Synthesis and in Vitro Biological Evaluation of Mannose-Containing

Bioconjugate Chem. 2006, 17, 1568−1581

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Synthesis and in Vitro Biological Evaluation of Mannose-Containing Prodrugs Derived from Clinically Used HIV-Protease Inhibitors with Improved Transepithelial Transport Dominique Roche,† Jacques Greiner,† Anne-Marie Aubertin,‡ and Pierre Vierling*,† Laboratoire de Chimie des Mole´cules Bioactives et des Aroˆmes, UMR 6001 CNRS, Universite´ de Nice Sophia-Antipolis, Parc Valrose, F-06108 Nice Ce´dex 2, France, and INSERM U74, Institut de Virologie, 3 rue Koeberle´, F-67000 Strasbourg, France. Received July 12, 2006; Revised Manuscript Received September 22, 2006

In an approach to improve the pharmacological properties, safety and pharmacokinetic profiles, and their penetration into HIV reservoirs or sanctuaries, and consequently, the therapeutic potential of the current protease inhibitors (PIs) used in clinics, we investigated the synthesis of various mannose-substituted saquinavir, nelfinavir, and indinavir prodrugs, their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells used as a model of the intestinal barrier. Mannose-derived conjugates were prepared in two steps, in good yields, by condensing an acid derivative of a protected mannose with the PIs, followed by deprotection of the sugar protecting group. With respect to hydrolysis, these PI prodrugs are chemically stable with half-life times in the 50-60 h range that are compatible with an in vivo utilization aimed at improving the absorption/penetration or accumulation of the prodrug in specific cells/tissues and liberation of the active free drug inside HIV-infected cells. These stabilities correlate closely with the low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of mannose to the PIs was performed through the peptidomimetic PI’s hydroxyl. Importantly, mannose conjugation to the PIs was further found to improve the absorptive transepithelial transport of saquinavir and indinavir but not of nelfinavir across Caco-2 cell monolayers, by contrast to glucose conjugation which had the opposite effect. The mannose-linked prodrugs of saquinavir and indinavir display therefore a most promising therapeutic potential provided that bioavailability, penetration into the HIV infected macrophages, and HIV-reservoirs of these PIs are improved.

INTRODUCTION Combination therapy against AIDS using two types of inhibitors, HIV reverse transcriptase and protease inhibitors (PIs), has been remarkably successful in reducing viral load below the threshold of detectability and increasing T4 lymphocytes, leading to a decline in morbidity and mortality (1). However, despite these improvements, viral replication in infected patients persists, indicating the existence of reservoirs or sanctuaries for the virus, such as the lymphatic system and central nervous system (2-4), wherein the antivirals, and more particularly the PIs, do not penetrate at an inhibitory level (5, 6). To reduce total body viral replication, alternatives are to develop new more efficient PIs or to improve the pharmacological properties, safety and pharmacokinetic profiles, and, consequently, the therapeutic potential of the PIs already used in clinics. Aiming at this latter goal, we adopted the very efficient prodrug approach (7-14). This approach has been widely used for improving HIV reverse transcriptase inhibitors (8, 15, 16). It also has been applied to PIs (9, 17-19), and successful results were obtained with the disclosure of the recently FDA-approved fosamprenavir, a phosphate ester prodrug of amprenavir (20-24). However, a number of shortcomings have still to be overcome for the PIs. Among others, the generation of “modified” PIs should display higher water solubility, increased bioavailability (plasma concentration, blood circulation time), and/or improved delivery into HIV sanctuaries. One should also be able with these “modified” PIs to (i) circumvent their * To whom correspondence should be addressed. † UMR 6001 CNRS, Universite´ de Nice Sophia-Antipolis, Parc Valrose. ‡ INSERM U74, Institut de Virologie.

inactivation resulting from their in vivo binding to mainly the plasma R1-acid glycoprotein, (ii) limit their rapid metabolization and inactivation by the liver cytochrome P450 enzymes (mainly the 3A4 isoform), and/or (iii) inhibit their possible transport by the multi-drug-resistant P-glycoprotein (P-gp) responsible for their limited oral bioavailability and brain penetration (25, 26). In continuation of our previous studies that already showed the benefit of the prodrug approach (27-30), one of the remaining challenge is the targeting of HIV reservoirs and particularly the lymphatic system. It is well-known that macrophages and dendritic cells are professional antigen presenting cells, unique in their ability to initiate naive T cells, and primary targets for HIV-1 infection. The regeneration of these cells is of particular interest for helping the immune system to overcome the virus. Macrophages and dendritic cells abundantly express mannose receptors (31-33). Thus, receptor-mediated PIs transfer via mannose receptor offers a versatile tool for targeting PIs toward these cells (34-37). Therefore, we tagged PIs used in clinics, saquinavir (Saq), indinavir (Ind), and nelfinavir (Nelf), with a mannose residue. Although for successful and efficient targeting one should conjugate these entities to multivalent cluster glycoside moieties to obtain high-affinity ligands for the mannose receptor, as reported by several groups (31-33), we connected nevertheless the PIs to only a single and/or two mannose residues (in the case of indinavir and nelfinavir). Indeed, recognition of low molecular weight mannosides, even of monomers, by the mannose receptor was shown to be possible, albeit weak (38). This could be nevertheless sufficient to improve their absorption/penetration or accumulation into those reservoirs of HIV. Moreover, the development of cluster glycoside structures is a highly demanding and challenging strategy, leading to high-molecular weight compounds. This

10.1021/bc060210m CCC: $33.50 © 2006 American Chemical Society Published on Web 11/01/2006

Evaluation of Mannose-Containing Prodrugs

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Figure 1. Chemical structures and code names of the mannose-containing protease inhibitor (PI ) saquinavir, indinavir, and nelfinavir) prodrugs described in this study and atom numbering used in the description of their NMR spectra.

constitutes a drawback when the use of such high-value prodrugs as oral therapeutics against AIDS is contemplated and is also a reason for selecting mannose monovalent/divalent PI conjugates. The PI and mannose moieties were linked together through a hydrolyzable ester function. As esterases are ubiquitous in cells, in vivo hydrolysis of the ester prodrugs is expected to release the active parent drug. Although the mannose receptor tolerates ligands with large substituents at the mannose C-1 anomeric position (39), a spacer between the PI and mannose units was nevertheless introduced for the modulation of the chemical and biological stability of the prodrugs and for the accessibility of mannose, thus allowing a better recognition by its cellular receptors. For synthetic purposes, and as the impact of the anomeric mannosyl carbon atom on its recognition by the cellular receptor is not well established, the mannose unit was joined to the spacer through a glycoside bond of R/β configuration. This should further preserve its recognition (39-42). This paper is dedicated to the synthesis of various mannosederived PI prodrugs (Figure 1), their chemical stability with respect to hydrolysis under physiological conditions, and their in vitro anti-HIV activity. Using an in vitro model (i.e., Caco-2 cell monolayer) that mimics the human intestinal epithelium, we report also on their transepithelial transport. All these features are some of the prerequisites for further in vitro and in vivo investigations, including binding to and uptake into mannose receptor expressing cells and reservoirs of HIV, and pharmacokinetic studies.

EXPERIMENTAL PROCEDURES Chemical Section. General. Unless otherwise indicated, the reactions were performed under anhydrous nitrogen using dry solvents and reagents. Anhydrous solvents were prepared by standard methods. Chlorotrimethylsilane, 2,2-dimethoxypropane, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP), lithium hydroxide, pyridine, triethylamine, p-toluenesulfonic acid (p-TsOH), and N,O-bis(trimethylsilyl)acetamide δ-valerolactone were purchased from Aldrich, and ammonium iodide, 2-chloropyridine, 2,6-dimethylpyridine, diphenyl sulfoxide, iodotrimethylsilane, mannose, 2-methoxypropene, trifluoroacetic acid (TFA), and trifluoromethanesulfonic anhydride were from Fluka. All these materials were used without further purification. Saquinavir, indinavir, and nelfinavir (as their methanesulfonate salt or sulfate

salt) were a gift from Hoffmann-La Roche, E. Merck, and Agouron, respectively, and were deprotonated prior to their use in the synthetic processes (CHCl3 or EtOAc extraction of the free base from a NaHCO3 or Na2CO3 10% solution of the antiprotease). Using previously described procedures, methyl 5-hydroxypentanoate was quantitatively prepared by the acid-catalyzed transesterification of δ-valerolactone with methanol (43, 44). Because of its easy relactonization, the product was used without purification immediately after preparation [Rf 0.63 (AcOEt); 1H NMR (CDCl3): δ 3.80 (s, 1 H, OH), 3.38 (s, 3 H, OCH3), 3.31 (t, J 6.1 Hz, 2 H, H-5), 2.07 (t, J 7.3, 2 H, H-2), 1.49-1.21 (m, 4 H, H-3, H-4); 13C NMR (CDCl3): δ 173.91 (C-1), 61.2 (C-5), 51.0 (OCH3), 33.2 (C-2′), 31.4 (C-4), 20.8 (C-3)]. 2,3;4,6-Di-O-isopropylidene-R-D-mannopyranoside 3r (HOManP) was prepared from D-mannose in three steps consisting of the synthesis of benzyl R-D-mannopyranoside, then protection as the benzyl 2,3;4,6-di-O-isopropylidene-R-D-mannopyranoside, then benzyl deprotection (nearly 55% yields), as described in the literature (45-47) [Rf 0.26 (7:3 hexane-AcOEt); 1H NMR (DMSO-d6): δ 6.47 (s, 1 H, OH), 5.13 (s, 1H, H-1′), 4.70 (dd, 1 H, J 5.9, J 3.6 Hz, H-3′), 4.44 (d, 1H, J 5.9 Hz, H-2′), 4.24 (∼ q, 1 H, J 6.4, 5.8 Hz, H-5′), 4.01 (dd, 1 H, J 6.4, 3.6 Hz, H-4′), 3.96 (dd, 1 H, J 8.3, J 6.4 Hz, H-6′a), 3.82 (dd, 1 H, J 8.3, J 5.8 Hz, H-6′b), 1.34, 1.32, 1.26, and 1.23 (4 s, 12 H, CH3C); 13C NMR (DMSO-d6): δ 111.3 and 107.9 (CH3C), 100.4 (C-1′), 85.5 (C-2′), 79.4 (C-3′), 79.2 (C-4′), 72.8 (C-5′), 65.9 (C-6′), 26.6, 25.7, 25.2, and 24.3 (CH3C)]. If not specified, column chromatography purifications were carried out on Silica Gel 60 (E. Merck, 70-230 mesh). The purity of all new compounds was checked by TLC, NMR, MS, and HPLC. TLC analyses were performed on precoated Silica Gel F254 plates (E. Merck) with detection by UV and by charring with 50% methanol-sulfuric acid solution, ninhydrin or KMnO4. HPLC analyses of the synthesized prodrugs (flow of 1 mL/min) were performed using a HP1100 apparatus using a Lichrospher 100 RP-18 (5 µm) column (250 × 3.2 mm) with gradient A: H2O-CH3CN (v:v) 0.1% TFA gradient as eluent (from 80:20 to 0:100) over 30 min; UV detection at 210 nm. With these conditions, retention times (Rt) of indinavir, nelfinavir, and saquinavir are 10.4, 10.8, and 15.3 min, respectively. 1H, 13C, and 19F NMR spectra were recorded with a Brucker AC 200 or AC 500 spectrometer at 200 (or 500), 50.3 (or 125.8),

