Novel phosphorus antigen prodrug compounds and uses thereof

Abstract

The present invention relates to a novel phosphate antigen (PAg) prodrug compound that provides potent activation of gamma delta T cells with therapeutic activity. The PAg prodrug compounds of the present invention are useful for the preparation of gamma delta T cells with therapeutic activity and for the immunotherapy of various diseases, and in particular of different types of cancers. The invention further relates to a method for expanding gamma delta T cells in vitro by using the PAg prodrug compound of the invention. Also provided are gamma delta T cells expanded with the PAg prodrug compounds of the invention, as well as pharmaceutical compositions comprising such expanded cells and / or the PAg prodrug compounds of the invention.

Description

[0001] The present invention relates to a novel phosphoantigen (PAg) prodrug compound that provides potent activation of therapeutically active γδ T cells. The PAg prodrug compound of the present invention is useful for the preparation of therapeutically active γδ T cells and for the immunotherapy of a variety of diseases, and in particular different types of cancer. The present invention further relates to a method for expanding γδ T cells in vitro using the PAg prodrug compound of the present invention. Also provided are γδ T cells that have been expanded with the PAg prodrug compound of the present invention, as well as pharmaceutical compositions comprising such expanded cells and / or the PAg prodrug compound of the present invention. BACKGROUND

[0002] Vγ9 / Vδ2 T cells are present from birth and are the predominant subtype of human γδ T cells in the adult peripheral blood. Vγ9 / Vδ2 T cells have now been established as a key subset of γδ T cells that participate in protection against a number of diseases such as tuberculosis, leprosy, typhoid fever, malaria and toxoplasmosis, see for example Morita, C. T.; 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 Morita et al. 2007, 215, 59-76 (hereinafter "Morita et al. 2007").

[0003] Vγ9 / Vδ2 T cells have also been shown to be involved in immunity to Mycobacterium tuberculosis in primate model studies, 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, P. K.; Li, Y.; Zeng, X.; Morita, C. T.; Brenner, M. B.; Letvin, N. L.; Chen, Z. W. Adaptive immune response of Vgamma2Vdelta2+ T cells during mycobacterial infections. Science (New York, N.Y.) 2002, 295, 2255-8.

[0004] These cells have also been shown to target and lyse a variety of cancer cells in vitro, either spontaneously or following activation with small molecule activators, see Morita et al, Immunol Rev 2007, 215, 59-76. Taken together, these observations have led the Vγ9 / Vδ2 subset to become a major focus of development for γδ T cell therapy, particularly in autologous or allogeneic immunotherapy, see Fisher, J. P.; 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 using small molecule activators can be performed in vivo or in vitro.

[0005] Treatment of patients with gd T cells usually involves the procurement of T cells from a donor blood. For example, T cells can be procured from peripheral blood mononuclear cells (PBMCs), which are isolated from peripheral blood. T cells can also be procured from cord blood mononuclear cells (CBMCs), which are isolated from cord blood. Another source of suitable T cells is non-hematopoietic tissue. After isolation of T cells, the T cells are cultured to increase their number, which is referred to as expansion. To enhance the growth and / or function of the T cells to be expanded, it is known to culture the T cells in the presence of small molecule activators. For example, the in vitro expansion of PBMCs in the presence of small molecule activators such as phosphoantigens or aminobisphosphonates provides gd T cells of the Vγ9 / Vδ2 phenotype; see, e.g., WO 2016 / 166544 or WO 2018 / 055191.

[0006] To date, a variety of small molecule activators of Vγ9 / Vδ2 T cells have been reported. Among them are naturally occurring Pag (E) - 4-hydroxy-3-methylbut-2-enylpyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP), as well as the two synthetic molecules risedronate and zoledronate (Zometa®) (E) - 4-hydroxy-3-methylbut-2-enylpyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP), as well as the two synthetic molecules risedronate and zoledronate (Zometa®) Figure 1These are currently used clinically to treat osteoporosis and certain types of cancer; see Maraka, S.; Kennel, KABisphosphonates for the prevention and treatment of osteoporosis. BMJ (Clinical Research Editor.) 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) AryloxyTriester Phosphoramidate Prodrugs as Activators of Vgamma9 / Vdelta2 T-CellImmune Responses. J Med Chem 2018, 61, 2111-2117 (hereinafter referred to as "Mehellou et al. 2018").

[0007] The most potent Vγ9 / Vδ2 T cell activator reported to date is HMBPP, whose EC50... 50 = 0.00051 μM, which activates T cells by binding to the type 1 transmembrane protein lactolipin 3A1. Although the binding sites of these PAgs remain unclear, and reports on whether they bind to the extracellular or intracellular domains of this transmembrane protein are conflicting, there is currently compelling evidence to support the idea that HMBPPs bind to the intracellular B30.2 domain of lactolipin 3A1.

[0008] Inspired by the efficacy of HMBPP in activating Vγ9 / Vδ2 T cells, aryloxytrypter phosphoramidate prodrug technology has recently been applied to HMBPP monophosphate derivatives (i.e., HMBP) as a means to improve their drug-like properties (see Mehellou et al., 2018). In this prodrug approach, the monophosphate group is masked by an aryl and / or amino acid ester. Figure 2 The monophosphate groups are enzymatically cleaved within the cell to release monophosphates or monophosphonates. Because these compounds are prodrugs of phosphate antigens (PAg), they are named "ProPAgens" to distinguish them from nucleotide prodrugs "ProTides".

[0009] Although these HMBP ProPAgens exhibit potent activation of Vγ9 / Vδ2 T cells (EC) 50 = 0.45-11nM), but their serum stability is quite low (t 1 / 2 (<30 minutes), mainly due to the breaking of the -PO- bonds in these active compounds, as above.

[0010] Against this backdrop, aryloxy diester phosphonamidates (ProPAgens) were designed. Figure 3A These compounds provide serum stability (in human serum t). 1 / 2 >12 h) and potent activation of Vγ9 / Vδ2 T cells (EC) 50 (Range from 5 fm to 73 nM), see Kadri, H.; Taher, TE; Xu, Q.; Sharif, M.; Ashby, E.; Bryan, RT; Willcox, BE; Mehellou, Y. AryloxyDiester 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"). Aryloxydiester phosphonamidates ProPAgens are also disclosed in WO2020 / 008189.

[0011] However, these aryloxydiester phosphonamides, ProPAgens (2020), by Mehellou et al., may potentially be associated with in vivo toxicity due to the aryl ester group on the phosphorus atom. Furthermore, the phosphorus atom in these aryloxydiester phosphonamides, ProPAgens, is chiral, initiating a mixture of diastereomers. Disadvantageously, different diastereomers may be associated with different biological characteristics in terms of pharmacology, toxicology, pharmacokinetics, and metabolism, necessitating the separation of diastereomer mixtures. The separation of stereoisomers is typically cumbersome and generally inefficient, costly, time-consuming, and difficult to scale up.

[0012] Therefore, the object of the present invention is to provide ProPAgens that maintain potent activation of Vγ9 / Vδ2 T cells while overcoming the challenges associated with the stereochemistry and in vivo toxicity of previously reported HMBP ProPAgens active compounds. Summary of the Invention

[0013] This invention relates to novel symmetrical amino acid ester-derived phosphonodiamidate (ProPAgens) according to the claims set forth below, and pharmaceutical compositions comprising ProPAgens. The invention also relates to the synthesis of symmetrical amino acid ester-derived methyl and difluoromethylphosphonodiamidate (ProPAgens).

[0014] This invention relates to the application of symmetrical amino acid ester groups to phosphate antigens. The resulting products exhibit advantageous properties in terms of stability, metabolism, and pharmacological activity.

[0015] The ProPAgens of this invention represent a novel class of small-molecule activators for Vγ9 / Vδ2 T cells, with potentially improved in vivo safety and efficacy. The phosphonidyldiamine ProPAgens of this invention, upon metabolism, release alcohols and natural amino acids, which are relatively less toxic compared to the phenols released by aryloxy-based ProPAgens. Furthermore, because the ProPAgens of this invention use relatively hydrophilic amino acid ester groups instead of lipophilic aromatic rings (aryloxy groups) as phosphate ester masking groups, they provide improved water solubility. Moreover, due to their symmetrical molecular structure, the number of stereocenters is reduced, which advantageously simplifies the synthesis and isolation of the target compound. This is accompanied by the beneficial effect of avoiding undesirable mixtures of product isomers with potentially different biological characteristics, thereby increasing the reliability of the target compound when used to treat subjects in need.

[0016] The ProPAgens of this invention exhibit excellent stability in human serum and induce potent activation of Vγ9 / Vδ2 T cells, which translates into potent in vitro killing of the bladder cancer cell line T24. Surprisingly, compared to prior art compounds such as zoledronic acid and HMBPP, the ProPAgens of this invention provide superior activation of Vγ9 / Vδ2 T cells.

[0017] The combination of high specificity, serum stability, and potency of these novel phosphonates (ProPAgens) makes them suitable for development as novel immunotherapies to treat a variety of conditions, including proliferative diseases such as cancer, osteoporosis, various infectious diseases such as tuberculosis, leprosy, typhoid fever, malaria, and toxoplasmosis, and / or inflammatory diseases. The phosphonidamides (ProPAgens) of this invention can be used in monotherapy regimens and as part of or following clinical regimens to expand γδ T cells in vivo; alternatively, they can be administered to patients receiving in vitro expanded γδ T cell adoptive cell therapy to directly enhance Vγ9 / Vδ2 T cell-mediated activity, particularly antitumor activity.

