Design, synthesis, and biological evaluation of estrone-derived hedgehog signaling inhibitors

Abstract

The design, synthesis and biological evaluation of new analogs of the naturally occurring compound cyclopamine, a Hedgehog signaling inhibitor, are described. Stucture-activity relationship studies lead to an evolving model for the pharmacophore of this medically promising compound class of anti-cancer chemotherapeutic agents.

1. Introduction

More than forty years ago, Binns, Keeler and coworkers established that the alkaloid cyclopamine1 (Figure 1a) was responsible for the birth defects observed in calves from livestock that were fed diets rich in the corn lily, Veratrumcalifornicum (Figure 1b).^i,ii The observed phenotype included anophthalmia, cyclopia, and severe craniofacial effects (Figure 1c).

Figure 1.

a) Structure of Cyclopamine1; b) Veratrumcalifornicum^x; c) cyclopia;^xi and d) A simplified schematic of the SHH signaling showing the effect of 1 on SMO.

It was later established that cyclopamine1 acts by inhibiting the Sonic Hedgehog (SHH) cellular signaling pathway, which is critical for tissue growth and differentiation, thus playing a pivotal role in embryogenesis.^iii,iv Activation of the SHH-signal transduction pathway is initiated by the binding of the SHH ligand to the cellular membrane receptor Patched (PTCH1), which relieves the PTCH1-mediated inhibition of the transmembrane protein Smoothened (SMO) (Figure 1d).^v,vi Activated SMO transduces the signal to the nucleus to regulate gene expression via Gli transcription factors. Beachy and coworkers have established that 1 disrupts this pathway by inhibition of SMO.^vii

SHH signaling was first linked to cancer with the identification of mutations in the PTCH1 gene in Gorlin syndrome patients.^viii It was subsequently shown that this pathway is also active in the majority of sporadic basal cell carcinomas.^ix In addition, activation of the SHH pathway has been linked to brain tumors, including medulloblastomas and gliomas,^xii melanoma,^xiii lung adenocarcinoma,^xiv as well as prostate,^xv small cell lung,^xvi and pancreatic cancer.^xviiTreatment of cancer cells with cyclopamine1 induces a decrease in proliferation, an increase of apoptosis and/or a decrease of metastasis.^{10, xviii} The teratogenicity associated with cyclopamine has not hampered interest in this natural product as an important lead structure in the development of cancer chemotherapeutic agents that act via inhibition of SHH signaling.^xix

In spite of the attractive pharmacological profile against a number of cancer xenografts, in vivo evaluation of cyclopamine has been hampered by its poor aqueous solubility (ca. 5 mg/ml) and acid lability. Under acidic conditions, cyclopamine1 readily converts to veratramine2, via cleavage of the spirotetrahydrofuran ring, followed by aromatization of the D ring.^xx Unlike cyclopamine, 2 does not act as an SHH antagonist, and causes hemolysis by targeting other receptors.^xxi

Two strategies have been reported to address the issues of water solubility and acid lability of 1: 1) the covalent modification of 1 to produce structurally related and metabolically stable lead structures, i.e., IPI-926 3,^xxii in which the D ring of 1 is expanded to a seven-membered ring, as pioneered by Tremblayand coworkers at Infinity Pharmaceuticals; and 2) thescreening of libraries of diverse chemical structures in the hope of discovering drug-like structures that will interfere with SHH signaling.^xxiii The most noteworthy success to date using this approach is GDC-0449 4 (Figure 3), a compound that is currently in Phase II clinical trials.^xxiv The first approach, however, relies on the availability of the natural product 1, which is expensive, and recent results indicate an acquired resistance to inhibition of SHH signaling in a MB patient treated with the GDC-0449, providing the impetus for the development of new SHH signaling antagonists.^xxv

Figure 3.

Structures of Hedgehog Signaling Inhibitors IPI-926 (Infinity Pharmaceuticals) 3 and GDC-0449 (Curis/Genentech) 4

Consequently, there is an urgent need to identify readily available potent inhibitors of SHH as lead structures for the development of new cancer chemotherapies. We report herein the design and synthesis of cyclopamine-like structures derived from readily available steroidal precursors that function as potent cyclopaminemimetics. Outlined herein are the results of our structure-activity relationship studies on this novel compound class.

