Synthesis and Evaluation of Triazole-Containing Aryl/Acyloxy Prodrugs of a BTN3A1 Ligand

Abstract

The most effective natural ligand for the butyrophilins is (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate. However, due to its susceptibility to plasma hydrolysis and its high charge that limits passive diffusion across cell membranes, its potential as a drug is limited. Our efforts to identify compounds that stimulate γδ T cell proliferation have been focused on phosphonates to gain metabolic stability and phosphonate prodrugs to improve diffusion into cells. To identify potential prodrugs that are soluble, relatively stable in plasma, and undergo facile hydrolysis once inside the cell, we have prepared a series of aryl acyloxyesters where the acyl group includes a triazole moiety. Several of these novel prodrug forms have been shown to demonstrate nanomolar potency for T cell activation and relatively long half-lives in plasma. Interestingly, compound 26b stimulated T cells at sub-nanomolar levels (proliferation EC₅₀ = 0.49 nM) while achieving a half-life of 63 minutes in human plasma. The details of these syntheses and the biological evaluation are presented here.

Graphic Abstract

INTRODUCTION

Phosphonate prodrugs are useful during drug development due to their potential to overcome the inherent limitations of phosphates, such as poor cell permeability and rapid metabolic degradation.^1–3 Conversion of phosphonates into phosphonate prodrugs can improve their pharmacokinetic properties, allowing for faster uptake into cells and better stability within the body.⁴ In addition, phosphonate prodrugs can improve the bioavailability of the drug.⁵ These advantages make phosphonate prodrugs a valuable strategy in the design and development of new therapies. To our knowledge, at least six phosphate/phosphonate prodrugs have reached clinical use.⁴

Butyrophilin (BTN) proteins are promising therapeutic targets due to their roles in modulating immune responses, particularly the activation of Vγ9Vδ2 T cells.^6–8 These proteins, including BTN2A1, BTN3A1, BTN3A2 and BTN3A3,⁹ are involved in the recognition of phosphoantigens (pAgs) by γδ T cells and contribute to immune surveillance and the elimination of tumor cells and pathogens.^10–12 We currently believe that pAgs such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (1, HMBPP, Figure 1) promote a conformational change in the tetrameric butyrophilin complex consisting of a BTN2A1 homodimer and a BTN3A1 heterodimer (also known as the HMBPP receptor).^13–14 This involves HMBPP acting like a molecular glue to allow the BTN3A1 internal domain monomer to interact with the BTN2A1 internal domain homodimer.^{13, 15} The extracellular interaction between the HMBPP receptor complex and γδ T cells facilitates the activation and proliferation of these immune cells, leading to a robust and targeted immune response.^16–19 Agonists of the receptor have the potential to improve efficacy of immunotherapy, providing a novel approach to treating cancers.²⁰

Figure 1.

A natural ligand for BTN3A1 (1) and some phosphonate analogs (2 – 5).

Our research has focused on development and evaluation of phosphonate analogs of BTN ligands (e.g. 2) to overcome the limitations of natural diphosphate phosphoantigens like HMBPP, especially metabolic instability.^21–22 Then, to address poor cell permeability, we have explored a variety of prodrug forms, including stable aryl amidates (e.g. 3).^23–24 More recently we have become interested in improving the plasma stability of ester-based forms (e.g. 4), which are capable of greater potency and faster activation kinetics.²⁵ To this end, we recently synthesized a series of diesters bearing one aryl and one acyloxyalkyl group (e.g. 5), which were evaluated for their ability to stimulate Vγ9Vδ2 T cell proliferation, increase interferon γ production, resist plasma metabolism, and internalize into leukemia cells.²⁶ These studies revealed that varying the aryl and acyloxyalkyl groups significantly impacted bioactivity and stability, with some compounds still demonstrating sub-nanomolar potency. This research has provided valuable insights into the structure-activity relationships of mixed aryl/acyloxyalkyl phosphonate prodrugs and their potential as effective immunotherapeutic agents targeting butyrophilin proteins. However, the ester-based prodrugs could be improved further by increasing their water solubility and improving their plasma stability, of course while retaining the sub-nanomolar potency.

Triazoles are used in drug discovery due to their unique chemical properties and versatility. These five-membered heterocyclic compounds exhibit significant stability and resistance to metabolic degradation, which enhances the pharmacokinetic profiles of drug candidates. Triazoles can form strong hydrogen bonds and participate in π-stacking interactions, contributing to favorable water solubility and high binding affinity with biological targets.²⁷ In addition, the triazole ring can be easily synthesized and modified, allowing for the creation of diverse libraries of compounds with varying biological activities. Triazoles are valuable scaffolds in the design of drugs for various therapeutic areas, including anticancer and anti-inflammatory agents.

Our prior studies have demonstrated that the acyloxyester position is the rate limiting step to payload release from aryl acyloxyester prodrugs and that structural changes to this ester can dramatically impact the ratio of desirable cellular metabolism to undesirable plasma metabolism.^{26, 28} Therefore, we desired to more fully explore the range of tolerated structures at this position and their impact on metabolism. We hypothesized that incorporation of triazole into the ester prodrug moiety would not only improve its water solubility but also alter its geometry in a way that may beneficially shift the ratio of cellular to plasma metabolism. Here, we report the synthesis and evaluation of triazole-containing aryl/acyloxyalkyl phosphonate prodrugs applied to a key butyrophilin ligand.

RESULTS

Chemistry.

The 1,2,3-triazole system has been positioned in a wide variety of structures²⁹ including carbon skeletons leading to biologically active bisphosphonates.³⁰ In some cases the triazole system even is a viable replacement for an isoprene unit.^31–32 However the small size of the natural BTN3A1 ligand HMBPP (1) and its phosphonate analog 2 encouraged exploration of structures where a triazole was incorporated into a component of the prodrug form, rather than into the carbon skeleton of the ligand itself. Compound 2 was chosen as the payload for this study because it binds directly to the pAg pocket of BTN3A1³³ and its activity is abrogated in BTN3A1 knockout cells.³⁴ Use of compound 2 as a payload also allows for direct comparison to prodrug forms found in prior studies, so that the impact of the prodrug form rather than the pAg scaffold can be assessed.

Based on past experience with aryl/acyloxy esters,^{26, 28} the prodrug form 6 was particularly attractive (Figure 2). The essential ligand bearing an allylic alcohol could be derived in principle from compound 7 by selenium dioxide oxidation and the most appealing route to triazoles such as compound 7 would involve a late-stage diversification via click reactions on the acetylene 8 with varied azides (9). Our standard approach to acyloxy esters has been based on reaction of a phosphonate monoacid (10) with a chloromethyl ester (11), and thus preparation of the ester 11 became the initial goal.

Figure 2.

Retrosynthesis for the triazole prodrug 6.

Various chloromethyl esters have been prepared by reaction of chloromethyl chlorosulfate with the corresponding carboxylic acid.³⁵ Unfortunately, efforts to prepare the chloromethyl ester of propiolic acid went unrewarded, which may be due to the sensitivity of this conjugated acetylene and its esters.^36–37 A dibromoacrylate has been employed as a protected form of propiolic acid,³⁸ and exploration of this approach proved somewhat more successful. The dibromoacrylate 3,3-dibromoprop-2-enoic acid (13) can be readily prepared by Favorski rearrangement³⁹ of tetrabromoacetone (12), and this carboxylic acid reacted with chloromethyl chlorosulfate to provide the desired ester 14 (Scheme 1). When the mixed phosphonate ester 15⁴⁰ was treated with the chloromethyl ester 14 reaction proceeded as expected to afford the desired aryl/acyloxy ester 16. Selenium dioxide oxidation of phosphonate 16 gave the necessary allylic alcohol 17 albeit in modest yield. This yield may be due to competitive oxidation adjacent to the phosphonate, given that selenium dioxide oxidation of active methylene positions is known (a Riley oxidation)⁴¹ and the resulting acyl phosphonate may undergo hydrolysis under the reaction conditions.⁴² In any event, the desired product 17 was obtained in sufficient quantity to allow bioassay as well as a final reaction with acetic anhydride to obtain the corresponding acetate 18. While efforts to generate the corresponding acetylene from this dibromoacrylate were not successful, both compounds 17 and 18 were appropriate for examination as prodrugs of the desired ligand and so they were carried into the biological evaluations.

Scheme 1.

Synthesis of the dibromoacrylate prodrug of a BTN3A1 ligand.

^aReagents and conditions: (a) NaHCO₃, H₂O, 2 days, 60%; (b) 13, NaHCO₃, Bu₄NHSO₄, chloromethyl chlorosulfate, CH₂Cl₂:H₂O 1:1, 89%; (c) 15, NaI, CH₃CN (anhyd), reflux, 48 h, 38%; (d) SeO₂, t-BuOOH, 4-hydroxybenzoic acid, CH₂Cl₂, 0 °C, 3 days, 27%; (e) Ac₂O, Et₃N, rt, overnight, 92%.

Given the challenges encountered in preparation of the chloromethyl ester of propiolic acid, a more linear synthetic sequence was developed. For this approach, propiolic acid (prop-2-ynoic acid, 19) was allowed to react with an aromatic azide (20, Scheme 2) to form a triazole carboxylic acid (21). After conversion of this acid to the corresponding chloromethyl ester (22), reaction with a mixed methyl aryl phosphonate diester (23) gave the desired aryl acyloxy diester (24). Oxidation of these diesters introduced the allylic alcohol in the desired E configuration in generally low yields but sufficient quantity to proceed with the desired biological assays. Finally, the allylic alcohols 25 could be converted to the corresponding acetates 26 in a straightforward reaction. All told, these efforts gave a total of 16 new aryl acyloxy esters. The molecular weights of these compounds ranged from 457.32 to 593.51 g/mol while the cLogP values ranged from 3.51 to 6.37 (Table 1).

Scheme 2.

Synthesis of a family of triazole-based prodrugs.

^aReagents and conditions: (a) CuSO₄·5 H₂O, sodium ascorbate, ^tBuOH, H₂O, 60 – 65%; (b) 21, NaHCO₃, Bu₄NHSO₄, chloromethyl chlorosulfate, CH₂Cl₂:H₂O 1:1, 84 – 95%; (c) 23, NaI, CH₃CN (anhyd), reflux, 24 to 48 h, 25 – 46%; (d) SeO₂, t-BuOOH, 4-hydroxybenzoic acid, CH₂Cl₂, 0 °C, 3-4 days, 9 – 27%; (e) Ac₂O, Et₃N, rt, overnight, 53 – 94%.

Table 1.

Compound characteristics.

