Patent Application: US-201213979447-A

Abstract:
a styrylquinoline derivative of structure i or iv or a benzodioxol isoquinoline of structure ii or iii for use in the treatment of an angiogenesis - related disease or disorder . the invention also provides a composition comprising a styrylquinoline derivative of structure i or iv or a benzodioxol isoquinoline of structure ii or iii for use as a medicament .

Description:
we have identified small molecule compounds that exhibit an anti - angiogenic effect , in vivo . the anti - angiogenic compounds may be used to treat inappropriate blood vessel formation ( neovascularisation ) such as the neovascularisation associated with debilitating forms of human blindness , including age - related macular degeneration ( amd ) and diabetic retinopathy ( dr ). additionally , this compound may have therapeutic benefits in cancer , by cutting off the blood supply to tumours or by inhibiting the secretion of angiogenic and / or inflammatory factors from a tumour . the compound may be administered to patients with diseases characterised by neovascularisation such as forms of progressive blindness that would benefit from stunting the growth of inappropriate new blood vessels , or cancer patients in which tumour growth can be halted by cutting off blood supply or by inhibiting the secretion of angiogenic and / or inflammatory factors from the tumour . the anti - angiogenic compounds described herein have the potential to offer patients effective , easily administered , safe and cost - effective treatments to prevent vision loss and tumour growth the compounds described herein effectively inhibit new vessel growth . in the case of anti - angiogenic treatments for the eye , the compounds have the potential to be administered in the conventional manner as an injection or as eye drops as their small chemical size facilitates absorption from the cornea unlike antibodies which require intravitreal injection . similar - sized small molecules have been shown to exhibit anti - angiogenic efficacy in the eye upon topical administration ( doukas et al ., 2008 ). topical administration of the compound , such as through eye drops , will eliminate the repeated injections that are required for the administration vegf antibodies will reduce the safety risks associated with repeated intra vitreal injections . furthermore , small molecule compounds will be cheaper to manufacture than antibodies and unlike antibodies , no potentially hazardous biological components are required to synthesise the compounds which will reduce the manufacturing costs and regulatory safety requirements . we have used the zebrafish model as an in vivo screen for chemical libraries as the small size and transparency of the zebra fish enables high - content screens in multi - well plate formats ( macrae and peterson , 2003 ; pichler et al ., 2003 ; peterson et al ., 2004 ; den hertog , 2005 ; zon and peterson , 2005 ). furthermore , many drugs have been shown to have comparable actions in humans and zebrafish including aspirin , warfarin , l - name , carbachol and diazepam ( goldsmith , 2004 ). to identify anti - angiogenic drugs we used a transgenic line of zebrafish that expresses a fluorescent reporter ( egfp ) specifically in vasculature ( tg ( fli1 : efgp )). this line was obtained from the zebrafish international resource center . our assay involved screening the effect of drugs in the library on the development of blood vessels in zebrafish . specifically , we looked at the integrity of vessels developing in the eye ( hyaloid vessels attached to the lens ) and in the trunk . from these screens we have identified lead compounds from a library of about 1600 compounds that exhibited reproducible anti - angiogenic activity in vivo . our characterisation of lead compounds was based on significant inhibition of hyaloid vessel formation in terms of pattern or primary branch number . the invention will be more clearly understood from the following examples thereof . all experiments were carried out under ethical approval granted by the ucd animal research ethics committee . tg ( fli1 : egfp ) zebrafish were maintained according to standard procedures on a 14 hr light / 10 hr dark cycle at 28 ° c . referring to the schematic of fig1 a , embryos were obtained by natural spawning and developmental stages established by time and morphological criteria . a chemical library containing numerous compounds was screened ( fig1 a ). in this specific example , the chemical library contained 5000 compounds of the diverset ™ collection from chembridge corp ., usa . at 24 hours post fertilisation ( hpf ), 5 embryos per well were placed in 400 ml of embryo medium / 1 % dmso and incubated with drug at 28 ° c . on a 14 h light / 10 h dark cycle . larvae were euthanised , and fixed in 4 % pfa at 4 ° c . overnight before analysis . ( fig1 a ). prior to analysis of the intraocular vasculature , the control and treated larvae were observed under an olympus szx16 stereo zoom microscope and screened for general malformations . overall patterning of the vasculature ( fin , gut and intersegmental vessels ) was examined for abnormalities ( fig7 b and 9a ). right lenses were dissected from the larvae and transferred to depression slides for observation under epi - fluorescence in the olympus szx16 and to cover - slip bottom petri - dishes for confocal microscopy in a zeiss uv510 meta lsm system ( 20 × and 40 × inverted objectives ). to achieve appropriate orientation , lenses were embedded in 10 % methyl - cellulose and manipulated with a tungsten needle ( 0 . 