Patent Application: US-8200102-A

Abstract:
the invention relates to compounds for topically influencing the activity of dipeptidyl peptidase of the general formula wherein a is an amino acid having at least one functional group in the side chain ; b is a chemical compound covalently bound to a functional group of the side chain of a , chosen from the group consisting of oligopeptides having a chain length of up to 20 amino acids , homopolymers of glycine consisting of up to 6 glycine monomers , and polyethylene glycols having molar masses of up to 20 000 g / mol ; and c is a group amide - bonded to a chosen from the group consisting of thiazolidine , pyrrolidine , cyanopyrrolidine , hydroxyproline , dehydroproline or piperidine . the invention further relates to the use of said compounds for targeted intervention in local immunological processes , as well as effective and targeted treatment of pathophysiological and physiological processes related thereto , inter alia .

Description:
throughout the description and the claims , the expression “ alkyl ” can denote a c 1 - 50 alkyl group , preferably a c 1 - 30 alkyl group , especially a c 8 - 12 alkyl group ; for example , an alkyl group may be a methyl , ethyl , propyl , isopropyl or butyl group . the expression “ alk ”, for example in the expression “ alkoxy ”, and the expression “ alkan ”, for example in the expression “ alkanoyl ”, are defined as for “ alkyl ”. aromatic compounds are preferably substituted or optionally unsubstituted phenyl , benzyl , naphthyl , biphenyl or anthracene groups , which preferably have at least 8 c atoms . the expression “ alkenyl ” can denote a c 2 - 10 alkenyl group , preferably a c 2 - 6 alkenyl group , which has the double bond ( s ) at any desired location and may be substituted or unsubstituted . the expression “ alkynyl ” can denote a c 2 - 10 alkynyl group , preferably a c 2 - 6 alkynyl group , which has the triple bond ( s ) at any desired location and may be substituted or unsubstituted . the expression “ substituted ” or substituent can denote any desired substitution by one or more , preferably one or two , alkyl , alkenyl , alkynyl , mono - or multi - valent acyl , alkanoyl , alkoxyalkanoyl or alkoxyalkyl groups ; the afore - mentioned substituents may in turn have one or more ( but preferably zero ) alkyl , alkenyl , alkynyl , mono - or multi - valent acyl , alkanoyl , alkoxyalkanoyl or alkoxyalkyl groups as side groups . organic amines , amides , alcohols or acids , each having from 8 to 50 c atoms , preferably from 10 to 20 c atoms , can have the formulae ( alkyl ) 2 n — or alkyl - nh —, — co — n ( alkyl ) 2 or — co — nh ( alkyl ), - alkyl - oh or - alkyl - cooh . despite an extended side chain function , the compounds according to the invention can still bind to the active center of the enzyme dipeptidyl peptidase iv and analogous enzymes but are no longer actively transported by the peptide transporter pept1 . the resulting reduced or greatly restricted transportability of the compounds according to the invention leads , in ideal manner , to local , topical inhibition of dp iv and of analogous enzymes . the compounds according to the invention or compounds used in accordance with the invention can be present or used , respectively , in the form of racemates or in the form of enantiomerically pure compounds , preferably in the l - threo or l - allo form with respect to a . local intervention in the regulation of peptide hormones by means of topical administration of specific dp iv - inhibitors accordingly makes it possible to avoid systemic side - effects caused by enteral or parenteral administration of dp iv - inhibitors , because only locally limited inhibition of dp iv activity takes place . systemic regulation processes or regulation processes in other tissues remain unaffected to a very considerable extent because rapid systemic distribution of the compounds is avoided . by extending / expanding the side chain modifications , for example beyond a number of seven carbon atoms , it is accordingly possible in accordance with the invention to obtain a dramatic reduction in transportability ( table 1 ). the examples in table 1 clearly show that , with increasing spatial size of the side chains , there is a reduction in the transportability of the substances . by spatially and sterically expanding the side chains , for example beyond the atom group size of a monosubstituted phenyl radical , hydroxylamine radical or amino acid residue , it is possible according to the invention to modify or suppress the transportability of the target substances . it is accordingly possible to influence dp iv activity in the living body in discriminating manner . by means of the invention , it is accordingly possible , on the one hand , to achieve effective action of the inhibitors in the tissue to be treated and , on the other hand , by virtue of locally limited , that is to say topical , administration of dp iv - inhibitors it is possible to avoid systemic actions of the inhibitors to a very considerable extent . it is accordingly possible to influence local physiological and pathophysiological processes ( inflammation , psoriasis , arthritis , autoimmune diseases , allergies ) effectively and with few side - effects . dp iv - inhibitors administered enterally and parenterally , that is to say orally and intravenously or subcutaneously , are distributed systemically and inhibit dp iv and analogous activities throughout the body . a series of bioactive peptide substrates of dp iv are , however , involved in the regulation of local signal cascades ( chemotaxis , inflammation , neurotransmission ). the side chain - modified dp iv - inhibitors according to the invention exhibit , surprisingly , high inhibitory potency , but are absorbed and