1570 Bioconjugate Chem., Vol. 17, No. 6, 2006

and 188.3 MHz, respectively. Chemical shifts (δ) are given in ppm relative to the signal (i) of remaining CHCl3 (δ 7.26) for 1H, (ii) of CDCl (δ 77.16) for 13C, or of remaining water for 3 1H (δ 4.87) and CD OD for 13C (δ 49.00) when recorded in 3 CD3OD. Concerning the description of the prodrug NMR spectra, the atoms of the PI part are depicted as C-x and H-y, whereas those of the sugar part are depicted as C-x′ and H-y′ according to their standard nomenclature numbering and those of the linker part are depicted as C-x′′ and H-y′′ (see Figure 1 for numbering). COSY 1H/1H, 1H/13C NMR correlation (on Brucker AC 500 spectrometer), 13C DEPT, and/or mass spectrometry data fully confirm the signal assignments and structure of the isolated materials. The mannose-deprotected target compounds consisted of TFA salts, and the TFA anion quantification was assessed by 19F NMR using 3,3,3-trifluoroethanol as an internal standard. Electron-spray ionization mass spectra (ESI-MS) were run on a Finnigan MAT TSQ 7000 apparatus equipped with an atmospheric pressure ionization source. This method used in positive mode gives either M+, [M + H]+ and/or [M + Na]+ signals. Syntheses. Methoxycarbonylbutyl-R-D-mannopyranoside, 7r,β and 7r. D-Mannose (5.0 g, 25 mmol) and N,O-bis(trimethylsilyl)acetamide (30 mL, 125 mmol) in anhydrous CH2Cl2 were stirred at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure. The resulting mixture diluted in Et2O (100 mL) was washed with 5% aq HCl, saturated aq NaHCO3, and water. The organic layer was dried over Na2SO4 and concentrated giving a mixture of trimethylsilyl 2,3,4,6-tetraO-trimetylsilyl-D-mannopyranoside 6r,β as an oil (15 g, R:β ) 79:21) [Rf 0.82 (CH2Cl2); 1H NMR (CDCl3): δ 4.88 (m, 1 H, H-1R); 4.62 (m, 0.27 H, H-1β); 13C NMR (CDCl3): δ 95.8 (C-1R), 95.7 (C-1β)]. Freshly distilled iodotrimethylsilane (1.5 mL, 11.0 mmol) was added to a solution of 6r,β (5 g, 2.13 mmol) in dry CH2Cl2 (100 mL). After the sample was stirred at room temperature for 45 min, tetrabutylammonium iodide (6.75 g, 10.7 mmol) was added and stirred for 15 min more before adding a CH2Cl2 solution (50 mL) containing methyl 5-hydroxypentanoate (2.35 g, 16 mmol) and 2,6-lutidine (1.25 mL, 16 mmol). The reaction mixture was stirred at room temperature for 6 h, and then desilylation was performed in 30 min with MeOH (200 mL). After addition of Et3N, the resulting solution was concentrated and purified by column chromatography (9:1 CH2Cl2-MeOH) affording 7r,β as a yellow solid (2.3 g, 30% with R:β ) 60:40). Rf 0.56 (9:1 CH2Cl2-MeOH); 1H NMR (CDCl3): δ 5.15 (bs, 3 H, 3 OH), 4.78 (s, 0.6 H, H-1′R), 4.60 (s, 1 H, OH), 4.48 (s, 0.4 H, H-1′β), 4.10-3.72 (m, 5 H, H-2′, H-3′, H-4′, H-6′), 3.64 (s, 3 H, OCH3), 3.64-3.18 (m, 3 H, H-5′, H-5′′), 2.31 (t, J 7.1 Hz, 2 H, H-2′′), 1.62 (m, 4 H, H-3′′, H-4′′); 13C NMR (CDCl3): δ 174.2 (C-1′′), 100.2 (C-1′R,β), 77.4 (C-5′β), 74.0 (C-3′β), 72.5 (C-5′R), 71.6 (C-2′β), 71.3 (C-3′R), 71.0 (C-2′R), 69.4 (C-5′′β), 67.3 (C-5′′R), 66.4 (C-4′R), 66.3 (C-4′β), 61.0 (C-6′R,β), 51.7 (OCH3), 33.8 (C-2′′), 28.9 (C-4′′), 21.7 (C-3′′). ESI-MS (positive mode): (M + Na)+ ) 316.9 in agreement with the mass calculated for M ) C12H22O8 (294.13). The same experimental procedure when applied to 1.0 g of 6r, β gave, after several purifications by column chromatography (9:1 CH2Cl2-MeOH), 7r as a yellow solid (0.46 g, 30%). Rf 0.56 (9:1 CH2Cl2-MeOH); 1H NMR (CDCl3): δ 5.06 (bs, 1 H, OH), 5.03 (bs, 1 H, OH), 4.95 (bs, 1 H, OH), 4.79 (s, 1 H, H-1′), 4.49 (bs, 1 H, OH), 3.94-3.88 (m, 3 H, H-2′, H-3′, H-6′a), 3.80-3.73 (m, 2 H, H-4′, H-6′b), 3.65 (bs, 4 H, H-5′′a, OCH3), 3.47 (m, 1 H, H-5′), 3.39 (m, 1 H, H-5′′b), 2.32 (t, J 7.1 Hz, 2 H, H-2′′), 1.66-1.57 (m, 4 H, H-3′′, H-4′′); 13C NMR (CDCl3): δ 174.2 (C-1′′), 100.2 (C-1′), 72.4 (C-5′), 71.7 (C-3′), 71.1 (C-2′), 67.3 (C-5′′), 66.4 (C-4′), 61.1 (C-6′), 51.7 (OCH3), 33.8 (C-2′′), 28.9 (C-4′′), 21.8 (C-3′′); ESI-MS (positive

Roche et al.

mode): (M + Na)+ ) 316.9 in agreement with the mass calculated for M ) C12H22O8 (294.13). 2,3,4,6-Di-O-isopropylidene-1-(4-methoxycarbonylbutyl)-RD-mannopyranoside, 4r. Path A. Trifluoromethanesulfonic anhydride (24 µL, 0.16 mmol) was added to a solution of 3r (30 mg, 0.11 mmol) and diphenyl sulfoxide (65 mg, 0.32 mmol) in a mixture of 1:3 toluene-dichloromethane (4 mL) at -78 °C, and stirred for 5 min, then at -40 °C for 1 h. Chloropyridine (54 µL, 0.57 mmol) and methyl 5-hydroxypentanoate (43.6 mg, 0.33 mmol) were then added successively. The resulting solution was stirred at -40 °C for 1 h, at 0 °C for 30 min, and finally at 23 °C for 1 h before the addition of excess Et3N (153 µL, 1.09 mmol). The solution diluted with CH2Cl2 (50 mL) was successively washed with saturated aq Na2CO3 and saturated aq NaCl. The organic layer was dried over Na2SO4 and concentrated, and the residue was purified by column chromatography (gradient elution: 10:0 to 1:1 hexane-ethyl acetate) to afford 4r as a white solid (10 mg, 4%). Path B. p-Toluenesulfonic acid (p-TsOH) (20 mg, 0.1 mmol) was added to a solution of 7r (0.5 g, 1.70 mmol) and 2-methoxypropene (0.64 mL, 6.7 mmol) solubilized in CH2Cl2 (20 mL). After the sample was stirred for 6 h at room temperature, the resulting solution was made neutral with NaHCO3 (8.4 mg, 0.1 mmol) and concentrated, and purification by column chromatography (98:2 CH2Cl2-AcOEt) afforded compound 4r (400 mg, 63%). Rf 0.71 (98:2 CH2Cl2-AcOEt); 1H NMR (CDCl3): δ 4.89 (s, 1 H, H-1′), 4.08-4.04 (m, 2 H, H-2′, H-3′), 3.81-3.57 (m, 7 H, H-4′-6′, OCH3), 3.50-3.41 (m, 1 H, H-5′′a), 3.37-3.27 (m, 1 H, H-5′′b), 2.25 (t, J 7.0 Hz, 2 H, H-2′′), 1.70-1.52 (m, 4 H, H-3′′, H-4′′), 1.44, 1.41, 1.32, and 1.25 (4 s, 12 H, CH3C); 13C NMR (CDCl ): δ 173.6 (C-1′′), 109.2 and 99.5 (CH C), 3 3 97.7 (C-1′), 76.0 (C-2′), 74.8 (C-3′), 72.7 (C-5′), 67.2 (C-5′′), 62.0 (C-6′), 61.2 (C-4′), 51.4 (OCH3), 33.5 (C-2′′), 28.7 (C-4′′), 29.0, 28.1, 26.1, and 18.7 (CH3C), 21.6 (C-3′′). 2,3;4,6-Di-O-isopropylidene-1-(4-methoxycarbonylbutyl)-R,βD-mannopyranoside, 4r,β. The same experimental procedure described above in path B for the preparation of 4r, when applied to p-TsOH (86 mg, 0.5 mmol), 7r,β (2.30 g, 7.82 mmol) and 2-methoxypropene (2.94 mL, 30.7 mmol) gave, after purification by column chromatography, compound 4r,β (2.08 g, 71% with R:β ) 53:47). Rf 0.71 (98:2 CH2Cl2-AcOEt); 1H NMR (CDCl3): δ 4.96 (s, 0.6 H, H-1′R), 4.89 (s, 0.4 H, H-1′ β), 4.13-4.07 (m, 2 H, H-2′), 3.91-3.83 (m, 1 H, H-6′a), 3.793.65 (m, 7 H, H-3′, H-4′ β, H-5′R, H-6′b, H-5′′a, OCH3), 3.583.49 (m, 1 H, H-4′R, H-5′ β), 3.46-3.38 (m, 1 H, H-5′′b), 2.32 (t, J 7.0 Hz, 2 H, H-2′′), 1.92-1.80 (m, 2 H, H-3′′), 1.73-1.59 (m, 2H, H-4′′), 1.52, 1.49, 1.40 and 1.32 (4 s, 12 H, CH3C); 13C NMR (CDCl ): δ 174.0 (C-1′′), 109.5 and 99.8 (CH C), 3 3 98.9 (C-1′ β), 97.9 (C-1′R), 76.2 (C-3′ β), 76.1 (C-2′R), 75.0 (C-3′R, C-2′ β), 72.9, 72.8 (C-5′R, C-4′ β), 67.4 (C-5′′), 62.2 (C-6′R,β), 61.4, 61.3 (C-4′R, C-5′ β), 51.6 (OCH3), 33.8 (C-2′′), 28.9 (C-4′′), 29.1, 28.3, 26.2, and 18.8 (CH3C), 21.8 (C-3′′). 2,3;4,6-Di-O-isopropylidene-1-(4-carboxybutyl)-R-D-mannopyranoside, 5r. A 1 N LiOH aq solution (2 mL) was added to 4r (200 mg, 0.53 mmol) in THF (2 mL) cooled at 0 °C. After 12 h at room temperature, the resulting solution was made neutral with 1 N HCl. After evaporation of THF, the product was extracted with CH2Cl2, dried over Na2SO4, filtered, and concentrated. 5r was obtained almost quantitatively as a colorless oil (187 mg, 98%); Rf 0.38 (96:4 CH2Cl2-AcOEt); 1H NMR (CD OD): δ 4.94 (s, 1 H, H-1′), 4.11 (d, J 5.7 Hz, 1 3 H, H-2′), 4.01 (dd, J 7.6, 5.7 Hz, 1 H, H-3′), 3.82-3.64 (m, 4 H, H-4′-6′), 3.50-3.38 (m, 2 H, H-5′′), 2.28 (t, J 6.9 Hz, 2 H, H-2′′), 1.58-1.55 (m, 4 H, H-3′′, H-4′′), 1.43 (s, 6 H, CH3C), 1.26, and 1.24 (2 s, 6 H, CH3C); 13C NMR (CD3OD): δ 177.6



