PHOSPHOANTIGEN PRODRUG COMPOUNDS

Abstract

The invention relates to a compound of General Formula (I): wherein R1, R2, R3, R4 and R5 are as defined herein. Compounds and compositions comprising the same exhibit high serum stability and potent activation of the γδ T-cell immune response, so are suitable for use in immunotherapy. The invention also relates to methods of immunotherapy using the compounds of General Formula (I).

##STR00001##

Claims

1. A compound according to General Formula (I) including all tautomers thereof: ##STR00009## wherein R1 and R2 each independently represents an amino acid ester radical according to General Formula (II) or an aryloxy radical according to General Formula (III): ##STR00010## wherein R6 represents H, or a saturated or unsaturated and optionally substituted hydrocarbon chain; R7 represents a saturated or unsaturated and optionally substituted hydrocarbon chain; and R8 represents an optionally substituted C.sub.5-25 aryl or a 5 to 25 membered heteroaryl group; R3 represents an optionally substituted C.sub.2-20 alkyl, C.sub.4-20 alkenyl or C.sub.2-20 alcohol radical; and each of R4 and R5 independently represent H or halo; or a salt thereof.

2. A compound according to claim 1, wherein said compound is of General Formula (IV), wherein R3, R4, R5, R6, R7 and R8 are as defined in claim 1: ##STR00011##

3. A compound according to claim 1, wherein R3 is a radical according to Formula (V), Formula (VI) or Formula (VII), wherein R9 is selected from OH, OR.sup.10, SH, SR.sup.10, NH.sub.2 or NHR.sup.10, and wherein R.sup.10 represents C.sub.1-4 alkyl: ##STR00012##

4. A compound according to claim 3, wherein R9 is OH.

5. A compound according to claim 1, wherein one or both, of R4 and R5 represent halo.

6. A compound according to claim 5, wherein said halo substituents is/are fluoro.

7. A compound according to claim 1, wherein R6 is a C.sub.1-4 alkyl chain.

8. A compound according to claim 1, wherein R7 is an unsubstituted C.sub.1-4 alkyl chain or an unsubstituted benzyl group.

9. A compound according to claim 1, selected from: ##STR00013## ##STR00014## ##STR00015##

10. A pharmaceutical composition comprising a compound as defined in claim 1 and a pharmaceutically acceptable excipient or carrier.

11. (canceled)

12. (canceled)

13. (canceled)

14. A method of immunotherapy, the method comprising administering to a subject an effective amount of a compound as defined in claim 1 to activate a γδ T-cell immune response.

15. A method according to claim 14, wherein said compound is administered ex vivo to a sample obtained from an individual in need of treatment to induce proliferation of γδ T cells prior to said sample being returned to the body.

16. A method according to claim 14, wherein said compound is administered to treat an infection, a proliferative disease or osteoporosis.

17. A method according to claim 16, wherein said proliferative disease is a cancer.

18. (canceled)

19. (canceled)

20. (canceled)

21. A method of immunotherapy, the method comprising administering to a subject an effective amount of a pharmaceutical composition as defined in claim 10 to activate a γδ T-cell immune response.

22. A method according to claim 21, wherein said composition is administered to treat an infection, a proliferative disease or osteoporosis.

23. A method according to claim 22, wherein said proliferative disease is a cancer.

24. A method according to claim 21, wherein said composition is administered ex vivo to a sample obtained from an individual in need of treatment to induce proliferation of γδ T cells prior to said sample being returned to the body.

25. A method according to claim 24, wherein said composition is administered to treat an infection, a proliferative disease or osteoporosis.

26. A method according to claim 25, wherein said proliferative disease is a cancer.

27. A method according to claim 15, wherein said compound or composition is administered to treat an infection, a proliferative disease or osteoporosis.

28. A method according to claim 27, wherein said proliferative disease is a cancer.

Description

[0066] The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

[0067] FIG. 1: Chemical structures of reported small molecule Vγ9/Vδ2 T-cells activators: Naturally-occurring PAgs (E)-4-hydroxy-3-methyl but-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP); Synthetic molecules risedronate and zoledronate.

[0068] FIG. 2: Application of the aryloxy triester phosphoramidate prodrug technology to the monophosphate derivative of HMBPP (HMBP). The monophosphate group is masked by an aryl group and an amino acid ester, which are both enzymatically cleaved off inside cells to release the active monophosphate species. Instability was observed due to the cleavage of the —P—O— bond of these compounds (shaded).

[0069] FIG. 3: pKA values of phosphate and different phosphonate groups.

[0070] FIG. 4: Synthesis of (A) aryloxy phospharamidate ProPAgens of HMBP methylphosphonate (4a-d); and (B) HMBP difluoromethylphosphonate (9a-d). Reagents and conditions: (i) TMSBr, DCM, rt, 2 h then (COCl).sub.2, DMF cat, DCM, rt, 18 h; ii) a. Phenol, Et.sub.3N, DCM, −78° C. for 30 mins then rt, 3 h; b. Substituted L-alanine ester hydrochloride, Et.sub.3N, DCM, rt, 12 h, yields: 38-61%; (iii) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda-Grubbs Catalyst 2.sup.nd generation, DCM, rt, yields: 57-64%; (iv) Diethyl (bromodifluoromethyl)phosphonate, DMF, zinc powder, rt, N.sub.2, 3h then CuBr, allyl bromide, rt, 40 h; (v) (i) TMSBr, DCM, rt, 2 h then (COCl).sub.2, DMF cat, DCM, rt, 18 h; ii) a. Phenol, Et.sub.3N, DCM, −78° C. for 30 mins then rt, 3 h; b. Substituted L-alanine ester hydrochloride, Et.sub.3N, DCM, rt, 12 h, yields: 24-46%; (iii) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda-Grubbs Catalyst 2nd generation, DCM, rt, yields: 58-69%.

[0071] FIG. 5: Stability of HMBP phosphonate ProPAgen 4d in human serum at 37° C. for 12 hours as monitored by .sup.31P NMR. Prodrug 4d (5.0 mg) was dissolved in DMSO-d.sub.6 (0.10 mL) and D.sub.2O (0.15 mL). All .sup.31P NMR spectra were recorded at 37° C. Initially, a .sup.31P NMR scan of prodrug 4d (5.0 mg) in DMSO-d.sub.6 (0.10 mL) and D.sub.2O (0.15 mL) was recorded (shown as compound 4d alone in the figure). Following this, a previously defrosted human serum (0.30 mL) was added to the NMR tube and a spectrum immediately run. Spectra were recorded at 30 min after the addition and then at even time intervals over 12 hr.

[0072] FIG. 6: Stability of HMBP phosphonate ProPAgen 9a in human serum at 37° C. for 12 hours as monitored by .sup.31P NMR. Same as for compound 4d above. The .sup.31P NMR of prodrug 9a shows six phosphorous peaks because of the coupling between the fluorine-phosphorous. In fact, these are eight peaks as predicted and two extra peaks are seen when zoomed into these peaks to make the predicted eight phosphorous peaks of such compounds.

