US17/270,063 priority Critical patent/US11572382B2/en
Publication of WO2020038278A1 publication Critical patent/WO2020038278A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/056—Triazole or tetrazole radicals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C07H11/04—Phosphates; Phosphites; Polyphosphates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a novel camptothecin derivative and its application, a tumor cell growth inhibitor and a ternary complex, and a method for improving the solubility of a camptothecin derivative.
  • the camptothecin derivative can prepare an antitumor drug.
  • Camptothecin is an alkaloid containing a quinoline ring first extracted from the skin and fruits of Camptotheca acuminata by the American chemist Wall in 1966.
  • CPT Camptothecin
  • CPT was an exclusive inhibitor of topoisomerase I (ToPoI), and only then brought CPT research back to life.
  • Irinotecan hydrochloride CPT-11
  • CPT-11 is a water-soluble camptothecin derivative that is commonly used in the treatment of colorectal cancer, lung cancer, and breast cancer, but its antitumor activity is still not high and its side effects are large.
  • the development of various camptothecin derivatives and improvement of their medicinal properties is a challenge for tumor drug development.
  • Camptothecin derivatives such as topotecan, beloticam, and 10-hydroxycamptothecin have been developed and applied to the human body, but the "compatibility" of the above derivatives with the human body has always been a major challenge in the pharmaceutical industry.
  • SN-38 As a metabolite of irinotecan, SN-38 has 100 to 1000 times the antitumor activity of irinotecan in vitro, but its solubility is extremely poor, and it is almost insoluble in commonly used drug solvents and water, so it has poor drug-making properties (Santi et al., J. Med. Chem. 2014, 57 (6): 2303-2314), which has great limitations in clinical application.
  • one object of the present invention is to provide a camptothecin derivative which has high water solubility.
  • the present invention adopts the following scheme:
  • the camptothecin derivative is modified from a substance represented by Formula 1 at the R 3 position through glycosylated triazole; in the structural formula represented by Formula 1, R 1 represents an alkane of H, C 1-10 Group, C 1-10 deuterated alkyl group or C 1 -C 10 haloalkyl group; R 2 represents H, CH 2 N (CH 3 ) 2 or CH 2 N (CD 3 ) 2 ; R 4 represents Or H, where X represents N, O, or S; L represents a polypeptide, a C 1-20 linear alkyl or its derivative, a C 1-20 linear or branched acyl derivative, a C 2-100 ethylene glycol, or Its derivatives,
  • the R 3 represents Or H, where R 4 represents Or H, but the R 3 and R 4 are not both H; the R 5 represents a monosaccharide residue or an oligosaccharide residue; L represents a polypeptide, a C 1 -C 20 linear alkyl group or a derivative thereof, C 1- C 20 linear or branched acyl derivative, C 1 -C 20 ethylene glycol or derivative thereof, Where Y is a is an integer from 0 to 100; b is an integer from 1 to 100; c is an integer from 0 to 100; and d is an integer from 0 to 100.
  • R 1 represents H or -CH 2 CH 3 ; the R 2 represents H, -CH 2 N (CH 3 ) 2 or -CH 2 N (CD 3 ) 2 ; R 3 stands for Or H, where R 4 represents Or H, but R 3 and R 4 are not both H; R 5 is selected from monosaccharide or oligosaccharide residue; X is selected from N or O; L is selected from C 1-20 straight or branched Paraffins and their derivatives, C 1-20 linear or branched acyl derivatives, C 2-100 ethylene glycol and its derivatives, Where Y is a is an integer from 0 to 100; b is an integer from 1 to 100; c is an integer from 0 to 100; and d is an integer from 0 to 100.
  • a is 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100.
  • the b is 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100.
  • c is 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100.
  • d is 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100.
  • R 1 represents -CH 2 CH 3 ;
  • R 2 represents H; and
  • R 3 represents The R 4 represents H;
  • the X represents N or O; and
  • the L represents Where Y is The a is an integer of 1-20, b is an integer of 1-20, c is an integer of 0-20, and d is an integer of 0-20.
  • camptothecin derivative is a derivative of 7-ethyl-10-hydroxycamptothecin, and its structural formula is shown in Formula II,
  • glycosylated triazole is a structural formula described in Formula III or IV, wherein Y is a is an integer of 0-100, b is an integer of 1-100, c is an integer of 0-100, d is an integer of 0-100, and R 5 is a sugar residue or an oligosaccharide residue;
  • R 5 is selected from any one of the monosaccharide residues of Formula 5-28:
  • the a or the b is an integer of 1-20.
  • R 5 is selected from any one of monosaccharide residue formulae 5, 6, 18 and 19:
  • the derivative is one of the following compounds:
  • X is selected from N or O.
  • d is an integer of 0-1.
  • a is 1 and b is an integer of 1-4.
  • Another object of the present invention is to provide a method for synthesizing the camptothecin derivative, which can be applied to industrial production.
  • the present invention adopts the following scheme:
  • the method includes the following steps:
  • the azide compound is Where Z is none or O and e is 0-20.
  • Z is none or O, and e is 0-1.
  • a third object of the present invention is to provide a method for improving the solubility of a camptothecin derivative, which method is suitable for industrial application.
  • the present invention adopts the following scheme:
  • the method is to modify the 7-ethyl-10-hydroxycamptothecin derivative with glycosylated triazole, the structural formula of the 7-ethyl-10-hydroxycamptothecin derivative is shown in Formula 2, and the sugar group is Triazole is modified at the position of R 3 ; in the structural formula shown in Formula 2, R 3 represents Or H; where X represents N, O, or S; L represents a polypeptide, a C 1 -C 20 linear alkyl group or a derivative thereof, a C 1 -C 20 linear or branched acyl derivative, C 1 -C 20 ethyl Diol or its derivative,
  • glycosylated triazole is a structural formula described in Formula 3 or 4, wherein Y is a is an integer of 0-100, b is an integer of 1-100, c is an integer of 0-100, d is an integer of 0-100, and R 5 is a sugar residue or an oligosaccharide residue.
  • R 5 is as shown in any one of Formulas 5-28.
  • R 5 is selected from any one of monosaccharide residue formulae 5, 6, 18, and 19.
  • a is 1 and b is an integer of 1-4.
  • d is an integer of 0-1.
  • the fourth object of the present invention is to provide a tumor cell growth inhibitor, which has an anti-cancer effect.
  • the present invention adopts the following scheme:
  • a tumor cell growth inhibitor the inhibitor is prepared from the camptothecin derivative.
  • the camptothecin derivative may contain any pharmaceutically acceptable carrier and adjuvant.
  • the tumor cell growth inhibitor can form a ternary complex with topoisomerase I and DNA to break the DNA strand, inhibit fine growth of tumors and promote tumor cell apoptosis.
  • the tumor is colorectal tumor, lung tumor and breast tumor, liver cancer, gastric cancer, esophageal cancer, leukemia, prostate cancer, osteosarcoma, cervical cancer, thyroid cancer, ovarian cancer or pancreatic cancer.
  • a fifth object of the present invention is to provide a ternary complex, which can inhibit tumor cell growth.
  • the ternary complex is formed by the tumor cell growth inhibitor through topoisomerase I and DNA.
  • the ternary complex breaks the DNA strand, inhibits fine tumor growth and promotes tumor cell apoptosis.
  • a sixth object of the present invention is to provide an application of the camptothecin derivative, which provides a new idea for cancer treatment.
  • the present invention adopts the following scheme:
  • camptothecin derivative in preparing anticancer medicine.
  • the cancer is colorectal cancer, lung cancer and breast cancer, liver cancer, gastric cancer, esophageal cancer, leukemia, prostate cancer, osteosarcoma, cervical cancer, thyroid cancer, ovarian cancer or pancreatic cancer.
  • the anti-cancer drug can break the DNA chain by forming a ternary complex with topoisomerase I and DNA, inhibit the fine growth of tumors and promote tumor cell apoptosis to achieve anti-cancer effects.
  • the tumor cells are SW- 480 and / or HT-29 and / or HCT-116 and / or A549 and / or H1975 and / or HepG2 and / or BGC-823 and / or ECA-109 and / or K562 and / or PC3 and / or 143B and / Or MDA-MB-231 and / or Hela and / or TPC-1 and / or SKOV-3 and / or PANC-1.
  • a seventh object of the present invention is to provide a preparation which can effectively inhibit the growth of tumor cells.
  • the preparation contains a pharmaceutically acceptable carrier and / or an adjuvant.
  • the pharmaceutically acceptable dosage form includes the traditional Chinese medicine composition of the present invention and optionally one or more pharmaceutically acceptable carriers, diluents or excipients, and is added in a suitable step in the above preparation process.
  • pharmaceutically acceptable refers to compounds, raw materials, compositions and / or dosage forms which are within the scope of sound medical judgment and are suitable for contact with patient tissues without excessive toxicity or irritation , Allergies, or other problems and complications commensurate with a reasonable benefit / risk ratio, and effectively used for the intended purpose.
  • compositions are suitable for administration by any suitable route, such as by oral (including oral or sublingual), rectal, nasal, topical (including oral, sublingual, or transdermal), vaginal or parenteral (including subcutaneous, intradermal) , Intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous or subdermal injection or infusion) route.
  • Such formulations may be prepared by any method known in the pharmaceutical arts, for example by mixing the active ingredient with a carrier or excipient.
  • Oral, topical or injection administration is preferred.
  • Pharmaceutical preparations suitable for oral administration are provided in separate units, such as solutions or suspensions in aqueous or non-aqueous liquids; capsules or tablets; powders or granules; edible foam preparations or foaming preparations, and the like.
  • the active pharmaceutical ingredient may be mixed with a pharmaceutically acceptable oral non-toxic inert carrier such as ethanol, glycerol, water, and the like.
  • a pharmaceutically acceptable oral non-toxic inert carrier such as ethanol, glycerol, water, and the like.
  • the powder is prepared by pulverizing the compound to a suitable fine size and mixing it with a similarly pulverized pharmaceutical carrier (such as starch or edible sugars such as mannitol). Flavoring agents, preservatives, dispersants and colorants may also be present.
  • Capsules are prepared by preparing a powdery mixture as described above and filling it into a shaped gelatin shell.
  • glidants and lubricants e.g., colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol
  • glidants and lubricants can be added to the powder mixture.
  • Disintegrating or solubilizing agents such as agar, calcium carbonate, or sodium carbonate
  • agar, calcium carbonate, or sodium carbonate that will improve the availability of the drug when the capsule is taken may also be added.
  • suitable binders include starch, gelatin, natural sugars (e.g. glucose or ⁇ -lactose), corn sweeteners, natural and synthetic gums (e.g. gum arabic, tragacanth or sodium alginate), carboxymethyl cellulose , Polyethylene glycol, etc.
  • Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like.
  • Disintegrating agents include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • a tablet is made by making a powdery mixture, granulating or pre-pressing, adding a lubricant and a disintegrant, and pressing into tablets.
  • a suitably comminuted compound with a diluent or base as described above optionally with a binder (e.g., carboxymethyl cellulose, alginate, gelatin, or polyvinylpyrrolidone), a dissolution inhibitor (e.g., paraffin), An absorption accelerator (quaternary salt) and / or an absorbent (such as bentonite, kaolin or dicalcium phosphate) are mixed to prepare a powdery mixture.
