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PRO-CYCLIC DINUCLEOTIDES AND PRO-CYCLIC DINUCLEOTIDE CONJUGATES FOR CYTOKINE INDUCTION

20220339287 · 2022-10-27

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention provides a Pro-cyclic dinucleotide (Pro-CDN) comprising a STING agonist cyclic dinucleotide which is coupled to a linker system. The Pro-CDNs of the present invention can be metabolized at a targeted site into CDNs and exert their full immunomodulatory effects at said targeted site. The present invention also provides conjugates wherein a Pro-CDN is conjugated to a Biologically Active Molecule (BAM) such as e.g. a cytotoxic molecule, a lipid, a protein, a peptide, a nucleic acid, a sugar or a PRR ligand. The invention provides also methods related to the use of such compounds to perform their activities at their targeted sites, to exert cytotoxic, cytostatic or immunomodulatory effects, to treat or to prevent diseases such as cancers, immunological disorders or infections.

    Claims

    1. A Pro-CDN compound of Formula (I): ##STR00106## wherein: The CDN unit is a cyclic dinucleotide monophosphorothioate or diphosphorothioate of Formula (II.sub.a): ##STR00107## wherein: X.sub.1 and Y.sub.1 are independently H or F; X.sub.2 and Y.sub.2 are independently H or F; Z.sub.1 is O or S; R.sub.1 is H when Z.sub.1 is O; R.sub.1 is H or a linker system when Z.sub.1 is S; B.sub.1 and B.sub.2 are purine bases chosen from: ##STR00108## or pharmaceutically acceptable salts, stereoisomers, tautomers or solvates thereof; The linker system comprises: A connector which is a spontaneous self-eliminating group of Formulae (III.sub.a) to (III.sub.g) able to link with the CDN and the specifier: Formula (III.sub.a) is a Para-Amino-Benzvl (PAB) group: ##STR00109## Formula (III.sub.b) is a Para-Hydroxy-Benzyl (PHB) group: ##STR00110## Formula (III.sub.c) is a Para-Hydroxy-Meta-Trifluoromethyl-Benzyl (PHMTB) group: ##STR00111## Formula (III.sub.d) is a Para-Hydroxy-Meta-Nitro-Benzyl (PHMNB) group: ##STR00112## Formula (III.sub.e) is a Para-Hydroxy-Meta-Amino-Benzyl (PHMAB) group: ##STR00113## A group of Formula (III.sub.f) ##STR00114## wherein X.sub.3 is —O— or —NH—, and Y.sub.3 is —NO.sub.2, —NH.sub.2 or —CF.sub.3; A group of Formula (III.sub.g): ##STR00115## wherein X.sub.3 and Y.sub.3 are as defined above; A specifier which is an enzymatically cleavable unit.

    2. The Pro-CDN compound according to claim 1, wherein the CDN is a (3′,3′) CDN of Formula (II.sub.b): ##STR00116## wherein X.sub.1, Y.sub.1, X.sub.2, Y.sub.2, B.sub.1, B.sub.2, and R.sub.1 are as defined in claim 1.

    3. The Pro-CDN compound according to claim 1, wherein the CDN is a (2′,3′) CDN of Formula (II.sub.c) or a CDN of Formula (II.sub.d): ##STR00117## wherein X.sub.1, Y.sub.1, X.sub.2, Y.sub.2, B.sub.1, B.sub.2 and R.sub.1 are as defined in claim 1.

    4. The Pro-CDN compound according to claim 1, wherein the CDN is a (3′,2′) CDN of Formula (II.sub.e) or a CDN of Formula (II.sub.f): ##STR00118## wherein X.sub.1, Y.sub.1, X.sub.2, Y.sub.2, B.sub.1, B.sub.2 and R.sub.1 are as defined in claim 1.

    5. The Pro-CDN compound according to claim 1, wherein the CDN is a (3′,3′) CDN of Formulae (II.sub.g): ##STR00119## wherein B.sub.1, B.sub.2, and R.sub.1 are as defined in claim 1.

    6. The Pro-CDN compound according to claim 1, wherein the CDN is a (2′,3′) CDN of Formula (II.sub.h) or a CDN of Formula (II.sub.i): ##STR00120## wherein B.sub.1, B.sub.2 and R.sub.1 are as defined in claim 1.

    7. The Pro-CDN compound according to claim 1, wherein the CDN is a (3′,2′) CDN of Formula (II.sub.j) or a CDN of Formula (II.sub.k): ##STR00121## wherein B.sub.1, B.sub.2, and R.sub.1 are as defined in claim 1.

    8. The Pro-CDN compound according to claim 1, wherein the connector is a Para-Amino-Benzyl or a Para-amino-benzyl-carbonyl.

    9. The Pro-CDN compound according to claim 1, wherein the specifier is a peptide sequence or a sugar.

    10. The Pro-CDN compound according to claim 1, said Pro-CDN further comprising a spacer directly linked to the specifier or to the connector.

    11. The Pro-CDN compound of Formula (I) according to claim 1, wherein said Pro-CDN compound is chosen from: ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128##

    12. A BAM-CDN conjugate comprising the Pro-CDN compound of Formula (I) according to claim 1 and a biologically active molecule linked directly to the Pro-CDN compound of Formula (I) or through a spacer.

    13. The BAM-CDN conjugate according to claim 12, wherein the spacer is a hydrophilic group selected from: a polyethylene glycol (PEG); a polyamine; a compound of Formula (IV.sub.a): ##STR00129## wherein X.sub.3 is —O— or —NH—, m, n and p are an integer ranging from 0 to 12; a comnruind of Formula (IV.sub.b): ##STR00130## wherein q is an integer ranging from 1 to 6; r is an integer ranging from 1 to 6; s is an integer ranging from 1 to 6; a compound of Formula (IVc): ##STR00131## wherein each t and u is independently an integer ranging from 1 to 10; k is 0 or 1; w is an integer ranging from 0 to 6; or a compound of Formula (IV.sub.d): ##STR00132## wherein R.sub.2 and R.sub.3 and independently selected from H and an alkyl optionally substituted with an amino group.

    14. The BAM-CDN conjugate according to claim 12, wherein the biologically active molecule is chosen from: a protein for example an antigen or an antibody molecule, such as a monoclonal antibody, chimeric, a humanized or a human antibody or an antigen-binding fragment thereof; a peptide for example extracellular matrix (ECM)—super-affinity peptide derived from placenta growth factor-2 (P1GF-2.sub.123-144); a lipid to form for example a liposome; a fluorescent probe (FAM, HEX, TET, Cyanine dyes, JOE, ROX, TAMRA, Texas red . . . ); a PRR ligand (TLR, NOD ligands . . . ); a cytotoxic agent; a radio-sensitizing element; a small molecule inhibitor of protein for example a selective tyrosine kinase inhibitor or an Hsp90 inhibitor or IDO inhibitor or Carbonic Anhydrase IX/XII Inhibitor; a small molecule antagonist targeting PD-1/PD-L1 or other immune checkpoint; an activatory small molecule; a heterocycle molecule like folic acid; a particle like a liposome, a polymer based vehicles, or hyaluronic acid based delivery vehicles;

    15. BAM-CDN conjugate according to claim 12, wherein said conjugate is chosen from the compounds of Formulae (V.sub.a) to (V.sub.f): ##STR00133## ##STR00134## wherein: BAM is a biologically active molecule; The connector, connector 1 and connector 2 are spontaneous self-eliminating groups able to link the CDN and the specifier, independently chosen from groups of Formulae (III.sub.a) to (III.sub.g); Formula (III.sub.a) is a Para-Amino-Benzyl (PAB) group: ##STR00135## Formula (III.sub.b) is a Para-Hydroxy-Benzyl (PHB) group: ##STR00136## Formula (III.sub.c) is a Para-Hydroxy-Meta-Trifluoromethyl-Benzyl (PHMTB) group: ##STR00137## Formula (III.sub.d) is a Para-Hydroxy-Meta-Nitro-Benzyl (PHMNB) group: ##STR00138## Formula (III.sub.e) is a Para-Hydroxy-Meta-Amino-Benzyl (PHMAB) group: ##STR00139## wherein Y.sub.3 is —O— or —NH— and Y.sub.4 is —NO.sub.2, —NH.sub.2 or —CF.sub.3; ##STR00140## wherein Y.sub.3 and Y.sub.4 are as defined above; The specifier, specifier 1 and specifier 2 are enzymatically cleavable units, identical or different; The spacer is a hydrophilic group either polyethylene glycol (PEG) or a polyamine or a compound of Formula (IV.sub.a), (IV.sub.b), (IV.sub.c) or (IV.sub.d); ##STR00141## wherein X.sub.3 is —O— or —NH—, m, n and p are an integer ranging from 0 to 12; ##STR00142## wherein q is an integer ranging from 1 to 6; r is an integer ranging from 1 to 6; s is an integer ranging from 1 to 6; ##STR00143## wherein each t and u is independently an integer ranging from 1 to 10; k is 0 or 1; w is an integer ranging from 0 to 6; or ##STR00144## wherein R.sub.2 and R.sub.3 and independently selected from H and an alkyl optionally substituted with an amino group; v is 1 or 0; The CDN unit is a compound of Formula (II.sub.a): ##STR00145## wherein: X.sub.1 and Y.sub.1 are independently H or F; X.sub.2 and Y.sub.2 are independently H or F; Z.sub.1 is O or S; R.sub.1 is H when Z.sub.1 is O; R.sub.1 is H or a linker system when Z.sub.1 is S; B.sub.1 and B.sub.2 are purine bases chosen from: ##STR00146##

    16. The BAM-CDN conjugate compounds of Formulae (V.sub.a) to (V.sub.f) according to claim 15,wherein said conjugate is chosen from: ##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152##

    17. A pharmaceutical composition comprising a Pro-CDN according to claim 1 and a pharmaceutically acceptable excipient.

    18. (canceled)

    19. Method of treatment of a disease that may be alleviated by the induction of an immune response via the STING pathway comprising the administration to a patient of an effective amount of a Pro-CDN according to claim 1.

    20. An immunomodulatory agent comprising Pro-CDN according to claim 1.

    21. Method of treatment of cancer or pre-cancerous syndromes or infectious diseases, comprising the administration to a patient of an effective amount of a Pro-CDN according to claim 1.

    22. An immunoadjuvant comprising a Pro-CDN according to claim 1.

    23. A therapeutic combination comprising a Pro-CDN according to claim 1 and a therapeutic agent.

    24. A method for treating cancer, said method comprising administering to a patient in need thereof: a Pro-CDN according to claim 1 and a chemotherapeutic agent.

    25. A pharmaceutical composition comprising a BAM-CDN conjugate according to claim 12 and a pharmaceutically acceptable excipient.

    26. Method of treatment of a disease that may be alleviated by the induction of an immune response via the STING pathway comprising the administration to a patient of an effective amount of a BAM-CDN conjugate according to claim 12.

    27. An immunomodulatory agent comprising a BAM-CDN conjugate according to claim 12.

    28. Method of treatment of cancer or pre-cancerous syndromes or infectious diseases, comprising the administration to a patient of an effective amount of a BAM-CDN conjugate according to claim 12.

    29. An immunoadjuvant comprising a BAM-CDN conjugate according to claim 12.

    30. A therapeutic combination comprising a BAM-CDN conjugate according to claim 12 and a therapeutic agent.

    31. A method for treating cancer, said method comprising administering to a patient in need thereof: a BAM-CDN conjugate according to claim 12 and a chemotherapeutic agent.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0377] FIG. 1. Comparison of ISG54 (A-B, D-E) and NFκB (C and F) activity induced (time indicated above) by Pro-CDN (CDN-linker system: CL793 vs CL702, CL802 and CL804 vs CL797) of the present invention in a murine (A, D) or a human (B-C and E-F) reporter cell.

    [0378] FIG. 2: Dose-response curves representing induction of Type I IFN for Pro-CDNs compounds CL793 (A) or CL802 and CL804 (B) in whole-blood assay in comparison to parental molecules CL702 or CL797 and the endogenous STING agonist 2′3′-cGAMP. The data represent the mean OD±sem from ten healthy donors (FIG. 2A) or the mean OD from two healthy donors (FIG. 2B).

    [0379] FIG. 3: Comparison of ISG54 (A-B) and NF-κB (C) activity induced (time indicated above) by a BAM-CDN (CL808) of the present invention in a murine (A) or a human (B and C) reporter cell.

    [0380] FIG. 4: Dose-response curves representing induction of type I IFN (A), IL1α/β (B), IL6 (C) and TNFα (D) for BAM-CDN compound (CL808 full black triangle) relative to the unmodified corresponding CDN (CL702 empty square) in whole-blood assay. The data represent the mean OD±sem from two healthy donors.

    [0381] FIG. 5: Theoretical example of deconjugation through a retro-Michael pathway and comparison with CL874 pro-CDN compound.

    [0382] FIG. 6: Comparison of ISG54 activity induced by lipidated-CDN with the unmodified CDNs (CL797 left part, CL845 in the middle section and CL656 right part) in a human (above panel) or a murine (below panel) reporter cell.

    [0383] FIG. 7: BAM-CDN dependent ISG Activity. BAM-CDN dependent ISG activity from the present invention was assessed in RAW ISG in comparison with the unmodified CDN (CL797 upper panels, CL845 in the middle section and CL656 lower part).

    [0384] FIG. 8: BAM-CDN dependent ISG activity and internalization quantitation. A- BAM-CDN dependent ISG activity from the present invention was assessed in THP1 Dual in comparison with a commercially available fluorescent-CDN B-D: Fluorescence intensity in the intracellular compartment of THP1-Dual cells stimulated with CL832 was measured by flow cytometry in a dose dependent manner (B) or time dependently (C and D) at 10 μM.

    [0385] FIG. 9: BAM-CDN dependent ISG and TLR7 activity. A—BAM-CDN dependent ISG activity from the present invention was assessed in THP1 Dual (circle) or Raw ISG (square) after 24h of stimulation. B—In comparison with a free TLR7 agonist (CL264), CL264-CDN dependent TLR7 activity from the present invention was assessed in HEK Blue TLR7 cell line after 24h of stimulation.

    [0386] FIG. 10: Folic acid-CDN dependent ISG activity. FA-CDN dependent ISG activity from the present invention was assessed in RAW ISG (black) or THP1 Dual (grey) after 24h or 48h of stimulation using 10 or 30 μM dose.

    [0387] FIG. 11: Adjuvantation potential of compound from the present invention. Mice were subcutaneously immunized with 10 μg of Ovalbumin alone or linked with CDN of the present invention. Antibody title reacting against Ova was quantified 2 weeks after the first immunization in comparison with the positive control (Ova+Alum) or with OVA in combination with an equivalent amount of free CDN (CL656 eq.1 for OVA-CL862 and CL656 eq.2 for OVA-CL855) based on the DAR estimated by UV. ** means significantly different from Ova.

    EXAMPLES

    [0388] Specific compounds that are representative of this invention were prepared as per the following examples and are offered by way of illustration to aid in the understanding of the invention. They should not be construed to limit in any way the invention set forth in the claims that follow thereafter. The present compounds can also be used as intermediates in subsequent examples to produce additional compounds of the present invention. No attempt has necessarily been made to optimize the yields obtained in any of the reactions. One skilled in the art would know how to increase such yields through routine variations in reaction times, temperatures, solvents, reagents and chemical synthesis other parameters.

    [0389] The present invention is further illustrated in Example 1, which shows preparative methods for synthesizing the conjugates, and in Example 2, which shows methods for the biological evaluation of these conjugates. It will be understood by one of ordinary skill in the art that these examples are in no way limiting and that variations of details can be made without departing from the spirit and scope of the present invention.

    [0390] The terms used in describing the invention are commonly used and known to those skilled in the art. As used herein, the following abbreviations have the indicated meanings:

    [0391] ° C. for degrees Celsius; A for adenosine; Aca for 6-Aminocaproic acid; ACN for acetonitrile; AEEA for [2-(2-aminoethoxy)ethoxy]acetic acid; AEEEEP for 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propandic add; Ala for Alanine; AP-1 for Activator protein 1; Arg for arginine; AU for Arbitrary Unit; BF.sub.3.Et.sub.2O for Boron trifluoride ethyl etherate; Boc for tert-butoxycarbonyle; Bz for benzoyl; CE for cyanoethyl; C.sub.18 for octadecyl carbon chain bonded silica; C.sub.14/C.sub.14—CO— for 3-(tetradecanoyloxy)tetradecanoate; CHCl.sub.3 for chloroform; Cit for citrulline; CMV for cytomegalovirus; CO.sub.2 for carbon dioxide; 2-CTC resin for 2-Chlorotrityl chloride resin ; CTLA4 for cytotoxic T-lymphocyte-associated protein 4 ; Da for Dalton; dA for deoxyadenosine; Dap for 2,3-diaminopropionic acid; dAMP for deoxyadenosine monophosphate; DAR for Drug Antibody Ratio; ICl for immune checkpoint inhibitors; dl for deoxyinosine; dIMP for deoxyinosine monophosphate; D.sub.2O for deuterium oxide; DCA for dichloroacetic acid; DCM (or CH.sub.2Cl.sub.2) for dichloromethane; DIEA for diisopropyl-ethyl-amine; DMAP for Dimethyl aminopyridine; DMF for dimethyl formamide, DMSO for dimethylsulfoxide; DMSO-d.sub.6 for deuterated dimethylsulfoxide; DMTrCI for 4;4′-dimethoxytrityl chloride; DTT for Dithiothreitol; EC.sub.50 for Half maximal effective concentration; equiv. for equivalent; EDCI for N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; ES for electrospray ionization; Et.sub.2O for diethyl ether; EtOAc for ethyl acetate; EtOH for ethanol; FITC for Fluorescein isothiocyanate; Fmoc for fluorenylmethyloxycarbonyl; βGAL for beta-galactosidase; g for grams; Gp75 for glycoprotein 75; .sup.1H for proton; h for hours; HATU for 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid-hexafluoro-phosphate; HBV for hepatitis B virus; HCV for hepatitis C virus; HEK for Human embryonic kidney cells; Hz for Hertz; HER.sub.2 for human epidermal growth factor receptor 2; HPLC for high-performance liquid chromatography; I for inosine; HIV for human immunodeficiency virus; HTLV for Human T cell Leukemia/lymphoma Virus; IFNAR1 for interferon alpha receptor 1 ; IFNAR2 for interferon alpha receptor 2; IFN for interferon; IFN-α for interferon alpha; IFN-β for interferon beta; IL for Interleukin; IRF3 for interferon regulatory factor 3; ISG (or ISG54) for interferon-stimulated gene; ISAC for immunostimulatory antibody conjugate; ISRE for interferon-stimulated response element; JAK1 for Janus kinase 1; kD for kilo Dalton; Lev for levulinic; LC for liquid chromatography; Luc for luciferase; m for multiplet; M for molar; mAb for monoclonal antibody; Mal for maleimide; m/z for mass-to-charge ratio; MeOH for methanol; mg for milligrams; Me-Su- for 4-methoxy-4-oxobutanoic acid; MgSO.sub.4 for magnesium sulfate; MHz for megahertz; min for minutes; mL (or ml) for milliliters; mmol for millimoles; mol/L for mole/liter; MS for mass spectrometry; MSNT for 1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole; MyD for Myeloid differentiation primary response; NaCl for sodium chloride; NaHCO.sub.3 for sodium bicarbonate; Na.sub.2HPO.sub.4 for disodium hydrogen phosphate; NaHSO.sub.3 for sodium thiosulfate; Nal for sodium iodide; NEM for N-ethyl maleimide; NF-κB for nuclear factor kappa-light-chain-enhancer of activated B cells; NMR for nuclear magnetic resonance; Ova for ovalbumin; .sup.31P for phosphorus; PAB for para-amino-benzyl; PADS for phenylacetyl disulfide; Pd for Palladium; Pdl for polydispersity index determination; PD-1 for Programmed cell death 1; PDL1 for Programmed death-ligand 1; PEG for polyethylene glycol; Phe for phenylalanine; ppm for parts per million; Piv for pivaloyl; POM for pivaloyl-oxy-methyl; PS for phosphorothioate; rt or RT for room temperature; Rt for retention time; SEAP for secreted embryonic alkaline phosphatase; s for singlet; KO for knockout; SPPS for solid phase peptide synthesis; STAT1 for Signal transducer and activator of transcription 1; STING for stimulator of interferon genes; SOCl.sub.2 for thionyl chloride; TEAB for triethyl ammonium carbonate; THF for tetrahydrofuran; TBDMSCI for tert-butyldimethylsilyl chloride; TFA for trifluoroacetic acid; TLR for toll like receptor; TNF for tumor necrosis factor; TyK2 for Tyrosine Kinase 2; UV for ultra-violet; Val for valine; μg for microgram; μL (or μl) for microliter; μm for micrometer; μmol for micromole; δ for chemical shift; ε for extinction coefficients, λmax for maximum absorbance wavelengths.