2. Results and Discussion

We have opted to explore a third approach to the identification of novel SHH signaling inhibitors, which is not dependent on the availability of 1, and yet generates new lead compounds that closely resemble 1 in both structure and function. The difference in teratogenicity between cyclopamine1 and the close structural analog tomatidine5 (Figure 4; non-teratogenic) has been attributed to the difference in the orientation of the nitrogen atom relative to the steroid plane in 1 and 5. The C-nor-D-homo framework of 1 can thus be viewed as a scaffold that orients the E/F hetero-bicyclic moiety orthogonal to the steroidal ring system, with the F-ring nitrogen atom on the α-face of the steroid plane.^xxvi In contrast, the tetrahydrofuran ring of 5 lies in the steroid plane and the nitrogen atom is on the β-face of the steroid plane.

Figure 4.

Structures and energy-minimized structures of cyclopamine1, tomatidine5, estrone-derived analog 6, and 7, the C-17 epimer of 6.²⁷

We reasoned that the C-nor-D-homo steroidal ring system of 1functions as a scaffold for the orientation of the heterobicyclic framework of the EF rings relative to the C-3β oxygen functionality in 1. Replacement of the C-nor-D-homo steroidal system with the androstane ring system and further stereochemical simplification via aromatization of rings A and F leads to the novel estrone-derived analog 6.²⁷

The energy-minimized structures in Figure 4 suggest an important role for the C-17 stereochemistry common to both 1 and 3, which, unlike 5 (and 7, the C-17 epi analog of 6), share the orientation of the C-17 oxygen substituent on the β-face of the steroid plane. In contrast, the C-17 oxygen atom of 7, the C-17 epimer of 6, is oriented on the α-face of the steroid plane, which leads to the orientation of the F-ring nitrogen atom of 7 on the β-face of the steroid plane, the same orientation that is found in tomatidine5, a naturally occurring compound which displays no activity as a Hedgehog signaling inhibitor.

2. a. Synthesis and Biological Evaluation of Estrone-Derived Analogs 6, 7, and 8 of Cyclopamine 1

To test the hypothesis that the three-point recognition of the C-3 oxygen, C-17 oxygen and C-21 nitrogen heteroatoms as oriented in 1 is required for recognition at SMO, we have synthesized both 6^xxvii and 7.^xxviiiAs previously described, both 6 and 7 are potent inhibitors of SHH signaling as evaluated by inhibition of ligand-induced SHH signaling activity in a luciferase-based assay and by inhibition of SHH-induced proliferation of mouse granule neuron precursors, with activities comparable or superior to that of cyclopamine1 at concentrations as low as 5 µM.

These results, the comparable potency of 6 and the C-17 epimer7, challenged our hypothesis that the three-point recognition of the C-3 oxygen and each of the other E and F ring heteroatom functionalities in 1 (and 6)is required for recognition at SMO, the cellular target of cyclopamine, since structures with either orientation at C-17, i.e., both 6 and 7, are potent inhibitors of SHH signaling. The relative orientations of the tetrahydrofuran oxygen and pyridine nitrogen relative to the steroid plane do not appear to be important features for recognition of these cyclopamine analogs at SMO, suggesting that the C-3 oxygen functionality may not be required for recognition at SMO.

To establish the role, if any, of the C-3 oxygen functionality that is present in 6 and 7 on the biological activity of these estrone-derived analogs of cyclopamine1, we prepared the C-3 deoxy compound 8 (Figure 5) as previously described.²⁸Biological evaluation of 8 using the same GLI-luciferase assay^xxix described for 6 and 7 revealed that 8 is apotent inhibitor of SHH signaling. In this assay, the C-3 deoxy analog 8 led to a strong inhibition of SHH signaling activity (80% inhibition at 5 µM; compared to 70% inhibition in the same assay with 6).We have also reported that the C-3 deoxy analog 8 is ca. 2 × more potent than cyclopamine1 at reducing DAOY medulloblastoma cell viability, an important measure of SHH inhibitory activity, and a significant illustration of the potential of these structures for the development of brain cancer chemotherapeutics.²⁸

Figure 5.

Structures of estrone-derived cyclopamine analog 6, the C-17 epi analog 7, and the C-3 deoxy analog 8

2. b. Synthesis and Biological Evaluation of Truncated Analogs of Cyclopamine 1

These results necessitate a revision of the binding model that we originally advanced (Figure 4).^{27, 28} The potent activity of 8 suggests that the C-3 hydroxyl common to both 6 and 7 is not required for biological activity, and brings into question the importance of the intact steroidal framework. To examine the effect of truncating the steroid, we have examined the deletion of portions of the tetracyclic steroidal ring system common to 6, 7, and 8.