  • the powdery mixture can be granulated by being wetted with a binder (for example, syrup, starch syrup, gum arabic or a solution of a cellulose material or a polymeric material) and then pressed through a sieve.
  • a binder for example, syrup, starch syrup, gum arabic or a solution of a cellulose material or a polymeric material
  • An alternative method of granulation is to pass the powdery mixture through a tablet press, with the result that the poorly formed agglomerates are crushed and granulated.
  • the granules can be lubricated by adding stearic acid, stearates, talc or mineral oil to prevent sticking to the die of the tablet press.
  • the lubricated mixture is then compressed into tablets.
  • the compounds of the present invention can also be mixed with a free-flowing inert carrier and compressed into tablets without the need for granulation or pre-pressing steps.
  • Transparent or opaque protective coating materials consisting of shellac, sugar or polymeric coatings and waxy polishing coatings are available. Dyes can be added to these coating materials to distinguish different unit doses.
  • Oral liquid preparations such as solutions, syrups and elixirs can be prepared in dosage unit form so that a given amount contains a predetermined amount of the compound.
  • Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution
  • elixirs can be prepared by using a non-toxic vehicle.
  • Preservatives, flavoring additives such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners can also be added.
  • compositions suitable for transdermal administration can be used as discrete patches to maintain close contact with the epidermis of the recipient for a long time.
  • the active ingredient can be delivered by an iontophoretic patch, generally see Pharmaceutical Research, 1986, 3 (6), 318.
  • compositions suitable for topical administration can be prepared as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oil preparations or transdermal patches .
  • Pharmaceutical formulations suitable for nasal administration include coarse powders with a particle size in the range of, for example, 20-500 microns, which are administered by nasal suction, that is, quickly from a coarse powder container near the nose through the nasal passage Inhale.
  • Suitable formulations in which the carrier is a liquid and suitable for administration as a nasal spray or nasal drop include aqueous solutions or oily solutions of the active ingredient.
  • compositions suitable for administration by inhalation include fine particle powders or fine mists, which can be prepared in different types of metered doses of compressed aerosols, nebulizers, insufflators or other devices that deliver aerosol sprays.
  • Pharmaceutical formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions and aqueous and non-aqueous sterile suspensions.
  • Aqueous and non-aqueous sterile injectable solutions may contain antioxidants, buffers, and bacteriostatic agents. Agents and solutes that render the formulation isotonic with the blood of the recipient.
  • the formulations can be provided in unit-dose or multi-dose containers, such as sealed Ankai and vials, and can be stored under freeze-dried (lyophilized) conditions by adding a sterile liquid carrier, such as water for injection, immediately before use.
  • a sterile liquid carrier such as water for injection
  • injectable solutions for immediate use can be prepared from sterile powder injections, granules, and tablets.
  • compositions may be in unit dosage form, each unit dose containing a predetermined amount of the active ingredient.
  • the method of administration can be used as a long-term or short-term therapy.
  • the amount of active ingredient mixed with the carrier material to produce a single dosage form will depend on the disease to be treated, the severity of the disease, the time of administration, the route of administration, the excretion rate of the compound used, the time of treatment and the age, sex, weight and condition of the patient And change.
  • a preferred unit dosage form is a unit dosage form containing a daily or divided dose of the above-mentioned active ingredient or a suitable fraction thereof. Treatment can be initiated with small doses that are clearly below the optimal dose of the compound. Thereafter, the dose is increased in smaller increments until the best effect is achieved in this case. In general, it is most desirable to administer a compound at a concentration level that generally provides effective results in terms of antivirals without causing any harmful or toxic side effects.
  • the present invention provides a novel camptothecin derivative, and the solubility is significantly higher than that of SN38;
  • the tumor cell growth inhibitor and the prepared anticancer drug of the present invention have a broad anticancer spectrum, and the anticancer activity is better than that of irinotecan hydrochloride;
  • the tumor cell growth inhibitor and the prepared anticancer drug have high drug safety, and there are no obvious side effects when used at high doses.
  • FIG. 1 Tumor volume-time change map of nude mice
  • Figure 2 Plot of body weight versus time in nude mice.
  • Figure 3 Tumor volume-time plot of Compound 58 nude mice in a multidose compound in vivo antitumor activity study.
  • Figure 4 Tumor volume-time plot of Compound 67 nude mice in multidose compound in vivo antitumor activity studies.
  • Figure 5 A graph of time-to-body weight changes in multidose compounds in vivo antitumor activity studies.
  • the invention generally and specifically describes the materials and experimental methods used in the tests. Although many materials and methods of operation used to achieve the purpose of the present invention are well known in the art, the present invention is described here in as much detail as possible. It is clear to a person skilled in the art that in the following, unless otherwise specified, the materials and methods of operation used are well known in the art.
  • Solubility experiments were performed in equilibrium using the UV-Vis spectrophotometry of the fourth edition of the Pharmacopoeia of the People's Republic of China, 2015.
  • the UV-visible spectrophotometry was used to determine the solubility of the compounds in water, physiological saline, PBS at pH 7.4, and 0.1% Tween 80 aqueous solution. Solubility.
  • An appropriate amount of the compound was weighed and dissolved in 1 mL of a methanol solution to prepare a reference solution of a certain concentration, that is, C 0 .
  • C X (A X / A 0 ) C 0 ; where C X is the concentration of the test solution; A X is the absorbance of the test solution; C 0 is the concentration of the reference solution; A 0 is the absorbance of the reference solution.
  • the experimental results are shown in Table 1.
  • mice 4-5 week old Balb / c mice are housed in a specific pathogen-free (SPF) environment.
  • SPF pathogen-free
  • mice were inoculated with colon cancer cells SW-480 subcutaneously at a density of 1.0 ⁇ 10 7 each. After the tumors grew to about 100 mm 3 , they were randomly divided into 4 groups: a normal saline control group (Control group), Irinotecan hydrochloride group (CPT-11 group, 15 mg / kg or 0.024 mmol / kg), compound 58 group (13.73 mg / kg or 0.016 mmol / kg), compound 67 group (12.30 mg / kg or 0.016 mmol / kg)
  • the method of administration is to dissolve the compound in physiological saline, intravenously, once every three days, and continuously for 7 times. Tumor size and volume were measured every three days until dosing was complete. After the experiment, the nude mice were sacrificed by severing the neck, and the nude mice
  • Table 3 and Table 4 are mouse tumor volume-time change data and mouse weight-time change data, respectively.
  • Figures 1 and 2 are mouse tumor volume-time change maps and mouse weight-time change maps, respectively.
  • Table 3 and Figure 1 at a dose of two thirds of the moles of CPT-11, the inhibitory rates of compounds 58 and 67 on colon cancer cells SW-480 were significantly greater than those of CPT-11.
  • Table 4 and Figure 2 no weight loss was caused in the mice at the administered dose, indicating that the compound did not show significant toxicity at the administered dose.
  • mice Successfully vaccinated mice were randomly divided into 8 groups of 6 mice each: saline group (NS), CPT-11 group, compound 58 low-dose group, compound 58 medium-dose group, compound 58 high-dose group, and compound 67 low-dose Group, compound 67 middle dose group, and compound 67 high dose group.
  • the mice were administered via the tail vein of the mice every other day for 7 times.
  • the dose was CPT-11 group: 10 mg / kg; the high, middle and low dose groups of compounds 58 and 67 had molar ratios to irinotecan of 1/1, 1/2, and 1/4, respectively, ie compound 58 High dose group (H): 13.73 mg / kg, compound 58 medium dose group (M): 6.865 mg / kg, compound 58 low dose group (L): 3.433 mg / kg; compound 67 high dose group (H): 12.30 mg / kg, compound 67 medium dose group (M): 6.15 mg / kg, compound 67 low dose group (L): 3.075 mg / kg.
  • Tumor volume is measured every 2 days.
  • the maximum diameter (a) of the tumor is measured first, and then the longest diameter (b) perpendicular to the maximum diameter is measured.
  • the unit is mm.
  • the orbital venous blood of the mice was taken for routine blood tests, and then the mice were sacrificed by cervical dislocation, and the tumors were immediately removed and photographed. From day 0, the weight of each group of mice was measured and recorded every 2 days, the average weight of each group was calculated, and the curve of mouse body weight was plotted according to statistical data. After the end of the treatment period, the mice are anesthetized, the tumors are immediately removed, the tumor weights of each group are weighed and recorded, the average tumor weight of each group is calculated, and the tumor suppression rate (%) is calculated according to the following formula:
  • mice were harvested for H & E staining of the organs and immunohistochemistry of tumor tissue.
  • the curve of tumor volume growth was plotted with time as the abscissa and tumor volume as the ordinate; the curve of mouse body weight was plotted with time as the abscissa and mouse weight as the ordinate.
  • Changes in tumor volume growth and changes in body weight and time in nude mice are shown in Figure 5 and Table 6.
  • the growth curve of tumor volume and the change of body weight and time in nude mice are shown in Figure 3-5.

