Novel phosphoantigen prodrug compounds and their use

Abstract

The present invention relates to novel phosphoantigen (PAg) prodrug compounds that provide potent activation of therapeutically active γδ T cells. The PAg prodrug compounds of the present invention can be used for the preparation of therapeutically active γδ T cells and for immunotherapy of different diseases, particularly different types of cancer. The present invention further relates to a method for proliferating γδ T cells ex vivo using the PAg prodrug compounds of the present invention. Also provided are γδ T cells proliferated with the PAg prodrug compounds of the present invention, as well as pharmaceutical compositions containing such proliferated cells and / or the PAg prodrug compounds of the present invention.

Description

[Technical Field]

[0001] This invention relates to novel phosphoantigen (PAg) prodrug compounds that potently activate therapeutically active γδ T cells. The PAg prodrug compounds of this invention can be used for the preparation of therapeutically active γδ T cells and for immunotherapy of different diseases, particularly different types of cancer. This invention further relates to a method for proliferating γδ T cells ex vivo using the PAg prodrug compounds of this invention. Also provided are γδ T cells proliferated with the PAg prodrug compounds of this invention, as well as pharmaceutical compositions containing such proliferated cells and / or the PAg prodrug compounds of this invention. [Background technology]

[0002] Background of the Invention Present from birth and the major subtype of human γδ T cells in adult peripheral blood, Vγ9 / Vδ2 T cells are now established as an important subset of γδ T cells, involved in the fight against many diseases such as tuberculosis, leprosy, typhoid fever, malaria, and toxoplasmosis (see, for example, Morita, CT; Jin, C.; Sarikonda, G.; Wang, H. Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev 2007, 215, 59-76 (hereinafter "Morita et al. 2007")).

[0003] Studies in primate models also suggest that Vγ9 / Vδ2 T cells are involved in immunity against Mycobacterium tuberculosis (see Shen, Y.; Zhou, D.; Qiu, L.; Lai, X.; Simon, M.; Shen, L.; Kou, Z.; Wang, Q.; Jiang, L.; Estep, J.; Hunt, R.; Clagett, M.; Sehgal, PK; Li, Y.; Zeng, X.; Morita, CT; Brenner, MB; Letvin, NL; Chen, ZW Adaptive immune response of Vgamma2Vdelta2+ T cells during mycobacterial infections. Science (New York, NY) 2002, 295, 2255-8).

[0004] These cells also demonstrate the ability to target and lyse a variety of cancer cells spontaneously or after activation with small molecule activators in vitro (see Morita et al, Immunol Rev 2007, 215, 59-76). These observations make the Vγ9 / Vδ2 subset a major focus in the therapeutic use of γδ T cells, particularly in autologous or allogeneic immunotherapy (see Fisher, JP; Heuijerjans, J.; Yan, M.; Gustafsson, K.; Anderson, J. gammadelta T cells for cancer immunotherapy: A systematic review of clinical trials. Oncoimmunology 2014, 3, e27572 (hereinafter "Fisher et al. 2014")). Activation of γδ T cells with small molecule activators can be performed in vivo or ex vivo.

[0005] The treatment of patients with γδ T cells generally involves collecting T cells from donor blood. For example, T cells can be collected from peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood. T cells can also be collected from umbilical cord blood mononuclear cells (CBMCs) isolated from umbilical cord blood. Another suitable source of T cells is non-hematopoietic tissue. After isolation, T cells are cultured to increase their number; this is called expansion. It is known that T cells are cultured in the presence of small molecule activators to enhance cell growth and / or function of the T cells being expanded. For example, ex vivo expansion of PBMCs in the presence of small molecule activators such as phosphoantigens or aminobisphosphonates yields γδ T cells of the Vγ9 / Vδ2 phenotype; see, e.g., WO 2016 / 166544 or WO 2018 / 055191.

[0006] Numerous small molecule activators for Vγ9 / Vδ2 T cells have been reported to date. These include the naturally occurring PAg molecules (E)-4-hydroxy-3-methylbuta-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP), as well as two synthetic molecules, risedronate and zoledronate (Figure 1), which are currently used clinically for the treatment of osteoporosis and several types of cancer (Maraka, S.; Kennel, KA Bisphosphonates for the prevention and treatment of osteoporosis. BMJ (Clinical research ed.) 2015, 351, h3783; and Roelofs, AJ; Jauhiainen, M.; Monkkonen, H.; Rogers, MJ; Monkkonen, J.; Thompson, K. Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP / DMAPP. British journal of haematology). 2009, 144, 245-50; and Davey, MS; Malde, R.; Mykura, RC; Baker, AT; Taher, TE; Le Duff, CS; Willcox, BE; Mehellou, Y. Synthesis and Biological Evaluation of (E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate Prodrugs as Activators of Vgamma9 / Vdelta2 T-Cell Immune Responses. J Med Chem 2018, 61, 2111-2117 (see “Mehello et al. 2018”).

[0007] The most potent Vγ9 / Vδ2 T cell activator reported to date is HMBPP (EC 50 HMBPP (=0.00051 μM) activates T cells by binding to the type 1 transmembrane protein butyrophyllin 3A1. The binding site of these PAg was unclear, with conflicting reports regarding whether they bind to the extracellular or intracellular domain of this transmembrane protein, but there is now strong evidence supporting the idea that HMBPP binds to the intracellular B30.2 domain of butyrophyllin 3A1.

[0008] Driven by HMBPP's ability to activate Vγ9 / Vδ2 T cells, aryloxytriester phosphoramidate prodrug technology has recently been applied to monophosphate derivatives of HMBPP, i.e., HMBP, as a means of improving their drug-like properties (see Mehellou et al. 2018). In this prodrug approach, the monophosphate group is masked by an aryl group and / or an amino acid ester (Figure 2), both of which are enzymatically cleaved in the cell to release a monophosphate or monophosphonate species. These compounds PAg of professional Because it is a drug, nucleo Chido of professional It is called ProPAgen to distinguish it from the drug ProTide.

[0009] These HMBP ProPAgens show potent activation of Vγ9 / Vδ2 T cells (EC 50 (=0.45~11nM), and it has the disadvantage of extremely low serum stability (t 1 / 2 <30 minutes), this is mainly because the -PO- bonds of these active compounds are cleaved (from the same book (ibid.)).

[0010] Against this backdrop, aryloxydiester phosphonamidate ProPAgen was designed (Figure 3A), which has serum stability (in human serum). 1 / 2 (>12 hours) and potent activation of Vγ9 / Vδ2 T cells (EC in the range of 5-73 nM) 50 (See Kadri, H.; Taher, TE; Xu, Q.; Sharif, M.; Ashby, E.; Bryan, RT; Willcox, BE; Mehellou, Y. Aryloxy Diester Phosphonamidate Prodrugs of Phosphoantigens (ProPAgens) as Potent Activators of Vγ9 / Vδ2 T-Cell Immune Responses. J. Med. Chem. 2020, 63, 11258-11270 (hereinafter referred to as "Mehellou et al. 2020")). These aryloxy diester phosphoamidates (ProPAgens) are also disclosed in WO 2020 / 008189.

[0011] However, these aryloxydiester phosphonamidates ProPAgen (Mehellou et al. 2020) may potentially be associated with in vivo toxicity due to the aryl ester group at the phosphorus atom. Furthermore, the phosphorus atom in these aryloxydiester phosphonamidates ProPAgen is chiral, resulting in diastereomer mixtures. A drawback is that different diastereomers may be associated with different biological profiles from a pharmacological, toxicological, pharmacokinetic, and metabolic standpoint, in which case separation of the diastereomer mixture is necessary. Separation of stereoisomers is often cumbersome, generally inefficient, costly, time-consuming, and difficult to scale up.

[0012] Therefore, the object of the present invention is to provide a ProPAgen that maintains potent activation of Vγ9 / Vδ2 T cells while overcoming the stereochemistry and in vivo toxicity issues associated with conventionally reported HMBP ProPAgen active compounds. [Overview of the project]

[0013] Summary of the Invention The present invention relates to novel phosphonodiamidates ProPAgen derived from symmetric amino acid esters as described in the following claims, and to pharmaceutical compositions comprising ProPAgen. The present invention also relates to the synthesis of methyl and difluoromethyl phosphonodiamidates ProPAgen derived from symmetric amino acid esters.

[0014] The present invention involves applying symmetrical amino acid ester groups to a phosphoantigen. The resulting product exhibits advantageous properties in terms of stability, metabolism, and pharmacological activity.

[0015] The ProPAgen of this invention represents a novel class of small molecule activators for Vγ9 / Vδ2 T cells, which have the potential to improve in vivo safety and efficacy. The phosphonodiamidate ProPAgen of this invention releases alcohol and native amino acids upon metabolism, which are relatively less toxic compared to the phenols released from aryloxy ProPAgen. Furthermore, the ProPAgen of this invention achieves improved water solubility by using a relatively hydrophilic amino acid ester group as a phosphate masking group instead of a lipophilic aromatic ring (aryloxy group). In addition, their symmetrical molecular structures reduce the number of stereocenters, thereby advantageously simplifying the synthesis and separation of the target compound. This has the beneficial effect of avoiding undesirable mixed products of stereoisomers that may have different biological profiles, thereby improving the reliability of the target compound when used for the treatment of targets that require it.

[0016] The ProPAgen of the present invention exhibits excellent stability in human serum and induces potent activation of Vγ9 / Vδ2 T cells, which translates to potent in vitro killing ability of the bladder cancer cell line T24. Remarkably, the ProPAgen of the present invention provides superior activation of Vγ9 / Vδ2 T cells compared to prior art compounds such as zoledronate and HMBPP.

[0017] The combination of high specificity, serum stability, and capacity profiles of these novel phosphonate-based ProPAgens makes them suitable for development as novel immunotherapies for treating a variety of conditions, including proliferative disorders (such as cancer), osteoporosis, various infectious diseases (such as tuberculosis, leprosy, typhoid fever, malaria, and toxoplasmosis), and / or inflammatory diseases. The phosphonodiamidate ProPAgens of the present invention can be used in vivo for γδ T cell proliferation in monotherapy regimens, as part of clinical regimens, or after clinical regimens, or they can be administered to patients receiving adoptive cell therapy with γδ T cells proliferated ex vivo to directly enhance Vγ9 / Vδ2 T cell-mediated activity, particularly antitumor activity.

[0018] The present invention further relates to a method for ex vivo proliferation of γδ T cells, wherein the γδ T cells are cultured in the presence of ProPAgen according to the present invention or a pharmaceutical composition containing such ProPAgen. A population of proliferationd γδ T cells or a pharmaceutical composition containing such proliferationd γδ T cells is also provided. [Brief explanation of the drawing]

[0019] [Modes for carrying out the invention]

[0020] Detailed description of the invention According to a first aspect of the present invention, general formula (I):

[0021] [ka]

[0022] (In the formula, R1 is a general formula (II):

[0023] [Chemical formula]

[0024] (In the formula, R3 represents H, or a saturated or unsaturated hydrocarbon chain which may be substituted; R4 represents a saturated or unsaturated hydrocarbon chain which may be substituted.) represents an amino acid ester group of, and both R1s are the same; and R2 is optionally substituted C 2-20 alkyl, C 4-20 alkenyl, or C 2-20 alcohol group; and X and Y each independently represent H or halo.) A compound of or a salt thereof is provided, the compound including all of its tautomers, and R1 is an amino acid ester group of general formula (II) derived from L-alanine, L-leucine, L-isoleucine or L-methionine, most preferably L-alanine, and R2 is of formula (III) or formula (IV):

[0025] [Chemical formula] (In the formula, R5 is selected from OH, OR6, SH, SR6, NH2 or NHR6, preferably OH, and R6 represents C 1-4 alkyl.) is a group of.

[0026] These R2 substituents are similar to those found in naturally-occurring PAg IPP (formula (III)) and HMBPP (formula (IV)) respectively.

[0027] In contrast to the conventional phosphate group-containing ProPAgens mentioned above, the compound of general formula (I) contains a phosphonate group masked by two identical amino acid ester groups, which are enzymatically cleaved in cells to release an active phosphonate compound. Substitution of the unstable -OP- bond with a -CC- bond significantly improved the stability of the active phosphonate compound, demonstrating its potent activating effect on Vγ9 / Vδ2 T cells. Furthermore, this activity translated into potent lysis of bladder cancer cells in vitro.

