EP0862578A1 - Analogues de l'actinomycine d - Google Patents

Analogues de l'actinomycine d

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Publication number
EP0862578A1
EP0862578A1 EP96929190A EP96929190A EP0862578A1 EP 0862578 A1 EP0862578 A1 EP 0862578A1 EP 96929190 A EP96929190 A EP 96929190A EP 96929190 A EP96929190 A EP 96929190A EP 0862578 A1 EP0862578 A1 EP 0862578A1
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EP
European Patent Office
Prior art keywords
alkyl
group
immobihsed
array
optionauy
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EP96929190A
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German (de)
English (en)
Inventor
Glenn Tong
John Nielsen
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Phytera Symbion ApS
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Phytera Symbion ApS
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries

Definitions

  • the present mvention relates to new compounds bemg structurally and functionally similar to Actinomycin D
  • the present mvention also relates to the preparation of smgle species and libraries of such compounds usmg sohd phase peptide synthesis methodology
  • a new double-combinatorial technique which can also be apphed in the preparation of libraries of other classes of compounds, has been developed
  • Molecules which bmd m a highly specific manner to nucleic acid hybrids have found important apphcations as probes, primers (Tong, G Ph D Thesis, University of Melbourne (Australia), 1994), and more recently, as anti-sense and anti-gene mhibitors (Uhlmann, E , Peyman, A.
  • nucleic acid based drugs mclude (a) admmistration is problematic due to instability to enzymes and also the anionic nature of the phosphate backbone renders penetration of the cell membrane problematic, (b) analogues which solve the above problems are often either not as effective as natural nucleic acids and/or have side-effects e g non-specific bmdmg to cellular protems, and (c) the mechanism of anti-sense inhibition is now thought to be not totally sequence specific but is also dependent on the conformation of the target
  • Actmomycm D and tnostin are potent antibiotics with characteristics that provided us with some important "design features" for a new class of nucleic acid bmders Both are conjugates of cyclic peptides with an mtercalatmg moiety The cyclic peptides are usually very hydrophobic and contain N-methyl ammo acids which mask the hydrophihcity of the peptide bonds Drug-D ⁇ A mteraction is enhanced by minimising solvent-D ⁇ A, solvent-drug and unfavourable drug-D ⁇ A mteractions (Takasugawa, F The Journal of Antibiotics 1985, 38, 1596-1604 and Chu, W , Kamitori, S , Shmo
  • the present mvention provides a novel class of compounds of the followmg general formula I
  • each of P 1 and P 2 independently designates a linear or cychc moietv comprising 1-20 units, preferably 2-20 units, of the general formula II
  • R 6 is selected from hydrogen and C ⁇ 4 -alkyl
  • one of the substituent pairs R ! /R 2 , R'/R 3 , and R 3 R 4 may form a biradical which, together with the atoms located between these substituents (C ⁇ and or Cp), forms a ring; or one of the substituent pairs R 2 /R 6 and R 4 R B may form a biradical which, together with the atoms located between these substituents (C ⁇ , Cp, and/or N), forms a ring;
  • the cychc character arises from the presence of a linkage between two of the side chains, R 1 , R 2 , R 3 , and R 4 , of two units of the formula II within a moiety P 1 and/or P 2 .
  • the present invention -dso provides a method for the preparation of such compounds using sohd phase peptide synthesis methodology.
  • the present invention provides a method for the preparation of combinatorial hbraries of compound of the general formula I , e. g. , by using a novel double-combinatorial methodology.
  • This methodology may also be used to in the preparation of combinatorial hbraries of other classes of compounds.
  • the compound hbraries prepared may be used for screening purposes, and the individual compounds may be used in therapy.
