Patent Application: US-65689800-A

Abstract:
the invention relates to non - peptidic compounds that possess bioactive properties , such as the ability to protect neuronal cells from otherwise lethal treatments or the ability to promote the growth or regeneration of neuronal cells . in part , the invention provides compounds that interact with or bind to a cyclophilin and compounds that have activity towards neuronal cells . methods for using the compounds , such as administering them to cells or animals or using them to treat neurodegenerative conditions , are specifically included .

Description:
one skilled in the art can refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques . these texts include current protocols in molecular biology ( ausubel , et al ., eds ., john wiley & amp ; sons , n . y ., and supplements through june 1999 ), current protocols in immunology ( coligan , et al ., eds ., john wiley and sons , n . y ., and supplements through june 1999 ), and current protocols in pharmacology ( enna et al ., eds ., john wiley & amp ; sons , n . y ., and supplements through june 1999 ) for example , each of which are specifically incorporated by reference in their entirety . these texts can also be referred to in making or using an aspect of the invention . as noted above , cyclosporin a was the first compound identified to bind a cyp . based on the cyclic structure of cyclosporin a , a number of large , usually cyclic peptides were developed as immunosuppressive compounds that bind cyp . now , unexpectedly , the inventors have found a non - peptidic class of cyp binding compounds with activity in neuronal cells . each of compounds 1 - 4 , 6 , 7 , and 10 - 31 significantly inhibit cyclophilin rotamase activity at a concentration of 10 μm or below , and many inhibit 50 % of cyclophilin rotamase activity at a concentration lower than 5 μm ( ic 50 ), some lower than 1 um . compounds 6 and 7 possess neurotrophic or neuroprotectant activity . these data demonstrate the broad range of possibilities for a number of structural elements in the compounds of the invention . indeed , a number of substituents are well tolerated . accordingly , the scope of the invention is not limited to those compounds specifically described by formulae i and ii and those depicted in this specification . by performing any one or more of the assays for detecting cyp binding , one skilled in the art can determine whether or not modifications to the r 1 - 5 groups , x or y groups , or the value of n for formulae i and ii , result in a cyp binding compound of this invention . the compounds of the invention can be prepared by a number of synthetic routes . the examples below detail schemes 1 to 4 and the preparation of specific compounds . however , one skilled in the art can modify the steps , reactants , and reaction conditions in the examples and schemes to arrive at numerous examples of compounds of the invention . in addition , if particular stereoisomers or mixtures are desired , the starting materials and / or reactants in the preparatory scheme can be selected and used accordingly . alternatively or in addition , particular intermediates can be purified or enriched by chromatographic or enzymatic methods , or by manipulating reaction conditions or selective crystallization , to generate particular final products or mixtures . one skilled in the art is familiar with numerous methods to selectively produce or enrich for desired stereoisomers or mixtures . all of the compounds of the examples , including the intermediates , are specifically included in the compounds of the invention and can be used in the methods of the invention . the compounds of the invention may be prepared as a salt or derivative . various salts and derivatives are known in the art and a non - limiting list of possible choices includes acid salts : acetate , adipate , alginate , aspartate , benzoate , benzenesulfonate , bisulfate butyrate , citrate , camphorate , camphorsulfonate , cyclopentanepropionate , digluconate , dodecylsulfate , ethanesulfonate , fumarate , glucoheptanoate , glycerophosphate , hemisulfate , heptanoate , hexanoate , hydrochloride , hydrobromide , hydroiodide , 2 - hydroxyethanesulfonate , lactate , maleate , methanesulfonate , 2 - naphthalenesulfonate , nicotinate , oxalate , mesylate , dimesylate , pamoate , pectinate , persulfate , 3 - phenylpropionate , phosphates , picrate , pivalate , propionate , succinate , sulfates , tartrate , thiocyanate , tosylate , and undecanoate . base salts may include : amine salts , ammonium salts , alkali metal salts such as sodium and potassium salts , alkaline earth metal salts such as calcium and magnesium salts , salts with organic bases such as dicyclohexylamine salts , n - methyl - d - glucosamine , and salts with amino acids , for example arginine or lysine . nitrogen - containing groups of the compound can be quaternized with agents as : alkyl halides , for example methyl , ethyl , propyl , and butyl chlorides , bromides , or iodides ; dialkyl sulfates , for example dimethyl , diethyl , dibutyl and diamyl sulfates , long chain halides , for example decyl , dodecly , lauryl , myristyl , or stearyl chlorides , bromides , or iodides ; and aralkyl halides , for example benzyl and phenethyl bromides , chlorides , or iodides . the skilled artisan is familiar with methods for producing and testing any suitable salt or derivative . ( see , for example , remington &# 39 ; s pharmaceutical sciences , mack publishing co ., easton , pa ., 18 th edition , specifically incorporated herein by reference .) in general , activity in the nervous system for a particular compound can be identified by assaying for the ability to promote neurite outgrowth , protect neurons from damage by chemical treatments , promote the growth of neurons or neuronal cells , recover lost or damaged motor , functional or cognitive ability associated with nervous tissue or organs of the nervous system , or regenerate neurons . these activities can be useful in treating , diagnosing , or prognosing a number of human disease conditions , including , but not limited to , parkinson &# 39 ; s disease , alzheimer &# 39 ; s disease , amyotrophic lateral sclerosis ( als ), traumatic injury , spinal cord injury , multiple sclerosis , diabetic neuropathy , neuropathy associated with medical treatments such as chemotherapy , ischemia or ischemia - induced injury , stroke , oxygen deprivation , retinopathies , peripheral neuropathies , and neuropathies associated with viral infection . a number of animal model assays and cell culture assays have been developed and can be relied on for their clinical relevance to disease treatments , including the human diseases noted above . each of the following references can be used as a source for these assays , and all of them are specifically incorporated herein by reference in their entirety for that purpose : steiner , et al ., pnas 94 : 2019 - 2024 ( 1997 ); hamilton , et al ., bioorgan . med . chem . lett . 7 : 1785 - 1790 ( 1997 ); mcmahon , et al ., curr . opin . neurobiol . 5 : 616 - 624 ( 1995 ); gash , et al ., nature 380 : 252 - 255 ( 1996 ); gerlach , et al ., eur . j . pharmacol .- mol . pharmacol . 208 : 273 - 286 ( 1991 ); apfel , et al ., brain res . 634 : 7 - 12 ( 1994 ); wang , et al ., j . pharmacol . exp . therap . 282 : 1084 - 1093 ( 1997 ); gold , et al ., exp . neurol . 147 : 269 - 278 ( 1997 ); hoffer et al ., j . neural transm . [ suppl .] 49 : 1 - 10 ( 1997 ); and lyons , et al ., pnas 91 : 3191 - 3195 ( 1994 ). preferred methods for detecting neuronal activity include a neuroprotective assay , in which a compound is tested for the ability to protect against treatment causing glutamate neurotoxicity . sensory neuronal cultures ( drg ) can also be assayed for neurite outgrowth , an assay for neurotrophic activity . cultured cells are treated with a compound of the invention and later assayed for the presence of new neurite fibers . immunohistochemistry can aid in the visualization and quantitation of neurites as compared to control . the compounds of the invention can also be used to promote the establishment or maintenance of tissue or cell cultures . similar to the use for promoting neuronal cell growth , the compounds can be added to primary , transformed , or established cell cultures . particularly in the case of neuronal cells , the compounds can induce growth in culture and extend the culture lifetime of cells . in addition to or in the alternative to the activity in neuronal or nervous system cells , the compounds of the invention bind cyp . a recognized method for assessing the affinity of the compound to cyclophilin is the rotamase inhibition assay . for this purpose , the following references are specifically incorporated by reference and can be relied on to make assays of rotamase inhibition : fischer , et al ., biomed . biochem . acta 43 : 1101 - 1112 ( 1984 ); kofron , et al ., biochem . 