    [0392] Anhydrous solvents and reagents suitable for nucleoside and nucleotide synthesis were purchased and were handled under dry argon or nitrogen using anhydrous technique. Amidite coupling reactions and cyclizations were performed in anhydrous acetonitrile or pyridine under dry argon or nitrogen. The starting materials for all reactions in dry pyridine were dried by concentration (three times) from pyridine. Preparative silica-gel flash chromatography was performed using Fluka 60 Å high-purity grade or Merck Grade 9385 silica using gradients of methanol in dichloromethane. Analytical LC/ES MS was performed on an Agilent 1290 Infinity UHPLC system coupled to a diode array detector (DAD) Agilent 1260 Infinity and an Agilent 6130 Quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) and controlled by Chemstation software. The LC system was equipped with an Aquity CSH C18 50×2.1 mm 1.7 μm column using gradients of 10 mM ammonioum formate and acetonitrile at 300 μl/min flow. The UV detection wavelength was 254 nm. The mass spectrometer was operated in positive and negative ESI modes Preparative HPLC was performed on a Waters preparative 150Q HPLC system monitoring at 254 nm on a SunFire Prep C18 5 μm OBD 30×150mm column using gradients of 10 mM ammonium formate and acetonitrile at a flow rate 60 mL/min. The .sup.1H NMR spectra were acquired on either a Bruker 300 MHz (Fourrier 300) at room temperature and reported in ppm downfield. Molecular sieves (MS) 3Å were employed after drying the commercially supplied product at 250° C. for 12 h under vacuum. The commercial nucleoside phosphoramidites were supplied from Chemgenes.

    [0393] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

    Example 1: Synthesis of the Compounds of the Invention

    Example 1.1: Synthesis of CDNs of the Invention

    Intermediate 1.1: 3′-O-(Allyl,CE)phosphotriester-N.SUP.6.-(Bz)-2′-fluoro-2′-deoxy-Adenosine

    [0394] Commercially available 2′-fluoro-2′-deoxy-Adenosine phosphoramidite (30.0 g, 34.2 mmol) was co-evaporated with dry ACN three times, and the resulting solid was dissolved in a solution of Activator 42® (0.1 M in ACN) (685.0 mL, 22.8 mmol) in the presence of molecular sieves (3 Å). Allyl alcohol (4.66 mL, 68.5 mmol) was added, and the resulting mixture was stirred for 30 min. Then, 5.5 M tert-butyl hydroperoxide in decane (12.45 ml, 68.5 mmol) was added, and the mixture was stirred for 40 min. The solution was filtered, the molecular sieves were washed with DCM, and the filtrate was concentrated in vacuo. The residue was treated with 3% DCA in CH.sub.2Cl.sub.2 (750 mL), and water (10 equiv.), for 15 min. The reaction was quenched with MeOH and pyridine. The solvents were removed in vacuo, and the residue was purified by silica-gel column chromatography, using CH.sub.2Cl.sub.2/MeOH (5% to 20%) as eluent, to give 13.0 g (69% yield) of Intermediate 1.1 LC-MS: Rt=4.50 min, m/z=547 [M+H].sup.+, m/z=545 [M−H].sup.−.

    Intermediate 1.2: [3′-O-(CE)phosphorothioate triester-2′-deoxy-Inosine]-(3′,5′)-[3′-O-(Allyl,CE) phosphotriester-N.SUP.6.-(Bz)-2′-fluoro-2′-deoxy-Adenosine]

    [0395] Intermediate 1.1 (13.0 g, 23.79 mmol) was dissolved in a solution of Activator 42® (0.1 M in ACN) (476 mL, 47.6 mmol) in the presence of molecular sieves (3 Å). Commercially available phosphoramidite of 2′-deoxy-Inosine (18.85 g, 38.91 mmol) was added to the solution in one portion. The mixture was stirred for 30 min at RT. Then, a solution of PADS (23.8 g, 47.6 mmol) in dry pyridine (23.8 mL) was added, and the mixture was stirred for 1 h. The resulting solution was filtered, the molecular sieves were washed with CH.sub.2Cl.sub.2, and the filtrate was concentrated in vacuo. The residue was treated with 3% DCA in CH.sub.2Cl.sub.2 (500 mL) and water (10 equiv.), for 15 min. The reaction was quenched with MeOH and pyridine. The solvents were removed in vacuo, and the residue was purified by silica-gel column chromatography, using CH.sub.2Cl.sub.2/MeOH (5% to 20%) as eluent, to provide 10.62 g (59% yield) of Intermediate 1.2. Rt=4.12 min, m/z=930 [M+H].sup.+, m/z=928 [M−H].sup.−.

    Intermediate 1.3: [3′-O-(CE)phosphorothioate triester-2′-fluoro-2′-deoxy Inosine]-(3′,5′)-[3′-O-(CE)phosphodiester-N.SUP.6.-(Bz)-2′-deoxyAdenosine]

    [0396] To a solution of Intermediate 1.2 (10.6 g, 15.42 mmol) in dry THF (100 mL) N-methyl aniline (3.70 g, 34.26 mmol) and tetrakis(triphenylphosphine)palladium(0) (2.64 g, 2.28 mmol) were added. The resulting suspension was stirred at rt for 2 h. Then, the solvent was removed in vacuo, and the residue was triturated with diethyl ether. The resulting colorless precipitate was collected by filtration, and then washed with chilled diethyl ether. The crude product was purified by silica-gel column chromatography, using DCM/MeOH (0% to 10%) as eluent, to give 8.18 g (80% yield) of Intermediate 1.3. LC-MS: Rt=2.92 min, m/z=890 [M+H].sup.+, m/z=888 [M−H].sup.−

    Intermediate 1.4: (3′,3′)Cyclic-[3′-O-(CE)phosphotriester-N.SUP.6.-(Bz)2′-deoxy-Adenosine]-[3′-O-(CE)phosphorothioate triester-2′-fluoro-2′-deoxy-Inosine]

    [0397] Intermediate 1.3 (10.74 g, 12.08 mmol) was co-evaporated with dry pyridine three times. The residue was suspended in dry pyridine (1320 mL), and 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) (17.88 g, 60.4 mmol) was added to the resulting solution. The resulting mixture was stirred at 25° C. for 1 h. Then, the solvent was removed in vacuo the residue was suspended in EtOAc and was washed with water and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude product was purified by silica-gel column chromatography, using DCM/MeOH (0% to 10%) as eluent, to give 4.20 g (40% yield) of Intermediate 1.4. LC-MS: Rt=3.59 and 3.80 min, m/z=872 [M+H].sup.+, m/z=870 [M−H].sup.−.

    Intermediate 1.5:

    [0398] ##STR00060##

    [0399] Intermediate 1.4 (1.0 g, 1.15 mmol) was treated with a solution of methylamine in EtOH (33%), and the resulting mixture was stirred at rt for 4 h. The reaction mixture was concentrated. The crude was subjected to preparative HPLC using a C.sub.18 Sunfire column (19×150 mm, 51.1.m) and ammonium formate/ACN as eluent. The fractions containing the desired compound were pooled and lyophilized to provide 0.68 g; (89% yield) of Intermediate 1.5. LC-MS: Rt=0.590 and 0.501 min, m/z=662 [M+H].sup.+, m/z=660 [M−H].sup.−. .sup.1H NMR (D.sub.2O, 300 MHz) δ (ppm) 7.79-8.44 (m, 5H), 6.23-6.34 (m, 2H), 5.55 (m, 1H), 5.36-4.90 (m, 3H), 4.51 (m, 2H), 4.32 (m, 2H), 4.08 (m, 2H), 3.59 (m, 1H), 3.02 (m, 1H), 2.69 (m, 1H).

    Intermediate 1.6: 5′-O-DMTr-2′-deoxy-Inosine

    [0400] The commercially available 2′-deoxy-Inosine (10.0 g, 39.6 mmol) was co-evaporated three times with dry pyridine. The residue was suspended in dry pyridine. DMAP (7.27 g, 59.5 mmol) and DimethoxyTritryl chloride (DMTrCI; 20.15 g, 59.5 mmol) were added to the resulting solution. The resulting mixture was stirred at 40° C. overnight. Then, the reaction was stopped by addition of methanol, the reaction mixture was diluted with EtOAc and was washed with saturated NaHCO.sub.3, water and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude compound was purified by silica-gel column chromatography, using DCM/MeOH with 0.5% pyridine as eluent to provide 10.0 g (45% yield) of Intermediate 1.6. LC-MS: Rt=4.91 min, m/z=555 [M+H].sup.+, m/z=553 [M−H].sup.−.

    Intermediate 1.7: 5′-O-DMTr-3′-O-Lev-2′-deoxy-Inosine

    [0401] To a solution of Intermediate 1.6 (9.79 g, 17.65 mmol) in dry DMF (100 mL) EDCI (5.08 g, 26.5 mmol), DMAP (2.59 g, 21.18 mmol) and levulinic acid (2.89 mL, 28.2 mmol) were added. The resulting mixture was stirred at rt overnight. Then, the reaction mixture was diluted with EtOAc and was washed with saturated NaHCO.sub.3, water and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude compound was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 6.16 g (53% yield) of Intermediate 1.7. LC-MS: Rt=5.37 min, m/z=653 [M+H].sup.+, m/z=651 [M−H].sup.−.

    Intermediate 1.8: 5′-O-DMTr-3′-O-Lev-N.SUP.1.-POM-2′-deoxy-Inosine

    [0402] To a solution of Intermediate 1.7 (6.16 g, 9.44 mmol) in dry DMF (120 mL) K.sub.2CO.sub.3 (3.91 g, 28.3 mmol), and POM-CI (1.70 g, 11.3 mmol) were added. The resulting mixture was stirred at rt overnight. Then, the reaction mixture was diluted with EtOAc and was washed with saturated NaHCO.sub.3, water and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude compound was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 5.33 g (74% yield) of Intermediate 1.8, which was used in the next step without any further purification. LC-MS: Rt=6.36 min, m/z=767 [M+H].sup.+, m/z=765 [M−H].sup.−.

    Intermediate 1.9: 3′-O-Lev-N.SUP.1.-POM-2′-deoxy-Inosine

    [0403] Intermediate 1.8 (5.30 g, 6.91 mmol) was treated with a solution of DCA/DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched by addition of MeOH and pyridine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 3.03 g (94% yield) of Intermediate 1.9. LC-MS: Rt=4.03 min, m/z=465 [M+H].sup.+, m/z=463 [M−H].sup.−.

    Intermediate 1.10: [N.SUP.6.-Bz-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-(3′,5)-[N.SUP.1.-POM-3′-O-Lev-2′-deoxy-Inosine]

    [0404] Intermediate 1.9 (3.01 g, 6.48 mmol) and commercially available phosphoramidite of 2′-fluoro-2′-deoxy-Adenosine (5.90 g, 6.74 mmol) was co-evaporated three times with dry ACN, and the resulting solid was dissolved in a solution of Activator 42® (0.1 mol/L, 2 equiv.; 130 mL) in the presence of molecular sieves (3Å). The resulting mixture was stirred for 45 min. Then, a solution of PADS (3.91 g, 12.96 mmol) in pyridine was added to the mixture, which was stirred for 35 min. The solution was filtered and the molecular sieves were washed with DCM. The filtrate was concentrated in vacuo. The residue was treated with a solution of DCA/DCM (3%) in the presence of water (10 equiv.) for 15 min. The reaction was quenched with addition of MeOH and pyridine. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 4.90 g (78% yield) of Intermediate 1.10. LC-MS: Rt=5.17 min, m/z=969 [M+H].sup.+, m/z=967 [M−H].sup.−.

    Intermediate 1.11: [N.SUP.6.-Bz-5′-O-H-phosphonate-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-(3′,5)-[N.SUP.1.-POM-3′-O-Lev-2′-deoxy-Inosine]

    [0405] To a solution of Intermediate 1.10 (4.78 g, 4.93 mmol) in dry pyridine (100 mL) was added diphenyl phosphite (1.91 mL, 9.87 mmol), the resulting mixture was stirred at room temperature for 2 hours, quenched by an addition of triethylammonium acetate (3.78 mL, 4 equiv.), and then concentrated under reduced pressure. The residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 2.76 g (54% yield) of Intermediate 1.11. LC-MS: Rt=4.27 min, m/z=1033 [M+H].sup.+, m/z=1031 [M−H].sup.−.

    Intermediate 1.12: [N.SUP.6.-Bz-5′-O-H-phosphonate-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-(3′,5)-[N.SUP.1.-POM-3′-O-Lev-2′-deoxy-Inosine]

    [0406] Intermediate 1.11 (2.75 g, 2.66 mmol) was treated with a solution of 0.5 M hydrazine (0.99 mL, 13.31 mmol) in a mixture of pyridine/acetic acid (3:2) (27 mL) for 15 min. The reaction was quenched by addition of pentanedione (2.73 mL, 26.62 mmol). The solvents were removed in vacuo and the residue was triturated with diethyl ether to provide 2.45 g (99% yield) of Intermediate 1.12, which was used in the next step without any further purification. LC-MS: Rt=4.02 min, m/z=935 [M+H].sup.+, m/z=933 [M−H].sup.−.

    Intermediate 1.13: (3′,3′)Cyclic-[N.SUP.6.-Bz-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-[N.SUP.1.-POM-3′-O-phosphorothioate-diester-2′-deoxy-Inosine]

    [0407] Intermediate 1.12 (2.50 g, 2.67 mmol) was coevaporated with dry pyridine three times. The residue was suspended in dry pyridine (150 mL) and PivCl (1.02 mL, 8.29 mmol) was added to the resulting solution. The resulting mixture was stirred at 25° C. for 30 min. Then, sulfur (0.26 g, 8.29 mmol) was added and the new suspension was stirred for 45 min at rt. The solvent was removed in vacuo to give 2.54 g (100% yield) of crude Intermediate 1.13. This compound was used in the next step without any further purification. LC-MS: Rt=4.62 and 4.78 min, m/z=949 [M+H]+, m/z=947 [M−H]−

    Intermediate 1.14:

    [0408] ##STR00061##

    [0409] Intermediate 1.14 (1.68 g, 94% yield) was prepared from Intermediate 1.13 (2.50 g, 3.69 mmol) using a similar procedure to that described for Intermediate 1.5. LC-MS: Rt=0.844, 0.946, 2.28, 2.39, 2.72, 2.68 min, m/z=678 [M+H].sup.+, m/z=676 [M−H].sup.−. .sup.1H NMR (D.sub.2O, 300 MHz) δ (ppm) 8.48 (s, 1H), 8.24 (s, 1H), 8.21 (s, 1H), 8.04 (s, 1H), 7.96 (s, 1H), 7.90 (s, 1H), 6.30 (m, 2H), 5.70 (m, 1H), 5.53 (m, 1H), 5.02 (m, 2H), 4.45 (m, 1H), 4.21 (m, 1H), 4.09 (m, 2H), 4.02 (m, 2H), 3.94 (m, 2H), 3.10 (m, 1H), 2.90 (m, 1H)..sup.311.sup.3 NMR (D.sub.2O, 202 MHz) δ (ppm) 55.95, 55.71 and 54.76, 54.29.

    Intermediate 1.15: 5′-O-DMTr-2′-deoxy-2′-fluoro-Inosine

    [0410] Intermediate 1.15 (52.0 g, 98% yield) was prepared from commercially available 2′-deoxy-2′-fluoro-Inosine (25.0 g, 92.5 mmol) using a similar procedure to that described for Intermediate 1.6. LC-MS: Rt=5.13 min, m/z=573 [M+H].sup.+, m/z=571 [M−H].sup.−.

    Intermediate 1.16: 5′-O-DMTr-3′-O-Lev-2′-deoxy-2′-fluoro-Inosine

    [0411] Intermediate 1.16 (57.8 g, 95% yield) was prepared from Intermediate 1.15 (52.0 g, 91.2 mmol) using a similar procedure to that described for Intermediate 1.7. LC-MS: Rt=5.59 min, m/z=671 [M+H].sup.+, m/z=669 [M−H].sup.−.

    Intermediate 1.17: 5′-O-DMTr-3′-O-Lev-N.SUP.1.-POM-2′-deoxy-2′-fluoro-Inosine

    [0412] Intermediate 1.17 (67.8 g, 99% yield) was prepared from Intermediate 1.16 (57.8 g, 86.5 mmol) using a similar procedure to that described for Intermediate 1.8. LC-MS: Rt=6.60 min, m/z=785 [M+H].sup.+, m/z=783 [M−H].sup.−.

    Intermediate 1.18: 3′-O-Lev-N.SUP.1.-POM-2′-deoxy-2′-fluoro-Inosine

    [0413] Intermediate 1.18 (35.7 g, 82% yield) was prepared from Intermediate 1.17 (67.8 g, 86.5 mmol) using a similar procedure to that described for Intermediate 1.9. LC-MS: Rt=4.17 min, m/z=483 [M+H].sup.+, m/z=481 [M−H].sup.−.

    Intermediate 1.19: [N.SUP.6.-Bz-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-(3′,5)-[N.SUP.1.-POM-3′-O-Lev-2′-deoxy-2′-fluoro-Inosine]

    [0414] Intermediate 1.19 (10.7 g, 99% yield) was prepared from Intermediate 1.18 (9.5 g, 10.8 mmol) and commercially available phosphoramidite of 2′-fluoro-2′-deoxy-Adenosine (5.23 g, 10.8 mmol) using a similar procedure to that described for Intermediate 1.10. LC-MS: Rt=5.28 min, m/z=987 [M+H].sup.+, m/z=985 [M−H].sup.−.

