NZ246473A

NZ246473A - Nucleoside base oligomer having at least one purine base bound to a backbone having at least one peptide bond and compositions thereof

Info

Publication number: NZ246473A
Application number: NZ246473A
Authority: NZ
Inventors: Stephen A Thomson; Stewart A Noble; Daniel J Ricca
Original assignee: Glaxo Inc
Priority date: 1991-12-18
Filing date: 1992-12-17
Publication date: 1996-05-28
Also published as: MX9207334A; NO942327L; CA2125966A1; ZA929764B; WO1993012129A1; FI942935A0; EP0618923A4; JPH07502509A; FI942935A; NO942327D0; EP0618923A1; AU7032596A; AU3325493A

Description

New Zealand No. 246473 International No.

TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: i8Mqt International filing date: l~)j 13-19 2.

Classification: lftV.Co-?k«/©q.; Coiio/om.-, CoIHtfl/ofc, Jfa", fltol K31/S2, SoS-, Pubfication date: «<<" Journal No.: in,o u- NEW ZEALAND < ■» PATENTS ACT 1953 COMPLETE SPECIFICATION Title of invention: Peptide nucleic acids and their effect on genetic material Name, address and nationality of applicant(s) as in international application form: GLAXO INC, of Five^Moore Drive, Research Triangle Park, North Carolina 27709, United States of America. 9 l.lfollof+ef (*y pcufife I A) WO 93/12129 PCT/ Peptide Nucleic Acids and Their Effect on Genetic Material l&M 73 Background of the Invention J- The prevention of gene transcription and/or gene translation at the DNA/mRNA level is attractive for many reasons. Classical approaches to drug discovery involve the design and identification of compounds directed against unrelated proteins such as enzymes, receptors or ion channels, the structure and mode of 10 action of which are usually very complicated and often poorly understood. Conversely, the potential for therapeutic intervention at the nucleic acid level follows a well ordered, generalizable strategy which is targeted at the initiating events of an amplifying cascade; thus transcription of a gene gives rise to many copies of mRNA which on translation affords an even greater number of protein 15 molecules. Inhibition of gene expression ought, therefore, to be more efficient than inhibition of the gene product.

Anticancer therapy using DNA binding or modifying drugs is well established, however, current agents such as doxorubicin, mitoxantrone and cisplatin (Scrip's 20 Cancer Chemotherapy Review 1991) are not capable of recognizing specific gene sequences, and therefore, lack selectivity, discriminating only poorly between cancer and normal cells. A synthetic oligodeoxynucleotide (ODN), can provide absolute specificity of action since statistically the sequence defined by any linear combination of the four heterocyclic bases, adenine (A), guanine (G), cytosine (C), 25 and thymine (T), to form an oligonucleotide of 17 residues in length, occurs just once In the entire sequence of the human genome. Thus, the ODN can bind via Watson-Crick or Hoogsteen base pairing to its complementary base sequence which could, for example, be part of an oncogene implicated in tumorigenesis or an element of genetic material implicated as the dominant cause of a disease 30 phenotype, for instance, a sequence which comprises an essential target within a viral genome.

The potential of such 'antisense' (AS) oligodeoxynucleotides to serve as code blocking therapeutic principles was recognized by Zamecnik and Stephenson 35 (Proc. Natl. Acad. Sci. USA, 1978, 75,280) who demonstrated the inhibition of Rous sarcoma virus replication in chick embryo fibroblasts on addition of a tridecamer ODN with sequence complementarity to reiterative sequences in the viral (+) RNA genome. Duplex formation, i.e. the interaction of an AS ODN with an 243 47 ^WO 93/12129 PCT/US92/10921 RNA molecule, as a means to achieve inhibition of gene function has received considerable attention of late. The precise mechanism by which inhibition occurs is dependent on the micro-environment of the target sequence. Thus an AS ODN directed against a ribosome binding site would be expected to competitively inhibit 5 translation initiation whereas those complementary to a coding sequence might cause hybridization arrest of translation by preventing ribosomal translocation along the RNA (Bacon and Wickstrom, Oncogene Research, 1991, fi, 13). Alternatively, complexation might lead to cleavage of the target RNA via the intermediacy of RNase H (Shuttleworth and Colman, EMBO J., 1988, 7, 427). 10 Interception of post-transcriptional processes such as RNA splicing is also possible by judicious choice of sequence and has proved particularly effective against viral targets, e.g. Herpes simplex Virus (HSV) (Smith et al., Proc. Natl. Acad. Sci. USA, 1986, fiS. 2787) and Human Immunodeficiency Virus (HIV) (Goodchild et al., Proc. Natl. Acad. Sci. USA, 1988, fiS, 5507), where alternative splicing of precursor 15 RNAs is commonly used as a strategy to achieve control of viral replication.

The therapeutic potential of AS ODNs is the subject of several recent reviews (Zon, Nucleoside Analogs as Antiviral Agents, ACS Symposium Series, Martin, J.C. Ed., American Chemical Society, Washington, DC. 1989, p170; Weintraub, Scientific 20 American, 1990, 40: Uhlmann and Peyman, Chemical Reviews, 1990, 2Q (4), 543; Matteucci and Bischofberger, Annual Reports in Medicinal Chemistry, 1991. 26. 287). This therapeutic potental is further highlighted by a recent publication citing the selective action of unmodified AS ODNs targetted to c-myb on leukemic hematopoetic cells (Calabretta et al, Proc. Natl. Acad Sci. USA, 1991, £3. 2351). 25 The authors claim that perturbation of c-myb function with AS ODNs might form the basis of a molecular approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agents. Another paper (Calabretta et al, Proc. Natl. Acad. Sci. USA, 1991, 88) details experiments in which unmodfied AS ODNs were directed against the bcr-abl splice to beneficial effect. A patent by the same 30 authors describe AS ODNs against the c-abl proto-oncogene (WO 91/03260). It is of note that Genta, Inc. of San Diego, California, recently filed an Investigational New Drug (IND) application with the Food and Drug Administration to begin trials of an AS ODN to treat Chronic Myelogenous Leukemia (Sci Com Cancer Report, 1991,1Q). Also of note are the applications of unmodified oligomers to address 35 cystic fibrosis (Sorscher et al., Proc. Natl. Acad. Sci, USA, 1991, 88, 7759), Herpes Simplex Virus (HSV) (WO 90/07409) and Human Papillomavirus (HPV) (WO 91/08313). 246473 WO 93/12129 PCT/US92/10921 The inhibition of transcription by direct action on DNA itself, where copy number is restricted to two per cell, is an even more attractive target than inhibition of translation by therapeutic intervention at the RNA level. The sequence specific recognition of double helical DNAJ^synthetic ligands is the subject of recent s reviews (Dervan, Nucleic Acids ariu Molecular Biology, Vol 2, ed., F. Eckstein and D.M. Lilley, Springer-Verlag, 1988, p49; Nielsen, Bioconjugate Chemistry, 1991, 2 (1), 1). Of particular interest to the current invention is the design of ODNs and their analogues which bind to ds DNA forming triple stranded structures, i.e. a "triplex", using the structural motifs first described by Hoogsteen (Acta. Cryst., 1959,12, 10 822). It is recognized that the precise binding motif adopted by the ligand might vary from that cited above as postulated by Birg et al., Nucleic Acidi Research, 1990,18 (10), 2901. Furthermore interactive or reactive groups might be appended to the ligand to beneficial effect (Shaw et al., J. Amer. Chem. Soc,, 1991, 113, 7765). That such an approach has utility with regard to therapeutic 15 intervention is evidenced by several recent publications.

Thus triple helix formation has been shown to inhibit the function of DNA binding proteins (Maher et al., Science 1989, 245, 725, Orson et al., Nucleic Acids Research 1991, 19(12) 3435) and to effect inhibition of transcription elongation in 20 vitro (Young et al., Proc. Natl. Acad. Sci. USA 1991, 88, 10023). Most recently it has been shown that an ODN binds to the promoter region of c-myc in HeLa cells, thereby selectively reducting c-myc RNA levels. The therapeutic potential of this approach has also been highlighted in the recent patent literature (WO 90/15884, EP 0375408).

Most recently reports of analogues containing amide bonds have appeared in the art (Weller et al., J. Org. Chem., 1991, 56, 6000; Huang et al., ibid., 1991, 56, 6007). At the Twelth American Peptide Symposium at the Massachusetts Institute of Technology in Cambridge, Massachusetts on June 17,1991, Rolf Berg of the RISO 30 National Laboratory in Roiskilde, Denmark presented work on modified peptides with nucleoside side chains which were called peptide nucleic acids (PNAs). However, only PNAs from the T monomer could be made. Presentations by this group on July 8,1991 at the University of California at Berkeley set forth descriptions of certain PNAs. A publication of their work is by Peter E. Nielsen et al. 35 in Science, Vol. 254, pages 1497-1500 (6 December 1991). 3 Simnary of the Invention 24 6 4 7 3 In a first aspect the present invention provides a nucleoside base oligomer of the following formula (I): B r V QN 0 N R3 B rV N R3 (D J n wherein Q is an N-terminal blocking group; J is a C-terminal blocking group or Q and J may together be a single bond; n is at least 1; R1 is independently hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2CCX)Hi -CH2COO(Ci.4 alkyl), -CH2CH2COOH, -CH2CH2COO(C1.4 alkyl), -CH2CONH2. -CH2CH2CONH2. -CH2SH, CH2CH2SCH3, C'i.12 alkyl, C2-8 alkenyl, C2-8 alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl, ordi-(C-j-4 alkyl) substituted amino; R3 is independently hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2. -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO (C1.4 alkyl), -CH2CH2COOH, -CH2CH2COO(Ci.4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C-M2 alkyl, C2-8 alkenyl, C2-8 alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl, ordi-(C<|.4 alkyl) substituted amino; and B is independently a purine or pyrimidine nucleoside base providing that one B is a purine nucleoside base, or an acicfror base addition salt thereof. 4 (followed by page 4a) 24 6 4 7 In a further aspect the present invention provides a nucleoside monomer of the following formula (X): R1 is hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2. -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(Ci.4 alkyl), -CH2CH2COOH, ?CH2CH2COO(C-|_4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, Ci_i2 alkyl, C2-8 alkenyl, C2-8 alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1-4 alky!, C1.4 alkoxy, trifluoromethyl or di-(Ci-4 alkyl)-substituted amino; R2 is an amino protecting group; R3 is hydrogen, benzyl, -CH2-P-C6H40H, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2. -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(Ci.4 alkyl), -CH2CH2COOH, -CH2CH2COO(Ci-4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C1-12 alkyl, C2-8 alkenyl, C2.8 alkynyl, C5.0 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl.

R4 is hydrogen or a carboxylic acid protecting group; and B is a purine or pyrimidine nucleoside base provided that if R1 and R3 are hydrogen, B is a purine nucleoside base, or an acid or base addition salt thereof.

B (X) wherein (followed by page 4b) 4a 24 6 4 In a still further aspect the present invention provides a pharmaceutical formulation comprising a compound o£ the invention together with a pharmaceutical^ acceptable carrier therefor.