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Abstract

本发明涉及新型喜树碱衍生物及其应用、肿瘤细胞生长抑制剂和三元复合物以及提高喜树碱衍生物溶解性的方法。所述的喜树碱衍生物由式(I)所示的物质经糖基化三氮唑在R3位置修饰而成；所述式(I)所示的结构式中R1代表H、C1-10的烷基、C1-10的氘代烷基或C1-C10的卤代烷基；R2代表H、CH2N(CH3)2或CH2N(CD3)2；R4代表式(II)或H，其中X代表N，O或S；L代表多肽、C1-20直链烷基或其衍生物、C1-20直链或支链酰基衍生物、C2-100乙二醇或其衍生物。所述喜树碱衍生物溶解度较高，制备的抗癌药物具有抗癌谱广、安全性高的优点，体内抗癌活性优于盐酸伊立替康。

Description

新型喜树碱衍生物及其制备方法和应用 技术领域
本发明涉及新型喜树碱衍生物及其应用、肿瘤细胞生长抑制剂和三元复合物以及提高喜树碱衍生物溶解性的方法。所述喜树碱衍生物可制备抗肿瘤药物。
背景技术
喜树碱(Camptothecin，CPT)是最早由美国化学家Wall等于1966年从中国珙桐科植物喜树的皮、果实中提取出来的一种含有喹啉环的生物碱。早期研究发现CPT具有抗肿瘤的活性，但是，CPT在水中很差的溶解性却大大降低了其临床应用价值，之后，科学家虽然通过成盐的方法水解其分子中的酯环，增加了水溶性，但却伴随着其抗肿瘤活性的下降。此外，CPT自身有较强的副作用，如出血性膀胱炎，严重的骨髓抑制等。由于以上缺陷，20世纪70年代，科学家不得不停止CPT的临床研究。1985年，Hsiang等人发现CPT是拓扑异构酶I(ToPoI)的专属抑制剂，才使CPT的研究重获新生。国内外研究者在对其药理机制和构效关系研究的基础上，开发出一系列的喜树碱衍生物。盐酸伊立替康(Irinotecan hydrochloride，CPT-11)是一种水溶性喜树碱衍生物，临床上常用于治疗结直肠癌、肺癌和乳腺癌等，但其抗肿瘤活性依旧不高，且副作用大。开发各类喜树碱衍生物并提升其药用性能是肿瘤药物开发的挑战。
拓扑替康、贝洛替康、10-羟基喜树碱等喜树碱衍生物陆续被开发并运用于人体，但上述衍生物与人体的“兼容性”一直是制药界的重大挑战。比如，SN-38作为伊立替康的代谢产物，体外抗肿瘤活性是伊立替康的100～1000倍，但其溶解度极差，几乎不溶于常用的药物溶剂和水，因此成药性差(Santi等,J Med Chem.2014,57(6):2303-2314)，在临床上应用具有很大的局限性。
发明内容
有鉴于此，本发明的目的之一在于提供了一种喜树碱衍生物，其具有较高的水溶性。
为了实现上述目的，本发明采用以下方案：
所述的喜树碱衍生物由式1所示的物质经糖基化三氮唑在R 3位置修饰而成；所述式1所示的结构式中R 1代表H、C 1-10的烷基、C 1-10的氘代烷基或C 1-C 10的卤代烷基；R 2代表H、CH 2N(CH 3) 2或CH 2N(CD 3) 2；R 4代表
Figure PCTCN2019100802-appb-000001
或H，其中X代表N，O或S；L代表多肽、C 1-20直链烷基或其衍生物、C 1-20直链或支链酰基衍生物、C 2-100乙二醇或其衍生物，
Figure PCTCN2019100802-appb-000002
进一步，所述式I中，所述R 3代表
Figure PCTCN2019100802-appb-000003
或H，所述R 4代表
Figure PCTCN2019100802-appb-000004
或H，但所述R 3和R 4不同时为H；所述R 5代表单糖残基或寡糖残基；L代表多肽、C 1-C 20直链烷基或其衍生物、C 1-C 20直链或支链酰基衍生物、C 1-C 20乙二醇或其衍生物、
Figure PCTCN2019100802-appb-000005
其中Y为
Figure PCTCN2019100802-appb-000006
a为0-100的整数；b为1-100的整数；c为0-100的整数；d为0-100的整数。
进一步，所述式I中的所述R 1代表H或-CH 2CH 3；所述R 2代表H、-CH 2N(CH 3) 2或—CH 2N(CD 3) 2；所述R 3代表
Figure PCTCN2019100802-appb-000007
或H，所述R 4代表
Figure PCTCN2019100802-appb-000008
或H，但所述R 3和R 4不同时为H；所述R 5选自单糖残基或寡糖残基；X选自N或O；L选自C 1-20直链或支链烷烃及其衍生物、C 1-20直链或支链酰基衍生物、C 2-100乙二醇及其衍生物、
Figure PCTCN2019100802-appb-000009
其中Y为
Figure PCTCN2019100802-appb-000010
a为0-100的整数；b为1-100的整数；c为0-100的整数；d为0-100的整数。
进一步，所述a为0、2、4、6、8、10、12、14、16、18、20、30、40、50、60、70、80、90、100。
进一步，所述b为1、2、4、6、8、10、12、14、16、18、20、30、40、50、60、70、80、90、100。
进一步，所述c为0、2、4、6、8、10、12、14、16、18、20、30、40、50、60、70、80、90、100。
进一步，所述d为0、2、4、6、8、10、12、14、16、18、20、30、40、50、60、70、80、90、100。
进一步，所述R 1代表-CH 2CH 3；所述R 2代表H；所述R 3代表
Figure PCTCN2019100802-appb-000011
所述R 4代表H；所述X代表N或O；所述L代表
Figure PCTCN2019100802-appb-000012
其中所述Y为
Figure PCTCN2019100802-appb-000013
所述a为1-20的整数，b为1-20的整数，c为0-20的整数，d为0-20的整数。
进一步，所述的喜树碱衍生物为7-乙基-10-羟基喜树碱的衍生物，其结构式如式II所示，
Figure PCTCN2019100802-appb-000014
进一步，所述糖基化三氮唑如式III或IV所述的结构式，其中Y为
Figure PCTCN2019100802-appb-000015
Figure PCTCN2019100802-appb-000016
a为0-100的整数，b为1-100的整数，c为0-100的整数，d为0-100的整数，所述R 5为糖残基或寡糖残基；
Figure PCTCN2019100802-appb-000017
进一步，所述R 5选自式5-28的单糖残基中的任一：
Figure PCTCN2019100802-appb-000018
进一步，所述a或所述b为1-20的整数。
进一步，所述R 5选自单糖残基式5、6、18和19中的任一：
Figure PCTCN2019100802-appb-000019
进一步，所述衍生物为下列化合物之一：
Figure PCTCN2019100802-appb-000020
Figure PCTCN2019100802-appb-000021
进一步，所述X选自N或O。
进一步，所述Y为
Figure PCTCN2019100802-appb-000022
进一步，所述d为0-1的整数。
进一步，所述a为1，b为1-4的整数。
本发明的目的之二在于提供一种所述喜树碱衍生物的合成方法，该方法可应用到工业生产中。
为了实现上述目的，本发明采用以下方案：
所述方法包括以下步骤：
1)化学反应合成叠氮化合物；
2)化学反应合成端基炔；
3)将叠氮化合物和端基炔溶于THF-H 2O中，依次加入无水硫酸铜和抗坏血酸钠进行点击反应，然后室温搅拌过夜，浓缩后柱层析分离得所述喜树碱衍生物；
所述叠氮化合物为
Figure PCTCN2019100802-appb-000023
其中Z为无或O，e为0-20。
优选的，Z为无或O，e为0-1。
本发明的目的之三在于提供一种提高喜树碱衍生物溶解性的方法，该方法适用于工业化运用。
为了实现上述目的，本发明采用以下方案：
所述方法为对7-乙基-10-羟基喜树碱衍生物进行糖基化三氮唑修饰，7-乙基-10-羟基喜树碱衍生物的结构式如式2所示，糖基化三氮唑在R 3的位置进行修饰；所述式2所示的结构式中R 3代表
Figure PCTCN2019100802-appb-000024
或H；其中X代表N，O或S；L代表多肽、C 1-C 20直链烷基或其衍生物、C 1-C 20直链或支链酰基衍生物、C 1-C 20乙二醇或其衍生物、
Figure PCTCN2019100802-appb-000025
进一步，所述糖基化三氮唑如式3或4所述的结构式，其中Y为
Figure PCTCN2019100802-appb-000026
Figure PCTCN2019100802-appb-000027
a为0-100的整数，b为1-100的整数，c为0-100的整数，d为0-100的整数，所述R 5为糖残基或寡糖残基。
进一步，所述R 5的结构式如式5-28任一所示。
进一步，所述R 5选自单糖残基式5、6、18和19中的任一。
进一步，所述a为1，b为1-4的整数。
进一步，所述Y为
Figure PCTCN2019100802-appb-000028
进一步，所述d为0-1的整数。
本发明的目的之四在于提供一种肿瘤细胞生长抑制剂，该肿瘤细胞生长抑制剂具有抗癌作用。
为实现上述目的，本发明采用以下方案：
肿瘤细胞生长抑制剂，所述抑制剂由所述的喜树碱衍生物制备而成。
所述所述的喜树碱衍生物可含有药学上接受的任何载体和助剂。
进一步，所述肿瘤细胞生长抑制剂可通过和拓扑异构酶I、DNA形成三元复合物使DNA链断裂，抑制肿瘤细生长和促进肿瘤细胞凋亡。
进一步，所述肿瘤为结直肠瘤、肺瘤和乳腺瘤、肝癌、胃癌、食管癌、白血病、前列腺癌、骨肉瘤、宫颈癌、甲状腺癌、卵巢癌或胰腺癌。
本发明的目的之五在于提供一种三元复合物，其可以抑制肿瘤细胞生长。
为实现上述目的，本发明的技术方案为：
所述三元复合物由所述的肿瘤细胞生长抑制剂通过和拓扑异构酶I、DNA形成所述的三元复合物。所述三元复合物使DNA链断裂，抑制肿瘤细生长和促进肿瘤细胞凋亡。
本发明的目的之六在于提供所述喜树碱衍生物的应用，该应用为癌症治疗提供了新思路。
为实现上述目的，本发明采用以下方案：
所述的喜树碱衍生物在制备抗癌药物中的应用。
进一步，所述癌为结直肠癌、肺癌和乳腺癌、肝癌、胃癌、食管癌、白血病、前列腺癌、骨肉瘤、宫颈癌、甲状腺癌、卵巢癌或胰腺癌。
进一步，所述抗癌药物可通过和拓扑异构酶I、DNA形成三元复合物使DNA链断裂，抑制肿瘤细生长和促进肿瘤细胞凋亡以达到抗癌作用，所述肿瘤细胞 为SW-480和/或HT-29和/或HCT-116和/或A549和/或H1975和/或HepG2和/或BGC-823和/或ECA-109和/或K562和/或PC3和/或143B和/或MDA-MB-231和/或Hela和/或TPC-1和/或SKOV-3和/或PANC-1。
本发明的目的之七在于提供一种制剂，该制剂可以有效的抑制肿瘤细胞的生长。
为实现上述目的，本发明的技术方案为：
所述的喜树碱衍生物制备的制剂。
进一步，所述制剂中含有药学可接受载体和/或助剂。
所述药学可接受剂型包括本发明的中药组合物以及可选的一种或多种药学上可接受的载体、稀释剂或赋形剂并在上述制备过程中合适的步骤加入。本发明所使用的术语“药学上可接受的”是指这样的化合物、原料、组合物和/或剂型，它们在合理医学判断的范围内，适用于与患者组织接触而无过度毒性、刺激性、变态反应或与合理的利益/风险比相对称的其他问题和并发症，并有效用于既定用途。
药物制剂适于通过任何合适的途径给药，例如通过口服(包括口腔或舌下)、直肠、鼻、局部(包括口腔、舌下或经皮)、阴道或胃肠外(包括皮下、皮内、肌内、关节内、滑膜内、胸骨内、鞘内、病灶内、静脉内或者真皮下注射或输注)途径。可按药剂学领域的任何已知方法制备这类制剂，例如通过将活性成分与载体或赋形剂混合。优选口服给药、局部给药或注射给药。适于口服给药的药物制剂按独立的单位提供，例如水性或非水性液体中的溶液剂或混悬剂；胶囊剂或片剂；散剂或颗粒剂；可食用泡沫制剂或起泡制剂等。