[0028] Compounds of general formula (I) have been shown to activate Vγ9 / Vδ2 T cells with high specificity, making them ideal candidates for development as immunotherapies. In particular, compounds of general formula (I) are useful in the treatment of proliferative disorders (such as different types of cancer), osteoporosis, infections, and / or inflammatory diseases, as will be further outlined below.

[0029] Compounds of general formula (I) represent a novel class of small molecule activators for Vγ9 / Vδ2 T cells, which have the potential to improve in vivo safety and efficacy. Their symmetrical molecular structure reduces the number of stereocenters, thus favorably simplifying the synthesis and separation of the target compounds. This has the beneficial effect of avoiding undesirable mixed products of stereoisomers with potentially different biological profiles, thereby improving the reliability of the target compounds when used for the treatment of targets requiring them.

[0030] General and preferred aspects of the compounds of the present invention are disclosed below and in the appended claims.

[0031] As used herein, the term "halo" refers to substituents of a halogen group, particularly fluoro, chloro, bromo, or iod, more appropriately fluoro or chloro, and most appropriately fluoro.

[0032] Terms used in this specification include "C 2-20"Alkyl" refers to a linear or branched saturated hydrocarbon chain containing 2 to 20 carbon atoms. Examples include ethyl, n-propyl, isopropyl (iPr), n-butyl (nBu), s-butyl, t-butyl (tBu), n-hexyl, n-octyl, and n-decyl.

[0033] Terms used in this specification include "C 4-20 An "alkenyl" refers to a straight or branched unsaturated hydrocarbon chain containing 4 to 20 carbon atoms. Examples include butenyl and pentenyl.

[0034] Terms used in this specification include "C 2-20 "Alcohol" refers to a straight-chain or branched saturated or unsaturated hydrocarbon chain containing 2 to 20 carbon atoms and containing one or more hydroxyl (OH) functional groups.

[0035] As used herein, the term “saturated or unsaturated hydrocarbon chain” refers to a straight or branched aliphatic or aromatic hydrocarbon group, which may or may not contain one or more double or triple bonds. Thus, the term encompasses alkyl, alkenyl, alkynyl, or aryl groups. Aliphatic or aromatic hydrocarbon chains may contain heteroatoms within the chain or as substituents.

[0036] Terms used in this specification include "C 5-25 The term "aryl" refers to any hydrocarbon group containing 5 to 25 carbon atoms, including one or more carbocyclic aromatic rings. More appropriately, an aryl group is C 6-14 And preferably C 6-10 It is an aryl group.

[0037] As used herein, the term “heteroaryl” refers to any hydrocarbon group that comprises one or more aromatic rings and contains one or more heteroatoms (e.g., N, O, or S) as part of the rings. Particularly suitable examples of heteroaryl groups are pyridine, furan, thiophene, and indole groups. A 5- to 25-membered heteroaryl group refers to a group in which the total number of ring-forming atoms (carbon and heteroatoms) is between 5 and 25.

[0038] Other alkyl, alkenyl, aryl, and / or alcohol groups are as defined, but have different numbers of carbon atoms. For example, C 1-4 Alkyls have 1 to 4 carbon atoms, for example, 1, 2, 3, or 4 carbon atoms.

[0039] Hydrocarbon chains and / or aryl, heteroaryl, alkyl, alkenyl, or alcohol groups may be substituted with functional groups containing one or more heteroatoms (e.g., O, S, or N). Suitable examples of heteroatom-containing groups include, but are not limited to, nitro, nitrone, halo, amino, amide, cyano, carboxyl, sulfonyl, hydroxyl, alkoxy, ketone, aldehyde, thiol, thioether, and non-aromatic heterocyclic groups. Hydrocarbon chains may or may not contain saturated or unsaturated cyclic structures. As used herein, any number of carbon atoms in a hydrocarbon chain, alkyl, alkenyl, aryl, or alcohol group includes any carbon atoms present in the substituent. In preferred embodiments, hydrocarbon chains and / or aryl, heteroaryl, alkyl, alkenyl, or alcohol groups may be substituted with one or more heteroatom-containing functional groups selected from thiol, thioether, alkoxy, and amino (which may be primary or secondary amino groups).

[0040] Salts of compounds of general formula (I) are, appropriately, pharmaceutically or veterinarily acceptable salts. Depending on the properties of R1-R6, these may be sodium, potassium, calcium, aluminum, zinc, magnesium and other metal salts, as well as basic addition salts such as choline, diethanolamine, ethanolamine, ethyl diamine, meglumine, and well-known basic addition salts, and / or those known to those skilled in the art, as summarized in Paulekuhn et al., (2007) J. Med. Chem. 50: 6665-6672. Alternatively, if the compound of general formula (I) contains an amino group, it may be quaternized to form salts with counterions such as halides, hydroxides, sulfates, nitrates, phosphates, formates, acetates, trifluoroacetates, fumarates, citrates, tartrates, oxalates, succinates, mandelates, methanesulfons, and p-toluenesulfons.

[0041] In compounds of general formula (I), R2 is C 4-16 And preferably C 4-8 For example, C 5-7 , or a C4, C5, C6, C7, C8 alkyl, alkenyl, or alcohol group. More preferably, R2 is an alcohol group as defined above.

[0042] In certain embodiments, R2 is a group of formula (IV), and more preferably a group of formula (IV) in which R5 is OH.

[0043] As described above, the compounds of general formula (I) contain a masked phosphonate group including a -C(X)(Y)-P- bond, which has been shown to be more stable than the -OP- bond of conventional ProPAgen. In preferred embodiments, at least one, more preferably both, of X and Y represent a halo, preferably a fluoro.

[0044] The change from an -OP- bond to a -CH2-P- bond has been shown to achieve improved serum stability of the active compound, but the pK of the phosphonate group's second deprotonation a Value (pK a =7.49) is the one with a phosphate group (pK a This is significantly different from =6.31) (Figure 4). As a result, it affects the complete ionization of the active compound and its binding affinity to the target protein under physiological pH (<7.4), which requires the complete ionization of the phosphate group to bind to the positively charged pocket (arginine-rich) on the intracellular domain of butyrophyllin 3A1.

[0045] However, by providing a difluoromethylphosphonate (-CF2-P-) bond, the active compound exhibits excellent stability in physiological environments and a pK value for second deprotonation that is very close to that of the original (native) phosphate compound itself. a It has been found to possess both a value of (6.7) and (Figure 4). Therefore, mono and / or dihalomethyl, and especially mono and / or difluoromethylphosphonate derivatives of compounds of general formula (I), possess both excellent stability and potent activation of Vγ9 / Vδ2 T cells.

[0046] Compounds of general formula (I) are prodrugs in which a monophosphonate group is masked by two identical amino acid ester moieties R1, both of which are enzymatically cleaved in cells to release active monophosphonate species.

[0047] Suitable amino acid ester masking groups include a variety of amino acid side chain (R3) groups. Typically, R3 represents the side chain of an amino acid, preferably a nonpolar side chain. Particularly preferred R3 groups are C 1-4 This includes alkyl chains. Examples of such R3 groups include -CH3 (alanine), -CH2CH(CH3)2 (leucine), -CH(CH3)CH2CH3 (isoleucine), and -CH2CH2SCH3 (methionine). Compounds in which R3 represents a methyl group (CH3) are particularly suitable.

[0048] Therefore, R1 is an amino acid ester group of general formula (II) derived from an amino acid selected from L-amino acids and D-amino acids, preferably alanine, leucine, isoleucine, and methionine. For example, the amino acid can be selected from L-alanine, L-leucine, L-isoleucine, and L-methionine. In other cases, the amino acid can be selected from D-alanine, D-leucine, D-isoleucine, and D-methionine. In a preferred embodiment, the amino acid ester group of general formula (II) is derived from an L-amino acid selected from the group of L-alanine, L-leucine, L-isoleucine, or L-methionine, and most preferably the amino acid ester group of general formula (II) is derived from L-alanine.

[0049] R4 is a saturated or unsaturated linear or branched hydrocarbon chain that may be substituted, and may be aliphatic or aromatic. Preferably R4 is C 1-6 Alkyl or C 6-14 It is an aryl group. In a particularly preferred embodiment, R4 is C 6-10 The group is an aryl group, more preferably selected from methyl (Me), isopropyl (iPr), tert-butyl (tBu), and benzyl (Bn), and most preferably a benzyl (Bn) group. Compounds of general formula (I) containing benzyl esters have a higher degradation rate and improved lipophilicity (and therefore improved cellular uptake) compared to aliphatic ester compounds.

[0050] In a preferred embodiment, R2 is a group of formula (IV), preferably R5 is an OH group, R1 is an amino acid ester group of general formula (II) derived from L-alanine, L-leucine, L-isoleucine, or L-methionine, most preferably L-alanine, and R4 is selected from methyl, isopropyl, tert-butyl, and benzyl, more preferably R4 is isopropyl or benzyl, most preferably R4 is benzyl.

[0051] In other preferred embodiments, R2 is a group of formula (IV), preferably R5 is OH, R1 is an amino acid ester group of general formula (II) derived from L-alanine, L-leucine, L-isoleucine, or L-methionine, most preferably L-alanine, R4 is selected from methyl, isopropyl, tert-butyl, and benzyl, more preferably R4 is isopropyl or benzyl, most preferably R4 is benzyl, and both X and Y represent halo, preferably fluoro.

[0052] In a more preferred embodiment, in the compound according to either of the two embodiments, R1 is an amino acid ester group of general formula (II) derived from L-alanine.

[0053] In a more preferred embodiment, in the compound according to any of the three embodiments, R2 is a group of formula (IV) in which R5 is OH.

[0054] In a more preferred embodiment, R4 is benzyl (Bn) in the compound according to any of the four embodiments described above.

[0055] Some particularly suitable compounds of general formula (I) are the following:

[0056] [ka]

[0057] [ka]

[0058] [ka]

[0059] [ka]

[0060] [ka]

[0061] [ka]

[0062] [ka]

[0063] [ka]

[0064] [ka]

[0065] [ka]

[0066] [ka]

[0067] and

[0068] [ka]

[0069] Includes. In a preferred embodiment, the compound is

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] and

[0074] [ka]

[0075] Selected from, and more preferably, the compound is

[0076] [ka]

[0077] and

[0078] [ka]

[0079] Selected from, most preferably the compound is

[0080] JPEG2026516043000023.jpg4160

[0081] That is the case. According to the second aspect, general formula (I):

[0082] [ka]

[0083] (In the formula, R1 is the general formula (II):

[0084] [ka]

[0085] (In the formula, R3 represents H, or a saturated or unsaturated hydrocarbon chain; R4 represents a saturated or unsaturated hydrocarbon chain.) It represents the amino acid ester group, and both R1s are identical; and R2 may be replaced by C 2-20 Alkyl, C 4-20 Alkenyl, or C 2-20 Represents an alcohol group; and X and Y each independently represent H or halo, where at least one, preferably both, of X and Y represents a halo, preferably a fluoro. Compounds or salts thereof are disclosed, encompassing all of its tautomers.

[0086] At least one, preferably both, of X and Y, and further preferred aspects of the compound representing a halo, preferably a fluoro, are the same as those described for the previously disclosed embodiments, which are incorporated herein by reference to the embodiments disclosed herein.

[0087] The compounds of the present invention can be obtained by a method comprising: (i) supplying an alkyl phosphonate; (ii) removing the ester group and then subjecting it to a halogenation reaction to convert the alkyl phosphonate to a phosphonate halide; (iii) subjecting the phosphonate halide to an esterification reaction with an amino acid ester hydrohalogenide to obtain an amino acid ester derivative; and (iv) subjecting the amino acid ester derivative from step (iii) to olefin metathesis while preventing alkene isomerization.

[0088] In particular, compounds of general formula (I) where both X and Y represent F (referred to herein as compounds 9a-d) are prepared by the synthetic route summarized in Figure 5(A), which begins with the reaction of commercially available α,α-difluorophosphonate 5 with allyl bromide in THF and in the presence of lithium diisopropylamine (LDA) and hexamethylphosphoramide (HMPA) to obtain compound 6 in 44% yield. Compound 6 is then treated with trimethylsilyl bromide (TMSBr) at room temperature to remove the ethoxy group and produce a phosphonic acid derivative. Subsequently, a chlorination reaction with oxalyl chloride in the presence of a catalytic amount of DMF is carried out to produce compound 7, which is used in the next reaction without purification. Compound 7 is then treated with 2.5 equivalents of a suitable amino acid ester in the presence of triethylamine to produce the desired phosphonodiamidates 8a-d in good yield (23-49%). Finally, these compounds are subjected to Grubbs olefin metathesis with 2-methyl-2-propenol using a Hoveyda-Grubbs second-generation catalyst in the presence of 1,4-benzoquinone to prevent alkene isomerization. This yields the desired phosphonodiamidates ProPAgen 9a-d in good yield (38-67%) with a purity of ≥95%.

