Synthesis and Biological Evaluation of Trehalose-based Bi-aryl Derivatives as C-type Lectin Ligands

Abstract

The identification of Mincle as the C-type lectin receptor on innate immune cells responsible for binding TDM and the realization that this receptor could be key to productive vaccines for mycobacterial infection has raised interest in the development of synthetic Mincle ligands as novel adjuvants. We recently reported on the synthesis and evaluation of Brartemicin analog UM-1024 that demonstrated Mincle agonist activity, exhibiting potent Th1/Th17 adjuvant activity that was greater than that of trehalose dibehenate (TDB). Our pursuit to understand Mincle/ligand relationships and improve the pharmacologic properties of the ligands has expanded and continues to reveal new and exciting structure activity relationships. Herein we report the synthesis of novel bi-aryl trehalose derivatives in good to excellent yields. These compounds were evaluated for their ability to engage the human Mincle receptor and tested for the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel bi-aryl derivatives revealed that bi-aryl trehalose ligand 3D showed relatively high potency in cytokine production in comparison to trehalose glycolipid adjuvant TDB and the natural ligand TDM and induced dose-dependent, Mincle selective stimulation in hMincle HEK reporter cells. Also, through computational studies, we provide an insight into the potential mode of binding of 6,6′-Biaryl trehalose compounds on human Mincle receptor.

Graphical Abstract

1. Introduction

There is increasing interest in developing new vaccines against tuberculosis and other infectious diseases with subunit antigens.^[1] The reduced immunogenicity of the refined and homogenous subunit antigens necessitates the use of immune adjuvants to enhance the vaccines ability to elicit strong and durable cell-mediated immune responses to a specific antigen.^[2] Adjuvants are playing a key role in this endeavor however, the number of adjuvants approved for human use and receptors they target are limited. A breakthrough was the identification of the macrophage inducible C-type lectin (Mincle, Clec4e, or Clecf9) as a pattern recognition receptor (PRR) on the surface of macrophages and dendritic cells which are activated through ligation of trehalose dimycolate (TDM), a major component of Mycobacterium cell wall, and its synthetic analog trehalose dibehenate (TDB) (Figure 1).^[3,4] TDM and TDB have the ability to stimulate the innate and early adaptive immune response that has led to an interest in the potential of these molecules as adjuvants.^[5] In particular, TDB has shown promise as a vaccine adjuvant for both tuberculosis and HIV when formulated in the CAF01 liposome system.^[6]

Figure 1:

Representative Mincle ligands TDM, TDB, Brartemicin, UM-1024 and C18dMeBrar.

In addition to TDM and TDB, several synthetically derived ligands have been identified as Mincle agonists.^[7,8] In general these ligands are highly lipidated and lack favorable pharmacologic properties for incorporation into vaccines. In 2015, 6,6′-bis(2,4-dihydroxy-6-methylbenzoyl)-α,α-D-trehalose (Brartemicin), a trehalose-based natural product, was reported as a high-affinity ligand of the carbohydrate recognition domain (CRD) of Mincle.^[9] Furthermore, an in-silico docking study of brartemicin with bovine Mincle positions aryl substituents of the ligand in close proximity to Phe198 in the hydrophobic groove adjacent to the CRD and the other aryl group interacts with Arg182.^[10] Recently, we also reported in silico docking study of UM-1024, which showed no interaction of pendant aryl group with Arg182.^[8,11] Instead, the poses indicated a possible interaction with a small hydrophobic pocket adjacent to Phe198, shifting Phe198 towards significant pi-pi interaction of UM-1024 aryl side chain in bovine and human Mincle crystal structures. Given the surprising and improved activity of our mono-aryl trehalose derivatives, we were curious if the interaction of an extended aryl chain could take advantage of the potentially important pi-pi interaction of the aryl rich region adjacent to the CRD of bovine and human Mincle, F189 and Y201 in particular. Bovine Mincle was selected for docking of the bi-aryl trehalose derivative 3d to provide a core for the trehalose bound in the Phe198 in the hydrophobic groove adjacent to the CRD and the other aryl group interacts with Arg182.^[10] Recently, we also reported in silico docking study of UM-1024, which showed no interaction of pendant aryl group with Arg182.^[8,11] Instead, the poses indicated a possible interaction with a small hydrophobic pocket adjacent to Phe198, shifting Phe198 towards significant pi-pi interaction of UM-1024 aryl side chain in bovine and human Mincle crystal structures.

2. Results and Discussion

Given the surprising and improved activity of our mono-aryl trehalose derivatives, we were curious if the interaction of an extended aryl chain could take advantage of pi-pi interactions of the aryl rich region adjacent to the CRD of bovine and human Mincle, F189 and Y201 in particular. Bovine Mincle was selected for docking of the bi-aryl trehalose derivative 3d to provide a core for the trehalose bound in the CRD of the solved structure, and the high protein homology of bovine and human Mincle. The study places Ar² in close proximity to F198, a potential edge-face pi interaction in addition Y201 and Ar1 the vicinity of the F202 residue (Figure 2). We postulated that an extension of the ligand side chain through acylation of trehalose with commercially available bi-aryl benzoic acids would provide insight toward the importance of the potentially strong aryl interactions with substituents in this aryl-rich region adjacent to the CRD.

