BIOORTHOGONAL COMPOUNDS COMPRISING A PROPARGYL GROUP FOR TREATING CANCER

Abstract

A method of preparing an active agent or a salt thereof from a pro-drug first compound (1) comprising a propargyl group connected to an oxygen that is directly or indirectly connected to the active agent is provided, wherein the bond between the propargyl group and the oxygen is cleaved by reacting the first compound with palladium or gold, thereby releasing the active agent. Prodrug compositions suitable for use in the method are also provided.

Claims

1. A method of preparing an active agent or a salt thereof, the method comprising the steps: a) providing a first compound defined according to formula (1): ##STR00053## and b) cleaving the bond (*) between the oxygen and the propargyl group under biologically compatible conditions by reacting the first compound with palladium or gold; wherein R.sup.1 and R.sup.2 are independently selected from the group consisting of H, optionally substituted C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.3-C.sub.10 cycloalkyl, optionally substituted C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.3-C.sub.10 cycloalkenyl, optionally substituted C.sub.2-C.sub.10 alkynyl, optionally substituted C.sub.2-C.sub.10 heteroalkyl, optionally substituted C.sub.3-C.sub.10 heterocycloalkyl, optionally substituted C.sub.2-C.sub.10 heteroalkenyl, optionally substituted C.sub.3-C.sub.10 heterocycloalkenyl, optionally substituted C.sub.2-C.sub.10 heteroalkynyl, optionally substituted C.sub.6-C.sub.14 aryl, optionally substituted C.sub.5-C.sub.14 heteroaryl, wherein XO comprises at least one aryl group or heteroaryl group directly connected to the oxygen (O) of the XO substituent, and comprises the active agent or a salt thereof, and optionally comprises a linker between the oxygen and the active agent.

2. The method of claim 1, wherein the XO group comprises a derivative of the active agent.

3. (canceled)

4. The method of claim 1, wherein the method is performed in a biological environment, such as in a cell, a tissue and/or a subject using a suitable palladium source and/or a suitable gold source.

5. The method of claim 1, wherein R.sup.1 and R.sup.2 are independently selected from the group consisting of H, optionally substituted C.sub.1-C.sub.5 alkyl, optionally substituted C.sub.3-C.sub.6 cycloalkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.3-C.sub.6 cycloalkenyl, optionally substituted C.sub.2-C.sub.5 alkynyl, optionally substituted C.sub.2-C.sub.5 heteroalkyl, optionally substituted C.sub.3-C.sub.6 heterocycloalkyl, optionally substituted C.sub.2-C.sub.5 heteroalkenyl, optionally substituted C.sub.3-C.sub.6 heterocycloalkenyl, optionally substituted C.sub.2-C.sub.5 heteroalkynyl, optionally substituted C.sub.6-C.sub.12 aryl, optionally substituted C.sub.5-C.sub.11 heteroaryl.

6. (canceled)

7. The method of claim 1, wherein the first compound has a general formula selected from the group comprising: ##STR00054## where the XO group comprises an active agent and a linker, the active agent may be connected to the linker via an amine, hydroxyl, hydroxamic acid or carbonyl group of the active agent.

8. The method of claim 1, wherein the active agent contains a hydroxamic acid group connected to the propargyl group directly or via a linker.

9. The method of claim 8, wherein the active agent is vorinostat, belinostat, panobinostat, or derivatives thereof.

10. The method of claim 1, wherein the active agent contains one or more primary or secondary amino groups connected to the oxypropargyl group directly or via a linker.

11. The method of claim 10, wherein the active agent is doxorubicin, gemcitabine, histamine, mitoxantrone, panobinostat, hydroxyurea, paclitaxel, phosphoramide mustard, procarbazine, 5-(monomethyl triazine)-imidazole-4-carboxamide, dasatinib, erlotinib, bosutinib, gefitinib, lapatinib, vandetanib, pazopanib, crizotinib, ceritinib, afatinib, ibrutinib, dabrafenib, trametinib, palbociclib, spanisertib or derivatives thereof.

12. The method of claim 1, wherein the active agent comprises a phenolic OH connected to the oxypropargyl group directly or via a linker, including the equivalent lactam tautomers.

13. The method of claim 12, wherein the active agent is 5-fluorouracil (5-FU or 5FU), floxuridine, olaparib, permetrexed, sunitinib, nintedanib, doxorubicin, mitoxantrone, 4-hydroxytamoxifen, etoposide, duocarmycin or derivatives thereof.

14. The method of claim 1, wherein the compound according to formula (1) is selected from the following group: ##STR00055## ##STR00056## ##STR00057## ##STR00058##

15. A first compound according to the general formula (1): ##STR00059## wherein R1 and R2 are independently selected from the group consisting of H, optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C2-C10 alkenyl, optionally substituted C3-C10 cycloalkenyl, optionally substituted C2-C10 alkynyl, optionally substituted C2-C10 heteroalkyl, optionally substituted C3-C10 heterocycloalkyl, optionally substituted C2-C10 heteroalkenyl, optionally substituted C3-C10 heterocycloalkenyl, optionally substituted C2-C10 heteroalkynyl, optionally substituted C6-C14 aryl, optionally substituted C5-C14 heteroaryl, wherein XO comprises at least one aryl group or heteroaryl group directly connected to the oxygen (O) of the XO substituent, and comprises an active agent or a salt thereof, and optionally comprises a linker between the oxygen and the active agent; wherein the carbon-oxygen bond (*) is cleaved under biological conditions to release the active agent when the compound of formula (1) is reacted with palladium or gold.

16. The first compound of claim 15, wherein R1 and R2 are independently selected from the group consisting of H, optionally substituted C1-C5 alkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C3-C6 cycloalkenyl, optionally substituted C2-C5 alkynyl, optionally substituted C2-C5 heteroalkyl, optionally substituted C3-C6 heterocycloalkyl, optionally substituted C2-C5 heteroalkenyl, optionally substituted C3-C6 heterocycloalkenyl, optionally substituted C2-C5 heteroalkynyl, optionally substituted C6-C12 aryl, optionally substituted C5-C11 heteroaryl.

17. The first compound of claim 15, wherein the first compound has a general formula selected from the group comprising: ##STR00060## where the XO group comprises an active agent and a linker, the active agent may be connected to the linker via an amine, hydroxyl or carbonyl group of the active agent.

18. The first compound of claim 15, wherein the linker is selected from the group ##STR00061## ##STR00062## wherein Z.sup.1 and Z.sup.2 are independently selected from N, CH, C; Y.sup.1 and Y.sup.2 are independently selected from H, NO.sub.2, halogen, COOR.sup.3, OR.sup.4; R.sup.3 and R.sup.4 are independently selected from the group consisting of H, optionally substituted C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.3-C.sub.10 cycloalkyl, optionally substituted C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.3-C.sub.10 cycloalkenyl, optionally substituted C.sub.2-C.sub.10 alkynyl, optionally substituted C.sub.2-C.sub.10 heteroalkyl, optionally substituted C.sub.3-C.sub.10 heterocycloalkyl, optionally substituted C.sub.2-C.sub.10 heteroalkenyl, optionally substituted C.sub.3-C.sub.10 heterocycloalkenyl, optionally substituted C.sub.2-C.sub.10 heteroalkynyl, optionally substituted C.sub.6-C.sub.14 aryl, optionally substituted C.sub.5-C.sub.14 heteroaryl; and n is 1-10, preferably 1, 2 or 3.

19. The first compound according to claim 1 selected from the following group: ##STR00063## ##STR00064## ##STR00065## ##STR00066##

20. A method of treatment of disease by inserting an implant that comprises palladium and/or gold in a target area to be treated, and then delivering the first composition according to claim 15 to the target area, optionally wherein the disease is cancer.

21-23. (canceled)

24. An implant comprising palladium and/or gold for use in a method of treatment, wherein the method comprises administering a first compound or salt according to claim 1 or a pharmaceutically acceptable salt thereof and the implant to the subject.

25. The implant of claim 24 comprising palladium in particulate form and/or gold in particulate form embedded in a matrix.

26-31. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0203] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.

[0204] FIG. 1 shows the relationship between active agent/prodrug concentrations for Vorinostat and Vorinostat prodrugs and cell viability (%) for lung cancer A549 cells (FIG. 1A), glioblastoma U87G cells (FIG. 1B) and glioblastoma T98 cells (FIG. 1C) and the respective calculated EC.sub.50 values.

[0205] FIG. 2 shows High Performance Liquid Chromatography (HPLC) chromatograms (UV detector 254 nm) of Pd.sup.0-mediated conversion of propargyloxybenzyl-Vorinostat (POB-Vor) into Vorinostat performed in phosphate buffered saline (PBS, pH=7.3) and incubated for 24 h at 37 C. (Thermomixer, shaker speed: 1,200 rpm). The HPLC chromatograms for the conversion of POB-Vor to Vorinostat at 0 h(A), 3 h(B) and 6 h(C).

[0206] FIG. 3 shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against A549 cells of prodrug-palladium combinations for POB-Vor and Benzyl-Vor compared to Vorinostat.

[0207] FIG. 4 shows the dose dependent toxicology data (bar graph) indicated by % cell viability (A549 cells in FIG. 4A, U87G cells in FIG. 4B, and T98 cells in FIG. 4C) for conversion of POB-Vor into Vorinostat using extracellular palladium resins.

[0208] FIG. 5 shows the phase-contrast images of A549 cells after 5 days of treatment (120 h). Cell proliferation was monitored using the high-content live-cell imaging system Incucyte (Essen BioScience) placed in an incubator (5% CO.sub.2, 37 C.). POB-Vor and Vorinostat were used at 100 M.

[0209] FIG. 6 shows the relationship between active agent/prodrug concentrations for Doxorubicin and Doxorubicin prodrugs and cell viability (%) for lung cancer A549 cells (FIG. 6A), prostate cancer DU145 cells (FIG. 6B) and glioblastoma T98 cells (FIG. 6C) and the respective calculated EC.sub.50 values.

[0210] FIG. 7 shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against A549 cells (FIG. 7A), DU145 cells (FIG. 7B) and T98 cells (FIG. 7C) of prodrug-palladium combinations for oPOBC-Dox, pPOBC-Dox or Cbz-Dox compared to Doxorubicin.

[0211] FIG. 8 shows the dose dependent toxicology data (bar graph) indicated by % cell viability (A549 cells in FIG. 8A, DU145 cells in FIG. 8B and T98 cells in FIG. 8C) for conversion of Doxorubicin prodrugs into Doxorubicin using extracellular palladium resins.

[0212] FIG. 9 shows the relationship between active agent/prodrug concentrations for Gemcitabine and Gemcitabine prodrugs and cell viability (%) for pancreatic cancer MiaPaCa2 cells and the respective calculated EC.sub.50 values.

[0213] FIG. 10 shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against MiaPaCa2 cells of prodrug-palladium combinations for pPOBC-Gem or Cbz-Gem compared to Gemcitabine.

[0214] FIG. 11 shows the dose dependent toxicology data (bar graph) indicated by % cell viability (MiaPaCa2 cells) for conversion of pPOBC-Gem into Gemcitabine using extracellular palladium resins.