[0089] The synthesis of compounds of general formula (I) where both X and Y are H (referred to herein as compounds 14a-d) is summarized in Figure 5(B) and is achieved in the same manner as used for the preparation of compounds 8a-d, except for the preparation of compound 11. This is achieved by first reacting 3-butenoic acid (10) with oxalyl chloride in the presence of DMF to produce 3-butenoyl chloride, which is then reacted with triethyl phosphite to obtain compound 11 in good yield (51%). Phosphonodiamidates ProPAgen 14a-d are obtained in good yield (33-63%) and with a purity of ≥95%.

[0090] It is understood that the compounds of the first and second aspects of the present invention may be administered as part of a pharmaceutical composition. Accordingly, according to a third aspect of the present invention, a pharmaceutical is provided comprising the compounds of the first and / or second aspects of the present invention. The pharmaceutical composition of the present invention preferably comprises a pharmaceutically acceptable excipient or carrier.

[0091] Suitable pharmaceutical excipients are well known to those skilled in the art. Pharmaceutical compositions can be formulated for administration by any suitable route, e.g., orally, rectally, nasally, bronchially (inhalation), topically (including ophthalmos, buccally, and sublingually), vaginally, or parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally), and can be prepared by any method well known in the pharmaceutical field.

[0092] The composition can be prepared by binding the compound of the first aspect of the present invention to a carrier. Generally, the formulation is prepared by uniformly and closely binding the compound to a liquid carrier or a finely divided solid carrier, or both, and then shaping the product as needed.

[0093] The oral formulations of the present invention may be provided as: individual units such as capsules, pouches, or tablets containing a predetermined amount of the compound; as powder or granules; as a solution or suspension of the compound in an aqueous or non-aqueous liquid; or as a water-in-oil or oil-in-water liquid emulsion; or as a bolus, etc.

[0094] The term "acceptable carrier" for compositions for oral administration (e.g., tablets and capsules) includes common excipients, such as binders, vehicles such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose, and starch; fillers and carriers, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, glycerol stearate, stearic acid, silicone fluids, talc wax, oils, and colloidal silica. Flavorings such as peppermint, wintergreen oil, and cherry flavoring may also be used. The addition of colorants may be desirable to make the dosage form easily identifiable. Tablets may be coated in methods well known in the art.

[0095] Tablets can be manufactured by compression or molding with any one or more adjuncts. Compressed tablets can be prepared by mixing a compound in a fluid form, such as powder or granules, with optionally a binder, lubricant, inert diluent, preservative, surfactant, or dispersant, and compressing it in a suitable machine. Molded tablets can be manufactured by molding a mixture of powder compounds moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or notched, and can be formulated to provide sustained or controlled release of the activator.

[0096] Other formulations suitable for oral administration include lozenges containing the activator in a flavored base, usually sucrose and acacia or tragacanth; pastels containing the activator in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the activator in a suitable liquid carrier. Parenteral formulations are generally sterile.

[0097] Compositions for topical application to the skin can be prepared as creams, ointments, gels, solutions, or suspensions. Cream or ointment formulations that can be used for such drugs are conventional formulations well known in the field, as described in standard pharmaceutical textbooks such as the British Pharmacopoeia. In preferred embodiments of this aspect of the present invention, the composition is formulated for oral administration.

[0098] The therapeutically effective and precise amounts of the compositions defined herein, and the optimal routes for administering such compounds, can be readily determined by those skilled in the art. Naturally, these amounts depend on the specific condition being treated, the severity of the condition, the patient's parameters (including age, physical condition, build, and weight), the duration of treatment, the nature of any concomitant therapy, the specific route of administration, and similar factors within the scope of the healthcare professional's knowledge and expertise. These factors are well known to those skilled in the art and can be addressed through simple routine experimentation. Generally, it is preferable to use the maximum dose of individual components or their combinations, i.e., the safest maximum dose based on normal medical judgment. However, those skilled in the art will know that a patient may request a lower or tolerable dose for medical, psychological, or any other factual reason.

[0099] The dose of the compound or composition of the present invention administered to the subject can be selected depending on different parameters, particularly the mode of administration used and the condition of the subject. Other factors include the desired duration of treatment. If the subject's response is insufficient with the initial dose applied, a higher dose (or a substantially higher dose via a different, more localized delivery route) may be used, to the extent that the patient's tolerance allows.

[0100] As described above, the compounds of the first and second aspects of the present invention are prodrugs of highly stable and potent activators of Vγ9 / Vδ2 T cells. Accordingly, according to the fourth aspect of the present invention, compounds of the first and / or second aspects, or pharmaceutical compositions of the third aspect, are provided for use in medicine, more preferably immunotherapy. In a preferred embodiment, the immunotherapy comprises the activation of T cells, preferably γδ T cells, more preferably Vγ9Vδ2 T cells. In another preferred embodiment, the immunotherapy is directed toward the treatment of proliferative disorders, infections, inflammatory diseases, and / or osteoporosis. The immunotherapy typically comprises administering a therapeutically effective amount of the compounds of the present invention to a subject in need.

[0101] The compounds of the present invention can be administered to subjects in need of treatment, such as those suffering from proliferative disorders such as cancer, infections, inflammatory diseases, and / or osteoporosis. Upon administration, the compounds induce the activation of T cells, preferably γδ T cells, more preferably Vγ9Vδ2 T cells, in the subject's body. The activated T cells exert potent effector responses, such as cytotoxicity, and the immune response is then directed towards their respective target cells, such as tumor cells. The subjects may be mammals, preferably humans.

[0102] The immunotherapy may further include the administration of T cells, preferably γδ T cells, preferably Vγ9Vδ2 T cells. The cells may be autologous, meaning that they originate from the patient being treated and were obtained from that patient before the start of the immunotherapy. Alternatively, the cells may be allogeneic, in which case they are obtained from a donor, for example, from a blood sample of a blood donor. The administration of autologous or allogeneic T cells may be performed before, after, or concurrently with the administration of ProPAgen of the present invention.

[0103] Immunotherapy may also include the administration of interleukins, preferably interleukin-2 (IL-2), more preferably human IL-2. Again, the administration of autologous or allogeneic T cells can be performed before, after, or concurrently with the administration of ProPAgen according to the present invention.

[0104] In one embodiment, the immunotherapy is directed towards the treatment of proliferative disorders. Preferably, the proliferative disorder is a cancerous disease. The cancerous diseases treated with the compounds or pharmaceutical compositions of the present invention are not limited to any hematological and non-hematological cancers. In a preferred embodiment, the immunotherapy is directed towards the treatment of leukemia, i.e., malignancies of hematopoietic tissues such as the bone marrow or lymphoid system. The leukemias treated with the compounds or pharmaceutical compositions of the present invention may include any subclass of leukemia, including lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). In one embodiment, the immunotherapy is directed towards the treatment of ALL, more specifically, precursor B-cell acute lymphoblastic leukemia, precursor T-cell acute lymphoblastic leukemia, Burkitt's leukemia, or acute mixed-type leukemia. In another embodiment, the immunotherapy is directed towards the treatment of CLL, more specifically, B-cell prolymphoblastic leukemia. In yet another embodiment, immunotherapy is directed towards the treatment of hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic (LGL) leukemia, clonal eosinophilia, or adult T-cell leukemia.

[0105] Instead, immunotherapy can be directed towards treating solid tumors. Tumors treated with the compounds or pharmaceutical compositions of the present invention include, but are not limited to, bladder cancer, prostate cancer, lung cancer, cervical cancer, skin cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, ovarian cancer, brain tumor, gastric cancer, kidney cancer, uterine cancer, bone cancer, esophageal cancer, Kaposi's sarcoma, oropharyngeal cancer, testicular cancer, thyroid cancer, lymphoma, adrenocortical carcinoma, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, endometrial cancer, ependymoma, cardiac tumor, cholangiocarcinoma, germ cell tumor, germ blastoma, lip cancer, oral cancer, multiple myeloma, small intestine cancer, nasal cancer, paranasal sinus cancer, anal cancer, Burkitt lymphoma, bile duct cancer, cervical cancer, laryngeal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Wilms' tumor, plasma cell myeloma, retinoblastoma, or mesothelioma.

[0106] In a particularly preferred embodiment, the cancers treated by the immunotherapy of the present invention are selected from the group consisting of blood cancers such as leukemia, bladder cancer, prostate cancer, lung cancer, cervical cancer, skin cancer, breast cancer, and mesothelioma.

[0107] In another embodiment, immunotherapy is directed towards the treatment of infectious diseases. Infectious diseases suitable for treatment with the compounds or pharmaceutical compositions of the present invention include bacterial infections, viral infections, and fungal infections. Particularly suitable infections are bacterial infections such as infections caused by pathogenic bacteria of the genus Mycobacterium, including M. africanum, M. canettii, M. caprae, M. bovis, M. microti, M. mungi, M. pinnipedii, M. tuberculosis, M. genavense; M. leprae; M. immunogenicum; M. conspicuum; M. mucogenicum; M. ulcerans; M. xenopi, M. shottsii, M. avium, and M. paratuberculosis. Accordingly, in a preferred embodiment, immunotherapy of the present invention is directed towards the treatment of tuberculosis or leprosy.

[0108] The compounds or pharmaceutical compositions of the present invention may also be used to treat bacterial infections of the Salmonella genus, which include S. enteric species, preferably S. enterica subsp. enterica, S. enterica ssp. arizonae, and one of the S. enterica ssp. diarizona subspecies. In particularly preferred embodiments, the compounds of the present invention may also be used to treat bacterial infections caused by pathogenic S. enterica subsp. enterica strains that cause typhoid fever. Therefore, compounds of general formula (I) are particularly useful for the treatment of typhoid fever.

[0109] In another embodiment, immunotherapy is directed towards the treatment of fungal infections. Fungal infections that can be treated with the compounds and pharmaceutical compositions of the present invention include infections caused by Aspergillus fungi, particularly pulmonary infections. In one embodiment, the infection treated is aspergillosis caused by species selected from A. fumigatus, A. flavus, A. terreus, A. nidulans, and A. niger. Infections caused by Blastomyces fungi can also be treated with the compounds and pharmaceutical compositions of the present invention. In one embodiment, the infection treated is blastomycosis caused by the B. dermatitidis species. Infections caused by Candida fungi, particularly invasive infections, can also be treated. In one embodiment, the infection treated is candidiasis caused by a species selected from C. albicans, C. auris, C. blankii, C. stellatoidea, C. dubliniensis, C. famata, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. Infections caused by fungi of the genus Cryptococcus can also be treated. In one embodiment, the infection treated is cryptococcosis caused by a species selected from C. neoformans and C. gattii. The compounds and pharmaceutical compositions of the present invention are also suitable for the treatment of fungal infections of the nails, skin, or eyes.

[0110] In yet another embodiment, immunotherapy is directed towards the treatment of viral infections. The types of viral infections that can be treated with the compounds and pharmaceutical compositions of the present invention include viral infections caused by a wide range of RNA and DNA viruses, including orthomyxoviruses, paramyxoviruses, flaviviruses, pestiviruses, hepaviruses, arenaviruses, herpesviruses, adenoviruses, poxviruses, and retroviruses.

[0111] In another embodiment, immunotherapy is directed towards the treatment of parasitic infections. Particularly suitable parasitic infections that can be treated with the compounds or pharmaceutical compositions of the present invention include infections caused by organisms of the genus Plasmodium, such as P. vivax, P. falciparum, P. malariae, P. ovale, and P. knowlesi. In a particularly preferred embodiment, the compounds of the present invention or pharmaceutical compositions containing the same are used to treat Plasmodium species that cause malaria. Therefore, compounds of general formula (I) or pharmaceutical compositions containing the same are particularly useful for the treatment of malaria. Other suitable parasitic infections that can be treated include infections caused by organisms of the genus Toxoplasma, such as T. gondii. In a particularly preferred embodiment, the compounds of the present invention are used to treat Toxoplasma species that cause toxoplasmosis. Therefore, compounds of general formula (I) or pharmaceutical compositions containing the same are particularly useful for the treatment of toxoplasmosis.

