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CAS No. : | 59-05-2 | MDL No. : | MFCD00064370 |
Formula : | C20H22N8O5 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | FBOZXECLQNJBKD-ZDUSSCGKSA-N |
M.W : | 454.44 | Pubchem ID : | 126941 |
Synonyms : |
Amethopterin;CL14377;NCI-C04671;WR19039
|
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P280-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H317-H319-H351-H361 | Packing Group: | N/A |
GHS Pictogram: |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With cesium bicarbonate; In water; for 4h; | EXAMPLE 1 Preparation of di-cesium salt of (+)-amethopterin (methotrexate). <strong>[59-05-2]Methotrexate</strong> (0.75 mmole) was dissolved in 200 ml of water to which was added cesium bicarbonate (1.5 mmol). The yellow solution was stirred for 4 hours, evaporated under reduced pressure, and the oily residue dissolved in ethanol. This was evaporated from anhydrous ethanol 3 times to remove residual water. |
Yield | Reaction Conditions | Operation in experiment |
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With dicyclohexyl-carbodiimide; In DMF (N,N-dimethyl-formamide); at 20℃; for 4h; | To 57 mg (0.125 mmol) of methotrexate (Sigma) was added 1 mL of anhydrous DMF followed by 22 mg (0.15 mmol) of 4-nitrophenol and 27mg (0.13 mmol) of N,N'-dicyclohexylcarbodiimide. The resulting reaction mixture was allowed to stir at room temperature for 4 hours and the resulting methotrexate activated ester (compound 20) was used in situ in the next step without isolation. To 100 mg (0.14 mmol) of osmium dibipyridyl t-Boc histamine (compound 4) was added 1 mL of trifluoroacetic acid. The resulting reaction mixture was allowed to stir at room temperature 30 minutes and concentrated under reduced pressure. To the residue 5 mL of methylene chloride was added and concentrated. The addition of 5 mL methylene chloride and concentration process was repeated four more times. To the residue 1 mL of DMF was added followed by 200 muL (1.43 mmol) of triethylamine. The reaction mixture was allowed to stir at room temperature under argon atmosphere and the activated ester of methotrexate prepared as above (compound 20) was added dropwise. The reaction was allowed to stir for 18 hours at room temperature under argon atmosphere and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC to give 52.5 mg (0.046 mmol, 32%) of the osmium methotrexate conjugate (compound 21) as a brown powder, LC/MS M+H 1086.2. Osmium PEG <strong>[59-05-2]Methotrexate</strong> Conjugate (Compound 38): To 19 mg (0.041 mmol) of methotrexate (Sigma) was added 0.4 mL of anhydrous DMF followed by 8 mg (0.06 mmol) of 4-nitrophenol and 9 mg (0.043 mmol) of N, N'-dicyclohexylcarbodiimide. The resulting reaction mixture was allowed to stir at room temperature for 4 hours and the resulting activated ester (compound 20) was used in situ in the next step without isolation. To 22.9 mg (0. 019 mmol) of osmium PEG linker (compound 36) was added 500 muL of DMF followed by 100 muL (0.71 mmol) of triethylamine. The reaction mixture was allowed to stir at room temperature and the solution of methotrexate activated ester prepared above (compound 20) was added dropwise. The reaction mixture was allowed to stir at room temperature for 18 hours and concentrated. The residue was purified by preparative reverse phase HPLC to give 7.8 mg (4.9 x 10-3 mmol, 25%) of osmium PEG methotrexate conjugate (compound 38), LC/MS M+H 1554.5. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
99% | EXAMPLE 8 3.4 g (0.009 mole) of the 2,4-diamino-6-bromomethylpteridine hydrobromide from Example 4 was stirred overnight at ambient temperature with 4.5 g (0.01 mole) of the barium salt dihydrate of p-(N-methyl)-aminobenzoyl-L-glutamic acid in 100 ml of a mixture of DMF and H2 O (1 to 1 according to volume). After that the pH of the solution was adjusted to pH 4 by means of diluted HCl. The mixture was evaporated to dryness under vacuum. The residue was absorbed (received) in 100 ml of H2 O. The mixture was stirred for 15 minutes at ambient temperature and then was filtered. The filtration residue was dried at 100 C. under house vacuum. Methotrexate was obtained in the form of yellow crystals, which still contained, according to NMR, about 7 percent of dimethylformamide. After purification according to Example 7, 3.6 g (87.5 percent) of L-methotrexate was obtained as yellow crystals (>99 percent purity according to DC-electrophoresis). | |
87% | The synthesis of MTX with a protected alpha-carboxylic acid was initiated by adding a solution of L-Glu(OH)-OtBu (0.132 mmol) in 5 mL of DMF dropwise into a mixture of 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid hemihydrochloride dihydrate (0.132 mM), HBTU, and DIEA in 5 mL of DMF. The reaction mixture was stirred 2 h at room temperature under nitrogen. The crude product was concentrated under reduced pressure to yield MTX-(OtBu) as a yellow oil. The product was further purified by preparative HPLC to give 87% yield. Mass spectroscopy (FAB) analysis indicated the product had the expected MW of 511 (M+1). Next, a solution of peptide (0.098 mmol) was added dropwise to a solution of HBTU, MTX-(OtBu), and DIEA (0.098: 0.098: 0.198 mmol) in 5 mL of DMF. The mixture was stirred for 3 h at room temperature under nitrogen. The reaction was concentrated under reduced pressure to give an oily residue. The resulting residue was dissolved in 3 mL of CH2Cl2 followed by addition of 3 mL of TFA. After the solution was stirred for 1 h at room temperature, the solvents were evaporated and the crude product purified by semi-preparative HPLC using a C-18 column to give MTX-peptide conjugates in 60-70% yield. | |
A method according to claim 10wherein the antimetabolite is selected from the group consisting of: mitomicyn C; methotrexate; 6-mercaptopurine; thioguanine; 5-fluorouracil; cytosine arabinoside; 5-azacytidine; hydroxyurea; |
The following antineoplastic agents may be compatible for combination with Taurolidine and/or Tarultam: PCV-Chemotherapy: Combination of: procarbazine HCI ... vincristine sulfate Cisplatin Methotrexate | ||
A 130 mg portion of the latter complex was subjected to acid treatment to remove the Boc groups therefrom and prepare a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-H, (110 mg). This complex (100 mg) was reacted with the active ester of MTX (90 mg), whereby a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX (111 mg, with an MTX content of 17.4 wt. %) was obtained. | ||
130 mg of the latter complex was subjected to acid treatment to remove the Boc groups therefrom and prepare a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-H, (114 mg). This complex (100 mg) was reacted with the active ester of MTX (90 mg), whereby a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala--MTX (111 mg, with an MTX content of 17.4%) was obtained. | ||
The resulting precipitate was collected, washed with methylene chloride and then dried in vacuo, whereby a complex of N-acetylcarboxymethylchitosan-(PEG1)--Gly-Phe-Phe--MTX (17 mg, with the PEG content of 6.1% and an MTX content of 8.5%) was obtained. | ||
R--COO-- is the acyloxy residue of one of the following bio-affecting carboxylic acid agents (compounds B) 2. ... Valproic acid Tranexamic acid 715. Furosemide 722. Methotrexate 727. Chlorambucil Clofibric acid Amphotericin B ... | ||
The following lysine transporter compounds are exemplary, in which (1) therapeutic compounds are indicated by the symbol "X", where "X" is either 1. vincristine 2. vinblastine 3. methotrexate 4. daunorubicin 5. bleomycin 6. cytosine arabinoside 7. thiotepa 8. dactinomycin 9. doxorubicin ... | ||