Figure 2:

High-ranking docking pose of 3d (purple) in bovine Mincle (Protein data bank 4KZV) overlays with trehalose (yellow) and maintains similar interactions with bound calcium (green).

We prepared these analogs via two synthetic routes as shown in Scheme 1. The TMS-protected 6,6′-trehalose diol^[8c,12] 1 was utilized for the efficient double ester coupling with various aryl carboxylic acids (2.5 equiv. per 1 equiv. of TMS-protected trehalose) using an excess of EDCI-MeI or DCC and DMAP. Under these optimized reaction conditions, coupled products were synthesized in a range of isolated yields (40–90%) after purification. We also optimized reaction conditions for coupling aryl acids containing an unprotected hydroxyl moiety (3e, 3i, 3j, 3k, and 3m) that are inaccessible via carbodiimide strategies. These were synthesized in three steps from diol in 80–90% overall yield by employing the highly reactive 6,6′-bis-O trifluoromethanesulfonate (triflate) in conjunction with potassium salts of aryl carboxylic acids. These salts of the aryl carboxylic acids were generated in quantitative yield in THF using potassium trimethylsilanoate. A solution of the acid (1 equiv.) and KOSiMe₃ (1.2 equiv.) were stirred for 30 min at room temperature and then solvent was removed in vacuo to obtain the precipitated salt which was used without further purification. The crude ditriflate (1 equiv.) was condensed with the potassium salts (2.2 equiv.) of aryl carboxylic acid and 18-crown ether in toluene at 80 °C to give symmetrical hexa-O-trimethylsilyl 6,6′ diesters 2 in good to excellent yield (65–80%).^[13] Having accomplished the synthesis of the protected diesters, global deprotection of TMS groups using Dowex-H⁺ resin was used to prepare the targeted compounds (Scheme 1).^[14] These reactions do not require the use of strictly anaerobic or scrupulously anhydrous reaction conditions. Accordingly, we set out to synthesize a variety of analogs with different functionalities on the aromatic ring so that these could be tested in functional assays. All these novel compounds were fully characterized by spectroscopic techniques.

Scheme 1:

Synthesis of Trehalose derivatives 3(a-p).

Testing the activity of compounds on primary human PBMCs is a sensitive and convenient tool for the identification and comparison of new Mincle ligands. This also provides useful information on the production of cytokines, such as IL-6 and IL-1β which promote the development of a Th1/Th17 immune response.^[15] Therefore, for preliminary screening, the synthesized compounds were assayed for IL-6, TNFα, and IL-1β production in human PBMC’s (Figure 3 & Figure 4).^[16] TDB and TDM were used as positive controls in the assays.^[17] Briefly, compounds were serially diluted in ethanol, applied to tissue culture plates and the solvent was evaporated. Freshly isolated human PBMCs were added and incubated with the compound for 18–24 hours. Supernatants were collected and evaluated for IL-6 levels by ELISA. A small library of commercially available bi-aryl benzoic acids was used explore the postulate that the extended aryl compounds may be useful as Mincle ligands through induction of cytokines in human PBMCs in comparison with our previously reported highly active Mincle ligand UM-1024 (Figure 1; Figure 3).^[11] Cellular assays are monitored for cell viability and potential toxicology of new compounds. No indication of compound related cell toxicity throughout the doses evaluated was observed (data not shown). Consistent with our previous findings, the 2′-methylpheyl group at 3 position of Ar¹ (3d) instead of tert-butyl exhibited similar activity to UM-1024 (Figure 3). Notably, the compound without any substituent on Ar², 3c, resulted in no significant production of IL-6. This stressed the subtle bulkiness requirement at the 3 position of Ar² in combination with the position of Ar² on Ar¹. Investigation of the surprising activity 3d, we synthesized 3i in which the methyl is replaced with a hydroxyl group. This resulted in a complete loss of activity. On the other hand, 4-hydroxy phenyl acid (3e) resulted in a low but measurable production of IL-6, while its methylated analog (3g) resulted in a complete loss of activity. Additionally, it was found that the presence of hydroxyl group at 2 position (3l) resulted in decreased human IL-6 production. Also, moving the hydroxyl position to the 4′- position, in 3J resulted in no IL-6 production. The 3′-methoxy-4′-fluoro substituted (3h) and meta hydroxy-substituted compound (3j) exhibited little to no activity. Furthermore, we have demonstrated that the phenyl group at ortho (3b) and para position (3a) of the aromatic ring resulted in modest IL-6 production at higher concentrations. We further explored the effect of substituents on the phenyl ring at the para position of the AR¹ by synthesizing 3K, 3M and 3N. These demonstrated an improved IL-6 response compared to 3A. Interestingly, phenyl anthranilic acid derivatives (3o & 3p) showed no significant response.