Cmpd	R ester in 5 or Ar1 in 25/26	R’ ester in 5 or Ar2 in 25/26	OH/AC	MW	cLogP
5	OCH2OC(O)C(CH3)3	OPh	OH	370.38	3.67
5	OCH2OC(O)C(CH3)3	O-p-iPrPh	OH	412.46	4.90
5	OCH2OC(O)Ph	OPh	OH	390.37	4.03
17	OCH2OC(O)CHCBr2	Ph	OH	498.10	4.39
18	OCH2OC(O)CHCBr2	Ph	Ac	540.14	4.80
25a	Ph-triazole	Ph	OH	457.42	3.51
26a	Ph-triazole	Ph	Ac	499.46	3.92
25b	p-F-Ph-triazole	Ph	OH	475.41	3.65
26b	p-F-Ph-triazole	Ph	Ac	517.45	4.06
25c	p-iPr-Ph-triazole	Ph	OH	499.50	4.73
26c	p-iPr-Ph-triazole	Ph	Ac	541.53	5.15
25d	Ph-triazole	p-iPrPh	OH	499.50	4.74
26d	Ph-triazole	p-iPrPh	Ac	541.54	5.15
25e	p-FPh-triazole	p-iPrPh	OH	517.49	4.88
26e	p-FPh-triazole	p-iPrPh	Ac	559.53	5.29
25f	p-iPr-Ph-triazole	p-iPrPh	OH	541.58	5.96
26f	p-iPr-Ph-triazole	p-iPrPh	Ac	583.62	6.37
25g	2,4-diFPh-triazole	p(CH2)2OCH3-Ph	OH	551.48	3.89
26g	2,4-diFPh-triazole	p(CH2)2OCH3-Ph	Ac	593.51	4.30

Biological Evaluation

The compounds were first tested for their ability to stimulate the proliferation of human Vγ9Vδ2 T cells (Table 2). All compounds were active in this regard, with potencies ranging from 0.49 nM to 7.4 nM, a 15-fold range of activity. The most potent compounds were 26b with an EC₅₀ of 0.49 nM and 25a with an EC₅₀ of 0.51 nM. These potencies are among the most potent of all the prodrug forms we have investigated to date, suggesting that the triazole substructure is beneficial. The least potent compounds were 17/18 and 25f/26f. The lower activity of 17/18 was not surprising, but it does illustrate the importance of alkyl groups at this position of the molecule. The lower activity of 25f/26f was somewhat surprising, and suggests that addition of p-iPr groups to both sides of the molecule might interfere with cellular metabolism. Compounds 25f/26f were also the most hydrophobic compounds, which also may have contributed to their decreased activity. The average EC₅₀ of the six phenyl-containing compounds was 0.71 nM while that of the six p-iPr-Ph-containing compounds was 2.4, a 3.4-fold difference, which suggested that in the class of compounds the p-iPr-Ph may mildly reduce activity.

Table 2.

Vγ9Vδ2 T cell Proliferation.

Cmpd	R ester in 5 or Ar1 in 25/26	R’ ester in 5 or Ar2 in 25/26	OH/Ac	72 h EC50 (nM)	n
1 33	NA	NA	OH	0.51
2 33	NA	NA	OH	4000
5 26, 40	OCH2OC(O)C(CH3)3	OPh	OH	1.0
5	OCH2OC(O)C(CH3)3	O-p-iPrPh	OH	1.9
5	OCH2OC(O)Ph	OPh	OH	0.34
17	OCH2OC(O)CHCBr2	Ph	OH	7.4 (2.6 to 21)	3
18	OCH2OC(O)CHCBr2	Ph	Ac	6.0 (3.9 to 9.0)	3
25a	Ph-triazole	Ph	OH	0.51 (0.30 to 0.88)	3
26a	Ph-triazole	Ph	Ac	0.57 (0.29 to 1.1)	3
25b	p-FPh-triazole	Ph	OH	0.78 (0.62 to 0.98)	3
26b	p-FPh-triazole	Ph	Ac	0.49 (0.26 to 0.91)	3
25c	p-iPrPh-triazole	Ph	OH	0.71 (0.21 to 2.4)	3
26c	p-iPrPh-triazole	Ph	Ac	1.2 (0.45 to 3.0)	3
25d	Ph-triazole	p-iPrPh	OH	1.7 (0.61 to 4.5)	3
26d	Ph-triazole	p-iPrPh	Ac	0.75 (0.27 to 2.1)	3
25e	p-FPh-triazole	p-iPrPh	OH	1.5 (1.1 to 2.0)	4
26e	p-FPh-triazole	p-iPrPh	Ac	1.3 (0.76 to 2.3)	4
25f	p-iPrPh-triazole	p-iPrPh	OH	4.5 (2.8 to 7.5)	4
26f	p-iPrPh-triazole	p-iPrPh	Ac	4.7 (0.68 to 32)	4
25g	2,4-diFPh-triazole	p(CH2)2OCH3-Ph	OH	1.6 (0.66 to 3.9)	3
26g	2,4-diFPh-triazole	p(CH2)2OCH3-Ph	Ac	1.6 (0.92 to 2.6)	3

^*Human PBMCs stimulated with test compounds for 3 days and quantified on day 14.

The test compounds were next assessed for their ability to stimulate Vγ9Vδ2 T cell interferon γ secretion following their exposure to compound-loaded K562 or U937 cells (Table 3). This assay normally serves as a secondary readout of cellular phosphoantigen activity, and it tends to correlate with the proliferation numbers, albeit with slightly lower potency due to the shorter incubation time. Here, in K562 cells the potencies ranged from 0.6 nM to 77 nM, a 130-fold range of activity, while in U937 cells they ranged from 1.4 nM to 45 nM, a 32-fold range of activity. Compound 25b was the most potent in K562 cells with an EC₅₀ of 0.6 nM while compound 25d was the most potent in U937 cells with an EC₅₀ of 1.4 nM. As expected, compounds 17/18 were again the least potent compounds of the group, in both cell lines. The cytokine production activities generally correlated with the proliferation activities, but were more correlated in the U937 cells (r = 0.91) than the K562 cells (r = 0.65). Based on potency across both assays, compounds 25b/26b and 25d/26d were judged to be the most active compounds in the set, suggesting that the p-F-Ph-triazole ester and the Ph-triazole are the most promising triazole-containing protecting groups.

Table 3.

Vγ9Vδ2 T cell interferon γ response.

Cmpd	R ester in 5 or Ar1 in 25/26	R’ ester in 5 or Ar2 in 25/26	OH/AC	1h EC50 [nM] K562	n	1h EC50 [nM] U937	n
1 25	NA	NA	OH	>100,000		NA
5 26	OCH2OC(O)C(CH3)3	OPh	OH	11		NA
5	OCH2OC(O)C(CH3)3	O-p-iPrPh	OH	3.8		NA
5	OCH2OC(O)Ph	OPh	OH	4.1		NA
17	OCH2OC(O)CHCBr2	Ph	OH	77 (67 to 89)	4	45 (25 to 81)	3
18	OCH2OC(O)CHCBr2	Ph	Ac	11 (2.2 to 53)	4	20 (10 to 39)	3
25a	Ph-triazole	Ph	OH	2.3 (1.5 to 3.7)	4	1.8 (0.21 to 15)	3
26a	Ph-triazole	Ph	Ac	2.7 (2.2 to 3.3)	4	10 (3.9 to 26)	3
25b	p-FPh-triazole	Ph	OH	0.60 (0.35 to 1.2)	4	1.6 (0.31 to 8.3)	3
26b	p-FPh-triazole	Ph	Ac	2.5 (1.6 to 3.8)	4	6.3 (2.3 to 17)	3
25c	p-iPrPh-triazole	Ph	OH	11 (5.6 to 20)	3	1.8 (0.43 to 7.9)	3
26c	p-iPrPh-triazole	Ph	Ac	8.1 (6.6 to 10)	3	2.1 (0.58 to 7.5)	3
25d	Ph-triazole	p-iPrPh	OH	1.9 (0.54 to 6.9)	4	1.4 (0.14 to 14)	3
26d	Ph-triazole	p-iPrPh	Ac	1.7 (0.47 to 6.1)	4	4.4 (0.52 to 38)	3
25e	p-FPh-triazole	p-iPrPh	OH	2.3 (1.3 to 4.0)	4	3.6 (0.38 to 35)	3
26e	p-FPh-triazole	p-iPrPh	Ac	2.7 (1.1 to 6.5)	4	6.0 (1.6 to 22)	3
25f	p-iPrPh-triazole	p-iPrPh	OH	2.6 (1.7 to 4.0)	4	25 (6.7 to 90)	3
26f	p-iPrPh-triazole	p-iPrPh	Ac	1.7 (1.1 to 2.6)	4	26 (17 to 39)	3
25g	2,4-diFPh-triazole	p-(CH2)2OCH3-Ph	OH	10 (7.0 to 15)	3	13 (3.6 to 47)	3
26g	2,4-diFPh-triazole	p-(CH2)2OCH3-Ph	Ac	9.3 (5.3 to 16)	3	7.9 (2.7 to 23)	3

When both activity assays are considered, there are clear differences in activity dependent upon the four triazole substituents that were tested. Compounds 25g/26g containing the 2,4-fluoro phenyl triazole were among the least potent of the group, so that additional analogs of these compounds were not evaluated. Compounds containing the para-isopropyl triazole structure (25c/26c/25f/26f) also had lower activities. On the other hand, compounds containing the phenyl triazole (25a/26a/25d/26d) or the para-fluoro phenyl triazole (25b/26b/25e/26e) were generally more potent. This suggests that the bulkier triazole esters reduced the activity relative to the more compact structures.

Notably, in both K562 and U937 cells, minimal toxicity was observed following a 72-hour incubation with 10 µM of each compound (Figure S1). As this is a much higher dose and much longer incubation period than the functional assays, it is likely that minimal cytotoxicity is observed from either the payload, the protecting groups, or their metabolites.

The compounds were further assessed for stability in plasma using positive mode LC-MS. (Table 4). We evaluated human, mouse and rat plasma, which are important for future in vivo evaluation. Here, half-lives in human plasma ranged from 16 minutes to 106 minutes, a 6.6-fold range. The most stable compounds in human plasma were 25c with a half-life of 106 minutes, 26f with a half-life of 103 minutes, and 26c with a half-life of 89 minutes. All three of these compounds were more stable than the comparator compounds 5b. Both 25c/25d and 26f contained the p-iPr-Ph triazole, suggesting this group is the most optimal for stability. At the same time, the smaller phenyl triazole and para-fluoro phenyl triazole esters generally displayed lower plasma stability, which is the opposite pattern relative to their activity. Stability in mouse plasma was considerably lower than in human plasma, with an observed 20-fold decrease in the average half-life. This is consistent with prior studies which have also found higher esterase activity in mouse versus human plasma.^43–45 Correlation with the mouse plasma was found to be moderate (r = 0.52), suggesting that there are slightly different structure-activity relationships controlling human and mouse plasma stability. In the mouse plasma, compound 26b was the most stable, with a half-life of 8.8 minutes. Rat plasma gave intermediate values.

