WO2017193562A1 - 水溶性雷帕霉素类衍生物 - Google Patents

水溶性雷帕霉素类衍生物 Download PDF

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WO2017193562A1
WO2017193562A1 PCT/CN2016/105178 CN2016105178W WO2017193562A1 WO 2017193562 A1 WO2017193562 A1 WO 2017193562A1 CN 2016105178 W CN2016105178 W CN 2016105178W WO 2017193562 A1 WO2017193562 A1 WO 2017193562A1
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compound
group
formula
everolimus
tumor
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PCT/CN2016/105178
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English (en)
French (fr)
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汪海波
况洪福
张伟
蔡正江
朱天民
郑晓鹤
吴忠伟
杨志清
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浙江海正药业股份有限公司
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Priority to US15/553,088 priority Critical patent/US10442835B2/en
Priority to CN201680006268.3A priority patent/CN107949566B/zh
Priority to JP2017568325A priority patent/JP6770008B2/ja
Priority to EP16901517.9A priority patent/EP3299381B1/en
Publication of WO2017193562A1 publication Critical patent/WO2017193562A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the technical field of organic chemistry and medicinal chemistry, and relates to the preparation of water-soluble rapamycin derivatives and their use in inducing immunosuppression, treating transplant rejection, solid tumors, fungal infections and vascular diseases. More particularly, the present invention relates to a class of glutathione-modified rapamycin-based water-soluble derivatives, processes for their preparation, and their inducing immunosuppression, treatment of transplant rejection, solid tumors, fungal infections, and blood vessels Application in disease.
  • Rapamycin is a novel macrocyclic triene lactone compound, which is Streptomyces hygroscopicus (Vezina C, et al, J. Antibiot, 1975, 10: 721-726.) or Actinoplane sp. is produced (Nakao K, et al, EP022446, 1993-11-11).
  • Typical rapamycin compounds mainly include sirolimus, Everolimus, and Temsirolimus, and the structures are as follows:
  • Rapamycins were first discovered to have antifungal activity, particularly against Candida albicans.
  • rapamycins are a mammalian target of rapamycin (mTOR) inhibitors that can be used as immunosuppressive agents.
  • mTOR mammalian target of rapamycin
  • the immunosuppressive effects of rapamycins and the prevention of immune rejection following organ transplantation were first published in the Journal of the American Society for Experimental Biology (The FASEB Journal, 3, 3411, 1989). Its mechanism of action is to block signaling through different cytokine receptors, thereby Blocks the progression of T lymphocytes and other cells from the G1 phase to the S phase, thereby exerting an immunosuppressive effect.
  • Rapamycins have been clinically approved for the treatment of multiple tumor indications, such as everolimus has been approved by the FDA for advanced breast cancer, renal cell carcinoma, pancreatic cancer, angiomyolipoma and other entities. Treatment of the tumor. Rapamycins have also proven to be effective in therapeutic models for diseases such as multiple sclerosis, rheumatoid arthritis, anemia, etc. (Can J. Physiol. Pharmcol. 1997, 55, 48-52). It has also been reported that rapamycin has a potential role in extending the life cycle of mammals (Harrison DE, et al, Nature, 2009, 460, 392-395).
  • Rapamycin compounds have multiple indications and have great application value in clinical treatment. However, due to the poor water solubility and poor stability of the rapamycin compounds, the absorption in the body is low and the bioavailability is low, only 15%-30% (Guy Jerusalem, et al, Breast Cancer Research and Treatment, 2010, 125:2447-2455), while high doses cause more side effects.
  • Polypeptide is an endogenous substance in human body. It is composed of multiple amino acids. It has good water solubility and wide biological activity. On the one hand, it can improve the solubility of small molecule drugs through the combination of peptide and small molecule drugs. On the other hand, in vivo targeted release, sustained release or improved biological activity can be achieved with the aid of polypeptides.
  • glutathione is a common endogenous polypeptide, which is bound by glutamic acid, cysteine and glycine. Glutathione contains sulfhydryl groups, which have antioxidant effects and integrated detoxification. Glutathione is also a nutrient for cell growth and is easily taken up by cells, and is particularly easily taken up by tumor cells that multiply rapidly.
  • the complex prepared by coupling glutathione with small molecule drugs is highly likely to be selective for rapidly multiplying tumor cells, and at the same time can reduce the toxicity of antitumor drugs to normal human cells, and at the same time realize small molecule drugs. Targeted release.
  • glutathione is a water-soluble tripeptide that forms a complex with a small fraction of drugs, which can greatly improve the water solubility of small molecule drugs.
  • the invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:
  • R 1 is H or R 3 ;
  • R 2 is H or R 4 -R 5 ;
  • R 1 and R 2 are not H at the same time
  • R 3 is R 4 -R 5 , -CH 2 CH 2 OR 4 -R 5 ,
  • R 4 is a carbonyl C 2 -C 6 alkenylene group or a carbonyl C 2 -C 6 alkynylene group
  • n is an integer less than or equal to 6; that is, n is 1, 2, 3, 4, 5 or 6;
  • n is an integer less than or equal to 6; that is, m is 1, 2, 3, 4, 5 or 6;
  • R 5 is a polypeptide group, preferably a glutathione group formed by dehydrogenation of a thiol group (-SH) as shown in Formula II:
  • R 6 is a C 1 -C 6 alkylene group, a C 2 -C 6 alkenylene group or a C 2 -C 6 alkynylene group.
  • R 3 is preferably R 4 -R 5 or -CH 2 CH 2 OR 4 -R 5 , wherein R 4 and R 5 are as defined in formula I.
  • R 4 is preferably
  • R 1 is -CH 2 CH 2 OR 4 -R 5 ; further, R 4 is preferably Wherein n is an integer less than or equal to 6, and R 5 is preferably a glutathionyl group formed by dehydrogenation of a mercapto group (-SH) represented by formula II:
  • R 2 is preferably H.
  • the compound of formula I according to the invention is selected from the group consisting of
  • Another aspect of the invention provides a method of preparing a water soluble rapamycin derivative of formula I, the method comprising the steps of:
  • R 7 is H, -CH 2 CH 2 OH or
  • R 8 is H, R 4 X, -CH 2 CH 2 OR 4 X,
  • R 9 is H or R 4 X
  • R 8 and R 9 are not H at the same time
  • R 10 is a C 1 -C 6 alkylene group, C 2 -C 6 alkenylene or C 2 -C 6 alkynylene;
  • X is a halogen atom, preferably an I or Br atom
  • step (b) reacting the compound of formula IV prepared in step (a) with a polypeptide to provide a compound of formula I:
  • R 1 is H or R 3 ;
  • R 2 is H or R 4 -R 5 ;
  • R 1 and R 2 are not H at the same time
  • R 3 is R 4 -R 5 , -CH 2 CH 2 OR 4 -R 5 ,
  • R 4 is independently a carbonyl C 2 -C 6 alkenylene group or a carbonyl C 2 -C 6 alkynylene group;
  • n is an integer less than or equal to 6, that is, n is 1, 2, 3, 4, 5 or 6;
  • n is an integer less than or equal to 6, that is, m is 1, 2, 3, 4, 5 or 6;
  • R 5 is a polypeptide group, preferably a glutathione group formed by dehydrogenation of a thiol group (-SH) as shown in Formula II:
  • R 6 is a C 1 -C 6 alkylene group, a C 2 -C 6 alkenylene group or a C 2 -C 6 alkynylene group.
  • steps (a) and (b) refer to the method of patent WO0224706 and are partially modified.
  • step (b) the compound of the formula IV and the polypeptide are reacted in a mixed solvent of a N,N-dimethylformamide-alcohol-water mixed solvent, wherein the alcohol is preferably For ethanol.
  • the volume ratio of N,N-dimethylformamide-alcohol-water is from 1:1 to 5:1 to 5, preferably 1:2:1.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, preferably a citrate thereof, and a pharmaceutically acceptable excipient, wherein
  • the pharmaceutical excipients include pharmaceutically acceptable carriers, excipients, or combinations thereof.
  • the compound of formula I provided by the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is an immunosuppressive agent for treating or inhibiting transplant rejection; the compound of formula I of the present invention has an inhibitory effect on tumor cell growth It can be used for the treatment of tumors, preferably for the treatment of renal cell carcinoma, renal epithelial renal cell carcinoma, breast cancer, pancreatic cancer, lung cancer, prostate cancer, subependymal giant cell astrocytoma or renal angiomyolipoma.
  • the compounds of formula I of the invention are also useful in the treatment of fungal infections and vascular diseases.
  • the invention also provides a formulation comprising a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof, which is a tablet, a capsule, an injection, a powder, a granule, an applicator, a pill Or a film.
  • the preparation is an injection
  • the vehicle is water for injection
  • the injection is a lyophilized powder injection
  • the reconstituted solvent is physiological saline.
  • the invention also provides a method of administration comprising administering to a patient an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • the method of administration is administered by administering to the patient an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, once a week.
  • the present invention provides polypeptide modified rapamycin compounds of Formula I, particularly glutathione modified rapamycin compounds.
  • the compound of the formula I of the present invention is easily soluble in physiological saline; moreover, it is structurally stable after being dissolved in physiological saline, and no significant degradation is observed within 24 hours; the compound of the formula I of the present invention is a prodrug of a rapamycin compound in animals.
  • the corresponding rapamycin-like compound can be gradually released from the serum to have a sustained release effect; the compound of the formula I of the present invention exhibits better tumor suppressing activity than the rapamycin-like compound.
  • Alkyl as a group or as part of a group means a straight-chain or branched saturated aliphatic hydrocarbon group, preferably a C 1 -C 10 alkyl group, more preferably a C 1 -C 6 alkane base.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethyl Propyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1- Ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl Base, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. .
  • Polypeptide group as a group or a part of a group refers to a group formed by the removal of one or more hydrogen atoms from a polypeptide or protein, preferably a polypeptide group containing a thiol group or a protein group formed by deactivating H at a thiol group.
  • Examples of polypeptide groups include, but are not limited to, glutathione groups.
  • “Pharmaceutically acceptable salt” refers to certain salts of the above compounds which retain their original biological activity and which are suitable for pharmaceutical use.
  • the pharmaceutically acceptable salt of the compound of formula I may be a salt formed with a suitable acid, including inorganic and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, Fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, malic acid, maleic acid, mandelic acid, methanesulfonic acid, nitric acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, P-toluenesulfonic acid and the like
  • Figure 1 is a standard curve of the area and concentration of everolimus peak measured in Example 11, the ordinate is the peak area; The coordinates are the concentration in mg/mL.
  • Example 2 is a graph showing the results of the test of the release of everolimus in the serum of the compound I-1 measured in Example 13, the ordinate is the molar concentration percentage; the abscissa is the time, the unit: minute.
  • Figure 3 is a graph showing the growth trend of NCI-460 tumor volume in nude mice measured in Example 14.
  • Figure 4 is a graph showing changes in body weight of nude mice measured in Example 14.
  • Figure 5 is a graph showing the NCI-460 tumor mass in nude mice measured in Example 14.
  • Figure 6 is a graph showing the tumor growth trend of DU145 volume in nude mice measured in Example 15.
  • Figure 7 is a graph showing the increase in volume of human renal cell tumor OS-RC-2 in nude mice measured in Example 16.
  • Fig. 8 is a graph showing changes in body weight of nude mice measured in Example 16.
  • Figure 9 is a graph showing the mean blood concentration-time curve in SD male rats measured in Example 17.
  • HPLC methods for detection mentioned in the following examples are as follows:
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • Ivemus 5.0 g, 5.2 mmol
  • iodoacetic acid (1.94 g, 10.4 mmol) were added to a 100 mL three-necked flask, and 20 mL of dichloromethane (DCM) was added and stirred until the solid was dissolved.
