EP0693081A1 - Composes et compositions cycliques inhibant la resorption osseuse - Google Patents
Composes et compositions cycliques inhibant la resorption osseuseInfo
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- EP0693081A1 EP0693081A1 EP94913070A EP94913070A EP0693081A1 EP 0693081 A1 EP0693081 A1 EP 0693081A1 EP 94913070 A EP94913070 A EP 94913070A EP 94913070 A EP94913070 A EP 94913070A EP 0693081 A1 EP0693081 A1 EP 0693081A1
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- This invention relates to new cyclic compounds that inhibit excess bone resorption and methods for the production of said compounds. Described are also compositions containing said compounds associated with pharmaceutical suitable carriers and methods for the treatment of diseases characterized by excess bone resorption and/or restricted calcium excretion, which method comprises administering to a subject in need of such treatment a bone resorption inhibitory effective amount of any of the novel cyclic compounds or a mixture of said compounds, optionally together with a pharmaceutical suitable cairier.
- Typical diseases associated with the stimulation of bone resorption are osteoporosis, Paget disease of bones, humoral hypercalcemia of malignancy and metastatic bone disease. Increased bone resorption leads to brittle and weak bones which become susceptible to fracture and deformation. Hypercalcemia may lead to deposition of calcium within the kidneys and ultimately to impairment of kidney function.
- diseases associated with the stimulation of bone resorption can be treated in 2 ways: either preventively by inhibiting bone resorption at a stage when bone is still sufficiently abundant with estrogens, bisphosphonates and calcitonins; or curatively, after substantial bone loss and fractures have occurred, by stimulating bone formation with fluoride.
- IGF insulin-like growth factor
- PTH human parathyroid hormone
- PTHrP human parathyroid hormone-related protein
- TRSAW pentapeptide Thr-Arg-Ser-Ala-Trp
- A cyclic radical having 12-17 ring forming atoms
- B a spacer group, linked to the cyclic radical A via a carbon or nitrogen atom, having 1 to 6 carbon atoms and 0 to 2 nitrogen and 0 or 1 oxygen atoms in the backbone of the chain; wherein the carbon atoms can be substituted by oxo, hydroxy, sulfo, C 1 -C 4 alkyl, morpholino, amino, carboxy and/or by a radical of a naturally occurring amino acid or a radical of a peptide containing 2-5 naturally occurring amino acids; and the nitrogen atoms can be substituted by C 1 -C 4 alkyl, C r C 4 alkanoyl, e.g. formyl and acetyl, and/or by a radical of a naturally occurring amino acid or a radical of a peptide containing 2-5 naturally occurring amino acids;
- D aryl-C 1 -C 4 alkyl wherein the aryl radical is mono- or bicyclic and unsubstituted or substituted with OH, SH, NH 2 or halogen, e.g. F, Cl, Br, I;
- E NH 2 ; NH 2 substituted with C ⁇ alkyl or amino-C ⁇ alkyl; C 1 -C 4 alkyl substituted with one or more, especially one or two, amino groups; a five- or six-membered one or two nitrogen containing heterocyclic radical; or NH 2 substituted by an acyl radical of a naturally occurring amino acid or an acyl radical of a peptide containing 2-5 naturally occurring amino acids;
- B-D radicals are located above the plane assumed by the cyclic radical A and the radical E is located below the plane assumed by the cyclic radical A; or a pharmacologically acceptable salt thereof; is distinguished by an excellent stability and an inhibition of excess bone resorption.
- Cyclic radicals A embraced by the invention include for example crown ethers, steroids, porphyrines and cyclic peptides.
- the distance between the binding sites of the B-D-group to the cyclic radical and the E-group to the cyclic radical is 0.50 to 0.65 nm, preferred 0.55 to 0.60 nm
- the distance between the binding sites of the (CH 2 ) y -group to the cyclic radical and the E-group to the cyclic radical is 0.50 to 0.62 nm, preferred 0.55 to 0.60 nm
- the distance between the binding sites of the (CH 2 ) x -group to the cyclic radical and the B-D-group to the cyclic radical is 0.55 to 0.75 nm, preferred 0.60 to 0.70 nm.
- cyclic peptides (arranged head to tail) or peptides wherein the side chains of two amino acids or the side chain of one amino acid and the N- or C-terminus of another amino acid are connected directly or via a linking group.
- one or more, e.g. 1-5, genetically encoded amino acids can be replaced by non genetically encoded amino acids; or two or more, e.g. 2-3, amino acids can be replaced by a suitable ⁇ -amino-carboxylic acid which is substituted to fulfill the steric requirements of formula (1).
- the cyclic radical comprises a mixture of D- and L-f orms of amino acids.
- Amino acids are for example all naturally occurring amino acids, e.g. genetically encoded amino acids like alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine and valine; the D-forms of said amino acids or amino acids like 2-aminoadipinic acid, 3-aminoadipinic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoiso- butyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid
- the spacer group B can be a C r C 6 alkyl radical, e.g. methylene, ethylene, propylene, butylene, pentylene or hexylene.
- the spacer group can comprise up to two nitrogen and/or one oxygen atom in the backbone of the chain, e.g. -CH 2 -NH-, -CH 2 -CH 2 -NH-, -CH 2 -NH-CH 2 -.
- the spacer group B can be a radical of the formula -N(H or C 1 -C 4 alkyl)-(C 1 -C 4 alkyl) radical, e.g. -NH-CH 2 -, -N(CH 3 )-CH 2 -, -NH-CH 2 -CH 2 -, -N(CH 3 )-CH 2 -CH 2 - and -NH-CH(CH 3 )-CH 2 -.
- the spacer group has 2 to 6 atoms in the backbone and in a more preferred embodiment the spacer group is the backbone of an amino acid chain.
- One or more carbon or nitrogen atoms of this spacer group can be substituted by C 1 -C 4 alkyl like methyl or ethyl; carboxy and/or a radical of a naturally occurring amino acid or a radical of a peptide containing 2-5 naturally occurring amino acids.
- the amino acid radicals and the peptide radicals are preferably bound via an amide linkage.
- the carbon atoms in the spacer group can be additionally substituted by oxo; hydroxy; sulfo and amino.
- a part of the first amino acid can also be a member of the heterocyclic radical A.
- the aryl radical in D is, e.g. a five- or six-membered monocyclic radical or a bicyclic radical consisting of two five- or two six-membered rings or one five- and one six- membered ring.
- aryl radicals are phenyl, biphenyl, isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl and naphthyl.
- aryl radicals are unsubstituted or substituted by C r C 4 alkyl like methyl or ethyl, hydroxy, sulfo, amino and/or carboxy to give, e.g. the corresponding radicals of phenol, aniline, napthoquinone, phthalimide, phthalic acid, salicylic acid, sulfanilic acid, picolinic or nicotinic acid.
- Preferred radicals D are, e.g.
- radical D is the side chain of an aromatic amino acid like Trp, Phe, Tyr and His, wherein the side chain of Trp is especially preferred.
- the radical E is for example an amino group optionally substituted by one or two methyl, ethyl, propyl, butyl, aminomethyl or aminoethyl; or a five- or six-membered one or two nitrogen containing heterocyclic radical linked via a nitrogen atom to the cyclic radical A, e.g. piperidyl, 2-oxo-pyrrolidyl or piperazyl.
- the radical E can also be NH 2 substituted by an acyl radical of a naturally occurring amino acid or an acyl radical of a peptide containing 2-5 naturally occurring amino acids.
- the (CH 2 ) X and (CH 2 ) y -groups are for example unbranched chains such as methylene, ethylene, propylene and butylene.
- the ring forming atoms of the cyclic radical A are unsubstituted or substituted by small substituents like C r C 4 alkyl groups.
- Preferably said ring forming atoms are unsubstituted or substituted by methyl or ethyl.
- the basic compounds of formula (1) can form acid addition salts.
- Pharmaceutical acceptable acid addition salts are, for example, salts of compounds of the formula (1) with suitable mineral acids, such as hydrohalic acids, sulfuric acid or phosphoric acid, e.g. hydrochlorides, hydrobromides, sulfates, hydrogen sulfates or phosphates, salts with suitable aliphatic or aromatic sulfonic acids or N-substituted sulfamic acids, e.g.
- methane- sulfonates benzenesulfonates, p-toluenesulfonates or N-cyclohexylsulfamates (cyclamates), or salts with organic acids, such as lower alkanecarboxylic acids or optionally unsaturated or hydroxylated aliphatic dicarboxylic acids, e.g. acetates, oxalates, maleates, malonates, fumarates, malates or citrates. If possible, the formation of an inner salt between an amino group and a carboxyl group of a compound of the formula (1) is preferred.
- preferred 3 of the ring forming C-atoms can be replaced by oxygen, sulfur or nitrogen, which is preferred; and the ring forming atoms can additionally be substituted by up to 6, preferred up to 3, C r C 4 alkyl, oxo, hydroxy, sulfo and amino radicals, wherein methyl, ethyl or oxo is preferred.
- X C 1 -C 4 alkylene or C 1 -C alkenylene wherein up to 2 C-atoms, prefered is one, can be replaced by oxygen, sulfur, disulf ⁇ de, sulfoxy, sulfonyl or nitrogene, preferred is oxigen, sufur and disulfide.
- the C ⁇ alkylene or alkenylene group can be substituted by methyl, ethyl, oxo or amino.
