WO1997010263A1 - Actinomycin d analogues - Google Patents

Abstract

The present invention relates to new compounds being structurally and functionally similar to Actinomycin D and to combinatorial libraries of such compounds. The Actinomycin D analogues according to the present invention comprise two linear or cyclic peptide moieties constituted by α-amino acids, β-amino acids and/or longer chain φ-amino acids, and a difunctional group which preferably is a cyclic entity, in particular, an aromatic or heteroaromatic entity acting as an intercalator group. Specific compounds and a library of such compounds may be prepared using conventional solid phase peptide synthesis (SPPS) methodologies. The novel compounds are expected to have affinity for DNA and RNA, and libraries thereof may therefore advantageously be used for screening purposes. Furthermore, a novel double-combinatorial technique that may be used in the preparation of librairies of broader classes of compounds has been developed.

Description

ACTINOMYCIN D ANALOGUES

FIELD OF THE INVENTION

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 Furthermore, a new double-combinatorial technique, which can also be apphed in the preparation of libraries of other classes of compounds, has been developed

BACKGROUND OF THE INVENTION

Molecules which bmd m a highly specific manner to nucleic acid hybrids (I e DNA, RNA and DNA RNA) 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. Chem Rev 1990, 90, 544) Most of the efforts to date have focused on compounds based on oligodeoxynucleotide analogues The mam advantage of this approach is the highly predictable way m which DNA can bmd to their complementary strands via either Watson-Crick or Hoogsteen hydrogen bonding Thus, m theory, this allows a totally novel concept of drug design to be explored smce traditionally, the design of drugs has been focused on small molecules which regulate enzyme activities or mteract with receptors

However, some drawbacks of 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 (Takasugawa, F The Journal of Antibiotics 1985, 38, 1596-1604, and Chu, W , Kamitori, S , Shmomiya, M , Carlson, R G and Takasugawa, F J Am Chem Soc 1994, 116, 2243-2253) 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-DΝA mteraction is enhanced by minimising solvent-DΝA, solvent-drug and unfavourable drug-DΝA mteractions (Takasugawa, F The Journal of Antibiotics 1985, 38, 1596-1604 and Chu, W , Kamitori, S , Shmomiya, M , Carlson, R G and Takasugawa, F J Am Chem Soc 1994, 116, 2243-2253) All synthetic schemes for Actinomycin D analogues reported to date use solution phase strategies (Chu, W et al J Am Chem Soc 1994, 116, 2243, Meienhofer, J and Atherton, E Structure Activity Relationships among the Semisynthetic Antibiotics Perlman D Ed , Academic Press New York, San Francisco, London, 1974, 427, Mauger A et al J Am Chem Soc 1985, 107 7154, Mauger, A et al J Med Chem 1991, 54 1297, and Nakajima K et al Bull Chem Soc Jpn 1982, 55, 3237)

There has now been discovered a new class of nucleic acid-interacting compounds, which furthermore can be prepared through sohd phase synthesis

SUMMARY OF THE INVENTION

The present mvention provides a novel class of compounds of the followmg general formula I

P - C ( -O) -A- C ( -O) - P'

wherem A designates a cychc or linear entity, and

each of P¹ and P² independently designates a linear or cychc moietv comprising 1-20 units, preferably 2-20 units, of the general formula II

wherem n is 0 or 1 , each of R¹, R², R³, R⁴ mdependently is a side cham selected from hydrogen, optionally substituted Ci 6-alkyl, optionally substituted Ci β-alkenyl, optionally substituted C2 β-alkadienyl, optionally substituted Ce β-alkatπenyl, hydroxy, -O-R⁶, formyl, -C(=0)-R⁶, -0-C(=0)-R⁶, carboxy, -C(=0)- O-R⁶, optionally substituted heteroaryl, (optionally substituted heteroaryl) -Ci 6-alkyl, (optionally substituted heteroaryloxy)-Cι 6-alkyl, optionally substituted aryl, (optionally substituted aryl)-Cι 6-alkyl, (optionally substituted aryloxy)-Cι 6-alkyl, halogen such as fluoro, chloro, bromo, and iodo, nitro, cyano, ammo, -NHR⁶, -N(R⁶)2, mono- or dι(Cι 6-alkyl)amino-Ci 6-alkyl, -NCRO-CO-R⁶, (Ci 2o-alkyl)carbonylammo-Cι 6-alkyl, carbamoyl, aminocarbonyl- Ci 6-alkyl, mono- or dι(Cι 20- alkyl)ammocarbonyl, mono- or dι(Cι 6-alkyl)amιnocarbonyl-Cι 6-alkyl, sulphanyl, optionally substituted Ci 20-alkylthιo, optionally substituted Ci 20-alkylthιo-Cι 6-alkyl, (optionally substituted aryl)thio, guanidino, guanidino-Cι-6-alkyl, sulphono (-SO3H), sulphino (-SO2H), halosulphonyl, -OS(0)m-R⁶ where m is 2 or 3, -N(R⁷)S(0)_m-R⁶ where m is 2 or 3, -S(0)_m-N(R⁷)₂ where m is 2 or 3, -S(0)m-NH(R⁷) where m is 2 or 3, -S(0)m-NH2 where m is 2 or 3, isocyano, isothiocyano, thiocyano, -OP(0)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and -N(R⁷)P(0)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5; wherein each R⁶ independently is selected from hydrogen, optionally substituted C1 20- alkyl, optionally substituted Ci 2o-alkenyl, optionally substituted Ci 2o-alkadienyl, optionally substituted Cι _o-alkatrienyl, optionally substituted aryl, and optionally substituted heteroaryl; and each R⁷ is selected from hydrogen and Cι-4-alkyl; and

R⁶ is selected from hydrogen and Cι ₄-alkyl;

and wherein one of the substituent pairs R^!/R², R'/R³, and R³ R⁴ may form a biradical which, together with the atoms located between these substituents (C_α and or Cp), forms a ring; or one of the substituent pairs R²/R⁶ and R⁴ R^B may form a biradical which, together with the atoms located between these substituents (C_α, Cp, and/or N), forms a ring;

and, if any of P¹ and P² is a cyclic moiety, the cychc character arises from the presence of a linkage between two of the side chains, R¹, R², R³, and R⁴, of two units of the formula II within a moiety P¹ and/or P².

The present invention -dso provides a method for the preparation of such compounds using sohd phase peptide synthesis methodology.

Furthermore, 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.

DETAILED DESCRIPTION OF THE INVENTION

In the compound of formula I, the moieties P¹ and P² independently designate a linear or cychc moiety comprising 1-20 units, preferably 2-20 units, of the general formula II

It is apparent from the composition of the mdividual units II that each of the moieties P¹ and P² has, at least on a superficial level as the units are α- (n=0) and β- (n=l) ammo acids, a structure similar or identical to a peptidic moiety Thus, the moieties P¹ and P² 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 It should be understood, that m the case where P¹ and P² only comprise one unit, the N-termmal unit and the C-termmal unit are the very same unit

Thus, it should be understood that the ammo group (an α- or β-ammo group) of the N-termmal unit of P¹ and P², respectively, is covalently linked to the carbonyl separatmg P^l and P², respectively, from the entity A

Furthermore, it should also be clear that each of the moieties P¹ and P², m addition to the 1-20 units, 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 CONHNHR¹") 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-alkadιenyl, optionally substituted Ce 20-alkatnenyl, optionally substituted aryl, or optionally substituted heteroaryl Furthermore, the C-termmal carboxyhc acid may be reduced to the correspondmg alcohol or to the correspondmg aldehyde in the cleavage step, or the carboxyhc acid may be removed m a cleavage/decarboxylation step, see, e g , Hermkens et al, Tetrahedron, 52, 13, 4527

Preferably, 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

In the present context, the term "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^rclohexyl, hexadecyl, heptadecyl, octadecyl, nonadecyl Analogously, the term "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, cyclohexyl, and the term "Ci 4-alkyl" is mtended to cover bnear, cychc or branched hydrocarbon groups havmg 1 to 4 carbon atoms, e g methyl, ethyl, propyl, iso-propyl, cydopropyl, butyl, iso-butyl, tert-butyl, cyclobutyl

Preferred examples of "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

Similarly, the terms "C₂20-alkenyl", "C₄20-alkadιenyl", and "Cε 20-alkatπenyl" 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 Examples of alkenyl groups are vmyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, heptadecaenyl Examples of alkadienyl groups are butadienyl, pentadienyi, hexadienyl, heptadienyl, heptadecadienyl Examples of alkatrienyl groups are hexatrienyl, heptatnenyl octatnenyl, and heptadecatπenyl Preferred examples of alkenyl are vmyl, allyl, butenyl, especially allyl

Similarly, the term "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

In the present context, 1 e m connection with the terms "alkyl", "alkenyl", "alkadienyl", "alkatπenyl", and "alkynyl" the term "optionally substituted" is mtended to mean that the group in question may be substituted one or several times, preferably 1-3 times, with group(s) selected from hydroxy, Ci β-alkoxy (1 e alkyl-oxy), carboxy, Ci β-alkoxycarbonyl, Ci 6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, ammo, mono- and dι(Cι 6-alkyl)amιno, carbamoyl, mono- and dι(Cι 6---lkyl)ammocarbonyl, ammo-Ci 6-alkyl-amιnocarbonyl, mono- and dι(Cι 6-alkyl)amιno-Cι β-alkyl-aminocarbonyl, Ci β-alkylcarbonylam o, guanidino, carbamido, Ci 6-alkanoyloxy, sulphono, Ci 6-alkylsulphonyloxy, nitro, sulphanyl, Ci β-alkylthio, trihalogenalkyl, halogen such as fluoro, chloro, bromo or iodo, where aryl and heteroaryl may be substituted with methyl, methoxy, nitro or halogen

Preferably, the substituents are selected from hydroxy, Ci β-alkoxy, carboxy, Ci 6-alkoxycarbonyl, Ci 6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, ammo, mono- and dι(Cι 6-alkyl)amιno, carbamoyl, mono- and dι(Cι 6-alkyl)amιnocarbonyl, ammo-Ci β-alkyl- ammocarbonyl, mono- and dι(Cι 6-alkyl)ammo-Cι 6-alkyl-amιnocarbonyl, Ci β-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 6-alkoxy, carboxy, aryl, heteroaryl, ammo, sulfanyl, mono- and dι(Cι-6- alkyl)ammo, mono- and dι(Cι 6-alkyl)amιno, and halogen such as fluoro, chloro, bromo or iodo In the present context the term "aryl" is mtended to mean an aromatic carbocyclic rmg or rmg system, such as phenyl, naphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xan thenyl, among which phenyl is a preferred example

The term "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 Examples of such 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