  • DCM dichloromethane
  • the mixture was cooled to 0-5 ° C, dicyclohexylcarbodiimide (DCC, 2.36 g, 11.4 mmol) was added, stirred at 0-5 ° C for 10-15 minutes, and 4-dimethylaminopyridine (DMAP) was added to the reaction mixture. , 0.63 g, 5.2 mmol).
  • DMAP 4-dimethylaminopyridine
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • Sirolimus (4.0 g, 4.4 mmol) and iodoacetic acid (1.64 g, 8.8 mmol) were added to a 100 mL three-necked flask, 20 mL of dichloromethane was added, and the mixture was stirred until the solid was dissolved. The mixture was cooled to 0-5 ° C, dicyclohexylcarbodiimide (2.0 g, 11.4 mmol) was added, stirred at 0-5 ° C for 10-15 minutes, and 4-dimethylaminopyridine (0.54 g) was added to the reaction mixture. , 4.4mmol). The reaction solution was warmed to room temperature and stirred for 16-24 hours.
  • Embodiment 5 is a diagrammatic representation of Embodiment 5:
  • TLC TLC was used to detect the reaction of temsirolimus.
  • the insoluble solid was filtered with a Buchner funnel. The filtrate was concentrated to dryness under reduced pressure at 30-40 ° C.
  • Tesirolimus trihaloacetate IV-7 (0.33 g, 0.22 mmol) and glutathione (0.27 g, 0.88 mmol) were added to a 50 mL round bottom flask and 3 mL of N,N-dimethyl was added. The carboxamide was stirred until the solid was suspended in N,N-dimethylformamide. To the suspension was added K 2 CO 3 (60.7 mg, 0.44 mmol). Subsequently, 3 mL of H 2 O and 6 mL of ethanol were further added to the reaction suspension, and after vigorously stirring for 30 minutes, the reaction solution became clear. The reaction solution was stirred at room temperature overnight, and the reaction of the compound IV-7 was analyzed by HPLC. The reaction mixture was concentrated to dryness under reduced pressure at 45-55 ° C, and the obtained residue was purified by preparative liquid chromatography to give compound I-7 (0.24 g, 0.12 mmol).
  • the following experimental sections include water-soluble research methods and research results, in vitro and in vivo activity research methods, and research results for the specific compounds to be invented. Compounds not listed in the experimental section below can also be used with the following The same research methods and ideas in the test section are carried out to carry out related research.
  • the following experimental sections are merely illustrative of the methods and results of the specific compounds invented, but are not limited to the compounds that have been used.
  • the solubility of the compounds of the invention can be confirmed by standard experimental procedures which measure the dissolution of the compounds of the invention in water.
  • the water solubility of compounds I-3 and I-4 was increased by at least 470 and 610 times, respectively, compared to sirolimus; whereas the compounds I-5, I-6, I-7 were relative to temsiro
  • the water solubility of Moss was increased by at least 580, 710 and 830 times, respectively.
  • the antitumor activity and toxicity of the compounds of the present invention can be confirmed by a standard pharmacological experimental procedure for measuring the compounds of the present invention against human hepatoma cells HepG2, lung cancer cells NCI460, prostate cancer cells DU145, prostate cancer cells PC3 Inhibition of growth of human breast cancer cell MDA-MB-435.
  • the following is a brief description of the procedure used for the HepG2 inhibitory activity test of human hepatoma cells.
  • HepG2 Human hepatoma cells HepG2 grow in the following media:
  • BRL minimum essential medium 500 ml
  • Earle salts 500 ml
  • reagents were added to the medium:
  • the resulting growth medium is prepared for use.
  • test procedure is as follows:
  • the compounds of the invention tested were dissolved in 10 microliters of phosphate buffered saline (PBS) and incubated at 37 degrees Celsius for 48 hours.
  • PBS phosphate buffered saline
  • each well of a 96-well plate was labeled with 1 microcurie T Thymidine (New England Nuclear thymidine). 1 microcurie was added to 10 ⁇ l of PBS (on the day of sample collection). The 96-well plate was returned to the incubator for the last 6 hours of incubation.
  • T Thymidine New England Nuclear thymidine
  • Example 13 to Example 17 only list the effects of the compound 1-1, but those skilled in the art have used the same research method according to what has been disclosed in the present invention, and have appropriate common sense where necessary. It is reasonable to expect that other compounds of the invention not listed will be able to produce test results similar to those of Compound 1-1.
  • Example 13 Test of prodrug releasing drug in rat serum
  • the compounds of the invention as prodrugs of rapamycins can be confirmed by standard pharmacological experimental procedures. The following is a brief description of the procedure used and the results obtained by taking the release of the everolimus test in the serum of the compound I-1.
  • Centrifuge tubes 1-7 were centrifuged at 12,000 rpm for 15 minutes, and the protein in the serum was removed by centrifugation. 500 ⁇ l of the supernatant was taken for HPLC analysis and detected for 10 min, 0.5 h, 1 h, 2 h, 3 h, 4 h, respectively.
  • the content of everolimus and the content of compound I-1 in the supernatant were as shown in Fig. 2.
  • Compound I-1 can slowly release everolimus in rat serum, and the basic release is complete in 3 hours. Compound I-1 can effectively prolong the duration of action of everolimus in rats.
  • Example 14 Inhibitory activity of tumor of compound I-1 in nude mice
  • the anti-tumor activity of the compounds of the present invention in nude mice can be confirmed by a standard pharmacological experimental procedure which demonstrates that the compounds of the present invention have an inhibitory effect on cancer cell growth in animals.
  • the NCI-H460 inhibitory activity test of human lung cancer cells inoculated under the armpit of a nude mouse is taken as an example to briefly describe the procedure used and the results obtained.
  • the experimental method is as follows:
  • the NCI-H460 (3rd generation) tumor mass inoculated in the rapid proliferative phase of the nude mouse was cut into 1mm*1mm*1mm tumor mass and inoculated subcutaneously in the right limb of the nude mouse under sterile conditions. .
  • the tumors were grouped when the tumors proliferated to 150-200 mm 3 .
  • the long diameter (a) and short diameter (b) of the tumor mass were measured, 2 to 3 times per week. Calculate tumor volume (TV) and relative tumor growth rate (T/C).
  • Grouping method According to the growth rule of the tumor block and the dosage regimen, the tumors were averaged up to about 195 mm 3 groups, divided into 3 groups, 6 in each group.
  • Mode of administration intragastric administration:
  • the decrease in body weight can indirectly indicate the toxicity and side effects of the drug on animals.
  • the weight gain of compound I-1 (G3 group) is greater than that of the blank control group and positive.
  • the drug everolimus group (G1, G2 group), and the weight of the positive drug everolimus group was smaller than the blank control group, so the toxic side effect of compound I-1 was significantly less than that of everolimus.
  • the NCI-H460 vaccination model of nude mice showed that the compound I-1 group was an effective dose group with a T/C value of less than 40% (35%) and a tumor inhibition rate of more than 60% (68%). It has a good inhibitory effect on NCI-H460 cells inoculated with tumors; the T/C value of the positive drug everolimus group is greater than 40% (42%), and the tumor inhibition rate is less than 60% (56%).
  • the inhibitory activity of compound I-1 against NCI-H460 cells was significantly better than that of everolimus; and the weight gain of compound I-1 (G3 group) was significantly greater than that of the positive drug everolimus group (G2 group), compound I-1
  • the toxic side effects in animals are significantly less than that of everolimus.
  • Example 15 Comparative study on tumor suppressive effect of compound I-1 by intragastric administration and injection administration in vivo
  • the antitumor activity of the compounds of the present invention in nude mice can be confirmed by a standard pharmacological experimental procedure which demonstrates the inhibitory effect of the compounds of the present invention on the growth of cancer cells in animals.
  • the DU145 (3rd generation) tumor mass inoculated in the subgingival of nude mice was cut into 1mm*1mm*1mm tumor mass, and inoculated subcutaneously in the right limb of nude mice under sterile conditions.
  • the tumors were grouped when the tumors proliferated to 150-200 mm 3 .
  • the tumor mass was removed, weighed, and the tumor inhibition rate was calculated.
  • the formula was: (control tumor weight - experimental group tumor weight) / control tumor weight ⁇ 100%.
  • the tumors were averaged to a length of about 185-200 mm 3 and divided into 3 groups of 4 animals each.
  • G1 positive drug everolimus group: Everolimus (5mg/kg, qw, 4w, 3 ⁇ /w), administration method: intragastric;
  • G2 Compound I-1 (3.4 mg/kg, qw, 4w, 3 ⁇ /w) (3.4 mg of I-1 is equivalent to 2.5 mg of everolimus), administration method: tail vein injection;
  • G3 Compound I-1 (1.7 mg/kg, qw, 4w, 3 ⁇ /w) (1.7 mg of I-1 is equivalent to 1.25 mg of everolimus), administration method: tail vein injection;
  • the decrease in body weight can indirectly indicate the toxicity and side effects of the drug on animals. It can be seen from the data in Table 10 that the weight-increasing tendency of the injection-administered group (G2, G3 group) is greater than that of the intragastric administration group (G1 group), so injection is given. Compound I-1 did not produce significant toxic side effects.
  • Compound I-1 has obvious improvement in water solubility, can be dissolved in physiological saline, and the aqueous solution obtained after being dissolved in physiological saline is stable, and the bioavailability of the drug in the body can be greatly improved by using injection.
  • the test results show that Compound I-1 only needs to use the equivalent of 1/4 dose of everolimus to achieve the same tumor suppressive effect as everolimus.
  • Example 16 Inhibitory activity of compound I-1 on human renal cell tumor OS-RC-2 in nude mice
  • the average tumor length is about 167mm 3 groups, each group is 6 and divided into 7 groups, respectively:
  • G2 everolimus (2mg/kg ig 3 ⁇ /qw)
  • G3 everolimus (6mg/kg ig 3 ⁇ /qw)
  • G4 Compound I-1 (8.12 mg/kg ig 3 ⁇ /qw) (Compound I-1 and G3 group are used in equimolar doses of everolimus, ie 8.12 mg of compound I-1 is equivalent to 6 mg Everolimus
  • G7 Compound I-1 (32.7 mg/kg iv 1 ⁇ /qw) (32.7 mg of Compound I-1 is equivalent to 24 mg of everolimus)
  • nude mice in the everolimus group were orally administered three times a week, whether it was a high dose 6 mg/Kg group or a low dose 2 mg/Kg group, nude mice.
  • the tumor volume in vivo was not effectively inhibited after 32 days, but the compound I-1 group (G5 group, G6 group and G7 group), whether it was a low dose (2.0 mg/Kg) 3 times a week, or a high dose ( 32.7 mg/Kg) showed a better tumor suppressing effect by one injection per week.
  • the data in Table 11 further showed that the final mass and tumor inhibition rate of tumors in nude mice showed that both the low-dose and high-dose injections of Compound I-1 (G5, G6 and G7 groups) showed good results.
  • the tumor suppressive effect and tumor inhibition rate were 57.1%, 59.9% and 60.6%, respectively, which were significantly better than the everolimus group (G2 and G3 groups), and the high-dose injection of compound I-1 was the best every week.
  • the tumor inhibition rate was as high as 60.6%.
  • the inhibition rate of compound I-1 was significantly higher than that of everolimus ( Compared with the G3 group, the tumor inhibition rates were 55.6% and 41.1%, respectively.
  • Body weight can indirectly reflect the toxicity of the drug.
  • the weight of the blank control group of nude mice decreased significantly, and the rate of decline reached 33.0%. This phenomenon may be caused by the lack of nutrition caused by tumor growth.