  • the moieties P 1 and P 2 independently designate a linear or cychc moiety comprising 1-20 units, preferably 2-20 units, of the general formula II
  • the moieties P 1 and P 2 can easily be mterpreted as havmg a C-termmal unit, correspondmg to the first (seen from the left, cf formula II) unit of the cham of units, and an N-termmal unit, corresponding to the last unit of the cham of units
  • ammo group (an ⁇ - or ⁇ -ammo group) of the N-termmal unit of P 1 and P 2 , respectively, is covalently linked to the carbonyl separatmg P l and P 2 , respectively, from the entity A
  • each of the moieties P 1 and P 2 comprises a group covalently bound to the carbonyl group of the C-termmal unit, wherebj the C-termmal carboxy groups of the compound I mdependently are m the free acid form (-COOH), carboxylate form (-COO ), or is denvatised as the amide (-CONH2, -CONHR, - CONRR'), the hydroxylamide (-CON(OH)H), the hydrazide (-CONHNH2 CONHNHR 1 ”) or the ester (-COOR”), where each of R, R ⁇ R", and R'" mdependently designates optionally substituted Ci 20-alkyl, optionally substituted C220-alkenyl, optionally substituted C.20-alkad ⁇ enyl, optionally substituted Ce 20-alkatnenyl, optionally substituted aryl, or optionally substituted heteroaryl Furthermore, the C-termmal carboxy groups of the compound I mdependently are m the free acid form
  • the C-termmal carboxyl groups are in the ester form, e g the methyl ester form, or m the amide form, e g the -CONH2 form
  • Ci 20-alkyl is mtended to mean a bnear, cychc or branched hydrocarbon group havmg 1 to 20 carbon atoms, such as methyl, ethyl propyl, iso-propyl cydopropyl, butyl, tert-butyl, iso-butyl, cyclobutyl, pentyl, cyclopentyl hexyl, C3 r clohexyl, hexadecyl, heptadecyl, octadecyl, nonadecyl
  • Ci 6-alkyl is mtended to mean a linear, cychc or branched hydrocarbon group havmg 1 to 6 carbon atoms, such as methyl, ethyl, propyl, iso-propyl, pentyl, cyclopentyl, hexyl
  • alkyl are methyl, ethyl, propyl, iso-propyl, butyl, tert-butyl, iso-butyl pentyl, cyclopentyl hexyl, cyclohexyl, particular methyl ethyl, propyl iso-propyl, tert-butyl, iso-butyl and cyclohexyl
  • C 2 20-alkenyl C 4 20-alkad ⁇ enyl
  • C ⁇ 20-alkat ⁇ enyl are mtended to mean a linear, cychc or branched hydrocarbon group havmg 2 to 20, 4 to 20, and 6 to 20, carbon atoms, respectively, and compnsmg one, two, and three unsaturated bonds, respectively
  • alkenyl groups are vmyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, heptadecaenyl
  • alkadienyl groups are butadienyl, pentadienyi, hexadienyl, heptadienyl, heptadecadienyl
  • alkatrienyl groups are hexatrienyl, heptatnenyl octatnenyl, and heptadecat ⁇ en
  • C220-alkynyl is mtended to mean a linear or branched hydrocarbon group havmg 2 to 20 carbon atoms and comprising a triple bond Examples hereof are ethynyl, propynyl, butynyl, octynyl, and dodecaynyl
  • alkyl 1 e m connection with the terms “alkyl”, “alkenyl”, “alkadienyl”, “alkat ⁇ enyl”, and “alkynyl”
  • group(s) selected from hydroxy, Ci ⁇ -alkoxy (1 e alkyl-oxy), carboxy, Ci ⁇ -alkoxycarbonyl, Ci 6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, ammo, mono- and d ⁇ (C ⁇ 6-alkyl)am ⁇ no, carbamoyl, mono- and d ⁇ (C ⁇ 6---lkyl)ammocarbonyl, ammo-Ci 6-alkyl-am ⁇ nocarbonyl, mono- and d ⁇ (C ⁇ 6-alkyl)am ⁇ no-C ⁇ ⁇ -alky
  • the substituents are selected from hydroxy, Ci ⁇ -alkoxy, carboxy, Ci 6-alkoxycarbonyl, Ci 6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, ammo, mono- and d ⁇ (C ⁇ 6-alkyl)am ⁇ no, carbamoyl, mono- and d ⁇ (C ⁇ 6-alkyl)am ⁇ nocarbonyl, ammo-Ci ⁇ -alkyl- ammocarbonyl, mono- and d ⁇ (C ⁇ 6-alkyl)ammo-C ⁇ 6-alkyl-am ⁇ nocarbonyl, Ci ⁇ -alkylcarbonylammo, guanidino, carbamido, trihalogenalkyl, halogen such as fluoro, chloro, bromo or iodo, where aryl and heteroaryl may be substituted with methyl, nitro or halogen Especially preferred examples are hydroxy, Ci