In the present context, l e m connection with the terms "aryl" and "heteroaryl", the term "optionally substituted" is mtended to mean that the group in question may be substituted one or several times, preferably 1-5 times, with group(s) selected from hydroxy (which when present an enol system may be represented m the tautomeric keto form), Ci 6-alkoxy, carboxy, Ci 6- alkoxycarbonyl, Ci 6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- and dι(Cι 6-alkyl)amιno; carbamoyl, mono- and dι(Cι 6-alkyl)amιnocarbonyl, amino- Ci 6-alkyl-ammocarbonyl, mono- and di(Cι 6-alkyl)ammo-Cι 6-alkyl-ammocarbonyl, Ci 6- alkylcarbonylamino, guanidmo, carbamido, Ci 6- alkanoyloxy, sulphono, Ci β-alkylsulphonyloxy, nitro, sulphanyl, trihalogenalkyl, halogen such as fluoro, chloro, bromo or iodo Preferred examples are hydroxy, Ci β-alkoxy, carboxy, Ci 6-alkoxycarbonyl, Ci 6-alkylcarbonyl, aryl, ammo, mono- and dι(Cι 6-a_kyl)amιno, aryl and halogen such as fluoro, chloro, bromo or iodo

As it will be evident from the formula II, there may be up to two asymmetric carbon atoms (C_α and Cp) present in each unit, and possibly even more, depending on the nature of the side chains R'-R⁴ The compounds of the mvention are mtended to mclude all stereoisomers arising from the presence of any and all isomers of the individual moieties as well as mixtures thereof, mcludmg racemic mixtures

In a preferred embodiment of the present mvention, each of R¹, R², R³, R⁴ independently is selected from hydrogen, optionally substituted Ci 6-alkyl, optionally substituted Ci β-alkenyl, optionally substituted C2 β-alkadienyl, optionally substituted Cβ β-alkatπenyl, hydroxy, -O-R⁶, formyl, -C(=0)-R⁶, -0-C(=O)-R⁶, carboxy, -C(=0)-0-R⁶, optionally substituted heteroaryl, (optio- nally substituted heteroaryl)-Cι 6-alkyl, (optionally substituted heteroaryloxy)-Cι 6-alkyl, optionally substituted aryl, (optionally substituted aryl)-Cι 6-alkyl, (optionally substituted aryl- oxy)-Cι 6-alkyl, halogen such as fluoro, chloro, bromo, and iodo, nitro, cyano, ammo, -NHR⁶, -N(R⁶)2, mono- or dι(Cι 6-alkyl)amιno-Cι 6-alkyl, -N O-CO-R⁶, (Ci 2o-alkyl)carbonylammo-Cι 6- alkyl, carbamoyl, ammocarbonyl-Ci 6-alkyl, mono- or dι(Cι 2o-alkyl)aminocarbonyl, mono- or dι(Cι 6-alkyl)ammocarbonyl-Cι 6-alkyl, sulphanyl, optionally substituted Ci 20-alkylthιo, optionally substituted Ci 20-alkylthιo-Cι 6-alkyl, (optionally substituted aryl)thιo, guanidmo, guanidino-Ci 6-alkyl, sulphono (-SO3H), sulphmo (-SO2H), halosulphonyl, -OS(0)m-R⁶ where m is 2 or 3,

where m is 2 or 3, -S(0)m-N(R⁷)₂ where m is 2 or 3, -S(0)_m-NH(R⁷) where m is 2 or 3, -S(0)_m-NH2 where m is 2 or 3, isocyano, isothiocyano, thiocyano, -OP(0)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and -NGIWO^OI⁶), where p is 1, 2, or 3, q is 1 or 2,

wherem each R⁶ mdependently is selected from hydrogen, optionally substituted Ci 20- alkyl, optionally substituted Ci 20-alkenyl, optionally substituted Ci 20-alkadιenyl, optionally substituted Ci 20-alkatπenyl, optionally substituted aryl, and optionally substituted heteroaryl, and each R⁷ is selected from hydrogen and Ci 4-alkyl

Especially preferred examples are hydrogen, optionally substituted Ci 6-alkyl, hydroxy, -O-R⁶, carboxy, -C(=0)-0-R⁶, optionally substituted aryl, optionally substituted heteroaryl, sulphanyl, carbanoyl, optionally substituted Ci 20-alkylthιo, optionally substituted Ci 20-alkylthιo-Cι 6-alkyl, guanidino, guanidino-Ci 6-alkyl, -0P(O)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and

where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, wherem each R⁶ mdependently is selected from hydrogen, optionally substituted Ci 20-alkyl, and optionally substituted aryl, and each R⁷ is selected from hydrogen and Ci 4-alkyl, preferably hydrogen and methyl

According to the definition of compound I, one of the side cham pairs R¹^², R' R³, and R³/R⁴ may form a biradical which, together with the atoms located between these substituents (C_α and/or Cp), form a rmg In pnnciple a wide range of rmgs may arise from the combmation of two side chams pairs, however, it is preferred that the rmg is a biradical of one of the followmg nngs cyclopropane, oxirane, aziridine, cyclopropene, azinne, cyclobutane, oxetane, azetidine, thietane, 2-azetιdιnone, 1,3-lactone, pyrohdine, pyrohne, pyrrole, cyclopenetene, cyclopentadiene, pyrollidione, pyrolhdone, cyclohexyl, oxirane, dioxirane morpholine, piperidine, 1,5-lactone, 1,5- lactam, cydohexene, cyclohexadiene, pipeπdione, tropane, l,6-lactone(tropolone), 1,6-lactam

Especially preferred examples are oxirane, aziridine, azinne, oxetane, 2-azetιdιnone, 1,3-lactone, pyrohdine, pyrohne, pyrrole, pyrolhdone, pyrolhdione, oxirane, dioxirane, morphohne, piperidine, 1,5-lactam (pipendone), 1,5-lactone, piperidione, tropolone, 1,6-lactam

The most preferred examples are 2-azetιdιnone, 1,3-lactone, pyrolhdone, 1,5-lactam, 1,5-lactone, tropolone and 1,6-lactam Alternatively, one of the side cham pairs R² R⁵ and R⁴ R⁶ may form a biradical which, together with the atoms located between these substituents (C_α, C_p and/or N), form a rmg In this case, it is preferred that the rmg is a biradical of one of the followmg rmgs 2-azetιdιnone, pyrohdine pyrohne, pyrrole, pyrolhdione, pyrolhdone, piperidine, 1,5-lactam, piperidione, and 1,6-lactam The rmg is preferably selected from 2-azetιdιnone, 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

It may be especially preferred to use α-ammo acids, 1 e where n is 0 m formula II, as units for the moieties P¹ and P² m that a large variety of α-amino acids are readilv avadable Thus, in this case, each of the units mdependently has the followmg formula Ila

R¹ I 1

C - ^• Cα - ^■ N Ila

0 II R i^» wherem each of the side chams R¹ and R² are as defined above

In a preferred embodiment, one of the side chams R¹ and R² is hydrogen and the other side cham is selected from hydrogen, optionally substituted Ci 6-alkyl, hydroxy, -O-R⁶, carboxy, -C(=0)- O-R⁶, optionally substituted aryl, optionally substituted heteroaryl, sulphanyl, carbanoyl, optionally substituted Ci 20-alkylthιo, optionally substituted Ci 20-alkylthιo-Cι 6-alkyl, guanidmo, guanidino-Ci 6-alkyl, -OP(0)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and

where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, wherem each R⁶ mdependently is selected from hydrogen, optionally substituted Ci 20-alkyl, and optionally substituted aryl, and each R⁷ is selected from hydrogen and Ci 4-alkyl, and R⁵ is hydrogen or methyl, or R² and R^ε together with the intervening carbon (C_) and nitrogen atoms form an optionally substituted pyrrohdine or piperidine rmg

In a preferred embodiment, 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-aminoisobutyπc acid (Aib), phosphoaminobutyπc acid (PAbu) asparagme, glutamine, aspartic acid, glutamic acid, ornithine, lysme, arginine, histidme, proline, 4-hydroxy -proline, and pipecohc acid, and the N-methyl analogues thereof N^a-methyl-glycιne (sarcosme), N^a-methyl- alanine, N^a-methyl-vahne, N^a-methyl-norvahne, N^a-methyl-ιsovahne, N^a-methyl-leucme, N^a- methyl-norleucine, N^a-methyl-ιsoleucme, N^a-methyl-methιonme, N^a-methyl-phenylalanme, N^a- methyl-tryptophan, N^a-methyl-serme, N^a-methyl-threonme, N^a-methyl-cysteme, N^a-methyl- penicillamme, N^a-methyl-tyrosme, N^a-methyl-asparagme, N^a-methyl-glutamme, N^a-methyl- aspartic acid, N^a-methyl-glutamιc acid, N^a-methyl-ornιthme, N^a -methy 1-lysme, N^a-methyl- arginme, N^a-methyl-hιstιdιne, aminobutync acid, ammohexanoic acid, ammoisobutync acid, aminosuberic acid, butylglycme, citrulline, homocitrulhne, cyclohexylalanme, homoserme, penicillamme, phenylglycme, statine and denvatives, tetrahydroιsoqumohne-3-carboxyhc acids, thienylalanme, pyroglutamic acid, L-azetidme-carboxyhc acid, L-carnitme, D-glucamme, 2R/S- (+ or -)-propanolol-hydrochlonde, L-thyroxme, 3,3',5-tnιodo-L-thyronme-sodιum salt

Furthermore, each of the moieties P¹ and P² may optionally mclude 1-18 units of the formula III

- C ( =0) - (CH₂ ) ₃.₆ -NR⁵ ' - II

wherem the methylene groups may be optionally substituted one or several times, preferably 1-3 time, with group(s) as defined above for Rⁱ-R , and wherem where R⁶ designates the same groups as defined above for R⁶

With respect to end groups and in the case where a unit of the formula III is the C-termmal unit the same groups as defined for the units of formula II apply

With respect to the hnkage between two units of the formula II withm a moiety P¹ and or P² providmg the cychc character of the moiety P¹ and or P², such a hnkage may be any chemical hnkage providing a covalent connection between the two units of formula II m question, such as a direct bond between functionalities m substituents R'-R⁴, e g an ether (-0-), an amide (-NR⁵- C(=O)-), an ester(-C(=0)-0-), phosphodiester (-O-P(0)₂-O-), a disulphide (-S-S-), or a sulphide (-S-) hnkage between two units of formula II 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 an ammo group in another unit, an ester between a carboxy group in one unit and an hydroxy group m another unit, a phosphodiester linkage between a phosphoramidite m one unit and a hydroxy m the other unit (m this case, the resultant tervalent phosphorous hnkage needs to be further oxidised by I2 to the desired pentavalent phosphodiester hnkage), and a disulphide between two thiol groups m two different units Preferably, the hnkage is not estabhshed between two neighbourmg units, and preferably the hnkage is an amide, an ester, a phosphodiester, or a disulphide hnkage, m particular an amide or ester hnkage