- R a or R b radical of a naturally occurring amino acid or a radical of a peptide containing 2-5 naturally occurring amino acids, wherein the first or second amino acid after the ring forming atom is an amino acid carrying an aromatic side chain, e.g. tryptophane, phenylalanine, tyrosine or histidine; or C 1 -C 7 alkyl-aryl, wherein the C-atoms of the C j - ⁇ alkyl spacer group can be replaced by up to 3 nitrogen or oxygen, wherein nitrogen is preferred.
- the spacer group can be substituted by oxo; C 1 -C 4 alkyl wherein methyl and ethyl are preferred; amino, oxo and carboxy.
- R c , R d or R e hydroxy or C r C 4 alkyl substituted with hydroxy; preferred hydroxy, hydroxymethyl and hydroxyethyl; more preferred hydroxy and hydroxymethyl; and most preferred hydroxymethyl;
- R. or R k NH 2 ; NH 2 substituted with C r C 4 alkyl or amino-C r C 4 alkyl; C r C 4 alkyl substituted with one or more, especially one or two, amino groups; a five- or six-membered one or two nitrogen containing heterocyclic radical; or a radical of a naturally occurring amino acid or of a peptide containing 2-5 naturally occurring amino acids;
- a more preferred cyclic compound is a heterocyclic compound of the formula (2)
- R 2 -C r C 6 alkyl-aryl or -CO-NH-CH(C 1 -C 3 alkyl-aryl)R 1 ⁇ ; preferred is
- R 3 , R 4 , R 6 and g independently of each other are hydrogen or - talkyl preferred hydrogen, methyl or ethyl; more preferred hydrogen or methyl; and most preferred hydrogen;
- R 5 hydroxy or C 1 -C 4 alkyl substituted with hydroxy; preferred hydroxy, hydroxymethyl and hydroxyethyl; more preferred hydroxy and hydroxymethyl; and most preferred hydroxymethyl;
- -NH-CO-CH 2 -NH 2 or -NH-CH 2 -CH 2 -NH 2 unsubstituted or substituted with methyl or ethyl; preferred is NH 2 ; NH 2 substituted with C r C 2 alkyl; C r C 2 alkyl substituted with NH 2 ; -NH-CO-CH 2 -NH 2 or -NH-CO-CH(CH 3 )-NH 2 ; more preferred NH 2 ; -NH-CH 3 ; -CH 2 -NH 2 ; -CH 2 -CH 2 -NH 2 ; -NH-CO-CH 2 -NH 2 or -NH-CO-CH(CH 3 )-NH 2 ; and most preferred NH 2 ; -NH-CO-CH 2 -NH 2 or -NH-CO-CH(CH 3 )-NH 2 ; R j 1 hydrogen, -COOH, -CONH 2 , - C - N O or
- R 12 a radical of a naturally occurring amino acid or a radical of a peptide containing
- R 13 phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl; preferred phenyl, hydroxyphenyl or indolyl; and most preferred indolyl.
- R CH 2 , C 2 H 4 , S, CH 2 -S, CH 2 -S-S, S-S, NH, NH-CO, O, O-CH 2 , H 2 C-O, Se;
- R 2 or -CO-NH-CH(C r C 3 alkyl-R 13 )R n ;
- R 3 , R 4 , R 6 and R independently of each other are hydrogen, methyl or ethyl
- R ⁇ hydrogen, -COOH, -CONH 2 or -CO-R 12 ;
- R 12 a radical of a naturally occurring amino acid;
- R 13 phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl.
- Rj CH 2 , CH 2 -CH 2 , S, CH 2 -S, CH 2 -S-S, S-S, NH-CO, O, O-CH 2 , CH 2 -O;
- R 2 -C 3 -C 6 alkyl-R 13 or -CO-NH-CH(C 1 -C 3 alkyl-R 13 )R 11 ;
- R 3 , R 4 , R 6 and Rg independently of each other are hydrogen, methyl or ethyl
- R ⁇ hydrogen, -COOH, -CONH 2 or -CO-R 12 ;
- R 12 a radical of a naturally occurring amino acid;
- R 13 phenyl, hydroxyphenyl or indolyl.
- R i CH 2 , CH 2 -CH 2 , S, CH 2 -S, CH 2 -S-S, S-S, NH-CO, O, O-CH 2 , CH 2 -O;
- R 2 -C 3 -C 6 alkyl-R i3 or -CO-NH-CH(C 1 -C 3 alkyl-R 13 )R 11 ;
- R 3 , R 4 , R 6 and Rg independently of each other are hydrogen or methyl
- R 10 NH 2 ; -NH-CH 3 , -NH-CH 2 -CH 3 ; -CH 2 -NH 2 ; -CH 2 -CH 2 -NH 2 ; -NH-CO-CH 2 -NH 2 or -NH-CO-CH(CH 3 )-NH 2 ;
- R ⁇ hydrogen, -COOH, -CONH 2 or -CO-R 12 ;
- R 12 a radical of a naturally occurring amino acid;
- R 13 phenyl, hydroxyphenyl or indolyl;
- Ri CH 2 , CH 2 -CH 2 , S, CH 2 -S, CH 2 -S-S, S-S, NH-CO, O-CH 2 ;
- R 3 hydrogen or methyl
- R 4 , R 6 and Rg are hydrogen
- R n hydrogen, -COOH or -CONH 2 ;
- R 13 phenyl, hydroxyphenyl or indolyl;
- R j CH 2 , CH 2 -CH 2 , S, CH 2 -S, CH 2 -S-S, S-S, NH-CO, O-CH 2 ;
- R 2 -CO-NH-CH(CH 2 -R 13 )R n ;
- R 3 hydrogen or methyl;
- R 4 , R 6 and R 9 are hydrogen;
- R 5 hydroxy or hydroxymethyl;
- R 10 NH 2 ; -NH-CO-CH 2 -NH 2 or -NH-CO-CH(CH 3 )-NH 2 ; wherein NH 2 is most preferred;
- R n hydrogen, -COOH or -CONH 2 ;
- R 13 indolyl;
- Examples of the compounds according to the invention are those of formula (3), (4), (5),
- Another preferred cyclic compound is a heterocyclic compound of the formula (2) wherein
- R 2 NH 2 , C r C 4 alkyl substituted with NH 2 , — NH ; 0 r -CO-R 12 -NH 2 ; preferred
- R ⁇ hydrogen, NH 2 , -NH-CO(C r C 4 a-kyl) or -NH-R 12 ; preferred hydrogen, NH 2 ,
- R 12 a radical of a naturally occurring amino acid or a radical of a peptide containing 2-5 naturally occurring amino acids; preferred is the radical of a naturally occurring amino acid; wherein the amino acid radical or the peptide radical is preferably bound via an amide linkage;
- R 13 phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl; preferred phenyl, hydroxyphenyl or indolyl; and most preferred indolyl.
- R 2 CH 2 , C 2 H 4 , S, CH 2 -S, CH 2 -S-S, S-S-CH 2 , S-S, NH, NH-CO, O, Se, O-CH 2 , CH 2 -O;
- R 2 NH 2 ; C r C 4 alkyl substituted with NH 2 ; - N NH; -CO-NH-CH 2 -CO-NH 2 ;
- R 7 hydroxy or C 1 -C 3 alkyl substituted with hydroxy
- R 10 -NH-C r C 6 alkyl-R 13 or -NH-CO-CH(C r C 3 alkyl-R 13 )R n
- R n hydrogen, NH 2 , -NH-COCH 3 , -NH-COCH 2 -CH 3 or -NH-R 12
- R 12 a radical of a naturally occurring amino acid
- R 13 phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl.
- R j CH 2 , CH 2 -CH 2 , S, CH 2 -S, CH 2 -S-S, S-S-CH 2 , S-S, NH-CO, O, O-CH 2 , CH 2 -O;
- R 2 NH 2 ; C r C 4 alkyl substituted with NH 2 ; -N NH; -CO-NH-CH 2 -CO-NH 2 ;
- R j CH 2 , CH 2 -CH 2 , S, CH 2 -S, CH 2 -S-S, S-S-CH 2 , S-S, NH-CO, O, O-CH 2 , CH 2 -O;
- R 2 NH 2 ; -CH 2 -NH 2 ; -CH 2 -CH 2 -NH 2 ; -CO-NH-CH 2 -CO-NH 2 ;
- R j S, CH 2 -CH 2 , CH 2 -S, S-S, CH 2 -S-S, NH-CO, O-CH 2 , CH 2 -O;
- R 2 NH 2 , -CH 2 -NH 2 , -CH 2 -CH 2 -NH 2 , -CO-NH-CH 2 -CO-NH 2 ,
- R 3 , R 4 and R 8 hydrogen; one of the residues R 5 and R 6 is hydrogen and the other is
- R 7 hydroxy, hydroxymethyl or hydroxyethyl
- R 9 hydrogen or methyl
- R 10 -NH-CO-CH(C 1 -C 3 alkyl-R 13 )R 11
- R ⁇ hydrogen, NH 2 or -NH-COCH 3
- R 13 phenyl, hydroxyphenyl or indolyl
- R 2 -CH 2 -NH 2 , -CO-NH-CH 2 -CO-NH 2 or -CO-NH-CH 2 -CH 2 -NH 2 ;
- R 3 , R 4 , R 6 and R 8 hydrogen;
- R 7 hydroxy or hydroxymethyl;
- Rg hydrogen or methyl;
- R 10 -NH-CO-CH(CH 2 -R 13 )R 11 ;
- R ⁇ NH 2 or -NH-COCH 3 ;
- R 13 indolyl.