Figure 3:

Cytokine production of UM-1024, TDB, TDM, 3D and synthesized compounds (3A-P). Cytokine production from primary human mononuclear cells in response to stimulation with synthetic derivatives. The indicated compound was dissolved in ethanol, serially diluted, and then dried to the bottom of a tissue culture plate. Fresh PBMCs were purified, applied to the compound-coated plates, and incubated at 37 °C; the supernatant was harvested 24 h later. Graphs are mean values from 3 donors +/− SEM.

Figure 4: Cytokine production of synthesized compounds (3A-P).

Cytokine production from primary human mononuclear cells in response to stimulation with synthetic derivatives. The indicated compound was dissolved in ethanol, serially diluted, and then dried to the bottom of a tissue culture plate. Fresh PBMCs were purified, applied to the compound-coated plates, and incubated at 37°C; the supernatant was harvested 24 h later. Graphs are mean values from 3 donors +/− SEM.

In addition to IL-6, we also examined the capability of the synthesized compounds to stimulate human peripheral blood monocytes to produce the inflammatory cytokines TNFα and IL-1β to determine if indeed we are potentially driving a Th1 or Th17 biased adaptive immune response. As illustrated, these compounds produce appreciable quantities of TNFα and IL-1β (Figure 4). 3d is used as a positive control for this analysis. Further investigations into immunomodulatory properties, adjuvant activity, and mode of action of 3d are currently underway. The limited SAR presented here and the remarkable activity of a single compound raises many questions.

To confirm the demonstrated cytokine activity was Mincle specific, the compounds were evaluated for their ability to signal through human Mincle using human embryonic kidney 293 (HEK) cells expressing the human Mincle receptor along with an NF-κb-driven secreted embryonic alkaline phosphatase (SEAP) reporter (see Supplementary Methods for details). Several of the analogs were observed to induce the production of SEAP in a dose-dependent manner. HEK null cells (HEK cells containing the NF-κb reporter without Mincle receptor) were used as negative controls to confirm receptor specificity of the compounds (Figure 5). 3d demonstrated a dose-dependent response in the hMincle HEK reporter cells. Similarly, 3a, 3b, showed a strong dose-dependent increase in SEAP production from hMincle HEK cells. The rest of the compounds with different substitutions on the aromatic ring were less active in hMincle HEK reporter cells. Further investigations into immunomodulatory properties and mode of action of these interesting new compounds are currently underway

Figure 5: Activation of human Mincle in response to TDM, TDB, and synthesized derivatives.

A) and B) The indicated compounds were plate coated and hMincle HEK NF-κB-SEAP reporter cells were incubated with the compounds for 24 h followed by assessment of the supernatants for SEAP levels. C) results in HEK null cells Data are represented as fold change in OD650 over vehicle-treated cells. Graphs are mean values from three independent experiments ± SEM.

3. Conclusion

In summary, we have expanded our understanding of Mincle ligand structural and functional requirements using in silico docking and validated the hypothesis that extension of the aryl functionality in the side chain is tolerated as demonstrated through ability of these compounds to stimulate human peripheral blood monocytes to produce inflammatory cytokines (IL-6, TNFα, and IL-1β) in a Mincle specific and non-toxic fashion. This work provides a significant new avenue for Mincle ligand structure exploration. Through this study we are beginning to better understand that Mincle activity is very sensitive to trehalose substitution patterns and a high degree of hydrophobicity may not be required to maintain Mincle selective activity as demonstrated for 6,6′-bezoate derivatives depicted here. These observations serve as a door toward the discovery of new and potent immunomodulators with greatly improved pharmacological properties with potential application in vaccine research and Immunotherapy.

4. Experimental

All reagents and solvents were used as received. Reactions were monitored by TLC-analysis on Merck Silica gel 60 F254 plates and visualized by UV at 254 nm and dipping in vanillin (vanillin/water/ethanol/sulfuric acid, 0.2 g:5 mL:5 mL:1 mL) or phosphomolybdic acid in ethanol (PMA) and developed with heat. All compounds were confirmed to be >95% pure by NMR and HPLC-CAD analysis. ¹H and ¹³C NMR spectra were recorded on an Agilent or Bruker 400 MHz instrument and were referenced to TMS or a solvent peak. High-resolution HPLC-MS analysis was obtained on an Agilent 6520 Q-TOF mass spectrometer utilizing an electrospray ionization source in positive or negative mode. Chromatography was performed on Grace or Biotage automated medium pressure chromatography instruments with preloaded Buchi silica gel cartridges. Human and mouse Mincle expressing HEK cells were obtained from Invivogen (San Diego, CA). Cells were cultured according to the manufacturer’s instructions in DMEM with 10% FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin, 2 mM L-glutamine, 30 μg/ml blasticidin, 1 μg/ml puromycin, and 1x HEK-Blue^™ CLR Selection. 2,2’,3,3’,4,4’-Hexa-trimethylsilyl-α,α-D-trehalose was prepared using the literature method without any modification. ^[18]

General procedure for esterification via carbodiimide mediated coupling:

To a stirred mixture of 2,2’,3,3’,4,4’-Hexa-trimethylsilyl-α,α-D-trehalose (1 eq.; 1 mmol), aryl carboxylic acid (2.2 eq.; 2.2 mmol) and DMAP (3 eq.; 3 mmol) in anhydrous DCM (10 mL) was added DCC (3 eq.; 3 mmol) or EDCI-MeI (5 eq.; 5 mmol) at 0 °C for 30 min and then at room temperature overnight. The reaction mixture was diluted with water and then extracted it with DCM. The combined organic layers were dried over MgSO₄ and reduced in vacuo. The crude mixture was subjected to chromatography using the Biotage system with a 12 g silica column and a zero to 20% ethyl acetate in heptane gradient. This yielded the silyl intermediate in good to excellent yields.

