CN111138435A - Modified methotrexate and preparation method and application thereof - Google Patents
Modified methotrexate and preparation method and application thereof Download PDFInfo
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- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical class C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims description 5
- 229960000485 methotrexate Drugs 0.000 claims abstract description 19
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 13
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- 239000003960 organic solvent Substances 0.000 claims description 8
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007821 HATU Substances 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 206010003246 arthritis Diseases 0.000 claims description 4
- XBXCNNQPRYLIDE-UHFFFAOYSA-M n-tert-butylcarbamate Chemical compound CC(C)(C)NC([O-])=O XBXCNNQPRYLIDE-UHFFFAOYSA-M 0.000 claims description 4
- LFKDJXLFVYVEFG-UHFFFAOYSA-N tert-butyl carbamate Chemical compound CC(C)(C)OC(N)=O LFKDJXLFVYVEFG-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- DQYBDCGIPTYXML-UHFFFAOYSA-N ethoxyethane;hydrate Chemical compound O.CCOCC DQYBDCGIPTYXML-UHFFFAOYSA-N 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000012535 impurity Substances 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 230000021615 conjugation Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000002202 Polyethylene glycol Substances 0.000 abstract description 12
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 12
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 abstract description 8
- 238000004132 cross linking Methods 0.000 abstract description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 5
- 125000003827 glycol group Chemical group 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 15
- 239000000047 product Substances 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000001808 coupling effect Effects 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- SVPBRIZYFJFLOL-UHFFFAOYSA-N 2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanamine Chemical compound NCCOCCOCCOCCOCCOCCN=[N+]=[N-] SVPBRIZYFJFLOL-UHFFFAOYSA-N 0.000 description 1
- VQNDBXJTIJKJPV-UHFFFAOYSA-N 2h-triazolo[4,5-b]pyridine Chemical compound C1=CC=NC2=NNN=C21 VQNDBXJTIJKJPV-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003432 anti-folate effect Effects 0.000 description 1
- 229940127074 antifolate Drugs 0.000 description 1
- 229940034982 antineoplastic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000004052 folic acid antagonist Substances 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- -1 hexafluorophosphate Chemical compound 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D475/00—Heterocyclic compounds containing pteridine ring systems
- C07D475/06—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4
- C07D475/08—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Physical Education & Sports Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides modified methotrexate, which is characterized in that a polyethylene glycol chain with adjustable length is introduced into the methotrexate, a maleimide group is introduced at the tail end of a polyethylene glycol linking agent, and the maleimide group can be directionally crosslinked with free sulfydryl of protein, so that the steric hindrance of the methotrexate is overcome, the non-directional selectivity of two carboxyl regions is solved, and a better crosslinking effect and crosslinking efficiency are obtained.
Description
Technical Field
The invention relates to the field of chemical drugs, and in particular relates to modified methotrexate and a preparation method and application thereof.
Background
Methotrexate is an anti-folate antineoplastic agent. The product is orange yellow crystalline powder. The synthesis of tumor cells is hindered mainly by inhibiting dihydrofolate reductase, so that the growth and the propagation of the tumor cells are inhibited.
12/2017, and 27, the national institutes of health international cancer research promulgated reference to the carcinogen list, methotrexate is in the category 3 carcinogen list.
Methotrexate, as a folate derivative, has been used primarily in chemotherapy for tumors, and subsequently in arthritis coupled to specific serum proteins (Rheumatology 2004; 43: 1067-1068, Right on target: coupling method to albumin to molecular arthritis), and therefore, a relatively simple and practical method for coupling methotrexate to proteins has been sought.
The 1983 US4699784A patent document has already described the treatment of cancer by methotrexate conjugated to antibodies recognizing tumors.
In the prior art, the method for crosslinking methotrexate to protein or other drug delivery carriers is mainly to form amide covalent bonds between two carboxylic acids on the methotrexate and amino groups on the protein or drug carriers, and because the steric hindrance around carboxyl groups of the methotrexate is large, the coupling efficiency is not high, and the coupling effect is not good due to the selectivity of two carboxyl groups. The above problems are problems that the art needs to solve.
