Patent Application: US-201414898955-A

Abstract:
the present invention concerns compounds that selectively inhibit the activity of the hedgehog pathway , their preparation and uses thereof . the compounds of the present invention are useful in treating hh - dependent tumors , such as medulloblastoma .

Description:
to discover natural compounds as inhibitors of the hh pathway ( targeting the smo receptor and / or the gli1 protein ), two distinct computational screening protocols were established , based on the availability of structural or molecular information . a ligand - based approach was followed to search for antagonists of the smo receptor , based on the chemical structure of potent smo antagonists described in the literature . moreover , a receptor - based approach was used to target gli1 , based on the availability of a crystallographic structure of gli1 - zf in complex with dna ( pdb id : 2gli ) ( pavletich and pabo 1993 ). a training set of 9 active smo antagonists , retrieved from the literature and from patents , was chosen to generate ligand - based pharmacophore models . since multiple alignment schemas are allowed for selected compounds in the 3d space , multiple pharmacophores were generated accordingly by means of the “ common feature pharmacophore generation ” protocol implemented in the molecular modeling suite discovery studio 2 . 5 from accelrys ( http :// accelrys . com /) ( barnum , greene et al . 1996 ). pharmacophores were then scored and ranked based on their ability to map the training set and the six top - ranking pharmacophores were selected . they belong to two different groups : type1 — three hydrogen bond acceptor ( hba ) and three hydrophobic ( hyd ) features are necessary to represent the interaction pattern of potent smo antagonists with the smo receptor ( fig1 a ); type2 — three hba , two hyd and a hydrogen bond donor ( hbd ) features are necessary to represent the interaction pattern of potent smo antagonists with the smo receptor ( fig1 b ). coordinates of pharmacophoric features and inter - feature distances are reported in tables 1 - 4 . it is to be specified that this training set has been already used for similar purposes ( manetti , faure et al . 2010 ). however , the use of multiple pharmacophores represents a unique feature of the present invention , since in the previous attempt a single pharmacophore was used for filtering chemical libraries in searching for smo antagonists ( manetti , faure et al . 2010 ). the use of multiple pharmacophores for representing ligand binding to smo better accounts for receptor flexibility , as well as enhances the research of chemically diverse candidate lead molecules with respect to the training set . moreover , a hbd feature was used for the first time to represent the ligand interaction to smo , as several known smo antagonists are endowed with at least one hbd group . the six pharmacophores described above were used as 3d query to filter the unique library of natural compounds , whose features are described in the example n . 10 . ligand conformational analysis was performed by the caesar algorithm . pharmacophore screening was performed with discovery studio 2 . 5 . the search 3d database and the “ ligand pharmacophore mapping ” protocols were subsequently used to filter the virtual library and to calculate the fitvalue for each ligand , as a measure of how well a ligand fits a pharmacophore . based on pharmacophore screening results , ligands were divided into three groups : 1 ) ligands that map all pharmacophores ; 2 ) ligands that map only type1 pharmacophores and 3 ) ligands that map only type2 pharmacophores . a consensus “ rank by rank ” procedure was applied to merge results obtained by filtering through each pharmacophore model . since the library included natural products with molecular dimension spanning from small to very large ( for example , molecular volume ranges from 465 . 9 to 2981 . 8 å 3 , polar surface area from 8 . 2 to 265 . 6 å ), with the aim of prioritizing small molecular compounds the ligand efficiency ( le ) was further calculated for each selected compound as the ratio between the fitvalue and the number of ligand heavy atoms ( le = fitvalue / no . heavy atoms ). 16 molecules endowed with the highest le values were deemed top priority and selected for in vitro studies . the alignment of one exemplified compound , glabrescione b , to a pharmacophore model is showed in fig2 . based on biological results from a luciferase assay where the gene reporter was gli1 and the pathway was activated by the sag molecule , a potent smo agonist , 2 molecules were classified as highly active hh inhibitors , showing more than 50 % inhibition at 5 μm ( glabrescione b and 2 , 4 , 5 , 3 ′, 4 ′- penta - ome chalcone ), 5 as moderately active , showing up to 30 % inhibition at 30 μm ( jaceidin , auriculasin , 3 , 4 - di - mdo - 2 ′, 4 ′, 5 ′- tri - ome dihydrochalcone , 2 , 3 , 4 , 6 , 3 ′, 4 ′- hexa - ome chalcone , martinoside ,) and 9 were not active ( barbinervic acid , kuwanol - e , myricetin , sorocein - a , sorocein - b , isosophoranon , veratrin , hesperidin , naringin ). notably , a highly comparable average molecular weight for highly active ( average mw = 404 . 45 ) and moderately active hh inhibitors ( average mw = 390 . 80 ) was observed , whereas a significantly higher molecular weight is showed by inactive compounds ( average mw = 503 . 51 ). since tested compounds were composed only by c , o and h atoms , molecular weight was considered in this case as proportional to the molecular dimension . therefore , the inventors envisioned that compounds having a high mw were not active probably because the ligand binding site on the smo receptor was not large enough to accommodate these substances , or they encounter steric hindrance within the smo binding site . reducing the steric accessibility of pharmacophore models may improve computational results . therefore , inactive compounds were used for the steric refinement of pharmacophores by means of the “ steric refinement with excluded volumes ” protocol of discovery studio 2 . 5 that places excluded volumes to pharmacophore models . based on the availability of a crystal structure of the zing - finger domain of gli1 - zf in complex with dna , a structure - based virtual screening protocol was established to screen the unique library of natural compounds , whose features are described in the example n . 10 . first , md simulations were performed to relax atoms coordinates in explicit solvent and to sample the conformational space . four different replicas of unrestrained md were simulated for 20 ns each , starting from slightly different initial coordinates . a representative structure was extracted from md trajectories at the convergence and further relaxed by means of energy minimization after removal of dna . subsequently , molecular docking was used to predict the theoretical binding mode and affinity of natural compounds towards the representative gli1 - zf structure refined by md . based on literature data , the binding site was centered on t374 that is within zf4 and zf5 ( sheng , et al . 2006 ). compounds of the unique library were docked to gli1 - zf by means of the goldscore function of the gold program ( version 5 . 0 . 1 ) ( verdonk , et al . 