Phosphoantigen prodrug compounds

Abstract

To provide alternative ProPAgens that retain potent activation of Vγ9 / Vδ2 T-cells while addressing the poor serum stability of HMBP ProPAgens active compounds.SOLUTION: The invention provides compounds of the general formula (I). The compounds and compositions comprising the compounds exhibit high serum stability and potent activation of the γδ T-cell immune response, and are suitable for use in immunotherapy. The invention also relates to methods of immunotherapy using the compounds of the general formula (I).SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] This invention provides a phosphoane that exhibits high serum stability and potent activation of the γδT cell immune response. This relates to zigen (PAg) prodrug compounds and pharmaceutical compositions. The compounds and compositions For use in immunotherapy, particularly in the treatment of infectious diseases, cancer, osteoporosis, and other proliferative disorders. Furthermore, to activate the γδ T cell immune response, the compounds or compositions of the present invention Immunotherapy, including the use of [specific substance / method], is also offered. [Background technology]

[0002] Currently, in adult peripheral blood, human γδT cells from birth to dominant subtypes, Vγ9 / Vδ2T cells are currently used to treat tuberculosis, leprosy, typhoid fever, malaria, and toxoplasmosis (non-patented). Established as an important subset of γδT cells involved in the fight against many diseases, such as those mentioned in Reference 1). Studies in primate models have also shown that Mycobacterium tuberculosis (non-patent document) They are involved in Vγ9 / Vδ2 T cells in immunity against (Reference 2). These cells also This demonstrates that it is possible to target and lyse various cancer cells in vitro (non- Patent Document 1). In addition, these observations suggest that the Vγ9 / Vδ2 subset is related to γδT cells. This was the primary focus in terms of therapeutic use (Non-Patent Document 3).

[0003] To date, numerous small molecule activators for Vγ9 / Vδ2 T cells have been reported. Among these are naturally occurring PAg(E)-4-hydroxy-3-methylbut-2- Enylpyrophosphate (HMBPP) and isopentenylpyrophosphate (IPP), and There are two synthetic molecules, risedronate and zoledrone (Figure 1), which are currently being developed. Used clinically to treat osteoporosis and several types of cancer (Non-patent documents 4-6) It is being used.

[0004] To date, the most potent Vγ9 / Vδ2 T cell activator reported is HMBPP, EC 50 = 0.00051 μM, which binds the type 1 transmembrane protein butyrophyllin 3A1. These T cells are activated by binding. The binding sites of these PAg molecules are transmembrane. There are conflicting opinions regarding whether a protein binds to its extracellular or intracellular domain. While there have been reports suggesting otherwise, the answer remains unclear. However, it is now understood that HMBPP is equivalent to butyrophylline 3A1. There is compelling evidence to support the concept that it binds to the intracellular B30.2 domain.

[0005] The activation of Vγ9 / Vδ2 T cells is promoted by the efficacy of HMBPP, Ally Oxytriester phosphoramide prodrug technology has recently been developing its drug-like properties. As a means to improve the properties, a monophosphate derivative of HMBPP, i.e., HM Applied to BP (Non-Patent Literature 6). In this prodrug approach, monophosphate The t group is masked by an aryl group and / or an amino acid ester (Figure 2), and these Both are enzymatically cleaved within the cell, releasing monophosphate or monophosphonate species. To release. Since such compounds are prodrugs of PAg, they are nucleotides. ProPAgens are used to distinguish them from ProTides, which are prodrugs. It is called [name]. [Prior art documents] [Non-patent literature]

[0006]

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[0007] These HMBP ProPAgens are used in Vγ9 / Vδ2 T cells (EC) 50 (= 0.45~11 nM) It showed strong activation, but mainly due to the cleavage of the -P-O bond in these active compounds, The serum stability of these was quite low (Non-Patent Literature 6). Therefore, the objective of the present invention has been previously reported. While addressing the reported poor serum stability of HMBP ProPAgens active compounds, Vγ9 / Vδ2 The goal is to design alternative ProPAgens that maintain potent T cell activation. [Means for solving the problem]

[0008] According to a first aspect of the present invention, a compound of general formula (I) including all of its tautomers or The salt is provided.

[0009] TIFF0007888651000001.tif20152 In the formula, R1 and R2 are each independently an amino acid ester group of general formula (II). This represents the aryloxy group of general formula (III), TIFF0007888651000002.tif30169 In the formula, R6 is H, or a saturated or unsaturated carbon that may be optionally substituted. R7 represents a hydrogen chain; R7 is a saturated or unsaturated, optionally substituted hydrocarbon chain. Represented by; R8 may be optionally substituted with C 5-25 Aryl or 5-25 member heterozygous Represents a reel base; R3 may be replaced with C as desired. 2-20 Alkyl alkyl group, C 4-20 Alkenyl group or C 2-20 Represents an alcohol group; each of R4 and R5 independently contains either a H or a halogen atom. represent.

[0010] In contrast to the conventional phosphate-containing pro-PAgen described above, the compound of general formula (I) is It contains a phosphonate group that is masked by a reel group and / or an amino acid ester group. This is enzymatically cleaved inside the cell to release active phosphonate compounds. Substituting a stable -OP bond with a -CC bond increases the stability of the active phosphonate compound. It has been shown to improve the condition, and this is a potent activator of Vγ9 / Vδ2 T cells. This was demonstrated. Furthermore, this activity was translated into potent lysis of bladder cancer cells in vitro. It was done. Compounds of general formula (I) have been shown to activate Vγ9 / Vδ2 T cells with high specificity. Therefore, they are ideal candidates for development as immunotherapeutic agents. In particular, general Compounds of formula (I) are used in the treatment of infections, proliferative disorders such as cancer, and / or osteoporosis. It is for use.

[0011] Infections suitable for treatment with Vγ9 / Vδ2 T cell activation immunotherapy include bacteria and viruses. And fungal infections. A particularly relevant infection is bacterial infection, especially Mycobacterium. It is a bacterial infection of the genus and / or species Salmonella enterica. Therefore, general formula (I) The compounds are particularly useful in the treatment of one or more of tuberculosis, leprosy, and typhoid fever.

[0012] Alternatively, the infection may be a parasitic infection. A particularly suitable parasitic infection is Plasma. It belongs to the genera *modium* and / or *Toxoplasma*. Therefore, generally Compounds of formula (I) are particularly useful in the treatment of one or both malaria and toxoplasmosis. It is for use.

[0013] Compounds of general formula (I) are effective against cancer, particularly bladder cancer, prostate cancer, lung cancer, cervical cancer, skin cancer, and breast cancer. Furthermore, it is particularly useful in the treatment of mesothelioma. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows the chemical structure of the reported small molecule Vγ9 / Vδ2 T cell activator. [Figure 2] Figure 2 shows the application of aryloxytriester phosphoramide prodrug technology to monophosphate derivatives of HMBPP (HMBP). [Figure 3] Figure 3 shows the pKA values ​​for phosphate groups and different phosphonic acid groups. [Figure 4] Figure 4A shows the synthesis of aryloxyphosphalamide pro PAgen of HMBP methylphosphonate (4a-d), and Figure 4B shows the synthesis of HMBP difluoromethylphosphonate (9a-d). [Figure 5] Figure 5 shows the spectrum obtained by monitoring the stability (37°C, 12 hours) of the HMBP phosphonate ProPAgen 4d in human serum using 31P NMR. [Figure 6] Figure 6 shows the spectrum of the stability of HMBP phosphonate pro PAgen 9a in human serum at 37°C for 12 hours, monitored by 31P NMR. [Figure 7] Figure 7 shows the activation of human Vγ9 / Vδ2+ T cells by the HMBP phosphonates ProPAgens 4d and 9d. Human peripheral blood mononuclear cells (PBMCs) were incubated with the indicated concentrations of ProPAgens 4d (left) and 9d (right) for 18 hours. [Figure 8] Figure 8 shows FACS data demonstrating dose-dependent activation of human Vγ9 / Vδ2+ T cells by the HMBP phosphonate ProPAgen 4d. Results were obtained by incubating human peripheral blood mononuclear cells (PBMCs) with the indicated concentrations of ProPAgens 4d for 18 hours (Figure 8A) or 20 hours (Figures 8B and 8C). [Figure 9]Figure 9 shows FACS data demonstrating dose-dependent activation of human Vγ9 / Vδ2+ T cells by the HMBP phosphonate ProPAgen 9d. Results were obtained by incubating human peripheral blood mononuclear cells (PBMCs) with the indicated concentrations of ProPAgens 9d for 18 hours (Figure 9A) or 20 hours (Figures 9B and 9C). [Figure 10] Figure 10 shows the dose-dependent activation of human Vγ9 / Vδ2+ T cells by HMBPP. Human peripheral blood mononuclear cells (PBMCs) were incubated with the indicated concentrations of HMBPP for 20 hours. Data were collected and analyzed separately from two different donors: results from donor (1) shown in Figure 10A, and results from donor (2) shown in Figure 10B. [Figure 11] Figure 11 shows FACS data demonstrating the lack of human CD8+ T cell activation by the HMBP phosphonate ProPAgen 4d. Data were collected and analyzed separately from two different donors: results from donor (1) shown in Figure 11A, and results from donor (2) shown in Figure 11B. [Figure 12] Figure 12 shows FACS data demonstrating the lack of human CD8+ T cell activation by the HMBP phosphonate ProPAgen 9d. Data were collected and analyzed separately from two different donors: results from donor (1) shown in Figure 12A, and results from donor (2) shown in Figure 12B. [Figure 13] Figure 13 shows the results of a cytotoxic assay demonstrating potent lysis of bladder cancer cells after incubation with the HMBP phosphonate ProPAgen 4d. [Modes for carrying out the invention]

[0015] As used herein, the term "halo" refers to fluoro, chloro, bromo, or iodine More appropriately, it means fluoro or chloro, and most appropriately, fluoro.

[0016] The term "C" used herein 2-20 The term "alkyl" refers to a molecule containing 2 to 20 carbon atoms. Refers to a straight-chain or branched-chain saturated hydrocarbon chain. Examples include ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-hexyl, n-octyl, and n-decyl.

[0017] As used herein, the term "C 4-20 alkenyl" refers to a straight-chain or branched-chain unsaturated hydrocarbon chain containing 4 to 20 carbon atoms. Examples include butenyl, pentenyl, etc.

[0018] As used herein, the term "C 2-20 alcohol" contains 2 to 20 carbon atoms and refers to a straight-chain or branched-chain, saturated or unsaturated hydrocarbon chain containing one or more hydroxyl (OH) functional groups.

