PHOSPHORAMIDATE DERIVATIVES OF 5 - FLUORO - 2' - DEOXYURIDINE FOR USE IN THE TREATMENT OF CANCER
20230165886 · 2023-06-01
Assignee
Inventors
- Christopher McGuigan (Cardiff, GB)
- Jan Balzarini (Heverlee, BE)
- Magdalena Slusarczyk (Cardiff, GB)
- Blanka Gonczy (Cardiff, GB)
- Paola Murziani (Cardiff, GB)
Cpc classification
C07F9/65515
CHEMISTRY; METALLURGY
C07F9/65844
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07H19/10
CHEMISTRY; METALLURGY
C07F9/65586
CHEMISTRY; METALLURGY
A61K31/7072
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K31/7072
HUMAN NECESSITIES
C07F9/655
CHEMISTRY; METALLURGY
C07F9/6558
CHEMISTRY; METALLURGY
C07F9/6584
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
Phosphoramidate derivatives of 5-fluoro-2′-deoxyuridine are disclosed for use in the treatment of cancer, especially in the treatment of cancer where the patient shows resistance, for example, in a patient with cells with a lowered level of nucleoside transporter proteins and/or with nucleoside kinase-deficient cells and/or with mycoplasma-infected cells and/or with cells with a raised level of thymidylate synthase.
Claims
1. A method of treating cancer comprising administering intravenously to a human patient in need thereof an effective dose of 5-fluoro-2′-deoxyuridine-5′-O[1-naphthyl (benzoxy-L-alaninyl)] phosphate, or a pharmaceutically acceptable salt thereof; wherein the compound is provided in a sterile aqueous solution or suspension, or wherein the compound is presented as a liposome formulation; and wherein the cancer is responsive to 5-fluorouracil or 5-fluoro-2′-deoxyuridine.
2. The method of claim 1 wherein the cancer is selected from the group consisting of leukemia, pancreatic, prostate, lung, breast, and cervical cancer.
3. The method of claim 1 wherein the cancer is selected from the group consisting of esophageal; gastrointestinal, gastric, colon and rectum cancer; head and neck cancer; and ovarian cancer.
4. The method of claim 1 wherein the patient has developed or has the potential to develop resistance in tumor cells to the activity of 5-fluorouracil or 5-fluoro-2′-deoxyuridine.
5. The method of claim 1 wherein the patient has cells with a lowered than normal range of nucleoside transporter proteins.
6. The method of claim 1 wherein the patient has nucleoside kinase-deficient cells.
7. The method of claim 1 wherein the patient has mycoplasma-infected cells.
8. The method of claim 1 wherein the patient has cells with a raised level over the normal range of thymidylate synthase.
9. The method of claim 1 that circumvents susceptibility to nucleoside degradation by catabolic enzymes.
10. The method of claim 9 wherein the catabolic enzymes are selected from the group consisting of thymidine phosphorylase, uridine phosphorylase, and deoxycytidine deaminase.
Description
[0083] Examples of the present invention will now be described, by way of example only, with reference to the accompanying drawings comprising
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COMPOUND SYNTHESIS
[0104] With reference to
##STR00004##
##STR00005##
##STR00006##
[0105] Anhydrous solvents were obtained from Aldrich and used without further purification. All reactions were carried out under an argon atmosphere. Reactions were monitored with analytical TLC on Silica Gel 60-F254 precoated aluminium plates and visualised under UV (254 nm) and/or with .sup.31P NMR spectra. Column chromatography was performed on silica gel (35-70 μM). Proton (.sup.1H), carbon (.sup.13C), phosphorus (.sup.31P) and fluorine (.sup.19F) NMR spectra were recorded on a Bruker Avance 500 spectrometer at 25° C. Spectra were auto-calibrated to the deuterated solvent peak and all .sup.13C NMR and .sup.31P NMR were proton-decoupled. Analytical HPLC was conducted by Varian Prostar (LC Workstation-Varian prostar 335 LC detector) using Varian Polaris C18-A (10 μM) as an analytic column.
[0106] Low and High resolution mass spectra were performed as a service by Birmingham University, using electrospray (ES). CHN microanalysis was performed as a service by MEDAC Ltd., Surrey.
[0107] Standard Procedure A: Synthesis of Dichlorophosphate (2).
[0108] Phosphorus oxychloride (1.0 equiv) was added to a solution of 1-naphthol (1.0 equiv) in diethyl ether under argon atmosphere, then anhydrous triethylamine (1.0 equiv) was added dropwise at −78° C. and the resulting reaction mixture was stirred for 1 h. Subsequently the reaction mixture was allowed to slowly warm up to room temperature for 3 h. Formation of the desired compound was monitored by .sup.31P NMR. The resulting mixture was filtered and then evaporated in vacuo under nitrogen to afford the crude colourless oil as product, which was used without further purification in the next step.
[0109] Synthesis of 1-Naphthyl dichlorophosphate (2): Prepared according to Standard Procedure A, from 1-naphthol (3.00 g, 20.81 mmol), phosphorus oxychloride (1.94 mL, 20.81 mmol), triethylamine (2.9 mL, 20.81 mmol) and anhydrous diethyl ether (70 mL). After 1 h at −78° C. the reaction was left to rise to room temperature and stirred for 3 h. The crude product was obtained as an oil. The resulting mixture was filtered and then evaporated in vacuo, after purification by column chromatography eluting with hexane-EtOAc, (1:1) to afford a colorless oil (4.59 g, 84%) [R.sub.f=0.93 (hexane-EtOAc, 1:1)], .sup.31P NMR (202 MHz, CDCl.sub.3): δ.sub.P 5.07; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 7.52-7.71 (m, 4H, ArH), 7.86-7.89 (m, 1H, ArH), 7.95-7.98 (m, 1H, ArH), 8.16-8.19 (m, 1H, ArH).
[0110] Standard Procedure B: Synthesis of Phosphorochloridate (6).
[0111] A solution of aryl phosphorodichloridate (1.0 equiv.) and appropriate amino acid ester salt (1.0 equiv.) in dichloromethane under argon atmosphere was added dropwise to anhydrous triethyl amine (2.0 equiv.) at −78° C. After 1 h the reaction mixture was allowed to slowly warm to room temperature for 3 h and the formation of the desired compound was monitored by .sup.31P NMR. The reaction mixture was concentrated under reduced pressure, the residue was redissolved in diethyl ether, filtered and evaporated in vacuo under nitrogen to afford a crude colourless oil, which in some cases was used without further purification in the next step. The aryl phosphorochloridate synthesized was purified by column chromatography eluting with hexane-EtOAc, (7:3) to afford the title compound as a colorless oil.
[0112] Synthesis of 1-Naphthyl(benzyl-L-alaninyl) phosphorochloridate (6): The phosphorochloridate was prepared using 1-naphthyl dichlorophosphate (2.50 g, 9.57 mmol), alanine benzyl ester tosylate salt (3.36 g, 9.57 mmol), dry triethylamine (2.66 mL, 19.14 mmol) and dry dichloromethane (35.7 mL) according to the general procedure B. Purification by column chromatography eluting with hexane-EtOAc, (7:3) afforded the title compound as a colourless oil (1.82 g, 47%) [R.sub.f=0.90 (hexane-EtOAc, 7:3)], .sup.31P NMR (202 MHz, CDCl.sub.3, mixture of diastereoisomers): δ.sub.P 7.92, 8.14 (Int.: 1.00:1.00); .sup.1H NMR (500 MHz, CDCl.sub.3, mixture of diastereoisomers with a ratio of 1:1): δ.sub.H 1.42-1.45 (m, 3H, CHCH.sub.3), 4.20-4.23 (m, 11-1, CHCH.sub.3), 4.78-4.81 (m, 1H, NH), 5.09 (s, 2H, OCH.sub.2Ph), 7.09-7.73 (m, 11H, ArH), 7.97-8.12 (m, 1H, ArH).
[0113] Standard Procedure C: Synthesis of the Nucleoside Phosphoramidate (1).
[0114] A solution of the appropriate nucleoside (1.0 equiv.) in dry THF (10 mL) was added to NMI (5.0 equiv.) at room temperature under argon atmosphere. After 10 min the reaction mixture was added dropwise to a solution of phosphorochloridate (3.0 equiv) in anhydrous THF. The reaction was stirred at room temperature overnight and evaporated in vacuo. The oil obtained was dissolved in CH.sub.2Cl.sub.2, washed twice with H.sub.2O, then with HCl 0.5 M or in alternative the crude product was washed with diethyl ether. Then the crude product was purified by column chromatography on silica, eluting with CH.sub.2Cl.sub.2—MeOH as a gradient to afford the phosphoramidate.
Synthesis of 5-Fluoro-2′deoxyuridine-5′-O-[α-naphthyl (benzyl-L-alaninyl)] phosphate (1)
[0115] The phosphoramidate was prepared using 5-Fluoro-2′deoxyuridine (0.25 g, 1.01 mmol), NMI (0.40 mL, 5.07 mmol) and naphthyl(benzyl-L-alaninyl) phosphorochloridate (0.82 g, 3.04 mmol) according to the general procedure C. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (47.0 mg, 8%) [R.sub.f=0.19 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 636.1520. C.sub.29H.sub.29N.sub.3O.sub.9FNaP requires [MNa.sup.+], 636.1523); .sup.31P NMR (202 MHz, MeOD, mixture of diastereoisomers): δ.sub.P 4.24, 4.59; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.36, −167.18; NMR (500 MHz, MeOD): δ.sub.H 1.34-1.38 (m, 3H, CHCH.sub.3), 1.67-1.79 (m, 1H, H−2′), 2.08-2.17 (m, 1H, H−2′), 4.03-4.15 (m, 2H, CHCH.sub.3, H−4′), 4.24-4.36 (m, 3H, CH.sub.2OP, H−3′), 5.08 (d, 1H, J=12.0 Hz, OCHHPh), 5.13 (d, 1H, J=12.0 Hz, OCHHPh), 6.09-6.16 (m, 1H, H−1′), 7.27-7.45 (m, 6H, ArH); 7.47-7.55 (m, 3H, ArH), 7.67-7.72 (m, 2H, ArH, H−6), 7.86-7.90 (m, 1H, ArH), 8.12-8.18 (m, 114, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.3 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.3), 20.5 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 51.8 (CH), 51.9 (CH), 67.6 (d, .sup.2J.sub.C-P=5.3 Hz, CH.sub.2), 67.8 (d, .sup.2J.sub.C-P=5.2 Hz, CH.sub.2), 68.0 (CH.sub.2), 68.1 (CH.sub.2), 72.0 (CH), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=8.1 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.1 Hz, CH), 86.9 (CH), 87.0 (CH), 116.2 (d, .sup.3J.sub.C-P=3.3 Hz, CH), 116.5 (d, .sup.3J.sub.C-P==3.5 Hz, CH), 122.6 (CH), 125.3 (CH), 125.4 (CH), 125.6 (CH), 125.7 (CH), 126.2 (CH), 126.5 (CH), 126.6 (CH), 127.6 (CH), 127.7 (CH), 127.8 (C), 127.9 (C), 128.0 (CH), 128.1 (CH), 128.9 (CH), 129.0 (CH), 129.4 (CH), 129.5 (CH), 129.6 (CH), 129.7 (CH), 136.2 (C), 137.1 (C), 137.2 (C), 141.6 (d, .sup.1J.sub.C-F=233.8 Hz, C), 141.7 (d, .sup.1J.sub.C-F=233.9 Hz, C), 147.8 (d, .sup.2J.sub.C-P=7.7 Hz, C), 147.9 (d, .sup.2J.sub.C-P=7.4 Hz, C). 150.5 (d, .sup.4J.sub.C-F=4.0 Hz, C), 159.3 (d, .sup.2J.sub.C-F=26.1 Hz, C), 174.6 (d, .sup.3J.sub.C-P=5.0 Hz, C), 174.9 (d, .sup.3J.sub.C-P=4.3 Hz, C), m/z (ES) 636 (MH.sup.+, 100%), Reverse HPLC eluting with (H.sub.2O/MeOH from 100/0 to 0/100) in 45 min., showed two peaks of the diastereoisomers with t.sub.R 34.23 min, and t.sub.R 34.59 min. Anal. Calcd for C.sub.29H.sub.29FN.sub.3O.sub.9P: C, 56.77; H, 4.76; N, 6.85. Found: C, 56.57; H, 5.06; N, 6.72.
[0116] Radioactive Pyrimidine Deoxynucleosides
[0117] [5-.sup.3H]dCyd (radiospecificity: 22 Ci/mmol) and [5-.sup.3H]dUrd (radiospecificity: 15.9 Ci/mmol) were obtained from Moravek Biochemicals Inc. (Brea, Calif.).
[0118] Standard Procedure D: Synthesis of Phosphoramidates (NMI Method)
[0119] To a stirring solution of 5-F-dUrd (1.0 eq.) in anhydrous THF, an appropriate phosphorochloridate (3.0 eq.) dissolved in anhydrous THE was added dropwise under an Ar atmosphere. To that reaction mixture at −78° C. was added dropwise over 5 minutes NMI (5.0 eq.). After 15 minutes, the reaction mixture was let to rise to room temperature and stirred overnight. The solvent was removed under vacuum and the residue was re-dissolved in DCM and washed with 0.5 M HCl three times. The organic layer was dried over MgSO.sub.4, filtered, reduced to dryness and purified by column chromatography with gradient of eluent (DCM/MeOH 99:1 to 97:3 to 95:5).
[0120] Standard Procedure E: Synthesis of Phosphoramidates (tBuMgCl Method)
[0121] To a stirring solution of 5-FdUrd (1.0 eq.) dissolved in anhydrous THF, tBuMgCl (1.1 mol eq. 1M solution in THE) was added dropwise under an Ar atmosphere, followed by addition (after 30 min.) of the appropriate phosphorochloridate (2.0 mol eq.) dissolved in anhydrous THF. The resulting reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography using gradient of eluent (DCM/MeOH 99:1 to 97:3 to 95:5)
5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(benzoxy-L-alaninyl)] phosphate (CPF381)
[0122] ##STR00007##
[0123] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g, 1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (.sup.tBuMgCl) (1.0 M, 2.43 mL, 2.43 mmol) and phenyl(benzoxy-L-alaninyl) phosphorochloridate (1.08 g, 3.20 mmol) according to general procedure E. Purification by gradient column chromatography on silica, eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (71.0 mg, 8%) [R.sub.f=0.35 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 586.1360. C.sub.25H.sub.27N.sub.3O.sub.9NaPF requires [MNa.sup.+], 586.1367); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.74, 4.14; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.57, −167.46; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.35 (d, 3H, J=7.4 Hz, CHCH.sub.3, one diast.), 1.37 (d, 3H, J=6.9 Hz, CHCH.sub.3, one diast.), 1.96-2.32 (m, 2H, H−2′), 3.95-4.08 (m, 2H, CHCH.sub.3, H−4′), 4.23-4.34 (m, 3H, CH.sub.2OP, H−3′), 5.13 (br d, 1H, J=12.3 Hz, OCHHPh), 5.16 (br d, 1H, J=12.3 Hz, OCHHPh, one diast.), 5.17 (br d, 1H, J=12.2 Hz, OCHHPh, one diast.), 6.16-6.22 (m, 1H, H−1′), 7.17-7.25 (m, 3H, ArH), 7.26-7.40 (m, 7H, ArH), 7.81-7.85 (m, 1H, H−6); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.2 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3), 20.4 (d, .sup.3J.sub.C-P=6.2 Hz, CH.sub.3), 40.6 (CH.sub.2), 40.9 (CH.sub.2), 51.6 (CH), 51.8 (CH), 67.5 (d, .sup.2J.sub.C-P=5.3 Hz, CH.sub.2), 67.6 (d, .sup.2J.sub.C-P=5.5 Hz, CH.sub.2), 68.0 (CH.sub.2), 71.8 (CH), 71.9 (CH), 86.6 (d, .sup.3J.sub.C-P=8.0 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.3 Hz, CH), 86.9 (CH), 87.0 (CH), 121.4 (d, .sup.3J.sub.C-P=5.1 Hz, CH), 121.5 (d, 5.6 Hz, CH), 125.5 (d, .sup.5J.sub.C-P=3.2 Hz, CH), 125.8 (d, .sup.5J.sub.C-P=3.2 Hz, CH), 126.3 (CR), 129.0 (CH×2), 129.3 (CH×2), 129.6 (CH×2), 130.8 (CH×2), 140.9 (C), 141.6 (d, .sup.1J.sub.C-F=233.6 Hz, C), 141.7 (d, .sup.1J.sub.C-F=233.6 Hz, C), 150.7 (d, .sup.4J.sub.C-F=5.7 Hz, C), 152.1 (d, .sup.2J.sub.C-F=6.5 Hz, C), 159.2 (d, .sup.2J.sub.C-F=26.3 Hz, C), 174.6 (d, .sup.3J.sub.C-P=4.9 Hz, C), 174.7 (d, .sup.3J.sub.C-P=4.9 Hz, C), m/z (ES) 586 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed one peak of the mixture of diastereoisomers with t.sub.R 25.08 min. (97%).
5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(methoxy-L-alaninyl)] phosphate (CPF382) (Reference Example)
[0124] ##STR00008##
[0125] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and phenyl(methoxy-L-alaninyl) phosphorochloridate (0.84 g; 3.04 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (16.0 mg, 4%) [R.sub.f=0.30 (CH.sub.2Cl.sub.2—MeOH, 95:5)]; (Found: MNa.sup.+, 510.1045. C.sub.19H.sub.23N.sub.3O.sub.9NaPF requires [MNa.sup.+], 510.1054); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.79, 4.09; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.78, −167.72; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.34 (d, 3H, J=7.1 Hz, CHCH.sub.3, one diast.), 1.36 (d, 3H, J=7.1 Hz, CHCH.sub.3, one diast.), 2.02-2.16 (m, 1H, H−2′). 2.25-2.34 (m, H−2′), 3.69 (s, 3H, OCH.sub.3, one diast.), 3.70 (s, 31-1, OCH.sub.3, one diast.), 3.93-4.02 (m, 1H, CHCH.sub.3), 4.08-4.13 (m, 1H, H−4′), 4.27-4.45 (m, 3H, CH.sub.2OP, H−3′), 6.20-6.29 (m, 1H, H−1′), 7.18-7.28 (m, 3H, ArH), 7.35-7.40 (m, 2H, ArH), 7.85 (d, 1H, .sup.3J.sub.C-F=6.4 Hz, H−6); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.2 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3); 20.5 (d, 6.7 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 51.5 (CH.sub.3), 51.6 (CH.sub.3), 52.7 (CH), 52.8 (CH), 67.5 (d, .sup.2J.sub.C-P=5.5 Hz, CH.sub.2), 67.6 (d, .sup.2J.sub.C-P=5.1 Hz; CH.sub.2), 72.0 (CR), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=8.2 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.2 Hz, CH), 86.9 (CH), 87.0 (CH), 121.2 (d, .sup.3J.sub.C-P=4.5 Hz, CH), 121.4 (d, .sup.3J.sub.C-P=4.7 Hz, CH). 125.6 (d, .sup.5J.sub.C-P=2.9 Hz, CH), 125.9 (d, 2.9 Hz, CH), 126.2 (CH), 130.8 (CH), 130.9 (CH), 141.6 (d, .sup.1J.sub.C-F=233.8 Hz, C), 141.7 (d, .sup.1J.sub.C-F=233.9 Hz, C), 150.6 (d, .sup.4J.sub.C-F=3.6 Hz, C), 152.1 (d, .sup.2J.sub.C-P=6.8 Hz, C), 152.2 (d, .sup.2J.sub.C-F=6.8 Hz, C), 159.4 (d, .sup.2J.sub.C-F=26.0 Hz, C), 175.2 (d, .sup.3J.sub.C-P=4.8 Hz, C), 175.5 (d, .sup.3J.sub.C-P=3.7 Hz, C), m/z (ES) 510 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes; 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 23.11 min. and t.sub.R 24.11 min, (74%:24%).