  • the weight loss rate of the drug treatment group was smaller than that of the blank control group.
  • the high-dose group I-1 (G6, G7 group) weight loss rate was 11.7% and 10.7%
  • the oral everolimus group (G2, G3 group) weight loss rate was 17.4% and 11.7%
  • compound I- 1 The high-dose group did not show significant toxicity compared with the oral everolimus group. Therefore, the compound I-1 was administered by high-dose injection, and the mice were well tolerated, and no obvious toxicity was observed.
  • Compound I-1 can effectively inhibit the growth of human renal cell carcinoma in nude mice by high dose once a week.
  • the tumor inhibition rate is 60.6%; oral administration of 6mg/Kg everolimus three times a week.
  • the tumor inhibition rate in nude mice was only 41.1%, which was administered once a week, which greatly improved the compliance of patients with advanced cancer in the clinic.
  • high doses of compound I-1 mice were well tolerated and no significant toxicity was observed.
  • MRT represents the average dwell time
  • the modified compound can gradually release the original drug in the serum of the rat, which can play a sustained release effect and prolong the action time of the drug in the body;

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Abstract

一种如通式Ⅰ所示的谷胱甘肽修饰的雷帕霉素类水溶性衍生物及其制备方法，其中R1和R2的定义如说明书所述。通式Ⅰ所示的化合物可用于诱导免疫抑制，治疗移植排斥、实体肿瘤等疾病。

Description

水溶性雷帕霉素类衍生物 技术领域
本发明属于有机化学和药物化学技术领域，涉及水溶性雷帕霉素类衍生物的制备，以及它们在诱导免疫抑制、治疗移植排斥、实体肿瘤、真菌感染和血管疾病中的应用。更具体地说，本发明涉及一类经谷胱甘肽修饰的雷帕霉素类水溶性衍生物、它们的制备方法，以及它们在诱导免疫抑制、治疗移植排斥、实体肿瘤、真菌感染和血管疾病中的应用。
技术背景
雷帕霉素类是一种新型的大环三烯内酯类化合物，是由吸水链霉素(Streptomyces hygroscopicus)(Vezina C,et al,J.Antibiot,1975,10:721-726.)或游动放线菌(Actinoplane sp.)产生(Nakao K,et al,EP022446,1993-11-11)。雷帕霉素类典型化合物主要包括西罗莫司(Sirolimus)、依维莫司(Everolimus)、替西罗莫司(Temsirolimus)等，结构如下所示：
Figure PCTCN2016105178-appb-000001
雷帕霉素类化合物最早被发现具有抗真菌活性，特别是对白色念珠菌(Candida albicans)具有较强的抑制活性。
后期研究发现，雷帕霉素类化合物是一种哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)抑制剂，可用作免疫抑制剂。雷帕霉素类化合物的免疫抑制作用以及在预防器官移植后的免疫排斥作用最早被公开于美国实验生物学会杂志(The FASEB Journal，3，3411，1989)。其作用机制是通过不同的细胞因子受体阻断信号传导，从而 阻断T淋巴细胞及其他细胞由G1期至S期的进程，从而发挥免疫抑制效应。
雷帕霉素类化合物在临床上已被批准用于多个肿瘤适应症的治疗，如依维莫司已被FDA批准用于晚期乳腺癌、肾细胞癌、胰腺癌、血管平滑肌脂肪瘤等实体瘤的治疗。雷帕霉素类化合物在多发性硬化症、类风湿关节炎、贫血等疾病的治疗模型中也证明是有效的(Can J.Physiol.Pharmcol.1997,55,48-52)。另据报道，雷帕霉素在延长哺乳类动物的生命周期上具有一定的潜在作用(Harrison DE，et al,Nature,2009,460,392-395)。
雷帕霉素类化合物具有多个适应症，在临床治疗上具有非常大的应用价值。但由于雷帕霉素类化合物的水溶性极差、体内稳定性差，导致其体内吸收少、生物利用度低，只有15％-30％(Guy Jerusalem,et al,Breast Cancer Research and Treatment,2010,125:2447-2455)，而大剂量给药则导致更多的副作用。
鉴于此，提高雷帕霉素类化合物的水溶性，可以在很大程度上提高其生物利用度，改善该类化合物对相关疾病的治疗效果。
多肽是人体一种内源性的物质，由多个氨基酸组成，其具有较好的水溶性和广泛的生物活性，通过多肽与小分子药物的结合，一方面可改进小分子药物的溶解性能，另一方面可在多肽的协助下实现体内靶向释放、缓释作用或改善生物活性。如，谷胱甘肽是一个常见的内源性多肽，它由谷氨酸、半胱氨酸和甘氨酸结合，谷胱甘肽含有巯基，具有抗氧化作用和整合解毒作用。谷胱甘肽也是细胞生长的营养物质，易被细胞摄取，特别易被繁殖较快的肿瘤细胞摄取。利用谷胱甘肽同小分子药物偶联制备的复合物，极可能对快速繁殖的肿瘤细胞具有选择性，同时在一定程度上可以降低抗肿瘤药物对人体正常细胞的毒性，同时实现小分子药物的靶向释放作用。而且，谷胱甘肽是一种水溶性三肽，与小分药物形成复合物，在很大程度上可以改善小分子药物的水溶性。
发明内容
本发明提供一种式Ⅰ所示的化合物或其在药学上可接受的盐：
Figure PCTCN2016105178-appb-000002
其中
R1是H或R3；
R2是H或R4-R5；
但R1和R2不同时为H；
R3为R4-R5、-CH2CH2O-R4-R5、
Figure PCTCN2016105178-appb-000003
R4为
Figure PCTCN2016105178-appb-000004
羰基C2-C6亚烯基或羰基C2-C6亚炔基；
n为小于或等于6的整数；即，n为1，2，3，4，5或6；
m为小于或等于6的整数；即，m为1，2，3，4，5或6；
R5为多肽基，优选为如式Ⅱ所示的巯基(-SH)脱氢后形成的谷胱甘肽基：
Figure PCTCN2016105178-appb-000005
R6为C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基。
在优选的实施方案中，R3优选为R4-R5或-CH2CH2O-R4-R5，其中R4和R5如式Ⅰ中所定义。
在优选的实施方案中，其中，R4优选为
Figure PCTCN2016105178-appb-000006
在更优选的实施方案中，R1为-CH2CH2O-R4-R5；进一步地，R4优选为
Figure PCTCN2016105178-appb-000007
其中n为小于或等于6的整数，R5优选为式Ⅱ所示的巯基(-SH)脱氢后形成的谷胱甘肽基：
Figure PCTCN2016105178-appb-000008
进一步地，R2优选为H。
在更优选的具体实施方案中，本发明所述式Ⅰ化合物选自：
Figure PCTCN2016105178-appb-000009
Figure PCTCN2016105178-appb-000010
本发明的另一方面提供了制备式Ⅰ所示的水溶性雷帕霉素衍生物的方法，所述方法包括以下步骤：
(a)将如式Ⅲ所示的化合物与XR10COOH反应，制备得到如式Ⅳ所示的化合物：
Figure PCTCN2016105178-appb-000011
其中，
R7为H，-CH2CH2OH或
Figure PCTCN2016105178-appb-000012
R8为H，R4X，-CH2CH2OR4X，
Figure PCTCN2016105178-appb-000013
R9为H或R4X；
R8和R9不同时为H；
R10为C1-C6亚烷基、
Figure PCTCN2016105178-appb-000014
C2-C6亚烯基或C2-C6亚炔基；
X为卤素原子，优选I或Br原子；
(b)将步骤(a)制备得到的式Ⅳ化合物和多肽反应，得到式Ⅰ化合物：
Figure PCTCN2016105178-appb-000015
其中，
R1是H或R3；
R2是H或R4-R5；
但R1和R2不同时为H；
R3为R4-R5、-CH2CH2O-R4-R5、
Figure PCTCN2016105178-appb-000016
R4独立地为
Figure PCTCN2016105178-appb-000017
羰基C2-C6亚烯基或羰基C2-C6亚炔基；
n为小于或等于6的整数，即，n为1，2，3，4，5或6；
m为小于或等于6的整数，即，m为1，2，3，4，5或6；
R5为多肽基，优选为如式Ⅱ所示的巯基(-SH)脱氢后形成的谷胱甘肽基：
Figure PCTCN2016105178-appb-000018
R6为C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基。
步骤(a)、(b)的合成方法参考专利WO0224706中的方法，并进行部分改进。
进一步地，在步骤(b)中，式Ⅳ化合物和多肽反应是在混合溶剂中进行的，所述混合溶剂为N,N-二甲基甲酰胺-醇-水混合溶剂，其中所述醇优选为乙醇。
进一步地，在所述混合溶剂中，N,N-二甲基甲酰胺-醇-水的体积比为1:1-5:1-5，优选为1:2:1。
本发明的又一方面提供一种药物组合物，所述的药物组合物含有有效剂量的式Ⅰ所述的化合物或其药学上可接受的盐，优选其柠檬酸盐，及药用辅料，其中所述药用辅料包括药用载体、赋形剂或它们的组合。
本发明提供的式Ⅰ化合物或其药学上可接受的盐或者其药物组合物是一种免疫抑制剂，可用于治疗或抑制移植排斥反应；本发明的式Ⅰ化合物对肿瘤细胞的生长具有抑制作用，可用于肿瘤的治疗，优选用于肾细胞癌、肾脏上皮肾细胞癌、乳腺癌、胰腺癌、肺癌、前列腺癌、室管膜下巨细胞星形细胞瘤或肾血管平滑肌脂肪瘤的治疗。本发明的式Ⅰ化合物还可用于治疗真菌感染和血管疾病。
本发明的还提供一种含有式I化合物或其在药学上可接受的盐或其药物组合物的制剂，所述制剂为片剂、胶囊剂、注射剂、散剂、颗粒剂、涂药支架、丸剂或膜剂。
进一步地，所述制剂为注射剂，溶媒为注射用水。
更进一步地，所述注射剂为冻干粉针注射剂，复溶溶媒为生理盐水。