[0215] FIG. 12 shows the results of Ninhydrin test after incubation of histamine dihydrochloride (Hist), oPOBC-Hist and pPOBC-Hist in PBS with Pd.sup.0-functionalized resin at 37 C. for 24 h (Thermomixer, shaker speed: 1,200 rpm).

[0216] FIG. 13 shows Liquid Chromatography-Mass Spectroscopy (LCMS) chromatograms (microTOF II detector) of oPOBC-Hist incubated with Pd.sup.0-resins in PBS at 37 C. for 24 h (Thermomixer, shaker speed: 1,200 rpm). FIGS. 13A, 13B and 13C show the chromatograms at 0 h, 3 h and 6 h, respectively.

[0217] FIG. 14 shows LCMS chromatograms (microTOF II detector) of pPOBC-Hist incubated with Pd.sup.0-resins in PBS at 37 C. for 24 h (Thermomixer, shaker speed: 1,200 rpm). FIGS. 14A, 14B and 14C show the chromatograms at 0 h, 3 h and 6 h, respectively.

[0218] FIG. 15 shows the relationship between active agent/prodrug concentrations for 5-FU and 5-FU prodrug and cell viability (%) for pancreatic BxPC3 (FIG. 15A) and colorectal HCT116 cells (FIG. 15B) and the respective calculated EC.sub.50 values.

[0219] FIG. 16 shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against BxPC3 cells (FIG. 16A) and HCT116 cells (FIG. 16B) of prodrug-palladium combinations for bis-Pro-5-FU compared to 5-FU.

[0220] FIG. 17 shows the dose dependent toxicology data (bar graph) indicated by % cell viability (BxPC3 cells in FIG. 17A, HCT116 cells in FIG. 17B) for conversion of bis-Pro-5-FU into 5-FU using extracellular palladium resins.

[0221] FIG. 18 shows the relationship between drug/prodrug concentrations for Olaparib and Olaparib prodrug and cell viability (%) for ovarian A2780 cells and the respective calculated EC.sub.50 values.

[0222] FIG. 19 shows the relationship between active agent/prodrug concentrations for Panobinostat prodrug and cell viability (%) for lung cancer A549 cells (FIG. 19A) and the respective calculated EC.sub.50 values. FIG. 19B shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against A549 cells of prodrug-palladium combinations for POB-Panob compared to Panobinostat.

[0223] FIG. 20 shows the relationship between active agent/prodrug concentrations for SN-38 prodrug and cell viability (%) for glioblastoma U87G cells (FIG. 20A) and the respective calculated EC.sub.50 values. FIG. 20B shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against glioblastoma U87G cells of prodrug-palladium combinations for di-oPOB-SN-38 compared to SN-38.

[0224] FIG. 21 shows the relationship between active agent/prodrug concentrations for the Etoposide prodrug and cell viability (%) for glioblastoma U87G cells (FIG. 20A) and the respective calculated EC.sub.50 values.

[0225] FIG. 22 shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against A549 cells of prodrug and gold or palladium-gold resins combinations for POB-Vor compared to Vorinostat.

[0226] FIG. 23 shows the results of a BOOM conversion study showing relative toxicities (as indicated by % cell viability) against A549 cells of prodrug and gold or palladium-gold resins combinations for POB-Panob compared to Panobinostat.

DETAILED DESCRIPTION

[0227] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0228] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as a, an and the are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

##STR00024## ##STR00025## ##STR00026##

[0229] Hydroxamic AcidsVorinostat (Class II)

[0230] Cell viabilities for each prodrug provided as a combination with palladium is presented in FIG. 3 and gold is presented in FIG. 33 in the right of each set of two bars. Data for prodrug in the absence of palladium or gold catalyst is also provided (the left bar of each set of two bars) for comparison along with negative controls (from left to right: DMSO, Pd.sup.0 or Au and Vorinostat). Cells were incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0, Au, or Pd/Au-resins (1 mg/mL, negative control); 100 M of each prodrug (negative control); and Pd.sup.0, Au, or Pd/Au-resins (1 mg/mL)+100 M of each prodrug (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0231] Following 5 days treatment, cells were incubated with PrestoBlue Cell Viability Reagent (Life Technologies) for 60-90 min. Fluorescence intensity values were related to the untreated cells (100% cell viability). Data are provided in FIG. 4 for cells incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (0.8 mg/mL for U87G cells and 1 mg/mL for A549 and T98 cells, negative control); 1-100 M of POB-Vor (negative control); 1-100 M of Vorinostat (positive control); and Pd.sup.0-resin (0.8 mg/mL for U87G cells and 1 mg/mL for A549 and T98 cells)+POB-Vor (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0232] CarbamatesDoxorubicin (Class III)

[0233] Cell viabilities for each prodrug provided as a combination with palladium is presented in FIG. 7 in the right of each set of two bars. Data for prodrug in the absence of palladium catalyst is also provided (the left bar of each set of two bars) for comparison along with negative controls (from left to right: DMSO, Pd.sup.0 and Doxorubicin). Cells were incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 1 M of each prodrug (negative control); and Pd.sup.0-resins (1 mg/mL)+1 M of each prodrug (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0234] Following 5 days treatment, cells were incubated with PrestoBlue Cell Viability Reagent (Life Technologies) for 90 min. Fluorescence intensity values were related to the untreated cells (100% cell viability). Data are provided in FIG. 8 for cells incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 0.3-3 M of Doxorubicin prodrug for A549 and DU145 cell lines, 0.1-1 M of Doxorubicin prodrug for T98 cell line (negative control); 0.3-3 M of Doxorubicin for A549 and DU145 cell lines, 0.1-1 M of Doxorubicin for T98 cell line (positive control); and Pd.sup.0-resin (1 mg/mL)+Doxorubicin prodrug (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0235] CarbamatesGemcitabine (Class III)

[0236] Cell viabilities for each prodrug provided as a combination with palladium is presented in FIG. 10 in the right of each set of two bars. Data for prodrug in the absence of palladium catalyst is also provided (the left bar of each set of two bars) for comparison along with negative controls (from left to right: DMSO, Pd.sup.0 and Gemcitabine). Cells were incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 0.03 M of each prodrug (negative control); and Pd.sup.0-resins (1 mg/mL)+0.03 M of each prodrug (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0237] Following 5 days treatment, cells were incubated with PrestoBlue Cell Viability Reagent (Life Technologies) for 90 min. Fluorescence intensity values were related to the untreated cells (100% cell viability). Data are provided in FIG. 11 for cells incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 0.003-0.3 M of pPOBC-Gem (negative control); 0.003-0.3 M of Gemcitabine (positive control); and Pd.sup.0-resin (1 mg/mL)+pPOBC-Gem (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0238] Prop-O-Drug5-FU (Class I)

[0239] Cell viabilities for each prodrug provided as a combination with palladium is presented in FIG. 16 in the right of each set of two bars. Data for prodrug in the absence of palladium catalyst is also provided (the left bar of each set of two bars) for comparison along with negative controls (from left to right: DMSO, Pd.sup.0 and 5FU). Cells were incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 3 M (for BxPC3 cells) or 30 M (for HCT116 cells) of prodrug (negative control); and Pd.sup.0-resins (1 mg/mL)+3 M (BxPC3) or 30 M (HCT116) of each prodrug (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0240] Following 5 days treatment, cells were incubated with PrestoBlue Cell Viability Reagent (Life Technologies) for 90 min. Fluorescence intensity values were related to the untreated cells (100% cell viability). Data are provided in FIG. 17 for cells incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 0.03-3 M of bis-Pro-5FU for BxPC3, 0.3-30 M of bis-Pro-5FU for HCT116 (negative control); 0.03-3 M of 5FU for BxPC3, 0.3-30 M of 5FU for HCT116 (positive control); and Pd.sup.0-resin (1 mg/mL)+bis-Pro-5FU (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0241] Hydroxamic AcidsPanobinostat (Class II)

[0242] Cell viabilities for each prodrug provided as a combination with palladium is presented in FIG. 21 in the right of each set of two bars. Data for prodrug in the absence of palladium catalyst is also provided (the left bar of each set of two bars) for comparison along with negative controls (from left to right: DMSO, Pd.sup.0 and panobinostat). Cells were incubated in tissue culture media containing 0.1% (v/v) DMSO and: Pd.sup.0-resins (1 mg/mL, negative control); 1 M of each prodrug (negative control); and Pd.sup.0-resins (1 mg/mL)+1 M of each prodrug (BOOM reaction assay). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell control.

[0243] The invention is described in more detail by way of example only with reference to the following Examples.

[0244] General Methods

[0245] Materials Synthesis and Characterization

[0246] Chemicals and solvents were obtained from Fisher Scientific, Sigma-Aldrich or VWR International Ltd. Resins were purchased from Rapp Polymere GmbH and Merck Millipore. NMR spectra were recorded at ambient temperature on a 500 MHz Bruker Avance III spectrometer. Chemical shifts are reported in parts per million (ppm) relative to the solvent peak. High Resolution Mass Spectrometry was measured in a Bruker MicroTOF II. R.sub.f values were determined on Merck TLC Silica gel 60 F254 plates under a 254 nm UV source 0.1% ninhydrin solution in acetone for TLC staining. Purification of compounds was carried out by flash column chromatography using commercially available silica gel (220-440 mesh, Sigma-Aldrich).

[0247] Synthetic Procedure of Au Resins

[0248] TentaGel HL NH.sub.2 resins (250 mg, 0.4-0.6 mmol/g, particle size 110 m or 75 m) were added into a 25 mL Biotage microwave vial and suspended in THF (2.5 mL). A solution of gold(III) chloride hydrate (120 mg, 0.35 mmol) in distilled water (500 L) was basified with a 1 M NaOH aqueous solution (11 L). This freshly prepared solution was immediately added to the suspended resins and heated to 60 C. under stirring for 10 min. The mixture was then stirred at r.t. for additional 2 h. Subsequently, the solvents were filtered and the resins washed with DMF (310 mL), DCM (310 mL) and methanol (310 mL). Tetrakis(hydroxymethyl)phosphonium chloride (THPC) solution 80% in water (93 L) was diluted in distilled water (6 mL) and a 1 M NaOH aqueous solution (11 L) added. This solution was added to the gold(III)-treated resins and bubbled with a N.sub.2 flow at r.t. for 25 min. The solvents were then filtered off and the resins washed with methanol (310 mL) and DCM (310 mL). Resins were then added to a solution of Fmoc-Glu(OH)-OH (64 mg, 0.17 mmol), oxyma (50 mg, 0.35 mmol), N,N-diisopropylcarbodiimide (54 L, 0.35 mmol) and DCM/DMF (3:1, 8 mL) and stirred for 2 h at r.t. The solvents were filtered off and the resins washed with DMF (110 mL), DCM (310 mL) and methanol (310 mL). Finally, resins were dispersed and shaken in a solution of acetic anhydride (60 L) in DCM (10 mL) for 1h at r.t. The solvents were filtered and the resins were washed with DCM (310 mL) and methanol (310 mL). Resins were treated on the wheel overnight with methanol. The solvents were then filtered and resins were dried in an oven at 40 C. for 1 day.

[0249] Synthetic Procedure of AuPd Resins.

