5-FLUORO-2'-DEOXYURIDINE PHOSPHORAMIDATE DERIVATIVES, ITS USES AND PREPARATION PROCESS, PHARMACE

专利摘要:
5-fluoro-2'-deoxyuridine phosphoramidate derivatives for use in cancer treatment. The present invention relates to 5-fluoro-2'-deoxyuridine phosphoramidate derivatives are described for use in the treatment of cancer, especially in the treatment of cancer where the patient exhibits resistance, for example, in a patient with cells with a decreased level of nucleoside transporter proteins and/or with cells deficient in nucleoside kinase and/or with cells infected with mycoplasma and/or with cells with an increased level of thymidylate synthase.
公开号:BR112013021986B1
申请号:R112013021986-6
申请日:2012-02-29
公开日:2021-07-27
发明作者:Christopher Mcguican；Jan Balzarini；Magdalena Slusarczyk；Blanka Gonczy；Paola Murziani
申请人:Nucana Biomed Limited；
IPC主号:

专利说明:

[001] The present invention relates to chemical compounds useful in the treatment of cancer.
[002] In 1957, the antitumor activity of 5-Fluorouracil (5FU) was found. More than fifty years later, it was first synthesized, 5FU remains widely used in the treatment of solid tumors including breast, gastrointestinal system, head, neck and ovary and in particular colorectal cancer, as approved by the FDA in 1962. The fluoropyrimidine 5-fluoro -uracil (5FU) and 5-fluoro-2'-deoxyuridine (5-FdUrd) are used in combination with folic acid as standard treatment for a variety of carcinomas such as stomach, colon and breast. In addition, a combination of 5FU with leucovorin (LV) is considered standard chemotherapy for colon cancer. The 5FU drug is usually given by intravenous bolus or continuous infusion.
[003] The antitumor activity of 5FU is comparable to that of its 5-FdUrd analogue, which partially acts as a prodrug of 5FU. 5-FdUrd was approved by the FDA in 1970, and has been extensively employed for the clinical treatment of carcinoma of the ovary, breast, and gastrointestinal tract. Furthermore, due to extensive hepatic extraction 5-FdUrd is a useful drug for hepatic arterial chemotherapy of liver metastases as it is more efficiently metabolized by the liver than 5FU.
There is, therefore, a problem that activity of both the 5FU and 5-FdUrd agents can be impaired by the development of resistance in tumor cells. Cancer treatment with 5FU has also been found to cause neurotoxic and cardiotoxic side effects. Toxicity also derives from the loss of sensitivity of 5FU to tumors.
[005] It is an aim of the present invention to provide 5-fluoro-2'-deoxyuridine derivative compounds that exhibit enhanced activity and/or reduced toxicity in their cancer treatment, compared to that exhibited by 5-fluoro-2'-deoxyuridine or 5-fluoro-2 '-deoxyuridine per se.
It is another object of the present invention to provide 5-fluoro-2'-deoxyuridine derivative compounds that exhibit a low level of resistance in tumor cells, in particular a resistance in tumor cells that is less than that shown by 5FU or by 5-FdUrd.
[007] According to the present invention there is provided a compound of Formula (I):

[008] in which
[009] Ar is a monocyclic aryl moiety or fused bicyclic aryl moiety, any of which aryl moieties is carbocyclic or heterocyclic and is optionally substituted;
[0010] R3 is alkyl, which is optionally substituted;
[0011] R4 is H or alkoxy; and
[0012] R1 and R2 are independently selected from the group consisting of H and alkyl or R1 and R2 together form an alkylene chain in order to provide, together with the C atom to which they are attached, a cyclic system, or one of R1 and R2 comprises an alkylene chain bonded to the N atom, H bonded to the N is absent and one of R1 and R2 comprises H or alkyl, any of which alkyl moieties or alkylene chains may be substituted;
[0013] or a pharmaceutically acceptable metabolite or derivative of Formula I,
wherein the compound is not a compound having, in combination, Ar as unsubstituted phenyl, R3 as CH3, R4 as H and one of R1 and R2 as H and one of R1 and R2 as CH3.
[0015] It has been found that the compounds of the present invention exhibit activity that makes them useful in the prophylaxis or treatment of cancer in homo sapiens. In particular, the present compounds exhibit beneficial properties that indicate their ability to treat cancer in patients while exhibiting reduced resistance in tumor cells. Notably, the compounds of the present invention can exhibit cytoactivity comparable to or better than that of 5-fluoroacyl, but with a resistance that is comparable to or less than that of either 5-fluoroacyl and 5-fluoro-2'-deoxyuridine.
By "resistance" in the present application a low or diminished response to therapy is intended. Resistance can be innate or acquired. An innate resistance is a reduced sensitivity relative to other specimens or patients. An acquired resistance is a reduced efficacy over the course of time in a patient administered, whether or not acquired in conjunction with therapy comprising administering to the patient a drug regimen to treat cancer, for example, a drug regimen comprising 5FU and /or 5-FdUrd. Each innate resistance and acquired resistance may correspond to down-regulation or low activity of transporter proteins, including nucleoside transporter proteins, or required anabolic enzymes, or up-regulation of catabolic enzymes.
[0017] Although the applicant does not wish to be bound by any theory, it is postulated, as also discussed below, that the cause of resistance in tumor cells to the activity of 5FU and/or 5-FdUrd must be: a) deletion of activation of kinase such as thymidine kinase (TK), a major enzyme required for the initial phosphorylation step from 5-FdUrd to 5-FdUMP; b) overproduction of thymidylate synthase (TS); and/or c) defective transport in target cells.
Surprisingly it has now been found that compounds of the present invention can exhibit significant cytostatic activity in cells with a decreased level of nucleoside transporter proteins and/or with nucleoside kinase deficient cells and/or in mycoplasma infected cells.
[0019] The beneficial property of the compounds of the present invention to maintain enhanced cytostatic activity in nucleoside kinase deficient cells may confer in vivo a clinical advantage in cell media requiring nucleoside kinases or having decreased levels of nucleoside kinases and thus unable to efficiently activate 5-FdUrd.
Mycoplasma-infected cells greatly reduce the activity of nucleosides such as 5-FdUrd due to, it is believed, the overproduction of thymidylate synthase (TS). The currently proposed use of the present compounds in mycoplasma infected cells is thus postulated to derive from the beneficial property of the present compounds to further act as a TS inhibitor and thus allow the present compounds to maintain their cytostatic activity in mycoplasma infected cells. Prodrugs comprising the compounds of the present invention, due to their lipophilic nature, can be taken up by target cells in at least partially a nucleoside transport vehicle-independent form, and thus can avoid potential resistance mechanisms due to levels diminished from transport vehicles of nucleoside or nucleobases in the target cell membrane.
[0021] Additionally, the prodrugs comprising the compounds of the present invention are surprisingly insensitive to the action of the catabolic enzyme Thymidinephosphorylase (TP) which is often up-regulated in tumors, and thus, the prodrugs would be more independent of its presence. catabolic enzyme than 5-FdUrd.
[0022] It has been observed that mycoplasma infection of cells can greatly reduce the activity of nucleosides, including 5-FdUrd. Administration of a TP inhibitor restores the cytostatic activity of 5-FdUrd in mycoplasma-infected cell cultures, providing evidence of the deteriorating role of TP in the eventual cytostatic activity of 5-FdUrd. This may be a limitation in patients who are infected with mycoplasma. Unlike 5-FdUrd, the 5-FdUrd prodrugs of the present invention can maintain high activity in these mycoplasma infected cells.
The present compounds thus have the potential to overcome many of the limitations of 5-FU and 5-FdUrd.
[0024] 5-Fluorouracil (5FU) is one of the first examples of an anticancer drug. The purpose of 5-FU was based on available biochemical information: a fluorine atom and a hydrogen atom are similar in size, so a carbon-fluorine bond is stronger than a carbon-hydrogen bond. Thymidylate synthase acts by replacing the 5-hydrogen in deoxyuridine monophosphate with a methyl group obtained from methylene tetrahydrofolate to prepare thymidylate. 5FU exerts its cytotoxic effect through three different pathways. The nucleobase 5FU and the deoxyribonucleoside 5-FdUrd enter cells through facilitated nucleoside transport systems. One of the mechanisms of action of these agents is the inhibition of the enzyme thymidylate synthase (TS). The nucleobase 5FU is converted to the deoxynucleoside 5-fluoro-2'-deoxyuridine (5-FdUrd) by thymidine phosphorylase. Subsequent phosphorylation of the deoxynucleoside 5-FdURd by thymidine kinase results in the formation of the cytotoxic nucleotide 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP). In the presence of reduced folate, 5,10-methylene-tetrahydrofolate (mTHF), the nucleotide (5-FdUMP) inhibits thymidylate synthase (TS) due to the enzyme's inability to remove the 5-fluorine atom. Thus, primarily the important mechanism of action of 5FU and FDUR is the inhibition of the enzyme thymidylate synthase (TS). Thymidylate synthase (TS) presents two substrates for (dUMP and mTHF), both of which bind at the catalytic site to allow dTMP synthesis. 5-FdUMP forms a covalent ternary complex with thymidylate synthase (TS), inhibiting this enzyme activity and leading to depletion of deoxythymidine triphosphate, necessary for DNA synthesis. Alternatively, (5-FdUMP) is synthesized after conversion of 5FU to 5-FUMP by OPRT, to fluorouridine diphosphate (FUDP), fluorodeoxyuridine diphosphate (5-FdUDP) by ribonucleotide reductase (RR) and eventually to 5'-FdUMP. It was observed that after drug exposure to 5FU or 5-FdUrd, cells developed resistance to these chemotherapeutic agents. Overexpression of thymidylate synthase (TS) reduces the therapeutic effect of the TS inhibitory drug by inducing resistance. It has been observed that some individuals are more resistant to targeted therapy by TS than others. Second, the deoxynucleoside 5-fluoro-2'-deoxyuridine (5-FdUrd) can be converted to its triphosphate form 5-FdUTP which can be successively incorporated into DNA causing cell damage. Third, 5FU can also inhibit RNA synthesis by converting it to FUMP by OPRT and subsequently, in two steps, to fluorouridine triphosphate (FUTP) which is incorporated into RNA. This is believed to be another potential action of 5FU.
The 5FU molecule thus does not result in an ideal TS inhibitory drug because it is inefficiently converted to 5-FdUMP due to the various metabolic steps required for the metabolic activation of 5FU. Another resistance occurs if the cell produces excess amounts of dUMP to compete with the drug for the active site.
5-FdUrd is a relatively good substrate for thymidine kinase, which converts directly to 5-FdUMP. In vitro studies on various cancer cell lines have shown that 5-FdURd is about 5000 times more potent as a cell growth inhibitor than 5FU. Furthermore, the 5-FdURd prodrug does not exhibit any significant conversion to ribonucleotide metabolites at cytotoxic concentrations. In vivo studies have shown that a significant amount of 5-FdUrd is degraded to its relative base 5FU by thymidine phosphorylase, an enzyme for which 5-FdUrd exhibits good affinity. This rapid phosphorolytic cleavage of 5-FdUrd to 5FU in vitro and in vivo represents a major obstacle in releasing intact 5-FdUrd into cells for enhanced cytotoxic action. Furthermore, the degradation of 5-FdUrd in rat and human intestinal homogenates after oral administration suggests that 5-FdUrd would likely not be absorbed as intact 5-FdUrd.
[0027] According to another aspect of the present invention, the compound of the present invention is provided for use in a method of prophylaxis or treatment of cancer in homo sapiens. Suitably the cancer is selected from the group comprising leukemia, pancreatic, prostate, lung, breast and cervical cancer.
[0028] In particular, the compound of the present invention is for use in a method of prophylaxis or treatment of cancer in a patient who has developed or has the potential to develop resistance in tumor cells with respect to the activity of 5-fluoro or 5 -fluoro-2'-deoxyuridine for the prophylaxis or treatment of cancer. For example, the compound of the present invention may be for use in a method of prophylaxis or treatment of cancer in a patient with cells with a decreased level of nucleoside transporter proteins and/or with cells deficient in nucleoside kinase and/or with cells infected with mycoplasma, particularly where the cancer is leukemia. The compound of the present invention may instead of or as well be for use in a method of prophylaxis or treatment of cancer in a patient who presents cells with an increased level of thymidylate synthase (TS).
According to another aspect of the present invention, there is provided a method of prophylaxis or treatment of cancer comprising administering to a homo sapiens patient in need of such treatment an effective dose of a compound of the present invention. Suitably the cancer is selected from the group comprising leukemia, pancreatic, prostate, lung, breast and cervical cancer.
[0030] In particular, the present invention comprises a method for treating a patient who has developed or has the potential to develop resistance in tumor cells with respect to the activity of 5-fluoracyl or 5-fluoro-2'-deoxyuridine in a method of cancer prophylaxis or treatment. For example, the method of the present invention may comprise treating a patient with cells with a decreased level of nucleoside transporter proteins and/or with nucleoside kinase deficient cells and/or with mycoplasma infected cells, particularly where the cancer is leukemia. The method of the present invention for treating a patient may instead or as well be for treating a patient who presents cells with an increased level of thymidylate synthase (TS).
"Tumor" or "tumor cell" as used in the present application, unless otherwise indicated, refers to both solid tumors and cancers such as leukemia.
The compounds of the present invention can be used to treat a cancer patient alone again or in conjunction with another cancer therapy. For example, the compounds of the present invention can be used in a cancer treatment regimen in conjunction with other anti-cancer drugs, such as 5-FU and/or 5-FdUrd, with or without leucovorin (LV), and/or other drugs anticancer. Alternatively, the compounds of the present invention can be used where a patient has failed to respond to other anti-cancer drugs, such as, for example, 5FU and/or 5-FdUrd with or without leucovorin (LV), or where the patient has shown resistance to other anticancer drugs such as, for example, 5-FU and/or 5-FdUrd with or without leucovorin (LV).
[0033] Compounds of the present invention where Ar is 1-naphthyl, whether substituted or unsubstituted, are particularly suitable for use in the above methods and uses of the present invention, particularly in a patient who has developed, or who has the potential to develop , resistance in tumor cells, such as, for example, a patient with cells with a decreased level of nucleoside-carrying cells and/or with kinase deficient cells and/or with mycoplasma infected cells and/or a patient presenting cells with an increased level of thymidylate synthase (TS).
According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention in combination with a pharmaceutically acceptable excipient, diluent or carrier.
According to another aspect of the present invention, there is provided a method of preparing a pharmaceutical composition comprising the step of combining a compound of the present invention with a pharmaceutically acceptable diluent, carrier or excipient.
According to another aspect of the present invention, there is provided a process for the preparation of a compound of the present invention comprising reacting a compound of Formula (II)

[0037] with a compound of Formula (III)