Brief Description of the Drawings Fig. 1 depicts a schematic representation of a process used to make a particular peptide nucleic acid (PNA) of the invention.

Fig. 2 is a schematic of a test used to determine the degree of binding of a PNA according to the invention to genetic material.

Fig 3. is a graph showing the variation with increasing PNA concentration of binding to genetic material.

Detailed Description of the invention Nucleoside base oligomers which have at least one purine or pyrimidine nucleoside base bound to a backbone having at least one peptide bond constitute the present invention. Preferably, the backbone would have 1 peptide bond for each pendant base whereby the oligomer can be formed from monomers each having an A, T, G or C nucleoside base. By selecting the A,T, G or C amino acid monomers, each amino acid of the oligomer can be built up by successive peptide bond formations.

The particular number of nucleoside bases in a PNA of the invention will depend on the use to which the PNA is put, i.e. the target portion of genetic material. Below 6 nucleoside bases, there will usually be too many possible different targets within the genetic material, e.g. many different chromosomes have a portion with GATT as a subsequence. Above 16 bases, the additional specificity provided is unnecessary, i.e. there will only be 1 sequence with a particular 15 base arrangement and no further purpose is provided by the additional bases.

In addition to the backbone and bases, the peptide oligomers of the invention may have pendant groups, usually at the termini, to stabilize the end, to act as an intercalator, to facilitate cellular uptake or to increase solubility. 4b (followed by page 5) 246473 >VO 93/12129 A particular peptide oligomer of the invention is that of the following formula (I): wherein Q is an N-terminal blocking group; J is a C-terminal blocking group or Q and J may together be a single bond; n is at least 1; R1 is independently hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(Ci_4 alkyl), -CH2CH2COOH, -CH2CH2COO(Ci.4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C-I.-J2 alkyl, C2-8 alkynyl, C2.8 alkenyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C-j_4 alkyl, C1.4 alkoxy, trifluoromethyl, or di-(C^ .4 alkyl)substituted amino; R3 is independently hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yI, -CH2COOH, -CH2COO(C1.4 alkyl), -CH2CH2COOH, -CH2CH2COO(Ci.4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C-|.12 alkyl, C2-8 alkynyl, C2-8 alkenyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl, or di-(Ci .4 alkyl)substituted amino; B is independently a monovalent purine or pyrimidine nucleoside base,i.e. a base such as guanine without the hydrogen at the 9-position or an acid- or base- addition salt thereof.

Q is preferably an N-terminal blocking group which may stabilize that portion of the molecule, e.g. sterically hindered alkanoyl group whereby an amide is formed by the group QNH-. Another function of the N-terminal blocking group Q is as an intercalator to bind within the genetic material, e.g. to actually wedge itself within the B B (D n 246473 WO 93/12129 PCT/US92/10921, DNA double helix as described in Qlipodeoxvnucleotides-Antisense Inhibitors of Gene Expression, ed. by Jack S. Cohen, MacMillan Press, London (1989) (ISBN 0-333-49211-0). When used to increase cellular upake, Q may function to increase lipophilicity, e.g. a steriodal moiety, or to induce active uptake such as by having a 5 recognition moiety for the cell surface, e.g. a steroidal moiety or a saccharide moiety. To increase solubility, Q may contain an ionizable moiety such as a car boxy lie acid or an amine, e.g. the QNH moiety may be NH2(CH2)4CH(NH2)CONH-.

J may be any of the types of groups described above for Q. Specific examples include, as the -CO-J group, -COOt-butyl as a sterically hindered stabilizing group and -CONHCH(CONH2)CH2CH2CH2CH2NH2 as an ionizable moiety which functions as a solubizing moiety. n is at least 1 with compounds wherein n=1-4 being useful as intermediates for those wherein n is at least about 5, e.g. - about 5 to 20 although sequences where n is between 6 to 16 are particular aspects of the invention.

R"! is preferably hydrogen, benzyl, -CH2-p-C6H40H, -CH2-indol-3-yl 20 -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(C1.4 alkyl), -CH2CH2COOH, -CH2CH2COO(C1.4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C-M2 alkyl, C2-8 alkynyl, C2-8 alkenyl, e.g. -CH2CH=CHCH3, e.g. -(CH2)4CCH, C5.8 cycloalkyl, e.g. cyclohexyl aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted 25 independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl, or di-(C-|. 4 alkyl)substituted amino. As values for C1.4 alkyl in any of such definitions of R1, e.g. alkoxy, these may be methyl, ethyl, iso-propyl, n-propyl, n-butyl, sec-butyl, iso-butyl and tert-butyl. As values of aryl there are incuded phenyl and naphthyl and for heteroaryl, there are included 5- and 6-membered rings with 1,2 or 3 N,0 or S 30 heteroatoms with the proviso that two O or S atoms are not bonded to each other . with examples being pyridinyl, oxazolyl, thienyl, thiadiazolyl and triazolyl with attachment through a carbon or nitrogen atom, e.g. -N(CH=CH)2- Halogen includes chloro, bromo, iodo and fluoro.

R3 is preferably hydrogen, benzyl, -Ch2-p-C6H40H, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(C-|.4 alkyl), -CH2CH2CQOH, -CH2CH2COO(C1.4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C1-12 alkyl, C2-8 6 246473 WO 93/12129 PCT/US92/10921 alkenyl, C2-8 alkenyl, e.g. -CH2CH=CHCH3, e.g. -(ChteUCCH, C5.8 cycloalkyl, e.g. cyclopentyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl, or di-(Ci-4 alkyl)substituted amino. As values for C1.4 alkyl in any 5 of such definitions of R1, e.g. alkoxy, these may be methyl, ethyl, iso-propyl, n-propyl, n-butyl, sec-butyl, iso-butyl and tert-butyl. As values of aryl there are included phenyl and naphthyl and for heteroaryl, there are included 5- and 6-membered rings with 1,2 or 3, N,0 or S heteroatoms with the proviso that two O or S atoms are not bonded to each other with examples being pyridinyl, oxazolyl, thienyl, 10 thiadiazolyl and triazolyl with attachment through a carbon or nitrogen atom, e.g. -N(CH=CH)2- Halogen includes chloro, bromo, iodo and fluoro.

B is a purine or pyrimidine nucleoside base is preferably adenine, thymine, guanine or cytosine or an equivalent thereof which binds to its complement, i.e. adenine to 15 thymine and guanine to cytosine. Examples of such equivalents are 5- methylcytosine, 5-propynyluracil, 7-propyny!-7-deaza-adenine and 7-methyl-7-deaza-adenine. Preferably, the peptide oligomer of the invention has at least 3 different A, T, G and C bases or their equivalent, e.g. all four of such bases.

C 7 246473 Also part of the present invention are monomers of the following formula (X) which may be polymerized to yield the oligomer of formula (I) wherein R1 is as defined for formula (I); R2 is an amino protecting group; R3 is as defined for formula (I); R4 is a carboxylic acid protecting group; B is as defined for formula (I), or an acid-or base-addition salt thereof.

R2 is preferably t-butyloxycarbonyl, 9-fiuorenylmethoxycarbonyl, carbobenzoxy (i.e. benzyloxy carbonyl) trityl or dimethoxytrityl.

R4 is preferably alkyl, e.g. methyl, ethyl, tert-butyl, or (2-trimethylsilyl)ethyl, aryl, e.g. phenyl or benzyl.

Also part of the present invention are novel intermediates and processes, e.g. the di-, tri- and tetra- peptide oligomers which are used as intermediates to produce the AS oligomers of formula (I).

In the oligomer of formula (I) and the monomer of formula (X), several asymmetric centers are present. The present invention encompasses all isomers and mixtures thereof within the scope of all the formulae provided. For example, the carbon bearing the R1 and R3 groups may independently each be R or S to give the isomers RR, RS, SS and SR.

B (X) 8 .WO 93/12129 246473 Process The compounds of formula (I) may be prepared by the pathway outlined in Scheme 1.

Scheme 1 R1 R2HN (ID B-H + o B-H + (VD3) B rV QN N R3 r i B N R3 (D n 9 246473 k WO 93/12129 PCT/US92/10921 In step 1, an alpha-amino acid of formula (II) or a derivative thereof, wherein R"! is as defined above for formula (I) and R2 is defined for formula (X) is reduced by methods known in the literature (see Janusz Jurczak, Chem. Rev. 1989, 89,149) to yield a compound of formula (III). For example the ethyl ester of the compound of 5 formula (I) is treated with diisobutylaluminum hydride at -78 C to give the compound of formula (III).

R1 in formula (II) and R3 in formula (IV) may be used in a protected form to avoid reactivity of these groups during subsequent steps such as steps 1,2,6 and 7. Thus, 10 if R1 is CH2CH2CH2CH2NH2 forming a lysine sidechain, the starting material of formula (II) may be BocNHCH(CH2CH2CH2CH2NHCOOCH2C6H5)COOH, wherein the benzyloxycarbonyl group may be removed after preparation of the final compound of formula (I) by treatment with hydrogen fluoride, or hydrogenation with H2 over a noble metal catalyst.

In step 2, a compound of formula (III) is reacted with a compound of formula (IV) in a reductive amination to yield a compound of formula (V). In the compound of forrnula (IV), R3 is as defined above for formula (I) and R4 is a carboxylic acid protecting group as defined for formula (X) such as alkyl (e.g. methyl). The carboxylic acid 20 protecting group maintains the COO- group of formula (IV) through the reductive amination conditions of step 2 and the amide bond forming conditions of step 6. The reaction of step 2 is carried out in a solvent such as methanol, in the presence of a dehydrating agent, e.g. molecular sieves, and a reducing agent such as sodium cyanoborohydride at about 25 0 C as described by Zydowsky et al in J. Org. Chem. 25 1988, 53, 5607.

This route to the compounds of formula (V) has the advantage over other possible routes in that it allows for independent selection of R1 and R3 and independent control of the stereochemistry at the carbon atoms which bears R1 and R3. Since 30 the starting materials for this route to compounds of formula (V) are alpha-amino acids the chiral pool of natural and unnatural alpha-amino acids can be used to produce the oligomers of the invention.

In step 3, a compound B-H in which B is defined as in formula (X) or an 35 appropriately protected derivative thereof, for example N-6- benzyloxycarbonyladenine (Az), is reacted with a compound of formula (VI), wherein X is a leaving group, e.g. bromine and R5 is hydrogen or a commonly used carboxylic acid protecting group, such as tert-butyl, in a suitable solvent, such as 246473 , WO 93/12129 PCT/US92/10921 dimethylformamide, under basic conditions, e.g. potassium carbonate, to yield a compound of formula (VII). The attachment of the compound of formula (VI) to B-H is at the 1 position for T and C and at the 9 position for A and G, and at the corresponding positions when B is a nucleobase analog.