General procedure for SN₂ acylation:

To a stirred solution of protected trehalose in anhydrous DCM (10 mL/g) and pyridine (6 eq. for each OH) were added triflic anhydride (2.5 eq. for each OH) at − 5 °C dropwise. The reaction mixture was allowed to warm gradually at room temperature stirred for 30 min. After this the mixture was diluted with DCM and washed with cold 1 M HCl, aq. NaHCO₃ and then with water. The organic layer was evaporated under reduced pressure and the triflate was used it for next step without any purification. [¹H NMR (400 MHz, CDCl₃) δ 4.94 (d, J = 3.06 Hz, 1H), 4.61 (d, J = 1.96 Hz, 1H), 4.55 (d, J = 4.52 Hz, 1H), 4.07 – 4.14 (m, 1H), 3.87 (t, J = 9.05 Hz, 1H), 3.37 – 3.47 (m, 2H), 0.12 – 0.20 (m, 27H); ¹³C NMR (400 MHz, CDCl₃) δ 123.7, 120.5, 117.3, 114.2, 95.6, 75.4, 73.4, 72.7, 71.6, 70.8, 1.3, 1.1, 0.38].

The mixture of potassium salt of an aryl acid (2.2 eq.) [synthesis (2.5 mmol of acid and 1.5 mL of KOTMS were dissolved in THF and stirred for 10 min and then solvent was removed under reduced pressure and used without purification)] triflate (1 eq. ) and 18-crown-6 (1 eq. ) were heated in toluene at 70 °C for 12 hrs. The reaction mixture was diluted with DCM and washed with water. The organic layer was dried over MgSO₄ and concentrated. The crude mixture was subjected to chromatography using the Biotage system with a 12 g silica column and a zero to 20% ethyl acetate in heptane gradient.

General procedure for deprotection of silyl Ethers:

The silyl intermediate (0.333 mmol) was dissolved in equal amount of methylene chloride and methanol (8 mL) and treated with Dowex 50WX8 resin (668.4 mg) with magnetic stirring. Upon consumption of the starting material as determined by TLC (20% methanol in methylene chloride and charring with vanillin stain) the reaction was filtered, concentrated and chromatographed on a silica column eluting with a 40% to 80% methylene chloride to methanol gradient (Biotage system using a 12 g pre-packed column) to provided desired product. The analytical data of all final compounds are as follows:

Synthesis of 6-6-((([1,1’-biphenyl]-4-carbonyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl [1,1’-biphenyl]-4-carboxylate (3a): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (88%; 37 mg; white solid); ¹H NMR (400 MHz, DMSO-d₆) δ 7.76 – 7.82 (m, 1H), 7.60 – 7.65 (m, 1H), 7.48 – 7.54 (m, 1H), 7.34 – 7.44 (m, 3H), 7.29 (d, J = 7.21 Hz, 3H), 5.09 (d, J = 5.14 Hz, 1H), 4.89 (d, J = 5.01 Hz, 1H), 4.85 (d, J = 5.62 Hz, 1H), 4.80 (d, J = 3.55 Hz, 1H), 4.27 – 4.34 (m, 1H), 4.12 – 4.19 (m, 1H), 3.92 – 3.99 (m, 1H), 3.49 – 3.59 (m, 1H), 3.21 – 3.28 (m, 1H), 3.05 – 3.13 (m, 1H); ¹³C NMR(100 MHz, DMSO-d₆) δ168.00, 141.95, 140.83, 140.36, 131.88, 131.38, 131.15, 129.83, 128.71, 128.65, 127.86, 127.63, 94.12, 73.17, 71.81, 70.49, 70.13, 64.40; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₃ + H)⁺ requires 703.2385, found: 703.2380.

Synthesis of 6-6-((([1,1’-biphenyl]-2-carbonyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl [1,1’-biphenyl]-2-carboxylate (3b): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (85%; 33 mg; white solid); ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (d, J = 8.44 Hz, 2H), 7.84 (d, J = 8.44 Hz, 2H), 7.74 (d, J = 7.21 Hz, 2H), 7.48 – 7.53 (m, 2H), 7.41 – 7.46 (m, 1H), 5.21 – 5.25 (m, 1H), 4.97 – 5.00 (m, 2H), 4.92 – 4.95 (m, 1H), 4.44 – 4.50 (m, 1H), 4.32 – 4.38 (m, 1H), 4.09 – 4.17 (m, 1H), 3.58 – 3.67 (m, 1H), 3.35 – 3.41 (m, 1H), 3.27 – 3.32 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.96, 145.14, 144.66, 139.38, 134.32, 94.44, 73.35, 71.98, 70.66, 70.36, 64.59, 31.18; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₃ + H)⁺ requires 703.2385, found: 703.2382