5 mm diameter ). patterning of the hyaloid vessels on the treated larval lenses was compared to dmso controls and the archetypal pattern previously described ( alvarez et al ., 2007 ; alvarez et al ., 2009 ). the number of primary vessels radiating from the back of the lens ( 3 - 4 main branches at 5 dpf in controls and previously described ), was counted and the average number was graphed for each drug . from screens of ˜ 1600 small molecules , four “ hits ” that inhibited the number of primary hyaloids vessels were identified ( fig1 b ). the chemical structures of these compounds are shown in fig2 . at 6 hours post fertilisation , 5 embryos per well were placed in 400 μl of embryo medium / 1 % dmso and incubated with a range of drug concentrations ( 0 . 1 - 10 μm ) at 28 ° c . on a 14 h light / 10 h dark cycle . larvae were manually dechorionated , euthanised , and fixed in 4 % pfa at 4 ° c . overnight before analysis . the larvae were then washed with pbs and transferred to depression slides for observation under epi - fluorescence in an olympus szx16 fluorescent microscope . the number of intersegmental vessels was counted and the average number was graphed for each drug . 11b and 11f inhibit developmental angiogenesis of zebrafish intersegmental vessels in a dose - dependent and statistically significant manner ( fig4 and 10 ). at the highest concentration ( 10 μm ), 11b was seen to inhibit intersegmental vessel formation by 44 %. 11f inhibited intersegmental vessel formation by 16 % at the highest concentration ( 10 μm ). to validate the “ hits ” from the library screen , dose - dependence experiments were conducted by screening the drugs in the assay described in example 2 above but at a range of increasing concentrations between 1 - 10 μm . larvae were maintained under the drug treatments at 28 ° c . on 14 hr light / 10 hr dark cycle until 5 days post fertilisation ( dpf ), when they were euthanised and fixed in 4 % pfa at 4 ° c . overnight before analysis . the average number of primary hyaloids branches was graphed for each concentration of drug . compounds 3d and 3f , two of the “ hits ” from the primary library screen , did not exhibit a dose - dependent response and were discarded . however , compounds 11b and 11f did inhibit developmental angiogenesis of the zebrafish hyaloid vasculature in a dose - dependent and statistically significant manner and were therefore chosen as lead drugs for further characterisation ( fig3 and 7 ). one of the lead compounds identified by the screen of chemical library was 11b which has the chemical name 2 -[ 2 - 2 - quinolinyl ) vinyl ] phenol and its structure shown below : the other lead compound identified was 11f which has the chemical name 1 -( 1 , 3 - benzodioxol - 5 - yl methyl )- 6 , 7 - dimethoxy - 4 -( 1 - methyl - 2 - pyrrolidinyl ) isoquinoline and its chemical structure is shown below . the anti - angiogenic efficacy of compounds 11b and 11f has been demonstrated in the eye , in vivo . these compounds do not induce toxic effects on the larvae , the gross morphology of the larvae is normal ( fig7 b ). further characterisation of compound 11b shows that the gross morphology of fish treated from 1 - 5 dpf is normal , that the morphology ( layering of the retina , size of retina / lens , presence of optic nerve ) of eyes in transverse sections of treated larvae is normal and that 11b has no significant effect on visual function ( fig9 ). visual function was assessed using the optokinetic response , a behavioural assay that tests the ability of larval zebrafish to track moving stripes ( brockerhoff 2006 ). in addition , to a dose - dependent anti - angiogenic activity ( fig7 ), compound 11b exerted a time - dependent effect ( fig8 ). for these experiments the compounds were screened in zebrafish larvae as described above except that the drug ( compound 11b ) was added at either day 1 , 2 , 3 or 4 , and all treated larvae were analysed at day 5 . finally , analyses of the intersegmental vessels of the zebrafish trunk indicated that compound 11b added from day 1 - 5 had no effect on the earlier formed vessels but it did effect developmental angiogenesis of hyaloid vessels ( fig7 ). this indicated that compound 11b has an effect on newly forming , but not existing vessels . to further validate the anti - angiogenic activity of compounds 11b and 11f , we tested several structurally related compounds in the zebrafish angiogenesis assay described above . the structures of compounds related to 11f are shown in fig5 and the structures of the compounds related to 11b are shown