举例来说，对于以片剂或胶囊剂形式的口服给药，活性药物组分可与药学上可接受的口服无毒惰性载体(例如乙醇、甘油、水等)相混合。通过将化合物粉碎成合适的微细尺寸，并与被同样粉碎的药用载体(例如淀粉或甘露醇等可食用的糖类)混匀来制备散剂。还可存在矫味剂、防腐剂、分散剂和着色剂。通过制备如上所述的粉状混合物，并装填到成形的明胶壳内，来制备胶囊剂。在装填操作之前，可将助流剂和润滑剂(例如胶态二氧化硅、滑石粉、硬脂酸 镁、硬脂酸钙或固态聚乙二醇)加到粉状混合物中。还可加入当服下胶囊剂时将改进药物可利用性的崩解剂或增溶剂(例如琼脂、碳酸钙或碳酸钠)。
此外需要或必需时，也可将合适的粘合剂、润滑剂、崩解剂和着色剂掺到混合物中。合适的粘合剂包括淀粉、明胶、天然糖(例如葡萄糖或β-乳糖)、玉米甜味剂、天然和合成树胶(例如阿拉伯树胶、西黄蓍胶或藻酸钠)、羧甲基纤维素、聚乙二醇等。用于这些剂型的润滑剂包括油酸钠、氯化钠等。
崩解剂包括但并不限于淀粉、甲基纤维素、琼脂、皂土、黄原胶等。例如，通过制成粉状混合物，制粒或预压片，加入润滑剂和崩解剂，压制成片，从而制成片剂。将适当粉碎的化合物与如上述所述的稀释剂或基料、任选与粘合剂(例如羧甲基纤维素、藻酸盐、明胶或聚乙烯吡咯烷酮)、溶解阻止剂(例如石蜡)、吸收加速剂(季盐)和/或吸收剂(例如皂土、高岭土或磷酸二钙)混合，来制备粉状混合物。可用粘合剂(例如糖浆、淀粉浆、阿拉伯胶浆或纤维素材料或聚合材料溶液)润湿后加压过筛，将粉状混合物制粒。制粒的一个替代方法是，可将粉状混合物通过压片机，结果是将形成不佳的团块再击碎制成颗粒。可通过加入硬脂酸、硬脂酸盐，滑石粉或矿物油使颗粒润滑以防止粘到压片机的冲模上。然后将经润滑的混合物压制成片。本发明内容的化合物还可与自由流动的惰性载体混合，无需通过制粒或预压片步骤便可压制成片。可提供透明或不透明的由虫胶密封衣、糖衣或聚合材料衣和蜡质抛光衣组成的保护性包衣材料。可将染料加到这些包衣材料中以区分不同的单位剂量。
口服液体制剂例如溶液剂、糖浆剂和酏剂可以剂量单位形式制备，从而给定量含有预定量的化合物。糖浆剂可通过将化合物溶于适当调味的水溶液中来制备，而酏剂可通过使用无毒溶媒来制备。还可加入防腐剂、矫味添加剂(例如薄荷油或天然甜味剂或糖精或其他人造甜味剂)等。
适于经皮给药的药物制剂可作为离散的贴剂以在长时间内保持与接受者表皮密切接触。例如，活性成分可由通过离子导入贴剂递药，通常可参见Pharmaceutical Research,1986,3(6),318。
适于局部给药的药物制剂可制成软膏剂、乳膏剂、混悬剂、洗剂、散剂、 溶液剂、糊剂、凝胶剂、喷雾剂、气雾剂、油制剂或透皮贴剂。适于经鼻给药的药物制剂(其中载体为固体)包括粒径为例如20-500微米范围的粗粉剂，通过以鼻吸方式给药，即通过鼻通道从接近鼻子的粗粉剂容器中快速吸入。其中载体为液体、适于作为鼻腔喷雾剂或滴鼻剂给药的合适制剂包括活性成分的水性溶液剂或油性溶液剂。
适于通过吸入给药的药物制剂包括微细粒子粉剂或细雾剂，可用不同类型计量的剂量压缩气溶胶、雾化吸入器、吹入器或其他事宜递送气溶胶喷雾剂的装置中制备。适于胃肠外给药的药物制剂包括水性和非水性无菌注射溶液剂及水性和非水性无菌混悬剂，水性和非水性无菌注射溶液剂可含有抗氧化剂、缓冲剂、抑菌剂和使所述制剂与待接受者血液等渗的溶质。制剂可以单位剂量或多剂量容器提供，例如密封的安凯和小瓶，并可保存在冷冻干燥(冻干)条件下，只需在临用前加入无菌液体载体，例如注射用水。临用时配置的注射溶液剂可由无菌粉针剂、颗粒剂和片剂制备。
药物制剂可呈单位剂型，每个单位剂量含有预定量的活性成分。给药法可用作长期或短期疗法。与载体材料混合以制备单一剂型的活性成分的量将根据待治疗的疾病、疾病的严重程度、给药时间、给药途径、所用化合物的排泄速率、治疗时间和患者年龄、性别、体重和情况而改变。优选的单位剂型是含有上述活性成分的日剂量或分剂量或其适宜分数的单位剂型。可用显然低于化合物最佳剂量的小剂量开始治疗。此后，以较小的增量来加大剂量直到在这种情况下达到最佳效果。一般而言，最理想地给予化合物的浓度水平是通常可在抗病毒方面提供有效结果而又不至于引起任何有害或有毒的副作用。
本发明有益效果：
1)本发明提供了新型的喜树碱衍生物，且溶解度显著比SN38高；
2)本发明所述的肿瘤细胞生长抑制剂和制备的抗癌药物抗癌谱广，抗癌活性优于盐酸伊立替康；
3)所述肿瘤细胞生长抑制剂和制备的抗癌药物用药安全性高，高剂量使用也没有明显副作用产生。
附图说明
图1：裸鼠肿瘤体积-时间变化图；
图2：裸鼠体重-时间变化图。
图3：多剂量化合物体内抗肿瘤活性研究中化合物58裸鼠肿瘤体积-时间变化图。
图4：多剂量化合物体内抗肿瘤活性研究中化合物67裸鼠肿瘤体积-时间变化图。
图5：多剂量化合物体内抗肿瘤活性研究中体重时间变化图。
具体实施方式
下面通过具体实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细的具体说明用，而不应理解为用于以任何形式限制本发明。
本发明对试验中所使用到的材料以及实验方法进行一般性和具体性的描述。虽然为实现本发明的目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细的描述。本领域技术人员清楚，在下文中，如未特别说明，所使用的材料和操作方法是本领域公知的。
实施例1 化合物37的合成
Figure PCTCN2019100802-appb-000029
称量9g化合物29溶于60mL二氯甲烷中，加入25mL 33％氢溴酸的醋酸溶液，室温反应2小时，TLC监测反应完全，倒入50mL水中，二氯甲烷萃取(50mL×3)，合并有机层并用饱和碳酸氢钠洗涤一次，无水硫酸钠干燥，过滤，浓缩后，用石油醚/乙酸乙酯10/1的混合溶剂重结晶后得到8.2g化合物30，收率86.7％。通过核磁共振氢谱，鉴定化合物结构。
1H NMR(400Hz,CDCl 3)δ6.61(d,J＝4.4Hz,1H),5.56(t,J＝9.6Hz,1H),5.16(t,J＝9.8Hz,1H),4.84(dd,J＝10.0,4.0Hz,1H),4.28-4.35(m,2H),4.12-4.15(m,1H),2.10(s,3H),2.09(s,3H),2.05(s,3H),2.04(s,3H)。
将8.2g化合物30溶于40mL干燥的DMSO中，加入1.56g叠氮化钠，室温搅拌1小时，TLC监测反应完全，倒入100mL水中，乙酸乙酯萃取(50mL×3)，合并有机层并用无水硫酸钠干燥，过滤，浓缩后，用石油醚/乙酸乙酯10/1的混合溶剂重结晶后得到5.8g化合物31，收率77.7％。
1H NMR(400Hz,CDCl 3)δ5.22(t,J＝9.4Hz,1H),5.11(t,J＝9.6Hz,1H),4.96(t,J＝9.2Hz,1H),4.65(d,J＝8.8Hz,1H),4.26-4.30(m,1H),4.16-4.19(m,1H),3.78-3.82(m,1H),2.11(s,3H),2.08(s,3H),2.03(s,3H),2.01(s,3H)。
称量1g化合物31溶于5mL无水甲醇中，加入10mL 0.5N NaOMe-MeOH溶液，室温搅拌过夜，加入适量的强酸性阳离子交换树脂，继续搅拌15分钟，测量体系中pH为中性或弱酸性，过滤，滤液浓缩后得到420mg化合物32，收率76.4％。
1H NMR(400Hz,D 2O)δ4.68(d,J＝8.8Hz,1H),3.84-3.88(m,1H),3.66-3.71(m,1H),3.46-3.48(m,2H),3.29-3.38(m,1H),3.21(t,J＝9.0Hz,1H)。
将23mL乙二醇(化合物33)溶于100mL干燥的四氢呋喃中，冷却至0℃，分小批量加入5.5g氢化钠，加入完毕后，继续在此温度下反应2小时。称量10g 3-溴丙炔溶于30mL干燥的四氢呋喃中，并缓慢的滴加至上述反应中，完毕后室温反应过夜。向反应体系中加入20mL水，浓缩掉四氢呋喃，乙酸乙酯萃取(40mL×3)，合并有机层，无水硫酸钠干燥，过滤，浓缩后柱层析得5.5g化合物34，收率64.9％。
1H NMR(400Hz,DMSO-d 6)δ4.64(t,J＝5.6Hz,1H),4.14(d,J＝2.4Hz,2H),3.49-3.52(m,2H),3.44-3.47(m,2H),3.88(t,J＝2.4Hz,1H)。
称量1g化合物34溶于50mL二氯甲烷中，加入2g三乙胺和3g对硝基苯基氯甲酸酯，室温搅拌过夜，浓缩后柱层析得1g化合物35，收率37.7％。
称量392mg SN38溶于20mL 1:1 DCM-DMF的混合溶剂中，依次加入200mg三乙胺和310mg化合物35，室温搅拌过夜，减压真空浓缩，柱层析得300mg化合物36，收率57.9％。
1H NMR(400Hz,DMSO-d 6)δ8.22(d,J＝9.2Hz,1H),8.16(d,J＝2.8Hz,1H),7.78(dd,J＝9.2,2.4Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),4.41-4.43(m,2H),4.24(d,J＝2.4Hz,2H),3.77-3.79(m,2H),3.51(t,J＝2.4Hz,1H),3.18-3.21(m,2H),1.85-1.90(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.4Hz,3H)。
将157mg化合物36和63mg化合物32溶于10mL 1:1 THF-H 2O中，依次加入25mg无水硫酸铜和30mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到135mg化合物37，收率61.6％。
1H NMR(400Hz,DMSO-d 6)δ8.34(s,1H),8.23(d,J＝9.2Hz,1H),8.17(d,J＝2.4Hz,1H),7.77(dd,J＝9.2,2.8Hz,1H),7.34(s,1H),6.52(s,1H),5.55(d,J＝9.2Hz,1H),5.44(s,2H),5.38(d,J＝6.0Hz,1H),5.34(s,2H),5.27(d,J＝4.8Hz,1H),5.14(d,J＝5.6Hz,1H),4.62-4.63(m,3H),4.41-4.44(m,2H),3.69-3.82(m,4H),3.40-3.46(m,3H),3.19-3.24(m,3H),1.86-1.89(m,2H),1.29(t,J＝7.6Hz,3H),0.88(t,J＝7.2Hz,3H)。
实施例2 化合物42的合成
Figure PCTCN2019100802-appb-000030
将80mL四甘醇(化合物38)溶于200mL干燥的四氢呋喃中，冷却至0℃，分小批量加入5.5g氢化钠，加入完毕后，继续在此温度下反应2小时。称量10g 3-溴丙炔溶于30mL干燥的四氢呋喃中，并缓慢的滴加至上述反应中，完毕后室温反应过夜。向反应体系中加入20mL水，浓缩掉四氢呋喃，乙酸乙酯萃取(60mL×3)，合并有机层，无水硫酸钠干燥，过滤，浓缩后柱层析得11.7g化合物39，收率59.5％。
1H NMR(400Hz,CDCl 3)δ4.20-4.24(m,2H),3.67-3.71(m,14H),3.60-3.62(m,2H),2.46(t,J＝2.4Hz,1H)。
称量1g化合物39溶于50mL二氯甲烷中，加入870mg三乙胺和1.3g对硝基苯基氯甲酸酯，室温搅拌过夜，浓缩后柱层析得600mg化合物40，收率35.1％。
称量392mg SN38溶于20mL 1:1 DCM-DMF的混合溶剂中，依次加入200mg三乙胺和397mg化合物40，室温搅拌过夜，减压真空浓缩，柱层析得391mg化合物41，收率60.3％。