In certam interesting embodiments the hnkage is not a ester hnkage

In a preferred embodiment of the present invention, at least one of the moieties P¹ or P² is a cychc moiety, and more preferably, both of P¹ and P² are cychc moieties

In an embodiment of the present mvention, P^l and P² 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 P¹ is preferably identical to the number of units m the moiety P²

In -mother embodiment of the present, P¹ and P² 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 P¹ is preferably identical to the number of units m the moiety P²

It is particularly preferred, with reference to the method for synthesismg the compounds, that the moieties P¹ and P² are substantially identical, I e that the units of the moieties P¹ and P² 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 P¹ and P², respectively, from a sohd phase resm

Accordmg to the general formula I, 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

In the present context, the term "bnear entity" is intended to mean homobifunctional linking moieties, specifically dicarboxylic acids

In the present context, I e the entity A m the general formula I, the term "cychc entity" denotes a cychc or polycychc biradical The cychc entity is bound to the N-termmal ammo groups of the moieties P¹ and P² 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 moieties of the formulae m Figures 4 and 5 where the two radical positions formmg the connections to the two carbonyl groups may be located in any avadable position, or hetero-denvatives thereof arising when one or more appropriate carbon atoms is/are replaced by a hetero atom selected from nitrogen or ammonium (which m appropriate mstances may be carrying hydrogen or an alkyl substituent), or oxygen or sulphur

Specific examples of saturated or unsaturated carbocychc or heterocyclic entities are cyclopropane, oxirane, azrndme, cyclopropene, azinne, cyclobutane, oxetane, 2-azetιdιnone, 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-azetιdmone, 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

Specific examples of aromatic and polyaromatic entities, where one or more carbon atom(s) have been replaced with heteroatoms, are represented m figures 4 and 5

Preferred examples of 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

The 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

With respect to the carbonyl groups separation P¹ and A, and A and P² m formula I, it should be understood that these carbonyl groups may be an mtegral part of a startmg material for the mcorporation of B mto the compound I Thus, m several cases, it is advantageous to use a cychc entity carrying two carboxyhc acid groups, one of these groups optionally in protected form, and the other group in the free acid form or m activated form, as a startmg material In this way, the moieties P¹ and P² will be linked to the entity A via amide bonds Although, amide bonds are the most reahstic bonds in that position, also because of the compatibility with the remaining parts of the compound I due to its peptidic character, other bond types may be envisaged, e g urea (- NR^B-C(=0)-NH-) bonds or thiourea (-NR^B-C(=S)-NH-) bonds formed, e g , when reactmg the NHR⁶ group of the N-termmal unit of P¹ or P² with a isocyanate or isothiocyanate functionahsed cychc entity In the case where two two bonds different from amide bonds are used, it may be advantageous to use two different bond type linking P^l and A and A and P², respectively

Illustrative examples of dicarboxylic acid functional cychc entities, which after mcorporation the compound I will represent the fragment -C(=0)-A-C(=0)-, are

Aromatic and heteroaromatic moieties

2,2'-bιquιnohne-4,4'-dιcarboxyhc acid, 5-nιtro-ιsophthahc acid, 2-amιno-terephthahc acid, 2- bromo-terephthahc acid, 2-nιtro-terephthahc acid, 3,6-dιchloro-phthahc acid anhydnde, 4,5- dichloro-phthahc acid anhydnde, 3-nιtro-phthahc acid anhydnde, 4-nιtro-phthahc acid anhydride, homophthahc acid, 4,4'-bιphenyl-dιcarboxyhc acid, 2,2 -bιphenyl-dιcarboxyhc acid 2,3-naphthalene-dιcarboxyhc acid, 2,6-naphthalene-dιcarboxyhc acid, 1,8-naphthalene- dicarboxyhc acid anhydnde, 3-nιtro-l,8-naphthalene-dιcarboxyhc acid anhydride, 1,2-phenylen- dioxy-diacetic acid, embomc acid, 5,5'-dιthιobιs-(2-nιtrobenzoιc acid) (3, 3' -6), 2,2 -dιthιobenzoιc acid, glutamic acιc-5-(3-carboxy-4-nιtro-anιhde), ahzarm-3-methyhmmodιacetιc acid, 1,4- phenylene-diacetic acid, 2,4'-benzophenone-dιcarboxyhc acid, 2,4'-benzophenyl-dιcarboxyhc acid, chehdamic acid, 2,3-pyndιne-dιcarboxyhc acid, 2,4-pyrιdιne-dιcarboxyhc acid, 2,5-pyndιne- dicarboxyhc acid, 2,6-pyndιne-dιcarboxyhc acid, 3,4-pyπdιne-dιcarboxyhc acid, 3,5-pyrιdme- dicarboxyhc acid, 4,5-pyndιne-dιcarboxyhc acid, ιmιdazole-4,5-dιcarboxyhc acid, 3,4,5,6- tetrachloro-phthahc acid, 2-ammo-4,6-pyrmudιne-dιcarboxyhc acid, 9, 10-anthracene-dιcarboxyhc acid, l,4-dιhydroxy-naphthalene-2,3-dιcarboxyhc acid, benzιmιdazol-5,6-dιcarboxyhc acid, benzophenone-4,4'-dιcarboxylιc acid, 4-methoxy-phthahc acid, naphtidine- 3, 3' -dicarboxylic acid, naphthalene- 1,2-dιcarboxyhc acid, naphthalene- 1,3-dιcarboxyb.c acid, naphthalene- 1,4- dicarboxyhc acid, naphthalene- 1,5-dιcarboxyhc acid, naphthalene- 1 ,6-dιcarboxy he acid, naphthalene- 1,7-dιcarboxyhc acid, naphthalene- 1,8-dιcarboxyhc acid, naphthalene-2,3- dicarboxyhc acid, naphthalene-2,4-dιcarboxyhc acid, naphthalene-2 5- dicarboxylic acid, naphthalene-2,7-dιcarboxyhc acid, naphthalene-2,8-dιcarboxyhc acid, pynmιdme-4,6-dιcarboxyhc acid, pyrrazole-3,6-dιcarboxyhc acid, and l, 10-phenanthrohne-5,6-dιcarboxyhc acid, and

Lmear and non-aromatic cychc moieties oxahc acid, malonic acid, succmi acid, glutaric acid, adipic acid, pimehc acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid docosanedioic acid, trans.trans-mucomc acid, methylmaleic acid, methyhnaleic acid anhydnde, (+)- and (-)-camphorιc acid, 1,3-acetone dicarboxyhc acid, N-(acetamιdo)-ιmmodιacetιc acid, L-aspartic acid, S-carboxymethyl-L-cysteme, 2,2'-(ethylendιthιo)-dιacetιc acid, malic acid (+ and -), D-pemcillamine, phenylsuccmic acid, N-(phosphonomethyl)-ιmmodιacetιc acid tetrahydrofohc acid; (+ or -) 0,0'-dibenzyl-2-tartaric acid; (3-thienyl)-malonic acid; N-phtaloyl-1- glutamic acid; diphenyl maleic anhydride; cis-l,2,3,6-tetrahydrophthahc acid; 3,4,5,6-tetrahydro- phthahc acid; pyrazine-2,3-dicarboxyhc acid; ticarcillin; chehdonic acid; glycine cresol red; cyclopropane- 1, 1' -dicarboxylic acid; cyclobutane- 1, 1' -dicarboxylic acid; l-cyclopentene-1,2- dicarboxylic acid anhydride; l, l'-azobis-(cyclohexane-carboxyhc acid); cyclopropane- 1,2- dicarboxyhc acid; (cis+trans)-l,4-cyclohexandicarboxyhc acid; pyrazine-2,5-dicarboxyhc acid; piperidine-2,6-dicarboxyhc acid; piperidine-3,3-dicarboxyhc acid; pyrroline-N-oxide-5,5- dicarboxylic acid; gamma-pyrone-2,6-dicarboxyhc acid and piperazine-2,6-dicarboxylic acid.

Preparation

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 P¹ and P². 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. A factor which requires consideration is the efficiency of cyclisation (if required) after the linear peptide has been synthesised. In a prefened embodiment Tentagel resin with a large particle size (130 μ) and a low loading (150-160 μmol/g of the first amino acid) is preferably used to favour cychc peptide over dimer formation.

As an example, the compounds of the general formula (I) may be prepared by using the Fmoc strategy according to the following reaction protocol:

(1) synthesis of the linear moiety (P¹) using Fmoc sohd phase peptide synthesis: PyBOP/HOBt may be the condensation agent whilst Fmoc deprotection is effected by e.g. piperidine/DMF. The synthesis is performed in a manual glass sinter column, in a batch¬ wise manner. The methods described herein should in principle be easily adaptable for continuous-flow sohd phase synthesis on an automated machine; (2) if required, cychsation of the bnear moiety (P¹) Removal of the protectmg group (e g an allyl group) of e g the functional side chams of one of the moieties and the N-termmal moiety and addition of a suitable cychsation reagent,

(3) couplmg of the resm-bound moiety P¹ with a dicarboxylic acid functionahsed cychc entity The Fmoc group is removed usmg standard conditions, then treated with a monoprotected form of the diacid constituting the dicarboxylic acid functionahsed cychc entity (e g the monoallyl ester), PyBOP/HOBt, and DIEA,

(4) the protecting group (e g the allyl group) of the dicarboxylic acid is removed after which cleavage of the immobilised moiety P¹-C(=0)-A-COOH (e g usmg 10% pyπdine/methanol) is performed,

(5) synthesis and cychsation of an immobilised moiety P² in the same manner as m steps

(1) and (2),

(6) couplmg of the P¹-C(=0)-A-COOH fragment onto the immobilised moiety P² the free carboxyhc acid group on the diacid moiety is activated with PyBOP/HOBt, and the fragment P'-C(=0)-A-COOH is then coupled onto the immobilised moiety P², and

(7) the complete conjugate P¹-C(=0)-A-C(=0)-P² is then cleaved off the support (e g usmg 10% pyridine/methanol)

The scheme proposed above utilises well-established peptide synthesis techniques such as orthogonal protection and segment condensation