in fig1 . one of the compounds related to 11f , compound 11f - 522 , also inhibited developmental angiogenesis of the hyaloid vasculature in vivo ( fig6 ). the chemical name of 11f - 522 is 1 -( 1 , 3 - benzodioxol - 5 - yl )- 6 , 7 - dimethoxy - 2 - methyl - 1 , 2 , 3 , 4 - tetrahydroisoquinoline , the structure of 11f - 522 is shown below . compound 11b - 799 , related to 11b , also inhibits developmental angiogenesis of the hyaloid vasculature in vivo ( fig1 ). the chemical name of 11b - 799 is 4 -[ 2 -( 4 - quinolinyl ) vinyl ] phenol , the structure of 11b - 799 is shown below . to determine the anti - angiogenic activity of 11f - 522 enantiomers , we tested the r - and s - enantiomers in the zebrafish hyaloid angiogenesis assay described above . the structures of the 11f - 522 enantiomers are shown below . the chemical name of 11f - 522 is 1 -( 1 , 3 - benzodioxol - 5 - yl )- 6 , 7 - dimethoxy - 2 - methyl - 1 , 2 , 3 , 4 - tetrahydroisoquinoline , the structure of 11f - 522 is shown below . the structure of 11f - 522 enantiomer ( s )-(+)- 1 -( 1 , 3 - benzodioxol - 5 - yl )- 6 , 7 - dimethoxy - 2 - methyl - 1 , 2 , 3 , 4 - tetrahydroisoquinoline ( enantiomer 1 or 11f - 522s ) and the structure of 11f - 522 enantiomer ( r )-(−)- 1 -( 1 , 3 - benzodioxol - 5 - yl )- 6 , 7 - dimethoxy - 2 - methyl - 1 , 2 , 3 , 4 - tetrahydroisoquinoline ( enantiomer 2 or 11f - 522r ) are shown below . in the hyaloid angiogenesis assay 11f - 522 and 11f - 522r show a significant ( p & lt ; 0 . 05 ) reduction in the number of primary hyaloid vessels whereas 11f - 522s did not reduce primary hyaloid vessel number ( n ≧ 24 for all samples ) ( fig1 ). to demonstrate the anti - angiogenic potential of compounds 11b and 11f in human cells , we treated cultures of dermal - derived normal human microvascular endothelial cells ( hmvec - d ) with 11b or 11f or with the clinically used anti - angiogenic avastin . compounds 11b and 11f had no significant effect on the proliferation or migration potential of hmvec - d cultures ( fig1 ). however , compound 11b significantly inhibits the ability of hmvec - d cells to form tubules in vitro ( fig1 ). tubule formation is a key step in angiogenesis and this result indicates that compound 11b has anti - angiogenic activity upon human endothelial cells . hmvec - d were purchased from clonetics , san diego , calif ., usa . cells were maintained in complete endothelial cell medium ( clonetics ® egm ®- 2 - mv bulletkit ®) containing 500 ml of endothelial cell basal medium - 2 and supplemented with hegf , 0 . 5 ml ; hydrocortisone , 0 . 2 ml ; ga - 1000 , 0 . 5 ml ; fbs , 25 ml ; vegf , 0 . 5 ml ; hfgf - b , 2 . 0 ml ; r3 - igf - 1 , 0 . 5 ml ; ascorbic acid , 0 . 5 ml in a 37 ° c . humidified atmosphere of 5 % co2 / 95 % air . hmvec - d were used for experiments between passages 4 - 8 . the responses to different compound treatments were examined using a crystal violet - based proliferation assay to test the effects of the compounds on cell proliferation . hmvec - d were seeded in 96 - well plates in complete medium at a density of 25 , 000 cells / cm 2 and allowed to attach in a humidified atmosphere of 5 % co 2 , 95 % air and at 37 ° c . cells were treated with the compounds at different concentrations in replicates wells , and cell proliferation was evaluated after 24 h . at the specific time point medium was removed , cells were fixed with 1 % glutaraldehyde and stained with a 0 . 1 % crystal violet solution ( pro - lab diagnostics ) for 30 min . the plates were then washed extensively in water and air dried . cell - associated dye was extracted with a 1 % solution of triton x100 and the absorbance was determined at 550 nm using a plate reader ( multiskan ascent , lab systems ). similar to avastin , neither 11b nor 11f had a significant effect upon hmvec - d proliferation ( fig1 ). continuous monitoring of hmvec - d cultures cell migration was recorded using a cim - plate 16 ( rtca dp analyser , xcelligence system , roche ) with 5 % serum serving as chemoattractant and following the manufacturer &# 39 ; s protocol . each well is composed of an upper chamber , a membrane with an interdigitated gold electrode on the underside of the microporous membrane ( facing the lower chamber ) and a lower chamber . cells that have migrated to the lower chamber containing the chemoattractant attach to the gold electrodes and generate electrical impedance . impedance readings are taken from an electronic sensor plate and reflect their attachment levels . full strength egm growth medium was added as chemoattractant in the lower chamber , and serum free media was used instead for the negative controls wells . the upper chamber was then clicked into position and 30 μl of serum - free media were added to cover the membrane surface . the cim - plates were then placed in an incubator at 37 ° c . in 5 % co 2 humidified atmosphere for at least 1 h . in the meantime hmvec - d cultures were removed from culture , trypsinised , and resuspended in 1 % fbs egm growth medium . a background measurement was initially taken , then 75 × 10 4 cells / well were seeded with or without treatments in replicates and the plate left under a tissue culture hood at rt for 30 min to allow the cells to settle . the cim - plate was then loaded into a rtca dp analyser at 37 ° c . 