(C-1′′), 110.4 and 100.8 (CH3C), 99.1 (C-1′), 77.5 (C-2′), 76.2 (C-3′), 74.1 (C-5′), 68.4 (C-5′′), 63.0 (C-6′), 62.6 (C-4′), 34.8 (C-2′′), 29.9 (C-4′′), 29.4, 28.4, 26.5, and 19.2 (CH3C), 22.9 (C-3′′). 2,3;4,6-Di-O-isopropylidene-1-(4-carboxybutyl)-R,β-D-mannopyranoside, 5r,β. The same experimental procedure as described above for the preparation of 5r, when applied to 4r,β (2.0 g, 5.35 mmol) gave 5r,β (1.77 g, 92%) as a white solid; Rf 0.38 (96:4 CH2Cl2-AcOEt); 1H NMR (CD3OD): δ 4.09 (dd, J 5.5, 2.3 Hz, 1 H, H-2′), 4.00 (m, 1 H, H-6′a), 3.81-3.36 (m, 7 H, H-4′-5′, H-6′b, H-5′′), 2.13 (m, 2 H, H-2′′), 1.591.50 (m, 4 H, H-3′′, H-4′′), 1.32, 1.28, 1.26, 1.20 (4s, 12 H, CH3C); 13C NMR (CD3OD): δ 180.4 (C-1′′), 110.5 and 100.9 (CH3C), 100.1 (C-1′ β), 99.1 (C-1′R), 77.6 (C-3′ β), 77.4 (C-2′R), 76.3 (C-3′R, C-2′ β), 74.2, 74.1 (C-5′R, C-4′ β), 68.8 (C-5′′), 63.1 (C-6′R,β), 62.6, 62,5 (C-4′R, C-5′β), 38.9 (C-2′′), 30.5 (C-4′′), 29.4, 28.4, 26.5, 24.3, 19.1 and 18.4 (CH3C), 24.4 (C-3′′). Nelf(1)-C(O)C4Man and Nelf-[C(O)C4Man]2. General Esterification Method. EDC (101 mg, 0.53 mmol) was added to a solution of 5r,β (190 mg, 0.53 mmol), DMAP (65 mg, 0.53 mmol), and nelfinavir (300 mg, 0.53 mmol) in CH2Cl2 (10 mL) at 0 °C. Then, the mixture was stirred for 15 min at 0 °C then for 19 h at room temperature. The reaction mixture was washed, and the organic layer was dried over Na2SO4 filtered, and evaporated under reduced pressure. Then, the residue was chromatographed three times on silica gel (100:0 to 98:2 CH2Cl2-EtOH) to give Nelf(1)-C(O)C4ManP (130 mg, 27% with R:β ) 55:45) and Nelf-[C(O)C4ManP]2 (80 mg, 12% with R:β ) 55:45) as white solids. Nelf(1)-C(O)C4ManP. Rf 0.79 (92:8 CH2Cl2-EtOH); 1H NMR (CD3OD): δ 7.55 (m, 2 H, H-13, H-17), 7.40 (d, J 7.5 Hz, 1 H, H-4), 7.36-7.33 (m, 3 H, H-3, H-14, H-16), 7.23 (m, 1 H, H-15), 7.15 (m, 1 H, H-2), 5.09 (s, 0.6 H, H-1′R), 4.90 (d, J 2.2 Hz, 0.4 H, H-1′β), 4.51 (m, 1H, H-10), 4.35 (dd, J 2.5, J 6.1 Hz, 0.4 H, H-2′β), 4.25 (d, J 5.7 Hz, 0.6 H, H-2′R), 4.21 (m, 0.4 H, H-3′β), 4.09 (m, 2 H, H-3′R, H-5′β, H-18), 3.973.79 (m, 3.6 H, H-5′R, H-6′Rβ, H-5′′aRβ), 3.72-3.46 (m, 3.6 H, H-4′R, H-5′′bRβ, H-11), 3.35 (m, 0.4 H, H-4′β), 3.08 (d, J 11.5 Hz, 1 H, H-20a), 2.73 (t, J 7.1 Hz, 2 H, H-2′′), 2.65 (m, 2 H, H-19a, H-28), 2.31 (s, 3 H, H-7), 2.22 (m, 2 H, H-19b, H-20b), 2.07-1.55 (m, 16 H, H-3′′, H-4′′, H-21-27), 1.431,35 (m, 12 H, 4 CH3C), 1.21 (m, 9 H, H-32); 13C NMR (CD3OD): δ 176.0 (C-8), 173.0 (C-1′′), 172.3 (C-29), 150.9 (C-1), 140.0 (C-5), 137.5 (C-12), 130.6 (C-13, C-17), 129.9 (C-14, C-16), 129.4 (C-6), 127.5 (C-3), 126.9 (C-15), 126.0 (C-4), 124.5 (C-2), 111.7, 110.3, 100, 7, 100.6, (CH3C), 99.8 (C-1′β), 99.0 (C-1′R), 77.5 (C-3′β), 77.4 (C-2′R), 76.1 (C-3′R), 75.6 (C-2′β), 73.9 (C-5′R), 73.4 (C-5′β), 70.9 (C-28), 70.3 (C-18), 70.1 (C-5′′β), 68.3 (C-5′′R), 66.8 (C-4′β), 63.6 and 63.0 (C-6′Rβ), 62.6 (C-4′R), 60.3 (C-20), 59.5 (C-19), 54.3 (C-10), 51.7 (C-31), 37.4 (C-21), 35.4 (C-11), 35.0 (C-26), 34.5 (C-2′′), 32.0 (C-27), 30.0 and 29.8 (C-4′′Rβ), 28.9 (C-32), 31.6, 27.4, 27.0, and 21.6 (C-22-25), 29.5, 28.9, 28.5, 27.9, 26.6, 26.4, 19.2 and 19.3 (CH3C), 22.8 (C-3′′Rβ), 13.6 (C-7). Nelf-[C(O)C4ManP]2. Rf 0.89 (92:8 CH2Cl2-EtOH); 1H NMR (CD3OD): δ 7.58 (m, 2 H, H-13, H-17), 7.42 (m, 1H, H-4), 7.40-7.26 (m, 3 H, H-3, H-14, H-16), 7.16 (m, 1 H, H-2), 5.47 (m, 1 H, H-18), 5.10 (s, 0.6 H, H-1′R), 5.06 (s, 0.6 H, H-1′R), 4.93 (d, J 1.1 Hz, 0.4 H, H-1′β), 4.90 (bs, 0.4 H, H-1′β), 4.68 (m, 1H, H-10), 4.38 (m, 0.8 H, H-2′β), 4.28-4.06 (m, 6 H, H-2′R, H-3′Rβ, H-5′β), 3.99-3.73 (m, 7.2 H, H-5′R, H-6′Rβ, H-5′′aRβ), 3.68-3.46 (m, 5.2 H, H-4′R, H-5′′bRβ, H-11), 3.35 (m, 0.8 H, H-4′β), 3.08 (d, J 11.1 Hz, 1 H, H-20a), 2.76 (m, 3 H, H-2′′ext, H-19b), 2.66 (d, J 10.8 Hz, 1 H, H-28), 2.44 (m, 2 H, H-2′′int), 2.30 (m, 5 H, H-7, H-19b, H-20b), 2.09-1.23 (m, 44 H, H-3′′, H-4′′, H-21s27, 8 CH3C), 1.19 (s, 9 H, H-32);

13C

NMR (CD3OD): δ 175.8 (C-8), 174.0 (C-1′′int), 172.8 (C-29), 171.7 (C-1′′ext), 150.9 (C-1), 140.0 (C-5), 136.8 (C-12), 131.1, 130.6 (C-13, C-17), 129.9 and 129.8 (C-14, C-16), 129.1 (C-6), 127.5 (C-3), 127.2 (C-15), 125.8 (C-4), 124.5 (C-2), 111.6, 110.4, 100.7, and 100.6 (CH3C), 99.7 (C-1′β), 98.94 and 98.89 (C-1′R), 77.5 (C-3′β), 77.3 (C-2′R), 76.1 (C-3′R), 75.6 (C-2′β), 73.9 (C-5′R), 73.3 (C-5′β), 72.23 and 72.14 (C-18Rβ), 70.7 (C-28), 70.1 (C-5′′β), 68.3 (C-5′′R), 66.8 (C-4′β), 63.6, 62.9 (C-6′Rβ), 62.5 (C-4′R), 60.3 (C-20), 56.5 (C-19), 52.0 (C-10), 51.8 (C-31), 37.3 (C-21), 35.8 (C-11), 35.0 (C-26), 34.7 and 34.4 (C-2′′int,ext), 32.0 (C-27), 29.9 and 29.7 (C-4′′Rβ), 28.9 (C-32), 31.6, 27.4, 27.0, and 21.7 (C-22-25), 29.5, 28.9, 28.5, 28.0, 26.6, 26.4, 19.3 and 19.2 (CH3C), 22.79 and 22.76 (C-3′′Rβ), 13.6 (C-7). Nelf(1)-C(O)C4Man. General Deprotection Method. Nelf(1)-C(O)C4ManP (100 mg, 0.11 mmol) in a 1:9 TFA-CH2Cl2 v/v mixture (10 mL) was stirred for 2 h at room temperature. The residue obtained after evaporation of the solvents was purified by chromatography (10:0 to 4:1 CH2Cl2-MeOH) to give Nelf(1)-C(O)C4Man (2TFA) (105 mg, 90% with R:β ) 58:42) as a white solid; Rf 0.46 (4:1 CH2Cl2-MeOH); Rt 17.2 min (gradient A); 1H NMR (CD3OD): δ 7.45-7.41 (m, 2 H, H-13, H-17), 7.31-7.03 (m, 6 H, H-2-4, H-14-16), 4.73 (d, J 3.4 Hz, 0.6 H, H-1′R), 4.49 (s, 0.4 H, H-1′β), 4.25 (m, 1H, H-10), 4.13-3.40 (m, 12 H, H-2′-6′Rβ, H-5′′Rβ, H-11, H-18, H-20a), 3.22 (m, 1 H, H-20a), 2.82-2.60 (m, 4 H, H-2′′, H-19a, H-28), 2.19 (s, 3 H, H-7), 2.05-1.24 (m, 16 H, H-3′′, H-4′′, H-19b, H-20b, H-21-27), 1.16 (s, 9 H, H-32); 13C NMR (CD3OD): δ 173.4 (C-8), 173.2 (C-1′′), 172.3 (C-29), 151.1 (C-1), 140.0 (C-5), 137.5 (C-12), 130.9 (C-13, C-17), 130.1 (C-14, C-16), 129.5 (C-6), 127.6 (C-3), 127.3 (C-15), 126.1 (C-4), 124.8 (C-2), 101.6 (C-1′β), 101.5 (C-1′R), 78.1 (C-5′β), 75.1 (C-3′β), 74.5 (C-5′R), 72.6 (C-2′β), 72.5 (C-3′R), 72.2 (C-2′R), 70.2 (C-18, C-28), 70.0 (C-5′′β), 68.5 (C-4′R), 68.4 (C-4′β), 68.1 (C-5′′R), 62.7 (C-6′R), 62.6 (C-6′β), 59.8 (C-20), 58.3 (C-19), 54.3 (C-10), 52.2 (C-31), 36.8 (C-21), 35.5 (C-11), 34.5 (C-2′′), 34.3 (C-26), 31.7 (C-27), 29.9 (C-4′′Rβ), 28.8 (C-32), 31.1, 27.2, 26.5, and 21.6 (C-22-25), 22.9 and 22.7 (C-3′′Rβ), 13.6 (C-7). ESI-MS: m/z 830.4 [M + H]+, in agreement with the calculated mass for [M] ) C43H63N3O11S (829.42). Nelf-[C(O)C4Man]2. Deprotection of Nelf-[C(O)C4ManP]2 (14 mg, 0.008 mmol) afforded after purification by chromatography (10:0 to 4:1 EtOAc-MeOH) Nelf-[C(O)C4Man]2 (2TFA) (13 mg, 92% with R:β ) 60:40) as a white solid; Rf 0.25 (4:1 CH2Cl2-MeOH); Rt 16.1 min (gradient A); 1H NMR (CD3OD): δ 7.56 (m, 2 H, H-13, H-17), 7.41-7.23 (m, 5 H, H-3, H-4, H-14-16), 7.14 (m, 1 H, H-2), 5.43 (m, 1 H, H-18), 4.83 (s, 0.6 H, H-1′R), 4.78 (s, 0.6 H, H-1′R), 4.58 (s, 0.4 H, H-1′β), 4.56 (s, 0.4 H, H-1′β), 4.64 (m, 1H, H-10), 4.05-3.45 (m, 19 H, H-11, H-2′Rβ, H-3′Rβ, H-4′R, H-5′Rβ, H-6′Rβ, H-5′′Rβ), 3.26 (m, 0.8 H, H-4′β), 3.05 (d, J 11.1 Hz, 1 H, H-20a), 2.71 (m, 4 H, H-19b, H-28, H-2′′ext), 2.42-2.28 (m, 4 H, H-19b, H-20b, H-2′′int), 2.24 (m, 3 H, H-7,), 2.07-1.33 (m, 18 H, H-21s27, H-3′′, H-4′′), 1.16 (m, 9 H, H-32); 13C NMR (CD3OD): δ 176.1 (C-8), 174.4 (C-1′′int), 173.2 (C-29), 172.2 (C-1′′ext), 151.2 (C-1), 140.2, 137.0 (C-5, C-12), 131.2 (C-13, C-17), 130.0 (C-14, C-16), 129.1 (C-6), 127.7 (C-3), 127.4 (C-15), 125.9 (C-4), 124.7 (C-2), 101.6 (C-1′β), 100.7 (C-1′R), 78.1 (C-5′β), 75.1 (C-3′β), 74.8 (C-5′R), 74.6 (C-2′β), 74.5 (C-3′R), 72.6 (C-2′R), 72.2 (C-18), 70.8 (C-28), 70.1, 70.0 (C-5′′β), 68.5 (C-4′R), 68.4 (C-4′β), 68.1 (C-5′′R), 62.6 (C-6′R), 62.5 (C-6′β), 60.3 (C-20), 56.5 (C-19), 52.2 (C-10), 52.0 (C-31), 37.6 (C-21), 35.5 (C-11), 35.2 (C-26), 34.8, 34.5 (C-2′′), 32.1 (C-27), 30.0, 29.9 (C-4′′Rβ), 28.8 (C-32), 31.8, 27.5, 27.2, 21.7 (C-22-25), 22.9, 22.7 (C-3′′Rβ), 13.5 (C-7).