[0250] TentaGel HL NH.sub.2 resins (250 mg, 0.4-0.6 mmol/g, particle size 110 m or 75 m) were added into a 25 mL Biotage microwave vial and suspended in toluene (2.5 mL). Palladium(II) acetate (32.8 mg, 0.17 mmol) and gold(III) acetate (62.5 mg, 0.17 mmol) were added into the vial in one portion. The dispersion was immediately heated to 80 C. under stirring for 10 min. The mixture was then stirred at r.t. for additional 2 h. Subsequently, the solvents were filtered and the resins washed with DMF (310 mL), DCM (310 mL) and methanol (310 mL). A solution of 10% hydrazine monohydrate in methanol was added to the resin (5 mL). The suspension was then stirred at r.t. for 25 min. The solvents were then filtered off and the resins washed with methanol (310 mL) and DCM (310 mL). Resins were then added to a solution of Fmoc-Glu(OH)-OH (64 mg, 0.17 mmol), oxyma (50 mg, 0.35 mmol), N,N-diisopropylcarbodiimide (54 L, 0.35 mmol) and DCM/DMF (3:1, 8 mL) and stirred for 2 h at r.t. The solvents were filtered off and the resins washed with DMF (110 mL), DCM (310 mL) and methanol (310 mL). Finally, resins were dispersed and shaken in a solution of acetic anhydride (60 L) in DCM (10 mL) for 1 h at r.t. The solvents were filtered and the resins were washed with DCM (310 mL) and methanol (310 mL). Resins were treated on the wheel overnight with methanol. The solvents were then filtered and resins were dried in an oven at 40 C. for 1 day.

Synthesis of Vorinostat Prodrugs

Synthesis of 4-propargyloxy-benzyl bromide

[0251] 4-propargyloxy-benzyl alcohol was synthesised by propargylation of 4-hydroxylbenzyl alcohol using K.sub.2CO.sub.3 as base (Luo, J. et al. Chem Commun 21, 2136 (2007)). Alcohol was then converted in the corresponding halogenated intermediate using CBr.sub.4/PPh.sub.3 as previously described (Binauld, S. et al. Chem Commun 35, 4138 (2008)).

General Method for the Synthesis of Vorinostat Prodrugs

[0252] Vorinostat (60 mg, 0.23 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.27 mmol) were dissolved in dry acetonitrile (1 mL) under N.sub.2 atmosphere and cooled to 4 C. Either 4-propargyloxy-benzyl or benzyl bromide (0.23 mmol) were dissolved in dry acetonitrile (0.5 mL). The solution was added dropwise to the mixture and the resulting mixture stirred at room temperature for 24 h. Solvent was then removed under reduced pressure and the crude purified via flash chromatography eluting with AcOEt:Hexane (2:1).

Propargyloxybenzyl-Vorinostat (POB-Vor)

[0253] ##STR00027##

[0254] The synthetic method described above using 4-propargyloxy-benzyl bromide gave a white solid (30 mg, 32% yield). .sup.1H NMR (500 MHz, DMSO) 10.86 (s, 1H), 9.82 (s, 1H), 7.58 (d, J=7.7 Hz, 2H), 7.32 (d, J=8.6 Hz, 2H), 7.27 (m, 2H), 7.01 (t, J=7.4 Hz, 1H), 6.98 (d, J=8.6 Hz, 2H), 4.79 (d, J=2.4 Hz, 2H), 4.70 (s, 2H), 3.55 (t, J=2.4 Hz, 1H), 2.28 (t, J=7.4 Hz, 2H), 1.94 (t, J=7.3 Hz, 2H), 1.57 (m, 2H), 1.49 (m, 2H), 1.27 (m, 4H). .sup.13C NMR (126 MHz, DMSO) 171.18 (C), 169.26 (C), 157.19 (C), 139.33 (C), 130.43 (CH), 128.84 (C), 128.60 (CH), 122.88 (CH), 119.01 (CH), 114.56 (CH), 79.19 (C), 78.20 (C), 76.27 (CH.sub.2), 55.36 (CH.sub.2), 36.34 (CH.sub.2), 32.21 (CH.sub.2), 28.33 (d, J=10.4 Hz, CH.sub.2), 24.90 (d, J=17.4 Hz, CH.sub.2). HRMS (ESI) m/z [M+Na].sup.+ calculated for C.sub.24H.sub.28O.sub.4N.sub.2Na, 431.1941; found, 431.1949.

Benzyl-Vorinostat (Benzyl-Vor)

[0255] ##STR00028##

[0256] The synthetic method described above using benzyl bromide gave a white solid (21.5 mg, 27% yield). .sup.1H NMR (500 MHz, DMSO) 10.91 (s, 1H), 9.82 (s, 1H), 7.58 (d, J=7.6 Hz, 2H), 7.38 (d, J=4.4 Hz, 4H), 7.34 (m, 1H), 7.27 (m, 2H), 7.01 (t, J=7.4 Hz, 1H), 4.77 (s, 2H), 2.28 (t, J=7.4 Hz, 2H), 1.94 (t, J=7.3 Hz, 2H), 1.57 (m, 2H), 1.49 (m, 2H), 1.26 (m, 4H). .sup.13C NMR (126 MHz, DMSO) 171.17 (C), 169.32 (C), 139.33 (C), 136.09 (C), 128.66 (d, J=15.7 Hz, CH), 128.20 (d, J=11.8 Hz, CH), 122.87 (CH), 119.00 (CH), 76.71 (CH.sub.2), 36.33 (CH.sub.2), 32.19 (CH.sub.2), 28.31 (d, J=12.5 Hz, CH.sub.2), 24.89 (d, J=18.5 Hz, CH.sub.2). HRMS (ESI) m/z [M+Na].sup.+ calculated for C.sub.21H.sub.26O.sub.3N.sub.2Na, 377.1835; found, 377.1836.

Synthesis of Doxorubicin Prodrugs

Synthesis of Propargyloxy-benzyl Alcohols

[0257] 2-propargyloxy-benzyl alcohol and 4-propargyloxy-benzyl alcohol were synthesised according to literature procedure (Luo, J. et al. Chem Commun 21, 2136 (2007)).

Synthesis of 4-nitrophenyl-carbonate Promoieties

[0258] A solution of 4-nitrophenylchloroformate (0.48 g, 2.4 mmol) in dry DCM (8 mL) was added drop wise to a solution of either 2-propargyloxy-benzyl alcohol, 4-propargyloxy-benzyl alcohol or benzyl alcohol (2.2 mmol) and pyridine (0.19 mL, 2.4 mmol) in DCM (8 mL) at 0 C. under nitrogen in the dark. The mixture was stirred from 0 C. to room temperature overnight with TLC monitoring at t=0 hr, 0.5 hr, 2 hr and 20 hr, indicating full consumption of the alcohol. After concentrating in-vacuo, the crude residue was re-dissolved in ethyl acetate (70 mL), washed with water (250 mL) and brine (250 mL), dried over MgSO.sub.4 and re-concentrated in-vacuo, and the crude was purified via flash chromatography (50% DCM in hexane).

4-nitrophenyl-2-propargyloxy-benzyl Carbonate

[0259] ##STR00029##

[0260] The synthetic method described above using 2-propargyloxy-benzyl alcohol gave an off white oil that solidified to a white solid when below 20 C. (0.52 g, 1.59 mmol, 72% yield); R.sub.f 0.19 (50% DCM in hexane). .sup.1H NMR (500 MHz, CDCl.sub.3) 8.28-8.23 (m, 2H), 7.44-7.36 (m, 4H), 7.08-7.01 (m, 1H), 5.39 (s, 1H), 4.78 (d, J=2.4 Hz, 1H), 2.53 (t, J=2.4 Hz, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) 155.86 (s), 155.75 (s), 152.53 (s), 145.42 (s), 130.57 (d, J=12.8 Hz), 125.35 (s), 123.35 (s), 121.88 (s), 121.65 (s), 112.38 (s), 78.39 (s), 77.36 (s), 75.96 (s), 66.58 (s), 56.26 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.17H.sub.13O.sub.6N.sub.1 [M+Na].sup.+: 350.0635, found: 350.0590

4-nitrophenyl-4-propargyloxy-benzyl Carbonate

[0261] ##STR00030##

[0262] The synthetic method described above using 4-propargyloxy-benzyl alcohol gave an off white oil that solidified to a white solid when below 20 C. (0.54 g, 1.65 mmol, 75% yield); R.sub.f 0.20 (50% DCM in hexane). .sup.1H NMR (500 MHz, CDCl.sub.3) 8.28-8.24 (m, 2H), 7.42-7.38 (m, 2H), 7.38-7.35 (m, 2H), 7.03-6.98 (m, 2H), 5.24 (s, 2H), 4.71 (d, J=2.4 Hz, 2H), 2.53 (t, J=2.4 Hz, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) 158.30 (s), 155.68 (s), 152.57 (s), 145.51 (s), 130.72 (s), 127.39 (s), 125.39 (s), 121.88 (s), 115.27 (s), 78.38 (s), 77.37 (s), 75.90 (s), 70.87 (s), 55.95 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.17H.sub.13O.sub.6N.sub.1 [M+Na].sup.+: 350.0635, found: 350.0646

4-nitrophenyl-benzyl Carbonate

[0263] ##STR00031##

[0264] The synthetic method described above using propargyl alcohol alcohol gave fluffy white crystals (0.41 g, 1.5 mmol, 68% yield); R.sub.f 0.34 (50% DCM in hexane). .sup.1H NMR (500 MHz, CDCl.sub.3) 8.30-8.26 (m, 2H), 7.48-7.36 (m, 7H), 5.30 (s, 2H). .sup.13C NMR (126 MHz, CDCl.sub.3) 155.63 (s), 152.59 (s), 145.49 (s), 134.27 (s), 129.23 (s), 128.95 (s), 128.85 (s), 125.46 (s), 121.93 (s), 77.37 (s), 71.15 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.14H.sub.11O.sub.5N.sub.1 [M+Na].sup.+: 296.0529, found: 296.0507.

General Method for the Synthesis of Doxorubicin Prodrugs

[0265] A solution of the 4-nitrophenyl carbonate moiety (18 mg, 55.2 mol, 1.5 equiv) in anhydrous DMF (2 mL) was flushed with nitrogen after stirring for 10 minutes, then syringed into a flask containing a solution of Doxorubicin-hydrochloride (20 mg, 36.8 mol) and triethylamine (7.5 L, 55.2 mol) in anhydrous DMF under nitrogen at room temperature. The reaction was monitored by TLC (10% Methanol in DCM) at t=0 hr, 0.5 hr, 2 hr and 20 hr, monitoring for formation of product at R.sub.f 0.88 (10% Methanol in DCM). After 20 hr, the reaction was diluted with water (50 mL) and extracted with ethyl acetate (450 mL). The combined organic extracts concentrated down to a volume of -100mL, then washed successively with saturated NaHCO.sub.3 (250 mL), water (250 mL) and brine (250 mL), dried over MgSO4 and concentrated in-vacuo with the water bath kept below 40 C., and the crude was purified via flash chromatography (0.fwdarw.2% Methanol in DCM).

