PROCESS FOR PREPARING NUCLEOSIDE PRODRUGS

Abstract

A process for preparing phosphoramidates of nucleosides where a desired enantiomer, having regard to the asymmetric chiral center of the phosphorus atom P, is provided in an enriched amount. The process comprises admixing a nucleoside with a phosphorochloridate in the presence of a catalyst comprising a metal salt selected from the group consisting of salts of Cu, Fe, La and Yb.

Claims

1-37. (canceled)

38. A method of treating cancer, wherein the method comprises administering to a human patient in need thereof a therapeutically effective amount of a composition comprising a compound of the formula: ##STR00017## or a pharmaceutically acceptable salt thereof, wherein the ratio of the R.sub.P diastereomer to the Se diastereomer is in the range from 1:20 to 0:100.

39. The method of treatment of claim 38, wherein the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

40. The method of treatment of claim 38, wherein the cancer is breast cancer.

41. The method of treatment of claim 38, wherein the cancer is colon cancer.

42. The method of treatment of claim 38, wherein the cancer is prostate cancer.

43. The method of treatment of claim 38, wherein the cancer is leukemia.

44. The method of treatment of claim 38, wherein the composition is administered orally.

45. The method of treatment of claim 38, wherein the composition is administered parenterally.

46. The method of treatment of claim 45, wherein the composition is administered intravenously.

47. The method of treatment of claim 38, wherein the composition is administered topically.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0125] The present invention will now be described by way of example only with reference to the following examples and the following figures, wherein:

[0126] FIG. 1 shows the HPLC spectrum of the product of Example 1;

[0127] FIG. 2 shows the HPLC spectrum of the product of Example 42;

[0128] FIG. 3 shows the reaction scheme and HPLC spectrum of the product of Example 43;

[0129] FIG. 4 shows the reaction scheme and HPLC spectrum of the product of Example 44;

[0130] FIG. 5 shows the reaction scheme and HPLC spectrum of the product of Example 45;

[0131] FIG. 6 shows the reaction scheme and HPLC spectrum of the product of Example 46;

[0132] FIG. 7 shows the HPLC spectrum of the product of Example 47; and

[0133] FIG. 8 shows the HPLC spectrum of the product of Example 48.

[0134] The following example sets out the experimental procedure that was employed in each of the Examples for which data are set out below.

EXPERIMENTAL PROCEDURE

Example

[0135] ##STR00008##

[0136] A dry round bottomed flask is charged with a magnetic stirring bar, 2′-C methyl-6-O-methylguanosine (2′CMe6OMeG) (106.0 mg, 0.34 mmol.) and catalytic amount of copper (II) trifluoromethane sulfonate (12.32 mg, 0.34 mmol., 0.1 equiv.). The flask is sealed with a rubber septum and purged with dry argon. Anhydrous 1,2-dimethoxyethane (DME, 10 mL) is added via syringe and the resulting light blue solution is stirred at room temperature for 5-10 minutes. In a separate vial, a solution of naphthyl (Oneopentyl-L-alaninyl) phosphorochloridate (131 mg, 0.34 mmol., 1 equiv.) in 2-3 mL of anhydrous THF is prepared. To the nucleoside solution is then added N,N-diisopropylethylamine (DIPEA)(62.3 mg, 0.48 mmol., 84.0 μL, 1.5 equiv.) followed by the dropwise addition of the phosphorochloridate solution previously prepared. Upon addition of the base, the solution turned from light blue to dark green and a white precipitate appeared. Addition of the phosphorochloridate solution causes the disappearance of the precipitate and the color of the solution to turn to dark blue. The mixture is then stirred at room temperature for 12 hours. The reaction is monitored by HPLC, according to the following protocol:

[0137] a 0.1-0.2 mL aliquot of solution is withdraw from the flask, under argon, via syringe and diluted with HPLC grade methanol, filtered and further diluted with a mixture of acetonitrile/water 10:90. The resulting solution is then injected into HPLC and analyzed (Reverse-phase C-18 column, eluting with H.sub.2O/MeCN from 90/10 to 0/100 in 30 min, Flow=1 mL/min, λ=254 nm and λ=280 nm). A 38% yield and 1:8 Sp to Rp diastereomeric ratio are estimated by integration of the product and starting material peaks.