本发明还提供一种给药方法，包括给予患者有效剂量的式I化合物或其在药学上可接受的盐，或其药物组合物。
进一步地，该给药方法的给予方式为向患者每周注射一次有效剂量的式I化合物或其在药学上可接受的盐，或其药物组合物。
本发明提供了式Ⅰ的多肽修饰的雷帕霉素类化合物，尤其是谷胱甘肽修饰的雷帕霉素类化合物。本发明的式Ⅰ化合物易溶于生理盐水；而且，在溶于生理盐水后结构稳定，24小时内未见明显降解；本发明的式Ⅰ化合物是雷帕霉素类化合物的前药，在动物血清中可逐渐释放出相应的雷帕霉素类化合物，具有缓释作用；本发明的式Ⅰ化合物比雷帕霉素类化合物表现出更好的肿瘤抑制活性。
如无特别定义，本发明中所使用的术语具有本领域普遍所接受的含义，进一步地，本发明所使用的部分术语定义如下：
“烷基”当作一个基团或一个基团的一部分是指直链的或者带有支链的饱和脂肪烃基团，优选为C1-C10烷基，更优选为C1-C6烷基。烷基基团的例子包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。
“多肽基”当作一个基团或一个基团的一部分是指多肽或者蛋白质脱除一个或多个氢原子形成的基团，优选为含巯基的多肽或蛋白在巯基处脱H形成的多肽基。多肽基的实施例包括但不限于谷胱甘肽基。
“药学上可接受的盐”是指上述化合物能保持原有生物活性并且适合于医药用途的某些盐类。式Ⅰ化合物药学上可接受的盐可以为与合适的酸形成的盐，合适的酸包括无机酸和有机酸，例如乙酸、苯磺酸、苯甲酸、樟脑磺酸、柠檬酸、乙磺酸、富马酸、葡糖酸、谷氨酸、氢溴酸、盐酸、羟乙磺酸、乳酸、苹果酸、马来酸、扁桃酸、甲磺酸、硝酸、磷酸、琥珀酸、硫酸、酒石酸、对甲苯磺酸等。
附图说明
图1为实施例十一测得的依维莫司峰面积和浓度标准曲线图，纵坐标为峰面积；横 坐标为浓度，单位为mg/mL。
图2为实施例十三测得的化合物Ⅰ-1在大鼠血清中释放依维莫司检测结果图，纵坐标为摩尔浓度百分比；横坐标为时间，单位：分钟。
图3为实施例十四测得裸鼠体内NCI-460肿瘤体积增长趋势图。
图4为实施例十四测得裸鼠体重变化趋势图。
图5为实施例十四测得裸鼠体内NCI-460肿瘤质量。
图6为实施例十五测得的裸鼠体内DU145体积肿瘤增长趋势图。
图7为实施例十六测得的裸鼠体内人肾细胞瘤OS-RC-2体积增长趋势图。
图8为实施例十六测得的裸鼠体重变化趋势图。
图9为实施例十七测得的SD雄性大鼠体内平均血药浓度-时间曲线图。
具体实施方式
下面具体实施例可以使本领域技术人员更具体地理解本发明，但它们不应被理解为以任何方式限制本发明。
在下列实例中，除非另有指明，所有温度为摄氏温度，各种起始原料和试剂均来自市售，并且，市售原料和试剂均不经进一步纯化直接使用。
下列实施例中提到的制备液相色谱(制备HPLC)方法如下所述：
色谱柱Kromasil-C18柱，10μm，100DAC制备柱；流动相：47％乙腈-100mmol/L乙酸铵水溶液，等度洗脱40min；检测波长254nm；柱温25℃；流速200ml/min。
下列实施例中提到的检测用HPLC方法如下所述：
色谱柱Xselect CSH-C18柱(4.6mm×250mm，5μm)；流动相A：0.1％三氟乙酸水溶液，B：乙腈，梯度洗脱(0→10min，A∶B＝75∶25，10→15min，A∶B＝75∶25→65∶35，15→20min，A∶B＝5∶95，20→30min，A∶B＝5∶95)；检测波长280nm；柱温30℃；流速1ml/min，进样体积10μl。
下面的实例仅仅是用来说明所发明的具体化合物的合成方法。但并不表示对本发明的合成方法的任何限制。未在下面制备实施例中列出的化合物，也可以用与下面制备实施例中同样的合成路线与合成方法来制备，其中选择适当的起始原材料、在有必要的地方稍加适当的常识性的反应条件调整即可。
本发明代表性的实例制备过程及相关研究实例被描述如下：
实施例一：
Figure PCTCN2016105178-appb-000019
将依维莫司(5.0g,5.2mmol)和碘乙酸(1.94g，10.4mmol)加至100mL三颈瓶中，加入20mL二氯甲烷(DCM)，搅拌至固体溶清。混合液冷却至0-5℃，加入二环己基碳二亚胺(DCC，2.36g，11.4mmol)，0-5℃搅拌10-15分钟，向反应液中加入4-二甲氨基吡啶(DMAP，0.63g，5.2mmol)。反应液升温至室温，搅拌16-24小时。TLC检测依维莫司反应完毕，布氏漏斗抽滤不溶性固体，滤液于30-40℃减压浓缩至干，所得浆状物用硅胶柱分离纯化(正己烷/乙酸乙酯＝5:1-2:1洗脱)，分别得到依维莫司单卤代乙酸酯Ⅳ-1(2.15g,1.9mmol)和依维莫司双卤代乙酸酯Ⅳ-2(2.60g,2.0mmol)。
依维莫司单卤代乙酸酯Ⅳ-1：
1H NMR(400MHz,CDCl3)δ6.35(dt,J＝24.8,14.8Hz,1H),6.19–6.09(m,1H),5.93(dd,J＝30.1,10.5Hz,1H),5.60–5.45(m,1H),5.41(d,J＝9.9Hz,1H),5.27(t,J＝7.2Hz,1H),5.15(dt,J＝11.6,5.5Hz,1H),4.34–4.24(m,2H),4.16(ddd,J＝21.4,14.1,6.6Hz,2H),3.91–3.75(m,2H),3.73(d,J＝7.1Hz,2H),3.67(dd,J＝14.5,6.8Hz,1H),3.57(d,J＝13.5Hz,1H),3.44(d,J＝10.3Hz,4H),3.40–3.28(m,4H),3.21–3.00(m,5H),2.84(dd,J＝17.7,7.0Hz,1H),2.72(dd,J＝16.4,5.5Hz,2H),2.58(dd,J＝16.7,6.4Hz,1H),2.33(d,J＝12.9Hz,1H),2.10–1.89(m,6H),1.75(s,6H),1.71–1.57(m,8H),1.54–1.40(m,4H),1.38–1.19(m,8H),1.12(dd,J＝19.6,6.8Hz,4H),1.05(d,J＝6.3Hz,4H),0.99(d,J＝6.4Hz,2H),0.95(d,J＝6.5Hz,2H),0.93–0.82(m,4H),0.71(dt,J＝16.5,8.3Hz,1H).ESI-MS：[M+Na]+1149.58,C55H84INO15。
依维莫司双卤代乙酸酯Ⅳ-2：
1H NMR(400MHz,CDCl3)δ6.34(dt,J＝24.7,14.8Hz,1H),6.18–6.11(m,1H),5.93(dd,J＝29.8,10.2Hz,1H),5.62–5.48(m,1H),5.40(d,J＝9.9Hz,1H),5.28(t,J＝7.0Hz,1H),5.15(dt,J＝12.0,5.6Hz,1H),4.34–4.20(m,2H),4.16(ddd,J＝21.3,12.6,6.5Hz,2H),3.90–3.78(m,2H),3.75(d,J＝7.5Hz,2H),3.70(d,J＝13.5Hz,1H),3.67(dd,J＝14.8,6.3Hz,1H),3.44(d,J＝10.0Hz,4H),3.40–3.30(m,4H),3.21–2.98(m,5H),2.82(dd,J＝17.8,7.2Hz,1H),2.70(dd,J＝16.5,5.8Hz,2H),2.60(dd,J＝16.8,6.6Hz,1H),2.33(d,J＝12.8Hz,1H),2.12–1.91(m,6H),1.73(s,6H),1.70–1.58(m,8H),1.55–1.43(m,4H),1.37–1.21(m,8H),1.12(dd,J＝19.8,6.8Hz,4H),1.08(d,J＝6.5Hz,4H),0.98(d,J＝6.5Hz,2H),0.93(d,J＝6.5Hz,2H),0.92–0.80(m,6H),0.75(dt,J＝14.8,8.0Hz,1H).ESI-MS：[M+Na]+1317.41，C57H85I2NO16。
实施例二：
Figure PCTCN2016105178-appb-000020
将依维莫司单卤代乙酸酯Ⅳ-1(1g，0.9mmol)和谷胱甘肽(0.55g，1.8mmol)加至100mL三颈瓶中，加入5mL N,N-二甲基甲酰胺(DMF)，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(124.2mg，0.9mmol)。随后，再向反应混悬液中加入5mL H2O和10mL乙醇，剧烈搅拌10分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-1反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-1(0.98g，0.75mmol)。
化合物Ⅰ-1：1H NMR(400MHz,CD3OD+CDCl3)δ6.49–6.39(m,1H),6.19(ddd,J＝30.9,22.1,11.0Hz,3H),5.55–5.39(m,1H),5.26(d,J＝10.0Hz,1H),5.10(s,2H),4.64(s,1H),4.24(d,J＝24.1Hz,3H),4.08(d,J＝27.6Hz,2H),3.83(s,4H),3.73–3.51(m,3H),3.50–3.24(m,12H),3.12(d,J＝26.5Hz,7H),2.93(s,1H),2.79(d,J＝17.6Hz,1H),2.50(dd,J＝27.1,18.3Hz,4H),2.25(d,J＝13.3Hz,2H),2.15(s,2H),2.05(s,4H),1.93–1.53(m,16H),1.43(dd,J＝27.1,14.5Hz,5H),1.29(s,2H),1.25–1.11(m,4H),1.06(d,J＝4.6Hz,4H),1.02–0.79(m,13H),0.76(d,J＝11.9Hz,2H)。HR-ESI-MS：[M+H]+1305.6671，C65H100N4O21S。
实施例三：
Figure PCTCN2016105178-appb-000021
将依维莫司双卤代乙酸酯Ⅳ-2(1.5g，1.2mmol)和谷胱甘肽(1.17g，3.6mmol)加至250mL三颈瓶中，加入10mL N,N-二甲基甲酰胺，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(248.4mg，1.8mmol)。随后，再向反应混悬液中加入10mLH2O和20mL乙醇，剧烈搅拌15分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-2反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-2(0.51g，0.31mmol)。
化合物Ⅰ-2：1H NMR(400MHz,CD3OD+CDCl3)δ6.48–6.37(m,1H),6.19(ddd,J＝29.8,21.5,10.0Hz,3H),5.54–5.40(m,1H),5.26(d,J＝10.1Hz,1H),5.11(s,2H),4.62(s,1H),4.24(d,J＝23.5Hz,3H),4.10(d,J＝27.6Hz,2H),3.85(s,4H),3.74–3.55(m,3H),3.52–3.20(m,16H),3.10(d,J＝25.7Hz,7H),2.92(s,1H),2.81(d,J＝17.6Hz,1H),2.48(dd,J＝28.0,17.8Hz,4H),2.30(d,J＝13.5Hz,2H),2.17(s,2H),2.04(s,4H),1.97–1.48(m,21H),1.43(dd,J＝26.8,14.2Hz,5H),1.30(s,2H),1.27–1.10(m,4H),1.05(d,J＝4.6Hz,4H),1.01–0.74(m,17H),0.71(d,J＝12.0Hz,2H)。HR-ESI-MS：[M+H]+1640.5401，C77H117N7O28S2。