[0019] As used herein, the term "saturated or unsaturated hydrocarbon chain" refers to an aliphatic or aromatic hydrocarbon group that may or may not contain one or more double bonds or triple bonds within the chain. Thus, this term encompasses alkyl, alkenyl, alkynyl, or aryl groups.

[0020] As used herein, the term "C 5-25 aryl" contains 5 to 25 carbon atoms and refers to any hydrocarbon group containing one or more carbocyclic aromatic rings. More preferably, the aryl group is C C 6-14 and preferably a C 6-10 aryl group.

[0021] As used herein, the term "heteroaryl" refers to any hydrocarbon containing one or more aromatic rings containing one or more heteroatoms (e.g., N, O, or S) as part of the ring. It refers to a group. Particularly appropriate examples of heteroaryl groups are pyridine, furan, thiophene, and i It is an ndole group. A 5-25 member heteroaryl group is a ring-forming atom (carbon and heteroaryl group). This refers to a base where the total number of sub-units is between 5 and 25.

[0022] Other alkyl groups, alkenyl groups, aryl groups and / or alcohol groups are defined. However, they have different numbers of carbon atoms. For example, C 1-4 Alkyl groups consist of 1 to 4 carbon atoms. It contains elementary atoms.

[0023] Hydrocarbon chains and / or aryl, heteroaryl, alkyl, alkenyl or a The ethanol group may optionally be a functional group containing one or more heteroatoms (e.g., O, S, or N). They may be substituted with nitro, nitrone, halo. Suitable examples of heteroatom-containing groups include nitro, nitrone, and halo. , amino, amide, cyano, carboxyl, sulfonyl, hydroxyl, alkoxy, ke Examples include tons, aldehydes, thiols, thioethers, and non-aromatic heterocyclic groups, The hydrocarbon chain may contain saturated or unsaturated cyclic rings, and may also contain It does not have to be. When used herein, hydrocarbon chains, alkyls, alkenyls Any number of carbon atoms in an aryl or alcohol radical, any number of carbon atoms present in the substituent Contains atoms. In preferred embodiments, hydrocarbon chains and / or aryl, heteroaryl compounds. Alkyl, alkenyl, or alcohol groups are thiols, thioethers, alkoxy and One or more hete selected from aminos (may be primary or secondary amino groups) It can be optionally substituted with an atom-containing functional group.

[0024] Salts of compounds of general formula (I) are appropriately pharmaceutically or veterinarily acceptable salts. R 1 ~R 8 Depending on the type, these are sodium salts, potassium salts, calcium salts, and aluminum salts. Nium salts, zinc salts, magnesium salts and other metal salts, as well as choline salts and diethanol Amine salts, ethanolamine salts, ethyldiamine salts, meglumine salts, and Paulek This is summarized in uhn et al. (2007) J.Med.Chem. 50: 6665-6672. Other well-known basic addition salts and / or basic addition salts known to those skilled in the art Alternatively, if the compound of general formula (I) contains an amino group, it may be quaternized. chlorophosphates, hydroxides, sulfates, nitrates, phosphates, formates, acetates, trifluoroacetic acid Salt, fumarate, citrate, tartrate, oxalate, succinate, mandelate, meta Forming salts having counterions such as toluenesulfonate and p-toluenesulfonate. It is possible.

[0025] In a preferred subset of compounds according to general formula (I), the phosphonate group is an amino These compounds are masked by acid esters and aryl groups. Such compounds have the general formula (IV) The formula is as follows, where R3, R4, R5, R6, R7 and R8 are defined above. is: TIFF0007888651000003.tif35163 In some compounds of general formula (I) or general formula (IV), R3 is C 4-16 dea Preferably C 4-8 It is an alkyl, alkenyl, or alcohol group. More preferably In this case, R3 is an alcohol group as defined above.

[0026] In particularly preferred compounds, R3 is determined by formula (V), formula (VI), or formula (VII). It is a group, and in the formula, R9 is OH, OR 10 SH, SR 10 NH2 or NHR 10 Select from And preferably it is OH, R 10 C 1-4 Represents alkyl: TIFF0007888651000004.tif35170 These R3 substituents are naturally occurring PAgsIPP (formula (V)) and HMBPP (formula (VI)) ) and the IPP isomer dimethylallyl diphosphate DMAPP (found in formula (VII) It is similar to something else.

[0027] In a particularly preferred embodiment, R3 is the base of formula (VI).

[0028] As described above, compounds of general formula (I) or general formula (IV) are -C(R4)(R5) -Contains a masked phosphonate group including a Pbond, which is a conventional ProPAge It has been shown to be more stable than ns-O-Pbond. In a preferred embodiment, At least one, more preferably both, of R3 and R5 represents a halo.

[0029] It has been shown that the change from an -OP bond to a -CH2-P bond improves the stability of the active substance. However, the pKa value of the second deprotonation of the phosphonate group (pKa=7.49) This is significantly different from the phosphate group (pKa=6.31) (Figure 3). As a result, physiological Complete ionization of the active compound under pH (<7.4), and therefore against the target protein. This affects the binding affinity, which positively loads the intracellular domain of butyrophyllin 3A1. Complete ionization of the phosphate group is required to bind to the electrolyzed pocket (arginine-rich). Let's assume that.

[0030] However, to provide a difluoromethylphosphonate (-CF2-P-) bond As a result, the active compound exhibits excellent stability in physiological environments and possesses the same properties as the natural phosphate compound itself. The pKa (6.7) of the second deprotonation, which is very close to that of the first deprotonation, is coupled. It was found that (Figure 3). Therefore, the mono and (IV) of compounds of general formula (I) or (IV) The bi / or dihalomethylphosphonate derivatives exhibit excellent stability in Vγ9 / Vδ2 T cells. Combine with a powerful activation.

[0031] Compounds of general formula (I) or (IV) have a monophosphonate group with an aryl group and / or These are prodrugs masked by amino acid esters, and both of them It is enzymatically cleaved within the cell, releasing an active monophosphonate species.

[0032] Suitable amino acid ester masking groups include a wide variety of amino acid side chain (R6) groups. Typically, R6 is a side chain of a protein-producing amino acid, preferably a nonpolar side chain. A particularly preferred R6 group is C 1-4 It contains an alkyl chain. Examples of such R6 groups include -CH3 (alanine), -CH(CH3)2 (valine), -CH2CH(CH3)2 (royl Syn), -CH(CH3)CH2CH3 (isoroutine) and -CH2CH2SCH3 (Mesonine) is present. Compounds in which R6 represents methyl (CH3) are particularly preferred.

[0033] R7 is a hydrocarbon chain that is saturated or unsaturated and may be optionally substituted, and is a lipid. It may be aliphatic or aromatic. Preferably, R7 is C 1-6 alkyl or C 6-14 It is an aryl group. In a particularly preferred embodiment, R7 is C 6-10 It is an aryl group Most preferably, it is a benzyl group. General formula (I) or (I) including benzyl esters. The compound in V) shows a higher degradation rate and improved performance compared to aliphatic ester compounds. It has lipophilic properties (and therefore improved cell uptake).

[0034] Particularly suitable compounds of general formula (IV) include the following: TIFF0007888651000005.tif203166 TIFF0007888651000006.tif100168 Compounds of the first aspect of the present invention are generally understood to be administered as part of a pharmaceutical composition. It will be done. Therefore, according to the second aspect of the present invention, the transformation of the first aspect of the present invention A pharmaceutical product comprising a compound and pharmaceutically acceptable excipients or carriers is provided.

[0035] Suitable pharmaceutical excipients are well known to those skilled in the art. Pharmaceutical compositions can be transported via any suitable route, for example, Oral, rectal, nasal, bronchial (inhalation), topical (including eye drops, oral and sublingual), vaginal or Formulated for parenteral administration (including subcutaneous, intramuscular, intravenous, and intradermal). It can be prepared by any method well known in the field of pharmacy.

[0036] The composition is prepared by mixing the compound of the first aspect of the present invention with a carrier. This is possible. Generally, the formulation contains the compound in a liquid carrier or a finely divided solid carrier. It is then brought together uniformly and tightly with both of them, and the product is formed as needed. It is prepared by [method].

[0037] The oral formulations in this invention are capsules each containing a predetermined amount of the compound. , as separate units such as small packets or tablets; as powder or granules; as aqueous liquid or non-aqueous liquid. As a solution or suspension of the compound in a liquid; or as an oil-in-water liquid emulsion or oil It may be supplied as a neutral-water liquid emulsion; or as a bolus, etc.

[0038] For compositions intended for oral administration (e.g., tablets and capsules), the term "acceptable" is used. The "carrier" includes vehicles such as common excipients, for example, binders, for example, syrup, etc. Cassia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), Methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydro Xypropylmethylcellulose, sucrose, and starch; fillers and carriers, for example Corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kao Phosphorus, mannitol, dicalcium phosphate, sodium chloride, and alginic acid; and Lubricants, such as magnesium stearate, sodium stearate, and other metals Thearate, glycerol stearic acid, stearic acid, silicone fluid, talc Contains cinnamon oil and colloidal silica. Peppermint, wintergreen oil, cherry - Flavoring agents such as flavorings can also be used. This makes it easier to identify the dosage form. In some cases, it is desirable to add a coloring agent. Tablets are also well known in the field. It can be coated by a method.

[0039] Tablets are prepared by compression or molding, with one or more optional auxiliary components. It is possible to compress tablets using a suitable machine to transform the compound into a free-flowing form such as a powder or granules. In this state, it may be mixed with binders, lubricants, inert diluents, preservatives, surfactants, or dispersants. It can be prepared by combining and compressing. The molded tablets are moistened with an inert liquid diluent. It can be manufactured by molding a mixture of the processed powdered compounds using appropriate machinery. The tablets may be coated or notched as needed, and active It may be formulated to provide sustained or controlled release of the drug.

[0040] Other formulations suitable for oral administration include an activator in a flavored base, usually sucrose and Lozenges containing acacia or tragacanth; activators and inert bases, such as gelatin Pastels containing glycerin, sucrose, and acacia; and appropriate surfactants. This product contains mouthwash in a liquid carrier.

[0041] Oral preparations are generally sterile.

[0042] For topical application to the skin, the composition may be in the form of a cream, ointment, jelly, solution, or suspension. It can be prepared in various ways. For example, cream or ointment formulations that can be used in drugs are available in the UK. Conventional manufacturing methods well known in this field, as described in standard pharmaceutical textbooks such as pharmacopoeias. It is a drug.

[0043] In a preferred embodiment of this aspect of the present invention, the composition is formulated for oral delivery. ru.