Table 4.

Stability of triazole prodrugs in human and mouse plasma.

Cmpd	R ester in 5 or Ar1 in 25/26	R’ ester in 5 or Ar2 in 25/26	OH/Ac	Human t1/2 (min) (n=2)	Mouse t1/2 (min) (n=2)	Rat t1/2 (min) (n=2)
5	OCH2OC(O)C(CH3)3	OPh	OH	10	n.d.	n.d.
5	OCH2OC(O)C(CH3)3	O-p-iPrPh	OH	78	n.d.	n.d.
5	OCH2OC(O)Ph	OPh	OH	4	n.d.	n.d.
25a	Ph-triazole	Ph	OH	16	1.1	2.2
26a	Ph-triazole	Ph	Ac	32	3.1	12
25b	p-FPh-triazole	Ph	OH	28	2.0	3.6
26b	p-FPh-triazole	Ph	Ac	63	8.8	12
25c	p-iPrPh-triazole	Ph	OH	106	3.0	10
26c	p-iPrPh-triazole	Ph	Ac	89	3.6	12
25d	Ph-triazole	p-iPrPh	OH	46	1.0	<1
26d	Ph-triazole	p-iPrPh	Ac	30	1.0	1.3
25e	p-FPh-triazole	p-iPrPh	OH	35	1.1	9.8
26e	p-FPh-triazole	p-iPrPh	Ac	47	3.6	13
25f	p-iPrPh-triazole	p-iPrPh	OH	75	0.84	2.6
26f	p-iPrPh-triazole	p-iPrPh	Ac	103	5.8	43
25g	2,4-diFPh-triazole	p-(CH2)2OCH3-Ph	OH	29	<1	<1
26g	2,4-diFPh-triazole	p-(CH2)2OCH3-Ph	Ac	22	<1	<1

We evaluated the metabolism of the compounds in plasma and cells. Figure 3 shows the possible expected metabolites resulting from enzymatic removal of the acetate, the triazole ester, and the aryl ester. Through detection (or lack thereof) we could determine the preferred route(s) of metabolism in plasma and cells. We first used negative mode LC-MS to determine what metabolites were present in the plasma (Table 5). All acetylated compounds were readily converted to their non-acetylated, triazole ester prodrug forms. The acetylated compounds could also be converted to their acetylated aryl monoanionic form. This suggests that the rates of removal of the triazole ester and the allylic acetate are similar. Both metabolites are readily converted to the aryl monoanion form. No compound could be converted all the way to the dianion payload in human plasma, demonstrating that human plasma lacks the capability to hydrolyze the phospho-phenyl ester. Only the triazole esters could be cleaved, as neither the triazole monoanions nor the acetylated payload could be found. Although the peak intensities cannot be directly compared due to potential differences in ionization, the most stable compounds in the plasma metabolism study, 25c and 26f, produced the lowest amounts of aryl monoanion in this study as would be expected. The aryl monoanion form is still expected to retain some cell permeability.

Figure 3.

Metabolism of triazole phosphoantigen prodrugs. The central pathway represents the major pathway of metabolism, where all compounds were observed by LC-MS. The first two steps happen in both plasma and cells, while conversion to the dianion was only observed in cells. Compounds in grey are theoretically possible, but were not found in this study.

Table 5.

Human Plasma Metabolites Identified by Negative Mode LC-MS.

Cmpd	Ar1 in 25/26	Ar2 in 25/26	OH/AC	acetylated aryl monoanion	aryl monoanion
25a	Ph-triazole	Ph	OH		13000 ± 3000
26a	Ph-triazole	Ph	Ac	8800 ± 5400	1800 ± 1000
25b	p-FPh-triazole	Ph	OH		6000 ± 3900
26b	p-FPh-triazole	Ph	Ac	2100 ± 1500	410 ± 290
25c	p-iPrPh-triazole	Ph	OH		2700 ± 1900
26c	p-iPrPh-triazole	Ph	Ac	2400 ± 1100	80 ± 38
25d	Ph-triazole	p-iPr-Ph	OH		11000 ± 4100
26d	Ph-triazole	p-iPr-Ph	Ac	4200 ± 1800	730 ± 310
25e	p-FPh-triazole	p-iPr-Ph	OH		11000 ± 2500
26e	p-FPh-triazole	p-iPr-Ph	Ac	6800 ± 2300	1900 ± 720
25f	p-iPrPh-triazole	p-iPr-Ph	OH		15000 ± 6200
26f	p-iPrPh-triazole	p-iPr-Ph	Ac	880 ± 61	69 ± 19

We then evaluated the delivery of the payload into K562 cells (Figure 3, Table 6). In contrast to the plasma metabolism, the K562 cells were able to convert all of the compounds to the desired payload. Here, the peak intensities of the desired payload are directly comparable as the payload is identical in all twelve compounds. Compounds 26e, 26d, and 25f appeared to deliver the most desired payload into K562 cells. In contrast to our prior study, here we did not observe a clear correlation between payload delivery and activity in the ELISA as expected. It is possible that this class of compounds did not exhibit a wide enough range of activities in the metabolism study (8.9-fold) which obscured the structure activity relationships, or that the exposure time was not sufficiently long to fully assess conversion to the desired payload. Still, there is selectivity achieved as cells, but not plasma, can release the desired payload.

Table 6.

Delivery of Payload into K562 Cells after 1 Hour Exposure.

Cmpd	Ar1 in 25/26	Ar2 in 25/26	OH/AC	aryl monoanion (integrated peak intensity)	desired payload (integrated peak intensity)
25a	Ph-triazole	Ph	OH	10000 ± 4500	1500 ± 560
26a	Ph-triazole	Ph	Ac	7800 ± 4600	1200 ± 1300
25b	p-FPh-triazole	Ph	OH	11000 ± 6100	580 ± 250
26b	p-FPh-triazole	Ph	Ac	4700 ± 2700	400 ± 4.7
25c	p-iPrPh-triazole	Ph	OH	22000 ± 9000	900 ± 100
26c	p-iPrPh-triazole	Ph	Ac	7000 ± 1600	770 ± 110
25d	Ph-triazole	p-iPrPh	OH	17000 ± 11000	1700 ± 1300
26d	Ph-triazole	p-iPrPh	Ac	800 ± 600	2300 ± 2900
25e	p-FPh-triazole	p-iPrPh	OH	22000 ± 12000	1400 ± 700
26e	p-FPh-triazole	p-iPrPh	Ac	33000 ± 2400	2400 ± 330
25f	p-iPrPh-triazole	p-iPrPh	OH	21000 ± 19000	2300 ± 2300
26f	p-iPrPh-triazole	p-iPrPh	Ac	320 ± 200	270 ± 160

DISCUSSION

In this study, we describe the chemical synthesis, phosphoantigen activity, and stability/metabolism of a series of novel triazole-containing prodrugs. Although development of a synthetic sequence based on late-stage divergence through click reactions has proven elusive, the compounds were readily synthesized through a more linear sequence and obtained in acceptable yields. Several compounds exhibited sub-nanomolar phosphoantigen potency, and these are among the most potent phosphoantigen prodrugs described to date. The stability in human plasma is modest but nevertheless it represents a marked improvement over other ester prodrug forms. Based on studies by LCMS it is clear that although the parent prodrugs are partially metabolized by esterase enzymes found in plasma, they are not fully converted to the desired payloads until after they have entered cells. In contrast to metabolism in plasma, the K562 cells were able to take up all of these new prodrug forms and convert all of the compounds completely to the desired payload.

The synthesis of these compounds proved to be straightforward. The limiting factor in the synthetic sequence remains the allylic oxidation step mediated by selenium dioxide which gives modest yields. However, we continue to find its preference for the desired E configuration avoids the need for challenging separations of E- and Z-olefin isomers and that outweighs its modest yield.

When considering potency alone, a range of potency was observed. On the one hand are compounds 25f/26f. These compounds were among the least potent and least soluble. Compound 26f also delivered the lowest amount of free payload to the K562 cells. We suspect that its relatively high cLogP of 6.37 may have contributed to its poor performance. On the other hand, compounds 25b/26b containing the para-fluorophenyl triazole were the most active, followed closely by 25a/26a containing the undecorated phenyl ring. These triazoles also had the benefit of the lowest cLogP values of any aryl acyloxyesters prodrugs, which resulted in excellent water solubility. Although these compounds were less stable in plasma than some of their counterparts, we believe the potency and solubility outweigh the plasma stability, making these the most interesting compounds of the set to date. Thus, they may be appropriate for future studies in vivo, and serve as models for the design of still more efficacious prodrug forms.

To our knowledge this is the first report of triazole incorporation into the ester component of phosphonate ester prodrugs. We have identified the para-fluorophenyl triazole compounds 25b/26b as the most favorable of the group. Conclusions can also be drawn by comparison of this prodrug form to other known phosphonate prodrug forms, including other aryl esters and aryl amidates (also known as ProTides).⁴⁶ Under time-restricted conditions meant to model exposure after in vivo administration, aryl amidates have a lower rate of internalization into cells relative to aryl ester forms,²⁵ as the aryl amidate potency in 1 hour cytokine experiments is moderate (110-230 nM EC₅₀ range). By comparison compound 25b is more than 100-fold more active (EC₅₀ = 0.6 nM). This demonstrates that triazole esters can achieve faster cellular activation relative to aryl amidates and bisamidates. In fact, the 1-hour potency of the best triazole esters is similar to the best aryl ester forms.

At the same time, the aryl amidate forms exhibit high plasma stability exceeding 24 hours, while the bis-ester forms have very low plasma stability with half-lives under 5 minutes. The triazole esters described here represent a significant improvement in human plasma stability relative to bis-ester forms. For example, the most stabile triazole ester, 25c, had a half-life of 106 minutes, a marked improvement relative to the bis-POM form. Furthermore, we have recently found an aryl ester prodrug with plasma stability of 750 minutes,³² but unfortunately the cLogP of that compound was 5.7. The current compounds 25b/26b have cLogP values of 3.65/4.06, a marked improvement in solubility due to the triazole.