实施例四：
Figure PCTCN2016105178-appb-000022
将西罗莫司(4.0g,4.4mmol)和碘乙酸(1.64g，8.8mmol)加至100mL三颈瓶中，加入20mL二氯甲烷，搅拌至固体溶清。混合液冷却至0-5℃，加入二环己基碳二亚胺(2.0g，11.4mmol)，0-5℃搅拌10-15分钟，向反应液中加入4-二甲胺基吡啶(0.54g，4.4mmol)。反应液升温至室温，搅拌16-24小时。TLC检测西罗莫司反应完毕，布氏漏斗抽滤不溶性固体，滤液于30-40℃减压浓缩至干，所得浆状物用硅胶柱分离纯化(正己烷/乙酸乙酯＝10:1-2:1洗脱)，分别得到西罗莫司单卤代乙酸酯Ⅳ-3(1.8g,1.7mmol)和西罗莫司双卤代乙酸酯Ⅳ-4(1.0g,0.8mmol)。
西罗莫司单卤代乙酸酯Ⅳ-3：
1H NMR(400MHz,CDCl3)δ6.34(dt,J＝24.8,14.8Hz,1H),6.15–6.09(m,1H),5.95(dd,J＝30.0,10.2Hz,1H),5.58–5.46(m,1H),5.40(d,J＝9.9Hz,1H),5.28(t,J＝7.2Hz,1H),5.14(dt,J＝11.6,5.5Hz,1H),4.33–4.28(m,2H),4.18(ddd,J＝21.4,14.1,6.6Hz,2H),3.68(dd,J＝14.5,6.8Hz,1H),3.56(d,J＝13.5Hz,1H),3.44(d,J＝10.3Hz,4H),3.40–3.26(m,4H),3.20–3.00(m,5H),2.85(dd,J＝17.7,7.0Hz,1H),2.70(dd,J＝16.4,5.5Hz,2H),2.57(dd,J＝16.7,6.4Hz,1H),2.30(d,J＝12.9Hz,1H),2.12–1.90(m,6H),1.75(s,6H),1.71–1.58(m,8H),1.55–1.42(m,4H),1.38–1.20(m,8H),1.15(dd,J＝19.6,6.8Hz,4H),1.05(d,J＝6.3Hz,4H),0.98(d,J＝6.4Hz,2H),0.95(d,J＝6.5Hz,2H),0.95–0.85(m,4H),0.70(dt,J＝16.5,8.3Hz,1H).ESI-MS：[M+H]+1083.46,C53H80INO14。
西罗莫司双卤代乙酸酯Ⅳ-4：
1H NMR(400MHz,CDCl3)δ6.33(dt,J＝24.7,14.6Hz,1H),6.16–6.11(m,1H),5.98(dd,J＝25.2,12.0Hz,1H),5.62–5.49(m,1H),5.38(d,J＝9.8Hz,1H),5.26(t,J＝7.0Hz,1H),5.12(dt,J＝12.0,5.5Hz,1H),4.30–4.21(m,2H),4.15(ddd,J＝20.7,14.0,6.5Hz,2H),3.72(d,J＝13.5Hz,1H),3.68(dd,J＝14.2,7.2Hz,1H),3.45(d,J＝10.3Hz,4H),3.39–3.32(m,4H),3.21–2.99(m,5H),2.83(dd,J＝18.7,7.2Hz,1H),2.74(dd,J＝16.5,5.6Hz,2H),2.62(dd,J＝16.8,6.5Hz,1H),2.35(d,J＝12.6Hz,1H),2.10–1.95(m,6H),1.77(s,6H),1.70–1.62(m,8H),1.52–1.45(m,4H),1.38–1.23(m,8H),1.15(dd,J＝19.6,6.8Hz,4H),1.10(d,J＝6.2Hz,4H),0.97(d,J＝6.3Hz,2H),0.95(d,J＝6.5Hz,2H),0.92–0.82(m,6H),0.73(dt,J＝16.5,8.4Hz,1H).ESI-MS：[M+H]+1251.04，C55H81I2NO15。
实施例五：
Figure PCTCN2016105178-appb-000023
将西罗莫司单卤代乙酸酯Ⅳ-3(1.2g，1.1mmol)和谷胱甘肽(0.68g，2.2mmol)加至100mL三颈瓶中，加入6mL N,N-二甲基甲酰胺，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(151.8mg，1.1mmol)。随后，再向反应混悬液中加入6mL H2O和12mL乙醇，剧烈搅拌15分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-3反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-3(0.83g，0.75mmol)。
化合物Ⅰ-3：1H NMR(400MHz,CD3OD+CDCl3)δ6.48–6.40(m,1H),6.18(ddd,J＝25.8,20.5,10.8Hz,3H),5.56–5.41(m,1H),5.28(d,J＝12.0Hz,1H),5.17(s,2H),4.65(s,1H),4.20(d,J＝22.5Hz,3H),4.07(d,J＝26.0Hz,2H),3.80(s,4H),3.73–3.53(m,3H),3.45–3.22(m,12H),3.13(d,J＝25.5Hz,7H),2.95(s,1H),2.84(d,J＝17.8Hz,1H),2.55(dd,J＝26.0,18.2Hz,4H),2.26(d,J＝13.5Hz,2H),2.15(s,2H),2.01(s,4H),1.93–1.55(m,14H),1.47(dd,J＝27.3,15.5Hz,5H),1.38(s,2H),1.25–1.13(m,4H),1.07(d,J＝4.7Hz,4H),1.03–0.78(m,12H),0.76(d,J＝11.8Hz,2H)。ESI-MS：[M+H]+1261.52，C63H96N4O20S。
实施例六：
Figure PCTCN2016105178-appb-000024
将西罗莫司双卤代乙酸酯Ⅳ-4(0.8g，0.64mmol)和谷胱甘肽(0.59g，1.92mmol)加至250mL三颈瓶中，加入5mL N,N-二甲基甲酰胺，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(132.5mg，0.96mmol)。随后，再向反应混悬液中加入5mL H2O和10mL乙醇，剧烈搅拌15分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-4反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-4(0.35g，0.22mmol)。
化合物Ⅰ-4：1H NMR(400MHz,CD3OD+CDCl3)δ6.46–6.38(m,1H),6.16(ddd,J＝29.8,20.1,11.5Hz,3H),5.55–5.42(m,1H),5.25(d,J＝10.2Hz,1H),5.12(s,2H),4.63(s,1H),4.23(d,J＝24.5Hz,3H),4.03(d,J＝28.2Hz,2H),3.83(s,4H),3.75–3.58(m,3H),3.52–3.26(m,14H),3.16(d,J＝25.6Hz,7H),2.92(s,1H),2.78(d,J＝17.8Hz,1H),2.51(dd,J＝27.1,18.2Hz,4H),2.23(d,J＝13.5Hz,2H),2.17(s,2H),2.05(s,4H),1.95–1.49(m,20H),1.42(dd,J＝27.1,14.5Hz,5H),1.28(s,2H),1.25–1.17(m,4H),1.10(d,J＝4.6Hz,4H),1.05–0.79(m,18H),0.78(d,J＝11.9Hz,2H)。ESI-MS：[M+H]+1609.86，C75H113N7O27S2。
实施例七：
Figure PCTCN2016105178-appb-000025
将替西罗莫司(1.2g,1.2mmol)和碘乙酸(0.67g，3.6mmol)加至100mL三颈瓶 中，加入10mL二氯甲烷，搅拌至固体溶清。混合液冷却至0-5℃，加入二环己基碳二亚胺(0.74g，3.6mmol)，0-5℃搅拌10-15分钟，向反应液中加入4-二甲胺基吡啶(0.15g，1.2mmol)。反应液升温至室温，搅拌16-24小时。TLC检测替西罗莫司反应完毕，布氏漏斗抽滤不溶性固体，滤液于30-40℃减压浓缩至干，所得浆状物用硅胶柱分离纯化(正己烷/乙酸乙酯＝10:1-1:1洗脱)，分别得到替西罗莫司单卤代乙酸酯Ⅳ-5(0.31g,0.25mmol)、替西罗莫司双卤代乙酸酯Ⅳ-6(0.40g,0.36mmol)、替西罗莫司三卤代乙酸酯Ⅳ-7(0.36g,0.23mmol)。
替西罗莫司单卤代乙酸酯Ⅳ-5：
1H NMR(400MHz,CDCl3)δ6.38(dt,J＝25.1,14.6Hz,1H),6.16–6.08(m,1H),5.94(dd,J＝28.9,10.6Hz,1H),5.62–5.48(m,1H),5.42(d,J＝9.9Hz,1H),5.29(t,J＝7.3Hz,1H),5.18(dt,J＝11.8,5.5Hz,1H),4.36–4.25(m,2H),4.18(ddd,J＝20.8,14.3,6.8Hz,2H),3.90–3.78(m,2H),3.76(d,J＝7.4Hz,2H),3.68(dd,J＝14.6,6.8Hz,1H),3.59(d,J＝13.5Hz,1H),3.45(d,J＝10.3Hz,4H),3.42–3.29(m,2H),3.21–3.06(m,7H),2.86(dd,J＝17.8,7.2Hz,1H),2.74(dd,J＝16.5,5.8Hz,2H),2.57(dd,J＝16.6,6.2Hz,1H),2.30(d,J＝12.9Hz,1H),2.10–1.91(m,6H),1.76(s,6H),1.72–1.58(m,8H),1.54–1.44(m,4H),1.38–1.20(m,8H),1.14(dd,J＝19.8,6.7Hz,4H),1.06(d,J＝6.5Hz,4H),1.02(s,3H),0.99(d,J＝6.4Hz,2H),0.95(d,J＝6.5Hz,2H),0.93–0.82(m,4H),0.71(dt,J＝16.7,8.2Hz,1H).ESI-MS：[M+NH4]+1215.80,C58H88INO17。
替西罗莫司双卤代乙酸酯Ⅳ-6：
1H NMR(400MHz,CDCl3)δ6.37(dt,J＝24.6,14.8Hz,1H),6.15–6.09(m,1H),5.95(dd,J＝27.2,10.6Hz,1H),5.59–5.45(m,1H),5.38(d,J＝9.9Hz,1H),5.25(t,J＝7.2Hz,1H),5.10(dt,J＝11.3,5.9Hz,1H),4.38–4.29(m,2H),4.12(m,2H),3.88–3.76(m,2H),3.70(d,J＝7.1Hz,2H),3.63(m,6H),3.41(d,J＝10.3Hz,4H),3.38–3.25(m,2H),3.21–3.06(m,7H),2.83(dd,J＝17.8,7.0Hz,1H),2.77(dd,J＝16.3,5.5Hz,2H),2.55(dd,J＝16.8,6.0Hz,1H),2.32(d,J＝12.7Hz,1H),2.14–1.92(m,6H),1.78(s,6H),1.74–1.58(m,8H),1.55–1.39(m,4H),1.39–1.19(m,8H),1.10(dd,J＝182,6.8Hz,4H),1.06(d,J＝6.6Hz,4H),1.02(s,3H),0.98(d,J＝6.5Hz,2H),0.95(d,J＝6.5Hz,2H),0.90–0.80(m,4H),0.73(dt,J＝16.5,8.2Hz,1H).ESI-MS：[M+NH4]+1383.60,C60H89I2NO18。