  • heteroaryl is mtended to mean an aryl group where one or more of the carbon atoms have been replaced with heteroatoms, e g nitrogen, sulphur, and/or oxygen atoms
  • heteroaryl groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazmyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, indolyl, benzopyrazolyl, phenoxazonyl
  • Preferred heteroaryl groups are pyridmyl, benzopyrazolyl, and imidazolyl
  • one of the side cham pairs R 1 ⁇ 2 , R' R 3 , and R 3 /R 4 may form a biradical which, together with the atoms located between these substituents (C ⁇ and/or Cp), form a rmg
  • the rmg is a biradical of one of the followmg nngs cyclopropane, oxirane, aziridine, cyclopropene, azinne, cyclobutane, oxetane, azetidine, thietane, 2-azet ⁇ d ⁇ none, 1,3-lactone, pyrohdine, pyrohne, pyrrole, cyclopenetene, cyclopentadiene, pyrollidione, pyrolhdone, cycl
  • oxirane aziridine, azinne, oxetane, 2-azet ⁇ d ⁇ none, 1,3-lactone, pyrohdine, pyrohne, pyrrole, pyrolhdone, pyrolhdione, oxirane, dioxirane, morphohne, piperidine, 1,5-lactam (pipendone), 1,5-lactone, piperidione, tropolone, 1,6-lactam
  • one of the side cham pairs R 2 R 5 and R 4 R 6 may form a biradical which, together with the atoms located between these substituents (C ⁇ , C p and/or N), form a rmg
  • the rmg is a biradical of one of the followmg rmgs 2-azet ⁇ d ⁇ none, pyrohdine pyrohne, pyrrole, pyrolhdione, pyrolhdone, piperidine, 1,5-lactam, piperidione, and 1,6-lactam
  • the rmg is preferably selected from 2-azet ⁇ d ⁇ none, pyrolhdone 1,5-lactam and 1 6-lactam, in particular 1,5-lactam
  • the biradical may, just as the side chams from which they (hypothetically) arise, be substituted Possible and preferred substituents are those mentioned above as possible and preferred substituents for "alkyl” and “aryl”, respectively Furthermore, the rmg may be fused with one or more aromatic or heteroaromatic rmgs, such as a benzene rmg
  • the units of the general formula (II) are selected from naturally occurring or commercially available a-ammo acids (see formula Ila) and b-ammo acids as well as simple N-alkyl derivatives thereof, e g alanine, valine, norvahne, lsovahne, leucme, norleucme, isoleucine, methionme, phenylalanine, tryptophan, phosphotryptophan , serme, phosphoserme, threonme, phosphothreonme, cysteme, homocysteme, penicillamme, tvrosme, phosphotyrosme, a-aminoisobuty ⁇ c acid (Aib), phosphoaminobuty ⁇ c acid (PAbu) asparagme, glutamine, aspartic acid, glutamic acid, ornithine, lysme, arginine, histidme, proline, 4-hydroxy -proline,
  • each of the moieties P 1 and P 2 may optionally mclude 1-18 units of the formula III
  • the methylene groups may be optionally substituted one or several times, preferably 1-3 time, with group(s) as defined above for R i -R , and wherem where R 6 designates the same groups as defined above for R 6
  • Typical and preferred linkages which may be achieved through, e g , conventional sohd phase peptide synthesis methodology, are an amide between, on the one hand, a carboxy group one unit of the formula II and, on the other hand
  • At least one of the moieties P 1 or P 2 is a cychc moiety, and more preferably, both of P 1 and P 2 are cychc moieties
  • P l and P 2 each mdependently comprises 2-20, such as 4-15, preferably 4-10, m particular 5-8, units of the formula II
  • the number of units in the moiety P 1 is preferably identical to the number of units m the moiety P 2
  • P 1 and P 2 each mdependently comprises a total of 2-20 such as 4-15, preferably 4-10, m particular 5-8, units of the formula II and III
  • the number of units m the moiety P 1 is preferably identical to the number of units m the moiety P 2