In a specific example shown Scheme 1 for illustration purposes, 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 whύst 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 3 HATU activation (Carpmo, L A J Am Chem Soc , 1993, 115, 4397-4398, Angell, Y M , Garcia-Echeverna, 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 Soc 1995, 117, 7279-7280) was employed m this example Removal of the allyl group on the other naphthyl carboxyl group of 6 as above (Kates, S A , Daniels, S B and Albencio, F Analytical Biochemistry 1993, 212, 303-305) followed by MeOH/pyndme cleavage (Atherton, E and Sheppard, R C Solid Phase Peptide Synthesis- A Practical Approach, IRL Press Oxford 1989, pp 152-154) gave the naphthyl-cychc peptide segment 8 Small ahquots of sohd support were cleaved throughout this sequence of reactions and analysed by RP-HPLC (Figure 2) and products of high purity were observed An important pomt to note is that these cychsation conditions unhke others previously reported (Murray, J S Tetrahedron Lett 1991, 32, 7679- 7682, Kates, S A, Sole, N A , Johnson, C R , Hudson, D , Barany, G and Albencio, F

Tetrahedron Lett 1993, 34, 1549-1552, Bloomberg, G B , Askm, D , Gargaro, A R and Tanner, M J A Tetrahedron Lett 1993, 34, 4709-4712, and Eichler, J , Lucka, A W and Houghten, R A Peptide Research 1994, 7, 300-307), gives exclusively the desired cychc product in good yield with no dimeric side-product or the linear precursor This is very important smce there are two carboxyl groups m the support-bound peptide 7 and the α-carboxyl group of the C-termmal glutamic acid residue needs to be selectively blocked so that the naphthyl carboxyl can react with 6 Finally, the second portion of 6 was deprotected and a segment condensation (Carpino, L A , E1-Faham, A and Albencio, F Tetrahedron Lett 1994, 55, 2279-2282) of the second portion of 6 with the naphthyl-peptide segment gave the desired product 1

Other 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

The 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

In more general terms, a further aspect of the present mvention relates to a method for the preparation of a compound of the followmg formula I

pⁱ-C(=0)-A-C(=0)-P²

as defined above, compnsmg the followmg steps (A) providmg an optionally side cham protected moiety P¹, which may be linear or cychc immobihsed to a sohd support material, the α- or β-amino group of the N-termmal unit of the immobi sed moiety P¹ (the unit neighbourmg the carbonyl group (C(=0)) located between P¹ and A) bemg unprotected,

(B) couplmg a diacid HOOC-A-COOH, m the free acid, monoester or mternal anhydnde form, to the unprotected ammo group of the N-termmal unit of the immobihsed moiety P¹ for the formation of an immobihsed fragment P¹-C(=0)-A-COOH m the free acid or ester form,

(C) cleavmg the fragment P'-C(=0)-A-COOH optionally in the ester form (-A-COOR) from the sohd support material,

(D) providing an optionally side cham protected moiety P², which may be bnear or cychc immobi sed to a sohd support material, the α- or β-amino group of the N-terminal unit of the immobilised moiety P² (the unit neighbourmg the carbonyl group (C(=0)) located between P² and A) bemg unprotected,

(E) couplmg the fragment P¹-C(=0)-A-COOH to the immobihsed moiety P² for the formation of an immobihsed compound P¹-C(=0)-A-C(=0)-P², and

(F) cleavmg the compound P'-C(=0)-A-C(=0)-P² from the sohd support material

The optionally side cham protected moiety P¹ immobihsed to a sohd support material may be provided usmg standard sohd phase peptide synthesis methodologies Thus, if 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 Preferably, 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

The ammo group of the N-termmal unit of the immobihsed moietv P¹ (the unit neighbourmg the carbonyl group (C(=0)) located between P¹ and A) should be unprotected so that subsequent coupling of the entity A can be accomphshed Subsequently, a diacid HOOC-A-COOH, m the free acid, monoester, or mternal anhydnde form, to the unprotected ammo group of the N-termmal unit of the immobihsed moiety P¹ for the formation of an immobi sed fragment P¹-C(=0)-A-COOH m the free acid or ester form In this step 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-azabenzotrιazol-l- yl)-l,l,3,3-tetramethyluronιum hexafluorophosphate ((HATU)) may be used It will often be necessary to select a coupling reagent which is compatible with any side cham functionabty protectmg groups attached to P¹

After preparation of the fragment P¹-C(=0)-A-COOH on the sohd phase material, the fragment is cleaved from the sohd support material usmg cleavage reagent suited for the specific sohd phase material selected. As mentioned above, the C-terminal carboxyhc acid of the moiety P¹ may be derivatised m several way dunng cleavage, e.g. resultmg in the ester (-COOR) or amide (-

The optionally side chain protected moiety P², which, as P¹, may be linear or cychc, immobihsed to a sohd support material, is prepared m a way similar or identical to P¹. In the highly interesting instances where P¹ and P² are substantially identical, it may be advantageous to prepare the immobi sed moieties P¹ and P² m one batch

The fragment P^I-C(=0)-A-COOH is then coupled to the immobihsed moiety P² for the formation of an immobihsed compound P'-C(=0)-A-C(=0)-P² As for the couplmg of the entity A to P¹ a number of coupling reagents may be used

Fmally, the compound P¹-C(=0)-A-C(=0)-P² is cleaved from the sohd support matenal usmg standard methodologies In the case where P¹ and P² are identical, it is possible to mtroduce individuality m these moieties by usmg different cleavage conditions when cleavmg the compound from the sohd phase material compared to the conditions for cleavage of the fragment, P¹-C(=0)-A-COOH In this case the moieties P¹ and P² becomes substantially identical

The natural 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

With respect to the preparation of compounds I where one or more of the groups R⁶ are Ci 4- alkyl, e g. methyl, it is known that the coupling efficiency usmg conventional couplmg methods may be reduced due to stencal hmdrance However, the mcorporation of sterically hmdered ammo acids, e g N-methyl ammo acids and Aib, m a compound I can be achieved effectively usmg HATU activation Other possible methods for the synthesis of difficult peptides mclude the use of PyBrOP (Coste, J , Frerot, E , Joum, P and Castro, B Tetrahedron Lett 1991, 32, 1967- 1970), ammo acid fluorides (Carpino, L A , Sadat-Aalaee, D , Chao, H G and DeSelms, R H J Am Chem. Soc.) and 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 -4m Chem Soc 1995, 117, 7279-7280) PyBOP (Frerot, E , et al , Tetrahedron, 1991, 47(2), pp 259-270), and CF₃-N0₂-PyBOP (Wijkmans, J C H M , et al, Tetrahedron Lett 1995, 36(26), pp 4643-4646) The condensation reagents PyBrOP and HATU, and also the ammo acid fluoride (AAF) activation procedure have been shown to be effective

In order to be able to utilise a wide range of bifunctional aromatic and polyaromatic molecules m the synthesis of compounds I, the accessibility to synthetic routes towards, e g , aromatic dicarboxylic acid monoesters are advantageous Three different syntheses are shown m Scheme 2 The first method is a novel approach based on the esterification, m particular the allylation, of the monocaesium salt of a diacid, here the naphthalene diacid 2, with, e g , allyl bromide The second one is based on the use of anion exchange resms to block off one of the carboxyhc acid groups while the other undergoes esterification (Blankemeyer-Menge, B , Nimtz, M and Frank, R Tetrahedron Lett 1990, 57, 1701-1704) A third possible route it the selective deestenfication of dicarboxylic acid diesters A further possibility is using phase transfer chemistry (Friednch- Bochmtschek S , J Org Chem 54, 1989, 751-756)

Advantageous conditions for the production of the monoester with a mmimum yield of the diester were an equimolar amount of CS2CO3 added m small portions over an extended period such as 16h, and 10 equivalents of allyl bromide Obtamable yields of the purified monoester 3 are at least 30%

An alternative method (Scheme 2) based on the adsorption of the startmg diacid 2 onto an anion exchange resm followed by reaction with mesithylenesulphonylnitrotπazohde (MSNT), N- methyhmidazole and allyl alcohol produces the desired monoester product 3

The third method is based on the possibility of mono-deesteπfication 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

Adaptation of this method for the preparation of Actmomycm D analogues to combmatonal synthesis of a wide range of compounds of the formula I is possible Specifically, diversity can in principle be easily mtroduced withm the moiety P¹, the entity A, and the moiety P² Preferably the diversity is mtroduced m P¹ and/or P² smce production of peptide hbraries is a well- established technology Thus, the methodology allows any dicarboxylic acid monoester to be incorporated whύe any of the existmg methods for the generation of combmatonal hbraries of e g , peptides also may be used (Terrett, N K , Gardner, M , Gordon, D W , Kobylecki, R J and Steele, J Tetrahedron 1995, 51, 8135-8173)

The strategy and consideration with respect to the preparation of hbraries of Actmomycm D analogues will be exemplified m the followmg The strategy for synthesismg the cychc peptide- aromatic diacid conjugate 1 (Scheme 1) features the use of Fmoc sohd phase peptide synthesis (Atherton, E and Sheppard, R C Solid Phase Peptide Synthesis- A Practical Approach, IRL

Press Oxford, 1989), and orthogonal protection scheme and segment condensation The ultimate goal of combmatonal synthesis of these compounds can m principle be readily achieved dunng the synthesis of the linear peptide 5 usmg any of the standard techniques (Furka, A., Sebestyen, F , Asgedom, M and Dibo, G Abstr 14th Int Congr Biochem , Prague, Czechoslovakia 1988, 5 47, Furka, A, Sebestyen, F , Asgedom, M and Dibo, G Int J Peptide Protein Res 1991, 37, 487-493, Houghten, R A Proc Natl Acad Sci USA 1985, 82, 5131-5135, and Houghten, R A and Dooley, C T Bioorg Med Chem Lett 1993, 5, 405-412) and a second level of diversity can be mtroduced m the denvatisation of the support-bound cychc peptide 6 with the diacid monoester 3 Consequently the present concept can allow for double combmatonal synthesis of these analogues, with the first level of diversity resultmg from the combmatonal synthesis of the linear or cychc peptides bemg increased m multiples arismg from the consecutive segment condensation

In order to illustrate the pnnciple of preparmg hbraries of Actmomycm D analogues hbraries consistmg of compounds where P¹ and P² each consist of one optionally O-functionahsed tyrosme unit (number of units is 1), and where the entity A is 1,4-phenylene, have been prepared (see the examples)

Thus, m a further aspect the present mvention relates to a method for the preparation of a multi-dimensional array (combmatonal hbrary) of compounds, {P'}-{A}-{P²}, consistmg of at least three compounds each havmg the general formula I

P¹ - C ( =0) -A- C ( =0) - P² I s defmed above and m the claims, compnsmg the followmg steps

(A) providing an array {P¹} of at least two optionally side cham protected moieties P¹ immobihsed to a sohd support matenal, the ammo group of the N-termmal unit of the immobihsed moieties P¹ bemg unprotected,