[0112] In one embodiment, immunotherapy is directed towards the treatment of inflammatory diseases. Accordingly, the compounds of the present invention or pharmaceutical compositions comprising them are used to treat diseases or disorders characterized by dysregulation of the immune state or an excessive inflammatory response, particularly chronic inflammatory diseases. Such diseases or disorders include, for example, autoimmune diseases and hypersensitivity reactions. The inflammatory disease to be treated may be local or systemic. The types of inflammatory diseases or conditions that may benefit from treatment are not limited to, but include, arterial occlusive diseases such as peripheral artery occlusive disease (pAOD), severe limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by occlusion or constriction of arteries; systemic inflammation associated with metabolic disorders, including type II diabetes and obesity-related metabolic syndrome; and skin diseases, including eczema. In a preferred embodiment, the inflammatory disease is an autoimmune disease. The types of autoimmune diseases treated are not limited to ulcerative colitis, Crohn's disease, rheumatoid arthritis, autoimmune cardiomyopathy, autoimmune hepatitis, lupus erythematosus, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, juvenile idiopathic arthritis, myasthenia gravis, pemphigus vulgaris, psoriasis, Reiter's syndrome, scleroderma, Sjögren's syndrome, vasculitis, vitiligo, and Wegener's granulomatosis.

[0113] In another embodiment, immunotherapy is directed towards the treatment of hypersensitivity reactions. For example, hypersensitivity reactions may be selected from asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity, and mastocytosis.

[0114] In another embodiment, immunotherapy is directed towards the treatment of osteoporosis. The osteoporosis being treated may be primary or secondary osteoporosis.

[0115] A fourth aspect of the present invention also provides a method of immunotherapy comprising administering a therapeutically effective amount of a compound of the first or second aspect of the present invention, or a pharmaceutical composition of the third aspect of the present invention, to a subject in need (such as a human subject). By administering a compound of the first or second aspect of the present invention, or a pharmaceutical composition of the third aspect of the present invention, to the subject to be treated, the subject's γδ T cells are activated in vivo, and an immune response against the respective target cells, e.g., cancer cells, is formed. This therapy can be directed to the treatment of any of the above conditions or diseases, namely proliferative disorders, infections, inflammatory diseases, and / or osteoporosis. The therapy may comprise the administration of autologous or allogeneic T cells, preferably γδ T cells, more preferably Vγ9Vδ2 T cells, as otherwise referred to herein. The immunotherapy may further comprise the administration of interleukins, preferably IL-2, more preferably human IL-2.

[0116] In a fifth aspect, the present invention relates to an ex vivo method for preparing a population of therapeutic γδ T cells, wherein the method is: (i) supplying a population of γδ T cells; (ii) Culturing a population of γδ T cells in the presence of a compound according to the first or fifth aspect of the present invention as described above. Includes.

[0117] The method comprises contacting γδ T cells with the compound of the present invention to induce activation and / or proliferation. In the first step of the method, a population of γδ T cells is supplied. The population of γδ T cells can be obtained from various sources, such as blood samples from a donor or a patient being treated. γδ T cells can be obtained from peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood or umbilical cord blood mononuclear cells (CBMCs) isolated from umbilical cord blood. In the second step of the method, the population of γδ T cells is cultured in the presence of the compound according to the first or second aspect of the present invention for a period and under conditions that result in their activation and / or proliferation.

[0118] The sixth aspect of the present invention relates to a population of γδ T cells obtained by the method according to the fifth aspect. The cells treated by the method according to the fifth aspect of the present invention are preferably Vγ9 / Vδ2 T cells and can be advantageously used in medicine, particularly immunotherapy.

[0119] The seventh aspect of the present invention also provides pharmaceutical compositions comprising a population of γδ T cells according to the sixth aspect of the present invention. The above-described explanation relating to the pharmaceutical compositions of the third aspect of the present invention applies accordingly to these pharmaceutical compositions.

[0120] The eighth aspect of the present invention relates to a population of γδ T cells according to the sixth aspect of the present invention for medical use, particularly in immunotherapy. Preferably, the immunotherapy is directed toward the treatment of proliferative disorders, infections, inflammatory diseases and / or osteoporosis. For the medical use of cells according to the eighth aspect of the present invention, the above-described explanation with respect to the fourth aspect of the present invention applies accordingly. Thus, γδ T cells according to the sixth aspect of the present invention may be used to treat the same conditions and diseases discussed herein in relation to the fourth aspect of the present invention. In other words, γδ T cells according to the sixth aspect of the present invention obtained by the method of the fifth aspect of the present invention may be used to treat proliferative disorders, infections, inflammatory diseases and / or osteoporosis.

[0121] When using cells according to the sixth aspect of the present invention according to the eighth aspect of the present invention, the treatment may be autologous. This means that the γδ T cells subjected to the method of the fifth aspect of the present invention are obtained from the patient who will ultimately be treated with γδ T. In this case, the γδ T cells are obtained from the patient, then processed according to the method of the fifth aspect of the present invention, and then reintroduced into the patient. Alternatively, the treatment may be allogeneic therapy, which means that the γδ T cells subjected to the method of the fifth aspect of the present invention are obtained from a donor who is not the patient who will ultimately be treated.

[0122] The ninth aspect of the present invention relates to compounds of the first or second aspect of the present invention in the preparation of pharmaceutical compositions for immunotherapy. As otherwise outlined herein, the immunotherapy is preferably directed toward the treatment of proliferative disorders, infections, inflammatory diseases and / or osteoporosis.

[0123] Throughout this description and claims, the words “comprise” and “contain,” and their variations, e.g., “comprising” and “comprises,” mean “including but not limited to,” and do not exclude other parts, additives, components, integrals, or processes. Throughout this description and claims, unless the context requires otherwise, the singular form includes the plural form. In particular, where the indefinite article is used, unless the context requires otherwise, the specification should be understood to assume both the singular and plural forms.

[0124] All documents cited herein, including all patents and patent applications, are incorporated herein by reference. No document is acknowledged to constitute prior art. Furthermore, no document is acknowledged to constitute part of the common general knowledge in the art.

[0125] Preferred features of each aspect of the present invention may be as described in relation to any of the other aspects.

[0126] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention extends to any novel one or any novel combination of the features disclosed herein (including the appended claims and drawings). Accordingly, any features, wholes, properties, compounds, or chemical parts described in relation to a particular aspect, embodiment, or example of the present invention should be understood to be applicable to any other aspect, embodiment, or example described herein, insofar as they do not conflict.

[0127] Furthermore, unless otherwise specified, any feature disclosed herein may be replaced by an alternative feature that serves the same or similar purpose.

[0128] The present invention will be described only by reference to the following embodiments and drawings. Figure 1 shows the chemical structures of reported small molecule Vγ9 / Vδ2 T cell activators: naturally occurring PAg (E)-4-hydroxy-3-methylbuta-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP); synthetic molecules risedronate and zoledroneate.

[0129] Figure 2 shows the application of aryloxytriester phosphoramidate prodrug technology to the monophosphate derivative (HMBP) of HMBPP. The monophosphate group is masked by an aryl group and an amino acid ester, both of which are enzymatically cleaved in cells, releasing an active monophosphate species. Instability due to cleavage of the -PO- bond (shaded) of these compounds was observed.

[0130] Figure 3A shows the general chemical structure of the aryloxydiester phosphoneamide ProPAgen according to Mehellou et al. 2022. Figure 3B shows the general chemical structure of ProPAgen, a phosphonodiamidate derived from a symmetrical amino acid ester according to the present invention.

[0131] Figure 4A shows the pKa values ​​of the phosphate group and different phosphonate groups. Figure 4B shows the chemical structure of metabolite 15, which exhibits the electron-withdrawing effect of the fluorine atom in difluoromethylene phosphonate, resulting in a stronger electrophilicity (15a) of the phosphorus center compared to methylene phosphonate 15b. This also affects the strength of possible H-intramolecular bonds, which are stronger in 15b than in 15a due to the fluorine-attracting effect. Therefore, cyclization of methylene phosphonate may be slower than that of the unfluorinated form.

[0132] Figure 5 shows the synthesis of phosphonodiamidate prodrugs derived from symmetric amino acid esters of (A) HMBP methylphosphonate (14a-d) and (B) HMBP difluoromethylphosphonate (9a-d). Reagents and conditions: A. (i) LDA, HMPA, THF, allyl bromide, -78°C, yield 44%; (ii) TMSBr, DCM, 50°C, N2, 3 hours, then (COCl)2, DMF catalyst, DCM, room temperature, 2 hours; (iii) L-alanine hydrochloride, DCM, N2, -78°C, TEA, then room temperature, overnight, yield: 23-49%; (iv) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda-Grubbs catalyst (second generation). nd (second generation), DCM, 78°C, yield: 38-67%. B. (v) Oxalyl chloride (solvent and reagent), N2, 0°C-room temperature, DMF, then (EtO)3P, 0°C-room temperature, overnight, yield 51%; (vi) TMSBr, DCM, 50°C, N2, 3 hours, then (COCl)2, DMF catalyst, DCM, room temperature, 2 hours; (vii) L-alanine ester hydrochloride, DCM, N2, -78°C, TEA, then room temperature, overnight, yield: 23-44%; (vii) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda-Grubbs catalyst second generation, DCM, 78°C, yield: 33-63%.

[0133] Figure 6 shows the results at 37°C for 7.5 hours. 31 This shows the stability of HMBP phosphonodiamidate ProPAgen 9b in human serum, as monitored by 1P NMR.

[0134] Figure 7A shows the human Vγ9 / Vδ2 iontophoresis by ProPAgen 9a-d. + This demonstrates in vitro activation of T cells. The HMBP ProPAgen of the present invention was used at concentrations of 10 pM to 100 μM, and compound 9d was then used at 1 aM to 100 μM in a separate experiment (see Figure 7E). Figure 7B shows the human Vγ9 / Vδ2 iontophoresis by ProPAgen 14a-d. + This demonstrates in vitro activation of T cells. The HMBP ProPAgen of the present invention was used at concentrations of 10 pM to 100 μM, and compound 14d was subsequently used at concentrations of 1 aM to 100 μM in a separate experiment (see Figure 7E).

[0135] Figure 7C shows the human Vγ9 / Vδ2 levels induced by Zoledronate and HMBPP at concentrations of 10 pM to 100 μM. + This demonstrates in vitro activation of T cells. Zoledronate and HMBPP were used as prior art compounds for comparison with the HMBP ProPAgen of the present invention. Figure 7D shows EC 50 The values ​​were calculated using GraphPad Prism v9 based on the results of the activation assay. CLogP values ​​were calculated using ChemDraw Professional 16.0. EC14a 50 The value (nM) could not be determined because the exact ability level could not be obtained.

[0136] Figure 7E shows the activation of Vγ9 / Vδ2 T cells via phosphonodiamidate ProPAgen 9d and 14d after incubating PBMCs overnight with 1 attomolar (aM) to 100 μM 9d and 1 femtomolar (fM) to 100 μM 14d, and is intended to determine the EC50 values ​​of these compounds. Activation levels are expressed as the percentage (%) of CD69+CD25+Vγ9 / Vδ2 T cells. Data were analyzed with GraphPad Prism v9 software and are presented as mean ± SE (n=4).

[0137] Figure 8 shows a cytotoxicity assay demonstrating potent lysis of bladder cancer cells after incubation with the HMBP phosphonates ProPAgen 14d and 9d. ProPAgen 14d and 9d mediate specific lysis of T24 bladder cancer cells by Vγ9 / Vδ2 T cells. The % killing of T24 cells was calculated using the formula: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] × 100. Data are shown as mean ± SE (n=7). Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison tests in GraphPad Prism v9. **p<0.0063. Positive controls for cell death (target cells cultured in 10% v / v DELFIA lysis buffer) and controls treated with culture medium only (no drug) were also included.

[0138] Figure 9 shows the activation of CD8+ T cells mediated by phosphonodiamidate ProPAgen 9d and 14d in the same overnight culture as Vγ9 / Vδ2 T cells (left) (mean ± SE; n=4). The activation levels of Vγ9 / Vδ2 T cells with DMSO diluted similarly to ProPAgen (right) (mean ± SE; n=4). Data were analyzed using GraphPad Prism v9 software.