替西罗莫司三卤代乙酸酯Ⅳ-7：
1H NMR(400MHz,CDCl3)δ6.40(dt,J＝24.8,14.2Hz,1H),6.16–6.08(m,1H),5.90(dd,J＝28.1,10.3Hz,1H),5.60–5.48(m,1H),5.40(d,J＝9.9Hz,1H),5.28(t,J＝7.2Hz,1H),5.16(dt,J＝11.6,5.5Hz,1H),4.35–4.27(m,2H),4.16(m,2H),3.90–3.78(m,2H),3.75(d,J＝7.1Hz,2H),3.68(dd,J＝14.6,6.8Hz,2H),3.59(d,J＝13.5Hz,2H),3.44(d,J＝10.3Hz,4H),3.43–3.27(m,2H),3.21–3.05(m,7H),2.85(dd,J＝17.7,7.0Hz,1H),2.74(dd,J＝16.8,5.5Hz,2H),2.57(dd,J＝16.7,6.3Hz,1H),2.30(d,J＝12.8Hz,1H),2.12–1.90(m,6H),1.76(s,6H),1.73–1.56(m,8H),1.53–1.40(m,4H),1.38–1.20(m,8H),1.14(dd,J＝19.4,6.8Hz,4H),1.05(d,J＝6.3Hz,4H),1.01(s,3H),0.99(d,J＝6.5Hz,2H),0.93(d,J＝6.5Hz,2H),0.90–0.81(m,4H),0.70(dt,J＝16.7,8.0Hz,1H).ESI-MS：[M+NH4]+1551.09,C62H90I3NO19。
实施例八：
Figure PCTCN2016105178-appb-000026
将替西罗莫司单卤代乙酸酯Ⅳ-5(0.28g，0.23mmol)和谷胱甘肽(0.14g，0.46mmol)加至50mL圆底烧瓶中，加入3mL N,N-二甲基甲酰胺，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(19.1mg，0.14mmol)。随后，再向反应混悬液中加入3mL H2O和6mL乙醇，剧烈搅拌15分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-5反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-5(0.21g，0.15mmol)。
化合物Ⅰ-5：1H NMR(400MHz,CD3OD+CDCl3)δ6.47–6.37(m,1H),6.15(m,3H),5.50–5.33(m,1H),5.28(d,J＝10.2Hz,1H),5.12(s,2H),4.66(s,1H),4.23(d,J＝24.0Hz,3H),4.12(d,J＝26.8Hz,2H),3.85(s,4H),3.73–3.50(m,3H),3.49–3.22(m,12H),3.10(d,J＝26.5Hz,7H),2.90(s,1H),2.79(d,J＝17.7Hz,1H),2.52(dd,J＝26.2,18.0Hz,4H),2.26(d,J＝13.3Hz,2H),2.14(s,2H),2.08(s,4H),1.95–1.52(m,16H),1.45(dd,J＝27.1,14.5Hz,5H),1.27(s,2H),1.23–1.10(m,4H),1.08(d,J＝4.6Hz,4H),1.05(s,3H),1.01–0.79(m,13H),0.78(d,J＝11.9Hz,2H)。HR-ESI-MS：[M+H]+1378.52，C68H104N4O23S。
实施例九：
Figure PCTCN2016105178-appb-000027
将替西罗莫司双卤代乙酸酯Ⅳ-6(0.36g，0.26mmol)和谷胱甘肽(0.24g，0.78mmol)加至50mL圆底烧瓶中，加入3mL N,N-二甲基甲酰胺，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(39.5mg，0.29mmol)。随后，再向反应混悬液中加 入3mL H2O和6mL乙醇，剧烈搅拌20分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-6反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-6(0.30g，0.17mmol)。
化合物Ⅰ-6：1H NMR(400MHz,CD3OD+CDCl3)δ6.49–6.37(m,1H),6.14(m,3H),5.51–5.35(m,1H),5.27(d,J＝10.2Hz,1H),5.07(s,2H),4.60(s,1H),4.20(d,J＝24.3Hz,3H),4.06(d,J＝27.8Hz,2H),3.85(s,4H),3.73–3.48(m,8H),3.45–3.23(m,12H),3.12(d,J＝26.5Hz,7H),2.94(s,1H),2.73(d,J＝17.8Hz,1H),2.54(dd,J＝27.0,18.2Hz,4H),2.24(d,J＝13.2Hz,2H),2.15(s,2H),2.07(s,4H),1.96–1.50(m,16H),1.45(dd,J＝27.1,14.5Hz,5H),1.27(s,2H),1.24–1.10(m,6H),1.09(d,J＝4.7Hz,4H),1.05(s,3H),1.05–0.78(m,12H),0.78(d,J＝11.8Hz,2H)。HR-ESI-MS：[M+H]+1725.13，C80H121N7O30S2。：
实施例十：
Figure PCTCN2016105178-appb-000028
将替西罗莫司三卤代乙酸酯Ⅳ-7(0.33g，0.22mmol)和谷胱甘肽(0.27g，0.88mmol)加至50mL圆底烧瓶中，加入3mL N,N-二甲基甲酰胺，搅拌至固体悬浮至N,N-二甲基甲酰胺中。向悬浮液中加入K2CO3(60.7mg，0.44mmol)。随后，再向反应混悬液中加入3mL H2O和6mL乙醇，剧烈搅拌30分钟后，反应液变澄清。将反应液在室温下搅拌过夜，HPLC检测化合物Ⅳ-7反应完毕，45-55℃减压浓缩反应液至干，所得浆状物经制备液相色谱纯化，得化合物Ⅰ-7(0.24g，0.12mmol)。
化合物Ⅰ-7：1H NMR(400MHz,CD3OD+CDCl3)δ6.47–6.32(m,1H),6.15(m,3H),5.51–5.35(m,1H),5.21(d,J＝10.0Hz,1H),5.06(s,2H),4.57(s,1H),4.23(d,J＝24.3Hz,3H),4.05(d,J＝27.8Hz,2H),3.86(s,4H),3.73–3.48(m,10H),3.46–3.25(m,18H),3.12(d,J＝26.6Hz,7H),2.95(s,1H),2.78(d,J＝17.1Hz,1H),2.55(dd,J＝27.3,18.5Hz,4H),2.22(d,J＝13.5Hz,2H),2.12(s,2H),2.03(s,4H),1.93–1.53(m,20H),1.48(dd,J＝27.0,14.3Hz,5H),1.27(s,2H),1.23–1.14(m,8H),1.09(d,J＝4.6Hz,4H),1.03(s,3H),1.01–0.78(m,12H),0.76(d,J＝11.3Hz,2H)。HR-ESI-MS：[M+H]+2072.30，C92H138N10O37S3。
下面的实验部分包括关于所发明的具体化合物的水溶性研究方法及研究结果、体内外活性研究方法及研究结果。未在下面的实验部分列出的化合物，也可以用与下面的实 验部分中同样的研究方法及思路开展相关研究。下面的实验部分仅仅是用来示例说明所发明的具体化合物的研究方法和结果，但不仅仅限于已使用了的化合物。
实施例十一：水中溶解性试验
本发明化合物的溶解度可通过用标准的实验操作程序来证实，该实验可测量本发明的化合物在水中的溶解情况。
溶解度测定以化合物Ⅰ-1、Ⅰ-2为例简要地描述所用的操作程序。
取依维莫司25mg加至25ml容量瓶中，加乙腈定容至刻度线，摇匀至固体全部溶解，得1mg/mL依维莫司乙腈溶液；
取1mg/mL依维莫司乙腈溶液加至10ml容量瓶中，分别用乙腈稀释2倍、10倍、100倍、1000倍，得到浓度分别为0.5mg/mL、0.1mg/mL、0.01mg/mL、0.001mg/mL的依维莫司乙腈溶液；
1mg/mL、0.5mg/mL、0.1mg/mL、0.01mg/mL、0.001mg/mL浓度的依维莫司溶液用HPLC中主峰的峰面积做标准曲线(如图1所示)，得到线性公式：y＝26010x+18.338(R2＝1)
分别取10mg依维莫司、10mg化合物Ⅰ-1和10mg化合物Ⅰ-2，加至10mL容量瓶中，加纯化水定容至刻度线，充分摇匀，直至容量瓶中的固体不再溶解。取1mL溶液，用0.22μm滤头过滤，滤液进行HPLC分析，通过所得主峰的峰面积，利用标准曲线计算化合物的溶解度，结果如表1所示。
表1 化合物Ⅰ-1、Ⅰ-2和依维莫司溶解度测定结果
化合物 峰面积 水中溶解度 备注
依维莫司 21 ＜0.001mg/mL  
化合物Ⅰ-1 14138 0.54mg/mL 按含依维莫司量计
化合物Ⅰ-2 20896 0.80mg/mL 按含依维莫司量计
如表1所示：化合物Ⅰ-1在水中的溶解度相对于依维莫司至少提高了540倍，化合物Ⅰ-2在水中的溶解度相对于依维莫司至少提高了800倍。经谷胱甘肽修饰后的化合物的水溶性大大高于依维莫司。
根据同样的研究方法，分别对比西罗莫司、替西罗莫司用谷胱甘肽修饰后化合物的水溶性，结果如下表2所示：
表2 化合物Ⅰ-3、Ⅰ-4和西罗莫司以及Ⅰ-5、Ⅰ-6、Ⅰ-7和替西罗莫司溶解度测定结果
化合物 水中溶解度 备注
西罗莫司 ＜0.001mg/mL  
化合物Ⅰ-3 0.47mg/L 按含西罗莫司量计
化合物Ⅰ-4 0.61mg/L 按含西罗莫司量计
替西罗莫司 约0.001mg/mL  
化合物Ⅰ-5 0.58mg/mL  
化合物Ⅰ-6 0.71mg/mL 按含替西罗莫司量计
化合物Ⅰ-7 0.83mg/mL 按含替西罗莫司量计
如表2所示：化合物Ⅰ-3和Ⅰ-4的水溶性相对于西罗莫司分别提高了至少470和610倍；而化合物Ⅰ-5、Ⅰ-6、Ⅰ-7相对于替西罗莫司的水溶性分别至少提高了580倍、710倍和830倍。
结论：与修饰前的母体化合物依维莫司、西罗莫司、替西罗莫司相比较，按本发明经谷胱甘肽修饰后的本发明化合物的水溶性都得到了大大的改善。所以，谷胱甘肽用于改善雷帕霉素类化合物的水溶性作用显著。
实施例十二：体外活性测试
本发明的化合物的抗肿瘤活性及毒性，可通过用标准的药理实验操作程序来证实，该实验可测量本发明的化合物对人体肝癌细胞HepG2、肺癌细胞NCI460、前列腺癌细胞DU145、前列腺癌细胞PC3、人乳腺癌细胞MDA-MB-435生长的抑制情况。下面以人体肝癌细胞HepG2抑制活性测试为例简要地描述所用的操作程序。
人体肝癌细胞HepG2在以下介质中生长：
生长介质的制备：带厄尔(Earle)盐类的BRL最小必需介质(500毫升)，并向介质中分别加入下列试剂：
5毫升BRL MEM非必需的氨基酸(10mM)；
5毫升BRL青霉素-链霉素(10000国际单位/毫升，10000微克/毫升)；
5毫升BRL丙酮酸钠溶液(100mM)；
5毫升BRL L-谷氨酰胺(200mM)；
50毫升BRL小牛血清(合格品)；
制备所得的生长介质备用。
试验操作程序如下：
1、将细胞胰蛋白酶化并在96孔板上布板，使每个小孔中细胞浓度约为104/孔，并使之在200微升最终体积的介质中生长，布板在37摄氏度条件下允许粘附24小时；
