General experimental procedures
Positive or negative HR-FABMS were recorded on a Shimadzu LCMS-IT-TOF or a Joel SX-102 mass spectrometer. 1H and other NMR spectra were measured on a Varian Mercury 300 or 500 spectrometer. Samples were dissolved in CDCl3 or pyridine-d5 with TMS as an internal standard. Silica gel chromatography was carried out on a Biotage Horizon Flash chromatograph system with pre-packed Si gel column. The purity of BA derivatives was analyzed by using a Varian ProStar HPLC system with a PDA detector and Agilent Zorbax ODS or C-8 columns. The mobile phase was composed of solution A (5% acetonitrile in water with 0.045% trifluoroacetic acid) and solution B (water : methanol : acetonitrile = 5 : 10 : 85 with 0.045% trifluoroacetic acid). A linear gradient of 80% to 100% of solution B with a flow rate at 1 ml/min, or with flow rate at 4 ml/min was used to elute the compounds. The compounds were analyzed with the UV absorption displayed at 220 nm and recorded at a range from 200 to 250 nm. All the tested compounds have purity of 95% or above except for the inactive unmodified 1 (BA), which was purchased from Sigma-Aldrich with 90% purity.
Procedure for synthesizing type 1 compounds 2–5: A stirring solution of 3-O-Ac-BA (0.3 – 0.7 mmol) in dichloromethane (DCM, 2 ml) was added with oxalyl chloride (7 – 12 eq.). After 10 min of stirring, the organic solvent was removed under vacuum. The residue was dissolved in DCM and added with corresponding 1,ω-di-amino alkane (4 – 5 eq.) in DCM. The reaction was stirred overnight and then concentrated. The residue was washed with water and dissolved in ethanol. After filtration, the ethanolic solution was concentrated and the residue was chromatographed on Si-gel to yield the corresponding amine intermediate 2a–5a. To the corresponding amine intermediate 2a–5a in pyridine (anhydrous, 1 ml), acetic anhydride (0.5 ml) was added and stirred at room temperature overnight. The reaction mixture was concentrated and re-dissolved in DCM. After being washed with 1N HCl, water, and brine, the organic layer was concentrated and chromatographed on Si-gel to yield 2b–5b.
Procedure for saponification of ester at C-3 or/and C-28: Esters were hydrolyzed in a mixture of MeOH/THF/4N NaOH aq. (2:2:1). The intermediate 2b–5b was dissolved in organic solution (1 – 2 ml) and was added with aqueous NaOH solution (0.5 – 1 ml). After stirring overnight, the reaction mixture was neutralized with aqueous HCl (1 N). The resulted precipitate was washed with water and dried in vacuum to yield the final compound 2–5.
Procedure for synthesizing type 2 compound 6: The mixture of the above amine intermediate 3a (0.2 mmol) in DCM (2 ml) was added with malonic acid (10 eq.), EDC (2 eq.), and Et3N (10 eq.). After stirring at room temperature overnight, the reaction mixture was concentrated and re-dissolved in DCM, washed with water, brine, and was dried over Na2SO4. The organic layer was concentrated and chromatographed on Si-gel to yield the intermediate 6a. After the saponification procedure, as described above, 6 was obtained as a solid.
Procedure for synthesizing type 3 compounds 7–11: A stirring solution of 3-O-Ac-BA (0.1 – 0.6 mmol) in DCM (4 ml) was added with oxalyl chloride (10 eq.). After 10 min of stirring, the organic solvent was removed under vacuum. The residue was dissolved in DCM, then reacted with corresponding ω-amino alkanoate (1.2 – 1.5 eq.) in DCM and Et3N (6 eq.) overnight. The reaction mixture was diluted with DCM before washed with water, brine, and then dried over Na2SO4. After concentration in vacuum, the residue was chromatographed on Si-gel to yield the corresponding 7a–11a, which yielded the corresponding 7–11 after saponification.
Procedure for synthesizing type 4 compounds 12–16: To the solution of corresponding intermediates 7a–11a (0.2 mmol) in DCM (2 ml), was added glycine methyl ester hydrochloride (2.7 eq.), Et3N (7.5 eq), and EDC (3.0 eq.). After stirring overnight at room temperature, the reaction mixture was diluted with DCM, washed with water, brine, and was dried over Na2SO4. The organic layer was concentrated under vacuum and the residue was chromatographed on Si-gel. The pure intermediate was collected and subjected to saponification as described above to furnish the corresponding compounds 12–16.
Procedure for synthesis of type 4 compounds 18–21: The synthesis of these compounds was achieved by the same procedure as described above for 12–16, using intermediate 9 (for synthesis of 18–20) or 14 (for synthesis of 21) and the corresponding amino acid methyl ester hydrochloride.
HIV-1 and HIV-2 virus infection assay
Inhibition of HIV-1NL4-3, HIV-2 KR.X3 or HIV-2 KR.X3-YU2/V3 infection was measured through reduction in luciferase gene expression after a single round of virus infection of TZM-bl cells, as previously described (17). HIV-2 KR.X3 and HIV-2 KR.X3-YU2/V3 were generously provided by Dr. George Shaw, University of Alabama. For the anti-viral assays, 200 TCID50 of virus was used to infect TZM-bl cells in the presence of various concentrations of compounds. Two days after infection, the culture medium was removed from each well and 100 µl of Bright Glo reagent (Promega, Luis Obispo, CA) was added to the cells for measurement of luminescence using a Victor 3 luminometer. The 50% inhibitory concentration (IC50) was defined as the concentration that caused a 50% reduction of luciferase activity (Relative Light Units) compared to virus control wells.
A CytoTox-Glo™ cytotoxicity assay (Promega) was used to determine the cytotoxicity of the synthesized BA derivatives. TZM-bl cells were cultured in the presence of various concentrations of the compounds for 2 days. Percent of viable cells was determined by following the protocol provided by the manufacturer. The 50% cytotoxic concentration (TC50) was defined as the concentration that caused a 50% reduction of cell viability.