2003 ), which is generally recommended for binding sites particularly solvent - exposed or accounting for several h - bonding ligand - protein interactions , such as the zf4 . further , a rescoring procedure consisting on the calculation of the ligand delta energy of binding by means of the mm - gbsa methods was applied , with the aim of decreasing as much as possible the number of false positive identified by docking . after docking and rescoring , the le was calculated as the ratio between the delta energy of binding and the number of heavy atoms of each ligand . interestingly , glabrescione b that was already evaluated as smo antagonist was found within the top ranking positions of the structure - based virtual screening . for this reason , as well as to exploit the possibility to develop a multitarget inhibitor of the hh pathway , glabrescione b was deemed top priority for in vitro testing . the docking - based binding mode of glabrescione b to gli1 - zf is showed in fig3 . glabrescione b potency was evaluated by measuring its ability to inhibit hh pathway in a cellular context of hh signaling hyper - activation . this condition , leading to constitutive activation of gli transcription factors , can be triggered by overexpressing gli1 or treating hh - responsive cell lines with shh ligand or sag , a potent smo agonist or can occur in cells following mutations of key components of the pathway . the effects of glabrescione b as smo and gli1 antagonist have been examined by biochemical and in various cell - based assays . affinity of glabrescione b and its direct binding to smo receptor was quantified in a displacement assay . the assay is based on the use of the bodipy - cyclopamine ( bc ), a fluorescent derivative of cyclopamine , which interacts with smo at the level of its heptahelical bundle . to this end , hek293 cells were transfected with a vector expressing smo protein and then incubated with bc in the absence or presence of various concentrations of glabrescione b . this assay revealed that glabrescione b blocked bc binding to smo in a dose - dependent manner with an ic 50 of 1 μm ( fig5 ). kaad , used as positive control , abrogated bc binding to cell expressing smo . together , these results show that displacement assay is specific and demonstrate that glabrescione b binds to smo receptor at the level of the bodipy - cyclopamine binding site . to investigate the inhibitory properties of glabrescione b on hh signaling , the inventors examined its effects in nih 3t3 shh - light ii ( shh - lii ) cells stably incorporating an hh - responsive ( gli - re ) reporter , in which induction of the pathway occurs following treatment with the smo agonist sag . this in vitro test revealed that glabrescione b significantly reduced luciferase activity in cells treated with sag in a dose - dependent manner ( fig6 ) and suppressed the signaling . the ic 50 in this assay was 0 . 8 μm . efficacy of glabrescione b to directly target gli1 protein , the final effector of hh pathway , was also tested . hek293 cells transiently expressing gli1 and a gli - dependent luciferase reporter , showed that glabrescione b was capable of reducing gli1 - mediated transcription in a dose - dependent manner with an ic 50 of ≈ 15 μm ( fig7 ). gant61 , the only small - molecule described to inhibit gli1 function ( lauth et al ., pnas 104 : 8455 - 8460 ), was included as positive control . to analyze the antagonist properties of glabrescione b under more physiological conditions , the inventors used mef - ptch −/− cells , embryo fibroblasts derived from ptch −/− mouse , in which the activation of hh signaling is consequence of ptch1 deletion . since patched acts as upstream repressor of smo , inhibition of the hedgehog pathway from patched is prevented and the hedgehog pathway is constitutively activated in this cell lines , which turned out to be a reliable cellular model for studying the effect of hedgehog inhibitors . these cells showed strongly reduced mrna and protein levels of gli1 ( a readout of hh signaling ) when treated with glabrescione b at different concentrations ( fig8 a and b ). further , authors tested the inhibitory activity of glabrescione b on mef - sufu −/− cells , which represent another cellular model of activated hedgehog pathway . indeed , in this cell line aberrant hh pathway activation is due to genetic ablation of the downstream negative regulator sufu . treatment of mef - sufu −/− cells with glabrescione b led to significant reduction of the high expression levels of gli1 , as indicated by quantitative rt - pcr ( fig9 ). these results confirm that glabrescione b is a multitarget inhibitor of hh signaling , able to act both upstream by its direct binding to smo receptor and downstream of smo and sufu by suppressing gli1 transcription functions . a biological assay was performed in cerebellar neural progenitors cells obtained from 4 - days - old mice , when they are actively proliferating under sonic hedgehog ( shh ) stimulus . as expected , treatment of these cells with shh increased the proliferation rate , as evaluated by brdu - incorporation assay . importantly , glabrescione b antagonized this effect ( fig1 ). to further evaluate the antiproliferative effect of glabrescione b , human mb cell lines d283 were treated with glabrescione b at different concentrations . mts and brdu incorporation assay demonstrated a dose - dependent inhibitory effect on cell proliferation following 48 h of exposure to the dose range of 1 - 50 μm ( fig1 a and b ). the biomolecule was active , as shown by the growth rate inhibition ( 70 - 95 %). notably , the inhibitory effect of glabrescione b on mb d283 cell proliferation was coupled to an increase of the percentage of cell death , in a dose - dependent manner ( fig1 ). more important , glabrescione b was ineffective on unrelated signal transduction pathways ( wnt / bcatenin and jun / ap1activation ; 0 % inhibition at 30 μm of glabrescione b ), demonstrating a high degree of selectivity for hh signaling . hh activity preferentially associates to stemness features . for this reason , the hh pathway antagonist glabrescione b has also been investigated for its ability to modulate the behavior of mb cancer stem cells ( cscs ). analysis by quantitative rt - pcr revealed an inhibitory effect of glabrescione b on hh activity , as showed by reduction of gli1 mrna levels ( fig1 ). further , treatment with glabrescione b had a suppressive effect on mb cscs self - renewal , in particular on the percentage of neurospheres - forming cells derived from murine ptch +/− mb ( condition of hh pathway constitutive activation ). notably , this inhibitory effect was significantly higher than the effect achieved with known hh antagonist , as kaad ( fig1 ). abbreviations used in the description of the chemistry and in the examples that follow are : md : molecular dynamics ; mm - gbsa : molecular mechanics generalized born surface area ; gli1 - zf : zinc finger domain of gli1 ; brdu : 5 ′- bromo - 2ldeoxy - uridine ; dmso : dimethyl sulfoxide ; meoh : methanol ; etoh : ethanol ; etoac : ethyl acetate ; edta : ethylenediaminetetraacetic acid ; hbss : hank &# 39 ; s balanced salt solution ; hprt : hypoxanthine - guanine phosphoribosyl transferase ; cyclopamine - kaad : 3 - keto - n — ( aminoethyl - aminocaproyl - dihydrocinnamoyl )- cyclopamine ; m : molar ; min : minutes ; h : hour ( s ); g ( grams ); μl ( microlitres ); ml ( millilitres ); mmol ( millimoles ); nm ( nanometers ); μm ( micromolar ); r . t . : room temperature ; rt - qpcr : quantitative real time pcr ; gli - luc : gli - dependent luciferase reporter , esi ( electron spray ionization ). except where indicated otherwise , all temperatures are expressed in ° c . ( degrees centigrade ) or k ( kelvin ). 