[0018] The present invention further relates to a method for in vitro expansion of γδ T cells, wherein the γδ T cells are cultured in the presence of ProPAgens according to the invention or pharmaceutical compositions comprising such ProPAgens. Expanded populations of γδ T cells or pharmaceutical compositions comprising such expanded γδ T cells are also provided. Detailed Implementation

[0019] According to a first aspect of the invention, a compound according to general formula (I) is provided, comprising all its tautomers: (I) Where R1 represents an amino acid ester group according to general formula (II): (II) Where R3 represents H, or a saturated or unsaturated hydrocarbon chain with optional substitution; R4 represents a saturated or unsaturated hydrocarbon chain with optional substitution; and two of R1 are identical; and R2 represents C with optional substitution. 2-20 Alkyl, C 4-20 alkenyl or C 2-20 alcohol groups; and X and Y each independently represent H or halogen; or its salt, Furthermore, R1 is derived from L-alanine, L-leucine, L-isoleucine, or L-methionine, with the most preferred being an amino acid ester group of L-alanine according to general formula (II). Furthermore, R2 is a group according to formula (III) or formula (IV), R5 is selected from OH, OR6, SH, SR6, NH2 or NHR6, and preferably OH, and R6 represents C 1-4 alkyl: (III) (IV)

[0020] These R2 substituents are similar to those found in naturally occurring PAgs IPP (Formula (III)) and HMBPP (Formula (IV)).

[0021] Compared to the conventional phosphate-containing ProPAgens discussed above, compounds of general formula (I) contain phosphonate groups masked by two identical amino acid ester groups, which are enzymatically cleaved in cells to release the active phosphonate compound. It has been shown that replacing the unstable -OP- bond with a -CC- bond significantly improves the stability of the active phosphonate compound, which has been demonstrated as a potent activator of Vγ9 / Vδ2 T cells. Furthermore, this activity translates into potent lysis of bladder cancer cells in vitro.

[0022] Since compounds of formula (I) have been shown to activate Vγ9 / Vδ2 T cells with high specificity, they are ideal candidates for developing immunotherapies. In particular, compounds of formula (I) can be used to treat proliferative diseases such as different types of cancer, osteoporosis, infectious diseases, and / or inflammatory diseases, as further outlined below.

[0023] Compounds of general formula (I) represent a novel class of small-molecule activators of Vγ9 / Vδ2 T cells with potentially improved in vivo safety and efficacy. Their symmetrical molecular structure reduces the number of stereocenters, which advantageously simplifies the synthesis and isolation of the target compounds. This is accompanied by the beneficial effect of avoiding undesirable mixtures of product stereoisomers with potentially different biological characteristics, thereby increasing the reliability of the target compounds when used to treat subjects in need.

[0024] The general and preferred aspects of the compounds of the present invention are disclosed in the following and appended claims.

[0025] As used in this article, the term "halogen" refers to a halogen radical substituent, particularly fluorine, chlorine, bromine, or iodine, more preferably fluorine or chlorine, and most preferably fluorine.

[0026] As used in this article, the term "C" 2-20"Alkyl" refers to a straight-chain or branched saturated hydrocarbon chain containing 2 to 20 carbon atoms. Examples include ethyl, n-propyl, isopropyl (iPr), n-butyl (nBu), sec-butyl, tert-butyl (tBu), n-hexyl, n-octyl, and n-decyl.

[0027] As used in this article, the term "C" 4-20 "Alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon chain containing 4 to 20 carbon atoms. Examples include butenyl and pentenyl.

[0028] As used in this article, the term "C" 2-20 "Alcohol" refers to a straight or branched hydrocarbon chain containing 2 to 20 carbon atoms and one or more hydroxyl (OH) functional groups, and is either saturated or unsaturated.

[0029] As used herein, the term "saturated or unsaturated hydrocarbon chain" refers to a straight-chain or branched aliphatic or aromatic hydrocarbon group that may or may not contain one or more double or triple bonds within the chain. Therefore, the term encompasses alkyl, alkenyl, alkynyl, or aryl groups. Aliphatic or aromatic hydrocarbon chains may contain heteroatoms within the chain or as part of substituents.

[0030] As used in this article, the term "C" 5-25 "Aryl" refers to any hydrocarbon group containing 5 to 25 carbon atoms and comprising one or more carbocyclic aromatic rings. A more suitable aryl group is C1. 6-14 And C is preferred 6-10 Aryl.

[0031] As used herein, the term "heteroaryl" refers to any hydrocarbon group comprising one or more aromatic rings containing one or more heteroatoms (e.g., N, O, or S) as part of said ring. Particularly suitable strengths of heteroaryl groups are pyridine, furan, thiophene, and indole groups. 5- to 25-membered heteroaryls refer to groups in which the total number of ring-forming atoms (carbons and heteroatoms) is 5 to 25.

[0032] Other alkyl, alkenyl, aryl, and / or alcohol groups are as defined, but have different numbers of carbon atoms. For example, C 1-4 Alkyl groups have 1 to 4 carbon atoms, such as 1, 2, 3 or 4 carbon atoms.

[0033] The hydrocarbon chain and / or aryl, heteroaryl, alkyl, alkenyl, or alcohol groups may optionally be substituted with one or more heteroatom-containing functional groups (e.g., O, S, or N). Examples of suitable heteroatom-containing groups include, but are not limited to, nitro, nitroketone, halogen, amino, amide, cyano, carboxyl, sulfonyl, hydroxyl, alkoxy, ketone, aldehyde, thiol, thioether, and non-aromatic heterocyclic groups. The hydrocarbon chain may or may not contain saturated or unsaturated cyclic rings. As used herein, any number of carbon atoms in the hydrocarbon chain, alkyl, alkenyl, aryl, or alcohol group includes any carbon atom present in the substituent. In a preferred embodiment, the hydrocarbon chain and / or aryl, heteroaryl, alkyl, alkenyl, or alcohol groups may optionally be substituted with one or more heteroatom-containing functional groups selected from thiol, thioether, alkoxy, and amino groups (which may be primary or secondary amino groups).

[0034] Salts of compounds of formula (I) are suitable pharmaceutically or veterinary-acceptable salts. Depending on the nature of R1 through R6, these salts can be basic addition salts, such as sodium, potassium, calcium, aluminum, zinc, magnesium, and other metal salts, as well as choline, diethanolamine, ethanolamine, ethylenediamine, meglumine, and other known basic addition salts (as outlined in Paulekuhn et al., (2007) J. Med. Chem. 50: 6665-6672) and / or those known to those skilled in the art. Alternatively, when compounds of formula (I) contain an amino group, this can be quaternized to form a salt with a counterion such as a halide ion, hydroxide ion, sulfate ion, nitrate ion, phosphate ion, formate ion, acetate ion, trifluoroacetate ion, fumarate ion, citrate ion, tartrate ion, oxalate ion, succinate ion, mandelic acid ion, methanesulfonate ion, and p-toluenesulfonate ion.

[0035] In some compounds of general formula (I), R2 is C. 4-16 And C is preferred. 4-8 , such as 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.

[0036] In a particularly preferred embodiment, R2 is a group according to formula (IV), or even more preferably a group according to formula (IV), wherein R5 is OH.

[0037] As described above, compounds of general formula (I) contain a masked phosphonate group comprising a -C(X)(Y)-P- bond, which has been shown to be more stable than the -OP- bond of conventional ProPAgens. In a preferred embodiment, at least one of X and Y, and more preferably both, represents a halogen, preferably fluorine.

[0038] Although replacing the -OP-bond with the -CH2-P-bond has been shown to improve the serum stability of the active compound, the pKa value of the second deprotonation of the phosphonate group (pKa = 7.49) differs significantly from that of the phosphate group (pKa = 6.31) (Figure 4). This therefore affects the complete ionization of the active compound at physiological pH (<7.4), and thus its binding affinity to the target protein. Binding of the active compound to the target protein requires complete ionization of the phosphate group to bind to the positively charged pocket (rich in arginine) on the intracellular domain of lactolipoprotein 3A1.

[0039] However, it has been found that by providing a difluoromethylphosphonate (-CF2-P-) bond, the active compound exhibits excellent stability in the physiological environment, and its pKa value after second deprotonation (6.7) is very close to that of the natural phosphate compound itself after second deprotonation (Figure 4). Therefore, monohalomethyl and / or dihalomethyl compounds of the general formula (I), especially monofluoromethyl and / or difluoromethylphosphonate derivatives, possess both excellent stability and potent activation of Vγ9 / Vδ2 T cells.

[0040] Compounds of general formula (I) are prodrugs in which the monophosphonate group is masked by two identical amino acid ester moieties R1, both of which are enzymatically cleaved in the cell to release the active monophosphonate.

[0041] Suitable amino acid ester masking groups include various 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 contain C3. 1-4 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 (CH3) group are particularly suitable.

[0042] Therefore, R1 is an amino acid ester group according to general formula (II), which is selected from L-amino acids and D-amino acids, preferably alanine, leucine, isoleucine, and methionine. For example, the amino acid may be selected from L-alanine, L-leucine, L-isoleucine, and L-methionine. In other cases, the amino acid may be selected from D-alanine, D-leucine, D-isoleucine, and D-methionine. In a preferred embodiment, the amino acid ester group according to general formula (II) is derived from an L-amino acid selected from L-alanine, L-leucine, L-isoleucine, or L-methionine; more preferably, the amino acid ester group according to general formula (II) is derived from L-alanine.

[0043] R4 is a saturated or unsaturated straight-chain or branched hydrocarbon chain, optionally substituted, and can be aliphatic or aromatic. Preferably, R4 is C 1-6 Alkyl or C 6-14 Aryl. In a particularly preferred embodiment, R4 is C. 6-10 The aryl group is more preferably selected from methyl (Me), isopropyl (iPr), tert-butyl (tBu), and benzyl (Bn), with benzyl (Bn) being the most preferred. Compounds containing benzyl esters according to general formula (I) exhibit higher degradation rates and improved lipophilicity (and thus improved cellular uptake) compared to compounds based on aliphatic esters.