Disclosure of Invention
The technical problem to be solved by the invention is to provide modified methotrexate, which can effectively improve the coupling effect and the coupling efficiency of the methotrexate.
In order to solve the technical problem, the invention provides the following scheme: a modified methotrexate having the formula:
according to the method, the length of a polyethylene glycol chain can be adjusted by introducing one polyethylene glycol chain, and a maleimide group is introduced at the tail end of the polyethylene glycol linking agent, so that the maleimide group can be directionally crosslinked with free sulfydryl of protein, thereby overcoming the methotrexate steric hindrance and solving the problem of non-directional selectivity of two carboxyl regions, and further obtaining better crosslinking effect and crosslinking efficiency.
The application further provides a method for preparing the methotrexate: the method comprises the following steps:
s1, mixing (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamic acid tert-butyl ester and maleic anhydride in an organic solvent, reacting for more than 18h, removing the solvent, drying and purifying;
s2, dissolving the product prepared in the step S1 in acetic anhydride, adding sodium acetate, heating to 60-90 ℃, mixing for more than 18h, and then adding NaHCO3Until the pH value is 7 to 8, removing the solvent, drying and purifying;
s3, dissolving the product obtained in the step S2 in an organic solution of hydrochloric acid, reacting for 9-18h at room temperature, removing the solvent, drying and purifying,
and S4, mixing the methotrexate and the condensing agent in an organic solvent for 1-3h at room temperature, adding the product obtained in the step S3, continuously mixing for 9-18h, adding water to precipitate a solid, and filtering to remove impurities to obtain the modified methotrexate.
Further, the method comprises the following steps: in said step S1, (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamic acid tert-butyl ester is prepared by the following steps:
s01, dissolving 17-azido-3, 6,9,12, 15-pentaoxaheptadecane-1-amine in NaOH solution, mixing with di-tert-butyl dicarbonate solution at room temperature for more than 18h, removing the solvent, drying and removing dichloromethane;
s02, dissolving the product prepared in the step S01 in an ether water solution, mixing the solution with triphenylphosphine at room temperature for reacting for more than 18h, removing the solvent, purifying and drying to obtain (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) tert-butyl carbamate, wherein the structural formula of the tert-butyl carbamate is as follows:
further, the method comprises the following steps: the concentration of the NaOH solution was 1.3M.
Further, the method comprises the following steps: in the step S3, the organic solution of hydrochloric acid is an ethanol solution of hydrochloric acid, and the concentration of the ethanol solution of hydrochloric acid is 4M to 6M.
Further, the method comprises the following steps: in the step S4, the condensing agent is a mixture of HATU and DIPEA, and the molar ratio of the HATU to the DIPEA is 1: 1; the organic solvent is DMSO.
Further, the method comprises the following steps: in step S1, the organic solvent is acetonitrile.
The application further provides an application of the modified methotrexate: for coupling to a protein or drug carrier.
The application further provides an application of the modified methotrexate: it is used for treating cancer.
The application further provides an application of the modified methotrexate: it can be used for treating arthritis.
The invention has the beneficial effects that: the application prepares methotrexate modified by polyethylene glycol linker with maleimide group, and the modified methotrexate can be more easily crosslinked to a loose carrier of a medicament through reaction with sulfydryl.
Drawings
FIG. 1 is a composite circuit diagram of the present application;
FIG. 2 is a nuclear magnetic spectrum of the product prepared at step S2 of the present application;
FIG. 3 is a nuclear magnetic spectrum of the final product prepared in the present application;
Detailed Description
The present invention is further described below in conjunction with specific examples so that those skilled in the art may better understand the present invention and practice it. The following discloses many different embodiments or examples for implementing the subject technology described. Specific examples of one or more arrangements of features presented below are set forth to simplify the disclosure, but are not intended to limit the invention.