    Intermediate 1.20: [N.SUP.6.-Bz-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-(3′,5)-[N.SUP.1.-POM-2′-deoxy-2′-fluoro-Inosine]

    [0415] Intermediate 1.20 (6.46 g, 67% yield) was prepared from Intermediate 1.19 (10.7 g, 10.8 mmol) using a similar procedure to that described for Intermediate 1.11. LC-MS: Rt=4.89 min, m/z=889 [M+H].sup.+, m/z=887 [M−H].sup.−.

    Intermediate 1.21: (3′,3′)Cyclic-[N.SUP.6.-Bz-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-[N.SUP.1.-POM-3′-O-(CE)phosphorothioate-triester-2′-deoxy-2′-fluorolnosine]

    [0416] Intermediate 1.20 (10.3 g, 11.6 mmol) was co-evaporated three times with dry ACN, and the resulting solid was dissolved in a solution of Activator 42® (0.1 mol/L, 2 equiv.; 460 mL) in the presence of molecular sieves (3Å). Commercially available 2-Cyanoethyl-N,N-diisopropyl-chloro-phosphoramidite (4.2 g, 13.9 mmol) was added dropwise to the solution. The resulting mixture was stirred for 45 min. Then, a solution of PADS (7.01 g, 23.2 mmol) in pyridine was added to the mixture, which was stirred for 35 min. The solution was filtered and the molecular sieves were washed with DCM. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 11.0 g (98% yield) of Intermediate 1.21. LC-MS: Rt=5.55, 5.77, 5.89 and 6.06 min, m/z=1020 [M+H].sup.+, m/z=1018 [M−H].sup.−.

    Intermediate 1.22:

    [0417] ##STR00062##

    [0418] Intermediate 1.22 (5.3 g, 66% yield) was prepared from Intermediate 1.21 (11.0 g, 11.0 mmol) using a similar procedure to that described for Intermediate 1.5. LC-MS: Rt=3.41 min, m/z=696 [M+H].sup.+, m/z=694 [M−H].sup.−. .sup.1H NMR (D.sub.2O, 300 MHz) δ (ppm) 8.55 (s, 1H), 8.38 (s, 1H), 8.20 (s, 1H), 7.99 (s, 1H), 6.63 (dd, 1H), 6.15 (dd, 1H), 5.16-4.95 (m, 4H), 4.52 (m, 4H), 4.07 (m, 2H).

    Intermediate 1.23: (3′,3′)Cyclic-[N.SUP.6.-Bz-3′-O-(CE)phosphorothioate-triester-2′-fluoro-2′-deoxy-Adenosine]-[N.SUP.1.-POM-3′-O-(CE)phosphotriester-2′-deoxy-2′-fluorolnosine]

    [0419] Intermediate 1.20 (6.2 g, 7.3 mmol) was co-evaporated three times with dry ACN, and the resulting solid was dissolved in a solution of Activator 42® (0.1 mol/L, 2 equiv.; 290 mL) in the presence of molecular sieves (3Å). Commercially available 2-Cyanoethyl-N,N-diisopropyl-chloro-phosphoramidite (2.64 g, 8.74 mmol) was added dropwise to the solution. The resulting mixture was stirred for 45 min. Then, a solution of tert-Butyl hydroperoxide (5.5 M in decane) (5 eq.) was added to the mixture, which was stirred for 35 min. The solution was filtered and the molecular sieves were washed with DCM. The solvents were removed in vacuo and the residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 3.55 g (49% yield) of Intermediate 1.23. LC-MS: Rt=5.01, 5.13, 5.20, and 5.30 min, m/z=1004 [M+H].sup.+, m/z=1002 [M−H].sup.−.

    Intermediate 1.24:

    [0420] ##STR00063##

    [0421] Intermediate 1.24 (2.4 g, 99%) was prepared from Intermediate 1.23 (3.5 g, 3.5 mmol) using a similar procedure to that described for Intermediate 1.5. LC-MS: Rt=0.57 and 0.81 min, m/z=680 [M+H].sup.+, m/z=678 [M−H].sup.−. .sup.1H NMR (D.sub.2O, 300 MHz) δ (ppm) 8.34 (s, 1H), 8.27 (s, 1H), 8.20 (s, 2H), 7.99 (s, 1H), 7.92 (d, 1H), 6.35 (d, 1H), 6.24 (dd, 1H), 5.67 (m, 1H), 5.51 (m 1H), 5.06 (m, 4H), 4.51-4.41 (m, 8H) 4.07 (m, 4H).

    Example 1.2 : Synthesis of the Linkers of the Invention

    Intermediate 2.1: Boc-Val-Cit-PAB-Cl

    [0422] Commercially available dipeptide Boc-Val-Cit-PAB-OH (0.490 g, 1.02 mmol) was dissolved in dry Et.sub.2O (20 mL) and was cooled to 0° C. SOCl.sub.2 (82 μL, 1.12 mmol) was added dropwise to the solution and the mixture was stirred at rt overnight. The reaction was stopped by addition of a saturated solution of NaHCO.sub.3. The layers were separated and the organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was purified by silica-gel column chromatography, using DCM/MeOH as eluent to provide 0.260 g (51% yield) of Intermediate 2.1. LC-MS: Rt=4.76 min, m/z=499 [M+H].sup.+, m/z=497 [M−H].sup.−.

    Intermediate 2.2: Boc-Val-Cit-PAB-I

    [0423] Intermediate 2.1 (0.100 g, 0.20 mmol) was dissolved in dry ACN (10 mL) and Nal (0.06 g, 0.40 mmol) was added and the mixture was stirred at rt overnight in the dark. The reaction was stopped by addition of EtOAc and a solution of NaHSO.sub.3 (5%). The layers were separated and the organic layer was washed with water and brine dried over MgSO.sub.4, filtered and concentrated in vacuo to provide 0.145 g (51% yield) of Intermediate 2.2. This compound was used in the next step without any further purification. LC-MS: Rt=4.99 min, m/z=590 [M+H].sup.+, m/z=588 [M−H].sup.−.

    Intermediate 2.3: Solid phase synthesis of Fmoc-Val-Ala-OH

    [0424] A 150 mL peptide reactor was purged with nitrogen and then charged with 2 g of 2-CTC resin and 80 mL of DCM. The mixture was stirred for 15 min and the solvent was drained; this was repeated 2 times, then the resin was suspended in 80 mL of DMF. The resin-DMF mixture was stirred at rt for 30 min. Meanwhile, Fmoc-AlaOH (2.90 g, 9.30 mmol) in 40 mL DMF, and DIEA (162 mL, 9.30 mmol) were charged to a 100 mL flask. The contents of the flask were stirred at rt to dissolve the solid. After the DMF was drained from the reactor, the mixture containing the Fmoc-AlaOH was charged to the reactor with the resin and stirred. After 4 h the reactor was drained. Active sites on the resin were end-capped with a mixture of DIEA:MeOH (1:9 mL). This mixture was then stirred at rt for 1 h. The bed was drained, washed 2 times with 40 mL DMF, 2 times with 40 mL DCM, and one time with 40 mL DMF. The last wash demonstrated a negative UV test.

    [0425] To the reactor containing Fmoc-Ala-O-2-CTC resin was charged 60 mL of 20% piperidine in DMF which was then stirred at rt for 30 min. The reactor was drained and then charged with 60 mL of 20% piperidine in DMF. The mixture was stirred for 30 min at rt and the reactor drained. The resin bed was then washed 3 times with 40 mL DMF, 3 times with 40 mL of DCM, 3 times with 40 mL of MeOH and 3 times with 40 mL of DMF. The last wash was then sampled for piperidine levels by qualitative ninhydrin test.

    [0426] Then, Fmoc-Val-OH (3.16 g, 9.30 mmol), DIEA (1.62 mL, 9.30 mmol) and 15 mL DMF were charged to a flask. The contents were stirred at ambient temperature to dissolve the solid which were then cooled to 10° C. Then HATU (3.54 g, 9.30 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 3 times with 40 mL DMF, 3 times with 40 mL of DCM, 3 times with 40 mL of MeOH and 3 times with 40 mL of DMF.

    [0427] Cleavage from resin was achieved with 5 cycles of 15 min each at rt of 15 mL of a solution 2% TFA, 1% TES in dichloromethane. This solution was charged to the solid phase reactor containing the Intermediate 2.3, the reaction was stirred; the colour of the reaction changed from cycle to cycle from yellow/orange to brownish. After each cycle, cleavage reaction was directly quenched by pouring the reaction into dilute pyridine (pyridine/ethanol 1:9 (v/v)). Resin was then removed by filtration with a frit and subjected to the next cycle. All filtrates were pooled, concentrated to an orange semi-liquid under vacuo. The residue was purified on column of silica gel (5% MeOH/DCM) to provide 0.88 g (80% yield) of Intermediate 2.3. LC-MS: Rt=4.37 min, m/z=411 [M+H].sup.+, m/z=409 [M−H].sup.−.

    Intermediate 2.4: Fmoc-Val-Ala-PAB-OH

    [0428] Intermediate 2.3 (1.65 g, 4.02 mmol) was dissolved in dry DMF (10 mL). PAB-OH (0.49 g, 4.02 mmol), HATU (1.53 g, 4.02 mmol) and DIEA (2.11 mL, 12.06 mmol) were added to the solution and the mixture was stirred for 5 h. The reaction was stopped by addition of EtOAc and the mixture was washed with saturated NaHCO3, water and brine. The layers were separated and the organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was purified by silica-gel column chromatography, using DCM/Acetone and DCM/MeOH as eluent to provide 1.3 g (62% yield) of Intermediate 2.4. LC-MS: Rt=5.30 min, m/z=516 [M+H].sup.+, m/z=514 [M−H].sup.−.

    Intermediate 2.5: Fmoc-Val-Ala-PAB-Cl

    [0429] Intermediate 2.4 (0.100 g, 0.19 mmol) was dissolved in dry CHCl.sub.3 (10 mL) and was cooled to 0° C. SOCl.sub.2 (164, 0.213 mmol) was added dropwise to the solution and the mixture was stirred at rt for 1h. The reaction was stopped by addition of a saturated solution of NaHCO.sub.3. The layers were separated and the organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo to provide 99 mg (95% yield) of crude Intermediate 2.5 which was used for the next step without any further purification. LC-MS: Rt=5.76 min, m/z=535 [M+H].sup.+, m/z=533 [M−H].sup.−.

    Intermediate 2.6: Fmoc-Val-Ala-PAB-I

    [0430] Intermediate 2.5 (99.0 mg, 0.18 mmol) was dissolved in acetone (10 mL). NaCl (88.0 mg, 0.584 mmol) was added dropwise to the solution and the mixture was stirred at rt for 48h in the dark. The reaction was stopped by addition of EtOAc and was washed with a 5% solution of NaHSO3 and brine. The layers were separated and the organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo to provide 100 mg (82% yield) of crude Intermediate 2.6 which was used for the next step without any further purification. LC-MS: Rt=6.36 min, m/z=626 [M+H].sup.+, m/z=624 [M−H].sup.−.

    Intermediate 2.7: C.SUB.15.H.SUB.31.—CO-Val-Ala-OH

    [0431] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (0.682 g, 2.19 mmol) and Fmoc-Val-OH (0.743 g, 2.19 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. The reactor was drained and then the treatment was repeated one time. The resin bed was then washed with 3 times with 40 mL DMF, 3 times with 40 mL of DCM, 3 times with 40 mL of MeOH and 3 times with 40 mL of DMF. The last wash was then sampled for piperidine levels by qualitative ninhydrin test.

    [0432] Then, Palmitic acid (0.562 g, 2.19 mmol) and DIEA (0.381 mL, 2.19 mmol) in 15 mL DMF were charged to a flask. The contents were stirred at ambient temperature to dissolve the solid which were then cooled to 10° C. Then HATU (0.83 g, 2.19 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM.

    [0433] Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.35 g (31% yield) of Intermediate 2.7. LC-MS: Rt=7.19 min, m/z=427 [M+H].sup.+, m/z=425 [M−H].sup.−.

    Intermediate 2.8: C.SUB.15.H.SUB.31.—CO-Val-Ala-PAB-OH

    [0434] Intermediate 2.8 (0.14 g, 54% yield) was obtained from Intermediate 2.7 (0.215 g, 0.504 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=7.38 min, m/z=532 [M+H].sup.+, m/z=530 [M−H].sup.−.

    Intermediate 2.9: C.SUB.15.H.SUB.31.—CO-Val-Ala-PAB-Cl

    [0435] Intermediate 2.9 (0.14 g, 99% yield) was obtained from Intermediate 2.8 (0.14 g, 0.263 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=7.86 min, m/z=551 [M+H].sup.+, m/z=549 [M−H].sup.−.

    Intermediate 2.10: C.SUB.15 .H.SUB.31.—CO -Va I-Al a-PAB-I

    [0436] Intermediate 2.10 (0.14 g, 99% yield) was obtained from Intermediate 2.9 (0.14 g, 0.263 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=8.33 min, m/z=642 [M+H].sup.+, m/z=640 [M−H].sup.−.

    Intermediate 2.11: CH.SUB.3.—CO-Val-Ala-OH

    [0437] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (0.747 g, 2.40 mmol) and Fmoc-Val-OH (0.814 g, 2.40 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then Acetic acid (0.960 mL, 2.40 mmol) and DIEA (0.417 mL, 2.40 mmol) in 15 mL DMF were charged to a flask. Then HATU (0.912 g, 2.40 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.189 g (99% yield) of Intermediate 2.11. LC-MS: Rt=2.18 min, m/z=231 [M+H].sup.+, m/z=229 [M−H].sup.−.

    Intermediate 2.12: CH.SUB.3.—CO-Val-Ala-PAB-OH

    [0438] Intermediate 2.12 (0.15 g, 56% yield) was obtained from Intermediate 2.11 (0.184 g, 0.799 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=2.90 min, m/z=336 [M+H].sup.+, m/z=334 [M−H].sup.−.

    Intermediate 2.13: CH.SUB.3.—CO-Val-Ala-PAB-Cl

    [0439] Intermediate 2.13 (0.16 g, 99% yield) was obtained from Intermediate 2.12 (0.15 g, 0.447 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=4.15 min, m/z=354 [M+H].sup.+, m/z=352 [M−H].sup.−.

    Intermediate 2.14: CH.SUB.3.—CO-Val-Ala-PAB-I

    [0440] Intermediate 2.14 (0.11 g, 55% yield) was obtained from Intermediate 2.13 (0.158 g, 0.447 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=4.51 min, m/z=446 [M+H].sup.+, m/z=444 [M−H].sup.−.

    Intermediate 2.15: C.SUB.3.H.SUB.7.—CO-Val-Ala-OH

    [0441] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (0.747 g, 2.40 mmol) and Fmoc-Val-OH (0.814 g, 2.40 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then butyryl chloride (0.912 mL, 2.40 mmol) and DIEA (0.420 mL, 2.40 mmol) in 15 mL DMF were charged to a flask. Then HATU (0.912 g, 2.40 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.205 g (99% yield) of Intermediate 2.15. LC-MS: Rt=3.00 min, m/z=259 [M+H].sup.+, m/z=257 [M−H].sup.−.

    Intermediate 2.16: C.SUB.3.H.SUB.7.—CO-Val-Ala-PAB-OH

    [0442] Intermediate 2.16 (0.255 g, 88% yield) was obtained from Intermediate 2.15 (0.184 g, 0.799 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=3.49 min, m/z=364 [M+H].sup.+, m/z=362 [M−H].sup.−.

    Intermediate 2.17: C.SUB.3.H.SUB.7.—CO-Val-Ala-PAB-Cl

    [0443] Intermediate 2.17 (0.268 g, 99% yield) was obtained from Intermediate 2.16 (0.255 g, 0.702 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=4.72 min, m/z=382 [M+H].sup.+, m/z=380 [M−H].sup.−.

    Intermediate 2.18: C.SUB.3.H.SUB.7.—CO-Val-Ala-PAB-I

    [0444] Intermediate 2.18 (0.4 g, 57% yield) was obtained from Intermediate 2.17 (0.268 g, 0.702 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=5.03 min, m/z=474 [M+H].sup.+, m/z=472 [M−H].sup.−.

    Intermediate 2.19: C.SUB.7.H.SUB.15.—CO-Val-Ala-OH

    [0445] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (0.747 g, 2.40 mmol) and Fmoc-Val-OH (0.814 g, 2.40 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then octanoyl chloride (0.150 mL, 2.40 mmol) and DIEA (0.420 mL, 2.40 mmol) in 15 mL DMF were charged to a flask. Then HATU (0.912 g, 2.40 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.250 g (99% yield) of Intermediate 2.19. LC-MS: Rt=3.19 min, m/z=315 [M+H].sup.+, m/z=313 [M−H].sup.−.

    Intermediate 2.20: C.SUB.7.H.SUB.15.—CO-Val-Ala-PAB-OH

    [0446] Intermediate 2.20 (0.28 g, 83% yield) was obtained from Intermediate 2.19 (0.250 g, 0.795 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=4.90 min, m/z=420 [M+H].sup.+, m/z=418 [M−H].sup.−.

    Intermediate 2.21: C.SUB.7.H.SUB.15.—CO-Val-Ala-PAB-Cl

    [0447] Intermediate 2.21 (0.29 g, 99% yield) was obtained from Intermediate 2.20 (0.277 g, 0.660 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=5.85 min, m/z=439 [M+H].sup.+, m/z=437 [M−H].sup.−.

    Intermediate 2.22: C.SUB.7.H.SUB.15.—CO-Val-Ala-PAB-I

    [0448] Intermediate 2.22 (0.23 g, 65% yield) was obtained from Intermediate 2.21 (0.289 g, 0.660 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=6.11 min, m/z=530 [M+H].sup.+, m/z=528 [M−H].sup.−.

    Intermediate 2.23: C.SUB.14./C.SUB.14.—CO-Val-Ala-OH

    [0449] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (0.747 g, 2.40 mmol) and Fmoc-Val-OH (0.814 g, 2.40 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then 3-(tetradecanoyloxy)tetradecanoic acid (0.608 mL, 2.40 mmol) and DIEA (0.420 mL, 2.40 mmol) in 15 mL DMF were charged to a flask. Then HATU (0.912 g, 2.40 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.463 g (93% yield) of Intermediate 2.23. LC-MS: Rt=7.78 min, m/z=625 [M+H].sup.+, m/z=623 [M−H].sup.−.

    Intermediate 2.24: C.SUB.14./C.SUB.14.—CO-Val-Ala-PAB-OH

    [0450] Intermediate 2.24 (0.45 g, 61% yield) was obtained from Intermediate 2.23 (0.463 g, 0.741 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=8.82 min, m/z=731 [M+H].sup.+, m/z=729 [M−H].sup.−.

    Intermediate 2.25: C.SUB.14./C.SUB.14.—CO-Val-Ala-PAB-Cl

    [0451] Intermediate 2.25 (0.34 g, 99% yield) was obtained from Intermediate 2.24 (0.330 g, 0.452 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=9.14 min, m/z=749 [M+H].sup.+, m/z=747 [M−H].sup.−.

    Intermediate 2.26: C.SUB.14./C.SUB.14.—CO-Va I-Ala-PAB-I

    [0452] Intermediate 2.26 (0.21 g, 46% yield) was obtained from Intermediate 2.25 (0.338 g, 0.452 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=9.33 min, m/z=840 [M+H].sup.+, m/z=838 [M−H].sup.−.

