α-Amino bisphosphonate triazoles serve as GGDPS inhibitors.

Depletion of cellular levels of geranylgeranyl diphosphate by inhibition of the enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential strategy for disruption of protein transport by limiting the geranylgeranylation of the Rab proteins that regulate intracellular trafficking. As such, there is interest in the development of GGDPS inhibitors for the treatment of malignancies characterized by abnormal protein production, including multiple myeloma. Our previous work has explored the structure-function relationship of a series of isoprenoid triazole bisphosphonate-based GGDPS inhibitors, with modifications having impact on enzymatic, cellular and in vivo activities. We have synthesized a new series of α-amino bisphosphonates to understand the impact of modifying the alpha position with a moiety that is potentially linkable to other agents. Bioassays evaluating the enzymatic and cellular activities of these compounds demonstrate that incorporation of the α-amino group affords compounds with GGDPS inhibitory activity which is modulated by isoprenoid tail chain length and olefin stereochemistry. These studies provide further insight into the complexity of the structure-function relationship and will enable future efforts focused on tumor-specific drug delivery.

Diester Prodrugs of a Phosphonate Butyrophilin Ligand Display Improved Cell Potency, Plasma Stability, and Payload Internalization.

Activation of Vγ9Vδ2 T cells with butyrophilin 3A1 (BTN3A1) agonists such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) has the potential to boost the immune response. Because HMBPP is highly charged and metabolically unstable, prodrugs may be needed to overcome these liabilities, but the prodrugs themselves may be limited by slow payload release or low plasma stability. To identify effective prodrug forms of a phosphonate agonist of BTN3A1, we have prepared a set of diesters bearing one aryl and one acyloxymethyl group. The compounds were evaluated for their ability to stimulate Vγ9Vδ2 T cell proliferation, increase production of interferon γ, resist plasma metabolism, and internalize into leukemia cells. These bioassays have revealed that varied aryl and acyloxymethyl groups can decouple plasma and cellular metabolism and have a significant impact on bioactivity (>200-fold range) and stability (>10 fold range), including some with subnanomolar potency. Our findings increase the understanding of the structure-activity relationships of mixed aryl/acyloxymethyl phosphonate prodrugs.

Substitution of a triazole for the central olefin in biologically active stilbenes.

The stilbene moiety is commonly found in natural products and these compounds display an extraordinary range of biological activity. Efforts to derive useful drugs from stilbenes must address the potential liabilities of this structure, including a propensity for cis/trans isomerization. To identify olefin replacements that address this limitation while preserving biological activity we have prepared analogues of two bioactive stilbenes, a pawhuskin and a schweinfurthin, where a 1,2,3-triazole ring formally replaces the stilbene double bond. The new schweinfurthin analogue (23) has been tested for anti-proliferative activity against 60 cell lines, and shows a strong correlation of bioactivity when compared to the compound that inspired its synthesis (22).

Efficiency of bis-amidate phosphonate prodrugs.

Bis-amidate derivatives have been viewed as attractive phosphonate prodrug forms because of their straightforward synthesis, lack of phosphorus stereochemistry, plasma stability and nontoxic amino acid metabolites. However, the efficiency of bis-amidate prodrug forms is unclear, as prior studies on this class of prodrugs have not evaluated their activation kinetics. Here, we synthetized a small panel of bis-amidate prodrugs of butyrophilin ligands as potential immunotherapy agents. These compounds were examined relative to other prodrug forms delivering the same payload for their stability in plasma and cell lysate, their ability to stimulate T cell proliferation in human PBMCs, and their activation kinetics in a leukemia co-culture model of T cell cytokine production. The bis-amidate prodrugs demonstrate high plasma stability and improved cellular phosphoantigen activity relative to the free phosphonic acid. However, the efficiency of bis-amidate activation is low relative to other prodrugs that contain at least one ester such as aryl-amidate, aryl-acyloxyalkyl ester, and bis-acyloxyalkyl ester forms. Therefore, bis-amidate prodrugs do not drive rapid cellular payload accumulation and they would be more useful for payloads in which slower, sustained-release kinetics are preferred.

Synthesis and Metabolism of BTN3A1 Ligands: Studies on Diene Modifications to the Phosphoantigen Scaffold.

Phosphoantigens (pAgs) are small organophosphorus compounds such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that trigger an immune response. These molecules bind to butyrophilin 3A1 (part of the HMBPP receptor) and activate Vγ9Vδ2 T cells. To explore the structure-activity relationships underlying this process, we evaluated a series of novel diene analogs of HMBPP. Here we report that prodrug forms of [(1E)-4-methylpenta-1,3-dien-1-yl] phosphonic acid that lack the allylic alcohol of HMBPP but instead contained a diene scaffold exhibit mid-nanomolar potency for the activation of Vγ9Vδ2 T cells. The compounds also trigger the production of T-cell interferon γ upon exposure to loaded K562 cells. Although both the allylic alcohol and the diene scaffold boost pAg activity, the combination of the two decreases the activity and results in glutathione conjugation. Together, these data show that the diene scaffold results in intermediate pAgs that may have implications for the mechanisms regulating the HMBPP receptor.