- Examples of the compounds according to the invention are those of formula (10) and (11)
- the compounds according to the invention are synthesized in a manner known per se.
- the synthesis of the compounds depends on the cyclic radical used. Crown ethers, steroids and porphyrines are commercially available or can be isolated by standard methods.
- the functional groups of the compounds can be introduced by conventional methods of organic chemistry. Cyclic peptides consisting of various amino acids, amino acid analogs or modified amino acids can be synthesized in a manner known per se.
- the process for the production of a cyclic peptide according to the invention comprises, for example, reacting an amide bond forming first fragment of a compound of formula (1) with a second amide bond forming fragment of a compound of formula (1), said first fragment and said second fragment being complementary to one another such that an amide bond is formed between said first and second fragments to result in said compound of formula (1), one of said first and second fragments containing a reactive free carboxy group and sulfoxy group, respectively, or a reactive carboxylic acid or sulfonic acid derivative thereof, and the other of said first and second fragments containing a free amino group or a reactive derivative thereof, wherein free functional groups in the mentioned fragments, with the exception of the two groups participating in the reaction, are, if necessary, in protected form, and removing protecting groups which may be present, and, if desired, converting a salt obtainable in accordance with the process into the free compound and/or converting a free compound obtainable in accordance with the process into a salt.
- Reactive carboxylic acid and sulfonic acid derivatives are especially reactive activated esters or reactive anhydrides, and also reactive cyclic amides; as mentioned, reactive carboxylic acid derivatives can be formed in situ.
- Reactive carboxylic acid and sulfonic acid derivatives are especially reactive activated esters or reactive anhydrides, and also reactive cyclic amides; as mentioned, reactive carboxylic acid derivatives can be formed in situ, e.g. by the use of a suitable condensing agent.
- esters of acids are especially esters that are unsaturated at the linking carbon atom of the esterifying radical, for example of the vinyl ester type, such as true vinyl esters (which can be obtained, for example, by the transesterification of a corresponding ester with vinyl acetate; activated vinyl ester method), carbamoylvinyl esters (which can be obtained, for example, by treating the corresponding acid with an isoxazolium reagent; 1,2-oxazolium or Woodward method), or 1 -lower alkoxy vinyl esters (which can be obtained, for example, by treating the corresponding acid with a lower alkoxy acetylene; ethoxyacetylene method), or esters of the amidino type, such as N,N'-disubstituted amidino esters (which can be obtained, for example, by treating the corresponding acid with a suitable N,N'-disubstituted carbodiimide, for example N,N'-diiso
- Activated esters are also, for example, suitable aryl esters, especially phenyl esters suitably substituted by electronattracting substituents (which esters can be obtained, for example, by treating the corresponding acid with a suitably substituted phenol, for example 4-nitrophenol, 4-methylsulfonylphenol, 2,4,5-trichlorophenol, 2,3,4,5,6-pentachlorophenol, pentafluorophenol or 4-phenyldiazo- phenol, in the presence of a condensing agent, such as N,N'-dicyclohexylcarbodiimide; activated aryl esters method) cyanomethyl esters (which can be obtained, for example, by treating the corresponding acid with chloroacetonitrile in the presence of a base; cyanomethyl esters method), suitable thioesters, especially phenylthio esters that are unsubstituted or substituted, for example, by nitro (which can be obtained, for example, by treating the corresponding acid
- Reactive acid anhydrides may be symmetrical-or preferably mixed anhydrides of these acids, for example anhydrides with inorganic acids, such as acid halides, especially acid chlorides (which can be obtained, for example, by treating the corresponding acid, including sulfonic acid, with a suitable halogenating agent, such as thionyl chloride, phosphorus pentachloride or oxalyl chloride; acid chloride method); azides (which can be obtained, for example, from an acid ester by way of the corresponding hydrazide and treatment thereof with nitrous acid; azide method), anhydrides with carbonic acid semi- derivatives, such as with corresponding esters, for example carbonic acid lower alkyl semi-esters (which can be obtained, for example, by treating the corresponding acid with haloformic, such as chloroformic, acid lower alkyl esters or with a 1 -lower alkoxy- carbonyl-2-lower alkoxy- 1,2-dihydroquinoline,
- Suitable cyclic amides are especially amides with five-membered diazacycles of aromatic character, such as amides with imidazoles, for example imidazole (which can be obtained, for example, by treating the corresponding acid with N,N'-carbonyld ⁇ midazole; imidazolide method) or pyrazoles, for example 3,5-dimethylpyrazole (which can be obtained, for example, by way of the acid hydrazide by treatment with acetylacetone; pyrazolide method).
- imidazole which can be obtained, for example, by treating the corresponding acid with N,N'-carbonyld ⁇ midazole; imidazolide method
- pyrazoles for example 3,5-dimethylpyrazole (which can be obtained, for example, by way of the acid hydrazide by treatment with acetylacetone; pyrazolide method).
- the carboxylic acid derivatives can also be formed in situ.
- N,N'-disubstituted amidino esters can be formed in situ by reacting a mixture of the complementary fragment having the free amino group and the peptide fragment having a free carboxy group in the presence of a suitable N,N-disubstituted carbodiimide, for example N,N-diisopropyl- or N,N'-dicyclohexyl-carbodiimide.
- amino or amido esters of acids can be formed in the presence of the amine to be acylated by reacting a mixture of the corresponding acid and amino starting materials in the presence of an N,N'-disubstituted carbodiimide, for example N,N'-dicyclohexyl- or N,N'-diisopropyl- carbodiimide, and in the presence of an N-hydroxyamine or N-hydroxyamide, for example N-hydroxysuccinimide, optionally in the presence of a suitable base, for example 4-dimethylaminopyridine.
- an N,N'-disubstituted carbodiimide for example N,N'-dicyclohexyl- or N,N'-diisopropyl- carbodiimide
- an N-hydroxyamine or N-hydroxyamide for example N-hydroxysuccinimide
- a suitable base for example 4-dimethylaminopyridine.
- the process according to the invention can also be carried out by reacting a fragment having a free carboxy group with the complementary fragment in which the amino group is present in reactive form; the amino group can be activated, for example, by reaction with a phosphite, for example diethyl chlorophosphite, 1,1-phenylene chloro- phosphite, ethyl dichlorophosphite, ethylene chlorophosphite or tetraethyl pyrophosphite, or with a suitable silylation agent, such as an organic halosilane, for example trimethyl- chlorosilane.
- a phosphite for example diethyl chlorophosphite, 1,1-phenylene chloro- phosphite, ethyl dichlorophosphite, ethylene chlorophosphite or tetraethyl pyrophosphite
- a suitable silylation agent such as
- the amino group can also be activated by bonding to halocarbonyl, for example chlorocarbonyl, or can be activated in the form of an isocyanate group.
- Functional groups in the mentioned fragments which, if they are not to participate in the reaction, are advantageously in protected form, are especially carboxy, amino and hydroxy groups, and also carbamoyl and guanidino groups.
- Hydroxy-protecting groups are, for example, acyl radicals, such as unsubstituted or substituted, for example halo-substituted, lower alkanoyl, such as 2,2-dichloroacetyl, or acyl radicals of carbonic acid semi-esters, especially tert.-butoxycarbonyl, unsubstituted or substituted benzyloxycarbonyl, for example 4-nitrobenzyloxycarbonyl, or diphenyl- methoxycarbonyl, or 2-halo-lower alkoxycarbonyl, such as 2,2,2-trichloroethoxycarbonyl, or formyl.
- acyl radicals such as unsubstituted or substituted, for example halo-substituted, lower alkanoyl, such as 2,2-dichloroacetyl, or acyl radicals of carbonic acid semi-esters, especially tert.-butoxycarbonyl, unsubstituted or substituted
- hydroxy-protecting groups are, for example, suitable etherifying groups, such as trityl, tert.-lower alkyl, for example tert.-butyl, 2-oxa- or 2-thia-aliphatic or -cyclo- aliphatic hydrocarbon radicals, especially 1 -lower alkoxy-lower alkyl or 1 -lower alkyl- thio-lower alkyl, for example methoxymethyl, 1-methoxyethyl, 1-ethoxyethyl, methylthio- methyl, 1-methylthioethyl or 1-ethylthioethyl, or 2-oxa- or 2-thia-cycloalkyl having 5 or 6 ring atoms, for example 2-tetrahydrofuryl or 2-tetrahydropyranyl or corresponding thia analogues, and also unsubstituted or substituted 1-phenyl-lower alkyl, such as unsubsti ⁇ tuted or substitute
- hydroxy-protecting groups are also organic silyl or stannyl radicals that preferably contain lower alkyl, especially methyl, and/or aryl, for example phenyl, as substituents, especially tri-lower alkylsilyl, especially trimethylsilyl, and also dimethyl- tert.-butyl-silyl, or correspondingly substituted stannyl, for example tri-n-butylstannyl.
- Thiol groups are protected in analogous manner, for example, with trityl, acetamido- methyl or tert. -butyl.
- Carboxy groups are preferably protected in esterified form, such ester groupings being readily cleavable under mild conditions.