[0044] The exact amount of a composition defined herein that is therapeutically effective, and such compounds The best route of administration is easily determined by those skilled in the art. Such amounts are, of course, Hmm, including the specific condition being treated, the severity of the condition, age, physical condition, size and weight, etc. Each patient's parameters, duration of treatment, nature of concurrent treatments (if any), and specific administration cycles. It depends on the road, and similar factors within the knowledge and expertise of healthcare professionals. These factors are This is well known to those skilled in the art and can be addressed through routine experiments. Generally, the individual components... Or the maximum dose of those combinations, that is, the safest dose according to sound medical judgment. It is preferable to use it. However, the patient may not use it for medical, psychological, or substantial reasons. For any other reason, a lower dose or acceptable dose may be claimed, as a person skilled in the art would know. That's how it's understood.

[0045] The dose of the compound or composition of the present invention administered to the patient is determined according to different parameters. It may be selected according to the mode of administration used and the subject's condition. Other factors may include desired This includes the duration of treatment. If the patient's response is insufficient with the initial dose applied, Higher dose (or effectively higher dose via a different, more localized delivery route) It may be used to the extent that the patient's tolerance allows.

[0046] As described above, the compounds of the first aspect of the present invention are highly stable in Vγ9 / Vδ2 T cells. It represents a powerful activator prodrug. Therefore, according to the third aspect of the present invention A compound of the first embodiment or a pharmaceutical composition of the second embodiment is provided for use in immunotherapy. In particular, as already shown above, the compound or composition may cause infection, proliferative disease (e.g. For example, it is intended for use in the treatment of cancer and / or osteoporosis.

[0047] According to a fourth aspect, the present invention relates to a method of immunotherapy, the method of which γδT cells To activate the cellular immune response, a compound according to the first embodiment or a pharmaceutical compound according to the second embodiment This includes administering an effective dose of the substance.

[0048] In a preferred method, the compound or pharmaceutical composition of the present invention is obtained by any suitable means. It is then administered to patients who require such treatment. Activation of the γδ T cell immune response occurs in the body, leading to an immune response.

[0049] Alternatively, the compound or composition may be produced in a sample obtained from an individual requiring treatment. The sample is administered ex vivo, and the proliferation of γδT cells occurs before the sample is returned to the body. An ex vivo method is provided which may include inducing the following: For example, the The sample contains a sample of peripheral blood mononuclear cells (PBMCs) derived from an individual's blood sample. (but not limited to this), where the administration of the compound or composition is to the body of the individual. Before returning to the internal region, proliferation and activation of γδT cells are brought about.

[0050] Immunotherapy methods may be employed to treat infections, proliferative disorders, or osteoporosis. In particular, this method is used for the treatment of cancer, especially bladder cancer, and / or the following infectious diseases: tuberculosis, leprosy, and cystitis. It can be used to treat ointment, malaria, and toxoplasmosis.

[0051] Furthermore, for the treatment of infection, cancer or other proliferative disorders or osteoporosis in the subject Use of compounds according to the first phase or pharmaceutical compositions according to the second phase in the preparation of pharmaceuticals. It will be provided.

[0052] In a preferred embodiment of this aspect of the present invention, the patient is a mammal. Ideally, The aforementioned mammal is a human.

[0053] Throughout this description and claims, the terms “including” and “including” In terms of variations in terminology, for example, "includes" and "includes" can mean "includes but not limited to." This does not exclude other parts, additives, ingredients, integers or steps. The description and special features of this specification Throughout the scope of the request, the singular form includes the plural form unless the situation requires otherwise. In particular, when the indefinite article is used, this specification does not require anything else in that situation. Please understand that this is intended to refer to both singular and plural forms.

[0054] All references cited herein, including any patents or patent applications, are provided by reference. This is incorporated herein by reference. No reference is permitted to constitute prior art. Furthermore, none of the prior art constitutes part of the general knowledge in the relevant technical field. This does not constitute acceptance of that.

[0055] Preferred features of each aspect of the present invention are as described in relation to any of the other aspects. could be.

[0056] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention Any new features of the features disclosed herein (including the attached claims and drawings) This extends to standard or any novel combination. Therefore, in certain aspects of the present invention, Features, integers, properties, compounds, or chemical parts described in relation to the application form or example are as described in this document. Applicable unless otherwise specified in the details. It should be understood.

[0057] Furthermore, unless otherwise specified, any features disclosed herein are the same or similar in nature. It may be replaced by alternative features that fulfill the objective. [Examples]

[0058] The present invention is described with reference to the following embodiments and drawings, for illustrative purposes only. : Figure 1: Chemical structure of reported small molecule Vγ9 / Vδ2 T cell activator: Naturally occurring PAgs(E)-4-hydroxy-3-methylbut-2-enylpyrroline Acid (HMBPP) and isopentenyl pyrophosphate (IPP); synthetic molecule risedronate and zoledronate.

[0059] Figure 2: HMBPP(H)aryloxytriester phosphoramide prodrug technology Application of MBP to monophosphate derivatives: The monophosphate group is masked by the aryl group and the amino acid ester, and these Both are enzymatically cleaved within the cell, releasing active monophosphate species. The instability is This was observed due to the cleavage of the -P-O bond in these compounds (shading). Figure 3: pKA values ​​of phosphate groups and different phosphonic acid groups.

[0060] Figure 4: (A) Aryloxyphosphalamide of HMBP methylphosphonate (4a-d) (B) HMBP difluoromethylphosphonate (9a-d) Synthesis: Reagents and conditions: (i) TMSBr, DCM, rt, 2 hours, then (COCl)2, D MF catalog, DCM, rt, 18 hours; ii) a. Phenol, Et3N, DCM, -7 8°C for 30 minutes, then rt for 3 hours; b. Substitute L-alanine ester hydrochloride, Et3N , DCM, rt, 12 hours, yield: 38-61%; (ii) 2-methyl-2-propeno Lu, 1,4-benzoquinone, Hoveida-Grubbs catalyst 2 production, DCM, rt, yield: 57 ~64%; (iv) Diethyl (bromodifluoromethyl)phosphonate, DMF, zinc powder, rt, N2, 3 hours, then CuBr, allyl bromide, rt, 40 hours; (v)(i)TMSBr , DCM, rt, 2 hours, then (COCl)2, DMF cat, DCM, rt, 18 hours; ii) a. Phenol, Et3N, DCM, -78°C, 30 min, rt, 3 hours; b. Substituted L - Alanine ester hydrochloride, Et3N, DCM, rt, 12-hour yield: 24-46%; (i ii) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda-Gru BBS catalyst, second generation, DCM, rt, yield: 58-69%. Figure 5: Stability of HMBP phosphonate ProPAgen 4d in human serum (37°C, 1 2 hours 31 Monitored by P NMR: Prodrug 4d (5.0 mg) mixed with DMSO-d6 (0.10 mL) and D2O (0 Dissolved in 0.15 mL. All 31 The 3P NMR spectrum was recorded at 37°C. First, DM Prodrug 4d(5.5) in SO-d6 (0.10 mL) and D2O (0.15 mL). 0mg) 31 A 1P NMR scan was recorded (shown as compound 4d alone). Following this, Thawed human serum (0.30 mL) was added to an NMR tube, and the spectrum was measured immediately. The spectra were recorded at equal time intervals 30 minutes after addition, and then over a 12-hour period. .

[0061] Figure 6: Stability of HMBP phosphonate pro PAgen 9a in human serum at 37°C for 12 hours Sex, 31 Monitored by P NMR: This is the same as compound 4d above. Prodrug 9a 31 P NMR shows the bond between fluorine and phosphorus. This shows six phosphorus peaks. In fact, these are the eight peaks that would be predicted. Then, zoom in on these peaks to create the eight predicted phosphorus peaks for such compounds. Then, two extra peaks can be seen.

[0062] Figure 7. Human Vγ9 / Vδ ionization by HMBP phosphonate ProPAgens 4d and 9d. 2 + T cell activation: Human peripheral blood mononuclear cells (PBMCs) treated with ProPAgens 4d at the indicated concentrations (left). And 9d (right) were incubated for 18 hours. Then Vγ9 / Vδ2 + T cells As a readout for activation, TCR Vγ9 / Vδ2 + T cells, cell surface marker, CD6 We evaluated the upregulation of both ProPAgens 9 and CD25. EC 50 It is approximately 8 pM (picomoles).

[0063] Figure 8. Human Vγ9 / Vδ2 + T cells are stimulated by the HMBP phosphonate ProPAgen 4d FACS data showing dose-dependent activation: Human peripheral blood mononuclear cells (PBMCs) were treated with the indicated concentration of ProPAgens 4d. Incubated for 18 hours (Figure 8A) or 20 hours (Figures 8B and 8C). 20 hours During incubation, data was collected and analyzed separately from two different donors: Results for donor (1) shown in Figure 8B, and results for donor (2) shown in Figure 8C. Next, Vγ9 / Vδ2 + As a readout for T cell activation, TCR Vγ9 / Vδ2 + T cells, cell surface We evaluated the upregulation of the CD69 and CD25. The quantification is shown in Figure 7.

[0064] Figure 9. Human Vγ9 / Vδ2 + T cells are stimulated by the HMBP phosphonate ProPAgen 9d FACS data showing dose-dependent activation: Human peripheral blood mononuclear cells (PBMCs) were treated with the indicated concentration of ProPAgens 9d. Incubated for 18 hours (Figure 9A) or 20 hours (Figures 9B and 9C). 20 hours During incubation, data was collected and analyzed separately from two different donors: Results for donor (1) shown in Figure 9B, and results for donor (2) shown in Figure 9C. Next, Vγ9 / Vδ2 + As a readout for T cell activation, TCR Vγ9 / Vδ2 + T cells, cell surface We evaluated the upregulation of the CD69 and CD25. The quantification is shown in Figure 7.

[0065] Figure 10. Human Vγ9 / Vδ2 by HMBPP + The study demonstrated dose-dependent activation of T cells. Peripheral blood mononuclear cells (PBMCs) were incubated with the indicated concentration of HMBPP for 20 hours. te: Data was collected and analyzed separately from two different donors: the donors shown in Figure 10A (1 The result for donor (2) is shown in Figure 10B. Next, Vγ9 / Vδ2 + T cell activity As a readout for the chemical reaction, TCR Vγ9 / Vδ2 + T cells, cell surface markers, CD69 and We evaluated the CD25's upregulation.

[0066] Figure 11. Activation of human CD8+ T cells by HMBP phosphonate ProPAgen 4d. FACS data showing a lack: Human PBMCs were incubated with the indicated concentration of ProPAgen 4d for 20 hours. Next, TCR CD8 + T cells, CD8 + As a way of reading out T cell activation, The upregulation of surface markers, CD69 and CD25, was evaluated. Data was collected and analyzed separately from two different donors: Donor (1) shown in Figure 11A The result for donor (2) is shown in Figure 11B.