[0073] FIG. 7. Activation of human Vγ9/Vδ2.sup.+ T cells by HMBP phosphonate ProPAgens 4d and 9d. Human peripheral blood mononuclear cells (PBMC) were incubated with the indicated concentrations of ProPAgens 4d (left) and 9d (right) for 18 hr. TCR Vγ9/Vδ2.sup.+ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25, as a readout to the activation of Vγ9/Vδ2.sup.+ T cells. EC.sub.50 for both ProPAgens is ca. 8 pM (picomolar).

[0074] FIG. 8. FACS data showing the activation of human Vγ9/Vδ2.sup.+ T cells by the HMBP phosphonate ProPAgen 4d in a dose-dependent manner. Human peripheral blood mononuclear cells (PBMC) were incubated with the indicated concentrations of ProPAgens 4d for 18 h (FIG. 8A) or 20 h (FIGS. 8B and 8C). For 20 h incubations, data was collected and analysed individually from two separate donors: Donor (1) results shown in FIG. 8B, Donor (2) results shown in FIG. 8C. TCR Vγ9/Vδ2.sup.+ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25, as a readout to the activation of Vγ9/Vδ2.sup.+ T cells. A quantification of this is given in FIG. 7.

[0075] FIG. 9. FACS data showing the activation of human Vγ9/Vδ2.sup.+ T cells by the HMBP phosphonate ProPAgen 9d in a dose-dependent manner. Human peripheral blood mononuclear cells (PBMC) were incubated with the indicated concentrations of ProPAgens 9d for 18 h (FIG. 9A) or 20 h (FIGS. 9B and 9C). For 20 h incubations, data was collected and analysed individually from two separate donors: Donor (1) results shown in FIG. 9B, Donor (2) results shown in FIG. 9C. TCR Vγ9/Vδ2.sup.+ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25, as a readout to the activation of Vγ9/Vδ2.sup.+ T cells. A quantification of this is given in FIG. 7.

[0076] FIG. 10. FACS data showing the activation of human Vγ9/Vδ2.sup.+ T cells by HMBPP in a dose-dependent manner. Human peripheral blood mononuclear cells (PBMC) were incubated with the indicated concentrations of HMBPP for 20 h. Data was collected and analysed individually from two separate donors: Donor (1) results shown in FIG. 10A, Donor (2) results shown in FIG. 10B. TCR Vγ9/Vδ2.sup.+ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25, as a readout to the activation of Vγ9/Vδ2.sup.+ T cells.

[0077] FIG. 11. FACS data showing a lack of activation of human CD8+ T cells by the HMBP phosphonate ProPAgen 4d. Human PBMCs were incubated with the indicated concentrations of ProPAgen 4d for 20 h. TCR CD8.sup.+ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25, as a readout of the activation of CD8.sup.+ T cells. Data was collected and analysed individually from two separate donors: Donor (1) results shown in FIG. 11A, Donor (2) results shown in FIG. 11B.

[0078] FIG. 12. FACS data showing a lack of activation of human CD8+ T cells by the HMBP phosphonate ProPAgen 9d. Human PBMCs were incubated with the indicated concentrations of ProPAgen 9d for 20 h. TCR CD8.sup.+ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25, as a readout of the activation of CD8.sup.+ T cells. Data was collected and analysed individually from two separate donors: Donor (1) results shown in FIG. 12A, Donor (2) results shown in FIG. 12B.

[0079] FIG. 13. Cytotoxocity Assay showing potent lysis of bladder cancer cells following incubation with the HMBP phosphonate ProPAgen 4d. ProPAgen 4d mediates the specific lysis of T24 bladder cancer cells by Vγ9/Vδ2 T-cells. Human T24 urinary bladder carcinoma cell lines (target) were incubated for 4 hours with 10 μM zoledronate, 100 pM of HMBPP or 100 pM of ProPAgen 4d, before being washed five-times in medium and co-cultured with previously expanded Vγ9Vδ2 T cells in an effector target ratio of 10:1 for 18 hours.

MATERIALS AND METHODS

[0080] All reagents and solvents were of general purpose or analytical grade and were purchased from Sigma-Aldrich Ltd., Fisher Scientific, Fluorochem or Acros. .sup.31P, .sup.1H and .sup.13C NMR data were recorded on a Bruker Avance DPX500 spectrometer operating at 202, 500 and 125 MHz. Chemical shifts (δ) are quoted in ppm, and J values are quoted in Hz. In reporting spectral data, the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), and m (multiplet). All of the reactions were carried out under nitrogen atmosphere and were monitored with analytical thin layer chromatography (TLC) on pre-coated silica plates (kiesel gel 60 F254, BDH). Compounds were visualized by illumination under UV light (254 nm) or by the use of KMnO.sub.4 stain followed by heating. Flash column chromatography was performed with silica gel 60 (230-400 mesh) (Merck). Mass spectra (HRMS) were determined as a service by the School of Chemistry at Cardiff University.

Example 1: Synthesis of Aryloxy Phospharamidate ProPAgens of HMBP Methylphosphonates

[0081] Compounds of general formula (VI) in which R4 and R5 each represent H were prepared by a synthetic approach that employed Grubbs olefin metathesis.sup.7. The synthesis of ProPAgens 4a-d, which is summarised schematically in FIG. 4(A), was achieved by treating the commercially available diethyl 3-butenylphosphonate (1) with Bromotrimethylsilane (TMSBr) at room temperature to remove the ethoxy groups.sup.8. This was followed by chlorination reaction using oxalyl chloride in the presence of a catalytic amount of DMF. The product of this reaction, 2, was subsequently treated phenol in the presence of triethylamine and then with the appropriate amino acid ester to yield phosphonamidates 3a-d in moderate yields (38-61%). Subsequently, these compounds underwent Grubbs olefin metathesis with 2-methyl-2-propenol employing Hoveyda-Grubbs second generation catalyst in the presence of 1,4-benzoquinone. This gave ProPAgens 4a-d in good yields (57-64%).sup.9.

[0082] L-alanine was used as the amino acid of choice in the synthesis of ProPAgens 4a-d since it has historically shown the optimum biological activity, while the phenol motif was chosen for the aryloxy masking group as it has been used successfully in the discovery of two FDA-approved drugs; sofosbuvir and tenofovir alafenamide. Four different ester motifs were chosen in the synthesis of the ProPAgens—4a: methyl (Me); 4b: isopropyl (iPr); 4c: tert-butyl (tBu); and 4d: benzyl (Bn), because these show varying biological activities that vary from lowest activity (tBu) to highest activity (Bn).sup.6.

[0083] Further details of the above syntheses can be found in the appendix.