5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(ethoxy-L-alaninyl)] phosphate (CPF383)
[0126] ##STR00009##
[0127] The phosphoramidate was prepared using 5-fluoro-2′deoxyuridine (0.10 g, 0.40 mmol), N-methylimidazole (Mill) (0.16 mL, 2.03 mmol) and phenyl(ethoxy-L-alaninyl) phosphorochloridate (0.35 g, 1.21 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (10.0 mg, 5%) [R.sub.f=0.11 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 524.1202. C.sub.20H.sub.25N.sub.3O.sub.9NaPF requires [MNa.sup.+], 524.1210); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.83, 4.11; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.67, −167.61; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.25 (t, 3H, J=7.1 Hz, CH.sub.2CH.sub.3, one diast.), 1.26 (t, 3H, J=7.1 Hz, CH.sub.2CH.sub.3, one diast.), 1.34 (d, 3H, J=7.2 Hz, CHCH.sub.3, one diast.), 1.36 (d, 3H, 7.2 Hz, CHCH.sub.3, one diast.), 2.02-2.15 (m, 1H, H−2′), 2.24-2.34 (m, 1H, H−2′), 3.90-4.00 (m, 1H, JHCH.sub.3), 4.08-4.19 (m, 3H, CH.sub.2CH.sub.3, H−4′), 4.27-4.45 (m, 3H, CH.sub.2OP, H−3′), 6.20-6.28 (m, 1H, H−1′), 7.18-7.28 (m, 3H, ArH), 7.34-7.39 (m, 2H, ArH), 7.85 (d, 1H, .sup.3J.sub.H-F=6.4 Hz, H−6); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 14.4 (CH.sub.3), 15.4 (CH.sub.3), 20.3 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.3), 20.5 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 51.6 (CH), 51.7 (CH), 62.4 (CH.sub.2), 62.5 (CH.sub.2), 67.5 (d, 5.4 Hz, CH.sub.2), 67.6 (d, .sup.2J.sub.C-P=5.4 Hz, CH.sub.2), 72.0 (CH), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=8.1 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.3 Hz, CH), 86.9 (CH), 87.0 (CH), 121.3 (d, .sup.3J.sub.C-P=4.8 Hz, CH), 121.4 (d, .sup.3J.sub.C-P=4.6 Hz, CH), 125.6 (d, .sup.5J.sub.C-P=4.6 Hz, CH), 125.8 (d, .sup.5J.sub.C-P=4.8 Hz, CH), 126.3 (CH), 130.8 (CH), 130.9 (CH), 141.6 (d, .sup.1J.sub.C-P=233.7 Hz, C), 141.8 (d, .sup.1J.sub.C-P=233.8 Hz, C), 150.8 (br C), 152.0 (d, .sup.2J.sub.C-P=7.1 Hz, C), 152.1 (d, .sup.2J.sub.C-P=7.1 Hz, C), 159.6 (d, .sup.2J.sub.C-P=26.0 Hz, C), 174.8 (d, .sup.3J.sub.C-P=5.4 Hz, C), 175.1 (d, .sup.3J.sub.C-P=4.4 Hz, C), m/z (ES) 524 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 25.63 min. and t.sub.R 26.40 min. (71%:27%).
5-Fluoro-2′deoxyuridine-5′-O-[phenyl(isopropoxy-L-alaninyl)] phosphate (CPF384)
[0128] ##STR00010##
[0129] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and phenyl(isopropoxy-L-alaninyl) phosphorochloridate (0.93 g, 3.04 mmol) according to general procedure D. Purification by, gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2-MeOH (95:5) afforded the title compound as a colourless solid (31.0 mg, 6%) [R.sub.f=0.21 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 538.1370. C.sub.21H.sub.27N.sub.3O.sub.9NaPF requires [MNa.sup.+], 538.1367); .sup.31P NMR (202 MHZ, MeOD): δ.sub.P 3.87, 4.13; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.64, −167.56; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.22-1.26 (m, 6H, CH(CH.sub.3).sub.2), 1.33 (d, 3H, J=7.1 Hz, CHCH.sub.3, one diast.), 1.35 (d, 3H, 7.1 Hz, CHCH.sub.3, one diast.), 2.00-2.15 (m, 1H, H−2′), 2.23-2.34 (m, 1H, H−2′), 3.88-3.96 (m, 1H, CHCH.sub.3), 4.08-4.14 (m, 1H, H−4′), 4.27-4.45 (m, 3H, CH.sub.2OP, H−3′), 4.98 (hept, 1H, J=6.1 Hz, CH(CH.sub.3).sub.2), 6.20-6.29 (m, 1H, H−1′), 7.17-7.29 (m, 3H, Ar—H), 7.34-7.40 (m, 2H, Ar—H), 7.84 (d, 1H, .sup.3J.sub.H-F=6.4 Hz, H−6); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.3 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.3), 20.5 (d, .sup.3J.sub.C-P=6.4 Hz, CH.sub.3), 21.9 (CH.sub.3×2), 22.0 (CH.sub.3×2), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 51.7 (CH), 51.8 (CH), 67.5 (d, .sup.2J.sub.C-P=5.4 Hz, CH.sub.2), 67.6 (d, .sup.2J.sub.C-P=5.2 Hz, CH.sub.2), 70.2 (CH), 70.3 (CH), 72.0 (CH), 72.1 (CH), 86.6 (d, .sup.3J.sub.C-P=8.2 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.2 Hz, CH), 86.9 (CH), 87.0 (CH), 121.2 (d, .sup.3J.sub.C-P=4.7 Hz, CH), 121.4 (d, .sup.3J.sub.C-P=4.9 Hz, CH), 125.6 (d, .sup.5J.sub.C-P=7.1 Hz, CH), 125.9 (d, .sup.5J.sub.C-P=7.1 Hz, CH), 126.3 (CH), 130.8 (CH), 130.9 (CH), 141.8 (d, .sup.3J.sub.C-P=234.5 Hz, C), 141.9 (d, .sup.1J.sub.C-F=234.4 Hz, C), 150.7 (d, .sup.4J.sub.C-F=3.7 Hz, C), 152.0 (d, .sup.3J.sub.C-P=6.2 Hz, C), 152.1 (d, .sup.3J.sub.C-P=6.2 Hz, C), 159.3 (d, .sup.2J.sub.C-F=26.3 Hz, C), 159.4 (d, .sup.2J.sub.C-F=26.0 Hz, C), 174.3 (d, .sup.3J.sub.C-P=5.6 Hz, 0.3), 174.6 (d, .sup.3J.sub.C-P=4.6 Hz, C), m/z (ES) 538 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with to 28.93 min, and t.sub.R 29.45 min, (44%:52%).
5-Fluoro-2′deoxyuridine-5′-O-[phenyl (cyclohexoxy-L-alaninyl)] phosphate (CPF508
[0130] ##STR00011##
[0131] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.30 g, 1.21 mmol), N-methylimidazole (NMI) (0.48 mL, 6.09 mmol) and phenyl(cyclohexoxy-L-alaninyl) phosphorochloridate (1.02.6 g, 3.65 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2-MeOH (95:5) afforded the title compound as a colourless solid (6.7 mg, 3%) [R.sub.f=0.45 (CH.sub.2Cl.sub.2—MeOH, 95:5)]; (Found: MNa.sup.+, 565.48. C.sub.24H.sub.31N.sub.3O.sub.9NaPF requires [MNa.sup.+], 565.49); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.86, 4.15; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.68, −167.62; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.26-1.40 (m, 3H, CHCH.sub.3), 1.41-1.50 (m, 4H, CH(CH.sub.2).sub.5), 1.52-1.61 (m, 1H, CH(CH.sub.2).sub.5), 1.70-1.88 (m, 5H, CH(CH.sub.2).sub.5), 2.00-2.14 (m, 1H, H−2′), 2.23-2.34 (m, 1H, H−2′), 3.90-3.98 (m, 1H, CHCH.sub.3), 4.07-4.14 (m, 1H, H−4′), 4.29-4.39 (m, 2H, CH.sub.2OP), 4.40-4.45 (m, 1H, H−3′), 4.72-4.78 (m, 1H, CH(CH.sub.2).sub.5), 6.20-6.28 (m, 1H, H−1′), 7.18-7.29 (m, 3H, ArH), 7.34-7.39 (m, 2H, ArH), 7.85 (d, 1H, .sup.3J.sub.H-F=6.6 Hz, H−6); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.3 (d, .sup.3J.sub.C-P=7.3 Hz, CH.sub.3), 20.6 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3), 24.6 (CH.sub.2), 26.4 (CH.sub.2), 32.3 (CH.sub.2), 32.4 (CH.sub.2), 40.9 (CH.sub.2), 51.7 (CH), 51.9 (CH), 67.5 (d, .sup.2J.sub.C-P=5.3 Hz, CH.sub.2), 67.7 (d, .sup.2J.sub.C-P=5.3 Hz, CH.sub.2), 72.0 (CH), 72.1 (CH), 74.9 (CH), 86.6 (d, .sup.3J.sub.C-P=8.5 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.5 Hz, CH), 86.9 (CH), 87.0 (CH), 121.3 (CH), 121.4 (CH), 121.5 (CH), 121.6 (CH), 125.6 (CH), 125.7 (CH), 125.8 (CH), 125.9 (CH), 126.3 (CH), 130.1 (CH), 141.5 (d, .sup.1J.sub.C-F=234.0 Hz, C), 150.7 (d, .sup.4J.sub.C-P=4.0 Hz, C), 152.0 (d, .sup.2J.sub.C-P=7.2 Hz, C), 152.1 (d, .sup.2J.sub.C-P=7.2 Hz, C), 159.4 (d, .sup.2J.sub.C-F26.3 Hz, C). 174.3 (d, .sup.3J.sub.C-P=4.6 Hz, C), 174.5 (d, .sup.3J.sub.C-P=4.3 Hz, C); m/z (ES) 565 (MNa.sup.4, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 30.00 min. and t.sub.R 30.45 min. (33%:65%).
5-Fluoro-2′deoxyuridine-5′-O-[p-nitro-phenyl(ethoxy-L-alaninyl)] phosphate (CPF430)
[0132] ##STR00012##
[0133] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and p-nitro-phenyl(ethoxy-L-alaninyl) phosphorochloridate (1.02 g, 3.04 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (77.0 mg, 14%) [R.sub.f=0.24 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 569.1066. C.sub.20H.sub.24N.sub.4O.sub.11NaPF requires [MNa.sup.+], 569.1061); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.63, 3.67; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.89, −167.82; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.24 (t, 3H, J=7.0 Hz, CH.sub.2CH.sub.3), 1.25 (t, 3H, J=7.0 Hz, CH.sub.2CH.sub.3), 1.36-1.40 (m, 3H, CHCH.sub.3), 2.16-2.25 (m, 1H, H−2′), 2.30-2.38 (m, 1H, H−2′), 3.95-4.00 (m, 1H, CHCH.sub.3), 4.09-4.19 (m, 3H, CH.sub.2CH.sub.3, 4.32-4.48 (m, 3H, CH.sub.2OP, H−3′), 6.21-6.29 (m, 1H, H−1′), 7.46 (d, 1H, J=8.7 Hz, ArH), 7.49 (d, 1H, J=8.7 Hz, ArH), 7.85 (d, 1H, .sup.3J.sub.H-F=6.6 Hz, H−6), 7.87 (d, 1H, .sup.3J.sub.H-F=6.6 Hz, H−6), 8.2.9 (d, 2H, J=8.7 Hz, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 14.5 (CH.sub.3), 14.6 (CH.sub.3), 20.3 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3), 20.4 (d, .sup.3J.sub.C-P=6.4 Hz, CH.sub.3), 40.8 (CH.sub.2), 51.6 (CH.sub.3), 51.7 (CH), 62.5 (CH.sub.2), 67.8 (d, .sup.2J.sub.C-P=5.5 Hz, CH.sub.2), 68.0 (d, .sup.2J.sub.C-P=5.2 Hz, CH.sub.2), 71.8 (CH×2), 86.4 (CH), 86.5 (CH), 87.0 (d, .sup.3J.sub.C-P=7.5 Hz, CH), 122.1 (d, 5.2 Hz, CH), 122.5 (d, .sup.3J.sub.C-P=5.0 Hz, CH), 125.7 (CH), 126.0 (CH), 126.6 (CH), 141.3 (d, .sup.1J.sub.C-F=233.6 Hz, C), 141.5 (d, .sup.1J.sub.C-F=233.7 Hz, C), 146.2 (C), 150.6 (d, .sup.4J.sub.C-P=4.6 Hz, C), 156.9 (d, .sup.2J.sub.C-P=2.6 Hz, C), 157.0 (d, .sup.2J.sub.C-P=2.6 Hz, C), 159.3 (d, .sup.2J.sub.C-F=26.3 Hz, C), 174.6 (d, .sup.3J.sub.C-P=4.6 Hz, C), 174.9 (d, .sup.3J.sub.C-P=3.7 Hz, C), m/z (ES) 569 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 min., 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 31.63 min. and t.sub.R 31.89 min. (11%:85%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (benzoxy-L-alaninyl)] phosphate (CPF373)
[0134] ##STR00013##
[0135] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and 1-naphthyl(benzoxy-L-alaninyl) phosphorochloridate (0.82 g, 3.04 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (47.0 mg, 8%) [R.sub.f=0.19 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 636.1520. C.sub.29H.sub.29N.sub.3O.sub.9NaPF requires [MNa.sup.+], 636.1523); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 4.24, 4.59; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.36, −167.18; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.34-1.38 (m, 3H, CHCH.sub.3), 1.67-1.79 (m, 1H, H−2′), 2.08-2.17 (m, 1H, H−2′), 4.03-4.15 (m, 2H, CHCH.sub.3, H−4′), 4.24-4.36 (m, 3H, CH.sub.2OP, H−3′), 5.08 (d, 1H, 12.0 Hz, OCHHPh), 5.13 (d, 1H, J=12.0 Hz, OCHHPh), 6.09-6.16 (m, 1H, H−1′), 7.27-7.45 (m, 6H, ArH), 7.47-7.55 (m, 3H, ArH), 7.67-7.72 (m, 2H, ArH, H−6), 7.86-7.90 (m, 1H, ArH), 8.12-8.18 (m, 1H, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.3 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.3), 20.5 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 51.8 (CH), 51.9 (CH), 67.6 (d, .sup.2J.sub.C-P=5.3 Hz, CH.sub.2), 67.8 (d, .sup.2J.sub.C-P==5.2 Hz, CH.sub.2), 68.0 (CH.sub.2), 68.1 (CH.sub.2), 72.0 (CH), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=8.1 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.1 Hz, CM, 86.9 (CH), 87.0 (CH), 116.2 (d, .sup.3J.sub.C-P=3.3 Hz, CH), 116.5 (d, .sup.3J.sub.C-P=3.5 Hz, CM, 122.6 (CH), 125.3 (CH), 125.4 (CH), 125.6 (CH), 125.7 (CH), 126.2 (CH), 126.5 (CH), 126.6 (CH), 127.6 (CH), 127.7 (CH), 127.8 (C), 127.9 (C), 128.0 (CH), 128.1 (CH), 128.9 (CH), 129.0 (CH), 129.4 (CH), 129.5 (CH), 129.6 (CH), 129.7 (CH), 136.2 (C), 137.1 (C), 137.2 (C), 141.6 (d, .sup.1J.sub.C-F=233.8 Hz, C), 141.7 (d, .sup.1J.sub.C-F=233.9 Hz, C), 147.8 (d, .sup.2J.sub.C-P==7.7 Hz, C), 147.9 (d, .sup.2J.sub.C-P=7.4 Hz, C), 150.5 (d, .sup.4J.sub.C-F=4.0 Hz, C), 159.3 (d, .sup.2J.sub.C-F=26.1 Hz, C), 174.6 (d, .sup.3J.sub.C-P=5.0 Hz, C), 174.9 (d, .sup.3J.sub.C-P=4.3 Hz, C), m/z (ES) 636 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, 275 nm, showed two peaks of the diastereoisomers with t.sub.R 34.23 min. and t.sub.R 34.59 min. (23%:76%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (methoxy-L-alaninyl)] phosphate (CPF385)
[0136] ##STR00014##
[0137] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and 1-naphthyl(methoxy-L-alaninyl) phosphorochloridate (0.99 g, 3.04 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (7.0 mg, 1%) [R.sub.f=0.23 (C.sub.12C.sub.12—MeOH, 95:5)], (Found: MNa.sup.+, 560.1198. C.sub.23H.sub.25N.sub.3O.sub.9NaPF requires [MNa.sup.+], 560.1210); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 4.31, 4.56; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.51, −167.37; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.34 (d, 3H, J=6.7 Hz, CHCH.sub.3, one diast.), 1.36 (d, 3H, J=6.7 Hz, CHCH.sub.3, one diast.), 1.76-1.87 (m, 1H, H−2′), 2.12-2.22 (m, 1H, H−2′), 3.64 (s, 3H, OCH.sub.3, one diast.), 3.65 (s, 3H, OCH.sub.3, one diast.), 4.03-4.13 (m, 2H, CHCH.sub.3, H−4′), 4.30-4.38 (m, 2H, CH.sub.2OP), 4.41 (dd, 1H, 2.5 Hz, J=5.8 Hz, 11-3′), 6.12-6.19 (m, 1H, 1H′), 7.41-7.46 (m, 1H, ArH), 7.50-7.58 (m, 3H, ArH), 7.70-7.76 (m, 2H, H−6, ArH), 7.87-7.91 (m, 1H, ArH), 8.15-8.20 (m, 1H, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.3 (d, .sup.3J.sub.C-P=7.1 Hz, CH.sub.3), 20.4 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3), 40.7 (CH.sub.2), 40.8 (CH.sub.2), 51.6 (CH.sub.3), 51.7 (CH.sub.3), 52.7 (CH), 52.8 (CH), 67.8 (d, .sup.2J.sub.C-P=5.7 Hz, CH.sub.2), 67.5 (d, .sup.2J.sub.C-P=5.7 Hz, CH.sub.2), 72.0 (CH), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=7.9 Hz, CH), 86.9 (d, .sup.3J.sub.C-P=8.5 Hz, CH), 86.9 (CH), 87.0 (CH), 116.2 (d, .sup.3J.sub.C-P=3.1 Hz, CH), 116.5 (d, .sup.3J.sub.C-P=3.5 Hz, CH), 122.5 (cm, 122.6 (CR), 125.4 (CH), 125.5 (CH), 125.6 (cm, 125.7 (CH), 126.1 (CH), 126.2 (CH), 126.5 (CH), 126.6 (CH), 127.6 (CH), 127.7 (C×2). 127.8 (CH), 127.9 (CH), 128.9 (CH), 129.0 (CH), 136.3 (C), 141.6 (d, .sup.1J.sub.C-F=233.4 Hz, C), 141.7 (d, .sup.1J.sub.C-F=234.1 Hz, C), 147.8 (d, .sup.2J.sub.C-P=7.9 Hz, C), 148.0 (d, .sup.2J.sub.C-P=7.2 Hz, C). 150.6 (C), 159.4 (d, .sup.2J.sub.C-F=27.0 Hz, C). 175.2 (d, .sup.3J.sub.C-P=3.9 Hz, C), 175.5 (d, .sup.3J.sub.C-P=3.9 Hz, C), m/z (ES) 560 (Ma.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 28.45 min, and t.sub.R 28.85 min, (73%:25%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (ethoxy-L-alaninyl)] phosphate (CPF386)
[0138] ##STR00015##