2-proparglyoxybenzylcarbamoyl Doxorubicin (oPOBC-Dox)

[0266] ##STR00032##

[0267] The synthetic method described above using 2-nitrophenyl-4-propargyloxy-benzyl carbonate gave a dark red clumpy powder (12.1 mg, 16.5 mol, 45% yield); R.sub.f 0.25 (2% Methanol in DCM). .sup.1H NMR (500 MHz, CDCl.sub.3) 13.98 (s, 1H), 13.25 (s, 1H), 8.04 (dd, J=7.7, 1.0 Hz, 1H), 7.82-7.75 (m, J=8.4, 7.8 Hz, 1H), 7.39 (dd, J=8.5, 0.7 Hz, 1H), 7.33-7.27 (m, 1H), 7.01-6.93 (m, 2H), 5.51 (d, J=3.9 Hz, 1H), 5.33-5.27 (m, 1H), 5.15-5.08 (m, 3H), 4.76 (s, 2H), 4.70 (s, 2H), 4.55 (s, 1H), 4.14 (dd, J=12.3, 6.2 Hz, 1H), 4.08 (s, 3H), 3.92-3.82 (m, 1H), 3.68 (s, 1H), 3.28 (dd, J=18.8, 1.5 Hz, 1H), 3.03 (d, J=18.8 Hz, 2H), 2.48 (s, 1H), 2.34 (d, J=14.7 Hz, 1H), 2.17 (dd, J=14.7, 4.0 Hz, 1H), 1.88 (dd, J=13.5, 5.0 Hz, 1H), 1.77 (td, J=13.3, 4.2 Hz, 2H), 1.29 (d, J=6.6 Hz, 3H), 1.25 (s, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) 214.02 (s), 187.30 (s), 186.92 (s), 161.24 (s), 156.34 (s), 155.85 (s), 155.67 (d, J=26.0 Hz), 135.92 (s), 135.71 (s), 133.73 (s), 130.02 (s), 129.48 (s), 125.58 (s), 121.63 (s), 121.11 (s), 120.02 (s), 118.61 (s), 112.33 (s), 111.80 (s), 111.62 (s), 111.54-111.45 (m), 100.86 (s), 78.67 (s), 77.37 (s), 76.78 (s), 75.74 (s), 69.78 (s), 69.72 (s), 67.42 (s), 65.70 (s), 62.19 (s), 56.85 (s), 56.28 (s), 47.13 (s), 35.81 (s), 34.21 (s), 30.48-29.67 (m), 17.00 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.38H.sub.37O.sub.14N.sub.1+.sup.23Na.sub.1 754.2106 found 754.2130.

4-proparglyoxybenzylcarbamoyl Doxorubicin (pPOBC-Dox)

[0268] ##STR00033##

[0269] The synthetic method described above using 4-nitrophenyl-4-propargyloxy-benzyl carbonate gave a bright red clumpy powder (17.3 mg, 23.6 mol, 64% yield); R.sub.f 0.25 (2% Methanol in DCM). .sup.1H NMR (500 MHz, CDCl.sub.3) 13.96 (s, 1H), 13.23 (s, 1H), 8.03 (dd, J=7.7, 1.0 Hz, 1H), 7.81-7.76 (m, J=8.3, 7.8 Hz, 1H), 7.39 (dd, J=8.5, 0.7 Hz, 1H), 7.24 (s, 1H), 6.92 (d, J=8.0 Hz, 2H), 5.49 (d, J=3.9 Hz, 1H), 5.28 (s, 1H), 5.10 (d, J=8.3 Hz, 1H), 4.97 (s, 2H), 4.76 (s, 2H), 4.66 (s, 2H), 4.53 (s, 1H), 4.17-4.10 (m, 1H), 4.08 (s, 3H), 3.91-3.80 (m, 1H), 3.66 (s, 1H), 3.27 (dd, J=18.8, 1.8 Hz, 1H), 3.05-2.97 (m, 2H), 2.49 (t, J=2.4 Hz, 1H), 2.33 (dt, J=14.5, 1.8 Hz, 1H), 2.17 (dd, J=14.7, 4.0 Hz, 1H), 1.93 (d, J=4.2 Hz, 1H), 1.87 (dd, J=13.5, 4.8 Hz, 1H), 1.76 (td, J=13.3, 4.2 Hz, 1H), 1.28 (d, J=6.6 Hz, 3H), 1.25 (s, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) 213.99 (s), 187.26 (s), 186.87 (s), 161.22 (s), 157.63 (s), 156.33 (s), 155.81 (s), 155.69 (s), 135.92 (s), 135.67 (s), 133.71 (d, J=4.9 Hz), 130.05 (s), 129.58 (s), 121.05 (s), 120.01 (s), 118.61 (s), 115.05 (s), 111.77 (s), 111.59 (s), 100.88 (s), 78.56 (s), 77.37 (s), 76.77 (s), 75.74 (s), 69.84 (s), 69.73 (s), 67.41 (s), 66.60 (s), 65.69 (s), 56.83 (s), 55.94 (s), 47.11 (s), 35.79 (s), 34.17 (s), 30.53-29.46 (m), 16.98 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.38H.sub.37O.sub.14N.sub.1+.sup.23Na.sub.1 754.2106 found 754.2130.

Carboxybenzyl Doxorubicin (Cbz-Dox)

[0270] ##STR00034##

[0271] The synthetic method described above using 4-nitrophenyl-benzyl carbonate gave a dark red powder (17.3 mg, 25.5 mol, 94% yield); R.sub.f 0.25 (2% Methanol in DCM). .sup.1H NMR (500 MHz, CDCl.sub.3) 13.96 (s, 1H), 13.23 (s, 1H), 8.03 (dd, J=7.7, 1.0 Hz, 1H), 7.78 (dd, J=8.3, 7.9 Hz, 1H), 7.39 (dd, J=8.6, 0.7 Hz, 1H), 7.34-7.26 (m, 5H), 5.50 (d, J=3.9 Hz, 1H), 5.28 (s, 1H), 5.14 (d, J=8.4 Hz, 1H), 5.03 (s, 2H), 4.82-4.70 (m, 2H), 4.54 (s, 1H), 4.14 (dd, J=13.6, 7.2 Hz, 1H), 4.08 (s, 3H), 3.92-3.82 (m, 1H), 3.67 (s, 1H), 3.27 (dd, J=18.8, 1.9 Hz, 1H), 3.00 (d, J=18.8 Hz, 2H), 2.33 (d, J=14.7 Hz, 1H), 2.19-2.14 (m, 1H), 1.88 (dd, J=13.5, 4.8 Hz, 1H), 1.77 (td, J=13.2, 4.1 Hz, 1H), 1.29 (d, J=6.6 Hz, 3H), 1.25 (s, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) 213.99 (s), 215.75-206.09 (m), 187.25 (s), 186.85 (s), 161.22 (s), 156.33 (s), 155.81 (s), 155.66 (s), 136.48 (s), 135.92 (s), 135.66 (s), 133.77-133.65 (m), 128.65 (s), 128.26 (s), 121.04 (s), 120.01 (s), 118.61 (s), 111.76 (s), 111.58 (s), 100.88 (s), 77.37 (s), 76.77 (s), 69.84 (s), 69.73 (s), 67.41 (s), 66.93 (s), 65.69 (s), 56.83 (s), 47.14 (s), 35.79 (s), 34.16 (s), 31.06 (s), 30.45-29.69 (m), 29.42 (s), 16.98 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.35H.sub.35O.sub.13N.sub.1+.sup.23Na.sub.1 700.2001 found 700.1988.

Synthesis of Gemcitabine Prodrugs

Synthesis of tert-Butyldimethylsilyl-Gemcitabine (TSB-Gem)

[0272] Silylated derivative was synthesized as previously described (Weiss, J. T. et al. J Med Chem 57, 5395 (2014)).

Synthesis of carbamate Protected TBS-Gemcitabine

[0273] Dry pyridine (35.5 L, 440.7 mol, 2.7 equiv.) was added drop wise to a solution of TBS-Gemcitabine (60 mg, 162 mol, 1 equiv.) and the 4-nitrophenyl carbonate moiety (106 mg, 324 mol, 2 equiv.) in dry THF (2 mL) with rapid stirring, and the reaction monitored by TLC (5% Methanol in DCM) at t=1 hr, 6 hr and 24 hr, monitoring for formation of product at R.sub.f 0.13 (10% Methanol in DCM). After 24 hr the mixture was concentrated in-vacuo, and the crude was purified via flash chromatography (0 4 5% Methanol in DCM).

2-propargyloxybenzylcarbamoyl-tert-Butyldimethylsilyl-Gemcitabine

[0274] ##STR00035##

[0275] The synthetic method described above gave a white powder (6.4 mg, 11.3 mol, 6.9% yield); R.sub.f 0.28 (2.5% Methanol in DCM). .sup.1H NMR (500 MHz, MeOD) 8.26 (d, J=7.7 Hz, 1H), 7.43 (dd, J=7.5, 1.6 Hz, 1H), 7.39-7.32 (m, 2H), 7.16-7.12 (m, 1H), 7.03 (td, J=7.5, 1.0 Hz, 1H), 6.27 (t, J=6.8 Hz, 1H) 5.35-5.25 (m, 2H), 4.82 (d, J=2.4 Hz, 2H), 4.31 (td, J=12.5, 8.7 Hz, 1H), 4.12 (d, J=12.0 Hz), 4.03 (dt,J=8.7, 2.3 Hz, 1H), 3.95 (dd, J=12.1, 2.4 Hz, 1H), 2.96 (t, J=2.4 Hz, 1H), 1.00 (s, 9H), 0.19 (s, 6H). HRMS (m/z): [M+Si].sup.+ calcd for C.sub.26H.sub.34O.sub.7N.sub.3F.sub.2+Si.sup.28 566.21286 found 566.21470

4-propargyloxybenzylcarbamoyl-tert-Butyldimethylsilyl-Gemcitabine

[0276] ##STR00036##

[0277] The synthetic method described above gave a white powder (8.5 mg, 15 mol, 9% yield); R.sub.f 0.30 (2.5% Methanol in DCM). .sup.1H NMR (500 MHz, MeOD) 8.26 (d, J=7.7 Hz, 1H), 7.38-7.35 (m, 2H), 7.31 (d, J=7.7 Hz, 1H), 7.00-6.97 (m, 2H), 6.24 (t, J=6.7 Hz, 1H), 5.16 (d, J=1.4 Hz, 2H), 4.72 (d, J=2.4 Hz, 2H), 4.28 (td, J=12.5, 8.7 Hz, 1H), 4.09 (d, J=12.0 Hz, 1H), 4.00 (dt, J=8.7, 2.2 Hz, 1H), 3.92 (dd, J=12.1, 2.3 Hz, 1H), 2.92 (t, J=2.4 Hz, 1H), 0.97 (s, 9H), 0.16 (s, 6H). .sup.13C NMR (126 MHz, MeOD) 165.36 (s), 159.35 (s), 157.32 (s), 154.54 (s), 144.78 (s), 131.19 (s), 129.80 (s), 123.87 (t,J=258.8 Hz), 116.02 (s), 96.98 (s), 86.22 (t, J=32.5 Hz), 82.45 (s), 79.65 (s), 76.82 (s), 69.66 (d, J=22.9 Hz), 68.54 (s), 61.68 (s), 56.64 (s), 26.37 (s), 19.26 (s), 18.53 (s), 5.34 (s), 5.44 (s). HRMS (m/z): [M+Si].sup.+ calcd for C.sub.26H.sub.34O.sub.7N.sub.3F.sub.2+Si.sup.28 566.21286 found 566.21550.