[0038] wherein Ar, R3, R4, R1 and R2 have the meanings described above and in claim 1.
[0039] The Ar group comprises a substituted or unsubstituted aryl group, wherein the term "aryl group" and the possible substitution of said group is as defined herein. Suitably, Ar is a 5- to 14-membered aromatic ring moiety. Preferably Ar is carbocyclic. Alternatively, one or two rings may include 1, 2, 3 or 4 heteroatoms, preferably 1, independently selected from O, S and N. Preferably Ar is a fused carbobicyclic aryl moiety. More preferably Ar is naphthyl, even more preferably 1-naphthyl i.e. naphthyl linked to P through O linked at the 1-naphthyl position. Suitably Ar can alternatively be phenyl.
One, two, three or four substituents, which may be the same or different, may be present on Ar and are selected from the group comprising halogen, which may -F, -Cl, -Br or -I; -NO2; -NH2; optionally substituted -C1-3alkyl; optionally substituted -C 1-3 alkoxy, preferably methoxy (-OCH 3 ); optionally substituted -SC1-3alkyl; -CN; optionally substituted -COC1-3alkyl; and optionally substituted -CO2C1-3alkyl; wherein said optionally substituted groups may be substituted with one or more to six, preferably three, members independently selected from the group comprising halogen, which may be F, Cl, Br and I, and NO2. Particularly preferred substituents on Ar are electron removing groups such as halogen (preferably chlorine or fluorine), trihalomethyl (preferably trifluoromethyl), cyano and nitro groups.
The substituents may be at any position on the aryl portion Ar. Where Ar is 1-naphthyl, a single substituent at any of the 2, 3, 4, 5, 6, 7 or 8 positions is preferred. Where Ar is phenyl, a single substituent at the 2 (ortho) or 4 (para) position is preferred, more preferred at the 4 position. For example, where Ar is substituted phenyl, Ar may be 3,5-dichloro-phenyl, p- trifluoromethyl-phenyl, p-cyano-phenyl, or p-nitro-phenyl.
Suitably, R3 is a primary, secondary or tertiary C1-16 alkyl group and may include carbocyclic moieties; a cyclic C5-7 alkyl group; or a C1-6alkylC5-11aryl group. More suitably, R3 is a C1-10 alkyl group or C1-3 alkylC5-7 aryl group such as benzyl (-CH2-C6H5). A cyclic alkyl group can be carbocyclic or can contain in total one, two or three ring heteroatoms selected independently from O, N and S. Preferably R3 is unsubstituted. Where substituted, the substituents are shown below.
[0043] Suitably R4 is H or alkoxy, i.e. alkyl-C(=O)-, where alkyl is C1 to C10.
[0044] When R1 and/or R2 is alkyl, they are each independently suitably selected from C1 to C16, more suitably from C1 to C6. When R1 and R2 together comprise an alkylene chain, the chain is suitably C1 to C6 and may contain unsaturation and in total one, two or three heteroatoms in the chain independently selected from O, N and S. When one of R1 and R2 is N-linked, the total ring size including N and the C atom to which R 1 and R 2 are attached suitably comprises from 4 to 7 members, more suitably 5 members. Any alkyl or alkylene chain comprising R1 and/or R2 may be substituted with one or more substituents shown herein.
[0045] When R1 and R2 are different, the C atom to which they are attached is chiral. Preferably, the stereochemistry at an asymmetric center -CR1R2 corresponds to an L-amino acid. The stereochemistry at an asymmetric center -CR1R2 can, however, correspond to a D-amino acid. Alternatively, mixtures of compounds can be used having asymmetric centers corresponding to L and D amino acids.
Suitably, R1 and R2 may correspond to the portions attached to the alpha C atom in a naturally occurring alpha amino acid. By "naturally occurring alpha amino acid" is meant Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Cystine, Glycine, Glutamic Acid, Glutamine, Histidine, Hydroxylysine, Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine. One of R1 and R2 may thus be H and one of R1 and R2 may thus be H or alkyl selected from the following moieties or R1 and R2 together may form an alkylene chain selected from the following moieties: CH3- as shown in Alanine H2NC( =NH)NH[CH2]3- NH2C(O)CH2- as shown in Argenine as shown in Aspargine HO2CH2- as shown in Aspartic acid HSCH2- as shown in Cysteine HO2CH(NH2)CH2SSCH2- as shown in Cystine H- as shown in Glycine HO2CH2CH2- as shown in Glutamic acid H2N(O)CCH2CH2- C3N2HCH2- as shown in Glutamine as shown in Histidine H2NCH2CH(OH)CH2CH2- -CH2CH(OH)CH2-CH3CH2CH(CH3)- (CH3)2CHCH2- H2NCH2( CH2)3- CH3SCH2CH2- as presented in Hydroxylysine as presented in Hydroxyproline as presented in Isoleucine as presented in Leucine as presented in Lysine as presented in Methionine PhCH2- as presented in Phenylalanine -CH2CH2CH2- as presented in Proline OHCH2- as presented in Serine CH3CH(OH)- C8NH6CH2- as shown in Threonine as shown in Tryptophan HOC6H4CH2- as shown in Tyrosine (CH3)2CH- as shown in Valine.
By "a pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, ester, salt of such ester, hydrate, solvate, or crystalline form or metabolite or any other compound which on administration to a recipient is capable of providing (directly or indirectly) a compound of Formula (I).
Reference in the present specification to an alkyl group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical. Where the cyclic alkylene group is preferably C3 to C12, more preferably C5 to C10, most preferably C5 to C7. Where the acyclic alkyl group is preferably C1 to C16, more preferably C1 to C6.
Reference in the present specification to an aryl group means an aromatic group suitably containing 5 to 14 ring atoms. For example, Ar is phenyl or naphthyl. The aromatic group may be a heteroaromatic group containing one, two, three or four, preferably one, heteroatom independently selected from the group consisting of O, N and S. Examples of such heteroaromatic groups include pyridyl, pyrrolyl, furanyl and thiophenyl.
[0050] Alkyl and aryl groups can be substituted or unsubstituted. Where substituted, it will generally be from one to three substituents present, preferably one substituent. Substituents may include halogen atoms, by which F, Cl, Br and I atoms are intended, and halomethyl groups such as CF3 and CCl3; oxygen containing groups such as oxo, Hydroxy, carboxy, carboxyC1-16alkyl, alkoxy, alkoxy, alkoxy, aryloxy, arylyl and arylyloxy; nitrogen containing groups such as amino, C1-6alkylamino, diC1-6alkylamino, cyano, azide and nitro; sulfur containing groups such as thiol, C 1-6 alkylthiol, sulfonyl and sulfoxide; heterocyclic groups which may themselves be substituted; alkyl groups as defined herein which may themselves be substituted; and aryl groups as defined above which may themselves be substituted, such as phenyl and substituted phenyl. Substituents on said heterocyclic alkyl and aryl groups are as defined immediately above. Substituents on R1 and/or R2 include moieties to provide compounds in which R1 and R2 correspond to moieties attached to the alpha C atom in a naturally occurring alpha amino acid.
[0051] Reference in the present specification to alkoxy and aryloxy groups means, respectively, alkyl-O- (for example, where alkyl is C1 to C16, preferably C1 to C6) and aryl-O- (for example, where aryl is a 5- to 14-membered aromatic mono- or bifused ring moiety, optionally containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, preferably aryl is phenyl).
[0052] Reference in the present specification to alkoxy and arylyl groups means, respectively, alkyl-CO- (for example, where alkyl is C1 to C16, preferably C1 to C6) and aryl-CO- (for example, where aryl is a 5- to 14-membered aromatic mono- or bifused ring moiety, optionally containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, preferably aryl is phenyl).
[0053] Reference in the present specification to alcoyloxy and arylyloxy means, respectively, alkyl-CO-O (for example, where alkyl is C1 to C16, preferably C1 to C6) and aryl-CO-O (for example, where aryl is a 5- to 14-membered mono- or bifused aromatic ring system, optionally containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, preferably aryl is phenyl).
Reference in the present specification to heterocyclic groups means groups containing one or more, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl pyridazinyl, piperidyl, piperazinyl, morpholinyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxy-indolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, oxy-indolyl, pyrinolyl, pyrinolyl, pyrinolyl, isocumarinyl, isocarinyl, pyrinolyl, pyrinolyl benzoxazinyl, quinoxadinyl, cromenyl, chromanyl, isochromanyl and carbolinyl.
[0055] In one embodiment of the present invention, Ar is suitably naphthyl, especially 1-naphthyl, i.e. naphthyl linked to P via O-linked at the 1-naphthyl position.
[0056] In another embodiment of the present invention, Ar is suitably phenyl.
[0057] In one embodiment of the present invention, Ar is substituted. Suitable substituents are shown here.
[0058] In one embodiment of the present invention, Ar is unsubstituted 1-naphthyl.
[0059] In one embodiment of the present invention, Ar is unsubstituted phenyl.
[0060] In one embodiment of the present invention, R4 is selected from the group consisting of H and acetyl (CH3C(=O)-), especially R4is H.
[0061] In one embodiment of the present invention, R3 is selected from the group consisting of benzyl and members of the group comprising C1 to C10 alkyls, especially R3 is selected from n-propyl, n-butyl, n-pentyl and n-hexyl, plus especially R3 is n-pentyl.
[0062] In one embodiment of the present invention, R1 and R2 correspond to the portions linked to the alpha C atom in a naturally occurring alpha amino acid, as presented herein. A particularly suitable naturally occurring alpha amino acid is L-alanine such that suitably one of R1 and R2 is H, one of R1 and R2 is CH3 and the C atom to which they are attached has the chirality L. In other embodiments, R1 and R2 correspond to the alpha C atom bonded moieties in a non-naturally occurring alpha amino acid, for example R1 and R2 are both suitably CH3.
[0063] The specific features mentioned in the above embodiments are significantly described to be combined together in any and all combinations in the compounds of the present invention.
Particularly suitable compounds of the present invention are those compounds where Ar is 1-naphthyl, R3 is benzyl, one of R1 and R2 is H, one of R1 and R2 is methyl and the C atom to which R1 and R2 are attached have the chirality L and compounds where Ar is 1-naphthyl, R3 is n-pentyl, one of R1 and R2 is H, one of R1 and R2 is methyl and the C atom to which R1 and R2 are attached has a chirality L. For each compound, R4 is most suitably H.
[0065] Conventional cancer treatment using chemotherapeutics is largely based on the use of nucleoside analogues. These molecules are designated to mimic natural pyrimidine and purine nucleosides. After uptake by the cell, they are phosphorylated by cellular enzymes such as (deoxy)cytidine kinase (dCK), thymidine kinase (TK) and/or nucleo(s)(t)ide kinases. These antimetabolites can subsequently interfere with de novo DNA/RNA precursor synthesis to eventually inhibit DNA/RNA synthesis resulting in cytotoxic/static activity (Hatse et al., 1999; Galmarini et al., 2002).
[0066] Fluoropyrimidine-based antimetabolites such as fluorouracil (5-FU), capecitabine and 5-fluoro-2'-deoxyuridine (5-FdUrd) are primarily used in the treatment of ovarian, breast and colon (Bruin's) carcinoma and others, 2006; Ishikawa and others, 1998; Walko and others, 2005). Intracellularly, these drugs are metabolized to 5-FdUMP, which form a stable inhibitory complex with thymidylate synthase (TS) and the reduced 5,10-methylenetetrahydrofolate co-substrate, thereby blocking the binding of the normal substrate dUMP to the enzyme (Beck and other(s), 1994; Tanaka et al., 2000; Longley et al., 2003). TS is the enzyme responsible for converting dUMP to TMP and is therefore indispensable for cell proliferation, producing an interesting target for drug purposes. Among the aforementioned fluoropyrimidines, 5-FdUrd requires only a metabolic conversion, a TK catalyzed phosphorylation to generate 5-FdUMP (Longley et al., 2003). This mandatory phosphorylation is often the rate-limiting step in the metabolism of many anticancer drugs (including 5-FdUrd), and is therefore still one of the limiting factors for the therapeutic use of nucleoside analogues. Therefore, different strategies to improve the antitumor efficacy of nucleoside analogues have been investigated (Galmarini et al., 2002).
[0067] The charged nature of nucleoside monophosphates under physiological conditions results in poor penetration, if any, across the cell membrane (Mehellou et al., 2009). Therefore, direct administration of phosphorylated molecules to avoid the first stage of phosphorylation has little therapeutic advantage. Therefore, different strategies to bypass rate-limiting phosphorylation using various types of nucleoside 5'-monophosphate prodrugs for more efficient drug release have been explored (Hecker & Erion, 2008). The administration of lipophilic phosphoramidate nucleotide prodrugs (ProTides) has proven successful for several molecules with anti-viral/cancer activity (Harris et al., 2001; Congiatu et al., 2006; McGuigan and other(s), 2010). By masking the charges of the phosphate motif, good passive membrane diffusion of prodrugs can be completed after which the prodrug is rapidly converted intracellularly to nucleoside monophosphate by enzymatic cleavage (Mehellou et al., 2009).
[0068] Mycoplasmas are the smallest self-replicating organisms on earth and are characterized by the loss of a cell wall and a strongly reduced genome (600-1,200 kb). Many of these bacteria have a parasitic lifestyle and reside in the human body causing asymptomatic infections (Razin et al., 1998). These prokaryotes have been shown to preferentially colonize tumor tissue: Huang et al. (2001) reported that 39.7 56% of human gastric, colon, esophageal, lung and breast cancers are infected with mycoplasmas compared to 20.9-30% in non-tumorigenic tissue. Pehlivan et al. (2005) found >80% of kidney tissue samples from patients suffering from renal cell carcinoma to be infected with mycoplasmas compared to 14% in control tissue samples. Chan et al(s) (1996) reported an infection rate of 59% in ovarian cancer tissues and other studies also reported a high mycoplasma infection rate in cervical and gastric condyloma tissues (Sasaki et al.), 1995, Yang et al., 2010) (Kidder et al., 1998). Due to their reduced gene pool, mycoplasmas need the pathway for de novo pyrimidine and purine synthesis and therefore express a wide range of enzymes metabolizing rescue nucleo(s)(t)ide, such as thymidine phosphorylase (TP), deoxycytidine deaminase, etc. (Razin, 1978; Charron & Langelier, 1981; Neale et al., 1983; Tham et al., 1993). As early as 1985 it was observed that mycoplasma-encoded enzymes (eg, TP), present in contaminated cell cultures, induce decreased incorporation of dTTP into lymphocytes (Sinigaglia & Talmadge, 1985). Recently, it has been shown that these enzymes, in particular mycoplasma-encoded thymidinephosphorylase, also interfere with the cytostatic activity of several chemotherapeutics, including 5-trifluorothymidine, in vitro (Bronckaers et al., 2008; Jetté et al.), 2008; Liekens et al., 2009). For this reason it has been hypothesized that the elimination of mycoplasmas by antibiotics or suppression of mycoplasma-encoded enzymes in human tumor tissue may optimize the treatment of cancer patients using purine and pyrimidine antimetabolites (Liekens et al., 2009) .
[0069] The present invention is derived from the development and evaluation of TK-independent phosphoramidate prodrugs of 5-FdUrd and provides compounds that may also be insensitive to the TP-dependent inactivation of its free nucleoside analogue. The compounds of the present invention can thus provide mycoplasma insensitive nucleoside analogue prodrugs which can optimize the treatment of cancer patients using a pyrimidine antimetabolite. Among the phosphoramidate prodrugs currently synthesized from 5-FdUrd, CPF-373 (identified below and mentioned above as a particularly suitable compound of the invention with R4 as H) was selected for further in-depth studies. This molecule contains a naphthyl and benzylalaninyl group to mask the 5'-phosphate charged on 5-FdUMP.
[0070] Several tumor cell resistance mechanisms targeting fluoropyrimidines such as 5FU, 5-FdUrd and trifluorothymidine (TFT) have been described, including a decreased activity of drug activating enzymes (eg TK and phosphoribosyltransferase orotate), a increased activity of drug-inactivating enzymes (ie, thymidine phosphorylase) and/or an upregulation of target enzymes (eg, TS) (Agarwal et al., 1999; Murakami et al., 2000; Kosaka; Kosaka and other(s), 2004). Also, high TP levels found in various types of cancer tissue have been reported to predict a poorer prognosis of fluoropyrimidine treatment (Kamoshida et al., 2005; Ciaparrone et al., 2006; Koopman et al.( s), 2009), although other studies have not confirmed these findings (Ciccolini et al., 2004; Koopman et al., 2009). The present invention derives from the development of a prodrug for 5-FdUrd, to avoid possible resistance mechanisms and susceptibility to degradation by catabolic enzymes, present in the tumor microenvironment.
Compounds embodying the present invention, e.g., CPF-373, are 5-FdUrd phosphoramidate prodrugs and are described herein and can meet these objectives. After uptake into tumor cells, CPF-373, for example, generates 5-FdUMP intracellularly on enzymatic cleavage. Stability studies and enzymatic/serum studies by 31P NMR technology revealed that the prodrug CPF-373, for example, is completely stable in acid and alkaline conditions, however, subjected to hydrolysis in the presence of serum or carboxypeptidase Y, resulting in formation of the nucleoside 5'-phosphoramidate derivative. While TK is a major enzyme in activating 5-FdUrd, CPF-373, for example, was found to be much less dependent on TK to exert its cytostatic action in both murine (L1210) and human (CEM) cell cultures. Due to the lipophilic nature of ProTides, these molecules can release nucleoside monophosphates directly into the intact tumor cell after conversion to their nucleoside phosphoramidate derivative by enzymes such as carboxyesterases or carboxypeptidases (ie carboxypeptidase Y), eliminating the need for a phosphorylation by specific nucleoside kinases such as TK. In this regard, CPF-373, for example, may be a suitable tool for the treatment of tumor cells with a modified TK activity (either acquired or inherent). Also, since TK expression is S-phase dependent, it is expected that CPF-373, for example, can also efficiently release 5-FdUMP in tumor cells that are not in the S-phase of their replication cycle. TS activity studies revealed that, CPF-373, for example, was able to inhibit TS in both TK-deficient tumor cell lines, again pointing to an efficient intracellular release of 5-FdUrd 5'-monophosphate, and its virtual independence from cellular TK for metabolic activation.
The compounds of the present invention, such as CPF-373, are unlikely to be inactivated by catabolic enzymes involved in nucleoside metabolism. Indeed, while 5-FdUrd is highly susceptible to enzymatic hydrolysis by TP resulting in the formation of 5-FU and 2-deoxyribose-1-phosphate, its prodrug, eg, CPF-373, is not a substrate for prokaryotic (ie. E.coli) or mammalian (i.e., human erythrocyte) TP. Also, uridine phosphorylase does not recognize, for example, CPF-373 as a substrate, whereas 5-FdUrd is (poorly but measurably) hydrolyzed by this enzyme. Several studies have revealed that many tumor cells have elevated levels of TP, which also acts as an angiogenic factor (Koopman et al., 2009; Bronckaers et al., 2009). In addition, there are several reports on preferential colonization of tumor tissue by mycoplasmas (Sasaki et al., 1995; Chan et al., 1996; Huang et al., 2001; Pehlivan et al.), , 2005) that interfere with the cytostatic activity of several conventional chemotherapeutics in vitro despite their TP encoded (Bronckaers et al., 2008; Jetté et al., 2008; Liekens et al., 2009). The present observations that 5-FdUrd, but not, for example, CPF-373, markedly loses cytostatic activity when tumor cells are infected by (TP expressing) mycoplasmas, is in complete agreement with these observations. Therefore, administration of a TP-insensitive anticancer prodrug such as CPF-373, shown to be chemically stable under extreme pH conditions, may also improve cancer chemotherapy. In conclusion, ProTides, like CPF-373, provide an interesting new method aimed at developing more elastic anticancer drugs. For example, CPF-373 may have at least several advantages over its 5-FdUrd parent drug: it exerts its cytostatic activity independent of TK and is resistant to metabolic breakdown by TP, an enzyme that is often up-regulated in tumors or it may be externally expressed by mycoplasma infection of the tumor tissue.
The compound having the Formula I or the pharmaceutical composition according to the present invention can be administered to a homo sapiens patient by any suitable method.
The medicaments used in the present invention can be administered by oral or parenteral routines, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous suspension or solution.
Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents can include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption from the gastrointestinal tract.
[0077] Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or oil.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such vehicles as are known in the art to be appropriate.
[0080] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate isotonicity and pH. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as those derived from cellulose, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include n-propyl and ethyl p-hydroxybenzoate.
The compounds of the invention may also be presented as liposome formulations.
[0082] In general an adequate dose will be in the range of 0.1 to 300 mg per kilogram of recipient body weight per day. A preferred lower dose is 0.5 mg per kilogram of recipient body weight per day, a more preferred lower dose is 6 mg per kilogram of recipient body weight per day, an even more preferred lower dose is 10 mg per kilogram of body weight per recipient per day. A suitable dose is preferably in the range of 6 to 150 mg per kilogram of body weight per day, and more preferably in the range of 15 to 100 mg per kilogram of body weight per day. The desired dose is preferably presented as two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing from 10 to 1500 mg, preferably from 20 to 1000 mg, and more preferably from 50 to 700 mg of active ingredient per unit dosage form.
[0083] Examples of the present invention will now be described, by way of example only, with reference to the accompanying drawings comprising Figures 1 to 11, in which:
Figure 1 shows the Structural Formula of 5-FdUrd and its phosphoramidate prodrug CPF-373;
[0085] Figure 2 shows the effect of thymidine phosphorylase and uridine phosphorylase on dThd, Urd, 5-FdUrd and CPF-373, where data are the mean of at least 2 independent experiments (± S.D.);
[0086] Figure 3 shows TS inhibition by 5-FdUrd and CPF-373 as measured by tritium release from [5-3H]dUrd (panels A and B) and [5-3H]dCyd (panels C and D ) in L1210/0 cell cultures and by the release of tritium from [5-3H]dCyd (panels E and F) in L1210/TK- cell cultures, where data are the mean of 2 independent experiments (± SEM);
[0087] Figure 4 shows a putative proposed mechanism of activation of 5-FdUrd ProTides;
Figure 5 shows the carboxypeptidase-mediated cleavage of the CPF-373 prodrug monitored by 31 P NMR;
Figure 6 shows the 31 P NMR spectrum of compound CPF-373 in serum;
[0090] Figure 7 shows the 31P NMR spectrum of compound CPF-373 in buffer pH = 1;
[0091] Figure 8 shows the 31P NMR spectrum of compound CPF-373 in buffer pH = 8;
[0092] Figure 9 shows the nucleoside spectra and the relative base by 19F NMR: a) 5-FdUrd subjected to phosphorylase assay (A); b) 5-FdUrd and the base 5FU under the assay condition in the absence of enzyme (TP) (B);
[0093] Figure 10 shows the nucleoside and base spectra in potassium phosphate buffer (205 nM) by 19F NMR: a) 5-FdUrd subjected to phosphorylase assay in the absence of enzyme (A); b) Result after addition of enzyme (TP) (B); and
Figure 11 shows the spectra of the prodrug compound CPF373 in the phosphorylase assay: a) prodrug CPF373 under assay conditions in the absence of enzyme (TP) (A); b) CPF373 prodrug submitted to the action of thymidine phosphorylase (TP) (B). Compound Synthesis
[0095] Referring to Figure 1 and Schemes 1 to 3 below, the compounds of the present invention, as exemplified by compound CPF-373 (1), were synthesized using phosphorochloridate chemistry, which phosphorochloridate chemistry was previously reported by McGuigan et al. (1993, 1996, 1997). For example, arylphosphorodichlorophosphate (2) was prepared by coupling 1-naphthol (3) with phosphorus oxychloride (4) in the presence of Et3N (Scheme 1) and this was allowed to react with L-alanine benzyl ester tosylate (5) in the presence of Et3N to generate the phosphorochloridate derivative (6) (Scheme 2). The nucleoside 5-FdUrd (7) was converted to the 5' ProTide by coupling with the phosphorochloridate derivative (6) in THF, in the presence of N-methyl imidazole (NMI) to provide the target compound CPF-373 (1) ( Scheme 3). The sample was obtained as a mixture of two diastereoisomers as confirmed by the presence of two peaks in the 31P NMR.
Scheme 1. Reagents and conditions: (i) 1-naphthol (3), phosphorus oxychloride (4) dry Et2O, dry Et3N, -78°C, 30 minutes, then RT, 3 hours
Scheme 2. Reagents and conditions: (i) Dry Et3N, CH2Cl2, -78°C, one hour then RT, 3 hours.
Scheme 3. Reagents and conditions: (i) NML, dry THF, 10 minutes, then phosphorochloridate (6), RT, overnight.
[0096] 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 aluminum plates pre-coated with silica gel 60-F254 and visualized under UV (254 nm) and/or with 31P NMR spectra. Column chromatography was performed on silica gel (35-70 µM). Proton (1H), carbon (13C), phosphorus (31P) and fluorine (19F) NMR spectra were recorded on a Bruker Avance 500 spectrometer at 25°C. Spectra were autocalibrated to the deuterated solvent peak and all 13C NMR and 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 analytical column.
[0097] Low and high resolution mass spectra were performed as a service by the University of Birmingham, using (ES) electrospray. CHN microanalysis was performed as a service by MEDAC Ltd., Surrey. Standard Procedure A: Dichlorophosphate Synthesis (2).
[0098] 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 one hour. Subsequently the reaction mixture was allowed to slowly warm to room temperature over 3 hours. The formation of the desired compound was monitored by 31 P NMR. The resulting mixture was filtered and then evaporated in vacuo under nitrogen to give the crude colorless oil as product, which was used without further purification in the next step. Synthesis of 1-Naphthyl dichlorophosphate (2):
[0099] Prepared according to Standard Procedure A, of 1-naphthol (3.00 g, 20.81 mmols), phosphorus oxychloride (1.94 mL, 20.81 mmols), triethylamine (2.9 mL, 20.81 mmols) and anhydrous diethyl ether (70 mL). After 1 hour at -78°C the reaction was allowed to rise to room temperature and stirred for 3 hours. 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 give a colorless oil (4.59 g, 84%) [Rf = 0.93 (hexane-EtOAc, 1:1)], 31 P NMR (202 MHz, CDCl3): δP 5.07; 1H NMR (500 MHz, CDCl 3 ): δH 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). Standard Procedure B: Synthesis of Phosphorochloridate (6).
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 triethylamine (2.0 equiv. ) at -78°C. After one hour the reaction mixture was allowed to slowly warm to room temperature over 3 hours and the formation of the desired compound was monitored by 31 P NMR.
[00101] The reaction mixture was concentrated under reduced pressure, the residue was redissolved in diethyl ether, filtered and evaporated in vacuo under nitrogen to provide a colorless crude oil, which in some cases was used without further purification in the next step. The synthesized aryl phosphorochloridate was purified by column chromatography eluting with hexane-EtOAc (7:3) to furnish the title compound as a colorless oil. Synthesis of 1-Naphthyl(benzyl-L-alaninyl) Phosphorochloridate (6):
[00102] The phosphorochloridate was prepared using 1-Naphthyl dichlorophosphate (2.50 g, 9.57 mmols), L-alanine benzyl ester tosylate salt (3.36 g, 9.57 mmols), dry triethylamine (2.66 mL, 19.14 mmols) and dry dichloromethane (35.7 mL) according to general procedure B. Purification by column chromatography eluting with hexane-EtOAc, (7:3) provided the compound title as a colorless oil (1.82 g, 47%) [Rf = 0.90 (hexane-EtOAc, 7:3)], 31 P NMR (202 MHz, CDCl3, mixture of diastereoisomers): δP 7.92. 8 .14 (Int.: 1.00:1.00); 1H NMR (500 MHz, CDCl3, mixture of diastereoisomers with a ratio of 1:1): δH 1.42-1.45 (m, 3H, CHCH3), 4.20-4.23 (m, 1H, CHCH3) , 4.78-4.81 (m, 1H, NH), 5.09 (s, 2H, OCH2Ph), 7.09-7.73 (m, 11H, ArH), 7.97-8.12 ( m, 1H, ArH). Standard Procedure C: Synthesis of Nucleoside Phosphoramidate (1).
[00103] 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 minutes 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 CH2Cl2, washed twice with H2O, then with 0.5 M HCl or alternatively the crude product was washed with diethyl ether. Then, the crude product was purified by column chromatography on silica, eluting with CH 2 Cl 2 -MeOH as a gradient to furnish the phosphoramidate. Synthesis of 5-Fluoro-2'deoxyuridine-5'-O-[α-naphthyl (benzyl-L-alaninyl)] Phosphate (1):
The phosphoramidate was prepared using 5-Fluoro-2'deoxyuridine (0.25 g, 1.01 mmol), NMI (0.40 mL, 5.07 mmols) and naphthyl phosphorochloridate(benzyl-L- alaninyl) (0.82 g, 3.04 mmols) according to general procedure C. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) gives the title compound as a colorless solid ( 47.0 mg, 8%) [Rf = 0.19 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 636.1520. C29H29N3O9FNaP requires [MNa+], 636.1523); 31P NMR (202 MHz, MeOD, mixture of diastereoisomers): δP 4.24, 4.59; 19F NMR (470 MHz, MeOD): δF -167.36, -167.18; 1H NMR (500 MHz, MeOD): δH 1.34-1.38 (m, 3H, CHCH3), 1.67-1.79 (m, 1H, H-2'), 2.08-2.17 (m, 1H, H-2'), 4.03-4.15 (m, 2H, CHCH3, H-4'), 4.24-4.36 (m, 3H, CH2OP, 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, 1H, ArH); 13C NMR (125 MHz, MeOD): δC 20.3 (d, 3JC-P = 7.6 Hz, CH3), 20.5 (d, 3JC-P = 6.5 Hz, CH3), 40.8 ( CH2), 40.9 (CH2), 51.8 (CH), 51.9 (CH), 67.6 (d, 2JC-P = 5.3 Hz, CH2), 67.8 (d, 2JC- P = 5.2 Hz, CH2), 68.0 (CH2), 68.1 (CH2), 72.0 (CH), 72.1 (CH), 86.7 (d, 3JC-P = 8, 1 Hz, CH), 86.8 (d, 3JC-P = 8.1 Hz, CH), 86.9 (CH), 87.0 (CH), 116.2 (d, 3JC-P = 3, 3 Hz, CH), 116.5 (d, 3JC-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, 1JC-F = 233.8 Hz, C), 141.7 (d, 1JC-F = 233.9 Hz, C), 147.8 (d, 2JC-P = 7.7 Hz, C), 147.9 (d, 2JC-P = 7 .4 Hz, C), 150.5 (d, 4JC-F = 4.0 Hz, C), 159.3 (d, 2JC-F = 26.1 Hz, C), 174.6 (d, 3JC -P = 5.0 Hz, C), 174.9 (d, 3JC-P = 4.3 Hz, C), m/z (ES) 636 (MH+, 100%), reverse HPLC eluting with (H2O/ MeOH from 100/0 to 0/100) in 45 min. u two peaks of diastereoisomers with tR 34.23 min. and tR 34.59 min. Anal. Calcd for C29H29FN3O9P: C, 56.77; H, 4.76; N, 6.85. Found: C, 56.57; H, 5.06; N, 6.72. Radioactive Pyrimidine Deoxynucleosides
[00105] [5-3H]dCyd (radiospecificity: 22 Ci/mmol) and [5-3H]dUrd (radiospecificity: 15.9 Ci/mmol) were obtained from Moravek Biochemicals Inc. (Brea, CA). Standard Procedure D: Synthesis of Phosphoramids (NMI Method)
To a stirred solution of 5-F-dUrd (1.0 eq.) in anhydrous THF, an appropriate phosphorochloridate (3.0 eq.) dissolved in anhydrous THF was added dropwise under an atmosphere of Ar. The reaction mixture at -78°C was added dropwise over 5 minutes NMI (5.0 eq.). After 15 minutes the reaction mixture was allowed 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 MgSO4, filtered, reduced to dryness and purified by column chromatography with gradient eluent (DCM/MeOH 99:1 to 97:3 to 95:5). Standard Procedure E: Synthesis of phosphoramidates (Method t BuMgCl)
[00107] To a stirred solution of 5-FdUrd (1.0 eq.) dissolved in anhydrous THF, tBuMgCl (1.1 mol eq. 1M solution in THF) was added dropwise under an atmosphere of Ar, 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 eluent gradient (DCM/MeOH 99:1 to 97:3 to 95:5) 5-Fluoro-2'-deoxyuridine-5'-O -[henyl(benzoxy-L-alaninyl)] phosphate (CPF381)
[00108] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.40 g, 1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (tBuMgCl) (1.0 M, 2, 43 mL, 2.43 mmols) and phenyl(benzoxy-L-alaninyl) phosphorochloridate (1.08 g, 3.20 mmols) according to general procedure E. Purification by gradient column chromatography on silica, eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (71.0 mg, 8%) [Rf = 0.35 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 586.1360. C25H27N3O9NaPF requires [MNa+], 586.1367)); 31 P NMR (202 MHz, MeOD): ™P 3.74, 4.14; 19F NMR (470 MHz, MeOD): ™F -167.57, - 167.46; 1H NMR (500 MHz, MeOD): ™H 1.35 (d, 3H, J = 7.4 Hz, CHCH3, one diast.), 1.37 (d, 3H, J = 6.9 Hz, CHCH3, one diast.), 1.96 2.32 (m, 2H, H-2'), 3.95-4.08 (m, 2H, CHCH3, H-4'), 4.23-4.34 ( m, 3H, CH2OP, H-3'), 5.13 (br d, 1H, J = 12.3 Hz, OCHHPh), 5.16 (br d, 1H, J = 12.3 Hz, OCHHPh, um 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); 13C NMR (125 MHz, MeOD): ™C 20.2 (d, 3JC-P = 7.5 Hz, CH3), 20.4 (d, 3JC-P = 6.2 Hz, CH3), 40.6 (CH2), 40.9 (cH2), 51.6 (cH), 51.8 (cH), 67.5 (d, 2JC-P = 5.3 Hz, cH2), 67.6 (d, 2JC -P = 5.5 Hz, cH2), 68.0 (cH2), 71.8 (cH), 71.9 (cH), 86.6 (d, 3JC-P = 8.0 Hz, cH), 86.8 (d, 3JC-P = 8.3 Hz, cH), 86.9 (cH), 87.0 (cH), 121.4 (d, 3JC-P = 5.1 Hz, cH), 121.5 (d, 3JC-P = 5.6 Hz, cH), 125.5 (d, 5JC-P = 3.2 Hz, cH), 125.8 (d, 5JC-P = 3.2 Hz , cH), 126.3 (cH), 129.0 (cHx2), 129.3 (cHx2), 129.6 (cHx2), 130.8 (cHx2), 140.9 (c), 141.6 ( d, 1JC-F = 233.6 Hz, C), 141.7 (d, 1JC-F = 233.6 Hz, C), 150.7 (d, 4JC-F = 5.7 Hz, C), 152.1 (d, 2JC-F = 6.5 Hz, C), 159.2 (d, 2JC-F = 26.3 Hz, C), 174.6 (d, 3JC-P = 4.9 Hz , C), 174.7 (d, 3JC-P = 4.9 Hz, C), m/z (ES) 586 (MNa+, 100%); Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited a peak of the mixture of diastereoisomers with tR 25.08 min. (97%). 5-Fluoro-2'-deoxyuridine-5'-O -[henyl(methoxy-L-alaninyl)] phosphate (CPF382) (Reference example)