Toward that end, we have prepared 13 (Scheme 1), an analog lacking the AB ring system present in 6, 7, and 8. Using the same annelation strategy that was employed for the syntheses of the previously described analogs, addition of the conjugate base of 10 to the known thioketal9^xxx led to the formation of carbinol11, which on Buchwald-Hartwig cyclization generated 12, containing the dihydrofuropyridine that constitutes the EF ring system of cyclopamine1. Dithioketaldeprotection of 12 gave the desired truncated analog 13 in good yield.

Scheme 1.

Syntheses of des-AB Analog 13

Biological evaluation of 13 using the same SHH-Light2 cells luciferase-based assay²⁹ that was used to evaluate the biological activity of 6, 7, and 8 reveals that the tetracyclic analog 20, lacking the steroidal A and B rings contained in all of the previously described analogs, has no effect on SHH signaling, and suggests that the AB ring system is important for SHH signaling inhibitory activity. This finding prompted us to examine the preparation of analogs that would more closely resemble the structures of the previously prepared estrone-based systems, i.e., containing the aromatic A ring that is present in 6, 7, and 8. The synthesis of des-B (lacking the steroidal B ring common to 6, 7, and 8) analogs 15 and 16 is outlined in Scheme 2.

Scheme 2.

Syntheses of des-B Analogs 15 and 16

Reaction of the dienoltriflate14,derived from 13, via Suzuki coupling of 14 with both phenyl boronic acid and p-hydroxyphenylboronic acid, respectively, provided 15 and 16, both of which lack the B-ring present in cyclopamine. Based on the observation that the C-3 deoxy analog 8 (Figure 5) was more potent than the C-3 hydroxy compound 6 (and 7),²⁸ we were surprised to find that 16 (R=OH) is more potent than 15 (lacking the C-3 hydroxy group) in the previously described luciferase-based assay for Hedgehog signaling activity, as shown in Figure 6.

Figure 6.

Luciferase Based Assay for SHH Activity: Treatment of SHH-Light2 cells with recombinant SHH (200 ng) resulted in the strong induction of reporter activity, which was blocked by co-treatment with either cyclopamine1 or with 15 or 16 at 5 µM, both P<0.001 [SHH vs. SHH + 1; SHH vs. SHH + 15/16].

Further investigation with 16 revealed that it is a potent SHH signaling inhibitory compound, as demonstrated in the GNP proliferation assay as shown in Figure 7, where it is ca. 3× more potent than cyclopamine1. We have also established that 16 is ca. equipotent with cyclopamine1 in the DAOY medulloblastoma cell viability assay, as illustrated in Figure 8.

Figure 7.

Estrone-Derived Analog 16 Inhibits SHH-Induced Proliferation of Granule Neuron Precursors (GNPs)

Figure 8.

Analog 16 reduces DAOY medulloblastoma cell viability. DAOY human medulloblastoma cells were treated with either carrier DMSO (Control), cyclopamine1 (10µM) or 16 (10µM) for 3 days. The histogram measures cell viability assessed by the MTT assay (absorbance at 570nm) (asterisk indicates p<0.05). Similar results were obtained with U87GBM cells (not shown).

3. Conclusions

We have demonstrated that it is possible to replace the C-nor-D-homo ring system of cyclopamine1 with an estrone-derived steroidal ring system and to prepare a cyclopamine analog 6 that is a potent SHH signaling inhibitor as measured in both the luciferase and GNP (granule neuron precursor) assays.²⁷

Evaluation of the SAR of this lead compound by examination of the C-17 epi compound 7 and the C-3 deoxy analog 8 established that the two-point binding model (using the C-3β hydroxyl and the heterobicyclic EF ring system of 1 (and 6) at the cellular receptor SMO is not sufficient to explain the surprising level of potency observed for 7 and 8.²⁸

This important finding led us to examine the synthesis and biological evaluation of truncated structures, such as 13, lacking both the A and B rings common to the previously described structures. We have found such a structural modification too extreme to retain SHH inhibitory activity, but we report that the addition of the aromatic A ring to 13 leads to potent SHH signaling inhibitors.