1 h nmr and 13 c nmr spectra were recorded using a bruker 400 ultra shield ™ spectrometer ( operating at 400 mhz for 1 h and 100 mhz for 13 c ) using tetramethylsilane ( tms ) as internal standard . chemical shifts are reported in parts per million ( ppm ). signals for — nch3 carbon are not present in the 13 c nmr data . the chemical shifts are expressed in parts per million ( ppm , δ units ). the coupling constants are expressed in hertz ( hz ) and the splitting patterns are described as s ( singlet ), bs ( broad signal ), d ( doublet ), t ( triplet ), q ( quartet ), quint ( quintet ), m ( multiplet ). mass spectrometry was performed using a thermo finnigan lxq linear ion trap mass spectrometer , equipped with an electrospray ionization ( esi ) ion source . high - resolution mass spectra ( hrms ) were obtained using a bruker bioapex fourier transform ion cyclotron resonance ( ft - icr ) spectrometer fitted with an esi source . to investigate the binding properties of glabrescione b to smo receptor , a competition assay was carried out . in this experiment it was analyzed whether glabrescione b could compete with bodipy - cyclopamine ( bc ; 5 nm usbiological , cat . # b2527 ), a fluorescent derivative of cyclopamine , which is known to interact with the smo receptor at the level of its heptahelical bundle . to this aim , hek293 cells ( atcc , cat . # crl - 1573 ) were transfected with smo expression vector ( chen et al , 2002 ) by using lipofectamine 2000 ( invitrogen , cat # 11668 - 019 ); 24 h after transfection the cells were fixed in paraformaldehyde 4 % and incubated with 5 nm bc alone or in presence of increasing amounts of glabrescione b for 2 h at 37 ° c . the cells were also treated with the steroidal alkaloid smo antagonist cyclopamine - kaad ( 3 - keto - n -( aminoethyl - aminocaproyl - dihydrocinnamoyl )- cyclopamine , 1 μm calbiochem , cat . # 239804 ) a chemical analogous of cyclopamine , included as positive control . cells were analyzed with a carl zeiss microscope ( axio observer zi ). cell cultures images were taken with a 20 × objective for the analysis . bodipy - cyclopamine ( green ) and dapi ( blue ) signals were analyzed in 3 - 4 representative fields for coverslips . the result showed in fig5 indicates that glabrescione b abrogated bc binding to cells expressing smo with an ic 50 of 1 μm by interacting with smo receptor . the ability of glabrescione b to suppress hh signaling was assessed in a cellular context of hh pathway induction . to this end , the antagonist properties of glabrescione b were determined in shh light ii ( atcc , cat . # crl - 2795 ), a murine fibroblast nih 3t3 cell line . these cells , stably incorporating the gli - luc reporter and the prl - tk renilla reniformis ( as described in taipale , chen et al . 2000 ) are a standard tool used to assay hh pathway activity . the cells that reached the confluence were treated for 48 h with the smo agonist sag ( 200 nm , alexis , cat . # alx - 270 - 426 - m001 ) in absence or in presence of glabrescione b at the amount indicated in fig6 . sag is a potent cell - permeable benzothiophene compound that promotes the coupling of smo with its downstream effector by interacting with the smo heptahelical domain ( k d = 59 nm ) and shown to induce hedgehog pathway activation . control cells were treated with only dmso ( 0 . 5 %, serva , cat . # 39757 . 02 ) determination of luciferase activities was carried out using the dual - luciferase assay according to manufacturer &# 39 ; s instructions ( dual - luciferase ® reporter assay system ; promega , cat . # e1980 ). all presented data are firefly luciferase activity reported to the renilla control activity . as shown in fig6 , glabrescione b inhibits hh signaling upon sag activation , as evidenced by the dose - dependent decrease in luciferase reporter activity . ic 50 of 0 . 8 μm , represents the concentration of glabrescione b necessary to have fifty percent of inhibition . to investigate the ability of glabrescione b to block downstream hh signaling by targeting gli1 protein functions , the inventors performed a luciferase assay in hek293 cells transiently expressing gli1 . the cells were seeded in 24 - multiwell plates and the day after transfected with gli1 expression vector together with the reporter plasmids 12 × glibs - luc and prl - tk renilla reniformis ( kogerman p et al , 1999 ; everett l et al , 1999 ). twenty - four hours later , glabrescione b was added at different concentration as indicated and gant61 ( included as positive control , enzo life science , cat . # alx - 270 - 482 ) was added at the final concentration of 20 μm in dmso ( 0 . 5 %, serva , cat . # 39757 . 02 ). after 24 h of treatment cells were lysed and analyzed by using the dual luciferase kit according to manufacturer &# 39 ; s instructions ( dual - luciferase ® reporter assay system ; promega , cat . # e1980 ). all presented data are firefly luciferase activity reported to the renilla control activity . as shown in fig7 , glabrescione b was capable of interfering with gli1 - induced transcription in a dose - dependent manner and similarly to gant61 . in order to determine whether glabrescione b regulates cells with a constitutive hh pathway activation , the mef - ptch −/− cell line ( a kind gift by m . scott , stanford university , ca , in which the activation of hh signaling is consequence of ptch1 deletion , goodrich , l . v . et al , 1997 ) was treated with glabrescione b at different concentrations and the effect on expression of hh / gli1 pathway was determined by quantitative rt - pcr and western blot analysis . total rna was isolated with tri reagent ( invitrogen , cat . # am9738 ) and reverse transcribed with superscript ii reverse transcriptase ( invitrogen , cat . # pn100004925 ) and random hexamers ( invitrogen , cat . # pn58875 ). quantitative pcr ( qpcr ) analysis of gli1 mrna expression was performed on each cdna sample using the abi prism 7900 sequence detection system employing assay - on - demand reagents ( life technologies ). a reaction mixture containing cdna template , taqman universal pcr master mix ( abi ) and primer probe mixture was amplified using standard qpcr thermal cycler parameters . each amplification reaction was performed in triplicate and the average of the three threshold cycles was used to calculate the amount of transcript in the sample ( using sds version 2 . 3 software ). all values were normalized with hprt ( hypoxanthine - guanine phosphoribosyl transferase ) endogenous controls ( life technologies , cat . # mm 00494645_m1 gli1 ; mm 01545399_m1 ). for western blot analysis , cells were lysed in tris - hcl ( ph 7 . 6 ) 50 mm , nacl 150 mm , np - 40 1 %, edta 5 mm , deoxycholic acid sodium salt 0 . 5 %, naf 100 mm and protease inhibitors . the lysates were centrifuged at 13 , 000 g for 20 minutes , separated by a 8 % sds - page and transferred to a nitrocellulose membrane . immunoblotting , was performed with a mouse monoclonal antibody against gli1 ( cell signaling , cat . # l42b10 ) or goat polyclonal antibody against actin ( santa cruz biotechnology , cat . # 11012 ), used as loading control . hrp - conjugated secondary antibody anti - mouse or anti - goat igg ( santa cruz biotechnology , cat . # sc - 2005 , # sc - 2020 ) were used , and immunoreactive bands were visualized by enhanced chemiluminescence ( perkin elmer , cat . # nel105001ea ). kaad ( 1 μm , calbiochem , cat . # 239804 ) was used as control . as shown in fig8 , glabrescione b down - regulates gli1 expression compared to the untreated control . to further elucidate the concept of downstream pathway inhibition by glabrescione b , the inventors used mef - sufu −/− cells ( a kind gift by r . toftgard , karolinska institutet , sweden , svärd , j . et al , 2006 ), in which the downstream hh pathway activation is consequence of sufu genetic ablation . confluent cells were treated with glabrescione b at the amount indicated in the fig9 and gli1 expression levels were determined by quantitative rt - pcr as previously described . as shown in fig9 , glabrescione b promoted a significant downregulation of mrna gli1 levels . data were normalized with hprt endogenous controls , as described above . these findings confirm that glabrescione b is indeed inhibitor of hh signaling downstream of smo and sufu . another experiment to assess whether glabrescione b also regulates hh pathway in a physiological context was carried out in cerebellar granule cells precursors ( gcps ) isolated from 4 - days - old mice according to established protocols ( wechsler - reya and scott , 1999 ). briefly , cerebella were removed aseptically , cut into small pieces , and incubated at room temperature for 15 min in digestion buffer [ dulbecco &# 39 ; s pbs ( invitrogen , gaithersburg , md .) with 0 . 