Example 2: Synthesis of Aryloxy Phospharamidate ProPAgens of HMBP Difluoromethylphosphonate

[0084] Compounds of general formula (VI) in which R4 and R5 each represent F were also prepared using a synthetic approach that employed Grubbs olefin metathesis.sup.7. The synthesis of ProPAgens 9a-d, which is summarised schematically in FIG. 4(B), was achieved by first reacting the commercially available diethyl (bromodifluoromethyl)phosphonate (5) with allyl bromide in the presence of zinc and copper bromide in DMF as reported.sup.10. The generated compound, 6, was subsequently chlorinated and then treated with phenol and the appropriate amino acid ester to yield phosphoramidates 8a-d in good yields (24-46%) as described for the synthesis of compounds 3a-d. Subsequently, these phosphonamidates were treated with 2-methyl-2-propenol in the presence of 1,4-benzoquinone and Hoveyda-Grubbs second generation catalyst.sup.9. The final ProPAgens 9a-d were generated in good yield (58-69%). L-alanine was used as the amino acid of choice in the synthesis of these prodrugs since it has historically shown the optimum biological activity, while the phenol motif was chosen as it has been used successfully in the discovery of two FDA-approved drugs; sofosbuvir and tenofovir alafenamide.

[0085] As for ProPAgens 4a-d, L-alanine was used as the amino acid of choice in the synthesis of ProPAgens 9a-d and the phenol motif was chosen for the aryloxy masking group. Again, four different ester motifs were chosen in the synthesis of the ProPAgens—9a: methyl (Me); 9b: isopropyl (iPr); 9c: tert-butyl (tBu); and 4d: benzyl (Bn).

[0086] Further details of the above syntheses can be found in the enclosed appendix.

Example 3: Stability Studies of HMBP Methylphosphonates and HMBP Difluoromethylphosphonates

[0087] As it was a primary objective to address the poor stability previously observed for HMBP phosphate ProPAgens.sup.6, Serum stability studies were carried out upon the completion of the synthesis of the novel compounds in Examples 1 and 2.

[0088] ProPAgen 4d with human serum at 37° C. for 12 h and monitored the sample by .sup.31P-NMR as reported previously.sup.11. As shown in FIG. 5, ProPAgen 4d had two singlets at dP=33.60 and 34.05 ppm, on the .sup.31P-NMR corresponding to the two diastereoisomers that arise from the chiral phosphorous center, which is typical of these prodrugs. Following the addition of human serum and monitoring of the sample by .sup.31P-NMR, there was no degradation observed since no new phosphorous peaks were detected for the period studied (12 h).

[0089] A similar stability profile was observed for the difluoromethyl phosphonate ProPAgen 9a (FIG. 6). Together, these data indicate the superior stability of these prodrugs in comparison to the HMBP phosphate ProPAgens that we previously disclosed.sup.6.

Example 4: Activation of Human Vγ9/Vδ2.SUP.+ T Cells by ProPAgens 4a-d and 9a-d

[0090] To demonstrate that the ProPAgens of Examples 1 and 2 retained potent activation of Vγ9/Vδ2 T-cells, peripheral blood mononuclear cells (PBMCs) containing Vγ9/Vδ2 T-cells derived from healthy donors were incubated with increasing concentrations of ProPAgens 4d and 9d (FIGS. 7, 8 and 9).

[0091] Peripheral blood γδ T-cells lack appreciable levels of surface CD69 or CD25 under steady state conditions, but upon T-cell receptor (TCR) stimulation upregulate both T-cell activation markers within 72 hours. PAg responsive Vγ9/Vδ2 T-cells were then distinguished by TCR Vγ9 and Vδ2 expression and assessed for the upregulation CD69 and CD25.

[0092] As shown in FIGS. 7, 8, and 9, HMBP phosphonate ProPAgens 4d and 9d, as representatives of these classes of prodrugs, exhibited potent Vγ9/Vδ2 T-cells activation that is far superior to that reported for HMBP phosphate ProPAgens.sup.6.

Example 5 (Comparative): Activation of Human Vγ9/Vδ2.SUP.+ T Cells by HMBPP

[0093] To demonstrate the enhanced Vγ9/VO2 T-cell activation potency of the ProPAgens of Examples 1 and 2, peripheral blood mononuclear cells (PBMCs) containing Vγ9/Vδ2 T-cells derived from healthy donors were incubated for comparative purposes with increasing concentrations of HMBPP (FIG. 10).

[0094] As is evident upon comparison of FIGS. 8 and 9 with FIG. 10, the T-cell activation potency of HMBPP is inferior to that activation potency of ProPAgens 4d and 9d.

Example 6: Lack of Activation of Human CD8+ T Cells by ProPAgens 4a-d and 9a-d

[0095] To demonstrate that the ProPAgens of Examples 1 and 2 are Vγ9/Vδ2 T cell—specific activators, PBMCs containing CD8+ T-cells derived from donors were incubated with increasing concentrations of ProPAgens 4d and 9d (FIGS. 11 and 12).

[0096] Like peripheral blood γδ T-cells, peripheral blood CD8+ T-cells lack appreciable levels of surface CD69 or CD25 under steady state conditions, but upon T-cell receptor (TCR) stimulation upregulate both T-cell activation markers within 72 hours. PAg responsive CD8 T-cells were then distinguished by TCR CD8 expression and assessed for the upregulation CD69 and CD25.

[0097] As shown in FIGS. 11 and 12, HMBP phosphonate PropAgens 4d and 9d, as representatives of these classes of prodrugs, did not exhibit activation of CD8+ T cells, even upon incubation at a concentration of 1 μM, i.e. approx. 100,000 times greater than the Vγ9/Vδ2 T-cell activation EC.sub.50 values calculated for both ProPAgens.

Example 7: Lysis of T24 Bladder Cancer Cells by Vγ9/Vδ2 T-Cells is Mediated and Enhanced by ProPAgens 4a-d and 9a-d

[0098] As a further proof of principle, and to demonstrate that the superior Vγ9/Vδ2.sup.+ T cell activation efficacy of ProPAgens of Examples 1 and 2 shown above does translate into a beneficial therapeutic effect, the specific lysis of cancer cells by Vγ9/Vδ2 T-cells was compared following incubation of human T24 urinary bladder carcinoma cell lines for 4 hours with 10 μM zoledronate, 100 μM of HMBPP or 100 pM of ProPAgen 4d.

[0099] Further details of this in vitro cytotoxicity assay can be found in the enclosed appendix.

[0100] As shown in FIG. 13, incubation with HMBP phosphonate PropAgen 4d resulted in a significant enhancement in T24 bladder cancer lysis in comparison not only to that observed upon incubation with zoledronate but also, surprisingly, compared to that observed upon incubation with HMBPP.