Step 4 depicts where, in certain cases, it is of advantage to use a masked equivalent of a compound of formula (VI) such as a compound of formula (VIII) wherein X is a leaving group such as bromine. In step 4, B-H is reacted with 3-bromopropene (formula (VIII) X = Br) to give a compound of formula (IX), e.g. at 27°C in dimethylformamide under basic conditions e.g. potassium carbonate. Step 5 shows the conversion of (IX) to a compound of formula (VII) by oxidative cleavage of the double bond, for example by treatment with sodium periodate in the presence of ruthenium tetraoxide at about 25 ° C as described by Carlsen et al in J. Org. Chem. 1981, 46, 3936.

In step 6, a compound of formula (VII) in which R5 is H is reacted with a compound of formula (V) under conditions known in the art for forming amide bonds to yield a compound of formula (X) (see Miklos Bodanszky; Peptide Chemistry, A Practical Textbook, Springer-Verlag 1988). This may involve conversion of the carboxyl 20 moiety of a compound of formula (VII) to an activated form such as an activated ester, acid chloride, or mixed anhydride, and reaction of this activated form with a compound of formula (V) to give a compound of formula (X). For example, a compound of formula (V!l) in which R§ is hydrogen is activated with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-25 hydroxybenzotriazole (HOBt), in dimethylformamide in the presence of diisopropylethylamine, is reacted with a compound of formula (V) in which R2 is Boc, and R4 is methyl to yield a compound of formula (X) in which R2 is Boc, R4 is methyl.

In step 7, compounds of formula (X) can be converted to a compound of formula (I) by reacting a compound of formula (X) in which R2 is hydrogen with a compound of formula (X) in which R4 is hydrogen under conditions known in the art for forming amide bonds (cf. Miklos Bodanszky; Peptide Chemistry, A Practical Textbook, Springer-Verlag 1988). This may involve conversion of the carboxyl moiety of a 35 compound of formula (X) where R4 is hydrogen to an activated form such as an activated ester, acid chloride, or mixed anhydride, and reacting this activated form with a compound of formula (X) where R2 is hydrogen. This coupling reaction can be repeated with monomers of Formula (X) with different B groups to give oligomers li Z4 to WO 93/12129 PCT/US92/10921 and polymers of a compound of formula (I). The reaction of step 7 can be done using standard solution phase reaction conditions, for example a compound of formula (X) in which R4 is hydrogen, and R2 is Boc is reacted with a compound of formula (X) in which R2 is hydrogen, and R4 is methyl, in dimethylformamide in the 5 presence of the coupling reagents O-benzotriazoM-yl-N.N.N'.N',- tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), and diisopropylethylamine to yield a compound of formula (I). The coupling can also be perfonned by anchoring one of the reaction components on a solid support, such as a polystyrene resin and then performing a repetitive cycle of coupling and 10 deprotection steps which allows for the rapid preparation of compounds of formula (I) in which n is greater than 1. This method is commonly known as solid phase synthesis (see Merrifield, J. Am. Chem. Soc. 1963, 85, 2149, and Science 1986, 232, 341). For example, a compound of formula (X) in which R4 is hydrogen, and R2 is Boc is coupled to a MBHA resin to which is anchored a lysine (with the epsilon 15 amino group protected) through the carboxyl group in dimethylformamide in the presence of the coupling reagents HBTU, HOBt, and diisopropylethylamine.

After the coupling is complete the Boc group is removed by strong acid which reveals a free amino group to which a second residue can be coupled. Repeating this coupling-deprotection cycle five more times and cleaving the chain from the 20 solid support with hydrogen fluoride yields a compound of formula (I) in which n is five, J is lysine, and Q is hydrogen. In many cases some of the functional groups on the bases will be protected to avoid undesired side reactions during the synthesis of the compounds of formula (I).

Protecting groups on the nucleobases must be removed so that they will be able to bind to the target genetic material. The protecting groups can be removed by methods such as treatment with fluoride ion, hydrofluoric acid, or by hydrogenation with H2 in the presence of a noble metal catalyst. This deprotection can be preformed with the chain attached to the solid support, or after it has been removed. 12 Scheme 2 MM 7 3 ^ ^ step 8 H step 9 ^ ^ ^ ^ H2N" *— BocHN IT ► BocHN OCH, (XI) (Ilia) (Va) Scheme 2 depicts a method to make the compound of formula (V) in which R1 and R3 are hydrogen, R2 is Boc, and R4 is methyl (formula (Va)). In step 8 of this Scheme allyl amine (formula (XI)) is converted to the aldehyde of formula (III) where R1=H and R2=Boc (formula (Ilia)), N-tert-butyloxycarbonylglycinal, as described by 10 S.A.Thompson et al in J. Med. Chem. 1986,29,104. In step 9 the N-tert- butyloxycarbonylglycinal is reacted with glycine methyl ester hydrochloride, in the presence of sodium acetate, 4 A molecular sieves, and sodium cyanoborohydride in methanol to give the compound of formula (Va). is Scheme 3 r1 T.g step 10^ 0^^ step n —BocHN OH (XII) (Xa) (Vila) Scheme 3 is a more detailed description of steps 3 and 6 of Scheme 1, and depicts 20 a method for making the compound of formula (X) in which R1, R3 and R4 are hydrogen, R2 is Boc, and B is thymine (formula (Xa)). In step 10 of this scheme thymine (T-H, Formula (XII)) is reacted with chloroacetic acid in aqueous potassium hydroxide as described by A. S. Jones et al, in Tetrahedron, 1973, 29, 2293, to give the compound of formula (VII) where B=T and R5=H, formula (Vila). In step 11 the 25 compound of formula (Vila) is activated with BOP in dimethylformamide and reacted with the compound of formula (Va), followed by hydrolysis of the resulting methyl ester by treatment with aqueous lithium hydroxide to give the compound of formula (Xa). The compound of formula (Xa) is referred to as the Teg monomer. 13 Sgheme 4 C-H (XIII) step 12 (XIV) step 13 step 14 24647 PCT/IJS92/10921, (Vllb) NHZ NHZ step 15 Scheme 4 depicts a synthesis of the monomer of formula (X) in which R1, R3 and R4 are hydrogen, R2 is Boc, and the nucleobase, B, is 4-N- benzyloxycarbonylcytosine (formula (Xc)). In step 12 the exocyclic amino group of cytosine, formula (XIII), is protected with the benzyloxycarbonyl group (Z) to give the compound of formula (XIV). In step 13 the compound of formula (XIV) is reacted with tert-butyl bromoacetate, which is followed by removal of the t-butyl group with strong acid (trifluoroacetic acid in methylene chloride) to give the compound of formula • (VII) where R5=H and B=protected cytosine, formula (Vllb). In step 14 the compound of formula (VII) where R5=H and B=protected cytosine, formula (Vllb) is activated with BOP in dimethylformamide and reacted with the compound of formula (Va) to give the compound of formula (X) where R1=R3=H, (Xb). In step 15 the methyl ester of the compound of formula (Xb) is hydrolyzed by treatment with aqueous lithium hydroxide to give the compound of formula (Xc), which is referred to as the Z protected Ceg monomer. 14 kWO 93/12129 246473 PCT/IJS92/10921 CI xX'y hsN-^n'^-n Ph step 16 (XV) ZHN H,N xx> H (XVI) step 17 step 18 step 19 ZHN BocHN Ph &¥> . \ OCHa (Xd) Scheme 5 depicts a synthesis of the monomer of formula (X) in which R"! and R3 are hydrogen, R2 is Boc, R4 is methyl, and B is 6-0-benzyl-2-N-(benzyloxycarbonyl)-guanine (formula (Xd)). In step 16 the commercially available 2-amino-6-chloropurine (formula (XV)) is converted to the compound of (formula 10 (XVI)) as described by M. MacCoss et al. in Tetrahedron Lett. 1985, 26,1815. In step 17 the compound of formula (XVI) is alkylated with aiiyl bromide at the 9 position to give the compound of formula (IX) where B is protected guanine, formula (IXa). In step 18 the alkene moiety of the compound of (formula (IXa)) is oxidatively cleaved by treatment with sodium periodate in the presence ruthenium tetraoxide at 15 ca. 25°C as described by Carlsen et al in J. Org. Chem. 1981, 46, 3936, to give the carboxylic acid which is methylated with diazomethane to give the particular compound of formula (Vile). In step 19 the compound of formula (VII), i.e formula 246 473 k WO 93/12129 (VII), i.e. formula (Vile) is first hydrolysed to the carboxylic acid, then activated with BOP in dimethylformamide and reacted with the compound of formula (Va) to give the compound of formula (Xd). The compound of formula (Xd) is referred to as the Bn-Z protected Geg monomer methyl ester.

Scheme6 (XVII) step 20 NHZ step 21 (XVIII) step 22 tTX> °r (Vlld) 0H NHZ step 23 NHZ cx> BocHN 0 OH (Xf) 16 246 47 PCT/US9271095l ' Scheme 6 depicts a synthesis of the monomer (X) in which R1, R3 and R4 are hydrogen, R2 is Boc and B is 6-N-benzyloxycarbonyladenine (formula (Xf)). In step 20 the exocyclic amine group of adenine (A-H, formula (XVII)) is protected with the 5 benzyloxycarbonyl group (Z) to give the compound of formula (XVIII). In step 21 the compound of formula (XVIII) is reacted with tert-butyl bromoacetate, which is followed by removal of the t-butyl group with strong acid (trifluoroacetic acid) to give (Vlld). In step 22 the compound of formula (Vlld) is activated with BOP in dimethylformamide, and reacted with the compound of formula (Va) to give the 10 compound of formula (Xe). In step 23 the methyl ester of the compound of formula (Xe) is hydrolysed with aqueous sodium hydroxide to give the compound of formula (Xf), which is referred to as the Z protected Aeg monomer. 17 246473 Scheme 7 r1 BocHN step 24 OCH, - H,N OCHa (Xh) BocHN TFAH,N OCH, (la) Scheme 7 is a more detailed description of step 7 of scheme 1, and depicts a method for making the compound of formula (I) in which n is 1, and reading left to right Q is hydrogen, R1 is hydrogen, B is guanine, R3 is hydrogen, R1 is hydrogen, B is thymine, R3 is hydrogen, and J is methoxy (formula (1a)). In step 24 the compound of formula (Xg) is treated with hydrogen chloride in dioxane to give the compound of formula (Xh) as the hydrochloride sait. in step 25 the carboxyl group of the compound of formula (Xi), the Bn-Z protected Geg monomer, is activated with HBTU and reacted with the compound of formula (Xh). The protecting groups are removed by first treating with triflouroacetic acid, followed by hydrogen fluoride to give the compound of formula (la). The compound of formula (la) is referred to as the Geg-Teg methyl ester. 18 ,P an-, ? / WO 93/12129 PCT/US92/10921 The compounds of formula (I) may also be prepared by the solid phase method as described by Merrifield et al. in J. Am. Chem. Soc. 1963, 85, 2149, and Science 1986,232,341.