1 % trypsin , 0 . 2 % edta , and 100 μg / ml dnase ]. tissues were then triturated with fire - polished pasteur pipettes to obtain a single - cell suspension . cells were centrifuged , resuspended in neurobasal medium supplemented with b27 , penicillin - streptomycin , and l - glutamine ( 2 mm ) ( invitrogen ) and plated at a density of 8 × 10 5 cells / cm 2 on tissue - culture dishes or eight - well lab - tek chamber slides ( permanox slide ; nunc , cat . # 177445 ) coated with 1 mg / ml poly - l - lysine . these cells were treated with shh alone ( recombinant mouse sonic hedgehog , amino - terminal peptide ; 3 μg / ml , r & amp ; d system , cat . # 461 - sh ) or in combination with different concentration of glabrescione b for 48 h and growth inhibition ic 50 doses were determined by brdu ( bromodeoxyuridine , 5 ′- bromo - 2ldeoxy - uridine , roche , cat . # 11 296 736 001 ) labeling assay according to standard methods ( fig1 ). after brdu incorporation , gcps were fixed in 4 % paraformaldehyde for 20 min at room temperature , incubated in 0 . 2 % triton x - 100 to permeabilize cells and treated with 2n hcl to denature dna . brdu detection was performed according to the manufacturer &# 39 ; s instructions . in order to determine whether glabrescione b also inhibits the proliferation of cancer cells , human mb d283 cells ( atcc , cat . # htb - 185 ) were treated with glabrescione b and proliferation / viability rate were measured by brdu incorporation and mts assays , respectively . cells , as shown in fig1 , displayed a dose - dependent inhibitory effect on cell proliferation ( fig1 a ) and viability ( fig1 b ) following 48 h of exposure to the dose range of 1 - 50 μm . more important , glabrescione b was able to promote cell death of d283 mb cells , as reported by trypan blue count assay ( fig1 ). cell proliferation was evaluated by a brdu - labelling assay ( 8 h pulse ; roche , cat . # 11 296 736 001 ) as described above ; cell viability was evaluated by mts ( a tetrazolium compound [ 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 -( 4 - sulfophenyl )- 2h - tetrazolium , inner salt ; mts ; promega , cat . # g3580 ). cell death was measured by trypan blue ( sigma , cat . # t8154 ) exclusion counting at least 200 cells , in duplicate samples . all described assays were evaluated at 48 h after treatment . effects of glabrescione b on mb cancer stem cells and cancer - derived stem - like cells it has been described that hh activity preferentially associates to stemness features . in order to determine whether glabrescione b also modulates the behavior of mb cancer stem cells ( cscs ), neurospheres isolated from murine ptch +/− mb were treated with glabrescione b for 48 h and the levels of gli1 were determined by quantitative rt - pcr . murine mbs were isolated from ptch1 +/− mice ( goodrich , milenkivic et al . 1997 ). tissues were collected in hbss supplemented with 0 . 5 % glucose and penicillin - streptomycin , grossly triturated with serological pipette and treated with dnase i to a final concentration of 0 . 04 % for 20 min . finally , cells aggregates were mechanically dissociated using pipettes of decreasing bore size to obtain a single cell suspension . cscs were cultured as neurospheres in selective medium after centrifugation , dmem / f12 supplemented with 0 . 6 % glucose , 25 mg / ml insulin , 60 mg / ml n - acetyl - l - cystein , 2 mg / ml heparin , 20 ng / ml egf , 20 ng / ml bfgf , 1 × penicillin - streptomycin and b27 supplement without vitamin a . neurospheres were treated with the steroidal alkaloid smo antagonist , cyclopamine - kaad ( 1 μm calbiochem , cat . # 239804 ). rna extraction and mrna expression analysis were performed as described above . as shown in fig1 , gli1 expression was down - regulated compared to the untreated control . all values were normalized with hprt internal controls . another experiment was carried out to determine the ability of glabrescione b to suppress mb cscs self - renewal . to this aim neurospheres derived from murine ptch +/− mb were treated with glabrescione b at different concentrations and the percentage of neurospheres - forming cells was measured . for the neurosphere forming assay , cells were plated at clonal density ( 1 - 2 cells / mm2 ) into 96 - well plates and cultured in selective medium as described above . the results shown in fig1 indicate that glabrescione b has an inhibitory effect on self - renewal of cancer stem cells . notably , this inhibitory effect was significantly higher than the effect achieved with known hh antagonist kaad , thus reinforcing that glabrescione b is a potent inhibitor of tumor growth via inhibition of the hh signaling pathway . similar results were observed in asz001 basal cell carcinoma cells ( bcc ), previously characterized as an hh / gli - dependent tumor cell line harbouring ptch1 deletion ( aszterbaum et al , 1999 ). total rna was isolated with trizol ( invitrogen , eugene , or , usa ) and reverse transcribed with superscript ii reverse transcriptase and random hexamers ( invitrogen , eugene , or , usa ). quantitative real - time pcr ( qrt - pcr ) analysis of gli1 , gli2 , ptch1 , ptch2 , nanog , oct - 4 , cyclin d1 , cyclin d2 , nmyc , hip1 , sfrp1 , bmp2 , igf2 , β - 2 microglobulin , hprt , mrna expression was performed on each cdna sample using the abi prism 7900ht sequence detection system employing assay - on - demand reagents ( applied biosystems , foster city , calif ., usa ). a reaction mixture containing cdna template , taqman universal pcr master mix ( applied biosystems , foster city , calif ., usa ) and primer probe mixture was amplified using standard qrt - pcr thermal cycler parameters . each amplification reaction was performed in triplicate and the average of the three threshold cycles was used to calculate the amount of transcript in the sample ( using sds version 2 . 3 software ). mrna quantification was expressed , in arbitrary units , as the ratio of the sample quantity to the quantity of the calibrator . all values were normalized with two endogenous controls , β - 2 microglobulin and hprt , which yielded similar results . bcc cell proliferation was impaired by in vitro treatment with glabrescione b together with a suppression of gli1 mrna before a drug - induced cell death occurred ( fig1 ). notably , no effect was observed in hh - independent hepg2 hepatocellular carcinoma cells or jurkat t leukemia cells , which display undetectable levels of gli1 ( lauth et al , 2007 ). in summary , the inhibitory activity of glabrescione b on the subset of both normal and tumor progenitor / stem cells as well as the whole tumor cell populations , is restricted to hh / gli - dependent cells . to assess the in vivo activity of glabrescione b , we first tested its ability to suppress hh signaling in 6 - day old mouse cerebellar progenitors , considered the cell of origin of mb . 