- Carboxy groups protected in this manner contain as esterifying groups especially lower alkyl groups that are branched at the 1 -position or suitably substituted at the 1- or 2-position.
- Preferred carboxy groups in esterified form are, inter alia, tert.-lower alkoxycarbonyl, for example tert.-butoxycarbonyl, ⁇ -aryl-lower alkoxycarbonyl having one or two aryl radicals, these being phenyl radicals that are unsubstituted or substituted, for example, by lower alkyl, such as tert.-lower alkyl, for example tert.-butyl, lower alkoxy, such as methoxy, hydroxy, halogen, for example chlorine, nitro and or by phenyl, such as benzyloxycarbonyl that is unsubstituted or substituted, for example, as mentioned above, for example 4-methoxybenzyloxycarbonyl or 4-nitrobenzyloxycarbonyl, biphenylyl-lower alkoxycarbonyl in which biphenylyl substitutes the ⁇ -position, for example 2-(p-biphenylyl)-2-propoxycarbonyl
- Preferred protected carboxy groups are, for example, tert. -lower alkoxycarbonyl, such as tert.-butoxycarbonyl, and benzyloxycarbonyl that is unsubstituted or substituted, for example, as mentioned above, such as 4-methoxy- or 4-nitrobenzyloxycarbonyl, or diphenylmethoxycarbonyl, and also 2-(trimethylsilyl)-ethoxycarbonyl.
- a protected amino group can be, for example, in the form of a readily cleavable acyl- amino, arylmethylamino, etherified mercaptoamino, 2-acyllower alk-1-enylamino, silylamino or stannylamino group or in the form of an azido group.
- acyl is, for example, the corresponding radical of an organic carboxylic acid having, for example, up to 18 carbon atoms, especially an alkanecarboxylic acid that is unsubstituted or substituted, for example, by halogen or aryl, or of benzoic acid that is unsubstituted or substituted, for example, by halogen, for example chlorine, lower alkoxy, for example methoxy, nitro, and/or by phenyl, or of a carbonic acid semi-ester.
- halogen for example chlorine, lower alkoxy, for example methoxy, nitro, and/or by phenyl, or of a carbonic acid semi-ester.
- Such acyl groups are, for example, lower alkanoyl, such as formyl, acetyl or propionyl, halo-lower alkanoyl, such as 2-haloacetyl, especially 2-chloro-, 2-bromo-2-iodo-, 2,2,2-trifluoro- or 2,2,2-trichloroacetyl, benzoyl that is unsubstituted or substituted, for example, by halogen, lower alkoxy, nitro and/or by phenyl, for example benzoyl, 4-chlorobenzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl, or lower alkoxycarbonyl that is branched at the 1 -position of the lower alkyl radical or suitably substituted at the 1-or 2-position, especially tert.-lower alkoxycarbonyl, for example tert.-butoxycarbonyl, lower alkenyloxycarbonyl, for example allyloxycarbonyl,
- phenyl such as unsubstituted or substituted benzyloxycarbonyl, for example 4-nitrobenzyloxycarbonyl, biphenylyl-lower alkoxycarbonyl in which biphenylyl substitutes the ⁇ -position, for example 2-(p-biphenylyl)-2-propoxycarbonyl), or substituted diphenylmethoxycarbonyl, for example benzhydryloxycarbonyl or di-(4-methoxyphenyl)-methoxycarbonyl, 2,2-diarylethoxycarbonyl in which aryl is phenyl that is unsubstituted or substituted, for example, by nitro, such as 4-nitrophenyl, such as 2,2-di-(4-nitrophenyl)-ethoxycarbonyl, wherein the two aryl, for example phenyl, radicals
- acyl radicals suitable as amino-protecting groups are also corresponding radicals of organic phosphoric, phosphonic or phosphinic acids, such as di-lower alkylphosphoryl, for example dimethylphosphoryl, diethylphosphoryl, di-n-propylphosphoryl or d ⁇ sopro- pylphosphoryl, dicycloalkylphosphoryl, for example dicyclohexylphosphoryl, unsubsti ⁇ tuted or substituted diphenylphosphoryl, for example diphenylphosphoryl, di-(phenyl- lower alkyl)-phosphoryl that is unsubstituted or substituted, for example, by nitro, for example dibenzylphosphoryl or di-(4-nitrobenzyl)-phosphoryl, unsubstituted or substituted phenoxyphenylphosphonyl, for example phenoxyphenylphosphonyl, di-lower alkylphos- phinyl, for example diethylphosphinyl, or unsubstit
- arylmethylamino group which may be a mono-, di- or especially tri-arylmethyl- amino group
- the aryl radicals are especially unsubstituted or substituted phenyl radicals.
- Such groups are, for example, benzylamino, diphenylmethylamino and especially trityl- amino.
- An etherified mercapto group in an amino group protected by such a radical is especially arylthio or aryl-lower alkylthio in which aryl is especially phenyl that is unsubstituted or substituted, for example, by lower alkyl, such as methyl or tert.-butyl, lower alkoxy, such as methoxy, halogen, such as chlorine, and/or by nitro.
- a corresponding amino-protecting group is, for example, 4-nitrophenylthio.
- acyl is, for example, the corresponding radical of a lower alkanecarboxylic acid, of a benzoic acid that is unsubstituted or substituted, for example, by lower alkyl, such as methyl or tert.-butyl, lower alkoxy, such as methoxy, halogen, such as chlorine, and/or by nitro, or especially of a carbonic acid semi-ester, such as a carbonic acid lower alkyl semi-ester.
- lower alkyl such as methyl or tert.-butyl
- lower alkoxy such as methoxy
- halogen such as chlorine
- nitro or especially of a carbonic acid semi-ester, such as a carbonic acid lower alkyl semi-ester.
- Corresponding protecting groups are especially 1-lower alkanoylprop-l-en-2-yl, for example l-acetylprop-l-en-2-yl, or 1-lower alkoxycarbonylprop-l-en-2-yl, for example 1 -ethoxycarbonyl-prop- 1 -en-2-yl.
- An amino group can also be protected in protonated form; as corresponding anions there come into consideration especially those of strong inorganic acids, such as hydrohalic acids, for example the chloride or bromide anion, or organic sulfonic acids, such as p_-toluenesulfonic acid.
- Preferred amino-protecting groups are acyl radicals of carbonic acid semi-esters, especially tert.-butoxycarbonyl, allyloxycarbonyl, or benzyloxycarbonyl that is unsubstituted or substituted, for example as indicated, for example 4-nitrobenzyloxy ⁇ carbonyl, or diphenylmethoxycarbonyl, 9-fluorenyl-methoxycarbonyl, or 2-halo-lower alkoxycarbonyl, such as 2,2,2-trichloroethoxycarbonyl, and also trityl or formyl.
- Unsubstituted carbamoyl groups are protected, for example, in the form of N-(9- xanthenyl) derivatives or in the form of N-(mono-, di- or tri-arylmethyl) derivatives, in which aryl is especially phenyl that is unsubstituted or contains up to 5 identical or different substituents, preferably lower alkyl, such as methyl, or lower alkoxy, such as methoxy.
- arylmethyl protecting groups 4-methoxybenzyl, 2,4,6-trimethoxybenzyl, diphenylmethyl, di-(4-methoxy- phenyl)-methyl, di-(4-methylphenyl)-methyl and (4-methylphenyl)-([polymeric carrier]- phenyl)-methyl.
- a preferred carbamoyl-protecting group is trityl.
- Guanidino groups may be protected, for example, by protonation, or by nitro or by means of suitably substituted sulfonyl groups, such as arylsulfonyl in which aryl is phenyl that is unsubstituted or contains, for example, lower alkyl, such as methyl, or benzoheterocyclyl, such as chromanyl, that is unsubstituted or substituted, for example, by lower alkyl, such as methyl, and bonded by way of an aromatic carbon atom, such as 4-methoxy-2,3,5(tri- methyl)-phenylsulfonyl or 2,2,5,7,8-pentamethyl-6-chromanylsulfonyl.
- sulfonyl groups such as arylsulfonyl in which aryl is phenyl that is unsubstituted or contains, for example, lower alkyl, such as methyl, or benzoheterocyclyl
- a protecting group especially a carboxy- protecting group
- a polymeric carrier that is bonded to the functional group to be protected, especially to a carboxy group, which carrier is suitable especially for the so-called Merrifield peptide synthesis and can be readily removed.
- a polymeric carrier is, for example, preferably a polystyrene resin weakly crosslinked by copoly- merisation with divinylbenzene, which resin carries bridging members suitable for the reversible bonding of amino acid and peptide residues.
- these bridging members are especially methylene groups that are unsubstituted or substituted and that are bonded directly to aromatic radicals of the polystyrene resin.
- Substituents of the methylene groups are bonded to the methylene groups preferably by ether or ester groupings and contain suitable functional groupings that together with functional groups, especially carboxy groups, of the amino acid or peptide fragment, can form protected groups, especially corresponding carboxy groups, such as esterified carboxy groups.
- Such bridging members are, for example, the divalent radicals of 4-methoxybenzyl alcohols optionally containing in the ⁇ -position lower alkoxy, such as methoxy, or phenyl that is unsubstituted or substituted, for example in the o- and/or Deposition, for example by lower alkoxy, such as methoxy, in which 4-methoxybenzyl alcohols the carbon atom of the 4-methoxy group is bonded directly to a phenyl radical of the polystyrene resin, and the benzylic hydroxy group esterifies the carboxy function of the amino acid or of the peptide fragment.