  • the moieties P 1 and P 2 are substantially identical, I e that the units of the moieties P 1 and P 2 are identical and occur m the same order, but where the groups linked to the carbonyl group of the C-termmal units may be different due to, e g , the use of different reagents for cleavmg the P 1 and P 2 , respectively, from a sohd phase resm
  • the central entity A of the compound is either a cychc entity or a linear entity It is preferred that the entity is a cychc entity
  • the term “bnear entity” is intended to mean homobifunctional linking moieties, specifically dicarboxylic acids
  • cychc entity denotes a cychc or polycychc biradical
  • the cychc entity is bound to the N-termmal ammo groups of the moieties P 1 and P 2 via the two carbonyl groups of the general formula I
  • the cychc molecular moiety may, m principle, be any moiety which contams or consists of one or more cychc elements, m particular 5- or 6-membered cychc elements Each such cychc element may mdependently be saturated, unsaturated or aromatic, it may be carbocychc, or it may be heterocyclic by incorporating 1, 2, 3, or 4 hetero atoms, typically selected from nitrogen, oxygen or sulphur In the case of several cychc elements bemg present, these may be connected through smgle or double bonds, or they may be fused, or combinations thereof It is particularly preferred that the cychc entity A is an aromatic or polyaromatic moiety, m that the role of the moiety A m Actmomycm D is as an mtercalator and the vast majority of effective mtercalators are either aromatic, polyaromatic, heteroaromatic or polyheteroaromatic molecules Examples of such moieties which, however, should not be considered hmitmg, are the
  • saturated or unsaturated carbocychc or heterocyclic entities are cyclopropane, oxirane, azrndme, cyclopropene, azinne, cyclobutane, oxetane, 2-azet ⁇ d ⁇ none, 1,3- lactone, pyrohdine, pyrohne, pyrrole, cyclopentene, cyclopentadiene, pyrolhdione, pyrolhdone, cyclohexyl, oxirane, dioxirane morpholine, pipendme, 1,5-lactone, 1,5-lactam, cy ohexene, cyclohexadiene, pipendione, tropane, 1,6-lactone (tropolone), and 1,6-lactam
  • the nng is preferably selected from 2-azet ⁇ dmone, 1,3-lactone, pyrohdine, pyrohne, pyrrole, pyrolhdone, pyrollidione, oxirane, dioxirane, morpholine, piperidine, 1,5-lactam, 1 5-lactone, pipendione, tropolone, 1,6-lactam Especially preferred examples are 1,5-lactam and 1,5-lactone
  • aromatic and polyaromatic entities are phenyl, naphthyl, anthracyl, pyrenyl, benzopyrenyl, phenoxazonyl, N8-phenoxazonyl, quinolyl, benzophenazmyl, ethidium and fluorenyl
  • Especially preferred examples are naphthyl, benzopyrenyl, phenoxazonyl, N8- phenoxazonyl, quinolyl, benzophenazmyl, ethidium and fluorenyl
  • aromatic and polyaromatic entities may be substituted with one or more groups selected from the same groups as defined above as substituents for the aryl and heteroaryl groups
  • Aromatic and heteroaromatic moieties are aromatic and heteroaromatic moieties
  • 2,2'-b ⁇ qu ⁇ nohne-4,4'-d ⁇ carboxyhc acid 5-n ⁇ tro- ⁇ sophthahc acid, 2-am ⁇ no-terephthahc acid, 2- bromo-terephthahc acid, 2-n ⁇ tro-terephthahc acid, 3,6-d ⁇ chloro-phthahc acid anhydnde, 4,5- dichloro-phthahc acid anhydnde, 3-n ⁇ tro-phthahc acid anhydnde, 4-n ⁇ tro-phthahc acid anhydride, homophthahc acid, 4,4'-b ⁇ phenyl-d ⁇ carboxyhc acid, 2,2 -b ⁇ phenyl-d ⁇ carboxyhc acid 2,3-naphthalene-d ⁇ carboxyhc acid, 2,6-naphthalene-d ⁇ carboxyhc acid, 1,
  • the compounds of the invention may be prepared by any well known methods or coupling reactions for the preparation of peptides, in combination with well known methods for establishing the amide bonds between the moiety A and the moieties P 1 and P 2 .