Figure 1 outlines a synthesis of the compound of formula (I) in which n is 5, Q is hydrogen, all R1 an R3 are hydrogen and all B are thymine, and J is lysine (C-terminal amide). In step a of figure 1 the Teg monomer is coupled to the free alpha-amino group of lysine which is bound to a MBHA resin. After coupling is complete the resin is washed. In step b the Boc group is removed by treatment of the resin 10 with trifluoroacetic acid in methylene chloride. After the de-Boc reaction is complete the resin is washed and a second coupling can take place. After a total of six coupling and deprotection cycles, the resin is dried under vacuum, and in step c the resin is treated with hydrogen fluoride which cleaves the product from the resin to give the compound of formula (lb).

Figure 2 depicts an assay to show effective binding of a test compound nucleoside base oligomer of the invention of formula (I) to genetic material employing the enzyme RNase H. In this assay 3H labeled poly rA (RNA strand) is allowed to bind to its complementary DNA strand, dT (25 to 30 bases in length). After ca. 30 20 minutes the compound of formula (I), in particular where n=at least 5, is added at various concentrations, and the mixture is incubated for ca. 30 minutes, at which time the compound of formula (I), in particular where n= at least 5, binds to the poly rA strand by displacing the poly dT strand. The enzyme RNase H (from Hela cells) is then added to the mixture. RNase H will cleave the RNA strand of a RNA-DNA 25 duplex, but not the RNA strand of a RNA-(la) duplex. Therefore only the portion of the poly rA strand which is bound to the dT strands will be cleaved into smaller fragments, and the portion of poly rA which is bound to the nucleoside base oligomer (formula (la)) will remain in tact. After ca. 30 minutes t-RNA and acid is added which precipitates the larger pieces of the poly rA, and the radioactivity 30 remaining in the supernatant is counted. A decrease in radioactivity in the supernatant is a measure of the binding of the nucleobase oligomer of the invention over dT.

Figure 3 shows the results for the assay of Figure 2 for the nucleoside base 35 oligomer of formula (la). In this graph the Y axis is the radioactivity in the supernatant and the X axis is the concentration of the compound of formula (la). As can be seen from the graph increasing the concentration of the compound of formula (la) results in a strong decrease in the radioactivity in the supernatant. At a / 0 19 24 6 4 ^wo 93/12129 pct/us92/10921 concentration of 2 jj.M the compound of formula (la) has nearly totally displaced the dT strand from the poly rA strand. The addition of the compound of formula (la) had no effect on the RNase H cleavage of the RNA strand of an RNA-DNA duplex in which the RNA strand was not complementary to the base sequence of the 5 compound of formula (la). Therefore the binding of the compound of formula (la) to RNA is sequence dependent.

Another assay can be used to measure the binding of the test compound nucleoside base oligomer of the invention of formula (I) to double stranded (ds) 10 DNA by measuring inhibition of a restriction enzyme which cleaves the ds DNA near or within the binding site of the test comound. In this assay the test compound of formula (I) is allowed to bind to the target ds DNA which contains a complementary sequence to the test compound. After the test compound has been given time to bind to the ds DNA a restriciton enzyme is added and the amount of cleavage is 15 measured. A second restriction enzyme site removed from the test compound binding site is also within the ds DNA sequence and this site is used as an internal control. A decrease in ds DNA cleavage near the test compound binding site is a measure of increased binding of the test compound.

Pharmaceutical Compositions It will further appreciated that the amount of a compound of the invention required for use in treatment will vary not only with the particular compound selected but also with the route of administration, the nature of the condition being treated and the 25 degree and condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. In general however a suitable dose will be in the range of from about 1 to 75 mg/kg of bodyweight per day, such as about 0.01 to about 50 mg per kiolgram body weight of the recipient per day, preferably in the range of 0.025 to 40 mg/kg/day.

The desired dose may be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.

The compound is conveniently administered in unit dosage form. For example containing 0.1 to 1000, conveniently 0.2 to 500, most conveniently 0.4 to 250 mg of active ingredient per unit dosage form.

S T =q» .v a a WO 93/12129 PCT/US92/10921 Formulations of the present invention, for medical use, comprise an active compound, i.e., a compound of formula (I), together with an acceptable carrier therefof and optionally other therapeutically active ingredients. The carrier must be pharmaceutical^ acceptable in the sense of being compatible with the other 5 ingredients of the formulation and not deleterious to the recipient thereof. The present invention, therefore, further provides a pharmaceutical formulation comprising a compound of formula (I) together with a pharmaceutical^ acceptable carrier thereof. The formulations include those suitable for oral, rectal or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred 10 are those suitable for oral or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients, in general, the formulations are prepared by uniformly, and 15 intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier and then, if necessary, shaping the product into desired unit dosage form.

Formulations of the present invention suitable for oral administration may be 20 presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by 30 molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.

A syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be 35 added any accessory ingredients. Such accessory ingredient(s) may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol. 21 246473 kWO 93/12129 PCT/US92/10921 Formulations for rectal or vaginal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol 155 (trademark of Dynamite Nobel Chemical, Germany, for a suppository base).

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution or suspension of a pharmaceutical^ and pharmacologically acceptable acid addition salt of a 10 compound of the formula (I) that is isotonic with the blood of the recipient. Thus, such formulations may conveniently contain distilled water, 5% dextrose in distilled water or saline and a pharmaceutical^ and pharmacologically acceptable acid addition salt of a compound of the formula (I) that has an appropriate solubility in these solvents, for example the hydrochloride. Useful formulations also comprise 15 concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration above.

In addition to the aforementioned ingredients, the formulations of this invention may 20 further include one or more optional accessory ingredient(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like. 22 246 47 pct/us92/10921 EXAMPLES Example 1 Methvl N-f2-tert-butvloxvcarbonvlaminoethvnalvcinate (Formula fVl R1 = r3 = H. Rg = Boc. R4 = methyl! To a solution of N-tert-butyloxycarbonylglycinal (15 g, 97 mmol, freshly prepared according to Thompson et al) in 400 mL of methanol (anhydrous) under nitrogen atmosphere is added glycine methyl ester hydrochloride (15.4 g, 122 mmol), sodium acetate (16.9g, 205.6 mmol), and 80 g of powdered 4 A molecular sieves. After stirring for ca. 5 minutes sodium cyanoborohydride (12.9 g, 205.6 mmol) is added in about 4 portions, and the reaction mixture is stirred for ca 6 hours. The mixture is filtered and the filtrate concentrated. The residue is partitioned between Chloroform (250 mL) and half saturated aqueous sodium bicarbonate (250 mL). The aqueous phase is extracted with chloroform, and the combined organics are washed with brine, dried over sodium sulfate, and concentrated. The residue is purified by high vacuum kugelrohr distillation to give the title compound as an oil; 9.37 g, 41% yield. 1 H NMR (300MHz, DCI3): 4.97 (br s, 1 H), 3.71 (s, 3 H), 3.38 (s, 2 H), 3.18 (q, J = 6.5, 2 H), 2.72 (t, J = 6.5, 2 H), 1.42 (s, 9 H). mass spectrum: m/e calculated (M+H) = 233, observed = 233.

Example 2 (SVMethvl N-(2-tert-butoxvcarbonvlaminoethvn-2-faminotoropionate (Formula M: R1 = H. R2 =: Boc. R3 = (SVmethvl. R* = methvl To a solution of N-tert-butoxycarbonylglycinal (6.33 g, 39.7 mmol, freshly prepared according to Thompson et al.) in 160 mL of methanol (anhydrous) under a nitrogen atmosphere is added (L)-alanine methyl ester hydrochloride (5.55 g, 39.7 mmol), sodium acetate (6.51 g, 79.4 mmol) and 40 g of freshly activated powdered 4 A molecular sieves. After stirring for ca. 2 min, sodium cyanoborohydride (5.00 g, 79.5 mmol) is added in one portion. The reaction mixture is stirred at room temperature for 1.5 h then filtered and concentrated to a solid. The solid is partitioned between ethyl acetate (500 mL) and half saturated aqueous sodium bicarbonate (200 mL). The organics are dried 23 246473 ^wo 93/12129 pct/us92/10921 over sodium sulfate, filtered then concentrated to an oil. The oil is purified by flash chromatography to afford the title compound (3.60 g, 37% yield). 1 H NMR (300MHz, DCI3): 5.04(br s, 1H), 3.73 (s, 3H), 3.35 (q, J = 7 Hz, 1H), 3.30-3.11 (m, 2H), 2.80-2.72 (m, 1H), 2.63-2.55 (m, 1H), 1.45 (s, 9H), 1.30 (d, J = 5 7Hz, 3H); mass spectrum: m/e calculated (M+H) = 247, observed = 247.

Example 3 N-(2-tert-butvloxvcarbonvlaminoethv0-N-(1-thvminvlacetvhaminoacetic acid 10 (Formula 1X1: R1 = R3 . H. R2 = Boc. R4 = H. B = thvminal (a) Methvl N-(2-tert-butvloxvcarbonvlaminoethvn-N-( 1 -thym inylacetyQam inoacetate (Formula (X) R1 =R3 = h. r2 = boc. r4 = methvl. b = thymine)- To a solution of the compound of formula (V) (R1 = R3 = H, R2 = Boc, R4 = methyl, 3.00g, 12.91 mmol) in 90 mL of DMF (anhydrous) is added 1-carboxymethylthymine (formula(VII)), R5 = H, B = T, 2.97 g, 16.14 mmol, see A. S. Jones et al Tetrahedron 1973, 29, 2293), BOP (7.13 g, 16.14 mmol), 20 HOBt (2.18 g, 16.14 mmol), and triethylamine (3.00 mL, 25.83 mmol). After stirring for ca. 4 h the reaction mixture is diluted with 200 mL of half saturated brine and extracted with ethyl acetate. The combined organics are washed with 1 N aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, brine, dried over magnesium sulfate, and concentrated. The resulting residue 25 is chromatographed on silica gel (9:1 ethyl acetate:hexane) to give the title compound as a white solid: 3.65 g, 71 % yield. 1H NMR (300 MHz, CDCI3): 7.02 (s, 0.25 H), 6.95 (s, 0.75 H), 5.51 (br s, 1 H), 4.5 (s, 1.5 H), 4.42 (s, 0.5 H), 4.20 (s, 0.5 H), 4.05 (s, 1.5 H), 3.81 (s, 0.75 H), 3.75 (s, 2.25 H), 3.52 (t, J = .7, 2 H), 3.39 (m, 2 H), 1.91 (s, 3 H), 1.44 (s, 9H); mass spectrum: m/e 30 calculated (M+H) = 399, observed = 399. 24 kWO 93/12129 246473 (b) N-(2-tert-butvloxvcarbonvlaminoethvlVN-(1- thvminvlacetvnaminoacetic acid (Formula (X): R1 = R3 = H. R2 = Boc. R4 = H. B = thymine).