1H NMR(400Hz,DMSO-d 6)δ8.22(d,J＝9.2Hz,1H),8.16(d,J＝2.4Hz,1H),7.78(dd,J＝9.2,2.8Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.33(s,2H),4.38-4.41(m,2H),4.14(d,J＝2.4Hz,2H),3.73-3.76(m,2H),3.51-3.57(m,12H),3.40(t,J＝2.4Hz,1H),3.18-3.21(m,2H),1.86-1.90(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.4Hz,3H)。
将160mg化合物41和65mg化合物32溶于10mL 1:1 THF-H 2O中，依次加入20mg无水硫酸铜和24mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到60mg化合物42，收率28.5％。
1H NMR(400Hz,DMSO-d 6)δ8.27(s,1H),8.23(d,J＝9.2Hz,1H),8.17(d,J＝2.8Hz,1H),7.78(dd,J＝9.2,2.8Hz,1H),7.34(s,1H),6.52(s,1H),5.51(d,J＝9.6Hz,1H),5.44(s,2H),5.36(d,J＝6.0Hz,1H),5.34(s,2H),5.26(d,J＝4.8Hz,1H),5.13(d,J＝5.6Hz,1H),4.62(t,J＝5.6Hz,1H),4.53(s,2H),4.38-4.40(m,2H),3.72-3.76(m,4H),3.53-3.57(m,10H),3.42-3.46(m,2H),3.18-3.28(m,4H),1.84-1.90(m,4H),1.29(t,J＝7.6Hz,3H),0.87(t,J＝7.6Hz,3H)。
实施例3 化合物47的合成
Figure PCTCN2019100802-appb-000031
将46mL二甘醇(化合物43)溶于100mL干燥的四氢呋喃中，冷却至0℃，分小批量加入5.5g氢化钠，加入完毕后，继续在此温度下反应2小时。称量10g 3-溴丙炔溶于30mL干燥的四氢呋喃中，并缓慢的滴加至上述反应中，完毕后室温反应过夜。向反应体系中加入20mL水，浓缩掉四氢呋喃，乙酸乙酯萃取(50mL×3)，合并有机层，无水硫酸钠干燥，过滤，浓缩后柱层析得5.53g化合物44，收率45.3％。
1H NMR(400Hz,DMSO-d 6)δ4.56(t,J＝5.6Hz,1H),4.14(d,J＝2.4Hz,2H),3.51-3.59(m,4H),3.46-3.49(m,2H),3.40-3.44(m,3H)。
称量1g化合物44溶于50mL二氯甲烷中，加入1.4g三乙胺和2.1g对硝基苯基氯甲酸酯，室温搅拌过夜，浓缩后柱层析得603mg化合物45，收率28.1％。
1H NMR(400Hz,DMSO-d 6)δ7.56-7.58(m,2H),6.91-6.95(m,2H),4.37-4.39 (m,2H),4.16(d,J＝2.4Hz,2H),3.70-3.72(m,2H),3.59-3.62(m,4H),3.42(t,J＝2.4Hz,1H)。
称量392mg SN38溶于20mL 1:1 DCM-DMF的混合溶剂中，依次加入200mg三乙胺和309mg化合物45，室温搅拌过夜，减压真空浓缩，柱层析得357mg化合物46，收率63.6％。
1H NMR(400Hz,DMSO-d 6)δ8.23(d,J＝9.2Hz,1H),8.16(d,J＝2.4Hz,1H),7.78(dd,J＝9.2,2.8Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),4.38-4.41(m,2H),4.18(d,J＝2.4Hz,2H),3.72-3.75(m,2H),3.60-3.62(m,4H),3.44(t,J＝2.4Hz,1H),3.18-3.21(m,2H),1.86-1.90(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.4Hz,3H)。
将95mg化合物46和35mg化合物32溶于10mL 1:1 THF-H 2O中，依次加入27mg无水硫酸铜和33mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到80mg化合物47，收率61.6％。
1H NMR(400Hz,DMSO-d 6)δ8.31(s,1H),8.22(d,J＝9.2Hz,1H),8.17(d,J＝3.0Hz,1H),7.78(dd,J＝9.2,2.8Hz,1H),7.34(s,1H),6.52(s,1H),5.53(d,J＝9.2Hz,1H),5.44(s,2H),5.13-5.33(m,5H),4.57-4.63(m,3H),4.38-4.40(m,2H),3.67-3.79(m,4H),3.64(s,4H),3.37-3.45(m,3H),3.17-3.25(m,3H),1.82-1.91(m,2H),1.29(t,J＝7.6Hz,3H),0.89(t,J＝7.6Hz,3H)。
实施例4 化合物52的合成
Figure PCTCN2019100802-appb-000032
将69mL三甘醇(化合物48)溶于150mL干燥的四氢呋喃中，冷却至0℃，分小批量加入5.5g氢化钠，加入完毕后，继续在此温度下反应2小时。称量10g 3-溴丙炔溶于30mL干燥的四氢呋喃中，并缓慢的滴加至上述反应中，完毕后室温反应过夜。向反应体系中加入20mL水，浓缩掉四氢呋喃，乙酸乙酯萃取(50mL×3)，合并有机层，无水硫酸钠干燥，过滤，浓缩后柱层析得7.6g化合物49，收率47.8％。
1H NMR(400Hz,DMSO-d 6)δ4.54(t,J＝5.4Hz,1H),4.14(d,J＝2.4Hz,2H),3.47-3.53(m,10H),3.36-3.43(m,3H)。
称量1g化合物49溶于50mL二氯甲烷中，加入1.1g三乙胺和1.6g对硝基苯基氯甲酸酯，室温搅拌过夜，浓缩后柱层析得685mg化合物50，收率36.5％。
称量392mg SN38溶于20mL 1:1 DCM-DMF的混合溶剂中，依次加入200mg三乙胺和353mg化合物50，室温搅拌过夜，减压真空浓缩，柱层析得375mg化合物51，收率61.9％。
1H NMR(400Hz,DMSO-d 6)δ8.22(d,J＝9.2Hz,1H),8.16(d,J＝2.4Hz,1H),7.78(dd,J＝9.2,2.4Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),4.38-4.41(m,2H),4.15(d,J＝2.4Hz,2H),3.73-3.75(m,2H),3.56-3.62(m,8H),3.41(t,J＝2.4Hz,1H),3.17-3.23(m,2H),1.84-1.91(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.4Hz,3H)。
将146mg化合物51和50mg化合物32溶于10mL 1:1 THF-H 2O中，依次加入39mg无水硫酸铜和48mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到75mg化合物52，收率38.5％。
1H NMR(400Hz,DMSO-d 6)δ8.29(s,1H),8.19(d,J＝9.2Hz,1H),8.17(d,J＝2.8Hz,1H),7.78(dd,J＝9.2,2.8Hz,1H),7.34(s,1H),6.52(s,1H),5.52(d,J＝9.6Hz,1H),5.44(s,2H),5.36(d,J＝6.0Hz,1H),5.34(s,2H),5.26(d,J＝4.8Hz,1H),5.13(d,J＝5.6Hz,1H),4.62(t,J＝5.6Hz,1H),4.55(s,2H),4.38-4.40(m,2H),3.67-3.77(m,4H),3.56-3.61(m,8H),3.36-3.46(m,3H),3.16-3.24(m,3H),1.86-1.90(m,2H),1.30(t,J＝7.2Hz,3H),0.89(t,J＝7.2Hz,3H)。
实施例5 化合物56的合成
Figure PCTCN2019100802-appb-000033
称量18g化合物29溶于150mL二氯甲烷，加入6.3g溴乙醇和8.7mL三氟化硼乙醚，室温反应过夜。加入饱和碳酸氢钠直至没有气体产生，用DCM萃取三次，有机层合并干燥，浓缩，柱层析得到10g化合物53，收率47.7％。
1H NMR(400Hz,CDCl 3)δ5.25(t,J＝9.6Hz,1H),4.87-4.93(m,2H),4.77-4.81(m,1H),4.15-4.20(m,1H),3.96-4.00(m,2H),3.77-3.82(m,1H),3.55-3.62(m,2H),2.02(s,3H),2.00(s,3H),1.98(s,3H),1.94(s,3H)。
称量10g化合物53溶于150mLDMF中，加入2.86g叠氮化钠。60℃反应过夜。TLC监测反应完全。冷却至室温，加入适量的水，乙酸乙酯萃取两次(100mL×3)，浓缩掉有几层和残留的少量DMF，加入适量乙醇析出固体，过滤得到6.9g产物54,收率75.2％。
1H NMR(400Hz,CDCl 3)δ5.22(t,J＝9.4Hz,1H),5.10(t,J＝9.6Hz,1H),5.00-5.04(m,1H),4.60(d,J＝8.0Hz,1H),4.24-4.28(m,1H),4.14-4.17(m,1H),4.02-4.05(m,1H),3.67-3.74(m,2H),3.46-3.53(m,1H),3.27-3.32(m,1H),2.09(s,3H),2.05(s,3H),2.03(s,3H),2.01(s,3H)。
称量1g化合物54溶于5mL无水甲醇中，加入10mL 0.5N NaOMe-MeOH溶液，室温搅拌过夜，加入适量的强酸性阳离子交换树脂，继续搅拌15分钟，测量体系中pH为中性或弱酸性，过滤，滤液浓缩后得到430mg化合物55，收率 72.0％。
1H NMR(400Hz,D 2O)δ4.44(d,J＝8.0Hz,1H),3.97-4.02(m,1H),3.84-3.88(m,1H),3.76-3.80(m,1H),3.64-3.69(m,1H),3.38-3.51(m,4H),3.29-3.35(m,1H),3.24(t,J＝8.0Hz,1H)。
将170mg化合物36和82mg化合物55溶于16mL THF-H 2O中，依次加入26mg无水硫酸铜和33mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到110mg化合物56，收率43.8％。
1H NMR(400Hz,DMSO-d 6)δ8.22(d,J＝9.2Hz,1H),8.20(s,1H),8.17(d,J＝3.0Hz,1H),7.78(dd,J＝9.2,3.0Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),4.91-5.01(m,3H),4.57-4.61(m,4H),4.40-4.42(m,2H),4.24(d,J＝8.0Hz,1H),4.07-4.11(m,1H),3.89-3.95(m,1H),3.78-3.80(m,2H),3.68(d,J＝5.6Hz,1H),3.42-3.46(m,1H),2.96-3.17(m,7H),1.82-1.91(m,2H),1.31(t,J＝7.4Hz,3H),0.89(t,J＝7.4Hz,3H)。
实施例6 化合物57的合成
Figure PCTCN2019100802-appb-000034
将80mg化合物46和36mg化合物55溶于14mL THF-H 2O中，依次加入23mg无水硫酸铜和28mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到50mg化合物57，收率43.3％。