General Method for the Synthesis of Gemcitabine Prodrugs

[0278] The carbamate protected TBS-Gemcitabine (6.4 mg, 11.3 molexample for o-derivative; 8.5 mg, 15 mol -example for p-derivative) was dissolved in dry THF (2 mL) and TBAF (30 L, 101.8 mol) was added. The solution was stirred rapidly for 24 hr, then concentrated in-vacuo and the crude was purified via flash chromatography (0.fwdarw.5% Methanol in DCM).

2-proparglyoxybenzylcarbamoyl Gemcitabine (oPOBC-Gem)

[0279] ##STR00037##

[0280] The synthetic method described above gave a white powder (5.1 mg, 11.3 mol, 99% yield); R.sub.f 0.40 (5% Methanol in DCM). .sup.1H NMR (601 MHz, MeOH) 8.31 (d, J=7.7 Hz, 1H), 7.40 (dd, J=7.5, 1.3 Hz, 1H), 7.38-7.29 (m, 2H), 7.11 (d, J=8.3 Hz, 1H), 7.00 (t, J=7.5 Hz, 1H), 6.33-6.18 (m, 1H), 5.28 (s, 2H), 4.80 (d, J=2.4 Hz, 2H), 4.36-4.24 (m, J=12.1, 8.8 Hz, 1H), 4.03-3.92 (m, 2H), 3.87-3.77 (m, 1H), 2.94 (t, J=2.4 Hz, 1H). .sup.13C NMR (151 MHz, MeOH) 165.64 (s), 157.64 (s), 157.30 (s), 155.33 (s), 145.75 (s), 131.17 (s), 131.07 (s), 125.80 (s), 124.46 (s), 124.07 (s), 122.51 (s), 113.64 (s), 97.23 (s), 83.01-82.91 (m), 79.77 (s), 77.12 (s), 70.86-69.91 (m, J=24.2, 21.6 Hz), 64.37 (s), 60.43 (s), 57.21 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.20H.sub.19O.sub.7N.sub.3F.sub.2+Na.sup.23 474.10833 found 474.11050.

4-proparglyoxybenzylcarbamoyl Gemcitabine (pPOBC-Gem)

[0281] ##STR00038##

[0282] The synthetic method described above gave a white powder (6.7 mg, 15 mol, 99% yield); R.sub.f 0.42 (5% Methanol in DCM). .sup.1H NMR (601 MHz, MeOD) 8.30 (d, J=7.7, 1H), 7.39-7.36 (m, 2H), 7.35 (d, J=7.6, 1H), 7.01-6.97 (m, 2H), 6.29-6.19 (m, 1H), 5.17 (s, 2H), 4.73 (d, J=2.4, 2H), 4.30 (td, J=12.1, 8.7, 1H), 4.02-3.91 (m, 2H), 3.81 (dd, J=12.7, 3.0, 1H), 2.93 (t, J=2.4, 1H). .sup.13C NMR (151 MHz, MeOD) 165.61 (s), 159.52 (s), 157.62 (s), 154.66 (s), 145.78 (s), 131.34 (s), 130.04 (s), 125.78 (s), 124.07 (s), 116.17 (s), 97.18 (s), 83.11-82.88 (m), 79.81 (s), 76.95 (s), 70.89-70.00 (m), 68.64 (s), 60.44 (s), 56.79 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.20H.sub.19O.sub.7N.sub.3F.sub.2+Na.sup.23 474.10833 found 474.10830.

Synthesis of Histamine Derivatives

General Method for the Synthesis of Carbomate-Protected Histamine Derivatives

[0283] 2-(4-Imidazolyl)ethylamine dihydrochloride (25 mg, 0.14 mmol) was dissolved in dry DMF (1 mL) with triethylamine (28 L, 0.21 mmol) under N.sub.2 atmosphere. O- or p-(2-propynyloxy)phenyl)methyl 4-nitrophenyl carbonate (60 mg, 0.21 mmol) was added dropwise to the mixture. The mixture was stirred overnight at room temperature (r.t.). The solvents were then removed in vacuo, the crude redissolved with CH.sub.2Cl.sub.2 (5 mL), and washed with H.sub.2O (5 mL). The aqueous layer was washed with CH.sub.2Cl.sub.2 (35 mL) and the combined organic layers dried over MgSO.sub.4, filtered, and concentrated under reduce pressure. The crude was purified by column chromatography using 20% MeOH in DCM.

2-proparglyoxybenzylcarbamoyl Histamine (oPOBC-Hist)

[0284] ##STR00039##

[0285] The synthetic method described above gave a white solid (15 mg, 37% yield); Rf 0.39 (10% MeOH in DCM). .sup.1H NMR (500 MHz, MeOD) 8.74 (s, 1H), 7.30 (m, 3H), 6.98 (d, J=8.5, 2H), 5.00 (s, 2H), 4.75 (d, J=2.0, 2H), 3.45 (t, J=6.6, 2H), 2.98 (s, 1H), 2.93 (t, J=6.6, 2H). .sup.13C NMR (126 MHz, MeOD) 157.58-157.51 (d, CO), 133.38 (CH), 131.63 (C).sub.2, 129.75 (C), 129.24 (CH).sub.2, 116.25 (CH), 114.55 (CH).sub.2, 78.39 (CH), 75.55 (C), 65.84 (CH.sub.2), 55.32 (CH.sub.2), 39.34-39.22 (d, CH.sub.2), 24.96 (CH.sub.2). HRMS (ESI) m/z [M+H]+ calcd for C.sub.16H.sub.18O.sub.3N.sub.3, 300.13427; found, 300.13760.

4-proparglyoxybenzylcarbamoyl Histamine (pPOBC-Hist)

[0286] ##STR00040##

[0287] The synthetic method described above gave a white solid (35 mg, 85% yield); Rf 0.44 (10% MeOH in DCM). .sup.1H NMR (500 MHz, MeOD) 7.70 (s, 1H), 7.31 (dd, J=11.2, 7.7, 2H), 7.09 (d, J=8.1, 1H), 6.99 (t, J=7.4, 1H), 6.91 (s, 1H), 5.13 (s, 2H), 4.78 (d, J=2.2, 2H), 3.39 (t, J=7.1, 2H), 2.96 (t, J=2.3, 1H), 2.81 (t, J=7.0, 2H). .sup.13C NMR (126 MHz, MeOD) 157.55 (CO), 155.31 (C), 128.73-128.55 (d, CH).sub.2, 125.66 (C).sub.2, 120.91 (CH).sub.2, 111.98 (CH).sub.2, 78.36 (CH), 75.49 (C), 61.35 (CH.sub.2), 55.63 (CH.sub.2), 40.31 (CH.sub.2), 26.76 (CH.sub.2). HRMS (ESI) m/z [M+H]+ calcd for C.sub.16H.sub.18O.sub.3N.sub.3, 300.13427; found, 300.13830.

Synthesis of 5FU Prodrug

General Method for Synthesis of 5FU Prodrugs

[0288] Sodium hydride (60% in mineral oil), (120 mg, 3 mmol, 5 equiv.) was added to THF (10 mL) at 4 C. and stirred rapidly for 30 minutes. Propargyl alcohol (97 L, 1.8 mmol 3 equiv.) was mixed in THF (5 mL) and added dropwise to the stirring mixture. An evolution of gas was observed with a slight exotherm. After stirring for an additional 10 minutes, the flask was sealed and flushed with nitrogen. A gas-tight syringe containing 5-fluoro-2,4-dichloropyrimidine (100 mg, 0.6 mmol, 1 equiv.) mixed in dry THF (5 mL) was added dropwise at 4 C. with rapid stirring, and allowed to warm to room temperature. The reaction mixture was monitored at t=1 hr, t=3 hr and t=24 hr, then diluted in DCM (50 mL), and the organic layer was washed twice with water (50 mL) and acidified with acetic acid. The organic layers were then concentrated in-vacuo and the crude was purified via flash chromatography (12.5% Ethyl Acetate in Hexane).

2,4-bispropargyl-5-fluorouracil (bis-Pro-5FU)

[0289] ##STR00041##

[0290] The synthetic method described above gave a white powder (45 mg, 0.22 mmol, 36.4% yield); R.sub.f 0.40 (12.5% Ethyl Acetate in Hexane). .sup.1H NMR (500 MHz, CDCl3) 8.15 (d, J=2.3 Hz, 1H), 5.08 (d, J=2.4 Hz, 2H), 4.96 (d, J=2.4 Hz, 2H), 2.55 (t, J=2.4 Hz, 1H), 2.47 (t, J=2.4 Hz, 1H). .sup.13C NMR (126 MHz, CDCl3) 158.61 (d, J=7.5 Hz), 144.20 (s), 144.10 (s), 143.94 (s), 142.17 (s), 78.27 (s), 76.15 (s), 75.00 (s), 55.73 (s), 55.09 (s). HRMS (m/z): [M+Na].sup.+ calcd for C.sub.10H.sub.7O.sub.2N.sub.2F.sub.1+Na.sup.23 229.03838 found 229.03760.

Synthesis of Olaparib Prodrug

General Method for the Synthesis of Olaparib Prodrug

[0291] Olaparib (AZD2281, MedChem Express LLC (MCE)) (10 mg, 0.03 mmol) was dissolved in dry DMF (1 mL) under N.sub.2 atmosphere. The mixture was then cooled to 4 C. in an ice bath. Propargyl bromide solution 80 wt. % in toluene (8 L, 0.05 mmol) was diluted in dry DMF (250 L) and added to the mixture. Then, DBU (9 L, 0.06 mmol) in dry DMF (250 L) was added dropwise to the mixture. The mixture was stirred overnight and allowed to warm up to room temperature (r.t.). The solvents were then removed in vacuo, the crude redissolved with CH.sub.2Cl.sub.2 (5 mL), and washed with H.sub.2O (5 mL). The aqueous layer was washed with CH.sub.2Cl.sub.2 (35 mL) and the combined organic layers dried over MgSO.sub.4, filtered, and concentrated in vacuo. The crude was purified by Merck TLC Silica gel 60 F254 plates semipreparative using 5% MeOH in DCM.