A solution of the compound of formula_(X) (R1 = R3 = H, R2 = Boc, R4 = methyl, B = thymine, 3.10 g, 7.71 mmol) in 70 mL of THF and 20 mL of water is cooled on ice. To this is added 1 N aqueous lithium hydroxide (15 mL, 15 mmol) and the reaction mixture is stirred for ca. 30 minutes. The pH of the mixture is the adjusted to ca. 2 with solid sodium bisufate. The solution is diluted with 15 mL of water, saturated with sodium chloride, and extracted with ethyl acetate. The combined organics are dried over magnesium sulfate and concentrated. The residue is dissolved in 50 mL of 1:1 acetonitrile:water and lyophilized to give the title compound as a white powder; 2.41 g, 81 % yield. 1H NMR (300 MHz, d4-methanol): 7.30 (s, 0.66 H), 7.26 (s, 0.33), 4.74 (s, 1.33 H), 4.56 (s, 0.66 H), 4.27 (s, 0.66 H), 4.10 (s, 1.33 H), 3.51 (m, 2H), 3.20 (m, 2H), 1.87 (s, 3H), 1.44 (s, 9H), spectrum m/e calculated (m + H) = 385, observed (m + H) = 385.

Example 4 Z-Cep Monomer (Formula (XV R1 = R3 = r4 = h, r2~ boc. B = 4-N-benzvloxvcarbonvlcvtosine^ (a) 4-N-Benzvloxvcarbonvlcvtosine (Formula fXM where Z=4-NI-benzovloxv-carbonvlV To a suspension of cytosine (5 g, 45 mmol) in 90 ml of pyridine (anhydrous), under a nitrogen atmosphere, in an ice bath, is added benzyichloroformate (8 mL, 56 mmol) dropwise. The mixture is brought to room temperature and stirred for ca. 16 hours. To the mixture is then added 4-dimethylaminopyridine (2.75 g, 22.5 mmol) and more benzyichloroformate (8 mL, 56 mmol). After stirring a total of ca. 40 hours, the reaction mixture is poured into 200 mL of ice-water and stirred for 5 minutes. The resulting white solid is filtered, washed with water, dichloromethane, and dried under vacuum, to give the title compound as a white solid: 7.66 g, 69% yield. 1H NMR (300 MHz, d6-DMSO): 11.1 (br s, 1 H), 7.80 (d, 1 H), 7.37 (m, 5H), 6.92 (d, 1H), 5.18 (s, 2H). 246473 (b) 1-rftert-Butoxvcarbonvnmethvn-4-N-benzvloxvcarbonvlcvtosine (Formula (VIIVRS = t-butvl. B = 4-N-benzvloxvcarbonvlcvtosinel To a suspension of 4-N-benzyloxycarbonylcytosine (3.39 g, 13.84 mmol) in 25 ml of DMF (anhydrous), under a nitrogen atmosphere, is added cesium carbonate (4.96 g, 15.22 mmol). The mixture is stirred for ca. 13 min. and then tert-butyl bromoacetate (2.46 mL, 15.22 mmol) is added dropwise, and the mixture is stirred for ca. 4 hours. The resulting solids are filtered and washed with ethyl acetate. The filtrate is partially concentrated and then partitioned between ethyl acetate and dilute brine. The organic phase is washed with dilute brine, water, saturated aqueous brine, dried over sodium sulfate and concentrated, to yield a yellow foam, which is purified by crystallization (dichloromethane/hexane) to give the title compound as white crystals: 2.75 g, 55% yield. 1NMR (300 MHz, CDCI3): 7.52 (s, 1 H), 7.50 (d, 1 H), 7.36 (s, 5 H), 7.22 (d, 1 H), 5.20 (s, 2 H), 4.50 (s, 2 H), 1.4(s, 9 H). (c) 1-(Hvdroxvcarbonvlmethvn-4-N-benzvloxvcarbonvlcvtosine (Formula (Vin:R5 = H. B = 4-N-benzvloxvcarbonvlcvtosine).

To a solution of the compound of formula (VII) (6 g, 16.71 mmol, R5 = t-butyl, B = 4-N-benzyloxycarbonylcytosine) in 45 ml of dichloromethane (anydrous), under a nitrogen atmosphere, is added anisole (20 mL, 184 mmol), followed by trifluoroacetic acid (50 mL, 649 mmol). The reaction mixture is stirred for ca. 4 hours, and then concentrated to dryness. To the residue is added toluene, and the solution is concentrated to dryness. This process is repeated two more times. The residue is dried under vacuum, and then triturated with dichloromethane. The resulting solids are filtered and washed with dichloromethane to give the title compound as a solid; 6.38 g, 81% yield, as a 1:1 complex with trifluoroacetic acid. 1H*fJMR (300 MHz, d6-DMSO): 8.02 (d, J = 7.3, 1 H), 7.38 (m, 5 H), 7.01 (d, J = 7.3,1 H), 5.18 (s, 2 H), 4.51 (s, 2 H); mass spectrum: m/e calculated (M + H) =304, observed (M + H) = 304. 26 24647 WO 93/12129 PCT/US92/10921 (d) Z-C Monomer methvl ester (Formula (XI: R1 = R3 = H. R2 = Bnc. r4 = methvl.

B = 4-N-benzvloxvcarbonvlcvtosine^.

To a solution of the compound of formula (V) (0.59 g, 2.54 mmol, R1 = R3 = H, r2 - Boc, R4 = methyl) in 8 ml of DMF (anhydrous), under a nitrogen atmosphere, is added BOP (0.94 g, 2.1 mmol), HOBt (0.29 g, 2.1 mmol) and a solution of the compound of formula (VII) (0.93 g, 2.23 mmol, R5 = H, B = 4-N-10 benzyloxycarbonylcytosine) and triethylamine (1.41 mL, 10.15 mmol) in 8 ml of DMF (anhydrous). After stirring for ca. 3 hours the reaction mixture is concentrated, and the residue partitioned between 100 mL of ethyl acetate, and 50mL of 0.5 N aqueous hydrochloric acid. The organic phase is washed with half saturated aqueous sodium bicarbonate and saturated aqueous is brine. Crystallization from the ethyl acetate solution affords the title compound as white crystals:0.62 g, 59% yield. 1H NMR (300 MHz, CDCI3): 7.65 (br s, 1 H), 7.62 (d, 0.3 H), 7.58 (d, 0.7 H), 7.35 (s, 5 H), 7.22 (d, 1 H), 5.55 (t, 1 H), 5.19 (s, 2H), 4.70 (s, 1.4H), 4.55 (s, .6H), 4.30 (s, .6H), 4.05 (S, 1.4 H), 3.78 (s, 0.8 H), 3.70 (s, 2.2 H), 3.55 (t, 1.4 H), 3.50 (t, 0.6 H), 3.32 (q, 20 1.4 H), 3.22 (q, 0.6 H), 1.40 (s, 9 H); mass spectrum: m/e calculated (M + H) = 518, observed (M + H) = 518. (e) Z-Cea Monomer (Formula IX): r1 = r3 = r4 - h. r2 = Boc. b = 4-n-benzvloxvcarbonvlcvtosine).

To a suspension of the compound of formula (X) (2.36 g, 4.56 mmol, R1 = R3 = H, R2 = Boc, R4 a methyl, B = 4-N-benzyloxycarbonylcytosine) in 70 mL of THF-water (1/1), in an ice bath, is added 1 N aqueous sodium hydroxide (14 mL, 14 mmol). After stirring ca. 10 minutes, the reaction mixture is partitioned 30 between 80 mL of ethyl acetate, and 90 mL of dilute brine. The aqueous phase is washed with ethyl acetate, acidified with saturated aqueous sodium bisulfate, saturated with sodium chloride, and extracted ethyl acetate. The organic phases are dried over magnesium sulfate and partially concentrated. Crystallization occurs and the heterogenous solution is diluted with 35 chloroform, ethyl acetate, and filtered to give the title compound as white crystals: 2.02 g, 88% yield. 1H NMR (300 MHz, d6-DMSO): 10.78 (br s, 1 H), 7.89 (d, 0.65 H), 7.86 (d, 0.35 H), 7.36 (m, 5 H), 7.02 (d, 0.65 H), 6.99 (d, 0.35 H), 6.93 (t, (s, 0.7 H), 6.74 (t, .35H), 5.18 (s, 2H), 4.80 (s, 1.3H), 4.60 (s, .7H), 27 246473 ^WO 93/12129 PCT/US92/10921 4.20 (s, 0.7 H), 3.97 (s, 1.3 H), 3.38 (br t, 1.3 H), 3.28 (br t, 0.7 H), 3.18 (br q, 1.3 H), 3.00 (br q, 0.7 H), 1.35 (s, 9 H); mass spectrum: m/e calculated (M + H) = 504, observed (+ H) = 504.

Example 5 Bn-Z-Geo monomer methvl ester (Formula (X): R1 = R3 = H. Rg = Boc. R4 = methvl.

B = 6-Q-benzvl-2-N-(benzvloxvcarbonvnouanine^ (a) 9-N-allvl-6-Q-benzvlauanine (Formula (IXal To a solution of 6-O-benzylguanine (0.930 g, 3.85 mmol) (MacCoss) in 10 mL of dry DMF at room temperature is added potassium carbonate (2.66 g, 19.3 mmol) and 18-crown-6 (1.02 g, 3.85 mmol). After 0.25 h, ally) bromide (0.367 mL, 4.24 mmol) is added in one portion. The resulting solution is vigorously stirred for 1 h. The mixture is partitioned between 125 mL of ethyl acetate and 50 mL of water. The organics are washed with brine, dried over sodium sulfate, filtered and concentrated. The residue is purified by radial chromatography on silica gel (hexane/ethyl acetate, gradient elution) to afford the title compound as an oil (0.582 g, 54%). 1H NMR (300 MHz, CDCI3): 7.60 (s, 1H), 7.52-7.48 (m, 2H), 7.38-7.7 (m, 3H), 6.05-5.92 (m, 1H), 5.56 (s, 2H), 5.26 (dd, J = 3.4, 0.3 Hz, 1H), 5.13 (dd, J = 5.7, 0.3 Hz, 1H), 5.05 (br s, 2H), 4.65 (ddd, J = 1.9, 0.3,0.3 Hz, 2H); mass spectrum: m/e calculated (M+H) = 282, observed = 282. (b) 9-N-alM-6-Q-benzvl-2-N-(benzvloxvcarbonvhpuanine (Formula (IXVB = 6-0-benzvl-2-N-(benzvloxvcarbonvnouaninel To a solution of 9-N-allyl-6-0-benzylguanine (0.170 g, 0.604 mmol) in 5 mL of THF at room temperature is added 18-crown-6 (0.319 g, 1.21 mmol) and N-(benzyloxycarbonyl)imidazole (0.611 g, 3.02 mmol, Watkins). After 5 min, potassium hydride (35% in oil, 0.173 g, 1.51 mmol) is added dropwise. The resulting solution is maintained at room temperature for 1 h. The mixture is then partitioned between 100 mL of ethyl acetate and 50 mL of water. The organics are washed with brine, dried over sodium sulfate, filtered then concentrated. The residue is purified by radial chromatography on silica gel . (hexane/ethyl acetate, gradient elution), to afford the title compound as an oil; 28 246 4 JWO 93/12129 PCT/US92/10921 ().236 g, 93%). 1H NMR (300 MHz, CDCI3): 7.82 (br s, 1H), 7.76 (s, 1H), 7.53-7.42 (m, 2H), 7.42-7.28 (m, 8H), 6.04-5.91 (m, 1H), 5.59 (s,2H), 5.28 (dd, J = 3.5, 0.4 Hz, 1H), 5.25 (s, 2H), 5.20 (dd, J = 5.6, 0.3 Hz, 1H), .73 (ddd, J=1.9, 0.4, 0.4 Hz, 2H). (c) 6-Q-benzvl-2-N-(benzvloxvcarbonvh-9-N- Kmethoxvcarbonvnmethvllauanine (Formula JVIIVRS = methvl.