ESI-MS: m/z 1092.5 [M + H]+, 1114.5 [M + Na]+, in agreement with the calculated mass for [M] ) C54H81N3O18S (1091.52). Saq-C(O)C4ManP. The general esterification method applied to 5r,β (160 mg, 0.45 mmol), DMAP (55 mg, 0.45 mmol), saquinavir (300 mg, 0.45 mmol), and EDC (86 mg, 0.45 mmol) afforded, after workup and purification by chromatography on silica gel (100:0 to 98:2 CH2Cl2-MeOH), Saq-C(O)C4ManP (320 mg, 71% with R:β ) 64:36) as a white solid; Rf 0.72 (98:2 CH2Cl2-MeOH) 1H NMR (CD3OD): δ 8.46 (d, J 8.5 Hz, 1 H, H-6), 8.17 (d, J 8.5 Hz, 1 H, H-7), 8.14 (d, J 8.6 Hz, 1 H, H-1), 8.00 (d, J 8.5 Hz, 1 H, H-4), 7.83 (m, 1 H, H-2), 7.69 (m, 1 H, H-3), 7.25 (d, J 7.2 Hz, 2 H, H-21, H-25), 7.04 (m, 2 H, H-22, H-24), 6.90 (m, 1 H, H-23), 5.33 (m, 1 H, H-26), 5.00 (s, 0.6 H, H-1′R), 4.90 (m, 1 H, H-12), 4.84 (d, J 2.4 Hz, 0.4 H, H-1′β), 4.46 (m, 1 H, H-18), 4.30 (dd, J 2.5, J 6.1 Hz, 0.4 H, H-2′β), 4.17 (m, 0.6 H, H-2′R), 4.14 (m, 0.4 H, H-3′β), 4.05 (m, 0.6 H, H-3′R), 3.99 (m, 0.4 H, H-5′β), 3.89-3.68 (m, 3.6 H, H-5′R, H-6′Rβ, H-5′′aRβ), 3.53 (m, 1 H, H-4′R, H-5′′bβ), 3.45 (m, 0.6 H, H-5′′bR), 3.26 (m, 0.4 H, H-4′β), 3.06 (dd, J 14.0, J 3.5 Hz, 1 H, 19a), 3.00 (d, J 11.5 Hz, 1 H, H-29a), 2.80-2.66 (m, 5 H, H-13, H-19b, H-27a, H-37), 2.41 (m, 3 H, H-2′′, H-27b), 2.24 (m, 1 H, H-29b), 2.04-1.32 (m, 28 H, H-3′′, H-4′′, H-3036, 4 CH3C), 1.36 (s, 9 H, H-41); 13C NMR (CD3OD): δ 175.0, 174.7 (C-14, C-16, C-1′′), 172.3 (C-38), 166.1 (C-10), 150.3 (C-8), 147.8 (C-9), 139.3 (C-20), 138.9 (C-6), 131.5 (C-2), 130.8 (C-5), 130.7 (C-1), 130.3 (C-21, C-25), 129.4 (C-3), 129.2 (C-22, C-24), 129.0 (C-4), 127.2 (C-23), 119.7 (C-7), 111.7, 110.4, 100.8, 100.7 (CH3C), 100.0 (C-1′β), 99.1 (C-1′R), 77.7 (C-3′β), 77.5 (C-2′R), 76.3 (C-3′R), 75.8 (C-2′β), 74.7 (C-26), 74.1 (C-5′R), 73.6 (C-5′β), 70.9 (C-37), 70.3 (C-5′′β), 68.4 (C-5′′R), 67.0 (C-4′β), 63.7 (C-6′β), 63.0 (C-6′R), 62.6 (C-4′R), 59.9 (C-29), 56.8 (C-27), 53.0 (C-12), 52.0 (C-40), 51.5 (C-18), 38.0 (C-13), 37.0 (C-30), 35.5 (C-19), 34.9 (C-2′′), 34.8 (C-35), 31.8 (C-36), 30.1 and 30.0 (C-4′′Rβ) 29.0 (C-41), 31.4, 27.1 (2C), and 22.0 (C-31-34), 29.5, 28.4, 28.0, 26.5, 26.4, 19.3, and 19.2 (CH3C), 22.63 and 22.59 (C-3′′Rβ). Saq-C(O)C4Man. Deprotection of Saq-C(O)C4ManP (150 mg, 0.15 mmol) led, after chromatography (10:0 to 4:1 EtOAcMeOH), to Saq-C(O)C4Man (1TFA) (149 mg, 95% with R:β ) 64:36) as a white solid; Rf 0.23 (9:1 EtOAc-MeOH); Rt 16.7 min (gradient A); 1H NMR (CD3OD): δ 8.52 (d, J 8.5 Hz, 1 H, H-6), 8.20 (d, J 8.4 Hz, 1 H, H-7), 8.19 (d, J 7.8 Hz, 1 H, H-1), 8.05 (d, J 8.2 Hz, 1 H, H-4), 7.88 (m, 1 H, H-2), 7.74 (m, 1 H, H-3), 7.26 (d, J 7.5 Hz, 2 H, H-21, H-25), 7.06 (t, 2 H, J 7.5 Hz, H-22, H-24), 6.91 (m, 1 H, H-23), 5.33 (m, 1 H, H-26), 4.92 (t, J 13.0 Hz, 1 H, H-12), 4.78 (d, J 1.5 Hz, 0.6 H, H-1′R), 4.55 (s, 0.4 H, H-1′β), 4.48 (dt, J 10.7, J 1.4 Hz, 1 H, H-18), 3.92-3.38 (m, 7.6 H, H-2′Rβ, H-3′Rβ, H-4′R, H-5′Rβ, H-6′Rβ, H-5′′Rβ), 3.26 (m, 0.4 H, H-4′β), 3.05 (m, 2 H, 19a, H-29a), 2.74 (m, 5 H, H-13, H-19b, H-27a, H-37), 2.41 (m, 3 H, H-27b, H-2′′), 2.26 (m, 1 H, H-29b), 2.04-1.32 (m, 16 H, H-30-36, H-3′′, H-4′′), 1.37 (s, 9 H, H-41); 13C NMR (CD3OD): δ 175.0, 174.9 (C-14, C-16, C-1′′), 172.4 (C-38), 166.2 (C-10), 150.3 (C-8), 148.0 (C-9), 139.3 (C-20), 139.0 (C-6), 131.6 (C-2), 130.9 (C-5), 130.8 (C-1), 130.3 (C-21, C-25), 129.5 (C-3), 129.2 (C-22, C-24), 129.0 (C-4), 127.2 (C-23), 119.7 (C-7), 101.7 (C-1′β), 101.5 (C-1′R), 77.7 (C-5′β), 75.2 (C-3′β), 74.6 (C-5′R, C-26), 72.6 (C-2′β), 72.5 (C-2′R), 72.2 (C-2′R), 70.8 (C-37), 70.2 (C-5′′β), 68.6, 68.5 (C-4′Rβ), 68.1 (C-5′′R), 62.8 (C-6′R), 62.7 (C-6′β), 59.9 (C-29), 56.8 (C-27), 53.1 (C-12), 52.0 (C-40), 51.5 (C-18), 38.0 (C-13), 37.1 (C-30), 35.5 (C-19), 34.9 (C-2′′), 34.8 (C-35), 31.8 (C-36), 30.0 (C-4′′), 29.0 (C-41) 31.8, 31.0, 31.4, 27.1, and 22.0 (C-31-34), 22.7 and 22.6 (C-3′′). ESI-MS: m/z 933.5 [M + H]+, 955.5 [M + Na]+, in agreement with the calculated mass for [M] ) C49H68N6O12 (932.49).

Roche et al.