5 % co 2 humidified atmosphere and the cell index ( ci ) was recorded every 15 min . on the second day the cell index were checked and plotted using the rtca software . similar to avastin , neither 11b nor 11f had a significant effect upon hmvec - d cell migration in vitro . matrigel ( becton dickenson ) basement membrane matrix was used to examine hmvec - d tube formation . matrigel ( 50 μl ) was plated in 96 well culture plate wells after thawing on ice and allowed to polymerise at 37 ° c . 5 % co 2 humidified atmosphere for 1 hr . hmvec - d were removed from culture , trypsinised , and resuspended in full strength egm growth medium . cells were seeded at a density of 50 × 10 4 cells / cm 2 and incubated for 20 hr at 37 ° c . 5 % co 2 humidified atmosphere . endothelial cell tubule formation was assessed using phase contrast microscopy and photographed . a connecting branch between two discrete ecs was counted as one tube . the tube analysis was determined from 3 sequential fields ( magnification × 10 ) focusing on the surface of the matrigel . avastin ( av ), the clinically used anti - angiogenic , and 11b significantly inhibited (˜ 20 - 24 % reduction ) tubule formation compared to their controls whereas 11f reduced tubule formation by 9 % compared to its control ( fig1 ). to assess the ability of compound 11b to inhibit ocular neovascularisation in mammalian eyes , 11b was tested in the mouse model of oxygen induced retinopathy ( oir ). 11b inhibited angiogenesis in a mouse model of ocular neovascularisation ( fig1 ). 11b was injected intravitreally into a mouse model of oxygen - induced retinopathy ( oir ). the oir model was generated by placing postnatal day 7 ( p7 ) mouse pups into hyperoxia ( 75 % oxygen ) for 5 days and then removing them to normoxia ( 21 %) on postnatal day 12 ( p12 ). the hyperoxic ( high oxygen ) environment causes retinal blood vessels to regress between p7 and p12 . the normoxic environment on p12 is a relatively hypoxic ( low oxygen ) environment for the mice and causes the growth of new blood vessels ( angiogenesis ). for all samples , eyes were enucleated , fixed , flat mounted and then stained with an isolectin antibody to visualise the blood vessels . blood vessels were quantified and those treated with lead compounds compared to controls . relative to the avascular area in the p17 control , intraocular injections of 11b show an ˜ 2 . 4 fold increase in avascular retina ( i . e . inhibition of ocular neovascularisation ) compared to the ˜ 1 . 6 fold increase in avascular retina observed with avastin ( fig1 ). 11b and 11f significantly reduce angiogenic / inflammatory factor secretion from human colorectal tumour explants to assess the anti - cancer potential of compounds 11b and 11f , we tested their ability to modulate the levels of angiogenic / inflammatory factors secreted from explants cultures of human colorectal cancers . human colorectal tumour samples were taken directly from the pathology laboratory after surgery once adequate material was taken for diagnostic testing . the tumour samples were washed and stored in dmso / tumour conditioning media ( tcm ). the samples were snap - frozen in liquid nitrogen and stored at − 80 ° c . until compound testing was performed . prior to compound testing , the tumours were thawed and incubated in fresh tcm for 24 hours . the explants were then treated with 11b , 11f and avastin at 1 μm and 10 μm concentrations for 72 hours . the tcm solutions were collected and stored at − 20 ° c . and the remaining tumour explants were snap - frozen in liquid nitrogen and stored at − 80 ° c . the protein content of each tumour sample was determined using the bca protein assay . elisa was used to determine the levels of vegf , il - 8 , mcp - 1 , gro - α , il - 1β and il - 6 . the secretion data were normalised according to the tumour sample &# 39 ; s protein content . avastin , a clinically used anti - angiogenic , has significant effects on the levels of vascular endothelial growth factor ( vegf ) and interleukin 6 ( il - 6 ) secreted by the human tumour explants ( fig1 ). 11b significantly reduced the levels of vegf , il - 6 and interleukin 1 beta ( il - 1β ) secreted by the human tumour explants ( fig1 ). 11f significantly reduced the levels of vegf and il - 1β , secreted by the human tumour explants ( fig1 ). the invention is not limited to the embodiment hereinbefore described , with reference to the accompanying drawings , which may be varied in construction and detail . alvarez y , astudillo o , jensen l , reynolds a l , waghorne n , brazil d p , cao y , o &# 39 ; connor j j , kennedy b n . 2009 . selective inhibition of retinal angiogenesis by targeting p13 kinase . plos one 4 : e7867 . alvarez y , cederlund m l , cottell d c , bill b r , ekker s c , torres - 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