[0138] When the reaction is completed, the solvent is evaporated under reduced pressure, and the residue is purified by column chromatography on silica gel with gradient elution DCM: MeOH 98:2 to 94:6. The residue from the column is taken up in dichloromethane and washed with 0.5 M HCl (3×10 mL). The organic layer is separated, dried over sodium sulfate, filtered and evaporated to give the title compound as white solid (isolated yield: 40 mg, 20%). The isomer ratio obtained was 1:5 in favor of the R.sub.p isomer as judged from the HPLC of the pure compound, as shown in FIG. 1.

[0139] The procedure outlined above was followed in the following examples.

Examples 1 to 5

[0140] The above procedure was followed employing 2′CMe6OMeG, as set out above, as the nucleoside and each of the phosphorochloridates whose structures are set out immediately below, arranged in order of Example 1 to Example 5, and the following experimental conditions: Nucleoside. 100 mg, phosphorochloridate 1 equiv., Cu(OTf).sub.2 0.1 equiv., DIPEA 1.5 equiv., DME 10 mL, room temperature, 12-18 hours. Example 5 is a reference example.

##STR00009##

[0141] The results of the preparative processes in terms of the R.sub.P:S.sub.P ratio of enantiomers isolated and the HPLC yield achieved are given in Table 1 below. “2′CMeG” in Table 1 stands for “2′CMe6OMeG”, as set out above.

TABLE-US-00001 TABLE 1 Variation of the phosphorochloridate Example Nucleoside Phosphorochloridate Ratio Yield 1 2′CMeG L-Ala neopentyl, naphthyl 1:8.sup.  38% 2 2′CMeG L-Ala neopentyl, phenyl 1:3.sup.  32% 3 2′CMeG D-Ala benzyl, naphthyl 1:1.1  8% 4 2′CMeG L-Val benzyl, naphthyl 1:7.5 41% 5 2′CMeG Sarcosine ethyl, naphthyl — trace

Examples 6 to 12

[0142] Following the experimental procedure set out above, one equivalent of naphthyl (Oneopentyl-L-alaninyl)phosphorochloridate was reacted with a range of nucleosides under the following conditions: Nucleoside 100 mg, phosphorochloridate 1 equiv., Cu(OTf).sub.2 0.1 equiv., DIPEA 1.5 equiv., DME 10 mL, Room Temperature, 12-18 hours. Example 8 is a reference example.

[0143] The structure of naphthyl(Oneopentyl-L-alaninyl)phosphorochloridate and the structures of the nucleosides, arranged in order of Example 6 to Example 12, are given below:

##STR00010## ##STR00011##

[0144] The results in terms of the ratio of R.sub.P:S.sub.P and yield achieved are given in Table 2 below.

TABLE-US-00002 TABLE 2 Nucleoside variation Example Nucleoside Phosphorochloridate Ratio Yield 6 2′CMeG L-Ala neopentyl, naphthyl 1:8.sup.  38% 7 2′,3′iPr A L-Ala neopentyl, naphthyl 1:1.1 12% 8 Abacavir L-Ala neopentyl, naphthyl — — 9 Gemcitabine L-Ala neopentyl, naphthyl 1:1.sup.  Trace 10 Boc Gemcitabine L-Ala neopentyl, naphthyl 1:2.2  5% 11 FUDR L-Ala neopentyl, naphthyl 1:2.5 30% 12 d4t L-Ala neopentyl, naphthyl 1:1.8 50%

Examples 13 to 14

[0145] Copper Catalyst

[0146] Following the experimental procedure set out above and the following experimental conditions: Nucleoside 100 mg, phosphorochloridate 1 equiv., Cu(X).sub.Y 0.1 equiv., NEt.sub.3 1.5 equiv., THF 20 mL, Room Temperature, 12-18 hours, other copper salts in place of Cu(OTf).sub.2 were tested as the catalyst. The copper salts employed and the results in terms of the ratio R.sub.P:S.sub.P of enantiomers and the yield achieved are given in the Table 3 below.