1H NMR(400Hz,DMSO-d 6)δ8.22(d,J＝9.2Hz,1H),8.16(s,2H),7.78(dd,J＝9.2,2.4Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),5.07(d,J＝4.8Hz,1H),4.93(dd,J＝12.8,4.8Hz,2H),4.49-4.57(m,5H),4.39(t,J＝4.4Hz,2H),4.23(d,J＝7.6Hz,1H),4.07-4.10(m,1H),3.89-3.92(m,1H),3.62-3.75(m,7H),3.42-3.45(m, 1H),3.10-3.19(m,4H),2.97-3.05(m,2H),1.86-1.90(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.2Hz,3H)。
实施例7 化合物58的合成
Figure PCTCN2019100802-appb-000035
将98mg化合物51和40mg化合物55溶于14mL THF-H 2O中，依次加入26mg无水硫酸铜和32mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到65mg化合物58，收率48.5％。
1H NMR(400Hz,DMSO-d 6)δ8.22(d,J＝9.2Hz,1H),8.16(d,J＝2.4Hz,1H),8.15(s,1H),7.78(dd,J＝9.2,2.4Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),5.07(d,J＝3.2Hz,1H),4.92(dd,J＝12.8,4.8Hz,2H),4.49-4.58(m,5H),4.38-4.39(m,2H),4.22(d,J＝8.0Hz,1H),4.07-4.09(m,1H),3.89-3.92(m,1H),3.73-3.75(m,2H),3.65-3.69(m,1H),3.56-3.61(m,8H),3.42-3.45(m,1H),3.10-3.18(m,4H),2.96-3.05(m,2H),1.86-1.90(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.2Hz,3H)。
实施例8 化合物59的合成
Figure PCTCN2019100802-appb-000036
将316mg化合物41和121mg化合物55溶于16mL THF-H 2O中，依次加入78mg无水硫酸铜和96mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到100mg化合物59，收率22.9％。
1H NMR(400Hz,DMSO-d 6)δ8.23(d,J＝9.2Hz,1H),8.16(s,1H),8.14(s,1H),7.78(d,J＝9.2Hz,1H),7.34(s,1H),6.52(s,1H),5.44(s,2H),5.34(s,2H),5.06(d,J＝4.8Hz,1H),4.92(dd,J＝12.8,4.8Hz,2H),4.51-4.56(m,5H),4.39(s,2H),4.22(d,J＝7.6Hz,1H),4.06-4.09(m,1H),3.89-3.91(m,1H),3.66-3.74(m,3H),3.53-3.55(m,11H),3.37-3.44(m,1H),3.13-3.18(m,5H),2.96-3.04(m,2H),1.86-1.90(m,2H),1.30(t,J＝7.2Hz,3H),0.89(t,J＝6.8Hz,3H)。
实施例9 化合物66的合成
Figure PCTCN2019100802-appb-000037
称量5g化合物60和16.6g三乙胺溶于150mL二氯甲烷中，冷却至0℃,缓慢的滴加11.8g二碳酸二叔丁酯，滴加完毕后，室温反应过夜，反应液用1N HCl洗涤(100mL×3),有机层用无水硫酸钠干燥，过滤，浓缩得到5g化合物61，收率37.9％。
1H NMR(400Hz,DMSO-d 6)δ6.45(s,1H),4.57(t,J＝5.6Hz,1H),3.33-3.38(m,2H),2.95-2.99(m,2H),1.37(s,9H)。
称量2g化合物61溶于40mL四氢呋喃中，加入458mg四丁基碘化铵，280mg碘化钠和1.77g 3-溴丙炔，室温搅拌下分小批量加入7g氢氧化钾，完毕后室温反应过夜，浓缩掉四氢呋喃，加入30mL水，乙酸乙酯萃取(30mL×3),有机层 用无水硫酸钠干燥，过滤，浓缩柱层析得到1.9g化合物62，收率76.9％。
称量1.8g化合物62溶于20mL甲醇中，加入20mL 3N HCl溶液，室温搅拌过夜，浓缩得到900mg化合物63，收率73.8％。
1H NMR(400Hz,D 2O)δ4.22(s,2H),3.78(t,J＝4.2Hz,2H),3.18(t,J＝4.0Hz,2H)。
称量490mg SN38溶于30mLDMF中，冷却至0℃,加入320mg DIPEA和330mg对硝基苯基氯甲酸酯，在此温度下继续反应半小时，室温反应3小时得到中间体64，向其中加入320mg DIPEA和253mg化合物63。加入完毕后室温反应过夜，浓缩掉DMF，TLC监测反应良好。柱层析分离得到420mg粗品产物65。
将210mg化合物65和160mg化合物32于16mL THF-H 2O中，依次加入124mg无水硫酸铜和153mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到60mg化合物66，两步收率13.3％。
1H NMR(400Hz,DMSO-d 6)δ8.33(s,1H),8.17(d,J＝8.8Hz,1H),8.04-8.05(m,1H),7.96(s,1H),7.64(d,J＝9.2Hz,1H),7.33(s,1H),6.51(s,1H),5.54(d,J＝9.2Hz,1H),5.44(s,2H),5.38(d,J＝6.0Hz,1H),5.33(s,2H),5.28(d,J＝4.8Hz,1H),5.14(d,J＝5.2Hz,1H),4.60-4.62(m,3H),3.68-3.78(m,2H),3.60(t,J＝5.0Hz,2H),3.37-3.46(m,4H),3.16-3.26(m,4H),1.86-1.90(m,2H),1.30(t,J＝7.4Hz,3H),0.89(t,J＝7.2Hz,3H)。
实施例10 化合物67的合成
Figure PCTCN2019100802-appb-000038
将210mg粗品化合物65和160mg化合物55溶于16mL THF-H 2O中，依次 加入124mg无水硫酸铜和153mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到60mg化合物67，收率12.5％。
1H NMR(400Hz,DMSO-d 6)δ8.16-8.19(m,2H),8.01(t,J＝1.6Hz,1H),7.95(d,J＝2.4Hz,1H),7.64(dd,J＝9.2,2.4Hz,1H),7.33(s,1H),6.51(s,1H),5.44(s,2H),5.34(s,2H),5.08(d,J＝4.8Hz,1H),4.94(dd,J＝14.4,4.8Hz,2H),4.50-4.58(m,5H),4.23(d,J＝7.6Hz,1H),4.07-4.11(m,1H),3.90-3.93(m,1H),3.66-3.70(m,1H),3.58(t,J＝5.6Hz,2H),3.41-3.46(m,2H),3.10-3.20(m,5H),2.95-3.07(m,2H),1.84-1.91(m,2H),1.30(t,J＝7.4Hz,3H),0.88(t,J＝7.2Hz,3H)。
实施例11 化合物73的合成
Figure PCTCN2019100802-appb-000039
称量10g化合物68和19.2g三乙胺溶于150mL二氯甲烷中，冷却至0℃,缓慢的滴加22.9g二碳酸二叔丁酯，滴加完毕后，室温反应过夜，反应液用1N HCl洗涤(100mL×3),有机层用无水硫酸钠干燥，过滤，浓缩得到7.5g化合物69，收率38.5％。
1H NMR(400Hz,DMSO-d 6)δ6.75(s,1H),4.55-4.58(m,1H),3.46-3.50(m,2H),3.36-3.40(m,4H),3.05-3.09(m,2H),1.38(s,9H)。
称量2g化合物69溶于40mL四氢呋喃中，加入360mg四丁基碘化铵，220mg碘化钠和1.39g 3-溴丙炔，室温搅拌下分小批量加入5.5g氢氧化钾，完毕后室温反应过夜，浓缩掉四氢呋喃，加入30mL水，乙酸乙酯萃取(30mL×3),有机层用无水硫酸钠干燥，过滤，浓缩柱层析得到1.4g化合物70，收率59.1％。
称量1.3g化合物70溶于20mL甲醇中，加入20mL 3N HCl溶液，室温搅拌过夜，浓缩得到600mg化合物71，收率62.7％。
1H NMR(400Hz,D 2O)δ4.21(d,J＝2.4Hz,2H),3.68-3.74(m,6H),3.16-3.18(m,2H),2.87(t,J＝2.2Hz,1H)。
称量490mg SN38溶于30mLDMF中，冷却至0℃,加入320mg DIPEA和251mg对硝基苯基氯甲酸酯，在此温度下继续反应半小时，室温反应3小时后得到中间体64，向其中加入320mg DIPEA和269mg化合物71。加入完毕后室温反应过夜，浓缩掉DMF，TLC监测反应良好。柱层析分离得到330mg粗品产物72。将165mg化合物72和117mg化合物32溶于16mL THF-H 2O中，依次加入91mg无水硫酸铜和113mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到60mg化合物73，两步收率12.5％。
1H NMR(400Hz,DMSO-d 6)δ8.31(s,1H),8.18(d,J＝9.2Hz,1H),7.95-8.02(m,2H),7.64(dd,J＝9.2,2.0Hz,1H),7.33(s,1H),6.52(s,1H),5.53(d,J＝9.2Hz,1H),5.44(s,2H),5.34-5.38(m,3H),5.27(d,J＝4.8Hz,1H),5.14(d,J＝5.6Hz,1H),4.63(t,J＝5.6Hz,1H),4.58(s,2H),3.52-3.78(m,8H),3.36-3.45(m,4H),3.17-3.24(m,4H),1.86-1.90(m,2H),1.30(t,J＝7.4Hz,3H),0.88(t,J＝7.4Hz,3H)。
实施例12 化合物74的合成
Figure PCTCN2019100802-appb-000040
将165mg粗品化合物72和125mg化合物55溶于16mL THF-H 2O中，依次加入91mg无水硫酸铜和113mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到50mg化合物74，两步收率9.9％。
1H NMR(400Hz,DMSO-d 6)δ8.16-8.18(m,2H),8.00(d,J＝5.6Hz,1H),7.95(s,1H),7.63(d,J＝8.8Hz,1H),7.33(s,1H),6.51(s,1H),5.44(s,2H),5.33(s,2H),5.07(d,J＝4.8Hz,1H),4.93(dd,J＝13.2,4.4Hz,2H),4.49-4.58(m,5H),4.23(d,J＝6.8Hz,1H),4.07-4.09(m,1H),3.89-3.92(m,1H),3.60-3.70(m,5H),3.53(t,J＝6.0Hz,2H),3.39-3.43(m,1H),3.28-3.31(m,2H),3.10-3.20(m,4H),2.96-2.99(m,2H),1.84-1.89(m,2H),1.29(t,J＝7.6Hz,3H),0.89(t,J＝7.2Hz,3H)。