30 : 6127 - 6134 ( 1991 ); kofron et al ., j . am . chem . soc . 114 : 2670 - 2675 ( 1992 ); harrison et al ., biochem . 29 : 3813 - 3816 ( 1990 ); lang et al ., nature 329 : 268 - 270 ( 1987 ); mucke et al ., biochem . 31 : 7848 - 7854 ( 1992 ); schonbrunner et al ., j . biol . chem . 266 : 3630 - 3635 ( 1991 ); hsu et al ., j . am . chem . soc . 112 : 6745 - 6747 ( 1990 ); and justice et al ., biochem . biophys . res . commun . 171 : 445 - 450 ( 1990 ). additional uses for the compounds , which may or may not relate to cyp binding , are also included in the methods of the invention . for example , the compounds may be used to promote hair growth ( see , for example , maurer , et al . am . j . pathol . 150 ( 4 ): 143 - 341 ( 1997 )). the compounds may also be used to treat or effect mitochondrial disorders , metabolic disorders , diabetes , or vision loss . also , the compounds can be used to treat viral infections , such as with an hiv virus or influenza virus . the compounds of the invention have utility in pharmacological compositions for the treatment and prevention of various neurodegenerative conditions or for various in vitro and cell culture treatments . the compounds may also have utility in pharmacological compositions for the treatment and prevention of hiv - infection , promotion of hair growth , immunosuppression , mitochondrial disorders , traumatic injury to nervous tissue , or conditions associated with optic nerve damage . the compounds of the invention may be prepared as a salt or derivative , as described above . a compound of the invention can be administered to an animal or human patient by itself or in pharmaceutical compositions where it is mixed with suitable carriers or excipients , at doses to treat or ameliorate various conditions . a therapeutically effective dose refers to that amount of the compound sufficient to effect an activity in a nerve or neuronal cell , or produce a detectable change in a cell or organism . therapeutically effective doses may be administered alone or as adjunctive therapy in combination with other treatments for hiv infection or associated diseases . techniques for the formulation and administration of the compounds of the instant application may be found in remington &# 39 ; s pharmaceutical sciences , mack publishing co ., easton , pa ., 18 th edition ( 1990 ). suitable routes of administration may , for example , include oral , rectal , transmucosal , buccal , or intestinal administration ; parenteral delivery , including intramuscular , subcutaneous , intramedullary injections , as well as intrathecal , direct intraventricular , intravenous , intraperitoneal , intranasal , or intraocular injections , and optionally in a depot or sustained release formulation . furthermore , one may administer the agent of the present invention in a targeted drug delivery system , for example in a liposome coated with an antibody . the liposomes will be targeted to and taken up selectively by cells expressing the appropriate antigen . the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known , e . g ., by means of conventional mixing , dissolving , emulsifying , encapsulating , entrapping , or lyophilizing processes . pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries , which facilitate processing of the active compounds into preparations , which can thus be used pharmaceutically . for injection , the agents of the invention may be formulated in aqueous solutions , preferably in physiologically compatible buffers , such as hank &# 39 ; s solution , ringer &# 39 ; s solution , or physiological saline buffer . for transmucosal or buccal administration , penetrants appropriate to the barrier to be permeated may be used in the formulation . such penetrants are known in the art . for oral administration , the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers , well known to those in the art . such carriers enable the compounds of the invention to be formulated