There are several key challenges to the future translation of these compounds to in vivo and clinical use. Because BTN3A1 and Vγ9Vδ2 T cells are expressed by primates but not rodents, in vivo models are difficult. Currently, monkey models or humanized mouse models appear to be the best options for in vivo evaluation. Future advancements in the development of pAg-responsive humanized mouse tumor models would enhance the assessment of these prodrug forms. The field would also benefit from a better understanding of how pAgs and Vγ9Vδ2 T cells can effectively be used for cancer therapy, whether as small-molecule therapies, small-molecule vaccines, or cell-based therapies. Regardless of the treatment modality, it is believed that a balance of potency, solubility, high plasma stability, and rapid cellular delivery are the most critical factors to optimize for synthetic pAgs. This assumes that the compounds would be administered similarly to remdesivir, via IV administration.

In conclusion, the triazole-containing phosphonate ester prodrugs have an improved prodrug scaffold which provides excellent potency and solubility combined with good plasma stability. Future studies may find even further improvements to this scaffold.

5. Experimental

5.1. General Procedures.

Diethyl ether and tetrahydrofuran were freshly distilled from sodium/benzophenone, while acetonitrile and methylene chloride were distilled from calcium hydride prior to use. All other reagents were purchased from commercial sources and used without further purification. All the reactions in nonaqueous solvents were conducted in flame-dried glassware under a positive pressure of nitrogen and with magnetic stirring. All NMR spectra were obtained at 400 or 500 MHz for ¹H, and 101 or 125 MHz for ¹³C, 161 or 203 MHz for ³¹P and 471 MHz for ¹⁹F with internal standards of (CH₃)₄Si (¹H, 0.00) or CDCl₃ (¹H, 7.27; ¹³C, 77.2 ppm) for non-aqueous samples. The ³¹P chemical shifts were reported in ppm relative to 85% H₃PO₄ (external standard). High resolution mass spectra (HRMS) were obtained at the University of Iowa Mass Spectrometry Facility (Thermo Q-Exactive Orbitrap). Silica gel (60 Å, 0.040–0.063 mm) was used for flash chromatography. The purity profile of each assayed compound was evaluated with an Agilent 1220 series HPLC (100% methanol, Column: Restek ultrasilica, 5 μm, C18, dimensions 250 x 4.6 mm (analytical), flow rate: 1.0 mL/min.) and all assayed compounds had a purity > 99%.

5.2. Chemical Synthesis

General synthetic procedure A:

The mixed aryl methyl ester was dissolved in freshly distilled acetonitrile and concentrated in vacuo 3 times and then added to a solution of the chloromethyl (2.5 eq) and sodium iodide (1.5 eq) in acetonitrile. The solution was heated to reflux and maintained for 3 days while monitored by TLC analysis. The reaction then was allowed to cool to room temperature, extracted with diethyl ether, and the combined extracts were washed with brine. The organic portions were combined, dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified via flash chromatography (silica, 100% hexanes to 30% EtOAc in hexanes), and the resulting product was isolated as an oil.

General synthetic procedure B:

The monocid was added to a flask with tetrabutylammonium hydrogen sulfate (0.1 eq), and sodium bicarbonate (4.0 eq), followed by addition of CH₂Cl₂ (20 mL) and H₂O (20 mL) to provide a biphasic mixture. The reaction mixture was stirred at room temperature for 2 h. After it was cooled to 0 °C, chloromethyl chlorosulfate (1.4 eq) was added. The mixture was vigorously stirred for 24 h before extraction with CH₂Cl₂ (3 × 10 mL). The extract was dried (Na₂SO₄) and concentrated in vacuo.³⁸

Chloromethyl-3,3-dibromo acrylate (14).

Following general procedure B, the 3,3-dibromo acrylic acid 13 (1.2 g, 5.2 mmol) afforded chloromethyl ester 14 (1.3 g, 89%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 7.01 (s, 1H), 5.74 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 166.3, 132.3, 99.3, 69.3.

(((4-Methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-3,3-dibromoacrylate (16).

Following general procedure A, the mixed aryl methyl ester 15 (0.6 g, 2.36 mmol) afforded the mixed acyloxy ester 16 (480 mg, 38%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 40% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 7.34 – 7.29 (m, 2H), 7.20 – 7.16 (m, 3H), 6.70 (s, 1H), 5.73 (dd, J_PH = 16.0 Hz, J_HH = 5.3 Hz, 1H), 5.60 (dd, J_PH = 11.7 Hz, J_HH = 5.3 Hz, 1H), 5.10 (td, J = 7.1, 1.1 Hz, 1H), 2.40 – 2.31 (m, 2H), 2.02 – 1.94 (m, 2H), 1.67 (s, 3H), 1.60 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 159.8, 148.8 (d, J_PC = 9.9 Hz), 136.3, 132.6, 130.1 (2C), 123.2, 122.8 (2C), 122.7 (d, J_PC = 17.2 Hz), 108.6, 82.2 (d, J_PC = 6.7 Hz), 25.3 (d, J_PC = 139.3 Hz), 25.5, 20.6 (d, J_PC = 4.8 Hz), 17.5; ³¹P NMR (162 MHz, CDCl₃) 30.0 ppm.

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-3,3-dibromoacrylate (17).

The olefin 16 (360 mg, 0.74 mmol) was added to a suspension of selenium dioxide (66 mg, 0.59 mmol) and 4-hydroxybenzoic acid (15 mg, 0.10 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.411 mL, 2.98 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 15 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 17 as an oil in 27% yield (100 mg): ¹H NMR (400 MHz, CDCl₃) δ 7.37 – 7.33 (m, 2H), 7.23 – 7.29 (m, 3H), 6.73 (s, 1H), 5.76 (dd, J_PH = 16.0 Hz, J_HH = 5.3 Hz, 1H), 5.63 (dd, J_PH = 11.7 Hz, J_HH = 5.3 Hz, 1H), 5.44 (td, J = 7.0, 1.1 Hz, 1H), 4.0 (s, 2H), 2.50 – 2.41 (m, 2H), 2.09 – 2.01 (m, 2H), 1.68 (s, 3H): ¹³C NMR (101 MHz, CDCl₃) δ 161.0, 149.6 (d, J_PC = 9.7 Hz), 136.6, 131.5, 129.6 (2C), 126.2, 125.1, 122.7 (d, J_PC = 16.2 Hz), 120.5 (d, J_PC = 4.2 Hz), 109.9, 81.3 (d, J_PC = 6.8 Hz), 67.9, 25.9 (d, J_PC = 140.0 Hz), 20.2 (d, J_PC = 5.0 Hz), 13.5; ³¹P NMR (162 MHz, CDCl₃) 29.5 ppm. HRMS (ESI+) m/z calculated for C₁₆H₂₀Br₂O₆P (M+H)⁺ 498.9344, found 498.9335. HPLC purity >99% (t_R = 3.9).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-3,3-dibromoacrylate (18).

Alcohol 17 (25 mg, 0.05 mmol), acetic anhydride (7.6 mg, 0.074 mmol) and triethylamine (10 mg, 0.099 mmol) were dissolved in freshly distilled methylene chloride (4 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (5 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 18 was isolated as an oil in 92% yield (25 mg): ¹H NMR (400 MHz, CDCl₃) δ 7.33 – 7.26 (m, 2H), 7.16 – 7.11 (m, 3H), 6.65 (s, 1H), 5.68 (dd, J_PH = 16.3 Hz, J_HH = 5.2 Hz, 1H), 5.56 (dd, J_PH = 11.6 Hz, J_HH = 5.3 Hz, 1H), 5.40 (td, J = 7.1, 1.1 Hz, 1H), 4.4 (s, 2H), 2.44 – 2.35 (m, 2H), 2.02 – 1.93 (m, 2H), 2.01 (s, 3H), 1.61 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.0, 161.3, 149.9 (d, J_PC = 9.5 Hz), 132.2, 129.6 (2C), 126.8 (d, J_PC = 17.3 Hz), 126.5 (2C), 125.5, 120.8 (d, J_PC = 4.3 Hz), 110.2, 81.7 (d, J_PC = 6.9 Hz), 69.6, 26.1 (d, J_PC = 140.8 Hz), 21.1, 20.6 (d, J_PC = 4.8 Hz), 14.1; ³¹P NMR (162 MHz, CDCl₃) 29.2 ppm. HRMS (ESI+) m/z calculated for C₁₈H₂₂Br₂O₇P (M+H)⁺ 540.9449, found 540.9450. HPLC purity >99% (t_R = 3.3).

Chloromethyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (22, Ar¹ = phenyl).

Following general procedure B, the 1-phenyl-1H-1,2,3-triazole-4-carboxylic acid 21 (1.8 g, 9.5 mmol) afforded chloromethyl ester 22 (1.9 g, 84%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 8.67 (s, 1H), 7.69 – 7.64 (m, 2H), 7.46 – 7.37 (m, 3H), 5.86 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 158.6, 140.5, 139.1, 130.2 (2C), 129.9, 127.0, 120.9 (2C), 69.3.

Chloromethyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (22, Ar¹ = p-fluorophenyl).

Following general procedure B, the 1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylic acid 21 (1.8 g, 8.7 mmol) afforded chloromethyl ester 22 (2.0 g, 90%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 8.58 (s, 1H), 7.78 – 7.75 (m, 2H), 7.30 – 7.25 (m, 2H), 5.99 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 162.9 (d, J_CF = 251.6 Hz), 158.4, 150.5, 139.3, 126.6, 122.9 (d, J_CF = 8.8 Hz, 2C), 117.1 (d, J_CF = 23.5 Hz, 2C), 69.1.

Chloromethyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (22, Ar¹ = p-isopropylphenyl).

Following general procedure B, the 1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylic acid 21 (2.2 g, 9.5 mmol) afforded the chloromethyl ester 22 (2.5 g, 95%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 6.01 (s, 2H), 2.80 – 2.73 (m, 1H), 1.29 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 158.4, 150.8, 133.8, 127.8 (3C), 126.3, 120.7 (2C), 68.9, 33.7, 23.6 (2C).

Chloromethyl-1-(2,4-difluorophenyl)-1H-1,2,3-triazole-4-carboxylate (22 Ar¹ = 2,4-difluorophenyl).

Following general procedure B, the 1-(2,4-difluorophenyl)-1H-1,2,3-triazole-4-carboxylic acid 21 (1.4 g, 6.2 mmol) afforded the chloromethyl ester 22 (1.6 g, 94%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H), 7.97 – 7.91 (m, 1H), 7.13 – 7.06 (m, 2H), 5.97 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 162.8 (d, J_CF = 255.3 Hz), 162.7 (d, J_CF = 255.3 Hz), 154.8 (d, J_CF = 12.5 Hz), 152.3 (d, J_CF = 12.4 Hz), 138.8, 129.2 (d, J_CF = 10.2 Hz), 126.2 (d, J_CF = 12.4 Hz), 112.7 (d, J_CF = 22.9 Hz), 105.4 (d, J_CF = 23.6 Hz), 68.9.