Summary

[0101] We report the synthesis of novel methyl and difluoromethyl phosphonate ProPAgens. These ProPAgens exhibited superior serum stability compared to their phosphate ProPAgens derivatives, which were previously reported.sup.6. These prodrugs were shown not only to be specific activators of Vγ9/Vδ2 T-cells, but also to be far more potent activators of Vγ9/Vδ2 T-cells than the previously reported HMBP phosphate ProPAgens. This increase in Vγ9/Vδ2 T-cells activation efficacy was also shown to translate to highly potent lysis of cancer cells in vitro.

[0102] The combination of high, specificity, serum stability and potency profiles of these new phosphonate ProPAgens makes them suitable for development as new immunotherapeutics for treating a variety of conditions, including proliferative diseases such as cancer, osteoporosis and/or various infections such as tuberculosis, leprosy, typhoid, malaria, and toxoplasmosis.

[0103] Appendix: Synthesis and Evaluation of ProPAgen Compounds 4a-d and 9a-d

[0104] But-3-en-1-ylphosphonic dichloride (2). Trimethylsilylbromide (13.72 mL, 104.06 mmol, 10 eq.) was slowly added over 30 min to diethylbut-3-en-1-yl phosphonate 1 (2 g, 10.40 mmol, 1 eq.) in CH2Cl2 (50 mL) under nitrogen at room temperature. The mixture was stirred for 2 h followed by the removal of volatiles under reduced pressure to obtain a yellow liquid δ.sub.P NMR (202 MHz, CDCl.sub.3): 24.70. The was then dissolved in 50 mL CH.sub.2Cl.sub.2 and two drops of dry DMF were added and the mixture was cooled to 0° C. Oxalyl chloride (2.68 mL, 31.20 mmol, 3 eq.) was then added dropwise and the reaction mixture was allowed to warm to room temperature and stirred for 18 h. The volatiles were evaporated and additional CH.sub.2Cl.sub.2 (10 mL) was evaporated three more times to give the crude product (1.79 g, 100%) as a brown liquid which was used in the next step without further purification. δ.sub.P NMR (202 MHz, CDCl.sub.3): 49.66.

[0105] General procedure 1. Synthesis of allylphosphonoamidates 3a-d. The crude product but-3-en-1-ylphosphonic dichloride (2) was dissolved in 5 mL CH.sub.2Cl.sub.2 and added dropwise to a solution of phenol (1 eq.), dry Et.sub.3N (2 eq.) and CH.sub.2Cl.sub.2 (10 mL) at −78° C. After stirring at −78° C. for 30 min, the reaction mixture was allowed to warm to room temperature and stirring was continued for another 3 h. Once the reaction is complete as indicated by .sup.31P NMR [op NMR (202 MHz, CDCl.sub.3): ˜39.93], the mixture was filtered, and the volatiles were removed under reduced pressure, washed twice with Et.sub.2O, which was subsequently removed under reduced pressure to give a crude oil. This product was then dissolved in CH.sub.2Cl.sub.2 (10 mL) and was added dropwise over 15 min to a stirring mixture of L-alanine ester hydrogen chloride (1 eq.) and dry Et.sub.3N (2 eq.) in dry CH.sub.2Cl.sub.2 (10 mL) under nitrogen at −78° C. After stirring at −78° C. for 30 mins, the reaction was allowed to warm to room temperature and was left stirring overnight. The solvents were removed under reduced pressure, and the mixture was filtered and washed with Et.sub.2O, which was then removed under reduced pressure to give a crude oil. The final products were then purified by column chromatography (6:4 Hex/EtOAc) as colorless oils.

[0106] Methyl (but-3-en-1-yl(phenoxy)phosphoryl)-L-alaninate (3a). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine methyl ester hydrogen chloride (0.373 g, 2.23 mmol, 1 eq.) to give product 3a (0.248 g, 38%) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 30.88, 31.22. OH NMR (500 MHz, CDCl.sub.3): 7.30 (m, 2H, Ar), 7.10 (m, 3H, Ar), 5.82 (m, 1H, CH.sub.2═CH), 5.00 (m, 2H, CH.sub.2═CH), 4.11-3.86 (m, 1H, CH—NH), 3.60 (d, J=6.6 Hz, 3H, OCH.sub.3), 3.18 (m, 1H, NH), 2.48-2.27 (m, 2H, ═CH—CH.sub.2), 1.92 (m, 2H, CH.sub.2—P), 1.21 (2 d, J=7.1 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 174.68 (d, J=6.3 Hz, C═O), 174.38 (d, J=5.1 Hz, C═O), 150.72 (d, J=9.1 Hz), 150.51 (d, J=9.4 Hz), 129.78, 124.78 (d, J=5.5 Hz, CH═CH.sub.2), 120.86 (d, J=4.6 Hz, C—Ar), 120.71 (d, J=4.7 Hz), 115.56, 52.52 (d, J=3.1 Hz, CH.sub.3—O), 49.58 (d, J=14.7 Hz, CH—NH), 27.88 (d, J=130.9 Hz, CH.sub.2—P) 27.60 (d, J=131.6 Hz, CH.sub.2—P), 26.59 (d, J=4.1 Hz, CH.sub.2—CH.sub.2—P), 21.68 (2 d, J=4.3 Hz, CHCH.sub.3).

[0107] Isopropyl (but-3-en-1-yl(phenoxy)phosphoryl)-L-alaninate (3b). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine isopropyl ester hydrogen chloride (0.373 g, 2.23 mmol, 1 eq.) to give product 3b (0.348 g, 48%) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 30.93, 31.24. δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.30 (m, 2H, Ar), 7.21 (m, 3H, Ar), 5.81 (m, 1H, CH.sub.2═CH), 5.11 (m, 2H, CH.sub.2═CH), 4.96 (m, 1H, CH-iPr), 4.04-3.94 (m, 1H, CH—NH), 3.21 (m, 1H, NH), 2.45 (m, 2H, ═CH—CH.sub.2), 2.02-1.88 (m, 2H, CH.sub.2—P), 1.29-1.18 (m, 9H, CHs—CH—NH, CH-iPr). 6c NMR (126 MHz, CDCl.sub.3): 173.67 (d, J=6.3 Hz, C═O), 150.74 (d, J=9.1 Hz), 129.76 (d, J=6.7 Hz), 124.73 (d, J=5.0 Hz), 120.86 (d, J=4.6 Hz), 120.69 (d, J=4.6 Hz), 115.50, 69.23 (d, J=5.6 Hz, CHiPr), 49.75 (d, J=9.5 Hz, CH—NH), 27.89 (d, J=130.9 Hz, CH.sub.2—P), 27.54 (d, J=131.4 Hz, CH.sub.2—P), 26.74 (d, J=4.3 Hz, CH.sub.2—CH.sub.2—P), 26.57 (d, J=4.0 Hz, CH.sub.2—CH.sub.2—P), 21.58 (2 d, J=4.4 Hz, CHCH.sub.3).