TABLE-US-00003 TABLE 3 Screening of copper salts Example Cu salt (equiv.) Ratio (HPLC) (HPLC yield) 13 Cu(OAc).sub.2•H.sub.2O (0.1) 1:2.1 (34%) 14 CuI (0.1) 1:3.2 (22%)

Examples 15 to 18

[0147] Using the experimental procedure set out above, 2′CMe6OMeG as the nucleoside, naphthyl(oneopentyl-L-alaninyl)phosphorochloridate as the phosphorochloridate and the following experimental conditions: Nucleoside 100 mg, phosphorochloridate 1 equiv., Me(OTf)y 0.1 equiv., NEt.sub.3 1.5 equiv., THF 20 mL, room temperature, N.sub.2 atmosphere, 12-18 hours, metal triflates other than copper triflate were screened. The reaction is set out below. Example 15 is a reference example.

##STR00012##

[0148] The results in terms of the ratio R.sub.P:S.sub.P of enantiomers achieved and the yield achieved are set out in Table 4 below.

TABLE-US-00004 TABLE 4 Screening of other metal tritiates; * the isomers ratio of R.sub.P:S.sub.P was slightly reversed (results of two runs). Example Me(OTf).sub.2 (equiv.) Isomer Ratio (% yield) 15 Ag(OTf) 1 equiv. / No reaction 16 Yb(OTf).sub.3(0.1) 1:2 22% 17 Fe(OTf).sub.3(0.1) 1:2 13% 18 La(OTf).sub.3(0.1) * 1.1:1.sup.  19%

[0149] In addition, TiCl.sub.4 as well as B(C.sub.6F.sub.5) were also tested as catalyst. With titanium tetrachloride no diastereoselectivity was observed (1:1 ratio between the two isomers in 11% yield), meanwhile with tris(pentafluorophenyl)boron no reaction was observed.

Examples 19 to 22

[0150] Using the experimental procedure set out above, 2′CMe6OMeG as the nucleoside, naphthyl (oneopentyl-L-alaninyl) phosphorochloridate as the phosphorochloridate and the following experimental conditions Nucleoside: 100 mg, 1 equivalent; Cu(OTf).sub.2 0.1 equivalents; phosphorochloridate 1 equivalent; base 1.5 equivalents; THF 20 mL, Room Temperature, 12 hours, different bases were screened. The bases used and the results achieved in terms of R.sub.P:S.sub.P ratio of enantiomers and yield achieved are set out in Table 5 below. Example 22 uses DMAP, which is 4-dimethylaminopyrimidine, as the base, and is a reference example.

TABLE-US-00005 TABLE 5 Variation of base Cu(OTf).sub.2 Base Ratio Example (Equiv.) (1.5 equiv.) (yield) 19 0.1 DIPEA 1:2.5 (47%) 20 0.1 (i-pr).sub.2NH 1:2.9 (42%) 21 0.1 DBU B. 1:3.3 (5%) 22 0.1 DMAP Traces (1:2.5)

Examples 23 to 28

[0151] Solvent Screening

[0152] Following the experimental procedure above and using 2′CMe6OMeG as the nucleoside and naphthyl(onepentyl-L-alaninyl)phosphorochloridate as the phosphorochloridate and the following experimental conditions: Nucleoside: 100 mg, 1 equivalent; Cu(OTf).sub.2 0.1 equivalents; Phosphorochloridate 1 equivalent; NEt.sub.31.5 equivalents; solvent 20 mL, Room Temperature, 12 hours, varying solvent media for use in step (i) to dissolve the nucleoside compound and the metal salt catalyst were investigated. The results in terms of the ratio R.sub.P:S.sub.P of enantiomers and the yield achieved are set out in Table 6 below. Examples 24, 25, 27 and 28 are reference examples. “DCM” stands for dichloromethane (CH.sub.2Cl.sub.2).