The introduction of the aromatic A ring, that is present in 6, 7, and 18, leads to novel des-B structures 15 and 16, i.e., lacking the steroidal B ring. Biological evaluation of 15 and 16 reveals that, in contrast to 6 (C-3 hydroxy)and 8 (C-3 deoxy), in which removal of the C-3 hydroxyl leads to more potent inhibitory activity, the C-3 hydroxylated analog 16 is decidedly more potent than the C-3 deoxy compound 15. The basis for this difference is currently under investigation in our laboratories. Biological evaluation of 16 establishes that it is more potent than cyclopamine1 in the inhibition of SHH-induced proliferation of GNPs (Figure 7) and ca. equipotent with 1 in the DAOY medulloblastoma cell viability assay (Figure 8).

These findings suggest that partial structures of estrone-based analogs of 1 are sufficient to generate potent SHH signaling inhibitors. Further work on the development of more potent compounds is currently underway in our laboratory and our results will be reported in due course.

4. Experimental section

General Methods

Solvents used for extraction and purification were HPLC grade from Fisher. Unless otherwise indicated, all reactions were run under an inert atmosphere of Argon. Anhydrous tetrahydrofuran, ethyl ether and toluene were obtained via passage through an activated alumina column.^xxxi Commercial reagents were used as received. Deuterated solvents were obtained from Cambridge Isotope labs. Merck pre-coated silica gel plates (250µm, 60 F254) were used for analytical TLC. Spots were visualized using 254 nm ultraviolet light, with either anisaldehyde or potassium permanganate stains as visualizing agents. Chromatographic purifications were performed on Sorbent Technologies silica gel (particle size 32–63 microns). ¹H and ¹³C NMR spectra were recorded at 500 MHz and 125 MHz, respectively, in CDCl₃ on a Bruker AM-500 or DRX-500 spectrometer. Chemical shifts are reported relative to internal chloroform (δ 7.26 for ¹H, δ 77.0 for ¹³C). Infrared spectra were recorded on a NaCl plate using a Perkin-Elmer 1600 series Fourier transform spectrometer. High resolution mass spectra were obtained at the University of Pennsylvania Mass Spectrometry Service Center on an Autospec high resolution double-focusing electrospray ionization/chemical ionization spectrometer with either DEC 11/73 or OPUS software data system. Melting points were obtained on a Thomas Hoover capillary melting point apparatus and are uncorrected.

6. 1.(1'R,7a'S)-1'-((3-bromopyridin-2-yl)methyl)-7a'-methyl-1',2',3',6',7',7a'-hexahydrospiro[[1,3]dithiolane-2,5'-inden]-1'-ol (11)

To a solution of diisopropylamine (2.6 mL, 18.6mmol) in dry Et₂O (6 mL) stirred at 0 °C under argon was added dropwise a solution of 2.5 M n-BuLi in hexanes (7.4 mL, 18.6mmol). The solution was stirred at 0 °C for 30 min. The flask was cooled to − 20 °C and a solution of 2-methyl-3-bromopyridine (3.19 g, 18.6mmol) in Et₂O (9 mL) was added dropwise. The resulting red mixture was stirred at −20 °C, under argon, for 2 h. A solution of thiolane9 (1.78 g, 7.42 mmol) in THF (11 mL) was added dropwise and kept stirring at −20 °C for 1 h. The reaction flask was allowed to warm up to room temperature and was quenched slowly with H₂O (30 mL). The mixture was extracted with EtOAc (3× 75 mL), washed with saturated NH₄Cl (30 mL), saturated NaHCO₃ (30 mL), brine (30 mL) and dried with Na₂SO₄. The solvent was removed under reduced pressure and the crude product was purified by silica gel chromatography (10% ethyl acetate in hexanes) to yield11 as an off-white solid (2.66 g, 87%, mp71–73°C.). [α]^21.2D = −91.9 (c = 1.0, CHCl₃). ¹H NMR (CDCl₃): δ = 8.41 (dd, J = 1.5, 4.5 Hz, 1H), 7.88 (dd, J = 1.5, 8.0 Hz, 1H), 7.06 (dd, J = 4.5, 8.0 Hz, 1H), 6.31 (s, 1H), 5.58 (s, 1H), 3.43–3.35 (m, 3H), 3.25–3.19 (m, 1H), 3.11 (d, J = 15.5 Hz, 1H), 2.98 (d, J = 15.5 Hz, 1H), 2.50–2.44 (m, 1H), 2.34 (m, 1H), 2.26–2.17 (m, 2H), 1.89–1.82 (m, 1H), 1.77 (m, 1H), 1.61–1.58 (m, 1H), 1.48 (m, 1H), 1.18 (s, 3H). ¹³C NMR (CDCl₃): δ = 159.3, 147.4, 146.6, 140.8, 124.4, 122.7, 122.6, 82.7, 66.0, 47.1, 40.4, 40.2, 39.7, 38.9, 33.5, 29.6, 26.0, 19.8. FTIR (thin film) 3362, 2922, 1428, 1066, 1033 cm⁻¹.HRMS (ES) Calcd.for C₁₈H₂₂BrNOS₂: 411.0326 (M⁺), found 412.0388 (MH⁺).