- lower alkoxy such as methoxy
- the reaction to form the amide bond can be carried out in a manner known per se, the reaction conditions depending especially on whether and how the carboxy group that participates in the reaction has been activated, customarily in the presence of a suitable solvent or diluent or a mixture thereof, and, if necessary, in the presence of a condensing agent which, for example, if the carboxy group that participates in the reaction is present in the form of an anhydride, may also be a suitable acid-binding agent, with cooling or heating, for example in a temperature range of from approximately -30°C to approximately +150°C, especially from +10°C to +70°C, preferably from room temperature (approximately +20°C) to +50°C, in a closed reaction vessel and/or in the atmosphere of an inert gas, for example nitrogen.
- Customary condensing agents are, for example, carbodiimides, for example N,N'-diethyl-, N,N'-diisopropyl-, N,N'-dicyclohexyl- or N-ethyl-N'-(3-dimethylaminopropyl)-carbo- diimide, suitable carbonyl compounds, for example carbonyldiimidazole, or 1,2-oxa- zolium compounds, for example 2-ethyl-5-phenyl-l,2-oxazolium-3'-sulfonate and 2-tert.- butyl-5-methylisoxazolium perchlorate, a suitable acylamino compound, for example 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline, furthermore a uronium compound, for example 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium t
- Customary acid-binding agents are, for example, alkali metal carbonates or bicarbonates, for example sodium or potassium carbonate or bicarbonate (customarily together with a sulfate), or organic bases, such as customarily sterically hindered tri-lower alkylamines, for example N,N-diisopropyl-N-ethylamine.
- the above-mentioned Merrifield peptide synthesis is suitable especially for a semi-auto ⁇ matic or fully automatic synthesis of compounds of formula (2a), wherein amino acids and/or peptide fragments and/or other non-peptidic moieties in which functional groups not participating in the reaction are usually in protected form are linked to one another by way of amide groupings without isolation of the peptide fragments formed.
- One of the functional groups normally the terminal carboxy group present in the end peptide, is optionally bonded to a suitable polymeric carrier by a bridging member, as described.
- this process variant is carried out analogously to the customary synthesis of peptides, care being taken that, in the already synthesized (peptide) fragment that contains the polymeric carrier moiety, the freeing, from the protected group, of the functional group that participates in the reaction, usually the terminal amino group, is in each case carried out under conditions in which the protecting groups of the functional groups not participating in the reaction are retained.
- the removal of carboxy-, amino-, hydroxy-, carboxylic acid amide-, carbamoyl- and/or guanidino-protecting groups is carried out in a manner known per se, for example by means of ⁇ -elimination, solvolysis, especially hydrolysis (under acid or basic conditions), alcoholysis, acidolysis or treatment with a base, or by means of reduction, especially hydrogenolysis or chemical reduction, optionally in stages or simultaneously, it also being possible to use enzymatic methods.
- tert.-lower alkoxycarbonyl, or lower alkoxycarbonyl substituted in the 2-position by an organic silyl group or in the 1-position by lower alkoxy or lower alkylthio, or unsubstituted or substituted diphenylmethoxycarbonyl can be converted into free carboxy by acidolysis, for example by treatment with a suitable acid, such as a lower alkane ⁇ carboxylic acid which may contain halogen, for example formic acid or trifluoroacetic acid, with or without the addition of a nucleophilic compound, such as phenol or anisole.
- a suitable acid such as a lower alkane ⁇ carboxylic acid which may contain halogen, for example formic acid or trifluoroacetic acid
- Unsubstituted or substituted benzyloxycarbonyl can be freed, for example, by hydro ⁇ genolysis, that is to say by treatment with hydrogen in the presence of a metallic hydrogenation catalyst, such as a palladium catalyst
- a metallic hydrogenation catalyst such as a palladium catalyst
- suitably substituted benzyloxycarbonyl such as 4-nitrobenzyloxycarbonyl
- 2,2-Diarylethoxycarbonyl or 2-(9-fluorenyl)-ethoxycarbonyl groups can be cleaved under mild basic conditions, for example by treatment with piperidine.
- a reducing metal or metal salt as described above, it is also possible to convert 2-halo-lower alkoxycarbonyl (optionally after conversion of a 2-bromo-lower alkoxycarbonyl group into a corresponding 2-iodo-lower alkoxycarbonyl group) or aroylmethoxycarbonyl into free carboxy, it being possible to cleave aroyl- methoxycarbonyl also by treatment with a nucleophilic, preferably salt-forming, reagent, such as sodium thiophenolate or sodium iodide.
- Substituted 2-silylethoxycarbonyl can also be converted into free carboxy by treatment with a salt of hydrofluoric acid yielding the fluoride anion, such as an alkali metal fluoride, for example sodium or potassium fluoride, in the presence of a macrocyclic polyether ("crown ether"), or with a fluoride or an organic quaternary base, such as tetra-lower alkylammonium fluoride or tri-lower alkyl- arylammonium fluoride, for example tetraethylammonium fluoride or tetrabutyl- ammonium fluoride, in the presence of an aprotic polar solvent, such as dimethyl sulf oxide or N,N-dimethylacetamide.
- a salt of hydrofluoric acid yielding the fluoride anion, such as an alkali metal fluoride, for example sodium or potassium fluoride
- a macrocyclic polyether such as te
- a protected amino group is freed in a manner known per se and, depending on the nature of the protecting groups, by various methods, but preferably by solvolysis or reduction.
- 2-Halo-lower alkoxycarbonylamino (optionally after conversion of a 2-bromo-lower alkoxycarbonylamino group into a 2-iodo-lower alkoxycarbonylamino group), aroyl- methoxycarbonylamino or 4-nitrobenzyloxycarbonylamino can be cleaved, for example, by treatment with a suitable chemical reducing agent, such as zinc in the presence of a suitable carboxylic acid, such as aqueous acetic acid.
- a suitable chemical reducing agent such as zinc in the presence of a suitable carboxylic acid, such as aqueous acetic acid.
- Aroylmethoxycarbonylamino can also be cleaved by treatment with a nucleophilic, preferably salt-forming, reagent, such as sodium thiophenolate, and 4-nitrobenzyloxycarbonylamino also by treatment with an alkali metal dithionite, for example sodium dithionite.
- a nucleophilic, preferably salt-forming, reagent such as sodium thiophenolate
- 4-nitrobenzyloxycarbonylamino also by treatment with an alkali metal dithionite, for example sodium dithionite.
- Unsubstituted or substituted diphenylmethoxycarbonylamino, tert.-lower alkoxycarbonylamino or 2-trisubstituted silylethoxycarbonylamino can be cleaved by treatment with a suitable acid, such as a lower alkanecarboxylic acid that is unsubstituted or substituted, for example, by halogen, such as fluorine, for example formic acid or trifluoroacetic acid, and 2,2-diarylethoxy- carbonylamino, such as 2,2-di-(4-nitrophenyl)-ethoxycarbonylamino, 2-(4-methylsulfonyl- phenylsulfonyl)-ethoxycarbonyl and also 9-fluorenyl-methoxycarbonylamino by treatment with a suitable base, such as an aliphatic, preferably secondary, amine, for example piperidine.
- a suitable base such as an aliphatic
- the amino group can be freed from unsubstituted or substituted benzyloxycarbonylamino, for example, by hydrogenolysis, that is to say by treatment with hydrogen in the presence of a suitable hydrogenation catalyst, such as a palladium catalyst, from unsubstituted or substituted triarylmethylamino or formylamino, for example, by treatment with an acid, such as a mineral acid, for example hydrochloric acid, or an organic acid, for example formic, acetic or trifluoroacetic acid, in the presence or absence of water, and from an organic silylamino group, for example, by hydrolysis or alcoholysis.
- a suitable hydrogenation catalyst such as a palladium catalyst
- an acid such as a mineral acid, for example hydrochloric acid
- an organic acid for example formic, acetic or trifluoroacetic acid
- An amino group protected by 2-haloacetyl for example 2-chloroacetyl
- 2-chloroacetyl can be freed, for example, by treatment with thiourea in the presence of a base, or with a thiolate salt such as an alkali metal thiolate, of thiourea, and by subsequent solvolysis, such as alcoholysis or hydrolysis, of the resulting condensation product
- An amino group protected by 2-substituted silylethoxycarbonyl can also be converted into the free amino group by treatment with a salt of hydrofluoric acid yielding fluoride anions, as described above in connection with the freeing of a correspondingly protected carboxy group.
- Amino protected in the form of an azido group can be converted into free amino, for example, by reduction, for example by catalytic hydrogenation with hydrogen in the presence of a hydrogenation catalyst such as platinum oxide, palladium or Raney nickel, or alternatively by treatment with zinc in the presence of an acid, such as acetic acid.
- the catalytic hydrogenation can preferably be carried out in an inert solvent, such as an alcohol, for example methanol, tetrahydrofurane, or alternatively in water or a mixture of water and an organic solvent, such as an alcohol or dioxan, at approximately from 20°C to 25°C, or alternatively with cooling or heating.