    Intermediate 2.27: Me-Su-Val-Ala-OH

    [0453] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (0.747 g, 2.40 mmol) and Fmoc-Val-OH (0.814 g, 2.40 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then 4-methoxy-4-oxobutanoic acid (0.317 mL, 2.40 mmol) and DIEA (0.420 mL, 2.40 mmol) in 15 mL DMF were charged to a flask. Then HATU (0.912 g, 2.40 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.241 g (100% yield) of Intermediate 2.27. LC-MS: Rt=4.55 min, m/z=303 [M+H].sup.+, m/z=301 [M−H].sup.−.

    Intermediate 2.28: Me-Su-Val-Ala-PAB-OH

    [0454] Intermediate 2.28 (0.68 g, 87% yield) was obtained from Intermediate 2.27 (0.240 g, 0.794 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=3.38 min, m/z=731 [M+H].sup.+, m/z=729 [M−H].sup.−.

    Intermediate 2.29: Me-Su-Val-Ala-PAB-Cl

    [0455] The Intermediate 2.29 (0.29 g, 99% yield) was obtained from Intermediate 2.28 (0.280 g, 0.687 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=4.54 min, m/z=426 [M+H].sup.+, m/z=424 [M−H].sup.−.

    Intermediate 2.30: Me-Su-Val-Ala-PAB-I

    [0456] Intermediate 2.30 (0.14 g, 41% yield) was obtained from Intermediate 2.29 (0.293 g, 0.688 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=4.83 min, m/z=518 [M+H].sup.+, m/z=516 [M−H].sup.−.

    Intermediate 2.31: Fmoc-(AEEA).SUB.4.-Val-Ala-OH

    [0457] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (4.483 g, 14.4 mmol) and Fmoc-Val-OH (4.89 g, 14.4 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then Fmoc-(NH—CH.sub.2—CH.sub.2—O—CH.sub.2—CH.sub.2—O—CH.sub.2—CO).sub.4—OH (3.70 g, 9.60 mmol) and DIEA (1.67 mL, 9.60 mmol) in 15 mL DMF were charged to a flask. Then HATU (3.65 g, 9.60 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. This operation was repeated 4 times. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 4.07 g (86% yield) of Intermediate 2.31. LC-MS: Rt=4.09 min, m/z=991 [M+H].sup.+, m/z=989 [M−H].sup.−.

    Intermediate 2.32: Fmoc-(AEEA).SUB.4.-Val-Ala-PAB-OH

    [0458] Intermediate 2.32 (2.75 g, 67% yield) was obtained from Intermediate 2.31 (4.07 g, 4.11 mmol) using a similar procedure to that described for Intermediate 2.4 LC-MS: Rt=4.79 min, m/z=1097 [M+H].sup.+, m/z=1095 [M−H].sup.−.

    Intermediate 2.33: Fmoc-(AEEA).SUB.4.-Val-Ala-PAB-Cl

    [0459] Intermediate 2.33 (0.99 g, 99% yield) was obtained from Intermediate 2.32 (0.954 g, 0.870 mmol) using a similar procedure to that described for Intermediate 2.5 LC-MS: Rt=6.24 min, m/z=1115 [M+H].sup.+, m/z=1113 [M−H].sup.−.

    Intermediate 2.34: Fmoc-(AEEA).SUB.4.-Val-Ala-PAB-I

    [0460] Intermediate 2.34 (0.69 g, 66% yield) was obtained from Intermediate 2.33 (0.970 g, 0.870 mmol) using a similar procedure to that described for Intermediate 2.6 LC-MS: Rt=5.47 min, m/z=1207 [M+H].sup.+, m/z=1205 [M−H].sup.−.

    Intermediate 2.35: C.SUB.15.H.SUB.31.—CO-AEEEEP-Val-Ala-OH

    [0461] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.7 with Fmoc-Ala-OH (1.28 g, 4.10 mmol) and Fmoc-Val-OH (0.926 g, 4.10 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then Fmoc-NH—(CH.sub.2—CH.sub.2—O).sub.4—CH.sub.2—CH.sub.2—COOH (1.0 g, 2.05 mmol) and DIEA (0.360 mL, 2.05 mmol) in 15 mL DMF were charged to a flask. Then HATU (0.780 g, 2.05 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then Palmitic acid (1.0 g, 2.05 mmol) and DIEA (0.714 mL, 0.410 mmol) in 15 mL DMF were charged to a flask. Then HATU (1.56 g, 4.10 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 0.327 g (71% yield) of Intermediate 2.35. LC-MS: Rt=4.19 min, m/z=674 [M+H].sup.+, m/z=672 [M−H].sup.−.

    Intermediate 2.36: C.SUB.15.H31—CO-AEEEEP-Val-Ala-PAB-OH

    [0462] Intermediate 2.36 (0.3 g, 70% yield) was obtained from Intermediate 2.35 (0.320 g, 0.475 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=6.82 min, m/z=780 [M+H].sup.+, m/z=778 [M−H].sup.−.

    Intermediate 2.37: C.SUB.15 .H.SUB.3 .i—CO -A EEEEP -Val-Ala-PAB-C1

    [0463] Intermediate 2.37 (0.26 g, 99% yield) was obtained from Intermediate 2.36 (0.260 g, 0.334 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=7.45 min, m/z=798

    [0464] [M+H].sup.+, m/z=796 [M−H].sup.−.

    Intermediate 2.38: C.SUB.15 .H.SUB.31.—CO-AEEEEP-Val-Ala-PAB-I

    [0465] Intermediate 2.38 (0.24 g, 84% yield) was obtained from Intermediate 2.37 (0.260 g, 0.326 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=7.63 min, m/z=889 [M+H].sup.+, m/z=887 [M−H].sup.−.

    Intermediate 2.39: Fmoc-AEEA-Val-Ala-OH

    [0466] The dipeptide Fmoc-Val-Ala-2CTC was prepared using a similar procedure to that described for Intermediate 2.3 with Fmoc-Ala-OH (5.98 g, 19.2 mmol) and Fmoc-Val-OH (6.52 g, 19.2 mmol). Then the Fmoc protecting group was cleaved by addition of 60 mL of 20% piperidine in DMF under stirring at rt for 30 min. Then Fmoc-NH—(CH.sub.2—CH.sub.2—O).sub.2—CH.sub.2—COOH (4.93 g, 12.8 mmol) and DIEA (2.23 mL, 12.8 mmol) in 15 mL DMF were charged to a flask. Then HATU (4.87 g, 12.8 mmol) was charged into the flask. The mixture was stirred at 10° C. to dissolve solid. The cooled solution was charged to the solid phase reactor. The flask was then washed with 5 mL of DMF and the wash charged to the SPPS reactor. The mixture was stirred at rt for 4 h. The resin beads were then sampled for the completion of the reaction by a Keiser Test. After the reaction was complete, the reactor was drained and the resin bed washed 4 times with 20 mL DMF, 3 times with 20 mL of DCM, 4 times with 20 mL of MeOH, 3 times with 20 mL of DMF and 8 times with 20 mL of DCM. Cleavage from resin was achieved using a similar procedure to that described for Intermediate 2.3 to provide 3.25 g (91% yield) of Intermediate 2.39. LC-MS: Rt=4.15 min, m/z=556 [M+H].sup.+, m/z=554 [M−H].sup.−.

    Intermediate 2.40: Fmoc-AEEA-Val-Ala-PAB-OH

    [0467] Intermediate 2.40 (3.82 g, 98% yield) was obtained from Intermediate 2.39 (3.21 g, 5.78 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=5.21 min, m/z=661 [M+H].sup.+, m/z=559 [M−H].sup.−.

    Intermediate 2.41: Fmoc-AEEA-Val-Ala-PAB-Cl

    [0468] Intermediate 2.41 (3.6 g, 99% yield) was obtained from Intermediate 2.40 (3.5 g, 5.30 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=5.92 min, m/z=680 [M+H].sup.+, m/z=678 [M−H].sup.−.

    Intermediate 2.42: Fmoc-AEEA-Val-Ala-PAB-I

    [0469] Intermediate 2.42 (3.0 g,73% yield) was obtained from Intermediate 2.41 (3.6 g, 5.30 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=6.15 min, m/z=771 [M+H].sup.+, m/z=769 [M−H].sup.−.

    Intermediate 2.43: Methyl (2,3,4-tri-O-acetyl-D-glucopyranosyl bromide)uronate

    [0470] Methyl 1,2,3,4-tetra-O-acetyl-D-glucopyranouronate (5.0 g, 13.3 mmol) was treated with a solution of hydrobromic acid in acetic acid 33% (98 mL, 597.9 mmol) at 0° C. After stirring overnight, the mixture was diluted with DCM and successively washed with cold water, saturated aqueous NaHCO.sub.3 and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo to provide 4.78 g (91% yield) of Intermediate 2.43 which is used for the next step without any further purification. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 6.64 (d, 1H), 5.61 (t, 1H), 5.24 (m, 2H), 4.84 (dd, 1H), 4.56 (d, 1H), 3.76 (s, 3H), and 2.05 (m, 9H).

    Intermediate 2.44: 1-(4-formyl-2-nitrophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0471] For the Koenigs-Knorr reaction, the Intermediate 2.43 (4.78 g, 12.0 mmol) was coupled to 4-hydroxy-3-nitrobenzaldehyde (2.61 mg, 15.6 mmol) using Ag.sub.2O (8.37 g, 36.1 mmol) in 30 mL of anhydrous ACN. The mixture was stirred under argon atmosphere at rt overnight. The reaction was filtered and diluted with EtOAc. The organic layer was washed with saturated aqueous NaHCO.sub.3 and applied to flash chromatography (stepwise elution with hexane/EtOAc; 20, 30, and 40% EtOAc) to afford 5.22 g (90%, yield) of Intermediate 2.44. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 9.98 (s, 1H), 8.44 (s 1H), 8.23 (d, 1H), 7.62 (d, 1H), 5.93 (d, 1H), 5.45 (t, 1H), 5.16 (q, 2H), 4.80 (d, 1H), 3.63 (s, 3H), and 2.02 (m, 9H). LC-MS: Rt=3.82 min, m/z=486 [M+H].sup.+, m/z=484 [M−H].sup.−.m/z=484 [M+H].sup.+, m/z=482 [M−H].sup.−.

    Intermediate 2.45: 1-(hydroxymethyl-2-nitrophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0472] Intermediate 2.44 (5.22 g, 10.1 mmol) was reduced with sodium borohydride (4.90 g, 130.0 mmol) in 65 mL of 1:5 2-propanol/CHCl.sub.3 (volume ratio). The mixture was stirred under for 1h at 0° C. The reaction was filtered over Celite, the filtrate was concentrated in vacuo to afford 4.40 g (84%, yield) of Intermediate 2.45. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 7.99 (s, 1H), 7.62 (d, 1H), 7.38 (d, 1H), 5.72 (m, 1H), 5.45 (m, 1H), 5.09 (q, 2H), 4.73 (d, 1H), 4.51 (s, 2H), 3.65 (s, 3H), and 2.02 (m, 9H). LC-MS: Rt=3.33 min, m/z=486 [M+H].sup.+, m/z=484 [M−H].sup.−.

    Intermediate 2.46: 1-(Chloromethyl-2-nitrophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0473] Intermediate 2.46 (0.5 g, 99% yield) was obtained from Intermediate 2.45 (0.5 g, 1.03 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=4.86 min, m/z=504 [M+H].sup.+, m/z=502 [M−H].sup.−.

    Intermediate 2.47: 1-(lodomethyl-2-nitrophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0474] Intermediate 2.47 (0.4 g, 72% yield) was obtained from Intermediate 2.46 (0.5 g, 1.03 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=5.62 min, m/z=596 [M+H].sup.+, m/z=594 [M−H].sup.−.

    Intermediate 2.48: 1-(hydroxymethyl-2-aminophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0475] Intermediate 2.45 (4.52 g, 9.31 mmol) was hydrogenated in presence of Pd/Carbon (0.99 g, 0.93 mmol) in 40 mL of a mixture of EtOAc/MeOH (1/1). The mixture was stirred under for 1h at rt. The reaction was filtered over Celite, the filtrate was concentrated in vacuo to afford 2.97 g (70%, yield) of Intermediate 2.48. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 6.80 (d, 1H), 6.62 (s, 1H), 6.45 (d, 1H), 5.48 (t, 1H), 5.39 (d, 1H), 5.07 (q, 2H), 4.95 (t, 1H), 4.66 (d, 2H), 4.58 (sl, 2H), 4.31 (d, 2H), 3.65 (s, 3H), and 2.02 (m, 9H). LC-MS: Rt=4.33 min, m/z=456 [M+H].sup.+, m/z=454 [M−H].sup.−.

    Intermediate 2.49: 1-(hydroxymethyl-2-palmitamidophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0476] To a solution of Intermediate 2.48 (0.50 g, 1.10 mmol) in dry DCM (15 mL) palmitoyl chloride (0.4 mL, 0.1.32 mmol) and DIEA (0.38 mL, 2.20 mmol) were added. The mixture was stirred at rt overnight. The reaction mixture was washed with saturated aqueous NaHCO.sub.3 and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was applied to flash chromatography to afford 0.645 g (85%, yield) of Intermediate 2.49. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 8.52 (s, 1H), 7.87 (d, 1H), 7.02 (s, 2H), 5.46 (m, 2H), 5.12 (m, 3H), 4.72 (d, 1H), 4.40 (m, 2H), 3.69 (s, 3H), 2.31 (t, 2H), 2.02 (s, 9H), 1.57 (m, 2H), 1.24 (m, 24H) and 0.85 (s, 3H). LC-MS: Rt=6.33 min, m/z=694 [M+H].sup.+, m/z=692 [M−H].sup.−.

    Intermediate 2.50: 1-(Chloromethyl-2-palmitamidophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0477] Intermediate 2.50 (0.6 g, 99% yield) was obtained from Intermediate 2.49 (0.645 g, 0.93 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=7.86 min, m/z=713 [M+H].sup.+, m/z=711 [M−H].sup.−.

    Intermediate 2.51: 1-(Iodomethyl-2-palmitamidophenoxy)-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0478] Intermediate 2.51 (0.6 g, 80% yield) was obtained from Intermediate 2.50 (0.6 g, 0.93 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=8.27 min, m/z=804 [M+H].sup.+, m/z=802 [M−H].sup.−.

    Intermediate 2.52: C.SUB.15.H.SUB.31.—CO-Val-Cit-OH

    [0479] Intermediate 2.52, (4.2 g, 97% yield) was obtained from Fmoc-Ala-OH (8.1 g, 24.0 mmol), Fmoc-Cit-OH (9.5 g, 24.0 mmol) and Palmitic acid (6.1 g, 24.0 mmol) using a similar procedure to that described for Intermediate 2.7. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 8.13 (d, 1H), 7.72 (d, 1H), 4.22 (m, 1H), 4.12 (m, 1H), 2.88 (t, 2H), 2.08 (m, 2H), 1.95 (m, 1H), 1.68 (m, 1H), 1.41 (m, 4H), 1.38 (s, 24H), and 0.85 (t, 3H). ESI-MS: m/z=694 [M+H].sup.+, m/z=692 [M−H].sup.−. ESI-MS: m/z=513 [M+H].sup.+, m/z=511 [M−H].sup.−.

    Intermediate 2.53: C.SUB.15.H.SUB.31.—CO-Val-Cit-PAB-OH

    [0480] Intermediate 2.53 (4.6 g, 90% yield) was obtained from Intermediate 2.52 (4.2 g, 8.2 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=6.64 min, m/z=618 [M+H].sup.+, m/z=616 [M−H].sup.−.

    Intermediate 2.54: C.SUB.15.H.SUB.31.—CO-Val-Cit-PAB-Cl

    [0481] Intermediate 2.54 (1.9 g, 84% yield) was obtained from Intermediate 2.53 (0.14 g, 0.263 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=7.38 min, m/z=637 [M+H].sup.+, m/z=635 [M−H].sup.−.

    Intermediate 2.55: C.SUB.15.H.SUB.31.-CO-Val-Cit-PAB-I

    [0482] Intermediate 2.55 (0.8 g, 86% yield) was obtained from Intermediate 2.54 (0.8 g, 1.25 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=7.37 and 7.58 min, m/z=728 [M+H].sup.+, m/z=726 [M−H].sup.−.

    Intermediate 2.56: 1-[hydroxy-methyl-2-(6-maleimido-hexanamido)-phenoxy]-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0483] To a solution of Intermediate 2.48 (0.20 g, 0.4 mmol) in dry DMF (10 mL) 6-Maleimidohexanoic acid (0.1 mL, 0.48 mmol), HATU (0.18 g, 0.48 mmol) and DIEA (0.23 mL, 1.32 mmol) were added. The mixture was stirred at rt overnight. The reaction was stopped by addition of EtOAc (20 mL) and the mixture was washed with saturated aqueous NaHCO.sub.3, water and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was applied to flash chromatography to afford 0.21 g (72%, yield) of Intermediate 2.56. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 8.55 (s, 1H), 7.84 (s, 1H), 7.00 (m, 1H), 5.84 (m, 2H), 5.13 (m, 3H), 4.70 (d, 1H), 3.63 (s, 3H), 3.40 (m, 2H), 2.30 (t, 2H), 2.01 (s, 9H), 1.52 (m, 2H) and 1.33 (m, 2H). ESI-MS: m/z=649 [M+H].sup.+, m/z=647 [M−H].sup.−.

    Intermediate 2.57: 1-[chloro-methyl-2-(6-maleimido-hexanamido)-phenoxy]-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0484] Intermediate 2.57 (0.21 g, 99% yield) was obtained from Intermediate 2.56 (0.21 g, 0.32 mmol) using a similar procedure to that described for Intermediate 2.5. ESI-MS: m/z=668 [M+H].sup.+, m/z=666 [M−H].sup.−.

    Intermediate 2.58: 1-[iodo-methyl-2-(6-maleimido-hexanamido)-phenoxy]-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0485] Intermediate 2.58 (0.05 g, 22% yield) was obtained from Intermediate 2.57 (0.2 g, 0.32 mmol) using a similar procedure to that described for Intermediate 2.6. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 8.59 (1H, s), 7.96 (s, 1H), 7.16 (m, 1H), 7.00 (m, 3H), 5.61 (m, 1H), 5.58 (m, 1H), 5.24 (m, 1H), 5.06 (m, 1H), 4.72 (m, 1H), 4.59 (s, 2H), 3.63 (s, 3H), 3.40 (t, 2H), 2.30 (m, 2H), 2.01 (s, 9H), 1.52 (m, 4H) and 1.23 (m, 2H). ESI-MS: m/z=759 [M+H].sup.+, m/z=757 [M−H].sup.−.

    Intermediate 2.59: 6-Maleimido-(hexanamido)-hexanoic acid

    [0486] Intermediate 2.59 (1.84 g, 99%) was obtained from Fmoc-6-amino-hexanoic acid (5.09 g, 14.40 mmol) and 6-Maleimido-hexanoic acid (3.04 g, 14.4 mmol) using a similar procedure to that described for Intermediate 2.3. LC-MS: Rt=2.44 min, m/z=325 [M+H].sup.+, m/z=323 [M−H].sup.−.

    Intermediate 2.60: 1-[hydroxy-methyl-2-(6-maleimido-di-(hexanamido))-phenoxy]-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0487] Intermediate 2.60 (92.8 mg, 28%) was obtained from Intermediate 2.48 (5.09 g, 14.40 mmol) and Intermediate 2.59 (3.04 g, 14.4 mmol) using a similar procedure to that described for Intermediate 2.56. LC-MS: Rt=4.67 min, m/z=762 [M+H].sup.+, m/z=760 [M−H].sup.−.