[0139] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), triethylimidazole (NMI) (0.40 mL, 5.07 mmol) and 1-naphthyl(ethoxy-L-alaninyl) phosphorochloridate (1.04 g, 3.04 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (47.0 mg, 4%) [R.sub.f=0.25 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 574.1360. C.sub.24H.sub.2-7N.sub.3O.sub.9NaPF requires [MNa.sup.+], 574.1367); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 4.34, 4.55, .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.31, −167.16; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.20 (t, 3H, J=7.0 Hz, CH.sub.2CH.sub.3, one diast.), 1.21 (t, 3H, J=7.0 Hz, CH.sub.2CH.sub.3, one diast.), 1.33-1.37 (m, 3H, CHCH.sub.3), 1.73-1.86 (m, 1H, H−2′), 2.12-2.21 (m, 1H, H−2′), 4.01-4.07 (m, 1H, CHCH.sub.3), 4.08-4.13 (m, 3H, CH.sub.2CH.sub.3, H−4′), 4.31-4.43 (n, 3H, CH.sub.2OP, H−3′), 6.11-6.19 (m, H−1′), 7.39-7.46 (m, 1H, ArH), 7.50-7.57 (n, 3H, ArH), 7.68-7.75 (m, 2H, ArH, H−6), 7.86-7.91 (m, 1H, ArH), 8.15-8.20 (m, 1H, ArH); NMR (125 MHz, MeOD): δ.sub.C 14.4 (CH.sub.3), 20.3 (d, .sup.3J.sub.C-P=7.4 Hz, CH.sub.3), 20.5 (d, .sup.3J.sub.C-P=6.2 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.3), 51.8 (CH), 51.9 (CH), 62.4 (CH.sub.2), 62.5 (CH.sub.2), 67.8 (d, .sup.2J.sub.C-P=4.6 Hz, CH.sub.2), 67.9 (d, .sup.2J.sub.C-P=4.6 Hz, CE1.sub.2), 72.0 (CH), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=8.4 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=8.4 Hz, CH), 86.9 (CH), 87.0 (CH), 116.1 (d, .sup.3J.sub.C-P=3.5 Hz, CH), 116.5 (d, .sup.3J.sub.C-P=3.5 Hz, CH), 122.6 (CH), 125.4 (CH), 125.5 (CH), 125.7 (CH), 125.8 (CH), 126.1 (CH), 126.2 (CH), 126.5 (CH), 126.6 (CH), 127.5 (CH), 127.6 (C), 127.7 (C), 127.8 (CH), 127.9 (CH), 128.9 (CH), 129.0 (CH), 136.3 (C), 141.6 (d, 233.3 Hz, C), 141.7 (d, 233.4 Hz, C), 147.8 (d, .sup.2J.sub.C-P6.9 Hz, C), 148.0 (d, .sup.2J.sub.C-P=6.9 Hz, C), 150.6 (C), 159.3 (d, .sup.2J.sub.C-F=26.3 Hz, C), 174.8 (d, .sup.3J.sub.C-P=4.8 Hz, C), 175.1 (d, .sup.3J.sub.C-P=4.0 Hz, C); m/z (ES) 574 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 30.77 min. and t.sub.R 31.20 min. (51%:48%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (isopropoxy-L-alaninyl)] phosphate (CPF387)
[0140] ##STR00016##
[0141] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.10 g, 0.40 mmol), tert-butylmagnesium chloride in tetrahydrofuran (.sup.tBuMgCl) (1.0 M, 0.61 mL, 0.61 mmol) and 1-naphthyl(isopropoxy-L-alaninyl) phosphorochloridate (0.31 g, 0.89 mmol) according to general procedure E. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH, (95:5) afforded the title compound as a colourless solid (71.0 mg, 17%) [R.sub.f=0.21 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 588.1521. C.sub.25H.sub.29N.sub.3O.sub.9NaPF requires [MNa.sup.+], 588.1523); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 4.38, 4.58; .sup.19F NMR (470 MHZ, MeOD): δ.sub.F −167.43, −167.26; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.19-1.23 (m, 6H, CH(CH.sub.3).sub.2), 1.34-138 (m, 3H, CHCH.sub.3), 1.68-1.84 (m, 1H, H−2′), 2.09-2.20 (m, 1H, H−2′), 3.96-4.05 (m, CHCH.sub.3), 4.07-4.12 (m, 1H, H−4′), 4.29-4.38 (m, 2H, CH.sub.2OP), 4.39-4.42 (m, 1H, H−3′), 4.93-5.01 (m, 1H, CH(CH.sub.3).sub.2), 5.10-6.18 (m, H−1′), 7.40-7.46 (m, 1H, ArH), 7.50-7.57 (m, 3H, ArH), 7.70-7.75 (m, 2H, H−6, ArH), 7.87-7.92 (m, 1H, ArH), 8.16-8.20 (m, 1H, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C, 20.3 (d, 7.1 Hz, CH.sub.3), 20.5 (d, .sup.3J.sub.C-P=6.6 Hz, CH.sub.3), 21.8 (CH.sub.3), 21.9 (CH.sub.3), 22.0 (CH.sub.3), 22.1 (CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 51.9 (CH), 52.0 (CH), 67.8 (d, .sup.2J.sub.C-P=4.5 Hz, CH.sub.2), 67.9 (d, .sup.2J.sub.C-P=4.8 Hz, CH.sub.2), 70.2 (CH), 70.3 (CH), 72.0 (CH), 72.1 (CH), 86.6 (CH), 86.7 (CH), 86.9 (d, .sup.3J.sub.C-P=8.6 Hz, CH), 87.0 (d, .sup.3J.sub.C-P=8.6 Hz, CH), 116.2 (d, .sup.3J.sub.C-P=2.5 Hz, CH), 116.5 (d, .sup.3J.sub.C-P=2.7 Hz, CH), 122.6 (CH), 125.5 (CH), 125.7 (CH), 126.1 (CH), 126.2 (CH), 126.5 (CH), 127.5 (CH), 127.6 (C), 127.7 (C), 127.8 (CH), 127.9 (CH), 128.9 (CH), 129.0 (CH), 136.3 (C), 141.6 (d, .sup.1J.sub.C-F=233.2 Hz, C), 141.7 (d, .sup.1J.sub.C-F=233.4 Hz, C), 147.7 (d, .sup.2J.sub.C-P=7.6 Hz, C), 147.9 (d, .sup.2J.sub.C-P=7.7 Hz, C), 150.5 (C), 159.4 (d, .sup.2J.sub.C-F=26.2 Hz, C), 174.4 (d, .sup.3J.sub.C-P=5.0 Hz, C), 174.7 (d, .sup.3J.sub.C-P=5.1 Hz, C); m/z (ES) 588 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 32.20 min. and t.sub.R 32.80 min. (27% 69%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (cyclohexoxy-L-alaninyl)] phosphate (CPF509)
[0142] ##STR00017##
[0143] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.30 g, 1.21 mmol), N-methylimidazole (NMI) (0.48 mL, 6.09 mmol) and phenyl(cyclohexoxy-L-alaninyl) phosphorochloridate (1.45 g, 3.65 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (9:5:5) afforded the title compound as a colourless solid (6.7 mg, 3%) [R.sub.f=0.47 (CH.sub.2Cl.sub.2—MeOH, 95:5)]; (Found: MNH.sub.4.sup.+, 623.2261. C.sub.28H.sub.37N.sub.4O.sub.9NaPF requires [MNH.sub.4.sup.+], 623.2282); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 4.35, 4.52; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.31, −167.17; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.30-1.43 (m, 3H, CHCH.sub.3), 1.44-1.56 (m, 4H, CH(CH.sub.2).sub.5), 1.57-1.66 (m, 1H, CH(CH.sub.2).sub.5), 1.67-1.83 (m, 5H, CH(CH.sub.2).sub.5), 1.84-1.93 (m, 1H, H−2′), 2.09-2.20 (m, 1H, H−2′), 3.98-4.06 (m, 1H, CHCH.sub.3), 4.07-4.15 (m, 1H, H−4′), 4.29-4.38 (m, 2H, CH.sub.2OP), 4.39-4.44 (m, 1H, H−3′), 4.67-4.76 (m, 1H, CH(CH.sub.2).sub.5). 6.09-6.19 (m, 1H, H−1′), 7.38-7.57 (m, 5H, ArH), 7.68-7.75 (m, 1H, ArH), 7.79-7.92 (m, 1H, ArH), 8.17 (d, 1H, .sup.3J.sub.H-F=6.6 Hz, H−6); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 20.4 (d, .sup.3J.sub.C-P=8.0 Hz, CH.sub.3), 20.6 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3), 24.5 (CH.sub.2), 26.3 (CH.sub.2), 32.3 (CH.sub.2), 40.8 (CH.sub.2), 51.8 (CH), 51.9 (CH), 67.8 (CH.sub.2), 72.0 (CH), 72.2 (CH), 75.0 (CH), 86.7 (d, .sup.3J.sub.C-P=8.2 Hz, CH), 87.0 (CH), 116.1 (d, .sup.3J.sub.C-P=2.5 Hz, CH), 116.4 (d, .sup.3J.sub.C-P=3.0 Hz, CH), 122.6 (CH), 124.8 (CH), 125.9 (C H), 126.1 (CH), 126.2 (CH), 126.4 (CH), 126.5 (CH), 126.6 (CH), 127.6 (CH), 127.7 (C×2), 127.8 (CH), 127.9 (CH), 128.9 (CH), 129.0 (CH), 136.3 (C), 141.6 (C), 148.0 (d, .sup.2J.sub.C-P=7.2 Hz, C), 150.6 (C), 159.4 (d, .sup.2J.sub.C-F=27.0 Hz, C), 175.2 (d, .sup.3J.sub.C-P=3.9 Hz, C), 175.5 (d, .sup.3J.sub.C-P=3.9 Hz, C); m/z (ES) 623 (MNH.sub.4.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 30.50 min. and t.sub.R 31.48 min. (27%:69%).
5-Fluoro-2′deoxyuridine-5′-O-[phenyl (benzoxy-α,α-dimethylglycine)] phosphate (CPF393)
[0144] ##STR00018##
[0145] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g, 1.62 mmol), tea-butylmagnesium chloride in tetrahydrofuran (.sup.tBuMgCl) (1.0 M, 2.43 mL, 2.43 mmol) and phenyl(benzoxy-α,α-dimethylglycine) phosphorochloridate (1.17 g, 3.20 mmol) according to general procedure E. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (69.0 mg, 7%) [R.sub.f=0.27 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 600.1527. C.sub.26H.sub.29N.sub.3O.sub.9NaPF requires [MNa.sup.+], 600.1523); .sup.31P NMR (202 MHZ, MeOD): δ.sub.P 2.42, 2.47; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.80, −167.62; NMR (500 MHz, MeOD): δ.sub.H 1.51-1.60 (m, 6H, C(CH.sub.3).sub.2), 1.89-1.97 (m, 1H, H−2′ one diast.), 2.07-2.15 (m, 1H, H−2′, one diast.), 2.21 (ddd, 1H, J=3.4 Hz, 5.9 Hz, 13.5 Hz, H−2′, one diast.), 2.29 (ddd, 1H, J=3.2 Hz, 6.1 Hz, 13.5 Hz, H−2′, one diast.), 4.00-4.07 (m, 1H, H−4′), 4.22-4.31 (m, 2H, CH.sub.2OP), 4.32-4.36 (m, 1H, H−3′, one diast.), 4.37-4.41 (m, 1H, H−3′ one diast.), 5.08-5.18 (m, 2H, OCH.sub.2Ph), 6.19-6.25 (m, 1H, H−1′), 7.20-7.26 (m, 3H, ArH), 7.27-7.39 (m, 7H, ArH), 7.74 (d, .sup.3J.sub.H-F=6.4 Hz, H−6, one diast.), 7.80 (d, .sup.3J.sub.H-F=6.4 Hz, H−6, one diast.); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 27.5 (CH.sub.3), 27.7 (d, .sup.3J.sub.C-P=7.1 Hz, CH.sub.3), 27.8 (d, .sup.3J.sub.C-P=7.1 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 58.2 (C), 58.3 (C), 67.6 (d, .sup.3J.sub.C-P=5.5 Hz, CH.sub.2), 67.7 (d, .sup.2J.sub.C-P=5.5 Hz, CH.sub.2), 68.3 (CH.sub.2), 71.9 (CH), 72.0 (CH), 86.6 (d, .sup.3J.sub.C-P=8.1 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=7.3 Hz, CH), 86.9 (CH), 121.4 (d, .sup.3J.sub.C-P=4.8 Hz, CH), 121.6 (d, .sup.3J.sub.C-P=4.5 Hz, CH), 125.6 (CH), 125.8 (CH), 125.9 (CH), 126.1 (CH), 126.2 (CH), 129.3 (CH), 129.4 (CH), 129.6 (CH), 130.7 (CH), 130.8 (CH), 137.2 (C), 137.3 (C), 141.8 (d, .sup.1J.sub.C-F=233.7 Hz, C), 150.6 (C), 152.1 (d, 7.0 Hz, C). 152.1 (d, 7.6 Hz, C), 159.3 (d, .sup.1J.sub.C-F=26.1 Hz, C), 159.4 (d, .sup.2J.sub.C-F=26.1 Hz, C), 176.5 (d, .sup.3J.sub.C-P=4.0 Hz, C), 176.6 (d, .sup.3J.sub.C-P=3.8 Hz, C), m/z (ES) 600.1 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 35 minutes, 1 ml/min, λ=275 nm, showed one peak of the mixture of diastereoisomers with t.sub.R 17.71 (96%).
5-Fluoro-2′deoxyuridine-5′-O-[phenyl (ethoxy-α,α-dimethylglysine)] phosphate (CPF394)
[0146] ##STR00019##
[0147] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.20 g, 0.80 mmol), N-methylimidazole (NMI) (0.31 mL, 4.0 mmol) and phenyl(ethoxy-α,α-dimethylglycine) phosphorochloridate (0.73 g, 2.40 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (25.0 mg, 6%) [R.sub.f=0.24 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 538.1367. C.sub.21H.sub.27N.sub.3O.sub.9NaPF requires [MNa.sup.+], 538.1367); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 2.49, 2.52; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.62, −167.58; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.24 (t, 3H, 7.1 Hz, CH.sub.2CH.sub.3, one diast.), 1.26 (t, 3H, J=7.1 Hz, CH.sub.2CH.sub.3, one diast.), 1.44-1.54 (m, 6H, C(CH.sub.3).sub.2), 1.95-2.04 (m, 1H, H−2′, one diast.), 2.13-2.21 (m, 1H, H−2′, one diast.), 2.24 (ddd, 1H, J=3.1 Hz, J=6.3 Hz, J=13.5 Hz, H−2′, one diast.), 2.31 (ddd, 1H, J=3.2. Hz, J=6.1 Hz, J=13.7 Hz, H−2′, one diast.), 4.08-4.19 (m, 3H, CH.sub.2CH.sub.3, H−4′), 4.33-4.49 (m, 3H, CH.sub.2OP, H−3′), 6.20-6.30 (m, 1H, H−1′), 7.23-7.28 (m, 3H, ArH), 7.33-7.40 (m, 2H, ArH), 7.80 (d, .sup.3J.sub.H-F=6.4 Hz, H−6, one diast.), 7.88 (d, .sup.3J.sub.H-F=6.4 Hz, H−6, one diast.); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 14.4 (CH.sub.3), 14.5 (CH.sub.3), 27.5 (d, .sup.3J.sub.C-P=7.3 Hz, CH.sub.3), 27.7 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.3), 27.8 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.3), 40.8 (CH.sub.2), 40.9 (CH.sub.2), 58.1 (C), 62.6 (CH.sub.2), 62.7 (CH.sub.2), 67.6 (d, .sup.2J.sub.C-P=6.7 Hz, CH.sub.2), 67.7 (d, .sup.2J.sub.C-P=5.8 Hz, CH.sub.2), 71.9 (CH), 72.0 (CH), 86.6 (d, .sup.3J.sub.C-P=8.1 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=7.6 Hz, CH), 86.9 (CH), 121.4 (d, .sup.3J.sub.C-P=4.4 Hz, CH), 121.6 (d, .sup.3J.sub.C-P=4.4 Hz, CH), 125.6 (CH), 125.8 (CH), 125.9 (CH), 126.1 (CH), 126.2 (CH), 130.7 (CH), 130.8 (CH), 130.9 (CH), 141.8 (d, .sup.1J.sub.C-F=233.5 Hz, C), 150.6 (C), 150.7 (C), 152.2 (d, .sup.4J.sub.C-F=7.3 Hz, C), 152.3 (d, .sup.4J.sub.C-F=6.9 Hz, C), 159.2 (d, .sup.2J.sub.C-F=20.3 Hz, C), 159.4 (d, .sup.2J.sub.C-F=20.4 Hz, C), 176.6 (d, .sup.3J.sub.C-P=4.2 Hz, C), 176.8 (d, .sup.3J.sub.C-P=4.6 Hz, C), m/z (ES) 538.1 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with to 18.76 min. and t.sub.R 20.44 min. (68%:30%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (benzoxy-α,α-dimethylglyeine)] phosphate (CPF395)
[0148] ##STR00020##
[0149] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g, 1.62 mmol), N-methylimidazole (NMI) (0.64 mL, 8.0 mmol) and 1-naphthyl(benzoxy-α,α-dimethylglycine) phosphorochloridate (2.00 g, 4.80 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.3—MeOH (95:5) afforded the title compound as a colourless solid (16.4 mg, 6%) [R.sub.f=0.15 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 650.1678. C.sub.30H.sub.31N.sub.3O.sub.9NaPF requires [MNa.sup.+], 650.1680); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 2.87, 3.03; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.95, −167.13; .sup.1H NMR (500 MHz, MeOD): δ.sub.H 1.37-1.42 (m, 6H, C(CH.sub.3).sub.2), 1.61-1.69 (m, 1H, H−2′, one diast.), 1.79-1.87 (m, 1H, one diast.), 2.06 (ddd, 1H, J=3.0 Hz, J=6.1 Hz, J=13.6 Hz, H−2′, one diast.), 2.15 (ddd, 1H, J=3.2 Hz, J=5.9 Hz, J=13.7 Hz, H−2′, one diast.), 3.98-4.04 (4.19-4.35 (m, 3H, CH.sub.2OP, H−3′), 5.09-5.13 (m, 1H, OCHHPh), 5.18-5.19 (m, 1H, OCHHPh), 6.05-6.15 (m, 1H, H−1′), 7.28-7.40 (m, 7H, ArH), 7.48-7.55 (m, 3H, ArH), 7.62 (d, .sup.3J.sub.H-F=6.4 Hz, H−6, one diast.), 7.70 (d, .sup.3J.sub.H-F=6.4 Hz, H−6, one diast.), 7.86-7.90 (m, 1H, ArH), 8.17-8.22 (m, 1H, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 27.5 (d, .sup.3J.sub.C-P=4.4 Hz, CH.sub.3), 27.9 (d, .sup.3J.sub.C-P=7.3 Hz, CH.sub.3), 28.0 (d, .sup.3J.sub.C-P=7.3 Hz, CH.sub.3), 40.7 (CH.sub.2), 40.8 (CH.sub.2), 65.2 (C), 67.8 (d, .sup.2J.sub.C-P=6.5 Hz, CH.sub.2), 68.3 (CH.sub.2), 72.0 (CH), 72.1 (CH), 86.6 (d, .sup.3J.sub.C-P=8.2 Hz, CH), 86.8 (d, .sup.3J.sub.C-P=7.8 Hz, CH), 86.9 (CH), 116.3 (d, .sup.3J.sub.C-P=3.2 Hz, CH), 116.7 (d, .sup.3J.sub.C-P=2.9 Hz, CH), 122.8 (CH), 122.9 (CH), 125.4 (CH), 125.5 (CH), 125.6 (CH), 126.0 (CH), 126.1 (CH), 126.4 (CH), 126.5 (CH), 127.4 (CH), 127.5 (CH), 127.7 (CH), 127.8 (CH), 127.9 (C), 128.0 (CH), 128.9 (CH), 129.3 (CH), 129.4 (CH), 129.6 (CH), 136.2 (C), 137.3 (C), 141.8 (d, .sup.1J.sub.C-F=234.4 Hz, C), 147.9 (d, .sup.1J.sub.C-P=7.7 Hz, C), 148.0 (d, .sup.3J.sub.C-P=8.2 Hz, C), 150.7 (d, .sup.4J.sub.C-F=3.7 Hz, C), 159.5 (d, .sup.2J.sub.C-F=25.8 Hz, C), 159.6 (d, .sup.2J.sub.C-F=25.8 Hz, C), 176.5 (C), 176.6 (C), m/z (ES) 650.0 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, λ=275 nm, showed two peaks of the diastereoisomers with t.sub.R 2.0.80 min. and t.sub.R 21.00 min. (72%:24%).