[0067] Figure 12. Activation of human CD8+ T cells by HMBP phosphonate ProPAgen 9d. FACS data showing a lack: Human PBMCs were incubated with the indicated concentration of ProPAgen 9d for 20 hours. Next, TCR CD8 + T cells, CD8 + As a way of reading out T cell activation, The upregulation of surface markers, CD69 and CD25, was evaluated. Data was collected and analyzed separately from two different donors: Donor (1) shown in Figure 12A The result for donor (2) is shown in Figure 12B.

[0068] Figure 13. Bladder cancer after incubation with HMBP phosphonate ProPAgen 4d A cytotoxic assay showing strong cell lysis. ProPAgen 4d is a specific lysis of T24 bladder cancer cells by Vγ9 / Vδ2 T cells. It mediates the transmission of the human T24 bladder cancer cell line (target) to 10 μM zoledronate, 100 Incubate for 4 hours with 100 pM HMBPP or 100 pM ProPAgen 4d After fermentation, the cells were washed five times in culture medium and then mixed with pre-grown Vγ9Vδ2 T cells in a 10:1 ratio. Co-cultured for 18 hours at the target ratio of the genotype.

[0069] Materials and methods: All reagents and solvents are general-purpose or analytical grade, and are manufactured by Sigma-Aldrich Ltd., Fis. I purchased it from Her Scientific, Fluorochem, or Acros. 31 P, 1 H call 13 ¹³C NMR data, Blue Caravan operating at 202, 500 and 125 MHz. The data was recorded using a DPX500 spectrometer. Chemical shifts (δ) are shown in ppm, and J values ​​are shown in Hz. The following abbreviations were used when reporting spectral data: s (singlet), d (double). doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets) , and m (multitative). All reactions were carried out under a nitrogen atmosphere and on a pre-coated silica plate. Analysis on Kiersel gel 60 F254, BDH by thin-layer chromatography (TLC) The compound was monitored by irradiation with ultraviolet light (254 nanometers), or by K Visualization was performed using MnOOOA staining followed by heating. Flash Column Lammagraphy was performed using silica gel 60 (230-400 mesh) (Merck). The mass spectrum (HRMS) was obtained by the School of Chemistry at Cardiff University. It was decided as a service.

[0070] Example 1: HMBP methylphosphonate aryloxyphosphalamide ProPA Gens synthesis Compounds of general formula (VI) where R4 and R5 each represent H, are processed by Grubbs Olefin Me It was prepared by a synthesis method using tathesis (Non-Patent Document 7). A schematic summary is shown in Figure 4(A). The synthesis of ProPAgens4A~d is carried out using commercially available diethyl 3-butenylphosphonate. The ethoxy group is removed by treating (1) with bromotrimethylsilane (TMSBr) at room temperature. This was achieved by (Non-Patent Document 8). Subsequently, in the presence of a catalytic amount of DMF Next, the chlorination reaction was carried out using oxalyl chloride. Then, product 2 of this reaction was treated with triethyl In the presence of luamine, then treated with phenol with a suitable amino acid ester to obtain a moderate yield. Phosphonone amides 3a-d were obtained at concentrations of 38-61%. Subsequently, these compounds were... Using the Hoveyda-Grubbs second-generation catalyst in the presence of 1,4-benzoquinone, 2 It underwent Grubbs olefin metathesis with methyl-2-propenol. Therefore, ProPAgens 4a-d were obtained in good yield (57-64%) (non-patent document) Reference 9).

[0071] L-alanine is the amino acid selected in the synthesis of ProPAgens 4a-d. We used this because it historically showed optimal biological activity, while pheno The motif is the development of two FDA-approved drugs, sofosbuvir and tenofovir alafenamide. The aryloxy masking group was selected because it was successfully used in the study. Four different ester motifs in ProPAgens-4a:methyl (Me); 4b: Isopropyl (iPr); 4c: tert-butyl (tBu); and 4d: benzyl (Bn Although selected in the synthesis of ), these range from the lowest activity (tBu) to the highest activity (Bn). This is because it exhibits a variety of biological activities that change up to that point (Non-Patent Literature 6).

[0072] Further details of the above synthesis can be found in the Appendix.

[0073] Example 2: Aryloxyphosphalamide of HMBP difluoromethylphosphonate Propane synthesis Compounds of the general formula (VI) where R4 and R5 each represent F, as well as Grubbs olefins. Metathesis 7 It was prepared using a synthesis method employing [the specified material]. A schematic summary is shown in Figure 4(B). The synthesis of ProPAgens 9a-d was initially carried out according to previously reported methods, using zinc and copper bromide in DMF. In the presence of [unclear], commercially available diethyl (bromodifluoromethyl)phosphonate (5) is bromide [unclear]. This was achieved by reacting it with (Non-Patent Document 10). The resulting compound 6 was then salted Compounds 3a-d are ionized and then treated with phenol and appropriate amino acid esters. As described in the synthesis instructions, phospholamidates 8a-d were synthesized in good yield (24-46%). These were obtained by [method]. Subsequently, these phosphoamidates were [method] with 1,4-benzoquinone and Hovey Treated with 2-methyl-2-propenol in the presence of a second-generation da-Grubbs catalyst. Non-patent document 9). Final ProPAgens 9a-d were produced in good yield (58-69%). L-alanine has historically shown optimal biological activity, so these prodrugs It is used as an amino acid selected in the synthesis of, while the phenol motif has two Successfully discovered FDA-approved drugs, sofosbuvir and tenofovir alafenamide. It was selected because it was used.

[0074] Regarding ProPAgens 4a-d, in the synthesis of ProPAgens 9a-d L-alanine is used as the amino acid selected, and the aryl oxy masking group... Then the phenol motif was selected. Again, four different ester motifs were selected, ProPA In the synthesis of gens-9a, the following were selected: methyl (Me); 9b: isopropyl (iPr );9c: tert-butyl (tBu); and 4d: benzyl (Bn).

[0075] Further details of the above synthesis can be found in the Appendix.

[0076] Example 3: HMBP methylphosphonate and HMBP difluoromethylphosphonate Stability testing Instability previously observed in stability tests of HMBP phosphate propagence (Non-Patent Literature 6) The primary objective was to address the issue, and therefore the synthesis of novel compounds in Examples 1 and 2 was It was implemented once it was completed.

[0077] ProPAgen 4d was performed using human serum at 37°C for 12 hours, and the previously reported results were obtained. As such, 31 The sample was monitored by P-NMR. As shown in Figure 5, ProPAg en 4d is a prodrug that has two dias originating from a chiral phosphate center typical of these prodrugs. Corresponding to teleosomers 31 On P-NMR, dP = 33.60 and 34.05 ppm It had two singlet cells. Human serum was added to the sample. 31 After monitoring with P-NMR, No new phosphorus peaks were detected during the test period (12 hours), so degradation was not observed. I couldn't do it.

[0078] The same stability profile applies to difluoromethylphosphonate ProPAgen 9a. A field was observed (Figure 6). In summary, these data were previously observed by the authors. . Disclose Compared to HMBP phosphate proPAgen (Non-Patent Literature 6), these prodrugs have superior This indicates stability.

[0079] Example 4: Human Vγ9 / Vδ2 by ProPAgens 4a-d and 9a-d + T Cell activation ProPAgen in Examples 1 and 2 maintained potent activation of Vγ9 / Vδ2 T cells. To demonstrate this, peripheral cells containing Vγ9 / Vδ2T cells derived from a healthy donor were used. Blood mononuclear cells (PBMCs) were introduced with gradually increasing concentrations of ProPAgen 4d and 9d. The cells were incubated (Figures 7, 8, and 9).

[0080] Peripheral blood γδ T cells exhibit considerable surface CD69 or CD2 under steady-state conditions. Although it lacks cell 5, both T cells are activated within 72 hours by T cell receptor (TCR) stimulation. The marker is upregulated. Next, PAg-responsive Vγ9 / Vδ2 T cells are... Identify by TCR Vγ9 and Vδ2 expression, and upregulate CD69 and C I evaluated the D25.

[0081] As shown in Figures 7, 8, and 9, HMBP phosphonate pro PAgens4d 9d is a representative of these classes of prodrugs, HMBP phosphate pro PA The potent Vγ9 / Vδ2 T-filament, far superior to what has been reported for gensOOB. It showed cell activation.

[0082] Example 5 (Comparison): Human Vγ9 / Vδ2 ionization with HMBPP + T cell activation The enhanced Vγ9 / Vδ2 T cell activation efficacy of ProPAgen in Examples 1 and 2 To demonstrate this, peripheral blood mononuclear cells containing Vγ9 / Vδ2 T cells derived from a healthy donor were used. For comparison purposes, the PBMCs were incubated with increasing concentrations of HMBPP. (Figure 10)

[0083] As is clear from the comparison between Figures 8 and 9 and Figure 10, the T cell activating ability of HMBPP is Its activation ability is inferior to that of ProPAgens 4d and 9d.

[0084] Example 6: Activation of human CD8+ T cells by ProPAgen4a-d and 9a-d lack of The progens in Examples 1 and 2 are Vγ9 / Vδ2 T cell-specific activators. To demonstrate this, PBMCs containing donor-derived CD8+ T cells were subjected to Progen 4d and The concentration of 9d was increased and incubated (Figures 11 and 12).

[0085] Similar to peripheral blood γδ T cells, peripheral blood CD8+ T cells are at a considerable level under steady-state conditions. Although lacking surface CD69 or CD25, it responds to T cell receptor (TCR) stimulation. Both T cell activation markers are upregulated within 72 hours. Subsequently, PAg response Responsive CD8 T cells are distinguished by TCR CD8 expression, and upregulation CD69 is used. We evaluated CD25.

[0086] As shown in Figures 11 and 12, HMBP phosphonate PropAgens 4d and 9d is representative of these classes of prodrugs, at 1 μM, i.e., both Pro Vγ9 / Vδ2 T cell activation EC calculated for PAgens 50 Approximately 100 from the value, Even when incubated at a concentration 000 times higher, it did not show activation of CD8+ T cells. It was.

[0087] Example 7: Lysis of T24 bladder cancer cells by Vγ9 / Vδ2 T-cells is performed using ProPAgen Mediated and enhanced by 4a-d and 9a-d As further evidence of the above principle, and of the ProPA of Examples 1 and 2 shown above, gen's excellent Vγ9 / Vδ2 + The T-cell activation effect is translated into beneficial therapeutic effects. To demonstrate this, human T24 bladder cancer cell line was treated with 10 μM zoledronate and 100 pM H24. After incubating with MBPP or 100 pM ProPAgen 4d for 4 hours, V We compared the specific lysis of cancer cells by γ9 / Vδ2 T cells.