as tablets , pills , capsules , liquids , quick - dissolving preparations , gels , syrups , slurries , suspensions and the like , for oral ingestion by a patient to be treated . pharmaceutical preparations for oral use can be obtained solid excipient , optionally grinding a resulting mixture , and processing the mixture of granules , after adding suitable auxiliaries , if desired , to obtain tablets . suitable excipients are , in particular , fillers such as sugars , including lactose , sucrose , mannitol , or sorbitol ; cellulose preparations such as , for example , maize starch , wheat starch , rice starch , potato starch , gelatin , gum tragacanth , methyl cellulose , hydroxypropylmethyl - cellulose , sodium carboxymethylcellulose , and / or polyvinylpyrrolidone ( pvp ). in general , the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients . examples of such carriers or excipients include but are not limited to calcium carbonate , calcium phosphate , various sugars , starches , cellulose derivatives , gelatin , and polymers such as polyethylene glycols . if desired , disintegrating agents may be added , such as the cross - linked polyvinyl pyrrolidone , agar , or alginic acid or a salt thereof such as sodium alginate or a number of others disintegrants ( see , for example , remington &# 39 ; s pharmaceutical sciences , mack publishing co ., easton , pa ., 18 th edition ( 1990 )). for administration by inhalation , the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer , with the use of a suitable propellant , e . g ., dichlorodifluoromethane , trichlorofluoromethane , dichlorotetrafluoroethane , carbon dioxide , pressurized air , or other suitable gas or mixture . in the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount . capsules and cartridges of e . g . gelatin for use in an inhaler or insulator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch . the compounds may be formulated for parenteral administration by injection , e . g ., by bolus injection or continuous infusion . pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water - soluble form . additionally , suspensions of the active compounds may be prepared as appropriate oily injection suspensions . suitable lipophilic solvents or vehicles include fatty oils such as sesame oil , or synthetic fatty acid esters , such as ethyl oleate or triglycerides , or liposomes . aqueous injection suspensions may contain substances , which increase the viscosity of the suspension , such as sodium carboxymethyl cellulose , sorbitol , or dextran . optionally , the suspension may also contain suitable stabilizers or agents , which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions . alternatively , the active ingredient may be in powder form for constitution with a suitable vehicle , e . g ., sterile pyrogen - free water , before use . the compounds may also be formulated in rectal compositions such as suppositories , e . g ., containing conventional suppository bases such as cocoa butter or other glycerides . in addition to the formulations described previously , the compounds may also be formulated as a depot preparation . such long acting formulations may be administered by implantation ( for example subcutaneously or intramuscularly ) or by intramuscular injection . thus , for example , the compounds may be formulated with suitable polymeric or hydrophobic materials ( for example as an emulsion in an acceptable oil ) or ion exchange resins , or as sparingly soluble derivatives , for example , as a sparingly soluble salt . liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs . certain organic solvents such as dimethylsulfoxide also may be employed , although usually at the cost of greater toxicity . additionally , the compounds may be delivered using a sustained - release system , such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent . various of sustained - release materials have been established and are well known by those skilled in the art . sustained - release capsules may , depending on their chemical nature , release the compounds for a few weeks up to over 100 days . depending on the chemical nature and the biological stability of the therapeutic reagent , additional strategies for stabilization may be employed . pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose , to effect a therapeutic benefit , or to effect a detectable change in the function of a cell , tissue , or organ . more specifically , a therapeutically effective amount means an amount effective to prevent the development of or to alleviate the existing symptoms of the subject being treated . determining the effective amount is well within the capability of those skilled in the art , especially in light of the detailed disclosure provided herein . toxicity and therapeutic efficacy of the compounds or compositions can be determined by standard pharmaceutical , pharmacological , and toxicological procedures in cell cultures or experimental animals . for example , numerous methods for determining the ld 50 ( the dose lethal to 50 % of the population ) and the ed 50 ( the dose therapeutically effective in 50 % of the population ) exist . the dose ratio between toxic and therapeutic effects is the therapeutic index , which can be expressed as the ratio between ld 50 and ed 50 . compounds and compositions exhibiting high therapeutic indices are preferred . the data obtained from cell culture assays or animal studies can be used in formulating a range of dosages for use in humans . ( see , for example , fingl et al ., in the pharmacological basis of therapeutics , ch . 1 p . 1 ( 1975 ).) a subset of the compounds of formula i may be prepared by reacting isocyanates with amines , as depicted in scheme 1 below . one skilled in the art is familiar with suitable reaction conditions and parameters . the synthesis of compound 9 , detailed below , illustrates . a mixture of phenyl - 1 , 3 - diisocyanate ( 0 . 1 mmol ), cyclohexylamine ( 0 . 25 mmol ), and diisopropylethylamine ( 0 . 1 mmol ) in 1 ml dichloromethane was stirred overnight . the resulting precipitate was washed with water and ether to provide ( cyclohexylamino )- n -{ 3 -[( cyclohexylamino ) carbonylamino ] phenyl } formamide ( gpi 7104 ) as a white solid , having 1 h nmr ( cdcl 3 , 400 mhz ) peaks as follows : δ0 . 88 ( m , 6h ); 1 . 07 ( m , 4h ); 1 . 28 ( m , 2h ); 1 . 41 ( m , 4h ); 1 . 59 ( m , 4h ); 6 . 73 ( m , 3h ); 7 . 17 ( s , 1h ); 7 . 52 ( m , 3h ); 7 . 78 ( m , 1h ). another subset of compounds of formula i may be prepared by the route depicted in scheme 2 below . one skilled in the art is familiar with suitable reaction conditions and parameters . the synthesis of compound 14 , detailed below , illustrates . a solution of 1 , 3 - bis - benzoyl chloride ( 0 . 99 g , 4 . 9 mmol ), 3 , 5 - dichloroaniline ( 1 . 58 g , 9 . 75 mmol ), and triethylamine ( 2 ml , 14 . 3 mmol ) in 50 ml of dichloromethane was stirred at room temperature overnight . the reaction mixture was washed with water and the resulting precipitated solid was collected by filtration to deliver 1 . 94 g of crude solid . recrystallization from acetone furnished analytically pure material with a mp = 260 - 262 ° c . and 1 h nmr ( dmso , 400 mhz ) peaks at : δ7 . 37 ( m , 2h ); 7 . 76 ( t , 1h , j = 7 . 8 ); 7 . 93 ( d , 4h , j = 1 . 8 ); 8 . 18 ( dd , 2h , j = 1 . 7 , 7 . 8 ); 8 . 52 ( d , 1h , j = 1 . 5 ); 10 . 73 ( s , 2h ). the theoretical atomic composition for c 20 h 12 n 2 o 2 cl 4 [ c , 52 . 90 ; h , 2 . 66 ; n , 6 . 17 ; cl , 31 . 23 ], compares favorably with that found experimentally [ c , 53 . 04 ; h , 2 . 72 ; n , 6 . 11 ; cl , 31 . 35 ]. a subset of the compounds of the invention with unsymmetrical substituents off of the cyclohexyl or phenyl ring structure of formulae i or ii may be prepared by scheme 3 , below . one skilled in the art is familiar with suitable reaction conditions and parameters . the synthesis of compounds 13 and 15 , detailed below , illustrates . synthesis of 1 - nitro - 3 -( 2 - phenylethoxy ) benzene . a stirred solution of 3 - nitrophenol ( 1 . 39 g , 10 mmol ), 1 - naphthaleneethanol ( 1 . 89 g , 11 mmol ), and triphenylphosphine ( 2 . 9 g , 11 mmol ) in 100 ml of tetrahydrofuran was treated with a solution of 2 . 22 g ( 11 mmol ) of diisopropylazodicarboxylate added dropwise . the resulting mixture was stirred overnight , and then concentrated and redissolved in a minimum amount of ethyl acetate . purification on a silica gel column , eluting with 10 % ethyl acetate in hexane , delivered 2 . 