[0108] tert-Butyl (but-3-en-1-yl(phenoxy)phosphoryl)-L-alaninate (3c). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine tert-Butyl ester hydrogen chloride (0.405 g, 2.23 mmol, 1 eq.) to give product 3c (0.461 g, 61%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 30.95, 31.21. δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.32 (m, 2H, Ar), 7.20 (m, 3H, Ar), 5.87 (m, 1H, CH.sub.2═CH), 5.12 (m, 2H, CH.sub.2═CH), 4.00-3.86 (m, 1H, CH—NH), 3.26 (m, 1H, NH), 2.45 (m, 2H, ═CH—CH.sub.2), 2.04-1.85 (m, 2H, P—CH.sub.2), 1.42 (s, 9H, tBu-H), 1.22 (2 d, J=7.2 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 173.13 (d, J=5.5 Hz, C═O), 150.77 (d, J=9.2 Hz), 137.43 (d, J=7.1 Hz), 137.32 (d, J=7.6 Hz), 129.76 (d, J=6.2 Hz), 124.71 (d, J=4.0 Hz), 120.88 (d, J=4.6 Hz), 120.72 (d, J=4.7 Hz), 115.47, 81.99 (d, J=8.2 Hz), 60.54, 50.18 (d, J=4.0 Hz, CH—NH), 28.05, 27.91 (d, J=131.6 Hz, CH.sub.2—P), 27.20 (d, J=130.2 Hz, CH.sub.2—P), 21.82 (2 d, J=4.2 Hz, CHCH.sub.3).

[0109] Benzyl (but-3-en-1-yl(phenoxy)phosphoryl)-L-alaninate (3d). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine benzyl ester hydrogen chloride (0.405 g, 2.23 mmol, 1 eq.) to give product 3d (0.461 g, 55%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 30.85, 31.21. δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.38-7.27 (m, 7H, Ar), 7.21-7.18 (m, 2H, Ar), 7.16-7.09 (m, 1H, Ar), 5.85 (m, 1H, CH.sub.2═CH), 5.04 (m, 4H, CH.sub.2═CH, —OCH.sub.2), 4.28-4.03 (m, 1H, CH—NH), 3.24 (m, 1H, NH), 2.56-2.34 (m, 2H, ═CH—CH.sub.2), 2.08-1.82 (m, 2H, P—CH.sub.2), 1.24 (2 d, J=7.1 Hz, 3H, CH—CH.sub.3). 6c NMR (126 MHz, CDCl.sub.3): 173.74 (d, J=5.2 Hz, C═O), 150.70 (d, J=9.1 Hz), 137.35, 135.40 (d, J=6.7 Hz), 129.79 (d, J=6.1 Hz), 128.79 (d, J=2.8 Hz), 128.64 (d, J=6.4 Hz), 128.33, 120.86 (d, J=4.6 Hz), 120.68 (d, J=4.7 Hz), 115.55, 67.31 (d, J=3.0 Hz, CH.sub.2—O), 49.72 (d, J=11.1 Hz, CH—NH), 27.88 (d, J=130.7 Hz, CH.sub.2—P), 27.57 (d, J=131.3 Hz, CH.sub.2—P), 26.74 (d, J=4.3 Hz, CH.sub.2—CH.sub.2—P), 26.57 (d, J=4.1 Hz, CH.sub.2—CH.sub.2—P), 21.70 (2 d, J=4.4 Hz, CHCH.sub.3).

[0110] General procedure 2. Synthesis of phosphonoamidates 4a-d through Hoveyda-Grubbs cross metathesis. To a solution of allylphosphonoamidates 3a-d (1 eq.) and 2-methyl-2-propen-1-ol (85 μL, 1 mmol, 2 eq.), 1,4-benzoquinone (5.40 mg, 10 mol %) in dry DCM (10 mL) was added Hoveyda-Grubbs catalyst 2.sup.nd generation (23.5 mg, 0.038 mmol, 7.5 mol %). The catalyst was added in three equal portions of 2.5 mol % (7.8 mg, 0.013 mmol) at t=0, 2 and 4 h over the course of the reaction. The solution was then heated to reflux at 45° C. under nitrogen atmosphere for 18 h. After cooling to room temperature, a scoop of activated carbon was added, and the mixture stirred for another 2 hr then filtered through a Celite pad. Volatiles were evaporated and the residue was purified by extensive silica gel column chromatography (Hexane/EtOAc, 7:3 to 0:10) to give 4a-d as colorless oils.

[0111] Methyl (((E)-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (4a). Synthesised following general procedure 2 using 3a (150 mg, 0.5 mmol, 1 eq.) to give 4a (91 mg, 57%) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 30.89, 31.31. δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.31 (m, 2H, Ar), 7.18 (m, 3H, Ar), 5.48 (m, 1H, ═CH), 4.24-3.98 (m, 3H, CH.sub.2OH, CH—NH), 3.68 (d, J=7.9 Hz, 3H, OCH.sub.3), 3.39-3.18 (m, 1H, NH), 2.55-2.41 (m, 2H, ═CH—CH.sub.2), 2.12-1.87 (m, 2H, CH.sub.2—P), 1.71 (d, J=6.6 Hz, 3H, CH.sub.3(CH.sub.2)C═), 1.27 (2×d, J=7.1 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 176.71 (d, J=5.9 Hz, C═O), 150.72, 129.81 (d, J=6.9 Hz), 124.81 (d, J=8.6 Hz, CH═CH.sub.2), 124.17, 124.04, 120.94, 120.69 (d, J=4.6 Hz), 68.56 (d, J=9.1 Hz, CH.sub.2—OH), 52.64 (d, J=3.5 Hz, CHs-0), 49.51 (d, J=4.1 Hz, CH—NH), 28.33 (d, J=129.6 Hz CH.sub.2—P), 28.06 (d, J=130.2 Hz, CH.sub.2—P), 21.86 (2 d, J=4.9 Hz, CHCH.sub.3), 21.00 (d, J=4.8 Hz, CH.sub.2—CH.sub.2—P), 20.89 (d, J=4.4 Hz, CH.sub.2—CH.sub.2—P), 13.87. HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.16H.sub.24NO.sub.5NaP: 364.1290; found: 364.1293.