(B) coupling an array {A} of one or more diacids, HOOC-A-COOH, in the free acid, monoester or mternal anhydride form, to the unprotected ammo groups of the N-termmal unit of the immobihsed moieties P¹ for the formation of an array {P'}-{A> of immobihsed fragments P¹-C(=0)-A-COOH, optionally m the ester form,

(C) cleavmg the array {P'}-{A} of fragments P¹-C(=0)-A-COOH, optionally the ester form, from the sohd support material,

(D) providing an array {P²} of at least two optionally side cham protected moieties P² immobi sed to a sohd support material, the α- or β-amino group of the N-termmal unit of the immobihsed moieties P² bemg unprotected,

(E) couplmg the array {P^]}-{A} of fragments P¹-C(=0)-A-COOH, optionally m the ester form, to the array {P²} of immobihsed moieties P² for the formation of an array {P'}-{A}-{P²} of immobihsed compounds P¹-C(=0)-A-C(=0)-P², and

(F) cleavmg the array {P'}-{A}-{P²} of immobi sed compounds P'-C(=0)-A-C(=0)-P² from the sohd support material

Preferably, the array {P²} of immobihsed moieties P² is substantially identical to the array {P¹} of immobihsed moieties P¹ This can easdy be obtamed when the array {P¹} of immobihsed moieties P¹ and the array {P²} of immobilised moieties P² is prepared in one batch

The conditions m each of the synthetic steps m the method for the preparation of compound hbraries are simύar to the correspondmg conditions m the method for the preparation of smgle compounds However, 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

It is bebeved that the principle of preparmg a compound hbrary, e g P¹ and P², m batch and subsequently reacting a first part thereof with a compound or an array of compounds, e g the entity HOOC-A-COOH, cleavmg the fragment obtamed, e g P¹-C(=0)-A-COOH, and couphng this fragment back onto a second part of the origmal batch, is novel itself

Thus, a further aspect, the present mvention relates to a method for the preparation of a multi-dimensional array {HHBML²} of at least three compounds each havmg the formula L'-B- L², wherem the arrays {L¹} and {L²} are of similar chemical composition, each of L¹ and L² mcludes a fragment l¹ and l², respectively, of a chemical functionabty, B is an entity which mcludes two fragments b¹ and b² of chemical functionalities, each of the fragment sets Pb¹ and b²l² formmg a covalent hnkage between L¹ and B and between B and L², 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

(A) providing an array {L} of at least two moieties L immobihsed to a sohd support matenal, where the array {L} has similar chemical composition to the arrays {L¹} and {L²}, each of the moieties L mcludes a group conesponding to a fragment 1 of a chemical functionabty, where the fragment 1 is identical to the fragments l¹ and l², the groups corresponding to the fragment 1 bemg in protected or unprotected form and any other groups of L sensitive to b/1 bond formation reaction conditions bemg optionally protected,

(B) dividing the sohd support matenal representmg the array {L} mto two parts to give arrays {L¹} and {L²}, respectively, the array {L¹} compnsmg immobihsed moieties designated L¹ and the array {L²} compnsing immobihsed moieties designated L²,

(C) m the case where the group conesponding to the fragment l¹ is protected, deprotecting said group,

(D) under b/1 bond formation reaction conditions, couphng an array {B} of one or more compounds conesponding to the entity/entities B to the anay {L¹} for the formation of an array {L'}-{B} of immobihsed compound fragments L'-B, where the b/1 bond formation reaction mvolves the group corresponding to b¹ of the compounds correspondmg to the entity/entities B and the group correspondmg to l¹ of the immobihsed moieties L¹, the group conesponding to b² of the entity/entities B and any other groups sensitive to bΛ bond formation reaction conditions optionally bemg protected, (E) cleavmg the array {L'}-{B} of immobihsed compound fragments L'-B from the sohd support material,

(F) if necessary, deprotectmg the group correspondmg to the fragment b²,

(G) in the case where the group correspondmg to the fragment l² is protected, deprotectmg said group,

(H) under b/1 bond formation reaction conditions, couphng the array {L'}-{B} of compound fragments L*-B to the array {L²} for the formation of a multi-dimensional array {L¹}-{B}- {L²} of immobilised compounds L'-B-L², the b/1 bond formation reaction mvolvmg the group corresponding to l² and the group corresponding to b², and

(J) cleavmg the multi-dimensional anay {L¹}-{B}-{L²} of immobihsed compounds U-B-U from the sohd support material

In the case where the bonds between the L's and B are ester bonds l¹ may be a carbonyl group (- C(=0)-) and b¹ may be a oxy group (-0-), or, alternatively, b¹ may be a carbonyl group (-C(=0)-) and l¹ may be a oxy group (-0-)

The prmciples for the preparation of hbraries {L'}-{B}-{L²} of compounds U-B-U follows the prmciples described for the Actmomycm D analogue hbraries

Method of screening

By usmg the strategy described above, it is possible to produce a very diverse range of compounds all based on the Actmomycm D principle Thus, 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 In the event that 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 easdy and rapidly for large numbers of samples at a time on an ELISA plate reader However, the compound accordmg to the mvention may have biologically effects different from that of Actmomycm D thus other assays could include screenmg for antiviral, anticancer, antibiotic (such as antibacterial and antifungicidal), osteoclast inhibitory, protease inhibitory or herbicidal activity

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)

Thus, 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

DESCRIPTION OF THE DRAWINGS

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/pyπdine, (h) HATU/DIEA

Scheme 2 shows the three different method (A, B, and C) for the preparation of a monoester of a dicarboxylic acid Experimental details are given m the examples

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

1 1 H 19 benzyl benzyl

12 benzyl 20 benzyl propyl

13 propyl 21 benzyl 2-Ph-Et-

14 2-Ph-Et- 22 propyl propyl

15 benzyl 23 propyl 2-Ph-Et-

16 propyl 24 2-Ph-Et- 2-Ph-Et-

17 2-Ph-Et- 25 benzyl H

18 H 26 propyl H

27 2-Ph-Et- H

28 H H

Figure 1 shows HPLC chromatograms of intermediates m the synthesis of 1 All peptides were cleaved from the sohd support after removal of the Fmoc group as described m Atherton, E and Sheppard, R C Solid Phase Peptide Synthesis - A Practical Approach, IRL Press Oxford, 1989, pp 152-154 Column Merck 50983 LiChrospher 100 RP-18, 5 mm, 250 X 4 mm Buffer A, 0 1% TFA/H2O Buffer B, 0 1% TFA, 80% CH3CH/H2O Elution gradient for (a) and (b) 0% B (5 mm) 0-20% B over 40 mm, 20-100% B over 10 mm, then 100% B (5 mm) Monitored at l=220nm Elution gradient for (c) 0% B (5 mm), then 0-100% B over 50 mm (a) Linear peptide cleaved from resm 5 (b) Cychc peptide cleaved from resm 6 (c) Naphthoic acid cychc peptide segment 8

Figure 2 shows a HPLC chromatogram of crude conjugate 1 All conditions identical to those of Figure 1 (c)

Figure 3 shows the UV spectrum of conjugate 1

Figures 4 and 5 show structures of examples of the aromatic or polyaromatic part of the carbocychc entities

EXAMPLES

Synthesis of the model compound (Scheme 1)

The synthetic strategy was tested by synthesismg a model compound consistmg of a naphthalene dicarboxylic acid core (-C(=0)-A-C(=0)-) with two cychc peptides (P¹ and P²) attached to the carboxyhc acid groups of the naphthalene dicarboxylic acid

Fmoc solid phase peptide synthesis

One synthesis cycle is as follows Fmoc deprotection with 20% pipeπdine/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)

Peptide cyclisation on the solid support (6)

The allyl group was removed usmg Pd(0) (Kates, S A, Daniels, S B and Albencio, F Analytical Biochemistry 1993, 212, 303-305 The conditions were Pd(0) (0 14 M solution m 5% acetic acid and 2 5% N-methylmorphohne m chloroform, 3 4 eq) 2 h under argon)

The followmg procedure was used for the removal of the Boc group The resm was rmsed with 50% TFA/dichlorom ethane prior to a (2 + 18 mm) treatment The resm was then rinsed with dichloromethane and 10% DIEA N-methylpyrolhdone followed by N-methylpyrolhdone) respectively, PyBOP-mediated condensation (Our conditions are similar to those described m Kates, S A, Daniels, S. B and Albencio, F Analytical Biochemistry 1993, 212, 303-305 except the followmg modifications were mcluded to maximise the yield of cychc peptide over unwanted side-products, large particle size TentaGEL (130μ) resm with low loading (approximately 160 μmol/g of ammo groups compared to the usual 240 μmol/g), PyBOP (4 eq)/HOBt (4 eq) DIEA (8 eq) m N-methylpyrolhdone for 1 h) then gives the cychc peptide 6, which was shown to have the correct molecular weight (525) by ion-spray mass spectrometry No other peaks were observed m either the LC-ion-spray MS or the PD-MS spectra An isolated yield of 30% was obtamed

Derivatization of the support-bound cyclic peptide (6) with the monoester (3)

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. Soc 1995, 7/ 7, 7279-7280 One representative example is monoester (2 eq), HATU (2 eq), DIEA (4 eq) m a dry N-methylpyrrohdone solution, 0 13 M) was employed Removal of the allyl group on the other naphthyl carboxyl group of 6 as above

Cleavage and condensation of the naphthyl-cyclic peptide segment (8)

The support-bound naphthyl-cychc peptide segment (7) was cleaved usmg 90% methanol/pyπdine (17 h) (Atherton, E and Sheppard, R C Solid Phase Peptide Synthesis- A Practical Approach, IRL Press Oxford, 1989) The usual base employed m this cleavage is tnethylamme but we found that the quahty of the products varied significantly Replacement of piperidine gave consistently good results A second noteworthy observation is that the MeOH/pyridine cleavage gives exclusively the C-termmal Glu methyl ester with none of the free acid bemg produced from any residual moisture in the cleavage mixture (the segment 8 was compared by RP-HPLC with another sample cleaved with 1 M NaOH from the same support 7 to a peptide segment with the Glu as a free acid)

A segment condensation between 8 and 6 (Carpino, L A , El-Faham, A and Albencio, F Tetrahedron Lett 1994, 35, 2279-2282) was performed with several major modifications After Fmoc deprotection, the resm 6 (0 167g, 22 9μmol of ammo groups determmed by quantitation of the Fmoc group prior to deprotection (Milhgen 9050 PepSynthesizer Technical Note 3 10,