[00109] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 ml, 5.07 mmols) and phosphorochloridate of phenyl(methoxy-L-alaninyl) (0.84 g, 3.04 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides compound title as a colorless solid (16.0 mg, 4%) [Rf = 0.30 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 510.1045. C19H23N3O9NaPF requires [MNa+], 510.1054) ; 31 P NMR (202 MHz, MeOD): ™P 3.79, 4.09; 19F NMR (470 MHz, MeOD): ™F -167.78, - 167.72; 1H NMR (500 MHz, MeOD): ™H 1.34 (d, 3H, J = 7.1 Hz, CHCH3, one diast.), 1.36 (d, 3H, J = 7.1 Hz, CHCH3, a diast.), 2.02 2.16 (m, 1H, H-2'), 2.25-2.34 (m, 1H, H-2'), 3.69 (s, 3H, OCH3, one diast.), 3.70 (s, 3H, OCH3, one diast.), 3.93-4.02 (m, 1H, CHCH3), 4.08 4.13 (m, 1H, H-4' ), 4.27-4.45 (m, 3H, CH2OP, 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, 3JH-F = 6.4 Hz, H-6); 13C NMR (125 MHz, MeOD): ™C 20.2 (d, 3JC-P = 7.5 Hz, CH3), 20.5 (d, 3JC-P = 6.7 Hz, CH3), 40.8 (CH2), 40.9 (CH2), 51.5 (CH3), 51.6 (cH3), 52.7 (cH), 52.8 (cH), 67.5 (d, 2JC-P = 5 .5 Hz, cH2), 67.6 (d, 2JC-P = 5.1 Hz, CH2), 72.0 (CH), 72.1 (CH), 86.7 (d, 3JC-P = 8 .2 Hz, CH), 86.8 (d, 3JC-P = 8.2 Hz, CH), 86.9 (CH), 87.0 (CH), 121.2 (d, 3JC-P = 4 .5 Hz, CH), 121.4 (d, 3JC-P = 4.7 Hz, CH), 125.6 (d, 5JC-P = 2.9 Hz, CH), 125.9 (d, 5JC -P = 2.9 Hz, CH), 126.2 (CH), 130.8 (CH), 130.9 (CH), 141.6 (d, 1JC-F = 233.8 Hz, C), 141.7 (d, 1JC-F = 233.9 Hz, C), 150.6 (d, 4JC-F = 3.6 Hz, C), 152.1 (d, 2JC-P = 6.8 Hz , C), 152.2 (d, 2JC-P = 6.8 Hz, C), 159.4 (d, 2JC-F = 26.0 Hz, C), 175.2 (d, 3JC-P = 4.8 Hz, C), 175.5 (d, 3JC-P = 3.7 Hz, C), m/z (ES) 510 (MNa+, 100%); Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 min, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 23.11 min. and tR 24.11 min. (74%: 24%). 5-Fluoro-2'-deoxyuridine-5'-O -[henyl(ethoxy-L-alaninyl)] phosphate (CPF383)

[00110] The phosphoramidate was prepared using 5-fluoro-2'deoxyuridine (0.10 g, 0.40 mmol), N-methylimidazole (NMI) (0.16 ml, 2.03 mmols) and phenyl phosphorochloridate (ethoxy-L-alaninyl) (0.35 g, 1.21 mmol) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (10.0 mg, 5%) [Rf = 0.11 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 524.1202. C20H25N3O9NaPF requires [MNa+], 524.1210); 31 P NMR (202 MHz, MeOD): ™P 3.83, 4.11; 19F NMR (470 MHz, MeOD): ™F -167.67, - 167.61; 1H NMR (500 MHz, MeOD): ™H 1.25 (t, 3H, J = 7.1 Hz, CH 2 CH 3 , one diast.), 1.26 (t, 3H, J = 7.1 Hz, CH 2 CH 3 , one diast.), 1.34 (d, 3H, J = 7.2 Hz, CHCH3, one diast.), 1.36 (d, 3H, J = 7.2 Hz, CHCH3, one diast.), 2 .02-2.15 (m, 1H, H-2'), 2.24-2.34 (m, 1H, H-2'), 3.90-4.00 (m, 1H, CHCH3,) , 4.08-4.19 (m, 3H, CH2CH3, H-4'), 4.27-4.45 (m, 3H, CH2OP, 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, 3JH-F = 6.4 Hz, H-6); 13C NMR (125 MHz, MeOD): ™C 14.4 (CH3), 15.4 (CH3), 20.3 (d, 3JC-P = 7.6Hz, CH3), 20.5 (d, 3JC -P = 6.5 Hz, CH3), 40.8 (CH2), 40.9 (CH2), 51.6 (CH), 51.7 (CH), 62.4 (CH2), 62.5 ( CH2), 67.5 (d, 2JC-P = 5.4 Hz, CH2), 67.6 (d, 2JC-P = 5.4 Hz, CH2), 72.0 (CH), 72.1 ( CH), 86.7 (d, 3JC-P = 8.1 Hz, CH), 86.8 (d, 3JC-P = 8.3 Hz, CH), 86.9 (CH), 87.0 ( CH), 121.3 (d, 3JC-P = 4.8 Hz, CH), 121.4 (d, 3JC-P = 4.6 Hz, CH), 125.6 (d, 5JC-P = 4 .6 Hz, CH), 125.8 (d, 5JC-P = 4.8 Hz, CH), 126.3 (CH), 130.8 (CH), 130.9 (CH), 141.6 ( d, 1JC-F = 233.7 Hz, C), 141.8 (d, 1JC-F = 233.8 Hz, C), 150.8 (br C), 152.0 (d, 2JC-P = 7.1 Hz, C), 152.1 (d, 2JC-P = 7.1 Hz, C), 159.6 (d, 2JC-F = 26.0 Hz, C), 174.8 (d, 3JC-P = 5.4 Hz, C), 175.1 (d, 3JC-P = 4.4 Hz, C), m/z (ES) 524 (MNa+, 100%); Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 min, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 25.63 min. and tR 26.40 min. (71%: 27%). 5-Fluoro-2'deoxyuridine-5'-O -[henyl(isopropoxy-L-alaninyl)] phosphate (CPF384)