[0139] Figure 10A shows the hypothetical mechanism of phosphonodiamidate prodrugs as suggested by McGuigan et al. 2018.

[0140] Figure 10B shows the in vitro degradation of the phosphonodiamidate prodrug ProPAgen 9b via carboxypeptidase Y. 31 ProPAgen 9b alone 31 P-NMR spectra and the results at different time points (shown) after incubation at 37°C for 6.5 hours using recombinant carboxypeptidase Y. 31 P-NMR spectrum. [Examples]

[0141] Examples The present invention is further described by the following examples, which are provided solely for illustrative purposes and should not be construed as limiting the scope of the invention. The following materials and methods were used in the examples.

[0142] Example 1 Synthesis of HMBP methylphosphonate aryloxyphosphoamide ProPAgen All reagents and solvents were of general-purpose or analytical grade and were purchased from Sigma-Aldrich Ltd., Fisher Scientific, Fluorochem, or Acros. 31 P, 1 H, 19 F, and 13¹³C NMR data were recorded using a Bruker Avance DPX500 spectrometer operating at 202, 500, and 125 MHz. Chemical shifts (δ) are expressed in ppm, and J values ​​are expressed in Hz. The following abbreviations were used in reporting the spectral data: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), and m (multiplet). All reactions were carried out under a nitrogen atmosphere and monitored by analytical thin-layer chromatography (TLC) on pre-coated silica plates (Kiesel gel 60 F254, BDH). Compounds were visualized under UV light (254 nm) or by KMnO4 staining and subsequent heating. Flash column chromatography was performed using silica gel 60 (230-400 mesh) (Merck). HPLC was performed using a SHIMADZU Prominence-i quaternary low-pressure gradient pump with a Prominence-i UV detector (190-700 nm). All solvents used for HPLC were HPLC-grade and purchased from Fisher Scientific. HPLC data analysis was performed using the SHIMADZU Lab Solutions software package. The purity of the tested ProPAgen was measured by HPLC, and all were ≥95%.

[0143] Diethyl (1,1-difluorobuta-3-en-1-yl)phosphonate (6): To a solution of lithium diisopropylamine (LDA) (1.0 M, 7.97 mL, 1 equivalent, 7.97 mmol) and hexamethylphosphoramide (HMPA) (1.38 mL, 1 equivalent, 7.97 mmol) in 5 mL of THF at -78°C, a chilled solution of diethyl α,α-difluorophosphonate (1.7 mL, 1 equivalent, 7.97 mmol) in 3 mL of THF was added. After stirring for 2 minutes, allyl bromide (5 mL, 1.2 equivalents, 9.56 mmol) was rapidly added under vigorous stirring. After 10 minutes, the reaction was quenched with NH4Cl and extracted with diethyl ether (10 mL) and ethyl acetate (2 × 20 mL). The combined organic phase was dried over MgSO4 and concentrated under reduced pressure. The crude product was separated by flash column chromatography (siRNA:hexane = 4:6). Yield: 793 mg (44%). 1 H NMR (500 MHz, CDCl3): 5.83-5.91 (m, 1H, CH=CH2), 5.31 (s, 1H, CH=CH2, trans), 5.29 (d, J = 5.05 Hz, 1H, CH=CH2, cis), 4.26-4.32 (m, 4H, 2 × CH2CH3), 2.80-2.91 (m, 2H, CH2CH=CH2), 1.40 (t, J = 7.07 Hz, 6H, 2 × CH2CH3). 13 C NMR (125 MHz, CDCl3): 127.0, 126.9, 121.34, 64.42 (d, J = 6.87 Hz), 38.49-38.96 (m), 16.39 (d, J = 5.48 Hz). 31 P NMR (202 MHz, CDCl3): 6.94 (t, J = 107.43 Hz). 19 F NMR (470 MHz, CDCl3): -111.23 (d, J = 108.42 Hz).

[0144] Diethyl buta-3-enoylphosphonate (11): This product was synthesized in two steps. First, 3-butenoic acid (1.5 mL, 1 equivalent, 6 mmol) and oxalyl chloride (1 mL, 2 equivalents, 12 mmol) were added to a round-bottom flask under a nitrogen-inert atmosphere. The mixture was cooled to 0°C and 3 drops of DMF were added to catalyze the reaction. The mixture was warmed to room temperature and monitored by TLC. When the starting material spots disappeared, excess oxalyl chloride was removed under reduced pressure, and the crude 3-butenoyl chloride was used in the next step without purification. In the second step, the crude 3-butenoyl chloride from the previous step was packed into a dry round-bottom flask and cooled to 0°C. Triethyl phosphite was then added dropwise. The mixture was warmed to room temperature and stirred overnight. After removing the solvent under reduced pressure, the crude product was purified by column chromatography (Â:hexane 1:1). Yield: 626 mg (51%). 1 H NMR (500 MHz, CDCl3): 5.92-6.03 (m, 1H, CH=CH2), 5.26-5.31 (m, 2H, CH=CH2), 4.08-4.22 (m, 4H, 2 × CH2CH3), 3.31 (dt, J = 6.95, 1.43, 2H, COCH2), 1.35 (t, J = 7.08, 6H, 2 × CH2CH3). 13 C NMR (125 MHz, CDCl3): 173.90, 134.92, 119.53, 62.84, 38.54, 16.20. 31 P NMR (202 MHz, CDCl3): 9.07 (s).

[0145] General procedure for synthesizing compounds 7 and 12. These compounds were synthesized in two steps. First, compound 6 or 11 (1 equivalent) was dissolved in 5 mL of DCM, and TMSBr (10 equivalents) was added under a nitrogen-inert atmosphere. The reaction was refluxed at 50°C for 3 hours, after which the solvent and excess TMSBr were removed under reduced pressure. The crude product was dissolved in 10 mL of DCM and chlorinated, and 3 drops of DMF were added as a catalyst under a nitrogen atmosphere. Then, oxalyl chloride (10 equivalents) was added dropwise. The reaction was stirred at room temperature for 2 hours, after which the solvent and excess oxalyl chloride were removed under reduced pressure. Crude product 7 or 12 was used in the next step without further purification.

[0146] General procedure for synthesizing 8a-d and 13a-d: Compound 7 or 12-18 (1 equivalent) was dissolved in 10 mL of DCM under a nitrogen-inert atmosphere with a suitable L-alanine ester hydrochloride (2.5 equivalents). The mixture was then cooled to -78°C, and TEA (4 equivalents) was added dropwise. The reaction was warmed to room temperature and stirred overnight. The solvent was removed under reduced pressure, and the crude product was dissolved in ethyl acetate. After filtration, the filtrate was concentrated, and the crude product was purified by flash column chromatography (ethyl acetate:hexane 1:1) to obtain the desired product.

[0147] Dimethyl 2,2'-(((1,1-difluorobuta-3-en-1-yl)phosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (8a). Yield: 144 mg (36%). 1 H NMR (500 MHz, CDCl3): 5.81-5.89 (m, 1H, CH=CH2), 5.26-5.31 (m, 2H, CH=CH2), 4.05-4.15 (m, 2H, 2×NHCH), 3.75 (d, J = 4.15 Hz, 6H, 2×OCH3), 3.53 (t, J = 10.57 Hz, 1H, NH), 3.34 (t, J = 11.70 Hz, 1H, NH), 2.85 (tt, J = 6.40 Hz, 29.09 Hz, 2H, CH2CF2), 1.44 (t, J = 7.20 Hz, 6H, 2×NHCHCH3).13 C NMR (125 MHz, CDCl3): 176.6, 127.4, 121.5, 52.6, 48.8, 38.1, 21.6. 31 P NMR (202 MHz, CDCl3): 12.82 (t, J = 95.58 Hz). 19 F NMR (470 MHz, CDCl3): -110.08 (d, J = 96.29 Hz), -110.66 (d, J = 54.14 Hz), -110.86 (d, J = 54.18 Hz), -111.44 (d, J = 96.12 Hz).

[0148] Diisopropyl 2,2'-(((1,1-difluorobuta-3-en-1-yl)phosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (8b). Yield: 209.4 mg (49%). 1 H NMR (500 MHz, CDCl3): 5.81-5.89 (m, 1H, CH=CH2), 5.27-5.30 (m, 2H, CH=CH2), 5.01-5.06 (m, 2H, OCH(CH3)2), 3.99-4.12 (m, 2H, 2×NHCH), 3.57 (t, J = 10.57 Hz, 1H, NH), 3.34 (t, J = 11.50 Hz, 1H, NH), 2.85 (tt, J = 6.23 Hz, 28.97 Hz, 2H, CH2CF2), 1.40-1.44 (m, 6H, 2×NHCHCH3), 1.24-1.28 (m, 12H, OCH(CH3)2). 13 C NMR (125 MHz, CDCl3): 173.3, 127.5, 121.4, 69.2, 48.9, 37.9, 21.6, 21.9. 31 P NMR (202 MHz, CDCl3): 12.78 (t, J = 95.37 Hz). 19F NMR (470 MHz, CDCl3): -110.18 (d, J = -110.18 Hz), -110.74 (d, J = 31.24 Hz), -110.94 (d, J = 30.16 Hz), -111.49 (d, J = 94.82 Hz).

[0149] Di-tert-butyl 2,2'-(((1,1-difluorobuta-3-en-1-yl)phosphoryl)bis(azandiyl))(2S,2'S)-dipropionate(8c) Yield: 113 mg (25%). 1 H NMR (500 MHz, CDCl3): 5.81-5.89 (m, 1H, CH=CH2), 5.26-5.29 (m, 2H, CH=CH2), 3.92-4.01 (m, 2H, 2×NHCH), 3.54 (t, J = 10.65 Hz, 1H, NH), 3.31 (t, J = 11.52 Hz, 1H, NH), 2.84 (tt, J = 6.01 Hz, 29.49 Hz, 2H, CH2CF2), 1.46 (d, J = 4.95 Hz, 18H, 2×OC(CH3)3), 1.39 (t, J = 7.47 Hz, 6H, 2×NHCHCH3). 13 C NMR (125 MHz, CDCl3): 173.0, 127.5, 121.3, 82.0, 49.4, 37.9, 27.9, 21.7. 31 P NMR (202 MHz, CDCl3): 12.80 (t, J = 94.97 Hz). 19 F NMR (470 MHz, CDCl3): -110.35 (d, J = 94.45 Hz), -110.89 (d, J = 11.55 Hz), -111.09 (d, J = 11.95 Hz), -111.62 (d, J = 96.38 Hz).

[0150] Dibenzyl 2,2'-(((1,1-difluorobuta-3-en-1-yl)phosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (8d). Yield: 122 mg (23%). 1 H NMR (500 MHz, CDCl3): 7.30-7.37 (m, 10H, Ph), 5.77-5.82 (m, 1H, CH=CH2), 5.22-5.27 (m, 2H, CH=CH2), 4.11-4.16 (m, 2H, 2×NHCH), 3.52 (t, J = 10.60 Hz, 1H, NH), 3.33 (t, J = 11.55 Hz, 1H, NH), 2.82 (tt, J = 6.44 Hz, 29.45 Hz, 2H, CH2CF2), 1.44 (d, J = 7.10 Hz, 3H, NHCHCH3), 1.36 (d, J = 7.10 Hz, 3H, NHCHCH3). 13 C NMR (125 MHz, CDCl3): 176.6, 135.0, 134.6, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 127.4, 121.5, 67.3, 48.7, 37.9, 21.3. 31 P NMR (202 MHz, CDCl3): 12.76 (t, J = 95.62 Hz). 19 F NMR (470 MHz, CDCl3): -109.99 (d, J = 96.26 Hz), -110.56 (d, J = 39.79 Hz), -110.76 (d, J = 39.38 Hz), -111.32 (d, J = 94.75 Hz).

[0151] Dimethyl 2,2'-((buta-3-en-1-ylphosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (13a). Yield: 424 mg (44%). 1H NMR (500 MHz, CDCl3): 5.86 (m, 1H, CH2=CH-), 5.07 (m, 2H, CH2=CH-), 4.03 (m, 2H, 2×CHNH), 3.74 (d, J = 2.3 Hz, 6H, OCH3), 3.03, 2.93 (m, 2×1H, 2×NH), 2.36 (m, 2H, PCH2CH2), 1.81 (m, 2H, PCH2CH2), 1.38 (d, J = 7.2 Hz, 6H, 2×NHCHCH3). 13 C NMR (125 MHz, CDCl3): 174.5, 156.25, 137.55, 115.47, 52.36, 48.81, 48.42, 28.94, 28.04, 26.86, 21.56, 18.99. 31 P NMR (202 MHz, CDCl3): 28.78 (s).