Milhpore Corporation, 1987)) was treated with the naphthyl segment 8 (0 026 g, 34 4 μmol), HATU (0 014g, 34 4 μmol) and DIEA (11 8 μL, 68 8 μmol) m iV-methylpyrolhdone (350 μL) for 17h The reaction was shown to be mcomplete at this stage by TNBSA assay (Benjamin, D M , McCormack, J J and Gump, D W Anal Chem 1973, 45, pp 1531-1534) More DIEA (50 μL 291 5 μmol) was added and the reaction allowed to proceed for another 24 h, at which stage the TNBSA assay showed no residual ammo groups) was performed with 8, giving the bis-cychc peptide-aromatic diacid conjugate 1 The HPLC chromatogram of 1 (Figure 2) showed complete conversion of the cychc peptide precursor 6 to one major peak (67%) corresponding to the desired conjugate (compare with Figure 1 b), this m turn corresponds to a very good yield of 59% for the segment condensation LC-MS (LC/Ion-spray MS for 1 1230 8 (M + H) and 1198 4 (M - 2 X CHa) Interestingly, the latter peak (about the same intensity as the other peak) is consistent with the product of hydrolysis of 1 whilst bemg concentrated m an aqueous buffer (8 mbar, 40° C, 3 h) There is no detectable hydrolysis (HPLC, MS) after standmg in aqueous buffers for days at 25°C lyophihzation or concentration in low-boihng pomt organic solvents such as MeOH and acetonitnle This suggests that the methyl esters can be either removed from the conjugate by hydrolysis under mild conditions or left intact if the appropriate precautions are adhered to) The UV spectrum of 1 (Figure 3) is characteristic of a conjugate between a peptide (band at 210 nm) and a naphthyl moiety (the other bands), the naphthyl bands are blue-shifted by 40 nm on attachment of the peptides Chromatograms shown (Figures 1 and 2) are of crude products

Synthesis of aromatic dicarboxylic acids monoesters (Scheme 2)

Method A

Allylation of the Cs salt of the diacid 2 with allyl bromide

CS2CO3 (1 60 g, 5 00 mmol) was added in two equal portions over 0 5 h to a solution of 2 (2 16 g, 10 0 mmol) m anhydrous DMF (97 mL) Allyl bromide (17 3 mL, 200 mmol) was then added and the reaction mixture stirred vigorously for 24 h The same amount of CS2CO3 was then added in two equal portions over 8 h and the reaction allowed to proceed for a further 17 h The precipitate was filtered and rinsed with DMF (3 X 20 mL) The combmed filtrate was then concentrated in vacuo and extracted with hot 50% MeOH acetone (2 X 100 mL) After evaporation of the solvent, residual startmg material 2 was removed by sihca gel chromatography The resultmg fractions which contamed a mixture of the monoester 3 and the diester was concentrated, redissolved m 10% MeOH/dichloromethane, and treated with Amberlyst A-26 (hydroxide form) resm (24 0 g, 96 meq) for 17 h The resm was then filtered and washed with fresh solvent untύ no diester was detected m the effluent by TLC (about 3000 mL) The desired compound 3 was then eluted with MeOH dichloromethane/acetic acid (10 80 10) After solvent removal, recrystallization from acetone gave 3 as a fine colourless sohd (0 77 g, 30%), mp 217-218°C d (250 MHz, DMSO-d6) 4 88 (d, 2H, CH₂-CH=CH₂ J = 5 1 Hz), 5 30 (m, 1 H, CH2-CH-CH2), 5 45 (m, 1 H, CH₂-CH=CH₂))

Method B Anion exchange

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

Tetrahedron Lett 1990, 31, 1701-1704) with allyl alcohol (Amberlyst A-26 (hydroxide form) resm (0 150 g, 0 6 meq) was added to a solution of 2 (0 108 g, 0 5 mmol) m DMF (5 mL) and the mixture vortexed for 17 h The loading of the resm was determmed to be 1 28 mmol of 2 per gram by monitoring the UV absorbance of the supernatant before and after treatment with the resm After filtration and washing with DMF and dichloromethane, the resm was dried under high vacuum (17 h) Anhydrous dichloromethane was then added to swell the resm under an argon atmosphere (15 mm), followed by N-methyhm dazole (158 μL, 2 0 mmol), allyl alcohol (1 7 mL, 25 0 mmol) and MSΝT (0 181 g, 0 6 mmol) The reaction was vortexed (1800 rpm) for 24 h Elution m the same manner as in (a) gave a mixture of the startmg material 2 (87%) and the desired product 3 (13%) as observed by HPLC The former results m a 30% yield after facde and efficient purification by sihca and ion-exchange chromatography The conditions were optimised the most influential factor bemg the amount of CS2CO3 which had to be equimolar with the diacid 2 and added m small portions of 0 25 eq over 24 h In another method only the monoester 3 is formed The only other side product is the startmg material 2 Amberlyst A-26 (hydroxide form) resm (0 150 g, 0 6 meq) was added to a solution of 2 (0 108 g, 0 5 mmol) m DMF (5 mL) and the mixture vortexed for 17 h The loading of the resm was determmed to be 1 28 mmol of 2 per gram by monitoring the UV absorbance of the supernatant before and after treatment with the resm After filtration and washmg with DMF and dichloromethane, the resm was dried under high vacuum (17 h) Anhydrous dichloromethane was then added to swell the resm (15 mm) (Ar) followed by N-methyhmidazole (158 μL, 2 0 mmol), allyl alcohol (1 7 mL, 25 0 mmol) and MSNT (0 181 g, 0 6 mmol) The reaction was vortexed (1800 rpm) for 24 h Elution the same manner as m (a) gave a mixture of the startmg material 2 (87 %) and the desired product 3 (13 %) as observed by HPLC

Method C

Selective hydrolysis of dicarboxylic acid diesters

Synthesis of Terephthalic acid dimethylester

16 6 g (100 mmol) of Terephthahc acid was dissolved in methanol (250 ml) Concentrated sulphuric acid (2 ml) was added and the mixture refluxed over night The methanol was removed in. vacuo and the residue redissolved m dichloromethane The organic phase was washed with saturated aqueous sodium bicarbonate solution (3 x 50 ml), dried (magnesium sulphate), filtered and concentrated to give a white crystalline compound (18 4 g, 95 %, m p = 141-142 °C)

Synthesis of Terephthalic acid monomethylester

1.94 g (10 mmol) of Terephthahc acid dimethylester was dissolved m DMF/water (95 5, 60 ml, small amount of precipitate m the solution) Potassium hydroxide (1 12 g, 20 mmol) was dissolved m 5 ml of water yielding a 4 M solution which was mtroduced m 0 25 ml portions at intervals of 10 mm for 160-170 mm The mixture was concentrated and the residue redissolved in saturated aqueous sodium bicarbonate and extracted with dichloromethane (3 x 20 ml) Durmg cooling m an ice-water bath, cone HCL was added to the aqueous phase to pH = 3 and 20 % citric acid to pH = 1 The mixture is filtered, the precipitate washed with cold water to neutral outflow and dried (1 67 g, 92 %, m p = 214-215 °C)

Synthesis of Terephthalic acid methyl-2-propenylester Terephthahc acid monomethylester (1 80 g, 10 mmol) was mixed with 100 ml of dry DMF Cesium carbonate (3 57 g, 11 mmol, 1 1 eq ) was addded and the mixture was stirred o n The mixture was filtered and the filtrate was concentrated to dryness The oily residue was redissolved dichloromethane, filtered, washed with saturated aqueous sodium bicarbonate (3 x 50 ml) dried (magnesium sulphate) and filtered Activated carbon black was added and the solution stirred for 15 mm After numerous filtrations the organic solution was almost colourless Upon evaporation a colourless oil was obtamed which freezes upon cooling

Η-NMR (CDCls) d 4,9 d, 2 H Jab - 5,6 (OCH_CH=CH₂), 5,4 m, 2 H (=CH₂), 6,1 m, 1 H (- CH=CH₂), 8,2 s, 4 H (arom ), '^C-NMR (CDCb) d 66 (OCHa), 119 (=CH₂), 129,6 (4 * CH, arom ), 130, 1 (CH=)

The allyl methyl ester of lsothphalic acid was also prepared m good yield

Synthesis of Terephthalic acid mono-2-propenylester (9)

Terephthahc acid methyl-2-propenylester (1 6 g, 7 3 mmol) was dissolved m HMPA (20 ml) and heated to 65 °C whde stirring Sodium cyanide (062 g, 1 75 eq ) was added and the mixture was stirred for 2 h at 65 °C The mixture was cooled to T < 10 oC , and HCl (aq , 10%) was added

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 organic phase was dried (magnesium sulphate), and extracted with saturated aqueous sodium bicarbonate (3 x 50 ml) HCl (10%) was added until pH = 4, and then citric acid (20%) was added untd pH = 1 The precipitate was collected, washed with water untd the effluent was neutral and dried (0 67 g,

45%, 149-151 °C)

»H-NMR(CDCl3) d 3 95 s, 3 H (OCHa), 4 85 dt, 2 H J._b = 5 7, <.«/-+- = 1 4

5 3 dq, 1 H Jbc = 10 2, J-/_₊d = 1 7 45 dq, 1 H J-a = 17 1, Jd/.-c = 1 7

6 05 m,

8 1 sφred), 4 H(arom), ¹³C-NMR(CDCl3) d

52 335(OCH₃), 65 866(OCH₂), 118 571( =CH₂), 129 471(4 * CH, arom), 132(CH=), 134 1(2 * C, arom, Cl + C4), 165 3, 166 5(2* C=0)

The monoester of isophthahc acid were also prepared in good yield

Synthesis of iV-methylated peptides

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

This sequence contams most of the ammo acids m the natural product without the N-methyl groups The N-termmal Lys and the C-termmal Glu are convenient handles on which the cychc peptide bond can be formed The mvention makes it possible to use the powerful technique of combmatonal synthesis to generate a hbrary of compounds which fit the "double-binding" theme of natural Actmomycms I e compounds which can both hydrogen bond and intercalate with nucleic acids

Three test peptides were used to ascertam the efficiency of the acylation procedures

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 These two peptide sequences were used to compare the efficacy of couphng procedures for mcorporation of N-methylammo acids 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 The PyBrOP and ammo acid fluoride procedures were compared

As an illustrative example, 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) and long reaction tunes (2 X 17h couplings) were found to give quantitative couplings It was also found that an equimolar (with respect to the ammo acid fluoride) amount of diisopropylethylamine gives excellent results

Conditions for the HATU activation were ammo acid (10 eq), HATU (10 eq) and DIEA (20 eq) in anhydrous -V-methylpyrolhdone (0 2 M) with two consecutive 17 h couplings The coupling yield was 89%, and the product was homogeneous by RP-HPLC, there was no sign of racemization in this case Attempts at repeatmg the conditions described elsewhere (Coste, J , Frerot E , Journ, P and Castro, B Tetrahedron Lett 1991 32 1967-1970, Carpino, L A , Sadat-Aalaee, D , Chao, H G and DeSelms, R H J. Am Chem. Soc 1990, 112, 9652-9653, 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, Angell, Y M , Thomas, T L , Flenkte, G R and Rich, D R J Am Chem Soc 1995, 117, 7279-7280) gave low couplmg yields)