[00111] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 ml, 5.07 mmols) and phosphorochloridate. phenyl(isopropoxy-L-alaninyl) (0.93 g, 3.04 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides compound title as a colorless solid (31.0 mg, 6%) [Rf = 0.21 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 538.1370. C21H27N3O9NaPF requires [MNa+], 538.1367) ; 31 P NMR (202 MHz, MeOD): ™P 3.87, 4.13; 19F NMR (470 MHz, MeOD): ™F -167.64, - 167.56; 1H NMR (500 MHz, MeOD): ™H 1.22-1.26 (m, 6H, CH(CH3)2), 1.33 (d, 3H, J = 7.1Hz, CHCH3, one diast. ), 1.35 (d, 3H, J = 7.1 Hz, CHCH 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, CHCH3), 4.08-4.14 (m, 1H, H-4'), 4.27-4.45 (m, 3H, CH2OP, H-3'), 4.98 (hept, 1H, J = 6.1 Hz, CH(CH3)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, 3JH-F = 6 .4 Hz, H-6); 13C NMR (125 MHz, MeOD): ™C 20.3 (d, 3Jc-p = 7.6Hz, CH3), 20.5 (d, 3JC-P = 6.4Hz, CH3), 21.9 (CH3x2), 22.0 (CH3x2), 40.8 (cH2), 40.9 (cH2), 51.7 (cH), 51.8 (cH), 67.5 (d, 2JC-P = 5 0.4 Hz, cH2), 67.6 (d, 2JC-P = 5.2 Hz, cH2), 70.2 (cH), 70.3 (cH), 72.0 (cH), 72.1 ( cH), 86.6 (d, 3JC-P = 8.2 Hz, cH), 86.8 (d, 3JC-P = 8.2 Hz, cH), 86.9 (cH), 87.0 ( cH), 121.2 (d, 3JC-P = 4.7 Hz, cH), 121.4 (d, 3JC-P = 4.9 Hz, cH), 125.6 (d, 5JC-P = 7 .1 Hz, cH), 125.9 (d, 5JC-P = 7.1 Hz, cH), 126.3 (cH), 130.8 (cH), 130.9 (cH), 141.8 ( d, 1JC-F = 234.5 Hz, c), 141.9 (d, 1JC-F = 234.4 Hz, c), 150.7 (d, 4JC-F = 3.7 Hz, c), 152.0 (d, 3JC-P = 6.2 Hz, c), 152.1 (d, 3JC-P = 6.2 Hz, c), 159.3 (d, 2JC-F = 26.3 Hz , c), 159.4 (d, 2JC-F = 26.0 Hz, c), 174.3 (d, 3JC-P = 5.6 Hz, c), 174.6 (d, 3JC-P = 4.6 Hz, c), m/z (ES) 538 (MNa+, 100%); Reversed phase HPLc eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 28.93 min. and tR 29.45 min. (44%: 52%). 5-Fluoro-2'deoxyuridine-5'-O -[henyl(cyclohexoxy-L-alaninyl)] phosphate (CPF508

[00112] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.30 g, 1.21 mmol), N-methylimidazole (NMI) (0.48 ml, 6.09 mmols) and phosphorochloridate of phenyl(cyclohexoxy-L-alaninyl) (1.026 g, 3.65 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) gives the title compound as a colorless solid (6.7 mg, 3%) [Rf = 0.45 (CH 2 Cl 2 -MeOH, 95:5)]; (Found: MNa+, 565.48. C24H31N3O9NaPF requires [MNa+], 565.49); 31 P NMR (202 MHz, MeOD): ™P 3.86, 4.15; 19F NMR (470 MHz, MeOD): ™F -167.68, -167.62; 1H NMR (500 MHz, MeOD): ™H 1.26-1.40 (m, 3H, CHCH3), 1.41-1.50 (m, 4H, CH(CH2)5), 1.52-1 .61 (m, 1H, CH(CH2)5), 1.70-1.88 (m, 5H, CH(CH2)5), 2.00-2.14 (m, 1H, H-2') , 2.23-2.34 (m, 1H, H-2'), 3.90-3.98 (m, 1H, CHCH3), 4.07-4.14 (m, 1H, H-4' ), 4.29-4.39 (m, 2H, CH2OP), 4.40-4.45 (m, 1H, H-3'), 4.72-4.78 (m, 1H, CH(CH2) )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, 3JH-F = 6.6 Hz, H-6); 13C NMR (125 MHz, MeOD): ™C 20.3 (d, 3Jc-p = 7.3 Hz, CH3), 20.6 (d, 3JC-P = 6.5 Hz, CH3), 24.6 (CH2), 26.4 (CH2), 32.3 (cH2), 32.4 (cH2), 40.9 (cH2), 51.7 (cH), 51.9 (cH), 67.5 ( d, 2JC-P = 5.3 Hz, cH2), 67.7 (d, 2JC-P = 5.3 Hz, cH2), 72.0 (cH), 72.1 (cH), 74.9 ( cH), 86.6 (d, 3JC-P = 8.5 Hz, cH), 86.8 (d, 3JC-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, 1JC-F = 234.0 Hz, c), 150.7 (d, 4JC-P = 4.0 Hz, c), 152.0 (d, 2JC-P = 7.2 Hz, c), 152.1 (d, 2JC-P = 7.2 Hz, c), 159.4 (d , 2JC-F = 26.3 Hz, c), 174.3 (d, 3JC-P = 4.6 Hz, c), 174.5 (d, 3JC-P = 4.3 Hz, c); m/z (ES) 565 (MNa+, 100%); Reversed phase HPLc eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 30.00 min. and tR 30.45 min. (33% : 65%). 5-Fluoro-2'deoxyuridine-5'-O-[-nitro-phenyl(ethoxy-L-alaninyl)] phosphate (CPF430)

[00113] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmols) and phosphorochloridate of p-nitro-phenyl(ethoxy-L-alaninyl) (1.02 g, 3.04 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5 ) furnish the title compound as a colorless solid (77.0 mg, 14%) [Rf = 0.24 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 569.1066. C20H24N4O11NaPF requires [MNa+], 569,1061); 31 P NMR (202 MHz, MeOD): ™P 3.63, 3.67; 19F NMR (470 MHz, MeOD): ™F -167.89, - 167.82; 1H NMR (500 MHz, MeOD): ™H 1.24 (t, 3H, J = 7.0 Hz, CH2CH3), 1.25 (t, 3H, J = 7.0 Hz, CH2CH3), 1.36 -1.40 (m, 3H, CHCH3), 2.16-2.25 (m, 1H, H-2'), 2.30-2.38 (m, 1H, H-2'), 3, 95-4.00 (m, 1H, CHCH3), 4.09-4.19 (m, 3H, CH2CH3, H-4'), 4.32-4.48 (m, 3H, CH2OP, 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 0.7 Hz, ArH), 7.85 (d, 1H, 3JH-F = 6.6 Hz, H-6), 7.87 (d, 1H, 3JH-F = 6.6 Hz, H-6) , 8.29 (d, 2H, J = 8.7 Hz, ArH); 13C NMR (125 MHz, MeOD): ™C 14.5 (CH3), 14.6 (CH3), 20.3 (d, 3JC-P = 7.5Hz, CH3), 20.4 (d, 3JC -P = 6.4 Hz, CH3), 40.8 (CH2), 51.6 (CH), 51.7 (CH), 62.5 (CH2), 67.8 (d, 2JC-P = 5 .5 Hz, cH2), 68.0 (d, 2JC-P = 5.2 Hz, cH2), 71.8 (cHx2), 86.4 (cH), 86.5 (cH), 87.0 ( d, 3JC-P = 7.5 Hz, cH), 122.1 (d, 3JC-P = 5.2 Hz, cH), 122.5 (d, 3JC-P = 5.0 Hz, cH), 125.7 (cH), 126.0 (cH), 126.6 (cH), 141.3 (d, 1JC-F = 233.6 Hz, c), 141.5 (d, 1JC-F = 233 .7 Hz, c), 146.2 (c), 150.6 (d, 4JC-P = 4.6 Hz, c), 156.9 (d, 2JC-P = 2.6 Hz, c), 157.0 (d, 2JC-P = 2.6 Hz, c), 159.3 (d, 2JC-F = 26.3 Hz, c), 174.6 (d, 3JC-P = 4.6 Hz , c), 174.9 (d, 3JC-P = 3.7 Hz, c), m/z (ES) 569 (MNa+, 100%); Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 min., 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 31.63 min. and tR 31.89 min. (11% : 85%). 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)] phosphate (CPF373)

Phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 ml, 5.07 mmols) and phosphorochloridate. 1-naphthyl(benzoxy-L-alaninyl) (0.82 g, 3.04 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (47.0 mg, 8%) [Rf = 0.19 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 636.1520. C29H29N3O9NaPF requires [MNa+], 636, 1523); 31 P NMR (202 MHz, MeOD): ™P 4.24, 4.59; 19F NMR (470 MHz, MeOD): ™F -167.36, - 167.18; 1H NMR (500 MHz, MeOD): ™H 1.34-1.38 (m, 3H, CHCH3), 1.67-1.79 (m, 1H, H-2'), 2.08-2, 17 (m, 1H, H-2'), 4.03-4.15 (m, 2H, CHCH3, H-4'), 4.24-4.36 (m, 3H, CH2OP, 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, 1H, ArH); 13C NMR (125 MHz, MeOD): ™C 20.3 (d, 3Jc-p = 7.6 Hz, CH3), 20.5 (d, 3Jc-p = 6.5 Hz, CH3), 40.8 (CH2), 40.9 (CH2), 51.8 (CH), 51.9 (CH), 67.6 (d, 2Jc-p = 5.3 Hz, cH2), 67.8 (d, 2Jc -p = 5.2 Hz, cH2), 68.0 (cH2), 68.1 (cH2), 72.0 (cH), 72.1 (cH), 86.7 (d, 3Jc-p = 8 .1 Hz, cH), 86.8 (d, 3Jc-p = 8.1 Hz, cH), 86.9 (cH), 87.0 (cH), 116.2 (d, 3Jc-p = 3 .3 Hz, cH), 116.5 (d, 3JC-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, 1JC-F = 233.8 Hz , C), 141.7 (d, 1JC-F = 233.9 Hz, C), 147.8 (d, 2JC-P = 7.7 Hz, C), 147.9 (d, 2JC-P = 7.4 Hz, C), 150.5 (d, 4JC-F = 4.0 Hz, C), 159.3 (d, 2JC-F = 26.1 Hz, C), 174.6 (d, 3JC-P = 5.0 Hz, C), 174.9 (d, 3JC-P = 4.3 Hz, C), m/z (ES) 636 (MNa+, 100%); Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 34.23 min. and tR 34.59 min. (23%: 76%). 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl (methoxy-L-alaninyl)] phosphate (CPF385)

[00115] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 ml, 5.07 mmols) and phosphorochloridate of 1-naphthyl(methoxy-L-alaninyl) (0.99 g, 3.04 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (7.0 mg, 1%) [Rf = 0.23 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 560.1198. C23H25N3O9NaPF requires [MNa+], 560, 1210); 31 P NMR (202 MHz, MeOD): ™P 4.31.4.56; 19F NMR (470 MHz, MeOD): ™F -167.51, - 167.37; 1H NMR (500 MHz, MeOD): ™H 1.34 (d, 3H, J = 6.7 Hz, CHCH3, one diast.), 1.36 (d, 3H, J = 6.7 Hz, CHCH3, a diast.), 1.76-1.87 (m, 1H, H-2'), 2.12-2.22 (m, 1H, H-2'), 3.64 (s, 3H, OCH3 , one diast.), 3.65 (s, 3H, OCH3, one diast.), 4.03-4.13 (m, 2H, CHCH3, H-4'), 4.30-4.38 (m , 2H, CH2OP), 4.41 (dd, 1H, J = 2.5 Hz, J = 5.8 Hz, H-3'), 6.12-6.19 (m, 1H, H-1' ), 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); 13 C NMR (125 MHz, MeOD): ™C 20.3 (d, 3Jc- p = 7.1 Hz, CH3), 20.4 (d, 3JC-P = 6.5 Hz, CH3), 40.7 (CH2), 40.8 (CH2), 51.6 (CH3), 51 0.7 (cH3), 52.7 (cH), 52.8 (cH), 67.8 (d, 2JC-P = 5.7 Hz, cH2), 67.5 (d, 2JC-P = 5. 7 Hz, cH2), 72.0 (cH), 72.1 (cH), 86.7 (d, 3JC-P = 7.9 Hz, cH), 86.9 (d, 3JC-P = 8, 5 Hz, cH), 86.9 (cH), 87.0 (cH), 116.2 (d, 3JC-P = 3.1 Hz, cH), 116.5 (d, 3JC-P = 3, 5 Hz, cH), 122.5 (cH), 122.6 (cH), 125.4 (cH), 125.5 (cH), 125.6 (cH), 125.7 (cH), 126, 1 (cH), 126.2 (cH), 126.5 (cH), 126.6 (cH), 127.6 (cH), 1 27.7 (cx2), 127.8 (cH), 127.9 (cH), 128.9 (cH), 129.0 (cH), 136.3 (c), 141.6 (d, 1JC- F = 233.4 Hz, c), 141.7 (d, 1JC-F = 234.1 Hz, c), 147.8 (d, 2JC-P = 7.9 Hz, c), 148.0 ( d, 2JC-P = 7.2 Hz, c), 150.6 (c), 159.4 (d, 2JC-F = 27.0 Hz, c), 175.2 (d, 3JC-P = 3 0.9 Hz, c), 175.5 (d, 3JC-P = 3.9 Hz, c), m/z (ES) 560 (MNa+, 100%); Reversed phase HPLc eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 28.45 min. and tR 28.85 min. (73% : 25%). 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(ethoxy-L-alaninyl)] phosphate (CPF386)

[00116] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmols) and phosphorochloridate of 1-naphthyl(ethoxy-L-alaninyl) (1.04 g, 3.04 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (47.0 mg, 4%) [Rf = 0.25 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 574.1360. C24H27N3O9NaPF requires [MNa+], 574, 1367); 31 P NMR (202 MHz, MeOD): ™P 4.34, 4.55; 19F NMR (470 MHz, MeOD): ™F -167.31, - 167.16; 1H NMR (500 MHz, MeOD): ™H 1.20 (t, 3H, J = 7.0 Hz, CH 2 CH 3 , one diast.), 1.21 (t, 3H, J = 7.0 Hz, CH 2 CH 3 , a diast.), 1.33 1.37 (m, 3H, CHCH3), 1.73-1.86 (m, 1H, H-2'), 2.12-2.21 (m, 1H, H -2'), 4.01-4.07 (m, 1H, CHCH3), 4.08-4.13 (m, 3H, CH2CH3, H-4'), 4.31-4.43 (m, 3H, CH2OP, H-3'), 6.11-6.19 (m, 1H, H-1'), 7.39-7.46 (m, 1H, ArH), 7.50-7.57 (m, 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); 13C NMR (125 MHz, MeOD): ™C 14.4 (CH3), 20.3 (d, 3JC-P = 7.4 Hz, CH3), 20.5 (d, 3JC-P = 6.2 Hz , CH3), 40.8 (cH2), 40.9 (cH2), 51.8 (cH), 51.9 (cH), 62.4 (cH2), 62.5 (cH2), 67.8 ( d, 2JC-P = 4.6 Hz, cH2), 67.9 (d, 2JC-P = 4.6 Hz, cH2), 72.0 (cH), 72.1 (cH), 86.7 ( d, 3JC-P = 8.4 Hz, cH), 86.8 (d, 3JC-P = 8.4 Hz, cH), 86.9 (cH), 87.0 (cH), 116.1 ( d, 3JC-P = 3.5 Hz, cH), 116.5 (d, 3JC-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, 1JC-F = 233.3 Hz, c), 141.7 (d, 1JC-F = 233.4 Hz, c), 147.8 (d, 2JC-P = 6.9 Hz, c), 148.0 (d, 2JC-P = 6.9 Hz, c), 150.6 (c), 159.3 (d, 2JC-F = 26.3 Hz, c), 174, 8 (d, 3JC-P = 4.8 Hz, c), 175.1 (d, 3JC-P = 4.0 Hz, c); m/z (ES) 574 (MNa+, 100%); Reversed phase HPLc eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 30.77 min. and tR 31.20 min. (51%: 48%). 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(isopropoxy-L-alaninyl)] phosphate (CPF387)

The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.10 g, 0.40 mmol), tert-butylmagnesium chloride in tetrahydrofuran (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 2 Cl 2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (71.0 mg, 17%) [Rf = 0.21 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 588 1521. C25H29N3O9NaPF requires [MNa+], 588.1523); 31 P NMR (202 MHz, MeOD): ™P 4.38, 4.58; 19F NMR (470 MHz, MeOD): ™F -167.43, - 167.26; 1H NMR (500 MHz, MeOD): ™H 1.19-1.23 (m, 6H, CH(CH3)2), 1.34-1.38 (m, 3H, CHCH3), 1.68-1 .84 (m, 1H, H-2'), 2.09-2.20 (m, 1H, H-2'), 3.96-4.05 (m, 1H, CHCH3), 4.07- 4.12 (m, 1H, H-4'), 4.29-4.38 (m, 2H, CH2OP), 4.39-4.42 (m, 1H, H-3'), 4.93 -5.01 (m, 1H, CH(CH3)2), 5.10-6.18 (m, 1H, 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); 13C NMR (125 MHz, MeOD): ™C 20.3 (d, 3Jc-p = 7.1 Hz, CH3), 20.5 (d, 3JC-P = 6.6 Hz, CH3), 21.8 (CH3), 21.9 (CH3), 22.0 (CH3), 22.1 (cH3), 40.8 (cH2), 40.9 (cH2), 51.9 (cH), 52.0 ( cH), 67.8 (d, 2JC-P = 4.5 Hz, cH2), 67.9 (d, 2JC-P = 4.8 Hz, cH2), 70.2 (cH), 70.3 ( cH), 72.0 (cH), 72.1 (cH), 86.6 (cH), 86.7 (cH), 86.9 (d, 3JC-P = 8.6 Hz, cH), 87 .0 (d, 3JC-P = 8.6 Hz, cH), 116.2 (d, 3JC-P = 2.5 Hz, cH), 116.5 (d, 3JC-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, 1JC-F = 233.2 Hz, c), 141.7 (d, 1JC-F = 233.4 Hz, c), 147.7 (d, 2JC-P = 7.6 Hz, c), 147.9 (d, 2JC-P = 7.7 Hz, c), 150.5 (c), 159.4 (d, 2JC-F = 26.2 Hz, c), 174, 4 (d, 3JC-P = 5.0 Hz, c), 174.7 (d, 3JC-P = 5.1 Hz, c); m/z (ES) 588 (MNa+, 100%); Reversed phase HPLc eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 32.20 min. and tR 32.80 min. (27%: 69%). 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(cyclohexoxy-L-alaninyl)] phosphate (CPF509)

