A Photochemical Route to Optically Active Hexahydro-4H-furopyranol, a High-Affinity P2 Ligand for HIV-1 Protease Inhibitors

Abstract

We describe here the syntheses of optically pure (3aS,4S,7aR)-hexahydro-4H-furo[2,3-b]pyran-4-ol and (3aR,4R,7aS)-hexahydro-4H-furo[2,3-b]pyran-4-ol. These stereochemically defined heterocycles are important high-affinity P2 ligands for a variety of highly potent HIV-1 protease inhibitors. The key steps involve an efficient Paternò–Büchi [2 + 2] photocycloaddition, catalytic hydrogenation, acid-catalyzed cyclization to form the racemic ligand alcohol, and an enzymatic resolution with immobilized Amano Lipase PS-30. Optically active ligands (−)-6 and (+)-6 were obtained with high enantiomeric purity. Enantiomer (−)-6 has been converted to potent HIV-1 protease inhibitor 3.

Graphical Abstract

Structure-based design is enabling innovations in modern drug discovery and medicinal chemistry.^1,2 This is particularly evident in the design and syntheses of HIV-1 protease inhibitor drugs.^3,4 HIV-1 protease inhibitors are an important component of combined antiretroviral therapy which has dramatically transformed HIV/AIDs from a fatal disease to a manageable chronic disorder.^5,6 In our efforts to develop resistance-proof protease inhibitors (PIs), we have designed a range of exceedingly potent PIs incorporating bicyclic polyether templates that are inherent to many bioactive natural products.^7,8 Many such PIs exhibited broad-spectrum activity against multidrug-resistant HIV-1 variants. For example, we have designed and developed fused bicyclic (3R,3aS,6aR)-bis-tetrahydrofuran (bis-THF) as a nonpeptide, high-affinity P2 ligand, and utilized this ligand to create Darunavir (1, Figure 1), a highly potent FDA approved PI drug.^9,10

Figure 1.

Structures of HIV-1 protease inhibitors 1–3.

Darunavir has emerged as a frontline therapy for HIV/AIDS patients.^11,12 The X-ray structures of Darunavir (1) and its methoxy derivative (2) bound to HIV-1 protease revealed that both PIs are involved in extensive interactions, particularly with the backbone atoms throughout the enzyme active site.¹³ The bis-THF P2 ligand of Darunavir is an intriguing pharmacophore that is responsible for its robust drug resistance profiles.⁷ To further improve binding properties, particularly to enhance both backbone binding and van der Waals interactions, we sought to fine-tune ligand interactions based upon X-ray structures. Therefore, we designed a stereochemically defined hexahydrofuropyranol-based P2 ligand that improved the dihedral angle of the bicyclic acetal to form enhanced hydrogen bonding interactions with the backbone NHs of Asp29 and Asp30. Indeed, X-ray structural analysis of inhibitors bearing the (3aS,4S,7aR)-hexahydro-4H-furo[2,3-b]pyran-4-ol ligand indicated stronger hydrogen bonding interactions with the backbone atoms in the S2 pocket.^14,15 Furthermore, the extra methylene group appears to have favorable van der Waals interactions with hydrophobic residues in the S2 subsite.¹⁴

The hexahydro-4H-furopyranol ligand has been incorporated in the design and synthesis of a host of potent PIs.¹⁴ Our initial synthesis of optically active ligand alcohol 6 involved known, enantiomerically pure lactone 4 as the key starting material.^14,16,17 However, the synthetic route is lengthy, and the synthesis of lactone 4 requires several steps. Our more recent synthesis of optically active ligand alcohol utilized optically active alcohol 5 that was obtained by an enzymatic desymmetrization strategy using 1,2-di(acetoxymethyl)-cyclohex-4-ene as the key starting material.¹⁸ To further establish structure–activity relationships, we planned to synthesize both ligand enantiomers efficiently. We planned to utilize a photocycloaddition of furan and an aldehyde as the key step. Similar photocycloadditions were recently reported for the synthesis of bis-THF ligands.^19,20 Herein, we describe an efficient photochemical route to the hexahydro-4H-furopyranol ligand in racemic form followed by enzymatic resolution to provide optically active ligands with high enantiomeric purity. The route is amenable to gram quantities of enantiomeric ligands. We have converted the optically active ligand (−)-6 to potent HIV-1 protease inhibitor 3.