[0044] In a preferred embodiment, R2 is a group according to formula (IV), preferably, wherein R5 is OH, and R1 is an amino acid ester group derived from L-alanine, L-leucine, L-isoleucine or L-methionine, most preferably L-alanine according to general formula (II), and R4 is selected from methyl, isopropyl, tert-butyl and benzyl, more preferably, wherein R4 is isopropyl or benzyl, most preferably, wherein R4 is benzyl.

[0045] In other preferred embodiments, R2 is a group according to formula (IV), preferably, wherein R5 is OH, and R1 is an amino acid ester group according to general formula (II) derived from L-alanine, L-leucine, L-isoleucine or L-methionine, more preferably L-alanine, and R4 is selected from methyl, isopropyl, tert-butyl and benzyl, more preferably, wherein R4 is isopropyl or benzyl, most preferably, wherein R4 is benzyl, and both X and Y represent halogens, preferably fluorine.

[0046] In a more preferred embodiment, in the compound according to either of the two foregoing embodiments, R1 is an amino acid ester group derived from L-alanine according to general formula (II).

[0047] In a more preferred embodiment, in the compound according to any one of the three foregoing embodiments, R2 is a group according to formula (IV), wherein R5 is OH.

[0048] In a more preferred embodiment, in the compound according to any one of the four foregoing embodiments, R4 is benzyl (Bn).

[0049] Some particularly suitable compounds of general formula (I) include the following compounds: ; ; ; ; ; ; ; ; ; ; ;and .

[0050] In a preferred embodiment, the compound is selected from... ; ; ;and , More preferably, the compound is selected from: ;and , Most preferably, the compound is: .

[0051] According to the second aspect, a compound according to general formula (I) is disclosed, including all its tautomers: (I) Where R1 represents an amino acid ester group according to general formula (II): (II) Where R3 represents H, or a saturated or unsaturated hydrocarbon chain with optional substitution; R4 represents a saturated or unsaturated hydrocarbon chain with optional substitution; and two of R1 are identical; and R2 represents C with optional substitution. 2-20 Alkyl, C 4-20 alkenyl or C 2-20 alcohol groups; and Each of X and Y independently represents H or a halogen, wherein at least one of X and Y, and preferably both, represents a halogen, preferably fluorine; Or its salt.

[0052] Further and preferred aspects of compounds having at least one of X and Y, and preferably both representing halogens, preferably fluorine, are as described in the previously disclosed embodiments, which are incorporated herein by reference.

[0053] The compounds of the present invention can be obtained by a method comprising the following steps: (i) providing an alkyl phosphonate; (ii) converting the alkyl phosphonate into a phosphonate halide by removing the ester group and subsequently undergoing a halogenation reaction; (iii) subjecting the phosphonate halide to an esterification reaction with an amino acid ester hydrogen halide to obtain an amino acid ester derivative; and (iv) subjecting the amino acid ester derivative of step (iii) to olefin metathesis to prevent olefin isomerization.

[0054] Specifically, where X and Y each represent compounds of general formula (I) of F (referred to herein as compounds 9a-d) are obtained via, for example... Figure 5 (a) The synthetic route outlined in this paper first involves reacting commercially available α,α-difluorophosphonate 5 with allyl bromide in THF in the presence of lithium diisopropylamine (LDA) and hexamethylphosphonamide (HMPA) to obtain compound 6 in 44% yield. Subsequently, compound 6 is treated with trimethylsilyl bromide (TMSBr) at room temperature to remove the ethoxy group and generate a phosphonic acid derivative. This is then followed by chlorination with oxalyl chloride in the presence of a catalytic amount of DMF to generate compound 7, which is used in the next reaction without purification. Next, compound 7 is treated with 2.5 equivalents of a suitable amino acid ester in the presence of triethylamine, which yields the desired phosphonidic diamines 8a-d in good yields (23-49%). Finally, these compounds undergo Grubbs olefin metathesis with 2-methyl-2-propenol in the presence of 1,4-benzoquinone using a Hoveyda-Grubbs second-generation catalyst to prevent olefin isomerization. This yielded the desired phosphonodiamine ProPAgens 9a-d in good yield (38-67%) with a purity of ≥95%.

[0055] Figure 5 (B) outlines the synthesis of compounds of general formula (I) (referred to herein as compounds 14a-d), in which X and Y each represent H, and is carried out in the same manner as for the preparation of 8a-d, with the sole exception of the preparation of compound 11. This is achieved by first reacting 3-butenoic acid (10) with oxalyl chloride in the presence of DMF to generate 3-butenoyl chloride, followed by reaction with triethyl phosphite to give compound 11 in good yield (51%). Phosphonidamides ProPAgens 14a-d were obtained in good yield (33-63%) and with a purity of ≥95%.

[0056] It will be understood that the compounds of the first and second aspects of the present invention can be administered as part of a pharmaceutical composition. Therefore, according to a third aspect of the present invention, a pharmaceutical composition is provided comprising the compounds of the first and / or second aspects of the present invention. The pharmaceutical compositions of the present invention preferably comprise pharmaceutically acceptable excipients or carriers.

[0057] Suitable pharmaceutical excipients are well known to those skilled in the art. Pharmaceutical compositions can be formulated for administration via any suitable route, such as oral, rectal, nasal, bronchial (inhalation), topical (including eye drops, oral and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, and can be prepared by any method known in the field of pharmaceutics.

[0058] This composition can be prepared by combining a compound from the first aspect of the invention with a carrier. Typically, the formulation is prepared by uniformly and tightly combining the compound with a liquid carrier or a finely dispersed solid carrier, or both, and then shaping the product if necessary.

[0059] The formulations for oral administration in this invention may be presented in the following forms: discrete units such as capsules, sachets, or tablets, each 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 an oil-in-water emulsion or water-in-oil emulsion; or as pills, etc.

[0060] For oral administration (e.g., tablets and capsules), the term "acceptable carrier" includes a variety of media, such as common excipients like binders (e.g., syrups, gum arabic, gelatin, sorbitol, tragacanth gum, 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 alginate; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, glyceryl stearate, stearic acid, silicone oil, talc, oils, and colloidal silica. Flavorings such as peppermint, wintergreen oil, and cherry flavoring may also be used. Coloring agents may be added to make the dosage form easily identifiable. Tablets may also be coated using methods known in the art.

[0061] Tablets can be made by compression or molding, optionally using one or more excipients. Compressed tablets can be prepared by compressing a free-flowing compound, such as powder or granules, in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets can be made by molding a mixture of powdered compounds wetted with an inert liquid diluent in a suitable machine. Tablets can optionally be coated or scored and can be formulated to provide a slow or controlled release of the active agent.

[0062] Other formulations suitable for oral administration include: lozenges containing the active ingredient in a flavoring matrix (usually sucrose and gum arabic or tragacanth); tablets containing the active ingredient in an inert matrix such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid carrier.

[0063] Parenteral preparations are usually sterile.

[0064] For topical application to the skin, the composition can be formulated as a cream, ointment, gel, solution, or suspension. Cream or ointment formulations that can be used with this drug are conventional formulations known in the art, for example, as described in pharmaceutical standards textbooks (such as the British Pharmacopoeia).

[0065] In a preferred embodiment of this aspect of the invention, the composition is formulated for oral delivery.

[0066] One skilled in the art can readily determine the precise amount of a therapeutically effective composition as defined herein, as well as the optimal route of administration of such compound. Of course, such amounts will depend on the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, and weight), duration of treatment, the nature of any concomitant treatments, the specific route of administration, and similar factors within the knowledge and expertise of the healthcare provider. These factors are well known to those skilled in the art and can be determined using only routine experimental methods. Generally, the maximum dose of a single ingredient or combination thereof is preferred, i.e., the highest safe dose based on reasonable medical judgment. However, those skilled in the art will understand that patients may adhere to lower or tolerable doses for medical, psychological, or virtually any other reason.

[0067] The dosage of the compound or composition according to the invention administered to the subject can be selected based on various parameters, particularly the method of administration and the subject's condition. Other factors include the required duration of treatment. If the subject does not respond adequately to the initial dose, a higher dose may be used (or a higher dose may be used effectively via a different, more localized route of delivery), to the extent tolerated by the patient.

[0068] As described above, the compounds of the first and second aspects of the present invention represent prodrugs of highly stable and potent activators of Vγ9 / Vδ2 T cells. Therefore, according to a fourth aspect of the present invention, a compound of the first and / or second aspects, or a pharmaceutical composition according to the third aspect, is provided for medical use, and more preferably for immunotherapy. In a preferred embodiment, the immunotherapy comprises activating T cells, preferably activating γδ T cells, and even more preferably activating Vγ9 / Vδ2 T cells. In another preferred embodiment, the immunotherapy comprises treatment for proliferative diseases, infectious diseases, inflammatory diseases, and / or osteoporosis. The immunotherapy will generally comprise administration of a therapeutically effective amount of the compound of the present invention to a subject in need.

[0069] The compounds of the present invention can be administered to subjects requiring treatment, such as those suffering from proliferative diseases like cancer, infectious diseases, inflammatory diseases, and / or osteoporosis. Upon administration, the compounds result in the activation of T cells, preferably γδT cells, and more preferably Vγ9Vδ2 T cells, in the subject's body. The activated T cells exert potent effector responses, such as cytotoxicity, which then direct the immune response toward appropriate target cells, such as tumor cells. The subject can be a mammalian subject, preferably a human subject.