TABLE-US-00006 TABLE 6 Solvent screening Cu(OTf).sub.2 Solvent Ratio Example (Equiv.) (20 mL) (Yield) 23 0.1 DME 1:5 (14%) 24 0.1 DCM Trace 25 0.1 Ethylene glycol No reaction 26 0.1 1,4 dioxane 1:2.5 (38%) 27 0.1 Toluene 1:1.5 (traces) 28 0.1 Pyridine No Reaction

Examples 29 to 35

[0153] Using the experimental procedure set out above, gemcitabine (100 mg) was employed as the nucleoside and naphthyl(isopropyl-L-alaninyl)phosphorochloridate (1 molar equiv) was employed as the phosphorochloridate. For each example, the catalyst (MX), base and solvent were employed, as set out in the reaction scheme and in Table 7 below. Example 29 employed 10 ml THF and 2 ml MeCN. Table 7 gives, for each example, the total yield and the ratio of R.sub.P:S.sub.P enantiomers achieved. Example 31, employing Ti(OiPr).sub.4 as the catalyst, is a reference example. In Table 7, AA indicates the amino acid moiety corresponding to —CNHCR.sub.1R.sub.2CO.sub.2—

##STR00013##

TABLE-US-00007 TABLE 7 Gemcitabine as the nucleoside MX Base Yield Ratio Ex. AA OR.sub.3 (eq.) 1.5 eq. Solvent (%) R.sub.P:S.sub.P 29 L-Ala iPr Cu(I)OAc NEt.sub.3 THF/2 mL 10 1:5.sup.  (0.2) MeCN 30 L-Ala iPr Cu(I)OAc NEt.sub.3 MeCN 4-5 1:2.8 (0.2) 31 L-Ala iPr Ti(OiPr).sub.4 NEt.sub.3 MECN 2 1:3.5 32 L-Ala iPr Cu(I)OAc NEt.sub.3 THF/MeCN 10 1:4.1 (0.2) 1:1 33 L-Ala iPr Cu(I)OAc NEt.sub.3 DME 10 1:5.2 (0.6) 34 L-Ala iPr Cu(I)OAc DIPEA THF/MeCN 12 1:8.4 (0.2) 10 mL/2 mL 35 L-Ala iPr Cu(I)OAc NEt.sub.3 THF 4 1:5.3 (0.5)

Examples 36 to 42

[0154] Using the experimental procedure set out above, 2′deoxy-2′fluorouridine (100 mg) was employed as the nucleoside and naphthyl(iso-propyl-L-alaninyl)phosphorochloridate (1 molar equiv) was employed as the phosphorochloridate. The catalyst, base and solvent for each example are set out in Table 8 below. In each case, the reaction took place at room temperature under nitrogen and for 24 hours. Examples 38 and 41 are reference examples.

TABLE-US-00008 TABLE 8 2′deoxy-2′ fluorouridine as the nucleoside Isomer M(X).sub.n Base solvent ratio Example (eq.) (1.5 eq.) 10 mL (yield %) 36 CuSO.sub.4 NEt.sub.3 THF 1:3 (1) (50%) 37 CuSO.sub.4 NEt.sub.3 THF 1:2.6 (1) (50%) 38 CuSO.sub.4 Ag.sub.2CO.sub.3 THF / (1) 39 Cu(MeCN)•.sub.4CF.sub.3SO.sub.3 DIPEA DME 1:2.3 (0.5) (56%) 40 Cu(OTf)•C.sub.6H.sub.6 DIPEA DME 1:2.1 (0.5) (34%) 41 Ti(OiPr).sub.4 DIPEA DME 42 Cu(I)OAc DIPEA DME 1:5.6 (0.5) (36%)

[0155] The HPLC spectrum for the product of Example 42 is shown in FIG. 2.

Example 43

[0156] Using the experimental procedure set out above, nelarabine was employed as the nucleoside (100 mg) and naphthyl(oneopentyl-L-alaninyl)phosphorochloridate (1 molar equiv) was employed as the phosphorochloridate. Cu(OTf).sub.2 (0.1 equiv) was employed as the catalyst. NEt.sub.3 (1.5 equiv) was employed as the base and 10 ml of THF were employed as the solvent. The reaction took place at room temperature under argon for 12 hours.

[0157] The phosphoramidated nelarabine reaction product was produced in a yield of 80% and comprised a ratio of R.sub.P:S.sub.P enantiomers of 3.6:1.

[0158] The reaction scheme for the diastereoselective synthesis of protide via metal catalysis with respect to nelarabine of the present example and the HPLC of the reaction product are set out in FIG. 3. A/B in FIG. 3 refers to R.sub.P/S.sub.P.