6 - day old cd1 mice were randomly divided into two groups ( n = 6 ) and injected s . c . with solvent only ( 2 - hydroxypropyl - β - cyclodextrin : ethanol , 3 : 1 ) or glabrescione b in solvent ( 100 μmol / kg ) for 2 days ( 2 - hydroxypropyl - β - cyclodextrin was purchased from sigma aldrich , st . louis , mo , usa ). cerebella were collected and mrna levels were determined by qrt - pcr . glabrescione b treatment reduced significantly the cerebellar levels of hh target genes ( fig1 ). therefore , to verify the glabrescione b efficacy to inhibit hh - dependent tumor cell growth in vivo , we turned to an allograft model of mb cells . spontaneous medulloblastomas from ptch1 +/− mice were isolated , minced , pipetted to obtain a single - cell suspension and grafted s . c . at the posterior flank of female balb / c nude mice ( nu / nu ) ( charles river laboratories , lecco , italy ). tumors were grown until a median size of ˜ 100 mm 3 . animals were randomly divided into two groups ( n = 6 ) and treated with solvent only ( 2 - hydroxypropyl - β - cyclodextrin : ethanol , 3 : 1 ) or glabrescione b in solvent ( 75 μmol / kg ) for 18 days ( 2 - hydroxypropyl - β - cyclodextrin was purchased from sigma aldrich , st . louis , mo ., usa ). 2 × 10 6 asz001 bcc cells were resuspended in an equal volume of 154cf medium and matrigel ( bd biosciences , heidelberg , germany ) and injected s . c . at the posterior flank of female nod / scid mice ( charles river laboratories , lecco , italy ), as previously described ( eberl et al , 2012 ). tumors were grown until a median size of ˜ 200 mm 3 . animals were randomly divided into two groups ( n = 6 ) and treated with solvent only ( 2 - hydroxypropyl - β - cyclodextrin : ethanol , 3 : 1 ) or glabrescione b in solvent ( 100 μmol / kg ) for 18 days . tumor volumes change was calculated by the formula length × width × 0 . 5 ×( length + width ) ( lauth et al , 2007 ). nude mice were grafted with spontaneous primary mb from ptch1 +/− mice and treated every second day with s . c . injections of glabrescione b at a concentration of 75 μmol / kg or solvent only ( n = 6 for each group ). during an 18 - day treatment period glabrescione b significantly suppressed tumor mass compared with controls , as confirmed by in vivo decreased ki67 staining in glabrescione b - treated tumors together with a reduction of gli1 mrna levels ( fig1 ). notably , in vivo glabrescione b - induced tumor growth inhibition was also observed in bcc s . c . allografts . a significant reduction of tumor growth , as well as gli1 mrna levels were observed 18 days after administration of glabrescione b ( 100 μmol / kg ) compared to solvent alone ( fig1 ). the total synthesis of the isoflavone glabrescione b is a six - step synthetic route , with an overall yield of 7 %. it also permits the preparation of numerous derivatives of glabrescione b . the use of pd encat ™ 40 , as catalyst , introduces an aspect of green chemistry into the synthesis . the yields were calculated assuming that products were 100 % pure if not stated otherwise . a flame - dried flask was charged with 2 , 4 , 6 - trimethoxyacetophenone ( 23 . 8 mmol , 5 g ) ( sigma - aldrich 630594 ) and dry ch 2 cl 2 ( 75 ml ) under argon . bbr 3 ( 87 . 3 mmol , 15 ml ) was added drop - wise . the resulting solution was stirred at room temperature for 2 h before adding naoh 4m ( 90 ml ) and allowed to stand for 30 min . the solution was extracted with ch 2 cl 2 and combined organic layers were washed with brine , dried over na 2 so 4 and finally concentrated under reduced pressure . the resulting solid was recrystallized from etoh and white crystals of 2 - hydroxy - 4 , 6 - dimethoxyacetophenone 2 ( 4 . 43 g , 98 % yield ) were obtained . white solid , ( 4 . 43 g ) 98 % yield . mp : 81 - 82 ° c . 1 h nmr ( 400 mhz , acetone - d6 ): δ ( ppm ) 13 . 8 ( s , 1h , oh ), 5 . 94 ( d , 1h , j = 2 . 4 hz , arh ), 5 . 91 ( d , 1h , j = 2 . 4 hz , arh ), 3 . 80 ( s , 3h , och 3 ), 3 . 72 ( s , 3h , och 3 ), 2 . 43 ( s , 3h , ch 3 ); 13 c nmr ( 400 mhz , acetone - d6 ): δ ( ppm ) 202 . 5 ( s , c ═ o ), 167 . 36 ( s , c - 4 ), 166 . 34 ( s , c - 6 ), 163 . 15 ( s , c - 2 ), 105 . 5 ( s , c - 1 ), 93 . 42 ( d , c - 3 ), 90 . 43 ( d , c - 5 ), 55 . 20 , 55 . 00 ( q , 2 × och 3 ), 31 . 94 ( q , ch 3 ). esi - ms ( positive ): m / z calcd for c 10 h 12 o 4 + h + : 197 . 080800 [ m + h ] + ( monoisotopic mass ). found : 197 . 080848 . a mixture of 2 - hydroxy - 4 , 6 - dimethoxyacetophenone 2 ( 23 . 1 mmol , 4 . 5 g ) and n , n - dimethylformamide dimethylacetal ( 97 . 1 mmol , 13 ml ) was stirred at 95 ° c . for 3 h , then concentrated in vacuo to give enamino ketone 3 ( 5 . 8 g ) in quantitative yield . ( biegasiewicz , st denis et al . 2010 ) compound 3 was dissolved in meoh ( 450 ml ) and i 2 ( 46 . 2 mmol , 11 . 7 g ) was added to the solution . the mixture was stirred at room temperature for 1 h , then the solvent was evaporated . to remove residual i 2 , the crude was treated with a saturated aqueous na 2 s 2 o 3 solution until the mixture became clear . the mixture was then extracted with chcl 3 , the combined organic layers were dried over na 2 so 4 and concentrated under reduced pressure . the residue was purified by column chromatography using hexane - etoac as eluent to obtain 3 - iodo - 5 , 7 - dimethoxy - 4h - chromen - 4 - one 4 ( 2 . 5 g , 33 % yield ) as a white powder . red solid , ( 5 . 8 g ) quantitative yield . mp : 145 - 147 ° c . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 15 . 65 ( s , 1h , oh ), 7 . 92 ( d , 1h , j = 12 hz , ═ ch — n ), 6 . 25 ( d , 1h , j = 12 . 0 hz , ═ ch —( co )), 6 . 07 ( d , 1h , j = 2 . 4 hz , arh ), 5 . 91 ( d , 1h , j = 2 . 4 hz , arh ), 3 . 84 ( s , 3h , och 3 ), 3 . 80 ( s , 3h , och 3 ), 3 . 15 ( s , 3h , nch 3 ), 2 . 92 ( s , 3h , nch 3 ); 13 c nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 191 . 64 ( s , c ═ o ), 169 . 00 ( s , c - 4 ), 165 . 54 ( s , c - 6 ), 162 . 86 ( s , c - 2 ), 155 . 83 ( d , ═ ch — n ), 106 . 58 ( s , c - 1 ), 96 . 27 ( d , ═ ch —( co )), 95 . 59 ( d , c - 3 ), 92 . 06 ( d , c - 5 ), 57 . 09 , 56 . 57 ( q , 2 × och 3 ). esi - ms ( positive ): m / z calcd for c 13 h 17 o 4 + h + : 252 . 1 [ m + h ] + ( monoisotopic mass ). found : 252 . 1 . white solid , ( 2 . 5 g ) 33 % yield . mp : 156 - 157 ° c . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 8 . 02 ( s , 1h , h - 2 ), 6 . 37 ( d , j = 2 . 0 hz , 1h , arh ), 6 . 32 ( d , j = 2 . 0 hz , 1h , arh ), 3 . 87 ( s , 3h , och 3 ), 3 . 82 ( s , 3h , och 3 ); 13 c nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 183 . 43 ( s , c ═ o ), 164 . 28 ( s , c - 7 ), 160 . 97 ( s , c - 5 ), 159 . 81 ( s , c - 9 ), 155 . 33 ( d , c - 2 ), 107 . 53 ( s , c - 10 ), 96 . 59 ( d , c - 8 ), 92 . 44 ( d , c - 6 ), 89 . 71 ( d , ═ ch — i ), 56 . 41 , 55 . 79 ( q , 2 × och 3 ). esi - ms ( positive ): m / z calcd for c 11 h 9 o 4 i + h + : 332 . 961800 [ m + h ] + ( monoisotopic mass ). found : 332 . 961633 . to a solution of 4 ( 7 . 5 mmol , 2 . 5 g ) in 1 , 2 - dimethoxyethane / h 2 o = 50 : 50 ( 150 ml ) were added na 2 co 3 ( 30 mmol , 3 . 18 g ), 3 , 4 -( methylenedioxy )- phenylboronic acid ( 11 mmol , 1 . 8 g ), and pd encat ™ 40 ( 937 mg , 5 %). the resulting mixture was stirred at 45 ° c . for 2 h and then filtered . the catalyst was washed with h 2 o and ch 2 cl 2 . the aqueous phase was extracted with ch 2 cl 2 . the combined organic layers were dried over na 2 so 4 and concentrated under reduced pressure . the crude residue was purified by flash chromatography to give 3 -( 3 ′, 4 ′- methylendioxyphenyl )- 5 , 7 - dimethoxy - 4h - chromen - 4 - one 5 ( 1 . 