[00118] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.30 g, 1.21 mmol), N-methylimidazole (NMI) (0.48 ml, 6.09 mmols) and phosphorochloridate of phenyl(cyclohexoxy-L-alaninyl) (1.45 g, 3.65 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides compound title as a colorless solid (6.7 mg, 3%) [Rf = 0.47 (CH 2 Cl 2 -MeOH, 95:5)]; (Found: MNH4+, 623.2261. C28H37N4O9NaPF requires [MNH4+], 623.2282); 31 P NMR (202 MHz, MeOD): ™P 4.35, 4.52; 19F NMR (470 MHz, MeOD): ™F -167.31, - 167.17; 1H NMR (500 MHz, MeOD): ™H 1.30-1.43 (m, 3H, CHCH3), 1.44-1.56 (m, 4H, CH(CH2)5), 1.57-1 .66 (m, 1H, CH(CH2)5), 1.67-1.83 (m, 5H, CH(CH2)5), 1.84-1.93 (m, 1H, H-2') , 2.09-2.20 (m, 1H, H-2'), 3.98-4.06 (m, 1H, CHCH3), 4.07-4.15 (m, 1H, H-4' ), 4.29-4.38 (m, 2H, CH2OP), 4.39-4.44 (m, 1H, H-3'), 4.67-4.76 (m, 1H, CH(CH2 )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, 3JH-F = 6.6 Hz, H-6); 13C NMR (125 MHz, MeOD): ™C 20.4 (d, 3JC-P = 8.0 Hz, CH3), 20.6 (d, 3JC-P = 6.5 Hz, CH3), 24.5 (CH2), 26.3 (CH2), 32.3 (CH2), 40.8 (CH2), 51.8 (CH), 51.9 (cH), 67.8 (cH2), 72.0 ( cH), 72.2 (cH), 75.0 (cH), 86.7 (d, 3Jc-p = 8.2 Hz, cH), 87.0 (cH), 116.1 (d, 3Jc- p = 2.5 Hz, cH), 116.4 (d, 3Jc-p = 3.0 Hz, cH), 122.6 (cH), 124.8 (cH), 125.9 (cH), 126 ,1 (cH), 126.2 (cH), 126.4 (cH), 126.5 (cH), 126.6 (cH), 127.6 (cH), 127.7 (cx2), 127, 8 (cH), 127.9 (cH), 128.9 (cH), 129.0 (cH), 136.3 (c), 141.6 (c), 148.0 (d, 2Jc-p = 7.2 Hz, c), 150.6 (c), 159.4 (d, 2Jc-F = 27.0 Hz, c), 175.2 (d, 3Jc-p = 3.9 Hz, c) , 175.5 (d, 3Jc-p = 3.9 Hz, c); m/z (ES) 623 (MNH4+, 100%); Reversed phase HPLC eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 30.50 min. and tR 31.48 min. (27%: 69%). 5-Fluoro-2'deoxyuridine-5'-O-[henyl(α,α-dimethylglycine)] phosphate (CPF393)

[00119] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.40 g, 1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (tBuMgCl) (1.0 M, 2, 43 mL, 2.43 mmols) and phenyl(α,α-dimethylglycine) phosphorochloridate (1.17 g, 3.20 mmols) according to general procedure E. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) gives the title compound as a colorless solid (69.0 mg, 7%) [Rf = 0.27 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 600 1527. C26H29N3O9NaPF requires [MNa+], 600.1523); 31 P NMR (202 MHz, MeOD): ™P 2.42, 2.47; 19F NMR (470 MHz, MeOD): ™F -167.80, - 167.62; 1H NMR (500 MHz, MeOD): ™H 1.51-1.60 (m, 6H, C(CH3)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, CH2OP), 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, OCH2Ph), 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, 3JH-F = 6.4Hz, H- 6, one diast.), 7.80 (d, 3JH-F = 6.4 Hz, H-6, one diast.); 13C NMR (125 MHz, MeOD): ™C 27.5 (CH3), 27.7 (d, 3JC-P = 7.1 Hz, CH3), 27.8 (d, 3JC-P = 7.1 Hz , CH3), 40.8 (CH2), 40.9 (CH2), 58.2 (C), 58.3 (c), 67.6 (d, 2JC-P = 5.5 Hz, cH2), 67.7 (d, 2JC-P = 5.5 Hz, cH2), 68.3 (CH2), 71.9 (CH), 72.0 (CH), 86.6 (d, 3JC-P = 8 .1 Hz, CH), 86.8 (d, 3JC-P = 7.3 Hz, CH), 86.9 (CH), 121.4 (d, 3JC-P = 4.8 Hz, CH), 121.6 (d, 3JC-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, 1JC-F = 233.7 Hz, C), 150.6 (C), 152.1 (d, 4JC-F = 7.0 Hz, C), 152.1 (d , 4JC-F = 7.6 Hz, C), 159.3 (d, 2JC-F = 26.1 Hz, C), 159.4 (d, 2JC-F = 26.1 Hz, C), 176 .5 (d, 3JC-P = 4.0 Hz, C), 176.6 (d, 3JC-P = 3.8 Hz, C), m/z (ES) 600.1 (MNa+, 100%) ; Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 35 minutes, 1 mL/min, | = 275 nm, exhibited a peak of the mixture of diastereoisomers with tR 17.71 (96%). 5-Fluoro-2'deoxyuridine-5'-O-[henyl(ethoxy-a,a-dimethylglycine)] phosphate (CPF394)

Phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.20 g, 0.80 mmol), N-methylimidazole (NMI) (0.31 ml, 4.0 mmols) and phosphorochloridate. phenyl(ethoxy-α,α-dimethylglycine) (0.73 g, 2.40 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (25.0 mg, 6%) [Rf = 0.24 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 538.1367. C21H27N3O9NaPF requires [MNa+], 538, 1367); 31 P NMR (202 MHz, MeOD): ™P 2.49, 2.52; 19F NMR (470 MHz, MeOD): ™F -167.62, -167.58; 1H NMR (500 MHz, MeOD): ™H 1.24 (t, 3H, J = 7.1 Hz, CH 2 CH 3 , one diast.), 1.26 (t, 3H, J = 7.1 Hz, CH 2 CH 3 , one diast.), 1.44-1.54 (m, 6H, C(CH3)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, CH2CH3, H-4'), 4.33-4.49 (m, 3H, CH2OP, 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, 3JH-F = 6.4Hz, H -6, one diast.), 7.88 (d, 3JH-F = 6.4 Hz, H-6, one diast.); 13C NMR (125 MHz, MeOD): ™C 14.4 (CH3), 14.5 (CH3), 27.5 (d, 3JC-P = 7.3 Hz, CH3), 27.7 (d, 3JC -P = 7.6 Hz, CH3), 27.8 (d, 3JC-P = 7.6 Hz, CH3), 40.8 (CH2), 40.9 (CH2), 58.1 (C), 62.6 (CH2), 62.7 (CH2), 67.6 (d, 2JC-P = 6.7 Hz, CH2), 67.7 (d, 2JC-P = 5.8 Hz, CH2), 71.9 (CH), 72.0 (CH), 86.6 (d, 3JC-P = 8.1 Hz, CH), 86.8 (d, 3JC-P = 7.6 Hz, CH), 86.9 (CH), 121.4 (d, 3JC-P = 4.4 Hz, CH), 121.6 (d, 3JC-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, 1JC-F = 233.5 Hz, C), 150.6 (C), 150.7 (C), 152.2 (d, 4JC-F = 7.3 Hz, C), 152 .3 (d, 4JC-F = 6.9 Hz, C), 159.2 (d, 2JC-F = 20.3 Hz, C), 159.4 (d, 2JC-F = 20.4 Hz, C), 176.6 (d, 3JC-P = 4.2 Hz, C), 176.8 (d, 3JC-P = 4.6 Hz, C), m/z (ES) 538.1 (MNa+ , 100%); Reversed phase HPLC eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 18.76 min. and tR 20.44 min. (68%: 30%). 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl (benzoxy-a,a-dimethylglycine)] phosphate (CPF395)

[00121] The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.40 g, 1.62 mmol), N-methylimidazole (NMI) (0.64 ml, 8.0 mmols) and phosphorochloridate of 1-naphthyl(benzoxy-α,α-dimethylglycine) (2.00 g, 4.80 mmols) according to general procedure D. Purification by gradient column chromatography eluting with CH 2 Cl 2 until CH 2 Cl 2 - MeOH (95:5) furnish the title compound as a colorless solid (16.4 mg, 6%) [Rf = 0.15 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 650.1678. C30H31N3O9NaPF requires [MNa+], 650 1680); 31 P NMR (202 MHz, MeOD): ™P 2.87, 3.03; 19F NMR (470 MHz, MeOD): ™F -167.95, - 167.13; 1H NMR (500 MHz, MeOD): ™H 1.37-1.42 (m, 6H, C(CH3)2), 1.61-1.69 (m, 1H, H-2', one diast. ), 1.79-1.87 (m, 1H, H-2', 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 (m, 1H, H-4'), 4.19-4.35 (m, 3H, CH2OP, 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, 3JH-F = 6.4Hz, H-6, one diast.), 7.70 (d, 3JH- F = 6.4Hz, H-6, one diast.), 7.86-7.90 (m, 1H, Ar H), 8.17-8.22 (m, 1H, Ar H); 13C NMR (125 MHz, MeOD): ™C 27.5 (d, 3JC-P = 4.4 Hz, CH3), 27.9 (d, 3JC-P = 7.3 Hz, CH3), 28.0 (d, 3JC-P = 7.3 Hz, cH3), 40.7 (cH2), 40.8 (cH2), 65.2 (c), 67.8 (d, 2JC-P = 6.5 Hz , cH2), 68.3 (cH2), 72.0 (cH), 72.1 (cH), 86.6 (d, 3JC-P = 8.2 Hz, cH), 86.8 (d, 3JC -P = 7.8 Hz, cH), 86.9 (cH), 116.3 (d, 3JC-P = 3.2 Hz, cH), 116.7 (d, 3JC-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, 1JC-F = 234.4 Hz, c), 147.9 (d, 3JC-P = 7.7 Hz, c), 148.0 (d, 3JC-P = 8.2 Hz, c), 150.7 (d, 4JC-F = 3.7 Hz, c), 159.5 (d, 2JC-F = 25.8 Hz, c), 159.6 (d, 2JC-F = 25.8 Hz, c), 176.5 (c), 176.6 (c), m/z (ES) 650.0 (MNa+, 100%); Reversed phase HPLc eluting with H2O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited two peaks of the diastereoisomers with tR 20.80 min. and tR 21.00 min. (72%: 24%). 5-Fluoro-2'deoxyuridine-5'-O -[1-naphthyl (ethoxy-g,g-dimethylglycine)] phosphate (CPF396)

The phosphoramidate was prepared using 5-fluoro-2'-deoxyuridine (0.40 g, 1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (tBuMgCl) (1.0 M, 2, 43 mL, 2.43 mmols) and 1-naphthyl(ethoxy-a,a-dimethylglycine) phosphorochloridate (1.14 g, 3.20 mmols) according to general procedure E. Purification by gradient column chromatography eluting with CH2Cl2 until CH2Cl2-MeOH (95:5) provides the title compound as a colorless solid (54.0 mg, 2%) [Rf = 0.10 (CH2Cl2-MeOH, 95:5)], (Found: MNa+, 588.1528. C25H29N3O9NaPF requires [MNa+], 588.1523); 31 P NMR (202 MHz, MeOD): ™P 2.91, 3.03; 19F NMR (470 MHz, MeOD): ™F -167.38, - 167.21; 1H NMR (500 MHz, MeOD): ™H 1.24 (t, 3H, J = 7.1 Hz, CH 2 CH 3 , one diast.), 1.25 (t, 3H, J = 7.1 Hz, CH 2 CH 3 , one diast.), 1.50 1.55 (m, 6H, C(CH3)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, J = 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, J = 7.1 Hz, CH2CH3), 4.36-4.41 (m, 3H, CH2OP, 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); 13C NMR (125 MHz, MeOD): ™C 14.4 (CH3), 27.5 (br s, CH3), 27.9 (d, 3JC-P = 6.1 Hz, CH3), 28.0 ( d, 3JC-P = 6.1 Hz, CH3), 40.7 (CH2), 40.8 (CH2), 58.2 (C), 58.3 (C), 62.6 (CH2), 67 .8 (d, 2JC-P = 4.9 Hz, CH2), 67.9 (d, 2JC-P = 4.5 Hz, CH2), 72.0 (CH), 72.1 (CH), 86 .7 (d, 3JC-P = 7.7 Hz, CH), 86.9 (d, 3JC-P = 7.3 Hz, CH), 87.0 (CH), 116.3 (d, 3JC- P = 3.2 Hz, CH), 116.6 (d, 3JC-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, 1JC-F = 233.5 Hz, C), 148.0 (d, 2JC-P = 7.3 Hz, C), 148.1 (d, 2JC-P = 7.6 Hz, C), 150.5 (C) , 150.6 (C), 159.3 (d, 2JC-F = 26.2 Hz, C), 159.4 (d, 2JC-F = 26.6 Hz, C), 176.8 (C) , 176.9 (C); m/z (ES) 588.1 (MNa+, 100%); Reversed phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 minutes, 1 mL/min, | = 275 nm, exhibited a peak of the mixture of diastereoisomers with tR 16.05 min. (96%). 5-Fluoro-2'-deoxyuridine-5'-O -[henyl(benzoxy-L-prolinyl)] phosphate (CPF583)

[00123] Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and phenyl(benzoxy-L-prolinyl)-phosphochloridate (0.77 g, 2.03 mmols) in THF (10 mL). Column purification followed by two preparative TLC purifications provided the product as a white solid (0.010 g, 2%).
[00124] 31P-NMR (MeOD, 202 MHz) δ 1.82
[00125] 19F-NMR (MeOD, 470 MHz) δ - 167.91
[00126] 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, H-1'), 5.22 - 5.13 (m, CH2Ph ester), 4 .40 - 4.35 (m, 3H, NCH, 2 x H-5'), 4.33 - 4.28 (m, 1H, H-3'), 4.06 - 4.04 (m, 1H , H-4'), 3.36 - 3.32 (m, 2H, NCH2), 2.26 - 2.19 (m, 1H, H-2'), 2.18 - 2.13 (m, 1H, C H2-L-Pro), 2.00 - 1.81 (m, 4H, 3 x H, C H2-L-Pro, 1 x H, H-2')
[00127] 13C-NMR (MeOD, 125 MHz) δ 174.81 (C=O, ester), 159.40 (C=O, base), 152.0 (d, 2JC-P = 6.32 Hz, OC-Ar), 150.71 (C=O, base), 141.88 (1JC-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 (2xd, 2JC-F = 29.0 Hz, CH-base), 121.00, 120 .96 (CH-Ar), 87.80 (C-1'), 86.80 (C-4'), 72.02 (C-3'), 68.16 (CH2Ph), 67.64 (d , 2JC-P = 4.65 Hz, C-5'), 62.40 (d, 2JC-P = 5.60 Hz, NCH), 48.03 (d, 2JC-P = 4.80 Hz, NCH2 ), 41.07 (C-2'), 32.18, 32.11 (CH2-L-Pro), 26.29, 26.21 (CH2-L-Pro).
[00128] MS (ES+) m/e: 612 (MNa+, 100%), 590 (MH+, 1%) Exact mass: C27H29FN3O9P required 589.51 5-Fluoro-2'-deoxyuridine-5'-O- phosphate [1-naphthyl(benzoxy-L-prolinyl)] (CPF577)

[00129] Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and 1-naphthyl(benzoxy-L-prolinyl)-phosphochloridate (0.84 g, 2.03 mmols) in THF (10 mL). Column purification followed by two preparative TLC purifications provided the product as a white solid (0.006 g, 1%).
[00130] 31P-NMR (MeOD, 202 MHz) δ 2.27
[00131] 19F-NMR (MeOD, 121 MHz) δ - 167.46
[00132] 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 x H-Ar, 1 x 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, CH2Ph), 4.49 - 4.46 (m, 1H, NCH), 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, NCH2), 2.23 - 2.15 (m, 1H, CH2-L-Pro), 2.10 - 2.02 (m, 2H, 1xH, CH2-L-Pro, 1xH, H-2'), 1.97 - 1.77 (m, 2H, CH2-L-Pro), 1.63 - 1, 57 (m, 1H, H-2')
[00133] 13C-NMR (MeOD, 125 MHz) δ 174.82 (C=O, ester), 159.52 (C=O, base), 150.54 (C=O, base), 147.84, 147.78 (d, 2JC-P = 6.03 Hz, OC-Ar), 141.75, 139.97 (2xd, 1JC-F = 232 Hz, CF, 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, 2JC-F = 30.0 Hz, CH-base), 122.13 (CH-Ar), 115.76 (d, 3JC-P = 3.3 Hz, CH-Ar), 87.06 (C 1'), 86.79 (C-4'), 72.23 (C-3'), 68.15 (d, 2JC-P = 5.46 Hz, C-5'), 68.08 (CH2Ph), 62.53 (d, 2JC-P = 5.60 Hz, NCH), 48.26 (d, 2JC-P = 5.34 Hz, NCH2), 40.97 (C-2 '), 32.16, 32.09 (CH2-L-Pro), 26.22, 26.15 (CH2-L-Pro). 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(3,3-dimethyl-1-butoxy-L-alaninyl)] phosphate (CPF585)

Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and 1-naphthyl-(3,3-dimethyl-1-butoxy-L-alaninyl)-phosphochloridate (1.21 g, 3.04 mmols) in THF (10 mL). Column purification followed by two preparative TLC purifications provided the product as a white solid (0.010 g, 2%).
[00135] 31P-NMR (MeOD, 202 MHz) δ 4.48, 4.33
[00136] 19F-NMR (MeOD, 470 MHz) δ - 167.30, - 167.47
[00137] 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, 3H, H-base, 2 x H-Ar), 7.46 - 7.41 (2 xt, 1H, J = 7.8 Hz, H-Ar), 6.19 - 6.13 (m, 1H, H-1'), 4.42 - 4.40 (m, 1H, 1 x H-5'), 4.38 - 4.32 (m, 2H, H-3', 1 x H-5'), 4.14 - 4.00 (m, 4H, H-4', CHCH3, OCH2CH2(CH3)3) , 2.21 - 2.13 (m, 1H, 1 x H-2'), 1.91 - 1.76 (m, 1H, 1 x H-2'), 1.52 - 1.48 (m , 2H, OCH2CH2(CH3)3), 1.37 - 1.35 (m, 3H, CHCH3), 0.92, 0.91 (2xs, 9H, OCH2CH2(CH3)3)13C-NMR (MeOD, 125 MHz) δ 175.16, 174.84 (2 xd, 3JC-P = 4.75 Hz, C=O, ester), 159.56, 159.35 (C=O, ester), 150.61 ( C=O, ester), 148.00, 147.86 (2xd, 2JC-P = 6.25Hz, OC-Ar), 141.78, 141.73 (2xd, 1JC-F = 232Hz, 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 (2xd, 2JC-F = 34Hz, CH, base), 122.65, 122.63 (CH Ar), 116.48, 116.15 (2xd, 3JC-P = 3.0 Hz, CH-Ar), 87.01, 86.94 (C-1'), 86.73, 86.68 (d, 3JC-P = 7.75 Hz, C-4'), 72.18, 72.07 (C-3'), 67.87, 67.85 (2xd, 2JC-P = 5.0 Hz, C-5'), 64.08, 64.05 (OCH2CH2( CH3)3), 51.86 (d, 3JC-P = 5.5 Hz, CHCH3), 42.74 (OCH2CH2(CH3)3), 40.91, 40.83 (C-2'), 29, 96 (OCH2CH2(CH3)3), 20.50, 20.34 (2xd, 3JC-P = 6.5Hz, CHCH3).
[00138] MS (ES+) m/e: 630 (MNa+, 100%), 608 (MH+, 10%) Exact mass: C28H35FN3O9P required 607.56 5-Fluoro-2'-deoxyuridine-5'-O phosphate - [1-naphthyl-(cyclobutoxy-L-alaninyl)] (CPF578)