5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (ethoxy-α,α-dimethylglycine)] phosphate (CPF396)
[0150] ##STR00021##
[0151] The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g, 1.62 mmol), ten-butylmagnesium chloride in tetrahydrofuran (.sup.tBuMgCl) (1.0 M, 2.43 mL, 2.43 mmol) and 1-naphthyl(ethoxy-α,α-dimethylglycine) phosphorochloridate (1.14 g, 3.20 mmol) according to general procedure E. Purification by gradient column chromatography eluting with CH.sub.2Cl.sub.2 until CH.sub.2Cl.sub.2—MeOH (95:5) afforded the title compound as a colourless solid (54.0 mg, 2%) [R.sub.f=0.10 (CH.sub.2Cl.sub.2—MeOH, 95:5)], (Found: MNa.sup.+, 588.1528. C.sub.25H.sub.29N.sub.3O.sub.9NaPF requires [MNa.sup.+], 588.1523); .sup.31P NMR (202 MHz, MeOD): δ.sub.P 2.91, 3.03; .sup.19F NMR (470 MHz, MeOD): δ.sub.F −167.38, −167.21; NMR (500 MHz, MeOD): δ.sub.H 1.24 (t, 3H, J=7.1 Hz, CH.sub.2CR.sub.3, one diast.), 1.25 (t, 3H, J=7.1 Hz, CH.sub.2CH.sub.3, one diast.), 1.50-1.55 (m, 6H, C(CH.sub.3).sub.2), 1.68-1.76 (m, 1H, H−2′, one diast.), 1.87-1.94 (m, 1H, H−2′, one diast.), 2.09 (ddd, 1H, 2.9 Hz, J=6.3 Hz, J=13.4 Hz, H−2′, one diast.), 2.19 (ddd, 1H, J=3.0 Hz, J=6.3 Hz, J=13.8 Hz, H−2′, one diast.), 4.07-4.10 (m, 1H, H−4′), 4.16 (q, 2H, 7.1 Hz, CH.sub.2CH.sub.3), 4.36-4.41 (m, 3H, CH.sub.2OP, H−3′), 6.10-6.18 (m, 1H, H−1′), 7.40-7.46 (m, 1H, ArH), 7.50-7.59 (m, 3H, ArH), 7.66-7.72 (m, 2H, ArH, H−6), 7.85-7.91 (m, 1H, ArH), 8.18-8.24 (m, 1H, ArH); .sup.13C NMR (125 MHz, MeOD): δ.sub.C 14.4 (CH.sub.3), 27.5 (br s, CH.sub.3), 27.9 (d, .sup.3J.sub.C-P=6.1 Hz, CH.sub.2), 28.0 (d, .sup.3J.sub.C-P=6.1 Hz, CH.sub.3), 40.7 (CH.sub.2), 40.8 (CH.sub.2), 58.2 (C), 58.3 (C), 62.6 (CH.sub.2), 67.8 (d, .sup.2J.sub.C-P=4.9 Hz, CH.sub.2), 67.9 (d, .sup.2J.sub.C-P=4.5 Hz, CH.sub.2), 72.0 (CH), 72.1 (CH), 86.7 (d, .sup.3J.sub.C-P=7.7 Hz, CH), 86.9 (d, .sup.3J.sub.C-P=7.3 Hz, CH), 87.0 (CH), 116.3 (d, .sup.3J.sub.C-P=3.2 Hz, CH), 116.6 (d, .sup.3J.sub.C-P=2.9 Hz, CH), 122.8 (CH), 122.9 (CH), 125.4 (CH), 125.6 (CH), 125.7 (CH), 126.0 (CH), 126.1 (CH), 126.5 (CH), 127.4 (CH), 127.5 (CH), 127.7 (CH), 127.8 (CH), 127.9 (C), 128.0 (C), 128.9 (CH), 136.2 (C), 141.8 (d, .sup.1J.sub.C-F=233.5 Hz, C), 148.0 (d, .sup.2J.sub.C-P=7.3 Hz, C), 148.1 (d, .sup.2J.sub.C-P=7.6 Hz, C), 150.5 (C), 150.6 (C), 159.3 (d, .sup.2J.sub.C-F=26.2 Hz, C), 159.4 (d, .sup.2J.sub.C-F=26.6 Hz, C), 176.8 (C), 176.9 (C); m/z (ES 588.1 (MNa.sup.+, 100%); Reverse-phase HPLC eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 nil/min, λ=275 nm, showed one peak of the mixture of diastereoisomers with t.sub.R 16.05 min. (96%).
5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(benzoxy-L-prolinyl)] phosphate (CPF583)
[0152] ##STR00022##
[0153] Prepared according to the standard procedure 1) from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and phenyl(benzoxy-L-prolinyl)-phosphochloridate (0.77 g, 2.03 mmol) in THF (10 mL). Column purification followed by two preparative TLC purifications gave the product as a white solid (0.010 g, 2%).
[0154] .sup.31P-NMR, (MeOD, 202 MHz) δ 1.82
[0155] .sup.19F-NMR (MeOD, 470 MHz) δ −167.91
[0156] .sup.1H-NMR (MeOD, 500 MHz) δ 7.84 (d, J=7.18 Hz, 1H, H-base), 7.39-7.33 (m, 7H, H—Ar), 7.22-7.19 (m, 3H, H—Ar), 6.26-6.23 (m, 1H.sub.2, H−1′), 5.22-5.13 (m, CR.sub.2Ph ester), 4.40-4.35 (m, 3H, NCH, 2×H−5′), 4.33-4.28 (m, 1H, H−3′), 4.06-4.04 (m, 1H, H−4′), 3.36-3.32 (m, 2H, NCH.sub.2), 2.26-2.19 (m, 1H, H−2′), 2.18-2.13 (m, 1H, CH.sub.2-L-Pro), 2.00-1.81 (m, 4H, 3×H, CH.sub.2-L-Pro, 1×H, H−2′)
[0157] .sup.13C-NMR (MeOD, 125 MHz) δ 174.81 (C═O, ester), 159.40 (C═O, base), 152.0 (d, .sup.2J.sub.C-P=6.32 Hz, OC—Ar), 150.71 (C═O, base), 141.88 (.sup.1J.sub.C-F=232 Hz, CF, base), 137.23 (C—Ar), 131.33, 129.70, 129.48, 129.45, 129.30, 126.45 (CH—Ar), 125.80, 125.53 (2×d, .sup.2J.sub.C-F29.0 Hz, CH-base), 121.00, 120.96 (CH—Ar), 87.80 (C−1′), 86.80 (C−4′), 72.02 (C−3′), 68.16 (CH.sub.2Ph), 67.64 (d, .sup.2J.sub.C-P=4.65 Hz, C−5′), 62.40 (d, .sup.2J.sub.C-P=5.60 Hz, NCH), 48.03 (d, .sup.2J.sub.C-P=4.80 Hz, NCH.sub.2), 41.07 (C−2′), 32.18, 32.11 (CH.sub.2-L-Pro), 26.29, 26.21 (CH.sub.2-L-Pro).
[0158] MS (ES+) m/e: 612 (MNa.sup.+, 100%), 590 (MH+, 1%) Accurate mass: C.sub.27H.sub.29FN.sub.3O.sub.9P required 589.51
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzoxy-L-prolinyl)] phosphate (CPFS77)
[0159] ##STR00023##
[0160] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and 1-naphthyl(benzoxy-prolinyl)-phosphochloridate (0.84 g, 2.03 mmol) in THF (10 mL). Column purification followed by two preparative TLC purifications gave the product as a white solid (0.006 g, 1%).
[0161] .sup.31P-NMR (MeOD, 202 MHz) δ 2.27
[0162] .sup.19F-NMR (MeOD, 121 MHz) δ −167.46
[0163] .sup.1H-NMR (MeOD, 500 MHz) δ 8.14-8.12 (m, 1H, H—Ar), 7.90-7.89 (m, 1H, H—Ar), 7.74-7.71 (m, 2H, 1×H—Ar, 1×H-base), 7.56-7.42 (m, 4H, H—Ar), 7.36-7.33 (m, 5H, H—Ar), 6.13 (t, J=6.38 Hz, H−1′), 5.22-5.13 (m, 2H, CH.sub.2Ph), 4.49-4.46 (m, 1H, NCR), 4.42-4.33 (m, 2H, H−5′), 4.25-4.23 (m, 1H, H−3′), 4.06-4.04 (m, 1H, H−4′), 3.36-3.34 (m, 2H, NCH.sub.2), 2.23-2.15 (m, 1H, CH.sub.2-L-Pro), 2.10-2.02 (m, 2H, 1×H, CH.sub.2-L-Pro, 1×H, H−2′), 1.97-1.77 (m, 2H, CH.sub.2-L-Pro), 1.63-1.57 (m, 1H, H−2′)
[0164] .sup.13C-NMR (MeOD, 125 MHz) 174.82 (C═O, ester), 159.52 (C═O, base), 150.54 (C═O, base), 147.84, 147.78 (d, .sup.2J.sub.C-P=6.03 Hz, OC—Ar), 141.75, 139.97 (2×d, .sup.1J.sub.C-F=232 Hz, CT, base), 137.20, 136.34 (C—Ar), 129.76, 129.65, 129.44, 129.36, 129.27, 129.06, 128.95, 128.04, 128.75, 126.56 (CH—Ar), 125.41 (d, .sup.2J.sub.C-F=30.0 Hz, CH-base), 122.13 ((′H—Ar), 115.76 (d, .sup.3J.sub.C-P=3.3 Hz, CH—Ar), 87.06 (C−1′), 86.79 (C−4′), 72.23 (C−3′), 68.15 (d, .sup.2J.sub.C-P=5.46 Hz, C−5′), 68.08 (CH.sub.2Ph), 62.53 (d, .sup.2J.sub.C-P=5.60 Hz, NCH), 48.26 (d, .sup.2J.sub.C-P=5.34 Hz, NCH.sub.2), 40.97 (C−2′), 32.16, 32.09 (CH.sub.2-L-Pro), 26.22, 26.15 (CH.sub.2-L-Pro).
[0165] Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(3,3-dimethyl-1-butoxy-L-alaninyl)]phosphate (CPF585)
##STR00024##
[0166] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and 1-naphthyl-(3,3-dimethyl-1-butoxy-L-alaninyl)-phosphochloridate (1.21 g, 3.04 mmol) in THF (10 mL). Column purification followed by two preparative TLC purifications gave the product as a white solid (0.010 g, 2%).
[0167] .sup.31P-NMR (MeOD, 202 MHz) δ 4.48, 4.33
[0168] .sup.19F-NMR (MeOD, 470 MHz) δ −167.30, −167.47
[0169] .sup.1H-NMR (MeOD, 500 MHz) δ 8.20-8.17 (m, 1H, H—Ar), 7.91-7.89 (m, 1H, H—Ar), 7.77-7.72 (m, 2H, H—Ar), 7.58-7.51 (m, H-base, 2×H—Ar), 7.46-7.41 (2×t, 1H, J=7.8 Hz, H—Ar), 6.19-6.13 (m, 1H, H−1′), 4.42-4.40 (m, 1H, 1×H−5′), 4.38-4.32 (m, 2H, H−3′, 1×H−5′), 4.14-4.00 (m, 4H, H−4′, CHCH.sub.3, OCH.sub.2CH.sub.2(CH.sub.3).sub.3), 2.21-2.13 (m, 1H, 1×H−2′), 1.91-1.76 (m, 1H, 1×H−2′), 1.52-1.48 (m, 2H, OCH.sub.2CH.sub.2(CH.sub.3).sub.3), 1.37-1.35 (m, 3H, CHCH.sub.3), 0.92, 0.91 (2×s, 9H, OCH.sub.2CH.sub.2(CH.sub.3).sub.3).sup.13C-NMR (MeOD, 125 MHz) δ 175.16, 174.84 (2×d, .sup.3J.sub.C-P=4.75 Hz, C═O, ester), 159.56, 159.35 (C═O, ester), 150.61 (C═O, ester), 148.00, 147.86 (2×d, .sup.2J.sub.C-P=6.25 Hz, OC—Ar), 141.78, 141.73 (2×d, .sup.3J.sub.C-P=232 Hz, CF, base), 136.28 (C Ar), 128.98, 128.95, 127.92, 127.90, 127.58, 126.57, 126.20, 126.14 (CH—Ar), 125.63, 125.55 (2×d, .sup.2J.sub.C-F=34 Hz, CH, base), 122.65, 122.63 (CH—Ar), 116.48, 116.15 (2×d, .sup.3J.sub.C-P=3.0 Hz, CH—Ar), 87.01, 86.94 (C−1′), 86.73, 86.68 (d, .sup.2J.sub.C-P=7.75 Hz, C−4′), 72.18, 72.07 (C−3′), 67.87, 67.85 (2×d, .sup.2J.sub.C-P=5.0 Hz, C−5′), 64.08, 64.05 (OCH.sub.2CH.sub.2(CH.sub.3).sub.3), 51.86 (d, .sup.3J.sub.C-P=5.5 Hz, CHCH.sub.3), 42.74 (OCH.sub.2OH.sub.2(CH.sub.3).sub.3), 40.91, 40.83 (C−2′), 29.96 (OCH.sub.2CH.sub.2(CH.sub.3).sub.3), 20.50, 20.34 (2×d, .sup.3J.sub.C-P=6.5 Hz, CHCH.sub.3).
[0170] MS (ES+) m/e: 630 (MNa.sup.+, 100%), 608 (MH+, 10%) Accurate mass: C.sub.28H.sub.35FN.sub.3O.sub.9P required 607.56
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclobutoxy-L-alaninyl)] phosphate (CPF578)
[0171] ##STR00025##
[0172] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.23 g, 0.93 mmol), NMI (0.38 g, 4.67 mmol, 037 mL) and 1-naphthyl-(cyclobutoxy-L-alaninyl)-phosphochloridate (0.85 g, 2.33 mmol) in THF (10 mL). Column purification followed by preparative TLC purification gave the product as a white solid (0.010 g, 2%).
[0173] .sup.31P-NMR (MeOD, 202 MHz) δ 4.54, 4.36
[0174] .sup.19F-NMR (MeOD, 470 MHz) δ −167.12, −167.29
[0175] .sup.1H-NMR (MeOD, 500 MHz) δ 8.18-8.17 (m, 1H, H—Ar), 7.81-7.87 (m, 1H, R—Ar), 7.74-7.71 (m, 2H, 1×H—Ar, 1×H-base), 7.60-7.53 (m, 3H, H—Ar), 7.46-7.43 (2×t, J=8.0 Hz, 1H, H—Ar), 6.18-6.12 (m, 11-1, H−1′), 5.00-4.95 (m, 1H, OCR ester), 4.41-4.36 (m, 3H, 2×H−5′, H−3′), 4.11-4.00 (m, CHCH.sub.3), 2.36-2.27 (m, 2H, CH.sub.2), 2.18-1.98 (m, 3H, CH.sub.2 ester, 1×H−2′), 1.82-1.56 (m, 3H, CR.sub.2 ester, 1×H−2′), 1.36-1.34 (m, 3H, CHCH.sub.2)
[0176] .sup.13C-NMR (MeOD, 125 MHz) δ 175.97, 173.34 (C═O, ester), 159.88 (C═O, base), 151.64 (C═O, base), 146.58 (OC—Ar), 141.15 (d, .sup.1J.sub.C-F=220 Hz, CF, base), 136.28 (C—Ar), 128.93, 127.89, 127.54, 126.52, 126.18, 126.14 (CH—Ar), 125.53, 125.44 (2×d, .sup.2J.sub.C-F=32.5 Hz, CH-base), 122.63 (CH—Ar), 116.46, 116.44 (2×d, .sup.3J.sub.C-P=2.5 Hz, CH—Ar), 86.98 (d, .sup.3J.sub.C-P=6.25 Hz, C−4′), 86.71 (C−1′), 72.14, 72.04 (C−3′), 71.07 (OCH ester), 67.83 (d, .sup.2J.sub.C-P=7.38 Hz, C−5′), 51.66 (d, .sup.2J.sub.C-P=8.75 Hz, CHCH.sub.3), 40.89, 40.83 (C−2′), 31.03 (OCHCH.sub.2) 20.43 (CHCH.sub.3), 14.23 (CH.sub.2 ester).
[0177] MS (ES+) m/e: 600 (MNa.sup.+, 100%), 578 (MHT, 10%) Accurate mass: C.sub.26H.sub.29FN.sub.3O.sub.9P required 577.50
5-Fluoro-r-deoxyuridine-5′-O-[1-naphthyl-(cyclopropylmethanoxy-L-alaninyl)]phosphate (CPF579)
[0178] ##STR00026##
[0179] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and 1-naphthyl-(cyclopropylmethanoxy-L-alaninyl)-phosphochloridate (0.93 g, 2.54 mmol) in THF (10 mL). Column purification gave the product as a white solid (0.056 g, 10%).
[0180] .sup.31P-NMR (MeOD, 202 MHz) δ 4.58, 4.30
[0181] .sup.19F-NMR (MeOD, 470 MHz) δ −167.18, −167.22
[0182] .sup.1H-NMR (MeOD, 500 MHz) δ 8.18 (d, J=7.0 Hz, 1H, H—Ar), 7.89-7.87 (m, 1H, H—Ar), 7.73 (m, 2H, H—Ar), 7.58-7.53 (m, 3H, H—Ar), 7.45-7.40 (2×t, J=8.0 Hz, 1H, H—Ar), 6.17-6.11 (m, 1H, H−1′), 4.43-4.41 (m, H−5′), 4.38-4.32 (m, 2H, H−5′, H−3′), 4.11-4.04 (m, 2H, H−4′, CHCH.sub.3), 3.95-3.85 (m, 2H, OCH.sub.2 ester), 2.19-2.11 (m, 1H, H−2′), 1.84-1.72 (m, H−2′), 1.38, 1.36 (2×d, J=5.0 Hz, 3H, CHCH.sub.3), 1.15-1.07 (m, OCH.sub.2CH ester), 0.59-0.50 (m, 2H, CH.sub.2 ester), 0.30-0.24 (m, 2H, CH.sub.2 ester)
[0183] .sup.13C-NMR (MeOD, 1.25 MHz) δ 175.25, 174.94 (2×d, .sup.3J.sub.C-P=4.75 Hz, C═O, ester), 159.54, 159.35 (C═O, base), 150.60, 150.56 (C═O, base), 148.05, 147.86 (2×d, .sup.2J.sub.C-P=7.5 Hz, OC—Ar), 141.79, 141.73 (2×d, .sup.1J.sub.C-F=232 Hz, CF, base), 136.29 (C—Ar), 128.94 (d, .sup.3J.sub.C-P=4.4 Hz, CH—Ar), 127.89 (d, .sup.4J.sub.C-P=3.7 Hz, CH—Ar), 127.56, 126.55, 126.52, 126.19, 126.16 (CH—Ar), 125.64, 125.53 (.sup.2J.sub.C-F=34 Hz, CH-base), 122.65 (CH—Ar), 116.54, 116.24 (2×d, .sup.4J.sub.C-P=2.6 Hz, CH—Ar), 87.04, 86.99 (C−1′), 86.90, 86.73 (2×d, .sup.3J.sub.C-P=7.1 Hz, C−4′), 72.18, 72.07 (C−3′). 71.21, 71.18 (OCH.sub.2, ester), 67.87, 67.84 (apparent t, .sup.2J.sub.C-P=5.0 Hz, C−5′), 51.88 (d, .sup.2J.sub.C-P=10.0 Hz, CHCH.sub.3), 40.91, 40.83 (C−2′), 20.60, 20.46 (2×d, .sup.3J.sub.C-P=6.5 Hz, CHCH.sub.3), 10.69 (OCH.sub.2CH ester), 3.70, 3.65 (2×CH.sub.2, ester).