[0088] Further details of this in vitro cytotoxicity assay can be found in the Appendix. Cut.

[0089] As shown in Figure 13, incubation with HMBP phosphonate PropAgen 4d The results were also surprising when compared to what was observed in incubation with zoledronate. What should have been observed was the T24 bladder compared to what was observed in incubation with HMBPP. This resulted in a significant enhancement of bladder cancer lysis.

[0090] summary: This report describes the synthesis of novel methyl and difluoromethyl phosphonates (ProPAgens). It is announced that these ProPAgens are different from the previously reported ProPAgens phosphate. These prodrugs showed superior serum stability compared to ns derivatives (Non-Patent Literature 6). It is not only a specific activator of Vγ9 / Vδ2 T cells, but has also been reported in the past. HMBP phosphate pro-PAgens are far more potent in activating Vγ9 / Vδ2 T cells. It was shown to be a tivator. This increased Vγ9 / Vδ2 T cell activation effect was in It has also been shown that this translates to very potent lysis of cancer cells in vitro.

[0091] These new phosphonate progens offer high specificity, serum stability, and potency. The combination of fields includes cancer, osteoporosis and / or tuberculosis, leprosy, typhoid fever, malaria, and To treat various conditions, including proliferative disorders such as various infections including toxoplasmosis. It is suitable for development as a new immunotherapy drug.

[0092] Appendix: Synthesis and Evaluation of ProPAgen Compounds 4a-d and 9a-d But-3-en-1-ylphosphonic acid dichloride (2) Trimethylsilyl bromide (13.72 mL, 104.06 mmol, 10 equivalents) is oxidized to nitrogen Below, at room temperature, diethyl but-3-en-1-ylphospho nate 1 (2 g, 10.40 mmol, 1 equivalent) was slowly added over 30 minutes. The mixture was stirred for 2 hours and then the volatiles were removed under reduced pressure, giving a yellow liquid δ P NMR (202 MHz, CDCl3): 24.70. This was then dissolved in 50 ml of CH2Cl2 and 2 drops of dry DMF were added, and the mixture was cooled to 0 °C. Next, oxalyl chloride (2. 68 mL, 31.20 mmol, 3 equivalents) was added dropwise, and the reaction mixture was warmed to room temperature and stirred for 18 hours. The volatiles were evaporated and additional CH2Cl2 (10 mL) was evaporated 3 times to give the crude product (1.79 g, 100%) as a brown liquid, which was used in the next step without further purification. δ NMR (202 MHz, CDCl3): 49.6 P 6.

[0093] General procedure 1: Synthesis of allyl phosphonoamidates 3a-d The crude product, but-3-en-1-ylphosphonodichloride (2), was dissolved in 5 mL of CH2C l2 and added dropwise at -78 °C to an aqueous solution of phenol (1 equivalent), dry Et3N (2 equivalents) and CH2Cl2 (10 mL). After stirring at -78 °C for 30 minutes, the reaction mixture was warmed to room temperature and stirring was continued for a further 3 hours. 31 P NMR [δ P NMR (202MHz, CDCl3) : ~39.93] indicated that the reaction was complete, the mixture was filtered, and the volatiles were removed under reduced pressure, washed twice with Et2O and then removed under reduced pressure to give a crude oil. Next, this product was dissolved in CH2Cl2 (10 mL) and L-alanine ester hydrochloride ​(1 equivalent) and dry Et3N (2 equivalents) are stirred in dry CH2Cl2 (10 mL) The mixture was added dropwise at -78°C for 15 minutes. After stirring at -78°C for 30 minutes, the reaction mixture was warmed to room temperature. The mixture was left to stir overnight. The solvent was removed under reduced pressure, filtered, washed with Et2O, and then reduced. The crude oil was removed under pressure. The final product was then collected by column chromatography (6: It was refined into a colorless oil using 4 Hex / alkyl (alkyl) phosphate.

[0094] Methyl(but-3-en-1-yl(phenoxy)phosphoryl)-L-alanic acid (3a ) Phenol (0.210 g, 2.23 mmol, 1 equivalent) and L-alanine methyl ester Using sterol hydrochloride (0.373 g, 2.23 mmol, 1 equivalent), follow general procedure 1. The compound was synthesized, and product 3a (0.248g, 38%) was obtained as a colorless oil.

[0095] δ P NMR (202 MHz, CDCl3): 30.88, 31.22. δ H NMR (500 MHz, CDCl3): 7.30 (m, 2H, Ar), 7.10 ( m, 3H, Ar), 5.82 (m, 1H, CH2=CH), 5.00 (m, 2H, CH 2=CH), 4.11-3.86 (m, 1H,CH-NH), 3.60 (d, J = 6. 6 Hz, 3H, OCH3), 3.18 (m, 1H,NH), 2.48-2.27 (m, 2H,=CH-CH2), 1.92 (m, 2H, CH2-P), 1.21 (2d, J = 7.1 Hz, 3H, CH-CH3). δ C NMR (126 MHz, CDCl3): 174.68 (d, J = 6.3 Hz, C= O), 174.38 (d, J = 5.1 Hz, C=O), 150.72 (d, J = 9. 1 Hz), 150.51 (d, J = 9.4 Hz), 129.78, 124.78 (d , J = 5.5 Hz, CH=CH2), 120.86 (d, J = 4.6 Hz, C-Ar) , 120.71 (d, J = 4.7 Hz), 115.56, 52.52 (d, J = 3. 1 Hz, CH3-O), 49.58 (d, J = 14.7 Hz, CH-NH), 27. 88 (d, J = 130.9 Hz, CH2-P) 27.60 (d, J = 131.6 H z, CH2-P), 26.59 (d, J = 4.1 Hz, CH2-CH2-P), 21. 68 (2d, J = 4.3 Hz, CHCH3).

[0096] Isopropyl(but-3-en-1-yl(phenoxy)phosphoryl)-L-alanic acid (3b) Phenol (0.210 g, 2.23 mmol, 1 equivalent) and L-alanine isopropyl alcohol Use pyr ester hydrogen chloride (0.373 g, 2.23 mmol, 1 equivalent) in the general procedure The compound was synthesized according to 1, and product 3b (0.348 g, 48%) was obtained as a colorless oil.

[0097] δ P NMR (202 MHz, CDCl3): 30.93, 31.24. δ H NMR (500 MHz, CDCl3): 7.30 (m, 2H, Ar), 7.21 ( m, 3H, Ar), 5.81 (m, 1H, CH2=CH), 5.11 (m, 2H, CH 2=CH), 4.96 (m, 1H, CH-iPr,), 4.04- 3.94 (m, 1 H, CH-NH), 3.21 (m, 1H, NH), 2.45 (m, 2H, =CH-CH 2), 2.02 - 1.88 (m, 2H, CH2 - P), 1.29 - 1.18 (m, 9H , CH3 - CH - NH, CH - iPr). δ C NMR (126 MHz, CDCl3): 173.67 (d, J = 6.3 Hz, C = O), 150.74 (d, J = 9.1 Hz), 129.76 (d, J = 6.7 Hz) , 124.73 (d, J = 5.0 Hz), 120.86 (d, J = 4.6 Hz), 1 20.69 (d, J = 4.6 Hz), 115.50, 69.23 (d, J = 5.6 H z, CHiPr), 49.75 (d, J = 9.5 Hz, CH - NH), 27.89 ( d, J = 130.9 Hz, CH2 - P), 27.54 (d, J = 131.4 Hz, C H2 - P), 26.74 (d, J = 4.3 Hz, CH2 - CH2 - P), 26.57 (d, J = 4.0 Hz, CH2 - CH2 - P), 21.58 (2d, J = 4.4 Hz , CHCH3).

[0098] tert - butyl (but - 3 - en - 1 - yl(phenoxy)phosphoryl) - L - alan ine (3c) Phenol (0.210 g, 2.23 mmol, 1 eq) and L - alanine tert - butyl ester hydrochloride (0.405 g, 2.23 mmol, 1 eq) were used to synthesize according to the general procedure 1, and the product 3c (0.461 g, 61%) was obtained as a colorless oil.

[0099] δ P NMR (202 MHz, CDCl3): 30.95, 31.21. δ H NMR (500 MHz, CDCl3): 7.32 (m, 2H, Ar), 7.20 ( m, 3H, Ar), 5.87 (m, 1H, CH2=CH), 5.12 (m, 2H, CH2 =CH), 4.00-3.86 (m, 1H, CH-NH), 3.26 (m, 1H, N H), 2.45 (m, 2H,=CH-CH2), 2.04-1.85 (m, 2H, P- CH2), 1.42 (s, 9H, tBu-H), 1.22 (2d, J = 7.2 Hz, 3 H, CH-CH3). δ C NMR (126 MHz, CDCl3): 173.13 (d, J = 5.5 Hz, C= O), 150.77 (d, J = 9.2 Hz), 137.43 (d, J = 7.1 Hz) , 137.32 (d, J = 7.6 Hz), 129.76 (d, J = 6.2 Hz), 1 24.71 (d, J = 4.0 Hz), 120.88 (d, J = 4.6 Hz), 120 .72 (d, J = 4.7 Hz), 115.47, 81.99 (d, J = 8.2 Hz) , 60.54, 50.18 (d, J = 4.0 Hz, CH-NH), 28.05, 27. 91 (d, J = 131.6 Hz, CH2-P), 27.20 (d, J = 130.2 H z, CH2-P), 21.82 (2d, J = 4.2 Hz, CHCH3).

[0100] Benzyl(but-3-en-1-yl(phenoxy)phosphoryl)-L-alanine(3 d) Phenol (0.210 g, 2.23 mmol, 1 equivalent) and L-alanine benzyl Using esterified hydrogen chloride (0.405 g, 2.23 mmol, 1 equivalent), proceed to general procedure 1. Therefore, the product was synthesized, and product 3d (0.461 g, 55%) was obtained as a colorless oil.