- a hydroxy group protected by a suitable acyl group, an organic silyl group or by unsubstituted or substituted 1-phenyl-lower alkyl can be released analogously to a correspondingly protected amino group.
- a hydroxy group protected by 2,2-dichloroacetyl can be released, for example, by basic hydrolysis, and a hydroxy group etherified by tert- lower alkyl, for example tert-butyl, or by a 2-oxa- or 2-thia-aliphatic or -cycloaliphatic hydrocarbon radical can be released by acidolysis, for example by treatment with a mineral acid or a strong carboxylic acid, for example trifluoroacetic acid.
- a carboxylic acid amide group protected by 9-xanthenyl can be released, for example, by treatment with hydrogen bromide in glacial acetic acid or with hydrogen fluoride in the presence of anisole.
- a carboxylic acid amide group protected by mono-, di- or tri-aryl- methyl can be released, for example, by treatment with hydrogen fluoride in the presence of anisole; furthermore, a diphenylmethyl protecting group can be removed, for example, by hydrogenolysis in the presence of a palladium-on-carbon catalyst, and a di-(4-methoxy- phenyl)-methyl protecting group or a 2,4,6-trimethoxybenzyl protecting group can be removed, for example, by treatment with trifluoroacetic acid.
- Guanidino groups protected by organic sulfonyl groups such as 4-methylphenylsulfonyl- guanidino or 2,2,5,7,8-pentamethyl-6-chromanylsulfonylguanidino, can be released, for example, by treatment with a suitable acid, such as trifluoroacetic acid.
- a suitable acid such as trifluoroacetic acid.
- Guanidino groups protected by nitro can be released, for example, by hydrogenolysis in the presence of palladium-on-carbon catalyst or cationic reduction.
- a protected functional group especially a corresponding carboxy group, in which the protecting group simultaneously acts as a carrier material in the mentioned Merrifield peptide synthesis, can be cleaved in a manner known per se, for example as described above.
- a correspondingly esterified carboxy group that is bonded to the polymeric carrier material by way of a suitable bridging member is cleaved in accordance with the nature of the bridging member.
- a carboxy group bonded to the polymeric carrier material by way of an ester grouping having an activated benzylic bridging member for example a 4-methoxybenzyloxycarbonyl group, in which the carbon atom of the methoxy group is bonded, for example, to a phenyl radical of the polystyrene resin weakly crosslinked with divinylbenzene, can be released analogously to the above-mentioned unsubstituted or substituted benzyloxycarbonyl groups, for example by treatment with a suitable acid, such as trifluoroacetic acid.
- a suitable acid such as trifluoroacetic acid
- the protecting groups can be selected in order to facilitate the removal of more than one of these protecting groups at the same time, for example by acidolysis, such as by treatment with trifluoroacetic acid or formic acid, or by reduction, such as by treatment with zinc and acetic acid, or with hydrogen and a hydrogenation catalyst, such as a palladium-on-carbon catalyst.
- the cyclization is normally performed after the complete synthesis of the linear precursor, e.g. by formation of a peptide bond, as described above, between the corresponding amino acids and subsequent removal of the remaining protecting groups. If the cyclization is based on a disulfide or sulfide bond, the protecting groups are removed usually before the oxidative ring formation, e.g. via oxygen, normal air, iodine, disodiumdisulfide, 1,2-diiodo-ethane or other oxidizing agents.
- the formed disulfide bond can be modified further, e.g. by desulfurization to a thioether radical and oxidation.
- the invention relates also to a pharmaceutical composition which consists of one or more of the compounds of formula (1) in a pharmaceutically effective amount, or which comprises said compounds together with conventional auxiliaries, typically carriers and diluents.
- compositions of this invention are those for enteral, oral, rectal and parenteral administration, as for subcutaneous, intravenous or intraperitoneal administration to warm blooded animals, said compositions containing the pharmacological active compound alone or together with a pharmacologically acceptable carrier.
- the daily dosage, the carrier, the frequency and route of administration will depend on the age and individual condition of the patient as well as on the mode of administration and the individual activity of the selected compound.
- a bone resorption inhibitory effective amount of a compound of the formula (1) for a patient with of about 70 kg weight may comprise from 1 ⁇ g to 1 g or more.
- the dosage units may comprise about 10 ⁇ g to 10 mg, preferably 10 ⁇ g to 500 ⁇ g and more preferably 10 ⁇ g to 100 ⁇ g of the active compound.
- novel pharmaceutical compositions contain from about 10 to 80 %, preferably from 20 to about 60 %, of the active compound.
- Pharmaceutical compositions for enteral or parenteral administration are typically those in dosage unit forms such as drag ⁇ es, tablets, capsules or suppositories, and also ampoules. These dosage forms are prepared in a manner known per se, typically by conventional mixing, granulating, confectioning, dissolving or lyophilising methods.
- Suitable carriers are especially fillers such as sugars, conveniently lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, typically tricalcium phosphate or calcium hydrogen phosphate, and also binders such as starch pastes, conveniently using maize, corn, rice or potato starch, gelatin, tragacanth, methyl cellulose and/or poly vinyl pyrrolidone, and/or, if desired, disintegrators such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
- fillers such as sugars, conveniently lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, typically tricalcium phosphate or calcium hydrogen phosphate, and also binders such as starch pastes, conveniently using maize, corn, rice or potato
- Excipients are in particular glidants, flow control agents and lubricants, conveniently silica, talcum, stearic acid or salts thereof, typically magnesium stearate or calcium stearate, and/or polyethylene glycol.
- Drag ⁇ e cores can be provided with suitable non-enteric or enteric coatings, typically using concentrated sugar solutions which may contain gum arabic, talcum, polyvinyl- pyrrolidone, polyethylene glycol and/or titanium dioxide, shellac solutions in suitable organic -solvents or mixtures of solvents or, for the preparation of enteric coatings, solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxy- propyl methyl cellulose phthalate. Dyes or pigments can be added to the tablets or drag ⁇ e coatings, conveniently to identify or indicate different doses of active compound.
- compositions for oral administration are dry-filled capsules made of gelatin and also soft-sealed capsules consisting of gelatin and a plasticiser such as glycerol or sorbitol.
- the dry-filled capsules can contain the active ingredient in the form of granules, conveniently in admixture with fillers such as lactose, binders such as starches, and/or glidants such as talcum or magnesium stearate, and with or without stabilisers.
- the active ingredient is preferably dissolved or suspended in a suitable liquid, typically a fatty oil, paraffin oil or a liquid polyethylene glycol, to which a stabiliser can also be added.
- Suitable pharmaceutical compositions for rectal administration are typically suppositories, which consist of a combination of the active compound with a suppository base.
- suitable suppository bases are natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols and higher alkanols. It is also possible to use gelatin capsules for rectal administration that contain a combination of the active compound with a base substance.
- Suitable base substances are typically liquid triglycerides, polyethgylene glycol or paraffin hydrocarbons.
- compositions for parenteral administration are preferred, conveniently in the form of aqueous solutions of non toxic salts or suspensions of the active compound, for example oily injection suspensions using suitable lipophilic solvents or vehicles such as fatty oils, typically sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or aqueous injection suspensions which may contain viscosity increasing substances, conveniently sodium carboxymethyl cellulose, sorbitol and/or dextran, and also with or without stabilisers.
- suitable lipophilic solvents or vehicles such as fatty oils, typically sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides
- viscosity increasing substances conveniently sodium carboxymethyl cellulose, sorbitol and/or dextran, and also with or without stabilisers.
- the invention also relates to the use of the compound of formula (1), preferably in the form of pharmaceutical compositions.
- the dosage of the active compound will depend on the species of the warm-blooded animal, on the age and individual condition of the patient, and also on the mode of application.
- Another embodiment of the invention relates to compound of formula (1) for use in a method for the therapeutic treatment of the human or animal body.
- the inventive compounds can be used for the treatment of excess bone resorption e.g. osteoporosis, Paget' s disease of bone, humoral hypercalcemia or malignancy as well as metastic bone diseases.
- excess bone resorption e.g. osteoporosis, Paget' s disease of bone, humoral hypercalcemia or malignancy as well as metastic bone diseases.
- DCCI dicyclohexylcarbodiimide
- DIB AH diisobutyl aluminum hydride
- DICD diisopropylcarbodiimide
- DIPE diisopropylether
- DIPEA diisopropylethylamine
- HOBt 1 -hy droxy-benzotriazol
- NArg glycine derivative N-substituted with an arginine-side chain
- TBTU 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborat
- TCA trichloroacetic acid
- Trt trityl
- MALDI matrix-assisted laser desorption ionization mass spectrometry
- Tlc Thin-layer chromatography on silica (Merck-Kieselgel 60, F254 plates),
- CM mixture of chloroform and methanol (85:15 v/v);
- the synthesis starts with a Fmoc-'C-terminal amino acid'-p-benzyl oxybenzyl ester polystyrene resin (1 % crosslinked, 'Wang-resin, Novabiochem, Laeufelfingen, Switzerland).
- a fully automatic peptide synthesis apparatus is used, that is suitable for alternate removal of the amino-protecting groups, in the present case the Fmoc-group, and coupling of the Fmoc-amino acid derivatives without isolation of the peptide/resin intermediates obtainable at each step.
- Trifunctional amino acids are introduced as correspondingly protected derivatives of L- or D-configuration: Fmoc-Ser(But), Fmoc-Cys(Trt), Fmoc-Arg(Pmc), Boc-Dpr(Fmoc), Fmoc-Trp(Boc) or Fmoc-Trp.