[0070] The immunotherapy may further include the administration of T cells, preferably γδ T cells, and preferably Vγ9Vδ2 T cells. These cells may be autologous cells, meaning they are derived from the patient to be treated and have been obtained from the patient prior to the start of immunotherapy. Alternatively, these cells may be allogeneic, in which case the cells have been obtained from a donor, such as from a blood sample from a blood donor. The administration of autologous or allogeneic T cells may be performed before, after, or simultaneously with the administration of the ProPAgens of this invention.

[0071] Immunotherapy may also include the administration of interleukins, preferably interleukin-2 (IL-2), and more preferably human IL-2. Similarly, the administration of autologous or allogeneic T cells may be performed before, after, or simultaneously with the administration of ProPAgen of the present invention.

[0072] In one embodiment, the immunotherapy relates to treating a proliferative disease. Preferably, the proliferative disease is a cancer. The cancer treated with the compounds or pharmaceutical compositions of the present invention is not limited to any type, including hematologic and non-hematologic cancers. In a preferred embodiment, the immunotherapy relates to treating leukemia, a malignant disease of hematopoietic tissues such as the bone marrow or lymphatic system. Leukemia treated with the compounds or pharmaceutical compositions of the present invention can include any subtype of leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). In one embodiment, the immunotherapy relates to treating ALL, and more particularly to treating precursor B-cell acute lymphoblastic leukemia, precursor T-cell acute lymphoblastic leukemia, Burkitt's leukemia, or acute biphenotypic leukemia. In another embodiment, the immunotherapy relates to treating CLL, and more particularly to treating B-cell prolymphocytic leukemia. In yet another implementation, the immunotherapy relates to 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.

[0073] Alternatively, immunotherapy can also be used to treat solid tumors. Tumors treated with the compounds or pharmaceutical compositions of this invention include, but are not limited to: bladder cancer, prostate cancer, lung cancer, cervical cancer, skin cancer, breast cancer, colon cancer, liver cancer, pancreatic cancer, ovarian cancer, brain cancer, stomach 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 tumors, bile duct cancer, germ cell tumors, embryonal tumors, lip cancer, oral cancer, multiple myeloma, small intestine cancer, nasal cavity cancer, 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.

[0074] In a particularly preferred embodiment, the cancer treated with the immunotherapy of the present invention is selected from the group consisting of hematologic cancers, such as leukemia, bladder cancer, prostate cancer, lung cancer, cervical cancer, skin cancer, breast cancer, and mesothelioma.

[0075] In another embodiment, the immunotherapy relates to the treatment of infectious diseases. Infectious diseases suitable for treatment using the compounds or pharmaceutical compositions of the present invention include bacterial infections, viral infections, and fungal infections. Particularly suitable are bacterial infections, such as those caused by pathogenic bacteria of the genus *Mycobacterium*, including *Mycobacterium africanum*, *Mycobacterium cannibalum*, *Mycobacterium capsulatum*, *Mycobacterium bovis*, *Mycobacterium vulgare*, *M. mungi*, *Mycobacterium pinnipeds*, *Mycobacterium tuberculosis*, *Mycobacterium Genevai*; *Mycobacterium leprae*; immunogenic mycobacteria; *Mycobacterium chrysogenum*; *Mycobacterium myxogenum*; *Mycobacterium ulcerans*; *Mycobacterium bufo*, *Mycobacterium cholestianum*, *Mycobacterium avium*, and *Mycobacterium paratuberculosis*. Therefore, in a preferred embodiment, the immunotherapy of the present invention relates to the treatment of tuberculosis or leprosy.

[0076] The compounds or pharmaceutical compositions of the present invention can also be used to treat Salmonella infections, including *Salmonella enterica* species, and preferably one of the following subspecies: *Salmonella enterica* subsp. *enteroides*, *Salmonella enterica* subsp. *Arizona*, and *Salmonella enterica* subsp. *double Arizona*. In a particularly preferred embodiment, the compounds of the present invention can also be used to treat bacterial infections caused by pathogenic *Salmonella enterica* subsp. *enteroides* strains that cause typhoid fever. Therefore, compounds of general formula (I) are particularly useful for treating typhoid fever.

[0077] In another implementation, immunotherapy involves treating fungal infections.

[0078] Fungal infections that can be treated with the compounds and pharmaceutical compositions of the present invention include infections caused by Aspergillus fungi, particularly lung infections. In one embodiment, the infection to be treated is aspergillosis caused by Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidus, and Aspergillus niger. The compounds and pharmaceutical compositions of the present invention can also be used to treat infections caused by Blastomycetes fungi. In one embodiment, the infection to be treated is blastomycosis caused by Blastomycetes dermatitidis. Infections caused by Candida fungi, particularly invasive infections, are also treatable. In one embodiment, the infection to be treated is candidiasis caused by Candida albicans, Candida auris, Candida blankii, Candida asteroides, Candida dublin, Candida famata, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. Infections caused by Cryptococcus fungi are also treatable. In one embodiment, the infection to be treated is cryptococcosis caused by Cryptococcus neoformans and Cryptococcus gutterusii. The compounds and pharmaceutical compositions of the present invention are also suitable for treating fungal infections of the nails, skin or eyes.

[0079] In yet another embodiment, immunotherapy involves treating viral infections. Types of viral infections that can be treated with the compounds and pharmaceutical compositions of the present invention include viral infections caused by a variety of RNA and DNA viruses, including: orthomyxoviruses, paramyxoviruses, flaviviruses, plague viruses, hepatoviruses, arenaviruses, herpesviruses, adenoviruses, poxviruses, and retroviruses.

[0080] In another embodiment, the immunotherapy relates to the treatment of parasitic infections. Parasitic infections particularly suitable for treatment with the compounds or pharmaceutical compositions of the present invention include infections caused by organisms of the genus *Plasmodium*, such as *Plasmodium vivax*, *Plasmodium falciparum*, *Plasmodium malariae*, *Plasmodium ovale*, and *Plasmodium norocioli*. In a particularly preferred embodiment, the compounds of the present invention or pharmaceutical compositions comprising them are used to treat *Plasmodium* species that cause malaria. Therefore, compounds of formula (I) or pharmaceutical compositions comprising them are particularly useful for treating malaria. Other suitable parasitic infections that can be treated include infections caused by organisms of the genus *Toxoplasma*, such as *Toxoplasma gondii*. In a particularly preferred embodiment, the compounds of the present invention are used to treat *Toxoplasma* species that cause toxoplasmosis. Therefore, compounds of formula (I) or pharmaceutical compositions comprising them are particularly useful for treating toxoplasmosis.

[0081] In one embodiment, immunotherapy relates to treating inflammatory diseases. Therefore, the compounds of the present invention or pharmaceutical compositions comprising them can be used to treat diseases or disorders characterized by immune dysregulation or 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 can be localized or systemic. The types of inflammatory diseases or conditions that may benefit from treatment are not limited and include, but are not limited to: arterial occlusive diseases such as peripheral arterial occlusive disease (pAOD), severe limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or constriction; inflammation associated with systemic metabolic disorders, including type II diabetes and obesity-related metabolic syndrome; and dermatological diseases, including eczema. In a preferred embodiment, the inflammatory disease is an autoimmune disease. There are no restrictions on the types of autoimmune diseases to be treated, and these include 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.

[0082] In another implementation, immunotherapy involves treating hypersensitivity reactions. For example, hypersensitivity reactions may be selected from asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity reactions, and mastocytosis.

[0083] In another implementation, immunotherapy involves treating osteoporosis. The osteoporosis to be treated can be primary or secondary osteoporosis.

[0084] A fourth aspect of the invention also provides an immunotherapy method comprising administering to a subject in need, such as a human subject, a therapeutically effective amount of a compound of the first or second aspect of the invention or a pharmaceutical composition of the third aspect of the invention. By delivering the compound of the first or second aspect of the invention or the pharmaceutical composition of the third aspect of the invention to the subject to be treated, the subject's γδ T cells are activated in vivo, forming an immune response against corresponding target cells (e.g., cancer cells). The treatment method may involve treating any of the aforementioned conditions or diseases, i.e., treating proliferative diseases, infectious diseases, inflammatory diseases, and / or osteoporosis. The treatment method may include administering autologous or allogeneic T cells, preferably γδ T cells, and more preferably Vγ9Vδ2 T cells, as mentioned elsewhere herein. Immunotherapy may further include administering interleukins, preferably IL-2 and more preferably human IL-2.

[0085] In a fifth aspect, the present invention relates to an in vitro method for preparing a population of γδ T cells for therapeutic purposes, the method comprising: (i) Provide a population of γδ T cells; (ii) As described above, γδ T cell populations are cultured in the presence of compounds according to the first or fifth aspect of the invention.

[0086] This method involves contacting γδ T cells with compounds of the present invention to induce their activation and / or expansion. In the first step of the method, a population of γδ T cells is provided. This population of γδ T cells can be obtained from various sources, such as blood samples from donors or patients to be 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 for a period of time in the presence of compounds of the first or second aspect of the present invention, and cultured under conditions that lead to their activation and / or expansion.

[0087] According to a sixth aspect, the present invention relates to a population of γδ T cells obtained by the method according to the fifth aspect. Cells treated by the method described above according to the fifth aspect of the invention are preferably Vγ9 / Vδ2 T cells, and can be advantageously used in medicine, particularly in immunotherapy.

[0088] A seventh aspect of the invention also provides a pharmaceutical composition comprising a population of γδ T cells according to a sixth aspect of the invention. The explanations given above regarding the pharmaceutical compositions of the third aspect of the invention apply accordingly to these pharmaceutical compositions.