  • Such coupling reactions for establishing peptides may be carried out in solution or, preferably, through sohd phase synthesis, e.g. by using the well-established Merrifield sohd phase synthesis methodology (e.g. Barany, G., and Merrifield, R.B. in The Peptides, Vol. 2, Academic Press, New York, 1979, pp. 1-284).
  • the sohd phase peptide synthesis is preferably performed by using either the Boc (tert-butoxycarbonyl) or the Fmoc (9-fluorenylmethyloxycarbonyl) protection strategy, or combinations thereof.
  • Other possible bonds may be formed under conditions know to the person skilled in the art, see e.g. Hermkens et al. Tetrahedron, 52, 13, 4527.
  • Fmoc sohd phase peptide synthesis may be used to assemble the conjugates of formula (I).
  • the sohd support may be a polyethylene glycol-polystyrene copolymer (Tentagel) which has been shown to be very effective for the synthesis of difficult peptides.
  • Tegel polyethylene glycol-polystyrene copolymer
  • a factor which requires consideration is the efficiency of cyclisation (if required) after the linear peptide has been synthesised.
  • Tentagel resin with a large particle size (130 ⁇ ) and a low loading (150-160 ⁇ mol/g of the first amino acid) is preferably used to favour cychc peptide over dimer formation.
  • the compounds of the general formula (I) may be prepared by using the Fmoc strategy according to the following reaction protocol:
  • the startmg pomt for the synthesis is the support-bound glutamic acid derivative 4, which after conventional Fmoc sohd phase peptide synthesis results m the Imear peptide 5
  • the C-termmal Glu and the N-termmal Lys residues serve as convenient handles upon which cychsation can take place wh ⁇ st leaving the a-amino group of the Lys free to react m high yields with the naphthyl derivative 3
  • the orthogonal protection scheme allows the g-carboxyl of the Glu and the e-ammo groups of the Lys to be consecutively and selectively removed usmg a palladium-complex catalyst (Kates, S A Daniels, S B and Albencio, F Analytical Biochemistry 1993, 212, 303-305) and TFA
  • the polymer support carrymg the cychc peptide 6 is divided mto two portions, a first and a second portion The first portion is then acylated with
  • cyc sations may also be performed by usmg standard methodologies, e g , the phosphoramidite strategy for the preparation of phosphodiester linkages (Beaucage, S , Caruthers, M H , Tetrahedron Lett 1981, 22, 1859-1862), and disulfide bridge formation via oxidation, e g anal oxidation, of two thiol containing units, e g , two cystemes
  • methyl ester is often advantageous because the cleaved peptide can be isolated simply by evaporation of the cleavage solution Formation of amides via cleavage with ammes as nucleophiles will result in a more chemically and biologically stable bond but isolation of the peptide may in some cases be more difficult In any case, end group (here the methyl ester) should be stable enough to stay intact durmg the coupling of the naphthyl-peptide conjugate with the support-bound peptide
  • a further aspect of the present mvention relates to a method for the preparation of a compound of the followmg formula I
  • the optionally side cham protected moiety P 1 immobihsed to a sohd support material may be provided usmg standard sohd phase peptide synthesis methodologies
  • any of the side chams functionalities of the mdividual ammo acid units are susceptible to undergo degradation or reactmg during the coupling steps, or if the mtegnty of such function ahties are expected to endangered m any of the subsequent steps of the synthesis of the compound I, e g m any segment coupling or cleavage step