[0101] δ PNMR(202 MHz、CDCl3): 30.85、31.21. δ H NMR(500 MHz、CDCl3): 7.38–7.27 (m、7H、Ar)、 7.21–7.18 (m、2H、Ar)、7.16–7.09 (m、1H、Ar)、5 .85 (m、1H、CH2=CH)、5.04 (m、4H、CH2=CH、-OCH2 )、4.28–4.03 (m、1H、CH-NH)、3.24 (m、1H、NH)、2 .56–2.34 (m、2H、,=CH–CH2)、2.08–1.82 (m、2H、 P-CH2)、1.24 (2d、J = 7.1 Hz、3H、CH-CH3). δ C NMR(126 MHz、CDCl3): 173.74 (d、J = 5.2 Hz、C= O)、150.70 (d、J = 9.1 Hz)、137.35、135.40 (d、J = 6.7 Hz)、129.79 (d、J = 6.1 Hz)、128.79 (d、J = 2 .8 Hz)、128.64 (d、J = 6.4 Hz)、128.33、120.86 ( d、J = 4.6 Hz)、120.68 (d、J = 4.7 Hz)、115.55、67 .31 (d、J = 3.0 Hz、CH2-O)、49.72 (d、J = 11.1 Hz、 CH-NH)、27.88 (d、J = 130.7 Hz、CH2-P)、27.57(d 、J = 131.3 Hz 、CH2-P)、26.74 (d、J = 4.3 Hz、CH2- CH2-P)、26.57 (d、J = 4.1 Hz、CH2-CH2-P)、21.70 (2d、J = 4.4 Hz、CHCH3).

[0102] General procedure 2: Phosphonoamide 4a- by Hoveyda-Grubbs cross-decomposition Synthesis of d Allylphosphonoamidate 3a-d (1 equivalent) and 2-methyl-2-propene-1-O (85 μL, 1 mmol, 2 equivalents), 1,4-benzoquinone (5.40 mg, 10 mg) 1%) of dried DCM (10 mL) solution containing the second generation Hoveyda-Grubbs catalyst. 23.5 mg, 0.038 mmol, 7.5 mol) was added. 2.5 mol of catalyst was added. Three equivalent doses of % (7.8 mg, 0.013 mmol) were administered at t = 0, 2, and 4 hours, and the reaction was observed. It was added over a certain period of time. Then, this solution was heated under a nitrogen atmosphere at 45°C for 18 hours under reflux. After cooling to room temperature, the activated carbon scoop was added, and the mixture was stirred for another 2 hours. The mixture was then filtered through a Celite pad. The volatile substances evaporated, and the residue was spread across a wide area of ​​silica gel. Purified by column chromatography (hexane / siRNA, 7:3~0:10) Then, 4a to d were obtained as colorless oil.

[0103] Methyl(((E)-5-hydroxy-4-methylpent-3-en-1-yl)(pheno Xy(phosphoryl)-L-alanine (4a) Synthesize using 3a (150 mg, 0.5 mmol, 1 equivalent) according to general procedure 2, 4 a (91 mg, 57%) was obtained as a colorless oily substance.

[0104] δ P NMR (202 MHz, CDCl3): 30.89, 31.31. δ H NMR (500 MHz, CDCl3): 7.31 (m, 2H, Ar), 7.18 ( m, 3H, Ar), 5.48 (m,1H, =CH), 4.24 - 3.98 (m, 3H , CH2OH, CH-NH), 3.68 (d, J = 7.9 Hz, 3H, OCH3), 3 .39 - 3.18 (m, 1H, NH), 2.55 - 2.41 (m, 2H, =CH-CH 2), 2.12 - 1.87 (m, 2H, CH2-P), 1.71 (d, J = 6.6 Hz , 3H, CH3(CH2)C=), 1.27 (2x d, J = 7.1 Hz, 3H, CH-C H3). δ C NMR(126 MHz, CDCl3): 176.71 (d, J = 5.9 Hz, C= O), 150.72, 129.81 (d, J = 6.9 Hz), 124.81 (d, J = 8.6 Hz, CH=CH2), 124.17, 124.04, 120.94, 120. 69 (d, J = 4.6 Hz), 68.56 (d, J = 9.1 Hz, CH2-OH), 52.64 (d, J = 3.5 Hz, CH3-O), 49.51 (d, J = 4.1 Hz , CH-NH), 28.33 (d, J = 129.6 Hz CH2-P), 28.06 (d, J = 130.2 Hz, CH2-P), 21.86 (2d, J = 4.9 Hz, CHCH3 ), 21.00 (d, J = 4.8 Hz, CH2-CH2-P), 20.89 (d, J = 4.4 Hz, CH2-CH2-P), 13.87. HRMS (ES+, m / z) calculation: (M+Na)+ C 16 H 24 NO5NaP: 364.1290; detected : 364.1293

[0105] Isopropyl (((E)-5-hydroxy-4-methylpent-3-en-1-yl)( (phenoxy)phosphoryl)-L-alanine (4b) Using compound 3b (0.150 g, 0.46 mmol, 1 eq), the synthesis was carried out according to General Procedure 2 and the product 4b (97 mg, 59%) was obtained as a colorless oil.

[0106] δ P NMR (202 MHz, CDCl3): 31.11, 31.49. δ H NMR (500 MHz, CDCl3): 7.28 (m, 2H, Ar), 7.20 ( m, 3H, Ar), 5.46 (m, 1H, =CH), 4.95 (m, 1H, CH-iP r), 4.08 - 3.79 (m, 3H, CH2OH, CH-NH), 3.47-3. 25 (m, 1H, NH), 2.51-2.35 (m, 2H, =CH-CH2), 2.0 2-1.85 (m, 2H, CH2-P), 1.73-1.56 (d, J = 6.0 Hz , 3H, CH3(CH2)C=), 1.35-1.08 (m, 9H, CH3-CH-NH , CH-iPr). δ C NMR (126 MHz, CDCl3): 173.62 (d, J = 5.8 Hz, C= O), 150.74 (d, J = 9.0 Hz), 136.76, 124.75, 123. 95 (d, J = 5.4 Hz), 123.84 (d, J = 6.9 Hz), 120.90 (d, J = 4.5 Hz), 120.67 (d, J = 4.6 Hz), 119.83, 1 15.58, 69.41 (d, J = 4.7 Hz, CHiPr) 68.39 (d, J = 7.0 Hz, CH2-OH), 49.65, 28.36 (d, J = 129.7 Hz, CH2-P), 27.92 (d, J = 131.1 Hz, CH2-P), 21.83 ( 2d, J = 6.2 Hz, CHCH3), 20.90 (d, J = 4.4 Hz, CH2-C H2-P), 20.84 (d, J = 4.4 Hz, CH2-CH2-P),13.84. HRMS (ES+, m / z) calculated value: (M+Na)+ C 18 H 28 NO5NaP: 392.16 03; Detected: 392.1613

[0107] tert-butyl(((E)-5-hydroxy-4-methylpent-3-en-1-yl (Phenoxy)phosphoryl-L-alanine (4c) Using compound 3c (0.150 g, 0.44 mmol, 1 equivalent), follow general procedure 2. The product was synthesized, and product 4c (108 mg, 64%) was obtained as a colorless oil.

[0108] δ P NMR (202 MHz, CDCl3): 31.05, 31.42. δ H NMR (500 MHz, CDCl3): 7.30 (m, 2H, Ar), 7.17 (m, 3H,Ar), 5.46 (m, 1H, =CH), 4.17-3.78 (m, 3H , CH2OH, CH-NH), 3.41-3.18 (m, 1H, NH), 2.60-2 .30 (m, 2H, =CH-CH2), 2.01-1.87 (m, 2H, CH2-P) , 1.69 (d, J = 6.8 Hz, 3H, CH3(CH2)C=), 1.42 (s, 9 H, tBu), 1.27 (2d, J = 7.9 Hz, 3H, CH-CH3). δ C NMR (126 MHz, CDCl3): 173.34 (d, J = 6.0 Hz, C= O), 150.79 (d, J = 9.0 Hz), 136.81 (d, J = 9.6 Hz) , 115.59, 82.22 (d, J = 5.6 Hz), 68.45 (d, J = 10. 4 Hz, CH2-OH), 50.07 (d, J = 4.5 Hz, CH-NH), 28. 01 (d, J = 129.7 Hz, CH2-P), 27.26 (d, J = 130.4 H z, CH2-P), 21.98 (d, J = 3.8 Hz, CHCH3), 20.97 ( d, J = 4.7 Hz, CH2-CH2-P), 20.88 (d, J = 4.4 Hz, C H2-CH2-P), 13.86(CH3). HRMS (ES+, m / z) calculation: (M+Na)+ C 19 H 30 NO5NaP: 406.1759; detected :​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 33 (2d, J = 7.0 Hz, 3H, CH-CH3) δ C NMR (126 MHz, CDCl3): 176.76, 129.81 (d, J = 7 .4 Hz), 128.75 (d, J = 14.9 Hz), 128.39, 124.77 , 124.15 (d, J = 15.0 Hz), 120.93-120.84, 120.6 7 (d, J = 4.7 Hz), 68.57 (d, J = 9.6 Hz), 67.42 (d , J = 2.9 Hz), 49.65, 28.05 (d, J = 129.9 Hz), 21. 77 (d, J = 4.1 Hz), 20.87 (d, J = 4.4 Hz), 13.87. HRMS (ES+, m / z) calculation: (M+Na)+ C 22 H 28 NO5NaP: 440.1603; detected : 440.1609

[0111] Diethyl(1,1-difluorobuto-3-en-1-yl)phosphonate (6) (unpatented) Reference 10) 250 mL round-bottom flask containing activated zinc powder (2.50 g, 38.23 mmol, 1 equivalent) Anhydrous DMF (20 mL) was added to the Scot under nitrogen. Subsequently, diethyl (bromodifier) ​​was added. Fluoromethyl phosphonate (6.80 mL, 38.23 mmol, 1 equivalent) slowly The mixture was added dropwise and stirred at room temperature for 3 hours. CuBr (5.48 g, 38.23 mmol, Add 1 equivalent, then add allyl bromide (3.96 mL, 45.87) to prevent the exothermic reaction. mmol (1.2 equivalents) was slowly added dropwise. After stirring for 40 hours, the mixture was filtered, and then... The solution was partitioned between DCM and a 10% NH4Cl aqueous solution. The aqueous phase was extracted three times with DCM. The separated organic phase was dried with anhydrous MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by column chromatography using 20% EtOAc in hexane to give 6 (5.41 g, 62%) as a pale yellow oil.

[0112] δ P NMR (202 MHz, CDCl₃): 6.93 (t, J = 107.4 Hz). [[ID=1X]]δ H NMR (500 MHz, CDCl₃): 5.84 (m, 1H, =CH), 5.26 (m, 2H, CH₂=), 4.32 - 4.21 (m, 4H, 2x OCH₂CH₃), 2.8 2 (m, 2H, =CH - CH₂), 1.37 (t, J = 7.xx Hz, 6H, 2 X OCH₂ CH₃)

[0113] (1,1 - Difluorobut - 3 - en - 1 - yl)phosphonic dichloride (7) Using compound 6 (2.5 g, 10.95 mmol, 1 eq), it was synthesized as described for 2 to give crude product 7 (2.28 g, 100%) as a brown liquid, which was used in the next step without further purification.