[00139] Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.23 g, 0.93 mmol), NMI (0.38 g, 4.67 mmols, 0.37 mL) and 1-naphthyl-(cyclobutoxy-L-alaninyl)-phosphochloridate (0.85 g, 2.33 mmols) in THF (10 mL). Column purification followed by preparative TLC purification provided the product as a white solid (0.010 g, 2%).
[00140] 31P-NMR (MeOD, 202 MHz) δ 4.54, 4.36
[00141] 19F-NMR (MeOD, 470 MHz) δ - 167.12, - 167.29
[00142] 1H-NMR (MeOD, 500 MHz) δ 8.18 - 8.17 (m, 1H, H-Ar), 7.81 - 7.87 (m, 1H, H-Ar),
7.74 - 7.71 (m, 2H, 1 x H-Ar, 1 x H-base), 7.60 - 7.53 (m, 3H, H-Ar), 7.46 - 7 .43 (2xt, J = 8.0Hz, 1H, H-Ar), 6.18 - 6.12 (m, 1H, H-1'), 5.00 - 4.95 (m, 1H, OCHester), 4.41 - 4.36 (m, 3H, 2 x H-5', H-3'), 4.11 - 4.00 (m, 2H, H-4', CHCH3), 2, 36 - 2.27 (m, 2H, CH2), 2.18 - 1.98 (m, 3H, CH2 ester, 1 x H-2'), 1.82 - 1.56 (m, 3H, CH2 ester , 1 x H-2'), 1.36 - 1.34 (m, 3H, CHCH3)
[00144] 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, 1JC-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 (2xd, 2JC-F = 32.5 Hz, CH-base), 122.63 (CH-Ar), 116 .46, 116.44 (2xd, 3JC-P = 2.5 Hz, CH Ar), 86.98 (d, 3JC-P = 6.25 Hz, C-4'), 86.71 (C- 1'), 72.14, 72.04 (C-3'), 71.07 (OCH ester), 67.83 (d, 2JC-P = 7.38 Hz, C-5'), 51.66 (d, 2JC-P = 8.75 Hz, CHCH3), 40.89, 40.83 (C-2'), 31.03 (OCHCH2), 20.43 (CHCH3), 14.23 (CH2 ester) .
[00145] MS (ES+) m/e: 600 (MNa+, 100%), 578 (MH+, 10%) Exact mass: C26H29FN3O9P required 577.50 5-Fluoro-2'-deoxyuridine-5'-O- phosphate [1-naphthyl-(cyclopropylmethaneoxy-L-alaninyl)] (CPF579)

Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and 1-naphthyl-(cyclopropylmethaneoxy-L-alaninyl)-phosphochloridate (0.93 g, 2.54 mmols) in THF (10 mL). Column purification provided the product as a white solid (0.056 g, 10%).
[00147] 31P-NMR (MeOD, 202 MHz) δ 4.58, 4.30
[00148] 19F-NMR (MeOD, 470 MHz) δ -167.18, -167.22
[00149] 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 - 7.70 (m, 2H, H-Ar), 7.58 - 7.53 (m, 3H, H-Ar), 7.45 - 7.40 (2 xt, J = 8.0 Hz, 1H, H-Ar), 6.17 - 6.11 (m, 1H, H-1'), 4.43 - 4.41 (m, 1H, H-5'), 4.38 - 4 .32 (m, 2H, H-5', H-3'), 4.11 - 4.04 (m, 2H, H-4', CHCH3), 3.95 - 3.85 (m, 2H, OCH2 ester), 2.19 - 2.11 (m, 1H, H-2'), 1.84 - 1.72 (m, 1H, H-2'), 1.38, 1.36 (2 xd , J = 5.0 Hz, 3H, CHCH 3 ), 1.15 - 1.07 (m, 1H, OCH 2 CHester), 0.59 - 0.50 (m, 2H, CH 2 ester), 0.30 - 0. 24 (m, 2H, CH2 ester)
[00150] 13C-NMR (MeOD, 125 MHz) δ 175.25, 174.94 (2xd, 3JC-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 (2xd, 2JC-P = 7.5 Hz, OC-Ar), 141.79, 141 .73 (2xd, 1JC-F = 232 Hz, CF, base), 136.29 (C-Ar), 128.94 (d, 3JC-P = 4.4 Hz, CH-Ar), 127.89 (d, 4JC-P = 3.7 Hz, CH-Ar), 127.56, 126.55, 126.52, 126.19, 126.16 (CH-Ar), 125.64, 125.53 ( 2JC-F = 34 Hz, CH-base), 122.65 (CH-Ar), 116.54, 116.24 (2xd, 4JC-P = 2.6 Hz, CH-Ar), 87.04, 86.99 (C-1'), 86.90, 86.73 (2xd, 3JC-P = 7.1 Hz, C-4'), 72.18, 72.07 (C-3'), 71.21, 71.18 (OCH2, ester), 67.87, 67.84 (apparent t, 2JC-P = 5.0 Hz, C-5'), 51.88 (d, 2JC-P = 10 0.0Hz, CHCH3), 40.91, 40.83 (C-2'), 20.60, 20.46 (2xd, 3JC-P = 6.5Hz, CHCH3), 10.69 (OCH2CH ester ), 3.70, 3.65 (2 x CH 2 , ester).
[00151] MS (ES+) m/e: 600 (MNa+, 100%), 578 (MH+, 15%) Exact mass: C26H29FN3O9P required 577.50.
[00152] HPLCb (H2O/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)

[00153] Prepared according to standard procedure E of 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 mmols) in THF (10 mL). Column purification followed by two preparative TLC purifications provided the product as a white solid (0.010 g, 1.6%).
[00154] 31P-NMR (MeOD, 202 MHz) δ 3.77, 3.22
[00155] 19F-NMR (MeOD, 470 MHz) δ - 168.27, -168.35
[00156] 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, CHCH3), 3.88 - 3.66 (m, 4H, 2 x OCH2a'ester, 2 x H-5'), 3.55 - 3.50 (m, 2H, 2 x OCH2a''ester), 2.63 - 2.30 (m, 2H, H-2'), 1.91 - 1 .85 (m, 2H, 2 x CH2b'ester), 1.65 - 1.54 (m, 2H, CH2b''ester), 1.39 - 1.35 (m, 3H, CHCH3).
[00157] 13C-NMR (MeOD, 125 MHz) δ 174.34 (C=O, ester), 159.24 (C=O, base), 150.76 (C=O, base), 148.03 ( OC-Ar), 141.97 (d, 1JC-F = 238 Hz, CF, base), 136.37 (C-Ar),
[00158] 128.97, 128.56, 127.61, 127.57, 126.58, 126.23, 126.16, 126.12, 125.84 (CH-Ar), 122.70 (d, 2JC-F = 24.0 Hz, CH-base), 116.62, 116.37 (CH-Ar), 87.54 (d, 3JC-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 x OCH2a ester), 66 .02 (C-5'), 51.83 (CHCH3), 39.97, 39.94 (C-2'), 32.65, 32.57 (2 x CH2bester), 20.45, 20.30 (CHCH3).
[00159] MS (ES+) m/e: 630 (MNa+, 100%), 608 (MH+, 10%) Exact mass: C27H31FN3O10P required 607.52. 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(pentoxy-L-alaninyl)] phosphate (CPF581)

[00160] Prepared according to standard procedure E of 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 mmols) in THF (10 mL). Column purification provided the product as a white solid (0.047 g, 8%).
[00161] 31P-NMR (MeOD, 202 MHz) δ 4.48, 4.32
[00162] 19F-NMR (MeOD, 470 MHz) δ -167.18, -167.29
[00163] 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 x H-5', H-3'), 4.28 - 4.00 (m, 4H, H-4', CHCH3, OCH2CH2CH2CH2CH3), 2.32 - 2.12 (m, 1H, H-2'), 1.95 - 1.75 (m, 1H, H-2'), 1.70 - 1.55 (m, 2H, OCH2CH2CH2CH2CH3) , 1.50 - 1.28 (m, 7H, 4xH OCH2CH2CH2CH2CH3, CHCH3), 0.83, 0.82 (2xd, J=7.9Hz, 3H, OCH2CH2CH2CH2CH3)
[00164] 13C-NMR (MeOD, 125 MHz) δ 175.22, 174.91 (C=O, ester), 159.5 (C=O, base), 150.54 (C=O, base), 147.90, 147.88 (OC-Ar), 141.75 (d, 1JC-F = 225 Hz, CF, base), 136.37 (C-Ar), 128.95, 127.90, 127, 56, 126.55, 126.19 (CH-Ar), 125.64, 125.53 (2xd, 2JC-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, 2JC-P = 5.0 Hz, C 5'), 66.54 (OCH2), 51.87, 51.81 (d, 2JC-P = 7.5 Hz, CHCH3 ), 40.87, 40.80 (C-2'), 29.35, 29.10 (CH2 ester), 23.33 (CH2 ester), 20.60, 20.43 (2xd, 3JC-P =6.5 Hz, CHCH3), 14.28 (CH3, ester).
MS (ES+) m/e: 616 (MNa+, 100%), 594 (MH+, 10%) Exact mass: C27H33FN3O9P required 593.54.
[00166] HPLCb (H2O/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)

Prepared according to standard procedure E of 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 mmols) in THF (10 mL). Column purification provided the product as a white solid (0.030 g, 5%).
[00168] 31P-NMR (MeOD, 202 MHz) δ 4.53, 4.37
[00169] 19F-NMR (MeOD, 470 MHz) δ - 167.07, -167.19
[00170] 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 x H-Ar, H-base), 7.45 - 7.40 (m , 1H, H-Ar), 6.16 - 6.11 (m, 1H, H-1'), 5.15 - 5.09 (m, 1H, OCH ester), 4.41 - 4.30 ( m, 3H, 2 x H-5', H-3'), 4.11 - 4.08 (m, 1H, H-4'), 4.04 - 3.98 (m, 1H, CHCH3), 2.19 - 2.10 (m, 1H, H-2'), 1.86 - 1.73 (m, 3H, OCHCH2 ester), 1.73 - 1.56 (m, 6H, H-2' , CH2 ester), 1.35, 1.34 (2xd, J = 6.57 Hz, CHCH3)
[00171] 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, 2JC-P = 7.5 Hz, OC-Ar), 141.78, 141.72 (2xd, 1JC-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 (2xd, 2JC-F = 34Hz, CH-base), 122.65, 122.61 (CH-Ar), 116.53, 116.22 (2xd, 4JC- P = 3.75 Hz, CH-Ar), 86.99, 86.96 (C-1'), 86.70 (d, 3JC-P = 7.50 Hz, C-4'), 79.64 , 79.61 (OCH ester), 72.21, 72.07 (C-3'), 67.89, 67.85 (2xd, 2JC-P = 5.0 Hz, C-5'), 51 .92 (d, 2JC-P = 5.0 Hz, CHCH3), 40.92, 40.86 (C-2'), 33.65, 33.61, 33.52, 33.47 (2 x CH2 ) ester), 24.68, 24.66 (CH2 ester), 20.45, 20.30 (2xd, 3JC-P=6.25 Hz, CHCH3).
[00172] MS (ES+) m/e: 614 (MNa+, 100%), 592 (MH+, 30%) Exact mass: C27H31FN3O9P required 591.52
HPLCb (H2O/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)

Prepared according to standard procedure E of 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 mmols) in THF (20 mL). Column purification provided the product as a white solid (0.045 g, 6%).
[00175] 31P-NMR (MeOD, 202 MHz) δ 4.62, 4.30
[00176] 19F-NMR (MeOD, 470 MHz) δ - 167.14, - 167.34
[00177] 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 x H-Ar, H-base), 7.40 - 7.37 (m, 1H, H-Ar), 7.20 - 7.12 (m, 4H, H-Ar, Ph), 6.14 - 6.08 (m, 1H, H-1'), 5.49 - 5.46 (m, 1H, OCHester), 4.32 - 4.26 (m, 3H, 2 x H-5', H-3'), 4.04 - 3.98 (m, 1H, H -4', CHCH), 3.30 - 3.24 (m, 2H, 2 x CHester), 2.99 - 2.91 (m, 2H, 2 x CH ester), 2.14 - 2.07 ( m, 1H, H-2'), 1.75 - 1.64 (m, 1H, H-2'), 1.33 - 1.29 (m, 3H, CHCH3)
[00178] 13C-NMR (MeOD, 125 MHz) δ 175.02, 174.66 (2xd, 3JC-P = 3.75 Hz, C=O, ester), 159.48 (2JC-F = 25, 0 Hz, C=O, base), 150.57 (C=O, base), 147.97, 147.80 (2xd, 2JC-P = 7.5 Hz, OC-Ar), 141.73, 141.68 (2xd, 1JC-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 (2xd, 2JC-F = 25 Hz, CH-base), 122.64, 122.60, 121.85 (CH-Ar) , 116.57, 116.26 (2xd, 4JC-P = 2.5Hz, CH-Ar), 86.96 (C-1'), 86.87, 86.66 (2xd, 3JC-P = 7.50 Hz, C-4'), 77.85, 79. (OCH ester), 72.21, 72.07 (C-3'), 67.77, 67.75 (2xd, 2JC- P = 6.25 Hz, C-5'), 51.97, 51.82 (CHCH3), 40.91, 40.86 (C-2'), 40.44, 40.43, 40.38, 40.34 (2 x CH2 ester), 20.30, 20.16 (2 xd, 3JC-P = 6.25 Hz, CHCH3) 5-Fluoro-2'-deoxyuridine-5'-O-[henyl-phosphate (Benzoxy-L-methioninyl)] (CPF586)

[00179] Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and phenyl-(benzoxy-L-methioninyl)-phosphochloridate (0.7 g, mmol) in THF (10 mL). Column purification provided the product as a yellowish solid (0.014 g, 2%).
[00180] 31P-NMR (MeOD, 202 MHz) δ 4.34, 3.94
[00181] 19F-NMR (MeOD, 470 MHz) δ - 167.40, - 167.69
[00182] 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, JAB = 12.0 Hz, 2H, CH2Ph), 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 (2xt, J = 7.50Hz, CH2S), 2.16 - 2.08 (m, 1H, 1xH-2'), 1.98 - 1.82 (m, 6H, 1 x H-2', NHCHCH2CH2SCH3),
[00184] MS (ES+) m/e: 646 (MNa+, 100%), 624 (MH+, 10%) Exact mass: C27H31FN3O9PS required 623.56 5-Fluoro-2'-deoxyuridine-5'-O- phosphate [1-naphthyl-(benzoxy-L-phenylalaninyl)] (CPF587)

Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and 1-naphthyl-(benzoxy-L-phenylalaninyl)-phosphochloridate (1.45 g, mmol) in THF (10 mL). Column purification provided the product as a white solid (0.007 g, 1%).
[00186] 31P-NMR (MeOD, 202 MHz) δ 4.27, 4.14
[00187] 19F-NMR (MeOD, 470 MHz) δ - 166.99, - 167.18
[00188] 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 x 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, 1H, H-1'), 5.11 - 5.01 (m , 2H, CH2Ph), 4.29 - 4.18 (m, 1H, CHCH3), 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 x NHCHCH2Ph), 2.91 - 2.82 ( m, 1H, 1 x NHCHC H2Ph), 2.12 - 2.06, 2.00 - 1.95 (2 xm, 1H, H-2'), 1.68 - 1.62, 1.42 - 1 .36 (2 xm, 1H, H-2') 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)] phosphate (CPF588)

[00189] Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and 1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)-phosphochloridate (0.77 g, mmol) in THF (10 mL). Column purification provided the product as a white solid (0.006 g, 1%).
[00190] 31P-NMR (MeOD, 202 MHz) δ 4.56, 4.33
[00191] 19F-NMR (MeOD, 470 MHz) δ - 167.32, - 167.43
[00192] 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 x 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 x H-5'), 4 .13 - 4.07 (m, 2H, H-4', CHCH3), 3.86, 3.75 (AB system, JAB = 10.50 Hz, 2H, CH2C(CH3)3), 2.18 - 2.10 (m, 1H, H-2'), 1.81 - 1.70 (m, 1H, H-2'), 1.41 - 1.38 (m, 3H, CHCH3), 0.95 , 0.94 (2xs, 9H, CH2C(CH3)3) 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(butoxy-L-alaninyl)] phosphate (CPF589)

[00193] Prepared according to standard procedure D of 5-Fluoro-2'-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmols, 0.40 mL) and 1-naphthyl-(butoxy-L-alaninyl)-phosphochloridate (0.75 g, mmol) in THF (10 mL). Column purification provided the product as a white solid (0.006g, 1%).
[00194] 31P-NMR (MeOD, 202 MHz) δ 4.52, 4.35
[00195] 19F-NMR (MeOD, 470 MHz) δ - 167.36, - 167.49
[00196] 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 x 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 x H-5'), 4, 37 - 4.32 (m, 2H, 1 x H-5', H-3'), 4.12 - 4.01 (m, 4H, H-4', CHCH3, OCH2 CH2CH2CH3), 2.20 - 2.12 (m, 1H, H-2'), 1.85 - 1.73 (m, 1H, H-2'), 1.61 - 1.54 (m, 2H, OCH2CH2CH2CH3), 1.39 - 1.31 (m, 5H, OCH2CH2CH2CH3, CHCH3), 0.93 - 0.89 (m, 3H, OCH2CH2CH2CH3) Assays Biological
[00197] Experimental data considering the compounds incorporating the present invention are described below. Cell cultures
[00198] L1210/0 murine leukemia and CEM/0 human T-lymphocyte 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). The thymidine kinase deficient CEM/TK- cells were a kind of donation from Prof. S. Eriksson (currently at Uppsala University, Uppsala, Sweden) and Prof. A. Karlsson (Karolinska Institute, Stockholm, Sweden). Thymidine kinase deficient L1210/TK- were derived from L1210/0 cells after selection for resistance against 5-bromo-2'-dUrd (Balzarini et al(s), 1982). Infection of relevant cell lines with Mycoplasma hyorhinis (ATCC) resulted in chronically infected cell lines as referred to as L1210.Hyor and U87.Hyor. All cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), 10 mM Hepes and 1 mM Pyruvate of sodium (Invitrogen). Cells were grown at 37°C in an incubator moistened with a gas phase of 5% CO2. Cytostatic Tests
Monolayer cells (U87 and U87.Hyor) were seeded in 48-well microtiter plates (NuncTM, Roskilde, Denmark) at 10,000 cells/well. After 24 hours, an equal volume of fresh medium containing test compounds was added. On day 5, cells were trypsinized and counted in a Coulter counter (Analis, Suarlée, Belgium). Suspension cells (L1210/0, L1210/TK-, L1210.Hyor, CEM/0, CEM/TK-) were seeded in 96-well microtiter plates (NuncTM) at 60,000 cells/well in the presence of a supplied amount of the test compounds. Cells were allowed to pre-polyferate for 48 hours (L1210) or 72 hours (CEM) and were then counted in a Coulter counter. The 50% inhibitory concentration (IC50) was defined as the concentration of compound required to reduce the number of viable cells by 50%.
Assay 1. Samples were assayed for biological activity versus a series of tumor cell lines with the data reported in Table 1 below. Data are expressed as CC50 in µM, i.e. cytostatic concentration required to inhibit cell proliferation by 50%. The cell lines used 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) .
[00201] Table 1 also contains comparative data for 5FU, 5-FdUrd and the 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:

As can be seen from the data in Table 1, the compounds of the present invention can exhibit cytostatic activity that is comparable to or better than that of 5-FU, while exhibiting marked cytostatic activity in nucleoside kinase deficient cells. Such cytostatic activity in nucleotide kinase deficient cells is in direct contrast to that of 5-FdUrd.
[00203] As can be seen from Table 1, the activity in TK- cells of compounds incorporating the present invention may be markedly higher than that of the reference compounds CPF 382, CPF 437 and CPF 438.