[0184] MS (EST) m/e: 600 (MNa.sup.+, 100%), 578 (MH+, 15%) Accurate mass: C.sub.26H.sub.29FN.sub.3O.sub.9P required HPLC.sub.b (H.sub.2O/Acetonitrile from 100/0 to 0/100 in 35 min) Rt 12.91 min.
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(tetrahydropyroxy-L-alaninyl)]phosphate (CPF580)
[0185] ##STR00027##
[0186] Prepared according to the standard procedure E from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), tBuMgCl (1.1 mL, 1.1 mmol) and 1-naphthyl-(tetrahydropyroxy-L-alaninyl)-phosphochloridate (0.80 g, 2.03 mmol) in THF (10 mL). Column purification followed by two preparative TLC purifications gave the product as a white solid (0.010 g, 1.6%).
[0187] .sup.31P-NMR (MeOD, 202 MHz) δ 3.77, 3.22
[0188] .sup.19F-NMR (MeOD, 470 MHz) δ −168.27, −168.35
[0189] .sup.1H-NMR (MeOD, 500 MHz) δ 8.60 (d, J=7.0 Hz, 2H, H—Ar), 8.22-8.19 (m, 1H, H—Ar), 7.92-7.91 (d, J=5.50 Hz, 1H, H—Ar), 7.60-7.45 (m, 4H, H—Ar, H-base), 6.29-6.25 (m, 1H, H−1′), 5.25-5.17 (m, 1H, H−3′), 4.96-4.87 (m, 1H, CH-ester), 4.28-4.26 (m, 1H, H−4′), 4.11-4.03 (m, 1H, CHCH.sub.3), 3.88-3.66 (m, 4H, 2×OCH.sub.2b′ ester, 2×H−5′), 3.55-3.50 (m, 2H, 2×OCH.sub.2a″ ester), 2.63-2.30 (m, 2H, H−2′), 1.91-1.85 (m, 2H, 2×CH.sub.2b′ ester), 1.65-1.54 (m, 2H, CH.sub.2b″ ester), 1.39-1.35 (m, 31-1, CHCH.sub.3).
[0190] .sup.13C-NMR (MeOD, 125 MHz) δ 174.34 (C═O, ester), 159.2, (C═O, base), 150.76 (C═O, base), 148.03 (OC—Ar), 141.97 (d, J=238 Hz, CF, base), 136.37 (C—Ar), 128.97, 128.56, 127.61, 127.57, 126.58, 126.23, 126.16, 126.12, 125.84 (CH—Ar), 122.70 (d, .sup.2J.sub.C-F=24.0 Hz. CH-base), 116.62, 116.37 (CH—Ar), 87.54 (d, .sup.3J.sub.C-P=5.40 Hz, C−4′), 86.60, 86.57 (C−1′), 79.82, 79.47 (C−3′), 71.45 (CH-ester), 66.12, 66.08 (2×OCH.sub.2a ester), 66.02 (C−5′), 51.83 (CHCH.sub.3), 39.97, 39.94 (C−2′), 32.65, 32.57 (2×CH.sub.2b ester), 20.45, 20.30 (CHCH.sub.3).
[0191] MS (ES+) m/e: 630 (MNa.sup.+, 100%), 608 (MH+, 10%) Accurate mass: C.sub.27H.sub.31FN.sub.3O.sub.10P required 607.52.
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(pentoxy-L-alaninyl)] phosphate (CPF581)
[0192] ##STR00028##
[0193] Prepared according to the standard procedure E from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), tBuMgCl (1.1 mL, 1.1 mmol) and 1-naphthyl-(pentoxy-L-alaninyl)-phosphochloridate (0.78 g, 2.03 mmol) in THF (10 mL). Column purification gave the product as a white solid (0.047 g, 8%).
[0194] .sup.31P-NMR (MeOD, 202 MHz) δ 4.48, 4.32
[0195] .sup.19F-NMR (MeOD, 470 MHz) δ −167.18-167.29
[0196] .sup.1H-NMR (MeOD, 500 MHz) δ 8.25-8.17 (m, 1H, H—Ar), 8.05-7.95 (m, 2H, H—Ar), 7.85-7.60 (m, 2H, H—Ar, H-base), 7.65-7.48 (m, 3H, H—Ar), 6.30-6.18 (m, 1H, H−1′), 4.60-4.37 (m, 3H, 2×H−5′, H−3′), 4.28-4.00 (m, 4H, H−4′, CHCH.sub.3, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.32-2.12. (m, 1H, H−2′), 1.95-1.75 (m, 1H, H−2′), 1.70-1.55 (m, 2H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.50-1.28 (m, 7H, 4×H OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3, CHCH.sub.3), 0.83, 0.82 (2×d, J=7.9 Hz, 3H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3)
[0197] .sup.13C-NMR (MeOD, 125 MHz) δ 175.22, 174.91 (C═O, ester), 159.5 (C═O, base), 150.54 (C−4 base), 147.90, 147.88 (OC—Ar), 141.75 (d, .sup.1J.sub.C-F=225 Hz, CF, base), 136.37 (C—Ar), 128.95, 127.90, 127.56, 126.55, 126.1.9 (CH—Ar), 125.64, 125.53 (2×d, .sup.2J.sub.C-F=34.0 Hz, CH-base), 122.65 (CH—Ar), 116.51, 116.21 (CH—Ar), 87.03, 86.96 (C−1′), 86.85, 86.74 (C−4′), 72.16, 72.05 (C−3′), 67.87 (d, .sup.2J.sub.C-P=5.0 Hz, C−5′), 66.54 (OCH.sub.2), 51.87, 51.81 (d, .sup.2J.sub.C-P=7.5 Hz, CHCH.sub.3), 40.87, 40.80 (C−2′), 29.35, 29.10 (CH.sub.2 ester), 23.33 (CH.sub.2 ester), 20.60, 20.43 (2×d, .sup.3J.sub.C-P=6.5 Hz, CHCH.sub.3), 14.28 (CH.sub.3 ester).
[0198] MS (ES+) m/e: 616 (MNa.sup.+, 100%), 594 (MH+, 10%) Accurate mass: C.sub.27H.sub.33FN.sub.3O.sub.9P required 593.54.
[0199] HPLC.sub.b (H.sub.2O/Acetonitrile from 100/0 to 0/100 in 35 min) Rt 15.56 min.
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclopentoxy-L-alaninyl)] phosphate (CPF582)
[0200] ##STR00029##
[0201] Prepared according to the standard procedure E from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), tBuMgCl (1.1 mL, 1.1 mmol) and 1-naphthyl-(cyclopentoxy-L-alaninyl)-phosphochloridate (0.77 g, 2.03 mmol) in THF (10 mL). Column purification gave the product as a white solid (0.030 g, 5%).
[0202] .sup.31P-NMR (MeOD, 202 MHz) δ 4.53, 4.37
[0203] .sup.19F-NMR (MeOD, 470 MHz) δ −167.07, −167.19
[0204] .sup.1H-NMR (MeOD, 500 MHz) δ 8.18-8.16 (m, 1H, H—Ar), 7.89-7.85 (m, 1H, H—Ar), 7.70 (apparent t, J=6.50 Hz, 2H, H—Ar), 7.57-7.50 (m, 3H, 2×H—Ar, H-base), 7.45-7.40 (m, 1H, H—Ar), 6.16-6.11 (m, 0.114, 11-1′), 5.15-5.09 (m, 1H, OCH ester), 4.41-4.30 (m, 3H, 2×H−3′), 4.11-4.08 (m, 1H, H−4′), 4.04-3.98 (m, 1H, CHCH.sub.3), 2.19-2.10 (m, 1H, H−2′), 1.86-1.73 (m, 3H, OCHCH.sub.2 ester), 1.73-1.56 (m, 6H, H−2′, CH.sub.2 ester), 1.35, 1.34 (2×d, J=6.57 Hz, CHCH.sub.3)
[0205] .sup.13C-NMR (MeOD, 125 MHz) δ 174.68, 174.64 (C═O, ester), 159.27 (C═O, base), 150.51 (C═O, base), 147.86 (d, .sup.2J.sub.C-P=7.5 Hz, OC—Ar), 141.78, 141.72 (2×d, .sup.1J.sub.C-F==232 Hz, CF-base), 136.30 (C—Ar), 128.95, 128.54, 127.94, 127.80, 127.60, 127.56, 127.17, 126.80, 126.54, 126.19, 126.16 (CH—Ar), 125.66, 125.53 (2×d, .sup.2J.sub.C-F34 Hz, CH-base), 122.65, 122.61 (CH—Ar), 116.53, 116.22 (2×d, .sup.4J.sub.C-P=3.75 Hz, CH—Ar), 86.99, 86.96 (C−1′), 86.70 (d, .sup.3J.sub.C-P=7.50 Hz, C−4′), 79.64, 79.61 (OCH ester), 72.21, 72.07 (C−3′), 67.89, 67.85 (2×d, .sup.2J.sub.C-P=5.0 Hz, C−5′), 51.92 (d, .sup.2J.sub.C-P=5.0 Hz, CHCH.sub.3), 40.92, 40.86 (C−2′), 33.65, 33.61, 33.52, 33.47 (2×CH.sub.2 ester), 24.68, 24.66 (CH.sub.2 ester), 20.45, 20.30 (2×d, .sup.3J.sub.C-P=6.25 Hz, CHCH.sub.3).
[0206] MS (ES+) m/e: 614 (MNa.sup.+, 100%), 592 (MH+, 30%) Accurate mass: C.sub.27H.sub.31FN.sub.3O.sub.9P required 591.52
[0207] HPLC.sub.b (H.sub.2O/Acetonitrile from 100/0 to 0/100 in 35 min) Rt 14.03 min.
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(2-indanoxy-L-alaninyl)] phosphate (CPF597)
[0208] ##STR00030##
[0209] Prepared according to the standard procedure E from 5-Fluoro-2′-deoxyuridine (0.30 g, 1.22 mmol), tBuMgCl (1.34 mL, 1.34 mmol) and 1-naphthyl-(2-indanoxy-L-alaninyl)-phosphochloridate (1.06 g, 2.43 mmol) in THF (20 mL). Column purification gave the product as a white solid (0.045 g, 6%).
[0210] .sup.31P-NMR (MeOD, 202 MHz) δ 4.62, 4.30
[0211] .sup.19F-NMR (MeOD, 470 MHz) δ −167.14, −167.34
[0212] .sup.1H-NMR (MeOD, 500 MHz) δ 8.15-8.12 (m, 1H, H—Ar, Naph), 7.89-7.87 (m, 1H, H—Ar, Naph), 7.72-7.67 (m, 2H, H—Ar, Naph), 7.56-7.46 (m, 3H, 2×H—Ar, H-base), 7.40-7.37 (m, 1H, H—Ar). 7.20-7.12 (m, 4H, Ph), 6.14-6.08 (m, 1H, H−1′), 5.49-5.46 (m, 1H, OCR ester), 4.32-4.26 (m, 3H, 2×H−5′, H−3′), 4.04-3.98 (m, 1H, H−4′, CHCH.sub.3), 3.30-3.24 (m, 2H, 2×CH ester), 2.99-2.91 (m, 2H, 2×CH ester), 2.14-2.07 (m, 1H, H−2′), 1.75-1.64 (m, 1H, H−2′), 1.33-1.29 (m, 3H, CHCH.sub.3)
[0213] .sup.13C-NMR (MeOD, 125 MHz) δ 175.02, 174.66 (2×d, .sup.3J.sub.C-P=3.75 Hz, C═O, ester), 159.48 (.sup.2J.sub.C-F=25.0 Hz, C═O, base), 150.57 (C═O, base), 147.97, 147.80 (2×d, .sup.2J.sub.C-P=7.5 Hz, OC—Ar), 141.73, 141.68 (2×d, .sup.1J.sub.C-F=232.5 Hz, CF-base), 141.54, 141.49, 141.48, 139.10, 136.27, 136.26 (C—Ar), 129.01, 128.94, 128.91, 127.91, 127.87, 128.85, 127.80, 127.77, 127.60, 127.57, 127.50, 126.20, 126.18, 125.69 (CH—Ar), 125.50, 125.43 (2×d, .sup.2J.sub.C-F=25 Hz, CH-base), 122.64, 122.60, 121.85 (CH—Ar), 116.57, 116.26 (2×d, .sup.4J.sub.C-P=2.5 Hz, CH—Ar), 86.96 (C−1′), 86.87, 86.66 (2×d, .sup.3J.sub.C-P=7.50 Hz, C−4′), 77.85, 79. (OCH ester), 72.21, 72.07 (C−3′), 67.77, 67.75 (2×d, .sup.2J.sub.C-P=6.25 Hz, C−5′), 51.97, 51.82 (CHCH.sub.3), 40.91, 40.86 (C−2′). 40.44, 40.43, 40.38, 40.34 (2×CH.sub.2 ester), 20.30, 20.16 (2×d, .sup.3J.sub.C-P=6.25 Hz, CH(CH.sub.3)
5-Fluoro-2′-deoxyuridine-5′-O-[phenyl-(benzoxy-L-methioninyl)] phosphate (CPF586)
[0214] ##STR00031##
[0215] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and phenyl-(benzoxy-L-methioninyl)-phosphochloridate (0.7 g, mmol) in THF (10 mL). Column purification gave the product as a yellowish solid (0.014 g, 2%).
[0216] .sup.31P-NMR (MeOD, 202 MHz) δ 4.34, 3.94
[0217] .sup.19F-NMR (MeOD, 470 MHz) δ −167.40-167.69
[0218] .sup.1H-NMR (MeOD, 500 MHz) δ 7.83-7.80 (m, 1H, H—Ar), 7.74-7.72 (m, 1H, H—Ar), 7.64-7.62 (m, 1H, H—Ar), 7.37-7.32 (m, 6H, H—Ar, H-base), 7.26-7.17 (m, 2H, H—Ar), 6.25-6.17 (m, 1H, H−1′), 5.18, 5.13 (AB system, J=12.0 Hz, 2H, CH.sub.2Ph), 4.40-4.35 (m, 1H, H−3′), 4.32-4.22 (m, 2H, H−5′), 4.16-4.03 (m, 2H, NHCH, H−4′), 2.44, 2.36 (2×t, J=7.50 Hz, CH.sub.2S), 2.16-2.08 (m, 1H, 1×H−2′), 1.98-1.82 (m, 6H, 1×H−2′, NHCHCH.sub.2CH.sub.2SCH.sub.3), MS (ES+) m/e: 646 (MNa.sup.+, 100%). 624 (MH+, 10%) Accurate mass: C.sub.27H.sub.31FN.sub.3O.sub.9PS required 623.56
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(benzoxy-L-phenylalaninyl)] phosphate (CPF587)
[0219] ##STR00032##
[0220] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and 1-naphthyl-(benzoxy-L-phenylalaninyl)-phosphochloridate (1.45 g, mmol) in THF (10 mL). Column purification gave the product as a white solid (0.007 g, 1%).
[0221] .sup.31P-NMR (MeOD, 202 MHz) δ 4.27, 4.14
[0222] .sup.19F-NMR (MeOD, 470 MHz) δ −166.99, −167.18
[0223] .sup.1H-NMR (MeOD, 500 MHz) δ 8.11-8.00 (m, 1H, H—Ar, Ar), 7.89-7.85 (m, 1H, H—Ar), 7.69-7.67 (m, 1H, H—Ar), 7.60-7.49 (m, 3H, 2×H—Ar, H-base), 7.37-7.33 (m, 2H, H—Ar), 7.25-7.12 (m, 10H, H—Ar), 6.09-6.04 (m, 11-1′), 5.11-5.01 (m, 2H, CH.sub.2Ph), 4.29-4.1.8 (m, 1H, CHCH.sub.3), 4.15-4.08 (m, 1H, H−3′), 4.02, 3.95 (m, 2H, H−5′), 3.86-3.67 (m, 1H, H−4′), 3.14-3.10 (m, 1H, 1×NHCHCH.sub.2Ph), 2.91-2.82 (m, 1H, 1×NHCHCH.sub.2Ph), 2.12-2.06, 2.00-1.95 (2×m, 1H, H−2′), 1.68-1.62, 1.42-1.36 (2×m, 1H, H−2′)
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)] phosphate (CPF588)
[0224] ##STR00033##
[0225] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NCH (0.41 g, 5.07 mmol, 0.40 mL) and 1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)-phosphochloridate (0.77 g, mmol) in THF (10 mL). Column purification gave the product as a white solid (0.006 g, 1%).
[0226] .sup.31P-NMR (MeOD, 202 MHz) δ 4.56, 4.33
[0227] .sup.19F-NMR (MeOD, 470 MHz) δ −167.32, −167.43
[0228] .sup.1H-NMR (MeOD, 500 MHz) δ 8.19-8.16 (m, 1H, H—Ar, Ar), 7.91-7.89 (m, 1H, H—Ar), 7.74-7.71 (m, 2H, H—Ar), 7.57-7.51 (m, 3H, 2×H—Ar, H-base), 7.46-7.41 (m, 1H, H—Ar), 6.17-6.10 (m, 1H, H−1′), 4.42-4.30 (m, 3H, H−3′, 2×H−5′), 4.13-4.07 (m, 2H, H−4′, CHCH.sub.3), 3.86, 3.75 (AB system, J.sub.AB=10.50 Hz, 2H, CH.sub.2C(CH.sub.3).sub.3), 2.18-2.10 (m, 1H, H−2′), 1.81-1.70 (m, 1H, H−2′), 1.41-1.38 (m, 3H, CHCH.sub.3), 0.95, 0.94 (2×s, 9H, CH.sub.2C(CH.sub.3).sub.3)
5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(butoxy-L-alaninyl)] phosphate (GPF589)
[0229] ##STR00034##
[0230] Prepared according to the standard procedure D from 5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol, 0.40 mL) and 1-naphthyl-(butoxy-L-alaninyl)-phosphochloridate (0.75 g, mmol) in THF (10 mL). Column purification gave the product as a white solid (0.006 g, 1%).
[0231] .sup.31P-NMR (MeOD, 202 MHz) δ 4.52, 4.35
[0232] .sup.19F-NMR (MeOD, 470 MHz) δ −167.36, −167.49
[0233] .sup.1H-NMR, (MeOD. 500 MHz) δ 8.19-8.16 (m, 1H, H—Ar, Naph), 7.1-7.89 (m, 1H, H—Ar, Naph), 7.75-7.72 (m, 2H, H—Ar, Naph), 7.58-7.51 (m, 3H, 2×H—Ar, H-base), 7.46-7.41 (m, 1H, H—Ar), 6.18-6.11 (m, 1H, H−1′), 4.42-4.40 (m, 1H, 1×H−5′), 4.37-4.32 (m, 2H, 1×H−5′, H−3′), 4.12-4.01 (m, 4H, H−4′, CHCH.sub.3, OCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.20-2.12 (m, 1H, H−2′), 1.85-1.73 (m, 1H, H−2′), 1.61-1.54 (m, 2H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.39-1.31 (m, 5H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.32CHCH.sub.3), 0.93-0.89 (m, 3H, OCH.sub.2CH.sub.2CH.sub.2CH.sub.3)
[0234] Biological Assays
[0235] Experimental data having regard to compounds embodying the present invention are described below.