2、小心地通过抽吸除去介质，不扰动单细胞层。在每个孔中加入200微升的新鲜介质，允许有足够多的孔被点样以便进行3个平行的试验。
3、被测试的本发明中的化合物被溶解到10微升磷酸盐缓冲溶液中(PBS)，并在37摄氏度孵育48小时。
4、在孵育最后6小时期间，96孔板的每个小孔用1微居里T胸腺嘧啶脱氧核苷标记(New England Nuclear thymidine)。1微居里被加进10微升PBS中(采集试样测试当天)。96孔板放回孵育器中孵育最后的6小时。
5、在不扰动单细胞层的前提下，通过抽吸除去带放射性的介质。然后往每个小孔中加入50微升BRL 10×胰蛋白酶，并继续在37摄氏度的条件下孵育10分钟或直到单细胞层从孔底(或孔壁)松脱。用Skatron 96孔板采集器把试样采集在玻璃纤维簇上。纤维簇在Wallac Betaptate计数器中计数，体外活性检测结果如表3至表5所示。
表3 化合物Ⅰ-1和化合物Ⅰ-2体外活性测试结果
Figure PCTCN2016105178-appb-000029
Figure PCTCN2016105178-appb-000030
由表3的数据可见，化合物Ⅰ-1对人体肝癌细胞HepG2、肺癌细胞NCI460、前列腺癌细胞DU145、前列腺癌细胞PC3、人乳腺癌细胞MDA-MB-435生长的抑制活性均优于或相当于依维莫司。化合物Ⅰ-2的体外癌细胞抑制活性相当于或稍差于依维莫司。
表4 化合物Ⅰ-3和Ⅰ-4体外活性测试结果
Figure PCTCN2016105178-appb-000031
由表4的数据可见，化合物Ⅰ-3对人体肝癌细胞HepG2、肺癌细胞NCI460、前列腺癌细胞DU145、前列腺癌细胞PC3、人乳腺癌细胞MDA-MB-435生长的抑制活性均优于或相当于西罗莫司。化合物Ⅰ-4的体外癌细胞抑制活性相当于或稍差于西罗莫司。
表5 化合物Ⅰ-5、Ⅰ-6和Ⅰ-7体外活性测试结果
Figure PCTCN2016105178-appb-000032
由表5的数据可见，化合物Ⅰ-5、Ⅰ-6对人体肝癌细胞HepG2、肺癌细胞NCI460、前列腺癌细胞DU145、前列腺癌细胞PC3、人乳腺癌细胞MDA-MB-435生长的抑制活性均优于或相当于替西罗莫司。化合物Ⅰ-7的体外癌细胞抑制活性相当于或稍差于替西罗莫司。
结论：体外活性筛选实验表明，仅在大环的第42位被修饰后得到的化合物的体外癌细胞抑制活性均相当于或稍优于母体化合物，第42位和第31位同时被修饰后得到的化合物的体外癌细胞抑制活性均相当于或稍差于母体化合物。这种差异可能是由于第31位被修饰后，影响化合物与靶点的结合能力，导致癌细胞抑制活性出现下降；而抑制活性并未出现大幅降低，可能是在培养液中，第31位羟基可以被释放出来。基于以上的体外活性筛选实验结果，化合物Ⅰ-1的体外癌细胞抑制活性效果最好。
因此，我们对化合物Ⅰ-1进行了更为详细的研究，包括血清中药物释放、体内活性和体内药代学研究。以下实施例十三至实施例十七仅列出化合物Ⅰ-1的效果，但是本领域技术人员根据本发明已经公开的内容，采用相同的研究方法，在有必要的地方稍加适当的常识性的调整，可以合理预期其他未列出的本发明化合物均能够产生与化合物Ⅰ-1类似的试验结果。
实施例十三：前药在大鼠血清中释放药物的试验
本发明的化合物作为雷帕霉素类的前药可以通过标准的药理实验操作程序来证实。下面以化合物Ⅰ-1在大鼠血清中释放依维莫司试验为例，简要地描述所用的操作程序和获得的结果。
配置1mg/mL化合物Ⅰ-1的水溶液10mL备用；
配置0.10mol/L ZnCl2溶液10mL备用；
取大白鼠眼动脉血液1.5mL至1.5mL离心管中，12000转离心10分钟，吸取上清液(血清)300μl至1.5mL离心管中，同法制备7份血清溶液备用；
向装有血清液的1-6号离心管中加入100μl化合物Ⅰ-1的水溶液，7号离心管加入100μl水作为空白对照，恒温培养箱中37℃恒温培养；
分别在10min、0.5h、1h、2h、3h、4h向1-6号离心管中加入300μl乙腈和300μl 0.1mol/LZnCl2溶液，涡旋振荡5分钟混匀，以终止血清中的酶对化合物的作用，4h向7号离心管中加入300μl乙腈和300μl 0.1M ZnCl2溶液，涡旋振荡5分钟混匀；
1-7号离心管分别在12000转的转速下离心15分钟，离心除去血清中的蛋白，取上清液500μl，进行HPLC分析，分别检测10min、0.5h、1h、2h、3h、4h时，上清液中的依维莫司含量和化合物Ⅰ-1的含量，检测结果如图2所示。
结论：化合物Ⅰ-1在大鼠血清中可缓慢地释放出依维莫司，3小时基本释放完全，化合物Ⅰ-1可以有效地延长依维莫司在大鼠体内的作用时间。
实施例十四：化合物Ⅰ-1裸鼠体内对肿瘤的抑制活性试验
本发明的化合物的裸鼠体内抗肿瘤活性，可通过用标准的药理实验操作程序来证实，该实验可证明本发明的化合物在动物体内对癌细胞生长具有抑制作用。下面以裸鼠腋下接种的人体肺癌细胞NCI-H460抑制活性测试为例，简要地描述所用的操作程序和获得的结果。实验方法如下：
取接种于裸鼠腋下处于快速增殖期的NCI-H460(3代)瘤块，将其切成1mm*1mm*1mm的瘤块，无菌条件下用套管针于裸鼠右肢皮下接种。待肿瘤增殖至150～200mm3时分组。分组后给药4周，测量瘤块的长径(a)、短径(b)，每周2～3次。计算肿瘤体积(tumor volume，TV)和相对肿瘤增值率(T/C)，肿瘤体积计算公式为：TV＝1/2×a×b2；相对肿瘤增值率(T/C)的计算公式为：T/C＝TRTC/CRTV(TRTC：治疗组平均相对肿瘤体积，CRTV：溶媒对照组平均相对肿瘤体积)。Day 28剥取瘤块，称重，计算抑瘤率，抑瘤率计算公式为：(对照组瘤重量-实验组瘤重量)/对照组瘤重量×100％。
分组方法：根据瘤块生长规律及给药方案，待肿瘤平均长至约195mm3分组，分为3组，每组6只，给药方式：灌胃给药：
G1：Control(vehicle，qw 4×，iv)
G2：阳性药依维莫司(Everolimus)(5mg/kg，qw 4×，iv)
G3：化合物Ⅰ-1(6.8mg/kg，qw 4×，iv)(化合物Ⅰ-1与依维莫司采用的是等摩尔给药，即6.8mg的化合物Ⅰ-1相当于5mg的依维莫司)
试验结果如表6至表8，图3至图5所示。
1、瘤块体积变化
表6 瘤块体积变化
Figure PCTCN2016105178-appb-000033
Figure PCTCN2016105178-appb-000034
从表6的数据和图3所示的裸鼠体内肿瘤体积增长曲线可见：28天时，化合物Ⅰ-1在裸鼠体内对NCI-H460表现出明显的抑制作用，其T/C值为35％(小于40％)，而等物质的量的依维莫司在裸鼠体内对NCI-H460表现出的T/C值为42％(大于40％)。可见，化合物Ⅰ-1组对肿瘤的抑制作用优于依维莫司组。
2、裸鼠体重变化
表7 裸鼠体重变化
Figure PCTCN2016105178-appb-000035
体重的下降可间接表明药物对动物的毒性反应和副作用，从表7中的数据和图4所示的裸鼠体重变化趋势可见：化合物Ⅰ-1(G3组)体重增长大于空白对照组和阳性药依维莫司组(G1、G2组)，而阳性药依维莫司组体重增加小于空白对照组，所以，化合物Ⅰ-1在体内毒副作用明显小于依维莫司。
3、瘤块重量(Tumor Weight)及抑瘤率
表8 瘤块重量及抑瘤率
组别 Tumor Weight(g)±SD 抑瘤率(％)
control 2.6691±0.6117  
依维莫司5mg/kg 1.1741±0.3687 56.01％
化合物Ⅰ-1 6.8mg/kg 0.8463±0.1582 68.29％
表8数据和图5所示的裸鼠体内瘤块重量与抑瘤率大小表明，化合物Ⅰ-1在裸鼠体内的抑瘤率明显大于依维莫司。
结论：至实验终点，NCI-H460接种裸鼠模型数据表明：化合物Ⅰ-1组为有效剂量组，其T/C值小于40％(35％)、其抑瘤率大于60％(68％)，对NCI-H460细胞接种肿瘤有良好的抑制作用；阳性药依维莫司组的T/C值均大于40％(42％)、抑瘤率小于60％(56％)。化合物Ⅰ-1体内对NCI-H460细胞的抑制活性明显优于依维莫司；且化合物Ⅰ-1(G3组)体重增长明显大于阳性药依维莫司组(G2组)，化合物Ⅰ-1动物体内的毒副作用明显小于依维莫司。
实施例十五：化合物Ⅰ-1灌胃给药和注射给药体内肿瘤抑制效果对比研究
本发明的化合物的裸鼠体内抗肿瘤活性，可通过用标准的药理实验操作程序来证实，该实验可证明本发明的化合物在动物体内对癌细胞生长的抑制作用。
取接种于裸鼠腋下处于快速增殖期的DU145(3代)瘤块，将其切成1mm*1mm*1mm的瘤块，无菌条件下用套管针于裸鼠右肢皮下接种。待肿瘤增殖至150～200mm3时分组。分组后给药4周，测量瘤块的长径(a)、短径(b)，每周2～3次。肿瘤体积(tumor volume，TV)计算公式为：TV＝1/2×a×b2。第28天剥取瘤块，称重，计算抑瘤率，公式为：(对照组瘤重量-实验组瘤重量)/对照组瘤重量×100％。
根据瘤块生长规律及给药方案，待肿瘤平均长至约185-200mm3分组，分为3组，每组4只。
G1：阳性药依维莫司组：Everolimus(5mg/kg，qw，4w，3×/w)，给药方式：灌胃；
G2：化合物Ⅰ-1(3.4mg/kg，qw，4w，3×/w)(3.4mg的Ⅰ-1相当于2.5mg的依维莫司)，给药方式：尾静脉注射；
G3：化合物Ⅰ-1(1.7mg/kg，qw，4w，3×/w)(1.7mg的Ⅰ-1相当于1.25mg的依维莫司)，给药方式：尾静脉注射；
试验结果如表9至表10，图6所示：
1、瘤块体积变化
表9 瘤块体积变化
Figure PCTCN2016105178-appb-000036
从以上表9数据和肿瘤体积增长趋势图6可看出，至试验终点，化合物Ⅰ-1采用相当于依维莫司1/4剂量尾静脉注射给药，即可达到与灌胃给予依维莫司的肿瘤抑制效果基本一致，化合物Ⅰ-1采用相当于依维莫司1/2剂量尾静脉注射给药，肿瘤抑制效果明显优于灌胃给予依维莫司。
2、裸鼠体重变化
表10 裸鼠体重变化
Figure PCTCN2016105178-appb-000037
Figure PCTCN2016105178-appb-000038
体重的下降可间接表明药物对动物的毒性反应和副作用，从表10中数据可见：注射给药组(G2、G3组)体重增长趋势大于灌胃给药组(G1组)，所以，注射给予化合物Ⅰ-1并未产生明显的毒副作用。
结论：化合物Ⅰ-1由于水溶性得到明显的改善，可以溶于生理盐水，而且溶于生理盐水后所得的水溶液稳定，可以通过采用注射给药大大提高药物在体内的生物利用度。试验结果表明，化合物Ⅰ-1仅需采用相当于1/4剂量的依维莫司，即可达到灌胃给予依维莫司一样的肿瘤抑制效果。
实施例十六：化合物Ⅰ-1裸鼠体内对人肾细胞瘤OS-RC-2的抑制活性试验
取接种于裸鼠腋下处于快速增殖期的OS-RC-2(3-10代)瘤块，将其切成1mm*1mm*1mm的瘤块，无菌条件下用套管针于裸鼠右肢皮下接种。待肿瘤增殖至167mm3时分组。分组后给药，测量瘤块的长径(a)、短径(b)，每周2～3次。肿瘤体积(tumor volume，TV)计算公式为：TV＝1/2×a×b2。实验终止剥取瘤块，称重，计算抑瘤率。
分组方法：待肿瘤平均长至约167mm3分组，每组6只，分为7组，分别为：
1)G1：Control(vehicle)
2)G2：依维莫司(2mg/kg ig 3×/qw)
3)G3：依维莫司(6mg/kg ig 3×/qw)
4)G4：化合物Ⅰ-1(8.12mg/kg ig 3×/qw)(化合物Ⅰ-1与G3组依维莫司采用的是等摩尔给药，即8.12mg的化合物Ⅰ-1相当于6mg的依维莫司)