is administered intravenously as in Test Procedure A, supra, in conjunction with a chemotherapeutic amount of one of the following antineoplastic agents capable of causing emesis: ... dactinomycin, 6-thioguanidine, carmustine, lomustine, methotrexate, 5-fluorouracil, vinblastine, aminoglutethimide, ... | ||
The carboxylic acid group in compound 1 was initially activated with benzotriazolyloxytetramethylivonium hexafluorophosphate (HBTU) in the presence of N,N-diisopropylethyl amine (DIEA) in N,N-dimethyl formamide (DMF), and followed by the reaction with amine group of Glu(O-tBu)-OH (compound 2) to give a selectively protected alpha-carboxylic acid MTX (compound 3). | ||
wherein the agent is an antimetabolite selected from the group consisting of: mitomicyn C; methotrexate; 6-mercaptopurine; thioguanine; 5-fluorouracil; cytosine arabinoside; 5-azacytidine; hydroxyurea; | ||
Chemotherapeutic agents that are bound to monoclonal antibodies of the present invention to drive the cytotoxic activity include the following: ... medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, ... | ||
Illustrative examples of chemotherapeutic agents which may be conjugated with the antibody of the invention and have a cytotoxic effect include: ... medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, ... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80 mg portion of the latter complex was subjected to acid treatment to remove the Boc groups therefrom and give a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly-H, (71 mg). This complex (50 mg) was reacted with the active ester of MTX (45 mg), whereby a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly--MTX complex (50 mg, with an MTX content of 7.0%) was obtained. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
51.4% | With pyridine; hydrogenchloride; p-toluenesulfonic acid monohydrate; 1,1,1,3,3,3-hexamethyl-disilazane; | EXAMPLE 1 Methopterin hydrate (360 mg; 0.75 mmol), anhydrous pyridine (3.6 cc), p-toluenesulfonic acid monohydrate (21 mg; 0.11 mmol) and hexamethyldisilazane (1 cc; 4.74 mmol) are introduced into a 10-cc Hastelloy autoclave. The contents are heated at 100 C. for 21.5 hours, with stirring. After cooling to 20 C., the pyridine solution is concentrated to dryness. The residue obtained is taken up with water (10 cc) and acidified to pH 3.3 by adding normal hydrochloric acid. The yellow precipitate formed is separated by filtration, rinsed with water and washed with ethanol (3 cc). After drying under reduced pressure (0.2 torr) for 15 hours at 20 C., crude methotrexate (340 mg) is obtained. Analysis by high performance liquid chromatography shows that the degree of conversion is 100% and that the yield is 51.4%. The product obtained previously (230 mg) is taken up with distilled water (5 cc). |
48% | With pyridine; hydrogenchloride; 1,1,1,3,3,3-hexamethyl-disilazane; | EXAMPLE 2 Methopterin hydrate (360 mg; 0.75 mmol), anhydrous pyridine (3.6 cc) and hexamethyldisilazane (1 cc; 4.75 mmol) are introduced into a 10-cc autoclave. The contents are stirred at 100 C. for 21 hours 30 minutes. After cooling to a temperature of about 20 C., the reaction mixture is concentrated to dryness. The residue obtained is taken up with water (10 cc) and then acidified to pH 3.3 by adding N hydrochloric acid. The yellow precipitate formed is separated by filtration, rinsed with water and then washed with ethanol (3 cc). After drying at 20 C. under reduced pressure (1 mm Hg; 0.13 kPa), methotrexate (300 mg) is obtained. Analysis by high performance liquid chromatography shows that the degree of conversion of methopterin is 88% and that the reaction yield is 48%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
E.2 Production of tPA Using DHFR Protein with a Low Binding Affinity for MTX |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
potassium hydroxide; In N-methyl-acetamide; methanol; ethanol; chloroform; water; | EXAMPLE 3 Diethyl N-[p[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]glutamate (Amethopterin Diethyl Ester; Methotrexate Diethyl Ester). A solution containing 4.8 g. (10.2 mmole) of diethyl N-[p[[(2-amino-3-cyano-5-pyrazinyl)methyl]methylamino]benzoyl] glutamate and 5 g. (42 mmole) of guanidine acetate in 40 ml. of dimethylformamide was stirred under notrogen at 120 C. for six hours. The resulting solution was cooled to room temperature, filtered and evaporated to a glassy product using a rotary evaporator and a mechanical vacuum pump to insure a better vacuum. The residual glass was taken up in 500 ml. of chloroform, the resulting suspension filtered using Celite, and the filtrate washed with water, dried using anhydrous MgSO4, and evaporated to dryness. (The residual material was chromatographed rapidly on a column prepared from 250 g of Baker silica gel using, initially, 2% ethanol in chloroform, and then 5% ethanol in chloroform as eluents.) The material obtained by evaporation of the eluates was crystallized from ethanol-chloroform (4:1) to give small, pale yellow lustrous platelets, mp. 142-154 C.; yield, 3.8 g. (73%). Further crystallization of this material from ethanol-chloroform (4:1) raised the mp. to 153-155 C. The compound is completely racemic. A sample of this product was hydrolyzed in a mixture of water and methanol in the presence of potassium hydroxide. Essentially pure methotrexate was thus obtained. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With caesium carbonate; In dimethyl sulfoxide; at 70℃; for 40h; | To a solution of 5 g of HA-CI TBA salt from example 5 in 150 ml of anhydrous DMSO under stirring and under nitrogen, a solution of 9 g of MTX in 100 ml of DMSO was added; then 6.43 g of solid caesium carbonate were added in portions. The resulting mixture was stirred and heated at 70C for 4Oh. After this time the mixture was cooled at room temperature and poured into 200 ml ice water. The pH was decreased to 4.75 with HCI 1 N and then adjusted to 7 with saturated NaHCO3 solution (final volume of 500 ml). It was then dialysed against 1 M NaCI (2x2.5L), the insoluble material was filtered through sintered glass filters (class II, III and then IV), and the solution was ultrafiltered against a 10 KDa membrane and then filtered through 1.2 micron, 0.45 micron and 0.22 micron pore size filters. The yellow solution was concentrated in vacuum and freeze-dried to obtain a yellow fluffy solid (0.8 g).1H NMR DOSY and 13C-NMR data confirmed the linkage of MTX on position 6 of the D-glucosamine residue. The MTX content was 20% w/w (HPLC); free MTX was below 0.5% w/w (HPLC); Water content: 1 1.7% w/w. MW: 39000; Pl: 3.3. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With caesium carbonate; In dimethyl sulfoxide; at 65℃; for 40h; | To a solution of 3.5 g of HA-CI TBA salt from Example 12 in 175 ml of anhydrous DMSO under stirring and under nitrogen, a solution of 2eq (per repeating unit of HA backbone) of methotrexate (5.14 g) in DMSO (51 ml) was added; then 2eq (per repeating unit) of solid cesium carbonate (3.68 g) were added in portions. The resulting mixture was stirred and heated at 65C for 4Oh. After this time the mixture was cooled to room temperature and poured into ice water. The pH was adjusted to 7 with dilute HCI solution and the mixture stirred at room temperature for 4 hours. It was then dialysed against 1 M NaCI (2 x 2.5L), the insoluble material was filtered through a sintered glass filter (class IV), and the solution was ultrafiltered against a 10 KDa membrane and then filtered through 1.2mum, 0.45mum and 0.22mum pore size filters. The yellow solution was concentrated in vacuum and freeze-dried to obtain a yellow fluffy solid (0.8 g). EPO <DP n="17"/>The 13C-NMR spectrum confirmed the occurrence of the linkage in position 6 of D- glucosamine residue: the peak at 64 ppm is assigned at CH2O-MTX and its intensity corresponds to the decrease of the peak at 44 ppm (CH2CI) compared to the parent chlorine derivative. The MTX content, was 4.2% w/w (HPLC); content of free MTX was below 0.5% w/w (HPLC); Water content: 10.5% w/w; MW: 63.000; Pl: 2.8. Residual chlorine: 0.8 % w/w. | |