Ind(8)-C(O)C4ManP and [Ind-[C(O)C4ManP]2]. The general esterification method applied to 5r,β (176 mg, 0.49 mmol), DMAP (71 mg, 0.58 mmol), indinavir (300 mg, 0.49 mmol), and EDC (112 mg, 0.58 mmol) afforded, after workup and three successive chromatographies on silica gel (10:0 to 98:2 CH2Cl2-EtOH) Ind(8)-C(O)C4ManP (102 mg, 22% with R:β ) 52:48) and Ind-[C(O)C4ManP]2 (95 mg, 15% with R:β ) 62:38) as white solids. Ind(8)-C(O)C4ManP. Rf 0.58 (92:8 CH2Cl2-EtOH); 1H NMR (CD3OD): δ 8.51(s, 1 H, H-29), 8.47 (d, J 4.8 Hz, 1 H, H-28), 7.84 (d, J 7.6 Hz, 1 H, H-26), 7.43 (dd, J 7.6, J 4.8 Hz, 1 H, H-27), 7.29-7.19 (m, 9 H, H-2-5, H-32-36), 5.60 (d, J 5.1 Hz, 1 H, H-9), 5.40 (m, 1 H, H-8), 4.96 (s, 0.5 H, H-1′R), 4.77 (s, 0.5 H, H-1′β), 4.26 (dd, J 6.0, J 2.2 Hz, 0.5 H, H-2′β), 4.164.13 (m, 1 H, H-2′R, H-3′β), 4.05-3.98 (m, 1 H, H-3′R, H-5′β), 3.89-3.72 (m, 3 H, H-2′R, H-6′Rβ, H-5′′aβ, H-14), 3.64 (m, 0.5 H, H-5′′aR), 3.59 (s, 2 H, H-24), 3.52-3.47 (m, 1 H, H-4′R, H-5′′bβ), 3.39 (m, 0.5 H, H-4′β), 3.29-3.20 (m, 2 H, H-7a, H-19), 3.10-2-97 (m, 4 H, H-7b, H-12, H-18a, H-30a), 2.88 (m, 0.5 H, H-5′′bR), 2.75-2.66 (m, 3 H, H-16a, H-17a, H-30b), 2.50-2.35 (m, 5 H, H-15, H-16b, H-17b, H-18b), 2.20 (m, 2 H, H-2′′), 2.11 (m, 1 H, H-13a), 1.67-1.55 (m, 4 H, H-3′′, H-4′′), 1.54-1.33 (m, 13 H, H-13b, CH3C), 1.33 (s, 9 H, H-23); 13C NMR (CD OD): δ 177.6 (C-11), 174.3, (C-1′′), 172.9 3 (C-20), 150.7 (C-29), 149.0 (C-28), 141.6 (C-31), 140.7, 140.6 (C-1, C-6), 139.1 (C-26), 135.0 (C-25), 130.1 (C-32, C-36), 129.4 (C-33, C-35), 129.1 (C-2), 128.0 and 127.3 (C-3, C-4), 125.9 (C-34), 125.0 (C-5), 124.8 (C-27), 111.6, 110.4, 100.7, and 100.6 (CH3C), 99.8 (C-1′β), 98.9 (C-1′R), 77.6 (C-3′β), 77.3 (C-2′R), 76.7 (C-8), 76.1 (C-3′R), 75.6 (C-2′β), 74.0 (C-5′R), 73.4 (C-5′β), 70.2 (C-5′′β), 68.3 (C-5′′R), 68.2 (C-4′β), 67.6 (C-14), 66.8 (C-19), 63.6 (C-15), 63.2, 62.9 (C-6′Rβ), 62.5 (C-4′R), 60.4 (C-24), 56.5 and 53.1 (C-16, C-17), 56.4 (C-9), 52.1 (C-22), 51.9 (C-18), 46.1 (C-12), 40.6 (C-30), 38.5 (C-13), 38.2 (C-7), 34.5 (C-2′′), 29.9 and 29.8 (C-4′′Rβ), 29.0 (C-23), 29.4, 28.4, 28.0, 26.4, and 19.2 (CH3C), 22.5 and 22.4 (C-3′′Rβ). Ind-[C(O)C4ManP]2. Rf 0.79 (9.2:0.8 CH2Cl2-EtOH); 1H NMR (CD3OD): δ 8.53 (s, 1 H, H-29), 8.49 (d, J 3.9 Hz, 1 H, H-28), 7.85 (d, J 7.9 Hz, 1 H, H-26), 7.46 (dd, J 7.7, J 4.9 Hz, 1H, H-27), 7.41 (s, 1 H, H-21), 7.32-7.25 (m, 10 H, H-2-5, H-10, H-32-36), 5.66 (m, 1 H, H-9), 5.52 (m, 1 H, H-8), 5.21 (m, 1 H, H-14), 5.04 (s, 0.6, H-1′Rint), 4.98 (s, 0.6 H, H-1′Rext), ∼4.87 (hindered by water H-1′Rint), 4.78 (1s, 0.4 H, H-1′Rext), 4.34 (dd, J 6.0, J 2.5 Hz, 0.4 H, H-2′β), 4.27 (dd, J 6.0, J 2.4 Hz, 0.4 H, H-2′β), 4.22-3.99 (m, 4 H, H-2′Rβ, H-3′Rβ), 3.953.65 (m, 8 H, H-5′Rβ, H-6′Rβ, H-5′′aRβ), 3.60 (s, 2 H, H-24), 3.58-3.39 (m, 3.2 H, H-4′R, H-5′′bRβ), 3.55-3.25 (m, 2 H, H-7a, H-19), 3.04-2.90 (m, 4.4 H, H-7b, H-12, H-18a, H-30a, H-4′β), 2.77-2.20 (m, 13 H, H-13a, H-15, H-16, H-17, H-18b, H-30b, H-2′′), 1.79-1.36 (m, 33 H, H-13b, H-3′′, H-4′′, CH3C), 1.34 (s, 9 H, H-23); 13C NMR (CD3OD): δ 177.4 (C-11), 174.6 (C-1′′int), 174.2 (C-1′′ext), 172.4 (C-20), 150.6 (C-29), 148.9 (C-28), 141.3 (C-31), 141.7, 140.0 (C-1, C-6), 138.9 (C-26), 134.8 (C-25), 129.9 (C-32, C-36), 129.3 (C-33, C-35), 129.1 (C-2), 128.0, 127.4 (C-3, C-4), 125.7 (C-34), 125.0 (C-5, C-27), 111.6, 110.3, 100.7, and 100.6 (CH3C), 99.78 and 99.70 (C-1′R), 98.83 and 98.77 (C-1′β), 77.5 (C-3′β), 77.3 (C-2′R), 76.75 and 76.71 (C-8), 76.0 (C-3′R), 75.5 (C-2′β), 73.8 (C-5′R), 73.3 (C-5′β), 70.84 and 70.77 (C-14), 70.24 and 70.16 (C-5′′β), 68.29 and 68.19 (C-5′′R), 68.1 (C-4′β), 66.7 (C-19), 63.5 and 62.9 (C-6′Rβ), 62.4 (C-4′R), 60.4 (C-24), 59.6 (C-15), 56.4 (C-9), 56.3 (C-18), 52.9 and 51.9 (C-16, C-17), 51.5 (C-22), 45.0 (C-12), 40.5 (C-30), 38.2 (C-7), 34.9 (C-13), 34.7 and 34.5 (C-2′′), 30.0, 29.8, and 29.7 (C-4′′), 29.0 (C-23), 29.4, 28.4, 28.0, 26.4, and 19.2 (CH3C), 22.5 and 22.4 (C-3′′Rβ).


Ind(8)-C(O)C4Man. Deprotection of Ind(8)-C(O)C4ManP (90 mg, 0.09 mmol) yielded, after chromatography (10:0 to 4:1 EtOAc-MeOH), Ind(8)-C(O)C4Man (2TFA) (90 mg, 91% with R:β ) 58:42) as a white solid. Rf 0.62 (7:3 EtOAc-MeOH); Rt 9.5 min (gradient A); 1H NMR (CD3OD): δ 8.52-8.44 (m, 2 H, H-28, H-29), 7.86 (d, J 7.9 Hz, 1 H, H-26), 7.43 (m, 1 H, H-27), 7.30-7.15 (m, 9 H, H-2-5, H-32-36), 5.50 (d, J 4.8 Hz, 1 H, H-9), 5.30 (m, 1 H, H-8), 4.63 (s, 0.6 H, H-1′R), 4.33 (s, 0.4 H, H-1′β), 4.00-2.55 (m, 25 H, H-7, H-12, H-14-19, H-24, H-30, H-2′-6′, H-5′′), 2.11 (m, 2 H, H-2′′), 1.88 (m, 1 H, H-13a), 1.49 (m, 5 H, H-3′′, H-4′′, H-13b), 1.24 (s, 9 H, H-23); 13C NMR (CD OD): δ 177.5 (C-11), 174.6, 174.5 (C-20, C-1′′), 3 150.8 (C-29), 149.5 (C-28), 141.5 (C-31), 140.9, 140.5 (C-1, C-6), 139.9 (C-26), 135.0 (C-25), 130.1 (C-32, C-36), 129.4 (C-33, C-35), 129.2 (C-2), 128.1, 127.5 (C-3, C-4), 125.9 (C-34), 125.5 (C-5), 125.0 (C-27), 101.6 (C-1′R), 101.4 (C-1′β), 78.1 (C-5′β), 76.8 (C-8), 75.1 (C-3′β), 74.5 (C-5′R), 72.6 (C-2′β), 72.4 (C-3′R), 72.1 (C-2′R), 70.0 (C-5′′β), 68.4 (C-4′Rβ), 68.0 (C-5′′R), 67.2 (C-14), 66.4 (C-19), 62.7 and 62.6 (C-6′Rβ, C-15), 59.3 (C-24), 58.3 and 54.7 (C-16, C-17), 56.6 (C-9), 52.5 (C-22), 51.3 (C-18), 45.8 (C-12), 40.5 (C-30), 38.2 and 38.4 (C-7, C-13), 34.6 (C-2′′), 29.9, 29.8 (C-4′′Rβ), 28.7 (C-23), 22.6, 22.4 (C-3′′Rβ). ESI-MS: m/z 876.4 [M + H]+, 898.5 [M + Na]+, in agreement with the calculated mass for [M] ) C47H65N5O11 (875.47). Ind-[C(O)C4Man]2. Deprotection of Ind-[C(O)C4ManP]2 (80 mg, 0.06 mmol) led, after chromatography (10:0 to 4:1 EtOAcMeOH), to Ind-[C(O)C4Man]2 (2TFA) (78 mg, 95% with R:β ) 60:40) as a white solid; Rf 0.48 (7:3 EtOAc-MeOH); Rt 9.0 min (gradient A); 1H NMR (CD3OD): δ 8.50 (m, 2 H, H-28, H-29), 7.85 (d, J 7.7 Hz, 1 H, H-26), 7.45 (s, 1 H, H-21), 7.38 (m, 1H, H-27), 7.29-7.22 (m, 10 H, H-2-5, H-10, H-32-36), 5.62 (d, J 4.8 Hz, 1 H, H-9), 5.48 (m, 1 H, H-8), 5.16 (m, 1 H, H-14), 4.76 (1s, 0.6 H, H-1′Rint), 4.71 (1s, 0.6 H, H-1′Rext), 4.51 (1s, 0.4 H, H-1′βint), 4.39 (1s, 0.4 H, H-1′βext), 3.943.41 (m, 19.2 H, H-2′Rβ, H-3′Rβ, H-4′R, H-5′Rβ, H-6′Rβ, H-5′′Rβ, H-15, H-24), 3.26-3.17 (m, 2 H, H-7a, H-19), 3.002.87 (m, 4.8 H, H-4′β, H-7b, H-12, H-18a, H-30a), 2.72-2.39 (m, 13 H, H-2′′, H-13a, H-15, H-16, H-17, H-18b, H-30b), 1.72-1.53 (m, 9 H, H-3′′, H-4′′, H-13b), 1.30 (s, 9 H, H-23); 13C NMR (CD OD): δ 177.4 (C-11), 175.1 (C-1′′int), 174.7 3 (C-1′′ext), 172.8 (C-20), 150.9 (C-29), 149.0 (C-28), 141.5 (C-31), 141.1, 140.3 (C-1, C-6), 139.3 (C-26), 132.4 (C-25), 130.2 (C-32, C-36), 129.5 (C-33, C-35), 129.3 (C-2), 128.2, 127.6 (C-3, C-4), 125.9 (C-34), 125.3 (C-5, C-27), 101.68, 101.60, 101.56, and 101.48 (C-1′R and C-1′β), 78.22 and 78.16 (C-5′β), 76.9 (C-8), 75.31 and 75.23 (C-3′β), 74.62 and 74.55 (C-5′R), 72.68, 72.65, 72.56, and 72.48 (C-2′β, C-3′R), 72.2 (C-2′R), 71.1 (C-14), 70.1 and 70.0 (C-5′′β), 68.65, 68.61, and 68.51 (C-19, C-4′Rβ), 68.1 and 68.0 (C-5′′R), 62.9, 62.8, and 62.7 (C-6′Rβ), 60.4 (C-24), 59.9 (C-15), 56.8 (C-9), 56.3 (C-18), 53.1, and 52.1 (C-16, C-17), 51.8 (C-22), 45.2 (C-12), 40.7 (C-30), 38.3 (C-7), 35.1 (C-13) 34.9 and 34.7 (C-2′′), 30.1 and 30.0 (C-4′′Rβ), 29.0 (C-23), 22.8, 22.6, and 22.5 (C-3′′Rβ). ESI-MS: m/z 1138.5 [M + H]+, 1160.6 [M + Na]+, in agreement with the calculated mass for [M] ) C58H83N5O18 (1137.57). Biological Section. Materials. 1-Pentanesulfonic acid sodium salt, sodium acetate trihydrate, and acetonitrile were highperformance liquid chromatography (HPLC) grade. Foetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), penicillin-streptomycin solution (5000 U/mL:5000 µg/mL), trypsin-EDTA 0.25% solution (2.5 g trypsin and 0.2 g EDTA in 1 L DPBS), D-(+)-glucose, and N-[2-hydroxyethyl]piperazine-N-ethanesulfonic acid (HEPES) were purchased from Sigma Chemical (St Quentin Fallavier, France). Dulbecco’s modified eagle’s medium (DMEM) and nonessential amino