  • such side chams are advantageously protected by usmg protectmg groups known m the art
  • such protectmg groups are compatible
  • I e forms an orthogonal protectmg scheme, with either the Boc peptide synthesis scheme or the Fmoc peptide synthesis scheme mciuding any cychsation steps
  • any couplmg reagent know m the art of peptide synthesis, e g of the carbodiimide type (e g dicydohexylcarbodiimide) or benzotriazole type, uronium salt type (e g 0-(7-azabenz
  • the fragment is cleaved from the sohd support material usmg cleavage reagent suited for the specific sohd phase material selected.
  • the C-terminal carboxyhc acid of the moiety P 1 may be derivatised m several way dunng cleavage, e.g. resultmg in the ester (-COOR) or amide (-
  • the optionally side chain protected moiety P 2 which, as P 1 , may be linear or cychc, immobihsed to a sohd support material, is prepared m a way similar or identical to P 1 .
  • P 1 and P 2 are substantially identical, it may be advantageous to prepare the immobi sed moieties P 1 and P 2 m one batch
  • a number of coupling reagents may be used
  • Actmomycms contain -V-methylamino acids which confer the configuration and also the hydrophobicity necessary for biological activity (Takasugawa, F The Journal of Antibiotics 1985, 38, 1596-1604)
  • Other highly sterically hmdered and hydrophobic ammo acids like a- ammoisobutync acid (Aib) may also be a useful addition to the pool of ammo acid budding blocks
  • the third method is based on the possibility of mono-deeste ⁇ fication of certain aromatic dimethyl esters Synthesis of the mixed methyl-allyl-diester followed by selective demethylation (NaCN/HMPA) yields the correspondmg monoallyl ester Combinatorial libraries of Actinomycin D analogues
  • the present mvention relates to a method for the preparation of a multi-dimensional array (combmatonal hbrary) of compounds, ⁇ P' ⁇ - ⁇ A ⁇ - ⁇ P 2 ⁇ , consistmg of at least three compounds each havmg the general formula I
  • the array ⁇ P 2 ⁇ of immobihsed moieties P 2 is substantially identical to the array ⁇ P 1 ⁇ of immobihsed moieties P 1 This can easdy be obtamed when the array ⁇ P 1 ⁇ of immobihsed moieties P 1 and the array ⁇ P 2 ⁇ of immobilised moieties P 2 is prepared in one batch
  • each of the synthetic steps m the method for the preparation of compound hbraries are sim ⁇ ar to the correspondmg conditions m the method for the preparation of smgle compounds
  • m steps where a mixture of two or more compounds are used, e g two different diacids it may be advantageous to adjust the molar ratio between these compounds in order to compensate for any difference m reactivity between such compounds Double combinatorial principle
  • the present mvention relates to a method for the preparation of a multi-dimensional array ⁇ HHBML 2 ⁇ of at least three compounds each havmg the formula L'-B- L 2 , wherem the arrays ⁇ L 1 ⁇ and ⁇ L 2 ⁇ are of similar chemical composition, each of L 1 and L 2 mcludes a fragment l 1 and l 2 , respectively, of a chemical functionabty, B is an entity which mcludes two fragments b 1 and b 2 of chemical functionalities, each of the fragment sets Pb 1 and b 2 l 2 formmg a covalent hnkage between L 1 and B and between B and L 2 , respectively, said covalent linkages bemg substantially identical and bemg formed under b/1 bond formation reaction conditions (e g amide, ester, ether, phosphate bond formation reaction conditions), the method compnsmg the followmg operations