4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl-phthalazin-1-oxypropargyl (Propargyl Olaparib, or Prop-Olap)

[0292] ##STR00042##

[0293] The synthetic method described above gave a white solid (6 mg, 47% yield); Rf 0.39 (5% MeOH in DCM. .sup.1H NMR (500 MHz, CDCl3) 8.51 (m, 1H), 7.77 (m, 2H), 7.71 (m, 1H), 7.36 (m, 2H), 7.07 (t, J=8.9, 1H), 5.04 (d, J=2.5, 2H), 4.33 (s, 2H), 3.79 (m, 4H), 3.62 (m, 2H), 3.35 (m, 2H), 2.35 (t, J=2.4, 1H), 1.72 (m, 1H), 1.28 (t, J=7.1, 2H), 1.03 (dd, J=4.6, 2.9, 2H). .sup.13C NMR (126 MHz, CDCl3) 172.35 (CO), 171.15 (CO), 158.73 (C), 158.02 (C), 156.05 (C), 145.01 (C), 134.44-134.41 (d, C), 133.33 (CH), 131.69-131.61 (d, CH).sub.2, 129.15 (C), 128.28 (C), 127.65 (CH).sub.2, 124.93 (CH), 116.31-116.14 (d, CH), 77.22 (CH), 72.43 (C), 60.39 (CH.sub.2).sub.2, 53.49 (CH.sub.2).sub.2, 40.73 (CH.sub.2), 37.81 (CH.sub.2), 11.04 (CH), 7.70 (CH.sub.2).sub.2. HRMS (ESI) m/z [M+H]+ calcd for C.sub.27H.sub.26O.sub.3N.sub.4F.sub.1, 473.19835; found, 473.19410.

Synthesis of Panobinostat Prodrug

Synthesis of N-[4-(propargyloxy)benzyloxy]phthalimide

[0294] N-[4-(propargyloxy)benzyloxy]phthalimide was synthesised by alkylation of N-hydroxyphthalimide with 4-propargyloxy-benzyl bromide using NaH as previously described for others O-alkylhydroxylamines (High, A et al. J Pharmacol Exp Ther. 1999, 288, 490-501).

##STR00043##

[0295] The synthetic method described above gave a pale-yellow solid (534 mg, 79% yield). .sup.1H NMR (500 MHz, DMSO) 7.86 (s, 4H), 7.45 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H), 5.10 (s, 2H), 4.81 (d, J=2.4 Hz, 2H), 3.56 (t, J=2.4 Hz, 1H). .sup.13C NMR (126 MHz, DMSO) 163.12 (C2), 157.78 (C), 134.77 (CH2), 131.35 (CH2), 128.51 (C2), 126.92 (C), 123.22 (CH2), 114.70 (CH2), 79.06 (C), 78.78 (CH.sub.2), 78.29 (CH), 55.41 (CH.sub.2). HRMS (ESI) m/z [M+Na].sup.+ calcd for C.sub.18H.sub.13O.sub.4NNa, 330.0737; found, 330.0785.

Synthesis of O-[4(propargyloxy)benzyl]hydroxylamine hydrochloride

[0296] O-[4(propargyloxy)benzyl]hydroxylamine was obtained by removal of the phthaloyl group by hydrazinolysis in diethyl ether and converted to the hydrochloride salt with an ethereal hydrochloric acid solution as reported by Bindman, N. A. et al. J Am Chem Soc. 2013, 135, 10362-10371.

##STR00044##

[0297] The synthetic method described above gave a white solid (190 mg, 77% yield). .sup.1H NMR (500 MHz, DMSO) 10.94 (s, 3H), 7.37 (d, J=8.7 Hz, 2H), 7.03 (d, J=8.7 Hz, 2H), 4.95 (s, 2H), 4.82 (d, J=2.4 Hz, 2H), 3.58 (t, J=2.4 Hz, 1H). .sup.13C NMR (126 MHz, DMSO) 157.83 (C), 131.05 (CH2), 126.29 (C), 114.92 (CH2), 79.06 (C), 78.36 (CH), 75.39 (CH.sub.2), 55.43 (CH.sub.2). HRMS (ESI) m/z [MCl].sup.+ calcd for C.sub.10H.sub.12O.sub.2N, 178.0863; found, 178.0885.

Synthesis of (E)-3-(4-{[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl}phenyl)acrylic acid

[0298] (E)-Methyl 3-(4-{[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl}phenyl)acrylate was synthesised according to literature procedure (Wang, H. et al. J Med Chem. 2011, 54, 4694-4720) and hydrolyzed to the corresponding carboxylic acid by treatment with NaOH.

General Method for the Synthesis of Panobinostat Prodrug

[0299] (E)-3-(4-{[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl}phenyl)acrylic acid (25 mg, 0.075 mmol) and O-[4(propargyloxy)benzyl]hydroxylamine hydrochloride (24 mg, 0.112 mmol) were added to a 25 mL round-bottom flask and partially dissolved in distilled water (1 mL). N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (43 mg, 0.225 mmol) was then added to the mixture in one portion. The pH was monitored and adjusted to 4.5 with a NaOH and/or HCl aqueous solution (1 M). The reaction was stirred for 6 hours at room temperature and constant pH (4.5). Water was removed under reduced pressure, and crude suspended in acetonitrile and vacuum filtered. Solution was purified by semi-preparative TLC eluting with DCM:MeOH/7:3.

Propargyloxybenzyl-Panobinostat (POB-Panob)

[0300] ##STR00045##

[0301] The synthetic method described above gave a yellow solid (9.3 mg, 25%). .sup.1H NMR (500 MHz, DMSO) 11.18 (s, 1H), 10.67 (s, 1H), 7.49 (m, 3H), 7.36 (m, 5H), 7.21 (d, J=7.9 Hz, 1H), 7.00 (d, J=8.7 Hz, 2H), 6.95 (m, 1H), 6.89 (m, 1H), 6.41 (d, J=15.8 Hz, 1H), 4.80 (s, 4H), 3.80 (s, 2H), 3.56 (s, 1H), 2.81 (m, 2H), 2.72 (m, 2H), 2.30 (s, 3H). LC-MS (m/z): 494.1749.

Synthesis of SN-38 Prodrug

Synthesis of Benzoic Acid, 2,6-bis(2-propyn-1-yloxy)-2-propyn-1-yl Ester

[0302] 2,6-Dihydroxybenzoic acid (6.96 g, 45 mmol) and potassium carbonate (30.5 g, 220 mmol) were suspended in dry DMF (40 mL) and stirred for 30 mins at 0 C. Propargyl bromide (21 mL, 80% in toluene, 16.8 141 mmol) was added dropwise and the reaction was warmed to ambient temperature and stirred for three days. The reaction was diluted with water (300 mL) and extracted with diethyl ether (6200 mL). The combined organic phases were washed with brine, dried over MgSO.sub.4 and concentrated in vacuo to yield the title compound.

##STR00046##

[0303] The synthetic method described above gave a brown oil (5.37 g 20.1 mmol, 44%), used without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.32 (t, J=8.4 Hz, 1H), 6.76 (d, J=8.4 Hz, 2H), 4.91 (d, J=2.5 Hz, 2H), 4.71 (d, J=2.5 Hz, 4H), 2.51 (t, J=2.4 Hz, 2H), 2.50 (t, J=2.5 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3) 165.1, 155.9, 131.3, 114.0, 106.8, 78.2, 77.7, 76.2, 75.2, 57.0, 52.9.

Synthesis of (2,6-Bis(prop-2-yn-1-yloxy)phenyl)methanol

[0304] 2,6-Bis(2-propyn-1-yloxy)-, 2-propyn-1-yl ester benzoic acid (5.37 g, 20 mmol) was dissolved in THF and cooled to 0 C. for the addition of LiAlH.sub.4 (1 M in THF, 24 mL, 24 mmol) before warming to ambient temperature and stirring overnight. The reaction was quenched at 0 C. with 10% NaOH (40 mL), stirring for 30 mins. The aqueous phase was extracted with CH.sub.2Cl.sub.2 (370 mL) and the combined organic phases washed with brine (40 mL), dried over MgSO.sub.4 and concentrated in vacuo. The crude alcohol was purified by flash column chromatography (30% AcOEt/hexane).

##STR00047##

[0305] The synthetic method described above gave a white solid (2.74 g, 12.6 mmol, 63%). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.24 (t, J=8.4 Hz, 1H), 6.72 (d, J=8.4 Hz, 2H), 4.81 (d, J=6.7 Hz, 2H), 4.74 (d, J=2.4 Hz, 4H), 2.51 (t, J=2.4 Hz, 2H), 2.37 (t, J=6.7 Hz, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) 162.5, 155.7, 129.1, 106.6, 78.5, 75.82, 56.6, 54.4.

Synthesis of 2-(chloromethyl)-1,3-bis(prop-2-yn-1-yloxy)benzene

[0306] Cyanuric chloride (180 mg, 1.00 mmol) was stirred as a suspension in DMF (0.1 mL) for one hour. (2,6-Bis(prop-2-yn-1-yloxy)phenyl)methanol (194 mg, 0.90 mmol) in CH.sub.2CL.sub.2 (1 mL) was added and the reaction stirred at ambient temperature overnight. The reaction was diluted with CH.sub.2CL.sub.2 (25 mL) and washed with sat. bicarb. The aqueous phase was extracted with CH.sub.2CL.sub.2 (215 mL), dried over MgSO.sub.4 and concentrated in vacuo. The crude product was further purified with column chromatography (40% AcOEt/hexane).

##STR00048##

[0307] The synthetic method described above gave a white solid (161 mg, 0.69 mmol, 77%). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.28 (t, J=8.4 Hz, 1H) 6.72 (d, J=8.4 Hz, 2H), 4.78 (d, J=2.3 Hz, 4H), 4.78 (s, 2H), 2.51 (t, J=2.4 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) 156.7, 123.0, 115.9, 106.2, 78.4, 75.7, 56.0, 35.3.

General Method for the Synthesis of SN-38 Prodrug

[0308] 10-Hydroxy-7-ethylcamptothecin (SN-38, 40 mg, 0.10 mmol) and potassium carbonate (21 mg, 0.15 mmol) were dissolved in dry DMF (2 mL) and stirred for 10 minutes at 0 C. under nitrogen. 2,6-Bis(propargyloxy)benzyl chloride (28.1 mg, 0.21 mmol) in DMF (0.5 mL) was added and the reaction warmed to ambient temperature and stirred overnight. Solvent was evaporated in vacuo and the crude prodrug purified by semi-preparative TLC (3% MeOH/CH.sub.2Cl.sub.2).

(S)-9-((2,6-bis(prop-2-yn-1-yloxy)benzyl)oxy)-4,11-diethyl-4-hydroxy-1,12-dihydro-14H-pyrano[3,4:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (di-oPOB-SN-38)

[0309] ##STR00049##

[0310] The synthetic method described above gave a white solid (2.7 mg, 3% yield). .sup.1H NMR (500 MHz, MeOD) 8.05 (d, J=9.3 Hz, 1H), 7.97 (s, 2H), 7.65 (s, 1H), 7.52 (dd, J=9.3, 2.6 Hz, 1H), 7.34 (t, J=8.5 Hz, 1H), 6.84 (d, J=8.5 Hz, 2H), 5.60 (d, J=16.2 Hz, 1H), 5.37 (s, 2H), 5.37 (d, J=16.2 Hz, 1H), 5.30 (s, 2H), 4.81 (d, J=2.4 Hz, 4H), 3.26 (q, J=7.6 Hz, 2H), 2.88 (t, J=2.4 Hz, 2H), 2.03-1.90 (m, J=7.0 Hz, 2H), 1.43 (t, J=7.6 Hz, 3H), 1.02 (t, J=7.4 Hz, 3H); .sup.13C NMR (126 MHz, DMSO) 173.0, 158.4, 157.6, 156.4, 150.5, 150.0, 146.8, 145.1, 144.5, 131.9, 130.9, 130.3, 128.8, 128.3, 123.1, 118.6, 113.4, 106.8, 105.1, 96.5, 79.6, 78.9, 72.9, 65.7, 56.7, 50.0, 30.7, 22.8, 14.0, 8.2.