[0236] Cell Cultures
[0237] Murine leukaemia L1210/0 and human T-lymphocyte CEM/0 cells were Obtained from the American Type Culture Collection (ATCC) (Rockville, Md.). Human glioblastoma U87 cells were kindly provided by Dr. E. Menue (Institut Pasteur, Paris, France). Thymidine kinase-deficient CEM/TK.sup.− cells were a kind gift from Prof. S. Eriksson (currently at Uppsala University, Uppsala, Sweden) and Prof. A. Karlsson (Karolinska Institute, Stockholm, Sweden). Thymidine kinase-deficient L1210/TK.sup.− were derived from L1210/0 cells after selection for resistance against 5-bromo-2′-dUrd (Balzarini et al., 1982). Infection of relevant cell lines with Mycoplasma hyorhinis (ATCC) resulted in chronically-infected cell lines further referred to as L1210.Hyor and U87.Hyor. All cells were maintained in Dulbecco's modified. Eagle's medium (DMEM) (Invitrogen, Carlsbad, Calif.) with 10% foetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), 10 mM Hepes and 1 mM Sodium Pyruvate (Invitrogen). Cells were grown at 37° C. in a humidified incubator with a gas phase of 5% CO.sub.2.
[0238] Cytostatic Assays
[0239] Monolayer cells (U87 and U87.Hyor) were seeded in 48-well microtiter plates (Nunc™, Roskilde, Denmark) at 10,000 cells/well. After 24 hours, an equal volume of fresh medium containing the test compounds was added. On day 5, cells were trypsinized and counted in a Coulter counter (Analis, Suarlée, Belgium). Suspension cells (L1210/0, L1210/TIC, L1210.Hyor, CEM/0, CEM/TK.sup.−) were seeded in 96-well microtiter plates (Nunc™) at 60,000 cells/well in the presence of a given amount of the test compounds. The cells were allowed to proliferate for 48 h (L1210) or 72 hours (CEM) and were then counted in a Coulter counter. The 50% inhibitory concentration (IC.sub.50) was defined as the compound concentration required to reduce the number of viable cells by 50%.
[0240] Assay 1. The samples were assayed for biological activity versus a range of tumour cell lines with data recorded in Table 1 below. Data are expressed as CC.sub.50 in μM, i.e. cytostatic concentration required to inhibit cell proliferation by 50%. The cell lines employed were L1210/0 (a leukemia cell line), FM3A/0 (a breast cancer cell line), Cem/0 (an acute lymphoblastic leukemia cell line) and HeLa (a cervical cell line).
[0241] Table 1 also contains comparative data for 5FU, 5-FdUrd and reference compounds CPF 382, CPF 437 and CPF 438. The structure of CPF 382 is given above. The structure of each of CPF 437 and CPF 438 is as follows:
##STR00035##
[0242] As can be seen from the data in Table 1, compounds of the present invention can exhibit cytostatic activity that is comparable to or better than that of 5-FU, whilst exhibiting marked cytostatic activity in nucleoside kinase-deficient cells. Such a cytostatic activity in nucleotide kinase-deficient cells is in direct contrast to that of 5-FdUrd.
[0243] As can also be seen from Table 1, the activity in TK.sup.− cells of compounds embodying the present invention can be markedly greater than that of reference compounds CPF 382, CPF 437 and CPF 438.
TABLE-US-00002 TABLE 1 L1210/0 L1210/TK.sup.− FM3A/0 FM3A/TK.sup.− Cem/0 Cem/TK.sup.− HeLa HeLa/TK.sup.− 5-FdUrd 0.00082 ± 0.00008 3.1 ± 0.2 0.028 ± 0.002 1.5 ± 0.1 5-FdUrd (2) 0.0010 ± 0.0001 4.8 ± 4.0 0.0065 ± 0.0055 0.70 ± 0.02 0.026 ± 0.000 4.4 ± 2.9 0.029 ± 0.007 1.4 ± 0.5 5-FdUrd (3) 0.0011 ± 0.0002 3.0 ± 0.1 0.022 ± 0.006 3.0 ± 0.4 0.050 ± 0.011 1.4 ± 0.4 FU 0.33 ± 0.17 0.32 ± 0.31 0.18 ± 0.02 18 ± 5 0.54 ± 0.12 CPF 382(1) 0.0255 37.8 0.346 32.7 CPF 382(2) 0.0271 39.3 0.21 29.2 CPF 437 36 ± 5 >100 >100 >100 >100 >100 CPF 438 0.12 ± 0.02 51 ± 9 2.1 ± 0.6 32 ± 2 3.7 ± 0.5 72 ± 0 CPF 373 0.015 ± 0.007 0.027 ± 0.004 0.089 ± 0.043 0.32 ± 0.07 CPF 373(2) 0.0061 ± 0.0043 0.064 ± 0.028 0.059 ± 0.046 0.74 ± 0.18 0.046 ± 0.010 0.74 ± 0.63 0.065 ± 0.013 2.5 ± 1.3 CPF 381 0.028 ± 0.007 13 ± 8 0.18 ± 0.03 22 ± 7 CPF 383 0.13 ± 0.04 0.94 ± 0.18 0.64 ± 0.57 4.1 ± 2.0 0.92 ± 0.11 14 ± 0 0.48 ± 0.19 9.8 ± 1.4 CPF 384 0.076 ± 0.022 1.1 ± 0.1 0.36 ± 0.25 13 ± 1 1.0 ± 0.1 30 ± 10 0.71 ± 0.15 25 ± 11 CPF 386 0.031 ± 0.005 0.36 ± 0.01 0.25 ± 0.04 1.6 ± 0.2 0.22 ± 0.04 2.8 ± 0.0 CPF 393 0.017 ± 0.003 0.18 ± 0.05 0.23 ± 0.04 4.8 ± 0.7 CPF 394 0.039 ± 0.001 4.6 ± 0.0 0.65 ± 0.16 22 ± 1 CPF 395 0.011 ± 0.005 0.13 ± 0.04 0.16 ± 0.02 2.4 ± 0.8 CPF 396 0.064 ± 0.008 0.82 ± 0.16 0.36 ± 0.05 6.9 ± 1.8 CPF 508 0.039 ± 0.001 0.14 ± 0.02 0.18 ± 0.00 0.17 ± 0.07 0.18 ± 0.05 CPF 509 0.043 ± 0.023 0.15 ± 0.00 0.31 ± 0.06 0.057 ± 0.055 0.090 ± 0.014 CPF 576 1.1 ± 0.5 35 ± 8 0.80 ± 0.28 46 ± 14 0.67 ± 0.03 27 ± 6 CPF 577 0.21 ± 0.08 25 ± 8 0.89 ± 0.35 32 ± 9 1.2 ± 0.0 26 ± 1 CPF 578 0.014 ± 0.003 0.088 ± 0.038 0.073 ± 0.018 1.5 ± 0.3 0.069 ± 0.003 1.5 ± 0.6 CPF 579 0.017 ± 0.007 0.12 ± 0.06 0.059 ± 0.017 1.1 ± 0.2 0.068 ± 0.001 1.4 ± 0.4 CPF 580 0.038 ± 0.014 27 ± 6 0.11 ± 0.02 43 ± 12 0.13 ± 0.04 15 ± 7 CPF 581 0.0028 ± 0.0010 0.13 ± 0.13 0.015 ± 0.006 0.28 ± 0.04 0.029 ± 0.023 0.44 ± 0.35 CPF 582 0.031 ± 0.010 0.13 ± 0.02 0.035 ± 0.025 0.92 ± 0.007 0.071 ± 0.036 2.2 ± 1.3 CPF 583 0.35 ± 0.07 31 ± 5 0.98 ± 0.40 28 ± 8 1.1 ± 0.4 20 ± 11 CPF 585 0.016 ± 0.006 0.062 ± 0.009 0.053 ± 0.021 0.19 ± 0.04 0.078 ± 0.018 1.3 ± 0.9 CPF 586 0.073 ± 0.035 4.1 ± 1.2 0.28 ± 0.03 25 ± 0 0.15 ± 0.02 11 ± 7 CPF 587 0.012 ± 0.007 5.6 ± 1.3 0.10 ± 0.03 7.2 ± 0.1 0.16 ± 0.08 6.8 ± 1.5 CPF 588 0.27 ± 0.11 1.2 ± 0.7 0.49 ± 0.05 6.7 ± 1.0 0.70 ± 0.11 32 ± 26 CPF 589 0.022 ± 0.004 0.11 ± 0.06 0.064 ± 0.007 0.84 ± 0.60 0.12 ± 0.02 2.7 ± 1.5
[0244] Assay 2. Samples were also assayed for their % retention of activity in mycoplasma infected cells. The results are set out in Table 2 below. The results show that compounds of the present invention can retain high activity in mycoplasma infected cells, in contrast to the activity shown by 5-FdURD. Administration of a Thymidine Phosphorylase (TP) inhibitor restores the cytostatic activity of 5-FdUrd in myocoplasma infected cell cultures, providing evidence of the deteriorating role of TP in the eventual cytostatic activity of 5-FdUrd. As mycoplasma infection of cells is known to greatly reduce the activity of nucleosides, including 5-fdUrd, the activity of some nucleosides in mycoplasma infected cells provides a potential benefit in patients that are mycoplasma infected.
TABLE-US-00003 TABLE 2 CC50 values in μM for 5-FdUrd and compounds embodying the present invention in mycoplasma negative and positive cells, and % retention of activity on mycoplasma infection. Cpd L1210 L1210/Hyor % Retention 5-FdUrd 0.00051 0.278 0.2 CPF 373 0.011 0.025 44 CPF 381 0.026 0.15 18 CPF 393 0.029 0.02 145 CPF 394 0.030 0.26 12 CPF 395 0.019 0.045 42 CPF 396 0.056 0.17 33 CPF 576 1.4 2.73 51 CPF 577 0.23 0.63 36 CPF 578 0.015 0.048 31 CPF 579 0.019 0.045 42 CPF 580 0.048 0.41 12 CPF 581 0.0037 0.017 22 CPF 582 0.035 0.042 83 CPF 583 0.387 11.9 3.3 CPF 585 0.021 0.051 41 CPF 586 0.1 0.87 11 CPF 587 0.022 4.2 0.5 CPF 588 0.237 0.39 61 CPF 589 0.02 0.063 32 “% retention” is a measure of the ratio of the CC50 values measured with respect to L1210 with respect to those for L1210/Hyor and is calculated as CC50.sub.L1210 × 100 ÷ CC50.sub.L1210/Hyor.
[0245] Further experiments (Assays 3 to 8 below) were carried out with respect to the compound CPF 373 embodying the present invention.
[0246] Assay 3. Cytostatic Activity of 5-FdUrd and its Prodrug CPF-373 Against TK-Competent and TK-Deficient Tumour Cell Lines
[0247] The cytostatic activity of 5-FdUrd and CPF-373 was determined in different TK-expressing and TK-deficient tumour cell lines. As shown in Table 3, 5-FdUrd is strongly dependent on the expression of TK for its cytostatic activity. Its IC.sub.50 increased by 4,000-fold for L1210/TK.sup.− cells (IC.sub.50: 3.1 μM) versus wild-type L1210/0 cells (IC.sub.50: 0.0008 μM) and by 50-fold for CEM/TK.sup.− cells (IC.sub.50: 1.5 μM) versus CEM410 cells (IC.sub.50: 0.028 μM). In contrast, the cytostatic activity of the 5-FdUrd prodrug CPF-373 remained virtually unchanged in TK-deficient cells when compared with wild-type cells (IC.sub.50: 0.027 and 0.011 μM for L1210/TK.sup.− and L1210/0, and 0.32 and 0.089 μMAl for CEM/TK.sup.− and CEM/0 cells, respectively). Although the cytostatic activity of CPF-373 was 3- to 10-fold inferior to 5-FdUrd against wild-type L1210/0 and CEM/0 cells, it proved 5- to 100-fold superior to 5-FdUrd in the TK-deficient tumour cell lines (see Table 3).
TABLE-US-00004 TABLE 3 Cytostatic activity of 5-FdUrd and CPF-373 as represented by the IC.sub.50 value in different cell lines IC.sub.50.sup.a (μM) Cell lines 5-FdUrd CPF-373 L1210/0 0.0008 ± 0.000095 0.011 ± 0.0065 L1210/TK− 3.1 ± 0.14 0.027 ± 0.0028 L1210.Hyor 0.24 ± 0.054 0.025 ± 0.0073 CEM/0 0.028 ± 0.0014 0.089 ± 0.030 CEM/TK− 1.5 ± 0.071 0.32 ± 0.049 U87 0.007 ± 0.001 0.035 ± 0.0005 U87.Hyor 3.0 ± 0.55 0.039 ± 0.0025 .sup.a50% Inhibitory concentration or compound concentration required to inhibit tumour cell proliferation by 50%
[0248] Assay 4. Effect of Mycoplasma Infection of Tumour Cell Cultures on the Cytostatic Activity of 5-FdUrd and Its Prodrug CPF-3.73
[0249] The L1210/0 cell cultures were infected with the mycoplasma species M. hyorhinis (cells designated: L1210.Hyor). 5-FdUrd markedly lost its cytostatic activity against the mycoplasma-infected L1210.Hyor cells by 300-fold (IC.sub.50: 0.24 μM). Also, 5-FdUrd lost its cytostatic activity by 400-fold in U87.Hyor cell cultures when compared with uninfected U87 cells (see Table 3). In sharp contrast, the 5-FdUrd prodrug CPF-373 kept a similar cytostatic potential in both L1210/0 and L1210.Hyor cell cultures (IC.sub.50: 0.011 and 0.025 μM, respectively). A similar observation was made for this prodrug when evaluated for its cytostatic activity in U87 and U87.Hyor cell cultures (IC.sub.50: 0.035 and 0.039 PA, respectively). Thus; whereas the free nucleoside 5-FdUrd markedly lost its cytostatic potential against Mycoplasma hyorhinis-infected tumour cell lines, the antiproliferative potential of its prodrug CPF-373 was independent of the mycoplasma infection.
[0250] Assay 5. Experiments were carried out to assess the stability of CPF 373 in the presence of Thymidine Phosphorylase (TP). The experiments are illustrated with reference to
[0251] Phosphorylase Assay on 5-TdUrd and its ProTide Compound CPF 373 by Thymidine Phosphorylase (TP) Purified from Escherichia Coli.
[0252] Nucleoside 5-FdUrd can be degraded to its relative base 5FU by a phosphorolytic reaction, using thymidine phosphorylase purified from Escherichia coli as well as uridine phosphorylase purified from Ehrlich ascite tumor. This breakdown has been suggested to be one of the reasons for the limited therapeutic effectiveness of 5-FdUrd according to the following scheme:
##STR00036##
[0253] The phosphorylase assay was carried out towards phosphorolysis by Thymidine Phosphorylase purified from Escherichia coli using in situ .sup.19F NMR. The application to the ProTide compound CPF 373 was an attempt to prevent the cleavage of the structure and thus circumvent the action of the enzyme.
[0254] Two potassium phosphate buffers at pH 7.4, 200 nM solution and 300 nM solution respectively, were used as phosphate donor. Units of enzyme were defined as the amount of enzyme required to hydrolyse about 0.25 mg of inosine per min used as standard. Assays were conducted for 30 minutes.
[0255] Phosphorylase Assay on 5-FdURd
[0256] Initially, .sup.19F NMR (470 MHz) spectra of 5-FdUrd and 5FU previously dissolved in deuterated methanol, were recorded. 5-FdUrd showed a singlet at ˜□−167.21 ppm and 5FU at ˜□−169.30 ppm. Thus, the phosphorylase assay was carried out by dissolving 5-FdUrd in deuterated methanol, in the presence of potassium phosphate buffer (200 nM solution; pH=7.4), recording the blank before of the addition of the enzyme thymidine phosphorylase (TP) (20.7 UNI). .sup.19F NMR spectra, recorded at 25° C., showed the singlet of 5-FdUrd at ˜□−165.17 ppm and a new peak at ˜□−169.50 ppm, attributed to 5FU, as shown in
[0257] Then, to prove the cleavage of the nucleoside into the relative base, a new experiment was performed by dissolving equal moles of the nucleoside analogue 5-FdUrd and the relative base 5FU, at the same condition described above without the TS enzyme, as shown in
[0258] When the initial concentration of potassium phosphate buffer was increased from 200 nM up to 205 nM, substrate 5-FdUrd was fully converted into the base 5-FU as shown in the
[0259] Phosphorylase Assay on ProTide Compound CPF 373
[0260] Phosphorylase assay was applied to benzyl L-alanine phenyl derivative CPF 373 in order to investigate the stability, following the procedure and the conditions above described. ProTide compound CPF 373 proved to be completely stable as showed by comparing chemical shifts of sample analysed without TP enzyme, as shown in
[0261] These experiments confirmed that the nucleoside 5-FdUrd is rapidly degraded into its relative base 5FU by a phosphorolytic reaction, in the presence of thymidine phosphorylase, with a half-life of less than 30 minutes, while prodrug compound CPF 373 showed an evident stability against TP enzymatic activity, at longer time exposure up to 3 days. This important result showed that 5-FdUrd Protides derivatives embodying the present invention could favor the therapeutic effect of 5-FdUrd.
[0262] Assay 6. Exposure of 5-FdUrd and CPF-373 to E. coli-Encoded TP and Human-Encoded TP and UP
[0263] The substrate specificity of thymidine phosphorylase towards natural thymidine (dThd), uridine (Urd), 5-FdUrd and CPF-373 was investigated by high pressure liquid chromatography (HPLC). Reaction mixtures containing 100 μM test compound and recombinant TP or UP (human TP: 8.6 ng/μL; E. coli TP: 3.0 ng/μL; human UP: 4 ng/mL) in a total volume of 500 μL reaction buffer (10 mM TrisHCl; 300 μM NaCl; 1 mM EDTA; 2 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4) were incubated at room temperature. At different time points (i.e. 0, 20, 40 min) 100 μL aliquots of the reaction mixtures were withdrawn and heated at 95° C. for 3 min to inactivate the enzyme. The resulting reaction products were separated on a reverse-phase RP-8 column (Merck, Darmstadt, Germany) and quantified by HPLC analysis (Alliance 2690, Waters, Milford, Mass.). The separation of dThd from thymine was performed by a linear gradient from 98% separation buffer (50 mM NaH.sub.2PO.sub.4 and 5 mM heptane sulfonic acid, pH 3.2) and 2% acteonitrile, to 20% separation buffer+80% acetonitrile (8 min 98% separation buffer+2% acetonitrile; 5 min linear gradient of 98% separation buffer+2% acetonitrile to 20% separation buffer+80% acetonitrile; 10 min 20% separation buffer+80% acetonitrile, followed by equilibration at 98% separation buffer+2% acetonitrile). UV-based detection was performed at 267 nm. The separation of Urd from uracil was performed by a linear gradient from 100% separation buffer (see above) to 60% separation buffer 40% acetonitrile (3 min 100% separation buffer; 6 min linear gradient of 100% separation buffer to 60% separation buffer+40% acetonitrile; 6 min 60% separation buffer+40% acetonitrile, followed by equilibration at 100% separation buffer). UV-based detection was performed at 258 nm.
[0264] Phosphorolysis of 5-FdUrd and CPF-373 by Thymidine and Uridine Phosphorylases
[0265] 5-FdUrd and its prodrug CPF-373 were exposed to purified thymidine phosphorylase derived from E. coli or human erythrocytes, and to purified uridine phosphorylase derived from human tumors. Whereas E. coli and human TP rapidly converted dThd and 5-FdUrd to their free bases, CPF-373 kept fully stable in the presence of these enzymes (
[0266] Assay 7. Thymidylate Synthase (TS) Activity Measurements
[0267] The activity of TS in intact L1210/0 and L1210/TK.sup.− cells was measured by evaluation of tritium release from [5-.sup.3H]dUMP (formed in the cells from [5-.sup.3H]dUrd or [5-.sup.3H]dCyd) in the reaction catalysed by TS. This method has been described in detail by Balzarini &. De Clercq (1984). Shortly, cell cultures (500 μL DMEM culture medium) were prepared containing ˜3×10.sup.6 L1210 cells and appropriate amounts of the test compounds (5-FdUrd and CPF-373). After 30 min, 2 h and 4 h pre-incubation of the cells with the compounds at 37° C., 1 μCi of [5-.sup.3H]dUrd or [5-.sup.3H]dCyd was added to the cell cultures. After 30 min incubation, 100 μL of the cell suspensions were withdrawn and added to a cold suspension of 500 μL activated charcoal (VWR, Haasrode, Belgium) (100 mg/ml in TCA 5%). After 10 min, the suspension was centrifuged at 13,000 rpm for 10 min, after which the radioactivity in 400 μL supernatant was counted in a liquid sinctillator using OptiPhase HiSafe (Perkin Elmer, Waldham, Mass.).