(((4-Methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (24a).

Following general procedure A, the mixed aryl methyl ester 23 (1.1 g, 4.3 mmol) afforded the mixed acyloxy ester 24a (630 mg, 33%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 70% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.72 – 7.69 (m, 2H), 7.58 – 7.49 (m, 3H), 7.22 – 7.18 (m, 4H), 6.98 – 6.94 (m, 1H), 5.99 (dd, J_PH = 15.4 Hz, J_HH = 5.2 Hz, 1H), 5.81 (dd, J_PH = 11.9 Hz, J_HH = 5.2 Hz, 1H), 5.12 (td, J = 7.2, 1.1 Hz, 1H), 2.42 – 2.33 (m, 2H), 2.06 – 1.97 (m, 2H), 1.64 (s, 3H), 1.57 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 158.3, 149.4 (d, J_PC = 9.7 Hz), 138.6, 135.8, 133.3, 129.7 (2C), 129.4 (2C), 126.0, 124.7, 122.8 (d, J_PC = 15.5 Hz), 120.4, 120.3 (4C), 81.3 (d, J_PC = 6.7 Hz), 25.9 (d, J_PC = 140.0 Hz), 25.3, 20.4 (d, J_PC = 5.0 Hz), 17.3; ³¹P NMR (162 MHz, CDCl₃) 29.7 ppm.

(((4-Methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (24b).

Following general procedure A, the mixed aryl methyl ester 23 (1.45 g, 5.7 mmol) afforded the mixed acyloxy ester 24b (1.0 g, 38%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 70% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.73 – 7.69 (m, 2H), 7.26 – 7.15 (m, 6H), 6.97 – 6.93 (m, 1H), 5.97 (dd, J_PH = 15.2 Hz, J_HH = 5.2 Hz, 1H), 5.80 (dd, J_PH = 12.0 Hz, J_HH = 5.3 Hz, 1H), 5.08 (td, J = 7.2, 1.1 Hz, 1H), 2.40 - 2.32 (m, 2H), 2.08 – 1.97 (m, 2H), 1.63 (s, 3H), 1.56 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 162.9 (d, J_CF = 251.6 Hz), 158.5, 149.7 (d, J_PC = 9.7 Hz), 138.9, 133.3, 132.2, 129.5 (3C), 126.1, 124.8, 122.6 (d, J_PC = 15.6 Hz), 122.5 (d, J_CF = 8.8 Hz, 2C), 120.5 (d, J_CF = 4.2 Hz), 116.8 (d, J_CF = 23.4 Hz, 2C), 81.6 (d, J_PC = 6.6 Hz), 25.9 (d, J_PC = 139.9 Hz), 25.3, 20.1 (d, J_PC = 5.0 Hz), 17.4; ³¹P NMR (162 MHz, CDCl₃) 30.1 ppm.

(((4-Methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (24c).

Following general procedure A, the mixed aryl methyl ester 23 (1.15 g, 4.4 mmol) afforded the mixed acyloxy ester 24c (592 mg, 27%) as a colorless oil after purification by column chromatography (silica, 100% hexane – 50% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.6 Hz, 2H), 7.24 – 7.19 (m, 4H), 7.01 – 6.97 (m, 1H), 5.99 (dd, J_PH = 15.2 Hz, J_HH = 5.2 Hz, 1H), 5.82 (dd, J_PH = 11.9 Hz, J_HH = 5.2 Hz, 1H), 5.10 (td, J = 7.1, 1.2 Hz, 1H), 3.03 – 2.96 (m, 1H), 2.43 – 2.34 (m, 2H), 2.06 – 1.96 (m, 2H), 1.65 (s, 3H), 1.58 (s, 3H), 1.29 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 159.2, 151.3, 150.3 (d, J_PC = 9.8 Hz), 139.4, 134.5, 134.0, 130.1 (2C), 128.4 (3C), 126.4, 125.4, 122.7 (d, J_PC = 17.3 Hz), 121.2 (3C), 82.2 (d, J_PC = 6.8 Hz), 34.4, 26.9 (d, J_PC = 139.9 Hz), 26.1, 24.3 (2C), 21.2 (d, J_PC = 4.9 Hz), 18.1; ³¹P NMR (162 MHz, CDCl₃) 30.0 ppm.

((4-Isopropylphenoxy)(4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (24d).

Following general procedure A, the mixed aryl methyl ester 23 (1.33 g, 4.49 mmol) afforded the mixed acyloxy ester 24d (830 mg, 38%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 70% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 8.43 (s, 1H), 7.70 – 7.67 (m, 2H), 7.52 – 7.42 (m, 3H), 7.08 – 7.04 (m, 4H), 5.95 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.84 (dd, J_PH = 12.4 Hz, J_HH = 5.2 Hz, 1H), 5.05 (td, J = 7.2, 1.1 Hz, 1H), 2.75 – 2.68 (m, 1H), 2.38 - 2.29 (m, 2H), 2.01 – 1.92 (m, 2H), 1.59 (s, 3H), 1.52 (s, 3H), 1.07 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 158.5, 147.4 (d, J_PC = 9.4 Hz), 145.3, 138.8, 135.8, 133.0, 129.6 (3C), 129.3, 127.2 (2C), 126.8, 122.0 (d, J_PC = 15.7 Hz), 120.3 (2C), 120.0, 81.5 (d, J_PC = 6.4 Hz), 33.0, 25.9 (d, J_PC = 139.2 Hz), 25.2, 23.5 (2C), 20.5 (d, J_PC = 5.0 Hz), 17.3; ³¹P NMR (162 MHz, CDCl₃) 29.8 ppm.

(((4-Isopropylphenoxy)(4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (24e).

Following general procedure A, the mixed aryl methyl ester 23 (1.1 g, 3.71 mmol) afforded the mixed acyloxy ester 24e (850 mg, 46%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 70% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 8.40 (s, 1H), 7.71 – 7.68 (m, 2H), 7.24 – 7.20 (m, 2H), 7.08 (s, 4H), 5.96 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.86 (dd, J_PH = 12.6 Hz, J_HH = 5.2 Hz, 1H), 5.07 (td, J = 7.2, 1.1 Hz, 1H), 2.78 – 2.71 (m, 1H), 2.39 – 2.30 (m, 2H), 2.02 – 1.94 (m, 2H), 1.62 (s, 3H), 1.54 (s, 3H), 1.10 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 162.9 (d, J_CF = 251.5 Hz), 158.7, 147.8 (d, J_PC = 9.4 Hz), 145.7, 139.4, 133.5, 132.4, 127.6 (3C), 126.3, 122.7 (d, J_PC = 15.7 Hz), 122.6 (d, J_CF = 8.8 Hz, 2C), 120.4 (d, J_CF = 4.2 Hz), 117.1 (d, J_CF = 23.4 Hz, 2C), 81.9 (d, J_PC = 6.3 Hz), 33.4, 25.9 (d, J_PC = 139.1 Hz), 25.6, 23.9 (2C), 20.8 (d, J_PC = 5.0 Hz), 17.7; ³¹P NMR (162 MHz, CDCl₃) 28.8 ppm.

(((4-Isopropylphenoxy)(4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (24f).

Following general procedure A, the mixed aryl methyl ester 23 (1.2 g, 4.0 mmol) afforded the mixed acyloxy ester 24f (770 mg, 36%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 70% ethyl acetate in hexane); ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H), 7.63 – 7.60 (m, 2H), 7.41 – 7.38 (m, 2H), 7.10 (s, 4H), 5.99 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.88 (dd, J_PH = 12.4 Hz, J_HH = 5.2 Hz, 1H), 5.10 (td, J = 7.3, 1.2 Hz, 1H), 3.03 – 2.96 (m, 1H), 2.80 – 2.73 (m, 1H), 2.42 – 2.33 (m, 2H), 2.06 – 1.97 (m, 2H), 1.65 (s, 3H), 1.57 (s, 3H), 1.28 (d, J = 6.9 Hz, 6H), 1.12 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 158.9, 151.0, 147.8 (d, J_PC = 9.4 Hz), 145.8, 139.2, 134.2, 133.5, 128.0 (3C), 127.7 (2C), 126.2, 122.5 (d, J_PC = 15.7 Hz), 120.8 (2C), 120.5, 82.0 (d, J_PC = 6.3 Hz), 34.0, 33.5, 26.4 (d, J_PC = 139.2 Hz), 25.7, 24.0 (2C), 23.9 (2C), 20.9 (d, J_PC = 5.0 Hz), 17.7; ³¹P NMR (162 MHz, CDCl₃) 29.8 ppm.

4-(2-Methoxyethyl)phenyl methyl (4-methylpent-3-en-1-yl)phosphonate (23).

Following a known procedure,^{26, 28} the dimethyl phosphonate ester²⁶ (1.98 g, 10.1 mmol) afforded the phosphonate 23 (2.1 g, 67%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 60% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 7.13 – 7.03 (m, 4H), 5.07 (td, J = 7.2, 1.1 Hz, 1H), 3.74 (d, J_PH = 11.0 Hz, 3H), 3.51 (t, J_HH = 7.0 Hz, 2H), 3.28 (s, 3H), 2.79 (t, J_HH = 6.92 Hz, 2H), 2.34 – 2.26 (m, 2H), 1.89 – 1.81 (m, 2H), 1.63 (s, 3H), 1.56 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 148.7 (d, J_PC = 8.4 Hz), 135.2, 132.8, 129.7 (3C), 122.4 (d, J_PC = 17.3 Hz), 119.9 (d, J_PC = 4.2 Hz), 73.1, 58.3, 52.2, 35.1, 25.2 (d, J_PC = 138.7 Hz), 25.3, 20.6 (d, J_PC = 4.8 Hz), 17.3; ³¹P NMR (162 MHz, CDCl₃) 30.1 ppm.

(((4-(2-Methoxyethyl)phenoxy)(4-methylpent-3-en-1-yl)phosphoryl)oxy)methyl-1-(2,4-difluorophenyl)-1H-1,2,3-triazole-4-carboxylate (24g).