With caesium carbonate; In dimethyl sulfoxide; at 70℃; for 40h; | The conjugate was prepared as in Example 16 with the difference that a solution of HA-CI TBA salt (1 g, from example 10) in 50 ml anhydrous DMSO was treated with methotrexate (1.65 g) and cesium carbonate (1.13 g) at 70C for 40 hour, to afford, after the work up as described in Example 12, a yellow solid (0.7 g). The 1 H-NMR DOESY and 13C-NMR data were similar to those of the compound prepared in Example 16 confirming the presence of MTX covalently bound on C-6 of HA. The MTX content was 10.0% w/w (HPLC); free MTX was 0.17%w/w (HPLC); Water content was 10% w/w, MW: 94.000; Pl: 5.2. | |
With caesium carbonate; In dimethyl sulfoxide; at 70℃; for 40h;Product distribution / selectivity; | The conjugate was prepared as in Example 16 with the difference that 1.52 g of HA-CI-TBA salt from Example 1 1 in DMSO (75 ml) was treated with 2.4g MTX and 1.63g cesium carbonate at 70C for 40 h, to give, after the usual work up, 450 mg of a yellow solid.The structure was confirmed by 1H-NMR DOESY and 13C-NMR. MTX content was 16% w/w (HPLC); free MTX was <0.5% (HPLC); Water content: 12% w/w, MW: 63.000, Pl: 3.2. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With caesium carbonate; at 80℃; for 40h; | 10 g of HA-CI from Example 8 were reacted with 2 eq of MTX and 2 eq of Cs2CO3 at 80C for 40 hours. 1.7 g of the resulting HA-MTX-CI with a MTX content of 24%w/w, were suspended in 150 ml of dry DMSO in the presence of 2.5 eq of CsBut at 85 C for 21 hours. The reaction was worked up as described in Example 22 to give 1.2 g of HA-MTX-But. MTX content was 12.7% w/w (HPLC). Water content: 1 1.5% w/w. Butyrate content was determined by the integration of signals at 23 ppm (CH3 of acetamido group) and 13.3 ppm (CH3 of butyryl group) and resulted equal to 1.7 %w/w. |
Yield | Reaction Conditions | Operation in experiment |
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With caesium carbonate; In dimethyl sulfoxide; at 80℃; for 40h;Product distribution / selectivity; | The conjugate was prepared as in Example 16 with the difference that a solution of HA-CI TBA salt (20 g, from Example 14) in DMSO (1.25 L) is treated with MTX (29.3g) and cesium carbonate (21 g) at 80C for 40 h, giving 5.6 g of a yellow solid. The structure of the conjugate was supported by NMR. 13C-NMR spectrum (Figure 3) confirmed the occurrence of the linkage in position 6 of N-acetyl-D- glucosamine: the peak at 64 ppm is assigned at CH2O-MTX and its intensity corresponds to the decrease of the peak at 44 ppm QH2CI) compared to the EPO <DP n="18"/>parent chlorine derivative. The MTX content was 18.8% w/w (HPLC); free MTX was 0.1 % w/w, water content: 8.2% w/w; MW: 1 1.000; Pl: 1.4; residual chlorine content: 1.76% w/w (NMR). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With caesium carbonate; at 80℃; for 40h; | 21 g of HA-CI from Example 14 were reacted with 2.67 eq of MTX and 2.67 eq of Cs2CO3 at 80C for 40 hours. 200 mg of the resulting HA-MTX-CI with a MTX content of 30%w/w, were suspended in 20 ml of DMSO in the presence of 5 eq of cesium butyrate psBut) at 80C for 21 hours. The reaction was worked up as described in Example 22 to give 0.195 g of HA-MTX-But MTX content was 20% EPO <DP n="20"/>w/w (HPLC). Mw= 13000, Pl = 1.5, water content: 1 1.5% w/w. Butyrate content was determined by the integration of signals at 23 ppm (CH3 of acetamido group) and 13.3 ppm (CH3 of butyryl group) and resulted of 3.5 % w/w. The structure of HA-MTX-But:Na is supported by its 13C-NMR spectra. The disappearance of the signal at 44 ppm confirms the complete displacement of chlorine groups and the presence of the signals at 63.8 ppm (CH2-OBut) and at 13.3 ppm (CH3 of butyrate group) confirmed the presence of butyrate groups in position 6 of glucosamine. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
HA:TBA salt (250mg; 0.403mmol; MW 20,000) was dissolved in DMSO (10ml) by stirring and gentle heating under nitrogen; thethylamine (452muL; 3.22mmol) was then added at room temperature followed by dropwise addition of MsCI (157muL; 2.02mmol), whereupon a yellow solution formed. After 1 h stirring at room temperature, further 0.50ml of thethylamine were added, the reaction flask was connected to the vacuum and gently heated up to 50 C (bath temperature), until gas evolution ceased. Then 1.1 Og (2.43mmol) of methotrexate and 792mg (2.43mmol) of cesium carbonate were added and the mixture was stirred at 80 C overnight. Half of the reaction mixture was quenched by pouring into water (20ml); pH 6.3. The pH was adjusted to 6.8 with saturated NaHCO3 solution and then 10ml of saturated NaCI solution were added. After stirring for 10min, the solution was ultrafiltered, concentrated in a rotary evaporator and freeze-dried to give 60mg of a yellow solid. The DS of MTX was found to be 1 1.6% w/w by HPLC and was confirmed by NMR analysis (12% mol/mol), which also showed a small percentage of left mesylates on the polymer. The rest of the reaction mixture was worked up after further 24h at 80 C (40h overall) as described above, to afford 103mg of a yellow solid. DS in MTX 14.4% w/w by HPLC, confirmed by NMR analysis (15% mol/mol), which did not show any mesylate left on the polymer. MW 269610, Pl 10.5. Cross-linking ester bonds were cleaved by hydrolyzing the freeze dried product in 10ml of a carbonate buffer (pH 10) for 8h. After neutralization and dialysis, freeze-drying afforded 96 mg of a yellow solid. The DS of MTX in the product was 12.6% w/w by HPLC, which was confirmed by NMR analysis (13% mol/mol); MW 27,120, Pl 1.9. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
52% | With 3-chloro-benzenecarboperoxoic acid; In methanol; water; N,N-dimethyl-formamide; at 20℃; for 1h;Product distribution / selectivity; | Procedure 1. MCPBA (0.011 g, 0.064 mmol) was dissolved in 1 mL of methanol. To this solution 4 mL of water was added. In a separate flask 0.020 g (0.044 mmol) of methotrexate was dissolved in 0.3 ml of dimethylformamide (DMF) and then portions were added dropwise to a stirred solution of MCPBA at room temperature. <strong>[59-05-2]Methotrexate</strong> initially precipitates out of the methanol/water/DMF solution after being added. When the mixture becomes clear, another portion of methotrexate/DMF solution was added. The reaction was complete after 1 h. The resulting solution was filtered to remove any traces of solid unreacted methotrexate and then it was extracted with methylene chloride (3 x 5 mL) and ether (1 x 5 mL) to remove m-chiorobenzoic acid. The aqueous phase was lyophilized to give 0.020 g (97%) of methotrexate N-oxide as a yellow-orange powder. The crude N-oxide product is reasonably clean and typically contains about 10% of impurities. The crude product was loaded onto a silica gel column as a suspension in water/acetonitrile (1 :1, 1 mL) and then it was chromatographed using an acetonitrile/water/acetic acid (5:2:2) mixture as eluent. The desired fractions were collected and extracted with methylene chloride (1OmL x 3) and ether (1OmL x 1) to remove some of the acetonitrile and acetic acid. Note that extraction is preferable to evaporation due to sensitivity of the product to light. Lyophilization of the aqueous phase gave 0.01 1 g (52% yield) of methotrexate N-oxide as a greenish-yellow powder with an estimated purity of >90% based on the 1H NMR spectrum. |