4 g , 57 % yield ) as gray powder . gray solid , ( 1 . 4 g ) 57 % yield . mp : 155 - 156 ° c . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 75 ( s , 1h , h - 2 ), 7 . 1 ( d , j = 2 . 0 hz , 1h , h - 2 ′), 6 . 94 ( dd , j = 8 . 0 hz and 2 . 0 hz , 1h , h - 6 ′), 6 . 83 ( d , j = 8 . 0 hz , 1h , h - 5 ′), 6 . 44 ( d , j = 2 . 2 hz , 1h , h - 6 ), 6 . 37 ( d , j = 2 . 2 hz , 1h , h - 8 ), 5 . 97 ( broad s , 2h , o — ch 2 — o ), 3 . 94 ( s , 3h , och 3 ), 3 . 89 ( s , 3h , och 3 ); 13 c nmr ( 400 mhz , cdcl 3 ): δ ( ppm )= 175 . 09 ( s , c ═ o ), 164 . 02 ( s , c - 7 ), 161 . 54 ( s , c - 5 ), 160 . 30 ( s , c - 9 ), 150 . 05 ( d , c - 2 ), 147 . 58 ( s , c - 3 ′), 147 . 58 ( s , c - 4 ′), 126 . 5 ( s , c - 3 ), 126 . 04 ( s , c - 1 ′), 122 . 84 ( d , c - 6 ′), 110 . 50 ( d , c - 2 ′), 110 . 0 ( s , c - 10 ), 108 . 35 ( d , c - 5 ′), 101 . 30 ( t , o — ch 2 — o ), 96 . 74 ( d , c - 6 ), 92 . 73 ( d , c - 8 ), 56 . 41 , 55 . 79 ( q , 2 × och 3 ). esi - ms ( positive ): m / z calcd for c 18 h 14 o 6 + h + : 327 . 086300 [ m + h ] + ( monoisotopic mass ). found : 327 . 086206 . a mixture of 5 ( 4 . 3 mmol , 1 . 4 g ) and pb ( oac ) 4 ( 17 mmol , 7 . 5 g , freshly recrystallized from acoh ) in dry c 6 h 6 ( 100 ml ) was stirred at 80 ° c . under argon overnight . after , being cooled to room temperature , the reaction mixture was filtered through a pad of celite , washed with ch 2 cl 2 and concentrated under reduced pressure . the crude was diluted with thf / h 2 o = 5 : 1 ( 50 ml ) and ch 3 cooh ( 50 ml ) and the resulting mixture was stirred at room temperature for 6 h . ( ye , koshino et al . 2009 ) afterwards , a saturated aqueous nahco 3 solution was added until ph 8 and extracted with etoac . to the combined organic layers was added a solution of naoh 0 . 1 m . water layer was treated with ch 3 cooh and then extracted with etoac . the combined organic layers were dried over na 2 so 4 and concentrated under reduced pressure to obtain 3 -( 3 ′, 4 ′- dihydroxyphenyl )- 5 , 7 - dimethoxy - 4h - chromen - 4 - one 6 ( 570 mg , 42 % yield ). yellow solid , ( 570 mg ) 42 % yield . mp : 127 - 129 ° c . 1 h nmr ( 400 mhz , meod ): δ ( ppm ) 7 . 86 ( s , 1h , h - 2 ), 6 . 88 ( broad s , 1h , h - 2 ′), 6 . 70 ( broad s , 2h , h - 5 ′ and h - 6 ′), 6 . 50 ( d , j = 2 . 0 hz , 1h , h - 6 ), 6 . 40 ( d , j = 2 . 0 hz , 1h , h - 8 ), 3 . 80 ( s , 3h , och 3 ), 3 . 79 ( s , 3h , och 3 ); 13 c nmr ( 400 mhz , meod ): δ ( ppm ) 175 . 00 ( s , c ═ o ), 165 . 08 ( s , c - 7 ), 161 . 13 ( s , c - 5 ), 159 . 91 ( s , c - 9 ), 151 . 28 ( d , c - 2 ), 145 . 12 ( s , c - 3 ′), 145 . 0 ( s , c - 4 ′), 126 . 49 ( s , c - 3 ), 123 . 12 ( s , c - 1 ′), 120 . 50 ( d , c - 6 ′), 116 . 99 ( d , c - 2 ′), 110 . 00 ( s , c - 10 ), 114 . 48 ( d , c - 5 ′), 95 . 86 ( d , c - 6 ), 92 . 82 ( d , c - 8 ), 55 . 09 ( q , 2 × och 3 ). esi - ms ( positive ): m / z calcd for c 17 h 14 06 + h + : 315 . 086300 [ m + h ] + ( monoisotopic mass ). found : 315 . 086363 . to a solution of 6 ( 61 . 8 mmol , 570 mg ) in acetone ( 100 ml ) was added k 2 co 3 ( 5 . 4 mmol , 7 . 4 g ) and , after 10 minutes of stirring at room temperature , was added 3 , 3 - dimethylallyl bromide ( 6 . 5 mmol , 968 mg ). then , the mixture was stirred at 80 ° c . overnight . afterwards , the solvent was evaporated . the resulting solid was dissolved in etoac and extracted with water . the combined organic layers were dried over na 2 so 4 and finally concentrated under reduced pressure . the crude was purified by column chromatography using hexane - etoac as eluent to obtain compound 1 as white powder . the powder was recrystallized from hexane resulting with white crystals . white solid , ( 696 mg ) 86 % yield . mp 102 - 104 ° c . 1 h nmr ( 400 mhz , acetone - δ 6 ): δ ( ppm ) 7 . 94 ( s , 1h , h - 2 ), 7 . 16 ( d , 1h , j = 1 . 6 hz , h - 2 ′), 6 . 99 ( dd , 1h , j = 8 . 0 hz and 1 . 6 hz , h - 6 ′), 6 . 89 ( d , 1h , j = 8 . 0 hz h - 5 ′), 6 . 50 ( d , 1h , j = 2 . 0 hz , h - 8 ), 6 . 42 ( d , 1h , j = 2 . 0 hz , h - 6 ), 5 . 43 ( m , 2h , 2 ×= ch ), 4 . 51 ( d , j = 6 . 8 hz , 4h , 2 × och 2 ), 3 . 86 ( s , 3h , och 3 ), 3 . 81 ( s , 3h , och 3 ), 1 . 70 ( s , 6h , 2 × ch 3 ), 1 . 67 ( s , 6h , 2 × ch 3 ); 13 c nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 175 . 59 ( s , c ═ o ), 164 . 02 ( s , c - 7 ), 161 . 23 ( s , c - 5 ), 159 . 67 ( s , c - 9 ), 150 . 20 ( d , c - 2 ), 148 . 89 ( s , c - 3 ′), 148 . 51 ( s , c - 4 ′), 136 . 85 ( s , 2 × c =), 126 . 50 ( s , c - 3 ), 125 . 00 ( s , c - 1 ′), 121 . 44 ( d , c - 6 ′), 120 . 65 ( d , 2 ×= ch ), 115 . 50 ( d , c - 2 ′), 110 . 00 ( s , c - 10 ), 114 . 00 ( d , c - 5 ′), 96 . 47 ( d , c - 6 ), 92 . 60 ( d , c - 8 ), 65 . 95 ( t , 2 × och 2 ), 56 . 44 ( q , och 3 ), 55 . 93 ( q , och 3 ), 25 . 62 ( q , 2 × ch 3 ), 18 . 13 ( q , 2 × ch 3 ). esi - ms ( positive ): m / z calcd for c 27 h 30 o 6 + h + : 451 . 211500 [ m + h ] + ( monoisotopic mass ). found : 451 . 211495 . the 1 h - nmr data of this product were identical to an authentic sample of glabrescione b . a mixture of 3 , 5 - dimethoxyphenol ( 1 . 3 mmol , 463 mg ) ( sigma - aldrich 132632 ), 3 , 4 - dihydroxyphenylacetic acid ( 2 , 3 mmol , 504 . 5 mg ) ( sigma - aldrich 850217 ) and bf 3 . et 2 o ( 15 . 3 mmol , 1 . 94 ml ) was stirred at 90 ° c . for 90 min under argon . the reaction mixture was poured into 10 % aqueous naoac solution ( 100 ml ) and allowed to stand 4 h . the solution was extracted with etoac . the combined organic layers were washed with saturated solution of nahco 3 , dried over na 2 so 4 and finally concentrated under reduced pressure . the residue was purified by column chromatography using hexane - etoac mixture as eluent to obtain 1 -( 2 - hydroxy - 4 , 4 - dimethoxy - phenyl )- 2 -( 3 - hydroxy - 4 - methoxy - phenyl )- ethanone 7 . a mixture of 7 ( 3 mmol ) and bf 3 . et 2 o ( 9 mmol , 1 . 2 ml ) was cooled to 10 ° c . and dmf ( 4 . 6 ml ) was added drop wise . in another flask , dmf ( 8 ml ) was cooled to 10 ° c . and pcl 5 ( 4 . 5 mmol ) was added . the mixture was then allowed to stand to 55 ° c . for 20 min . the light yellow colored solution containing n , n ′- dimethyl ( chloromethylene ) ammonium chloride was then added to the above reaction mixture at 20 - 25 ° c . the mixture was stirred at r . t . for 2 h then poured into methanolic hcl ( 0 . 1n ) and allowed to stand at 700 for 2 h . after removing the solvent the solution was extracted with etoac ( 3 × 100 ml ) and the combined organic layer was washed with brine , dried over na 2 so 4 , and finally concentrated under reduced pressure . the residue was purified by column chromatography using hexane - etoac as eluent to give 3 -( 3 ′- hydroxy - 4 ′- methoxy - phenyl )- 5 , 7 - dimethoxy - chromen - 4 - one 8 . to a solution of 8 ( 0 . 18 mmol , 60 mg ) in acetone ( 5 ml ) at 45 ° c . was added solid k 2 co 3 ( 8 eq ). r 5 br ( 1 . 5 eq ) was added drop wise to the mixture and stirred at 45 ° c . for 3 h . the progress of the reaction was monitored by thin layer chromatography ( sio 2 gel , developing solvent v exane : v etoac = 3 : 7 ). when only one spot was on the tlc the reaction was quenched , extracted with etoac , the combined organic layer was dried over na 2 so 4 and evaporated under reduced pressure . yellow oil , 47 % yield . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 76 ( s , 1h , h - 2 ), 7 . 22 ( d , 1h , j = 1 . 6 hz , h - 2 ′), 7 . 01 ( dd , 1h , j = 8 hz and j = 1 . 6 hz , h - 6 ′), 6 . 88 ( d , 1h , j = 8 . 4 hz , h - 5 ′), 6 . 44 ( d , 1h , j = 2 hz , h - 8 ), 6 . 