acids DMEM 100× (NEAA) were purchased from Gibco-Life Technologies (Cergy-Pontoise, France). Transport Medium (TM) consisted of DPBS containing 25 mM glucose and 10 mM HEPES (pH 7.4). Cell culture medium consisted of DMEM supplemented with 20% foetal bovine serum, 1% NEAA, and 2% penicillin-streptomycin. HPLC Conditions for Chemical Stability and Transport Experiments. All HPLC analyses used for chemical stability and transport experiments were performed using a HP1100 apparatus with a Lichrospher 100 RP-18 (5 µm)-packed column (250 × 3.2 mm) with a flow rate of 1 mL/min. The isocratic mobile phase consisted of CH3COONa‚3H2O (15 mM) and CH3(CH2)4SO3Na (15 mM), 5 mM pH 6 buffer and CH3CN: 59:41 for Ind, Ind(8)-C(O)C4Man and Ind-[C(OC4Man]2, 41:59 for Saq and Saq-C(O)C4Man, and 45:55 for Nelf, Nelf(1)-C(O)C4Man and Nelf(1)-[C(O)C4Man]2. The prodrugs and/or drugs were detected by measuring their UV absorption at 210 (Ind, Nelf, and their derivatives) or 254 nm (Saq and its derivative) and the signals (peak integration) were interpreted by the software provided. Under their respective HPLC conditions, the retention times measured for the different compounds were of 4.9 min for Ind, 3.5 min for Ind(8)-C(O)C4Man, 3.3 min for Ind[C(OC4Man]2, 5.6 min for Saq, 5.0 min for Saq-C(O)C4Man, 12.5 min for Nelf, 5.2 min for Nelf(1)-C(O)C4Man, 4.8 min for Nelf-[C(OC4Man]2. The prodrug and/or drug concentration was determined from HPLC calibration curves. These curves were established under the same HPLC conditions, using standard calibrated prodrug and drug solutions that were prepared in the same hydrolysis medium as the sample under investigation. The calibration curves were linear (correlation coefficients from 0.9993 to 0.9998) in a concentration range of 0.4 to 1143 µM for Ind and its prodrugs, 0.1 to 300 µM for Saq and its prodrug, and 0.1 to 301 µM for Nelf and its prodrugs, the lower limit corresponding to the limit of detection that can be quantified with accuracy. Above the lower concentration limit, the analytical method was reproducible. Hydrolysis. Hydrolysis experiments were performed by incubating 20 mL of a DMEM-MeOH solution (pH 7.3) of the prodrug (250 µg/mL) at 37 °C with stirring. The amount of MeOH (v/v) in these solutions was 4% for Ind(8)-C(O)C4Man, 5% for Ind-[C(OC4Man]2, 3% for Saq-C(O)C4Man, and 5% for Nelf(1)-C(O)C4Man. Hydrolysis was followed by HPLC monitoring of either the disappearance of the prodrug and appearance of the parent drug by injecting 40 µL of the solution onto the HPLC column (for HPLC conditions see above). Plots of ln([prodrug]o - [prodrug(t)]) and of ln[drug(t)] against time were linear within the concentration range studied, indicating that the hydrolysis is a first-order process with respect to the prodrug. The half-lives of hydrolysis (t1/2) were measured, when possible, or calculated from these plots; t1/2 is related to the slope, K, of these curves by the relation t1/2 ) (ln 2)/K. Caco-2 Cell Monolayers. Caco-2 cells, clone TC7, were kindly provided by Dr. A. Zweibaum (INSERM U178, Villejuif, France). The cells were routinely maintained in 75 cm2 culture flasks at 37 °C in an atmosphere containing 5% CO2 and 95% relative humidity. Cells were split every 7 days at a density of 1.5 × 106 cells/flask. For the transepithelial transport experiments, Caco-2 cells (passage 60-65) were harvested with trypsin-EDTA and seeded on Anopore membrane inserts (0.2 µm pore diameter, 25 mm diameter; Nunc, Roskilde, Denmark) at a density of 5 × 105 cells/insert (cm2). Apical (AP) and basolateral (BL) chamber volumes were maintained at 2 mL. Culture medium was changed every 3-4 days and cells were used for the experiments between days 14 and 27 post-seeding. Monolayer formation was monitored by measurement of transepithelial electrical resistance (TEER) using a Millicel ERS apparatus (Millipore).


Transport Experiments. Before the transepithelial transport experiments, the Caco-2 monolayers were rinsed twice with the transport medium (TM) (both chambers) and preincubated for 30 min in the TM. After this equilibration period, the monolayer integrity was checked by measuring its TEER. For the transport experiments, only monolayers displaying TEER values above 176 Ω‚cm2 and for which TEER values fell by less than 15% from the value measured at the end of the equilibration period were used. Under these conditions, the age of the cell monolayer within the 14-27 day’s postseeding range did not affect the transport results. Transport was initiated by replacing the TM in the AP or in the BL “donor” compartment with 2 mL of the drug or prodrug solution. AP and BL chamber volumes were maintained at 2 mL. The test solutions were prepared by mixing a known amount of TM with a concentrated MeOH stock solution of the drug or prodrug under investigation to reach a final concentration of the drug or prodrug in the 230-300 µM, 280 µM, and 252-344 µM range for the indinavir, saquinavir, and nelfinavir derivatives, respectively (the exact concentrations used for each derivative are given in the figures showing their transepithelial transport). The final MeOH concentration in the drug or prodrug solutions in contact with the monolayers never exceeded 3% MeOH (TEER monitoring and transport experiments have shown that MeOH at concentrations up to 5% did not affect cell monolayer integrity during the 3-h period of transport experiment nor the amount of drug and/or prodrug transported, respectively). 200 µL samples were withdrawn from the “acceptor” compartment (opposite to the addition “donor” chamber) every 1 h over a period of 3 h and replaced by the same amount of fresh transport medium to maintain the same volume. The dilution was taken into account for the calculations. To prevent hydrolysis of the prodrugs, all these samples were stored at 4 °C awaiting prodrug and/or parent drug HPLCanalysis (see above section: HPLC conditions). At the end of the experiments (3 h), samples were also taken from the “donor” compartment for HPLC analysis, and the monolayers were checked for integrity by measuring TEER values. The concentrations of the prodrug and parent drug that were measured in the donor and acceptor chamber at the end of the transport experiment indicated that no nonspecific adsorption on glass or on plastic had occurred. The experiments for which TEER has decreased by more than 8% from the value measured at the beginning of the experiment were discarded. Transport was expressed as a percentage of the initial amount added to the donor compartment. All flux experiments were conducted at least in triplicate in the AP to BL and BL to AP directions. If possible, a concentration of the prodrug in the donor chamber the closest to that of the parent drug was preferred, but solubility issues of the prodrug (which needed in some cases the use of MeOH) and detection issues of the prodrug and/or parent drug in the acceptor chamber were also considered for the selection of the prodrug concentration. Data Analysis for the Transport Experiments. The apparent permeability coefficients Papp (cm/s) were calculated by linear regression analysis on the time course plot of amount of (pro)drug transported from equation Papp ) k(VD/A), where k is the slope of the linear curve ln(Ct/Co) ) -kt, Ct being the (pro)drug concentration in the receiver chamber at time t, Co is the initial concentration in the donor chamber, A is the membrane surface area (4.52 cm2), and VD is the volume of the donor chamber (2 cm3). These calculations were performed for each transport experiment, and the values that are reported in Table 3 represent the means ((SD) of at least three independent experiments.

RESULTS AND DISCUSSION Synthesis. The mannose-derived PI prodrugs shown in Figure 1 were prepared in two steps by condensing the saquinavir,

Roche et al.

indinavir, or nelfinavir with the key acid synthon 5r,β, a protected mannosylated derivative, followed by deprotection of the sugar protecting group (Scheme 1). Owing to the difficulties to prepare large amounts of pure 5r or 5β, the synthon 5 was used as a mixture of R/β anomers. The esterification (first step) of the PI hydroxyl(s) was the limiting step. Indeed, the deacetalation of the protected mannose unit in the various saquinavir, indinavir, or nelfinavir was performed with 90-95% yields. Note that all the mono- and diester PI-prodrugs were isolated as mixtures of R,β-D-mannose anomers (see below). Concerning saquinavir, acylation of its unique hydroxyl was performed in 71% yield using conventional EDC/DMAP as coupling reagent (48). Concerning indinavir and nelfinavir, which both contain two hydoxyls, and in line with previous work (27-29), a stochastic esterification, one to one equivalent, was preferred instead of a more tedious and time-consuming protection/deprotection strategy of one of these two hydoxyls. The stochastic esterification led into the formation of a mixture of mainly the monoester and diester resulting from the esterification of the C1-hydroxyl of nelfinavir and C8-hydroxyl of indinavir, and of both hydroxyls, respectively. Only tiny amounts (less than 2%) of the monoesters corresponding to the esterification of the C18-hydroxyl of nelfinavir and C14-hydroxyl of indinavir were detected. In the case of indinavir and nelfinavir esters, monoester versus diester formation was favored but only slightly by contrast with what we noticed for all nelfinavir or indinavir prodrugs synthesized so far, using the same reaction conditions (27-29). Indeed, acylation of indinavir (respectively nelfinavir) with acid 5r,β led to a 1.5:1 (respectively 2.2:1) mixture of monoester Ind(8)-C(O)C4ManP (respectively Nelf(1)-C(O)C4ManP) and diester Ind-[C(O)C4ManP]2 (respectively Nelf-[C(O)C4ManP]2). By contrast, and as reported elsewhere, reaction of indinavir with 1,2:5,6-di-O-isopropylidene-3-O-(4carboxybutyl)-R-D-glucofuranose, a close analogue of the mannosylated acid 5r,β, produced a 4:1 mixture of the monoester and diester adducts (29). Deprotection of the mannose unit in the saquinavir, indinavir, or nelfinavir conjugates was performed using aqueous TFA and afforded almost quantitatively the target Saq-C(O)C4Man(TFA), Ind(8)-C(O)C4Man(2TFA), Ind-[C(O)C4ManP]2(2TFA), Nelf(1)-C(O)C4Man(2TFA), and Nelf-[C(O)C4ManP]2(2TFA) prodrugs, respectively. Concerning the synthesis of the starting mannose-derived building block 5 and owing to the presence of several hydroxyl groups on mannose with close reactivity, protection/deprotection steps were needed to prepare this key synthon. Moreover, as this synthon was linked to the PI via an ester linkage, the use of protecting groups for the sugar part that are labile under basic conditions was precluded. Therefore, we selected the isopropylidene acid-labile acetal protection, which has further been shown to be highly compatible for similar glucosylated derivatives (29). To the best of our knowledge, examples of glycosylation with saccharides containing a 2,3-isopropylidene protecting group used as a glycoside donor are few (49-54), and only one concerns the mannopyranose moiety (53). In our hands, this latter strategy failed. Indeed, the glycosylation of methyl 5-hydroxypentanoate with 2,3;4,6-di-O-isopropylidene R-Dmannopyranose derivative 3r (HO-ManP) (Scheme 1, path A) gave the target molecule 4r in 4% yield only. To overcome this drawback, another strategy was attempted where the diisopropylidene protection was performed after the glycosylation step using a more conventional procedure (Scheme 1, path B). The glycosylation was achieved using the very labile trimethylsilyl group as transient intermediate protecting group adapting a process described in literature for the preparation of an analogous mannosylated derivative (55). The transitory persilylated mannopyanose 6r,β (obtained as a 80:20 R/β



Scheme 1. Synthetic Pathway to the Mannose-Containing Prodrugs of Saquinavir, Indinavir, and Nelfinavira

a (i) PhCH2OH (reactive and solvent), p-TsOH; (ii) 2,2-dimethoxypropane, acetone, p-TsOH; (iii) H2/Pd/C, 2:2:1 THF/EtOH/H2O; (iv) HO(CH2)4CO2Me, Ph2SO, (CF3SO2)2O, 2,6-dimethylpyridine; (v) MeC[)NSi(Me)3]OSiMe3, CH2Cl2, rt or Me3SiCl, (Me3Si)2NH, pyridine, rt; (vi) (a) Me3SiI, NH4I, CH2Cl2, rt, (b) HO(CH2)4CO2Me, rt, (c) MeOH; (vii) 2-methoxypropene, p-TsOH, CH2Cl2; (viii) 1 M LiOH, THF; (ix) EDC/ DMAP, CH2Cl2; (x) TFA, CH2Cl2.