0 g of the ether . synthesis of 3 -( 2 - phenylethoxy ) phenylamine . to a refluxing suspension of 150 mg “ wet ” raney - nickel in 100 ml of ethanol containing 1 . 70 g ( 34 mmol ) of hydrazine hydrate was added the nitro compound . after refluxing for an additional 15 minutes , the mixture was cooled and filtered through celite to remove solids . removal of the solvent furnished the product as an orange oil , which crystallized on standing and was used without further purification for the next step . synthesis of naphthyl - n -[ 3 -( 2 - naphthylethoxy ) phenyl ] formamide , compound # 15 . a solution of 3 -( 2 - phenylethoxy ) phenylamine ( 200 mg , 0 . 76 mmol ), 1 - naphthoyl chloride ( 160 mg ; 0 . 84 mmol ), and triethylamine ( 0 . 2 ml , 1 . 43 mmol ) in 50 ml of dimethylacetamide was stirred overnight . the solvent was removed and the residue dissolved in ethyl acetate and washed with water and brine . after concentration , a clear oil was obtained that crystallized on standing . this was purified on a silica gel column , eluting with methylene chloride , to obtain 200 mg of compound # 15 as a white solid , mp = 191 - 193 ° c ., and 1 h nmr ( dmso , 400 mhz ) peaks of : δ3 . 56 ( t , 2h , j = 6 . 8 ); 4 . 31 ( t , 2h , j = 6 . 9 ); 6 . 71 ( dd , 1h , j = 2 . 1 , 8 . 1 ); 7 . 25 ( t , 1h , j = 8 . 0 ); 7 . 34 ( bd , 1h , j = 8 . 4 ); 7 . 47 - 8 . 22 ( m , 15h ); 10 . 52 ( s , 1h ). the theoretical atomic composition for c 29 h 23 no 2 [ c , 83 . 43 ; h , 5 . 55 ; n , 3 . 35 ] compares favorably to that found experimentally [ c , 83 . 29 ; h , 5 . 69 ; n , 3 . 39 ]. synthesis of [ 3 -( 2 - naphthylethoxy ) phenyl ]( naphthylsulfonyl ) amine , compound # 13 . a solution of 3 -( 2 - phenylethoxy ) phenylamine ( 200 mg , 0 . 76 mmol ), 1 - naphthylsulfonyl chloride ( 190 mg , 0 . 84 mmol ), and triethylamine ( 0 . 2 ml , 1 . 43 mmol ) was stirred overnight and worked up as described in the previous example . purification of the crude product delivered 210 mg of compound 13 , mp = 165 - 167 ° c ., and 1 h nmr ( dmso , 400 mhz ) peaks of : δ3 . 42 ( t , 2h , j = 6 . 8 ); 4 . 10 ( t , 2h , j = 6 . 9 ); 6 . 48 - 6 . 60 ( m , 3h ); 7 . 01 ( t , 1h , j = 8 . 1 ); 7 . 40 - 8 . 20 ( m , 13h ); 8 . 70 ( d , 1h , j = 8 . 6 ); 10 . 68 ( s , 1h ). the theoretical atomic composition for c 28 h 23 nso 3 [ c , 74 . 15 ; h , 5 . 11 ; n , 3 . 09 ; s , 7 . 07 ] compares favorably with that found experimentally [ c , 73 . 88 ; h , 5 . 05 ; n , 3 . 06 ; s , 7 . 03 ]. additional examples of compounds of the invention may be prepared as depicted in scheme 4 below . synthesis of 3 -[( tert - butoxy ) carbonylamino ] benzoic acid . 3 - aminobenzoic acid ( 5 . 0 g , 36 . 5 mmol ) was dissolved in 150 ml of 2n naoh . dioxane ( 100 ml ) was added , followed by 9 . 6 g ( 44 mmol ) of tert - butyl dicarbonate added slowly , with stirring . after the addition was complete , the mixture was stirred overnight . it was diluted with water and washed with ether ( 3 portions ). the aqueous phase was acidified with 20 % citric acid , and the resulting purplish solid was collected by filtration and recrystallized from ethyl acetate to obtain 1 . 6 g of the boc - protected amine . synthesis of { 3 -[( tert - butoxy ) carbonylamino ] phenyl }- n -( naphthylmethyl ) formamide . a solution of 3 -[( tert - butoxy ) carbonylamino ] benzoic acid ( 250 mg , 1 . 05 mmol ), 1 - naphthylmethylamine ( 170 mg , 1 . 05 mmol ), diethyl cyanophosphonate ( 260 mg , 1 . 6 mmol ), and triethylamine ( 0 . 22 ml , 1 . 6 mmol ) in acetonitrile was stirred overnight . the solvent was evaporated , and the residue was partitioned between ethyl acetate and 1n hcl . the layers were separated , and the organic phase was washed twice more with 1n hcl , then 3 times each with water and brine . the solvent was removed in vacuo , and the crude product was purified on a silica gel column , eluting with 20 % ethyl acetate in hexane , to deliver 270 mg of the amide . synthesis of { 3 -[( 3 , 5 - dichlorophenyl ) carbonylamino ] phenyl }- n -( naphthylmethyl ) formamide , compound 16 . { 3 -[( tert - butoxy ) carbonylamino ] phenyl }- n -( naphthylmethyl ) formamide ( 270 mg , 0 . 72 mmol ) was dissolved in 25 ml of dichloromethane and treated with 7 ml of 2n hcl in ether . after stirring overnight , the precipitate was collected by filtration and dried under vacuum . the aniline ( 190 mg , 0 . 