37 ( d , 1h , j = 2 hz , h - 6 ), 5 . 55 ( t , 1h , j = 6 . 4 hz , ═ ch ), 4 . 59 ( d , 2h , j = 6 . 8 hz , ch 2 ), 3 . 94 ( s , 3h , och 3 ), 3 . 88 ( s , 6h , 2 × och 3 ), 1 . 76 ( s , 3h , ch 3 ), 1 . 71 ( s , 3h , ch 3 ). white powder , 44 % yield . mp : 126 ° c . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 71 ( s , 1h , h - 2 ), 7 . 46 ( dd , 2h , j = 7 . 6 and j = 1 . 6 hz , arh ), 7 . 36 ( t , 2h , j = 7 . 6 hz , arh ) 7 . 31 ( dd , 1h , j = 7 . 2 and j = 2 hz , arh ), 7 . 29 ( d , 1h , j = 2 hz , h - 2 ′) 7 . 07 ( dd , 1h , j = 8 . 4 and j = 2 hz , h - 6 ′), 6 . 92 ( d , 1h , j = 8 . 4 hz , h - 5 ′), 6 . 44 ( d , 1h , j = 2 hz , h - 8 ), 6 . 37 ( d , 1h , j = 2 hz , h - 6 ), 5 . 17 ( s , 2h , och 2 ), 3 . 95 ( s , 3h , och 3 ), 3 . 90 ( s , 3h , och 3 ), 3 . 89 ( s , 3h , och 3 ). white powder , 44 % yield 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 76 ( s , 1h , h - 2 ), 7 . 22 ( d , 1h , j = 1 . 6 hz , h - 2 ′), 7 . 01 ( dd , 1h , j = 8 hz and j = 1 . 6 hz , h - 6 ′), 6 . 88 ( d , 1h , j = 8 . hz , h - 5 ′), 6 . 44 ( d , 1h , j = 2 hz , h - 8 ), 6 . 37 ( d , 1h , j = 2 hz , h - 6 ), 5 . 55 ( t , 1h , j = 6 . 4 hz , ═ ch ), 5 . 39 ( t , 1h , j = 7 hz , ═ ch ), 4 . 59 ( d , 2h , j = 6 . 4 hz , och 2 ), 3 . 94 ( s , 3h , och 3 ), 3 . 88 ( s , 6h , 2 × och 3 ), 2 . 14 ( t , 2h , j = 6 . 9 hz ch 2 ), 2 . 00 ( q , 2h , j = 7 hz , ch 2 — ch 2 — ch ═), 1 . 76 ( s , 3h , ch 3 ), 1 . 71 ( s , 6h , 2 × ch 3 ). white powder , 78 % yield . mp : 128 . 6 - 129 . 1 ° c . 1 h nmr ( 400 mhz , acetone - d6 ): δ ( ppm ) 8 . 01 ( s , 1h , h - 2 ), 7 . 78 ( m , 4h , arh ), 7 . 36 ( d , 1h , j = 2 . 1 hz , h - 2 ′), 7 . 15 ( dd , 1h , j = 8 . 3 hz and j = 2 . 1 hz , h - 6 ′), 7 . 02 ( d , 1h , j = 8 . 4 , h - 5 ′), 6 . 57 ( d , 1h , j = 2 . 3 hz , h - 8 ), 6 . 49 ( d , 1h , j = 2 . 3 hz , h - 6 ), 5 . 27 ( s , 2h , och 2 ), 3 . 93 ( s , 3h , och 3 ), 3 . 89 ( s , 3h , och 3 ), 3 . 88 ( s , 3h , och 3 ). a mixture of 3 , 5 - dimethoxyphenol ( 1 . 3 mmol , 463 mg ), 3 , 4 - dihydroxyphenylacetic acid ( 2 , 3 mmol , 504 . 5 mg ) and bf 3 . et 2 o ( 15 . 3 mmol , 1 . 94 ml ) was stirred at 90 ° c . for 90 min under argon . the reaction mixture was poured into 10 % aqueous naoac solution ( 100 ml ) and allowed to stand 4 h . the solution was extracted with etoac . the combined organic layers were washed with saturated solution of nahco 3 , dried over na 2 so 4 and finally concentrated under reduced pressure . the residue was purified by column chromatography using hexane - etoac mixture as eluent to obtain 9 . a mixture of 9 ( 3 mmol ) and bf 3 . et 2 o ( 9 mmol , 1 . 2 ml ) was cooled to 10 ° c . and dmf ( 4 . 6 ml ) was added drop wise . in another flask , dmf ( 8 ml ) was cooled to 10 ° c . and pcl 5 ( 4 . 5 mmol ) was added . the mixture was then allowed to stand to 55 ° c . for 20 min . the light yellow colored solution containing n , n ′- dimethyl ( chloromethylene ) ammonium chloride was then added to the above reaction mixture at 20 - 25 ° c . the mixture was stirred at r . t . for 2 h then poured into methanolic hcl ( 0 . 1n ) and allowed to stand at 700 for 2 h . after removing the solvent the solution was extracted with etoac ( 3 × 100 ml ) and the combined organic layer was washed with brine , dried over na 2 so 4 , and finally concentrated under reduced pressure . the residue was purified by column chromatography using hexane - etoac as eluent to give 10 . to a solution of 10 ( 0 . 18 mmol , 60 mg ) in acetone ( 5 ml ) at 45 ° c . was added solid k 2 co 3 ( 8 eq ). r 5 br ( 1 . 5 eq ) was added drop wise to the mixture and stirred at 45 ° c . for 3 h . the progress of the reaction was monitored by thin layer chromatography ( sio 2 gel , developing solvent v exane : v etoac = 3 : 7 ). when only one spot was on the tlc the reaction was quenched , extracted with etoac , the combined organic layer was dried over na 2 so 4 and evaporated under reduced pressure . yellow powder , 45 % yield . mp : 130 . 7 - 131 . 8 ° c . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 78 ( s , 1h , h - 2 ), 7 . 23 ( d , 1h , j = 2 . 0 hz , h - 2 ′), 6 . 96 ( dd , 1h , j = 8 . 0 and j = 2 . 0 hz , h - 6 ′), 6 . 88 ( d , 1h , j = 8 . 0 hz , h - 5 ′), 6 . 44 ( d , 1h , j = 2 . 4 hz , h - 8 ), 6 . 37 ( d , 1h , j = 2 . 4 hz , h - 6 ), 5 . 53 ( m , 1h , ═ ch ), 4 . 60 ( d , 2h , j = 6 . 8 hz , och 2 ), 3 . 94 ( s , 3h , och 3 ), 3 . 89 ( s , 6h , 2 × och 3 ), 1 . 77 ( s , 3h , ch 3 ), 1 . 73 ( s , 3h , ch 3 ). white powder , 56 % yield . mp : 142 . 8 - 143 . 7 ° c . 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 77 ( s , 1h , h - 2 ), 7 . 45 ( dd , 2h , j = 8 . 0 hz and j = 2 . 0 hz , arh ), 7 . 37 ( t , 2h , j = 8 . 0 hz , arh ), 7 . 31 ( dd , 1h , j = 8 hz and j = 1 . 6 hz , arh ), 7 . 30 ( d , 1h , j = 2 . 0 hz , h - 2 ′), 6 . 89 ( m , 2h , h - 6 ′ and h - 5 ′), 6 . 45 ( d , 1h , j = 2 hz , h - 8 ), 6 . 38 ( d , 1h , j = 2 hz , h - 6 ), 5 . 19 ( s , 1h , och 2 ), 3 . 94 ( s , 3h , och 3 ), 3 . 93 ( s , 3h , och 3 ), 3 . 90 ( s , 3h , och 3 ) white powder , 44 % yield 1 h nmr ( 400 mhz , cdcl 3 ): δ ( ppm ) 7 . 76 ( s , 1h , h - 2 ), 7 . 22 ( d , 1h , j = 1 . 6 hz , h - 2 ′), 7 . 01 ( dd , 1h , j = 8 hz and j = 1 . 6 hz , h - 6 ′), 6 . 88 ( d , 1h , j = 8 hz , h - 5 ′), 6 . 44 ( d , 1h , j = 2 hz , h - 8 ), 6 . 37 ( d , 1h , j = 2 hz , h - 6 ), 5 . 42 ( t , 1h , j = 6 . 4 hz , ═ ch ), 5 . 12 ( t , 1h , j = 7 hz , ═ ch ), 4 . 41 ( d , 2h , j = 6 . 4 hz , och 2 ), 3 . 94 ( s , 3h , och 3 ), 3 . 90 ( s , 3h , och 3 ), 3 . 88 ( s , 3h , och 3 ), 2 . 14 ( t , 2h , j = 6 . 9 hz ch 2 ), 2 . 00 ( q , 2h , j = 7 hz , ch 2 — ch 2 — ch ═), 1 . 76 ( s , 3h , ch 3 ), 1 . 71 ( s , 6h , 2 × ch 3 ). white powder , 25 % yield . mp : 128 . 4 - 130 . 2 ° c . 1 h nmr ( 400 mhz , acetone - d6 ): δ ( ppm ) 8 . 04 ( s , 1h , h - 2 ), 7 . 76 ( m , 4h , arh ), 7 . 29 ( d , 1h , j = 2 . 1 hz , h - 2 ′), 7 . 06 ( m , 2h , h - 5 ′ and h - 6 ′), 6 . 58 ( d , 1h , j = 2 hz , h - 8 ), 6 . 50 ( d , 1h , j = 2 hz , h - 6 ), 5 . 28 ( s , 2h , och 2 ), 3 . 94 ( s , 3h , och 3 ), 3 . 89 ( s , 6h , 2 × och 3 ). the in house unique library was composed of 816 different natural products . single molecular entries were generated in smiles format and then transformed in the 3d sdf format . the ligprep application of the schrodinger maestro suite ( https :// www . schrodinger . com ) was used for ionizing compounds at ph = 7 . 5 ± 1 , for generating tautomers and for energy minimization with the opls2005 force field ( jorgensen , maxwell et al . 1996 ). ionization and tautomerization states endowed with a normalized probability higher than 0 . 6 were retained in the library . after this step the library was composed of 1111 individual entries . the qikprop application of the maestro suite was used to predict chemical and chemico - physical features of all compounds . the conformational analysis was carried out by means of the “ build 3d database ” protocol of discovery studio 2 . 5 using the caesar conformation method with default options and keeping up to 600 conformers for each ligand . a training set of 9 potent smo antagonists was used to generate pharmacophore models according with a ligand - based procedure . the “ common feature pharmacophore generation ” protocol implemented in discovery studio 2 . 5 from accelrys was used . up to 20 pharmacophores were generated , composed by a maximum of 10 features . the minimum inter - feature distance was set at 2 . 