    Intermediate 2.61: 1-[chloro-methyl-2-(6-maleimido-di-(hexanamido))-phenoxy]-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0488] Intermediate 2.61 (95.0 mg, 99% yield) was obtained from Intermediate 2.60 (92 mg, 0.12 mmol) using a similar procedure to that described for Intermediate 2.5. LC-MS: Rt=5.86 min, m/z=781 [M+H].sup.+, m/z=779 [M−H].sup.−.

    Intermediate 2.62: 1-[iodo-methyl-2-(6-maleimido-di-(hexanamido))-phenoxy]-(Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate

    [0489] Intermediate 2.62 (62.7 mg, 59% yield) was obtained from Intermediate 2.61 (95.0 mg, 0.12 mmol) using a similar procedure to that described for Intermediate 2.6. LC-MS: Rt=6.33 min, m/z=872 [M+H].sup.+, m/z=870 [M−H].sup.−.

    Intermediate 2.63: C.SUB.14./C.SUB.14.—CO-Val-Cit-OH

    [0490] Intermediate 2.63 (2.2 g, 87% yield) was obtained from Fmoc-Val-OH (3.26 g, 9.6 mmol), Fmoc-Cit-OH (3.820 g, 9.6 mmol) and 3-(tetradecanoyloxy)tetradecanoic acid (C.sub.14/C.sub.14—COOH) (2.73 g, 6.4 mmol) using a similar procedure to that described for Intermediate 2.23. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 8.16 (d, 1H), 7.86 (d, 1H), 7.19 (m, 3H), 5.12 (m, 1H), 4.23 (m, 3H), 4.10 (t, 3H), 2.93 (m, 2H), 2.16 (m, 2H), 1.92 (m, 1H), 1.71 (m, 1H), 1.47 (m, 6H), 1.21 (m, 36H) and 0.83 (m, 12H). ESI-MS: m/z=711 [M+H].sup.+, m/z=709 [M−H].sup.−.

    Intermediate 2.64: C.SUB.14./C.SUB.14.—CO-Val-Cit-PAB-OH

    [0491] Intermediate 2.64 (0.98 g, 50% yield) was obtained from Intermediate 2.63 (1.71 g, 2.4 mmol) using a similar procedure to that described for Intermediate 2.4. ESI-MS: m/z=817 [M+H].sup.+, m/z=815 [M−H].sup.−.

    Intermediate 2.65: C.SUB.14./C.SUB.14.—CO-Val-Cit-PAB-I

    [0492] To a suspension of Intermediate 2.64 (0.40 g, 0.49 mmol) in dry ACN (20 mL) KI (0.134 g, 0.81 mmol) and BF.sub.3.Et.sub.2O (0.102 mL, 0.81 mmol) were added. The solution was stirred at RT for 1H00, then water was added to stop the reaction. The mixture was exacted 3 times with EtOAc. The organic layer was washed with a solution of NaHCO.sub.3, NaHSO.sub.3 and brine, dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was applied to flash chromatography to afford 0.42 g (86%, yield) of Intermediate 2.65. LC-MS: Rt=9.21 min, m/z=927 [M+H].sup.+, m/z=925 [M−H].sup.−.

    Intermediate 2.66: Mal-Aca-Aca-OH

    [0493] Intermediate 2.66 (4.1 g, 90% yield) was obtained from Fmoc-Aca-OH (10.2 g, 28.8 mmol) and 6-Maleimido-Aca-OH (4.06 g, 19.2 mmol) using a similar procedure to that described for Intermediate 2.3. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 7.69 (m, 2H), 7.17 (m, 1H), 7.00 (s, 2H), 3.36 (t, 2H), 2.98 (m, 2H), 2.18 (t, 2H), 2.00 (t, 2H), 1.48 (m, 8H), 1.35 (m, 2H) and 1.20 (m, 2H). ESI-MS: m/z=325 [M+H].sup.+, m/z=323 [M−H].sup.−.

    Intermediate 2.67: Solid phase synthesis of Fmoc-Val-Cit-OH

    [0494] Intermediate 2.67, (9.1 g, 80% yield) was obtained from Fmoc-Val-OH (8.15 g, 24.0 mmol) and Fmoc-Cit-OH (9.54 g, 24.0 mmol) using a similar procedure to that described for Intermediate 2.3. .sup.1H NMR (DMSOd.sub.6, 400 MHz) δ 8.15 (d, 1H), 7.88 (d, 1H), 7.74 (m, 2H), 7.43-7.15 (m, 8H), 4.25-4.21 (m, 4H), 3.92 (m, 1H), 2.95 (t, 2H), 2.29 (m, 2H), 2.00 (m, 1H), 1.68 (m, 1H), 1.41 (m, 4H), 1.40 (m, 2H), and 0.87 (m, 6H). ESI-MS: m/z=711 [M+H].sup.+, m/z=709 [M−H].sup.−.

    Intermediate 2.68: Fmoc-Val-Cit-PAB-OH

    [0495] Intermediate 2.68 (2.79 g, 94% yield) was obtained from Intermediate 2.67 (2.45 g, 4.93 mmol) using a similar procedure to that described for Intermediate 2.4. LC-MS: Rt=4.82 min, m/z=602 [M+H].sup.+, m/z=600 [M−H].sup.−.

    Intermediate 2.69: Fmoc-Val-Cit-PAB-I

    [0496] Intermediate 2.69 (0.96 g, 77% yield) was obtained from Intermediate 2.68 (1.06 g, 1.76 mmol) using a similar procedure to that described for Intermediate 2.65. LC-MS: Rt=577 min, m/z=712 [M+H].sup.+, m/z=710 [M−H].sup.−.

    Example 1.3 : Synthesis of Pro-CDNs of the Invention

    [0497] ##STR00064##

    [0498] Intermediate 1.14 (20 mg, 0.03 mmol) was dissolved in deionized water (2 mL). Intermediate 2.2 (61 mg, 0.103 mmol) in a mixture of acetone/dioxane/water (9/9/2) was added. The mixture was stirred overnight at rt. Then NaHSO.sub.3 (6.14 mg, 0.06 mmol) was added and the compound was precipitated by addition of EtOAc. The precipitate was filtered, washed with EtOAc and water and dried in vacuo to provide 15 mg (32% Yield) of Compound 1. LC-MS: Rt=4.57, 4.65 min, m/z=801 [M+2H].sup.2+, m/z=799 [M−2H].sup.2−.

    ##STR00065##

    [0499] Intermediate 1.5 (40 mg, 0.06 mmol) was dissolved in deionized water (2 mL). A solution of Intermediate 2.6 (132 mg, 0.132 mmol) in acetone was added. The mixture was stirred overnight at rt in the dark. The solvents were removed in vacuo and the residue was applied to silica-gel column chromatography, using DCM/MeOH as eluent to provide 12 mg (17% yield) of Compound 2. LC-MS: Rt=3.89, 4.11, 4.21 min, m/z=1160 [M+H].sup.+, m/z=1158 [M−H].sup.−.

    ##STR00066##

    [0500] Compound 2 (8 mg, 6.90 μmol) was dissolved in DMF (2 mL). A solution of piperidine 20% in DMF was added and the mixture was stirred for 1 h. Then the solvent was removed in vacuo and the residue was triturated with Et.sub.2O to provide 6 mg (99% yield) of Compound 3. LC-MS: Rt=3.89, 4.11, 4.21 min, m/z=1160 [M+H].sup.+, m/z=1158 [M−H].sup.−.

    Intermediate 3.1: c-[2′FdAM(PS-PAB-Ala-Val-(AEEA).SUB.4.-Fmoc)-dIMP]

    [0501] Intermediate 1.5 (100 mg, 0.151 mmol) was dissolved in in deionized water (500 ΞL). A solution of Intermediate 2.34 (274 mg, 0.227 mmol) in DMF was added. The mixture was stirred overnight at rt in the dark. The solvents were removed and the residue was applied to a flash chromatography to provide 160 mg (61% yield) of Intermediate 3.1. LC-MS: Rt=4.31 min, m/z=1740 [M+H].sup.+, m/z=1738 [M−H].sup.−.

    ##STR00067##

    [0502] Intermediate 1.5 (110 mg, 0.166 mmol) was dissolved in deionized water (2 mL). A solution of

    [0503] Intermediate 2.30 (129 mg, 0.249 mmol) in DMF was added. The mixture was stirred overnight at rt in the dark. The solvents were removed in vacuo and the residue was applied to silica-gel column chromatography, using DCM/MeOH as eluent to provide 105 mg (60% yield) of Compound 4. LC-MS: Rt=3.35 and 3.53 min, m/z=1051 [M+H].sup.+, m/z=1049 [M−H].sup.−.

    ##STR00068##

    [0504] To a solution of Compound 3 (45 mg, 0.048 mmol) in DMF (1 mL) at 10° C. DIEA (0.025 mL, 0.144 mmol), 6-Maleimidohexanoic acid (12 mg, 0.053 mmol) and HATU (20 mg, 0.053 mmol) were added. The mixture was stirred at rt for 4 h. Then the solvent was removed in vacuo and the residue was applied to a C18 chromatography with TEANACN as eluent to provide 33 mg (61% yield) of Compound 5. LC-MS: Rt=3.61 and 3.77 min, m/z=1131 [M+H].sup.+, m/z=1129 [M−H].sup.−.

    ##STR00069##

    [0505] Compound 6 (120 mg, 92% yield) was obtained from Intermediate 3.1 (150 mg, 0.086 mmol) using a similar procedure to that described for Compound 3. LC-MS: Rt=3.34 and 3.48 min, m/z=1518 [M+H].sup.+, m/z=1516 [M−H].sup.−.

    Intermediate 3.2: c-[2′FdAM(PS-PAB-Ala-Val-AEEA-Fmoc)-dIMP]

    [0506] Intermediate 1.5 (200 mg, 0.302 mmol) was dissolved in in deionized water (2 mL). A solution of Intermediate 2.42 (280 mg, 0.363 mmol) in THF was added. The mixture was stirred overnight at 40° C. in the dark. The solvents were removed and the residue was applied to a flash chromatography to provide 267 mg (68% yield) of Intermediate 3.2. LC-MS: Rt=4.65 and 4.71 min, m/z=1305 [M+H].sup.+, m/z=1303 [M−H].sup.−.

    Intermediate 3.3: c-[2′FdAM(PS-PAB-Ala-Val-AEEA)-dIMP]

    [0507] Intermediate 3.3 was obtained from Intermediate 3.2 (267 mg, 0.205 mmol) using a similar procedure to that described for Compound 3 to provide 222 mg (100% yield) of Intermediate 3.3. LC-MS: Rt=3.20 and 3.41 min, m/z=1083 [M+H].sup.+, m/z=1081 [M−H].sup.−.

    ##STR00070##

    [0508] Compound 7 (97 mg, 90% yield) was obtained from Intermediate 1.5 (63 mg, 0.095 mmol) and Intermediate 2.47 (85 mg, 0.140 mmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=3.78 and 3.88 min, m/z=1129 [M+H].sup.+, m/z=1127 [M−H].sup.−.

    ##STR00071##

    [0509] Intermediate 1.24 (1.0 g, 1.4 mmol) was dissolved in deionized water (2 mL). A solution of Intermediate 2.6 (1.4 g, 2.2 mmol) in THF was added. The mixture was stirred overnight at rt in the dark. The solvents were removed in vacuo and the residue was treated with 60 mL of 20% piperidine in DMF and stirred at rt for 30 min. The solvent was removed in vacuo and applied to silica-gel column chromatography, using DCM/MeOH as eluent, to provide 0.92 g (68% yield) of Compound 8. LC-MS: Rt=3.11 and 3.16 min, m/z=955 [M+H].sup.+, m/z=957 [M−H].sup.−.

    ##STR00072##

    [0510] Compound 9 40.0 mg (57% yield) was obtained from Intermediate 1.22 (53.0 mg, 70.0 μmol) and Intermediate 2.58 (53.0 mg, 54 μmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=4.08 and 4.16 min, m/z=1310 [M+H].sup.+, m/z=1308 [M−H].sup.−.

    Compound 10: c-[2′FdAM(PS-(3-(Maleimido-di-hexanamido)-4-(Methyl-2,3,4-tri-O-acetyl-D-glucopyranouronate)-benzyl)-2′FdIMP]

    [0511] ##STR00073##

    [0512] Compound 10 (4.0 mg, 5% yield) was obtained from Intermediate 1.24 (62.7 mg, 71.9 μmol) and Intermediate 2.62 (37.6 mg, 55.3 μmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=4.09 min, m/z=1423 [M+H].sup.+, m/z=1421 [M−H].sup.−.

    ##STR00074##

    [0513] Compounds 11 and 12 were obtained from Intermediate 1.22 (72.0 mg, 0.10 mmol) and Intermediate 2.62 (90.0 mg, 0.10 mmol) in DMF using a similar procedure to that described for Compound 4 to provide: 23.0 mg (15% yield) of Compound 11. LC-MS: Rt=4.32 min, m/z=1439 [M+H].sup.+, m/z=1437 [M−H].sup.−; 24.0 mg (12% yield) of Compound 12. LC-MS: Rt=5.41 min, m/z=2182 [M+H].sup.+, m/z=2180 [M−H].sup.−

    ##STR00075##

    [0514] Compounds 13 and 14 were obtained from Intermediate 1.22 (300 mg, 0.431 mmol) and Intermediate 2.69 (338 mg, 0.474 mmol) in DMF using a similar procedure to that described for Compound 2 to provide 287.0 mg (52% yield) of Compound 13 : LC-MS: Rt=4.65, 4.50 and 4.83 min, m/z=1279 [M+H].sup.+, m/z=1277 [M−H].sup.−, and 130 mg (16% yield) of Compound 14 : LC-MS: Rt=5.66 min, m/z=1863 [M+H].sup.+, m/z=1861 [M−H].sup.−.

    ##STR00076##

    [0515] Compound 15 (115 mg, 50% yield) was obtained from Compound 13 (280 mg, 0.219 mmol) using a similar procedure to that described for Compound 3. LC-MS: Rt=4.48, 4.65 and 4.77 min, m/z=1057 [M+H].sup.+, m/z=1055 [M−H].sup.−.

    ##STR00077##

    [0516] Compound 16 (55 mg, 58% yield) was obtained from Compound 14 (125 mg, 0.067 mmol) using a similar procedure to that described for Compound 3. LC-MS: Rt=4.91, 5.07 and 5.21 min, m/z=1419 [M+H].sup.+, m/z=1417 [M−H].sup.−.

    ##STR00078##

    [0517] Compound 17 (16 mg, 30% yield) was obtained from Compound 15 (40 mg, 0.038 mmol) and Intermediate 2.66 (14 mg, 0.042 mmol) using a similar procedure to that described for Compound 5. LC-MS: Rt=4.48, 4.65 and 4.77 min, m/z=1057 [M+H].sup.+, m/z=1055 [M−H].sup.−.

    ##STR00079##

    [0518] Compounds 18 and 19 were obtained from Intermediate 1.22 (300 mg, 0.431 mmol) and Intermediate 2.6 (351 mg, 0.561 mmol) in DMF using a similar procedure to that described for Compound 2 to provide 150.0 mg (50% yield) of Compound 18 : LC-MS: Rt=4.62, 4.73 and 4.90 min, m/z=1194 [M+H].sup.+, m/z=1192 [M−H].sup.−, and 170 mg (45% yield) of Compound 19 : LC-MS: Rt=6.17, 6.32 and 6.33 min, m/z=1691 [M+H].sup.+, m/z=1689[M−H].sup.−.

    ##STR00080##

    [0519] Compound 20 (95 mg, 76% yield) was obtained from Compound 18 (150 mg, 0.127 mmol) using a similar procedure to that described for Compound 3. LC-MS: Rt=3.23, 3.39 and 3.62 min, m/z=971 [M+H].sup.+, m/z=969 [M−H].sup.−.

    ##STR00081##

    [0520] To a solution Compound 20 (58 mg, 0.060 mmol) in dry DMF, Mal-L-Dap(Boc)-OH (28 mg, 0.060 mmol), DIEA (314, 0.18 mmol) and HATU (25 mg, 0.066 mmol) were added. The solution was stirred for 3h at rt. Then the solution was diluted with AcOEt washed with water, NaHCO.sub.3 and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude was treated with a solution of TFA and p-toluenethiol (16 mg, 0.128 mmol). The solution was stirred 5 min at rt, the solution was concentrated, the residue was triturated with acetone. The crude was purified on C18 column to provide Compound 21 (6.7 mg, 46% yield). LC-MS: Rt=3.72, 3.53 and 3.37 min, m/z=1137 [M+H].sup.+, m/z=1135 [M−H].sup.−.

    Example 1.4: Synthesis of BAM-CDN Conjugates of the Invention

    [0521] ##STR00082##

    [0522] Intermediate 1.5 (40 mg, 0.06 mmol) was dissolved in deionized water (2 mL). A solution of Intermediate 2.10 (85 mg, 0.132 mmol) in acetone was added. The mixture was stirred overnight at rt in the dark. The solvents were removed in vacuo and the residue was applied to silica-gel column chromatography, using DCM/MeOH as eluent to provide 12 mg (25% yield) of Compound 22. LC-MS: Rt=7.97 min, m/z=1176 [M+H].sup.+, m/z=1174 [M−H].sup.−.

    [0523] Compound 22 (CL808) belongs to Formula (V.sub.a) and comprises a monophosphorothioate CDN (CL797), a PAB moiety as a connector, a Val-Ala specifier and a BAM consisting of a saturated fatty acid (C.sub.16 carbon chain) to facilitate CDN penetration into cell.

    ##STR00083##

    [0524] Compound 23 was obtained from Intermediate 1.5 (60 mg, 0.091 mmol) and Intermediate 2.14 (101 mg, 0.227 mmol) in DMF using a similar procedure to that described for Compound 4 to provide 12.8 mg (14% yield) of Compound 23. LC-MS: Rt=3.22 min, m/z=979 [M+H].sup.+, m/z=977 [M−H].sup.−.

    ##STR00084##

    [0525] Compound 24 was obtained from Intermediate 1.5 (60 mg, 0.091 mmol) and Intermediate 2.18 (107 mg, 0.227 mmol) in DMF using a similar procedure to that described for Compound 4 to provide 12.0 mg (13% yield) of Compound 24. LC-MS: Rt=3.46 min, m/z=1007 [M+H].sup.+, m/z=1005 [M−H].sup.−.

    ##STR00085##

    [0526] Compound 25 was obtained from Intermediate 1.5 (60 mg, 0.091 mmol) and Intermediate 2.22 (120 mg, 0.227 mmol) in DMF using a similar procedure to that described for Compound 4 to provide 12.0 mg (12% yield) of Compound 25. LC-MS: Rt=4.35 min, m/z=1064 [M+H].sup.+, m/z=1065 [M−H].sup.−.