[0112] Isopropyl (((E)-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (4b). Synthesised following general procedure 2 using 3b (0.150 g, 0.46 mmol, 1 eq.) to give product 4b (97 mg, 59%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 31.11, 31.49. OH NMR (500 MHz, CDCl.sub.3): 7.28 (m, 2H, Ar), 7.20 (m, 3H, Ar), 5.46 (m, 1H, ═CH), 4.95 (m, 1H, CH-iPr), 4.08-3.79 (m, 3H, CH.sub.2OH, CH—NH), 3.47-3.25 (m, 1H, NH), 2.51-2.35 (m, 2H, ═CH—CH.sub.2), 2.02-1.85 (m, 2H, CH.sub.2—P), 1.73-1.56 (d, J=6.0 Hz, 3H, CH.sub.3(CH.sub.2)C═), 1.35-1.08 (m, 9H, CH.sub.3—CH—NH, CH-iPr). δ.sub.C NMR (126 MHz, CDCl.sub.3): 173.62 (d, J=5.8 Hz, C═O), 150.74 (d, J=9.0 Hz), 136.76, 124.75, 123.95 (d, J=5.4 Hz), 123.84 (d, J=6.9 Hz), 120.90 (d, J=4.5 Hz), 120.67 (d, J=4.6 Hz), 119.83, 115.58, 69.41 (d, J=4.7 Hz, CHiPr) 68.39 (d, J=7.0 Hz, CH.sub.2—OH), 49.65, 28.36 (d, J=129.7 Hz, CH.sub.2—P), 27.92 (d, J=131.1 Hz, CH.sub.2—P), 21.83 (2 d, J=6.2 Hz, CHCH.sub.3), 20.90 (d, J=4.4 Hz, CH.sub.2—CH.sub.2—P), 20.84 (d, J=4.4 Hz, CH.sub.2—CH.sub.2—P), 13.84. HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.18H.sub.28NO.sub.5NaP: 392.1603; found: 392.1613.

[0113] tert-Butyl (((E)-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (4c). Synthesised following general procedure 2 using 3c (0.150 g, 0.44 mmol, 1 eq.) to give product 4c (108 mg, 64%) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 31.05, 31.42. δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.30 (m, 2H, Ar), 7.17 (m, 3H, Ar), 5.46 (m, 1H, ═CH), 4.17-3.78 (m, 3H, CH.sub.2OH, CH—NH), 3.41-3.18 (m, 1H, NH), 2.60-2.30 (m, 2H, ═CH—CH.sub.2), 2.01-1.87 (m, 2H, CH.sub.2—P), 1.69 (d, J=6.8 Hz, 3H, CH.sub.3(CH.sub.2)C═), 1.42 (s, 9H, tBu), 1.27 (2 d, J=7.9 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 173.34 (d, J=6.0 Hz, C═O), 150.79 (d, J=9.0 Hz), 136.81 (d, J=9.6 Hz), 115.59, 82.22 (d, J=5.6 Hz), 68.45 (d, J=10.4 Hz, CH.sub.2—OH), 50.07 (d, J=4.5 Hz, CH—NH), 28.01 (d, J=129.7 Hz, CH.sub.2—P), 27.26 (d, J=130.4 Hz, CH.sub.2—P), 21.98 (d, J=3.8 Hz, CHCH.sub.3), 20.97 (d, J=4.7 Hz, CH.sub.2—CH.sub.2—P), 20.88 (d, J=4.4 Hz, CH.sub.2—CH.sub.2—P), 13.86 (CH.sub.3). HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.19H.sub.30NO.sub.5NaP: 406.1759; found: 406.1762.

[0114] Benzyl ME)-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (4d). Synthesised following general procedure 2 using 3d (0.150 g, 0.4 mmol, 1 eq.) to give product 4d (100 mg, 59%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 30.90, 31.32, 6H NMR (500 MHz, CDCl.sub.3): 7.39-7.28 (m, 7H, Ar), 7.22-7.17 (m, 2H, Ar), 7.12 (m, 1H, Ar), 5.44 (m, 1H, ═CH), 5.09 (m, 2H, OCH.sub.2), 4.22-3.86 (m, 3H, m, 3H, CH.sub.2OH, CH—NH), 3.49-3.17 (m, 1H, NH), 2.64-2.36 (m, 2H, ═CH—CH.sub.2), 2.04-1.80 (m, 2H, CH.sub.2—P), 1.69 (d, J=6.8 Hz, 3H, CH.sub.3(CH.sub.2)C═), 1.33 (2 d, J=7.0 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 176.76, 129.81 (d, J=7.4 Hz), 128.75 (d, J=14.9 Hz), 128.39, 124.77, 124.15 (d, J=15.0 Hz), 120.93-120.84, 120.67 (d, J=4.7 Hz), 68.57 (d, J=9.6 Hz), 67.42 (d, J=2.9 Hz), 49.65, 28.05 (d, J=129.9 Hz), 21.77 (d, J=4.1 Hz), 20.87 (d, J=4.4 Hz), 13.87. HRMS (ES+, m/z) calcd. for (M+Na)+C.sub.22H.sub.28NO.sub.5NaP: 440.1603; found: 440.1609.

[0115] Diethyl (1,1-difluorobut-3-en-1-yl)phosphonate (6)..sup.10 Anhydrous DMF (20 mL) was added to a 250 mL round bottom flask containing activated zinc powder (2.50 g, 38.23 mmol, 1 eq.) under nitrogen. This was followed by slow dropwise addition of diethyl (bromodifluoromethyl)phosphonate (6.80 mL, 38.23 mmol, 1 eq.) and the mixture was stirred for 3 h at room temperature. CuBr (5.48 g, 38.23 mmol, 1 eq.) was added followed by slow dropwise addition of allyl bromide (3.96 mL, 45.87 mmol, 1.2 eq.) to prevent exothermic reaction. After stirring for 40 h, the mixture was filtered and then partitioned between DCM and 10% aqueous NH.sub.4Cl. The aqueous phase was extracted three times with DCM. The combined organic phases were dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure and the obtained residue was purified by column chromatography using 20% EtOAc in hexane to give 6 (5.41 g, 62%) as a pale-yellow oil. Op NMR (202 MHz, CDCl.sub.3): 6.93 (t, J=107.4 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 5.84 (m, 1H, ═CH), 5.26 (m, 2H, CH.sub.2═), 4.32-4.21 (m, 4H, 2×OCH.sub.2CH.sub.3), 2.82 (m, 2H, ═CH—CH.sub.2), 1.37 (t, J=7.1 Hz, 6H, 2×OCH.sub.2CH.sub.3).

[0116] (1,1-Difluorobut-3-en-1-yl)phosphonic dichloride (7). Synthesised as described for 2 using 6 (2.5 g, 10.95 mmol, 1 eq.) to give the crude product 7 (2.28 g, 100%) as a brown liquid which was used in the next step without further purification. δ.sub.P NMR (202 MHz, CDCl.sub.3): 31.56 (t, J=138.8 Hz).