Our strategy for the synthesis of optically pure (3aS,4S,7aR)-hexahydro-4H-furo[2,3-b]pyran-4-ol (−)-6 is outlined in Scheme 1. Enantiomerically pure ligand alcohol (−)-6 would be obtained from an enzymatic resolution of the racemic hexahydro-4H-furopyranol 6. The synthesis of a racemic 6,5-fused ring heterocycle is envisioned from racemic oxetane 7. An appropriate acid-catalyzed reaction is expected to promote deprotection of the THP-ether followed by transacetalization to provide racemic bicyclic alcohol 6. The racemic saturated oxetane 7 would be obtained by a catalytic hydrogenation of oxetane derivative 8. Oxetane 8 can be prepared by the Paternò–Büchi photocycloaddition of inexpensive furan 9 and (tetrahydropyranyloxy)propionaldehyde 10.

Scheme 1.

Photochemical Route to Optically Active Hexahydro-4H-furopyranol

Our key strategy for the synthesis of hexahydro-4H-furopyranol involved the Paternò–Büchi reaction with furan.^21,22 Photoadditions of aldehydes and furan leading to substituted oxetanes have been investigated by Sakurai and coworkers.^23,24 Schreiber and co-workers demonstrated the utility of such photocyclizations of aldehydes and furan during the synthesis of (+)-avenaciolide.^25,26 The exo-oxetane was formed exclusively, and three contiguous chiral centers were formed in the reaction. The Paternò–Büchi reaction was recently utilized for the synthesis of a related bis-THF ligand. Initially, we considered CBz and TBS protecting groups for this synthetic route due to their prior use in photochemical routes for the synthesis of the bis-THF ligand.^19,20 However, we chose to use a THP protecting group due to the stability of the THP-ether as well as the low cost of the requisite precursor, 3,4-dihydro-2H-pyran. Thus, (tetrahydropyranyloxy)propionaldehyde 10 was prepared on multigram scale by monoprotection of propane-1,3-diol with 3,4-dihydro-2H-pyran in the presence of a catalytic amount of ptoluenesulfonic acid (p-TsOH·H₂O) in CH₂Cl₂ followed by Swern oxidation of the resulting alcohol.²⁷ The Paternò–Büchi reaction was carried out as shown in Scheme 2. A solution of furan 9 and aldehyde 10 was stirred vigorously and irradiated with UV light (300 nm) for 48 h in a Rayonet photochemical reactor to afford the racemic photoadduct 8 in 91% yield. The ¹H NMR (400 MHz) analysis revealed 99% conversion after 48 h. It is important to note that the reaction mixture must be degassed by sparging with argon prior to irradiation. Additionally, high yields were obtained only after purification of aldehyde 10 by chromatography. Photocyclization provided exclusively the exo-oxetane 8.^21,22 The product was isolated and analyzed as a 1:1 mixture of inconsequential diastereomers due to the presence of the stereocenter in the THP protecting group. The photoadduct was dissolved in ether and hydrogenated over a catalytic amount of Rh on Al₂O₃ under a hydrogen-filled balloon at 23 °C for 6 h. The resulting saturated oxetane 7 was not purified due to instability on silica gel and Al₂O₃. The crude saturated oxetane was then dissolved in MeOH and heated to 55 °C in the presence of a catalytic amount of p-TsOH to afford racemic hexahydro-4H-furopyranol 6 in 63% yield over 2 steps after chromatography on Al₂O₃. The racemic alcohol was then subjected to enzymatic resolution with immobilized Amano Lipase PS-30 on Celite¹⁴ in the presence of 10 equiv of vinyl acetate in THF at 23 °C. The reaction was monitored by TLC and by ¹H NMR analysis of a small aliquot until a 50% conversion of acylation was observed (120 h). The enzymatic resolution afforded (3aS,4S,7aR)-hexahydro-4H-furo[2,3-b]pyran-4-ol ligand (−)-6 $\left[{\left(\alpha \right)}_{\text{D}}^{23}-31.1\left(c0.52,{\text{CHCl}}_3\right)\right]$ in 49% yield. Acylated ligand (+)-11 was isolated in 50% yield. The optical purity of alcohol (−)-6 was determined after its conversion to p-nitrocarbonate 12.¹⁴ Thus, exposure of (−)-6 to p-nitrophenylchloroformate in the presence of pyridine in CH₂Cl₂ at 0 to 23 °C for 2 h provided nitrocarbonate 12 in near-quantitative yield.