The following terms appearing in the present application are the proper names in the art, and those skilled in the art can unambiguously identify the meaning of a term:
room temperature: 25 +/-5 ℃;
HATU: 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate;
DIPEA: n, N-diisopropylethylamine;
DMSO, DMSO: dimethyl sulfoxide;
Boc2o: di-tert-butyl dicarbonate.
A modified methotrexate having the formula:
according to the method, the length of a polyethylene glycol chain can be adjusted by introducing one polyethylene glycol chain, and a maleimide group is introduced at the tail end of the polyethylene glycol linking agent, so that the maleimide group can be directionally crosslinked with free sulfydryl of protein, thereby overcoming the methotrexate steric hindrance and solving the problem of non-directional selectivity of two carboxyl regions, and further obtaining better crosslinking effect and crosslinking efficiency. Meanwhile, due to the replaceability of the polyethylene glycol linking agent, the degradable polyethylene glycol linking agent can be selected as the cross-linking agent, so that the modulatable property of the coupling effect is improved.
In addition, the present application also provides a method for preparing modified methotrexate as described above, wherein the reaction steps are shown in fig. 1, and for the convenience of understanding, a specific example for preparing modified methotrexate is provided below.
Example 1:
the method comprises the following steps:
s01, 17-azido-3, 6,9,12, 15-pentaoxaheptadecan-1-amine (40g, 0.13mol) was dissolved in 200mL of aqueous NaOH (1.3M) and Boc was added at room temperature2Dissolving O (31.3g, 0.14mol) in 200mL of acetone solution, slowly adding the reaction solution dropwise, stirring at room temperature for 18 hours, detecting by TLC, removing acetone by spinning off, extracting the remaining aqueous phase with dichloromethane for 3 times, collecting the organic phase, adding anhydrous sodium sulfate, drying, filtering, and removing dichloromethane. 53g of crude product are obtained, and after column purification, 41g of colorless liquid are obtained, and the yield is as follows: 77%, the structural formula of the compound is:
s02, mixing(41g, 0.1mol) was dissolved in 200mLAfter stirring at room temperature, triphenylphosphine (37.4g, 0.11mol) was added in portions to the mixed solution of ether and 200mL water, and then stirred at room temperature for 18 hours, after the TLC detection reaction was completed, ether was removed, the remaining aqueous phase was extracted with ethyl acetate 3 times, and the aqueous phase was collected and directly dried by spin-drying to obtain 31g of pale yellow liquid, which was obtained in the following yield: 81% of a compound which is tert-butyl (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamate having the formula:
s1, dissolving (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamic acid tert-butyl ester (5g, 13.1mmol) and maleic anhydride (1.29g, 13.1mmol) in 50mL acetonitrile, stirring at room temperature for 18 hours, directly removing the acetonitrile after TLC detection reaction to obtain 6.1g of product, wherein the yield is as follows: 99%, the structural formula of the product is:
the tert-butyl (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamate used above may be prepared by referring to steps S01 to S02 in the present example, or may be purchased as it is.
S2, mixing(6.1g, 12.8mmol) is dissolved in 50mL of acetic anhydride, sodium acetate (1.03g, 12.8mmol) is added, the temperature is raised to 60 ℃, the stirring is carried out for 18h, after the TLC detection reaction is finished, saturated NaHCO3 which is twice as much as acetic anhydride is slowly dripped into the reaction liquid while stirring, the pH value is detected to be alkalescent, namely between 7 and 8, then ethyl acetate is used for extraction for 3 times, the organic phase is collected, anhydrous sodium sulfate is added for drying, the filtration and the desolventization are carried out, 6.3g of crude product is obtained, the crude product is purified by a column to obtain 4.2g of light yellow liquid, and the yield is as follows: 71.3%, the structural formula of the product is:
the nuclear magnetic spectrum comprises:1H NMR(400MHz,CDCl3) 1.43(9H, s),3.29-3.31(2H, m),3.51-3.73(22H, m),5.09(1H, s),6.69(2H, s), as shown in FIG. 2.