67/102
[00204] Assay 2. Samples were also assayed for their % activity retention in mycoplasma infected cells. The results are shown in Table 2 below. The results show that the compounds of the present invention can maintain high activity in mycoplasma infected cells, in contrast to the activity exhibited by 5-FdURD. Administration of a thymidine phosphorylase (TP) inhibitor restores the cytostatic activity of 5-FdUrd in mycoplasma-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 who are infected with mycoplasma. Table 2.
The CC50 values in µM for 5-FdUrd and compounds incorporating the present invention on mycoplasma negative and positive cells, and % retention of activity on mycoplasma infection. "% Retention" is a measure of the ratio of the evaluated CC50 values with respect to L1210 with respect to those for L1210/Hyor and is calculated as CC5OLI2IO x 100 - CC50L1210/Hyor.


[00206] Other experiments (Assays 3 to 8 below) were carried out with respect to the compound CPF 373 incorporating the present invention. Assay 3. Cytostatic activity of 5-FdUrd and its prodrug CPF-373 against TK-deficient and TK-apt tumor cell lines
The cytostatic activity of 5-FdUrd and CPF-373 was determined in different TK-deficient and TK expressing tumor cell lines. As shown in Table 3, 5-FdUrd is strongly dependent on TK expression for its cytostatic activity. Its IC50 increased by 4,000 times for L1210/TK- cells (IC50: 3.1 μM) versus wild type L1210/0 cells (IC50: 0.0008 μM) and by 50 times for CEM/TK- cells (IC50: 1.5 µM) versus CEM/0 cells (IC50: 0.028 µM). In contrast, the cytostatic activity of the prodrug 5-FdUrd CPF-373 remained virtually unchanged in TK-deficient cells when compared to wild-type cells (IC50: 0.027 and 0.011 μM for L1210/TK- and L1210/0, and 0.32 and 0.089 µM for CEM/TK- and CEM/0 cells, respectively). Although the cytostatic activity of CPF-373 was 3- to 10-fold lower than 5-FdUrd against wild-type L1210/0 and CEM/0 cells, it was found to be 5- to 100-fold greater than 5-FdUrd in L1210/0 and CEM/0 cell lines. TK-deficient tumor (see Table 3). Table 3. Cytostatic activity of 5-FdUrd and CPF-373 as represented by IC50 value in different cell lines
at 50% Inhibitory concentration or concentration of compound required to inhibit tumor cell proliferation by 50% Assay 4. Effect of mycoplasma infection of tumor cell cultures on the cytostatic activity of 5-FdUrd and its prodrug CPF-373
L1210/0 cell cultures were infected with the mycoplasma species M. hyorhinis (cells designated: L1210.Hyor). 5-FdUrd markedly loses its cytostatic activity against 300-fold mycoplasma infected L1210.Hyor cells (IC50: 0.24 μM). Also, 5-FdUrd loses its cytostatic activity 400-fold in U87.Hyor cell cultures when compared to uninfected U87 cells (see Table 3). In contrast, the prodrug 5-FdUrd CPF-373 maintained a similar cytostatic potential in both L1210/0 and L1210.Hyor cell cultures (IC50: 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 (IC50: 0.035 and 0.039 μM, respectively). Thus, while free nucleoside 5-FdUrd markedly loses its cytostatic potential against Mycoplasma hyorhinis-infected tumor cell lines, the antiproliferative potential of its prodrug CPF-373 was independent of mycoplasma infection.
[00209] Assay 5. Experiments were performed to assess the stability of CPF 373 in the presence of Thymidine Phosphorylase (TP). The experiments are illustrated with reference to Figures 9 to 11, each of which comprises NMR spectra, as discussed below. The present test shows that the insensitivity of compounds incorporating the present invention to the action of the catabolic enzyme TP, which is often upregulated in tumors, makes the compounds of the present invention more independent of the catabolic enzyme TP than 5-FdUrd. Phosphorylase Assay on 5-FdUrd and its compound ProTide CPF 373 by Thymidine Phosphorylase (TP) purified from Escherichia Coli.
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 ascites tumor. This disorder has been suggested to be one of the reasons for the limited therapeutic efficacy of 5-FdUrd according to the following scheme:
Scheme
[00211] The phosphorylase assay was performed facing phosphorolysis by Thymidine Phosphorylase purified from Escherichia coli using in situ 19F NMR. Application to the ProTide CPF 373 compound was an attempt to prevent structure cleavage and thereby involve enzyme action.
[00212] Two potassium phosphate buffers at pH = 7.4, 200 nM of solution and 300 nM of solution respectively, were used as phosphate donors. Enzyme units were defined as the amount of enzyme required to hydrolyze about 0.25 mg of inosine per minute used as a standard. Tests were conducted for 30 minutes. Phosphorylase Assay on 5-FdUrd
[00213] Initially, 19F NMR spectra (470 MHz) of 5-FdUrd and 5FU previously dissolved in deuterated methanol were recorded. 5-FdUrd exhibited a singlet at ~δ-167.21 ppm and 5FU at ~δ-169.30 ppm. Thus, the phosphorylase assay was performed by dissolving 5-FdUrd in deuterated methanol, in the presence of potassium phosphate buffer (200 nM of solution; pH = 7.4), recording the space before the addition of the enzyme thymidine phosphorylase ( TP) (20.7 UNI). The 19F NMR spectra, recorded at 25°C, exhibited the 5-FdUrd singlet at ~ δ-165.17 ppm and a new peak at ~ δ-169.50 ppm, assigned to 5FU, as shown in Figure 9 in the spectrum THE.
[00214] Then, to prove the nucleoside cleavage in the relative base, a new experiment was performed by dissolving equal moles of the nucleoside analogue 5-FdUrd and the relative base 5FU, in the same condition described above without the TS enzyme, as shown in Figure 9 in spectrum B. This spectrum exhibited two singlets with the same chemical changes previously observed in Figure 9 spectrum A. These data confirmed that 5FU presents a chemical change at ~ δ-169.50 ppm and thus the phosphorolytic action of enzyme (TP). The conversion of the nucleoside 5-FdURd to the free base 5FU was 66%.
When the initial concentration of potassium phosphate buffer was increased from 200 nM to 205 nM, 5-FdUrd substrate was completely converted to 5-FU base as shown in Figure 10. Phosphorylase Assay on ProTide Compound CPF 373
[00216] The phosphorylase assay was applied to the benzyl L-alanine phenyl derivative CPF 373 in order to investigate the stability, after the procedure and conditions described above. The compound ProTide CPF 373 proved to be completely stable as shown comparing the chemical changes of the analyzed sample without the TP enzyme, as shown in Figure 11 spectrum A, and in the presence of TP, as shown in Figure 11 spectrum B. 19F NMR was repeated 4 days and the ProTide compound CPF 373 was again shown to be stable.
[00217] These experiments confirmed that the nucleoside 5-FdUrd is rapidly degraded to its relative base 5FU by a phosphorolytic reaction, in the presence of thymidine phosphorylase, with a half-life of less than 30 minutes, whereas the prodrug compound CPF 373 exhibited an evident stability against TP enzymatic activity, in longer exposure time up to 3 days. This important result showed that 5-FdUrd ProTides derivatives incorporating the present invention should favor the therapeutic effect of 5-FdUrd. Assay 6. Exposure of 5-FdUrd and CPF-373 to E. coli encoded TP eTP and human encoded UP
[00218] The substrate specificity of thymidine phosphorylase directed towards natural thymidine(dThd), uridine (Urd), 5-FdUrd and CPF-373 was investigated by high pressure liquid chromatography (HPLC). Reaction mixtures containing 100 μM of test compound and TP or recombinant UP (human TP: 8.6 ng/μL; E. coli TP: 3.0 ng/μL; human PU: 4 ng/mL) in a total volume of 500 µL of reaction buffer (10 mM TrisHCl; 300 µM NaCl; 1 mM EDTA; 2 mM KH2PO4/K2HPO4) were incubated at room temperature. At different time points (ie, 0, 20, 40 min) 100 µL aliquots of the reaction mixtures were removed and heated at 95°C for 3 minutes to inactivate the enzyme. The resulting reaction products were separated on an RP-8 reversed-phase column (Merck, Darmstadt, Germany) and quantified by HPLC analysis (Alliance 2690, Waters, Milford, MA). The separation of dThd from thymine was performed by a linear gradient of 98% separation buffer (50 mM NaH2PO4 and 5 mM heptane sulfonic acid, pH 3.2) and 2% acteonitrile, to 20% separation buffer + 80% acetonitrile (8 minutes 98% separation buffer + 2% acetonitrile; 5 minutes linear gradient from 98% separation buffer + 2% acetonitrile to 20% separation buffer + 80% acetonitrile; 10 min 20% separation buffer + 80% acetonitrile, followed by equilibration to 98% separation buffer + 2% acetonitrile). UV-based detection was performed at 267 nm. Urd separation of 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 to 100% separation buffer). UV-based detection was performed at 258 nm.
[00219] Phospholysis of 5-FdUrd and CPF-373 by thymidine and uridine phosphorylases
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. While E.coli and human TP quickly converted dThd and 5-FdUrd to their free bases, CPF-373 remained completely stable in the presence of these enzymes (Figure 2). Under similar experimental conditions, uridine was converted to uracil by human UP, but not by E.coli TP, or human TP. When both compounds were exposed to UP, dThd and CPF-373 were not affected by the enzyme, whereas 5-FdUrd was slightly hydrolyzed (Figure 2, panel C). Assay 7. Measurements of thymidylate synthase (TS) activity
[00221] TS activity in intact L1210/0 and L1210/TK- cells was measured by evaluating the release of tritium from [5-3H]dUMP (formed in [5-3H]dUrd or [5-3H] cells dCyd) in the TS-catalyzed reaction. This method has been described in detail by Balzarini & De Clercq (1984). Briefly, cell cultures (500 µL of DMEM culture medium) were prepared containing ~ 3 x 106 L1210 cells and the appropriate amounts of test compounds (5-FdUrd and CPF-373). After 30 min, two hours and 4 hours of pre-incubation of cells with compounds at 37°C, 1 μCi of [5-3H]dUrd or [5-3H]dCyd was added to cell cultures. After 30 minutes of incubation, 100 µL of cell suspensions were removed and added to a cold suspension of 500 µL of activated charcoal (VWR, Haasrode, Belgium) (100 mg/ml in 5% TCA). After 10 minutes, the suspension was centrifuged at 13,000 rpm for 10 min, after which the radioactivity in 400 µL of supernatant was calculated in a liquid scintillator using OptiPhase HiSafe (Perkin Elmer, Waldham, MA). Inhibition of thymidylate synthase (TS) by 5-FdUrd and CPF-373
[00222] The main target for the cytostatic activity of 5-FdUrd is thymidylate synthase (TS). TS activity in intact tumor cells can be directly monitored by measuring tritium release in intact L1210/0 cell cultures that have been exposed to [5-3H]deoxyuridine ([5-3H]dUrd) or [5-3H ]deoxycytidine ([5-3H]dCyd). In fact, after the intracellular conversion of [5-3H]dUrd or [5-3H]dCyd to [5-3H]dUMP, the C-5 tritium atom in the pyrimidine base is released during TS-catalyzed reductive methylation. The ability of 5-FdUrd and its prodrug CPF-373 to inhibit tritium release from [5-3H]dUrd and [5-3H]dCyd was therefore evaluated in L1210/0 cell cultures at a variety of compound concentrations. 5-FdUrd has proven to be a potent inhibitor of TS in situ. Its IC50 for the release of tritium from [5-3H]dCyd and [5-3H]dUrd was about 0.0007 - 0.0009 μM (see Table 4). Table 4. IC50 values of 5-FdUrd and CPF-373 against TS in L1210/0 intact tumor cells (as determined by tritium release of [5-3H]dUrd and [5-3H]dCyd after 30 minutes of exposure to drugs).
at 50% inhibitory concentration or compound concentration required to inhibit the release of tritium from [5-3H]dUrd or [5-3H]dCyd in L1210/0 cell cultures exposed to the drug by 50%.
[00223] 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 of preincubation of cells with the drugs (IC50: 0.16- 0.19 µM). However, longer cell preincubation times (up to 4 hr) with 5-FdUrd and CPF-373 prior to measuring TS activity in intact tumor cells revealed a much more pronounced inhibitory activity of the prodrug against TS in situ (Figure 3 ). In fact, while the inhibition of 3H release was only increased twice at the longer preincubation times of 5-FdUrd, the inhibitory potential of CPF-373 increased 10-fold (Figure 3, panels A and B, and C and D) .
[00224] Preincubation of tumor cells with 5-FdUrd and CPF-373 for at least 4 hours results in inhibition of TS in intact tumor cells at drug concentrations that are very comparable to the 50% cytostatic activity concentrations of these pharmaceuticals.
The present observations thus indicate that the 5-FdUrd prodrug requires several metabolic conversion steps before reaching TS as the target enzyme for inhibition, and supports the view that CPF-373 acts as an efficient 5-FdUrd prodrug to exert its eventual cytostatic activity.
[00226] TS activity in the presence of 5-FdUrd and CPF-373 was also measured in intact L1210/TK- cells using [5-3H]dCyd as an externally supplied substrate (due to TK deficiency, [5] -3H]dUrd cannot be used). As demonstrated in Table 5 and Figure 3 (panels E and F), the concentration of 5-FdUrd required to cause 50% inhibition of TS decreased by a factor 5,700 in TK-deficient L1210/TK- cells (IC50: 1.4 µM) when compared to wild type L1210/0 cells (IC50: 0.0003 µM). In contrast, the inhibitory activity of CPF-373 against TS remained virtually unchanged in L1210/TK- cells (IC50: 0.053 μM in L1210/TK- cells versus 0.013 μM in L1210/0 cells). Table 5. IC50 values of 5-FdUrd and CPF-373 against TS in L1210/0 and L1210/TK- intact cells (as determined by tritium release from [5-3H]dCyd after 4 hours of preincubation with products)
at 50% inhibitory concentration or compound concentration required to inhibit tritium release from [5-3H]dCyd in drug-exposed L1210 cells by 50% on pre-exposure of tumor cells for 4 hours to drugs. Test 8. Stability tests Carboxypeptidase Y assay (EC 3,4.16.1)
[00227] The enzymatic stability of the prodrug CPF-373 facing carboxypeptidase Y was studied using in situ31P NMR (202 MHz). The experiment was performed by dissolving CPF-373 (3.0 mg) in d6-acetone (150 mL) and adding TRIZMA buffer pH 7.6 (300 mL). The resulting solution was placed in an NMR tube and a 31P-NMR experiment at 25°C was recorded as the blank experiment. Carboxypeptidase Y enzyme (0.2 mg) was solubilized in TRIZMA (150 ml) and added to the phosphoramidate derivative solution in the NMR tube. Next, the tube was placed in the NMR machine, which was set to perform a 31P-NMR experiment (64 scans) every 4 minutes for 14 hours at 25°C. Data were processed and analyzed using the Bruker Topspin 2.1 program. Carboxypeptidase Y and TRIZMA buffer were purchased from Sigma-Aldrich. human serum
[00228] The stability of the prodrug CPF-373 in the presence of human serum was studied using in situ31P NMR (202 MHz). ProTide CPF-373 (1) (5.0 mg) was dissolved in DMSO (0.05 mL) and D2O (0.15 mL). The sample was then transferred into an NMR tube, which was inserted into the NMR chamber at 37°C (with enough solvent to obtain a blank control NMR reading). Then, 0.3 ml of human serum was quickly added to the sample in the NMR tube. The NMR experiments were programmed to record data every 15 minutes for 12 hours and 30 minutes. Because of excess noise and poor scattered light profiles (most likely due to biological media and concentration), individual spectra were also processed. After normal Fourier transform processing, each spectrum was deconvoluted (Lorentz-Gauss deconvolution) to reveal only the frequency and area of the spectral peaks without the parameter. The recorded data were processed and analyzed using the Bruker Topspin 2.1 program. Buffer at pH 1
[00229] The stability of the prodrug CPF-373 facing hydrolysis at pH = 1 was studied using in situ31P NMR (202 MHz). ProTide CPF-373 (1) (2.6 mg) was dissolved in MeOD (0.1 mL) after which 0.5 mL of buffer (pH = 1) (prepared from equal parts of 0.2 M HCl and 0.2 M KCl) was added. Then, the sample was transferred into an NMR tube, and a 31P NMR experiment was performed at 37°C recording data every 12 minutes for 14 hours. Data were processed and analyzed using the Bruker Topspin 2.1 program. Buffer at pH 8
[00230] The stability of the prodrug CPF-373 facing hydrolysis at pH = 8 was studied using in situ31P NMR (202 MHz). ProTide CPF-373 (1) (4.9 mg) was dissolved in MeOD (0.1 ml) after which 0.5 ml of buffer (pH = 8) (prepared from a 0.1 M solution of Na2HPO4 , which was adjusted by 0.1 M HCl) was added. Then, the sample was transferred into an NMR tube, and a 31P NMR experiment was performed at 37°C recording data every 12 minutes for 14 hours. Data were processed and analyzed using the Bruker Topspin 2.1 program. Stability Studies
[00231] Chemical stability studies on the prodrug CPF-373 (1) were performed by exposing the compound to human serum and aqueous buffers (pH 1.0 and 8.0) using in situ 31P NMR. Each experiment was performed by dissolving ProTide in the appropriate deuterated solvent and analyzing the samples at 37°C for approximately 14 hours, obtaining scans at regular time intervals. For a better resolution of the original spectra (lower graphs) and deconvoluted ones (upper graphs) are recorded. The CPF-373 phosphoramidate stability assay (1), after incubation in human serum, exhibited 73% unchanged compound after 12 hours and 30 minutes as shown in Figure 6.
The spectra showed a singlet peak inherent in human serum at ~ δ2.00 and the doublet peak of origin at ~ δ4.50 which after 4 hours and 15 minutes was hydrolyzed to the aminoacyl phosphoromidate intermediate displayed as a peak singlet at δ7.20.
[00233] When chemical hydrolysis was evaluated under extreme experimental conditions, ie at pH 1.0 and pH 8.0 at 37°C, a complete stability of prodrug CPF-373 (1) in both acidic and basic buffer conditions was observed. Spectra were recorded for 14 hours taking scans every 12 minutes at regular intervals as shown in Figures 7 and 8. ProTide (1) examined at pH 1.0 exhibited doublet constant peaks of diastereomers at δ4.35; 4.50 during the entire trial period (Figure 7).
[00234] Also, at pH 8.0 the spectra exhibited a persistent peak of prodrug (1) at δ4.48 and a singlet peak at δ2.55 corresponding to a buffer peak (Figure 8). Metabolism of 5-FdUrd phosphoramidates
[00235] As shown in Figure 4, the putative mechanism of activation of ProTides within the cell, after uptake, involves a first step of enzymatic activation mediated by a carboxypeptidase-like enzyme that hydrolyzes the ester of the aminoacyl moiety (step a) followed by spontaneous cyclization and subsequent spontaneous displacement of the aryl group (step b) and water-mediated opening of the unstable ring (step c). The last step involves a phosphoramidase-mediated enzyme-mediated hydrolysis of the PN bond (step d) with the release of nucleoside monophosphate in the intact cell (Fig 4) (McGuigan et al., 2009; Mehellou et al.) , 2010).
[00236] To prove the proposed metabolic scheme for CPF-373 (1) and whether the ester motif of the 5-FdUrd phosphoramidate derivative is cleaved or not, an enzyme incubation experiment was performed that was assigned to the mimic of the first stages of putative activation in intact tumor cells. Compound (1) was incubated with carboxypeptidase Y (also known as cathepsin A) in TRIZMA buffer and the conversion of (1) was monitored by 31 P NMR. The spectra were recorded for 14 hours getting scans at periodic intervals every 4 minutes as shown in Figure 5. For better resolution the original spectra (lower graphs) and deconvoluted ones (upper graphs) are shown.
[00237] At 31P NMR the prodrug CPF-373 (1) appeared as two δ4.07 peaks; 4.23 corresponding with the two diastereoisomers observed as origin with the characteristic of doubling the chiral phosphate center of the phosphoramidate. After addition of cathepsin A the compound was rapidly hydrolyzed after 4 minutes to intermediates δ4.95; 5,16 that need an ester motif and this intermediate does not persist as it was successively rapidly metabolized to the aminoacyl phosphoramidate intermediate, the final product in this assay, through loss of the aryl group (steps a to c in Figure 4). The intermediate appeared as a singlet peak at δ6.85 due to the achiral phosphate center. Thus, the enzyme assay spectra exhibited rapid metabolism of the ~δ4.00 origin with complete conversion to the putative intermediate within 26 minutes, which was also consistently present throughout the entire 14 hours of the assay. Cleavage of the P-N bond releasing the nucleoside monophosphate was not detected in the enzyme experiment, as expected. This experiment indicates the first-step activation of ProTide CPF-373 (1) may be sufficiently efficient, and therefore, may allow the eventual release of the nucleoside monophosphate metabolite into intact tumor cells. Conclusion
[00238] In conclusion, the present invention provides novel phosphoramidate nucleotide prodrugs of the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (5-FdUrd), which have been synthesized and evaluated for their cytostatic activity. While 5-FdUrd substantially loses its cytostatic potential in thymidine kinase (TK) L1210 deficient murine leukemia and human lymphocyte CEM cell cultures, the compounds of the present invention, for example CPF-373, markedly maintain their antiproliferative activity in both cells of tumor-deficient and wild-type and are thus largely independent of intracellular TK activity to exert its cytostatic action. CPF-373, for example, has been found to inhibit thymidylate synthase (TS) in wild-type and TK-deficient cell lines at drug concentrations that well correlated with their cytostatic activity in these cells. CPF-373 did not appear to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These results are in line with the observations that 5-FdUrd, but not CPF-373, substantially loses its cytostatic potential in the presence of mycoplasmas expressing TP in tumor cell cultures. Therefore, compounds of the present invention such as CPF-373 are novel 5-FdUrd phosphoramidate prodrugs which (i) can avoid potential tumor cell resistance mechanisms (e.g., decreased TK activity) and (ii) ) not be degraded by catabolic enzymes such as TP whose activity is often up-regulated in tumor cells or expressed in mycoplasma infected tumor tissue. Incorporated by reference in its entirety is Vande Voorde, J. and other(s) Biochemical Pharmacology 82 (2011) 441-452.
[00239] Embodiments of the present invention, as set forth below, are described in McGuigan, C. et al. J. Med. Chem. 2011, 54 7247-7258 (posted September 5, 2011), the contents of which in their entirety are incorporated herein by reference.
[00240] Table 6 below records the cytostatic activity of 5-FU, 5-FdUrd, Reference example CPF382 and compounds incorporating the present invention against tumor cell lines in terms of IC50 or the concentration of compound required to inhibit tumor 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 the compounds embodying the present invention with respect to: "aryl", which corresponds to Ar of Formula I and is phenyl (Ph) or 1-naphthyl (Nap); "ester" which corresponds to R3 of Formula I; and "AA", which represents the amino acid whose C alpha atom and the substituents on the alpha C atom correspond to CR1R2 of Formula I. Table 6 describes the compounds embodying the present invention not previously mentioned above in Table 1, as well as those additional data for some compounds mentioned in Table 1. Table 6