Scheme 2. Synthesis of Optically Active Hexahydro-4H-furo [2,3-b]pyran-4-ol^a.

^aReagents and conditions: (a) furan, UV light (300 nm), 23 °C, (91%); (b) H₂, Rh/Al₂O₃, Et₂O, 23 °C; (c) p-TsOH·H₂O, MeOH, 55 °C, (63% for 2 steps); (d) immobilized Amano Lipase PS-30, vinyl acetate, THF, 23 °C, ((−)-6, 49%), acetate 11, (50%); (e) K₂CO₃, MeOH, 23 °C, (97%); (f) p-(NO₂)PhOCOCl, pyridine, CH₂Cl₂, 0 to 23 °C, 2 h, (99%).

Chiral HPLC analysis of 12 on a CHIRALPAK IC-3 column revealed an enantiomeric purity of 99% ee. Acetate derivative 11 was converted to the enantiomeric ligand alcohol (+)-6 by treatment with K₂CO₃ in MeOH at 23 °C for 2 h to provide (+)-6 in 97% yield. Enantiomerically pure ligand alcohol (−)-6 was converted into HIV-1 protease inhibitor 3.¹⁴ As shown in Scheme 3, treatment of carbonate 12 with the known (R)-(hydroxyethyl)-sulfonamide isostere 13¹⁴ in the presence of diisopropylethylamine (DIPEA) at 23 °C for 5 days furnished HIV-1 protease inhibitor 3 in 76% yield. Inhibitor 3 showed a K_i value of 10 pM in the HIV-1 protease inhibitory assay.²⁸

Scheme 3. Synthesis of HIV Protease Inhibitor (−)-3^a.

^aReagents and conditions: (a) DIPEA, MeCN, 23 °C, 5 days (76%).

In summary, we described an efficient synthesis of racemic hexahydro-4H-furopyranol, a high affinity P2 ligand for a variety of potent HIV-1 protease inhibitors. The racemic ligand alcohol was synthesized using a Paternò–Büchi reaction of (tetrahydropyranyloxy)propionaldehyde and furan as the key step. The photocycloaddition reaction was carried out on a multigram scale. Racemic ligand alcohol 6 was resolved by an enzymatic resolution using immobilized Amano Lipase PS-30 on Celite. This provides convenient access to optically active ligand alcohols (−)-6 and (+)-6 in high enantiomeric purity (99% ee). The overall process utilized inexpensive starting materials and has the potential for large-scale synthesis of optically pure ligands for medicinal chemistry development. Further applications of this photochemical cyclization are in progress.

EXPERIMENTAL SECTION

General Methods.

All reactions were carried out under an atmosphere of argon in oven-dried (120 °C) glassware with magnetic stirring unless otherwise noted. Solvents, reagents and chemicals were purchased from commercial suppliers. Solvents were purified as follows: CH₂Cl₂ was distilled from calcium hydride or purified using a solvent purification system; furan was shaken with aqueous 5% KOH, dried with Na₂SO₄, and distilled from KOH immediately before use; ether was purified using a solvent purification system; methanol was used without further purification; tetrahydrofuran was distilled from sodium/benzophenone; acetonitrile was purified with a solvent purification system. Purification of reaction products was carried out by flash chromatography using either silica gel 230–400 mesh (60 Å pore diameter) or alumina 80–200 mesh. Photochemical reactions were carried out in a Rayonet photochemical reactor (RPR-100) equipped with 300 nm bulbs (lamp model: RPR-3000A) and a cooling fan. Analytical thin layer chromatography was performed on glass-backed silica gel thin-layer chromatography plates (0.25 mm thickness, 60 Å, F-254 indicator) or alumina thin-layer chromatography plates (0.25 mm thickness, UV254). Optical rotations were measured by using a digital polarimeter with a sodium lamp. ¹H NMR spectra were recorded at 23 °C on a 400 MHz spectrometer and are reported in ppm relative to solvent signals (CDCl₃ at δ = 7.26 ppm) as an internal standard. Data are reported as (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dddd = doublet of doublet of doublets of doublets, td = triplet of doublets, qd = quartet of doublets, dt = doublet of triplets, dq = doublet of quartets, brs = broad singlet; coupling constant(s) in Hz; integration). Proton-decoupled ¹³C NMR spectra were recorded on a 100 MHz spectrometer and are reported in ppm by using the solvent as the internal standard (CDCl₃ at δ = 77.16 ppm). Low resolution mass spectra were obtained using a Quadrupole LCMS instrument under ESI+. High resolution mass spectra were obtained by the Mass Spectrometry Center at Purdue University. These experiments were performed under ESI+ and APCI + conditions using an Orbitrap XL instrument.

exo-6-(2-((Tetrahydro-2H-pyran-2-yl)oxy)ethyl)-2,7-dioxabicyclo[3.2.0]hept-3-ene (±)-(8).