实施例13 化合物80的合成
Figure PCTCN2019100802-appb-000041
称量5g化合物75和13.4g三乙胺溶于150mL二氯甲烷中，冷却至0℃,缓慢的滴加14.5g二碳酸二叔丁酯，滴加完毕后，室温反应过夜，反应液用1N HCl洗涤(100mL×3),有机层用无水硫酸钠干燥，过滤，浓缩得到7g化合物76，收率60.3％。
1H NMR(400Hz,DMSO-d 6)δ6.72(s,1H),4.37(t,J＝9.2Hz,1H),3.36-3.41(m,2H),2.93-2.98(m,2H),1.48-1.55(m,2H),1.37(s,9H)。
称量3.5g化合物76溶于60mL四氢呋喃中，加入1.09g四丁基碘化铵，450mg碘化钠和2.85g 3-溴丙炔，室温搅拌下分小批量加入11.2g氢氧化钾，完毕后室温反应过夜，浓缩掉四氢呋喃，加入30mL水，乙酸乙酯萃取(30mL×3),有机层用无水硫酸钠干燥，过滤，浓缩柱层析得到2.4g化合物77，收率56.3％。
称量1.3g化合物77溶于20mL甲醇中，加入20mL 3N HCl溶液，室温搅拌过夜，浓缩得到620mg化合物78，收率68.2％。
称量392mg化合物SN38溶于30mLDMF中，冷却至0℃,加入320mg DIPEA 和266mg对硝基苯基氯甲酸酯，在此温度下继续反应半小时，室温反应3小时后得到中间体64，然后加入320mg DIPEA和224mg化合物78。加入完毕后室温反应过夜，浓缩掉DMF，TLC监测反应良好。柱层析分离得到440mg粗品产物79。将220mg化合物79和214mg化合物32溶于16mL THF-H 2O中，依次加入166mg无水硫酸铜和206mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到70mg化合物80，两步收率19.0％。
1H NMR(400Hz,DMSO-d 6)δ8.30(s,1H),8.16(d,J＝9.2Hz,1H),7.94-7.97(m,2H),7.64(dd,J＝9.2,2.4Hz,1H),7.32(s,1H),6.51(s,1H),5.53(d,J＝9.2Hz,1H),5.44(s,2H),5.37(d,J＝5.6Hz,1H),5.31(s,2H),5.28(d,J＝4.8Hz,1H),5.15(d,J＝5.6Hz,1H),4.62(t,J＝5.6Hz,1H),4.55(s,2H),3.68-3.79(m,2H),3.55-3.58(m,2H),3.37-3.41(m,3H),3.17-3.27(m,5H),1.77-1.91(m,4H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.4Hz,3H)。
实施例14 化合物81的合成
Figure PCTCN2019100802-appb-000042
将220mg粗品化合物79和224mg化合物55溶于16mL THF-H 2O中，依次加入166mg无水硫酸铜和206mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到100mg化合物81，两步收率25.6％。
1H NMR(400Hz,DMSO-d 6)δ8.16-8.18(m,2H),7.96-7.99(m,2H),7.65(dd,J＝9.0,2.2Hz,1H),7.32(s,1H),6.54(s,1H),5.44(s,2H),5.34(s,2H),5.11(d,J＝4.8Hz,1H),4.97(dd,J＝15.2,4.8Hz,2H),4.52-4.57(m,5H),4.23(d,J＝7.6Hz,1H),4.07-4.12(m,1H),3.89-3.92(m,1H),3.66-3.70(m,1H),3.54(t,J＝6.0Hz,2H), 3.40-3.46(m,1H),3.10-3.18(m,6H),2.94-3.06(m,2H),1.84-1.91(m,2H),1.75-1.80(m,2H),1.29(t,J＝7.6Hz,3H),0.88(t,J＝7.2Hz,3H)。
实施例15 化合物87的合成
Figure PCTCN2019100802-appb-000043
称量2.09g化合物82和5.6mL三乙胺溶于50mL二氯甲烷中，冷却至0℃,缓慢的滴加4.43g二碳酸二叔丁酯，滴加完毕后，室温反应过夜，反应液用1N HCl洗涤(100mL×3),有机层用无水硫酸钠干燥，过滤，浓缩得到4g化合物83，收率97.3％。
1H NMR(400Hz,CDCl 3)δ4.56(s,1H),3.64(t,J＝6.4Hz,2H),3.10-3.15(m,2H),1.27-1.61(m,15H)。
称量4g化合物83溶于60mL四氢呋喃中，加入812mg四丁基碘化铵，660mg碘化钠和3.92g 3-溴丙炔，室温搅拌下分小批量加入2.48g氢氧化钾，完毕后室温反应过夜，浓缩掉四氢呋喃，加入30mL水，乙酸乙酯萃取(30mL×3),有机层用无水硫酸钠干燥，过滤，浓缩柱层析得到2.8g化合物84，收率59.1％。
1H NMR(400Hz,CDCl 3)δ4.54(s,1H),4.13(d,J＝2.4Hz,2H),3.51(t,J＝6.4Hz,2H),3.09-3.14(m,2H),2.42(t,J＝2.4Hz,1H),1.58-1.63(m,2H),1.35-1.54(m,13H)。
称量2.8g化合物84溶于40mL甲醇中，加入40mL 3N HCl溶液，室温搅拌过夜，浓缩得到1.7g化合物85，收率86.7％。
1H NMR(400Hz,D 2O)δ4.09(d,J＝2.4Hz,2H),3.52(t,J＝6.8Hz,2H),2.89(t,J＝7.6Hz,2H),2.77(t,J＝2.2Hz,1H),1.48-1.58(m,4H),1.24-1.36(m,2H)。
称量392mg化合物SN38溶于30mL DMF中，冷却至0℃,加入320mg DIPEA 和266mg对硝基苯基氯甲酸酯，在此温度下继续反应半小时，室温反应3小时得到中间体64，然后加入320mg DIPEA和266mg化合物85。加入完毕后室温反应过夜，浓缩掉DMF，TLC监测反应良好。柱层析分离得到400mg粗品产物86。将200mg化合物86和200mg化合物32溶于16mL THF-H 2O中，依次加入166mg无水硫酸铜和206mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到70mg化合物87，两步收率18.3％。
1H NMR(400Hz,DMSO-d 6)δ8.29(s,1H),8.17(d,J＝9.2Hz,1H),7.93-7.95(m,2H),7.64(dd,J＝9.0,2.2Hz,1H),7.33(s,1H),6.51(s,1H),5.52(d,J＝9.2Hz,1H),5.44(s,2H),5.36(d,J＝6.0Hz,1H),5.33(s,2H),5.26(d,J＝4.8Hz,1H),5.13(d,J＝5.6Hz,1H),4.62(t,J＝5.6Hz,1H),4.52(s,2H),3.68-3.78(m,2H),3.36-3.51(m,6H),3.20-3.25(m,2H),3.10-3.15(m,2H),1.85-1.92(m,2H),1.51-1.59(m,4H),1.36-1.42(m,2H),1.30(t,J＝7.6Hz,3H),0.89(t,J＝7.2Hz,3H)。
实施例16 化合物88的合成
Figure PCTCN2019100802-appb-000044
将200mg粗品化合物86和210mg化合物55溶于16mL THF-H 2O中，依次加入166mg无水硫酸铜和206mg抗坏血酸钠，室温搅拌过夜，浓缩后柱层析分离得到70mg化合物88，两步收率17.3％。
1H NMR(400Hz,DMSO-d 6)δ8.16(d,J＝9.2Hz,1H),8.14(s,1H),7.91-7.94(m,2H),7.63(dd,J＝9.2,2.4Hz,1H),7.32(s,1H),6.51(s,1H),5.44(s,2H),5.33(s,2H),5.06(d,J＝4.8Hz,1H),4.93(dd,J＝13.2,5.2Hz,2H),4.56(t,J＝5.2Hz,2H),4.49-4.52(m,3H),4.23(d,J＝8.0Hz,1H),4.07-4.08(m,1H),3.89-3.92(m,1H),3.65-3.67(m,1H),3.42-3.48(m,3H),3.09-3.20(m,6H),2.96-3.06(m,2H),1.86-1.89(m,2H), 1.50-1.58(m,4H),1.34-1.38(m,2H),1.29(t,J＝7.6Hz,3H),0.89(t,J＝7.4Hz,3H)。
实施例17 溶解度测定
溶解度实验采用平衡法，根据中华人民共和国药典2015版第四部0 4 0 1紫外-可见分光光度法，检测所得化合物在水、生理盐水、pH7.4的PBS及0.1％吐温80水溶液中的溶解度。称取适量化合物溶解至1mL甲醇溶液中，配制成一定浓度的对照品溶液，即C 0。按下式计算供试品中被测溶液的浓度：C X＝(A X/A 0)C 0；式中C X为供试品溶液的浓度；A X为供试品溶液的吸光度；C 0为对照品溶液的浓度；A 0为对照品溶液的吸光度。实验结果见表1。
表1 化合物溶解度试验结果
Figure PCTCN2019100802-appb-000045
由表1结果可知，SN38在水、生理盐水、pH7.4的PBS及0.1％吐温80水溶液中的溶解度均非常低。通过结构改造后，所得化合物溶解度比SN38大大 提高，且大部分化合物在生理盐水中的溶解度显著高于或接近CPT-11。
实施例18 细胞毒性试验
为了考察所得化合物的抗肿瘤活性，我们选择了多种肿瘤细胞株进行试验。取对数生长的细胞，接种于96孔板中过夜，然后加入一定梯度浓度的药物，置于细胞培养箱(5％CO 2，37℃)孵育72h，采用MTT法进行药效评价，考察药物对不同细胞的半数生长抑制浓度IC 50值，以CPT-11作为对照。评价结果见表2。根据体外实验结果，在所测试的肿瘤细胞株中，合成的SN38衍生物均显示出优异的抗肿瘤活性，且对大部分肿瘤的生长抑制作用强于CPT-11。
表2 体外细胞毒性试验结果
Figure PCTCN2019100802-appb-000046
Figure PCTCN2019100802-appb-000047
实施例19 体内抗肿瘤活性研究
综合考虑溶解性和体外细胞毒性结果，我们对化合物58和67的体内抗肿瘤活性进行研究。步骤如下：
将4～5周龄的Balb/c小鼠饲养于无特定病原体(SPF)的环境中。饲养一周后，在小鼠皮下接种结肠癌细胞SW-480，接种密度为1.0×10 7个每只，待肿瘤长至约100mm 3后，随机分成4组：生理盐水对照组(Control组)、盐酸伊立替康组(CPT-11组,15mg/kg即0.024mmol/kg)、化合物58组(13.73mg/kg即0.016mmol/kg)、化合物67组(12.30mg/kg即0.016mmol/kg)，给药方式为将化合物溶解于生理盐水，静脉注射，每三天给药一次，连续给药7次。每三天测量一次肿瘤大小和体积，直至给药完成。实验结束后，断颈处死裸鼠，对裸鼠进行解剖，取重要脏器保存待用。
表3和表4分别是小鼠肿瘤体积-时间变化数据和小鼠体重-时间变化数据，图1和图2分别是小鼠肿瘤体积-时间变化图和小鼠体重-时间变化图。根据表3和图1，化合物58和67在剂量为CPT-11摩尔数的三分之二时，对结肠癌细胞 SW-480的抑制率均显著大于CPT-11。根据表4和图2，在给药剂量下，没有引起小鼠体重下降，表明化合物在给药剂量下没有表现出明显的毒性。
表3 裸鼠肿瘤体积-时间变化数据(平均值，n＝6)
Figure PCTCN2019100802-appb-000048
表4 裸鼠体重-时间变化数据
Figure PCTCN2019100802-appb-000049