[0089] According to an eighth aspect, the present invention relates to the use of γδ T cell populations according to the sixth aspect of the invention for medical purposes, and particularly for immunotherapy. Preferably, the immunotherapy relates to the treatment of proliferative diseases, infectious diseases, inflammatory diseases, and / or osteoporosis. The explanations given above regarding the fourth aspect of the invention apply accordingly to the medical use of cells according to the eighth aspect of the invention. Therefore, γδ T cells according to the sixth aspect of the invention can be used to treat the same conditions and diseases discussed herein and related to the fourth aspect of the invention. In other words, γδ T cells according to the sixth aspect of the invention (which have been obtained by the method of the fifth aspect of the invention) can be used to treat proliferative diseases, infectious diseases, inflammatory diseases, and / or osteoporosis.

[0090] According to the sixth aspect of the invention and the eighth aspect of the invention, when using cells, the treatment can be autologous. This means that the γδ T cells undergoing the method of the fifth aspect of the invention are obtained from a patient who is ultimately treated with γδ T cells. In this case, the γδ T cells are obtained from the patient, subsequently processed by the method of the fifth aspect of the invention, and then reintroduced into the patient. Alternatively, the treatment can be allogeneic, meaning that the γδ T cells 2 undergoing the method of the fifth aspect of the invention are obtained from a donor who is not ultimately a patient being treated.

[0091] According to a ninth aspect, the present invention relates to the use of compounds from the first or second aspect of the invention for the preparation of pharmaceutical compositions for immunotherapy. As described in other parts herein, the immunotherapy preferably relates to the treatment of proliferative diseases, infectious diseases, inflammatory diseases, and / or osteoporosis.

[0092] Throughout the description and claims of this specification, the terms “comprise” and “containing,” and variations thereof, such as “comprising” and “comprises,” mean “including, but not limited to,” and do not exclude other parts, additives, components, integers, or steps. Throughout the description and claims of this specification, unless the context otherwise requires, the singular encompasses the plural. In particular, where indefinite articles are used, unless the context otherwise requires, the specification should be understood to encompass both the plural and the singular.

[0093] All references cited in this specification, including any patents or patent applications, are incorporated herein by reference. No reference is acknowledged to constitute prior art. Furthermore, no prior art is acknowledged to be part of common general knowledge in the art.

[0094] Preferred features of various aspects of the present invention may be described in conjunction with any other aspects.

[0095] Other features of the invention will become apparent from the following embodiments. Generally, the invention extends to any novel one or any novel combination of features disclosed in this specification (including the appended claims and drawings). Therefore, features, integers, properties, compounds, or chemical portions described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible with it.

[0096] Furthermore, unless otherwise stated, any feature disclosed herein may be replaced by an alternative feature serving the same or similar purpose.

[0097] The invention will now be described by way of example only with reference to the following embodiments and the accompanying drawings: Figure 1 Showing the chemical structure of reported small molecule Vγ9 / Vδ2 T cell activators: naturally occurring Pags (E) -4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP); synthetic molecules risephosphonate and zoledronic acid.

[0098] Figure 2 This demonstrates the application of aryloxytriester phosphonamide prodrug technology in HMBPP monophosphate derivatives (HMBP). The monophosphate group is masked by an aryl group and an amino acid ester, which are enzymatically cleaved within the cell to release the active monophosphate. Instability was observed due to the breaking of the -PO- bond in these compounds (shaded area).

[0099] Figure 3A This shows the general chemical structure of aryloxydiester phosphonamides ProPAgens, based on Mehellou et al., 2022.

[0100] Figure 3B The general chemical structure of ProPAgens, a symmetric amino acid ester-derived phosphonodiamine, is shown according to the present invention.

[0101] Figure 4A The pKa values ​​are shown for phosphate and different phosphonate groups.

[0102] Figure 4BThe chemical structure of metabolite 15 is shown, revealing that the electron-withdrawing effect of the fluorine atom in difluoromethylenephosphonate makes the phosphorus center a stronger electrophile compared to methylenephosphonate 15b (5a). This also affects the strength of any possible intramolecular H- bonds—these bonds are stronger in 15b than in 15a due to the attracting effect of fluorine. This makes the cyclization of methylenephosphonate potentially slower than its nonfluorinated counterpart.

[0103] Figure 5 This paper shows the synthesis of symmetric amino acid ester-derived phosphonidyl diamine prodrugs of (A) HMBP methylphosphonate (14a-d) and (B) HMBP difluoromethylphosphonate (9a-d). Reagents and conditions: A. (I) LDA, HMPA, THF, allyl bromide, -78 °C, 44% yield; (ii) TMSBr, DCM, 50 °C, N2, 3 h, then (COCl)2, DMF catalyst, DCM, room temperature, 2 h; (iii) L-alanine ester hydrochloride, DCM, N2, -78 °C, TEA, then room temperature, overnight, yield: 23-49%; (iv) 2-methyl-2-propenol, 1,4-benzoquinone, second-generation Hoveyda-Grubbs catalyst, DCM, 78 °C, yield: 38-67%. B. (v) Oxaloyl chloride (solvent and reagent), N2, 0°C to room temperature, DMF, then (EtO)3P, 0°C to room temperature, overnight, yield 51%; (vi) TMSBr, DCM, 50°C, N2, 3 h, then (COCl)2, DMF catalyst, DCM, room temperature, 2 h; (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, second-generation Hoveyda-Grubbs catalyst, DCM, 78°C, yield: 33-63%.

[0104] Figure 6 Display as passed 31 The stability of HMBP phosphonidamide ProPAgen 9b in human serum at 37°C for 7.5 hours was monitored by PNMR.

[0105] Figure 7A The results showed that ProPAgens 9a-d was effective against human Vγ9 / Vδ2. + In vitro activation of T cells. The HMBPProPAgens of this invention were used at concentrations from 10 pM to 100 μM, and then in separate experiments, compound 9d was used at concentrations from 1 aM to 100 μM, see [link to relevant documentation]. Figure 7E .

[0106] Figure 7BThe results showed that ProPAgens 14a-d was used to treat human Vγ9 / Vδ2. + In vitro activation of T cells. The HMBPProPAgens of this invention were used at concentrations from 10 pM to 100 μM, and then in separate experiments, compound 14d was used at concentrations from 1 aM to 100 μM, see [link to relevant documentation]. Figure 7E .

[0107] Figure 7C The study showed that zoledronic acid and HMBPP at concentrations ranging from 10 pM to 100 μM had an effect on human Vγ9 / Vδ2. + In vitro activation of T cells. Zoledronic acid and HMBPP were used as prior art compounds for comparison with HMBPProPAgens according to the present invention.

[0108] Figure 7D The EC50 calculated using GraphPad Prism v9 is shown based on the results of activation determination. 50 Values. CLogP values ​​were calculated using ChemDraw Professional 16.0. EC14a was not determined because accurate efficacy levels were not readily available. 50 Value (nM).

[0109] Figure 7E The activation of Vγ9 / Vδ2 T cells mediated by phosphonidamide ProPAgen was demonstrated after 9 days and 14 days of overnight incubation of PBMCs with 1 atomolar (aM) to 100 μM, respectively, to determine the EC50 of these compounds. 50 Values. Activation levels are expressed as % CD69+ CD25+ Vγ9 / Vδ2 T cells. Data were analyzed using GraphPad Prism v9 software and are shown as mean ± standard error (n=4).

[0110] Figure 8Cytotoxicity assays showed potent lysis of bladder cancer cells after incubation with HMBP phosphonates ProPAgens for 14 and 9 days. ProPAgen 14 and 9 days mediated specific lysis of T24 bladder cancer cells by Vγ9 / Vδ2 T cells. The % killing rate of T24 cells was calculated using the following formula: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] × 100. Data are presented as mean ± standard error (n = 7). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test on GraphPad Prism v9 software. **p < 0.0063. Also included were a positive control for cell death (target cells incubated with 10% v / v DELFIA lysis buffer) and a control treated only with culture medium (no drug).

[0111] Figure 9 The activation of CD8+ T cells mediated by phosphonamide (ProPAgen) at 9d and 14d under the same overnight culture conditions as Vγ9 / Vδ2 T cells is shown in the left panel (mean ± standard error; n=4). The activation levels of Vγ9 / Vδ2 T cells diluted with DMSO in the same manner as ProPAgens are shown in the right panel (mean ± standard error; n=4). Data were analyzed using GraphPadPrism v9 software.

[0112] Figure 10A This demonstrates the hypothetical mechanism of phosphonidamide prodrugs as proposed by McGuigan et al. in 2018.

[0113] Figure 10B This demonstrates the in vitro degradation of phosphatidyldiamine ProPAgen 9b mediated by carboxypeptidase Y. ProPAgen 9b alone and at different time points (as shown in the figure) after incubation with recombinant carboxypeptidase Y at 37°C for 6.5 hours... 31 P-NMR nuclear magnetic resonance spectroscopy. Example

[0114] The present invention is further described in more detail through the following embodiments, which are provided only for illustrative purposes and should not be construed as limiting the scope of the invention. The following materials and methods are used in the embodiments.

[0115] Example 1 Synthesis of aryloxyphosphonamides ProPAgens from HMBP methylphosphonates All reagents and solvents were of general grade or analytical grade and were purchased from Sigma-Aldrich Ltd., Fisher Scientific, Fluorochem or Acros. 31 P, 1 H, 19F and 13 C10 NMR data were recorded on a Bruker Avance DPX500 spectrometer operating at 202, 500, and 125 MHz. Chemical shifts (δ) are expressed in ppm, and J values ​​in Hz. When reporting spectral data, the following abbreviations are used: s (singleton), d (doublet), t (triplet), q (quartet), dd (doublet), td (doubletuplet), and m (multiplex). All reactions were performed under a nitrogen atmosphere and monitored by analytical thin-layer chromatography (TLC) on pre-coated silica gel plates (Kiesel gel60F 254, BDH). Compounds were visualized by irradiation with UV light (254 nm) or by staining with KMnO4 followed by heating. Rapid column chromatography was performed using silica gel 60 (230–400 mesh) (Merck). HPLC was performed on a SHIMADZU Prominence-i four-stage low-pressure gradient pump using a Prominence-i UV detector (190–700 nm). All solvents used in HPLC were HPLC-grade solvents purchased from Fisher Scientific. HPLC data analysis was performed using Shimadzu laboratory solution software. The purity of the tested ProPAgens was determined by HPLC, and all were ≥95% pure.