    ##STR00086##

    [0527] Compounds 26, 27 and 28 were obtained from Intermediate 1.22 (51 mg, 0.073 mmol) and Intermediate 2.22 (39 mg, 0.073 mmol) in DMF using a similar procedure to that described for Compound 4 to provide: 5.0 mg (6% yield) of Compound 26. LC-MS: Rt=4.44 min, m/z=1098 [M+H].sup.+, m/z=1096 [M−H].sup.−; 1.4 mg (1.8% yield) of Compound 27. LC-MS: Rt=4.27 min, m/z=1081 [M+H].sup.+, m/z=1079 [M−H].sup.−; 8.4 mg (10% yield) of Compound 28. LC-MS: Rt=6.05 min, m/z=1498 [M+H].sup.+, m/z=1496 [M−H].sup.−

    ##STR00087##

    [0528] Compound 29 was obtained from Intermediate 1.5 (80 mg, 0.121 mmol) and Intermediate 2.26 (152 mg, 0.181 mmol) in DMF using a similar procedure to that described for Compound 4 to provide 17.0 mg (10% yield) of Compound 29. LC-MS: Rt=7.05 and 7.26 min, m/z=1374 [M+H].sup.+, m/z=1372 [M−H].sup.−.

    ##STR00088##

    [0529] Compound 30 was obtained from Intermediate 1.5 (30 mg, 0.045 mmol) and Intermediate 2.38 (121 mg, 0.136 mmol) in Acetone using a similar procedure to that described for Compound 4 to provide 33 mg (51% yield) of Compound 30. LC-MS: Rt=5.66 min, m/z=1423 [M+H].sup.+, m/z=1421 [M−H].sup.−.

    ##STR00089##

    [0530] To a solution of Compound 6 (66 mg, 0.043 mmol) in DMF (1 mL) DIEA (0.023 mL, 0.130 mmol), D-Biotin (12 mg, 0.053 mmol) and HATU (18 mg, 0.048 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed in vacuo and the residue was applied to a flash chromatography to provide 57 mg (75% yield) of Compound 31. LC-MS: Rt=3.48 and 3.59 min, m/z=1744 [M+H].sup.+, m/z=1742 [M−H].sup.−.

    ##STR00090##

    [0531] To a solution of Compound 6 (40 mg, 0.026 mmol) in DMF (1 mL) TEA (21.7 μL, 0.156 mmol), FITC (20.6 mg, 0.052 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed in vacuo and the residue was applied to a flash chromatography to provide 34 mg (68% yield) of Compound 32. LC-MS: Rt=3.60 and 3.65 min, m/z=1907 [M+H].sup.+, m/z=1905 [M−H].sup.−.

    ##STR00091##

    [0532] To a solution of Compound 6 (40 mg, 0.026 mmol) in DMF (1 mL) DIEA (14.0 μL, 0.079 mmol), N-hydroxy-succinyl folate (21.0 mg, 0.040 mmol) were added. The mixture was stirred at rt for 4h. Then the solvent was removed in vacuo and the residue was applied to a flash chromatography with to provide 36 mg (70% yield) of Compound 33. LC-MS: Rt=3.20 and 3.31 min, m/z=1941 [M+H].sup.+, m/z=1939 [M−H].sup.−.

    ##STR00092##

    [0533] To a solution of Intermediate 3.3 (55 mg, 0.051 mmol) in DMF (1 mL) TEA (42.54 μL, 0.306 mmol), FITC (40.0 mg, 0.102 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed in vacuo and the residue was applied to a flash chromatography with to provide 25 mg (33% yield) of Compound 34. LC-MS: Rt=3.02 min, m/z=1472 [M+H].sup.+, m/z=1470 [M−H].sup.−.

    ##STR00093##

    [0534] To a solution of Intermediate 3.3 (55 mg, 0.051 mmol) in DMF (1 mL) DIEA (27 μL, 0.153 mmol), D-Biotin (14 mg, 0.053 mmol) and HATU (21 mg, 0.056 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed and the residue was applied to a flash chromatography to provide 12 mg (18% yield) of Compound 35. LC-MS: Rt=3.45 and 3.58 min, m/z=1309 [M+H].sup.+, m/z=1307 [M−H].sup.−.

    ##STR00094##

    [0535] To a solution of Intermediate 3.3 (55 mg, 0.051 mmol) in DMF (1 mL) DIEA (27 μL, 0.153 mmol), CL264 (InvivoGen) (21 mg, 0.051 mmol) and HATU (21 mg, 0.056 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed and the residue was applied to a flash chromatography to provide 60 mg (83% yield) of Compound 36. LC-MS: Rt=3.94 min, m/z=1478 [M+H].sup.+, m/z=1476 [M−H].sup.−.

    ##STR00095##

    [0536] Compound 37 (85 mg, 77% yield) was obtained from Intermediate 1.5 (55 mg, 0.082 mmol) and Intermediate 2.51 (100 mg, 0.124 mmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=6.03 and 6.14 min, m/z=1337 [M+H].sup.+, m/z=1335 [M−H].sup.−.

    ##STR00096##

    [0537] Compound 38 (2.5 mg, 2.2% yield) was obtained from Intermediate 1.5 (60.0 mg, 91.0 .sub.limo!) and Intermediate 2.55 (60.0 mg, 0.140 mmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=5.46 and 5.53 min, m/z=1262 [M+H].sup.+, m/z=1260 [M−H].sup.−.

    ##STR00097##

    [0538] To a solution of Compound 8 (100.0 mg, 0.105 mmol) in DMF (1 mL) DIEA (55.0 μL, 0.314 mmol), Palmitic acid (29.5 mg, 0.115 mmol) and HATU (43.8 mg, 0.115 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed and the residue was applied to a flash chromatography to provide 11 mg (9% yield) of Compound 39. LC-MS: Rt=5.67 and 5.73 min, m/z=1193 [M+H].sup.+, m/z=1191 [M−H].sup.−.

    ##STR00098##

    [0539] To a solution of Compound 8 (100.0 mg, 0.105 mmol) in DMF (1 mL) DIEA (55.0 μL, 0.314 mmol), Stearic acid (32.8 mg, 0.115 mmol) and HATU (43.8 mg, 0.115 mmol) were added. The mixture was stirred at rt overnight. Then the solvent was removed and the residue was applied to a flash chromatography to provide 1.9 mg (2% yield) of Compound 40. LC-MS: Rt=6.15 and 6.21 min, m/z=1121 [M+H].sup.+, m/z=1119 [M−H].sup.−.

    ##STR00099##

    [0540] Compound 41 12.5 mg (13% yield) was obtained from Intermediate 1.24 (50.0 mg, 74.0 μmol) and Intermediate 2.55 (80.0 mg, 110.0 μmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=5.49 and 5.59 min, m/z=1280 [M+H].sup.+, m/z=1281 [M−H].sup.−.

    ##STR00100##

    [0541] Compound 42 7.1 mg (10% yield) was obtained from Intermediate 1.22 (50.0 mg, 72.0 μmol) and Intermediate 2.55 (105.0 mg, 144.0 μmol) in THF using a similar procedure to that described for Compound 4. LC-MS: Rt=5.62 and 5.70 min, m/z=1296 [M+H].sup.+, m/z=1294 [M−H].sup.−.

    ##STR00101##

    [0542] Compound 43 (12.0 mg, 36% yield) was obtained from Intermediate 1.5 (15 mg, 0.023 mmol) and Intermediate 2.65 (42 mg, 0.042 mmol) in DMF using a similar procedure to that described for Compound 4. LC-MS: Rt=7.23 and 7.12 min, m/z=1460 [M+H].sup.+, m/z=1458 [M−H].sup.−.

    ##STR00102##

    [0543] Compound 44 (24.0 mg, 26% yield) was obtained from Intermediate 1.24 (40 mg, 0.059 mmol) and Intermediate 2.65 (109 mg, 0.118 mmol) in DMF using a similar procedure to that described for Compound 4. LC-MS: Rt=7.07 and 7.17 min, m/z=1478 [M+H].sup.+, m/z=1476 [M−H].sup.−.

    ##STR00103##

    [0544] Compound 45 (30.0 mg, 35% yield) was obtained from Intermediate 1.22 (40 mg, 0.059 mmol) and Intermediate 2.65 (80 mg, 0.086 mmol) in DMF using a similar procedure to that described for Compound 4. LC-MS: Rt=7.19 and 7.31 min, m/z=1494 [M+H].sup.+, m/z=1492 [M−H].sup.−.

    ##STR00104##

    [0545] Compound 46 (21.0 mg, 24% yield) was obtained from Intermediate 1.22 (72 mg, 0.050 mmol) and Intermediate 2.10 (60 mg, 0.093 mmol) in DMF using a similar procedure to that described for Compound 4. LC-MS: Rt=5.83, 5.92, 6.09 and 6.20 min, m/z=1210 [M+H].sup.+, m/z=1208 [M−H].sup.−.

    [0546] General Procedure for Coupling CDN to an Antibody:

    [0547] Intact and reduced antidody, with or without deglycosylation, were processed using the procedure as follow:

    [0548] An antibody solution in phosphate buffer (Na.sub.2HPO.sub.4 0,1 M+0,1 M NaCl+50 mM borax pH8) was reacted with 0.5 M dithiothreitol (DTT) Ratio DTT/Ac 250/1, the mixture was incubated at 37° C. for 35min. The reduced antibody solution was cooled in an ice-bath at around 0° C. for 15 minutes. Then stock solution of Pro-CDN with a maleimido group in DMSO was added (Ratio Pro-CDN/Ac 15/1) and incubated on a roller-plate in a refrigerator at 4° C. for 3 hours. A solution of N-ethyl maleimide NEM (20 equiv.) in water was added and was incubated at RT for 35 min. The crude coupled antibody was purified using Zeba™ Spin desalting Columns (7kD) with phosphate buffer (Phosphate buffer 0,05 M+0,15 M NaCl pH7,2). The coupled antibody concentration was obtained via the NanoDrop spectrophotometer. The coupled antibody was stored at 4° C.

    [0549] The reduced coupled Antibody (i.e the de-glycosylated coupled Antibody) can be analyzed using LC/MS spectrometry. The Payload Distribution Analysis can be then determined with the DAR (Drug Antibody Ratio) calculator. Raw data obtained from LC/MS were deconvoluted. For complete coupled Antibody (i.e glycosylated coupled Antibody), UV spectroscopy is the simplest and most convenient approach for DAR determination and works well with a wide range of conjugation methods, including cysteine-linked ISAC. As the UV spectrum of the antibody and the payload (here the CDN) have different maximum absorbance wavelengths (λmax) this method can be used to approximate the DAR. After measuring the extinction coefficients (ε) of the antibody at

    [0550] λ=280 nm and that of the Pro-CDN at λ=260 nm, the individual concentrations of mAb and Pro-CDN can be determined by the solution of two simultaneous equations, from which the molar ratio (moles of Pro-CDN per mole of antibody) can be calculated.

    TABLE-US-00001 Antibody Isotype mAb1 hIgG1 N298A mAb2 mIgG2a mAb3 mIgG1 mAb4 hIgG1

    [0551] Compound 47 was obtained from Anti-PDL1-mAb1 (32.7 nmol) and Compound 11 (491.25 nmol) using the general procedure described above to obtain 21.3 nmol (65% Yield) of the conjugate. DAR determination:

    [0552] The deconvolution parameters for this conjugate were set as follows:

    TABLE-US-00002 Mass (Da) Payload Distribution analysis CL855  1 438.0 Reduced Antibody 144 518.0 Coupled Reduced Antibody 155 783.0 7.8 157 259.0 8.8 UV 260 nm 280 nm DAR Anti-PDL1-mAb3 0.152 0.283 Anti-PDL1-mAb3-CL855 0.436 0.418 7.2

    [0553] Compound 48 was obtained from Anti-GP75-mAb2 (23.4 nmol) and Compound 5 (250 nmol) using the general procedure described above to obtain 15.1 nmol (64% Yield) of the conjugate.

    [0554] DAR determination:

    TABLE-US-00003 UV 260 nm 280 nm DAR Anti-PDL1-mAb3 2.220 3.930 Anti-PDL1-mAb3-CL843 3.204 4.387 1.81

    [0555] Compound 49 was obtained from Anti-GP75-mAb2 (11.5 nmol) and Compound 10 (145 nmol) using the general procedure described above to obtain 7.1 nmol (62% yield) of the conjugate.

    [0556] DAR determination:

    TABLE-US-00004 UV 260 nm 280 nm DAR Anti-PDL1-mAb3 0.171 0.312 Anti-PDL1-mAb3-CL851 0.406 0.414 8.7

    [0557] Compound 50 was obtained from Anti-CTLA4-mAb2 (26.5 nmol) and Compound 11 (422 nmol) using the general procedure described above to obtain 12.5 nmol (47% yield) of the conjugate.

    [0558] DAR determination:

    TABLE-US-00005 UV 260 nm 280 nm DAR Anti-PDL1-mAb3 0.139 0.264 Anti-PDL1-mAb3-CL855 0.506 0.457 9.8

    [0559] Compound 51 was obtained from Anti-PDL1-mAb3 (61.4 nmol) and Compound 11 (1242 nmol) using the general procedure described above to obtain 10 nmol (16% yield) of the conjugate.

    [0560] Compound 52 was obtained from Ova (16.1 nmol) and Compound 11 (226.8 nmol) using the general procedure described above to obtain 15.41 nmol (95% yield) of the conjugate.

    [0561] DAR determination:

    TABLE-US-00006 UV 260 nm 280 nm DAR Ova 0.233 0.394 Ova-CL855 1.961 0.997 7.1

    [0562] Compound 53 was obtained from Anti-PDL1-mAb1 (4.4 nmol) and Compound 17 (61.98 nmol) using the general procedure described above to obtain 3.79 nmol (86% Yield) of the conjugate.

    [0563] DAR determination:

    TABLE-US-00007 UV 260 nm 280 nm DAR Anti-PDL1-mAb1 0.530 0.933 Anti-PDL1-mAb1-CL862 0.712 0.793 4.3

    [0564] Compound 54 was obtained from Anti-PDL1-mAb3 (6.79 nmol) and Compound 17 (60.07 nmol) using the general procedure described above to obtain 4.25 nmol (73% yield) of the conjugate.

    [0565] DAR determination:

    TABLE-US-00008 UV 260 nm 280 nm DAR Anti-PDL1-mAb3 0.573 0.983 Anti-PDL1-mAb3-CL862 0.550 0.674 3.0

    [0566] Compound 55 was obtained from Anti-GP75-mAb2 (6.73 nmol) and Compound 17 (62.52 nmol) using the general procedure described above to obtain 4.78 nmol (79% yield) of the conjugate.

    [0567] DAR determination:

    TABLE-US-00009 UV 260 nm 280 nm DAR Anti-GP75-mAb2 0.545 0.975 Anti-GP75-mAb2-CL862 0.577 0.750 2.7

    [0568] Compound 56 was obtained from Anti-CTLA4-mAb2 (6.56 nmol) and Compound 17 (61.08 nmol) using the general procedure described above to obtain 4.88 nmol (82% yield) of the conjugate.

    [0569] DAR determination:

    TABLE-US-00010 UV 260 nm 280 nm DAR Anti-CTLA4-mAb2 0.528 0.950 Anti-CTLA4-mAb2-CL862 0.639 0.782 3.4

    [0570] Compound 57 was obtained from Anti-HER.sub.2-mAb4 (7.10 nmol) and Compound 17 (65.42 nmol) using the general procedure described above to obtain 5.04 nmol (82% yield) of the conjugate.

    [0571] DAR determination:

    TABLE-US-00011 UV 260 nm 280 nm DAR Anti-HER2-mAb4 0.577 1.028 Anti-HER2-mAb4-CL862 0.649 0.803 3.2

    [0572] Compound 58 was obtained from Anti-pGAL-mAb4 (6.70 nmol) and Compound 17 (61.17 nmol) using the general procedure described above to obtain 4.07 nmol (68% yield) of the conjugate.

    [0573] DAR determination:

    TABLE-US-00012 UV 260 nm 280 nm DAR Anti-βGAL-mAb4 0.531 0.970 Anti-βGAL-mAb4-CL862 0.786 0.865 4.5

    [0574] Compound 59: Ova-C1862

    [0575] Compound 59 was obtained from Ova (7.89 nmol) and Compound 17 (198.0 nmol) using the general procedure described above to obtain 4.30 nmol (79% yield) of the conjugate.

    [0576] DAR determination:

    TABLE-US-00013 UV 260 nm 280 nm DAR Ova 0.348 0.584 Ova-CL862 0.690 0.471 2.3

    [0577] Compound 60 was obtained from PE (7.89 nmol) and Compound 17 (198.0 nmol) using the general procedure described above to obtain 4.30 nmol (79% yield) of the conjugate.

    Example 2: Biological assays for Linked CDNs

    [0578] In the present invention, the immunomodulatory activity of CDN analogs modified with linker systems (Pro-CDN) and the CDN-BAM conjugates described in Formula (I) and in Formulae (V.sub.a) to (V.sub.f), was ascertained in in vitro-cell based assay and in live mammalian cells. These compounds induced the production of multiple cytokines, specifically the production of Type I interferons and/or pro-inflammatory cytokines, as indirectly determined by an ISG54 (interferon-stimulated gene) reporter assay (Fenster) et al., 2008).

    [0579] The in vitro cytokine-induction activity of a representative set of both Pro-CDN and BAM-CDN conjugates is reported here to require the presence of the eukaryotic cellular receptor known as “stimulator of interferon genes” (STING).

    [0580] These experiments were performed as described below.

    Example 2.1: Evaluation of Pro-CDN for their Ability to Activate STING-Dependent Cytokine Induction in vitro in a Human or Murine Reporter Cell Line

    [0581] Cytokine reporter cell line used: THP1-Dual™ and RAWLucia™ ISG cells (InvivoGen cat code: thpd-nfis, rawl-isg) [0582] Compounds tested: [0583] CL793 (Compound 1) vs CL702 (Intermediate 1.14) [0584] CL802 (Compound 2) and CL804 (Compound 3) vs CL797 (Intermediate 1.5) [0585] Reference compounds: 2′3′-cGAMP (InvivoGen catalog code: tlrl-nacga23-5) [0586] Cytokines evaluated: IFN-α/β and NF-κB activity

    [0587] The in vitro STING agonist activity disclosed in the present invention has been measured by monitoring of the IRF pathway. The IRF pathway has been investigated by using the two following ISG reporter cell lines (described here and provided with their corresponding InvivoGen catalog code).

    [0588] RAW-Lucia™ ISG (InvivoGen catalog code: rawl-isg): These cells were generated from the RAW 264.7 murine macrophage cell line (ATCC® TIB-71™). They enable study of IRF signaling pathway, by assessing the activity of a secreted luciferase (Lucia), measured in cell culture supernatant by using QUANTI-Luc™ (InvivoGen; catalog code: rep-qIc1), a luminometric enzyme assay that measures luciferase expression.

    [0589] THP1-Dual™ (InvivoGen catalog code: thpd-nfis): These cells were derived from the human monocytic cell line THP-1 by stable integration of two inducible reporter constructs. They enable simultaneous study of two signaling pathways: the NF-κB pathway, by monitoring the activity of secreted embryonic alkaline phosphatase (SEAP); and the IRF pathway, by assessing the activity of a secreted luciferase (Lucia). Both reporter proteins can be readily measured in the cell culture supernatant by using QUANTI-Blue™ (InvivoGen catalog code: rep-qb1), a SEAP detection reagent that turns purple/blue in the presence of SEAP (quantified by measuring the optical density from 620 nm to 655 nm), and QUANTI-Luc™ (InvivoGen; catalog code: rep-qIc1), a luminometric enzyme assay that measures luciferase expression to report on ISG54 expression (as an indicator of IFN-α/β production).