6.2. (1'R,7a'S)-7a'-methyl-2',3',7',7a'-tetrahydro-3Hspiro[furo[3,2-b]pyridine-2,1'-inden]-5'(6'H)-one (13)

A resealableSchlenk tube was charged with alcohol 11 (90 mg, 0.2mmol), Pd(OAc)₂ (10 mg, 0.04 mmol), BINAP (27 mg, 0.04 mmol) and Cs₂CO₃ (107 mg, 0.3mmol). Dry toluene (3 mL) was added and the tube was capped under argon and the resulting mixture was allowed to stir at 80 °C for 3 h. The mixture was allowed to cool to room temperature, filtered through Celite, concentrated under reduced pressure and purified by silica gel chromatography (15% ethyl acetate in hexanes) to yield 12as a white solid (43 mg, 60%): mp 134–136 °C. [α]^19.2D = −29.7 (c = 1.0, CHCl₃). ¹H NMR (CDCl₃): δ = 8.02 (dd, J = 2.0, 4.0 Hz, 1H), 6.98 (m, 2H), 5.63 (s, 1H), 3.42–3.33 (m, 4H), 3.22–3.16 (m, 1H), 2.90 (d, J = 17.0 Hz, 1H), 2.60–2.54 (m, 1H), 2.45–2.39 (m, 1H), 2.31–2.15 (m, 3H), 1.95–1.91 (m, 1H), 1.75 (td, J = 3.5, 13.5 Hz, 1H), 1.33 (dt, J = 3.5, 13.5 Hz, 1H), 1.22 (s, 3H). ¹³C NMR (CDCl₃): δ = 153.5, 150.8, 144.0, 141.4, 125.8, 122.2, 114.9, 96.1, 65.3, 46.4, 40.5, 40.0, 39.7, 38.3, 34.7, 29.2, 24.9, 19.8. FTIR (thin film) 2923, 1429, 1001 cm⁻¹.HRMS (ES) Calcd.for C₁₈H₂₁NOS₂: 331.1064 (M⁺), found 332.1129 (MH⁺).

To a solution of thioketal12(35 mg, 0.11 mmol) in MeOH (1.4 mL), H₂O (0.2 mL) and CH₂Cl₂ (0.7 mL) was added THF (0.1 mL) followed by bis(trifluoroacetoxy)iodobenzene (68 mg, 0.16 mmol) at room temperature. After 10 min, the solution was poured into a saturated NaHCO₃ solution (5 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (50% ethyl acetate in hexanes) yielded 13 as a white solid (23 mg, 85%): mp 133–135 °C. [α]^19.6D = − 56.4 (c = 1.0, CHCl₃). ¹H NMR (CDCl₃): δ = 8.04 (s, 1H), 7.02 (m, 2H), 5.88 (s, 1H), 3.41 (d, J = 17.0 Hz, 1H), 2.98 (d, J = 17.0 Hz, 1H), 2.82 (m, 1H), 2.53 (m, 3H), 2.37 (dd, J = 5.0, 13.0 Hz, 1H), 2.05 (m, 2H), 1.63 (m, 1H), 1.40 (s, 3H). ¹³C NMR (CDCl₃): δ = 198.1, 172.1, 153.2, 150.0, 141.9, 124.3, 122.6, 115.3, 95.7, 47.9, 39.1, 34.7, 33.0, 28.9, 26.3, 18.7. FTIR (thin film) 3428, 2930, 1666, 1430, 1258, 1004 cm⁻¹.HRMS (ES) Calcd.for C₁₈H₁₇NO₂: 255.1259 (M⁺), found 256.1332 (MH⁺).