transported hardly at all or not at all and consequently do not result in demonstrable systemic effects . the invention therefore makes available new dp iv - inhibitors and a novel approach for the use of dp iv - inhibitors in vivo . such inhibitors can be matched to the type of use by means of chemical modifications and / or formulations . for example , systemic distribution is made difficult or prevented by means of voluminous hydrophilic substitutions on the side chain . the inhibitors can be administered in pharmaceutical and cosmetic preparations . topical use encompasses local use of the inhibitors by direct application to the tissue to be treated ( e . g . skin , wounds , tumours ) by means of ointments , creams or cosmetics , and indirect application by means of effector - containing patches , dressings or the like , by application in parts of the body ( mouth , nose , ears , eyes , lungs ) in the form of drops , sprays , inhalations or the like , by direct injection into or around the tissue to be treated and by implantation of effector - containing materials . topical use further encompasses oral or anal administration of non - absorbable or not readily absorbable effectors of dipeptidyl peptidase iv or of dp iv - analogous sequences for the purpose of selectively influencing gastrointestinal dp iv . in accordance with the invention , there are especially used compounds wherein the oligopeptides have chain lengths of from 3 to 15 , especially from 4 to 10 , amino acids , and / or the polyethylene glycols have molar masses of at least 250 g / mol , preferably of at least 1500 g / mol and up to 15 , 000 g / mol , and / or the optionally substituted organic amines , amides , alcohols , acids or aromatic compounds have at least 12 c atoms and preferably up to 30 c atoms . furthermore , there are disclosed pharmaceutical and cosmetic compositions that comprise at least one compound according to the invention , optionally in combination with carriers or adjuvants customary per se . the compounds or pharmaceutical or cosmetic compositions according to the invention can be used for topically influencing the activity of dipeptidyl peptidase iv or of analogous enzymes , especially for the prophylaxis or therapy of diseases of the skin or mucosa , autoimmune diseases and inflammation such as , for example , psoriasis , allergies , arthritis , tumours or autoimmune diseases . the compounds and pharmaceutical or cosmetic compositions can be formulated and used in the form of an ointment , cream , cosmetic , patch , dressing , drops , spray , inhalation , implant or injection solution . the adjuvants used in accordance with the invention are known per se . the invention accordingly relates to the topical use of effectors of dipeptidyl peptidase iv and of dp iv - analogous enzyme activities and of dp iv - like proteins . topical use allows local modification of the activities of the afore - mentioned highly specific enzymes which are crucially involved in inactivation and activation of biologically active peptides ( chemokines , substance p , vip , phm , pacap , growth factors , inter alia ). targeted intervention in local immunological processes ( chemotaxis , inflammatory processes , autoimmune diseases ) is accordingly possible , as well as effective and targeted treatment of pathophysiological and physiological processes related thereto ( psoriasis , periodontitis , arthritis , allergies , inflammation ). the invention makes it possible for the inhibitors to be used simply and in high local concentrations . as a result of low systemic loading with the corresponding effectors , an influence on the incretin system or systemic immune response is avoided . side chain - modified glutamylthiazolidines having a structure h - glu ( x )- thia were synthesized , with polyethylene glycol or glycine oligomers of various chain lengths being used as x ( see method a for description of synthesis ). the binding characteristics of those derivatives and their transportability by the peptide transporter pept1 were investigated and the k i values with respect to dp iv were determined ( table 1 ). it was found , surprisingly , that the side chain modifications modify the binding characteristics to the compound only to a slight extent . in contrast , the ability of the inhibitors to be transported by the peptide transporter is dramatically diminished by the side chain modification . the said dp iv - inhibitors are therefore excellently suited to achieving locally limited ( topical ) inhibition of dp iv in the body . although the inhibitors have approximately equal k i values to dp iv ( table 1 ), plasma - dp iv is inhibited by the novel side chain - modified inhibitors much more slowly and to a much lesser extent overall . this means that the inhibitors are absorbed from the intestine much less readily or not at all . in the case of glu ( gly ) 5 - thia , especially , no systemic action of the orally administered active ingredient is detectable . those inhibitors may consequently act as basic structures for the synthesis of novel topically administrable dp iv - inhibitors without systemic action . reaction of boc - glu ( ome )— oh with thia * hcl according to method b ( see section 3 . 4 for methods ), hydrolysis of boc - glu ( ome )- thia according to method g . boc - glu - thia was modified at the y - carboxylic acid function by introducing radicals of varying size . the radicals were coupled by way of their amino group by forming an amide bond to the y - carboxylic acid function , with a variety of coupling methods being used depending on the radical . the following amino components were attached to boc - glu - thia using the method stated : coupling amino component methods ( see § 3 . 