Conjugate reduction of vinyl bisphosphonates.

Vinyl bisphosphonates can be readily prepared by condensation of an aromatic aldehyde with the tetraester of a methylenebisphosphonate, and reduction of the resulting olefin is an attractive strategy for the preparation of monoalkyl geminal bisphosphonates. Conjugate reduction through use of variations on the Stryker approach has proven to be an efficient method for that reduction, even in the presence of aromatic substituents that also could be reduced. Furthermore, remote olefins in an isoprenoid chain survive this conjugate reduction unaffected, allowing access to isoprenoid-substituted triazole bisphosphonates of interest as potential inhibitors of terpenoid biosynthesis.

Synthesis of a Coumarin-Based Analogue of Schweinfurthin F.

The natural schweinfurthins are stilbenes with significant antiproliferative activity and an uncertain mechanism of action. To obtain a fluorescent analogue with minimal deviation from the natural structure, a coumarin ring system was annulated to the D-ring, creating a new analogue of schweinfurthin F. This stilbene was prepared through a convergent synthesis, with a Horner-Wadsworth-Emmons condensation employed to form the central stilbene olefin. After preparation of a tricyclic phosphonate via a recent and more efficient modification of the classic Arbuzov reaction, condensation was attempted with an appropriately substituted bicyclic aldehyde but the coumarin system did not survive the reaction conditions. When olefin formation preceded generation of the coumarin, the stilbene formation proceeded smoothly and ultimately allowed access to the targeted coumarin-based schweinfurthin analogue. This analogue displayed the desired fluorescence properties along with significant biological activity in the National Cancer Institute's 60-cell line bioassay, and the pattern of this biological activity mirrored that of the natural product schweinfurthin F. This approach gives facile access to new fluorescent analogues of the natural schweinfurthins and should be applicable to other natural stilbenes as well.

Impact of α-modifications on the activity of triazole bisphosphonates as geranylgeranyl diphosphate synthase inhibitors.

Agents that inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS) have anti-cancer activity and our prior studies have investigated the structure-function relationship for a family of isoprenoid triazole bisphosphonates as GGDPS inhibitors. To further explore this structure-function relationship, a series of novel α-modified triazole phosphonates was prepared and evaluated for activity as GGDPS inhibitors in enzyme and cell-based assays. These studies revealed flexibility at the α position of the bisphosphonate derivatives with respect to being able to accommodate a variety of substituents without significantly affecting potency compared to the parent unsubstituted inhibitor. However, the monophosphonate derivatives lacked activity. These studies further our understanding of the structure-function relationship of the triazole-based GGDPS inhibitors and lay the foundation for future studies evaluating the impact of α-modifications on in vivo activity.

Incorporation of a FRET pair within a phosphonate diester.

Cell-cleavable protecting groups are an effective tactic for construction of biological probes because such compounds can improve problems with instability, solubility, and cellular uptake. Incorporation of fluorescent groups in the protecting groups may afford useful probes of cellular functions, especially for payloads containing phosphonates that would be highly charged if not protected, but little is known about the steric or electronic factors that impede release of the payload. In this report we present a strategy for the synthesis of a coumarin fluorophore and a 4-((4-(dimethylamino)phenyl)diazenyl)benzoic acid (DABCYL) ester chromophore incorporated as a FRET pair within a single phosphonate. Such compounds were designed to deliver a BTN3A1 ligand payload to its intracellular receptor. Both final products and some synthetic intermediates were evaluated for their ability to undergo metabolic activation in γδ T cell functional assays, and for their photophysical properties by spectrophotometry. One phosphonate bearing a DABCYL acyloxyester and a novel tyramine-linked coumarin fluorophore exhibited strong, rapid, and potent cellular activity for γδ T cell stimulation and also showed FRET interactions. This strategy demonstrates that bioactivatable phosphonates containing FRET pairs can be utilized to develop probes to monitor cellular uptake of otherwise charged payloads.

Synthesis and Metabolism of BTN3A1 Ligands: Studies on Modifications of the Allylic Alcohol.