B = 6-0-bQnzvl-2-N-fbenzvloxvcarbonvh<?uanin6^.

To a solution of the compound of formula (IX) (0.230 g, 0.554 mmol, B = 6-0- benzyl-2-N-(benzyloxycarbonyl)- guanine) in 2 mL of carbon tetrachloride, 2 mL of acetonitrile and 3 mL of water is added sodium periodate (0.474 g, 2.21 mmol) followed by ruthenium(lll) chloride hydrate (0.010 g, 0.048 mmol). After 5 h at room temperature an additional amount of ruthenium (III) chloride 15 hydrate (0.010 g, 0.048 mmol) is added. The resulting solution is vigorously stirred at room temperature for 15 h. The mixture is partitioned between 100 mL of methylene chloride and 25 mL of water. The organics are dried over sodium sulfate, filtered then concentrated. The residue is partially purified by radial chromatography on silica gel (methanol/methylene chloride/acetic 20 acid, gradient elution) to afford the free acid. The free acid is azeotropically dried with toluene and the residue dissolved in methanol. A solution of diazomethane in ethyl ether is added dropwise until the yellow color persists. The volatiles are removed under reduced pressure and the resulting oil is purified by radial chromatography on silica gel (hexane/ethyl acetate, 25 gradient elution) to afford the title compound as an oil (0.061 g, 25%). 1H NMR (300 MHz, CDCI3): 7.85 (s, 1H), 7.54-7.51 (m, 2H), 7.45-7.30 (m, 8H), 5.61 (s, 2H), 5.27 (s, 2H), 4.97 (s, 2H). 3.79 (s, 3H). (d) Bn-Z-Geo monomer methvl ester (Formula 00: R1 = R3 = H. R2 = Boc. R4 = methvl. B = 6-Q-benzvl-2-N- (benzvloxvcarbonvnauanineV To a solution of the compound of formula (VII) (0.055 g, 0.12 mmol, R5 = methyl, B = 6-0-benzyl-2-N-(benzyloxycarbonyl)guanine) in 2 mL of THF 35 and 1 mL of water at room temperature is added lithium hydroxide monohydrate (0.015 g, 0.37 mmol). After 0.5 h the reaction is acidified with 2 mL of 5% aqueous hydrochloric acid and extracted with ethyl acetate. The organics are dried over sodium sulfate, filtered then concentrated to a white 29 246473 .WO 93/12129 PCT/US92/10921 powder (0.050 g, 94%). To a solution of the resulting free acid and the compound of formula (V) (0.050 g, 0.21 mmol, R1 = R3 = H, R2 - Boc, R4 = methyl) in 2 mL of dry DMF is added BOP (0.076 g, 0.17 mmol) and HOBt (0.023 g, 0.17 mmol). After 5 min, triethylamine (0.048 mL, 0.35 mmol) is 5 added in one portion. The resulting solution is stirred at room temperature for 2 h. The mixture is partitioned between 100 mL of ethyl acetate and 50 mL of brine. The organics are washed with brine, dried over sodium sulfate, filtered then concentrated tc. ~n oil. The oil was purified by radial chromatography on silica gel (methanol/methylene chloride, gradient elution), to afford the title 10 compound as an oil; 0.85 g, quantitative yield). 1H NMR (250 MHz, CDCI3): 7.96 (s, .75H), 7.89 (s, .25H), 7.69 (s, 1H), 7.49-7.46 (m, 2H), 7.38-7.27 (m, 8H), 6.27 (dd, J = 5.1, 5.1 Hz, 1H), 5.55 (s, 2H), 5.22 (s, 2H), 5.03 (s, 1.5H), 4.86 (s, .5H), 4.37 (s, .5H), 4.09, (s, 1.5H), 3.78 (s, .75H), 3.70 (s, 2.25H), 3.63 (m, 1.5H), 3.51 (m, .5H), 3.39 (m, 1.5H), 3.23 (m, .5H), 1.20 (s, 9H); mass 15 spectrum: m/e calculated (M+H) = 648, observed = 648.

Example 6 Z-Aeo Monomer (Formula 00: r1 =r3= r4 = h. r2 = Boc. B = 6-N-enzvloxvcarbonvladenine> (a) 9-f(tQrt-Butvloxvcarbonvnmethvn-adenine (Formula (vip: r5 = tert-butvl. b a adenine^ To a suspension of adenine (3 g, 22.2 mmol) in 60 .nL of DMF (anhydrous), under a nitrogen atmosphere, is added cesium carbonate (7.96 g, 24.42 mmol), followed by the dropwise addition of tert-butyl bromoacetate (4.3 mL, 26.64 mmol). The reaction mixture is stirred at room temperature for ca. 14 hours, concentrated to half the volume and partitioned between 125 mL of 30 chloroform and 100 mL of dilute brine. The aqueous phase is extracted with chloroform, and the combined organic phases are washed with 30 mL of saturated brine. Upon partial concentration, crystallization occurs. The white crystals were washed with ethyl acetate and dried to afford the title compound: 3.05 g, 55% yield. 1H NMR (300 MHz, d6-DMSO): 8.12 (s, 1 H), 35 8.09 (s, 1 H), 7.24 (br s, 2 H), 4.93 (s, 2 H), 1.41 (s, 9 H). 246 47 3 JVO 93/12129 PCT/IJS92/10921 (b) 6-N-(Benzvloxvcarbonvn-9-r(tert-butvloxvcarbonvhmethviy adenine (Formula fVlh r5 = tsrt-butvl. B = 6-N-benzvloxvcarbonvladeninel To a solution of N-(benzyloxycarbony I) imidazole (0.16 g, 0.8 mmol, see J. Am. Chem. Soc., 1982, 104, 5702-5708) in 1.5 mLof 1,2-dichloroethane (anhydrous), under a nitrogen atmosphere, in an ice bath, is added tristhyloxonium tetrafluoroborate (0.82 mL, 1.0 M in methylene chloride, 0.82 10 mmol). After the addition, the ice bath is removed and the mixture is stirred at room temperature for ca. 2 hours. To the reaction mixture is then added the compound of formula (VII) (0.050 g, 0.20 mmol, R5 = tert-butyl, B = adenine) as a solid. The reaction mixture is heated at 82 C for ca. 5 hours, and allowed to stand at room temperature for ca. 2 days. The mixture is 15 concentrated and chromatographed twice on silica gel (first;ethyl acetate- hexane gradient, second; ethyl acetate-chloroform gradient) to give the title compound as a white solid: 0.040g, 53% yield. 1H NMR (300 MHz, CDC 3): 8.73 (s, 1H), 8.46 (br s, 1H), 7.96 (s, 1 H), 7.37 (m, 5 H), 5.28 (s, 2 H), 4.84 (s, 2 H), 1.47 (s, 9 H); mass spectrum: m/e calculated (M + H) = 384, observed 20 (m + H) = 384. (c) 6-N-(Benzvloxvcarbonvn-9-r(hvdroxvcarbonvnmethvn-adenine (Formula (VIIVRS = H. B = 6-N-benzvloxvcarbonvladenine^.

To a solution of the compound of formula (VII) (0.115 g, 0.30 mmol, R5 = tert- butyl, B = 6-N-benzyloxycarbonyladenine) in 5 mL of dichloromethane (anhydrous), under a nitrogen atmosphere, is added anisole (2.5 mL, 23 mmol), followed by trifluoroacetic acid (7 mL, 91.0 mmol). The reaction mixture is stirred for ca. 4 nr, and concentrated to dryness. The residue is 30 azeotroped five times from chloroform, methanol and ethyl ether mixtures and dried under high vacuum overnight, to give the title compound as a 1:1 complex with trifluoroacetic acid: 0.134 g, 90% yield. 1H NMR (300 MHz, d4- 3 H), 8.67 (s, 1H), 8.52 (s, 1H), 7.45 (dd, 2H), 7.38 (m, 3H), 5.35 (s, 2 H), 5.18 (s, 2 H); mass spectrum: m/e calculated (M + H) = 328, observed (m + H) = 328. 31 246473 .WO 93/12129 PCT/US92/10921/ (d) Z-Aeo Monomer methvl ester (Formula (X): R"1 = r3 = H. R2 = Boc. R4 = methvl.

B = 6-N-benzvloxvcarbonvladenine).

To a solution of BOP (0.124 g, 0.28 mmol), HOBt (0.038g, 0.28mmol) and the compound of formula (VII) (0.130 g, 0.28 mmol, R5 = H, B = 6-N-benzyloxycarbonyladenine) in 1.5 mL of DMF (anhydrous), under a nitrogen atmosphere, is added triethylamine (0.188 mL, 1.35 mmol). After stirring the 10 mixture for ca. 1.5 min, it is transferred via syringe into a solution of the compound of formula (V) (0.07 g, 0.27 mmol, R1 = R3 = H, R2 = Boc, R4 = methyl) in 1 mL of DMF (anhydrous), and the mixture is stirred for ca. 2 hours. To the reaction mixture is added BOP (0.124 g, 0.28 mmol) and the mixture is stirred for ca. 3 hours. The mixture is concentrated to dryness and partitioned 15 between 25 mL of ethyl acetate, and 10 mL of 0.5 N hydrochloric acid containing 3 mL of saturated aqueous brine. The organic phase is washed with aqueous 0.5 N hydrochloric acid-brine, dilute aqueous sodium bicarbonate-brine, and saturated aqueous brine. The organics are concentrated and chromatographed on silica gel (first; ethyl acetate-hexane, 20 second; methanol-ethyl acetate) to give the title compound as a white foam: 0.078 g, 53% yield. 1H NMR (300 MHz, CDCI3): 8.72 (s, 3 H), 8.14 (br s, 1H), 8.02 (s, 1H), 7.42 dd, 2H), 7.36 (m< 3H), 5.60 (br t, 1 H), 5.28 (s, 2 H), 5.14 (s, 1.6 H), 4.97 (s, 0.4 H), 4.29 (s, 0.4 H), 4.05 (s, 1.6 H), 3.82 (s, 0.75 H), 3.73 (s, 2.25 H), 3.64 (t, 1.6 H), 3.54 (t, 0.4 H), 3.38 (q, 1.6 H), 3.26 (q, 0.4H), 1.4 (s, 25 9H); mass spectrum: m/e calculated (m +H) = 541, observed (m + H) = 541. (e) Z-Aea Monomer (Formula fflO: rl = r3 = r4 = H. R2 = Boc.

B = 6-N-benzvloxvcarbonvladeninel.

