1200 series system

HPLC spectra for all compounds were acquired using an Agilent 1200 series system with DAD detector. Chromatography was performed on a 2.1 mm × 150 mm Zorbax 300SB-C₁₈ 5 μm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0–1 min), 1–99% B (1–4 min), and 99% B (4–8 min). High-resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DRX-600 spectrometer (600 MHz ¹H, 150 MHz ¹³C) or a Varian Mercury spectrometer (400 MHz ¹H, 100 MHz ¹³C). Chemical shifts are reported in ppm (δ). Preparative HPLC was performed on Agilent Prep 1200 series with UV detector set to 254 nm. Samples were injected into a Phenomenex Luna 750 mm × 30 mm, 5 μm, C₁₈ column at room temperature. The flow rate was 40 mL/min. A linear gradient was used with 10% (or 50%) of MeOH (A) in H₂O (with 0.1% TFA) (B) to 100% of MeOH (A). HPLC was used to establish the purity of target compounds. All final compounds had >95% purity using the HPLC methods described above.

Analytical HPLC was carried out on an Agilent 1200 series system with UV detection at 214 nm. Methods used are summarized as follows, referenced by number in the text and in figures:
HPLC Method 1: Column - Phenomenex Aeris Widepore C4 column, 150 × 4.6 mm, 3.6 μm, 200 Åsilica; flow rate 0.8 mL/minute; Solvent System - A = water with 0.1% TFA, B = acetonitrile with 0.08% TFA; Gradient − 3 minute hold 1% B, 1–61% B gradient over 60 minutes, 3 minute hold 61% B, 10-minute post run 1% B; Flow Rate − 0.8 mL/minute
HPLC Method 1a: As in Method 1, but with a 1–61% B gradient over 30 minutes.
HPLC Method 2: Column - Phenomenex Luna C18(2 ) column, 100 × 4.6 mm, 3 μm, 100 Å silica; flow rate 1.0 mL/minute; Solvent System - A = water with 0.1% TFA, B = acetonitrile with 0.08% TFA; Gradient - 3 minute hold 1% B, 1–61% B gradient over 60 minutes, 3 minute hold 61% B, 10-minute post run 1% B
Integrals of HPLC peaks were calculated automatically with Agilent ChemStation software with subsequent manual inspection of the magnified baseline and modification of the automated calls - most commonly removal of erroneous peaks more consistent with background variation or splitting of a major peak to reflect tailing.