Synthesis of 6-6-((([1,1’-biphenyl]-3-carbonyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl [1,1’-biphenyl]-3-carboxylate (3c): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (84%; 35 mg; yellowish powder); ¹H NMR (400 MHz, DMSO-d₆) δ 8.17 – 8.20 (m, 1H), 8.02 – 8.06 (m, 1H), 7.94 – 7.98 (m, 1H), 7.81 – 7.86 (m, 1H), 7.72 – 7.76 (m, 1H), 7.60 – 7.71 (m, 2H), 7.41 – 7.54 (m, 2H), 5.18 – 5.28 (m, 1H), 4.92 – 5.01 (m, 3H), 4.43 – 4.56 (m, 1H), 4.26 – 4.39 (m, 1H), 4.05 – 4.19 (m, 1H), 3.58 – 3.68 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆)δ 166.86, 140.81, 139.69, 139.22, 130.74, 130.25, 130.01, 128.69, 127.51, 126.72, 126.49, 92.98, 72.03, 70.67, 69.36, 68.99, 63.26; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₃ + H)⁺ requires 703.2385, found: 703.2389.

6-6-(((2’-methyl-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 2’-methyl-[1,1’-biphenyl]-3-carboxylate (3d): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (86%; 36 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 7.97 (td, J = 1.65, 7.09 Hz, 1H), 7.88 (s, 1H), 7.58 – 7.66 (m, 2H), 7.19 – 7.31 (m, 4H), 5.23 (d, J = 5.38 Hz, 1H), 4.97 (d, J = 4.89 Hz, 1H), 4.85 – 4.92 (m, 2H), 4.49 (d, J = 10.03 Hz, 1H), 4.32 (dd, J = 5.69, 11.68 Hz, 1H), 4.04 – 4.12 (m, 1H), 3.59 (td, J = 4.57, 9.08 Hz, 1H), 3.22 – 3.30 (m, 2H), 2.47 – 2.55 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.28, 141.36, 139.91, 134.42, 133.56, 130.20, 129.66, 129.23, 129.17, 12 8.57, 127.54, 127.49, 125.86, 93.28, 72.54, 71.26, 69.90, 69.51, 63.85, 19.77; HRMS (ESI+, m/z) calcd for (C₄₀H₄₂O₁₃ + H)⁺ requires 731.2697, found: 731.2691.

6-6-(((4’-hydroxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4’-hydroxy-[1,1’-biphenyl]-3-carboxylate (3e): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (75%; 32 mg; white powder); ¹H NMR (400 MHz, DMSO-d6) δ 8.08 – 8.21 (m, 1H), 7.83 – 7.95 (m, 2H), 7.51 – 7.67 (m, 3H), 6.99 – 7.12 (m, 2H), 5.09 (d, J = 6.11 Hz, 1H), 4.89 (d, J = 5.01 Hz, 1H), 4.85 (d, J = 5.87 Hz, 1H), 4.80 (d, J = 3.55 Hz, 1H), 4.27 – 4.36 (m, 1H), 4.11 – 4.21 (m, 1H), 3.91 – 4.00 (m, 1H), 3.48 – 3.60 (m, 1H), 3.20 – 3.28 (m, 1H), 3.03 – 3.14 (m, 1H)^{; 13}C NMR (100 MHz, DMSO-d6) δ165.87, 159.15, 140.43, 131.48, 131.13, 130.60, 129.66, 128.00, 126.72, 116.17, 115.45, 93.93, 73.08, 71.71, 70.48, 70.02, 67.60, 64.57; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2282.

6-6-(((3’-hydroxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3’-hydroxy-[1,1’-biphenyl]-3-carboxylate (3f): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (20%; 12 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 9.61 (br. s., 1H), 8.13 (s, 1H), 7.95 (d, J = 7.83 Hz, 1H), 7.88 (d, J = 7.82 Hz, 1H), 7.61 (t, J = 7.76 Hz, 1H), 7.23 – 7.30 (m, 1H), 7.02 – 7.11 (m, 2H), 6.78 (dd, J = 1.71, 8.07 Hz, 1H), 5.25 (d, J = 5.26 Hz, 1H), 4.91 – 5.02 (m, 3H), 4.51 (d, J = 10.27 Hz, 1H), 4.33 (dd, J = 5.93, 11.68 Hz, 1H), 4.14 (dd, J = 5.75, 9.78 Hz, 1H), 3.64 (dt, J = 4.40, 8.99 Hz, 1H), 3.36 – 3.41 (m, 1H), 3.29 (dt, J = 5.44, 9.32 Hz, 1H); ¹³CNMR (400 MHz, DMSO-d₆)δ 165.56, 157.94, 140.69, 140.45, 131.36, 130.39, 130.11, 129.47, 128.10, 127.03, 117.36, 114.97, 113.49, 93.66, 72.83, 71.50, 70.22, 69.82, 64.31; HR MS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2280.