- the penultimate Fmoc amino acid is coupled with the last amino acid on the resin, and then the other Fmoc amino acids are coupled in steps, in the appropriate sequence.
- the individual steps are carried out in accordance with the following scheme, approximately 20 ml of the washing liquids being used in each case, with the individual operations, and the reaction mixture being shaken regularly.
- TAB hydroxy-trialkoxy-benzhydryl-resin
- Fmoc-Cys(Trt)-Arg(Pmc)-Ser(But)-D-Cys(Trt)-Trp-resin by sequential coupling of Fmoc-D-Cys(Trt), Fmoc-Ser(But), Fmoc-Arg(Pmc) and Fmoc-Cys(Trt); wherein "resin” denotes the carboxy group-esterifying polystyrene (1 % crosslinked with divinylbenzene)-methoxy-4-phenylmethoxy radical.
- the resin After removal of the Fmoc protecting group at the N-terminal cysteine residue, and extensive washing with DMA and isopropanol as described above, the resin is dried in vacuo (1.45 g).
- the combined filtrates and washing liquids are concentrated under reduced pressure to a volume of about 2 ml, and the crude peptide is precipitated by the addition of 15 ml of a 1:1 mixture (v/v) of DIPE and petroleum ether (low-boiling).
- the precipitate is isolated by filtration, washed with 5 ml of the precipitation mixture and dried under reduced pressure.
- the solid 64 mg is dissolved in 4 ml of the above mentioned cleavage mixture and kept for two hours, precipitated and dried under reduced pressure.
- the main fraction with a retention time of approximately 11 minutes, is collected, concentrated under reduced pressure and filtrated through an ion-exchange column (15 ml), AG1-X8 (Bio-Rad), preloaded with AcOH, for exchanging TFA with AcOH.
- the column is extensively washed with water and the combined fractions (40 ml) are lyophilized.
- the compound with the formula (3) is obtained as a colorless, fluffy powder.
- HPLC column measuring 4.6x250 mm, Nucleosil 7C 18 (10 nm) manufactured by
- 0.1 % TFA is used as eluant (A) and a 0.1 % solution of TFA in CH 3 CN is used as eluant (B).
- the linear gradient is 10 % B to 90 % B in
- linear intermediate of the compound of the formula (4) is prepared in an analogues manner as described in example 1.1 and 1.2, but L-homo-cysteine is incorporated in place of L-cysteine (Fmoc-homo-Cys(Trt) from Bachem).
- N-benzyl-N-3-phthalimidopropylglycinebenzylester 17.4 g (39.3 mmol) of N-benzyl-N-3-phthalimidopropylglycinebenzylester is suspended in 100 ml of 6N HC1. The suspension is stirred under reflux for 6 hours. On cooling phthalic acid separates from the reaction mixture. The phthalic acid is filtered off and washed with 4N HC1. The filtrate is evaporated to a small volume (about 20 ml) and several times coevaporated with methanol. The oily residue is dissolved with 60 ml of ethanol and the excess HC1 is removed by the addition of propylene oxide. N-3-aminopropyl-N-benzylglycine is obtained by the addition of acetone. The colorless solid is filtered and washed with acetone and diethyl ether and dried in a desiccator over potassium hydroxide.
- the obtained foam (N-(2,2,5,7,8-pentamethylchroman- 6-sulfonyl)-N'-(3-glycinyl)propylguanidine) is dried under high vacuum and is used for the next step without further purification.
- the remaining aqueous phase is acidified to pH 2 with 4N HC1 and again extracted with ethyl acetate (3x 30 ml).
- the combined organic phases are dried over sodium sulfate and evaporated to dryness.
- the purified product is obtained by chromatography on 20 g silica gel (70 - 230 mesh) with dichloromethane/methanol 100:1 to 100:10 to give N-(2,2,5,7,8-pentamethylchroman-6- sulfonyl)-N'-(3-N-9-fluorenylmethoxycarbonylglycinyl)propylguanidine (Fmoc-NArg(Pmc)).
- the preparation of Fmoc-NArg(Pmc) has been described by Simon et al., Proc. Natl. Acad. Sci USA (1992), 89, 9367-9371.
- linear intermediate of the compound of the formula (5) is prepared in an analogues manner as described in example 1.1 and 1.2, but NArg is incorporated in place of Arg.
- 70 mg of the linear precursor are dissolved in 1000 ml water and the pH of the solution is adjusted to 8.0 by addition of concentrated ammonia (ca. 0.5 ml). Air is bubbled through the solution for 22 hours. After addition of 15 ml AcOH the solution is concentrated and lyophilized.
- concentrated ammonia ca. 0.5 ml
- the crude material is purified with HPLC and converted to the acetate as described in example 1.3.
- the compound of the formula (5) is obtained as a fluffy white powder.
- Fluorenyl-methoxy- carbonyl-N-hydroxy-succinimidate (Novabiochem) is dissolved in 250 ml DMA and this solution is added to the copper-complex and slow stirring during 0.5 hour.
- the pH is kept at 8-9 by addition of triethylamine (total 6 ml).
- the solution is kept for 1.5 hour.
- the mixture is concentrated to 40 ml under reduced pressure at 30-40°C and under stirring slowly added to 950 ml water (0-5°C). After stirring for 1 hour, the precipitate is filtered, washed with water (180 ml) and dried over P 2 O 5 : 10.3 g. After treatment with 50 ml DIPE, the solid is filtrated and dried under reduced pressure: 8.9 g of blue-colored of Fmoc-Dpr-copper-complex is obtained.
- the complex (12.46 mmol) is dissolved in 1100 ml of a mixture of ethanol and water (1:1 v/v), and 4.0 g ethylene-diamino-tetra-acetic acid (EDTA) (13.71 mmol) are added.
- EDTA ethylene-diamino-tetra-acetic acid
- the solution is cooled to 0-5°C and after 15 hours, the precipitate is isolated by filtration and dried under reduced pressure. After treatment with 40 ml isopropanole at 50°C, the suspension is maintained at 0-5°C for 1 hour and filtrated. The solid is dried under reduced pressure: 2.76 g white powder.
- Tic CMW main spot with one minor by-component R f ca. 0.25.
- Tic CMW single spot R f ca. 0.55.
- ⁇ -NMR is compatible with the structure, chemical shifts in ppm: tert. butyl (Boc) 1.1,
- the compound is synthesized starting from 1.36 g Fmoc-TAB-amide-resin (Novabiochem) (0.60 mmol) by sequential coupling of Fmoc-Trp(Boc) and Fmoc-D-Asp(OBut) as described in example 1.1.
- the resulting resin is dried under reduced pressure: 4.7 g.
- Example 4.4 Cleavage of Fmoc-D-Asp-Trp-amide from the TAB-amide-resin and subsequent removal of the tert-butyl protecting groups
- the colorless solid is treated with a mixture of 20 ml TFA and ethanedithiole (98:2 v/v) for 20 minutes.
- the solution is concentrated to ca. 10-15 ml and the product is precipitated and isolated as described above.
- the crude material (ca. 0.836 g) is dissolved in 120 ml of a mixture of CH 3 CN and DMF (1:1 v/v) and kept for 15 hours. After concentration under reduced pressure, the product is precipitated as described above, and dried under reduced pressure: 0.663 g.
- Tic CMW single spot R f ca. 0.5. Analytical HPLC as described, retention time 24.0 min. MALDI, positive mode, 539.9 (calculated: 541.6).
- Example 4.5 Coupling of the Fmoc-D-Asp-Trp-amid to the TAB -ester-resin via the Asp-side chain.
- the reaction mixture is then shaken for 4 hours.
- the resin is washed as follows: 6 times with DMA (30 ml each), 5 times with isopropanole (30 ml each).
- the resin is dried under reduced pressure: 2.5 g (with traces of solvents).
- the solid-phase attached protected peptide is synthesized in analogy to example 1.1 by sequential coupling of Fmoc-D-Cys(Trt), Fmoc-Ser(But), Fmoc-Arg(Pmc) and Boc-Cys(Acm).
- the cleavage at the aforementioned resin is performed in an analogous manner as described in example 1.2 to give 90 mg.
- Purification with HPLC is accomplished as in example 1.3, except that the gradient is running from 10% to 60% during 30 minutes.
- the peak with the retention time of 10.5 minutes is collected, concentrated and filtrated through an acetate ion-exchanger as described in example 1.3.
- After lyophilization the compound of the formula (7) is obtained as a white powder.
- the sightly yellow enolate solution is stirred for 5 min at -78°C and then 0.568 g of the iodinated compound according to example 6.1.6, dissolved in 1 ml of THF is added.
- the reaction mixture is warmed to -40°C and stirred at this temperature for 4 hours, then partitioned between saturated ammonium chloride solution and ether, the aq. layer is separated and extracted twice with ether, the combined ether extracts are dried over sodium sulfate and concentrated in vacuo. Purification of the residue is carried out by flash chromatography (hexane/ether 8:2).