[0268] Inhibition of Thymidylate Synthase (TS) by 5-FdUrd and CPF-373
[0269] The major target for the cytostatic activity of 5-FdUrd is thymidylate synthase (TS). The activity of TS in intact tumour cells can be directly monitored by measuring the tritium release in intact L1210/0 cell cultures that were exposed to [5-.sup.3H]deoxyuridine ([5-.sup.3H]dUrd) or [5-.sup.3H]deoxycytidine ([5-.sup.3H]dCyd). Indeed, after intracellular conversion of [5-.sup.3H]dUrd or [5-.sup.3H]dCyd to [5-.sup.3H]dUMP, the C−5 tritium atom on the pyrimidine base is released during the TS-catalysed reductive methylation. The ability of 5-FdUrd and its prodrug CPF-373 to inhibit tritium release from [5-.sup.3H]dUrd and [5-.sup.3H]dCyd was therefore evaluated in L1210/0 cell cultures at a variety of compound concentrations. 5-FdUrd proved to be a potent inhibitor of TS in situ. Its IC.sub.50 for tritium release from [5-.sup.3H]dCyd and [5-.sup.3H]dUrd was around 0.0007-0.0009 μM (see Table 4).
TABLE-US-00005 TABLE 4 IC.sub.50 values of 5-FdUrd and CPF-373 against TS in intact L1210/0 tumour cells (as determined by tritium release from [5-.sup.3H]dUrd and [5-.sup.3H]dCyd after 30 min exposure to the drugs). IC.sub.50.sup.a (μM) Compound Tritium release from dUrd* Tritium release from dCyd* 5-FdUrd 0.0009 ± 0.0002 0.0007 ± 0.003 CPF-373 0.16 ± 0.05 0.19 ± 0.08 .sup.a50% Inhibitory concentration or compound concentration required to inhibit tritium release from [5-.sup.3H]dUrd or [5-.sup.3H]dCyd in drug-exposed L1210/0 cell cultures by 50%.
[0270] The inhibitory activity of CPF-373 on tritium release was much less 200-fold) pronounced than that of 5-FdUrd, especially after only 30 min preincubation of the cells with the drugs (IC.sub.50: 0.16-0.19 μM). However, longer preincubation times of the cells (up to 4 hr) with 5-FdUrd and CPF-373 before measuring TS activity in the intact, tumour cells revealed a much more pronounced inhibitory activity of the prodrug against TS in situ (
[0271] Preincubation of the tumour cells with 5-FdUrd and CPF-373 for at least 4 hrs results in TS inhibition in the intact tumour cells at drug concentrations that are very comparable with the 50% cytostatic activity concentrations of these drugs.
[0272] The present observations thus indicate that the 5-FdUrd prodrug needs several metabolic conversion steps before reaching TS as the target enzyme for inhibition, and support the view that CPF-373 acts as an efficient prodrug of 5-FdUrd to exert its eventual cytostatic activity.
[0273] The activity of TS in the presence of 5-FdUrd and CPF-373 was also measured in intact L1.210/TK.sup.− cells using [5.sup.3H]dCyd as an externally supplied substrate (due to TK deficiency, [5-.sup.3H]dUrd cannot be used). As demonstrated in Table 5 and
TABLE-US-00006 TABLE 5 IC.sub.50 values of 5-FdUrd and CPF-373 against TS in intact L1210/0 and L1210/TK.sup.− cells (as determined by tritium release from [5-.sup.3H]dCyd after 4 hours of preincubation with the products) IC.sub.50.sup.a (μM) Compound L1210/0 L1210/TK.sup.− 5-FdUrd 0.0003 ± 0.00003 1.42 ± 0.09 CPF-373 0.013 ± 0.008 0.053 ± 0.0009 .sup.a50% Inhibitory concentration or compound concentration required to inhibit tritium release from [5-.sup.3H]dCyd in drug-exposed L1210 cells by 50% upon pre-exposure of the tumour cells for 4 hrs to the drugs.
[0274] Assay 8. Stability Assays
[0275] Carboxypeptidase Y (EC 3.4.16.1) Assay
[0276] The enzymatic stability of the prodrug CPF-373 towards carboxypeptidase Y was studied using in situ .sup.31P NMR (202 MHz). The experiment was carried out by dissolving CPF-373 (3.0 mg) in do-acetone (150 μL) and adding TRIZMA buffer pH 7.6 (300 μL). The resulting solution was placed in an NMIR tube and a .sup.31P-NMR experiment at 25° C. was recorded as the blank experiment. The enzyme Carboxypeptidase Y (0.2 mg) was solubilised in TRIZMA (150 μL) and added to the solution of the phosphoramidate derivative in the NMR tube. Next, the tube was placed in the NMR machine, which was set to run a .sup.31P-NMR experiment (64 scans) every 4 minutes for 14 hours at 25° C. Data were processed and analysed with the Bruker Topspin 2.1 program. Carboxypeptidase Y and TRIZMA buffer were purchased from Sigma-Aldrich.
[0277] Human Serum
[0278] The stability of the prodrug CPF-373 in the presence of human serum was studied using in situ .sup.31P NMR (202 MHz). The ProTide CPF-373 (1) (5.0 mg) was dissolved in DMSO (0.05 mL) and D.sub.2O (0.15 mL). Then the sample was transferred into an NMR tube, which was inserted in the NMR chamber at 37° C. (with enough solvent to obtain a control NMR reading of the blank). Then 0.3 ml human serum was quickly added to the sample in the NMR tube. NMR experiments were programmed to record data every 15 minutes for 12 hours and 30 minutes. Because of excess noise and poor shimming profiles (most likely due to the biological media and concentration), individual spectra were further processed. After normal Fourier transform processing, each spectrum was deconvoluted (Lorentz-Gauss deconvolution) to reveal solely the frequency and area of spectral peaks without the baseline. Data recorded were processed and analysed with the Bruker Topspin 2.1 program.
[0279] Buffer at pH 1
[0280] The stability of the prodrug CPF-373 towards hydrolysis at pH=1 was studied using in situ .sup.31P NMR (202 MHz). The ProTide CPF-373 (1) (2.6 mg) was dissolved in MeOD (0.1 mL) after which 0.5 mL buffer (pH=1) (prepared from equal parts of 0.2 M HCl and 0.2 NI KCl) was added. Then the sample was transferred into an NMR tube, and a .sup.31P NMR experiment was performed at 37° C. recording the data every 12 minutes for 14 hours. Data were processed and analysed with the Bruker Topspin 2.1 program.
[0281] Buffer at pH 8
[0282] The stability of the prodrug CPF-373 towards hydrolysis at pH=8 was studied using in situ .sup.31P NMR (202 MHz). The ProTide CPF-373 (1) (4.9 mg) was dissolved in MeOD (0.1 mL) after which 0.5 mL buffer (pH=8) (prepared from a solution of 0.1 M Na.sub.2HPO.sub.4, which was adjusted by 0.1 M HCl) was added. Then the sample was transferred into an NMR tube, and a .sup.31P NMR experiment was performed at 37° C. recording the data every 12 minutes for 14 hours. Data were processed and analysed with the Bruker Topspin 2.1 program.
[0283] Stability Studies
[0284] Chemical stability studies on the prodrug CPF-373 (1) have been performed by exposing the compound to human serum and to aqueous buffers (pH 1.0 and 8.0) using in situ .sup.31P NMR. Each experiment has been carried out dissolving the ProTide in the suitable deuterated solvent and analysing the samples at 37° C. for about 14 hours, acquiring scans at the regular time intervals. For a better resolution original spectra (lower graphs) and deconvoluted ones (upper graphs) are reported. The stability assay of the phosphoramidate CH-373 (1), after incubation in human serum, showed 73% of unchanged compound after 12 hours and 30 minutes as shown in
[0285] The spectra displayed a singlet peak inherent to the human serum at 62.00 and the double peak of the parent at 64.50 which after 4 hours and 15 minutes was hydrolyzed to the aminoacyl phosphoramidate intermediate shown as a singlet peak at 67.20.
[0286] When chemical hydrolysis was evaluated at extreme experimental conditions, i.e. at pH 1.0 and pH 8.0 at 37° C., a full stability of prodrug CPF-373 (1) in both acidic and basic buffer conditions was observed. Spectra were recorded for 14 hours acquiring scans every 12 minutes at regular intervals as shown in the
[0287] Also, at pH 8.0 the spectra displayed a persistent peak of the prodrug (1) at 64.48 and a singlet peak at 62.55 corresponding to a buffer peak (
[0288] Metabolism of 5-FdUrd Phosphoramidates
[0289] As shown in
[0290] To prove the proposed metabolic scheme for CPF-373 (1) and whether the ester motif of the 5-FdUrd phosphoramidate derivative is cleaved-off or not, an enzyme incubation experiment was carried out that was designed to mimic the first stages of the putative activation in the intact tumour cells. The compound (1) was incubated with carboxypeptidase Y (also known as cathepsin A) in TRIZMA buffer and the conversion of (1) was monitored by .sup.31P NMR. Spectra were recorded for 14 h acquiring scans at the periodic intervals every 4 minutes as shown in
[0291] At the .sup.31P NMR the prodrug CPF-373 (1) appeared as two peaks 64.07; 4.23 corresponding with the two diastereoisomers noted as parent with the characteristic doubling-up of the chiral phosphate centre of the phosphoramidate. After the addition of cathepsin A the compound was quickly hydrolyzed after 4 minutes to intermediates 64.95; 5.16 which lack the ester motif and this intermediate did not persist as it was in turn quickly metabolized to the aminoacyl phosphoramidate intermediate, the final product in this assay, via the loss of the aryl group (steps a to c in
CONCLUSION
[0292] In conclusion, the present invention provides novel phosphoramidate nucleotide prodrugs of the anticancer nucleoside analogue 5-fluoro-2′-deoxyuridine (5-FdUrd), which were synthesized and evaluated for their cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, compounds of the present invention, for example CPF-373, markedly kept their antiproliferative activity in both the wild-type and TK-deficient tumour cells and are thus largely independent of intracellular TK activity to exert their cytostatic action. CPF-373, for example, was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. CPF-373 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with the observations that 5-FdUrd, but not CPI′-373, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, compounds of the present invention such as CPF-373 are novel 5-FdUrd phosphoramidate prodrugs that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP whose activity is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue. Incorporated in by reference in its entirety is Vande Voorde, J. et al Biochemical Pharmacology 82 (2011) 441-452.
[0293] Embodiments of the present invention, as set out below, are disclosed in McGuigan, C. et al J. Med. Chem. 2011, 54 7247-7258 (published Sep. 5, 2011), the contents of which in their entirety are hereby incorporated by reference.
[0294] Table 6 below records the cytostatic activity of 5-FU, 5-FdUrd, reference example CPF382 and compounds embodying the present invention against tumour cell lines in terms of IC.sub.50 or compound concentration required to inhibit tumour cell proliferation by 50%. Data are the mean (±SD) of at least two to four independent experiments. Table 6 identifies the phosphoramidate motif of reference example CPF382 and of compounds embodying the present invention with respect to: “aryl”, which corresponds to Ar of Formula I and is either phenyl (Ph) or 1-naphthyl (Nap); “ester”, which corresponds to R.sub.3 of Formula and “AA”, which sets out the amino acid whose alpha C atom and substituents on the alpha C atom correspond to CR.sub.1R.sub.2 of Formula I. Table 6 discloses compounds embodying the present invention not previously mentioned above in Table 1, as well as additional data for some of the compounds mentioned in Table 1.
TABLE-US-00007 TABLE 6 IC.sub.50 (μM) Compd aryl Ester AA L1210/0 L1210/TK.sup.− Cem/0 Cem/TK.sup.− HeLa HeLa/TK.sup.− 5-FU 0.33 ± 0.17 0.32 ± 0.31 18 ± 5 12 ± 1 0.54 ± 0.12 0.23 ± 0.01 5-FdUrd 0.0011 ± 0.0002 3.0 ± 0.1 0.022 ± 0.006 3.0 ± 0.4 0.050 ± 0.011 1.4 ± 0.4 Ph Me Ala 0.022 ± 0.007 41 ± 3 0.70 ± 0.37 35 ± 12 0.28 ± 0.14 4.7 ± 0.4 Ph Et Ala 0.13 ± 0.04 0.94 ± 0.18 0.92 ± 0.11 14 ± 0 0.48 ± 0.19 9.8 ± 1.4 Ph i-Pr Ala 0.076 ± 0.022 1.1 ± 0.1 1.0 ± 0.1 30 ± 10 0.71 ± 0.15 25 ± 11 Ph c-Hex Ala 0.039 ± 0.001 0.14 ± 0.02 0.17 ± 0.07 1.2 ± 0.01 0.18 ± 0.05 5.9 ± 0.4 Ph Bn Ala 0.028 ± 0.007 13 ± 8 0.18 ± 0.03 22 ± 7 0.13 ± 0.01 19 ± 2 Ph Et Val 0.16 ± 0.05 42 ± 2 1.0 ± 0.1 >250 1.2 ± 0.3 27 ± 7 Ph Bn Leu 0.044 ± 0.025 2.0 ± 0.3 0.24 ± 0.04 16 ± 1 0.067 ± 0.042 5.6 ± 0.3 Ph Bn Ile 0.076 ± 0.022 1.1 ± 0.1 1.0 ± 0.1 30 ± 10 0.71 ± 0.15 25 ± 11 Ph Bn Phe 0.036 ± 0.010 39 ± 4 0.25 ± 0.02 11 ± 3 0.014 ± 0.007 12 ± 2 Ph Pnt Met 0.11 ± 0.06 2.2 ± 0.5 0.35 ± 0.13 13 ± 1 0.15 ± 0.00 7.1 ± 1.2 Ph Bn Met 0.073 ± 0.035 4.1 ± 1.2 0.28 ± 0.03 25 ± 0 0.15 ± 0.02 11 ± 7 Ph Bn Pro 0.35 ± 0.07 31 ± 5 0.98 ± 0.40 28 ± 8 1.1 ± 0.4 20 ± 11 Ph Et DMG 0.039 ± 0.001 4.6 ± 0.0 0.65 ± 0.16 22 ± 1 0.59 ± 0.09 17 ± 2 Ph Bn DMG 0.017 ± 0.003 0.18 ± 0.05 0.23 ± 0.04 4.8 ± 0.7 0.24 ± 0.07 3.7 ± 0.1 Nap Et Ala 0.031 ± 0.005 0.36 ± 0.01 0.25 ± 0.04 1.6 ± 0.2 0.22 ± 0.04 2.8 ± 0.0 Nap Pr Ala 0.021 ± 0.012 0.16 ± 0.07 0.14 ± 0.01 1.1 ± 0.2 0.11 ± 0.03 2.5 ± 0.1 Nap Butyl Ala 0.022 ± 0.004 0.11 ± 0.06 0.064 ± 0.007 0.84 ± 0.60 0.12 ± 0.02 2.7 ± 1.5 Nap Pnt Ala 0.0028 ± 0.0010 0.13 ± 0.13 0.015 ± 0.006 0.28 ± 0.04 0.029 ± 0.023 0.44 ± 0.35 Nap Hex Ala 0.0072 ± 0.0000 0.076 ± 0.015 0.080 ± 0.020 0.65 ± 0.34 0.039 ± 0.018 1.8 ± 0.1 Nap c-Bu Ala 0.014 ± 0.003 0.088 ± 0.038 0.073 ± 0.018 1.5 ± 0.3 0.069 ± 0.003 1.5 ± 0.6 Nap c-Pnt Ala 0.031 ± 0.010 0.13 ± 0.02 0.35 ± 0.025 0.92 ± 0.007 0.071 ± 0.036 2.2 ± 1.3 Nap c-Hex Ala 0.043 ± 0.023 0.15 ± 0.00 0.057 ± 0.055 1.0 ± 0.1 0.090 ± 0.014 ND Nap CH.sub.2-t-Bu Ala 0.27 ± 0.11 1.2 ± 0.7 0.49 ± 0.05 6.7 ± 1.0 0.70 ± 0.11 32 ± 26 Nap CH.sub.2CH.sub.2-t-Bu Ala 0.016 ± 0.006 0.062 ± 0.009 0.053 ± 0.021 0.19 ± 0.04 0.078 ± 0.018 1.3 ± 0.9 Nap CH.sub.2-c-Pr Ala 0.017 ± 0.007 0.12 ± 0.06 0.059 ± 0.017 1.1 ± 0.2 0.068 ± 0.001 1.4 ± 0.4 Nap 2-Ind Ala 0.021 ± 0.002 40 ± 0 0.079 ± 0.018 1.0 ± 0.2 0.10 ± 0.06 7.1 ± 2.1 Nap Bn Ala 0.011 ± 0.007 0.045 ± 0.027 0.068 ± 0.035 0.31 ± 0.06 0.065 ± 0.013 2.5 ± 1.3 Nap THP Ala 0.038 ± 0.014 27 ± 6 0.11 ± 0.02 43 ± 12 0.13 ± 0.04 15 ± 7 Nap c-Hex Val 1.1 ± 0.5 35 ± 8 0.80 ± 0.28 46 ± 14 0.67 ± 0.03 27 ± 6 Nap Pnt Leu 0.017 ± 0.001 1.2 ± 0.4 0.071 ± 0.008 15 ± 4 0.039 ± 0.014 7.5 ± 0.4 Nap Bn Leu 0.028 ± 0.004 1.5 ± 0.6 0.13 ± 0.00 30 ± 6 0.080 ± 0.022 9.4 ± 1.4 Nap Pnt Ile 0.22 ± 0.12 12 ± 2 0.46 ± 0.11 17 ± 1 0.30 ± 0.02 11 ± 1 Nap Pnt Phe 0.026 ± 0.001 2.9 ± 1.2 0.10 ± 0.00 8.3 ± 1.0 0.040 ± 0.000 6.6 ± 0.5 Nap Bn Phe 0.012 ± 0.007 5.6 ± 1.3 0.10 ± 0.03 7.2 ± 0.1 0.16 ± 0.08 6.8 ± 1.5 Nap Bn Met 0.072 ± 0.001 1.9 ± 0.2 0.19 ± 0.10 11 ± 1 0.087 ± 0.017 8.3 ± 0.0 Nap Bn Pro 0.21 ± 0.08 25 ± 8 0.89 ± 0.35 35 ± 9 1.2 ± 0.0 26 ± 1 Nap Et DMG 0.064 ± 0.008 0.82 ± 0.16 0.36 ± 0.05 6.9 ± 1.8 0.20 ± 0.12 3.2 ± 0.0 Nap Pnt DMG 0.037 ± 0.010 0.30 ± 0.13 0.14 ± 0.00 5.4 ± 1.1 0.12 ± 0.03 2.3 ± 0.1 Nap Bn DMG 0.011 ± 0.005 0.13 ± 0.04 0.16 ± 0.02 2.4 ± 0.8 0.078 ± 0.020 3.1 ± 0.6
[0295] Table 7 below records the cytostatic activity of 5-FdUrd, reference example CPF382 and compounds embodying the present invention in wild type murine leukemia. L1210 cell cultures (L1210/0) and L1210 cell cultures, infected with Mycoplasma hyorhinis (L1210.Hyor) in terms of IC.sub.50 or compound concentration to inhibit cell proliferation by 50%. Data are mean (±SD) of at least two to four independent experiments. Table 7 identifies the phosphoramidate motif of reference example CPF382 and of compounds embodying the present invention, as discussed above with respect to Table 6, but with “Naph” standing for 1-naphthyl. Table 7 discloses compounds embodying the present invention not previously mentioned above in Table 2, as well as additional data for some of the compounds mentioned in Table 2,
TABLE-US-00008 TABLE 7 IC.sub.50 (μM) IC.sub.50(L1210.Hyor)/ Compd aryl ester AA L1210/0 L1210.Hyor IC.sub.50(L1210/0) 5-FdUrd 0.0009 ± 0.0003 0.34 ± 0.13 378 Ph Me Ala 0.040 ± 0.016 0.87 ± 0.28 22 Ph Et Ala 0.11 ± 0.0021 0.54 ± 0.12 5 Ph i-Pr Ala 0.050 ± 0.013 0.70 ± 0.10 14 Ph c-Hex Ala 0.032 ± 0.0050 0.040 ± 0.016 1.25 Ph Bn Ala 0.026 ± 0.008 0.15 ± 0.006 5.8 Ph Et Val 0.20 ± 0.033 4.4 ± 1.1 22 Ph Bn Leu 0.054 ± 0.0021 0.17 ± 0.047 3.2 Ph Bn Ile 0.98 ± 0.39 2.2 ± 0.031 2.2 Ph Bn Phe 0.016 ± 0.0014 0.56 ± 0.023 35 Ph Pnt Met 0.13 ± 0.0078 0.41 ± 0.21 3.2 Ph Bn Met 0.058 ± 0.035 0.76 ± 0.18 13 Ph Bn Pro 0.35 ± 0.022 18 ± 0.71 51 Ph Et DMG 0.030 ± 0.0005 0.26 ± 0.01 8.7 Ph Bn DMG 0.029 ± 0.001 0.02 ± 0.002 0.69 Naph Et Ala 0.028 ± 0.0021 0.095 ± 0.0028 3.4 Naph Pr Ala 0.030 ± 0.00035 0.036 ± 0.0064 1.2 Naph butyl Ala 0.0095 ± 0.0021 0.021 ± 0.0071 2.2 Naph Pnt Ala 0.0021 ± 0.00007 0.006 ± 0.0014 2.9 Naph Hex Ala 0.0032 ± 0.00035 0.0022 ± 0.00028 0.69 Naph c-Bu Ala 0.011 ± 0.0014 0.024 ± 0.00014 2.2 Naph c-Pnt Ala 0.016 ± 0.0007 0.024 ± 0.005 1.5 Naph c-Hex Ala 0.036 ± 0.017 0.049 ± 0.004 1.4 Naph CH.sub.2-t-Bu Ala 0.093 ± 0.033 0.18 ± 0.069 1.9 Naph CH.sub.2 CH.sub.2-t-Bu Ala 0.012 ± 0.0018 0.032 ± 0.0088 2.7 Naph CH.sub.2-c-Pr Ala 0.014 v 0.0042 0.031 ± 0.0064 2.2 Naph 2-Ind Ala 0.039 ± 0.019 0.042 ± 0.040 1.08 Naph Bu Ala 0.011 ± 0.009 0.025 ± 0.01 2.27 Naph THP Ala 0.041 ± 0.0028 0.48 ± 0.11 11.7 Naph c-Hex Val 1.2 ± 0.17 1.29 ± 0.29 1.08 Naph Pnt Leu 0.031 ± 0.0020 0.035 ± 0.010 1.13 Naph Bn Leu 0.029 ± 0.0021 0.048 ± 0.020 1.7 Naph Pnt Ile 0.42 ± 0.021 0.70 ± 0.074 1.67 Naph Pnt Phe 0.030 ± 0.0039 0.14 ± 0.007 4.67 Naph Bn Phe 0.021 ± 0.0061 0.23 ± 0.078 11 Naph Bn Met 0.054 ± 0.013 0.20 ± 0.098 3.7 Naph Bn Pro 0.26 ± 0.055 0.65 ± 0.070 2.5 Naph Et DMG 0.056 ± 0.04 0.17 ± 0.03 3 Naph Pnt DMG 0.045 ± 0.0021 0.019 ± 0.0028 0.42 Naph Bn DMG 0.019 ± 0.004 0.045 ± 0.004 2.4
[0296] Table 8 below records the cytostatic activity of 5-FdUrd and compounds embodying the present invention in CEM cell cultures containing (Cem/hEnt-1) or lacking (Cem/hEnt-0) the hEnt1 transporter in terms of IC.sub.50 or compound concentration required to inhibit tumour cell proliferation by 50%. Data are mean (±SD) of at least two to four independent experiments. Table 8 identifies the phosphoramidate motif of compounds embodying the present invention, as discussed above with respect to Table 6, but with “Naph” standing for 1-naphthyl. The data of Table 8 show that compounds embodying the present invention are less dependent on the presence of the hENT1 transporter, than 5-FdUrd, since they lose only 7- to 15-fold antiproliferative activity against the hENT1-deficient CEM cells. These observations are in agreement with an only 2- to 7-fold decreased cytostatic activity of compounds embodying the present invention in the presence of transport inhibitors (i.e., dipyridamole and NBMPR), compared to a 20- to 60-fold loss of antiproliferative activity of 5-FdDrd and HUMP under similar experimental conditions.