    [0590] To each well of a flat-bottom 96-well plate were added 20 μL of a solution a CDN (50 μM in saline solution), followed by 180 pi of a suspension of a single cell line (100,000 cells per well). The plate was incubated for 18 h to 48 h at 37° C. in 5% CO.sub.2. The level of ISG response in each well was indirectly quantified using QUANTI-Luc™ (as an indicator of ISG/IFN-β production), which was prepared and used according to the manufacturer's instructions (InvivoGen).

    [0591] The results from this experiment are shown above in FIG. 1 (A-F). FIGS. 1A and 1D illustrate the fold induction for tested compound (CL793, CL802 and CL804) relative to untreated cells and compared to the reference compound (2′3′-cGAMP and unmodified CDN CL702 or CL797) when used at the same concentration in an ISG murine reporter cell line. FIGS. 13 and 1E depict the ISG response in a human reporter cell line while FIGS. 1C and 1F the STING-dependent NF-κB response. The different dose response curves show that for each one of the read out (ISG or NF-κB), the tested CDNs (CL793, CL802 or CL804) induce a similar to a greater response than the 2′3′-cGAMP (the endogenous STING agonist in mammals) does. The modified Pro-CDN compounds display the same activity than the unmodified CDN CL702 or CL797. This findings demonstrate that modification of STING ligands to promote their binding to any biologically active molecules are able to keep on their ability to induce ISG and NF-κB pathways.

    Example 2.2: Evaluation of Pro-CDNs for their Ability to Induce Cytokines ex vivo in Whole Blood from Healthy Human Donors

    [0592] Compounds tested: [0593] CL793 (Compound 1) vs CL702 (Intermediate 1.14) [0594] CL802 (Compound 2) and CL804 (Compound 3) vs CL797 (Intermediate 1.5) [0595] Reference compound: 2′3′-cGAMP (InvivoGen catalog code: tlrl-nacga23-5) [0596] Cytokines evaluated: Type I IFNs (using HEK-Blue™ IFN-α/β cells KO STING)

    [0597] Reporter cell lines:

    [0598] Type I IFNs: HEK-Blue™ IFN-α/β-KO-STING: These cells, in which the STING gene has been inactivated, are derived from HEK293 cell line known as HEK-Blue™ IFN-α/β (InvivoGen catalog code: hkb-ifnab). HEK-Blue™ IFN-α/β cells enable detection of bioactive human type I IFNs through monitoring of activation of the ISG3 pathway. These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active Type-I IFN signaling pathway. The other genes of the pathway (IFNAR.sub.1, IFNAR.sub.2, JAK1, TyK2 and STAT1) are naturally expressed in sufficient amounts. The cells were further transfected with a SEAP reporter gene under control of an IFNα/β-inducible ISG54 promoter. This promoter comprises five IFN-stimulated response elements (ISREs) fused to a minimal promoter of the human ISG54 gene, which is unresponsive to activators of the NF-κB or AP-1 pathways. Stimulation of HEK-Blue™ IFN-α/β cells with human IFN-α or IFN-β activates the JAK/STAT/ISGF3 pathway and subsequently induces production of SEAP. Production of type I IFNs in these cells is measured using QUANTI-Blue™.

    [0599] Acquisition and Handling of Human Blood Samples

    [0600] Human blood samples were acquired from healthy donors at the Etablissement Français du Sang (EFS Pyrénées Méditerranée, Toulouse, France; per agreement #21/PLER/TOU/2016--0082). Briefly, the samples were collected by venipuncture into sodium heparin tubes at the time of donation. The samples were analyzed for rhesus (Rh), blood group, hematocrit and serological status (AgHBS, HIV, HCV, HTLV, HBC, CMV, Syph). The tubes were picked up on the day of collection and subsequently tested (blood analysis, and treatment with test items) on the same day.

    [0601] Treatment of Human Blood Samples

    [0602] Each blood sample was diluted (1:2 [v/v]) in RPMI medium and aliquoted into 96-well plates (180-μL wells) containing either CDN (√10-fold serial dilution starting from 50 μM). The plates were incubated at 37° C. in a 5% CO.sub.2 incubator for 18 hours. Then, the supernatants were collected, transferred into the corresponding wells of round-bottom 96-well plates, and either stored at −80° C., or immediately tested in the appropriate reporter cell line.

    [0603] Testing of Human Blood Samples

    [0604] A new 96-wells plate was prepared for each reporter cell lines tested, as follows: 10 μL of supernatant from the previous plate were added to the corresponding well in the new reporter cell plate. Then, a 190-μL aliquot of cells of the desired reporter cell line, previously harvested in medium containing heat-inactivated serum and counted, was added to each well (approximately 50,000 cells/well), and the plate was incubated for approximately 20 hours. The desired cytokine induction activity was determined using the QUANTI-Blue™ assay, as previously described. Briefly, 20 μL of supernatant from the previously incubated plate was transferred to the corresponding well of a new 96-well plate in which 180 μL of QUANTI-Blue™ reagent had previously been added.

    [0605] Results presented in FIG. 2 summarize the induction of type I IFNs for CDN-linker system compounds (CL793, CL802 and CL804) relative to the reference compound (2′3′-cGAMP) or unmodified CDN (CL702 or CL797 respectively) in the whole-blood assay for each compound in this assay. As shown in FIG. 2A, the tested Pro-CDN CL793 provides, like the unmodified corresponding CDN (CL702), superior induction of type I interferons compared to the reference compound 2′3′-cGAMP. The dose-response curve in FIG. 2B shows that the tested Pro-CDN CL804 induce a type I IFNs production similar to the one induced by the unmodified corresponding CDN, CL797. This findings corroborate in vitro observations presented in FIG. 1 and demonstrate that modifications of STING ligands to promote their linkage to any biologically active molecules are able to keep on their ability to induce type I IFN pathway.

    Example 2.3: Evaluation of BAM-CDN Conjugates for their Ability to Activate STING-Dependent Cytokine Induction in vitro in a Human or Murine Reporter Cell Line

    [0606] Cytokine reporter cell line used: THP1-Dual™ and RAW-Lucia™ ISG cells [0607] Compounds tested: CL808 (Compound 22) vs CL797 (Intermediate 1.5) [0608] Reference compounds: 2′3′-cGAMP (InvivoGen catalog code: tlrl-nacga23-5) [0609] Cytokines evaluated: IFN-α/β and NF-κB activity

    [0610] The in vitro STING agonist activity of BAM-CDN conjugates has been measured by monitoring of the IRF pathway as described above in Example 2.1.

    [0611] The results from this experiment are shown above in FIG. 3 (A-C). FIG. 3A illustrates the fold induction for tested compounds (CL794 and CL808) relative to untreated cells and compared to the reference compound (2′3′-cGAMP and unmodified corresponding CDN CL797) when used at the same concentration in an ISG murine reporter cell line. FIG. 4B shows the ISG response in a human reporter cell line while FIG. 3C the STING-dependent NF-κB response. The different dose response curves (FIGS. 3 A-C) show that the BAM-CDN CL808 induces an ISG or NF-κB response to a greater extent than does 2′3′-cGAMP, the endogenous STING agonist in mammals. The BAM-CDN compound CL808 displays a much higher activity compared to the unmodified corresponding CDN, CL797. It has to be noted that we observed toxicity at higher concentration of CL808 for both cell lines. This finding demonstrates that the coupling of STING ligands to a biologically active molecules does not modify their ability to induce ISG and NF-κB pathways. In this example, BAM is a lipid moiety that is able to form liposomal structure with CDN or a nanoparticle to protect and to increase the uptake of CDN. The strong gain in activity compare to unmodified CDN could be explained by a better penetration of the CDN to reach its intracellular target.

    Example 2.4: Evaluation of BAM-CDNs for their Ability to Induce Cytokines ex vivo in Whole Blood from Healthy Human Donors

    [0612] Compound tested: CL808 (Compound 22) [0613] Reference compound: unmodified CDN CL797 (Intermediate 1.5) [0614] Cytokines evaluated: Type I IFNs, IL1α/β, IL6 and TNFα (using HEK cytokine reporter cell lines)

    [0615] Reporter Cell Lines:

    [0616] Type I IFNs: HEK-Blue™ IFN-α/β-KO-STING: These cells, in which the STING gene has been inactivated, are derived from HEK293 cell line known as HEK-Blue™ IFN-α/β (InvivoGen catalog code: hkb-ifnab). HEK-Blue™ IFN-α/β cells enable detection of bioactive human type I IFNs through monitoring of activation of the ISG3 pathway. These cells were generated by stable transfection of

    [0617] HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active Type-I IFN signaling pathway. The other genes of the pathway (IFNAR.sub.1, IFNAR.sub.2, JAK1, TyK2 and STAT1) are naturally expressed in sufficient amounts. The cells were further transfected with a SEAP reporter gene under control of an IFN-α/β-inducible ISG54 promoter. This promoter comprises five IFN-stimulated response elements (ISREs) fused to a minimal promoter of the human ISG54 gene, which is unresponsive to activators of the NF-κB or AP-1 pathways. Stimulation of HEK-Blue™ IFN-α/β cells with human IFN-α or IFN-β activates the JAK/STAT/ISGF3 pathway and subsequently induces production of SEAP. Production of type I IFNs in these cells is measured using QUANTI-Blue™.

    [0618] IL1α/β: HEK-Blue™ IL-1R (InvivoGen catalog code: hkb-il1r): The HEK293 cell line known as HEK-Blue™ IL-1R was designed to detect bioactive human and murine IL-1 through monitoring of activation of the NF-κB and AP-1 pathways. Additionally, these cells detect bioactive IL-1 from cynomolgus monkeys, dogs, hamsters and rats. In fact, HEK-Blue™ IL-1R cells can detect IL-1α and IL-1β, as these cytokines bind to the same receptor, IL-1R. These cells derive from HEK-Blue™ IL-1β cells (InvivoGen catalog code: hkb-il1b), in which the TNF-α response is blocked. Therefore, HEK-Blue™ IL-1R cells respond specifically to IL-1. These cells endogenously express the human IL-1 receptor and were stably transfected with the murine IL-1 receptor, rendering them sensitive to both human and murine IL-1β. HEK-Blue™ IL-1R cells express a SEAP reporter gene under control of an IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites. Binding of IL-1β to IL-1R on the surface of HEK-Blue™ IL-1R cells triggers a signaling cascade that leads to the activation of NF-κB and subsequent production of SEAP. Production of IL-1β in these cells is measured using QUANTI Blue™.

    [0619] IL6: HEK-Blue™ IL-6 (InvivoGen catalog code: hkb-hil6): HEK-Blue™ IL-6 cells allow the detection of bioactive human IL-6 by monitoring the activation of the STAT-3 pathway. These cells were generated by stable transfection of HEK293 cells with the human IL-6R gene and a STAT3-inducible SEAP reporter gene. Upon IL-6 stimulation, HEK-Blue™ IL-6 cells trigger the activation of STAT3 and the subsequent secretion of SEAP. Levels of STAT3-induced SEAP can be readily monitored using QUANTI-Blue™.

    [0620] TNFα: HEK-Blue™ TNF-α (InvivoGen catalog code: hkb-tnfdmyd): HEK-Blue™ TNF-α cells are a HEK293 cell line that enables detection of bioactive human and murine TNF-α through monitoring of activation of the NF-κB pathway. These cells were generated by stable transfection of HEK293 cells with a SEAP reporter gene under control of an IFN-β minimal promoter fused to five NF-κB and five AP-1 binding sites. They were further rendered unresponsive to IL-1β by knocking out the MyD88 gene. Stimulation of HEK-Blue™ TNF-α cells with TNF-α triggers activation of the NF-κB-inducible promoter and production of SEAP. Production of TNF-α in these cells is measured using QUANTI-Blue™.

    [0621] The ex vivo activity of BAM-CDN conjugates has been measured by monitoring the production of type I IFNs, and pro-inflammatory cytokines IL1α/β, IL6 and TNFα, in whole blood assays as described above in Example 2.2.

    [0622] The results presented in FIG. 4 confirms findings observed in previous Example 2.3. Whatever the cytokine tested, CL808 displays a stronger activity than the unmodified corresponding CDN CL797 (significant shift of the dose-response curve to the left). Of note the C.sub.16 carbon chain moiety by itself doesn't have ISG or NFκB activity. The chemistry coupling of a biologically active molecule (here a lipid) on the parent CDN CL797 does not alter the STING-dependent cytokine induction and strongly increase the basal activity due to a better penetration/uptake of the CDN.

    Example 3: Biological Assays for Pro-CDNs

    [0623] In the present invention, the immunostimulatory activity of these CDN analogs modified with linker systems (Pro-CDN) described in Formula (I) and in Formulae (V.sub.a) to (V.sub.f), was ascertained in in vitro-cell based assays. These compounds induced the production of multiple cytokines specifically the production of Type I interferons, as determined by a proprietary IRF (interferon regulatory factor) reporter cell based assay. The in vitro cytokine-induction activity of a representative set of Pro-CDN is depicted here after. [0624] Cytokine reporter cell line used: THP1-Dual™ cells (InvivoGen catalog code: thpd-nfis) [0625] Compounds tested: CL804 (Compound 3), CL822 (Compound 6), CL831 (Compound 7), CL843

    [0626] (Compound 5), CL846 (Compound 8), CL847 (Compound 9), CL851 (Compound 10), CL750 (see formula below), CL855 (Compound 11), CL856 (Compound 12), CL862 (Compound 17), CL868 (Compound 15), CL869 (Compound 16), CL 873 (Compound 20) and CL 874 (Compound 21)

    ##STR00105## [0627] Reference compounds: 2′3′-cGAMP (InvivoGen catalog code: tlrl-nacga23-5), CL797 (Intermediate 1.5), CL845 (Intermediate 1.24), CL656 (Intermediate 1.22) also known as 3′3′-cAlM(PS)2 Difluor (Rp/Sp) (InvivoGen catalog code: tlrl-nacairs) [0628] Cytokines evaluated: IFN-α/β

    [0629] The in vitro STING agonist activity disclosed in the present invention has been measured by monitoring of the IRF pathway. The IRF pathway has been investigated by using InvivoGen's THP1-Dual reporter cell line as described in Example 2.

    TABLE-US-00014 TABLE ISG activity of pro-CDN described in the present invention. Fold ISG % activity induction vs Pro-CDN/ LINKER EC50 (vs unstimulated Compound CDN Connector Specifier Spacer (μM) cGAMP) @10 μM CL804 CL797 PAB Val-Ala / 11.77 174 110 CL822 CL797 PAB Val-Ala (AEEA).sub.4 n/a 29 CL831 CL797 PHMNB GLU / n/a 6 CL843 CL797 PAB Val-Ala Mal-Aca n/a 16 CL797 / / / 8.15 251 181 (Reference) CL846 CL845 PAB Val-Ala 4.21 486 269 CL847 CL845 PHMAB GLU Mal-Aca n/a 132 CL851 CL845 PHMAB GLU Mal-Aca-Aca 12.83 160 37 CL845 / / / 4.57 448 227 (Reference) CL750 CL614 1-Amino-pentanol n/a n/a 1 (Comparative) (POM) CL855 CL656 PHMAB GLU Mal-Aca-Aca 7.136 287 115 CL856 CL656 PHMAB × 2 GLU Bis(Mal-Aca- 3.097 661 59 Aca) CL862 CL656 PAB Val-Cit Mal-Aca-Aca 6.688 306 198 CL868 CL656 PAB Val-Cit / 1.764 1161 291 CL869 CL656 PAB × 2 Bis(Val- / 1.359 1507 231 Cit) CL873 CL656 PAB Val-Ala / 1.025 1998 211 CL874 CL656 PAB Val-Ala Dap-Mal 6.812 297 156 CL656 / / / 1.004 2040 215 (Reference) n/a: not applicable, AEEA for [2-(2-aminoethoxy)ethoxy]acetic acid, GLU = Methyl-2,3,4-tri-O-acetyl)-D-glucopyranouronate, Aca = Aminocaproate, Mal = Maleimido, Dap = 2,3-diaminopropionic acid. EC.sub.50 2′3′cGAMP = 20.48 μM (Lioux et al., 2016) (48 h post-stimulation). % activity (vs cGAMP) = (EC.sub.50 2′3′cGAMP/EC.sub.50 compound) × 100 Fold ISG induction vs unstimulated 2′3′-cGAMP at 10 μM = 93.

    [0630] The results from this experiment are summarized above in Table above. First, each CDN-linker system or Pro-CDN described in the present invention displayed a significant ISG response from a fold of 6 to 291 versus unstimulated, when they are used at 10 μM. Then, in comparison with 2′3′-cGAMP, most of pro-CDNs have a greater ISG activity and those derived from CL656 display a better potency than the pro-CDNs derived from unmodified CL797 or CL845, as observed with the CDN from which they are derived. Lastly, we can conclude that in order to have an ISG activity, the moiety added to the CDN phosphorous group to transform it into a pro-CDN has to be cleavable as the CL750 which comprises an uncleavable moiety does not display any activity.

    [0631] In the present invention we also describe the use of two types of maleimide group to form pro-CDNs.

    [0632] Maleimide groups have been extensively used for coupling on cysteine because they react rapidly and selectively with thiols. It has recently come to light that the thioether linkage undergoes deconjugation through a retro-Michael pathway, leading to loss of cargo and reduction in efficacy (FIG. 5). The maleimide-cargo conjugate can then be bound to other plasma thiols (e.g. human serum albumin) leading to off-site toxicity and reduced efficacy.

    [0633] ‘Selfhydrolysing maleimide reagents undergo rapid hydrolysis to the corresponding succinamic acid post-conjugation, thus eliminating the retro-Michael deconjugation pathway and resulting in more effective/stable bioconjugates. Pro-CDN CL874 is an example of compounds that can be coupled to a thiol of a cystein present in an antibody or protein and can be rapidly hydrolyzed to obtain a stable conjugated compound. This pro-CDN retains full ISG activity.

    Example 4: Biological assays for BAM-CDNs

    Example 4.1: BAM=Lipid

    [0634] The in vitro STING agonist activity of BAM-CDN conjugates (with BAM=lipid) has been measured by monitoring the IRF pathway as described above in Example 2.3. [0635] Cytokine reporter cell lines used: THP1-Dual™ cells and RAW-ISG Lucia ™ (InvivoGen catalog code: thpd-nfis and rawl-isg) [0636] Compounds tested: CL808 (Compound 22), CL821 (Compound 25), CL820 (Compound 24), CL819 (Compound 23), CL850 (Compound 40), CL849 (Compound 39), CL841 (Compound 27), CL840 (Compound 26), CL842 (Compound 28), CL863 (Compound 46) [0637] Reference compounds: CL797 (Intermediate 1.5), CL845 (Intermediate 1.24) and CL656 (Intermediate 1.22) also known as 3′3′-cAIM(PS)2 Difluor (Rp/Sp) (InvivoGen catalog code: tlrl-nacairs) [0638] Cytokines evaluated: IFN-α/β

    [0639] The results shown in FIG. 3 (A-C) in Example 2.3 show that the BAM-CDN compound CL808 displays a much higher activity compared to the unmodified corresponding CDN, CL797. This finding demonstrates that lipid moiety is able to form liposomal structure with CDN or a nanoparticle to protect and to increase the uptake of CDN. In this example and as described in Example 2.3., we further evaluated the role of the lipid chain in terms of IRF pathway and particle formation.