5)G5：化合物Ⅰ-1(2.0mg/kg iv 3×/qw)(2.0mg的化合物Ⅰ-1相当于1.5mg的依维莫司)
6)G6：化合物Ⅰ-1(8.12mg/kg iv 3×/qw)(8.12mg的化合物Ⅰ-1相当于6mg的依维莫司)
7)G7：化合物Ⅰ-1(32.7mg/kg iv 1×/qw)(32.7mg的化合物Ⅰ-1相当于24mg的依维莫司)
试验结果如表11、12和图7、8所示：
1、裸鼠体内瘤块重量及抑制率
表11 瘤块重量及抑瘤率
Figure PCTCN2016105178-appb-000039
从图7裸鼠体内肿瘤体积增长趋势图可见，每周3次口服依维莫司组(G2和G3组)裸鼠，不管是高剂量6mg/Kg组还是低剂量2mg/Kg组，裸鼠体内肿瘤体积在32天后均未得到有效抑制作用，而注射化合物Ⅰ-1组(G5组、G6组和G7组)，不管是低剂量(2.0mg/Kg)每周3次，还是高剂量(32.7mg/Kg)每周一次注射给药均表现出较好的肿瘤抑制效果。
表11的数据进一步通过裸鼠体内肿瘤最终质量大小及抑瘤率的计算结果表明，无论是低剂量还是高剂量注射给药化合物Ⅰ-1(G5、G6和G7组)均表现出很好的肿瘤抑制效果，抑瘤率分别为57.1％、59.9％和60.6％，均显著优于依维莫司组(G2和G3组)，且其中每周一次高剂量注射化合物I-1效果最好，抑瘤率高达60.6％；同时，即使将化合物Ⅰ-1采用等同于依维莫司的摩尔数且采用相同的给药方式，化合物Ⅰ-1的抑瘤率也明显高于依维莫司(G4和G3组相比，两者抑瘤率分别为55.6％和41.1％)。
2、裸鼠体重变化
表12 裸鼠体重变化
Figure PCTCN2016105178-appb-000040
体重可间接反应药物毒性大小，从表12和图8可见，空白对照组裸鼠体重大幅下降，下降率达到33.0％，这种现象可能是肿瘤增长导致营养缺失所致。而药物治疗组的体重下降率均比空白对照组小。其中，化合物Ⅰ-1高剂量组(G6、G7组)体重下降率为11.7％和10.7％；口服依维莫司组(G2、G3组)体重下降率为17.4％和11.7％，化合物Ⅰ-1高剂量组与口服依维莫司组相比较，并未显示出明显的毒性。所以，高剂量注射给予化合物Ⅰ-1，小鼠的耐受性较好，未见明显毒性反应。
结论：化合物Ⅰ-1通过每周一次高剂量给药，可以有效的抑制裸鼠体内人肾细胞癌肿瘤的生长，抑瘤率达60.6％；每周3次口服6mg/Kg依维莫司组裸鼠体内抑瘤率仅41.1％，每周一次注射给药，这在很大程度上可以提高临床晚期癌症患者治疗的依从性。而且，高剂量注射化合物Ⅰ-1，小鼠的耐受性较好，未见明显毒性反应。
实施例十七：化合物Ⅰ-1大鼠体内药代动力学研究
为进一步探讨化合物Ⅰ-1在大鼠体内药代动力学特征，对SD大鼠静脉给予化合物Ⅰ-1后，测定释放出来的依维莫司在大鼠体内的药代动力学特性及生物利用度，并与口服直接给予依维莫司后的药代动力学特性进行比较。给药方式及取样频率如下表13所示。
表13 大鼠体内注射化合物Ⅰ-1药代动力学对比研究
Figure PCTCN2016105178-appb-000041
观察给药后2小时内动物的健康状况，随后每次取血观察一次动物并记录，直至最终样品收集完。血样检测结果如表14至表16，图9所示：
表14 雄性SD大鼠单次静脉给予化合物Ⅰ-1(2.72mg·kg-1)后血浆中依维莫司浓度
Figure PCTCN2016105178-appb-000042
表15 雄性SD大鼠口服给予依维莫司(10.000mg·kg-1)后血浆中依维莫司的浓度
Figure PCTCN2016105178-appb-000043
NA：Not Available
表16雄性SD大鼠分别单次静脉给予化合物Ⅰ-1(2.72mg·kg-1)或单次口服给予依维莫司(10.000mg·kg-1)后血浆中依维莫司的药代动力学参数
Figure PCTCN2016105178-appb-000044
Figure PCTCN2016105178-appb-000045
注：MRT表示平均驻留时间。
从上表14至16以及血药浓度-时间曲线图(图9)来看，口服10mg/Kg依维莫司的大鼠平均血药浓度较低，而在注射相当于口服依维莫司五分之一剂量(2mg/Kg)的化合物Ⅰ-1大鼠体内，其大鼠体内释放出的依维莫司平均血药浓度远远大于口服依维莫司。由注射2.72mg/Kg化合物Ⅰ-1(相当于2mg/Kg依维莫司)在大鼠体内释放出来的依维莫司血药浓度-时间曲线(AUC)面积约为2500，口服依维莫司大鼠体内依维莫司血药浓度-时间曲线面积仅约为450。可见，在注射五分之一剂量的化合物Ⅰ-1情况下，其体内释放出来的依维莫司生物利用度是口服依维莫司的5-6倍。
结论：口服依维莫司生物利用度极低，而注射化合物Ⅰ-1可以完全释放出相当量的依维莫司，解决了依维莫司的生物利用度不高的问题。
综合以上研究可见：
1)由于雷帕霉素的水溶性差，进入体内之后其结构不稳定等特征是导致其生物利用度差的主要原因，所以改善水溶性，提高体内结构稳定性是提高其生物利用度的关键点；用谷胱甘肽修饰雷帕霉素类化合物，可以显著地改善其水溶性，修饰后的化合物可以较好地溶于生理盐水，并且结构稳定，可以用于注射给药，显著提高了雷帕霉素类化合物体内的生物利用度；
2)修饰后的化合物可以在大鼠血清中逐渐地释放出原药，可以起到缓释作用，延长药物在体内的作用时间；
3)动物体内外试验表明，修饰后的化合物比原化合物均表现出更好的肿瘤抑制活性和更低的体内毒性。

Claims (19)

  1. 式Ⅰ所示的化合物或其在药学上可接受的盐：
    Figure PCTCN2016105178-appb-100001
    其中
    R1为H或R3；
    R2为H或R4-R5；
    但R1和R2不同时为H；
    R3为R4-R5、-CH2CH2O-R4-R5、
    Figure PCTCN2016105178-appb-100002
    R4为
    Figure PCTCN2016105178-appb-100003
    羰基C2-C6亚烯基或羰基C2-C6亚炔基；
    n为小于或等于6的整数，即，n为1，2，3，4，5或6；；
    m为小于或等于6的整数，即，m为1，2，3，4，5或6；
    R5为多肽基，优选为如式Ⅱ所示的巯基(-SH)脱氢后形成的谷胱甘肽基：
    Figure PCTCN2016105178-appb-100004
    R6为C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基。
  2. 根据权利要求1所述的化合物，其中，R3为R4-R5或-CH2CH2O-R4-R5，其中R4和R5如权利要求1所定义。
  3. 根据权利要求1或2所述的化合物，其中，R4为
    Figure PCTCN2016105178-appb-100005
  4. 根据权利要求1所述的化合物，其中，R1为-CH2CH2O-R4-R5。
  5. 根据权利要求4所述的化合物，其中，
    R4为
    Figure PCTCN2016105178-appb-100006
    n为小于或等于6的整数；
    R5为式Ⅱ所示的巯基(-SH)脱氢后形成的谷胱甘肽基：
    Figure PCTCN2016105178-appb-100007
  6. 根据权利要求4或5所述的化合物，其中，R2为H。
  7. 根据权利要求1-6任一项所述的化合物，其中所述的式Ⅰ化合物选自：
    Figure PCTCN2016105178-appb-100008
    Figure PCTCN2016105178-appb-100009
    Figure PCTCN2016105178-appb-100010
  8. 制备权利要求1-7任一项所述的式Ⅰ化合物的方法，所述方法包括以下步骤：
    (a)将式Ⅲ所示的化合物与XR10COOH反应，制备得到如式Ⅳ所示的化合物：
    Figure PCTCN2016105178-appb-100011
    其中，
    R7为H，-CH2CH2OH或
    Figure PCTCN2016105178-appb-100012
    R8为H，R4X，-CH2CH2OR4X，
    Figure PCTCN2016105178-appb-100013
    R9为H或R4X；
    R8和R9不同时为H；
    R10为C1-C6亚烷基、
    Figure PCTCN2016105178-appb-100014
    C2-C6亚烯基或C2-C6亚炔基；
    X为卤素原子，优选I或Br原子；
    (b)将步骤(a)制备得到的式Ⅳ化合物和多肽反应，得到式Ⅰ化合物：
    Figure PCTCN2016105178-appb-100015
    其中，
    R1是H或R3；
    R2是H或R4-R5；
    但R1和R2不同时为H；
    R3为R4-R5、-CH2CH2O-R4-R5、
    Figure PCTCN2016105178-appb-100016
    R4为
    Figure PCTCN2016105178-appb-100017
    羰基C2-C6亚烯基或羰基C2-C6亚炔基；
    n为小于或等于6的整数，即，n为1，2，3，4，5或6；
    m为小于或等于6的整数，即，m为1，2，3，4，5或6；
    R5为多肽基，优选为如式Ⅱ所示的巯基(-SH)脱氢后形成的谷胱甘肽基：
    Figure PCTCN2016105178-appb-100018
    R6为C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基。
  9. 根据权利要求8所述的方法，其中在步骤(b)中，式Ⅳ化合物和多肽反应是在混合 溶剂中进行的，所述混合溶剂为N,N-二甲基甲酰胺-醇-水混合溶剂，其中所述醇优选为乙醇。
  10. 根据权利要求9所述的方法，其中在所述混合溶剂中，N,N-二甲基甲酰胺-醇-水的体积比为1:1-5:1-5，优选为1:2:1。
  11. 一种药物组合物，所述的药物组合物含有有效剂量的权利要求1-7任一项所述的化合物或其药学上可接受的盐，优选其柠檬酸盐，及药用辅料，其中所述药用辅料包括药用载体、赋形剂或它们的组合。
  12. 权利要求1-7任一项所述的化合物或其在药学上可接受的盐、或权利要求11所述的药物组合物在制备用于治疗或抑制移植排斥反应的药物中的用途。
  13. 权利要求1-7任一项所述的化合物或其在药学上可接受的盐、或权利要求11所述的药物组合物在制备用于治疗肿瘤、真菌感染或血管疾病的药物中的用途。
  14. 如权利要求13所述的用途，其中所述的肿瘤选自肾细胞癌、肾脏上皮肾细胞癌、乳腺癌、胰腺癌、肺癌、前列腺癌、室管膜下巨细胞星形细胞瘤或肾血管平滑肌脂肪瘤。
  15. 含有权利要求1-7任一项所述的化合物或其在药学上可接受的盐或权利要求11所述的药物组合物的制剂，所述制剂为片剂、胶囊剂、注射剂、散剂、颗粒剂、涂药支架、丸剂或膜剂。
  16. 根据权利要求15所述的制剂，其中所述制剂为注射剂，溶媒为注射用水。
  17. 根据权利要求16所述的制剂，其中所述注射剂为冻干粉针注射剂，复溶溶媒为生理盐水。
  18. 一种给药方法，包括给予患者有效剂量的权利要求1-7任一项所述的化合物或其在药学上可接受的盐，或权利要求11所述的药物组合物。
  19. 根据权利要求18所述的给药方法，其中给予方式为向患者每周注射一次有效剂量的权利要求1-7任一项所述的化合物或其在药学上可接受的盐、或权利要求11所述的药物组合物。
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