To a solution of the compound of formula (X) (0.070 g, 0.129 mmol, R1 = R3 = H, R2 = Boc, R4 = methyl, B = 6-N-benzyloxycarbonyladenine) in 3 mL of 1:1 THF-water, in an ice bath, is added 1 N aqueous sodium hydroxide (0.388 mL, 0.388 mmol). After stirring for ca. 1 hour the reaction mixture is 35 partitioned between 5 mL of ethyl acetate and 10 mL of water. The aqueous phase is washed with ethyl acetate, acidified with 3 mL of saturated aqueous sodium bisulfate, saturated with sodium chloride and extracted with ethyl acetate. The combined organic phases are dried over sodium sulfate and 32 ™93/12129 246473 concentrated to give the title compound as a white solid: 0.065 g, 95% yield. 1H NMR (300 MHz, d4 methanol): 8.58, (s, 0.6H), 8.57 (s, 0.4H), 8.27 (s, 0.6 H), 8.26 (s, 0.4 H), 7.46 (d, 2 H), 7.36 (m, 3 H), 5.39 (s, 1.4 H), 5.30 (s, 2 H), 5.21 (s, 0.6 H), 4.40 (s, 0.6 H), 4.12 (s, 1.4 H), 3.64 (t, 1.4 H), 3.47 (t, 0.6 H), 3.38 (t, 1.4H), 3.20 (t, 0.6H), 1.41 (s, 9H); mass spectrum: m/e calculated (m + H) = 527, observed (m + H) = 527.

Example 7 (SVN-(2-tert-butoxvcarbonvlaminoethvn-N-M-thvminvlacetvn-2-(aminotoropionic acid (Formula (X) R1 = H, R2 = Boc. r3 = (SWnethvl. r4 = h. b = thymine^ (a) (S)-Methvl N-(2-tert-butoxvcarbonvlaminoethvn-N-M-thvminvlacetvn- 2-(aminp)propipnat<=> (Formula 00 R1 = H. R2 = Boc. R3 = (SVmethvl.

R4 =: methvl. B = thvminel To a solution of the compound of formula (V) (R1 = H, R2 = Boc, R3 = (S)- • methyl, R4 = methyl, 1.7 g, 6.9 mmol) in 20 mL of DMF (anhydrous) is added 1-carbomethoxythymine (formula (VII) R5 = H, B = thymine, 1.4 g, 7.6 mmol) and 1,3-dicyclohexylcarbodiimide (1.5 g, 7.6 mmol). After stirring at room temperature for ca. 20 h, the reaction mixture is filtered through celite. The filtrate is concentrated then partitioned between ethyl acetate (300 mL) and a 1:1 mixture of brine and saturated aqueous sodium bicarbonate (150 mL). The organics are dried over sodium sulfate, filtered then concentrated on ca. 10 g of silica gel. The silica gel is placed on the top of a flash column packed , with silica and then the column is eluted with methanol/methylene chloride (gradient elution) to afford the title compound (2.6 g, 91%): 1H NMR (300 MHz, CDCI3): 9.3 (br s, 0.25H), 8.97 (br s, .75H), 6.99 (s, .25H), 6.93 (s, .75H), 5.57 (br t, J = 5Hz, 1H), 4.54 (m, 2H), 4.34 (q, J = 7Hz, 1H), 3.78 (s, .25H), 3.73 (s, .75H), 3.67-3.22 (m, 4H), 1.90 (s, 3H), 1.60 (d, J = 7Hz, 3H). 1.43 (s, 9H); mass spectrum: m/e calculated (M+H) = 413, observed = 413. (b) (S)-N-(2-tert-butoxvcarbonvlaminoethvh-N-(1-thvminvlacetvn-2- fcminotoropionic acid (Formula tXY. R1 = h. r2 = Boc. r3 = (SVmethvl.

R4 = h. B = thymine). 33 246473 /O 93/12129 PCT/US92/10921 A solution of the compound of formula (X) (R1 = H, R2 = Boc, R3 = (S)-methyl, R4 = methyl, B = thymine, 2.50 g, 6.06 mmol) om 30 mL of methanol and 15 mL of water is treated with sodium hydroxide water (0.29 g, 7.3 mmol). The reaction mixture is left at room temperature. After ca. 60 h, 5 additional sodium hydroxide (0.12 g, 3.0 mmol) is added. After ca. 24 h, the methanol is removed under reduced pressure. The residual aqueous mixture is diluted with ethyl acetate (300 mL) and brine (100 mL). The pH of the aqueous phase is adjusted to ca. 2 with solid sodium bisulfate and the layers separated. The aqueous layer is back extracted with ethyl acetate 10 (150 mL). The combined organics are dried over sodium sulfate, filtered then concentrated to a foam. The foam is dissolved in methylene chloride then added dropwise to vigorously stirred hexane (300 mL). The resulting precipitate is filtered and dried to afford the title compound as a white powder (2.12 g, 88% yield). 1H NMR (300 MHz, d6-DMSO): 11.29 (s, 1H), 7.35 (s, 15 1H), 6.93 (br s, .67H), 6.86 (br s, .33H), 4.68-4.56 (m, 2H), 4.31 (q, J = 7Hz, 7Hz, 3H), 3.2-2.93 (m, 4H), 1.73 (s, 1H), 1.39 (s, 9H), 1.34 (d, J=7Hz, 3H); mass spectrum: m/e calculated (M+H) = 399, observed = 399. 34 <^0 4/0 fc.wo 93/12129 pct/us92/10921 Exgnml6_a Gea-Teo dimer (Formula ffl: R11 = R3l = r12 = r32 = H. B1 = T. B2 = 6-Q-benzvl-2-N-(benzvloxvcarbonvhauanine. J = methoxv. Q = Bort (a) Tea monomer amine hydrochloride (Formula (X); R1^_B3-=B2 = H, R4 = methyl, P = T).

The compound of formula (X) (0.093 g, 0.237 mmol, R1 = R3 » H, R2 = Boc, B = T) is dissolved in 6 mL of 4 N hydrochloric acid in dioxane (Pierce). After 0.25 h, the reaction is concentrated to afford a white powder of the title compound (0.081 g, quantitative yield). 1H NMR (300 MHz, CDCI3): 7.33 (s, 15 .33H), 7.26(s, .67H), 4.71 (s, .33H), 4.54 (s, .67H), 4.34 (s, .67H), 4.12 (s, .33H), 3.82-3.49 (m, 2H), 3.76 (s, 3H), 3.29-3.03 (m, 2H), 1.80 (s, 3H); mass spectrum: m/e calculated (M+H) = 299, observed = 299. (b) Gea-Teg dimer (Formula (bin which n is 1. and reading left to right Q is Boc. R1 is hvdroaen. B is 6-Q-benzvl-2-N-(benzvloxvcarbonvnguanine. R3 is hydrogen. R"! is hydrogen. B is thymine. R3 is hvdroaen. and J is methoxv.

To a room temperature solution of the compound of formula (IX) (0.081 g, 0.12 mmol, R1 = R3 = H, R2 = Boc, R4 = methyl, B = 6-0-benzyl-2-N-(benzyloxycarbonyl)guanine) in 5 mL of THF and 2 mL of water is added lithium hydroxide monohydrate (0.008 g, 0.2 mmol). After 1 h, the reaction is quenched with solid sodium bisulfate then partitioned between 75 mL of ethyl 30 acetate and 25 mL of brine. The aqueous phase is back extracted with 50 mL of ethyl acetate. The combined organics are dried over sodium sulfate, filtered then concentrated to a yellow powder (0.083 g, 0.13 mmol). To a solution of the resulting free acid (0.083 g, 0.13 mmol) and the compound of formula (V) (0.040 g, 0.12 mmol, R1 = R3 = R2 = H, R4 = methyl, B = T) in 1 35 mL of dry DMF is added a solution of HOBT/HBT (.45 M, 0.18 mmol). After 1 min, DIEA (0.063 mL, 0.36 mmol) is added in one portion. After 1 h at room temperature, the mixture is partitioned between 100 mL of ethyl acetate and 50 mL of brine. The aqueous layer is back extracted with an additional 50 24647 kWO 93/12129 PCT/US92/109 1 mL of ethyl acetate. The combined organics are dried over sodium sulfate, filtered and concentrated. Radial chromatography on silica gel (methanol/methylenechloride, gradient elution) affords the title compound (0.065 g, 60%); mass spectrum: m/e calculated (M+H) = 914, observed = 914. (c) Geo-Teg dimer (Formula (h in which n is 1. and reading left to right Q is hvdroaen.

R1 is hvdroaen. B is guanine. R3 is hydrogen.

R1 is hvdrooen. B is thymine. R3 is hydrogen, and J is methoxv.^ To a solution of trifluoroacetic acid (0.5 mL) and methylene chloride (1.0 mL) under a nitrogen atmosphere is added the fully protected guanine-thymine dimer (.044 g, .048 mmol). After 0.5 h at room temperature, the volatiles are removed in vacuum. After ca. 16 h under high vacuum, the resulting powder 15 is dissolved in anisole (.020 mL) and hydrogen fluoride (ca. 5 mL) is added.

After 1 h in an ice bath, the volatiles are removed under reduced pressure and the residue is dissolved in trifluoroacetic acid (ca. 5 mL) then concentrated. The residue is dissolved in water (5 mL) and acetonitrile (5 mL) then lyophilized to afford the guanine-thymine dimer methyl ester as a 20 white powder (.028 g, quantitative); mass spectrum: m/e calculated (M+H) = 590, observed = 590.

Example 9 Ceo-Tea dimer (Formula (W in which n is 1. and reading left to right 0 is Boc. Rl is hydrogen. B is 4-N-benzvloxvcarbonvlcvtosine. R3 is hydrogen. R1 is hydrogen. B is thymine. r3 is hydrogen and J is methoxv.

To a solution of Ceg monomer (0.030 g, 0.060 mmol) in 0.5 mL of anhydrous DMF is added HBTU and HOBt (0.133 mL of a 0.45 M solution in DMF, 0.60 mmol each), followed by DIEA (0.017 mL, 0.10 mmol). After stirring for 15 minutes the mixture is transferred, via syringe, to a solution of the compound 35 of formula (V) (0.040 g, 0.12 mmol, R1 = R3 = r2 = H, R4 = methyl, B = T, 0.016 g, 0.050 mmol) and DIEA (0.017 ml, 0.100 mmol) in 1 ml of anhydrous DMF that was premixed for 25 minutes. The reaction is followed by HPLC and judged to be complete afer 60 minutes. The mixture is concentrated and 36 £ . f (T U 4 k WO 93/12129 PCT/US92/10921 the residue dissolved in 15 mL of chloroform and washed with 0.3N hydrochloric acid, dilute sodium bicarbonate and saturated brine. The organic phase is dried over sodium sulfate, filtered and concentrated. Preparative thin layer chromatography on silica gel (3 plates, 20X20 cm, 5 2000 micron thickness, 11% methanol-chloroform as eluent) affords the title compound (0.019 g, 49%). 1H-NMR (300 MHz, CDCI3): Rotamers observed. Major rotamen 7.6 (broad signal, 1H), 7.55 (broad signal, H), 7.35 (m, 5H), 7.2 (broad signal, 1H), 7.15 (s, 1H), 5.70 (broad signal, 1H), 5.15 (s, 2H), 4.7 (s, 2H), 4.6 (s, 2H), 4.05 (s, 4H), 3.75 (s, 3H), 3.7-3.15 (m, 8H), 1.75 (s, 3H), 10 1.35(s, 9H).