6. 3. (1'R,7a'S)-7a'-methyl-2',6',7',7a'-tetrahydro-3Hspiro[furo[3,2-b]pyridine-2,1'-inden]-5'-yl trifluoromethanesulfonate (14)

A solution of 13 (64 mg, 0.25 mmol) in dry CH₂Cl₂ (1.2 mL) was cooled to −20°C and triethylamine (42 µL, 0.30mmol) was added dropwise to the stirring solution. After a period of 5 min, trifluoromethanesulfonic anhydride (50 µL, 0.30mmol) was added dropwise and the solution was allowed to warm up to 0°C over a 1 h period. The reaction mixture was diluted with CH₂Cl₂ (10 mL) and quenched with brine (5 mL). The layers were separated and the aqueous layer was extracted with CH₂Cl₂ (10 mL). The combined organic layers were dried with Na₂SO₄ and the solvent was removed under reduced pressure. Purification by silica gel chromatography (33% ethyl acetate in hexanes) gave 14as an orange oil (65mg, 67%). ¹H NMR (CDCl₃): δ = 8.03 (m, 1H), 7.02 (m, 2H), 6.23 (J = 3.0 Hz, 1 H), 5.64 (s, 1H), 3.51 (d, J = 16.5 Hz, 1 H), 3.08 (d, J = 17.0 Hz, 1H), 2.96 (d, J = 17.0 Hz, 1H), 2.64 (dd, J = 3.0, 17.0 Hz, 2H), 2.43 (dd, J = 5.5, 18.0 Hz, 1 H), 1.89, (dt, J = 5.5, 12.0 Hz, 1 H), 1.54 (dd, J = 5.5, 12.0 Hz, 1 H), 1.19 (s, 3H). ¹³C NMR (CDCl₃): δ = 153.1, 150.7, 150.6, 141.9, 141.7, 123.7, 122.5, 115.4, 115.1, 97.1, 47.1, 44.6, 39.2, 27.6, 26.1, 17.6. FTIR (thin film) 2935, 1423, 1212 cm⁻¹.HRMS (ES) Calcd.for C₁₇H₁₆F₃NO₄S: 387.0752 (M⁺), found 388.0821(MH⁺).

6. 4. (1'R,7a'S)-7a'-methyl-5'-phenyl-2',6',7',7a'-tetrahydro-3H-spiro[furo[3,2-b]pyridine-2,1'-indene] (15)

To a solution of triflate14(16 mg, 0.04 mmol) in a 600 µL of a 1:1 THF:PhCH₃ mixture was added phenylboronic acid (5 mg, 0.04 mmol), Pd(PPh₃)₄ (2.4 mg, 0.002 mmol) followed by a 0.5N solution of Na₂CO₃ (80 µL, 0.04 mmol). The mixture was heated to reflux for 3 h. The reaction flask was allowed to cool to room temperature and then diluted with H₂O (5 mL). The mixture was partitioned with CH₂Cl₂ (3 × 10 mL) and the combined organic layers were washed with brine (10 mL). The organic layer was dried with Na₂SO₄and the solvent was removed under reduced pressure. Purification by silica gel chromatography (33% ethyl acetate in hexanes) yielded 15as a yellow oil (12 mg, 92%). [α]^23.6D = −111.2 (c = 0.36, CHCl₃). ¹H NMR (CDCl₃): δ = 8.03 (t, J = 2.8 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.26 (m, 1H), 7.02 (m, 2H), 6.63 (s, 1H), 5.52 (s, 1H), 3.61 (d, J = 17.0 Hz, 1H), 3.11 (d, J = 16.5 Hz, 1H), 2.98 (d, J = 17.0 Hz, 1H), 2.66 (m, 2H), 2.61 (dd, J = 3.0, 16.5 Hz, 1H), 1.88 (m, 1H), 1.63 (m, 1H), 1.22 (s, 3H). ¹³C NMR (CDCl₃): δ = 153.4, 151.4, 146.0, 141.3, 140.8, 138.5, 128.4, 127.5, 125.2, 122.3, 120.0, 119.4, 115.2, 98.2, 47.0, 44.1, 39.2, 28.4, 25.6, 17.9. FTIR (thin film) 2928, 1429, 993cm⁻¹. HRMS (ES) Calcd.for C₂₂H₂₁NO: 315.1623 (M⁺), found 316.1719 (MH⁺).