TABLE-US-00009 TABLE 8 IC.sub.50 (μM) Cem/hEnt-1 + Cem/hEnt-1 + compd aryl Ester AA Cem/hEnt-1 Cem/hEnt-0 dipyridamole NBMPR 5-FdUMP 0.05 ± 0.02 3.6 ± 0.69 1.74 1.06 5-FdUrd 0.04 ± 0.02 2.5 ± 0.65 1.36 0.80 Ph Bn Ala 0.13 ± 0.05 1.4 ± 0.65 0.66 0.72 Ph Et DMG 0.37 ± 0.14 5.8 ± 0.50 2.35 2.56 Ph Bn DMG 0.17 ± 0.06 1.2 ± 0.11 0.26 0.61 Naph Bn Ala 0.05 ± 0.02 0.6 ± 0.11 0.13 0.26 Naph Et DMG 0.21 ± 0.07 1.4 ± 0.20 0.52 0.62 Naph Bn DMG 0.05 ± 0.03 0.4 ± 0.13 0.16 0.28
[0297] Studies were performed on compound CPF 381 as follows:
[0298] An enzymatic phosphorylase assay was carried out using thymidine phosphorylase (TP, purified from Esherichia coli) in the presence of potassium phosphate buffer (300 nM solution, pH 7.4). The .sup.19F NMR spectrum after 5 min, 14 h and 72 h did not show any evidence of phosphorolysis. In contrast to 5-FdUrd, CPF 381 is at best a very poor, if any, substrate for thymidine phosphorylase.
[0299] A chemical hydrolysis was evaluated under experimental conditions at pH 1 and pH 8 and monitored by .sup.31P NMR. During the assay (14 h) under acidic conditions (pH 1) only two peaks representing the two diastereoisomers were recorded. Lack of formation of new signals in the .sup.31P NMR spectrum indicates that compound CPF 381 is highly stable in acidic medium. The same result was observed when compound CPF 381 was subjected to the assay under mild basic conditions (pH 8).
[0300] Studies were performed on compound CPF 581 as follows:
[0301] A enzymatic study using a carboxypeptidase Y assay was performed in which compound CET 581, carboxypeptidase Y, and Trizma buffer (pH 7.6) were dissolved in acetone-d.sub.6 and .sup.31P NAIR spectrum (202 MHz) spectra were recorded at regular intervals (every 7 min) over 14 h. Compound CPF 581 was rapidly hydrolyzed to a first metabolite lacking the ester (R.sub.3) moiety, both diastereoisomers being processed at roughly similar rate. Further processing of the first metabolite led to the formation of an anionic second metabolite, lacking Ar, within about 45 min with an estimated half life of less than 5 min. The rate of the initial activation step might thus be considered in general as one of requirements for good biological activity of phosphoramidates. Chemical hydrolysis of compound CPF 373 in the presence of triethylamine and water produced the diammonium salt of the anionic second metabolite, which was added to the final assay sample derived from compound CPF 373, i.e. containing only the enzymatic second metabolite derived from compound CPF 581 in Trizma. The sample had a .sup.31P NMR spectrum showing only, a single peak at δ.sub.P 6.85 ppm, strongly supporting this part of the metabolic pathway and activation of the phosphoramidate compounds of the present invention.
[0302] Studies were performed on compound CPF 386 as follows:
[0303] The stability of compound CPF 386 in the presence of human serum was investigated using in situ .sup.31P NMR. A control .sup.31P NMR data of compound CPF 386 in DMSO and D.sub.2O were recorded. The NMR sample was then treated with human serum and immediately subjected to further .sup.31P NMR experiments at 37° C. The .sup.31P NMR data were recorded every 15 minutes over 14 h. The spectra displayed a single peak inherent to human serum at ˜δ.sub.P 2.00 ppm and two peaks corresponding to compound CPF 386 at −δ.sub.P 4.59 and 4.84 ppm. After about 6 h and 45 min the compound was hydrolyzed partly to an intermediate, lacking R.sub.3, (Et), as a single peak at δ.sub.P 5.79 ppm. After 11 h and 30 min, the formation of the second metabolite, lacking Ar (1-naphthyl), shown as single peak at δ.sub.P 7.09 ppm was observed. After 13 h and 30 min the reaction mixture contained 96% of the parent compound CPF 386 together with the proposed first metabolite (3%) and second metabolite (1%).
REFERENCES
[0304] Agarwal R P, Han T, Fernandez M, Collateral resistance of a dideoxycytidine-resistant cell line to 5-fluoro-2′-deoxyuridine. Biochem Biophys Res Commun 1999; 262:657-60, [0305] Ayusawa D, Koyama H, Iwata K, Seno T, Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase. Somatic Cell Genet 1980; 6:261-70, [0306] Ayusawa D, Koyama H, Seno T, Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FM3A cells. Cancer Res 1981; 41:1497-501. [0307] Balzarini J, De Clercq Torrence P F, Mertes M P, Park E S, Schmidt C L, Shugar D, Barr P J, Jones A S, Verhelst G, Walker R T, Role of thymidine kinase in the inhibitory activity of 5-substituted-2′-deoxyuridines on the growth of human and murine tumor cell lines. Biohcem Pharmacol 1982:31:1089-1095 [0308] Balzarini J and De Clercq E, Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells. Biochim Biophys Acta 1984; 785:36-45. [0309] Beck A, Etienne M C, Cheradame S. Fischel J L, Fomento P, Renee N, Milano G, A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil. Eur J Cancer 1994; 30A:1517-22. [0310] Bronckaers A, Balzarini J, Liekens S, The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: Implications for cancer therapy, Biochem Pharmacol 2008; 76:188-97. [0311] Bronckaers a, Gago Balzarini J, Liekens S, The dual role of thymidine phosphorylase in cancer development and chemotherapy. Med Res Rev 2009; 29:903-53. [0312] Chan P J, Seraj I M, Kalugdan T H, King A, Prevalence of mycoplasma conserved DNA in malignant ovarian cancer detected using sensitive PCR-ELISA. Gynecol Oncol 1996; 63:258-60. [0313] Charron J and Langelier Y, Analysis of deoxycytidine (dC) deaminase activity in herpes simplex virus-infected or HSV TK-transformed cells: association with mycoplasma contamination but not with virus infection. J Gen Virol. 1981; 57:245-50, [0314] Ciaparrone M. Quirino M, Schinzari G, Zannoni G, Corsi D C, Vecchio F M, Cassano A, La Torre G, Barone C. Predictive role of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase expression in colorectal cancer patients receiving adjuvant 5-fluorouracil. Oncology 2006; 70:366-77. [0315] Ciccolini J, Evrard A, Cuq P. Thymidine phosphorylase and fluoropyrimidines efficacy: a Jekyll and Hyde story. Curr Med Chem Anticancer Agents 2004; 4:71-81. [0316] Congiatu C, Brancale A, Mason M D, Jiang W G, McGuigan C, Novel potential anticancer naphthyl phosphoramidates of BVdU separation of diastereoisomers and assignment of the absolute configuration of the phosphorus center. J Med Chem 2006; 49:452-5, [0317] de Bruin M, van Capel T, Smid K, van der Born K, Rikushima M, Hoekman K, Pinedo H M, Peters G J, Role of platelet derived endothelial cell growth factor/thymidine: phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1-phosphate. Nucleosides Nucleotides Nucleic Acids 2004; 23:1485-90. [0318] Cialmarini C M, Mackey J R, Dumontet C, Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 2002; 3:415-24. [0319] Harris S A, McGuigan C, Andrei G, Snoeck R, De C E, Balzarini J, Synthesis and antiviral evaluation of phosphoramidate derivatives of (E)-5-(2-bromovinyl)-2′-deoxyuridine. Antivir Chem Chemother 2001; 12:293-300. [0320] Hatse 5, De C E, Balzarini J, Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. Biochem Pharmacol 1999; 58:539-55. [0321] Hecker S J and Erion M D, Prodrugs of phosphates and phosphonates. J Med Chem 2008; 51:2328-45. [0322] Huang S, Li J Y, Wu J, Meng L, Shou C C, Mycoplasma infections and different human carcinomas. World J Gastroenterol 2001; 7:266-9. [0323] Ishikawa T, Ltoh M, Sawada N, Nishida M, Fukase Y, Sekiguchi F, Ishitsuka H, Tumor selective delivery of 5-fluorouracil by capecitabine, a new oral fluoropyrimidine carbamate, in human cancer xenografts. Biochem Pharmacol 1998; 55:1091-7, [0324] Jetté L, Bissoon-Haqqani S, Le F B, Maroun J A, Birnboim H C, Resistance of colorectal cancer cells to 5-FUdR and 5-FU caused by Mycoplasma infection, Anticancer Res 2008; 28:2175-80. [0325] Kamoshida S, Shiogama K, Shimomura R, Inada K, Sakurai Y, Ochiai M. Matuoka H, Maeda K, Tsutsumi Y. Immunohistochemical demonstration of fluoropyrimidine-metabolizing enzymes in various types of cancer. Oncol Rey. 2005, 14:1223-30, [0326] Kidder M, Chan P J, Seraj I M, Patton W C, King A. Assessment of archived paraffin-embedded cervical condyloma tissues for mycoplasma-conserved DNA using sensitive PCR-ELISA. Gynecol Oncol 1998; 71:254-757, [0327] Kinsella A R and Smith D, Tumor resistance to antimetabolites. Gen Pharmacol 1998; 30:623-6. [0328] Koopman M, Venderbosch S. Nagtegaal I D, van Krieken J H, Punt C J. A review on the use of molecular markers of cytotoxic therapy for colorectal cancer, what have we learned? Eur J Cancer 2009; 45: 1935-49. [0329] Kosaka T, Usami K, Ueshige N, Sugaya J. Nakano Y, Takashima S. Effects of thymidine phosphorylase levels in cancer, background mucosa, and regional lymph nodes on survival of advanced gastric cancer patients receiving postoperative fluoropyrimidine therapy. Oncol Rep 2004; 12:1279-86. [0330] Liekens S, Bronckaers A, Balzarini J, Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasma-encoded catabolic enzymes. Lancet Oncol 2009; 10:628-35, [0331] Longley D B, Harkin D P, Johnston P G, 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003; 3:330-8, [0332] McGuigan, C.; Pathirana, R. N.; Balzarini, J.; De Clercq, E. Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. J. Med. Chem. 1993, 36, 1048. [0333] McGuigan, C.; Cahard, D.; Hendrika, D.; Sheeka, M.; De Clercq, E.; Balzarini, J. Aryl phosphoramidate derivatives of d4T have improved anti-HIV efficacy in tissue vulture and may act by the generation of a novel intracellular metabolite. J. Med. Chem. 1996, 39, 1748. [0334] McGuigan, C.; Tsang, H. W.; Cahardr, D. Turner, K.; Velazquez, S.; Salgado, Bidois, L.; Naesens, L.; De Clercq, E.; Balzarini, J. Phosphoramidate derivatives of d4T as inhibitors of HIV: The effect of amino acid variation. Antiviral Res. 1997, 35, 195, [0335] McGuigan, C.; Mehellou, Balzarini, J. Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. Chem. Med. Chem., 2009, 4, 11, 1779. [0336] McGuigan C, Gilles A, Madela K, Aljarah M, Holl S; Jones S, Vernachio J, Hutchins J, Ames B, Bryant K B, Gorovits E, Ganguly B, Hunley D, Hall A, Koiykhalov A, Liu Y. Muhammad J, Raja N, Walters R, Wang J, Chamberlain 5, Henson G, Phosphoramidate Protides of 2′-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties. J Chem 2010; 53:4949-57. [0337] Mehellou Y, Balzarini J, McGuigan C, Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. ChemMedChem 2009; 4:1779-91. [0338] Mehellou, Y.; Valente, R.; Mottram, H.; Walsby, E.; Mills, K. I.; Balzarini, J.; Mcgugan, C. Phosphoramidates of 2′-β-d-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism. Bioorg. Med. Chem. 2010, 18, 2439 [0339] Moertel C G, Chemotherapy for colorectal cancer. N Engl J Med 1994; 330:1136-42. [0340] Murakami Y, Kazuno H, Emura T, Tsujimoto H, Suzuki N, Fukushima M, Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells. Int J Oncol 2000; 17:277-83. [0341] Neale G A, Mitchell A, Finch L R, Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides. J Bacteriol 1983; 156:1001-5. [0342] Pehlivan M, Pehlivan S, Onay H, Koyuncuoglu M, Kirkali Z, Can mycoplasma-mediated oncogenesis be responsible for formation of conventional renal cell carcinoma? Urology 2005; 65:411-414 [0343] Peters G J and Kohne C H, Resistance to antimetabolites. In: Fluoropyrimidines as Antifolate Drugs in Cancer Therapy. Jackman A L (ed). Humana Press Inc 1999; pp 101-145. [0344] Razin S, The mycoplasmas. Microbiol Rev 1978; 42:414-70. [0345] Razin S, Yogev D, Naot Y, Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 1998; 62:1094-156. [0346] Sasaki H, Igaki H, Ishizuka. T, Kogoma Y, Sugimura T, Terada Ail, Presence of Streptococcus DNA sequence in surgical specimens of gastric cancer. Jpn J Cancer Res 1995; 86:7914. [0347] Seno T, Ayusawa D, Shimizu K, Koyama H, Takeishi K, Hori T, Thymineless death and genetic events in mammalian cells. Basic Life Sci 1985; 31:241-63. [0348] Seno T, Ayusawa D, Shimizu K, Koyama H, Takeishi K, Hori T, Thymineless death and genetic events in mammalian cells. Basic Life Sci 1985; 31:241-63. [0349] Sinigaglia F and Talmadge K W, Inhibition of [3H]thymidine incorporation by Mycoplasma arginini-infected cells due to enzymatic cleavage of the nucleoside. Eur J Immunol 1985; 15:692-6. [0350] Sotos G A, Grogan L, Allegra C J, Preclinical and clinical aspects of biomodulation of 5-fluorouracil. Cancer Treat Rev 1994; 20:11-49. [0351] Tanaka F, Fukuse T, Wada H, Fukushima M, The history, mechanism and clinical use of oral 5-fluorouracil derivative chemotherapeutic agents. Curr Pharm Biotechnol 2000; 1:137-64. [0352] Tham T N, Ferris S, Kovacic R, Montagnier L, Blanchard A, Identification of Mycoplasma pirum genes involved in the salvage pathways for nucleosides. J Bacteriol 1993:175:5281-5. [0353] Walko C M and Lindley C, Capecitabine: a review. Clin Ther 2005; 27:23-44. [0354] Yang H, Qu L, Ma H, Chen L, Liu W, Liu C, Meng L, Wu J, Shou C, Mycoplasma hyorhinis infection in gastric carcinoma and its effects on the malignant phenotypes of gastric cancer cells. BMC Gastroenterol 2010; 10:132