0 å while the number of leads that may miss was kept at the default value ( 1 ). conformational analysis of the training set was carried out with the caesar method . the maximum number of omitted features during the alignment of the training set to pharmacophores was set at 1 . this procedure generated twenty pharmacophores ( maximum allowed value , according to custom settings ). the six top - ranking pharmacophores are divided into two groups , based on the pharmacophoric feature composition . type1 pharmacophores have three hydrogen bond acceptor ( hba ) and three hydrophobic ( hyd ) features . type2 pharmacophores have three hba , two hyd and a hydrogen bond donor ( hbd ) features ( fig1 ). coordinates of pharmacophores and inter - feature distances are given in tables 1 and 2 for the representative type1 pharmacophore model ( 3 hyd ; 3 hba ) and in tables 3 and 4 for the representative type2 pharmacophore ( 2 hyd ; 1 hbd ; 3 hba ). the in house unique library of natural products , generated as described in example n . 10 was screened through the six pharmacophores . first , the “ search 3d database ” protocol implemented in discovery studio 2 . 5 was used to select molecules able to map the pharmacophores . the “ best ” search method was used , which perform a flexible fit of the ligand conformations against the pharmacophore . the “ ligand pharmacophore mapping ” protocol was subsequently used to fit the selected conformations to the pharmacophores and to calculate the fitvalue . no omitted features were allowed during ligand fitting to pharmacophores . the flexible fitting method was used , which permit a slight modification of each ligand conformation to better fit the pharmacophore . by pharmacophore screening , three groups of molecules were selected from the initial library : 1 ) ligands that map all pharmacophores ; 2 ) ligands that map only type1 pharmacophores and 3 ) ligands that map only type2 pharmacophores . with the aim of prioritizing small molecular compounds , the ligand efficiency ( le ) was further calculated for each compound as the ratio between its fitvalue and the number of heavy atoms ( le = fitvalue / no . heavy atoms ). 16 natural compounds endowed with the highest le were selected for in vitro studies ( see above ). initial coordinates of the gli1 - zf / dna complex were retrieved from the protein data bank ( http :// www . rcsb . org / pdb / home / home . do ) under the pdb accession code 2gli . coordinates of crystal water molecules were manually removed from the complex , while cobalt ions were manually replaced with zinc ions within the coordination system of each zinc finger . the amber11 program ( case , cheatham et al . 2011 ) was used for generating md trajectories and performing energy calculations . the ambertools1 . 5 software was used for preparing input coordinates and topology files , and for performing preliminary analysis on md trajectories by the ptraj and cpptraj modules . the ff99bsc0 and general amber ( gaff ) force fields were used for parameterizing protein and ligands , respectively . zinc ions were treated following a bound approach ; parameters for the zinc ion and zinc - coordinating residues were adapted from a previous qm calculation ( mori , dietrich et al . 2010 ). the gli1 - zf / dna complex was solvated in a rectilinear box of explicit water molecules , buffering 8 å from the macromolecular system . the tip3p water model was used . the total charge of the system was neutralized by the addition of sodium counterions ( na +). the solvated macromolecular system was first energy minimized as follows : the water solvent and counterions were first minimized for 250 steps by using a steepest descent algorithm ( sd ) and for 750 steps by using a conjugate gradient algorithm ( cg ), while keeping the gli1 - zf / dna coordinates as frozen ; then , the solvated system was energy minimized for 1000 steps sd and further 4000 steps cg without positional restraints . before the final production of trajectories , the energy minimized system was gradually heated from 0 to 300 k for 50 ps using the langevin control of the temperature at constant pressure and constraining the gli1 - zf backbone and the dna phosphate backbone with a harmonic force constant of 5 . 0 kcal · mol − 1 · å − 2 . then , the density of the system was equilibrated for 50 ps , by applying the same constraints mask used in the heating step . restrained md trajectory were produced for 3 ns while the force constant applied to gli1 - zf and dna backbone was gradually decreasing from 5 to 2 to 1 kcal · mol − 1 · å − 2 every 1 ns . after this step , unrestrained md trajectories were generated for 20 ns by using sander . during all md simulations , a time step of 0 . 001 ps was used . a representative gli1 - zf structure was extracted from md trajectories and used as rigid receptor for docking simulations . the gold docking program ( version 5 . 0 . 1 ) was used to dock the in house library towards the binding site centered on the side chain of thr374 and having a radius of 18 å . the goldscore function was used whit genetic algorithm ( ga ) efficacy set at 150 % and generating 50 runs for ligands , while other parameters were kept at their default values . docking poses were rescored by means of the mm - gbsa method implemented in amber11 . nmr experiments were carried out to probe the direct interaction of glabrescione b to gli1 - zf and to support computational and biological data . a sample of glabrescione b 0 . 412 mm in dmso - d 6 was prepared and added to 20 μl of a solution of gli1 - gst ( glutathione s - transferase ) fusion protein at 5 μg / μl , providing a molar ratio of glabrescione b / gli1 - gst of 150 : 1 . relaxation speeds of glabrescione b protons were monitored via nmr ( 600 mhz ), results are showed in table 5 . in experimental conditions , glabrescione b protons experiencing the most significant perturbation of the relaxation speed , due to the presence of gli1 - gst , belong to the ring a of the isoflavone nucleus , namely aromatic protons h 1 , h 3 and those belonging to methoxyl groups 2 ′ and 4 ′. in addition , protons h 11 and h 15 belonging to the ring c showed a significant perturbation of the relaxation speed in presence of gli1 , while h 8 showed only a small variation . it is worth noting that the same experiments were conducted also in presence of gst alone to monitor the influence of gst in the fusion protein gli1 - gst to the binding of glabrescione b . results showed that the normalized perturbation of the relaxation speed is significantly lower than that observed in presence of gli1 - gst ( table 5 ) and suggested that glabrescione b interacts most strongly with gli1 than gst . moreover , glabrescione b protons involved in binding to gst are significantly different from those involved in binding to gli1 . finally , since gli1 is a zinc binding protein , it was monitored via nmr the direct interaction between glabrescione b and zn 2 + , showing that the molecule is not capable of binding to the metal ions . since glabrescione b was the most potent hh inhibitor identified by the screening , a number of glabrescione b analogues ( namely nt8 , nt9 , nt10 , nt 11 , nt12 , nt13 , nt14 and nt15 ) were synthesized and tested in vitro to improve inhibitory potency against the hh pathway and to afford structure - activity relationships ( sar ) for the congeneric series ( see examples 8 and 9 ). evaluation of the hh inhibitory activity of these molecules at 5 μm was preliminarily conducted in vitro on shh light ii cells ( see example 2 ). preliminary results showed that some of these molecules were at least as active as glabrescione b , with nt8 and nt9 being more potent than glabrescione b ( see fig1 ). ahn , s . and a . l . joyner ( 2005 ). nature 437 ( 7060 ): 894 - 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