To a flame-dried quartz flask were added aldehyde 10 (3.36 g, 21.24 mmol) and furan 9 (53 mL, 728.8 mmol) at 23 °C. The mixture was degassed by sparging with argon for 15 min, and then the flask was quickly sealed. The flask was placed in the photoreactor. The reaction was run in a Rayonet photoreactor equipped with 300 nm bulbs and a cooling fan. The distance from lamps to irradiation vessel was approximately 3.5 in., and no additional filter was used. The reaction mixture was irradiated and stirred vigorously for 48 h at 23 °C. The reaction mixture was concentrated under reduced pressure to afford a crude oil. Analysis by ¹H NMR shows 99% conversion of the aldehyde substrate. The crude oil is then purified by flash chromatography on alumina using 20% EtOAc/hexanes as the eluent to yield the purified photoadduct (±)-8(4.36 g, 91%) as a white amorphous solid. R_f = 0.50 (25% EtOAc/hexanes, alumina). ¹H NMR (400 MHz, CDCl₃) δ 6.61 (d, J = 2.7 Hz, 2H), 6.34–6.26 (m, 2H), 5.32 (q, J = 2.8 Hz, 2H), 4.75–4.65 (m, 2H), 4.59 (t, J = 3.2 Hz, 1H), 4.54 (t, J = 3.4 Hz, 1H), 3.94–3.75 (m, 4H), 3.60–3.42 (m, 6H), 2.18–2.05 (m, 4H), 1.86–1.64 (m, 4H),1.62–1.44 (m, 8H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 148.2, 108.3, 104.5, 99.4, 98.7, 90.3, 90.1, 63.3, 63.1, 62.6, 62.1, 49.4, 49.2,37.2, 37.1, 30.8, 30.7, 25.5, 19.8, 19.5. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₂H₁₈O₄Na 249.1097, found 249.1100.

6-(2-((Tetrahydro-2H-pyran-2-yl)oxy)ethyl)-2,7-dioxabicyclo-[3.2.0]heptane (±)-7.

To a flame-dried flask was added photoadduct (±)-8 (4.36 g, 19.27 mmol) in ether (96 mL) followed by 5% Rh/Al₂O₃ (872 mg, 20% w/w). The reaction flask was flushed with argon and evacuated under vacuum three times. The flask was then flushed with H₂ and evacuated under vacuum three times before leaving the mixture to stir under a hydrogen-filled balloon at 23 °C for 6 h. The reaction mixture was then filtered through a small Celite plug and concentrated under reduced pressure to yield racemic oxetane 7 (4.3 g) as a colorless oil. The crude oxetane was used for the next reaction without further purification due to its instability on silica gel or alumina. R_f = 0.70 (50% EtOAc/hexanes, alumina). ¹H NMR (400 MHz, CDCl₃) δ 5.88 (d, J = 3.8 Hz, 2H), 4.57 (t, J = 3.4 Hz, 1H), 4.54 (t, J = 3.6 Hz, 1H), 4.32–4.18 (m, 6H), 3.92–3.76 (m, 4H), 3.55–3.41 (m, 4H), 3.20–3.10 (m, 2H), 2.14–1.43 (m, 20H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 106.3, 99.4, 98.9, 79.8, 79.5,67.7, 67.7, 63.5, 63.41, 62.6, 62.3, 46.5, 46.4, 37.2, 37.1, 30.78, 30.77,28.8, 28.8, 25.6, 19.8, 19.6; HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₂H₂₀O₄Na 251.1254, found 251.1253.

Hexahydro-4H-furo[2,3-b]pyran-4-ol [(±)-6].