[0116] (6) Diethyl α,α-difluorophosphonate: At -78 °C, a cooled solution of α,α-difluorophosphonate (1.7 mL, 1 equivalent, 7.97 mmol) in 3 mL THF was added to a solution of lithium diisopropylamine (LDA) (1.0 M hexane / THF, 7.97 mL, 1 equivalent, 7.97 mmol) and hexamethylphosphonamide (HMPA) (1.38 mL, 1 equivalent, 7.97 mmol) in 5 mL THF. After stirring for 2 min, allyl bromide (5 mL, 1.2 equivalent, 9.56 mmol) was rapidly added under vigorous stirring. After 10 min, the reaction was quenched with NH4Cl and extracted with diethyl ether (10 mL) and ethyl acetate (2 × 20 mL). The combined organic phases were dried over MgSO4 and concentrated under reduced pressure. The crude product was separated by rapid column chromatography (EtOAc:hexane 4:6). Yield: 793 mg (44%). 1 ¹H 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).

[0117] Diethyl butyronitrile (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 equivalent, 12 mmol) were added to a round-bottom flask under a nitrogen-inert atmosphere. The mixture was cooled to 0 °C and three drops of DMF were added to catalyze the reaction. The mixture was allowed to be heated to room temperature and monitored by TLC. When the starting material spot disappeared, excess oxalyl chloride was removed under reduced pressure, and crude 3-butenoic chloride was used in the next step without purification. In the second step, crude 3-butenoic chloride from the previous step was added to a dry round-bottom flask and cooled to 0 °C. Triethyl phosphite was then added dropwise. The mixture was allowed to be heated to room temperature and stirred overnight. After solvent removal under reduced pressure, the crude product was purified by column chromatography (EtOAc: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). 13C 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).

[0118] The general procedure for synthesizing compounds 7 and 12 is as follows: These compounds are synthesized in two steps. First, compound 6 or 11 (1 equivalent) is dissolved in 5 mL of DCM, and TMSBr (10 equivalents) is added under a nitrogen inert atmosphere. The reaction mixture is refluxed at 50 °C for 3 hours, and then the solvent and excess TMSBr are evaporated under reduced pressure. The crude product is then dissolved in 10 mL of DCM for chlorination, and three drops of DMF are added as a catalyst under a nitrogen inert atmosphere. Subsequently, oxalyl chloride (10 equivalents) is added dropwise. The reaction mixture is stirred at room temperature for 2 hours, and the solvent and excess oxalyl chloride are removed under reduced pressure. Crude product 7 or 12 is used in the next step without further purification.

[0119] The general procedure for synthesizing 8a-d and 13a-d is as follows: Compound 7 or 12-18 (1 equivalent) was dissolved in 10 mL of DCM with an appropriate amount of L-alanine ester hydrochloride (2.5 equivalent) under a nitrogen-inert atmosphere. The mixture was then cooled to -78 °C and TEA (4 equivalent) was added dropwise. The reactants were allowed to be heated to room temperature and stirred overnight. The solvent was removed under reduced pressure, and the crude product was dissolved in EtOAc. After filtration, the filtrate was concentrated, and the crude product was purified by rapid column chromatography (EtOAc:hexane 1:1) to obtain the desired product.

[0120] Dimethyl 2,2'-(((1,1-difluorobut-3-en-1-yl)phosphoryl)bis(azonyl))(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).

[0121] 2,2'-(((1,1-difluorobut-3-en-1-yl)phosphoryl)bis(azonyl))(2S,2'S)-diisopropyl 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). 19 F NMR (470MHz, 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).

[0122] 2,2'-(((1,1-difluorobut-3-en-1-yl)phosphoryl)bis(azonyl))(2S,2'S)-ditert-butyl 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 = 11.95Hz), -111.62 (d, J = 96.38 Hz).

[0123] 2,2'-(((1,1-difluorobut-3-en-1-yl)phosphoryl)bis(azonyl))(2S,2'S)-dibenzyl 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 PNMR (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).

[0124] Dimethyl 2,2'-((but-3-en-1-ylphosphoryl)bis(azonyl))(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).

[0125] 2,2'-((but-3-en-1-ylphosphoryl)bis(azonyl))(2S,2'S)-diisopropyl 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).

[0126] Di-tert-butyl 2,2'-((but-3-en-1-ylphosphoryl)bis(azonyl))(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).

[0127] 2,2'-((but-3-en-1-ylphosphoryl)bis(azonyl))(2S,2'S)-dibenzyl 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. 31 P NMR (202 MHz, CDCl3): 28.71 (s).

[0128] The general procedure for synthesizing 9a-d and 14a-d was as follows: Compounds 8a-d and 13a-d (1 equivalent, 0.335 mmol) were dissolved in 10 mL of DCM under an inert nitrogen atmosphere. 2-Methyl-2-propen-1-ol (0.056 mL, 2 equivalent, 0.67 mmol), 1,4-benzoquinone (3.6 mg, 10% mol, 0.0335 mmol), and Hoveyda-Grubbs second-generation 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 the mixture was stirred for 1 hour to absorb inorganic Ru from the catalyst. The mixture was then filtered through diatomaceous earth, concentrated under reduced pressure, and purified by rapid column chromatography (EtOAc:hexane, gradient 20:80 to 100:0). The geometric configuration was confirmed by NOESY.

[0129] Dimethyl 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azonyl))(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. 31 P NMR (202 MHz, CDCl3): 13.31 (t, J = 96.83 Hz). 19 F 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): Calculated value (M + Na) + C 14 H 25 F2N2O6PNa, 409.1317; Measured value, 409.1316. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30, 12 min, λ = 210 nm, tRt = 5.82 min (100%).

[0130] 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azonyl))(2S,2'S)-diisopropyl dipropionate (9b). Yield: 174 mg (52%). 1 H 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 C 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 P NMR (202 MHz, CDCl3): 13.18 (t, J = 96.56 Hz). 19 F NMR (470 MHz, CDCl3): -108.85 (d, J = 11.95 Hz), -109.06 (d, J = 11.58 Hz). HRMS (ES+, m / z): Calculated value (M + Na) + C 18 H33 F2N2O6Pna, 465.1950; Measured value, 465.1942. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30, 12 min, λ = 210 nm, tRt = 5.82 min (100%).

[0131] 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azine))(2S,2'S)-ditert-butyl dipropionate (9c). Yield: 81 mg (67%). 1 H 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 C NMR (125 MHz, CDC13): 173.35, 142.15, 113.62, 82.38, 68.09, 49.47, 32.67, 27.92,21.85, 14.04. 31 P NMR (202 MHz, CDCl3): 13.19 (t, J = 96.31 Hz). 19 F NMR (470 MHz, CDCl3): -108.85 (d, J = 7.72 Hz), -109.06 (d, J = 8.39 Hz). HRMS (ES+, m / z): Calculated value (M + Na) + C 20 H 37 F2N2O6PNa, 493.2260; Measured value, 493.2255. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30, 12 min, λ = 210 nm, tRt = 5.81 min (100%).

[0132] 2,2'-((((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)-bis(azine))(2S,2'S)-dibenzyl propionate (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 PNMR (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): Calculated value (M + Na) + C 26 H 33 F2N2O6PNa, 561.1940; Measured value, 561.1942. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30, 12 min, λ = 210 nm, tRt = 5.53 min (100%).

[0133] Dimethyl 2,2'-((((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azonyl))-(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 CNMR (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): Calculated value (M+ Na). + C 14 H 27 N₂O₆PNa, 373.1498; Measured value, 373.1504. HPLC (reversed phase) 0.5 mL / min MeOH / H₂O 70:30, 12 min, λ = 210 nm, tRt = 5.80 min (98%).

[0134] 2,2'-((((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azonyl))-(2S,2'S)-diisopropyl dipropionate (14b). Yield: 40 mg (33%). 1H 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): Calculated value (M+ Na). + C 18 H 35 N2O6PNa, 429.2143; Measured value, 429.2130. HPLC (reversed phase) 0.5 mL / min MeOH / H2O 70:30, 12 min, λ = 210 nm, tRt = 5.81 min (100%).

[0135] 2,2'-((((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azonyl))-(2S,2'S)-ditert-butyl dipropionate (14c). Yield: 43 mg (33%). 1 H 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): Calculated value (M+ Na). + C 20 H 39 N₂O₆PNa, 457.2453; Measured value, 457.2443. HPLC (reversed phase) 0.5 mL / min MeOH / H₂O 70:30, 12 min, λ = 210 nm, tRt = 5.54 min (98%).

[0136] 2,2'-((((E)-5-hydroxy-4-methylpent-3-en-1-yl)phosphoryl)bis(azonyl))-(2S,2'S)-dibenzyl propionate (14d). Yield: 128 mg (59%). 1 H 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): Calculated value (M+ Na).+ C 26 H 35 N₂O₆PNa, 525.2127; Measured value, 525.2130. HPLC (reversed phase) 0.5 mL / min MeOH / H₂O 70:30, 12 min, λ = 210 nm, tRt = 5.82 min (100%).

[0137] To ensure that the samples were not damaged during dissolution and storage, they were analyzed again by mass spectrometry and HPLC to confirm that their structure was correct and that they were pure.

[0138] Example 2 Stability study of ProPAgen HMBP difluoromethylphosphonate 9b according to the present invention To demonstrate the stability of the phosphonidamides ProPAgens according to the invention in human serum, a representative serum stability study was conducted with difluoromethylphosphonate 9b, which has iPr esters such as two FAD-approved phosphonidamide prodrugs, sofosbuvir and tenofovir alafenamide.