4 ) yields polyethylene glycol amine ( m r ≈ 8000 ) c 93 % h - gly - gly - gly - oh d + e 49 % h - gly - gly - gly - gly - gly - oh d + e 86 % in 2 cases , purification of the reaction products differs from the general description of synthesis : 1 . boc - glu ( glys )- thia : the product already precipitates out from the mixture on stirring overnight ; it is subsequently filtered off and washed with 0 . 1n hcl and copious amounts of water . it is then dried over p 4 0 10 in vacuo . 2 . boc - glu ( peg )- thia : in contrast to the general procedure , the starting materials for the synthesis are dissolved in a 500 - fold excess of dmf . after the reaction is complete , the dmf is completely removed in vacuo and the residue is dissolved in a large amount of methanol . after ether is poured on , to form an upper layer , the product precipitates out together with the unreacted peg . fine purification was carried out by preparative hplc separation on a gel filtration column ( pharmacia , sephadex g - 25 , 90 μm , 260 mm - 100 mm ; separating conditions : eluant : water ; flow rate : 5 ml / min ; λ = 220 nm .) the n - terminal boc protecting groups were cleaved off the compounds described in table 3 using method f . the substances modified with gly derivatives were purified by preparative hplc separation and are present as trifluoroacetates . the h - glu ( peg )- thia was purified on a gel filtration column in the same manner as the boc - protected precursor . 10 mmol of n - terminally protected amino acid or peptide are dissolved in 20 ml of absolute thf . the solution is cooled to − 15 ° c .± 2 ° c . with stirring in each case , 10 mmol of n - mm and 10 mmol of chloroformic acid isobutyl ester are added in succession , the stated temperature range being strictly adhered to . after approximately 6 min , 10 mmol of the amino component are added . when the amino component is a salt , a further 10 mmol of n - mm are then added to the reaction mixture . the reaction mixture is then stirred for 2 h in the cold state and overnight at room temperature . the reaction mixture is concentrated using a rotary evaporator , taken up in ea , washed with 5 % kh2s0 4 solution , saturated nahc0 3 solution and saturated nacl solution and dried over naso 4 . after removal of the solvent in vacuo , the compound is recrystallised from ea / pentane . method b : peptide bond attachment by the mixed anhydride method pivalic acid chloride as activation reagent [ 0079 ] 10 mmol of n - terminally protected amino acid or peptide are dissolved in 20 ml of absolute thf . the solution is cooled to 0 ° c . with stirring in each case , 10 mmol of n - mm and 10 mmol of pivalic acid chloride are added in succession , the stated temperature range being strictly adhered to . after approximately 6 min , the mixture is cooled to − 15 ° c . and , once the lower temperature has been reached , 10 mmol of the amino component are added . when the amino component is a salt , a further 10 mmol of n - mm are then added to the reaction mixture . the reaction mixture is then stirred for 2 h in the cold state and overnight at room temperature . further working up is carried out as in method a . 10 mmol of the n - terminally protected amino acid or peptide and 10 mmol of the c - terminally protected amino component are dissolved in 20 ml of absolute dmf . the solution is cooled to 0 ° c . with stirring in each case , 10 mmol of dipea and 10 mmol of tbtu are added in succession . the reaction mixture is stirred for one hour at 0 ° c . and then overnight at room temperature . the dmf is completely removed in vacuo and the product is worked up as described in method a . 10 mmol of n - terminally protected amino acid or peptide and 10 mmol of n - hydroxy - succinimide are dissolved in 20 ml of absolute thf . the solution is cooled to 0 ° c . and 10 mmol of dicyclohexylcarbodiimide are added , with stirring . the reaction mixture is stirred for a further 2 h at 0 ° c . and then overnight at room temperature . the resulting n , n ′- dicyclohexylurea is filtered off , the solvent is removed in vacuo and the remaining product is recrystallised from ea / pentane . 10 mmol of the c - terminally unprotected amino component are introduced into an nahco 3 solution ( 20 mmol in 20 ml of water ). at room temperature and with stirring , 10 mmol of the n - terminally protected n - hydroxysuccinimide ester dissolved in 10 ml of dioxane are slowly added dropwise . stirring of the reaction mixture is continued overnight and the solvent is then removed in vacuo . further working up is carried out as in method a . 3 ml of 1 . 1n hcl / glacial acetic acid ( method f1 ) or 3 ml of 1 . 1n hcl / dioxane ( method f2 ) or 3 ml of 50 % tfa in dcm ( method f3 ) are added to 1 mmol of boc - protected amino acid pyrrolidide , thiazolidide or peptide . the cleavage at rt is monitored by means of tlc . after the reaction is complete ( approximately 2 h ), the compound is precipitated out in the form of the hydrochloride using absolute diethyl ether , is isolated with suction and dried over p 4 o 10 in vacuo . using methanol / ether , the product is recrystallized or reprecipitated . 1 mmol of peptide methyl ester is dissolved in 10 ml of acetone and 11 ml of 0 . 1m naoh solution and stirred at rt . the course of the hydrolysis is monitored by means of tlc . after the reaction is complete , the acetone is removed in vacuo . the remaining aqueous solution is acidified , using concentrated kh2so4 solution , until a ph of 2 - 3 is reached . the product is then extracted several times using ea ; the combined ethyl acetate fractions are washed with saturated nacl solution and dried over naso 4 , and the solvent is removed in vacuo . crystallization from ea / pentane is carried out . ansorge , s ., schön , e ., and kunz , d . 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