    [0640] The results presented in FIG. 6 confirm findings observed in previous Example 2.3. CL808 displays a stronger activity than the unmodified corresponding CDN CL797 (significant shift of the dose-response curve to the left). The chemistry coupling of a biologically active molecule (here a lipid) on the parent CL797, CL845 or CL656 strongly increases the basal activity due to a better penetration/uptake of the CDN and the improvement in potency is significant with C8 or longer chain (e.g. EC50 (CL808=C16-CL797)<EC50 (CL821=C8-CL797)<EC50 (CL820=C4-CL797)=EC50 (CL819=C2-CL797)=EC50 (CL797)).

    [0641] As shown in the Table below, using hydrophobic lipid chains as a BAM (a C14 C14 for example), the modified CDN can self-organize/assemble in lipid-based nanoparticles (CL838, CL859, CL860, CL842 or CL861). Then, when the specifier is the dipeptide Valine-Citrulline (CL860 or CL861) the size tends to be smaller, around the size of Small Unilamellar Vesicle (aka SUV<40 nm).

    [0642] This organization in lipid-based particles suggests that the CDN is protected from the environment and allows a better penetration and a sustained release mechanism into the cell.

    TABLE-US-00015 EC.sub.50 EC.sub.50 (μM) (μM) IFN Pro-CDN/ BAM = Z Ave THP1- in Compound CDN Connector Specifier Spacer lipid PdI (nm) Dual WBA CL821 CL797 PAB Val-Ala / C.sub.8 0.27 54.9 0.23 7.1 CL838 CL797 PAB Val-Ala / C.sub.14/C.sub.14 0.214 91 0.09 0.009 CL859 CL797 PAB Val-Cit / C.sub.14/C.sub.14 0.372 39.2 1.35 0.59 CL808 CL797 PAB Val-Ala / C.sub.16 0.382 173.9 0.006 0.24 CL848 CL797 PAB Val-Cit / C.sub.16 0.18 84.3 0.01 0.11 CL840 CL656 PAB Val-Ala / C.sub.8 0.08 168.9 0.005 0.13 CL842 CL656 PAB × 2 bis(Val-Ala) / C.sub.8 × 2 0.086 184 0.006 1.4 CL861 CL656 PAB Val-Cit / C.sub.14/C.sub.14 0.183 36.8 0.27 0.01 CL863 CL656 PAB Val-Ala / C.sub.16 0.416 106.6 0.04 0.15 CL853 CL656 PAB Val-Cit / C.sub.16 0.188 155.9 0.02 0.14 CL841 CL845 PAB Val-Ala / C.sub.8 0.475 264.3 0.19 3.03 CL849 CL845 PAB Val-Ala / C.sub.16 0.56 152.4 0.12 0.12 CL852 CL845 PAB Val-Cit / C.sub.16 0.468 100.7 1.01 0.41 CL860 CL845 PAB Val-Cit / C.sub.14/C.sub.14 0.248 33 0.44 0.009

    Example 4.2: Dipeptide Specifier: Comparison Val-Citrulline vs Val-Alanine

    [0643] A general substrate for cathepsins is Z-Val/Phe-Arg-X (Barrett, A. J. Biochem. J. 1980, 187, 909-912). Over decades, protected substrates have been developed and substrates containing citrulline (Cit), which is isosteric and isoelectronic like Arg but much less basic, display a superior systemic stability over other cleavable linkers (Dubowchik et al, Bioorganic & Medicinal Chemistry Letters 1998, 3341-3346). The widely used Val-Cit dipeptide linker has been popularized as a way to maintain a stable covalent attachment of the drug to the antibody that could be preferentially cleaved by intracellular protease(s) of the lysosomal degradation pathway (Dubowchik GM et al Bioconjug Chem 2002, 13:855-69 or Junutula JR et al Nat Biotechnol 2008; 26:925-32). Based on this literature, in this application we describe the use of Val-Cit as a specifier to connect CDN to a BAM (a C.sub.16 lipid chain in this example) using the methodology described in Example 2.1 and we compare it to the Val-Ala specifier described in patent WO2018/100558. [0644] Cytokine reporter cell lines used: RAW-ISG Lucia™ (InvivoGen ca code: rawl-isg) [0645] Compounds tested: CL848 (Compound 38), CL808 (Compound 22), CL852 (Compound 41), CL849 (Compound 39), CL853 (Compound 42), CL863 (Compound 46) [0646] Reference compounds: CL797 (Intermediate 1.5), CL845 (Intermediate 1.24) and CL656 (Intermediate 1.22) [0647] Cytokines evaluated: IFN-α/62

    [0648] The results presented in FIG. 7 show that a dipeptide specifier used to link STING agonists to a biologically active molecule (here a lipid moiety) is highly efficient to induce an ISG response. In THP1 Dual and RAW ISG cells, CL848 and CL808, CL852 and CL849, CL853 and CL863, exert higher ISG activity than the unmodified parental CDN, CL797, CL845 and CL656 respectively. The cathespins-cleavable linkers allow the release of the active form of CDN and the use of Valine-Citrulline appears better than Valine-Alanine to improve the ISG response.

    Example 4.3 BAM =Detection System

    [0649] Defining, visualizing and sorting the cells that are able to uptake a functional CDN is a challenge for researchers and pharmaceutical companies. Several fluorescent cGAMP analogs are commercially available but none of them have been reported to have a STING dependent ISG activity. In the present invention, we evaluated the immunostimulatory properties of a novel chimeric compound designed that have both a STING agonist activity and a detection system (Fluorescent tag or biotin). [0650] Cytokine reporter cell line used: THP1-Dual™ cells (InvivoGen cat code: thpd-nfis) [0651] Compounds tested: CL832 (Compound 34), CL833 (Compound 35) [0652] Reference compounds: CL797 (Intermediate 1.5) and fluorescent-CDN (Fluorescent cGAMP available from www.biolog.de, cat n° C. 178-001) [0653] Cytokines evaluated: IFN-α/β

    [0654] The results presented in FIG. 8 show that STING agonists linked to a detection system have the same ISG activity than the unmodified corresponding CDN CL797, while a commercially available fluorescent CDN (www.biolog.de, cat n° C. 178-001) is not able to induce an IRF response. The chemistry coupling of a detectable biologically active molecule allows to visualize and quantify STING agonists internalization in a dose and time-dependent manner.

    Example 4.4 BAM=PRR Ligand (TLR)

    [0655] For vaccination purpose, potent new adjuvants are required to facilitate the development of more effective vaccines. PRR (including TLRs) ligands have distinct functions in the innate immune system and are commonly used as vaccine adjuvants. Synergistic crosstalk between TLRs has been in the spotlight recently (Goff et al 2015. J. Virol. 89: 3221-3235). Among them, TLR7, a nucleic acid sensor, plays an important role in the immune response to viral infection by recognizing ssRNAs. In the present invention, we evaluated the immunostimulatory properties of a novel chimeric compound designed to stimulate both intracellular STING and TLR7 pathways. [0656] Reporter cell lines used: THP1-Dual™ cells, RAW-ISG-Lucia™ or HEK-Blue TLR7™ (InvivoGen cat code: thpd-nfis, rawl-isg and hkb-htlr7) [0657] Compounds tested: CL834 (Compound 36) [0658] Reference compounds: CL804 (Compound 3) and CL264 (InvivoGen cat code tlrl-c264e) [0659] Cytokines evaluated: IFN-α/β

    [0660] The results presented in FIG. 9 show that STING agonists linked to a TLR7 agonist have similar ISG activity than the unmodified corresponding CDN CL797 (in comparison with data presented in Example 4.2) or and similar TLR7 activity than the corresponding TLR7 ligand (CL264). The chemistry coupling of a PRR ligand to a CDN allows to simultaneously stimulate in the same cell different innate immune signaling pathways.

    Example 4.5 BAM=Heterocyclic Molecule (Folic Acid)

    [0661] The selective delivery of cytotoxic agent has been widely investigated including linking folic acid to a suitable heterobifunctional PEG coupled to a cytotoxic agent like Gemcitabine (G. Pasut et al., Journal of Controlled Release 127 (2008) 239-248). Folic acid (FA) was chosen because its cell surface receptor GP38 is generally over-expressed in several types of human epithelial cancers, especially ovarian, but also kidney, uterus, brain, colon and lung and has been established as a tumor cellular-surface marker for targeted drug delivery. In normal human tissues GP38 has limited distribution, mainly in kidneys, lungs, choroids plexus and placenta (Weitman et al., Cancer Res. 52 (12) (1992) 3396-3401).

    [0662] In addition, previous works have also demonstrated that a subset of macrophages expresses folate receptors that can mediate internalization of folate-linked molecules and that can be used to target drugs to activated macrophages (W. Xia et al., Blood (2009) 113:438-446).

    [0663] In the present invention, we evaluated the ability to use folic acid as a way to target CDN compound to cancer cells or tumor-infiltrated macrophages using the methodology described in Example 2.1. [0664] Cytokine reporter cell lines used: THP1-Dual™ cells and RAW-ISG-Lucia™ (InvivoGen cat code: thpd-nfis, rawl-isg) [0665] Compounds tested: CL826 (Compound 33) [0666] Cytokines evaluated: IFN-α/β

    [0667] The results presented in FIG. 10 show that STING agonists linked to folic acid have the ability to induce an ISG response in immortalized monocyte/macrophage-like cell lines. The chemistry coupling of folic acid to a CDN (FA-small molecule immunoconjugate) with a releasable linker might be a way to vector STING agonists into cells (including tumoral or antigen-presenting cells) expressing folate receptor.

    5. Example BAM=Protein

    [0668] 5.1 BAM=Immune Checkpoint Inhibitor (ICI) Antibody

    [0669] Therapeutic blockade of T-cell coinhibitory receptors such as CTL-associated antigen 4 (CTLA-4, programmed death 1 (PD-1) and programmed death ligand 1 (PDL-1) is sufficient to promote durable immune-mediated tumor regression across multiple cancers in mouse and man. However in multiple applications the efficacy is limited to a percentage of patients. Thus strategies involving simultaneous manipulation of multiple, non-redundant immune regulatory pathways to activate potent antitumor immune responses in mouse and man are currently evaluated. Several reports showed that an optimized cocktail of immunomodulators engaging both innate (using PRR ligand) and adaptive immunity (using immune checkpoint inhibitors aka ICI) mediates efficient rejection of poorly immunogenic cancers (Fu, et al 2015. Sci. Trans!. Med. 7, 283ra52; Charlebois, et al (2017). Cancer Res. 77, 312-319. Corrales et al. (2015). Cell Rep. 11, 1018-1030. Kranz, L et al. (2016). Nature 534, 396-401. Shekarian, et al (2017). Ann. Oncol. 28, 1756-1766). While combination therapies are extensively investigated, linking immunomodulators to a functional immune checkpoint antibody is poorly described. In this application we describe the functionality of a single molecule consisting of an ICI:CDN conjugate which exhibits several advantages over mixtures of both individual molecules. An ICI-conjugated-CDN is expected to gain the pharmacodynamic properties of the antibody and in addition may be administered systemically due to a new PK profile. Further, ICI:CDN conjugates are simultaneously delivered to the same cell in a fixed molecular ratio, thereby preventing potentially detrimental bystander effect when the antibody used also targets STING expressing cells.

    [0670] In the present invention, we evaluate the IFN activity and antibody recognition property of a CDN compound linked to an immune checkpoint inhibitor antibody (here PDL-1 antibody). We aimed to use this compound to boost both the innate and adaptative immune system and to target CDN delivery into the vicinity of PDL-1 expressing cells like tumoral cells or antigen presenting cells (APCs) which are known to express STING.

    [0671] We performed Whole Blood Assay on healthy human donors to evaluate the ability of the anti-PDL1mAb:CDN conjugate (Compound 47) to induce type I Interferons as described previously in Example 2.2. The binding properties of the anti-PDL1mAb:CDN conjugate have been evaluated using

    [0672] ELISA.

    [0673] The results presented in the table above show that the Pro-CDN CL855 (Compound 11) has been conjugated with the anti-PDL1 mAb with a CDN load of ˜8 (DAR 7.8) and the mAb:CDN conjugate is as efficient as the unmodified CDN CL656 (Intermediate 1.22) to induce type I IFNs. The mAb:CDN conjugate retains its antigen binding capacity compared to the anti-PDL1 mAb alone.

    [0674] 5.2 BAM=Protein (including Antibody)

    [0675] In this application, we describe the generation of novel conjugates consisting of a CDN and a protein (that can include any antibody). Protein:CDN conjugates have several advantages over simple non-conjugated mixtures of both components. Similarly to the ICI:CDN conjugate, Protein:CDN conjugates have a new pharmacokinetic profile. If the protein is an antibody, the new compound will be part of the ADC (antibody drug conjugate) family. Such immunoconjugates combine the immunomodulatory potency of CDNs with the high selectivity, stability and favorable pharmacokinetic profile of mAbs.

    [0676] In the present invention, we evaluated using reporter cell lines the ISG activity of protein:CDN conjugates using in vitro conditions favorable to the release of both components.

    [0677] The results presented in the table above show that, using in vitro conditions favorable to the release of both components of the conjugate, released CDNs are able to induce an ISG activity and retain their capacity to bind to the target receptor STING. Indeed, none of the tested Protein:CDN conjugates displayed ISG activity on STING KO reporter cells. Moreover, CDN linkage using Glucuronic acid (Valine-Alanine) or Valine-Citrulline as specifiers is feasible. Notably, the complex consisting of Anti-CTLA4-mAb2-CL862 (Compound 56) has a surprisingly greater ISG activity than the parental CDN. This observation might be due to a nanocomplex formation with compound 56 that allows an optimal uptake by the target cells.

    [0678] 5.3 BAM=Antigen

    [0679] In this application, we describe the generation and immunological characterization of a novel vaccine candidate consisting of the CDN as an adjuvant and an Antigen such as the model allergen Ovalbumin. Antigen:adjuvant conjugates have several advantages over simple non-conjugated mixtures of both components: (1) they target the conjugate to the respective immune cells by binding to specific immune receptors (in this case STING); (2) adjuvant and allergen are simultaneously delivered to the same cell in a fixed molecular ratio, thereby preventing potentially detrimental bystander activation or adjuvant leakage to other cells. Kastenmuller and colleagues reported a conjugate vaccine combining a TLR7/8-ligand and Ova and showed that this conjugate elicits potent Th1-biased T cell responses by activation and recruitment of dendritic cells to draining lymph nodes and the subsequent induction of type I interferon production (Kastenmuller et al., The Journal of Clinical Investigation, vol.121, no. 5, pp. 1782-1796, 2011).

    [0680] The adjuvant potency of STING agonist linked to the antigen disclosed in the present invention has been measured by monitoring the OVA specific immunoglobulin title according the protocol described below.

    [0681] Immunization Protocol:

    [0682] Swiss mice were immunized with a total dose of 10 μg of OVA protein with or without uncoupled STING agonist or a conjugate vaccine (CL855-OVA-Compound 52, CL862-OVA-Compound 59). The regimens were administered sub-cutaneously in a volume of 200 μL at day 0 and challenged on day 15. For analysis of OVA specific immunoglobulin title, blood withdrawal was performed 15 days after primary or secondary immunization.

    [0683] First, we evaluated the ISG activity of antigen:CDN conjugates using in vitro condition favorable to release of both components in order to ensure that we had a linkage.

    [0684] The results presented in the table above show that, using in vitro condition favorable to release of both components of the conjugate, released CDN are able to induce an ISG activity and to retain their capacity to bind to target receptor STING. The Compound 52 (Ova-CL855) and Compound 59 (Ova-CL862) exhibit a similar ISG activity in comparison with the unmodified CDN CL656.

    [0685] Then we measured the ability of Compound 52 and Compound 59 to induce an immune response against ovalbumin by assessing the antibody title reacting against the antigen. We compared Compound 52 and Compound 59 to the combination of both molecules (OVA and CL656) by adapting the amount of CL656 injected based on the DAR (Drug Antigen Ratio) described in table above. A positive control consisting of Alum as adjuvant for ovalbumin was used.

    [0686] As shown in FIG. 11, the coupling of a CDN to an antigen is efficient to induce a significant immunization by day 15 which is in the same range than the one induced by Alum-complexed ovalbumin. The example described above is not limited to OVA antigen. Trying to overcome issues related to tumor escape and immunosuppression is the current challenge for the development of more efficient immunotherapeutic vaccines. Pro-CDN linkage to tumoral antigen can be a way to boost immunity against cancer cell. Several studies have produced database referencing all human tumor antigens recognized by T lymphocytes and usable in cancer immunotherapy approaches according to the invention (Van der Bruggen et al. Cancer Immun. 2013; 13: 15. Novellino L et al, Cancer Immunol Immunother, 2005 Mar;54(3):187-207). The most well-known (but not exhaustive) tumor antigens that can be easily coupled to pro-CDN can be selected from the group consisting of alpha-actinin-4; ARTC1; BCR-ABL fusion protein (b3a2); B-RAF; CASP-5; CASP-8; beta-catenin; Cdc27; CDK4; CDKN2A; COA-1; dek-can fusion protein; EFTUD2; Elongation factor 2; ETV6-AML1 fusion protein; FN1; GPNMB; LDLR-fucosyltransferaseAS fusion protein; HLA-A2d; HLA-A11d; hsp70-2; KIAA0205; MART2; ME1; MUM-If; MUM-2; MUM-3; neo-PAP; Myosin class I; NFYC; OGT; OS-9; pml-RARalpha fusion protein; PRDXS; PTPRK; K-ras; N-ras; RBAF600; SIRT2; SNRPD1; SYT-SSX1 or -SSX.sub.2 fusion protein; Thosephosphate Isomerase; BAGE-1; GAGE-1,2,8; GAGE-3,4,5,6,7; GnTVf; HERV-K-MEL; KK-LC-1; KM-HN-1; LAGE-1; MAGE-A1; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-A10; MAGE-Al2; MAGE-C2; mucin k; NA-88; NY-ESO-1/LAGE-2; SAGE; Sp17; SSX-2; SSX-4; TRAG-3; TRP2-INT2g; CEA; gp100/Pnne117; Kallikrein 4; mammaglobin-A; MeIan-A/MART-1; NY-BR-1; 0A1; PSA; RAB38/NY-MEL-1; TRP-1/gp75; TRP-2; tyrosinase; adipophilin; AIM-2; BING-4; CPSF; cyclin D1; Ep-CAM; EphA3; FGF5; G250/MN/CAIX; HER-2/neu; IL13Ralpha2; Intestinal carboxyl esterase; alpha-foetoprotein; M-CSF; mdm-2; MMP-2; MUC1; p53; PBF; PRAME; PSMA; RAGE-1; RNF43; RU2AS; secernin 1; SOX10; STEAP1; survivin; Telomerase; WT1; FLT3-ITD; BCLX(L); DKK1; ENAH(hMena); MCSP; RGS5; Gastrin-17; Human Chorionic Gonadotropin, EGFRvlll, HER.sub.2, HER.sub.2/neu, P501, Guanylyl Cyclase C, and PAP. The chemistry coupling of a CDN to an antigen (viral, bacterial, tumor antigens) with a releasable linker is a suitable way to improve the quality of the innate immune response because of the co-delivery of both adjuvant and antigen to the same antigen-presenting cell.