Following general procedure A, the mixed aryl methyl ester 23 (1.5 g, 4.80 mmol) afforded the mixed acyloxy ester 24g (630 mg, 25%) as a colorless oil after purification by column chromatography (silica, 100% hexane to 70% ethyl acetate in hexane): ¹H NMR (400 MHz, CDCl₃) δ 8.44 (s, 1H), 7.93 – 7.88 (m, 1H), 7.09 – 7.04 (m, 6H), 5.93 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.83 (dd, J_PH = 12.4 Hz, J_HH = 5.2 Hz, 1H), 5.05 (td, J = 7.2, 1.1 Hz, 1H), 3.45 (t, J_HH = 7.0 Hz, 2H), 3.25 (s, 3H), 2.68 (t, J_HH = 6.92 Hz, 2H), 2.38 – 2.29 (m, 2H), 2.01 – 1.93 (m, 2H), 1.60 (s, 3H), 1.53 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 162.7 (d, J_CF = 255.2 Hz), 162.6 (d, J_CF = 255.4 Hz), 158.2, 154.7 (d, J_CF = 12.4 Hz), 152.2 (d, J_PC = 12.4 Hz), 147.9 (d, J_PC = 9.5 Hz), 138.8, 135.6, 133.0, 129.7 (2C), 128.7 (d, J_CF = 7.6 Hz), 126.0 (d, J_CF = 10.0 Hz), 122.0 (d, J_PC = 15.7 Hz), 120.1 (d, J_CF = 4.2 Hz), 112.7 (d, J_CF = 3.7 Hz), 105.3 (d, J_CF = 23.7 Hz), 81.6 (d, J_PC = 6.3 Hz), 72.9, 58.2, 35.0, 26.1 (d, J_PC = 138.1 Hz), 25.2, 20.4 (d, J_PC = 5.1 Hz), 17.3; ³¹P NMR (162 MHz, CDCl₃) 29.8 ppm.

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (25a).

The olefin 24a (630 mg, 1.42 mmol) was added to a suspension of selenium dioxide (126 mg, 1.13 mmol) and 4-hydroxybenzoic acid (27 mg, 0.19 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.788 mL, 5.70 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25a as an oil in 24% yield (160 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.72 – 7.70 (m, 2H), 7.58 – 7.49 (m, 3H), 7.24 – 7.19 (m, 4H), 7.01 – 6.98 (m, 1H), 5.98 (dd, J_PH = 15.4 Hz, J_HH = 5.2 Hz, 1H), 5.82 (dd, J_PH = 11.9 Hz, J_HH = 5.2 Hz, 1H), 5.42 (td, J = 7.2, 1.2 Hz, 1H), 3.96 (s, 2H), 2.50 – 2.40 (m, 2H), 2.11 – 2.03 (m, 2H), 1.62 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 158.5, 149.6 (d, J_PC = 9.7 Hz), 138.8, 136.6, 135.9, 129.8 (2C), 129.5 (2C), 126.0, 124.9, 122.7 (d, J_PC = 15.5 Hz), 120.5 (5C), 81.5 (d, J_PC = 6.7 Hz), 67.9, 25.9 (d, J_PC = 140.0 Hz), 20.2 (d, J_PC = 5.0 Hz), 13.5; ³¹P NMR (162 MHz, CDCl₃) 29.7 ppm. HRMS (ESI+) m/z calculated for C₂₂H₂₅N₃O₆P (M+H)⁺ 458.1481, found 458.1461. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (26a).

Alcohol 25a (35 mg, 0.076 mmol), acetic anhydride (10 mg, 0.098 mmol) and triethylamine (13 mg, 0.13 mmol) were dissolved in freshly distilled methylene chloride (4 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (5 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26a was isolated as an oil in 84% yield (32 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.67 – 7.65 (m, 2H), 7.52 – 7.43 (m, 3H), 7.16 – 7.11 (m, 4H), 6.94 – 6.91 (m, 1H), 5.93 (dd, J_PH = 15.9 Hz, J_HH = 5.2 Hz, 1H), 5.75 (dd, J_PH = 11.6 Hz, J_HH = 5.2 Hz, 1H), 5.40 (td, J = 7.2, 1.1 Hz, 1H), 4.36 (s, 2H), 2.46 – 2.36 (m, 2H), 2.09 – 1.99 (m, 2H), 1.59 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.3, 159.1, 150.1 (d, J_PC = 9.7 Hz), 139.5, 136.6, 132.4, 130.5 (2C), 130.2 (2C), 127.1 (d, J_PC = 17.1 Hz), 126.5, 125.5, 121.2 (5C), 82.2 (d, J_PC = 6.7 Hz), 69.9, 26.4 (d, J_PC = 140.5 Hz), 21.4, 21.0 (d, J_PC = 4.8 Hz), 14.4; ³¹P NMR (162 MHz, CDCl₃) 29.3 ppm. HRMS (ESI+) m/z calculated for C₂₄H₂₇N₃O₇P (M+H)⁺ 500.1587, found 500.1570. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (25b).

The olefin 24b (635 mg, 1.38 mmol) was added to a suspension of selenium dioxide (122 mg, 1.09 mmol) and 4-hydroxybenzoic acid (27 mg, 0.19 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.763 mL, 5.5 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25b as an oil in 12% yield (80 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.76 – 7.71 (m, 2H), 7.29 – 7.18 (m, 6H), 7.04 – 7.01 (m, 1H), 5.98 (dd, J_PH = 15.2 Hz, J_HH = 5.2 Hz, 1H), 5.84 (dd, J_PH = 12.0 Hz, J_HH = 5.3 Hz, 1H), 5.44 (td, J = 7.2, 1.1 Hz, 1H), 3.98 (s, 2H), 2.51 – 2.42 (m, 2H), 2.13 – 2.04 (m, 2H), 1.64 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 162.9 (d, J_CF = 251.6 Hz), 158.4, 149.5 (d, J_PC = 9.7 Hz), 138.9, 136.6, 132.2, 129.5 (3C), 126.1, 124.8, 122.6 (d, J_PC = 15.6 Hz), 122.5 (d, J_CF = 8.8 Hz, 2C), 120.5 (d, J_CF = 4.2 Hz), 116.8 (d, J_CF = 23.4 Hz, 2C), 81.6 (d, J_PC = 6.6 Hz), 67.9, 25.9 (d, J_PC = 139.9 Hz), 20.1 (d, J_PC = 5.0 Hz), 13.4; ³¹P NMR (162 MHz, CDCl₃) 29.6 ppm. HRMS (ESI+) m/z calculated for C₂₂H₂₄FN₃O₆P (M+H)⁺ 476.1387, found 476.1382. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (26b).

Alcohol 25b (25 mg, 0.076 mmol), acetic anhydride (8 mg, 0.078 mmol) and triethylamine (11 mg, 0.10 mmol) were dissolved in freshly distilled methylene chloride (4 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (5 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26b was isolated as an oil in 92% yield (25 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.25 (s, 1H), 7.72 – 7.69 (m, 2H), 7.29 – 7.20 (m, 6H), 7.02 – 6.97 (m, 1H), 5.99 (dd, J_PH = 15.7 Hz, J_HH = 5.2 Hz, 1H), 5.82 (dd, J_PH = 11.5 Hz, J_HH = 5.2 Hz, 1H), 5.46 (td, J = 7.1, 1.1 Hz, 1H), 4.42 (s, 2H), 2.52 – 2.43 (m, 2H), 2.12 – 2.06 (m, 2H), 2.05 (s, 3H), 1.65 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 170.7, 162.8 (d, J_CF = 251.8 Hz), 158.3, 149.6 (d, J_PC = 9.7 Hz), 139.0, 131.8, 129.5 (3C), 126.5 (d, J_PC = 17.0 Hz), 126.1, 124.8 (2C), 122.5 (d, J_CF = 8.8 Hz, 2C), 120.6 (d, J_CF = 4.2 Hz), 116.9 (d, J_CF = 23.4 Hz, 2C), 81.6 (d, J_PC = 6.5 Hz), 69.3, 25.8 (d, J_PC = 140.4 Hz), 20.8, 20.3 (d, J_PC = 4.7 Hz), 13.8; ³¹P NMR (162 MHz, CDCl₃) 29.2 ppm. HRMS (ESI+) m/z calculated for C₂₄H₂₆FN₃O₇P (M+H)⁺ 518.1492, found 518.1491. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (25c).

The olefin 24c (586 mg, 1.2 mmol) was added to a suspension of selenium dioxide (108 mg, 0.97 mmol) and 4-hydroxybenzoic acid (23 mg, 0.17 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.670 mL, 4.9 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25c as an oil in 12% yield (71.6 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 7.63 – 7.60 (m, 2H), 7.40 – 7.37 (m, 2H), 7.24 – 7.16 (m, 4H), 7.01 – 6.98 (m, 1H), 5.97 (dd, J_PH = 15.2 Hz, J_HH = 5.2 Hz, 1H), 5.81 (dd, J_PH = 11.9 Hz, J_HH = 5.2 Hz, 1H), 5.41 (td, J = 7.1, 1.1 Hz, 1H), 3.96 (s, 2H), 3.01 – 2.94 (m, 1H), 2.49 – 2.40 (m, 2H), 2.10 – 2.02 (m, 2H), 1.62 (s, 3H), 1.28 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 159.2, 151.3, 150.1 (d, J_PC = 9.7 Hz), 139.3, 137.2, 134.4, 130.2 (2C), 128.4 (3C), 126.5, 125.5, 123.3 (d, J_PC = 15.5 Hz), 121.2 (3C), 82.2 (d, J_PC = 6.5 Hz), 68.5, 34.3, 26.6 (d, J_PC = 139.9 Hz), 24.2 (2C), 20.8 (d, J_PC = 5.0 Hz), 14.1; ³¹P NMR (162 MHz, CDCl₃) 29.6 ppm. HRMS (ESI+) m/z calculated for C₂₅H₃₁N₃O₆P (M+H)⁺ 500.1950, found 500.1949. HPLC purity >99% (t_R = 3.3).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(phenoxy)phosphoryl)oxy)methyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (26c).

Alcohol 25c (17.6 mg, 0.035 mmol), acetic anhydride (5.0 μL, 0.053 mmol) and triethylamine (9.8 μL, 0.070 mmol) were dissolved in freshly distilled methylene chloride (3 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (4 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26c was isolated as an oil in 53% yield (10.1 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.19 – 7.12 (m, 4H), 6.96 – 6.92 (m, 1H), 5.94 (dd, J_PH = 15.6 Hz, J_HH = 5.2 Hz, 1H), 5.74 (dd, J_PH = 11.6 Hz, J_HH = 5.2 Hz, 1H), 5.40 (td, J = 7.2, 1.1 Hz, 1H), 4.4 (s, 2H), 2.97 – 2.90 (m, 1H), 2.45 – 2.36 (m, 2H), 2.05 – 1.98 (m, 2H), 1.98 (s, 3H), 1.59 (s, 3H), 1.23 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 171.0, 158.8, 151.0, 149.9 (d, J_PC = 9.8 Hz), 139.0, 134.2, 132.1, 129.9 (2C), 128.1 (3C), 126.8 (d, J_PC = 17.0 Hz), 126.1, 125.2, 120.8 (3C), 81.9 (d, J_PC = 6.7 Hz), 69.6, 34.0, 26.1 (d, J_PC = 140.5 Hz), 24.0 (2C), 21.1, 20.7 (d, J_PC = 5.0 Hz), 14.1; ³¹P NMR (162 MHz, CDCl₃) 29.2 ppm. HRMS (ESI+) m/z calculated for C₂₇H₃₃N₃O₇P (M+H)⁺ 542.2056, found 542.2054. HPLC purity >99% (t_R = 3.3).