6. 5. 4-((1'R,7a'S)-7a'-methyl-2',6',7',7a'-tetrahydro-3Hspiro[furo[3,2-b]pyridine-2,1'-inden]-5'-yl)phenol (16)

To a solution of triflate14 (19 mg, 0.05 mmol) in a 740 µL of a 1:1 THF:PhCH₃ mixture was added phenylboronic acid (7 mg, 0.05 mmol), Pd(PPh₃)₄ (3 mg, 0.003mmol) followed by a 0.5N solution of Na₂CO₃ (100 µL, 0.05 mmol). The mixture was heated to reflux for 3 h. The reaction flask was allowed to cool to room temperature and then diluted with H₂O (5 mL). The mixture was partitioned with CH₂Cl₂ (3 × 10 mL) and the combined organic layers were washed with brine (10 mL), dried with Na₂SO₄ and the solvent was removed under reduced pressure. Purification by silica gel chromatography (33% ethyl acetate in hexanes) yielded 16as a yellow oil (9 mg, 56%). [α]^23.9D = −35.2 (c = 0.64, CHCl₃). ¹H NMR (CDCl₃): δ = 8.04 (t, J = 3.0 Hz, 1H), 7.30 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 3.0 Hz, 2H), 6.77 (d, J = 8.5 Hz, 2H), 6.49 (s, 1H), 5.43 (s, 1H), 3.57 (d, J = 17.0 Hz, 1H), 3.17 (d, J = 16.5 Hz, 1H), 3.00 (d, J = 17.0 Hz, 1H), 2.57 (dd, J = 3.0, 16.5 Hz, 1H), 2.52 (m, 2H), 1.71 (m, 1H), 1.45 (dt, J = 2.5, 12 Hz, 1H), 1.16 (s, 3H). ¹³C NMR (CDCl₃): δ = 156.5, 154,1, 151.2, 145.7, 140.4, 137.6, 132.3, 126.3, 122.7, 118.3, 117.8, 115.7, 115.6, 98.6, 47.5, 44.0, 39.2, 28.2, 25.3, 17.9. FTIR (thin film) 2926, 1513, 1433, 1278, 992cm⁻¹. HRMS (ES) Calcd.for C₂₂H₂₁NO₂: 331.1572 (M⁺), found 332.1661 (MH⁺).

6.6 MTT cell viability assay

DAOY medulloblastoma or U87 glioma cells were plated in 96 well-plates at 3000 cells/well in DMEM/0.5% serum media. Cyclopamine or compounds to test were added at a concentration of 10 µM to the cells. As control, cells were treated with media containing DMSO only. Seventy-two hours later, cell viability was assayed using the MTT cell survival kit (Chemicon; cat#CT01) following the manufacturer protocol. For each assay, the measurement was done in triplicate. Each compound was tested at least in 3 independent experiments. A T-test was applied for statistical analysis.

6.7. Granule neuron progenitor proliferation assay

Granule neuron progenitors (GNPs) were purified from P5 mouse cerebella.^xxxii Cells were plated in 24-well plates with 800000cells/well and cultured in DMEM/F12 (Gibco, 11330), B27 (Gibco, 1X), N2 (Gibco, 1X), Glutamine (Cellgro, 2mM) and Penicillin (50units/ml)/ Streptomycin (50mg/ml) media. The day following plating, SHH (600ng/ml, R&D) and/or compounds to test were added to the cells. After 24h in culture with SHH and/or compounds, BrdU was added to the media at a final concentration of 12mg/ml for 5 hours. The cells were then rinsed with PBS and fixed on ice for 1 hour and washed in PBS/0.1% TritonX-100 (PBT) before HCl treatment was carried out. Cells were treated with HCl 2N for 30min at 37C, then with 0.1M of sodium borate, pH8.5 for 20min. at room temperature. and washed 5 times with PBT. Cells were incubated with a blocking solution (PBT with 10% goat serum) at room temperature for 1h and then incubated with an anti-BrdU antibody (Becton Dickinson, 1:400). The cells were then washed and incubated with a secondary antibody anti-mouse FITC (Vector Lab, 1:500) for 1hour at room temperature. The nuclei were counterstained with Hoechst (Sigma) and then mounted in Mowiol/ Dabco solution. BrdU-positive cells were counted with a fluorescence microscope using a 20X objective (Axioskop, Zeiss). At least 5 independent fields for each culture condition were counted. Statistical analysis was performed with the Student t test.

Figure 2.

Conversion of Cyclopamine1 to the D-ring aromatic compound veratramine2 under acidic conditions.