61 mmol ) was dissolved in dimethylacetamide ( 10 ml ), and 3 , 5 - dichlorobenzoyl chloride ( 130 mg , 0 . 61 mmol ) and triethylamine ( 0 . 5 ml ) were added and the resulting mixture was stirred overnight . the product was worked up as described above and recrystallized from ethyl acetate to provide compound 16 as a white crystalline solid , mp = 205 - 208 ° c ., and 1 h nmr ( dmso , 400 mhz ) peaks of : δ4 . 97 ( d , 2h , j = 5 . 76 ); 7 . 45 - 8 . 26 ( m , 14h ); 9 . 10 ( t , 1h , j = 5 . 76 ); 10 . 57 ( s , 1h ). the theoretical atomic composition for c 25 h 18 n 2 o 2 cl 2 [ c , 66 . 83 ; h , 4 . 04 ; n , 6 . 23 ; cl ; 15 . 78 ] compares favorably with that found experimentally [ c , 66 . 73 ; h , 4 . 15 ; n , 6 . 16 ; cl , 15 . 81 ]. a number of substrates for rotamase are known in the art or can be derived from those known . typically , the substrate contacts a sample containing a protein with rotamase activity and the conversion of the substrate is detected after a period of time . the method for detecting conversion of the substrate will vary with the particular substrate chosen . one method has been termed the k i test ( see harding , et al ., nature , 341 : 758 - 760 ( 1989 ); and holt et al ., j . am . chem . soc ., 115 : 9923 - 9938 ). the cis - trans isomerization of an alanine - proline bond in a model substrate , n - succinyl - ala - ala - pro - phe - p - nitroanilide , is monitored spectrophotometrically in a chymotrypsin - coupled assay . the action of chymotrypsin releases p - nitroaniline from only the trans form of the substrate . the amount of p - nitroaniline can be monitored in a spectrophotometer , for example . other methods of detecting the presence of p - nitroaniline can also be used . the inhibition of this reaction caused by different concentrations of inhibitor is determined and the data is analyzed as a change in first - order rate constant as a function of inhibitor concentration , which yield the k i value . the following are added to a plastic cuvette : 950 ml of ice cold assay buffer ( 25 mm hepes , ph 7 . 8 , 100 mm nacl ), 10 μl of cyp a ( 2 . 5 μm in 10 mm tris - cl ph 7 . 5 , 100 mm nacl , 1 mm dithiothreitol ), 25 μl of chymotrypsin ( 50 mg / ml in 1 mm hcl ), and 10 μl of test compound , at various concentrations , in dimethyl sulfoxide . the reaction is initiated by the addition of 5 μl of substrate ( succinyl - ala - phe - pro - phe - para - nitroanilide , 5 mg / ml in 470 mm licl in trifluoroethanol ). the absorbance at 390 nm versus time is monitored for 90 seconds using a spectrophotometer and the rate constants are determined from the absorbance versus time data files . data obtained for representative compounds are presented in the following table . the inhibition values refer to the percent of rotamase activity that is inhibited by the compound when the compound is present at a concentration of 10 μm . the higher the percentage , the more the compound inhibits rotamase , which in turn means the more active the compound is at binding or interacting with cyp . the ic 50 values refer to the concentration that inhibits 50 % of the rotamase activity in a sample . the lower the value , the more active the compound is at binding or interacting with cyp . while cyp a is used in these examples , other cyp proteins can be substituted . similar methods can be used with other immunophilins , such as the fkbps , to demonstrate the presence or absence of fkbp binding activity . preferred compounds have an ic 50 ≦ 1 μm for inhibition of cyclophilin rotamase activity . epecially preferred compounds may also have an ic 50 ≧ 10 μm , or ≧ 50 μm , for inhibition of fkbp rotamase activity . as noted above , a number of methods can be used to assay for the bioactivity of the compounds of the invention . these assays can be in vivo or in vitro methods . the examples below illustrate assays for the ability of the compounds to protect neuronal cells from toxic treatments and the ability of the compounds to elicit neuronal cell growth , regeneration , or neurite extension . spinal cord and dorsal root ganglion ( drg ) cells from adult mice can be isolated by micro - dissection . the spinal cord with attached drgs from an adult mouse ( 15 - 10 g ) is removed . spinal nerves are cut away using micro - dissection scissors and