30% | With magnesium monoperoxyphthalate hexahydrate; In methanol; water; for 2h;Product distribution / selectivity; | Procedure 3. A suspension containing 0.010 g (0.022 mmol) of methotrexate, 0.5 mL water and 0.5 mL methanol was stirred for 20 minutes. To this suspension magnesium bis(monoperoxyphthalate) hexahydrate (0.02 g, 0.044 mmol) was added. The methotrexate dissolved over 1 hour. The mixture was stirred additional hour and solvents were evaporated until about 0.5 mL of water remains. This solution was transferred on silica gel column chromatography and eluted with acetonitrile/water/acetic acid mixture. Magnesium phthalate eluted with acetonitrile. Other impurities eluted with 5:0.5:0.5 acetonitrile/water/acetic acid mixture and the desired methotrexate /V-oxide eluted with 5:1:1 acetonitrile/water/acetic acid mixture. Appropriate fractions were collected and solvents were evaporated to give yellow powder <n="59"/>(3 mg, 30%) pure by TLC and 1H NMR. MS (m/e): Calculated for methotrexate C20H22N8O5 (M): 454.17; found (M): 454.8. Calculated for methotrexate N-oxide C20H22N8O6 (M): 470.17; found (M): 470.8. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
54% | Procedure 2. The sodium salt of methotrexate was generated by adding 1.5-1.8 equivalent of aqueous sodium hydroxide to a suspension of methotrexate in water. An aqueous solution of methotrexate sodium salt (0.011 g, 0.022 mmol) in 2 mL of water was added dropwise to a solution of w-chloroperoxybenzoic acid (0.0060 g, 0.035 mmol) dissolved in a mixture of <n="58"/>methanol (0.5 mL) and water (0.5 mL). The reaction was complete in 30 min. The m-chlorobenzoic acid byproduct was removed from the reaction mixture by extraction with methylene chloride (5 mL x 3) and ether (5mL x 1). The aqueous phase was lyophilized to give 0.011 g (97%) of methotrexate sodium salt N-oxide as a yellow-orange powder. The crude product was loaded on a silica gel column as a suspension in water/acetonitrile mixture (1 :1, ImL) and then chromatographed using acetonitrile/water/acetic acid (5:2:2) mixture as eluent. The desired fractions were collected and extracted with methylene chloride (10 mL x 3) and ether (1OmL x 1) to remove some of the acetonitrile and acetic acid. Liophilization of the aqueous phase gave 0.006 g (54% yield) of the sodium salt of methotrexate N-oxide as a greenish-yellow powder with an estimated purity of >90% based on the 1H NMR spectrum.[00136] 1H NMR (D2O) delta 1.95 (m, IH), 2.12 (m, IH), 2.34 (t, J= 7.2, 2H), 3.72(s, 3H), 4.28-4.33 (m, IH), 5.09 (AB system, J= 12.6, 2H), 7.68-7.79 (m, 4H), 8.48 (s, 0.5H), 8.49 (s, 0.5H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With caesium carbonate; In dimethyl sulfoxide; at 80℃; for 18h; | A solution of HA-OMs:TBA salt from Example 10 (500mg; 0.73mmol) in DMSO (15 ml) was treated with a solution of methotrexate (833mg; 1.83mmol) in DMSO (10ml) in the presence of solid cesium carbonate (596mg; 1.83mmol). The mixture was stirred under nitrogen at 80C for 18h, whereupon it darkened with formation of solids. It was then cooled to ambient temperature, poured into 100ml of water (pH 6.5), treated with 15ml of saturated NaCI solution, and stirred for 1.5h. Then solids were filtered off and the solution was ultrafiltered, concentrated and freeze- dried to give 131 mg of a yellow-brownish solid. DS of MTX by NMR: 40% mol/mol; 13C NMR shows that 40% of C6 is modified. HPLC analysis gave 32% w/w, corresponding to 40% mol/mol. In addition, the NMR revealed the absence of any residual secondary mesylate group and that the basic structure of HA was <n="21"/>unchanged, except some of the C-6 position because of the substitution by MTX. This demonstrates that any possible leaving (mesylate) groups introduced at the secondary positions (C-4,C-2',C-3') during the mesylation reaction have been hydrolysed during the displacement reaction under the basic conditions either directly or by way of 2',3'-anhydhde formation followed by hydrolysis with the retention of configuration at those positions. The NMR spectrum was repeated on the same sample after 3-months storage at room temperature, it provides the same peaks and the same intensity as those obtained on the freshly prepared product, thus indicating that the substitution degree is maintained and no by- products are formed. | |
With caesium carbonate; In dimethyl sulfoxide; at 75℃; for 18h; | A solution of HA-OMs:TBA salt from Example 37 (7.Og; 11.3mmol) in DMSO (670 ml) was treated with a solution of methotrexate (12.79g; 28.1 mmol) in DMSO (120ml) in the presence of solid cesium carbonate (9.15g; 28.1 mmol). The mixture was stirred under nitrogen at 75 C for 18h. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 8.8 and the volume to 2.5L. After stirring for 24h the solution was neutralized, filtered, ultrafiltered, concentrated and freeze-dried to give 4.3Og of a yellow solid. DS of MTX by NMR: 13% mol/mol; MW 208,400, P.I. 2.18. | |
With caesium carbonate; In dimethyl sulfoxide; at 75℃; for 18h; | A solution of HA-OMs:TBA salt from Example 38 (13.2g; 21.3mmol) in DMSO (1270 ml) was treated with a solution of methotrexate (24.13g; 53.1 mmol) in DMSO (120ml) in the presence of solid cesium carbonate (17.26g; 53.1 mmol). The mixture was stirred under nitrogen at 75 C for 18h. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 10.0 and the volume to 5L. After stirring for 18h the solution was neutralized, filtered, <n="28"/>ultrafiltered, concentrated and freeze-dried to give 6.52g of a yellow solid. DS of MTX by NMR: 20% mol/mol; MW 217,300, P.I. 2.02. |
With caesium carbonate; In dimethyl sulfoxide; at 80℃; for 20h; | A solution of HA-OMs:TBA salt from Example 34 (8.Og; 12.9mmol) in DMSO (250 ml) was treated with a solution of methotrexate (14.66g; 32.3mmol) in DMSO (150ml) in the presence of solid cesium carbonate (10.5g; 32.2mmol). The mixture was stirred under nitrogen at 80 C for 2Oh. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 9.7 and the volume to 2L. After stirring for 18h the solution was neutralized, filtered, ultrafiltered, concentrated and freeze-dried to give 4.7Og of a yellow solid. DS of MTX by NMR: 7.5% mol/mol; MW 27,960, P.I. 1.92. | |
With caesium carbonate; In dimethyl sulfoxide; at 80℃; for 20h; | A solution of HA-OMs sodium salt from Example 33 (1.Og; 2.0 mmol) in DMSO (40 ml) was treated with a solution of methotrexate (1.83 g; 4mmol) in DMSO (40ml) in the presence of solid cesium carbonate (1.30; 2mmol). The mixture was stirred under nitrogen at 80 C for 2Oh. The solution was neutralized using Na2CO3 saturated solution bringing the final volume to 500 mL, filtered, ultrafiltered, concentrated and freeze-dried to give 500 mg of a yellow solid. DS of MTX by HPLC: 7.8% w/w; MW 16,000, P.I. 2.4. | |
With caesium carbonate; In dimethyl sulfoxide; at 80℃; for 22h; | A solution of HA-OMs:TBA salt from Example 39 (500mg; 0.74mmol) in DMSO (80ml) was treated with a solution of methotrexate (1.01 g; 2.23mmol) in DMSO (10ml) in the presence of solid cesium carbonate (726mg; 2.23mmol). The mixture was stirred under nitrogen at 80 C for 22h. It was then cooled to ambient temperature and poured into a carbonate buffer, adjusting the pH to 10.0 and the volume to 40OmL. After stirring for 18h the solution was neutralized, filtered, ultrafiltered, concentrated and freeze-dried to give 260mg of a yellow solid. DS of MTX by NMR: 1 1 % mol/mol; MW 460,100, P.I. 2.21. |