To a stirred solution of crude oxetane 7 (4.34 g, 19.01 mmol) in MeOH (152 mL) at 23 °C p-TsOH·H₂O (362 mg, 1.90 mmol) was added. The flask was then equipped with a reflux condenser, and the reaction mixture was stirred under argon for 12 h at 55 °C (heat source, oil bath). After this period, the reaction mixture was concentrated under reduced pressure to yield a crude residue. The residue was adsorbed onto alumina and purified by flash chromatography on alumina using 66% EtOAc/hexanes as the eluent to yield the racemic alcohol 6 (1.75 g, 63% over 2 steps) as an amorphous white solid. R_f = 0.36 (100% EtOAc, alumina). ¹H NMR (400 MHz, CDCl₃) δ 4.97 (d, J = 3.4 Hz, 1H),4.24–4.14 (m, 2H), 3.98–3.86 (m, 2H), 3.33 (td, J = 12.0, 2.6 Hz, 1H), 2.50 (dddd, J = 11.6, 8.7, 5.9, 3.5 Hz, 1H), 2.12–1.97 (m, 1H),1.97–1.84 (m, 2H), 1.80–1.64 (m, 2H). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 101.5, 68.6, 67.5, 61.2, 46.5, 29.5, 22.0.

(3aS,4S,7aR)-Hexahydro-4H-furo[2,3-b]pyran-4-ol [(−)-6].

Immobilized Amano Lipase PS-30 was prepared according to a known procedure.¹⁴ To a flame-dried flask containing distilled THF (173 mL) at 23 °C racemic alcohol 6 (1.75 g, 12.14 mmol), vinyl acetate(11.2 mL, 121.4 mmol), and immobilized Amano Lipase PS-30 (875 mg, 50% w/w) were added. The mixture was stirred under argon at 23 °C for 120 h. Aliquots of the reaction were analyzed by ¹H NMR, and the reaction was stopped once 50% of the alcohol was acylated (about 120 h). The mixture was then filtered through a small plug of Celite and concentrated under reduced pressure to yield a crude oil. The crude mixture was purified by flash chromatography on silica gel using 50% EtOAc/hexanes to 100% EtOAc as the eluent to afford enantiomerically pure ligand alcohol (−)-6 (849 mg, 49%) and acylated ligand (+)-11 (1.13 g, 50%) as amorphous solids. Ligand alcohol (−)-6: ¹H and ¹³C NMR spectra are identical to those of the racemic ligand alcohol. R_f = 0.36 (100% EtOAc, alumina). ${\left(\alpha \right)}_{\text{D}}^{23}-31.3(c0.52,{\text{CHCl}}_3)$. An enantiomeric purity of 99% ee for the alcohol was determined by analysis of the corresponding carbonate 12 on chiral HPLC (CHIRALPAK IC-3 column; 15% IPA/hexanes; 1.0 mL/min; 254 nm; 23 °C; t_R minor = 89.0 min, t_R major = 81.9 min).

Acylated Ligand Alcohol (+)-11.

¹H NMR (400 MHz, CDCl₃) δ 5.23 (dt, J = 11.5, 5.9 Hz, 1H), 5.01 (d, J = 3.4 Hz, 1H), 4.27–4.16 (m, 1H), 3.97–3.87 (m, 2H), 3.47–3.31 (m, 1H), 2.59 (dddd, J =11.6, 8.8, 6.0, 3.4 Hz, 1H), 2.28–2.08 (m, 1H), 2.06 (s, 3H), 1.87–1.70 (m, 3H). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.5, 101.3,69.8, 68.6, 61.0, 43.4, 26.2, 22.7, 21.3. TLC: R_f = 0.90 (100% EtOAc, alumina). ${\left[\alpha \right]}_{\text{D}}^{23}-54.0\left(c0.32,{\text{CHCl}}_3\right)$. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₉H₁₄O₄Na 209.0784, found 209.0786.

(3aR,4R,7aS)-Hexahydro-4H-furo[2,3-b]pyran-4-ol (+)-6.

To a flask was added (3aR,4R,7aS)-hexahydro-4H-furo[2,3-b]pyran-4-yl acetate (+)-11 (1.13 g, 6.04 mmol). Methanol (121 mL) was added, followed by addition of anhydrous K₂CO₃ (1.25 g, 9.1 mmol). The solution was stirred at 23 °C under argon for 2 h, and then the reaction mixture was filtered and concentrated to yield a crude white solid that was purified by column chromatography on silica using 50% EtOAc/hexanes and then 100% EtOAc/hexanes as the eluent to afford ligand alcohol (+)-6 (844 mg, 97%) as a white solid. ¹H and ¹³C NMR spectra are identical to those of the racemic Tp-THF ligand. R_f = 0.30 (100% EtOAc). ${\left[\alpha \right]}_{\text{D}}^{23}-28.9\left(c0.36,{\text{CHCl}}_3\right)$.