[0139] This experiment is similar to previous work by Mehelllou et al. 2020 and Slusarczyk, M.; Ferrari, V.; Serpi, M.; Gönczy, 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 (hereinafter referred to as “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 The process of developing agent (NUC-1031) in clinical development was reported in J. Med. Chem. 2014, 57, 1531-1542 (hereinafter referred to as "McGuigan et al. 2014").

[0140] In short, 5.0 mg of phosphonamide diamine ProPAgen 9b was dissolved in a mixture of 0.05 mL DMSO and 0.15 mL D2O. (The first record...) 31 After obtaining the pNMR data, 0.3 mL of human serum (Merck Life Sciences) was added and monitored by NMR. The experiment was conducted using NMR... 31 The system was run in P mode, scanning every half hour for 7.5 hours. The incubation temperature was 37°C. The recorded data were processed and analyzed using Bruker Topspin 2.1 software.

[0141] ProPAgen 9b was incubated with human serum at 37°C for 7.5 hours, and then... 31 Monitoring was performed using P NMR spectroscopy. For example... Figure 6As shown, due to coupling with fluorine atoms, ProPAgen 9b's 31 The P NMR spectrum showed three phosphorus peaks (δP = 14.87, 15.37, and 15.87 ppm), which are the prodrugs. 31 Typical and expected spectra of P-NMR. Notably, human serum also shows a p-NMR spectrum at δP = 1.82. 31 P-NMR peak. Incubation of phosphonidamide ProPAgen 9b with human serum supplemented with human serum and... 31 After P-NMR monitoring of the samples, these original 9b 31 The P-NMR peak remained intact during the 7.5-hour study, and no new peaks were observed. 31 P-NMR peaks. These data indicate that 9b has excellent serum stability (t). 1 / 2 >7.5 hours).

[0142] This stability profile is consistent with the aryloxy diester phosphoramidate prodrugs of these monophosphonates reported by Mehellou et al. in 2020.

[0143] Example 3 Human Vγ9 / Vδ2 + T cell activation Overview: As described by Morita et al. 2007, peripheral blood mononuclear cells (PBMCs) were collected from healthy donors. Specifically, blood was obtained from consenting healthy donors (approved by the NRES Committee West Midlands-Solihull Ethical Board; REC reference 14 / WM / 1254) in the presence of a mixture of heparin and EDTA as anticoagulants (2 U / ml heparin, 1.5 mM EDTA). The blood was then separated into lymphocyte separation medium (lymphoprep) (stem cell technique) and peripheral blood mononuclear cells (PBMCs) 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 bovine serum.

[0144] To assess the activation of Vγ9 / Vδ2 T cells, PBMCs were seeded at a cell density of 500,000 cells per well in 96-well U-shaped plates that had been treated with tissue culture. Cells were incubated overnight at 37°C / 5% CO2 in the presence of zoledronic acid and HMBPP at concentrations of 10 pM to 100 µM, and with HMBP ProPAgens of the present invention (i.e., ProPAgens 9a-d and 14a-d, initially at concentrations of 10 pM to 100 µM, and then at 1 aM to 100 µM for compounds 9d and 14d in separate experiments), and stained by flow cytometry for the following markers: 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 LSL Fortessa X20 (BDBiosciences), and the data were analyzed using FlowJo v10 and GraphPad Prism v9 software.

[0145] Under steady-state conditions, peripheral blood γδ T cells lacked significant levels of surface CD69 or CD25, but T cell receptor (TCR) stimulation upregulated these two T cell activation markers within 72 hours. PAg-responsive Vγ9 / Vδ2 T cells were then distinguished by TCR Vγ9 and Vδ2 expression, and the upregulation of CD69 and CD25 was assessed.

[0146] Example 3-1: Effects of ProPAgens 9a-d and 14a-d on human Vγ9 / Vδ2 + T cell activation To demonstrate the activation of Vγ9 / Vδ2 T cells by ProPAgens 9a-d and 14a-d and subsequent in vitro lysis of cancer cells, peripheral blood mononuclear cells (PBMCs) containing Vγ9 / Vδ2 T cells derived from healthy donors were incubated with gradually increasing concentrations of phosphonamide HMBP ProPAgens 9a-d and 14a-d (up to 100 μM) as described above. See [link to relevant documentation]. Figure 7A , 7B And 7D.

[0147] Regarding the activation of Vγ9 / Vδ2 T cells by phosphonamides ProPAgens (9a-d and 14a-d), these were initially tested using concentrations ranging from 0.1 nM to 100 µM. Figure 7A and 7BThe results showed that these phosphonidamides, ProPAgens, exhibited varying levels of activation, ranging from extremely potent activation of EC24. 50 = 0.0000136 nM to 6.1 µM. In the fluorinated (9a-d) and non-fluorinated (14a-d) series, phosphonidamides ProPAgens with tert-butyl esters (9c and 14c) exhibited minimal potent activation of Vγ9 / Vδ2 T cells, respectively, EC 50 = 1.5 and 6.1 µM ( Figure 7A , 7B And 7D). This was followed by a methyl phosphonic diamine prodrug, of which 9a showed good potency, EC 100%. 50 = 191 nM, although we cannot obtain the exact potency level of 14a.

[0148] Of the two series of phosphonidamides ProPAgens 9a-d and 14a-d, those containing isopropyl ester or benzyl ester showed the most potent activation of Vγ9 / Vδ2 T cells in vitro. Phosphonidamides ProPAgens 9b and 14b showed good activation of Vγ9 / Vδ2 T cells, respectively, EC... 50 = 167 and 87 nM. However, among the eight ProPAgens studied in this work, phosphonidamide ProPAgens 9d and 14d showed the most potent activation of Vγ9 / Vδ2 T cells, and ProPAgen 9d was the most potent in activating Vγ9 / 1Vδ2 T cells, EC 167 and 87 nM. 50 = 13.6 fM ( Figure 7A , 7B And 7D).

[0149] To determine the accurate potency (EC) of two benzylphosphonic diamine prodrugs at 9 days and 14 days. 50 We conducted activation tests at concentrations ranging from 10 aM to 100 µM. Figure 7E ).

[0150] Notably, the extremely high Vγ9 / Vδ2 T cell activation potency of phosphorylated diamine ProPAgen 9d is comparable to that of its corresponding aryloxydiester phosphonamide derivatives, EC 50 = 9.15 fM. It is noteworthy that at the highest concentration studied (100 μM), phosphorylated diamine ProPAgen 9d and 14d showed less activation of Vγ9 / Vδ2 T cells than activation achieved using 10 μM. Figure 4A and 4BThis can be explained by a negative feedback mechanism induced by antigen overstimulation, which leads to TCR downregulation, subsequently resulting in decreased expression of CD25 activation markers. This has also been observed in aryloxydiester phosphonamide prodrugs of HMBP methylene and difluoromethylene monophosphonates.

[0151] Example 3-2 (Comparison): HMBPP and zoledronic acid on Vγ9 / Vδ2 + T cell activation In the positive control experiment, activation assays were performed using HMBPP and zoledronic acid (Zol) as described above. Both HMBPP and zoledronic acid (Zol) showed significant activation of Vγ9 / Vδ2 T cells. Specifically, EC... 50 = 181 nM and 18.5µM ( Figure 7C and 7D It is worth noting that, compared to the previously reported potency of these compounds (HMBPP EC50), 50 = 60-500 pM, and zoledronic acid is 0.003-0.5 µM. Compared with these, HMBPP and zoledronic acid have lower potency (see 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 tocompetitive antagonism, Bioorg. Med. Chem. Lett. 2003, 13, 1257-1260.

[0152] Compared to those observed in this study, such differences between previously reported HMBPP and zoledronic acid are most likely due to modest inter-individual variability in Vγ9 / Vδ2 T cell activity.

[0153] As from Figures 7A-7E The comparison clearly shows that, except for compounds 9a, 9c and 14c, the T-cell activation efficacy of HMBPP and zoledronic acid is not as good as that of ProPAgens according to the present invention.

[0154] Example 3-3: ProPAgens 4a-d and 9a-d deficiency activates human CD8+ T cells To demonstrate that ProPAgens of the present invention is a Vγ9 / Vδ2 T cell specific activator, CD8 derived from the donor is included. + αβ T cells were incubated with gradually increasing concentrations of ProPAgen at 9d and 14d. Figure 9 ).

[0155] Similar to peripheral blood γδ T cells, peripheral blood CD8 + T cells lacked significant levels of surface CD69 or CD25 under homeostatic conditions, but both of these T cell activation markers were upregulated within 72 hours upon stimulation by the T cell receptor (TCR). PAg-responsive CD8 T cells were then distinguished by TCR CD8 expression, and the upregulation of CD69 and CD25 was assessed.

[0156] like Figure 9 As shown, HMBP phosphonates PropAgens 9d and 14d, which are representative prodrugs of this type according to the present invention, even at a concentration of 1 µM (i.e., at a concentration of Vγ9 / Vδ2 T cell activation ECGs as determined for both ProPAgens 9d and 14d), 50 Even when incubated at concentrations approximately 100,000 times higher than the effective concentration, no activation of CD8+ T cells was observed.

[0157] Example 4 Cytotoxicity assays showed that the potent lysis of T24 bladder cancer cells by Vγ9 / Vδ2 T cells was mediated and enhanced by ProPAgens 9d and 14d. As further proof of the principle, and to demonstrate the superior Vγ9 / Vδ2 of the ProPAgens of the present invention shown above. + The activation efficacy of T cells does indeed translate into beneficial therapeutic effects. Studies investigated the specific lysis of cancer cells by in vitro expanded Vγ9 / 1Vδ2 T cells. (See [link to relevant documentation]). Figure 8 : To assess the killing levels of tumor cells treated with drugs and those not treated with drugs, a europium-based cytotoxicity assay (DELFIA, Perkin-Elmer) was performed, as described by Fisher et al. in 2014.