综上所述，通过对SN38的结构进行改造，大大增加了SN38的水溶性，体内外实验结果表明，所得衍生物抗肿瘤活性优于盐酸伊立替康。
实施例20 多剂量体内抗肿瘤活性研究
试验分组及治疗方案
将接种成功的小鼠随机分成8组，每组6只：生理盐水组(NS)、CPT-11组、化合物58低剂量组、化合物58中剂量组、化合物58高剂量组、化合物67低剂量组、化合物67中剂量组和化合物67高剂量组。通过小鼠尾静脉给药，每隔2天给药一次，共给药7次。
给药剂量为CPT-11组：10mg/kg；化合物58和67的高、中、低剂量组，分别与伊立替康的摩尔比为1/1、1/2和1/4，即化合物58高剂量组(H):13.73mg/kg，化合物58中剂量组(M)：6.865mg/kg、化合物58低剂量组(L)：3.433mg/kg；化合物67高剂量组(H)：12.30mg/kg、化合物67中剂量组(M)：6.15mg/kg、化合物67低剂量组(L)：3.075mg/kg。
从0天起，每隔2天测量1次肿瘤体积，先测量肿瘤的最大径(a)，再测量与最大径线垂直的最长径线(b)，单位为mm，按以下公式，计算肿瘤体积：
Figure PCTCN2019100802-appb-000050
治疗周期结束后，取小鼠眼眶静脉血用于后期的血常规检测，随后采用颈椎脱臼法处死小鼠，立即剥除肿瘤，拍照。从第0天起，每隔2天称量并记录各组小鼠体重，计算各组体重平均值，根据统计数据绘小鼠体重变化曲线。待治疗周期结束后，麻醉小鼠，立即剥除肿瘤，称量并记录各组肿瘤重量，计算各组肿瘤瘤重平均值，根据以下公式计算肿瘤抑制率(％)：
Figure PCTCN2019100802-appb-000051
然后摘取小鼠的心、肝、脾、肺、肾，以备后期脏器的H&E染色以及肿瘤组织的免疫组化。以时间为横坐标，肿瘤体积为纵坐标绘制肿瘤体积生长变化曲线；以时间为横坐标，小鼠体重为纵坐标绘制小鼠体重变化曲线。肿瘤体积生长变化和裸鼠体重时间变化见图表5和表6。肿瘤体积生长变化曲线和裸鼠体重时间变化见图3-5。
由表5和图3，4肿瘤体积生长曲线可知，化合物58和化合物67对肿瘤的抑制均具有剂量依耐性，末次给药后各治疗组与对照组相比均存在统计学差异(p<0.05)。化合物58和化合物67高剂量组在同等给药剂量下(10mg/kg)肿瘤抑制效果强于阳性对照组CPT-11组(p<0.001)，其中，化合物58高剂量组肿瘤体积增长最缓慢，且末次给药后的肿瘤体积与其他制剂组均具有显著性 差异(p<0.001)，说明该制剂组抑瘤药效果最强。由表6和图5可得，各剂量组药物均未对小鼠体重产生影响，表明化合物具有较高的安全性。
表5 多剂量化合物58和化合物67的体内抗肿瘤活性研究肿瘤体积-时间变化数据
Figure PCTCN2019100802-appb-000052
表6 多剂量化合物体内抗肿瘤活性研究中体重时间变化数据
Figure PCTCN2019100802-appb-000053

Claims (31)

  1. 喜树碱衍生物，其特征在于，所述的喜树碱衍生物由式1所示的物质经糖基化三氮唑在R 3位置修饰而成；所述式1所示的结构式中R 1代表H、C 1-10的烷基、C 1-10的氘代烷基或C 1-C 10的卤代烷基；R 2代表H、CH 2N(CH 3) 2或CH 2N(CD 3) 2；R 4代表
    Figure PCTCN2019100802-appb-100001
    或H，其中X代表N，O或S；L代表多肽、C 1-20直链烷基或其衍生物、C 1-20直链或支链酰基衍生物、C 2-100乙二醇或其衍生物，
    Figure PCTCN2019100802-appb-100002
  2. 根据权利要求1所述的喜树碱衍生物，其特征在于，所述式1中，所述R 3代表
    Figure PCTCN2019100802-appb-100003
    或H，所述R 4代表
    Figure PCTCN2019100802-appb-100004
    或H，但所述R 3和R 4不同时为H；所述R 5代表单糖残基或寡糖残基；L代表多肽、C 1-C 20直链烷基或其衍生物、C 1-C 20直链或支链酰基衍生物、C 1-C 20乙二醇或其衍生物、
    Figure PCTCN2019100802-appb-100005
    其中Y为
    Figure PCTCN2019100802-appb-100006
    a为0-100的整数；b为1-100的整数；c为0-100的整数；d为0-100的整数。
  3. 权利要求1或2所述的喜树碱衍生物，其特征在于：所述式1中的所述R 1代表H或-CH 2CH 3；所述R 2代表H、-CH 2N(CH 3) 2或—CH 2N(CD 3) 2；所述R 3代表
    Figure PCTCN2019100802-appb-100007
    或H，所述R 4代表
    Figure PCTCN2019100802-appb-100008
    或H，但所述R 3和R 4不同时为H；所述R 5选自单糖残基或寡糖残基；X选自N或O；L选自C 1-20直链 或支链烷烃及其衍生物、C 1-20直链或支链酰基衍生物、C 2-100乙二醇及其衍生物、
    Figure PCTCN2019100802-appb-100009
    其中Y为
    Figure PCTCN2019100802-appb-100010
    a为0-100的整数；b为1-100的整数；c为0-100的整数；d为0-100的整数。
  4. 根据权利要求3所述的喜树碱衍生物，其特征在于：所述R 1代表-CH 2CH 3；所述R 2代表H；所述R 3代表
    Figure PCTCN2019100802-appb-100011
    所述R 4代表H；所述X代表N或O；所述L代表
    Figure PCTCN2019100802-appb-100012
    其中所述Y为
    Figure PCTCN2019100802-appb-100013
    所述a为1-20的整数，b为1-20的整数，c为0-20的整数，d为0-20的整数。
  5. 根据权利要求1所述的喜树碱衍生物，其特征在于，所述的喜树碱衍生物为7-乙基-10-羟基喜树碱的衍生物，其结构式如式2所示，
    Figure PCTCN2019100802-appb-100014
  6. 根据权利要求1所述的喜树碱衍生物，其特征在于，所述糖基化三氮唑如式3或4所述的结构式，其中Y为
    Figure PCTCN2019100802-appb-100015
    a为0-100的整数，b为1-100的整数，c为0-100的整数，d为0-100的整数，所述R5为糖残基或寡糖残基；
    Figure PCTCN2019100802-appb-100016
  7. 根据权利要求6所述的喜树碱衍生物，其特征在于，所述R 5选自式5-28的单糖残基中的任一：
    Figure PCTCN2019100802-appb-100017
  8. 根据权利要求6或7所述的喜树碱衍生物，其特征在于，所述a或所述b为1-20的整数。
  9. 根据权利要求7所述的喜树碱衍生物，其特征在于，所述R 5选自单糖残基式5、6、18和19中的任一：
    Figure PCTCN2019100802-appb-100018
  10. 根据权利要求1-9任一项所述的喜树碱衍生物，其特征在于，所述衍生物为下列化合物之一：
    Figure PCTCN2019100802-appb-100019
    Figure PCTCN2019100802-appb-100020
  11. 根据权利要求10所述的喜树碱衍生物，其特征在于，所述X选自N或O。
  12. 根据权利要求10所述的喜树碱衍生物，其特征在于，所述Y为
    Figure PCTCN2019100802-appb-100021
  13. 根据权利要求12所述的喜树碱衍生物，其特征在于，所述d为0-1的整数。
  14. 根据权利要求8所述的喜树碱衍生物，其特征在于，所述a为1，b为1-4的整数。
  15. 权利要求1-14任一项所述的喜树碱衍生物的合成方法，其特征在于，包括以下步骤：
    1)化学反应合成叠氮化合物；
    2)化学反应合成端基炔；
    3)将叠氮化合物和端基炔溶于THF-H 2O中，依次加入无水硫酸铜和抗坏血酸钠进行点击反应，然后室温搅拌过夜，浓缩后柱层析分离得所述喜树碱衍生物；
    所述叠氮化合物为
    Figure PCTCN2019100802-appb-100022
    其中Z为无或O，e为0-20。
  16. 提高喜树碱衍生物溶解性的方法，其特征在于，所述方法为对7-乙基-10-羟基喜树碱衍生物进行糖基化三氮唑修饰，7-乙基-10-羟基喜树碱衍生物的结构式如式2所示，糖基化三氮唑在R 3的位置进行修饰；所述式2所示的结构式中R 3代表
    Figure PCTCN2019100802-appb-100023
    或H；其中X代表N，O或S；L代表多肽、C 1-C 20直链烷基或其衍生物、C 1-C 20直链或支链酰基衍生物、C 1-C 20乙二醇或其衍生物、
    Figure PCTCN2019100802-appb-100024
  17. 根据权利要求16所述的方法，其特征在于，所述糖基化三氮唑如式3或4所述的结构式，其中Y为
    Figure PCTCN2019100802-appb-100025
    a为0-100的整数，b为1-100的整数，c为0-100的整数，d为0-100的整数，所述R 5为糖残基或寡糖残基。
  18. 根据权利要求17所述的方法，其特征在于，所述R 5的结构式如式5-28任一所示。
  19. 根据权利要求18所述的方法，其特征在于，所述R 5选自单糖残基式5、6、18和19中的任一。
  20. 根据权利要求17所述的方法，其特征在于，所述a为1，b为1-4的整数。
  21. 根据权利要求17所述的方法，其特征在于，所述Y为
    Figure PCTCN2019100802-appb-100026
  22. 根据权利要求21所述的方法，其特征在于，所述d为0-1的整数。
  23. 肿瘤细胞生长抑制剂，其特征在于，所述抑制剂由权利要求1-14任一项所述的喜树碱衍生物制备而成。
  24. 根据权利要求23所述的肿瘤细胞生长抑制剂，其特征在于，所述肿瘤细胞生长抑制剂可通过和拓扑异构酶I、DNA形成三元复合物使DNA链断裂，抑制肿瘤细生长和促进肿瘤细胞凋亡。
  25. 根据权利要求23或24所述的肿瘤细胞生长抑制剂，其特征在于，所述肿瘤为结直肠瘤、肺瘤和乳腺瘤、肝癌、胃癌、食管癌、白血病、前列腺癌、 骨肉瘤、宫颈癌、甲状腺癌、卵巢癌或胰腺癌。
  26. 三元复合物，其特征在于，由权利要求22-24任一项所述的肿瘤细胞生长抑制剂通过和拓扑异构酶I、DNA形成所述的三元复合物。
  27. 权利要求1-14任一项所述的喜树碱衍生物在制备抗癌药物中的应用。
  28. 根据权利要求27所述的应用，其特征在于，所述癌为结直肠癌、肺癌和乳腺癌、肝癌、胃癌、食管癌、白血病、前列腺癌、骨肉瘤、宫颈癌、甲状腺癌、卵巢癌或胰腺癌。
  29. 根据权利要求27所述的应用，其特征在于，所述抗癌药物可通过和拓扑异构酶I、DNA形成三元复合物使DNA链断裂，抑制肿瘤细生长和促进肿瘤细胞凋亡以达到抗癌作用，所述肿瘤细胞为SW-480和/或HT-29和/或HCT-116和/或A549和/或H1975和/或HepG2和/或BGC-823和/或ECA-109和/或K562和/或PC3和/或143B和/或MDA-MB-231和/或Hela和/或TPC-1和/或SKOV-3和/或PANC-1。
  30. 权利要求1-14任一项所述的喜树碱衍生物制备的制剂。
  31. 根据权利要求30所述的制剂，其特征在于，所述制剂中含有药学可接受载体和/或助剂。
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