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(4-isopropylphenoxy)phosphoryl)oxy)methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (25d).

The olefin 24d (820 mg, 1.69 mmol) was added to a suspension of selenium dioxide (150 mg, 1.35 mmol) and 4-hydroxybenzoic acid (32 mg, 0.23 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.935 mL, 6.79 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25d as an oil in 12% yield (100 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.49 (s, 1H), 7.73 – 7.71 (m, 2H), 7.56 – 7.46 (m, 3H), 7.10 (s, 4H), 5.97 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.86 (dd, J_PH = 12.4 Hz, J_HH = 5.2 Hz, 1H), 5.41 (td, J = 7.2, 1.1 Hz, 1H), 3.95 (s, 2H), 2.80 – 2.73 (m, 1H), 2.48 – 2.39 (m, 2H), 2.09 – 2.01 (m, 2H), 1.61 (s, 3H), 1.12 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 158.6, 147.4 (d, J_PC = 9.4 Hz), 145.5, 138.9, 136.4, 135.9, 129.7 (3C), 129.4, 127.4 (2C), 126.0, 122.6 (d, J_PC = 15.7 Hz), 120.5 (3C), 81.6 (d, J_PC = 6.4 Hz), 67.8, 33.1, 25.9 (d, J_PC = 139.2 Hz), 23.6 (2C), 20.1 (d, J_PC = 5.0 Hz), 13.4; ³¹P NMR (162 MHz, CDCl₃) 29.5 ppm. HRMS (ESI+) m/z calculated for C₂₅H₃₁N₃O₆P (M+H)⁺ 500.1950, found 500.1943. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(4-isopropylphenoxy)phosphoryl)oxy)methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (26d).

Alcohol 25d (20 mg, 0.04 mmol), acetic anhydride (6 mg, 0.058 mmol) and triethylamine (8 mg, 0.079 mmol) were dissolved in freshly distilled chloride (4 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (5 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26d was isolated as an oil in 83% yield (18 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.44 (s, 1H), 7.74 – 7.71 (m, 2H), 7.58 – 7.49 (m, 3H), 7.11 (s, 4H), 6.01 (dd, J_PH = 14.3 Hz, J_HH = 5.2 Hz, 1H), 5.87 (dd, J_PH = 12.2 Hz, J_HH = 5.2 Hz, 1H), 5.45 (td, J = 7.2, 1.1 Hz, 1H), 4.41 (s, 2H), 2.81 – 2.74 (m, 1H), 2.50 – 2.41 (m, 2H), 2.10 – 2.02 (m, 2H), 2.04 (s, 3H), 1.63 (s, 3H), 1.13 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 158.6, 147.4 (d, J_PC = 9.4 Hz), 145.6, 139.0, 136.0, 131.7, 129.8 (3C), 129.5, 127.4, 126.5 (d, J_PC = 17.3 Hz), 125.9, 120.5 (3C), 120.1, 81.7 (d, J_PC = 6.2 Hz), 69.3, 33.2, 25.9 (d, J_PC = 139.6 Hz), 23.7 (2C), 20.7, 20.4 (d, J_PC = 4.8 Hz), 13.7; ³¹P NMR (162 MHz, CDCl₃) 29.1 ppm. HRMS (ESI+) m/z calculated for C₂₇H₃₃N₃O₇P (M+H)⁺ 542.2056, found 542.2051. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(4-isopropylphenoxy)phosphoryl)oxy)methyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (25e).

The olefin 24e (840 mg, 1.67 mmol) was added to a suspension of selenium dioxide (148 mg, 1.33 mmol) and 4-hydroxybenzoic acid (32 mg, 0.23 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.925 mL, 6.70 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25e as an oil in 12% yield (100 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.44 (s, 1H), 7.72 – 7.69 (m, 2H), 7.26 – 7.21 (m, 2H), 7.10 (s, 4H), 5.96 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.86 (dd, J_PH = 12.6 Hz, J_HH = 5.2 Hz, 1H), 5.41 (td, J = 7.2, 1.1 Hz, 1H), 3.94 (s, 2H), 2.81 – 2.74 (m, 1H), 2.48 – 2.38 (m, 2H), 2.08 – 2.01 (m, 2H), 1.60 (s, 3H), 1.13 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 162.7 (d, J_CF = 251.7 Hz), 158.5, 147.5 (d, J_PC = 9.4 Hz), 145.6, 139.1, 136.5, 132.2, 127.4 (3C), 126.1, 122.7 (d, J_PC = 15.7 Hz), 122.5 (d, J_CF = 8.8 Hz, 2C), 120.5 (d, J_CF = 4.2 Hz), 116.8 (d, J_CF = 23.4 Hz, 2C), 81.7 (d, J_PC = 6.3 Hz), 67.9, 33.2, 25.9 (d, J_PC = 139.1 Hz), 23.7 (2C), 20.2 (d, J_PC = 5.0 Hz), 13.4; ³¹P NMR (162 MHz, CDCl₃) 29.5 ppm. HRMS (ESI+) m/z calculated for C₂₅H₃₀FN₃O₆P (M+H)⁺ 518.1856, found 518.1841. HPLC purity >99% (t_R = 3.3).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(4-isopropylphenoxy)phosphoryl)oxy)methyl-1-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate (26e).

Alcohol 25e (19 mg, 0.04 mmol), acetic anhydride (5.6 mg, 0.054 mmol) and triethylamine (7.4 mg, 0.073 mmol) were dissolved in freshly distilled methylene chloride (3 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (4 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26e was isolated as an oil in 94% yield (19.5 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.35 (s, 1H), 7.67 – 7.64 (m, 2H), 7.21 – 7.17 (m, 2H), 7.04 (s, 4H), 5.93 (dd, J_PH = 14.2 Hz, J_HH = 5.2 Hz, 1H), 5.81 (dd, J_PH = 12.1 Hz, J_HH = 5.2 Hz, 1H), 5.41 (td, J = 7.2, 1.1 Hz, 1H), 4.34 (s, 2H), 2.75 – 2.68 (m, 1H), 2.44 – 2.34 (m, 2H), 2.03 – 1.95 (m, 2H), 1.97 (s, 3H), 1.57 (s, 3H), 1.08 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 162.7 (d, J_CF = 251.8 Hz), 158.5, 147.5 (d, J_PC = 9.3 Hz), 145.6, 139.1, 131.6, 127.4 (3C), 126.5 (d, J_PC = 17.1 Hz), 126.1 (2C), 122.5 (d, J_CF = 8.8 Hz, 2C), 120.1 (d, J_CF = 4.2 Hz), 116.8 (d, J_CF = 23.5 Hz, 2C), 81.7 (d, J_PC = 6.4 Hz), 69.3, 33.2, 25.7 (d, J_PC = 139.7 Hz), 23.7 (2C), 20.7, 20.3 (d, J_PC = 4.8 Hz), 13.7; ³¹P NMR (162 MHz, CDCl₃) 29.1 ppm. HRMS (ESI+) m/z calculated for C₂₇H₃₂FN₃O₇P (M+H)⁺ 560.1962, found 560.1945. HPLC purity >99% (t_R = 3.3).

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(4-isopropylphenoxy)phosphoryl)oxy)methyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (25f).

The olefin 24f (740 mg, 1.40 mmol) was added to a suspension of selenium dioxide (125 mg, 1.12 mmol) and 4-hydroxybenzoic acid (27 mg, 0.19 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.777 mL, 5.63 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25f as an oil in 9% yield (70 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H), 7.64 – 7.60 (m, 2H), 7.41 – 7.37 (m, 2H), 7.10 (s, 4H), 5.98 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.87 (dd, J_PH = 12.4 Hz, J_HH = 5.2 Hz, 1H), 5.42 (td, J = 7.2, 1.1 Hz, 1H), 3.95 (s, 2H), 3.02 – 2.95 (m, 1H), 2.81 – 2.74 (m, 1H), 2.49 – 2.39 (m, 2H), 2.10 – 1.99 (m, 2H), 1.62 (s, 3H), 1.28 (d, J = 6.9 Hz, 6H), 1.13 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 159.3, 151.3, 148.1 (d, J_PC = 9.4 Hz), 146.2, 139.4, 137.1, 134.4, 128.4 (3C), 128.0 (2C), 126.6, 123.4 (d, J_PC = 15.7 Hz), 121.2 (2C), 120.7, 82.3 (d, J_PC = 6.3 Hz), 68.6, 34.3, 33.8, 26.5 (d, J_PC = 139.2 Hz), 24.4 (2C), 24.2 (2C), 20.1 (d, J_PC = 5.0 Hz), 14.1; ³¹P NMR (162 MHz, CDCl₃) 29.4 ppm. HRMS (ESI+) m/z calculated for C₂₈H₃₇N₃O₆P (M+H)⁺ 542.2420, found 542.2418. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(4-isopropylphenoxy)phosphoryl)oxy)methyl-1-(4-isopropylphenyl)-1H-1,2,3-triazole-4-carboxylate (26f).