[0117] Methyl ((1,1-difluorobut-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (8a). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine methyl ester hydrogen chloride (0.261 g, 1.87 mmol, 1 eq.) to give product 8a (0.150 g, 24%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 9.05 (dd, J=101.1, 49.9), 8.41 (dd, J=100.0, 51.1 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.35 (m, 2H, Ar), 7.21 (m, 3H, Ar), 5.89 (m, 1H, CH.sub.2═CH), 5.29 (m, CH.sub.2═CH), 4.14 (m, 1H, CH—NH), 3.69 (d, J=6.6 Hz, 3H, OCH.sub.3), 3.64 (m, 1H, NH), 3.13-2.84 (m, 2H, ═CH—CH.sub.2), 1.38 (2×d, 7.1 Hz, 3H, CH—CH). δ.sub.C NMR (126 MHz, CDCl.sub.3): 173.76 (d, J=4.1 Hz, C═O), 130.02 (d, J=4.1 Hz), 127.40-127.07 (m), 125.64, 121.54 (d, J=9.3 Hz), 120.54 (t, J=4.8 Hz), 52.69, 50.09 (d, J=6.6 Hz), 39.39-37.22 (m), 21.74 (2d, J=3.5 Hz, CH.sub.3).

[0118] Isopropyl ((1,1-difluorobut-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (8b). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine isopropyl ester hydrogen chloride (0.314 g, 1.87 mmol, 1 eq.) to give product 8b (0.165 g, 25%) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 9.17 (dd, J=101.1, 30.3 Hz), 8.46 (dd, J=99.9, 38.0 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3). 7.34 (m, 2H, Ar), 7.20 (m, 3H, Ar), 5.89 (m, 1H, CH.sub.2═CH), 5.29 (m, 2H, CH.sub.2═CH), 4.99 (m, 1H, CH-iPr), 4.14-3.99 (m, 1H, CH—NH), 3.68 (m, 1H, NH), 3.00-2.89 (m, 2H, ═CH—CH.sub.2), 1.35-1.17 (m, 9H, CH.sub.3—CH—NH, CH-iPr). δ.sub.C NMR (126 MHz, CDCl.sub.3): 172.68 (d, J=5.9 Hz, C═O), 149.46, 129.88 (d, J=4.2 Hz), 127.11 (d, J=5.4 Hz), 125.46, 121.36 (d, J=8.9 Hz), 120.40 (t, J=4.9 Hz), 69.39 (d, J=2.8 Hz, CHiPr), 50.15, 38.63-37.99 (m), 21.58 (d, J=1.6 Hz), 21.44 (2 d, J=3.2 Hz, CHCH.sub.3).

[0119] tert-Butyl ((1,1-difluorobut-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (8c). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine tertbutyl ester hydrogen chloride (0.340 g, 1.87 mmol, 1 eq.) to give product 8c (0.320 g, 46%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 9.23 (dd, J=101.0, 34.4 Hz), 8.52 (dd, J=99.6, 36.0 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.34 (m, 2H, Ar), 7.21 (m, 3H, Ar), 5.89 (m, 1H, CH.sub.2═CH), 5.28 (m, 2H, CH.sub.2═CH), 4.08-3.98 (m, 1H, CH—NH), 3.67 (m, 1H, NH), 3.01-2.84 (m, 2H, ═CH—CH.sub.2), 1.42 (d, J=5.2 Hz, 9H, tBu), 1.30 (d, J=7.2 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 172.48 (d, J=4.5 Hz, C═O), 149.72, 129.99 (d, J=2.1 Hz), 128.59-126.95 (m), 125.54, 121.46 (d, J=9.2 Hz), 120.54 (t, J=4.8 Hz), 82.32 (d, J=5.8 Hz), 50.69 (d, J=2.8 Hz, CH—NH), 38.80-38.12 (m), 27.99, 21.85 (2 d, J=3.1 Hz, CHCH.sub.3).

[0120] Benzyl ((1,1-difluorobut-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (8d). Synthesised following general procedure 1 using phenol (0.210 g, 2.23 mmol, 1 eq.) and L-alanine benzyl ester hydrogen chloride (0.403 g, 1.87 mmol, 1 eq.) to give product 8d (0.350 g, 45% yield) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 9.08 (dd, J=101.2, 45.4 Hz), 8.39 (dd, J=100.1, 46.5 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.41-7.29 (m, 7H, Ar), 7.25-7.17 (m, 3H, Ar), 5.87 (m, 1H, CH.sub.2═CH), 5.27 (m, 2H, CH.sub.2═CH), 5.11 (m, 2H, —OCH.sub.2), 4.33-4.09 (m, 1H, CH—NH), 3.68 (m, 1H, NH), 2.93 (m, 2H, P—CH.sub.2), 1.36 (d, J=7.2 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 173.15 (d, J=3.2 Hz, C═O), 149.68, 135.26 (d, J=6.4 Hz), 130.01 (d, J=4.1 Hz), 128.87-128.52 (m), 128.35 (d, J=1.1 Hz), 127.38-127.03 (m), 125.62, 122.24-120.38 (m), 120.51, 67.49, 50.20, 38.74-38.07 (m), 21.96 (2 d, J=3.5 Hz, CHCH.sub.3).

[0121] Methyl (((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (9a). Synthesised following general procedure 2 using 8a (0.150 g, 0.45 mmol, 1 eq.) to give product 9a (97 mg, 58%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3) δ 9.27 (dd, J=112.1, 18.4 Hz), 8.44 (dd, J=126.4, 20.2 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.35 (m, 2H, Ar), 7.21 (m, 3H, Ar), 5.56 (m, 1H, ═CH), 4.18 (m, 1H, CH—NH), 4.07 (m, 2H, ═CH—CH.sub.2), 3.75-3.46 (m, 4H, NH, OCH.sub.3), 3.10-2.79 (m, 2H, ═CH—CH.sub.2), 1.83 (m, 1H, OH), 1.76 (s, 3H, CH.sub.3(CH.sub.2)C═CH), 1.37 (2×d, J=7.1 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 176.76, 141.38 (d, J=7.6 Hz), 130.04 (d, J=3.2 Hz), 125.65 (d, J=4.0 Hz), 120.56 (d, J=4.6 Hz), 120.47 (d, J=4.7 Hz), 68.25 (d, J=1.9 Hz), 52.80 (CH.sub.3—O), 50.09 (d, J=4.1 Hz, CH—NH), 32.82, 21.82 (2 d, J=2.6 Hz, CHCH.sub.3), 14.15 (CH.sub.3). HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.16H.sub.22F.sub.2NO.sub.5NaP: 400.1101; found: 400.1109.