[0114] δ P NMR (xxx MHz, CDCl₃): 31.56 (t, J = 138.8 Hz) .

[0115] Methyl ((1,1 - difluorobut - 3 - en - 1 - yl)(phenoxy)phosphoryl) -L - alaninate (8a) Using phenol (0.210 g, 2.23 mmol, 1 eq) and L - alanine methyl ester hydrochloride (0.261 g, 1.87 mmol, 1 eq), it was synthesized according to General Procedure 1 to give product xa (0.150 g, 24%) as a colorless oil. Note: There are some unclear data in the original text such as "J = 7.xx Hz" which is retained as "J = 7.xx Hz" in the translation for the lack of specific value in the original. Also, "xa" in the last line is likely a typo in the original, assumed to be "8a" in the translation.​​​​

[0116] δ P NMR(202MHz, CDCl3): 9.05 (dd, J = 101.1, 49. 9), 8.41 (dd, J = 100.0, 51.1Hz) δ H NMR (500 MHz, CDCl3): 7.35 (m, 2H, Ar), 7.21 ( m, 3H, Ar), 5.89 (m, 1H, CH2=CH), 5.29 (m, CH2=C H), 4.14 (m, 1H, CH-NH), 3.69 (d, J = 6.6 Hz, 3H, OCH3),3.64 (m, 1H, NH), 3.13-2.84 (m, 2H, =CH- CH2), 1.38 (2x d, 7.1 Hz, 3H, CH-CH) δ C NMR (126 MHz, CDCl3): 173.76 (d, J = 4.1 Hz, C= O), 130.02 (d, J = 4.1 Hz), 127.40-127.07 (m), 125.64, 121.54 (d, J = 9.3 Hz), 120.54 (t, J = 4. 8 Hz), 52.69, 50.09 (d, J = 6.6 Hz), 39.39-37.2 2 (m), 21.74(2d, J = 3.5 Hz, CH3).

[0117] Isopropyl((1,1-difluorobuto-3-en-1-yl)(phenoxy)phospho Lyl)-L-alanine (8b) Phenol (0.210 g, 2.23 mmol, 1 equivalent) and L-alanine isopropyl alcohol Use pyr ester hydrogen chloride (0.314 g, 1.87 mmol, 1 equivalent) in the general procedure The compound was synthesized according to 1, and product 8b (0.165 g, 25%) was obtained as a colorless oil.

[0118] δP NMR(202MHz, CDCl3): 9.17 (dd, J = 101.1, 30. 3Hz), 8.46 (dd, J = 99.9, 38.0Hz) δ H NMR (500MHz, CDCl3). 7.34 (m, 2H, Ar), 7.20 (m , 3H, Ar), 5.89 (m, 1H, CH2=CH), 5.29 (m, 2H, CH2 =CH), 4.99 (m, 1H, CH-iPr), 4.14-3.99 (m, 1H, C H-NH), 3.68 (m, 1H, NH), 3.00-2.89 (m, 2H, =CH- CH2), 1.35-1.17 (m, 9H, CH3-CH-NH, CH-iPr). δ C NMR (126 MHz, CDCl3): 172.68 (d, J = 5.9 Hz, C= O), 149.46, 129.88 (d, J = 4.2 Hz), 127.11 (d, J = 5.4 Hz), 125.46, 121.36 (d, J = 8.9 Hz), 120.40 (t, J = 4.9 Hz), 69.39 (d, J = 2.8 Hz, CHiPr), 50. 15, 38.63-37.99 (m), 21.58 (d, J = 1.6 Hz), 21. 44 (2d, J = 3.2 Hz, CHCH3).

[0119] tert-Butyl((1,1-difluorobast-3-en-1-yl)(phenoxy )phosphoryl-L-alanine (8c) Phenol (0.210 g, 2.23 mmol, 1 equivalent) and L-alanine tert Using butyl ester hydrogen chloride (0.340 g, 1.87 mmol, 1 equivalent) in a general procedure The product was synthesized according to step 1, and product 8c (0.320 g, 46%) was obtained as a colorless oil.

[0120] δ P NMR(202MHz, CDCl3): 9.23 (dd, J = 101.0, 34. 4Hz), 8.52 (dd, J = 99.6, 36.0Hz) δ H NMR (500 MHz, CDCl3): 7.34 (m, 2H, Ar),7.21 ( m, 3H, Ar), 5.89 (m, 1H, CH2=CH), 5.28 (m, 2H, CH 2=CH), 4.08 - 3.98 (m, 1H, CH-NH), 3.67 (m, 1H, NH), 3.01-2.84 (m, 2H, =CH-CH2), 1.42 (d, J = 5. 2 Hz,9H, tBu), 1.30 (d, J = 7.2 Hz, 3H, CH-CH3). δ C NMR (126 MHz, CDCl3): 172.48 (d, J = 4.5 Hz, C= O), 149.72, 129.99 (d, J = 2.1 Hz), 128.59-126. 95 (m), 125.54, 121.46 (d, J = 9.2 Hz), 120.54 ( t, J = 4.8 Hz), 82.32 (d, J = 5.8 Hz), 50.69 (d, J = 2.8 Hz, CH-NH), 38.80-38.12 (m), 27.99, 21.85 (2d, J = 3.1 Hz, CHCH3).

[0121] Benzyl((1,1-difluorobuto-3-en-1-yl)(phenoxy)phosphoryl )-L-alanine (8d) Phenol (0.210 g, 2.23 mmol, 1 equivalent) and L-alanine benzyl Using esterified hydrogen chloride (0.403 g, 1.87 mmol, 1 equivalent), proceed to general procedure 1. Therefore, the product was synthesized, and product 8d (0.350 g, 45% yield) was obtained as a colorless oil.

[0122] δ P NMR(202MHz, CDCl3): 9.08 (dd, J = 101.2, 45. 4Hz), 8.39 (dd, J = 100.1, 46.5Hz) δ H NMR (500 MHz, CDCl3): 7.41-7.29 (m, 7H, Ar), 7.25-7.17 (m, 3H, Ar), 5.87 (m, 1H, CH2=CH), 5. 27 (m, 2H, CH2=CH), 5.11 (m, 2H, -OCH2), 4.334. 09 (m, 1H, CH-NH), 3.68 (m, 1H, NH), 2.93 (m, 2H, P-CH2), 1.36 (d, J = 7.2 Hz, 3H, CH-CH3). δ C NMR (126 MHz, CDCl3): 173.15 (d, J = 3.2 Hz, C= O), 149.68, 135.26 (d, J = 6.4 Hz), 130.01 (d, J = 4.1 Hz), 128.87-128.52 (m), 128.35 (d, J = 1.1 Hz), 127.38-127.03 (m), 125.62, 122.24-120. 38 (m), 120.51, 67.49, 50.20, 38.74-38.07 (m) , 21.96 (2d, J = 3.5 Hz, CHCH3).

[0123] Methyl(((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-ene -1-yl)(phenoxy)phosphoryl)-L-alanine (9a) Using compound 8a (0.150 g, 0.45 mmol, 1 equivalent), follow general procedure 2. Synthesized, and product 9a (97 mg, 58%) was obtained as a colorless oil.

[0124] δ P NMR (202 MHz, CDCl3) δ 9.27 (dd, J = 112.1, 18.4 Hz), 8.44 (dd, J = 126.4, 20.2 Hz) δ H NMR (500 MHz, CDCl3): 7.35 (m, 2H, Ar), 7.21 ( m, 3H, Ar), 5.56 (m, 1H, =CH), 4.18 (m, 1H, CH-NH ), 4.07 (m, 2H, =CH-CH2), 3.75 - 3.46 (m, 4H, NH, OCH3), 3.10 - 2.79 (m, 2H, =CH-CH2), 1.83 (m, 1H, OH), 1.76 (s, 3H, CH3(CH2)C=CH), 1.37 (2x d, J = 7 .1 Hz, 3H, CH-CH3). δ C NMR (126 MHz, CDCl3): 176.76, 141.38 (d, J = 7 .6 Hz), 130.04 (d, J = 3.2 Hz), 125.65 (d, J = 4.0 Hz), 120.56 (d, J = 4.6 Hz), 120.47 (d, J = 4.7 H z), 68.25 (d, J = 1.9 Hz), 52.80 (CH3-O), 50.09 (d, J = 4.1 Hz, CH-NH), 32.82, 21.82 (2d, J = 2.6 Hz, CHCH3), 14.15 (CH3). HRMS (ES+, m / z) calculated: (M + Na)+ C 16 H 22 F2NO5NaP: 400.1101; detected: 400.1109

[0125] Isopropyl(((E)-1,1-difluoro-5-hydroxy-4-methylpent-3 -en-1-yl)(phenoxy)phosphoryl)-L-alanine(9b) Using compound 8b (0.150 g, 0.41 mmol, 1 equivalent), follow general procedure 2. The product was synthesized, and product 9b (117 mg, 69%) was obtained as a colorless oil.

[0126] δ P NMR(202MHz, CDCl3): 9.30 (dd, J = 109.1, 34. 4Hz), 8.42 (dd, J = 111.1, 38.4Hz) δ H NMR (500 MHz, CDCl3): 7.35 (m, 2H, Ar), 7.21 ( m, 3H, Ar), 5.56 (m, 1H, =CH), 5.00(m, 1H, CH-iP r), 4.17-3.91 (m, 3H, CH-NH, =CH-CH2), 3.67 (m , 1H, NH), 3.08-2.75 (m, 2H, =CH-CH2), 1.86 (m, 1H, OH), 1.70 (s, 3H, CH3(CH2)C=CH), 1.34-1.19 (m, 9H, CH3-CH-NH, CH-iPr). δ C NMR (126 MHz, CDCl3): 173.09 (d, J = 6.8 Hz, C= O), 141.37 (d, J = 7.9 Hz), 130.03 (d, J = 2.6 Hz) , 125.60 (d, J = 4.4 Hz), 120.50 (dd, J = 10.6, 4. 7 Hz), 113.79 - 113.43 (m), 69.71 (d, J = 4.5 Hz ), 68.25, 50.29 (d, J = 9.6 Hz), 30.88-28.72 (m ), 21.73 (d, J = 8.2 Hz), 21.84 (2d, J = 4.1 Hz, CH CH3), 14.18 (CH3). HRMS (ES+, m / z) calculation: (M+Na)+ C 18 H 26 F2NO5NaP: 428.1414; Detected: 428.1414

[0127] tert-butyl(((E)-1,1-difluoro-5-hydroxy-4-methylpent -3-en-1-yl)(phenoxy)phosphoryl)-L-alanine (9c) Using compound 8c (0.150 g, 0.39 mmol, 1 equivalent), follow general procedure 2. The compound was synthesized, and product 9c (105 mg, 63%) was obtained as a colorless oil.