(3aS,4S,7aR)-Hexahydro-4H-furo[2,3-b]pyran-4-yl (4-Nitrophenyl) Carbonate 12.

To a flame-dried flask were added optically active alcohol (−)-6 (31 mg, 0.22 mmol) and CH₂Cl₂ (1.8 mL) followed by addition of pyridine (64 μL, 0.80 mmol). The mixture was stirred under argon and cooled to 0 °C. To the mixture was quickly added 4-nitrophenyl chloroformate (130 mg, 0.65 mmol), and the resulting reaction was stirred at 0 to 23 °C for 2 h. After this period, the mixture was concentrated under reduced pressure and purified by flash chromatography (15% EtOAc/hexanes, then 33% EtOAc/hexanes) to yield carbonate 12 (70 mg, 99% yield) as an amorphous white solid. R_f = 0.50 (50% EtOAc/hexanes, silica). ${\left[\alpha \right]}_{\text{D}}^{23}-41.8\left(c0.12,{\text{CHCl}}_3\right)$. ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 9.0 Hz, 2H), 5.31–5.17 (m, 1H), 5.07 (d, J = 3.2 Hz, 1H), 4.28 (td, J = 10.6, 10.1, 2.4 Hz, 1H), 4.09–3.94 (m, 2H),3.42 (td, J = 12.6, 4.1 Hz, 1H), 2.83–2.66 (m, 1H), 2.28–2.09 (m, 1H), 2.03–1.91 (m, 3H). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 155.5, 151.9, 145.6, 125.5, 121.9, 101.32, 75.6, 68.7, 60.7, 43.3, 26.0, 22.6.

(3aS,4S,7aR)-Hexahydro-4H-furo[2,3-b]pyran-4-yl((2S,3R)-3-hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)carbamate 3.

To a flame-dried flask was added a solution of amine 13 (30 mg, 0.07 mmol) in MeCN (1 mL). The reaction mixture was placed under argon and cooled to 0 °C. To the flask was added DIPEA (42 μL, 0.24 mmol) followed by carbonate 12 (15 mg, 0.05 mmol). The resulting mixture was stirred at 0 °C for 10 min, and then it was stirred for 5 days at 23 °C. After this period, the reaction mixture was concentrated under reduced pressure to afford a crude solid that was purified by flash chromatography using 33% EtOAc/hexanes and then 50% EtOAc/hexanes as the eluent to yield inhibitor 3 (21.3 mg, 76% yield) as an amorphous white solid. R_f = 0.25 (50% EtOAc/hexanes, silica). ${\left[\alpha \right]}_{\text{D}}^{23}-13.9\left(c0.12,{\text{CHCl}}_3\right)$. ¹3 H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 8.9 Hz, 2H), 7.32–7.18 (m, 5H), 6.98 (d, J = 8.9 Hz, 2H), 4.98 (dt, J = 11.5, 5.8 Hz, 1H), 4.93 (d, J = 3.4 Hz, 1H), 4.84 (d, J = 8.7 Hz, 1H), 4.15 (td, J = 9.7, 2.5 Hz, 1H),3.95–3.88 (m, 1H), 3.87 (s, 3H), 3.86–3.71 (m, 3H), 3.32 (td, J =11.8, 2.2 Hz, 1H), 3.15 (dd, J = 15.2, 8.2 Hz, 1H), 3.09–2.93 (m, 3H), 2.90–2.74 (m, 2H), 2.49–2.35 (m, 1H), 1.91–1.44 (m, 5H),0.92 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H). ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.2, 155.7, 137.8, 129.9, 129.6, 128.6, 126.7, 114.5, 101.3, 73.0, 70.3, 68.6, 60.9, 59.0, 55.8, 55.0, 53.9, 43.6, 35.6,27.4, 26.3, 22.4, 20.3, 20.0. LRMS-ESI: m/z = 577.2 [M + H]⁺, 599.2 [M + Na]⁺. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₂₉H₄₀N₂O₈NaS 599.2403, found 599.2406; the purity of the inhibitor was determined to be 98.1% by HPLC (YMC-Pack ODS-A column; 20 min gradient, MeCN/H₂O/TFA 20:80:0.1 to MeCN/H₂O/TFA 90:10:0.1, then 20 min MeCN/H₂O/TFA 90:10:0.1; 1.5 mL/min; 254 nm; 25 °C; t_R =14.4 min).