[0122] Isopropyl (((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (9b). Synthesised following general procedure 2 using 8b (0.150 g, 0.41 mmol, 1 eq.) to give product 9b (117 mg, 69%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 9.30 (dd, J=109.1, 34.4 Hz), 8.42 (dd, J=111.1, 38.4 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.35 (m, 2H, Ar), 7.21 (m, 3H, Ar), 5.56 (m, 1H, ═CH), 5.00 (m, 1H, CH-iPr), 4.17-3.91 (m, 3H, CH—NH, ═CH—CH.sub.2), 3.67 (m, 1H, NH), 3.08-2.75 (m, 2H, ═CH—CH.sub.2), 1.86 (m, 1H, OH), 1.70 (s, 3H, CH.sub.3(CH.sub.2)C═CH), 1.34-1.19 (m, 9H, CH.sub.3—CH—NH, CH-iPr). 6c NMR (126 MHz, CDCl.sub.3): 173.09 (d, J=6.8 Hz, C═O), 141.37 (d, J=7.9 Hz), 130.03 (d, J=2.6 Hz), 125.60 (d, J=4.4 Hz), 120.50 (dd, J=10.6, 4.7 Hz), 113.79-113.43 (m), 69.71 (d, J=4.5 Hz), 68.25, 50.29 (d, J=9.6 Hz), 30.88-28.72 (m), 21.73 (d, J=8.2 Hz), 21.84 (2d, J=4.1 Hz, CHCH.sub.3), 14.18 (CH.sub.3). HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.18H.sub.26F.sub.2NO.sub.5NaP: 428.1414; found: 428.1414.

[0123] tert-Butyl (((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (9c). Synthesised following general procedure 2 using 8c (0.150 g, 0.39 mmol, 1 eq.) to give product 9c (105 mg, 63%) as a colorless oil. δ.sub.P NMR (202 MHz, CDCl.sub.3): 9.36 (dd, J=109.1, 34.4 Hz), 8.48 (dd, J=111.1, 36.4 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.36 (m, 2H, Ar), 7.22 (m, 3H, Ar), 5.56 (m, 1H, ═CH), 4.16-3.93 (m, 3H, CH—NH, ═CH—CH.sub.2), 3.63 (m, 1H, NH), 3.08-2.77 (m, 2H, ═CH—CH.sub.2), 1.90 (m, 1H, OH), 1.71 (s, 3H, CH.sub.3(CH.sub.2)C═CH), 1.43 (d, J=5.2 Hz, 9H, t Bu), 1.30 (2×d, J=7.1 Hz, 3H, CH—CH.sub.3). δ.sub.C NMR (126 MHz, CDCl.sub.3): 172.79 (d, J=6.8 Hz, C═O), 130.02, 125.56 (d, J=5.8 Hz), 120.55 (d, J=4.6 Hz), 120.47 (d, J=4.4 Hz), 82.59, 68.26, 50.72 (d, J=7.4 Hz, CH—NH), 32.97-32.80 (m), 28.02 (d, J=1.1 Hz, 3×CH.sub.3), 21.98 (2 d, J=3.8 Hz, CHCH.sub.3), 14.16. HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.19H.sub.28F.sub.2NO.sub.5NaP: 442.1571; found: 442.1578.

[0124] Benzyl WE)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)-L-alaninate (9d). Synthesised following general procedure 2 using 8d (0.150 g, 0.36 mmol, 1 eq.) to give product 9d (101 mg, 61%) as a colorless oil. Op NMR (202 MHz, CDCl.sub.3): 9.21 (dd, J=113.2, 22.3 Hz), 8.37 (dd, J=111.1, 24.3 Hz). δ.sub.H NMR (500 MHz, CDCl.sub.3): 7.39-7.28 (m, 7H, Ar), 7.24-7.16 (m, 3H, Ar), 5.55 (m, 1H, ═CH), 5.13 (m, 2H, OCH.sub.2), 4.27-4.14 (m, 1H, CH—NH), 4.06 (m, 2H, ═CH—CH.sub.2), 3.68 (m, 1H, NH), 3.05-2.73 (m, 2H, ═CH—CH.sub.2), 1.80 (m, 1H, OH), 1.69 (s, 3H, CH.sub.3(CH.sub.2)C═CH), 1.35 (2×d, J=7.1 Hz, 3H, CH—CH.sub.3). δ.sub.C (126 MHz, CDCl.sub.3): 173.38 (d, J=7.9 Hz, C═O), 149.71, 141.37 (d, J=8.3 Hz), 130.04 (d, J=3.1 Hz), 128.76 (d, J=12.8 Hz), 128.35 (s), 125.63 (d, J=5.5 Hz), 120.49 (dd, J=13.7, 4.6 Hz), 113.75-113.34 (m), 68.25 (d, J=2.4 Hz), 67.61, 50.22 (d, J=8.0 Hz), 34.32-31.18 (m), 21.84 (d, J=3.8 Hz, CHCH.sub.3), 14.15 (CH.sub.3). HRMS (ES+, m/z) calcd. for (M+Na)+ C.sub.22H.sub.26F.sub.2NO.sub.5NaP: 476.1414; found: 476.1421.

[0125] Cell Isolation

[0126] Blood was obtained in the presence of a mixture of Heparin and Ethylenediaminetetraacetic acid (EDTA) as anticoagulants (2 U/ml heparin, 1.5 mM EDTA) from consented healthy donors (approved by the NRES Committee West Midlands ethical board; REC reference 14/WM/1254). Blood was then layered on a density gradient medium, lymphoprep (Stem Cell Technologies) and Peripheral blood mononuclear cells (PBMCs) were purified by gradient centrifugation. The cells were washed 2 times with Phosphate Buffered Saline (PBS), then resuspended in RPMI-1640 media supplemented with 2 mM L-glutamine, 25 mM HEPES, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin (Invitrogen) and 10% foetal calf serum.

[0127] Flow Cytometric Analysis

[0128] Untreated and treated PBMCs were labelled with Zombie aqua viability dye (Biolegend) and subsequently were stained with a mixture of BV421-conjugated anti-CD3 (UCHT1, Biolegend), BV650-conjugated anti-CD8 (SK1; BD Bioscience), FITC-conjugated anti-CD25 (M-A25, Biolegend), PE-conjugated anti-CD69 (TP1.55.3; Beckman Coulter) and PE Cy5-conjugated anti-Vγ9 TCR (IMMU360, Beckman Coulter) and APC-conjugated anti-Vδ2 TCR (123R3, Miltenyi Biotech) antibodies. The percentages of CD69.sup.+CD25.sup.+ within CD8.sup.+ T cell subset or Vγ9Vδ2 T-cell population were measured using flow cytometer. Data were analysed using FlowJo V10 software.

[0129] Cytotoxicity Assay

[0130] Vγ9Vδ2 T cells were expanded from PBMCs in the presence of 5 μM zoledronate for 14 days and 100 U/ml IL-2 (Peprotech) was added into the media every 2-3 days, yielding ˜85% Vγ9Vδ2 T cells. Bladder carcinoma cell line, T24 (ATCC HTB4) were labelled with 0.1 μM CFSE and incubated for 4 hours with 10 μM zoledronate, 100 μM of HMBPP or 100 μM of the indicated prodrugs, before being washed five-times in medium and co-cultured with previously expanded Vγ9Vδ2 T cells in an effector target ratio of 10:1 for 18 hours. All cells were then labelled with eFluor780 viability dye and CFSE.sup.+ eFluor780 viability dye.sup.+ cells were measured using flow cytometry. Data were analysed using FlowJo V10.

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