Yield | Reaction Conditions | Operation in experiment |
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With caesium carbonate; In dimethyl sulfoxide; at 80℃; for 40h; | To a solution of HATBA (5Og, Mw 70,000) in 1000 ml of dry DMF under stirring, mesylchloride (10 eq) was added dropwise in 1 hr time at -10C under N2 flow. The mixture was maintained for 1 hour at room temperature and then heated at 60 C for 16 hr. The work-up allow the obtainment of 46.9 g of HA-CL having chlorine content 4.2% w/w (13C-NMR). <n="24"/>A solution of the HA-CI TBA salt (20 g) in DMSO (1.25 L) is treated with MTX (29.3g) and cesium carbonate (21 g) at 80 C for 40 h, giving 5.6 g of a yellow solid.13C-NMR spectrum confirmed the occurrence of the linkage in position 6 of N- acetyl-D-glucosamine: the peak at 64 ppm is assigned at CH2O-MTX and its intensity corresponds to the decrease of the peak at 44 ppm (CH2CI) compared to the parent chlorine derivative. The MTX content was 18.8% w/w (HPLC); free MTX was 0.1 % w/w, water content: 8.2% w/w; MW: 11.000; Pl: 1.4. In addition, the NMR reveals the presence of residual chlorine which amounts to 1.76% w/w. |
Yield | Reaction Conditions | Operation in experiment |
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35% | A solution comprising the corresponding 1-stearoyl-2-omega-aminoacyl-sn-glycero-3-phosphocholine of stage 4, acetic acid (0.589 g., 0.9 mmol) and triethylamine (0.182 g., 239 mul, 1.8 mmol) in 30 ml of dry freshly distilled chloroform is added dropwise to the reaction mixture of stage 5 for 30 min at -25 C. The reaction mixture is stirred for additional one hour at -25 C., and then for overnight at room temperature. The solvents are removed in evaporator under reduced pressure. The obtained residue is a thick viscous liquid. Diethyl ether (50 ml) is added to this liquid and the mixture is stirred. The product is gradually transformed into a yellow powder which is filtered and washed with diethyl ether (3×20 ml). The crude product is purified by column chromatography as follows: 450 g. crude product is dissolved in 50 ml of methanol, followed by addition of 11.0 g. dry silica gel. The mixture is swirled, and then the volatile liquid is evaporated under reduced pressure to yield free-flowing yellow finely divided solid. The obtained solid is packed on top of a silica column (3×30 cm) (100 g. silica gel per 1 g of crude product). The product is eluted in succession with the solution MeOH:H2O of variable composition: first fraction is 100:0 (v/v) (1L), second fraction is 99:1 (v/v) (1L), third fraction is 98:2 (v/v) (1L), and fourth fraction is 98:3 (v/v) (2L). The fractions, which contain the product (the determination is carried out by TLC analysis), are combined and the solvent is evaporated under reduced pressure. The obtained product (about 250 mg) is dissolved in mixture of methanol (10 ml) and chloroform (250 ml). The solution is washed with 1% HCl (3×20 ml) and then with water (3×20 ml). To achieve better separation of the aqueous and organic phases, isopropanol (about 25% of the volume of solution) is added. Isopropanol addition also promotes transition of the product into the organic phase. The organic layers are combined and dried over sodium sulfate. The sodium sulfate is filtered off and the solvent is distilled under reduced pressure. The obtained product is dried under vacuum for 3 hours. [00146] Examples of resulted final products and their analyses are: [00147] 1-Stearoyl-2-[3-(alpha-MTX amido)]-proponoyl-sn-glycero-3-phosphocholine, C49H79N10O12P. [00148] Yield 35%. Yellow solid. Decompose at 200 C. without melting. pH 5.1. TLC analysis: Silica gel on aluminium plates. Eluent is CH3OH:H2O (98:2, v/v). Indication by UV-Vis spectrum. One spot. Rf0.16. MS(FAB). C49H79N10O12P. Main peak (+FAB) is 1031.2. 1H NMR (CD3OD), delta (ppm): 0.89 (t, 3H), 1.22-1.29 (broad s, 30H), 1.53 (m, 2H), 2.08 (m, 2H), 2.35 (m, 2H), 2.60 (t, 2H), 2.50 (t, 2H), 3.21 (s, 3H), 3.38 (s, 9H), 3.47 (t, 2H), 3.93-4.31 (m, 8H), 4.41 (m, 1H), 4.80 (s, 2H), 5.21 (m, 1H), 6.84 (d, 2H), 7.76 (d, 2H), 8.60 (s, 1H). 31P NMR (CD3OD), delta (ppm): -3.3 (s). Chemical analysis: C49H79N10O12P.HCl.3H2O. Calculated: C 52.47%, H 7.55%, N 12.49%, P 2.76%, Cl 3.16%. Found: C 52.92% , H 7.76%, N 12.21%, P 2.46%, Cl 30 3.08%. [00149] 1-Stearoyl-2-[6-(alpha-MTX amido)]-hexanoyl-sn-glycero-3-phosphocholine C25H85N10O12P. [00150] Yield 40%. Yellow solid. Decomposes without melting at 200 C. pH 5.0. TLC analysis: Silica gel on aluminium plates. Eluent is MeOH:H2O (98:2, v/v). Indication is UV-Vis spectra. One spot Rf0.18. MS (FAB): C52H85N10O12P 1073.3 (+FAB) is main peak. 1H NMR (CD3OD), delta (ppm): 0.86-0.89 (t, 3H), 1.22-1.30 (broad s, 32H), 1.51-1.55 (m, 4H), 2.06-2.10 (m, 4H), 2.33-2.36(m, 2H), 2.58-2.62 (t, 2H), 2.49-2.52 (t, 2H), 3.22 (s, 3H), 3.38 (s, 9H), 3.45-3.48 (t, 2H), 3.92-4.35 (several m, 8H), 4.40-4.42 (m, 1H), 4.79 (s, 2H), 5.20 (n, 1H), 6.82-6.84 (d, 2H), 7.74-7.76 (d, 2H0, 8.58 (s, 1H). 31P NMR (CD3OD), delta (ppm): -3.7 (s). Chemical analysis: C52H85N10O12P.2HCl.5H2O: Calculated: C 50.48%, H 7.84%, N 11.33%/, P 2.51%, Cl 5.74%. Found: C 50.45%, H 7.220/a, N 11.46%, P 2.43%, Cl 5.43%. [00151] 1-Stearoyl-2-[3-(alpha-dodecylate-gamma-MTX amido)]-proponoyl-sn-glycero-3-phosphocholine. C61H103N10O12P. [00152] Yield 40%. Yellow solid. Decompose at 200 C. without melting. TLC analysis: Silica gel on aluminium plates. Eluent is CHCl3:CH3OH:H2O (65:35:5, v/v). Indication by UV-Vis spectrum. One spot. Rf0.17. MS(FAB). C61H103N10O12P. Main peak (+FAB) is 1198.4. 1H NMR (CD3OD+CDCL3), delta (ppm): 0.82-0.85 (t, 3H), 1.23 (broad s, 46H), 1.53-1.58 (m, 2H), 2.24-2.34 (m, 3H), 2.50-2.52 (m, H), 3.17 (s, 9H), 3.41 (t, 1H) 3.54 (s, 1H), 4.04-4.18 (m, 4H), 4.32-4.47 (m, 1H), 4.80 (s, 2H), 5.21 (m, 1H), 6.79-6.82 (d, 2H), 7.76 (d, 2H), 8.54 (s, 1H). 31P NMR (CD3OD+CDCL3), delta (ppm): +0.71 (s). Chemical analysis: C61H103N10O12P.3H2O. Calculated: C 58.47%, H 8.71%, N 11.18%, P 2.47%. Found: C 58.67%, H 8.88%, N 11. 15%, P 2.47%. |
Yield | Reaction Conditions | Operation in experiment |
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With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In aq. phosphate buffer; at 20℃; for 1h;pH 7.4;Inert atmosphere; Darkness; | In order to develop MTX conjugated Alg-Ccm AuNPs, at first MTX was conjugated to Bis (aminopropyl) PEG to generate MTX-PEG conjugate (MP conjugate). For MP conjugate synthesis, MTX was dissolved in PBS (pH 7.4) and was activated using EDC and NHS (MTX:EDC:NHS = 1:1.3:1.1 molar ratio) at room temperature for one hour under the blanket of N2 (g) in dark. Then solution of bis(aminopropyl) PEG (MTX:PEG = 1:1 molar ratio) in PBS (pH 7.4) was gradually added to the activated MTX solution and the mixture was allowed to stir at room temperature for around four hours in dark. Next, the MP conjugate was dialyzed (using dialysis mem-brane of MWCO 1000) against PBS (pH 7.4) for one day and against distilled H2O for one day and was dried by lyophilization. In the second step, MP conjugate was conjugated to the Alg-Ccm AuNPs utilizing the EDC chemistry. The free carboxyl functionality on Alg-Ccm AuNPs (15 mg) was activated with EDC/NHS (in aqueous carbonate/bicarbonate buffer medium with pH 8.2) for one hour at 25 C followed by the addition of aqueous solution of MP conjugate (25 mg) and the reaction mixture was moderately stirred overnight at 25 C. The reaction mixture was then dialyzed (MWCO 3500) and lyophilized to get MPAlg-Ccm AuNPs. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