(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and its phosphonate analogs are potent phosphoantigens. HMBPP contains an (E)-allylic alcohol which interacts with the molecular target BTN3A1 giving an antigenic signal to activate Vγ9Vδ2 T cells. As probes of BTN3A1 function, we prepared prodrug derivatives of the HMBPP analog C-HMBP that lack the (E)-allylic alcohol or have modified it to an aldehyde or aldoxime and evaluated their biological activity. Removal of the alcohol completely abrogates phosphoantigenicity in these compounds while the aldoxime modification decreases potency relative to the (E)-allylic alcohol form. However, homoprenyl derivatives oxidized to an aldehyde stimulate Vγ9Vδ2 T cells at nanomolar concentrations. Selection of phosphonate protecting groups (i.e., prodrug forms) impacts the potency of phosphoantigen aldehydes, with mixed aryl acyloxyalkyl forms exhibiting superior activity relative to aryl amidate forms. The activity correlates with the cellular reduction of the aldehyde to the alcohol form. Thus, the functionality on this ligand framework can be altered concurrently with phosphonate protection to promote cellular transformation to highly potent phosphoantigens.

Potent double prodrug forms of synthetic phosphoantigens.

Phosphoantigens are ligands of BTN3A1 that stimulate anti-cancer functions of γδ T cells, yet the potency of natural phosphoantigens is limited by low cell permeability and low metabolic stability. Derivatives of BTN3A1 ligand prodrugs were synthesized that contain an acetate-protected allylic alcohol and act as doubly protected prodrugs. A novel set of phosphonates, phosphoramidates, and phosphonamidates has been prepared through a new route that simplifies synthesis and postpones the point of divergence into different prodrug forms. One of the new prodrugs, compound 11, potently stimulates γδ T cell proliferation (72 h EC50 = 0.12 nM) and interferon γ response to loaded leukemia cells (4 h EC50 = 19 nM). This phosphonamidate form was > 900x more potent than the corresponding phosphoramidate, and the phosphonamidate form was also significantly more stable in plasma following acetate hydrolysis. Therefore, prodrug modification of phosphonate butyrophilin ligands at the allylic alcohol can both facilitate chemical synthesis and improve potency of γδ T cell stimulation.

Amides as bioisosteres of triazole-based geranylgeranyl diphosphate synthase inhibitors.

Geranylgeranyl diphosphate synthase (GGDPS) inhibitors are of potential therapeutic interest as a consequence of their activity against the bone marrow cancer multiple myeloma. A series of bisphosphonates linked to an isoprenoid tail through an amide linkage has been prepared and tested for the ability to inhibit GGDPS in enzyme and cell-based assays. The amides were designed as analogues to triazole-based GGDPS inhibitors. Several of the new compounds show GGDPS inhibitory activity in both enzyme and cell assays, with potency dependent on chain length and olefin stereochemistry.

Novel benzimidazole phosphonates as potential inhibitors of protein prenylation.

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.

A luciferase lysis assay reveals in vivo malignant cell sensitization by phosphoantigen prodrugs.

Human Vγ9Vδ2 T cells respond to small phosphorus-containing compounds, often called phosphoantigens, which are now known to be intracellular ligands of the immune receptor butyrophilin 3A1 (BTN3A1). In order to compare the efficiency of butyrophilin ligands, we developed a luciferase-based lysis assay that measures the direct cytolysis by Vγ9Vδ2 T cells of luciferase-expressing K562 leukemia cells sensitized by phosphoantigen prodrugs. Our results show that the luciferase-based lysis assay allows in vitro and in vivo assessment of phosphoantigen activity in a way that does not require the extensive processing of flow cytometry or ELISA based approaches. In cellular assays, the structure activity relationships of phosphoantigen prodrugs correlate with ELISA-based activation assays, though phosphoantigen induced target cell lysis occurs at lower concentrations relative to T cell interferon γ production measured by ELISA. In mice dosed with phosphoantigens, a racemic aryl phosphonamidate prodrug, methyl 2-[[[(E)-5-hydroxy-4-methyl-pent-3-enyl]-(1-naphthyloxy)phosphoryl]amino]acetate (1-Nap/GlyOMe C-HMBP, 5), sensitized subcutaneous K562 tumors within minutes, and this effect was maintained at least four hours after treatment. In vivo activity of compound 5 was stronger than that of an equivalent dose of zoledronate. This luciferase lysis assay can be used for evaluation of phosphoantigens due to its time efficiency, high sensitivity, and in vivo compatibility and demonstrates rapid in vitro and in vivo sensitization of tumor cells by phosphoantigen prodrugs.

Synthesis and Bioactivity of the Alanyl Phosphonamidate Stereoisomers Derived from a Butyrophilin Ligand.

Aryloxy phosphonamidate derivatives of a butyrophilin 3A1 ligand are stimulants of Vγ9 Vδ2 T cells. However, when bonded to an aryl ester and an amine, the phosphorus is stereogenic, and past compounds were studied as racemates. To determine the impact of stereochemistry on the activity, we now have prepared phosphonate derivatives of l- and d-alanine ethyl ester, separated the diastereomers, and evaluated their biological activity as single stereoisomers. The results demonstrate that phosphonamidates substituted with l-alanine stimulate Vγ9 Vδ2 T cells at lower concentrations than the racemic glycine counterpart, while those derived from d-alanine require higher concentrations. All four diastereomers are more active than charged phosphoantigens such as HMBPP. Surprisingly, only a 2-fold difference was observed between the l-alanine phosphorus isomers, with the R P isomer more potent. This suggests that the small phosphoantigen scaffold reduces but does not eliminate dependence upon phosphorus stereochemistry for cellular activity.

ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.

Stability and Efficiency of Mixed Aryl Phosphonate Prodrugs.

A set of phosphonate prodrugs of a butyrophilin ligand was synthesized and evaluated for plasma stability and cellular activity. The mixed aryl acyloxy esters were prepared either via a standard sequence through the phosphonic acid chloride, or through the more recently reported, and more facile, triflate activation. In the best of cases, this class of prodrugs shows cellular potency similar to that of bis-acyloxyalkyl phosphonate prodrugs and plasma stability similar to that of aryl phosphonamidates. For example, {[((3E)-5-hydroxy-4-methylpent-3-en-1-yl) (naphthalen-2-yloxy)phosphoryl]oxy}methyl 2,2-dimethylpropanoate can activate BTN3A1 in K562 cells after just 15 minutes of exposure (at an EC50 value of 31 nm) and is only partially metabolized (60 % remaining) after 20 hours in human plasma. Other related novel analogues showed similar potency/stability profiles. Therefore, mixed aryl acyloxyalkyl phosphonate prodrugs are an exciting new strategy for the delivery of phosphonate-containing drugs.

Phosphonamidate Prodrugs of a Butyrophilin Ligand Display Plasma Stability and Potent Vγ9 Vδ2 T Cell Stimulation.

Small organophosphorus compounds stimulate Vγ9 Vδ2 T cells if they serve as ligands of butyrophilin 3A1. Because the most potent natural ligand is ( E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), which is the last intermediate in bacterial biosynthesis of isoprenoids that is not found in mammalian metabolism, activation of these T cells represents an important component of the immune response to bacterial infections. To identify butyrophilin ligands that may have greater plasma stability, and clinical potential, we have prepared a set of aryl phosphonamidate derivatives (9a-i) of the natural ligand. Testing of these new compounds in assays of T cell response has revealed that this strategy can provide compounds with high potency for expansion of Vγ9 Vδ2 T cells (9f, EC50 = 340 pM) and interferon γ production in response to loaded K562 cells (9e, EC50 = 62 nM). Importantly, all compounds of this class display extended plasma stability ( t1/2 > 24 h). These findings increase our understanding of metabolism of butyrophilin ligands and the structure-activity relationships of phosphonamidate prodrugs.

Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine monophosphate.

Exocyclic olefin variants of thymidylate (dTMP) recently have been proposed as reaction intermediates for the thymidyl biosynthesis enzymes found in many pathogenic organisms, yet synthetic reports on these materials are lacking. Here we report two strategies to prepare the exocyclic olefin isomer of dTMP, which is a putative reaction intermediate in pathogenic thymidylate biosynthesis and a novel nucleotide analog. Our most effective strategy involves preserving the existing glyosidic bond of thymidine and manipulating the base to generate the exocyclic methylene moiety. We also report a successful enzymatic deoxyribosylation of a non-aromatic nucleobase isomer of thymine, which provides an additional strategy to access nucleotide analogs with disrupted ring conjugation or with reduced heterocyclic bases. The strategies reported here are straightforward and extendable towards the synthesis of various pyrimidine nucleotide analogs, which could lead to compounds of value in studies of enzyme reaction mechanisms or serve as templates for rational drug design.

Quantitative determination of a potent geranylgeranyl diphosphate synthase inhibitor using LC-MS/MS: Derivatization and application.

An isomeric mixture of homogeranyl/homoneryl triazole bisphosphonates (VSW1198) has previously been shown to be a potent inhibitor of geranylgeranyl diphosphate (GGDP) synthase (GGDPS) and of therapeutic interest for the treatment of multiple myeloma. We have developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of both the E- and Z- isomers of VSW1198 in cell culture media, mouse plasma and tissues. VSW1198 and internal standard are extracted from the bio-matrices by solid-phase extraction, followed by derivatization using trimethylsilyldiazomethane. The chromatographic separation of analytes was achieved on a Phenomenex Gemini NX column (150 mm * 2.0 mm, 5 µ) with gradient elution using 0.1% acetic acid and methanol/acetonitrile (1:1) as the mobile phase at a flow rate of 0.2 mL/min. Derivatized analytes were ionized with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode and quantitated using MS/MS. The MS/MS response was linear over the concentration range from 0.38-1500 and 0.13-500 ng/mL for the E- and Z-isomers, respectively. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was used for quantitative determination of the compounds in preclinical studies focused on the development of VSW1198 as a novel anti-cancer agent.