[0152] Diisopropyl 2,2'-((buta-3-en-1-ylphosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (13b). Yield: 300 mg (26.5%). 1 H NMR (500 MHz, CDCl3): 5.86 (m, 1H, CH2=CH-), 5.03 (m, 4H, CH2=CH-, 2×CH(CH3)2), 3.96 (m, 2H, 2×CHNH), 3.07, 2.98 (m, 2×1H, 2×NH), 2.36 (m, 2H, PCH2CH2), 1.80 (m, 2H, PCH2CH2), 1.37 (m, 6H, 2×NHCHCH3), 1.25 (m, 12H, OCH(CH3)2). 13 C NMR (125 MHz, CDCl3): 174.27, 137.49, 115.36, 68.95, 49.00, 48.63, 29.05, 28.15, 26.88, 21.69, 19.16. 31 P NMR (202 MHz, CDCl3): 28.59 (s).

[0153] Di-tert-butyl 2,2'-((buta-3-en-1-ylphosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (13c). Yield: 284 mg (23%). 1 H NMR (500 MHz, CDCl3): 5.84 (m, 1H, CH2=CH-), 5.06 (m, 2H, CH2=CH-), 3.90 (m, 2H, 2×CHNH), 3.03, 2.91 (m, 2×1H, 2×NH), 2.35 (m, 2H, PCH2CH2), 1.78 (m, 2H, PCH2CH2), 1.46 (m, 18H, 2×OC(CH3)3), 1.36 (m, 6H, 2×NHCHCH3). 13 C NMR (125 MHz, CDCl3): 173.8, 137.56, 115.24, 81.63, 49.26, 29.11, 28.22, 27.97, 26.89, 21.76. 31 P NMR (202 MHz, CDCl3): 28.53 (s).

[0154] Dibenzyl 2,2'-((buta-3-en-1-ylphosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (13d). Yield: 400 mg (28%). 1 H NMR (500 MHz, CDCl3): 7.35 (m, 10H, Ph), 5.79 (m, 1H, CH2=CH-), 5.15 (m, 4H, 2×OCH2C6H5), 5.01 (m, 2H, CH2=CH-), 4.05 (m, 2H, 2×CHNH), 3.00, 2.93 (m, 2×1H, 2×NH), 2.31 (m, 2H, PCH2CH2), 1.76 (m, 2H, PCH2CH2), 1.42 (m, 6H, 2×NHCHCH3). 13 C NMR (125 MHz, CDCl3): 174.53, 137.54, 128.64, 128.45, 128.24, 115.44, 67.06, 48.76, 46.78, 28.98, 28.08, 26.84, 21.41, 8.63. 31P NMR (202 MHz, CDCl3): 28.71 (s).

[0155] General procedure for synthesizing 9a-d and 14a-d. Compounds 8a-d and 13a-d (1 equivalent, 0.335 mmol) were dissolved in 10 mL of DCM under a nitrogen-inert atmosphere. 2-Methyl-2-propen-1-ol (0.056 mL, 2 equivalents, 0.67 mmol), 1,4-benzoquinone (3.6 mg, 10% mol, 0.0335 mmol), and second-generation Hoveyda-Grubbs catalyst (5.22 mg added three times at 0, 3, and 6 hours (total 15.7 mg), 7.5% mol, 0.025 mmol) were added. The mixture was refluxed at 45°C for 18 hours and then cooled to room temperature. Activated carbon was added and stirred for 1 hour to adsorb inorganic Ru from the catalyst. The mixture was then filtered through Celite, concentrated under reduced pressure, and purified by flash column chromatography (siRNA:hexane, 20:80-100:0 gradient). The geometric configuration was confirmed by NOESY.

[0156] Dimethyl 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis(azandiyl))(2S,2'S)-dipropionate (9a). Yield: 62 mg (38%). 1 H NMR (500 MHz, CDCl3): 5.53 (m, 1H, CH2CH=CCH3CH2OH), 4.11 (m, 2H, 2×NHCH), 4.04 (s, 2H, CH2OH), 3.76 (s, 6H, 2×OCH3), 3.62(m, 1H, NHCH), 3.42 (m, 1H, NHCH), 2.91 (m, 2H, CF2CH2), 1.72 (s, 3H, CH=CCH3CH2OH), 1.44 (t, J = 6.83 Hz, 6H, 2×NHCHCH3). 13 C NMR (125 MHz, CDCl3): 176.58, 142.01, 113.34, 67.95, 52.69, 48.78, 32.56, 21.77, 21.41, 14.00. 31³¹P NMR (202 MHz, CDCl₃): 13.31 (t, J = 96.83 Hz). 19 ¹⁹F NMR (470 MHz, CDCl₃): -108.32 (d, J = 96.68 Hz), -108.91 (d, J = 66.49 Hz), -109.11 (d, J = 66.42 Hz), -109.70 (d, J = 96.49 Hz). HRMS (ES⁺, m / z): calcd for (M + Na) + C 14 H 25 F₂N₂O₆PNa, 409.1317; found, 409.1316. HPLC (reverse phase) 0.5 mL / min MeOH / H₂O 70:30 in 12 min, λ = 210 nm, tRt = 5.82 min (100%).

[0157] Diisopropyl 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis-(azanediyl))(2S,2'S)-dipropionate (9b). Yield: 174 mg (52%). 1 ¹H NMR (500 MHz, CDCl₃): 5.54 (m, 1H, CH=C), 5.04 (m, 2H, 2 × OCHCH₃), 4.05 (m, 4H, CH₂OH, 2 × CHNH), 3.57, 3.32 (m, 2 × 1H, 2 × NH), 2.89 (m, 2H, POCF₂CH₂), 2.30 (s, 1H, OH), 1.73 (s, 3H, CH₃(CH₂OH)C=CH), 1.42 (t, J = 6.7 Hz, 6H, 2 × NHCHCH₃), 1.26 (m, 12H, OCH(CH₃)₂). 13 ¹³C NMR (125 MHz, CDCl₃): 173.6, 142.2, 113.5, 69.5, 68.0, 49.0, 32.7, 22.0, 21.6, 21.4, 14.0. 31 ³¹P NMR (202 MHz, CDCl₃): 13.18 (t, J = 96.56 Hz). 1919F NMR (470 MHz, CDCl3): -108.85 (d, J = 11.95 Hz), -109.06 (d, J = 11.58 Hz). HRMS (ES+, m / z): calcd for (M + Na) + C 18 H 33 F2N2O6PNa, 465.1950; found, 465.1942. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.82 min (100%).

[0158] Di-tert-butyl 2,2'-(((E)-1,1-difluoro-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis(azanediyl))(2S,2'S)-dipropionate (9c). Yield: 81 mg (67%). 1 1H NMR (500 MHz, CDCl3): 5.54 (m, 1H, CH=C), 4.05 (s, 2H, CH2OH), 3.97 (m, 2H, 2 × CHNH), 3.53, 3.30 (m, 2 × 1H, 2 × NH), 2.89 (m, 2H, POCF2CH2), 2.30 (s, 1H, OH), 1.73 (s, 3H, CH3(CH2OH)C=CH), 1.47 (d, J = 4.0 Hz, 18H, 2 × OC(CH3)3), 1.40 (m, 6H, 2 × NHCHCH3). 13 13C NMR (125 MHz, CDCl3): 173.35, 142.15, 113.62, 82.38, 68.09, 49.47, 32.67, 27.92, 21.85, 14.04. 31 31P NMR (202 MHz, CDCl3): 13.19 (t, J = 96.31 Hz). 19 19F NMR (470 MHz, CDCl3): -108.85 (d, J = 7.72 Hz), -109.06 (d, J = 8.39 Hz). HRMS (ES+, m / z): calcd for (M + Na)+ C 20 H 37 F2N2O6PNa, 493.2260; found, 493.2255. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.81 min (100%).

[0159] Dibenzyl 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)-bis(azandiyl))(2S,2'S)-dipropionate (9d). Yield: 76 mg (57%). 1 H NMR (500 MHz, CDCl3): 7.34 (m, 10H, Ph), 5.51 (m, 1H, CH=C), 5.15 (m, 4H, 2 × OCH2C6H5), 4.13 (m, 2H, 2 × CHNH), 4.03 (s, 2H, CH2OH), 3.55, 3.35 (m, 2 × 1H, 2 × NH), 2.87 (m, 2H, POCF2CH2), 2.16 (s, 1H, OH), 1.70 (s, 3H, CH3(CH2OH)C=CH), 1.44, 1.37 (2m, 2 ×3 H, 2 × NHCHCH3). 13 C NMR (125 MHz, CDCl3): 173.76, 142.07, 135.15, 128.66, 128.54, 128.27, 113.38, 67.70, 64.38, 60.41, 48.83, 32.56, 31.34, 14.02. 31 P NMR (202 MHz, CDCl3): 13.14 (t, J = 96.70 Hz). 19 F NMR (470 MHz, CDCl3): -108.36 (d, J = 96.43 Hz), -108.94 (d, J = 59.26 Hz), -109.15 (d, J = 58.87 Hz), -109.72 (d, J = 96.68 Hz). HRMS (ES+, m / z): calcd for (M + Na) + C26 H 33 F2N2O6PNa, 561.1940; found, 561.1942. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.53 min (100%).

[0160] Dimethyl 2,2'-((((E)-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis(azandiyl))-(2S,2'S)-dipropionate (14a). Yield: 66 mg (63%). 1 H NMR (500 MHz, CDCl3): 5.45 (m, 1H, CH=C), 4.05 (m, 2H, 2 × CHNH), 4.01 (s, 2H, CH2OH), 3.74, 3.73 (2×s, 2 × 3H, 2 × OCH3), 3.02 (m, 2H, 2 × NH), 2.39 (m, 2H, PCH2CH2), 2.01 (s, 1H, OH), 1.79 (m, 2H, PCH2CH2), 1.65 (s, 3H, CH3(CH2OH)C=CH), 1.40 (m, 6H, 2 × NHCHCH3). 13 C NMR (125 MHz, CDCl3): 175.33, 137.04, 124.07, 68.41, 52.44, 48.62, 29.64, 28.75, 21.62, 21.06, 13.80. 31 P NMR (202 MHz, CDCl3): 29.05 (s). HRMS (ES+, m / z): calcd for (M + Na) + C 14 H 27 N2O6PNa, 373.1498; found, 373.1504. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.80 min (98%).

[0161] Diisopropyl 2,2'-((((E)-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis(azandiyl))-(2S,2'S)-dipropionate (14b). Yield: 40 mg (33%). 1 H NMR (500 MHz, CDCl3): 5.46 (m, 1H, CH=C), 5.01 (m, 2H, 2 × OCH(CH3)2), 4.00 (s, 2H, CH2OH), 3.97 (m, 2H, 2 × CHNH), 3.05 (m, 2H, 2 × NH), 2.38 (m, 2H, PCH2CH2), 1.80 (m, 2H, PCH2CH2), 1.70 (s, 3H, CH3(CH2OH)C=CH), 1.38 (m, 6H, 2 × NHCHCH3), 1.25 (m, 12H, 2 × OCH(CH3)2). 13 C NMR (125 MHz, CDCl3): 174.39, 137.13, 124.14, 69.09, 68.44, 48.90, 29.78, 28.88, 21.89, 21.67, 21.12, 13.81. 31 P NMR (202 MHz, CDCl3): 28.95 (s). HRMS (ES+, m / z): calcd for (M + Na) + C 18 H 35 N2O6PNa, 429.2143; found, 429.2130. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.81 min (100%).

[0162] Di-tert-butyl 2,2'-((((E)-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis(azandiyl))-(2S,2'S)-dipropionate (14c). Yield: 43 mg (33%). 1H NMR (500 MHz, CDCl3): 5.45 (m, 1H, CH=C), 4.01 (s, 2H, CH2OH), 3.92 (m, 2H, 2 × CHNH), 3.02 (m, 2H, 2 × NH), 2.38 (m, 2H, PCH2CH2), 1.77 (m, 2H, PCH2CH2), 1.71 (s, 3H, CH3(CH2OH)C=CH), 1.46 (d, J = 4.6 Hz, 18H, 2 × OC(CH3)3), 1.36 (dd, J = 13.7, 7.1 Hz, 2×NHCHCH3). 13 C NMR (125 MHz, CDCl3): 174.12, 137.18, 124.27, 81.82, 68.52, 49.23, 29.82, 28.94, 27.98, 22.08, 21.16, 13.82. 31 P NMR (202 MHz, CDCl3): 28.90 (s). HRMS (ES+, m / z): calcd for (M + Na) + C 20 H 39 N2O6PNa, 457.2453; found, 457.2443. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.54 min (98%).