22% | To a solution of methotrexate hydrate (0.684 mg, 1.45 mmol) in anhydrous DMF (35 mL) was added cesium carbonate (708 mg, 2.17 mmol), and potassium carbonate (0.3 g, 2.17 mmol). While stirring this mixture under nitrogen atmosphere, N-(bromoacetyl)-N'-Boc-1,3-diaminopropane 3[69] (0.47 g, 1.59 mmol) and sodium iodide (0.217 g, 1.45 mmol) were added to it. The final mixture was stirred at room temperature for 48 h under nitrogen atmosphere. The mixture was concentrated in vacuo, yielding yellow oily material. It was diluted with 15 mL of 10% MeOH/CH2Cl2, and purified by flash column chromatography on silica gel (100 g) by eluting with 10% MeOH/CH2Cl2 to 2% AcOH/15% MeOH/CH2Cl2. Fractions with an Rf value of 0.27 (2% AcOH in 20% MeOH/CH2Cl2) were combined and evaporated to afford the desired product 4 as yellow solid (213 mg, 22%). An HPLC analysis of the purified product suggested that the regioselectivity of the O-alkylation is 10.2 (gamma-ester to alpha-ester). 1H NMR (400 MHz, DMSO-d6): delta 8.53 (s, 1H), 8.45-8.44 (d, NH-Glu, 1H, J = 7.2 Hz), 7.82-7.80 (t, 1H, J = 5.6 Hz), 7.70-7.68 (d, 2H, J = 8.8 Hz), 6.78-6.76 (d, 2H, J = 8.8 Hz), 6.73-6.70 (m, 1H), 4.74 (s, 2H), 4.42 (s, 2H), 4.36-4.35 (m, 1H), 3.17 (s, 3H), 3.02-2.88 (m, 2H), 2.86-2.83 (m, 1H), 1.94-1.85 (m, 1H), 1.48-1.44 (quin, 2H, J = 6.8 Hz), 1.30 (s, 9H) ppm. 13C NMR (100 MHz, DMSO-d6): delta 174.38, 172.23, 167.36, 166.90, 163.15, 151.58, 149.54, 129.53, 122.0, 120.88, 111.54, 77.94, 62.94, 55.28, 52.69, 36.63, 30.59, 29.90, 28.70, 26.11, 14.36 ppm. MS (ESI): m/z (relative intensity, %) = 669.3 (100) [M + H]+, 569.3 (56) [M-Boc+H]+. HRMS (ESI) calcd for C30H41N10O8 669.3109, found 669.3123. |
Yield | Reaction Conditions | Operation in experiment |
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193 mg | With caesium carbonate; sodium iodide; In N,N-dimethyl-formamide; at 20℃; for 24h;Inert atmosphere; | The ortho-nitrobenzyl (ONB) linker 2 was prepared by using 4-formyl-2-methyoxyphenol (vanillin) as the starting material as reported elsewhere2. Its benzylic alcohol was converted to the methanesulfonate derivative, and covalently coupled to MTX in situ as follows. To a cold solution of linker 2 (0.4 g, 1.0 mmol) in CHCl3 cooled with an ice bath was added triethylamine (0.29 mL, 2.1 mmol), and methanesulfonyl chloride (0.082 mL, 1.1 mmol). The mixture was stirred for 90 min at 0oC, and evaporated in vacuo, yielding the methanesulfonate derivative as pale beige solid (Rf = 0.5 in 10% MeOH/CHCl3). This crude product was dissolved in DMF (4 mL), and used immediately for its alkylation with MTX. In a separate round bottomed flask was prepared the solution of DMF (25 mL) that contains MTX hydrate (0.54 g, 1.1 mmol) and cesium carbonate (0.65 g, 2.0 mmol). To this stirred mixture was added the methanesulfonate product prepared earlier in DMF solution, and followed by the addition of sodium iodide (0.15 g, 1.0 mmol). The final mixture was stirred at rt for 24 h under nitrogen atmosphere in the dark. It was evaporated in vacuo, yielding a pale yellow residue. The material was dissolved in a small volume (~5 mL) of 5% MeOH/CHCl3, loaded onto a silica gel column, and fractionated by eluting with 5% MeOH/CH2Cl2 to 1% AcOH/20% MeOH/CH2Cl2. The desired product was contained in the fraction that displays an Rf value of 0.53 (1% AcOH in 20% MeOH/CHCl3). Evaporation of the desired fractions afforded solid residue. It was rinsed with acetonitrile and dried in vacuo, yielding 9 as pale yellow solid (193 mg, 23%). 1H NMR (400 MHz, DMSO-d6): d = 8.49 (s, 1H), 7.67 (m, 2H), 7.63 (s, 1H), 7.15 (s, 1H), 6.76 (m, 2H), 5.39 (s, 2H), 4.72 (s, 2H), 4.54 (s, 2H), 4.43 (br m, 1H), 3.83 (s, 3H), 3.15 (s, 3H), 3.10 (br, 2H), 2.94 (br, 2H), 2.24 (br, 2H), 1.80 (br, 2H), 1.29 (s, 9H) ppm; MS (ESI): m/z (relative intensity, %) = 836.4 (100) [M+H]+, 736.4 (19) [M+H-Boc]+, 1672.7 (2) [2M+H]+; HRMS (ESI) calcd for C37H46N11O12 [M+H]+ 836.3327, found 836.3325.The MTX-linker 4 was prepared by deprotecting the N-Boc group of 9. In a typical condition, 9 (31 mg, 37.1 mmol) was treated with a mixture of CHCl3 (1.0 mL) and TFA (0.5 mL) for 15 min at rt. The reaction mixture was evaporated to dryness in vacuo, yielding 4 as pale yellow oil (TFA salt). 1H NMR (300 MHz, DMSO-d6): d = 8.70 (d, 1H), 7.80 (m, 1H), 7.68-7.67 (m, 2H), 7.20 (m, 1H), 6.80 (t, 2H), 5.40-5.35 (m, 2H), 4.85 (d, 2H), 4.62 (br s, 3H), 3.94 (s, 3H), 3.36 (t, 2H), 3.24 (d, 3H), 2.87 (t, 2H), 2.68 (m, 2H), 2.20-2.10 (m, 2H) ppm; MS (ESI): m/z (relative intensity, %) = 736.2 (100) [M+H]+, 1471.5 (3) [2M+H]+, 308.1 (61); HRMS (ESI) calcd for C32H38N11O10 [M+H]+ 736.2803, found 736.2824. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With water; In methanol;UV-irradiation; | Photolysis experiments were carried out using Spectroline UV bench lamps (XX-15A; power = 1.1 mW/cm2), either at the UV-B (312 nm) or UV-A (365 nm) wavelength. As a representative photolysis method, the linker 3 was dissolved in an aqueous medium (30 muM, 2% MeOH/H2O), and the solution (20 mL) was loaded onto a glass Petri dish. The linker solution was exposed to the UV lamps irradiated at the distance of ?5 cm at 312 nm over up to 15 min. Progress of the photolysis was monitored by UV/vis spectrometry, and for the analysis, each aliquot (700 muL) was taken out during the exposure at a specific time point as indicated in Figure 4. Photolysis of the linker 4 was performed similarly in the same aqueous medium (30 muM) at 312 or 365 nm, and its reaction aliquots were analyzed by both the UV/vis (Fig. 4) and analytical HPLC method (Fig. 5). |
Yield | Reaction Conditions | Operation in experiment |
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With caesium carbonate; In N,N-dimethyl-formamide; at 20℃; for 24h; | Step (i): 3-[(Bromoacetyl)amino]propyl]-carbamic acid 1,1-dimethylethyl ester 6 was prepared as described elsewhere with slight modifications1. To a cold solution of 3-(N-tert-butoxycarbonylamino)propylamine 5 (1.1 g, 6.31 mmol) in CHCl3 (50 mL) cooled with an ice bath was added N,N-diisopropylethylamine (1.1 mL, 2.03 mmol), and bromoacetyl chloride (526 mL, 6.32 mmol) as neat liquid. The reaction mixture was stirred at 5oC for 3 h under nitrogen atmosphere. The mixture was diluted with dichloromethane (200 mL), and it was washed with 1M H3PO4 (50 mL) solution, and a saturated sodium bicarbonate solution (50 mL). The organic layer was dried over Na2SO4, and evaporated to dryness in vacuo yielding colorless syrup. The product was gradually solidified to white crystals (1.9 g). It was used immediately for the next step withought further purification. Rf (50% EtOAc/hexane) = 0.40.Step (ii): To a solution of 3-hydroxymethyl-4-nitrophenol (1.05 g, 6.21 mmol) dissolved in acetonitrile (50 mL) was added 6 (1.75 g, 5.93 mmol) and potassium carbonate (1.71 g, 12.4 mmol). The mixture was stirred at rt for 24 h under nitrogen atmosphere, and refluxed for 12 h. It was filtered through a Buechner funnel, and the filtrate was concentrated in vacuo. The residue was dissolved in ethyl acetate (150 mL), and the solution was washed with a saturated sodium bicarbonate solution (200 mL). The organic layer was dried over Na2SO4, and evaporated to dryness, yielding pale amber oil. This crude product was purified by flash silica column chromatography, eluting with 2:1 to 1:2 hexane/ethyl acetate mixture. The desired product 1 was obtained as pale yellow foam (0.73 g, 31%). Rf (30% EtOAc/hexane) = 0.78. 