Preparation of a compound library

Synthesis of comp. 11

0 519 g (0 23 mmol) of Rmk resm was swollen in DMF (5 ml) The resm was treated twice with 20% pipendme/DMF for 20 mmutes Fmoc-tyrosine (98 mg 1 1 eq ) DMF (1ml) and DIPEA (42 ml, 1 1 eq ) was added A solution of HBTU (84 8 mg 1 05 eq ) m DMF (1 ml) was then added After standmg for 2 mmutes the solution was added to the resm along with DMF (1 ml) The mixture was shaken for 30 mm , and then washed with DMF (2 x 3 ml) The couphng procedure was repeated, and the resm washed with DMF (2 x 3 ml) THF (3 ml) and DCM (3 x 3 ml) (HPLC 2-3 % dimer, Fmoc-test Loading = 0 43 mmol/g)

Synthesis of comp. 12-14

50 mg (0 02 mmol) of Rink-Fmoc-tyrosine was swollen in THF/DCM (1 1) Ten mmutes appart diethyl-azo-dicarboxylate (DEAD) (31 2 ml, 10 eq ), alcohol (BnOH 13 7 ml, 3 3 eq , propanol 10 ml, 3 3 eq , 2-Ph-EtOH 15 9 ml, 3 3 eq ) and TPP (62 mg, 10 eq ) was added and the mixture was shaken for 28 h The resm was washed with DMF (2 ml), THF (2 ml) and DCM (4 x 2 ml)

Synthesis of comp. 15

Approximately 50 mg (0 02 mmol) of the resm compnsmg of comp 1-4 was washed with DMF (2 ml) and subsequently the Fmoc-group was removed under standard conditions A solution of 1 ,4- benzenedicarboxyhc acid monopropenylester (32 mg 8 eq ) and DIPEA (50 ml, 20 eq ) m DMF (0 4 ml) was added A solution of HATU (52 mg, 7 2 eq ) in DMF (0 4 ml) was then added and after standmg for 2 mmutes the mixture was added to the resm, which was stirred for 2 h Subsequently the resm was washed with DMF (5 x 2 ml), THF (2 x 2ml), 1% DIPEA/DCM (2 x 2 ml), DCM (5 x 2 ml) and 5% AcOH/2 5% NMM/CHCla (2 2 ml) (PPh3)4Pd(0) (113 mg, 5 eq ) was dissolved m 5% AcOH 2 5% NMM/CHCI3 (2 ml) m the dark and under argon atmosphere The resm was treated with the mixture 0 n in the dark and under an argon atmosphere Subsequently the resm was washed with 5% AcOH 2 5% NMM/CHC13 (2 x 2 ml), 0 5% DIPEA 0 5% sodium-diethyl-dithiocarbamate DMF (10 x 2 ml), DMF (3 x 2 ml) and DCM (5 x 2 ml) 50% TFA/DCM was added to approximately 15 mg (6 mmol) of resm and the mixture was shaken for 40 mm The resm was washed with DCM (2 x 0 5 ml) and the combmed organic phases was concentrated (speed-vac)

Synthesis of 19 Approximately V- of the amount of resm cleaved was washed with DMF (2 x 1 ml), the Fmoc- group was removed under standard conditions and washed with DMF (5 x 2 ml) Comp 15 were dissolved m DMF (0 1 ml) and DIPEA (9 ml, 18 eq ) was added A solution of HATU (2 2 mg, 1 8 eq ) m DMF (0 1 ml) was then added and after standmg for 2 mm the mixture was added to the resm which was then shaken 1 5 h The resm was washed with DMF (4 x 2 ml) and DCM (5 x 2 ml) and stored wet at 0-5 °C

Synthesis of 15-18

Approximately 2/3 (0 013 mmol) of the resm compnsmg of comp 11- 14 was washed with DMF (2 ml) and subsequently the Fmoc-group was removed under standard conditions 1,4- benzenedicarboxyhc acid monopropenylester (22 mg, 8 eq ) m DMF (0 3 ml) and DIPEA (45,6 ml 20 eq ) was added A solution of HATU (36 5 mg, 7 2 eq ) m DMF (0 2 ml) was then added and after standing for 2 mmutes the mixture was added to the resm, which stirred o n SubsequentH the resm was washed with DMF (2 x 2 ml), THF (2 x 2ml), 1% DIPEA/DCM (2 x 2 ml) DCM (5 x 2 ml) and 5% AcOH/2 5% NMM/CHC13 (2 x 2 ml)

(PPh3)4Pd(0) (43 mg, 3 eq ) was dissolved m 5% AcOH/2 5% NMM/CHC13 (2 ml) m the dark and under argon athmosphere The resm was treated with the mixture o n in the dark and under an argon athmosphere The resm was then washed with 5% AcOH/2 5% NMM/CHC13 (2 x 2 ml), 0 5% DIPEA 0 5% sodium-diethyl-dithiocarbamate/DMF (10 x 2 ml), DMF (3 x 2 ml) and DCM (5 x 2 ml) 50% TFA/DCM was added to the resm and the mixture was shaken for 40 mm The resm was washed with DCM (2 x 0 5 ml) and the combmed organic phases were concentrated (speed-vac) to an oily residue

Synthesis of 19-27 The remammg 1/3 of the resm from C was washed with DMF (2 x 1 ml) the Fmoc-group was removed under standard conditions and the resm was then washed with DMF (5 x 2 ml) Comp 15-18 m DMF (0 2 ml) and DIPEA (23 ml, 10 eq ) was added A solution of HATU (4 9 mg, 1 9 eq ) m DMF (0 1 ml) was then added and after standmg for 2 mm the mixture was added to the resm which was shaken o n The resm was washed with DMF (4 x 2 ml) and DCM (5 x 2 ml) and stored wet at 0-5 °C

Claims

1 A compound of the followmg general formula I

P¹ - C ( =0) -A- C ( -O ) - P²

wherem A designates a cychc or bnear entity, and

each of P¹ and P² mdependently designates a bnear or cychc moiety compnsmg 1-20 units preferably 2-20 units, of the general formula II

wherem n is 0 or 1 , each of R¹, R², R³, R⁴ mdependently is a side cham selected from hydrogen, optionaUy substituted Ci e-alkyl, optionaUy substituted Ci β-alkenyl, optionaUy substituted C_ β-alkadienyl, optionaUy substituted Ce 8-alkatrιenyl, hydroxy, -O-R⁶, formyl, -C(=0)-R⁶, -0-C(=0)-R⁶, carboxy, -C(=0)- O-R⁶, optionaUy substituted heteroaryl, (optionaUy substituted heteroaryl)- Ci 6-alkyl, (optionaUy substituted heteroaryloxy)-Cι β-alkyl, optionaUy substituted aryl, (optionaUy substituted aryl)-Cι 6-alkyl, (optionaUy substituted aryloxy)-Cι 6-alkyl, halogen such as fluoro, chloro, bromo, and iodo, nitro, cyano, ammo, -NHR⁶, -N(R⁶)2, mono- or dι(Cι 6-alkyl)ammo-Cι 6-alkyl, -N^O-CO-R⁶, (Ci 2o-alkyl)carbonylamιno-Cι 6-alkyl, carbamoyl, ammocarbonyl-Ci 6-alkyl, mono- or dι(Cι 20- alkyl)amιnocarbonyl, mono- or dι(Cι 6-alkyl)ammocarbonyl-Cι β-alkyl, sulphanyl, optionaUy substituted Ci 20-alkylthιo, optionaUy substituted Ci 20-alkylthιo-Cι 6-alkyl, (optionaUy substituted aryl)thιo, guanidmo, guanidino-Ci 6-alkyl, sulphono (-SO3H), sulphmo (-SO2H), halosulphonyl, -OS(0)m-R⁶ where m is 2 or 3,

where m is 2 or 3, -S(0)_m-N(RO₂ where m is 2 or 3,

where m is 2 or 3, -S(0)_m-NH2 where m is 2 or 3, isocyano, isothiocyano, thiocyano, -OP(0)_P(R⁶)„ where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and

where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, wherem each R⁶ mdependently is selected from hydrogen, optionaUy substituted Ci 20- alkyl, optionaUy substituted Ci 20-alkenyl, optionaUy substituted Ci 20-alkadιenyl, optionaUy substituted Ci 20-alkatπenyl, optionaUy substituted aryl, and optionaUy substituted heteroaryl, and each R⁷ is selected from hydrogen and Ci 4-alkyl, and R⁶ is selected from hydrogen and Ci 4-alkyl,

and wherem one of the substituent pairs R'/R², R'/R³, and R³ R⁴ may form a biradical which, together with the atoms located between these substituents (C_α and or C_p) forms a rmg, or one of the substituent pairs R² R⁵ and R /R^B may form a biradical which, together with the atoms located between these substituents (C_α, C_p, and/or N), forms a rmg,

and, if any of P¹ and P² is a cychc moiety, the cychc character arises from the presence of a hnkage between two of the side chams, R¹, R², R³, and R⁴. of two units of the formula II withm a moiety P¹ and/or P²

2 A compound accordmg to claim 1, wherem at least one of P¹ and P² is a cychc moiety

3 A compound according to claim 2, wherem both of P¹ and P² are cychc moieties

4 A compound according to any of the claims 2-3, wherein the hnkage comprises a group selected from an amide group (-NR^B-C(=0)-), a disulphide group (-S-S-), an ester group (-C(=0)- 0-), an ether group (-0-), phosphodiester group (-0-P(0)₂-0-), and a sulphide group (-S-)

5 A compound according to any of the precedmg claims, wherem the moieties P¹ and P² each comprises 4-20, such as 4-15, preferably 4-10, m particular 5-8 units of the formula II

6 A compound according to any of the precedmg claims, wherem the number of units m the moiety P¹ is identical to the number of units m the moiety P²