[0128] δ P NMR(202MHz, CDCl3): 9.36 (dd, J = 109.1, 34. 4Hz), 8.48 (dd, J = 111.1, 36.4Hz) δ H NMR (500 MHz, CDCl3): 7.36 (m, 2H, Ar), 7.22 ( m, 3H, Ar), 5.56 (m, 1H, =CH), 4.16-3.93 (m, 3H, CH-NH, =CH-CH2), 3.63 (m, 1H, NH), 3.08-2.77 ( m, 2H, =CH-CH2), 1.90 (m, 1H, OH), 1.71 (s, 3H, C H3(CH2)C=CH), 1.43 (d, J = 5.2 Hz,9H, t Bu), 1.30 (2x d, J = 7.1 Hz, 3H, CH-CH3). δ C NMR (126 MHz, CDCl3): 172.79 (d, J = 6.8 Hz, C= O), 130.02, 125.56 (d, J = 5.8 Hz), 120.55 (d, J = 4.6 Hz), 120.47 (d, J = 4.4 Hz), 82.59, 68.26, 5 0.72 (d, J = 7.4 Hz, CH-NH), 32.97-32.80 (m), 2 8.02 (d, J = 1.1 Hz, 3x CH3), 21.98 (2d, J = 3.8 Hz, CHCH3), 14.16. HRMS (ES+, m / z) calculation: (M+Na)+ C 19 H 28 F2NO5NaP: 442.1571; Detected: 442.1578

[0129] Benzyl(((E)-1,1-difluoro-5-hydroxy-4-methylpent-3-e (Phenoxyphosphoryl)-L-alanine (9d) Using compound 8d (0.150 g, 0.36 mmol, 1 equivalent), follow general procedure 2. The compound was synthesized, and product 9d (101 mg, 61%) was obtained as a colorless oil.

[0130] δ P NMR(202MHz, CDCl3): 9.21 (dd, J = 113.2, 22. 3Hz), 8.37 (dd, J = 111.1, 24.3Hz) δ H NMR (500 MHz, CDCl3): 7.39-7.28 (m, 7H, Ar), 7.24-7.16 (m, 3H, Ar), 5.55 (m, 1H, =CH), 5.13 (m, 2H, OCH2), 4.27-4.14 (m, 1H, CH-NH), 4.06 ( m, 2H, =CH-CH2), 3.68 (m, 1H, NH), 3.05 -2.73 (m , 2H, =CH-CH2), 1.80 (m, 1H, OH), 1.69 (s, 3H, CH3 (CH2)C=CH), 1.35 (2x d, J = 7.1 Hz, 3H, CH-CH3). δ C (126 MHz, CDCl3): 173.38 (d, J = 7.9 Hz, C=O) , 149.71, 141.37 (d, J = 8.3 Hz), 130.04 (d, J = 3 .1 Hz), 128.76 (d, J = 12.8 Hz), 128.35 (s), 125 .63 (d, J = 5.5 Hz), 120.49 (dd, J = 13.7, 4.6 Hz) , 113.75-113.34 (m), 68.25 (d, J = 2.4 Hz), 67. 61, 50.22 (d, J = 8.0 Hz), 34.32-31.18 (m), 21. 84 (d, J = 3.8 Hz, CHCH3), 14.15 (CH3). HRMS (ES+, m / z) calculation: (M+Na)+ C 22 H 26 F2NO5NaP: 476.1414; detected : 476.1421

[0131] Cell isolation The blood is mixed with heparin and ethylenediaminetetraacetic acid (EDTA) as anticoagulants. In the presence of substances (2U / ml heparin, 1.5mM EDTA), a consenting healthy donor (NRES Co) Approved by the West Midlands Ethics Committee; REC Reference 14 / WM / 125 4) was obtained from. Next, the blood was layered on density gradient medium and lymphoprep (Stem Cell Tech Peripheral blood mononuclear cells (PBMCs) were purified by gradient centrifugation. Wash twice with phosphate-buffered saline (PBS), then rinse with 2 mM L-glutamine and 25 mM H2. EPES, 1% sodium pyruvate, 50 μg / ml penicillin / streptomycin (I The RPMI-1640 medium was refilled with nvitrogen and 10% fetal bovine serum. It was suspended.

[0132] Flow cytometry analysis Untreated and treated PBMCs were labeled with Zombie aqua survival dye (Biolegend). Next, BV421-bound anti-CD3 (UCHT1, Biolegend), BV650-bound anti-CD3 CD8 (SK1; BD Bioscience), FITC-conjugated anti-CD25 (M-A25, Bioleg end), PE-bound anti-CD69 (TP1.55.3; Beckman Coulter) and PE-Cy5 binding Conjugated anti-Vγ9 TCR (IMMU360, Beckman-Coulter) and APC-conjugated anti-Vδ2 TC Stained with a mixture of R (123R3, Miltenyi Biotech) antibodies. CD8 + T cell subset or CD69 in the Vγ9Vδ2 T cell population + CD25 + The percentage of , Measurements were taken using an itch meter. The data was analyzed using FlowJo V10 software. Ta.

[0133] Cytotoxicity assay Vγ9Vδ2T cells were grown from PBMCs in the presence of 5 μM zoledronate for 14 days. Next, add 100 U / ml of IL-2 (Peprotech) to the culture medium every 2-3 days. Approximately 85% of Vγ9Vδ2 T cells were obtained. Bladder cancer cell line T24 (ATCC HTB4) was added at 0.1 μM. Labeled with CFSE, then treated with 10 μM zoledronate, 100 pM HMBPP, or 10 After incubation for 4 hours with the indicated prodrug at 0 pM, wash 5 times in culture medium. Then, using pre-proliferated Vγ9Vδ2 T cells and an effector target ratio of 10:1 for 18 hours... The cells were cultured. Then, all cells were labeled with the eFluor780 survival dye and subjected to CFSE. + e Fluor780 viability dye + The cells were measured using flow cytometry. The data was obtained from F The analysis was performed using lowJo V10.

[0134] References (non-patent literature) 1. Morita, CT; Jin, C.; Sarikonda, G.; Wang, H. Nonpeptide antigens, prese ntation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: di discriminating friend from foe through the recognition of prenyl pyrophosphate ant Immunol Rev 2007, 215, 59-76. 2. Shen, Y.; Zhou, D.; Qiu, L.; Lai, X.; Simon, M.; Shen, L.; Kou, Z.; Wang , Q.; Jiang, L.; Estep, J.; Hunt, R.; Clagett, M.; Sehgal, PK; Li, Y.; Zeng, X.; Morita, CT; Brenner, MB; Letvin, NL; Chen, ZW Adaptive immune re sponse of Vgamma2Vdelta2+ T cells during mycobacterial infections. Science (New York, NY) 2002, 295, 2255-8. 3. Fisher, J. P.; Heuijerjans, J.; Yan, M.; Gustafsson, K.; Anderson, J. ga mmadelta T cells for cancer immunotherapy: A systematic review of clinical trial s. Oncoimmunology 2014, 3, e27572. 4. Maraka, S.; Kennel, K. A. Bisphosphonates for the prevention and treatme nt of osteoporosis. BMJ (Clinical research ed.) 2015, 351, h3783. 5. Roelofs, A. J.; Jauhiainen, M.; Monkkonen, H.; Rogers, M. J.; Monkkonen, J.; Thompson, K. Peripheral blood monocytes are responsible for gammadelta T ce ll activation induced by zoledronic acid through accumulation of IPP / DMAPP. Brit ish journal of haematology 2009, 144, 245-50. 6. Davey, M. S.; Malde, R.; Mykura, R. C.; Baker, A. T.; Taher, T. E.; Le D uff, C. S.; Willcox, B. E.; Mehellou, Y. Synthesis and Biological Evaluation of ( E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramid ate Prodrugs as Activators of Vgamma9 / Vdelta2 T-Cell Immune Responses. J Med Che m 2018, 61, 2111-2117. 7. Vougioukalakis, G. C.; Grubbs, R. H. Ruthenium-based heterocyclic carben e-coordinated olefin metathesis catalysts. Chem Rev 2010, 110, 1746-87. 8. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M.-C. The facile de alkylation of phosphonic acid dialkyl esters by bromotrimethylsilane. Tetrahedro n Letters 1977, 18, 155-158. 9. Bessieres, M.; Sari, O.; Roy, V.; Warszycki, D.; Bojarski, A. J.; Nolan, S. P.; Snoeck, R.; Andrei, G.; Schinazi, R. F.; Agrofoglio, L. A. Sonication-As sisted Synthesis of (E)-2-Methyl-but-2-enyl Nucleoside Phosphonate Prodrugs. Che mistrySelect 2016, 1, 3108-3813. 10. Mehellou, Y. The ProTides Boom. ChemMedChem 2016, 11, 1114–6. 11. Osgerby, L.; Lai, YC; Thornton , PJ ; Amalfitano, J.; In Duff, CS ; Strong, I.; Kadri, H.; Miccoli , A. ; Tucker , JHR ; Muqit, MMK; Mehello u, Y. Activation of Kinetin Riboside and Its ProTides in Parkinson's Disease Assoc ted PTEN-Induced Putative Kinase 1 (PINK1) Independent of Mitochondrial Depolarity zation. J Med Chem 2017, 60, 3518–3524.

Claims

1. Compounds or salts of general formula (IV) including all tautomers: In the formula, R3 is a base of formula (V), formula (VI), or formula (VII), where R9 is OH; Each of R4 and R5 independently represents either H or a halo (except when both R4 and R5 are H, or when both are a halo); R6 is C 1-4 It is an alkyl chain; R7 is unsubstituted C 1-4 It is an alkyl chain or an unsubstituted benzyl group; R8 is C 5-25 Represents an aryl or 5- to 25-membered heteroaryl group.

2. The compound according to claim 1, wherein the halo substituent is a fluoro group.

3. A compound according to claim 1, selected from the following group.

4. The compound according to claim 1, which is the following compound.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient or carrier.

6. A compound according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 7, for use in immunotherapy.

7. The compound or pharmaceutical composition according to claim 6, for use in the treatment of infectious diseases, proliferative disorders, or osteoporosis.

8. The compound or pharmaceutical composition for use according to claim 7, wherein the proliferative disorder is cancer.

9. The use of a compound according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5 in the preparation of a drug that activates the γδ T cell immune response in a patient.

10. The use according to claim 9 in the preparation of a drug for the treatment of infection, proliferative disorders or osteoporosis in a patient.

11. The use according to claim 10, wherein the proliferative disease is cancer.

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