1H NMR (300 MHz, CD3OD): d 8.18-8.15 (d, 1H, J = 8.8 Hz), 7.48 (d, 1H; J = 2.8 Hz), 7.06-7.03 (dd, 1H, J = 8.8, 2.8 Hz), 4.97 (s, 2H; -CH2OH), 4.66 (s, 2H; -PhOCH2CO-), 3.29 (m, 2H; -CONHCH2CH2CH2NHBoc), 3.06 (m, 2H; -CONHCH2CH2CH2NHBoc), 1.64 (m, 2H, -CONHCH2CH2CH2NHBoc), 1.28 (s, 9H, -NHBoc) ppm. 13C NMR (100 MHz, CD3OD): d 170.13, 163.42, 143.11, 128.55, 114.73, 114.43, 68.37, 62.17, 38.52, 37.37, 30.78, 28.76 ppm. HRMS (ESI, positive ion mode): calcd for C17H26N3O7 [M+H]+, m/z = 384.1765, found 384.1765; calcd for C17H25N3O7Na [M+Na]+, m/z = 406.1585, found 406.1583.Steps (iii, and iv): To a cold solution of 1 (0.63 g, 1.64 mmol) dissolved in chloroform (20 mL) was added triethylamine (252 mL, 1.8 mmol) and then bromoacetyl chloride (144 mL, 1.73 mmol). The mixture was stirred under nitrogen atmosphere at 5oC for 2 h, and then overnight at ambient temperature. The reaction mixture was washed with 1M H3PO4 solution (30 mL), a saturated NaHCO3 solution (30 mL), and the brine solution. The organic layer was dried over Na2SO4, and the filtrate was evaporated to dryness, yielding pale brown foam (0.75 g). This product 7 was used without further treatment in the next step, an alkylation reaction with MTX. Rf (50% EtOAc/hexane) = 0.56. MTX hydrate (0.42 g, 0.86 mmol) was loaded into a round bottomed flask containing DMF (15 mL), and followed by the addition of cesium carbonate (0.30 g, 0.92 mmol). The mixture was sonicated for 10 min until the MTX solid was fully dissolved. The linker 7 (0.54 g, 0.11 mmol), prepared freshly as described above, was dissolved in DMF (5 mL), and this solution was added to the MTX mixture. The final mixture was stirred at rt for 24 h. After concentration in vacuo, the residue was purified by flash silica column chromatography by eluting with 5% MeOH/CH2Cl2 to 1% AcOH/20% MeOH/CH2Cl2. Fractions having the Rf value of 0.79 (0.5% AcOH/10%MeOH/CH2Cl2) were combined and evaporated to dryness, yielding the N-Boc protected MTX-linker 8 as pale yellow solid (195 mg, 26%). The isolated material comprises of two regioisomers, each resulting from the O-alkylation at the a- (~37%) and g-carboxylate (~63%) of the L-glutamate. Separation of these isomers was not attempted because upon UV irradiation, MTX is released to an equal extent regardless of the nature of the isomer. 1H NMR (400 MHz, DMSO-d6): d 8.73 (br), 8.56 (s), 8.25 (br), 8.22 (d), 7.74-7.73 (br), 7.43 (br), 7.19 (s), 7.12 (d), 6.81 (br s), 6.62 (br), 5.52 (s), 4.93-4.82 (dd), 4.78 (s), 4.67 (s), 4.49 (br), 4.10 (m), 3.20 (br), 3.20-3.16 (m), 2.90 (br), 2.50 (s), 2.33 (br s), 2.07 (br), 2.00-1.88 (m), 1.51 (s), 1.35 (s), 1.22 (m) ppm. MS (ESI, positive ion mode): m/z (relative intensity, %) = 878.3 (100) [M+H]+, 778.3 (52) [MBoc+H]+. HRMS (ESI) calcd for C39H48N11O13 [M+H] + 878.3433, found 878.3410.Step (v): MTX-ONB linker 3 was synthesized by deprotecting the N-Boc group of the compound 8. In this process, 8 (50 mg, 57 mmol) was suspended in chloroform (1 mL) and treated with trifluroacetic acid (1 mL). The mixture was stirred at rt for 20 min, and evaporated to dryness yielding pale yellow oil. It was dissolved in methanol (15 mL), and the solution was concentrated to dryness to remove any remaining volatiles including trifluoroacetic acid. The product w... |
Yield | Reaction Conditions | Operation in experiment |
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Step (i): 3-[(Bromoacetyl)amino]propyl]-carbamic acid 1,1-dimethylethyl ester 6 was prepared as described elsewhere with slight modifications1. To a cold solution of 3-(N-tert-butoxycarbonylamino)propylamine 5 (1.1 g, 6.31 mmol) in CHCl3 (50 mL) cooled with an ice bath was added N,N-diisopropylethylamine (1.1 mL, 2.03 mmol), and bromoacetyl chloride (526 mL, 6.32 mmol) as neat liquid. The reaction mixture was stirred at 5oC for 3 h under nitrogen atmosphere. The mixture was diluted with dichloromethane (200 mL), and it was washed with 1M H3PO4 (50 mL) solution, and a saturated sodium bicarbonate solution (50 mL). The organic layer was dried over Na2SO4, and evaporated to dryness in vacuo yielding colorless syrup. The product was gradually solidified to white crystals (1.9 g). It was used immediately for the next step withought further purification. Rf (50% EtOAc/hexane) = 0.40.Step (ii): To a solution of 3-hydroxymethyl-4-nitrophenol (1.05 g, 6.21 mmol) dissolved in acetonitrile (50 mL) was added 6 (1.75 g, 5.93 mmol) and potassium carbonate (1.71 g, 12.4 mmol). The mixture was stirred at rt for 24 h under nitrogen atmosphere, and refluxed for 12 h. It was filtered through a Buechner funnel, and the filtrate was concentrated in vacuo. The residue was dissolved in ethyl acetate (150 mL), and the solution was washed with a saturated sodium bicarbonate solution (200 mL). The organic layer was dried over Na2SO4, and evaporated to dryness, yielding pale amber oil. This crude product was purified by flash silica column chromatography, eluting with 2:1 to 1:2 hexane/ethyl acetate mixture. The desired product 1 was obtained as pale yellow foam (0.73 g, 31%). Rf (30% EtOAc/hexane) = 0.78. 1H NMR (300 MHz, CD3OD): d 8.18-8.15 (d, 1H, J = 8.8 Hz), 7.48 (d, 1H; J = 2.8 Hz), 7.06-7.03 (dd, 1H, J = 8.8, 2.8 Hz), 4.97 (s, 2H; -CH2OH), 4.66 (s, 2H; -PhOCH2CO-), 3.29 (m, 2H; -CONHCH2CH2CH2NHBoc), 3.06 (m, 2H; -CONHCH2CH2CH2NHBoc), 1.64 (m, 2H, -CONHCH2CH2CH2NHBoc), 1.28 (s, 9H, -NHBoc) ppm. 13C NMR (100 MHz, CD3OD): d 170.13, 163.42, 143.11, 128.55, 114.73, 114.43, 68.37, 62.17, 38.52, 37.37, 30.78, 28.76 ppm. HRMS (ESI, positive ion mode): calcd for C17H26N3O7 [M+H]+, m/z = 384.1765, found 384.1765; calcd for C17H25N3O7Na [M+Na]+, m/z = 406.1585, found 406.1583.Steps (iii, and iv): To a cold solution of 1 (0.63 g, 1.64 mmol) dissolved in chloroform (20 mL) was added triethylamine (252 mL, 1.8 mmol) and then bromoacetyl chloride (144 mL, 1.73 mmol). The mixture was stirred under nitrogen atmosphere at 5oC for 2 h, and then overnight at ambient temperature. The reaction mixture was washed with 1M H3PO4 solution (30 mL), a saturated NaHCO3 solution (30 mL), and the brine solution. The organic layer was dried over Na2SO4, and the filtrate was evaporated to dryness, yielding pale brown foam (0.75 g). This product 7 was used without further treatment in the next step, an alkylation reaction with MTX. Rf (50% EtOAc/hexane) = 0.56. MTX hydrate (0.42 g, 0.86 mmol) was loaded into a round bottomed flask containing DMF (15 mL), and followed by the addition of cesium carbonate (0.30 g, 0.92 mmol). The mixture was sonicated for 10 min until the MTX solid was fully dissolved. The linker 7 (0.54 g, 0.11 mmol), prepared freshly as described above, was dissolved in DMF (5 mL), and this solution was added to the MTX mixture. The final mixture was stirred at rt for 24 h. After concentration in vacuo, the residue was purified by flash silica column chromatography by eluting with 5% MeOH/CH2Cl2 to 1% AcOH/20% MeOH/CH2Cl2. Fractions having the Rf value of 0.79 (0.5% AcOH/10%MeOH/CH2Cl2) were combined and evaporated to dryness, yielding the N-Boc protected MTX-linker 8 as pale yellow solid (195 mg, 26%). The isolated material comprises of two regioisomers, each resulting from the O-alkylation at the a- (~37%) and g-carboxylate (~63%) of the L-glutamate. Separation of these isomers was not attempted because upon UV irradiation, MTX is released to an equal extent regardless of the nature of the isomer. 