Synthesis of Etoposide Prodrugs

General Method for the Synthesis of Etoposide Prodrugs

[0311] Etoposide (20 mg, 0.034 mmol) and potassium carbonate (7 mg, 0.051 mmol) were stirred in dry DMF (1 mL) at 0 C. for five mins. Alkyl halides (9 mg, 0.076 mmolexample for propargyl bromide; 9 mg, 0.050 mmolexample for 2-(Chloromethyl)-2-(prop-2-yn-1-yloxy)benzene; 12 mg, 0.051 mmolexample for (Chloromethyl)-1,3-bis(prop-2-yn-1-yloxy)benzene) in dry DMF (1 mL) was added and the reaction stirred at ambient temperature 6h for propargyl bromide; overnight for 2-(Chloromethyl)-2-(prop-2-yn-1-yloxy)benzene; and five days for (Chloromethyl)-1,3-bis(prop-2-yn-1-yloxy)benzene. Solvent was removed in vacuo and the crude compound purified by semi-preparative TLC (4% MeOH/CH.sub.2Cl.sub.2 for propargyl and 2-(methyl)-2-(prop-2-yn-1-yloxy)benzene; or 5% MeOH/CH.sub.2Cl.sub.2 for (methyl)-1,3-bis(prop-2-yn-1-yloxy)benzene).

Propargyloxy Etoposide (Pro-Etoposide)

(5R,5aR,8aR,9S)-9-(((2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-5-(3,5-dimethoxy-4-(prop-2-yn-1-yloxy)phenyl)-5,8,8a,9-tetrahydrofuro[3,4:6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one

[0312] ##STR00050##

[0313] The synthetic method described above gave a white solid (14 mg, 0.022 mmol, 79%). .sup.1H NMR (500 MHz, MeOD) 6.99 (s, 1H), 6.52 (s, 1H), 6.32 (s, 2H), 5.98-5.95 (m, 2H), 5.03 (d, J=3.3 Hz, 1H), 4.76 (q, J=5.0 Hz, 1H), 4.65 (d, J=7.7 Hz, 1H), 4.61 (d, J=5.5 Hz, 1H), 4.57 (d, J=2.5 Hz, 2H), 4.43 (dd, J=10.7, 8.6 Hz, 1H), 4.29 (dd, J=8.7, 7.6 Hz, 1H), 4.17 (dd, J=10.3, 4.8 Hz, 1H), 3.71 (s, 6H), 3.58 (t, J=10.1 Hz, 1H), 3.54 (t, J=9.1 Hz, 1H), 3.48 (dd, J=14.2, 5.5 Hz, 1H), 3.30-3.23 (m, 2H), 2.99-2.91 (m, 1H), 2.81 (t, J=2.4 Hz, 1H), 1.32 (d, J=5.0 Hz, 3H); .sup.13C NMR (126 MHz, MeOD) 177.6, 154.1, 150.1, 148.4, 138.1, 135.9, 133.9, 130.4, 111.2, 110.9, 109.4, 103.2, 102.9, 100.8, 81.8, 80.3, 76.14, 75.9, 74.5, 74.0, 69.7, 69.2, 67.6, 60.7, 56.6, 45.2, 42.2, 39.3, 20.6.

Propargyloxybenzyl Etoposide (oPOB-Etoposide)

(5R,5aR,8aR,9S)-9-(((2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methyl hexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-5-(3,5-dimethoxy-4-((2-(prop-2-yn-1-yloxy)benzyl)oxy)phenyl)-5,8,8a,9-tetrahydrofuro[3,4:6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one

[0314] ##STR00051##

[0315] The synthetic method described above gave a white solid (4.7 mg, 0.0065 mmol, 19%). .sup.1H NMR (400 MHz, MeOD) 7.45 (dd, J=7.8, 1.2 Hz, 1H), 7.27 (td, J=8.3, 1.8 Hz, 1H), 7.06-7.03 (m, 1H), 7.01 (s, 1H), 6.96 (td, J=7.4, 0.9 Hz, 1H), 6.54 (s, 1H), 6.31 (s, 2H), 5.99 (dd, J=3.3, 1.0 Hz, 2H), 5.05 (d, J=3.4 Hz, 1H), 4.99 (s, 2H), 4.79 (q, J=5.0 Hz, 1H), 4.68 (d, J=2.5 Hz, 1H), 4.67 (m, 3H), 4.62 (d, J=5.6 Hz, 1H), 4.45 (dd, J=10.7, 8.6 Hz, 1H), 4.32 (t, J=8.2 Hz, 1H), 4.19 (dd, J=10.3, 4.8 Hz, 1H), 3.64-3.45 (m, 4H), 3.30-3.24 (m, 2H), 2.95 (t, J=2.4 Hz, 1H), 1.34 (d, J=5.0 Hz, 3H); .sup.13C NMR (126 MHz, MeOD) 180.4, 155.7, 153.7, 147.8, 146.7, 137.8, 135.3, 132.1, 130.1, 128.8, 128.5, 126.6, 120.8, 112.1, 108.8, 107.3, 105.5, 101.8, 101.1, 99.3, 80.2, 78.1, 75.0, 74.5, 73.4 69.1, 68.7, 67.8, 66.1, 55.9, 55.3, 45.0, 43.5, 39.3, 29.4, 19.2.

[0316] Bis-propargyloxybenzyl Etoposide (di-oPOB-Etoposide)

[0317] (5R,5aR,8aR,9S)-5-(4-((2,6-bis(prop-2-yn-1-yloxy)benzyl)oxy)-3,5-dimethoxyphenyl)-9-(((2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-5,8,8a,9-tetrahydrofuro[3,4:6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one

##STR00052##

[0318] The synthetic method described above gave a white solid (7 mg, 0.009 mmol, 23%). .sup.1H NMR (500 MHz, MeOD) 7.23 (t, J=8.4 Hz, 1H), 7.12 (s, 1H), 6.73 (d, J=8.4 Hz, 2H), 6.45 (s, 3H), 5.93 (q, J=1.2 Hz, 2H), 5.18 (dd, J=11.3, 6.7 Hz, 2H), 4.80 (d, J=4.8 Hz, 1H), 4.74 (q, J=5.0 Hz, 1H), 4.58 (qd, J=15.7, 2.4 Hz, 4H), 4.43-4.33 (m, 3H), 4.26 (d, J=7.8 Hz, 1H), 4.09 (dd, J=10.4, 4.6 Hz, 1H), 3.66 (s, 6H), 3.66-3.54 (m, 2H), 3.54 (dd, J=10.7, 9.1 Hz, 1H), 3.46 (t, J=8.9 Hz, 1H), 3.29-3.16 (m, 4H), 2.94 (t, J=2.4 Hz, 2H), 1.30 (d, J=5.0 Hz, 3H); .sup.13C NMR (126 MHz, MeOD) 181.9, 159.6, 155.4, 149.2, 148.0, 138.9, 136.4, 133.7, 131.0, 129.8, 116.7, 110.2, 108.7, 107.4, 106.7, 103.2, 102.5, 100.7, 81.6, 80.2, 76.8, 76.6, 75.9, 74.8, 70.1, 69.2, 67.6, 63.6, 57.7, 56.7, 46.4, 44.7, 40.7, 20.6.

[0319] Experimental Data

[0320] Cell-Free Palladium Mediated Deprotection of Prodrugs

[0321] To recreate a biocompatible scenario, prodrug-into-active agent conversion was carried out at 37 C. in an isotonic solution with a physiologic pH. POB-Vor, oPOBC-Hist and pPOBC-Hist (100 M) were incubated in phosphate buffered saline (PBS, 1 ml) with 1 mg of Pd.sup.0 resin for 24 h at 37 C. (Thermomixer, shaker speed: 1,200 rpm). Reaction crudes were monitored at 0 h, 3h, 6 h by analytical HPLC using an UV-VIS detector (for POB-Vor) and analytical LCMS using a microTOF II detector (for oPOBC-Hist and pPOBC-Hist). HPLC method: eluent A: water and trifluoroacetic acid (0.4%); eluent B: acetonitrile; A/B=95:5 to 20:80 in 6 min, isocratic 1 min, 20:80 to 95:5 in 0.1 min, and isocratic 2 min with the UV detector at 254 nm. LCMS method: eluent A: water and formic acid (0.1%); eluent B: acetonitrile and formic acid (0.1%); A/B=95:5 isocratic 0.5 min, 95:5 to 0:100 in 4.5 min, isocratic 2 min, 0:100 to 95:5 in 0.5 min, and isocratic 2.5 min (flow=0.2 mL/min).

[0322] FIG. 2 shows the HPLC chromatograms for the Pd-catalysed deprotection of POB-Vor at times of 0 h, 3 h and 6 h. As seen in FIG. 2C, POB-Vor completely disappeared from the crude mixture after 6 h, with Vorinostat being the major reaction product.

[0323] FIGS. 13 and 14 show the LCMS chromatograms for the Pd-catalysed deprotection of oPOBC-Hist and pPOBC-Hist. As seen in FIGS. 13 and 14, oPOBC-Hist and pPOBC-Hist completely disappeared from the crude mixture after 3 h of incubation with Pd.sup.0-beads.

[0324] Ninhydrin Test for Detection of Pd.sup.0-Mediated Deprotected Histamine

[0325] Histamine, oPOBC-Hist and pPOBC-Hist (100 M) were incubated in phosphate buffered saline (PBS, 1 ml) with 1 mg of Pd.sup.0 resin for 24 h at 37 C. (Thermomixer, shaker speed: 1,200 rpm). After 24 h, PBS was removed and 300 L of solution A (described below) and then, 100 L of solution B (described below) were added to the Pd.sup.0-functionalized resins. Eppendorfs were then heated at 95 C. for 5 min. A negative test, indicating the absence of free primary amine (histamine), was communicated by a light yellow/orange solution. A positive test was indicated by a dark purple solution. Variations in the darkness of the solution reflect variations in amine concentration. Optical density (O.D.) was measured at the maximum of absorbance for ninhydrin purple-blue complex at 570 nm.

[0326] Reagent solution A. Phenol (40 g) is added to EtOH (10 mL) and the mixture was heated until complete dissolution of the phenol. A solution of KCN (65 mg) in water (100 mL) was added to pyridine (100 mL). Reagent solution B. A solution of ninhydrin (2.5 g) in absolute EtOH (50 mL) was prepared and maintained in a light-proof container, preferably under inert atmosphere.

[0327] FIG. 12 shows the optical density of the solution mixture for control, Histamine, oPOBC-Hist and pPOBC-Hist by ninhydrin test. As expected, primary amines were detected in all samples due to the histamine release from the Pd-beads compared to the negative control (Pd-resin+DMSO).