  • b/1 bond formation reaction conditions e g amide, ester, ether,
  • the utility of the compounds of the mvention can be demonstrated through the screenmg of hbraries of such compounds for strength and specificity of bindmg to smgle-stranded DNA and RNA, and also DNA/DNA and RNA/DNA duplexes, m particular by usmg combmatonal compound hbraries
  • the screenmg may be done by immobilising synthetic nucleic acids (e g on an ELISA plate) and treatmg with solutions of the compound hbraries
  • the entity A has a specific absorption, especially when A is an aromatic entity, which is not only different to that of DNA and RNA, but is also very sensitive to the environment of the molecule (I e the wavelength of absorption changes significantly on binding to the nucleic acid)
  • successful candidates can be selected by measurmg parameters such as hght absorption and emission, this can be done very e
  • the compound of the present mvention may bmd to smgle or double stranded DNA and RNA or DNA/RNA hybrids Smce a large number or serious ailments such as AIDS and hepatitis are gene-based, the targetmg of genetic materials usmg these compounds has potential apphcations in the study, diagnosis, and treatment of gene-based diseases
  • Compounds of the present mvention are expected to have the same effects as the naturally occurring Actmomycms (Meienhofer, J and Atherton, E "Structure-activity Relationships m the Actmomycms" Structure-activity Relationships among the Semisynthetic Antibiotics, Perlman, D ed , Academic Press, N Y , 1974, pp 427-529)
  • the mvention also relates to the use of a compound of the general formula (I) as defined herem for antiviral, anticancer, antibiotic (such as antibacterial and antifungicidal), or herbicidal purposes
  • Scheme 1 shows schematically the complete synthetic route for the preparation of an example of a compound of the mvention HMB designate a hydroxymethylbenzoyl linker (see the examples)
  • the reaction conditions are as follows (a) 20% piperidine/DMF, (b) Fmoc ammo acid, PyBOP HOBt/DIEA, (c) tetrakistnphenylphosphme Pd(0), (d) 50% TFA/dicloromethane, (e) PyBOP/HOBt/DIEA, (f) 3, HATU/DIEA, (g) 90% MeOH/py ⁇ dine, (h) HATU/DIEA
  • Scheme 2 shows the three different method (A, B, and C) for the preparation of a monoester of a dicarboxylic acid
  • A, B, and C for the preparation of a monoester of a dicarboxylic acid
  • Scheme 3 shows the double combmatonal principle exemplified for compounds of the Actmomycm D analogue type
  • Scheme 4 describes compounds synthesized durmg hbrary synthesis The numbering of the compounds are as foUows Comp number R Comp number Ra Rb
  • Figure 2 shows a HPLC chromatogram of crude conjugate 1 All conditions identical to those of Figure 1 (c)
  • Figures 4 and 5 show structures of examples of the aromatic or polyaromatic part of the carbocychc entities
  • One synthesis cycle is as follows Fmoc deprotection with 20% pipe ⁇ dine/DMF (2 X 2 5 mm), DMF wash (5 X column volumes), couphng with Fmoc ammo acid (3 eq), PyBOP (3 eq), HOBt (3 eq) and DIEA (6 eq) for 0 5 h, DMF wash (5 X column volumes)
  • the Fmoc group of the support-bound cychc peptide 6 was removed as described above After washing with DMF, the resm was treated with 3 after prior activation with HATU (Carpino, L A. J. Am Chem. Soc, 1993, 115, 4397-4398, Angell, Y M , Garcia-Echevema, C and Rich, D H Tetrahedron Lett 1994, 35, 5981-5984, and Angell, Y M , Thomas, T L , Flenkte, G R and Rich, D R J Am Chem.