[0163] Dibenzyl 2,2'-((((E)-5-hydroxy-4-methylpenta-3-en-1-yl)phosphoryl)bis(azandiyl))-(2S,2'S)-dipropionate (14d). Yield: 128 mg (59%). 1H NMR (500 MHz, CDCl3): 7.34 (m, 10H, Ph), 5.40 (m, 1H, CH=C), 5.14 (m, 4H, 2 × OCH2C6H5), 4.06 (m, 2H, 2 × CHNH), 3.99 (s, 2H, CH2OH), 3.02 (m, 2H, 2 × NH), 2.34 (m, 2H, PCH2CH2), 1.74 (m, 2H, PCH2CH2), 1.67 (s, 3H, CH3(CH2OH)C=CH), 1.41, 1.32 (2d, 2 × 3H, J = 7.0 Hz, 2 × NHCHCH3). 13 C NMR (125 MHz, CDCl3): 174.59, 136.99, 135.29, 128.64, 128.48, 128.25, 124.11, 68.41, 67.77, 48.76, 29.66, 28.75, 21.63, 21.05, 13.79. 31 P NMR (202 MHz, CDCl3): 29.10 (s). HRMS (ES+, m / z): calcd for (M + Na) + C 26 H 35 N2O6PNa, 525.2127; found, 525.2130. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30 in 12 min, λ = 210 nm, tRt = 5.82 min (100%).

[0164] To ensure that the samples used during dissolution and storage were not contaminated, the samples were re-analyzed by mass spectrometry and HPLC to confirm that they were structurally correct and pure.

[0165] Example 2 Stability test of HMBP difluoromethylphosphonate 9b, which is a ProPAgen according to the present invention. To demonstrate the stability of the phosphonodiamidate ProPAgen according to the present invention in human serum, representative serum stability studies were conducted using difluoromethylphosphonate 9b, which has an iPr ester, similar to sofosbuvir and tenofovir alafenmide, two FAD-approved phosphoramidate prodrugs.

[0166] This experiment was previously performed in Mehellou et al. 2020, and Slusarczyk, M.; Ferrari, V.; Serpi, M.; Goenczy, B.; Balzarini, J.; McGuigan, C; Symmetrical Diamidates as a Class of Phosphate Prodrugs to Deliver the 5'-Monophosphate Forms of Anticancer Nucleoside Analogues; ChemMedChem 2018, 13, 2305-2316 (“McGuigan et al. 2018”), and Slusarczyk, M.; Lopez, MH; Balzarini, J.; Mason, M.; Jiang, WG; Blagden, S.; Thompson, E.; Ghazaly, E.; McGuigan, C. Application of ProTide technology to gemcitabine: a successful approach to overcome the key cancer resistance mechanisms leads to a new agent resistance mechanism The procedure was carried out as reported in (NUC-1031) in clinical development. J. Med. Chem. 2014, 57, 1531-1542 (hereinafter referred to as "McGuigan et al. 2014").

[0167] In short, 5.0 mg of phosphonodiamidate ProPAgen 9b was dissolved in a mixture of 0.05 mL of DMSO and 0.15 mL of D2O.31 After recording the PNMR data, 0.3 mL of human serum (Merck Life Sciences) was added and monitored by NMR. The experiment was performed using NMR. 31 The experiment was conducted in P-mode, with scans performed every 30 minutes for 7.5 hours. The incubation temperature was 37°C. The recorded data was processed and analyzed using Bruker Topspin 2.1 software.

[0168] ProPAgen 9b was incubated with human serum at 37°C for 7.5 hours. 31 The results were monitored by 1P NMR spectroscopy. As shown in Figure 6, the amount of ProPAgen 9b 31 The P NMR spectrum shows three phosphorus peaks (δ) due to coupling with fluorine atoms. P =14.87, 15.37, and 15.87 ppm) (of the three prodrugs) 31 P-NMR showed typical and expected patterns. In particular, human serum also showed δ P = 1.82 31 It showed a P-NMR peak. Phosphonodiamidate ProPAgen 9b was incubated with human serum. 31 After monitoring the samples with P-NMR, these original 9b 31 The P-NMR peak remained present throughout the 7.5-hour study, and a new 31 No P-NMR peaks were observed. These data suggest excellent human serum stability (t) of 9b. 1 / 2 This indicates >7.5 hours.

[0169] This stability profile is consistent with the aryloxydiester phosphoramidate prodrugs of these monophosphonates reported by Mehellou et al. 2020.

[0170] Example 3 Human Vγ9 / Vδ2 + T cell activation General: Peripheral blood mononuclear cells (PBMCs) from healthy donors were collected as described in Morita et al. 2007. In particular, blood was obtained from informed healthy donors in the presence of a mixture of heparin and ethylenediaminetetraacetic acid (EDTA) as anticoagulants (2 U / ml heparin, 1.5 mM EDTA) (approved by the NRES Committee West Midlands-Solihull Ethical Board, REC Reference 14 / WM / 1254). The blood was then layered on density gradient medium lymphoprep (Stem Cell Technologies), and peripheral blood mononuclear cells (PBMCs) were purified by gradient centrifugation. The cells were washed twice with phosphate-buffered saline (PBS) and then resuspended in RPMI-1640 medium supplemented with 2 mM L-glutamine, 25 mM HEPES, 1% sodium pyruvate, 50 μg / ml penicillin / streptomycin (Invitrogen), and 10% fetal calf serum.

[0171] To evaluate the activation of Vγ9 / Vδ2 T cells, PBMCs were seeded at a cell density of 500,000 cells per well in U-bottomed, tissue-culture-treated 96-well plates. The cells were incubated in medium alone (control) in the presence of zoledronate and HMBPP at concentrations of 10 pM to 100 μM, as well as with the HMBP ProPAgen of the present invention, i.e., ProPAgen 9a-d and 14a-d, initially at a concentration of 10 pM to 100 μM, and then in other experiments, at a concentration of 1 μM to 100 μM for compounds 9d and 14d. Cells were incubated overnight at 37°C / 5% CO2 and stained with the following markers by flow cytometry: viability (Zombie Aqua 1:400), CD3 (BV421 1:100), CD8 (BV650 1:200), Vγ9 (PEcy5 1:400), Vδ2 (APC 1:200), CD69 (PE 1:25), and CD25 (FITC 1:100). Samples were acquired via LSRFortessa X20 (BD Biosciences), and the obtained data were analyzed using FlowJo v10 and GraphPad Prism v9 software.

[0172] Peripheral blood γδ T cells lack the ability to sense surface CD69 or CD25 under steady-state conditions, but T cell receptor (TCR) stimulation upregulates both T cell activation markers within 72 hours. PAg-responsive Vγ9 / Vδ2 T cells were then identified by TCRVγ9 and Vδ2 expression and evaluated for upregulation of CD69 and CD25.

[0173] Example 3-1: Human Vγ9 / Vδ2 by ProPAgen 9a-d and 14a-d + T cell activation To demonstrate the activation of Vγ9 / Vδ2 T cells by ProPAgen 9a-d and 14a-d, and the subsequent in vitro lysis of cancer cells, peripheral blood mononuclear cells (PBMCs) containing Vγ9 / Vδ2 T cells from a healthy donor were incubated with increasing concentrations (up to 100 μM) of phosphonodiamidate HMBP ProPAgen 9a-d and 14a-d as described above (see Figures 7A, 7B, and 7D).

[0174] Regarding the activation of Vγ9 / Vδ2 T cells by phosphonodiamidates ProPAgen (9a-d and 14a-d), these were first tested using concentrations ranging from 0.1 nM to 100 μM (Figures 7A and 7B). The results showed that these phosphonodiamidates ProPAgen exhibited varying levels of activation, ranging from extremely potent to variable (EC). 50 (=0.0000136nM~6.1μM). In both the fluorinated (9a~d) and non-fluorinated (14a~d) series, phosphonodiamidates ProPAgen (9c and 14c) containing tert-butyl esters had the lowest activation ability of Vγ9 / Vδ2 T cells (EC, respectively). 50 (=1.5 and 6.1 μM) (Figures 7A, 7B and 7D). This was followed by methyl ester phosphonodiamidate prodrugs, where 9a showed good ability (EC 50 (=191nM), we were unable to obtain an accurate capability level of 14a.

[0175] In the two series of phosphonodiamidates, ProPAgen 9a-d and 14a-d, those containing isopropyl or benzyl esters showed the most potent activation of Vγ9 / Vδ2 T cells in vitro. Phosphonodiamidates ProPAgen 9b and 14b showed good activation of Vγ9 / Vδ2 T cells (EC). 50(=167 and 87 nM). However, phosphonodiamidates ProPAgen 9d and 14d showed the most potent activation of Vγ9 / Vδ2 T cells, and ProPAgen 9d was the most potent among the eight ProPAgens investigated in this study for Vγ9 / Vδ2 T cell activation (EC 50 = 13.6 fM) (Figures 7A, 7B, 7D).

[0176] The precise capabilities of two benzylphosphonodiamidate prodrugs, 9d and 14d (EC 50 To determine this, activation assays were performed in the concentration range of 10 aM to 100 μM (Figure 7E).

[0177] In particular, the extremely high Vγ9 / Vδ2 T cell activation ability of phosphonodiamidate ProPAgen 9d is equivalent to that of the corresponding allyloxydiester phosphoramidate derivative (EC 50 (=9.15 fM). In particular, at the highest concentration studied (100 μM), activation of Vγ9 / Vδ2 T cells by phosphonodiamidate ProPAgen 9d and 14d was lower than that achieved at 10 μM (Figures 4A and 4B). This can be explained by a negative feedback mechanism induced by antigen overstimulation. This mechanism leads to downregulation of the TCR, resulting in decreased expression of the CD25 activation marker. This was also observed with allyl oxydiester phosphoramidate prodrugs of HMBP methylene and difluoromethylene monophosphonate.

[0178] Example 3-2 (Comparison): Vγ9 / Vδ2 by HMBPP and Zoledronate + T cell activation In the positive control experiment, HMBPP and zoledronate (Zol) were used in the activation assay as described above. HMBPP and zoledronate (Zol) showed significant activation of Vγ9 / Vδ2 T cells (EC, respectively). 50=181nM and 18.5μM) (Figures 7C and 7D). In particular, these capabilities by HMBPP and zoledronate are comparable to the capabilities previously reported for these compounds (EC 50 Compared to HMBPP (60-500 pM) and zoledronate (0.003-0.5 μM), the potential is lower (Hsiao, CH; Lin, X.; Barney, RJ; Shippy, RR; Li, J.; Vinogradova, O.; Wiemer, DF; Wiemer, AJ Synthesis of a phosphoantigen prodrug that potently activates Vgamma9Vdelta2 T-lymphocytes. Chem. Biol. 2014, 21 (8), 945-954, and Reichenberg, A.; Hintz, M.; Kletschek, Y.; Kuhl, T.; Haug, C.; Engel, R.; Moll, J.; Ostrovsky, DN; Jomaa, H.; Eberl, M. Replacing the pyrophosphate group of HMB-PP by a diphosphonate function abrogates Its potential to activate human gammadelta T cells). but does not lead to competitive antagonism, see Bioorg. Med. Chem. Lett. 2003, 13, 1257-1260).

[0179] Compared to those observed in this study, such differences in the efficacy of HMBPP and zoledronate reported previously are very likely due to modest inter-individual variability in Vγ9 / Vδ2 T cell activity.

[0180] As is clear from the comparison in Figures 7A-7E, the T-cell activating ability of HMBPP and zoledronate is inferior to that of ProPAgen according to the present invention, with the exception of compounds 9a, 9c, and 14c.

[0181] Example 3-3: Absence of human CD8+ T cell activation by ProPAgen 4a-d and 9a-d To demonstrate that the ProPAgen of this invention is a specific activator of Vγ9 / Vδ2 T cells, donor-derived CD8 + PBMCs containing αβ T cells were incubated with increasing concentrations of ProPAgen 9d and 14d (Figure 9).