Example 44

[0159] Using the above experimental procedure, clofarabine (100 mg) was employed as the nucleoside and naphthyl(oneopentyl-L-alaninyl)phosphorochloridate (1 molar equiv) was employed as the phosphorochloridate. Cu(OTf).sub.2 (0.1 equiv) was employed as the catalyst. NEt.sub.3 (1.5 equiv) was employed as the base and 10 ml of THF were employed as the solvent. The reaction took place at room temperature under argon for 12 hours.

[0160] The phosphoramidated clofarabine reaction product was achieved in a yield of about 40% and comprised a ratio of R.sub.P:S.sub.P enantiomers of 1:1.5.

[0161] The reaction scheme for the diastereoselective synthesis of protide via metal catalysis with respect to clofarabine of the present example and the HPLC spectrum of the reaction product are set out in FIG. 4. A/B in FIG. 4 refers to R.sub.P/S.sub.P.

Example 45

[0162] Using the experimental procedure set out above, 2′deoxy-2′fluorouridine (100 mg) was employed as the nucleoside and naphthyl(iso-propyl-L-alaninyl)phosphochloridate (1 molar equiv.) was employed as the phosphorochloridate. 0.2 molar equivalents of Cu(OC(O)CF.sub.3).sub.2 were used as the catalyst. 1.5 molar equivalents of NEt.sub.3 were employed as the base. 10 ml of DME were employed as the solvent. The reaction took place at room temperature under nitrogen for 12 hours.

[0163] The phosphoramidated 2′deoxy-2′fluorouridine reaction product was produced in a yield of 35% and comprised a ratio of R.sub.P:S.sub.P enantiomers of 1:5.3.

[0164] The reaction scheme of the present example and the HPLC spectrum of the reaction product are set out in FIG. 5.

Example 46

[0165] Using the experimental procedure set out above, Boc gemcitabine (100 mg) was employed as the nucleoside and naphthyl(iso-propyl-L-alaninyl)phosphorochloridate (1 molar equiv.) was employed as phosphorochloridate. 0.2 molar equivalents of Cu(OC)(O)CF.sub.3).sub.2 were used as the catalyst. NEt.sub.3 (1.5 equiv) were employed as the base. 50 ml DME were employed as the solvent. The reaction took place under nitrogen at room temperature for 24 hours.

[0166] The phosphoramidated gemcitabine reaction product was produced in a yield of 9% and comprised a ratio of R.sub.P:S.sub.P enantiomers of 1:9.

[0167] The reaction scheme of the present example and the HPLC spectrum of the reaction product are set out in FIG. 6.

##STR00014##

Example 47

[0168] Using the experimental procedure set out above, 4-amino-1-((2R,3R,4R5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one (100 mg) was employed as the nucleoside and 2 molar equivalents of phenyl(isopropyl-L-alaninyl)phosphorochloridate (150 mg) were employed as the phosphorochloridate. 0.5 molar equivalents of Cu(CF.sub.3CO.sub.2).sub.2 (30 mg) were employed as the catalyst. 1.5 molar equivalents of DIPEA (55 microlitres) were employed as the base and 10 ml of DME were employed as the solvent. The reaction took place at room temperature for 24 hours.

[0169] The reaction scheme of the present example is set out below.

##STR00015##

[0170] The phosphoramidated reaction product was produced in a yield of 20% and comprised a ratio of R.sub.P:S.sub.P enantiomers of 1:66.

[0171] The HPLC of the phosphoramidated reaction product is set out in FIG. 7.

Example 48

[0172] Using the experimental procedure set out above, 3′-boc gemcitabine (100 mg) was employed as the nucleoside and 2 molar equivalents of phenyl(benzyl-L-alaninyl)phosphoramidate (150 mg) were employed as the phosphorochloridate. 0.5 molar equivalents of tris(acetylacetonato)FeIII (56 mg) were employed as the catalyst. 1.5 molar equivalents DIPEA (55 microlitres) were employed as the base and 10 ml of THF were employed as the solvent. The reaction took place at room temperature under nitrogen for 24 hours.

[0173] The reaction scheme of the present example is set out below.

##STR00016##

[0174] The phosphoramidated reaction product was produced in yield of 45% and comprised a ratio of R.sub.P:S.sub.P enantiomers of 3:1.

[0175] The HPLC of the phosphoramidated reaction product is set out in FIG. 8.