[0328] Biological Activity

[0329] Human lung adenocarcinoma A549 cells, human glioblastoma U87G cells and T98 cells were chosen as models for the Vorinostat and Panobinostat antiproliferative studies; A549 cells, human prostate carcinoma DU145 cells and T98 cells were chosen as models for Doxorubicin; human pancreatic carcinoma MiaPaCa2 cells were chosen as a model for Gemcitabine; human pancreatic adenocarcinoma BxPC3 and human colorectal carcinoma HCT116 cells were chosen as models for 5FU; human glioblastoma U87G cells cells were chosen as models for the SN-38 and Etoposide antiproliferative studies; and human ovarian carcinoma A2780 cells were chosen as model for Olaparib. These cell lines were selected as these are primary malignancies against which the parental drugs are currently prescribed.

[0330] Prodrug Safety Studies

[0331] The toxicities of each active agent and prodrugs were compared by performing dose-response studies. Doses of Vorinostat and Vorinostat prodrugs (10, 30, 100, 200, 300, 400, 500 M for A549, U87G and T98 cell lines); Panobinostat and POB-Panob (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 M for A549 cells); Doxorubicin and Doxorubicin prodrugs (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 M for A549 and DU145 cell lines, and an additional dose of 100 M for T98 cell line); Gemcitabine and Gemcitabine prodrugs (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 M for MiaPaCa2 cells); 5FU and Bis-Pro-5FU (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 M for BxPC3 and HCT116 cells); SN-38 and di-oPOB-SN-38 (0.03, 0.1 and 0.3 M for U87G cells); Etoposide and Etoposide prodrugs (0.3, 1, 3, 10, 30 and 100 M for U87G cells); and Olaparib and Prop-Olap (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 M for A2780 cells) were incubated with cells for 5 days and cell viability measured to determine the corresponding EC.sub.50 values. Cell viability was analysed by fluoresce intensity (.sub.ex=540 nm; .sub.em=590 nm) after 60-90 min incubation with PrestoBlue Reagent (Life Technologies).

[0332] The cell viability data for A549, U87G and T98 cells are provided in FIG. 1. EC.sub.50 values calculated for Vorinostat were 4.85 M, 11 M and 12.4 M, respectively. Both POB-Vor and Benzyl-Vor display a significant reduction in the cytotoxic effect, showing an EC.sub.50 value>500 M for each cell line tested. The cell viability data for A549 cells are provided in FIG. 19 with EC.sub.50 values calculated for Panobinostat and POB-Panob were 16 nM and 758 nM, respectively.

[0333] The cell viability data for A549, DU145 and T98 cells are provided in FIG. 4. EC.sub.50 values calculated for Doxorubicin were 105 nM, 23 nM and 320 nM, respectively. Fold reduction ratios against Doxorubicin for pPOBC-Dox, oPOBC-Dox and Cbz-Dox were calculated as (1:66, 1:259, 1:89 [A549 cells]); (1:240, 1:310, 1:116 [DU145 cells]), (1:312, 1:312, 1:15 [T98]), respectively.

[0334] Cell viability data for MiaPaCa2 is provided in FIG. 9. EC.sub.50 value calculated for Gemcitabine was 13 nM. pPOBC-Gem displays an intermediate reduction in the cytotoxic effect, showing a 27-fold reduction relative the parent active agent.

[0335] FIG. 15 shows the cell viability data for BxPC3 and HCT116 cells. EC.sub.50 values calculated for 5FU were 140 nM and 1.5 M, respectively. Bis-Pro-5FU shows a significant reduction in cytotoxic effect for both BxPC3 and HCT116 cells (EC.sub.50 values=>100 M).

[0336] FIG. 18 shows the cell viability data for A2780 cells. EC.sub.50 value calculated for Olaparib was 1.87 M. Prop-Olap displays a significant reduction in the cytotoxic effect, showing an EC.sub.50 value>100 M for the cell line tested.

[0337] The cell viability data for U87G cells are provided in FIGS. 20-21. EC.sub.50 values calculated for SN-38 and Etoposide were 24.6 nM and 4.97 M, respectively. SN-38 prodrug shows an EC.sub.50 value 3.15 M for U87G cells. Etoposide prodrugs display a significant reduction in the cytotoxic effect, showing an EC.sub.50 value>100 M for each prodrug tested.

[0338] Generation of Drug from Prodrug in Cell Culture and Cytotoxic Effects

[0339] The toxigenic effect as a result of in situ generation of parental drug in cell culture was determined by incubating all cells in tissue culture media containing 0.1% (v/v) DMSO and a) Pd.sup.0, Au, or Pd/Au-resin (1 mg/mL for all cells tested, negative control); b) prodrug (negative control); or c) Pd.sup.0, Au, or Pd/Au-resin (1 mg/mL for all cells)+prodrug (reaction assay). Cells incubated in 0.1% (v/v) DMSO in media was used as an untreated cell reference standard (100% viability). A PrestoBlue cell viability assay as described above was carried out and fluorescent intensities compared to the untreated cell control. Prodrug concentrations were 100 M for Vorinostat prodrugs, 0.3 M for Panobinostat prodrug, 1 M for Doxorubicin prodrugs, 0.03 M for Gemcitabine prodrugs for A549 cells and 3 M (BxPC-3 cells) or 30 M (HCT116 cells) for 5FU prodrugs or 100 M (U87G cells) for SN-38 prodrug.

[0340] The combination of POB-Vor+Pd.sup.0 resins, oPOBC-Dox+Pd.sup.0 resins, pPOBC-Dox+Pd.sup.0 resins or pPOBC-Gem+Pd.sup.0 resins showed a strong toxigenic effect in A549 (POB-Vor); A549, DU145 and T98 (oPOBC-Dox and pPOBC-Dox) and MiaPaCa2 cell lines (pPOBC-Gem) A549 (POB-Panob) and U87G (di-oPOB-SN-38) as shown in FIGS. 4, 8, 11, 19 and 20 respectively, indicating the generation of Vorinostat, Doxorubicin or Gemcitabine to significant levels. Benzyl-Vor, Cbz-Dox or Cbz-Gem combined with Pd.sup.0-resins showed only low levels of toxicity in the different cell lines, which suggests the generation of low levels of parental drug.

[0341] Bis-Pro-5FU+Pd.sup.0 resins showed a strong toxigenic effect in both BxPC3 and HCT116 cells (FIG. 16), indicating the generation of 5FU to significant levels.

[0342] Combination of POB-Vor (100 M)+Au or Pd/Au resins showed a strong toxigenic effect in A549 cells (FIG. 22), indicating the generation of Vorinostat to significant levels.

[0343] Dose Response Cell Viability Assay

[0344] To show extracellular efficacy of the palladium-mediated dealkylation of Vorinostat, Doxorubicin, Gemcitabine or 5FU prodrugs, a range of concentrations of POB-Vor, oPOBC-Dox, pPOBC-Dox, pPOBC-Gem, Bis-Pro-5FU and di-oPOB-SN-38 and Pd.sup.0-resins were incubated independently (negative controls) and in combination (BOOM conversion assay) at varying doses to study of proliferation cells in comparison to unmodified Vorinostat, Doxorubicin, Gemcitabine or 5FU, respectively (positive control). A dose response study was performed for each cell line keeping the quantity of Pd.sup.0 resin constant (0.8 mg/mL for U87G and 1 mg/mL for the rest of cell lines). All cells were plated in Dulbecco's Modified Eagle Media (DMEM) supplemented with serum (10% FBS) and L-glutamine (2 mM). Cells were seeded in a 96 well plate format (density: 1500 cells/mL for A549, 2000 cells/mL for U87G, 1000 cells/mL for T98, 2000 for DU145 cells, 1000 for MiaPaca2 cells, 2500 for BxPc3 cells and 3000 for HTC116 cells) and incubated for 48 h at 37 C. and 5% CO.sub.2 before treatment. Each well was then replaced with fresh media containing: Pd.sup.0-resins (0.8 mg/mL for U87G and 1 mg/mL for the rest of cell lines, negative control); prodrug (1 M to 100 M for POB-Vor; 0.03 M to 3 M for Doxorubicin prodrugs in A459 and DU145 cells and 0.1 M to 10 M for T98 cells; 0.003 M to 0.3 M for Gemcitabine prodrugs; 0.03 M to 3 M for 5FU prodrug in BxPC3 cells and 0.3 M to 30 M in HTC116 cells and 0.03 M to 0.3 M for SN-38 prodrug in U87G) in DMSO (0.1% v/v) (negative control); active agent (concentrations as above) in DMSO (0.1% v/v), (positive control); or a combination of Pd.sup.0 resin+prodrug (concentrations as above in 0.1% v/v DMSO). Cells incubated in 0.1% (v/v) DMSO in media were used as untreated cell reference standard (i.e. 100% cell viability). Cells were incubated in the fresh media for 5 days. PrestoBlue cell viability reagent (Life Technologies) (10% v/v) was then added to each well and the plate incubated for 60-90 min. Fluorescence intensity values (detected using a PerkinElmer EnVision 2101 multilabel reader with excitation filter at 540 nm and emissions filter at 590 nm) were determined relative to the untreated cell control.

[0345] As shown in FIGS. 1, 4, 6, 8, 9 and 20, the prodrug/catalyst system for each Vorinostat, Doxorubicin, Gemcitabine, 5FU and SN-38 prodrugs showed significant cytotoxic effects at each concentration tested, and calculated EC.sub.50 values similar to those calculated above for free active agent, as shown in Tables 1-3 (below):

TABLE-US-00001 TABLE 1 EC.sub.50 POB-Vor + Cell line Vorinostat Pd-resins A549 4.85 M 10.49 M U87G 11 M 24.70 M T98 12.4 M 16.77 M

TABLE-US-00002 TABLE 2 EC.sub.50 oPOBC-Dox + pPOBC-Dox + Cell line Doxorubicin Pd-resins Pd-resins A549 105 nM 81 nM 382 nM DU145 23 nM 2.6 M 1.1 M T98 320 nM 603 nM 360 nM

TABLE-US-00003 TABLE 3 EC.sub.50 pPOBC-Gem + Cell line Gemcitabine Pd-resins MiaPaCa2 13 nM 226 nM

Conclusions

[0346] The data show that the compounds (i.e. prodrugs) of the invention can be deprotected in a controlled manner using biocompatible palladium and/or gold catalyst to generate free active agent in situ, which exhibits the desired biological activity. The data show that prodrugs of the invention are suitably non-toxic and do not interfere with the active agent pathway, thus providing ideal active agent precursors. Furthermore, the by-products produced in the deprotection reaction are also biocompatible (e.g. propargyl groups provide 1-hydroxyacetone as by-product, benzyl groups provide 1,2 or 1,4 hydroxybenzyl alcohol as by-products).

[0347] The precise spatial control of prodrug deprotection provided by palladium nad/or gold implants, along with lack of toxicity of the prodrug compounds means that prodrugs of the invention can be deprotected specifically at the disease site, which should thus reduce general systemic concentration of the free active agent. This is especially desirable in cancer treatments where side-effects resulting from the active agent acting non-specifically on other organs in the body can be severe. This may also in turn allow prodrugs of the invention to be administered in higher doses, providing higher concentrations of active agent at the disease site than would have been tolerated through general systemic administration of the active agent due to risk of the side-effects mentioned above.

[0348] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and the spirit of the invention.