any excess material is trimmed until the drg is free . using sharp micro - dissecting scissors , a transverse cut is made in the peripheral nerve , leaving 1 - 2 mm attached , and the explant placed into petri dish and covered with plating media . when finished collecting all drgs , the spinal nerve is trimmed to about 1 mm in length . then embed the explant in 30 μl of reduced growth factor matrigel on a circular coverslip , and place in a 35 mm culture dish . cover the sensory ganglion explant with 2 mls of media . compounds , drugs or control solutions are added from 10 × stocks , and incubated at 37 ° c ., 5 % co 2 , 95 % humidity for 48 hrs . wash cultures twice with pbs , and fix with 10 % formalin for 30 minutes . wash the fixed cultures twice with pbs and store refrigerated in pbs . place cultures in block buffer ( 5 % horse serum , 5 % goat serum , 1 % triton x , pbs ph = 7 . 4 ) overnight , while rotating , at a temperature of 4 ° c . add primary antibody ( beta tubulin , sigma chemical co .) diluted in block buffer and incubate overnight at 4 ° c . wash 5 times with pbs and apply secondary antibody ( alexa 488 goat anti - mouse ) diluted in block buffer . incubate overnight at 4 ° c . wash 5 times with pbs and leave overnight at 4 degrees . coverslip the cultures and measure total neurite length from the end of the attached spinal nerve . lengths of all neurites are quantitated and compared to those present in vehicle - treated control drgs . all cultures were derived from postnatal day 8 ( p8 ) sprague - dawley rat lumbar spinal cord slices of 325 micron thickness . each experiment consisted of two 6 - well plates with 5 slices from 4 different animals per well . media changes were performed every 3 to 4 days . cultures were treated with tha [ l (−)- threo - 3 - hydroxyaspartic acid ; tocris cookson inc ., ballwin , mo .] at 200 μm + compound ( 10 μm ) after one week in culture . the control was an untreated sample with 0 . 1 % dmso as vehicle . the tha control was a tha treated sample with 0 . 1 % dsmo as vehicle . two wells were used per condition . one media change with new tha and compounds was performed . the experiment was stopped 6 to 8 days following drug treatment ( 13 - 15 total days in vitro , div ) as dictated by visual assessment of lesion , by fixation with 4 % paraformaldehyde / 0 . 1 m phosphate buffer for 30 minutes . slices were permeabilized with 100 % cold methanol for 10 minutes . slices were transferred to staining wells . the slices were blocked with 10 % hs / tbs . primary antibody incubation was overnight at 4 ° c . with smi - 32 antibody 1 : 5000 in 2 % hs / tbs . smi - 32 was specific towards unphosphorylated h neurofilament subunit . vectastain abc elite kit with rat absorbed anti - mouse secondary antibody was used with dab to stain the slices . the slices were mounted onto a slide and a coverslip was sealed with dpx mounting solution . quantification of surviving neurons was performed on a ziess axiovert microscope . neuronal survival was determined by observing an intact neuronal cell body with processes located ventrally of the central canal in each hemisphere . this correlated to laminae vii , viii and ix . each hemisphere was counted individually . the statistics were performed with statview software on a minimum of three different experiments per condition and significance was determined as compared to tha control . the percent of protection was determined from the average number of living neurons by the following equation : ( drug treatment condition − tha control )/( untreated control − tha control ). as noted above , the specific examples should not be interpreted as a limitation to the scope of the invention . instead , they are merely exemplary embodiments one skilled in the art would understand from the entire disclosure of this invention . each of the references cited below or in the text above can be relied on to make and use any aspect of this invention . while particular uses and references are discussed above , this should not be taken as a limitation on the use of any particular reference . all the references are specifically included into this text by reference , in their entirety . holt et al ., bioorg . med . chem . letters , 4 : 315 - 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