[0182] Similar to peripheral blood γδ T cells, peripheral blood CD8 + Under steady-state conditions, T cells lack detectable levels of surface CD69 or CD25, but T cell receptor (TCR) stimulation upregulates both T cell activation markers within 72 hours. PAg-responsive CD8 T cells were then identified by TCR CD8 expression and evaluated for CD69 and CD25 upregulation.

[0183] As shown in Figure 9, HMBP phosphonates ProPAgen 9d and 14d, as representative examples of this class of prodrugs according to the present invention, activated Vγ9 / Vδ2 T cells at a concentration of 1 μM, i.e., both ProPAgen 9d and 14d. 50 Even incubation at concentrations approximately 100,000 times greater than the measured concentration did not show activation of CD8+ T cells.

[0184] Example 4 A cytotoxicity assay demonstrating the potent lytic effect of Vγ9 / Vδ2 T cells on T24 bladder cancer cells is mediated and enhanced by ProPAgen 9d and 14d. As further proof of the principle, and as shown above, the superior Vγ9 / Vδ2 of ProPAgen of the present invention + To demonstrate that the activating effect of T cells can be converted into beneficial therapeutic effects, we studied the specific lysis of cancer cells by Vγ9 / Vδ2 T cells proliferated in vitro (see Figure 8).

[0185] To evaluate the killing levels of drug-treated and untreated tumor cells, europium-based cytotoxicity assays (DELFIA, Perkin Elmer) were performed as described in Fisher et al. 2014.

[0186] Human T24 bladder cancer cell lines were cultured at 37°C / 5% CO2 for 2 hours in PBS containing ProPAgen-free (i.e., untreated), 10 μM zoledronate (i.e., a small molecule drug used clinically), or the HMBP phosphonodiamidate ProPAgen of the present invention (i.e., 10 nM ProPAgen 9d and 10 nM ProPAgen 14d). Positive controls for cell death (target cells incubated in 10% v / v DELFIA lysis buffer) and controls treated with medium only (no drug) were also included. The cells were then washed three times in PBS at 600 × g for 5 minutes to remove any excess drug, and incubated again at 37°C for 20 minutes in PBS containing BATDA labeling agent (1 μl per ml) as before.

[0187] Meanwhile, Vγ9 / Vδ2 T cells grown ex vivo, specifically those grown for 14 days using 5 μM zoledronate and 100 U / ml IL2, were thawed, the cell count was measured, and the cells were resuspended at a cell concentration of 4 × 10⁶ cells / ml. After BATDA labeling, the T24 cells were washed three times in culture medium at 4°C and resuspended at a concentration of 5 × 10⁴ cells / ml.

[0188] Next, 100 μl of T24 cells were seeded into U-bottomed 96-well tissue culture plates and co-cultured with 100 μl / well of Vγ9 / Vδ2 T cells (i.e., an effector:target ratio of 80:1), as previously described in Fisher et al. 2014. In particular, drug-treated and untreated T24 cells were seeded individually without Vγ9 / Vδ2 effectors, with 100 μl of medium added to each well instead. For a positive killing control, 10% v / v lysis buffer was added to untreated T24 cells. The plates were centrifuged at 200 × g for 2 minutes to allow cells to come into contact in co-culture. Plates containing all samples were incubated at 37°C / 5% CO2 for 1 hour. After incubation, the plate was centrifuged again at 600×g for 2 minutes, and 25 μl of the supernatant was transferred to a flat-bottomed 96-well optical plate, to which 200 μl of europium solution was added per well.

[0189] Next, the killing level of T24 cells was measured via time-resolved fluorescence spectroscopy using a PHERAstar microplate reader (BMG Labtech). Specific lysis (T24 cell killing %) was calculated as follows: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] × 100. Data were processed and analyzed using Microsoft Excel and GraphPad Prism v9 software. The data are shown in Figure 8 as mean ± SE (n=7). Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison tests in GraphPad Prism v9. **p<0.0063.

[0190] The data show that the sensitizing effect of 10 nM phosphonodiamidates ProPAgen 9d and 14d was significantly stronger compared to 10 μM zoledronate.

[0191] As shown in Figure 8, the sensitizing effect of 10 nM phosphonodiamidates ProPAgen 9d and 14d was clearly much stronger compared to 10 μM zoledronate.

[0192] Example 5 : Metabolic studies using carboxypeptidase Y assay The in vitro metabolism of phosphonodiamidate ProPAgen according to the present invention was investigated by carboxypeptidase Y assay.

[0193] Based on the metabolic studies reported by McGuigan et al. 2018 for phosphorodiamidate ProTide, without wishing to be bound by theory, the proposed metabolism of ProPAgen according to the present invention is suggested to be initiated by an esterase (i.e., carboxypeptidase Y), which removes the ester motif from the amino acid ester and liberates the carboxyl group (metabolite 15 in Figure 10A). Subsequently, a spontaneous nucleophilic attack from one of the carboxyl groups to the phosphorus center then occurs, which causes the elimination of the second amino acid and the formation of an unstable five-membered ring (metabolite 16 in Figure 10A). The next metabolic step involves a nucleophilic attack from a water molecule to the phosphorus or carbonyl group to generate a phosphoramidate (metabolite 17 in Figure 10A). Finally, a phosphoramidase-type enzyme (e.g., Hint-1) cleaves the P-N bond in 17, leading to the release of the unmasked monophosphonate species (metabolite 18 in Figure 10A).

[0194] The carboxypeptidase Y assay was performed as previously reported by Mehellou et al. 2020, McGuigan et al. 2018, and McGuigan et al. 2014. 5.0 mg of phosphonodiamidate ProPAgen 9b was dissolved in 0.2 mL of acetone, 0.4 mL of Trizma buffer (pH 7.4) was added, and then 0.5 mg of carboxypeptidase Y in 0.2 mL of Trizma buffer (pH 7.4) was added. Phosphonodiamidate ProPAgen 9b was incubated with recombinant carboxypeptidase Y at 37 °C, 31 and the reaction was monitored by ³¹P-NMR for 12 hours. The recorded data were processed and analyzed with Bruker Topspin 2.1 software.

[0195] In the reaction buffer, at t=0, ProPAgen 9b reacted as expected with three 31 The 1P NMR peaks (δP = 14.16, 14.67, and 15.14 ppm) were observed (Figure 10B, recorded at 0 hours of incubation). 31 (See P NMR spectrum). When carboxypeptidase Y is added, three new molecules are detected within 0.5 hours as the assay progresses. 31 PNMR peaks (δP = 6.82, 7.27, 7.72 ppm) became prominent, and these new peaks became most pronounced within 6.5 hours of this study, while those in 9b were smaller. 31 This was a 1P NMR peak (Figure 10B, recorded during 6.5 hours of incubation). 31 (See P NMR spectrum). New peaks appear. 31 The 1P NMR shift is consistent with that of metabolite 17 shown in Figure 10A, and is similar to those previously observed for this metabolite (δP = 6.50, 6.90, and 7.20 ppm).

Claims

1. General formula (I): 【Chemistry 1】 (In the formula, R1 is the general formula (II): 【Chemistry 2】 (wherein R3 represents H, or a saturated or unsaturated optionally substituted hydrocarbon chain; R4 represents a saturated or unsaturated, possibly substituted hydrocarbon chain. Represents the amino acid ester group, and both R1s are identical: and R2 may be substituted with C ２－２０ Alkyl, C ４－２０ Alkenyl or C ２－２０ Represents an alcohol group; and X and Y each independently represent H or Halo. A compound or a salt thereof, wherein the compound encompasses all of its tautomers, R1 is an amino acid ester group of general formula (II) derived from alanine (R3 = -CH ３ ), leucine (R3 = -CH ２ CH(CH ３ ), ２ isoleucine (R3 = -CH(CH ３ CH ２ CH ３ ), or methionine (R3 = -CH ２ CH ２ SCH ３ ), and most preferably R1 is an amino acid ester group of general formula (II) derived from alanine (R3 = -CH ３ ); and R2 is given by formula (III) or formula (IV) (where R5 is given by OH, OR6, SH, SR6, NH) ２ Alternatively, selected from NHR6, where R6 is C １－４ Represents alkyl groups): 【Transformation 3】 It is a base, and preferably R2 is a base of formula (IV). A compound or its salt.

2. The compound according to claim 1, wherein R2 is a group of formula (IV) and R5 is an OH group.

3. A compound according to any of the prior claims, wherein at least one, preferably both, of X and Y represents a halogen.

4. The compound according to claim 3, wherein one or both halo substituents are fluoro.

5. R4 is an unsubstituted C １－４ The compound according to any of the prior claims, wherein the alkyl chain or unsubstituted benzyl group is preferably selected from methyl, isopropyl, tert-butyl and benzyl, more preferably R4 is isopropyl or benzyl, and most preferably R4 is benzyl.

6. The compound according to any one of claims 1 to 5, wherein R1 is an amino acid ester group of general formula (II) derived from L-alanine, L-leucine, L-isoleucine, or L-methionine, most preferably L-alanine.

7. The compound according to claim 6, wherein R4 is selected from methyl, isopropyl, tert-butyl, and benzyl, preferably R4 is isopropyl or benzyl, and most preferably R4 is benzyl. 【Request Item 8】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 and 【Chemistry 15】 Selected from, Preferably, the compound is 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] and 【Chemistry 19】 Selected from, More preferably, the compound is 【Chemistry 20】 and 【Chemistry 21】 Selected from, Most preferably, the compound is 【Chemistry 22】 The compound according to claim 1. 【Request Item 9】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 and 【Chemistry 26】 Selected from, Preferably, the compound is 【Chemistry 27】 The compound according to claim 1.

10. The compound is 【Chemistry 28】 The compound according to claim 1.

11. A pharmaceutical composition comprising a compound defined in any one of claims 1 to 10, preferably comprising a pharmaceutically acceptable excipient or carrier.

12. A compound according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11, for medical use.

13. A compound according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11, for use in immunotherapy.

14. A compound or pharmaceutical composition for use according to claim 13, wherein the immunotherapy is directed to the treatment of proliferative disorders, infections, inflammatory diseases and / or osteoporosis, the proliferative disorder is preferably cancer, and the cancer is preferably selected from the group consisting of hematological cancers, bladder cancers, prostate cancers, lung cancers, cervical cancers, skin cancers, breast cancers and mesotheliomas.

15. The aforementioned immunotherapy (a) comprising administering a therapeutically effective amount of any one of claims 1 to 10 to a subject in need; (b) comprising administering autologous or allogeneic T cells, preferably γδ T cells, more preferably Vγ9Vδ2 T cells; (c) further comprising administering interleukin, preferably IL-2, more preferably human IL-2; or (d) Activation of T cells, preferably γδ T cells, more preferably Vγ9Vδ2 T cells A compound or pharmaceutical composition for use according to claim 13 or 14.

16. An ex vivo method for preparing a population of therapeutic γδ T cells, wherein the method is (i) Supplying a population of γδ T cells; (ii) Culturing a population of γδ T cells in the presence of the compound according to any one of claims 1 to 10. A method that includes this.

17. A population of γδ T cells obtained by the method of claim 16, wherein the γδ T cells are preferably Vγ9 / Vδ2 T cells.

18. A pharmaceutical composition comprising a population of γδ T cells as described in claim 17.

19. A population of γδ T cells according to claim 17 or a pharmaceutical composition according to claim 18, for medical use.

20. A population of γδ T cells according to claim 17 or a pharmaceutical composition according to claim 18 for use in immunotherapy.

21. A population of proliferated γδ T cells or a pharmaceutical composition for use according to claim 20, wherein the immunotherapy is directed to the treatment of proliferative disorders, infections, inflammatory diseases and / or osteoporosis, the proliferative disorder being preferably cancer, and the cancer being preferably selected from the group consisting of hematological cancer, bladder cancer, prostate cancer, lung cancer, cervical cancer, skin cancer, breast cancer and mesothelioma.

22. (i) A step of supplying alkyl phosphonate; (ii) A step of converting an alkyl phosphonate ester to a phosphonate halide by removing the ester group and then subjecting it to a halogenation reaction; (iii) A step of subjecting a phosphonic acid halide to an esterification reaction with an amino acid ester hydrohalide to obtain an amino acid ester derivative; and (iv) A step in which the amino acid ester derivative from step (iii) is subjected to olefin metathesis while preventing alkene isomerization. A method for synthesizing a compound defined in any one of claims 1 to 10, including the above.

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