will be provided with(4.2g, 9.13mmol) was dissolved in 80mL of ethanol (4M) hydrochloride, stirred at room temperature for 9-18h, and after TLC detection, the reaction was removed to give 3.6g of a yellow liquid with a yield of 99%, the product having the formula:
s4, methotrexate (454mg, 1mmol) is dissolved in 10mL DMSO, HATU (1.18g, 3mmol) and DIPEA (380mg, 3mmol) are added in sequence, the mixture is stirred at room temperature for 1h to 3h, and slowly added dropwise(430mg, 1.1mmol), stirring at room temperature for 6-18h, detecting by TLC after the reaction is finished, adding water, precipitating solid, filtering, collecting filter cake, adding a small amount of DMSO, and passing through a reverse phase column to obtain 30mg of a product, wherein the yield is as follows: 3.7 percent.
The product is subjected to nuclear magnetic resonance test, and the nuclear magnetic spectrum of the product is as follows:1h NMR (400MHz, d-DMSO)3.22-3.51(20H, m),4.82-4.84(2H, m),6.80-6.83(2H, m),7.00(1H, s),7.51-7.52(1H, m),7.73-7.74(1H, m),8.18-8.28(2H, m),8.53-8.59(1H, m), as shown in FIG. 3, whereby it was confirmed that the structural formula was:
the application prepares methotrexate modified by polyethylene glycol linker with maleimide group, and the modified methotrexate can be more easily crosslinked to a loose carrier of a medicament through reaction with sulfydryl.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
2. a method of preparing modified methotrexate as claimed in claim 1, comprising the steps of:
s1, mixing (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamic acid tert-butyl ester and maleic anhydride in an organic solvent, reacting for more than 18h, removing the solvent, drying and purifying;
s2, dissolving the product prepared in the step S1 in acetic anhydride, adding sodium acetate, heating to 60-90 ℃, mixing for more than 18h, and then adding NaHCO3Until the pH value is 7 to 8, removing the solvent, drying and purifying;
s3, dissolving the product obtained in the step S2 in an organic solution of hydrochloric acid, reacting for 9-18h at room temperature, removing the solvent, drying and purifying,
and S4, mixing the methotrexate and the condensing agent in an organic solvent for 1-3h at room temperature, adding the product obtained in the step S3, continuously mixing for 9-18h, adding water to precipitate a solid, and filtering to remove impurities to obtain the modified methotrexate.
3. The method according to claim 2, wherein tert-butyl (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamate in step S1 is produced by:
s01, dissolving 17-azido-3, 6,9,12, 15-pentaoxaheptadecane-1-amine in NaOH solution, mixing with di-tert-butyl dicarbonate solution at room temperature for more than 18h, removing the solvent, drying and removing dichloromethane;
s02, dissolving the product prepared in the step S01 in an ether water solution, mixing the solution with triphenylphosphine at room temperature for reacting for more than 18h, removing the solvent, purifying and drying to obtain (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) tert-butyl carbamate, wherein the structural formula of the tert-butyl carbamate is as follows:
4. the preparation method according to claim 3, wherein in the step S01, the concentration of the NaOH solution is 1.3M.
5. The method according to claim 2, wherein in step S3, the organic solution of hydrochloric acid is an ethanol solution of hydrochloric acid, and the concentration of the solution is 4M to 6M.
6. The preparation method according to claim 2, wherein in step S4, the condensing agent is a mixture of HATU and DIPEA in a molar ratio of 1: 1; the organic solvent is DMSO.
7. The production method according to claim 2, wherein the organic solvent is acetonitrile in step S1.
8. Use of the modified methotrexate of claim 1 for conjugation to a protein or drug carrier.
9. Use of the modified methotrexate of claim 1 for the treatment of cancer.
10. Use of the modified methotrexate of claim 1 for the treatment of arthritis.
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