Alcohol 25f (16 mg, 0.029 mmol), acetic anhydride (4.5 mg, 0.044 mmol) and triethylamine (5.9 mg, 0.058 mmol) were dissolved in freshly distilled methylene chloride (3 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (4 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26f was isolated as an oil in 87% yield (15 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H), 7.64 – 7.61 (m, 2H), 7.42 – 7.37 (m, 2H), 7.11 (s, 4H), 6.01 (dd, J_PH = 14.3 Hz, J_HH = 5.2 Hz, 1H), 5.88 (dd, J_PH = 12.1 Hz, J_HH = 5.2 Hz, 1H), 5.45 (td, J = 7.2, 1.1 Hz, 1H), 4.4 (s, 2H), 3.03 – 2.97 (m, 1H), 2.81 – 2.73 (m, 1H), 2.51 – 2.42 (m, 2H), 2.10 – 2.01 (m, 2H), 2.04 (s, 3H), 1.64 (s, 3H), 1.29 (d, J = 6.9 Hz, 6H), 1.14 (d, J = 6.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 171.3, 159.3, 151.4, 148.1 (d, J_PC = 9.4 Hz), 146.3, 139.5, 134.5, 132.3, 128.4 (3C), 128.1 (2C), 127.2 (d, J_PC = 17.3 Hz), 126.5, 121.2 (2C), 120.7, 82.4 (d, J_PC = 6.4 Hz), 70.0, 34.3, 33.8, 26.4 (d, J_PC = 139.9 Hz), 24.4 (2C), 24.3 (2C), 21.4, 21.0 (d, J_PC = 4.7 Hz), 14.4; ³¹P NMR (162 MHz, CDCl₃) 29.1 ppm. HRMS (ESI+) m/z calculated for C₃₀H₃₉N₃O₇P (M+H)⁺ 584.2526, found 584.2519. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Hydroxy-4-methylpent-3-en-1-yl)(4-(2 methoxyethyl)phenoxy)phosphoryl)oxy)methyl-1-(2,4-difluorophenyl)-1H-1,2,3-triazole-4-carboxylate (25g).

The olefin 24g (630 mg, 1.17 mmol) was added to a suspension of selenium dioxide (104 mg, 0.93 mmol) and 4-hydroxybenzoic acid (23 mg, 0.16 mmol) in dichloromethane (10 mL). At 0 °C tert-butyl hydroperoxide (70% wt. in H₂O, 0.650 mL, 4.70 mmol) was added slowly to the stirred reaction mixture. The resulting reaction mixture was stirred at 0 °C and was allowed to react for 3 days. The reaction mixture was diluted by the addition of water (5 mL), quenched with Na₂SO₃ (5 mL), and extracted with dichloromethane (3 x 20 mL). The combined extracts were dried (Na₂SO₄), filtered through celite, and the filtrate was concentrated in vacuo. The resulting oil was purified by column chromatography (silica, 100% ether to 3% MeOH in ether) to give the desired allyl alcohol 25g as an oil in 19% yield (130 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.50 (s, 1H), 7.98 – 7.92 (m, 1H), 7.11 – 7.07 (m, 6H), 5.96 (dd, J_PH = 13.9 Hz, J_HH = 5.2 Hz, 1H), 5.86 (dd, J_PH = 12.4 Hz, J_HH = 5.2 Hz, 1H), 5.41 (td, J = 7.2, 1.1 Hz, 1H), 3.95 (s, 2H), 3.50 (t, J_HH = 7.0 Hz, 2H), 3.30 (s, 3H), 2.73 (t, J_HH = 6.92 Hz, 2H), 2.49 – 2.39 (m, 2H), 2.10 – 2.02 (m, 2H), 1.62 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 162.8 (d, J_CF = 255.2 Hz), 162.7 (d, J_CF = 255.4 Hz), 158.4, 154.8 (d, J_CF = 12.5 Hz), 148.0 (d, J_PC = 9.5 Hz), 138.9, 136.5, 135.9, 129.9 (3C), 128.9 (d, J_CF = 7.7 Hz), 126.0 (d, J_CF = 10.0 Hz), 122.7 (d, J_PC = 15.7 Hz), 120.2 (d, J_CF = 4.2 Hz), 112.8 (d, J_CF = 3.7 Hz), 105.4 (d, J_CF = 23.7 Hz), 81.7 (d, J_PC = 6.3 Hz), 73.1, 67.9, 58.4, 35.1, 25.9 (d, J_PC = 139.3 Hz), 20.2 (d, J_PC = 5.0 Hz), 13.4; ³¹P NMR (162 MHz, CDCl₃) 29.4 ppm. HRMS (ESI+) m/z calculated for C₂₅H₂₉F₂N₃O₇P (M+H)⁺ 552.1711, found 552.1707. HPLC purity >99% (t_R = 3.4).

(E)-(((5-Acetoxy-4-methylpent-3-en-1-yl)(4-(2-methoxyethyl)phenoxy)phosphoryl)oxy)methyl-1-(2,4-difluorophenyl)-1H-1,2,3-triazole-4-carboxylate (26g).

Alcohol 25g (28 mg, 0.05 mmol), acetic anhydride (7.8 mg, 0.076 mmol) and triethylamine (10 mg, 0.099 mmol) were dissolved in freshly distilled methylene chloride (3 mL) and the resultant reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted by the addition of water (4 mL), quenched with sodium bicarbonate (2 mL), and extracted with methylene chloride (3 x 5 mL). The combined extracts were dried (Na₂SO₄), and filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, 100% hexane to 50% EtOAc in hexane) and the resulting acetate 26g was isolated as an oil in 86% yield (26 mg): ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 7.99 – 7.93 (m, 1H), 7.14 – 7.08 (m, 6H), 5.98 (dd, J_PH = 14.3 Hz, J_HH = 5.2 Hz, 1H), 5.86 (dd, J_PH = 12.2 Hz, J_HH = 5.2 Hz, 1H), 5.45 (td, J = 7.2, 1.1 Hz, 1H), 4.41 (s, 2H), 3.50 (t, J_HH = 6.7 Hz, 2H), 3.30 (s, 3H), 2.74 (t, J_HH = 6.92 Hz, 2H), 2.50 – 2.41 (m, 2H), 2.10 – 2.02 (m, 2H), 2.04 (s, 3H), 1.64 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 162.8 (d, J_CF = 253.0 Hz), 162.7 (d, J_CF = 255.1 Hz), 158.4, 154.8 (d, J_CF = 12.4 Hz), 148.0 (d, J_PC = 9.4 Hz), 138.9, 135.9, 131.7, 129.9 (3C), 128.9 (d, J_CF = 7.8 Hz), 126.5 (d, J_PC = 17.2 Hz), 126.0 (d, J_CF = 10.2 Hz), 120.2 (d, J_CF = 4.2 Hz), 112.8 (d, J_CF = 3.9 Hz), 105.6 (d, J_CF = 23.6 Hz), 81.7 (d, J_PC = 6.2 Hz), 73.1, 69.3, 58.4, 35.1, 25.9 (d, J_PC = 139.8 Hz), 20.7, 20.3 (d, J_PC = 4.8 Hz), 13.7; ³¹P NMR (162 MHz, CDCl₃) 29.1 ppm. HRMS (ESI+) m/z calculated for C₂₇H₃₁F₂N₃O₈P (M+H)⁺ 594.1817, found 594.1820. HPLC purity >99% (t_R = 3.4).

5.3. Biological evaluation

Proliferation assay.

The diphosphates were tested for their ability to promote growth of human Vγ9Vδ2 T cells.²¹ PBMCs in T cell media were stimulated with test compounds (up to 10 μM) for 3 days using 10-fold dilutions, washed, and allowed to grow for another 11 days. Fresh interleukin 2 (5 ng/mL) was added at the start and every 3 days. The percentage of Vγ9Vδ2 T cells was measured by flow cytometry using CD3 (Clone UCHT1, PE conjugated, Biolegend) and γδ-TCR (Clone B1, APC conjugated, Biolegend) staining. HMBPP (100 nM) was the positive control. EC₅₀ values were calculated as the concentration inducing 50% of the maximum proliferative effect.

ELISA.

A cellular dose-response curve for the samples was prepared using an IFN-γ ELISA. Different dilutions of synthesized novel compounds were prepared and added separately to either K562 cells or U937 cells for one hour. The loaded cells were then washed twice with T cell media and mixed with purified Vγ9Vδ2 T cells in a 96-well plate, using 4,000 target cells and 12,000 T cells per well. The following day, IFN-γ release was measured by ELISA according to our reported protocol protocol.⁴⁷

Viability assay.

K562 or U937 cells were plated in fresh media in a 96-well plate at a density of 5,000 cells per well, and compounds were added at a concentration of 10 μM. After an incubation period of 71 hours at 37°C and 5% CO₂, 10 μL of CellQB reagent was added to each well. The plate was then quantified by spectrophotometry after 1 hour.

Plasma stability studies.

Compounds were tested for plasma stability in 50% human, mouse, or rat plasma at 37 °C. Plasma was diluted to 50% with tris-buffered saline (pH 7.5) and compounds were added to 100 μM. Samples were incubated at 37 °C, and 50 μL aliquots were extracted with 150 μL of LCMS grade acetonitrile at each time point.

Metabolite analysis.

Cell and plasma metabolism of the prodrugs was examined. Plasma metabolites were produced as described. For cellular metabolites, K562 cells were concentrated to 5 x 10⁶ per mL in media and treated with 100 μM of the compound for 1 hour. The cells were then centrifuged at 600 x g for 3 minutes and washed with cold phosphate-buffered saline to remove unabsorbed compounds. Metabolites were extracted using 200 μL of extraction solvent (75% LC-MS grade acetonitrile, 25% 75 mM NH₄OH) with vigorous mixing for 30 seconds. Cellular debris was removed by centrifugation at 10,000 x g for 2 minutes.

LC-MS System.

For stability studies, 10 μL of the extract was analyzed using a Waters Synapt G2-Si mass spectrometer in positive polarity mode. The gradient began at 50% acetonitrile and increased to 90% over 9 minutes. The analysis focused on the precursor ion [M+H]+ and the sodium adduct [M+Na]+. The integrated peak values of these ions were combined to calculate the fraction remaining relative to the initial value. Metabolism studies were conducted in negative polarity mode. A volume of 10 μL of the diphosphate was evaluated using a C18 column in negative mode. The mobile phase involved a gradient of 10 mM triethylammonium acetate (A) and methanol/10 mM triethylammonium acetate pH 6 (90/10, v/v) (B). The initial gradient was 10% B which increased to 80% B over 4 min and held there for 2.5 min before re-equilibration.^48–49

Statistics.

All experiments were performed 2-4 times as indicated in the figure legends (e.g., n=3). The number of replicates varied by technique. Results were analyzed using GraphPad Prism 10. In dose-response assays, EC₅₀ values were determined using the log agonist vs. response model and listed with 95% confidence intervals as indicated.

Highlights.

• Novel phosphoantigen prodrugs have been prepared based on a 1,2,3-triazole system

• These compounds stimulate proliferation of Vγ9Vδ2 T cells (EC₅₀ = 490 pM to 7.4 nM)

• These compounds stimulate interferon γ production (EC₅₀ = 600 pM to 77 nM)

• These prodrugs have improved stability in human plasma (T_1/2 = 16 to 106 min)

• Cells, but not plasma, release the desired dianionic phosphoantigen payload

ABBREVIATIONS:

BTN

butyrophilin

HMBPP

(E) 4-Hydroxy-3-methyl-but-2-enyl diphosphate

TCR

T cell receptor