7 A compound according to any of the precedmg claims, wherem the moieties P¹ and P² are substantiaUy identical

8 A compound according to any of the preceding claims, wherem each of the side chams R¹, R² R³, and R⁴ mdependently is selected from hydrogen, optionaUy substituted Ci 6-alkyl hydroxy, -O-R⁶, carboxy, -C(=0)-0-R⁶, optionaUy substituted aryl, optionaUy substituted heteroaryl, sulphanyl, carbamoyl, optionaUy substituted Ci 20-alkylthιo, optionaUy substituted Ci 20-alkyl- thio-Ci e-alkyl, guanid o, guaniάino-Ci 6-alkyl, -OP(0)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and

where p is 1, 2, or 3 q is 1 or 2, and p+q is 3, 4, or 5, wherem each R⁶ mdependently is selected from hydrogen, optionaUy substituted Ci 20-alkyl, and optionaUy substituted aryl, and each R⁷ is selected from hydrogen and Ci 4-alkyl 9 A compound accordmg to any of the precedmg claims, where one of the substituent pairs R'/R², R'/R³, and R³/R⁴, together with the atoms located between these substituents (C_α and/or C_p), forms a rmg of one of the types cyclopropane, oxirane, aziridine, cyclopropene, azinne, cyclobutane, oxetane, azetidine, thietane, 2-azetιdιnone, 1,3-lactone, pyrohdine, pyrohne, pyrrole, cyclopenetene, cyclopentadiene, pyroUidione, pyroUidone, cyclohexyl oxirane, dioxirane morpholine, piperidine, 1,5-lactone, 1,5-lactam, cydohexene, cyclohexadiene, pipendione, tropane, 1,6-lactone (tropolone), or 1,6-lactam which may be optionaUy substituted, or one of the substituent pairs R²/R^B and R /R⁵, together with the atoms located between these substituents (C_α, Cp, and/or N), forms a rmg of one of the types 2-azetιdιnone, pyrohdine, pyrohne, pyrrole, pyroUidione, pyroUidone, pipendme, 1,5-lactam, pipendione, and 1,6-lactam

10 A compound accordmg to any of the claims 1-8 wherem none of the substituent pairs R'/R², R'/R³, R³/R⁴, and R⁴/R⁵ are mvolved in rmg(s)

11 A compound according to any of the precedmg claims, wherein the units of the moieties P¹ and P² have the formula Ila

R¹ - C - C I _a - N - Ha

O II R l ₂ R l₅

wherem each of the side chams R¹ and R² mdependently is selected from hydrogen, optionaUy substituted Ci 6-alkyl, hydroxy -O-R⁶, carboxy, -C(=0)-0-R⁶, optionaUy substituted aryl, optio¬ naUy substituted heteroaryl, sulphanyl, carbanoyl, optionaUy substituted Ci 20-alkylthιo, optionally substituted Ci _o-alkylthιo-Cι 6-alkyl, guanidino, guanidino-Ci 6-alkyl, -OP(0)_P(R⁶)_q where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, and

where p is 1, 2, or 3, q is 1 or 2, and p+q is 3, 4, or 5, wherein each R⁶ independently is selected from hydrogen, optionaUy substituted Ci 20-alkyl, and optionaUy substituted aryl, and each R⁷ and R^ε independently are selected from hydrogen and Ci 4-alkyl

12 A compound accordmg to any of the precedmg claims, wherem one of the side chams R¹ and R² is hydrogen and the other side cham is selected from hydrogen optionaUy substituted Ci 6- alkyl, hydroxy, -O-R⁶, carboxy -C(=0)-0-R⁶, optionaUy substituted aryl, optionaUy substituted heteroaryl, sulphanyl, carbanoyl, optionaUy substituted Ci 20-alkylthιo, optionaUy substituted Ci 20-alkylthιo-Cι e-alkyl, guanidmo, guamdmo-Ci 6-alkyl, -OP(0)_P(R⁶)_q where p is 1, 2, or 3 q is 1 or 2, and p+q is 3, 4, or 5, and

where p is 1, 2, or 3, q is 1 or 2, and p+q is 3 4, or 5, wherem each R⁶ mdependently is selected from hydrogen, optionaUy substituted Ci _o-alkyl and optionaUy substituted aryl and each R⁷ is selected from hydrogen and Ci 4-alkyl R⁵ is hydrogen, or the substituent pair R /R⁶ together with the intervening carbon (CJ) and nitrogen atoms form an optionaUy substituted pyrrolidine or piperidine rmg

13 A compound accordmg to any of the preceding claims, wherem the entity A is a cychc entity

14 A compound accordmg to claim 13, wherem the cychc entity A is an optionaUy substituted aromatic or polyaromatic entity

15 A compound accordmg to any of the claims 1- 12, wherein the entity A is a linear or carbocychc entity

16 A compound accordmg to any of the preceding claims, wherem at least one of the moieties P¹ and P² mcludes 1- 18, preferably 1-2, units of the formula III

- C ( =0 ) - ( CH₂ ) 3 - 6 - NR⁵ ' - II

wherem the methylene groups are optionaUy substituted one or several tunes, preferably 1-3 times, with group(s) as defined m claim 1 for R'-R⁴, and wherem where R⁵' designates the same groups as defmed in claim 1 for R^ε

17 A method for the preparation of a compound of the foUowmg formula I

P¹ - C ( =0 ) - A- C ( =0 ) - P² I

as defined m any of the claims 1- 16, compnsmg the foUowing steps

(A) providmg an optionaUy side cham protected moiety P' immobihsed to a sohd support material, the α- or β-ammo group of the N-termmal unit (the unit neighbourmg the carbonyl group (C(=0)) located between P¹ and A) of the immobihsed moiety P¹ bemg unprotected,

(B) couphng a diacid HOOC-A-COOH, in the free acid, monoester, or mternal anhydride form, to the unprotected ammo group of the N-termmal unit of the immobihsed moiety P¹ for the formation of an immobihsed fragment P'-C(=0)-A-COOH the free acid or ester form,

(C) cleavmg the fragment P'-C(=0)-A-COOH, optionaUy in the ester form , from the sohd support material, (D) providmg an optionaUy side cham protected moiety P² immobihsed to a sohd support material, the α- or β-ammo group of the N-termmal unit of the immobihsed moiety P² (the unit neighbourmg the carbonyl group (C(=0)) located between P² and A) bemg unprotected,

(E) coupling the fragment P'-C(=0)-A-COOH to the immobilised moiety P² for the formation of an immobihsed compound P'-C(=0)-A-C(=0)-P², and

(F) cleavmg the compound P'-C(=0)-A-C(=0)-P² from the sohd support material

18 A method for the preparation of a multi-dimensional array of compounds, {P'}-{A}-{P²}, consisting of at least four compounds each havmg the general formula I

P¹ - C ( =0) -A- C ( =0 ) - P² I

as defmed in any of the claims 1-16, compnsmg the foUowing steps

(A) providmg an array {P¹} of at least two optionaUy side cham protected moieties P' immobihsed to a sohd support material, the α- or β-ammo group of the N-termmal unit of the immobihsed moieties P' bemg unprotected,

(B) coupling an array {A} of one or more diacids, HOOC-A-COOH, the free acid, monoester or mternal anhydride form, to the unprotected ammo groups of the N-termmal unit of the immobihsed moieties P¹ for the formation of an array {P'}-{A} of immobihsed fragments P'-C(=0)-A-COOH, optionaUy m the ester form

(C) cleavmg the array {P'}-{A} of fragments P'-C(=0)-A-COOH, optionaUy in the ester form from the sohd support mateπal,

(D) providing an array {P²} of at least two optionaUy side cham protected moieties P² immobihsed to a sohd support material, the α- or β-ammo group of the N-termmal unit of the immobihsed moieties P² bemg unprotected,

(E) couphng the array {P'HA} of fragments P'-C(=0)-A-COOH, optionaUy m the ester form, to the array {P²} of immobihsed moieties P² for the formation of an array {P'}-{A}-{P²} of immobihsed compounds P'-C(=0)-A-C(=0)-P², and

(F) cleavmg the array {P'}-{A}-{P²} of immobihsed compounds P'-C(=0)-A-C(O)-P² from the sohd support material 19 A method according to claim 18, wherem the array {P²} of immobihsed moieties P² is substantiaUy identical to the array {P¹} of immobi sed moieties P'

20 A method according to claim 19, wherem the array {P¹} of immobihsed moieties P¹ and the array {P²} of immobihsed moieties P² are prepared m one batch

21 A method for the preparation of a multi-dimensional array {L'MBHL²} of at least four compounds each havmg the formula L'-B-L², wherem the arrays {L¹} and {L²} are of sumlar chemical composition, each of L¹ and L² mcludes a fragment 1' and l², respectively, of a chemical functionabty, B is an entity which mcludes two fragments b¹ and b² of chemical functionalities each of the fragment sets l'b' and b²l² formmg a covalent linkage between L' and B and between B and L², respectively, said covalent hnkages/hnks bemg substantiaUy identical and bemg formed under b/1 bond formation reaction conditions, the method compnsmg the foUowmg operations

(A) providing an array {L} of at least two moieties L immobihsed to a sohd support material, where the array {L} has simdar chemical composition to the arrays {L'} and {L²}, each of the moieties L mcludes a group compnsmg a fragment 1 of a chemical functionahtj where the fragment 1 is identical to the fragments l¹ and l². the groups correspondmg to the fragment 1 bemg in protected or unprotected form and any other groups of L sensitive to b/1 bond formation reaction conditions bemg optionaUy protected

(B) dividing the sohd support material representmg the array {L} mto two parts to give arrays {L'} and {L²}, respectively, the array {L¹} compnsmg immobihsed moieties designated L' and the array {L²} compnsmg immobihsed moieties designated L²,

(C) in the case where the group corresponding to the fragment l¹ is protected, deprotectmg said group,

(D) under b 1 bond formation reaction conditions, couphng an array {B} of one or more compounds correspondmg to the entity/entities B to the array {L¹} for the formation of an array {L'}-{B} of unmobihsed compound fragments L'-B where the b 1 bond formation reaction mvolves the group corresponding to b' of the compounds corresponding to the entity/entities B and the group correspondmg to 1' of the immobihsed moieties L¹, the group corresponding to b² of the entity/entities B and any other groups sensitive to b/1 bond formation reaction conditions optionaUy bemg protected (E) cleavmg the array {L'}-{B} of immobihsed compound fragments L'-B from the sohd support material,

(F) if necessary, deprotectmg the group correspondmg to the fragment b²,

(G) m the case where the group correspondmg to the fragment l² is protected, deprotectmg said group,

(H) under b/1 bond formation reaction conditions, couphng the array {L'}-{B} of compound fragments L'-B to the array {L²} for the formation of a multi-dimensional array {L'}-{B}-

{L²} of immobihsed compounds L'-B-L², the b 1 bond formation reaction involving the group correspondmg to l² and the group correspondmg to b², and

(J) cleavmg the multi-dimensional array {L'MBHL²} of immobihsed compounds L'-B-L² from the sohd support matenal

22 The use of a hbrary of compounds as defmed in any of the claims 1-16 for screenmg purposes

23 The use of a compound of the general formula (I) as defmed m any of the claims 1- 16 for antiviral, anticancer, antibiotic such as antibacterial and antifungicidal or herbicidal purposes