  • An anion exchange resm is used to block off one of the carboxyl groups while the other undergoes MSNT-mediated esterification (Blankemeyer-Menge, B , Nimtz, M and Frank, R
  • the acidic solution was extracted with dichloromethane (3 x 75 ml)
  • the combmed organic phases were washed 1% aq HCL (3 x 100 ml) and water (4 x 250 ml)
  • the precipitate was collected, washed with water untd the effluent was neutral and dried (0 67 g,
  • the natural product has peptides contammg N-methylammo acids (NAA) In the first mstance all the N-methylammo acids are omitted and the test sequence synthesised was H-Lys-Val-Pro-Gly-Glu-OH
  • H-Lys-Val-Pro-MeGly-Glu-OH (sequence 1) H-Lys-MeVal-Pro-MeGly-Glu-OH (sequence 2) H-Lys-MeVal-Glu-OH (sequence 3)
  • Sequence 1 is identical with sequence 2 except that Val is replaced by a MeVal
  • Sequence 3 was used to find the optimal conditions for N-methylammo acid couphng MeVal was chosen smce it is a highly sterically hmdered N-methylammo acid, and therefore optimal conditions for these peptides should be apphcable to the efficient synthesis of hbraries of peptides contammg other sterically hmdered N-methylammo acids
  • PyBrOP and ammo acid fluoride procedures were compared
  • ammo acid fluoride is very effective for the mcorporation of N- methylammo acids although forcmg conditions are often advantageous
  • Anhydrous conditions dry N-methylpyrolhdone as solvent and reaction done under an argon atmosphere
  • high concentrations of the ammo acid fluoride between 0 7 and 1 0 M
  • large excesses of ammo acid fluoride (10 eq)
  • long reaction tunes (2 X 17h couplings) were found to give quantitative couplings
  • an equimolar (with respect to the ammo acid fluoride) amount of diisopropylethylamine gives excellent results

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Abstract

L'invention concerne des nouveaux composés structurellement et fonctionnellement similaires à l'actinomycine D et des banques combinatoires desdits composés. Les analogues de l'actynomycine D selon l'invention, comprennent deux fragments peptidiques linéaires ou cycliques constitués par des acides α-aminés, des acides β-aminés et/ou des acides φ-aminés à chaîne plus longue, ainsi qu'un groupe bifonctionnel qui est de préférence une entité cyclique, en particulier, une entité aromatique ou hétéroaromatique faisant office de groupe intercalaire. On peut préparer des composés spécifiques et une banque desdits composés en utilisant des méthodes classiques de synthèse de peptides en phase solide. Les nouveaux composés ont une affinité pour l'ADN et l'ARN, et les banques de ceux-ci peuvent être ainsi utilisées de manière avantageuse à des fins de criblage. De plus, on a mis au point une nouvelle technique combinatoire double pouvant être utilisée dans la préparation de banques de classes de composés plus vastes.
EP96929190A 1995-09-12 1996-09-12 Analogues de l'actinomycine d Withdrawn EP0862578A1 (fr)

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DE19942624A1 (de) * 1999-08-28 2001-03-08 Chemotopix Gmbh Verfahren zur Herstellung von zyklischen Peptidomimetika
CA2983201A1 (fr) * 2015-04-23 2016-10-27 VIIV Healthcare UK (No.5) Limited Inhibiteurs de la replication du virus d'immunodeficience humaine

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US4562176A (en) * 1985-10-03 1985-12-31 Sengupta Sisir K Method and composition for treating cancer
US4680382A (en) * 1986-02-03 1987-07-14 Trustees Of Boston University Analogues of actinomycin D
EP0593618B1 (fr) * 1991-06-27 1998-04-22 Genelabs Technologies, Inc. Methode de tri pour la detection de molecules se liant a adn
AU5733594A (en) * 1992-11-30 1994-06-22 Bulk Chemicals, Inc. A method and composition for treating metal surfaces

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