6,6-(((4’-methyl-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4’-methyl-[1,1’-biphenyl]-3-carboxylate (3g): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (57%; 26 mg; yellowish powder); ¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (s, 1H), 7.92 (t, J = 7.64 Hz, 2H), 7.53 – 7.62 (m, 3H), 7.26 (d, J = 8.07 Hz, 2H), 5.25 (d, J = 5.38 Hz, 1H), 4.98 – 5.01 (m, 2H), 4.95 (d, J = 5.99 Hz, 1H), 4.51 (d, J = 10.27 Hz, 1H), 4.33 (dd, J = 6.17, 11.68 Hz, 1H), 4.08 – 4.18 (m, 1H), 3.64 (dt, J = 5.01, 9.05 Hz, 1H), 3.36 – 3.41 (m, 1H), 3.28 (dt, J = 5.44, 9.32 Hz, 1H), 2.30 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.57, 140.47, 137.33, 136.16, 131.19, 130.44, 129.66, 129.44, 127.82, 12 6.85, 126.49, 93.53, 72.84, 71.57, 70.30, 69.82, 64.35, 20.62; HRMS (ESI+, m/z) calcd for (C₄₀H₄₂O₁₃ + H)⁺ requires 731.2697, found: 731.2698.

6,6-(((4’-fluoro-3’-methoxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4’-fluoro-3’-methoxy-[1,1’-biphenyl]-3-carboxylate (3h): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (76%; 40 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (s, 1H), 7.96 (dd, J = 1.65, 7.76 Hz, 2H), 7.62 (t, J = 7.76 Hz, 1H), 7.40 (dd, J = 2.08, 8.31 Hz, 1H), 7.17 – 7.31 (m, 2H), 5.25 (d, J = 5.50 Hz, 1H), 4.99 (d, J = 5.01 Hz, 1H), 4.91 – 4.97 (m, 2H), 4.50 (d, J = 10.03 Hz, 1H), 4.34 (dd, J = 6.05, 11.68 Hz, 1H), 4.10 – 4.17 (m, 1H), 3.92 (s, 3H), 3.63 (dt, J = 5.01, 9.11 Hz, 1H), 3.22 – 3.32 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.71, 152.94, 150.51, 147.74, 140.15, 136.38, 131.90, 130.64, 129.60, 12 8.34, 127.45, 119.27, 116.54, 116.36, 112.65, 93.85, 73.03, 71.76, 70.46, 70.0 3, 64.55, 58.30; HRMS (ESI+, m/z) calcd for (C₄₀H₄₀F₂O₁₅ + H)⁺ requires 799.2408, found: 799.2411.

6-6-(((2’-hydroxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 2’-hydroxy-[1,1’-biphenyl]-3-carboxylate (3i): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (81%; 47 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 9.54 (br. s., 1H), 8.14–8.17 (m, 1H), 7.94 – 8.05 (m, 1H), 7.81 – 7.88 (m, 3H), 7.47 – 7.55 (m, 3H), 5.18 (d, J = 5.26 Hz, 1H), 4.86 – 4.95 (m, 3H), 4.44 (d, J = 10.27 Hz, 1H), 4.27 (dd, J = 5.93, 11.68 Hz, 1H), 4.08 (dd, J = 5.75, 9.78 Hz, 1H), 3.57 (dt, J = 4.40, 8.99 Hz, 1H), 3.30 – 3.35 (m, 1H), 3.23 (dt, J = 5.44, 9.32 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) d 164.94, 140.07, 139.83, 129.77, 129.49, 128.85, 127.48, 126.41, 116.74, 114.35, 112.87, 93.04, 72.21, 70.88, 69.60, 69.20, 63.69; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2281.

6-6-(((5-hydroxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 5-hydroxy-[1,1’-biphenyl]-3-carboxylate (3j): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (82%; 49 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 10.04 (br. s., 1H), 7.64 – 7.66 (m, 1H), 7.61 (d, J = 7.21 Hz, 2H), 7.44 (t, J = 7.70 Hz, 2H), 7.36 – 7.38 (m, 1H), 7.27 – 7.34 (m, 2H), 5.24 (d, J = 5.38 Hz, 1H), 4.98 – 5.03 (m, 2H), 4.91 (d, J = 5.87 Hz, 1H), 4.51 (d, J = 10.27 Hz, 1H), 4.28 (dd, J = 5.93, 11.68 Hz, 1H), 4.06 – 4.16 (m, 1H), 3.64 (d, J = 4.65 Hz, 1H), 3.35 – 3.39 (m, 1H), 3.24 – 3.31 (m, 1H); ¹³C NMR (400 MHz, DMSO-d₆) d 164.64, 157.14, 141.04, 138.36, 130.71, 128.12, 126.98, 125.68, 117.32, 113.88, 92.61, 71.94, 70.65, 69.35, 68.88, 63.37; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2279.