1H NMR (400 MHz, DMSO-d6): d 8.73 (br), 8.56 (s), 8.25 (br), 8.22 (d), 7.74-7.73 (br), 7.43 (br), 7.19 (s), 7.12 (d), 6.81 (br s), 6.62 (br), 5.52 (s), 4.93-4.82 (dd), 4.78 (s), 4.67 (s), 4.49 (br), 4.10 (m), 3.20 (br), 3.20-3.16 (m), 2.90 (br), 2.50 (s), 2.33 (br s), 2.07 (br), 2.00-1.88 (m), 1.51 (s), 1.35 (s), 1.22 (m) ppm. MS (ESI, positive ion mode): m/z (relative intensity, %) = 878.3 (100) [M+H]+, 778.3 (52) [MBoc+H]+. HRMS (ESI) calcd for C39H48N11O13 [M+H] + 878.3433, found 878.3410.Step (v): MTX-ONB linker 3 was synthesized by deprotecting the N-Boc group of the compound 8. In this process, 8 (50 mg, 57 mmol) was suspended in chloroform (1 mL) and treated with trifluroacetic acid (1 mL). The mixture was stirred at rt for 20 min, and evaporated to dryness yielding pale yellow oil. It was dissolved in methanol (15 mL), and the solution was concentrated to dryness to remove any remaining volatiles including trifluoroacetic acid. The product w... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The linear protected GnRH-III derivatives (Glp-His(Trt)-Trp(Boc)-Ser(tBu)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Mtt)-Pro-Gly-R, Glp-His(Trt)-Trp(Boc)-Lys(Dde)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Mtt)-Pro-Gly-R, Glp-His(Trt)-Trp(Boc)-Lys(Dde)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Boc)-Pro-Gly-R; where R = resin) were prepared manually by solid phase peptide synthesis according to Fmoc/tBu chemistry on a Rink-Amide MBHA resin (0.38 mmol/g coupling capacity). The following Fmoc-protected amino acid derivatives were used: Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH and Fmoc-Ser(tBu)-OH. Pyroglutamic acid (Glp or |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The linear protected GnRH-III derivatives (Glp-His(Trt)-Trp(Boc)-Ser(tBu)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Mtt)-Pro-Gly-R, Glp-His(Trt)-Trp(Boc)-Lys(Dde)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Mtt)-Pro-Gly-R, Glp-His(Trt)-Trp(Boc)-Lys(Dde)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Boc)-Pro-Gly-R; where R = resin) were prepared manually by solid phase peptide synthesis according to Fmoc/tBu chemistry on a Rink-Amide MBHA resin (0.38 mmol/g coupling capacity). The following Fmoc-protected amino acid derivatives were used: Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH and Fmoc-Ser(tBu)-OH. Pyroglutamic acid (Glp or |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The linear protected GnRH-III derivatives (Glp-His(Trt)-Trp(Boc)-Ser(tBu)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Mtt)-Pro-Gly-R, Glp-His(Trt)-Trp(Boc)-Lys(Dde)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Mtt)-Pro-Gly-R, Glp-His(Trt)-Trp(Boc)-Lys(Dde)-His(Trt)-Asp(OtBu)-Trp(Boc)-Lys(Boc)-Pro-Gly-R; where R = resin) were prepared manually by solid phase peptide synthesis according to Fmoc/tBu chemistry on a Rink-Amide MBHA resin (0.38 mmol/g coupling capacity). The following Fmoc-protected amino acid derivatives were used: Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH and Fmoc-Ser(tBu)-OH. Pyroglutamic acid (Glp or |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With dmap; dicyclohexyl-carbodiimide; In dimethyl sulfoxide; at 20℃; for 24h; | 2-(4-(2-(2-aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethanol (5); 2-(4-(2-(2-aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethylMTX (6); Folic acid (FA) (195 mg, 0.44 mmol) in DMSO (10 mL) was added to a solution of 4 (170 mg, 0.44 mmol) and (DCC) (182 mg, 0.88 mmol) in DMSO (5 mL). The reaction was stirred at room temperature for 24 hours. The reaction mixture was filtered. H2O (30 mL) was added to the filtrate. The resulting precipitate was filtered, washed with H2O and acetone, and dried under reduced pressure. The cruder product was further purified by HPLC. Compound 5 was obtained as a yellow solid (147 mg, 41%): ESI-MS m/z 808.3 (M+H+) calculated for C33H46N17O8 808.4.<strong>[59-05-2]Methotrexate</strong> (MTX) (25 mg, 0.055 mmol) in DMSO (5 mL) was added to a solution of 5 (40 mg, 0.50 mmol), N,N'-dicyclohexylcarbodiimide (DCC) (204 mg, 1.0 mmol), and trace amount 4-(dimethylamino)pyridine (DMAP) in DMSO (5 mL). The reaction was stirred at room temperature for 24 hours. H2O (30 mL) was added to the reaction mixture. The solid was collected by centrifugation, washed with H2O and acetone, and dried under vacuum to yield 6 as a yellow solid.Two isomers 5 and 6 were isolated and purified as yellow solids, 5 (94 mg 26%), 6 (53 mg, 15%): 1H NMR (500 MHz, d6-DMSO) 5 delta 1.23 (m, 1H), 1.77 (m, 2H), 1.85 (m, 1H), 1.96 (m, 1H), 2.26 (m, 2H), 3.49 (m, 25H), 4.35 (m, 1H), 4.50 (s, 1H), 6.63 (d, J=8.5 Hz, 2H), 7.28 (m, 2H), 7.65 (d, J=8.5 Hz, 2H), 7.87-7.98 (m, 2H), 8.10 (m, 1H), 8.27 (m, 1H), 8.66 (s, 1H); 6 delta 1.23 (m, 1H), 1.76 (m, 2H), 1.85 (m, 1H), 2.04 (m, 1H), 2.19 (m, 2H), 3.17 (t, J=6.0 Hz, 1H), 3.35-3.61 (m, 24H), 4.28 (m, 1H), 4.50 (s, 1H), 6.64 (d, J=8.5 Hz, 2H), 7.22 (m, 2H), 7.65 (d, J=8.5 Hz, 2H), 7.89 (m, 1H), 8.11 (m, 1H), 8.19 (d, J=7.5 Hz, 1H), 8.27 (m, 1H), 8.66 (s, 1H); ESI-MS m/z 808.3 (M+H+) calculated for C33H46N17O8 808.4. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
50% | <strong>[59-05-2]Methotrexate</strong> trihydrate (500 mg) was placed in a ChemDry apparatus attached to a vacuum pump (?100 milliTorr), the temperature was set to 80 C, and the material dried overnight. After 22 h, 448.2 mg of a light orange solid remained, consistent with loss of three water molecules (MW trihydrate 508; MW anhydrous 452). HPLC displayed essentially a single peak at 3.1 min (20% acetonitrile/80% 0.1% aqueous trifluoroacetic acid), indicating that the methotrexate was dry without decomposition. Anal. Calcd for C22H24N6O5: C, 52.86; H, 4.88, N, 24.66. Found: C, 52.65; H, 4.90; N, 24.41. Dry methotrexate (400 mg, 0.88 mmol) was dissolved in anhydrous degassed DMF (5.0 mL), HBTU (334 mg, 0.88 mmol) was added, and the reaction was stirred for 2 min. 5-aminofluorescein (320 mg, 0.92 mmol) was then added to the clear red solution, and the reaction stirred at ambient temperature protected from light for 120 h. The reaction was purified directly via preparative reversed phase HPLC on a 47 × 300 mm 15 muM ODS AQ column, eluting each of the twelve 0.40 mL injections with 26% acetonitrile/74% 0.1% aqueous trifluoroacetic acid, collecting from 0.2 to peak top to 0.2 AU. The column was cleaned after every three injections by washing with 80% acetonitrile/20% 0.1% aqueous trifluoroacetic acid until AU was L solution) were concentrated to ?100 mL on the rotovap and lyophilized. An orange yellow solid (512 mg) was produced, which was redissolved in aqueous acetonitrile and relyophilized overnight, to produce 502 mg (50%) of an orange yellow solid. 1H NMR (DMSO-d6) delta 10.55 (s, 1H), 10.06 (br, 1H), 9.29 (s, 1H), 9.08 (s, 1H), 8.72 (s, 1H), 8.33 (m 2H), 7.84 (dd, 1H, J = 8.6, 1.7 Hz), 7.81 (d, 2H, J = 8.7 Hz), 7.20 (d, 1H, J = 8.2 Hz), 6.83 (d, 2H, J = 9.0 Hz), 6.65 (d, 2H, J = 2.2 Hz), 6.58 (dd, 2H, J = 8.4, 2.5 Hz), 6.51 (m, 2H), 4.88 (s, 2H), 4.51 (q, 2H, J = 7.2 Hz), 3.15 (s, 3H), 2.47 (q, 2H, J = 8.2 Hz), 2.06 (m, 3H). ESMS m/z 784.5 (M+H+). Analytical HPLC (4.6 × 150 mm YMC ODS-AQ, 28:72 CH3CN:0.05% aqueous trifluoroacetic acid, 254 nm) 98.8% purity. Anal. Calcd for C40H33N9O9 2.5 CF3COOH: C 50.57, H 3.35, N 11.79. Found: C, 50.53; H, 3.51; N, 12.10. Anal. (dried material). Calcd for C40H33N9O9 1.5 CF3COOH: C, 54.09; H, 3.64; N, 13.20. Found: C, 54.41; H, 3.48; N, 13.41. |
Tags: 59-05-2 synthesis path| 59-05-2 SDS| 59-05-2 COA| 59-05-2 purity| 59-05-2 application| 59-05-2 NMR| 59-05-2 COA| 59-05-2 structure
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