Example 10 Aeg-Teo dimer (Formula mi in which n is 1. and reading left to right 0 is Boc. R1 is hydrogen. B is 6-N-benzvloxvcarbonvladenine. R3 is hydrogen. R1 is hvdrooen. B is thymine. R3 is hydrogen and J is methoxv.

To a solution of Aeg monomer (0.012 g, 0.023 mmol) in 1 mL of anhydrous DMF is added HBTU and HOBt (0.050 mL of a 0.45 M solution in DMF, 0.023 mmol each), followed by DIEA (0.015 mL, 0.086 mmol). After stirring for 3 minutes, the mixture is transferred, via syringe, to a solution of the compound of formula (v) (R1 = r3 = r2 + h, r4 = methyl, b =T, 0.009 g, 0.022 mmol), 25 and DIEA (0.015 ml, 0.086 mmol) in 0.8 mL of DMF that was premixed for 10 minutes. The reaction is followed by HPLC and judged to be complete after 30 minutes. The mixture is concentrated, the residue dissolved in chloroform and washed with dilute sodium bicarbonate and saturated brine. The organic phase is dried over sodium sulfate, filtered and concentrated. 30 Preparative TLC chromotography on silica gel (2 plates, 20x20 cm, 2000 micron thickness, 15% methanol-chloroform as eluent) affords the title compound (0.008 g, 44%). 1H-NMR (300 MHz, MeOH-d4): Four rotamers observed. Major rotamer: 8.56 (s, 1H), 8.19 (s, 1H), 7.5-7.3 (m, 5H), 7.08 (d, J=1.2 Hz, 1H), 5.42 (s, 2H), 5.30 (s, 2H), 4.55 (s, 2H), 4.12 (s, 4H), 3.72 (s. 35 3H), 3.7-3.2 (m, 8H), 1.6 (s, 3H), 1.4 (s, 9H). 37 246 4 kWO 93/12129 PCT/US92/10921 Example 11 (Teg> 6-1vs-NH£ (Formula (W in which n is 5. 0 is hydrogen, all Rl and r3 are hydrogen, all B are thymine, and J is NHo.

To MBHA resin 91.00 g, 0.25 meq/g, Peptides Interantional) prewashed with 10 DMF is added t-butyloxycarbonyl-Ne-2-chlorobenzyloxycarbonyl-L-lysine (0.416 g, 1.00 mmol) in 2.22 mL of a HBTU/HOBt DMF solution 90.45 M in both HBTU and HOBt, referred to as the coupling solution) and DIEA (0.3 mL). The mixture is shaken gently for ca. 6 hours at room temperature. The reaction solution is removed by filtration, and the resin is washed with DMF. 15 To the resin is added 2 mL of an acetic anhydride, DIEA, DMF solution (0.4/0.7/1.5 ratio, referred to as the capping solution). This is shaken gently for 0.5 hours. The resin is washed with DMF and methylene chloride, and is then treated wth 2 mL of a 1/1 trifluoroacetic acid/methylene chloride solution (referred to as teh de-Bocing solution) for 30 minutes. The reaction solution 20 is removed by filtration and the resin is washed with a 15% solution of DIEA in methylene chloride, methylene chloride, and dried under vacuum, to give 1.101 g of dry C1Z-lys-MBHA resin.

To the Teg monomer (0.193 mg, 0.50 mmol) is added 1.1 mL of the coupling 25 solution , 1 mL of DMF, and 0.15 mL of DIEA, and this mixture is allowed to stand for ca. 3 minutes. This solution is then added to the above resin (0.50 g, prewashed with DMF). The mixture is removed by filtration and the resin is washed with DMF, and treated with 2 mL of the capping solution for ca. 30 minutes. The reaction solution is removed by filtration and the resin is 30 washed with DMF and methylene chloride. The resin is then treated with 2 mL of the de-Bocing solution for ca. 30 minutes and washed with 15% DIEA in methylene chloride, methylene chloride and DMF. This coupling-capping-de-Bocing cycle is repeated a total of six times. After the final de-Bocing step the resin is washed with methylene chloride and dried under vacuum to give 35 674 mg of resin. A portion of this resin (50 mg) is treated with hydrofluoric acid (ca. 5 mL) in the presence of anisole (ca. 0.5 mL) for ca. 50 minutes. The hydrofluoric acid is removed under vacuum, and the residue is taken up . in trifluoroacetic acid and filtered. The trifluoroacetic acid solution is 38 246 473 i^WO 93/12129 PCT/US92/10921 concentrated and the residue purified by reverse phase HPLC (acetonitrile/water gradient) to give the title compound as a white solid (12 mg, 55% yield), mass spectrum: m/e (electrospray) calculated (M+H)=1743, observed = 1743.

Example 12 Rate of Strand Invasion into a poly r/V-125-30 heterodupiex bv the compound of (Formula (m in which n is 5. 0 is hvdroaen. all Rl and Fft are hydrogen, all B are 10 thymine and J is NHg.

The 3H poly rA-T25-30 heterodupiex was prepared as follows: 50 ml 3H poly rA (5 nCi, 940 pmol nucleotide) and 100 pmol T25.30 (2500-3000 pmol nucleotide) were incubated in buffer A (40 mM Tris-HCI pH 7.5, 50 mM NaCI, 8 mN NaCI, 8 mM MgCl2, 2 mM spermidine) in a 480 p.1 reaction at 70°C for minutes followed by slow cooling to room temperature over ca. 1 hour and then placed at 15°C for 15 minutes. To the above solution 10 (J.I of the compound of formula (I) (in which n is 5, Q is hydrogen, all R1 and R3 are 20 hydrogen, all B are thymine, and J is NH2,1000 pmol ODN, 6000 pmol nucleotide) is added and at various times (0, 5,10, 15, 20, 30, 40, 50, 60 minutes) 40 \i\ are removed and 1 nl Hela cell nuclear extract (Bethesda Research Laboratories), as a source of human RNase H, is added. The reaction is incubated at 15C for 5 minutes and then terminated by the 25 addition of 50 (il of 1|ig/ml tRNA and 100 fxl of 2 M HCI, 0.2 M sodium pyrophosphate. The solutions are placed on ice for ca. 10 minutes, and then centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant is removed and the amount of 3H determined by scintillation counting. The extent of strand invasion is determined by comparing the 3H in the supernatant for 30 each time point to that of control reactions. A reaction which contained none of the compound of formula (I) was performed as above to determine the maximum amount of 3H in the supernatant. A reaction, in which the PNA and T25-30 were added simultaneously to the reaction, was performed to determine the minimum amount to 3H released (an additional way to 35 determine the minumum 3H released was to conduct the reaction in the absence of both T25-30 and the PNA; both approaches gave essentially identical amounts of 3H in the supernatant. 39

Claims

WHAT WE CLAIM IS: 1. A nucleoside base oligomer of the following formula (I): B r V QN N R3 R1 T I 0 n 24 6 4?3 PCT/US92/10921 (D 5 wherein 10 15 20 Q is an N-terminal blocking group; J is a C-terminal blocking group or Q and J may together be a single bond; n is at least 1; R1 is independently hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2. -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(Ci.4 alkyl), -CH2CH2COOH, -CH2CH2COO(C1.4 alkyl), -CH2CONH2. -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C1.12 alkyl, C2-8 alkenyl, C2-8 alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, trifluoromethyl, or di-(C-|.4 alkyl) substituted amino; R3 is independently hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO (C1.4 alkyl), -CH2CH2COOH, -CH2CH2COO(Ci.4 a'M). -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C-J.-J2 alkyl, C2-8 25 alkenyl, C2-8 alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, Cf .4 alkoxy, trifluoromethyl, or di-(C-|_4 alkyl) substituted amino; and B is independently a purine or pyrimidine nucleoside base providing that at least 30 one Bisa purine nucleoside base, or an acid «f base addition salt thereof. 24 6 4 7 J WO 93/12129 PCT/US92/10921 5 2 • The nucleoside base oligomer of Claim 1 wherein at least one B is adenine. 3 • The nucleoside base oligomer of Claim 1 wherein at least one B is guanine. 4 • The nucleoside base oligomer of Claim 1 wherein at least one B is thymine. 5. The nucleoside base oligomer of Claim 1 wherein at least one B is cytosine. 10 6. The nucleoside base oligomer of Claim 1 wherein n is at least substantially 5. 7. A nucleoside monomer of the following formula (X): „J ® B' ^ o 15 R2HN N OR4 R3 wherein R1 is hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, 20 -CH2CH2CH2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(Ci.4 alkyl), -CH2CH2COOH, rCH2CH2COO(C1-4 alkyl), -CH2CONH2, -CH2CH2CONH2, -CH2SH, CH2CH2SCH3, C-j.12 alkyl, C2-8 alkenyl, C2-8 alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl which is mono, di, or trisubstituted independently with halogen, nitro, C1.4 alkyl, C1.4 alkoxy, 25 trifluoromethyl or di-(C-|.4 alkyl)-substituted amino; R2 is an amino protecting group; R3 is hydrogen, benzyl, -CH2-P-C6H4OH, -CH2-indol-3-yl, -CH2CH2CH2CH2NH2, 30 -CH2CH2Ch2NHC(NH)NH2, -CH2-imidazol-4-yl, -CH2COOH, -CH2COO(Ci_4 alkyl), -CH2CH2COOH, -CH2CH2COO(C-|.4 alkyl), -CH2CONH2, -CH2CH2CONH2. -CH2SH, CH2CH2SCH3, C-j.12 alkyl, C2-8,aMi alkynyl, C5.8 cycloalkyl, aryl, heteroaryl, or aryl or heteroaryl yftitch is monaj^i, or 41 °eC'S9s 245 473 WO 93/12129 PCT/US92/10921 trisubstituted independently with halogen, nitro, C1-4 alkyl, C1.4 alkoxy, trifluoromethyl. R4 is hydrogen or a carboxylic acid protecting group; and B is a purine or pyrimidine nucleoside base provided that if R1 and R3 are hydrogen, B is a purine nucleoside base, or an acid or base addition salt thereof. 8. The monomer of Claim 1, wherein B is a purine nucleoside base. 9- A pharmaceutical formulation comprising a compound as claimed in any one of claims i to 6 together with a pharmaceutical^ acceptable carrier therefor. 10. a compound as claimed in any one of claims 1 to 6 for use in medicine. • A compound as claimed in any one of claims 1 to 6 for use in the manufacture of a medicament for the treatment of a condition which may be ameliorated by affecting genetic material. 12. A nucleoside base oligomer of the formula (X) as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 13. A nucleoside monomer of the formula (X) as defined in claim 7 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 14. A pharmaceutical formulation as claimed in claim 9 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. C? //WO IJOC- By the authoriserfSgante, A J park&sdn