[00241] Table 7 below records the cytostatic activity of 5-FdUrd, Reference example CPF382 and compounds incorporating the present invention in L1210 wild-type murine leukemia cell cultures (L1210/0) and L1210 cell cultures, infected with Mycoplasma hyorhinis (L1210.Hyor) in terms of IC50 or compound concentration to inhibit cell proliferation by 50%. Data are the mean (±SD) of at least two to four independent experiments. Table 7 identifies the phosphoramidate motif of Reference Example CPF382 and compounds embodying the present invention, as discussed above in relation to Table 6, but with "Naf" meaning 1-naphthyl. Table 7 describes 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 7.



[00242] Table 8 below records the cytostatic activity of 5-FdUrd and compounds incorporating the present invention in CEM cell cultures containing (Cem/hEnt-1) or needing the hEnt1 transporter (Cem/hEnt-0) in terms of IC50 or the concentration of compound required to inhibit tumor cell proliferation by 50%. Data are the 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 "Naf" meaning 1-naphthyl. The data in Table 8 show that compounds embodying the present invention are less dependent on the presence of the hENT1 transporter than 5-FdUrd, as they only lose 7- to 15-fold anti-proliferative activity against CEM-deficient cells. hENT1. These observations are in agreement with only a 2- to 7-fold decreased cytostatic activity of compounds incorporating the present invention in the presence of transport inhibitors (ie, dipyridamole and NBMPR), compared to a 20- to 60-fold loss of antiproliferative activity of 5-FuDrd and FdUMP under similar experimental conditions. Table 8.

[00243] Studies were performed on the compound CPF 381 as follows:
[00244] An enzyme phosphorylase assay was performed using thymidine phosphorylase (TP, purified from Esherichia coli) in the presence of potassium phosphate buffer (300 nM solution, pH 7.4). The 19F NMR spectrum after 5 min, 14 hours and 72 hours did not show any evidence of phosphorolysis. Unlike 5-FdUrd, CPF 381 is at best very poor substrate, if at all, for thymidine phosphorylase.
[00245] A chemical hydrolysis was evaluated under experimental conditions at pH 1 and pH 8 and monitored by 31P NMR. During the test (14 h) under acidic conditions (pH 1) only two peaks representing the two diastereoisomers were recorded. The lack of new signal formation in the 31P NMR spectrum indicates that the compound CPF 381 is slightly acidic stable. The same result was observed when compound CPF 381 was tested under mild basic conditions (pH 8).
[00246] Studies were performed on the compound CPF 581 as follows:
[00247] An enzymatic study using a carboxypeptidase Y assay was carried out in which the compound CPF 581, carboxypeptidase Y, and Trizma buffer (pH 7.6) were dissolved in acetone-d6 and 31P NMR spectrum (202 MHz) spectra were recorded at regular intervals (every 7 min) for 14 hours. Compound CPF 581 was rapidly hydrolyzed to a first metabolite without the ester moiety (R3), both diastereoisomers being processed at approximately similar rate. Also processing the first metabolite induced the formation of a second anionic metabolite, without Ar, within about 45 minutes with an estimated half-life of less than 5 minutes. The rate of the initial activation step can thus be considered in general as one of the 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 second anionic metabolite, which was added to the final test sample derived from compound CPF 373, i.e., containing only the second enzymatic metabolite derived from the compound CPF 581 in Trizma. The sample had a 31 P NMR spectrum showing only a single peak at δP 6.85 ppm, strongly supporting this part of the metabolic pathway and activation of the phosphoramidate compounds of the present invention.
[00248] Studies were performed on the compound CPF 386 as follows:
[00249] The stability of the compound CPF 386 in the presence of human serum was investigated using 31P NMR in situ. Control 31P NMR data of compound CPF 386 in DMSO and D2O were recorded. The NMR sample was then treated with human serum immediately subjected to further 31P NMR experiments at 37°C. 31P NMR data were recorded every 15 minutes for 14 hours. The spectra exhibited a single peak inherent to human serum at ~δP 2.00 ppm and two peaks corresponding to compound CPF 386 at ~δP 4.59 and 4.84 ppm. After about 6 hours and 45 minutes the compound was partially hydrolyzed to an intermediate, without R3 (Et), as a single peak at δP 5.79 ppm. After 11 hours and 30 minutes, the formation of the second metabolite, without Ar (1-naphthyl), displayed as a single peak at δP 7.09 ppm was observed. After 13 hours and 30 minutes the reaction mixture contained 96% of the parent compound CPF 386 together with the proposed first metabolite (3%) and the second metabolite (1%). 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Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. J. Med. Chem. 1993, 36, 1048. 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 vulnerability and may act by the generation of a novel intracellular metabolite. J. Med. Chem. 1996, 39, 1748. McGuigan, C.; Tsang, H.W.; Cahardr, D. Turner, K.; Velazquez, S.; Salgado, A.; 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. McGuigan, C.; Mehello, J.; Balzarini, J. Aryloxy phosphoramidate triesters: a technology for delivering mono phosphorylated nucleosides and sugars into cells. Chem.Med.Chem., 2009, 4, 11, 1779. 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权利要求:
Claims (17)
[0001]
1. Compound, characterized by the fact that it has Formula (I):
[0002]
2. Compound according to claim 1, characterized in that Ar is naphthyl and is preferably 1-naphthyl.
[0003]
3. Compound according to claim 1, characterized in that Ar is phenyl.
[0004]
4. Compound according to any one of claims 1 to 3, characterized in that Ar is substituted.
[0005]
5. A compound according to any one of claims 1 to 4, characterized in that R4 is selected from the group consisting of H and acetyl and is preferably H.
[0006]
6. A compound according to any one of claims 1 to 5, characterized in that R3 is selected from the group consisting of benzyl and members of the group comprising C1 to C10 alkyls, is preferably ethyl, n-propyl, n-butyl, n-pentyl, or n-hexyl and is more preferably n-pentyl.
[0007]
7. Compound according to any one of claims 1 to 6, characterized in that R1 and R2 are different and the stereochemistry at the asymmetric center -CR1R2 corresponds to a member selected from the group consisting of an amino acid-L, an amino acid- D and mixtures of L and D amino acids.
[0008]
8. A compound according to any one of claims 1 to 7, characterized in that R1 and R2 correspond to the portions linked to the alpha C atom in a naturally occurring amino acid and preferably the naturally occurring alpha amino acid is L-alanine .
[0009]
9. Compound according to claim 1, characterized in that Ar is 1-naphthyl, R3 is benzyl or n-pentyl, and one of R1 and R2 is H, one of R1 and R2 is methyl and the atom of C to which R1 and R2 are attached has L-chirality.
[0010]
10. Compound according to claim 1, characterized in that it is selected from the group comprising: 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(benzoxy-L-alaninyl)] phosphate] (CPF 381) 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(ethoxy-L-alaninyl)] phosphate (CPF383) 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl phosphate (isopropoxy-L-alaninyl)] (CPF384) 5-Fluoro-2'deoxyuridine-5'-O-[phenyl(cyclohexoxy-L-alaninyl)] phosphate (CPF508) 5-Fluoro-2'deoxyuridine-5 phosphate '-O-[p-nitro-phenyl(ethoxy-L-alaninyl)] (CPF430) 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)] phosphate (CPF373 ) 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(methoxy-L-alaninyl)] phosphate (CPF385) 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl phosphate (ethoxy-L-alaninyl)] (CPF386) 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(isopropoxy-L-alaninyl)] (CPF387) 5-Fluoro-2'deoxyuridine phosphate -5'-O-[1-naphthyl(cyclohexoxy-L-alaninyl)] (CPF509) 5-Fluoro-2'deoxyuridine-5'-O-[phenyl(benzox) phosphate ia,α-dimethylglycine)] (CPF393) 5-Fluoro-2'deoxyuridine-5'-O-[phenyl(ethoxy-α,α-dimethylglycine)] phosphate (CPF394) 5-Fluoro-2'deoxyuridine-phosphate 5'-O-[1-naphthyl(benzoxy-α,α-dimethylglycine)] (CPF395) 5-Fluoro-2'deoxyuridine-5'-O-[1-naphthyl(ethoxy-a,a-dimethylglycine) phosphate ] (CPF396) 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(benzoxy-L-prolinyl)] phosphate (CPF583) 5-Fluoro-2'-deoxyuridine-5'-O-[ phosphate 1-naphthyl(benzoxy-L-prolinyl)] (CPF577) 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(3,3-dimethyl-1-butoxy-L-alaninyl)] phosphate (CPF585) 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(cyclobutoxy-L-alaninyl)] phosphate (CPF578) 5-Fluoro-2'-deoxyuridine-5'-O-[ 1-Naphthyl-(cyclopropylmethaneoxy-L-alaninyl)] phosphate (CPF579) 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(tetrahydropyroxy-L-alaninyl)] phosphate phosphate (CPF580) Phosphate 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(pentoxy-L-alaninyl)] (CPF581) 5-Fluoro-2'-deoxyuridine-5'-O-[1- naphthyl-(cyclopentoxy-L-alaninyl)] (CPF582) 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(2-indanoxy-L-alaninyl)] phosphate (CPF597) 5-Fluoro-2'-deoxyuridine-5' phosphate -O-[Phenyl-(Benzoxy-L-methioninyl)] (CPF586) 5-Fluoro-2'-Deoxyuridine-5'-O-[1-Naphthyl-(Benzoxy-L-phenylalaninyl)] Phosphate (CPF587) Phosphate of 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)] (CPF588) 5-Fluoro-2'-deoxyuridine-5'-O phosphate -[1-naphthyl-(butoxy-L-alaninyl)] (CPF589) 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(ethoxy-L-valinyl)] phosphate 5-fluoro-2 '-deoxyuridine-5'-O-[phenyl(benzoxy-L-leucinyl)] 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(benzoxy-L-isoleucinyl)] 5-fluoro phosphate -2'-deoxyuridine-5'-O-[phenyl(benzoxy-L-phenylalaninyl)] 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(pentoxy-L-methioninyl)] Phosphate 5 -fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(hexoxy-L-alaninyl)] 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(cyclohexoxy-L-) phosphate valinyl)] 5-Fluoro-2'-deoxyuridine-5'-O- phosphate [1-naphthyl(pentoxy-L-leucinyl)] 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-leucinyl)] 5-fluoro-2'-deoxyuridine phosphate -5'-O-[1-naphthyl(pentoxy-L-isoleucinyl)] 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(pentoxy-L-phenylalaninyl)] phosphate 5- fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-methioninyl)] 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(pentoxy-α,α) phosphate -dimethylglycine)], or a pharmaceutically acceptable derivative of any thereof, wherein said pharmaceutically acceptable derivative is any pharmaceutically acceptable salt thereof.
[0011]
11. Compound, characterized in that it is 5-fluoro-2'deoxyuridine-5'-0-[1-naphthyl(benzoxy-L-alaninyl)] phosphate (CPF373) or a pharmaceutically acceptable derivative thereof, being that said pharmaceutically acceptable derivative is any pharmaceutically acceptable salt thereof.
[0012]
12. Compound, characterized in that it is 5-fluoro-2'deoxyuridine-5'-0-[1-naphthyl(benzoxy-L-alaninyl)] phosphate (CPF373).
[0013]
13. Use of a compound as defined in any one of claims 1 to 12, characterized in that it is in the manufacture of a medicine for prophylaxis or cancer treatment comprising administering to a homo sapiens patient in need of such treatment an effective dose of the compound, preferably the cancer being selected from the group comprising leukemia, pancreatic, prostate, lung, breast and cervical and esophageal cancer; gastrointestinal, including gastric, small bowel, colon, and rectal cancer; head and neck cancer; and ovarian cancer.
[0014]
14. Use of a compound as defined in any one of claims 1 to 12, characterized by the fact that it is in the manufacture of a drug for the prophylaxis or treatment of a patient: (i) that has developed or has the potential to develop resistance in tumor cells with respect to 5-fluoro- or 5-fluoro-2'-deoxyuridine activity in a method of cancer prophylaxis or treatment; (ii) with a decreased level of nucleoside transporter proteins; (iii) which has nucleoside kinase deficient cells; (iv) that has mycoplasma-infected cells; (v) which presents cells with an increased level of thymidylate synthase (TS); and/or (vi) to avoid susceptibility to nucleoside degradation by catabolic enzymes, preferably wherein the catabolic enzymes are selected from the group comprising thymidine phosphorylase, uridine phosphorylase and deoxycytidine deaminase.
[0015]
15. Pharmaceutical composition, characterized in that it comprises a compound as defined in any one of claims 1 to 12, in combination with a pharmaceutically acceptable excipient, diluent or carrier.
[0016]
16. Method for preparing a pharmaceutical composition, characterized in that it comprises the step of combining a compound, as defined in any one of claims 1 to 12, with a pharmaceutically acceptable diluent, carrier or excipient.
[0017]
17. Process for preparing a compound of Formula (I), as defined in claim 1, characterized in that it comprises reacting a compound having Formula (II) with a compound having the Formula (III) in which Ar, R3, R4, R1 and R2 are as defined in claim 1.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-12-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-16| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-27| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/02/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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GB1103582.1|2011-03-01|

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GBGB1103582.1A|GB201103582D0|2011-03-01|2011-03-01|

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GB1105660.3|2011-04-01|

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GBGB1105660.3A|GB201105660D0|2011-04-01|2011-04-01|Chemical compounds|

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PCT/GB2012/050457|WO2012117246A1|2011-03-01|2012-02-29|Phosphoramidate derivatives of 5 - fluoro - 2 ' - deoxyuridine for use in the treatment of cancer|

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