Scheme 2. Chemical Structures and Code Names of the Antiprotease Inhibitor (PI ) Saquinavir, Indinavir, and Nelfinavir) Prodrugs Already Publisheda

a

Refs 28 and 29.

anomeric mixture, according to 1H NMR) was prepared from D-mannose using either N,O-bis(trimethylsilyl)acetamide in dichloromethane or a mixture of chlorotrimethylsilane and 1,1,1,3,3,3-hexamethyldisilazane in pyridine, the former conditions being preferred. Then, action of iodotrimethylsilane gave the in situ persilylated mannopyranosyl iodide which, by reaction with methyl 5-hydroxypentanoate followed by desilylation, afforded methyl ester 7r,β in 30% yield as a 60:40 R/β mixture according to 1H NMR (see below). Pure 7r could be separated and isolated from this mixture after several column chromatographies. The acetalation protection was then performed by action of 2-methoxypropene on 7r,β giving 4r,β in 71% yield. Saponification released almost quantitatively the acid intermediate 5r,β (20% overall yield from D-mannose). In the same way, the anomeric material 5r was obtained from 7r. The chemical structures of (i) all protected and deprotected PI prodrugs, which consist into mixtures of R,β-D-mannose anomers, (ii) anomeric mixtures of 4r,β/5r,β/7r,β, and (iii) pure 4r/5r/7r were unambiguously ascertained by 1H,13C NMR and mass spectrometry. That each of the protected and deprotected PI prodrugs consisted of a mixture of R,β-D-mannose anomers was shown by the presence of anomeric H-1R and H-1β singlets in approximately 64:36 to 52:48, which are close to that of the 53:47 protected material 4r,β, and of anomeric C-1R

and C-1β resonances in its 1H and 13C NMR spectrum, respectively (see Table 1). The R/β assignment was attested by comparison of these NMR signals with those collected for pure 4r, 5r, and anomeric 4r,β or 5r,β mixtures (see Table 1) and by comparison with the 1H (55-57) and 13C NMR data for similar derivatives (55, 56, 58). For instance, the R and β configurations of 4 are confirmed by the anomeric H-1R and H-1β singlets at 4.96 and 4.89 ppm, respectively, the C-13R and C-13β resonances determined by 2D experiments being at 97.9 and 98.9 ppm, respectively (55, 59). The variations of R:β ratios of the various protected and deprotected PI prodrugs were essentially due to the purification steps which were performed by column chromatography. That formation of the monoesters occurred on the C-26 saquinavir, C-8 indinavir, and C-1 nelfinavir hydroxyls is also unambiguously established by 1H and 13C NMR. As expected, the resonances of the corresponding H-26 (respectively H-8) proton and C-26 (respectively C-8) carbon atoms of the saquinavir (respectively indinavir) derivative, are deshielded (|∆δ| ) 1.25-1.38 ppm and |∆ δ| ) 3.7-7.6 ppm, respectively) in comparison with those of the parent PI. For nelfinavir, acylation of its aromatic C-1 hydroxyl resulted in the shielding (|∆δ| ) 5.9 ppm) of the C-1 carbon resonance (60, 61), as expected for phenyl esters. Furthermore, the resonances of the


Roche et al.

Table 1. r:β Anomer Ratio of the Mannosylated Starting Building Blocks 4/5 and PI-Prodrugs, and NMR Data of Their Anomeric H- and C-Atom Signalsa compound

R:β ratio

H-1′Rb

4r 4r,β 5r 5r,β Saq-C(O)C4ManP Saq-C(O)C4Man Ind(8)-C(O)C4ManP Ind(8)-C(O)C4Man Ind-[C(O)C4ManP]2d

100:0 53:47 100:0 64:36 64:36 52:48 58:42 62:38

Ind-[C(O)C4Man]2d

60:40

Nelf(1)-C(O)C4ManP Nelf(1)-C(O)C4Man Nelf-[C(O)C4ManP]2f

55:45 58:42 55:45

[Nelf-C(O)C4Man]2f

60:40

4.89 4.96 4.94 c 5.00 4.78 (d, J 1.5) 4.96 4.63 5.04 (C-14) 4.98 (C-8) 4.76 (C-14) 4.71 (C-8) 5.09 4.73 (d, J 3.4) 5.10 5.06 4.83 4.78

H-1′βb 4.89 c 4.84 (d, J 2.4) 4.55 4.77 4.33 4.87 (C-14) 4.78 (C-8) 4.51 (C-14) 4.39 (C-8) 4.90 (d, J 2.2) 4.49 4.93 (d, J 1.1) 4.90 4.58 4.56

C-1′R 97.7 97.9 99.1 99.1 99.1 101.5 98.9 101.6 99.78 (C-14) 99.70 (C-8) 101.68e 101.60e 99.0 101.5 98.94 98.89 100.7

C-1′β 98.9 100.1 100.0 101.7 99.8 101.4 98.83 (C-14) 98.77 (C-8) 101.56e 101.48e 99.8 101.6 99.7 101.6

a δ in ppm, J in Hz. b Singlet unless otherwise stated. c Hindered by the residual peak of water. d C-8/C-14 means that the mannosylated residues are connected to the indinavir C-8/C-14 atom, respectively. e Could not be assigned to the indinavir C-8 or C-14 connecting position f Could not be assigned to the nelfinavir C-1 or C-18 connecting position.

H-14/C-14 indinavir and H-18/C-18 nelfinavir atoms bearing the remaining “free” hydroxyl and of their vicinal β-carbon atoms are almost not affected. These resonances are deshielded in the diesters, confirming that acylation of the two hydroxyls of indinavir and nelfinavir has occurred. All these trends are in line with those reported for the C-14/C-8 indinavir and C-18/ C-1 nelfinavir prodrugs (27-29). Concerning the protected and deprotected diester prodrugs derived from indinavir or nelfinavir, their 1H/13C NMR spectra are very complex. This is due to the presence of four isomers consisting of the R/R-, R/β-, β/R-, and β/β- connection of the mannose moiety to each of the two hydroxyl groups of the PI. Indeed, their 1H/13C NMR spectra exhibit four signals for the R/β anomeric H/C atoms, two sets for each of the C14R/β- and C8-R/β-indinavir (respectively, C18- and C1-nelfinavir) R/βanomers, as expected (see Table 1). Mannose-deprotection is confirmed among others by the absence of the characteristic isopropylidene resonances in the NMR spectra of the isolated deprotected PI prodrugs. Biological Activity and Chemical Stability. The saquinavir hydroxyl, the indinavir C-14 but not the C-8 hydroxyl, and the nelfinavir C-18 but not the C-1 hydroxyl, are involved in the peptidomimetic noncleavable transition state isostere responsible for the protease inhibitory potency of saquinavir, indinavir, and nelfinavir, respectively (62). Therefore, it is most important that these hydroxyls be accessible for antiviral activity. In the previous studies dedicated to the ester prodrugs of saquinavir, indinavir, and nelfinavir (27, 28), a close correlation between their anti-HIV activity and the hydrolysis of their acylated “peptidomimetic” hydroxyl, hence the liberation of the active free drug during the time of incubation, was found: the faster the hydrolysis, the closer the anti-HIV activity level to that of the respective parent drug. Concomitantly, the level of HIV inhibition was very low for the prodrugs for which hydrolysis and release of this peptidomimetic hydroxyl was very slow. On the other hand, no correlation was found between the hydrolysis rate of the acylated C-8 indinavir prodrugs (C-8 hydroxyl is not part of the transition state isostere) and their anti-HIV activity (27). The HIV inhibition levels and cytotoxicities of D-mannose-containing saquinavir, indinavir, and nelfinavir prodrugs (as their TFA salt) were evaluated in vitro in CEM-SS and MT4 cells against HIV-1 according to published procedures (63-65). The data are collected in Table 2 together with those of saquinavir and nelfinavir (66) (as their methanesulfonate salt), and indinavir (as its sulfate salt).

The sensitivity to hydrolysis of the mannose prodrugs was checked using the same hydrolysis protocol as that described in previous studies from our laboratory. These hydrolysis experiments were performed in a pH 7.3 buffer at 37 °C and in the absence of serum, cells and virus using a prodrug concentration in the 476-1015 µM range. All compounds were hydrolyzed within a close interval of time, their hydrolysis t1/2 being comprised between 30 and 60 h (Table 2). These results are close to those established for glucose prodrug analogues containing the same spacer with the striking exception of the glucose analogue Saq-C(O)C4Glc (Scheme 2) which was found to be quite unstable (t1/2 ) 3 h) (29). No obvious explanation may be found for the higher resistance to hydrolysis of the mannosylated saquinavir conjugates as compared with the glucosylated one. The anti-HIV efficiency of saquinavir, indinavir, and nelfinavir is substantially reduced when D-mannose is conjugated to the peptidomimetic hydroxyl of these protease inhibitors as well as to the hydroxyl of indinavir and nelfinavir, which is not part of the transition state isostere. This is illustrated by the large R values (from g44 to 1060), which correspond to the ratio of the IC50 of the PI prodrug versus that of the parent PI (see Table 2). The lower anti-HIV activities of these PI prodrugs reflect to some extent their relative stabilities with respect to hydrolysis during the 4 day time-span of the antiviral assays. Considering the sole masking of the “non-peptidomimetic hydroxyl” C-8 of indinavir and C-1 of nelfinavir, this modified also drastically the antiviral activity of nelfinavir (R ∼ 170 and ∼ 120 for Nelf(1)-C(O)C4Man) but not that of indinavir (R ∼ 7 and ∼ 12 for Ind(8)-C(O)C4Man). The activity found for this latter prodrug is in the same range than that of its glucose analogue, Ind(8)-C(O)C4Glc (Scheme 2) (29). These data indicate likely that C-8 indinavir conjugates themselves may possess an antiviral activity, which is lower than that of indinavir, and that the hydrolysis rate may also, to some extent, contribute to increasing their intrinsic antiviral potency. In addition, it appears that the nature of the substituent at the other end of the C(O)C4 spacer linked to the non-peptidomimetic C-8 indinavir or C-1 nelfinavir hydroxyl is also of importance for the anti-HIV activity. Indeed, anti-HIV activity is increasing along the mannose, glucose, and tyrosine series (Scheme 2), with these derivatives displaying comparable chemical stability (t1/2 in the 40-60 h range, Table 2). Transepithelial Transport. There is currently a considerable interest in increasing the intestinal absorption of the HIV



Table 2. Anti-HIV Activity (IC50) and Cytotoxicity (CC50) Data for Mannosylated Saquinavir, Indinavir and Nelfinavir Prodrugsa in CEM-SS and MT-4 Cell Cultures Infected with HIV-1 LAI and HTLV IIIB, Respectively, Together with Their Hydrolysis Half-Life (t1/2), as Compared with Glucose- and Tyrosine-Based Analogous Prodrugsb compoundc

IC50 (nM)

Saquinavir* Saq-C(O)C4Man Indinavir** Ind(8)-C(O)C4Man Ind-[C(OC4Man]2 Nelfinavir* Nelf(1)-C(O)C4Man Nelf-[C(OC4Man]2 Saq-C(O)C4Tyri Saq-C(O)C4Glcj Ind(8)-C(O)C4Tyrh,i Ind(8)-C(O)C4Glcj Nelf(1)-C(O)C4Tyri

CEM-SS 9 484 e10 71 440 2f 339 2120 39 CC50 160 62 47 190 193

∼54

Rd

∼120 ∼12 ∼102 120 ∼9 3.4 4.7 9

CC50 (M)

CC50 (M)

CEM-SS >10-5 >5 × 10-5 >10-4 >10-4 >5 × 10-5

MT-4 > 10-5 >5 × 10-5 >10-4 >10-4 > 5 × 10-5

>5 × 10-5 >5 × 10-5

>5 × 10-5 >5 × 10-5

t1/2e hydrolysis (h)

50 50 60 48 g 40 3 50 k 60

a These data reflect the amount of parent drug released during the 4-day period of the experiments. b See Scheme 2 for the structures of glucose (Glc) and tyrosine (Tyr) prodrugs. *, as its mesylate and **, sulfate salt. c Prodrugs are tested as their TFA salt. d R is the ratio of the prodrug IC50 to that of its parent compound. e t1/2, which corresponds to the time at which 50% of hydrolysis is observed, has been determined from hydrolysis experiments performed by incubating the prodrugs in a pH 7.3 DMEM solution at 37 °C. f Data from ref 66. g Not tested. h Data from ref 27. i Data from ref 28. j Data from ref 29. k 5% of hydrolysis in 3 h of incubation in the transepithelial transport assay across Caco-2 cell monolayers (culture cell media close to those used for hydolysis experiments) (data from ref 30).