6-6-(((3-hydroxy-[1,1’-biphenyl]-4-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-hydroxy-[1,1’-biphenyl]-4-carboxylate (3k): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (76%; 39 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 10.56 (s, 1H), 7.90 (d, J = 8.19 Hz, 1H), 7.69 – 7.74 (m, 2H), 7.40 – 7.53 (m, 3H), 7.24 – 7.32 (m, 2H), 5.24 (d, J = 5.38 Hz, 1H), 4.98 – 5.03 (m, 2H), 4.91 (d, J = 5.87 Hz, 1H), 4.51 (d, J = 10.27 Hz, 1H), 4.28 (dd, J = 5.93, 11.68 Hz, 1H), 4.05 – 4.18 (m, 1H), 3.64 (d, J = 4.65 Hz, 1H), 3.36 – 3.40 (m, 1H), 3.23 – 3.32 (m, 1H); ¹³C NMR (400 MHz, DMSO-d₆) δ 168.30, 160.33, 147.02, 138.31, 130.49, 128.82, 128.44, 126.73, 117.85, 11 4.78, 111.54, 93.82, 72.56, 71.24, 69.90, 69.44, 64.22, 48.37; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2285.

6,6-(((2-hydroxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 2-hydroxy-[1,1’-biphenyl]-3-carboxylate (3l): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (73%; 33 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 11.11 (s, 1H), 7.88 (dd, J = 1.71, 7.95 Hz, 1H), 7.60 (dd, J = 1.77, 7.52 Hz, 1H), 7.52 – 7.56 (m, 2H), 7.39 – 7.46 (m, 2H), 7.31 – 7.38 (m, 1H), 7.08 (t, J = 7.76 Hz, 1H), 5.29 (d, J = 5.38 Hz, 1H), 5.02 (t, J = 5.81 Hz, 2H), 4.93 (d, J = 3.55 Hz, 1H), 4.38 – 4.58 (m, 2H), 4.17 (ddd, J = 2.02, 5.17, 9.93 Hz, 1H), 3.63 (dt, J = 4.89, 9.11 Hz, 1H), 3.36 – 3.41 (m, 1H), 3.24 – 3.32 (m, 1H); ¹³CNMR (100 MHz, DMSO-d₆) δ 169.65, 157.84, 136.75, 129.66, 129.17, 128.10, 127.30, 119.63, 112.57, 94.09, 72.76, 71.46, 70.08, 69.63, 64.71; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2280.

6-6-(((4’-hydroxy-[1,1’-biphenyl]-4-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4’-hydroxy-[1,1’-biphenyl]-4-carboxylate (3m): The diaryl ester was prepared according to the general procedure for esterification via triflate coupling and silyl deprotection to obtain the desired compound (83%; 44 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (d, J = 3.91 Hz, 2H), 7.73 – 7.83 (m, 2H), 7.65 – 7.72 (m, 2H), 7.06 (d, J = 2.81 Hz, 2H), 5.20 (d, J = 5.01 Hz, 1H), 5.11 (d, J = 5.01 Hz, 1H), 4.90 – 4.97 (m, 4H), 4.76 – 4.85 (m, 1H), 4.42 – 4.51 (m, 1H), 4.29 – 4.38 (m, 1H), 4.15 (br. s., 2H), 3.62 (d, J = 5.26 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) 165.8, 159.5, 131.2, 130.1, 128.3, 128.2, 126.4, 115.4, 102.3, 95.5, 94.2, 94.1, 73.1, 71.6, 70.7, 70.1; HRMS (ESI+, m/z) calcd for (C₃₈H₃₈O₁₅ + H)⁺ requires 735.2283, found: 735.2282.

6-6-(((4’-methoxy-[1,1’-biphenyl]-3-carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4’-methoxy-[1,1’-biphenyl]-3-carboxylate (3n): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (88%; 49 mg; white powder); ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 8.19 Hz, 2H), 7.78 (d, J = 8.31 Hz, 2H), 7.69 (d, J = 8.68 Hz, 2H), 7.04 (d, J = 8.68 Hz, 2H), 5.24 (d, J = 4.77 Hz, 1H), 4.88 – 5.05 (m, 3H), 4.42 – 4.51 (m, 1H), 4.35 (dd, J = 5.50, 11.62 Hz, 1H), 4.08 – 4.18 (m, 1H), 3.57 – 3.70 (m, 1H), 3.32 (d, J = 10.15 Hz, 2H); ¹³CNMR (100 MHz, DMSO-d₆) δ 165.57, 159.66, 144.30, 131.08, 129.87, 128.18, 127.18, 126.28, 114.54, 93.90, 72.88, 71.52, 70.18, 69.90, 64.04, 55.25; HRMS (ESI+, m/z) calcd for (C₄₀H₄₂O₁₅ + H)⁺ requires 763.2596, found: 763.2599.