[0158] Human T24 bladder cancer cells were cultured in PBS containing the following substances at 37°C / 5% CO2 for 2 hours: no ProPAgen (i.e., untreated); containing 10 µM zoledronic acid (i.e., a clinically used small molecule drug); or containing HMBP phosphonidamide ProPAgens as specified in this invention (i.e., 10 nM ProPAgen 9d and 10 nM ProPAgen 14d). Cell death positive controls (target cells incubated with 10% v / v DELFIA lysis buffer) and a control treated only with culture medium (no drug) were also included. Cells were then washed three times in PBS at 600 xg for 5 minutes each time to remove any excess drug, and incubated for 20 minutes at 37°C in PBS containing BATDA labeling agent (1 μl / ml), as before.

[0159] During this period, in vitro expanded Vγ9 / Vδ2 T cells (i.e., Vγ9 / Vδ2 T cells expanded over a period of 14 days with 5 μM zoledronic acid and 100 U / ml IL-2) were thawed, counted, and resuspended to a cell concentration of 4 × 10⁶ cells / mL. After BATDA labeling, T24 cells were incubated at 4... o Wash three times in medium C and resuspend to a concentration of 5 × 10⁴ cells / mL.

[0160] 100 μl of T24 cells were then seeded into 96-well U-bottomed plates prepared for tissue culture and co-cultured with 100 μl / well of Vγ9 / Vδ2 T cells (i.e., at an effector:target ratio of 80:1), as previously described by Fisher et al. 2014. Specifically, drug-treated and untreated T24 cells were also seeded separately without Vγ9 / Vδ2 effectors, with 100 μl of culture medium added to each well instead. For the positive killing control, 10% v / v lysis buffer was added to the untreated T24 cells. The plate was centrifuged at 200 × g for 2 min to allow cell contact in the co-culture. Plates containing all samples were incubated at 37°C. o Incubate at 5% CO2 for 1 hour. After this incubation, centrifuge the plate again at 600 × g for 2 minutes, and transfer 25 μl of the supernatant to a flat-bottomed 96-well optical plate. Add 200 μl of europium solution to each well of the plate.

[0161] The killing level of T24 cells was then measured by time-resolved fluorescence using a Pherastar microplate reader (BMG Labtech). Specific lysis (T24 cell killing rate %) 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. Data are in... Figure 8 Data are presented as mean ± standard error (n = 7). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test on GraphPad Prism v9. **p < 0.0063.

[0162] Data showed that 10 nM phosphonamide ProPAgens 9d and 14d had significantly stronger sensitizing effects compared to 10 µM zoledronic acid.

[0163] like Figure 8 As shown, the sensitizing effect of 10 nM phosphonidamide ProPAgens 9d and 14d is significantly stronger than that of 10 µM zoledronic acid.

[0164] Example 5 Metabolic studies using carboxypeptidase Y assay The in vitro metabolism of phosphonyl diamine ProPAgens according to the present invention was studied using the carboxypeptidase Y assay.

[0165] Based on the metabolic study of phosphorodiamidate ProTides reported by McGuigan et al. in 2018, and not wanting to be bound by theory, it is proposed that the hypothetical metabolism of ProPAgens according to the present invention is initiated by an esterase (e.g., carboxypeptidase Y) that removes the ester moiety from the amino acid ester and releases the carboxylate group. Figure 10A Metabolite 15). This is followed by a spontaneous nucleophilic attack on the phosphorus center by one of the carboxylate groups, which triggers the departure of the second amino acid and the formation of an unstable five-membered ring (…). Figure 10A Metabolite 16). The next metabolic step involves a nucleophilic attack of water molecules on phosphorus or carbonyl groups to generate phosphonamides ( Figure 10A Metabolite 17). Finally, phosphonamide enzymes (e.g., Hint-1) cleave the PN bond in 17, leading to the release of unmasked monophosphonates (…). Figure 10A Metabolites 18 in the middle.

[0166] As previously reported by Mehellou et al. (2020), McGuigan et al. (2018), and McGuigan et al. (2014), a method for the assay of carboxypeptidase Y was performed. 5.0 mg of phosphonidyldiamine ProPAgen 9b was dissolved in 0.2 mL of acetone, and 0.4 mL of Trizma buffer (pH 7.4) was added, followed by 0.5 mg of carboxypeptidase Y in 0.2 mL of Trizma buffer (pH 7.4). The offline diamine ProPAgen 9b and recombinant carboxypeptidase Y were incubated at 37°C, and the assay was performed using... 31 The reaction was monitored by p-NMR for 12 hours. The recorded data were processed and analyzed using Bruker Topspin 2.1 software.

[0167] The results showed that, in the reaction buffer at t = 0, ProPAgen 9b exhibited the expected three [responses / effects]. 31 P NMR peaks (δP = 14.16, 14.67 and 15.14 ppm) (see...) Figure 10B Recorded at 0 hours of incubation time 31 (P NMR spectrum). Following the addition of carboxypeptidase Y and within 0.5 hours, three new [results / phases] were observed during the assay. 31 The P NMR peaks (δP = 6.82, 7.27, and 7.72 ppm) became significant, and these new peaks were most significant within 6.5 hours of this study, while the 9b peak was smaller. 31 PNMR peak (see) Figure 10B Recorded during an incubation period of 6.5 hours 31 (P NMR spectrum). New peaks appear. 31 P NMR displacement corresponds to Figure 10A The shifts of metabolite 17 shown are similar to those previously observed for this metabolite (δP = 6.50, 6.90, and 7.20 ppm).

Claims

1. A compound according to the general formula (I) including all tautomers thereof: (I) wherein R1represents an amino acid ester group according to the general formula (II): (I) wherein R3represents H, or a saturated or unsaturated and optionally substituted hydrocarbon chain; R4represents a saturated or unsaturated and optionally substituted hydrocarbon chain; and wherein both R1are identical; and R2represents optionally substituted C 2-20 alkyl, C 4-20 alkenyl or C 2-20 alcohol group; and each of X and Y independently represents H or halogen; or a salt thereof, and wherein R1is an amino acid ester group according to the general formula (II) derived from alanine (R3= -CH3), leucine (R3= -CH2CH(CH3)2), isoleucine (R3= -CH(CH3)CH2CH3) or methionine (R3= -CH2CH2SCH3), more preferably R1is an amino acid ester group according to the general formula (II) derived from alanine (R3= -CH3); and wherein R2is a radical according to formula (III) or formula (IV), wherein R5in formula (IV) is selected from OH, OR6, SH, SR6, NH2or NHR6, and wherein R6denotes C 1-4 alkyl: (III) (IV), preferably wherein R2is a group according to formula (IV).

2. The compound according to claim 1, wherein R2is a group according to formula (IV) and R5is OH.

3. The compound according to any one of the preceding claims, wherein at least one, and preferably both, of X and Y represent halogen.

4. The compound of claim 3, wherein, one or both halogen substituents are fluorine.

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

6. The compound according to any one of claims 1 to 5, wherein R1is an amino acid ester group according to the 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 R4is selected from the group consisting of methyl, isopropyl, tert-butyl and benzyl, preferably wherein R4is isopropyl or benzyl, most preferably wherein R4is benzyl.

8. The compound according to claim 1, selected from the group consisting of: ； ； ； ； ； ； ； ； ； ； ； and preferably the compound is selected from the group consisting of: ； ； ； and ， more preferably the compound is selected from the group consisting of: ; and ， most preferably the compound is 。 9. The compound according to claim 1, selected from the group consisting of: ； ； ; and ， preferably the compound is 。 10. The compound according to claim 1, wherein the compound is 。 11. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 10, wherein the pharmaceutical composition preferably comprises a pharmaceutically acceptable excipient or carrier.

12. The compound according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 for medical use.

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

14. The compound or pharmaceutical composition for use according to claim 13, wherein the immunotherapy involves the treatment of a proliferative disease, an infectious disease, an inflammatory disease and / or osteoporosis, and wherein the proliferative disease is preferably a cancer, and wherein the cancer is preferably selected from the group consisting of a hematological cancer, a bladder cancer, a prostate cancer, a lung cancer, a neck cancer, a skin cancer, a breast cancer and a mesothelioma.

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

16. An in vitro method of preparing a population of gd T cells for therapeutic use, the method comprising: (i) providing a population of gd T cells; (ii) culturing the population of gd T cells in the presence of a compound according to any one of claims 1 to 10.

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

18. A pharmaceutical composition comprising a population of gd T cells according to claim 17.

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

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

21. An expanded population of gd T cells or a pharmaceutical composition for use according to claim 20, wherein the immunotherapy involves the treatment of a proliferative disease, an infectious disease, an inflammatory disease and / or osteoporosis, wherein the proliferative disease is preferably a cancer, and wherein the cancer is preferably selected from the group consisting of a hematological cancer, a bladder cancer, a prostate cancer, a lung cancer, a neck cancer, a skin cancer, a breast cancer and a mesothelioma.

22. A method of synthesis of a compound as defined in any one of claims 1 to 10, comprising the steps of: (i) providing an alkyl phosphonate; (ii) converting the alkyl phosphonate to a phosphonic halide by removing the ester group and subsequently undergoing a halogenation reaction; (iii) subjecting the phosphonic halide to an esterification reaction with an amino acid ester hydrohalide to obtain an amino acid ester derivative; and (iv) subjecting the amino acid ester derivative of step (iii) to an olefin metathesis, preventing olefin isomerization.

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