Table 3. Percentages of Prodrug and PI Transport Across a Monolayer of Caco-2 Cells from the Donor Chamber after 3 h of Experiment and Apparent Permeability Coefficients Papp absorptive direction (AP to BL)

secretion direction (BL to AP)

compounda

conc in donor chamber (µM)

percent ((SD) in receiver chamber

Papp ((SD) (cm/s, × 10-8)

percent ((SD) in receiver chamber

Papp ((SD) (cm/s, × 10-8)

Saquinavir* Saq-C(O)C4Man Indinavir** Ind(8)-C(O)C4Man Ind-[C(OC4Man]2 Nelfinavir* Nelf(1)-C(O)C4Man Nelf-[C(OC4Man]2 Saq-C(O)C4Glcb Ind(8)-C(O)C4Glcb Nelf(18)-C(O)NC4Glcb Nelf(1)-C(O)C4Tyrb

9.4 280 175 300 230 8 344 247 12.5 265 29 15

2.6 (0.4) 6.8 (0.3) 2.6 (0.3) 14.3 (2.5) 19 (2.3) 2.7 (1.1) ndc nd nd 2.7 (1.3) 1.6 (0.2) nd

110 (31) 321 (73) 103 (10) 526 (66 761 (71) 103 (66)

10.3 (1.9) 43.1 (0.4) 38 (2) 17.9 (0.6) 23.5 (2.6) 6.3 (1.4) nd nd 22.1 (7.4) 6.2 (1.5) 8.5 (0.8) nd

398 (117) 1622 (36) 1550 (234) 662 (25) 947 (102) 251 (68)

66 (31) 44 (6)

950 (720) 251 (110) 339 (51)

a *, as its mesylate MeSO H and **, sulfate salt. Prodrugs are tested as their TFA salt. b Data from ref 30; see Scheme 2 for the structures of glucose (Glc) 3 and tyrosine (Tyr) prodrugs. c nd ) not detected, i.e., below the detection limit which is 0.4, 0.1, and 0.1 µM for the indinavir, saquinavir, and nelfinavir derivatives, respectively.

protease inhibitors and in reducing their recognition by the efflux P-gp carrier (67, 68) which is a possible reason for their poor or variable uptake from the intestinal lumen into the blood circulation. In this regard, esterase-rich and efflux carrierexpressing Caco-2 cell line monolayers are widely accepted in vitro models of the intestinal epithelium for screening drug and prodrug candidates and thus for evaluating prodrug approaches for enhanced intestinal drug absorption (69-73). This human cell line forms indeed polarized monolayers, which have been shown to express most of the enzymatic, functional, and morphologic characteristics of the intestinal mucosa (69, 74). They express, on the apical side of the monolayer, efflux carrier systems, such as the multidrug-resistant P-gp, which is responsible for the limited oral bioavailability and brain penetration of the protease inhibitors (67, 68). Caco-2 cell line monolayers have already been used in numerous studies to characterize the permeation of various drugs and prodrugs, including indinavir, saquinavir, and nelfinavir (67, 68) and prodrugs deriving therefrom which have been developed in our laboratory (30). Translocation of the various mannosylated protease inhibitor prodrugs across Caco-2 cell monolayers was evaluated at a concentration where they are soluble. Transepithelial electrical resistance after confluence has been used to monitor the integrity of the cell layer (75). Translocation was then initiated by adding the test solution to the apical (AP) or basolateral (BL) side of

the monolayer (donor chamber). Investigation of the transport across the polarized Caco-2 cell monolayer in the absorptive AP to BL direction and in the secretory BL to AP one constitutes a mean of evaluating the influence of P-gp and related efflux carriers which are located on the AP side of the monolayer. As Caco-2 cells are also rich in esterases, the hydrolysis of the ester prodrugs during their permeation across Caco-2 monolayers was carefully checked. Only the prodrugs were detected either in the donor or acceptor chamber, indicating that no hydrolysis occurred during the transport experiments. The results of the bidirectional transport studies of D-mannose conjugated prodrugs and of their parent protease inhibitor are presented in Figure 2 (which illustrates the transport profiles) and Table 3. This table collects the percentages of (pro)drug that have been transported after the 3 h experiment and the apparent permeability coefficients Papp that were calculated from the slope of a plot of the cumulative receiver concentration with time. In addition, the Papp of mannose-conjugated derivatives are presented in Figure 3 in comparison with those of the closely related glucose-conjugated ones (29, 30). The bidirectional transport results of D-mannose-conjugated prodrugs were very contrasting depending on the PI concerned. Moreover, they contrast also very strongly with those reported for their D-glucose analogues (30). Most importantly, the mannosylated saquinavir and indinavir (but not nelfinavir)


Roche et al.

Figure 2. Bidirectional transepithelial transport across a monolayer of Caco-2 cells of mannose-conjugates of PIs (panels B, D, E, G, H) in comparison with their parent drugs (panels A, C, F) (data from ref 30). Absorptive translocation [apical (AP) to basolateral (BL) compartment]: filled lozenges. Secretory translocation (BL to AP): filled squares. The results are expressed as prodrug transport percentages versus time. The percentage values represent the ratios of (pro)drug concentration in receiver vs donor chamber × 100. The initial concentration of the prodrug in the donor compartment is indicated on each panel. Results are means ( SD from three experiments. All incubations were performed at 37 °C and pH 7.4.

prodrugs were found to display very promising transport profiles that could improve the intestinal absorption of these two protease inhibitors. Concerning the saquinavir derivative (Figure 2B), Saq-C(O)C4Man showed an asymmetric permeation profile indicating that, as its parent PI, it is substrate of the apically localized efflux P-gp carrier (67, 68). This highly asymmetric permeation profile was already observed for the D-glucose conjugate analogues [(i.e., Saq-C(O)C4Glc and Saq-C(O)NC4Glc (Scheme 2))]. However, it is noteworthy that the permeation of Saq-C(O)C4Man in the absorptive AP to BL direction is significantly much higher than those of its glucose analogues for which no transport across the Caco-2 cell monolayer was observed (Table 3 and Figure 3). In comparison with that of its parent drug, permeation of Saq-C(O)C4Man is enhanced in the two directional transport: ∼3-fold in the absorptive AP to BL direction and ∼4-fold in the secretory BL to AP efflux (Table 3, Figure 2A). These absorptive and secretory transport enhancements are not only attributable to a lower affinity of the efflux carriers for this conjugate but also to an active transport mechanism involving a mannose carrier system located at the brush border

side of the Caco-2 cell monolayer, if present. It could also arise from an improvement of passive permeation. However, this seems unlikely since conjugation of mannose to saquinavir induced a hydrophilicity increase, though tiny, as shown by the slight decrease of HPLC mobility and logP coefficient76 (Table 4). For the indinavir prodrugs, the monoester and diester conjugates, Ind(8)-C(O)C4Man and Ind-[C(OC4Man]2, presented similar and almost superimposable absorptive and secretory permeation profiles (Figure 2D,E). It appears also that, as for saquinavir, conjugation of indinavir to D-mannose improves substantially the absorptive permeation of indinavir (Figure 2C) across Caco-2 cell monolayers (∼5- and 7-fold increase, respectively), by contrast to its conjugation to Dglucose (Table 3 and Figure 3). It is further noticeable that the secretory permeations of these indinavir prodrugs are lower than that of indinavir, indicating that conjugation of D-mannose to indinavir decreases its passive diffusion, as expected from an increase of its hydrophilicity76 (see Table 4). Therefore, the absorptive transport enhancements are more likely attributable to a substantial decrease of their efflux owing to a lower affinity



crease of its hydrophilicity. This is indeed reflected by the substantial much larger decrease of HPLC mobility and logP coefficient observed for nelfinavir (which of the three PIs investigated is the most lipophilic) than for saquinavir and indinavir76 (Table 4).

CONCLUSION

Figure 3. Caco-2 cell monolayer apparent permeation coefficients (Papp) in the absorptive (AP to BL; gray bars) and secretion (BL to AP; dark gray bars) direction of indinavir, saquinavir, nelfinavir, and of their mannose conjugates displayed in Scheme 1, and of their glucose conjugates (data from ref 30), structures in Scheme 2. Table 4. HPLC Mobility and LogP Data of PIs and Prodrugs compounda

HPLC mobilitya (in min)

log Pb

Saquinavir

5.6

Saq-C(O)C4Man Indinavir

5.0 4.9

4.727 (4.73)c 4.218 3.681 (3.68)c 3.216 2.062 5.842 (5.84)c 4.490 3.659

Ind(8)-C(O)C4Man Ind-[C(OC4Man]2 Nelfinavir

3.5 3.3 12.5

Nelf(1)-C(O)C4Man Nelf-[C(OC4Man]2

5.2 4.8

a Determined on a HP1100 apparatus with a Lichrospher 100 RP-18packed column. The isocratic mobile phase consisted of a pH 6 buffer/ CH3CN mixture (41:59 for saquinavir and its prodrug; 59:41 for indinavir and its two prodrugs; 45:55 for nelfinavir and its two prodrugs (for more details, see experimental section). b Calculated using Chem 3D Ultra 8.0, CambridgeSoft, Cambridge, MA. c Data taken from ref 76 and calculated using CQSAR program.

of the efflux carriers for these conjugates and/or to an active transport mechanism involving a mannose carrier system located at the brush border side of the Caco-2 cell monolayer, if present. Such an absorptive transport improvement was already detected for valine conjugates of indinavir (30). Concerning the mannose-nelfinavir prodrugs, the conjugation of D-mannose to nelfinavir, Nelf(1)-C(O)C4Man and Nelf[C(OC4Man]2, was highly detrimental to its permeation across the Caco-2 cell monolayers. No detectable transport, at least above the detection limit for nelfinavir derivatives (0.1 µM), was observed whatever the AP to BL or BL to AP directional transport (Figure 2F-H). A similar behavior was also found for Nelf(1)-C(O)C4Tyr (30) (Table 3, Scheme 2), a tyrosine analogue prodrug of Nelf(1)-C(O)C4Man. However, it is in sharp contrast with a D-glucose analogue prodrug, i.e., Nelf(18)-C(O)NC4Glc (Table 3, Figure 3, Scheme 2), for which transport in both direction was observed. These results indicate that the mannose-nelfinavir conjugates are not recognized and actively transported by a mannose carrier system, if present on the Caco-2 cells. The absence of any secretory transport supports further that conjugation of D-mannose to nelfinavir reduces drastically its passive permeation, as expected from an in-

Mannose-derived conjugates were prepared in two steps, in good yields, by condensing an acid derivative of the appropriate protected mannose with the PI, followed by deprotection of the sugar protecting group. With respect to hydrolysis, these PI prodrugs are chemically stable with half-life times in the 50-60 h range that are further compatible with an in vivo utilization aimed at improving the absorption/penetration or accumulation of the prodrug in specific cells/tissues and liberation of the active free drug inside HIV-infected cells. These stabilities correlate closely with the low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of mannose to the PIs was performed through the peptidomimetic PI’s hydroxyl. Ind(8)-C(O)C4Man, wherein the coupling of the mannosylated residue was performed onto the non-peptidomimetic PI hydroxyl, displayed a quite high antiviral activity and stability, indicating that this prodrug is also to some extent an antiviral drug by itself. Mannose conjugation to the PIs was further found to improve the absorptive transepithelial transport of saquinavir and indinavir, but not of nelfinavir, across Caco-2 cell monolayers used as a model of the intestinal barrier. These data indicate also that the conjugation of mannose to saquinavir and indinavir could constitute a mean to improve their intestinal absorption. It is noteworthy that mannose conjugation improved the absorptive transepithelial transport of saquinavir and indinavir by contrast to glucose conjugation which had the opposite effect. The mannose-linked prodrugs of saquinavir and indinavir display therefore a most promising therapeutic potential provided that bioavailability, penetration into the HIV infected macrophages and HIV-reservoirs of these PIs are improved. Further work aimed at a better understanding of the impact of Dmannose on the absorptive transepithelial transport of the PIs and of the transport mechanism of the mannosylated PI prodrugs, and at evaluating their uptake by macrophages is under progress and will be reported in due course.

ACKNOWLEDGMENT This work was supported by research funds provided by the Fondation de la Recherche Me´dicale (Sidaction), the Agence Nationale de la Recherche sur le SIDA (ANRS) and the Centre National de la Recherche Scientifique (CNRS) and by a Conseil Re´gional de la Martinique grant (D.R.).

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Synthesis and in Vitro Biological Evaluation of Mannose-Containing

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