General comments
All extractions were carried out in a closed system consisting of a Soxhlet extractor equipped with a chilled condensor (-4°C). Benzene was determined to be an efficient extraction solvent and was easily recaptured and reused. The extraction apparatus was fitted into a large walk-in hood to ensure safe ventilation of any benzene vapor that might escape from the extraction unit. For the first flash chromatography separation, Fisher 70–230 mesh silica gel with a 60 Å pore size was used; the second chromatographic step employed J.T. Baker 40 μm silica gel flash chromatography packing with a 60 Å pore size. The progress of each separation was followed by TLC on 5 × 10 cm, 250 μm TLC plates (EMD Chemicals, Darmstadt, Germany) using EtOAc-isopropanol-NH4OH, 80:14:1 (rf of cyclopamine 0.24). After development, the spots were visualized by staining with phosphomolybdic acid.
HPLC-MS
Cyclopamine samples were analyzed via LC-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) on a quadrupole ion trap mass spectrometer (LCQ Classic, Thermo-Finnigan, San Jose, CA) coupled to a Hewlett-Packard 1100 HPLC system. A 1.2 × 100 mm XTerra MS C-18 3.5 μm C-18 reversed-phase LC column (Waters Corporation, Milford, MA) was utilized. Cyclopamine was eluted employing a 30 min gradient starting at 10% acetonitrile, 0.2% aqueous formic acid to 70% acetonitrile, 0.2% aqueous formic acid with a flow rate of 100 μL/min and a 1:10 post-column split. A blank was analyzed between samples to limit carryover. Some mass spectra were also obtained either on a Waters Micromass ZQ Mass detector equipped with a Waters 600 HPLC pump and photodiode array detector or on a Thermo-Finnegan TSQ Quantum equipped with an Agilent 1100 series HPLC system.
Nuclear magnetic resonance spectroscopy
Initial NMR spectra of cyclopamine were acquired on a 400 MHz spectrometer (Varian UNITYINOVA, Palo Alto, CA) equipped with a 5 mm indirect detect z-gradient probe in 700 μL deuteromethylene chloride (CD2Cl2). Partial assignments of the proton spectra were achieved by employing a DQCOSY experiment acquired in the phase-sensitive mode. The chemical shifts of unresolved multiplets were based on the chemical shifts of the cross-peaks. Partial assignments of the carbon resonances were made using gradient versions of multiplicity-edited HSQC, and gradient HMBC experiments were used for long-range correlations. Some proton assignments were made and stereochemical relationships were established by employing NOESY.
High-resolution NMR spectra were collected on a 700 MHz spectrometer (Avance II, Bruker Biospin, Inc., Billerica, MA) equipped with a triple-resonance, z-gradient cryogenic probe (5 mm TCI Cryoprobe, Bruker Biospin, Inc.). A 6.8 mg sample was dissolved in 600 μL CD2Cl2 and analyzed at 298°K. In addition to standard proton and carbon spectra, a number of two-dimensional experiments were conducted in order to fully assign the resonances. Homonuclear assignments were aided by gradient phase-sensitive, DQCOSY spectra and gradient phase-sensitive NOESY spectra. Heteronuclear correlations were determined by using gradient multiplicity-edited, sensitivity-enhanced HSQC correlation spectroscopy, and long-range correlations were determined using gradient HMBC. Multiplicity (CHn, n = 0–3) was confirmed through one-dimensional DEPT pulse sequences. Spectra were processed using the instrument software (TopSpin, Bruker Biospin, Inc.), and analysis was conducted using iNMR (Nucleomatica, Molfetta, Italy; http://www.inmr.net), a computer program designed to aid in resonance assignments.
Cyclopamine isolation
Veratrum californicum is an abundant, robust perennial herb 1.5 to 2.0 meters in height that is commonly found at elevations of 1500 to 4000 meters along open watercourses and in moist meadows of the northern Rocky and Pacific coastal mountains. Roots and rhizomes from this plant, wherein steroidal alkaloids are concentrated, were collected in Idaho's Clearwater National Forest during September 2005. They were thoroughly air dried, milled to a coarse powder (Triarco Industries, Green Pond, SC), and stored in sealed containers with desiccant at 0°C. For each 130 × 300 mm Soxhlet extractor (Southeastern Lab Apparatus, North Augusta, SC), 1.45 kg of powdered plant that had been wet with 800 mL of 7.5% aqueous NH4OH (3.4 mole) was placed in a 110 × 280 mm 100% cotton cloth bag. Each bag was extracted for 14 hrs with 7.5 L of refluxing benzene. After cooling to room temperature, the combined benzene solutions were chromatographed on 1.5 kg of silica gel in a 16 cm column and the solvent pulled through with vacuum. Evaporation of 100 mL aliquots from 10 runs indicated that an average of 140 g of alkaloids was put on the column. The column was freed of benzene, washed with 4 L of CH2Cl2-isopropanol (97:3), and then washed again with 10 L of CH2Cl2-isopropanol (93:7). Fractions highly enriched in cyclopamine were then eluted with CH2Cl2-isopropanol (75:25) in the next 10 L. After the solvents were removed at reduced pressure, an average of 50 g of semi-pure cyclopamine per extraction cycle was obtained. For the second chromatography step, 100 g of semi-pure cyclopamine dissolved in 500 mL of CH2Cl2 was absorbed on 300 g of silica gel, and the solvent was removed at reduced pressure. The resulting dry power was placed on top of 2.5 kg of dry silica gel in a 16 cm column, and the column was wet with 3 L of EtOAc. After washing the column with an additional 3 L of EtOAc, cyclopamine was eluted with EtOAc-isopropanol-NH4OH (85:14:1), with 1 L fractions being collected. Concentration of the fractions containing cyclopamine gave an average 23.3 g of a yellow-glass. After triturating with cold acetone (1.5 mL/g) and cooling at 0°C for at least 18 hrs, filtration yielded an average of 10.1 g of a cream solid. Two recrystallizations of 29.0 g from EtOH-H2O (10:1), 8 ml/g, gave 21.7 g of cyclopamine with a purity >99%. From the primary isolation and purification, 1.3 g per kilogram of dried root, or 55% of the available cyclopamine was recovered. Repurification of all of the acetone wash filtrates as well as the filtrates from the ethanol recrystallizations would substantially increase the percent recovery.
The 400 MHz NMR spectrum and the ESI mass spectrum of this isolated product were identical to those of reference cyclopamine (a kind gift from Dale R. Gardner, Poisonous Plants Research Lab, Logan, UT). Benzene recovered from the column could be used repeatedly for subsequent extractions before purification by redistillation was required. The methylene chloride was also reused after drying over calcium sulfate and fractional distillation.