- ⁇ -NMRtCDClg 8.92 (s, 1H);7.89 (d, 2H); 7.60 (d, 2H); 7.45-7.30 (m,2H); 7.20 (t,lH); 7.12 (t, IH); 7.00 (s, IH), 6.28 (s, IH); 5.85 (s, IH); 4.35 (d, 2H); 4.15 (t, IH); 3.83 (m, 3H); 3.65 (m, 2H); 3.00 (t, 2H); 1.80-1.00 (m, 8H); 1.62 (s, 3H); 1.46 (s, 9H).
- the compound of the formula (8.1) is attached to the resin in an analogue manner as described in example 4.5.
- ⁇ -NMRtCD ⁇ D 4.32 (m, IH); 4.18 (m, IH); 3.90 (m, IH); 3.60 (m, 2H); 3.20 (m, 2H); 2.68 (t, 2H), 2.57 (s, 3H); 2.56 (s, 3H); 2.10 (s, 3H); 1.80 (t, 2H); 1.80-1.20 (m, 10H); 1.43 (s, 3H); 1.38 (s, 9H); 1.30 (s,9H); 1.18 (m, 8H).
- the catalyst is removed by filtration and the filtrate is evaporated to give the pure product as a colorless oil.
- Fmoc-D-Cys(Trt) (0.24 mmol) are dissolved in 0.4 ml DMA (dest), 2 ml DCE and 0.40 g TAB-ester-resin (0.24 mmol) (Novabiochem) are added under slow stirring. The mixture is cooled to 0-5°C and a solution of 0.1 g DCCI (0.48 mmol) in 0.2 ml DMA and 0.06 ml DCE is added. After 5 minutes at 0-5°C a solution of 5.9 mg DMAP (0.048 mmol) in 0.4 ml DCE is added. After 20 minutes at 0-5°C 27.3 ⁇ l N-methylmorpholine is added.
- the Fmoc-group is removed and the terminal amino group is acetylated with the capping agent as described above. After final washings with isopropanole, the resin is dried under reduced pressure: 0.38 g.
- MALDI, positive mode, 1746.5 (calculated: 1746.0).
- the solid is dissolved in 4 ml of the above mentioned cleavage mixture and kept for two hours, precipitated and dried under reduced pressure: 43 mg white powder.
- the crude peptide is dissolved in a mixture of 4.5 ml CH 3 CN/water (1:9 v/v) and 0.1 ml AcOH and subjected to high pressure liquid chromatography (HPLC) under the following conditions: the column, measuring 20x250 mm, Nucleosil 7C 18 (10 nm) manufactured by Machery-Nagel, Dueren, Germany; 0.1 % TFA is used as eluent (A) and a 0.1 % solution of TFA in CH 3 CN is used as eluent (B).
- the linear gradient is 10 % B to 90 % B in 30 minutes, the throughput speed 18 ml min and the detection is at 215 nm wavelength.
- the main fraction with a retention time of approximately 16 minutes, is collected concentrated under reduced pressure and filtrated through an ion-exchange column (15 ml), AG1-X8 (Bio-Rad), preloaded with AcOH, for exchanging TFA with AcOH.
- the column is extensively washed with water and the combined fractions (40 ml) are lyophilized.
- the compound with the formula (10) is obtained as a colorless, white powder.
- Fmoc-Arg(Pmc) is attached to the resin in an analogue manner as described in example 8.1.
- Fmoc-D-Arg(Pmc)-TAB-ester-resin 0.30 g Fmoc-D-Arg(Pmc)-TAB-ester-resin (0.19 mmol) is subjected to a solid-phase synthesis analogues to process example 8.2, attaching in a sequential manner the following amino acid derivatives: Fmoc-D-Ser(But), Fmoc-Cys(Trt) and Fmoc-D-Trp. At the end of the synthesis the Fmoc-group is removed and the terminal amino group is acetylated with the capping agent described in example 8.2. After final washings with isopropanole, the resin is dried under reduced pressure: 0.47 g.
- Example 9.5 Coupling of the linear protected peptide to compound of the formula (11.1)
- TRSAW and analogues were incubated at 37°C in rat serum at a concentration of 200 ⁇ M. Aliquots of the incubation mixture (100 ⁇ l) were withdrawn at various times, mixed with 60 ⁇ l of 20 % perchloric acid to precipitate protein and centrifuged at 12000 g for 8 min. Residual starting material and degradation products remaining in the serum were determined by reversed phase HPLC using a Nucleosil C 18 and Nucleosil C 8 column with UV monitoring at 216 nm, flow rate 1.0 ml/min and a gradient of 10-90 % CH 3 CN over 30 min. (table 1).
- mice Female rats of the Sprague-Dawley-derived strain TifiRAIF (Sisseln) weighing 130-150 g body weight were used. The animals were fed ad libitum a standard rat breeding diet (NAFAG, Grossau, Switzerland) containing 1.2 % calcium, 0.9 % phosphorus and 24.8 % protein. Ovariectomy (OVX) was performed by electrotomy via the dorsal route in anesthesia with vetanarcol (Veterinaria, Zurich, Switzerland). The animals were randomly divided into groups of 8.
- Treatment was started on the day of ovariectomy.
- TRASW or its analogues were dissolved in phosphate buffered saline containing 0.1 % rat serum albumin.
- the test compounds were administered by daily subcutaneous injection on 5 days per week. Intact and ovariectomized control animals received the appropriate vehicle. Treatment lasted for 3 weeks.
- Both femurs were freed of connective tissue and the amount of trabecular bone was determined according to Gunness-Hey and Hock (Metab. Bone Dis. & Rel. Res. (1984), 5, 177-181).
- the femurs were cut in half at the mid-diaphysis using a dental saw and the proximal halves were discarded. With a scalpel the epiphysis of the distal half was cut off and the bone was split into saggital halves. The marrow was flushed out with water.
- a dental curette the trabecular bone was scraped out of both cortical shells, combined and put into 5 % TCA. After standing 16 hours at room temperature, the TCA extract was removed and used for the determination of calcium by atomic adsorption spectroscopy.
- Test compound Dose + Calcium content of the trabecular bone of the femur [mg] ⁇ g/ml Intact control OVX control OVX + test compound
- Example 12 Influence on protein kinase-C activation
- PKC protein kinase-C
- Serum free cells (osteosarcoma cell line UMR 106) are stimulated with the compound in question (10 minutes at 30°C, concentrations see table 1)
- Example 12.2 Separation of cytosolic and membrane associated PKC
- the cells are washed with cold saline twice (4°C) and scraped with 800 ⁇ l of a buffer (A) consisting of:
- the cells are centrifuged for 60 min at 100,000 g and the supernatant (cytosolic PKC) is separated.
- the pellet is suspended using 776 ⁇ l of buffer A; 25 ⁇ l triton (10%) is added; the tubes are shaken for 1 h at 4°C and again centrifuged for 60 min at 100,000 g.
- the supernatant contains the membrane associated PKC.
- Tris (0.2 M; pH 7.5) 25 ⁇ l 25 ⁇ l Mg acetate (0.5 M) 5 ⁇ l 5 ⁇ l leupeptin (0.5 mg/ml) 25 ⁇ l 25 ⁇ l radioactive ATP (100 ⁇ m) 25 ⁇ l 25 ⁇ l CaCl 2 (2.5 mMol) 2.5 ⁇ l 2.5 ⁇ l phosphatidyl-serine mix - 80 ⁇ l Tris (0.02 M) 80 ⁇ l - H 2 O 30.5 ⁇ l 30.5 ⁇ l peptide (7 mM) 7 ⁇ l 7 ⁇ l
- Compound (3) shows an even increased activity compared to also active TRSAW-amide.
- Acetyl-TRSAW shows no characteristic activity as expected.
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
L'invention concerne des nouveaux composés cycliques inhibant la résorption osseuse excessive ainsi que leurs procédés de production. L'invention porte également sur des compositions contenant lesdits composés associés avec des excipients pharmaceutiques appropriés ainsi que sur des méthodes de traitement de maladies se caractérisant par une résorption osseuse excessive. Ladite méthode consiste à administrer à un sujet ayant besoin de ce traitement une dose efficace pour inhiber la résorption osseuse d'un des nouveaux composés cycliques ou d'un mélange de ces derniers éventuellement combinés avec un excipient pharmaceutique approprié.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP94913070A EP0693081A1 (fr) | 1993-04-08 | 1994-03-28 | Composes et compositions cycliques inhibant la resorption osseuse |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP93810254 | 1993-04-08 | ||
EP93810255 | 1993-04-08 | ||
EP93810254 | 1993-04-08 | ||
EP93810255 | 1993-04-08 | ||
EP94913070A EP0693081A1 (fr) | 1993-04-08 | 1994-03-28 | Composes et compositions cycliques inhibant la resorption osseuse |
PCT/EP1994/000977 WO1994024153A1 (fr) | 1993-04-08 | 1994-03-28 | Composes et compositions cycliques inhibant la resorption osseuse |
Publications (1)
Publication Number | Publication Date |
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EP0693081A1 true EP0693081A1 (fr) | 1996-01-24 |
Family
ID=27235582
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP94913070A Ceased EP0693081A1 (fr) | 1993-04-08 | 1994-03-28 | Composes et compositions cycliques inhibant la resorption osseuse |
Country Status (1)
Country | Link |
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EP (1) | EP0693081A1 (fr) |
-
1994
- 1994-03-28 EP EP94913070A patent/EP0693081A1/fr not_active Ceased
Non-Patent Citations (1)
Title |
---|
See references of WO9424153A1 * |
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