6-6-(((2-((2,3-dimethylphenyl)amino)benzoyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 2-((2,3-dimethylphenyl)amino)benzoate (3O): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (56%; 34 mg; yellow powder);¹H NMR (400 MHz, DMSO-d₆) δ 9.10 (s, 1H), 7.92 (dd, J = 1.41, 8.01 Hz, 1H), 7.28 – 7.36 (m, 1H), 7.09 – 7.13 (m, 2H), 7.04 (d, J = 3.42 Hz, 1H), 6.73 (t, J = 7.58 Hz, 1H), 6.64 (d, J = 8.56 Hz, 1H), 4.90 (d, J = 3.67 Hz, 1H), 4.41 – 4.48 (m, 1H), 4.33 – 4.40 (m, 1H), 4.12 (dd, J = 2.69, 9.90 Hz, 1H), 3.61 (t, J = 9.17 Hz, 1H), 3.22 – 3.31 (m, 4H), 2.27 (s, 3H), 2.08 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) 154.9, 148.8, 138.1, 131.8, 126.9, 126.3, 122.8, 116.7, 113.5, 110.8, 94.1, 73.0, 69.9, 63.9, 20.4, 13.8; HRMS: (C₄₂H₄₈N₂O₁₃ + H)⁺ requires 789.3229 Found 789.3229

6,6-(((2-(phenylamino)benzoyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 2-(phenylamino)benzoate (3P): The diaryl ester was prepared according to the general procedure for esterification via carbodiimide-mediated coupling and silyl deprotection to obtain desired compound. (61%; 34 mg; yellow powder); ¹H NMR (400 MHz, DMSO-d₆) δ 9.26 (s, 1H), 7.94 (dd, J = 1.59, 7.95 Hz, 1H), 7.38 – 7.43 (m, 1H), 7.31 – 7.37 (m, 2H), 7.18 – 7.26 (m, 3H), 7.01 – 7.10 (m, 1H), 6.76 – 6.86 (m, 1H), 5.23 (d, J = 5.38 Hz, 1H), 4.96 (dd, J = 3.61, 5.20 Hz, 2H), 4.92 (d, J = 3.67 Hz, 1H), 4.32 – 4.48 (m, 2H), 4.13 (ddd, J = 2.20, 4.83, 9.96 Hz, 1H), 3.62 (dt, J = 4.95, 9.08 Hz, 1H), 3.34 – 3.37 (m, 1H), 3.28 (td, J = 4.71, 9.41 Hz, 1H); ¹³C NMR (400 MHz, DMSO-d₆) δ 167.39, 146.71, 140.34, 134.45, 131.55, 129.47, 123.28, 121.56, 117.71, 11 4.03, 112.15, 93.93, 72.79, 71.49, 70.14, 69.72, 63.78; HRMS: (C₃₈H₄₀N₂O₁₃ + H)⁺ requires 733.2605 Found 733.2607.

Biological Activity

Transgenic HEK cell SEAP assays.

Human Mincle expressing HEK cells were obtained from Invivogen (San Deigo, CA). Cells were cultured according to the manufacturer’s instructions in DMEM with 10% FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin, 2 mM L-glutamine, 30 μg/ml blasticidin, 1 μg/ml puromycin and 1x HEK-Blue^™ CLR Selection. For assay, indicated compounds were serially diluted in diluent (EtOH), 20 μl of a 10x final concentration were applied to the bottom of a 96-well tissue culture plate and the solvent was evaporated for > 1 hr in a biosafety hood. HEK cells were applied to the plates at a density of 3×10⁵ cells/well and incubated for 18–24 hour at 37 °C. Cell supernatants were harvested and analyzed via the manufacturer’s instructions using Hek-Blue^™ Detection. SEAP activity was assessed by reading the optical density (OD) at 620–655 nm with a microplate reader; data are expressed as the fold change in OD over vehicle treated cells.

Isolation of PBMC.

Peripheral blood samples were collected from healthy adult donors after approval by the University of Montana Institutional Review Board (43–16) and signed written informed consent was obtained from each donor. Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood using Ficoll-Paque. Briefly, heparin-anticoagulated blood was diluted with an equal volume of Dulbeccos phosphate-buffered saline, pH 7.4 (DPBS), 35 mL of diluted blood was layered over 15 ml of the Ficoll-Paque (Sigma). Gradients were centrifuged at 400 × g for 30 min at room temperature in a swinging-bucket rotor without the brake applied. The PBMC interface was carefully removed by pipetting and washed with PBS-5%FBS by centrifugation at 250 × g for 10 min. This was followed by a second wash with PBS-5%FBS. Cells were resuspended in RPMI media with 5% autologous plasma. Cell number and viability were determined using a hemocytometer. Non-viable cells were identified by staining with trypan blue and cell counts calculated on viable cells only.

Cytokine analysis.

Compounds were serially diluted in 100% Ethanol to the bottom of a tissue culture plate and solvent was allowed to fully evaporate (i.e. plate coating). Freshly prepared PBMCs purified via Ficoll gradient in RPMI/5% autologous plasma were added to the indicated compound concentrations by addition to plates containing plate coated compounds. Cells were incubated at 37 °C/5% CO₂.

Supernatants were harvested from treated cells 18–24 hour post cell application. Supernatants were analyzed using either a DuoSet IL6 ELISA (R&D Systems, Minneapolis, MN) ELISAs were read on a plate reader at 450 nm and raw OD values potted using Prism software.